key: cord- - rfbmi q authors: nan title: news date: - - journal: aust vet j doi: . /avj. sha: doc_id: cord_uid: rfbmi q nan please email emma.malcolm@ava.com.au a small version of your photo and confirm that you also have a high resolution to send us. final images have to be in crystal-clear focus, at a minimum of dpi and at least mm at the longest edge. for us to publish an image on the cover it needs to be at least x mm at dpi. we had some lovely photographs that we couldn't publish this year because they were taken on a camera phone and the resolution was too low. as good as phone cameras can be, many still aren't going to be able to take a photograph that is high resolution enough to print at a reasonable size. so get your 'old fashioned' digital camera out! if you would like to try to take a cover shot, keep in mind that we have to be able to add the avj masthead at the top and highlights down one or both sides. it's also important to send us an accurate description of the photo, otherwise we might get eagle-eyed members calling to tell us we've got our cows confused -thank you robert mills. where to on welfare? julia nicholls, president f ootage of australian animals being mistreated during slaughter in kuwait, jordan and gaza prompted our members to respond in various ways to the confronting images and to ask what the ava is doing in response. it was hard not to be moved and horrified by the footage, and members have questioned the currency of our policies and position statements on live animal export and humane slaughter. the details of what ava can do are relevant to the debate; we can only comment and question. these breaches of the exporter supply chain assurance system are being investigated, export of animals to gaza is suspended and the department of agriculture keeps us informed of developments in this regard. enormous efforts have been made to improve the welfare of exported animals and it is remarkable that so much has changed for the better in destination countries given that australia has no jurisdictional control. despite this, i appreciate that there are many of us who would like to see an end to live export and others whose livelihood depends on the trade. our membership reflects the diversity of opinion of australian society and this makes it difficult for the ava to take sides in some circumstances. our membership is diverse, but our opinions are reinforced by a deeper knowledge of animal science and physiology. the ava can and does comment on issues of the day relevant to our expertise, and our profile and influence are steadily increasing. we have invested significantly in our public affairs program, which includes setting goals and tracking our achievements on behalf of members. however, i think many members overestimate the ava's ability to influence public opinion and bring about change. just because we are best qualified to comment doesn't mean that we enjoy the most attentive listeners to those comments! part of the art of advocacy is to understand what you can change, as well as how you can change it. there are many animal welfare issues of concern to both the community and members. what are the most important ones for our members and the long-term benefits for animals? how do we reflect your views and represent you to stakeholders and politicians? is there a common thread or philosophy on animal welfare across all members? can we better articulate a more robust and satisfying basis for the ava's policy and advocacy work on animal welfare issues? these are the questions we are hoping to explore and develop further within our public affairs program over the next year. stay tuned to ava communications for opportunities to be part of the conversation. other veterinary associations are doing a range of activities in relation to animal welfare issues. late last year, the american veterinary medical association held a forum called 'the conversation' , which involved veterinarians, ethicists and animal scientists who presented on the scientific, social, political, market, and legal aspects of how and why animal welfare decisions are made. the papers and discussion are being collated into a resource. the nzva has an animal welfare strategy with a stand-alone vision: "our members, using a science-based and ethically principled approach to the humane treatment of animals in new zealand, are respected and recognised for their leadership and educative role in animal welfare and well-being" and mission: "to enable and promote our membership as having the knowledge, skills and leadership in the field of animal welfare and wellbeing". the strategy also emphasises the need to be proactive in relation to animal welfare. the canadian veterinary medical association (cvma) has an animal welfare committee whose purpose is to advocate and promote animal welfare within the animal industry, to government and the public, and to advise the cvma on animal welfare issues and develop pertinent position statements. animal welfare advocacy is a priority of the cvma. should ava hold a conversation-type event? should we have a stand-alone strategy? should we have a welfare committee? perhaps all of these? i'd be interested to hear your thoughts in the discussion forums or as a comment on this article on the website. finally, i wish each and every one of you a very enjoyable festive season, filled with catching up with friends and family and not too many calls on your work time. this is a time of year when we take stock and reflect on the past while making resolutions for the next year. the ava is no different and we are looking ahead to planning our new -year strategy for - in the first months of next year. this will involve input from as many of you as possible, so start thinking about what you expect from your association. i wish you all the best for in your work and personal lives, may you find that elusive balance and continue to enjoy your chosen profession. financial products and services described in this document are provided by boq specialist bank limited abn (boq specialist). boq specialist is a wholly owned subsidiary of bank of queensland limited abn (boq). boq and boq specialist are both authorised deposit taking institutions in their own right. neither boq nor boq specialist guarantees or otherwise supports the obligations or performance of each other or of each other's products. the issuer of these products is boq specialist afsl and australian credit licence . all finance is subject to our credit assessment criteria. terms and conditions, fees and charges and eligibility criteria apply. equipment and fit-out finance / home loans / commercial property finance / car finance / practice purchase loans / smsf lending and deposits / transactional banking and overdrafts / savings and deposits / foreign exchange stress, insomnia, anxiety. we're expert in handling these conditions. veterinary professionals know just how important it is to put an owner's mind at ease. it takes expertise to understand the problem and the ability to clearly explain the options to patient and owner. at boq specialist, we operate in much the same way. over the last years, we've developed a profound understanding of how the veterinary profession works. you are our area of expertise and we've developed an extensive range of products to meet your banking and finance needs. so, if there's anything on your mind talk to us, and then enjoy a good night's sleep. visit us at boqspecialist.com.au/ava or speak to our financial specialists on . boqs e ducation is the cornerstone of every profession. the ava has close ties to australia's veterinary schools and we provide input where appropriate to help universities produce the veterinarians of the future. as you know if you read these columns, one of the ava's key strategic priorities is workforce planning. we want to develop and advocate for good evidence-based policies that will provide the right number of veterinarians, with the right skills, in the right places, to meet australia's need for veterinary services into the future. when the commonwealth government announced significant changes to higher education in the may budget, we were concerned that this could affect the veterinary workforce. after more analysis, our modelling showed that the proposed changes had dire implications for the debt accrued by a veterinary student and its repayment. our detailed submission to the senate inquiry into the legislation was followed by an invitation to speak at a public hearing. national strategy manager, dr debbie neutze, spoke passionately and persuasively about the potential effects on the profession and answered a range of questions about the profession. the submission we made to the senate and the communications we've been sending to the media have emphasised that veterinarians provide services that no-one else can provide. these include essential services to biosecurity, public health and food safety. the higher education changes in the proposed bill, we believe, could create unintended and inequitable outcomes for the veterinary profession that threaten those vital services. the changes will affect veterinary students more than other student groups because of the length of study and the high cost of delivering the course combined with the significantly lower average lifetime earnings than graduates of comparable courses. we are already witnessing market distortions that will potentially affect the viability of the veterinary profession and its ability to provide essential services. our view is that that the proposed changes to higher education are almost certain to distort the market further. at the time of writing, the senate had not voted on the bill. we are expecting the vote to happen at the beginning of december -close to the time you'll be reading this article. however, we anticipate at least one favourable outcome -the interest rate increases are unlikely to be passed. because of the attention ava focussed on this aspect, particularly for female graduates, we can claim some of the credit for this amendment. over the past few weeks, ava president julia nicholls and i have been meeting with each veterinary school dean or head to discuss our position on the proposed changes. whatever the outcome of the proposed higher education changes, it is critical that we maintain open and honest dialogue with the universities and educators. our meetings have been very positive and i'd like to thank each of the deans for their time and candour. early next year, we are looking forward to reporting on the workforce modelling we've been doing throughout this year. the stock and flow model of the veterinary workforce currently in preparation will analyse both supply and demand factors for veterinary services. the ultimate aim is to create policies that are relevant to the current political and policy environment to ensure a healthy future for the profession. we want to ensure appropriate levels of supply and demand for both current and future veterinarians, and also ensure student debt doesn't become something that veterinarians have to service for decades in their careers. the number and skills of new entrants to the workforce will be part of this analysis, so we anticipate further cooperation and conversations with the veterinary school deans as we respond to the workforce modelling findings. the only person who is educated is the one who has learned how to learn and change. international veterinary, biomedical and business journals at your fingertips ebola, dogs and a vaccine i n october, a nurse in spain tested positive for ebola virus after caring for an infected patient in hospital. people who were in contact with this nurse were quarantined, and the madrid regional government obtained a court order to euthanase her pet dog against her wishes, on the grounds that available scientific information could not rule out a risk of contagion. wsava is strongly of the view that available technology should allow for testing and quarantine, rather than automatic euthanasia of exposed animals. professor michael day, chairman of the wsava's one health committee, noted: "zoonotic diseases, particularly those transmitted through pets, are concerning to the pet-owning public, but there have been no scientific reports indicating that ebola virus has been isolated from or directly transmitted by dogs." the virus that causes ebola is not airborne and can only be spread through direct contact with the bodily fluids of an infected person who is showing symptoms or who has died. dogs appear to be the first animal species shown to be naturally and asymptomatically infected by ebola virus. during the - ebola outbreak in gabon, one group sampled dogs, following the observation that several dogs were highly exposed to ebola virus by eating infected dead animals. dogs were screened using ebola virus-specific immunoglobulin (ig) g assay, antigen detection, and viral pcr. the serological report showed a significant positive association between seroprevalence and the distances to the ebola virusepidemic area, with seroprevalence up to . % in dogs from villages with both infected animal carcasses and human cases. the authors did not detect any circulating ebola antigens or viral dna sequences (tested by pcr), and were unable to isolate any virus. they suggest that this indicated either old, transient ebola infection of the tested dogs, or antigenic stimulation. none of these highly exposed dogs during the outbreak showed any clinical signs. it is possible that dogs may excrete infectious viral particles in urine, faeces, and saliva for a short period before virus clearance, as observed experimentally in other animals. further work on experimental canine infection is needed to establish the potential human risk of ebola virus-infected dogs, including the mechanisms of viral excretion. the main concern in west africa is infection via fruit bats and non-human primates, and by eating bush meat that may include fruit bats and primates infected with ebola. a house pet that may potentially be exposed in developed countries represents a very different scenario to those in epidemic west africa. early human testing of an investigational ebola vaccine co-developed by the national institute of allergy and infectious diseases (niaid) and glaxosmithkline (gsk) began in early september. initial data on safety and immunogenicity (the capacity to generate an immune response) from clinical trials of the niaid/gsk ebola vaccine are expected by the end of . human testing of a second ebola vaccine candidate is under way at the national institutes of health's clinical center in bethesda, maryland. one group is conducting a trial to evaluate the vaccine, called vsv-zebov, for safety and its ability to generate an immune system response in healthy adults who are given two intramuscular doses. this nih phase placebo-controlled clinical trial of the vsv-zebov vaccine candidate will enrol healthy adults. enrolment at each dosing level is staggered, so interim safety assessments of vaccinated individuals can be conducted. vsv-zebov is based in part on a genetically engineered version of vesicular stomatitis virus (vsv), which primarily affects rodents, cattle, swine and horses. the gene for the outer protein of the vesicular stomatitis virus has been replaced with a segment of the gene for the outer protein of the zaire ebola virus species, and the vaccine cannot cause a vaccinated individual to become infected with ebola. a second group at the walter reed army institute of research is simultaneously testing the vaccine candidate as a single dose, to evaluate in real time the safety profile at different dosages and compare the immune responses induced by one injection versus two injections. initial safety and immune response data on the vsv-zebov vaccine are expected by the end of . this vaccine candidate was developed by researchers at the public health agency of canada's national microbiology laboratory, and has been developed from preclinical to clinical testing stage remarkably quickly. this story can be compared to the extraordinary work of dr deborah middleton's group, which developed the hendra virus vaccine so quickly in australia. medivet animal health, a world leader in veterinary regenerative medicine, has a comprehensive product offering for animals including stem cell therapy, platelet rich plasma and nanofiber technologies, amongst others. • improves the quality of life of your patients t he ava is seeking nominations for its board. can you join the leadership crew and help to steer the profession? here are some tips and information to help you decide if joining the ava board might be the right thing for you. the ava has nine directors. six are elected directly by the general membership and three are nominated and appointed by the three largest special interest groups (cattle, equine and asava). each director has a -year term, with a maximum of two successive terms. the president, vice president and treasurer are elected each year by the board. nominations are invited for two elected positions on the board of directors for a -year term to take office in may . these elected positions in are in addition to a position to be nominated by asava. the first requirement is that candidates have prior experience as an office holder within a special interest group or division committee. "to be a good board member, you need to be a good listener and then you need to take the information given to you and be a good advocate for the profession, " dr gilkerson said. some additional skills and experience that are useful include being a member of community organisations, being a member of other boards and committees, a commitment to animal health and welfare, and the ability to prepare reports for the ava board. with a lot of the work involving the ava's groups and committees, communication and team work are key skills to bring to the ava board. "it helps to be patient, diplomatic and to communicate clearly. directors need to have a broad knowledge of the different groups within the ava and how they work together, " board member dr robert johnson said. "if you are interested in helping our profession, joining the board is a great way to do that. you soon learn what matters to your fellow members, and it is a privilege to be a voice for them. " the board has a charter and a code of conduct that outline the expectations and operational details of the board. all election candidates need to agree to abide by the charter, the code and the ava constitution before their nominations are accepted. directors have legal responsibility for the australian veterinary association limited (acn ) under the corporations act and they undertake mandatory training to help them understand and fulfil their legal responsibilities. these include ensuring that the ava complies with all aspects of the law, risks are managed appropriately and all parts of the organisation are doing what they should be doing for the benefit of members. the board sets the direction for the ava, makes the big strategic decisions and ensures effective risk management. the board appoints the chief executive officer and is responsible for managing his or her performance. the ceo is responsible for managing the ava's employees and ensuring the board's strategy is realised. if you haven't already, you can put some faces to names by viewing a video of the ava board meeting earlier this year: http://ow.ly/e rmf. t he object of the ava's animal welfare trust (awt) is to provide small grants for research, education or promotion and action programs that lead to improvements in the welfare and wellbeing of animals. in october , awt's grants committee critically evaluated and ranked eight worthy research proposals involving a range of species. awt trustees accepted the committee's recommendations and offered two grants for the following research projects. yu zhang, phd candidate, university of queensland this project focuses on how ammonia accumulation affects sheep's feed intake during simulated sea export by studying their physiology, behaviour and emotional state. as the largest live export industry in the world, livestock shipments from australia mostly involve sheep, especially to the middle east. during a voyage, ammonia is released from their excreta, which accumulates as a pad that the sheep have to lie on. ammonia especially accumulates when there is insufficient ventilation, high temperatures and humidity. this is a recognised welfare problem and causes sheep to stand more, feed and ruminate less, hold their heads high for fresher air and suffer conjunctivitis. the university of queensland recently conducted research on gaseous ammonia accumulation on livestock ships that has been used by rspca to seek the establishment of legal limits. however, it is unknown how sheep's feed intake is affected by ammonia on ships. using a simulation, the research objectives are to: • understand how high concentrations of gaseous ammonia affect feed intake • study the sheep's emotional state after exposure to gaseous ammonia. project results will increase our understanding of the welfare implications of long-distance sheep export. in the longer term, they will contribute to modification of sea transport standards to improve sheep welfare and reduce lower returns caused by weight loss. outcomes may also help improve welfare issues associated with ammonia accumulation in intensive farming. effect of shearing in pre-embarkation feedlots on sheep feeding behaviour before export awt will fund an honours project under associate professor anne barnes at murdoch university. inappetence late in feedlotting has been shown to increase the risk of shipboard death and consistent feed intake is the key to preventing shipboard deaths from inanition and salmonellosis. therefore, any interference with consistent intake might have poor health and welfare outcomes. sheep exported live from australia are required to spend time in registered premises before shipping. during this time they are fed pelleted feed similar to that provided on the ship so that they can adapt to it, which may take several days. some sheep may never adapt to the different feed, leading to inappetence, which is associated with salmonellosis and eventual starvation. disruption and moving may delay feed adaptation, increasing the risk of inappetence and disease at the feedlot and/or on board. sheep are often shorn during their pre-embarkation preparation to limit wool cover and enhance heat loss. there are no specific standards for the timing of shearing in sheds in relation to the time of embarkation. previous studies report that short periods of restraint, isolation and shearing are acute stressors for sheep. superimposing these on animals undergoing adaptation to a novel environment and feed could affect their continued intake of feed. the research objective is to determine whether moving and handling sheep for shearing at the pre-embarkation feedlot interferes with their feeding pattern. video footage of the sheep's initial entry to the shed, after shearing and before exit will be analysed using qualitative behavioural assessment (qba). using this, observers score descriptors of how animals behave to create an integrated measure of behaviour for comparison of treatments. qba scores are correlated to physiology and ethology and are a non-invasive means of assessing livestock welfare. project results will provide guidance for the industry on whether and when shearing should take place during pre-embarkation. it will inform best practice in the intensive sheep industry regarding the effects of shearing on feeding behaviour during feedlotting. outcomes of both projects will be disseminated via refereed journals, conference presentations and advice to industry. of the european countries that provided data over this timespan, observed decreases ranging from . % to %. "these latest figures, which suggest a positive trend in terms of the responsible use of antibiotics in animals in europe, are highly welcome, " explains david mackay, the head of the ema's division on veterinary medicines. "however, the report also shows that there is scope for further decrease. "measures to promote the rational use of antibiotics in animals need to continue as part of the european commission's action plan against antimicrobial resistance, " he said. public health authorities worldwide are confronted with increasing levels of resistance to antibiotics in humans and animals and are engaged in actions at various levels to fight this issue. the responsible use of antibiotics is a key factor to minimise the risk of resistance. the ava has been working closely with the australian department of agriculture and human health groups to join this global campaign to promote responsible use of antibiotics. • national programs and campaigns on the responsible use of antimicrobials • restrictions on the use of certain antimicrobials • increased awareness of the threat of antimicrobial resistance • reduction targets for the use of antimicrobials in animal production in certain member states • fluctuations in size and types of animal populations. while additional analysis is needed to confirm the main reasons for this decline, the reduction in the use of antibiotics is a positive sign. l ife for women was very different when judith went to school. she had dreamed of being a veterinarian while spending her school holidays on her uncle's property in guyra. attending a private girls' school in the s and early s put her at a disadvantage for pursuing a career as a veterinarian, as physics and chemistry were not deemed important areas of study for young girls. luckily, she was able to do biology and latin, which were approved prerequisites for veterinary science at the time. "my school leaving certificate marks were good enough to receive a commonwealth scholarship to study at the university of sydney, " she said. "i accepted it and indicated that i planned to study veterinary science. "i was called for an interview at which it was strongly recommended that i use the scholarship to do an arts degree, particularly as i was so disadvantaged, not having studied physics or chemistry. "i stood firm -i had my scholarship and i had my opportunity, " she said. in her first year at vet school, she was the only woman and for whatever reason, her male counterparts decided to elect her as the year representative. this role gave her valuable contact with staff through vet school meetings and allowed her greater access to the vet school area of the university than other first years who had very limited access because they were 'lumped in' with medicine and science students. "i loved the vet school building; the people, the work, the smells and the lifestyle. "elbert was a good teacher, and would be a boss i enjoyed and respected but to my horror he went on holiday a few days after i started work… "how to make a nervous wreck of a new graduate!" "in those early days i remember monitoring a sick heifer in my backyard, which died and whose autopsy revealed one of the early cases of sbe. calvings were chain and rope pull jobs, distemper was rife and haemonchus in lambs was devastating, " she said. after marrying rod, the couple moved to armidale. he had become a jackaroo after deciding not to complete veterinary science. the local veterinarian, dr joe o'brien, had heard that she was coming. "very soon after arrival, joe arrived on my doorstep, announced who he was, checked who i was and asked why the [expletive] was i sitting up reading a book when there was vet work to be done?" "i learnt much from joe, who calls a spade a spade and was an innovative, real-thinking vet and excellent surgeon. we had an excellent ava branch and meeting up with colleagues was a vital way for us all to learn and share information, " she said. however, the erratic hours and unpredictable life of a veterinarian in general practice and marriage were not sitting well and she reluctantly applied for a lecturing position in rural science at the university of new england. everything looked great until the fur started to grow. a siamese with a fluffy hindleg with the fur growing the wrong way made a good talking point," australian veterinary journal volume , no , december news n "i was told that as a female i was not a likely long-term proposition for the lecturing position in parasitology. prejudice withstanding, i became demonstrator in anatomy, histology and parasitology and enrolled to do my phd, working on ovine brucellosis. "i enjoyed the teaching, but research and the experimental work were not really my thing. i yearned for general practice over those years, and by the time i submitted my thesis, i had four children. "further experimental work was requested from a biochemist examiner of my thesis. so, i decided to call it quits. this was a huge decision as i had invested so much time and my 'no more research' stand represented failure. but i took great delight in being a mother and knew that my priorities would always be with family, " she said. the tb and brucellosis eradication scheme gave dr grieve her next veterinary venture. "i was often working with four children and a surrogate grandmother as baby sitter travelling to remote properties and 'interesting' yards, like one that was constructed of iron bedheads. "over this time i made good friends with many of the land holders and found myself doing more small jobs and bringing the odd cat or dog back with me to return on the next visit having done whatever treatment it required, " she said. dr grieve's next career move was to buy an old building, which she moved to her property and set up a small surgery. "with my children at the local school, i met more of the locals and built up my small practice, and then had another baby. "mixed practice hours didn't work with school hours and i started having to exploit friends and neighbours for school pick-ups and drop-offs. to prioritise my children, it became obvious that i should be keeping close to home (and the school) by doing small animal and 'anything that could be brought to the surgery' work, with limited local large animal cases, " she said. w ith ava special interest groups (sigs), there's one for everyone. you bring your own expertise, skills and experience to join others with similar interests to exchange ideas, inspire and support each other. here's a rundown of the different sig opportunities available to ava members. our largest sigs are for veterinarians working with cattle (acv), horses (eva) and small animals (asava) and in practice management (avapm). "you can really benefit from connecting with your like-minded veterinarians through the ava's sigs, " ava president julia nicholls said. "the big sigs offer outstanding value if you're involved in these areas. they keep you on the cutting-edge of your field by providing you with a range of member benefits. from clinical journals, to tailored continuing professional development and a novel strain of coronavirus that causes a rapid onset of severe respiratory disease in humans was first identified in saudi arabia in . as of november , the world health organization (who) has been notified of laboratoryconfirmed human cases of mers-cov, including at least related deaths. the virus appears to be circulating widely throughout the arabian peninsula, and travel-related cases have been reported in europe, africa, asia and the united states. there have been no cases reported in australia. mers-cov is genetically and biologically distinct from other known coronaviruses such as the virus causing severe acute respiratory syndrome (sars) in humans. it is considered to be a serious public health threat, because the infection can cause severe disease in humans and coronaviruses may adapt to new hosts and become more easily transmittable between humans. the epidemiology of mers-cov infection is not yet fully understood. similar strains of mers-cov have been identified in samples taken from camels and humans in the middle east and in some cases there has been an association between infections in humans and camels, suggesting that camels are a likely primary source of the mers-cov infection in humans. viruses genetically related to mers-cov have also been detected in bat species around the world, and a fragment of viral genetic material matching the mers-cov was found in one bat from saudi arabia. however according to the world organisation for animal health (oie), the current evidence does not indicate a direct link between bats and mers-cov in humans and further research is warranted. other species of animals, including sheep, goats, cattle, water buffalo and wild birds, have tested negative for the presence of antibodies to mers-cov. nevertheless, owing to the relatively small sample sizes, the results of these studies cannot exclude infection in other animal species. the oie, who and fao emphasise that joint human health and animal health investigations are needed to develop a better understanding of the overall epidemiology of mers and the role of camels and potentially other animal species in the maintenance and transmission of mers-cov. clusters of confirmed human cases have occurred in healthcare settings and households, but there is no evidence of sustained human to human transmission in the community. immunocompromised persons and those with diabetes, cancer and chronic lung disease are considered at high risk of severe disease from mers-cov infection. the who recommends that as a general precaution anyone visiting farms or other places where camels are present should practise general hygiene measures, including regular hand washing before and after touching animals, avoiding contact with sick animals and following food hygiene practices. for latest information on mers-cov, visit the who website: www.who.int/ resolutions are generally of two types -special resolutions and ordinary resolutions. any resolution to amend the ava constitution requires a special resolution. in order to be passed special resolutions require the approval of at least % of the members entitled to vote on that resolution. ordinary resolutions are for matters relating to the accounts or reports of officers of the ava. in order to be passed an ordinary resolution requires the approval of a simple majority of more than % of members entitled to vote on the resolution. the wording of a proposed resolution may require amendment after submission (in consultation with the proposers of the resolution). this is to ensure the resolution is in an acceptable form and provides sufficient clarity, particularly if the resolution involves an amendment to the ava constitution. before submitting a resolution, please contact the company secretary who may assist with preparation of an acceptable form of wording of the resolution. it is also appropriate to discuss any proposed resolution with a wide range of members prior to formally lodging it with the company secretary to ensure that the proposed wording of the resolution clearly conveys your objectives. all proposed resolutions will be considered by the ava board, and if they are of the view they are in the best interests of the ava, put to the annual general meeting. for more information contact john robb, company secretary at ava national office on or corporate@ava.com.au. chief executive officer comment on this article at www.ava.com.au/ member communications, information resources and guidelines, client-centred materials and accredited programs, your sig keeps you at the forefront of your field. "the collegiality that comes with joining your sig is invaluable. it provides you with support, and enables you to support your colleagues, inspires new ways of thinking and doing things, and may even provide some much needed light relief from time to time, " she said. not involved with cows, horses, small animals or practice management? then we have boutique sigs to cater for nearly everyone. these sigs have fewer members but no less collegiality or passion for their work. most communicate with members via email or through the ava's discussion forums. like the larger sigs, these smaller ones bring together leading veterinarians in a particular field to exchange knowledge and support each other. "the ava's sigs are representative of the diversity within our profession, " dr nicholls said. "whether these smaller sigs represent your daily working activity or they cover something that you're interested in, it's well worth the nominal investment to connect with your colleagues in these groups, " she said. learn the ins and outs of all the unusual and exotic pets or explore some new territory through the acupuncture or integrative medicine group. the public health vets share important information about government issues, while the animal welfare and ethics group explores the philosophical and practical aspects of welfare. the behaviour and dental groups are growing quickly as practitioners understand the value of broadening their skills and knowledge into new areas to meet the demands of modern practice. pig, sheep, reproductive, alpaca, greyhound and poultry vets are active in creating networks and sharing the latest information within their defined fields of practice. conservation biologists come together to explore the latest in wildlife health, disease and management and species conservation. the education and research sig represents veterinarians working in educational institutions and research bodies, while the history group produces the australian veterinary history record, which chronicles the rich and diverse history of veterinary medicine in australia. you can view the array of sigs on offer at www.ava.com.au/about-us/who-does-what/groups. w ith the christmas holiday season fast approaching, it's a good time to stop and think about the forthcoming social events that will be organised within workplaces for staff, clients and visitors. such celebrations provide a great opportunity to bond with colleagues and clients, as you can get to know them more in an informal setting. it's not an opportunity to lower your conduct standards such that you end up embarrassed to turn up to work on monday. together with employees, employers have a responsibility to take all reasonable steps to ensure the safety and health of all who attend work-related functions. this includes a duty of care under work (occupational) health and safety, a range of antidiscrimination legislation, including provision for dealing with sexual and racial harassment, and the criminal code, which includes assault of either of a physical or verbal nature. all of which promote an environment free of discrimination, harassment, bullying and violence, whether or not the function is held offsite or onsite. we have all heard those embarrassing stories of staff doing things they should not have done at christmas parties, only to find photos that you did not want people to see on social media and the professional and social fallout that comes from such posts. this should be enough to make you stop and think about the photo you would prefer to see of yourself on social media. although there are great advantages and value to holding workrelated functions, there are also risks, which, if not managed appropriately, can quickly turn a joyous and enriching time into a nightmare. as many social functions are provided as an extension of the workplace, it is important that employees are aware of the inappropriate behaviour that can surface, especially when functions are highly charged with alcohol. if you're planning to attend an upcoming work function, be aware that you have a responsibility to take reasonable precautions for your own health and safety and that of others. you are expected to conduct yourself in a respectable manner that leaves the workplace function free from harassment and other offensive behaviour. avoid becoming intoxicated to the point where your behaviour causes risks to health and safety. such behaviour includes unwanted touching and kissing, derogatory comments and humour, violence, inappropriate gifts and send-ups, oversharing and illegal drug taking. strategies to minimise the risk of staff social functions getting out of control may include the following: • before the function, remind all staff of company policies and standards of behaviour in relation to drugs, smoking and alcohol, dress code, bullying and harassment, particularly sexual harassment • discourage excessive drinking and remind all staff of the dangers of drink-driving • take reasonable steps to ensure the consumption of alcohol is limited and make sure non-alcoholic drinks are also available • ensure staff make arrangements to get home safely • provide plenty of food, especially when serving alcohol • have set start and finish times for the function • have a grievance handling procedure in place to deal with any complaints, should they arise • remind management and senior staff that they are role models for expected appropriate behaviour • raise staff awareness of the consequences of breaching of company policy, such as warnings and possible dismissal. the best functions are those that are well thought out and fundamentally underpinned by clear, concise and reasonable policies, procedures and expectations in relation to the acceptable codes of conduct of all who attend. such planning will go a long way to making sure the work function is successful and enjoyed by all. comment on this article at www.ava.com.au/ the material contained in this article is general comment and is not intended as advice on any particular matter. no reader should act or fail to act on the basis of any material contained herein. the material contained in this publication should not be relied on as a substitute for legal or professional advice on any particular matter. the veterinary nurse council of australia and hill's pet nutrition presented the award, which recognises qualified veterinary nurses employed within a veterinary clinic, who provide exceptional service and deliver the highest possible standard of patient care. editor in chief t ri-solfen (t-s) was developed in response to animal welfare concerns about the animal husbandry practice of mulesing of lambs intended for wool production. many woolproducing sheep are excessively wrinkly in the breech area, which predisposes them to blowfly strike. removal of this wrinkly skin in the breech area by a process known as 'mulesing' produces a plainer skin profile, which in turn mitigates the occurrence of flystrike. the active ingredients of t-s are the local anaesthetics lignocaine (lig) and bupivacaine (bup), together with adrenaline as a vasoconstrictor and the antiseptic cetrimide. these are formulated together in t-s and the product is used as a post-mulesing dressing in lambs. the recommended age for mulesing lambs is - weeks, with the maximum age allowed under the present commonwealth government model code of practice for welfare of animals being months. mulesing is only carried out on young sheep that are destined for wool production. in reality, any concerns about residues from the four actives contained in t-s would be essentially zero, as any traces would have disappeared long before the sheep entered the human food chain. the interval between application and slaughter in almost all cases is likely to be many years, with some sheep dying or being used for pet food. to further reduce human health concerns, all four active ingredients were chosen by the developers because they already had widespread and safe use in human and veterinary medicine. three of the active components are used topically in humans, and lig and bup are also administered parenterally. the hazard is known and is essentially zero. the australian medicines handbook (amh) recommends that the maximum single injected dose of lig in humans is mg/kg body weight (bw) when used alone or mg/kg bw when administered with adrenaline. the injected dose for bup with or without adrenaline is recommended at mg/kg bw. the amh also lists a number of human preparations of lig used in the nasopharyngeal area or gastrointestinal tract, which results in oral intake of lig. the amh does not recognise therapeutic doses of lig or bup in humans as a particular hazard. lig and bup do not persist in mammals because they are rapidly metabolised, particularly in the liver. the plasma halflives of lig and bup in all species are very short (range - minutes), depending on the species, age, dose and route of administration. the metabolic pathways are common to humans and other mammalian species, including sheep, cattle and pigs. excretion of parent compounds and their metabolites is rapid and predominantly in the urine. , metabolism results in degradation of the compounds, which is responsible for the loss of local anaesthetic action. the australian office of chemical safety has reviewed the toxicology and occupational safety of t-s and established an acceptable daily intake (adi) for its active ingredients: . mg/ kg bw/day for lig, . mg/kg bw/day for bup and . mg/kg bw/day for cetrimide. an adi for adrenaline was not set because residues are not expected to be measurable above endogenous levels normally found in the tissues. the biotransformation of lig and bup produces , -xylidine as a minor metabolite that has been shown to be a weak mutagenic agent in vitro and to have genotoxic characteristics in vivo. this metabolite produced nasal tumours in a -year oral toxicity study in rats receiving daily doses of the metabolite equivalent to mg/kg bw/day. the incidence of these tumours was dosedependent and there was no significant increase in nasal tumours at either of the two lower dose rates of and mg/kg bw/ day. in this rat study it was reported that some of the , -xylidine evaporated from the feed and was inhaled by the rats throughout the study period. the nasopharangeal anatomy of rats is unlike that of humans, which leads to differences in the amount and distribution of inhaled materials. rodents are also obligate nose breathers with a highly efficient nasal filtering capacity. a further complication in rats is spontaneous respiratory infections and their sequelae. rats appear to be uniquely susceptible to chronic inflammation and tumours from insoluble cytotoxic particles. , overall, this makes the rat an unsuitable model for predicting the significance of chemicals on the induction of nasal tumours in humans. the unique characteristics of the rat and the volatility of the , -xylidine make it difficult to place any significance on the appearance of nasal tumours in the chronic rat study. the results of the -year rat study using , -xylidine have been extensively reviewed and it is accepted that this metabolite of lig and bup does not represent a hazard to human health. , the end result is that when meat-producing animals are treated with t-s, the possibility of , -xylidine residues being present and causing problems in humans is not an issue. furthermore, human pharmaco-vigilance studies over a long period have not revealed any reports of carcinogenicity following the widespread therapeutic use of lig or bup. the lower limits for finding or detecting (lod) lig and bup and their metabolites in tissues and bodily fluids using contemporary mass spectrometric based methods are in the range of - ng/ml or gram. the regulatory methods actually used to measure the amount of residues in meat and offal have limits of quantification (loq) of . mg/kg for bup, . mg/kg for lig and . mg/kg for cetrimide. in the case of t-s, in order to keep residues of lig, bup and cetrimide as low as practicable, the australian pesticides and veterinary medicines authority (apvma) originally used the loqs for these active constituents to set the maximum residue limit (mrl). continued on page n australian veterinary journal volume , no , december news n based on published methods of calculation and data, , if the adi for lig of . mg/kg bw/day is used, the acceptable intake of lig and its metabolites is . mg/day for a -kg human ( . x = . ). similarly, the acceptable intake of bup plus metabolites is . mg/day. thus, the total acceptable maximum daily intake of the parent local anaesthetics and their metabolites, expressed as parent drug is . mg ( . + . = . ) for a -kg human. the standard intake of meat is a total of . kg/day, made up of the sum of muscle, liver, kidney and fat. the most conservative mrl set for either of the local anaesthetics is for bup at . mg/ kg meat. therefore, the highest acceptable daily consumption of meat from treated animals can be estimated by dividing the total acceptable drug intake of . mg by the conservative maximum amount of drug permitted in . kg meat of . mg. this results in a theoretical maximum daily intake of kg of meat, which would be unrealistic. it must also be stressed that adis and the resultant mrls are levels for regulatory action and are not human health levels. in the case of lig and bup for t-s, the adis have been calculated using -fold safety factors. this means that the occasional consumption of meat containing residues that marginally exceed the mrl does not translate into a human health hazard. the management of any risks associated with the use of t-s for mulesing lambs has been achieved by standard regulatory procedures. the risk of residues in food and the associated risk to trade have been addressed by the apvma. the active ingredients are included in table of the australian maximum residue limit standard, where substances for which mrls are not necessary are listed. the entry for t-s in the mrl standard also contains the qualifier "as a component in a post-mulesing treatment of lambs that are kept for wool production". as a further safeguard, a meat withholding period (whp) of days after treatment has been established. at this time, more than million sheep have been treated with t-s and there have been no reported residue violations in australia, or in any other country. the safety of t-s has also been assessed by the australian national drugs and poisons scheduling committee and as a result has recently been placed in schedule , which identifies t-s as having "low potential for causing harm, the extent of which can be reduced through the use of appropriate packaging with simple warnings and safety directions on the label". the proposed new australian animal welfare standards include the requirement for pain relief for many routine animal husbandry practices in sheep and cattle. the result of these proposed changes will be to place ongoing pressure to make it a requirement to provide pain relief in sheep and cattle undergoing procedures such as castration, dehorning and tail docking. additional pressures to improve farm animal welfare are also being applied by processors of human food and by consumers of animal products. in order to meet these welfare requirements, there is a need for products, such as t-s, that can provide pain relief and infection control. however, as a consequence of these pressures, there have been reports of the use of t-s for purposes other than mulesing of lambs, which is the only registered use at this time. although it may be possible for a veterinary practitioner to use t-s 'off-label' for individual animals, this usage pattern was never intended to accommodate the mass medication of foodproducing animals. there is currently development work under way to provide data to support the extension of the registration of t-s to support other husbandry procedures in livestock. such work should provide a registered product that will meet the new and additional requirements of the livestock industries, through expanded therapeutic claims and associated appropriate usage patterns. it will also ensure that the risk of violative residues from all registered uses will not be an issue. this outcome will also continue to protect the use of t-s for mulesing of lambs. i came across a book recently by psychologist daniel kahneman, titled thinking fast and slow. for veterinary practitioners, this book makes for interesting reading. thinking fast and slow has become a bestseller and in it the author describes two different modes of thinking and decision-making. system operates automatically, with little or no effort, and no sense of voluntary control. system involves effortful mental activities, including complex computations. a punter may make a selection in a race because he thinks he is a good judge of horse flesh and this is a system or a heuristic selection. alternatively, a punter may spend a week studying form before he makes his selection and this is a system judgement. kahneman argues that although the heuristic process is useful, there are biases and traps that intelligent people and even statisticians fall into. system performs better, if established protocols and formulae can be set up. much of the book is about the biases of intuition. kahneman's stated aim is to improve our ability to identify and understand errors of judgment and choice, in others, and eventually in ourselves, by providing a richer and more precise language to discuss them. consider an anorexic cat presented to a practitioner. practitioner a: palpates pain in the anterior abdomen and quizzes the owner, who advises that the cat is fed a high-fat diet. a lipase test may or may not be performed. acute pancreatitis is diagnosed, which is a heuristic decision, especially if pathology tests are not performed. practitioner b: finds the same clinical signs but orders bloods, radiology and sonography. based on those results, a system decision is made. kahneman describes the traps and biases made by practitioner a as follows. . insensitivity to prior probabilities of outcomes. available textbooks indicate that pancreatitis in cats is rare. . insensitivity to sample size. even if pancreatitis was correctly diagnosed in two cats last year, that sample is too small to state that it is a common condition. . misconceptions of chance. there hasn't been a case of a cat with pancreatitis so far this year, so it is about time one showed up. this is using the gambler's fallacy -three tosses of a coin landing heads does not mean that the next one is any more likely to be tails. . availability. the client comes in with a preconceived diagnosis, usually made by an internet search. the client is often wrong, because of not having available experience or data. "i think my dog has ringworm, " for example. practitioner b is a big fan of evidence-based medicine, which seems to be gaining popularity in the veterinary as well as the medical profession. system is continually reinforced by system , so a more experienced practitioner will need less evidence-based medicine than a new graduate. in the emergency room, there is often no time for system , so an experienced practitioner with welldeveloped system knowledge will be more effective. our clients may not be able to afford evidence-based medicine, in which case, they will favour an intuitive practitioner. some practices may find it more profitable to use evidence-based medicine, particularly those with in-house pathology. fear of litigation may encourage some practitioners to use evidence-based medicine. i recommend this book to practitioners. it's pretty heavy going, but repays persistence. comment on this article at www.ava.com.au/ in tasmanian dogs t he paper by david jenkins and others and an associated news article published in the august edition of the journal remind us of the need for ongoing management of tapeworms in dogs, especially hydatids. i believe, however, that the present situation in tasmania has been misrepresented, especially in the news article, which concluded that "hydatid tapeworm (echinococcus granulosus) is still present in australian rural dogs, most commonly in tasmania, despite 'provisional eradication' having been declared in tasmania in ". tasmania ran an effective campaign to control hydatids from the s until provisional freedom was declared in . since then all detections of hydatids in livestock at slaughter have been investigated. although many of these detections can be explained by the animals having been infected on the mainland, there are occasional animals that have only ever resided in tasmania, so must have been infected locally. this indicates a low level of transmission of hydatids in tasmania, but the level of detection in abattoir suggests that transmission is a rare event. continued on page n avma seeks to promote intraprofessional dialogue about animal welfare issues ebola virus antibody prevalence in dogs and human risk world health organization (who) frequently asked questions on middle east respiratory syndrome coronavirus (mers-cov questions & answers on middle east respiratory syndrome coronavirus (mers-cov) update on mers-cov transmission from animals to humans, and interim recommendations for at-risk groups model code of practice for the welfare of animals: the sheep. primary industries ministerial council publication no. australian medicines handbook. australian medicines handbook pty ltd local anaesthetics that metabolize to , -xylidine or o-toluidine: final review of toxicological literature opinion of the scientific committee of the norwegian scientific committee for food safety. norwegian scientific committee for food safety australian government department of health and ageing office of chemical safety. health risk assessment for tri-solfen topical anaesthetic and antiseptic solution for pain relief in sheep hayes principles and methods of toxicology toxicology: the basic science of poisons. th edn mcgraw-hill the european agency for the evaluation of veterinary medicinal products, veterinary medicines evaluation unit. lidocaine summary report tri-solfen: anaesthetic and antiseptic solution for pain relief in sheep research permit (rp ) hayes principles and methods of toxicology national drugs and poisons schedule committee. scheduling proposals, final decisions and reasons australian animal welfare standards and guidelines echinococcus granulosus and other intestinal helminths: current status of prevalence and management in rural dogs of eastern australia publishing asia pty ltd, cremorne street, richmond vic. , australia. tel + ( ) . fax + ( ) . corporate citizenship wiley's corporate citizenship initiative seeks to address the environmental, social, economic, and ethical challenges faced in our business and which are important to our diverse stakeholder groups. we have made a long-term commitment to standardize and improve our efforts around the world to reduce our carbon footprint. follow our progress at www. wiley.com/go/citizenship journal customer services for ordering information, claims and any enquiry concerning your journal subscription please go to www.wileycustomerhelp.com/ask or contact your nearest office. prices include delivery of print journals to the recipient's address. delivery terms are delivered at place (dap); the recipient is responsible for paying any import duty or taxes. title to all issues transfers fob our shipping point, freight prepaid. we will endeavour to fulfil claims for missing or damaged copies within six months of publication, within our reasonable discretion and subject to availability. ltd. all journals are normally despatched direct from the country in which they are printed by surface air-lifted delivery. copyright © australian veterinary association. all rights reserved. no part of this publication may be reproduced, stored or transmitted in any form or by any means without the prior permission in writing from the copyright holder. this consent does not extend to other kinds of copying such as copying for general distribution, for advertising or promotional purposes, for creating new collective works or for resale. authorisation to photocopy items for internal and personal use is granted by the copyright holder for libraries and other users registered with their local reproduction rights organisation (rro), eg. copyright clearance center (ccc), rosewood drive, danvers, ma , usa (www.copyright.com), provided the appropriate fee is paid directly to the rro. this consent does not extend to other kinds of copying such as copying for general distribution, for advertising or promotional purposes, for creating new collective works or for resale. special requests should be addressed to permissionsuk@wiley.com disclaimer the publisher, the australian veterinary association and editors cannot be held responsible for errors or any consequences arising from the use of information contained in this journal; the views and opinions expressed do not necessarily reflect those of the publisher, the australian veterinary association and editors, neither does the publication of advertisements constitute any endorsement by the publisher, the australian veterinary association and editors of the products advertised. submission of photographs for publication will be held to imply that permission for publication has been obtained from the photographer and from the subject(s) of the image.avj.pi.feb trademarks ava and the ava logo are registered trademarks of the australian veterinary association limited. there is no evidence of local transmission of hydatids to people in tasmania. the tasmanian department of health and human services advises there has not been any detection of hydatids in people that can be related to infection acquired in tasmania after and evidence suggests that transmission to people ceased in tasmania in the early s. the tasmanian dogs reported by jenkins et al. were sampled as part of investigations into cattle infections detected at slaughter and as such comprise a sample that is heavily biased towards high-risk dogs. the prevalence detected in these dogs cannot be considered an indication of the broader situation in tasmanian dogs and also cannot be reliably compared with the results from a volunteer sample of dogs tested in other states. it is illegal in tasmania to feed livestock offal to dogs unless it has been cooked to the point of being commercially sterile. unfortunately, the section in the paper dealing with dog feeding practices did not differentiate tasmanian results from other states, so does not provide any indication of the level of compliance with this requirement.we are fortunate in tasmania that, unlike on the mainland, hydatids have never been detected in wildlife. this means that by continuing to educate at-risk groups not to feed livestock offal to dogs we have an effective tool for the control of the disease.tasmania will continue to follow up all detections of hydatids in livestock at slaughter to investigate and try to eliminate sources of infection. we will also continue to provide targeted information to dog and livestock owners about the steps they should take to prevent the disease. comment on this article at www.ava.com.au/ vic div hits a century! t he ava victorian division and more than of its members recently celebrated years with a dinner at parliament house. the premier of victoria, dr denis napthine also attended and is pictured below with victorian division president dr trish stewart.anne jackson editor in chief key: cord- -qw e cof authors: cantas, leon; suer, kaya title: review: the important bacterial zoonoses in “one health” concept date: - - journal: front public health doi: . /fpubh. . sha: doc_id: cord_uid: qw e cof an infectious disease that is transmitted from animals to humans, sometimes by a vector, is called zoonosis. the focus of this review article is on the most common emerging and re-emerging bacterial zoonotic diseases. the role of “one health” approach, public health education, and some measures that can be taken to prevent zoonotic bacterial infections are discussed. key points: a zoonotic bacterial disease is a disease that can be very commonly transmitted between animals and humans. global climate changes, overuse of antimicrobials in medicine, more intensified farm settings, and closer interactions with animals facilitate emergence or re-emergence of bacterial zoonotic infections. the global “one health” approach, which requires interdisciplinary collaborations and communications in all aspects of health care for humans, animals, and the environment, will support public health in general. new strategies for continuous dissemination of multidisciplinary research findings related to zoonotic bacterial diseases are hence needed. zoonotic diseases are those infections that can be transmitted between animals and humans with or without vectors. there are approximately pathogens, which are known to infect humans and % of these cause zoonotic diseases ( ) . the unique dynamic interaction between the humans, animals, and pathogens, sharing the same environment should be considered within the "one health" approach, which dates back to ancient times of hippocrates ( , ) . bacterial zoonotic diseases can be transferred from animals to humans in many ways ( ): (i) the transfer may occur through animal bites and scratches ( ) ; (ii) zoonotic bacteria originating from food animals can reach people through direct fecal oral route, contaminated animal food products, improper food handling, and inadequate cooking ( ) ( ) ( ) ; (iii) farmers and animal health workers (i.e., veterinarians) are at increased risk of exposure to certain zoonotic pathogens and they may catch zoonotic bacteria; they could also become carriers of the zoonotic bacteria that can be spread to other humans in the community ( ) ; (iv) vectors, frequently arthropods, such as mosquitoes, ticks, fleas, and lice can actively or passively transmit bacterial zoonotic diseases to humans. ( ) ; (v) soil and water recourses, which are contaminated with manure contains a great variety of zoonotic bacteria, creating a great risk for zoonotic bugs and immense pool of resistance genes that are available for transfer of bacteria that cause human diseases ( , ) . bacterial zoonotic infections are one of the zoonotic diseases, which can, in particular, re-emerge after they are considered to be eradicated or under control. the development of antimicrobial resistance due to over-/misuse of antibiotics is also a globally increasing public health problem. these diseases have a negative impact on travel, commerce, and economies worldwide. in most industrialized countries, antibiotic resistant zoonotic bacterial diseases are of particular importance for at-risk groups such as young, old, pregnant, and immune-compromised individuals ( ) . almost years ago, prior to application of hygiene rules and discovery of neither vaccines nor antibiotics, some bacterial zoonotic diseases such as bovine tuberculosis, bubonic plague, and glanders caused millions of human deaths. the spread and importance of some bacterial zoonoses are currently globally increasing. that is precisely why most of the developing countries are sparing more resources for a better screening of animal products and bacterial reservoirs or vectors for an optimal preventative public health service ( ) . improvements in surveillance and diagnostics have caused increased recognition of emerging zoonotic diseases. herein, changes in our lifestyles and closer contacts with animals have escalated or caused the re-emergence of some bacterial infections. some studies lately have revealed that people have never been exposed to bacterial zoonotic infection risks as high as this before ( ) . it is probably due to closer contact with adopted small animals, which are accepted and treated as a family member in houses. on the other hand, more intensified animal farms, which have a crucial role in the food supply, are still one of the greatest sources of food-borne bacterial zoonotic pathogens in today's growing world ( , ) . people who have closer contact with large numbers of animals such as farmers, abattoir workers, zoo/pet-shop workers, and veterinarians are at a higher risk of contracting a zoonotic disease. members of the wider community are also at risk from those zoonoses that can be transmitted by family pets. the immune-suppressed people are especially at high risk for infection with zoonotic bacterial diseases. people can be either temporarily immuno-suppressed owing to pregnancy, infant age, or long-term immuno-suppressed as a result of cancer treatment or organ transplant, diabetes, alcoholism or an infectious disease (i.e., aids). this manuscript reviews the most common bacterial zoonoses and practical control measures against them. companion animals are increasingly treated as family members, and pets have many bacteria that may infect their owners. the human population of the european union (eu) was approximately million in . the number of pet owning households was estimated at around million in . the most commonly suffered zoonotic bacterial infections in humans are transmitted via animal bites and scratches. various dog breeds have been characterized for their role in killing dog bite attacks, such as pit bull breeds, malamutes, chows, rottweiler, huskies, german shepherds, and wolf hybrids ( ) ( ) ( ) . in usa, pit bull breeds accounted for almost half of the dog bite-related zoonotic infections, three times more than german shepherds ( ) . the oral cavity of healthy dogs and cats contains hundreds of different pathogenic bacteria including pasteurella sp. ( ) . only % of dog bites get infected overall compared with % in cats. there are times higher pasteurella multocida infection risks after a cat bite than a dog bite ( , ) . p. multocida infected bite wounds appear usually within h. it is estimated that approximately % of animal bites or scratches get infected in humans ( ) . bacterial culturing from pet bite infections in humans is found to be smilar to the oral microbiota of the pets. infections in dog bite wounds are usually dominated by aerobic bugs: p. multocida ( %), alpha-hemolytic streptococcus ( %), staphylococcus ( %), neisseria ( %), and corynebacterium ( %). however, following anaerobic bacteria are also isolated from infected wounds: fusobacterium nucleatum ( %), prevotella heparinolytica ( %), propionibacterium acnes ( %), prevotella intermedia ( %), and peptostreptococcus anaerobius ( %) ( ) . normal human skin bacteria or other environmental microorganisms are scarcely isolated from infected wounds in bitten person ( ) ( ) ( ) . usually, infection occurs within - h after the animal attack, with variable pain on the site of the injury. the cellulitis might be followed by discharge that contains pus, which can sometimes be foul-smelling. immuno-suppressed patients with diabetes or liver dysfunction are frequently predisposed to develop serious infections after animal bites. in those cases, they may develop bacteremia faster and pass away in a shorter period of time ( ) . a penetrating bite close to the joints and bones may cause septic arthritis and osteomyelitis. knowing the microbial composition of dental plaque biofilm formation in pets' mouth is a key factor in wound chronicity in humans ( , ) . cat-scratch disease is a clinical syndrome that has been reported in people for over years. yet, the etiological agent bartonella henselae, which was transmitted by cat scratches and bites, was only identified in ( ) . however, contact with cat saliva on broken skin or sclera can also cause bartonellosis. a person who has had a cat scratch may show papules and pustules at the site of injury (the first initial sign). the disease may progress with a chronic non-healing wound, fever (sometimes), weak regional lymph circulation, and abscession. cat owners and veterinarians are most at risk ( ) . systematic medical treatment is usually needed in people with suppressed immune systems. otherwise, encephalopathy, osteomyelitis, and granulomatous conjunctivitis might develop. horses and humans have always shared a close relationship due to recreation, sporting, and occupational reasons, for over thousands of years. in europe, the number of horses per capita remained relatively stable during the past decade. germany and great britain have the largest horse populations in the eu, whereas sweden has the highest number of horses per capita. the frequency of infected horse bite wounds is estimated to be - % in europe ( , ) . however, it has been roughly estimated that the horse bites account for as high as % of overall animal bites in turkey, which comes after dog bites ( %) ( ) . more extensive muscle damage may develop in most of the horse attacks, which is different from small animal bites. a mixture of aerobic and anaerobic organisms has been isolated from horse bites in humans, which are frequently dominated by actinobacillus lignieresii ( , ) . escherichia coli and bacteroides species have also been isolated from foul-smelling infections and pus drainage after horse bites in humans ( ) . infectious diarrhea in companion animals is caused by salmonella sp., escherichia coli, shigella sp., and campylobacter sp. can also be transmitted to people through fecal oral route. it is difficult to estimate the distribution of these ubiquitous microorganisms. but it is known that they can be isolated from many healthy animals, which can be shed in their feces for long periods of time. campylobacteriosis were the most frequently reported zoonotic bacterial diseases in among the eu member countries in humans ( ) . like many other enteropathogens, they can cause gastroenteritis (diarrhea, vomiting), headaches, and depression, sometimes even leading to death. it is obvious that raw food diets for pets dramatically increase the risk of human exposure to such zoonotic bacterial enteropathogens, which cause gastrointestinal diseases. although pet birds, also called songbirds (e.g., canaries, finches, sparrows) and psittaciformes (e.g., parrots, parakeets, budgerigars, love birds) are a small fraction of adopted pets, they are widely popular in europe and they are potential carriers of zoonotic diseases ( ) . some of them could have an important impact on human health, such as chlamydophilosis ( ) , campylobacteriosis ( ) , and salmonellosis ( ) . parrot fever (chlamydophilosis), which is caused by intracellular bacteria, chlamydia psittaci, lives within the frontiers in public health | infectious diseases respiratory system of birds. inhalation of dust, dander, and nasal secretions of infected birds is the main way of transmission to humans ( , ) . the mild to severe flu-like illnesses may develop and infected people might be misdiagnosed as influenza. there is unfortunately a lack of quantitative research into the antimicrobial susceptibility of bacterial zoonotic organisms isolated from bite/scratch wounds or companion animal associated gastroenteritis. zambori et al. ( ) revealed an increased prevalence of drug resistance in animal bite isolates from people. furthermore, methicillin-resistant staphylococcus aureus (mrsa) or extended-spectrum beta-lactamases (esbl) producing enterobacteriaceae, which are known as nosocomial infections have been frequently isolated in companion animals ( ) , including horses ( ) . it might be one of the main reasons for the rising prevalence of these potential zoonotic pathogens in human clinical samples. food producing animals in stock has reached a total of more than million (cattle, pigs, sheep, goats, and chicken) living on farms in europe (see text footnote ). it has been demonstrated that farm animals are reservoirs of many zoonotic pathogens to humans ( , ) . however, annually, a large amount of drugs are being used worldwide to sufficient quantities of food to feed a rapidly growing world human population ( ) ( ) ( ) ( ) . the farm animals consume worldwide approximately eight million kilograms of antibiotics annually ( % of which is used for non-therapeutic purposes such as growth promotion; forbidden in the eu from january , and disease prevention) compared with only approximately one million kilogram per year used in human medicine ( ) . antibiotics are routinely fed to livestock as growth promoters to increase profits and to ward off potential bacterial infections in the stressed and crowded livestock factory environment ( ) ( ) ( ) ( ) ( ) . despite large differences in methodology, most results demonstrate that not so long after the introduction of an antibiotic in veterinary practice, resistance in pathogenic zoonotic bacteria and/or the fecal flora increases. in particular, the wide-spread use of antibiotics in animals has resulted in an increased emergence of bacterial resistance to antibiotics, in zoonotic organisms such as salmonella, campylobacter, shigella, yersinia, listeria, and enterococcus genera, as well as the e. coli species. these zoonotic bacterial organisms are propagated primarily among animals and subsequently infect people ( ) ( ) ( ) ( ) . humans can be infected by contact with animals and their dung or droppings or consumption of infected food. severe diarrhea may develop, sometimes with blood in the feces. at all ages, but especially in children under years and adults over years, very serious illnesses often occur. these complications can result in loss of life or permanent kidney damage. according to the latest epidemiological trends, salmonellosis and campylobacteriosis are indicated as the most frequent food-borne bacterial zoonoses in europe. the main food sources were eggs and mixed foods ( ) . furthermore, the recent emergence of esbl-producing and carbapenemase positive enterobacteriaceae bacteria in animal production ( ) , the emergence of farm associated mrsa st (the main pig associated clone) ( ) ( ) ( ) , and of plasmid-mediated quinolone resistance in animal isolates and food products ( , ) are great threat for public health. unfortunately, these antimicrobial resistant "superbugs" are not only confined to hospital environments where antimicrobial use was high and many pathogens were prevalent. they are already widespread in the european community and animal populations that have a great hazard on public health ( , ) . the causative agent of bovine tuberculosis, mycobacterium bovis (m. bovis) has been identified worldwide. thanks to decades of disease control measures that the occurrence of the infection has been greatly reduced. but there are still hundreds of new cases of human tuberculosis reported in the usa ( ) . experience in europe and the usa, has shown that m. bovis can be controlled when it is restricted in livestock; however, eradication is almost impossible once it has spread into wildlife as follows; the european badger in the united kingdom ( ), elk in canada ( ) and white tailed deer in the usa ( ) . in the last decade, q fever caused by coxiella burnetii was one of the most devastating farm animal originated bacterial zoonotic bacteria in europe. the netherlands, in particular, has experienced several outbreaks from to following identification of a q fever outbreak on various dairy farms in . infected humans were mainly located within the intensive dairy goat farms (< km) ( ) . the infection is spread by ticks, inhalation of the organism from the placental fluids, urine, and consumption of unpasteurized milk -meat products of sheep, goats, and cattle. the clinical signs in humans might be severe flu-like syndrome that may last for months ( ) . in the eu, many vector-borne zoonotic diseases are considered as emerging infectious diseases, which either appear in a population for the first time or may have existed previously but spreading rapidly. the ecology of vector-borne zoonotic bacterial diseases is complex where climate and weather may influence the transmission cycles. milder winters, earlier start of spring or long intervals between winters cause extended seasonal tick activity and hence pathogen transmission between hosts in new regions of the world ( , ) . many vector-borne infections occurred in new regions in the past two decades, while many endemic diseases have increased in incidence ( ) . the following bacterial pathogens were most frequently identified as the causes of emerging vector-borne infections in the last decades in the eu: rickettsiae spp., anaplasma phagocytophilum, borrelia burgdorferi, bartonella spp., and francisella tularensis ( , ) . rickettsia rickettsii causes rocky mountain spotted fever and spreads to humans by ticks. the signs of this disease are fever, headache, muscle pain, and spots with very dark rash. hiking in an area with many infested ticks is a great risk factor. a tick bite of < h is usually not enough to transfer these bacteria to a person ( ) . ehrlichiosis (anaplasma phagocytophilum) and lyme disease (borrelia burgdorferi) have emerged as an important vector-borne zoonotic disease since s ( , ) . hard ticks are principal vectors, whereas small rodents are known as their natural vertebrate reservoir. a wide variety of signs including rash, joint pains, fever, enlarged lymph nodes, and some neurological signs may www.frontiersin.org develop. the trend of house buildings in woodlots where humans share the same ecology with reservoirs and vectors was found to be correlated with the increased frequency of such diseases in humans ( ) . bartonella spp. is transferred to humans via fleas, lice, and sand flies ( ) . however, recent studies have shown the importance of tick exposure in human bartonellosis ( ) . as previously mentioned elsewhere in this article, bartonellosis are usually associated with cat-scratch diseases. lately, researchers have revealed that bartonella spp. can be transmitted via cat fleas without any scratches to humans ( ) . symptoms include fever, enlarged lymph nodes (after - weeks), and a papule at the inoculation site. etiological agent of tularemia, f. tularensis, is a rare disease in europe ( ) . bacteria are usually transferred by slaughtering (hunters are at a higher risk), eating of infected hares, respiration of dust, or drinking of contaminated water ( ) . the prevalence of f. tularensis was found to be - % from dog ticks in north america ( ) . clinical symptoms depend on how the organism is acquired: erythematous papule at inoculation side within h, pneumonia (the most serious form), endotoxemia, which gives continuous fever, acute pharyngotonsillitis, mucopurulent conjunctivitis (rarest form) ( ) . among many others, brucellosis, which is not an emerging disease, has a significant impact on the endemic southern european countries with sporadic outbreaks. fortunately, the impact on humans has not increased since ( ) . however, the cross border tracing of some brucella strains isolated in germany revealed concordance with sheep isolates originating from eastern anatolian, turkey. it is a characteristic example for the global spread of such diseases, in that case most probably by turkish immigrants living in germany ( ) . plague, caused by yersinia pestis, is the most important reemergent bacterial wild rodent borne disease. the current case reports of plague are primarily limited to africa. however, it is a great potential public health hazard for europe due to increased traveler mobility or a potential bioterrorist attack ( ) . bacterial zoonoses have a major impact on global public health. both emerging and re-emerging bacterial zoonoses have gained increasing national and international attention in recent years. the closer contact with companion animals and rapid socioeconomic changes in food production system has increased the number of animal-borne bacterial zoonoses. animal bite injuries in daily human-animal contact are not surprising, especially for the school-aged children. most of these wounds are medicated by patients as first aid and not registered in health systems. in more developed countries, most of the victims with moderate to severe bite injuries will seek professional medical treatment. regardless, all bites should be treated as serious, especially if the skin is broken. prompt diagnostic and treatment can prevent wound complications. the possibility to form biofilms by previously mentioned wound microorganisms is quite high, may cause severe tissue damage and protect the bacteria from innate-immune response and antimicrobials. the most of the commercial topical agents and wound dressings are ineffective against the biofilm matrix. surgical repair (for example, co surgical laser techniques, leon cantas, personal research notes ), which is usually used to obtain a better cosmetic result might be needed to remove biofilm formed bite infections. this mechanical debridement is essential in the eradication of a wound biofilm. antimicrobials may be more effective in the treatment of the wound after debridement in the prevention of biofilm reformation. despite the use of currently optimal culturing methods, approximately % of infected wounds yield no bacterial growth. in such cases, some other fastidious pathogens, i.e., chlamydia spp., mycoplasma spp., and even viruses should be investigated. new advanced molecular diagnostic techniques are needed. prevention strategies for animal bites include close supervision of child-animal interactions, stronger animal control laws, better reporting of animal bites, and public education for better ownership of pets. regular nail trimming, routine oral examinations under annual health checks and comprehensive dental treatments of the companion animals (i.e., routine removal of the teeth tartar and plaques) by veterinarians will reduce the bacterial mass exposure to humans in case of direct contacts or animal bites. it is important to realize that enteropathogenic zoonoses may be contracted from both clinically sick and apparently healthy companion animals. feeding of pets with raw food diets is a potential source of salmonella, campylobacter, and other important bacterial zoonoses; however, some recalls of commercial pet food diets have also occurred as a result of contamination with those microorganisms. pig ear dog treats, in particular, have been implicated as an important source of salmonella infection for dogs, which can also serve as a source of infection to humans. nevertheless, it can be said that easy-to-use personal hygiene rules should be applied by companion animal owners. thorough hand washing with soap after handling of a companion animal and before eating or drinking, avoiding mouth-to-mouth contact, avoiding aerosolization of dusty fecal matter will help to prevent transmission of the zoonotic disease to humans. the animals with diarrhea should be isolated immediately and veterinary advice should be sought. the household should be cleaned with agents and kept as clean as possible. on the other hand, the prevalence of antimicrobial resistance in small animal pathogens is increasing globally due to overuse of broad spectrum antibiotics by veterinarians. there is an immediate need for worldwide smarter use of antimicrobials that have some positive effect on the recovery of animals from life threatening diseases. national veterinary antimicrobial treatment guidelines should be established by the local authorities according to the updated routine surveillance results. chronic diarrhea, dermatitis, ear and eye infections of pets caused by microbes demand longer durations of antimicrobial remedies at home. more frequent use of advanced laboratory tests, such as; feed/insect/mould allergy tests and differential diagnosis of the other relevant auto-immune disorders may help to investigate the main underlying cause of the such reactions which can be managed in various alternative treatment methods (i.e., hypoallergenic diets) rather than antibiotics solely. herein, pet specific auto-immune vaccines against allergens and auto-lactobacillales (auto-lac, leon cantas, personal research notes, - ) as dietary supplements can also be more frequently administered within the preventative veterinary practice measures. owners should be encouraged to insure their family animals to afford such costly veterinary services contradictory to the cheaper and sometimes life-long medical (i.e., antibiotic) treatment demanding options. veterinarians should also spear more time to educate the pet owners under consultations to handle infected-antimicrobial treated animals with precaution due to irreversible consequences of the antimicrobial resistance development and its spread in households. proper hand washing and use of gloves are strictly recommended while handling antimicrobial in veterinary clinics. veterinarians should prescribe broad spectrum and synthetic antimicrobials preferably after culturing with extreme precautions (i.e., dosage, dosing intervals and length of the treatment). reduced antibiotic use will hinder the development of antibiotic resistance in animal microbiota which might cause zoonotic infections in humans ( , ) . food-borne zoonoses are an important public health concern worldwide and every year a large number of people affected by diseases due to contaminated animal originated food consumption. food hygiene education of the consumers is an important competent of food-borne diseases prevention. however, main prevention of food-borne zoonoses must begin at the farm level with in the concept of "one health." herein, control of the production stress especially in intensive livestock industry, with the development of better animal health management routines (i.e., routine vaccinations, immune stimulants: pre-, probiotic feed additives) and the increased animal welfare programs, will contribute eventually to an optimal production of animal health. increased antimicrobial resistance among emerging and re-emerging farm-borne bacterial pathogens in crowded settings (i.e., poultry, pig farms) is a growing problem. restrictive antimicrobial choice with better animal welfare managements are needed to control the spread of antibiotic resistance elements. in the eu, the use of avoparcin was banned in and the use of spiramycin, tylosin, and virginiamycin for growth promotion were banned in . all other growth promoters used in feeding of food producing animals were banned from january , after a few national bans the years ahead . in the u.s., politicians are still discussing to introduce a similar ban (s- , th u.s. congress (preservation of antibiotics for medical treatment act). despite the ban on the use of all antibiotics as growth promoters in the eu and a ban on the use of quinolones as growth promoters in the poultry feed in the us medical, important antibiotics are still routinely fed to livestock prophylactically to increase profits and to ward-off potential bacterial infections in the stressed and crowded livestock and aquaculture environments in some parts of the world ( , , ) . because stress lowers the immune system function in animals, antibiotics are seen as especially useful in intensive animal confinements ( ) . the non-therapeutic use of antibiotics involves low-level exposure in feed over long periods -an ideal way to enrich resistant bacterial population ( , ) . moreover, antibiotic resistance has been detected in different aquatic environments ( ) . fish pathogenic bacteria often produce devastating infections in fish farms where dense populations of fish are intensively reared. bacterial infections in fish are regularly treated with antibiotics in medicated feed. so far, most of the fish pathogenic bacteria with a http://europa.eu history in diseased fish farms have developed drug resistance ( ) . modern fish farming relies increasingly on vaccination procedures and improved management to avoid infections ( ) . for example, the norwegian aquaculture industry has produced over one million tons farmed fish by using improved vaccines, management techniques, and only kg of antimicrobials in ( ) . vector-borne and zoonotic bacterial pathogens are a major source of emerging diseases, and since the time of hippocrates, weather and climate are linked to the incidence of such infectious diseases. complexity of epidemiology and adoptive capacity of microorganisms and the arthropods make the vector-borne disease almost impossible to eradicate. insect repellants, routine tick checks after outdoor activity in risk regions, prompt-proper tick removal, use of long sleeves and trousers (light-colored), and routine insecticide treatment of pets are recommended as general preventative measures ( ) . herein, lyme disease, tick-borne illness, is vastly underestimated over past decades and clearly the urgent prevention is needed. besides individual awareness of such vector-borne diseases, better national surveillance and reporting programs will contribute to improved the disease control strategies. clinicians have an important role in the effective management of vector-borne zoonotic diseases, with enhanced differential diagnostic skills based on clinical symptoms and rapid molecular identification techniques ( ) ( ) ( ) ( ) . most of the time, the clinicians are on the first line of detection of these epidemics due to large group of patients with novel sets of similar symptoms. increased medical networking via online databases offer a broad overview to followers with regard to changes in temporal patterns of illness in real time, which helps faster detection of new epidemics ( ) . identification and control of emergent zoonotic bacterial diseases require a "one health" approach, which demands combined efforts of physicians, veterinarians, epidemiologists, public health workers, and urban planners. collaborative international routine surveillance strategies, prompt -reliable agent identification techniques, and optimization of the treatment regiments will ensure the prevention and management of such infections. leon cantas defined the review theme, manuscript design, established the coordination and the collaborations, designed the manuscript, contributed to the data collection, data analysis, and drafting, writing and editing of the manuscript. kaya suer contributed to drafting and editing of the manuscript. all authors have seen, and approved the manuscript. risk factors for human disease emergence professor emeritus of veterinary epidemiology the components of 'one world -one health' approach animal associated opportunistic infections among persons infected with the human immunodeficiency virus biofilms in oral cavity of dogs and implication in zoonotic infections sensitive populations: who is at the greatest risk? monitoring and identifying antibiotic resistance mechanisms in bacteria emerging foodborne diseases: an evolving public health challenge spread of antibiotic resistance plasmids from chicken to chicken and from chicken to man emergence and prevalence of human vector-borne diseases in sink vector populations an overview of public health and urban agriculture: water, soil and crop contamination and emerging urban zoonoses analysis, fate and effects of the antibiotic sulfadiazine in soil ecosystems paho. zoonoses and communicable diseases common to man and animal emerging and re-emerging bacterial zoonoses: factors of emergence, surveillance and control reverse zoonotic disease transmission (zooanthroponosis): a systematic review of seldom-documented human biological threats to animals dog-bite-related fatalities-united states fatal dog attacks dog, cat, and human bites: a review human and animal bite wounds hospital management of animal and human bites dog bites microbiology of animal bite wound infections bacteriological analysis of infected dog and cat bites bite infections. in infectious diseases of the dog and cat chronic wound colonization, infection, and biofilms nelson textbook of pediatrics principles and practice of infectious diseases mammalian bites of children; a problem in accident prevention horse injuries animal-related injuries: epidemiological and meteorological features infection due to actinobacillus lignieresii after a horse bite actinobacillus spp. and related bacteria in infected wounds of humans bitten by horses and sheep actinobacillus lignieresii infection after a horse bite zoonoses in the european union: origin, distribution and dynamics -the efsa-ecdc summary report zoonotic diseases of common pet birds: psittacine, passerine, and columbiform species chlamydophila psittaci transmission from pet birds to humans incidence of campylobacter species in hobby birds efficacy of european starling control to reduce salmonella enterica contamination in a concentrated animal feeding operation in the texas panhandle chlamydia psittaci infection in canaries heavily infested by dermanyssus gallinae bacterial and parasitic diseases of passerines methicillinresistant staphylococci (mrs) and extended-spectrum beta-lactamases (esbl)-producing enterobacteriaceae in companion animals: nosocomial infections as one reason for the rising prevalence of these potential zoonotic pathogens in clinical samples emergence o f mrsa infections in horses in a veterinary hospital strain characterisation and comparison with mrsa from humans fecal shedding of salmonella spp. by dairy cows on farm and at cull cow markets prevention of immunologic stress contributes to the growth-permitting ability of dietary antibiotics in chicks the use of feed medications in swine and poultry facilities in the weser-ems region autoactivation of the marrab multiple antibiotic resistance operon by the mara transcriptional activator in escherichia coli persistence of antibiotic resistant bacteria antimicrobial resistance: responsible and prudent use of antimicrobial agents in veterinary medicine heavy use of prophylactic antibiotics in aquaculture: a growing problem for human and animal health and for the environment the european ban on growth-promoting antibiotics and emerging consequences for human and animal health a brief multi-disciplinary review on antimicrobial resistance in medicine and its linkage to the global environmental microbiota antibiotic resistance from food antibiotic resistant bacteria in food of man and animals inter-and intraspecies spread of escherichia coli in a farm environment in the absence of antibiotic usage distribution of the streptomycinresistance transposon tn among phylloplane and soil bacteria from managed agricultural habitats the european union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in fecal carriage and shedding density of ctx-m extended-spectrum ß-lactamaseproducing escherichia coli in cattle, chickens, and pigs: implications for environmental contamination and food production emergence of methicillin-resistant staphylococcus aureus (mrsa) in different animal species methicillin-resistant staphylococcus aureus in food products: cause for concern or case for complacency? methicillin-resistant staphylococcus aureus in animals does broad-spectrum β-lactam resistance due to ndm- herald the end of the antibiotic era for treatment of infections caused by gram-negative bacteria? vibrionaceae as a possible source of qnr-like quinolone resistance determinants the shared antibiotic resistome of soil bacteria and human pathogens human health consequences of use of antimicrobial agents in aquaculture mycobacterium bovis (bovine tuberculosis) infection in north american wildlife: current status and opportunities for mitigation of risks of further infection in wildlife populations tuberculosis: the disease and its epidemiology in the badger, a review bovine tuberculosis in canadian wildlife: an updated history managing the wildlife reservoir of mycobacterium bovis: the michigan, usa, experience epidemic q fever in humans in the netherlands the - . q fever epidemic in the netherlands: characteristics of notified acute q fever patients and the association with dairy goat farming tick-borne encephalitis in sweden and climate change impact of climatic change on the northern latitude limit and population density of the disease-transmitting european tick ixodes ricinus drivers, dynamics, and control of emerging vector-borne zoonotic diseases emerging zoonoses and vectorborne infections affecting humans in europe present and future arboviral threats rickettsia rickettsii and other spotted fever group rickettsiae (rocky mountain spotted fever and other spotted fevers) ehrlichioses in humans: epidemiology, clinical presentation, diagnosis, and treatment the emergence of lyme disease vector transmission of bartonella species with emphasis on the potential for tick transmission concurrent infection of the central nervous system by borrelia burgdorferi and bartonella henselae: evidence for a novel tick-borne disease complex cat scratch disease and arthropod vectors: more to it than a scratch? worldwide genetic relationships among francisella tularensis isolates determined by multiple-locus variable-number tandem repeat analysis tularaemia outbreak in hare hunters in the darmstadt-dieburg district nonrandom distribution of vector ticks (dermacentor variabilis) infected by francisella tularensis current, comprehensive information on pathogenesis, microbiology, epidemiology, diagnosis, treatment, and prophylaxis. center for infectious disease research and policy brucellosis outbreak due to unpasteurized raw goat cheese in andalucia (spain) isolation of brucella melitensis biotype from epidural empyema in a bosnian immigrant in germany plague in india: world health organization team executive report antibiotic resistance in animals -the australian perspective guide to antimicrobial use in animals the price we pay for corporate hogs sublethal antibiotic treatment leads to multidrug resistance via radical-induced mutagenesis diversity and distribution of commensal fecal escherichia coli bacteria in beef cattle administered selected subtherapeutic antimicrobials in a feedlot setting antibiotic resistance of gram-negative bacteria in rivers, united states antibiotic resistance associated with veterinary drug use in fish farms fish vaccination, an overview usage of antimicrobial agents and occurrence of antimicrobial resistance in norway tick-borne disease curtailing transmission of severe acute respiratory syndrome within a community and its hospital strategies for mitigating an influenza pandemic identification of a novel polyoma virus from patients with acute respiratory tract infections microbe hunting digital disease detection: harnessing the web for public health surveillance the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- - g dc z authors: mcintyre, k. marie; setzkorn, christian; hepworth, philip j.; morand, serge; morse, andrew p.; baylis, matthew title: a quantitative prioritisation of human and domestic animal pathogens in europe date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: g dc z disease or pathogen risk prioritisations aid understanding of infectious agent impact within surveillance or mitigation and biosecurity work, but take significant development. previous work has shown the h-(hirsch-)index as an alternative proxy. we present a weighted risk analysis describing infectious pathogen impact for human health (human pathogens) and well-being (domestic animal pathogens) using an objective, evidence-based, repeatable approach; the h-index. this study established the highest h-index european pathogens. commonalities amongst pathogens not included in previous surveillance or risk analyses were examined. differences between host types (humans/animals/zoonotic) in pathogen h-indices were explored as a one health impact indicator. finally, the acceptability of the h-index proxy for animal pathogen impact was examined by comparison with other measures. pathogens appeared solely in the top highest h-indices ( ) human or ( ) animal pathogens list, and occurred in both. of human pathogens, were zoonotic and were emerging, compared to and for animals. there were statistically significant differences between h-indices for host types (humans, animal, zoonotic), and there was limited evidence that h-indices are a reasonable proxy for animal pathogen impact. this work addresses measures outlined by the european commission to strengthen climate change resilience and biosecurity for infectious diseases. the results include a quantitative evaluation of infectious pathogen impact, and suggest greater impacts of human-only compared to zoonotic pathogens or scientific under-representation of zoonoses. the outputs separate high and low impact pathogens, and should be combined with other risk assessment methods relying on expert opinion or qualitative data for priority setting, or could be used to prioritise diseases for which formal risk assessments are not possible because of data gaps. disease or pathogen risk prioritisation exercises are used by organisations charged with providing surveillance and mitigation measures including disease management and control, and biosecurity measures. qualitative, semi-quantitative or quantitative approaches can be used, but most take significant time to develop, so their use is limited, and when research involving the study of multiple diseases or pathogens is planned, agents are rarely systematically selected. quantitative measures for risk prioritisation include the calculation of epidemiological parameters such as disease incidence, prevalence, mortality and morbidity rates, costs of prevention, treatment or control, and for human disease, years lived with disability (yld) and disability-adjusted-life-year estimates (daly). additional measures for animals include losses to production. for many diseases, robust estimates of these measures do not exist. semi-quantitative and qualitative risk assessments are less demanding of data than quantitative approaches. nevertheless, they require significant time and physical resources (for example, to obtain parameters and effect sizes from the scientific literature), need updating regularly, and they usually require expert-opinion, adding subjectivity [ , , , , ] . the h-index is an alternative approach to disease prioritisation. it objectively and rapidly provides a quantitative proxy of human disease or pathogen impact [ ] , (mcintyre, unpublished) . the hindex captures scientific interest in a disease by deriving a metric from the number of papers published and how many citations each receives. combining scientific impact (citations) with technical productivity (papers published) is useful as, individually, total papers does not account for the quality of publications, while citation count may be influenced by a small number of seminal papers or if a disease becomes 'fashionable' briefly. the h-index method is significantly correlated with more comprehensive measures of human infectious disease impact, including dalys [ ] , and deaths from disease (mcintyre, unpublished data). it can be rapidly obtained at low cost, attained automatically, and repeated regularly to reflect changes in impact, serving as a generic tool to assess the relative bearing of diseases or pathogens, in an easier, timelier manner than traditional risk assessments. while the h-index method undoubtedly has limitations, these tend to be different to those of other approaches; its use could be a step forward in separating high and low priority diseases or pathogens, in combination with other risk assessment methods. the enhanced infectious diseases (eid ) database integrates published data sources on pathogens, their hosts (including vectors) and geographic ranges [ ] . by coupling the h-index method with the eid , the primary aim of this study was to establish priority lists of human and domestic animal pathogens (including zoonoses) present in europe. we then consider reasons for the omission of some pathogens in our lists from those of other disease prioritisations: the global burden of disease (gbd) estimates [ ] , communicable human diseases reportable in the european community [ ] , the oie list of notifiable animal diseases, infections and infestations [ ] , and the eu fp discon-tools project [ ] . the gbd study was a large collaborative five year project which used all relevant published and unpublished evidence to create the strongest evidence-based epidemiological assessment of people's infectious and non-infectious health problems around the world [ ] . the discon-tools project, funded by the european commission over five years, investigated the impacts of domestic animal diseases, to focus and prioritise future research [ ] . as the zoonotic and emerging status of pathogens as well as their taxonomic division could affect the likelihood of their inclusion in surveillance and impact quantification work, these factors were also investigated as reasons for omission from the other disease prioritisations. the h-index can be obtained in the same way for both human and animal pathogens. it therefore has potential as a single metric for prioritising across both host groups. its potential as a quantitative one health indicator (i.e. a single measure applicable to both human and animal diseases) was investigated by comparing scores for human-only, zoonotic, and animal-only pathogen groups, including emerging status as this would likely drive research impact. previous work has shown that the h-index is a proxy for human disease impact [ ] , (mcintyre, unpublished) . we investigated its value as a proxy for animal disease impact by comparing domestic animal pathogen h-indices with other measures of impact including presence on the oie list [ ] , and inclusion in discontools [ ] . the eid database collates data on human and domestic animal pathogens: where, when, and in which hosts there is evidence of their occurrence. the database is built largely using automated procedures to interrogate publicly available databases. an eid background has been described previously [ ] ; here, we used similar criteria to define pathogens, including pathogenic status (frequently pathogenic: a pathogen which frequently causes a clinically pathogenic effect -morbidity or mortality -in humans or domestic animals; non-pathogenic: an organism which causes no clinical signs within any of its hosts; unknown pathogenicity: an organism for which there is insufficient evidence to decide), evidence of pathogens affecting hosts ('host-pathogen interactions': evidence from at least one piece of meta-data uploaded with dna or rna sequence information to [ ] , which describes where, when and from which host the pathogen came, or specific scientific publications [ ]), and evidence of pathogens occurring within countries (evidence from at least one piece of meta-data [ ] , or at least five publications in [ ] where pathogen name and a country mesh-term [ ] co-occurred in the title/abstract). information on host-pathogen interactions was collated when there was evidence of a pathogen occurring in at least one host of interest to the study (including humans and european domestic animals; see table ). further information about each organism, such as their taxonomic division for pathogens (bacteriaincluding rickettsia, fungi -including algal pathogens, helminths -including thorny-headed worms and pentastomids, protozoa, and viruses -including prion agents) or their taxonomic rank (genus, species, etc.) is stored using a series of statements. previously, we examined characteristics of pathogen species [ ] ; here, we include sub-species, to account for important strains e.g. escherichia coli o :h . information on whether pathogens were zoonotic, non-zoonotic, emerging and not emerging was examined based upon previously published information [ , ] . if not included in earlier work or if their status had changed due to more recent scientific evidence, updated pathogen information was based upon the previous definitions. zoonotic pathogens were classified as those naturally transmitted between vertebrate (non-human) animals and humans (as the definitive host), not including species which have recently evolved from animal pathogens but are no longer transmitted between animals and humans [ , ] . emerging pathogens are those that have appeared in a host population for the first time (including newly-evolved strains), or have occurred previously but are increasing in incidence or expanding into areas where they had not previously been reported [ , ] . pathogens needed to have emerged in several geographically distinct areas to be 'emerging'. information sources. h-index searches were undertaken in january using web of science (wos) [ ] . previous work established that results of h-index searches for pathogens undertaken using different bibliographic sources (e.g. wos, scopus, google scholar) are not identical but are highly correlated [ ] . eligibility criteria. searches were restricted to the years to , inclusive. english is used in wos, however searches also include foreign-language publication title translations. all literature in the wos database has been published. searches. searches were undertaken using search phrases specified in quotation marks (''''), the 'topic' search field and with no lemmatization. phrases were compiled including pathogen scientific name, alternative names, synonyms and alternative spellings according to ncbi taxonomy [ ] . h-indices for clinical diseases used clinical terms as well as pathogen phrases for the main pathogens of disease. virus searches also included synonyms and acronyms from the ncbi taxonomy database and international committee on taxonomy of viruses [ , ] , and the term 'virus', and excluded other entities (viral or non-viral) which shared acronyms. the boolean operators 'and', 'or', and 'not' linked multiple search phrases. example of a search phrase. (''mycobacterium tuberculosis'' or ''bacillus tuberculosis'' or ''bacterium tuberculosis'' or ''mycobacterium tuberculosis typus humanus'' or ''mycobacterium tuberculosis var. hominis''). a full list of human and domestic animal pathogens frequently causing pathogenic effects and for which there was evidence of european occurrence was created using eid information [ ] , and defined criteria, see figure . the relative impact of pathogens in this full list was assessed by calculating h-indices using the specified search protocol; high impact pathogens had the highest h-indices. the list was split according to host-pathogen interaction information, into two directories, one including pathogens with evidence of their occurrence in humans, and the second including domestic animal occurrence; zoonotic pathogens appeared in both lists. information was manually obtained on whether these pathogens cause diseases featuring in other prioritisation lists [ , , ] , by examining pathogens listed under each disease's details in the ncbi mesh library [ ] ; specific lists of diseases had been provided in other work [ , ] . these additional pieces of information are included in the results (tables and ). finally, information on the pathogenic status of each pathogen, whether they frequently occurred in the relevant hosts and in europe was verified by the study authors using manual literature searches of the scientific literature, for the pathogens with the highest h-indices. h-indices and previous prioritisations. pearson's chisquared tests with yates' continuity correction and fisher's exact tests (fet) were used to test for differences in counts of pathogens included in previous work [ , , ] , according to outcomes including their taxonomic division, zoonotic and emerging status. where appropriate, odds ratios (or) and % confidence intervals (ci) are presented. h-indices for one health. differences in h-indices for human-only, zoonotic, and animal-only pathogens were examined using a two-way analysis of variance (anova) with log transformation of the response, including emerging status as an explanatory covariate. post-hoc tukey multiple comparisons of treatments were undertaken using the hsd.test [ ] . h-indices for domestic animal pathogens. -oie list. homogeneity of variances of h-indices for animal-only (and not zoonotic) pathogens included or not within the oie list of notifiable animal diseases was examined using the fligner-killeen (median) test. one-way anova thereafter established differences in the (log -transformed) h-indices of pathogens included or not in the oie list. -discontools. h-indices and discon-tools scores were compared using spearman's rank correlations. if more than one pathogen had been included within disease information for the discontools rankings (for campylobacter, leishmaniasis, and salmonellosis), the higher h-index score was used for analyses. table . animal species including humans for which pathogens have been studied, including domestic animals we eat or companion animals we keep as pets, and exotic animals also used as food sources or as pets. all analyses were undertaken using the statistical software package r [ ] , with statistical significance determined by a pvalue of less than . . two lists each including the top human ( table ) and domestic animal pathogens (table ) which cause significant clinical disease and which therefore need consideration from a health and well-being perspective were short-listed using the hindex prioritisation method (for alternative names and synonyms see [ ] ). when combined, ( . %) pathogens appeared solely in the human or animal list, and ( . %) were in both lists. of the top human pathogens, were classed as zoonotic and were emerging, compared to and for domestic animal pathogens, respectively. of the top human pathogens identified, were either included in the gbd [ ] , or are reportable to the ec [ ] , or both. reasons for failure to include pathogens may be that pathogenic agents cause rarely diagnosed disease (e.g. human t-lymphotropic virus , lymphocytic choriomeningitis virus, and moraxella catarrhalis), or because disease agents are diverse, e.g. pneumonia or other lung infections (aspergillus niger, chlamydophila pneumonia, cryptococcus neoformans, klebsiella pneumonia, and mycoplasma pneumoniae) and gastro-intestinal (gi) symptoms or gi-tract infections (aeromonas hydrophila, bacillus cereus, bacteroides fragilis, clostridium species, vibrio parahaemolyticus, and yersinia enterocolitica). the impact of chronic disease or diseases causing low morbidity may be difficult to quantify or seen as less important (bartonella henselae, borrelia burgdorferi, human enterovirus c, human herpesvirus group, human papillomavirus, human parvovirus b , mycobacterium bovis, mycobacterium avium, and mycoplasma genitalium). in addition, some pathogens may generally be commensals or natural biota (aggregatibacter actinomycetemcomitans, candida species, enterobacter, enterococcus, staphylococcus species, candida tropicalis, helicobacter pylori, and porphyromonas gingivalis) or species existing in the environment (acinetobacter baumannii, burkholderia cepacia, candida glabrata, entamoeba histolytica, fusarium oxysporum, gibberella moniliformis, proteus mirabilis, pseudomonas species, rhizopus oryzae, serratia marcescens, staphylococcus aureus, and stenotro-phomonas maltophilia) causing opportunistic infections in immunecompromised individuals (including those young, old or pregnant); their impact upon the general population may not be quantified. of the top domestic animal pathogens described, were either notifiable according to the oie [ ] , or included in discontools [ ] , or both. reasons for failure to include may be similar to for human pathogens (only pathogens not previously mentioned are cited: multiple disease symptoms or lack of diagnosis -ascaris suum, feline immunodeficiency virus, feline leukemia virus, gallid herpesvirus , haemonchus contortus, and yersinia pseudotuberculosis; causes of specific disease being diverse, for respiratory infection -feline calicivirus, and gi symptoms -campylobacter fetus, cryptosporidium parvum, and listeria monocytogenes,; and existing in the environment and opportunistic -pseudomonas aeruginosa). in addition, some omitted pathogens may be production issues with impact difficult to quantify (streptococcus agalactiae causing mastitis in cattle and neospora caninum causing abortion in cattle and dogs) and some may be issues of pets (canine parvovirus, neospora caninum, and parainfluenza virus ). for human pathogens in our list (table ) , fungi and helminths are particularly under-represented in gbd assessments (percentage included in gbd: bacteria, %; fungi, %; helminths, %; protozoa, %; viruses, . %; fet, p = ? ). there was no difference between taxonomic divisions in the percentage reportable to the ec (fet, p = . ). human pathogens classed as emerging (compared to not emerging) were statistically more likely to have gbd estimates (fet, p, . , or = . , ci = . - . ) and be ec reportable (fet, p, . , or = . , ci = . - . ), but not more likely to have gbd estimates or be ec reportable if they were zoonotic compared to nonzoonotic (pearson's x , p = . and fet, p = . , respectively). for domestic animal pathogens in our list (table ) , fungi were particularly under-represented in the oie list (percentage included in oie: bacteria, %; fungi, %; helminths, . %, protozoa, . %, viruses, . %; fet, p = . ). by contrast, viruses are particularly under-represented in discontools (percentage included in discontools: bacteria, . %; fungi, %; helminths, . %; protozoa, . %; viruses, %; fet, p = . ). animal pathogens classed as zoonotic (compared to non-zoonotic) were (of borderline statistical significance) less likely to be included in the oie list (pearson's x , p = . , effect size(ø) = . , or = . ) but there was no difference in the percentage included in discontools, (pearson's x , p = . ) nor any effect of being emerging (versus not emerging) there was a statistically significant difference between the hindices of zoonotic, human-only or animal-only pathogens (twoway anova, f , = ? , p, . ); h-indices were significantly higher for human-only (untransformed mean = . . and lower for animal-only pathogens ( . . ) compared to zoonotic ( . . ). h-indices were higher (with borderline statistical significance) for emerging ( . . ) compared to not emerging ( . . ) pathogens (two-way anova, f , = . , p = . ). the interaction between zoonotic and emerging factors was not significant (p = . ). -discontools. there were significant correlations between h-indices and discontools estimates of public (human) health (zoonotic and animal pathogens) and impact on wider society (animal-only pathogens), and a further relationship of borderline significance between h-indices and the discon-tools overall result; no other correlations were significant (table ). the european commission has outlined measures to strengthen coordinated approaches to health security at eu level, including monitoring, early warning and combating specific threats of a cross-border nature. these measures could be for climate change resilience [ ] or for biosecurity, particularly for infectious diseases including communicable diseases, antimicrobial resistant and healthcare-associated infections related to communicable diseases, and biotoxins or other biological agents [ ] . in this study, we implement a number of previously defined actions [ ] , including presenting a quantitative evaluation for the impact of infectious pathogens affecting human health and well-being (via effects upon domestic animals) [ ] . the work is unique, starting with all known infectious pathogens, and then objectively and systematically deciding which occur in relevant hosts in europe using a transparent process. the study establishes priority lists of human and domestic animal pathogens (including zoonoses) present in europe, using the h-index as a proxy measure for impact. previous work suggests that higher h-indices indicate higher impact for a pathogen relative to lower h-indices [ ] , (mcintyre, unpublished material). the h-index method has both strengths and weaknesses. the strengths include that it is much more evidence-based and objective than semi-quantitative and qualitative approaches, and the results provide an easily understood quantitative estimate of impact. h-indices estimates can be simply and rapidly calculated, and they can therefore be repeatedly obtained to reflect changes in status, with the potential for automation of this process. the results are available for all pathogens at a global scale, and the scores reflect the wider scientific interest that would be expected to follow from a pathogen being either zoonotic or emerging [ ] . most importantly, within a study of human diseases, h-indices were correlated with daly estimates [ ] , (mcintyre, unpublished material). dalys are an accepted measure of true disease burden in humans which accounts for the years of healthy life lost as a result of poor health or disability as well as the potential years of life lost due to premature death [ ] . in further work, h-indices were also correlated with the number of human deaths (mcintyre, unpublished material). the weaknesses of the h-index method include that calculations need some manual oversight, as false positives can occur for instance when pathogens are used as model organisms; biases in results may happen because of trends in interest in specific pathogens, diseases or research fields or in certain regions; and estimates are subject to biases in funding (mcintyre, unpublished material) and research publication. h-indices are likely to underestimate the contribution of scientific literature published in non-english languages, although after translation some publications are included in wos and consequently in our calculations of h-indices. the literature searching method also doesn't account for the quality of publications in which pathogen names appear and the typical number of citations within different fields, and all bibliographic software packages incorporate newly published literature from different literature sources into their databases at different rates. finally, h-indices are only a proxy for impact, with the results susceptible to a lag in time-to-publication, and newly emerging pathogens likely to be under-represented. as the strengths and weaknesses of using the h-index method are different to those of other prioritisation methods, it is probably best used in combination with other approaches, for example, to shortlist a set of pathogens for more detailed risk assessment relying on expert opinion or qualitative data. it may also be used to prioritise diseases for which formal risk assessments are not possible because of data gaps. our priority lists of pathogens enabled investigation of why infectious pathogens are omitted from disease surveillance and impact quantification work [ , , , ] . we considered several reasons for exclusion, including lack of diagnosis or misdiagnosis [ ] , because the impact of particularly chronic infections is difficult to quantify or they are seen as less important, and because some pathogens are commensals or natural biota causing opportunistic infections in immune-compromised individuals; their pathogens include those which are zoonotic (z), non-zoonotic (nz), emerging (e) and not emerging (ne) [ , ] , or given a new status (ns) in this work. pathogens also included in the list of top animal pathogens are noted (a). the major pathogens causing diseases included within the global burden of disease (gbd) report are noted [ , ] , as are those reportable in the ec (ec) [ ] . doi: . /journal.pone. .t table . top domestic animal pathogens in europe, prioritised according to the h-index methodology [ ] with the same emerging and zoonotic definitions as for table . [ ] ; for some animal pathogens, this is the first time that emerging status has been examined. methods to assess disease impacts use metrics capturing either human or animal host effects; they neither measure the magnitude in all hosts nor take account of scientific knowledge and tools for control. it is hard to prioritise human and animal diseases, because of the different metrics used (health or societal impacts versus welfare or economic impacts). significant differences between hindices mean values for human-only, zoonotic, and animal-only pathogens provide evidence that this single measure may have some use as a one health metric accounting for such factors. for example values for zoonotic pathogens were higher than for animal-only, suggesting that they account for human as well as animal-impact. higher values for human-only compared to other pathogen groups suggests that zoonoses may be under-represented due to underestimation of their global burden [ , ] , or research impact [ ] , or because of biases in research impact and funding for chronic human pathogens [ ] . in addition, lower animal-only hindices may be due to funding biases. finally, there was limited evidence that the h-index method is a reasonable proxy for the impact of animal pathogens; animal pathogen h-indices were significantly positively correlated with subsections of discontools [ ] , including impact on public (human) health and overall results (borderline significance). if animal-only (not zoonotic) diseases were included, there was a significant positive relationship with impact on wider society. as the more animal-focussed subsections (disease knowledge, impact on animal health and welfare, impact on trade, and available control tools) were not correlated with h-indices, and h-indices were not affected by inclusion in the oie list [ ] , this suggests a human-centric bias in h-indices; for example, a pathogen causing little impact in animals may nevertheless have a high h-index if zoonotic. the priority lists presented in this work should be used by agencies and research organisations in combination with other risk assessment methods to identify gaps in working for priority setting. it has been suggested that zoonoses must be dealt with at the interface of human and animal health using all available information [ ] ; this work, combining the eid and h-index technique, demonstrates such 'big-data' approaches. pathogens also included in the list of top human pathogens are noted (h). the major pathogens causing diseases included within the oie list of notifiable terrestrial and aquatic animal diseases (oie) are noted [ ] , as are those included in the discontools project (disc) [ ] . doi: . /journal.pone. .t table . results of spearman's rank correlations between h-indices and the discontools prioritisation of major animal diseases [ ] . fungi ascaris suum, z, ne, ns helminths yersinia enterocolitica, z, e, h bacteria borna disease virus, z, e, ns, disc viruses bacillus anthracis evidence-based semiquantitative methodology for prioritization of foodborne zoonoses global change: impact, management, risk approach and health measures -the case of establishing priorities for national communicable disease surveillance prioritizing emerging zoonoses in the netherlands establishing priorities for european collaboration in communicable disease surveillance the h-index as a quantitative indicator of the relative impact of human diseases using open-access taxonomic and spatial information to create a comprehensive database for the study of mammalian and avian livestock and pet infections disability-adjusted life years (dalys) for diseases and injuries in regions, - : a systematic analysis for the global burden of disease study commission implementing decision of august amending decision / /ec laying down case definitions for reporting communicable diseases to the community network under decision no. / /ec of the european parliament and of the council oie-listed diseases, infections and infestations in force the ncbi nucleotide database homepage the ncbi medical subject headings (mesh) database homepage risk factors for human disease emergence host range and emerging and reemerging pathogens global trends in emerging infectious diseases overview -web of science the ncbi taxonomy database homepage agricolae package cran . - ed r: a language and environment for statistical computing adapting to climate change: towards a european framework for action decision no / /eu of the european parliament and of the council of october on serious cross-border threats to health and repealing decision no / /ec monitoring eu emerging infectious disease risk due to climate change quantifying the burden of disease -the technicial basis for diability-adjusted life years emerging infectious diseases: vulnerabilities, contributing factors and approaches neglected and endemic zoonoses the socioeconomic burden of parasitic zoonoses: global trends emerging zoonoses: the challenge for public health and biodefense years lived with disability (ylds) for sequelae of diseases and injuries - : a systematic analysis for the global burden of disease study thanks to helen roberts (uk department for environment, food and rural affairs) and john stephenson (institute of infection and global health, university of liverpool) for input during study development. key: cord- -kruy eex authors: weiner, h. richard title: diagnosis and prevention of rabies date: journal: compr ther doi: . /s - - - sha: doc_id: cord_uid: kruy eex over a million americans are bitten by animals every year. since the rabies vaccine is uniformly effective and the disease is uniformly fatal when the vaccine is not given, management decisions must be made promptly. year. since the rabies vaccine is uniformly effective and the disease is uniformly fatal when the vaccine is not given, management decisions must be made promptly. the risk of rabies exposure can often be assessed by understanding the local reservoirs of the disease. in locales where domestic animal rabies has not been controlled, dog bites are by far the most common mode of transmission. where a thorough program of immunizing domestic animals has been implemented, such as north america and western europe, dogs are responsible for less than % of cases. in these developed regions, other domestic animals, including cows, cats, horses, and other farm animals are occasionally found to have the virus. the risk conferred by wild animal bite varies widely, depending on species. foxes, coyotes, wolves, and jackals are highly susceptible to rabies, whereas skunks, raccoons, bats, bobcats, mongooses, and monkeys have somewhat less susceptibility. birds and reptiles, small rodents such as mice, chipmunks, and squirrels seldom carry the virus or transmit it to humans. transmission by bat is especially worrisome for several reasons. bat teeth, which are the size of to -gauge needles, inflict wounds, which are more difficult to detect than those of other animals. in a significant number of cases, bites or other significant exposure to bats cannot be recalled. , whether this reflects the disease's long incubation period or another mode of transmission, such as aerosolization, is unclear. occasionally wild animals may convey the disease to domestic pets. the area animal control, police, and health departments are useful resources for local patterns of infection. live virus enters the host through an epidermal puncture or exposure of mucous membrane. viral replication begins within striated muscle cells at the site of inoculation, after which, the virus enters the nervous system through unmyelinated sensory and motor terminals. the infection travels along peripheral nerves to the central nervous system (cns), where it replicates in the gray matter almost exclusively. then, it extends centrifugally to skin, salivary glands, heart, lung, liver, kidney, and skeletal muscle. manifestations of rabies arise from the involved tissue. five clinical stages may occur in rabies infections. , , incubation. the period of viral replication within peripheral tissue varies considerably, from four days to many years. on average, it ranges from to days. during this time, the only symptoms may reflect local wound healing. the victim may not recall exposure because it occurred so long before. different incubation periods may reflect size of inoculum, size and depth of injury, and distance to the cns. thus, a scratch on the hand may take longer to develop symptoms of rabies than a bite on the head. prodrome. for one to four days patients experience fever, headache, myalgias, malaise, fatigue, anorexia, nausea and vomiting, sore throat, and nonproductive cough. these symptoms, which occur when the virus first reaches the central nervous system, are so nonspecific that only careful history-taking would suggest the disease. paresthesias or fasciculations in the region of inoculation, which occur in about % of cases, may result from involvement of the dorsal root ganglion in the affected dermatome. other clues to infection include neurologic symptoms such as anxiety, agitation, irritability, insomnia, and depression. acute rabies encephalitis. the viral syndrome described above leads to further symptoms that clearly reflect cns involvement. these manifest themselves in two forms, furious and paralytic rabies. the more common furious rabies begins with hyperactivity and psychiatric symptoms such as disorientation, bizarre behavior, and hallucinations. episodes of running, biting and thrashing may be precipitated by sensory stimuli, such as touch, sight, or sound; hyperesthesia is common. combativeness, seizures, and focal paralysis may occur. during lucid periods subjects are cooperative and oriented, but these intervals grow shorter until coma and paralysis occur. autonomic dysfunction, with anisocoria and mydriasis, lacrimation, salivation, perspiration, and postural hypotension are seen during this period. cranial nerve involvement with diplopia, facial palsy, and optic neuritis may be noted; most significant is impaired deglutition, since salivation and poor swallowing results in "foaming at the mouth." about half of patients experience extremely painful spasms of the pharynx, larynx and diaphragm when attempting to swallow. even the sight of water may cause this pain and results in hydrophobia. about one-fifth of patients experience paralysis as the principal symptom throughout their infection. paralysis may occur at the site of inoculation, or may be diffuse and symmetric, or ascending; in the last case, symptoms may suggest guillain-barré syndrome. the agitation and mental derangement of furious rabies may be absent. this is termed apathetic rabies. coma and complications. within a week of the onset of neurologic symptoms, the patient lapses into coma. unless supportive measures are employed, respiratory failure quickly leads to death. the majority of complications occur while the patient is receiving intensive support during the coma phase. hyperventilation and respiratory alkalosis, often seen in the prodrome, are replaced by hypoventilation and metabolic acidosis. autonomic dysfunction, resulting in wide variations in blood pressure, cardiac dysrhythmias, and hypothermia has been observed. supraventricular rhythms, which occur early in the disease, turn to marked bradycardia and cardiac arrest with the onset of hypoxemia. hypotension may result from congestive heart failure, fluid depletion, or autonomic dysfunction. involvement of the hypothalamus, resulting in the syndrome of inappropriate secretion of antidiuretic hormone or diabetes insipidus, may occur. adult respiratory distress syndrome (ards) and intravascular thrombotic phenomena, common findings in patients requiring intensive support, are seen. recovery. every report on rabies describes it as almost uniformly fatal. the scrupulously noted exceptions are four cases, all of which occurred before . in every case, the survivor received pre-or postexposure prophylaxis. despite aggressive medical care, no person infected with rabies since has recovered. history of exposure to a potentially rabid animal limits what may otherwise become a diagnosis of exclusion, since rabies causes few findings that distinguish it from other viral encephalitides. guillain barré syn-drome, poliomyelitis, and allergic (i.e., postvaccinial) encephalomyelitis may all result in a clinical picture that would be difficult to distinguish from rabies. time-course and exposure history are helpful. hydrophobia and acrophobia (choking and gagging induced by blowing air on the patient's face) strongly suggest the diagnosis and separate it from tetanus. in the latter disease, mental status is usually normal. pseudohydrophobia, the hysterical fear of rabies, may occur in those with a possible history of exposure. they may have learned about the disease, and may have received postexposure prophylaxis. fear of the disease can induce anxiety, agitation, bizarre behavior, and pseudoseizures. standard laboratory and radiologic studies are not helpful in the diagnosis of rabies infection. wide variations in the peripheral white blood cell count have been observed, but most measurements are normal or slightly elevated with a mild monocytosis. red blood cell count decreases slowly, while platelet numbers are normal. chest roentgenography may be normal, or may reflect complications such as pneumonia, congestive heart failure, or ards. cerebral spinal fluid (csf) may show leukocytosis with mononuclear predominance, suggesting an encephalitis; protein and glucose levels are often normal, as are opening pressures. electroencephalography is not diagnostic while computed tomography and magnetic resonance imaging often do not reveal any abnormality. the failure of conventional studies to identify the disease underscores the importance of a carefully obtained history. during the first week of the illness, immunofluorescent staining of hair follicle biopsies taken from the back of the neck is the most reliable test of infection. isolation of virus from saliva, brain, csf, and urine may occur, but false negatives limit the usefulness of such studies. the virus is not found in blood and stool. in the unvaccinated patient, rapid fluorescent antibody testing will demonstrate a rapid rise in neutralizing antibody in serial serum specimens. previously vaccinated subjects will demonstrate a rise in antibody titer as well as presence of antibody in the csf. with the onset of symptoms, no treatment has been shown to reduce the % fatality rate of this infection. immune globulin fragments, interferon and numerous other antiviral agents have not altered the course of the disease. prevention is the only means of avoiding death. primary prevention and preexposure prophylaxis. measures that decrease exposure to the virus, such as mandatory animal vaccination, animal control measures, and public education are fundamental to reducing the incidence. those who cannot avoid possible exposure (e.g., veterinarians, spelunkers and laboratory workers) should be given preexposure prophylaxis according to their level of risk. those who face the continuous risk of exposure, such as research laboratory personnel and vaccine production workers, should receive the primary preexposure course with serologic testing every six months. booster doses should be given when antibody levels fall below acceptable values. persons who face episodic and usually recognized exposure to the virus, a category which includes spelunkers, veterinarians, animal control and wildlife workers, and travelers to foreign areas booster* hdcv, human diploid cell vaccine; pcec, purified chick embryo cell vaccine; rva, rabies vaccine, adsorbed. *depends on exposure risk category and immune status. in occupations where exposure risk is infrequent and comes from recognized sources (animal workers in areas of low enzooticity) should receive the primary vaccination course without further testing or booster. the general population of the us does not require preexposure vaccination. their exposures are rare and are always episodic. (table every animal bite should be evaluated for potential to transmit this deadly virus. the u.s. centers for disease control and prevention recommendations for evaluating risk are listed in table . if the exposure risk is sufficient to warrant treatment, therapy should begin with chemical and mechanical cleansing of the wound site using soap and water. the likelihood of infection is reduced up to % by vigorous cleaning. tetanus toxoid and antimicrobial drugs are often indicated. passive antibody with antirabies antiserum (human rabies immune globulin, hrig) and active immunization with antirabies vaccine together constitute the balance of postexposure prophylaxis. dosage and sites of administration are given in table . local reactions, including swelling, erythema, and induration are common ( % to %); systemic reactions such as fever, headache and nausea occur in % to %; and immediate hypersensitivity with urticaria is seen in far fewer than % of cases. prompt identification of rabies exposure, along with appropriate preventive therapy, can prevent infection and death from rabies. ct new aspects of rabies with emphasis on the epidemiology, diagnosis, and prevention of the disease in the united states human deaths from cryptogenic bat rabies in the usa epidemiology of human rabies in the united states rabies in humans features and treatment of rabies recommendations of the immunization practices advisory committee (acip). mmwr morb mortal wkly rep recommendations of the immunization practices advisory committee (acip). mmwr morb mortal wkly rep recommendations of the immunization practices advisory committee (acip). mmwr morb mortal wkly rep key: cord- -q s fkh authors: roth, james a. title: mechanistic bases for adverse vaccine reactions and vaccine failures date: - - journal: adv vet med doi: . /s - ( ) - sha: doc_id: cord_uid: q s fkh nan vaccines have proven to be very beneficial for controlling diseases in domestic animals. their widespread use has dramatically reduced the incidence of severe and fatal diseases in companion animals (canine distemper, canine parvovirus, infectious canine hepatitis, and feline panleukopenia). they have also enabled the intensification of livestock production, thus enabling great increases in efficiency in animal origin food and fiber production. in addition, animal vaccines have improved human health through control of zoonotic diseases such as rabies, brucellosis, and leptospirosis. indeed, it can be argued that animal vaccines have had a profound impact on modern society. without effective rabies vaccines many people would not opt to keep companion animals in their homes, and without effective vaccines for controlling major diseases in food-producing animals the availability of animal proteins for human consumption would be greatly reduced. however, in spite of the success of animal vaccines, vaccines sometimes induce adverse reactions in animals and sometimes they fail to protect animals. when making decisions regarding vaccination programs for animals, veterinarians and animal owners must weigh the risks of vaccinating vs. the risks of not vaccinating. they must also use vaccines in a manner that induces optimal protection. this article provides an overview of some of the reasons why vaccines occasionally produce adverse reactions (table i) and reasons why vaccines sometimes fail to protect animals from disease (table ii) . to produce protective immunity, a vaccine must stimulate a reaction in the animal. there usually must be a reaction both at the site of injection and systemically in order to produce an effective immune response. this reaction involves extensive activity by antigen-presenting cells, production of a variety of cytokines, and alterations in the trafficking of lymphocytes within the body. in addition, if the vaccine contains live organisms, they probably need to replicate to induce effective immunity. live viruses must infect and replicate within cells. these essential reactions to a vaccine may induce observable clinical signs. hopefully, the reaction to the vaccine will be mild and either unnoticeable or acceptable to the animal owner. to understand vaccine safety and efficacy, it is important to understand the process by which vaccines are developed and tested by vaccine producers, and licensed by the united states department of agriculture (usda), animal and plant health inspection service (aphis), center for veterinary biologics (cvb). the federal government regulations for the united states of america regarding veterinary vaccines contamination with extraneous agents failure to inactivate agent in killed vaccine residual virulence of vaccine organisms vaccination of immunosuppressed animal immune suppression induced by the vaccine excessive induction of cytokine release multiple vaccines administered concurrently hypersensitivity to vaccine antigens type i--immediate type type iimcytotoxic type type iiimimmune complex type type ivmdelayed type triggering or exacerbation of hypersensitivity to nonvaccine antigens allergies autoimmune disease induction of neoplastic changes mlv bvd vaccine triggering mucosal disease in persistently infected cattle are found in the virus serum toxin act (vsta) in title of the code of federal regulations ( cfr). the vsta gives the usda the authority to regulate veterinary vaccines in the united states. according to the cfr a usda licensed biological must be "pure, safe, potent, and efficacious, and not be worthless, contaminated, dangerous, or harmful." to understand this statement, it is important to understand what is meant by safe and efficacious. the definition found in the cfr for safe or safety regarding veterinary biologics is "freedom from proper ties causing undue local or systemic reactions when used as recom-mended or suggested by the manufacturer." this definition has two important qualifiers for the term safety. it does not state that a vaccine should produce no reaction, rather it states that a vaccine should not cause "undue local or systemic reactions." this is a recognition of the fact that stimulating a potent immune response is likely to produce at least a mild local and systemic reaction in the animal. the second important point is that according to the definition the safety of the vaccine is only ensured when it is used as recommended or suggested by the manufacturer. the recommendations and suggestions can be found on the label for the vaccine. most vaccine label statements will indicate that a particular vaccine is only for use in healthy animals of a particular species. healthy is defined as "apparently normal in all vital functions and free of signs of disease." the cfr definition for efficacious or efficacy is "specific ability or capacity of the biological product to effect the result for which it is offered when used under the conditions recommended by the manufacturer." the label found on the vaccine will indicate the "result for which the vaccine is offered" and will also indicate the conditions under which the vaccine is recommended for use. therefore, it is very important to read and follow label instructions in order to achieve maximum safety and efficacy from vaccine usage. when animals develop adverse clinical signs within a few days to weeks after vaccination it is important to determine whether those clinical signs were vaccine induced or were not due to vaccination and only coincidentally occurred after the vaccine was administered. animals commonly experience adverse clinical signs from a wide variety of causes and animals are commonly vaccinated. therefore, it is to be expected that occasionally adverse clinical signs will occur after animals have been vaccinated for reasons unrelated to vaccine administration. there are also many reasons why vaccines may induce adverse reactions in the animal. it is important to differentiate true adverse vaccine reactions from false adverse vaccine reactions. some of the causes of true adverse vaccine reactions are summarized in table i and explained next. a prominent example of this occurred when it was discovered that some lots of the live oral human poliomyelitis vaccine were contami-nated with live simian virus (sv ) in the s (pennisi, ; shah and nathanson, ) . millions of people were potentially exposed to live sv through administration of polio vaccine. to date, there is no solid epidemiologic evidence that any adverse health affects can be attributed to exposure to this agent. the sv virus had not yet been discovered when the human polio vaccine was produced. this raises the question of how does one test for all potential known and unknown viruses in each production lot of modified live virus vaccines. (gard and lycke, ; nathanson and langmuir, ) . this resulted in several cases of poliomyelitis in people that had received the vaccine. there have also been cases where formaldehyde failed to inactivate the foot-and-mouth disease virus (beck and strohmaier, ; king et al., ) and the venezuelan equine encephalitis virus (kinney et al., ) in their respective vaccines. in both of these cases the vaccine was shown to induce disease because of the lack of complete inactivation of the virus by the formaldehyde (brown, ) . an example of a failure to completely inactivate a bacterial pathogen in a killed bacterin occurred when thimerosol was used to inactivate haemophilus somnus in an h. somnus vaccine. the thimerosol failed to kill the h. somnus. approximately half the ani- mals on one farm that were injected with vaccine shortly after its production developed thromboembolic meningoencephalitis and died. modified live vaccine organisms have been attenuated to have reduced virulence. the attenuation must be shown to be stable when passaged through animals; therefore, reversion to virulence is thought to be a rare event. however, the attenuated vaccine strains may be capable of producing disease in immunosuppressed animals. induction of disease by the vaccine organism has occasionally been reported when modified live virus (mlv) vaccines have been administered to healthy animals. however, it has occurred much more frequently when mlv vaccines are administered to unhealthy animals, by a nonrecommended route of exposure, to animals younger than the intended age for use of the vaccine, or when the vaccine is used in other than the intended species. examples of mlv vaccines occasionally causing disease in healthy animals of the recommended species without apparent predisposing causes include the induction of rabies in dogs and cats after administration of an mlv rabies vaccine (bellinger et al., ; esh et al., ; erlewein, ; whetstone et al., ; pedersen et al., ) and the induction of ovarian lesions and infertility in seronegative heifers administered mlv bovine herpesvirus (bhv ) vaccine during estrus chiang et al., ; miller et al., ; van der maaten et al., ) . since most heifers already have antibody to bhv due to either vaccination or previous exposure, this is thought to be a rare occurrence. an example of vaccine-induced disease resulting from administration of vaccine to unhealthy animals is the induction of encephalitis by mlv canine distemper virus vaccine in dogs infected with canine parvovirus (krakowka et al., ) . an example of adverse vaccine reaction after exposure of an animal to an mlv vaccine by a nonrecommended route of exposure is the induction of clinical feline viral rhinotracheitis after inadvertent exposure by the intranasal route to an mlv vaccine that was intended for intramuscular administration only (povey and wilson, ) . mlv vaccines that have been shown to be safe in older animals may not be safe in neonatal animals. an mlv bhv- vaccine induced fatal bhv infection in neonatal purebred salers calves (bryan et al., ) . this may have been partially due to the breed of the animals since there are other reports that mlv bhv vaccines are apparently safe in neonatal calves (schuh and walker, ). there have been several examples of mlv vaccines inducing lethal disease when administered to a species other than the target species. an mlv pseudorabies virus vaccine produced fatal pseudorabies in lambs (clark et al., ; van alstine et al., ) . this occurred when a syringe that had been used to administer the pseudorabies vaccine to pigs was used without proper disinfection to vaccinate lambs with another vaccine days later. the mlv canine distemper virus vaccine has been shown to induce canine distemper infection in gray foxes (halbrooks et al., ) , kinkajous (kazacos et al., ) , and lesser pandas (bush et al., ). an mlv rabies vaccine has been shown to induce rabies in a pet skunk (debbie, ). an mlv feline panleukopenia vaccine induced cerebellar hypoplasia when given experimentally to neonatal ferrets (duenwald et al., ). an mlv bovine viral diarrhea (bvd) virus vaccine has been shown to suppress neutrophil function and lymphocyte blastogenesis in cattle (roth and kaeberle, ) . this correlates with the observation that cattle tend to be somewhat more susceptible to bacterial pneumonia after administration ofmlv bvd vaccines, especially if the animals are stressed at the time of vaccination. several commercially available canine vaccines have been shown to be capable of inducing lymphopenia and suppressing blastogenesis of peripheral blood lymphocytes (phillips et al., ; mastro et al., ; kesel and neil, ) . lymphopenia and suppression of blood lymphocyte blastogenesis must be interpreted with caution, however, because it may only be an indication of changes in lymphocyte trafficking between the blood and lymphatic systems rather than an indication of depressed lymphocyte function. vaccination with an mlv bhv vaccine has been shown to exacerbate the lesions of infectious bovine keratoconjunctivitis after experimental intraocular challenge with moraxella bovis (george et al., ). interleukin (il- ), il- , and tumor necrosis factor ~ (tnf-~) are potent proinflammatory cytokines that are released by macrophages and other cells in response to infection, endotoxin and other bacterial components, and some vaccine adjuvants. these proinflammatory cytokines can induce a wide range of clinical signs. they may induce acute inflammation at the local site of production, they may induce rapid synthesis and secretion of acute phase proteins by the liver, they may act on the hypothalamus to induce fever and malaise, they may reduce rate of gain and feed efficiency, and in sufficiently high concentrations they may induce hypoglycemia, reduce cardiac output, cause hypovolemic shock, and cause disseminated intravascular coagulation. lipopolysaccharide (or endotoxin) from gram-negative bacteria is one of the most potent inducers of the proinflammatory cytokines (cullor, ; ellis and yong, ; galanos and freudenberg, ) . a number of other bacterial components, listed in table iv , have also been shown to induce proinflammatory cytokine production (erdos et al., ; henderson and wilson, ; allison and eugui, ) . these components are generally the most active if they are released from the degraded bacterial cell. killed bacterins that contain excessive amounts of these bacterial components can induce clinical signs due to excessive induction of cytokine release. this is more likely to occur if multiple killed bacterins are administered at the same time and if these bacterins contain adjuvants that also induce cytokine release. the production of small amounts of proinflammatory cytokines is beneficial to the induction of a protective immune response. however, overproduction of the proinflammatory cytokines can have mild to very severe adverse side affects. (bonin et al., ; wilson et al., ; erdos et al., ) . as with all of the hypersensitivity reactions, the animal will not react on first exposure to an antigen (unless it has received passive antibody responsible for the reaction). it will only react after there has been sufficient time to produce the sensitizing antibody or memory t cells. a local type i hypersensitivity reaction may occur due to ige induced against infectious agents by the vaccine. immunization against bovine respiratory syncytial virus under experimental conditions was shown to induce ige antibodies specific for brsv which apparently contributed to the development of symptoms following aerosol challenge with brsv (stewart and gershwin, a,b) . vaccine-induced type ii (cytotoxic type) hypersensitivity reactions can occur when vaccines are used that contain normal cell antigens. for example, vaccines that contain erythrocyte antigens may induce anti-erythrocyte antibodies leading to immune-mediated hemolytic anemia. type iii (immune complex type) hypersensitivity can occur when circulating antibody specific for vaccine antigens is present at the time of vaccination. this can lead to an arthus reaction at the site of injection due to complement fixation and neutrophil recruitment to the site. this mechanism is commonly responsible for the local inflammatory reaction at the site of injection, especially when administering booster vaccinations with killed vaccines. sometimes, hypersensitivity can be one component of a more complex adverse vaccine reaction. antibody induced by the vaccine may lead to immune complex type hypersensitivity reactions after the animal becomes infected when the antibody binds to replicating infectious agents. examples include anterior uveitis and corneal edema (blue eye) after vaccination with canine adenovirus (carmichael et al., ; wright, ) and the sensitization to the effusive form of feline infectious peritonitis after vaccination with experimental killed vaccines (pedersen and black, ) . sometimes, hypersensitivity may be one component of a more complex adverse vaccine reaction. bacterins for pasteurella haemolytica which were marketed and widely sued for several years were of marginal efficacy and were even capable of increasing the severity of lesions in animals either experimentally (wilkie et al., ) or naturally exposed (bennett, ) to the p. haemolytica. there are at least two hypothesized mechanisms by which the immune response induced by the bacterin could potentiate pneumonia after p. haemolytica challenge. first, the high concentration of complement-fixing antibody in-duced by vaccination with a bacterin could rapidly activate complement if a large number of p. haemolytica organisms were introduced into the lung either naturally or artificially. this could cause a type iii hypersensitivity response leading to acute inflammation in the lung and severe pneumonia. second, antibody against cell surface antigens will opsonize the p. haemolytica in the lung and enhance phagocytosis by alveolar macrophages and neutrophils. because there may be insufficient leukotoxin-neutralizing antibody or cell-mediated immunity to activate phagocytes, the bacteria present in the alveoli and ingested by phagocytes are not efficiently killed and may produce leukotoxin that could destroy the phagocytes. this destruction would cause the phagocytes to release their hydrolytic enzymes into the lung. in the last few years concern has been expressed that vaccination may trigger or exacerbate autoimmune disease or allergies (hypersensitivities), especially in dogs and cats (see article by dr. jean dodds in this volume). vaccination has been shown to augment production of ige antibody to pollen in inbred atopic dogs (frick and brooks, ) . remember that animals with allergies or autoimmune diseases are not healthy animals, and that vaccines are only recommended for use in healthy animals. dr. harm hogenesch addresses the topic of vaccineinduced autoimmunity in another article in this volume. in recent years, an increased incidence of fibrosarcoma occurring at sites commonly used for vaccination in cats has been observed (hendrick et al., (hendrick et al., , kass et al., ) . the causal relationship and mechanistic basis for vaccine-associated fibrosarcomas in cats has not been firmly established (ellis et al., ) . shortly after mlv bvd vaccines were introduced, it was recognized that a very small percentage of cattle developed a syndrome - days after vaccination that closely resembled bvd mucosal disease (lambert, ; peter et al., ) . based on the current understanding of the pathogenesis of mucosal disease (bolin et al., ; brownlie et al., ) this was almost certainly due to the cytopathic bvd virus in the vaccine triggering mucosal disease in calves that were immunotolerant to, and persistently infected with, a noncytopathic bvd virus. the mechanistic basis for the induction of the lesions of mucosal disease is not clearly understood. this unique syndrome is primarily due to abnormalities in the animal rather than to a defect in the vaccine. vaccines are tested for safety and efficacy when administered to healthy animals in the formulation in which they are packaged to be sold. vaccines are not required to be tested for safety and efficacy when administered concurrently with other vaccines. this would not be practical since there are too many possible vaccines that may potentially be used in combination. an example of a safety problem that occurred when two different vaccines were administered concurrently involved a newly developed mlv canine coronavirus and parvovirus vaccine given at the same time as an mlv canine distemper-hepatitis virus vaccine. the evidence indicated that the other mlv components allowed the canine coronavirus in the vaccine to induce neurologic disease in some vaccinated animals (wilson et al., ) . injection site lesions are a common occurrence and are of great concern in food-producing animals. they may lead to unacceptable blemishes in, or decreased quality of, meat intended for human consumption. there are many possible causes of injection site lesions, including organisms introduced with a contaminated needle, live contaminating organisms in the vaccine, adjuvant induced reactions, cytokine release, hypersensitivity reactions (types i, ii, iii, or iv), trauma, and hemorrhage (straw et al., (straw et al., , droual et al., ; littledike, ; stokka et al., ; dexter et al., ; apley et al., ; straw, ) . vaccines that are licensed by the usda have been tested to determine that they are safe and effective. however, "effective" is a relative term. it does not mean that the vaccine must be able to induce complete immunity under all conditions which may be found in the field. this would not be realistic since the immune system is not capable of such potent protection under adverse conditions. to be federally licensed, the vaccine must have been tested under controlled experimental conditions. the vaccinated group must have had significantly less disease than the nonvaccinated control group. this testing is typically done on healthy, nonstressed animals under good environmental conditions and with a controlled exposure to a single infectious agent. vaccines may be much less effective when used in animals that are under stress, incubating other infectious diseases, or exposed to a high dose of infectious agents due to overcrowding or poor sanitation. it is important to remember that for most diseases the relationship between the infectious agent and the host is sufficiently complicated that vaccination cannot be expected to provide complete protection. the vaccine can increase the animal's resistance to disease, but this resistance can be overwhelmed if good management practices are not followed. some of the causes for vaccine failure are summarized in table ii and explained next. the host requires several days after vaccination before an effective immune response will develop. if the animal encounters an infectious agent near the time of vaccination, the vaccine will not have had time to induce immunity. the animal may come down with clinical disease resulting in apparent vaccination failure. in this situation, disease symptoms will appear shortly after vaccination and may be mistakenly attributed to vaccine virus causing the disease (mckercher et al., ) . improperly handled and administered vaccines may fail to induce the expected immune response in normal, healthy animals. modified live bacterial and viral vaccines are only effective if the agent in the vaccine is viable and able to replicate in the vaccinated animal. observing proper storage conditions and proper methods of administration are very important for maintaining vaccine viability. failure to store the vaccine at refrigerator temperatures, or exposure to light, may inactivate the vaccine. even when stored under appropriate conditions, the vaccine loses viability over time. therefore, vaccines that are past their expiration date should not be used. the use of chemical disinfectants on syringes and needles can inactivate modified live vaccines if there is any residual disinfectant. the use of improper diluent or the mixing of vaccines in a single syringe may also inactivate modified live vaccines. diluents for lyophilized vaccines are formulated specifically for each vaccine. a diluent that is appropriate for one vaccine may inactivate a different vaccine. some vaccines and diluents contain preservatives that may inactivate other modified live vaccines. for these reasons, multiple vaccines should not be mixed in a single syringe unless that particular combination has been adequately tested to ensure there is no interference. vaccine failures may occur because a vaccinated animal is not able to respond appropriately to the vaccine. vaccine failure in young animals may be due to the presence of maternal antibody which prevents adequate response to vaccination. it can also be due to immunosuppression from a variety of causes. maternal antibodies derived from colostrum are a well-known cause of vaccine failure (greene, ) . these antibodies in the young animal's circulation may neutralize or remove the antigen before it can induce an immune response. typically, virulent infectious agents are capable of breaking through maternal immunity earlier than modified live or killed vaccines. this means that even if young animals are immunized frequently, there still may be a period when they are vulnerable to infection. vulnerability occurs between the time that young animals lose their maternal antibody and before they develop their own active immune responses. this period can be shortened by the use of less-attenuated and/or higher titered modified live vaccines or the use of killed vaccines with high antigenic mass and strong adjuvants (smith-carr et al., ; larson and schultz, ) . a high challenge dose of infectious agents will break through maternal immunity sooner than low exposure to infectious agents. therefore, overcrowding and poor sanitation exacerbate the problem of inducing immunity in young animals before they come down with clinical disease. veterinarians commonly recommend that puppies and kittens be vaccinated every weeks between approximately and weeks of age. however, for large domestic animals, a single vaccination is commonly recommended to induce immunity during the first few weeks or months of life. there is no inherent difference between large and small domestic animals in their responses to vaccination in the face of maternal immunity. the frequent vaccinations recommended in pup-pies and kittens minimizes the period of vulnerability to infectious diseases. because only one vaccination is commonly recommended for large domestic animals, the timing of vaccination is important. if the vaccine is administered too soon, it may be ineffective because of the presence of maternal antibody. if the vaccine is administered after all maternal antibodies are gone from animals in the group, there may be a prolonged period of vulnerability before they develop their own immune response. the optimal age to vaccinate young animals is highly variable. it will depend on the antibody titer of the mother and the amount of colostrum ingested. it is impossible to predict an optimal age to vaccinate a young animal, unless its antibody titers are determined. most veterinarians and producers decide that because of time and expense considerations it is impractical to vaccinate young food-producing animals frequently to minimize their period of vulnerability to infection. however, frequent vaccination may be justified in cases of unusually high disease incidence in young animals. immunosuppression due to a variety of factors including stress, malnutrition, concurrent infection, or immaturity or senescence of the immune system may also lead to vaccination failure. if the immunosuppression occurs at the time of vaccination, the vaccine may fail to induce an adequate immune response. if the immunosuppression occurs sometime after vaccination, then disease may occur due to reduced immunity in spite of an adequate response to the original vaccine. therapy with immunosuppressive drugs (e.g, glucocorticoids) may also cause this to occur. most vaccines do not produce complete immunity to disease. they provide an increased ability to resist challenge by infectious agents. if a high-challenge dose of organisms is present due to overcrowding or poor sanitation, the immune system may be overwhelmed, resulting in clinical disease. the peak response to a vaccine typically occurs - weeks after vaccination. the level of immunity then begins to gradually decline. a common recommendation is to revaccinate annually. however, if the animal did not have a strong initial immune response due to stress at the time of vaccination, or if it is stressed and exposed to a highchallenge dose several months after vaccination, there may not be enough residual immunity to protect the animal. this is especially true for certain killed vaccines. under these circumstances, it may be necessary to revaccinate more frequently than once per year. for certain types of infectious agents, particularly bacteria that are vulnerable to control by the development of antibodies against surface components and viruses which use rna as their genetic material and consequently have high mutation rates, there are often several antigenic variants of each agent. for antibody-mediated protection to be effective, the antibodies formed must bind the important strain-specific antigens on the surface of the bacteria or virus. cell-mediated immunity is usually not as strain specific as antibody-mediated immunity. to determine if a vaccine's failure to protect is due to antigenic differences between the vaccine and field strains it is necessary to isolate the field strain and compare it to the vaccine strain. antigenic differences between strains leading to lack of vaccine efficacy are usually more of a problem with killed vaccines than modified live vaccines. as mentioned earlier, vaccines are tested for safety and efficacy when administered singly to animals. however, multiple vaccines are commonly administered concurrently to animals. very little published data are available concerning the efficacy of vaccines when used in combination. one study demonstrated that there was no detrimental effect on the antibody response to a bovine respiratory syncitial virus vaccine when administered in combination with up to different immunogens (carmel et al., ) . in contrast, an mlv bhv vaccine when administered in combination with an experimental pasteurella haemolytica vaccine containing outer membrane proteins and genetically attenuated leukotoxin significantly reduced the antibody response to the leukotoxin and the efficacy of the p. haemolytica vaccine in preventing morbidity and mortality due to bovine respiratory disease (harland et al., ) . mild local and systemic reactions to vaccines are to be expected as a natural consequence of vigorously stimulating the immune system. dramatic adverse reactions to vaccines are occasionally due to mistakes during the production or handling of vaccines. more often, they are due to not following label instructions, particularly the restriction to only use vaccines in healthy animals. it is important to publish welldocumented instances of adverse vaccine reactions so that producers and users of vaccines can all learn from the experience and avoid similar problems. vaccine failure to protect from disease is usually due to problems with either client education or compliance with good animal management practices. it is important for clients to understand the proper timing and method of vaccine administration, what to realistically expect for vaccine efficacy, and the importance of minimizing immunosuppressive factors and exposure to high doses of infectious agents in vaccinated animals. veterinary vaccines have produced dramatic benefits in terms of animal health, human health, and efficiency of food production. advances in research and the accumulating experience with vaccines are leading to safer and more effective vaccines. proper usage of vaccines and adherence to good management practices will continue to be essential to achieve maximal vaccine safety and efficacy. live sv in human polio vaccine (pennisi killed hog cholera virus in pseudorabies vaccine live mycoplasma in multiple live virus veterinary vaccines live border disease virus in orf vaccine live bovine leukemia virus in babesiosis and anaplasmosis vaccines live bovine viral diarrhea virus in hog cholera vaccine live border disease virus in pseudorabies vaccine live blue tongue virus in a canine vaccine live bovine viral diarrhea virus in bovine vaccines induction of cytokine formation by bacteria and their products. in 'virulence mechanisms of bacterial pathogens subcutaneous injection site comparison of two multiple valent clostridial bacterin/toxoids in feedlot cattle subtyping of european foot-and-mouth disease virus strains by nucleotide sequence determination rabies induced in a cat by high-egg-passage flury strain vaccine efficacy ofpasteurella bacterins for yearling feedlot cattle severe clinical disease induced in cattle persistently infected with noncytopathic bovine viral diarrhea virus by superinfection with cytopathic bovine viral diarrhea virus sensitization against calf serum proteins as a possible cause of allergic reactions after vaccination review of accidents caused by incomplete inactivation of viruses experimental production of fatal mucosal disease in cattle fatal, generalized bovine herpesvirus type- infection associated with a modified-live infectious bovine rhinotracheitis parainfluenza- vaccine administered to neonatal calves vaccine-induced canine distemper in a lesser panda effects of vaccination against immunogens in beef replacement heifers at weaning viral-antibody complexes in canine adenovirus type (cav- ) ocular lesions: leukocyte chemotaxis and enzyme release the effect of infectious bovine rhinotracheitis vaccine on reproductive efficiency in cattle vaccinated during estrus pathogenicity of a modified-live pseudorabies vaccine virus in lambs safety and efficacy of gram-negative bacterial vaccines vaccine-induced rabies in a pet skunk incidence of injection-site blemishes in beef top sirloin butts local reaction and serological responses in commercial layer chickens injected intramuscularly in the leg with oiladjuvanted mycoplasma gallisepticum bacterin feline panleukopenia: experimental cerebellar hypoplasia produced in neonatal ferrets with live virus vaccine systemic adverse reactions in young simmental calves following administration of a combination vaccine use of immunohistochemistry and polymerase chain reaction for detection of oncornaviruses in formalin-fixed, paraffin-embedded fibrosarcomas from cats the demonstration of the sensitizing effect of the residual animal serum content of vaccines post-vaccinal rabies in a cat vaccine-induced rabies in four cats canine fatalities associated with the use of a modified live vaccine administered during late stages of pregnancy immunoglobulin e antibodies to pollens augmented in dogs by virus vaccines mechanisms of endotoxin shock and endotoxin hypersensitivity inactivation of poliovirus by formaldehyde. analysis of inactivation curves enhancement of infectious bovine keratoconjunctivitis by modified-live infectious bovine rhinotracheitis virus vaccine infectious diseases of the dog and cat response of gray foxes to modified live-virus canine distemper vaccines the effect of subunit or modified live bovine herpesvirus- vaccines on the efficacy of a recombinant pasteurella haemolytica vaccine for the prevention of respiratory disease in feedlot calves modulins: a new class of cytokine-inducing, proinflammatory bacterial virulence factor postvaccinal sarcomas in the cat: epidemiology and electron probe microanalytical identification of aluminum comparison of fibrosarcomas that developed at vaccination sites and at nonvaccination sites in cats: cases ( - ) hog cholera antibodies in pigs vaccinated with an aujeszkyvaccine based on antigen produced in ib-rs- cells epidemiologic evidence for a causal relation between vaccination and fibrosarcoma tumorigenesis in cats vaccinationinduced distemper in kinkajous combined mlv canine parvovirus vaccine: immunosuppression with infective shedding. vm / sac biochemical identification of viruses causing the outbreaks of foot-and-mouth disease virus in the u.k molecular evidence for the origin of the widespread venezuelan equine encephalitis epizootic of to canine parvovirus infection potentiates canine distemper encephalitis attributable to modified live-virus vaccine bovine viral diarrhea: prophylaxis and postvaccinal reactions high-titer canine parvovirus vaccine: serologic response and challenge-of-immunity study variation of abscess formation in cattle after vaccination with a modified-live pasteurella haemolytica vaccine investigation of dams and their offspring inoculated with a vaccine contaminated by bovine viral diarrhea virus. vm/ sac outbreaks of border disease in goats induced by a pestivirus-contaminated orf vaccine, with virus transmission to sheep and cattle repeated suppression of lymphocyte blastogenesis following vaccination of cpv-immune dogs with modified-live cpv vaccines complications in cattle following vaccination with a combined bovine viral diarrhea-infectious bovine rhinotracheitis vaccine infertility in heifers inoculated with modified-live bovine herpesvirus- vaccinal strains against infectious bovine rhinotracheitis on postbreeding day the cutter incident. poliomyelitis following formaldehyde-inactivated poliovirus vaccination in the united states during the spring of which bvd vaccine should i use? attempted immunization of cats against feline infectious peritonitis, using avirulent live virus or sublethal amounts of virulent virus rabies vaccine virus infection in three dogs monkey virus dna found in rare human cancers characteristics of a condition following vaccination with bovine virus diarrhea vaccine effects of vaccines on the canine immune system a comparison of inactivated feline viral rhinotracheitis and feline caliciviral disease vaccines with live-modified viral vaccines bovine leucosis virus contamination of a vaccine produced in vivo against bovine babesiosis and anaplasmosis suppression of neutrophil and lymphocyte function induced by a vaccinal strain of bovine viral diarrhea virus with and without the concurrent administration of acth outbreaks of neonatal infectious bovine rhinotracheitis human exposure to sv : review and comment nicrotic oophoritis in heifers vaccinated intravenously with infectious bovine rhinotracheitis virus vaccine during estrus canine parvovirus. part i. pathogenesis and vaccination role of ige in the pathogenesis of bovine respiratory syncytial virus in sequential infections in vaccinated and nonvaccinated calves detection of ige antibodies to bovine respiratory syncytial virus inflammatory response to clostridial vaccines in feedlot cattle injection reactions in swine comparison of tissue reactions produced by haemophilus pleuropneumoniae vaccines made with six different adjuvants in swine antibody production and tissue irritation in swine vaccinated with actinobacillus bacterins containing various adjuvants a survey of mycoplasma detection in veterinary vaccines vaccineinduced pseudorabies in lambs ovarian lesions induced in heifers by intravenous inoculation with modified-live infectious bovinerhinotracheitis virus on the day after breeding contamination of a live virus vaccine against pseudorabies (aujeszky's disease) by an ovine pestivirus pathogen for the pig bovine viral diarrhoea virus infections in piglets born to sows vaccinated against swine fever with contaminated vaccine use of monoclonal antibodies to confirm vaccine-induced rabies in ten dogs, two cats, and one fox abortion and death in pregnant bitches associated with a canine vaccine contaminated with bluetongue virus response of calves to lung challenge exposure with pasteureua haemolytica after parenteral or pulmonary immunization a neurologic syndrome associated with use of a canine coronavirus-parvovirus vaccine in dogs detection of sensitizing tissue culture components in viral vaccines canine adenovirus: its role in renal and ocular disease: a review key: cord- - cty s o authors: merten, o.-w. title: virus contaminations of cell cultures – a biotechnological view date: - - journal: cytotechnology doi: . /a: sha: doc_id: cord_uid: cty s o in contrast to contamination by microbes and mycoplasma, which can be relatively easily detected, viral contamination present a serious threat because of the difficulty in detecting some viruses and the lack of effective methods of treating infected cell cultures. while some viruses are capable of causing morphological changes to infected cells (e.g. cytopathic effect)which are detectable by microscopy some viral contaminations result in the integration of the viral genome as provirus, this causes no visual evidence, by means of modification of the cellular morphology. virus production from such cell lines, are potentially dangerous for other cell cultures (in research labs)by cross contaminations, or for operators and patients (in the case of the production of injectable biologicals) because of potential infection. the only way to keep cell cultures for research, development, and the biotech industry virus-free is the prevention of such contaminations. cell cultures can become contaminated by the following means: firstly, they may already be contaminated as primary cultures (because the source of the cells was already infected), secondly, they were contaminated due to the use of contaminated raw materials, or thirdly, they were contaminated via an animal passage. this overview describes the problems and risks associated with viral contaminations in animal cell culture, describes the origins of these contaminations as well as the most important virsuses associated with viral contaminations in cell culture. in addition, ways to prevent viral contaminations as well as measures undertaken to avoid and assess risks for viral contaminations as performed in the biotech industry are briefly described. since the development of viral vaccines, animal cell technology has been used for the production of biologicals for prophylaxis and therapy of humans and animals. as many of these products are injectables, the microbiological safety is of particular importance and is a permanent concern. whereas microbial contaminations (fungi, yeasts, and bacteria) can be rather easily detected via cultivation methods, the detection of mycoplasma and viruses is more difficult because they are not observable by routine light microscopy. however, fluorescence microscopy, mycoplasma amplification and culture techniques, elisa and pcr are well developed for determining the extent of mycoplasma contamination. viral contamination, on the other hand, represents a greater concern because viruses require more complex and frequently need highly sophisticated detection methods (see later). in addition the potential for viruses to cause silent infection of cell culture needs to be addressed, negative results do not always signify that there is no virus contamination. viral infection can originate from contaminated cell lines, contaminated raw materials, or from a gmp breakdown in the production and purification process (minor, ) leading to a virus-contaminated final product. in addition, although downstream processing is able to eliminate or inactivate certain viruses, not all viruses can be eliminated in such a way because they can be resistant to elimination and/or inactivation steps. viral infection can be highly pathogenic, and in contrast to microbial infection, there are frequently no effective treatments available, this requires serious consideration to be given to the prevention of contamination. as a result of this everything possible has to be done in order to maintain the entire manufacturing process, and thus the final product, virus-free. in this review, the problem of viral contaminations in animal cell culture will be presented with special emphasis on animal cell technology used for the production of biologicals for prophylaxis and therapy. in addition, this article will suggest actions which can be taken, in order to assure the absence of viral contaminations. these may include the use of production media devoid of animal derived substances, validation of viral clearance in downstream processing or analytics for detecting adventitious viruses in cell culture and final biological product. at the end of this article, the implications for the more basic research laboratory will be discussed. viral contamination of cultured cells is associated with several problems: -in contrast to bacterial and fungal contamination, viral contamination cannnot be easily detected, because they cannot be observed by normal light microscopy. it is only when a viral contamination leads to a morphological modification of the cultured cells, such as a cytopathic effect, that a contamination by a virus can be suspected. silent infection by viruses with no observable morphological modification of the infected cell are clearly of greater concern. -a restricted number of viruses can infect cells and can integrate as a provirus, as in the case of adeno-associated virus (aav), for instance. in this case, the provirus is present, but cannot replicate without the assistance of a helper virus, when one is present both virus species will be produced, together (mayor and melnick, ; mayor et al., ; hoggan et al., ; berns et al., ) . -virus contaminated cell cultures represent a risk for the operators, collaborators, patients, as well as for non-contaminated cell cultures. -cells contaminated with some viruses can show a change to their susceptibility to infection by other viruses. for example some safety testing proto-cols require indicator cells to be used to show the presence of virus, if these are chronically infected by viruses this reduces their susceptibility to other virus species, this in turn, can lead to false negative results, because the virus to be detected can no longer infect the indicator cells. -as a general statement, cell lines contaminated by viruses cannot be treated to become virus free. the result of this is that potentially valuable cell lines will have to be discarded and replaced by new, non-contaminated cells. one of the few exceptions to this rule is the case of lactate dehydrogenase virus (ldv). cultures of cells recovered from a passage in infected animals contain this virus; however, as this virus cannot infect the cells, it will be diluted during subsequent in vitro passaging and thus will be lost (nicklas et al., ; nakai et al., ) . in order to avoid viral contamination or reduce the incidence it is helpful to know the source of the contamination. viruses can be introduced by a limited number of different routes and knowing this provides the possibility to avoid infection. the identified routes of infection are: (i) the cells used to produce the production cells are already contaminated by exogenous virus because the cells were already contaminated at source, e.g., the donor animal from which the cells were explanted (see 'contamination via the cell source'), the pre-culture (in vitro), or the in vivo passage in an animal led to the virus contamination (see 'passages via virus infected animals'). (ii) endogenous viruses, such as retroviruses are a particular concern. several cell lines of biotechnological importance, such as murine hybridomas or chinese hamster ovary (cho) cells, contain endogenous retroviruses and can produce retroviral particles during production. in the case of murine retroviruses these can be capable of replication, as observed with hybridoma cells, or which are incapable of replication, as in the case of cho-cells (see 'cell lines of biotechnological interest-endogenous retroviruses and other cell associated/latent viruses'). (iii) the cell cultures can be contaminated by viruses which were present in the animal derived materials used in the manipulation or for the growth of the cells. these types of materials include serum or trypsin (see 'use of contaminated raw materials'). animal derived raw materials are of particular concern as many different animal viruses can potentially be present originating from the use of infected source animals. non-animal derived raw materials can also be contaminated by viruses due to contact with virus shed from animals or man from production until its eventual use in the medium of which they are a component. (iv) finally, errors made by the operator can also result in viral contamination of animal cells (see 'handling errors of the operator'). in the following paragraph, these issues will be described in more detail. primary cells derived from explants or continuous cell lines immortalised/transformed by viruses can be contaminated by adventitious viruses. primary cell cultures derived from animal tissues are seldom used for the production of vaccines for humans but are more frequently applied for veterinary use. when the donor animals are already latently infected with viruses the subsequent in vitro cultures of cells derived from these animals may be infected. if such primary cells are then used for the production of viral vaccines, these vaccines are likely to be contaminated with the adventitious virus. in the period between and , when primary kidney cells from macaque or rhesus monkeys were used for the production of poliovirus vaccine, this vaccine was frequently contaminated by sv . the source of this virus was from the kidney cells of infected monkeys (sweet and hilleman, ; shah and nathanson, ) (for details, see 'passages via viruses infected animals'). young immunocompetent rhesus or macaque monkeys can readily be infected with sv by the oral, intranasal, and subcutaneous routes, and viremia and viruria occur in these animals (shah et al., ) . the use of such animals as source for kidney cells may lead automatically to a sv contaminated primary kidney cell culture because the virus may persist in the kidneys in a latent form (shah et al., ) . similar observations were made with secondary lamb kidney cells, which are widely used for the production of veterinary vaccines. in the case described, an attenuated aujeczky's disease viral vaccine was produced on lamb kidney cells. the master virus stock used for the production had become contaminated with the border disease virus due to the contamination of the cell culture used for its production. the vaccination of the sows with this vaccine during the first third of their pregnancy led to the infection of the fetuses which led to a disease similar to classical swine fever (vannier et al., ) . leiter et al. ( ) reported on the establishment of a mouse epithelial pancreatic cell line which was persistently infected by a polyoma virus. the origin of this infection was not completely clear, but it seemed probable that the mouse which was the source of these cells was also the source of the virus. finally, all cell lines which have been established by using a virus transformation (e.g. ebv-transformed b-lymphocytes, such as the namalva cell line (klein et al., ; butler, ) ) are potentially able to produce the virus used for the transformation and therefore also represent a potential infection risk for the operators, the cell culture lab, and the patients receiving a biological produced with such cell lines. the in vitro production of viral vaccines began with the demonstration that explanted embryonic tissue could be used for the production of poliovirus (enders et al., ) . subsequently primary cells, in particular primary monkey kidney cells, were used for the production of this virus. although the use of such cells was very convenient and the first accepted way to produce a viral vaccine, the source of these cells (the primary monkey kidney cells) was associated with a number of problems particularly with the introduction of a number of viral contaminats. the advantage of the use of primary cells for the vaccine production system was that it was the first in vitro system for the production of viral vaccines, that high titers of viruses could be obtained, and that the system could be scaled up to a reactor stage by using microcarriers . however, it has been recognised that, this system could be prone to contamination originating from the donor animals. the frequency of contaminants may have been due initially because, the animals were caught in the wild with no control of the disease risks with these animals, leading to a high incidence of contaminations by adventitious viruses. stones ( ) reported that - % of the cultures of kidney cells from vervet monkeys were positive for adventitious agents. as mentioned, many of the viruses which infect primates are pathogenic for humans, the species barrier is being able to be crossed (eloit, ) . the notion of the species barrier is sometimes seen as the ultimate rampart that will protect humans against animal viruses, and this can be true for a number of animal viruses which are not able to intitate infection in human cells. however, the species barrier relies on a number of inate features of the immune system which are bypassed in the case of medicinal products (injectables). in addition, the barrier may only be a quantitative issue (quantity of active virus, e.g. the ld of rabies virus in mice is times higher by the oral than by the intracerebral route) and is a question of the route of administration (e.g. the mucous membrane is a rather efficient barrier, however, when a virus is inoculated parenterally, the species barrier is no longer valid). finally, even in the case of an abortive replication cycle, cell transformation via the expression of early viral genes is still possible, eventually leading to a transformed phenotype of non-permissive cells (e.g., mouse cells are transformed by sv , which in monkey cells leads only to a lytic cycle, or non-permissive cells transformed by bovine polyomavirus) (eloit, (eloit, , . zoonotic infection where humans are infected by animal pathogens is frequently observed in nature. only two examples are described here: minor ( ) reported on cases of infections occuring between and in which individuals who had close contacts with macaque monkeys infected with a highly pathogenic virus (herpes simiae or b virus) fell ill. this virus is latent in these monkeys, but causes fatal disease in man. twenty cases were fatal and seven had severe sequelae. the second example concerns the contamination by the marburg virus. smith et al. ( ) reported that the contamination of monkeys with this filovirus led to an infection of operators/monkey handlers of which died. these monkeys had been imported into germany for vaccine production by using their kidney cells. more details can be found in peters et al. ( ) . a further relevant example from the production of poliovirus vaccine is provided by sv , a polyomavirus which is an extremely common infection of macaques and rhesus monkeys (shah and nathanson, ) where it persists in the kidneys in a latent form without causing a cytopathic effect (sweet and hilleman, ) . this virus grows much more slowly than poliovirus and thus an infection might not be observed during the vaccine production. it is estimated that almost everybody who was vaccinated against poliovirus between and also recieved viable sv together with the poliovirus vaccine. this is true for the live attenuated as well as for the inactivated poliovaccine, because, in the first case, no inactivation step was applied and, in the second case, the formalin inactivation step was insufficient to inactivate sv (sweet and hilleman, ) . whereas in the case of the live vaccine, the route by mouth is a poor route for infection with sv (sweet and hilleman, ) , others were injected with inactivated poliovaccine together with infectious sv . a long-term follow-up study with a small number of individuals, as well as the observation that, in spite of the large number of vaccinees ( - millions of millions who were vaccinated, or almost % of individuals under yr in (shah and nathanson, ) ) which are believed to have received infectious sv , showed no corresponding increase in related cancers (shah and nathanson, ) . it should be mentioned, however, that (i) dna-sequences of sv have been detected in association with different human tumors and at an higher incidence in mesotheliomas (horaud, ) , (ii) that sv isolated from primary monkey kidney cells by sweet and hilleman ( ) induced sarcomas in newborn hamsters (eddy et al., ) , and (iii) that sv is oncogenic for laboratory rodents (magrath, ) . in , hoggan reported the detection of latent infections of aav in human and monkey kidney cells. he and his coworkers screened cell lines intended for vaccine production and found that approximately % of human embryonic kidney cells and % of african green monkey kidney cells produced aav when infected with helper adenovirus. these results suggested that aav stayed in an integrated form in the absence of helper virus and that this inapparent infection was a rather frequent natural occurrence. hoggan et al. ( ) could prove by using detroit cells that a deliberate infection with aav in the absence of helper virus led to a latent infection. a superinfection with helper adenovirus led to the induction of aav replication. it is evident that steps can be taken to reduce the risk of contamination risk in primary cell cultures. in practice, different actions are possible: use of virus free animals, use of kidney cells from animals which are less susceptible to virus infections, establishment of veterinary examination and quarantine of animals intended for use, and/or use of diploid or continuous cell lines (use of the cell bank concept/seed stock concept); all reduce the frequency of contamination, use of captive-bred monkeys versus use of imported wild-caught animals. besides the advantage of independence from the diminishing wild populations and the sparing of these, it is evident that breeding under controlled conditions led to the production of better quality animals especially in respect of virus infections (van steenis et al., ) . van wezel et al. ( ) could show, by performing serological tests on captive-bred cynomolgous monkeys and imported wild caught parent animals that most of the wild caught animals were positive for antibodies against herpes simplex b, parainfluenza , or measles virus, whereas two thirds of the captive bred animals were only positive for antibodies against rotaviruses. twenty out of imported animals were positive for foamy virus antibodies whereas these antibodies were not observed in the animals bred in captivity (table i) . during production, control cultures are generally established in parallel to the cultures used for the production of polio vaccine. van steenis et al. ( ) compared the frequency of contaminated primary, secondary, tertiary, and quaternary cultures (flasks as well as microcarrier cultures) and stated that all cultures derived from captive-bred animals were free of virusinduced cytopathic effects or hemadsorption, whereas out of kidney cultures from single imported wild caught animals and all of cultures derived from multiple animals showed cytopathology (table ii) . reduction of the frequency of contaminations. statistically, the use of fewer animals (kidneys) will increase the probability to establish virus free cultures. as primary kidney cells from monkeys can be amplified to reactor scale cultures by using microcarriers, the number of animals (kidneys) per batch could be considerably reduced (van wezel et al., , . the calculations done by van wezel et al. ( ) in- dicated very clearly that this approach could reduce the number of animals necessary for production purposes of polio virus vaccine by - fold. shah and nathanson ( ) calculated the probability to obtain kidney cultures free from sv with respect to the number of animals used per production batch. by using one animal the frequency of sv contamination was about %. however, the frequency increased to % when the kidneys of two to three animals were pooled and was % when the kidneys of more than animals were pooled, indicating very clearly that the increase of the number of animals per batch increased considerably the probability of the presence of virus contamination. use of kidney cells from animals, which are less susceptible for virus infections. one way to reduce the contamination by human pathogenic virus is to change the species of the animal as donor of the primary cells. shah and nathanson ( ) proposed that new world spider monkeys should be used instead of the rhesus monkeys or macaques because sv does not readily multiply in cells from spider monkeys. on the other hand, macaque monkeys can be infected with herpes simiae or b viruses, which are highly pathogenic for humans. the replacement of the macaques as donors by african green monkeys, which are not susceptible to infection by herpes simiae virus, would be the best precaution in this case (minor, ) . other means. in addition to the above mentioned measures, there are some other measures which can be performed in order to increase biological safety. the animals intended for use should be examined for their health status and must pass through a quarantine regime. for safety reasons, there has to be routine use of in vitro and in vivo culture systems for detection of viruses in any case. however, the best means to increase the biological safety of the produced viral vaccines is the use of diploid or continuous cell lines, because it can be determined that such cells are free of animal derived viruses: this can be achieved by establishing master (seed stock) and working (distribution and user stocks) cell banks which have been rigorously tested and validated for the absence of microbial as well as viral contaminants (see chapter by freshney and the section on 'testing-virus screening in cell banks' of this article). by this means producers of viral vaccines and all other biotech products can make use of a homogeneous pool of characterized cells from which each production run will be started, in the knowledge that they are free of any contaminant (because they have been tested) (berthold et al., ) . in addition, by using the seed stock/working stock concept for the viral inoculum the manufacturer of viral vaccines can use a tested and validated stock of virus inocula of which one aliquot is used for the infection of each production run. many contaminating murine viruses, such as minute virus of mice (mvm), k virus, mouse encephalomyelitis virus, and mouse adenovirus have been isolated from contaminated virus pools. viruses, such as a similar study was published by nicklas et al. ( ) , however, revealing a lower rate of contamina- (nicklas et al., ) origin tions probably due to the improvement of the microbiological quality of the laboratory rodents. of tumors examinated, ( . %) were contaminated. considerable differences were observed for in vivo ( . % positive of tumors) and in vitro ( . % positive of tumors) passaged transplantable tumors. mouse transplantable tumors showed the highest frequency of contamination, whereas tumors of other species showed much lower frequencies (table iv) . contamination with reovirus and mvm was found in ( . %) of cell lines tested, and in of monoclonal antibody bulk preparations. with respect to lcmv, bhatt et al. ( ) reported its isolation from transplantable tumor cell lines. the testing of tumor cell lines revealed that out of in vivo tumor samples and one out of eight in vitro samples were positive. a similar situation was found in a new jersey research institute, where human cell lines and tumor cell lines were passaged via nude mice for the development of monoclonal immunodiagnostics and immunotherapeutic agents. this lcmv contamination led to the outbreak of laboratory-acquired human lcmv infection (mahy et al., ) . lcmv contaminated hamster tumor cell lines have also been responsible for an outbreak of infections occurred in medical center personal at the university of rochester (hay, ) . mouse hybridomas are of particular concern because, first, these cells have been created by fusion of mouse spleenocytes with mouse myeloma cells, second, many hybridoma cells have been cultivated in animals, and third they are used for the produc-tion of injectables. this signifies that mouse viruses are potential contaminants of these cell lines and their products. these viral contaminants can be divided into two groups; group contains viruses which are also known to cause human diseases or to be able to infect human cells, while group contains other mouse viruses (table v) (minor, ) . although ectromelia virus is listed in group , cultures infected with this virus are only processed in the special p unit available at the nih (hay, ) . ectromelia, a member of the orthopoxviurs group, is a natural pathogen in mice, and is able to replicate in all mouse lymphoma lines, in some hybridoma cell lines, and in bs-c- cells (buller et al., ) . consequently, the atcc has screened its collection of murine cell lines but no characteristic cytopathic effects have been observed (hay, ) . moore ( ) listed the mouse viruses which had been detected in production cell banks of hybridomas: lmcv, mvm, sendai, ldh, and epizootic diarrhea virus of infant mice. the present view is that hybridoma cell lines should be tested for the viruses indicated in table v , and only those should be used for biotechnological applications which are free of these viruses. the only acceptable viral particles in bulk supernatants from hybridoma cell lines are those of endogenous origin. the passage of human tumor cells in nude mice can also lead to the infection of these cells by murine endogenous retroviruses. crawford et al. ( ) reported the contamination of a human nasopharyngeal carcinoma with murine endogenous xenotropic retroviruses after a passage in a nude mouse. as these table v . viruses potentially infecting rodent cells (minor, ) group contaminations are of animal origin, it is necessary to verify the contamination status of laboratory animals. minor ( ) indicated that all mouse strains, which were received at nibsc from breeders of laboratory animals, were tested positive for mvm and sendai virus. finally working with rat cell lines, which have been passaged via rats, is also a concern because they can be contaminated by different rat viruses, in general, and by hantaan virus, in particular. hantaan virus has been isolated in cell culture from rat immunocytomas. transplantation into lou/m/wsl rats and storage of passaged immunocytomas at - • c over a period of - yr did not eliminate the virus. lloyd and jones ( ) also showed that the passage of rat immunocytomas in infected lou/wsl rats led to a contamination of these cell lines. hantaan virus is a silent pathogen in rats and mice, but causes disease in humans (hemorrhagic fever with renal syndrome) after infection (leduc et al., ) . laboratory animal care workers working with infected animals as well as persons handling contaminated rat immunocytoma cell lines have been infected with this virus (leduc et al., ; lloyd and jones, ; mahy et al., ) . for this reason, all atcc certified cell lines of rat origin have been screened for the presence of hantavirus. the following rat cell lines and rat hybridomas appeared free of hantavirus infection: ccl , , , , , . , , , , , , , ; crl crl , crl , crl , crl , crl , crl , crl , crl , crl , crl , crl , crl , crl , crl , crl , crl , crl , crl , crl , crl , crl , crl , crl , crl , crl , crl , hb , , , , , ; and tib , , , , , , , , , , , , , , , , , , , , , , , , , (leduc et al., ) . it should also be mentioned that cell lines of other species can also be contaminated by hantavirus. this virus had been detected in a human lung carcinoma cell line (a ) (french et al., ) and in vero e cells (mccormick et al., ) . precautions: the precautions which should be taken to reduce the risk of contamination of cells during passage in animals: (i) cells which have been once passaged in animals have to be screened for the absence of microbial and viral contaminants normally found in the animal species; (ii) animal passages should be avoided as much as possible, the contamination risk by mouse pathogens is reduced -fold when these cells are cultivated in vitro (nicklas et al., ) ; and (iii) only tested laboratory-bred animals (virus-defined, specific pathogen free), and no wild caught animals, should be used for animal passages of mammalian cells. the most important cell lines of biotechnological interest, mouse hybridomas and cho cells, are known to contain endogenous retroviruses (ervs) and are known to produce retroviral particles. ervs exist in forms, a viral form and a proviral form. retroviral proviruses are transmitted through germ cells and are present in the genomes of almost all vertebrates thus far studied. humans possess many erv genomes related to mammalian c type retroviruses and to a, b and d types of this family of viruses. the presence of erv-like sequences in a cell line to be used in the production of a biological is a potential cause for concern because of the possibility that the endogenous retrovirus may be activated and result in infectious virus being present. particles (iap) as well as budding c-type particles (table vi) . with respect to iaps, anderson et al. ( ) demonstrated, that the cho cells' genome contained approximately copies of viral sequence per haploid genome. no intact open reading frame for gag, pol, or env could be detected in clones of either family (anderson et al., ) . in addition, no infectivity has been associated with a-type retroviruses from cho cells (kuff and lueders, ; adamson, ) . cho cells also produce c-type particles at concentrations of < - ml − . the presence of virus particles was correlated with detectable reversed transcriptase activity (a retrovirus specific enzyme) (dinowitz et al., ) (table vii) . as for the iaps, each cho cell contains between and copies/genome (dinowitz et al., ) . no evidence of infectivity could be detected (dinowitz et al., ; adamson, ) , this may be due to the lack of functional open reading frames, rendering the retroviruses incapable of encoding an intact endonuclease (dinowitz et al., ) . hybridoma and murine plasmacytoma cell lines. as for cho cells, plasmacytoma and hybridoma cells produce iaps and c-type particles (table vi) (spriggs and krueger, ; weiss, ) . as for cho iaps, hybridoma iaps stay inside the cells and are noninfectious because they are devoid of intact open reading frames. however, unlike cho c-type retroviral particles, hybridoma c-type particles have the ability to replicate in several different cell lines including a small number of human cell lines (lung fibroblasts, rd cells (weiss, ; levy, ; adamson, ) ). by using electron microscopy, the retroviruses present in cell culture supernatant have been as high as particles per ml (moore, ) . about one in - particles is infectious. in this context, froud et al. ( ) presented data from lonza biologics (former celltech biologics), indicating that all hybridoma cell lines processed by the company produced retroviral particles. five to six percent of the cells produced infectious mouse ecotropic retrovirus, whereas almost all (about %) mouse cell lines tested produced low levels of infectious mouse xenotropic retrovirus (x-mlv) when the cell banks were tested. all mouse cell lines commonly used for antibody or recombinant protein production are derived from the mopc tumor of a female balb/c mouse. this indicates that all cell lines, clones, and subclones which are derived from the mopc tumor (the plasmacytomas p x .ag . , ns , ns , and the hybridoma sp / .ag ) produce x-mlv (froud et al., ) . infectious retrovirus has also been found in mouse/human hybridomas. in co-cultivation studies it could be established that these retroviruses were of the x-mlv type, and no human retroviruses have been detected in any mouse/human hybridoma or genetically engineered human cell line (moore, ) . although not detected so far, the possibility of molecular recombinations leading to pseudotyped particles is a concern. other cell associated viruses and safety considerations mouse/human hybridomas can be established by fusing human ebv transformed lymphoblastoid cells with mouse plasmacytomas. as these cells were ebv transformed, the hybridoma cells are potential ebv producers. cells transformed by ebv are potential ebv producers (e.g. namalva), and the downstream processing protocol as well as the safety testing have to take this fact into account (cartwright, ; robertson, ) (see section on 'process validation -downstream -processing -viral clearance'). bhk cells are also transformed rodent cells and it was possible to induce production of r-type particles in these cells (moore, ) . table viii presents a short résumé on cell lines of biotechnological interest which contain endogenous viruses or latent proviruses and are therefore producers or potential producers of these viruses. whereas plasmacytomas, hybridomas, cho and bhk cells contain endogenous viruses which integrated into the genome table vii . characteristics of c-type particles isolated from cho cells (dinowitz et al., ) reversed transcriptase activity detected in highly concentrated ( - -fold) culture fluids from some cell lines; mn + preferring. similar to other c-type particles in sucrose density gradients (about . - . g ml − ). nucleotide homology to other c-type endnuclease region contains significant homology particles with mammalian c-type retroviruses. no intact open reading frames detected in cloned cdna sequences. about - copies per cho genome. proteins p core protein is related to murine and other c-type retroviruses. no infectivity detected by direct inoculation of reverse transcriptase-containing concentrates or cocultivation of cho cells with a battery of cell lines. of the respective species after an infection millions of years ago, the presence of ebv (due to ebv transformation of human b-lymphocytes) or sequences of parvoviruses (e.g. aav (latent infection leading to integration) due to natural contamination of human embryonic kidney cells and african green monkey kidney cells (hoggan, ) , or procine parvovirus (latent infection) (fikrig and tattersall, ) (see section on 'trypsin')) are due to recent events and can eventually be avoided by using tested cell lines (absence of the respective sequences). it is evident that all biological products derived from cell lines containing endogenous retroviruses or other latent viruses have to be characterized for the presence of virus. in addition, in order to increase their biological safety, first, all biotech products derived from such cell cultures have to be rigorously tested for the absence of retroviral activities/viruses or latent viruses, and second, the purification protocols for biotech products derived from these cells have to be validated for their capacity to eliminate or inactivate retroviruses/latent viruses. an important potential source of viral contaminations are raw materials used for the preparation of culture media in animal cell technology. although any component of the culture medium can theoretically by contaminated by viruses, the materials with the highest probability of viral contamination are those derived from animal origin. the classical animal cell culture technology currently makes use of several raw materials of animal or human origin. this is true for the production of viral vaccines for human or veterinary use or many other biological therapeutic. animal sera as medium additive is the most widespread animal derived material used today. fetal, new born or adult bovine sera and in some cases also horse sera are used. trypsin, mainly from pig pancreas, is a very important detachment agent for all adherent cells. other animal or human derived substances are often used in association with the replacement of serum by serumfree media, but which can eventually also be found as excipients: human albumin, protein hydrolysates (casein, gelatin, etc.), and human transferrin. other substances of animal origin are some amino acids, which are derived from complete hydrolysis of proteins. however, because of the chemical conditions used in their production, there is less risk than that of materials prepared without this process. although the use of entirely chemically defined media devoid of any animal derived substance reduces the risk of viral contaminations, it is important to mention that this risk will never be zero. several zoonotic viruses are known and can be transmitted from animal sources (eloit, ) . because of the recognised risks from these agents careful sourcing and screening can easily prevent the risk of their transmission. however, it should be noted that other viruses not known to be harmful for humans might be infectious and might lead to severe disease. contamination problems associated with the use of serum bovine serum might be contaminated by many different bovine viruses. although theoretically qualitycontrolled serum should have been tested for all possible viruses, this is not possible for economic reasons and may not even be necessary. it is evident that each serum batch has to be tested for those viruses which are ubiquitous and known risks, such as the bovine viral diarrhoea virus (bvdv). however, there is also the question of the geographical origin of the serum which may indicate the need for additional virus tests. it is only necessary to test for those viruses, which are present in the geographical region from which the serum is coming. table ix presents bovine viruses for which tests have to be performed depending on the geographical origin of the serum. the testing table ix . specific tests applicable to the screening of calf serum and porcine trypsin used for the production of medicinal products for human use (eloit, ) calf serum trypsin (table x) . in the following, the most important bovine viruses are presented in more detail. bvdv. bovine viral diarrhoea/mucosal disease is one of the most important viral diseases of cattle. the natural prevalence is very high with approximately % of cattle being seropositive and - % of these animals being persistently viremic animals due to immune tolerance which occurs after infection of the fetus (kniazeff, ) . the infection rate has been increased by the uncontrolled use of live vaccines and by heterologous vaccines fortituously contaminated with bvdv virus (kreeft et al., ) . together with hog cholera and border disease virus of sheep, bvdv constitutes the pestivirus group. bolin et al. ( ) have studied the frequency of contamination of fetal calf serum with bvdv and reported that of raw fetal serum samples ( . %) derived from the abbatoirs were positive for this virus, of these samples ( . %) contained antibodies against bvdv and . % of the samples ( / ) were positive for both, bvdv and antibodies against bvdv (table xi ). they have also tested commercial fetal calf serum for cell culture and detec-ted bvdv in % of the samples ( / ): contained non-cytolytic and only two contained cytolytic bvdv isolates. two percent of these samples ( / ) were positive for infectious bovine rhinothracheitis virus isolates. wessman and levings ( ) have reported similar results, indicating that to % of fetal bovine serum samples (pooled one liter lots from two bovine fetuses) were rejected for presence of bvdv or antibodies against bvdv, in the period of - . these studies indicate the importance of the problem of bvdv contamination in fetal calf serum and several conclusions can be made: first, veterinary diagnostic laboratories should avoid the use of fetal calf serum in diagnostic procedures for pestivirus infections, second, there is a significant risk that adventitious bvdv from fetal calf serum may lead to contaminations in the veterinary biologicals industry (see section on 'other substances of animal/human origin and non-animal derived substances'), and third, the results indicate a very high rate of fetal infection with bvdv, possibly reflecting a failure in hygiene issues or in control measures (bolin et al., ) . finally the most important question concerns the contamination status of cell lines in culture collections, because many cell lines have existed for many years in these collections and might have been contaminated in periods when no or fewer tests were performed for proving the absence of bvdv. the question most relevant to the use of fetal calf serum for animal cell culture and animal cell culture technology is: which cells are/were contaminated by bvdv and are the cells from different species as easily contaminated as bovine cells or is there any species barrier. in this context, bolin et al. ( ) performed a survey of cell lines from the american type culture collection and observed the following contamination status: using immunocytochemical procedures and pcr amplification, of atcc cell lines were tested bvdv positive: these cell lines were derived from cattle, sheep, goat, deer, bison, rabbit, and domestic cat. attemps to experimentally infect different cell lines from animals, which were not found positive in the survey of the atcc cell lines, led to the result that all swine cell lines ( / : mpk, esk- , and one other) and most rabbit ( out of : sf ep, r ab, rab- ) and cat cell lines ( out of : crfk, ak-d, nce-f ) became infected with bvdv, whereas hamster (bhk- ), human (imr- ), dog (mdck), rabbit (sirc) and cat (fc tg) cells were refractory to bvdv infections. the results concerning monkey cells (llc mk ) were variable -no clear answer was obtained. wessman and levings ( ) reported that the following cell lines could be infected with bvdv: bovine cells (ebk, mdbk, botur, primary and continuous kidney cell lines, lung, trachea, and aortic endothe-lium), sheep choroid plexus and lamb kidney cells, monkey kidney cells (vero and others), mosquito cells, porcine cells (pk- and others, testis, minipig kidney cells), goat cells (kidney and oesophagus), cat cells (lung, crfk, tongue, feline embryo), rabbit kidney cells (rk- ), and others. harasawa and mizusawa ( ) published a study on the pestivirus contamination of cell stocks of the japanese cancer research resources bank. fifteen out of cell lines ( %) were positive using rt-pcr. whereas bovine cell lines (hh, mdbk, cpa, cpae, ebtr, ch es) were contaminated with genotypes i, ii, and iii, cell lines of dog, cat, and primate origin were contaminated with genotype ii of bvdv (hela, molt- , u , wi- , widr, cv- , vero, mdck, crfk). roehe and edwards ( ) assessed the ability of pestiviruses from pig, eight from cattle, and five from sheep to replicate in cells of porcine (pk- ), bovine (bt) and ovine (scp) origin. the pattern of replication in different cell types varied between different isolates of the same virus species. these results indicate that the virus suceptibilities of a species are not completely predictible and that many cells derived from other species than cattle can be infected by bvdv. of importance. viral infections lead to respiratory syndrome in cattle (shipping fever) and abortion in bovines. in % of calves significant antibody levels against pi- have been detected. the virus can be easily replicated in primary (bovine kidney cells) and established bovine cells (ebtr, mdbk) (kniazeff, ) . tracheitis -a very common infection of cattle -, abortion, pustular vulvovaginitis, meningoencephalitis of calves, and conjunctivitis. the incidence of infection is high with viremia a common feature. it replicates in leukocytes and can stay there latently. it is never found free in the bloodstream. it replicates in bovine cells, but also in cell cultures from: elk, mule deer, sheep, l cells, chick embryo cells, pig, human (amnion (probably also a clon of hela cells (see chapter by masters)), hela), and primary monkey kidney cells (kniazeff, ) . bpyv. bovine polyomavirus belongs to the family of the polyomaviruses. these viruses have been isolated by several laboratories (e.g., from monkey kidney and other cells cultured in the presence of bovine serum. by infecting permissive cells, this virus leads to a cytopathic effect, whereas non-permissive cells are transformed and they acquire certain properties of a malignant cell. as for the other bovine derived viruses, bpyv is an ubiquitous virus and about % of the calves are seropositive in the first month after birth. in the subsequent months this seropositivity decreases to about % at an age of one year, however, the older the animals become seropositive again and the final percentage of seropositivity is beyond %. it was also shown that bovine fetuses were infected in utero, leading to the presence of antibodies against this virus in fetal bovine serum batches (in about % of the tested batches). despite this rather high incidence of infection in fetuses, no known disease is associated with this virus, neither for cattle nor for humans. using pcr, / european serum batches ( %) contained bpyv dna sequences . a similar frequency of contamination was observed in north american serum batches (kappler et al., ; van der noordaa et al., ) . there was no correlation between the pcr results and the presence/absence of antibodies against bpyv, however, could show that all pcr-positive sera contained infectious bpyv. ibr or bhv- . bpyv can infect calf kidney cells, and monkey kidney cells (vero, bsc- , cv- , rita) but also human embryonic kidney cell cultures (waldeck and sauer, ; wognum et al., ) . the virus does not seem to replicate in mouse t cells, nor in the human embryonic lung cell wi- (waldeck and sauer, ) . the bpy virus is known to lead to cell transformation and tumorigenesis, which is induced by the expression of the large-t antigen . all adherently growing cells have to be detached for passaging from time to time. to facilitate this the enzyme trypsin is frequently used. as for serum, trypsin is an animal derived product, generally from porcine pancreas. therefore, similar safety criteria as for serum have to be applied for trypsin. a special concern is porcine parvovirus (ppv). latent parvovirus contamination has been found in many permanent human cell lines. the first contamination was observed by hallauer and kronauer ( ) when they observed that some control cultures (non-infected with yellow fever virus) yielded a different hemagglutinating agent, unrelated to yellow fever virus, when subjected to their isotonic, high ph glycine extraction buffer (= physiological stress). further studies identified this infectious agent as a member of the parvovirus group. following this, hallauer et al. ( ) isolated parvoviruses in permanent human cell lines obtained from laboratories. some cell lines showed signs of degenerescence when arriving into the laboratory of hallauer, others appeared completely normal. three different serotypes of parvovirus could be identified, the origin of them is not really known. however, the recovery of the same serotypes from different laboratories suggests a common source, such as a reagent used in cell cultivation. one of the serotypes had been identified as ppv, indicating that the use of contaminated trypsin lots was the probable source of contamination (fikrig and tattersall, ) . the definite proof that porcine trypsin was the source for cell culture contaminations by ppv was apported by croghan et al. ( ) , because they detected the same serotype in commercial trypsin lots. it could be shown that various cell lines from different species can be infected by ppv, such as human continuous cell lines (lu , hela, and the following hela clones (see chapter by masters): kb, amnion, and hep- ) or swine kidney cells (pk ) (hallauer et al., ) . recently, a new group of viruses, the circoviridae, was described. the circoviridae are very small viruses (non-enveloped, circular single stranded dna, diameter of nm) and are very resistant against most of the inactivation methods currently used. this group of viruses was found in japanese patients suffering from non a to g hepatitis, described as tt virus (nishizawa et al. ) , and also found in chickens, where it is described as chicken anemia virus (yuasa et al., ) . this virus seems to be ubiquitous in humans because dna of the tt virus was identified in plasma of % of french blood donors (biagini et al., ) . it is evident that such a virus might be a problem when human derived proteins are used, because this it is very resistant against most inactivation methods. tischer et al. ( ) described a circovirus in pigs, and it has been reported that the swine cell line pk- was chronically contaminated by this type of virus. a serological study showed that out of randomly collected pig sera contained specific antibodies against the virus, whereas no specific antibodies could be detected in sera from rabbits, mice, calves, and man, including the laboratory staff working with this virus (tischer et al., ) . the virus exists as subtypes, type porcine circovirus replicates actively in porcine fetuses (sanchez et al., ) and is associated with abortions, reproductive failure and postweaning multisystemic wasting disease in swine (o'connor et al., ; ellis et al., ) , however, it seems that this virus is very porcine specific . although there is no record of porcine circovirus being able to infect man, precautions should be taken when porcine derived substances, such as trypsin, are used in animal cell technology. as this type of virus is very resistant it is preferable to avoid contaminations of the cell culture, and thus of the biotech product, than to try to separate the product from the virus (due to stability reasons autoclaving the final product is not possible). for the moment, these viruses are most frequently detected by pcr, presently there is no good culture method available. the highest risk is associated with the use of human or animal derived substances. with respect to material of human origin, it is evident that there exists an important risk of viral transmission, because of the absence of any species barrier to infection. human sourced raw materials should be checked for the absence of viruses, like hepatitis b virus (hbv), hepatitis c virus (hcv), human immunodeficiency virus (hiv), and ebv, or cmv (committee for proprietary medicinal products: ad hoc working party on biotechnology/pharmacy and working party on safety medicines, ). the testing for these viruses is most frequently accomplished by pcr assay. the extent of the use of human derived materials is limited with only human transferrin and human serum albumin still in use. the development of serum-and protein-free media leads more and more to media devoid of any animal/human derived substance. other substances of animal origin, such as certain amino acids, lipids, or other protein additives (peptones) are also potentially contaminated by viruses and have to be rigorously tested for virus absence or should be replaced by non-animal derived compounds (merten, ; jayme, ) . finally it should be mentioned that adventitious viruses can also be introduced via contaminated nonanimal derived substances (medium components), as observed by the contamination of a manufactured material by viruses of extraneous origin. the most widely reported case was that occuring with the manufacturer genentech (garnick, ) (see next section). the scientific literature gives few descriptions of viral contamination in biotechnological productions of recombinant proteins or viral vaccines (table x) . nine cases have been published indicating that viral contamination can be acquired via the serum source (in out of cases) or the culture medium (in out of cases). biotechnological products were contaminated by different viruses, by those leading to a cytopathic effect (epizootic haemorrhagic disease virus) but in most cases by those which have no effect on the morphology of the contaminated cells (minute virus of mice (mvm), bvdv, bpyv, bluetongue virus, reovirus). in cases where the viral infection leads to observable morphological effects, the contamination is easily detectable. however, those viruses which do not lead to a modification of the morphology and/or growth characteristics of the cells require methods such as rt-pcr, pcr, bulk in vitro testing. or alternatively the onset of diseases in animals administered with the test material. these virus positive products were not delivered in the case of the products destined for human use, because the virus detection was performed before product release (garnick, ; rabenau et al., ; harasawa and sasaki, ) , however, with respect to the live attenuated veterinary vaccines, several incidents of disease in vaccinated animals were reported (kreeft et al., ; wilbur et al., ; falcone et al., ) . it should be mentioned here, that the case of mvm contamination of cho cultures for the production of tpa, did not lead to a cytopathic effect and could not be detected without specific virus tests (garnick, ) . in contrast, nettleton and rweyemamu ( ) and hughes ( ) reported on a mvm contamination of bhk- cells for veterinary vaccine production, which was detected via persistent cell deaths of these cells. it could be shown that the serum batch was the origin of this contamination. it is evident that rigourous testing of raw materials is necessary because of the following: -in the case of the production of recombinant proteins for human use, virus contaminations can only be eliminated with difficulty from the bulk product. should a virus present in the bulk product be able to be eliminated during downstream processing, the fda will generally not accept the final product after purification (burstyn, ) . -the problem associated with live attenuated virus vaccines is that these products cannot be treated for virus inactivation because the active ingredient would be inactivated at the same time. such products require a more extended quality control testing for the raw materials. for instance, new serum batches should be tested for a more extensive range of bovine viruses and in particular for those viruses, which are of relevance for the final application of the product. for instance, in the case of the contamination of a canine vaccine by bluetongue virus which lead to the death of some bitches (table x) (wilbur et al., ) , the application of a specific test would have avoided this incidence. -in conclusion, the best solution for reducing the risks of viral contaminations is the use of raw materials which are not of animal or human origin, but of plant or microbial origin or produced by chemical synthesis. it is evident that such an approach will not eliminate the risk of viral contaminations, but represents an important step towards risk minimization. sourcing. this approach is clearly limited to agents for which there is a well-documented specific geographical distribution. such examples are quite rare and only the case of tse agents will be mentioned here, where sourcing of bovine serum from diseasefree countries (a geographical choice) is possible. this approach, however, can also be used for viruses such as bluetongue virus. screening. in principle, all raw materials, of animal origine or produced by chemical synthesis, have to be rigourously tested and should fulfill certain quality attributes, when used for the production of biological injectables (gmp-guidelines). the characteristics which are frequently required to be described in raw materials are identity (testing, tracability, labels), purity (testing, inspection, vendor certificate of analysis), suitability for intended use (process validation, vendor audit programme, performance testing if needed), tracability (vendor audit programme, vendor certification, certificates of analysis, contractual obligations under change control, labelling, control). for more details, consult lubiniecki and shadle ( ) . although it would be desirable that all raw materials should be tested for the absence of adventitious agents, in order to be sure that they are safe, this is often impracticable. therefore there are two approaches: first, tests are employed which are based on the detection of general characteristics of viruses (cytopathic effects, haemadsorption) and, second, specific tests using imunological and/or pcr methods are employed for detecting virus antigens or specific viral sequences after amplification in permissive cells (see table ix for testing of bovine serum and porcine trypsin, see section on 'testing -virus screening in cell banks'). however, such a screening gives only a limited guarantee of safety because of the following: -complete testing can be impracticable on a batch to batch basis. in most cases, screening will only be done for certain viruses, e.g. for bvdv, ibr, and pi- in the case of bovine serum, for porcine parvovirus in the case of trypsin, because these are the most probable viral contaminants. however, depending on the geographical origin of the serum or the trypsin, additional tests for viruses which are present in that geographical area from where the raw material is coming may have to be performed (table ix) . if a raw material is of animal origin, screening tests should also include the use of cells of the species of origin. with respect to the use of serum for the gmp production of biologicals for human use, the emea proposes in a draft that more viral screening tests should be performed for proving the absence of bvdv, ibr, pi- , bovine adenovirus, bovine parvovirus, bovine respiratory syncytial virus, bovine re- virus tests, otherwise they pass undetected. -due to sampling size, low titers of some adventitious viruses can remain undetected but may be amplified during the manufactureing process. in this context it should be remarked that screening methods are not always sufficient because contaminated serum batches which had passed as uncontaminated have been detected by and yanagi et al., ( ) ; contamination of serum samples with bpyv and bvdv, respectively. recommendations for fetal bovine serum quality (hansen and foster, ) . although the best choice would be a serum-free cell culture process which is devoid of any animal or human derived substances, this is not always possible. where serum supplementation is necessary, the serum should be of high quality. in addition to the absence of viruses the hemoglobin level should be < mg, the endotoxin level should be below eµ ml − , and there should be a reliable tracability to countries without bse nor foot and mouth disease. a serial filtration using nm poresize filters should be used and the serum should be γ irradiated with > kgy using validated procedures. for veterinary use, the radiation dose should be kgy. more on the quality control of bovine serum used for the production of viral vaccines for human use can be found in mareschal ( ) . other precautions: the screening of animal derived raw materials for the presence of adventitious viruses is of utmost importance, however, as already mentioned, screening has its limits, because it is impractical to screen for all theoretical viruses, and other new viruses might emerge for which no tests are available. because of this supplementary precautions have to be undertaken for reducing the risk of viral contamination, by inactivating or eliminating at least viruses of families, which are susceptible for inactivation and/or elimination. with respect to the treatment of fetal bovine serum for animal cell technology, γ irradition, and uvc irradiation are used. heat treatment as well as treatment with peracetic acid are possible, however, they are not really used. some treatments are presented in the following: (a) nanofiltration (troccoli et al., ; aranha-creado et al., ; graf et al., ) . if the size difference (molecular weight) between the raw material and the virus is large enough, viruses can be removed by nanofiltration, which makes use of pore cut offs of , nm, and even nm. filters with a pore cut offs of nm can be used to eliminate viruses which have a diameter larger than nm, such as retroviruses or influenza a virus ( - nm) (typical log titer reduction in a validation study: > . ). however, such filters only partly reduce the quantity of poliovirus ( - nm), and viruses of a size of nm (model particle: bacteriophage pp ) pass without any significant retention (log titer reduction: < - . , depending on the buffer system used) (graf et al., ) . it should be mentioned that the composition of the medium/buffer system in which the virus is placed, has an effect on the log titer reduction of viruses which are below the molecular weight cut-off of the membrane used. an example of typical virus retention data for a commercial hydrophilic pvdf membrane filter is shown in table xii . improved virus retention can be obtained by using pore cut offs of nm. using a nm membrane in line with two prefilters (one nm filter followed by a first nm filter) led to log titer reductions of > . for hepatitis a virus (hav) and encephalomyocarditis virus, although both viruses are smaller ( - nm) than the cut-off of the filters (troccoli et al., ) . all viruses larger than nm were completely removed. finally the use of a nm membrane filter followed by a nm pore size membrane filter assures a log reduction factor of > . and > . for hav and bvdv, respectively, signifying that in principle all potential adventitious viruses (also the small ones) can be removed from the product (johnston et al., ) . nanofiltration is mainly used as a final step in the production of biologicals purified from human plasma and of recombinant dna-derived products. fetal calf serum is often filtered three times using a cut-off of nm (removal of, for instance, ibr and pi- , but not of bvdv). only some companies provide fetal bovine serum which is serially filtered through nm pore size filters (hanson and foster, ) , because this cut-off allows also the elimination of, for instance, bvdv, which has a size of - nm (see table xiii ). (b) γ irradition (plavsic et al., ) . γ irradition (using a co source) is a very efficient and straightforward means for inactivating many different virus types. as animal derived substances such as serum can be contaminated by adventitious viruses, γ irradition is, after routine quality control for virus detection, the best method to increase the safety of using serum in the production of animal cell culture derived biologicals. using petg (polyethylene terephthalate g copolymer) bottles with ml of frozen serum (- • c) inoculated with model virus (hanson and foster, ) , validation experiments have been performed to determine the optimal radiation dose to inactivate relevant bovine and porcine viruses, and in parallel to assure that the irradiated serum has still a sufficient growth supporting ability. plavsic et al. ( ) could show that at a radiation dose of kgy, all tested viruses (bovine reovirus, porcine parvovirus, canine adenovirus, ibr, and bvdv) showed a significant decline in titer. an exposure of kgy led to titers of all viruses tested falling below the detection level (≤ . tcid ml − ). even very resistant viruses, such as the porcine parvovirus, could be reduced to below the detection level. for all viruses tested the log reduction factor was at least . (table xiv) . willkommen et al. ( ) reported an overview on virus inactivation and removal from serum and serum substitutes. with respect to the efficiency of ppv inactivation by γ irradition they indicated that even after application of a radiation dose of kgy, a tcid of . per ml was observed, indicating that the log reduction was only about . this difference with data published by plavsic et al. ( ) might be due to differences in the design of the respective studies. however, with respect to the other viruses tested (bvdv, ibr, pi- , reovirus ), no differences in the inactivation doses were observed. it should be mentioned here, that sera are normally irradiated using a dose of to kgy. for veterinary use, the radiation dose has to be kgy. a very important consideration is the capacity of the irradiated serum to support cell growth. by performing long-term standard cultures (three passages in a medium supplemented with % of the irradiated sera), plavsic et al. ( ) were able to show that in principle all tested cell lines could be cultivated, but also that different cells reacted relatively differently on the radiation doses used. whereas low passage bhk cells, vero (only slightly), and cho cells displayed an inverse relationship between growth and radiation dose, high passage bhk cells and the human diploid fibroblasts, wi- and mrc- -the latter are of special interest for vaccine production and virology -did not display growth decline as a function of radiation dose (table xv ). none of the tested cell lines showed a modifed morphology. the advantages of irradiation is that it is easy and safe and does not leave residual molecules in the final product as when chemical inactivation methods are used. today γ irradition is mandatory in europe for fetal bovine serum. validation experiments of the γ irradiation (dose: - kgy) of porcine pancreatic trypsin powder indicated a . log reduction of the median tissue culture infective dose (tcid ) (erickson et al., ) . (c) uvc irradiation (kurth et al., ) : a second rather easy and safe method is uvc irradiation for inactivating adventitious agents. as distinct from γ irradition, the uvc irradiation has to be performed by using a continuous flow through irradiator. an irradiation time of ± s and a fluence of . j cm − are used normally. the principle of this type of irradiation is a dna excitation leading to electron transfer (→ -hydroxoy-guanine), photohydration (→ cytosin hydrates), photoaddition and dimerization (→ pyr -pyr, thy -ade, pyrimidine ( - ) pyrimidone). uvc irradiation is very effective for selected virus groups, especially for those with single-stranded nucleic acids. the data shown in table xiii indicate that all tested single-stranded viruses were rather efficiently inactivated with a log clearance ranging from > . to . only the reovirus which has a double stranded rna shows a lower log reduction ( ). long term growth assays did not reveal any reduction in the ability of the uvc irradiated sera (used at %) to support cell growth. (d) other treatments: substances of animal origin, such as serum or trypsin, or final biotech products can also be treated by other methods for reducing the eventual viral burden. these treatments can be of chemical nature, such as treatment with peracetic acid (hughes, ) , solvents (e.g. % tween and . % tri-n-butyl-phosphate at • c for . h for the treatment of plasma derived factor ix, works only for lipid enveloped viruses) (johnston et al., ) , or imines (brown et al., ) , or physical methods, such as heating (hughes, ) or the reduction of the ph to . . with respect to heat treatment, it is less effective than irradiation methods (willkommen et al., ) and the serum composition is too much altered (hanson and foster, ) , leading to a rather weak growth promotion. the addition of a chemical substance, such as peracetic acid, is not ideal because a chemically reactive substance is added which might also lead to an inactivation of some medium compounds. in spite of this, virus inactivation based on the treatment with peracetic acid is rather effective for inactivating resistant virus, such as poliovirus table xv . the effect of increasing doses of gamma radiation on the ability of fetal bovine serum to support the long term growth of selected cell lines in culture (expressed as percent of growth of control cultures) (plavsic et al., ) (sprössig and mücke, ) , as well as for maintaining the growth supporting ability of the serum. other adventitous agents like mycoplasmas and bacteria are also efficiently inactivated (schweizer et al., ) . (e) replacement of animal derived substances by non-animal/non-human derived substances: although viral screening tests are efficient for detecting adventitious viruses, the best remedy for avoiding the presence of these viruses is the use of non-animal/nonhuman derived raw materials. this is not a complete assurance for the absence of virus, but a considerable risk reduction, since adventitious viruses might also be introduced via non animal derived raw materials, such as medium components as shown by garnick ( ) . in this context it should be mentioned that most of the recent biologicals based on the use of animal cell technology are produced in serum-free or protein-free media (froud, ; merten, ) . with respect to the production of viral vaccines, the first serum-free viral vaccines were developed and tested in clinical studies (brands et al., ; kistner et al., ) , and are going to be put on the market. recently, a study concerning a veterinary live virus vaccine, which was produced under protein-free conditions (devoid of any animal or human derived substances), showed that such a vaccine was as efficacious and safe as a classically produced vaccine (under serum-conditions) (makoschey et al., ) . this indicates very clearly that the use of serum for the production of viral vaccines, in particular, and of biologicals, in general, is an anachronism and that the efficient replacement of non-animal derived serum-supplements is feasible. operator induced biological contaminations in cell culture is a multifaceted problem involving the unex-pected introduction of other animal cells (see chapter by masters), microbial (see chapter by drexler and uphoff), and viral contaminants. there are few reports on operator induced viral contaminations. the potential exists, however, as reports have appeared documenting the considerable stability of rhinoviruses, respiratory syncytial virus, rotaviruses, and others, in aerosols on worker's hand and safety hood surface (for more details, see hay, ) . in general, viral contaminations of cell lines cannot be treated and contaminated cultures should be discarded, with the exception of ldv. this virus causes a life long viremia in infected mice without any clinical signs, and each sample of these animals is virus contaminated. because ldv requires primary mouse macrophages for replication it cannot survive repeated in vitro subcultivations, leading to a loss of this virus in infected in vitro cultures. another elimination method is the passage of the contaminated cell line/tumor in another species, for example nude rats (nicklas et al., ; nakai et al., ) . the absence of virus can only be assured by performing a rigorous testing programme, which includes all steps in a bioprocess: master cell bank, working cell bank, the raw materials, the unprocessed bulk harvest, late expanded cells, and the final product. a summary on the tests to perform is presented in table xvi . whereas research cell banks are mostly tested for sterility and absence of mycoplasmas, gmp cell banks a used for preparation of wcb or in production runs starting from mcb or wcb. b cells at the end of a typical production run are tested to determine the virus load in case of retrovirus like particle bearing production cells. only few harvests need to be tested (validation). c old cells may be from production runs (as post production cells) or from a separate culture kept in continuous culture for a long period and prepared for this analysis of 'limit of cell age' only (as late expanded cells). extensive testing performed as part of the qualification of the mcb regarding absence of latent virus, inducible by cultivation on production conditions. d a very large sample volume for testing would be required for statistics of a sufficiently sensitive detection of low virus titers. for the production of biologicals for parenteral applications have to be tested much more rigorously. ich topic q a ( ) suggests the following virus tests for different cell banks: 'a master cell bank has to be extensively screened for both endogeneous and non-endogeneous viral contaminants. for heterohybridoma cell lines in which one or more partners are human or non-human primate in origin, tests should be performed in order to detect viruses of human or nonhuman primate origin as viral contaminants arising from these cells may pose a particular hazard. testing for non-endogeneous viruses should include in vitro and in vivo inoculation tests and any other specific tests, including species-specific tests such as mouse antibody (map) test, that are appropriate, based on the passage history of the cell line, to detect possible contaminating viruses.' 'the working cell bank as a starting cell substrate for drug production should be tested for adventitious viruses either by direct testing or by analysis of cells at the limit of in vitro cell age, initiated from the wcb. when appropriate non-endogenous virus tests have been performed on the mcb and cells cultured up to or beyond the limit of in vitro age have been derived from the wcb and used for testing for the presence of adventitious viruses, similar tests need not to be performed on the initial wcb. antibody production tests (map, rap, or hap) are usually not necessary for the wcb. an alternative approach in which full tests are carried out on the wcb rather than on the mcb would also be acceptable. ' 'the limit of in vitro cell age used for production should be based on data derived from production cells expanded under pilot-plant scale or commercial-scale conditions to the proposed in vitro cell age or beyond. generally, the production cells are obtained by expansion of the wcb; the mcb could also be used to prepare the production cells. cells at the limit of in vitro cell age should be evaluated once for those endogenous viruses that may have been undetected in the mcb and wcb. the performance of suitable tests (e.g. in vitro and in vivo) at least once on cells at the limit of in vitro cell age used for production would provide further assurance that the production process is not prone to contamination by adventitious virus. if any adventitious viruses are detected at this level, the process should be carefully checked in order to determine the cause of the contamination, and completely redesigned if necessary.' the detection of adventitious viruses in cell banks has to follow two principles -use of detection methods for specific viruses such as map, hap, rap (mouse, hamster, rat antibody production tests -examination of serum antibody levels against specific viruses or enzyme activity after a specified period), and different specific pcrs, as well as the use of general tests which may indicate the presence of one or more of a large variety of different viruses. general tests include the in vitro test for adventitous virus. this test involves the inoculation of different cell lines -a human, a primate and a bovine (if bovine material was used for the production, otherwise a cell line from the species of origin of the cell substrate), and the production cell line (co-cultivation test). the rtase assay is a general test which will detect the presence of all viruses which contain reverse transcriptase enzyme. the last general test which is normally applied is the, in vivo tests where animals are used to identify the presence of virus. the animals are treated using different inoculation routes (the health of these animals should be monitored and any abnormality should be investigated to establish the cause of the illness). finally, electron microscopic examination is also a general test which can be used for detecting adventitious viruses in the case of rather high virus loads. more details can be found in the articles by poiley ( ) , by werz et al. ( ) , and in the ich topic q a ( ). when a producer cell line of murine origin is used, the consensus opinion among regulators is that all known murine viruses should be tested for. if a cell of human origin is involved in production, then there should be tests for human viruses, such as hiv, htlv, ebv, cmv, hhv and hhv . human-mouse heterohybridoma cell lines have to be tested for both human and murine viruses (robertson, ) . in general, the unprocessed bulk material (pool of harvests of cells and culture media) should be tested for viruses after the end of production and before any downstream processing. the scope, extent, and frequency of virus testing on the unprocessed bulk should be determined by taking several points into consideration including the nature of the cell lines used to produce the desired product, the results and extent of virus tests performed during the qualification of the cell line, the cultivation method, raw material sources and results of viral clearance studies. in vitro screening tests, using several cell lines, are generally employed for testing. if appropriate, a pcr test or other suitable methods may be used. the presence of an adventitious virus will block the further use and processing of the harvest material. for screening of raw materials, mainly serum, see section on 'sourcing, screening and other precautions'. in summary general virus tests are vital because past incidents of viral contaminations have derived from viruses not known to be present within the production systems. therefore the approach involving a variety of both general and specific tests applied at more than one stage of manufacture in combination with viral elimination steps during subsequent processing to assure the safety of the product is of utmost importance. the use of modern biotechnology for the production of biopharmaceuticals allows the treatment of diseases, which could not be treated previously. however, virus infection and replication is an inherent risk during cultivation of mammalian cells. raw material testing, rigorous characterization of the master and the working cell bank as well as testing of the final bulk product (before downstream processing and virus inactivation steps have been performed) lead to a considerable increase in the viral safety. although viral contamination might happen during production only preventive measures can be taken in fermentation and product biosynthesis. thus the downstream processing is an integral part of the manufacturing process and has to be validated in order to assess its potential to eliminate, clear, or inactivate viruses. the downstream processing has two, sometimes contradictory aims: (i) purify the product to the required purity at high recovery using the lowest number of steps possible, and (ii) eventually increase the number of purification steps in order to assure the elimination of potential viral contaminants. the general difficulty resides in the physico-chemical properties of the product. the properties that maintain the beneficial effects of a product are often very similar to those carried by all viruses, in particular when the bioproduct is a live virus (for vaccination or for gene therapy purposes). therefore only a limited spectrum of techniques can be used for virus inactivation/elimination. to assess the capability of individual process steps to remove viruses these steps have to be tested with live model viruses for which clearing factors can then be calculated. these, so called, viral clearance studies have the objective to demonstrate the capacity of different steps of the purification process to eliminate or inactivate adventitious agents acquired during the production process (contaminated cells, raw materials, process failure, etc.). they are performed via spiking experiments. as viruses vary greatly in properties such as size, resistance to inactivation, type of envelope, type and structure of their genome, the model viruses used for these spiking experiments have to be selected in order to cover the whole spectrum of potentially present viruses. however, in order to assure absence of viral contaminations derived from the producer cell line in the product, the downstream protocol has also to be validated for its capacity to inactivate or eliminte these specific viruses (e.g. retroviruses derived from hybridoma and cho cells, or ebv derived from human lymphoblastoid cell lines). in general, it is difficult to show more than a five log removal on any given step due to the titers of the model virus used. more details can be found in the following references: fritsch ( ) , cartwright ( ) , werz et al. ( ) , ich topic q a ( ), and larzul ( ) . it is evident that a research laboratory cannot afford all tests, which have to be performed by the biotech industry in the case of gmp production. in addition, there is no need for such exhaustive testing unless the materials are to be used in the treatment of human patients. however, most of the issues described in the chapters on 'problems associated with viral contaminations' and on 'origin of viral contaminations' are valid for everyone working in the field of animal cell culture. the use of validated cell lines, shown to be 'virus-free', is the best choice because cell collections, such as the atcc (www.atcc.org), perform entrance tests for new cell lines for assuring the absence of mycoplasma, bacteria, fungi, protozoa, and cytopathic viruses and can guarantee a certain microbial quality for the delivered cell lines. the german cell line bank (www.dsmz.de) provides cells, most of them have been tested for the absence of hiv-i, htlv-i and ii, ebv, hbv, hcv, and hhv- by using pcr or rt-pcr. the absence of retroviruses is proven by performing a reverse transcriptase assay. however, cell lines obtained from such collections, can still be contaminated by viruses because viruses, which do not lead to a cytopathic effect, are not detected by the tests commonly used, on one side (atcc), or only tests for detecting human pathogenviruses have been performed on the other side (dsmz). if a very important cell line of a research laboratory has to be more rigorously screened, commercial screening services are needed and their use is recommended. the use of controlled animals, free of microbial contaminants, for animal passages of cells and of controlled raw materials derived from accredited dealers, who are performing viral screens (e.g. for virus absence in serum and trypsin preparations), for the preparation of culture media are steps in the right direction for reducing the risk for viral contaminations. the rules concerning the general cell culture operation procedures for avoiding viral contaminations are largely the same as for preventing mycoplasmal contaminations (see chapter by drexler and uphoff). viral contaminations are a serious threat for animal cell cultures and may lead to false results in research, development, and virus screening, to viral contaminations in the biologicals derived from the contaminated cultures and finally to an infection of the treated patient. fortunately due to rigorous testing of the animals used as the source of explants, the production of ascites or the passage of cells, of raw materials, of the cell strains and cell lines in use, and finally of bulk and the final product can prevent potentially dangerous viral contaminations. existing data demonstrate that contamination of cells and harvests by viruses can occur for products of biotechnology, and while the frequency may be low it is not zero. for instance, routine testing of cell lines of biotech interest revealed a contamination frequency with adventitious viruses of less than % (moore, ) . however, the possibility of new emerging viruses and the permanently existing risk of contaminations by adventitious agents and viruses leads to the conclusion that the user of animal cells as well as the producer of biotech products by using animal cells have to be attentive to this possible threat and that they have to assure the absence of adventitious agents/viruses by any mean. only then, animal cell technology biotech products can be used for the benefit of everyone. experiences of virus, retrovirus and retroviruslike particles in chinese hamster ovary (cho) and hybridoma cells used for production of protein therapeutics presence and transcription of intracisternal a-particle related sequences in cho cells virus retention by a hydrophilic triple-layer pvdf microporous membrane filter detection of adeno-associated virus (aav)-specific nucleotide sequences in dna isolated from latently infected detroit cells relationship between nature and source of risk and process validation contamination of transplanatble murine tumors with lymphocytic choriomeningitis virus high prevalence of tt virus infection in french blood donors revealed by the use of three pcr systems methods for detection and frequency of contamination of fetal calf serum with bovine viral diaahea virus and antibodies against bovine viral diarrhea virus survey of cell lines in the american type culture collection for bovine viral diarrhea virus influvac tc : a safe madin darby canine kidney (mdck) cell culture-based influenza vaccine a universal virus inactivant for decontaminating blood and biopharmaceutical products observations on the replication of ectromelia virus in mousederived cell lines: implications for emidemiology of mousepox contamination of genetically engineered chinese hamster ovary cells the characteristics and growth of cultured cells murine virus contaminants of leukemia viruses and transplantable tumors guidelines for medicinal products derived from human blood and plasma note for guidance on the use of bovine serum in the manufacture of human biological medicinal products identification of murine endogenous xenotropic retrovirus in cultured multicellular tumour spheroids from nude-mouse-passaged nasopharyngeal carcinoma isolation of porcine parvovirus from commercial trypsin recent studies on retrovirus-like particles in chinese hamster ovary cells tumors induced in hamsters by injection of rhesus monkey kidney cell extracts lack of antibodies to porcine circovirus type virus in beef and dairy cattle and horses in western canada risques virologiques associés aux matières premières extraites de tissus animaux utilisées dans la fabrication des médicaments risks of virus transmission associated with animal sera or substitutes and methods of control cultivation of lansing strain of poliomyelitis virus in culture of various human embryonic tissues detection and elimination of adventitious agents in continuous cell lines bovine viral diarrhea disease associated with a contaminated vaccine latent parvoviral infection of continuous cell lines korean haemorrhagic fever. propagation of the etiologic agent in a cell line of human origin validation of virus removal in large scale purification processes viral contaminants found in mouse cell lines used in the production of biological products experience with viral contamination in cell culture h ( ) virus removal by filtration nachweis von gelbfiebervirus-haemagglutinin in menschlichen explantaten parvoviruses as contaminants of permanent human cell lines. i. virus isolations from - viral safety in serum for cell culture use adventitious pestivirus rna in live virus vaccines against bovine and swine diseases demonstration and genotyping of pestivirus rna from mammalian cell lines sequence analysis of the ' untranslated region of pestivirus rna demonstrated in interferons for human use operator-induced contamination in cell culture systems adenovirus associated virus continuous 'carriage' of adenovirus-associated virus genome in cell culture in the absence of helper adenovirus la sécurité virale des produits biologiques: aspects historiques et conceptuels. virologie quality of biotechnological products: viral safety evaluation of biotechnology products derived from cell lines of human or animal origin an animal origin perspective of common constituents of serum-free medium formulations inactivation and clearance of viruses during the manufacture of high purity factor ix detection of bovine polyomavirus contamination in fetal bovine sera and modified live viral vaccines using polemerase chain reaction development of a vero cell derived influenza whole virus vaccine sensitivity of epstein-barr virus producer and non-producer human lymphoblastoid cell lines to superinfection with eb-virus endogenous virus contaminants in fetal bovine serum and their role in tissue culture contamination attempts to characterize bovine viral diarrhea virus isolated from cattle after immunization with a contaminated vaccine the intracisternal a-particle gene family; structure and functional aspects efficient inactivation of viruses and mycoplasma in animal sera using uvc irradiation viral validation design of a manufacturing process certified cell lines of rat origin free of infection with hantavirus an epithelial cell line with chronic polyoma infection established from a spontaneous mouse pancreatic adenocarcinoma mouse plasmacytoma cells produce infectious type c viruses. the lancet ii infection of laboratory workers with hantavirus acquired from immunocytomas propagated in laboratory rats raw material considerations safety of vaccines produced in continuous cell lines virus zoonosis and their potential for contamination of cell culture serum-free produced bovine herpesvirus type and bovine parainfluenza type virus vaccines are efficacious and safe quality control of bovine serum used for vaccine production small deoxyribonucleic acidcontaining viruses (picodnavirus group) morphological studies on the replication of a defective satellite virus and its helper adenovirus morphological identification of the agent of korean haemorrhagic fever (hantaan virus) as a member of the bunyaviridae. the lancet i safety issues of animal products used in serumfree media mammalian cells and their contaminants experience in cell line testing detection and elimination of contaminating microorganisms in transplantable tumors and cell lines the association of calf serum with the contamination of bhk clone suspension cells by a parvovirus serologically related to the minute virus of mice (mvm) contamination of transplantable tumors, cell lines, and monoclonal antibodies with rodent viruses a noveal dna virus (ttv) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology multiple porcine circovirus -associated abortions and reproductive failure in a multisite swine production unit filovirus contamination of cell cultures gamma irradiation of bovine sera methods for the detection of adventitious viruses in cell cultures used in the production of biotechnology products contamination of genetically engineered cho-cells by epizootic haemorrhagic disease virus (ehdv) strategy for adventitious agent assays comparison of pestivirus multiplication in cells of different species bovine polyomavirus, a frequent contaminant of calf serum porcine circovirus infection in swine foetuses inoculated at different stages of getation frequent detection of bovine polyomavirus (bpyv) in commercial batches of calf serum, using the polymerase chain reaction peracetic acid as sterilizing agent for culture serum human exposure to sv : review and comment experimental infection of rhesus with simian virus fatal human disease from vervet monkeys. the lancet ii identification of the major structural proteins of two balb/c myeloma c-type viruses Über die antimikrobielle wirkung der peressigsäure. . mitteilung: untersuchung zur viruziden wirkung production and control of live oral poliovirus vaccine in wi- human diploid cells the vacuolating virus, s.v. a very small porcine virus with circular single-stranded dna removal of viruses from human intravenous immune globulin by nm nanofiltration bovine polyomavirus, a frequent contaminant of calf sera contamination of a live virus vaccine against pseudorabies (aujeszky's disease) by an ovine pestivirus pathogen for the pig use of captive-bred monkeys for vaccine production the production of inactivated poliovaccine on serially cultivated kidney cells from captive-bred monkeys new approach to the production of concentrated and purified inactivated polio and rabies tissue culture vaccines new oncogenic papovavirus from primate cells retroviruses produced by hybridomas strategies to avoid virus transmissions by biopharmaceutic products benefits and risks due to animal serum used in cell culture production abortion and death in pregnant bitches associated with a canine vaccine contaminated with bluetongue virus serum and serum substitutes: virus safety by inactivation or removal isolation and characterization of a papovavirus from cynomolgus macaque kidney cells contamination of commercially available fetal bovine sera with bovine viral diarrhea virus genomes: implications for the study of hepatitis c cirus in cell cultures isolation and characteristics of an agent inducing anemia in chicks i would like to thank drs. i. freshney and d. galbraith for correcting and improving the english formulation of the text. key: cord- - nj f authors: ambrose, rebecca k.; gravel, jennifer l.; commins, margaret a.; fowler, elizabeth v.; mahony, timothy j. title: in vivo characterisation of five strains of bovine viral diarrhoea virus (subgenotype c) date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: nj f bovine viral diarrhoea virus (bvdv- ) is strongly associated with several important diseases of cattle, such as bovine respiratory disease, diarrhoea and haemoragic lesions. to date many subgenotypes have been reported for bvdv- , currently ranging from subgenotype a to subgenotype u. while bvdv- has a world-wide distribution, the subgenotypes have a more restricted geographical distribution. as an example, bvdv- subgenotypes a and b are frequently detected in north america and europe, while the subgenotype c is rarely detected. in contrast, bvdv- subgenotype c is by far the most commonly reported in australia. despite this, uneven distribution of the biological importance of the subgenotypes remains unclear. the aim of this study was to characterise the in vivo properties of five strains of bvdv- subgenotype c in cattle infection studies. no overt respiratory signs were reported in any of the infected cattle regardless of strain. consistent with other subgenotypes, transient pyrexia and leukopenia were commonly identified, while thrombocytopenia was not. the quantity of virus detected in the nasal secretions of transiently infected animals suggested the likelihood of horizontal transmission was very low. further studies are required to fully understand the variability and importance of the bvdv- subgenotype c. bovine respiratory disease (brd) is the most important disease of intensively finished cattle. while multiple factors contribute to the likelihood of cattle developing brd, a generally accepted model for brd development is a primary viral infection predisposing cattle to more severe secondary bacterial infections. several viruses have been associated with an increased risk of brd, including bovine herpesvirus (bohv- ), bovine viral diarrhoea virus (bvdv- ), bovine respiratory syncytial virus and bovine parainfluenza virus. of the key brd associated viruses, bvdv- is the most genetically diverse with subgenotypes, bvdv- a to bvdv- u, having been reported [ ] . yesilbag et al. [ ] recently reviewed the geographical distribution of the bvdv- subgenotypes. this analysis highlighted several unusual trends in the distribution of the bvdv- subgenotypes. as an example, the vast majority of genotyped strains identified in australia have been classified within the subgenotype c [ , ] . this is in contrast to the usa where the subgenotype b is dominant, but the subgenotype a is also frequently reported [ ] . while in europe the most common subgenotypes are a and b, also common are subgenotypes d, e, f and h [ ] . however, the distribution of subgenotypes can vary between countries within europe, for example subgenotype b and d were recently reported to be the most frequently identified subgenotypes in germany [ ] . strains of the subgenotype c have been rarely reported in the usa or europe. the drivers of these distributions are unclear, but do not appear to be influenced by vaccine use [ ] . several studies have suggested that the subgenotypes, at least in part, also reflect the antigen diversity between strains using cross-neutralisation assays [ , , ] . clearly the degree of cross-protection afforded by the subgenotype(s) included in a bvdv- in a vaccine against the subgenotypes circulating in a cattle population is an important issue. the overall biological importance of the bvdv- subgenotypes remains to be fully elucidated since the majority of published studies have focused on the bvdv- a and bvdv- b genotypes. it is reasonable to accept that the subgenotypes of bvdv- share similar properties with respect to their capacity to infect susceptible ruminants, and their contribution to the development of more severe clinical disease outcomes such as brd and the birth of persistently infected calves following transplacental infection. all of these outcomes are reported in cattle populations regardless of what subgenotype is present. the evaluation of any variation of the in vivo properties of bvdv- subgenotypes has received little attention. to assist in developing a better understanding of the importance of the bvdv- subgenotypes, the aim of this study was to assess the in vivo properties of five bvdv- strains from the poorly studied subgenotype c. no overt clinical scores were recorded for any of the trial animals, challenged or unchallenged with the bvdv- c strains used in this study (data not shown). for all groups, except the cattle infected with bvdv- c strain ns , there was a general trend for an increase in the average rectal temperature on day of the experiment, prior to any viral exposure, compared to the temperatures from day to day (figure a -f). the reason(s) for this increase are unclear. the cattle had been inducted into the containment facility seven days prior to the commencement of the trial and were therefore considered to have been well adapted/acclimatised to the environment. however, on day there were extra staff and equipment present while the cattle were being inoculated which included extra handling of the animals/cattle potentially contributing to this apparent increase. due to this anomaly, the day temperatures were used as the reference point in the subsequent statistical comparisons within each treatment group. the daily rectal temperatures were monitored for the trial cattle for days following experimental infection. overall the mean rectal temperature results were variable ( figure ). animals (n = ) infected with bvdv- c strain pi exhibited a significant temperature elevation on day post infection (p < . , figure a ). for animals infected with bvdv- c strain trangie (n = ), the temperature increase was gradual from day to day post infection, although the increase was only statistically significant on day (p < . , figure b ). animals infected with bvdv- c strain ao (n = ) exhibited elevated mean temperatures on day and day post infection, with only the day mean temperature being statistically significant (p < . , figure d) . a similar temperature profile was observed for bvdv- c strain ns (n = ), with elevated temperatures on day and day , with only day being statistically significant (p < . , figure d ). although there was a suggestion of an elevated mean temperature on day post-infection for the cattle (n = ) infected with vr this was not statistically significant ( figure e ). the rectal temperatures of the contact animals (n = ) did not exceed . • c during the experiment (figure f ). no statistical comparisons were undertaken for this group due the low animal numbers. animals infected with bvdv- c strain trangie (n = ); (c) animals infected with bvdv- c strain ao (n = ); (d) animals infected with bvdv- c strain ns (n = ); (e) animals infected with bvdv- c strain vr (n = ); (f) uninfected animals (n = ). the asterisks above selected days indicate significant differences compared to day post-infection for that group of cattle. level of significance; * p < . ; ** p < . ; **** p < . . the course of the bvdv- c nasal shedding by the trial cattle was assessed by testing extracts from nasal swabs collected from day to day and serum samples collected on day and day post-infection with a bvdv- specific quantitative real-time (qpcr). a summary of these results expressed as the threshold cycle (ct) is shown in table . overall, the distribution of positive nasal swabs was variable between and within the groups of cattle infected with the different strains of bvdv- c. the earliest that virus was detected in nasal swabs was day post-infection for animals infected with pi ( of animals), trangie ( of animals) and ao ( of animals). one nasal swab from an animal infected with strain pi tested positive at day post infection. the bvdv- c strain ao was the virus most consistently detected in the nasal swabs with all infected animals reacting with the qpcr at day and day post infection, and three of the four animals also reacted in the qpcr on day ( table ). the serum samples collected from animals infected with ao at day post-infection also reacted and were deemed to be positive for bvdv- c (table ). (b) animals infected with bvdv- c strain trangie (n = ); (c) animals infected with bvdv- c strain ao (n = ); (d) animals infected with bvdv- c strain ns (n = ); (e) animals infected with bvdv- c strain vr (n = ); (f) uninfected animals (n = ). the asterisks above selected days indicate significant differences compared to day post-infection for that group of cattle. level of significance; * p < . ; **** p < . . the course of the bvdv- c nasal shedding by the trial cattle was assessed by testing extracts from nasal swabs collected from day to day and serum samples collected on day and day post-infection with a bvdv- specific quantitative real-time (qpcr). a summary of these results expressed as the threshold cycle (ct) is shown in table . overall, the distribution of positive nasal swabs was variable between and within the groups of cattle infected with the different strains of bvdv- c. the earliest that virus was detected in nasal swabs was day post-infection for animals infected with pi ( of animals), trangie ( of animals) and ao ( of animals). one nasal swab from an animal infected with strain pi tested positive at day post infection. the bvdv- c strain ao was the virus most consistently detected in the nasal swabs with all infected animals reacting with the qpcr at day and day post infection, and three of the four animals also reacted in the qpcr on day ( table ). the serum samples collected from animals infected with ao at day post-infection also reacted and were deemed to be positive for bvdv- c (table ) . table . detection of bovine viral diarrhoea virus subgenotype c in extracts from cattle samples using quantitative real time pcr (qpcr). nasal swabs (n) and serum (s) samples were analysed using qpcr. reactive samples were deemed positive for bvdv- c with the cycle threshold value shown (shaded cells). samples which did not react (> ) with the qpcr were deemed to be negative (−) for the virus. with respect to the other groups, bvdv- c was consistently detected in nasal swabs collected on day , day and day post-infection from two of the six animals infected with bvdv- c strain pi ( table ) . one of the animals in this group tested positive (nasal swabs) from day to day post-infection and the swabs from day and day were also positive. for cattle infected with bvdv- c strain trangie, one of the four animals tested positive on day post-infection, while all the other samples were negative throughout the sampling period (table ) . of the animals infected with bvdv- c strain ns , two animals had positive nasal swabs. one of these animals was positive on day . the strain ns was detected sporadically between day and in samples from animal . the serum samples from day and day from this animal were both reactive with the qpcr assay (table ) . two animals infected with bvdv- c strain vr tested positive for the virus in a sporadic manner. the remaining animals did not return any positive results ( table ) . none of the sample extracts from the two contact animals reacted with the qpcr during the sampling period (table ) . bvdv- c was not detected via qpcr in the nasal swab or serum samples collected from all animals on day , day , day and day post-infection and were deemed to be negative (data not shown). attempts were made to isolate the bvdv- c strains from the nasal swabs collected at day post-infection from selected animals. no bvdv- c was detected in any of the culture supernatants by qpcr after three passages of animal (pi infected and tested positive from day to day ), animal (trangie infected), animal (ao infected) or animal (ao infected). with respect to the other groups, bvdv- c was consistently detected in nasal swabs collected on day , day and day post-infection from two of the six animals infected with bvdv- c strain pi ( table ) . one of the animals in this group tested positive (nasal swabs) from day to day post-infection and the swabs from day and day were also positive. for cattle infected with bvdv- c strain trangie, one of the four animals tested positive on day post-infection, while all the other samples were negative throughout the sampling period (table ) . of the animals infected with bvdv- c strain ns , two animals had positive nasal swabs. one of these animals was positive on day . the strain ns was detected sporadically between day and in samples from animal . the serum samples from day and day from this animal were both reactive with the qpcr assay (table ) . two animals infected with bvdv- c strain vr tested positive for the virus in a sporadic manner. the remaining animals did not return any positive results ( table ) . none of the sample extracts from the two contact animals reacted with the qpcr during the sampling period (table ) . bvdv- c was not detected via qpcr in the nasal swab or serum samples collected from all animals on day , day , day and day post-infection and were deemed to be negative (data not shown). attempts were made to isolate the bvdv- c strains from the nasal swabs collected at day postinfection from selected animals. no bvdv- c was detected in any of the culture supernatants by qpcr after three passages of animal (pi infected and tested positive from day to day ), animal (trangie infected), animal (ao infected) or animal (ao infected). monocytes: seven days post-infection, animals infected with bvdv- c strain pi had significantly reduced concentration of monocytes compared to the pre-infection sample (p < . , figure a ). no significant changes in the numbers of monocytes were detected for any of the animals infected with the remaining bvdv- c strains or the contact animals ( figure ). (e) (f) (e) (f) (e) (f) platelets: overall, the concentration of platelets in infected animals were reduced for most bvdv- c strains on day and/or day post infection compared to day . the only significant reduction was for the animals infected with bvdv- c strain ns on day (p < . , figure d) . a reduction in platelet numbers was also apparent on day for animals infected with strain pi , trangie and vr , but these differences were not statistically significant (figure a,b,e) . in contrast to other infected groups, the concentration of platelets in cattle infected with bvdv- c strain ao appeared stable for the duration of the experiment, apart from a significant increase on day postinfection (p < . , figure c ). platelets: overall, the concentration of platelets in infected animals were reduced for most bvdv- c strains on day and/or day post infection compared to day . the only significant reduction was for the animals infected with bvdv- c strain ns on day (p < . , figure d) . a reduction in platelet numbers was also apparent on day for animals infected with strain pi , trangie and vr , but these differences were not statistically significant (figure a,b,e) . in contrast to other infected groups, the concentration of platelets in cattle infected with bvdv- c strain ao appeared stable for the duration of the experiment, apart from a significant increase on day post-infection (p < . , figure c ). platelets: overall, the concentration of platelets in infected animals were reduced for most bvdv- c strains on day and/or day post infection compared to day . the only significant reduction was for the animals infected with bvdv- c strain ns on day (p < . , figure d) . a reduction in platelet numbers was also apparent on day for animals infected with strain pi , trangie and vr , but these differences were not statistically significant (figure a,b,e) . in contrast to other infected groups, the concentration of platelets in cattle infected with bvdv- c strain ao appeared stable for the duration of the experiment, apart from a significant increase on day postinfection (p < . , figure c ). (a) (b) (c) (d) all trial animals were monitored for the development of bvdv- specific antibodies throughout the course of the experiment. virus specific antibody was first detected on day post-infection with of the infected animals testing positive. however, none of the animals infected with strains trangie or ao had detectable antibodies by this time point. by day post-infection, of the animals had detectable bvdv- specific antibody ( table ). all the bvdv- c challenged animals had detectable bvdv- antibodies by day post-infection ( table ). one of the animals infected with strain ao was positive on day but subsequently tested negative on day (table ) . no bvdv- antibody was detected in the serum samples from either of the two contact animals at any of the sampling time points (table ) . table . serological responses (igg) of cattle challenged with one of five strains of bovine viral diarrhoea virus genotype c. the level of virus specific igg were determined using a commercial elisa and assigned to one of six arbitrary categories, negative (−) or positive (+, ++, +++, ++++, or +++++). all trial animals were monitored for the development of bvdv- specific antibodies throughout the course of the experiment. virus specific antibody was first detected on day post-infection with of the infected animals testing positive. however, none of the animals infected with strains trangie or ao had detectable antibodies by this time point. by day post-infection, of the animals had detectable bvdv- specific antibody ( table ). all the bvdv- c challenged animals had detectable bvdv- antibodies by day post-infection ( table ). one of the animals infected with strain ao was positive on day but subsequently tested negative on day (table ) . no bvdv- antibody was detected in the serum samples from either of the two contact animals at any of the sampling time points (table ) . table . serological responses (igg) of cattle challenged with one of five strains of bovine viral diarrhoea virus genotype c. the level of virus specific igg were determined using a commercial elisa and assigned to one of six arbitrary categories, negative (−) or positive (+, ++, +++, ++++, or +++++). animal id infection pi − − − + ++ +++ ++ − − + +++ ++++ +++++ ++++ − − + +++ ++++ ++++ +++ − − − ++ +++ ++++ ++++ − − − ++ ++ ++++ ++++ − − − +++ +++ ++++ ++++ trangie − − − +++ ++++ ++++ +++ − − − ++ +++ +++ +++ − − − +++ +++ ++++ +++++ − − ++ +++ +++++ +++++ +++++ ao − − ++ +++ ++++ +++++ ++++ − − +++ − +++++ +++++ +++++ − − +++ ++++ +++++ +++++ +++++ − − − ++ ++++ +++++ ++++− − + + +++++ +++++ ++++ − − − − ++++ ++++ ++++ − − + ++++ +++++ +++++ +++++ vr − − − − ++ ++ ++ − − +++ +++ +++++ +++++ +++++ − − − + + ++ ++ contact − − − − − − − − − − − − − − several studies have explored the potential links between the subgenotypes and antigenic variation through cross neutralisation studies [ , , ] . clearly, knowledge of any such relationship is important as it would facilitate the selection of vaccine components to match the circulating bvdv- subgenotypes, while also enabling the ongoing monitoring of field strains to detect any change in the dominant subgenotype. the importance of the bvdv- subgenotypes with respect to the in vivo biology has received minimal attention and more research is required to define commonalities and divergences between each group such as virulence. several of the parameters measured in this study showed similar effects of the bvdv- c strains on their bovine hosts. these commonalities were not unexpected as the strains used in this study were all bvdv- subgenotype c. currently, there are no specific criteria proposed to evaluate bvdv- virulence, however clinical signs (respiratory and/or digestive), biphasic pyrexia, biphasic leukopenia and thrombocytopenia have been reported for bvdv- subgenotypes a, b, d, e and k from various countries [ ] [ ] [ ] [ ] [ ] . in the current study, no respiratory or digestive clinical signs were observed in the bvdv- c inoculated cattle. while a significant pyrexia was identified in cattle infected with four of the five bvdv- strains, however, there was no evidence of a biphasic pyrexia (figure ). four of the five bvdv- infected groups exhibited significant leukopenia at day post-infection, three of which were biphasic (figure ). only the group infected with vr did not have detectable leukopenia. as vr was the only cytopathic bvdv- strain included in the current study, further research is required to determine why this was the case. with respect to thrombocytopenia, only the cattle infected with strain ns had a significant loss of platelets that was detected days after infection (figure d ). collectively these data suggest the bvdv- subgenotypes c evaluated in this study have low virulence in transiently infected animals under the experimental conditions utilised. one additional parameter which is commonly considered in the assessment of viral virulence is transmission capacity [ ] . there is general agreement that there is either no or limited horizontal transmission of bvdv- between transiently infected cattle [ ] [ ] [ ] . sarrazin et al. [ ] concluded that the field strains of bvdv- subgenotype a and b evaluated in their study were unlikely to play an important role in transmission. the detection of bvdv- c in the nasal swabs of infected cattle the current study was sporadic and where detected the results suggested low quantities of virus ( table ). the range of ct values from nasal swabs and serum samples in the current study were . to . and . to . respectively (table ) . previous studies have evaluated the use of qpcr to differentiate persistently and transiently infected animals. hanon et al. [ ] estimated that a ct value below . from a blood sample would identify all persistently infected animals, although this value was likely to misclassify some transiently infected animals as being persistently infected. while hay et al. [ ] estimated that a ct value below from serum was indicative of an animal being persistently infected with bvdv- . the results of the current study, suggest a ct value of is too high for the differentiation of transiently and persistently infected animals based on a single sample. noting that both prior studies utilised field samples for these estimates, thus direct comparison to the current study requires caution. the use of these estimates, would also require sample preparation and analyses to be comparable, particularly volume of sample extracted and subsequently used in the qpcr assay. associated with these results, the two uninfected control animals included in the study did not test positive for bvdv- or seroconvert to bvdv- over the course of the experiment. unchallenged animals could only be included in one of the containment rooms of the study for logistical reasons. consequently, there were no uninfected animals penned with the animals infected with bvdv- c strain ao or strain pi , the viruses most consistently detected in nasal swabs and in the highest quantities (table ) . collectively, these results suggest that minimal amounts of the challenge viruses were present in the nasal secretions of the infected animals and as a result the risk of virus transmission to other animals was very low for these bvdv- c strains. evans et al. [ ] recently reported the absence of horizontal transmission from sheep experimentally infected with an australian strain of bvdv- subgenotype c to sentinel sheep. the possibility of animals transiently infected with the bvdv- c strains used in these studies producing and shedding sufficient quantities of virus to facilitate transmission if subjected to stressful conditions cannot be excluded. it has recently been demonstrated that the bvdv- strain h (subgenotype a) was only transmitted to sentinel animals when the infected animals were immunosuppressed with dexamethasone [ ] . if the low risk if transmission from transiently infected animals extends to all bvdv- subgenotype c it could have important implications in the implementation of effective bvdv- control plans with persistently infected animals as the sole source of virus [ , [ ] [ ] [ ] . further research is required to determine if any bvdv- subgenotype c strains replicate sufficiently in the nasal epithelia at levels to facilitate transmission to susceptible sentinel animals. the bvdv- c strains pi and ao would be excellent candidate viruses for these studies. while the detection of virus in nasal swab and serum samples was sporadic, the cattle were clearly infected as demonstrated by the serological analyses. infected animals started to seroconvert by day post-infection with of the infected animals testing positive for bvdv- specific antibody. the number of positive animals increased by day , with all infected animals being antibody positive by day . these data are consistent with other bvdv- infection studies [ , ] . there did not appear to be anything specific to the bvdv- c strains used in this study in relation to the serological data with animals from all groups becoming seropositive in a fourteen day period and all animals being seropositive by day . the impacts of some bvdv- c strains on cattle in the current study may have been under-estimated due to the smaller number of cattle in each group, particularly for strain vr where one animal was withdrawn from the experiment immediately prior to commencement of the trial for ethical reasons (lameness). the number of cattle in this study was constrained by the capacity of the facility and need to evaluate the properties of several bvdv- subgenotype c strains. another potential limitation of the current study was the viral inoculums used were quantified using rt-qpcr of the final cell culture supernatants. while previous studies have demonstrated correlations between rt-qpcr results and measures of in vitro infectivity such as plaque forming units and/or % cell culture infectious dose (tcid ) for other viruses, such relationships have not been reported for bvdv- as yet [ ] [ ] [ ] [ ] . future studies which aim to directly compare the in vivo properties of the bvdv- c strains used in the study would need to establish the relationship between rt-qpcr results and measures of in vitro infectivity to enable the standardisation of the challenge doses. future studies will be required to better understand the relationships between the bvdv- subgenotypes and virulence. strong et al. [ ] also identified that the challenge dose can influence the clinical outcomes of cattle challenged with bvdv- a. data was also reported which suggested an influence of calf age on clinical outcome. as a consequence, future studies aiming to characterise the interactions of bvdv- and its bovine host should aim to do so under a standardised challenge system, including route of infection, challenge dose (where possible multiple doses) and age of animals. it is imperative that the subgenotype of the bvdv- isolate(s) used also be included. while the current study has focused on the respiratory component of the bvdv- c infection, it is well accepted the virus can have profound impacts on the reproductive capacity of individual animals and cattle herds overall. the bvdv- c isolate trangie was used in several earlier studies that reported the capacity of this virus to significantly impair bovine reproductive function [ ] [ ] [ ] [ ] . the capacity of specific bvdv- strains and/or subgenotype groups to cause both respiratory and reproductive disease are yet to be investigated, and may be required to fully understand this important cattle pathogen. this study is the first to characterise the in vivo properties of bvdv- strains confirmed as belonging to the subgenotype c. interestingly, the overall impacts of the infection of the different strains on the infected cattle were in general similar to those reported for other bvdv- subgenotypes with transient pyrexia, leukopenia, and quantities of virus in nasal swabs which are unlikely to facilitate horizontal transmission [ ] [ ] [ ] [ ] [ ] ] . of the bvdv- c strains examined in this study pi had the most consistent impact on the experimentally infected cattle and is a strong candidate for use in cattle studies to further define the in vivo properties of the subgenotype c, including direct comparisons to strains of other subgenotypes. all experimental procedures involving animals were reviewed and approved by the university of queensland animal ethics committee, approval number qaafi/ / /mla. the bvdv- c isolates used in the cattle trial are described in table . viral inoculums were prepared by adding µl of primary stock of each bvdv- c strain to culture medium of subconfluent monolayers of mdbk cells in tissue culture flasks ( cm ) and incubated at • c in a % co atmosphere for seven days. the supernatants were clarified at g, aliquoted and stored at − • c until required. as the aims of this study did not include intergroup statistical comparisons, the viral supernatants were used as harvested to inoculate cattle at the maximum possible titre. cattle were sourced by veterinary health research ltd. pty (armidale, nsw, australia). the animals were black angus and to months of age. prior to enrolment into the study, cattle were tested multiple times and confirmed negative for serological evidence of prior bvdv- exposure/infection. elisas were performed using the bio k elisa, as described by the manufacturer (bio-x diagnostics, jemelle, belgium). seven days prior to commencement of the trial, cattle (n = ) were moved into the large animal pc facility at the queensland animal science precinct (gatton, qld, australia). the cattle were randomly assigned to one of four pens ( × ) with two pens per room ( table ). the rooms are operated independently, including separate air handling systems. to minimise the risk of virus transmission between rooms, separate teams of staff were used to maintain/care for and collect samples from the animals in each room daily. on day , the rectal temperature for each animal was recorded and two blood samples collected via the jugular vein. the cattle groups were inoculated with one of the bvdv- viral strains as shown in table . briefly, the animal was restrained with the nose elevated and the viral inoculum ( ml) was slowly dripped into each nostril. the nose was held in this position for to s and the animal then released. two animals in room were not inoculated with virus. clinical assessments: cattle were monitored from day to day post-infection for clinical signs in respect to nasal discharge, coughing, behavior/demeanour and loss of appetite (feed residue). temperature: the rectal temperatures for each animal was recorded from day to day . the expected rectal temperature of healthy cattle was . • c [ ] . nasal swabs were collected from day to day , day , day , day , day and day . nasal swabs were immediately placed on ice for transport back to the laboratory for storage at • c until required. nasal swabs were immersed in µl of pbs containing × antibiotic-antimycotic (thermofisher scientific, waltham, ma, usa) and gently agitated. the swab was subsequently removed and discarded. a µl aliquot of this resuspension was used for total nucleic acid extraction using the dneasy blood & tissue kit (qiagen, hilden, germany) as described by the manufacturer, except for the exclusion of rnase a. total nucleic acid extracts were also prepared from aliquots ( µl) of cattle serum samples, collected as described below, using the same methodology. sample extracts prepared from nasal swabs and sera were analysed by qpcr for the presence of bvdv- rna as previously described [ ] . samples yielding a ct value ≥ were deemed to be negative for bvdv- . an aliquot of the resuspended nasal swab from day post-infection from selected animals were utilised for virus isolation. aliquots, µl and µl of the nasal swab resuspension diluted : with pbs were added directly to culture medium of subconfluent monolayers of mdbk cells in well plates and incubated at • c in a % co atmosphere for seven days. the monolayers were freeze/thawed once and a µl aliquot of the culture supernatant added to new subconfluent monolayers of mdbk cells in well plates and incubated at • c in a % co for seven days. this process was repeated three times. total nucleic acids were extracted from a µl aliquot of each culture supernatant and tested using qpcr for the presence of bvdv- as described previously. blood sampling: blood for serum harvesting ( ml bd vacutainers™, bd biosciences, franklin lakes, nj, usa) and blood cell count analyses ( ml edta bd vacutainers™, bd scientific, franklin lakes, nj, usa) were collected on day , day , day , day , day , day , day and day post infection. serum samples were tested for the presence of bvdv- specific antibodies using the bio k elisa, as described by the manufacturer (bio-x diagnostics, jemelle, belgium). the level of virus specific igg in each serum sample was assigned to one of six arbitrary categories, negative (−) or positive (+, ++, +++, ++++, or +++++) according to the manufacturer's instructions. whole blood samples were submitted to the veterinary science diagnostic services (school of veterinary science, university of queensland, gatton, qld, australia) for analyses of the cell populations using standard blood smearing and cell counting methodologies. data generated from the animal trial were analysed using a one-way analysis of variance (anova) with dunnett's multiple comparisons test and statistical significance attributed where p < . within graphpad prism™ (version . , graphpad software, inc., la jolla, ca, usa). variability and global distribution of subgenotypes of bovine viral diarrhea virus genetic analysis of bovine viral diarrhoea viruses from australia prevalence and antigenic differences observed between bovine viral diarrhea virus subgenotypes isolated from cattle in australia and feedlots in the southwestern united states bovine viral diarrhea virus (bvdv) b: predominant bvdv subtype in calves with respiratory disease eradication of bovine viral diarrhea virus in germany-diversity of subtypes and detection of live-vaccine viruses bovine viral diarrhea virus: global status genetic and antigenic characterization of bovine viral diarrhea viruses isolated from cattle in hokkaido genomic and serological diversity of bovine viral diarrhea virus in japan pathogenicity of an indian isolate of bovine viral diarrhea virus b in experimentally infected calves virulent properties of russian bovine viral diarrhea virus strains in experimentally infected calves kinetics of single and dual infection of calves with an asian atypical bovine pestivirus and a highly virulent strain of bovine viral diarrhoea virus impact of variation in acute virulence of bvdv strains on design of better vaccine efficacy challenge models a bovine viral diarrhea virus type a strain in china: isolation, identification, and experimental infection in calves epidemiological features and economical importance of bovine virus diarrhoea virus (bvdv) infections lack of virus transmission from bovine viral diarrhoea virus infected calves to susceptible peers failure to spread bovine virus diarrhoea virus infection from primarily infected calves despite concurrent infection with bovine coronavirus infectivity of pestivirus following persistence of acute infection virulence comparison and quantification of horizontal bovine viral diarrhoea virus transmission following experimental infection in calves distinction between persistent and transient infection in a bovine viral diarrhoea (bvd) control programme: appropriate interpretation of real-time rt-pcr and antigen-elisa test results effects of exposure to bovine viral diarrhoea virus on risk of bovine respiratory disease in australian feedlot cattle the risk of transmission from sheep experimentally infected with bvdv- c during the acute phase to bvdv naïve sheep viral dose and immunosuppression modulate the progression of acute bvdv- infection in calves: evidence of long term persistence after intra-nasal infection practical significance of heterogeneity among bvdv strains: impact of biotype and genotype on u.s. control programs bovine viral diarrhoea virus (bvdv) subgenotypes in diagnostic laboratory accessions: distribution of bvdv a, b, and a subgenotypes characteristics in the epidemiology of bovine viral diarrhea virus (bvdv) of relevance to control level and duration of serum antibodies in cattle infected experimentally and naturally with bovine virus diarrhoea virus real-time polymerase chain reaction as a rapid and efficient alternative to estimation of picornavirus titers by tissue culture infectious dose % or plaque forming units quantitative pcr: a quality control assay for estimation of viable virus content in live attenuated goat pox vaccine quantification system for the viral dynamics of a highly pathogenic simian/human immunodeficiency virus based on an in vitro experiment and a mathematical model development and validation of a q-pcr based tcid( ) method for human herpesvirus studies of the pathogenesis of bovine pestivirus-induced ovarian dysfunction in superovulated dairy cattle early reproductive loss due to bovine pestivirus infection increased reproductive losses in cattle infected with bovine pestivirus around the time of insemination a field investigation of the effects of bovine viral diarrhea virus infection around the time of insemination on the reproductive performance of cattle a single amino acid is critical for the expression of b-cell epitopes on the helicase domain of the pestivirus ns protein a manual for the primary animal health care worker; food and agriculture organization (fao multiplex real-time rt-pcr detection of three viruses associated with the bovine respiratory disease complex the authors declare no conflict of interest. the funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results". key: cord- -ldkjqco authors: nan title: news date: - - journal: aust vet j doi: . /avj. sha: doc_id: cord_uid: ldkjqco nan r eports of psychological distress, occupational stress and burnout and an increased risk of suicide in the veterinary profession are urgent reminders that veterinary schools, professional organisations and also employers should continue to address these issues. , managerial aspects of the job, long working hours, heavy workload, poor work-life balance, difficult client relations and performing euthanasias have been consistently associated with increased levels of occupational stress. chronic work stress can lead to emotional exhaustion and burnout. female veterinarians, younger veterinarians and those working on their own are at greatest risk of stress and mental health difficulties. , results from a recent observational study conducted at veterinary practices in canada suggest that team effectiveness can improve an individual team member's job satisfaction and offer protection against stress and burnout. the study found that % of veterinary team members were considered to be at a high risk of burnout. the study recommendations are that practice managers: • ensure all team members are kept abreast of changes in the clinic and given opportunities to provide suggestions to improve patient care and client service • recognise team members for their contributions • provide all staff with adequate resources and guidance to complete their jobs in a meaningful fashion and provide opportunities for growth and professional development • ensure all team members have clearly defined roles and are given autonomy to make decisions consistent with their position in the practice • encourage colleagues and supervisors to provide guidance and social support to help team members develop coping skills • take steps to create and maintain a positive work environment -this may include addressing conflicts among co-workers, ensuring all employees are treated equally and fairly, promoting civility and collegiality • consider current staff numbers and individual workloads to prevent excessive workload. providing communication and coping skills training and improving cognitive skills for young professionals may also help to decrease depression, anxiety, stress and burnout. if you are concerned that a colleague, close friend or family member is experiencing mental health difficulties, try talking to them in a supportive manner and encourage them to consult their gp or mental health professional. a -hour telephone counselling service is available for ava members on . this service can also arrange a counsellor to attend veterinary workplaces to help support staff after a traumatic incident. lifeline is another excellent support option: . dogs suffering from osteoarthritis need proven relief from pain and infl ammation. julia nicholls, president a s i write, our policy advisory council (pac) is voting on policies that are under review or newly drafted. so it may be a good time to reflect on why we have these polices and how they are written, as there has also been some debate about the function and process of the pac. ava policies are used as a reference point for the public, the media and members. many of our members sit on external boards and committees and the policies are key documents in these forums. i recently had to respond to a good question as to why our policy on equine dentistry did not completely align with the recommended key principles for veterinary practice acts in australia, which were recently circulated to members (www.ava.com.au/node/ ). the crux of this is that the policy reflects an ideal world, but the veterinary practice acts, which are written for the protection of the public, reflect current reality. we have other policies that do not align with those of key stakeholders or with current legislation. this is healthy and reflects our independence and the democratic process. the ava has approximately policies and position statements. subjects range from live animal export to genetic defects in domestic animals, and from puppy socialisation to cane toad euthanasia. position statements replace policies where there is a significant diversity of opinion among members. many policies reflect our stance on animal welfare issues and this is something to be proud of. the pac consists of a representative from every division and special interest group and meets once a year face-to-face but engages in healthy debate online all year. the full process of policy development may seem long-winded to an outsider. an initial draft is created by a working group and is then discussed by all the councillors until the majority are happy that the policy is suitable to go to all members for comment. members have a chance to comment on draft policies twice each year. the councillors work very hard to gain consensus from the members and to work with each other to get a policy ready for the vote. the october voting round is an electronic vote for policies that have been discussed online or have been out for general member comment. councillors vote whether to move each policy to the next stage in the process, send it back to a working group or abandon it altogether. anything requiring more debate is referred to the face-to-face meeting in may. the final sign off is by the ava board. the structure of the ava's policies is important. they should start with a short statement suitable for a media release or sound bite and then have a section on the underlying philosophy, the evidence and some scientific references. operational matters or procedures linked to the policy are usually included as guidelines. each policy undergoes regular review to maintain currency. some are deleted and others added as the environment changes. the policy compendium is a living document and something that is the envy of other veterinary associations. it is hard to see any shortcuts that would improve the process for member involvement. yet situations occur where we have no policy to fall back on. in the world of the -hour news cycle, topics arise that require an immediate response or at least one faster than the formal policy development process. the changes to higher education funding are a case in point. we do not have a policy on this, but it was clear that the proposed changes pose a threat to the future viability of our profession. we believe that we have to oppose this government policy shift, so we are, hopefully with your support. another example is where we have a policy but it is silent on the particular aspect of the topic under debate. in these circumstances, the board or a consultative group delegated by the board will arrive at an official position in keeping with the spirit of our policies. ideally, a policy will eventually flow from experiences like these. members and board directors have asked a range of questions about our policy development process in recent years: • should there be more scanning and identification of futureproofing policies? pet insurance is an increasingly important element of economic sustainability for many members. it means that clinical veterinarians can provide the best care for their patients and clients, regardless of the cost. unfortunately, there have been a number of teething problems with policies and claims, which led us to form a pet insurance taskforce in . the taskforce's role is to work with the profession and pet insurance companies to identify and then address issues of concern for veterinarians, pet owners and the insurance companies. our ultimate goal is to ensure that pet owners and veterinarians have confidence in pet insurance as a trustworthy and valuable element of pet care. one of the key activities the taskforce has undertaken is to develop a veterinarians guide to pet health insurance. the guide includes: • general information on what pet insurance is (and isn't) • useful facts about the australian market • reasons why more clients don't use pet insurance • techniques for making pet insurance work in your practice and for your clients. it also includes our guidelines for promoting pet insurance, your legal obligations, and what you can and can't say when talking to clients. we've heard from many members who are confused about this area, and the guidelines provide some much needed clarity. a veterinarians guide to pet health insurance is available for download now from the ava website: www.ava.com.au/node/ . on the back of member a survey conducted earlier this year, the taskforce is now gathering more detailed feedback including case studies from members at conferences and other forums. the feedback highlights several areas we believe could be addressed to remove barriers to take-up of pet insurance in australia, including: • the need for a cooling off period so that the pet owner can withdraw from an insurance policy if they are dissatisfied with the exclusions • the need for clearer terms and conditions, and explanations of any exclusions at the time of policy uptake • removing policy exclusions for pre-existing conditions based on entire body systems and unrelated to previous conditions -for example, removal of a toe lump cannot be considered a pre-existing condition just because a dog had a superficial skin infection a year before • making it easier for clients to know if their policy provides cover for particular treatments and diagnostic tests • an improved review of disputed claims by australianregistered veterinarians -we think that an industry review panel of ava members would offer an alternative pathway for appeal to the financial industry ombudsman • moving to electronic submission of claims, and making interactions with the insurers more efficient for veterinarians. we're looking forward to engaging with the insurance companies over these and other issues in the coming weeks. we'll keep you up-to-date with any new developments, and we also plan to develop some more educational resources for veterinarians shortly. with changes to the way the insurance policies and claims process works, we anticipate that more and more pet owners will choose to insure their pets, resulting in more treatment options. it will also mean that veterinarians are working with a system that supports good outcomes for all involved -pets, owners and veterinarians. fun is like life insurance; the older you get, the more it costs. international veterinary, biomedical and business journals at your fingertips vet ed library: www.ava.com.au/library as a vet, you are used to handling your patients with care and understanding, (especially if their bite is worse than their bark). at boq specialist, we adopt the same approach. we've spent nearly years working closely with vets and we've come to know your world as well as we do our own. we know your idiosyncrasies and we can anticipate your needs. so, unlike a conventional bank, we've developed products and services that are carefully designed to meet your business and personal banking requirements. when you call us, you'll always feel you are dealing with one of your own breed. crayfish plague is caused by a water mould, aphanomyces astaci. its native hosts are north american freshwater crayfish and although it produces some minor pathology, does not usually cause death. however, in naïve crayfish (never exposed to the pathogen), the disease causes death and effectively exterminates native crayfish from infected waters in as little as a couple of weeks. historically, north american crayfish were first introduced into europe in the late s for culture and fishery purposes. they carried crayfish plague with them. crayfish plague travelled around europe, with american crayfish that were translocated into european waterways. however, once crayfish plague was introduced to a country, it would also spread rapidly via contaminated fishing gear and water. crayfish plague continues to spread to previously unaffected areas of europe. the effect was severe in areas with a culture of consuming crayfish, particularly scandinavia. national declines in crayfish populations vary from % to % and lakes where crayfish were eliminated became overgrown with aquatic plants. in finland, the estimated cumulative direct loss from crayfish plague over the past years is € million. australia's freshwater crayfish fauna is diverse, with over described species, including a large number that are rare and endangered. crayfish play a crucial role as predators, herbivores and in breaking down detritus in freshwater ecosystems, and are often the largest animals in australian mountain streams. australian crayfish can be split into two broad groups: spiny and smooth-shelled crayfish. spiny crayfish are slow growing, have very large claws and usually prefer clear, cool water. because of their low meat yield and slow growth, they are not farmed. they are important fauna of streams where habitat is suitable, and many have highly restricted distributions, low fecundity and are listed as endangered. the best known of the spiny crayfish is the murray river cray (euastacus armatus). smooth-shelled crayfish include commercially and recreationally important species such as marron (cherax cainii), redclaw crayfish (cherax quadricarinatus), and the ubiquitous 'yabby' (cherax spp.). marron, redclaw and yabbies are farmed in queensland, new south wales, south australia and western australia. marron are primarily farmed in western australia and south australia. annual production varies, but in - it was valued at a$ . million and a$ , , respectively. redclaw production in queensland has declined over the past years and is now valued at a$ , . yabby production for food is limited, mainly from farms in western australia ($ , ) and new south wales ($ , ). redclaw and yabbies have been translocated (legally and illegally) for recreational fishing. many species have substantial cultural importance, and have been a source of food for indigenous people for millennia. crayfish are 'keystone species' in aquatic environments, acting as major processors of organic materials, facilitating the release of energy and nutrients, turning over substrates and aerating soil, and can reach very high biomasses in some systems. some smooth-shelled crayfish are remarkably persistent in dry environments and can survive drought in deep burrows for extended periods. aphanomyces astaci spreads by means of motile zoospores released from mature filaments in infected crayfish. the zoospores are attracted to crayfish cuticle, and the filaments penetrate immediately. zoospores can remain motile for up to days and cysts can survive for weeks (in distilled water). zoospores can re-encyst three times if they do not encounter a host. it is recommended to wait months before attempting to re-stock waters in which crayfish have been killed by crayfish plague, to allow all zoospores to die out. crayfish plague is also spread via contaminated fishing equipment or zoospores in water. american crayfish, including red swamp crawfish (procambarus clarkii) and signal crayfish (pacifastacus leniusculus) are tolerant of infection. they can remain carriers for life, and may exhibit little or no sign of infection. until recently, crayfish plague was only known in temperate to cool climates, but a spanish strain is active at temperatures of - °c. the strain that affects the invasive red swamp crawfish can sporulate at temperatures up to . °c. red swamp crawfish have established in japan, china and taiwan, and populations are sometimes maintained in ornamental fish outlets in the region. thus there is potential for spread of the pathogen throughout the region, either in contaminated water or with live or dead crayfish. freshwater crabs are susceptible to crayfish plague and may act as reservoirs for the pathogen. the chinese mitten crab (eriocheir sinensis) and the european crab (potamon potamios) are both susceptible to infection with crayfish plague. the chinese mitten crab is listed as one of the most invasive species on the planet and has established successfully in europe and north america. australia has very rich and diverse freshwater crab fauna. if infected chinese mitten crabs established in australia, their extensive migration habits (migrating upstream from estuary spawning grounds) could spread crayfish plague inland to crayfish and crab populations. australian crayfish are known to be highly susceptible to crayfish plague. eight species of four genera (including yabbies, cherax destructor) of australian crayfish were experimentally exposed to zoospores of a. astaci. they showed limited or little effective host response to invading filaments. the usual response of crayfish to infection is encapsulation and melanisation of invading filaments. redclaw crayfish and marron have been introduced into many countries for farming purposes. in late , crayfish plague was detected in farmed redclaw crayfish in taiwan. it was detected in five widely spread locations, four of which experienced % mortality, while one experienced % mortality and % morbidity. pathology was not described in the report. as demonstrated by its ability to infect multiple crayfish species, chinese mitten crabs and european crabs, crayfish plague is not very species-specific and could potentially infect most or all australian freshwater crayfish and crab species. it can cause disease and death in crabs, although the effects are not as immediate or dramatic as in crayfish. often the first sign of crayfish plague is the sudden mass death of crayfish in a water body. however, some symptoms do appear, including behavioural changes and external signs. affected crayfish may become active in the day and appear uncoordinated and lethargic. whitish areas in the muscle may be visible through the cuticle under the tail. at very low temperatures (< °c) effects may be slower to emerge, and the disease may be chronic rather than lethal in susceptible species. when american crayfish species (i.e. tolerant of infection but carry it) become infected, the shell may develop dark patches, as the reaction by crayfish to damage is to lay down melanin around the pathogen. these patches are easiest to see on the underside along the abdomen. the identification field guide to aquatic animal diseases of significance to australia provides details of clinical signs and diagnosis for crayfish plague. the aquavetplan disease strategy manual for crayfish plague provides details on gross and clinical signs of the disease. crayfish plague should be differentiated from other possible causes of mass death, such as insecticide poisoning. pcr techniques for detection of the pathogen have been developed. there is an australian and new zealand standard diagnostic procedure detailing methods for diagnosing crayfish plague. the oie manual of diagnostic tests for aquatic animals also provides details on diagnostics to confirm the presence of the pathogen. australian freshwater crayfish are known to be susceptible to crayfish plague fungus. freshwater crayfish are important and dominate in many australian ecosystems, and many species are rare or threatened. many species have restricted distributions, so could become extinct very rapidly if exposed to the disease. crayfish are the basis of an aquaculture industry, as well as supporting recreational and cultural fisheries. the potential consequences of entry of this disease into australia could be severe. the outbreak in taiwan is the first reported case of australian native crayfish being naturally infected by a. astaci and demonstrates the high susceptibility of this tropical crayfish to the pathogen. it caused very high mortalities, suggesting that a similar outbreak in australia could have devastating consequences. some strains of crayfish plague fungus are temperate but others transmit in temperatures up to . °c, suggesting that crayfish plague could infect large areas of australia. american crayfish are widespread in asia. they are regarded as pests in some countries, and until recently crayfish plague had not been reported from asia. the report from taiwan indicates that plague is present and could, potentially, be widespread in the region in feral populations of north american crayfish or chinese mitten crabs. australia has import conditions to restrict the entry of freshwater crayfish or crabs for both environmental and biosecurity reasons. these conditions limit the risk of entry of crayfish plague into australia. however, vigilance for diseases should always be maintained. if an exotic disease is suspected, please call the disease watch hotline for advice and assistance. brett herbert australian government department of agriculture comment on this article at www.ava.com.au/ r abies is present in more than countries across the world, causing more than , human deaths each year, almost all of them in africa and asia and many of them young children. although rabies can infect all warm-blooded animals, the most important source of human infection around the world is an infected dog bite or scratch. australia is one of the few countries that claims freedom from the disease, but as the rabies virus spreads in nearby countries, it is becoming more likely that it will eventually reach our shores. honorary associate professor at the university of sydney, dr helen scott-orr, has been involved in rabies programs in flores, as well as early response and control programs when rabies was first diagnosed in bali in . "rabies is a terrible and terrifying disease when it occurs. it exists in canine populations on several indonesian islands close to our northern shores. in recent years there have been outbreaks on eastern islands, including bali, flores, ambon and the tanimbar islands, which have increased the risk of the disease entering australia. "if it were to reach our border, the most likely place for an incursion is northern australia and the most likely scenario is an illegally imported, infected animal arriving by boat, " dr scott-orr said. dr ted donelan is president of animal management in rural and remote indigenous communities (amrric), which delivers animal health programs in rural and remote aboriginal and torres strait islander communities. dr donelan says that people in isolated indigenous communities are most at threat from a rabies outbreak. "large portions of the northern australian coastline are very sparsely populated. there are significant populations of semi-free-ranging camp dogs, dingos and other wild dogs in and around these communities. if these dogs became infected, they would pose a huge threat to the health and safety of the aboriginal and torres strait islander people living in those areas, " dr donelan said. the australian government has been active in rabies preparedness and prevention. the minister for agriculture, barnaby joyce, recently said, "the work offshore, at our borders and on shore -including the department's engagement with indigenous communities in northern australia as part of the northern australia quarantine strategy (naqs) -is vitally important to ensuring australia maintains its freedom from rabies and other exotic diseases. " since , the department of agriculture has been working in partnership with the indonesian ministry of agriculture to improve the management of emerging infectious diseases. the australia indonesia partnership for emerging infectious diseases is funded by the australian government, with a$ million allocated to developing a more integrated veterinary service capable of preventing, detecting and controlling important endemic and emerging infectious diseases. dr scott-orr and dr donelan agree that the naqs is doing a great deal of work on rabies surveillance, education and preparedness to prevent its introduction to australia. they also believe that australia's ongoing work with neighbouring countries is playing an important part in our protection from the deadly disease. "spending resources on helping our neighbours control rabies helps reduce the risk to australia. it also helps build the pool of professionals with experience in fighting an outbreak, " dr donelan said. preventing the spread of rabies is best achieved through vaccination. dr scott-orr says the most universal knee-jerk reaction from authorities is to kill dogs, but this has proven to be ineffective in containing the virus. "in new incursions, it often spreads insidiously for several months in the dog population before one or more human deaths occur, causing panic. the way to control rabies is to vaccinate dogs. the target for control is at least % of dogs vaccinated every year with a vaccine that has a -year duration of immunity. in addition to this, preventing rapid turnover of the dog population by fertility control is strongly advisable, " dr scott-orr said. • rabies is a vaccine-preventable viral disease that occurs in more than countries and territories. • infection causes tens of thousands of deaths every year, mostly in asia and africa. • % of people who are bitten by suspect rabid animals are children under years of age. • dogs are the source of the vast majority of human rabies deaths. • immediate wound cleansing and immunisation within a few hours after contact with a suspect rabid animal can prevent the onset of rabies and death. • every year, more than million people worldwide receive a post-exposure vaccination to prevent the disease -this is estimated to prevent hundreds of thousands of rabies deaths annually. within australia, dr donelan is encouraging veterinary practitioners who regularly visit remote communities to provide dog health programs to be more proactive in rabies preparedness. "relationships are everything in working with indigenous communities and people who are known, trusted and respected by the locals are far more likely to be given information about sick dogs and gain their cooperation with control measures and vaccination programs, than are outsiders. there is often a healthy mistrust of authority, stemming from experience that usually the prime goal of dog 'management' is to kill the dogs, " dr donelan said. both dr scott-orr and dr donelan advise veterinarians to consider getting vaccinated against rabies for personal protection and to enable them to assist in an outbreak if one was to occur. amrric is currently establishing a register of rabies-vaccinated practitioner members currently servicing or prepared to volunteer in northern australia. "the prevalence of the closely related australian bat lyssavirus (ablv) in both flying foxes and some insectivorous bats, with three human and two equine fatalities recorded, presents another reason for australian vets to consider being vaccinated against rabies. ablv has been found in all states except tasmania and the act, " dr scott-orr said. the media space is becoming more competitive, with more pressure on fewer journalists to file stories in a shorter space of time and when big stories overtake the news, such as the recent terrorist threat, these pressures intensify. total times the ava and its spokespeople feature in the media each year. ava news is not only covered in print, radio and television but increasingly online as well. in fact, between january and september this year, % of our media coverage appeared in online news sites. this reflects people's demand for immediate, up-to-date news and growing preferences to receive information from online sources. you can view some of the online news coverage we've received on the ava website at www.ava.com.au/news- . the ava media program involves identifying and telling positive stories to raise the ava's profile and promote the veterinary profession. the topics we cover include topical policies, response to disease outbreaks and the advocacy work the ava does to support our five strategic priorities. an example of coverage from our advocacy work is the higher education reforms campaign where media coverage has supported our efforts to communicate our position to decision makers in person. significant media coverage was received on this issue. julia nicholls conducted several interviews about the impact the reforms would have on veterinary students and the profession. the story was covered in the sun herald, the sunday age and the sunday canberra times. flow on coverage included wsfm, ue in sydney, ec bega and pr perth, as well as the rural report on abc online. a successful program of stories throughout the year for many of our special interest groups has helped maintain our media profile. these stories have covered heat waves, tick paralysis, parvovirus, behavioural issues, farm safety, dental health, distemper, heartworm, parvovirus, horse heart murmurs, hendra virus, bvdv, pollen, snake bites and swooping magpies. you can view all the ava media releases on the website at www.ava.com.au/mediareleases. ava members also have regular pet advice columns in the west australian and the adelaide advertiser, and a weekly pet radio segment on nm muswellbrook. although stories focussing on companion animals tend to be popular with the media, we continue to increase our influence in the rural media and promote the great work veterinarians do with producers and livestock.  % pets  % livestock  % equine  % legislation / advocacy  % other media releases have been distributed throughout the year to promote sig and division conferences. the publicity program for the ava annual conference was a great success this year and generated media hits. one of the biggest drawcards was the media conference on antimicrobial resistance, which generated more than media hits alone, including radio national and abc radio stations around australia. a panel of experts, who presented papers on the topic at the conference, spoke at the media conference about the facts on antimicrobial resistance in animals and the potential impact on people. although the situation in australia is better than in many other parts of the world, the take home message was that we can't be complacent. australia needs a long-term national surveillance program of antibiotic resistance before any problems get out of hand. you can view some of the coverage at yahoo news (aap) and abc rural. this year's asava conference resulted in a tv news piece on nbn and an interview with david neck on abc brisbane. several media releases to support the world buiatrics congress delivered media impressions in queensland country life, abc rural, the veterinarian and southern cross radio among others. every year the ava conducts media training sessions using professional media trainers to ensure we have a pool of spokespeople to cover every division, sig and potential topic of media interest. we have been very fortunate with the calibre of ava volunteer spokespeople over the years and they have been instrumental in helping us to achieve such excellent exposure for the profession and the ava in the media. comment on this article at www.ava.com.au/ protection against hendra at equestrian events e quine veterinarians australia (eva) has recently advised members on the best-practice approach to managing hendra risk at equestrian events. "managing hendra biosecurity risk at events is extremely challenging due to the nature of the disease and the large number of horses and people intensively interacting, " said nathan anthony, president of eva. "event organisers have a responsibility to ensure a safe environment for horses and people. however, not all event organisers are willing or able to implement and adhere to hendra biosecurity essentials during an equestrian event, " dr anthony said. veterinarians providing services at equestrian events have obligations in relation to work health and safety, and they are expected to provide accurate advice on biosecurity threats. eva has provided recommendations and resources to help veterinarians meet these obligations. "for events in queensland and new south wales, eva advises organisers to implement a mandatory hendra vaccination requirement for all event participants. this is to protect both people and horses from the extremely serious effects of an outbreak at an event, " dr anthony said. "for events in other states and territories, we advise event organisers to require horses travelling from queensland or new south wales to be vaccinated against hendra virus. "eva has developed resources to help vets communicate this advice and to ensure that event organisers acknowledge their responsibility to manage the risk of a hendra outbreak, " he said. a ntimicrobial resistance threatens human and animal health worldwide. antibiotic awareness week australia ( - november) forms part of a global campaign to increase awareness of antimicrobial resistance and to promote responsible use of antimicrobials. the veterinary profession is involved this year through the ava's strategic priority program 'fighting antimicrobial resistance' . the veterinary profession has adopted the one health concept of expanding interdisciplinary collaboration and communications in all aspects of health care for humans and animals. veterinarians are seen by other health professions and the public as key players in the responsible use of antimicrobials, because of our stewardship over a considerable amount of antimicrobial use in companion and food animal species. participating in antibiotic awareness week alongside human health professionals is a key way for veterinarians to demonstrate their commitment to working with others to help solve this global crisis. the focus on antimicrobial resistance and use in humans and animals is increasing around the world, and both the world health organization and world organisation for animal health (oie) are leading policy to extend the useful life of antimicrobials. here in australia, the departments of agriculture and health are developing a national strategy on antimicrobial resistance similar to canada, the united states and the united kingdom. resistance to antibiotics is found in australian hospitals and increasingly in the community. multidrug-resistant bacterial pathogens are becoming more prevalent. patients with resistant infections experience delayed recovery and treatment failure and are more likely to die than patients with non-resistant infections. the profession and the ava have been proactive in addressing antimicrobial resistance concerns, naming 'fighting antimicrobial resistance' as one of the ava's five strategic priorities. recent new resources have included simple one-page guidelines on the principles of responsible prescribing of antimicrobials by veterinarians and a client fact sheet on safe handling of animals being treated with antibiotics. the ava is also currently exploring options to develop new national guidelines to help veterinarians ensure they're making the best decisions when prescribing antibiotics. the australian veterinary journal has recently published several articles on antimicrobial resistance and its effect on our patients and ourselves, including articles on methicillin-resistant staphylococcus aureus (mrsa) in horses, vet hospitals and its carriage by veterinarians. australia is in an enviable position with regard to antimicrobial resistance -we have never allowed the use of fluoroquinolones in food-producing animals, and a recent survey by meat and livestock australia showed low levels of resistance in australian beef. over time, the one health focus on antimicrobial resistance will increase and it's important that veterinarians are proactively involved and informed of their responsibilities when using antimicrobials in all areas of practice, both companion and food animals. in the future, veterinarians may well be more involved in human cases of antimicrobial resistance. for example, family pets may be identified as reservoirs of infection. veterinarians may have to make decisions on whether to use the newest 'big gun' antimicrobial or more conservative treatment. off-label prescribing and compounding practices in food-producing animals may also come under increasing scrutiny. these challenges will provide an opportunity to underscore the importance of veterinary science to the overall health of both humans and animals. veterinarians are not unique in facing this challenge. more than health professionals have already taken a pledge to support the mind me principles: microbiology guides therapy wherever possible indications should be evidence based narrowest spectrum required dosage appropriate to the site and type of infection minimise duration of therapy ensure monotherapy in most cases. indeed, clients may come to expect this approach from all health professions, veterinarians included. jonathan taylor amrric is an independent group of veterinarians, academics and health professionals, both indigenous and non-indigenous, working in the one health framework to improve the health and well-being of companion animals and their communities. since its inception, amrric has focused on developing and implementing sustainable, culturally-sensitive programs in rural and remote aboriginal and torres strait islander communities. this program has seen dozens of veterinarians, nurses and other volunteers from around australia and overseas donate their time and skills in desexing, worming and treating animals in some of the country's most remote communities. the conference, held in darwin in conjunction with the international fund for animal welfare (ifaw), attracted over delegates from australia and around the world. following a welcome to country by larrakia elder bilawara lee, amrric president ted donelan and ifaw regional director, isabel mccrea, opened proceedings. keynote speakers included kate nattrass atema, ifaw's program director companion animals, who discussed the logistics of animal welfare programs in countries ranging from bosnia to bali. dr frank ascione, from the university of denver, has performed extensive research on the link between animal abuse and interpersonal violence. he discussed the relationship between mental health and animal abuse, as well as animal abuse as an indicator of family violence. he also discussed initiatives that had been developed as a result of this research, including pet-friendly shelters for victims of domestic violence. with the threat of a rabies incursion into northern australia presenting a real risk to remote communities, the topic of rabies detection and control was a key theme of the conference. a panel of experts, including nt chief veterinary officer dr malcolm anderson, nt centre for disease control public health physician dr charles douglas, northern australia quarantine strategy veterinary officer joe schmidt and former nsw chief veterinary officer helen scott-orr, participated in a simulated rabies incursion to assess australia's preparedness for such an outbreak. issues raised included the number of vaccinated veterinarians available to assist in such an event, the need for existing relationships with and experience with communities, and understanding of dog population dynamics in remote communities. above all, the conference provided an opportunity for those working at the coalface to connect with others in academia and government and ensure that community animal health and welfare are on the agenda. "what i have learnt there was very motivating and made me see my community from another perspective, " said torres strait island regional council animal management worker, william bero. "it has motivated me to continue in promoting healthy and happy animals equals healthy and safe community. " the study reaffirms much of what we already know, but it is an australian study -which is important, as we don't have much local data, " said dr andrew carter, ava sa division president. "interestingly, the study also noted that two-thirds of incidents involved the dog being provoked. this is significant, as it again points to the need for children to be taught about appropriate interaction with pets, but also highlights the need for adults to step in, " dr carter said. in regard to breed, the study finds that breed-specific legislation is not necessarily effective and proposes the development of a set of well-defined criteria that enables early identification of dangerous dogs on an individual basis. "this is very much in line with our own ava research, which emphasises the importance of education coupled with identification and control of individual 'potentially dangerous' dogs along with 'dangerous' dogs, " dr carter said. the results mirror another recent study of dog bite-related fatalities in the united states. that study, released late last year, looked at fatalities from to . although calculated risk factors could not be specified by the study, the key factors that were found to be present in fatal attacks were: • absence of someone to intervene • the dog and victim were unknown to each other • the dog was not desexed • compromised ability of victims to interact appropriately with dogs • the dogs were isolated from regular positive human interactions (possibly kept outside) • owners' prior mismanagement of dogs • owners' history of abuse or neglect of dogs. importantly, in the majority of fatalities at least four of these factors were in place. the study reinforced other findings relating to breed, stating that dog bite-related fatalities "were characterized by coincident, preventable factors; breed was not one of these. " the paper also notes that although desexing appeared to be a factor, it is uncertain whether this is causal or coincidental. they note that past research "…suggests that owner failure to have their dog spayed or castrated may co-occur with other factors that more directly influence a dog's social competence. " executive officer -sa and nt division comment on this article at www.ava.com.au/ the results support the existing understanding of dog bite incidents: dog bites are most likely to involve children aged - years, who are bitten on the face by a familiar dog and in a familiar environment. esutures is a discount distributor of ethicon, covidien, synthes, bard and arthrex suture, mesh and surgical devices. we specialize in selling brand name products at below market prices in quantities you decide. we stock thousands of surgical devices available by the box or by the individual item, and ready to ship today! no contracts. no minimum orders. fast shipping. all orders ship global priority from the u.s. at a flat rate. order today: info@esutures.com www.esutures.com use promo code: avj for $ off your next order of $ or more.* a ccording to the food and agriculture organisation of the united nations (fao), a recent strain of avian influenza virus in poultry in southeast asia needs to be closely monitored. known as a(h n ), the fao says it represents a new threat to animal health, with recent detection in poultry in the lao pdr and vietnam after first being reported in poultry in china in april . it also represents a threat to the poultry-related livelihoods that contribute to the incomes of hundreds of millions of people in the region. fao's chief veterinary officer, dr juan lubroth, said that influenza viruses are constantly mixing and recombining to form new threats. "h n is particularly worrisome as it's been detected in several places so far from one another. and because it's so highly pathogenic, meaning infected poultry become sick and, within hours, death rates are very high, " dr lubroth said. the world health organisation for animal health (oie), which works together with the fao and the world health organization (who) to support countries' responses to animal and human disease threats, is also monitoring the situation closely. only one case of h n has been reported in humans after contact with exposure to poultry shortly after its detection in china. the person later died. according to the who, although the dynamics of the new strain are still not fully understood, it's unlikely that h n represents an immediate and significant threat to human health. both the who and fao have advised that even if the public health risks posed by h n currently appears to be low, it is still of concern and they recommend that consumers follow appropriate hygiene, food preparation and food safety guidelines. these include washing hands often, cleaning utensils and surfaces used during food preparation and eating only well-cooked poultry meat products. people should also avoid handling sick birds or those that have died of illness. the fao is also urging countries to remain vigilant to prevent further spread of the virus and is recommending that governments in the area support poultry producers in following essential biosecurity measures and standard hygiene precautions. this includes early detection, immediate reporting and rapid response. comment on this article at www.ava.com.au/ d r russell dickens oam is a trailblazer and mentor to countless young and now a bit older veterinarians. dr robert johnson suggested we shine the avj member spotlight on his mentor, dr dickens, so we can share his story with ava members. dr dickens started a veterinary practice in western sydney in and has been protecting australian wildlife and caring for animals, farmers and pet owners ever since. he has seen western sydney transition from a rural, farming community to a burgeoning, densely-populated metropolis and has evolved his practice to meet the changing needs of his community. beyond his practice and research work, dr dickens is a primary producer of angus beef at cullen bullen and has also placed his considerable energy into community activities. he has been a councillor at blacktown council for over years, served as mayor and is currently deputy mayor, while still working at his practice. he is a foundation member of the animal ethics committee at westmead hospital and millennium institute, a rotary paul harris fellow, on the salvation army's advisory board and this year was lauded with the university of sydney's alumni award, which places him among some of the sharpest minds in australia. dr johnson recounted a recent saturday afternoon, when he thought he'd done a stellar day's work after a full week. he finished by pm and thought he'd pop in to visit dr dickens' practice. "i found a packed waiting room with russ methodically managing the clients by himself. when he saw the look on my face, he smiled and said, 'i'm also acting mayor today' , " dr johnson recalled. he also still -almost exclusively -runs his practice's out-of-hours service and has done so since the s. "age is a matter of mind. if you don't mind it doesn't matter, " he quipped. when asked to recall a time he had shared with his mentor that had a particular effect on him, robert chose to share the aftermath of being sent to help a friesian cow with a dislocated hip, some years ago. "russ sent me out to attend to a large heifer and i managed to sedate her, get her to lie down and with the farmer's help and some impressive force, to reduce the dislocation. triumphant, i headed back to the practice where russ looked at me with some surprise. i asked, "why the funny look?" russ said that he had expected me to be covered from head to toe in mud and to provide him with his entertainment for the afternoon. like most other days, we laughed. "russ has taught me the two most important things in my career: that it's fun to be a veterinarian and you should always be prepared to laugh at yourself, " dr johnson said. e ach year the ava recognises those who contribute to and serve the veterinary profession or the association. nominations are now open for the following prizes and awards: this is the ava's most prestigious award for outstanding service by a member or a non-member to veterinary science in australia. nominees who were unsuccessful from the previous two years are automatically included. issued jointly with the australian college of veterinary scientists, the kesteven medal is awarded to members for distinguished contributions to international veterinary science through technical and scientific assistance to developing countries. nominees who were unsuccessful from the previous two years are automatically included. awarded to members for outstanding service to the association. the award is given either to members who have rendered meritorious service to the association, regional divisions, branches or special interest groups, or to persons who are not eligible for membership of the association but have clearly provided meritorious service to those bodies. awarded to eminent non-veterinarians for services to the ava or the veterinary profession. animal health australia, the australian veterinary association and guild insurance have put together a unique framework to cover private practising vets like you to assist in an emergency animal disease response. no other insurer offers this unique cover. for leading veterinary business insurance and professional indemnity insurance call us today for a quote. insurance issued by guild insurance ltd (gil) abn , afsl and subject to terms, conditions and exclusions. gil supports your association through the payment of referral fees. gil will assess an eadr outbreak and make a decision to provide cover on a case by case basis. gil will need to be contacted for the cover to be activated. additional premiums may apply depending on existing cover with gil. for information on the guild veterinary business insurance policy, refer to the product disclosure statement (pds) and policy wording. you can get a copy of the pds by calling . gld avj eadr ad / while you're taking care of an emergency, who's taking care of you? make the right choice. freecall guildinsurance.com.au/eadr d r alex rosenwax from waterloo in sydney, reports that he has had five unrelated unusual cases of rabbit deaths over the past few weeks. all presented with gastrointestinal stasis and were febrile with anorexia. the signs appeared to be consistent with calicivirus and all died within - hours, despite intensive treatment. all came from one general area of sydney, and they had been vaccinated in the past. "i have contacted the dpi and they have confirmed that a new strain of calicivirus has been documented in sydney by elizabeth macarthur agricultural institute (emai) in the past year. that strain is resistant to the vaccine. their signs were similar to the ones in the rabbits we have seen, " said dr rosenwax. rabbit haemorrhagic disease virus (rhdv) is a calicivirus that usually kills % of susceptible adult rabbits within hours, but the molecular mechanisms for this virulence are unknown. it has been used in australia as a biological control agent to reduce rabbit numbers since it was released in . there is also an endemic non-pathogenic australian rabbit calicivirus, rcv-a , that is known to provide some cross-protection to lethal infection with rhdv, and pet rabbits are usually vaccinated against the endemic strain to protect them. australia's chief veterinary officer, dr mark schipp, notified oie of the new strain in january this year. the notification related to the outbreak in sydney where sudden deaths occurred in show rabbits of various ages and both sexes. there were very few clinical signs prior to death. the rabbits had previously been in good health and were vaccinated against the endemic strain of rabbit calicivirus. of the susceptible rabbits, there were cases, and deaths, equating to an apparent morbidity rate of . %, mortality rate of . %, and case fatality rate of . %. gross necropsy findings showed little signs of the heavy haemorrhage usually seen with rhdv. they had more cranial changes. emai reported that the normal elisa test for calicivirus was negative whereas pcr was positive. some of the rabbits were presented to dr rosenwax because they were apparently having seizures. he noted that although his cases were highly suspicious of calicivirus, as the appropriate samples had not been taken, the cases could not be confirmed. a nonformalin-fixed, frozen -g liver sample is required for submission to emai to confirm the presence of the virus. tracing and surveillance of the new strain is underway to determine how many rabbits have been exposed and how widespread the virus is. this may have implications for pet rabbits, as evidence suggests the current vaccine may not be effective against this new strain. this new strain also has implications for rabbit control. the invasive animal cooperative research centre (ia crc) research shows that australian native vegetation is very sensitive to rabbit damage, and as few as . rabbits per hectare can remove all seedlings of the more palatable native trees and shrubs, so delaying natural regeneration. rabbits are australian agriculture's most costly pest animal with the annual cost of over $ million. according to the ia crc, the use of myxomatosis and calicivirus is still limiting rabbit numbers and without them, the annual cost to agriculture from the imported pest would exceed $ billion. however, they report that rabbit numbers are increasing, with research also showing there are a number of rabbit colonies that are immune to the calicivirus. without this virus, other control techniques would need to be tried, including warren ripping, fumigating, shooting and baiting. the ia crc is now involved in studies to evaluate other rhdv strains, mainly because there is increasing genetic resistance in the rabbits to current rhdv strains and young rabbits are acquiring immunity. dr rosenwax reports that in the absence of a definitive diagnosis, his practice has instituted quarantine for all rabbits with suspicious clinical signs. we heard about the contrasting aspects of rural practice in three different areas of australia. all seemed to share similar problems of beef and dairy profitability, increased numbers of graduating veterinarians, competition from paraprofessionals, the cost of travel and the loss of populations from rural towns. david petersen from deniliquin, told us about his practice in the dairy area of the riverina in victoria. there is no water except for irrigation -the gates on the channels are opened at the top levels, and the water 'wooshes' down for about hours per day. they see american holstein cattle, which are big cows with lots of milk, that have poor reproductive function, in an area where daytime temperatures are regularly between and ºc, and ºc at am. the veterinarians keep temperature logs in their cars, and have to throw away medications if they get too hot. sam mcmahon told us of her experiences in setting up the northern territory veterinary services in katherine. with an area of . million km , slightly bigger than nz, uk, ireland and france combined, but with a population of only , , they need a fleet of wd to cover the area. their practice includes going out to stations and indigenous communities and their new branch in alice springs is a . hour road trip away from their main centre. brahman cattle are most common in the humid katherine, and british cattle breeds in the drier alice springs area. comment on this article at www.ava.com.au/ workshop host bill tranter's practice covers km to the north, south and west, with two small animal and equine practices in mareeba. they cover extensive beef in the west of the region as well as herd health management for the more intensive dairy industry. the practice is an integral part of clinical training at james cook university. our first visit was to a relatively small feedlot, with heat-tolerant brahman-cross cattle. the property has . metres of rain annually, and also produces silage. the next stop was at a dairy of between and holstein cows. the tropical grasses grow very slowly except for a flush when it rains in december. they have a year-round calving pattern, and have to produce the same amount of milk throughout the year. their best conception rates are in the cooler winter months, and they also produce silage and grow ryegrass to boost production at that time. bull and heifer reproductive examination was next, with andrew hoare demonstrating the ultrasound probe and showing us ovarian anatomy, and enoch bergman demonstrating the intimacies of bull examination. andrew hoare multitasking i wasn't the only one taking photos communicationthe essential ingredient w ith the increasing amount of technology changing the way we communicate, it can be difficult to find the best medium to engage with all of your stakeholders. despite all these advancements, in particular the role of the internet, text messaging and social media in our lives, there is still no replacement for verbal communication in a work environment. it is verbal communication that is the essential ingredient in building an efficient and harmonious workplace. the key to an efficient workplace is to create an environment that is conducive to open and honest communication. this creates a positive relationship between employee and employer and while this relationship remains positive, then the workplace will usually stay productive. most issues with employees can be resolved through a face-to-face discussion where both parties are able to voice concerns, resolve misunderstandings and get a clear understanding of each party's motivations and the solutions required to overcome any grievances. communication is a two-way process. it involves both the dissemination of information, but even more importantly, the receipt of information through genuine listening. both are equally important for the employee and the employer. firstly, managers need to explain to employees what is expected of them and not simply rely on job descriptions to explain this. employees also have an obligation to ask questions if they are unsure about work requirements and advise managers of any changes that may affect the way they perform the job, or improve the process to deliver expected outcomes. it has often been said that the employment relationship is similar to a marriage. both parties enter into the relationship with the best of intentions, but throughout the relationship there will be good and bad times, which put the relationship to the test. once communication becomes difficult, or either party looks for ways to avoid verbal communication, it is a strong sign that there is a problem. the only way to resolve these problems is to address them as soon as the issue is identified and do this face-to-face. just because an employee chooses to communicate via text, it is never a good idea for a manager to start texting anything more than confirming receipt of the message. it is always best to make a phone call and talk to the employee directly. there can never be too much communication. managers do not have to wait for that annual review, or monthly meeting, to raise a concern or discuss a matter with their employees on any matter. frequent communication on any matter will assist in building relationships. communication must be kept simple and relevant with employees -don't start communicating information that does not affect the employee. importantly, effective communication does not mean gossiping about an employee's personal life; it simply means communicating what is expected of employees and issues that might be affecting their performance and the effect on other stakeholders. in summary, there are three things that are critical to build an efficient workplace ... communication, communication, communication. if you do this well, you will reduce and avoid most workplace relations issues. michelle eamer ava hr advisory service comment on this article at www.ava.com.au/ the material contained in this article is general comment and is not intended as advice on any particular matter. no reader should act or fail to act on the basis of any material contained herein. the material contained in this publication should not be relied on as a substitute for legal or professional advice on any particular matter. suicidal behaviour and psychosocial problems in veterinary surgeons: a systematic review veterinary surgeons and suicide: a structured review of possible influences on increased risk the role of veterinary team effectiveness in job satisfaction and burnout in companion animal veterinary clinics references . nobanis. invasive alien species fact sheet: -aphanomyces astaci. www.nobanis.org the effects of crayfish plague on finland's crayfish economy australian fisheries statistics manual of diagnostic tests for aquatic animals cultured aquatic species information programme: procambarus clarkii (girard, ) the crayfish plague pathogen can infect freshwater-inhabiting crabs defence reactions in and susceptibility of australian and new guinean freshwater crayfish to european-crayfish-plague fungus event summary: crayfish plague (aphanomyces astaci) australian government department of agriculture, fisheries and forestry. identification field guide to aquatic animal diseases of significance to australia. th edn. www.daff.gov.au/animal-plant-health/pests-diseases-weeds/ aquatic-animal-diseases-significant-to-australia-identification-field-guide australian government department of agriculture, fisheries and forestry. aquavetplan disease strategy manual for crayfish plague world organisation for animal health. oie manual of diagnostic tests for aquatic animals. www.oie.int/international-standard-setting/aquaticmanual/access-online/ accessed conservation of freshwater crayfish in australia australian commission on safety and quality of health care. antibiotic awareness week australian veterinary association. fighting antimicrobial resistance international organisation for animal health, oie. managing antimicrobials use in animals emergence of multi-resistant pseudomonas aeruginosa in a western australian hospital methicillin-resistant staphylococcus aureus in a population of horses in australia methicillin-resistant staphylococcus aureus: an issue for veterinary hospitals carriage of methicillin-resistant staphylococcus aureus by veterinarians in australia antimicrobials and the cattle industry methicillin-resistant staphylococcus aureus in a family and its pet cat nps medicinewise: resistance fighter pledge first-time study of women's and children's hospital bite victims shows that nine in attacks is by a familiar dog retrospective review of dog bite injuries in children presenting to a south australian tertiary children's hospital emergency department policy and model legislative framework co-occurrence of potentially preventable factors in dog bite-related fatalities in the united states rabbit haemorrhagic disease invasive animals cooperative research centre. landscape control -rabbits prices include delivery of print journals to the recipient's address. delivery terms are delivered at place (dap); the recipient is responsible for paying any import duty or taxes. title to all issues transfers fob our shipping point, freight prepaid. we will endeavour to fulfil claims for missing or damaged copies within six months of 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other kinds of copying such as copying for general distribution, for advertising or promotional purposes, for creating new collective works or for resale. special requests should be addressed to permissionsuk@wiley.com disclaimer the publisher, the australian veterinary association and editors cannot be held responsible for errors or any consequences arising from the use of information contained in this journal; the views and opinions expressed do not necessarily reflect those of the publisher, the australian veterinary association and editors, neither does the publication of advertisements constitute any endorsement by the publisher, the australian veterinary association and editors of the products advertised. submission of photographs for publication will be held to imply that permission for publication has been obtained from the photographer and from the subject(s) of the image.avj.pi.feb trademarks ava and the ava logo are registered trademarks of the australian veterinary association limited. key: cord- -cdt wcr authors: van der logt, j. t. m. title: microbiological effects and quality control in laboratory rodents date: - - journal: aging clin exp res doi: . /bf sha: doc_id: cord_uid: cdt wcr numerous viruses, mycoplasmas, bacteria and parasites have been associated with infectious diseases in laboratory animals. it is clear that pathogenic agents causing overt disease represent a serious hazard to research results in both short- as well as long-term studies. however, these organisms may contaminate colonies without causing any clinical or pathological symptom. this makes research less reliable because of the more subtle effects of the silent infections, especially in long-term studies as in aging research. the establishment of animal colonies that were free from these (micro-) organisms has increased substantially the value of animals used in biomedical research. characterization of the health status and microbiological monitoring of the animals in experiments are particularly important. this paper reviews many of the major considerations in the efforts to maintain animals free of unwanted organisms, including quality and sources of animals, transportation and quarantine, maintenance during experimentation, microbiological characterization and monitoring of animals and environment. (aging clin. exp. res. : – , ) the significance of biomedical research using laboratory animals has increased substantially because of the increased awareness of an-imal producers and investigators of the need to use high-quality animals with standardized microbiological status, under environmentally defined conditions, in their experiments. the data emanating from animal experiments may be affected profoundly by a number of environmental and biological factors. these complicating factors could be categorized as physical, chemical, and microbial. there is a wealth of published information from which we have learned that not only obvious, but also subtle changes in physical and chemical factors can influence the generation and interpretation of experimental results ( , ) . control and standardization of these factors, and environmental monitoring have improved the quality of the animals, as well as the reproducibility and reliability of experiments ( , ) . a range of environmental recommendations for rat and mouse rooms from different countries has been published, and was summarized by clough ( ) . this chapter will deal further with the main microbiological aspects in laboratory animal research, especially in relation to long-term studies with mice and rats. it should be emphasized that these microbiological and environmental aspects are each only one facet of quality assurance programs both for breeding and research facilities. the establishment and management of a full quality assurance program for rodent health surveillance have been covered in a recent review ( ) to which the reader is directed_ it is common knowledge that certain microorganisms including bacteria, protozoa, yeasts, fungi, viruses, rickettsia, mycoplasmas, and agents, such as helminths and arthropods, have the capacity to cause biological alterations that can have great influence on the course and results of experiments, and can even lead to premature termination of a study ( ) ( ) ( ) . however, it is frequently overlooked that it is a common characteristic of potentially highly pathogenic organisms to persist in a colony without causing any clinical sign or pathological symptom. hsu ( ) and others ( ) ( ) ( ) ( ) summarized much of these data. a great number of infectious agents can reduce the mean lifetime of animals, and alter some organ systems, more or less, in their specific functions. during the past years, much progress has been made in identifying and controlling intercurrent diseases and latent infections. during the early s, the combination of caesarean-derivation (and the more recently used embryo transfer), establishment of a non-pathogenic gastrointestinal microflora, and the production of these animals in isolators or under strict barrier conditions has been successful in excluding adventitious pathogens. programs to assess the health status of rodents, and characterize their microbiological status were improved, and monitoring data thereafter showed that breeders were able to produce and supply clean, specified pathogen free (spf) animals ( , ) . however, there was only a little improvement in the ability of a great number of institutional and research facilities to maintain their laboratory animals free from infectious agents. many investigators still ignored the tremendous number of reports describing experimental complications associated with infections. new legislation on animal welfare, the introduction of teaching programs on good laboratory animal science, and opposition from antivivisectionists have forced experimenters to use high quality animals under standardized conditions. this trend would be greatly abetted if editorial boards of scientific journals would request authors of scientific papers to provide details concerning maintenance and welfare of animals during the experiments. when aging clin. exp. res., vol. , no. studies are reviewed and approved by institutional animal care and use committees, the design of the studies should be tested for aspects of health status and maintenance. in long-term studies, as in aging research, spf animals are widely used. spf animals were defined in by an icla (international committee on laboratory animals) subcommittee as animals that are free of specified microorganisms and parasites, but not necessarily free of other unnamed contaminants ( ) . this definition implies that the microbiological status of such animals can differ considerably among research groups. the term should, in the strictest sense, be related to a specific list of organisms, and to a specific set of methods used to detect these organisms. further, the program used for determining the microbiological status should also be applied for monitoring the animals during the experiment to ensure that they remain free of the specified pathogens. the term spf, however, often has been misused. in practice, many socalled spf colonies are not as healthy as they purport to be, since only a low number of pathogens is screened for. it is abundantly clear that for good comparison of experimental results obtained by different research groups, international standardization of programs used for microbiological characterization of spf laboratory rodents is indispensable. rodents for long-term studies must be obtained from a reliable source. production of clean spf animals is preferably performed in specialized breeding units, or by commercial breeders. breeding and maintenance of clean caeserean-derived animals in isolators, which minimize the chance that they are being exposed to unwanted organisms, offers the highest degree of security but has some disadvantages, especially higher daily costs and reduced animal capacity. spf barrier units ( ) are frequently used as the next best solution for keeping out specific rodent organisms. many commercial breeders will supply details of the microbiological profile of their animals and the environmental control. no health report can be an absolute guarantee of quality, as any screening test reflects only the status of the colony at the time it was carried out. the breeders' standards of husbandry and management, however, do offer guidance on the probability that the state of the animals will be maintained for long periods of time. however, because of lack of standardized (often incomplete) health reports of the suppliers, which makes reading and interpretation sometimes nearly impossible, there exists a lot of mistrust on the part of the investigators concerning the reliability of these reports. a critical period during which the quality of the animal can be negatively affected occurs during the transportation of animals from the breeding to the experimental facilities. drastic alterations in environment during transport, and entering the new facilities may affect behaviour and health status of the animals. there is a high risk of environmental or mutual contamination of the animals. for further reading on transport of animals, the reader is directed to a review on this subject ( ) . quarantine of newly arrived animals provides an opportunity to examine them for the presence of pathogens, and to characterize their health status by laboratory and clinical diagnosis. this reduces the possibility of mutual infection with established colonies of animals. for reliable serological laboratory diagnosis, the quarantine period should take at least - weeks before testing. during long-term experiments, animals should ideally be held in a full spf barrier system until the conclusion of the experimental procedure or observation period. however, there are some disadvantages which include the costs and the fact that carrying out frequent experimental procedures on the animals is often limited or almost impossible. experience shows ( ) ( ) ( ) that mice and rats born and reared under strict spf conditions can be kept pathogen and disease free for a long time in modest, sensibly improvised barrier systems, under high standards of hygiene, sometimes called clean conventional ( ) . the building should be rodent-proofed, visitors should be kept out, and only a minimal number of highly qualified staff should attend the colony. they should stick to a set of carefully devised, practical but microbiology and laboratory rodents strict rules and routines. it must be emphasized that strict precautionary measures must be taken when animals or biological materials are introduced into the animal facility from elsewhere, and that appropriate quarantine procedures be available. in designing such a regimen, a continual surveillance of the health status of the animals must be included. routine monitoring of the animals and the environment of a barrier system is essential to assure that microbial exclusion is being achieved. environmental monitoring includes testing of sterilization procedures, and culturing of room surfaces, equipment, food, bedding, water, air, etc. this has been reviewed by others to whom readers are directed for further reading ( ). monitoring the animal in the barrier gives perhaps the most sensitive and relevant indication of change in the microbial environment, and the presence of unwanted organisms, and the approaches used are retrospective health monitoring and prospective routine health monitoring. programs used for characterization of the microbiological status and routine monitoring should include daily clinical observation of the animals, histopathological searches for lesions of infectious diseases, cultures for bacteria and fungi, serological tests especially for viruses, and parasitological tests. some agents are infrequently encountered, and therefore do not represent significant problems to breeders or investigators, whereas others are frequently found. in the latter case, limited programs, rather than total microbiological analyses, may be used for controlling microbiological status during the course of an experiment, or to control breaks in a barrier system. general information on the protocols of various diagnostic methods used are readily available ( ) , but there is lack of standard methods for performing the test, or reporting the results among different laboratories. standard operation procedures (sop) for methodology used by testing laboratories, and the availability of standardized reagents and reference sera would be of great help to increase the interlaboratory stan-dardization. in north america, committees of aclad (american committee on laboratory animal diseases) and in europe, a working group of felasa (federation of european laboratory animal science associations), are actively developing standard formulas for laboratory animal health monitoring, as well as a quality assurance program. the purpose of the next part of this chapter is to address only basic matters concerning the characterization and monitoring of the microbiological status without the intention of being complete. clinical observation is performed by daily observation; diseased animals should be investigated by necropsy and appropriate laboratory methods, including histopathology. if macroscopic changes are found in animals submitted for routine monitoring, they should be examined as for diseased animals. it should be emphasized, however, that observation of clinical signs is not a reliable method by itself; infections, especially viral infections are often subclinical morphological changes in organs are primarily observed in animals that are already seriously ill; such changes may not be specific, so pathology also has limited value. laboratory methods ) bacterial, mycoplasmal and fungal infections. a. cultural methods: samples must always be investigated with nonselective media, e.g. , blood agar, and selective media should be used only for special or confirmatory investigations. aerobic culture conditions are sufficient for most bacteria; however, some bacteria require conditions, such as anaerobic incubation or micro-aerophilic conditions. for further identification, biotests are often used. b. serological methods: serological methods have been described for the detection of antibodies against various bacterial pathogens. these serological tests are, at present, insufficiently standardized. it is important that serological results be confirmed by cultural methods. in case of bacillus pili/ormis and mycoplasma, serology is frequently used as the method of choice. in certain cases, pathological methods are aging clin. exp. res. , vol. , no. used for the detection of car bacillus ( ) or to establish whether the presence of antibodies against b. pili/ormis is accompanied by the pathological signs of tyzzer's disease ( ) . c. sampling: samples for culture in general are taken by swabbing the nasopharynx, trachea, prepuce/vagina, small intestine, caecum or any suspect organ. for the detection of specific antibodies, the serum (from animals less than weeks old) may be screened serologically. in table a list of organisms is shown as a guide in establishing screening programs for bacteria, mycoplasma and fungi. ) parasitology routine diagnostic methods used at necropsy are low power microscopy/stereomicroscopy examination of the pelt for ectoparasites. for endoparasites, fecal flotation , fresh wet mount of intestinal wall scrapings, and adhesive strip preparations are used. an extensive list of parasites detected in mice and rats is given by kunstyr ( ) and sebesteny ( ) . ) viral infections a. virus isolation: virus isolation, often termed the "gold standard" ( ) to which all other methods should be compared, results in the identification of the etiological agent. unsuspected viruses may also be detected. however, the active phase of infection in individual animals is often short, and virus isolation therefore is an insensi- tive method which may give false negative results. the same holds, in addition, for other methods for virus detection by direct assays, for viral antigens, or nucleic acid in tissue specimens. new techniques like the polymerase chain reaction (pcr) are now available for the detection of nucleic acid by amplification of specific sequences ( ) . this method seems to be a very promising diagnostic tool, especially for the detection of persistent microorganisms like ldv (lactic dehydrogenase elevating virus) and mycoplasmas. b. serological methods: the most generally used method to characterize the viral status of rodents is the demonstration of virus specific immunity, by detection of antibodies by serological tests. these antibodies are specific and persistent, and can be detected rapidly and reliably with simple inexpensive techniques. the major disadvantages of the serological test are that unknown viruses will not be detected, and that for certain viruses there is no serological test available (e.g., ldv). during the last decade, there has been a change in the methods used in serology. new techniques have been introduced. the different test systems have been reviewed exten-sively ( , ) . it is well accepted now that elisa (enzyme-linked immunosorbent assay) and fa (fluorescence assay) are the primary techniques of choice, and that hal (hemagglutination) may sometimes be used as an alternative method to confirm primary test results, or specify strains. another new test, the western blotting technique ( ) , identifies the viral proteins to which antibody is produced; it provides reliable confirmation of primary positive test results. c. sampling: blood may be obtained aseptically and without additives from the brachial vessels, jugular vein, periorbital venus sinus, or heart. serum can be stored at °c before testing, and should be frozen at - °c for longer periods of time. table shows screening lists for the assessment of the total profile of viruses for mice and rats, and limited lists. these smaller lists of viruses, which are frequently encountered, and for which antibodies can easily be detected, might be used as a tracking profile to control the microbiological status of animals more frequently during the course of an experiment, or for barrier breaks. for purposes of serology, retired breeders and - week-old animals of immunologically competent strains are generally recommended for monitoring. for bacteriology, parasitology and pathology, the sampling of weanlings is also recommended. sample size is based on statistical considerations, which have been discussed elsewhere ( ) . there is no single answer regarding test frequency, which is governed by a number of factors. a single sampling reflects the actual status in a colony, but this may change rapidly. sampling intervals reported in the literature for serological screening range from six weeks to one year for infections that are not very likely to occur. when it is difficult to obtain samples from animals in study, one might use sentinel animals. these are animals of the same strain or with particular sensitivity to the pathogenic effect of undesirable microorganisms, with good immune competence. these animals are to be placed at strategic points in unfiltered cages in the animal unit to maximize transmission. there is an increasing awareness of the necessity of laboratory animals of high quality for long-term studies. scientific and economic setback by overt disease and mortality in experimental animals due to infections, as well as by more subtle interference after subclinical infections, and the influence of environmental factors is well documented. for these reasons researchers are striving to use clean animals under standardized conditions, which will also reduce the variability of results obtained by different research groups. environmental recommendations have been formulated by different countries and international working groups. since the early 's, when caesarian-derived colonies were transferred into barrier type facilities designed to exclude adventious (micro)organisms, breeders have been able to produce and supply clean animals. these spf animals have been shown to be free of a number of common (micro)organisms. it is clear that a program for monitoring the microbiological status needs to be an integral part of a quality assurance program, not only for breeding colonies, but also to assess the health status during experimentation and control for a break in a barrier system. to be able to compare interlaboratory results, it is necessary to characterize and control the spf status with well standardized monitoring programs. this has forced the professional community to contribute to increasing such standardization, which will be one of the main goals to realize in the near future. c: factors that complicate animal research environmental effects on animals used in biomedical research the ufa w handbook on the care and management of laboratory animals environmental and equipment monitoring environmental requirements for laboratory animal breeding and experimentation rodent and lagomorph health surveillance quality assurance ectromelia (mousepox) in the united states a naturally occurring epizootic caused by sendai virus infection in breeding and aging rodent colonies ii. infection in the rat a naturally occurring epizootic caused by sendai virus infection in breeding and aging rodent colonies i. infection in the mouse quality assurance of rodent models viral and mycoplasma infections of laboratory rodents. effects on biomedical research a: viruses can cause disease in the absence of morphological evidence of cell injury. implication for uncovering new diseases in the future parasitic and mycotic infections in laboratory animals prevalence of viral and mycoplasmal infections in laboratory rodents serological study on the prevalence of murine viruses in laboratory animal colonies in france and the netherlands ( - ) long-term holding of laboratory rodents the ufaw handbook on the care and management of laboratory animals immunological techniques applied to aging research a: types and quality of animals in cancer research necessity of a more standardized microbiological characterization of rodents for aging studies current experience using the laboratory rat in aging studies isolation of a newly recognized respi-microbiology and laboratory rodents ratory pathogen of laboratory rats: the car bacillus natural disease of laboratory animals with emphasis on subclinical forms publication no. of the gv-solas rapid viral diagnosis polymerase chain reaction the rodent parvovirus serological tests for detection of antibody to rodent viruses electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. procedure and some applications key: cord- -gw cow d authors: gray, darren w.; welsh, michael d.; mansoor, fawad; doherty, simon; chevallier, olivier p.; elliott, christopher t.; mooney, mark h. title: diva metabolomics: differentiating vaccination status following viral challenge using metabolomic profiles date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: gw cow d bovine respiratory disease (brd) is a major source of economic loss within the agricultural industry. vaccination against brd-associated viruses does not offer complete immune protection and vaccine failure animals present potential routes for disease spread. serological differentiation of infected from vaccinated animals (diva) is possible using antigen-deleted vaccines, but during virus outbreaks diva responses are masked by wild-type virus preventing accurate serodiagnosis. previous work by the authors has established the potential for metabolomic profiling to reveal metabolites associated with systemic immune responses to vaccination. the current study builds on this work by demonstrating for the first time the potential to use plasma metabolite profiling to differentiate between vaccinated and non-vaccinated animals following infection-challenge. male holstein friesian calves were intranasally vaccinated (pfizer rispoval(®)pi +rsv) and subsequently challenged with bovine parainfluenza virus type- (bpi v) via nasal inoculation. metabolomic plasma profiling revealed that viral challenge led to a shift in acquired plasma metabolite profiles from day to p.i., with metabolites identified whose peak intensities were significantly different following viral challenge depending on vaccination status. elevated levels of biliverdin and bilirubin and decreased -indolepropionic acid in non-vaccinated animals at day p.i. may be associated with increased oxidative stress and reactive oxygen scavenging at periods of peak virus titre. during latter stages of infection, increased levels of n-[( α, β, α)- , -dihydroxy- , -dioxocholan- -yl]glycine and lysophosphatidycholine and decreased enterolactone in non-vaccinated animals may reflect suppression of innate immune response mechanisms and progression to adaptive immune responses. levels of hexahydrohippurate were also shown to be significantly elevated in non-vaccinated animals from days to p.i. these findings demonstrate the potential of metabolomic profiling to identify plasma markers that can be employed in disease diagnostic applications to both differentially identify infected non-vaccinated animals during disease outbreaks and provide greater information on the health status of infected animals. introduction bovine respiratory disease (brd) is a multifactorial disease characteristic of a viral-bacterial synergistic infection with predisposition from environmental stressors [ ] . the disease constitutes a major source of economic loss through mortality, clinical disease and the associated treatments and long lasting reduced growth performance of infected young stock [ , ] . the annual cost of brd is estimated at $ billion in the usa, with preventative measures contributing a further $ billion [ , ] . vaccines are commonly used for controlling brd viral pathogens [ ] , but despite seasonal vaccination, animals can become infected with each new outbreak [ ] , maintaining the infection within the population. the viral pathogens associated with brd [bovine parainfluenza virus type- (bpi v), bovine respiratory syncytial virus, bovine viral diarrhoea virus and bovine herpes virus- ] impair immune responses in infected animals and damage the respiratory tract allowing the establishment of secondary infections, that may develop further into bacterial pneumonia [ ] . however, vaccinated animals can successfully clear viral infections faster than non-vaccinated animals through immune memory response, reducing the associated viral tissue damage or impairment of immune functions preventing the establishment of secondary bacterial and mycoplasma infections [ ] . during disease outbreaks, identification of unvaccinated animals at the early stages of infection could provide a window for effective treatment and facilitate the removal of animals that pose a greater risk of becoming infected and transmitting the infection to more susceptible juvenile stock. furthermore, halting viral disease progression to more severe and costly secondary bacterial infections through the identification of vaccine failure animals during infection outbreaks would reduce the level of antibiotic use in the agricultural industry. the only definitive method for successfully identifying vaccinated animals in the presence of an active viral infection is to determine the rate of viral shedding by virus isolation, cytokine/interleukin profiling or virus neutralization assay [ ] . these types of analysis require repeated sampling, a period for seroconversion and are expensive compared to serology based elisa, and are therefore not routinely employed during endemic viral infection outbreaks. differentiating infected from vaccinated animals (diva) marker vaccines (e.g. a modified wild type virus with a gene deletion resulting in the absence of a particular diagnostic antigen) can be employed to differentiate vaccine antibody responses from that of wild type virus. companion serology based tests rely on seroconversion, and upon exposure to wild type virus the antibody response to diva vaccines will be masked by that of the wild type virus. vaccine diva functionality is often limited to large viruses with increased potential for gene deletion and removal of redundant expressed antigens. therefore, for viruses with small genomes such as paramyxoviruses (e.g. bpi v and bovine respiratory syncytial virus of the brd complex) where gene deletion of neutralizing antigens may reduce vaccine efficacy, alternative approaches are required to provide diva functionality. one approach is to design molecular diva vaccines that contain a marker nucleotide sequence differing from the wild type virus that can be employed in combination with pcr-based molecular diagnostics to differentiate between vaccine and wild virus strains [ , ] . successful differentiation of vaccinated from non-vaccinated animals using this technique requires concurrent vaccination and infection [ , ] , with a narrow diagnostic window post-infection for detection of diva vaccine and viral genetic material. furthermore, detection of vaccine genetic material only demonstrates exposure to the vaccine and not the successful generation of immune protection, limiting functionality in assessment of herd level immunity. consequently, there is a clear need for alternative diagnostic methods that can assess efficacy of vaccines and vaccination status of animals exposed to brd viral pathogens at the early stages of infection prior to seroconversion and which do not require repeated sampling. additionally, the lower initial exposure rates to viral infections in field settings combined with variation in strain nucleotide sequences and short periods of virus secretion highlights the requirement for a diva approach with a long diagnostic window which is not strain specific. a potential approach that can meet these needs is based on the application of metabolomics to identify metabolites or 'small molecules' in biological samples that are signatures that correlate or provide some evidence of immune protection. these metabolites are often the end stage products of biological processes and therefore provide an accurate representation of an organism's homeostatic status at time of sampling [ , ] . metabolomic analysis of bio-fluids has provided new insights to the understanding of the patho-physiological processes involved in disease establishment, development and diagnosis [ ] [ ] [ ] [ ] . whilst metabolomics has had limited application in the field of veterinary research, several studies have demonstrated the potential of this technique in the prediction of brd disease outcome [ ] , differentiation of stress from viral infection responses [ ] , and characteristic of immune responses following vaccination [ ] . this study focuses specifically on bpi v due to its endemnicity within cattle populations and absence of clinical symptoms which still predispose animals to more severe bacterial infections [ ] . due to its small genome and absence of non-redundant proteins suitable for removal in diva vaccines, bpi v is an excellent model for assessing the potential of metabolomics to establish vaccination status in infected animals. the aims of the current study were therefore to assess the performance of reverse phase (rp) and hydrophobic interaction liquid chromatography (hilic) separation methods for ultra performance liquid chromatography-mass spectrometry (uplc-ms) metabolomic profiling of bovine plasma and identify plasma metabolomic markers capable of differentiating between vaccinated and nonvaccinated calves following intranasal challenge with bpi v. this work for the first time reports the metabolomic responses following challenge with bpi v and demonstrates how the application of metabolomic profiling may help overcome current limitations in diva diagnostics by identifying markers capable of differentiating between vaccinated and non-vaccinated animals, and importantly allow the development of better tools to assess the performance of vaccines. assessment of clinical findings of animals post bpi v vaccination and challenge have been reported in detail previously [ ] . briefly, animals were healthy throughout the duration of the study with no clinical signs of disease in vaccinated or non-vaccinated study groups. calves were sourced from respiratory disease free farms with no history of vaccination against brd. prior to the commencement of vaccination all calves tested seropositive for anti-bpi v igg. these residual levels of maternally derived anti-bpi v immunoglobulin are in keeping with other studies employing calves of the same age [ ] . at the commencement of vaccination there was no significant difference in anti-bpi v igg observed between treatment groups. vaccination with rispoval pi +rsv resulted in a significant increase in anti-bpi v igg with no significant increase in non-vaccinated controls. following bpi v challenge vaccinated calves had significantly higher plasma anti-bpi v igg relative to non-vaccinated animals. post-bpi v challenge, anti-bpi v igg remained elevated in vaccinated animals. although no significant differences in anti-bpi v igg levels were observed in non-vaccinated animals at days - post-infection (p.i.), igg was elevated in / animals relative to day levels, with the remaining animal demonstrating elevated levels from days to day p.i. there was a significant increase in lymphocyte counts in non-vaccinated animals from day to p.i., and a significant decrease from days to , with no significant variations observed in vaccinated animals. there were no significant differences in neutrophil counts between study groups at sampling points post-infection, however significant (p < . ) temporal variations were observed with a decrease from day to in non-vaccinated animals, and an increase from day to in both study groups. at day p.i., animals per group were prepared for gross postmortem analysis. two of animals in the non-vaccinated group showed gross inflammatory lesions, with some peri-vascular cuffing consistent with pneumonia. no significant gross / histological abnormalities were detected in / of the vaccinated calves at day p.i. at postmortem. preliminary metabolomic analysis was performed to select a suitable method for the comprehensive profiling of metabolites within bovine plasma. rp and hilic chromatographic methods were compared using day p.i. samples (n = ), which corresponds to peak viral titre [ ] . despite improved resolution of poorly retained hydrophobic compounds eluting between - min by hilic separation, increased chromatographic peaks corresponding to plasma derived compounds (relative to blank injections) were observed in rp acquired profiles (fig a and b) relative to hilic profiles (fig c and d ). observable differences between plasma samples from vaccinated and non-vaccinated animals within chromatogram profiles were only present using rp (s fig) . furthermore, upon data extraction and processing (metabolites with cv > % in qc pools), increased numbers of accurate mass retention time pairs (amrtps) were observed following rp (n = ) relative to hilic (n = ) separation of plasma. unsupervised pca analysis of plasma profiles acquired by rp-uplc-ms resulted in clear separation between study groups when assessing principle components (pc) and ( . % of systemic (r x) variation within the dataset) as illustrated in fig a. in contrast, hilic-uplc-ms metabolomic profiles facilitated only partial separation between study groups based on the pca scores plot (pc and pc , . % of systemic (r x) variation) ( fig b) . due to the small number of chromatographic peaks, fewer amrtps and poor unsupervised pca separation of plasma profiles acquired following hilic-uplc-ms chromatography, rp-uplc-ms was selected as the method of choice for metabolomic profiling of remaining study plasma samples. plasma samples from days , , and p.i. were extracted, analysed and combined with day p.i. data for multivariate analysis. mass accuracy and retention time deviation of reference compounds between analysis runs was excellent (s table) and within marker lynx extraction parameters ( . da and . min respectively) ensuring accurate peak matching. amrtps (n = ) with %cv less than % in inter-run quality control plasma pools were used to construct of pca models (pre-filtered from amrtps extracted from raw data). the effect of bpi v challenge on the metabolite profile of all animals irrespective of vaccination status, was illustrated in un-supervised pca scores plot ( fig a) by separation in pre-(day p.i.) and post-bpi v challenge samples (days - p.i.) when observing pc and pc ( . % r x). the greatest separation was observed between day pre-and day p.i. post-challenge stages in vaccinated and non-vaccinated animals. differentiation of vaccinated from non-vaccinated animals based on metabolite profiles following challenge with bpi v was investigated using un-supervised (pca) and supervised (opls-da) multivariate analysis. prior to bpi v challenge metabolite profile variation between vaccinated and non-vaccinated animals was low, evidenced by no separation at day ( fig b) when observing all pcs. with study progression to post-challenge stages, the variation between vaccinated and non-vaccinated animal metabolite profiles increased from partial separation at day p.i. (fig c, . % r x with pcs and ) to clear separation at days , and , with % (fig a) , . % ( fig d) and . % ( fig e) variation (r x) respectively in pcs and . opls-da analysis was employed for the selection of marker amrtps that could discriminate between vaccinated and non-vaccinated animals post-infection. fig a- d illustrates opls-da score (inset) and s-plots for supervised discriminate analysis of plasma metabolite profiles from bpi v infected vaccinated and non-vaccinated animals at days , , and p. i. respectively. the amount of variation responsible for differentiation of animals of different vaccination status (r x) was . %, . %, . % and . % in models generated for days , , and p.i. respectively, with excellent fit (r y > . %) and good cross-validated prediction (q > %). as opls-da is known to over-fit, particularly in megavariate datasets where the number of variables are higher than the sample size we employed permutation and false discovery rate testing to reduce the chances of selecting false positives. leave-one-out cross validation was performed to assess the performance of the models generated. briefly, all technical replicates from a single biological sample (test sample) were removed and the remaining dataset employed to generate a predictive opls-da model (vaccinated vs non-vaccinated). this predictive opls-da model was employed to predict the vaccination treatment group of the test sample. this cross-validation was permeated until all biological samples had been assessed for treatment classification. the results (s table) indicated that all biological replicates were accurately classified to their respective treatment groups upon cross validation prediction model testing. amrtps which contributed to class discrimination were selected on a criterion of a variable importance score (v.i.p.) score > from opls-da discriminate analysis. amrtps were further filtered to select only those with a fold change (fc) > . and significant difference (anova with bonferroni post-hoc test) p < . between study groups. final selection of amrtps was performed by assessment of raw mass spectrometric data to determine those with good peak shape and consistent peak intensity (height) in replicate injections. unique potential markers combined from all opls-da analysis ( at day p.i., at day p.i., at day p.i., and at day p.i.) were selected for further refinement to remove fragments and adducts for parent ion identification. the selected panel of unique amrtps (s table) differentiating animals of different vaccination status at various time-points post-bpi v challenge were deconvoluted to identify parent ion mass, adducts and low energy fragments using low and high energy data (function and respectively), yielding parent ions for elemental composition determination. nonparametric mann whitney was also employed on the selected panel of metabolites from day , and p.i. time points, with all markers attaining significance levels within thresholds comparative with anova. the false discovery rate was calculated from the cv ( %) filtered dataset via the benjamini-hochberg procedure with a threshold of %. all selected markers passed this threshold and met further selection criteria (v.i.p. score > and fc > . ) were selected as potential markers. retention times and accurate masses of potential markers post-bpi v challenge (days , , and p.i.) are shown in table this is the first study to report the potential to differentiate between infected animals with differing vaccination status through the use of metabolomic profiling techniques (diva metabolomics). previous work [ ] by the authors has demonstrated that metabolomics can identify metabolites associated with immune responses to vaccination, and the current study has advanced this concept by applying metabolomics analysis of plasma to identify unique metabolite marker profiles that are capable of distinguishing between vaccinated and non-vaccinated animals following infection. calves vaccinated (rispoval pi +rsv) and infection-challenged (bpi v) demonstrated a significantly stimulated anti-bpi v igg post-vaccination response which remained elevated post-challenge. lung histology and haematology confirmed that the bpi v inoculum employed for viral challenge was sufficient to induce some evident respiratory pathology in non-vaccinated animals, with an absence in vaccinated animals. the subsequent challenge in primed vaccinated animals also resulted in maintenance of pre-challenge elevated anti-bpi v igg (indicating response to inoculum), with absence of respiratory pathology. following preliminary analysis of a subset of samples (day p.i.), rp-uplc-ms analysis demonstrated increased capacity to profile bovine plasma metabolites relative to hilic-based chromatographic separation and was subsequently used for extensive metabolomic plasma profiling. unsupervised pca analysis of acquired metabolomic profiles of preand post-challenge plasma revealed clear and distinguishable variation in plasma metabolites between vaccinated and non-vaccinated animals. considering that elevated anti-bpi v igg typically occurs at weeks post-challenge [ , ] , variation in plasma metabolite profiles as early as day p.i. illustrates the capacity of metabolomic profiling methods to detect changes stimulated by immune responses at early post-infection phases. supervised multivariate discriminant analysis of plasma metabolomic profiles yielded potential metabolites (amrtps) that were significantly different in the plasma of vaccinated compared to non-vaccinated animals following bpi v challenge. following de-convolution (with removal of adducts and fragment ions), parent metabolite ions were identified and shown to be present at different levels within plasma from vaccinated and non-vaccinated animals from days - p.i. despite no significant differences in the parent ion intensity between treatment groups at day p.i. plasma metabolite profiles differed between vaccinated and nonvaccinated animals through supervised opsl-da. identities of of these parent metabolites were revealed via database searching, in silco fragmentation and spectral matching. divergent plasma metabolite profiles have previously [ ] been reported following vaccination, with altered metabolites shown to be associated with primary or secondary immune responses to vaccination. the metabolomic profiling performed here in this study on post-bpi v challenge acquired samples, has identified a unique panel of plasma metabolites which differ between vaccinated and non-vaccinated animals, and significantly are involved in recognised immune response mechanisms. at day p.i., increased biliverdin (fc = . ), bilirubin (fc = . ) and decreased -indolepropionic acid (fc = - . ) levels were observed in plasma of non-vaccinated animals. biliverdin is degraded from heme by heme-oxygenases, and is further reduced to bilirubin by bilirubin reductase [ ] . heme-oxygenase- exerts anti-inflammatory effects as demonstrated by reduced tumour necrosis factor alpha release in lipopolysaccharide-stimulated macrophages [ ] . -indolepropionic acid is a reactive oxygen species scavenger [ ] , produced in the microbiome [ ] . phagocyte activation by rna viruses results in both increased reactive oxygen species release and the production of pro-oxidant cytokines (tumour necrosis factor alpha and interleukin- ) which promote iron uptake by the mononuclear phagocyte system [ , ] . decreasing plasma -indolepropionic acid levels in concert with elevated biliverdin and bilirubin levels (via heme-oxygenase- action) in non-vaccinated animals at day p.i. maybe indicative of -indolepropionic acid scavenging of reactive oxygen species produced by phagocytes, or increased downstream ros production from cytokine signalling. fluctuations in the levels of a number of bile acids ( -oxocholic acid, cholic acid and ndgca) were observed in plasma of animals at various stages in the study. -oxocholic acid and ndgca were found to be significantly increased (fc = . and . respectively) in the plasma of non-vaccinated animals at day and p.i respectively, whereas cholic acid was significantly higher in vaccinated animals (fc = - . ) at day p.i. altered plasma bile acid profiles at distinct study phases suggest that bile acid metabolism and conjugation is associated with different immune response mechanisms. bile acid driven farnesoid x receptor activation (expressed in pulmonary endothelial cells [ , ] ) enables a regulatory immune response as demonstrated by dendritic cell modulation [ ] , natural killer t-cell inhibition, reduced proinflammatory osteopontin production [ ] and reduced lung permeability, suppressing leukocyte movement to sites of tissue inflammation [ ] . when transported into cells ndgca is a potent farnesoid x receptor activator [ ] and elevated ndgca at day in non-vaccinated animals suggests an association with the switching and/or suppression of innate inflammatory responses towards adaptive immune responses. cholic acid, the primary metabolite of cholesterol, was found to significantly increase from day to p.i. in vaccinated animals. cholesterol, a component of lymphocyte lipid rafts, supports b-and t-cell receptor signalling [ ] . reverse cholesterol transport is associated with immunosuppression via reduction in b-and t-cell receptor signalling, lymphocyte activation and proliferation [ , ] , and elevated cholic acid may indicate increased cholesterol metabolism via reverse cholesterol transport in proliferating lymphocytes, down-regulating the secondary immune response to viral challenge during later stages of infection (day p.i.). lysophosphatidylcholine produced from phosphatidylcholine [ ] [ ] [ ] stimulates dendritic cell maturation through the action of a g-protein-coupled receptor with further ability to stimulate interleukin- and interferon-γ production in t cells [ ] indicating a role in innate-toadaptive immune response progression. plasma levels of six lysophosphatidylcholine derivatives were significantly up-regulated (fc > . ) in non-vaccinated animals at day p.i. significantly higher plasma levels suggest involvement in systemic immune responses to primary bpi v antigen stimulation with dendritic cell recruitment to lymph nodes and subsequent dendritic cell driven t-cell activation. decreased plasma peak intensity of enterolactone (fc = - . ), a lignan metabolite formed in ruminants through the action of colonic bacteria on secoisolariciresinol diglucoside [ ] , was observed in non-vaccinated animals at days and p.i. enterolactone crosses the intestinal barrier and exerts anti-inflammatory functions by suppressing nuclear factor-κb signalling, tumour necrosis factor alpha production [ ] and interleukin- β release [ ] . as enterolactone is dietary derived it cannot be readily replenished or up-regulated during periods of high demand, therefore decreased enterolactone in non-vaccinated animals during the latter stages of bpi v infection (days and post-challenge) may reflect its metabolism to induce anti-inflammatory effects associated with the transition of acute to adaptive immune responses. from a diagnostic perspective, those plasma metabolite markers found to be altered at early stages of infection prior to sero-conversion and which persist to the later stages of infection are of particular interest. hexahydrohippurate, despite not showing significantly altered plasma levels until day p.i., remained elevated in vaccinated animals (fc > - . ) for the duration of the study. significantly lower levels of n-methylhippuric acid (fc = . ) and higher n-(cyclohex- -en- -ylcarbonyl)glycine (fc = - . ) were also observed in vaccinated calves at day p.i. n-(cyclohex- -en- -ylcarbonyl)glycine, n-methylhippuric acid and hexahydrohippurate are formed through the action of glycine n-acyltransferase on cyclohexanecarboxy-coa, cyclohexene- -carboxyl-coa or benzoyl-coa, produced during shikimate and phenylalanine metabolism [ , [ ] [ ] [ ] [ ] . with no significant variation in the plasma levels of phenylalanine (which would attribute post-bpi v challenge acyl-glycine conjugate to dietary variations), elevated hexahydrohippurate and n-(cyclohex- -en- -ylcarbonyl)glycine levels may be a consequence of an increased demand for coa in the liver due to the need to free coa otherwise sequestered in cyclohexanecarboxy-coa or cyclohexene- -carboxyl-coa. increased coa demand may therefore be associated with increased rate of b-and t-cell proliferation and antibody production following an adaptive memory response to bpi v challenge (as illustrated previously by post-parental immunization [ ] ). assessment of hexahydrohippurate concentrations in plasma could allow for differentiation of vaccination status in infected calves with a wider diagnostic window to that observable currently through monitoring differences in anti-bpi v igg levels. hexahydrohippurate occurs at high abundance in plasma offering potential for use as a realistic marker that could be measured using on-site based testing methods. in conclusion, this study highlights the potential of untargeted uplc-ms metabolomics to differentiate the vaccination statuses of virus challenged animals (i.e. diva metabolomics). the differential pre-challenge immune status of vaccinated and non-vaccinated animals resulted in divergent plasma metabolite profiles following bpi v challenge, evident as early as days p.i., with increasing variation from time post challenge. the metabolites identified were associated with immune cell regulation mechanisms, including t/b-cell proliferation and phagocyte activation and maturation. the wide diagnostic window for hexahydrohippurate combined with metabolite markers altered at distinct time periods associated with specific immune response mechanism (e.g. lysopc, biliverdin, bilirubin or -indolepropionic acid), could find application in staging of infection. the effectiveness of these efforts is reflected in the large magnitude fold-change and low intra-group variation of statistically significantly metabolites identified at days and p.i. similar to cytokine profiling, metabolomic based diagnostics are unlikely to match the pathogen specificity of molecular and serological testing but instead provide greater information on physiological health status, with future disease diagnostics potentially employing multiple methods to improve disease management decisions. a limitation of this study, as with many other biomarker discovery investigations involving large bovine animals, is the small cohort size (i.e. n = per group; n = at days and p.i.) which can be incorporated effectively into experimental groups. this potentially may impact on how findings from resulting analysis can be accurately translated to that observed within the wider more variable herd population. to mitigate against such issues, animals were extensively screened with regards to health and maternal antibody status prior to study group inclusion, and post-metabolomics profiling, stringent metabolite selection criteria (fc > . ; p < . ; v.i.p. score > ; % false discovery rate) were applied to select only the most robust metabolites as marker candidates. as such, further investigation using a larger, more comprehensive sample set (differing sexes, breeds and ages of animals) with alternative routes of vaccination, vaccination failure (degradation or immunosuppression) and challenge (with multiple pathogens) may determine a panel or 'fingerprint' of metabolites with greater diagnostic specificity. a number of unidentified markers showed promising expression profiles and as metabolomics is still a relatively new field, lacking the level of database curation compared to genomics and proteomics for marker identification, these markers may be identified in the future. further studies are required to expand upon this initial proof-of-concept to determine if the observed diva metabolite markers are robust. importantly these studies have identified potential markers that would also be of benefit in screening and assessing new vaccine formulations and allow identification of trails indicative of protective immune responses. hplc grade acetone and analytical standards were purchased from sigma aldrich (dorset, uk). lc-ms grade acetonitrile, water, methanol and chloroform were purchased from fisher scientific (loughborough, uk). all animal studies were carried out in accordance with the uk animals (scientific procedures) act and with the approval of the agri-food and biosciences institute northern ireland ethical review committee. calves were vaccinated with rispoval pi +rsv as previously reported [ ] . briefly, male holstein friesian calves aged between and weeks were sourced commercially from farms with no history of prior respiratory disease outbreaks. the calves had no prior vaccination for brd and were clinically examined and declared fit for the study on day by the named veterinary surgeon. claves were divided into two study groups (n = ) and assigned as non-vaccinated and vaccinated calves. vaccinated calves were treated with pfizer rispo-val pi +rsv intranasal vaccine (designated vaccinated animals) as per manufacturer's instructions, and non-vaccinated calves treated with empty poly-(lactic-co-glycolic) acid nanoparticles (designated non-vaccinated) prepared using standard double emulsification solvent evaporation technique (w/o/w) [ ] . calves received two dosages of vaccine formulation at and days prior to intranasal bpi v challenge (post-infection = p.i.) (inoculation with ml of virus suspension (tcid of . /ml) per nostril). calves were screened weekly following vaccination and at days , , , and post-bpi v infection (p.i.) for the presence of bpi v igg in blood serum using svanovir-pi v-ab kit (boehringer ingelheim svanovir, uppsala, sweden) as per manufacturer's instructions. at day p.i. animals per group were sacrificed for viral isolation and histology after sampling. blood samples drawn via jugular venepuncture at days , , , and p.i. into ml plastic k edta vacuette tubes (greiner bio-one, stroudwater, uk) were processed at random to platelet poor plasma via a double centrifugation method [ ] optimized for metabolomic analysis within hours of initial blood drawing and plasma stored at - ˚c prior to use. samples processing order was randomized to negate processing bias on sample metabolite profiles. μl of plasma was added to . ml of ice cold acetone, vortexed for sec and placed on ice for min. the sample was then deproteinated by centrifugation at , g at ˚c for min. . ml of supernatant was removed and dried under nitrogen for min at ˚c using turbovap lv (caliper life sciences, hopkinton, usa). resulting residue was reconstituted in μl of ultra-pure h o and liquid/liquid extraction of lipids performed by addition of μl of ice-cold methanol:chloroform ( : v/v) and vortexing for sec followed by centrifugation at , g at ˚c for min. liquid/liquid extraction was repeated and after centrifugation μl of the aqueous layer was removed and dried under nitrogen. the residue was reconstituted in μl ultra-pure h o and filtered by centrifugation at , g, ˚c using . μm costar spin-x centrifuge tube filter for mins. μl of plasma was added to a well in an ostro protein precipitation and phospholipid removal plate (waters corporation, milford, ma, usa). μl of % formic acid/acetonitrile (v/v) was added to the sample well, the plate shaken for sec and extracted metabolites drawn through under vacuum into a well ml collection plate. rp-uplc-ms analysis was performed using an acquity uplc system coupled to a xevo g q-tof (waters corporation, milford, ma, usa). a test mix of acetominophen, sulfaguanidine, sulfadimethoxine, val-tyr-val, verapmil, terfenadine, leucine-enkephalin, reserpine and erythromycin was injected to ensure calibration of mass spectrometer mass accuracy and uplc performance prior to analysis. pooled samples (comprising a μl aliquot from all study samples) were injected times before the start of each run for column conditioning [ ] and intermittently throughout the run to validate instrument performance. the run-order of samples entering the mass spectrometer was constructed using a randomized sample list comprising technical replicates per biological sample. no other samples were injected during the analysis run. μl of prepared sample extracts were injected onto an acquity uplc hss-t column ( mm x . mm i.d., . μm; waters corporation, milford, ma, usa). column and autosampler temperature were maintained at ˚c and ˚c respectively. chromatographic separation was carried out at a flow rate of μl/min with mobile phase consisting of . % h o/ . % formic acid (a) and . % acetonitrile/ . % formic acid (b). the elution gradient was as follows: - min isocratic at % of b, - . min linear gradient form - % of b, . - . min isocratic at % of b, and finally . - min linear gradient at - % of b. mass spectrometry was performed in positive-ion mode (esi+) with the capillary voltage set to v and the sampling cone voltage v. the desolvation and cone gas flows were set at l/h and l/h respectively. source and desolvation temperatures were ˚c and ˚c respectively. leucine enkephalin ([m+h] + = . da, and [m+h] + = . da) was used for accurate lockmass calibration during data acquisition. lockmass acquisition setting were: . sec scan time, sec interval, scan average, mass window +/- . da. centroid data were acquired in positive mode using resolution mode. collision energy was only applied on function , with ramping between ev and ev. for hilic-uplc-ms/ms analysis the test mix was cytosine, o-acetyl-l-carnitine and lvaline. pooled samples (comprising a μl aliquot from all study samples) were injected times before the start of each run for column conditioning [ ] and intermittently throughout the run to validate instrument performance. the run-order of samples entering the mass spectrometer was constructed using a randomized sample list comprising technical replicates per biological sample. no other samples were injected during the analysis run. μl of prepared sample extracts were injected onto an acquity uplc beh hilic column ( . mm x mm i.d., . μm; waters corporation, milford, ma, usa). column and autosampler temperature were maintained at ˚c and ˚c respectively. chromatographic separation was carried out at a flow rate of μl/min with mobile phase consisting of mm ammonium formate ph . (a) and acetonitrile with . % formic acid (b). the elution gradient was as follows: - min isocratic at % of a, - min linear gradient form - % of a, - min linear gradient from - % of a, - min isocratic at % of a, - . min linear gradient from - % of a, . - min isocratic at % of a. mass spectrometry was performed using a waters xevo g q-tof (milford, ma) operating in positive-ion mode (esi+) with the capillary voltage set to v and the sampling cone voltage v. the desolvation and cone gas flows were set at l/h and l/h respectively. source and desolvation temperatures were ˚c and ˚c respectively. leucine enkephalin was used for accurate lockmass calibration during data acquisition, with acquisition settings the same as with rp-uplc-ms analysis. centroid data were acquired in positive mode using resolution mode. collision energy was only applied on function , with ramping between v and v. total ion count (tic) chromatograms and spectra were acquired with masslynx version . (waters corporation, milford, ma, usa) in centroid format and metabolite data was processed using the markerlynx software. the markerlynx method for data extraction and deconvolution was as follows. ions were extracted from function data using peak detection analysis of retention time window . - . min, with a mass range of da to da. the xic window for data collection was . da and apex peak tracking parameters were set to automatic with no smoothing. data collection parameters consisted of an intensity threshold (counts) of , a mass window of . da with a retention time window of . min. a noise elimination level of was applied and isotopes were removed. peak heights for extracted ions were normalized against the total peak height of all extracted ions and standardized to a total ion count of , . the results were exported in .csv format as a two dimensional data table in which rows and columns respectively represented analysed samples and the relative normalized peak heights of each detected mass spectrometric signal, i.e. as an accurate mass (m/z) and retention time (min) pair (amrtp). extracted and processed data submitted for downstream multivariate and statistical analysis for metabolite marker selection can be found in supplementary data (s table) . temporal changes in bpi v antibody titre were analysed using two-tailed paired t-test, and significant differences between treatment groups at sampling stages was assessed using two-tailed heteroscedastic t-test. simca-p+ version . (umetrics, sweden) was used for multivariate metabolite marker selection. the dataset was pre-filtered to exclude amrtps with coefficient of variation greater than % in inter-run quality control pools (generated from equal aliquots of all extracted samples and injected intermittently throughout the analysis run). all centroid data were pareto scaled and analysed by unsupervised principle component analysis (pca) and supervised discriminatory analysis by orthogonal projections of latent structures-discriminant analysis (opls-da). unsupervised pca models were generated at each sampling day to reveal potential relationships between treatment groups. supervised analysis by opls-da was performed to reveal potential markers of response to treatment in vaccinated calves compared to non-vaccinated calves at each sampling day. robustness of final opls-da discriminative models was assessed by setting a predictive model of each case in which / of the data (known treatment) was used to predict the remaining / (unknown treatment). significance of the identified markers at all time points was determined using anova with bonferroni post-hoc test and non-parametric mann whitney for day and p.i. time points. the elemental composition of selected parent compounds was determined in masslynx using both positive and negative mode data. mass uncertainty was set to mda, odd and even electron state, carbon isotope filter of +/- % and elements included were c, h, o, n, p and s. where applicable na and k adduct elemental composition were determined with the respective element included in the analysis parameters. elemental compositions were searched against pubchem and chemspider online databases, and where possible function fragments were matched against metlin, hmdb or massbank databases. where fragmentation spectra for the analyte in question was not available, in silico fragmentation was performed using metfrag and function fragmentation data was validated against potential in silico fragments. identified compounds were validated using mass spectrum and retention time relative to authentic analytical standards analysed under identical experimental conditions (s fig). pooled plasma samples and individual analytical standards ( μm) were analysed under identical uplc and mass spectrometric run conditions as utilized previously [ ] , and metabolite identities confirmed by matching retention time and function and function spectra (including low and high energy fragments and adducts). putative annotations were acquired for compounds with spectral similarity to public spectral libraries (metlin, hmcb or massbank). table. cross-validation of opls-da models. leave-one-out (loo) cross validation was performed to assess the performance of the models generated. all technical replicated from a single biological sample were removed and an opls-da model containing the remaining biological and technical replicates employed to predict class representation. (xlsx) s table. selected amrtps of plasma metabolomic markers of response bpi v challenge in vaccinated and non-vaccinated animals. combined list of amrtps selected using opls-da at days , , and post-bpi v infection filtered to exclude those with p < . , fc < . and v.i.p. score < . (xlsx) s table. processed raw data employed for downstream metabolomic analysis. markerlynx was employed for data extraction and processing. amrtp levels are reported as peak height. the medicine and epidemiology of bovine respiratory disease in feedlots factors of respiratory disease: review of management factors. bovine respiratory disease: total health management use of treatment records and lung lesion scoring to estimate the effect of respiratory disease on growth during early and late finishing periods in south african feedlot cattle economic impact associated with respiratory disease in beef cattle. veterinary clinics of north america-food animal practice a dynamic range compression and three-dimensional peptide fractionation analysis platform expands proteome coverage and the diagnostic potential of whole saliva duration of immunity of a quadrivalent vaccine against respiratory diseases caused by bhv- , pi v, bvdv, and brsv in experimentally infected calves iowa: iowa state university press pathogenesis and pathology of bovine pneumonia. the veterinary clinics of north america food animal practice comparison of cytokine measurements using elisa, elispot and semi-quantitative rt-pcr differentiation of c-strain "riems" or cp _e alf vaccinated animals from animals infected by classical swine fever virus field strains using real-time rt-pcr escape of classical swine fever cstrain vaccine virus from detection by c-strain specific real-time rt-pcr caused by a point mutation in the primer-binding site laboratory test descriptions for bovine respiratory disease diagnosis and their strengths and weaknesses: gold standards for diagnosis, do they exist? canadian veterinary journal-revue veterinaire canadienne a comparison of diagnostic methods for the detection of bovine respiratory syncytial virus in experimental clinical specimens. canadian journal of veterinary research-revue canadienne de recherche veterinaire towards quantitative metabolomics of mammalian cells: development of a metabolite extraction protocol metabolic profiling of serum using ultra performance liquid chromatography and the ltq-orbitrap mass spectrometry system identification of potential biomarkers for ovarian cancer by urinary metabolomic profiling high performance liquid chromatographymass spectrometry for metabonomics: potential biomarkers for acute deterioration of liver function in chronic hepatitis b investigation of the human brain metabolome to identify potential markers for early diagnosis and therapeutic targets of alzheimer's disease lc-ms based serum metabolomics for identification of hepatocellular carcinoma biomarkers in egyptian cohort biomarkers for prediction of bovine respiratory disease outcome comparative approaches to the investigation of responses to stress and viral infection in cattle identification of systemic immune response markers through metabolomic profiling of plasma from calves given an intra-nasally delivered respiratory vaccine infectious bovine rhinotracheitis, parainfluenza- , and respiratory coronavirus. veterinary clinics of north america-food animal practice comparison of levels and duration of detection of antibodies to bovine viral diarrhea virus , bovine viral diarrhea virus , bovine respiratory syncytial virus, bovine herpesvirus , and bovine parainfluenza virus in calves fed maternal colostrum or a colostrum-replacement product efficacy of a quadrivalent vaccine against respiratory diseases caused by bhv- pi( )v, bvdv and brsv in experimentally infected calves effect of parainfluenza- virus challenge on cell-mediated immune function in parainfluenza- vaccinated and non-vaccinated calves heme oxygenase- /carbon monoxide: from basic science to therapeutic applications carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway indole- -propionic acid attenuates neuronal damage and oxidative stress in the ischemic hippocampus metabolomics analysis reveals large effects of gut microflora on mammalian blood metabolites human leukocyte pyrogen induces release of specific granule contents from human neurtophils oxidative stress during viral infection: a review. free radical biology and medicine downregulation of endothelin- by farnesoid x receptor in vascular endothelial cells fxr-mediated regulation of enos expression in vascular endothelial cells ursodeoxycholic acid suppresses eosinophilic airway inflammation by inhibiting the function of dendritic cells through the nuclear farnesoid x receptor the bile acid sensor farnesoid x receptor is a modulator of liver immunity in a rodent model of acute hepatitis fxr protects lung from lipopolysaccharide-induced acute injury endogenous bile acids are ligands for the nuclear receptor fxr bar lipid rafts and signal transduction high-density lipoproteins and the immune system hdl in innate and adaptive immunity hydrolysis of phosphatidylcholine during ldl oxidation is mediated by platelet activating factor acetylhydrolase serum lysophosphatidic acid is produced through diverse phospholipase pathways differential hydrolysis of molecular species of lipoprotein phosphatidylcholine by groups iia, v and x secretory phospholipases a mature dendritic cell generation promoted by lysophosphatidylcholine weekly excretion of the mammalian lignan enterolactone in milk of dairy cows fed flaxseed meal enterodiol and enterolactone modulate the immune response by acting on nuclear factor-kappa b (nf-kappa b) signaling flaxseed, and the lignan enterolactone increase stroma-and cancer cell-derived il- ra and decrease tumor angiogenesis in estrogen-dependent breast cancer metabolism of cyclohexanecarboxylate in rat glycine conjugation activity of benzoic-acid and its acinar locaalization in the perfused-rat-liver benzoic acid glycine conjugation in the isolated perfused rat-kidney. drug metabolism and disposition the origin of urinary aromatic compounds excreted by ruminants. . the metabolism of quinic, cyclohexanecarboxylic and non-phenolic aromatic acids to benzoic acid the preparation and characterization of poly(lactide-co-glycolide) microparticles. . the entrapment of a model protein using a (water-in-oil)-in-water emulsion solvent evaporation technique peptidomic analysis of human blood specimens: comparison between plasma specimens and serum by differential peptide display global metabolic profiling procedures for urine using uplc-ms we acknowledge the staff at afbi animal services unit vsd (stormont) for their help in the animal study and the staff at the advanced asset centre igfs (qub) for their excellent technical assistance. key: cord- -o cw ufy authors: horby, peter w.; hoa, ngo thi; pfeiffer, dirk u.; wertheim, heiman f. l. title: drivers of emerging zoonotic infectious diseases date: - - journal: confronting emerging zoonoses doi: . / - - - - _ sha: doc_id: cord_uid: o cw ufy this chapter discusses drivers of emerging infectious diseases (eid) of humans that have an origin in other vertebrate animals (zoonoses). this is a broad topic, worthy of a book in its own right. this chapter will therefore provide only an overview of key concepts of drivers of the emergence of zoonotic diseases, and particularly infectious diseases with a major disease burden in humans. as the authors mainly work in asia, the focus of this chapter is asia, but many of the lessons learned in this region are likely to apply elsewhere. more than % of the world population live in asia, a region with some of the fastest developing economies in the world. yet, despite tremendous advances, infectious diseases still remain a major burden for the human population in asia. of the estimated . million deaths in children aged less than years in southeast asia in , % are attributable to infectious causes (liu et al., lancet : – , ). as such, asia is both vulnerable to imported eids and a global focus of major social and environmental change that may facilitate the emergence and dissemination of new pathogens. however, it would be too simplistic to present the extensive changes in asia as inevitably increasing the risk of eids. some aspects of socio-economic change might serve to reduce the overall risk of infectious disease emergence, but all ecosystem changes have the potential to provide new opportunities for microorganisms to spill-over into human populations. the conditions or events (the 'drivers') that result in the successful cross-over of an animal microbe into humans are not well characterised, but emergence is often precipitated by changes to ecological or biological systems (wilcox and colwell ) . such changes include altered patterns of contact between wild and domestic animals (e.g. nipah virus), of direct human and wild animal contact (e.g. hiv, ebola), and changes in species abundance or diversity (e.g. hantavirus; lyme disease). species diversity, including the diversity of insect vectors and pathogenic microorganisms, increases towards the equator (guernier et al. ) , and jones et al. found a correlation between the emergence of zoonotic pathogens and the diversity of mammalian wildlife species (jones et al. ) . whilst high animal host and pathogen species diversity may be associated with a high burden of infectious diseases and an increased risk of disease emergence, biodiversity loss may, perhaps counter-intuitively, be associated with increased disease transmission. biodiversity loss directly disrupts the functioning and stability of ecosystems, producing effects that can extend well beyond the particular lost species (hooper et al. ) . changes in species abundance and diversity may favour pathogen amplification and 'spill-over' through a variety of mechanisms, including reduced predation and competition resulting in increased abundance of competent hosts, and the loss of 'buffering species' leading to increased contact between amplifying host species and compatible pathogens (keesing et al. ) . tropical regions with a rich pool of existing and potential pathogens that are increasingly connected, but also experiencing high rates of ecosystem disruption and biodiversity loss, may therefore be at a particularly high risk of disease emergence. human pathogens occasionally re-emerge as a result of dynamics that are beyond the control of humans. for example the hantavirus outbreaks in the southwestern u. s. in - and - have been attributed to changes in the abundance of infected rodents following periods of heavy snow and rainfall and vegetation growth leading to abundant production of rodent food. however, many ecological disturbances resulting in an eid seem to originate in the direct actions of humans. a wide range of anthropogenic factors have been linked to infectious disease emergence, including changes in land-use, travel, trade, and demographics. notably, most of these associations are speculative and supported (wolfe et al. ). stage : the pathogen is still exclusively infecting animals. stage : the pathogen has been transmitted from animals to humans under natural conditions but not yet from human to human. stage : there is limited transmission from animals to humans and between humans. these are often severe and lethal diseases due to for instance filoviruses (e.g. ebola). stage : animal disease that have sustained transmission between humans (e.g. influenza). stage : a microbe that exclusively infects humans (e.g. measles, syphilis) by little hard data because ecological and biological systems are highly complex and multi-layered (woolhouse ) . demonstrating or predicting the impact of particular conditions or events on the functioning of a system is difficult, with further inference of the impact of any changes on the risk of pathogen emergence posing a formidable challenge. socioeconomic development is associated with large increases in demand for natural resources. the demand for water, wood, pulp, agricultural land, living space, roads, minerals and power has had an enormous impact on the landscapes of asia. deforestation occurred throughout the s and the area of primary forest in asia has continued to decline (fao ) . deforestation, forest fragmentation, and afforestation are all alterations in habitat, which change species composition and the interaction between wild animals, domestic animals, insect vectors and humans, providing new opportunities for microbial transmission and potential emergence. there are well-documented examples of deforestation and forest encroachment resulting in increases in infectious diseases, such as yellow fever, mayaro, and chagas disease in the americas (saker et al. ). the clearing of forest and planting of large cacao plantations was linked to the emergence of oropouche virus in brazil. in asia there are already very high pressures on productive land, and the peak in land-use change in asia has probably passed. many areas are now in an era of increasing intensification of land productivity. this intensification is driven largely by demographic pressures, which are predicted to result in a % increase in food production by , with decreased consumption of grains and increased demand for meats, fruits and vegetables (fao a, b) . the increased demand for food, and meat in particular, when combined with demands for natural resources from industry and domestic consumers, and river damming for hydroelectric power, is resulting in a large increase in stress on water resources (fao b). the consequences of intensified agricultural production include the depletion and degradation of river and groundwater, reduced soil quality, and biodiversity loss. a direct and predictable effect of reduced access to clean water for low-income families is an increase in the risk of water-washed diseases (diseases that increase when the availability of water for personal hygiene is limited e.g. diarrhoeal and respiratory infections, trachoma), and water-borne diseases such as typhoid and hepatitis e. however, unquantified risks arising from the intensification of agriculture are pollution of freshwater with pesticides and fertilizers, loss of biodiversity, and land abandonment by small-scale farmers. the potential consequences of these changes on the risk of emergence of zoonotic infections have not been assessed. wild animals are an important source of food (bush meat) in some developing countries and bush meat has been implicated in the emergence of hiv, and the spill over of monkeypox, nipah and ebola virus (brashares et al. ) . whilst the reservoir of the sars coronavirus is thought to be bats, wild civet cats traded for food are thought to have acted as an intermediate host, transmitting the sars virus to humans through live animal markets (li et al. ) . wild animal products are popular in asia as traditional medicines, tonics, delicacies, or as symbols of wealth. although all ten countries in the association of southeast asian nations (asean) are signatories to the convention on international trade in endangered species of wild fauna and flora (cites), asia continues to host the largest illegal wildlife trade in the world (rosen and smith ) . the smuggling of h n -infected birds of prey into europe, the frequent smuggling of bush meat from africa into the u.s., and the importation into the u.s. of pet rodents infected with monkeypox show that both the legal and illegal trade in wild animals and wild animal products is a potential conduit for the international spread of zoonotic pathogens (bair-brake et al. ; van borm et al. ) between and the world population is expected to increase by . billion (a % increase), and the increase will be concentrated in urban areas of developing countries (un-desa ). whilst mega-cities (cities with a population of at least ten million) receive a lot of attention, most urban dwellers live in small cities, with half of the global urban population in living in cities of less than , people (un-desa ). this can be perceived positively as cities generally offer better economic opportunities, better educational opportunities, better living conditions, better nutrition, better sanitation, and therefore better health than underdeveloped rural areas. at the same time it is likely to mean that in many low to middle income countries, health and veterinary infrastructure in rural areas will not improve, or may even deteriorate, adversely affecting the likelihood of the early detection of eid. the demand for food in urban centres will increase and result in livestock and their products being transported over large distances from a wider catchment area, and thereby increase risk of spread and amplification of eid. urbanisation is one facet of changing human sociocultural systems, which also includes changing consumer demands and dietary habits (janes et al. ). the consequence is a spatial concentration of people and animals; not necessarily co-located, but connected through increasingly complex networks of rural and peri-urban farms and markets, distributors, agricultural workers, and consumers. due to the increase in global human population and economic development, demand for livestock products has risen dramatically over the last years, with the per capita consumption of meat in developing countries more than tripling since the early s and egg consumption increasing fivefold (fao a, b) . the increased demand for meat has been met by more intensive and geographically concentrated production of livestock, especially pigs and poultry (steinfeld et al. ). much of this has been through expansion of both the number of small-scale production units and large commercial farms. high-density monoculture of domestic animals is a form of low biodiversity that poses a particular threat for the spread of infectious diseases from farmed animals to humans. where domesticated animals are a conduit of spread from wild animals to humans, high density livestock production may promote spread of zoonotic diseases. genetic diversity within an individual host species is important since genetic diversity limits the potential for devastating epidemics (king and lively ) . the nipah virus outbreak in malaysia and singapore in - is a good example. once nipah virus crossed from wild bats to domestic swine, an explosive outbreak in high-density swine farms resulted in widespread exposure of humans and over human cases of encephalitis (pulliam et al. ) . other examples where intensified livestock production practices may have led to emergence of a zoonosis include: • streptococcus suis causes severe sepsis and meningitis in humans and is associated with areas of intensive pig production (see fig. . ). risk factors for human infection include swine slaughtering and the eating of undercooked pig products (wertheim et al. ). outbreaks in swine herds of porcine reproductive and respiratory syndrome virus also potentially increases the rate of invasive s. suis infection in swine, which in turn leads to an increased risk of s. suis infection in humans (hoa et al. ). • highly pathogenic avian influenza a subtype h n crossed-over from wild aquatic birds (the natural reservoir of influenza a viruses) to humans via massive amplification in domestic poultry. • the human q-fever epidemic in the netherlands during - caused by the bacterium coxiella burnetii is thought to have arisen when economic drivers led to an increased density of dairy goat farming, which resulted in amplification of coxiella burnetii prevalence and consequent increased spill-over to humans (roest et al. ; georgiev et al. ). the classical foodborne diseases such as e. coli, campylobacteriosis and salmonellosis associated with livestock products have been a significant problem in highincome countries for some time (cdc ; painter et al. ). one of the key factors, in particular in the case of campylobacter, has been the integration of highly intensive poultry production with poultry processing systems facilitating cross-contamination of meat (moore et al. ; nyachuba ) . it is notable that some foodborne pathogens cause subclinical infections in their animal hosts (e.g., campylobacter in poultry); therefore there is no direct incentive for farmers to control them. in addition, subsequent attribution of the source of foodborne disease in an affected human is notoriously difficult. it is likely that the global incidence of foodborne illness will rise as a result of increased industrial production of poultry in the emerging economies. the situation will be exacerbated by the emergence of antimicrobial resistant pathogens of foodborne pathogens (sahin et al. ; luangtongkum et al. ; altekruse and tollefson ) . despite the examples above, the intensification of livestock farming often entails more effective separation of domestic and wild animals, improved standards of animal health and welfare, reduced movement and species mixing: all of which reduce the risk of eids. reducing contact between domestic and wild animals, whether the wild animals are in their natural habitat or captive, is a key strategy of the food and agriculture organization (fao) to reduce risk to human health, and is part of the wider fao strategy of enhancing 'biosecurity'. improving biosecurity in farms is a major challenge since a large proportion of farming in asia is based on small-scale backyard production, and there is often a mix of commercial and backyard farming in any one location. achieving improvements in biosecurity without adversely affecting the livelihoods of small-scale farmers requires an approach to risk management that is adapted to their socio-economic context. the longer-term vision is to restructure the livestock production sector towards a more commercialised and controlled system, where controls benefit animal health, human health, and commercial profitability. but it has also been recognised that small-scale producers will continue to have a key role in providing food security in developing economies, and in fact their importance may increase due to their more efficient use of natural resources compared with large-scale industrial production (sjauw-koen-fa ; quan ). food production has become a complex national, regional and global network of food value chains (ercsey-ravasz et al. ; dickson-hoyle and reenberg ). many countries have begun to more tightly regulate live animal trade in the wake of bovine spongiform encephalopathy (bse), sars and avian influenza a/h n , but complex and poorly regulated food manufacturing and distribution chains still offer ample opportunities for disease outbreaks. in endemic regions, live bird markets are frequently contaminated with highly pathogenic avian influenza a subtype h n (davis et al. ; samaan et al. ; wan et al. ) . contaminated markets can become reservoirs and the source of infection for poultry and humans. measures to reduce the risk in live animal markets include the separation of species, not allowing animals to remain in the market more than h, not allowing poultry to exit the market alive, improved cleaning and disinfection, and weekly rest-days, when all animals are removed and the market is thoroughly cleaned. the re-emergence of brucellosis, one of the world's most common zoonoses, is thought to be the result of higher risk of transmission through increased within-and between country trade of susceptible livestock and their products (seleem et al. ) . the development of denser regional road transport networks may be especially important since, compared to air travel, roads offer a more egalitarian form of connectivity that includes animals and goods as well as humans. this provides opportunities for pathogens to disperse beyond their traditional niche by increasing opportunities for informal trade in live animals and their products (eisenberg et al. ) . veterinary medical authorities often struggle to enforce compliance with regulations, and it is common practice for farmers and traders to adapt their production and trading behaviors to avoid adverse economic consequences of regulations aimed at controlling zoonotic diseases. as a result, informal trade networks may intensify and re-structure in unpredictable ways during disease outbreaks. increasing intensification of animal husbandry in asia may be a trade-off between a lower risk of emergence events, as animals will be 'healthier' and better isolated, but should an eid event occur, an increased risk of massive amplification in large, naïve monocultures. perhaps the greatest risk arises when a limited number of intensive livestock production units are surrounded by substantial backyard farming with little or no biosecurity, thereby linking extensive and weakly regulated value chains with global food systems. livestock production practices can have major unintended and unanticipated impacts on the risk of zoonotic infections. perhaps the most notable example is bse. the feeding of ruminant-derived meat and bone meal to cattle, and possibly changes in practices for rendering animal tissue, led to an epidemic of bse in cattle, that was followed by an epidemic of a fatal neurological disease (variant creutzfeldt-jacob disease) in humans (taylor and woodgate ) . in contemporary livestock production, the use of antibiotics is a significant concern. almost % ( / ) of eids identified in asia since represent the emergence of a new pattern of antimicrobial resistance (jones et al. ) . bacteria in some areas show alarmingly high rates of resistance to anti-bacterial agents, and the recent emergence of the new-delhi metallo-beta-lactamase- (ndm- ) resistance gene, conferring resistance to a last resort antibiotic, and its rapid dissemination to other regions highlights the serious threat to human health of antimicrobial resistance (khan and nordmann ) . antibiotics are used extensively in the livestock and aquaculture sector to treat or prevent infections, or as growth promoters. non-therapeutic use of antibiotics as growth promoters involves the prolonged administration of sub-therapeutic doses. this practise has a demonstrable effect on the emergence and prevalence of resistant microorganisms in food animals and their environment, as well as resulting in the excretion of antibiotics into the environment, where environmental bacteria may be subject to antibiotic selection pressures (marshall and levy ) . synthetic antibiotics such as quinolones are quite stable in the environment over long periods of time. heavy metal contamination of animal food may also play a role in selection of antimicrobial resistance. whilst there remains some debate about the overall impact of these findings on human health, it is clear that the continued use of non-therapeutic antibiotics in an agriculture industry that is rapidly increasing in scale and intensity, has potential for becoming a very real threat through the inability to prevent/cure disease in production animals and the consequences for human food security as well as the transmission, for example, of resistant food-borne bacterial pathogens to humans. some recent examples concern transmission of methicillin resistant staphylococcus aureus (mrsa) from pigs to humans and esbl (extended spectrum betalactamase, an important resistance mechanism against most beta-lactam antibiotics) positive e. coli on chicken meat for human consumption (verkade et al. ; kluytmans et al. ) . swine-associated mrsa is now contributing significantly to invasive disease in patients in the netherlands (verkade et al. ) . antibiotic production and consumption continues to increase, often in an uncontrolled way, accelerating the evolution of antibiotic resistance, which then spreads rapidly across the globe. since the s few new antibiotics have been developed and we are on the cusp of returning to an era of untreatable infections. the consequences of antibiotic use in animals therefore require better surveillance, research and regulation. infectious diseases continue to be an important cause of human and animal morbidity and mortality worldwide. whilst important health advances (e.g. hygiene and vaccination) have been made, infectious diseases are dynamic and resilient, and continue to challenge local, national and global public health systems (fauci and morens ). the recognition of the linkage between anthropogenic changes, animals, and disease emergence has resulted in repeated calls for a more holistic, interdisciplinary, and integrated approach to the study of infectious diseases. the one health approach is one initiative aimed at realizing this aspiration. the task ahead is to understand how social, economic, and environmental changes are altering the landscape of infectious disease risks for both animals and humans, and how future emerging risks may be mitigated. a more analytical approach to these emergence events requires characterisation of the attributes of natural and artificial ecological systems before and after disturbance, and the functional relationship between changes in system attributes and pathogen emergence (woolhouse ) . human campylobacteriosis: a challenge for the veterinary profession is that a rodent in your luggage? a mixed method approach to describe bushmeat importation into the united states economic and geographic drivers of wildlife consumption in rural africa vital signs: incidence and trends of infection with pathogens transmitted commonly through food-foodborne diseases active surveillance network detection and characterization of clade high pathogenicity avian influenza h n viruses in chickens seized at ports of entry and live poultry markets in vietnam the shrinking globe: globalisation of food systems and the changing geographies of livestock production environmental change and infectious disease: how new roads affect the transmission of diarrheal pathogens in rural ecuador the state of food and agriculture - . rome: food and agriculture organisation of the united nations fao ( ) the state of the world's land and water resources for food and agriculture. rome: food and agriculture organization of the united nations and earthscan fao ( ) state of the world's forests, rome. rome: food and agriculture organisation of the united nations fauci as, morens dm ( ) the perpetual challenge of infectious diseases q fever in humans and farm animals in four european countries ecology drives the worldwide distribution of human diseases streptococcus suis and porcine reproductive and respiratory syndrome a global synthesis reveals biodiversity loss as a major driver of ecosystem change emerging infectious diseases: the role of social sciences global trends in emerging infectious diseases ecology of zoonoses: natural and unnatural histories impacts of biodiversity on the emergence and transmission of infectious diseases spread of carbapenemase ndm- producers: the situation in india and what may be proposed does genetic diversity limit disease spread in natural host populations extended-spectrum beta-lactamaseproducing escherichia coli from retail chicken meat and humans: comparison of strains, plasmids, resistance genes, and virulence factors bats are natural reservoirs of sars-like coronaviruses global, regional, and national causes of child mortality: an updated systematic analysis for with time trends since antibiotic resistance in campylobacter: emergence, transmission and persistence food animals and antimicrobials: impacts on human health atlas communicable diseases vietnam from attribution of foodborne illnesses, hospitalizations, and deaths to food commodities by using outbreak data agricultural intensification, priming for persistence and the emergence of nipah virus: a lethal bat-borne zoonosis a future for small-scale farming the q fever epidemic in the netherlands: history, onset, response and reflection summarizing the evidence on the international trade in illegal wildlife molecular evidence for zoonotic transmission of an emergent, highly pathogenic campylobacter jejuni clone in the united states globalization and infectious diseases; a review of the linkages. world health organization critical control points for avian influenza a h n in live bird markets in low resource settings brucellosis: a re-emerging zoonosis framework for an inclusive food strategy. cooperatives -a key for smallholder inclusion in valeu chains rendering practices and inactivation of transmissible spongiform encephalopathy agents highly pathogenic h n influenza virus in smuggled thai eagles recent emergence of staphylococcus aureus clonal complex in human blood cultures indications that live poultry markets are a major source of human h n influenza virus infection in china streptococcus suis: an emerging human pathogen emerging and reemerging infectious diseases: biocomplexity as an interdisciplinary paradigm origins of major human infectious diseases how to make predictions about future infectious disease risks key: cord- - jh lvm authors: loureiro, natália i. v.; viana, henrique v.; rodrigues, carlos r.; cabral, lúcio mendes; silva, thaís d. n.; cardoso, fernanda serpa; santos, dilvani oliveira; castro, helena c. title: solving an ethical issue involved in experimentation with animals in a brazilian teaching laboratory date: - - journal: biochem mol biol educ doi: . /bmb. . sha: doc_id: cord_uid: jh lvm changes are occurring within brazilian institutes of higher education; currently several universities are reviewing their course offerings and teaching approaches to determine if they meet the needs of today's undergraduate students. when changes are made to the curriculum of experimental courses, there should be an understood guarantee that all efforts to avoid ethical and biosafety issues have been diligently considered. ethical considerations lead us to create an alternative experimental session to be conducted that eliminated the use of rats, the conventional in vivo model employed for learning metabolism of glycogen in our university. to avoid possible biosafety issues, we prepared an alternative sample to simulate human urine, which we called guarurine. using our new method, it is possible to verify positive results imitating a diabetic and starving people samples for detection of glucose and ketone. the alternative tool described herein is not only particularly suited to bypass the ethics of using animals for teaching, but also permits the discussion of significant aspects of pathological and physiological situations such as diabetics and starvation in a simple, safe, and interesting way. the use of animals for research and teaching is an issue of great concern worldwide. in contrast to the legislative systems in united states, britain, germany, scandinavia, and many other countries [ ] [ ] [ ] [ ] [ ] [ ] , brazilian scientists and teachers still can pursue research projects and teaching class with relative freedom [ ] . in brazil, animal research ethical committees were created only in the s. the federal law , which provides for the didactic-scientific practice of animal vivisection, was approved in may but still waits for regulation. in addition, some drafts that provide regulation for the use of animals for teaching and research purposes are still being analyzed by the brazilian congress [ , ] . in the brazilian fluminense federal university, the study of biochemistry in the department of molecular and cell biology includes laboratory activities, which mainly allow students to actively explore key concepts in biochemistry in greater depth and acquire skill in scientific reasoning [ - ] . animals (rats) have been widely used as models for teaching the catabolism of glycogen (glycogenolysis) in this discipline. however, in the last years, the student teaching assistants and some students of several different undergraduate courses that attend this class had considered this experimental session as inappropriate. according to them, there was an "unnecessary sacrifice" of rats to demonstrate the use of glycogen as a fuel by an organism. thus, the refusal to attend this specific session by some of these undergraduate students, including those from the faculty of veterinary school, became common. because ethics is not a matter of adhering to absolute rules, but rather of doing what will have best consequences, given the constraints under which we act, the ethics of using a specific experimentation will depend on if the goal can be reached while causing the animal less suffering, using fewer animals, or without using animals at all. anyway, due to ethical and others concerns, we were facing the challenge of changing this teaching session. in resolving this issue, understanding the reasons why students object to animal laboratories was an important step. in their point of view, this experimental class represented a serious religious/ethical dilemma because the life and death of laboratory animals becomes trivialized in the process of exemplifying only one topic of metabolism. therefore, based on their opinion and using the principle of our self-regulation, rules were set up to conduct the selection of a future practical laboratory experiment. these were i) avoid the use of laboratory animals that would be sacrificed; ii) the inclusion of other topics of metabolism such as glycolysis, citric acid cycle, fatty acid and amino acid synthesis and catabolism, and ketogenesis in the experimental discussion; iii) the experiment should have low cost and be performed after the relevant theory material is studied; and finally iv) it also should be easy and fast, due to the limited time of the practical class. consequently, the whole laboratory experience should not last more than h, according to the scheduled time available for the class. given these limitations, a previous laboratory class was considered to be an ideal candidate for replacement of the class in question. "urinalysis" is a nonanimal laboratory experiment basically consisting of assays to determine glucose and ketone levels in the urine from a diabetic and a healthy people. its experimental work does not last more than h, and all necessary reagents were already part of the stockroom, avoiding any further expenses. this class permits the discussion of several topics of metabolism including those previous established; and, most of all, it allows aligning classroom practices with professional practices. hence "urinalysis" met the main criteria that had been determined. however, despite passing these rules, "urinalysis" raises a biosafety issue, e.g. the risk of using body fluids at the undergraduate level. according to the world health organization, the body fluids and substances of all persons should be considered to contain potentially infectious agents [ ] . no distinction may be made between body fluids and substances from individuals with a known disease or infection and those from asymptomatic or undiagnosed individuals [ ] . consequently, infection control practices shall be present in the urinalysis class including the use of gloves and masks, frequent hand washing, proper cleaning, and good disinfectant practice. these procedures greatly raise the expense of the exercise, which could be a problem in less economically developed countries such as brazil [ ] . but an extreme example of the necessity for these protocols is the sars virus, which can survive in urine for at least h [ ] . another virus that can also be transmitted by this body fluid is the cytomegalovirus [ ] . in fact, the risk factors due to using body fluids were the reason for the replacement of the "urinalysis" session in this discipline in the first place. however, due to the several advantages of the "urinalysis" session theme, we decided to keep it, creating a harmless substitute for urine, averting the original animal ethical and biosafety issues. additionally, we also planned a different way of class presentation using a problembased learning-like approach, stimulating the student to be more active in the laboratory class [ , ] . in this article, we will present the protocol and approach used in this practice class, also including the evaluation by student teaching assistants and undergraduate students from nine different courses ("biological science," "pharmacy," "medicine," "veterinary medicine," "nutrition," "nursing," "odontology," "chemistry," and "industrial chemistry"). normal urine is a clear straw-colored liquid, which under normal conditions does not contain sugars, yeast cells, protein, ketones, bacteria, or parasitic organisms. in order to select a substance that imitates perfectly the visual aspect of urine, the famous brazilian guarana (paullinia spp.) was tested. the guarana seeds are used mainly to produce a soft drink that present similar visual appearance of urine [ ] . hence the soft drink in its diet version was utilized to prepare samples to be tested on the urine diagnostic assays. these assays include the detection of glucose by the classic benedict's copper reduction reaction and of acetoacetic acid (the physiological ketone) by the reaction with sodium nitroprusside in a strongly basic medium. in order to prepare the guarurine samples, a -ml cup of diet guarana was diluted twice with distilled and deionized water and three samples of ml were obtained, which we named guarurines (fig. ) . on the first sample, glucose ( % w/v) and acetone ( % v/v) were added, representing the diabetic urine (fig. ) . to the second sample, only acetone ( % v/v) was used to reproduce the urine from a starving person with prolonged diarrhea and vomiting (fig. ) . the last ml was considered the normal urine, therefore no glucose or acetone were added (fig. ) . usually the urine specimens should not remain unrefrigerated for longer than h, but in the case of guarurine samples they resisted several weeks out of the fridge, probably due to the presence of preservative in its formula. this aspect permits the storage of guarurine samples for longer periods than urine. classical benedictЈs copper reaction (glucose)-in the glass tubes, ml of benedict's reagent was added to ml of the guarurine samples. the test tubes were immediately placed into a beaker of boiling water and left for min. when heated with benedict's reagent, guarurine sample containing glucose (sample ) formed an orange-brown precipitate similar to the diabetic urine (fig. ) . sodium nitroprusside reaction in a strongly basic medium (ketone)-two drops of sodium nitroprusside reagent were added in ml of the guarurine samples in a glass tube. the test tube was mixed and tilted to about a °angle, and the samples were slowly overlaid with % ammonium hydroxide. a pink ring appeared at the junction of the two liquids in the guarurine samples containing acetone (samples and ) after min, similar to that observed for starving and diabetic people's urine. therefore, guarurine samples showed to be adequate as a harmless substitute for human urine to be used in the "urinalysis" session, because they act similar to the respective human samples (fig. ) . after the preparation of the protocol and arranging all necessary laboratory material including the guarurine, it was possible to evaluate this new practical class with the group of student teaching assistants from the biochemistry discipline (n ϭ ). the new problem-based learning-like approach used consisted of the presence of an initial situation where three unlabeled "urine" samples from three different people (a diabetic, a starving, and a healthy person) needed to be identified (fig. ) . the main purpose was to correctly identify this "biological" material according to the testing results that would be obtained during the experimental class. this approach would lead to the discussion about the initial expected results and those finally found. by providing specific reagents and the three guarurine samples, the students were invited to use the laboratory reagents in order to find out about the origin of these samples. the goal of this practical class was mainly to provide to these students the opportunity to develop their own reasoning and acquire knowledge from the experi-mental results obtained. the support given during this experimental session at this moment consists of aiding in clarifying the objectives of the experiments, organizing the content, and in the "diagnosing" the clinical cases. consequently, these student teaching assistants were also being prepared to coordinate future "guarurinalysis" session for undergraduate students. in the end, all six students were able to complete the whole session on time and also identify correctly the physiological "source" of the given material. an interview at the end of the experimental session revealed that all six student teaching assistants voted unanimously to the inclusion of "guarurinalysis" in the discipline. aspects such as students safety; the nonobligatory use of gloves and mask but keeping other safety procedures such as frequent hand washing, which would permit the discussion about biosafety issues; and most of all, the opportunity of simulating a "body fluid" clinical analysis in a safer way, were pointed as the main advantages in this experimental session during their interview. this experimental protocol was also tested with students from different undergraduate courses ("biological science," "pharmacy," "medicine," "veterinary medicine," "nutrition," "odontology," "chemistry," and "industrial chemistry") using the help of the students teaching assistants. at the beginning, undergraduate students were divided into groups for the organization of the experiments. however, everyone was stimulated to participate of the discussion during the whole teaching session. at the end, most of students could correctly identify the samples, increasing their knowledge about the biochemistry topics discussed. in case of the students, this practical session was evaluated by the application of a questionnaire consisting of simple and objective questions raised about four important issues; the acceptance level of the practical session; its permanence in the discipline for the next years; its direct correlation with biochemical topics discussed on the lecture part; and the clear applicability of this knowledge on their future professional career ( table i) . the survey results indicated that nearly all students find this experimental session interesting because all of them attributed good ( %) or very good ( %) acceptance level for it. in accordance with this data, % of the students voted for the permanence of this practical session in the biochemistry discipline for the next years (table i) . the undergraduate students noticed the direct correlations of this experimental simulation with the biochemical theoretical topics ( %) and their future professional life ( %) ( table i) . although these are positive results, the opinion about the professional-related topic radically changes if the "odontology" undergraduate course is analyzed individually. surprisingly, "odontology" students did not agree ( %) or partially agree ( %) that the knowl-edge offered by this practical session will be useful on their professional career, seeing no direct correlation between them (not shown). this result emphasizes the necessity of an improvement of the lectures, including professional themes in the biochemistry discipline to the understanding of the undergraduate student [ ] . in case of the "odontology" class, clinical cases involving topics such as the problems with the healing process in teeth extraction procedures on diabetic persons could be a good example on how important is the knowledge about this disease and consequently of this experiment for the dentist. it is important to emphasize the major improvement in the student participation during the learning process using this specific practical approach. in fact, some students asked during the class for testing their own urine ( - %), which was not allowed. this specific students behavior indicate a direct interpretation about the applicability of those assays in quotidian. this is probably due to the fact that they were more required to do critical analysis of experiment's result when a problem-based learning-like approach is used. conclusion various pressures are driving changes on brazilian higher education. they are from government, students, professional bodies, and employers; from changes in teaching styles and in the discipline itself; and from ethical/ animal rights considerations. as these demanded changes occur, an alteration in the knowledge, skills, and attitudes of academic professor will be required, which includes the way teaching is delivered and learning is facilitated [ , ] . herein we described a laboratory class experiment that can be applied in any biochemistry course avoiding ethical and biosafety issues. through the correct application of our established alternative tool and the understanding of the technical and clinical details of this practical session, the students could acquire knowledge not only about biochemistry but also about specific problems related to handling of the samples and reagents and how clinical results are obtained and interpreted. other advantages of this practical class are that it is fast, reasonably safe, and have low cost, requiring fewer resources to simulate clinical cases. this economical advantage is important to a country that struggles to maintain its own public academic institutions such as brazil. historical issues concerning animal experimentation in the united states research with animals: requirement, responsibility, welfare guidelines for animal surgery in research and teaching animal ethical committees the regulations concerning animal experiments in education by the german animal rights law animal experimentation in europe: from its origins to its future aspectos é ticos da pesquisa com animais normas de pesquisa em saú de ethics and animal experiments skills and processes in science education laboratory work in biochemical education: purpose and practice the link between laboratory and learning summit to put education at the heart of brazil's future severe acute respiratory syndrome (sars) multi-country outbreak update ; studies of sars virus survival, situation in china cytomegalovirus (cmv) infection, available at /www the boyer commission on educating undergraduates in the research university for the carnegie foundation for the advancement of teaching ( ) reinventing undergraduate education: a blueprint for america's research universities approaches to cell biology teaching: learning content in context-problem-based learning species of paullinia with economic potential, available at www the integrative nature of biochemistry: challenges of biochemical education in the u learning to become a teacher: the wheelock way successful teacher education programs in the era of reform (an essay review of studies of excellence in teacher education ( vols.): preparation in the undergraduate years; preparation in a five-year program; preparation at the graduate level acknowledgments-we thank the fluminense federal university for the honorable mention for practical and teaching approach development in the coordinators awards. we also thank professor cícero carlos de freitas, mr. hugo rodolfo de oliveira barbosa filho and mrs. dione m. silva for their help and technical assistance. key: cord- -e kuwf authors: nan title: opinion of the scientific panel on animal health and welfare (ahaw) on a request from the commission related with the risks of poor welfare in intensive calf farming systems date: - - journal: efsa j doi: . /j.efsa. . sha: doc_id: cord_uid: e kuwf nan summary efsa has been requested by the european commission to issue a scientific opinion on animal health and welfare aspects of intensive calf farming systems and their ability to comply with the requirements of the well-being of calves from the pathological, zootechnical, physiological and behavioural points of view. in particular the commission asked efsa to update the findings of the scientific veterinary committee (animal welfare section) report, on the welfare of calves of november , in the light of more recent data on this issue. where relevant the possible food safety implications of different farming systems should also be considered. in this report a risk assessment was made and the relevant conclusions and recommendations are forming the scientific opinion by the ahaw panel. the svc ( ) report contains information on measurements of welfare, needs of calves, descriptions of current housing systems, chapters on types of feed and feeding systems, weaning of calves, housing and pen design, climate, mananimal relationships, dehorning and castration. further chapters covered economical considerations of systems and for improving welfare. in the report conclusions were made on general management, housing, food and water and economics. the present report "the risks of poor welfare in intensive calf farming systems" is an update o the previous svc report with the exception of economical aspects which are outside of the mandate for this report. the various factors potentially affecting calves' health and welfare, already extensively listed in the report of the scientific veterinary committee animal welfare section (svc, ) , are updated and subsequently systematically determined whether they constitute a potential hazard or risk. to the latter end their severity and likelihood of occurrence in animal (sub) populations were evaluated and associated risks to calf welfare estimated, hence providing the basis for risk managers to decide which measures could be contemplated to reduce or eliminate such risks. in line with the terms of reference the working group restricted itself to (in essence a qualitative) risk assessment although it is agreed that welfare and health of calves can be substantially affected in the course of and as a result of transport and slaughter, this report does not consider animal health and welfare aspects of calves during transport and slaughter but such information can be found in a recently issued comprehensive report of the scientific committee on animal health and animal welfare (scahaw), on "the welfare of animals during transport (details for horses, pigs, sheep and cattle)" which was adopted on march (dg sanco, ) and in the efsa report "welfare aspects of animal stunning and killing methods" (ahaw / ). in relation with the food safety aspects, main foodborne hazards associated with calf farming are salmonella spp., human pathogenic-verotoxigenic escherichia coli (hp-vtec), thermophilic campylobacter spp., mycobacterium bovis, taenia saginata cysticercus and cryptosporidium parvum/giardia duodenalis. present knowledge and published data are insufficient to produce a universal risk assessment enabling quantitative food safety categorization/ranking of different types of calf farming systems. nevertheless, the main risk factors contributing to increased prevalence/levels of the above foodborne pathogens, as well as generic principles for the risk reductions are known. the latter are based on the implementation of effective farm management (e.g. qa, husbandry, herd health plans, biosecurity) and hygiene measures (e.g. gfp-ghp). in general, the conclusions made in the previous svc report remain. however, recent research has provided for some additional conclusions. the risk analysis is presented in the tables of annex . the graphics in this table are not intented to represent numerical relationships but rather qualitative relations. in some instances the exposure could not be estimated due to lack of data, in which cases the risks where labelled "exposure data not available". the following major and minor risks for poor animal health and welfare have been identified for one or several of the various husbandry systems considered: the hazards of iron deficiency and insufficient floor space are considered to be very serious, the hazard of inadequate health monitoring is considered to be serious and the hazards of exposure to inadequate hemoglobin monitoring, allergenic proteins and too rich diet are considered to be moderately serious. for these hazards, there is no consensus on the exposure of calves mainly due to lack of data and that is why it is recommended that further studies should be made to provide evidence for an exposure assessment. regarding castration and dehorning (and disbudding) without anaesthetic drugs, there is a variation in relation to national legislation why the risk of poor welfare in relation to castration and dehorning has a wide range between countries. council directive / /eec laying down minimum standards for the protection of calves as amended by council directive / /ec requires the commission to submit to the council a report, based on a scientific opinion, on intensive calf farming systems which comply with the requirements of the wellbeing of calves from the pathological, zootechnical, physiological and behavioural points of view. the commission's report will be drawn up also taking into account socio-economic implications of different calf farming systems. it should be noted that the scientific veterinary committee (animal welfare section) adopted a report on the welfare of calves on november which should serve as background to the commission's request and preparation of the new efsa scientific opinion. in particular the commission requires efsa to consider the need to update the findings of the scientific veterinary committee's opinion in light of the availability of more recent data on this issue. where relevant the possible food safety implications of different farming systems should also be considered. efsa has been requested by the european commission to issue a scientific opinion on animal health and welfare aspects of intensive calf farming systems and their ability to comply with the requirements of the well-being of calves from the pathological, zootechnical, physiological and behavioural points of view. in particular the commission requires efsa to update the findings of the scientific veterinary committee (animal welfare section) report on the welfare of calves of november in light of more recent data on this issue. where relevant the possible food safety implications of different farming systems should also be considered. the mandate outlined above was accepted by the panel on animal health and welfare (ahaw) at the plenary meeting, on / march . it was decided to establish a working group of ahaw experts (wg) chaired by one panel member. therefore the plenary entrusted a scientific report and risk assessment to a working group under the chairmanship of prof. bo algers. the members of the working group are listed at the end of this report. the scientific report is considered for the discussion to establish a risk assessment and the relevant conclusions and recommendations forming the scientific opinion by the ahaw panel. according to the mandate of efsa, ethical, socio-economic, cultural and religious aspects are outside the scope of this scientific opinion. . . the working group set out to produce a document in which the various factors potentially affecting calves' health and welfare [already extensively listed in the report of the scientific veterinary committee animal welfare section (svc, ) , are updated and subsequently to systematically determine whether these factors constitute a potential hazard or risk. to the latter end their severity and likelihood of occurrence in animal (sub) populations were evaluated and associated risks to calf welfare estimated, hence providing the basis for risk managers to decide which measures could be contemplated to reduce or eliminate such risks. it should be noted, however, that this does not imply that a hazard that has a serious effect on just a few animals should not be dealt with by managers on farm level as the suffering imposed on some animals constitute a major welfare problem for those individuals. in line with the terms of reference the working group restricted itself to (in essence qualitative) risk assessment, i.e. only one of three elements essential to risk analysis a risk assessment approach was followed, similar to the one generally adopted when assessing microbiological risks, i.e. along the lines suggested at the nd session of the codex alimentarius commission (cac, ) . incidentally, these guidelines have been characterized by the cac as 'interim' because they are subject to modifications in the light of developments in the science of risk analysis and as a result of efforts to harmonize definitions across various disciplines. cac's guidelines are in essence exclusively formulated for the purpose of assessing risks related to microbiological, chemical or physical agents of serious concern to public health. consequently -considering their disciplinary focus -the working group had to adapt the cac definitions to serve their purpose. these adapted definitions, have, in alphabethical order, been included in chapter (see risk analysis terminology) the objectives of this report are to review and report recent scientific literature on the welfare including the health of intensively reared calves, to report on recent findings as an update to the scientific veterinary committee's previous report, to make a qualitative risk assessment concerning the welfare of intensively kept calves. where relevant, food safety implications of different farming systems are also considered. the report is structured in five major parts. the first three follow the scientific veterinary committee's previous report "on the welfare of calves" with introductory chapters - on background, measurements and needs in relation to calf welfare, chapter describing housing, diet and management and chapter describing comparison of systems and factors. in chapter common disease and use of antibiotics is described. the other two parts involve aspects of meat quality and food safety (chapter ) and the risk assessment (chapters ). conclusions and recommendations from the previous svc document together with updated conclusions derived from recent research findings are presented in chapter . effect of transport and slaughter on calves' health and welfare although it is agreed that welfare and health of calves can be substantially affected in the course of and as a result of transport, this report does not consider animal health and welfare aspects of calves during transport because there is already a comprehensive recent report of the scientific committee on animal health and animal welfare (scahaw), on "the welfare of animals during transport (details for horses, pigs, sheep and cattle)" which was adopted on march (dg sanco, . the report takes into account all aspects related with transport that could affect the health and welfare of cattle and calves, including the direct effects of transport on the animals and the effects of transport on disease transmission. the loading methods and handling facilities for cattle, the floor space allowance, the relationships of stocking and the density requirements, the vehicle design, space requirements and ventilation for cattle transporters (see also the ahaw scientific opinion related to standards for the microclimate inside animal road transport vehicles, efsa-q- - ), the behaviour of cattle during road transport, the road conditions, long distance transport and the travel times are also reviewed. recommendations for all these aspects are also given in that report. feeding and housing systems, weaning strategies and quality of solid and liquid feed . . . feeding systems and weaning strategies recommendations without a fully functional rumen, calves will be unable to utilise nutrients provided in the post-weaning dry feed diet. attention must paid to type of forage and consistent of particle size of starter grain in order to achieve a proper rumen development. calf weaning should be based on the amount of dry feed calves ingest per day, not on their age or weight, and calf starter should be made available five to days after birth. a calf consuming . kg of dry feed or more on three consecutive days is ready for weaning. when calves are fed low levels of milk to encourage early consumption of dry food, weaning can be done abruptly. in contrast, if milk is given in large amounts, weaning may require two to three weeks of slow transition to avoid a setback in growth. the provision of solid feeds with adequate content and balance to veal calves is a prerequisite for the development of a healthy and functional rumen, the prevention of abnormal oral behaviours, and the stimulation of normal rumination activity. although some solid feeds may exacerbate problems with abomasal ulcers in milk-fed veal calves, properly balanced rations seem to moderate this effect. nutritional factors are clearly involved in the etiology of abomasal ulcers in veal calves. important elements include the consumption of large quantities of milk replacer and the interaction between a milk replacer diet and the provision of roughage. if vegetable proteins are not properly treated, milk replacers may cause hypersensitivity reactions in the gut, which may compromise calf welfare. it is recommended that solid feeds provided to veal calves, in addition to milk replacer, are adequately balanced in terms of the amount of fibrous material, which will promote rumination, and other components such as proteins and carbohydrates, which stimulate rumen development and support a healthy function of the digestive system. since milk replacer formulations are frequently changing, it is recommended to carefully and consistently examine allergenic properties and other possibly detrimental effects of all milk replacers before they are used on a large scale. if the concentration of haemoglobin in the blood of calves drops below . mmol l - , the ability of the calf to be normally active as well as lymphocyte count and immune system function are substantially impaired, and there is reduced growth rate. below . mmol l - , veal calves exhibit a number of adaptations to iron deficiency, including elevated heart rate, elevated urinary noradrenaline and alterered reactivity of the hpa axis. there is a lack of data on the variability in groups of calves. hence, when haemoblogin levels are found to be below . mmol l - in groups of young veal calves, it is field practice to give supplementary iron. for older calves, including those in the last four weeks before slaughter, efficient production is possible in individual calves whose haemoglobin concentration is above . mmol l - . if the concentration of haemoglobin in blood is not checked at all, there is a high risk of anaemia that is associated with poor welfare, for all calves fed a diet with a very low iron content. anaemia can be identified and quantified adequately if checks are carried out on veal production calves of - weeks, for example, when the calves are brought into a unit, between - weeks of fattening, and during the last four weeks before slaughter. if the concentration of haemoglobin in the blood of a group of calves during the last four weeks before slaughter is a mean of . mmol l - , some calves may have a concentration substantially lower than the group-mean, and hence their welfare may be poor. in order to avoid anaemia levels that are associated with poor welfare because normal activity is difficult or not possible and other functions are impaired, it is advisable that diets should be provided that result in blood haemoglobin concentrations of at least . mmol l - throughout the life of the calf. in order to avoid serious impairment of immune system function and hence poor welfare, no individual calf should have a blood haemoglobin concentration lower than . mmol l - . in most cases this is achieved by adjusting the concentration of iron in the diet and having an adequate checking system so that the above condition is avoided. other treatment may be needed for calves with clinical conditions which cause anaemia but which are not related to diet. since the lowest haemoglobin concentrations in the blood of veal calves are usually reached during the last four weeks before slaughter, these blood concentrations should be checked at this time. such controls would help to see if measures are necessary to be taken or not. a checking system using a mean level, but whose aim is to avoid the risk of a low haemoglobin concentration in any individual lower than . mmol l - would have to use a mean substantially higher than , mmol l - , probably mmol l - , and an appropriate sample size. in order to avoid poor welfare associated with anaemia, as explained in the conclusions (above), measurements of average blood haemoglobin concentration are not a satisfactory means of avoiding poor welfare but the use of a minimum level of . mmol l - for individual calves would achieve this. there is a lack of data on the haemoglobin levels and variation in groups in slaughtering calves. to gain more information as a basis for further actions and recommendations, it is advisable to perform sampling of calves at slaughter, by checking the haemoglobin level on a random basis in groups of calves. space and pen design recommendations space should be enough to allow animals to fulfill their needs for social behaviour, lying and grooming. as the pen shape affects the use of space by animals, pens should be rectangular rather than square and pen space should be divided into different usable areas. as the floor type affects the resting and lying postures of calves it should be comfortable. wet floors should be avoided due to thermal and resting problems. degree of social contact conclusions group housing can help calves to acquire social skills. some experience of mixing is important as calves that have been reared for a while in groups dominate calves that have always been in individual crates. when calves are mixed together in the first few days of life, and then kept for some weeks in a social group, there may be poor welfare because of the following risks: . especially when individuals are provided with inadequate access to teats and roughage in the diet, cross-sucking and other abnormal sucking behaviour may occur. . some individuals may be unaccustomed to the food access method, for example they may have only received food via a teat, and may find it difficult to drink from a bucket. . calves coming from different buildings, perhaps from different farms, may carry different pathogens and hence there is a risk of disease spread in all the calves that are put in the same airspace or are otherwise exposed to the pathogens. since calves are social animals, they should be kept in social groups wherever possible. these groups should be stable with no mixing or not more than one mixing. it is advisable for calves in the first two weeks of life not to be mixed with other animals. if calves from different buildings, perhaps different farms, are to be mixed in a pen or are to be put in different pens in the same airspace, quarantining animals for - weeks can reduce disease in the calves and hence prevent poor welfare. although cross-sucking can sometimes be minimised by provision of teats, water and roughage, if this is not possible, mixing into groups could be delayed for three to four weeks. calves fed by various means may require careful supervision after being put into groups in order that they learn how to feed effectively. temperature, ventilation and air hygiene calf rearing causes significant emissions of substances such as nitrate, phosphate, heavy metals and possibly antibiotics in manure and liquid effluents. in addition, there are odours, gases, dusts, micro-organisms and endotoxins in the exhaust air from animal houses. also in the handling of manure in storage and during application of manure and during grazing. these effluents can have distinct impacts on air, water, soil, biodiversity in plants, forest decay and also on animals and including humans. calf houses possess a high potential for emissions of ammonia and other gases. dust, endotoxins and micro-organisms are emitted in lower amounts than from pig or poultry production. odour, bioaerosols, ammonia, nitrogen, phosphorous and heavy metals may either have a local or a regional impact. gases such as methane and nitrous oxide contribute to global warming. respiratory disorders are the second largest reason for morbility and mortality in calf rearing. the most important causes are environmental conditions such as hygiene, management and the physical, chemical and biological factors in the environment. ventilation plays a decisive role in reducing the incidence of respiratory disease. temperatures below ºc can compromise lung function. ammonia concentrations of more than ppm seem to increase respiratory infections. relative humidity of more than % bear the risk of increased heat dissipation and can help bacteria to survive in airborne state. air velocities close to the animals of more than . m/s can significantly increase respiratory sounds in calves. sufficient air space in confined buildings can help to reduce the concentration of airborne bacteria. calf houses contain relatively high amounts of endotoxins ( eu) (eu: endotoxin unit, see scientific report, www.efsa.eu.int) there is concern that antibiotic residues may contribute to the development of bacterial resistance. local and regional environmental problems are enhanced by high animal densities and insufficient distances between farms and residential areas. the exact quantitative contribution of calf rearing to environmental pollution and its impact on water, air, soil vegetation and nearby residents is not yet well understood. when housing systems are compared, although dust emission levels will seldom pose problems for the health of calves, ammonia emission levels may be high enough to exacerbate calf disease, especially when calves are kept in slatted floor units. the development of low emission production systems should be encouraged including mitigation techniques, e.g. biofilters, bioscrubbers, covered manure pits and shallow manure application. in particular there is need to reduce ammonia emissions from slatted floor units or to reduce the usage of such systems. adequate and efficient feeding regimes are required with minimal wastage of nitrogen and phosphorous and limited use of growth promoters and drugs. there is an urgent need for cooperative research to design appropriate ventilation systems to improve health and welfare of calves kept in confined rearing conditions. temperatures for young calves should range between and ºc. ammonia concentrations should be kept as low as possible preferably not more than ppm. housing and management should aim a reducing dust, bacteria and endotoxin concentrations in the animal house air. minimum ventilation rates of m per kg live weight should be applied. human-animal relationships recommendation stockpersons should be appropriately trained so that they have sufficient skills in rearing calves. they should have a positive attitude towards animals and work with them in order to minimise stress and to maintain a high quality of health control. rough contact (e.g. use of painful device such as an electric prod, loud noises) should be avoided and gentle contacts (e.g. talking softly, stroking, offering food) should be encouraged. this sort of contact is of particular importance for calves in groups or with their dam that tend not to approach humans readily. dehorning and castration if cattle are to be dehorned, it is recommend to disbud young cattle rather than to dehorn older ones. disbudding by cautery is recommended over other methods. local anaesthesia (e.g. - ml lidocaïne or lignocaïne % around the corneal nerve) and analgesia with an nsaid (e.g. ml flunixin meglumine or - . mg ketoprofen % / kg body weight) should be given - min before disbudding. if cattle are to be castrated, it is recommended to castrate calves as early as possible (no later than . mo and preferably at wk of age), to use the burdizzo method, and to provide appropriate anaesthesia and analgesia (e.g. ml lignocaine % in each testicle through the distal pole and mg ketoprofen % / kg body weight injected intravenously both min before castration). prevention of typical calf diseases in the first months of life such as diarrhoea and enzootic bronchopneumonia requires a systematic approach by improving management and housing conditions, specifically the preparation of the cow, hygiene of the calving environment, including dry clean bedding and high air quality, immediate supply with maternal antibodies, no mixing with older animals and careful attention and a rapid response to any sign indicating disease. main foodborne hazards associated with calf farming are salmonella spp., human pathogenic-verotoxigenic escherichia coli (hp-vtec), thermophilic campylobacter spp., mycobacterium bovis, taenia saginata cysticercus and cryptosporidium parvum/giardia duodenalis. the prevalence-level of infection and/or contamination of calves with, and further spread of, foodborne pathogens on farms depend on the status and the inter-relationship of different contributing factors that are inherently highly variable. present knowledge and published data are insufficient to produce a universal risk assessment enabling quantitative food safety categorization/ranking of different types of calf farming systems. nevertheless, generic principles for risk reductions for the main foodborne pathogens at calf farm level are known and are based on the implementation of effective farm management (e.g. qa, husbandry, herd health plans, biosecurity) and hygiene measures based on gfp-ghp. for quantitative food safety risk categorization of farming systems individually, and/or their related ranking, further scientific information is needed. accordingly, related research should be encouraged. the conclusions of the scientific veterinary committee report on the welfare of calves are presented in table below together with additions relevant in the light of this update of the svc report. the best conditions for young rearing calves involve leaving the calf with the mother in a circumstance where the calf can suckle and can subsequently graze and interact with other calves.c agreed where the calf will be separated from its mother at an early age, evidence suggests that it is normally beneficial for the calf if the mother is allowed to lick the calf thoroughly for a few hours after birth.c agreed r whenever possible, cows should be given the opportunity to lick the calf during at least three hours after parturition. it is important that the calf should receive sufficient colostrum within the first six hours of life and as soon as possible after birth, in conditions which facilitate antibody absorption, preferably by suckling from the mother, so as to ensure adequate immunoglobulin levels in the blood. r where necessary, suckling assistance or additional colostrum should be provided for calves left to suckle from the dam.r agreed agreed calves need resources and stimuli which are normally provided by their mothers. all calves should be given adequate food and water, appropriate conditions of temperature and humidity, adequate opportunities to exercise, good lying conditions, appropriate stimuli for sucking during the first few weeks of life and social contacts with other calves from one week of age onwards. specific aspects of housing and management which fulfill these conditions are detailed. agreed young calves reared without their mothers should receive considerate human contact, preferably from the same stockperson throughout the growing period.r agreed stockpersons should be appropriately trained so that they have sufficient skill in the rearing of calves. they should have positive attitudes towards animals and to working with them in order to handle them while minimising stress and to maintain a high quality of health control. rough contacts (e.g. use of a painful device such as an electric prod, or loud noises) should be avoided and gentle contacts (e.g. talking softly, petting, offering food) should be encouraged. these contacts are of particular importance for calves in groups or with their dam that may tend not to approach humans easily. where calves cannot be kept with their mother, the system where welfare is best is in groups with a bedded area and an adequate space allowance available to them.c agreed. r see below. the welfare of calves is very poor when they are kept in small individual pens with insufficient room for comfortable lying, no direct social contact and no bedding or other material to manipulate. c agreed r as the floor affects the resting and lying posture of calves they should be useful to have a comfortable floor. wet floors should be avoided due to thermal and resting problems. tethering always causes problems for calves. calves housed in groups should not be tethered except for periods of not more than one hour at the time of the feeding of milk or milk substitute. individually housed calves should not be tethered. r calves are vulnerable to respiratory and gastro-intestinal disease and welfare is poor in diseased animals. better husbandry is needed to minimize disease in group housing conditions but results that are as good as those from individual housing can be obtained. c every calf should be able to groom itself properly, turn around, stand up and lie down normally and lie with its legs stretched out if it wishes to do so. r in order to provide an environment which is adequate for exercise ,exploration and free social interaction, calves should be kept in groups. calves should never be kept at too high stocking density. the following requirements are based on evidence of increasingly poor welfare as space allowance decreases. the space allowance should provide, especially for allowing resting postures, an area for each calf of at least (its height at the withers) x (its body length from the tip to its nose when standing normally to the caudal edge of the tuber ischii or pin bone x . ). the length measurements takes account of the forward and backward movements involved in standing up and lying down. this calculation takes account of differences in size amog breeds and with age. as a guideline, for holstein calves this area is . m at weeks, . m at weeks and . m at weeks. r r since calves are social animals, they should be kept in social groups wherever possible. these groups should be stable with no mixing or not more than one mixing. it is advisable for calves in the first two weeks of life not to be mixed with other animals. c when calves are mixed together in the first few days of life, and then kept for some weeks in a social group, there may be poor welfare because of the following risks: -especially when individuals are provided with inadequate access to teats and roughage in the diet, cross-sucking and other abnormal sucking behaviour may occur. -some individuals may be unaccustomed to the food access method, for example they may have only received food via a teat, and may find it difficult to drink from a bucket. -calves coming from different buildings, perhaps from different farms, may carry different pathogens and hence there is a risk of disease spread in all the calves that are put in the same airspace or are otherwise exposed to the pathogens. r if calves from different buildings, perhaps different farms, are to be mixed in a pen or are to be put in different pens in the same airspace a quarantine situation should be used in order to reduce disease in the calves and hence prevent poor welfare. for a given space allowance per calf, increasing group size results in a larger total area and hence better possibilities for exercise, social interaction and improved environmental complexity. c larger groups are preferred because of the better possibilities for providing an adequate environment but there are limits to the numbers of animals which should be in one building section and risks associated with mixing of calves from different sources should be considered. r agreed r the space provided for calves should be enough to allow animals to fulfill their needs for social behaviour, lying and grooming. space allowance per animal should be greater for groups of - animals and for feeding systems, and pen shapes or flooring materials that necessitate extra space availability. if the preferred system, group housing, is not possible then individual pens whose width is at least the height of the calf at the withers and whose length is at least the length of the calf from the tip of its nose when standing normally to the caudal edge of the tuber ischii or pin bone x . should be used. this space requirement is calculated on the basis of the space required for normal agreed r as the pen shape affects the use of space by animals, pens should maximize the perimeter and pen space should be divided into different usable areas. movements and evidence of increasingly poor welfare r appropriate bedding for example straw is recommended. bedding must be changed at appropriate intervals and every calf should have access to a dry lying area. slatted floors must not be slippery and must not be a cause of tail tip necrosis. r agreed see above buildings should be adequately ventilated taking into account of the number of animals present and the external conditions. the air space in the building should be m per calf up to weeks of age and an amount of air space which increases with age is needed for older calves.r agreed c -calf rearing causes significant emissions such as nitrate, phosphate, heavy metals and possibly antibiotics in manure and liquid effluents as well as odour, gases, dusts, micro-organisms and endotoxins in the exhaust air from animal houses, from manure storage facilities, during application of manure and during grazing. -these effluents can have distinct impacts on air, water, soil, and thus also on animals. -calf houses possess a high potential for emissions of ammonia and other gases. dust, endotoxins and micro-organisms are emitted in lower amounts than from pig or poultry production. -respiratory disorders are the second largest reason for morbidity and mortality in calf rearing. the most important reason are environmental conditions such as hygiene, management and the physical, chemical and biological factors of the aerial environment. -ventilation plays a decicive role in reducing the incidence of respiratory diseases. temperatures below c can compromise lung function. -ammonia concentrations of more than ppm seem to increase respiratory affections. relative humidity of more than % bear the risk of increased heat dissipation and can help bacteria to survive in airborne state. -air velocities close to the animals of more than . m/s can increase respiratory sounds in calves significantly. -sufficient air space in confined buildings can help to reduce the concentration of airborne bacteria. -calf houses contain relatively high amounts of endotoxins. -there is concern that antibiotic residues may contribute to the development of bacterial resistance. -environmental problems in calf houses are enhanced by high animal densities, insufficient distances between farms. -when housing systems are compared, although dust emission levels will seldom pose problems for the health of calves, ammonia emission levels may be higher enough to exacerbate calf disease, especially in slatted floor units. r -the development of low emission production systems should be encouraged including mitigation techniques, e.g. biofilters, bioscrubbers, covered manure pits and shallow manure application. in particular there is need to reduce ammonia emissions from slatted floor units or to reduce the usage of such systems. -adequate and efficient feeding regimes are required with minimal wastage of nitrogen and phosphorous and limited use of growth promoters and drugs. -there is an urgent need for cooperative research to design appropriate ventilation systems to improve health and welfare of calves kept in confind rearing conditions. -temperatures for young calves should range between and c. -ammonia concentrations should be kept as low as possible, preferably not more than ppm. -housing design and management procedures should aim to reduce dust, bacteria and endotoxin concentrations in the animal house air. -minimum ventilation rates of c per kg live weight should be applied. calves which lack specific nutrients, including iron, which are given poorly balanced diet, and which are not provided with adequate roughage in the diet after four weeks of age can have serious health problems, can show serious abnormalities of behaviour, and can have substantial abnormalities in gut development. c every calf should receive a properly balanced diet with adequate nutrients.r agreed r it is recommended that solids feeds provided to veal calves, in addition to milk replacer, are adequately balanced in terms of the amount of fibrous material, which will promote rumination, and other components such as proteins and carbohydrates, which stimulate rumen development and support a healthy function of the digestive system. c if the concentration of haemoglobin in the blood of calves drops below . mmol l - , the ability of the calf to be normally active as well as the lymphocyte count and immune system function are substantially impaired, and there is reduced growth rate. below . mmol l - , veal calves exhibit a number of adaptations to iron deficiency, including elevated heart rate, elevated urinary noradrenaline and alterered reactivity of the hpa axis. hence it is normal practice to identify young veal production calves with less than . mmol l - haemoglobin in plasma and to provide supplementary iron in addition to that normally included in the diet. for older calves, including those in the last four weeks before slaughter, efficient production is possible in individual calves whose haemoglobin concentration is above . mmol l - . if the concentration of haemoglobin in blood is not checked at all, there is a high risk of anaemia that is associated with poor welfare, for all calves fed a diet with very low iron content. anaemia can be identified and quantified adequately if checks are carried out on veal production calves of - weeks, for example, when the calves are brought into a unit, between - weeks of fattening, and during the last four weeks before slaughter. if the concentration of haemoglobin in the blood of a group calves during the last four weeks before slaughter is a mean of . mmol l - , some calves may have a concentration substantially lower than the group-mean, and hence their welfare may be poor. r in order to avoid anaemia levels that are associated with poor welfare because normal activity is difficult or not possible and other functions are impaired, it is advisable that diets should be provided that result in blood haemoglobin concentrations of at least . mmol l - throughout the life of the calf. in order to avoid serious impairment of immune system function and hence poor welfare, no individual calf should have a blood haemoglobin concentration lower than . mmol l - . in most cases this is achieved by adjusting the concentration of iron in the diet and having an adequate checking system so that the above condition is avoided. other treatment may be needed for calves with clinical conditions which cause anaemia but which are not related to diet, r since the lowest haemoglobin concentrations in the blood of veal calves are usually reached during the last four weeks before slaughter, these blood concentrations should be checked at this time. such controls would help to see if measures are necessary to be taken or not. a checking system using a mean level, but whose aim is to avoid the risk of a low haemoglobin concentration in any individual lower than . mmol l- , would have to use a mean substantially higher than , mmol l- , probably mmol l- . in order to avoid poor welfare associated with anaemia, as explained in the conclusions (above), measurements of average blood haemoglobin concentration are not a satisfactory means of avoiding poor welfare but the use of a minimum level of . mmol l - for individual calves would achieve this. some non-milk proteins are inappropriate for use in a milk substitute fed to calves because they produce allergenic reactions. some carbohydrates cannot be easily or properly digested by calves and they may cause digestive upset. no milk substitute should be fed to calves unless it can be easily digested and does not cause harmful reactions in the calves. r acidification of milk can reduce the incidence of diarrhoea, but any forms of acidified milk which are unpalatable to calves or which harm the calves should not be used. r every calf should be fed fermentable material, appropriate in quality and sufficient in quantity to maintain the microbial flora of the gut and sufficient fibre to stimulate the development of villi in the rumen. roughage, in which half of the fibre should be at least mm in length, should be fed to calves. they should receive a minimum of g of roughage per day from to weeks of age, increasing to g per day from to weeks of age but it would be better if these amounts would be doubled. the development of the rumen should be checked by investigating villi development in a proportion of calf guts after slaughter. r agreed r without a fully functional rumen, calves will be unable to utilise nutrients provided in the post-weaning dry feed diet. attention should paid to type of forage and consistent of particle size of starter grain in order to achieve a proper rumen development. calf weaning should be based on the amount of dry feed calves ingest per day, not on their age or weight, and calf starter should be made available five to days after birth. a calf consuming . kg of dry feed or more on three consecutive days is ready for weaning. when calves are fed low levels of milk to encourage early consumption of dry food, weaning can be done abruptly. in contrast, if milk is given in large amounts, weaning may require two to three weeks of slow transition to avoid a setback in growth. there are clear signs of increased disease susceptibility and immunosuppression in calves up to weeks of age, whose blood haemoglobin concentration is below . mmol/liter. however, in some studies the antibiotic treatment was not higher in calves whose haemoglobin was near to mmol/litre than in calves whose level was near to mmol/litreat weeks of age. studies of exercise in anaemic calves show that there can be problems during exercise at a level of , mmol/litre. c all calves should be fed in such a agreed see way that their haemoglobin level does not fall below a minimum of . mmol/litre. r where calves are fed a diet which is lower in iron than mg/kg, an adequate sample of animals should be checked at and weeks of age in order to find out whether the blood haemoglobin concentration is too low. r agreed see young calves have a very strong preference to suck a teat or teat-like object. it is preferable for calves to be fed milk or milk substitute from a teat during the first four weeks of life. calf welfare is improved if a non-nutritive teat is provided during the first four weeks of life especially if they are not fed from a teat. c when young group-housed calves are fed milk or milk substitute, the social facilitation effects of having a group of teats close together are beneficial. it is also advisable for several teats to be provided in groups of older calves. transponder controlled feeder systems have been found to work well. c the feeding to calves of large quantities of milk or milk substitute in a single daily meal can cause digestive problems. hence when calves are fed more than % of body weight in milk or milk substitute each day, this should be fed in at least two meals per day. r calves fed ad libitum, or close to this level should not be weaned off milk or milk replacer until they are consuming a minimum of g of concentrates per head per day in the week prior to weaning. where calves are fed restricted quantities of milk or milk replacer before weaning they should not be weaned until they are consuming a minimum of g of concentrates per head per day in the week prior to weaning. r calves which are diseased and calves which are in hot conditions often need to drink water as well as milk or milk substitute and all calves drink water if it is available. the provision of milk or milk substitute is not an adequate alternative for provision of water. hence calves should be provided daily with water to drink. it is recommended that drinkers be provided in all pens. r agreed r prevention of typical calf diseases in the first months of life such as diarrhoea and enzootic bronchopneumonia requires a systematic approach by improving management and housing conditions, specifically the preparation of the cow, hygiene of the calving environment, including dry clean bedding and high air quality, immediate supply with maternal antibodies, no mixing with older animals and careful attention and early reaction of all signs of any beginning diseases. dehorning calves between - weeks by cauterisation with adequate anaesthesia and analgesia (no precision given) castrate calves at months with adequate anaesthesia and analgesia (no precision given) r dehorning: if cattle are to be dehorned, it is recommended to disbud young cattle rather than to dehorn older ones. disbudding by cauterisation is recommended over other methods. local anaesthesia (e.g. - ml lidocaïne or lignocaïne % around the corneal nerve) and analgesia with a non steroidal anti-inflammatory drug ( ml flunixin meglumine or - . mg ketoprofen % / kg body weight) shall be performed - min before disbudding. r castration:if cattle are to be castrated, it is recommended to castrate calves as early as possible (no later than . mo and preferably at wk of age), to use the burdizzo method, and to provide appropriate anaesthesia and analgesia (e.g. ml lignocaine % in each testicle through the distal pole and mg ketoprofen % / kg body weight injected intravenously both min before castration). -main foodborne hazards associated with calf farming are salmonella spp., human pathogenic-verotoxigenic escherichia coli (hp-vtec), thermophilic campylobacter spp., mycobacterium bovis, taenia saginata cysticercus and cryptosporidium parvum/giardia duodenalis. -the prevalence-level of infection and/or contamination of calves with, and further spread of, foodborne pathogens on farms depend on the status and the inter-relationship of different contributing factors that are inherently highly variable. -present knowledge and published data are insufficient to produce a universal risk assessment enabling quantitative food safety categorization/ranking of different types of calf farming systems. -nevertheless, generic principles for risk reductions for the main foodborne pathogens at calf farm level are known and are based on the implementation of effective farm management (e.g. qa, husbandry, herd health plans, biosecurity) and hygiene measures based on gfp-ghp recommendations for future research . it is recommended that future research should be conducted within the following areas: -hemoglobin levels and iron deficiences of veal calves aged - weeks. -the monitoring of haemoglobin in groups of calves using representative samples -exposure to allergenic proteins -solid and liquid food balance. exposure to too rich diets and changes in feed composition. -space requirements -health monitoring systems and the effect of such on clinical health in calves -infection transmission (respiratory and digestive diseases) due to direct contact between calves in relation to social benefits of mixing -pain relief when disbudding, dehorning and castrating calves -design of appropriate ventilation systems for calves in confined rearing conditions -health and environmental effects of feeding minerals as antimicrobial agents -for quantitative food safety risk categorization of farming systems individually, and/or their related ranking, further scientific information is needed. accordingly, related research should be encouraged. references used in this scientific opinion are available and listed in the scientific report published at the efsa web (www.efsa.eu.int). the ahaw panel wishes to thank the members of the working group chaired by panel member summary efsa has been requested by the european commission to issue a scientific opinion on animal health and welfare aspects of intensive calf farming systems and their ability to comply with the requirements of the well-being of calves from the pathological, zootechnical, physiological and behavioural points of view. in particular the commission asked efsa to update the findings of the scientific veterinary committee (animal welfare section) report on the welfare of calves of november in light of more recent data on this issue. where relevant the possible food safety implications of different farming systems should also be considered. in this report a risk assessment was made and the relevant conclusions and recommendations are forming the scientific opinion by the ahaw panel. the svc ( ) report contains information on measurements of welfare, needs of calves, descriptions of current housing systems, chapters on types of feed and feeding systems, weaning of calves, housing and pen design, climate, mananimal relationships, dehorning and castration. further chapters covered economical considerations of systems and for improving welfare. in the report conclusions were made on general management, housing, food and water and economics. the present report "the risks of poor welfare in intensive calf farming systems" is an update o the previous svc report with the exception of economical aspects which are outside of the mandate for this report. the various factors potentially affecting calves' health and welfare, already extensively listed in the report of the scientific veterinary committee animal welfare section (svc, ) , are updated and subsequently systematically determined whether they constitute a potential hazard or risk. to the latter end their severity and likelihood of occurrence in animal (sub) populations were evaluated and associated risks to calf welfare estimated, hence providing the basis for risk managers to decide which measures could be contemplated to reduce or eliminate such risks. in line with the terms of reference the working group restricted itself to (in essence a qualitative) risk assessment although it is agreed that welfare and health of calves can be substantially affected in the course of and as a result of transport and slaughter, this report does not consider animal health and welfare aspects of calves during transport and slaughter but such information can be found in a recently issued comprehensive report of the scientific committee on animal health and animal welfare (scahaw), on "the welfare of animals during transport (details for horses, pigs, sheep and cattle)" which was adopted on march (dg sanco, ) and in the efsa report "welfare aspects of animal stunning and killing methods" (efsa, b) . in relation with the food safety aspects, main foodborne hazards associated with calf farming are salmonella spp., human pathogenic-verotoxigenic escherichia coli (hp-vtec), thermophilic campylobacter spp., mycobacterium bovis, taenia saginata cysticercus and cryptosporidium parvum/giardia duodenalis. present knowledge and published data are insufficient to produce a universal risk assessment enabling quantitative food safety categorization/ranking of different types of calf farming systems. nevertheless, the main risk factors contributing to increased prevalence/levels of the above foodborne pathogens, as well as generic principles for the risk reductions are known. the latter are based on the implementation of effective farm management (e.g. qa, husbandry, herd health plans, biosecurity) and hygiene measures (e.g. gfp-ghp). in general, the conclusions made in the previous svc report remain. however, recent research has provided for some additional conclusions. the risk analysis is presented in the tables of annex . the graphics in this table are not intented to represent numerical relationships but rather qualitative relations. in some instances the exposure could not be estimated due to lack of data, in which cases the risks where labelled "exposure data not available". the following major and minor risks for poor animal health and welfare have been identified for one or several of the various husbandry systems considered: the hazards of iron deficiency and insufficient floor space are considered to be very serious, the hazard of inadequate health monitoring is considered to be serious and the hazards of exposure to inadequate hemoglobin monitoring, allergenic proteins and too rich diet are considered to be moderately serious. for these hazards, there is no consensus on the exposure of calves mainly due to lack of data and that is why it is recommended that further studies should be made to provide evidence for an exposure assessment. regarding castration and dehorning (and disbudding) without anaesthetic drugs, there is a variation in relation to national legislation why the risk of poor welfare in relation to castration and dehorning has a wide range between countries. tables which clarify the risk assessment have been included in annex . calf a calf is a young bovine which is significantly younger and smaller in size than an adult of the same species and breed and which is not reproductively active. there is a gradual transition from a newborn animal, dependent on milk, to an animal with many adult characteristics. few people would use the term calf for domestic cattle of - months whilst most would call an animal of months or somewhat older a calf. in this report, calf is used for animals of up to months of age. however, in deciding on the end of the calf stage, any definition based on age or weight is arbitrary. the term calf is not normally restricted to animals that are unweaned or monogastric rather than having some degree of development of the rumen for its specialist function. the removal of the horn bud or the actual horn depending on the breed and the age of the animal. endotoxin unit (eu) endotoxin activity of . ng of reference endotoxin standard, ec- or eu/ng (fda). to convert from eu's into ng, the conversion is eu/ng. a process where water bodies receive excess nutrients that stimulate excessive plant growth (i.e. water pollution). intensively reared calf a calf which is not kept extensively at pasture. according to the council of europe european convention for the protection of animals kept for farming purposes, (chapter i, article ), "modern intensive animal farming systems are systems in which mainly technical facilities are used that are primarily operated automatically and in which the animals depend on the care of and supply from the farmer". nsaid non-steroidal anti-inflammatory drug. the process by which a mother mammal allows a young animal to obtain milk from its teats. odds ratio (or) the odds ratio is a measure of effect size particularly important in bayesian statistics and logistic regression. infection of the navel. meat produced from animals slaughtered at - weeks of age and supplied with roughage from at least months of age onwards. there is not any classification system for veal carcasses agreed across the eu. the only existing classification system would rather relate to a general beef carcass classification system, which comprises the following categories: however, these categories are valid for cattle having a live weight of more than kg. consequently, some member states have issued their own national schemes for veal carcass classification. in trade, there is agreement between importing and exporting countries that veal originates from calves which were fed predominantly milk replacers, and which displays a light colour. the age limit is around months. some countries such as the netherlands market meat of animals of the age of to months, as pink veal. the eu subsidies scheme represents an important incentive for pink veal production. the determination of the relationship between the magnitude of exposure of calves to a certain hazards and the severity and frequency of associated adverse effects on calf welfare. the quantitative and qualitative evaluation of the likelihood of hazards to welfare occurring in a given calf population. any factor, occurring from birth to slaughter, with the potential to cause an adverse effect on calf welfare. the qualitative and quantitative evaluation of the nature of the adverse effects associated with the hazard. considering the scope of the exercise of the working group the concerns relate exclusively to calf welfare. the identification of any factor, from birth to slaughter, capable of causing adverse effects on calf welfare. a function of the probability of an adverse effect and the severity of that effect, consequent to a hazard for calf welfare. the process of determining the qualitative or quantitative estimation, including attendant uncertainties, of the probability of occurrence and severity of known or potential adverse effects on welfare in a given calf population based on hazard identification, hazard characterisation, and exposure assessment. unaltered remain the following cac (codex alimentarius commission) definitions (note: for completeness all definitions used by cac -while not necessarily used in this document -have been included): a risk assessment that provides numerical expressions of risk and an indication of the attendant uncertainties (stated in the expert consultation definition on risk analysis). a risk assessment based on data which, while forming an inadequate basis for numerical risk estimations, nevertheless, when conditioned by prior expert knowledge and identification of attendant uncertainties, permits risk ranking or separation into descriptive categories of risk. a process consisting of three components: risk assessment, risk management and risk communication. a scientifically based process consisting of the following steps: i) hazard identification, ii) hazard characterisation, iii) exposure assessment and iv) risk characterisation. the interactive exchange of information and opinions concerning the risk and risk management among risk assessors, risk managers, consumers and other interested parties. output of risk characterisation. the process of weighing policy alternatives in the light of the results of risk assessment and, if required, selecting and implementing appropriate control options (i.e. prevention, elimination, or reduction of hazards and /or minimization of risks) options, including regulatory measures. a method to examine the behaviour of a model by measuring the variation in its outputs resulting from changes to its inputs. characteristics of a process where the rationale, the logic of development, constraints, assumptions, value judgements, decisions, limitations and uncertainties of the expressed determination are fully and systematically stated, documented, and accessible for review. a method used to estimate the uncertainty associated with model inputs, assumptions and structure/form. the process by which a young mammal obtains milk from the teat of its mother or another lactating female by sucking. the term veal refers to the meat produced from calves, principally those of the species bos taurus and bos indicus. there are several meat products from calves. they are generally distinguished by their colour: "pale" or "white" veal is generally produced from an animal under months of age and fed mostly milk or milk replacer; "pink" veal is generally produced from an animal of up to months fed larger amounts of solid foods and possibly weaned. meat from calves of - months is called young beef. weaning, weaned in mammals, weaning is a gradual process during which the young animal receives less and less milk from its dam and consumes more and more solid food. it is accompanied by changes in the dam-offspring relation. in farming, calves are often separated from their dams soon after birth and receive milk (or milk replacer) from humans or a machine. although separated from the dam, calves are considered as un-weaned as long as they are fed milk. suckler calves are left with their dam for some months and are generally weaned some time before the next calving by separating them suddenly from the dam. calves normally commence eating solid food at - weeks, although some start earlier, and they eat enough solid food for development of a functional rumen to start by about weeks of age. a weaned animal is one that no longer needs to suckle and so does not consume milk in any significant quantity indicating that the weaning process has finished. council directive / /eec laying down minimum standards for the protection of calves as amended by council directive / /ec requires the commission to submit to the council a report, based on a scientific opinion, on intensive calf farming systems which comply with the requirements of the wellbeing of calves from the pathological, zootechnical, physiological and behavioural points of view. the commission's report will be drawn up also taking into account socio-economic implications of different calf farming systems. it should be noted that the scientific veterinary committee (animal welfare section) adopted a report on the welfare of calves on november (svc, ) which should serve as background to the commission's request and preparation of the new efsa scientific opinion. in particular the commission requires efsa to consider the need to update the findings of the scientific veterinary committee's opinion in light of the availability of more recent data on this issue. where relevant the possible food safety implications of different farming systems should also be considered. efsa has been requested by the european commission to issue a scientific opinion on animal health and welfare aspects of intensive calf farming systems and their ability to comply with the requirements of the well-being of calves from the pathological, zootechnical, physiological and behavioural points of view. in particular the commission requires efsa to update the findings of the scientific veterinary committee (animal welfare section) report on the welfare of calves of november in light of more recent data on this issue. where relevant the possible food safety implications of different farming systems should also be considered. the mandate outlined above was accepted by the panel on animal health and welfare (ahaw) at the plenary meeting, on / march . it was decided to establish a working group of ahaw experts (wg) chaired by one panel member. therefore the plenary entrusted a scientific report and risk assessment to a working group under the chairmanship of prof. bo algers. the members of the working group are listed at the end of this report. this report is considered for the discussion to establish a risk assessment and the relevant conclusions and recommendations forming the scientific opinion by the ahaw panel. according to the mandate of efsa, ethical, socio-economic, cultural and religious aspects are outside the scope of this scientific opinion. in , the scientific veterinary committee of the european commission published the report on the welfare of calves. the svc ( ) report contains information on measurements of welfare, needs of calves, descriptions of current housing systems, chapters on types of feed and feeding systems, weaning of calves, housing and pen design, climate, mananimal relationships, dehorning and castration. further chapters covered economic considerations of systems and for improving welfare. in the report conclusions were made on general management, housing, food and water and economics. the present report "the risks of poor welfare in intensive calf farming systems" is an update of the previous svc report with the exception of economic aspects which are out of the mandate for this report. this report represents an update of the previous svc report ( ) with a risk assessment perspective. factors which are important for calf welfare include housing (space and pen design, flooring and bedding material, temperature, ventilation and air hygiene), feeding (liquid feed, concentrates, roughage) and management (grouping, weaning, human-animal relations). the measures used to assess welfare include behavioural and physiological measures, patho-physiological measures and clinical signs as well as production measures. as explained in the glossary, in this report young bovines are called calves up to a maximum of eight months of age and veal is the meat of a calf. countries with substantial production of veal are france, italy, the netherlands, belgium, spain and germany. significant veal production exists also in portugal, austria and denmark, the production of white veal, from calves that have been fed predominantly milk replacer and which has a light colour, takes place largely in france, the netherlands, belgium and italy. the eu subsidies scheme represents an important incentive for pink veal production. most calves produced for further rearing are in france, germany, uk, ireland and italy. the ways of keeping calves vary considerably from country to country and between breeds. most dairy calves are separated from their dam at birth and artificially fed whereas calves from beef breeds generally suckle their dam. according to eu statistics, in in the eu ( ) , , calves were reared for slaughter (table ) and , , calves were reared for other reasons than slaughter (table ). in total (table ) , tonnes of calf meat were produced in eu ( ) which probably implies that about , tonnes were produced in eu ( ) during . human consumption of meat from calves decreased slightly from to in eu ( ) ( table ). : item and element selected in the eurostat database to make the query and extract the data the working group set out to produce a document in which the various factors potentially affecting calves' health and welfare [already extensively listed in the report of the scientific veterinary committee animal welfare section (svc, ) , are updated and subsequently to systematically determine whether these factors constitute a potential hazard or risk. to the latter end their severity and likelihood of occurrence in animal (sub) populations were evaluated and associated risks to calf welfare estimated, hence providing the basis for risk managers to decide which measures could be contemplated to reduce or eliminate such risks. it should be noted, however, that this does not imply that a hazard that has a serious effect on just a few animals should not be dealt with by managers on farm level as the suffering imposed on some animals constitute a major welfare problem for those individuals. in line with the terms of reference the working group restricted itself to (in essence qualitative) risk assessment, i.e. only one of three elements essential to risk analysis a risk assessment approach was followed, similar to the one generally adopted when assessing microbiological risks, i.e. along the lines suggested at the nd session of the codex alimentarius commission (cac, ) . incidentally, these guidelines have been characterized by the cac as 'interim' because they are subject to modifications in the light of developments in the science of risk analysis and as a result of efforts to harmonize definitions across various disciplines. cac's guidelines are in essence exclusively formulated for the purpose of assessing risks related to microbiological, chemical or physical agents of serious concern to public health. consequently -considering their disciplinary focus -the working group had to adapt the cac definitions to serve their purpose. these adapted definitions, have, in alphabethical order, been included in chapter (see risk analysis terminology). the objectives of this report are to review and report recent scientific literature on the welfare including the health of intensively reared calves, to report on recent findings as an update to the scientific veterinary committee's previous report, to make a qualitative risk assessment concerning the welfare of intensively kept calves. where relevant, food safety implications of different farming systems are also considered. the report is structured in five major parts. the first three follow the scientific veterinary committee's previous report "on the welfare of calves" with introductory chapters - on background, measurements and needs in relation to calf welfare, chapter describing housing, diet and management and chapter describing comparison of systems and factors. in chapter common disease and use of antibiotics is described. the other two parts involve aspects of meat quality and food safety (chapter ) and the risk assessment (chapter ). conclusions and recommendations from the previous svc document together with updated conclusions derived from recent research findings are presented in the scientific opinion (www.efsa.eu.int). effect of transport and slaughter on calves' health and welfare although it is agreed that welfare and health of calves can be substantially affected in the course of and as a result of transport, this report does not consider animal health and welfare aspects of calves during transport because there is already a comprehensive recent report of the scientific committee on animal health and animal welfare (scahaw), on "the welfare of animals during transport (details for horses, pigs, sheep and cattle)" which was adopted on march (dg sanco, . the report takes into account all aspects related with transport that could affect the health and welfare of cattle and calves, including the direct effects of transport on the animals and the effects of transport on disease transmission. the loading methods and handling facilities for cattle, the floor space allowance, the relationships of stocking and the density requirements, the vehicle design, space requirements and ventilation for cattle transporters (see also the ahaw scientific opinion related to standards for the microclimate inside animal road transport vehicles; efsa, ), the behaviour of cattle during road transport, the road conditions, long distance transport and the travel times are also reviewed. recommendations for all these aspects are also given in that report. the following general requirements in relation to animal welfare were annexed as a protocol to the eu treaty of amsterdam in : "in formulating and implementing the community's agriculture, fisheries, transport, and internal market policies, the community and the member states shall pay full regard to the welfare requirements of animals, while respecting the legislative provisions and customs of member states relating to religious rites, cultural traditions and regional heritage." in the introduction to the proposed eu constitution, the following extended wording is included: "in formulating and implementing the european union's agriculture, fisheries, transport, internal market, research and technological development and space policies, the union and the member states shall pay full regard to the welfare requirements of animals, as sentient beings, while respecting the legislative provisions and customs of member states relating to religious rites, cultural traditions and regional heritage." this wording reflects the ethical concerns of the public about the quality of life of the animals. it also takes into account customs and cultural traditions. farm animals are subject to human imposed constraints and for a very long time the choice of techniques has been based primarily on the efficiency of production systems for the provision of food. however it is an increasingly held public view that we should protect these animals against mistreatment and poor welfare. in order to promote good welfare and avoid suffering, a wide range of needs must be fulfilled. these needs may require the animal to obtain resources, receive stimuli or express particular behaviours (hughes and duncan, ; jensen and toates, ; vestergaard, ) . to be useful in a scientific context, the concept of welfare has to be defined in such a way that it can be scientifically assessed. this also facilitates its use in legislation and in discussions amongst farmers and consumers. welfare is clearly a characteristic of an individual animal and is concerned with the effects of all aspects of its genotype and environment on the individual (duncan, ) . broom ( ) defines it as follows: the welfare of an animal is its state as regards its attempts to cope with its environment. welfare therefore includes the extent of failure to cope, which may lead to disease and injury, but also ease of coping or difficulty in coping. furthermore, welfare includes pleasurable mental states and unpleasant states such as pain, fear and frustration (duncan, ; fraser and duncan, ) . feelings are a part of many mechanisms for attempting to cope with good and bad aspects of life and most feelings must have evolved because of their beneficial effects (broom, ) . although feelings cannot be measured directly, their existence may be deduced from measures of physiology, behaviour, pathological conditions, etc. feelings cannot be directly measured and therefore care is necessary to avoid uncritical anthropomorphic interpretations (morton et al., ) . good welfare can occur provided the individual is able to adapt to or cope with the constraints to which it is exposed. hence, welfare varies from very poor to very good and can be scientifically assessed. measures which are relevant to animal welfare during housing, i.e. largely longterm problems, are described by broom and johnson ( ) and by broom ( broom ( , a . production criteria have a place in welfare assessment. however, although failure to grow, reproduce etc. often indicates poor welfare, high levels of production do not necessarily indicate good welfare. physiological measurements can be useful indicators of poor welfare. for instance, increased heart-rate, adrenal activity, or adrenal activity following acth challenge, or reduced heart-rate variability, or immunological response following a challenge, can all indicate that welfare is poorer than in individuals which do not show such changes. the impaired immune system function and some of the physiological changes can indicate the pre-pathological state (moberg, ) . in interpreting physiological measurements such as heart rate and adrenal activity it is important to take account of the environmental and metabolic context, including activity level. behavioural measures are also of particular value in welfare assessment (wiepkema, ) . the fact that an animal avoids an object or event, strongly gives information about its feelings and hence about its welfare (rushen, ) . the stronger the avoidance the worse the welfare whilst the object is present or the event is occurring. an individual, whom is completely unable to adopt a preferred lying posture despite repeated attempts will be assessed as having poorer welfare than one which can adopt the preferred posture. other abnormal behaviour which includes excessively aggressive behaviour and stereotypes, such as tongue-rolling in calves, indicates that the perpetrator's welfare is poor. very often abnormal activities derive from activities that cannot be expressed but for which the animal is motivated. for example, calves deprived of solid foods and hence lacking the possibility of nutritive biting, develop non-nutritive biting. whether physiological or behavioural measures indicate that coping is difficult or that the individual is not coping, the measure indicates poor welfare. studies of the brain inform us about the cognitive ability of animals and they can also tell us how an individual is likely to be perceiving, attending to, evaluating, coping with, enjoying, or disturbed by its environment so can give direct information about welfare (broom and zanella, ) . in studies of welfare, we are especially interested in how an individual feels. as this depends upon highlevel brain processing, we have to investigate brain function. abnormal behaviour and preferred social, sexual and parental situations may have brain correlates. brain measures can sometimes explain the nature and magnitude of effects on welfare. the word "health", like "welfare", can be qualified by "good" or "poor" and varies over a range. however, health refers to the state of body systems, including those in the brain, which combat pathogens, tissue damage or physiological disorder (broom and kirkden, ; broom, ) . welfare is a broader term than health, covering all aspects of coping with the environment and taking account of a wider range of feelings and other coping mechanisms than those associated with physical or mental disorders. disease, implying that there is some pathology, rather than just pathogen presence, always has some adverse effect on welfare (broom and corke, ). the pain system and responses to pain are part of the repertoire used by animals to help them to cope with adversity during life. pain is clearly an important part of poor welfare (broom, b) . however, prey species such as young cattle and sheep may show no behavioural response to a significant degree of injury (broom and johnson, ) . in some situations responses to a wound may not occur because endogenous opioids which act as analgesics are released. however, there are many occasions in humans and other species when suppression of pain by endogenous opioids does not occur (melzack et al., ) . studies of the brain inform us about the cognitive ability of animals and they can also tell us how an individual is likely to be perceiving, attending to, evaluating, coping with, enjoying, or disturbed by its environment so can give direct information about welfare (broom and zanella, ) . in studies of welfare, we are especially interested in how an individual feels. as this depends upon high-level brain processing, we have to investigate brain function. abnormal behaviour and preferred social, sexual and parental situations may have brain correlates. brain measures can sometimes explain the nature and magnitude of effects on welfare. the majority of indicators of good welfare which we can use are obtained by studies demonstrating positive preferences by animals (dawkins, ) . methods of assessing the strengths of positive and negative preferences have become much more sophisticated in recent years. the price which an animal will pay for resources, or pay to avoid a situation, may be, for example, a weight lifted or the amount of energy required to press a plate on numerous occasions. the demand for the resource, i.e. the amount of an action which enables the resource to be obtained, at each of several prices can be measured experimentally. this is best done in studies where the income available, in the form of time or energy, is controlled in relation to the price paid for the resource. when demand is plotted against price, a demand curve is produced. in some studies, the slope of this demand curve has been measured to indicate price elasticity of demand but in recent studies (kirkden et al., ) it has become clear that the area under the demand curve up to a particular point, the consumer surplus, is the best measure of strength of preference. once we know what animals strongly prefer, or strongly avoid, we can use this information to identify situations which are unlikely to fulfil the needs of animals and to design better housing conditions and management methods (fraser and matthews, ) . however, as pointed out by duncan ( duncan ( , , all data from preference studies must be interpreted taking account of the possibilities that, firstly, an individual may show a positive preference for something in the shortterm which results in its poor welfare in the long-term, and secondly, that a preference in a simplified experimental environment needs to be related to the individual's priorities in the more complicated real world. each assessment of welfare will pertain to single individual and to a particular time range. in the overall assessment of the impact of a condition or treatment on an individual, a very brief period of a certain degree of good or poor welfare is not the same as a prolonged period. however, a simple multiplicative function of maximum degree and duration is often not sufficient. if there is a net effect of poor welfare and everything is plotted against time, the best overall assessment of welfare is the area under the curve thus produced (broom, c) . . the needs and functioning of calves . . the concept of needs in assessing the needs and functioning of calves, many different approaches can be taken. one is to study, at a fundamental level, the physiology and behaviour of cattle and the ways in which they have evolved, in order to try to understand their causation and function. needs are in the brain but may be fulfilled by obtaining resources, physiological change, or carrying out a behaviour. in order to conclude that a need exists to show certain behaviour, it is necessary to demonstrate that the calves used in modern production systems are strongly motivated to show the behaviour and that, if the need is not provided for, there are signs of poor welfare such as abnormal behaviour or physiology or pathological effects (see chapter ). where the housing design allows the animals to show the behaviour that they need to show, this will promote the avoidance of poor welfare. a need is a requirement, which is a consequence of the biology of the animal, to obtain a particular resource or respond to a particular environmental or bodily stimulus. an animal may have a need that results in the existence at all times of mechanisms within the brain and abilities to perceive stimuli and respond appropriately. however, this does not mean that every individual at all times needs to carry out the response. for example, a calf has a need to avoid attack by a predator but it does not need to carry out anti-predator behaviour if no individual perceived as a predator is present. there are some needs which require urgent fulfilment, otherwise the body functioning will be impaired and in the medium or long term, the animal may suffer. for example, an adequate amount of an essential nutrient or avoidance of exposure to a serious disease. there are other needs which, if not fulfilled lead to frustration and excessive activities in an attempt to fulfil the need. the resulting poor welfare may be extreme and prolonged. needs to avoid predation and other danger mean that animals have a negative experience in some situations. close human presence and handling of animals may elicit physiological and behavioural anti-predator responses. the avoidance of such situations can also be considered as a need. calves require space to perform activities such as resting, feeding, exploring, interacting and escaping from perceived danger. to assess what risks of poor welfare are involved when the housing circumstances do not allow certain activities, it can be helpful to consider why the calves are intrinsically motivated to perform the activities. the selection criteria applied to modern cattle genotypes have resulted in changes in morphological phenotype. although these have not altered the categories of needs of calves, they may have altered rates of growth and energy partitioning so that the timing of problems and the probability that they will arise may be changed. the overall need of calves is to maintain bodily integrity while growing and preparing for adult life. in order to do this, calves have a series of needs that are relevant to the housing and management conditions imposed upon them by humans. the needs of calves are described in detail by broom ( broom ( , . in listing needs and in later consideration of how to provide for them, it is assumed that extreme human actions, such deliberately creating a large wound or infecting an animal with a dangerous pathogen, will not occur. the list of needs is not in order of importance. some of the needs mentioned here are discussed at greater length in the previous report. . . . to breathe calves need air that has sufficient oxygen and a low level of noxious gases in it. calves may be adversely affected by some of the gaseous products of the breakdown of animal faeces and they show preferences that help them to avoid any harm that they may cause. calves need to rest and sleep in order to recuperate and avoid danger. they need to use several postures which include one in which they rest the head on the legs and another in which the legs are fully stretched out (de wilt, ; smits, , ) . sleep disruption may occur if comfortable lying positions cannot be adopted or if there is disturbance to lying animals because they are trodden on or otherwise disturbed by other calves. exercise is needed for normal bone and muscle development. calves choose to walk at intervals if they can, show considerable activity when released from a small pen and have locomotor problems if confined in a small pen for a long period (warnick et al., ; dellmaier et al., ; trunkfield et al., ) . calves living in natural conditions would be very vulnerable to predation when young. as a consequence, the biological functioning of calves is strongly adapted to maximise the chance of recognition of danger and escape from it. calves respond to sudden events and approaches by humans or other animals perceived to be potentially dangerous with substantial sympathetic nervous system and hypothalamic-pituitary-adrenocortical (hpa) changes. these physiological changes are followed by rapid and often vigorous behavioural responses. fear is a major factor in the life of calves and has a great effect on their welfare. . . . to feed and drink . . . . sucking the calf needs to attempt to obtain nutrients at a very early stage after birth and shows behavioural responses that maximise this chance. as a consequence, from an early age, calves have a very strong need to show sucking behaviour and if a calf is not obtaining milk from a real or artificial teat, it sucks other objects (broom, (broom, , metz, ; hammell et al., ; jung and lidfors, ) . the need of the calf is not just to have the colostrum or milk in the gut but also to carry out the sucking behaviour on a suitable object (jensen, ) . further, the sucking is of importance for the release of gastrointestinal hormones. it has been shown in calves that oxytocin is released during milk ingestion. the amount released, however, was less in calves drinking their milk from a bucket compared with calves suckling the dam (samuelsson, ) . peripheral oxytocin stimulates the release of glucagon from the pancreas whereas central oxytocin increases hunger and the release of gastrointestinal hormones promoting growth (stock et al., ; björkstrand, ) . in the early days after birth, calves are motivated to suck and obtain milk. however, calves also have a need to obtain sufficient water and will drink water even when fed milk. if the temperature is high, calves will drink water if it is available and sick calves will also choose to drink water. if water is not available, over-heated calves and sick calves may become dehydrated. sick calves may become dehydrated even when water is offered. calves with acidosis with or without diarrhoea often lose their suckling reflex. this may also happen in calves with hypoglycaemia and septicaemia (berthold, pers. com). after the first few weeks of life, calves attempt to start ruminating. if they have received no solid material in their diet, calves still try to ruminate but cannot show the full rumination behaviour. in addition to the need to suck when young, calves need to manipulate material with their mouths. they try to do this whether or not they have access to solid material and they will seek out solid material that they subsequently manipulate. (van putten and elshof, ; webster, ; webster et al., ) . calves eat solid food better when water is offered simultaneously. certain rapidly digestible carbohydrates are necessary for the development of ruminal papillae with associated physiological development and fibrous roughage helps the anatomical development of the rumen. so it is clear that calves need appropriate solid food in their diet after the first few weeks of life; first, food that is digested rapidly and provide fatty acids; then fibrous foods. rumen development is enhanced when calves are fed with concentrates, water and roughage such as hay. . . . to explore exploration is important as a means of preparing for the avoidance of danger and is a behaviour shown by all calves (kiley worthington and de la plain, ; fraser and broom, ) . exploration is also valuable for establishing where food sources are located. calves need to explore and it may be that higher levels of stereotypes (dannemann et al., ) and fearfulness (webster and saville, ) in poorly lit buildings or otherwise inadequate conditions are a consequence of inability to explore. . . . to have social contact . . . . maternal contact the needs of young calves are met most effectively by the presence and actions of their mothers. in the absence of their mothers, calves associate with other calves if possible and they show much social behaviour. the need to show full social interaction with other calves is evident from calf preferences and from the adverse effects on calves of social isolation (broom and leaver, ; dantzer et al., ; friend et al., ; lidfors, ) . to minimise disease during the first few hours of life, the vigorous attempts of the calf to find a teat and suckle should result in obtaining colostrum from the mother. this colostrum includes immunoglobulins that provide passive protection against infectious agents. hence the needs of the calf have an evident function that is not just nutritional. calves also show preferences to avoid grazing close to faeces. they also react to some insects of a type which may transmit disease. if infected with pathogens or parasites, calves will show sickness behaviour that tends to minimise the adverse effects of disease (broom and kirkden, ) . young calves, less than four weeks of age, are not well adapted to cope with stressful events such as handling and transport, often suffering very high rates of mortality and the younger the calves are, the higher their mortality (staples and haugse, ; mormède et al., ) many succumbing to pneumonia or scouring, within four weeks of arrival at the rearing unit (staples and haugse, ). an inability to mount an effective glucocorticoid response, which is adaptive in the short term, may be a contributing factor to the high levels of morbidity and mortality which occur in young calves (knowles et al., ) as may neutrophilia (simensen et al., ; kegley et al., ) , lymphopaenia (murata et al., ) and suppression of the cell mediated immune response (kelley et al., ; mackenzie et al., ) . to groom grooming behaviour is important as a means of minimising disease and parasitism and calves make considerable efforts to groom themselves thoroughly (fraser and broom, ). calves need to be able to groom their whole bodies effectively. to thermoregulation calves need to maintain their body temperature within a tolerable range. they do this by means of a variety of behavioural and physiological mechanisms. . . . . selection of location when calves are over-heated, or when they detect that they are likely to become over-heated, they move to locations that are cooler. if no such movement is possible, the calf may become disturbed, thus exacerbating the problem and other changes in behaviour and physiology will be employed. responses to a temperature that is too low will also involve location change if possible. . . . . body position over-heated, or potentially over-heated, calves adopt positions that maximise the surface area from which heat can be lost. such positions often involve stretching out the legs laterally if lying and avoiding contact with other calves and with insulating materials. if too cold, calves fold the legs and lie in a posture that minimises surface area. . . . . water drinking over-heated calves will attempt to drink in order to increase the efficiency of methods of cooling themselves. to avoid harmful chemical agents calves need to avoid ingesting toxic substances and to react appropriately if harmful chemical agents are detected within their bodies. . . . to avoid pain calves need to avoid any environmental impact or pathological condition that causes pain. the text in this section refers to current situation in eu countries. calf housing in other countries may be different. replacement dairy calves . . . diet brief description of the diet of replacement heifer calves. this has not really changed since the report. following birth, calves receive (or should receive) colostrum and are than reared with whole milk or milk replacer. calves are weaned; weaning ages and weaning strategies may differ according to region or country. briefly mention current weaning strategies. calves receive starter and, for example, hay and maize silage to promote rumen development. according to the latest eu regulation on the housing of calves (council directive eu / /ec), group housing is compulsory for calves older than weeks, unless there is any need for isolation certified by a veterinarian. individual housing of rearing calves younger than weeks, is quite common in the european dairy industry. below, the most important housing systems for replacement heifer calves are briefly listed. . . . . hutches: partially closed, outside area hutches are made of plywood, plastic or fibre glass. if hutches are made from a synthetic opaque material, this prevents the greenhouse effect inside the hutch and reduces heat stress. if reflective material is used (light coloured), the sun rays are reflected which reduces the risk for overheating. the size of hutches may vary from . - . m width and . - . m length. a layer of sand, e.g. cm gravel or crushed stone can be placed under the calf hutch. litter may be provided preferably as straw, as it provides the warmest surface temperature (panivivat et al., ) , but also wood shavings, sawdust or newspapers are used and the layer should be thick enough to provide a comfortable and dry bed. calf hutches provide three different environments, as the inside is dry and protected from the weather and outside the calf is able to get limited exercise and sunlight. the calf can be also position itself half in and half out, getting sunlight and being protected from wind. hutches should be placed where they catch the most sunlight and avoiding hot, windy and wet locations. nevertheless, during hot summer conditions hutches should be placed in a shady area to avoid overheating. in the rear wall, a hole that can be closed provides better air ventilation within the hut in warm weather. in the hutches, the calf can be kept using wire panels in a building with an outdoor run, preferably of more than . m , enabling some contact to other calves. calves can also be fed outside using a milk bucket support, a dry feed recipient support and a hay rack. other hutch types locate feed and water pails inside the hutch. individual pens are situated in a roofed building. the area should be wellventilated so that the air is dry and fresh, but draught has to be avoided. separation from adult cows is advantageous with respect to disease prevention. pens are either made from hard material with concrete walls or dismountable with three solid sides (i.e. plywood) and an open front (see figure ). walls have to be perforated according to council directive eu / /ec in holdings with more than five calves, which allow at least limited social contact with other calves, one of the key needs of calves. the open front gets fresh air to the calf and makes them easier to feed through a bucket support provided on the front. hardwood is normally used for the floor, which is covered with a litter that is thick enough, dry and clean. totally slatted floors are in use also, made of wood, plastic or metal, but require more care for air temperature. the . - . m x . - . m pen can be put mm above the ground allowing for draining and the removal of urine. dismountable individual pens should be designed in such a way that they can be taken apart and stored when they are not needed, and also easily cleaned with a skid-steer loader or small bucket tractor. in case of cold weather, a plywood cover can be placed over the rear portion of the pen to preserve heat produced by the calf. in hot weather, a removable panel at the rear of the shelter can be opened to provide additional air exchange. collective hutches may house a group of between and calves. the hutches are made of synthetic materials or wood. the inside of the hutch is provided with litter and some hay may be put in a rack. roughage is distributed at a feeding barrier and anti-freeze drinking devices are needed if freezing temperatures may occur. with collective hutches fastened on concrete, a good outdoor run has a non-slippery surface. manure and bedding have to be removed manually or the collective hutch has to move over a few metres distance by means of a tractor and guide-blocks. as for the individual hutches, the location has to be chosen carefully to avoid overheating during summer and provide protection from wind and rain entering the hutch during cold seasons, but give as much sunlight as possible. when sufficient straw and proper ventilation is provided these are the most suitable facilities for young replacement heifer calves. if the calves stay there for several months it is necessary to provide a passage on slippery free concrete. if the floor of this passage is quite rough this will prevent slipping. the concrete floor may be replaced by a slatted floor provided that the spacing between slats agrees with the age of the animals. the lying area can be built in different ways and littered with different materials. in the deep litter system, the dung is removed at regular intervals from every few weeks to twice per year. . . . . group pens inside another common system for group housing of replacement heifer calves is group housing inside, in straw littered pens usually with - calves per pen. calves may enter such group housing already after weeks of individual housing. the regulatory change with regard to calf housing together with a general trend towards larger dairy farms has increased the interest in group housing systems for rearing calves during the milk feeding period (hepola, ; jensen, ) . in addition to systems with small groups of calves ( - animals per group) kept on straw and usually bucket-fed, calves are increasingly kept in larger groups ( up to about calves) with computer-controlled automatic milk feeders. an automatic milk feeder may contain two milking dispensers, and each milking dispenser can be used for about calves. to prevent hierarchic and health problems within the group, calves are grouped with a limited age difference between the animals. calves receive milk replacer according to their needs or ad libitum. when calves are fed according to their needs, a radio-frequency electronic identifier can be used, with a transponder inserted in the collar, in an ear tag, injected under the skin or inside a ruminal bolus swallowed by the animal. the diet of the vast majority of veal calves in the european union is determined by the market demand for "white meat", i.e. meat with low myoglobin content. the production of white veal meat comes from a tradition of fattening calves thanks to a diet based on milk, which is naturally poor in iron, and slaughtering the animals when they are young. nowadays most veal calves are fed milk replacers that contains a variable proportion of milk powder and which iron content is maintained at a low level. this results in relatively low blood haemoglobin levels. an average blood haemoglobin level at slaughter between . and . mmol/l is compatible with an acceptable meat colour. as haemoglobin levels increase, the number of animals whose meat is darker in colour increases. in order to prevent calves from having haemoglobin levels that are too low, early in the production phase, the iron supply in the milk replacer fed during the first - weeks of the fattening period (starter) is usually about ppm, whereas iron supply in the milk replacer fed during the remainder of the fattening period (fattener) is ppm. moreover, blood haemoglobin levels are generally monitored, most intensively upon arrival at the fattening unit, and calves with levels below age-dependent thresholds are treated with iron, either individually or group-wise. thus, blood haemoglobin levels usually gradually decline across the fattening period, and the lowest average levels are supposed to be reached during the last four weeks prior to slaughter. some veal calves are still fed raw milk. in case of dairy breeds, the cows are generally milked and the milk is given to calves in buckets. in case of beef breeds, the calves are led twice a day for suckling their dam or another cow. according to the latest amendment to the annex of council directive / /eec (commission decision / /ec) calves should receive sufficient iron to ensure an average blood haemoglobin of at least . mmol/l, and calves over two weeks old should be provided daily with some fibrous feed which should increase from to a minimum of grams per day from the beginning to the end of the fattening period. the main types of solid feed given to veal calves differ somewhat between the veal producing countries in europe. in france and italy solid feeds for veal calves usually consist of chopped straw or pelleted dry feed consisting of both fibrous (e.g. straw) and concentrate-like (e.g. cereal) materials. in the netherlands, maize silage is a popular roughage source for white veal calves, provided that the iron content is not too high (an upper limit of - pp/kg dry matter is generally imposed). maize silage is usually fed in relatively high amounts, with maximum daily amounts of up to . kg ( gr dry matter)/calf/day. other feeds used in the veal industry include chopped straw and rolled barley. white veal calves are fattened for approximately weeks in italy and the netherlands, and for - weeks in france. besides the production of white veal meat, several systems exist across europe that lead to the production of so-called "pink veal meat". the main differences from the more conventional production of white veal are that the calves are reared for a longer period and they receive higher amounts of solid foods. as a consequence the muscles have a higher content of myoglobin, hence the darker colour of the meat. in france, the calves are most often from suckler beef breeds; they are reared with their dam and may be weaned before the end of rearing. in the netherlands, pink veal meat is generally produced from calves of dairy breeds. pink veal calves are weaned at - weeks of age. after weaning, they receive a diet of ad lib roughage (frequently maize silage) and by-products. pink veal calves are not restricted with regard to dietary iron supply and, consequently, develop normal haemoglobin levels and the associated "red" (pink) meat colour. the age at slaughter can vary from calves of - months to young bred animals of - months with the slaughter age of individuals depending on the production rate. these products are labelled to help consumers to distinguish them from white veal meat. in line with the latest eu regulation (council directive eu / /ec), individual housing of veal calves has been officially abolished in the european union. already in the s extensive studies were initiated with the aim to develop a practically feasible husbandry system for group housing of veal calves. at present the systems involve both large and smaller groups. housing of calves is in groups of - animals, with a slight trend towards larger group sizes ( - calves per group). the floor can be bedded with straw or wood shavings but is more commonly made of wooden slats. wooden slats require less labour and straw or woodshavings easily become dirty and wet. calves are kept in individual pens, sometimes called "baby-boxes" for a period of - weeks upon arrival at the fattening unit to prevent overt preputial sucking thereafter and to be able to monitor more closely the health of calves. baby boxes are usually made of galvanised or wooden partitions placed inside the group pen. in these boxes, calves are bucket-fed individually. after - weeks, these temporary partitions are removed and calves are free to move around in the pen. calves are fed milk replacer in a trough or in individual buckets. a crucial management procedure associated with trough feeding is the regular re-grouping of calves, to maintain homogeneous groups in terms of calf weight and particularly drinking speed throughout fattening. experimental work confirmed the feasibility of this procedure in that calves could be repeatedly regrouped without effects on their health, growth rate and a number of physiological measures of stress . in this latter study, aggression between calves was rare, and calves seemed to habituate to repeated mixing. individual calves not thriving on milk replacer because of drinking problems, are provided with floating teats or with a teat-bucket. veal calves are sometimes kept in pairs. this type of housing results in less availability of space for movement and social opportunities than in larger groups of calves but is reported to have no disadvantages in health, weight gain and the occurrence of cross-sucking (chua et al., ) . suckling veal calves are generally accomodated in small groups. as in the rearing of dairy calves, automatic feeding systems have been extended to veal production systems, particularly since increasingly sophisticated computer technology is becoming available for sensor-aided recognition of individual animals, and to control feeding times and intake. calves are usually housed in large groups ( - calves) and receive milk replacer via an automatic feeding machine. with such feeders, calves suck to obtain their milk. the floor generally consists of wooden slats, or concrete in combination with wooden slats. some veal calves are kept on straw bedded floors, or have access to rubber mats or concrete covered by rubber. calf rearing and animal environmental pollution . . . general introduction in the report there was a short chapter on calf production and environmental pollution referring to gases (ammonia, nitrous oxide, carbon dioxide). manure resulting from calf production was seen as a fertiliser only. this chapter briefly describes in a condensed way the impact of modern animal calf production affects the environment of the animals. modern animal production is a source of solid, liquid and gaseous emissions which i.a. can be harmful to the animals. solid and liquid manure and waste water contain nitrogen and phophorus which are the most important plant nutrients, but are harmful when applied to agricultural land in excess amounts thereby leading to pollution of ground water by nitrates, surface water with phosphorous causing eutrophication and soil with heavy metals such as zinc and copper which are used as growth promotors in the feed stuff, all of which can affect the animals if returned to them. a third group of potentially hazardous effluents are drug residues, such as antibiotics, which may be present in the excreta of farm animals after medical treatment and which are passed to the environment during grazing or spreading of animal manure where they may conceivably contribute to the formation of antibiotic resistance in certain strains of bacteria. the same risk arises when sludge and waste water from sewage plants containing residues of antibiotics and other drugs from human consumption are discharged as fertiliser in the soil and water body of agricultural land. the most important aerial pollutants from calf rearing systems are odours, some gases, dust, micro-organisms and endotoxins, together also addressed as bioaerosols (seedorf and hartung, ) , which are emitted by way of the exhaust air into the environment from buildings and during manure storage, handling and disposal. aerial pollutants can give cause for concern for several reasons. e.g. an animal's respiratory health may be compromised by these pollutants. in fattening units, up to % of all calves may show signs of pneumonia, pleuritis or other respiratory disease within the first three weeks of housing when the calves come together from different herds (see chapter on temperature, ventilation and air hygiene). the travel distance of viable bacteria from animal houses via the air is presently estimated at m (müller and wieser, ) downwind why there is a possible transmission between animal houses. very little is known about the distribution characteristics of dust particles, endotoxins, fungi and their spores, in the air surrounding animal houses. recent investigations showed dispersion of staphylococcae sp. (bioaerosols) up to m (schulz et al., ) from a broiler barn. the contribution of calf production is presently unknown. it is estimated that calves produce about . kg fresh manure and . kg slurry per animal and day. this is a share of . % in the total amount of fresh manure produced in cattle farming (richter et al., ) . manure suspected to contain pathogens such as salmonella should be stored for at least months without adding or removing material and subsequently applied to arable land where it is ploughed in, or it is disinfected before any further use. the second area of concern is the emission compounds such as gases, odours, dust, micro-organisms and other compounds like endotoxins which are regularly present in calf house air where they can cause or exacerbate respiratory disorders in animal and work force. the quantities emitted from calf houses are summarised in seedorf et al. ( b) there are considerable emission amounts from calf husbandry. the emissions of micro-organisms are higher than from dairy or beef barns but distinctly lower than from pig or poultry production (seedorf et al., a) . the same is true for endotoxins which are one log lower in cow barns but distinctly higher in pig and poultry houses. the dust emissions can be times higher in piggeries and times higher in broiler barns . the ammonia concentration is usually lower than in piggeries or laying hen houses. however this depends greatly on the housing and manure management system. in a us study johnson et al. ( ) reported that cow-calf, stocker and feedlot phases contribute considerable amounts of nitrous oxide and methane to the emissions from cattle production. . comparison of systems and factors . . feeding and housing systems, weaning strategies and quality of solid and liquid feed . . . feeding systems the main potential problems associated with the housing of calves in large groups with automatic feeders include: cross sucking, i.e., non-nutritive sucking of parts of another calf's body (in particular the ears, mouth, navel, udder-base and, in case of bull calves, the scrotum and prepuce) (plath et al., ; bokkers and koene, ; jensen, ) , competition for access to the feeder (jensen, ; , and health problems, in particular a high incidence of respiratory disease (maatje et al., ; plath et al., ; svensson et al., svensson et al., , hepola, ; engelbrecht pedersen et al., ) . a number of factors have been identified that are likely associated with some of these problems, although conflicting results have been reported. cross-sucking is linked with the sucking motivation of calves and, hence, measures to reduce the motivation of calves for non-nutritive sucking may reduce the occurrence of cross-sucking (de passillé, ). an increased milk allowance also reduced non-nutritive sucking on a teat as well as cross-sucking in group-housed calves in one experiment (jung and lidfors, ), but did not affect cross-sucking in another (jensen and holm, ) . reducing the milk flow rate decreased nonnutritive sucking on a teat in individually housed calves (haley et al., ) , but failed to influence cross-sucking in group-housed ones (jung and lidfors, ; jensen and holm, ) . alternatively, it has been suggested that hunger may also control the level of non-nutritive sucking and possibly cross-sucking (jensen, ) . this idea is consistent with the observations that the duration of unrewarded visits to an automatic feeding station increased during gradual weaning (jensen and holm, ) , and that under practical farm conditions the frequency of cross-sucking among dairy calves around weaning is increased with decreasing availability or energy density of solid feeds (keil et al., ; keil and langhans, ) . in contrast to other calves, white veal calves are not weaned, receive large amounts of milk replacer and usually obtain only restricted amounts of solid food. these additional factors may also affect and perhaps exacerbate crosssucking in systems with an automatic feeder (jensen, ) . results by veissier et al. ( ) showing that bucket-fed group-housed veal calves show less cross-sucking than those fed by an automatic feeder again seem to implicate factors other than sucking motivation per se in the development and expression of cross-sucking. on the other hand, rearing calves in large groups with an automatic feeder allows more interactions between calves and offers calves the possibility to suck milk. competition for access to an automatic milk feeder was increased in groups of or calves in comparison with groups of or , respectively (herrmann and knierim, ; jensen, ) , and under dietary conditions of relatively low milk allowance and reduced milk flow rate (jensen and holm, ) . protecting calves from displacement at the feeder may also be accomplished by fitting a closed feeding stall to the station (weber and wechsler, ) . in comparison with the usual setup, this modification increased the duration of visits to the feeder as well as the duration of non-nutritive sucking on the teat after milk ingestion, and significantly reduced the frequency of cross-sucking within minutes after milk ingestion. however, the incidence of cross-sucking performed without prior milk ingestion was not affected by the design of the feeder (weber and wechsler, ) . in a recent comprehensive review, jensen ( ) observes that there is a lack of knowledge on the effect of different weaning methods on cross-sucking. she also concludes that future research should focus on preventive measures to reduce cross-sucking and problems with aggression in automatically fed calves, including the establishment of appropriate numbers of calves per feeder. the apparent increase in health problems of calves kept in large groups with automatic feeders might be related to group size rather than to feeding system. a comparison of two different group sizes of calves fed by an automatic milk feeder showed that calves housed in groups of - had a higher incidence of respiratory illness and grew less than calves housed in groups of - (svensson and liberg, ) . similarly, placement of preweaning heifer calves in groups of or more was associated with high calf mortality in a large scale epidemiological survey (losinger and heinrichs, ) . interestingly, in a study by kung et al. ( ) , group-housed calves fed by an automatic feeding system for milk supply had fewer days of medication than those kept individually in separate calf hutches. these authors also emphasize the importance of good management and frequent observations of calves as an integral part of a successful rearing program. likewise, howard ( ) specifically links good and correct management practices with the prevention of disease and successful group housing of dairy calves. natural weaning in cattle takes place when young animals are around - months of age. depending on productive system, weaning can usually occur between and months of age. dairy calves are usually reared away from their dams and they are given milk or milk replacer until weaning at to weeks of age. however holstein calves can be weaned at to weeks of age (early weaning). beef calves are usually weaned at to months of age depending on season of birth. early weaning of beef calves may be considered as a management practice in poor climate conditions and where forage quality is poor later in the grazing season. several studies have shown that it is possible to wean calves at very young ages based on concentrate intake (svc, ) . however, regardless of the productive system, weaning is effective and does not cause health and welfare problems to calves when it occurs as a smooth transition from an immature to mature ruminant with an adequate size and development of the reticulo-rumen for efficient utilisation of dry and forage based diets. at birth, the reticulum, rumen, and omasum of the calf are undeveloped, nonfunctional and small in size compared to the abomasum and rumen remains underdeveloped during the first - months of age. calves being ruminant animals require a physically and functionally developed rumen to consume forages and dry feeds. however, the rumen will remain undeveloped if diet requirements for rumen development are not provided. solid feed intake stimulates rumen microbial proliferation and production of microbial end products, volatile fatty acids, which initiate rumen epithelial development . solid feeds are preferentially directed to the reticulo-rumen for digestion, however they differ in efficacy to stimulate rumen development. recent studies have shown that addition of yeast culture ( %) increased calf grain intake, but did not affect rumen development in young calves ; while papillae length and rumen wall thickness were significantly greater in week old calves fed calf starters containing steamflaked corn over those fed dry-rolled and whole corn when these corn supplements made up % of the calf starter showing that the type of grain processing can influence rumen development in young calves. forages seem to be the primary stimulators of rumen muscularization development and increased rumen volume (zitnan et al., ) . large particle size, high effective fibre content, and increased bulk of forages or high fibre sources physically increase rumen wall stimulation, subsequently increasing rumen motility, muscularization, and volume coverdale et al., ) . besides, solid feeds other than forages or bulky feedstuffs can be effective in influencing rumen capacity and muscularization. coarsely or moderately ground concentrate diets have been shown to increase rumen capacity and muscularization more than finely ground or pelleted concentrate diets, indicating that extent of processing and/or concentrate particle size affects the ability of concentrates to stimulate rumen capacity and muscularization (beharka et al., ; greenwood et al., ) . therefore, it seems that concentrate diets with increased particle size may be the most desirable feedstuff for overall rumen development, due to their ability to stimulate epithelial development, rumen capacity, and rumen muscularization . calf weaning should be based on the amount of dry feed calves ingest per day, not on their age or weight, and calf starter should be made available five to days after birth. but, as pointed out from recent research attention must paid to type of forage and consistent of particle size of starter grain in order to achieve a proper rumen development. a calf consuming . kg of dry feed or more on three consecutive days is ready for weaning. when calves are fed low levels of milk to encourage early consumption of dry food, weaning can be done abruptly. in contrast, if milk is given in large amounts, weaning may require two to three weeks of slow transition to avoid a setback in growth. early weaning systems should not be used if the animals are in a negative energy balance. . . . quality of solid and liquid feed . . . . solid feed: concentrates and roughage traditionally, veal calves were fattened on a diet consisting exclusively of milk replacer. calves fed in this manner show a number of welfare problems (reviewed in the previous report), including abnormal behaviours and disease associated with lack of rumen development. to better safeguard the welfare of calves, provision of (some) solid feed to veal calves has become compulsory according to the latest amendment to the annex of council directive / /eec (commission decision / /ec). however, provision of roughage to veal calves fed a regular milk replacer diet, has clearly been demonstrated to increase the incidence of abomasal ulcers, in particular in the pyloric part (which connects to the duodenum) (wensink et al., ; welchman and baust, ; breukink et al., ) . thus, recent studies have largely focussed concurrently on the effects of provision of roughage on calf behaviour, abomasal lesions and rumen development, in an attempt to identify feeds that may benefit veal calf welfare without compromising abomasal integrity. in a comprehensive eu-funded project, a range of different types of roughage/solid feeds (straw, maize silage, maize cob silage, rolled barley and beet pulp) in different amounts ( versus gr dry matter) and of different particle sizes and physical characteristics (i.e., chopped versus ground, dried versus fresh, un-pelleted versus pelleted) were given to veal calves in addition to milk replacer in large-scale multifactorial trials (chain management of veal calf welfare, ; cozzi et al., ; mattiello et al., ) . control treatments consisted of milk replacer only, and milk replacer with ad lib access to hay. another control group consisted of bull calves reared in a similar way to normal dairy calves, i.e. the animals received ad libitum hay and concentrates and were weaned at weeks of age. in comparison with milk replacer only, those types of roughage that were richest in fibrous material, i.e. straw (regardless of amount and physical structure) and hay, significantly reduced the level of abnormal oral behaviours (composed of tongue rolling, tongue playing and compulsive biting/sucking of substrates), and concomitantly increased the level of rumination. weaned calves exhibited no abnormal oral behaviours. higher levels of rumination in veal calves as a function of the fibre content of the solid feed were also reported by morrisse et al. ( morrisse et al. ( , . in line with these findings, veissier et al. ( ) , observed reduced levels of biting at substrate and more chewing behaviour in veal calves provided with straw compared with un-supplemented controls. previously, it has been suggested that a sucking deficit causes abnormal oral behaviours in calves (sambraus, ) . more recent data, however, clearly identify the lack of appropriate roughage as a major determinant of abnormal oral behaviours in veal calves. correspondingly, bokkers and koene ( ) found no differences in abnormal oral behaviours between group-housed veal calves fed either by bucket or by an automatic feeder. results obtained in veal calves are also fully consistent with data in cows (redbo et al., ; redbo and nordblad, ) and other ruminants such as giraffes (baxter and plowman, ) , which all link increased levels of abnormal oral behaviours with feeds poor in fibre. in agreement with previous data, most roughages provided to milk-fed veal calves significantly increased the incidence of abomasal lesions, particularly ulcers in the pyloric region, in comparison with the feeding condition without additional roughage (chain management of veal calf welfare, ; . incidences of abomasal ulcers (expressed as the percentages of calves with one or more lesions) among weaned bull calves, calves fed milk only, and veal calves given supplemental roughages were , between - , and between - %, respectively. this suggests that the interaction between roughage and a milk replacer diet rather than roughage per se, is involved in the etiology of abomasal ulcers in veal calves. these findings support the hypothesis that pyloric ulcers in milk-fed veal calves may be caused by local ischaemia followed by focal necrosis as a consequence of strong contractions of the pyloric wall when large volumes of milk are consumed. provision of roughage, in turn, would then exacerbate an existing problem in that roughage particles exert a mechanically abrasive effect on a sensitive abomasal mucosa, and delay the healing of any lesions already present (unshelm et al., ; dämmrich, ; krauser, ; welchman and baust, ; breukink et al., ) . this explanation may also fit the observations that veal calves fed either hay or a combination of concentrates and straw exhibited similar incidences of abomasal ulcers to those fed milk replacer only (chain management of veal calf welfare, ; veissier et al., ) . these roughages represent more balanced feeds, accompanied by better rumen fermentation. this may have improved ruminal digestion of fibres, thereby preventing sharp undigested particles entering the abomasum. other factors proposed or examined in relation to the pathogenesis of abomasal ulcers in calves include stress, infection with bacteria, trace mineral deficiencies, and prolonged periods of severe abomasal acidity (lourens et al., ; mills et al., ; jelinski et al., ; de groote et al., ; palmer et al., ; ahmed et al., ahmed et al., , ahmed et al., , . however, so far none of these factors have been convincingly related to abomasal ulcers in veal calves. calves fed milk only, showed a high incidence of ruminal hairballs. in different experiments between - % of milk-fed veal calves had hairballs (chain management of veal calf welfare, ; cozzi et al., ) . feeding roughage gave a profound reduction of hairballs; depending on the type of roughage the incidence varied between - %. similarly, morisse et al. ( ) reported a marked reduction of ruminal hairballs in calves fed pelleted straw and cereals. this reduction was thought to result from a continuous elimination of ingested hair by improved ruminal motility. however, it may well be that abnormally high self-licking behaviour is reduced when roughage is provided. it is suggested that further optimising the composition of roughage in terms of adequate rumen development and rumen function, may eventually result in feeds that promote rumination and reduce abnormal oral behaviours without damaging the digestive apparatus (chain management of veal calf welfare, ; morisse, ; mattiello et al., ) for all newborn calves, receiving an adequate amount of high quality colostrum is essential for their health and survival. in comparison with mature milk, colostrum contains greater concentrations of total solids and of fat, protein, vitamins and minerals. most importantly, colostrum provides the calf with immunoglobulins (igg), which are vital for its early immune protection. in addition, colostrum contains a range of other non-nutrient and bioactive components including various types of cells, peptide hormones, hormone releasing factors, growth factors, cytokines and other bioactive peptides, oligosaccharides and steroid hormones. these factors modulate the microbial population in the gastrointestinal tract, have profound effects on the gastrointestinal tract itself (e.g. cell proliferation, migration, differentiation; protein synthesis and degredation; digestion, absorption, motility; immune system development and function), and in part exert systemic effects outside the gastrointestinal tract on metabolism and endocrine systems, vascular tone and hemostasis, activity and behaviour, and systemic growth (waterman, ; blum, ) . the highest quality colostrum, or true colostrum, is obtained from the very first milking after parturition. thus, provision of first colostrum to newborn calves is one critical factor for successful calf rearing. the timing of provision of colostrum is also crucial since the ability of the calf's small intestine to absorb large proteins such as igg decreases rapidly following birth. consumption of sufficient colostrum within the first h of life is needed not only for an adequate immune status but also to produce the additional important and favourable effects on metabolic and endocrine traits, and on vitality. finally, colostrum should be regularly provided for a sufficient length of time, preferably for the first three days after birth (hadorn et al., ; waterman et al., ; rauprich et al, ) . although the importance of colostrum for calf health and survival is generally recognized, actual practices in calf rearing do not always favour adequate colostrum intake in newborn calves, and may therefore pose a risk for their welfare. after the period of colostrum feeding, calves can be switched to whole milk or a high quality milk replacer. in the case of rearing calves, both sources of liquid feed are used, although the majority of dairy calves are currently reared on a milk replacer diet. milk replacers are usually less costly than saleable whole milk, and the feeding of raw waste milk may pose several health and contamination risks, including the transfer of infectious diseases to the calf, and problems with antibiotic residues or overdoses (wray et al., ; selim and cullor, ; waltz et al., ) . at present, good quality milk replacers may provide comparable performance to whole milk. however, pasteurization of waste milk prior to feeding it to calves may also represent an effective and viable alternative for minimizing health risks (stabel et al., ) . results from a recent clinical survey by godden et al. ( ) even suggested that dairy calves fed pasteurized waste milk have a higher growth rate and lower morbidity and mortality rates than do calves fed conventional milk replacer. with the exception of production systems involving suckler cows, veal calves are generally fattened on milk replacer diets. over time, formulations of commercially available milk replacers for veal calves (as well as those for dairy calves) have become more and more sophisticated. at the same time, economic pressures continuously prompt the industry to reduce feeding costs and to consider alternative components and raw materials. originally, proteins in milk replacers were milk-based, and skim milk powder constituted the major protein source. subsequently, milk replacers based on whey powder became available. approximately during the last two decades, attempts have been made to replace animal-based proteins in milk replacers by vegetable proteins, mainly from soybean and wheat and, to a lesser extent, pea and potato. initially, some of these attempts met with little success because of health problems in the calves. for example, compared to calves fed diets based on skim milk powder, calves fed milk replacers containing heated soybean flour developed severe immunemediated gut hypersensitivity reactions characterized by partial atrophy of the small intestinal villi, malabsorption, diarrhoea, and large infiltrations of the small intestine by immune cells, accompanied by the presence of high antibody titres against soy antigens in plasma and intestinal mucous secretions (lalles et al., a (lalles et al., , b (lalles et al., , (lalles et al., , dreau et al., ; dreau and lalles, ) . however, the nutritional utilization of vegetable proteins can be improved by a variety of technological treatments including, for example, heating, protein hydrolysis, and ethanol extraction. such treatments reduce anti-nutritional factors and antigenic activity, and increase protein digestibility by denaturing three-dimensional structures (lalles et al., c (lalles et al., , d . at present a number of processed plant proteins are successfully applied in combination with milkbased protein sources in milk replacers for (veal) calves, including hydrolysed soy protein isolate and hydrolysed wheat gluten. recent research in the area of plant proteins in milk replacer formulas is focussed on understanding mechanisms underlying the flow of proteins in duodenal digesta, and the interaction of dietary peptides with the gut, in particular at the level of the mucus layer (montagne et al., (montagne et al., , (montagne et al., , . results of this type of work may further enhance the use of plant proteins in milk replacers for calves. in addition to an enhanced risk for gut problems, low quality milk replacers may also cause dysfunction of the oesophageal groove reflex, which may result in ruminal acidosis. in this respect, temperature is also an important quality feature; a too cold drinking temperature of the milk replacer attenuates the oesophageal groove reflex (gentile, ) . if vegetable proteins are not properly treated, milk replacers may cause hypersensitivity reactions in the gut, which may compromise calf welfare. low iron dietary supply is a prerequisite for the production of white veal. the blood haemoglobin level in veal calves towards the end of fattening (between . and . mmol/l), is generally considered a threshold below which iron deficiency anaemia occurs (bremner, ; sprietsma, a, b; postema, ; lindt and blum, a) , although some authors have argued that this level is already below a critical value (welchman et al., ) . when calves were forced to walk on a treadmill, those with a mean haemoglobin level of . mmol/l consumed more oxygen and exhibited higher cortisol levels after walking than calves whose haemoglobin level was . . or . mmol/l (piguet et al., ) . on the other hand, blood lactate after transport was not significantly different between groups of calves with average haemoglobin levels of . and . . mmol/l, respectively (lindt and blum, b) . there is a large body of evidence showing that iron deficiency anaemia may compromise immunocompetence, in particular cellular immune function, in a range of species including laboratory rodents and humans (dallman, ; dhur et al., ; galan et al., ; latunde-dada and young, ; ahluwalia et al., ) . in human children, iron-deficiency states have been epidemiologically associated with increased morbidity due to respiratory infection and diarrhoea (keusch, ; de silva et al., ; levy et al., ) . this justifies the question of whether dietary iron supply and associated haemoglobin levels are sufficient to guarantee adequate health in white veal calves. previous results concerning the relationship between clinical health and anaemia in veal calves are scarce, and were inconclusive. using very small numbers of calves, möllerberg and moreno-lopez ( ) found no difference between iron anaemic and normal calves in the clinical response to infection with an attenuated parainfluenza- virus strain, whereas sárközy et al. ( ) reported a depressed immune response as reflected in significantly lower antibody levels in anaemic calves compared with controls following inoculation with a live adenovirus. in a study by gygax et al. ( ) , cellular immune function was depressed, and disease incidence, especially of respiratory infections, was increased in calves fed low amounts of iron. however, in this particular study, haemoglobin levels dropped considerably below the value of . mmol/l. a more recent study (van reenen et al., ) , therefore, aimed to examine immunocompetence in a bovine herpes viral (bhv ) infection model in white veal calves with blood haemoglobin levels maintained at all times above or just at . mmol/l. calves daily supplemented with extra iron exhibited normal haemoglobin levels across the entire experiment (average approximately . mmol/l), whereas white veal calves had average haemoglobin levels at the time of bhv infection and at slaughter of approximately . and . mmol/l, respectively. dietary iron supply did not affect the reactions of calves to bhv infection (clinical signs, viral excretion in nasal fluid, antibody reponse), white blood cell and lymphocyte counts, and growth rate. by contrast, in comparison with calves with high haemoglobin levels, white veal calves exhibited a higher heart rate during milk intake, had consistently elevated levels of urinary noradrenaline, and showed enhanced plasma acth and reduced plasma cortisol responses in a number of hpa axis reactivity tests. these latter findings concur with increased heart rate and catecholamines in urine, and altered responsiveness of the hpa axis in iron-deficient or anaemic humans and laboratory rodents (voorhess et al., ; dillman et al., ; dallman et al., ; groeneveld et al., ; saad et al., ) . these physiological changes are part of an elaborate adaptive response to iron deficiency (beard, ; rosenzweig and volpe, ) , which also involves alterations in glucose metabolism (blum and hammon, ) . veal calves with blood haemoglobin levels clearly below . mmol/l demonstrated reduced growth rates as well as a large depression in white blood cell and lymphocyte counts (reece and hotchkiss, ; gygax et al., ) . thus, it is suggested that maintaining blood haemoglobin in individual veal calves over . mmol/l induces a number of physiological adaptations which seem universal for iron-deficient mammals in general, but do not harmfully compromise biological capacities in terms of growth and immunocompetence. in actual practice, however, the haemoglobin threshold of . mmol/l is currently considered at the group rather than at the individual level. for example, an average haemoglobin level of . mmol in a group of finished veal calves is assumed to be exactly at the lower threshold value. however, depending on the variation between individuals, if a group of calves has an average haemoglobin level of . mmol/l, then some individuals within that group may have levels well below this lower threshold value. in fact, based on an analysis of the variation between calves in blood haemoglobin levels, it has been argued that the haemoglobin threshold for anaemia of a group of veal calves should be higher than that of an individual calf, i.e. an average level of . rather than mmol/l (van hellemond and sprietsma, a) . in order to prevent anaemia during fattening, blood haemoglobin levels are monitored to some extent in white veal calves, and animals are treated with supplemental iron according to age-dependent haemoglobin thresholds. however, systematic monitoring generally occurs only on two occasions: within the first - weeks upon arrival at the fattening unit, in all animals, and between - weeks of fattening, in a sample of calves. outside these instants, individual calves may receive iron supplementation in the presence of clinical signs of iron deficiency. but once clinical signs are apparent, haemoglobin levels are usually well below . mmol l - (blaxter et al., ; bremner et al., ) . since blood haemoglobin levels are not routinely monitored in veal calves beyond the th week of fattening, there is a likelihood of too low haemoglobin levels occurring in part of the animals, in particular towards the end of fattening, when low haemoglobin levels are most likely to occur. general housing calves kept indoors are housed in an environment where several important factors interact such as space, pen design, social contacts, flooring and bedding material as well as climate. in experimental studies, usually one or a few of these factors are varied and the others controlled for. however in larger epidemiological studies many of these factors vary and their interaction can be measured. in a study of heifer calves in swedish dairy herds the effect of draught, cleanliness of the animals, hygiene level of the farm, placing of the calf pens, nature of the pen walls, air volume per animal, management factors such as status of the caretaker and feeding routines was evaluated by means of a two-level variance component logistic model. the placing of calf pens along an outer wall was significantly associated with the risk of diarrhoea (odds ratio (or): . , p< . ), the risk for respiratory disease was significantly associated with an ammonia concentration below ppm (or: . , p< . ) while the or for moderately to severely increased respiratory sounds was significantly associated with draught (or: . , p< . ) (lundborg et al., ) . odds ratios for respiratory disease were increased in calves housed in large-group pens with an automatic milk-feeding system (or: , ) . the report highlights that the housing systems of calves and the available space affect the development and determine which behaviours the animals are able to perform. the report (svc, ) recommends the minimum space for both single crate and group pen and it points out how lack of space can affect health and welfare of reared calves (maatje and verhoeff, ; dantzer et al., ; friend et al., ) . the report also suggests that shape of the pen can be important to the animal. recent studies confirmed that the space available can affect both behavioural and physiological traits and productive performances of cattle. however, the majority of them compare behaviour, production or other indicators of calves reared in individual crates versus group pens (vessier et al., ; andrighetto et al., ; jensen, ; verga et al., ; cozzi et al., ; bokkers and koene, ) or tethered or single pen (terosky et al., ; wilson et al., ) which were already discussed in chapter . little research has been done to directly compare behavioural and physiological indicators of welfare in calves reared in pens of various space allowances. in dairy calves it has been shown that spatial environment stimulated play: calves in small group pens performed less locomotory play that the ones kept in larger pens (jensen et al., ; jensen and kyhn, ) . it has been reported in a preliminary study that dairy calves kept, from birth to month of life in larger stalls ( . m x . m) showed a higher percentage of lying behaviour and grooming than calves kept in smaller stalls ( . m x . m); besides, lymphocyte proliferation was significantly higher in calves reared in large stalls (ferrante el al., ) . it is known that cattle prefer to use the perimeter of pens rather than the central area (stricklin et al., ; hinch et al., ; fraser and broom, ) . the ratio between the number of corners in the pen and number of animals seems to influence the individual space, the space that calves try to keep to other calves, as showed by simulation models (stricklin et al., ) . therefore pen shapes maximising the perimeter to area ratio might be preferable for cattle (jóhannesson and sørensen, ) . for this reason it has been pointed out that measurements such as pen perimeter, the number of corners and the diagonal distance of the pen could be important for dairy cattle (jóhannesson and sørensen, ) . however there is a lack of knowledge on this topic on calves. in a study on veal calves most of the animals lying next to the wall, the quieter and drier part of the pen, stood more on the side of the far pen and eliminated in the feeding area (stefanowska et al., ) . calves kept in a large group ( animals) and fed using an automatic milk replacer showed an elevated use of the area around the partition of the pen and they spent little time in the centre of the area. (morita et al., ) , this use of the pen space could lead to a pen design functionally divided into a walking and feeding area and a lying area. the report concludes that slatted floors must not be slippery, it also recommends appropriate bedding, for example straw, and that every calf should have access to a dry lying area. the report highlights that housing and management conditions can affect the posture adopted when lying and resting in calves. . . . recent findings regarding importance of floor and bedding materials slatted floors have been used for many years as convenient for intensive housing for beef cattle but concerns have been expressed about their effects on animal welfare (scahaw, ) . the type of surface not only affects the movements of getting up and lying down, lying and resting behaviour of the fattening animals but also other behavioural traits and physiological indicators of stress (scahaw, ) . moreover when cattle can choose between different floor types they prefer deep litter to slatted floor especially for resting. many studies were conducted in order to analyse the floor comfort in the lying area in dairy and in beef cattle (for a list of references see tuyttens, ; scahaw, ) . the group pens for veal calves do not have separate lying areas and therefore the animals spend all their time on the same surface. if the floor is too hard for lying or too slippery, discomfort, distress and injury may result. a suitable floor is very important for calves as adequate rest is essential for the good welfare of young growing animals, moreover a positive correlation between the amount of rest and growth rates has been observed for growing cattle (mogensen et al., ; hanninen et al., ) . adequate resting is important both for sleep and temperature regulation. veal calves are often housed on slatted floors, commonly made of hardwood, a product that is controversial because it often comes from unsustainable forestry in tropical countries (stefanowska et al., ) , or on concrete floors due to the fact that bedding material is costly and requires more labour and can cause problems in manure handling systems. wooden slatted floors can absorb liquid from manure and a wet surface is not comfortable for moving and lying (verga et al., ) . even if straw bedding provides better floor comfort to animals than slatted or concrete floors, suitable alternatives to reduce or eliminate the use of straw bedding are available for cattle (tuyttens, ) . recent studies have investigated the effect of the texture (how soft) and the thermal properties of floor on lying postures and resting behaviour of calves. in cool or drafty floors calves spent less time resting on the side and rest curled up in order to conserve heat (hanninen et al., ) . in contrast with adult dairy cows which rested longer and lay down more frequently on softer floors, there was no effect of type of floor (concrete floor or rubber mats) on resting behaviour of dairy calves (hanninen et al., ) . in another experiment where veal calves could choose to use a hardwood slatted floor surface or a synthetic rubber coated floor surface the calves preferred the wooden floor for lying (stefanowska et al., ) . moreover the animals rested in the drier part of the pen (stefanowska et al., ) . from these studies it seems that the texture of the floor is not as important to calves as to older animals, whereas thermal comfort seems to affect lying and lying postures. panivivat et al. ( ) investigated growth performance and health of dairy calves bedded with five different types of materials (granite fines, sand, rice hulls, long wheat straw, wood shavings) for days during august to october from birth. overall average daily gain and dry matter intake of calves did not differ with bedding type, although during week , calves housed on rice hulls had the greatest dry matter intake and those housed on wood shavings had the lowest. during week , calves housed on granite fines and sand were treated more often for scours, and calves housed on long wheat straw received the fewest antibiotic treatments (week by bedding material interaction). granite fines formed a harder surface than other bedding, and calves housed on granite fines scored the dirtiest. long wheat straw had the warmest surface temperature, and rice hulls and wood shavings were warmer than granite fines and sand. serum cortisol, alpha ( )-acid glycoprotein, immunoglobulin g concentrations, and the neutrophil:lymphocyte ratio were not affected by bedding type. on day , coliform counts were greatest in rice hulls. after use, coliform counts were greatest in long wheat straw (week by bedding material interaction). growth rates of calves bedded for d with bedding types did not differ; however, the number of antibiotic treatments given for scours was greatest on granite fines and sand; coliform counts in the bedding were highest in rice hulls before use and in long wheat straw after days of use. degree of social contact the report recommends that calves are cared for by their dam after birth so that they are licked and receive colostrum and that calves are not deprived of social contact, especially with other calves because ) calves for social contacts; ) calves isolated from other calves express more abnormal activities (e.g. excessive grooming, tongue rolling), are hyper-reactive to external stimuli and their subsequent social behaviour is impaired; and ) in combination with restricted space or lack of straw, individual housing induces a chronic stress state as assessed through enhanced responses to an acth challenge. . . . recent findings regarding contacts with the dam the bond between dam and calf is likely to develop very soon after birth: calves separated from their dam at h can recognise the vocalizations of their own dam one day later (marchant-forde et al., ) in their review about early separation between dairy cows and calves, flower and weary ( ) conclude that, on the one hand, behavioural reactions of cows and calves to separation increase with increased contacts but, on the other hand, health and future productivity (weight gain for the calf, milk production for the cow) are improved when the two animals have spent more time together. calves reared by their dam do not develop cross-sucking while artificially reared calves do so (margerison et al., ) . the provision of milk through a teat, a long milk meal, and the possibility to suck a dry teat can decrease non-nutritive sucking in artificially reared calves but do not abolish it (review by jensen, ; lidfors and isberg, ; veissier et al., ) . the presence of adult cows other than the dam do not help calves to get accustomed to new rearing conditions, as observed by schwartkorf-genswein et al. ( ) for calves submitted to feedlot conditions. . . . recent findings regarding contacts with other calves recent studies confirmed that calves are motivated for social contact. such a motivation was shown using operant conditioning by holm et al. ( ) ; furthermore calves that are housed individually engage in more contacts with their neighbours than calves housed in pairs (raussi et al., ) . the presence of a companion can reduce emotional responses of calves. this, for instance, is the case when group housed calves are exposed to a novel situation like a novel object (boivin et al., ) , a novel arena (jensen et al., ) , a sudden event (veissier et al., ) , or a lorry (lensink et al., ) . humans are not a good substitute for social contacts. individually housed calves interact more with their neighbours compared with pair-housed calves, even when they receive additional contacts from the stockperson (e.g. stroking, letting suck fingers, speaking softly) (raussi et al., ) . (see section on humananimal relationships). . . . comparison between individual housing vs. group housing individual housing can be stressful to calves as measured by adrenal responses to acth (raussi et al., ) . group housed calves are generally more active than individually housed calves as far as gross activity is concerned (more time spent moving or eating, less time spent idling or lying) (babu et al., ; raussi et al., ) . group housing can benefit production: xiccato et al. ( ) found that calves housed in fours put on more weight than calves tethered in individual crates. however, this seems not to be the case when the calves are not tethered in individual crates (veissier et al., ) . group housed calves are less easy to handle. human contact is thus essential for them to become accustomed to humans and to react less to handling (lensink et al., ; mogensen et al., ) . group housing can help calves acquiring social skills (boe and faerevik, ) . some experience of mixing is of particular importance: calves that have been reared for a while in a group dominate calves that have always been in individual crates (veissier et al., ) . by contrast, it is not clear whether repeated mixing would be beneficial or harmful to calves veissier et al., ) . recent research (e.g. svenson et al., and svensson and liberg, ) suggests that transfer from individual pen to group-housing during the second week of life is disadvantageous for health reasons (see chapter ) and, that a delay in mixing until the calf is weeks old may be preferable. additional research seems necessary to establish what mixing age would be preferable from a health and welfare perspective. . . temperature, ventilation and air hygiene the importance of the aerial environment inside a calf house for the health status of the animals was stressed in the report, and it still seems to be one of the major factors which cause morbidity and mortality (svensson et al., ) . bioaerosols (micro-organisms, dust), low air temperatures together with high air humidity, gases such as ammonia, draught, insufficient air space and poor ventilation form a complex environmental situation which can be detrimental particularly for the respiratory health of young calves (lundborg et al. ; svc, ) . . . . temperature and relative humidity a healthy calf consuming a sufficient amount of feed has a wide zone of thermal neutrality. there is no difference in the performance of healthy, normal eating calves at temperatures ranging between ºc and ºc provided it is dry and not exposed to draughts. above ºc conditions in confined calf houses can start to become uncomfortable. moran ( ) suggests that the ideal temperature and relative humidity for calves are ºc and %, respectively. however, there is a large number of influencing factors to consider which can alter the situation for a calf substantially. lower critical temperatures for a calf in calm air and full feed are different whether it stands (- ºc) or is lying on dry concrete (- ºc) or on dry straw (- ºc) (thickett et al. ). the younger the animal the higher is its demand for the thermal environment. by week of age, the lower critical temperature in still air is approximately ºc (webster, ) . this temperature can be significantly changed by draught, wet coat and feeding level. young calves start to shiver at ºc when they are exposed to draught even if their coat is dry and they are fed sufficient feed. when fed on maintenance only level shivering starts at ºc. if their coat is wet and they are exposed to draught shivering starts at ºc when on full feed and ºc when fed on a low level (moran, ) . no signs of shivering are observed at ºc when the coat is dry with no draught and feeding is at a normal level. cold stress in calves can be prevented by providing dry lying areas, appropriate feeding and draught free ventilation. dry bedding such as straw significantly improves thermal comfort for the lying calf. in summer situations reduced feeding (but with sufficient water supply!) or feeding calves in the cooler evening or at a reduced animal density per pen and increased ventilation rates can help to lower heat stress. heat stress can also be reduced through constructing sheds with insulated roofs and well ventilated walls. calf houses with a solid wall construction and a high capacity to store energy combined with an efficient ventilation system can also contribute to create comfortable environmental temperatures for young calves kept indoors all year (din , ) . the preferred environmental temperature for calves is not fixed, it largely depends on management and other environmental factors such as wind speed and humidity of the air. the generally accepted range of relative humidity for calf barns is between and % with an optimum around % which is not too humid to dissipate excess heat and not too dry to dry out the respiratory pathways predisposing the mucous membranes to infectious and noxious agents present in the inhaled air. air humidities of more than % can lead to condensation on the walls and ceiling increasing the risk of wetting the animals by water dripping off these surfaces. high relative humidities can also impair the insulation properties of the walls increasing heat losses. cold and humid air at high velocities can considerably increase the heat loss of animals. lundborg et al. ( ) showed that draughts greater than . m/s measured close to the animal, significantly increased the odds ratio for moderate to severe respiratory sounds. the higher the humidity the higher the risk of wet skin and cooling and shivering. high air humidity increases the probability that bacteria survive in an airborne state and are transmitted between animals in the same pen and between animal pens (wathes, ) . there are existing numerous reports from s onwards of the survival of bacteria and viruses employing simple regression models to describe the loss of viability of microbes over time. however, these models lack an insight into biophysical and biochemical mechanisms of cell and virus death. as long as we do not understand these mechanisms, the measures to reduce the air pollutants are limited to either increased ventilation or increased air space. the smaller the air space per animal the more sophisticated the ventilation system must be. the influence of air space was demonstrated by wathes ( ) showing that doubling of the air space in a calf barn from to m³ per calf had the same effect on the concentration of airborne bacteria as a six fold increase in ventilation rate (air exchange rate). an air space of to m³ per calf was recommended by hilliger ( ) from experience. . . . air quality aerial pollutants in confined animal houses are widely recognised as detrimental for respiratory health. primary and opportunistic microbial pathogens may cause directly infectious and allergic diseases in farm animals, and chronic exposure to some types of aerial pollutants may exacerbate multi-factorial environmental diseases, such as enzootic bronchopneumonia. the factors can be inadequate environmental conditions, e.g. too low temperatures, high ammonia concentrations and poor ventilation resulting in low air quality. poor air and surface hygiene in calf buildings are nearly always associated with intensive systems of husbandry, poor standards of management and high stocking densities (wathes ) . the most common aerial pollutants in calf housing are summarised in table . . gases such as ammonia (nh ), hydrogen sulphide (h s), carbon dioxide (co ), and more than hundred trace gases form an airborne mixture of bioaerosols composed of about % organic compounds and can also contain endotoxin, antibiotic residues and further trace components. significantly high amounts of endotoxins were found in calf house air while bacteria and dust are relatively low compared with pig and poultry houses and suggest that a high number of gram-negative bacteria are present in the air. the average concentration of ng/m³ endotoxin given in table . represents about eu (endotoxin units) according to the new nomenclature. it seems rather high in comparison to a formerly proposed occupational threshold of eu for humans at the work place (rylander and jacobs, ) . it can be assumed that high endotoxin concentrations in calf house air may substantially contribute together with the other bioaerosol compounds and the physical environment to the high level of respiratory disorders in young calves up to days (assie et al., ) . in general, there is little detailed knowledge on the specific composition of bioaerosols in calf keeping systems and which factors cause respiratory diseases. assie et al. ( ) found e.g. a tendency to higher repiratory disorders in non-weaned calves reared in loose-housing yards compared with tied-cow stalls. the highest incidence rates of cases were observed between november and january, while daily meteorological conditions obviously did not influence incidence rates. one of the most detrimental gases in calf barns is ammonia which is formed by bacterial degradation of nitrogen containing compounds in urine and faeces. it is the most widespread naturally occurring alkaline gas in the atmosphere and a strong irritant in animals and humans. concentrations in the air of more than ppm can impair the proper functioning of the mucous membranes of the respiratory tract and predispose to infection. in a recent study lundborg et al. ( ) found that the risk for respiratory disease was significantly associated with an ammonia concentration below ppm (or: . ; p< . ). high concentrations of hydrogen suphide can be released in high amounts from stored liquid calf manure when it is stirred up before removal from the slurry pit of the barn. concentrations of about ppm are acute fatal. the composition of the inhalable and respirable particles in animal houses is associated with compounds such as dried dung and urine, skin dander and undigested feed. the majority of bacteria found in shed airspace have been identified as gram-positive organisms, with staphylococci spp. predominating (cargill et al., ) . a survey by heinrichs et al. ( ) showed the importance of good ventilation which removes dust and other respirable particles as well as noxious gases and is essential for calf health. adequate ventilation is seen as vital to help to reduce the incidence of respiratory disease. air inlets should be above calf height and the outlet at least . m above the inlet (howard, ) . however, heating and ventilation in combination with an air filtration system significantly improved the environment in a calf house but did not completely eliminate pneumonia (bantle et al., ) . this may have to do with other factors such as ambient temperature. in a recent study by reinhold and elmer ( ) some compromise in lung function (compared with controls) was seen in calves exposed to an ambient temperature of ºc. . . . . light in the past, veal calves were often kept in dark to reduce muscle activity but the requirements for light have increased over the last years from to lux (bogner and grauvogel, ) , to over lux (irps, ) to lux for at least h and according to daylight circadian rhythm. (tierschutz-nutztierhaltungsverordnung germany, ) . there is wide agreement that calves need light for orientation in their boxes or pens and for social contact. a precise threshold has not been determined. there is a need for air movement around calves to supply fresh air and to remove excessive heat, moisture and air pollutants (gases, dust, microorganisms). good ventilation systems provide this exchange. however, high air speeds close to the animals can lead to draughts and should be avoided. draughts happen when part of an animal is hit by an air stream with a higher velocity than the ambient air movement and which has a substantially lower temperature than the surrounding air, causing a feeling of cold and physiological reaction in that particular part of the body. draught can lead to poor welfare and disease when it continues and the animal cannot escape, e.g. when it is tethered. it is generally recommended that wind speeds around animals should be between . m/s in winter (least value) and about . m/s in summer. these values strongly depend on relative humidity and temperature of the air and whether the skin of the animals is dry or wet, full fleece or shorn. in confined buildings this complex relationship between the various factors is strongly influenced by the ventilation system and the ventilation rate which is necessary for the number of animals kept in the animal house. it seem useful to develop a more comprehensive model for the interaction of the different air quality compounds and the air exchange rate to improve our understanding of the welfare and health impacts arising from the air environment. . . . ventilation ventilation plays an essential role in improving air quality in calf barns. this applies to free ventilated and forced ventilated houses. calculations of ventilation rates are usually based on heat removal in summer and moisture removal in winter, and give some guideline temperatures and humidities of the air which should not be exceeded (e.g. cigr, ; din , ) . ventilation rates in calf barns can only be calculated satisfactorily for confined buildings. minimum ventilation rates around m³ per kg live weight should be sufficient to keep the air quality within acceptable limits if the air distribution system ensures an even air exchange in all parts of the building. such guidelines (cigr) and norms (din , ) cannot guarantee healthy calves but they can substantially help in designing confined calf houses. it seems useful to standardise the air quality and ventilation requirements for confined calf houses in europe in order to reduce respiratory disorders, suffering of the animals and economic losses. the report highlights two aspects of human-animal relationships: the skills and motivations of caretakers to raise healthy calves, which are of particular importance for indoor calves and in large groups and are linked to the health status of calves; the physical contacts between caretakers and calves to improve subsequent reactions of calves to humans. it recommends careful monitoring (by the same person throughout rearing) and careful handling to habituate calves to human contacts. recent studies confirmed that the stockpersons have a great impact on both the productivity (e.g. growth) and the welfare of farm animals (stress responses, fear reactions during handling) (boivin et al., ; reviewed by hemsworth and coleman, ) . the effect of stockmanship is two-fold: good stockmanship leads to healthy animals and less stressful human-animal interactions. stockman skills are associated with positive attitudes towards work and towards animals. in calf production, a better health status is observed on farms where the caretaker (also the owner) believes that calves are sensitive animals and he/she has a positive attitude towards farming tasks in calf production (lensink et al., b) contacts given by stockpersons to animals depend also on human attitudes. stockmen that are positive towards gentle contacts with calves (e.g. stroking, talking) are more likely to provide calves with such contacts (lensink et al., a) . it is not only the duration of contact but also its nature that plays a role. gentle contacts (e.g. stroking, talking, letting a calf suck fingers, offering food) lead to calves approaching humans as they have less fear of handling (boivin et al., ; jago et al., ; lensink et al., b) . whereas rough contacts (e.g. hitting with a stick, use of nose tongs or an electric prod) lead to fear reactions in presence of humans (rushen et al., ) . the electric prod seems particularly stressful to calves (croney et al., ) and noises (metal clanging, shouts by humans) will also increase stress during handling (waynert et al., ) . during transport to slaughter, less fear responses to handling (e.g. due to regular previous experience of gentle contact) not only improves the welfare of calves but also improves meat quality (lower ph and lighter colour) (lensink et al., c; lensink et al., a) . cattle are able to distinguish between familiar and unfamiliar persons (rybarczyk et al., ; taylor and davis, ) . among familiar persons, they distinguish between those who have been rough with them and those who have been gentle (e.g. stroking, brushing, and offering food) (de passillé et al., ) . compared with individually housed calves, calves housed in groups tend not to approach humans and are more difficult to handle (lensink et al., b) . the presence of the dam can lower the effectiveness of gentle contacts with animals (boivin et al., ) . contact early after birth can be more effective that contact provided later; however, regular contact is necessary to maintain a lower level of fear responses to humans (boivin et al., ) . the report recommended: to dehorn calves between - weeks by cauterisation with adequate anaesthesia and analgesia (no details given) to castrate calves at months with adequate anaesthesia and analgesia (no details given) . . . dehorning dehorning means the removal of horns while disbudding (on young animals) corresponds to the removal of horn buds. disbudding can be performed by cautery, or by rubbing or covering the horn buds with a chemical (naoh, koh or colloidon), or by amputation with a specifically designed sharp tool, a scoop. recent publications confirmed that disbudding and dehorning are painful to cattle (stafford and mellor, ) . the existence of pain is deduced from observations of an increase in blood cortisol for several hours after dehorning and from specific pain related behaviour: head shaking, ear flicking (faulkner and weary, ) disbudding without anaesthesia or analgesia is painful to calves, even when young, and dehorning with a wire-saw is painful to cows even if anaesthesia is carried out (taschke and folsch, ) . disbudding by cautery (hot iron, electric tool) and chemical disbudding (naoh) are less painful than disbudding with a scoop (stilwell g. et al., a; stilwell g. et al., b; sylvester et al., ) . local anesthesia ( - ml lidocaine or lignocaine % around the corneal nerve - min before disbudding) can abolish the pain that immediately follows cautery and largely diminishes the pain caused by disbudding by other methods; the effects last for the few hours and when the nerve block has lost its effect, pain ensues stilwell et al., b; sutherland et al., b) . local anesthesia plus analgesia with a non-steroidal anti-inflammatory drug (nsaid)(e.g. ml flumixin meglubine or - . mg/kg ketoprofen ( %) min before disbudding) abolish pain caused by cautery but only reduces it in the case of disbudding with a scoop (unless it is followed by cautery) (sylvester et al., ; faulkner and weary, ; sutherland et al., a; stilwell et al., b) . in their review, stafford and mellor ( ) concluded that cautery is the less painful method for disbudding and that optimal pain relief is obtained with xylazine sedation, local anaesthesia and analgesia with a non-steroidal antiinflammatory drug. the report recommend that when cattle are to be castrated this should be done at around months of age and under appropriate anaesthesia and analgesia. methods to castrate cattle are: clamping (generally with a burdizzo clamp), constriction of the blood supply with a rubber ring, and surgical removal (cutting of the scrotum then traction on the testes and spermatic cords or cutting across the spermatic cross). calves are castrated as early as week up to over months (see review of practices used in uk by kent et al., ) . castration is painful whatever the method used and whatever the age of the calf (molony et al., ; robertson et al., ) . acute pain is deduced from the observation of increases in blood cortisol and abnormal postures (immobility), and behaviours such as foot stamping and kicking. chronic pain is deduced from the observation of activities targeting at the site of castration (e.g. licking, head turning, alternate lifting of the hind legs, and slow movements of the tail) as well as abnormal standing. burdizzo clamping and surgery induce acute pain for at least h (molony et al., ; obritzhauser et al., ; robertson et al., ) . burdizzo is less painful than surgery but may also cause pain for longer (at least h) due to scrotal inflammation (stilwell, pers. comm.) . castration with a rubber ring causes both acute and chronic pain for at least . mo (molony et al., ) castration is less painful for wk old calves than for - wk old calves (robertson et al., ) and it is less painful at . months than at older ages (ting et al., ) . local anesthesia ( ml lignocaine % into each testicle through the distal pole) abolishes the acute pain associated with castration by a ring or a band (stafford k.j. et al., ) . it reduces but does not abolish acute pain associated with castration by surgery or clamping (fisher et al., ; stafford et al., ) . analgesia with a nsaid drug (e.g. ketoprofen % mg/kg body weight) reduces the pain associated with clamping (ting et al., ) . some analgesics (e.g. caprofen, , mg/kg body weight) are effective for longer than h and are thus more likely to provide more effective pain relief (stilwell, comm. pers.) . local anaesthesia plus analgesia appears to eliminate the acute pain due to castration by surgery or clamping . the most important diseases in young calves are diarrhoea and respiratory disease (olsson et al., ; sivula et al., ; virtala et al., a , donovan et al., lundborg, ) . a prospective study was carried out on randomly selected beef herds in the midi-pyrenees region in france (bendali et al., ) . the objective was to describe diarrhoea and mortality in beef calves from birth to days of age. calves ( , ) were followed from december to april , and a total of visits allowed records of herd management practices, individual data and environmental conditions to be collected. the incidence rate for diarrhoea during the neonatal period was . %, and varied markedly between herds. eighteen herds did not suffer from diarrhoea, while five herds had an incidence of more than %. results indicate that % of diarrhoea appears during the first week and only % after the second week of life. the greatest risk of diarrhoea for a calf was during the first and second weeks of life ( . and . times, respectively). the month of birth was also significantly associated with morbility, the highest incidence was observed in december and march ( . % and . %, respectively) . the global mortality rate was . % and was two-times higher in december than in other months. forty per cent of herds did not exhibit mortality, and % had mortality rates greater than %. in a study of calf health in cow-calf herds in switzerland, busato et al. ( ) found that of calves included in the study % of the calves had been treated by a veterinarian. of those, % of the treatments were given because of diarrhoea and % because of respiratory disease. another swiss study (frei et al., ) showed that in swiss dairy herds, the incidence density (id) per animal-years of diarrhoea, omphalitis (infection of the navel), respiratory diseases and other diseases were . , . , . and . respectively. in a study of nine herds and calves with swedish red and whites, swedish holsteins and some crossbreeds the effect of group size on health was studied (svensson and liberg, ) . after transfer to group pens (at - days of age) . % of the calves had diarrhoea, . % had omphalophlebitis/umbilical abscess, . % had a clinical respiratory-tract disease and . % had weak calf syndrome. of all calves, in . % there was associated general condition impairment. in . % of the diarrhoea cases antibiotics were used as treatment and of the clinical respiratory-tract cases % were treated with antibiotics. several factors have been associated with an increased risk of infectious disease during the first days of life, particularly factors affecting serum immunoglobulin concentration. in a study of dairy herds in south-west sweden, svensson et al. ( ) clinically monitored the health of heifer calves from birth until days of age. % of the calves developed one or more diseases during this period. most of the diarrhoea cases were mild ( %) whereas of the cases of respiratory disease % were severe. the total morbidity was . cases per calf-month at risk and the incidence rates of arthritis, diarrhoea, omphalophlebitis, respiratory disease and ringworm were . , . , . , . and . cases per calf-month at risk respectively. odds ratios were calculated for severe diarrhoea in calves born in the summer (or: . ) and receiving colostrums through suckling instead of a bucket or nipple (or: . ). it has been shown that calves left with their mothers have a delayed/longer time to ingest colostrum and often fail to ingest adequate volumes (rajala and castrén, ) . svensson and liberg ( ) found that the health status of the mother cow - days before calving, length of dry period, retained placenta and somatic cell count were predisposing risk factors for respiratory disease in the calf. svensson et al. ( ) were also able to demonstrate that the odds ratios for respiratory disease and increased respiratory sounds were increased in calves housed in large group pens with an automatic milk-feeding system (or: . and . ). similar results have been reported by maatje et al. ( ) and plath ( ) . there was a decreased odds ratio for respiratory disease if calving was supervised (or: . ) . if birth was taking place in individual maternity pens or in tie stalls instead of in cubicle or group maternity pen, the odds ratio for increased respiratory sounds was . or . respectively. % of the diarrhoea cases were treated with antibiotics whereas % of the respiratory cases were treated using antibiotics. in another study of nine farms, svensson and liberg ( ) found that in pens for six to nine calves there was a significantly reduced risk of clinical respiratory tract disease (or: . - . ) compared with pens with - calves and there was also an association with the age at transfer to the group pen. the risk of diarrhoea was not affected by housing the calves in differently sized groups. however, calves housed in large sized groups grew significantly less quickly (approximately g/day) than calves housed in groups of six to nine. serological responses to respiratory viruses (e.g. bovine respiratory syncytical virus, parainfluenza virus, corona virus and viral diarrhoea virus) showing that animals within a herd are usually either all seropositive or all seronegative, indicate that infections spread to all calves in the herd when introduced or activated (hägglund et al., in press) and hence that aerosol is an important means for the spread of viruses. however, an infected animal is not equivalent to a diseased animal. it has been shown that calves housed in adjacent pens can maintain quite different levels of disease. svensson and liberg ( ) reported that calves in a group of had a significantly higher incidence of clinical respiratory disease that calves in an adjacent pen kept in groups of . engelbrecht ( ) reported calves transferred to group pens in a batchwise manner had significantly higher prevalence of diarrhoea and respiratory disease than calves in adjacent pens that were transferred to and from the group pen continuously. in both studies calves had no direct contact with calves in adjacent pens. these results indicate an important role of direct contact for the transmission of respiratory disease and hence the importance in disease control of decreasing direct contact between calves within the same building by means of solid walls. svensson and liberg ( ) also reported that the age at transfer from single pens to group pens was associated with the risk of respiratory disease, indicating that delaying transfer to after two weeks of age might be preferable for health reasons. enteritis is the most common disease in calves less than a month old (virtala et al., b; wells et al., ; radositis et al., ) . diarrhoea is caused by dietary factors or caused by infections due to viruses, bacteria or parasites. routines in distributing colostrum to the calf are crucial for transferring immunoglobins to the calf and to obtain a good health. (rajala and castrén, ; liberg and carlsson, ) . enteritis is clinically recognized by the observation of faeces with a looser consistency than normal. colour as well as smell of the faeces might be affected. diseased animals exhibit fever and may be inactive usually as a result of dehydration and possibly acidosis (radositis et al., ) . usually it is not possible to differentiate between different agents causing the diarrhoea by clinical findings. rotavirus is worldwide a major cause of diarrhoea and it is an often detected agent among young calves with enteritis (e.g. björkman et al., ) . rotavirus affects calves usually between and weeks of age and the diarrhoea can vary from very mild to lethal (de leeuw et al., ) . the virus is excreted through faeces of infected animals and is very resistant for several months, that is why cleaning of pens is necessary to break the infectious path (saif and theil, ) . bovine coronavirus is most commonly seen in calves at about week of age (fenner et al., ) . escherichia coli k + may cause diarrhoea in young calves although it is a part of the normal intestinal flora. poor routines for transferring colostrum to the calf, stress etc. might trigger a diarrhoea outbreak (wray and thomlinson, ) . severity of the disease may vary but with a high proportion of mortality (radositis et al., ) . only amoxicillin is recommended for the treatment of diarrhoea caused by e. coli bacteria associated by systemic illness. in calves with diarrhoea and no systemic illness (normal appetite for milk, no fever), the health of the calves should be monitored carefully and no antibiotics should be administered (constable, ) . bovine viral diarrhoea may occur at any age. the infection causes an immunosuppression in infected animals which may lead to infections with other intestinal or respiratory pathogens (elvander et al., ; de verdier klingenberg, ) and it may increase the mortality rate in the herd (ersböll et al., ) . salmonella spp, mainly s dublin and s typhimurium can affect calves usually between and weeks of age. the pathogen is introduced into the herd via infected feed, water, pastures, cattle or humans or via other animals entering the herd. calves are infected orally and clinical signs are fever and bloody diarrhoea (carter and chengappa, ) . clostridial infections in the gastrointestinal tract are sometimes a problem in calves. usually, the calf, less than days of age, develops haemorrhagic, necrotic enteritis and enterotoxemia, often associated with clinical abdominal pain. affected calves exhibit tympany, hemorrhagic abomasitis and ulcerations in abomasum (songer and miskimins, ) . as yet, relatively little is known about the etiology aside of the participation of c. perfringens type a. overfeeding or feeding which decreases gut motility is suggested to contribute to the occurrence of the disease (songer and miskimins, ) eimeria spp. are frequently present in cattle in europe (bürger, ) . predominantly e. ellipsoidalis was found in housed calves in east germany (hiepe et al., ) and the distribution may differ from country to country. svensson ( ) found a predominance of e. alabamensis in swedish dairy calves. clinically, signs are rarely seen but diarrhoea can occur usually as a result of exposure at the first grazing season in areas contaminated with oocysts (svensson, ) . there is evidence that infection rates have increased since the prohibition of tethering (berthold, pers. com.). emeria bovis and emeria zuernii are other intracellular protozoan parasites belonging to the same group and with a worldwide distribution (urquhart et al., ) . it is often seen in calves between - months of age (holliman, ) . the disease is triggered by stress such as very cold or hot climate (urquhart et al., ) . cryptosporidial infection in calves less than days old is significantly associated with the risk of infection in the dairy herd. the risk increases when animals are grouped together and when hygiene and management practices are deficient (attwill et al., ; mohammed et al., ) . factors associated with a decreased risk of infection in preweaning calves were shown to be use of ventilation in calf rearing areas, daily addition of bedding, feeding of milk replacer, daily disposal and cleaning of bedding and use of antibiotics. in addition, postweaning moving of animals was also associated with a decreased risk of infection with c. parvum (mohammed et al., ) . perryman et al. ( ) showed that with appropriate supply of immune colostrum, diarrhoea can be prevented. two species are distinguished: c. parvum and c. andersoni, although only c. parvum has been shown to be associated with diarrhoea . cryptosporidia parvum is an intracellular protozoan parasite belonging to coccidiae. in the uk c. parvum has been considered to be one of the most common agents in neonatal diarrhoea in calves (reynolds et al., ) . in denmark it was found mixed with other enteropathogens in % of diseased calves (krogh and henriksen, ) . in two recent swedish studies it was found in % and % respectively of calves with diarrhoea (de verdier klingenberg and svensson, ; björkman et al., ) . the most common form of respiratory disease affecting young calves is enzootic pneumonia (ames, ; radositis et al., ) . it is considered to be a multifactorial disease with causative agents, individuals and environmental factors as important components (ames, ) . enzootic pneumonia usually affects calves between and months of age (radositis et al., ) . the signs usually found are fever, nasal discharge, coughing and increased respiratory sounds when lung auscultation is performed. secondary bacterial infections may occur which might increase the fever. diagnosis of etiological factors may be achieved from serological examinations, viral examinations from nasal discharge or at autopsy. bovine respiratory syncytical virus (brsv) is a worldwide present agent with seasonal peaks during autumn and winter (baker and frey, ) . the virus is thought to be transmitted from infected animals, by transmission of humans or by airborne transmission (van der pohl et al., ; elvander, ) . morbidity can be high but mortality is usually low . another virus with a milder course of disease is para-influenza- virus but the virus can cause immunosuppression predisposing to secondary bacterial infections (adair et al., ) . the most common bacterial pathogens in calves with respiratory disease are pasteurella multocida and manheimia hemolytica (mosier, ; bengtsson and viring, ) . these agents are usually found in the bovine nasopharynx and may, as a result of viral disease proliferate and colonise the lungs of the calf (kiorpes et al., ) . haemophilus somnus was shown to be commonly present in danish calves (tegtmeier et al., ) where no such agents were found in swedish calves (bengtsson and viring, ) . arcanobacterium pyogenes and staphylococcus aureus (carter and chengappa, ) as well as mycoplasma spp. (ames, ) are other agents found in immune depressed calves with other infections. infections may occur in the umbilical cord (radositis et al., ) of newborn calves. various bacteria are found and through a bacteraemia infection may spread to to joints, meninges and internal organs (radositis et al., ) . omphalitis is painful in response to palpation of the umbilicus. arthritis is often secondary to an umbilical infection and usually affects the calf during its first month resulting in warm and swollen joints, fever and lameness (radositis et al., ) . the effect of environmental factors on the risk of diarrhoea and respiratory disease was studied by lundborg et al. ( ) in the same farms and calves as previously reported by svensson et al. ( ) . they found that the placing of calf pens along an outer wall was associated with a significantly higher risk of diarrhoea (or: . ). an ammonia level below ppm was significantly associated with the risk of respiratory disease (or: . ) but variations of ammonia levels were low, while the odds ratio for increased respiratory sounds was associated with a bvdv infection in the herd (or: . ) and draught (or: . ). absence of draught was associated with the risk for infectious diseases other than diarrhoea and respiratory disease (or: . ), a finding which could not be explained by the authors. an increased calf mortality rate in herds with a bvdv infection has also been reported by ersböll et al. ( ) and the eradication of bvdv infection in a dairy herd has been demonstrated to decrease the incidence of calf diarrhoea (de verdier klingenberg et al., ) . typically, clinical experience is that the incidence and prevalence of infectious respiratory disease is much higher in rearing systems where the calves have been bought and transported from several farms where they were born to a specific rearing farm than if they are reared on the farm they were born on. calves reared indoors commonly develop complex respiratory diseases. bergmann ( ) reported that % of calves of a large fattening unit with several thousand calves suffered from bronchopneumonia within the first six month of their life. similar figures were reported from herrmann ( ) with a prevalence of to %, lämke et al. ( ) % and busato et al. ( ) %. the disease seems to be continuously present and does not come or go in form of isolated outbreaks. therefore kielstein et al. ( ) called it enzootic pneumonia. it is a typical multifactorial disease caused by a variety of different types of micro-organisms which are always present but becoming a nuisance only when additional factors contribute (grunert, ) . the most prominent reasons for losses of calves in the first weeks of life are respiratory and digestive disorders (katikaridis ; girnus, ) . losses can reach to per cent in the first six months of rearing (berchtold et al. and others). estimations show that the financial losses are reaching million €/year in germany (biewer, ) . this sum does not cover the costs of veterinary treatment and reduced growth rates of the calves. there are several epidemiological studies on the different diseases in calves in the first couple of weeks of life (katikaridis, ; biewer, ; girnus , svenson et al., . heinrichs ( ) reported that % to % of , fallen calves coming for post-mortem dissection showed digestive disorders. calves weeks of age died predominantly of respiratory diseases ( % to %). an investigation of , calves less than months shows that enzootic bronchopneumonia can already start with the age of weeks (buhr, ) . the calves were not older than months. % displayed abomasum enteritis. % of the animals suffered from pneumo-abomasal enteritis. only . % of the fallen animals suffered from a distinct pneumonia. an epidemiological survey of calf losses in free range and suckling cow herds showed that the percentage of calf losses is increased with herd size. % of herds with less than suckling cows had a calf mortality of less than %. in herds with more than cows, these were % of all investigated farms, calf losses were higher than % (laiblin and metzner, ) . main disorders were again diseases of the digestive tract ( %) and respiratory tract ( %). the authors calculated that the disease risk for calves born from cows that were housed during the calving season was . times higher compared with cows kept the whole year on free-range. the epidemiological data from the vast majority of investigations suggest considerable differences in morbidity and mortality of calves among different farms. this implies that the management and housing conditions greatly influence heath, welfare and survival of calves in the first months of their life. the situation was not substantially improved by vaccination of cows against a cocktail of infectious agents causing diarrhoea. there are no experimental studies available to indicate whether or not there is any advantage to calf welfare of preventing individuals in separate pens from social contact as opposed to a disadvantage to calf welfare of greater spread of disease with housing where such social contact is possible. in general, disease spread occurs in buildings with continous air space and contact is not a clearly identified factor. however, recent results indicate an important role of direct contact for the transmission of respiratory disease and hence the importance in disease control of decreasing direct contact between calves within the same building by means of solid walls (see chapter / / ). antibiotic resistance although the use of antibiotics as growth promoters is being progressively restricted through eu regulations, they are still used in large quantities in calf rearing for both prophylactic and therapeutic purposes. in those instances where calves are not reared on site but transported to other locations and mixed in groups, the incidence of clinical illness is high and the use of antibiotics is frequent. in a study of antibiotic resistance, berge et al. ( ) found high levels of multiple resistance in calf commensal faecal escherichia coli both on farms with calf production and on dairy farms. the investigators found that escherichia coli from calves on dedicated calf-rearing facilities was more likely to be multiple-resistant than e. coli from dairy-reared calves (or: . ) (berge et al., a) . in her phd thesis, berge ( ) showed that both prophylactic use of antibiotics in milk replacer and individual antibiotic therapy increased the resistance of faecal e. coli in calves. e. coli isolates from calf ranches were the most resistant, with in order of decreasing levels, isolates from feedlots, dairies and beef cow-calf farms. on organic dairies fewer resistant e. coli isolates were found in comparison with conventional dairies. e. coli isolates from beef cow-calf farms were less resistant if the farms were on remote locations compared to those on locations close with dairy intense areas. the use of antibiotics to treat clinical illness will increase the welfare of the animal given that the drug has a beneficial clinical effect. however, the frequent use of antibiotics results in increasing resistance in bacteria such as e. coli and thus poses a threat to the welfare of calves in a longer perspective as well as to man (aarestrup and wegener, ) . in a clinical trial on a calf ranch in california, it was shown that the most important factor for decreasing morbidity and mortality was to ensure adequate passive transfer through colostrum (berge et al., c) . thereafter, the ability to use antibiotics for clinical treatment of disease was important to decrease morbidity and mortality. the use of antibiotics in the milk replacer had a minor protective effect on calf health. the authors concluded that in order to minimize prophylactic use of antibiotics, adequate passive transfer of colostrum needs to be assured. furthermore measures need to be taken to optimize nutrition, decrease environmental stress and pathogen load on the farms. in the same study, the antibiotic resistance patterns of the commensal faecal e. coli of calves receiving antibiotics in the milk-replacer, antibiotics for clinical disease, and no antibiotic therapy were compared (berge et al., ) . the study showed the emergence of highly multiple resistant e. coli in the calves receiving antibiotics in the milk-replacer. the commensal faecal e. coli were predominantly resistant to at least of antibiotics tested. the resistance covered the antibiotics available for clinical therapy. antibiotic treatments for clinical disease resulted in a transitory shift to more resistant faecal e. coli, but the effect was not detectable approximately days post-treatment. the effect of clinical therapy with antibiotics was similarly assessed in steers in south dakota. in a feedlot study a single dose injectable florfenicol to steers resulted in transitory shifts to increasing levels of multiple resistant e. coli in the faeces. the e. coli from the treated steers were not only more resistant to chloramphenicol (same antibiotic group as florfenicol), but were increasingly resistant to several other antibiotics in other antibiotic classes. (berge et al., b) in dairy cattle it has been estimated (kelton et al., ) that between and % of all lactations include a mastitis infection. most of these cases are treated with antibiotics. milk must be withheld from sale during the treatment and for the compulsory withdrawal period. such "waste milk" is often fed to calves as it is the most economical alternative from the farmer's perspective. earlier studies have previously given various results on how antibiotic resistance develops as a result of the use of this procedure. recently, a controlled, multiple-dose experiment by langford et al. ( ) found an increasing resistance of gut bacteria to antibiotics with increasing concentrations of penicillin in milk fed to dairy calves. in a multi-farm study in california (berge et al., ) including dairies, no association was found between increasing levels of antibiotic resistance in calf faecal e. coli and the consumption of waste milk. it should, however, be noted that mastitis in these dairies are predominantly treated with intra-mammary antibiotics (cephalosporins) and injectable antibiotics are rarely used for mastitis treatments (berge, non-published data) . the use of rearing systems for calves that increase the incidence of disease and thus the use of antibiotics for either preventive or clinical purposes should be avoided. further, there is a risk that the use of "waste milk" for calves will increase antibiotic resistance in gut bacteria in calves. . food safety aspects of calf farming foodborne hazards that can be present on calf farms include biological and chemical hazards. biological hazards associated with calf farming include following main examples: a) bacterial foodborne pathogens salmonella spp., human pathogenic vtec (hp-vtec), thermophilic campylobacter spp., and mycobacterium bovis; and b) parasitic foodborne pathogens tania saginata cysticercus and cryptosporidium/giardia. on-farm control of chemical foodborne hazards is outside the scope of this chapter and will not be considered. faecal shedding of foodborne pathogens can occur in calves and adult bovines without symptoms of disease; but the shedding pattern may differ between the two age categories. in the conventionally reared animal the intestinal tract becomes colonised from birth by combinations of bacterial species until the characteristic and complex flora in the adult animal is achieved. in the early stages of the process infections with bacterial pathogens are common. once the indigenous flora is established it resists colonization by pathogens and other 'foreign' strains by competitive exclusion . the gut flora of the bovine species changes with ruminal development and the population of faecal coliforms of the adult differ markedly, both qualitatively and quantitatively, from that of the young; particularly that of veal calves fed milk replacer (smith, some early studies (gronstol et al., a; gronstol et al., b) , based on experimental salmonella infection of calves, described virulence, spread of infection and the effects of stress on the carrier status. hinton et al. ( hinton et al. ( , a hinton et al. ( , b determined the incidence of salmonella typhimurium (dt and dt ) excretion by veal calves fed milk replacer and report that, while initially low on in-take at around days of age, its incidence rose to a peak by days. the level of salmonella contamination of the environment also affects the incidence of infection in housed animals (hinton et al., a (hinton et al., , b . provided calves are reared in separate fattening units and slaughtered on separate slaughterlines the incidence of salmonellae in calves can be maintained at a very low level (guinée et al., ) . during meat inspection clinical salmonellosis is sometimes diagnosed in a herd of veal calves; however, the prevalences are usually of the order of magnitude of per thousand and the strains isolated are generally restricted to salmonella typhimurium (occasionally) and more commonly, salmonella dublin (up to %). although salmonellae may not be isolated from faeces, significant proportions of calves slaughtered commercially ( . - . %) have contamination involving hepatic lymph nodes, liver, mesenteric lymph nodes and, because of cross contamination, they may ultimately also be isolated from the carcass surface (nazer and osborne, ; wray and sojka, ) . studies conducted in the netherlands in the late 's indicate that microorganisms may be released from lymph nodes through transport stress and may appear in the faeces. this results in young veal calves being cross-infected in transit and at markets; however, in dutch studies faecal samples from no more than . % of the animals were found to contain salmonellae on arrival at the fattening units (van klink and smulders, ) . moreover, within weeks of arrival, faeces samples become negative again (van zijderveld et al., ) . subsequent studies by the same workers (van zijderveld et al., unpublished, cited by van klink and smulders, ) indicate that faecal samples from calves which had survived clinical salmonellosis also become culture-negative, albeit only after weeks. these findings suggest that, provided stressful transport conditions are avoided and sufficient hygienic care is taken to avoid cross infection during transport to the abattoir, the extent of introduction of salmonellae to the veal slaughterline is indeed extremely low. this is substantiated by repeated failure to isolate salmonellae from carcass surfaces of veal calf populations, and from their livers and offal meats (van klink and smulders, ) . as with other bacterial foodborne pathogens, antimicrobial resistance in salmonella shed by calves represents an additional food safety risk. numerous studies have shown that use of antimicrobials in food producing animals selects for resistance in non-typhoid salmonella spp. and that such variants have been spread to humans (who, ; walker et al., ; fey et al., ) . in general, antimicrobial resistance in s. typhimurium isolates from bovines in the eu was widespread in , with highest prevalence of resistance to ampicilin, sulfonamide, tetracycline and streptomycin (efsa zoonosis report, b), but the data does not relate specifically to calves. vtec is a group of e. coli that produces one or more verocytotoxins (vt) also known as shiga toxins (stx), but not all members of this group cause foodborne disease in humans. in the opinion on verotoxigenic e. coli (vtec) in foodstuffs (scvph, c) , vtec that have been associated with causing human disease were referred to as human pathogenic vtec (hp-vtec). foods of bovine origin (e.g. beef, milk) have been implicated in a number of foodborne outbreaks caused by hp-vtec (borczyck et al., ; chapman et al., ; martin et al., ; pennington, ; scvph, a) ; these include several serotypes (e.g. o , o , o , o and o ). when adult cattle were inoculated with vtec o , they showed no outward signs of infection and the organism was cleared from the gastrointestinal tract within two weeks (wray et al., ) . the organism seems to be a constituent of their naturally-occurring microflora, and longitudinal studies show most cattle occasionally carry e. coli o in their faeces (hancock et al., ; lahti, ) . however, the prevalence of infection with hp-vtec of, and the shedding patterns in, cattle can vary due to variable factors including age, immunocompetence status, husbandry conditions, season and geographical areas. prevalence of vtec o is usually higher in younger animals (synge, ; cray and moon, ; hancock et al., ; mechie et al., ; van donkersgoed et al., ) . in calves, the prevalence of e. coli o :h can range from zero to . % prior to weaning, and often increases after weaning (bonardi et al., ; laegried et al., ) . calves normally show no, or little, sign of infection, perhaps only some excess faecal mucus (myers et al., ; synge and hopkins, ; brown et al., ; richards et al., ; wray et al., ) ; the shedding rate can fall rapidly in the first two weeks after inoculation and continue intermittently for several weeks. in the first three months of life on contaminated farms, faecal prevalence can increase from around % to %, possibly due to the decline in maternal immunity (busato et al., ) . fasting showed little effect on the carriage and excretion of e. coli o in calves (harmon et al., ) . less information is available on non-o hp-vtec in calves that have potential to cause enterohaemorrhagic disease in humans, so establishing indicators for virulence and clarifying the epidemiology of such serotypes is needed. thermophilic campylobacter spp. according to the biohazard scientific report on campylobacter in animals and foodstuffs (efsa, a) , the most important species of campylobacter are the thermophilic species c. jejuni ssp. jejuni, c. coli and c. lari; other species which are known to cause human illness are c. upsaliensis, c. fetus ssp. fetus and c. jejuni ssp. doylii. the most common species recovered in human disease is c. jejuni. campylobacter spp. can be found throughout the intestine of cattle, but the level of the organism in the rumen is significantly lower than that found in the small intestine and in faeces (stanley et al., ) . the class of cattle also has an effect on the level of campylobacter spp. found in the faeces; faeces may contain around cfu/g in calves, around cfu/g in beef cattle and around cfu/g in adult dairy cattle. campylobacter spp. are more often found in the faeces and intestine than in the rumen, while campylobacter jejuni prevalence is much less than that of campylobacter hyointestinalis (ataby and corry, ; grau, ) . campylobacter jejuni has been found in calves in % of beef farms, campylobacter coli in % and campylobacter hyointestinalis in % (busato et al., ) . within the herds, zero to over % of the calves may be excreting campylobacter spp.; % of the calves may be positive for campylobacter spp. in herds without evidence of diarrhoeal disease (myers et al., ) . in this study, % of the isolates were c. jejuni. in a study of veal calves at slaughter, c. jejuni was found in % of calf rumen samples (in low numbers; < /g) and in % of calf faecal samples (grau, ) , whilst c. hyointestinalis was found in % of calf rumen samples (in numbers > /g) and in % of faecal samples. the coats of the calves were also contaminated, as % were positive for c. jejuni and % for c. hyointestinalis. as with other bacterial foodborne pathogens, antimicrobial resistance in thermophilic campylobacter spp. shed by calves represents an additional food safety risk. in , although the total number of isolates was relatively small and the data are not related specifically to calves, some eu member states reported relatively high prevalence of resistance to quinolones, fluoroquinolones and tetracyclines in campylobacter spp. including c. jejuni from bovines (efsa zoonosis report, ) which can be an emerging public health concern. . . mycobacterium bovis efsa a, opinion on the risk assessment of a revised inspection of slaughter animals in areas with low prevalence of cysticercus); therefore, the parasite will not be further considered here. cryptosporidium parvum and giardia duodenalis are protozoan parasites that have caused disease in humans primarily via contaminated water or foods (e.g. salads), but also via chicken salad and milk drinks. high prevalences of cryptosporidium and giardia in veal calves (the age group - weeks) have been reported (van der giessen et al., ) . however, in this study, all isolates from the former group belonged to the pathogenic cryptosporidium parvum genotype , whilst only few isolates from the latter group showed similarities with giardia isolates from humans. other authors also reported presence of these protozoan parasites, cryptosporidium (de visser et al., ) and giardia (trullard ; mcdonough et al., ) in veal calves. risk evaluation and principles of food safety assurance at calf farm level the prevalence-level of infection and/or contamination of calves with, and further spread of, foodborne pathogens at calf farms depend on a large number of risk factors that are inherently variable even at single-farm level. the complexity of the problem is further exacerbated by the existence of a number of different farming systems for veal calves in the eu; and even within each of the main farming categories (e.g. intensive vs extensive) a large number of "epidemiological" subcategories exists that differ with respect to one or more risk factors. therefore, presently, both knowledge and published data are insufficient to produce a universal risk assessment enabling quantitative categorization of different types of calf farms and/or their quantitative comparison/ranking with respect to main foodborne pathogens. nevertheless, the role of some main factors contributing to an increase in prevalence and/or in levels of foodborne pathogens in food animals on farms (including calves) are reasonably well understood, as are the generic principles of their control. they are indicated in a condensed form in table . . it is logical that calves from farming systems in which fewer of the contributing factors exist and where the controls are more complete/efficient will represent lower foodborne pathogen-risk than calves from farming systems having opposite contributing factors-controls situation. therefore, future food safety risk categorization of individual farming system, or related between-systems comparisons, would be dependent upon obtaining and analysing quantitative information on: a) status/levels of contributing factors; b) status/levels of hazards of main concern; and c) existence and effectiveness of their controls. increased "importation" and spread of pathogens via animals "asymptomatic excretors" animal supply only from known, epidemiologically "equivalent" sources; "all in-all out" system presence of animal diseases spread of zoonotic agents in calves global disease control programmes; heard health plans *good farming practice-good hygiene practice . risk assessment . . introduction to risk assessment approach when the ahaw panel of efsa was confronted with the tasks of updating the report (svc, ) , the working group members were asked to make it on the basis of a risk assessment , and particularly to consider the possible effects on the calf and, where relevant, on food safety. it appeared entirely feasible for the working group members to follow this part of a risk analysis approach where risks were defined as those concerning the welfare of calves. the risk of concern in this report is that the welfare of the calves will be poor. this may involve an increased risk of injury, of disease, of negative feelings or of failure to cope. the time span of such poor welfare might vary from short to long and severity can vary from low to high. a member experienced in risk assessment procedures was included in the working group from the start. initially, the procedure adopted for the risk assessment was identified and presented to the participants of the whole working group. when identifying the hazards, it has been assumed that the managers of the farm and animal keepers have a basic knowledge, that they have undergone training, that they are aware that the particular constraints on the farm do not hamper their work (e.g. lack of facilities on a farm). however, it is pointed out that under practical conditions hazards may interact, e.g. inadequate air flow may interact with poor air quality, inadequate clinical health monitoring may interact with inadequate haemoglobin monitoring, etc. the identification of hazards and consequential risks to welfare, as well as the risk assessment approach, were agreed by the working group. steps of the risk assessment a. multidisciplinary approach the expert working group it was selected on the basis of having expertise in animal science, ethology, veterinary medicine, risk assessment and food safety. b. listing of potential hazards, hazard characterization and exposure assessment the first step was to describe the needs of calves (see chapter and listed below). then, hazards that might compromise those needs were identified (table . ) and related to each specific need (table . ). the hazards were characterized in relation to the impact they have on the animal. the exposure to the hazard might vary between different rearing systems. for this purpose a set of different rearing categories was developed (table . ) as well as scoring categories for the hazard characterisation, exposure assessment and risk evaluation (table . ). b.a. small groups, bucket fed (not suckling) + solid foods, weaned at - months b.b. groups with an automatic feeding system (not suckling) + solid foods, weaned at - months b.c. feed lots (high density groups within outside pens) b.d. hutches outside, bucket fed (not suckling) + solid foods, weaned at - months beef calves c.a. suckler calves in small groups kept inside, led twice a day to the dam for suckling up to - months the hazards were identified and characterized, as well as, an estimate of the probable exposure. however, to ensure that these estimates of exposure correspond with current practice in various european calf production systems, a group of veterinarians, experts in clinical practice in calf production, named the "consultation group", was identified. criteria for invitation were the following; predominantly engaged in clinical practice; extensive clinical experience in calf medicine; and covering various geographical areas where calf production is significant in the eu. another important criterion was that the consultant should not be affiliated with the calf production industry. in total veterinarians accepted an invitation to assist in the exposure assessment. the experience of the individuals covered the various husbandry systems and important veal producing countries in europe. the consultation group prompted that for the exposure assessment, a quintile distribution (i.e. five classes of % increments) of exposure classes be adopted. in some instances the estimates of the wg and the consultation group on exposure did not agree in which case the opinion of the consultation group was interpreted to represent the factual situation. in other instances the exposure could not be estimated due to lack of data, in which cases the risks were labelled "uncertain". for hazards characterized as moderately to very serious, this uncertainty is highlighted. table in annex show the agreed scoring between the wg members and the "consultation group" for the hazard characterisation, exposure assessment and risk evaluation. inadequate colostrum intake -quantity b) inadequate colostrum intake -quality c) inadequate colostrum intake -duration ) iron deficiency resulting in haemoglobin levels below . mmol/l ) deficiency of other minerals (cu, se) ) insufficient access to water (not milk) (especially during warm season) ) allergenic proteins ) insufficient appropriately balanced solid food ) overfeeding (too rich diet) ) underfeeding ) too low temperature of milk or milk replacer ) exposure to excessively contaminated feed that results in pathology ) no access to natural teat or artificial teat housing ) high humidity and too high or low a temperature ) indoor draughts ) inadequate ventilation, inappropriate airflow, and air distribution within the house, airspeed, temperature ) poor air quality (ammonia, bio-aerosols and dust)) ) poor air quality ( major risk ; is considered not applicable c. assessment of whether hazards pose risks (substantiation by scientific evidence) as a consequence of the hazard characterisation and exposure assessment, the risk for poor animal welfare and health was assessed by integrating the hazard character with exposure according to the table . below. the risk was assessed as "major" if the hazard was judged to have a very serious effect and the exposure was frequent or very frequent or if the hazard was serious and exposure was very frequent. the risk was assessed to be "minor" if the hazard was very serious and exposure was rare, if hazard was moderately serious and exposure was moderately frequent or if hazard was adverse and exposure was very frequent. the hazards of iron deficiency and insufficient floor space is considered to be very serious, the hazard of inadequate health monitoring is considered to be serious and the hazards of exposure to inadequate hemoglobin monitoring, allergenic proteins and too rich diet is considered to be moderately serious. for these hazards, there is not enough information on the exposure of calves mainly due to lack of data why it is recommended that further studies should be made to provide evidence for an exposure assessment. regarding the hazard ) castration and dehorning without anesthetic and analgesic drugs, there is a variation in relation to national legislation as to why the risk of poor welfare in relation to castration and dehorning is widely different between countries. further, there is a variation in the use of analgesia during the time after the surgery is carried out which also affects the welfare of the calf. the ahaw panel wishes to thank the members of the working group chaired by panel member the needs and functioning of calves the needs of calves hutches: partially closed, outside area individual pens: open front structure straw yard with bedded lying area . . . group pens with automatic milk feeder . calf rearing and animal environmental pollution . . the quantitative share of calf production in the pollution problem . calf diseases and use of antibiotics human pathogenic-verotoxigenic escherichia coli (hp-vtec) on calf farms risk evaluation and principles of food safety assurance at calf farm level listing of potential hazards, hazard characterization and exposure assessment assessment of whether hazards pose risks (substantiation by scientific evidence) annex . hazard characterisation and exposure assessment the effects of antibiotic usage in food animals on the development of antimicrobial resistance of importance for humans in campylobacter and escherichia coli cytotoxic interactions between bovine parainfluenza type virus and bovine alveolar macrophages immune function is impaired in iron-deficient, homebound, older women effect of orally administered cimetidine and ranitidine on abomasal luminal ph in clinically normal milk-fed calves effect of feeding frequency and route of administration on abomasal luminal ph in dairy calves fed milk replacer effect of orally administered omeprazole on abomasal luminal ph in dairy calves fed milk replacer dairy calf pneumonia. the disease and its impact effect of type of housing on veal calf growth performance, behavior and meat quality incidence of respiratory disorders during housing in non-weaned charolais calves in cow-calf farms of pays de la loire (western france) age, geographic, and temporal distribution of fecal shedding of cryptosporidium parvum oocysts in cow-calf herds effect of individual versus group rearing on ethological and physiological responses of crossbred calves bovine respiratory syncytical virus bovine respiratory syncytical virus ventilation system to reduce pneumonia in a warm calf house: a case study the effect of increasing dietary fibre on feeding, rumination and oral sterotypies in captive giraffes (giraffa camelopardalis) neuroendocrine alterations in iron deficiency effects of form of the diet on anatomical, microbial, and fermentative development of the rumen of neonatal calves pattern of diarrhoea in newborn beef calves in south-west france respiratory infections -project, panorama and treatment strategies kälberkrankheiten spatial, temporal and management-specific factors influencing antibiotic resistance and carbohydrate fermentation patterns in bovine enteric escherichia coli and the clinical consequences of limiting antibiotic use in pre-weaned calves assessing antibiotic resistance in fecal escherichia coli in young calves using cluster analysis techniques animal and farm influences on the dynamics of antibiotic resistance in faecal escherichia coli in young dairy calves assessing the effect of a single dose florfenicol treatment in feedlot cattle on the antimicrobial resistance patterns in fecal escherichia coli a clinical trial evaluating prophylactic and therapeutic antibiotic use on health and performance of calves a field trial evaluating the influence of prophylactic and therapeutic antibiotic administration on antibiotic resistance in fecal escherichia coli in dairy calves cryptosporidium parvum, giardia intestinalis and other enteric pathogens in swedish dairy calves role of oxytocin in glucose homeostasis and weight gain iron-deficiency anaemia in calves endocrine and metabolic aspects in milk-fed calves nutritional physiology of neonatal calves grouping and social preferences in calves, heifers and cows emotional reactivity affected by chronic stress: an experimental approach in calves submitted to environmental instability is gentling by people rewarding for beef calves? early contact with peers or a stockperson influences later emotivity and social behaviour of dairy calves the maternal environment limits the effect of early human nursing and contact on lambs' socialisation to the stockperson stockmanship and farm animal welfare activity, oral behaviour and slaughter data as welfare indicators in veal calves: a comparison of three housing systems isolation of verocytotoxin-producing escherichia coli o :h from cattle at slaughter in italy bovine reservoir for verotoxin-producing escherichia coli o :h . the lancet(i anaemia and veal calf production abomasal ulcers in veal calves: pathogenesis and prevention husbandry methods leading to inadequate social and maternal behaviour in cattle indicators of poor welfare needs and welfare of housed calves. in: new trends in veal calf production scientific research on veal calf welfare welfare, stress and the evolution of feelings welfare assessment and problem areas during handling and transport the use of the concept animal welfare in european conventions, regulations and directives. food chain evolution of pain coping, stress and welfare welfare behaviour and welfare in relation to pathology the effects of group-rearing or partial isolation on later social behaviour of calves approaching questions of stress and welfare effects of disease on farm animal welfare bem-estar animal: conceito e questões relacionadas -revisão brain measures which tell us about animal welfare experimental escherichia coli o :h carriage in calves untersuchung von todesursachen bei kälbern in schleswig-holstein in den jahren eimeria-infektionen beim rind calf health in cow-calf herds in swotzerland prevalence and infection risks of zoonotic enteropathogenic bacteria in swiss cow-calf farms principles and guidelines for the conduct of microbiological risk assessment. document cac/gl the influence of air quality on production increases associated with all-in/all-out management essentials of veterinary bacteriology and cattle as a possible source of verocytotoxin-producing escherichia coli o infections in man effects of pair versus individual housing on the behavior and performance of dairy calves antimicrobial use in the treatment of calf diarrhoea effects of various levels of forage and form of diet on rumen development and growth in calves somministrazione di un mangime solido a vitelli a carne bianca stabulati in gabbia individuale the provision of solid feeds to veal calves: i. growth performance, forestomach development, and carcass and meat quality experimental infection of calves and adult cattle with escherichia coli o :h effects of handling aids on calf behavior iron deficiency and the immune response the pituitary-adrenal response to stress in the iron-deficient rat indicators relevant to animal welfare: a seminar in the c.e.c. programme of coordination of research on animal welfare the behaviour of calves under four levels of lighting the effect of different housing conditions on behavioural and adrenocortical reactions in veal calves from an animal's point of view: motivation, fitness, and animal welfare phylogenetic characterization of 'candidatus helicobacter bovis', a new gastric helicobacter in cattle rotavirus infections in calves in dairy herds sucking motivation and related problems in calves dairy calves discrimination of people based on previous handling iron supplementation improves iron status and reduces morbidity in children with or without upper respiratory tract infections: a randomized controlled study in colombo, sri lanka enhancement of clinical signs in experimentally rotavirus infected calves by combined viral infections group a rotavirus as a cause of neonatal calf enteritis in sweden incidence of diarrhea among calves after strict closure and eradication of bovine viral diarrhea virus infection in a dairy herd enteric infections in veal calves: a longitudinal study on four veal calf units behaviour and welfare of veal calves in relation to husbandry systems comparison of four methods of calf confinement. ii report of the scientific committee on animal health and animal welfare. health and consumer protection, directorate-general, directorate c -scientific opinions iron status, immune capacity and resistance to infections planungs-und berechnungsgrundlagen für geschlossene zwangsbelüftete ställe (thermal insulation for closed livestock buildings. thermal insulation and ventilation forced. part : principles for planning and design for closed ventilated livestock buildings). deutsches institut für normung e assosiations between passive immunity and morbidity and mortality in dairy heifers in florida, usa contribution to the study of gut hypersensitivity reactions to soybean proteins in preruminant calves and early-weaned piglets igg, iga, igg and igg specific responses in blood and gut secretion of calves fed soyabean products the interpretation of preference tests in animal behaviour animal right-animal welfare. a scientist's assessment measuring preferences and the strength of preference animal welfare as defined in terms of feelings. acta agriculturae scandinavica section a opinion of the scientific panel on biological hazards of the european food safety authority on standards for the microclimate inside animal transport road vehicles opinion of the scientific panel on biological hazards of the european food safety authority on "the risk assessment of a revised inspection of slaughter animals in areas with low prevalence of cysticercus welfare aspects of the main systems of stunning and killing the main commercial species of animals opinion of the scientific panel on biological hazards of the european food safety authority on "campylobacter in animals and foodstuffs the community summary report on trends and sources of zoonoses, zoonotic agents and antimicrobial resistance in the european union in opinion of the scientific panel on biological hazards of the european food safety authority on "an assessment of the public and animal health risks associated with the adoption of a visual inspection system in veal calves raised in a member state (or part of a member state) considered free of tuberculosis an experimental study of a concurrent primary infection with bovine respiratory syncytical virus (brsv) and bovine viral diarrhoea virus (bvdv) in calves a study of bovine respiratory syncytical virus infections in swedish cattle performance and health of group-housed dairy calves fed milk automatically versus manually virkning av forskellige insaettelsestrategier på tillvaekst og sundhed hos gruppeopstaldede kalve increased mortality among calves in danish cattle herds during bovine virus diarrhoea infection reducing pain after dehorning in dairy calves veterinary virology the effect of the size of individual crates on the behavioural and immune reactions of dairy calves ceftriaxone-resistant salmonella infection acquired by a child from cattle effect of castration method and the provision of local anesthesia on plasma cortisol, scrotal circumference, growth, and feed intake of bull calves farm animal behaviour and welfare farm animal behaviour and welfare preference and motivation testing pleasures, "pains" and animal welfare: towards a natural history of affect the effects of early separation on the dairy cow and calf the production system and disease incidence in a national random longitudinal study of swiss dairy herds comparison of four methods of calf confinement. i interleukin production in iron-deficient children rumional acidosis in milk fed calves. large animal veterinary rounds inzidenz und verlauf von neugeborenendurchfall bei kälbern in einem praxisgebiet in oberbayern economic analysis of feeding pasteurized nonsaleable milk versus conventional milk replacer to dairy calves campylobacter jejuni and campylobacter hyointestinalis in the intestinal tract and on the carcasses of calves and cattle a new method of measuring diet abrasion and its effect on the development of the forestomach urinary catecholamines in iron-deficient rats at rest and following surgical stress experimental salmonella infection of calves. . the effect of stress factors on the carrier state experimental salmonella infection of calves. . virulence and the spread of infection concentrations and emissions of ammonia in livestock buildings in northern europe zur Äthiologie und prophylaxe der perinatalen mortalität beim kalb collegium veterinarium xxiv salmonella bij gezonde runderen en kalveren in nederland immune functions of veal calves fed low amounts of iron delaying colostrum intake by one day has important effects on metabolic traits and on gastrointestinal and metabolic hormones in neonatal calves dynamics of virus infections involved in the bovine respiratory disease complex in swedish dairy herds. the vet effects of resistance to milk flow and the provision of hay on non-nutritive sucking by dairy calves sucking behaviour of dairy calves fed milk ad libitum by bucket or teat a longitudinal study of escherichia coli o in fourteen cattle herds resting behaviour, growth and diarrhoea incidence rate of young dairy calves housed individually or in groups in warm or cold building the effect of flooring type and social grouping on the rest and growth of dairy calves fecal shedding and rumen growth of escherichia coli o :h in fasted calves feeding the newborn dairy calf calf and heifer rearing : principles of rearing the modern heifer from calf to calving the national dairy heifer evaluation project: a profile of heifer management practices in the united states human-livestock interactions: the stockperson and the productivity and welfare of intensively farmeed the relationship between the presence of helicobacter pylori, clostridium perfringens type a, campylobacter spp, or fungi and fatal abomasal ulcers in unweaned beef calves effects of confinement on rebound of locomotor behaviour of calves and heifers, and the spatial preferences of calves the effects of feeding method, milk allowance and social factors on milk feeding behaviour and cross-sucking in group housed dairy calves computer-controlled milk feeding of dairy calves: the effects of number of calves per feeder and number of milk portions on use of feeder and social behaviour who needs 'behavioural needs'? motivational aspects of the needs of animals play behaviour in group-housed dairy calves, the effect of space allowance the effect of milk flow rate and milk allowance on feeding related behaviour in dairy calves fed by computer controlled milk feeders effect of single versus group housing and space allowance on responses of calves during open-field tests play behaviuor in domestic calves kept in pens: the effect of social contact and space allowance evaluation of welfare indicators for social environment in cattle effect of amount of milk, milk flow and access to a rubber teat on cross-sucking and non-nutritive sucking in dairy calves epidemiologische erhebungen zur kälberdiarrhoe in einem praxisgebiet in oberbayern effect of shipping and chromium supplementation on performance, immune response, and disease resistance of steers the development of intersucking in dairy calves around weaning factors associated with intersucking in swiss dairy heifers whole blood leukocytes vs separated mononuclear cell blastogenesis in calves. time-dependent changes after shipping recommendations for recording and calculating the incidence of selected clinical diseases in dairy cattle castration of calves: a study of methods used by farmers in the united kingdom onderzoek naar de uit ethologisch oogpunt minimaal gewenstle boxmaten voor vleeskalveren met een gewicht van tot kg spatial requirements of individually housed veal calves of to kg nutritional effects on response of children in developing countries to respiratory tract pathogens: implications for vaccine development problems on the etiological importance of pasteurella, mycoplasma and parainfluenza- -virus in enzootic pneumonia of calves the behaviour of beef suckler cattle enzootic pneumonia in calves: clinical and morphological features theoretical comparison of the consumer surplus and elasticities of demand as measures of motivational strength effects on calves less than one month old of feeding or not feeding tham during road transport of up to hours untersuchungen zur pathogenese der pylorusulzera beim mestkalb bovine cryptosporidiosis in denmark. ii. cryptosporidia associated with neonatal calf diarrhoea an evaluation of two management systems for rearing calves fed milk replacer prevalence of escherichia coli o :h in range beef calves at weaning cattle and reindeer as possible sources of escherichia coli o infection in humans aktuelle probleme der tierärztlichen betreuung von mutterkuhherden mother-young behaviour in cattle prevention of microbial contamination of red meat in the ante mortem phase; epidemiological aspects small intestinal motility disorders in preruminant calves chronically fed a diet based on antigenic soya : characterization and possible mediators influence of soya antigen levels in milk replacers on the disruption of intestinal motility patterns in calves sensitive to soya systemic and local gutspecific antibody responses in preruminant calves sensitive to soya feeding heated soyabean flour increases the density of b and t lymphocytes in the small intestine of calves hydrolysed soy protein isolate sustains high nutritional performance in veal calves antigenicity and digestive utilization of four soya products by the preruminant calves antibiotic resistance in gut bacteria from dairy calves: a dose response to the level of antibiotics fed in milk iron deficiency and immune responses the relationship between farmers' attitude and behaviour towards calves, and productivity of veal units reducing veal calves' reactivity to people by providing additional human contact the influence of farmers' behavior towards calves on animals' responses to transport and quality of veal meat the farmers' influence on calves' behaviour, health and production of a veal unit the impact of gentle contacts on ease of handling, welfare, and growth of calves and quality of veal meat reactions of calves to handling depend on housing condition and previous experience with humans effects of supplemental yeast culture on rumen development, growth characteristics, and blood parameters in neonatal dairy calves effects of corn processing on growth characteristics, rumen development, and rumen parameters in neonatal dairy calves anemia as a risk factor for infectious diseases in infants and toddlers: results from a prospective study colostrum in swedish dairy cows: quality and effects on passive immunity in the calves cross-sucking in group-housed dairy calves before and after weaning off milk intersucking in dairy cattle--review and questionnaire cryptosporidium andersoni n. sp. (apicomplexa: cryptosporidiidae) from cattle, bos taurus occurrence of iron deficiency in growing cattle growth performance, haematological traits, meat variables, and effects of treadmill and transport stress in veal calves supplied different anounts of iron prevention of microbial contamination of red meat in the ante mortem phase; epidemiological aspects management practices associates with high mortality among preweaned dairy heifers the gastric mucosal barrier and abomasal ulcers in veal calves housing, management and health in swedish dairy calves. doctoral thesis herd-level risk factors for infectious diseases in swedish dairy calves aged - days automated feeding of milk replacer and health control of group-hiused veal calves automated feeding of milk replacer and health control of group-housed veal calves effect of transportation and weaning on humoral immune responses of calves responses of dairy cows and calves to each other's vocalisations after early separation cross-sucking and other oral behaviours in calves, and their relation to cow suckling and food provision isolation of escherichia coli o :h from dairy cattle associated with two cases of haemolytic uraemic syndrome the provision of solid feeds to veal calves: ii. behavior, physiology, and abomasal damage enteric pathogens in intensively reared veal calves a fifteen month study of escherichia coli o :h in a dairy herd a comparison of catecholamine and cortisol responses of young lambs and calves to painful husbandry procedures acute pain in an emergency clinic regulation of sucking behaviour of calves laboratory findings associated with abomasal ulcers/tympany in range calves biological response to stress: key to assessment of animal well-being? association between resting behaviour and live weight gain in dairy heifers housed in pens with different space allowance and floor type long-term effect of housing method during the first three months of life on human-animal relationship in female dairy cattle risk factors associated with cryptosporidium parvum infection in dairy cattle in southeastern new york state the response of normal and iron anemic calves to nasal infection with an attenuated strain parainfluenza- virus assessment of acute and chronic pain after different methods of castration of calves intestinal digestion of dietary and endogenous proteins along the small intestine of calves fed soybean or potato influence of dietary protein level and source on the course of protein digestion along the small intestine of the veal calf effect of diet on mucin kinetics and composition: nutrition and health implications calf rearing a practical guide, natural resource and environment influence of dry feed supplements on different parameters of welfare in veal calves the effect of four fibrous feed supplementations on different welfare traits in veal calves behavioural investigation of group rearing calves in automatic milk replacer feeding system effect of transportation on blood serum composition, disease incidence, and production traits in young calves. influence of the journey duration critical anthropomorphism, animal suffering and the ecological context bacterial pneumonia dust and microbiol emissions from animal production influence of truck transportation of calves on their cellular immune function prevalence of enteric pathogens in the feces of healthy beef calves salmonella infection and contamination of veal calves: a slaughterhouse survey comparison of two castration methods in cattle: plasma cortisol levels, leukocyte count and behavioral changes calf diseases and mortality in swedish dairy herds abomasal ulcers in captive white-tailed deer (odocoileus virginianus) growth performance and health of dairy calves bedded with different types of materials factors involved in recent outbreaks of escherichia coli o :h in scotland and recommendations for its control protection of calves against cryptosporidiosis with immune bovine colostrum induced by a cryptosporidium parvum recombinant protein treadmill exercise of calves with different iron supply, husbandry, and work load beurteilung verschiedener tränketechniken und betreuungsmassnahmen hinsichtlich ihrer auswirkungen auf die oralen aktivitäten, den gesundheitszustand und die mastleitung über zwei bis acht wochen alter mastkälber in gruppenhaltung. dissertation. institut für tierhygiene und tierschutz der tierärztlichen hochschule hannover group housing for two to eight week old fattening calves veterinary and zootechnical aspects of veal production veterinary medicine. a textbook of the diseases of cattle, sheep, pigs, goats and horses serum immunoglobulin concentrations and health of dairy calves in two management systems from birth to weeks of age influence of feeding different amounts of first colostrum on metabolic, endocrine, and health status and on growth performance in neonatal calves the impact of social and human contacts on calves' behaviour and stress responses stereotypies om heifers are affected by feeding regime feeding level and oral stereotypies in dairy cows blood studies and performance among calves reared by different methods consequences of short term fluctuations of the environmental temperatures in calves--part : effects on the health status of animals within three weeks after exposure microbiology of calf diarrhea in southern britain studies on the presence of verocytotoxic escherichia coli o in bovine faeces submitted for diagnostic purposes in england and wales and on beef carcases in abattoirs in the united kingdom effect of different methods of castration on behaviour and plasma cortisol in calves of three ages iron, thermoregulation, metabolic rate the validity of behavioural measures of aversion: a review fear of people by cows and effects on milk yield, behavior, and heart rate at milking recognition of people by dairy calves using colour of clothing endotoxins in the environment: a criteria document reduced cortisol secretions in patients with iron deficiency viral diarrhea in man and animals mouth-based anomalous syndromes the influence of management routines on endocrine systems involved in the control of lactation in dairy cattle immune response in anemic calves the welfare of cattle kept for beef production. directorate general health and consumer protection, directorate c -scientific health opinions, unit c -management of scientific committees staphylococci as an indicator for bacterial emissions from a broiler house the use of trainer cows to reduce stress in newly arrived feedlot calves scvph (scientific committee on veterinary measures relating to public health) scvph (scientific committee on veterinary measures relating to public health) scvph (scientific committee on veterinary measures relating to public health) bioaerosole in und aus der landwirtschaftlichen nutztierhaltung (vorläufiger titel). kuratorium für technik und bauwesen in der landwirtschaft (ktbl, hrsg.) concentrations and emissions of airborne endotoxins and microorganisms in livestock buildings in northern europe a survey of ventilation rates in livestock buildings in northern number of viable bacteria and presumptive antibiotic residues in milk fed to calves on commercial dairies effects of transportations, a high lactose diet and acth injections on the white blood cell count, serum cortisol and immunoglobulin g in young calves descriptive epidemiology of morbidity and mortality in minnesota dairy heifer calves the development of the bacterial flora of the faeces of animals and man: the changes that occur during aging clostridial abomasitis in calves: case report and review of the literature destruction of mycobacterium paratuberculosis, salmonella spp., and mycoplasma spp effects of local anaesthesia or local anaesthesia plus a non-steroidal anti-inflammatory drug on the acute cortisol response of calves to five different methods of castration dehorning and disbudding distress and its alleviation in calves seasonal variation of thermophilic campylobacters in lambs at slaughter losses in young calves after transportation reaction of claves to two flooring materials offered simultaneously in one pen effects of three different methods of dehorning on cortisol levels and behaviour of calves evaluation of the effect of local anaesthesia and local anaesthesia associated with analgesia on the levels of cortisol after hot-iron, chemical or scoop dehorning effects of in vivo release of insulin and glucagon studied by microdialysis in rat pancreas and audiographic evidence for ( h)-oxytocin binding sites within the islets of langerhans some theoretical and observed relationships of fixed and portable spacing behavior of animals selfish animats and robot ethology: using artificial animals to investigate social and spatial behavior effect of local anaesthetic combined with wound cauterisation on the cortisol response to dehorning in calves cortisol responses to dehorning of calves given a -h local anaesthetic regimen plus phenylbutazone, ketoprofen, or adrenocorticotropic hormone prior to dehorning scientific veterinary committee, animal welfare section. directorate generale for agriculture, vi/bii. . adopted the effect of group size on health and growth rate of swedish dairy calves housed in pens with automatic milk-feeders bovine coccidiosis with special reference to eimeria alabamensis infections in grazing calves morbidity in swedish dairy calves from birth to days of age and individual calf-level risk factors for infectious diseases health status of dairy calves kept in individual pens or in group pens with or without automatic milk feeder acute cortisol responses of calves to scoop dehorning using local anaesthesia and/or cautery of the wound verotoxigenic escherichia coli o in scottish calves verocytotoxin-producing escherichia coli: a veterinary view concentrations and emissions of airborne dust in livestock buildings in northern europe ethological, physiological and histological aspects of pain and stress in cattle when being dehorned individual humans as discriminative stimuli for cattle (bos taurus) pathological and microbiological studies on pneumonic lungs from danish calves effects of individual housing design and size on special-fed holstein veal calf growth performance, hematology, and carcass characteristics calf rearing verordnung zum schutz landwirtschaftlicher nutztiere und anderer zur erzeugung tierischer produkte gehaltener tiere bei ihrer haltung (tierschutz-nutztierhaltungsverordnung -tierschnutztv)*) vom geändert durch die erste verordnung zur Änderung der tierschutz -nutztierhaltungsverordnung vom effects of age of holstein-friesian calves on plasma cortisol, acute-phase proteins, immunological function, scrotal measurements and growth in response to burdizzo castration effect of ketoprofen, lidocaine local anesthesia, and combined xylazine and lidocaine caudal epidural anesthesia during castration of beef cattle on stress responses, immunity, growth, and behavior etude de la prévalence de l'infection des veaux par giardia duodenalis en pays de la loire effects of housing on responses of veal calves to handling and transport. in new trends in veal calf production the importance of straw for pig and cattle welfare: a review behavioural and physiological studies on rearing and veal calves molecular epidemiology of cryptosporidium parvum and giardia duodenalis to study the potential for zoonotic transmission dynamics of bovine respiratory syncytical virus: a longitudinal epidemiological study in dairy herds the prevalence of verotoxins, escherichia coli o :h and salmonella in the feces and rumen of cattle at processing haemoglobin and haematocrit levels in veal calves is anaemia of veal calves normochromic or hypochromic a comparison of different enrichment media for the isolation of salmonella dublin from livers, kidneys and muscles of salmonella-positive veal calves zusatzfuttering von stroh an mastkälber the effect of housing system and dietary iron supply on stress physiological measures and resistance against an experimental infection with bovine herpesvirus (bhv ) in veal calves enkele epidemiologische aspecten van salmonellose bij mestkalveren in nederland calves' responses to repeated social regrouping and relocation does nutritive and non-nutritive sucking reduce other oral behaviors and stimulate rest in calves nonnutritive oral activities and stress responses of veal calves in relation to feeding and housing conditions providing social contacts and objects for nibbling moderates reactivity and oral behavior in veal calves the effects of rearing in individual crates on subsequent social behaviour of veal calves somministrazione di un mangime solido a vitelli a carne bianca stabulati in gabbia individuale o in box di gruppo. . parametri comportamentali, fisiologici e patologici behaviour and performance of veal calves under different stabling conditions assessing animal welfare: the significance of causal studies of behaviour at the motivational level epidemiologic and pathological characteristics of respiratory tract disease in dairy heifers during the first three months of life morbidity from nonrespiratory diseases and mortality in dairy heifers during their first months of life iron deficiency anemia and increased urinary norepinephrine excretion decreased susceptibility to ciprofloxacin in outbreak-associated multiresistant salmonella typhimurium dt otitis media in preweaned holstein dairy calves in michigan due to mycoplasma bovis effects of group, individual, and isolated rearing of calves on weight gain and behavior colostrum -the beginning of a successful calf raising program. dairy feed facts air and surface hygiene the response of beef cattle to noise during handling reduction of cross-sucking in calves by the use of a modified automatic teat feeder calf husbandry, health and welfare rearing of veal calves the effect of different rearing systems on the development of calf behaviour a survey of abomasal ulceration in veal calves haematology of veal calves reared in different husbandry systems and the assessment of iron deficiency health status of preweaned dairy heifers in the united states the effect of feeding pellets of different types of roughage on the incidence of lesions in the abomasum of veal calves st joint fao/oie/who expert workshop on non-human antimicrobial usage and antimicrobial resistance: scientific assessment on the significance of ethological criteria for the assessment of animal welfare effects of individual housing design and size on behaviour and stress indicators of special-fed holstein veal calves feeding antibiotic-contaminated waste milk to calves--effects on physical performance and antibiotic sensitivity of gut flora reviews of the progress of dairy science: bovine salmonellosis factors influencing occurrence of colibacillosis in calves natural and experimental infection of normal cattle with escherichia coli o rearing veal calves with respect to animal welfare: effects of group housing and solid influence of dietary concentrate to forage ratio on the development of rumen mucosa in calves the members of the working group which authored this report were: animals. cab international, oxon/new-york pp.hepola, h., . milk feeding systems for dairy calves in groups: effects on feed intake, growth and health. applied animal behaviour science : - .herrmann, j., knierim, u., hinton, m., linton, a.h., . antibacterial drug resistance among escherichia coli isolated from calves fed on a milk substitute diet, vet. rec., , - .hinton, m., ali, e. a., allen, v., linton, a. h., . the excretion of salmonella typhimurium in the faeces of calves fed on a milk substitute diet. j. hyg. camb. , - .hinton, m., hedges, a. j., linton, a. h., a . the ecology of escherichia coli in calves fed on a milk substitute diet. j. appl. bacteriol. , - .hinton, m., linton, a. h., hedges, a. j., b . the ecology of escherichia coli in calves reared as dairy-cow replacers. j. appl. bacteriol. , - .hinton, m., rixson, p. d., allen, v., linton, a. h., a . the persistence of drugresistant escherichia coli strains in the majority faecal flora of calves, j. hyg. camb., , - .hinton, m., suleyman, i. o., allen, v., linton, a. h., b . further observations on the excretion of salmonella in the faeces of calves fed milk substitute, j. hyg. camb., , - .holliman, a., . overview of coccidiosis -recent observations. cattle practice ; - .holm, l., jensen, m. b., jeppesen, l. l., key: cord- -h hxroos authors: wielinga, peter r.; schlundt, jørgen title: one health and food safety date: - - journal: confronting emerging zoonoses doi: . / - - - - _ sha: doc_id: cord_uid: h hxroos many, if not most, of all important zoonoses relate in some way to animals in the food production chain. therefore food becomes an important vehicle for many zoonotic pathogens. one of the major issues in food safety over the latest decades has been the lack of cross-sectoral collaboration across the food production chain. major food safety events have been significantly affected by the lack of collaboration between the animal health, the food control, and the human health sector. one health formulates clearly both the need for, and the benefit of cross-sectoral collaboration. here we will focus on the human health risk related to zoonotic microorganisms present both in food animals and food derived from these animals, and typically transmitted to humans through food. some diseases have global epidemic—or pandemic—potential, resulting in dramatic action from international organizations and national agricultural- and health authorities in most countries, for instance as was the case with avian influenza. other diseases relate to the industrialized food production chain and have been—in some settings—dealt with efficiently through farm-to-fork preventive action in the animal sector, e.g. salmonella. finally, an important group of zoonotic diseases are ‘neglected diseases’ in poor settings, while they have been basically eradicated in affluent economies through vaccination and culling policies in the animal sector, e.g. brucella. here we will discuss these three different foodborne disease categories, paying extra attention to the important problem of antimicrobial resistance (amr). in addition, we present some of the one health inspired solutions that may help reduce the threat of several of the foodborne diseases discussed. people in ancient times already understood they could get sick from consumption of infected meat. by keeping their animals healthy, and by using dedicated methods of food preparation and conservation, ancient farmers learned how to improve health and prevent disease. probably the oldest written document about it is 'on airs, waters, and places', written by hippocrates, which describes how human health is influenced by its interaction with the environment. since then, our health situation has improved by applying these simple rules of thumb, and even more through improved technologies such as good animal management, hygiene and biosecurity, vaccination programs and prudent animal drug treatment. nowadays, some of the most feared zoonotic diseases such as anthrax or brucellosis are absent in many countries. however, there are still many important diseases that threaten human health and which have animals as their reservoir. these animal reservoirs range from wildlife to domestic animals, both in companionship and agricultural settings. by the obvious close contact and the sheer number of animals needed for consumption, the animals produced for food form the largest reservoir and production grounds for emerging zoonotic pathogens. actions of authorities to protect society from zoonotic diseases differ significantly according to socio-economic status and the zoonotic pathogen in question. basically, zoonotic diseases related to food animals can be separated into three groups. in the first group are diseases with a potential for global spread and with a dramatic public relations potential, often these diseases have a significant human reservoir showing human-human transmission, e.g. sars, avian influenza and certain types of amr bacteria. the second group is constituted by persistent zoonotic diseases related to the industrialized food production chain, such as salmonella and campylobacter, which are broadly distributed in the farm-to-fork chain. these human pathogens are often non-pathogenic in animals and seem to be distributed in all countries, both rich and poor. in the third group are the 'neglected zoonotic diseases'. they are zoonotic diseases which have been eradicated (or drastically reduced) in affluent economies through vaccination and culling policies, and through introduction of hygienic and animal biosafety management practices. however, in many poor settings these diseases are 'neglected diseases' and receive very little attention from national authorities or even international organizations. this group includes brucella, bovine tb (tuberculosis), i.e. mycobacterium bovis, and many parasitic diseases, e.g. leishmaniasis and cysticercosis. in addition to these traditional infectious diseases there is a new threat of antimicrobial resistant (amr) bacteria. caused by the use of antimicrobials both in human and veterinary medicine this problem has emerged and is now to be recognized as one of the most important threats to human health. although in the detail the control of these groups of diseases differ, they are all most efficiently prevented by a one health approach which considers the full farm-to-fork chain. such preventive and holistic approaches may reduce both the disease burden to human health and the economic burden to developing economies, and therefore represent a significant potential for improvement related to food safety as seen in a one health perspective. through food and feed, direct contact, and via the environment, the human-and the animal microbial flora are in contact with each other. figure . outlines the most important routes of transmission for infectious diseases between humans and animals. via these routes infectious diseases from (food-)animals may enter the human reservoir and vice versa. the foodborne transmission route is probably the most important gateway for this contact, and the vast majority of human infections with enteric zoonotic bacterial pathogens, such as salmonella enterica, campylobacter coli/jejuni, and yersinia enterocolitica, occur through this route. for other diseases there is evidence that transmission also occurs via direct contact between (food) animal and humans, e.g. live-stock associated methicillin resistant staphlylococcus aureus (mrsa) (graveland et al. ) . next, there is transmission via the environment (e.g. surface water or water used to irrigate plants) mainly as a result of spreading of manure into the environment (spencer and guan ; hutchison et al. ) . and though much less frequently reported, there may be transmission of pathogens from humans to animals, which most probably was the meat/milk/eggs etc. direct contact and products (eg.skin) veterinary medicine food & consumer authorities food environment indirect contact via water/air (faeces / urine / corpses) fig. . schematic presentation of important microbial transmission routes via which the human and (food) animals are in contact with each other. in blue control mechanisms are shown, and in red some of the transmission routes that are more difficult to control. via the environment transmission may take place of microorganisms present in excretion products, and in diseased animals and people. in addition, wildlife constitutes a risk, as it holds a broad spectrum of diseases, including many highly pathogenic diseases case for the staphylococcus aureus cc (price et al. ) . in many developing countries, wildlife forms an additional important reservoir for foodborne pathogens, not only through consumption of wildlife. because of often lower bio-safety levels in these countries, direct contact between humans and food animals is generally more frequent, and diseases from the wildlife community may cross over more easily to domestic animals. for instance, the general understanding now is that the sars epidemic in originated in direct human contact with and/or consumption of wildlife, or indirectly through contact between wildlife and domestic animals (guan et al. ; shi and hu ) . wildlife holds a broad spectrum of diseases including some of the most feared, such as ebola, rabies and anthrax and, and in contrast to other food sources, much of the consumption of wildlife goes undetected by food controlling agencies. for these reasons, and because of the global trade in wildlife derived food and other items (pavlin et al. ), consumption of wildlife animals, and the spillover of infectious diseases from wildlife to food/production animals, should not be overlooked. the spread of foodborne zoonoses through the food production chain is often referred to as the 'farm-to-fork ' (or 'farm-to-table' or 'boat-to-throat') chain. it should be noted that risk mitigation solutions under this framework typically have focused on a consideration of the full food production continuum, involving all relevant stakeholders, i.e. a typical one health framework invented before the one health paradigm was defined. figure . tries to capture a generalized picture of a farm-to-fork chain, starting with animal feed and ending in human consumption of animal food products. although a number of very important zoonoses are related to wildlife-and in some cases directly transmitted from wildlife animals-the vast majority of zoonotic disease cases in the world relate to animals that are bred for food purposes. such zoonotic pathogens include bacteria such as brucella, salmonella, campylobacter, verotoxigenic escherichia coli and leptospira; parasites, such as taenia, echinococcus and trichinella; and viruses, such as influenza a h n (avian influenza) and rift valley fever virus. next to these infectious diseases, derived agents such as (microbial) toxins and prions (prusiner ) form another important zoonotic subgroup. diseases originating on the farm can in many cases most efficiently be dealt with on the farm itself, thereby eliminating more complex measures or crosscontamination down the farm-to-fork chain. for example, brucellosis in animals (mainly cattle, sheep and goats) has been eliminated in many countries, thereby virtually eliminating the human disease burden (godfroid and käsbohrer ) . also, some of the main parasites can be effectively controlled at the farm level, and this could work for both taenia solium in pigs (defined by who/fao/oie as a 'potentially eradicable parasite'), as well as, trichinella spiralis which is found in many wild animals and importantly in pigs for human consumption. both parasites have essentially been eliminated from farmed pigs in most northern european countries (who/fao/oie ; gottstein et al. ). however, both diseases still form a serious 'neglected diseases' threat in settings where there is potential for contact between wild and domestic animals. it was primarily the outbreaks of sars and zoonotic influenza, amr (dealt with separately) and bse (bovine spongiform encephalopathy) which alerted the world to the need for a one health approach. outbreaks of viral diseases in humans, originating in or spreading through farm animals (avian flu-h n and swine flu-h n ) have caused major global alerts in the last decade. these influenza outbreaks spread very quickly, either in the animal population (h n ) or directly in the human population (h n ), and formed a global threat for human health. h n was therefore characterized by the world health organization (who) as a pandemic. although in total the human disease burden related to the endemic bacterial zoonoses is probably many fold higher than these influenza outbreaks, it is basically these relatively few but fast spreading outbreaks that have put one health on the global agenda. in addition, the failure to predict, monitor and control the spread of these diseases in animals presented regulators and politicians with a wake-up call, and made them demand (better) cross-sectoral collaboration between the animal and human health sectors. prions, non-living infectious agents, have been a significant burden of disease in animal and man. the most well-known zoonotic prion disease is probably the one causing bovine spongiform encephalopathy (bse) in cows, and new variant creutzfeldt-jakob disease in humans, as represented by the massive outbreak of 'mad cow disease' in the uk in the s and s. this agent, a mutant protein, which mainly sits in the brain, got into the (beef) food chain by the feeding of ruminant derived meat and bone meal to ruminants. prions (prusiner ) , scrapie (the disease in goats), spongiform encephalopathy of rocky mountain elk, transmissible mink encephalopathy, kuru and creutzfeldt-jakob disease were known before the large outbreak of bovine spongiform encephalopathy in the uk. it, however, took some time and great efforts, and an early one health approach, to establish the links between the different animal diseases and the human disease (hill et al. ; prusiner ; ghani et al. ). this insight created a background to efficiently stop the spread of this prion disease, by banning the use of animals in animal feed, and seeing a subsequent decrease of the disease in humans (hoinville ) . in the western world prion diseases have attracted much attention, and their control has resulted in a large economic burden to society. in developing countries, often with less strict rules about the re-use of dead animals, and more direct contact with wildlife, prion diseases may still be endemic though unrecognized. in contrast to the dramatic outbreaks discussed above, many food-related zoonoses are endemic in farm animals and some of the most important of these do actually cause disease in the animals. it should be realized that most countries-including most developing countries-produce large amounts of food animals, and most of the production takes place in some sort of industrialized setting. such settings are invariably linked to a number of important zoonotic pathogens. table . shows three lists of the most important food pathogens, as reported in studies published by the cdc in the usa and by rivm (havelaar et al. ) in the netherlands, as well as a list of pathogens recognized by ecdc as focus organisms for the eu. although widespread, these pathogens are often not recognized as important human pathogens because of their often mild disease syndromes in healthy persons (e.g. limited to diarrhea and vomiting) and because of the complexity of source attribution. however, they do form a serious threat to the vulnerable segments of our societies (i.e. the young, the elderly, the immune-compromised and recovering patients), and some patients may develop long-lasting chronic disorders (e.g. arthritis and neurological disorders) (mckenna ). these facts, together with the sheer number of infections they cause, results in a substantial total burden of disease for these pathogens as expressed in disability-adjusted life years (dalys) (see murray ) . for instance, the study of havelaar et al. ( ) showed that the total burden of the diseases he studied was , dalys, for a total of . million cases and deaths caused by these diseases (in million people). source attribution estimates showed that one-third could be attributed to foodborne transmission. similarly large numbers were reported for the usa (cdc ), where surveillance studies of known pathogens gave an estimated total of . million illnesses, , hospitalization and , deaths attributed to foodborne diseases (in million people). importantly, the latter report also showed that the pathogens studied make up only % of the foodborne diseases, and the majority of % is caused by unknown agents. this situation is probably not unique for the usa, and thus indicates that there is still much health to be gained from improved food safety. table . shows that an almost identical list of disability-adjusted life years (dalys), are a combined estimate of the burden of disease due to both death and morbidity. one daly can be thought of as year of healthy life lost and is often expressed in years of life lost on the population level, and can be thought of as a measure of the gap between current health status and an ideal situation where each individual in the population lives to old age, free from disease and disability (murray ) for the list of the cdc and the netherlands the ranking in terms of incidence, hospitalizations, deaths or daly is given. the list from ecdc was generated by expert consultation and for use as an eu focus list in future disease burden studies important foodborne pathogens are found in europe, and that toxoplasma gondii, listeria monocytogenes, campylobacter, rotaviruses, noroviruses and salmonella, should probably form the key targets for interventions. except for norovirus, these pathogens have been shown to be zoonotic, and find their way to humans via food and the environment ( fig. . ). a one health approach ensuring efficient crosssectoral collaboration and data-sharing, could lay the foundation for a realistic description of the situation, and could help implement sensible cross-sector solutions. building on the idea of one health to control these diseases, there are several countries (especially in northern europe and north america) that have instituted cross-sectoral data collection and collaboration. this is typically done through the construction of zoonosis centers or their equivalents. these centers aim to stimulate and facilitate the collaboration between human, veterinary and food institutes. some examples of such specialized centers are the us national center for emerging and zoonotic infectious diseases (http://www.cdc.gov/ncezid), the british national centre for zoonosis research (http://www.zoonosis.ac.uk/ zoonosis) and danish zoonosis centre which is part of the danish national food institute (http://www.food.dtu.dk/english/research/research_groups/zoonosis_ centre.aspx). two clear examples of what such centers can accomplish are: the reduction of salmonella in food animals in denmark, and the danish integrated approach to combat amr (described below in a separate section). in the danish salmonella reduction program, data sharing across animal, food and human health sectors has enabled science-based solutions, and has most noticeably resulted in significant reductions in human salmonellosis through lowering salmonella prevalence in animals (wegener et al. ) . in relation to laying hens the program started with a simple and inexpensive serological surveillance of egg producers. flocks found positive were either culled and repopulated, or used to produce heat-processed eggs, danish eggs are now considered free of salmonella. next to this arm, a program of surveillance and eradication of infected broiler flocks was setup. the effect of the whole program was measured in term of cases of human salmonellosis, which were found to be significantly reduced as the project progressed in time. the construction and solutions of this program clearly followed one health principles. food, veterinary and human health sciences worked together, using similar detection and (geno)typing techniques, which enabled comparison and sharing of data. this top-down selection of salmonella-free poultry could work in other countries with industrialized food animal production as well. in other countries-including most likely most developing countries, salmonella-positive animals have been imported from big producers in industrialized countries. one such documented example was the import into zimbabwe of salmonella enteritidis via live animals. salmonella entered the country through import of infected poultry in the commercial national production system around , and thereafter spread quickly to the communal sector (small-scale farming), as well as to the human population (matope et al. ). the most likely reason for the spread within zimbabwe was that old animals from the commercial sector were sold to small-scale communal production systems. as the trade of live animals is done on a global level and does not take into account whether the traded animals carry any of the diseases from table . , reducing the prevalence in the commercial sector in producing countries, may also lower the global spread and human disease burden in the rest of the world. the spectrum of neglected diseases is broad and includes diseases caused by bacteria, viruses and parasites. many are found world-wide but their prevalence in the human and animal populations varies according to the local agricultural, demographic and geographic conditions and tradition. for many of the neglected diseases solutions to dramatically decrease the disease burden are well-known, but action is lagging, this is the case for example for many of the parasitic zoonoses. this is the reason why the who refers to these diseases as 'neglected diseases' (who ; molyneux et al. ) . neglected diseases may be categorized into two (strongly overlapping) categories. in the first category are the neglected tropical diseases which include chagas disease, trypanosomiasis, leprosy, rabies, schistosomiasis and others, many of which are zoonotic and parasitic diseases. the second category are the neglected zoonotic diseases, covering many of the diseases above and also some bacterial diseases such as anthrax, bovine tuberculosis (tb), brucellosis, and also cysticercosis and echinococcosis. many of the neglected diseases are carried by wildlife and in poor and rural settings by livestock (e.g. brucellosis, anthrax, leptospirosis, q-fever and bovine tb). in addition, many are food-and waterborne (e.g. brucellosis, cysticercosis/ taeniasis and echinococcosis). in particular, the prevalence of bovine tb appears to be increasing in many poor settings and has been linked to hiv infections as an important factor for progression of a tb infection to an active tb disease (lobue et al. ) . brucellosis and bovine tb in cattle cause lowered productivity in the animal population, but seldom death, and both have largely been eradicated from the bovine population in the developed world by test-and-slaughter programs, which in effect has eliminated the human health problem (godfroid and käsbohrer ) . some of the parasitic diseases (e.g. schistosomiasis, cysticercosis, trematodiasis and echinococcosis) have high mortality rates and long-term sequelae including cancer and neurological disorders. cysticercosis is emerging as a serious public health and agricultural problem in poor settings (garcía et al. ) . humans acquire taenia solium tapeworms when eating raw or undercooked pork contaminated with cysticerci. the route of transmission is, pigs infected through taenia eggs shed in human faeces, and the disease is thus strongly associated with pigs raised under poor hygienic conditions. this means that the cycle of infection can be relatively easily broken by introducing efficient animal management, as has been done in most developed countries. given that % of the rural population in poor countries is dependent on livestock as working animals to survive (fao ) , the effect of these animals carrying a zoonotic disease can be dramatic, both relative to human health directly, but also as it affects the potential to earn an income. this also affects the potential mitigation action; for instance the large-scale culling of animals, which can be a viable solution in rich countries, might be problematic in the poorest countries. such solutions would not only mean loss of food, but also a serious socio-economic disruption, in some cases leading to national instability. some of the recent serious outbreaks of antibiotic resistant (amr) foodborne disease, such as ehec in germany (mellmann et al. ) , have shown us a new problem. there seems to be a global trend with the prevalence of amr rising (who ; danmap ; ecdc ; aarestrup ) . especially dangerous is the emergence of resistance against antimicrobials that are considered critically important in human medicine, and in multidrug resistant (mdr) infections (potron et al. ; kumarasamy et al. ). in the early s antibiotics were first introduced to control bacterial infections in humans. the success in humans led to their introduction in veterinary medicine in the s, where they were used in both production and companion animals. nowadays, antibiotics are also used with intensive fish farming and to control some infectious diseases in plants. their use is thus widespread. antibiotics in animals are mainly used in three ways: ( ) for therapy of individual cases, ( ) for disease prevention (prophylaxis) treating groups of animals, and ( ) as antibiotic growth promoters (agp) treating groups of healthy animals with sub-therapeutic concentrations to promote animal growth. when first introduced, the use of antibiotics led to improved animal health, and subsequently higher levels of both food safety and food security. all use, but in particular the use as agp, resulted in a dramatic rise in the use of antibiotics, and for instance, between and the use in the united states alone went from to , t (who ). however, the use of antibiotics in animals has over the years also resulted in a selective pressure for amr microorganisms, contributing significantly to the human health problem of amr bacteria. notably a number of bacterial strains that were previously susceptible to antibiotics are now in very high frequencies becoming resistant to various antibiotics, some of which are very important as last resort treatment potential for humans (bonten et al. ) . in particular the use as agp is questionable, as the concentrations used are sub-therapeutic which result in the selection for resistance but do not efficiently kill microorganisms. nowadays there are serious efforts by national authorities and some international organizations to reduce the antibiotic overuse in animals (food agricultural organization of the united nations (fao)/world organization for animal health (oie)/who ; who ; u.s. food and drug administration (fda) ( )), especially-but not only-through abolishing their use as agp. however, there seem to be major problems in ensuring cross-sectoral understanding, since the veterinary and medical professions are still in debate about how the amr problem has emerged (phillips et al. ; karp and engberg ; smith et al. ; price et al. ) . to achieve a science-based understanding of the problem, data on amr from both the animal and the human side should be compared, and both risk assessments and source attributions performed in an integrated way. in other words, a one health approach in which human and animal health sectors, including food and environmental sectors, work together, may help to deliver answers needed and suggest ways to reduce problems in both human and animal reservoirs (figs. . and . ). in addition to the factors described above, food production and food trade are now more and more global, and thus some of the food related problems are also global food problems. on the positive side, globalization has helped with some of the important global food issues: it raised food security, made our food more varied and tastier, and even including transport costs in the equation, still has global financial advantages. however, together with the food also the foodborne diseases now travel the globe. and if we do not stay on top of the problem, disease outbreaks might affect large parts of the global food sector negatively, in the end leading to negative health-but will also have financial and socio-economical consequences. a more holistic and pro-active approach to food safety may help prevent future food disasters and build healthy economies. one health approaches to combat zoonotic foodborne diseases need to consider at least three levels, the international level, national level and the farm level where the actual production takes place. to facilitate the work at all these levels, many countries have established specialized zoonosis centers. these centers focus their work on zoonotic diseases and promote collaboration between different sectors, and between different countries. they examine the prevalence of zoonotic diseases in humans and (food-)animals, their routes of transmission, the risk associated with their presence in our food chain, and the relation between human disease and zoonotic transmission. in addition, as our food production system has become increasingly dependent on global trade, the approaches taken by these zoonoses centers (should) also include a global angle. national zoonosis centers may also help tackle the global problems associated with zoonotic diseases. however, at the moment most of this work is done by international and global organization, such as the who, oie, and fao. these three international organizations have recognized that combating zoonoses is best achieved via a one health approach, as stated in their seminal paper 'a tripartite concept note' (fao/oie/who ), in which they express the need to collaborate for a common vision. given the impact zoonotic diseases have in socio-economical terms and on the vulnerable sectors in our societies, a one health vision is also endorsed by the world bank and the united nations children's fund (unicef) (world bank/who/unicef/oie/fao/unsic ). in their common vision, they say that a one health approach may lead to novel and improved solutions, including solutions that have not been considered before because of the high costs involved. for instance, while in some cases vaccination is the ultimate tool to prevent disease, it is not always considered because the costs of mass vaccination are higher than the public health benefit savings, or because of global trade regulations. under a one health approach sharing of costs, as well as other mitigation strategies could likely enable novel ways of reaching sensible solutions (narrod et al. ) . for global infectious disease safety national authorities report to who important outbreaks of human disease which have the potential of cross-border spread, under the auspices of the international health regulations (ihr) (who ) . these regulations also cover foodborne diseases associated with globally traded food. however, given the major impact that such announcements may have on global food trade, such as was the case with bse in the uk or the more recent trade barriers put up after the ehec outbreak in germany, national authorities may have become more careful and restricted in what they report. a global one health approach which both considers human health aspects and socio-economic consequences would therefore be a welcome improvement to the ihr of . next to who, other organizations are active in reporting global infectious disease outbreaks, most notably promed-mail (http://www.promedmail. org), which is an internet based program for monitoring emerging diseases worldwide, set up by the international society for infectious diseases. the program is dedicated to rapid global dissemination of information on outbreaks of infectious diseases and acute exposures to toxins that affect human health, including those in animals and in plants grown for food or animal feed, and thereby supports the one health principles. many of the (international) organizations and governing bodies named above have generated guidelines to control -and disseminate information about-food related zoonoses, such as for instance who's global foodborne infections network (gfn) (www.who.int/gfn), the european food safety authority (efsa) (www.efsa.europa.eu/en/topics/topic/zoonoticdiseases), foodnet from the us centers for disease control and prevention (www.cdc.gov/foodnet) and others. the goal of these networks is essentially the same: to help capacity-building and promote integrated, laboratory based surveillance and intersectional collaboration among human health, veterinary and food-related disciplines to reduce the risk of foodborne infections. the emergence of amr in food animals is a serious threat for modern human medicine. the risks exist that both (i) the overuse by mass prophylaxis and agp in animals, and (ii) the misuse of human critically important antibiotics in animals, will lead to the emergence of new amr organisms which may spread to the human reservoir, and via global food trade spread around the world. in the most critical scenario this will make our arsenal of antibiotics unfit to treat previously treatable infectious disease, and it might take us back to a situation as before world war ii, when antibiotics were not yet used in human medicine. one health principles may help mitigate this risk and deal with the amr problem in an efficient way. collaboration between the fao/who codex alimentarius commission and the oie have generated important guidance on how an integrated approach and the prudent use of antimicrobials may reduce the emergence of amr in (food-)animals and subsequently in humans. previous to this, in the who published the 'global principles for the containment of antimicrobial resistance in animals intended for food' (who ) which all countries should follow to reduce the risk of amr. the three major principles are: • use of antimicrobials for prevention of disease can only be justified where it can be shown that a particular disease is present on the premises or is likely to occur. the routine prophylactic use of antimicrobials should never be a substitute for good animal health management. • prophylactic use of antimicrobials in control programs should be regularly assessed for effectiveness and whether use can be reduced or stopped. efforts to prevent disease should continuously be in place aiming at reducing the need for the prophylactic use of antimicrobials. • use of antimicrobial growth promoters that belong to classes of antimicrobial agents used (or submitted for approval) in humans and animals should be terminated or rapidly phased-out in the absence of risk-based evaluations. these global principles have been supplemented with, ( ) guidance on the prudent use of antibiotics from the codex alimentarius commission together with oie, and ( ) six priority recommendations from who to reduce the overuse of antibiotics in food animals for the protection of human health (who ), being: ( ) require obligatory prescriptions for all antibiotics used for disease control in (food) animals; ( ) in the absence of a public health safety evaluation, terminate or rapidly phase out the use of antibiotics for growth promotion if they are also used for treatment of humans; ( ) create national systems to monitor antibiotic use in food-animals; ( ) introduce pre-licensing safety evaluation of antibiotics [intended for use in food animals] with consideration of potential resistance to human drugs; ( ) monitor resistance to identify emerging health problems and take timely corrective actions to protect human health; ( ) develop guidelines for veterinarians to reduce overuse and misuse of antibiotics in food animals. a recent publication (who ) covers the broader scope of amr in relation to both animals and humans. thus, a 'one health' approach has explicitly been proposed by these international organizations to mitigate the risk of amr. since the occurrence of amr in the food production sector, different programs to contain zoonoses and amr zoonoses have been developed following these principles and guidelines. the danish program to contain amr zoonoses, danmap, has in particular gained international attention and has been analyzed in different publications (who ; hammerum et al. ; aarestrup et al. ) . the reason for this was the early one health approach that the danish government and stakeholders proposed to combat amr. in , after publication of the finding that % of enterococci in all industrial produced chickens in denmark were highly resistant to vancomycin (a last resort drug for human therapy) the government decided that actions had to be taken (wegener et al. ) and set up the danish integrated antimicrobial resistance monitoring and research program (danmap). figure . shows the organization of danmap and how the animal health, food safety and public health sectors work together. the objectives of danmap are: ( ) to quantitatively monitor the consumption of antimicrobials used in (food) animals and humans, ( ) to quantitatively monitor the occurrence of amr in (zoonotic) bacteria in animals, food and humans, ( ) to study and describe the associations between antimicrobial consumption and antimicrobial resistance, and ( ) to identify routes of transmission and areas for further research. next to this an automated/ict program, called vetstat, was introduced to collect quantitative data on all prescribed medicine for animals from veterinarians, pharmacies and feed mills (stege et al. ) . vetstat data on drug usage proved important for understanding the different aspects of the antibiotic usage problem, and to provide tools to control the use. for instance, with the information from vetstat it has been possible for the danish veterinary and food authority (dvfa) to introduce "the yellow card initiative" (dvfa ) . this initiative works similarly to that in football, and farmers and veterinarians get a yellow card when their antimicrobial use is excessive as compared to similar farms. only by reducing the antibiotic use, which may be done for instance by adopting management practices from low users, the card can be retracted. this has not only worked as a stick, it also gives the farmers a sense of how they are doing compared to their colleagues. in the european union several countries have now also started to collect antibiotic usage data and to compare antibiotic use at country level (ema ). surveillance of foodborne infections, and infectious diseases in general, is important to understand the transmission of infectious diseases and identify risks. to do this efficiently data collection should be done in a harmonized way, so data can be compared and integrated. until now, this has been difficult because different human medical, veterinary medical, food and environmental laboratories have been using different techniques for surveillance, making it often almost impossible to compare data. with the introduction of whole genome sequencing (wgs) this problem may be solved. its unbiased way of detecting dna and its single platform (the dna code) for comparing genomic information gives wgs the potential to take disease diagnostics to a new level. some early uses showing the value of wgs for diagnostic and epidemiological purposes were the tracking of the massive cholera outbreak in haiti in (hendriksen et al. ) and by the ehec outbreak that was first detected in germany and later also found in other countries and which could be traced back to egyptian imported fenugreek seed using wgs (mellman et al. ). following up on these successes, an international group of scientists with representatives from oie, who, ec, usfda, us cdc, ecdc, universities and public health institutes, came together in brussels, september , to further discuss the possibilities of using wgs on a larger scale. their simple conclusion was that the technology to use wgs for diagnostic purposes is available, and its potential high, however, to make efficient use of the data, a global genomic database is needed (kupferschmidt ; aarestrup et al. ). such a database should be open to, and supported by, scientists from all fields: human health, animal health, environmental health and food safety, and should include genomic data for all types of microorganisms as well as meta-data to trace back the source of the microorganism. building such a database depends on a global one health approach, and in a one health manner both human health as well as other sectors will benefit from it. an important aspect of pursuing this initiative is that it will not only be beneficial for the developed world, but it may especially be beneficial for developing countries. for them genomic identification will mean a giant leap forward as they do not need to implement the wide variety of specialized methods that are nowadays used in the developed world. if set up in a sensible, inclusive, open-source framework wgs analysis will provide the world with a strong weapon in the fight to combat infectious diseases in all sectors. one health approaches may be synergistic in controlling foodborne zoonotic diseases to support both sufficient food safety, and sustainable food security. clearly, because of the unique situation of transmissibility between humans and animals, zoonoses control relies on the control of the microorganisms in ( ) animals, ( ) the food chain and in ( ) humans. in addition, as zoonoses originate in animals before being transmitted to humans, the most effective interventions may be achieved at the source, i.e. at the farm. to be most effective, approaches to reduce the risk of foodborne zoonoses should include all stakeholders from the human as well as the animal health side. at the transmission level, it will be of major importance to involve food and consumers authorities and related stakeholders (e.g. environmental specialist), to make sure the spillover from the animal reservoir is kept as low as possible. the exact solution will differ per country and type of disease (e.g. in many developing countries neglected diseases may still be of importance). given that % of the rural population in poor countries is still dependent on livestock as working animals to survive, the effect of these animals carrying a zoonosis will work out differently than in the industrialized settings. a number of the most important zoonoses relate directly to food production systems in poor settings which could be reduced dramatically through well-known interventions, such as has been the case for brucella, bovine tb and cysticercosis. furthermore, it is important to realize that much of the one health efforts until now have focused on zoonotic pathogens with a potential for dramatic global spread (such as avian influenza and bse). however, major health gains can be obtained with the endemic zoonotic pathogens. for instance salmonella causes a dramatic global disease burden because of the sheer number of cases and the global spread via food and live animal trade. for salmonella there are efficient methods to reduce the prevalence in food animals. one health approaches in the food sector are complex and involve both public and commercial stakeholders, which may put limitations on what can be done. on the one hand food should be nutritious and adding to one's health. on the other hand most food is commercially produced and traded. as food is a commercial product, one of the ways to make food producers (the supply-side) produce more healthy food, is if the public demands this (the supply-demand balance). therefore, educating the public to buy healthier food may be a way to make food manufacturers produce healthier food. next, there are other stakeholders and fields of science (e.g. industrial sciences or logistics) and policy (e.g. economics) that contribute to the food chain, but which may focus on other aspects than healthy food alone, and their conclusions may conflict with the food safety principles (e.g. the use of agp to improve animal growth). clearly food safety is a complex issue, and integration of all its problems and data is difficult and should best be limited to its essential components. for this reason, countries should learn from experiences abroad that have documented success. there are many examples of one health approaches that have helped lower the risk of zoonotic foodborne disease. key to all approaches has been surveillance of the farm-to-fork chain. surveillance should be done at relevant levels of the chain, at the farm level by the veterinary system, and at the food production stage by food-scientist. findings should be shared and compared with the findings in human medicine, to be able to make decisions about potential risks for human health. thus the animal, food and human sectors need working together, to collect and to share data in such a way that they may be compared. as there are still many different techniques used in all three fields, it is still difficult to compare data. an important development in infectious disease diagnostics will be the introduction of wgs techniques, and the construction of a global, open-access genomic database for microorganisms. in a one health manner, the latter would take diagnostics to a new level, and will greatly improve human, animal and food safety. sustainable farming: get pigs off antibiotics changes in the use of antimicrobials and the effects on productivity of swine farms in denmark integrating genomebased informatics to modernize global disease monitoring, information sharing, and response vancomycin-resistant enterococci: why are they here, and where do they come from? cdc estimates of foodborne illness in the us the danish integrated antimicrobial resistance monitoring and research programme the yellow card initiative annual report of the european antimicrobial resistance surveillance network (ears-net) methodology protocol for estimating burden of communicable diseases trends in the sales of veterinary antimicrobial agents in nine european countries improved animal health for poverty reduction and sustainable livelihoods. fao animal production and health paper the fao-oie-who collaboration -a tripartite concept note food agricultural organization of the united nations (fao)/world organization for animal health (oie)/who ( ) joint fao/oie/who expert workshop on non-human antimicrobial usage and antimicrobial resistance: scientific assessment cysticercosis working group in peru. taenia solium cysticercosis epidemiological determinants of the pattern and magnitude of the vcjd epidemic in great britain brucellosis in the european union and norway at the turn of the twenty-first century epidemiology, diagnosis, treatment, and control of trichinellosis persistence of livestock associated mrsa cc in humans is dependent on intensity of animal contact isolation and characterization of viruses related to the sars coronavirus from animals in southern china danish integrated antimicrobial resistance monitoring and research program disease burden of foodborne pathogens in the netherlands population genetics of vibrio cholerae from nepal in : evidence on the origin of the haitian outbreak the same prion strain causes vcjd and bse decline in the incidence of bse in cattle born after the introduction of the 'feed ban' analyses of livestock production, waste storage, and pathogen levels and prevalences in farm manures comment on: does the use of antibiotics in food animals pose a risk to human health? a critical review of published data emergence of a new antibiotic resistance mechanism in india, pakistan, and the uk: a molecular, biological, and epidemiological study outbreak detectives embrace the genome era tuberculosis in humans and animals: an overview salmonella enteritidis in poultry: an emerging zoonosis in zimbabwe food poisoning's hidden legacy prospective genomic characterization of the german enterohemorrhagic escherichia coli o :h outbreak by rapid next generation sequencing technology zoonoses and marginalised infectious diseases of poverty: where do we stand? quantifying the burden of disease: the technical basis for disability adjusted life years a one health framework for estimating the economic costs of zoonotic diseases on society risk of importing zoonotic diseases through wildlife trade, united states does the use of antibiotics in food animals pose a risk to human health? a critical review of published data european dissemination of a single oxa- -producing klebsiella pneumoniae clone staphylococcus aureus cc : host adaptation and emergence of methicillin resistance in livestock prion diseases and the bse crisis a review of studies on animal reservoirs of the sars coronavirus agricultural antibiotics and human health public health implications related to spread of pathogens in manure from livestock and poultry operations vetstat-the danish system for surveillance of the veterinary use of drugs for production animals food and drug administration (fda) ( ) years of resistance who global principles for the containment of antimicrobial resistance in animals intended for food: report of a who consultation with the participation of the food and agriculture organization of the united nations and the office international des epizooties who global strategy for containment of antimicrobial resistance impacts of antimicrobial growth promoter termination in denmark. the who international review panel's evaluation of the termination of the use of antimicrobial growth promoters in denmark international health regulations neglected tropical diseases: hidden successes, emerging opportunities tackling antibiotic resistance from a food safety perspective in europe guidelines for the surveillance contributing to one world, one health: a strategic framework for reducing risks of infectious diseases at the animal-human-ecosystems interface key: cord- -esnsa u authors: nan title: abstracts (th) tripartite meeting salzburg/austria, september – , date: journal: langenbecks arch chir doi: . /bf sha: doc_id: cord_uid: esnsa u nan the gastric secretion capacity increases during the first year after all types of vagotomy which is assumed to be important for the development of recurrent ulceration. however, the cause of this reestablishment of the preoperative gastric function is not adequately explained. the purpose of this study was to investigate the vagal innervation of the parietal cell mass in dogs / year after truncal vagotomy (tv), selective gastric vagotomy (sgv) and parietal cell vagotomy (pcv). material and methods: mongrel dogs supplied with a gastric fistula were used. the groups comprised dogs with tv, dogs with sgv and dogs with pcv. the acid secretion was measured before and one month and one year after vagotomy following insulin and pentagastrin stimulations. operative technique: a gastroduodenostomy was performed in addition to the gastric fistula operation in the dogs randomised for either tv or sgv. the vagotomy operations were performed after the prevagotomy secretion experiments. tv by a cm resection of the main truncs through a left side thoracotomy and sgv as described by amdrup [ ] . the pcv dogs had no gastroduodenostomy added but the technique at the lower esophagus was identical with the sgv and the dissection of the lesser curvature was extended cm distal of the sharp physiological and histologically determined borderline between the fundus and the antrum. about / year after the vagotomy the dogs were sacrificed by an acute experiment. the persistent or the regenerated vagal innervation of the parietal cell area was mapped out by neutral red excretion elicited by electrical stimulation of either the thoracic or the cervical vagal nerves [ ] . results: one year after vagotomy no increase in insulin response could be demonstrated in the tv and sgv groups, but a gradual increase to about % of prevagotomy output occured in all pcv dogs. reliable neutral red experiments were performed in tv, sgv and pcv dogs. the stimmulated neutral red excretion was scanty to the same extent at the cardia region in all three groups, but a distinct antralfundic border was outlined in all pcv dogs. the border was, however, only suggested in one of the sgv dogs. conclusion: pcv including denervation of cm distal of the antral-fundic border do not prevent a functional important reinnervation of the distal part of the parietal cell area. no important reinnervation seems to occur at the cardia region after any of the three vagotomy procedures. and insulin ( . ugkg - ) i.v. bolus; after uni-and then bilateral truncal vagotomy. each study was minutes and blood was taken at , , , and then min intervals. gastric acid was measured by autobiuret titration. plasma was stored at - °c until assayed for pp by ria using an antibody sensitive to fmol ml- . results are expressed as mean total increment _+ se. significance: p < . ". bombesin-stimulated gastric acid secretion was not significantly altered by vagotomy (p < . ), whereas that stimulated by insulin was significantly inhibited by bilateral truncal vagotomy (p < . ). bilateral and right hemi-vagotomy significantly inhibited pp release by bombesin ( < . ), however; only bilateral truncal vagotomy significantly inhibited pp release by insulin (p < . ). these results suggest that the measurement of pp release by insulin or bombesin is a sensitive index of vagal integrity and that bombesin-released pp may specifically delineate the integrity of the right vagus. since the measurement of gastric acid secretion after operation is both uncomfortable and often difficult to interpret, the value of a simple blood test to determine vagal integrity may be of considerable clinical relevance. glucose (g) or oleate (o) empty more slowly from the stomach than . m nac (s). this chemoregulation may be achieved by resistance beyond the proximal stomach [ ] . we sougth to define the site of this resistance. expt. : gastric emptying of s and mm g was measured in dogs with intestinal cannulas cm distal to the pylorus while gastric pressure was maintained at , or cm h with a barostat. the dogs were studied either with an uninterrupted intestinal stream or with duodenal effluent diverted through a second barostat prior to its return downstream. since the latter ensured a constant pressure at the ligament of treitz, any observed effects on emptying could not be due to resistance further distally. expt. : emptying of s, g, and mm oleate emulsion was measurd in dogs without cannulas after control antroduodenal transection and after antrectomy while gastric pressure was controlled at , and cm h . emptying rates expressed as the average of the volumes emptied at the pressures. under both conditions in expt. emptying rose with pressure yet s emptied faster than g. thus a major site of chemoselective resistance to emptying of liquids lies proximal to the ligament of treitz. after antrectomy s and o emptied faster than before, however, the differences between saline and nutrients were maintained. resistance, therefore, does not reside in the antrum or pylorus and clearly the duodenum is implicated. gastric emptying of liquids may be abnormally rapid in du. this abnormality my be due to a selective loss of inhibition in response to acid since previous studies [ ] have demonstrated rapid emptying of acid solutions but normal emptying of glucose and fat. previous investigations, however, used meals of only one concentration, so differences between dus and normals (n) may have been minimized by selection of a supra-maximal inhibitory dose. furthermore intragastric ph was not maintained constant so emptying could have been slowed by higher rates of acidification in du. we therefore studied emptying of graded concentrations of citric acid, glucose and oleate in ml meals. gastric volumes were measured by the george technique in dus and n at rain intervals for min after ingestion. gastric ph was maintained constant by intragastric titration ( . for acid; . for nutrients). subjects received one type of meal on separate days and the doses were randomly administered. dose dependent inhibition of emptying in response to acid and nutrients occured in n and du, but emptying was faster in du (p< . ). with glucose and acid the difference occured within - min whereas with fat the difference occurred between - min. thus ( ) dus do not have a selective loss of inhibition of emptying in response to acid, and ( ) the mechanisms which control emptying of fat and which are disturbed in du differ from those which control emptying of glucose and acid. gross ra, isenberg ji, hogan d, samloff im ( ) the effect of fat on meal-stimulated duodenal acid load, duodenal pepsin load and serum gastrin in duodenal ulcer and normal subjects. a means of reducing the requirement for postoperative nasogastric intubation would be advantageous. we report the effect of cimetidine on postoperative nasogastric aspirates. thirty patients undergoing elective abdominal aortic surgery or colonic resection were entered into a double blind randomised study, receiving cimetidine mg hourly or placebo (saline) by continuous intravenous infusion from the morning of the first postoperative day. volumes of hourly aspirates were recorded and ph and (h ÷) of samples measured. nasogastric tube removal accorded to standard clinical practice. it has been shown, that the duodenum has a better ability to resist acid than jejunum or ileum. this resistance was attributed to alkaline secretion (a.s.) of the mucosa, because pancreatic and bile secretion were excluded. recent in vitro studies demonstrated an energy dependent hco -transport (flemstroem am j physiol g : ). we were interested in studying the role of blood flow (bf) in the production of alkali in an in vivo preparation. min. three groups of animals were studied: i) controis with normovolemia for min. ii) normovolemia for min, thereafter min of shock induced by bleeding to mmhg mean arterial blood pressure. iii) min normovolemia and min vasopressin ( . i. u./kg/min) under normovolemia. in controls a small decrease in bfand a.s. as well was observed. both shock and vasopressin induced a significant reduction in a.s. and bf. the relation ofbf to a.s. was found to be exponential (y = . + . x- . x r= . ; p< . ). the reduction in a.s. during shock was much less, when shock induced acidosis was compensated by i.v.-infusion of hco ( . ,uequ/kg/min). conclusion: the alkaline secretion of the proximal duodenum is dependent on blood flow and the arterial [hco~. there has been considerable controversy as to the nature of the offending agent in the etiology in reflux oesophagitis in man. in rat studies, surgically induced reflux oesophagitis was shown to be correlated with the presence of active trypsin in the reflucting juice. reflux of bile and gastric juice with a ph of did not induce oesophagitis. even short periods of oesophagitis ( - weeks) showed an increasing amount ofpanmural fibrosis which even further increased after a reflux abolishing roux-y operation. the pattern of the collagen content of the full thikkness oesophageal wall was similar to that found in human reflux oesophagitis. the hypothesis that active trypsin is also in man responsible for reflux oesophagitis prompted to the following investigation. patients with typical gastrooesophageal reflux symptoms and control patients were endoscopically examined. on entering the stomach with the endoscope samples of gastric juice were taken for ph and active trypsin determinations. trypsin activity was determined with a kinetic method using s- (kabi vitrum b.v. amsterdam) as substrate. results: in all patients some trypsin could be detected in the gastric juice. in those patients with endoscopically erosive and/or ulcerative changes (n = ) the trypsin content was significantly elevated compared to the controls (/ < . wilcoxon). conclusions: the composition of gastric juice is determined by gastric secretion and duodenogastric reflux. duodenogastric reflux is increased in patients with symptoms of reflux oesophagitis [ ] . trypsin in gastric juice (ph . - ) will stable and partly active over prolonged periods [ ] . so from our study in man one may conclude that the high concentration of trypsin in the gastric juice in patients with reflux oesophagitis, may well be the etiological factor of the oesophagitis. disinfection, using artificial contamination of hands with escherichia coil and 'surgical' disinfection directed against the resident microflora of the hands. the authors who developed the procedure have reported unexpectedly high potency for n-propanol compared to iso-propanol, and, in turn, for iso-propanol compared to povidone-iodine and chlorhexidine preparations [ ] . however, using the same procedure we have been unable to find any significant differences in activity between these agents. we have identified several potential sources of error including the method of applying e. coliand its spontaneous loss of viability, the use of neutralizers before disinfection, the differing surfactant effects of the agents, and the absence both of a control untreated area and of a cross-over of disinfectants studied sequentially. in our parallel tests using an excision-sample technique [ ] which is considerably more sensitive than the dghm procedure, we have observed the following mean reductions in the counts of accessible bacteria: iodine in ethanol, %; povidone-iodine, %; chlorhexidine in ethanol, %; iso-propanol, the purpose of this study was to compare radiation injury in guinea pig small bowel ( ) devoid of contents ( ) containing bile ( ) containing pancreatic juice. one group was intact control animals not radiated. another was intact animals radiated. in group a roux y cholecystojejunostomy was constructed and the bile duct ligated. thus one limb contained bile, the other pancreatic juice. in group a blind roux y was constructed such that one limb was empty of contents and the other contained both bile and pancreatic juice. each animal was subjected to a single dose of rads via an abdominal port and sacrificed four days later. the severity of injury was judged by counting the number of surviving crypts per circumference. damage was further evaluated by histologic criteria -mucosal loss, edema, inflammation, hypervascularity and loss of mucus. the maximum histologlc grading score on this scale was . the microscopist was ,,blinded" as to the origin of each tissue section. for histologic grading all radiated groups were significantly different from control (p< . ) and from one another (/r< . ) except pancreatic juice vs. empty. intestine devoid of contents sustains a mild, but significant radiation injury. the presence of pancreatic juice enhances the damage. pure bile makes for yet more severe injury but still significantly less than normal whole intestinal contents which contain both bile and pancreatic juice. the main problem facing patients with ulcerative colitis after mucosal proctectomy and ileo-anal anastomosis is severe frequency of bowel action. our hypothesis was that either an artificial valve [ ] or reversed ileal loop might improve intestinal absorption and slow transit. we tested it in dogs after colectomy and low ileo-rectal anastomosis (ira). body weight, xylose absorption, serum albumin, folate, vitamin b , calcium, urea and electrolytes, full blood count, faecal weight and mouth to anus transit time [ ] were measured before operation. the dogs then randomly underwent either ira alone (control, c, n = ), ira with a cm reversed loop (ira + rl, n = ) or ira with an artifical valve (ira + v, n = ). body weight, haematological estimations and faecal chemistry were measured weekly for months post operatively. xylose absorption and transit time were measured a minimum of months after operation. body weight decreased significantly after each type of operation. there was no difference however, between (ira + rl) and c or (ira + v) and c. likewise, haematological and faecal measurements after both test operations did not differ significantly from c or from pre-op, measurements. the grosfeld valve prevented the reduction in transit time that occurred after the control operation, whereas the reversed loop did not. use of such a valve in combination with mucosal proctectomy might slow transit, but is unlikely to improve absorption. it seems worthy of further study. histologic grading___ sem crypt count-+-sem we have examined the effects of changes in intestinal blood flow induced by hypovolaemia, the mesenteric vasoconstrictors somatostatin and vasopressin and the mesenteric vasodilator prostaglandin e (pge ), on intestinal absorption, electrical activity and volume. in each of dogs a cm jejunal segment was isolated and serosal electrodes attached. in absorption experiments the segments were perfused at . ml/ rain with a solution of retool/ sodium and retool/ glucose. to measure segment volume, a solution of . g/ mannitol was perfused. this solution shows zero net absorption but does not alter electrical activity. intestinal motility and absolute volume were monitored by gamma camera. experiments were performed after a steady state was reached. absorption and transit time were measured using non-absorbable markers. electrical fast wave activity was reduced by (a) intravenous somatostatin . mcg/kg/h ( + . to _+ . ; mean_+sem/ rain) and abolished by (b) intravenous vasopressin . u/kg/h (p< . ). motility was inhibited and mean transit time was prolonged (a) . to . rain and (b) . to . rain (/r< . ). intestinal volume rose by (a) . ___ . ml and (b) . -+ . ml (p< , ). absorption was unchanged by somatostatin and fell with vasopressin ( . --- . to . -+ . ml/ min). acute blood loss sufficient to lower the central venous pressure by cm of water produced identical changes to somatostatin. pge produced no alteration in electrical activity or absorption. intestinal absorption is resistant to changes in intestinal blood flow whilst electrical activity is depressed and intestinal volume increased by mesentric vasoconstriction. the accepted views on the pathogenesis of amoebic lesions in complicated amoebic colitis are that amoebae produce a toxin resulting in cytolysis and necrosis. this concept may adversely affect the management of patients with complicated amoebic colitis by implying that once the amoebae are killed, the disease process is arrested and colonic perforation is unlikely. we present an alternative hypothesis that 'complicated amoebic colitis is the result of vascular compromise'. transmural disease is caused by amoebic invasion of vessels supplying a segment of the colon with subsequent thrombosis and ischaemic necrosis of the affected area. the ischaemic nature of the necrosis is suggested by its shape, and the demonstration of vascular thrombosis on dissection ofresected colons which have perforated. amoebic invasion of blood vessels can be demonstrated histologically. the ischaemic nature of the lesions can be confirmed by angiographic examination of the resected colon. vascular occlusion can be demonstrated pre-operatively with the use of selective mesenteric angiography, which clearly delineates the ischaemic segment of the colon. selective rnesenteric angiography in patients with fulminating amoebic colitis aided the preoperative diagnosis of colonic infarction before signs of visceral perforation had developed and permitted life saving surgical intervention. there is accurate correlation between angiographic and operative findings. in postdysenteric colitits associated with amoebic strictures of the colon, mesenteric angiography will demonstrate the ischaemic nature of the stricture and accurately define the extent of resection. after ca. the mean age at operation was and years respectively. anal canal length was . ___ . cm (mean + s.e.m.) after ia and . ___ . cm after ca. a resting tone of ___ cm water after ia and ----- cm water after cawas recorded. squeeze pressure was ___ cm water after ia and ----- cm water after ca. the recto-anal reflex was present in % of the ia patients and in % of the ca patients. a mean daily bowel frequency of . ___ . followed ia and +_ after ca. these findings show that after ia anal pressures are within the normal reference range for our laboratory [ ] . lower pressures were found after ca, probably due to the older age of the patients in this group [ ] . normal anal canal length, the integritiy of the striated sphincter and in most cases preservation of the recto-anal reflex contribute to satisfactory continence following peranal anastomoses. perineal descent is found in patients with idiopathic faecal incontinence (ifi) and patients presenting with symptoms of the descending perineum syndrome (dps), who have no incontinence. manometric, radiological and neurophysiological studies were performed in patients with ifi, all of whom leaked during rectal infusion of ml saline, patients with dps, who were continent of rectally infused saline, and matched controls. both patient groups exhibited similar degrees of perineal descent below the pubococcygeal line of straining (ifi, - . ___ . cm; dps, - . + . cm) compared with controis (- . __+ . cm;p< . in both cases), and similar prolongation of both the latency of the cutaneoanal reflex (ifi, ___ ms; dps, + ms; controls, + ms;/,< . in both cases), and motor unit potential duration (ifi, . ___ . ms; dps, . ___ . ms, control, . + . ms; p< . in both cases). moreover, both groups had an abnormal ano-rectal angle (/,< . ), though this was more obtuse in ifi than dps ( ___ ° vs ___ °;p< . ). however, while patients with ifi had lower sphincter pressures than normal (basal; ___ vs ___ cm water; / < . ; squeeze, ___ vs ___ cm water; / < . ), and required a lower rectal volume to inhibit sphincter tone for more than one minute ( + vs ___ ml; p< . l), these values were normal in patients with dps (basal; ___ cm water; squeeze, _+ cm water; rectal volume, ___ ml). these findings suggest that perineal descent and neuropathy are not necessarily associated with incontinence if sphincter pressures remain normal. aminoacids administered either enterally or intravenously stimulate gastric secretion in both dog and man. it has been suggested that absorption of amino acids from the gut into the circulation might contribute to the intestinal phase of gastric secretion. the mechanism of this stimulation by amino acids remains uncertain but in an earlier communication we reported the direct action on the parietal cell of phenylalanine, glutamine and alanine [ ] . in the present study using ( c) aminopyrine uptake as an index of dispersed parietal cell secretory function, the interaction between histamine and individual amino acids and the effect of the histamine h -receptor blocker ranitidine ( - molar) on these interactions have been examined. results (percentage of maximal stimulation produced by - molar histamine) results (percentage of maximal stimulation produced by - molar histamine) methionine glutamine alanine amino acids only ( - molar) amino acids and histamine ( - molar) - amino acids and ranitidine ( - molar) histamine and ranitidine -complete inhibition - . the response to the combination of amino acids and histamine was greater than the sum of the maximal responses to each of these agents alone. . the histamine h -receptor antagonist ranitidine completely inhibited the histamine stimulated response but only partially inhibited secretion stimulated by amino acids. we conclude that amino acids act directly on the parietal cell by a mechanism which is not solely dependent on histamine. sex related differences in gastric acid secretion have been documented (lilja et al, ) . it has been also reported that male rats have a significantly higher serum gastrin concentration than females and that oophorectomy increases serum gastrin to male levels (lichtenberger et al., ) . estimation of serum gastrin levels after administration of androgens has not been reported. aim. the aim of this study was to investigate the serum gastrin levels before and after oophorectomy and administration of estrogens and testosterone in female guinea pigs. method. groups of guinea pigs were used for this study, animals as controls, were given estrogens for weeks, were given testosterone for weeks and underwent oophorectomy. results. the mean serum (-+sem) gastrin value in controls was found + . pg/ml, after estrogens . -+ . pg/ml, after testosterone _+ . pg/ ml, after oophorectomy -+ . pg/ml. conclusion. it is concluded that estrogens decrease the serum gastrin, and that the increase of serum gastrin after administration of androgens and oophorectomy is statistically significant (/r< . ). the mechanism by which the ovarian hormones influence gastrin levels may be a sex-dependent change in the synthesis or secretion of gastrin. the reported inhibitory influence of estrogen on food consumption may be also the predominant factor in serum gastrin alterations. mean caloric intake was --+ kcal/day. moderate to severe dumping occurred in one patient. faecal fat was normal in while patients had greater than grams fat loss per day. weight loss was . _+ . % of recall weight. muscle mass measured by arm muscle circumference ( . _+ . cm) and creatinine heigth index ( ___ %) was normal for our patients but triceps skin fold was % less than normal ( . _+ . mm) indicating that in these patients fat stores were reduced. in six patients haemoglobin was less than grams per cent but serum iron, iron binding capacity and serum folate were normal. red cell folate was depressed in six patients whose haemoglobin values were normal. it is concluded that with this reconstruction total gastrectomy produces satisfactory digestive and "nutritional results. examination of factors affecting faecal fat loss and folate metabolism may help improve the nutritional status of these patients. in order to assess the optimal conditions for segmental pancreatic graft viability the following experiment was conducted. the duct obliterated splenic lobe of the canine pancreas was autotransplanted by end to side fashion to the femoral vessels (n = ). the graft was lodged in a subcutaneous pocket. a month later total pancreatectomy was completed. the dogs were supplemented with pancreatic enzymes (viokase). mean survival was ___ days. deaths were due to hyperglycemia when grafts lysed from local infection and thrombosis. in the second experimental group (n= ) the autografts were anastomosed to the iliac vessels and placed intraperitoneally followed by total pancreatectomy one month later. construction of a distal splenic arterio-venous fistula increased blood flow from . cc/min to cc/min. the pancreatic duct was obliterated with neoprene (n= ) silastic (n= ) or left open (n= ). three dogs died one month to four months due to graft fibrosis and failure. seven dogs had been followed from four months up to seven months when sacrificed. all were normoglycemic. mean k values for ivgtt were . . the normoglycemic dogs had mean plasma concentrations of amino acids similar to healthy controls. a two to threefold elevation was observed in pancreatectomized diabetic dogs for plasma leucine, isoleucine and valine. immunoreactivity of pancreatic polypeptide was measured by radioimmunoassay with an antibody raised against the human hormone by re. chance, lilly research laboratories. mean plasma levels rose form --+ s.e. pg/ml to over pg/ml following protein meal ( g ground lean beef/kg) in all normal control dogs (n= ), and to levels in the range of to over pg/ml in the same dogs following infusion ofbombesin ( pg/kg/ h) for min. mean basal levels were lower ( ___ . s.e. pg/ml) in dogs with autotransplants and did not increase significantly during any stimulatory test. the levels of pancreatic polypeptide did not distinguish between viable and failing grafts. in successful grafts histology showed fibrosis of the exocrine component. transmission electron microscopy revealed the presence of clusters of functional islet cells in the six-month implants. a and b cells were common. d cells were much less frequent. release of secretion granules into the perivascular connective tissue space was observed. the results indicate that vascularized segmental pancreatic graft comprising % to % of the pancreatic mass maintains normal carbohydrate and amino acid metabolism. graft survival was extended by intraperitoneal location and creation od distal splenic a-v fistula, but chronic graft fibrosis occurred regardless of the method of pancreatic duct treatment. the results further suggest the necessity of an intact vagal innervation for the physiological or pharmacological stimulation of pancreatic polypeptide release from thendocrine pancreas which is otherwise apparently functional. besides, these considerations rule out pancreatic polypeptide levels as a useful marker for evaluation of pancreas graft. polyisoprene ductal occlusion allows segmental pancreatic transplantation in man with satisfactory early islet cell function. however acute rejection remains a problem as the diagnosis, based on hyperglycaemia and glycosuria, represents a late manifestation of rejection. although llqndiumlabelled platelets have been used in the diagnosis of renal rejection [ ] , their use in pancreas transplants has not been studied. ~ in patients, autologous llqn-labelled platelets were injected on the second, sixth and tenth days following combined renal and pancreatic transplantation. graft radioactivity was expressed as the ratio of counts from the pancreas over a reference area on daily gamma images for days. one patient developed hyperglycaemia coinciding with renal rejection on day . over the previous h pancreas radioactivity had increased by %. in a further patient whose pancreas continued to function, a perigraft haematoma was recognised on the gamma image. the remaining patients had good pancreatic function and no xin-platelet accumulation: mean (_s.c. mean) pancreatic radioactivity was . + . on the third postoperative day and . _+ . at the end of study. these results demonstrate that ductal occlusion with polyisoprene does not cause significant platelet accumulation. hence min-platelets may potentially be used for the earlier diagnosis of pancreatic rejection. isograft models of pancreatic transplantation, methods involving closure of the duct system result in severe inflammatory changes and eventual fibrosis [ ] . these changes can be avoided by formal drainage of the duct into the bowel of urinary tract. inflammatory changes initiated by duct closure may contribute to and enhance allograft rejection. pancreas transplants were performed in rats using lewis (rt ) donors and streptozotocin-induced dia- betic da(rt a) recipients, using duct-ligation, open duct and ureteric duct drainage. cyclophosphamide produced significant prolongation ofnormoglycaemia in all groups and although there was a tendency towards longer function in the unligated groups there was not statistical difference (wilcoxon's unpaired test) between the methods of duct management. management of the pancreatic duct did not seem to have any immunological consequences for graft survival but septic complications, associated with the normoglycaemic deaths, were more common in immunosuppressed animals with draining ducts. between july , and march , combined pancreatic and kidney transplantation was performed in patients with type i diabetes who had all been on insulin therapy for at least years. age at the time of transplantation was to years. the pancreatic segment used for transplantation consisted of body and tail of the organ based on a vascular pedicle of the celiac axis and the splenic vein. imediately prior to intraperitoneal transplantation to the iliac vessels the ductal system was filled with to ml of prolamine, a rapidly solidifying alcoholic protein solution. simultaneous kidney transplantation was performed through a separate incision. five pancreas transplants were lost for non-immunological reasons. four of them never had any useful endocrine function, one graft was lost of venous thrombosis after hours of excellent function. in none of these patients did failure of the graft lead to any substantial complication. in the remaining patients initial graft function was excellent with plasma glucose normalizing within hours and subsequent normoglycemia on a regular diet without exogenous insulin administration. plasma glucose did not rise spontaneously during rejection episodes of the kidney and eleva-tions due antirejection treatment were promptly; reversed as high dose prednisolone was discontinued. in oral glucose tolerance test (ogtt) performed at to weeks in patients median glucose rose from a basal . mmol/l to . mmol/ and was back in normal range ( . mmol/ ) at h. basal insulin was - pmol/ and also returned to the normal range after h after a peak of - pmol/ at - rain. c-peptide showed a significant peaked rise over basal values ( , pmol/ ) in patients while all values were above pmol/l in the third. two of these patients have since rejected both organs. two tranplants still function very well after and months, respectively. in one of these patients the ogtt after months is even slightly better than the above -mentioned first test, and he shows a norreal insulin and c-peptide response. in the fourth patient an ivgtt performed at months and an ogtt at sixteen months were normal with insulin peaking at pmol/ and a c-peptide peak of pmol/ at min in the latter. -results at and months, respectively, will be presented. three problems persist in clinical organ preservation. these are failure of current systems to replenish the ischemically injured organ, to reliably extend the period of organ preservation and to definitely determine organ viability. previous studies have documented the value of electrochemical redox control in rejuvenation of the ischemically injured kidney during perfusion preservation. this study was undertaken to develop the cost effective technique applicable to current organ preservation systems, to test the reliability of redox measurement in prediction of organ viability and to determine the ability ofredox maintenance to safely extend the period of organ preservation. a disposable cell has been developed using reticulated vitreous carbon as an electrode which is driven by a potentiostat powered by a nine volt transistor battery. short term preservation studies using ischemically injured dog kidneys ( rain in-situ warm ischemia) were auto transplanted after h of pulsatile preservation to determine the optimum redox level of the hypothermic kidney. this was determined to be a - mv vs the standard calomel electrode. twenty-one adult mongrel dogs were equally divided into three groups. pulsatile perfusion preservation was extended to four days in group a with redox level monitored only. group b was treated with electrochemical reduction for four days and group c for six days. three of seven dogs survived in group a following auto transplantation and immediate contralateral nephrectomy. the kidneys of all survivors were able to bring perfusate redox potential under control and maintain this level throughout the preservation period. six of seven dogs in group b survived with a mean posttransplant peak serum creatinine of . rag/ ml. in group c four of seven kidneys supported life immediately. all redox controlled kidneys made copious amounts of urine. our data indicate that perfusate potential may be either monitored as a reliable index of organ viability or controlled to allow extended safe preservation. antithymocyte globulin (atg) has been shown to be an effective agent in combination with increased doses of steroids in reversing renal allograft rejection. since it is frequently undesirable to employ raised doses of steroids, the following study evaluated the effect of - i.v. daily doses of horse atg alone without additional steroids, on nd- th rejection episodes in recipients of cadaveric renal allografts ( rejections: biopsy-proven) detected within - mos. of transplantation. of rejection episodes, were successfully reversed, with return of serum creatinine to pre-rejection levels in episodes ( patients). three patients had primarily humoral rejections and returned to dialysis - mos. after treatment of the last rejection. levels of circulating horse immunoglobulins were obtained in all patients during and follwing administration of atg. recurrent rejections ( rd- th) following last treatment with atg ( - mos.) were seen in / patients. all patients had rapid immune eliminiation of atg ( / life - days) as compared to the patients who had no more than rejection episodes ( / life - days). atg without additional steroids is an effective agent for reversal of multiple renal allograft rejections which by biopsy are primarily cell-mediated. to be effective, heterologous atg must be given in adequate total doses and/or from appropriate heterologous source, to prevent rapid immune elimination by the recipient. the use of atg alone for treatment of recurrent allograft rejections is particularly recommended for its steroidsparing effect in treatment of multiple rejections and for those patients at high risk from steroid side effects. the significance of the monocyte crossmatch in lrd recipients of hla identical kidney grafts j. cerilli, l. brasile and s. rogers ohio state university hospitals, columbus, ohio, usa in a preliminary study from our transplant center, the presence of pre-formed antibody in recipient sera directed against monocytes from their respective living-related donors correlated with a poor clinical course. a poor prognosis for graft survival was found regardless of the hla match grade. to minimize the role of the hla system, only those living-related recipient/donor pairs who were hla identical at the a, b, c, d and dr antigen loci, and who exhibited severe immunological types of rejection were evaluated. due to the small numbers found in this category at any one center, this abstract represents an international study from different transplant centers. patients who met the criteria were studied for the presence of antibody directed against their respective donor's monocytes both pre-and posttransplant. in eighteen of these patients, cytotoxic antibody against their donor's monocytes was found in their pre-transplant sera. there was no detectable cytotoxic activity against their donor's t or b lymphocytes. two additional transplant recipients exhibited this antibody in post-transplant sera. again, no t or b lymphocyte cytotoxicity was detected. a control group of hla identically matched siblings who incurred no or minimal rejection demonstrated no anti-donor monocyte antibody. the results of this international study points towards a correlation between a high incidence of graft rejection and the presence of antibody directed against their respective donor's monocytes. therefore, in our view, the presence of anti-monocyte antibody to the prospective donor pre-transplant is a contraindication to transplantation. in patients with different liver diseases the reduced concentration of the peripheral blood t-cells and altered immune response were observed. the purpose of our studies was to investigate the mechanism of the immunological modification of the lymphoid system in the hepatic injury. studies were carried out in groups: ) lew rates were treated with dimethylnitrosamine (dmna), mg/kg b.w., i.v. for acute liver necrosis induction, ) cc ( . ml/ g b.w., i.v.) twice weekly, over weeks for chronic liver damage, ) ccl over weeks for liver cirrhosis (histologically examined). serum and thymus, spleen, mesenteric lymph nodes (ln) were removed and days after dmna treatment and and days after last cc injection. the total number of thymocytes and spleen cells was counted and the reactivity to pha and cona was measured. normal liver perfusate (lp) was prepared h after removing ( °c) by times perfusion in rain intervals (flow rate ml/g/min with ml/g of ringer). the effect oflp, sera, dmna and cc on the viability and pha response of normal thymocytes was tested in vitro. liver enzymes were evaluated, we found the decreased total number of thymocytes immediately after the stopping of medication (group : , ___ , %, group : , ___ %, group : less than % of control). proliferative response of the remaining cells in thymus to pha was much higher than normal thymocytes (group : ± % group : ___ %, group : nd). the total number of spleen cells was not changed but their response to cona was altered in the all groups and to pha only in group . pha response of ln-cells was decreased in the all groups. liver perfusate was cytotoxic ( ___ % of viable cells) and suppressive for pha response of thymocytes ( _+ % of control). the sera of rats with the hepatic damage showed an enhanced suppressive activity. cc and dmna did not have any effect on thymocyte in vitro. one month after last medication we observed the recovery of thymus involution (total i in the acute and partial in the chronic hepatic damage and cirrhosis). our results suggest that the liver origin cytotoxic and immunosuppressiv e factor(s) can be released from the damaged liver into the circulation (like to lp) and can destroy the thymus leading to the secondary changes in the other lymphoid organs. the grade of thymus alteration is dependent on the degree and the duration of hepatic damage and is reversable. the hepatic factor (s) showed the similar effect on the cortical population of thymocytes like the steroid immunosuppressants. liver grafts in the rat are in certain strain combinations not rejected and in this situation there is evidence for spontaneous donor specific tolerance [ ] . we have developed a model of auxiliary liver transplantation which would allow us to study the immunosuppressive properties apparently produced by a liver allograft. the portal vein is anastomosed to the left renal artery, the i.v.c. to the renal vein and the bile duct to the ureter. simultaneous kidney or heart allografts were performed. conclusions:auxiliary liver grafts are rejected. survival of heart or kidney grafts is not influenced by a simultaneous kidney or heart allograft. heart and kidney grafts are prolonged by simultaneous auxiliary liver grafts. [ ] . the role of suppressor cells in the initiation and propagation of malignant tumours in man, is less clearly defined. the present study, using in vitro mitogen assays (pha, cona, pwm) and various rosetting assays [ ] with specific monoclonal antibodies to lymphocytic helper (okt ) and suppressor (okt ) cells, has revealed the presence of such suppressor lymphocytes in women with clinically localised (breast and axilla) mammary carcinoma. lymplaocyte hyporeactivity to mitogens was found in % of lymphocyte preparations from blood and axillary lymph nodes of patients with breast cancer. in % of patients nodal lymphocytes were totally anergic. the most profound hyporeactivity, however, was detected in the lymphocyte subsets (< %) of specimens isolated from the breast carcinomas by collagenase digestion and sephadex g- column passage [ ] . the lymphocyte preparative techniques were not responsible for the low levels of responses detected. also, in situ prostaglandin synthesis and release did not appear to be involved in depressing lymphocyte reactivity [ ] . comparable percentages of suppressor cells (okt +) were detected within these different lymphocyte preparations. suppressor cells were not found in the lymphocyte preparations from the blood and lymph nodes of appropriate controls. from clinical and experimental studies it is known that blood transfusions may have immunosupressive as well as immunostimulating consequences. the effect of transfusions on graft survival has been extensively studied by our group in the bn to wag rat model. in this donor-host combination it was found that a donor specific pre-transplant blood transfusion could lead to a marked prolongation of heart and kidney graft survival, whereas the similar pretreatment resulted in accelerated rejection of bn skin allografts. this specific model was used to investigate the influence of a single bn transfusion on the growth of different syngeneic transplantable tumors in wag rats. the first tumor was a radiation induced basal cell carcinoma of the skin (t ), the second tumor was a chemically induced adenocarcinoma of the duodenum (t ). the antigenicity of both tumors was assessed in vivo, using classical challenge-protection experiments. it was observed that t i exhibited strong immunogenetic properties, whereas t was only weakly immunogenic. the doubling time oft i was . days, the doubling-time of the adenocarcinoma was days. intravenous inoculation of isolated t cells led to development of lung nodules which could be counted after days. wag rats were injected i.e. with ml of bn blood or syngeneic blood (controls) at - days before tumor challenge. t l was given in two different ways: a) sc. implantation of+ x mm pieces, b) i.e. injection of isolated tumor cells. t was implanted sc. only. each experimental group consisted of animals. for t it was found that allogeneic blood transfusions caused a slight (but not significant) inhibition of subcutaneous tumor growth. however, in the t lung-metastasis model it was observed that a single bn blood transfusion led to a % reduction of nodules, counted at weeks after inoculation. this reduction in number and size of nodules was highly significant (p< . ). for tumor t , the bn blood transfusions evoked a strong inhibitory effect on the growth of the sc. implanted tumor. at weeks after implantation all tumors in the control group had grown to a diameter of - rnm (average diameter . ram). in the group pretreated with bn blood, only of tumors were palpable at that time (average diamter mm). for t it was further investigated whether a single bn transfusion, given one week after i.e. tumor cell inoculation, would have any influence on tumor growth. no significant effect on number or size of the lung nodules could be noticed, if anything, the transfusion appeared to have a stimulatory effect. the results indicate that allogeneic transfusions can lead to a substantial modification of tumor growth, depending on tumor type and site of implantation. this observation may have important clinical implications. we report here the serial study of circulating immune complexes (cic) in two human tumor systems, colorectal cancer and gestational trophoblastic neoplasia (gtn). cic were assayed by antigen nonspecific insolubilization induced by . % polyethylene glycol (peg) and monitored as a od o changes by spectrophotometry. all of the serially studied colorectal cancer patients presented with elevated cic levels (mean = + zt od ) as compared to our standard cic level for pooled normal human sera ( --+_ aod , p< . ). initial values in these patients range from to a od with no correlation to tumor load, site of presentation, or subsequent clinical course. in / patients who underwent ,,curative" resection of primary or metastatic colorectal cancers, serial cic elevations occured only when antigen excess (measured by simultaneous carci-noembryonic antigen [cea] assay) decreased. immunoglobulin components of fractionated cic showed predominantly iga subclass. in gtn patients followed with serial cic and simultaneous human chorionic gonadotropin (hcg) assay, only those patients documented to enter hcg remission after molar evacuation showed significant elevation of cic. chromatographic fractionation of peak cic in one such patient defined three irnmunoglobulin containing fractions showing immunoreactivity to one of four paternal hla haplotypes (aw ). one ,,antigen" fraction (< . mw) from this complex completely inhibited reference anti-aw binding. as in the colorectal cancer patients, these data show that cic rise only when antigen excess decreases (reflected in the gtn patients by hcg normalization). in addition, some gtn patients may react to immunogenic paternal hla haplotypes as part of their response to molar pregnancy. dfmo, an enzyme-activated irreversible inhibitor of ornithine decarboxylase (odc), reduces tumor polyamine levels, inhibits growth of emt sarcomas and hepatomas in experimental animals, and induces remission in human leukemia. a renal adenocarcinoma (ra) cell suspension, or a mm segment ofwilms' (wm) tumor was transplanted intrarenally into balb/c mice (n-- ) or subcutaneously into wistar-furth rats (n= ) respectively. dfmo ( %) in drinking water was administered to half the animals in each group throughout the experiment. at days ra tumors in dfmo-fed mice weighed % less than tumors in control animals (p< . ); wm tumor weight at days was not affected by dfmo feeding. the mean number of lung metastases in dfmo-fed r.a-bearing mice was . and in ra-bearing control mice was . (p< . ). dfmo caused - % inactivation of tumor odc, reduced ra putrescine levels by /o (p< . ), reduced wm putrescine levels by % (d < . ), reduced wm spermidine levels by % (p< . ) and increased wm spermine levels by % (p< . ). dfmo feeding did not alter dna content of ra or wm tumors. although final carcass weight was similar in all animals, dfmo feeding progressively reduced total body weight (tbw) of mice, but not rats, until at day the tbw of dfmo-fed mice was . % less than tumor-bearing control mice (p< . ). dfmo-fed mice bearing ra tumors survived . -t- . days longer than control mice (p< . ). reduction of polyamine levels in wilms' tumors does not affect tumor growth. lowering of renal ade-nocarcinoma putrescine levels by continuous feeding of dfmo to tumor-bearing animals decreases tumor growth, reduces lung metastases, and increases host survival. we have previously demonstrated that vagal nerve stimulation releases -ht into the lumen of the feline gut. this study was initiated to: . determine if substance p (sp) and motilin (mt), other enterochromaffin cell products, are released simultaneously, . to evaluate if this release is under cholinergic or adrenergic control. in cats, cm isolated in situ segments of proximal jejunum were perfused with saline at ml/min ( ~c). perfusate samples were evaluated at rain intervals and concentrations of -ht, sp, motilin were measured by ria's developed in our laboratory. after two -min basal periods, the supradiaphragmatic sectioned vagus nerves were stimulated electrically ( v, m s, hz). the output from the loop in ng/ min ( -ht) and pg/ rain (sp and mt) was: introduced into the jejunum of conscious dogs through an external small bowel fistula. the gut was perfused at ml min- with a physiological electrolyte solution containing the non-absorbable marker polyethylene glycol (mol. wt. ; g - ); water and electrolyte absorption and transit time (tt) were measured during intravenous (i.v.) administration of each peptide, and during preceding and succeeding i.v. control infusions of . m naci. separate studies showed jejunal absorption and tt to be constant over prolonged periods during i.v. nac administration. bombesin ( pmol kg-lmin- ) and neurotensin ( pmol kg-~min ) significantly reduced jejunal water absorption; bombesin and enkephalin ( . nmol kag- rain- ) significantly prolonged tt; and enkephalin encreased water absorption (/ < . in all cases). measurement of plasma neurotensin during i.v. infusion indicated that physiological blood levels were not exceeded during these studies. conclusion: a number of peptides may be involved in the regulation of small bowel function. the effect of neurotensin on jejunal water transport provides a possible mechanism linking raised blood neurotensin levels with intestinal intraluminal fluid accumulation in the dumping syndrome. in order to establish whether this release was under adrenergic control, cats had cervical ganglionectomies. using the same electrical paramenters, stimulation of the cut cervical vagus nerves resulted in identical -ht responses as above. in additional cats atropine administration (lmg/kg iv) totally abolished the -ht responses to vagal nerve stimulation. the paralled release of -ht, sp, and mt following vagal nerve stimulation, strongly suggest that the ec cell is the source of these luminal hormones. this release appears to be under cholinergic control. since diabetes mellitus is markedly improved immediately after jejunoileal bypass before significant weight loss, but only gradually and often incompletely changed after gastric bypass, it seemed appropriate to investigate the effects of intestinal exclusion on experimental diabetes. studies were performed on alloxan diabetic sprague-dawley rats. two days after jejunal exclusion (je) in rats (resection of proximal / of small intestine), fasting blood sugars (fbs) decreased , from to mg/dl, and averaged mg/dl at weeks. after ileal exclusion (ie) in rats (resection of distal / of small intestine), fbs fell % in days, from to mg/dl, but increased % above preoperative levels to mg/ dl at weeks. sham operated rats responded similarly to ie rats. after alloxan and operations, all groups lost weight, but only je rats began to increase weight at a normal rate. increased water intake, polyuria ( ml/ h), and glycosuria ( rag/all), were present in ie rats; je rats were normal (urinary output < ml/ h, and urinary glucose mg/dl). oral glucose tolerance tests (gtt) were extremely abnormal in ie rats, similar to those in non-operated alloxan rats, while gtt curves in je rats were similar to normal animals, with some elevation at , and min. serum insulin levels remained low in all alloxan treated rats after jejunal exclusion. possible mechanisms, currently under study, relate to carbohydrate malabsorption and changes in enteric chemical mediators. the purpose of the present study was to investigate the changes in somatostatin release and somatostatin-containing cells of the pancreas and stomach of the streptozotocin (stz) -induced diabetic rat after the amelioration of diabetes by whole pancreatic transplantation. highly inbred lewis rats were divided into three groups: ( ) normal rats, ( ) stzinduced diabetic rats and ( ) transplanted rats. diabetes was induced by the administration of stz ( mg/kg). on the seventh day after stz treatment, pancreatic transplantation was performed. four weeks after the transplantation, in vivo and in vitro, studies were performed. pancreatic d cells and gastric somatostatin-containing cells were stained with antibody enzyme method. studies in vivo showed marked improvement of the impaired arginine-induced insulin release by the transplantation. studies in vitro employing isolated perfused rat pancreas and stomach revealed following results: mean basal pancreatic somatostatin release in normal, diabetic and transplanted rats were ___ , -t- , and __+ pg/ml, respectively. total amount of pancreatic somatostatin release in each group during arginine stimulation ( . . mm) were --+ , ___ , and -+ pg/ min, respectively. significantly higher somatostatin release was obtained from the diabetic pancreas, which was, however, reduced to normal after the whole pancreatic transplantation. on the other hand, insulin release from the diabetic pancreas was severly impaired and pancreatic transplantation had not effect on insulin release from the host pancreas in the transplanted rats. as to the glucagon release, there was not significant difference among them. mean basal gastric somatostatin release in normal, diabetic and transplanted rats were -t- , ___ , and + pg/ml, respectively. there was no significant difference between normal and diabetic rats, though the significant decreased value was obtained in the transplanted rats. (vs. normal;/ < . , and diabetes; / < . ). on the other hand, glucagon-stimulated peak values in these groups were _ , ___ , and + pg/ml, respectively. glucagon-stimulated gastric somatostatin release in diabetic rats was significantly increased, but reduced to normal value by pancreatic transplantation. also, a number of pancreatic d cells and gastric somatostatin-containing cells were markedly increased on the diabetic rats. on the other hand, a number of these cells in the transplanted rats were descreased to normal levels. in summary, enhanced pancreatic and gastric somatostatin release and cells in the diabetic rats were both normalized after the amelioration of diabetes by the whole pancreatic transplantation. from these results, it is suggested that pancreatic and gastric somatostation are regulated by circulation and/or metablic of nutrients. doppler velocity recordings are widely used for the non-invasive diagnosis of carotid arterial disease. although detailed analysis of carotid doppler spectral information has been suggested as a method for improving diagnostic sensitivity, the accuracy of the relationship between the doppler recording and the true instantaneous velocity profile has not been established. the purpose of this study is to determine if a cw doppler velocitymeter can accurately transduce the true instantaneous blood flow velocity information. methods: a pulsatile flow model has been constructed in which it is possible to record the instantaneous doppler spectrum and simultaneously photograph and measure the true velocity profile. a computer controlled pump generates a carotid waveform in tubes without stenoses and with, symmetrical stenoses. flow is visualized using the photochromic dye tracer technique. a short burst of uv light from a laser is passed across the tube. a narrow band of the fluid turns blue, its movement is photographed, and the instantaneous velocity profile determined every msec throughout the pulse cycle. at the same time, the instantaneous doppler spectral information is recorded by a frequency analyzer. the results follow. for pulsatile laminar flow, the doppler spectrum correctly recorded the true velocity spectrum, including the instant/~neous maximum velocity and mean velocity. for disturbed flow, it was not possible to show the same direct relationship between the doppler spectral recordings and the blood flow velocity. in conclusion doppler velocitymeters accurately transduce velocity information when flow is laminar but when flow is disturbed there is not a direct relationship between doppler recordings and the true velocity profile. consequently, one should be cautious in attempting to relate doppler measurements of disturbed flow directly to the true changes in the velocity pattern. early failure of arterial reconstruction may originate in poor patient selection. in aorto-iliac stenosis (ais) selection for operation relies upon clinical examination of the femoral pulse and radiology. since single plane arteriography is inadequate for accurate definition ofiliac stenosis [ , ] , this paper compares clinical examination, doppler ankle systolic pressure (aspi) and femoral signal analysis (laplace transform damping, ltd, and pulsatility index, pi) [ ] with biplanar contrast studies. i at biplanar angiography of ischaemic lower limbs had ais with diameter reduction from - %, of the remainder, were normal (< % stenosis) and were ,,occluded" ('_-- / ). nearly two thirds ( %) of limbs with a clinically normal femoral pulse had identifiable arteriographic stenosis (~-__. / ), upstream abnormality was predicted incorrectly in % and the overall accuracy of clinical examination was %, both for detecting stenosis and predicting its severtiy. aspi (median . ; % confidence ,limits . - . ) and pi ( . ; . - . ) although correlated with stenosis (aspir = . ; p = . , pir = . ; p= . variance analysis for linear regression), did not aid the clinician further (accuracy %). however ltd ( . ; . - . ) was well correlated (r= . , p= . ) and did improve assessment ofiliac stenosis (accuracy %). the need for biplanar arteriography is reiterated and its use with doppler signal analysis should improve the evaluation of aorto-iliac disease. in the absence ofa non-invasive method for estimating volume flow in an individual artery, local blood pressure measurement has proved, with certain limitations, useful in assessing the cardiovascular system. now ultrasound technology has progressed to enable blood flow in an artery to be measured noninvasively. we report the results o four evaluation ofa mhz, computerized, channel, pulsed doppler vessel imaging and flow measuring instrument in in-vivo experiments. computed blood flow was compared to actual blood flow (calculated by timed collection) in anaesthetised dogs. correlation between computed and actual blood flow was stronger in the larger abdominal aorta than in the smaller common carotid arteries. from the regression plot, the coefficients of determination, p, were: . (exposed aorta scans); . (transcutaneous carotid scans); and . (exposed carotid scans). stepwise regression analysis showed the computed flow values to be independent of probevessel angle, depth and lumen diameter for vessels greater than . mm in diameter. these results suggest that this pulsed doppler instrument has the versatility and accuracy essential for diagnostic flow measurements in the main conducting arteries of the neck and limbs and in vascular bypass grafts. in the assessment of patients undergoing carotid artery surgery, many laboratory methods are available in addition to angiography. in a series of patients experience has been gained with the use of eeg, tc m isotope scanning, opg, ct scanning and doppler. in a year follow up over % of patients had a satisfactory outcome. an early mortality of % in the beginning of the series has been eliminated due to improved selection. in this report the application of multi-gated pulsed doppler techniques is reported. this allows a non invasive measurement of mean volume flow in the common carotid artery with a method reproducibility of + %. mean volume flow in undiseased arteries ( subjects mean age years) was found to be ± (s.d.) ml/min. from this a lower range for normal flow of ml per minute ( x s.d.) was selected. patients were investigated before surgery and a follow up examination was performed at a mean interval of v months post operatively. groups were defined. group a > ml/min; group b - ml/min; group c < ml/min, of arteries in group a before surgery, remained in the group and dropped to group b. in group b, of arteries, go to group a, remain and dropped to group c. in group c, out of arteries moved to group a and to group b. thus of arteries with below normal volume flow before surgery, were returned to normal range and further improved. remain unchanged and disimproved. of the arteries examined ;/ months after surgery, are in the normal range and a further improved. remain unchanged and disimproved. of the arteries examined / months after surgery, are in the normal range in flow values and a further remain unchanged. the non-invasive and isotope techniques have a valuable and practical application in assessment of carotid artery surgery. timing is influenced by the finding fo infarction on ct or isotope scanning. doppler techniques are useful not only in defining severity of diseas and sub-radiological plaques, but valuable flow information can be obtained by pulsed doppler pre and post operatively. this may help in identifying patients who need further medical or surgical treatment. stepwise logistic regression-amodel for predicing success of femorai-popliteal bypass grafts the objectives of this study were to identify the preoperative factors that influenced postoperative patency of femoral-popliteal grafts and to develop a model that could be used prospectively to determine the probability of successful outcome. data base material consisting of history, physical examination, laboratory data, angiography, and operative findings in patients undergoing femoral-popliteal bypass grafting was entered into a computer programmed for stepwise logistic regression analysis. the computer identified and ranked factors that influenced outcome. the top five factors (other than technical problems) included quantity of runoff, previous ipsilateral femoral-popliteal bypass, preoperative prediction of potential amputation level, concurrent proximal vascular reconstruction, and the location for distal graft anastomosis. having established the computer data base, it is now possible to enter information from new patients into the computer which will weigh all factors and indicate the likelihood of surgical success. in addition, tables can be generated which will look at simple combinations of variables to predict patency. for example, in a patient about to undergo a primary femoral-popliteal bypass with no anticipated technical problems, the likelihood of success as a function of runoff and preoperative amputation level is as follows: irreversibility of shock and ischemic injury is generally considered a consequence of extensive cellular injury. to study the role of intravascular coagulation in shock, rats were bled to a mean arterial pressure of mm hg for hrs or % uptake of shed blood, whichever occured first. return of shed blood with these data provide the patient and the surgeon with a quantitative prediction for success and permit an informed decision when considering therapeutic alternatives. potential cytoprotection by heparin was studied by similarly bleeding additional rats; controls were only cannulated. twenty pairs were heparinized (h); were not (nh). paired-bled and control rats were sacrificed following hemorrhage, liver (l) and kidney (k) mitochondria were isolated, and the isolates were studied by the polarographic technic with glutamate and succinate to determine the respiratory control index (rci) as a measure of cellular injury. results were: an equal volume of isotonic saline resulted in a % survival; % uptake of blood during shock allowed prediction of survival. an additional rats were then randomized (coin toss) to heparinization versus no heparin prior to shock, and were similarly bled and resuscitated. significantly improved survival (p< . ) was seen in heparinized ( / ; / ) versus nonheparinized rats ( / ; %). uncoupling and inhibition of mitochondrail function were noted in both h and nh rats with rci being significantly reduced from control. however, there was no difference between h and nh compared to each other. heparin does not provide cytoprotection during shock; improved survival with heparin may rather be a consequence of improved reperfusion of tissues following the shock episode. fl-endorphin (b-end) has been postulated to play a role in the pathogenesis of shock because the opiate "antagonist naloxone improves the macrohemodynamics in various shock models [ ] . however, plasma levels ofopioid peptides have not been determined as yet. the aime of our study was to measure the plasma concentrations of various peptides and to evaluate the influence of naloxone particularly on the plasma concentration of r-end. in anesthetized foxhounds, the adrenolumbar vein was cannulated and hemorrhagic shock (map = mm hg for h) was induced according to wiggerstechnique. the plasma levels offl-end, methioninenkephalin (m-enk), and leucine-enkephaline (l-enk) were simultaneously determined in c.v. and/ or adrenal venous blood by a specific ria. crossreactivity of r-end withfl-lipotropin was about %. the enk-antibodies crossreacted with less than %. five dogs received an i.v. bolus of naloxone ( mg/ kg) and a subsequent naloxone infusion of mg/kg/ h after h of hypovolemia. eleven dogs served as control and received equivalent volumes ( mg/kg per h) of ringer solution. hemorrhage resulted in a sharp rise of central venous plasma levels particularly of m-enk and l-enic this effect was even more pronounced in the adrenal's effluent system.fl-endorphin levels remain elevated whereas the enk secretion began to decrease h after hemorrhage. naloxone treatment inhibited any spontaneous fall of adrenal enkephalin release during the shock phase and the values remained elevated - fold. volume substitution with autologous blood resulted in a normalization of all peptide levels. these data demonstrate that hemorrhagic shock will cause stimulation of endogenous opioid peptides. the high levels of enkephalins in the adrenolumbar vein indicate that the adrenal gland is the main source of these peptides in the circulation. in addition toil-end, the enk seem to play a role in the pathogenesis of shock as well. at our present state of knowledge, however, it is difficult to design a coherent concept of mechanisms involved. this shows that cp treated cells bound nearly as much [ t]-acth analog as control cells but there was very little specific binding to sp treated cells. low concentrations of acth effectively displaced the acth analog whereas exposure of adrenocortical cells to sp resulted in a significant decrease in acth receptors. this suggests that sp has a factor(s) that binds to acth receptors of adrenocortical ceils which may adversely affect the stress response of shock. f- has been proposed as superior to other asanguinous fluids due to increased oxygen carrying capacity. evaluation to date has been largely uncontrolled and at extremes of hemodilution (hct. < %) rarely seen clinically. near infrared spectrophotometric monitoring of brain cytochrome a, a redox state, a sensitive indicator ofintramitochondrial oxygen availability, offers a unique opportunity to contrast f- with balanced salt-albumin (bsa) and whole blood saline (wbs) as a resuscitative regimen in a clinically relevant model. fifteen rats were subjected to minutes of hypoxia (fio = , %) and hemorrhagic hypotension (map = mmhc), then randomly allocated to one of three groups and resuscitated by fio = % and infusion of either f- , bsa, or wbs. cytochrome a, a redox state was monitored continuously at run. thirty additional rats were sacrificed at baseline, end shock and and minutes post resuscitation for cerebral cortical atp and lactate assay. despite hematocrits as low as % in the bsa and f- groups, there were no significant differences / < . between groups in the parameters of oxygen sufficiency; atp, lactate, and cytochrome a, a redox state. we assume differences in cardiac output compensated for differences in arterial oxygen content. on this basis we suggest perfluorochemical utilization should be limited to situations in which hematocrits are < % and when cardiac reserve is limited. metabolites of the prostaglandin endoperoxide h (pgh ) affect both vascular tone and platelet aggregation and thereby may influence blood flow. we, therefore, determined the metabolites formed from pgh by microsomes isolated from human saphenous vein used for aortocoronary bypass surgery. in the absence of reduced glutathione (gsh), the enzymatic metabolism of c-pgh produced only prostacyclin (pgi ) as measured by the formation of its stable breakdown product -keto-pgfla. the amount of pgi formed varied from - % of the substrate depending upon the microsomal protein and pgh concentrations. in addition, the nonenzymatic breakdown of pgh resulted in the formation of pgf a, pge , pgd and heptadecatrienoic acid (hht). there was no formation of thromboxane a (txa ) as measured by the absence of its stable breakdown product txb . in the presence ofgsh, a required cofactor for microsomal pge isomerase activity, the formation of pge was augmented fold (up to % of the substrate) indicating enzymatic for-mation of pge . the gsh either did not alter or augmented (less than fold) the formation of pgi . the increased formation of pge in the presence of gsh was at the expense of decreased nonenzymatic breakdown of pgh to pgf a, pgd and hht. these data suggest that prostacyclin synthetase activity may serve to protect the vessel graft from platelet aggregation and/or vessel spasms and may possibly serve as an indicator of graft viability. thrombosis is a frequent cause of early arterial bypass graft failure and platelets are known to be major determinants ofthrombus formation on arterial surfaces. pgi and flbriolytic activators from the vascular wall counteract intravascular thrombosis. the aim of this work was to study the effect of arterial grafting on the aforementioned mechanisms. cm lengths of tanned human umbilical vein grafts (huvg) with an internal diameter of mm were interposed end-to-end in the carotid arteries (c.a.) and jugular veins (j.v.) of sheep. placed in the c.a.'s, grafts with restricted flow ( cc/min) were removed on the th postoperative day (group i); grafts with unrestricted flow ( ___ cc/min after placement) were taken out days later (group ii) and grafts placed in the j.v.'s were removed days after surgery (group iii). upon removal, the grafts were checked for patency and sections from the proximal and distal anastomoses and midgraft were obtained for determination of pgi production (ria) fibrinolysis activators activity (histochemical method) and for light and scanning electron microscopy. sections from the femoral arteries were also obtained. the results of pgi generation are expressed in ng/ml/cm% all grafts showed fibrinolytic activity in the adventitia but grafts in group ii also showed fibrinolytic activity in the intima. early neointimal fibrous hyperplasia (nfh) characterized by proliferation of smooth muscle cells was present in group ii. the, occluded grafts showed organizing thrombus material and inflammatory cells and the patent ones showed fibrin and scattered inflammatory cells. in groups i and iii, sem revealed numerous platelets and rbc's incorporated into a proteinaceous material overlying the anastomoses and in some specimens obvious thrombus material was present. in group ii, the anastomotic areas were covered with large endothelial cells, nonetheless, some areas were denuded and small thrombi were occasionally noticed. in conclusion: . anastomotic sites create a strong stimulus for thrombus formation despite a high production of pgi . this suggests that antithrombotic therapy may be necessary to prevent early failures. . huvg develop the capacity to produce pgi and fibrinolytic activators and . although the etiology of nfh remains obscure, the decreased levels of pgi in group ii suggest that exhaustion of pgi generation from the endothelium might occur leading to proliferation of smooth muscle cells (nfh). these cells will in turn supply pgi if a persistent stimulus exists. permeability of intestinal capillaries to fibrinolytic products d. manwaring and p. william curreri department of surgery, university of south alabama college of medicine, usa fibrin/fibrinogen degradation product d (fdp-d) is significantly elevated in the serum of patients after trauma or sepsis. purified fdp-d infused into nontraumatized rabbits precipitates thrombocytopenia, complement depletion, pulmonary dysfunction and increased permeability of lung capillaries to i s-albu -composite fibrin plate assay size oflytic zones (mmx) after h incubation at °c rain. in order to determin of products of fibrinolysis alter fluid filtration or permeability, either purified fdp-d or fdp-e were tested in an isolated, autoperfused cat ileum preparation. steady-state lymphatic: plasma protein concentration ratio (cl/cp) and lymph flows (ql) were measured at a venous outflow pressure of mmhg. data was analyzed for each animal group by the paired student t test for ql, cl/cp and protein clearance (ql x cl/cp). in cats which received fdp-d (n= ), ql and clearance increased five-fold (p< . ), but cl/cpwas not altered, which suggests a permeability change. ileal mucosal biopsies prepared for histology had villi that were de-epithelialized and platelet clots in blood vessels. fdp-e (n= ) provoked a slight increase in ql (p< . ), but not in cl/cp or clearance. histology was normal. (fdp-e causes no pathological lung change in awake rabbits). fdp-d may contribute to various organ pathologies after trauma. the effect of aspirin on the fibrinolytic activity of viable granulocytes r.c. franz, w.j.c. goetzee, b. rotunno and r. anderson department of surgery, university of pretoria, rsa although several influential authors have suggested that low dose ( rag) acetyl salicylic acid (asa) represents a balanced daily antithrombotic regimen probably by both inactivating thromboxane a production and enhancing prostacyclin synthesis little is known about the effect of aspirin on the fibrinolytic activity of live granulocytes [ ] . the present study was designed to evaluate this effect in vivo. methods: granulocytes from fasting samples of heparinized venous blood taken from male volunteers were separated from monomuclear cells and platelets by density gradient centrifugation (ficoll: sodium metrizoate). viable granulocyte suspensions and plasma samples were placed as drops on a coin- before aspirin after aspirin p = [ ] . the experiment was repeated h after each subject had ingested . g of aspirin. the results are summarized in table . . there appears to be a significant increase in granulocyte fibrinolytic activity h after the ingestion of .sg of aspirin. . this increment is insufficient to overcome the resting inhibitor potential of plasma on granulocyte fibrinolysis. . aspirin does not evoke a significant increase in plasminogen activator-(urokinase) induced flbrinolysis in platelet free plasma or in the combined system of granulocyte-plasma mictures. . the optimal dosage of aspirin as a fibrinolytic agent requires further study. the terminal vascular bed of malignant tumors is characterized by a lack of organization, differentiation and sufficient developement of nutritional capillaries. as a result, malignant tumors reveal consistently small regions of low or even no perfusion. pre-vious data in a melanoma indicate that due to the rarefication of capillaries, the full impact of tumor treatment ±st diminished by an elevated microvascu lar resistance, which could significantly affect the impact of tumor therapy. since the improvement of the blood's fluidity has been shown as one therapeutic modality to increase significantly the capillary blood flow, it was assumed that this measure might enhance the accessibility of tumor tissue for bloodborne drugs. this study was aimed to investigate the effects of the improvement,of microcirculatory flow on tumor growth and tissue oxygenation. moreover, the response of the melanoma to chemotherapy was evaluated when isovolemic hemodilution was employed in conjunction with chemotherapy. a transparent chamber technique, intravital microscopy, a platinum multiwire electrode (local po measurement) and quantitative television image analysis (capillary blood cell velocity and diameter) were employed to study the microvasculature in the amelanotic melanoma a-mel- of hamsters in the event of hemodilution without and in conjunction with chemotherapy ( mg/m dtic, dimethyl-triazeno-imidazol-carboxamid). permanent indwelling catheters in carotid artery and jugular vein served for measuring systemicpressures, heart rate, for withdrawing blood and the infusion of dtic and/or dextran . after inoculation of x cells of the amelanotic hamster melanoma a-mel- into s.c. tissue in the preparation, this tumor re~ched a diameter of approx mm within five days. the reduction of systemic hematocrit from . to . ( . ± . ml blood vs dextran , animals) at a tumor diameter of mm increased the growth rate of the melanoma by about % while enhancing significantly the volume flow through capillaries and the mean local po . table control hemodilution capillary velocity (ram/s) . _ . . ± . capillary blood flow (ml/min x - ) . ± . . ± . mean local po (mmhg) . ( - ) . ( - ) the frequency distribution of local po on the tumor's surface showed a distinct shift toward higher po values with still some hypoxic regions remaining. intravital microscopy, however, revealed petechial bleeding and localized, interstitial edema which compressed a small number of capillaries. by contrast, the tumor's diameter remained at app. mm for a period of ten days with chemotherapy alone ( animals). in one of the animals, a complete stop in the melanoma microcirculation was seen within four hours after infusion of dtic followed by a significant decrease of tumor diameter. when chemotherapy was initiated in hemodiluted animals, neither retardation of tumor developement nor vascular obstruction was observed ( animals). conclusion: capillary blood flow of the melanoma can be enhanced by hemodilution thus diminishing tissue hypoxia. this measure, however, was associat-ed with an increase in melanoma diameter of %. at the present, we investigate whether, in hemodiluted animals, a reduction of tumor size can be obtained with a higher dose of dtic. ). however, the etiology of stress hyperglucagonemia in the immobilized rat is only poorly defined. since during restraint stress, catecholamines (ca) are elevated and stimulation ofglucagon by ca is accepted (woods sc d jr [ ] physiol rev. : ), we decided to study by surgical means the the relative contribution to glucagonemia of different sources of ca, i.e. peripheral sympathetic nervous system and adrenal medulla. methods: male sprague-dawley gastric fistula rats (n= ), weight approx. g, were subjected to either sham-op or various sympathectomies [microsurgical splanchnicotomy = s-sx; chemical sympathectomy = c-sx ( mg/kg -oh-dopamine ip two days prior to the experiment); adrenal demedullation = amx; combinations: s-sx + amx; c-sx + amx]. gastric acid secretory trials (duration h), preceded by a h fasting period were carried out - days following the operation. at the start of the experiment an intraperitoneal polyethylene tube, was inserted into the abdominal cavity of the rats, allowing a constant infusion of physiological saline ( ml/ kg/h). in addition, stress was performed by pairwise restraint of the extremities and small electric shock waves applied by a tail electrode. at the end of the experiment, blood was drawn from the vena portae and the abdominal aorta for plasma and serum. hormones (glucagon, insulin) were measured by radioimmunoassay, glucose enzymatically, volume was read to the next . ml, acidity by microtitration. results (see table) : volume and acidity are not changed by the various sympathectomies, when compared to the sham group. the same is true for acid secretion, except in s-sx + amx, where it is elevated. glucagon in peripheral plasma is elevated in c-sx, amx and c-sx + amx. in the portal vein, glucagon is dccreased in s-sx + amx ( ___ pg/ ml) and elevated in c-sx + amx ( ___ pg/ml) when compared with sham rats ( --. pg/ml). the portal/aortal glucagon ratio is significantly decreased only in c-sx and amx ( . --+ . , . --. . , resp.) when compared with sham ( . --_ . ). insulin is increased only in amx, glucose decreased in amx, s-sx + amx and c-sx + amx, insulin and glucose are unchanged in the other groups. conclusion: . stress hyperglucagonemia in the rat is confirmed (levels during zero stress - pg/ml) and also the rise in insulin following removal ofadrenomedullary ca (but not other sympathectomy); . the blood glucose fall (amx; s-sx + amx; c-sx + amx) is not uniformly paralleled by hyperglucagonemia, but in the case of amx it may be secondary to relative stress hypoglycemia owing to removal of adrenal medullary ca or reactive insulin release; . mechanism underlying the increased systemic glucagon despite partial (c-sx; amx) or total (c-sx + amx) sympathectomy are yet unknown. . during stress the enterogastrone component of hyperglucagonemia may be of minor importance. evlw showed good agreement with gravimetric lung water determinations. significant lung water accumulation was produced by pressure elevations over mmhg. reductions in plasma oncotic pressure significantly increased transvascular fluxes at each level of pressure elevation. however, fluid accumulation was not significantly greater during hypoproteinemia. we conclude that a - % reduction in plasma oncotic pressure does not contribute to increased high pressure edema because the lymphatic safety factor is augmented. this phenomenon may explain the well tolerated state of hypoproteinemia in patients after hemorrhagic shock. computerized gamma scintigraphy is a new technique for the analysis of albumin flux in the acute respiratory distress syndrome (ards). the objectives of this study were to obtain normal control values and to determine the method's validity in patients with cardiogenic vs. permeability pulmonary edema. methods: following mci mtechnetium-human serum albumin, lung :heart radioactivity ratios were determined. this ratio remains constant unless there is a leak o falbumin, when a rising ratio is seen, called the ,,slope index" (si). si's were determined in control individuals who had :> % left ventricular ej ection fraction and < s pulmonary circulation. thirtythree studies were obtained in patients using a portable gamma camera. fourteen patients had clinical evidence of ards. results: studies were considered positive if the si was s.d. > control mean ( - . ___ . x - units/ min). among positive studies, all had diffuse air space disease on chest radiographs. their average pulmonary capillary wedge pressure (pcwp) was . _+ . mmhg. the average artertial: alveolar oxygen tension ratio (a/a)o was . +-- . on . cm h peep, which were both significantly (p< . ) different from patients with normal si's. positive si's were present from hours to days following the apparent onset of ards in patients. recovery of gas exchange was associated with normal si's on repeat studies in patients. of patients with cardiogenic pulmonary edema, had negative studies ( - mmhg pcwp) and i a positive study ( mmhg pcwp). conclusion: gamma scintigraphy was a sensitive, non-invasive tool for the detection of a pathological increase in pulmonary protein flux, which was usually normal in cardiogenic pulmonary edema. positive scintigraphy was associated with significantly impaired gas exchange. the method documented that the leak of albumin in ards may last for days but resolves with recovery. cancellous bone thermocoagulation ph. dumontier, r. benichoux and a. vidrequin institut de recheres chirurgicales -c.h.u. de brabois vandoeuvre les nancy cedex, france electrocoagulation can not stop bleeding from the cancellous part of a sectioned bone. therefore we tested the efficiency ofthermocoagulation by hot air. the hot air generator delivers a flow, of non illtercd air, at - /min at a fixed temperature of °c measured at the exit of a mm diameter pipe and °c at the site of bleeding. the generator sustains usual steam sterilization. dogs were operated on both patellae, femoral short segments and iliac crests giving different site of cancellous bone. in vitro sterility studies: the conduit of hot air was applied at various distances, from cm to meters, above a petri plate containing culture material. thus the turbulence in the atmosphere around the zone of thermocoagulation has been bacteriologically controlled and a particle counter used. in vivo thermocoagulation: three sites of cancellous bone were used in anesthetized dogs, using a sterile procedure: the iliac crest, a small segment of the femoral bone and the divided patella. . five iliac crests were divided and bleeding measured after thermocoagulation. . the segmental femoral resections were thermocoagulated. . the patellae were vertically divided and each section submitted separately either to thermo or electrocoagulation. the pipe of thermo- coagulation was directed to the bleeding surface at a cm distane, sweeping it during to seconds. the bleeding was compared by photography and the two fragments were approximated by a wire synthesis to provide a bone fusion. in few cases both sides were thermocoagulated. results: in vitro: no contamination was found in the atmosphere, up to a distance of meters. there was a significant decrease of particles around the operating site ( % less, at cm and % less, at meters). in vivo: the bleeding was weighted around % less than with conventional coagulation. thermocoagulation did not delay or disturb the healing of the patella after wire synthesis. the in vitro nucleation time of cholesterol crystals from gallbladder bile of patients with gallstones is more rapid than that from normal persons, (holan rt [ ] gastroenterology : ). this study determined whether this was due to a gallbladder or liver defect and wether the defect was the addition of a nucleating factor or the deletion of an antinucleating factor. hepatic and gallbladder bile were gathered at surgery in stone patients and gallbladder bile in patients with a normal biliary tract. after ultracentrifugation, the isotropic phase was observed daily by polarizing microscopy until cholesterol crystals appeared. in gallstone patients, the nucleation time of gallbladder bile was significantly more rapid, . days+ . sem, than that of hepatic bile . + . days, although hepatic bile was significantly more saturated with cholesterol [cholesterol saturation index (csi), . _+ . ], than gallbladder bile, (csi, . _+ . ). thus the characteristic short nucleation time of stone formers is due to an alteration in bile after it enters the gallbladder. to determine whether the gallbladder defect was due to addition of a nucleating factor or the deletion of an antinucleating factor, isotropic phases of normal gallbladder bile and that from stone formers were mixed and nucleation time determined. mixtures of up to % normal bile had pathological nucleation times demonstrating that the defect is the addition of a nucleating factor by the gallbladder, and that this factor is potent. the rate of formation ofgaustone precursor crystals in bile, although faster in gallstone patients than in controls, is unrelated to the degree of cholesterol supersaturation [ ] , implying that other factors are involved. two competing factors seem likely; (a) secondary seed crystals in bile may trigger and accelerate gallstone crystal formation from supersaturated solution; (b) "poisons" in bile may retard or inhibit crystal growth. because of the complexity of bile itself, experiments were performed in highly purified mixtures of bile salt, lecithin and cholesterol, in concentrations closely resembling those of gallbladder bile. (a) lipid solutions were seeded with calcium carbonate, hydroxy-apatite, calcium bilirubinate and biliary mucus, all of which are found in gallstones [ ] . cholesterol crystal formation was significantly faster in_ the presence of all of the seed compounds tested (x= . /~g ml-lh- ) than in unseeded controls ( . /ag ml-~h- ) (/ < . ). (b) substances with "crystal-poisoning" properties included heparin, chondroitin sulphate and bile salt. these and changes in ph altered the quantitiy ( - % decrease) and rate of calcium carbonate and calcium phosphate crystal formation. we suggest that gallstone precursor crystal formation may be affected by a subtle balance between crystal seeding and crystal growth-inhibition, both due to the presence of other compounds in bile. at the last tripartite meeting we reported experimental data on a new method to destroy concrements of the kidney in situ by shockwaves allowing for spontaneous excretion via the urinary tract [ ] . the shockwaves are generated externally by underwater discharge of a condensor with sparking electrodes which are localized in a focus o fan elliptic cavity. for treatment the renal concrement must be positioned exactly in the second, virtual focus opposite to the elliptic cavity. since then, altogether patients were subjected to this form of treatment in our institute by the colleagues of the department of urology at the university of munich. % of the patients got rid of their concrements within a few days, in . % small remnants remained in the renal pelvis, and in . % ( cases) additional surgery became necessary. meanwhile this technique is employed on a routine basis in the department of urology of the university of munich [ ] . the experimental as well as clinical results were considered encouraging enough to extend the technique for the treatment of biliary concrements. for this purpose, human gallbladder concrements of different composition (bilirubin, cholesterol) were implanted into the gallbladder of dogs for exposure to shockwave treatment after wound healing. under in vitro-conditions the biliary concrements could be crushed into any size desired, irrespective of their composition, while only in out of experiments this was accomplished under in vivo-conditions. blockage of the biliaiy duct after shockwave exposure was never observed. concrements which were experimentally implanted into the bile duct could be visualized without difficulties by contrast medium. here, destruction by shockwaves was accomplished as well. currently experiments are conducted to dissolve remnants of biliary concrements after treatment by administration of desoxycholic acid. precise positioning of the gallbladder concrements in the second virtual focus is a problem which has not been satisfactorily solved so far, because the concrements cannot be visualized by conventional x-ray techniques. alternatively it is attempted to employ ultrasound, or visualization by retrograde injection of xray contrast medium through a catheter. in experimental animals, we leave a t-formed drain in the gallbladder for injection of contrast medium. in animal experiments conducted so far, histological or clinical evidence for tissue damage has not been obtained, as is the case with shockwave treatment of kidney stones. we are convinced that treatment of biliary concrements by shockwave exposure can be employed under clinical conditions in the near future. exploration of the common bile duct (cbd) for calculi, particularly in the presence of obstructive jaundice, is a procedure with considerable mortality and morbidity. to avoid the problem of retained stones, choledochoduodenostomy and transduodenal sphincteroplasty have been recommended, but have their own complications. this morbidity might be reduced by removal of cbd calculi prior to surgery. endoscopic sphincterotomy (es) allows this. a review of cases of es performed for calculi indicated that this was a safe (complications % no deaths) and reliable procedure ( % success rate). a study was conducted of patients with known cbd stones who had either preliminary es followed by operation at a later date (group i) or operation alone (group ii). this study showed a lower morbidity in group i. a prospective randomised controlled study has begun on the basis of these findings and the data from both studies are shown in the table. these results suggest that pre-operative endoscopic sphincterotomy my reduce the morbidity of cbd stones. group ii n = two controversies regarding the physiology of the biliary sphincter (bs) concern its functional independence from the duodenum [ ] and those aspects of its acitivity which control bile flow [ ] . the rabbit was chosen as the experimental animal as it has an easily identifiable sphincter. during anaesthesia induced by intravenous pentobarbital sodium, recordings of the electrical and mechanical activity of the bs and duodenum were made from (a) starved, (b) fed and (c) starved animals during administration of cholecystokinin, pentagastrin, secretin and glucagon. spike complexes (sc) were ordinarily associated with mechanical acitivity of the sphincter and duode-num. of . sphincter sc, recorded in animals, ( %) were not associated with duodenal acitivity, whereas . of ( . %) duodenal sc were accompanied by synchronous bs activity. this supports the hypothesis that the rabbit's bs can contract independently of the duodenum but that duodenal contraction is usually accompanied by simultaneous contraction of the bs. sphincter scs correspond to its phasic acitivity. food and cholecystokinin increased the number of sc without altering the baseline pressure of the perfused common bile duct. pentagastrin produced a transitory increase in sphincter activity whereas :secretin and glucagon were without effect. phasic activity of the spincter may influence bile flow through the choledochoduodenal junction. natural blood coagulation finally results in the formation of fibrin, which is one of the most important components of hemostasis in the human organism and thus provides the basis of all reparative procedures that are part of wound healing. it stands to reason to utilize the properties of fibrin for hemostasis during surgery and for joining severed tissue. first attempts of this kind were made at the beginning of this century. but only after greater insight had been gained into the coagulation proc- ess and the manufacturing techniques of blood derivatives had become more sophisticated, the essential breakthrough was made. a biological adhesive system has been developed, which consists of highly concentrated fibrinogen, thrombin and clotting factor xiii. this tissue sealant is completely resorbable and of high adhesive property. further advantages are elasticity of consistence and excellent tissue compatibility. after extensive animal experimentation, first clinical experience was made in . in the meantime the fibrin-adhesive-system (fas) has been introduced into numerous surgical disciplines with excellent results. the outstanding properties are: atraumatic tissue synthesis; enhancement of fibroblast proliferation and promotion of rapid wound healing; obtaining of local hemostasis by sealing bleeding surfaces, which is of special importance in the treatment of patients suffering from hemophilia or during operations under heparinization. the authors experience in using the fas within the last years is reported and a review over indications, techniques and advantages of this method is given. bile salts have been shown to enhance the stability and prolong the activity of intraluminal pancreatic enzymes and may therefore influence the effects of impaired exocrine secretion in patients with pancreatitis [ ] . individual bile salts in the peak min collection of duodenal fluid following cck/secretin administration have been quantitated by high performance liquid chromatography in patients without pancreatic or hepatic impairment (group c), patients with acute pancreatitis (group ap) and patients with chronic pancreatitis (group cp) all with functioning gallbladders, and patients with gallstone related acute pancreatitis (group gs). the peak total bile salt output in moles and the trihydroxy: dihydroxy (tri:di), primary:secondary (p:s) and glycine:taurine (g:t) bile salt ratios are shown below (mean___ sem). the duodenal aspirates contained detectable amounts of taurine and glycine conjugates of cholate, chenodeoxycholate, deoxycholate and ursodeoxycholate but not lithocholate or free bile salts. the low total bile salt output in groups cp and gs were due to decreased levels of all the individual bile salts. although the bile salt pattern in groups c and ap were similar, the relative proportions of trihydfoxy and secondary bile salts were higher in groups cp and gs respectively. these results indicate that patients with chronic pancreatitis without obvious large bile duct obstruction have an impaired bile salt output into the duodenum and this may exacerbate the effects of pancreatic exocrine insufficiency. an elevated amylase creatinine clearance ratio (accr) was considered a specific test for the diagnosis of acute pancreatitis (ap). however, it has been found elevated in other diseases as well as after surgery. the aim of this study was to evaluate prospectively the accr levels in patients with ap and in several groups of surgical patients. we studied subjects divided into groups: group a: acute pancreatitis (n= ). group b: patients undergoing biliary tract surgery (n= ). group c: peptid ulcer patients undergoing gastric surgery (n= ). group e: patients undergoing cardiac surgery under extracorporeal circulation (n = ). group f: control group of healthy subjects (n = ). the accr was determinated using the levitt method. in the surgical groups the accr was measured before and after the operation. amylase was determinated by the phadebas amylase test. ap was diagnosed on the basis of both clinical and radiological findings and the presence of high serum amylase levels. this diagnosis was confirmed through laparotomy in cases ( %). the accr was . + . % (mean___ sd) in group f, control group, and . + . % in group a, acute pancreatitits p< . . accr values below % were found in cases of acute pancreatitis ( %). among those patients whose ap diagnosis was confirmed through surgery the accr was . + . % (mean_+sd), higher than in the rest of the ap patients, . ± . % (p< , ). in groups b,c,d and e (surgical groups) the accr before the operation was . ± . %, . ± . %, . + . % and . + . % (mean___ sd), respectively. after the operation it was: . + . %, . + %, . ± . % and . + . %, respectively. on the average, we found an increase in the accr levels after the operation in the biliary tract group (p< . ), but not in the other surgical groups. in patients ( %), the accr after operation was above the upper limit of normal. none of these patients had symptoms compatible with clinical pancreatitis. in conclusion: . the accr increase in acute pancreatitis (sensitivity: o/ ) . the average accr increase after biliary tract surgery, but not after either gastric or thyroid surgery or after cardiac surgery under extracorporeal circulation. however, it is possible to find isolated cases with high accr after any type of surgery without any symptoms of pancreatitis, suggesting that an increase of accr may be an unspecific finding in postoperative patients which require further investigations. out of patients with acute pancreatitis developed a fulminant type. were males and females, mean age years (range - years). all patients were primarily treated by peritoneal lavage applied at laparotomy. indication for laparotomy was sudden deterioration with ( ) or without ( ) organ failure. a necrotic or hemorrhagic pancreas was found in every patient. the pancreas was exposed and soft and large catheters were placed close to the pancreas. mean duration of lavage (ll/h) was days. due to secondary deterioration a pancreas resection was performed - days later in nine patients. patients with acute fulminant pancreatitis died, a mortality of . %. all patients with the mild type of acute pancreatitis survived, thus the overall mortality was . %. none treated by peritoneal lavage only developed diabetes mellitus, whereas out of surviving patients with an additional pancreas resection had this complication. patients with acute fulminant pancreatitis displayed or more ranson criteria [ ] and six died. however, no less than of those with a mild type of acute pancreatitis fulfilled or more of these criteria and they all survived. a laparotomy -not a laboratory test -is necessary to confirm the diagnosis of acute fulminant pancreatitis. the indication for laparotomy is mainly clinical and therefore such a patient should preferably be handled by surgeons or physicians experienced in this disease. after confirmation of a correct diagnosis peritoneal lavage is one of the methods by which the mortality of acute fulminant pancreatits -which by conservative means is - % -can be reduced. coincidences ofhyperparathyroidism and pancreatitis have been given up by different author varying between % and %. frequently a causal relationship has been defended. recently, however, any causality has been queried [ ] . we analysed our own series of patients with surgically and histopathologically confirmed primary hyperparathyroidism (phpt) (n= ) and found a coincidence with a coexisting or prior pancreatitis of . % (n = ). from this a causal relationship cannot be concluded. however, out of these patients (i.e. . %) had an acute onset or exacerbation of a pancreatitis immediately following the parathyroidectomy, which is strikingly more than one would expect after an operation without any anatomical relation to the pancreas (< . %) [ ] . none of these patients had another cause of the pancreatitis such as cholelithiasis or alcohol abuse. hence a causal relationship cannot be excluded. it is imaginable that excessive amounts of parathyroid hormone are released during the surgical manipulation of the pathologically altered parathyroids. the postoperative pancreatitis may be caused by the acute elevation of parathyroid hormone levels in the presence of hypercalcemia. in our series the parathyroidectomy was combined with a partial or total thyroidectomy in patients. in another patients a thyroid operation was performed prior to the parathyroidectomy. although calcitonin has been employed as a possible therapy in patients with acute pancreatitis (ap), the normal levels of this substance in ap are not well documented and have not been correlated with pth. furthermore no definitive role in human calcium homeostasis is accepted for calcitonin, whereas high pth levels have previously been correlated with hypocalcaemia [ ] . in patients with ap the mean serum calcitonin on admission was ng/ , and the peak level mean lag/ (upper limit of normal ng/ ). calcitonin levels were higher in severe than mild ap, and of patients with levels > ng/ , were objectively graded as severe ap [ ] . in the hypocalcaemic patients, with corrected serum calcium < . mmol/ all recorded calcitonin levels > ng/ and had elevated pth. nine of the patients had significantly elevated pth ('> rig/l) and of these only did not have an associated elevation in calcitonin. the high calcitonin levels in patients with ap suggest that supplementary calcitonin intended to inhibit pancreatic secretion is unnecessary. at present it is merely speculative to suggest a role for calcitonin in ap but intriguing to report a tendency to parallel the elevations of pth. nuclide labelled microspheres were used to measure pancreatic and other visceral blood flow in two groups of conscious dogs before and after intravenous alcohol infusion. blood alcohol concentrations at the time of blood flow measurements were similar to those encountered in intoxicated humans. thus dogs (groups a) were given a somewhat low dose of alcohol to produce a mean blood alcohol level of . gm d - , while dogs (group b), received substantially greater doses of alcohol, and reached a mean blood alcohol of . gm dl- . wilcoxon matched pairs signed rank test confirmed a biphasic, concentration-related, response of pancreatic blood flow after alcohol infusion; no such response was found in blood flow to other viscera. moderate alcohol levels (group a) were associated with a decrease in pancreatic blood flow (p< . ), while high blood alcohol concentrations resulted in increased pancreatic blood flow (p'< . ). colonic blood flow increased in both groups a and b, but blood flow to the gallbladder, small intestine and the parotid gland increased in group b only. gastric, duodenal, renal, hepatic (arterial) and cerebral perfusion did not change. in addition, direct observations of the surface of the pancreas showed occasional haemorrhagic areas and mottling. these findlings however could not be confirmed by objective attempts to measure blood flow in such discrete areas in conclusion pancreatic blood flow shows a biphasic, concentration-related, response shortly after intoxication. this response appears to be peculiar to the pancreas and does not occur in other viscera. we recently showed that acute ethanol (e) and/or aspirin (a) ingestion increased the permeability of the pancreatic duct to large molecules, this suggested that pancreatic enzymes might leak from the duct into the parenchyma, causing pancreatic disease. this is a new concept in the study ot he pathophysiology of this organ (to be presented at aga, may, , plenary session). in the present experiments wie studied the effects of chronic e/a ingestion on pancreatic function in dogs. methods: dogs were fitted with duodenal and gastric cannulas. after recovery, baseline secretory sutdies were performed by cannulating the pancreatic duct and collecting pancreatic juice during secretin infusion ( . (submax) or . u/kg-hr. (maximal) iv). at least studies were performed in each dog at each dose. after baseline studies, dogs (group e) were given daily intragastric ethanol ( gm/kg-day). dogs (group a/e) were given e and a ( mg/kgday). after - weeks, secretory studies were repeated. results: all dogs gained weight (x = . kg). the pancreases appeared normal by light microscopy. drug treatment increased volume and hco output by and %, respectively, in group e, submax secretin, but decreased them by and % in group a/e animals. (p= . vs. predrug values and group e vs. a/e values). drug treatment decreased volume and hco output in both groups by - °/c (p= . ) after maximal secretin stimulus. (manova test for all statistical analyses). conclusions: consumption of e alone increased volume and hco output after submaximal and decreased them after a maximal secretin stimulus. this confirms the work of sarles. consumption of e and a reduced volume an i-ico output after secretin at both doses. thus chronic a ingestion further impaired pancreatic function in these animals. since only a small proportion of chronic alcoholics develop clinically significant pancreatic disease, an aggravating "cofactor" may be operating in this group. chronic asa ingestion, not uncommon in alcoholics, may represent such a cofactor. the development of diabetes mellitus in pancreatic cancer is well known but the standard oral glucose tolerance test is not recognised as a useful diagnostic indicator. glucose homeostasis, insulin and c-peptide secretion in response to intravenous glucagon were studied prospectively in patients with suspected pancreatic cancer and assessed as to their diagnostic value. fasting patients were given glucagon, m.g., i.v., and serial measurements of blood glucose, plasma insulin and c-peptide concentrations made for min. subsequently it was shown that patients had pancreatic cancer and the remainder constituted a control group. there was not significant difference in the rise in blood glucose between the groups after glucagon. the mean plasma insulin concentrations rose rapidly in both groups peaking between and rain but the values were sginificantly lower in the pancreatic cancer group (p< . at min: p< . at min: p< . at min). a similar pattern was observed with c-peptide. in patients with obstructive jaundice the plasma insulin response was a better discriminator of pancreatic cancer. we conclude that abnormal pancreatic beta cell funktion exists in patients with pancreatic carcinoma, detectable before any change in glucose homeostasis, particularly in patients with obstructive jaundice. the glucagon stimulation test may have a useful role in the diagnosis of pancreatic cancer. t-suppressor cells (t~) have previously been implicated as mediators of graft surival in baboons tolerant to their renal grafts [ ] . in addition, it seems that t-helper cells (th) are also affected by totallymphoid irradiation in that they are unable to provide help in mitogen-induced t-cell responses or pokeweed mitogen induced immunoglobulin synthesis in bcells. in this study the evolution of t~ and th is followed in baboons undergoing graft rejection at different rates. peripheral blood was collected from the animals at weekly intervals after renal transplantation, defibrinated and the lymphoid cells isolated by flotation on ficol hypaque. the t-lymphocyte fraction was purified by filtration through a column packed with nylon wool. th, ts and total t-cells were enumerated using monoclonal antibodies okt , and respectively (ortho). the th/ts rations reported were taken immediately before a rapid rise in serum creatinine occurred. in longlived baboons who maintained normal creatinine values, a mean of the ratios over the last month was used. b rats are produced by sublethal ( rad) x-irradiation of thymectomized animals, reconstituted with bone marrow from syngeneic, thymectomized, thoracic duct drained donors. in these lew rats, (lew x bn)f cardiac allografts survice indefinitely (> days); unmodified lew rats acutely reject such allografts ( -+ days). in this study, we have tried to restore the processes of acute rejection in b recipients. graft survival appeared independent of blocking factors or suppressor cells, as transfer of serum or lymphocytes from b recipients into syngeneic normal animals failed to increase survival of test allografts, placed subsequently. similarly, immunogenicity of long surviving grafts was unchanged; such grafts functioning > days and retransplanted into normal animals were rejected acutely. adoptive transfer of unseparated spleen cells (sl) from nonimmune syngeneic animals produced slow rejection ( --+ days) in b rats; sensitized slwas somewhat more effective ( _+ day). transfer of x syngeneic peritoneal exudate (pe) cells plus sensitized sl caused acute rejection in % orb recipients ( _ days), the remainder experiencing rejection at c weeks. pe cells harvested from rats injected ip with thioglycollate days previously, were primarily macrophages/adherent cells, as the cells used were that fraction sticking to plastic dishes and removed with lidocaine (purity > %). in vitro, b rat macrophages were abnormal, having only % of capacity of normal macrophages to promote production of interleukin (il ) when co-cultured with purified t lymphocytes. however, b recipients experienced acute graft rejection ( -+ days) after transfer of sensitized sl plus semipurified il , thus bypassing the above defect. addition ofll to the t cell (purity > °/ ) equivalent of sl (purified over degalan bead columns coated with rabbit antirat lgg, nonadherent fraction) failed to reestablish acute rejection ( -+ days), while further addition of b lymphocytes (degalan bead adherent fraction, purity > %) or macrophages was uninfluential ( ___ days and ___ days, respectively). transfer of il- alone never produced rejection. acute rejection can be re-established, however, by increasing the number oft lymphocytes ( , transferred concomitantly with il ). thus, the state of unresponsiveness in b rats can be reversed in vivo by adoptive transfer of particular cellular elements in the presence of growth factors; increased graft survival seems dependent ultimately upon il- production by sensitized t cells, presumably t helper cells. the relative inability of b rat macrophages to promote production of il- by t cells may be primarily responsible for the immunological deficit of the b rat. one of the most intriguing findings ofcyclosporin a (cya) immunosuppression is that in some species a short course of treatment will produce very prolonged allograft survival. we have tested the ability of cya to prolong the survival ofvascularized heart, kidney and pancreas allografts by direct comparison in a da (rt a) to lew (rt ~) rat allograft model. accessory abdominal heart and orthotopic left kidney transplantation were performed using standard microsurgical techniques. in renal transplantation the left kidney was removed at the time of transplantation, the remaining right kidney days thereaf- ter. streptozotocin-diabetic animals received ductligated pancreas whole organ grafts isolated on the portal vein and a segment of the aorta giving offthe coeliac axis and the superior mesenteric artery. rejection was taken as complete stop of palpable pulsations in heart transplantation, the day of death in renal transplantation and recurrance of hyperglycemia above mmol/ in pancreas transplantation, respectively. cyclosporin a mg/kg body weight, dissolved in olive oil, was administered intramuscularly for days starting with the day of transplantation. in all instances functional demonstration of rejection was confirmed by histological examination. cyclosporin a is effective to prolong the survival of vascularized heart, kidney and pancreas allografts. while cya is administered none of the grafts has been rejected. however, following withdrawal of the drug pancreas grafts are rejected within days and heart grafts within days. none of the kidney grafts has been rejected so far. the differential susceptibility of vascularized heart, kidney and pancreas allografts to cya immunosuppression may be caused by differences in immunogenicity due to organ specific alloantigens or a differential representation of spezialized antigen presenting cells. it may also reflect different patterns of rejection of the various organs. during cya administration all rejection processes are effectively suppressed. in the maintaince phase after withdrawal of cya such immune responses may prevail and ultimately lead to rejection of pancreas and to a lesser degree of heart allografts. the venous allograft still remains an attractive alternative for the reconstruction of small caliber vessels. however, when the venous graft is introduced to a non-histocompatible host, rejection and early occlusion is the rule. this study evaluates the use of cyclosporin a (cya) as a graft pretreatment, or systemic immunosuppressant for venous allografts. in addi-tion, cryopreservation techniques for pretreated venous allografts was investigated. adult mongrel dogs, weighing between and kg, were used as recipients for donor jugular vein segments ( - cm) which had been excised and flushed with cc of plasma protein fraction (ppf) at °c. these venous allografts were anastamosed end-to-end into a carotid artery of the recipients. the animals were divided into five groups as follows: group i (n= ) received untreated venous allografts without subsequent immunosuppression, group ii (n = ) was the same as group i with minimal immunosuppression (azathioprine . mg/kg/day). in group iii (n= ) the animals were transplanted with venous grafts stored in cc of plasma protein fraction (ppf) containing cy a ( mg/ ) at ° c for hours, immunosuppression was as in group ii. in group v (n= ) the animals received allografts that had been cryopreserved in a % dmso solution at - ° c for - days and then the animals had azathioprine as in group ii. in group v (n= ) venous allografts recipients were treated with systemic cya ( mg/kg/day x weeks, followed by mg/kg/day x weeks) as the only immunosuppression. the patency of the allografts was evaluated at , , , and weeks post transplantation. patency results at one month showed that azathioprine alone failed to improve the patency rate (gr. i and ii = % patency). cya graft pretreatment, however, significantly improved the one month patency (gr. iii = %). in addition, cryopreservation appeared to enhance the graft pretreatment effect of cya (gr. iv; one month patency = . %) of the allografts. finally, systemic cya proved very effective in preventing rejection and occlusion (gr. v; one month patency = %). grafts that remained patent for a initial critical period of - weeks, all showed long term patency. the effect of cya in preventing graft rejection was further documented by histiological studies of the allografts which showed a marked cellular infiltration and degenerative changes in all the grafts of the control group as compared to minimal or no cell infiltration in the patent grafts of the treatment groups. in summary, it appears that cya used as a graft pretreatment with minimal immunosuppression of the recipient, in conjunction with cryopreservation or given systemically as the sole immunosuppressant can significantly improve the survival of venous allografts. in our previous reports, it was shown that isolated hepatocytes transplanted into the splenic parenchyma of syngeneic rats, proliferated markedly and recomposed the hepatic tissue. this experimental system provided a new model to elucidate the mechanism of hepatic regeneration which could not be obtained in in vitro cell culture experiments. in the present paper, fetal hepatic tissue instead of isolated adult rat hepatocytes were transplanted into the rat spleen. we document briefly long-term morphological observations on the transplanted fetal hepatic tissue with special reference to proliferation of the hepatocytes and bile ducts. materials andmethods:wistar rats were mated in our laboratory for a fetal liver source. gestation day was when a plug or sperm were observed in the vaginal smear. fetuses used were of to days gestation. about ten fetal livers which were obtained from one maternal rat, were minced with scissors. the liver fragments were washed three times with saline solution. transplantation was carried out by direct injection into the spleens of syngeneic adult rats using a gauge needle. half of the liver fragments obtained from one maternal rat were innoculated into the spleen of one animal. a total of approximately rats with transplanted liver fragments were killed , , , and days and then every two to three months until one year after transplantation. the spleens removed were stained by h.e., pas and silver nitrate for histological examination. results: fetal livers exhibited no lobular architecture or hepatic cord structure. the very sparse cytoplasm of the hepatocytes and many hemopoetic cells among the hepatocytes were characteristically found only in the fetuses. one week after transplantation, the survived hepatocytes revealed almost the same morphological features as in fetal liver except for the presence of several proliferated bile ducts around the hepatocytes. two weeks later, the hepatocytes formed apparent hepatic cord structures and the extoplasm of each hepatocyte increased abunduntly and became acidophilic as seen in normal neonatal hepatocytes. hemopoetic cells disappeared. four weeks later, hepatocytes began to proliferate sporadically among the markedly proliferated bile ducts, groups of survived hepatocytes with cord structure were very similar to a neonatal liver except for the lack of the glisson's area. two or three months later, proliferation of the hepatocytes became prominent. there seemed to be no interrelationship between proliferated hepatocytes and bile ducts. one year after transplantation, a white nodule was observed on the spleen macroscopically and it consisted of numerous bile ducts and hepatocytes with or without cord structure on histology. summary: . fetal hepatocytes transplanted into the spleen, differentiated to almost normal neonatal hepatocytes two weeks after transplantation. . hepatocytes began to proliferate about weeks after transplantation. . three days after transplantation, proliferation of bile ducts was already observed independent of the transplanted hepatic tissue. . when comparing the difference in proliferation between fetal hepatic tissue and isolated hepatocyte transplantation, marked proliferation of the bile ducts in fetal hepatic tissue was observed and fetal hepatocytes proliferated more rapidly, while there were no proliferated bile ducts in isolated hepatocyte transplantation. pretransplant splenectomy (sx) has been of disputed benefit since its introduction two decades ago. of patients with first cadaver transplants treated at our institution between dec. and dec. have had pretransplant sx. at six monts, sx patients had % better kidney survival, but this benefit was lost shortly after year and by and years was % and % worse in sx patients. patient survival for sx and no sx was identical for the first year but was % and % worse by and years respectively in sx patients. thus, the early improvement in kidney survival was more than offset by a late high mortality. a rational basis for selecting patients who might benefit most from pretansplant splenectomy is urgently needed. since july, , patients ages - have received first cadaver transplants after having been tested for reactivity to dncb. nine of dncb negative patients had splenectomy as did of dncb positive patients. kidney survival at year for dncb negative patients without sx was %; for dncb negative with sx, %; for dncb positive without sx, %; for dncb positive with sx, %. rejection was the sole cause for kidneyloss in dncb positive patients without sx. however, of dncb negative patients with splenectomy died, primarily of septic complications. since survival of sx patients has been % compared to % in non sx patients (p< . ). sx appears to be beneficial in dncb positive patients but has an adverse effect in dncb negative patients because of an increased susceptibility to fatal infections. prior blood transfusion improves renal graft survival [ ] . plasma from uraemic patients suppresses the in vitro responses of normal lymphocytes to antigen (plasma suppressive activity, psa) and this effect is mainly attributable to the plasma protein macroglobulin (a m) [ ] . the aims of the present study were: a) to identify changes in psa and a m concentration in uraemic subjects following primary blood transfusion. b) to correlate the psa of transfused renal transplant recipients with subsequent graft survival. a) ten potential transplant recipients were studied before and after their first blood transfusion. following blood transfusions the psa increased significantly (p< . ) reaching a maximum at two months. there was no significant change in the plasma a m concentration over the same period. b) the plasma of consecutive chronic renal failure patients was tested for psa prior to renal transplantation and before institution of immunosuppressive therapy. all but two patients had received previous blood transfusions. after transplantation patients were followed for a minimum of months and a maximum of months. grafts failed for non-immunological reasons and were excluded from the study group. patients were divided into two groups according to the degree of suppressive activity of their plasma. a volume of /t , producing a % inhibition of normal lymphocytes was used as a treshold to differentiate those with a high or low suppressive activity. graft survival in the first three months was significantly better, % (/ < . ) for those recipients with a high psa as compared to % for those with a low psa. we conclude that blood transfusion causes a significant increase in psa although not a m concentration and that patients with high psa have a better graft survival. the effect of in vitro steroid on antibody dependent cellular cytotoxicity (adcc) was studied in patients awaiting renal allotransplantation and the results were correlated with transplant outcome. recipients of primary cadaveric allografts were classified as steroidsensitive or steroid-resistant from the degree of adcc suppression induced in vitro by methylprednisolone, patients being steroid-sensitive and steroid-resistant. following transplantation patients received azathioprine and prednisone, and rejection crises were treated with bolus doses of methyl-prednisolone. graft failure occured in of the steroid-sensitive patients, and in of the steroid-resistant patients. the observed one year graft survival rate was . % for the whole group, . % for the patients with steroid-sensitive adcc and . % for those with steroid-resistant adcc, the difference between the two groups being highly significant (xz= . ). a high incidence of early graft failure was seen in steroid-resistant adcc patients, . % of grafts being lost in the three months after transplantation, as compared with only of graft failures in the steroid-sensitive adcc group in the same period. analysis of hla-a, hla-b and hla-dr incompatibilities showed no significant difference between the groups, and since all patients had received deliberate pregraft blood transfusion, the difference in survival rates between the two groups appears to be independent of these two variables. these findings confirm our preliminary observation that pregraft assay of adcc response to in vitro steroids identifies those patients who are unlikely to respond to steroid therapy in the treatment of rejection, and in whom alternative forms of therapy may be appropriate. post-operative dxt, whilst not influencing survival, protected patients from loco-regional recurrence < . , hazard ratio (hr) = . ). interestingly it was found to be most effective against axiallary node recurrence (p< . , hr = . ), reasonably effective against chest wall recurrence (/ < . , hr= . ) but conferred no protection against supraclavicular node recurrence (hr = . ) in spite of a supraclavicular field being routinely employed in the radiotherapy technique. with such large numbers involved, this trial has facilitated the study of the prognostic significance of sub-groups of patients with different patterns oflocoregional recurrence as first evidence of treatment failure (see table) . of those patients developing loco-regional recurrence who have since died ( out of in wp group; out of in dxt group) % in the wp group and % in the dxt group did so with evidence of persistent loco-reglonal disease. however, the incidence of uncontrolled local disease at death was higher in the wp group overall. stress as well as dietary fatty acids have been shown to prolong allograft survival in rats [ ] . poly unsaturated fatty acids (linoleic acid, arachnoidic acid) have been reported to depress immune response [ ] . depressed immune response was suggested to correlate with a higher incidence of spontaneous tumor [ ] as well as with an increased growth rate of inoculated tumors [ ] . the objective of this study was to elucidate the effect of two environmental factors i.e. chronic stress (change in light/dark pattern) and diets low and high in linoleic acid on immune response and growth of transplantable tumors in bn rats. immune response: four experimental groups (n > ) were used in immune response studies. group i: high linoleic acid dietl; group i : low linoleic diet , group iii: l/d shift weekly, normal diet and group iv: controls on normal diet, normal lighting. seven weeks after the start of the experiment the immune response was measured. the results showed that corticosterone levels were slightly increased in all experimental groups, although only the high linoleic group showed statistic significant difference with the control group. cellular immune response (con a stimulation and popliteal node assay) was decreased in all experimental groups compaired to controls. transplantable tumors: x leukemia cells were injected i.v. and pieces of mm of an spontaneous adrenal cortical carcinoma, a urethral squamous cell carcinoma and a round cell cervix sarcoma were implanted subcutaneously. all tumors were inoculated in groups of animals each. spleen weight as a measure of leukemia growth was high in the control group and low in the experimental group. the same pattern was seen in the growth of the subcutaneously implanted adrenal cortical carcinomas. both the urethral squamous cell carcinoma and the round cell cervix sarcoma, being non-immunogenic, did not show any difference in growth. so far, it can be concluded, that the immunosuppression as induced by mild chronic stress or dietary fatty acids does not lead to enhanced tumor growth. in contrary, the results of both leukemia and adrenal cortical carcinoma show a possible reserve effect. little is known of the derivation or content of human breast cysts. recent reports have shown wide variations in the content of steroid hormones, particularly dehydroepiandrosterone sulphate (dhas) [ , ] . no explanation for this is apparent. to confirm the large variation in dhas concentrations and to further define the contents of cyst fluids, cysts from patients have been analysed for dhas, sodium and potassium. dhas concentrations ranged from . - pmol/ . both sodium and potassium content also varied widely (sodium - pmol/ and potassium - ~umol/ ). there was a significant direct correlation between the content of potassium and dhas in cyst fluid (p< . ) and a significant negative correlation with sodium content (/ < . ). three separate subpopulations of cysts could be identified according to the sodium and potassium content and these were, predominantly potassium cysts ( ), predominantly sodium cysts ( ) and mixed cationic cysts ( ). the median dhas concentration of the potassium cysts was pmol/ similar to the levels found in human breast secretions [ ]. in contrast the median concentration of dhas in the predominantly sodium cysts was pmol/ and significantly different (p< . ), with many of these cysts having dhas concentrations in the same range as those found in plasma. the remaining mixed cysts had a median dhas concentration intermediate between the two main groups. it may be that the variation in cationic content and dhas concentration in these two major subpopula-tions of human breast cysts represents either, derivation from two different sources, namely breast secretions and plasma or marked differences in the secretory activity of the epithelium lining these two groups of cysts. there is no uniform agreement on the correct management of patients with invasive lobular carcinoma (ilc). it is widely considered that in ilc there is an increased risk of developing a contra-lateral carcinoma and the major controversy surrounds the management of the second breast. the survival of patients with ilc was significantly better than that of idc fp<: . ). six patients had bilateral carcinomata at diagnosis and a further developed a contra-lateral carcinoma during the period of follow-up ( to years). survival data showed poor survival for patients with simultaneous bilateral disease, but no difference in survival for patients with metachronous bilateral or unilateral disease. this suggests that the later development of a second carcinoma does not necessarily reduce the probability of survival for patients with ilc. the major factor predicting patients at risk of developing a contralateral carcinoma was histologi- cal type. of patients with a particular histological pattern of ilc [ ] with a classical pattern of spread but showing nuclear pleomorphism and cellular cohesion, developed a contralateral carcinome, compared with a further in the remaining patients (p< . ). if bilateral mastectomy is justified it ought to be restricted to patients with this histological type of ilc. both the anti-oestrogen tamoxifen and cyclical combined chemotherapy will provide significant palliation in advanced breast cancer. the optimal use of these agents requires further evaluation and thus this trial was designed to compare a combination ofcytotoxic therapy and tamoxifen, against cytotoxics alone in patients with advanced breast cancer. post-menopausal patients presenting with metastatic breast cancer, locally advanced cancer extending beyond the breast and regional nodes, or with tumor recurrence following primary local treatment were allocated to the treatment arms via sequential manner. doxorubicin, cyclophosphamide, -fluouracil, and vincristine were given intravenously once every weeks. tamoxifen was prescribed in a dose of mg. b.d. on failure or relapse from one of the single modality arms, a crossover of those arms occurred. the combination consisted of both the above therapies. assessment of therapies was made in terms of objective response (uicc criteria), duration of response, and survival. we have previously reported that the combination results in a significantly greater response rate [ ] . as a result of stenosis reducing flow or by platelet embolisation [ ] . as neither aniography nor ultrasound can identify thrombotic activity we have evaluated gamma camera neck imaging using n indium platelets. labelled platelets on endarterectomy specimens were also measured and the activities found were then examined in a theoretical model. twentyfive patients with tia received rain platelets and sequential gamma images were interpreted by two observers, carotid endarterectomy in patients allowed measurement of specimen radioactivities. angiography and doppler spectral analysis [ ] were also performed. all endarterectomy specimens contained labelled platelet deposits with the most active equivalent to platelets from . ml of blood. this activity level was at the threshold of resolution in the theoretical model. both observers agreed that of the carotid bifurcations showed platelet accumulation on imaging. of the atheromatous ulcers demonstrated by angiography were visualised, but only of stenoses greater than per cent were detectable~ since ultrasound identified all stenoses only one angiographically diseased carotid was not detected by combining doppler and platelet imaging. diseased carotids accumulate rain platelets with the more thrombogenic ulcerated plaques identified more frequently than stenoses. long term follow-up is required to establish the clinical relevance of platelet deposition. major problem in vascular endoscopy is the existence of blood which prevents clear visualization. we devised a new technique using a combination of balloon catheter and slender fiberoptic endoscope, by which clear visualization was obtained experimentally and clinically. three to four pairs of orifices of intercostal arteries were also visualized in one visual field. in some dogs, acute aortic dissection was experimentally created by means of blanton's method. the entry, which was located at the descending aorta just distal to the left subclavian artery, was clearly identified. complete occlusion of blood flow and clear visualization could be obtained when balloon pressure exceeded systemic blood pressure. clinical study: in six patients requiring major vascular reconstruction of the aorta (abdominal aneurysm , leriche's syndrome , dissecting aneurysm ), vascular endoscopy was performed intraoperatively. in five patients, balloon catheter was introduced through the one of the limbs of y graft after proximal anastomosis. in each case, orifices of the major abdominal aortic branches were clearly observed. irregular orifices and atheromatous plaque of the aortic intima which were not expected from aortogram, were also identified in all patients. intimal tears by vascular claps were more extensive than expected and anastomotic suture lines were able to be checked from inside. in a case of dissecting aneurysm, balloon catheter was advanced through the mm graft which was sutured to the common femoral artery with finding the entry just above the left renal artery. using fiberoptic endoscope and balloon catheter was useful to observe orifices of the major aortic branches, unexpected intimal tears by vascular clamps and atheromatus plaques. it was particularly usbful to check the anastomotic suture line from inside of the aorta and to identify the exact location of the entry in dissecting aneurysm. vascular endoscopy could be one of the invaluable methods to examine, diagnose and treat the patients requiring aortic, caval and other major vascular surgery. ( ) produced endothelial injury and a local increase in shear stress in cynomolgus monkeys by suture plicating and constricting the aorta and then feeding an atherogenic diet for months. our findings reveal that carotid plaques localize on the outer wall of the internal carotid (plaque thickness . --+ . mm) which is an area of low flow velocity ( - ___ cm/s at re ) and shear stress ( -+ dynes/cm ) and not at the flow divider (thickness . ___ . mm, p< . ) which is an area of high flow velocity ( --- cm/s) and shear ( -+ dynes/cm ). distal to the carotid bulb, velocity and shear increased on the outer wall and little or no plaque was observed. in experimental coarctations, no endothelial damage was observed (sem and tem) within the high-shear coarct channel and the channel was noted to be free ofatherosclerotic plaque despite the development of extensive diet-induced lesions proximally and distally. thus, high flow velocity and shear stress do not appear to produce endothelial damage in vivo. in addition, plaques were minimal in high shear areas in the human carotid bifurcation and high shear appears to have an inhibitory effect on experimental plaque formation. these data contradict previous investigations implicating high shear stress in plaque pathogenesis. in contrast, host aortic endothelium (ae) fails to cover large vp by pannus ingrowth even over much longer times. to see if iaes succeeds because of inherent differences in growth potential between ae and ve, we used ae to seed cm x mm diameter dacron velour infrarenal vp in dogs. an average of x cells obtained by trypsin/collagenase digestion of the bypassed aortic segment was used to seed each vp by a step preclotting method. the identity of ae was confirmed by stains for factor viii antigen. viability of seeded ae was verified by growth of subaliquots in tissue culture. six weeks after surgery central segments of aeseeded (n = ) and control unseeded (n = ) vp were compared by light and scanning electron microscopy using an endothelial coverage score range of - (for fibrin/platelet thrombi) to + (for confluent endothelial coverage). ae-seeded vp had a score of+ . ___ . (mean___ sd) versus. - . ___ . for controls (p< . ). in addition to endothelial coverage, the subluminal smooth muscle and intramural vasa vasorum previously reported in ve-seeded vp were also seen in ae-seeded vp. since ae and ve seeding give identical results, the success of iaes with ve cannot be due to inherent biological differences in mitotic potential between ae and ve. iaes must instead achieve additional endothelial growth either through a) the action of the proteolytic enzymes used for cell harvest or b) mitogenic stimuli to nonconfluent cells at the edges of seeded cell clusters on the vp. further improvement of the efficiency of iaes to allow use of less harvested vein per cm of vp should come from enhancing one or both of these effects. pyrolytic carbon is a crystalline form of carbon that has been extensively used in the construction of cardiac and bone prostheses. since it has also been suggested that pyrolytic carbon will prevent thrombosis from occuring in vascular prostheses, the aim of the present study performed in dogs was to test the immediate blood compatibility of this material and to evaluate its biocompatibility when inserted as vascular substitute. after pryolysis of a gazeous hydrocarbon, the carbone crystalite was deposited on a knitted textile surface or tube. its surface examined by scanning electron microscopy (sem) was rough and porous to a depth of p. this material was tested °) for immediate hemocompatibility as inserts within the vascular lumen (aorta and inferior vena cava). the specimens were examined sequentially by sem and histology at , , , s and min after reestablishment of the blood flow, ° ) for long term biocompatibility as vascular cylinders ( mm id) inserted either in the aorta or inferior vena cava or as intraatrial (left or right) implants. patency of vascular cylinders was tested during postoperative month by doppler ultrasound investigations, specimens were examined by histology, electron microscopy (scanning transmission) at , and days following implantation. satellite lymph nodes were examined by histology. already s after establishement of the blood flow, platelet adhesion and limited fibrin mesh with few erythrocytes developed on the material. platelet aggregates of limited extent were only observed on intravenous implants. plasmatic protein deposition, an early event on polymeric vascular material was not observed. after s a fibrino-erythrocytic membrane recovers completely the material. except in the case of intravenous insert, no thrombosis developed at the contact of intraarterial or intracardiac implant. after days it was completely recovered by a - fibrocellular layer consisting of large myofibroblasts with microfilaments, newly synthetized collagen and elastin. the blood interface was of fibrous nature. at one month by sem, endotheliallike cells developed in a mosaic-like pattern, characterized by transmission e.m., by microvillous projections, numerous pinocytic vesicles and intercellular tight junctions. this endothelial-like cell lining was complete months after implantation. their immunocytochemical properties are now under investigation using specific anti-dog factor viii-rag sera. although preliminary, the present results suggest that among the numerous vascular biomaterials tested, pyrolytic carbon may represent a unique feature of rapid cell development and differentiation of endothelial lining at the blood material interface. department of connective tissue biology, institute of anatomy, university of aarhus, aarhus, denmark in the surgical clinic a significant number of patients report that their incision wound has burst, even though the scar appears to be intact. by mechanical testing of strips from skin wounds we have noticed a breaking pattern, in which the deepest layer of the wound ruptures earlier than the superficial part. therefore, we have investigated the strength and extensibility of rat skin wounds at different levels (superficial-deep) of the epidermis ( . mm), dermis ( . - . mm) and m. panniculus carneous ( . - . mm), average thickness is indicated. , and day old standardized skin wounds from the dorsal region of rats have been used. strips were punched out at right angle to the wound line and mounted in a materials testing machine. the strips were stretched until rupture and load-strain curves registered continuously. simultaneously, the strips were transluminated and the breaking pattern was studied by taking photographs of the wound specimens during the mechanical testing ( - photographs of each specimen). the photographs were marked on the load-strain curve by means of a connection between camera and x-y-recorder. from the load-strain curves the maximum load and the failure energy were calculated. the breaking patterns of , and day old wounds were found to be similar. the deep part of the wounds ruptured first. the force required to break the deep part was less than that required to break the superficial part of the wounds. the musculus panniculus carneous was very extensible and did not break. however, it possessed only minimal strength. quantitative measurements of the strength of the combined superficial-deep layers were performed on mm wound strips. specimens contained the superficial . , . and . mm of the wound area and were produced by cutting off the deep layer parallel to the skin surface. specimens containing the total wound area down to the musculus panniculus carneous were produced by cutting off the muscular tissue. these specimens were mechanically tested as described above. the present studies demonstrate the mechanical inhomogeneity of incision wounds. a new method for testing the mechanical properties of the tissue of incision wounds at various levels (superficial-deep) is presented. the superficial layer of an incision wound contributes a major part of the strength of the wound and is more extensible than the deep layers. these results may explain the clinical observations. the effect on wound healing of different kinds of vitamins is worth investigating, since the efficiency of vitamin c has been dearly demonstrated. the possible action of vitamin bs-whose trophic effect on skin is well known -has been experimentally studied on skin and aponeurosis healing after a standard laparotomy. materials and methods: experiments were carried out on five months old rabbits which were randomly divided into three groups: in group i, animals served as controls ( animals in sequence of days from the th to the th post-operative day), group ii, animals injected with vit b ( mg/kg of body weight/ h) and group iii, animals injected with a placebo ( animals in sequence of days for each group). in each case four samples were tested of skin and aponeurosis for determinating tensile strength, directly recorded with an original technic [ ] : this new apparatus allowed us to obtain simultaneously two dynamic parameters, the healing tensile strength and stretching of the scar. results: . no significant difference was found between controls (group i) and the placebo group (group iii) both for resistance of skin and the aponeurosis. . as far as vitamin b treated animals were concerned (group ii) there was no significant difference regarding skin resistance when compared with the other two groups. . inversely aponeurosis resistance become significantly greater when measured on the th (p< . ), th (/r< . ) and th (p< . ) post-operative day. in mongrel dogs ( x cm cranial based rectus abdominis) mc and corresponding rp flaps were raised. in group i ( dogs) skin bf was determined from the clearance curve for ~ xenon injected intradermally and measured with a computer-linked gamma camera. in group ii ( dogs) subcutaneous pro was determined by a recently developed method using a silastic tonometer, subcutaneously implanted. the pro inside the tonometer was measured in infused saline, by a platinum oxygen needle electrode and a silver/silver chloride reference electrode. b f and pto were measured before and after the flaps were raised and on postoperative days (pod) , , and . pto were taken at various inspiratoric oxygen levels (f~o ) ranging from % (air) to % oxygen. intact areas lateral to the flaps and in flap regions prior to surgery served as controls. immediately after surgery bf in the mc increased while in the rp flaps was %, % and % of the flow in the mc flaps, in lateral intact area and in the preoperative areas (p< . ). during pod - bf in the rp flaps increased to the preoperative level, but not to the increased levels found in the mc flaps and the lateral intact areas. by pod there were no differences in bf between the two types of flaps and the lateral areas, but all were higher than corresponding preoperative values (/'< . ). tissue oxygen tension showed a dramatic fall pod , and in the rp flaps for all fio , and for all days the values were lower than the preoperative level (p< . ). one rp developed pod distal necrosis and the pro was then even with a fio of %. the mc flap showed an increased pro on the operative day but at pod the values were slightly lower than the preoperative level, but pod , and the values for all fio were higher than for rp flaps (p< , ). at pod the pro reached preoperative level for rp as well as mc flaps. lateral intact areas showed comparable changes to that observed in the mc flaps. it is concluded that the mc flap demonstrates superior bf as well as pro when compared to the rp flap. early postoperative pro in the distal part of the rp flaps is critically low despite of increasing f~o to % and increasing bf. differences in bf and pto may be the biologic factors responsible for the superior healing characteristics of the mc flap. ( ) atp~adp + pi (inorganic phosphate) ( ) pcr + adp~atp the net result ofreaotjoxas and is a fall in pcr and a rise in pi while atp ~'emains relatively constant. all of the phosphorus metabolites are easily measured in gastrocnemiaas muscle using pnmr spectroscopy. normal volunteers and patients with angiographically documented arterial occlusions were studied in a / " bore oxford research systems tmr- spectrometer at rest and after exercising each limb separately. normal resting values ofpcr/p iwere > and the nmr index = p/(p~+pcr) was . _+ . (s.d.). limbs with femoral arterial occlusions whose ankle systolic pressure index was < . had nmr index which was significantly elevated above norreals ( . + . p< . ) indicating a failure of metabolic compensation for reduced bloodflow and oxygen delivery, although atp concentration was norreal. exercise produced a five-fold rise in nmr index in both normal and diseased legs. spectra were taken over one minute intervals during the recovery period and in normal limbs returned to resting values within rain. the recovery period was considerably slower in the diseased limbs indicating abnormal mitochondrial oxygen delivery and impaired mitochondrial formation of atp. these data demonstrate the feasibility of using pnmr to non-invasively probe the biochemical abnormalities of energy metabolism in patients with peripheral vascular disease. the incidence of urinary calculous disease (ucd) in the south african black population is very low in comparison with the white population group. no biochemical differences in serum nor urine account for this discrepancy and no other measurable parameters have demonstrated any difference between the two groups. urinary particulate activity measurements have demonstrated differences between normal persons and those with ucd who are otherwise biochemically similar, and it would therefore seem rational to expect such measurements to demonstrate differences between the two population groups. urinary particulate activity was measured in the urin of normal whites and normal blacks, the two groups being matched for age, height and weight, and monitored under normal dietary hydrational and environmental conditions. the three parameters of particulate nucleation, growth and aggregation were measured and the two groups compared. particulate nucleation demonstrated the most significant contrast between the two groups with the production of new particles through nucleation being far greater in the white group than that which occurred in the black group (p< . ). particulate growth occurred at similar rates in the two groups although at slightly higher rates in the white group. particulate aggregation occurred at a greater rate in the white group but the difference between the two groups was not statistically significant. the differences between the two groups are shown to occur as a consequence of differing rates of particulate nucleation although the rates of particulate growth and aggregation are parallel. whilst the factors responsible for the low nucleation rate in black person remain unknown their effect can now be measured quantitatively through the parameters of urinary particulate activity. blood levels of ketone bodies appear to determine skeletal muscle amino acid release; high levels conserve protein and attenuate gluconeogenesis. starvation indt/ced ketosis is suppressed by infection [ ] . to determine if the relative hypoketonaemia following sepsi s in turn contributes to increased glucogenesis, arterial substrates and glucose production (constant infusion - h(n)-glucose) were measured before and after infusion of na-dl-./ -hydroxybutyrate (/ oh) to raise levels three-to fourfold in fed (n= ), in fasted (n = ) and in fasted-infected (n= ) animals. in fasted-infected animals before infusion ketosis did not occur (/ oh . ± . mm/ fasted; . ± . fasted-infected) and basal glucose turnover was increased ( . ± . /tm/kg/min fasted; . ± . fastedinfected). with infusion of glucose and alanine concentrations decreased as expected in fed and fasted animals but not in fasted-infected (glucose . ± . ram/ befor; . + . mm/ after). glucose production also fell significantly in the fed ( . ± . /~m/kg/min before; . ± . after) and fasted ( . ± . v. . ± . ) groups but was unaffected by infusion in the fasted-infected group ( . +- . v. . + . ). the accelerated rate of gluconeogenesis in infection is thus not a consequence of hypoketonaemia. the usual reciprocal relationship between glucose and ketone utilisation during feeding and fasting has not been demonstrated~in sepsis. preliminary experiments in a hindlimb model support the hypothesis that during infection amino acid release from muscle is not affected by ketone levels. we have developed a technique for measuring the total body carbon of the living subject which is suitable for measuring the critically-ill as well as the ambulatory patient. by combining this measurement with that of total body nitrogen and calcium [ ] an estimate of total body fat is derived. measurement at the beginning and end of a given period enables the changes in total body protein and fat to be obtained, as well as the patient's energy expenditure if energy intake is also known. the method is a radiation technique. the supine patient is irradiated laterally with a horizontal beam of fast neutrons and the resulting gamma rays from the body are detected by a radiation detector placed unterneath the subject. the nuclear reaction employed is the inelastic scattering of fast neutrons by the carbon nuclei of the body with the emitted gamma rays having an energy of . mevi in the initial application, measures of total body fat obtained using the technique were compared with those derived from skinfold thicknesses in six volunteers: there was no significant differences between the two measurements, (see table) . the method is being employed in studying the changes in total body protein and fat, and the energy requirements of surgical patients receiving nutrition. in order to investigate the mechanism of this effect, normal monocytes were incubated at °c for min (with intralipid /a/ml) and their function assessed by three different techniques (chemotaxis, phagocytosis and chemiluminescence). all three methods showed impairment of function following exposure to intralipid. in order to try and prevent this potentially damaging effect, heparin was added to the various in vitro tests and found to cause marked impairment of phagocytosis. (p< . ) to assess its effect in vivo, volunteers were given , units of subcutaneous heparin h prior to intravenous intralipid (as above). although the use of heparin did not affect either immunological function, it completely prevented the fall of monocyte chemotaxis following intralipid alone. these findings suggest that monocyte function may be impaired by the presence ofintracellular lipid particles. the use of s.c. heparin may help to alleviate this problem and could, therefore, be beneficial to ill and often septic patients requiring intravenous nutrition. to investigate the effect of elevated glucocorticoids of stress and trauma on peripheral glutamine metabolism, . mg/kg bw dexamethasone was injected daily intramuscularely in adult mongroel dogs over a period of weeks. at least weeks prior to the experiments catheters were placed into the animal's abdominal aorta ( ) and caval vein ( ) in order to measure a-v differences and hindquarter blood flow. during dexamethasone treatment nitrogen balances were negative, - . - - g n per day, whereas slightly positive n-balances were observed during the control period ( . +__ . g n/day). muscle glutarnine concentrations declined constantly from . + . mmol/ intracellular water to . - - . by % within two weeks. whole blood arterial and venous plasma concentrations remained constant. to test the hypothesis of increased peripheral glutamine utilisation or decreased glutamine formation, the activities of glutaminase and glutamine synthetase were measured in a muscle homogenate obtained before and days after dexamethasone treatment. both enzyme activities were found to be unchanged. hindquarter glutamine efflux increased from . + . pmol/min in the control state to . + . during dexamethasone treatment indicating a fold muscle glutamine output. this increased glutamine output was enirely due to increased a-v differences and despite decreased hindquaarter blood flow during dexamethasone. it is concluded that dexamethasone reproduces the metabolic response of trauma and sepsis in terms of negative nitrogen balance and muscle glutamine depletion. muscle glutamine is shifted from peripheral tissues to visceral organs with muscle compensating for visceral demands rather than skeletal muscle being the primary target of corticoid action. it has been suggested that there is abnormal glucose utilisation in malnourished patients and that this may explain the adverse clinical sequelae of high rates of glucose infusion during intravenous feeding. we have investigated the hypothesis that there is a depression of the key enzymes of glucose oxidation in the muscle of malnourished patients which is due to an alteration of muscle fibre type proportions. malnourished patients (p) ( m, f + yrs) our results demonstrate that there is a positive correlation between preoperative cp and stage of cancer ' = . +- . x; r= . ;/ < . ). nevertheless before surgery there is no difference between cp values in the two groups considered (g.c.= . ___ . mg %;p.u. = . ---- . mg%), but after surgical trauma cp presents a positive response in patients with p.u. (mean increase + . %), whereas it acts as a negative ap protein in g.c. patients (mean decrease - / ) (p< . ). moreover malnourished g.c. patients present a reduction of cp values ( - %) which is greater than g.c. patients with albumin > . g% ( - . %); this difference is not statistically significant. cancer patients undergoing palliative (n= ) or radical surgical procedure (n= ) show parallel decrease of preoperative cp ( - %), the first group presenting higher preoperative values in relation to the tumor diffusion. in conclusion our results demonstrate that cp is not only a positive ap protein, but in some circumstances it may act as a negative pa protein depending on the underlying disease and the preoperative nutritional status. in this study the free aa concentration in liver tissue of non septic patients (cholecystectomy) were compared with those of septic patients (abdominal sepsis). the liver specimens were taken intraoperatively..the nature and possible risk involved in this study were explained to the patients and their consent obtained. the data presented in this abstract are part of a metabolic screening program of septic patients including the determination of aa (plasma, muscle), hormones (insulin, glucagon, cortisol), nutritional parameters (prealbumin, retinol-binding protein, transferrin), and of energy metabolism (atp, adp, glucose, free fatty acids). for the determination of the free aa the intra-and extracellular water content (chlorid method) and of fat content of the liver specimen were analysed. a membrane potential o f - mv was assumed. the aa analysis were performed with an automatic aa analyser (kontron, svitzerland) by means of an ionexchange resin (durrum dc- ) and a lithium buffer system (durrum-pico buffers). conclusions: . this study reveals decreased concentrations of nearly all aa in liver tissue of septic patients (exception: phenylalanine, tyrosine, cystathionine). . the significantly decreased concentrations of the gluconeogenetic aa (thr, ser, ala) indicate that the gluconeogenetic capacity of the liver is not exhausted through an increased uptake of those aa as shown earlier by wilmore et al. [ ] . an increased administration of gluconeogenetic and basic aa (lys, his) may normalise the aa pattern in the liver of septic patients. the liver is being increasingly recognized as a critical organ in postoperative multiple organ failure. the principle factors precipitating postoperative multiple organ failure were sepsis, hypotension and injury to the liver. previous studies from our laboratory have shown that hepatic failure, which has a high mortality rate, is linked to the marked decrease in energy charge. in order to evaluate the possible presence of metabolic blocks, the changes in the ratio of acetoacetate to fl-hydroxybutyrate (ketone body ratio), which reflects the hepatic mitochondrial redox potential, were analyzed in relation to energy charge in hepatectomized, jaundiced, hemorrhagic-shokked and septic animals, as well as patients with postoperative multiple organ failure. experimental: . in hepatectomized rabbits, mitochondrial phosphorylative activity increased to % of the control and the energy charge level decreased from the normal level of . to . at h after hepatectomy (/ < . ). afterward, these values returned to preoperative levels within a week. the ketone body ratio in arterial blood was positively correlated with hepatic energy charge (r= . , p~ . ). . in jaundiced rabbits, the hepatic energy charge decreased rapidly after the bile duct ligation along with the decrease of mitochondrial pbospborylative activity. the hepatic energy charge fell from . to . at h postoperatively with a maximum incidence of mortality. moreover, changes in the blood ketone body ratio were positively correlated with the hepatic energy charge (r= . ,/~ . ). the decrease in the blood ketone body ratio was attributed to the restricted mitochondrial reoxidation of nadh due to an inhibition of oxidative phosphorylation. . in hemorrhagic-shocked rabbit with a mean arterial blood pressure of mmhg, the changes in the blood ketone body ratio were correlated with hepatic energy charge (r= . , p< . ). . in septic pigs subjected to the ligation and perforation of the cecum, the hepatic energy charge level decreased gradually from . to . and the mitochondrial phosphorylative activity was enhanced to % of controls in the hyperdynamic state. in the hypodynamic state, the hepatic energy charge level fell drastically . concomitant with the decrease in mitochondrial phosphorylative activity and blood ketone body ratio. from these results, the blood ketone body ratio may be regarded as a reliable indicator for assessing the degree of decreased energy charge. clinical: changes in the blood ketone body ratio were measured in patients who underwent major surgery such as hepatectomy. these patients were classified into groups according to the postoperative changes in blood ketone body ratio: group a without decrease to below . , group b with transient decrease to . , group c with progressive decrease to below . and group d with terminal decrease to below . . all group a and b patients tolerated the operation well. by contrast, the group c patients showed multiple organ failure with % mortality rate, which involved pulmonary failure ( %), hepatic failure ( %), gastrointestinal bleeding ( %), renal failure ( / ), cerebral failure ( %) and coagulopathy ( %). all patients who transitioned to the terminal stage of group d died of cardiogenic decompensation. in patients of group c, the decreased blood ketone body ratio was restored with the amelioration of clinical symptoms after ex vivo pig or baboon liver crosshemodialysis and patients of them were later discharged. evidence presented indicates that the decreased blood ketone body ratio has a direct bearing on multiple organ failure. conclusion: sepsis, hypotension or injury to the liver are a metabolic burden to the liver mitochondria which can result in mitochondrial impairment leading to a marked decrease in hepatic energy charge. such impairment ultimately leads to multiple organ failure as a result of the critically decreased energy and substrate store and the reduced protein synthesis relative to demand in the various organs. in interferon-treated cells, the '- 'a synthetase, activated by double stranded rna, polymerizes atp into a series of oligonucleotides characterized by '- ' phosphodiester bonds and collectively designated '- 'a. these activate an endoribonuclease which cleaves rna. other regulatory functions of this enzyme may be expected because of its wide occurence in mammalian cells (untreated with interferon), where its activity appears, in vitro, to be dependent on the growth conditions, hormone responses regenerating liver after partial hepatectomy is often used as a model for the study of control growth and cell proliferation in vivo. in order to evaluate the role of the '- 'a synthetase in the processes leading to initiation of cell division, we measured this enzymatic activity in the rat liver during the first h after partial hepatectomy. partial hepatectomy ( rats) was performed under neuroleptanalgesia by removing the median and the left lateral lobes of the liver according to the method of higgins and anderson. control animals ( rats) were subjected to a sham operation. after selected time intervals, the animals were sacrificed and the enzymatic activity in the regenerated liver was measured. the '- 'a synthetase activity present in the two first removed lobes was defined as %. we observe a very rapid decrease of enzymatic activity which reaches % already h after partial hepatectomy. the lower level of enzymatic activity ( %) is measured between and h after partial hepatectomy. this minimum is followed by a slow restoration of the activity (at h: %). during this early phase of liver regeneration, a maximal incorporation of tritiated thymidine in dna takes place h after surgery. so well differentiated liver cells have elevated levels of '- 'a synthetase. but, after partial hepatec-tomy, the '- 'a synthetase activity decreases dramatically before the first wave of cell mitosis. these observations clearly illustrate the relationship between '- 'a synthetase activity and the growth status. moreover, this drop of enzymatic activity may be a trigger for the initiation of cell division. the primary event or events setting in motion the process of liver regeneration after partial hepatectomy (ph) remain unsettled. regarding the so called hepatotrophic factors, i.e. insulin, glucagon and recently egf, present evidence suggests that they would play mainly a promoting rather than a initiating role. early changes such as glycogen breakdown, fat infiltration and changes in adenine nucleotides and mitochondrial phosphorylative activity are usually, at least the first two, ascribed to metabolic overload of the remaining liver (bucher et al ( ) johns hopkins med, j, : ). so far, however, little attention has been paid to a possible involvement of this phenomenon in the initiation of liver regeneration. attempts were therefore made to modify metabolic overload through early changes in energy metabolism in order to study their influence on the pattern of dna synthesis. fed or h fffsting male wistar rats weighing + g were examined after ph at , , , , and h for adenine nucleotides, oxidative phosphorylation and dna synthesis, based on the rate of~h thymidine incorporation. fasting animals received continuous infusion of % dextrose for h at the rate of . ml/ g/h. in addition to the above mentionned parameters hepatic glycogen and fatty acids were measured. within h partial hepatectomy caused a decrease in hepatic atp which was maximal at h (from . ___ . to . + . /~ moles/g p< . ); in energy charge (atp + . adp/atp + adp + amp) from . -+ . to . ___ . (p< . ) and increase in phosphorylation potential which was maximal at h (from + to _ p< . ). dna synthesis began at h reaching a peak by h. glucose infusion to ph rats suppressed the decrease of hepatic atp, . ___ . / mole/g at h vs . _ . in the control group, prevented glycogen depletion (histochemical estimation) and the increase in fatty acids (two folds increase in ffa and triglycerides (tg) at h vs folds in ffa and folds in tg in the control group),with little effect on mitochondrial activity. the initiation of dna synthesis was delayed and the whole pattern was considerably modified. cessation of glucose infusion restored the usual rate of h thymidine incorporation after a late fall of hepatic atp. in conclusion, glucose infusion was shown by one of us to modify the hormonal response to ph, but insulin and glucagon administration to glucose treated animals failed to normalize the pattern of dna synthesis. it is suggested that metabolic overload as estimated on the basis of early changes in energy matabolism may account for one of the events involved in the initiation of dna synthesis after ph. infusion on liver cell regeneration after partial hepatectomy in the rat b. de hemptinne, j.f. ngala and l. lambotte university of louvain, laboratory of experimental surgery ucl , brussels, belgium after partial hepatectomy (ph) the portal and the peripheral blood serum concentration of immunoreactive insulin suffers a drastic fall and levels ofglucagon show a rapid increase which is maximal h after the liver resection. as these changes appear closely correlated to the blood glucose levels which show a % decrease at h and progressive restoration towards normal values up to h, attempts have been made to alter the insulin/glucagon ratio by glucose infusion after ph and study its relation to liver regeneration. the purpose of this work is thus to determine after ph and hypertonic ( %) glucose infusion: . the effect of glucose on insulin and glucagon blood levels over h; . the repercussion of the insulin/glucagon modified ratio on dna synthesis; . the possible improvement of dna synthesis by extensive glucagon infusion. male fisher rats underwent a standard % ph. a % glucose solution or isotonic salin was infused at a constant rate of . ml/h through a cannula placed in the iliac vein. the rats were sacrified at , , , , and h. ( h) thymidyne ( .. /~ci/mmol) was injected h prior to sacrifice and the liver caudate lobes removed for analysis of ( h) thymidine uptake into nuclear dna. blood samples were withdrawn from the portal vein, the inferior vena cava and the aorta for glucose, insulin and glucagon assays. compared to the salin treated group, the infusion of glucose while keeping a normal steady blood glycemia was responsible of a marked increase of insulin ( . --+ . ng vs . _+ . ng,/ < . ) and decrease of glucagon ( . -+ . ng vs . + . ng, p< . ), with a major switch of the insulin/glucagon ratio at h after ph (from . to . ). dna synthesis started at h in both series, but was very significantly impaired at h in the glucose infused group ( -+ cpm/mg dna vs + cpm/mg dna, p< . ). infusion of increasing doses of glucagon (from . to mg/kg/day could not restore the impaired dna synthesis. only a slight improvement was recorded at . mg/kg/day as the insulin/glucagon ratio tended to approach that of the control group. fractionation of various doses of glucagon over the h perfusion time, in such a way that the changes in concentration of glucagon after partial hepatectomy was imitated, remained unsuccessful to improve thymidine incorporation. in conclusion: . the infusion of hypertonic glucose which impairs dna synthesis after ph, modifies markedly the insulin and glucagon secretion. . if a specific insulin/glucagon ratio after ph is important to sustain normal regeneration, its modification does not seem to be the major factor contributing to the blunted dna synthesis response in the hypertonic glucose infusion model. operative mortality of emergency shunt operations or esophageal transection during acute hemorrhage from ruptured esophageal varices in cirrhotic patients (child's category c) has been intolerably high. hence, the emergency operations in these patients should be avoided when the bleeding could be stopped by non-operative measures. when the emergency operation eventually becomes inevitable, the operative procedures should be simple and of short duration. in , we have introduced the endoscopic balloon tamponade method for the management of esophageal variceal bleeding. the new balloon tube used in this method has essentially the similar structure as the sengstaken-blakemore tube, but is made of translucent plastic materials, and has a larger internal diameter so that a small caliber optic fiberscope (ex. bronchofiberscope) can be passed through the tube. by this method, the endoscopic observation of the esophagus is possible through the translucent balloon tube during tamponade of the ruptured varices. it is possible to know directly whether the bleeding from varices has been successfully stopped or not, which seems to be an advantage over the blind tamponade method using the sengstaken-blakemore tube. this endoscopic tamponade method has been used for emergency hemostasis of acute bleeding from ruptured esophageal varices in patients. the mean initial tamponade pressure by the esophageal balloon necessary to stop bleeding was _+ mrnhg and the average duration of tamponade was h. the location of the ruptured v~ices observed by this method was in the lower esophagus within cm of the esophagocardiac junction in % of the cases. the bleeding has been stopped in occasions ( . %) by this method. no significant complications other than atelectasis in patients have been observed. in patients, the bleeding was initially stopped by the new translucent balloon tube, but recurred within h after decompression of the esophageal balloon. tamponade was repeated for several times since these patients were in the category c of the child's classification, however, the emergency operation eventually became necessary. transthoracic esophageal transection was performed using autosu-ture apparatus, eea, in these patients. one patient died of liver failure in the th postoperative day, but others survived the operation and recovered. the use of eea in transthoracic esophageal transection simplifies the esophageal anastomosis, and shortens the duration of operation by rain. we have so far used the autosuture apparatus, eea ( mm cartridge) in elective esophageal transection of patients. neither anastomotic bleeding or insufficiency has been encountered. acute variceal bleeding in category c patients should be treated initially by endoscopic balloon tamponade, when emergency operation is inevitable, transthoracic esophageal transection using eea is the operation of choice. arterialisation of the portal vein in conjunction with an end-to-side portacaval shunt has been proposed as a method of improving survival following shunting [ ] . however, there is little experimental evidence to support this suggestion and so we have examined the effects of arterialisation of the portal stump in cirrhotic rats. rats with dimenthylnitrosamine-induced cirrhosis were used in this study. of the rats received an end-to-side portacaval shunt, had a portacaval shunt and the portal stump arterialised with the left gastric artery, eight were sham-operated. liver blood flow (lbf) and wedged hepatic venous pressure (whvp) were measured before and after shunting. three weeks after surgery lbf and whvp measurements were repeated, the animals bled and the liver removed and weighed. an end-to-side portacaval shunt led to an immediate fall in lbf ( . _+ . to . _+ . mls/min/ g-~) and whvp ( . _+ . to . -+ . mmhg). however, if the portal stump was arterialised lbf ( . -+ . to . + . ) and whvp ( . _+ . to . _+ . ) did not change significantly. no further changes in lbf and whvp were observed three weeks after operation in any group of animals. arterialisation of the portal stump prevented the loss in body weight, loss in liver weight deterioration of liver function tests and hyperammonemia observed in animals with a portacaval shunt alone. these findings suggest that arterialisation of the portal stump may prevent some of the deleterious effects after shunting. departments of surgery and pathology, university of virginia hospital, charlottesville, virginia , usa we have hadexperience with operative restoration ofhepatopedal portal blood flow in five patients intolerant of total splanchnic shunting. hepatopedal flow was reestablished by takedown of the total shunt and construction of a selective, distal splenorenal shunt, or by isolation and arterialization of the hepatic limb of the shunted portal vein. in two patients, shunt revision was undertaken electively for chronic encephalopathy, unresponsive to low protein diet, intestinal antibiosis and oral lactulose. each individual had been hospitalized more than eight times for encephalopathy or coma. nine and months postoperatively, both patients have had no encephalopathy on unrestricted protein intake, and work. actively as homemakers. serial liver needle biopsies have shown bilobed nuclei and enhanced mitotic activity suggesting hepatocyte regeneration. in three patients, shunt conversion or arterialization was undertaken in desperate circumstances, characterized by liver failure (bilirubin > mg/dl, albumin < . girl, prothrombin time > " s)~ coma and respirator dependency. although two patients showed immediate, marked improvement in mentation, all three died of intraabdominal hemorrhage in the first few postoperative days in spite of prolonged attempts to achieve hemostasis and maximum blood product support. three conclusions can be drawn from this limited experience: . total shunt procedures which are anatomically suitable for subsequent conversion to a selective configuration or for hepatic limb arterialization should be favored over those not offering such potential; . at a time of election, restoration of hepatopedal portal flow can be accomplished in patients with side-to-side portacaval or hemodynamically equivalent shunts with considerable benefit; and . similar procedures in patients with fulminant liver failure are unlikely to succeed. peritoneal-venous (leveen) shunts are associated with a significant incidence of disseminated intravascular coagulation (d.i.c.). this study identifies a site of origin, a pathogenic mechanism, and the hemostatic pathway which accounts for the thrombogenicity of human ascites. ml of peritoneal fluid were removed percutaneously from individuals with malignant and cirrhotic ascites. ficoll-hypaque column chromatography and ultracentrifugation were utilized to prepare four fractions: cellular; a low speed cell-free fluid; a high speed supernatant; and the precipitate from the high speed centrifugation. the cellular fraction from both types demonstrated an ability to shorten a one stage clotting time by % relative to saline and endotoxin controls. similarly, low speed cell-free fluid shortened the clotting time of pooled normal plasma by %; was also effective in factor viii (required for intrinsic pathway of coagulation) deficient plasma; but had no effect on factor vii (required for extrinsic pathway of coagulation) deficient plasma or platelet aggregation and release. the high speed supernatant was demonstrably less thrombogenic. the resuspended precipitate shortened the clotting time of pooled normal plasman by % and of factor viii deficient plasma from infinity to s. in contrast, this material was ineffective on factor vii deficient plasma. we conclude that the thrombotic potential of human ascites derives from peritoneal cells, either leucocytes or malignant cells. consistently, the thrombotic factor exists in suspension and is thromboplastin-like in its behavior, operating through the extrinsic pathway of coagulation. thus, a site of origin and a pathway of activity for the thrombotic agent in human ascites is identified. the effect of somatostatin in hepatic haemodynamics in the cirrhotic rat s. jenkins, p. devitt and r. shields department of surgery, university of liverpool, liverpool, u.k. although somatostatin has been suggested as an alternative treatment to vasopressin in the emergency control ofbleeding oesophageal varices [ ] , recent studies on its effect on wedged hepatic venous pressure in cirrhotic patients have provided conflicting results [ , ] . therefore we have examined the effect of somatostatin on hepatic heamodynamics in cirrhotic rats. rats with dimethylnitrosamine-induced cirrhosis received a bolus injection of , or pg/kg body weight of somatostatin followed by a min infusion of either , or pg/h/kg body weight. control rats received saline only. portal venous flow (pvp) portal pressure (pp), liver blood flow (lbf) and wedged hepatic venous pressure (whvp) were measured before, during and after somatostatin infusion. at the lowest rate of somatostatin administration (bolus injection of pg/kg body weight followed by an infusion of pg/h/kg body weight)there was a rapid decrease in pp ( . + . to . --+ . mmhg), whvp ( . ___ . to . --- . mmhg) and pvf ( . ___ . to . ___ . ml/min). rain after the start of the infusion pp, whvp and pvf were still signaflcantly lower than preinfusion levels. at higher rates of somatostatin infusion there was no furhter decrease in pp, whvp and pvf. lbf was significantly reduced at all the doses of somatostatin. the highest rate of infusion of somatostatin ( pg) produced a significantly greater reduction in lbf than either or pg. these data suggest that somatostatin may have a role in the management of portal hypertension, but that higher doses may have a detrimental effect on lbf. er positive breast cancers preferentially metastasize to bone ( ) prostaglandin e (pgez) is synthesized by breast cancers and potentiates bone resorption in vitro ( ) . this study investigates the relationship between er status and pge synthesis in breast cancer cells. pge was measured by radioimmunoassay in primary breast cancers: a) after ethanol inhibition of further prostaglandin (pg) production -,,basal pg" b) after stimulating pg production with excess arachidonic acid -,,total pg". ,,total" minus ,,basal" = ,,synthesized pg". in order to relate pg production to breast cancer cells, measured pge values have been corrected for the epithelial cellularity of each tumour. pge x corrected pge = actual (%) cellularity cellularity was evaluated by a proportional count of cancer cells expressed as a percentage against non cellular material in all fields of - histological sections at x magnification. we conclude that er positive tumour cells synthesize greater amonts pge than er negative cells. this may account for the greater tendency of er positive cancers to recur in bone. oestrogen receptor activity (er) is of prognostic value in breast cancer [ ] . however, the chosen cutoff between er-negative and -positive is arbitrary and likely to affect its prognostic value. in patients with invasive breast cancer treated by mastectomy, tumour er was determined [ ] and the patients were followed up until first recurrence. using a computer program to perform repeated logrank analysis, the effect of varying the cut-off on prognostic value of erwas studied between and fmol/mg protein. er levels were higher in postmenopausal patients ( - , median , n= ) than in pre-menopausal patients ( - , median , n= ). for the whole group, optimal prognostic discrimination was achieved with a cut-off fmol/mg protein. in premenopausal patients, the effect of varying cut-off was complex, leading to an optimum of fmol/mg protein, whilst in post-menopausal patients the optimum was fmol/mg protein. these findings were unexpected and may relate to the relationship of er to tumour cellularity [ ] . it is concluded that: . the failure of some centres to relate er to prognosis may be due to the inappropriate cut-off point chosen; . in our patients, the optimal cut-off was fmol, which agrees with the level suggested by the british breast group [ ], though it differed between pre-and post-menopausal patients; . optimal cut-off for prognosis may differ from that for predicting response to endocrine therapy. previous studies have reported that the use of adjuvant chemotherapy improves relapse free survival following mastectomy. the effect on overall survival is less certain. patients with histologically confirmed axillary node metastases were randomised to receive postoperative radiotherapy (rt), chemoterapy (cmf) or radiotherapy plus chemotherapy (rt + cmf). patients have now been followed for between months and years. patients initially treated with rt received chemotherapy on failure where possible. of patients receiving rt alone, have developed distant recurrence and have died. of receiving cmf alone have developed distant recurrence and have died (see table) . although the use of adjuvant chemotherapy significantly prolongs the relaps free interval there is no corresponding improvement in overall survival. a sample of patients with histologically proven prostatic carcinoma underwent transrectal find needle aspiration at six month intervals, for a mean follow-up of months, as an out-patient procedure, without significant side effects. patients were followed from time of initial diagnosis. the others had been on treatment for a mean period of months before the study commenced. in of the new patients, cytology was positive at the time of histological diagnosis, and correlated with the histological grade. there were no false positives. repeat cytology on patients after months showed positive and in of these the disease was progressing, and in one it was stable. of the who were negative, were stable and one progressing. of the patients already on therapy had positive cytology at their initial aspiration - showing progressive disease and being stable. patients whose cytology was initially negative became positive months later and all had disease progression at this time_ patients had negative cytology on or more occasions and in only cases was there evidence of disease progression. cytology showed evidence of squamous metaplasia on follow-up in of the patients whose condition was stable. the prognosis for all patients was assessed on the basis of gleeson's score and aspiration biopsy has been shown to be a safe and useful procedure for monitoring disease activity and response to treatment. the optimum method of restoring the ability to swallow in patients with unresectable carcinoma of the oesophagus remains controversial. this study evaluates the palliative potential of pulsion intubation versus retrostemal gastric bypass of the excluded oesophagus in unresectable carcinoma of the upper thoracic segment ( - cm from the incisor teeth). patients and methods: patients were prospectively randomised for treatment by pulsion intubation ( patients) or gastric bypass ( patients). non-resectability was indicated by ( ) tumour lengths greater than cm, ( ) tracheal or bronchial invasion, ( ) disrant dissemination, ( ) mediastinal invasion. the operative mortality, morbidity, palliation of dysphagia and postoperative nutritional status was compared in the two groups. results: mortality: intubation resulted in deaths, a mortality rate of , %. gastric bypass resulted in deaths, a mortality rate of , %. complications: intubation was complicated by respiratory infection ( ), tube migration ( ), respiratory obstruction ( ), oesophageal perforation ( ), bleeding ( ). gastric bypass was complicated by chest infection ( ), pneumothorax ( ), wound infection ( ), subphrenic abscess ( ), anastomotic leak ( ), pulmonary embolism ( ), purulent neck discharge ( ). ability to swallow: palliation of dysphagia was achieved in % of patients following intubation and % of patients following bypass. postoperative nutritional status: improvement in nutritional status was more rapid following intubation. conclusion: pulsion intubation is the preferred palliative procedure because of fewer complications and lesser degree of postoperative catabolism. the current technique for investigating the response of vascular prosthetic materials to infection is by challenge with a sub-lethal dose of bacteria, usually an intravenous infusion of organisms in an animal model. this large bacterial innoculum, however, obscures any difference in the infectibility of prostheses that may be inherent in the material, its incorporation into host tissues, or its resistance to infection. we have developed a sensitive method for determining the susceptibility to infection of vascular prostheses based on calculation of the number of bacteria required to infect a specific prosthesis in % of trials (ids ). following implantation of the prosthesis to be tested in the canine infra-renal aorta, a dose-response curve was generated by the intravenous injection of known innocula of s. aureus (at log intervals from to bacteria). at six weeks the prosthesis was harvested and cultured to document infection with s. aureus. a characteristic sigmoid curve resulted from which the ids was determined. to test this method, a comparison was made between commercial human umbilical vein grafts (huvg) and huvg impregnated with silver sulfadiazine in dogs. although both prostheses were infected by the standard innoculum, a greater than tenfold increase in the resistance to infection by the treated huvg (< to ) was demonstrated in the dose-response curves. since the number of bacteria in a postimplant bacteremia rarely exceeds organisms/ml, such differences in infectibility are clinically significant. ids determination provides a sensitive, reproducible method for quantitating resistance to infection in vascular protheses. prosthetic infection following reconstructive vascular operations is an infrequent but often fatal complication which generally persists until the graft is removed. it is accepted that infection arises from operative contamination, bacteraemic seeding and abscesses or viscus eroding into the graft. this study investigates the role played by distal sepsis on groin grafts. ten dogs had a specific strain of staphyloccocus aureus inoculated onto a surgical wound in the right foot pad. five days later interposition mm dacron grafts were implanted into the ipsilateral and contralateral groin in continuity with the superficial femoral artery. ten days following this the grafts were removed for bacteriological and histological examination. blood cultures and lymphnode cultures were also taken at this time. in seven dogs the specific staph, aureus, was grown from both grafts. two dogs failed to grow the specific staph, aureus from either graft. these results are significant at the % level using fischer's exact test. blood cultures grew staph, aureus from only one dog. ipsilateral lymphnode cultures yielded the specific staph, aureus in seven dogs. we believe that distal sepsis plays an important role in the estabishment of graft sepsis. the mechanism of spread would appear to be via the lymphatics. the influence of tumor growth on wound healing p.w. de graaf and a. zwaveling laboratory experimental surgery, state university, leyden, the netherlands leakage of a colonic anastomosis is a complication each surgeon fears, because it usually leads to a prolonged hospital stay and is accompanied by a high mortality. carcinoma as an indication for operation and preoperative malnutrition are considered to be high risk factors. the subject of this study was to measure the influence of malignant tumor growth at a distant site on woundhealing in colon and skin, and to find a correlation between the influence of the nutritional state of the experimental animal, and the influence of malignant tumorgrowth on woundhealing. we also tried to find ways to compensate the possible unfavourable influence a malignant tumor might have on woundhealing. the study was performed in rats, the tumor used was a rhabdomyosarcoma, transplanted subcutaneously in the rats right flank. parameters for woundhealing were tensile strength of the skin-incision, and bursting pressure of a colonic anastomosis. six groups of rats were studied, each rat undergoing a standardized colonic operation after two or four weeks. the groups consisted of: control animals ( ); tumorbearing animals ( ), without tumor removal; malnutrltioned animals ( ); tumorbearing animals receiving intravenous hyperalimentation ( ), without tumor removal; animals undergoing tumor removal and colonic operation in one session ( ); animals undergoing the colonic operation two weeks after tumor removal ( ). woundhealing was negatively influenced in an early stage of tumorgrowth (f< . ). four weeks of tumor growth did not impair woundhealing to a greater degree than two weeks. laboratory determinations showed no deviations. malnutrition, leading to less weight gain than in control and tumor bearing animals took much longer to influence woundhealing than tumor pearing. this is in accordance with publications by irvin and hunt ( ), devereux et al ( ) and garattini and guaitani ( ) , which led us to the conclusion that the negative effect of tumor bearing on woundhealing was not solely the result of anorexia. hyperalimentation in tumorbearing rats resulted in undisturbed woundhealing, despite the fact that hyperalimentation as practised by us (with amino-acids and carbohydrates) stimulated tumorgrowth. we concluded from these experiments that tumorgrowth caused a metabolic chaos in the animal, which in turn led to a disturbance in woundhealing. intravenous hyperalimentation could apparently compensate this. woundhealing in rats operated in one session was significantly more disturbed than woundhealing in rats operated in one session was significantly more disturbed than woundhealing in rats operated in two sessions (p< . ). irvin an hunt ( ) have demonstrated however that extraabdominal trauma had no influence on colonic healing. this led to the conclusion that the diminished woundhealing found in rats operated in one session was an effect of the tumor. obviously this influence is not instantly removed with tumor excision. this study offers a theoretical foundation for the clinical observation that in operations for colonic carcinoma with a substantial operative trauma, the anastomosis needs extra protection and/or needs to be performed in a second session. the aorta of the blotchy mouse undergoes dilatation during adult life, resulting in the formation of fusiform aortic aneurysms. the mutation is x-linked, so only the males are affected. we have found a shift in the fluorescent spectrum of insoluble, hydrolyzed, dermal collagen in these mice; suggesting the presence of an abnormal crosslinkage compound. the purpose of this report is to describe additional studies carried out on the soluble collagen fraction in these animals, which lend support to the hypothesis of a crosslinkage abnormality. dermal collagen was prepared from one-monthold mutant mice and their normal male littermates by sequential extractions in salt solution, acetic acid, and urea. amino acid analyis and sds gel electrophoreses of the salt-soluble fractions revealed similar profiles of amino acids and collagen-chain types, suggesting that there is no abnormality of the basic building blocks of collagen in the mutants. fluorescent spectroscopy of the acid hydrolysates of these fractions also demonstrated similar absorption and emission peaks. however, reduction with sodium porohydride abolished fluorescence in the collagen fraction from the mutants. results ( emission m a x ± sem): normal blotchy before reduction . ± . . ----- . after reduction . + . none sodium borohydride is used experimentally to stabilize labile collagen cross-linkages. the present studies indicate that the salt-soluble collagen from the blotchy mice is borohydride-reactive and is thus chemically ,,unstable". these studies suggest a rational for developing probes based on these fluorescent croperties of investigate collagen from human subjects affected with aneurysms. one third of testes are removed after torsion ( , ) because of delay in presentation and diagnosis. in a retrospective study of patients with torsion our salvage rate was %. nineteen percent were misdiagnosed as epididymitis by a junior doctor and % by a surgical registrar. doctors could not distinguish between torsion of the testis and its appendages. in the literature, eight factors have been reported to be of discriminant value (table ) . a computer program, using these parameters and bayes' theorem, was written to calculate the most likely diagnosis. data from the patients with torsion and patients with acute epididymitis were analysed by this program ( table ) . the program correctly diagnosed all cases of epididymitis. of patients with torsion of the testis . in this study we determined the critical ischemic time for the development of an arrhythmogenic potential. egg and bipolar electrograms were recorded from the his bundle, ventricular endocardium and epicardium in twelve anesthetized dogs. short bursts ofventricular pacing ( beats; - beats/min) were used to induce ventricular arrhythmias before and after lidocaine administration ( , , mg/kg). previously, lidocaine has been shown to exacerbate conduction delay in ischemic myocardium leading to reentrant ventricular arrhythmias. in the control state, ventricular pacing with or without lidocaine induced either no response or single or repetitive ventricular responses. sustained ventricular tachycardia was never evoked in the control studies. in six dogs, after min of left anterior descending coronary artery ligation and reperfusion, ventricular pacing with and without lidocaine produced sustained ventricular tachycardia in one animal. in another six dogs, after rain ofligation and reperfusion, one dog showed sustained vertricular tachycardia in response to ventricular pacing alone. the other showed sustaine~l vertricular tachycardia after ventricular pacing with lidocaine. sustained ventricular tachycardia averaged /min and degenerated into ventricular fibrillation within - rain. in conclusion - min of ischemia appears to be the critical period for development of malignant arrhythmogenic potential in the canine heart. administration of lidocaine during this critical period enhances the inducibility of cardiac arrhythmias, which may have clinical relevance in the management of acute myocardial infarction. untreated coronary artery air embolism in the experiments without ecc (group i) resulted in a transmural myocardial ischemia with a mortality rate of % in contrast there were no death in the experiment when extracorporeal circulation was used (group ii to v). the best therapeutic effect with regard to the reversibility of temporary myocard ischemia following coronary artery air embolism was achieved by increasc of the postembolic perfusion pressure (group v), a finding being significantly different (p< . ) to groups i through iv. also volume depletion of the left ventricle on ecc (group ii) resulted in a faster regression of the left ventricular hypothermic area compared to group i (p<: . ). the application of dipyridamole (group iii) and st.thomas cardioplegic solution (group iv) was not able to produce a significant therapeutic effect. the increase of perfusion pressure following coronary artery air embolism has demonstrated to be the most effective procedure to achieve reversibility of myocardial ischemia. to transfer these experimental findings into clinical application is suggested because of its practicability and convenience. transmural biopsies were obtained before and , , , , , min after acc for biochemical and structural analysis. myocardial temperature (t) ph (mph), and pco ) were measured continously with a thermistor, a new ph electrode, and a mass spectrometer respectively. in group i (n= ) acc was under normothermia. in group ii mean t was reduced to °c by the administration of cold potassium cardioplegia immediately after acc and consecutively every min. mph at the end of acc reached . -t- . group i) and . ___ . (group ii) (/ < . ); pmco rose from ___ to _+ mmhg in group i and did not change in group ii. tissue content of atp decreases by % group i) and by °/ (group ii) (f< . ); tissue creatine phosphate fell by % (group i) and by % (group ii) (p< . ). changes in atp and creatine phosphate as well as in tissue glucose and glycogen related ton concomitant changes in ph. the degree of ischemic damage assessed histologically by a mean ischemic score was . +-- . in group i and . _+ . in grup ii (p< . ). irreversible structural demage assessed by electron microscopy occurred in group i - min after acc and was associated with a mph below . . no such damage was observed in group ii. we conclude: . irreversible damage during normothermic arrest occurs considerably later than has been reported for regional ischemia in the beating heart; . during h of acc cold potassium cardioplegia does not completely protect against id when mean t is °c; and . on-line oaeasurement of mph reflects the inadequacy of' mp more accurately than do either pmco or t and thus, provides a useful potential method for intraoperative monitoring of mp. in rabbits, infusion cardioplegia was installed at , , and °c respectively. the infusate contained na , k , , , or , ca , or mmol/ , and varying amounts of glucose. measured osmolality varied from to mosmol/kg. the hearts were excised at the end of a rain infusion period; one half was immediately frozen, the other half was incubated for varying periods of time. specimens of left ventricular myocardium were analyzed for metabolite and electrolyte contents. at termination of the cardiplegic perfusion, total adenine nucleotides (tan) and total creatine (tcr) were within control ranges under all conditions. however, atp and ecp = (atp + . adp)/tan as well as creatine phosphate (crp) and the ratio crp/tcr decreased significantly with increasing dosages of k and ca and higher temperatures. there were corresponding increases in adp, amp, and free creatine. at °c, there were no such changes except in hearts perfused with retool/ k and mmol/ ca. on the contrary, crp and the ratio crp/ tcr were elevated above the control values in hearts perfused with ca-free solutions containing between and mmol/ k. the decrease in atp served as the parameter of the ischemia-induced metabolic alterations and the energy deficit developing during cardiac arrest. at °c, it averaged . and . /zmol/g (p< . ) after min of arrest induced by and mmol/l k respectively. ca, or retool/ respectively, significantly increased the atp-decay to . and more than . pmol/g/lo min respectively (/ < . and p< . ). hypothermia of °c reduced the rate of metabolic deterioration and suspended the influence of the k-dosage. however, the ca-effect was still visible: induction of cardiac arrest by and mmol/l k without/with ca decreased atp by . / . pmol/g (p< . ) and . / . pmol/g (n.s.) respectively within rain. at °c, the rate of metabolic alterations was further reduced. the effects of k-dosage and of ca were completely abolished. the atp-decreased . pmol/g during min of arrest. although higher concentrated k-solutions for infusion cardioplegia do not enhance the ischemiainduced myocardial metabolic alterations in deep hypothermia, they are not recommended for practi-despite the advantages of cbk carrdioplegia, the severely impaired myocardium and/or long ischemia time continue to be a challenge. because of the association of ca ~ with cell injury and death the use of ca ~ channel blockers is logical. investigation of cbd revealied no advantages over cbk. the combination of k and d is appropriate because of their different mechanisms of action. ten dogs had one hour of myocardial ischemia (mi) with topical ice (temp ___ °c) after coronary perfusion with ml cb ( ___ l___c) containing k, meq/l and d, pg/kg. eight dogs had two hours of mi after perfusion with ml cb containing k, meq/l and d, /zg/kg. six dogs received the same treatment as the previous group except that d was increased to /zg/kg for all four perfusions. baseline studies were repeated after min ofreperfusion without the use of ca ~ or inotropic agents. heart rate, peak systolic pressure, vc¢, v~,~x, peak + dp/dt, peak -dp/dt, dp/dt over common peak isovolumic pressure, left ventricular compliance, stiffness and elasticity and great water were unchanged from control. coronary vascular resistance was unchanged in groups one and two but declined in group . creatine phosphate returned to control but atp, adp and the adenosine pool were depressed. ultrastructure was well preserved. in / dogs defibrillation was not required whereas / dogs with cbk and / with cbd required defibrillation. these data suggest that d is a worthwhile addition to cbk. early one-stage repair of some congenital vascular anomalies might be accompleshed readily if the material used for grafts grew along with the reconstructed tissues. we have evaluated the capacity of tubular grafts constructed from anterior rectus sheath to serve as growing segmental replacements of the descending thoracic aorta of puppies (age weeks). grafts measuring cm in length were placed in animals; were implanted with attention to tissue preservation (live grafts) while received grafts subjected to prior freezing and thawing in order to kill the donor tissue cells (devitalized grafts). grafts were measured initially and at sacrifice , or months later. each months animals with live grafts and with devitalized grafts were sacrificed. no aneurysmal dilatations or grafts ruptures were observed in any of the specimens. by months, the live grafts had increased . - - % in length and . ___ % in diameter, while length and diameter of the devitalized grafts showed minimal growth ( . ___ /o and . ___ o/o respectively). during the same period the length of the thoracic aorta increased . ___ % in the live graft group and . ___ % in the devitalized graft group. grafts were lined by endothelium and organized into transmural zones. the thickness of each zone in the live grafts remained static from to months, whereas thickness of the middle and outer zones in the devitalized grafts increased to fold. in living grafts the layers consisted principally of newly formed fibrocellular tissue; the rectus sheath was reduced to a atrophic fibrous band. by contrast, the rectus sheath remained prominent in the devitalized graft specimens. cellularity (cells/field) of the live grafts was much greater than that of the devitalized grafts (ratio . ___ : . ----- ). a newly formed, fibrocellular layered structure replaces the rectus sheath in the live grafts. these findings indicate that rectus sheath grafts are capable of growth in the young animal, providing that at implantation the tissue is well preserved. it is well known that durability of valvular bioprostheses (vbp) depends mainly on the structure of constituent biomaterial and long-term preservation of the collagen network. to our knowledge, no studies on ultrastructure of tissues currently used for vbp has been reported so far after a long time of chemical preservation. the presentation of a new anysotropic tissue, xenogenic cervical duramater (xcd), with a collagen tridimensional network, and a comparative study of scanning (sem) and transmission (tem) electron microscopy of bovine pericardium (bp), human duramater (hd) and procine aortic-cusp (pao) represent the purpose of the present communication. samples of the tissues were obtained in semisterile fashion, rinsed and fixed in karnovsky medium at °c for h. materials were then minced on paraphine plates and washed with . m cachodylate buffer for h and dehydrated in increasing concentrations of ethanol ( %- %) and intermediate exposure to % uranyl acetate for the in-bloc contrasts. were conducted, and results are exposed in table . as a general rule, histologic evaluation was by far, more satisfactory for tissues preserved with . % glutaraldehyde (xcd, pap) than those with % glycerol (hd) and % formaldehyde (bp) in combination. it is concluded that the new anysotropic tissue herein presented, xcd, can be appropriately preserved with . % glutaraldehyde as collagen preservation is concerned, and it's anysotropic and ultrastructural features, maintained for as long as months as assessed by sem and tem. albeit, clinical use of this new biomaterial is subjected to further experimental and animal trials. tetralogy of fallot and absent pulmonary valve (tapv) is associated with massively dilated pulmonary arteries which cause tracheobronchial compression in the newborn and heart failure and cyanosis in older patients. corrective operations have been attended by mortality rates exceeding % due to pulmonary insufficiency causing right heart failure (rhf) and pulmonary complications. pulmonic valve insertion (pvi) with complete repair has resulted in improved survival. the purpose of this paper is to assess the results of total correction and pvi in ten patients. during the last five years, patients with tetralogy were corrected. of these, ten patients (ages days to years) had absent pulmonary valve. one patient ( days) present with severe rhf and pul-monary insufficiency and nine patients presented with mild rhf and cyanosis. chest roentgenographs showed increased cardiothoracic ratio and pulmonary prominence in all. arteriography revealed massively enlarged pulmonary arteries with a mean right pulmonary artery to aorta size ratio of . to . associated pulmonic stenosis and insufficiency was present in all. seven patients underwent closure of ventricular septal defect (vsd) and pvi. of these, three had pvi ( tissue and prosthetic) with outflow patch and four had right ventricle to pulmonary artery (rv-pa) tissue valved conduits. two patients had repair without pvi, and one had repair with a monocusp pericardial valve patch. nine patients have done well with no episodes of thromboembolism or infection. death occurred in a day old infant who had vsd closure and relief of pulmonic stenosis. pulmonary valve insertion seems indicated in these patients at it lowers peak pulmonary artery pressure and thus reduces compression effects on the trachea and bronchi. as well, when pvi was used right heart failure was not noted postoperatively. although in experimental acute myocardial ischemia intraaortic balloon pumping (iabp) appears to increase regional contractility of ischemic segments of the left ventricle by increasing the collateral flow, evidence for this effect of iabp in patients with refreactory myocardial ischemia is not available. this study was done to analyze the effect of labp on global and segmental left ventricular performance in patients with refractory, acute myocardial ischemia using gated blood pool scanning with tc- albium tracer. eight patients were studied on-and-off iabp prior to and following coronary bypass surgery. left ventricular (lv) wall motion analysis was undertaken and both cardiac output (co) and left ventricular filling pressure (lvfp) were determined from thermistor-tipped pulmonary artery catheters. when placed on iabp, mean preoperative global ejection fraction (gef) increased ( . - . (off) to . - . (on); p< . ) abut no changes in co or lvfp were observed. when comparisons were made praend postoperatively, co increased after operation (p< . ), due chiefly to an increase in heart rate ( - to - beats/rain; p< . ), but gef was unchanged whether or not iabp was on. only segmental wall motion analysis pre-and postoperatively revealed that iabp increased regional ejection fraction (ref) and contraction velocity by % (mean) in the angiographically determined regions of myocardial ischemia and these regional changes were maximal during the last one-third of the lv contraction cycle. it appears that ref is a more sensitive indicator of changes in lv performance than co and gef in these patients. further, this study indicates that iabp does increase the regional wall contracility in the ischemic areas of the myocardium in man. the use of aspirin (asa) to inhibit platelet thromboxane synthesis (tbx ) in patients undergoing coronary artery bypass grafting (cabg) has been advocated to reduce the potential for graft occlusion. however, asa in conventional doses also inhibits the venous synthesis of prostacyclin (pgi ) a potent anti-thrombotic factor, and may contribute to excessive surgical bleeding. to investigate the relationship between asa dose, blood loss and inhibition of venous synthesis of pgi , patients undergoing cabg were studied. control patients (no asa) were compared to those receiving or mg. asa as a single dose - hours prior to surgery. production of pgi by saphenous vein specimens removed at the time of surgery was measured by radioimmunoassay (ria) of -keto pgf~a following incubation with um na arachidonate. blood loss was assessed by chest tube drainage and transfusion requirement in the perioperative period. serum tbx was measured by ria following asa ingestion. transfusion (units), drainage (cc), vein pgi (pg/mg tissue) and serum tbx (ng/ml) were (mean -sem): therefore, asa mg or mg did not increase blood loss during cabg. however, asa mg, significantly reduced saphenous vein pgi synthesis (/ < . ) while the lower dose asa mg spared the production of pgi . asa is both doses inhibited tbx (p< . ) and blocked platelet aggregation. low dose asa deserves further investigation as an anti-thrombotic agent since it does not appear to increase operative bleeding or significantly inhibit venous pgi production. transfusion postoperatively, igg increased progressively in patients in group a, reaching preoperative levels on the th or th day, whereas in patients in group b, igg remained at lower than preoperative levels during the first week. the number of patients with abnormally low lavels of igg during the whole po period was significantly higher in group b (p"~. ). no significant differences were found in the others studied variables. one patient in group a had infection. three patients in group b had infections. conclusion: high doses of fab igg administered during ohs and the first po days are effective in lessening the po decrease oflgg and thus may be beneficial in preventing po infection. a water insoluble but very hygroscopic polyvinyl alcohol powder, zy- ", is capable of significantly stimulating the cicatrization of open, contaminated skin wounds in rats. when it is compared to other substances in clinical use such as powders containing antibiotics, antiseptics, amino-acids, enzymes or a dextranomer, no other agent tested was capable of producing a similar beneficial effect. these excellent experimental result justified pilot clinical trials on patients: varicose vein ulcers, atherosclerotic leg ulcers, infected traumatic wounds and infected postoperative wounds. whether in rats or in patients, zy- powder removed secretion, bacteria and tissue debris; it produced an early and increased proliferation of fibroblasts with the appearance of dense granulation tissue; it reduced wound size after less than three applications permitting very early grafting of the wound of eventually, cicatrization advanced to complete epithellzation. regardless of whether the treatments were applied daily in the hospital bed or every other day in outpatients, there were not complaints of unpleasant sensations such as itching, burning or pain. few studies have dealt with long term healing in stomach and none is available for duodenum. this study evaluates morphology and development of mechanical strength and collagen concentration in and adjacent to wounds after to ]g days of healing. incisions were made in the non-glandular (rumen) and in the glandular oxyntic (corpus) part of the stomach and in duodenum of male wistar rats, intact rat served as controls. the wounds were dosed with - polypropylene sutures using a single sutur technique. after , and days of healing complete load deformation curves were determined on strips perpendicular to the incision line after the sutures were cut. collagen distribution was measured as hydroxyproline in strips parallel to the incision line. wound tissue continued to gain strength up to days of healing (breaking energy significantly increased in all tissues from to days of healing). no such increase was found for wounds tested togetherwith adjacent tissue, because the "point of maximum weakness" had moved laterally from the incision line with healing time. wound collagen concentration increased in corpus and duodenum between and days of healing, but was unchanged between and days. the biochemical active zone around the incision line was unchanged - mm on each side from day to . conclusion: while the wound tissue itself continues to gain strength during the days ofheaiing studied, the increase seen after days does hot,enhance the functional properties of the organ as a'whole. the "point of maximum weakness" has at that time moved to the borderline of the biochemically active zone, which lies outside the tissue area enclosed by sutures. myocutaneous flaps based on either the inferior or superior epigastric artery may be used to cover extensive soft tissue defects from the mid thigh to the clavicle. we report our experience with rectus abdominis flaps, based on the superior epigastric artery and based on the ingerior epigastric artery. the flap is easily elevated, no skin loss has occurred and primary closure of the donor defect has been achieved in every case. in a follow-up period of between month and months no patient has developed an incisional hernia. the flaps have been used to cover extensive chest wall resection for recurrent carcinoma of the breast ( patients), extensive radionecrosis of the chest wall ( case) as part of a primary breast reconstructive procedure ( patients) and to replace soft tissue overlying the femoral vessels after radical dissection of the groin for metastatic tumour ( patients). of the patients undergoing primary reconstruction required additional subpectoral prosthesis but in adequate bulk was provided by the flap itself. primary healing occurred in all cases. this easily raised and reliable flap will shorten hospital stay after major ablative surgery for recurrent disease and provides a useful addition to methods of breast reconstruction. a. watson department of surgery, royal lancester in girmary, lancaster, u.k. occult gastro-intenstinal bleeding which defies readiological and endoscopic diagnosis provides one of the greatest diagnostic challenges in surgery. lesions producing this clinical situation are often very small by definition and not infrequently mucosal angiodysplastic lesions, localisation of which may be extremely difficult pre-operatively and indeed at laparotomy. various methods have been described in an attempt to improve pre-operative localisation of such lesions. string tests an dthe detection of sicr labelled red cells in the faeces are notoriously inaccurate. arterioghraphy is invasive and usually necessitates a bleeding rate in excess of ml per day. scintiscanning after red cell tagging with mtc gastro-intestinal blood loss, but localisation is difficult. a method oflocalisation of such lesions using slcr labelled red cells and intestinal intubation is described. after labelling of the red cells, a miller-abbot intestinal tube with the balloon inflated and rendered radio-opaque is passed and samples of gastrointestinal secretions aspirated at each cm during passage down the gastro-intestinal tract. samples are counted on a well scintillator counter and when a sample of high radioactivity is obtained, localisation is assessed both by the length of tube passed and by instilling dilute barium down the miller-abbot tube underfluoroscopic control. this method has been used successfully in five patients which were subsequently treated surgically with localised resection resulting in cessation of bleeding. case histories together with photographs of the method and respected specimens will be presented in poster form. postoperative sepsis is tlae most frequent complication of surgery and is the commponest cause ofprolungation of hospital stay. purpose of the study is to prospectively evaluate incidence predisposing factors, bacteriology and costs of postoperative infections. consecutive surgical patients admitted to our institute from may to july were studied. patients undergoing minor surgical procedures (wound less than cm) were excluded from the study. patients were evaluated daily during hospital stay for onset of infection and results recorded in a data sheet. hemocultures in septic patients and free plasma, activated partial thromboplastin time, prothrombin time, and thrombin time, ristocetin test for soluble monomer complexes and fibrin degradation products (fdp, welco test) euglobulin lysis time (elt) and platelet counts. conclusion: . thrombocytopenia is not a typical feature of boomslang coagulopathy. . the demonstration of soluble monomer complexes is blood samples (positive ristocetin test) appears to be an early and consistent indication of intravascular coagulation. . despite unequivocal evidence of advanced consumption, platelet function seems to be maintained, thus preventing complete heamostatic failure. . support for this view is found in the clinical observation that bleeding is essentially mild and late in onset. . thrombelastographic hyperretractility the "spinning top" appearance is a constant feature and is probably mediated via the platelets. . although d. (vpus venom is extremely potent and often fatal, the antivenom is rapidly effective despite advanced consumption. this mechanism which may involve the platelet release reaction has not been fully elucidated and deserves detailed study. in most cases artificial ventricles are driven pneumatically with the advantage of easy handling and the disadvantages of regulation problems, high weight and volume of the driving units and the danger of air embolism in the case of membrane rupture. the driving unit developed at innsbruck universiy consists of a microprocessor-controlled electromagnet driving a pump that functions as a safety chamber (sk). force transmission to the artificial ventricle (ellipsoid heart-eh) is effected hydraulically. thus there is a fixed connection between armature stroke and membrane movement in the eh. the armature position is measured by the microprocessor by means of a position sensor with programmable switch-over points determining the change from systole to diastole so that idling and beat volumes, respectively, of the eh can be programmed. the sk is a newly designed double rolling membrane pump for pressure and suction with very low compliance. pressure and suction are determined by the armature force which is proportional to the microprocessor-controlled current in the solenoids. thus a very positive control of the pumping action is possible. in mock circulation experiments with the hydraulic safety driving unit the cardiac output (co) was between /min and /min at beat frequencies of to bpm. with constant piston-stroke a direct relation between frequency and co was observed. the influence of preload (starling's law) and afterload decreases with increasing armature force (decreasing periods of systole and diastole, respectively) which is due to friction in the hydraulic transmission system. improvements in the geometry of the next system should reduce these losses. in vivo experiments confirmed the hemodynamic efficiency of the system. applied as lvad the system proved to relieve the beating heart considerably. in the case of heart fibrilation the circulation could be maintained by the safety driving unit. the variable height differences between driving unit and eh as they occur in long-term experiments (animal lying or standing) affect only the ratio systole/diastole periods with the co remaining constant. mechanically assisted circulation is indicated in patients with cardiac failure after open heart operations. the clinical experiments show that left ventricular assist devices are capable only in a few cases to maintain full circulation. the limiting factor is the additional right heart failure syndrome. in patients with postoperative cardiac failure, a distinction must be made between isolated left heart failure and total heart failure. in patients with total heart failurebased on diffuse coronary sclerosis or on an increased pulmonary resistance -left ventricular assistance alone is not effective and right ventricular support is desired. therefore, a simple, quick and safe biventricular assist device is necessary. with the biventricular bypass it is possible to maintain complete circulation in cases of cardiac insufficiency. for a successful outcome in patients with low cardiac output after cardiopulmonary bypass, the e-bvad was evaluated in animal experiments (with calves in acute and survival experiments) in cardiac failure situations. the cannulas for the inflow are put into the left and right ventricle, the outflow tracts into the descending aorta and the pulmonary artery. both parts are connected with the ellipsoid hearts. with the e-bvad it is possible to have a replacement of the heart -a total heart assistance similar to the total artificial heart. in cases of clinical emergency this device can be recommended because of the satisfactory hemodynamic effects achieved and the small degree of traumatic hemolysis ( , mg% free hemoglobin). it represents an easy and quick implantable system for total functional heart replacement. the realtionship between acute pancreatitis (ap) and the enzyme and hormone level in blood and gastric and duodenal juices is the factor that had driven us to do this experiment that would complete the study of the physiopathology of this illness. material and methods. we used eight -year-old dogs. a) production of two antiperistaltic fistula in the mann and bullman way; one gastric and the other duodenal (first part). . blockage of the gastric and duodenal compartments: by the gastric fstula we introduced one foley's bucket with closed point and inflatable globe to block the pylorus. by means of the duodenal fistula we introduced another foley's bucket into the duodenum. once inflated, the ball blocks the first duodenal portion. . juice extraction: by adjacent openings in the gastric bucket blocking the pylorum, we extracted the gastric juice separately. with another thin foley's bucket, that was introduced by the duodenal fistula near the already blocked one, to block the third part of duodenum when the globes inflates. b) production of ap: days after the first surgical operation, by the morandeira method with intraparenchymal injection of a mixture of autologous bile and olive oil. results. one of the animals died on the fifth day after the first operation. + no animal died spontaneously after the second operation. they were killed on the th day. in each one acute necrotizing pancreatitis could be verified. + the radiograph showed that the gastric and duodenal compartments were sealed. -it was easy to separately extract blood and gastric and duodenal juices. conclusion. we believe that this method is complete, simple, with good results and very useful for the study of the physiopathology of ap. liver samples were taken form patients operated for cholelithiasis and common bile duct obstruction by gallstones or pancreatic tumour. early evident histoenzymatic deficiency was found even without jaundice or liver structural lesions. in the bilio-obstructive jaundice, the histochemical methods revealed more marked liver impairment, as compared to the histological picture. the degree of the enzymatic depression could explain the occurence of the postoperative liver failure in some patients. experimentally, the effect of common bile duct ligation was studied in rats. group i = healthy controls. group ii = aspartate ( ml % sol.) was injected i.p. in rats daily, for a week. group iii = the common bile duct was ligated under other anes-thesia, in rats h before killing, and group iv=in another rats days before killing. group iv =in rats aspartate was administered h before an twice after choledocus ligation, and group vi = in another rats aspartate was injected daily for a week after surgery. the liver slices frozen in liquid nitrogen were cut in a slee-type cryostat and the histochemical reactions were performed according to arnold, chayen-bitensky and lojda-gossrau-schiebler's methods. the biochemical reactions were performed using merckotests and merz-dade test-kits. after h, and still more after days, very severe structural, histochemical and enzymatic lesions developed. the liver cell glycogen and rna, as well as the "marker" enzymes of infrastructures markedly diminished: nach -tetrazolium reductase - . %, glucose- -phosphatase - . %, alphanaphthylacetat esterase - . %, atp-ase - . %, '-nucleotidase - . %, pas-reaction - . %, mgp-staining - . %. aspartate treatment seems to exert a protecting effect against the noxious action of retained bilirubin and conjugated bile salts upon the liver cells: nadh -tetrazolium reductase + . %, gsp-ase + . %, esterase + . %, atp-ase + . %, 'n-ase + . %, pas + . %, mgp + . % and lipids - . %. but it does not influence the biochemical signs ofcholestasis: bilirubinemia, alkaline phosphatase, leucinearylamidase, gamma-glutamyltransferase, lactate dehydrogenase. aspartate prevented partially but significantly only the increase of cytolysis enzymes: asat - . % and alat - . %. the histochemical and enzymatic results are in agreement with the severity ofmorphologic changes. therefore, aspartate treatment might be adequate for the preoperative improvement of the liver function in cases of obstructive-jaundice, in order to reduce the incidence of liver failure, without influencing cholestasis. oral glucose tolerance tests (ogtt) y. yamoaka, a. sugitani, ic kimura, ic ozawa and y. tobe department of surgery, kyoto university medical school, sakyo-ku, kyoto, japan two patients with cholecystographic evidence of septate gallbladder underwent dynamic hepatobiliary radionuclide scanning with mtc-ehida. when a steady state of emission had been reached from both gallbladder lobes, cholecystokinin was infused incrementally in four doses ( . , . , . and . ivy-dog-units kg-lmin - ) and counts from the gallbladder analysed by computer. in each an obvious functional difference was revealed between the lobes: both distal lobes ceased emptying abruptly during the third hormone infusion whereas the proximal lobes continued to empty predictably [ ] . in one case, histology revealed the septum to be a well-developed smooth muscle ring with cholecystitis glandularis proliferans confined to the distal lobe; the other case awaits surgery. the cause of the difference in response was probably contraction of the muscle in the septum acting as a sphincter. this study provides indirect confirmation that pain in septate gallbladder is due to septum contraction and raised distal intralobe pressure. it is known that the gallbladder stone passes into the common bile duct. furthermore, common bile duct stones pass to the duodenum. these gallstone movements were analysed in cases during the past six years. the phenomenon in which gallbladder stones pass through the cystic duct down to the common bile duct are seen in cases where stones are small and numerous, the cystic duct is wide and connected with the common bile duct angularly or in parallel. in . % of our cases common bile duct stones passed into the duodenum. there were neither inflammatory stenosis of the terminal choledouchous, high grade pappilitis nor severe obstruction of the common bile duct in these cases. it appears that the movement of gall stone is related to size and number of stone and morphological variations in the biliary system. the study was conducted in order to investigate whether intraoperative mano~etry of the bile ducts could be of additional valtie in diagnosis of afflictions of biliary tract or of the papilla. in contrast to current methods of water manometry it seemed important to the authors to use a simple and safe method which could deliver precise results. method: electromanometrics studies were carried out with a cannula placed in the common duct. three manometric parameters were obtained for evaluation. . resting pressure (rp); . pressure increase after injection of ml saline (lml/s) (pi); . time in seconds needed for return of initial pressure (bile flow) (tr). for pressure measurements a pressure transducer with a recorder was used. results: (table) manometry results in patients with calculi in the common duct or with organic stenosis of the papillawere significantly changed as compared with normal findings in the bile ducts. false positive results (proven by cholangiography and biopsies of the papilla) were found in . % of cases, false negatives in % % of all patients with patho-histological changes in the papilla had pathological bile pres-. sures. although false positive and negative results were mainly caused by methodical errors, the described method delivers a high percentage of correct results in agreement with the final diagnosis. it is avery sensitive method in indicating impaired and reduced bile flow and discovers early pathological changes in the papilla as proven by histological examinations. a functional anhepatic state in experimental animals for biochemical studies and as a model for treatment of acute liver failure can be achieved by different surgical procedures like portocaval shunt and complete arterial devascularization or by replacement of the liver with vascular prosthesis and protocaval shunt. we developed a simple hepatectomy procedure using t/ inch silastic tubing, carbon-y-connector and specially designed "vascular spirales" to restore normal portal and caval blood flow. this model was used for auxiliary liver perfusion studies in heparinized animals; the operation takes min, requires no surgical skill, vascular occlusion is less than min, no signs ofsplanchic hypertension are present and blood loss in fully heparinized animals over h is insignificant. one of the main problems in atypical and anatomical liver resections consists of achieving appropriate hemostasis. also because bile capillaries are opened when liver tissue is separated there is the danger of infection and subphrenic or parahepatic abscesses. the authors have used the human fibrinogen clue to seal the surface of the resected liver in cases ( hemihepatectomies and resections). even in anatomical resections exists, in the majority of cases, a diffuse bleeding or oozing of the resected area. this can be completely controlled by sealing the surface with the fihrinogen adhesive. also one of the main advantages of the fibrinogen consists of having the possibility to avoid the insertion of numerous tubes for drainage. all our cases were drained by a single silicon tube. the postoperative courses were uneventful in all cases no major complications were observed. liver regeneration is thought to be stimulated by changes in portal blood constituents, or flow or by a substance in the hepatocytes. rice et al. have documented increases in total hepatic perfusion in rats during liver regeneration. this study has measured the portal and arterial components sequentially in large animals. young pigs ( - weeks) were subjected to shamoperation or % partial hepatectomy (ph). blood flow in the portal vein or hepatic artery were measured pre-operatively and at intervals of h postoperatively using cuff probes of an s.e.m. electromagnetic flowmeter; these were positioned daily under light anaesthesia. systemic arterial and portal venous pressures were measured via indwelling catheters. previous studies have shown that the peak of regenerative response occurs in pigs to days after ph. the mean pre-operative total liver blood flow increased from . + . (s.e.m.) ml.g-lmin- to a peak of + % on day . thereafter flow declined towards normal by day . the pre-operative portal component was ___ %; this increased to + . % on day and returned to normal level within to days. it seems that both total hepatic flow and the portal component increase markedly just preceding the peak of regeneration. this response may stimulate regeneration, or merely accompany it, or may be proyoked by similar stimulators. the analysis of individual bile acids is relevant to several clinical investigations although established techniques have a number of disadvantages. to overcome these we have developed a simple hplc method using a waters associates rcm radial compression module with a radial-pak c , ,u, mm id, reverse-phase cartridge (column). bile acids were eluted from the column at a flow of . ml min- with a mobile phase of methanol:water ( : v/v) containing . % (v/v) acetic acid and adjusted to ph . with m naoh. a mixture of standards of cholic, chenodeoxycholic, deoxycholic, lithocholic and ursodeoxycholic acids and their glycine and taurine conjugates were resolved, while the conjugates alone were completely separated in under rain. the technique has been applied to the study of human bile from the common duct, gallbladder, tube and duodenal fluid, and serum from patients with hepatobiliary disorders. bile acids in these samples were rapidly extracted using a sep-pak c cartridge before being applied ( /a to pl) to the hplc system, although bile could be analysed directly without extraction. quantitation (~mol ml- sample) of separated bile acids was achieved by comparison with standards using an on-line integrating computer and less than nmol could be detected using a refractive index detector. acute hepatic failure induced by total liver devascularization in pigs -amino acid uptake by combined charcoal -resin hemoperfusion support system where losses averaged %. jib patients had progressively greater losses with increasing preoperative weight (/ lbs- %, - lbs- %, - lbs- %, - lbs- %, lbs- %). weight loss in patients dbs was statistically and clinically greater with jib. . diabetics, especially insulin-dependent, were rapidly cured after jib. normal plasma glucoses, serum insulins, and oral glucose tolerance curves were usually seen within l postoperative mouth, unrelated to weight loss. all patients requiring insulin or oral medication preoperatively could discontinue mediation usuallywithin a weekpostoperatively. improvement after gb was gradual, appeared related to weight loss, and was often incomplete. . hypercholesterolemic patients had % decreases, from to mg/dl, after jib, but only % decreases after gb. all serum cholesterols were normal after jib, but % remained elevated after gb. because of complications after jib, some needed reoperation and conversion to gb. fortunately, most benefits were retained after conversion to gb. we suggest considering jib for "superobese", diabetic and hypercholesterolemic patients. in the past years jenunoileal bypass procedures were performed. the most common complications and side effects were frequent abdominal pain, or discomfort and flatulence in almost half of the cases. in addition kidney stones, blind loop invagination, electrolyte and liver dysfunction problems could be observed. the over all complication rate was %, the postoperative mortality rate %. to eliminate the blind loop of the small intestine, to maintain enterohepatic circulation of bile, and to diminish undesirable side effects in the conventional jejunoileal bypass, biliointestinal bypass was introduced in . twenty patients with a mean weight of kg were subjected to primary biliointestinal bypass within the last two and a half years. three additional patients had secondary biliointestinal bypass due to side effects, especially diarrhea and flatulence, of jejunoileal bypass, performed two to four years previously. the surgical procedure entailed establishment of an end-to-side jejunoileostomy. cm of the jejunum and cm of the ileum were left in the continuity. the blind loop of the jejunum was anastomosed to a functioning gallbladder. so far the above mentioned complications of conventional jejunoileal bypass could be remarkebly diminished. there have been no deaths in this material and no metabolic side effects. frequent diarrhea is avoided by reduced bile spill-over to the colon. the weight reduction has been satisfactory. in summary: due to a lesser complication rate biliointestinal bypass seems to be superior to the simple jejunoileostomy in the treatment of morbid obesity. hyperplasia or neoplasia g.w. geelhoed, c.j. schaeffer and p. daudu department of surgery, george washington university medical center, washington, usa patients with various thyroid disorders who were undergoing thyroid operation were studied for the presence of absence of estradiol and progesteronebinding proteins in thyroid tissue. steroid receptor assays were carried out using similar techniques and standards as those routinely employed for study of breast cancer specimens. quantitative data were collected by coded specimen number by an observer unaware of the patients' clinical diagnoses, and diagnostic correltions were drawn following de-coding. there was no correlation with age or sex and the presence or quantitative value of steroid-binding site. specimens with the histologic diagnosis of follicular adenoma had estradiol binding sites, and had them at high levels ( . to . ) femtomoles/mg cystosol protein). patients with the histologic diagnosis of thyroid hyperplasia had marginally positive estradiol-binding and lacked progesterone binding. patients with adenocarcinoma of the thyroid exhibited neither estradiol nor progesterone-binding in significant quantity. presence or absence of steroid binding sites in thyroid tissue appears independent of sex or age, but correlates with benign neoplasia of the thyroid, suggesting a possible etiologic association for thyroid adenomas. patients with esophageal cancer underwent resection and primary reconstruction of the esophagus by posterior invagination esophagogastrostomy which we devised. the anastomosis was made in the cervical level in cases and in the left thoracic cavity in cases. functions of stomach placed in the posterior mediastinum were examined in patients surviving more than years and in patients surviving less than years. within one year postoperatively, the absorption ofvitamine b decreased markedly. one and a half year after operation, however, the secretion of castle's instrinsic factor recovered and it showed normal values in patients surviving over years. in secretion of gastric acid by tetragastrin, both total acidity and free hydrochloric acid increased in the progress of the postoperative period and showed normal values in all patients surviving over years. this fact was also confirmed by endoscopic observation of color development by congo-red in the gastric mucosa; its coloring area was scattered in patients surviving years, but extended to the whole surface in patients surviving over years. roentgenographycally in head-down position, the surgically created fornix with a sharp angle of his was effective in preventing gastroesophageal reflux completely. the strain combination of donor and recipients. the nature of the mechanisms determing the different fate of allografts remains obscure and all interpretations of the above findings must be speculative. however, the long-term acceptance of hepatic allografts in the rat appears to be similar to that in other species and emphasizes the role of the liver as an immunologically favoured organ. footnote: part of the work (intrahepatic bile duct proliferation!) contained in this abstract will be presented at the xvii essr meeting . peoples' friendship university, tamojenii pr. , department of operative surgery, moscow, ussr a transplanted kidney, besides tissue incompatibility reaction, is influenced by non-specific injuring factors, including decentralisation. for normal functioning of a kidney transplant over a long period of time we carried out an experimental study. the purpose of the study was the investigation of operative reinnervation possibilities and its influence upon the functional state of a transplanted kidney. for the kidney reinnervation kirpatovski's method for suturing perivascular fascial tissue flaps of anastomosed vessels with branches of vegetative plexus nervosus on them was used. experiments were carried out on mongrel dogs, both male and female. in the first group of experiments a kidney was transplanted into the pelvis while the opposite kidney was preserved or ablated. in the second group the kidney autotransplanted into pelvis was reirmerved by the described method with preserving the contralateral kidney in part of the animals and ablating the kidney in the rest of them. the third, fourth and fifth were control groups. in the third group the kidney was denerved, in the fourth it was denerved and reinnerved by the mentioned method, in the fifth group unilateral nephrectomy was performed. after the operation the animals, in different periods of time (from days to years), were studied by isotopic renography and by scanning kidney's by jbl-hippuran and neohydrin-hg ° intravenous injections. results of the isotopic renography were estimated qualitatively and quantitatively by universal renographic index (hirakawa-corcoran, ). results of the experiments indicate that autotransplantation and denervation of a kidney without its reinnervation were followed by lowering of a kidney vascularisation and its secretory and excretory functions. during the first or months after the operation a tendency to improvement of the kidney function took place; later those functions gradually oppressed and the renographic index lowered down to . +__ . , . +--. . (p< . ) respectively according to the groups. after operative reirmervation of a kidney autotransplant and denervation of a kidney without its transplantation gradual improvement of separate functions of the kidney took place: in or months the kidney circulation was restored; in or , and in or months respectively, secretory and excretory functions of the kidney restored; renographic index reached its norm after or months and remained stable during years (according to the groups . +___ . , . - - . , p> . respectively). thus, the operative reirmervation of a kidney prevented depression of its functions and provided its normal functioning during a long period of time. introduction of fine needle aspiration cytology in renal transplantation gives the possibility not only to distinguish acute tabular necrosis, arterial and venous obstruction and viral infection from acute rejection, fnc also seems to allow judgement of the effectiveness of various types ofimmunosuppressive therapy during rejection episodes. method: after sterile fine needle biopsy the cytological evaluation of the aspirate was performed on cytocentrifuged smears after staining the cells by the method of may-grtinwald-giemsa. thus normal and "activated" mononuclear cell populations as well as parenchymal cells such as tabular and endothelial cells and their pathological changes could easily be recognized. the white blood cell types in fnc were compared with those of the peripheral blood. the difference in number due to the graft invading cells was expressed as "increment". the findings of fnc were correlated with clinical signs and serum creatinine values. results: fnc allows to judge the in situ inflammation in the rejecting kidneys within h. non-rejection grafts show cell counts comparable to peripheral blood. with onset of rejection (grade ) t-and bblasts and monocytes appear in the graft. with sever-ity of the rejection (grade ) monocytes and lymphocytes as well as blood cells increase in numbers. acute rejection (grade ) is heralded by high numbers of monocytes and macrophages. acute tubular necrosis (atn) and virus infections can be distinguished from rejection episodes to be treated. in all cortisone and azathioprine resistant cases where alg was used in order to suppress inflammation during rejection episodes it reduced not only the peripheral white blood cells, but also within h after start of alg therapy the number of in situ cells. beside reduction of inflammatory cells typical changes in shape of the nucleus and density of chromatine in up to % of the lymphocytes and granulocytes could be detected. these changes look closest to what has been described as apoptoses. conclusion: cortisone and azathioprine resistant rejection episodes could be monitored within - h using fine needle aspiration cytology. this method is safe, cheep, and it provides good information about the in situ situation of the graft, it allows to distinguish acute rejection from atn and other situations where higher immuno-suppressive therapy, especially with cortisone, could only be harmful for the patient. criteria of the border of normothermic ischemic tolerance in dog kidneys are first normal pah-and exogenic creatinine-clearances compared with unilateral nephrectomized dogs in neuroleptic analgesia under excessive water and sodium load and second perfectly normal kidneys after two weeks in pathological examination. the protective solution htk ® by bretschneider has a superior buffering capacity compared to euro-collins-solution ® , which, by itself, enhances anaerobic glycolysis by substrate stimulation (glucose) especially between ° and °c. without preservation in normothermia oligo-anuric arf is observed at the border of ischemic tolerance ( - min; °c). the obligo-anuric arf is dependend on ischemia which will result in necrosis of the kidney after rain and °c ischemic temperature. the border ofischemic tolerance corresponds to an intrarenal ph of less than . (outer stripe of medulla), and medullary lactate levels of to pmol/gdw. substrate stimulation of anaerobic glycolysis limits the effectiveness of euro-collins-solution ® above °c earlier than anaerobic glycolysis in pure ischemia. after warm ischemia (>--__.. °c; min) all kidneys protected with euro-collins-solution ® show anuric arf, whereas all htk®-protected kidneys ( to min of warm ischemia; °c) are having polyuric arf. under these conditions, renal ph will not increase above nmol/ h+-concentra -tion. the border of ischemic tolerance for htk ®protection ofkidneys is rain- °c and seems to be limited by a transitory secondary postischemic oliguria ( - h). in summary: intrarenal anaerobic glycolysis limits tolerance of pure warm ischemia by inducing anuric arf. in contrast polyuric renal failure is observed at the border ofischemic tolerance by using the protective procedure with the htk®-solution mentioned above. at least % of unrelated pigs respond to liver allografting with a rejection episode which they overcome without immunosuppression; kidney transplants are rejected uniformly within to days. in this study, mlc responses were measured at weekly intervals in such animals. non-litter mate pigs were subjected to autograft, exchange liver allograft, or exchange renal allograft. blood from mlc responses were taken from unaesthetised pigs before and at weekly intervals after operemained the same for each pair of transplants. in of pairs of liver allografts, mlc responses were depressed up to fold for weeks post-operatively. in all these pigs, the mlc responses were initially high. in pairs where the mlc response was initially low, there was no change after the transplant, and in one pair with a low pre-operative response, there was a marked increase post-operatively. in pairs of kidney allografted pigs, there was a similar depression in the single post-operative sample available. in liver autografted pigs, there was a slight post-operative rise in mlc response. it appears that liver and kidney transplantation but not hepatic autografting, markedly depress sequential post-operative mlc responses. the process of ischaemic kidney degeneration was estimated by measuring the adenine nucleotide (atp)levels and the effect of inosine and naftidrofuryl (praxilene) was assessed. min ischaemia was induced on both dissected kidneys of wistar male rats. then, either both clamps were removed and the right kidney was excised after i rain reperfusion the role of mononuclear phagocytes in various disease states has been extensively studied by phagocytosis, fc and complement receptor sites, and enzyme content. chemotaxis of peripheral blood mononuclear phagocytes, however, has not been studied to any great extent. we have therefore investigated a method for quantifying chemotaxis by mononuclear phagocytes. mononuclear phagocytes were purified from peripheral blood by centrifugation over ficoll-hypaque and a discontinuous percol gradient. chemotaxis was quantified by the 'under-agarose method'. mononuclear phagocytes were allowed to migrate towards the chemotactic agent zymosan activated serum (zas), and the control non-chemotactic agent eagles medium. the distance of cell migration was measured after hours incubation at °c in % co with the aid of a microscope eyepiece graftcule. using non-sepcific esterase staining the purity of mononuclear phagocytes obtained was %___ % and the viability by trypan blue dye exclusion was at least % preliminary results have shown selective migration by purified human peripheral mononuclear phagocytes towards zas. this method may therefore represent a means of investigating an important role of these cells in disease states. the intrathoracic pressure rises when a person exhales into a manometer to such an extent that finally the venous blood-flow to the heart stops. it is supposed that this venous stop flow pressure (vsfp), measured by an ultrasound device, is equal to the central venous pressure. in a prospective clinical trial by two independant examiners in % of the cases (n= ) the correlation was within cm h . therefore, the central venous pressure (cvp) can be measured non-invasively with an ultrasound device. we have previously reported that opiate receptor blockade with naloxone (nal) significantly improves cardiovascular function and survival in canine hemorrhagic shock [ , ] . since species differences do exist, we investigated the effects of nal in cynomulgus monkeys anesthetized with n /o . blood was withdrawn to achieve a mean arterial pressure (map) of mmhg (t--- ) which was maintained until t= h when the reservoir was clamped and the animals treated with either nal at mg/kg bolus plus mg/kg.h infusion i.v. or . % nac in equivalent volumes. there were no significant differences between nal (n= ) and con (n= ) in map, left ventricular contractility (lv dp/dt max, mmhg. vs), and survival; the nal animals, however, were acidotic (pha . ----- . ) and colder ( . - . °c) than con ( . +__ . , . -+- . ). map responses to nal were proportional to ph a complications of vascular bypass grafts, especiallyin the femoro-popliteal region are common. the most frequent of these by far is occlusion. other late complications iclude leaking and false aneurysm formation at the anastomotic site, dilatation of the graft material and stenosis ofanastomotic sites. the most commonly employed non-invasive studies render little information of value in the diagnosis of these complications. we have recently undertaken a study to evaluate arterial grafts at various intervals after implantation with the use of a duplex ultrasonic scanner. imaging of graft material with this system ist excellent and patency of the lumen can easily be established. graft diameter can be measured and accumulation of material within the lumen can be measured and quantitated. although anastomoses are not always clearly visualised they are often seen satisfactorily for diagnostic purposes. graft dilatation, false aneurysm formation and occlusions can be accurately diagnosed with this method. we have also studied the behaviour of femoro-popliteal grafts across the knee joint using the duplex scanner. as a non-invasive technique scanning with a duplex system has many advantages enabling frequent investigations and therefore early recognition of complications where synthetic material vascular grafts have been used for arterial bypass. an experimental model for dissecting aortic aneurysm has been designed to obtain much better understanding of the disease, leading to more effective methods of surgical treatment. dissection of the descending thoracic aorta was successfully performed by modified blanton's prodedure in % of more than mongrel dogs. fals lumens ruptured distally into true lumens to form a doublebarreled aorta in % of the dogs. the method of surgical treatment performed were ( ) closure of entry by direct suture, ( ) closure of entry by inserting dacron vascular prosthesis with a stainless steel ring, and ( ) bypass grafting with vascular prosthesis and ligation of the thoracic aorta. in dogs treated by methods ( ) or ( ) at the time of performing the dissection, cine angiography revealed that false lumens hat thrombosed within month and dissection was found to have completely healed on autopsy. in chronic cases treated by methods ( ) or ( ), false lumens were all patent with various degrees of formation of mural thrombi at observation of to months after surgical treatment. these results indicate that closure of entry for acute dissecting aneurysm should be a curative procedure. studies are now continuing on chronic dissecting aneurysm. the lack of controlled trials not rarely resulted in the incorrect acceptance o fan ineffective operation as an effective one igation of internal mammary arteries for relief of angina pectoris, for example). this failure also explains the non-stop controversies over the appropriate operation (and whether the question operate at all) for various myocardial revascularization procedures, over the thymectomy for myasthenia gravis, the limited vs radical mastectomy for breast cancer etc. several clinicians and surgeons assert that controlled clinical trials for new operations are unrealistic and naive because of some important differences between operations and drugs. compared with medical trials surgical ones are often really more difficult to be carded out. they are subject to more restrictions, ethical, statistical and practical, and may require longer or even remarkably long times. in any case, patients must nevertheless be sheltered from harmful and ineffective surgical operations since luckily not all the undoubted difficulties are unsurmountable, and prospective controlled trials of surgery, including random allocation of patients, are quite feasible. comparison between surgical and non-surgical treatment is a really particularly suitable field for these studies. comparison of effectiveness of two operations of the same type or of different types can also be carried out, but only on certain conditions and using suitable and appropriate expedients. also the doubleblind approach may be feasible, although remarkable restrictions and adequate cooperation are necessary. acceptance in trials of surgery as placebo is objectively very difficult, and obviously it has to be considered only when an effective operation for the considered disease does not exist. the ethical sound of this delicate aspect of clinical research can be minimized enough only if ( ) there are substantial doubts about the effectiveness of the intended operation and ( ) requested placebo-surgery is of very slight importance. posters for scientific meetings mary evans and a.v. pollock scarborough hospital, north yorkshire y ql , u.k. the display of information in posters antecedes even the,art of writing. they have been used to advertise, to educate and to inform and their function is to disseminate information to a wide audience quickly, simply and effectively. in medicine they have been used since the thirteenth century for the promotion of health education. in recent years poster sessions have been introduced into scientific meetings as an alternative to the spoken paper for the presentation of original work. following this experimental procedure hepatic coma supervenes in - h and animals die after ca. h. five male pigs, aged - months, weighing - kg were studied ca. h after operation. the detoxifying system consisted of activated charcoal (norit ¢ (lmm x ram) and ionexchange resin (dowex lx ), subsequently inserted in an extracorporeal circulation. plasma amino acids were determined serially ( , , , rain) in and out the detoxifying system. plasma extraction of individual amino acids (means_ se in/zmol/min) is reported in the table a remarkable extraction was present in the first rain and chiefly involved aromatic amino acids and tryptophan which did not further increase. during hemoperfusion, the molar ratios between neutral amino acids improved. the ratio of branched-chain to aromatic amino acids increased from late hepatic effects of small intestinal bypass (sibp) for obesity plasmafl-endorphin (/ -end, pg/ml, by ria) was related to t:fl-end = (t- ) + , r= . , p< . . when acid-base balance and temperature were maintained, nal (n= ) significantly (*p< . ) improved map and lv dp pg/ml in nal and + pg/ml in con). r-end is released in and contributes to the cardiovascular depression of primate hemorrhagic shock. nal reverses this depression and improves survival but only when arterial ph and core temperature samples of exudates at site of infection were taken wherever possible for aerobic and anaerobic cultures. in following infections were recorded: wound, respiratory tract, thrombophlebitis, indwelling intravenous catheter, unidentified origin fever (> °c lasting more than days) (fuo) and miscellaneous. incidences: patients ( %) had postoperative septic complications. wound infection was recorded in patients ( . %), respiratory tract infection in ( %), urinary tract infection in ( %), fuo in ( . %) thrombophlebitis in ( . %) and miscellaneous infections in ( . o/ ). predisposing factors: wound infections were / ( . %) in clean operations, / ( . %) in potentially contaminated, / ( %) in contaminated and / ( . o/ ) in dirty.wound infections were diagnosed later (mean th day) in clean than in dirty operations (mean th day) (p< . ). a statistically significant correlation was found between wound infection and leght of preoperative hospital stay: from . % in patients operated on within - days to . % for patients operated after days or more (p< . ). no correlation was found between wound infection and age. urinary infections were more frequent when the patients were catheterized at least once in the postoperative period ( % vs %). a statistically significant correlation was found between the incidence of respiratory infections and duration of anaesthesia ( . %----< min, / > < min, / > min;/~ . ). bacterology: out of cultures gave positive results ( %): aerobes were isolated in samples ( / ); mixed aerobes and anaerobe in ( . o/ ); anaerobes in ( . %). bacteroides fr. was the commonest isolated anaerobe in all types of sample except hemocultures were propionibacterium aches was the most frequent. a statistically significant correlation was found between the incidence of recvery of anaerobes and intraoperative contamination ( . % in clean operations, . % in potentially contaminated, . % in contaminated and . % in dirty; p< . ).in wound infections the most frequent aerobes were staphylococcus in clean and e. coli in contaiminated operations. costs: mean postoperative hospital stay of patients with septic complications was days, whereas patients with no postoperative sepsis were discharged after an average of days (p< . ). mean daily cost in our hospital was extimated $ ; accordingly, the mean postoperative hospital stay of patients with sepsis costs $ vs $ of that o~ the patients without postoperative infections. overwhelming postsplenectomy sepsis/infection has been accepted the highest risk with more than percent mortality due to pneumococcus species. however, e. coli, pseudomonas and staph. aureus have been reported a deadful threat to the splenectomized individual also. pneumococcal challenge after splenectomy in animal experiment and after partial salvage has been well examined. staph. aureus-challenge has not been tried after splenic repair, so far. material and method: a total of nmri-mice ( - g) have been subjected to: sham-operation, splenectomy, peritoneal splenosis, controls as non-operated group.the technique was similar to the one performed in rabbits, prior reported. weeks postoperatively, the mice were exposed to intraperitoneal injection of staph.aureus concentrations of °c/ml down to c/ml. the peritoneal cavity of mice has a high resistance to staph.aureus, as only no. proved lethal. results: whereas the macroscopic view post mortem seemed promissing, showing almost total salvage of the splenic particles weeks postoperatively bacterial challenge with staph.aureus was contradictory: x s c/ml was survived by all splenectomized mice, by only percent of the splenosis micecand by none of the sham and nonoperated mice. due to the small number of only mice within this mice-pathologenic staph. aureus species, we may not draw definite conclusions. as for one, the intraperitoneal application may not be the natural way of septicemia. on the other hand, splenic remnants may not be as effective in the protection of staph.aureus-sepsis as in a pneumococcal challenge. therefore, further studies with staph.aureus species in other animals and by other application route will be needed! studies of the coagulant effects of boomslang (dispholidus typus) venom have indicated that the coagulant effect was mainly due to its ability to activate prothrombin. it also activates prethrombin i, factor x and possible factor ix as well [ ] . although boomslang envenomation is said to represent a classic model of disseminated intravascular coagulation, certain features are worthy of critical thought. the coagulation profile of a nine year old lad, who was admitted to the h.f. verwoerd-hospital, h after a reputed boomslang bite, provided the impetus to explore the in vivo effect ofcurde. d. typusvenom on the thrombelastographic and other haemostatic changes in the chacma beboon. methods: . mg, . rag and . mg respectively of crude. d. typus venom were injected subcutaneously in adult baboons. serial determinations of the flow parameters were done over - days. thrombelastography on whole blood an platelet this report presents the ten year survey of liver mitochondria analized in patients. . two mechanisms were of major importance in the augmentation of mitochondrial ability to synthesize atp: a) the enhancement of atp-generating capacity per unit of respiratory assemblies and b) the increase in respiratory enzyme contents. those compensatory mechanisms were functional within a certain range of contents in cytochrome a ( . - . nmol/mg protein, normal; . ). hepatic insufficiency was observed in patients with cytochrome a contents less than . or more than . . . in all patients with cytochrome a of . - . nmol/mg, the blood glucose level after an oral glucose load ( g) returned toward normal within h (parabolic pattern). in patients with cytochrome a of more than . , the blood glucose level did not return toward normal within h (linear pattern). parabolic and linear patterns were intermingled in the patients with cytochrome a form . to . .in this report, we will emphasize the importance of preoperative ogtt and measurement of cytochrome a contents during operation for the prediction of operative prognosis, better than any other currently used index of liver function, in the patients receiving major surgery such as hepatectomy or operation for esophageal varices in severe cirrhosis. the reticulo-endothelial system (res) has largely been ignored in studies of liver regeneration. in this study, the res was "blocked" with colloidal carbon, or was stimulated with olive oil or with glucan. indices of liver regeneration were the thymidine kinase acitivtiy (tk) and the number of mitotic figures (mi) in liver biopsies. fibronectin was measured as a putative index of kupffer cell function.four animals in each group were sacrified at , , , , , and h after sham-operation or % partial hepatectomy (ph). group : carbon suspension (pelikan ink, wagner) mg/ g rat single injection pre-operative; group : olive oil ( % in % dextrose water + . % tween ) . ml/ g rat single injection pre-operative; group : as group given at intervals of h; group : glucan . rag/ g rat pre-op, and at intervals of h.results: glucan or a single dose of oil increased tk in ph and sham-operated rats but did not influence m.i. carbon inhibited mi but did not influence tk. fibronectin levels were increased in sham-operated and ph rats after daily oil or glucan.in conclusion, no administration enhanced both tk and mi but stimulation of the res with glucan or oil did increase fibronectin levels in both sham and ph rats. it appears that the res does not have a role in liver regeneration as assessed above, but fibronectin appears to have been an indicator of res activity. anatomical abnormalities of the gallbladder include multiseptate and bilobed organs, and the more common transverse septate variant. patients with the latter type often have typical biliary symptoms which are thought to be caused initially by raised intracystic pressure and subsequently by inflammation and calculi formation in the distal lobe [ ] .this study evaluate~ the long term effects of sibp on hepatic lipid and fibrous tissue accumulation. liver was obtained at the time of abdominal operation from normal weight patients, morbidly obese patients (mean body weight + kg) and patients or more years (mean -+ months) postoperative from sibp. from wedge liver biopsies hepatic total lipid, triglyceride, cholesterol, phospholipid and protein contents were determined as well as the activities of acetyl coa carboxylase and fatty acid synthetase. histologic evaluation of needle biopsies of the liver was used to quantitatively estimate the amount of hepatic steatosis and fibrosis present.there was also a significant histopathologic increase in hepatic fibrosis present in the liver tissue from sibp patients when compared to hepatic fibrosis in liver from obese patients as well as when compared to liver specimens obtained from the sibp patients at the time of their sibp. the effect of reversal of the sibp on hepatic fat content and fibrosis was determined by transcutaneous liver biopsy - mos. following take down of sibp. post-reversal histologic quantitation of hepatic fibrosis and inflammation was significantly decreased from pre-reversal values and the improvement was greater in patients who lost weight. patients year after sibp have persistent hepatic steatosis with hepatic fibrosis and inflammation. improvement in these parameters may be anticipated following reconstruction of the intestinal tract particularly if weight loss is maintained. the aim of this paper is to study the metabolic effects of gastric bypass in dogs. we used dogs weighing between and kg. the animals were divided into three groups: a) five dogs without surgical operation (control); b) six dogs with a fundoj ejunal bypass, and c) six dogs with a gastroplasty. in group b and c the exclusion was / of the stomach. in all of the animals and groups were determined: cholesterol , live r function tests, calcium, magnesium-and electrolyte levels, the body weight evolution and the apparent-digestibility coefficient (adc) of fat, protein, minerals and glucosides for a type of diet. the composition of this diet in dry substance was: fat . %, protein . %, glucosides . % and minerals . %. these analyses were determined five times during the twelve months of control. the animals were kept in individual metabolism cages which allow feces and urine to be gathered separately as well as the food consumed to be controlled. the cages were housed in a room thermoregulated at °c± . the cholesterol, calcium, magnesium and electrolyte levels were normal during the twelve months of control in the three groups. the liver function tests were normal in all groups with the exception of serum aspartate transaminase initially deteriored in group b. the body weight evolution was significantly diminished in groups b and c compared with the control group (p< . and p< . ). the results of adc expressed in % ± sd were.by roviding a functional gastric capacity of less than ml and consequently a forced reduction in food intake the gastric bypass produces an effective loss of weigth. our results suggest that the gastric bypass can be considered as an effective and safe alternative to intestinal bypass for treatment of morbidly obese subjects who have failed nonsurgical treatment; however, a great and deeper research is necessary to discover the possible side effects of gastric bypass surgery; then its ultimate benefits will be fully understood. there was no operative mortality. the percent of the excess weight lost following the two operations is shown and compared with weight lost following gastric bypass. it is concluded that: . complications occured more often following gastroplasty, . % compared with . % of the patients treated by gastrogastrostomy. . the amount of excess weith lost was greater after gastroplasty, a mean of % for patients followed for two years, than after gastrogastrostomy, a mean of % for patients followed for two years. . because of the incidence of stomal obstruction requiring reoperation following gastroplasty, . % compared with . % after gastrogastrostomy, is our preferred operation. . to improve weight loss after gastrogastrostomy the ta- is now used instead of the ta- stapling instrument. the proximal gastric pouch is reduced in size to - ml and the stoma made smaller to ram. an artifical esophagus was made of silicone rubber tube covered with a dacron mesh. a segment of thoracic esophagus of dogs was replaced with this graft using three different types ofamastomosis, i.e., overlayer end-to-end anastomosis, two layer end-toend anastomosis both using a flanged tube, and monolayer end-to-end anastomosis with noflange tube. seven of dogs ( %) survived more than months without complications, and of them more than years. in of of the prolonged survivors, extrusion of the graft was recognized in the rd to th month after operation. esophageal stenosis increased slightly up to the th month after extrusion of the graft, but it did not further advance until sacrifice. in these dogs, mucosal regeneration of the neoesophagus was complete with muscle layers and mucous glands in the submucosa recognized microscopically. proximal esophagus from the replaced portion was apparently dilatated more than that of the distal portion. there was no definite difference between the anastomotic techniques with regard to complication or prognosis. these results suggest a possibility for clinical trials. paper withdrawn attempts to reduce nephrovascular hypertension by surgical techniques deviating renal venous blood and renin directly to the hepatic filter are at present discouraging. experimental data with portocaval transposition are contradictory, due mostly to the use of heterogeneous biological models (mongrel dogs) and collateral circulation developement. renal to portal vein end-to-side (ets) and end-to-end (ete) anastomoses in the rat were therefore tested as possible experimental models. we observed that even after right kidney decapsulation, collateral vein circulation develops through the periureteral plexus, particularly after ets shunts, one month after surgery. nevertheless, collateral cirulation in the male rat can be prevented by ligature and cutting offthis plexus near the kidney. in the female rat instead, collateral circulation is still possible. in fact, newly formed pericapsular veins join the right kidney to the right ovaric vein and therefore preventive ligature and eradication of these vessels is also necessary. in conclusion, both models (ete-ets shunt) appear feasible in the rat, and reliable for studies dealing with nephrovascular hypertension and hepatic metabolism of renin. the handling of the exocrine system is one of the main problems inpancreas transplantation. one trial to overcome this problem consists of the occlusion of the pancreatic duct system. a the theoretical disadvantage is the induction of a secretion oedema which may lead to blood flow disturbance followed by venous thrombosis. the aim of the present experiments was to study the effect of an anti-oedematous drug (cumarin and rutin sulphate (venalot ®) on the blood flow of autologous segmental pancreatic transplantation was tested by chosing the cervical region of the dog as the site of grafting. material and methods: dogs weighting - kg were used. these dogs were divided into two groups (control group, n= ; treated group, n= , venalot ® dose: rag, cumarin/kg/b.w.). the body and the left limb of the pancreas was removed, perfusion with eurocollins was started wia the splenic artery and the duct system was injected with prolamine. a distal a. v. fistula of the splenic vessels was performed. this conditioned graft was transplanted at the neck of the same dog, performing the anastomoses between the carotid and the splenic vessels. the blood flow of the pancreatic graft was measured with radioactively labelled microsperes of /~m immediately after, as well as days after grafting. the developing oedema of the graft was estimated by weight gain, histological and electron microscopic investigation. residual exocrine function of the graft was measured by determining the serum lipase and amylase levels.results: the autotransplantation of an segmental pancreatic graft to the neck of a dog is a feasable and technically easy surgical procedure, without major venalot -treated dogs local complications. in the ® there was a higher weight increase of the graft in comparison to the controls. as tested so far there was no difference in the behaviour of the i~lood flow of the grafts in both experimental groups. there was a significant increase of serum amylase and lipase values in the venalot®-trea'ted group. conclusion: the technique of cervical segmental pancreatic transplantation in dogs is recommended in cases where immunological monitoring (aspiration cytology, biopsies) is the aim of the study (good access to needles). the technique of microspheres injection is a useful method for the examination of the blood flow in segmental pancreatic grafts in dogs. the prolamine-induced secretion-oedema of ductoccluded pancreatic grafts is resistant to venalot ®treatment. graft pretreatment has been used in various organs to prolong auograft survival. we have recently demonstrated that graft pretreatment of canine renal allografts with cyclosporin a (ca a) led to improved animal and graft survival. our present study assesses the effect ofcy a as a graft pretreatment on pancreatic islet cell allografts. pancreases were removed from unrelated donor mongrel dogs and placed in iced saline ( °c) after the collagenase digestion. the tissue fragments were washed once more and then another mg cy a was added to the preparation prior to intrasplenic injection of the islet cell allografts. three groups were studied: group i (n= ) served as pancreatectomized non-transplanted controls, group ii (n = ) received a non-pretreated islet cell allograft after total pancreatectomy and group iii (n= ) received a cy a graft pretreated islet cell transplant after total pancreatectomy. all animals were given minimal immunosuppression with azathioprine ( rng/kg/day x , followed by , mg/kg/ day). blood glucose values were monitored to determine engraftment and subsequent rejection. hyperglycemia was considered when plasma glucose values rose above mg/dl. all pancreatectomized controls (group i) became hyperglycemic by the first post-operative day. non-pretreated islet cell allografts in group ii had variable function and became hyperglycemic between one and nine days after transplantation. only five of ten animals in group iii, receiving cy a pretreated islet cells, became hyperglycemic levels greater than days and one died of unknown causes immediately after transplantation. the following table presents the animal survival data for the day follow-up period.although the exact mechanism of action is not known, this study indicates that cy a graft pretreatmerit can be beneficial in prolonging pancreatic islet cell allograft survival, further studies will optimize the use ofcy a in this application and hopefully contribute to the improvement of pancreatic islet cell transplantation. grafting of vascularised organs has become a standard procedure in surgical research. however, only few data of the fate oforthotopicliver transplantation in the rat are available, probably because of the difficult ~iargery involved. this communication gives an account of the outcome of liver allografts and liver isografts in four inbred strains of rat. the following groups of allo and isografts are formed; the survival time is given in days postoperatively.the technical details of the operative procedure of orthotopic liver transplantation in the rat are described elsewhere (eur surg res : ( ). the distribution of death among allallograft groups show blocks of survivors. the first block includes animals surviving up to days. acute rejection and infection are the diagnosed causes of death in this compartment. the second block includes animals surviving up to days. in the grafts, which belong to this compartment a intrahepatic bile duct proliferation is a frequent and dominant histological feature. because of the development of identical lesions in isograft surviving the same periode and the induction of this lesions by common bile duct ligation experiments, chronic rejection is excludet as the cause of death. the third block includes all so called long-term survivors, in which the structure of the grafts are remarkably well preserved and very few abnormalities are present. the occurence of fatal acute rejection and long-term survival in each allograft group demonstrates, that the fate of the orthotopically transplanted livers in not dependent upon key: cord- -mcfnhscj authors: blecha, frank; charley, bernard title: rationale for using immunopotentiators in domestic food animals date: - - journal: adv vet sci comp med doi: . /b - - - - . - sha: doc_id: cord_uid: mcfnhscj nan to losses due to death, economic losses caused by bovine respiratory disease include reduced growth performance and increased treatment costs. these losses emphasize the need for alternative or comple mentary therapeutic approaches, such as immunomodulators, that may be well suited for the multifactorial etiology involved in the disease. in economical terms, mastitis is the most devastating disease affect ing dairy cows. in the united states, losses attributed to mastitis ap proach $ billion each year; % of this economic loss is due to a reduced milk yield as a result of subclinical mastitis (national mastitis council, ) . similarly, a french epidemiological survey found that mastitis was by far the most frequent pathology affecting dairy cows (barnouin et al., ) . vaccination against bacteria that cause intramammary infections has been attempted as a means of decreasing mastitis. however, even in studies that have shown beneficial effects of immuni zation against mastitis, vaccination did not prevent new intramam mary infections (pankey et al., ) . antibiotic therapy is used in the control of mastitis. however, because staphylococcus aureus mastitis responds poorly to antibiotic therapy and because of the problem of antibiotic residues in milk, the effectiveness of antibiotic therapy in mastitis prevention and treatment is limited. these specific examples emphasize the need to continue to search for more effective ways to minimize the impact of disease on animal pro duction. augmentation of the animal's immune response with the in tent of increasing resistance to disease-causing organisms should de crease the economic loss due to disease in food animal production. immunomodulation may provide an effective means of enhancing the ability of domestic food animals to withstand disease. when one considers the possibility of enhancing an animal's immune response, a question that must be addressed is whether specific or nonspecific immunomodulation is desired or required. specific immuno modulation involves the potentiation of the host's immune system toward a unique, specific antigen. vaccination programs are perhaps the best example of producing specific immunity in domestic food animals. nonspecific immunomodulation generally is an attempt to heighten immunologic capabilities at a time when an animal may be exposed to one or several pathogens and/or be immunocompromised. both of these concepts will be discussed further. the distinction between adjuvants and specific immunomodulators is blurred and may be only a matter of semantics. classical and new adjuvants offer the capability of enhancing specific immunity and are discussed in great detail in chapter of this volume. however, some substances that are not generally thought of as adjuvants, such as the interleukins and interferons, also induce a state of specific immuno modulation. for example, peripheral blood mononuclear cells from cat tle injected with recombinant bovine interleukin- display enhanced cytolytic capabilities against bovine herpesvirus-infected target cells (reddy et al., a) . however, protection against a bovine herpesvirus challenge was observed only in animals that received a vaccination against the virus in conjunction with injections of interleukin- . thus, in this case both nonspecific and specific immunomodulation was produced in cattle that were administered interleukin- , but only specific immunomodulation resulted in protection against a viral chal lenge. theoretically, the capability of potentiating the host's immune re sponse at a time when it might be immature, compromised, or overcome with pathogens should enhance the animal's ability to resist disease. this is the rationale for nonspecifically augmenting an animal's im mune response. nonspecific immunomodulation has potential in at least different conditions: ( ) during the neonatal period when the immune system may not be fully developed; ( ) during periods of stressinduced immunosuppression; and ( ) during virus-or bacteria-induced immunosuppression. because of a very efficient placental barrier, pig, horse, and ruminant fetuses are generally very well protected from in utero antigenic stim uli. therefore, although fully immunocompetent at birth, domestic food animal newborns differ from other mammalian neonates in being im munologically "virgin" (kim, ; salmon, ) and the development of totally effective immune defenses requires to weeks. during this critical neonatal period the young animal is highly susceptible to mi crobial infections. postnatal development of immune functions has been most exten sively studied in the pig (sterzl and silverstein, ; kim, ) . most immune parameters that have been studied appeared to be very low at birth and reached adult values at about month of age. thus, the percentage of Τ and Β lymphocytes in peripheral blood, as estimated by Ε-rosettes and anti-ig immunofluorescence techniques, was shown to increase from to % at birth to adult values by days of age (reyero et al., ) . a similar age-related increase has been described for serum concentrations of the third component of complement (c ) in pigs (tyler et al., ) . because of the high incidence and economic impact of respiratory and intestinal infections in young domestic animals, it is important to review studies related to the postnatal development of the mucosaassociated immune system in the pig. at birth, the intestinal, nasal, and tracheobronchial mucosa are devoid of plasma cells. plasma cells first appear in the respiratory tract at - days of age and reach adult values at - weeks of age. this postnatal development was described for cells containing iga as well as igm and igg (bradley et al., ) (chu et al., ; pabst et al., ) . inside the lung, residing at the air-tissue interface and directly exposed to inhaled microorganisms or air pollutants, the alveolar mac rophage functions as the primary defense against respiratory infections (hocking and golde, ) . functional properties of alveolar macro phages, including their immunological and antiinfectious features, have been studied in domestic food animals (khadom et al., ; charley, ) . rothlein et al. ( ) have studied the postnatal devel opment of alveolar macrophages in minnesota miniature swine. these researchers showed that lavage fluids from the lungs of newborn piglets were devoid of macrophages. however, within to days after birth, macrophages gradually appear inside the lung airspaces and adult values are reached at weeks of age. furthermore, macrophages col lected from piglets less than week old showed immature function, i.e., lower phagocytic capacity and enzyme content than adult cells. the postnatal development of lung macrophages appears to depend upon nonspecific antigenic stimulation since germ-free piglets have a much lower number of alveolar macrophages than specific-pathogen-free pig lets (rothlein et al., ) . additionally, alveolar macrophages from young piglets have been shown to be more permissive to pseudorabies virus, yielding higher virus progeny titers, than cells from older animals (iglesias et al., ) . a last example of an immune defect occurring during the neonatal period is given by studies on porcine natural killer (nk) cells. natural killer cell activity in the peripheral blood of newborn pigs is much lower (often undetectable) than the activity of adult cells. this nk cell defect has been observed regardless of the target cell system used: hu man tumor cells (huh et al., ) , virus-infected cells (cepica and derbyshire, a; yang and schultz, ), or porcine tumor b-cells (onizuka et al., ) . of particular interest are the observations that postnatal development of nk cells activity, which requires - weeks in specific-pathogen-free miniature swine (huh et al., ) and in conventionally reared large-white pigs , is de layed in germ-free miniature piglets (huh et al., ) . these data imply that microbial flora play a role in the maturation process of nk cell activity in neonates. , a) . this observation has led to the hypothesis that a nk cell defect could in part explain the great suscep tibility of piglets to coronavirus-induced transmissible gastroenteritis. indeed, adoptive transfer of adult pig leukocytes established functional nk cell activity in recipient piglets and reduced their susceptibility to a tgev challenge (cepica and derbyshire, b) . the examples described above illustrate the existence of several different immune defects (see table i ) in neonatal domestic food animals. this lower functional immune status during the neonatal period could explain some of the neonates' susceptibility to infectious diseases, especially intestinal infections. thus, the potential exists to increase the neonates' immune functions by using immunomodulators. a few studies have been conducted exploring means of enhancing the young animals' immune functions. for example, newborn piglets' nk cell activity was shown to be responsive in vitro to interferon (charley et al., ) , and in vivo to poly i : c (lesnick and derbyshire, ) or bacterial extracts (kim, ) . additionally, isoprinosine has been shown to enhance the immunocompromised immune status of artifi cially reared neonatal pigs (hennessy et al., ) . in the following chapters several immunomodulating strategies will be reviewed and should help to define possible immunotherapeutic approaches to en hance young domestic food animals' resistance to disease. (loan, ; filion et al., ) . the idea that stressed animals are more susceptible to disease generally relies on the assumption that alterations in immunocompetence have occurred (table ii) . indeed, some researchers have suggested that changes in immune function may be a useful indicator of stress in domestic food animals (kelley, ; siegel, ) . over the last decade several review articles have been written on the topic of stress and immunity in farm animals (kelley, (kelley, , (kelley, , (kelley, , observation reference blecha and minocha ( ) ; albani-vangili, ; siegel, ; blecha, a; griffin, ). if stress-induced changes in host immunity predisposes animals to disease, then methods of modulating the immune response in stressed animals should increase disease resistance (blecha, b) . when one attempts to intervene in an animal's response to a stressor, several different approaches can be envisioned (fig. ) . perhaps the best means of reducing the impact of stress on animal health is by providing a less stressful environment. however, deciding which envi ronment or management condition is the least stressful is not a simple or easy task (curtis et al., ; mcglone and hellman, ) . thus, several environments and management conditions have been evalu ated for their influence on immune function blecha et al., blecha et al., , blecha et al., , mcglone and blecha, ; minton etal, ) and for their impact on the physiology of the animal (dantzer and mormede, ) . another approach has been investigated as a method of reducing the influence of stress on susceptibility to disease: blocking the physiologic response to the stressor. the association between stress, neuroendo- crine responses, and alterations in immune function or disease sus ceptibility has been recognized for several years (munck et al., ; kelley, ; griffin, ) . when increased concentrations of glucocorticoids have been associated with lower immune responses, administration of drugs that block the synthesis of corticosterone, such as metyrapone, resulted in an abrogation of the stress-induced immu nosuppression (blecha et al., ) . recently, adrenal blocking chemicals (metyrapone and dichlorodiphenyldichlorethane) have been shown to increase the resistance of stressed chickens to viral and respi ratory infections (gross, ) . finally, when stress-induced immuno suppression has occurred, neurohormones, such as melatonin (maestroni et al., ) , immunomodulating drugs (hennessy et al., ; blecha, b; komori et al., ) , and cytokines (conlon et al., ) have been used to "up-regulate" or restore the immune response. it is likely that a combination of the approaches indicated above will pro vide the best means of reducing stress-induced disease problems in domestic food animals. c. pathogen-induced immunosuppression animals exposed to infectious disease often show depressed immune function. this is the case for several parasitic, bacterial, and viral infections. pathogenic bacteria have been shown to affect immune re sponsiveness of infected animals. thus, pasteurella hemolytica or hae mophilus pleur^pneumoniae, which both cause acute pneumonia in cattle and pigs, have been reported to exert toxic effects on lung macro phages and to alter macrophage phagocytic functions (markham and wilkie, ; bendixen et al., ) . during bacteria-induced mastitis, suppressed responses in lymphocyte proliferation and neutrophil phagocytic functions have been reported (nonnecke and harp, ; reddy et al., b) . immunosuppression of the host is also a frequent consequence of viral infections. several examples of virus-related im munosuppression are well documented in domestic food animals (table iii) , including viral diseases of great economic importance such as infectious bovine rhinotracheitis (bovine herpesvirus type- ) and pseudorabies, which cause severe pneumonia and death in cattle and pigs, respectively. as a consequence of virus-induced alteration of im mune function, animals become very susceptible to secondary bacterial infections. the detrimental effects of these virus-bacteria synergistic interactions are of particular importance in the case of respiratory infections. thus, following an initial viral multiplication in the lung, pathogenic bacteria proliferate, inducing the development of more se- in the production of domestic food animals several situations exist where disease decreases production efficiency. some of these diseases are exacerbated by a lowered or compromised immune response of the host. if immunomodulators can be used to augment immune function at critical periods during the production of food animals, such as the neonatal period, and prior to or during exposure to stressors or patho genic organisms, then the economic loss caused by infectious disease should be reduced. vere and acute lung lesions (jakab, ; babiuk et al., ) . if pathogen-induced immunosuppression can be moderated by immunomodulating substances, then the prospects for domestic food animals to withstand disease should be increased. stimulation of defense mecha nisms, especially lung immune defenses, will likely require activation of local lymphoid cells such as alveolar macrophages (charley, ) . targeting of immunomodulators to the critical organs will require special delivery systems, such as encapsulation in liposomes (fogler et al., ) , which should be considered in the field of domestic food animal immunoenhancement. stress e immunita. obiettivi vet the relative frequencies and distribution of immunoglobulin-bearing cells in the intestinal mucosa of neonatal and weaned pigs and their significance in the development of secretory immunity bovine respiratory disease: pathogenesis and control by interferon viral-bacterial synergistic interaction in respiratory disease enquêt e éco-pathologique continue toxicity of haemophilus pleuropneumoniae for porcine lung macrophages, peripheral blood monocytes, and testicular cells viral infections in domestic animals as models for studies of viral immunology and pathogenesis stress et immunité chez l'animal immunomodulation: a means of disease prevention in stressed live stock cold stress reduces the acquisition of colostral immunoglobulin in piglets suppressed lymphocyte blastogenic responses and enhanced in vitro growth of infectious bovine rhinotracheitis virus in stressed feeder calves adrenal involvement in the expression of delayed-type hypersensitivity to dnfb in stressed mice weaning pigs at an early age decreases cellular immunity shipping suppresses lymphocyte blasto genic responses in angus and brahman x angus feeder calves immunologic reactions of pigs regrouped at or near weaning decreased mononuclear cell re sponse to mitogens in artificially reared neonatal pigs influence of isoprinosine on bovine herpesvirus type- infection in cattle the respiratory tract immune system in the pig. i. distribution of immunoglobulin-containing cells in the respiratory tract mucosa effect of respiratory infections caused by bovine herpesvirus- or parainfluenza- virus on bovine alveolar macrophage functions inhibition of t-lymphocyte mitogenic responses and effects on cell func tions by bovine herpesvirus antibody-dependent and spontaneous cellmediated cytotoxicity against transmissible gastroenteritis virus infected cells by lymphocytes from sows, fetuses and neonatal piglets the effect of adoptive transfer of mononuclear leukocytes from an adult donor on spontaneous cell-mediated cytotocity and resistance to transmissible gastroenteritis in neonatal piglets le macrophage alvéolaire de porc: revue bibliographique effects of immunopotentiating agents on alveolar macrophage proper ties modifications des réactions immunitaires au cours de la peste porcine classique interferon-induced enhancement of newborn pig natural killing (nk) activity lymphoid tissues of the small intestine of swine from birth to one month of age the treatment of induced immune deficiency with interleukin- effects of sow-crate design on health and performance of sows and piglets stress in farm animals: a need for réévaluation new directions in bovine veterinary practice the possible role of stress in the induction of pneumonic pasteurellosis influence of isoprinosine on lymphocyte function in virus-infected feeder pigs in situ activation of murine macrophages by liposomes containing lymphokines bovine herpesvirus- and parainfluenza- virus interactions: clinical and immunological responses in calves stress and immunity: a unifying concept effect of social stress on the occurrence of marek's disease in chickens effect of adrenal blocking chemicals on viral and respiratory infec tions of chickens the effect of social isolation on resistance to some infectious diseases genetic and environmental effects on the response of chickens to avian adenovirus group ii infection air temperature selection guides for growingfinishing swine based on performance and carcass composition critère s biochmique s e t hématologique s d u stres s e t leur s relation s avec le s mécanisme s d e défens e reel immumoglobulin g, a and m antibody response in sow-reared and artificially reared pigs isoprinosine and levamisole immunomodulation in artificially reared neonatal pigs the pulmonary alveolar macrophage natural killing and antibody-dependent cellular cytotoxicity in specific-pathogen-free miniature swine and germ-free piglets. ii. ontogenic development of nk and adcc interactions of pseudorabies virus with swine alveolar macrophages i: virus replication viral-bacterial interactions in pulmonary infection stress and immune function: a bibliographic review immunobiology of domestic animals as affected by hot and cold weather stress et immunité des animaux domestiques. point vét immunological consequences of changing environmental stimuli cross-talk between the immune and endocrine systems whole blood leukocyte vs separated mononuclear cell blastogenesis in calves: timedependent changes after shipping bovine alveolar macrophages: a review developmental immunity in the piglet the effects of ok- on porcine nk and Κ cell system rapid effects of adrenocorticotropic hormone (acth) and restraint stressor on porcine lymphocyte function alleviation of depressed immunity caused by restraint-stress, by the immunomodulator, lobenzarit disodium (disodium -chloro- , '-iminodibenzoate) replication of transmissible gastroenteritis coronavirus (tgev) in swine alveolar macrophages activation of natural killer cells in newborn piglets by interferon induction bovine respiratory disease: a symposium role of the pineal gland in immunity. iii. melatonin antagonizes the immunosuppressive effect of acute stress via an opiatergic mechanism interaction between pasteurella hemolytica and bovine alveolar macrophages: cytotoxic effect on macrophages and impaired phagocytosis the welfare of sows and piglets: an examination of behavioral, immunological and productive traits in four management systems local and general effects on behavior and performance of two-and seven-week-old castrated and uncastrated piglets partial inhibition of the humoral immune response of pigs after early postnatal immunization fluctua ting ambient temperatures for weaned pigs: effects on growth performance and immu nological and endocrinological functions effect of transportation on blood serum composition, disease incidence, and production traits in young calves. influence of the journey duration physiological functions of glucocorticoids in stress and their relation to pharmacological actions the effects of road transportation on peripheral blood lymphocyte subpopulations, lymphocyte blastogenesis and neutro phil function in calves current concepts of bovine mastitis experimentelle untersuchungen zur wirkung einer chronischen aerogenen schadgasbelastung des saugferkels mit ammoniak unterschiedlicher konzentrationen. ii. die reaktion zellularer und humoraler infectionsabwehrmechanismen nh -exponieter saugferkel unter den bedingungen einer experimentallen pasteurella-multocidainfektion mit und ohne thermo-motorische belasturg effects of staphylococcus aureus on bovine mononuclear leukocyte proliferation and viability: modulation by phagocytic leuko cytes nonspecific cell-mediated cytotoxicity of peripheral blood lymphocytes derived from suckling piglets postnatal development and lymphocyte production of jejunal and ileal peyer's patches in normal and gnotobiotic pigs evaluation of protein a and commercial bacterin as vaccines against staphylococcus aureus mastitis by experimental challenge bovine recombinant interleukin- augments immunity and resistance to bovine herpesvirus infection bovine recombinant granulocyte-macrophage colony-stimulating factor aug ments functions of neutrophils from mastitic cows heat-and cold-stress suppresses in vivo and in vitro cellular immune responses of chickens development of peripheral Β and Τ lymphocytes in piglets suppression of neutrophil and lymphocyte function induced by a vaccinal strain of bovine viral diarrhea virus with and without the administration of acth development of alveolar macrophages in specific pathogen-free and germ-free minnesota miniature swine effect s o f ambien t temperature s o n induction o f transmissibl e gastroenteriti s i n feede r pigs immunologica l response s a s indicator s o f stress . world's losse s i n youn g calve s afte r transportation developmental aspects of immunity age-related variations in serum concentrations of the third component of complement in swine congenital infections with nonarbotogaviruses the effect of different climatic environment on metabolism and its relation to time of haemophilus pleurop neumonias infection in pigs physiologic concentrations of cortisol suppress cell-mediated immune events in the domestic pig ontogeny of natural killer cell activity and antibody dependent cell mediated cytotoxicity in pigs key: cord- -ukrk nej authors: bidewell, cornelia; carson, amanda; diesel, gillian; floyd, tobias title: suspected adverse reaction to erysipelas vaccine in sheep date: - - journal: vet rec doi: . /vr.m sha: doc_id: cord_uid: ukrk nej nan the apha and the veterinary medicines directorate (vmd) would like to draw vets' attention to potential adverse reactions associated with the use of porcine vaccines in sheep, especially if footrot vaccines have previously been administered. the apha bury st edmunds veterinary investigation centre was called on to investigate a number of sheep deaths in a lowland flock of mixed age crossbred ewes. lameness due to erysipelas had been diagnosed in a previous lamb crop, so in january , following cascade principles, ewes were vaccinated with porcilis ery (msd animal health) ( ml intramuscularly) six to seven weeks before the start of lambing. a group of triplet-bearing ewes were vaccinated on one day and a further twin-bearing ewes were vaccinated the following day. in the - hours after vaccination, ewes from the tripletbearing group presented with signs of malaise that were attributed to hypocalcaemia. they were treated with calcium, multivitamins and meloxicam. three died and seven recovered; two other sheep aborted. in the twin-bearing group, within hours of vaccination, four sheep had died, had aborted and showed signs of severe lethargy and anorexia. approximately half of the ewes in both groups showed mild lethargy and inappetence hours after vaccination with some mild respiratory signs observed within - hours. most recovered hours after vaccination. by one week after vaccination eight ewes had died and by the last prelambing week, ewes ( per cent) had aborted. a further ewes that lambed produced at least one mummified fetus -the size indicated fetal death around the time of erysipelas vaccination. over the same time period in a group of ewes with the same management that did not receive the porcilis ery vaccine, abortions were less than per cent. the ewes in the first flock had received a first dose of vaccination for clostridial disease (heptavac-p plus; msd animal health) two to three weeks before the incident. sheep entering the flock at months of age were vaccinated against enzootic abortion (cevac chlamydia; ceva), toxoplasmosis (toxovax; msd animal health) and campylobacter abortion (campyvax; msd animal health) and in december all ewes were vaccinated against footrot (footvax; msd animal health). three ewes and six fetuses were submitted for postmortem examination. the lungs of all the ewes were diffusely dark red with a rubbery texture. microscopically, there was pulmonary congestion and oedema; one ewe had disseminated intravascular coagulation. in conjunction with the clinical history, an anaphylactoid reaction to erysipelas vaccine was deemed a plausible explanation for the lung lesions. the abortions were attributed to the suspected adverse reaction and a report was submitted to the vmd. the case is similar to cases seen following the use of moxidectin (cydectin % injection for sheep; zoetis) in sheep that have previously been vaccinated against footrot. cydectin % has a contraindication for use in animals that have previously been vaccinated against footrot. it is postulated that the presence of polysorbate (ps- ), an excipient in cydectin % and the mineral oil base of footvax, produces a non-immunological anaphylactoid reaction. similar reactions have been reported in cattle and people. there have been a small number of adverse reaction reports made to the vmd following the use of porcine erysipelas vaccine in sheep. if sheep are reacting to ps- alone this would suggest they are more sensitive to it than other species in which it is widely used. since there have been no vaccines authorised in the uk to control erysipelas in sheep. subsequently, erysipelas vaccines authorised for use in other species have been used in sheep under cascade principles. while there is no proven link between anaphylaxis and the use of these porcine vaccines in sheep, veterinary surgeons should be cautious when prescribing unauthorised vaccines in sheep and determine whether footrot vaccines have been previously administered. in addition, veterinary surgeons are reminded to report any suspected adverse reactions to the vmd at www. gov.uk/report-veterinary-medicineproblem vaccines not authorised for use in sheep -the responsibilities of vets cydectin % w/v solution for injection for sheep. www.noahcompendium.co.uk/?id=- &template=template_printview polysorbate in medical products and nonimmunologic anaphylactoid reactions key: cord- -wymtn q authors: susanne, rensing; ann-kathrin, oerke title: husbandry and management of new world species: marmosets and tamarins date: - - journal: the laboratory primate doi: . /b - - / - sha: doc_id: cord_uid: wymtn q nan as well as rylands et al. ( ) . hershkovitz ( ) covers all aspects of biology in callitrichidae. selected topics can also be found in kleiman ( ) and rothe et al. ( ) . the callitrichid species that are mainly kept in laboratories are the common marmoset (callithrix jacchus), the saddle-back tamarin (saguinus fuscicollis), the moustached tamarin (saguinus mystax) and the cotton-top tamarin (saguinus oedipus). individuals of these four species are shown in figure . . like other primates, callitrichidae should be housed in social groups, either as pairs, family or peer groups. if animals have to be single-housed for experimental reasons, they should have at least visual and olfactory contact to a compatible animal. depending on the size of the group, callithrichids are usually kept in cages or rooms. cages might cover a part of the room or are more limited in size. in general, cages should be built and placed in a way that monkeys can go up or escape above human eye level. the cages should use the full measure of available height rather than width. cages are usually made from stainless steel or wire on a wooden frame. whilst steel cages are more resistant to regular cleaning, they are heavy to move around. wire on a wooden frame has to be rebuilt often due to frequent cleaning and gnawing and gouging by the animals. cage walls should be made from meshwire, instead of being solid, to allow animals to use them for climbing. the meshwire should not exceed a measure of × mm since very young babies might crawl out of, or get stuck in, the mesh. the cage base should be solid and covered with sawdust, woodchips or hay. animals frequently use the ground to forage for food, and to encounter in social play. the cage size depends on governmental regulations and has to be adapted to the number of inhabitants. the minimum size for a breeding couple should be m × m × . m high. each additional animal weaned needs an extra . m . cages can simply be extended by adding a second cage and removing the side walls or connected by simple walkways. the minimum equipment of a cage for callitrichids consists of a nest or sleeping-box, a sitting shelf, various branches of different size and, on different levels, a water bottle and a feeding bowl. nest-boxes can either be made of metal or wood. whilst metal is better for regular cleaning it needs holes for ventilation. nestboxes made out of wood are advisable as they absorb the moisture better. the nest-box should be placed higher in the cage than natural sleeping sites. it also has to be large enough to allow all animals of the group to sleep in it. if animals need to be caught regularly, it is worth using nest-boxes that can be closed by a sliding door in order to catch them inside. if possible, cages should have a maximum of furnishing that accommodates callitrichid locomotion and behaviour. branches have to be placed in both horizontal and vertical lanes and can be either fixed or swinging. runways on branches can be connected with ropes or hose pipes. shelves are usually used for resting, grooming and playing. although it is possible to keep callitrichids with access to the outside, they need environmental conditions comparable to their natural habitat. generally three factors are important: temperature: °c ( - ); humidity: %; light: hours light/dark. other important factors for callitrichid well-being are smell, noise and view. since marmosets and tamarins communicate through odour, cleaning should not be so intensive that it removes the family smell of the home cage. marmosets and tamarins vocalise a lot and it is attractive for them to hear other animals. the monkeys are usually interested in the voices of the personnel and might accept music of low volume. marmosets and tamarins are nosy and therefore will always try to see as much as they can. neighbouring groups should be visually separated by foliage, a hanging screen or a curtain, in order to avoid stress. however, single housed animals or young peer groups should be able to see each other. handling of animals is inevitable for quarantine, transport, health checks, routine examinations and experimental procedures. in general, no callitrichid species likes to be handled but can be trained to co-operate by receiving rewards like nutri-cal ® or marshmallows. as a rule, handling should only be done by skilled persons, in a calm and careful way, to avoid injuries to both animal and handler. all handling assumes trapping of the animals first. this can be done in several ways depending on cage design and group size. in larger cages and groups, there should be an area which can be used to separate individuals or, as mentioned before, the sleeping box. if there is a passage or corridor that the group has to use, this can also be used for separation of individuals. animals can also be trained to enter a smaller cage or their sleeping box which will then be closed. alternatively individuals have to be caught with a net. it is necessary to identify animals individually. microchip, tattoo, chain collar, cutting of hair at the tail or colouring of ear tufts can easily be maintained. microchips, usually placed under the soft skin in the neck, can get lost by allogrooming or may have to be removed in the case of mri. animals have to be restrained for reading both the microchip and the tattoo, since the numbers are not easily visible due to pigmentation of the skin or hair growing. whilst chain collars have to be checked frequently for the right size, in order to avoid injuries to the animals, cutting or colouring of hair also needs to be repeated on a regular basis. the nutritional status has a major influence on the growth, reproduction and longevity of nonhuman primates as well as their ability to resist pathogenic and other environmental stress (knapka et al., ) . due to their small body size, limited gut volume, and rapid rate of food passage (garber, ) callitrichids require a diet high in nutritional quality and available energy. to develop an appropriate diet, the following items should be considered: • information from feeding in the wild; • information from published nutrient requirements; • food available at the facility; • food preference of animals. callitrichids are omnivorous, consuming both insects and plant items such as flowers and fruits. garber ( ) reported that marmosets spend - % of their feeding time on exudates like gum arabic, with their specially adapted lower anterior dentition and v-shaped mandible. because of their "long-tusked" dentition, tamarins are called opportunistic exudate eaters which benefit from the bark gouging of marmosets (ferrari and martins, ) . the national research council (nrc) recommendations ( ) state that diet of tamarins should consist of % insects, % fruits, % exudates, % nectar and % seeds, whereas marmosets eat % exudates, % fruits, % insects. gum arabic provides the marmosets with a source of complex polysaccharides and carbohydrates, certain minerals, especially calcium (carroll, ) , and tannin. trials, in different callitrichid species, with two different diets with or without gum arabic, showed that the addition of gum slowed the gut passage rate in marmosets, without depressing the digestive efficiency (power and oftedal, ) . marmosets are able to ferment tree exudates with the help of micro-organisms in their bigger cecum (ferarri and martins, ) . the feeding schedule should be orientated to the activity pattern of callitrichidae. active periods occur early in the morning, at noon and during early afternoon. housing conditions are also known to influence the feeding modus like indoors/outdoors group or single housing. when primates are group housed, it is important to ensure that the lowest ranking individual has sufficient access to food and water. food should be offered in feeding bowls well above floor level. in larger groups several bowls should be offered. feeding bowls and water bottles have to be cleaned every day. pellets represent a combination of all essential nutritive compounds. they are best fed ad libitum and should add up to % of the total diet. marmosets eat an average of - g pellets/day. many facilities moisten the marmoset pellets with milk or juice, feeding this as "porridge" in the morning. different commercial pellets are available (ssniff, sds, mazuri, pmi) and are listed for comparison of ingredients in table . . a standard feeding regime in many colonies is a high calory vitamin porridge for breakfast, fruits, vegetables, and a protein source for lunch, and probably insects in the afternoon. varying extras are offered, like cooked rice, noodles, potatoes, rusks, hardboiled eggs, cottage cheese, boiled chicken, cat food, raisins, sunflower seeds, dried figs or dates, peanuts, carob, grasshoppers, mealworms and waxworms during the week. the feeding intervals should be . to . hours. it is difficult to determine how much food an individual consumes on average. on a dry matter basis, an active adult animal consumes approximately % of its bodyweight per day. the following is an overview of special nutrient requirements for nonhuman primates (nhp) with emphasis on callitrichid nutrition (nrc, ) . energy is required to support the basic life functions. the daily energy intake must be sufficient to meet requirements for basal metabolism and activity. the average amount for callitrichids is . to . kcal/g diet. adult cotton-top tamarins consume, on average, kcal/kg lbw and lactating females up to kcal/kg lbw (kirkwood and underwood, ) . fat is an important resource for energy and commercial diets contain about % of the essential fatty acids. dry skin and hair loss are evidence of deficiencies of unsaturated fatty acids in the diet. experimental diets with high saturated fat and cholesterol concentrations lead to arterioscleroses and a higher incidence of arterial aneurysm (mcintosh et al., ) . observations in the field, and also in the laboratory, have shown that callitrichids have a big demand for high quality proteins with up to % originating from an animal source. flurer and zucker ( ) fixed the daily protein requirements between . and . g/kg lbw. ausman et al. ( ) indicated that soy protein is half as effective as lactalbumin and also reduces the iron resorption. essential amino acid requirements for nonhuman primates have not been established (nrc, ) but, in , flurer and zucker reported that arginine and histidine are essential amino acids in adult callithrix jacchus. pathological findings due to protein deficiencies are alopecia, facial oedema, diarrhoea, fatty liver syndrome and anaemia. carbohydrates provide about % of the metabolised energy in the diet. crude fibre concentrations in commercial diets vary between and % but the addition of to % is recommended. clapp and tardiff ( ) described a diet for marmosets consisting of . to % fibre, while power and oftedal ( ) suggested an addition of % of total fibre to the diet. an increase in dietary fibre increases faecal volume and the digestive passage through the gastrointestinal tract, thus reducing the time for digestion. supplementation of fat soluble vitamins must be carried out carefully due to their toxicity in higher concentrations. the nrc recommendation for vitamin a is , to , iu/kg diet. like all other primates, callitrichids are dependent on an external supply of vitamin c of mg/kg metabolic bodyweight. with the lack of uv b light, callitrichids can only utilise vitamin d and so the daily requirement for marmosets is iu/ g lbw and iu/ g lbw for tamarins (knapka et al., ) . in power et al. in the wild, callitrichids have a huge home range. they live in social groups which can comprise up to family members. they spend about % of their day on foraging. in order to compensate for this high activity of the animals in the wild, it is necessary to provide their environment in captivity with some enrichment devices. a starting point for environmental enrichment is to reproduce some of the main features of their natural habitat and to create opportunities for captive animals to develop skills they might need in the wild. enrichment can be offered through several ways, like food, play or encounters. food can become more interesting when fruits and vegetables are offered as large pieces. these food items should ideally be distributed throughout the cage, even at places where animals have to find a way to reach it, e.g. in a container with drilled holes or hanging on a chain from the ceiling. another possibility is to offer live insects, like crickets and mealworms. vignes et al. ( ) described a mealworm feeder as a foraging enrichment device, made out of ml water bottles with holes of . cm diameter and hung horizontally. for species of the genera callithrix and cebuella, the mode of feeding of gum arabic can be altered to provide environmental enrichment. gum is usually provided as a powder to be mixed with water, or as crumbles of different sizes, but gum powder, mixed with water to a thick fluid, can also be painted on clean branches and shelves. more recently, ventura and buchanan-smith ( ) introduced artificial gum trees to stimulate the peculiar feeding skills of marmosets, and de rosa et al. ( ) have observed the use of puzzle feeders. in order to encourage play and explorative behaviour, toys can be provided easily in the form of available laboratory material that will be used and "destroyed" by the monkeys. paper rolls, cardboard boxes, plastic tubes or wooden blocks represent perfect toys for marmosets. majolo and buchanan-smith ( ) introduced different novel objects for enrichment such as film cases containing a marble, or a cup containing ten small plastic test tubes. a foraging tree was made from pvc pipe cut into sections and connected with t-shaped pvc tubes (byron, ) . toys that are built for cages have to be checked for safety to avoid injuries. free ranging callitrichids are usually living as monogamous groups, but can also be encountered as polyandrous or multi-male and multi-female groups. if there is a need for single housing, there should at least be the possibility of visual and olfactory contact with other conspecifics. different sex pairs can be housed together, e.g. with a vasectomised male. same sex pairs have to be introduced by giving them visual contact first and the new homecage should be the cage of the subordinate animal. females are more aggressive than males (scott, l., personal communications) and female-female pairs do not represent natural group compositions and are therefore less stable, with allogrooming being rarely observed (majolo and buchanan-smith, ) . for further information on environmental enrichment see heath and libretto ( ) , kitchen and martin ( ) , poole ( ) and schoenfeld ( ) . reproduction in callitrichids is characterised by several peculiarities: females show ovarian cycles all year round with a high rate of fecundity, males copulate throughout the cycle and, even during pregnancy, with a higher frequency around the time of ovulation (kendrick and dixson, ) . callitrichids are the only simian primates with multiple ovulation. births usually comprise twins, but increasingly triplets or even quadruplets in captivity. shortly after birth, and despite lactation, callitrichids ovulate ( to days post-partum) and can conceive again. in cotton-top tamarins, ziegler et al. ( ) determined different post-partum ovulation periods depending on litter size: . ± days after the birth of twins and only ± . days after giving birth to a singleton. information on the length of the ovarian cycle and pregnancy, as well as the time of occurence of post-partum ovulation, is given in table . . another characteristic of callitrichids is the inhibition of reproduction in sub-dominant females. a group usually consists of only one breeding pair. the presence of a dominant female (normally the mother) prevents lower ranking females (the daughters) from reproducing. however, when an unrelated animal is introduced to a group, polygyny or polyandry are increasingly observed. in the common marmoset, up to % of daughters can ovulate while living in their natal family, but sexual behaviour with the fathers does not occur (abbott, ; hubrecht, ; saltzmann et al., ) . in cotton-top tamarins, none of the daughters ovulates in the presence of the mother (french et al., ; tardif, ; ziegler et al., ) . pregnancy can be detected by abdominal palpation, ultrasonography with a . - mhz probe (jaquish et al., ) or by measuring hormones (e.g. progesterone) in urine, faeces or blood (harlow et al., ) . implantation of the early embryos is superficial. the placenta consists of two discoid parts connected by vascular anastomosese (placenta bidiscoidalis) and is permeable to antibodies (placenta haemochorialis). thus twins are immunological blood chimers. at conception the litter may comprise a higher number than will be born, since marmosets are capable of resorbing individual embryos without disturbance of the development of the others (jaquish et al., ) . resorption can only occur until the end of the embryonic period. death of the foetus in later gestation stages might either cause abortion of the whole litter or mummification of the dead foetus. birth usually takes place at night. observations in multiparous females show that the delivery occurs almost always at the same time (layne, d., personal communications). if the female shows signs of labour during the day something is usually wrong and veterinary examination necessary and a probable caesarian indicated. for many experimental studies, and also colony management, it is important to know about the reproductive status of the females. monitoring can be performed using invasive and non-invasive methods. hormone measurements in blood, urine or faeces are indirect methods since hormone values reflect the function of the reproductive organs. ultrasonography provides direct results through an immediate view of the ovaries and uterus (tarantal and hendrickx, ) . to determine the time of ovulation, all sample collection for hormone analysis, needs to be performed at least twice, but preferably three times, a week. figure . a,b progesterone profile of a common marmoset with natural cycle and with pgf α -application. new pairs should only be made up of fully grown animals in good condition, not younger than months. in immature females, pregnancy may restrict normal growth and very young mothers more often fail to rear their first offspring. it is advisable to place the new pair away from their natal groups in order to prevent stress and the suppression of ovarian cycles by the mother. infants are carried by all members of the family, but participation varies between the different species. this co-operative rearing system is important for the breeding success of the offspring. hearn and burden ( ) developed a rotation system of collaborative rearing of marmoset triplets without depriving them of the maternal and family influence. if available, a foster mother can also raise the new-borns, after marking the babies with urine of the new mother prior to presentation. infants start to eat solid food from week - , stealing the food from the mouths of family members. at the age of days, infants are completely weaned. twin fights can be observed with a higher incidence at the age of eight months. offspring should be removed from their family at the age of eighteen months. irreversible control of fertility can be performed by sterilisation or castration of the male or female. pregnancy can be prevented by injecting the female with prostaglandin f α every three weeks or implanting melengestrol-acetate between the shoulder blades. this mga implant can last for two years (moehle et al., ) . body mass differs significantly between species. marmosets are the smallest of these four laboratory species, weighing between and g, and moustached tamarins are the largest, weighing between and g. in comparison to free ranging animals, those in captivity are up to % bigger (kingston, ) . many new world primates are trained to allow routine blood sampling without anaesthesia. some values like ast, ldh and ck are elevated after ketamine application. the following gives an overview of blood chemistry values of different callitrichid species. urine and faeces can best be collected early in the morning when the light is switched on. a container or a mat can be placed under the cage where the sample will be collected. anzenberger and gossweiler ( ) described a procedure where animals are trained to go into a small compartment of the cage and pee for a reward. hearn ( ) collected -hour urine samples from marmosets, measuring - ml ( . ml). faeces samples can be coloured by feeding nutrient colour. if urine or faeces have to be sampled for hours, animals have to be single housed in a metabolism cage for this period. intramuscular injections, of no more than . ml, can be given into the quadriceps muscle. subcutaneous injections are ideally introduced under the skin of the dorsum, with a maximum volume of ml on each side. the average blood volume of animals is - % of the bodyweight and a maximum of % of the blood volume can be taken in days ( . ml/ g lbw) without expecting health problems. if more is required, up to % fluid has to be substituted. blood samples can be taken from alert animals from the femoral or lateral tail vein with a - ml syringe and a - gauge needle. hearn ( ) developed a restraining device to allow a single person to carry out routine procedures. intravenous injections or fluid administration can be given into the vena saphena, v. cephalica or v. coccygea, using a - gauge catheter. if the animal's veins are collapsed, fluid can be given intraosseally, directly into the bone-marrow of the tibia or femur, using a g disposable intraosseus infusion needle with a t-handle. this needle can also be used to collect bone marrow from the trochanteric fossa, iliac crest or proximal humerus. vascular access ports can also be implanted into marmosets to deliver compounds intravenously, e.g. the v. femoralis (dalton, ) . oral gavage can easily be maintained. semen can be collected by penile stimulation (kuederling et al., ) or electroejaculation (cui et al., ) . osmotic pumps can be placed subcutaneously between the shoulder blades, or intraperitoneally (fortman et al., ) to deliver drugs continuously for up to four weeks without restraining animals. geretschläger et al. ( ) described a method to collect cerebrospinal fluid from anaesthetised marmosets with a gauge scalp vein set, venofix ® , and a ml syringe, collecting . ml per puncture from the cisterna magna. health checks should be made by care takers during feeding schedules, once or, preferably, twice daily, but it should definitely be the first duty, in the morning, to see if an animal is down, in labour or healthy. observations should be made of behaviour and general condition of the animals, such as appetite, attentiveness, locomotion, bodyweight, coat quality, faecal quality, stress between group members and how the animals go to the food, which might be quite difficult in larger groups. if necessary, the veterinarian should be consulted to decide if the animal requires a proper physical examination, including body temperature, rectal and vaginal swab, faecal sample, blood sample, palpation of the abdomen, auscultation of heart and lungs, ultrasound and x-ray if indicated. sick animals should be kept warm (e.g. heating lamp or pad) and, if necessary, the individual should be isolated from its group for intensive care. drug of choice for mild anaesthesia is ketamine ( mg/kg), with a maximum of mg/animal because of its myotoxicity (davy et al., ) . saffan ® is also very safe ( - mg/kg). for longer surgery, a combination of saffan ( mg/kg) and valium ® ( . mg/animal) is reliable in common marmosets, and ketamine ( mg/kg) + midazolam ( mg/kg) for cotton-top tamarins. the combinations, with an inhalation narcotic (e.g. isoflurane) for longer lasting surgery is very effective either with a modified tubus ( . mm) or a face mask. most imported animals arrive in a stressed condition, dehydrated and underfed and should therefore be rested (deinhardt, ) . quarantine duration depends on national regulations but should be a minimum of days. animals should be totally isolated from the rest of the colony. two days after arrival, each animal should undergo a general health examination including a tuberculin-test. this examination is repeated at the end of the quarantine period, and can be done either with alert or anaesthetised animals, depending on the animal. only a short overview can be given about the most common diseases in callitrichids, and for further details see bennett et al. ( bennett et al. ( , , potkay ( ) and savage ( ) . the occurrence of diseases can differ a lot with different housing conditions, and whether animals are imported from the wild or bred in a laboratory, housed outdoors/indoors or behind a barrier system with restricted access of personnel. herpes simplex or hominis can be transmitted to all callitrichids from humans with an active herpes infection, e.g. via saliva. it produces severe ulceration in the facial area, oral cavity and oesophagus and can lead to death within hours with an incubation period of seven days. many facilities therefore recommend that people with cold sores should not work with the animals. the reservoir host for herpes tamarinus is the squirrel monkey. it is transmitted via saliva, bite wounds, capture nets and gloves. after an incubation period of - days, the animals develop multiple ulcerations of lips, eyes and oesophagus and nasal discharge, apathy and anorexia, death occurring from between and days. callitrichids and cebids should therefore not be housed together (king, ) . herpes saguinus was described by melendez ( ) as not leading to clinical symptoms. cytomegalovirus (cmv), which occurs very often as a latent infection in old world primates (owp), seems not to be relevant in marmosets, and has been isolated from the salivary gland of tamarins without clinical symptoms (nigida et al., ) . herpes saimiri is latent in squirrel monkeys without symptoms but leads to malignant lymphomas in callitrichids and old world primates. herpes ateles is also known to produce lymphomas. inoculation with varicella zoster evokes delayed antibody titres but no clinical symptoms. epstein-barr-virus is an established animal model in callitrichids but it does not occur naturally. ramer et al. ( ) described a syndrome of weight loss, loss of appetite, diarrhoea and palpable abdominal mass with grossly large mesenteric lymph nodes. cho et al. ( ) isolated, from these spontaneous b cell lymphomas, an ebv related lymphocryptovirus called calhv- . it is the third herpes virus isolated in callitrichidae. monkeypox is a zoonosis, transmitted through direct contact with lesions on the skin and mucosa of the oral cavity. smallpox, vaccinia and monkeypox infection can be fatal diseases in marmosets characterised by cutaneus erythemateous papules on the tail, hands and feet, anogenital region and abdomen. they are also associated with weight loss (gough et al., ) , and can even be lethal. the role in gastroenteritis of rotavirus, which occurs more often in captivity than in the wild, has not been verified (kalter, ) . mansfield et al. ( ) and thomson and scheffler ( ) , isolated coronavirus from animals with watery diarrhoea and an acute escherichia coli infection. para-influenza virus type i produces symptoms from nasal discharge to pulmonary lesions whereas type ii and iii have been isolated from cotton-top tamarins without symptoms (murphy et al., ) . paramyxovirus saguinus, in combination with gastroenterocolitis and a high mortality rate, in cotton-top tamarins have been described. clinical symptoms include apathy, kachexie, diarrhoea and death within hours. spread as an aerosol, measles is a very contagious disease with high morbidity and mortality up to %. this morbillivirus infection starts with swollen eyelids, fever, nasal discharge, facial oedema and exanthema, ending up with interstitial pneumonia. secondary complications include septicaemia, abortion, metritis, severe dysentery and disseminated intravascular coagulopathy. human measles vaccine and gamma globulin can be used prophylactically. christe et al. ( ) immunised juvenile rhesus monkeys successfully with a canine distemper vaccine. the incubation period for hepatitis a virus, a picornavirus, is - days but animals do not develop clinical symptoms. it is of more importance as an anthropozoonosis, transmitted via urine or faeces, and personnel should be vaccinated. callitrichid hepatitis virus (chv), transmitted by mice, is closely related to the murine lymphocytic choriomeningitis virus (lcmv). clinical findings are anorexia, lethargy and dyspnoea and levels of aspartate aminotransferase, alkaline phosphatase and bilirubin are elevated. outbreaks in different colonies have shown that it is connected with a high incidence of morbidity and mortality (asper et al., ; montali et al., ) . bordetella bronchiseptica causes death in juvenile marmosets and tamarins and clinical symptoms are mucopurulent nasal discharge, fever and pneumonia. antibiotic treatment with doxycycline works well. vaccination is indicated if there is a manifest infection in the colony (brack et al., ) . campylobacter spp. is one of the most frequently isolated organisms from nhp with diarrhoea, or even from asymptomatic animals. campylobacter jejuni is a cause of diarrhoea and enterocolitis in tamarins (paul-murphy, ) producing yellowish, soft mucoid faeces that can also contain occult blood. faecal-oral route is the primary mode of transmission. it is of higher prevalence in wild caught animals and is a sign of poor hygiene in captivity (gozalo et al., ) . erythromycin is the antibiotic of choice (johnson et al., ) . brack et al. ( ) reported cases of erysipelothrix insidiosa septicaemia in red bellied tamarins and common marmosets. pathological lesions were gastrointestinal haemorrhages, hepatitis and myocarditis. a vaccination with porcine erysipelothrix insisdiosa vaccine terminated the infection. pathogenic strains of escherichia coli are an important cause of diarrhoea although not well documented as a cause of diarrhoea in nhp. the enteropathogenic (epec) strain induces watery, non inflammatory, non bloody diarrhoea while the ehec (enterohaemorrhagic) strain induces life-threatening haemorrhagic diarrhoea due to the production of a shiga-like toxin, associated with anaemia and neutrophilic leucocytosis . animals become anorexic, inactive, lethargic and develop a recognised clinical dehydration (mansfield et al., ) . treatments are enrofloxacine and supportive fluids. klebsiella pneumoniae is an opportunistic pathogen that is very common in callitrichids, leading either to sudden death, without prior clinical signs, or pneumonia, septicaemia, peritonitis, lymph node abscessation and enteritis (berendt et al., ) . the strains can quickly develop multi drug resistance due to plasmid-transfer. bronchopneumonia can be related to a pseudomonas aeruginosa infection, with associated conditions including endocarditis, myocarditis, empyema and septicaemia (deinhardt, ) . salmonella spp. can be manifested as gastroenteritis with watery diarrhoea, anorexia and fever with severe dehydration. transmission is via the faecal-oral route or contaminated food and rodents or insects can also be vectors (savage, ) . shigellosis doesn't seem to play such as an important role in new world primates as it does in old world primates, but potkay ( ) mentioned shigellosisas as an important pathogen in callitrichids, seen in concurrent infections with salmonella. in comparison to old world primates, new world primates are not very sensitive to infections with mycobacterium tuberculosis. michel and huchzermeyer ( ) described a case of an anthropozoonosis in a common marmoset, kept as a pet in south africa, with loss of condition and palpable mass in the abdomen, identified as an abscessed mesenteric lymph node. yersinia pseudotuberculosis is of great importance in facilities with outdoor housing. this enterobacterium is spread by rodents. infected animals develop diarrhoea, ulcerative enterocolitis and mesenteric lymphadenitis, associated with hepatosplenic necrosis (mcclure et al., ) . a polyvalent vaccine should be administered in colonies at higher risk. the acanthocephalan prosthenorchis elegans penetrates into the wall of small and large intestines, mainly the lower ileum and caecum as far as the serosa, resulting in ulceration, necrosis, perforation and peritonitis. trichospirura leptostoma is a spiroid nematode that inhabits the pancreatic duct of common marmosets. animals with high parasitosis have moderate to severe fibrosis in the pancreas (hawkins et al., ) . clinical symptoms are weight loss and increased faecal volume. mg/kg fenbendazol sid for days is the most effective treatment. severe pterigodermatitis (rictularia nycticebus) is manifested by diarrhoea, weakness, hypoproteinaemia and anaemia. this spiroid is attached to the small intestines and may be treated with mebendazol or ivermectine. cockroaches are the reservoir hosts and should be eliminated (potkay, ) . giardia lamblia is a flagellate protozoan found world-wide, infecting humans and animals. clinical signs range from none to watery bloody mucoid diarrhoea, associated with abdominal cramps, bloating, anorexia and nausea (kalishman et al., ) . other protozoal infections are balantidium spp. and entamoeba spp. entamoeba histolytica produces cysts in the liver, whereas e. dispar is apathogenic but can also result in diarrhoea, anorexia, weakness, abdominal pain and nausea. natural infections are very uncommon. drug of choice is metronidazol or paramomycinsulfate. new world primates are very sensitive to infections with toxoplasma gondii. transmission is mostly by ingestion of sporulated oocysts shed by felidae, or diaplazentar (potkay, ) . death occurs after - days with non-specific clinical symptoms like anorexia, weakness, fever, coughing, dyspnoe , leukopenia and abortion. filariasis can be overwhelming with thrombus-like occlusions of small vessels in the lung, heart muscle and liver. there are not many case reports of mycotic infections in callitrichids. they can be observed after long and repeated antibiotic treatment. candida sp. is a normal inhabitant of mucous membranes and skin (savage, ) . juan- salles et al. ( ) described a case of intestinal cryptococcosis in a common marmoset with fibrinonecortizing enteritis. geula et al. ( ) reported that % of marmosets, older than seven years, have deposits of β-amyloid in the brain, liver and kidney, without having clinical symptoms. dental abscesses, recognised by a typical swelling beneath the eye, are very common and not only in aged monkeys. the oral cavity should be carefully examined for cracking, splitting or looseness of teeth, exposed pulp cavity or peridontitis. antibiotics should be administered prior to tooth extraction under general anaesthesia. in brack carried out a retrospective study of all callitrichidae at the german primate centre, revealing that % of tamarins and marmosets, older than six months, had an igm-mediated mesangioproliferative glomerulonephropathy. clinical signs can be proteinuria and haematuria. eitner et al. ( ) considered that it is an iga-mediated nephropathy. hemosiderosis is the deposition of the iron pigment, hemosiderin, in the liver, and is a common finding in many new world primates (miller et al., ) . they found % incidence of hemosiderosis in animals with wasting marmoset syndrome. there is also evidence of immune dysfunction and increased susceptibility to infection in affected individuals (desousa, ) . spelman et al. ( ) hypothesised that the clinical disease associated with hemosiderosis in captive lemurs is caused by excessive dietary iron intake, high dietary ascorbic acid and low amounts of tannin. sergejew et al. ( ) treated iron overloaded marmosets successfully with different chelations. osteomyelitis can occur after bite wounds on the digits or tail. disinfection with poviodine and antibiotic administration are the best prophylactic measures. amputation of digits may be necessary. wasting marmoset syndrome (wms) is a disease of poorly understood aetiology. it may be related to stress, malnutrition (e.g. protein deficiency, too much fruit in the diet), parasitic, bacterial or viral infections or colitis. clinical symptoms are rapid weight loss (> % of lbw/week), dull fur, alopecia, particularly on the tail and breast, muscle atrophy, diarrhoea or obstipation colitis. clinical pathology includes normochromic anaemia, thrombocytosis, hypoproteinaemia, hypoalbuminaemia, and elevated alkaline phosphatase levels (logan and khan, ; tucker, ) . lewis et al. ( ) compared the faecal microflora of healthy marmosets with animals who developed wms. the latter showed an increase of bacteriodes and fewer lactobacilli. the value of dietary lactobacilli in nhp is a largely unexplored area. a chronic inflammation may be the primary insult in the development of wms in many marmosets. treatment with antibiotics and supportive care with fluids, immune modulators, vitamin d and calcium, ensure ® , nutri-cal ® may help. there are very few cases of diabetes mellitus in nwp (howard and yasudu, ) . metabolic bone disease is mostly associated with a diet low in vitamin d and calcium and the lack of sunlight, followed by osteomalacia and secondary hyperparathyroidism (hatt and sainsbury, ) . clinical findings are bone fractures. multiparous females are of higher risk because of their high demand for calcium and d during pregnancy and lactation. nasopharyngeal squamous cell carcinoma was described in two related marmoset colonies by betton ( ) and mcintosh et al. ( ) , and characterised by conjunctivitis, mucoid nasal discharge and exophthalmos. gozalo et al. ( ) described a case of a renal hemangiosarcoma in a moustached tamarin. in captivity, cotton-top tamarins develop colon adenocarcinoma spontaneously, with a high incidence of up to % of adult animals aged - years. if chronic diarrhoea and weight loss are present, colon carcinoma should be considered. animals respond to sulfazalazine treatment given for more than two months (clapp, ) . dystocia is frequently observed in callitrichids and not only in primiparous animals. caesarean section is indicated if the female shows signs of labour for more than one hour. it is also possible that the females deliver one or two babies without problems and the last baby has to be delivered via c-section. retentio secundinaria has been observed very rarely. a case of placenta previa is described by lunn ( nonhuman primates in biomedical research: i biology and management nonhuman primates in biomedical research, ii. diseases exotic animal formulary handbook: marmosets and tamarins in biology and biomedical research proc. natl. acad. sci. usa a primate model for the study of colitis and colonic cancer: the cotton-top tamarin (saguinus oedipus) ecology and behavior of neotropical primates the laboratory nonhuman primate living new world primates (platyrrhini), with an introduction to primates the biology and conservation of the callitrichidae nonhuman primates in biomedical research: i biology and management primates: the road to self-sustaining populations nutrient requirements of nonhuman primates zoo and wild animal medicine: current therapy the ufaw handbook on the care and management of laboratory animals biology and behaviour of marmosets. proceedings of the marmoset workshop göttingen the cotton-top tamarin husbandry manuel any correspondence should be directed to susanne rensing, department of animal health, covance laboratories, kesselfeld , d- , muenster, germany. key: cord- - su uan authors: lynteris, christos title: introduction: infectious animals and epidemic blame date: - - journal: framing animals as epidemic villains doi: . / - - - - _ sha: doc_id: cord_uid: su uan the introduction to the edited volume summarises the chapters of the volume and discusses their contribution in the context of current historical and anthropological studies of zoonotic and vector-borne disease, with a particular focus on how epidemic blame is articulated in different historical, social and political contexts. of 'emerging infectious diseases' (eid), which configures the rise of new diseases as carrying with it a potential for human extinction. this volume examines the history of the emergence and transformation of epidemiological and public health framings of non-human disease vectors and hosts across the globe. providing original studies of rats, mosquitoes, marmots, dogs and 'bushmeat', which at different points in the history of modern medicine and public health have come to embody social and scientific concerns about infection, this volume aims to elucidate the impact of framing non-human animals as epidemic villains. underlining the ethical, aesthetic, epistemological and political entanglement of non-human animals with shifting medical perspectives and agendas, ranging from tropical medicine to global health, the chapters in this volume come to remind us that, in spite of the rhetoric of one health and academic evocations of multispecies intimacies, the image and social life of non-human animals as epidemic villains is a constitutive part of modern epidemiology and public health as apparatuses of state and capitalist management. whereas the above approaches (including microbiome studies, and 'entanglement' frameworks in medical anthropology) do contribute to a much-needed shift in the intellectual landscape as regards the impact of animals on human health, their practical and political limitations are revealed each time there is an actual epidemic crisis. then, all talk of one health, multispecies relationships and partnerships melts into thin air, and what is swiftly put in place, to protect humanity from zoonotic or vector-borne diseases, is an apparatus of culling, stamping out, disinfection, disinfestation, separation and eradication; what we may call the sovereign heart of public health in relation to animal-borne diseases. for the maintenance and operation of this militarised apparatus, the framing of specific animals as epidemic villains is ideologically and biopolitically indispensable, even when blame of the 'villain' in question lacks conclusive scientific evidence (see thys, this volume). going against the grain of scholarship that in recent years has sought to portray the vilification of animals as hosts and spreaders of disease as a thing of the past, histories of non-human disease hosts and vectors aims to illuminate the continuous importance of this ideological and biopolitical cornerstone of modern epidemiology and public health. representations of animals as enemies, antagonists or sources of danger have, in different forms, shapes and degrees, been part and parcel of human interactions with the non-human world across history. it is, however, only at the turn of the nineteenth century that, as a result of bacteriological breakthroughs, non-human animals began to be systematically identified and framed as reservoirs and spreaders of diseases affecting humans. to take one famous example, before the end of the nineteenth century, rats were not believed to be carriers or spreaders of plague or any other infectious disease. whereas rats had long been considered to be damaging to human livelihood, due to consuming and spoiling food resources, their only redeeming characteristic was, erroneously, widely believed to be their supposed disease-free nature. hence while mid-seventeenth-century plague treatises noted the rat's destructive impact on fabrics and food, no mention of its connection with the disease was made. equally, two centuries later, when in - british colonial officers in india observed that, at the first sight of rat epizootics, garhwali villagers fled to the himalayan foothills in fear of the 'mahamari' disease, they dismissed this behaviour as merely superstitious. however, the bacteriological identification of rats as carriers of plague or mosquitoes as carriers and spreaders of yellow fever and malaria, at the end of the nineteenth century, was itself enabled and indeed complicated by an already-existing stratum of signification which, by the mid-seventeenth century, had led to the introduction of new symbolic, ontological and legal frameworks of thinking about animals as 'vermin'. vermin, in mary fissell's definition, 'are animals whom it is largely acceptable to kill', not because of some inherent characteristic they possess, but because, in specific historical contexts, 'they called into question some of the social relations which humans had built around themselves and animals'. paraphrasing fissell, we may say that, arising in early modern europe, the category 'vermin' problematised animals which devoured or destroyed the products of human labour and the means of human subsistence in terms of an agency or intentionality that confounded human efforts to control them. departing from the structuralist influences of mary douglas, which dominated animal studies in the s (see, for example, robert danton's work on the great cat massacre in france), and from keith thomas' 'modernisation' reading of vermin as simply animals that were of no use in an increasingly utilitarian world, fissell's discourse analysis of popular texts on vermin from seventeenth-century england was the first to dwell in the social historical reality of the emergence of this notion. however, more recent studies have opposed fissell's idea that what made vermin a threat to 'human civility' was their perceived 'greed and cunning', or their overall 'trickster' character. lucinda cole's recent monograph imperfect creatures argues that, 'what made vermin dangerous was less their breedspecific cleverness or greed than their prodigious powers of reproduction through which individual appetites took on new, collective power, especially in relation to uncertain food supplies'. the two approaches are not mutually exclusive. indeed, if approached anthropologically, they point to an entanglement between symbolic and economic aspects of vermin as threats to 'social integrity', something that is further supported by the association of vermin at the time with vagrancy and the poor. medical historians have in turn noted the association of vermin with miasma in disease aetiologies and public health practices of early modern europe, especially in times of epidemics when extensive legislation against them and prescriptions for their destruction are recorded. this was particularly the case in the context of plague outbreaks that had long been associated with 'putrid' and 'corrupt' vapours, which certain animals, like dogs, pigs, cats and poultry (and their excrements and carcasses), were believed to emanate. as in the late middle ages, the fear of pestilential miasmata emanating from offal and other meat products had led to the spatial regulation of butchery in england and other parts of europe (cf concerns with 'bushmeat' in relation to ebola; thys, this volume), william riguelle has shown that, in the course of the seventeenth century, concerns with 'noxious' animals played an important role in instituting limits of where these could be kept and where they could be allowed to roam in urban environments. the idea of miasma would continue to impact medical thinking into the nineteenth century. as a part of ontologies that escape both the straightjacket of recent anthropological classifications and classical medicalhistorical dichotomies of contagionism/anti-contagionism, the idea of miasma was malleable, adaptable and ambiguous enough to be compatible with, rather than antagonistic to, that of infection and contagion. however, as new medical and biopolitical challenges arose in the context of colonial conquest, the problematisation of animal-derived miasma or 'febrile poison' gave way to concerns about the climate as the driving force of epidemic disease. thus while the dawn of bacteriology, by the s, did not introduce understandings of animals as sources of disease ex nihilo, it did mark a drastic return to this idea, and, at the same time, led to a significant conceptual shift as regards the ontology of the diseases transmitted, and the mechanism involved in this transmission. this transformation was catalysed by an intense medical, economic and political interest and concern over cattle epizootics, which, as historians have shown, catalysed both the emergence of veterinary medicine and the medicalisation of animals across the globe in the second half of the century. as regards infectious diseases affecting humans, the medicalisation of non-human animals and their transformation into 'epidemic villains' involved an interlinked, two-part framing of their epidemiological significance: on the one hand, as spreaders and, on the other hand, as reservoirs of diseases. the historiography of the identification and study of non-human animals as spreaders of infectious diseases has for some time now stopped being the foray of heroic biographies of men like ronald ross, paul-louis simond or carlos chagas. focused on the social, political and epistemological histories of scientific studies of zoonosis and vector-borne diseases, historians, anthropologists and sts scholars have underlined the ways in which, within epidemiology, bacteriology and parasitology, non-human animals constituted active agents in complex networks of power and knowledge, and how they assumed different epistemic value in diverse colonial and metropolitan contexts. framed as spreaders of infectious diseases, animals also came to play an important role in what charles rosenberg has famously described as the dramaturgy of epidemics. assuming a protagonistic role in a series of epidemic and public health dramas, animals came to be seen as the ultimate source of disease outbreaks. no longer simply a nuisance or 'pests', the transformed image of a series of animals (mosquitos, rats, ticks, lice and flies in particular) as enemies of humanity was invested with militaristic tropes and colonial moralities. these animals formed as it were a global repertoire of disease spreaders, while at the same time assuming importantly diverse local forms, often in interaction with concerns and social imaginaries about other, regionally specific, disease hosts and vectors (beetles, bats, sandflies, etc.). while it is not in the scope of this introduction to map these 'glocal' interactions, deborah nadal's chapter in this volume provides a detailed picture of the longue durée of dogs as spreaders of rabies in india. nadal's chapter underlines the complex and important semiotic and ontological workings and re-workings on dogs as spreaders of rabies from colonial india to our times. with dog-borne rabies being recognised as an important public health problem across the globe since the s, in india, where rabies is endemic, human understandings of the particular zoonosis were linked to practices of classifying dogs. for british colonials, distinguishing between rabies-prone and rabies-impervious dogs was key to the imperial project of mastery over both indian society and 'nature'. within the confines of tropical medicine and its biopolitical imperatives, the management of rabies made crucial the definition of dog-human relations in terms of ownership. believed to be able to spontaneously develop rabies, for the british, 'ownerless' dogs presented a distinct danger for the colony. seen as the source of infection amongst owned dogs (which were considered unable to develop spontaneous rabies), these animals, nadal argues, challenged victorian morality and were associated with two key notions: on the one hand the notion of 'stray', with its overtones of vagrancy, and, on the other hand, the notion of the 'pariah'-an anglicised caste term used by british colonials to refer to outcaste or untouchable communities. at the heart of these classifications lied ideas about domesticity and wildness, as well as a pervasive social hierarchical mentality. perceiving street life in general as a threat to colonial rule grouped dogs of distinct social status and social life under one, infectious category. transforming 'strays' from 'vermin' and 'nuisance' into epidemic villains that should be sacrificed in the name of human health was not, however, a frictionless process but, as nadal shows us, one that embroiled indian society in debates about the value of life and compassion (led by both anti-vivisectionists and mahatma gandhi). after , 'catch-and-kill' of dogs for the control of rabies continued unabated but also involved indian society in renewed debate involving civil society activists, animal welfarists and political parties. in nadal's reading, these dog-related conflicts underlined a lingering problem pertaining to the classification of dogs vis-à-vis rabies: the persistence of the term 'stray' (inclusive of its 'pariah' associations). the solution since , nadal argues, has been the emergence of a discourse around 'street dogs', which has marked a shift towards an accommodation between different attitudes towards the particular animals, allowing for the concept that they can be both masterless and hygienic. nadal's chapter thus points out that, at the same time as what we may call high-epidemiology redefined experiences of non-human animals as spreaders of disease, it also instituted regimes of hygienic hope. envisioning and putting in place programs of increasing separation between humans and non-human disease vectors became the hallmark of public health from onwards. whether this involved rat-proofing, ddt spraying, mosquito nets, the cleaning of streets from stray dogs or the drying of swamps, this sanitary-utopian aspiration to liberate humanity of zoonotic and vectorborne diseases was based on a vision of universal breaking of the 'chains of infection'; a separation and, at the same time, unshackling of humans from disease vectors that was aimed at confining pathogens in the animal realm. in this way, whereas separation from animals was seen as a sufficient means of protection of humans from zoonotic and vector-borne diseases, animals themselves were defined as ultimately hygienically unredeemable-they were, in other words, rendered indistinct from disease. hence, the naturalist ontology of the enlightenment, which in philippe descola's anthropological model defines humans and animals as unified under the rubric of nature, was unsettled by a radical divide that saw disease as a mode of being which was only inherently proper to non-human animals, and only tentatively, or, as sanitary utopians would have it, temporarily, part of the human species. sayer's chapter in this volume focuses on the - plague outbreak in freston (suffolk, uk)-the last outbreak of plague in the history of england-and excavates the epistemological, political, class and colonial history of such a regime of prevention and hope. analysing what she calls 'the vermin landscape' of the outbreak, sayer focuses on non-human animal actors so as to show that, in spite of the widespread epidemiological acceptance of the rat flea (xenopsylla cheopis ) as the true spreader of plague, ideas about locality and class created a medico-juridical matrix where it was the rat that constituted the main object of scientific investigation and public health intervention. situating the suffolk outbreak both within the third plague pandemic and within british imperial science politics, sayer stresses the ways in which suffolk was connected to india, as the prime locus of the pandemic and of plague science in the empire. as the outbreak in suffolk was experienced as an echo of the ongoing devastating epidemic in india, the rat became an object of epidemiological concern and fear. what if infected rats moved from the rural hotspots of the epidemic into urban areas, transforming them into the equivalents of plague-ravaged bombay on english soil? such fears were fostered not just by the perceived natural traits of rats (as invasive of migratory animals), but also through their association with the rural poor. tapping into complex imaginary registers involving victorian systems of class-related disgust, the english rural idyll, and the image of 'the labourer's country cottage […] as literal and figurative representation of the state of the nation', sayer argues that, 'because this rested in turn on the state of the rural labouring class, and that class were said here to be unsanitary and their cottages invaded by rat and plague, the indian racial other therefore ghosted a new category of (dead) undeserving poor'. as epidemic villains, in the eyes of epidemiologists and public health authorities, rats indianised the dwellings of rural labourers in suffolk. as 'plague was equated with "rat plague"', plague also became indian plague, and in turn necessitated control measures and legislation aimed at 'codif [ying] the rat in law and normalis[ing] its destruction'. formulated around an entanglement of class and interspecies relations, the suffolk plague crisis led, on the one hand, to an increasing medico-juridical investment of the rat in england, while, on the other hand, to a systematic neglect of 'the hares, cats, dogs that featured in gamekeepers' and labourers' narratives of the disease'. identifying and investing on a non-human epidemic protagonist (the rat) led to, and indeed required, a disinvestment and neglect of other species involved in the spread of the disease, and-perhaps most crucially-to overlooking the ecological complexity of disease persistence and transmission between different species in any given ecosystem. the rats and mice (destruction) act , 'which tasked every british citizen with a legal obligation to remove rats from their property', was the pinnacle of the configuration of the rat as an epidemic villain in england and of the institutionalisation of sanitary regimes of hope as regards the prevention of animal-borne infection. having conquered the globe by the mid- s, this regime of prevention and hope came to an end with the dawn of the emerging infectious diseases framework in the early s, when scientists began to focus on processes leading to new diseases, hitherto of non-human animals, infecting humans and to the 'specie-jump' processes (so-called spillover) leading to this phenomenon: 'rather than revolving around already-existing pathogens and how they circulate in specific ecological contexts, the focus on emergence required a shift of attention to what we may call "viral ontogenesis"'. over the past years, the rise of 'emergence' as the central framework of studying and understanding infectious diseases has led to a radical shift of scales and a reinvestment on zoonotic diseases that has been tied to a shift away from prevention towards preparedness. this is a regime of biosecurity that, as anthropologists like andrew lakoff, frédéric keck and carlo caduff have shown, is based on the anticipation of an unavoidable pandemic catastrophe, and which sets in place technologies of biosecurity that have come to increasingly dominate the realm of global health. envisioned as inevitable and catastrophic, 'emergence' has thus radically transformed the status of animals as epidemic villains. on the one hand, whereas in the sanitary-utopian framework of highepidemiology, animals were considered to be isolatable carriers of disease, in the eid framework infection is rendered inevitable. and, on the other hand, whereas for the sanitary-utopian framework, animal-human infection posed a limited threat to humanity, for eid it poses an unlimited one, or to be precise one associated with existential risk. it is telling that the mytho-historical event defining the conceptual horizon of the sanitaryutopian framework was the black death. believed by to have been rat-borne bubonic plague, the fourteenth-century pandemic was used by moderns as a key cautionary tale, and at the same time as a potent medical metaphor: black death was something that could 'return' (as hundreds of reports and news items made clear during the third plague pandemic) but whose impact would be effectively limited by grace of modern medicine and sanitation. on the other hand, as caduff has shown, the mytho-historical event defining the conceptual horizon of eid is the flu pandemic. the political ontology of this event for our contemporary pandemic imaginary is distinctly different from that of the black death for the early-tomid-twentieth-century public. for, as every contemporary epidemiological report and news broadcast makes clear, were an event like 'the spanish flu' to occur again today, globalisation and modern transport would transform it to an event of human extinction proportions; something not only nonpreventable, but whose control, once it has begun, is not guaranteed. both of these mytho-historical events have non-human animals at the heart of their causation narrative: the black death (at least so scientists believed at the time) rats, while the flu birds, probably chicken. however, while the sanitary myth of origin of the black death portrayed the rat as an ancient enemy of humanity whose days were numbered due to the advancement of science, the eid myth of origin frames chicken as just one example of a host of unknown species from which the 'killer virus' may emerge and against which the only action we can take is being prepared. séverine thys' chapter in this volume explores the consequences of the eid approach to non-human animals, as it applied to 'bushmeat' in the context of the recent ebola epidemic in west africa ( - ), with a focus on the impact of epidemiological and public health framings of 'bushmeat' hunting, butchering and consumption. especially affecting 'forest people' in macenta, guinea-conakry, the framing of a fluid host of animals as the source of epidemiologically illicit meat relies on persistent colonial tropes that imagine the 'tropical jungle' as an originally natural realm whose disturbance by human activity leads to the emergence of killer viruses. rehearsed time and again in films like outbreak ( ), this mortal link between nature and culture, thys reminds us, is currently being mediated by the figure of the bat-the in-between figure of a 'rogue' animal, which, james fairhead has shown, is being increasingly deployed as an epidemiological bridge in several zoonotic scenarios (ebola, mers, sars). thys follows other anthropologists in pointing out that this insistence on 'bushmeat' and contact with fruit-bats frames local cultures as pathogenic, in line with paul ewald's notion of 'culture vectors', and thus 'obscure[s] the actual, political, economic, and political-economic drivers of infectious disease patterns'. framed in terms of a 'transgression of species boundaries', ebola spillover events are thus pictured as resulting from a life led according to 'traditional' (and the implication is irrational) classificatory systems that fail to maintain 'us vs. them' boundaries. replete with visual and affective structures of disgust, this view, thys argues, is not challenged by the one health framework, which 'should provide a more nuanced and expanded account of the fluidity of bodies, categories and boundaries' so as to 'generate novel ways of addressing zoonotic diseases, which have closer integration with people's own cultural norms and understandings of human-animal dynamics'. key to this, according to thys, is to recognise and examine the historically dynamic nature of these classificatory and more broadly ontological systems (a view shared by nadal, this volume), and the explanatory models with which they are entangled. thys outlines the complex matrix of uses of non-farmed meat in the region (for nourishment, medicaments, trophies, etc.) and their transformation under the weight of regional and global commodity market networks. one may add that what is often neglected is the fact that 'bushmeat' was used by colonial authorities as a reward to local communities; in angola, for example, the portuguese rewarded local communities with 'bushmeat' for rat-catching in the colonial power's effort to contain plague during the s. the political investments of non-human animals as disease spreaders are further explored in gabriel lopes' and luísa reis-castro's chapter in this volume on the history of the aedes aegypti mosquito in modern brazil. following the social life of the particular mosquito species from the s until today, lopes and reis-castro stress that, while recognising that it has always constituted an 'epidemic villain', we need to pay closer attention to the particular diseases to which this villainous character has been linked to, and to the corresponding political system under which this identification has been undertaken, over the course of modern brazilian history. at the beginning of the twentieth century, aedes aegypti was associated with 'underdevelopment' as a key overarching ailment of brazil, with 'the image of a plagued country swarming with mosquitoes' filled with yellow fever playing an important role in bringing health under the rubric of the state and its modernising agenda. lopes and reis-castro follow gilberto hochman's classic work on the linkage between sanitation and nationbuilding in brazil in stressing that what began as a project of 'civilizing the tropics' by eliminating yellow fever across the country transformed by the early s into a more modest programme of preventing outbreaks in urban centres. by contrast to the liberal nation-building sanitary-utopian visions of oswaldo cruz and his collaborators in the first decades of the twentieth century, in the second half of the s a renewed focus on aedes aegypti was underscored by the politics of democratisation, following the end of the -year-long military dictatorship in . as by april it had become identified with dengue fever, as a new disease to plague urban 'areas marked by racialised histories of state abandonment and violence', the aedes aegypti became associated with a disease that was not as lethal as yellow fever, and which bore with it the sign of social, political and economic restitution. as public health had been the pejorative of left-wing and other democratic forces during the last decade of the dictatorship, calls to control dengue-carrying aedes aegypti as an embodiment of state violence and neglect contributed to the success of the 'sanitary reform movement' and the establishment, in , of brazil's sistema Único de saúde. lopes and reis-castro then turn their attention to the latest incarnation of aedes aegypti as a spreader of the zika virus. unfolding during the years of the impeachment (or judicial coup, depending on one's point of view) against dilma rousseff, the appearance of zika in brazil involved aedes aegypti in an international emergency. lopes and reis-castro examine the political struggles around zika-related mosquito control and argue that, focused on social inequality and the 'uneven effects of climate change', this new framing of the aedes aegypti on the one hand continues a longestablished practice of problematising it as a disease vector with specific political and political-economic parameters, while, on the other hand, introducing important gender-related critiques of public health. hence, while the authors claim that, 'the specific kind of virus in mosquitoes' bodies shaped what kind of epidemic villain the mosquito became', they also stress that, 'the mosquito as a vector carried not only three epidemiologically distinct viruses but very different political desires, struggles, and debates'. focusing on the recent zika crisis, in their chapter to this volume gustavo corrêa matta, lenir nascimento da silva, elaine teixeira rabello and carolina de oliveira nogueira in turn argue that the focus on mosquitoes' guilt and on the technological strategies developed to control these vectors unfolded within a context of profound political instability, and at the same time of epistemic uncertainty regarding key epidemiological traits of the disease. framing aedes aegypti as epidemic villains in this context, diverted attention from issues of social, economic and environmental injustice and inequality that were driving determinants of the outbreak, and legitimised the absence of governmental measures regarding the latter in response to the epidemic. the 'enactment of a global enemy, aedes aegypti, as the villain of the epidemic' thus allowed the brazilian government to paint an all-too-familiar and deceptive picture of a promethean struggle of the country as a unified whole (notwithstanding its enormous and often violent class, race, gender and ideological discrepancies and antagonisms) against a vile creature, which was solely held responsible for the disease. drawing on critical medical anthropological perspectives, matta et al. thus underline the structural violence inherent in both the discourse of epidemic villains and in the policies built and legitimated by this discourse. brazil's mosquitocentred policy in the face of zika, financially, politically and morally boosted by the declaration of public health emergency of international concern (pheic) by the who, relied on a securitisation framework that rhymed well with the broader neoliberal turn of the country and mobilised the image of the mosquito as a public enemy to create a spectacle of national unity that obscured 'iniquities, poverty, the skin colour of those bitten by mosquitoes, the house and streets where these fly, and the environment where they lay their eggs'. as mark honigsbaum has shown, disease ecology frameworks, arising in the usa in the s, framed non-human animals not simply as spreaders of infectious diseases but also as their 'reservoirs'. the 'great parrot fever epidemic' of - involved pet parrots in an epidemic panic across the globe, with a particular focus in the usa. as readers of the colonialist bande dessiné exemplar, tintin in the congo (published in the shadow of the epidemic in ), may remember, psittacosis (caused by chlamydia psittaci) is a zoonotic disease carried by parrots and parakeets that can infect humans. however, for karl f. meyer, a key contributor to the development of disease ecology, the ability of parrots and parakeets (popular pets at the time in the usa) to be asymptomatic carriers of the disease posed a more important problem that the immediate epidemic crisis; especially, honigsbaum explains, as '[t]hese latent infections were a particular problem in california where during the depression many people supplemented their incomes by breeding parakeets in backyard aviaries'. the discovery that psittacosis was not simply an 'exotic' disease imported to the usa by parrot traders, but one that had established itself endemically in american aviaries transformed the structure of epidemic blame from one focused on an outbreak to one focused on an endemic and, at the same time, from one revolving around an exotic invasion to one regarding unhygienic infrastructures at home. more profoundly, it also contributed to a shift towards a reframing of animal-borne disease in terms of disease ecology, a process which involved several decades of studies and interdisciplinary exchanges, but was ultimately triggered by an integration of charles elton's pathbreaking understanding of animal zoology in the realm of epidemiology. what is less well recognised historically is that the notion of the reservoir had a long history in epidemiological reasoning predating disease ecology. rats in particular were suspected, from as early as , as not only spreading plague (via their flea, xenopsylla cheopis ) but as also contributing to the maintenance of persistence of the disease in given urban settings. indeed, elton's interest in the role of disease in the regulation of animal populations was itself stimulated by earlier russian and chinese studies of the siberian marmot as a host of plague in the inner asian steppes. in chapter of this volume, christos lynteris returns to these studies to examine how the so-called tarbagan became the subject of investigations regarding plague's ability to survive the harsh winters of the region. the question was related to ideas about 'chronic plague', which in the case of the siberian marmot were linked to its hibernation between october and april. using an abundance of visual material, lynteris argues that, on the one hand, tarbagan burrows, which had been epistemic objects ever since the discovery of the species in , and, on the other hand, marmot hibernation, which had been the focus of scientific investigation in relation to host immunity already by , were tied together into an epidemiological duet as a result of the emergency of the manchurian plague epidemic of - . there is indeed a crucial metonymic work involved in this tying together the 'mystery of the survival of plague' over winter to marmot hibernation, and marmot underground dwellings. for the three actants in this network of what following genese sodikoff, we may call 'zoonotic semiotics'-latent plague, hibernating marmots, underground burrows-shared and maintained between them an image of 'mystery' and occultation which has been key both to epidemiological reasoning regarding infectious diseases and to the 'pandemic imaginary' underlying understandings of zoonosis. this image of plague taking advantage of unseen biological processes, materialities or infrastructures so it can assume an imperceptible form that would allow it to persevere over either human action against it or environmentally adverse conditions is of course reliant on pasteurian notions virulence, latency and attenuation. yet, more than simply illuminating a reiteration of bacteriological doctrine, what the tarbagan example points out to is a pervasive aspect of epidemiological reasoning; for the assumption that, when plague (or indeed any other disease) is not seen, this is because it is 'hiding', is part of what we may call a cynegetic complex in epidemiology. as john berger once noted, admittedly in a very different context, a key principle (and, one may add, a mythic structure) of cynegetic worlds is that, 'what has vanished has gone into hiding'. in the case of epidemiology, as with other cynegetic cosmologies, this implies an ambivalent relation. on the one hand, microbes are seen as predators of humanity, who lurk and hide so as to better ambush their prey. and on the other hand, as the enduring metaphor of 'virus hunters' amply illustrates, microbes are also seen as humanity's pray-which thus 'hide' to escape being caught and vanquished by us. as frédéric keck has stressed (following chamayou), '[w]hereas pastoral techniques are asymmetrical, relying on the pastor's superior gaze over the flock manifested by sacrifice, cynegetic techniques are symmetrical, as hunters and prey constantly change perspectives when displayed in rituals'. maurits meerwijk's chapter in the present volume shows that this is indeed a historically pervasive framework, which in the case of mosquitoes is carried over from tropical medicine into global health. comparing the discourses of ronald ross and bill gates, meerwijk shows how the cynegetic metaphor comes to encompass not only the pathogens in question but also their vectors. this points out at a transformative ontology underlying epidemiological reasoning, and its obsession with the 'invisibility' of disease, insofar as pathogens are seen, on the one hand, as able to persist by transforming themselves inside non-human animal hosts (by means of attenuation or mutation) and, on the other hand, as able to spread by transforming their hosts into bestial man-hunters. more than simply blaming non-human animals, in epidemiological reasoning, this double transformative ability configures the former into the loci par excellence of pathogenesis and, at the same time, necessitates techniques of rendering host-pathogen relations visible. visual images of non-human animals have played a historically important role in their configuration as epidemic villains. since the dawn of bacteriology, the scientific identification and examination of non-human hosts and vectors of infectious diseases have heavily relied on photographic technologies (including microphotography), diagrams and epidemic cartography. following sayer (this volume), animals have been 'fed into a data-focused visual regime', combining photography, mapping, diagrams and statistical graphs, that seeks to establish points of contact, habitats, interspecies boundaries and other forms of what hannah brown and ann h. kelly have called human/non-human 'material proximities'. in the context of high-modern epidemiology as well as in today's eid framework, these visualisations are part of a project of mastery aimed not so much at the subjugation of nature, as to the control of humanity's relations with nature. diagrammatic images of dissected mosquitoes played a key role in ronald ross' examination of the insects as malaria vectors, as, in later years, the microphotography of anopheles gambiae dissected ovaries would prove an indispensable, soviet-led method for identifying the capacity of a given mosquito to transmit the malaria plasmodium to humans. similarly, nicholas evans has shown, in the course of the third plague pandemic, comparative images between healthy and plague-infected rats became standard visual objects in epidemiological investigations and their published reports. but the visualisation of 'epidemic villains' did not always necessitate their direct representation. in her chapter for this volume, sayer draws an insightful comparison between two sets of visualising rat control, the first in the english port of liverpool and the second in british india. in both cases, the actual rats are imperceptible, with the photographic focus being on humans undertaking carefully orchestrated epidemiological work (rat dissection, flea collection); a fact which, in the case of liverpool, is underlined by the staged poses of the sanitary officers in questions, and, in the case of india, was permeated by colonial racial hierarchies in the representation of lab work. as representations of the relation between pandemic plague, medical science and empire, these images provide reassuring portraits of control in direct dialogue with the image of objectified rats, described by evans, thus 'making rats an integral part of plague'. similarly, with a focus on this relational aspect of human/non-human mastery and its visual regimes, in the second chapter of this volume lynteris illustrates how the epidemic framing of siberian marmots as reservoirs of plague in inner asia relied on photography and the diagrammatic cartography of their burrows. comprising in survey photographs of excavated marmot burrows and diagrammatic depictions of burrow systems, the visual regime constructed around this suspected host of plague following the manchurian plague outbreak of - comes to show, on the one hand, that intrusive practices of epidemiological visualisation were not limited to human dwellings, but also included those of non-human animals (photographing the marmot burrows required their prior excavation), and, on the other hand, that the visual framing of 'epidemic villains' is not limited to the representation of their role as spreaders of diseases. at the same time, the popularisation of the identity of specific mammals, birds and insects as disease spreaders has and continues to be mediated by their visual representation through photography, film and illustration. photographs of 'wet markets' in south china during and in the aftermath of the sars pandemic have been shown to incorporate a key principle of 'epidemic photography': the depiction of animal-related spaces as potential ground zeros of the 'next pandemic'. the practice of the public vilification of non-human animals and the framing of contact spaces between them and humans as infection hotspots was established for the first time in the course of anti-malarial and anti-yellow fever campaigns in the first decades of the twentieth century, but also during complex public health operations against plague in the context of the third plague pandemic ( - ) when the dreaded disease was often visually personified as the rat. indeed, quite often, the image of animals as enemies of humanity assumed anthropomorphic aspects, which under a colonialist gaze, involved racist inflections. in australian newspaper illustrations, for example, plague-carrying rats were depicted having chinese faces, thus both making an aetiological connection between plague and china (plague as an 'oriental disease' arriving from china, by chinese migrants) and fostering broader sinophobic bigotry at the time. in his examination of the framing of 'tiger mosquitoes' (aedes aegypti and aedes albopictus ) in this volume, meerwijk explores the rich visual culture supporting progressive framings of the specific mosquito species as infectious enemies of humanity. in a striking example, meerwijk shows how the diagrammatic juxtaposition of a mosquito and a tiger was used in a public health poster, meant to underline the predatory, man-eating qualities of aedes mosquitos. pointing at a pervasive tendency to talk about and visualise mosquitoes in terms of great predators (tigers, sharks) or 'enemies of humanity' (terrorists, vampires, prostitutes), meerwijk elucidates the work of the fusion between military, cynegetic and sexual metaphors and visual tropes employed in the depiction of mosquitoes across epidemiological paradigms. this is all the more important as the visualisation of animals as 'epidemic villains' was a trope that found application and success beyond epidemiology and public health. non-human animals were charismatic protagonists of political caricatures since the turn of the eighteenth century. in particular, lukas englemann notes, 'the "political bestiary", as gombrich calls the long tradition of depicting political issues through animal characters, acquired widespread popularity in the nineteenth century. the meaning many animals inhabited could be easily exploited to convey strong messages and almost always suggested degradation'. what changed at the turn of the nineteenth century was the introduction of a new aspect in the use of animals in caricature: their infectious nature. with political discourse utilising more and more medical terms at the time, the use of the visual form of the infectious animal to portray one's political enemies became an exemplary field of vilification. to mention only one example, in the course of the moscow trials, soon after the soviet state prosecutor, andrey vyshinsky, publicly pledged 'to stamp out the accursed vermin' who 'should be shot down like rabid dogs', the prolific cartoonist of the pravda, boris efimov (who was present at the trial), produced a striking caricature of leon trotsky and nikolai bukharin as a two-headed rabid dog held on the leash by the hand of the gestapo. however, as engelmann has shown in his examination of caricatures in the course of the plague outbreak in san francisco, the aim of depicting animals in the context of epidemic crises has not been limited to practices of blaming the former as spreaders or reservoirs of disease. in fact, animals were also used to critique and ridicule bacteriology itself. for example, in the case of san francisco, newspaper caricatures used animals to portray bacteriology 'as a science that formulated its judgments through experiments with animals, not in the treatment of people'. by visualising laboratory animals as 'vermin and pest', englemann argues, bacteriology was portrayed as 'a wasteful expenditure of public funds' and 'the medical laboratory was stripped of its progressive potential and instead appeared as an infliction of damage on the public good'. at the same time, as dawn day biehler has shown in her monograph on pests in twentieth-century us history, images of disease hosts, like rats, have also been used for subaltern purposes, such as the campaigns by the black panther party in the s- s against slumlords and the living conditions in african american neighbourhoods. for example, biehler argues, the well-known illustration by emory douglas, 'black misery! ain't we got right to the tree of life?', 'constrast[ed] with images of women afraid of rats; the woman's grip on the rat suggests determination, courage and fury'. here, the rat represented the unhygienic, exploitative and pestilential conditions imposed by white capital on working-class african americans, and the latter's determination to face up to this social injustice. the prolific use of images of non-human animals as 'epidemic villains' in diverse fields of social practice as public health campaigns, political propaganda, the critique of bacteriology and subaltern critiques of power and domination, points at the importance placed on the infectious nature or potential of animals both as a reality and as a metaphor in the modern world. however, whether it is to convey a threat to the national body, or to mock science, the use of these images also points at the fascination and discomfort of moderns towards non-human agency. underlining how epidemiology and public health emerged in relation to, and continue to be informed by framings of non-human animals as epidemic villains, the chapters in this volume explore the layered political, symbolic and epistemic investments of non-human animals, as these have become rhetorically and visually enabled in distinct ways over the past years. whether it is stray dogs as spreaders of rabies in colonial and contemporary india, bushmeat as the source of ebola in west africa, mosquitoes as vectors of malaria, dengue, zika and yellow fever in the global south, or rats and marmots as hosts of plague during the third pandemic, this volume shows framings of non-human animals to be entangled in local webs of signification and, at the same time, to be global agents of modern epidemic imaginaries. civet cats, fried grasshoppers, and david beckham's pajamas: unruly bodies after sars' the pandemic perhaps: dramatic events in a public culture of danger the scale politics of emerging diseases more than one world more than one health: reconfiguring inter-species health i am using animal-borne diseases here as a term inclusive of zoonotic and vector-borne diseases the rat-catcher's prank: interspecies cunningness and scavenging in henry mayhew's for an influential example of the rat being described as disease-free, see cristofano and the plague: a study in the history of public health in the age of galileo the rat would become suspect of carrying plague for the first time during the inaugural outbreak of the third plague pandemic, in hong kong, with another decade elapsing before the universal acceptance of the link between the animal and human plague. the first scientific study showing the role of the rat and its flea in the propagation of plague was: p. l. simond, 'la propagation de la peste imagining vermin in early modern england the great cat massacre and other episodes in french cultural history religion and the decline of magic: studies in popular beliefs in sixteenth and seventeenth century england imagining vermin in early modern england imperfect creatures: vermin, literature, and the sciences of life on vermin and the poor, see p. camporesi, bread of dreams: food and fantasy in early modern europe animal bodies, renaissance culture filth is the mother of corruption". plague, the poor and the environment in early modern florence que la peste soit de l'animal! la législation à l'encontre des animaux en période d'épidémies dans les villes des pays-bas méridionaux et de la principauté de liège ( - ) que la peste soit de l'animal!'; on ideas of miasma emanating from butchered meat see d. r. carr, 'controlling the butchers in late medieval english towns great stenches, horrible sights and deadly abominations": butchery and the battle against plague in late medieval english towns infection," and the logic of quarantine in the nineteenth century fractured states: smallpox, public health and vaccination policy in british india toxic histories: poison and pollution in modern india as kathleen kete has shown, the modern transformation of this connection, before the dawn of bacteriology, was fostered by a sexualisation of the disease, which rendered it comparable to uncontrollable impulses or lust. commenting on kete's work, linda kalof writes: 'since nymphomania and uncontrollable sexual desire in men were considered the result of prolonged sexual abstinence the beast in the boudoir: petkeeping in nineteenth-century paris looking at animals in human history healing the herds: disease, livestock economies, and the globalization of veterinary medicine veterinary research and the african rinderpest epizootic: the cape colony the great epizootic of - : networks of animal disease in north american urban environments' beastly encounters of the raj: livelihoods, livestock and veterinary health in india animals and disease: an introduction to the history of comparative medicine from coordinated campaigns to watertight compartments: diseased sheep and their investigation in britain, c. - unpacking the politics of zoonosis research and policy catching the rat: understanding multiple and contradictory human-rat relations as situated practices blaming the rat? accounting for plague in colonial indian medicine the bacteriological city and its discontents' malarial subjects: empire, medicine and nonhumans in british india what is an epidemic? aids in historical perspective wartime rat control, rodent ecology, and the rise and fall of chemical rodenticides urban mosquitoes, situational publics, and the pursuit of interspecies separation in dar es salaam the colonial disease: a social history of sleeping sickness in northern zaire the mobile workshop: the tsetse fly and african knowledge production cat and mouse: animal technologies, trans-imperial networks and public health from below building out the rat: animal intimacies and prophylactic settlement in s south africa'. american anthropological association (engagement modern" management of rats: british agricultural science in farm and field during the twentieth century of rats, rice, and race: the great hanoi rat massacre, an episode in french colonial history for a more detailed discussion of this process, see c. lynteris, 'zoonotic diagrams: mastering and unsettling human-animal relations' curing their ills: colonial power and african illness rethinking human-nonhuman primate contact and pathogenic disease spillover the scale politics of emerging diseases the pandemic perhaps avian preparedness: simulations of bird diseases and reverse scenarios of extinction in hong kong unprepared: global health in a time of emergency the pandemic perhaps great anticipations human extinction and the pandemic imaginary for discussion, see k. ostherr, cinematic prophylaxis: globalization and contagion in the discourse of world health inclusivity and the rogue bats and the war against "the invisible enemy as fairhead argues, this entanglement of 'native culture' with 'rogue animals' has the effect of transferring the status of the 'rogue' to the 'culture' in question; fairhead, 'technology, inclusivity and the rogue bats and the war against "the invisible enemy"'. see also m. leach and i. scoones, 'the social and political lives of zoonotic disease models: narratives for a discussion of disgust and animal disease, see a. l. olmstead, arresting contagion. science, policy and conflicts over animal disease control it needs to be noted here that, following fissell, the emergence of the early modern notion of 'vermin' was not associated with disgust-something that points to the introduction of this affective and sensory structure in the nineteenth century imagining vermin in early modern england serviço permanente de prevenção e combate à peste bubónica no sul de angola: relatório (lisboa: agência geral das colónias the sanitation of brazil: nation, state, and public health latent infections, and the birth of modern ideas of disease ecology' tipping the balance blaming the rat? plague and the regulation of numbers in wild mammals Évolution de la peste chez la marmotte pendant l'hibernation'. comptes rendus hebdomadaires des séances de l'académie des sciences for a more detailed examination of zoosemiotics in the case of marmots, see c. lynteris, 'speaking marmots, deaf hunters: animal-human semiotic breakdown as the cause of the manchurian pneumonic plague of - on the ambivalence as applies to hunters and gatherers, see r. willerslev lessons in medical nihilism. virus hunters, neoliberalism and the aids pandemic in cameroon on chamayou's theory, see grégoire chamayou, manhunts: a philosophical history for a discussion of the mythic ability of pathogens to transform their hosts into man-hunters, see c. lynteris, 'the epidemiologist as culture hero: visualizing humanity in the age of "the next pandemic on diagrams and the configuration of zoonosis, see lynteris the evolution of ebola zoonotic cycles'. contagion material proximities and hotspots: toward an anthropology of viral hemorrhagic fevers' human extinction and the pandemic imaginary seeing cellular debris, remembering a soviet method' blaming the rat? on the practice of intrusive epidemic photography as regards human dwellings, see r. peckham, 'plague views. epidemic, photography and the ruined city the prophetic faculty of epidemic photography: chinese wet markets and the imagination of the next pandemic this 'global visual economy' was so pervasive in fact so as to lead to a retrospective diagnosis of the presence of rats in paintings such as nicholas poussin's the plague of ashdod as evidence of a pre-bacteriological knowledge of this zoonotic connection; for a critique, see s. barker yellow peril epidemics: the political ontology of degeneration and emergence a plague of kinyounism: the caricatures of bacteriology in san francisco the sharp weapon of soviet laughter: boris efimov and visual humor a plague of kinyounism', p. . . ibid pests in the city: flies, bedbugs, cockroaches, and rats (washington key: cord- - vh jg authors: fortané, nicolas title: antimicrobial resistance: preventive approaches to the rescue? professional expertise and business model of french “industrial” veterinarians date: - - journal: nan doi: . /s - - - sha: doc_id: cord_uid: vh jg this article focuses on the development of veterinary medicine in the industrial pig and poultry production sector. in the current context of controversies over the public problem of antimicrobial resistance (amr), the veterinary profession is tending to promote a model of preventive medicine that is supposed to reduce the use of antibiotics in livestock farming. however, veterinarians specializing in pig and poultry production (“industrial vets”) have in fact been adopting such approaches to animal health for several decades. based on interviews with pig and poultry veterinarians practicing or having practiced in western france between the s and the s, the article aims to understand how such a form of professional expertise has developed, and the business model that underpins it. contrary to public discourses which promote preventive approaches as a way to diversify professional expertise and to disconnect veterinary incomes from drug sales, it is indeed this economic model that has allowed the development of such approaches within industrial livestock farming. modern strategies for reducing antibiotic use should therefore seek less to renew the professional expertise of veterinarians than to find new ways to valorize it economically. the french veterinary profession is currently undergoing major changes; or at least it tends to see itself as being at the heart of a period of major challenges that are pushing it to reinvent itself. this is not the first time that it has had to face such a reflexivity test, even in recent history (in the british case, some historians even see a cycle-woods a), but recent literature produced by professional veterinary organizations shows the importance of what is currently perceived as a need for self-analysis and change (ondpv ; vetfuturs france ) . there are several issues that might explain why this period is favourable to such a prospective assessment of veterinary futures. one of the most important concerns the controversies and public policies that have developed over recent years with regard to the issue of antimicrobial resistance (amr), which has directly challenged the economic and professional model of farm animal veterinary practices that were setting drugs (mostly antibiotics) up as a cornerstone of veterinary activity, as a source of both income and professional expertise. cross-fertilization of research on the veterinary profession and drug regulation is not common. although veterinarians have aroused the interest of certain historians and sociologists of professions, this has essentially been in relation to the analysis of this social group's process of professionalization (berdah ; mitsuba ) , its role in animal health or food safety policies (woods b; enticott et al. ; fortané , ) , the dynamics that contribute to its specialization (gardiner ) or feminization (surdez ), or finally to knowledge and professional practices in farm (shortall et al. ; ruston et al. ) and small animal medicine (sanders ; morris ) . as for the regulation of veterinary drugs, there are also several studies by historians on the vaccination of animals against major zoonoses or epizootic diseases (woods ; berdah ) , sometimes on the veterinary pharmaceutical industry (corley and godley ) , and more recently on the amr issue (kirchhelle ). yet unlike the uses of human medicines that medical anthropology has been able to theorize and document for many years (whyte et al. ) , the uses of veterinary medicines, i.e. the conditions under which they are prescribed, dispensed and used, are rarely studied, except in interdisciplinary literature from the field of veterinary sciences (speksnijder et al. ; coyne et al. ) . this article tries to open a way to cross-fertilize these reflections. using recent debates on the amr problem, it proposes to examine the relationship between the development of professional veterinary expertise and of the drug market, based on the case of a specific segment of the profession, namely veterinarians specializing in industrial poultry and pig production in western france. it thus puts the amr issue under a broader lens as it analyses ongoing changes within the veterinary profession not as potential consequence of recent measures aiming to reduce antibiotic use, but rather as a reason for the way the problem is now framed. indeed, it is common to hear professional organizations or public authorities state that in order to reduce their economic dependence on antibiotic sales, vets must rethink their activity by favouring preventive approaches to animal health which would involve a diversified range of services and would contribute to placing vets in an advisory role with a holistic vision of livestock farming or even of the food supply chain (vetfuturs france ) . however, such a form of professional expertise, combined with a particular business model for the practices promoting it, is not fundamentally new. if it is at the heart of contemporary debates, it is because it is based and supported by far earlier dynamics that initially had nothing to do with amr, but which used the opportunity of current controversies surrounding antibiotic use, sale and prescription to reinforce and legitimize a certain vision of veterinary medicine, based on preventive approaches to animal health. poultry and pig medicine in industrial production is an especially interesting area for an analysis of these dynamics. firstly, because approaches to animal health that have developed in this field are very singular and characteristic of the intensive farming methods used in these sectors, particularly in the brittany and pays de loire regions where a large proportion of the production is located. indeed, a certain vision of preventive medicine was developed by pig and poultry vets in the s and s, even if the issue of this form of veterinary expertise has not always been raised in these terms. this article therefore aims to understand the professional knowledge, practices and economic model upon which this kind of expertise is based, and why the current amr context is an opportunity to expand it (or at least attempt to). secondly, pig and poultry vets make up an extremely small and autonomous segment of the profession (which makes it possible to draw up a fairly representative picture) although its homogeneity should not be overestimated. this article thus seeks to provide a thorough analysis of this very particular part of the veterinary profession, the specificities of which have almost never been addressed by the literature. despite their small numbers, "industrial" vets are nevertheless an essential component of the profession, because they manage the health of an economic sector which supplies a considerable share of national animal production. the article opens with a brief presentation of the political context and controversies surrounding the amr problem, and how the french veterinary profession has faced up to this by defending the preventive medicine model. it then describes this form of expertise in the professional segment studied here, showing why pig and poultry vets chose this specialization. the article then looks at the origins of these preventive approaches to animal health, both in terms of knowledge and practices, and the economic model associated with it. finally, it reviews the strategies currently developed by industrial vets to adapt to the constraints of increased control of antibiotic use in livestock. the amr problem and the preventive "solution" the problem of antibiotic use in livestock farming is not new. as soon as these molecules were introduced in agriculture in the late s, there was controversy concerning the development of resistant bacteria in animals and food, and the risks of human contamination (bud ). yet for several decades, this issue has been eclipsed by the belief in a permanent renewal of the therapeutic arsenal, consisting in thinking that the continuous discovery of new antibiotics would compensate for the development of increasingly resistant bacteria (podolsky ) . after the swann report in , a series of measures to control the use of antibiotics as growth promoters was nevertheless adopted in europe, progressively separating the molecules used in agriculture and human medicine (kirchhelle ) . but years later, during the avoparcin crisis , , , these measures were considered ineffective (in the sense that they did not prevent the transmission of resistant bacteria between humans and animals) and the use of antibiotics as growth promoters was finally banned in the european union in (kahn ) . the problem of antibiotic use in livestock farming as we know it today reemerged in the late s, this time focusing on veterinary uses, i.e. on curative or preventive uses with veterinary prescription (fortané ) . veterinarians were directly accused of being responsible for the overuse and misuse of antibiotics (and therefore for the spread of resistant bacteria) on the basis of a fairly simple argument: their supposed professional "conflict of interest". indeed, in france, since the act on veterinary pharmaceuticals, vets have had a dual monopoly on the prescription and supply of medicines (hubscher ) . even if, in theory, delivery is shared between three beneficiaries (veterinarians, pharmacists and approved co-operatives), vets capture the vast majority of the curative drug market (of which antibiotics constitute the main category) (guillemot and vandaële ) . the argument that veterinarians over-prescribe antibiotics in order to increase their incomes then became the main framing of the amr problem. this construction of the problem was in reality carried by a coalition of human health actors (doctors, pharmacists, health administration) whose political agenda was twofold. on the one hand, they defended a measure that crystallized the debates around the years - : the "decoupling" of prescription and delivery, which consists in applying the professional and economic model that prevails on the human drug market, i.e. reserving prescriptions to physicians and sales to pharmacists. decoupling basically means forbidding veterinarians from selling pharmaceuticals, as is the case in countries such as sweden or spain (fortané ) . on the other hand, this coalition supported the concept of "critically important antibiotics", the principle of which is to reserve certain molecules, in particular the latest generations of antibiotics, for human medicine. from a political and institutional point of view, this period was extremely interesting because it put the spotlight on definitional and jurisdictional conflicts between different social groups for the control of the legitimate use of antibiotics. it finally ended in a relative victory for veterinarians who succeeded, at the end of an unprecedented mobilization, in reversing the stigma that human health stakeholders assigned to them. indeed, vets have been able to impose the image of a profession that is not guilty of overusing antibiotics but which is instead accountable for their proper use. the notions of prudent, judicious, rational or responsible use, now widely used in amr debates, are thus a social construct produced by conflicts between social groups for the definition of the legitimate use of antibiotics (fortané ) . this veterinarian victory led to the withdrawal of the two emblematic measures (decoupling of prescription and delivery; ban on critically important antimicrobials) supported by the coalition of human health actors. in return, a stricter framework for the use of antibiotics in animal husbandry was implemented between and : margins on the sale of antibiotics are now limited and the retail price of antibiotics must be the same for every client, and antimicrobial susceptibility tests are mandatory for the prescription of critically important antibiotics. but once again, the most important part of this victory certainly concerns the changes regarding the image of the profession. indeed, veterinarians not only reversed the stigma and positioned themselves as guardians of antibiotics, they were also able to re-appropriate the problem by highlighting the way they could solve it. without denying that the economic model of the profession was too financially dependent on antibiotic sales, and that antibiotic use may have been too prevalent in animal care in the past, vets started to defend the development of preventive approaches which would, according to them, be the only way to ensure the transition towards an economic and professional model guaranteeing responsible use of antibiotics. this view was reinforced and supported by farmers and public authorities who also called for the development of such approaches which are usually promoted by national amr policies ( fig. ) in france and in other countries (badau ; piquerez ) . at the heart of this new prospective narrative for the profession and its role in managing the amr problem, we can observe the construction of a (supposedly) new conception of animal health, which is not based on a strictly clinical approach to diseases but on a holistic vision of animals and livestock farming (biosecurity, hygiene, nutrition, good husbandry practices, etc.). in the posters above, veterinarians are portrayed as the gatekeepers of such an approach, through their transversal role as "health advisors". this professional model, based on the knowledge and practices of preventive approaches to animal health, goes hand law for food, agriculture and forestry n° - of october th, . decree no. - of march th, . when public authorities and the veterinary profession began to conceive this campaign, a third poster was designed, saying: "my vet is much more than a mere drug supplier, he is also a teacher! he prescribes the medicines i need … but above all he talks to my farmer to make sure that i am perfectly fed, sheltered and vaccinated". however this poster was not retained for the campaign because of its relatively sensitive headline. in hand with an economic model where the incomes of veterinary businesses would be more diversified since these new "health advisors" would be able to monetize a wider range of goods and services (hygiene and nutrition products, bacteriological analyses, livestock audits, etc.) than just pharmaceuticals. could we look beyond the symbols and images of professional and political discourses and see whether these preventive approaches that might change the way veterinary medicine is practiced, and how antibiotics are used, rely on actual knowledge and practices and, if so, where and since when? the fact is that this model of preventive veterinary medicine seems in reality to be quite typical of a very particular segment of the veterinary profession, the one this article proposes to describe, namely industrial vets. the remainder of the article thus seeks to address the following two questions: is this form of professional expertise really perceptible in the field, and is its development truly linked to the global context of the amr problem and to recently implemented policy measures, or does it have other origins and raisons d'être that might actually when public authorities and the veterinary profession began to conceive this campaign, a third poster was designed, saying: "my vet is much more than a mere drug supplier, he is also a teacher! he prescribes the medicines i need … but above all he talks to my farmer to make sure that i am perfectly fed, sheltered and vaccinated". however this poster was not retained for the campaign because of its relatively sensitive headline. these two posters were used in the french amr policy ("plan ecoanƟbio"). they are a perfect illustraƟon of how the veterinarian's role was reframed towards prevenƟve approaches to animal health (in parƟcular vaccinaƟon), as a means to reduce the use of anƟbioƟcs. the first poster (on the leŌ) says: "my vet is far more than a mere emergency doctor, he is an expert contribuƟng to good husbandry pracƟces". the second poster (on the right) says: "my vet is far more than a mere hands-on man, he is an advisor, always there to prevent and vaccinate". both conclude with: "ask your vet for advice" . when poultry or pig vets are asked why they chose this specialty, they often point out a huge contrast between what they do and the way they perceive cattle (i.e. "rural") or companion animal vets. they feel that being an "industrial vet" essentially relates to four characteristics. firstly, veterinarians specialized in poultry or pig medicine attach importance to working at the heart of the agri-food system, with livestock farming professionals. many of them have agricultural family origins and believe that they chose this profession in order to maintain a strong link with the rural world. they perceive farmers as animal experts, unlike pet owners, and see their activity as teamwork alongside skilled professionals, with whom it is easier to interact and who can also provide them with knowledge about animals. their clients are therefore also their partners in animal health management: they can trust them, rely on them and delegate tasks to them. secondly, pig and poultry vets consider their work to be a permanent renewal, as opposed to the repetitive and sometimes boring work of cattle vets who constantly reproduce the same gestures and who are rarely motivated or intellectually stimulated by new situations and new challenges. most of them, whether they are young vets or already have or years of experience, tend to compare their professional activity with the image of the "emergency vet" (or "fire brigade" vet), available day and night to care for sick animals. from their point of view, this traditional activity is typical of cattle vets, corresponds to the past and relates to a type of work which is limited to clinical diagnosis, drug prescription and/or surgery (e.g. midnight calving). in their opinion, the only objective of such a way of working is to ensure that clients are satisfied with an occasional intervention and that they will once again call upon veterinary services the next time health problems occur. the point here is not to claim that this is what cattle vets are actually doing, but simply to note that this narrative is widely used to define, by contrast, the professional identity of industrial vets. i am indirectly from a rural family, that is to say that my grand-parents, my uncles, they all worked as farmers, in mixed farming and mixed animal farming, from both sides of my family. it is just my parents who had access during the postwar period to national education programmes and became teachers or researchers. so i am from the third generation but i spent a lot of time at the farm. although some of the characteristics that pig and poultry vets tend to associate with cattle vets have actually been observed in other studies, in particular the fact that cattle vets do not have a high opinion of the technical expertise of the farmers they deal with (shortall et al. ) . these opinions of their clients nevertheless depend to a certain extent on the type of farm and farmers they relate to: cattle vets have better professional relationships with "commercial farmers" (managers of large, modern and business-oriented farms) as the latter tend "to understand the need to use the vet as a disease prevention consultant rather than to treat individual sick animals: i.e. part of vets' desired move from a 'test and treat' to a 'predict and prevent' model of veterinary intervention" (shortall et al. , p. ) . in addition, this shows that a tension between "fire brigade" work and advisory veterinary work is also present within a certain section of the cattle vet profession. i became a vet to take care of farm animals because i like being outside and having an intellectual occupation. so that is the reason why. so at vet school, you didn't think about working with dogs? certainly not (laughs)! that was my nightmare! when you work in the pig sector, what is good is that you work % on farms. because when you work with cattle, most of the time you have to do some small animal work as well. and what i want is to work with professionals who are producing the animals we eat. pig farmers (well, not all of them but most of them) have very good technical skills. pig farming is a very dynamic sector, you never get bored! we often have to upgrade the farms to new standards so it's always evolving. research also moves very fast, the pharmaceutical industry innovates a lot so it's interesting. (…) and to be honest, what interests me the most is this kind of follow-up, not being a "fire brigade" vet. but i can't talk very knowledgeably about the "classic" rural vet as i never was one. but what i do think about this kind of practice is that there is a lot of emergency and "fire brigade" work and i don't think that is very challenging. in pig production, we have been evolving for quite a long time because if we only do "fire brigade" work… well, farmers expect a lot more than that! and for us, that is also what's interesting. pig farming is a batch production, so we have this tendency to always try to do something for the next batch, try to prevent future batches from getting the disease we have now. so having to try this and that is always very dynamic and challenging. so we know that there is always a new batch to come, and that is not the same in cattle farming. thirdly, pig and poultry vets describe their work as being part of an epidemiological rather than clinical approach to animal health, which they associate with the idea of a preventive rather than curative or therapeutic approach to diseases. this conception is closely linked to the two previous points. on the one hand, it refers to the professional proximity that veterinarians maintain not only with their clients but also with technical advisors, often employees of the co-operative to which the farmer belongs or sometimes of a feed mill company. indeed, in pig and poultry farming, technical advisors play an important role as they visit the farms much more frequently than vets. although their role should not involve animal health issues (but rather feeding, housing or husbandry practices), they tend to conceive these aspects as a whole, as do vets when they look at the technical factors (rather than biological and medical ones) of diseases. animal health is thus the work of a professional trio, whose theoretically distinct roles often overlap, encouraging situations of cooperation and also sometimes conflict (adam et al. ). on the other hand, and consequentially, this so-called epidemiological conception of animal health establishes a form of activity which is not limited to a clinical and individual approach to pathology but which, on the contrary, opens up professional expertise to areas often considered as not specifically veterinary, such as hygiene, nutrition, zootechnics or biosecurity. the knowledge and the toolbox of industrial vets are thus extremely varied and cannot be limited to surgery tools or prescription booklets. the veterinarians interviewed often emphasized the "evidence-based" side of their professional expertise (and legitimacy), which is based on the collection and analysis of various data and promotes intellectual stimulations regularly renewed by the diverse situations they must face. they believe all these elements to be part of preventive approaches to animal health and they tend to perceive themselves as health advisers, or "health managers" as thoms ( ) has shown in the case of german poultry vets. i graduated from veterinary school in . then i got a degree in epidemiology. what i realized and particularly interested me during my studies was the difference between individual medicine and herd medicine. so with my education and also my family farming background i realized that individual medicine was more a cost than a profit. so i tried to develop this kind of herd care and preventive approach. i started my professional life in rural medicine in a region that was quite a pioneer in this type of approach, especially regarding reproduction. we have audit grids and we have exploration tools. for example, i have a device to measure co , to dose carbon monoxide. i have a ph-meter, a conductivity meter, a laser. i have a burette to measure the water flow in the feeding system. i have a light meter in case i need it. i have a camera to explore water pipes. i always have smoke bombs in my car to check ventilation in the buildings. so really, we have exploration tools in the form of scissors and gloves to perform autopsies but also equipment for managing the buildings. but aren't you stealing work from the technicians? technicians do autopsies (laughs)! no, it's true that controlling ventilation doesn't mean that i am able to deal with the farm it system. so technicians keep their skills. but i consider that it is my job to explain to the farmer that if he has a colibacillosis it's not because a germ fell from the sky but because his ventilation system doesn't work the way it should. that's part of a vet's job, even though it is the technician who is able to fix the problem. we are doing what i might call an etiological or epidemiological diagnosis. fourthly, the characteristics of pig and poultry medicine must also be related to the type of animals with which these vets work on a daily basis. without necessarily following the epistemological principles of the "animal turn" promoted by certain branches of the social sciences (guillo ) , which consists in analysing the way in which animals themselves shape human interactions or the socio-technical devices within which they take place (notion of "animal agency"), it must be noted that taking care of chickens or pigs has different implications than is the case with cattle, dogs or cats. two elements seem central here and are directly related to the characteristics of pigs and poultry and the farming systems to which they belong. on the one hand, these animals have a relatively short lifespan: only about days for a conventionally raised chicken (between and days in organic or label farming) and about months for pigs (the sows being slaughtered after or years). it is therefore very different from a dairy cow that must remain healthy and productive for an average of years, or pets that live or years, sometimes more. in these conditions, which obviously also depend on the economic structure of the agri-food industry, animal health becomes part of biological and temporal dynamics where the slightest disorder may have pathological consequences that might be seen as "just-in-time diseases", i.e. health issues directly related to, or shaped by, the sociotechnical and economic infrastructures (including the animal bodies themselves) within which they emerged (allen and lavau ) . on the other hand, the health of chickens and pigs is not considered individually, but rather from a population perspective. these are animals that are reared in batches and it is the group of animals that constitutes the epidemiological reference unit for both the vet and the farmer. in certain cases, particularly in poultry farms where the production cycle is very short, many decisions are therefore made not for the current batch but for the following one, in order to avoid repetition of the same problem. overall, all of these aspects are typical of industrial veterinary medicine and, therefore, of this form of expertise and professional legitimacy that can be qualified as preventive veterinary medicine. to sum up, and without prejudging whether or not these elements can be found in other segments of the veterinary profession, we can say that poultry and pig medicine is characterized by (i) a vocation; (ii) a technical and preventive approach; (iii) tripartite work; (iv) animals and farming systems that "call for", or co-construct, this form of expertise. however, when listening to veterinarians talk about their profession, this way of working on industrial farms does not seem to be linked to the rebuilding claimed by the profession since its incrimination in the amr problem, even though certain policy measures recently implemented may encourage the development of this preventive and evidence-based approach, such as the obligation to perform antimicrobial susceptibility tests before prescribing critically important antimicrobials (bourély et al. ) . indeed, the roots of this particular form of veterinary expertise can be traced back to the mid- s/early s, when some pioneers began to specialize in pig and poultry farms-at that time during a massive industrialization and intensification process. the origins of preventive approaches in "industrial" veterinary medicine during the s and s in france, particularly in brittany and pays de loire, poultry and pig farms expanded rapidly (nicourt ) . the mixed crop-livestock model was gradually being replaced by specialist farms which were developing through a twofold process of intensification (increased herd size, confinement and containment of animals, rationalization and (bio)technicization of husbandry methods such as genetics, feeding and pharmaceuticals) and industrialization (concentration and vertical integration of the food chain's stakeholders, and taylorization of farm labour) (diry ) . this movement led to the emergence of new needs in terms of health management. firstly, at that time veterinarians knew little about these animals in terms of medical knowledge or techniques (poultry and pig medicine was rarely touched upon in veterinary schools back then). secondly, the confinement of animals in enclosed buildings led to outbreaks of disease, especially-but not only-infectious diseases which were unknown or at least whose management methods were no longer appropriate. a small number of vets then began to specialize in this type of production, viewing it as a promising and developing market. poultry farming was not taught at all. poultry diseases even less so and the prevention of poultry diseases even less than that. so my gateway to poultry farming was the technical-economic approach to things, and not the purely pathological approach, which i could have done at the time on sheep or cattle. except that there, i was in a completely capitalist system. as you can see, one can adapt to anything (laughs). in the pig sector, it was mainly vets employed by agricultural co-operatives who embarked on the adventure, supported in particular by the creation of the station de pathologie porcine (spp) (national veterinary laboratory specializing in pig health) in ploufragan (north brittany) in (fortané ) . in the poultry sector, which is structured more around industrial groups than around co-operatives, there were more independent vets who were orienting their practices towards this type of clientele. what these veterinarians had in common was the conviction that they could no longer do their job in the traditional way, i.e. as emergency "rural" vets, alone or in partnership in small practices serving a diversified clientele (cattle farmers and pet owners in particular, with a few occasional interventions on poultry or pig farms). moreover, they were convinced that their job was to accompany the development of intensive and industrial livestock farming by providing services adapted to the specific animal health issues of this sector, and that their own organization had to follow and mimic the development of the industry they were working for. the term "industrial vets" therefore refers not only to the kind of clientele they were (and still are) working for, but also to their own state of mind and conception of veterinary medicine, as a profession and as a business. in fact, we were the defenders, the propagators of intensive livestock production. we never denied that, that's what we were employed for. we were just saying "ok, but the conditions for success are this and this and this". at the time, there was a double-digit growth in the sector, so we said: "we must stick to the development of this sector". we were focused on poultry, we had almost given up on pigs. we said: "we have to stick to the leaders". very quickly, in - , we said: "it's x [one of the biggest poultry industrial group], we'll stick to x, we'll stick to the growth of x, like a leech, we'll never let it go". that was our aim in - and it became concrete a little later when we were working for x, even though we weren't their official vets. (…) we had developed well in - , so how could we consolidate our system, how could we really sell it, how could we duplicate it? this is where the notion of "global offer" began. here, you find three activities: the veterinary practice, including advice and training, the medicines and the analysis. when you come here, you have this "global offer": the lab part, the drug part and the advisory part. the way this pioneering generation of pig and poultry vets worked was characterized by their capacity and interest in conceptual innovation and do-it-yourself techniques for responding to the often unknown situations they had to face. on the one hand, it concerned the development of pharmaceutical products adapted to these animals and their husbandry conditions, at a time when the pharmaceutical industry was producing very few medicines for this type of livestock, not considering it to be a worthwhile market. it was therefore not uncommon for veterinarians to order pharmaceutical raw materials (rather than manufactured drugs) in order to themselves prepare a product that could be effective in terms of both pharmacological (e.g. combining an antibiotic and an anti-inflammatory) and galenic properties (e.g. designing a drug in the form of a powder to be spread in bedding rather than to be mixed with food-for dermatological infections in particular). in - , we had outbreaks of staphylococci. obviously, the animals caught it very early because they already started to have small pimples at - days. so the mother was a carrier, so we thought: "what if we use an antiseptic powder?" then we thought: "we need a powder that is very powdery, that adheres well to the skin". we worked on the galenic, excipients and everything. we developed a powder and tried it and the farmers said: "this works well". who were you doing this with? we made the powder ourselves. we had a mixer. in our pharmacy, we had a manufacturing workshop. (...) it was common at the time. we had the right to make extemporaneous preparations that were the result of our prescription and our imagination. on the other hand, this broader conception of the veterinary role could be seen in the holistic vision of health that these professionals were promoting. most of the time, they combined therapeutic intervention with bacteriological analysis and research on animal nutrition (some of these vets were also employed by feed mills). all of the larger practices of this pioneering generation developed a laboratory, sometimes a makeshift one, in order to perform the autopsies necessary for bacteriological tests. x [a pioneering poultry vet] had a very solid clientele of horses and cattle at the time, but he was interested in birds. (...) when the first industrial poultry farms, the large flocks of to chickens, settled in, he moved towards that. very quickly, he realized that the bigger the batches of poultry became, the more the diagnosis had to go through the laboratory in order to be accurate -at least regarding bacteriology and parasitology. since he's a guy who doesn't want to do things by half, he thought: "i can't make a laboratory like that, we suck as vets, i need training". he said to his partner: "i will go to pasteur". further education at that time was unimaginable. (...) then, when he came back, he started his own lab and that was really the beginning of the adventure. in the pig sector, it is interesting to note the extent to which the history of the ploufragan station is still rooted in the north brittany territory, as many generations of veterinarians, still today, have done part of their training there or continue to rely on the expertise of spp's epidemiologists or microbiologists in their daily activity (especially in the case of outbreaks of infectious diseases or to set up clinical trials). oh yes, with regard to emerging diseases, i often contacted the station when we saw pathologies that seemed new to us. this was the case for many diseases. there was streptococcal, there was respiratory coronavirus. at the spp, i called [the vet in charge] and the others, they were not convinced because at that time there was still pseudorabies in the farms. i had to talk to [a pharmaceutical company] about it and then the spp became interested. indeed, veterinary researchers from the spp had developed a preventive approach to animal health called "ecopathology" that had considerable success among pig vets and farmers in the s (fortané ) . it was mostly based on an epidemiological conception of animal health (disease outbreaks are related to multiple variables, particularly "technical", i.e. non-clinical ones), and was adapted to the specific issues and husbandry conditions of intensive livestock farming. in this regard, the type of preventive veterinary medicine which was developed at that time in france would seem to have similar characteristics to that which had flourished in the uk slightly earlier (from the s to s) (woods ) : animal health has to be conceived at the scale of the herd (and not the individual animal), be articulated to non-medical matters such as feeding, housing and genetics, and integrate the economic issues of performance and profitability within the advisory support vets provide to their clients. the most important difference during the process of institutionalizing these preventive approaches in france and uk seems to be the role played by public authorities. while in the uk, the state firmly supported the development of preventive veterinary medicine as a way to accompany the modernization of british livestock farming, in france, except for the spp which was partially founded by local authorities and state veterinary services, most of the preventive approaches that flourished in the s were supported by the private engagement of pioneering vets trying to respond to the challenges of a growing industry and, in the meantime, to capture the market of "industrial" veterinary services. all in all, the point here is not to say that contemporary pig and poultry vets are nowadays using the exact same techniques and knowledge of the preventive approaches developed in the s and s, or even to suggest that all industrial vets of that time were doing exactly the same. it is rather to demonstrate that this type of veterinary medicine has at least a to -year history in these sectors and that some aspects of this can still be seen in the way that modern industrial vets continue to work (and to perceive their job and professional identity), such as the importance of technical expertise, epidemiological knowledge and bacterial laboratory-in other words, that a diversified form of veterinary expertise is required. even though this history has more or less vanished from the profession's official memory, a few unconscious vestiges of it can still be seen in recent amr debates. for example, during the campaign promoting the amr policy in the pig sector in , an implicit link was made between this pioneering medicine and the kind of expertise which is now considered to be the future of the profession and through which veterinarians tend to legitimize their role as the guardians of antibiotics. this is demonstrated by the utilization of the ecopathology icon which expresses the holistic conception of animal health (namely the so-called "ploufragan hexagon") to promote good veterinary practices in antimicrobial use in livestock (fig. ) . all these elements show that what is now called preventive veterinary medicine and which is perceived, within the political context of the amr problem, to be the model of professional expertise towards which veterinarians must turn, is not fundamentally new, at least within the knowledge and practices of pig and poultry vets. but what about the economic model on which this kind of expertise is based and how it is actually related to the use of antimicrobials? although it is in fine difficult to associate the development of preventive approaches with the reaction of veterinarians to their incrimination on the amr issue, contrary to what the sole examination of professional discourses in the press or in various it should also be noted that in the uk the development of preventive approaches concerned farm animal medicine in general (so mostly cattle medicine), while in france this movement was located in "industrial" pig and poultry medicine -although due to local history it would seem that in some regions other forms of preventive medicine were also developed in a small number of cattle veterinary practices as from the late 's (combettes et al. ) . this is indeed the purpose of talking about "preventive approaches" in the plural, i.e. to highlight the fact that these approaches could be rooted in different local histories or individual trajectories yet still share some common principles. while pig vets were mainly referring to ecopathology by virtue of their link with the spp, poultry vets did not use a specific term (preventive, holistic or epidemiological approach, global offer, etc.). nevertheless all industrial vets were (and still are) referring to what they were (are) doing with the same kind of contrasting schemes: epidemiological vs clinical, preventive vs curative, herd vs individual, technical vs medical, etc. which i consider to be the common ground of "preventive approaches to animal health" with, in addition (cf. next section), an economic model based on free-of-charge services funded by drug sales and aimed at "capturing" clients. institutional spaces relating to amr policy-making might suggest, nowadays this model has nevertheless become more visible and contributes to the re-legitimation of the veterinary profession (badau et al. forthcoming) . however, the idea that preventive approaches will make it possible to move away from the economic model placing antibiotic sales at the heart of veterinary income seems more dubious, as the development of said preventive approaches was in fact based on this very system. by the notion of economic model, we mean the way in which veterinary services are monetized. one of the key elements of the criticism against veterinarians with regard to the amr issue has been the fact that their sources of income are largely dependent on drug sales (antibiotics in particular). however, whereas professional discourses seek to explain that the development of a preventive medicine in which antibiotics no longer hold a central place will make it possible to change the "business model" of veterinary practices, it is on the contrary clear that this economic model based on drugs sales has in fact allowed these preventive approaches to flourish. even though it is difficult to concretely assess the accountability of veterinary practices due to a lack of available data (in particular if we wish to distinguish between different segments of the ploufragan hexagon: animal health is dependent on six variables (tillon et al., ) prevenƟon campaign against anƟbioƟc misuse in pig farming, fig. the common principles of past and present preventive approaches in industrial veterinary medicine. -ploufragan hexagon: animal health is dependent on six variables (tillon ) . this famous diagram represents the way animal health (and economic performance of the farm) is conceived within the ecopathological framework: it is correlated to six variables, namely the farmer, the animal, housing, feeding, microbes and husbandry practices. at that time, it was considered quite innovative to claim that veterinary expertise should take into consideration all these aspects and not just focusing on animals and microbes (fortané ). -prevention campaign against antibiotic misuse in pig farming, . this poster used in the amr campaign perfectly reproduces the six "ecopathological" variables (here referred to as the six "pillars" of animal health). it says: "no more antibiotics than needed. with my vet, i manage the health of my animals while limiting the use of antibiotics". this poster therefore illustrates how preventive approaches within veterinary medicine are deemed to be a solution to reduce antimicrobial use profession), we can nevertheless rely on estimates from various literatures. a veterinary thesis on the structure of the drug market considered that drug sales were generating to % of the income of farm animal practices (rivière ) . thirty years later, in the midst of the amr debates, a report by the french food, agriculture and rural areas council estimated this figure to be around % for farm animal vets in general, and up to % (including a high proportion of antibiotics) for industrial vets (dahan et al. , p. ) . so one cannot help but wonder what happened during the three last decades and to what extent this evolution of the economic model of veterinary activity has in fact supported the development of preventive approaches to animal health, contrary to the prospective narrative the profession has constructed to defend its role as the guardian of responsible use of antibiotics. the answer is actually quite simple, and is a classic case in agricultural economics: the sale of inputs funds the advice. in the case of industrial vets, the context in the s and s favoured the emergence of strong competition between practices seeking to capture the growing clientele of pig and poultry farmers. this competition was also heightened by the fact that the supply of veterinary goods and services was still highly heterogeneous, due to the non-existence of standardized pharmaceutical, hygienic or nutritional products adapted to the new health problems caused by the intensification of animal husbandry. consequently, pharmaceuticals, which are the only product on which veterinarians have a monopoly for prescribing (and therefore advice) and dispensing (and therefore sales), became the main means of ensuring client loyalty. since the act, the veterinary drug market has thus been developing into a captive market, where drug sales have gradually become the only monetized exchange between vets and farmers, funding all the services actually offered by veterinarians (visits, diagnosis, analyses, prescription, audit, etc.) (bonnaud and fortané ) . indeed, preventive veterinary medicine developed on the basis of this economic model which corresponded, on the one hand, to that of the few pioneering independent practices which owned bacteriology laboratories, small drug manufacturing factories or wholesale companies and, on the other hand, to that of the agricultural co-operatives which the act established as lawful drug suppliers and which, until , employed veterinary practitioners in this regard. indeed, it was precisely by providing a whole range of "free of charge" services (visits, advice, vaccination, etc.) that these vets were able to disqualify their competitors, i.e. their "emergency" or "fire brigade" colleagues who offered little advice but were able to monetize their occasional interventions. yet were the farmers prepared to pay for their visits, as they would have done with rural vets at the time? because, even the independent vets specializing in group pathologies, so pig and poultry, like those from x [one of the pioneering veterinary practices in industrial medicine], most of the time, they did not charge for their visit, their visit was paid through drug sales. in fact, most farmers were smart enough to understand that if they were satisfied with the services of a vet or a veterinary practice, they should buy drugs to pay them indirectly, even if there were price differences. i think it was like this: "you do me a favour, i need you, you treat, you prevent, we work together, i buy drugs". it was indirect payment. (…) i've always thought that the best way to ensure farmers' loyalty is to be efficient. then they will not even talk about drug prices. there was a time when vets were very active on the farms. if farmers were happy with our services, [they didn't care about] the price of the drugs. at one point, we were a little cheaper but we weren't always cheaper. we were also known for being reasonably priced, so that there wasn't too much competition. sure, we weren't the cheapest on the market, but that's not the issue. but i'm convinced, and i know that's the way it was for a lot of people, the best way to keep them was to meet their expectations. their expectations were simple: "i have problems, i have to solve them, help me solve them, if it takes time ok it takes time". it was up to us to meet farmers' expectations in terms of health. if the farmer was satisfied, the rest would follow, he would take the feed, the genetics, the drugs. we had an indirect commercial role, but we could only do it if we were efficient. (…) but some of them stopped buying our drugs. they told me: "you're too expensive". so i answered: "if we are too expensive, you go elsewhere, but you no longer have the services". what does he want? a cheap price, but with a service that will be what it will be, it may be very good, ok, but the farmer is free to choose. however, he fully understood that he could not ask us to come ten times a year or twenty times and then buy zero medicines. moreover, i tell most people: "we provide you with important services, we know that you also like to work with other people from time to time, at x for example, so don't buy everything from us, but buy a little from us, as we come to see you regularly and we need a return". and then everything was fine, they bought some elsewhere and some from us. the construction of a captive market is therefore a central dimension of what i call here "the economic structures of professional expertise". the development of preventive approaches within industrial veterinary medicine is not only the result of the emergence of new demands or needs from farmers, but it is also and above all the consequence of the intra-and extra-professional competition that veterinarians have had to face after the act. prescribing and dispensing pharmaceuticals, combined with a holistic expertise, is a means of capturing a clientele through a simple and exclusive form of economic contracting (the sale of drugs) while providing a wide range of services (diagnosis, advice, etc.). it is indeed the articulation of these two dimensions that makes it possible to prevent, or at least reduce the risk, that clients are captured by a competitor. in this sense, preventive approaches to animal health must be read simultaneously as a professional and a business model, these two dimensions being constitutive of each other. such economic structures of veterinary expertise have been observed in another context, that of the usa in the s and s. smith-howard ( ) shows how american veterinarians, who did not have a monopoly on the sale of medicines, gradually established themselves as the main distribution channel by coupling their prescriptions with preventive services that provided real added value to farmers, compared with pharmacists or other retailers who were merely supplying the drugs. a linguistic distinction has even emerged between "dispensing" and "merchandising", contributing to (re)establishing the legitimacy of veterinarians on a new basis. in the end, it is therefore in the light of this link between professional expertise (in the sense of type of knowledge and services) and business model (in the sense of how these services are economically valorized) that we must question the current context and controversies surrounding amr. it seems finally a little too simplistic to support the thesis of the professional conflict of interest that human health actors have mobilized to point out veterinarians' responsibility in the amr problem, because of their dual monopoly on the prescription and supply of medicines. in fact, the structure of the veterinary drug market has set up the sale of pharmaceuticals as a quasi-unique way of making professional expertise profitable, even though this expertise was already based on preventive approaches (and thus diversified services) for quite a long time. the current challenge for the veterinary profession is therefore less the development of a preventive medicine than the renewal of the economic model on which it has been based until now. in this regard, it is actually interesting to note that the vets i met in this study clearly mention this issue and have already started to develop strategies to deal with it. that's exactly the issue we now have with my colleague. i'm right thinking this. historically in poultry, no charge is really made for visits. overall, payment is made through [the purchase of] medicines, which is, for me, not a bad thing if it is not excessive. at some point, when we take the example of human medicine, you pay a doctor, you pay a pharmacist, everyone is happy because there is healthcare security. the day there's no healthcare security, i don't know what we're going to do. so that's what i explain to farmers: "the day you have to pay a vet and a pharmacist it's going to cost double". so at the moment, with this system, you only have one cost, so in theory that's good. indeed, there can be misuse, someone who would systematically prescribe medicines even if there is no need. this is clearly an issue and it is difficult to explain, that's our problem. but honestly, every morning when i stand up, i don't say to myself: "i will prescribe kilos there, kilos there and kilos there, and the day is done". that's absolutely not the way i think, but i can understand… and i understand when people are telling me: "yes, but if you sell, you earn". that's all there is to say. it's been on my mind for a year. firstly, particularly in a context where profits on medicines are now more strictly controlled (and therefore more limited), it seems essential for the veterinary profession to be able to charge for services that were previously funded by drug sales. this is the case in particular for visits, technical advice and bacteriological analyses (the latter were generally charged but often below the real cost-which could be high for the veterinary practice that has to employ laboratory technicians). secondly, there are strategies consisting in investing in hygiene and/or nutrition product factories (in particular disinfectants and food additives), in order to be able to sell products other than just pharmaceuticals. it is a strategy of diversification of the professional and economic activity that pioneering independent practices or certain cooperatives employing veterinarians have been using for many years, but which is now tending to become generalized among industrial vets due to the development of franchised practices. company x is a part of group y [a pig co-operative]. [we sell] zero drugs. there is hygiene, so disinfectants and detergents, and also nutrition and probiotics, i.e. products that can be added to food or that farmers can put in drinking water. you have suppliers for these products or you manufacture them yourselves? there are products that we make ourselves, otherwise we choose products from everything we know, and from people we work with. we are doing clinical and zootechnical trials as well. we've written some articles, i did one on swine haemorrhagic dysentery. we did an international article in which we showed that we can prevent it with a certain probiotic. it allowed me to go and see farms in portugal, spain and england as well. i even went to cuba for x. trying to sell products? to preach the good word! we hold meetings on a given theme, for example it might be digestive issues, and we talk about the ecopathological approach. rather than using this or that antibiotic, there are other things that work, and we know this because we have experience with farmers from y. thanks to this experience, and because there were disastrous situations where antibiotics no longer worked, we can tell other veterinary colleagues [that these products work well]. a third strategy consists in developing various forms of contractualization with the clients. for example, the vet interviewed below tries to imagine an annual flat-rate model that would cover all veterinary services, from advice to medicines. this would be based on a form of insurance, where some farmers would ultimately pay a higher price than the actual cost of the services they have received, while others, particularly those facing more health issues, would pay a lower price (although the reverse might be true from one year to the next). in fact, this system is similar to the one set up by the so-called "veterinary groups under contract" in central and south-eastern france, but which was only developed in areas where livestock farming is not widespread i.e. in small and medium-sized cattle or sheep farms connected to local markets; this system is mainly associated with alternative agricultural projects resulting from s protest movements (combettes et al. ) . it would be interesting to know the extent to which such a model might be easily generalizable in the heart of an industrialized agri-food system where farmers are involved in more complex and constraining chains of interdependence with upstream and downstream industries. this trend resonates with certain dynamics currently observable in the uk where farm animal vets are also concerned about the sustainability of their economic model and are trying to develop similar forms of contractual veterinary services, although less than % of the clientele of the practices trying to develop these kinds of contractual schemes have adopted it so far (ruston et al. ). the economic model and the type of goods and services that veterinarians can provide are nevertheless now considered to be key criteria for the sustainability of veterinary businesses (henry et al. ) . so i've been thinking for a year. now i'm going to test a new system, a comprehensive flat-rate package. i'm testing it, it's brand new, with two or three farmers. this is a complete veterinary follow-up, which includes visits and medicines. we don't talk about fees anymore. [...] so i don't know yet. my package system might be good in theory, maybe not good if farmers... because you know, on the farms, there are major variations in antimicrobial consumption. at some point the package is going to be an average. some of the farmers will find my services a little bit too expensive whereas others will like it, but yes perhaps major antibiotic users so it might be a bad influence for them. i'm not claiming victory yet, but i'm trying to find another system. a fourth strategy consists in monetizing certain veterinary services no longer to farmers, but to co-operatives. this relates to at least two types of activity. first of all, it concerns the follow-up of "herd health plans" that co-operatives are obliged to set up if they want to be approved for selling veterinary medicines, in accordance with the act. it is of course an activity that has existed for a long time, but that many cooperatives delegated to their employed vets until . the second activity relating to this strategy is linked with the development of new roles for veterinarians, in particular those of standards controllers or certifiers. indeed, with the development of "antibioticfree" labels, more and more co-operatives are asking vets to help them implement such standards. for example, vets have to perform "pharmaceutical audits" in an attempt to recruit farmers who can comply with such specifications, or to set up protocols to monitor antibiotic use and help farmers reduce it. when the co-op announced its project [antibiotic-free pigs], some farmers called me the day after, or i called them, to be part of this project. so for those who were still using antibiotics in feed there were still some stages to go through but when a farmer calls you, you think that you're going to need him for the project so you have to find alternatives. you have to know if he really needs antibiotics and how to reduce them. so this is where you have to set up a procedure, to find ways to identify the flaws… and in the end this is the perfect way to familiarize farmers with this issue. (…) well the farmer has to be motivated but this is where the bill of specifications helps, because there is an added-value. and there is also glory and self-satisfaction because the added-value isn't much, but there is both. yet these new roles for veterinarians, or more exactly these new ways of monetizing their expertise, are associated with a redefinition of their professional identity that vets are not always comfortable with. while being a health advisor rather than a clinician is perfectly in line with their preventive conception of veterinary medicine, becoming a sort of controller and sometimes even a sales representative in charge of enrolling and accompanying farmers in quality insurance schemes is not necessarily easy. this difficulty has also been noted among uk cattle vets who felt conflicted between their role of practitioner (within the framework of their relationship with their clients) and their role of "enforcers" of biosecurity practices (while implementing policy measures to prevent bovine tuberculosis), although in this case they were acting on behalf of the government and not for market schemes of private stakeholders (enticott ) . the vet interviewed below explains the practical and ethical adjustments he has had to make to accommodate this new way of valuing his professional expertise, and the limits he personally sets to remain in what he considers to be the role of a health advisor. so luckily we have some experience [with following up on bills of specifications], especially with welfare standards. in this standard, the farmers have to use the co-op's animal feed. so our experience allows us to recognize the feed bags. and one day i was on a farm visit, and i saw a feed bag. and i thought "but this is not the feed from the co-op?". because i know that it is normally small granules, and this time it was big granules. so here, clearly, the farmer is not complying with the bill of specifications! so you see, if you look carefully, you can see things. well you can't see everything, especially when farmers don't want you to know… because in this case he even didn't think about it. leaving the bag like that… it is sure that he's going to be in trouble with the co-op. but you won't see the ones who really want to cheat! and in this example, what happened in the end? you just let him know, asked him to be careful, and let it go? or might you impose sanctions at some point? well that's not for me to decide. but the question for me is whether or not i want to report someone. one day the co-op boss asked me [about the co-op's granules] but i replied that i didn't have time to check whether every farmer in the quality insurance scheme actually uses its granules. (…) but no, what i do, mostly, is warn the farmer. i tell him that he has been found out. and i tell him that the coop is fairly meticulous and that if they find out they will take him off the scheme. so for a few months he will lose cents and he won't be happy. so that's the situation. and they are absolutely capable of doing it, without any doubt. so this is what i do, i warn him but i don't think it is my role to report him, or to impose sanctions. conclusion "if i change, i may lose my clients. but if i don't change, i might lose my job" by cross-examining the development of the drug market and the professional expertise of french "industrial" vets, this article has aimed to articulate reflections from the sociology of professions and the anthropology of medicines. the amr problem is a very interesting case-study through which to make such an articulation fruitful. indeed, numerous works of research in the anthropology of medicines invite us to analyse drug use through the lens of the global circulation of pharmaceuticals and not just by focusing on the practices and knowledge of end-users, be they farmers or patients (whyte et al. ; petersen ) . antibiotic use cannot therefore be disconnected from upstream practices (or pharmaceutical "life stages", as anthropologists of medicines would call them) such as production, distribution, prescription and sale. though little is yet known about the structure of the veterinary drug market, we have shown that as key actors at the heart of the veterinary drug chain, involved in many areas of this global circulation of pharmaceuticals, veterinarians play a major role in the regulation of antibiotic use-particularly industrial vets who are not only involved in prescription and delivery but also in some cases in production and distribution. this result is therefore interesting from the point of view of the sociology of professions as it shows that the "jurisdiction" (abbott (abbott , of veterinarians is certainly more complex than them being mere practitioners of animal healthcare. indeed, when we look at the veterinary profession as a whole it becomes quite obvious that veterinary knowledge is involved in many domains of animal health and food safety, from farm to policy and industry fortané , ) . what this article shows, in addition to describing the specificities of a relatively unknown segment of the profession, is that this highly diversified form of professional expertise, and therefore the variety of areas in which it can be exerted, can also be concentrated within a tiny group of professionals, namely pig and poultry vets. moreover, even though it still has to be more broadly demonstrated, it would seem that the trajectory of this particular segment, characterized by the development of specific approaches and economic models, is somehow quite representative and/or influential of more global changes that are now affecting the profession as a whole (at least in farm animal medicine). the expansion of the activity of industrial vets within the drug chain and the "technical" domains of livestock farming indeed corresponds to a (conflicted) evolution of veterinary jurisdictions that echoes the profession's ongoing attempts to renew itself in the context of amr controversies. all in all, articulating reflections from the sociology of professions and the anthropology of medicines has allowed us to highlight the "economic structures of professional expertise" (that is to say that professional practices and knowledge are shaped by the way they are economically valued on a given market), which is of considerable importance for the analysis of the amr problem. indeed, it underlines the fact that amr cannot be addressed by simply focusing on antibiotic use or prescription and, more importantly, it shows that the framing of the amr problem cannot be understood without reconnecting it with the global transformations of the veterinary profession (both in terms of expertise and business model). in this regard, we have shown that the veterinary profession has developed a prospective narrative to defend its role as the guardian of antibiotics (i.e. moving towards preventive approaches in order to prescribe less antibiotics and be less dependent on drug sales) that does not fully correspond to either the history or the actual practices of industrial vets. such approaches have in fact already existed for many years and it is thanks to this diversified veterinary expertise that this segment of the profession has developed. however, the economic model upon which this expertise has been based is in fact the one which is nowadays criticized within amr controversies (i.e. the sustainability of veterinary businesses being highly dependent on antibiotic sales), so that it would appear relatively doubtful, if not ironic, that the profession now brandishes preventive approaches as the undisputable solution to antibiotic overuse or misuse. indeed, drug sales were almost the only way of monetizing veterinary services in the pig and poultry farming sectors and this is how the pioneering generation of industrial vets disqualified their traditional competitors, by "enrolling" clients through the development of a captive drug market. so although promoting preventive approaches is probably the right way to encourage veterinarians to move towards a "health advisor" role where antibiotics are neither the main tool of their activity nor the main source of their income, the profession still has to develop economic models (practice accountability, contractual arrangements with the clients, etc.) that do not rely on drug sales. recent policy measures and incentives are probably pushing in this direction, as are broader trends such as the development of franchised practices and new professional roles and status, but this still has to be empirically confirmed (even though we know that over recent years antibiotic use has already started to decrease). social sciences have a major role to play here, by being theoretically and methodologically innovative in order to grasp complex and entangled dynamics to which the amr problem has just begun to draw attention. conflict of interest the author declares no competing interests. the system of professions: an essay of the division of expert labor linked ecologies: states and universities as environments for professions autonomy under contract: the case of traditional free-range poultry farmers just-in-time' disease: biosecurity, poultry, and power l'antibiorésistance à l'épreuve des discours de la presse agricole. questions de communication les appropriations de l'antibiorésistance en france : la carrière d'un problème public au sein de la presse vétérinaire entre scientifisation et travail de frontières : les transformations des savoirs vétérinaires en france, xviiie-xixe siècles. revue d'histoire moderne et contemporaine abattre ou vacciner : la france et le royaume-uni en lutte contre la tuberculose et la fièvre aphteuse penicillin: triumph and tragedy pour une sociologie de l'action publique vétérinaire l'État sanitaire de la profession vétérinaire. action publique et régulation de l'activité professionnelle why do veterinarians ask for antimicrobial susceptibility testing? a qualitative study exploring determinants and evaluating the impact of antibiotic reduction policy la santé animale sociale et solidaire. le sociographe the veterinary medicine industry in britain in the twentieth century understanding the culture of antimicrobial prescribing in agriculture: a qualitative study of uk pig veterinary surgeons encadrement des pratiques commerciales pouvant influencer la prescription des antibiotiques vétérinaires l'industrialisation de l'élevage the changing role of veterinary expertise in the food chain the local universality of veterinary expertise & the geography of animal disease le problème de l'antibiorésistance en élevage : essai de généalogie et caractérisation. questions de communication naissance et déclin de l'écopathologie (années -années ). l'essor contrarié d'une médecine vétérinaire alternative veterinarian 'responsibility': conflicts of definition and appropriation surrounding the public problem of antimicrobial resistance in france the 'dangerous' women of animal welfare: how british veterinary medicine went to the dogs l'arrêt riaucourt et ses effets what is the place of animals in the social sciences? the limits to the recent rehabilitation of animal agency exploring the sustainability of small rural veterinary enterprise les maîtres des bêtes. les vétérinaires dans la société française one health and the politics of antimicrobial resistance pharming animals: a global history of antibiotics in food production entangled histories: german veterinary medicine, c. - managing pet owners' guilt and grief in veterinary euthanasia encounters Être agriculteur aujourd'hui. paris, editions quae: l'individualisation du travail des éleveurs observatoire national démographique de la profession vétérinaire (ondpv) speculative markets : drugs circuits and derivative in nigeria une frontière géographique, des frontières de compétences: le cas de l'antibiorésistance en santé animale en france et en suisse the evolving response to antibiotic resistance achat et vente en gros de médicaments : structures mises en place par la profession vétérinaire, thèse pour le doctorat vétérinaire challenges facing the farm animal veterinary profession in england: a qualitative study of veterinarians' perceptions and responses annoying owners: routine interactions with problematic clients in a general veterinary practice broken biosecurity? veterinarians' framing of biosecurity on dairy farms in england, preventive veterinary medicine true cowmen & commercial farmers: exploring vets' & dairy farmers' contrasting views of 'good farming' in relation to biosecurity healing animals in an antibiotic age. veterinary drugs and the professionalism crisis, - determinants associated with veterinary antimicrobial prescribing in farm animals in the netherlands: a qualitative study les bouleversements de la profession vétérinaire. lorsque la recherche d'une nouvelle légitimité sociale coïncide avec l'arrivée des femmes, revue d'études en agriculture et environnement handlanger der industrie oder berufener schützer des tieres? -der tierarzt und seine rolle in der geflügelproduktion synthèse des travaux réalisés depuis à partir de la station de pathologie porcine de ploufragan le livre bleu : comprendre et anticiper les mutations de la profession vétérinaire social lives of medicines a manufactured plague: the history of foot-and-mouth disease in britain the lowe report and its echoes from history. veterinary records a historical synopsis of farm animal disease and public policy in twentieth century britain is prevention better than cure? the rise & fall of veterinary preventive medicine, c. - publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -oipail l authors: wilkie, david a. title: the ophthalmic examination as it pertains to general ocular toxicology: basic and advanced techniques and species-associated findings date: - - journal: ocular pharmacology and toxicology doi: . / _ _ sha: doc_id: cord_uid: oipail l ocular toxicology pertains to toxicologic effects of drugs administered topically, intraocularly, or systemically. it should also include evaluation of adverse effects of ophthalmic devices such as contact lenses, intraocular lenses, and glaucoma implants. the ophthalmic examination is able to provide detailed in-life information and is used in combination with clinical observations, clinical pathology, and histopathology to assess potential toxicologic effects. the ophthalmologist must be familiar with the wide range of species used in the field of toxicology, be familiar with the anatomic variations associated with these species, be able to determine what is an inherited or a breed-related finding from a study-related effect, be competent with the required ophthalmic equipment, and be capable of examining this wide range of animals. the purpose of this chapter is to discuss laboratory animal ophthalmology as it pertains to industry, not to pocket pets. the industries of interest include contract toxicology laboratories and researchers in an academic environment that may require the expertise of a board-certified veterinary ophthalmologist. contract research organizations (cro) evaluate products for pharmaceutical and agricultural use and are governed by food and drug administration (fda) and environmental protection agency (epa) guidelines. in addition they may test cosmetics, contact lenses and associated materials, intraocular devices, and a host of other products that might have an ocular use, contact the eye or be applied topically, or be inhaled, ingested, or injected. the evaluation of potential drug effects and toxicity must integrate the disciplines of pharmacology, toxicology, pathology, and ophthalmology [ ] . the cro study is overseen by a study director and all study-associated personnel, including the consulting ophthalmologist, must be adequately trained and a quality assurance (qa) unit is responsible for monitoring each study to ensure compliance. toxicology studies conducted for regulatory purposes need to be conducted in compliance with good laboratory practice (glp). all personnel, including the consulting ophthalmologist, involved in animal studies will be expected to be familiar with glp and will usually be required to take annual glp-refresher courses. systemic and ocular toxicity studies require evaluation of both systemic toxicity using clinical observations, body weight, and clinical and histologic pathology and ocular toxicity using detailed ophthalmic examinations [ ] . the eye, as it pertains to toxicology, can be considered in one of the three ways. with respect to undesirable ophthalmic toxicologic effects the ophthalmologist is concerned with ( ) undesirable ocular effects when the eye is the target organ of interest with the drug of interest applied to the eye, ( ) undesirable systemic effects associated with an ocularly applied agent, and ( ) undesirable ocular effects from an agent applied in a systemic manner (oral, dermal, injection, inhalation) with resulting ocular effects [ ] [ ] [ ] [ ] . in addition to drug effects, animals may be used to evaluate the effects and side effects of a procedure or a device. with regard to the eye this may include evaluation of a new intraocular device such as an intraocular lens or viscoelastic agent or evaluation of a new surgical procedure. these studies are designed to evaluate the potential for ocular toxicity or other adverse effects arising from the systemic, topical, or other administration of drugs or compounds, the application of medical devices, or certain surgical procedures. while in some cases the studies are designed to provide proof of concept as regards therapeutic efficacy, in the majority of cases the studies are being conducted specifically to enable an adequate assessment of safety of test materials and devices in consideration of meeting fda and epa (or other similar regulating agencies) approval for initiation of human clinical trials (supporting an investigational new drug (ind) and/or investigational device (ide) application). the eye, because of its large blood flow by organ weight, is a prime target for various systemic toxicities. in addition to the adnexal structures, vascularized intraocular structures include the retina and uveal tissues (iris, ciliary body, and choroid) [ ] . the transparent nature of the eye and the ability to visualize arteries, veins, and neural tissue make the eye an organ where toxicities may be readily detectable. this makes the eye unique in that it is possible to conduct a detailed assessment during the in-life portion of a study [ ] . as ophthalmologists, we must be familiar with the normal interand intraspecies variations that occur between the species involved in toxicologic studies. we must be able to examine all of these species given the limitations of size, temperament, and restraint. finally we must be familiar with common naturally occurring abnormalities observed in each of these species and be able to differentiate these from toxicologic effects. a discussion of the anatomy and physiology of the most common animals used in ocular research, including mice, rats, rabbits, guinea pigs, dogs, cats, pigs, and primates, is found in chap. . this chapter emphasizes the routine ophthalmic examination of laboratory animals. it also provides information on more advanced ophthalmic diagnostic tools that are becoming more commonplace in the area of ocular toxicology. prior to study initiation, the ophthalmologist should review all ophthalmic procedures, discuss with the study director and/or sponsor any issues or concerns with the study design or the examination procedures, and then follow standard operating procedures (sops) when conducting their examinations. it is the position of the american college of veterinary ophthalmologists (acvo) that in order to ensure public safety the status of diplomate of the acvo is the minimum qualification for performing these ocular examinations and assessment of findings in a laboratory animal study that is intended to support applications to the fda (or other similar regulating agencies) for entry into human clinical trials. evaluation of toxicological effects of pharmaceutical agents involves assessment by a number of personnel, many of which are board-certified specialists, including pathologists, cardiologists, and others in addition to ophthalmologists. sponsors engaging the services of a cro must be advised of the participation of veterinary ophthalmologists and the potential limitations that may arise if such studies do not involve veterinary ophthalmologists. a board-certified veterinary ophthalmologist is uniquely qualified to consult in the development of the experimental design (including the species selected, appropriate diagnostic tests, and frequency of exams) and the assessment of ocular effects of test materials being evaluated. coordination between the testing agency and the veterinary ophthalmologist is essential throughout the process, to include protocol development, establishing sops, and the identification and assessment of ocular findings. if ocular abnormalities are identified, communication between the ophthalmologist and the pathologist will allow correlation of clinical and histopathologic findings. the components of an ophthalmic examination may vary depending on the species involved and the specific objective of the study. however, if the purpose of such a study is to screen for adverse effects on any ocular tissue including, at a minimum, the adnexal structures (eyelids and conjunctiva), anterior segment (cornea, anterior chamber, iris, and lens), and posterior segment (vitreous and fundus), the following must be included: additional procedures may be included depending on the objective of the examination. these may include, but are not limited to, corneal staining, corneal esthesiometry, pachymetry, tonometry, fundus photography, fluorescein angiography, optical coherence tomography (oct), and electrophysiological assessment of the visual system (e.g., electroretinography, multifocal electroretinography, visual evoked potentials (vep)). sedation or general anesthesia may or may not be required depending on the species, the procedure being performed, and individual animal. the routine ophthalmic examination for all animals used in toxicologic studies should begin with the minimum database of both biomicroscopy and indirect ophthalmoscopy. regardless of the species of interest, these two examination techniques are essential to ensure an accurate and complete examination of both the anterior and posterior segments of the eye. a comparison of techniques by bellhorn found that of rats with known lens abnormalities diagnosed by biomicroscopy only / of the lenticular lesions could be found using the direct ophthalmoscopy and only / were found using indirect ophthalmoscopy [ ] . together these two examinations must, at a minimum, include evaluation of the adnexal structures (eyelids and conjunctiva), anterior segment (cornea, anterior chamber, iris, and lens), and posterior segment (vitreous and fundus). ophthalmic examinations should be conducted on an eye that has been pharmacologically dilated and should be performed in a darkened examination room. while some have advocated the use of % phenylephrine to aid in dilation of rodents [ , ] , this is generally not required. pharmacologic dilation is most commonly performed using tropicamide at a concentration of . % for rodents and % for larger mammals. the ophthalmologist should be familiar with the length of time required to achieve mydriasis and the duration of the mydriasis for the species being examined. in general, - min is the minimum time required to achieve acceptable mydriasis and this may be slightly longer in heavily pigmented eyes. the duration of mydriasis is directly related to the amount of intraocular melanin. in albinotic rodents, mydriasis will last no more than h while in a pigmented eye of a dog or a primate the effect will persist for - h. this information is important so that the ophthalmologist knows when to begin dilation and how many animals should be dilated at one time. the later will depend on how many animals the ophthalmologist can examine in a given time period. for the basic examination, biomicroscopy, and indirect ophthalmoscopy, animals are either manually restrained (rodent, dog, rabbit, pig, guinea pig, cat) or sedated or anesthetized (primates). rats and mice may be held in a dose-hold and presented to the ophthalmologist with their heads restrained. some ophthalmologists prefer the eyes to be slightly proptosed in rodents. dogs and rabbits are most often examined on a table. rabbits seem to do best if there is a towel on the table as this decreases their movements and may provide some comfort. for rabbits, the ophthalmologist should be seated slightly below the level of the restraint table to allow easy visualization of the rabbits' optic nerve and retinal vasculature which are located in the superior fundus. for dogs, the examiner may be seated or standing. since primates will be anesthetized or heavily sedated, they may be manually restrained or placed on an examination table. if more extensive examinations such as electrodiagnostic testing or oct are required sedation or anesthesia may be required regardless of the species. additional examination procedures such as direct ophthalmoscopy, corneal staining, tonometry, pachymetry, fluorescein angiography, photographic documentation (anterior or posterior segment), electrodiagnostic testing, ultrasonography, oct, and other tests may be indicated depending on the study and toxicologic effects of interest. if any of these additional tests are required, the order in which tests are performed and when to perform pupil dilation must be considered. for example, determination of intraocular pressure (iop) should be performed prior to pupil dilation. in addition, if repeated iop measurements are required during a study they should be performed at the same time of day to avoid diurnal pressure fluctuation. when possible, examination of the cornea should be performed prior to procedures that may result in corneal changes as a result of corneal contact (pachymetry, tonometry) or the use of topical anesthesia. if sedation is required for the ophthalmic examination then consideration must be given to dosing and feeding schedules, clinical observations, and clinical pathology sampling. finally, from an animal well-being standpoint, a balance must be struck between multiple procedures performed on the same day as compared with multiple repeated days requiring sedation [ ] . animals will be most commonly be identified by tattoo, ear tag, or microchip. if a tattoo or an ear tag is used, the animal handler will need to have enough light during the examination to read the identification to ensure accurate data collection. this may be provided by a separate light source elsewhere in the examination room or if possible by performing the ophthalmic examinations in a darkened anteroom allowing the handlers to keep the main animal room lights on. when identified by microchip, a computer scanner will allow animal identification to be linked to the computer program for data entry. to ensure accuracy, each animal must be identified to both the ophthalmologist and the data entry person at the time of the examination. the data entry person must verify that the animal being examined and the animal for which the data is being entered correspond. depending on the compound being evaluated and the sop, the ophthalmologist will be expected to wear shoe covers, a lab coat, or surgical scrubs and gloves at a minimum and may be required to wear a tyvek ® suit, surgical cap, mask, and occasionally a respirator. when working with nonhuman primates (nhps), annual testing for tuberculosis (tb) using a tb intradermal ppd skin test or the new quantiferon ® -tb blood test will generally be required of all personnel including the ophthalmologist. the ophthalmologist must be familiar with what is normal for the species being examined and what are the common, spontaneous abnormalities for that species, age of animal, and breed/strain. albino vs. pigmented eye may be a factor as is the type of retinal vasculature, ranging from anangiotic to merangiotic to holangiotic. the presence or the absence of a tapetum and whether the animal has a fovea should be considered. in addition, the examination techniques to be used, type of biomicroscope and indirect ophthalmoscope, size and diopter of the indirect lens, and number of animals that can be examined in an hour must be understood. the role of the veterinary ophthalmologist is to perform a pretest examination designed to eliminate those animals not suited to the study and to establish a baseline database to compare interim and end-ofstudy findings. animals are then subsequently examined one or more times during the study, at the conclusion of the study, and possibly in a recovery phase depending on the study duration and design. typically studies are divided into acute, subacute, subchronic, and chronic depending on the duration. the ophthalmologist must then interpret findings in light of the species examined, pretest data, compound evaluated, additional study procedures performed (anesthesia, orbital blood collection), and dose group outcome. since most laboratory studies involve a significant number of animals, organization and efficiency are essential. in general, most canine, primate, swine, feline, and rabbit studies involve - animals while rats and mice may involve - , animals in a single study. a single ophthalmologist generally requires - animal handlers, a data entry individual, and in studies over animals - individuals to go ahead of the animal handlers to dilate the pupils. for efficiency, an animal should be in front of the ophthalmologist at all times. the ophthalmologist's findings are reported verbally to the data entry individual who then either enters it into a computer program or records on a paper record for later entry into a computer database. this data is then verified at the end of the examination and both the ophthalmologist and data entry individual date and initial the accuracy of the report. when an ophthalmic abnormality is observed, it must be characterized with respect to diagnosis, location, and severity. depending on the laboratory, some will utilize a standardized scheme for recording of clinical observations such as provantis ® that has a set of preloaded ophthalmic terms for organ location (cornea, lens, iris, etc.), clinical signs/diagnosis (opacity, coloboma, degeneration, hemorrhage, etc.), specific location (cortex, nucleus, tapetal, anterior, posterior, etc.), and severity (slight, moderate, severe). other cros may have their own in-house online or hand recording system. the ophthalmologist should be familiar with each laboratory's recording system and terminology to be consistent both within and between studies. when an abnormality is observed, correlation between dose groups is important when evaluating the incidence and severity of lesions so that any association with the test article can be assessed [ ] . while it would be best if animals were examined out of dosing order so as to mask the ophthalmologist with respect to dose group being examined, this is often not possible given the way animals are housed and entered into the data collection system. the ophthalmologist should also have a standardized scoring or grading scheme to assign a severity to any abnormalities seen. in general, a grading scheme of slight, moderate, and severe/marked is most common. when using this grading scheme for the transparent media (cornea, aqueous, lens, and vitreous) a grading of slight would imply a lesion that does not obstruct visualization of the deeper tissues past the lesion, a moderate grade implies a lesion that interferes with but does not fully obstruct the view of the tissues deep to the lesion, and a severe/marked lesion fully obstructs the view of structures deep to the lesion (table ). this is analogous to the terms incipient, immature, and mature when applied to a cataract. lesions may also be characterized with respect to the area of involvement with the terms most commonly used being focal, multifocal, and diffuse. for studies involving topical ophthalmic application of a drug or an ocular/intraocular device a specific more detailed biomicroscopic examination protocol with standardized scoring or grading criteria is frequently used. this is most commonly the modified hackett-mc-donald scoring system (tables and ) [ , ] . additionally, for studies involving intravitreal injection or intraocular procedures, the standardization of uveitis nomenclature (sun) grading system can be used or modified to evaluate aqueous flare and cells (tables and ) [ , , ] . when counting anterior chamber cells, the type of cell pigmented, white blood cell or red blood cell, should be noted [ ] . for grading of intravitreal inflammation and cells, the national eye institute system can be used (table ) [ , ] . biomicroscopy is used to examine the ocular anterior segment including the eyelids, conjunctiva, third eyelid, tear film, cornea, anterior chamber, iris, lens, and anterior vitreous. biomicroscopy provides a magnified view of the living eye using a light that can be varied in intensity, width, height, and color. in general for laboratory animals, this is performed using a handheld slit lamp of the ophthalmologist's preference. the slit lamp used for routine examination should be portable, lightweight, and easy to use on a variety of species. the two most common portable slit lamps used for laboratory animals are the zeiss hso- ( fig. ) and the kowa sl- / (fig. ) . the zeiss hso- provides a  magnification with a mm working distance and is both lightweight and easy to use on all species. the kowa sl- / has either a  or a  magnification with a mm working distance. unlike the zeiss, the kowa works from a battery pack and is re-charged in a stand. both have fixed slit widths ( . and . mm zeiss; . , . , and . mm kowa) and both have a cobalt blue filter for fluorescein. if higher magnification or photographic documentation are required, a table-mounted slit lamp may be used (fig. ) . table slit lamps provide higher quality optics, increased magnification, and variable width and height of the slit beam and with additional attachments can allow for photographic documentation, gonioscopy, or specular microscopy. table slit lamps are however significantly more expensive, less portable, and more difficult to use on a large number of animals or un-sedated animals. the slit lamp performs two major functions. first, it provides magnification for a more detailed examination of the eye. secondly it makes use of the slit beam, decreasing the beam of light to a slit allowing an optical cross section of the eye to be obtained (figs. and ) . this allows precise localization of the depth of a lesion and allows visualization of subtle changes that cannot be seen with full illumination (figs. , , , , and ) . the term for this type of illumination and examination is an optical section and it is the most table criteria used for a modified hackett-mcdonald scoring system [ ] conjunctival congestion normal. may appear blanched to reddish pink without perilimbal injection (except at : and : o'clock positions) with vessels of the palpebral and bulbar conjunctiva easily observed. a flushed, reddish color predominantly confined to the palpebral conjunctiva with some perilimbal injection but primarily confined to the lower and upper parts of the eye from the : , : , : , and : o'clock positions. bright red color of the palpebral conjunctiva with accompanying perilimbal injection covering at least % of the circumference of the perilimbal region. dark, beefy red color with congestion of both the bulbar and the palpebral conjunctiva along with pronounced perilimbal injection and the presence of petechia on the conjunctiva. the petechia generally predominate along the nictitating membrane and the upper palpebral conjunctiva. conjunctival swelling (there are five divisions from to ) normal or no swelling of the conjunctival tissue. swelling above normal without eversion of the lids (can be easily ascertained by noting that the upper and lower eyelids are positioned as in the normal eye); swelling generally starts in the lower cul-de-sac near the inner canthus, which needs slit lamp examination. swelling with misalignment of the normal approximation of the lower and upper eyelids; primarily confined to the upper eyelid so that in the initial stages the misapproximation of the eyelids begins by partial eversion of the upper eyelid. in this stage, swelling is confined generally to the upper eyelid, although it exists in the lower cul-de-sac (observed best with the slit lamp). swelling definite with partial eversion of the upper and lower eyelids essentially equivalent. this can be easily ascertained by looking at the animal head-on and noticing the positioning of the eyelids; if the eye margins do not meet, eversion has occurred. eversion of the upper eyelid is pronounced with less pronounced eversion of the lower eyelid. it is difficult to retract the lids and observe the perilimbal region. conjunctival discharge-discharge is defined as a whitish-gray precipitate, which should not be confused with the small amount of clear, inspissated, mucoid material that can be formed in the medial canthus of a substantial number of rabbit eyes. this material can be removed with a cotton swab before the animals are used. normal. no discharge. discharge above normal and present on the inner portion of the eye but not on the lids or hairs of the eyelids. one can ignore the small amount that is in the inner and outer canthus if it has not been removed prior to starting the study. discharge is abundant, easily observed, and has collected on the lids and around the hairs of the eyelids. discharge has been flowing over the eyelids so as to wet the hairs substantially on the skin around the eye. aqueous flare-the intensity of the tyndall phenomenon is scored by comparing the normal tyndall effect observed when the slit lamp beam passes through the lens with that seen in the anterior chamber. the presence of aqueous flare is presumptive evidence of breakdown of the blood-aqueous barrier. the absence of visible light beam in the anterior chamber (no tyndall effect). the ophthalmic examination as it pertains to general ocular toxicology. . . table (continued) the tyndall effect is barely discernible. the intensity of the light beam in the anterior chamber is less than the intensity of the slit beam as it passes through the lens. the tyndall beam in the anterior chamber is easily discernible and is equal in intensity to the slit beam as it passes through the lens. the tyndall beam in the anterior chamber is easily discernible; its intensity is greater than the intensity of the slit beam as it passes through the lens. pupillary light reflex-the pupillary diameter of the iris is controlled by the radial and sphincter muscles. contraction of the radial muscle due to adrenergic stimulation results in mydriasis while contraction of the sphincter muscle due to cholinergic stimulation results in miosis. as an ophthalmic drug can exert potential effects on these neural pathways, it is important to assess the light reflex of an animal as part of the ophthalmic examination. using full illumination with the slit lamp, the following scale is used. normal pupillary response. sluggish pupillary response. maximally impaired (i.e., fixed) pupillary response. iris involvement-in the following definitions, the primary, secondary, and tertiary vessels are utilized as an aid to determining a subjective ocular score for iris involvement. the assumption is made that the greater the hyperemia of the vessels and the more the secondary and tertiary vessels are involved, the greater the intensity of iris involvement. the scores range from to . normal iris without any hyperemia of the iris vessels. occasionally around the : - : o'clock position near the pupillary border and the : and : o'clock position near the pupillary border, there is a small area around - mm in diameter in which both the secondary and tertiary vessels are slightly hyperemic. minimal injection of secondary vessels but not tertiary. generally, it is uniform, but may be of greater intensity at the : or : o'clock position. if it is confined to the : or : o'clock position, the tertiary vessels must be substantially hyperemic. minimal injection of tertiary vessels and minimal to moderate injection of the secondary vessels. moderate injection of the secondary and tertiary vessels with slight swelling of the iris stroma (this gives the iris surface a slightly rugose appearance, which is usually most prominent near the : and : o'clock positions). marked injection of the secondary and tertiary vessels with marked swelling of the iris stroma. the iris appears rugose; may be accompanied by hemorrhage (hyphema) in the anterior chamber. cornea cloudiness-the scoring scheme measures the severity of corneal cloudiness and the area of the cornea involved. severity of corneal cloudiness is graded as follows: normal cornea. appears with the slit lamp adjusting to a narrow slit image as having a bright gray line on the epithelial surface and a bright gray line on the endothelial surface with a marble-like gray appearance of the stroma. some loss of transparency. only the anterior half of the stroma is involved as observed with an optical section of the slit lamp. the underlying structures are clearly visible with diffuse illumination, although some cloudiness can be readily apparent with diffuse illumination. (continued) involvement of the entire thickness of the stroma. with the optical section, the endothelial surface is still visible. however, with diffuse illumination, the underlying structures are just barely visible (to the extent that the observer is still able to grade flare and iritis, observe for pupillary response, and note lenticular changes). involvement of the entire thickness of the stroma. with the optical section, cannot clearly visualize the endothelium. with diffuse illumination, the underlying structures cannot be seen. cloudiness removes the capability for judging and grading flare, iritis, lenticular changes, and pupillary response. corneal area-the surface area of the cornea relative to the area of cloudiness is divided into five grades from to . normal cornea with no area of cloudiness. pannus-pannus is vascularization or the penetration of new blood vessels into the corneal stroma. the vessels are derived from the limbal vascular loops. pannus is divided into three grades. no pannus. vascularization is present but vessels have not invaded the entire corneal circumference. where localized vessel invasion has occurred, they have not penetrated beyond mm. vessels have invaded mm or more around the entire corneal circumference. fluorescein-for fluorescein staining, the area can be judged on a to + scale using the same terminology as for corneal cloudiness. the intensity of fluorescein staining can be divided into - scale. absence of fluorescein staining. (continued) the ophthalmic examination as it pertains to general ocular toxicology. . . common method of biomicroscopic examination. using a narrow slit beam, a highly magnified optical section of the eye is obtained. the direction of the slit beam may be varied so that the structures may be viewed using either direct illumination or retroillumination [ ] . this will allow the examiner to detect and localize, with respect to depth, abnormalities in the anterior segment of the eye. for example, a corneal lesion can be localized to superficial, stromal, or endothelial; aqueous opacities such as cells (aqueous, vitreous), flare, or hemorrhage are detectable and quantifiable; and lesions of the lens may be localized to anterior, posterior, equatorial, and further to capsular, cortical, or nuclear. interpretation of the findings on slit lamp biomicroscopy requires extensive knowledge of normal findings as well as background lesions that occur as incidental findings in the species and breed being examined [ ] . indirect ophthalmoscopy is the preferred technique of choice for routine screening of the posterior segment in all laboratory animal species. indirect ophthalmoscopy provides a binocular, inverted, and reversed aerial image with a wide field of view. it requires an indirect headset and a condensing lens. once perfected, the technique of indirect examination also allows for a more rapid examination of the entire posterior segment as it provides a wider panoramic field of view, allowing the examiner to evaluate more animals, more accurately in a shorter period of time. the indirect headset of choice should be lightweight, comfortable, and easy to manipulate out of the way with one hand and have a small pupil setting. the ease of manipulation will allow the examiner to move efficiently between indirect and biomicroscopic examination techniques. while several excellent choices are available, the keeler all pupil ® is best suited in the author's opinion (fig. ) . alternatively, the heine indirect ophthalmoscope also offers excellent optics and can be fitted with a portable power supply, but is slightly heavier and more cumbersome for large number of animals (fig. ) . the indirect condensing lens of choice varies by species examined and by the examiner's choice. in general, a . pan retinal or diopter lens works well for routine screening examination of most lens-the crystalline lens is readily observed with the aid of the slit lamp biomicroscope, and the location of lenticular opacity can readily be discerned by direct and retro illumination. the lens is normal (n) or abnormal (a) if abnormal, the location and severity of lenticular opacities are noted in the comments. table grading scheme for aqueous cells based on the sun grading system using a biomicroscope and a mm  mm slit beam cells should be counted at the same location, usually the central anterior chamber [ , ] table grading scheme for aqueous flare based on the sun grading system using a biomicroscope [ ] grade description table grading scheme for vitreous inflammation based on the national eye institutes grading system using an indirect ophthalmoscope [ ] grade description alternately, a direct ophthalmoscope (fig. ) can be used to examine the optic nerve head and fovea in nhps, but given its small field and monocular view, it is less than optimal in the author's opinion. in addition, when performing direct ophthalmoscopy any opacity of the cornea, aqueous, lens, or vitreous will severely impair or prevent visualization of the posterior segment. prior to indirect ophthalmoscopy, a short-acting mydriatic agent is required to dilate the pupil. tropicamide . - . % is the mydriatic of choice. when examining using indirect ophthalmoscopy, the examiner remains at arm's length and places the condensing lens just anterior to the cornea. this technique is used to examine the posterior vitreous, optic nerve, retinal vasculature, retina, and choroid. in addition, opacities of the clear media (cornea, aqueous, lens, and vitreous) are readily detectable using retroillumination. this technique can be used to grade vitreous inflammation/opacification (table ). given the wide variety of laboratory animals, the examiner must be familiar with the variation of normal anatomy and species variations. the retinal vasculature will vary with the species anangiotic (guinea pig), merangiotic (rabbit), and holangiotic (rodent, canine, swine, primate). the pigmentation of the retinal pigment epithelium (rpe) and the choroid vary between albinotic, sub-albinotic, and pigmented animals of the same species (mouse, rat, rabbit). some species such as the canine will have a tapetum located in the superior choroid, but this can be lacking in color dilute or lemon beagles. finally, nhps are foveated and this region must be examined carefully for abnormalities. additional techniques to evaluate the retina may include fluorescein angiography, oct, confocal scanning laser ophthalmoscopy, fundus photography, and electrodiagnostic testing. when these tests are used correctly and in combination they can provide additional en face, cross-sectional, and functional information of the retina that may be then correlated with histopathology. prior to study initiation, a pretest ophthalmic examination should be performed on all study animals. this is done for two reasons. the first is to eliminate from the study animals with current significant or potentially progressive ophthalmic abnormalities. the second is to establish a baseline of ocular findings to compare to as the study progresses and subsequent ophthalmic examinations are performed. examples of pretest abnormalities that should automatically result in an animal's elimination from the study would include all ocular findings with a severity score of moderate or higher and all abnormalities that currently prevent or may prevent if progressive complete examination of intraocular structures. examples of ocular findings that may be progressive during the course of the study and common background abnormalities will vary by species, but may include ocular trauma associated with shipping, congenital embryonic remnants such as persistent pupillary membrane (ppm) and persistent hyaloid artery (pha), extravasation of blood in association with a pha, corneal opacity/dystrophy, coloboma (iris, lens, choroid), cataract, micropapilla, optic nerve hypoplasia, and retinal dysplasia [ , - , , - ] . ppm and pha are commonly observed in rodents (figs. and ) and less frequently in the beagle (figs. and ). in general, they are noted and graded. however, if they result in significant opacity of the cornea, lens, or vitreous at pretest examination elimination of the affected animal from study should be considered. if a persistent hyaloid has a significant component of extravasated blood, elimination from study is advised (fig. ) . whenever possible, animals with ocular abnormalities are eliminated from inclusion in the study. this is especially true if the lesion may be progressive during the study. however, some abnormalities are so common as to preclude elimination. the most common example of this would be corneal dystrophy in the rat and mouse [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the prevalence of corneal dystrophy varies by stain, age, and sex of the rat. in the sprague-dawley, males are more commonly affected than females and prevalence will increase with age and may vary from to % (fig. ) . in the wistar, corneal rat with significant intravitreal extravasated blood associated with a persistent hyaloid. this was noted in pretest examination and the animal was eliminated from the study dystrophy has been reported to affect > % (fig. ) and in the fisher (fig. ) , the prevalence is generally %. as a result of such a high prevalence, affected animals cannot be eliminated from inclusion in study. however, animals affected with corneal dystrophy with a severity grade of greater than slight or with secondary keratitis should be eliminated at pretest. corneal dystrophy lesions are also observed in other species including the beagle (fig. ) and dutch belted rabbit (fig. ) . it should also be noted that lesions such as corneal dystrophy can be exacerbated in both prevalence and severity by corneal exposure caused by desiccation, sedation/anesthesia, orbital bleeding, environmental irritants, and the test article itself. the ophthalmologist must be familiar with what is normal for the species in question and what are the common spontaneous abnormalities for that species, age of animal, and breed/strain [ ] . common laboratory species of interest may include mice, rats, guinea pigs, cats, rabbits, dogs, gottingen mini-pigs, and nhps (cynomolgus, rhesus). for the common laboratory species ). an albinotic vs. pigmented eye may be a factor in clinical findings as well as response to mydriasis or test article effect. the presence of a nictitating membrane (third eyelid), number, location, and type of lacrimal/orbital glands, location and number or lacrimal puncta, presence or absence of a tapetum, myelination of the optic nerve, anatomy and physiology of aqueous outflow, and whether the animal has a fovea should all be understood and considered [ ] . for intraocular procedures or intraocular dosing the specific size of the globe and the size and volume of all intraocular structures and the aqueous and vitreous must be understood in order to avoid inadvertent trauma during the procedure [ ] . for topical ophthalmic dosing, the volume capacity of the conjunctival cul-de-sac and volume of the pre-corneal tear film must be known. in addition, the examination and restraint techniques, need for sedation, type of biomicroscope and indirect ophthalmoscope, diopter power of the indirect lens, and number of animals that can be examined in the time period that the mydriatic effect will persist must be understood. the role of the veterinary corneal dystrophy in a dutch belted rabbit (arrow). this was not present in pretest examination, but appeared during the study david a. wilkie ophthalmologist is to perform a pretest examination designed to eliminate those animals not suited to the study and to establish a baseline database to compare to. animals are then subsequently examined one or more times during and at the conclusion of the study depending on the study duration. typically studies are divided into acute, subacute, subchronic, and chronic depending on the duration. the ophthalmologist must then interpret findings in light of the species examined, pretest data, compound evaluated, and dose group outcome. the beagle is a canine like any other and most veterinary ophthalmologists are both familiar and comfortable with the examination and abnormalities of this breed [ ] [ ] [ ] ] . the breed-related abnormalities will vary according to the supplier and are often seen with a prevalence that waxes and wanes according to the current sires and dams. for example in the author's experience, micropapilla is more common in a specific supplier's beagles, but rare in beagles from other suppliers. keep in mind that these are tightly controlled breeding programs to yield a beagle with specific characteristics and their ocular lesions while similar in kind may differ in prevalence from the pet beagles seen in a clinical setting. in the author's experience, prolapse of the gland of the nictitans (fig. ) , retinal dysplasia (folds) (figs. and ), optic nerve micropapilla/hypoplasia (fig. ) , and incipient posterior cortical or anterior suture cataract (figs. , , and ) are the most common breed-related findings in the laboratory beagle [ , , , ] . these are similar to the previously reported prevalence of ophthalmic findings in - -month-old beagles (table ) [ ] . corneal dystrophy is also observed in the laboratory beagle [ , ] . it should be noted that corneal dystrophy, cataracts, and retinal degeneration may also be noted as a treatment-related finding (figs. , , and ) . the beagle has a holangiotic retina and, unlike most laboratory animal species, has a tapetum located in the superior choroid (fig. ) . the tapetum is cellular in nature and is located between the large choroidal vessels and the choriocapillaris in the dorsal aspect of the fundus. the tapetal color is variable with blue, green, orange, and yellow the most common variations. there are also atapetal beagles, usually color dilute, which are reported to have tapetal cells that are deficient in tapetal rodlets [ ] . the tapetum can have a role in ocular toxicity [ ] and in such instances atapetal beagles can be used. the tapetum of the dog contains a high concentration of zinc and as such zinc chelators may result in fig. prolapse of the gland of the nictitans with associated mucoid epiphora, conjunctival hyperemia, and a focal corneal opacity tapetal necrosis and secondary retinopathy [ ] . additional drugassociated lens and retinal changes have also been described in the beagle (figs. and ) [ , ] . in general, young animals are more likely to have ph or ppm (figs. , , and ). in one report the prevalence of hyaloid remnants may be as high as % at weeks of age, but decrease to less than % by year of age [ , , ] . in mice, the prevalence of a ph has been reported to range between and % with a higher prevalence in females [ ] . on a pretest examination, animals with ppm/ph and associated hemorrhage (vitreous, aqueous) should typically be eliminated from the study if possible (fig. ) . anterior synechia, microophthalmos, and anterior cleavage anomalies are also occasionally seen as congenital ocular anomalies in rats and mice. the term corneal dystrophy implies a bilateral, noninflammatory inherited, degenerative disorder and is a specific term. unfortunately, the terms corneal opacity or corneal crystals are also used and are less specific and more general terms. the term calcific band keratopathy has also been applied to the lesions observed in rodents [ ] . clinically, the lesions of corneal dystrophy most commonly appear as fine granular or linear opacities in the nasal and axial palpebral fissure (figs. - ) . they are most frequently bilateral. histologically, corneal dystrophy in rodents is typically a basement membrane, anterior stromal corneal defect resulting in deposits of mineral and phospholipid in and adjacent to the epithelial basement membrane (fig. ) [ , ] . corneal dystrophy, while common in a pretest examination, will be observed to increase in prevalence and severity with age. corneal dystrophy is common in rats and less common in mice, and varies in prevalence, appearance, and severity by strain of rat/mouse, sex, age, and shipment and supplier. it is more prevalent in males and increases in prevalence and in some species severity with age [ ] . the prevalence will vary from to % [ ] [ ] [ ] [ ] [ ] [ ] [ ] . it has been variously termed corneal dystrophy, calcific keratopathy, corneal opacity, corneal calcification, and spontaneous corneal degeneration. it has also been reproduced experimentally in rats by corneal desiccation which can occur secondary to dehydration, a decrease in blink rate, sedation, or anesthesia. as it increases in severity with age it can be associated with secondary keratitis (typically nasal), especially in the fischer rat (fig. ). in addition, keratitis can occur secondary to anesthesia, corneal exposure, keratoconjunctivitis sicca, sialodacryoadenitis (sda), environment (dust, irritants), and conjunctivitis. cataracts may be seen during pretest screening and are most often nuclear (fig. ) . they can be unilateral or bilateral and if possible, these animals are eliminated from the study. posterior cortical cataracts are also seen, often associated with a ph with or without hemorrhage. cataracts may also be spontaneous, associated with age, secondary to retinal degeneration, or associated with trauma, anesthesia, or other external factors (fig. ) [ , [ ] [ ] [ ] . acquired cataracts may also be treatment related and the result of a toxicologic effect. the examiner should always ascertain if orbital bleeding was performed during the study in question, especially if the cataracts are unilateral and typically in the same eye within the study animals. reversible lens opacities may be induced in the rat by temperature (cold), dehydration, anoxia, and specific drugs (opiates, opioids, phenothiazine) [ , , ] . buphthalmos and glaucoma have been observed and are typically congenital (fig. ) [ ] . unfortunately, the intraocular pressure is generally not determined in affected animals as this is most often noted in young rats in a pretest examination and they are usually eliminated from study without further diagnostics or follow-up. retinal dysplasia may be noted in rats and can be unilateral or bilateral [ ] . retinal degeneration may occur spontaneously, be associated with aging, occur as a result of orbital bleeding techniques, occur secondary to phototoxicity, or be inherited [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . it is reported that the prevalence of senile retinal degeneration in the -year-old wistar rat may be as high as % [ ] . in addition, retinal degeneration can be a toxicologic effect [ ] . care should be taken to evaluate retinal "blanching" in combination with the temperament and restraint required to examine a particular animal. excessive restraint will result in apparent retinal degeneration. as rats age, the prevalence of corneal, lenticular, and retinal abnormalities will increase and in a -year chronic study abnormalities may be found in > % of the animals examined [ , ] . there is also often a sex predilection with corneal and possibly lens abnormalities seen more often in males than females. the technique of orbital bleeding may be used to collect venous blood from the retrobulbar sinus of rodents during a study [ , ] . most often one side is preferred, usually the right. the side effects of orbital bleeding can be severe and include exophthalmos, corneal rupture, exposure keratitis, retinal degeneration, hyphema, cataract, and phthisis bulbi (figs. , , , and ). the frequency of these complications likely relates directly to the skill of the individual performing the procedure. however, complications are frequent enough that the technique should be discouraged, especially in studies that require ophthalmic examination. if a rodent study requires general anesthesia the ophthalmologist can expect exacerbation of corneal dystrophy and in some instances cataract formation. xylazine has been incriminated as being cataractogenic in one study [ ] . chromodacryorrhea appears as a red-brown periorbital discharge (fig. ) [ , , ] . ocular irritation, respiratory infection, stress, and sda (coronavirus) can all result in chromodacryorrhea [ ] . irritation can occur from something as simple as a diet formulation change in an effort to get a test article into the feed. corneal dystrophy is common affecting - % of animals in the author's clinical experience. this is higher than what is reported in the literature most likely due to the failure to use biomicroscopy in several published reports. corneal dystrophy appears more prevalent in males than females and increases in prevalence with age [ , , , ] . in the sprague-dawley, the lesions of corneal dystrophy involve the axial cornea and appear as fine granules or crystals in the subepithelial stroma (fig. ) . a basement membrane dystrophy and calcium deposits are noted histologically and ultrastructurally (fig. ) . the severity is generally mild, keratitis is uncommon, and prevalence increases with age. rats as young as a few weeks of age can be affected. lens opacities in the sprague-dawley aged - weeks have been reported to be - % [ , ] . a chorioretinal abnormality, termed linear retinopathy, retinochoroidal degeneration/ atrophy, retinal dysplasia, or choroidal coloboma, is described in - -week-old sprague-dawley rats (fig. ) [ , , , , ] . histologically the lesion is characterized by thinning of the outer retina and choroid [ ] . posterior segment vascular anomalies have been described in the sprague-dawley and may include a preretinal vascular loop and saccular aneurysms (fig. ) [ ] . in one study this was observed in % of rats examined [ ] . i have never examined a fisher that did not have corneal dystrophy (fig. ) ; in my clinical experience the prevalence is %. it should be noted that papers citing prevalence below this often fail to examine using biomicroscopy [ , ] . when compared to the sprague-dawley the lesions in the fischer are more numerous and larger and may have a linear pattern in the axial cornea. as the fischer rat ages, the corneal dystrophy severity score is expected to increase and keratitis, corneal vascularization, and corneal ulceration may occur in association with corneal dystrophy. in addition to corneal dystrophy, in one report affected rats were also found to have basement membrane changes in other organs [ ] . corneal changes are characterized histologically by thickened, fragmented, and mineralized corneal basement membranes. these lesions have also been described to be associated with limbal inflammatory reaction which may be the precursor to the keratitis and vascularization. it should be noted that as corneal dystrophy in the fisher worsens clinically the opacities increase in size and density. the overlying epithelium will become elevated, as the dystrophy becomes a space-occupying lesion. histologically, the epithelium overlying a severe area of dystrophy will be thin and possibly keratinizing indicating irritation. it is this spaceoccupying, foreign-body effect combined with the disruption of the corneal epithelial basement membrane that results in corneal vascularization and keratitis. it has been the author's experience that in some instances dystrophy will even result in the loss of the overlying corneal epithelium exposing the underlying basement membrane and possibly the corneal stroma. severity may be more severe in males. corneal dystrophy has been reported to affect > % of wistar rats and to be composed of extracellular calcium and phosphorous at the level of the corneal epithelial basement membrane (fig. ). in one clinical study, / males and / females were affected with an interpalpebral corneal opacity [ , , ] . histologically, the lesions are located at the level of the basement membrane and consist of calcium and phosphorus [ ] . clinically, when compared to the sprague-dawley rat the axial corneal lesions in the wistar are larger, linear, and more opaque. another biomicroscopy study found lenticular water clefts in and % of -year-old female and male wistar rats and in and % of -year-old female and male wistar rats. the same study found cortical/nuclear lens opacities in approximately % of -year-old male/female wistars and posterior capsular opacities in up to and % of -year-old female and male wistars [ ] . a retinal dystrophy/degeneration has been reported in the wistar rat [ ] . it is reported that the prevalence of senile retinal degeneration in the -year-old wistar rat may be as high as % [ ]. rabbits have long been used in ophthalmic and toxicologic research for topical irritancy testing (draize, modified hackett-mcdonald) [ , ] , contact lens evaluation, ocular pharmacology, intraocular device biocompatibility, and intravitreal injection protocols as well as in systemic toxicity studies. the most common rabbits examined in toxicologic studies are new zealand white (nzw) and american dutch belted. most rabbits are found to be normal on routine ophthalmic examination. the eye of the rabbit may be albinotic or pigmented and has a single nasolacrimal punctum, a merangiotic fundus, and deep physiologic optic disc cup with a heavily myelinated optic nerve termed a medullary ray [ ] . congenital and spontaneous sporadic findings include optic nerve coloboma, corneal dystrophy, cataract, glaucoma, epiphora, pseudopterygium, and dacryocytitis (figs. , , , and ) . corneal dystrophy has been reported in american dutch belted and nzw rabbit (fig. ) [ , ] and can also occur as a result of diet. in corneal dystrophy of the dutch belted rabbit there is a thickening of the corneal basement membrane and a thinning and disorganization of the overlying corneal epithelium [ ] . in the watanabe rabbit, altered lipid metabolism may be associated with corneal lipidosis especially when fed a high-fat diet (fig. ) [ , ] . inherited glaucoma and associated buphthalmia are seen in the nzw rabbit and occur as the result of goniodysgenesis with the mode of inheritance being autosomal recessive (bu/bu gene) [ ] [ ] [ ] . iop increases beginning at - months of age with resulting buphthalmia [ ] . the use of nhps in toxicologic research seems to have become more common over the past years with cynomolgus and rhesus monkeys the most commonly examined. the ocular anatomy of the nhp is similar to that of man with a canal of schlemm for aqueous outflow, well-developed accommodative abilities, a central retinal artery, and a fovea resulting in a visual acuity and color vision similar to humans (fig. ) [ , , ] . as with other species, there can be variations in pigment distribution and amount in the fundus and the examiner needs to be familiar with normal variations [ ] . for safety purposes, nhps are generally examined under a short-acting general anesthetic such as ketamine with the mydriatic administered following sedation. the examiner will generally be required to wear gloves (usually double glove), a tyvec ® jump suit, surgical bouffant, and a mask. safety goggles are advised until the animals are anesthetized and you are examining the eyes. traumatic lesions are most common and may include eyelid lacerations and corneal scars. in addition, traumatic cataract and retinal scars have been noted. focal iris nevi are not considered abnormal and are observed not infrequently. in one study of , wild-caught cynomolgus monkeys animals ( . %) had findings, the majority of which involved the posterior segment [ ] . the lesions of the posterior segment are predominately chorioretinal scars [ ] . glaucoma has been described in several species of nhps [ ] as has macular degeneration [ , ] and cataract [ , ] . there are numerous noninvasive ophthalmic diagnostic techniques that, depending on the tissue of interest, can provide both structural and functional information of both the anterior and posterior segments of the eye. some of the more common techniques are discussed below and a more detailed discussion of these and additional techniques as they apply to toxicologic, ophthalmic research and clinical application has recently been published [ , ] . tonometry is used to measure and obtain an indirect measurement of the iop. there are several methods that are considered portable and they include indentation, applanation, and rebound tonometry. of these, indentation tonometry, utilizing the schiotz tonometer, would be considered inaccurate and unreliable and so it should not be used for laboratory studies. as all tonometers are originally designed for the human cornea, readings in animals may be slightly inaccurate, but provided the same tonometer is used throughout a study, the changes in iop will still remain valid [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the iop in most laboratory animals will range between and mmhg and there should be mmhg difference between the two eyes. the iop can be affected by restraint techniques, animal stress, diurnal or circadian rhythm [ , ] , eye position, sedation or anesthesia, corneal thickness, and several other variables. if possible, the iop should be obtained for all animals at the same general time of day throughout the study and by the same examiner, using the same tonometer, same handling personnel, and same technique each time. determination of iop should be performed prior to pharmacologic dilation. when iop is a critical aspect of a study, it is also advisable for the animals to be acclimatized to both the procedure and restraint techniques prior to study initiation. applanation tonometry is most commonly performed using the tonpen xl ® , tonopen vet ® , tonopen avia ® , or pneumotonograph. it requires topical anesthesia of which . % proparacaine is the most common topical ophthalmic anesthetic of choice. this technique measures the force required to applanate or flatten a given area of cornea and then converts this into an iop value in mmhg. these tonometers have the advantage of being able to be self-calibrated for a glp study (fig. ) and the pneumotonograph can also provide a hard paper copy for record keeping. the tonpen xl and tonopen vet ® obtain four independent readings, average them, and indicate both the iop and the % error indicating the variability between the four readings obtained. the tonopen avia ® obtains ten independent readings, averages them, and reports the iop and reports the variability in readings as a % confidence. for the tonopen xl ® the % error should be < % and for the tonopen avia ® the % confidence should be > %. rebound tonometry determines iop by firing a small plastic tip against the cornea. the tip then rebounds back into the device creating an induction current from which the iop is calculated. the probe must be fired at the cornea in the horizontal position, parallel to the floor to be accurate. the most common rebound tonometer for laboratory animals is the tonovet ® (fig. ) . unfortunately the tonovet ® is most specifically calibrated for the dog and horse, but it has been used reliably in other species [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . it has the advantage of not requiring topical anesthesia and seems to obtain the iop more easily than the tonpen ® in many laboratory animals including the dog, rabbit, and rat. its disadvantage is that it cannot be self-calibrated prior to use. like the tonopen ® the tonovet ® also averages six readings and gives an indication of % error using a bar at the left of the iop value. there should be no error bar or the bar should be at the left ventral aspect of the screen for a reading to be acceptable. regardless of the tonometer used, typically a minimum of - final averaged readings per eye should be obtained and recorded. as both the tonpen ® and tonovet ® give only digital readouts, the iop must be either hand recorded or entered into a computer database as no permanent record is created by the device. pachymetry is the evaluation of corneal thickness. it is most commonly performed by use of a contact ultrasound specifically designed for this purpose, but the corneal thickness measurement can also be obtained by high-resolution ultrasound or oct. pachymetry allows evaluation of subtle changes in corneal thickness prior to the appearance of clinically detectable corneal edema on biomicroscopy. the corneal thickness varies between species, but also varies by region of the cornea (axial vs. peripheral). as a result of the regional variation readings must be obtained from the same region of the cornea, usually axially, at each time point. sodium fluorescein to evaluate the cornea is routinely used in studies involving topical ophthalmic drug administration and contact lens evaluation and other studies that use the modified hackett-mcdonald scoring system [ ] . fluorescein is a watersoluble dye that is retained by the hydrophilic corneal stroma, but not by the corneal epithelium. it is used to evaluate for corneal epithelial defects and can also be used in evaluation of the precorneal tear film. fluorescein is available in individual impregnated strips that are moistened at the time of use using sterile saline. the moistened strip is gently applied to the dorsal sclera taking care not to contact the cornea. the excess fluorescein is then gently irrigated from the eye using a gravity-fed stream of saline rather than a forced high-velocity stream. the eye is then examined by the ophthalmologist using a biomicroscope and the cobalt blue filter to excite the fluorescein should any remain following irrigation. ophthalmic photography may be used to demonstrate a lack of change in an area like the fundus, to document abnormalities, or to monitor progression of a lesion. serial photographs taken at various time points during a study will allow comparison to accurately establish whether an abnormality is static or progressive. as photography adds additional time, cost, and animal stress, it is not routinely performed in all studies. rather it is in a study protocol as an option to be used to document a lesion when observed or for studies where abnormalities are more likely to occur, such as with an intraocular implant, or for intravitreal injection studies. photography can be divided into external and intraocular. external photography can be performed using a standard slr digital camera with a macro lens or with a digital kowa genesis-d fundus camera with the diopter settings adjusted to allow external and anterior segment imaging (fig. ) . photography of the posterior segment requires some type of fundus camera and the digital kowa genesis-d camera is suitable for most routine laboratory animal photography. the kowa genesis-d can also be adapted for indirect ophthalmic photography, rodent fundus photography, and fluorescein angiography [ ] . in addition, alternate methods for fundus photography in small rodents have been described [ ] [ ] [ ] [ ] [ ] . the advantages of fundus photography are the ability to have a permanent stored record to compare potential studyrelated findings and if indicated to obtain an independent review by another ophthalmologist [ ] . when obtaining photographs, the magnification and illumination settings should be standardized for all images. in addition, the eyes (left vs. right) should be photographed in the same order and all photographs should be accompanied by an animal identification photograph and a photographic log should be maintained as part of sop. fluorescein angiography is used to evaluate the vascular integrity of the intraocular arteriolar and venous vasculature. while it is most commonly used for examination of the retinal and choroidal vessels, it can be applied to iris vasculature as well. it is most frequently used in toxicology studies to evaluate a compounds effect on neovascularization. it has been applied to various laboratory animals with % fluorescein most commonly used, but with the use of indocyanine green also described [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the technique of fluorescein angiography requires sedation or anesthesia and pupil dilation [ ] . the excitable compound, fluorescein, is injected intravenously and a series of timed images of the tissue of interest (chorioretinal, iris) are obtained. complications associated with fluorescein injection may include extravasation and tissue irritation, vomiting, and anyphylaxis. an excitation filter ( nm) and a barrier filter ( - nm) must be used on a fundus camera that is capable of taking multiple, rapid sequenced images. the kowa genesis-df is designed for fluorescein angiography and is portable. prior to injection a baseline color image is obtained and then sequential black and white images are taken every s. as the fluorescein fills the chorioretinal vasculature various phases of vascular filling are described. they include the prearteriolar, retinal arteriolar, capillary, early venous, late venous, and recirculation. abnormalities noted on fluorescein angiography may include vascular anomalies (aneurysms, neovascularization), blocked fluorescence, leakage of fluorescein, hypofluorescence, and hyperfluorescence. depending on the toxicologic study and the specific aspect of the visual system that may be affected, there are various electrophysiologic tests that are available to evaluate the retina and visual pathways. electroretinography (erg) is the measurement of the electrical potential generated by the retina when stimulated by light. the standard erg is a full-field stimulation that provides information about the retina as a whole and is a mass response of the retinal pigment epithelium, photoreceptors, and inner retinal layer [ ] . for localized retinal evaluation the multifocal electroretinogram (mferg) and for evaluation of macular ganglion cells the pattern reversal electroretinogram (perg) are indicated [ ] . to evaluate the entire visual pathway from the retina to the visual cortex, a vep is the technique of choice [ ] . of these tests, the full-field erg is most common for preclinical toxicologic testing. the erg provides a noninvasive means of repeatedly assessing retinal function that in combination with indirect ophthalmoscopy and histology provides integrated assessment of retinal anatomy and function. the erg should be conducted in a standardized manner following pupil dilation and there are standardized protocols developed for human and canine ergs that can serve as a study design guide [ , ] . the conditions for obtaining a full-field erg must be consistent with respect to room illumination, dark adaptation, flash intensity and frequency, and sedation or anesthetic used and their dosage. discussion of these details and protocols is provided elsewhere and is beyond the scope of this chapter [ , , ] . oct is a high-resolution, noninvasive imaging technique that can provide a real-time cross-sectional imaging of ocular structures, most commonly retina and optic nerve, at an axial resolution of - μm [ , [ ] [ ] [ ] . it can also be used to image the anterior segment of the eye. like many advanced imaging techniques it requires sedation or anesthesia, pupil dilation, and specialized equipment. when imaged by oct, all individual retinal layers can be seen and their thickness measured to allow a quantitative and repeated evaluation of all retinal layers over time. the optic disc can be measured with respect to the cup area, disc area, cup diameter, disc diameter, and rim area [ ] . evaluation of the anterior segment by oct provides structural information of the cornea, anterior chamber, iris, and iridocorneal angle without the need for corneal contact as is required for ultrasound biomicroscopy (ubm) [ ] . it also provides greater axial resolution than that provided by ubm [ ] . the use of oct in laboratory animals has been well described in a variety of species and its use is increasing in animal models of human disease and preclinical trials [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . a recent advancement, spectral-domain oct (sd-oct), uses a significantly faster, nonmechanical technology than traditional oct or time-domain oct (td-oct) [ , , ] . sd-oct simultaneously measures multiple wavelengths of reflected light across a spectrum, hence the name spectral-domain. sd-oct is times faster than td-oct and acquires , a-scans per second. the increased speed and number of scans translate into higher resolution. specular microscopy provides in vivo, noninvasive imaging of the corneal endothelial cells [ , ] . it can be performed using a contact or non-contact method. once visualized, the corneal endothelial cells can be evaluated with regard to cell morphology and can be quantified as to the number of cells per mm [ ] . normal corneal endothelial cells are regular in arrangement and hexagonal in shape. cells are evaluated for cell density, pleomorphism, and polymegethism. as cell counts vary by age of the animal and region of the cornea these variables must be standardized using animals of the same age and examining the axial cornea. animals must be sedated or anesthetized to obtain an accurate image and automated systems are available that simplify the technique. guidelines for specular microscopy in human fda clinical trials have been established and these can be used as a guideline for preclinical study design [ ]. in vivo confocal microscopy is a noninvasive method for the microscopic imaging of the living tissues that allows optical sectioning of almost any material with increased axial and lateral spatial resolution and better image contrast [ , , ] . it allows in vivo, noninvasive, real-time images of the eye at magnifications ( Â) which allow resolution of anatomical detail at the cellular level [ ] . three-dimensional confocal microscopy of the eye has also been described [ , ] . confocal microscopy has been used to image the cornea of various laboratory species including rabbits, rats, and mice [ ] . its use has also been described in dogs, cat, birds, guinea pigs, and horses [ ] [ ] [ ] [ ] [ ] . confocal microscopy can provide detailed imaging of the corneal architecture at the cellular level of each corneal epithelial cell layer, the epithelial basement membrane, corneal stroma including nerve fibers and keratocytes, descemet's membrane, and endothelium [ ] . while confocal microscopy is most commonly used in the clinical arena [ - ], its use may be indicated to evaluate the cornea and corneal thickness in contact lens studies, to evaluate the stromal keratocytes or corneal endothelium for toxicity, or to monitor wound healing [ , ] . confocal scanning laser ophthalmoscopy (cslo) is an ophthalmic imaging technology that uses laser light instead of a bright flash of white light to illuminate the retina en face [ ] . the advantages of using cslo over traditional fundus photography include improved image quality, small depth of focus, suppression of scattered light, patient comfort through less bright light, d imaging capability, video capability, and effective imaging of patients who do not dilate well. cslo has been used in several laboratory animal models [ ] . when cslo is combined with sd-oct the combination provides both en face and cross-sectional imaging of the retina [ ] . traditional ocular ultrasound uses frequencies ranging from . to mhz and is used to image the entire globe and orbit. ubm utilizes higher frequencies ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) to image the anterior segment of the eye, specifically the cornea, iridocorneal angle, iris, ciliary body, and lens ( fig. ) [ , [ ] [ ] [ ] . it can be used to determine corneal thickness, document, and monitor changes in the iridocorneal angle, ciliary cleft, angle opening distance, and anterior chamber depth in response to various pharmacologic agents and in studies of accommodation [ , ] . assessing ocular toxicity potential: basic theory and techniques ocular toxicology ocular toxicology ocular toxicology ocular adverse effects associated with systemic medications: recognition and management blood circulation and fluid dynamics in the eye laboratory animal ophthalmology laboratory animal ophthalmology laboratory animal ophthalmology ophthalmic toxicology and assessing ocular irritation veterinary ophthalmology in laboratory animal studies standardization of uveitis nomenclature for reporting clinical data. results of the first international workshop are the standardization of the uveitis nomenclature (sun) working group criteria for codifying the site of inflammation appropriate for all uveitis problems? limitations of the sun working group classification standardization of vitreal inflammatory activity in intermediate and posterior uveitis classification of uveitis-current guidelines survey of ocular findings in -to -week-old beagles a survey of ocular findings in eight-to ten-month-old beagles age changes in the eyes of the beagle dog oval lipid corneal opacities in beagles: ultrastructure of normal beagle cornea oval lipid corneal opacities in beagles unilateral focal retinal dysplasia in beagle dogs the incidence of ocular defects in a closed colony of beagle dogs spontaneous eye lesions in laboratory animals: incidence in relation to age prevalence of ophthalmic lesions in wild-caught cynomolgus monkeys anterior corneal dystrophy of american dutch belted rabbits: biomicroscopic and histopathologic findings persistent hyaloid vasculature and vitreal hemorrhage in albino rats: a morphological and histological study spontaneous retinal changes in sprague dawley rats ophthalmic lesions in non-human primates eye lesions in sprague-dawley rats: type and incidence in relation to age spontaneous development of corneal crystalline deposits in mrl/mp mice spontaneous corneal opacities in laboratory mice a spontaneous corneal change in juvenile wistar rats corneal dystrophy in fischer rats spontaneous corneal dystrophy and generalized basement membrane changes in fischer- rats spontaneous ophthalmic lesions in young sprague-dawley rats spontaneous corneal degeneration in the rat frequency of spontaneous opacities in the cornea and lens observed in chronic toxicity studies in wistar rats: experience with a standardized terminology glossary frequency and nature of spontaneous age-related eye lesions observed in -year inhalation toxicity study in rats comparative ocular anatomy in commonly used laboratory animals wilkie and integrative toxicology: assessing ocular toxicology in laboratroy animals a survey of ocular findings in -to -week-old beagles hereditary tapetal abnormality in the beagle retinopathy from administration of an imidazo quinazoline to beagles toxicologic pathology of the eye: preparation and anterior segment drug-induced retinopathies in the beagle dog drug-induced lenticular lesions in the dog some clinical observations on the eyes of sprague-dawley rats ocular disease in rats: a review study on the ophthalmic diseases in icr mice and balb/c mice buphthalmos (congenital glaucoma) in the rat ophthalmic findings in laboratory animals spontaneous ocular fundus abnormalities in the rat the laboratory rat toxicologic pathology of the eye: lens and posterior segment spontaneous lenticular changes in the rat a note on the lens in aging sprague-dawley rats cataracts in the royal college of surgeons rat: evidence for initiation by lipid peroxidation products toxicology of the eye acute reversible cataract induced by xylazine and by ketamine-xylazine anesthesia in rats and mice retinal vessel abnormalities of phototoxic retinopathy in rats special sense organs and associated tissues mouse fundus photography and angiography: a catalogue of normal and mutant phenotypes spontaneous retinal degeneration in mature osborne-mendel rats agerelated and light-associated retinal changes in fischer rats animal model: peripheral retinal degeneration in rats a functional analysis of the age-related degeneration in the fischer rat the influences of age, retinal topography, and gender on retinal degeneration in the fischer rat ophthalmic lesions and dacryoadenitis: a naturally occurring aspect of sialodacryoadenitis virus infection of the laboratory rat retinal dystrophy in sprague-dawley rats retinal dystrophy in wistar-furth rats method for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes the rabbit the ophthalmic examination as it pertains to general ocular toxicology two cases of corneal epithelial dystrophy in rabbits lipid keratopathy in the watanabe (whhl) rabbit optic nerve head axonal transport in rabbits with hereditary glaucoma ultrastructural studies of primary congenital glaucoma in rabbits buphthalmia in the rabbit. pleiotropic effects of the (bu) gene and a possible explanation of mode of gene action development of ocular hypertension in congenitally buphthalmic rabbits observations on the ocular fundus of primates, anthropoids and prosimians ophthalmoscopic observations of the ocular fundus in colony-born cynomolgus monkeys aged from days to years fundus pigment distribution in rhesus monkeys primary open angle glaucomas in the rhesus monkey macular disease in related rhesus monkeys visual function in cynomolgus monkeys with macular degeneration ocular findings in two colonies of gray mouse lemurs (microcebus murinus) eye diseases in two families of animals emerging imaging technologies for assessing ocular toxicity in laboratory animals emerging electrophysiological technologies for assessing ocular toxicity in laboratory animals accuracy of intraocular pressure measurements in new zealand white rabbits estimation of intraocular pressure in rabbits with commonly used tonometers comparison of three tonometers for measuring intraocular pressure in rabbits comparison of the rebound tonometer (icare) to the applanation tonometer (tonopen xl) in normotensive dogs comparison of a rebound and an applanation tonometer for measuring intraocular pressure in normal rabbits what are the optimal tonometers for different animals species: cynomologous monkey noninvasive measurement of rat intraocular pressure with the tono-pen the induction/impact tonometer: a new instrument to measure intraocular pressure in the rat non-invasive determination of intraocular pressure in the rat eye. comparison of an electronic tonometer (tonopen), and a rebound (impact probe) tonometer. graefe's method for the noninvasive measurement of intraocular pressure in mice circadian rhythm in intraocular pressure: a rabbit model twenty-fourhour measurement of iop in rabbits using rebound tonometer suitability and calibration of a rebound tonometer to measure iop in rabbit and pig eyes validation of the tonovet(r) rebound tonometer in normal and glaucomatous cats evaluation of a rebound tonometer (tonovet) in clinically normal cat eyes panretinal, high-resolution color photography of the mouse fundus a low-cost and simple imaging technique of the anterior and posterior segments: eye fundus, ciliary bodies, iridocorneal angle novel non-contact retina camera for the rat and its application to dynamic retinal vessel analysis a portable, contact animal fundus imaging system based on rol's grin lenses a new procedure for fundus photography and fluorescein angiography in small laboratory animal eyes ocular fundus abnormalities detected by fluorescein and indocyanine green angiography in the royal college of surgeons dystrophic rat angiographic and histological evaluation of porcine retinal vascular damage and protection with perfluorocarbons after massive air embolism fluorescein angiography of the normal and diseased ocular fundi of the laboratory dog vascular changes associated with chorioretinal and optic nerve colobomas in rats (crj: cd (sd), igs) fluorescein angiography of the canine ocular fundus in ketamine-xylazine anesthesia progressive retinal atrophy in the abyssinian cat anaesthesia for fluorescein angiography of the ocular fundus in the miniature pig indocyanine green fluorescence angiography injection technique for indocyanine green and sodium fluorescein dye angiography of the eye guidelines for clinical electroretinography in the dog: update. doc ophthalmol iscev standard for full-field clinical electroretinography optical coherence tomography: an emerging technology for biomedical imaging and optical biopsy optical coherence tomography: history, current status, and laboratory work optical coherence tomography for the evaluation of retinal and optic nerve morphology in animal subjects: practical considerations noninvasive, in vivo assessment of mouse retinal structure using optical coherence tomography spectral domain optical coherence tomography in mouse models of retinal degeneration monitoring mouse retinal degeneration with high-resolution spectral-domain optical coherence tomography noninvasive imaging by optical coherence tomography to monitor retinal degeneration in the mouse key: cord- -aods rf authors: lessenger, james e. title: diseases from animals, poultry, and fish date: journal: agricultural medicine doi: . / - - - _ sha: doc_id: cord_uid: aods rf nan a key problem is the lack of foot protection so that the unprotected feet of workers come in contact with feces of the animals. the fecal-hand route of transmission is also critical. perhaps the most insidious and difficult to control is the consumption of raw poultry and meat products by workers in farms and processing plants. many people in agriculture are living on subsistence or below-subsistence wages and consume products off the processing lines. many of these products are not fully processed and may contact pathogens that have not been killed through cooking or irradiation (see chapter ) (table . ) ( , ) . the improper handling of manure is a major source of disease, including the use of manure on food crops, the discharge of manure into community water sources, and the spread of manure onto areas where children play. in canada, an outbreak of escherichia coli o :h was traced to organic growers who contaminated their produce with cow manure containing e. coli. also in canada, an outbreak of citrobacter freundii infections was associated with parsley originating from an organic garden in which pig manure was used. other documented infections of humans from manure-contaminated foods includes listeria monocytogenes in cabbage contaminated by sheep waste, cryptosporidium spread by municipal water contaminated by cattle, salmonella hartford in food prepared by contaminated water from a shallow well polluted with poultry manure, and pleisomonas shigelloides infection associated with well-water contaminated by poultry manure ( ) . j.e. lessenger guan and holley ( ) , and weber and rutala ( ) . workers, visitors, inspectors, veterinarians, and people who live on or adjacent to farms, ranches, feedlots, processing plants, and other agricultural endeavors are at risk for contracting diseases from animals, poultry, or fish. one needs only to follow the animals from the farm to the feedlots, slaughter house, processing and sorting lines, and packaging plants to appreciate the large number of people who are at risk due to contact with animals and animal products. physicians and other health care professionals are also at risk as they visit farms and plants for inspections or orientations ( ) . methods of preventing the transmission of infectious material from animals and poultry to agricultural workers mirror in many ways the safety techniques for protection from chemicals, trauma and other hazards (see chapter ) . the methods are summarized in table . . key to the prevention of the transmission of animal disease to humans is the proper processing of food products. this includes proper cook times and temperatures, adequate refrigeration, and appropriate transportation, processing, and stocking in stores. personal protective equipment includes hats or head coverings and protective coats or uniforms that can be laundered and left at the plant or farm. boots should also be cleaned and left at the farm or plant. especially in poultry operations, protective particulate masks may be necessary. in some . diseases from animals, poultry, and fish protective physical barriers in farm, ranch, or plant design allow for the raising or processing of food products without actual contact of humans with the animals or products. built-in barriers, changing rooms, boot baths, and hand-free handling techniques allow for the safe and efficient handling of food. in british chicken hatcheries, an aggressive combination of egg sanitization and handling methods was successful in decreasing zoonotic infections and diseases spread through flocks. procedures included: . design changes in incubators . whole building ventilation systems . control of dust, fluff, and aerosol production . disinfection of surfaces and equipment . improved handling of wastes ( ). policies and procedures to limit or prevent physical contact with animals, feces, or urine prevent transmission. rules prohibiting the consumption of food products on farms and ranches or on production lines are especially important. not only can the production food product be infectious to workers, but food brought in by workers can become contaminated, which mandates eating areas for workers away from the livestock ( ) . aggressive veterinary monitoring of livestock can detect early evidence of disease outbreaks in herds. similarly, public health monitoring of disease in humans can detect and appropriately treat epidemics of food-borne disease in humans and trace the source to the food-processing breakdown that caused the disease. hazard analysis of critical control points (haccp) is crucial to the prevention of infections in herds. low cost, ease of performance, and rapidity of results are the key criteria for the tests, and are sometimes more important than the performance characteristics of sensitivity, specificity, and reproducibility. field test kits are available for bacterial, protozoa, antibiotic residue, and other parameters of animal health ( , , ) . medical monitoring can detect early disease and prevent its spread to other employees, the food product, and family members. pre-placement medical monitoring can identify people who are susceptible to infection, for example people with diabetes or immune diseases. in parts of the world where bovine tuberculosis is common, tb skin test monitoring can detect early infections and allow early treatment ( , ) . immunizations are expensive, unavailable in many parts of the world, and only recommended for areas of high infectivity or occupations of high risk such as veterinarians. three critical immunizations are tetanus, rabies, and influenza (see chapter ) . vaccines against salmonella, shigella, and other pathogens are in development or testing. training and education in proper handling techniques are important. proper ways of herding, handling, and caring for animals and poultry can prevent infection and the transmission of infectious material. see chapter for details of education and training. research and the development of new techniques to prevent transmission are critical. for example, airborne dust has been discovered to be a carrier of pathogens in broiler breeder pullets (chicken pens). the use of an electrostatic space charge system has decreased the particle concentration and, in the process, decreased the potential of disease transmission to other chickens and to poultry workers ( ) . hygiene, both in the person and in the workplace, is essential in preventing the transmission of disease. for example, in many german piggeries workers must shower and change clothing when they enter and leave the buildings. this technique prevents the infection of the pigs with outside pathogens, the transfer of pathogens from one piggery to another, and the transfer of pathogens to the home environment. especially important are the cleaning of machinery and the timely cleaning of animal and poultry urine and feces. not only can urine and feces be infectious but they can attract insects that can spread pathogens. as in medicine, the most important hygiene procedure is aggressive hand washing for all persons handling food products. in louisiana, for example, alligator farmers must wear rubber boots and waders to protect themselves from pathogens (but not from bites, which can go right through the protective ensembles). each day, the pens must be flushed and hosed off to remove the wastes that could harbor pathogens dangerous to the alligator colonies and workers. governmental regulations and oversight are important in providing standardization and systemization of methods and procedures to reduce the risk of infection to agricultural workers. good regulations and oversight are evidence-based and consistent with sound agricultural methods (see chapter ). it is not enough to have rules, regulations, equipment and techniques to prevent the spread of pathogens from animals and poultry to workers. fair and consistent supervision by knowledgeable managers is critical to see that the proper equipment and supplies are used and that handling and hygiene rules and regulations are carried out. game are mammals killed or captured in the field for human consumption or for their hides, including elk, boars, bison, and deer. production animals include cattle, pigs, goats, sheep, horses, dogs, deer, and other animals grown in small to large farms and ranches for human consumption. typically the animals are slaughtered and dressed in various cuts made from the different parts of the animal. in addition, many animals are raised and kept as pets. rabies is a common viral infection in children who live in rural areas and in people who handle un-immunized mammalian animals. the prophylaxis for rabies is discussed in chapter . with the exception of four cases where the disease was treated with intensive therapy, the disease is considered universally fatal. therefore, immunizations and prophylaxis are critical. during may and june , the first cluster of human monkeypox cases in the united states was reported. most patients with this febrile, vesicular rash illness presumably acquired the infection from prairie dogs. monkeypox virus was demonstrated by using polymerase chain reaction in two prairie dogs in which pathologic studies showed necrotizing bronchopneumonia, conjunctivitis, and tongue ulceration. immunohistochemical assays for orthopoxviruses demonstrated abundant viral antigens in surface epithelial cells of lesions in conjunctiva and tongue, with lesser amounts in adjacent macrophages, fibroblasts, and connective tissues. viral antigens in the lung were abundant in bronchial epithelial cells, macrophages, and fibroblasts. virus isolation and electron microscopy demonstrated active viral replication in lungs and tongue. both respiratory and direct mucocutaneous exposures are potentially important routes of transmission of monkeypox virus among rodents and to humans. prairie dogs can be studied for insights into transmission, pathogenesis, and vaccine and treatment trials, because they are susceptible to severe monkeypox infection ( ) . chronic wasting disease (cwd) in north american deer and elk has been associated with creutzfeldt-jakob disease (cjd) in hunters who killed, prepared, and ate their own game. an absolute association was not established, but further monitoring is ongoing (see chapter ) . creutzfeldt-jakob disease does not appear to be a problem with workers who raise cattle or dairy cows ( ) . champylobacter chlamydophila abortus is a well recognized pathogen causing abortions in cattle and goats. a recent report from germany cites a case where a pregnant woman became infected from farm animals and aborted. this rare zoonotic infection underlines the insidious and widespread problem of zoonotic infections on farms ( , ) . campylobacter jejuni and c. coli have recently become recognized as common bacterial causes of diarrhea. infection can occur at any age. sources of infection are typically mammalian and avian hosts. the usual incubation period of campylobacter enteritis is to days. fever, diarrhea and abdominal pain are the most common clinical features. the stools frequently contain mucus and, a few days after the onset of symptoms, frank blood. significant vomiting and dehydration are uncommon. a rapid presumptive laboratory diagnosis may be made during the acute phase of the illness by direct phase-contrast microscopy of stools. isolation of the organism from stools requires culture in a selective medium containing antibiotics and incubation under reduced oxygen tension at ˚c. the organism persists in the stools of untreated patients for up to weeks following the onset of symptoms. erythromycin may produce a rapid clinical and bacteriologic cure and should be used to treat moderately to severely ill patients as well as patients with compromised host defenses ( ) . salmonellosis is one of the most important public health disease problems, affecting more people and animals than any other single disease in agriculture. in canada, for example, there were , cases of food-borne salmonellosis in humans during . the native habitat of members of the genus salmonella is the intestinal tract of warm-blooded and many coldblooded vertebrates. in humans, the incubation period is to hours and produces headache, malaise, nausea, fever, vomiting, abdominal pain, and diarrhea (with and without blood). salmonella is also capable of invading the intestinal mucosa, entering the blood stream, and causing septicemia, shock, and death. the diagnosis is made through the clinical presentation and confirmation with blood and stool cultures and serology. treatment is first started empirically pending culture results and then adjusted if necessary. multi-drug resistant s. typhimurium bacteria have been documented to be present in milk after pasteurization ( , ) . listeria monocytogenes is a zoonotic food-born pathogen that is responsible for % of food-related deaths in the united states annually and that is a major cause of food recalls worldwide. agricultural exposure is through drinking unpasteurized milk or direct contact with the animal or manure. the disease pattern is similar to salmonella ( ). tuberculosis (tb) continues to be a worldwide infectious problem for humans. while human-to-human infection is of greatest concern, one infected dairy herd can infect hundreds, if not thousands, of people. potentially, tuberculosis can infect any mammal, although production cattle, especially dairy cattle, are at greatest risk. complicating efforts to combat the disease is the fact that deer, badgers, elk and other wild species have been found to harbor the mycobacterium. in england, badgers were found to be spreading the infection to herds of cattle. also, in england and ireland, herds of sheep were found to be infected. in new zealand, wild brush tail possums (trichosurus vulpecula) were discovered to be the main source of infection in livestock, including deer herds. in tanzania, tuberculosis-infected herds were found more often in small, pastoral farms that have little veterinary monitoring, as opposed to the large, commercial enterprises ( ) ( ) ( ) ( ) . in a los angeles zoo, tb was found in two asian elephants, three rocky mountain goats, and one black rhinoceros. an investigation found no active cases of tuberculosis in humans; however, tuberculin skin-test conversions in humans were associated with training the elephants and attending an elephant necropsy ( ) . human-to-animal transmission of tb has been documented. in an exotic animal farm in illinois, three elephants died of mycobacterium tuberculosis and a fourth tested culture-positive. twenty-two handlers were screened for tb; eleven had positive reactions to intradermal injection with purified protein derivative. one had a smear-negative, culture-positive active tb. dna comparisons by is and tbn typing showed that the isolates from the four elephants and the handler with active tb were the same strain, thus documenting that the infection of the elephants came from the handler ( ) . mycobacterium (tuberculosis) can infect agricultural workers in a number of ways: . human-to-human contact with co-workers through the inhalation of respiratory droplets . drinking contaminated, unpasteurized milk . direct contact with infected animals . direct contact with the secretions of infected animals such as respiratory droplets, milk, manure, urine, semen, and vaginal secretions . direct contact or inhalations of respiratory droplets during necropsy, slaughter, or processing of meat or dairy products ( ) ( ) ( ) ( ) ( ) . the clinical presentation is that of weight-loss, night sweats, a chronic cough, and hemoptysis. asymptomatic workers are typically discovered through public health surveys. diagnosis is through the purified protein derivative (ppd) skin test, smears of respiratory secretions demonstrating acid-fast bodies, cultures of respiratory secretions and other body fluids, radiographs demonstrating caseating granulomas, and other typical findings. treatment is by multidrug therapy, complicated by regional drug resistance patterns ( ) ( ) ( ) ( ) ( ) . giardia infections have been associated with contaminated sewage and water in agricultural environments, producing gastroenteritis. in the sierra foothills of california, cattle drink water contaminated by infected beavers. beaver-and cattle-contaminated water is then consumed by unsuspecting tourists who develop crampy abdominal pain, fevers, and a profuse bloody diarrhea. the giardia infections are easily treated with metronidazole ( ). fowl are birds that grow in the wild. nearly every bird found in the wild can be prepared for human consumption. poultry are birds grown in farm environments for human consumption. common poultry include: chickens, turkeys, ducks, pigeons, game hens, geese, doves, and peacocks. avian influenza a (h n ) first infected humans in , in hong kong. the virus was transmitted directly from birds to humans. eighteen people were admitted to hospitals, and died. in , cases of avian influenza a (h n ) infection occurred among members of a hong kong family, of whom had traveled to mainland china. one person died. how or where these people became infected was not determined. influenza a has the potential to cross species and has been implicated in the flu pandemics in the th century ( , and ). pandemics occur when conditions are met: . the emergence of influenza a virus with a hemagglutinin subtype is completely different from that of strains circulating in humans for many preceding years. . there is a high proportion of susceptible people in the community (i.e., a population with low antibody titers to the new strain). . efficient person-to-person transmissibility of the new virus is possible with accompanying human disease ( ) ( ) ( ) . the reported signs and symptoms of avian influenza in humans include: . typical flu-like symptoms such as fever, cough, sore throat, and muscle aches . eye infections . pneumonia . acute respiratory distress syndrome (ards) . multiple organ failure . lymphopenia . elevated liver enzyme levels . abnormal clotting profiles. physicians are advised to isolate the patient, initiate droplet precautions, and contact their local medical officer for further discussions if an epidemiological link is suspected. the world health organization (who) is moving to rapidly produce a new influenza vaccine capable of protecting people against the h n strain of avian influenza a. preliminary genetic tests conducted in cdc laboratories in atlanta, london, and hong kong suggest that the h n strain is resistant to amantadine and rimantadine but is believed to be susceptible to neuraminidase inhibitors. the who has recommended urgent, rapid culling of infected and exposed bird populations to eliminate the reservoir of the h n strain. in addition, who has discouraged the practice of marketing live poultry directly to consumers in areas currently experiencing outbreaks of avian influenza a (h n ). some countries have introduced trade restrictions to protect animal health. however, available data do not suggest that processed poultry products (i.e., refrigerated or frozen carcasses and products derived from them) or eggs from affected areas pose a public health risk. the virus is killed by cooking ( ) ( ) ( ) . newcastle disease is caused by virulent strains of apmv. death rates among naive bird populations can exceed %. the virus responsible for newcastle disease has been known to cause conjunctivitis and upper respiratory infections in humans since the s. the disease is self-limiting and does not have any permanent consequences ( ) . in , wisconsin public health officials were notified of two cases of febrile illness in workers at a commercial turkey breeder farm. a high prevalence of west nile virus antibody was found among workers and turkeys. an associated high incidence of febrile illness among farm workers also was observed. possible non-mosquito transmission among birds and subsequent infection of humans was postulated, but the mode of transmission was unknown ( ) . avian tuberculosis was diagnosed in two mature rheas on different ratite farms over a -year period. both birds died after weight loss and development of granulomas in the lungs of one bird and bilaterally in the cubcutis cranial to the shoulder in the other. smears and cultures of the granulomas were positive for acid-fast bacilli and tuberculosis ( ) . chlamydophila (chlamydia) psittaci, c. trachomatis, and c. pneumoniae can be passed from birds of all species to humans. wild pigeons and pheasants have been demonstrated to be a source. wild birds in captivity, pets (usually cockatiels, parakeets, parrots, and macaws), and production animals can infect workers, and there are reports of customs and health inspection workers becoming infected. infection is through contact with feces, urine, and oral secretions ( ) . mild infection produces a tracheobronchitis with flu-like symptoms of cough, congestion, myalgias, fatigue, and fever. in severe infections, untreated workers, and immunocompromised workers, pneumonia, sepsis, shock, and death can occur. radiographs reveal a lobar infiltrate ( ) . diagnosis is by detection of the s rrna gene of c. psittasi in sputum with a pcr analysis, and a typical radiographic appearance and culture. tetracyclines and erythromycin are effective for treatment. prevention is through close monitoring and culling flocks and pet birds and personal protection equipment ( ). raising poultry at home is common in low-income countries. studies demonstrate that proximity to free-range domestic poultry increases children's risk of infection with diarrhea-causing organisms such as campylobacter jejuni. corralling might reduce the risk, but research on the socioeconomic acceptability of corralling is lacking. many people report that home-grown poultry and eggs taste better and are more nutritious. they enjoy living around animals and want to teach their children about raising animals. to prevent theft, some residents shut their birds in provisional enclosures at night but allege that birds are healthier, happier, and produce better meat and eggs when let loose by day. many rural peoples view bird feces in the house and yard as dirty, but few see a connection to illness. residents consider chicks and ducklings more innocuous than adult birds and are more likely to allow them inside the house and permit children to play with them. additional food and water costs with corralling are a significant obstacle for some. adequate space and corral hygiene must also be addressed to make this intervention viable. developing a secure, acceptable, and affordable corral remains a challenge for rural populations ( , ) . although approximately % of disease caused by non-typhoidal salmonella is transmitted by food-borne vehicles, four documented salmonella outbreaks in the s have been traced to contact with young poultry. no environmental studies of source hatcheries were completed. a case-control study was performed by comparing culture-confirmed salmonella infantis in michigan residents, identified between may and july , with two age-and neighborhood-matched controls. eighty environmental and bird tissue samples were collected from an implicated hatchery; all salmonella isolates underwent pulsed-field gel electrophoresis (pfge) analysis. the study included case-patients sharing the same pfge subtype and matched controls. within days before illness onset, % of case-patients resided in households raising young poultry compared with % of controls (matched or . ; % ci . , . ). eight hatchery samples yielded s. infantis with pfge subtypes matching the patients' isolates. this investigation identified birds from a single hatchery as the source of human illness and confirmed the link by matching pfge patterns from humans, birds and the hatchery environment. subsequent public health interventions reduced, but did not eliminate, transmission of poultry-associated salmonellosis. five additional pfge-linked cases were identified in spring , necessitating quarantine of the hatchery for depopulation, cleaning and disinfection ( ) . fish farming, or aquaculture, for fish and shellfish is becoming more common and more internationalized with every passing year. in the united states, more than half the seafood consumption is imported, much of it from fish farming. the world's seafood trade is very complex, and if is often difficult or impossible to determine where the seafood is raised or harvested. for example, the united states imports salmon from switzerland and panama though neither country is known for large salmon fisheries ( ) . in general, farmed fish is as safe and nutritious as wild-caught species, but there are public health hazards associated with ignorance, abuse, and neglect of aquaculture technology. numerous small fish ponds increase the shoreline of ponds causing higher densities of mosquito larvae and cercaria, which can increase the incidence and prevalence of lymphatic filariasis and schistosomiasis. especially dangerous is the use of human waste draining to fertilize or create ponds. technology abuse includes the misuse of therapeutic drugs, chemicals, fertilizers and natural fish habitat areas. technology neglect includes the failure to pay attention to mosquito habitats and the concomitant increase in malaria, as well as the propagation of other organisms ( ) . human exposure can be through direct skin contact with fish or the consumption of contaminated fish or shellfish products or contaminated water. the main pathogens acquired topically from fish (through spine puncture or open wounds) are aeromonas hydrophila, edwardsiella tarda, erysipelothrix rhusiopathiae, mycobacterium marinum, streptococcus iniae, vibrio vulnificus, and vibrio damsela. s. iniae has recently emerged as a public health hazard associated with aquaculture, and m. marinum often infects home aquarium hobbyists. common zoonoses contracted through the consumption of contaminated products or water include salmonella, leptospirosis, yersiniosis, and tuberculosis ( ) . salmonellae species have been found associated with all of the poikilothermic vertebrate species studied, as well as the mollusks and crustaceans ( ) . leptospirosis does occur in the poikilothermic vertebrates, as evidenced by positive serological reactions and by the isolation of pathogenic leptospiral serovars. the finding of leptospirosis species in fish, mollosks and other aquatic species are of special importance in view of the increased worldwide interest in aquaculture farming. since , of the ( . %) reported human cases of leptospirosis in hawaii have been associated with aquaculture industries (taro farms, prawn farms and watercress farms) ( ) . species of yersinia are a particular problem in fish and in people involved in fish farming. workers who wade in fish ponds or drink drainage water are especially at risk. yersinia enterocolitica has been demonstrated to be a causative agent in acute diarrhea illness in humans after workers become infected through the feces-hand-oral route ( ) . tuberculosis has also been reported in freshwater and marine fish species (piscine tuberculosis), especially in those grown on fish farms. mycobacterium marinum and m. celonae have been demonstrated in fish farms ( ) . turtles, lizards, snakes, green iguanas (iguana iguana), alligators, and crocodiles are grown from eggs in farms for their hides and meat. some species are also grown for sale as pets. salmonella infections in persons who had contact with reptiles usually cause gastroenteritis but can result in invasive illness, including septicemia and meningitis, especially in infants and immunocompromised persons. for decades, reptiles have been known to be a source for salmonellosis; however, numerous reptile owners remain unaware that reptile contact places them and other household members, including children, at greater risk for infection. ( ) captive reptiles (such as iguanas) are routinely identified as reservoirs of salmonella and the number of reports about reptile-associated salmonellosis is increasing. in germany and austria, salmonella was detected in . % of fecal reptile samples cultured. the percentage of salmonella-positive samples was significantly lower in turtles as compared with lizards and snakes, as salmonella was only detected in one sample from a single turtle out of turtles investigated. in all, different salmonella serovars were found. all isolated salmonella belonged to the species enterica, predominantly to the subspecies i (n = ) and iiib (n = ) but also to subspecies ii (n = ), iiia (n = ), and iv (n = ). all isolates were sensitive to the antimicrobials examined. a significantly higher percentage of salmonella-positive reptiles was detected in the group of owners who purchased reptiles in comparison with pure breeders. the high percentage of salmonella in reptiles in the study confirms the risk for the transmission of the infection to humans ( ) . zoonoses as a risk when associating with livestock or animal products social and environmental risk factors in the emergence of infectious diseases public health implications related to spread of pathogens in manure from livestock and poultry operations occupational bio hazards: current issues pathogen survival in swine manure environments and transmission of human enteric illness: a review zoonotic infections an approach to reduction of salmonella infection in broiler chicken flocks through intensive sampling and identification of cross-contamination hazards in commercial hatcheries maff statuatory incident reports in surveillance, prevention, and control of human salmonella typhimurium infection surveillance and control of emerging zoonoses testing to fulfill haccp (hazard analysis critical control points) requirements: principles and examples effect of an electrostatic space charge system on airborne dust and subsequent potential transmission of microorganisms to broiler breeder pullets by airborne dust veterinary monkeypox virus working group. monkeypox transmission and pathogenesis in prairie dogs wild game feasts and fatal degenerative neurological illness abortion in humans caused by chlamydophila abortus chlamydophila abortus infection in a pregnant woman associated with indirect contact with infected goats human salmonellosis associated with exotic pets multidrugresistant salmonella typhimurium infection from milk contaminated after pasteurization dairy farm reservoir of listeria monocytogenes sporadic and epidemic strains a study of the foodborne pathogens: campylobacter, listeria and yersinia, in faeces from slaughter-age cattle and sheep in australia factors influencing the incidence and scale of bovine tuberculosis in cattle in southwest england diagnostic strategies and outcomes on three new zealand deer farms with severe outbreaks of bovine tuberculosis prevalence of bovine tuberculosis in cattle in different farming systems in the eastern zone of tanzania human exposure following mycobacterium tuberculosis infection of multiple animal species in a metropolitan zoo mycobacterium tuberculosis infection as a zoonotic disease: transmission between humans and elephants avian influenza: recent developments human health implications of avian influenza viruses and paramyxoviruses avian influenza outbreak: update phylogenetic relationships among virulent newcastle disease virus isolates from the - outbreak in california and other recent outbreaks in north america center for disease control and prevention. west nile virus infection among turkey breeder farm workers: wisconsin tuberculosis in farmed rheas (rhea americana) a flu like syndrome a woman with a lobar infiltrate due to psittacosis detected by polymerase chain reaction campylobacter, from obscurity to celebrity campylobacter enteritis human salmonellosis associated with young poultry from a contaminated hatchery in michigan and the resulting public health interventions public, animal, and environmental health implications of aquaculture topically acquired bacterial zoonoses from fish: a review epidemiologic aspects of salmonellosis in reptiles, amphibians, mollusks and crustaceans-a review leptospirosis in poikilothermic vertebrates. a review reptile-associated salmonellosis-selected states salmonella enterica in reptiles of german and austrian origin systemic infection with alaria americana (trematoda) amphibians include frogs, toads, newts, and salamanders that are caught in the wild or grown on farms for use as food or as pets. frogs are caught in the wild and grown in farms for their meat, primarily frog legs. eating inadequately cooked frog legs can lead to an infection of alaria americana, a trematode. increasing evidence suggests that amphibians can pose risks for salmonellosis in humans ( ) . key: cord- - cckyz authors: price, jason d. title: desire and the law: creative resistance in the reluctant passenger and the heart of redness date: - - journal: animals and desire in south african fiction doi: . / - - - - _ sha: doc_id: cord_uid: cckyz this chapter offers a critique of animal rights approaches for their weakness in relying on the passage of laws, and in depending upon their proper administration by legal authorities to attempt the protection of animals. where some thinkers espouse an animal rights perspective, this chapter argues that postcolonial desire is vital to protecting communities in ways that rights discourse and the law cannot in the context of the biopolitical workings of the state and globalized capitalism. drawing from deleuze and guattari’s work on desire and the law in kafka ( ), the chapter considers the potential of desire to offer creative alternatives, outside of legal discourse, toward the protection of animals and the larger community. additionally, it recognizes how indigenous environmental knowledge and notions of desire offer ways of relating to animals that can challenge capitalist instrumentalization. by zakes mda portray communities that struggle to protect themselves, their lands, and the animals with which they dwell from being used and abused to turn a profit for businesses. both novels portray competing claims for land as business proposals attempt to develop potential tourist locales by disenfranchising their current inhabitants through the rhetoric of western notions of "development." as in chap. , the role of desire as "eating" appears here as government officials are bribed by business owners to approve their land-development proposals, and at the cost of sacrificing the homes, protection, and interests of the local inhabitants of these lands. these stories perform what graham huggan and helen tiffin identify as " [o] ne of the central tasks of postcolonial ecocriticism" as they "contest-also provide viable alternatives to-western ideologies of development" ( ) . the capitalist proposals for wealthy tourist destinations continue in a colonialist view of these lands as blank spaces awaiting appropriation and transformation into capital. this chapter explores how characters in both novels successfully work within and beyond the law to prevent the destruction of particular environments which they have come to know intimately. the protagonist of the reluctant passenger, an environmental lawyer, critiques the political scene that the novel sets up involving unethical environmental rulings and other legislation influenced by big business and bribery. heyns' novel highlights many environmental concerns, particularly the legal status, or lack thereof, of animals (specifically baboons) in south africa. as the protagonist struggles to help maintain the nature preserve for the troop of baboons at the request of his client luc tomlinson, the novel portrays luc's experiences dwelling with the baboons, demonstrating his great respect for their lives and culture. similarly, camagu, the protagonist of the heart of redness, argues against the development of the small village he has grown to love into a gambling city. offering a more ecocritical alternative to the tourist town, camagu expresses his view, informed by qukezwa's political analysis and knowledge of local culture and nature, that the town developed by outside businesses will offer little work or profit for the townspeople and be detrimental to their environment. in contrast, he proposes a smaller measure of a resort built with local materials by the villagers appealing to a different type of tourist who "like[s] to visit unspoiled places for the sole purpose of admiring the beauty of nature and watching birds without killing them" ( ). these novels consider the available avenues for opposing the late capitalist thrust to transform all the world and its inhabitants into objects that are available for consumption and for turning a profit. the communities in these works offer ways of thinking about promoting sustainable futures against the overconsumption of the environment associated with most capitalist development projects. as animals are valued highly by the characters in both texts, heyns' and mda's novels argue persuasively for sustainable futures for the humans and animals (and in heart of redness also the flora) that are part of their communities. characters in both understand community in a broader sense, including humans and non-humans, and recognize that they are all potentially disposable in the logic of profit-seeking capitalists. where some approaches to the stories espouse an animal rights perspective, i argue that an ethics of sustainability and a biopolitics informed by deterritorialized or postcolonial desire, here specifically the desire for animals, is essential to protecting communities in ways that rights discourse and the law cannot guarantee. in the reluctant passenger, the protagonist, an environmental lawyer named nick morris, somewhat contradictorily dislikes the ungovernable or disorderly aspects of the environment and animals. for example, he has a discussion with his friend and fellow lawyer, gerhard, about masturbation that turns into an analysis of romantic poets where morris discloses that he masturbates to the lake district of england. in response, gerhard encourages him to consider blake's poetry and to "try the tiger next time" ( ). nick explains his disregard for unruly nature: i am not a tiger type of person, and such fantasies as i have tend towards the tame. for this reason my involvement in the ever-deepening intrigue surrounding luc tomlinson's baboons was as unusual as it was unwelcome. as far as i'm concerned, the environment should behave itself if it wants us to look after its interests. as a matter of fact, the lake district is just about my notion of an ideal environment: well-mannered, contained, placidly packaged, officially protected and signposted. ( ) as the novel progresses and nick becomes increasingly involved with luc tomlinson and the case for the baboons, the lawyer discover the law's limited ability to protect the animals. " [t] he rights of animals are a much debated area in law" ( ) is the best that nick can offer in response to luc's query about protecting the baboons in a legal manner. where the law fails them, i'm interested here in how and why the characters work to protect the baboons extralegally. in light of nick's discomfort with "wild" or untamable nature (or zoe) and his sexual fantasies about ordered and "contained" environments, his reassessment of the unmasterable aspects of the world including his own desires and "self" leads him to break several laws in the course of rescuing the baboons with luc later in the story. where nick once lived a life of abstinence to avoid the messiness and feelings associated with a sexual relationship, he ends up having sex with luc in his house while the rescued baboons they have secured in the second floor of his house dirty, rearrange, and otherwise mess up his home, which had once been so clean and ordered as to appear uninhabited. this scene indicates the importance of a positive appraisal of desire to protect animals in relation to the limits of legal or animal rights approaches, positions which i analyze below. in the animal gaze: animal subjectivities in southern african fiction, wendy woodward reflects on the interiority and sense of "self" of the animals in southern african fiction, including the reluctant passenger and the heart of redness. for her, these literary representations of animals do important work toward changing the way we think about animals and their rights. woodward argues that animals have subjectivities, so they should be recognized in the south african constitution and be accorded rights. in support of her animal rights approach, she draws from martha nussbaum's philosophy of "moral agency," a philosophy which, as i'll discuss later, braidotti criticizes heavily early on in transpositions. for example, woodward recounts how martha c. nussbaum critiques utilitarian approaches to rights which position animals as having "moral standing." she summarizes the utilitarian position through a quotation of nussbaum: because they are subjects of social justice "if a creature has either the capacity for pleasure and pain or the capacity for movement from place to place or the capacity for emotion and affiliation or the capacity for reasoning and so forth (we might add play, tool use, and others), then the creature has moral standing" ( ). nussbaum quite rightly argues for the importance of the agency of the nonhuman animal; in moral agency, then, the animal is active in this sphere, whereas "moral standing" is conferred on the nonhuman animal for the characteristics he or she embodies. (woodward ) this preference for active instead of passive qualities in "moral agency" over standing or capacity is certainly a more interesting approach regarding the question of animal morality because agency suggests a recognition of the moral life and behaviors of animals, yet "moral agency" still suggests that animals should be granted rights because they have similar agency to that of humans. Élizabeth de fontenay also critiques this position in without offending humans: a critique of animal rights: without useless brutality toward metaphysical and legal humanisms, a pathocentrist perspective does in effect allow us to establish the fact that the moral community is constituted not only by "moral agents" capable of reciprocity, apt to enter into contracts with full knowledge of what this means, but also by "moral patients," which includes certain categories of human beings and animals. ( ) fontenay's argument here is that this approach to rights from the perspective of "agency" leaves out some humans and animals from being protected because they do not possess this agency. she further critiques this in her response's to peter singer's philosophy, arguing that such rights approaches are "offensive" to humans as they run the risk of sanctioning the poor treatment of non-normative humans, such as those with different mental abilities who may not necessarily be included in the category of "moral agents." posthumanist accounts of subjectivity, such as rosi braidotti's nomadic subjectivity, that involve a radical immanence, offer a fluid notion of the subject where subjects are interdependent, existing in assemblages with other humans and the non-human, instead of a fixed view of subjectivity in the liberal individual tradition. the law constructs dominant, discursive subjects which fail to do justice to or fully account for the fluidity and complexity of our subjectivities. braidotti argues: the becoming-animal axis of transformation entails the displacement of anthropocentrism and the recognition of trans-species solidarity on the basis of our being environmentally based, that is to say: embodied, embedded and in symbiosis … "life," far from being codified as the exclusive property or the unalienable right of one species-the human-over all others or of being sacralized as a pre-established given, however, is posited as process, interactive and open-ended. ( ) while woodward does discuss deleuze and guattari's becoming-animal briefly at times, she looks for more human qualities or attributes of human subjectivity in the animals in the literature she analyzes, which approaches a kind of becoming-human of the animal. rights discourse, while oriented toward protecting animals from violence, ends up humanizing animals, leaving this political approach perhaps less effective than other creative options. braidotti also critiques martha nussbaum's universalism (after kant), which assumes a stable humanist subject. nussbaum's formulation seems to view animals as fixed, individual subjects who possess agency, whereas the vital materialist deleuze views both subjectivity and agency as dispersed, interdependent, and the subject as a process in assemblage. braidotti also critiques nussbaum's position for the way she tries to intimidate new or experimental approaches and philosophies by asserting that they are relativist. another problem with universalism is the failure to appreciate local knowledges and hence a tendency toward a monocultural, dominant view of the world. woodward, however, nicely avoids this by recognizing and analyzing the importance of shamanist traditions and the indigenous knowledges of south african peoples. in essence, woodward's somewhat humanist approach and the posthumanist approach i espouse are after the same goals-the protection of animals-although her project seems limited to that particular kind of life that possesses "moral agency," whereas mine seeks to protect the community in a broader sense. that is, i am concerned with the protection and improvement of the conditions and treatment of animals, the environment, and the others of man that have been excluded from man's central position in humanism and therefore viewed more easily as disposable because of the negative valuation of difference that results in racism, anthropocentrism, and phallogocentrism. while the reluctant passenger and the heart of redness offer a view of animals as deserving of rights, and the reluctant passenger engages in this discourse of rights more directly, both novels also offer more creative ways of thinking about sustainable futures. thus they call for working inside the law and also other creative ways of protecting the animals, human and non-human, of their communities. for braidotti, sustainability consists of multiplying subjectivities "not for profit" and increasing the possibilities of positive attachments. a significant part of her project entails establishing a positive view of zoe, in contrast to negative views of it espoused by agamben and others. for braidotti, the others of man-women, native others, animals, earth others, and so on-are closer to zoe, whereas man is closer to bios or discursive life ( ). she explains that [w] hereas "life" or bios has been conceptualized as a discursive and political notion ever since aristotle, zoe is the non-or pre-human "outside" of the polity. it has been rendered in figurations of pejorative alterity as the "other of the living human", which means the inhuman or divine and the dead … . against this forensic turn in contemporary philosophy [agamben's association of zoe with death, for example], … [is] the need to cultivate positive political passions and ethics of affirmation. (transpositions ) she also explains how zoe disrupts a "unitary" vision of the subject-a non-humanness at the heart of the human that flows through bodies. this sustainability perspective replaces one of rights, as she argues: "the notion of co-dependence replaces that of recognition, much as the ethics of sustainability replaces the moral philosophy of rights" ( ). in contrast to a rights perspective that argues that animals be included in the community and be granted protection because of their similarity to humans under our notion of humanism and the law, this approach of co-dependence recognizes that the "human" has never been human, never existed independently, but always depends on a relation with the non-human. part of nussbaum's approach to animals also includes the argument "that animals be recognized as subjects" ( ). thus woodward bases her "rights" approach to animals on their subjectivity, and therefore their being subjects in the law. cary wolfe argues that this approach to protecting animals is not sensible: i think we would all agree that an admirable desire of humanism would be to respect the standing of at least some nonhuman animals and to protect them from exploitation, cruelty, and so on. but the attempt to articulate that desire, which is an admirable one, in terms of the rights framework ends up foreclosing and undercutting that desire by reinstating a normative picture of the subject of rights that ends up being humanist and anthropocentric through and through, that ends up with a being that looks a lot like us, so that, in the end, nonhuman animals matter because they are just a diminished version of us. it seems to me self-evident that trying to think about the value of dolphins in terms of their being diminished versions of homo sapiens makes no sense. ("after animality" , emphasis added) for wolfe, rights approaches then inevitably begin to look for human characteristics in animals as a means of securing their protection. while his reading of the law and animal rights perspectives often focuses on the work of derrida, specifically derrida's essay "before the law" from which he derives the title of his book on biopolitics, wolfe's emphasis on the "undercutting" of desire is something worth taking up from deleuze and guattari's perspective. both derrida, and deleuze and guattari, write about kafka's the trial, which includes the story entitled "before the law," as a starting point for, or in the course of, their thinking about the law and what it means to be "before the law." wolfe spends much of his work on biopolitics describing derrida's position, noting, for example, the lack of response in law as, constructed in the technicity of language, its automatic nature leads merely to reaction. yet in this interview he emphasizes "desire" in relation to the law. as this project has explored the role of desire throughout, deleuze and guattari's writing about desire and the law, specifically their kafka: towards a minor literature, adds another fold to biopolitical thought. while he doesn't consider deleuze and guattari's writing about kafka, and therefore their specific writing about the relationship of their vitalist project to the law, wolfe does address how deleuze's work, which might seem at first to promote the equality of all life in an affirmative biopolitics, is useful in terms of biopolitical thought. he explains how a pragmatic application of deleuze's philosophy bypasses the potential problems of an affirmative biopolitics: "by a pragmatist account, philosophy for deleuze, as paul patton puts it, 'is the invention or creation of concepts, the purpose of which is not accurate representation' but rather to provide 'a form of description which is immediately practical,' one 'oriented toward the possibility of change'" (before the law ). in other words, while their ethics of affirming zoe might appear to promote the flourishing of all life, this is not an accurate portrayal of life but instead a practical politics for resisting dominant thought, capitalist logic, and the consumption of everything that lives. desire poses a more direct and revolutionary threat-one that works outside of the rationality of the law, an authority which currently excludes most animals from the community. that is, rather than appeal to the authority or work within the confines of the law which has rendered animals in their current position, which has, through its exclusionary violence, failed to protect them, staying with that desire and its productive nature offers opportunities for working toward this protection in new ways, outside of the law. in kafka: toward a minor literature, deleuze and guattari argue more specifically against the law as a means to justice. they offer a corrective to the view that the law secures justice: "where one believed there was law, there is in fact desire and desire alone. justice is desire and not law" (kafka ). this perspective on law agrees with wolfe's discussion of the "undercutting" of desire that pertains to rights approaches. the law, when viewed as the only outlet to protect animals, "undercuts" desire, then, by reterritorializing desire into the existing legal framework, appealing to its authority or authorities, thereby undermining its revolutionary potential. or more specifically, for deleuze and guattari, the law does not undercut desire but instead is one of two kinds of desire: the transcendental law or the schizo-law. they argue: we should emphasize the fact of these two coexistent states because we cannot say in advance, "this is a bad desire, that is a good desire." desire is a mixture, a blend, to such a degree that bureaucratic or fascist pieces are still or already caught up in revolutionary agitation. it is only in motion that we can distinguish the "diabolism" of desire and its "immanence," since one lies deep in the other. nothing preexists anything else. it is by the power of his noncritique that kafka is so dangerous. ( ) in other words, for them, the law itself doesn't necessarily undercut desire, but the law perhaps is one arena where desire is either reterritorialized or takes off on a line of flight. the non-critique here gestures to the potential of desires to transform to positive ends or conversely to become violent. continuing their reading of the trial, they argue: from this point on, it is even more important to renounce the idea of a transcendence of the law. if the ultimate instances are inaccessible and cannot be represented, this occurs not as a function of an infinite hierarchy belonging to a negative theology but as a function of a contiguity of desire that causes whatever happens to happen always in the office next door … if everything, everyone is part of justice, if everyone is an auxiliary of justice … this is not because of the transcendence of the law but because of the immanence of desire. (kafka ) this position radically calls into question nussbaum's moral universalism, or "universal rights," as it is revealed that law is just an arena of sorts for desire that springs forth from the immanence of the body. desires that are not reterritorialized, that remain schizo-law, are therefore specific to the singularities of the material assemblages "next door" and, as they privilege deterritorialized desire and schizophrenia throughout their work, schizolaw provides the possibility for political action against dominant thought and dominant constructions of desire which have resulted in the failure to protect particular members of the community. what's at stake in this understanding of law as nothing but desire is the realization that a rights approach needs to be backed up by the desire to enforce it for it to work toward the protection of animals or the environment. the import of deleuze and guattari's insight into the law, however, is that the law itself is empty, is nothing but desire. in other words, and i'll discuss this further in my treatment of the novels below, if the desire to protect animals, the environment, humans, and so forth does not exist in the exercising or administration of the law, the law itself (and the passing of more laws ad infinitum, even) will surely fail to protect them and ensure their futures. if those positioned as authorities of the law are colonized by oedipus and their desires are therefore reterritorialized by capitalism, the laws themselves will not stand a chance against the disposing of these others for the accumulation of pleasure and profit by the arbiters of the law. if the law is really desire and its officials are colonized by capitalism's definition of desire as lack that must be filled through consumption and accumulation, their authority in the law enables the potential of capitalist desire to render all that lives "disposable" to their personal interests and make everything available for consumption and profit in a logic of exchange. thus it becomes all the more important to theorize desire differently and to think outside of capitalist logic which defines desire as lack, viewing desire instead as an opportunity to make ethical attachments toward sustainable futures. since what appears to be law is really desire, if we take that observation seriously, decolonizing desire or resisting its colonization toward a postcolonial desire then becomes a significant intervention into the political field as part of a project to protect the others and the environments of our communities. since capitalism bombards us with its definition of desire constantly, portrayals of what i'd like to call "postcolonial desire" in these novels offers a line of flight away from capitalist logic: a field of desire which can reorient one's sense of self and relationships to others, animals, and the environment woodward argues that animals can be focused on in literature and writing because human rights have been secured in south africa. she writes: now that human rights appear to be in place in a democratic south africa-even while much of our racialised history remains intact-writers can represent animals more expansively without engendering criticism of foregrounding animals at the expense of humans. white writers in particular may have felt constrained not to portray animals as ethical subjects when the majority of south africans were without rights. ( ) to be sure, the putting in place of human rights is a significant achievement, and yet, as she seems to acknowledge here in recognizing that the "racialised history" has not changed much with the advent of rights, the securing of these rights doesn't radically alter the state of affairs or ensure the protection and improved treatment of those now granted rights. additionally, the secondary consideration of rights for animals continues to privilege the human over the animal, ensuring the continual deferment of protections for animals. similarly, calls for the addition of more kinds of right, like elke zuern's argument for the case of "socioeconomic rights' ( ) as part of human rights, while certainly important in their attempt to redress economic inequality and its violence, which much current rights discourse overlooks, require their enforcement by the authorities of the law. zuern summarizes her research: south africans argue "that freedom can only be realized when civil, political, and socioeconomic rights are protected and enforced" (xii). the phrasing of this observation gets to the heart of the matter in that the passage of the rights in law, if we agree with deleuze and guattari, guarantees nothing without an accompanying schizo-desire that would desire to protect the community. to further extend these rights to animals, then, without thinking desire differently toward a postcolonial desire does little to protect them or the locations where they dwell. where the recognition of rights is important in a legal sense, poor conditions obviously still persist for many of those who are now extended rights in south africa. for example, the poor conditions for miners which led to the strikes at many mines, including the marikana platinum mine in august where several strikers were shot by the police, are evidence that the putting in place of human rights has not guaranteed the protection of south africans formerly left outside the law's protection during apartheid. deterritorializing and decolonizing desire is a necessary part of any approach to protecting the inhabitants, human and non-human, of south africa. the law "manifestly lacks balls" ( ), as gerhard puts it in the reluctant passenger in his summary of luc's description of the law as a "eunuch." michelè pickover reveals evidence of this powerlessness of the law throughout animal rights in noting the complicity of the law with corporate interests. she explains how in many animal protection issues such as vivisection, factory farming, the trade in wild animals, and conservation, the government officials who oversee and make laws protecting animals are often also involved with the corporations; or indeed in many cases, such as factory farming, corporations are often left to follow the laws on their own, without any oversight. for example, she writes: "the south african government either lacks the political will or the resources to police and regulate the industry" of trophy hunting, and she notes that "[t]he truth is that trophy hunting promotes a culture of violence and guns. this is in direct opposition to the needs of south african society, which is desperately trying to free itself from its violent past" ( ). she also describes how this instrumentalization of animals continues in an apartheid legacy: "wild animals were exploited to fund the apartheid war, the secret agencies, the special forces and the individuals connected to them. it is no secret that the nationalist government and its military machinery were involved in the illegal trade in ivory" ( ). these obvious conflicts of interest in the form of a desire to extract a maximum profit from animal bodies and being charged with the task to protect them speaks to the many ways in which the law is compromised through its colonization by capitalist desire. as pickover's reporting on the exploitation of bodies during apartheid reveals, authorities often acknowledge or disavow the rights of others and choose to administer the law as it suits their agendas and financial interests. this problem-that the access to rights and to the protections of the law are made to depend on those administering it (perhaps best described in kafka's "before the law" with the countryman seeking access to the law from the doorkeeper)-was most obvious during the apartheid regime with the passing of laws denying the rights of black south africans. in the context of this discussion of the law being colonized by capitalist desire, perhaps the most pertinent of these laws limiting the rights of black south africans were those acts which prevented or hindered these communities from acquiring decent paying jobs, ensuring that the white community would benefit financially. in the reluctant passenger, the villainous judge conroy describes these financial benefits of apartheid to morris upon telling him of his former plan to hand over the fortune he collected from corrupt dealings as a lawyer: "you no doubt imagine yourself too morally fastidious to benefit by money derived from an evil regime. i need hardly point out that for decades every white south african to a greater or lesser degree benefited by the policies and practices of that regime" ( ). heyns' and mda's novels shed light on many of these problems as they portray the corruption that informs environmental rulings: the heart of redness describes the conflict of interests of the government official deciding on the development project for qolorha by the sea; heyns' novel in particular describes the government's collusion in the abuse of baboons in vivisection. to return briefly to mda's the mother of all eating, discussed in chap. , it is the messenger's desire which takes off on a line of flight and threatens the power and privilege exercised by the man and his fellow government's officials, who are colonized by the reterritorialized desire of capitalism. the man's attempt to stop the messenger's revolutionary behavior, which i've referred to in chap. , acknowledges the way in which capitalism colonizes desire: the people don't have any leadership that will create a critical awareness in them, that will open their eyes. whenever new leadership emerges, even if it begins as honest leadership, it is swallowed by the culture of eating, and becomes one with it […] the people are doomed to … [an obvious kick, and a scream] okay, okay, i admit … it will take a very small thing to spark action in them, and to arouse them to an anger that has not been seen before. ( ) the man tries to quell the messenger's revolutionary desire by attempting to convince him that his behavior and the position of the people is one of impotence, as is the case for humans in relation to the gods in oedipus. his descriptions of the people as "blind" recall the "blindspots" of plumwood's discussion of what happens as a result of the colonizing force of reason, and his observation that "honest" leadership may be colonized by the culture of eating confirms her argument about the colonization of political systems as well. after experiencing the violent kick from the messenger, however, the man acknowledges that the desire of the people is what he lives in fear of as it resists his easy management and control. indeed, it is what is threatening his life at this point in the play as the messenger refuses his bribes, and he fears that this desire has the potential to change the state of affairs, threatening to unseat and remove all the corrupt officials who "eat" the community. south african author michiel heyns, whose lost ground won him the sunday times fiction prize in , has also received accolades for his translations of south african literary criticism and literature from afrikaans into english, such as marlene van niekerk's agaat. he also currently maintains a blog entitled books and dogs, where he writes often about his dog simon and dogs in literature ("michiel heyns"). heyns has written previously about the potential of affect to challenge and threaten the cool reason often exercised in the violent treatment of animals and others in his earlier novel, the children's day ( ) . his second novel, the reluctant passenger ( ), features a white south african middle-class environmental lawyer, nick morris, who takes on a case for luc tomlinson and a troop of baboons. the subjects of law, rights, and desire discussed earlier come into focus in the story as the protagonist becomes more involved in the case for the baboons. at the beginning of the novel, morris detests the unmasterable and disorderly aspects of himself and the environment. heyns' offers a critique of reason and mastery as morris is frustrated by any disorganization or things that don't adhere to the norms of human rationality or reason. for example, morris wishes he was an accountant as he originally planned, where the world is neatly divided and ordered into columns of debits and credits. he also resents his neighbor's dog, who shits on his lawn, and he chooses to live a celibate life with his girlfriend so he won't have to deal with the messiness of sex or love. in an early scene, the dog actually knocks him into a pile of shit as morris attempts to clean it up to restore cleanliness to his yard. in response, morris violently wipes the shit all over the dog's body, viewing animals in a pejorative sense as mere producers of dirt and filth. morris prefers order and is a perfect employee under capitalism, an ideal manageable subject of capitalist biopolitics, as he is never late, is apolitical, and maintains neatness and order above all else. he makes all attempts to master and control his animality through regimes of culture, and he seeks to avoid encounters with his own nonhuman life or zoe (including his desires), as well as encounters with nonhumans and the nonhuman world. in addition, his concerns for the neatness of his lawn and property cause him, rather comically, to forget to vote in the democratic elections. almost all of his life is ordered and he attempts to master or control it completely. as braidotti says of the dominant understanding of consciousness, he lives in fear of zoe: relentlessly vital, zoe is endowed with endurance and resilience … zoe carries on regardless: it is radically immanent. consciousness attempts to contain it, but actually lives in fear of it. such a life force is experienced as threatening by a mind that fears the loss of control. (nomadic subjects ) he also lives a rather sedentary lifestyle alone in a large house in a gated community, and he seems to have little life or connection to the world outside of working, with the exception of meeting his one friend, gerhard, an openly homosexual fellow lawyer who embraces zoe by, for example, discussing his sexual encounters and desires openly, engaging in sexual acts in public, and often being late for meetings and work for love-related and other reasons. the novel centers on a case morris takes up when a kind of hippie wild-man character, who turns out to be the wealthy luc tomlinson, comes into his office asking him to take his case to protect the baboons and the private reserve where he lives with them. in animal rights in south africa, michelè pickover begins with a striking story about a baboon who worked for the railroad, and as a service animal for a disabled railway man, to counter the negative view of baboons and their limited protections in the law: in most provinces in south africa baboons are classified as vermin or 'problem animals'. taking advantage of this status, in several businessmen devised a plan to build a slaughterhouse in limpopo province to kill wild-caught baboons by electric shock and then process them into salami, polony, and tinned meat for consumption in central africa and eastern europe. baboon body parts, such as hands, teeth, and tails, would be exported to asia as aphrodisiacs. ( ) the baboons in heyns' novel are classified in a similar violent, disposable status and therefore luc and nick's initial attempts to protect them through legal means are fruitless. luc's father threatens to build a resort at the reserve partly because it is a business venture and he is a capitalist, and partly because of a long family feud. where nick morris initially wants nothing to do with the case, his friend gerhard encourages him to be less reserved and to experience more of the uncertain, unpredictable, and non-normative aspects of life, like the dirty, unkempt luc and the baboons. as the novel unfolds, the protagonist learns about himself as he learns more about baboons, appraising them as a worthy case, suggesting the interconnectivity of a positive appraisal of the non-humanness of subjectivity and of the non-human world. as he embraces zoe as a positive passion, instead of a negative one which disrupts his orderly world of progress as he had before, he comes to value the complexities of the world in its open-ended becoming. he moves from being someone who fails to ever examine his own sexual feelings for the sake of order and not wanting to be inconvenienced by them, and who detests the disorder and dirt that animals bring into his life, like his neighbors' dog, to a person who finds himself having sex with his client, luc, in his home while the troop of baboons they've saved from a vivisection lab tear apart his furniture and cause havoc to the upstairs of his house. the novel's title and image on the cover, explained in the first pages, offers a suggestive description of zoe. morris describes: i once saw a man transporting his rottweiler in a shopping trolley through a no dogs allowed area: the beast was clearly well trained, and stayed put, but you could see that all it really wanted to do was chew the wheels off all the trolleys in the universe. ( ) this image emphasizes the zoe of the animal and how it is maintained in consumerist culture or oedipalized for the benefits of human shoppersthe dog is thus a reluctant passenger. as braidotti writes, challenging the idea that dogs are only dirty, "[d]ogs are not only messy, but also openly sexual. they unleash a reservoir of images for sexual explicitness and even aggression, as well as unbridled freedom: they are a vehicle for zoe" ( ). like the dog in morris' story, he himself is also reluctant as he somewhat hesitantly moves in the other direction from a bios-centred life to embrace zoe: at first, he is discomforted and bothered by the presence of luc and the thought of taking on the case for the baboons. as morris ends up exploring his sexual desires with luc tomlinson, he recognizes that the messiness of life which is part of his own subjectivity, or the zoe which flows through him, is connected to this non-human force of life in animals. he adopts a view of life and of the subject that correlates with ecofeminism "which asserts the fundamental interconnectedness of all life … [and which] offers an … ecological ethical theory for women and men who do not operate on the basis of a self-other disjunction" ( - ). he begins to care for the baboons, luc, and his neighbors' dog, which he formerly detested, when they plan to euthanize the dog because they are leaving for australia and no longer have a use for it, like the guard dogs in disgrace, to protect their property. the reluctant passenger reveals throughout the legal case for the baboons and through the behavior of various judges, lawyers, and government officials that the workings of the law are really the workings of desire. for example, we learn that luc desires protection of the baboons, and the protagonist, morris, begins to care for the case at first because he desires luc as the narrative later confirms. and it is gerhard's embracing of zoe and his encouraging of the protagonist to take pleasure in the world in its becoming that leads him to take on the legal case for the baboons. in other words, morris doesn't take on the case at first because he legally has to-the law doesn't require that he accept the case or that the baboons be protected, and indeed he appraises it as not much of a case at first. yet his desire, which is before thought, compels him to venture out to the preserve to meet luc and the baboons. further evidence of the law actually being desire is the revelation toward the novel's end that the authoritative figure judge conroy has been controlling most of the plot through underhanded deals, bribes, and so on-many of the same tactics that he and other judges used during the apartheid regime. having lost the woman he loved, joyce tomlinson, to the wealthy brick tomlinson, luc's father, while living a modest life to complete his law degree, conroy spends the rest of his life seeking revenge for the lack of consummation of his desire for joyce. we learn that he manipulates brick to propose the business development of the baboon's land, persuades luc to seek out legal recourse to protect the reserve, and controls many of the other events of the plot. as he tells the protagonist of his manipulations, nick queries: "so even in the capacity of puppet i was not indispensible?" ( ). as conroy's actions reveal, he disposes of others consistently through his authority as judge for his own profit and to exact his private revenge. as these behaviors bear out, what appear to be the workings of the law are actually the workings of desire. another example of this occurs in the resignation of the director of nature conservation and development as he does so because he's blackmailed with the threat that his affair with his secretary will be revealed to his wife if he doesn't resign. thus the regulation of desire that pertains to the family is exploited here as capitalist desire attempts to reterritorialize the director's desire to protect the animals and environment. deleuze and guattari explain that [t] hese two coexistent states of desire are the two states of the law. on the one hand, there is the paranoiac transcendental law that never stops agitating a finite segment and making it into a completed object, crystallizing all over the place. on the other hand, there is the immanent schizo-law that functions like justice, an antilaw, a 'procedure' that will dismantle all the assemblages of the paranoiac law … to dismantle a machinic assemblage is to create and effectively take a line of escape that the becoming-animal could neither take nor create. thus the attempt to control or limit desires, to force people into dominant subjectivities and fit the world nicely into concepts or categories, becomes a paranoiac attempt to limit schizo-law. this desire to master the world is also something jane bennet challenges via adorno's critique in his "negative dialectics," where he theorizes that violent behavior results from the frustration that humans experience when the world does not fit their concepts ( ). conroy's behavior and description of his motivations confirm his paranoid and narcissistic attempts to reterritorialize desire via his position as authority and arbiter of the law. especially interesting here also is that like her son luc, joyce tomlinson is closer to zoe in her embracing of desires as she often promotes luc's free-spiritedness. in contrast, conroy is colonized by oedipus, which defines his desire as lack in that his life is devoted to filling the lack created by joyce's marriage to brick, and to a lesser extent his desire to acquire wealth for the manipulation of others. conroy acquires wealth mostly for the purpose of attempting to separate joyce and brick. joyce, however, largely disregard's capitalism's lure as it seems she loves brick for himself, not for his finances. of course, brick's extreme adoption of capitalist logic leads him to view the baboons and their land as an opportunity for profit, and, anticipating this behavior, conroy manipulates him into the business venture. the novel thus points to the precarious biopolitical situation for those seeking protection from the law when its arbiters are colonized by capitalist desire. in other words, conroy's lack of joyce in his life and his use of his position to accumulate wealth jeopardizes the protection and futures of south african communities, making "disposable" those his position was created to protect. the desires of luc, morris, and the other characters who assemble to save the baboons, however, work to challenge these capitalist and narcissistic desires outside of the limitations of the law. as the novel reveals towards the end, conroy occupies a god-like position in his manipulation of the plot. his desire for mastery suggests an extreme form of the kind of control and disgust for vagueness and untidiness that the protagonist exhibited earlier in the novel. as the protagonist and conroy approach a relationship of mentor and mentee, heyns positions the development of the protagonist's character as dependent on his relationship to desire or zoe. at one end of the spectrum, the side of transcendental law, sits conroy, whereas nick's best friend and fellow lawyer, gerhard, embodies "immanent schizo-law" or desire and zoe as positive. conroy describes his frustration with the failure of his plan, that even after rendering brick to a state of poverty he still has not separated joyce from brick, blaming this on the incalculable nature of humans: "[a]gain i failed to take into account the inconsistency of human beings. joyce … was moved by his destitution to side with him" ( ). joyce's exceeding the mastery of conroy's control suggests the ungovernability of foucault's homo oeconomicus from the birth of biopolitics, as cary wolfe describes: in opposition to what foucault calls homo juridicus (or homo legalis)the subject of law, rights, and sovereignty-we find in this new subject, homo oeconomicus, "an essentially and unconditionally irreducible element against any possible government," a "zone that is definitively inaccessible to any government action," "an atom of freedom in the face of all the conditions, undertakings, legislation, and prohibitions of a possible government." "the subject of interest," foucault writes, thus "constantly overflows the subject of right. he is therefore irreducible to the subject of right. he is not absorbed by him." (before the law ) conroy thought he could control her, assuming that she loved her husband for his money, that she was colonized by capitalist desire, but she desires her husband for himself. even from his position of authority in the law, conroy cannot master joyce's desire or tame it to direct it toward himself. the protagonist, morris, also presents challenges to power and government that pertain to foucault's homo oeconomicus as he has broken numerous laws and no longer attempts to master his own desires. in addition, conroy offers in this scene his desire for an oedipal relation to the protagonist based on their similar orderliness and strict discipline: as you know, i had been taken with your dissertation some years ago … and now, i thought … you could take the place of the son i never had … you reminded me of myself at your age: ascetic, high-principled, civilised … for the first time my sterile obsession with avenging myself on the world for what i had missed yielded to a vision of what i might yet have. ( ) here, conroy's knowledge of the protagonist's homosexual act with luc upsets his oedipal ambitions of fatherhood. more specifically, morris' acting on his sexual desires with luc caused him to miss his appointment with conroy-the first time the protagonist ever missed or was late for an appointment-frustrating conroy's ambitions of mastery. frida beckman's discussion of homosexuality in deleuze's thought acknowledges how in addition to non-human sexuality, "[a]nother way of exploding the anthropomorphic, heterosexual, familial, and oedipal organisations of sexuality is found in homosexuality" ( ). she continues explaining that "[h]omosexuality, as verena andermatt conley notes, is seen here not as an identity, but as a becoming" ( ). morris' now positive appraisal of desire, which also motivates his protection of the baboons (who express sexual desire themselves), therefore enables the freedom from conroy's complete mastery of most of the characters in the novel from his position as an authority of the law. morris embraces his homosexual desires, instead of repressing or avoiding desire as he does at the beginning of the novel, in a way that enables a becoming away from the fixed identity of the subject of rights or the subject of capitalism that conroy might have otherwise mastered. conroy's description of his ideal new society built on "wisdom of authority" also includes biopower in the form of "death squads" ( ), biopower being that which foucault argues derives from a desire to govern and control homo oeconomicus. indeed, conroy describes his position of mastery here in a way that confirms his paranoid attempts to maintain and control desire. after suggesting that he wasn't trying to get joyce back, he comments: what i resolved to do was to achieve mastery over myself and others, partly through self-control, partly through control of them. and whereas control of the self is a matter of discipline, control of others is a matter of money. of this, as i have remarked, i soon had large sums, thanks to my contacts in countries officially hostile to south africa. ( ) here, conroy confirms the manipulation of his position of authority to exact his revenge, and along the way treating others, the land, and indeed the country of south africa as disposable in service to exercising his personal grievances. in short, he adopts a relation to others of "eating," as discussed in chap. . while early on in the novel the protagonist, like conroy, attempts to live an exceedingly controlled and ordered lifestyle (and this is in large part why conroy views him as an ideal candidate for his mentorship), his embracing of zoe, perhaps especially his embracing of his homosexual desire, frustrates the mastery and control of conroy's plan. the protagonist's decision to embrace zoe as positive then becomes revolutionary as it creates a line of escape from the mastery and instrumentalization of the humans, animals, and environment of south africa exhibited in the apartheid regime's practices. baboons before the law at one point in heyns' novel, after the baboons have been stolen from their home, luc tomlinson appears at morris' office to discuss the legal case for saving the baboons and their land from his father's business plans to set up a luxury resort there at cape point. the conversation that the characters engage in about the legal status of the baboons reveals the problems that the law has in deciding on the status of animals, an issue fontenay discusses in her chapter entitled "between possessions and persons." luc expresses his desire to get a "court order" ( ) for the baboon,s to which morris responds: "before i can get a court order i need to establish that somebody's rights are being infringed" ( ). luc argues that it is obvious that the "chacs'" (chacma baboons') rights aren't being respected, noting how they've been taken away for medical testing and vivisection, an issue i'll return to later. the protagonist replies: "the rights of animals are a much debated area in law" ( ). at this, luc replies angrily at morris: "i'm not interested in any fucking debate. anybody who isn't unbelievably stupid or dishonest knows what we're doing to the animals … it just suits us to come up with debates. the law is … ." morris attempts to finish luc's sentence, offering "an ass?", to which luc replies: "i was thinking of something more useless, like a … a eunuch" ( ). their conversation about the law continues, describing how the law protects only certain people through their metaphor of eunuchs as morris remarks, "they guard the sultan's harem," and luc replies: "yeah. great if you're the sultan, not so great if you're not" ( ). the discussion of the law in terms of eunuchs and harems continues to develop the theme of sexuality in the novel, and also points to the exclusionary nature of the law, as it only protects those who are considered as belonging to the community. the discussion of the harem and the sultan also perhaps highlights the discriminatory nature of the law's privileging some and excluding others. the protagonist attempts to end the conversation about the law and what can be done for the stolen baboons in a way that suggests he's exhausted all available avenues to help them. he remarks: "well … for better or for worse, the law, for all its shortcomings, is all we have to help us here" ( ). such a position, that we can only work within legal discourse to improve the state of affairs for animals, greatly limits the political potential for addressing the problems facing them. as wendy brown and janet halley suggest, "rights cannot be fully saturated with the aims that animate their deployment … they retain a certain formality and emptiness which allow them to be deployed and redeployed by different political contestants" ( ). in this sense, the affect and enthusiasm for protecting gets cut short when rights are viewed as the only avenue of intervention. brown and halley further explain the potential of critical theory for considering opportunities for politics and justice outside of legalistic frameworks, which the characters in reluctant passenger eventually take up in seeking extralegal, creative approaches: "[c]ritique [in contrast to legalism] hazards the opening of new modalities of thought and political possibility" ( ). where the protagonist, who lives most of his life according to society's normative rules, who lives extremely discursively or "by the book" as it were, seems to be giving up on the case for the baboons when they reach a dead end legally, his friend gerhard intervenes, responding to morris' claim that the law is all we have: "not necessarily" ( ). gerhard continues critiquing the lack of power that pertains to the law: "but where, as mr. tomlinson has pointed out, the law so manifestly lacks balls, we may have to rely on our own … devices for a remedy" ( ). the characters' desires for one another-gerhard is sexually attracted to luc, flirting with him, and, as the consummation of luc and morris' relationship bears out later, they desire each other as well-and for the baboons, especially luc's, result in their hatching a plan to save the baboons, outside the parameters of the law. as a lawyer for luc and the baboons, morris employs some blackmailing of his own to win the case for the baboons and the preserved land, with the help of a fellow lawyer and her husband, and the rest of the group which have rallied round the cause, including gerhard, morris' former girlfriend, and others. as a result they are able also to reinstate mr. haartshorn, the original director of nature conservation and development, who testified in the case that he was blackmailed with evidence of his extramarital affair and bribed into resigning after writing a report denying brick tomlinson's business proposal for the land under question. morris and his team also use some compromising pictures of minister stanford from what are revealed to be sex parties for ministers during the apartheid regime, which involved their raping of young men in military uniforms at a building in rocklands, the preserve of the baboons ( ). the photos were given to morris by joyce tomlinson, and later we learn, at the recommendation of judge conroy, to influence the minister's testimony so that he confesses that haartshorn gave him the report denying development prior to resigning. the trial results in haartshorn unseating the man who, in the old regime's pocket, replaced him and approved the proposal. for conroy, this is a victory because it caused a large financial loss for brick tomlinson by preventing the development of the land. however, the baboons are still rendered unprotected after the ruling as they are stolen and taken to the vivisection laboratory, and bulldozers appear at the nature preserve. thus even the legal ruling that uncovers the corruption and decides in favor of the baboons cannot protect them from the extralegal means of the capitalist desire to dispose of and turn a profit from them and their land. heyns portrays in morris a character who transforms his negative feelings about sexuality and animality into positive passions to work for the protection of the baboons, even outside the realm of the law. how do we think outside of dominant thought and beyond its closures? when faced with the limits of law, the characters' desires compel them to think differently. braidotti explains that deleuze and irigaray bank on the affective as a force capable of freeing us from hegemonic habits of thinking. affectivity in this scheme stands for the preconscious and the prediscursive: desire is not only unconscious but remains nonthought at the very heart of our thought because it is what sustains the very activity of thinking. our desires are that which evades us, in the very act of propelling us forth. (nomadic subjects ) thus desire and affectivity enable gerhard, luc, and nick to think differently, outside the parameters of the law, to devise a plan to protect the baboons. woodward argues about luc's appeals for the rights of baboons throughout the narrative that "luc coaxes the reader to accept the concept of baboons as 'creature[s] … of moral standing' within modernity, to refer back to nussbaum's argument" ( ). she further concludes her chapter on baboons by noting that "the moral agency of baboons … has not been acknowledged by the south african constitution, which does not incorporate the subjectivity or even sentience of nonhuman animals" ( ). while heyns' novel certainly points to the lack of legal status for baboons, the reluctant passenger in my view more strongly argues that it is necessary to think beyond the law, and acknowledges the role that desire can play in this thinking differently. that is, by acting on their desires for the baboons' protection, and for nick morris by embracing zoe as positive instead of attempting to master it in a negative relation, these characters ensure the protection of the baboons that the law cannot always guarantee. as talal asad argues in "what do human rights do? an anthropological inquiry," rights do not always guarantee the protections they describe. he argues: human rights discourse may not … always be the best way (and it is certainly not the only way) to help remove oppression and relieve suffering among human animals, as well as non-human animals, or to preserve the world's natural and cultural inheritance. working in hospices, providing comfort for the traumatized, the sick, the destitute, helping to rejuvenate depressed neighborhoods, are among the activities that help to relieve human suffering. such commitments remain outside the imperative of the law. ( ) in light of heyns' novel, we might add rescuing baboons from vivisection and hiding them in one's house to asad's list of the ways we can protect and prevent the suffering of non-human animals. although the reluctant passenger focuses on the life of an environmental lawyer, it emphasizes that we have creative options for responding to animals outside of legal means, options that exist "outside the imperative of the law" (asad) . as i've discussed in previous chapters, desire works across species boundaries, and again in the reluctant passenger an animal's desire and a human's desire for animals leads to the protection of the animals and their habitat. after nick and the adonis-like luc tomlinson walk naked with no deodorant on to meet the baboons (so as not to scare them off), petrus, the alpha male chacma baboon, takes a sexual interest in the protagonist: "'bloody amazing,' said luc, almost admiringly. 'you must have really potent pheromones.' he added with an unconvincing affectation of concern, 'i'm afraid he wants to fuck you.'" ( ). as his attentions turn to another baboon, petrus does not consummate the sexual exchange in what would surely be his domination of morris, who fears petrus' great strength and doesn't want to engage in sex with a non-human animal, but the sexual arousal that the protagonist and his pheromones caused in petrus turns out to be the reason that luc, the self-proclaimed friend of the baboons, initially finds the protagonist attractive sexually as well. the passage in heyns' novel suggests the transversal nature of desire as it works across species and indicates the role that pheromones might play in sexual attraction. like the desires that involve lahnee o, minke, and corsicana in tanuki ichiban, and the whale caller and sharisha in the whale caller, here again animals' desire and humans' desire for animals ensures the protection of the animals. the desire that arises out of assemblages (and not transcendental law) enables justice: "transcendent and reified, seized by symbolical or allegorical exegeses, it [the abstract machine] opposes the real assemblages that are worth nothing except in themselves and that operate in an unlimited field of immanence-a field of justice as against the construction of the law" (deleuze and guattari ) . thus the law is not the house of justice; instead, justice dwells in the potential of desire that results from the immanence of our bodies and relations to others in assemblage. of importance here is that this assemblage includes "black" south africans as well in the figure of nick's colleague and her husband mhlobo, who works at the mail & guardian and who supports the case for the baboons as he helps to discover the underhanded deals of the government officials. this environmentalism of the novel is therefore not a "white" or colonial conservationism but a postcolonial ecocriticism that benefits the larger community of south africa. heyns' novel portrays how the desires of an assemblage of human and non-human actors works against the mastery and control of dominant logics-a postcolonial desire that presents lines of flight out of the paranoid and narcissistic managements of the law and against the "eating" or instrumentalization of the community. gerhard's plan to retrieve the baboons from the vivisection ward demonstrates the creative potential of desire to work for the protection of others beyond the realm of the law. the group learns that the evil former apartheid government scientist now turned scientist for a corporate interest, colonel and doctor johanna van der merwe, performs vivisection on the baboons and also has a project of "rehabilitating" homosexuals to heterosexuality. pickover also highlights the horrifying fact that this testing on animals was done often to develop drugs and chemical weapons to dispose of and control those humans who opposed the apartheid government, and woodward notes this from pickover's work as well. pickover explains that this information came out in the trc hearings as she quotes dumisa ntsebesa who chaired them: when animals are being used by scientists for experiments to manufacture chemical and biological weapons, then society should condemn such experimentation in the strongest terms … even more alarming is the fact that the people who were using their research skills and knowledge to manufacture murder weapons, were people in white coats with stethoscopes hanging out of their pockets. these people are normally associated with preservation of life. that is the most repulsive feature of the evidence that has come before us. ( ) ntsebeza's response to learning of the vivisection and the creation of weapons to destroy humans speaks to the biopolitics involved in this case as he notes that those who are supposed to aid in maintaining life end up being the administrators of death. in response to van der merwe's two projects, which render animals and non-dominant sexual desires (which upset her normative visions of the human and white nationalism) disposable to the control and mastery of science for the profit or benefit of the corporation and a particular vision of the nation-state, gerhard devises a plan to distract her by offering himself up as a victim for her experiments so that the other members of the assemblage might sneak into steal the baboons away. in offering his body, gerhard renders himself vulnerable and lets himself be "eaten" by the state, sacrificing his body in exchange for the protection of the baboons. gerhard explains that van der merwe "was a medical officer in the south african defence force" and during that time she "was in charge of some highly controversial experiments" ( ). after mhlobo informs nick about van der merwe's horrifying science experiments, and creation of drugs and weapons to harm the black community, nick meets van der merwe, dubbed "the black widow," in a gay bar to set up the appointment where gerhard will be "rehabilitated." van der merwe expresses her views on sexuality here in a way that confirms her attempts to limit desire and to exercise biopolitical control over the south african population: "[i]n terms of all three of these paradigms [science, her womanhood, and christianity] the function of the human species is to procreate itself responsibly" ( ). she continues offering here a speciesist perspective: "i believe, of course, that as the bible tells us, we have been instructed and empowered to rule over creation. the human being is the crown of creation, and as such is entitled to use the rest of creation for his benefit-within certain limits, of course" ( ). such a view of animals confirms religion's role in attempting to separate humans and animals, something i've also discussed as it is portrayed in the whale caller. van der merwe continues to explain her convoluted arguments, at times insisting on the "naturality" of her positions while at other points explaining the need for science to correct nature after the fall of man. immediately prior to nick's meeting with the black widow, mhlobo informed him that one of the scientist's projects was to sneak birth control into foods that black south africans commonly eat as a way to control their populations. here van der merwe explains her position after nick queries: "isn't breeding natural?" ( ). she replies: it's natural only in the sense that the procreation of rabbits and chickens is natural. nature in that sense is an accident, without plan or purpose. that is the creation over which the lord gave us dominion. the higher nature is guided by divine wisdom as manifested through science and technology, and informed by a sense of individual and national identity. here van der merwe clearly dehumanizes black south africans given that her eugenic project to control the population's sexual reproduction is discussed in terms of rabbits and chickens in this case, and she thereby establishes a hierarchy where whites are superior to black south africans. as the conversation turns to her project of making gay men straight, she explains that she takes it as her calling "to tell them what they should be, and to help them assume their rightful identities" ( ). such a perspective demonstrates an attempt to control desire and subjectivity, limiting sexuality to only normative and procreative practices that might benefit the state in a fantasy of white nationalism. thus human and animal sexualities-these desires-threaten her biopolitical ambitions and ideal vision of the nation. as it turns out, van der merwe, in a sense, rapes gerhard "in the interests of science and the nation" by using a probe on him that was used to give baboons erections to extract their semen for use in experiments to develop birth control for the black population. gerhard's risking of his life, body, and perhaps sexual orientation for the baboons demonstrates a personal sacrifice that results from his desire for their protection. we learn later that the "reorientation" is unsuccessful as he admits having sex with his lover, clive, directly after the experiment, and perhaps gerhard is not as distressed from the experience with the black widow as one might assume as he seems to view it as a challenge. as the assemblage of people risk their lives and wellbeing for the baboons, it's clear that they've gone well beyond the law to ensure the animals' protection. in addition, nick's development of character emphasizes the novel's perspective that embracing zoe and desire as positive is a necessary intervention toward the protection of communities. no longer the solitary, sedentary, order-loving, manageable subject of complicit consumerism and model capitalist laborer, morris becomes political. he deals with the complexities and ungovernable aspects of life, including his sexuality, and multiplies his attachments around a common goal of a sustainable future, not just for the environment and baboons, but also toward social reforms of the legal system through removing those "eaters" of the country from their positions of authority in the government-sustainable futures for the social conditions of south africa. like morris and the assemblage of characters who work against the business proposal to "develop" the land and remove the baboons from their home in the reluctant passenger, camagu, the outsider to the village of qolorha-by-sea and protagonist of zakes mda's the heart of redness ( ), works to protect the community and environment of the village. mda also won the sunday times fiction prize for this novel, which describes how camagu and many of the other villagers, especially qukezwa, argue against the gambling city that is proposed to develop the small village he has recently made his home and come to love. offering a more sustainable alternative to the tourist town, camagu expresses his opinion, which he learns from qukezwa, that the town developed by outside businesses will offer little work or profit for the townspeople and be detrimental to their environment. as in the reluctant passenger, the association of big business with the law is revealed as the developers' attempt to intimidate the villagers' opposition with claims of their connections to the government: "how will you stop us? the government has already approved this project. i belong to the ruling party. many important people in the ruling party are directors of this company" ( ). here we see another instance of the political systems that might check the instrumentalization of a community failing as a result of their compromised positions and colonization by a capitalist logic of desire. the audacity with which the developers assert their power demonstrates their impunity as they abuse their positions of authority and elite status in administrating the law: they do not seek to protect the people and animals of the village as the law might happen to prescribe. instead, the developers use their position of power to secure their personal wealth at the expense of the well-being of the community, continuing a practice of "eating" of the community in viewing it as disposable for their personal gains. somewhat similar to the reluctant passenger, where the character development of the protagonist is situated on a scale of zoe-between his embracing its uncertainties in the example of gerhard and controlling or mastering it in the example of conroy-camagu's character in the heart of redness also develops in relation to desire. in this case, how he apprehends it as oedipal, capitalist and as a lack or as positive, productive, and ethical is manifested through his thinking about his relationships with women and the environment: whether he sees the casino development's potential violence to the environment as totally beneficial to the community and views women as existing for his sexual pleasure, or by contrast, taking a more critical position, considers other more positive ways of relating to women and the environment. his interest in relationships with two women of the village indicate his growth as a character as he is at first interested in bhonco's daughter, xoliswa ximiya: a teacher who no longer lives in the village, fetishizes the us, deplores local traditions, such as the practice of dyeing the face with ochre ("the redness" of the novel's title), and sees the advancement of the community toward a greater likeness of western civilization as worth the sacrifice of the environment and animals of the village. as the novel progresses, camagu finds that he more strongly desires qukezwa, daughter of zim, who demonstrates an exceptional knowledge of and intimacy with the environment, the flora and fauna of the village, and local history and traditions. particularly important here as well is the believers' expanded notion of community beyond the "human" that is evidenced in particular characters' devotions to their horses, camagu's refusal to kill the snake of his totem that appears in his bedroom, and especially zim's love of and communication with weaverbirds. in contrast to the developers' plan, and based on the insight into "development" he learns from qukezwa, camagu proposes a smaller measure of a resort built with local materials by the villagers appealing to a different type of tourist who appreciates nature, but not for trophyhunting purposes. camagu's alternative proposal, importantly, does not disregard the fact that the villagers' lives would benefit from some increase in funds. that is, as i discussed in chap. by quoting huggan and tiffin ( ), he is not against "development" in and of itself. instead, he is against those forms of development that position the developers in a parasitic relation to the environments and communities they develop, where they can better control and manipulate the community through their inclusion into the global capital system. his proposal is certainly one of development as well. the important difference is that the proposal is developed so as to benefit the community as it values local flora and fauna, knowledge, and labor, rather than devastating it as the capitalist casino and theme park certainly would. the villagers of qolorha are largely divided into two groups: believers and unbelievers. the believers are descendants of the followers of the prophetess nongqawuse back in the times of the cattle-killing movement in the s. as the novel jumps back and forth from the colonial past to the present, at times seamlessly, colonial and neocolonial themes are developed in relation to the environment and sustainability. the believers are more traditional in their return to and appraisal of customs of the past and, as espoused by zim, a prominent believer, they are against the development of the tourist casino. however to be sure, mda does not portray these two groups as totally at odds or pure in their difference: the groups are more complex because some of the unbelievers value the traditions of others and have a mode of believing in unbelief, for example. these divisions along lines of belief, which were exploited by the british colonizers in a divideand-conquer strategy, offer a critique of the present political divisiveness that becomes a distraction and more about self-interest and pride than working toward a sustainable future and protections for the community. the heart of redness emphasizes the variety of temporalities and historical influences experienced in the present of south africa. as mbembe explains, "everything happening today is [not] simply a rerun of a scenario …. [t]oday's shift … is … toward the underground channels" ( ). indeed, we've seen this underground market of animal trafficking in tanuki ichiban in chap. . in this sense, while i've gestured throughout the project to how the "new" south africa continues to be similar to the past violent organizations of society, mbembe makes clear that the new negative modes of relation and extraction are somewhat different in adopting this underground fashion. mbembe continues to explain how, [i] n this intermeshing of temporalities, several processes co-exist; there are processes tending to make peoples view the world in increasingly like ways, and at the same time, processes producing differences and diversities. in short, contradictory dynamics are at work …; it is too easy to reduce these dynamics to simple antagonism between internal and external forces. ( ) this suggestion to avoid oversimplifying the more complex and nuanced interactions of the present organization of society is particularly useful for assessing the dynamics and influences at work in mda's novel: his observation that different, diverse perspectives are occurring, as well suggesting ways out of the culture of sameness and the status quo. as i've been describing how characters seek extralegal means to protect their communities, the heart of redness aligns with mbembe's conclusion about the dynamics of the current moment where the state is no longer viewed as "the best instrument "for ensuring the protection and safety of individuals, for creating the legal conditions for the extension of political rights" ( ). the leading unbeliever, bhonco, and his daughter view all signs of "development" and "progress" imposed by corporations as beneficial for the future of the village. as evidence of this, bhonco discontinues some of the adornments of his traditional clothing and begins wearing business-style suits. as braidotti describes, late capitalism endangers not only biodiversity but also human and cultural diversity: [disregard for biodiversity] also threatens cultural diversity by depleting the capital of human knowledge through the devalorization of local knowledge systems and world-views. on top of legitimating theft, these practices also devalue indigenous forms of knowledge, cultural and legal systems. eurocentric models of scientific rationality and technological development damage human diversity. the plans to make the village of qolorha by the sea into a casino would not only render the environment unsustainable but also dominate the culture and non-dominant, indigenous knowledge, such as the ways of fishing and other environmental lessons that the american-educated camagu is continually learning from the villagers throughout the novel. alongside this are the high esteem that bhonco's daughter holds for the us, the country where camagu got a degree, only to return for the elections of . camagu attempts to correct this view of the us as a land of progress by describing the prevalent racism and imperialist practices that he experienced in everyday american life and foreign policy. in his essay on the heart of redness, byron caminero-santangelo argues that the novel positions "cosmopolitan bioregionalism" as a mode of resistance to western development: if capitalism in its various phases has made space out of place, stripping away prior signification (deterritorializing) and reshaping in order to facilitate control and exploitation, then the process of imagining or reimagining a "place" entailed by bioregionalism can be one means of countering threats of exploitation, environmental degradation, and disempowerment. such a concept perhaps resonates with the idea of "smooth space" discussed earlier as a resistance to striated or mapped-out and managed space of the state. yet bioregionalism seems to offer a more specific, intimate knowledge of place and may therefore be a concept better suited to resisting the neocolonial power enacted against particular people, flora, and fauna. its cosmopolitanism challenges the isolationism or "ecoparochialism," to use nixon's term, of some bioregionalisms that adopt a view of place as separate from a broader world, or that ignore the historical and other conditions that are involved in the composition of place as a "process" ( ). this cosmopolitanism is especially important in a south african context as it takes note of larger world processes, upsetting particular nationalist imaginaries and their rhetorics of purity that might attempt to deny or elide past violences, histories, occupations of land, and so forth, such as afrikaner white nationalism in south africa. as caminero-santangelo argues, "[n]otions of purity are debunked in the heart of redness not only in respect to identity and culture, but also in terms of the category of nature" ( ). a cosmopolitan bioregionalist perspective makes clear how particular rhetorics of purity deployed to claim a place as home or to determine the limits of community are undermined by a broader view of international movements, material processes, relations, and so on. marisol de la cadena's concept of "indigenous cosmopolitics" presents perhaps an even more apt concept for considering the notions of politics at play in the heart of redness, given the emphasis on indigenous knowledge throughout the novel. much of her writing in her essay about differing worlds and worldviews concerning the prospect of a mining development and its potential devastation of a mountain called ausangate in the andes parallels the varied logics and arguments about development in qolorha in mda's novel. for example, bhonco shows disdain for many traditional practices and knowledges privileging western development, while qukezwa and her father zim have intimate relationships with non-human animals, and great respect for the environment and the indigenous knowledge of the xhosa. like caminero-santangelo's reading, which emphasizes the open-endedness of the novel, its privileging of continued dialogue and its refusal to uphold any one character's or group's-believers and unbelieversperspective as the truth or right view, de la cadena emphasizes plural perspectives on nature and politics rather than the one universalizing understanding that often elides other ways of knowing. she argues that despite indigenous knowledge that conceives of the "earth-being" (or mountain in non-indigenous parlance) called ausangate as significantly agentive, there are no guarantees that this knowledge will determine the decision people arrive at regarding the question of development: pluriversal politics add a dimension of conflict and they do not have guarantees-ideological or ethnic (cf. hall ) . people-indigenous or not, and perhaps ethnically unlabeled-could side with the mine, choosing jobs and money over ausangate, either because they doubt or even publicly deny its being a willful mountain, or because they are willing to risk its ire for a different living. ausangate's willfulness could be defeated in the political process-some would embrace it, others would not-but its being other than a mountain would not be silently denied anymore for a pluriversal politics would be able to recognize the conflict as emerging among partially connected worlds. and although i would not be able to translate myself into nazario's ontology, nor know with him that ausangate's ire is dangerous, i would side with him because i want what he wants, to be considered on a par with the rest, to denounce the abandonment the state has relegated people like him … to denounce the mining ventures that do not care about local life. ( ) de la cadena, while situated outside of the world of indigenous knowledge, nonetheless promotes respect for it as she recognizes its potential-in the threat of agency from ausangate's ire-to challenge the instrumentalist views of nature offered by western biopolitics and development rhetoric. the heart of redness offers a similar perspective on "earth-being's" agency through indigenous knowledge that bhonco and other developers often elide or dismiss as backwards. for example, the qukezwa who married twin in the past explains that "we never kill the snake of the spring. if we did, the spring would dry out" ( ). later, in the present of the novel, her namesake tells camagu about the agency of a specific snake, gqoloma, who lives in the prophet nongqawuse's pool: "when it pays a visit … moving from the pool at the gxarha river to another pool at the qolorha river … it causes havoc in its wake, like a tornado. it destroys houses. it uproots trees" ( ). camagu also respects his people's customs when he prevents hotel workers from killing the snake that appears in his room because it is his totem. in contrast, xoliswa, who adopts western knowledge as universal, dismisses indigenous knowledge as nonsense: "what can we say about a man who believes in a snake?" ( ). earlier her father bhonco and others too dismiss the believer's respect for indigenous trees and animals as "foolish" and "madness," and takes a homogenous view of nature in suggesting that native trees could easily be replaced with "civilized trees" ( - ) . the heart of redness's indigenous cosmopolitics positions indigenous knowledge as frustrating bhonco's attempt to deploy as a universal a western definition of politics as reserved exclusively for humans and his attempts to uphold a human/ nature binary. the "animist materialism," harry garuba's phrase, expressed in characters' spiritual views of these flora and fauna suggests a mode of resisting their instrumentalization. where bhonco reads them as lacking in use value-"these plants are of no use at all to the people. they are good neither as wood nor as food" ( )-qukezwa and other characters' animism values the animals and plants on their own accord. as garuba argues of animism, "objects thus acquire a social and spiritual meaning within the culture far in excess of their natural properties and their use value" ( ). the reading of desire in the novel offered here aligns with cosmopolitan bioregionalism, animism, and indigenous cosmopolitics. where cosmopolitan bioregionalism offers a notion of place as process rather than defined space, this project focuses on challenging the defining or limiting views of subjectivity, desire, and other concepts that are also more processual. indigenous cosmopolitics emphasizes "non-representational affect" as key to its expanded notion of politics; theorizing desire, as one mode of affect, as a political potential to resist such exploitation coincides with these other concepts' emphasis on place and indigenous knowledge as modes of resistance. the ethics of sustainability that informs camagu's development of a cooperative calls into question the capitalist notions of western development. he discusses his cooperative with dalton, the trade store owner: i am talking of self-reliance where people do things for themselves. you are thinking like the businessman you are … you want a piece of the action. i do not want a piece of any action. this project will be fully owned by the villagers themselves and will be run by a committee elected by them in the true manner of cooperative societies. ( ) his description confirms that his motive is "not for profit" but for creating more positive passions, more joyful lives for the villagers than the current work some of the villagers perform as workers in the mines, service workers at hotels, and the potential jobs that would come from the casino. the narrator describes these workers as receiving racist treatment at the hands of white tourists and also receiving poor wages. camagu makes his opinion of the casino proposal as an unsustainable option clear at one of the meetings the villagers have with the developers. "it is of national importance only to your company and shareholders, not to these people!" he yells. "jobs? bah! they will lose more than they will gain from jobs. i tell you, people of qolorha, these visitors are interested only in profits for their company. this sea will no longer belong to you. you will have to pay to use it" ( ). part of the loss from these new jobs will include the devaluing of local customs, knowledge, and labor, and the inclusion of the work done by villagers into the economy of exchange in capitalism that translates into profit for the western developers. camagu's desire is clearly for the people, and this desire for the community stems from his initial desire for qukezwa and his knowledge of its importance to her. his anti-oedipal desire therefore counters the capitalist desire of the developers. as mentioned earlier, camagu arrives at his position on the proposal for the casino resort after his discussions with qukezwa. indeed, the arguments he voices at the village meeting with the developers are those she expressed to him earlier. having a ph.d. in "communication and economic development" ( ) from the us, camagu at first finds the casino proposal unproblematic. in fact, early in the novel he adopts more of a capitalist logic of desire as lack and it is qukezwa's teachings that change his views about desire and his relation to others. at the novel's outset, camagu is about to leave south africa to return to the us for a job in economic development, and yet it is his desire that derails his trip, it is desire that brings him to qolorha. as in the whale caller, mda here distinguishes the protagonist's desire from narcissistic desire. after hearing the song of a funeral singer, a "makoti" ( ), he cannot get her out of his mind: he becomes breathless when he thinks of her. he is ashamed that the pangs of his famous lust are attacking him on such a solemn occasion. but he quickly decides it is not lust. otherwise parts of his body would be running amok. no, he does not think of her in those terms. she is more like a spirit that can comfort him and heal his pain. a mothering spirit. and this alarms him, for he has never thought of any woman like that before. ( ) while all of his relations to women previously are of a selfish nature and about accumulating sexual pleasure for himself, and he is ethically deficient in this regard as he pays his servant to have sex with him in a way that the narrator compares to the rape of a slave by her master ( ), his desire for this woman is not of a self-serving kind; it is this desire for her that motivates him to seek her out again in qolorha. thus, while he is self-interested and narcissistic in his use of others for sexual pleasure at the novel's beginning, he comes to approach desire differently after this experience with the makoti and after learning from qukezwa. in contrast to the earlier one-sided affairs of camagu's lust and use of women, both he and qukezwa have sexual dreams about each other: "mutual dreams. messy dreams" ( ). this transformation of his relation with women from a position of dominance to a mutual relationship brought about by qukezwa suggests that she is a feminist character. indeed, harry sewlall dubs her "the quintessential ecofeminist" ( ), for her thorough knowledge of the plants and animals of her habitat, her challenges to the patriarchal traditions of the villagers, her dressing in traditional garb, and her educating others in the village about the environment and living in a sustainable way. yet, as caminero-santangelo convincingly argues, the novel's characterization of qukezwa is not an idealization of the indigenous as some critics hold, since she is more complex, transformed by history and cultural contact: "such characterizations [of qukezwa as the idealized ecofeminist] suggest that mda reinforces the notion of an unchanged indigenous ecosystem that existed before the impact of colonialism and advocates for a return to an indigenous, properly ecological relationship between human and nonhuman nature." in other words, qukezwa sees the similar logics of oppression at work that seek to disenfranchise animals, women, and others from a position of full standing and as deserving of the protections of the community. her ecofeminism becomes clear, especially in the scene where she seeks to defend her environmentalist practices of killing some foreign water-depleting trees before the council of elders, challenging the lower legal status afforded to women in traditional xhosa laws by gesturing to the laws of the "new" south africa that prevent gender discrimination. qukezwa's role as a mother to heitsi and her teaching him to swim in the sea at the novel's end demonstrate a particularly south african feminism that embraces motherhood as a political act, a mode of feminism in the country that as zoë wicomb explains differs greatly from the largely negative view of family roles in western feminisms. her teaching her child to swim in the sea, in relation to which she has played perhaps the most significant role in protecting it from being stolen from the community by the developers, confirms her feminism as a robust ecofeminism. qukezwa also plays a role in deterritorializing camagu's once capitalist desire as he no longer narcissistically spends his time seeking his own pleasures and profits at the expense of others. animals seem to play a role in this transformation of sexual desire as he, rather like the whale caller, achieves orgasm seemingly incidentally in the presence of an animal and in response to song, in this case while he and qukezwa both ride on the horse gxagxa: "[h]is pants are wet … . it is not from sweat" ( ). at this point in the novel, camagu becomes enthralled with qukezwa, especially her singing rather than with her looks. in fact, the more "civilized" and western potential love interest xoliswa is known for her beauty which rivals that of models. after their ride on gxagxa, "[h]e remembers … when she sang him to an orgasm" ( ). like nick morris in the reluctant passenger, at first he fears this different kind of non-human desire and seeks to maintain control over this zoë: "he must get away from these surroundings that are haunted by qukezwa's aura. he must fight the demons … he must try to be in control" ( ). as he is "spellbound" and not in a dominant position of control as in his previous sexual experiences where he used women, the scene indicates a more positive experience of desire. this horseback ride, apparently, results in qukezwa's pregnancy-a strange, non-normative chance pregnancy that occurs incidentally during a fully clothed ride. conceived at this moment of non-human desire and intensity, without sexual intercourse, while atop a horse, their child heitsi figures as a future for the community where desire will continue to offer new ways of thought and creativity to resist neocolonial threats. while dominant culture might view such an act as dirty, weird, or something to be prevented, or even as a sort of violence to the horse, mda's novel indicates that the relationship between qukezwa and gxagxa is positive and ethical: "they love each other, gxagxa and qukezwa … . her father lives in this horse. she wouldn't dare do anything shameful in its presence … . she gives it the same kind of respect" ( ). her sensual encounter with camagu while riding gxagxa in this regard is not "shameful" or pejorative but perhaps a moment of shared intensity and love between humans and animals. earlier, qukezwa draws attention to the colonization of camagu's thought that results from his american phd in economic development and highlights how this colonization informs his initial reaction to the proposal for the casino resort: vathiswa says they made you a doctor in the land of the white man after you finished all the knowledge in the world. but you are so dumb. white man's education has made you stupid. this whole sea will belong to tourists and their boats and their water sports. those women will no longer harvest the sea for their own food and to sell at the blue flamingo. water sports will take over our sea! ( ) like nongqawuse, qukezwa desires to protect her community, a community which includes animals, plants, and the people of qolorha. after camagu argues that the village will be paid for this, and that the development will create jobs, she further challenges his thinking: what do villagers know about working in casinos? … . i heard one foolish unbeliever say men will get jobs working in the garden. how many men? and what do they know about keeping those kinds of gardens? what do women know about using machines that clean? well, maybe three or four women from the village will be taught to use them. three or four women will get jobs. as for the rest of the workers, the owners of the gambling city will come with their own people who are experienced in that kind of work. ( ) thus it is qukezwa who is responsible for protecting the community, alongside the changed camagu, who decides to stay in qolorha instead of leaving for the us for his own financial gains, a decision to stay in south africa that nick morris makes as well in the reluctant passenger. therefore anthony vital's claim that "the united states is represented also as providing the central character with the economic understanding that protects the local from outside exploitation" ( ) seems to fail to recognize qukezwa's knowledge and influence on camagu's thought and desire. qukezwa's assessment of the proposal is accurate, and the way in which the proposal is framed to benefit the community works just like traditional colonial logic of the civilizing mission, where the colonizer's behavior is presented as a benefit for the people, when really its goal is to further disenfranchise them so that the politicians and developers can "eat." mda points to this colonial history as well in portraying george gray's taking of the xhosa's land in return for "civilizing" them. this ethics of sustainability in the heart of redness is not one of moral universalism but instead one that acknowledges the importance of local or indigenous knowledge. harry sewlall describes the novel's representation of this knowledge by pointing to the importance of the local court's treatment of qukezwa for cutting down foreign trees: what emerges at the village trial of qukezwa is that the indigenous people of this land have always had their own laws to protect the environment. while qukezwa's actions are considered criminal because there are no laws proscribing wattle trees, there are traditional laws in place which allow the destruction of noxious weeds and plants, such as the mimosa. ( ) the novel highlights multiplicities of perspectives, times, and values concurrently, as mentioned in the contrasting positions of the believers and unbelievers, but also in dalton and camagu's different plans for the village as either a cultural tourism site or a nature tourist site. the administration of the local laws of the amaxhosa reveals that the elders of the village who make rulings do not view the law as static or transcendental. for example, although the "old law" ( ) regards any woman not married as a minor, they listen to the unwed twenty-year-old qukezwa's arguments against this law: "[i]n the new south africa where there is no discrimination, it does not work" ( ). at this, the chief replies: "now she wants to teach us about the law" ( ). while bhonco's daughter xoliswa ximiya is officially a teacher, it is qukezwa who becomes a more active teacher in the community. as mentioned earlier, bhonco and his daughter xoliswa eurocentrically denigrate most traditional customs and practices as "backward" and "uncivilized," embracing instead many western and american ideas about lifestyle, the natural world, and development. this extends to local flora and fauna, as bhonco asserts: "we want to get rid of this bush which is a sign of our uncivilized state" ( ). zim and his daughter qukezwa, by contrast, follow traditional xhosa customs, practice indigenous environmental knowledge, and greatly value local biota, rejecting many of the western ideas introduced into their community. qukezwa and zim's embracing of tradition challenges bhonco and xoliswa's eurocentricism, yet it too is not without problems. in her chapter that focuses in part on heart of redness from her "wilderness into civilized shapes:" reading the postcolonial environment, laura wright reads some characters' engagement with traditions in mda's novel in terms of bhabha's notion of "colonial mimicry," arguing that they "rely on imagined identification with a now inaccessible and uninhabitable past, while simultaneously subverting and parodying that past in an attempt to monetarily benefit from its value as a marketable commodity" ( - ). she explains the problems of this exploitation of tradition in john dalton's obviously business-oriented and self-interested plans for a cultural village as she notes camagu's view of such a packaging of tradition as "inauthentic" and as "deny[ing] the value of contemporary xhosa culture" ( ). wright also astutely observes how dalton's planned last-minute heroics by withholding the letter he secured declaring the village a protected heritage site is an example of a "colonial mentality that indigenous peoples were not capable of caring of themselves" ( ). however, she also has problems with qukezwa's drawing from xhosa traditions. wright views qukezwa's focus on tradition and removal of invasive plants as taking part in a postcolonial tendency of "ineffective … attempts at establishing various prelapsarian-and imaginary-african edens, impossible landscapes that are somehow uncompromised by their postcolonial status" ( ). while she argues that both the believers and unbelievers attempt to borrow and invent traditions in ways that are appropriative, qukezwa's attempts to "recreate" ( ) tradition seem to occur without such appropriation. like bhonco, zim seems somewhat extreme, but in the other direction, in his embracing of past traditions, selectively deploying them at times in the imaginary fashion that wright identifies in order to emphasize his difference and purity from bhonco as when, for example, he shaves off his eyebrows. however, qukeswa's drawing from tradition and indigenous knowledge, and perhaps from nongqawuse's future-oriented biopolitics, is free from this edenic imaginary and approaches instead an attempt to serve the best interests of the community by exercising an ethics of sustainability. in this sense, qukezwa's breaking the traditional laws to cut down certain invasive and water-extracting trees might be seen as more in line with the extralegal efforts of the characters in heyns' reluctant passenger to protect their community and the baboons. perhaps the difference in readings comes here from wright's attempts to read the plants in mda's novel as metaphor: "plants function as metaphors for colonizers and colonized peoples, and the invasive species that characters seek to eradicate generate no hybrid entities" ( ). the fact that there are no hybrid plants is surprising and supports wright's view nicely. however, there are issues with such a metaphorical reading. after acknowledging that the killing of invasive plants can be read in a literal sense as well, wright goes on to emphasize the plants as metaphor as she attempts to read qukezwa's killing of plants as a "shortsighted solution to white intrusion": "removing all invasive plant species not only would be impossible, but also such an action can only operate on a metaphoric level" ( ). reading the invasive plants in a literal sense instead of as a metaphor for colonizers, we might appreciate qukezwa's environmental conservation efforts to preserve her community's water further. reading these scenes of the killing of flora and of community exclusions in terms of biopolitics enables more careful attention to the different kinds of life under consideration and the ethics of hospitality that pertain to them. wright's reading of qukezwa's biopolitical management of flora as metaphor also seems to ignore differences between kinds of life and the idea that qukezwa does not seek to remove all whites from the community in a xenophobic fashion. in fact, it is bhonco who seeks to remove dalton and other non-xhosa people by deploying a purist and prelapsarian rhetoric, as i discuss below, when he attempts to expel those from the community who disagree with his position on the casino development or who otherwise frustrate his ambitions. if bhonco is extreme in his disdain for local flora and fauna, and in his willingness to sacrifice indigenous plants, animals, and knowledges for english ones, zim is somewhat extreme in his love of all that is traditional and disdain for the foreign. yet quekezwa does not kill some of the foreign trees to restore an eden or out of a xenophobic impulse but out of concern for the community's water supply. bhonco's concerns seem more self-centered and short sighted. he becomes a kind of nationalist manager when he threatens camagu's membership of the community in response to camagu's anti-development arguments: "who are you to talk for the people of qolorha? … you talk of our rivers and our ocean. since when do you belong here? or do you think that just because you run after daughters of believers, that gives you the right to think you belong here?" while seeking to win the argument for the development, bhonco attempts to do so by excluding camagu from the community and from being a subject of rights in the community, enacting a biopolitics that threatens to remove those who are not pure enough, not xhosa enough. qukezwa doesn't seek to banish all the plants from the region, just those that greatly diminish the water supply and threaten the ability of other plants to survive. while she initially emphasizes that the trees she cut down are "foreign trees!" she explains that she did so because they are like the inkberry, which "destroys everything before it!" ( ). bhonco attempts to discern and charge qukezwa with xenophobia by picking up on the "foreign" in her explanation: "are you going to cut down trees just because they are foreign trees?" asks bhonco indignantly ( ). in her reply, qukezwa explains that she is not against trees just because they are foreign; instead, she kills the trees that harm the community. making biopolitical decisions about what must be sacrificed for the future of the community, qukezwa explains to the elders why she cuts certain trees and doesn't cut others: the [foreign] trees in nogqoloza don't harm anybody, as long as they stay there … . they are bluegum trees. the trees that i destroyed are harmful as the inkberry. they are the lantana and wattle trees. they come from other countries … from central america, from australia … to suffocate our trees. they are dangerous trees that need to be destroyed. ( ) in reply, bhonco asserts that the traditional law doesn't allow for the killing of these foreign trees without permission, and qukezwa argues for the law to be changed because the wattle tree "uses all the water" ( ). the law doesn't allow for the killing of the inkberry without permission either, yet everyone knows its harmful qualities and allows its cutting anyway, without the law's approval. qukezwa argues that this is the extralegal precedent for her actions, explaining: "[the wattle] is an enemy since we do not have enough water in this country" ( ). in this scene, qukezwa decides to remove those plants that threaten the community's future, just as the earlier believers killed the cattle, effectively stopping the viral spread of the european lung sickness that was decimating their cattle and horses. the opening of the novel also confirms that the environmentalist efforts of the xhosa have been successful in securing a sustainable present: "indeed, qolorha-by-sea is a place rich in wonders. the rivers do not cease flowing, even when the rest of the country knells a drought. the cattle are round and fat" ( ). described here as wonders, perhaps alluding to the magical realism of the novel and its prophets, the report on the state of the animals and environment suggests that environmentalist practices have created a sustainable relationship with the land where other parts of the country suffer water shortages. the comment about the healthy cattle follows the positive assessment of waters in qolorha, suggesting that the cattle-killing movement and the sustained interest in conserving water has in some measure helped to sustain the environment and the future of the community. bhonco, conversely, upholds the law at all costs in this case, further proving that it is desire which is actually in the place of law. although the ruling of the elders is interrupted by a fire that burns a few of the villagers' homes, the elders mostly disagree with bhonco as it's clear that he is just interested in punishing qukezwa because she is a believer, and zim's daughter at that. rather like judge conroy in the reluctant passenger, who seeks to use the law to abuse his rival brick tomlinson, bhonco appeals to the law in hopes of seeing zim punished for his daughter's crimes: "why doesn't he stand up like a man and take the rap?" ( ). bhonco's desire is reterritorialized, seeing as he has no problem with the development of the casino city, which would break these laws, a project which the developers reveal involves the killing of many of the village's trees: "how can we call it a grove when we're going to cut down all these trees to make way for the rides?" in response, the other developer remarks: "[w]e'll plant other trees imported from england. we'll uproot a lot of these native shrubs and wild bushes and plant a beautiful english garden" . in contrast to the developers and bhonco, whose desire is self-interested, and who arbitrarily upholds the law when it suits him (and it is perhaps significant in this regard that bhonco's frustrations with his inability to secure his old-age pension from the government is a frequent sore spot for him throughout the novel), qukezwa's actions are motivated by a desire to protect her community; in this instance, specifically the local plants and water supply of qolorha. as the history of the amaxhosa wars with the british colonists and qukezwa's killing of colonizing plants make clear, the response to new things and deciding on their belonging to the community or not often involves a consideration of how these new people and things harm or "eat" the community, or if they further protect it. bhonco at times wants to expel dalton, descendent of a british colonial officer, and camagu, who at times he calls a foreigner. he decides that these "outsiders" do not belong to the village community in relation to how their presence or absence would personally benefit him, not the larger community. he has repeated arguments and later a violent encounter with dalton, for example. the racial inflections of dalton's status in the community highlight the various modes of biopolitical management at work in qolorha and demonstrate characters' anxieties about belonging and their relationship to the land as "home." these concerns about who belongs and who doesn't, how many, who belongs most, and the desire for a space that makes it feel homely according to a particular imaginary or fantasy recall the discussion of "nationalist managers" and homely space in disgrace in chap. . camagu has the most obviously ambiguous relationship with the community of qolorah as home and, indeed, with south africa. he doesn't feel at home in johannesburg, in part because he can't dance the toyi-toyi (the freedom dance of the struggle) because he was exiled for years and never learned it ( ), which results in his being excluded from a number of employment opportunities. in qolorha, he embraces his totem animal, a snake, in a way that impresses the villagers by showing them that he is like them and belongs. roman bartosch, through a citation of harry garuba's definition of "animist materialism," argues that camagu's respecting his totem animal is a "deliberate re-traditionalisation as described by garuba" and suggests that "he is calculating ('i have gained more respect […] since they saw i respect my customs')" (bartosch ) . while this is an interesting reading, it neglects to consider that camagu respects the snake first, and only later understands the influence it has on others' perceptions of him. a calculating gesture would suggest more deliberate and perhaps insincere performing of respect. camagu, by contrast, seems genuinely joyful to have encountered his totem animal: "camagu is beside himself with excitement. he had never been visited by majola, the brown mole snake that is the totem of his clan … . he is the chosen one today" ( ). where not knowing how to dance the toyi-toyi prevents camagu from belonging and imagining himself at home in the city, his ability to respect the customs of his people and his affective response enables a kind of assertion of belonging and imagining of a homeland in qolorha. the descendent of a colonial official, dalton too attempts a certain imagined relation to the community in denying this legacy. he replies "'it is not true! it is not true!' " to bhonco's point that "he is a descendant of headhunters" ( ). while he is a "white man of english stock," he has an "umxhosa heart" ( ), and zim argues that "dalton is not really white … it is just an aberration of his skin. he is more of an umxhosa than most of us" ( ). dalton's particular imagined relationship and belonging to the community, through knowledge of the language and participation in its rituals such as circumcision, performs a rhetoric of belonging that attempts to ignore or elide the colonial past. yet he still benefits from his position of white privilege in south africa in belonging to the "white" community, and even continues some racist practices against the villagers, as when he makes bhonco ride in the back of his truck, a common mode of segregated, racist travel practiced by white men during apartheid: "the old man [bhonco] struggles to climb into the back of the bakkie. even though dalton is alone in the front seat, customs do not die easily. dalton can see a hint of anger on the elder's face" ( ). in this sense, dalton imagines a relationship to both communities: the white nationalist community and the xhosa village. he also seeks to establish a connection to the land through his marriage to an afrikaner-the afrikaner "belongs to the soil. he is of africa"-and asserts to his friends who are leaving south africa: "this is my land. i belong here" ( ). these imagined relationships to land and community through certain kinds of belonging and nationalism often demonstrate the kind of willful cognitive dissonance that characters take on in order to feel at "home" as they navigate their relationships to the past, to the land, and to the community. the imagined relationships also reveal the violence that results from certain rhetorics of "home" as characters often seek to exclude or treat poorly those they deem not "pure enough" or foreign. foucault observes this violence in the racism of biopolitics: [r]acism makes it possible to establish a relationship between my life and the death of the other that is … a biological-type relationship: 'the more inferior species die out, the more abnormal individuals are eliminated, … the more ias species rather than individual-can live, the stronger i will be, the more vigorous i will be. i will be able to proliferate. the fact that the other dies does not mean simply that i live in the sense that his death guarantees my safety; the death of the other, the death of the bad race, of the inferior race (or the degenerate, or the abnormal) is something that will make life in general healthier: healthier and purer. ( ) foucault explains in this discussion that the death he's discussing is not just literal but also "indirect murder: the fact of exposing someone to death, increasing the risk of death for some people, or, quite simply, political death, expulsion, rejection, and so on" ( ). bhonco's seeking to expel certain "impure" characters from the community that thwart his ambitions of wealth and social climbing to an elite status through westernizing himself, the environment, and the community exhibit this kind of racist bipolitics. his attempt to get zim and qukezwa punished legally, weakening their political standing, and indeed his risking of the community's water and the lives of the animals through attempting to stop the environmentalists, evidences a violent biopolitics that continues in a racist, colonial legacy. camagu's presence in the village is obviously not a problem initially when bhonco welcomes him energetically as a westernized south african and because he thinks camagu might marry his daughter. as it becomes clear that camagu's desire is for qukezwa instead, bhonco argues that he and dalton should be expelled from the community for their not being "pure" amaxhosa or their not being born in qolorha. however, this logic of exclusion based on categories only suits his desire for "development" and what he thinks will be his personal benefit and profit. indeed, he often disavows the importance of local knowledge, allowing western notions of "development" and civilization to inform his thinking about biopolitical decisions. in building his case against qukezwa for destroying the water-depleting trees, he argues that it is unjust to absolve her when "white tourists" were recently arrested for "smuggling cycads" and boys were also punished "for killing the redwinged starling, the isomi bird" ( ). the elders reply to bhonco's argument by noting the difference of life under consideration here: "shall we now be required to teach revered elders like bhonco about our taboos? it is a sin to kill isomi. yes, boys love its delicious meat that tastes like chicken. but from the time we were young we were taught never to kill isomi … . we only desired them from a distance" ( , emphasis added). the traditional laws and words of the elders here express a desire for animals that respects their lives and membership in the community and results in the community protecting them from being eaten. the passage highlights the differing models of desire at work in qolorha as the indigenous notion of desire frustrates the view that capitalist desire is a universal or the only way to desire animals. this other way of desiring challenges the tourists and those who seek only a relation to the animals of eating, thereby showing capitalist desire to be but one way among other, different possibilities. the passage reveals that not only have the xhosa in the heart of redness long had their own environmental laws, as sewlall suggests, but also they've long had their own ways of desiring animals. in contrast to bhonco, qukezwa, evaluates newcomers to the community in relation to the harm they may cause or, alternatively, their ability to further protect and sustain the community. indeed, qukezwa works for the white dalton and begins a romantic relationship with the non-xhosa, camagu, that results in a hybrid child and future. bhonco's attempts to expel camagu and dalton from the community, as well as his approval of the sacrifice of the non-human life of qolorha for the casino city, suggests that his approach to the law relates to what derrida calls an autoimmunitary problem. wolfe writes about the need to deconstruct species and race as a way to prevent this problem: "[r]ace and species must, in turn, give way to their own deconstruction in favor of a more highly differentiated thinking of life in relation to biopower, if the immunitary is not to turn more or less automatically into the autoimmunitary" ( ). in other words, bhonco's use of these categories at times as a way to challenge the membership of these others in the community risks the sustainability of the community in the expelling of its diversity, especially considering that it is dalton and camagu who, with the assemblage of other community members, ultimately successfully resist and prevent the development of the casino city. wolfe discusses the desire for sameness that pertains to the law, and how derrida's notion of "hospitality" can challenge this problem in its ethical response to the other: [t] he reason that this [the ideal of unconditional hospitality] is crucial to biopolitical thought is that it keeps that zone of immunological protection from automatically turning to, as derrida puts it, an autoimmune disorder. the idea is that once you start drawing lines between humans and animals, aryans and jews, muslims and christians, that is always going to lead to the runaway train process of an autoimmune disorder. so eventually, you know, how aryan is aryan enough? how christian is christian enough? how human is human enough? how "proper," to go back to heidegger, is proper enough? the horizon of unconditional hospitality as something to strive for is precisely calculated to remind you that whatever those lines are that you are drawing have to be always taken under erasure, even as, pragmatically those lines have to be drawn and are drawn all the time. ("after animality" ) bhonco's desire to expel particular members out of the community then evidences this kind of autoimmune disorder as he accuses people of not being xhosa enough at times when it suits him and, at others, of not being civilized enough. in essence, bhonco seeks to improve himself, and to that end approves of the casino city, not recognizing its neocolonial nature, and thus advocating the sacrifice of the local land, animals, and other members of his community for the building of the casino. in contrast, the other characters consider sacrifice in terms of the protection of their community, including its human and non-human members. qukezwa responds differently to the biopolitical problems of protecting her community-with hospitality. wolfe quotes derrida's description of hospitality: "'pure and unconditional hospitality, hospitality itself, opens or is in advance open to someone who is neither expected nor invited, to whomever arrives as an absolutely foreign visitor, as a new arrival, nonidentifiable and unforeseeable, in short, wholly other'" (before the law ). rather than write off or automatically exclude foreigners from the community, qukezwa responds ethically to camagu and the other new arrivals to qolorha, including flora and fauna. to be sure, she does not welcome everything in an unconditional hospitality because to do so, as wolfe observes, would be to become apolitical and unethical: "[d]iscrimination, selection, self-reference, and exclusion cannot be avoided, and it is also why the refusal to take seriously the differences between different forms of life-bonobos versus sunflowers, let's say-as subjects of immunitary protection is, as they used to say in the s, a 'cop out'" ( - ). qukezwa clearly exercises discrimination in deciding which plants, animals, and people are welcome to be part of her community and which aren't, often on the basis of their colonizing or harming the life and futures of the other members of the community. toward the novel's end, the community stops the casino development when dalton gets a court order declaring qolorha a "national heritage site" ( ), something camagu had suggested earlier in response to the developers' "how will you stop us?" the location's history of nongqawuse's prophecies that led to the cattle-killing movement renders it worthy as a potential site deserving protection. as dalton protects the community in a roundabout legal way, mda's novel provides another example of how desire exists where we think there is law to protect communities. camagu and dalton, in their desire to protect the community, think that this alternative way to ensure the casino development will not go through. thus, in addition to qukezwa's ethical response to protecting her community, nongqawuse's prophecies, her desire to protect the community in thinking toward the future that led to the cattle-killing movement in the s in response to colonization that brought with it lung disease for the cattle, disease for the amaxhosa's crops, and the starvation of many of the amaxhosa, while tragic, continues to protect her people via the national heritage site enabling further sustainable futures. laura wright observes this as well: if there is any hope to be had, the polyphonic and temporally simultaneous structure of the text seems to suggest, it is in the cyclical and nonlinear nature of history wherein nongqawuse can be read at once as the cause of a people's destruction in the past and, through her cultural cachet as such a figure, their salvation in the present and as the girl whose failed prophecy in is fulfilled late in the twentieth century. ( ) although following the prophecy of nongqawuse was devastating for the amaxhosa, mda offers it as an ethical attempt to bolster the immunity of the community. j.b. peires explains the biological conditions that resulted from the colonial contact and led to the cattle killing: an important cause of the cattle-killing was the lungsickness epidemic which reached xhosaland in . cattle mortality was as high as one half to two thirds in some places, and many xhosa lost all their cattle. the great believer chief phatho, for example, lost percent of his cattle … the xhosa began to believe that their cattle were rotten and impure, and that they might as well kill them since they were probably going to die anyway. ( ) in this light, part of the logic that informs the movement is the attempt to limit the spread of the disease, to prevent it from attacking all the cattle. another aspect of the logic of sacrificing the cattle involved the hope that it would remove the british colonists: cattle-killing was born partly out of xhosa frustration at colonial domination and partly out of the hope awakened by the news that the russians had beaten the english … . among the many predictions that circulated at the time was one to the effect that the english, like all other evil things, would be swept away in the great storm which would precede the resurrection of the dead. (peires ) peires also explains that, alternatively, many also believed the sacrifice would amend the conflicts and bring peace between the british and the amaxhosa nation. perhaps the greatest reason for the british "success" in colonizing the amaxhosa, then, is this cattle disease that "was brought to south africa in september by a dutch ship carrying friesland bulls" (peires ) as it functioned essentially as a kind of biological weapon in the war between the british and the amaxhosa. the slaughtering of the cattle in an attempt to prevent the spread of this disease is an exercise in biopolitics. as wolfe notes in critiquing affirmative biopolitics that fail to consider the difference of life-those approaches that view all life as equal-such an approach isn't practical (or perhaps desirable) given the destructive nature of some life forms: "[d]o we extend 'unconditional hospitality' to anthrax and ebola virus, to sars?" (before the law ). in the heart of redness, environmentalist characters often perform a biopolitics and hospitality that is not always affirmative but discriminates and seeks to eliminate the lung sickness, water-depleting plants, and neocolonial developments that would decimate the community. in the face of such an attack on the immune system of the community, king sarhili's decision to follow the words of the prophetess exercises an ethical attempt to protect and sustain the community. of course, as history bears out, the situation and decision were devastating: one of the figures offered for the drop in the human population of the xhosa people from the cattle killing is an estimated loss of , over a twoyear period. also, , cattle were slaughtered and the xhosa lost , acres of land to the british (peires ). these tragic figures highlight the vulnerability of bodies that humans and animals share. they also point to the great dependence of the community on these non-human others for survival. emphasizing our limited knowledge, and that to act at all we can only respond with a conditional hospitality, wolfe writes of biopolitics: we must choose, and by definition we cannot choose everyone and everything at once. but this is precisely what ensures that, in the future, we will have been wrong. our 'determinate' act of justice now will have been shown to be too determinate, revealed to have left someone or something out. ( ) while the cattle killing may appear to have been "wrong" from the perspective of today as it can be seen as being responsible in part for the devastation of the amaxhosa community-peires notes that some view it as a mass suicide, although he clearly disagrees with this view-in light of the biopolitical decisions exercised by qukezwa in the present of the heart of redness, mda situates the movement as an attempt to act politically and ethically to protect the community. mda's novel emphasizes, as peires also argues, that the tragic deaths of the amaxhosa community were caused by the colonizing disease of lung sickness as well as the colonizing british, both of which established a relationship of "eating" these communities. just like the development project of the casino city, which attempts to colonize and devastate the community, the british colonization during the cattle-killing movement was geared towards ruining the amaxhosa nation: the cattle-killing cannot be divorced from the colonial situation which was imposed on the xhosa in by sir harry smith … it should be remembered that the essential objectives of grey [sir george grey] were identical to those of smith and of colonial rule generally: to destroy the political and economic independence of the xhosa, to bring them under british law and administration, to make their land and their labor available to white settlers, and to reshape their religious and cultural institutions on european and christian models. (peires ) peires' passage describes an organization of labor and the community that would be replicated in a slightly new form by the international development company's proposal. yet by deterritorializing desire away from the narcissism of colonization and global capitalism toward a postcolonial desire, one for the benefit of the members of the community, qukezwa and the others of the assemblage, including camagu and dalton, work to fend off the disposal of the community by an international development corporation, exercising an ethical biopolitics that continues in the tradition of the prophetess, and, for the time being, she temporarily secures her community's protection and its potential for a future. in addition to the government officials who support the casino in the heart of redness, chief xikixa, who is charged with protecting the land where it "is illegal to build within a kilometer of the coast," also puts his own profits and pleasure ahead of the community. he gives the land away to wealthy white people and "some well-to-do blacks," some of whom build "right on the seashore," for bribes such as a "bottle of brandy" and later on "cellphones and satellite dishes" ( ). these products of globalized capitalism bring the village of qolorha into the global economy, threatening the life and future of the community as it is rendered exchangeable for a cheap price, and for the pleasure and profit of a few. in both heyns and mda's novels, the commodification of the lands of local communities and the disposing or harming of the interests of their residents, human and non-human, are thwarted through what i'm describing as the postcolonial desire of an assemblage of characters, a desire for the community in a broad sense that disrupts and resists the colonizing ambitions of capitalism and its adherents. as braidotti argues, in the context of biotechnology, zoe exceeds capitalist control: "nature is more than the sum of its marketable appropriations: it is also an agent that remains beyond the reach of domestication and commodification" (transpositions ). if capitalism seeks to commodify all of life and proliferate its logic of desire of lack through colonizing the unconscious, postcolonial desire describes a potential of desiring machines that escapes, resists, or undoes this colonization in a line of flight away from capitalism's reterritorialization of desire, reassembling communities around an ethics of sustainability instead of an accumulation of personal profit. where some global capitalists see an opportunity in the "under-developed" lands or villages to secure a profit at the expense of destroying and devastating its inhabitants, the resistances offered by these human and non-human assemblages disrupt the capitalist machine and, for the time being, protect the futures of their homes. in both the reluctant passenger and the heart of redness, the laws passed to protect these communities repeatedly fail to do so, often as a result of the compromised position of those tasked with upholding the law. while these authorities, judges, and politicians seek their own profit at the expense of the community, they reveal their colonization by capitalist logic. through weaving the history of the cattle-killing movement into the narrative about the present-day proposed casino city, zakes mda emphasizes the colonial legacy in which global capitalism operates and continues in the practices of capitalists and government officials from developed countries and their counterparts in "developing nations." heyns' novel focuses more specifically on protecting a troop of baboons instead of protecting a larger community of human and non-humans, as in mda's novel. nonetheless, the critique of the capitalist development of the baboon's home in the nature preserve also undermines the workings of the apartheid regime more broadly, in this case the animal testing and other exercises in biopower of the apartheid state on south african bodies. for both novelists, then, the explicit violence exerted on communities, on bodies, and on the land in the pasts of colonialism and apartheid reappears in a subtler and more nuanced fashion in the present of late capitalism through the weakened and compromised administration of the law which fails to fully protect communities from the threats of disposal and violence. mda and heyns represent desire as a resistance to this neocolonial threat that might better protect these communities and their futures from being consumed by the capitalist machine. notes . see jacques derrida, "before the law," in acts of literature, ed. derek attridge (new york, ny: routledge, ), - . derrida also emphasizes this emptiness in noting the lack of an origin for the law in "before the law:" "what is deferred forever till death is entry into the law itself, which is nothing other than that which dictates the delay … what must not and cannot be approached is the origin of difference: it must not be presented or represented and above all not penetrated. that is the law of the law, the process of a law whose subject we can never say, 'there it is,' it is here or there" ( peires. in the dead will arise, peires details how although extremely tragic for the amaxhosa, nongqawuse's prophecies and the decision to slaughter cattle resulted from extreme circumstances of british colonization and disease. the slaughter of most of the amaxhosa cattle, then, offers an example of a biopolitical issue. for example, peires writes "that the cattle-killing was a logical and rational response, perhaps even an inevitable response, by a nation driven to desperation by pressures that people today can barely imagine. i further believe, and i trust that the book will demonstrate this too, that the cattle-killing would not have been so fatal an error had it not been for the measures of governor grey, which first encouraged and then capitalized on the movement" (x). the unfortunate coincidence of increased pressure from british colonization and the lung the amaxhosa's decision to slaughter their cattle as a way to preserve their remaining community. as peires and mda's novel bears out, the decision to slaughter the cattle after the telling of nongqawuse's prophecies therefore figured as an attempt to secure the futurity of the amaxhosa people, even while it ultimately caused so many of them to die their alternative proposals prevent the immediate "development" of the casino, which would surely spell the ruin of the community, rendering it unsustainable. mda seems to acknowledge that this approach of ecotourism is not exactly the most radical approach either, as his later novel, the whale caller what do human rights do? an anthropological enquiry environmentality: ecocriticism and the event of postcolonial fiction what is sex? an introduction to the sexual philosophy of gilles deleuze nomadic subjects: embodiment and sexual difference in contemporary feminist theory after animality, before the law: interview with cary wolfe left legalism/left critique in place: tourism, cosmopolitan bioregionalism, and zakes mda's heart of redness indigenous cosmopolitics in the andes: conceptual reflections beyond 'politics kafka: toward a minor literature. translated by dana polan before the law without offending humans: a critique of animal rights march lecture explorations in animist materialism: notes on reading/writing african literature, culture, and society the problem of ideology: marxism without guarantees the reluctant passenger. jeppestown: jonathan ball postcolonial ecocriticism: literature, animals, environment mda, zakes. fools, bells and the habit of eating: three satires mda, zakes. the whale caller in african literature: an anthology of criticism and theory the dead will arise: nongqawuse and the great xhosa cattle-killing movement of - animal rights in south africa. cape town: double storey books ecofeminism in zakes mda's the heart of redness situating ecology in recent south african fiction: j.m. coetzee's 'the lives of animals' and zakes mda's 'the heart of redness to hear the variety of discourses before the law: humans and other animals in a biopolitical frame the animal gaze: animal subjectivities in southern african narratives wilderness into civilized shapes: reading the postcolonial environment the politics of necessity: community organizing and democracy in south africa key: cord- -y vsc r authors: peiffer, robert l.; armstrong, joseph r.; johnson, philip t. title: animals in ophthalmic research: concepts and methodologies date: - - journal: methods of animal experimentation doi: . /b - - - - . - sha: doc_id: cord_uid: y vsc r nan the component tissues of the eye are both individually and collectively unique and fascinating to both clinician and researcher. we find in a single organ a variety of complex tissues each with its own particular vascular relationships, from the avascular cornea and lens to the extensively vascularized uvea. charac teristic biochemical features of the tissues exist as well, including the endothelial pump of the cornea, the anaerobic glycolytic pathway of the lens, and the poorly defined transport systems of aqueous humor production and outflow. besides the dynamic aqueous, we find the most substantial tissue of the eye, the vitreous humor, to be apparently a relatively stagnant blob of hydrated connective tissue whose physiology is still largely a mystery. the neurologic control mechanisms of such diverse processes as pupillary size, accommodation, and probably aque ous humor inflow and outflow add another dimension of potential inquiry. the eye is devoid of lymphatics, and the nature of its tissues further contributes to its immunologie uniqueness. the retina incorporates all the complexities of neuroperception and transmission. nowhere else in the body are such diverse structure and function so intimately and intricately related. an appreciation of these perspectives, the importance of vision as a cognitive sense, and consideration of the plethora of infectious, inflammatory, degenera tive, traumatic, toxic, nutritional, and neoplastic diseases that can affect any one or all of the component tissues of the eye, will elucidate the challenge the authors face in attempting to review pertinent conceptual and logistical approaches to eye research in the animal laboratory. while the majority of investigations have had as their objective ultimate correlation with normal and abnormal function and structure of the human eye, laboratory studies have provided an abundance of comparative information that emphasizes that while there are numerous and amazing similarities in the peripheral visual system among the vertebrate (and even the invertebrate) animals, significant differences exist that are important to both researcher and clinician in selection of a research model and in extrapolation of data obtained from one species to another, and even among different species subdivisions. details of comparative anatomy and physiology have been reviewed by several authors (prince, (prince, , a polyak, ; duke-elder, ; prince et al., ; walls, ) . these are classic works and invaluable references. one is reminded that "research" means "to look for again," and while some concepts presented in these earlier texts are indeed outdated, and some facts since disproven, phylogenetic perspectives acquired from familiarity with this literature arms the scientist with valuable information. throughout this chapter, we will dwell on species anatomic and physiologic differences when they have been specifically defined or where they are of value in regard to specific laboratory methodologies. while the subhuman primate is frequently described as the ideal laboratory model for eye research, many of these animals are threatened or endangered, and logistical aspects of procurement, maintenance, and restraint should en courage investigators to explore and define the validity of nonprimate animals. when deemed essential, primates should be utilized with maximum thrift and humane care in mind. the eye is a sensitive organ, and in all species adequate anesthesia for man ipulative procedures as well as postoperative analgesia should be considered in in vivo studies; the rabbit, for instance, is extremely sensitive to barbiturate anesthesia and prolonged procedures that require intraocular manipulations pose a challenging problem in terms of subject mortality. the in vitro culture of ocular tissue cell lines offers increasing potential in research as an alternative to animal models. the lens, retinal pigment epithelium, and corneal epithelium and fibrocytes may be readily maintained and manipu lated. current work with the corneal and trabecular endothelium is perhaps the most exciting advance in ophthalmic research in recent years. in vitro culture techniques offer a number of logistical advantages over animal models. a knowledge of spontaneous ocular diseases is essential to avoid interpreting pathology as experimental that may well be unrelated to the investigation. thorough preoperative ophthalmic examination is a prerequisite of any study, and the laboratory scientist will find it to his benefit to be associated with an interested human or veterinary clinical ophthalmologist to assist in defining and managing spontaneous ocular disease. for instance, electrophysiologic studies of the canine retina will be invalid if the subject has chorioretinal lesions of canine distemper, a common entity in laboratory dogs. examination beyond the target organ is also recommended to detect abnormalities that may directly or indirectly influence results. frequently encountered spontaneous infectious diseases are reviewed in a subsequent section. we have limited our discussions primarily to research methodologies involv ing the globe itself; a paucity of information is available regarding the adnexa, orbit, and extraocular muscles, and described techniques are refinements of general approaches rather than those specifically developed for ocular research. in the same perspective, topics of pure physiology or biochemistry are limited, and we did not embark into the unfamiliar and complex area of central visual mechanisms. our approach, we hope, if not all inclusive, has been comprehen sive, with adequate references to specific detail and additional information for the interested reader. spontaneous infectious diseases that involve the eyes of laboratory animals are significantly prevalent to warrent brief discussion. it is important to consider these entities, especially before using animals derived from poorly defined sources. propensity of a species toward the development of spontaneous infecti-ous diseases that might not only affect general health but also induce ocular pathology in both experimental and control subjects may affect selection of a model of procurement and isolation procedures utilized. preinvestigation ocular examinations are necessary to detect preexisting pathology. of the three large species used in ophthalmic research (cat, dog, and primate) , the primate appears to have a minimal predisposition for spontaneous infectious ocular disease. this may be a result, however, of a lack of ophthalmic examina tion in disease situations. it is also important to note that the hamster and guinea pig rarely develop eye infections. table i summarizes those infectious diseases that more commonly involve the eyes of laboratory species. the use of laboratory animals in the investigation of infectious ocular disease has included rats, hamsters, guinea pigs, rabbits, cats, dogs, and subhuman primates. the disease agents studied have ranged from viruses to protozoa, with the rabbit serving as the most popular model. these studies have been of value in defining pathogenesis and the effects of therapeutic agents. those disease agents known to cause conjunctivitis in man do not necessarily produce a similar disease in laboratory species. a particular species may require immunosuppression to allow the induction of a specific infection (payne et al., ; forster and rebell, ) . in other instances, a model may be specifically susceptible to an organism; for example, the chlamydial inclusion body con junctivitis agent in guinea pigs. an additional and somewhat unusual use of the guinea pig involves the sereny test (formal et al., ) . because their conjunctivae are so susceptible to shigella organisms, this animal, through the use of a drip apparatus, has been used to identify the pathogenic strains (mackel et al., ) . listeria monocytogenes produces a severe keratoconjunctivitis in the guinea pig, but in the rabbit and monkey the disease is much milder and more difficult to reproduce (morris and julianelle, ) . this concept has lent itself to studies concerning epithelial cell phagocytosis in the guinea pig (zimianski et al., ) . organism installation techniques involve either direct swabbing or syringe inoculation into the conjunctival sac. the owl monkey (actus trivirgatus) has proved to be the model of choice for the study of chlamydia trachomatis conjunctivitis; other primate species includ ing orangutans have proved to be only mildly susceptible (fraser, ) . a number of models are available for investigators interested in studying keratitis. the rabbit has proved to be the most feasible subject, especially in herpes simplex studies (kaufman and maloney, ) . many bacterial and fun gal diseases afflicting man have also been examined in this model, including the effects of antibiotics, steroids, interferon, vitamins, and other agents nesburn and ziniti, ; smolin et al., ; pollikoff et al., ) . herpesvirus studies can be significantly influenced by the particular strain of virus utilized. for example, some are more effective in producing stromal keratidities as compared to the epithelial condition (metcalf et al., ) . con comitant in importance may be the route of inoculation. in some studies viral suspensions are placed into the conjunctival sac (nesburn and ziniti, ) , while in others they are dropped onto the cornea . corneal preparation may be important; for example, some studies have employed chalazion currette (stern and stock, ) , while others have employed spatula scrap ing or circular trephine defects (kaufman and maloney, ) . deep inoculations are required to produce a stromal keratitis (fig. ) , while the epithelial disease may be induced through shallow scratches (polli koff et al., ) . the rabbit appears to be the species of choice for bacterial keratitis, although davis et al. ( ) feel that the guinea pig is of equal value. in one study an inbred animal was used (strain ) to reduce the inherent variability seen in some of these experiments (davis and chandler, ) . whether or not the rabbit model has any specific biologic advantage, the guinea pig would appear to be more feasible because of its smaller size, ease of handling, ease to anesthetize, cost of purchase and maintenance, and, perhaps most significantly, resistance to spontaneous pasteur ella conjunctivitis, which frequently occurs in laboratory rabbits. methods of bacterial inoculation usually involve an intracorneal injec tion using a microsyringe and small volumes of quantitated microorganisms. fungal keratidities have been studied in rats, rabbits, and owl monkeys. rats were initially popular (burda and fisher, ) , but rabbits and monkeys have become the select species. fusarium solarti keratitis was first studied in the subhuman primate, but, due to expense, forster and rebell ( ) utilized the pigmented rabbit. this model, having received germinating conidia (as opposed to spores) interlamellarly, developed a sustained progressive infection, but only after pretreatment with subconjunctival steroids. candida albicans keratitis has also been induced in the corticosteroid pretreated rabbit . inoculation techniques for these organisms are identical to those described for bacterial and viral keratidities. ishibashi ( ) suggests that backward flow from the inoculation wound and volume accuracy can be controlled by using a microsyringe and a -gauge flat-faced needle. recently, human corneal transplants have been incriminated in the transmis sion of rabies to recipients. a model has been developed in the guinea pig utilizing the slow virus agent of creutzfeldt-jacob disease (manuelidis et al., ) . using a method developed by greene ( ) , this study involved the heterologous transplantation of infected guinea pig cornea sections into the an terior chamber of six recipients with resultant development of spongiform encephalopathy. studies designed to produce anterior uveitis usually employ inoculation into the anterior chamber. this procedure is best performed with a -to -gauge needle directed obliquely through the limbus into the chamber (smith and singer, ) . ocular histoplasmosis was effectively studied in the rabbit using this method, the result being a well-defined granulomatous uveitis. the same proce dure may be applied to produce bacterial or viral anterior uveitis (kaufman et al., ) . it is advisable, however, to withdraw initially an equivalent amount of aqueous fluid to avoid intraocular pressure elevation (zaitseva et al., ) . aguirre et al. ( ) administered canine adenovirus type i intravenously to young beagles to produce the anterior uveitis characteristic of infectious canine hepatitis. carmichael et al. ( ) demonstrated that this was a complementmediated antigen-antibody complex disease. the induction of endophthalmitis may utilize direct transcorneal chamber in oculation as described above; this method was used in rabbits to produce a bacterial infection in order to assess diagnostic anterior chamber paracentesis technique (tucker and forster, ) . the same procedure was also used in the rat to create a fulminating fusarium solarti endophthalmitis (o'day et al., ) . the intravitreal route of inoculation is utilized to induce posterior segment infection. this technique requires toothed forcep stabilization of the eye and needle introduction through the pars plana (may et al., ) . indirect ophthalmoscopy may be employed to ensure accurate deposition. typically a tuberculin syringe with a /s-inch, -gauge needle is used with the subject under general anesthesia or deep tranquilization. the needle is inserted at o'clock and directed toward the posterior pole. small volumes may be injected without signif icant alteration in the intraocular pressure. avallone et al. ( ) used a similar technique in rabbits to demonstrate the effectiveness of the limulus lysate test for rapid detection of e. coli endophthalmitis. after producing a staphylococcol endophthalmitis by this method, michelson and nozik ( ) tested the perfor mance of a subcutaneously implanted minipump for antibiotic administration in the rabbit. ocular toxocariasis, a human disease caused by larval migration of toxocara canis, was produced in mice by olson et al. ( ) . freshly incubated ova were administered per os by stomach tube. anterior chamber hemorrhage and actual sightings of larval migration were reported. hobbs et al. ( ) found ocular mycobacterium leprae and m. lepraemurium in the nine-banded armadillo and mouse, respectively, following intravenous, intraperitoneal, or foot-pad inoculation of the organisms; the uveal tract was primarily involved, although lesions were observed in other ocular tissues. lesions were more dramatic in mice immunologically depressed by thymectomy and total body irradiation. dur to its susceptibility to toxoplasma gondii, the rabbit has been developed as a model for ocular toxoplasmosis. nozik and o'connor ( ) used the california pigmented rabbit and a variation of a method described by vogel ( ) to study the associated retinochoroiditis. this technique consists of prop osing the eye of an anesthetized rabbit and passing the needle retrobulbarly through the sclera to the suprachoroidal space (fig. ) . the authors point out that while the lesions are less active and heal spontaneously in weeks, features are of comparative value. for detailed information concerning other uses of this model the reader is referred to tabbara's organism recoverability experiments (tabbara et al., ) . histoplasmosis has been studied in mice, rats, rabbits, guinea pigs, dogs, pigeons, chickens, and primates. smith et al. ( ) point out that because it is essentially a macular disease in man, the ideal model should have similar anatomy. basically two orders of animals qualify: the avian species due to their dual fovea and the primates. the above authors used the stumptailed macaque, m. arctoides, to produce a focal choroiditis by inoculating yeast-phase histoplasma capsulatum into the internal carotid artery. they emphasized the desirabil ity to create a model that, in addition to focal choroiditis, also exhibits minimal involvement of the retina, no anterior segment disease, and a late macular lesion. experimental ocular cryptococcosis has been induced in rabbits, primates and cats, the latter species appearing to be the ideal choice because of its susceptibil ity and related high incidence of chorioretinal lesions. blouin and cello ( ) used an intracarotid injection technique which reproduced lesions identical to those seen in the naturally occurring disease. a septic choroiditis of bacterial origin was produced by meyers et al. ( ) by intracarotid injection of staphlococcal and streptococcal organisms. this in turn produced a serous retinal detachment, which was the primary objective of the study. animal studies have provided an abundance of information dealing with the pharmacodynamics of the eye and drugs used to treat ocular disease. because of the accessibility of the ocular structures, there are a number of routes of drug administration which are not available in treating other organ systems. on the other hand, there are barriers to drug penetration in the eye which must be considered, including a hydrophobic corneal epithelial layer over the external ocular structures and an internal blood-ocular barrier similar to the blood-brain barrier. the endothelial cells of the iris and retinal vessels, the nonpigmented epithelium of the ciliary body and the retinal pigment epithelium all contribute to this blood-ocular barrier (cunha-vaz, ) . various agents can compromise these barriers to drug penetration. inflamma tion can also affect any of the ocular barriers. in some experimental designs it may be desirable to create inflammation and/or minimize the barriers. tech niques include mechanical removal of the corneal epithelium with a scalpel blade, inducing infectious keratitis or treating the external eye with enzymes (barza et al., ) . a more generalized inflammation can be created by sen sitizing the animal to a specific antigen and then challenging the eye with that antigen (levine and aronson, ) . certain pharmacologie agents affect spe cific barriers; anticholinesterase inhibitors, for instance, can break down the blood aqueous barrier (von sallmann and dillon, ) , while agents with epithelial toxicity, such as benzalkonium chloride, are added to many topical preparations to increase the permeability of the corneal and conjunctival epithelium. all these factors regarding method of administration and barrier permeability must be considered in an experimental design. once again the rabbit has been most frequently chosen for pharmacologie experiments on the eye. although at first glance the anatomy of the rabbit eye would seem close,enough to the human to make this a valid experimental model, at least for drug penetration studies, significant differences do exist, as will be pointed out. drugs can usually be applied topically to the eye in concentrations much higher than could be tolerated systemically. topical application has many limita tions, however. if a topical drug is to be effective intraocularly it must traverse the hydrophobic epithelial layer and the hydrophilic corneal stroma; ideally, then, it should exist in an equilibrium between ionized and unionized forms with biphasic solubility characteristics. thus, both chemical configuration and ph of the vehicle are important. other important limitations are the small tear volume and rapid clearance of drugs from the tear film (janes and stiles, ) . rabbits tend to blink infrequently; consequently corneal contact time is prolonged com pared to humans. most nonprimate mammals possess a nictitating membrane which may affect tear dynamics. drugs may be applied with an ointment vehicle to prolong contact time, but bioavailability may be affected. finally, the position of the animal may be important. a tenfold difference in aqueous drug levels in rabbits has been reported depending on whether the animal was upright or re cumbent during the experiment (sieg and robinson, ) . injection of a drug under the conjunctiva or tenon's capsule theoretically allows delivery of greater amounts of drug, prolonged contact time, and bypass of the external epithelial barrier. subconjunctival injection may be given in two ways; by ( ) injecting directly through the bulbar conjunctiva or ( ) by inserting the needle through the skin of the lids, leaving the conjunctiva intact. the method of injection may be very important; one study showed that in rabbits leakage of hydrocortisone back through the injection site into the tear film ac counted for most of the intraocular absorption (wine et al., ) . although numerous articles have been written on the kinetics of drug absorption after subconjunctival injection (baum, ) , the subject is still controversial, and recent evidence presented by maurice and ota ( ) indicates that absorption into the anterior chamber by this route may be much lower in rabbits than in man. if so, the rabbit may be an inappropriate model for this type of research. the potential for serious complications makes intraocular injection of drugs a 'last resort" option in clinical practice. in many cases the maximum amount of drug tolerated within the eye is small (leopold, ) . good results have been reported, however, in the treatment of experimental bacterial endophthalmitis in rabbits with intravitreal injection of antibiotics (von sallmann et al., ; peyman, ) . usually . - . ml of antibiotic solution is injected into the nucleus of the vitreous through the pars plana. intramuscular and intravenous are the preferred routes for systemic administra tion of drugs in most laboratory animals. drugs may be given orally in food, water, or by stomach tube, or more reliably and precisely with pills or capsules in the case of cats and dogs. whatever the systemic route, intraocular levels are limited by the body's tolerance for the drug and by the blood-ocular barriers. one method of obtaining high ocular levels is arterial infusion, usually of the ipsilateral carotid artery. the highest levels have been obtained experimentally by retrograde perfusion of the intraorbial artery in dogs; details of the technique are discussed by o'rourke et al. ( ) . in general the assessment of penetration into the various intraocular compart ments is carried out using radio-labeled preparations of the drugs under study. the labeled drug is administered, and after a predetermined length of time samples of aqueous are removed by paracentesis or the whole eye is removed and standard size tissue samples taken for scintillation counting. o' brien and edelhouser ( ) utilized a direct chamber perfusion technique to study penetra tion of radio-labeled antiviral drugs through the excised cornea. in the case of antimicrobials, drug levels in ocular tissues may also be determined using a microbioassay such as the agar diffusion technique described by simon and yin ( ) . other methods have been used less commonly. autoradiography has been used to assess intraocular penetration (mccartney et al., ) , and distribution in the orbital compartments has been studied using diatrizoate, a radio-opaque contrast media (levine and aronson, ) . techniques used to assess drug efficacy depend primarily upon the type of drug under study. most drugs used in the treatment of glaucoma, for instance, work through complicated autonomie mechanisms which alter the inflow and outflow of aqueous humor. tonography, tonometry, and fluorophotometry are used to study these effects and are discussed elsewhere. methods used to study antibiotic efficacy, on the other hand, are usually less objective. results are often based on semiquantitative or subjective impressions of clinical response. actual bacterial counts on samples of tissue following treatment of experimental infec tions are feasible, but the organism and size of original inoculum must be strictly defined (kupferman and leibowitz, ) . steroid efficacy is even more difficult to assess objectively in experimental models. leibowitz et al. ( ) have reported a method of quantifying steroid response based on the number of polymorphonuclear leukocytes labeled with tritiated thymidine remaining in the tissue following treatment of inflammatory keratitis. successful organ and cell culture has been reported with a variety of ocular tissues, including whole eyes. the culture of lens, cornea, and corneal endothelium have become routine techniques in many ophthalmic laboratories. paul ( ) provides a good manual of basic tissue culture techniques. the early work in culture of ocular tissue is the subject of an excellent review by lucas ( ) . a number of significant advances have been made since that time, especially in the areas of retinal pigment epithelial and cornea culture. suspended between the aqueous and vitreous humors with no direct vascular supply, the lens is maintained in a type of natural organ culture. it has always been felt, therefore, that the lens should be ideal for in vitro culture, and lens culture experiments have been utilized extensively to study the metabolism of this tissue. a great deal of research into ideal media and culture conditions has been necessary, however. rabbit, mouse, and rat lenses have been most widely used, but culture of bovine lenses is also feasible despite their large size (owens and duncan, ) . the lens may be cultured in an open (continuous perfusion) or closed system. the advantages and disadvantages of each type of system are discussed by schwartz ( a) . closed systems patterned after that described by merriam and kinsey ( ) are by far the most popular, but for certain types of experiments more elaborate perfusion systems are necessary (schwartz, b; sippel, ) . the composition of the media for lens culture has been found to be very important. kinsey et al. ( ) have provided systematic studies on the optimum concentration of various culture media constituents. presently most lens culture is being done in a modified tci media which has been used by kinoshita and others with good success (von sallmann and grimes, ) . thoft and kinoshita ( ) have found that a calcium concentration somewhat higher than that in the aqueous is desirable in the culture media. more recently, chylack and kinoshita ( ) have shown that removal of the lens with part of the vitreous still attached to the posterior surface improves certain parameters of lens function in vitro. presumably, leaving vitreous attached to the lens prevents damage to the pos terior capsule. in addition to culture of whole lenses, cell cultures of pure lens epithelium have been important in the study of lens metabolism. most of the early work was done with chick epithelium, but more recently cultures of mouse lens epithelium have been shown to retain greater cellular differentation in culture (mann, ; russell et al., ) . three different cell types make up the layers of the cornea: epithelial cells, fibrocytic stromal cells, and a single layer of endothelial (mesothelial) cells. all three corneal cell types, as well as whole corneas, can be maintained in culture. the culture of whole corneas has been investigated as a means of corneal preser vation prior to transplantation. most of these studies have used human rather than animal tissues. endothelial cell culture has become an important technique in the study of this very metabolic ly active cell layer. stocker et al. ( ) described a technique for separating a corneal button by carefully peeling off descemet's membrane with endothelium from the posterior surface and peeling the epithelium from the opposite side to yield three relatively pure cell types which can be explanted onto separate cultures (fig. ). colosi and yanoff ( ) have used trypsin to obtain endothelial cells or epithelial cells from whole corneas. perlman and baum ( ) have used stacker's method to isolate endothelial cells from rabbits and have had good success in maintaining large endothelial cell cultures for several months using a modified eagle's mem supplemented with calf serum, bicarbo nate, glutamine, and kanamycin sulfate. most exciting are the reports of using tissue cultured endothelial cells, seeded on to donor corneas with endothelium removed, for transplantation in rabbits (jumblatt et al., ) . although the corneal stromal cells are the most thoroughly studied ocular fibrocytic cells, other fibrocytes have been successfully cultured, including goniocytes from the anterior chamber angle (francois, ) and hyalocytes from the vitreous (françois et al., ) . tissue culture of immature neural retina has been a popular subject. early experiments are summarized by lucas ( ) . recently, differentation of em bryonic neural retinal cells from the chick has been described both in cell aggre gate cultures (sheffield and moscona, ) and monolayer cultures (combes et al., ) . organ culture of mature neural retina has proved more difficult. mature retina tends to deteriorate rapidly in a convential warburg apparatus (lucas, ) . the organ culture technique of trowell ( ) has been used to maintain mature and nearly mature retina for several days (lucas, ) . this technique requires mechanical support of the retina on a metal grid with receptor cells uppermost, exposed to the gas phase; in this case air as % oxygen was found to be toxic. ames and hastings ( ) described a technique for rapid removal of the rabbit retina, together with a stump of optic nerve, for use in short-term culture experi ments including in vitro studies of retinal response to light (ames and gurian, ) . until recently only a few studies were available on the behavior of retinal pigment epithelium (rpe) cells in culture, but recent work on the multipotential nature of the rpe cell has aroused new interest in this area. as with neural retina, chick embryos have been most often used for rpe cultures. hayashi et al. ( ) used edta and trypsin to isolate chick rpe cells for culture on eagles mem supplemented with % fetal calf serum. a similar technique has been used to culture rpe cells from syrian hamsters (albert et al., a) . mandel-corn et al. ( ) have reported an ingenious experiment in which rpe cells from one eye of an owl monkey were transplanted into the vitreous of the fellow eye through the pars plana where the proliferation and metaplasia of those cells could be studied. the experiment described above is not the first time that investigators have attempted to take advantage of the eye's transparent media and large avascular spaces to create a type of in vivo tissue culture. many successes have been reported in transplanting homologous and autologous tissues into the anterior chamber. markee ( ) transplanted endometrium into the anterior chamber of guinea pigs, rabbits, and monkeys where it continued to undergo cyclic changes. goodman ( ) also reported ovulatory cycles of ovaries transplanted into the anterior chamber of rats. woodruff and woodruff ( ) successfully trans planted homologous thyroid tissue into the anterior chamber of guinea pigs. eifrig and prendergast ( ) found that autologous transplants of lymph node tissue in rabbits were well tolerated. a variety of embryonic tissues, with the exception of liver, have been successfully transplanted into the anterior chamber of rabbits (greene, ) . heterologous transplants of tissue from one species to another have more often than not been unsuccessful. greene ( ) reported good results in transplanting malignant tumors into the anterior chamber of different species. he observed that transplants from human to guinea pig and rabbit to mouse worked best. greene's technique of transplantation involves introducing a small piece of donor tissue in a small canula fitted with a stylet through a limbal incision and firmly implanting the tissue into the anterior chamber angle opposite the incision. morris et al. ( ) reported disappointing results using this technique for heterologous tumor transplants in guinea pigs, but obtained better results by transplanting the tissue into the lens beneath the lens capsule. the reader is referred to woodruff ( ) for further discussion of intraocular transplantation experiments. the eye is unique in that it is devoid of lymphatics and thus has no directly associated lymph nodes. classic animal studies have been utilized to define ocu lar mechanisms of immune response and have demonstrated that the uveal and limbal tissues play a role similar to that of secondary lymphoid tissue elsewhere in the body. bursuk ( ) injected typhoid bacilli and staphylococci into the cornea of rabbits and found that agglutinins and opsonins appeared in the corneal tissues earlier and in greater concentration than in the serum, suggesting local respon siveness. thompson and co-workers ( ) obtained identical results using crys talline egg albumin as the antigen, and further showed that when only one cornea of each animal was injected precipitating antibody could not be detected in the contralateral uninjected cornea. rabbit experiments in which ovalbumin was injected into the right cornea and human serum albumin was injected into the left cornea demonstrated that the respective cornea contained antibodies only to the antigen they had received and not to the antigen present in the contralateral cornea; specific antigen was detectable within days (thompson and olsen, ) . using a'fluorescent antibody technique, witmer ( ) demonstrated immunoglobulin cells in the uvea of the rabbit, and wolkowicz et al. ( ) showed that excised, sensitized uveal tissue in short-term culture would actively produce specific antibody. lymphoid cells migrate to the eye in the presence of antigenic stimulus. thus, x irradiation of the eyes of a rabbit does not impair the ability of either the limbal tissues or the uvea to form antibody, whereas irradiation of the peripheral lym phoid system does (thompson and harrison, ; silverstein, ) . when the primary ocular response to an antigenic challenge has subsided, sensitized 'memory" lymphocytes may persist in the uvea or limbal tissues, since a later exposure to the same antigen introduced at a distant site or injected directly into the circulation results in renewed antibody production within the eye (pribnow and hall, ; silverstein, ) . the dog, cat and monkey have all been used as animal models in cornea research. the rabbit, however, has been exploited to such a degree for this purpose that a few comments on the rabbit cornea are appropriate. the rabbit has a large cornea approximately the same diameter as the human cornea. it is significantly thinner, however, with an average central thickness of about . mm. the thin cornea and shallow anterior chamber have contributed to technical difficulties in performing intraocular lens implantation and penetrating keratoplasty in rabbits (mueller, ) . the corneal epithelium is also thin in rabbits, and the existence of bowman's layer in the rabbit has been disputed (prince, b) . one of the most important qualities of the rabbit cornea is the capacity of the endothelium for regeneration. following injury, rabbit endothelial cells are able to divide and cover the defect with minimal effect on the endothelial cell density. the dog is similar in this respect (befanis and peiffer, ) . cats and monkeys, on the other hand, have been shown to have very little potential for regeneration of endothelium. in these animals, as in man, remaining endothelial cells grow and spread to cover the defect (van horn, ) . thus cats and monkeys are probably better models in studies where response to endothelial injury is impor tant. the recent surge in interest in the nature of the corneal endothelium has been brought about largely by the demonstration that the endothelium is the site of the "fluid pump" transport mechanism essential for maintaining corneal dehydra tion and thus transparency (maurice, ) . localization of the fluid pump and many subsequent studies have been made possible by the endothelial specular microscope developed for viewing the endothelium of enucleated eyes and modi fied for use with excised corneas in the form of the perfusion specular micro scope (maurice, ) (fig. ) . later the endothelial microscope was adopted for in vivo examination of humans and animals, including cats, rabbits, and monkeys (laing et al., ) . there is nothing new about viewing the specular reflection from the corneal endothelium, a long established slit lamp technique. the specular microscope, however, is an instrument designed to optimize this type of illumination to produce a high magnification ( - x) image of the endothelium suitable for photomicrography, accurate endothelial cell counts, and detailed observation of individual cells (fig. ) . a discussion of the optical principles of specular mi croscopy is provided by laing et al. ( ) . commercially available endothelial microscopes are designed primarily for use with humans, but can be easily adapted to any laboratory animal with a sufficiently large cornea (which excludes rats and mice). the smallest eye movements blur the image of the endothelium, and, therefore, general anesthesia is usually required when working with ani mals. technique and observations in dogs (stapleton and peiffer, ) and cats (peiffer et al., a) have been described. in ussing and zehran described an apparatus consisting of two small lucite chambers separated by a piece of frog skin which could be used to measure transport of materials potential differences across the frog skin. this same technique was modified for study of the cornea, and dual chamber perfu sion experiments form the basis for much of what we know about corneal physiology (donn et al., ; mishima and kudo, ) . the perfusion specu lar microscope has added an extra dimension to this type of in vitro experimenta tion by allowing direct observation of endothelial cell shape and integrity during perfusion. atraumatic removal of the cornea avoiding contact with the en dothelium or excessive bending of the cornea is essential to the success of this type of experiment. the technique of dikstein and maurice ( ) for excision of the cornea with a scierai rim has been used very successfully. for longer term in vitro experiments the cornea can be cultured by convential closed organ culture techniques as described elsewhere in this chapter. the impetus for the intensive investigation of optimum conditions for corneal storage has resulted from the feasibility of corneal transplantation in humans. most of the research has been done using rabbits, however, and is especially applicable, therefore, to use and storage of animal corneas for whatever purpose. the method of corneal storage in most eye banks today is moist-chamber storage, storing the intact globe at °c in a glass bottle with saline-soaked cotton in the bottom. the stagnant fluid quickly becomes loaded with waste prod ucts, making this method unsuitable for long-term storage. a number of alterna tive techniques are used in laboratory storage including storage of excised cor neas at °c, organ culture at °c, and cryopreservation for long-term storage. several different media for storage of excised corneas have been evaluated. the most popular at present is mccary-kaufman (m-k) media, modified tissue culture media containing tc- , % dextran , and a mixture of streptomycin and penicillin (mccarey and kaufman, ) . storage in m-k media probably preserves corneal viability longer than moist chamber storage, endothelial dam age occurring at days in moist chamber stored rabbit corneas compared to - days for m-k stored corneas (geeraets et al., ) . stocker et al. ( ) reported good success with excised corneas stored in autologous serum at °c. subsequent studies, however, show conflicting results as to the advantage of serum storage over moist chamber storage (geeraets et al., ; van horn and schultz, ) . corneal organ culture represents an alternative method of prolonging corneal viability. it is similar to convential storage of excised corneas except for tempera ture, which is maintained at °c and may allow for preservation of endothelial viability for up to weeks (doughman et al., ) . the culture media most often used is eagles mem supplemented with fetal calf serum. cryopreservation is the only method which permits indefinite storage of whole viable corneas. the tissue is passed through a graded series of solutions contain ing dimethyl sulfoxide and frozen at a controlled rate over liquid nitrogen. studies indicate that some endothelial damage may occur during the freezing process but destruction is incomplete (basta et al., ) . in fact, survival of donor endothelial cells following transplantation of cryopreserved corneas has been demonstrated using sex chromatin markers in monkeys (bourne, ) . techniques of cryopreservation have been reviewed and evaluated by ashwood-smith ( ) . methods of estimating corneal viability after various storage regimens are all based on evaluating endothelial integrity. histochemical stains have been used extensively (stocker et al., ) . dye exclusion stains, such as . % trypan blue applied to the endothelium for seconds and then rinsed off with saline, can be used without apparent toxicity to the endothelium. the pattern obtained with trypan blue staining is more difficult to see in rabbits than other species. a modification of this technique using a combination of trypan blue and alizarin red on rabbit corneas has recently been reported (spence and peyman, ) (fig. ). electron microscopy, both scanning and transmission, are also used to assess the condition of the endothelium (fig. ) . the most innocuous method of estimating corneal viability is simply mea surement of corneal thickness at body temperature which is an indirect indication of the condition of the endothelium, healthy endothelium being able to deturgese the cornea to near-normal thickness. the measurement of corneal thickness, or pachometry, has traditionally been done using an optical technique developed by von bahr ( ) . the specular microscope, however, provides for a more direct method of measuring corneal thickness by registering the distance from the point of applanation at the corneal surface to the image of the endothelium. viraj and bacterial infections of the cornea have been studied mostly in rab bits, especially in the case of herpes simplex keratitis where attempts to study this disease in other animals have been unsuccessful. techniques for initiating these infections are discussed in section iii. noninfectious forms of keratitis may result from tear deficiencies, vitamin a deficiency, allergic phenomenon, and chemical or mechanical damage. keratoconjunctivitis sicca (kcs) resulting from tear deficiency has been studied in the dog, the disorder being surgically induced by removal of the lacrimai gland and nictitating membrane (helper et al., a; gelatt et al., ) or chemi cally induced with phenazopyridine (pyridium), - mg/day orally for - weeks (slatter, ) . françois et al. ( ) have used rabbits to study kcs. they found it necessary to remove all the accessory lacrimai tissue, including harder's glands and the nictitating membrane as well as the lacrimai gland proper to induce the disease in rabbits. interstitial stromal keratitis, thought to be an immune-mediated phenomenon, has been demonstrated in mice after intravenous and intracutaneous injections of bovine γ-globulin or bovine serum albumin (kopeloff, ) . a simple method of inducing an inflammatory stromal keratitis in rabbits is injection of . ml of clove oil into the corneal stroma (leibowitz et al., ) . phlyctenular keratitis, another allergic form of keratitis, has been reported in animal models, but thygeson et al. ( ) have failed to produce true phlyctenular disease in rabbits and questions whether the previously reported models are valid. most of the clinically significant lesions, hereditary or acquired, affecting the corneal endothelium result in visually disabling corneal edema. spontaneous dystrophies leading to corneal edema are described in the veterinary literature and are reviewed by dice ( ) . corneal edema can be induced by in vivo freezing with a metal probe to destroy endothelial cells. the edema may be temporary or permanent depending on the area, temperature, and duration of cold exposure (chi and kelman, ) . we have found that two successive freezes with a contoured -mm brass probe, cooled to - °c with liquid c , consis tently produces temporary corneal edema in dogs (befanis and peiffer, ) . a slightly smaller probe is used for rabbits. the probe is applied two times for - seconds with time for complete thawing allowed in between. corneal edema and scarring are often irreversible. corneal transplantation is one method of restoring a transparent visual axis, but a number of prosthetic devices, usually fashioned out of methyl methacrylate, have been used experi mentally over the last years and are now being used clinically on a limited basis. stone and herbert ( ) reported a two-stage procedure for implanting a plastic window in rabbit corneas. dohlman and refojo ( ) have reviewed the previous years experience with plastic corneal implants. many of these procedures require the use of a tissue adhesive to hold the plastic prosthesis in place. cyanoacrylate adhesives are used. methyl- cyanoacrylate creates the strongest bond, but is too toxic for use on the cornea. butyl- -cyanoacrylate can be used, and the longer chain substituted forms are even less irritating but do not form as strong a bond (havener, ) . gasset and kaufman ( ) have used octyl- -cyanoacrylate for gluing contact lenses to rabbit and monkey corneas with the epithelium removed. glued-on contact lenses have also been used for treatment of experimental alkali burns in rabbits (zauberman and refojo, ; kenyon et al., ) . conventional contact lenses without adhesive can be used in animal experi ments. most investigators have found that both hard lenses and silicone lenses are well-tolerated in the rabbit (thoft and friend, ) . hard lenses should be specifically fitted to the rabbits, however, and a partial tarsorraphy may be used if necessary to prevent rabbits from expelling lenses (enrich, ) . systemic connective tissue disorders may be associated with local ocular signs of diffuse, nodular, or necrotizing scleritis that can involve the cornea as well. hembry et al. ( ) sensitized rabbits by intradermal ovalbumin plus freund 's adjuvant followed by injection of ovalbumin at the limbus to produce a necrotiz ing corneoscleritis. animal studies of the composition of the aqueous humor, its function, and the processes controlling the dynamic state of its constituents and volume have two main objectives: ( ) appreciation of the physiology, biochemistry, and hence metabolism of the tissues of the anterior segment and ( ) definition of the mechanisms that control rate of aqueous humor production and outflow and hence intraocular pressure. definitive information is available for only a limited number of mammalian species, and these data suggest that species variation in aqueous humor composition and dynamics exists. in addition, anatomical charac teristics of the outflow pathways differ between species. excellent reviews by cole ( ) and tripathi ( ) detail these differences, which emphasize that making inferences from research results using nonprimate mammals to man requires appreciation of these species differences. the maintenance of normal intraocular pressure (iop) is dependent upon a critial balance of dynamic equilibrium between the processes of aqueous humor production and drainage. concepts of aqueous humor formation are based largely upon laboratory work with the rabbit. aqueous humor is produced by the ciliary processes as a result of active transport and passive ultrafiltration processes; approximately one-half of the aqueous is produced by active secretion across the two-layered epithelium. the exact mechanisms of transport have not been de-fined and may demonstrate species differences; active transport of sodium in the presence of a sodium-and potassium-activated adenosine triphosphatase located in the cell membrane of the nonpigmented epithelium is one of the main primary events in the formation of the aqueous fluid (cole, ) . the remaining % of aqueous humor is formed by passive processes, including diffusion, dialysis, and ultrafiltration. the composition of aqueous humor is essentially that of a plasma ultrafiltrate but varies between species. gaasterland et al. ( ) have studied the composition of rhesus monkey aqueous. differences between aqueous and plasma levels of potassium, magnesium, chloride, and bicarbonate have been demonstrated and suggest species differences in transport mechanisms and/or anterior segment metabolism. a saturable active transport system for ascorbate has been documented in the rabbit (barany and langham, ) . transport mechanisms for amino acids demonstrated in the rabbit may not be present in rat, cat, monkey, and dog (reddy, ) . total volume of the aqueous humor will vary among species with the size of the globe and relative proportions of the anterior segment (cole, ) . the rate of aqueous humor formation in the species studied, with the exception of the uniquely high-valued cat, is approximately . - . μ,Ι/minute (cole, ) and is dependent upon ciliary artery blood pressure, the pressure in the ciliary body stroma (essentially equal to iop), and the facility of flow through the ciliary capillary and capillary wall. because of these pressure gradients, passive aqueous humor production is decreased as iop increases; this pressure-dependent component or ' 'pseudofacuity ' ' has been demonstrated to account for up to % of total facility in monkeys (bill and barany, ; brubaker and kupfer, ) . the aqueous humor enters the posterior chamber, flows through the pupil into the anterior chamber due to thermal currents, and exits the globe by passing through the flow holes in the trabecular meshwork and reentering the peripheral venous circulation via thin-walled vascular channels. methodology utilized to quantitate aqueous humor production and flow rates involve the measurement of the turnover of a substance within the aqueous introduced by active and/or passive processes from the peripheral vasculature or intraocular injection. the ideal technique does not involve introduction of a needle into the globe, as this undoubtedly disrupts normal homeostatic mechanisms. man, guinea pig, rabbit, and cat have been studied utilizing fluorescein turnover with slit lamp fluorometry, a variety of isotopes, and other substances. reasonable correlation among investigators utilizing different tech niques within species has been observed (cole, ) . the comparative anatomy of the outflow pathways has been reviewed by calkins ( ) and tripathi ( ) . in man and the primates a well-defined trabecular meshwork spans the scierai sulcus from the termination of descemet's membrane to the base of the iris to the scierai spur. deep to the trabecular meshwork within the sulcus a single large channel, schlemm's canal, drains the aqueous into the episcleral veins (fig. ) . in nonprimate mammals, the scierai spur and sulcus are absent; the trabecular fibers span the ciliary cleft, a division of the ciliary body into inner and outer leaves, deep to the fibers of the pectinate ligament. these fibers consist of uveal tissue and extend from the termination of descemet's membrane to the iris base. aqueous drains into small saculated vessels, the aqueous or trabecular veins, which communicate with an extensive scierai venous plexus (figs. and ). van buskirk ( ) utilized plastic lumenal castings and scanning electron mi croscopy to study the canine vessels of aqueous drainage and to demonstrate that in this species the exiting aqueous mixes with uveal venous blood. the mechanisms of aqueous humor outflow through the trabecular meshwork are not completely understood. passive pressure mechanisms certainly play an important role; increases in episcleral venous pressure result in decreased outflow and increased iop. active transport mechanisms for both organic and inorganic anions have been demonstrated. transmission electron microscopic studies of the endothelium of the scierai venous plexus have revealed giant cytoplasmic vac uoles that are suggestive of transcellular transport mechanisms and indicate that this is the main site of resistance to outflow (tripathi, ) . some aqueous humor may exit through the ciliary body, entering the suprachoroidal space and into the choroidal circulation and sclera. this pressureindependent uveoscleral outflow accounts for up to - % of bulk flow in subhuman primates and has been demonstrated in cats, rabbits, and man to be quantitatively less than in two species of monkeys (bill, (bill, , (bill, , bill and phillips, ; fowlks and havener, ) . qualitative uveoscleral routes have been suggested in the dog utilizing dextran-fluorescein studies (gelati et al., b) . because of the radius of curvature of the cornea and the presence of an overlying scierai ledge, light rays from the base of the iris, the angle recess, and the trabecular meshwork undergo total internal reflection, preventing direct visualization of the outflow structures without the use of a contact lens to elimi nate the corneal curve. two general types of gonioscopic contact lenses are available; indirect lenses, which contain mirrors and allow examination of the angle by reflected light, and direct lenses, through which the angle is observed directly. a magnifying illuminated viewing system, ideally the slit-lamp biomicroscope, is essential for critical evaluation. gonioscopic examination of the canine iridocomeal angle was first reported by troncoso and castroviejo ( ) , although nicholas had previously depicted the canine angle by drawings in . troncoso in compared the gross and gonioscopic appearance of the angles of the dog, cat, pig, rabbit, rhesus monkey, and man. the clinical application of gonioscopy in comparative ophthalmology is rela tively recent. lescure and amalric ( ) described its use in the dog, and subsequently numerous investigators have stressed the value of the technique in the diagnosis of glaucoma in the dog (vainsi, ; gelatt and ladds, ; bedford, bedford, , . martin ( ) has correlated the microscopic structure and gonioscopic appearance of the normal and abnormal canine iridocorneal angle. the technique is straightforward and involves topical anesthesia and minimal physical restraint in dog, cat, and rabbit and ketamine sedation in primates (fig. ). the concave surface of the lens is filled with artificial tears and placed on the corneal surface; the franklin goniolens with a circumferential flange is re tained by the eyelids and enables the examiner to have both hands free. the troncoso or koeppe lens may also be utilized; vacuum lenses and the swan lens are smaller and thus adaptable to younger and smaller animals (fig. ) . the structures observed during gonioscopic examination of the nonprimate mammal, using the dog as an example, include, from posterior to anterior, the following: . the anterior surface and base of the iris . the pectinate ligament . deep to the pectinate ligament, the ciliary cleft, and the trabecular meshwork . the deep or inner pigmented zone representing the anterior extension of the outer leaf of the ciliary body . the outer or superficial pigmented zone which is variable in presence and density and represents melanocytes in the limbus . the corneal dome (fig. ) species variations in appearance do exist. the sub-human primates present a gonioscopic appearance of the iridocorneal angle identical to man. in the cat, the pectinate fibers are thin and nonbranching, while in the dog they tend to be deeply pigmented, stout and arbiform. in the rabbit the pectinate fibers are short and broad (fig. a-c) . these tests have been used in man to detect suspicious or borderline glaucoma patients as well as to investigate the heredity of open angle glaucoma by stressing the homeostatic mechanisms of the globe. provocation results in an increase in iop that can be characterized by extent and duration. the water drinking and corticosteroid provocative tests have been most useful in open angle glaucoma, whereas the mydriatic and dark room tests have been valuable in narrow angle glaucoma (kolker and hetherington, ) . the water provocative test in man, rabbits, and subhuman primates (macaca irus) has been studied with schiotz and applanation tonometry and in combina tion with tonography and constant pressure perfusion (swanlijung and biodi, ; galin et al., galin et al., , mcdonald et al., ; galin, ; thorpe and kolker, ; casey, ) . the procedure has been used in rabbits to test the effects of different drugs on pressure-regulating mechanisms (mcdonald et al., ) . the test in dogs has defined normal values and demonstrated significant dif ferences in american cocker spaniels and beagles with spontaneous glaucoma (lovekin, ; gelatt et al., a) (fig. ). it is generally accepted that the increase in intraocular pressure after the administration of a substantial volume of water is primarily related to a sudden decrease in the osmolarity of the blood; the related influx of water into the eye is presumed to increase intraocular pressure proportional to the volume of water administered (galin et al., galin, ) . hemodilution may not be solely responsible; in man the increase in iop in % of the patients occurs before the fall in serum osmolarity (spaeth, ) . the subsequent rate of decay of iop assesses the ability of the outflow system to cope with the increase inflow. clinical measurement of the facility of outflow is accomplished by raising iop by placing a tonometer on the eye and determining the subsequent rate of volume loss and pressure decrease; as resistance to outflow of aqueous humor increases, the pressure changes will decrease. the principle of the test may be traced to the massage effect, whereby pressure on the eye leads to a softening of the globe due to increased outflow. schiotz indentation tonography employs placement of an electronically recording schiotz tonometer on the eye; the weight of the tonome ter will indent the cornea, reducing ocular volume and increasing iop. the instrument is left on the cornea for a -minute period. tables are utilized to derive the rate of aqueous humor outflow based upon the observed changes in iop over the -minute period as recorded by the tonograph (grant, ; kronfeld kronfeld , garner, ; drews, ; podos and becker, ) . in , grant showed that the coefficient of outflow facility (c) is related to the change in ocular volume (Δν) occurring over the time interval (t), as a result of the difference between the average pressure during tonography (p t av) a n d the iop prior to placement of the tonometer on the globe (p ): c = av/t(p tav -p ). the coefficient is expressed in microliters of aqeous humor outflow per minute per millimeters of mercury pressure (grant, ) . tonography has certain limitations. the accuracy is dependent upon the accu racy of the tonometer calibration, since Δν, ptav» **nd pq are derived from this data. in addition, six physiologic assumptions are made upon which accurate quantitative results are dependent (grant, ) . . that there is a constant continuous flow of aqueous humor . that the process of tonography does not alter this flow . that the process does not alter outflow facility . that the process does not change the uveal vascular volume . that tonography does in fact measure the outflow of aqueous humor through the trabecular meshwork . that the eye exists in a steady state in regards to aqueous humor dynamics during the -minute period. previous discussions of pseudofacility-the decrease in aqueous humor pro duction that occurs with increased iop-and uveoscleral outflow demonstrate that assumptions and are not valid. evidence exists that outflow facility is depen dent upon iop. in addition, uveal blood volume is probably influenced by the placement of the tonometer on the cornea (podos and becker, ) . however, tonographic c values determined for the human eye correlated well with values obtained by constant pressure perfusion, aqueous humor fluorescein disappear ance time, and aqueous humor turnover of certain substances (becker and costant, ; françois et al., ; bill and barany, ) . additional factors warrant consideration when evaluating tonographic proce dures in species other than man; animal globes differ significantly from the human eye in terms of ocular volume, radius of corneal curvature, vascular dynamics, and tissue characteristics upon which the accuracy of schiotz identation tonometry or tonography is dependent; schiotz tonometric conversion tables are inaccurate if applied to the canine eye (peiffer et al., ) . the technique cannot be utilized in animals without pharmacologie restraint, and the effect of the drugs utilized on the steady state of iop must be taken into account. helper and his associates ( b) used xylazine and ketamine sedation and found c values from . to . in normal dogs, . to . in basset hounds, and . to . in a small number of glaucomatous patients. gelatt and his associates ( a) observed a combination of acetylpromazine and ketamine to have mini mal effect on the steady-state iop of the normal canine eye, and demonstrated impairment of outflow in beagles with inherited glaucoma. peiffer et al. ( ) reported mean tonographic values of . using this anesthetic combination in normal mongrel dogs (fig. ) . applantation tonography may prove more accurate and versatile than schiotz indentation tonography in the animal laboratory. a -minute tonography period is adequate, and the effect of anatomic variables is minimized ). invasive laboratory techniques that have been described to measure facility of outflow involve direct measurement of intraocular pressure via a cannula inserted into the globe and one of three techniques: ( ) injection of a known volume of fluid and observing the rapid increase and subsequent slow decrease in iop (pressure decay curves), ( ) perfusion of the globe with fluid at a constant rate and observing the related pressure changes (constant rate perfusion), and ( ) perfusion at a variable rate necessary to maintain a given iop (constant pressure perfusion) (barany and scotchbrook, ; grant and trotter, ; macri, ; melton and hayes, ; armaly, ; melton and deville, ; grant, ; langham and eisenlohr, ; barany, ; brubaker, ; wickham etal., ) (fig. ) . these invasive techniques eliminate the variables of scierai creep and changes in uveal blood volume and species differences in corneal anatomy. all require methods in which intraocular pressure, fluid volume in the eye, and flow reach steady state during the measurement. cannulation of the anterior chamber will induce qualitative and quantitative aqueous humor changes. despite mathemati cal equivalence, the methods are quite different in practice because they differ greatly in the time it takes for the eye to go from one steady state to another. the time required to reach steady state during constant pressure perfusion is less than minutes compared to much longer times for the other techniques. all are based on the assumptions of pressure-independent facility and secretion rate, which have been criticized (langham, ) . facility of outflow can be determined in constant pressure perfusion by measuring average flow from an external reservoir into the eye; time period is a compromise between the desire to achieve accuracy by using a long period and to achieve high temporal resolution by using a short one. a -minute averaging period is utilized arbitrarily to facilitate comparison to tonographic values. facil ity may be calculated at any given pressure utilizing the formula c = fip, where f is the rate of perfusion flow in microliters of mercury per minute, p is the intraocular pressure in millimeters of mercury, and c is facility expressed in microliters of fluid per millimeter of mercury per minute. in an in vivo system, however, this equation does not consider aqueous humor production and thus provides values lower than actual facility. barany showed that facility can be calculated at two levels of pressure, Ρ λ and p , utilizing the formula c = (f -f\)k? ~ p\)· this formula necessitates assumption of a constant episcleral venous pressure and aqueous humor production. if one estimates a value of mm hg for the former and ul/minute for the latter, similar results between the two equations are obtainable by dividing rate of secretion by the episcleral venous pressure and adding the result ( . ul/minute/mmhg) to the values obtained by the equation c = f ip . character of the perfusate can influence facility; . % unbuffered saline causes a decline in resistance on prolonged infusion of the anterior chamber. barany ( ) utilized phosphate-buffered saline with calcium and glucose added, and brubaker and kupfer ( ) perfused the heparinized mammalian tissue culture medium to minimize this "washout" factor. gaasterland et al. ( ) used pooled heterologous aqueous humor to perfuse rhesus monkey eyes in vivo. melton and deville ( ) studied enucleated canine eyes using constant pressure perfusion and found an average c value of . at mm hg iop. peiffer and his associates ( ) found the facility of outflow in normal dogs anesthetized with sodium pentobarbital to have a mean value of . ± . sd which increased as iop increased. perfusion values for outflow were less than those determined tonographically ( . ± . sd with acetylpromazineketamine hydrocholoride sedation, . ± . sd with pentobarbital anes thesia) or in the enucleated globe. van buskirk and brett ( a,b) perfused enucleated canine eyes and found a pressure-dependent facility of outflow of . ± . sd at mm hg iop which increased to . at mm hg iop. outflow increased significantly during perfusion with hyaluronidase-containing solution and with time in the non-hyaluronidase-perfused eyes. peiffer et al. ( a) perfused beagles with inherited glaucoma in vivo and found impairment of outflow facility compared to normal dogs (fig. ). the limitations of tonographic and perfusion techniques to quantitate aqueous humor outflow in animal species must be appreciated; neither is ideal. the refinement and development of more accurate methodology remains a challenge to the investigator. either technique is certain to provide more relevant informa tion if performed in vivo rather than on enucleated globes. the iop is a differential fluid pressure that measures the vector sum of the forces generated by the intraocular fluids acting at the interface between the fluid and the fibrous coats of the globe. the accurate determination of the iop is difficult because all the techniques utilized to measure it in some way necessitate altering the parmeter from its original value. in the laboratory, the anterior chamber can be cannulated and iop determined directly by the fluid level of an open-air manometer; this situation, of course, is not applicable to clinical situa tions where a noninvasive technique is required. any noninvasive technique, however, must ultimately be compared to simultaneous readings from the cannu lated globe. quantitative determination of iop is achieved by one of two types of tonometry. clinical estimations depend on subjecting the cornea to a force that either indents (impresses) or flattens (applanates) it. tonometers that indent the cornea are referred to as indentation tonometers, and those that flatten it are referred to as applanation tonometers. the cornea is utilized because other areas, such as the anterior sclera, have a nonuniform thickness and the variability of additional tissues including conjunctiva bulbar fascia and the underlying anterior uvea. normal iop in many of the animal species, notably the nonprimate mammals, appears to be higher and more variable than that observed in persons (bryan, ; heywood, ) . a number of physiologic variables may affect the iop. these include the nature of the subject, the time of day, and the position of the subject. intraocular pressure is related to blood pressure, and it has been demon strated that animals that are excited will have higher iop. accurate values are obtained in animals that have been handled and previously subjected to the technique. in persons, a diurnal variation of iop has been observed with the lowest iop occurring early in the morning. this variation is probably related to changes in endogenous corticosteriod levels. diurnal variation has been demon strated in the new zealand white rabbit (katz et al., ) and in beagles with inherited glaucoma, but not in normal dogs (gelatt et al., a) . in persons significant differences in iop are observed with the patient in a sitting position as compared to the prone position. in addition, the variable of technique may contribute to the wide range of normal intraocular pressures observed in animals. sedation and anesthesia, because of associated cardiovascular effects, are likely to affect iop. this is especially true of the barbiturates. bito and co-workers ( ) found that ketamine hydrochloride had minimal effect on iop of rhesus monkeys (mucaca mulatta) and reported a mean iop of . ± . sd with higher values and a greater diurnal variation in young animals. schiotz indentation tonometry estimates iop by applying a carefully stan dardized instrument on the cornea and measuring the depth of indentation of the cornea by a weighter plunger. the schiotz tonometer has the advantages of simple construction, reasonable cost, portability, and relative simplicity of tech nique. in schiotz tonometry, a force from a small curved solid surface with a spheri cal curvature which indents the cornea will be balanced by a fluid pressure separated from the solid surface by a thin flexible membrane, the cornea in this case, when the applied force or weight of the tonometer equals the resultant force from the fluid pressure measured in a direction parallel to the direction of the applied force times the area of distortion to the membrane. the surface tension of the tear film exerts a small force parallel to the corneal surface. the instrument consists of a footplate that approximates the radius of curvature of the human cornea, a plunger, a holding bracket, a recording scale, and . , . , . and occasionally . gm weights (fig. ) . the cornea is in dented with a relatively frictionless weighted plunger; the amount of plunger protruding from the plate depends upon the amount of indentation of the cornea. the tonometer scale is adjusted so that . mm of the plunger equals one scale unit. calibration tables are used to derive the actual iop from the observed tonometer reading. most accurate estimations of iop are obtained with the lighter weights within the middle scale ranges. the technique for schiotz tonometry is relatively simple (bryan, ; vainisi, ). the cornea is anesthetized with a drop of topical anesthesia. while allowing a few seconds for the anesthesia to take effect, check the recording arm on the tonometer by placing it on the solid convex metal surface provided. the tonometer should read , indicating that no indentation of the plunger is occur ring and that the plunger surface is flush with the footplate. with a bit of practice, schiotz tonometry can be performed without assistance in the dog and cat. rabbit tonometry is facilitated with an assistant holding the animal in lateral recumbancy. the animal should be relaxed, and care should be taken not to compress the jugular veins. the first, second, and/or third fingers of the left hand or the thumb may be used to simultaneously retract the lower eyelid of the right or left eyes, respectively. the tonometer is grasped between the thumb and first finger of the right hand and the tonometer scale rotated such that it is easily observed by the examiner. the fourth finger of the right hand is rested on the frontal bone to provide stability and retract the upper eyelid. care must be taken in retracting the lids so that pressure is applied only to the bony orbital rim and not the globe itself. excessive eyelid retraction may also create abnormal forces on the globe and should be avoided. the footplate is placed on the cornea as central as possible and gentle pressure applied until the holding bracket glides freely around the footplate shaft (fig. ) . the scale reading is noted, and the tonometer is removed. the procedure is repeated two more times; each scale readings should be within one full unit to another. if the readings are to the low end of the scale, additional weights may be applied to the tonometer and the procedure repeated to obtain midscale readings. one potential source of error is the position of the tonometer in relationship to the perpendicular; deviation from the perpendicular will result in an overestimation of iop directly proportional to the degree of deviation. the process of tonometry will induce an increase in aqueous humor outflow and each repeated measure ment may be slightly lower than the previous estimate of iop. following use, the tonometer should be disassembled and cleaned carefully with a pipe cleaner. free movement of the plunger within the casing is essential for proper function, as is smooth working of the lever system and the recording arm. the process of placing the tonometer on the eye will increase iop between to mm hg; the schiotz tonometric conversion tables enable the clinician to correlate the tonometer reading with the iop prior to the placement of the fig. . use of the schiotz tonometer in a dog. the instrument is applied in a perpendicular fashion to the central cornea. its use is limited to larger primates, dogs, cats, and rabbits, and calibration tables must be devised for each species for maximally accurate determination of intraocular pressure. instrument on the cornea. because of species differences in corneal curvature, ocular rigidity, and tissue characteristics, the use of a human conversion table will result in inaccurate estimation of iop. conversion tables have been de veloped for rabbit (best et al., ) and canine (peiffer et al., ) globes. limitations of the schiotz indentation technique depend upon the species, the clinician, and the instrument. use in animals is limited to those species with relatively large corneas and animals that can be adequately restrained and posi tioned. it is most useful in larger primates, dog, cat, and rabbit. ocular rigidity, or the ability of the cornea and sclera to stretch, will vary with age and from species to species and animal to animal (best et al., ; peiffer et al., b) . ocular rigidity also increases as the tonometer is placed closer to the limbus. increases in ocular rigidity provide schiotz recordings that are higher than actual iop; with increased ocular rigidity there is less indentation by the schiotz tonometer, creating a false impression of increased iop. applanation tonometry is based upon the principles of fluid pressures; pressure equals force divided by the area. a force from a plane solid surface applied to a fluid contained by a thin membranous surface will be balanced when the area of contact times the pressure of the fluid equals the force applied by the plane solid surface. this is known as the imbert-fick law. simply stated, in applanation tonometry one may either measure the force necessary to flatten a constant area of the corneal surface or measure the area of cornea flattened by the constant applied force. electronic applanation tonometers, notably the mackay-marg, are the most versatile and accurate in a wide variety of species. probe tips are smaller than indentation tonometers, a minimum of intraocular fluid is displaced, iop is not significantly increased by the procedure, and the technique is independent of ocular rigidity and corneal curvature. it is applicable to smaller laboratory species, and its use has been reported in the chinchilla (peiffer and johnson, experimental animal models of glaucoma have been developed to study the effects of elevated iop on other ocular tissues, to determine the efficacy of medical and/or surgical treatment in reducing iop, and to define mechanisms of the glaucomatous process itself. spontaneous animal models of glaucoma have been utilized for the above purposes in addition to investigation to determine their similarities to the disease in man. historically, experimental glaucoma has been produced in rabbits by the injec tion of % kaolin into the anterior chamber to obstruct the outflow channels, with iop reaching - mm hg within days (voronina, ) . skotnicki ( ) enclosed rabbit globes with cotton threads to induce glaucoma with resultant optic disc cupping. flocks and his associates ( ) utilized a similar technique and rubber bands; while initial increases in iop reached - mm hg, pres sures dropped to - mm hg within hours. one-third of the eyes developed panophthalmitis and only % showed cupping of the optic disc. kupfer ( ) threaded polyethylene tubing into rabbit iridocorneal angles; iop increase was observed within hours and remained elevated for months. loss of retinal ganglion cells and cupping of the optic disc occurred. samis ( ) and kazdan ( ) produced glaucoma in rabbits by the intraocular injection of methylcellulose. huggert ( ) blocked the outflow of aqueous humor in rabbits using three different techniques but failed to cause significant increases in iop. de carvalho ( ) injected cotton fragments into rabbit anterior chambers, which elevated iop to - mm hg; in those animals in which the glaucoma persisted longer than days, retinal and optic disc pathology was observed. injection of talcum powder or dental cement (kalvin et al., b) produced elevated iop in monkeys. the injection of the enzyme α-chymotrypsin into the globe will produce a variable increase in iop that may or may not be prolonged; the enzyme particles dissolve the zonules of the lens, the fragments of which collect in and obstruct the trabecular mesh work. the technique has been utilized in the rhesus and owl monkey to study optic nerve and ocular vascular changes (kalvin et al., a; zimmerman et al., ; lambert et al., ; lesseil and kuwabara, ) . the enzyme may have direct toxic effects on the retina which must be considered in studying the morphologic and functional effects of elevated iop on this tissue. cyclocryotherapy will cause an acute elevation of iop in rhesus monkeys (minckler and tso, ) , and the technique has been utilized to study axoplasmic transport of the axons of the ganglion cells and optic nerve (minckler et al., ) ; the same parameters have been studied by controlled elevation of iop by cannulation and perfusion (minckler et al., ) . the use of repeated circumferential argon laser photocoagulation of the iridocorneal angle as described by gaasterland and kupfer ( ) results in a predictable sustained elevation of iop, marked reduction of outflow facility, and progressive cupping of the optic nerve head. the technique has the advantages of being noninvasive and associated with minimal intraocular inflammation and unrelated pathology. in chickens raised under continuous light exposure from the day of birth onward, iop rises related to increased resistance to aqueous humor outflow that is detectable as early as weeks of age (lauber et al., ) . morphologic studies of the trabecular meshwork of affected animals reveals an increase of intercellular collagen and elastic trabecular tissue with resultant densification of the meshwork. there was an absence of endothelial vacuoles, pores, and microchannels observed by transmission electron microscopy in normal birds (tripathi and tripathi, a,b; rohen, ) . while sporadic spontaneous cases of animal glaucoma have been described in a variety of species, only two models are reliably producable by controlled breedings, inherited glaucoma in the rabbit and beagle. these two models will be discussed briefly emphasizing methodologies utilized to define the disease pro cesses. buphthalmia (hydrophthalmus, congenital infantile glaucoma) in rabbits is due to an autosomal recessive gene with incomplete prentrance (hanna et al., ) . histologie abnormalities of the eye are observed at birth; elevated iop and buphthalmus may be observed as early as to weeks of age in some animals with progressive clouding and flattening of the cornea; ectasia of the globe, particularly in the sclero-cornea region; deepening of the anterior chamber with detachment and fragmentation of the iris membrane; partial atrophy of the ciliary body; and glaucomatous excavation of the optic disk (hanna et al., ) . the primary defect responsible for the development of glaucoma probably in volves impairment of facility of outflow. it has been postulated that the glaucoma may be part of a primary systemic disorder (hanna et al., ) . the gross ocular enlargement which characterizes buphthalmia is accompanied by fibrosis of the filtering angle (mcmaster and macri, ) , a decrease in facility of aqueous humor outflow (mcmaster, ; kolker et al., ) , and a hyposecretion of aqueous humor (smith, ; auricchio and wistrand, ; mcmaster and macri, ; greaves and perkins, ) . anatomic studies have em phasized changes at the angle and have postulated that they constitute at least one site of obstruction to aqueous humor outflow. hanna et al. ( ) demonstrate an absence of the space of fontana, the iris pillars, and either total absence or a rudimentary development of the trabecular canals and intrascleral channels. mcmaster and macri ( ) observed that the obstruction to outflow lies be tween the trabeculum and the episcleral veins. the angle according to hanna et al. ( ) is open in the adult buphthalmia but appears closed in the newborn. the combination of hyposecretion and reduced outflow explains why buphthalmic rabbits may have a normal iop. rabbits that are genetically buphthalmic but phenotypically normal appear to have an iop approximately mm hg higher than normal, and rabbits with clinical signs of buphthalmia may have iop as high as mm hg greater. the iop tends to increase a few weeks prior to observable distention of the globe. elevated iop will subsequently return to normal levels. the cause and effect relationship of the striking inverse correlation of aqueous humor ascorbate concentration with the severity of the buphthalmia is not clear; a marked drop in ascorbate levels is present in early preclinical stages of buphthalmia (lam et al., ; fox et al., ; lee et al., ) . the actual sequence of events, involving alterations in iop, outflow facility, ascorbate concentration of the aqueous humor, and clinical signs are not clearly defined. the rate of progression has been shown to vary with the genetic background. sheppard et al. ( ) reported that the corneal endothelial cells in a flat preparation from a buphthalmic rabbit were enlarged and of variable size. he postulated that the cells expand to cover the increased corneal area. van horn et al. ( ) utilized scanning electron microscopy to confirm this report, but also indicated that there is a loss of endothelial cells in the disease as well. gelati ( ) published a report on a familial glaucoma in the beagle; pathologic increases in iop occurred from months to years of age, and affected dogs demonstrated open iridocorneal angles upon gonioscopy. additional observations were summarized in subsequent papers (gelatt et al., b (gelatt et al., , c . controlled breedings suggested an autosomal recessive mode of inheritance. the glaucomatous process was divided into early ( to months of age), moderate ( to months of age), and advanced ( months of age and greater) and was evaluated clinically by tonometry, gonioscopy, and anterior and posterior segment examination. in early glaucoma, the iridocorneal angle was open and without anomalies, and iop was elevated. with moderate glaucoma, variable optic disc atrophy, elongation of the ciliary processes, and focal disin sertion of the zonules from the lens were seen in addition to elevated iop and open iridocorneal angles. advanced glaucoma was characterized by increased iop, narrow to closed iridocorneal angles, lens dislocation, optic atrophy, and progression to phthisis bulbus. scanning electron microscopy was performed in a small number of dogs and correlated with the gonioscopy observations. affected dogs responded positively to water provocation (gelatt et al., a) and dem onstrated decreased facility of aqueous humor outflow at all stages of the glaucomatous process when compared to normal dogs, both tonographically (gelatt et al., c) and by constant pressure perfusion (peiffer et al., b) . responses to topical autonomie agents whitley et al., ) and carbonic anhydrase inhibitors (gelatt et al., ) have been studied, and histochemical studies of adrenergic and cholinergic receptor sites have been performed (gwin et al., a) . peiffer and gelatt ( ) described gross and light microscopic observations of the iridocorneal angle; data sup ported gonioscopic observations that the disease appeared to be an open angle glaucoma, with secondary pathology of the angle structures noted. inflammation of the uveal tract is a common and challenging enigmatic clini cal entity that encompasses the variables of inciting stimulus, host response, and associated alteration of ocular structure. the infections uveidites are discussed elsewhere; this section will review noninfectious uveal inflammation (primarily immune-mediated in nature) in animal models, which have proved useful in defining the etiopathogenesis of disease processes; enhancing of our understand ing of the immune response in general and specifically in regards to the eye, a rather unique organ, immunologically speaking; and investigating pharmaco logie mediation of the disease processes. limitations of these models should be noted: inflammation of the uveal tract in the animal model, regardless of species, tends to be an acute, self-limited dis ease. in addition to antigens derived from ocular tissue, complete freund 's adjuvant must be given to induce experimental allergic uveitis (eau). models of chronic uveitis have been particularly difficult to develop and require repeated immunizations with the inciting antigen. even in such models, the inflammation is usually restricted to the anterior segment, and there are minimal retrograde changes compared to chronic human uveitis. several studies have shown that the eye is not an immunological privileged site. allogeneic tissue implanted into the anterior chamber triggers an im munologie reaction. franklin and pendergrast ( ) observed that allogeneic thyroidal implants were rejected by the rabbit eye in a histologie manner and chronologic sequence similar to that for implants to other parts of the body. kaplan and his associates ( ; kaplan and streilein, ) have reported that although the rejection of allogeneic implants placed in the anterior chamber of inbred rats is delayed, the immunologie recog nition via the afferent limb of the immune response is intact. since ocular implant vascularization is coincident with that of implants in other areas of the body, immunologie suppression is probably responsible for the delay in the transplanta tion rejection observed (raju and grogan, ) . in studies of anterior chamber immunization, kaplan and streilein ( ) have demonstrated that the allogeneic antigens present in the anterior chamber are processed immunologically by the spleen via a vascular route, since afferent lymphatic channels do not drain the anterior chamber. they suggest that immune deviation occurs as a result of splenic suppressor factors which delay anterior chamber graft rejection. work by vessela and his co-workers ( ) in the rat has supported this theory. their model suggests that antigen processing and recognition occur, but that effector response is delayed. a possible delay mechanism could be low dose antigen sensitization with tolerance because of the lack of lymphatics, but a more likely explanation would be the presence of suppressor factors including nonspecific serologie blocking factors, specific antigen-antibody blocking complexes, sup pressor macrophages, or suppressor t cells. the majority of experimental animal investigations have been performed using models of sympathetic ophthalmitis or lens-induced uveitis. the role of immunologie mechanisms in the sympathetic ophthalmitis models has been fairly well characterized. the vast majority of this literature does not distinguish whether the immunologie response is primary or whether it is merely an epiphenomenon resulting from an alteration in the ocular antigens produced by another, nonimmunologic, insult. the physical location and biochemical characterization of antigens responsible for the induction of sympathetic ophthalmitis models of eau have been studied extensively. earlier studies dealt with the identification of uveal antigens; bilat eral uveitis can be produced by homologous immunization (aronson, ) . the inflammation tends to be nongranulomatous; however, occasional reports (col lins, (col lins, , describe lesions histopathologically similar to the granulomatous process seen in human sympathetic ophthalmitis. vannas et al. ( ) pro duced experimental uveitis in rabbits by enucleating one eye and implanting it in the peritoneal cavity. aronson and co-workers ( a,b,c) demonstrated that uveal preparations from albino guinea pigs are antigenic, suggesting that melanin is not a vital antigenic component of the disease process. a number of workers have demonstrated that antigens from the rod outer segments and retinal pigment epithelium can induce experimental allergic uveitis in primates, guinea pigs, and rabbits (wong et al., ; meyers and pettit, ; hempel et al., ) . while previous workers had demonstrated that crude extracts of uveal tissue can also produce eau, faure et al. ( ) have suggested that the activity observed with these uveal preparations was probably due to contamination by retinal antigens, and most investigators accept their hypothesis that retinal antigens are more important than uveal antigens in eau. wacker ( ) and wacker and his associates ( ) have demonstrated that antigens present in the photoreceptor and retinal pigment epithelial layers can elicit the development of chorioretinitis and have partially characterized the responsible antigens. there is a soluble antigen (s antigen) located throughout the photoreceptor layer. this s antigen is most active in the production of eau; animals with the disease often develop delayed hypersensitivity responses to it. the s antigen appears biochemically similar to a protein subunit of retinolbinding lipoglycoprotein present throughout the photoreceptor layer. s antigen is apparently tissue-specific; crude bovine s antigen was relatively ineffective in the induction of guinea pig eau. a more purified preparation, however, resulted in a histopathologic lesion, with cellular infiltration in the anterior uvea and destruction of the photoreceptor layer. thus a purified extract of zenogeneic antigenic material had characteristics similar to those of the tissue-specific allogeneic extract. the s antigen is probably not rhodopsin, since a number of physical characteristics, including solubility, molecular weight, amino acid se quence, specific location, and the absorption spectrum, are different. the authors also identified a particulate antigen (p antigen), located in the rod outer seg ments, that does not elicit a delayed hypersensitivity response and has a lower eau induction rate. it does elicit the development of antibodies, even in the absence of disease. in most models of experimental uveitis, cell-mediated, rather than humoral, immune responses are most important in the pathophysiology of these diseases. delayed hypersensitivity reactions to inciting antigens have correlated with the onset and course of disease in eau; passive transfer experiments using lymphoid cells have been conducted; cellular mediators (lymphokines) have produced var iations of eau; and lymphoid cell depletion experiments have been performed. friedlander and his associates ( ) defined a predominantly eosinphilic re sponse to delayed hypersensitivity in the guinea pig. meyers and pettit ( ) demonstrated in the guinea pig both cutaneous de layed hypersensitivity and macrophage migration inhibition reactions toward rod outer segments and retinal pigment epithelial antigens in eau. in their study, cellular immunity appeared to correlate with clinical disease; specific antibody to the inciting antigen was absent in a number of animals that developed eau, and there was no correlation with humoral immunity. in a similar study, wacker and lipton ( ) demonstrated that delayed hypersensitivity toward the inciting antigen is usually present when eau is induced; in a number of animals who developed uveitis, no antibody response was detected. experimental uveitis can be passively tranferred to normal animals with lym phoid cells but not with serum from animals with eau (arnonson and mcmaster, ). chandler and his co-workers ( ) demonstrated that an ocular inflammation resembling uveitis can be induced with lymphokines produced by sensitized lymphocytes, and other investigators have demonstrated that experi mental uveitis can be abrogated using anti-lymphocyte serum (bürde et ai, ) . in a mouse viral model the induction and maintenance of lymphocytic choriomeningitis (lcm) virus-induced uveitis is dependent on the thymusderived lymphocytes . immune spleen cells adaptively trans ferred to immunosuppressed animals did not receive immune lymphoid cells, no uveitis was produced; if the immune spleen cells were treated with anti-theta serum to eliminate the t cells, the uveitis was also aborted. while cell-mediated immunologie alteration appears to be responsible for the majority of experimental allergic uveitis models, in experimental lens-induced granulatomatous uveitis (elgu) humoral immunity is important. needling of the lens will provoke a uveitis in rabbits provided they also receive an intramus cular injection of freund's adjuvant (müller, ) . homologous lens antigens injected into the eye have minimal effect in the absence of adjuvant (müller, ; goodner, ) , and even animals previously sensitized by repeated injec tions of lens material into the footpad fail to mount more than a limited uveitis when challenged with an intravitreal injection of either homologous or heterologous lens antigens (selzer et al., ) . findings such as these prompted the search for a naturally occurring adjuvant which might account for the spon taneous disease; halbert et al. ( a,b) were able to show that streptococcal extracts are able to potentiate lens reactions, although to a lesser degree com pared to freund's adjuvant, while burky ( ) had already demonstrated a similar effect in rabbits with staphylococcal extracts. clinical lgu, however, is not associated with bacterial infection. as lens-induced uveitis is essentially a feature of old and cataractous lenses containing a high proportion of insoluble material, behrens and manski ( a) focused attention on the possible adjuvant effect of albuminoid lens protein. they found that a single injection of albuminoid into the vitreous of inbred rats produced after about days a uveitis characterized by an initial neutrophil and macrophage response, whereas a similar injection of crystallins was essentially without effect; this suggested that albuminoid may have an effect similar to freund's adjuvant. other experiments in rats previously sensitized with whole lens preparations showed that intraocular challenge with albuminoid evokes a cellular response consisting initially of neutrophils followed by macrophages whereas crystallins, particularly a-crystallin, give rise to round cell exudation (behrens and manski, b) . these findings suggest that the soluble antigens are responsible for humoral antibody formation and that insoluble albuminoid accounts for cell-mediated responses. müller ( ) has drawn attention to the enhancing effect of previous sensitization on the uveal response to lens proteins, having found that injection of homologous lens tissue in the presence of freund's adjuvant some time before needling the lens gives rise to a uveitis of marked proportions. marak and his associates ( ) have demonstrated that elgu can be transferred with serum. immunofluorescent studies of traumatized lenses of sensitized animals have demonstrated the presence of igg and complement, components of an immune complex-mediated reaction. depletion of the third component of complement using cobra venom factor or anti-leukocyte serum to inactivate polymorphonuclear lymphocytes (pmns) decreased the incidence of elgu. while it is most likely that elgu is an immune complex disease, immune complexes in the serum or aqueous of experimental animals have not been demonstrated. carmichael et al. ( ) reproduced the uveitis that accompanied infectious canine hepatitis adenovirus infection or vaccination demonstrating an immune complex disease resulting form soluble virus-antibody complexes and the asso ciated pmn cell response. there is significant variance in the incidence of experimental allergic uveitis produced in different species of animals. genetic influences can markedly alter immune responses; in congenic animals which differ only in the region of the chromosome containing immune response (ir) genes, marked differences in the incidence of autoimmune disease, malignancy, and infections have been noted, and it is possible that genetically determined differences in immunologie reactiv ity may be important in the development of uveitis in both humans and animals. there is a paucity of data using experimental animal models to determine the importance of immune response genes in the development of uveitis. in guinea pigs, the incidence of uveitis differs markedly between different strains. while it is relatively easy to induce uveitis in the hartley or nih strains, it is slightly more difficult to induce it in strain and almost impossible to induce it in strain (mcmaster et al., ) . mice that have well-characterized histocompatibility and immune response gene systems are a potential model to further study this phenomenon. (silverstein, ; hall and o'connor, ) . is there a specific immunologie reaction by these cells toward uveal antigens that is important in the production of human endogenous uveitis, or is the reactivity observed merely as an epiphenomenon? an equally feasible mechanism for the prolongation of an ocular inflammatory response could be the structural alteration induced by a nonspecific immunologie reaction. in the rabbit the development of systemic immune complex disease, with nonocular antigens, results in a change in ocular vascular permeability (gamble et al., howes and mckay, ) . it is conceivable that, once an animal receives this type of insult to its vascular system, the structural alteration of the ocular tissue is such that a chronic uveitis either develops or continues despite the lack of specific immune reactivity toward ocular antigens. the structure, composition, and physiology of the lens has been studied in a wide variety of animal species. investigators in specific areas of lens research, however, have tended to concentrate on one type of animal model. thus, much of what is known about the embryology of the lens is derived from research with chick embryos. likewise, the characterization of lens proteins is based largely on research with bovine lenses, and the physiology and metabolism of the lens has been studied mainly in rabbits and rats. some caution must be exercised, there fore, in extrapolating data from one animal model to another since significant differences undoubtedly exist. the chick lens, for instance, has a very different protein composition than that of the mammalian lens (rabaey, ) . there are a number of important differences between the lens of all the com monly studied laboratory animals and the lens of humans and other higher pri mates as have been pointed out by van heyningen ( ) . the rat, rabbit, and bovine lens all demonstrate minimal growth during adulthood, whereas the human lens continues to grow throughout life. also, the human lens tends to maintain its water content at about %, while lower animals have a decrease in water content during old age. the composition of the primate lens is different from all others in that it contains a group of tryptophan derivatives, hydroxykynurinines, which are highly absorbant in the ultraviolet range, and, fi nally, only the higher primates appear to have the ability to change the shape of their lens in accommodation. in addition to the higher vertebrates, the amphibians also deserve mention for their contribution to lens research. newts and salamanders have long been recog nized for their ability to regenerate a lens from the pigmented epithelium of the iris (reyer, ) . toads, on the other hand, demonstrate a similar ability to regenerate a lens from cells derived from the cornea. some of the techniques involved in this type of study are discussed by stone ( ) . more recently eguchi and okada ( ) have caused renewed interest in the relationship be tween pigmented epithelium and lens by demonstrating lenslike structures in cultures of retinal pigment epithelium from chick embryos. the dry weight of the lens is comprised almost entirely of proteins, the trans parency of which is the sine qua non of lens function. understandably, then, a large part of lens research has been aimed at analyzing these proteins. the techniques used include the complete armamentarium of sophisticated tools available to the protein chemist. as previously mentioned, the bovine lens, because of its large size and availability, has been most extensively used, al though the rabbit has also been studied in some depth. mammalian lenses consist of three major classes of soluble proteins, known as α-, ß, and γ-crystallins, and an insoluble fraction, predominantly albuminoid, most likely derived from a-crystallin. the separation of lens proteins can be achieved in a variety of ways depending on the objectives of the experiment. most recent studies, however, begin by separating the lens crystallins into major classes by gel filtration chromatography. specifically, sephadex g , ultragel ac a , and biogel p all yield four major protein peaks corresponding to a-, ßn-, ßh~y and γ-crystallin (bloemendal, ) . sephacryl s- has also been used to separate the / -crystallins into four subclasses (mostafapour and reddy, ) . further separation into subclasses is often done utilizing polycrylamide electrophoresis, and still more refined separations can be achieved using twodimensional electrophoresis and immunochemical techniques. an important concept of lens composition which has been elicited by the use of immunochemical identification of proteins is that of organ specificity, as opposed to the more usual rule of species specificity. immunoelectrophoresis can be used to demonstrate cross-reactions between lens proteins of species which are widely separated on the phylogenetic scale. in general, antibodies to the lens of one species will show extensive cross-reactivity to lens proteins of species lower on the evolutionary scale, indicating that these antigens have been carried over as evolution has progressed. this technique has been useful in defining some basic evolutionary relationships. immunoelectrophoresis and other modern methods for analysis of lens proteins are discussed by kuck ( ) . more recently a technique for radioimmunoassay of a-and γ-crystillin which is sensitive to very minute amounts of protein has been described (russell et al., ) . systems for in vitro study of the intact lens may be of the open type, where the lens is continually perfused with fresh media, or the closed system in which there is no exchange or only intermittent exchange of culture media. very elaborate open systems have been designed for accurately measuring such functions as metabolite production and oxygen consumption by the lens (schwartz, a ,b, sippel, . although the open systems theoretically approximate the physiologic state more closely, closed systems similar to the one described by merriam and kinsey ( ) continue to be much more widely used because of simplicity and the potential for maintaining several lenses simultaneously. since a long series of articles has been published by kinsey et al. describing a variety of in vitro studies primarily using closed culture systems. these articles may be traced by referring to a recent addition to the series (kinsey and hightower, ) . if measuring exchange of materials between lens and culture media is not an important part of the experimental design, a compromise between open and closed system has been described by bito and harding ( ) which involves culturing lenses in dialysis bags with intermittent changes of the outer media. there is an overwhelming amount of literature describing agents capable of inducing experimental cataracts in animals and the subsequent chemical and metabolic effects of those agents. an attempt will be made here to review some of the most important and interesting methods of producing experimental cataracts. for a more complete list of experimental cataractogenic agents the reader is referred to reviews by van heyningen ( ) and kuck ( kuck ( , . rabbits and rat have been the most popular animals for this type of research. it is interesting that despite the vast amount of information accumulated on the mechanisms of cataractogenesis, the etiopathogenesis of senile cataracts so com mon in man remains undefined. a variety of physical insults ranging from the relatively gross technique of sticking a needle through the lens capsule to bombarding the lens with a neutron beam will induce cataracts in animals. by far the most widely studied types of cataract under this category is that produced by ionizing radiation. radiation cataracts have been studied in many different laboratory animals, most of which make satisfactory models with the exception of the bird which seems to be resistant to radiation cataract (pirie, ) . the lenses of younger animals are generally more susceptible to radiation, and the periphery of the lens which contains the actively dividing cells is much more sensitive than the nucleus (pirie and flanders, ) (fig. ). x rays, y rays, and neutrons are all potent cataractogens. beta radiation in sufficient doses can also be used to cause cataracts (von sallmann et al., ) . much of the early work on ocular effects of ionizing radiation is reviewed by duke-elder ( ) . because of the recent boom in microwave technology, cataracts caused by this type of radiant energy have received increased attention. the cataractogenic potential for low doses of microwave radiation is probably minimal, however. hirsch et al. ( ) and appleton et al. ( ) found that lethal doses of microwaves were often required to produce cataract in rabbits. ultraviolet radiation, although not ordinarily thought of as cataractogenic, can cause cataracts experimentally in animals when a photosensitizing agent is ad ministered simultaneously (see toxic cataracts), which illustrates the important concept that two or more cataractogenic factors may act synergistically to pro duce lens opacities (hockwin and koch, ) . diets consisting of more than % galactose will consistently produce cataracts in young, rats. this type of experimental cataract has obvious clinical relevance to galactosemia in humans and the secondary cataract which often develops in this disease. the work of kinoshita et al. ( ) and others in explaining the mechanism of this cataract have demonstrated that this model is also applicable to the study of diabetic cataracts. effects of cataractogenic sugars (galactose, xylose, and glucose) have also been studied in vitro using rabbit lenses (chylack and kinoshita, ) . diabetic cataracts may also be studied more directly by surgical or chemical induction of diabetes in laboratory animals. patterson ( ) has used alloxan, mg/kg in a single intravenous dose, to induce diabetes in rats but other tech niques have also been effective including % pancreatectomy and intravenous dehydroascorbate (patterson, ) , and intravenous streptozotocin in some spe cies (white and Çinottim, ) . a large number of drugs and toxins have been demonstrated to cause cataracts in both man and animals. toxic cataracts are reviewed in depth by kuck ( ) . many of these agents have been of particular interest because of their therapeutic use in humans. metabolic inhibitors, not surprisingly, frequently cause cataracts. one such inhibitor, iodoacetate, has been shown to produce cataracts in rabbit lenses in vivo, in vitro, and, interestingly, by direct injection into the lens (zeller and shoch, ) . dinitrophenol, a metabolic inhibitor used for weight loss in humans until a high incidence of cataracts was recognized, will also induce cataracts in the chicken (buschke, ) . sodium cyanate, used experimentally in sickle cell anemia, has recently been shown to cause cataracts in the beagle (kern et al., ) . cytotoxin agents are another group of drugs associated with cataracts. busulfan and triethylene melamine cataracts have been studied in the rat as has dibromomannitol grimes, , ) . recently bleomycin has been added to the list of cytotoxins with cataractogenic potential, causing cataracts in to -day-old rats injected intraperitoneally with a dosage of - gm/logm (weill et al., ) . drugs which cause cataracts through more subtle effects on lens metabolism include corticosteroids, anticholinesterose inhibitors, and a number of drugs which interfere with lipid metabolism. steroid cataracts, although relatively common in humans, have been difficult to reproduce in animals, but tarkkanen et al. ( ) and wood et al. ( ) have reported success in rabbits using subconjunctival and topical steroid preparations. anticholinesterase cataracts have also proved challenging to reproduce in animals, with the monkey being the only animal model available at present (kaufman et al., ) . triparanol, which blocks cholesterol synthesis, and chloroquine and chlorphentermine, which also affect lipid metabolism, have all been shown to produce cataracts. a reversible cataract is seen in rats receiving a diet containing . % triparanol; the cataract could not be reproduced in rabbits (harris and gruber, ). an un usual anterior polar cataract is produced with chloraquine and chlorphentermine (drenckhahn, ) . a group of drugs with the potential to produce cataracts by photosensitizing the lens to ultraviolet light has generated considerable attention. chlorpromazine and methozyproralen have been used in rats to study this effect (howard et al., ; jose and yielding, ) . of the nontherapeutic agents known to cause cataract, naphthalene has been studied most thoroughly, and lens opacities have been induced in the rabbit, rat, and dog. the mechanism of this type of cataract has been studied extensively by van heyningen and pirie ( ) . naphthalene can be used to create congential cataracts in rabbits (kuck, ) . toxic congential cataracts have also been seen in mice secondary to corticosteroids (ragoyski and trzcinska-dabrowska, ) . congenital cataracts can be due to infections as well as toxic and metabolic insults in vitro. although no animal model of rubella cataract is available for study, three models of viral-induced cataracts in animals have been reported. the enders strain of mumps virus, injected into the chick blastoderm prior to dif ferentiation of the lens pläcode, has been reported to cause cataracts in the survivors (robertson et al., ) . hanna et al. ( ) showed that subviral particles of st. louis encephalitis virus injected intracerebrally into -day-old rats would cause cataracts in survivors. finally, a poorly categorized infectious agent believed to be a type of slow virus, the suckling mouse cataract agent (smca), produces cataracts when injected intracerebrally in mice less than -hours old (olmsted et al., ) . smc a has also been studied in vitro by infecting cultured rabbit lenses (fabiyi et al., ) . cataracts due to amino acid deficiencies can cause cataracts in rats (hall et al., ) . the most reproducable form of this type of cataract is that due to tryptophan deficiency which can be demonstrated in guinea pigs by feeding a diet containing less than . % tryptophan (von sallmann et al., ) . despite the skyrocketing proliferation of intraocular lenses for use in humans following cataract extraction, very little animal experimentation has been done in *ff:##p s *" this area. animal selection should consider size and shape of the anterior chamber, pupil, and lens as well as species-related reactivity to surgery and lens implantation. in general, nonprimate mammals respond to intraocular manipula tions with increased exudation and inflammation compared to primates and hu mans. sampson ( ) implanted ridley-type lenses in nine dogs undergoing cataract extraction. schillinger et al. ( ) reported disappointing results with crude lenses implanted in rabbit and dog eyes. eifrig and doughman ( ) used modern iris fixation lenses in rabbits (fig. ) . rabbits were prone to develop corneal edema after lens extraction with or without lens implantation, but edema was prolonged in eyes receiving lenses. cats have been used for implantation of lenses with slightly less technical difficulty than rabbits, but their vertical slit pupil is not ideal. because of their large anterior chamber, cats are not suitable for implantation of standard-sized anterior chamber lenses. degeneration of the retina has been a leading cause of visual impairment and partial or complete blindness in both man and animals. many causal factors have been defined and may be categorized as either genetic, chemical, developmen tal, or environmental. the latter category has received the majority of experi mental attention, being primarily concerned with the induction of retrograde changes by chemicals, radiant energy, physical trauma, nutritional deficiency, and viral infections. the multiplicity of genetic models will be dealt with briefly because rather than specific techniques they are entities within themselves. the wag/rij rat is described by lai ( ) as the "retinitis pigmentosa model," featuring a slowly progressive photoreceptor degeneration thought to be an autosomal dominant trait. at present the genetic model for human retinal dystrophy is the rca (tan-hooded) or 'dystrophie rat" (herron, ) . in this animal rod photorecep tor degeneration occurs because the retinal pigment epithelial (rpe) cells do not phagocytize shed rod outer segment photopigment discs. the (rd) mutant mouse has proved to be helpful to evaluate the effects of photoreceptor degeneration on the bipolar cell terminal synapse (blanks et al., ) . feline central retinal degeneration involving a diffuse atrophy of retinal cones has been described and is a potential model for human macular degeneration (bellhorn and fischer, ) (fig. ) ; the genetics have not been defined (fischer, ) . a tool for studying retinal degeneration in mice and rats involves the use of chimera pro- inherited canine retinal degenerative disease collectively described as "pro gressive retinal atrophy" has been recognized in a number of breeds (magnusson, ; hodgmen et al, , parry, barnett, a) (fig. ) (table ii) . genetic studies have revealed the majority to be inherited as a simple autosomal recessive trait, with three genotypes in the population: the homozygous normal, the heterozygous normal carrier, and the homozygous affected dog. clinical, electroretinographic, and transmission electron microscopic studies have been utilized to define distinctly different conditions in various breeds including rod-cone dystrophy in poodles (aguirre and rubin, ) and central progressive retinal atrophy in other species (parry, ; barnett, b; aguirre and laties, ). evans rats and produced a retinal degeneration that involved both photoreceptor cells and rpe. fluorescein angiography and electron microscopy were utilized to assess altered retinal vascularity. these findings were likened to retinitis pigmentosa in man. to evaluate retinal changes caused by phenylalinine, the drug was adminis- tered subcutaneously to newborn rats for week, producing profound damage to the bipolar and ganglion cell layers. the presence of the immature blood-brain barrier was suggested to be significant in the development of the lesions (colmant, ) . hamsters were used by another investigator to produce retinal pigmentary degeneration with yv-methyl-jz-nitrosourea, this model being suggested for use in screening the retinotoxicity of carcinogenic drugs. it should be pointed out, however, that the fundus was difficult to visualize, and the results were derived from histopathology (herrold, ) . male dutch-belted rabbits were given different levels of oxalate compounds subcutaneously to study the "flecked retina" condition seen in oxalate retinopathy. this model is also pro claimed valuable for assessing b vitamin deficiencies, ethylene glycol poisoning, and methoxyfluorane anesthesia toxicity (caine et al., ) . four-to month-old pigmented rabbits were used by brown in to study the effect of lead on the rpe. the electroretinogram, electrooculogram, and flat mount and histopathological preparations were utilized. the erg and eog recordings were normal, suggesting no major disturbance of the rpe. one of the smaller new world primate species, the squirrel monkey, was used by mascuilli et al. ( ) to evaluate the effects of various elemental iron salts on the retina. ocular siderosis with rpe and photoreceptor cell damage was evident as early as hours after intravitreal administration. the erg was used to study these effects. synthetic antimalarials have long been recognized as producing ocular com plications in humans, especially macular pigmentary disturbances. berson ( ) administered chloroquine to cats to assess acute damage to the retina, utilizing intravitreal and posterior ciliary artery injections (figure ). hey wood ( ) mentions the affinity of chloroquine and related drugs for melanin pig ment. miyata et al. ( ) used the long evans pigmented rat to evaluate the effects of intramuscular fenthion on the retina. erg as well as histopathologic changes were extensive months after administration. a brief review of retinotoxic drug effects illustrated in color is available in the "atlas of veterinary ophthalmoscopy" (rubin, ) . included are funduscopic representations of ethambutol effects in the dog, naphthalene in the rabbit, chloroquine and related drugs in rabbits and cats, and a selection of other re tinotoxic agents. in the study of induced retinal degeneration, especially these conditions with rpe involvement, pigmented animals are more desirable than albinos. the sub- human primate is the only species with a macula comparable to the human, although birds may possess dual foveae. some mammals have an area centralis, a zone of high cone concentration that, while similar, is neither functionally nor anatomically identical to the macula. species differences in relative numbers of rod and cone photoreceptors varies with the animals' ecological niche, with nocturnal creatures exhibiting a preponderance of rods and diurnal animals more likely to possess a cone dominant retina. with these exceptions (not including variations in retinal vascular patterns, discussed elsewhere), the structural and functional relationships of the neurosensory retina are remarkably similar thorughout the vertebrates. a multitude of variations producing this type of degeneration have been stud ied including fluorescent, incandescent, ultraviolet, infrared, and colored light excesses with continuous or interrupted exposure patterns, high and low level intensities, and other variations. additional factors such as age, sex, body tem perature, species, and nutrition have also been simultaneously evaluated. lai et al. ( ) used fisher albino rats exposed to a high-intensity fluorescent light to produce a severe peripheral retinal degeneration. a continuous highintensity fluorescent light exposure regimen for albino and hooded rats produced a more severe photoreceptor cell degeneration in the former (reuter and hobbelen, ). low-intensity continuous colored lights disclosed that white and blue bulbs produced the most damage in adult albino rats . it is apparent that continuous light sources are the most damaging to the albino animal, the higher the intensity the more severe the degeneration. newborn albino species, however, will show initial outer segment growth before damage eventually occurs (kuwabara and funahashi, ) . in , berso evaluated the -striped ground squirrel, which has an all-cone retina, under high intensity illumination. essentially times the amount of light required to produce cone degeneration in the albino rat produced "retinitis punctata albescens" in the ground squirrel. zigman et al. ( ) a more specific study was performed by tso et al. ( ) in rhesus monkeys to assess the effects of a retinally focused, low-intensity light beam under normal and hypothermie conditions. although no difference was detected due to body temperature, a progressive degeneration that involved the rpe and photoreceptor cells was identified in both study groups. the recent development of laser application in ocular therapy prompted study in laboratory species. gibbons et al. ( ) used rhesus monkeys to study visible and non visible lasers at high-and low-power levels and found that a -hour exposure of the latter was equivalent in rpe damage to hours of the former. in another study tso and fine ( ) exposed the foveolas of rhesus monkeys to an argon laser for - minutes and found cystoid separations of the rpe and bruchs membrane at - years post-insult. good anesthesia and subject stabiliza tion are obvious requirements for these procedures. the advent of microwave radiation for everyday use has initiated considerable evaluation of its safety for the human eye. retinal damage in the form of synaptic neuron degeneration was produced in rabbits by exposure to mhz of microwave radiation (paulsson et al., ) . ultrastructural changes were dis covered that had not previously been seen, and these resulted from lower levels of energy than anticipated. solar retinitis has long been a problem in man but recently has increased due to greater numbers of unprotected people being exposed to snow-reflected sunlight. ham et al. ( ) produced photochemical (short wavelength) and thermal (long wavelength) lesions in rhesus monkeys by exposure to a w xenon lamp, stimulating in the former case the solar spectrum at sea level. this model demon strated the short wavelength causal nature of sunlight overexposure, resulting in solar retinitis and eclipse blindness. higher energy radiation effects have been evaluated by other means including high-speed particle acceleration. proton irradiation of discrete areas of the retina in owl monkeys produced full thickness retinal damage (gragoudas et al., ) . rhesus monkey retinal irradiation by highly accelerated ( mev) oxygen nuclei produced extensive retinal vascular damage followed by degrees of degen eration and necrosis of the neural layers (bonney et al., ) . traumatic optic nerve injuries in humans are not so unusual as diseases that attack and destroy the optic nerve. in either case, however, retinal degeneration frequently follows. ascending and descending optic atrophy was produced in squirrel monkeys by anderson ( ) to study the degenerative process. total optic nerve neurectomy by razor blade knife via lateral orbitotomy was per formed to assess the descending condition, while xenon arc photocoagulation was performed to create the ascending condition. electron microscopy demon strated degenerative changes effecting the nerve fiber and ganglion cell layers. both methods produced morphologically similar retrograde processes; photocoagulation, however, provided evidence of ascending atrophy within weeks. commotio retinae or traumatic retinopathy was initially induced in rabbits by berlin ( ) who used an elastic stick to produce retinopathy, sipperley et al. ( ) employed a bb gun technique that delivered a standard sized metallic bb which struck the cornea between the limbus and pupillary axis to produce the desired contrecoup injury of the posterior pole. the segments of the outer nuclear photoreceptor layer were specifically effected, the authors using fluorescein an-giographic, histopathologic, and electron microscopic results to demonstrate the disease. retinal degenerative conditions caused by nutritionally related problems have been studied in rats, cats, and monkeys. these studies have mostly been con cerned with vitamin deficiencies, and for economic reasons have lent themselves well to the rat as a model. the subhuman primate may be geneologically ideal for certain investigations, but it is equally as impractical for fiscal reasons. the cat has been used because of a specific susceptibility to a deficiency of the amino acid taurine (fig. ) . in hayes et al. used young and adult cats to assess the effects of taruine deficiency by feeding a diet of casein, which contains very little cystine, the major precursor for taurine. in months a photoreceptor cell degeneration was produced, the initial signs including a hyperreflective granular zone in the area centralis. the erg recordings indicated a photoreceptor degenerative process, while morphologically the outer nuclear and plexiform layers were destroyed. recently, wen et al. ( ) discovered that taurine deficiency in cats produces in addition to the photoreceptor cell a tapetal cell degeneration, thus suggesting that this amino acid also plays a role in maintaining structural integrity in this tissue. these investigators employed the use of the visual evoked potential (vep) technique to arrive at their conclusions. due to its known unusually high concentration in the retina, numerous species, such as chickens, rabbits, rats, and frogs, have been used to define the biologic function of taurine. pourcho ( ) utilized intravenous and intravitreal radiolabeled s-taurine. the cat, mouse, rabbit, and monkey exhibited higher concentrations of taurine in müller cells, whereas, by comparison, the chick and frog showed very little. vitamin a deficiency has been studied in various species, the rat having been utilized most extensively because it is susceptible to night blindness as well as being less expensive and readily available. carter-dawson et al. ( ) used the offspring of vitamin a-deficient pregnant female rats to accelerate the desired condition of tissue depletion. under low levels of cyclic illumination ( . - ft-c) both rhodopsin and opsin levels decreased, the latter requiring a longer period of time. in addition it was determined that rod cells degenerated before the cone cells. in another experiment, autoradiographic techniques were used to assess the influence of vitamin a deficiency on the removal of rod outer segments in weanling albino rats (herron and riegel, ) . the process was distinctly retarded by the absence of the vitamin. in a combination study, robison et al. ( ) evaluated the effects of relative deficiencies of vitamin a and e in rats. autofluorescent and histochemical techniques revealed that the vitamin e-free diets produced significant increases in fluorescence and lipofucsin granule stain ing regardless of the vitamin a levels. in conclusion vitamin e was thought to prevent light damage by scavenging oxygen radicals and thereby providing pro tection via lipid peroxidation. two different species of subhuman primates (ce bus and m acaca) were used by hayes ( ) to compare (separately) vitamin a and e deficiencies. after years on a vitamin e-deficient diet a macular degeneration developed, charac terized by a focal massive disruption of cone outer segments. vitamin a defi ciency, on the other hand, produced xerophthalmia, keratomalacia, and impaired vision in six ce bus monkeys, the latter due to cone degeneration in the macula and midperipheral retina. information describing these techniques is available from a previous publication (ausman and hayes, ) . the ocular effects of two slow virus diseases have recently been studied: scrapie agent in hamsters (buyukmihci et al., ) and borna disease in rabbits (krey et al., ) . the scrapie agent when inoculated intracerebrally in young hamsters produced a diffuse thinning of the retina, the photoreceptor cell layer being most severely affected (fig. ) . volvement in each foci. viral antigen was detected in these lesions as well as in nonaffected areas, thus suggesting an immune component involvement. this condition was likened to ocular pathology seen in subacute sclerosing panencephalitis in humans. friedman et al. ( ) injected newborn albino rats with simian virus (sv ) and demonstrated viral antigen by immunofluorescense at hours. adult rats injected at birth developed retinal neovascularization, folds, and gliosis, features similar to retinal dysplasia. in comparison, an extensive retinopathy was produced by monjan et al. ( ) by infecting newborn rats with lymphocytic choriomenigitis virus. the outer nuclear layers of the retina were initially effected, followed by the inner nuclear and ganglion cell layers and finally total destruction. due to a modest inflammatory infiltrate, this condition was also suspected to be immunopathologic in nature. the critical assessment of retinal degeneration has been done through the implementation of five basic techniques: ( ) electroretinography (erg), ( ) electrooculography (eog), ( ) visual evoked response (ver), ( ) autoradiography, and ( ) light and electron microscopy. the concept of electroretinogram (erg) was first demonstrated by holmgren in using the enucleated eye of a frog. this technique is in principle applica ble to both animal and man and provides a detailed assessment of the rod-cone (visual cell) nature of the retina as well as its functional status. it is based upon the summation of retinal electrical potential changes which occur in response to light stimulation and are measured via corneal or skin surface electrodes (fig. ) . detailed analyses of the erg can be obtained from brown ( ) as well as information concerning the use of microelectrodes. additional information describing the local electroretinogram (lerg), a more precise but invasive technique, is available from rodieck ( ) . the organization of the vertebrate retina and the origin of the erg potential change has been elucidated by the use of intracellular readings. the mudpuppy, necturia maculosa, has been a favorite subject because of its large retinal cells. excellent reviews of comparative retinal organization and function are provided by dowling ( ) and witkovsky ( ) . the electrooculogram (eog) is a clinically applied test of retinal function that was first measured in the eye of a tench by du bois-reymond in . this test assesses the standing or corneofundal potential which exists between the anterior and posterior poles of the eye as a subject is taken from a dark to a light adapted state. for detailed information concerning this technique refer to krogh ( ) (rabbit) or arden and ikeda ( ) (rat) . a newer technique, the visual evoked potential (vep), has been adapted for use msec in animals by wen et al. ( ) . this enables the clinical assessment of visual function in the area centralis as compared to the peripheral retina by measuring potential changes in the visual cortex in response to focal or diffuse retinal stimulation. the techniques described for the cat are also applicable to other species. autoradiography has been available and successfully used in ophthalmologic research for several years (cowan et al. y ) . tritiated amino acids are readily available in various concentrations and forms and may be injected into the vitreous or other ocular tissues where they may become imcorporated into cellu lar protein metabolism pathways. distribution may be studied by light or electron microscopy. ogden ( ) utilized the concept of axoplasmic transport together with autoradiography to trace the course of peripheral retinal nerve fibers to the optic disc. others have utilized this technique to study outer segment photopigment metabolism. rods have been shown to regenerate photopigment discs con tinuously with the rpe participating in the process by phagocytizing the shed products. (young, ; norton et al., ; mandelcorn et al., ) . cones also have mechanisms for photopigment renewal. specific information concern ing autoradiographic techniques may be obtained from cowan et al. ( ) , rogers ( ) , and kopriwa and leblond ( ) . light and electron microscopy have expanded the morphologic study of the retina beyond the limitations of light microscopy. in laties and liebman discovered by chance that the chlortriazinyl dye, procion yellow, was a selective stain for the outer segments of retinal cones in the mud puppy and frog. further investigation by laties et al. ( ) demonstrated the value of this dye in distinguishing between the outer segments of rods and cones and its use in monitoring outer segment renewal. these investigators studied the dye in rat, dog, rabbit, and monkey eyes as well and discovered that the rod basal saccules could not be visualized in the rat and dog with the same certainty as the gekko (nocturnal lizard) and mud puppy. specimen preparation involves the injection of aqueous procion yellow ( . to %) into the vitreous using a /e-inch, -gauge needle. although originally used to described a syndrome consisting of cns and systemic anomalies in the human infant, retinal dysplasia (rd) now applies specifically to any abnormal differentiation of the retina after the formation of the anläge. the histopathologic features normally constituting rd include: ( ) rosette formation, ( ) retinal folds, and ( ) gliosis (lahav and albert, ) . table iii lists those agents that have either been associated with or used in the induction of rd. the administration of blue tongue virus vaccine to fetal lambs appears to be the one technique that offers all of the features described for rd. this technique involves the use of a live, attenuated virus vaccine, and it is effective only during the first half of gestation (silverstein, et al., ) . the rd inducing effects of x-ray exposure on the retina of the primate fetus have been described (rugh, ) . the erg was used in this experiment as well as histopathology to demonstrate the lesions. shimada et al. ( ) and used suckling rats to evaluate the effects of antitumor and antiviral drugs, respectively, the results in both being the induction of rosette formation. in silverstein used the fetal lamb and intrauterine trauma to demonstrate the relationship of the rpe in the organizational histogenesis of the retina (fig. ). experimental retinal detachment was first described by chodin in . the rabbit, dog, and subhuman primate have since become species of choice; the pigmented rabbit model probably the most practical, it being less expensive, more genetically uniform, and easier to manipulate. a spontaneous inherited detachment has been reported in the collie dog, ac companied by choroidal hypoplasia and posterior staphylomas (freeman etal., ) . experimental methods have utilized specifically designed techniques to induce detachment including: ( ) traction detachment by perforating injury (cleary and ryan, a,b) , ( ) intravitreal hyperosmotic injection (marmor, (norton et al., ) , ( ) detachment by experimental circulatory embolization (algvere, ) , and ( ) detachment by blunt needle rotation and suction without the use of hyaluronidase (johnson and foulds, ) . traction detachments are dependent on vitreal hemorrhage and scarring affects (cleary and ryan, a,b), the rabbit and rhesus monkey being successfully utilized in these studies. vitreal changes are significant in most of these techniques, producing detachments of varying dimensions and duration (marmor, ) . embolization of the retinal and choroidal circulations with resultant retinal ischemia produced a long-lasting retinal detachment (algvere, ) ; the technique used plastic (polystyrene) beads injected into the central retinal artery and the supratemporal vortex veins of owl monkeys (aotus trivirgatus). another approach which produced septic choroiditis and multifocal serous retinal detachments in dogs involved the intracarotid injection of pathogenic bacteria (meyers et al., ) . experimental studies of the optic nerve have explored two aspects of this tissue; the pathogenesis of the degenerative changes resulting from increased iop, and the development of an experimental model for allergic optic nearitis, similar to that seen in multiple sclerosis. the former topic is discussed in the section on glaucoma. in regards to the latter, several workers have observed optic neuritis as well as retinal vasculitis and uvertis in guinea pigs, rabbits, and monkeys affected with experimental allergic encephalomyelitis. (raine et al. t ; von sallmann et al., ; bullington and waksman, ) . rao et al ( ) induced papilledema and demyelination of the optic nerve in strain guinea pigs by sensitization with isogenic spinal cord emulsion in complete freunds adjuvant. unlike many other areas of ocular research where evidence obtained from animal models has been accepted with allowances for differences between the human and animal eye, the striking contrasts in ocular blood supply from one species to the next are impossible to ignore. as a result a variety of animal models have been studied in an attempt to sort out those aspects of the vascular anatomy and physiology which are highly variable from those which seem to be generally similar for a number of different species. the rabbit is sometimes used in this line of research, but more often it is supplanted by the monkey, cat, pig, or rat, all of which have an ocular blood supply which is more analogous to that of man. there are some general trends in the comparative anatomy of laboratory ani mals with respect to the ocular circulation. all of the commonly studied animals have a dual circulation to the eye consisting of ( ) the uveal and ( ) the retinal blood vessels. in higher primates, including man, both the uveal and retinal vessels are branches of the ophthalmic artery, itself a branch of the internal carotid; the external carotid system contributes very little to the ocular blood supply. in lower animals, on the other hand, the external carotid system, by way of the external ophthalmic artery, supplies the major portion of blood to the eye. many animals, including the dog, cat, and rat (but not the rabbit), have a strong anastomosing ramus between the external ophthalmic artery and the circle of willis, and since a relatively large part of their cerebral blood supply comes from the vertebral arteries, the common carotid artery may often be totally occluded with no apparent ill effects to the eye (jewell, ) . the retinal circulation in primates is supplied by the central retinal artery which branches off from the ophthalmic artery to enter the optic nerve close to the globe (fig. ) . in lower animals the retinal circulation is more often derived from a network of anastomosing branches of the short ciliary arteries usually referred to as the circle of haller-zinn. in fact, of the nonprimate mammals, the rat is the only animal with a central retinal artery homologous to that of the primates. the presence of a central retinal artery in dogs and cats is disputed (françois and neetens, ) , but even if present it is not the main source of blood to the retina (fig. ) . the extent and configuration of the retinal vessels is even more variable than their source. in most laboratory animals, including primates, dogs, cats, rats and mice, the retina is more or less completely vascularized, or holangiotic. rabbits, however, have only two wing-shaped horizontal extensions of vessels from the optic nerve which are accompanied by myelinated nerve fibers (the medullary rays), and do not actually extend into the retina at all. horses have only a few small retinal vessels scattered around the optic disc (paurangiotic), and birds have a retina which is completely devoid from blood vessels (anangiotic). in summary then the primates have an ocular blood supply which is almost identical to that of man. the rat should also be considered as an animal model in studies of the retinal circulation. the ocular blood supply in cats and dogs is somewhat less analogous to that of the human. the rabbit has some very atypical features, especially of the retinal circulation, which should be considered very carefully before including it in experiments on the ocular circulation. one feature of the orbital vascular anatomy in rabbits which has not been mentioned is a peculiar anastomosis between the two internal ophthalmic arteries which has been implicated in the consensual irritative response in rabbits following an injury to one eye (forster et al., ) . because of the complexity and small size of the intraocular blood vessels, dissection, even under the microscope, is usually inadequate to study the anatom ical relationships of these vessels. corrosion casting techniques, involving infu-sion of the eye with neoprene (prince, l' b) or plastic resin followed by digestion of the tissues, usually with concentrated sodium hydroxide, have been used for study of the uveal circulation and less often the retinal circulation. in our laboratory corrosion casting with a special low-viscosity plastic has been used with excellent results to study both the anterior and posterior uveal as well as the retinal circulation in cats and monkeys (risco and noapanitaya, ) . castings are studied with the scanning electron microscope (fig. ) . the retina, because it is thin and relatively transparent, is amenable to simpler studies of vascular morphology. the classic text on this subject is by michaelson ( ) , who based his observations primarily on flat mounts of retina perfused with india ink. other dyes have been utilized in a similar fashion (prince, b) . another popular technique introduced bykuwabara and cogan ( ) involves digestion of the retina in % trypsin at °c for - hours until the tissue shows signs of disintegration followed by careful cleaning away of the partially digested tissue from the vascular tree and then staining with periodic acid-schiff (pas). other investigators believe that trypsin digestion may destroy some of the more delicate or immature vessels and advocate pas staining alone to delineate retinal vessels in immature retina (engerman, a,b; henkind, et al., ) . so far we have mentioned only in vitro studies of the ocular circulation. in vivo studies can provide information about the dynamics of blood flow as well as morphology. the most widely used in vivo technique is fluorescein angiography. fluorescein dye is excited by blue light with a wavelength of mm, emitting a yellow-green light with a wavelength of mm. the blood vessels of the retina and iris are relatively impermeable to fluorescein because of their endothelial tight junctions. thus fluorescein angiography has been useful not only in the study of normal anatomy and time sequence of blood flow in these vessels as demonstrated by the excellent studies in the monkey by hayreh ( ; hayreh and baines, a,b) but also as a test of integrity of the blood-ocular barrier in certain pathological states, especially neovascular lesions. the diffusion of fluorescein across the blood ocular barrier can be assessed quantitatively by fluorophotometric techniques (cunha-vaz, ) . there is evidence that, in mammals at least, the blood ocular barriers are similar for different species (rodriguez-peralta, ) . the basic equipment required is a fundus camera capable of rapid sequence flash photographs and appropriate filters. satisfactory fluorescein angiograms can be obtained in most laboratory animals, even mice and rats, although general anesthesia is usually required. angiograms of dogs, cats, and other carnivora are technically more difficult because the reflection from the tapetum interferes with the observation of retinal blood flow. in these animals color fluorescein angiog raphy may give better results. many of the technical aspects of fluorescein angiography in animals are discussed by bellhorn ( ) . anterior segment angiography of the iris vessels has the advantage of requiring less expensive equipment; a mm camera with a macro lens and rapid recycling flash are essential (rumelt, ) . the rabbit, the most popular experimental animal for anterior segment angiograms, has an anterior segment circulation similar to humans, with the exception of the absence of a contribution from the anterior ciliary arteries to the anterior uveal blood supply, a situation possibly unique among mammals (ruskell, ) . the technique and interpretation of anterior segment angiography is discussed in detail by kottow ( ) . choroidal angiograms are also possible but present a technical problem in that in pigmented animals the retinal pigment epithelium (rpe) absorbs light strongly in both the excitation and emission wavelengths for fluorescein. the rpe is transparent to wavelengths in the infared range and the use of cyanine dyes for infared absorption and emission angiograms of the choroid have been described (hochheimer, ) as well as a technique for simultaneous angiography of the retinal and choroidal circulations using both fluorescein and indocyanine green (flower and hochheimer, ) . tsai and smith ( ) have used fluorescein for choridal angiograms by injecting dye directly into a vortex vein. angiograms of the ciliary circulation in rabbits have been obtained using conventional radio-opaque media and dental x-ray film (rothman et al., ) . the measurement of absolute and relative blood flow, velocity of blood flow, and oxygen content in various ocular tissues has been key to the understanding of nervous control of ocular physiology as well as the ocular effects of many drugs. unfortunately, these measurements have been fraught with technical difficulties. many of the techniques utilized have been indirect methods requiring compli cated mathematical analysis often based on tenuous assumptions. as a result large discrepancies have resulted from the use of different techniques or even the same technique in different hands. to complicate matters further different inves tigators have expressed results in different terms which are not easily interconver tible. the results of many of these studies have been reviewed and tabulated for comparison by henkind et al. ( ) . measurements of total or localized ocular blood flow have been made based on the washout of various gases including nitrous oxide, kr and xe (o'rourke, ) . a heated thermocouple has been used to measure relative blood flow (armaly and araki, ) , and bill ( a,b) measured choroidal flow directly in rabbits by cannulating a vortex vein and in cats by cannulation of the anterior ciliary vein. the most recent technique is the use of radio-labeled microspheres (o'day et al., ) . the labeled microspheres are injected into the arterial system, and shortly thereafter the animal is sacrificed and samples of ocular tissues are taken for quantitation of radioactivity. the microspheres are presumed to embolize in the small vessels of the various ocular tissues in amounts propor tional to the blood flow in that tissue. photographic analysis following injection of fluorescein and other dyes has been used to determine retinal oxygen saturation (hickam et al., ) , mean circulation time (hickam and frayser, ) , and choroidal blood flow (trokel, ; ernest and goldstick, ) . oxygen saturation has also been measured directly in monkeys using a microelectrode inserted through the pars plana (flower, ) . velocity of blood flow has been estimated using doppler tech niques (riva et al., ; stern, ) and thermistors (takats and leister, ) . while absolute measurements may not agree from one study to another, relative measurements may still be valid. most studies confirm that the choroid receives about % of the total ocular blood flow while the anterior uvea receives from to % and the retina about % of total ocular flow. one of the most important problems facing clinical human ophthalmologists is the control and treatment of vasoproliferative diseases in the eye. these neovascular proliferations may be confined to the retina or grow into the over lying vitreous and eventually result in retinal detachment or vitreous hemorrhage. they may also involve the iris (rubeosis iridis) and lead to neovascular glaucoma. diabetes, retrolental fibroplasia, and retinal vein occlusion are all commonly associated with ocular neovascularization. all of the above conditions have in common some degree of vasoobliteration followed by a period of retinal ischemia and subsequent vasoproliferative response. the most widely accepted theory is that growth of new vessels is stimulated by elaboration of a vasoproliferative factor from ischemie retina, a diffusable substance similar to the embryologie inducing agents discovered by spemann ( ) . this concept was supported by the isolation of a diffusable factor from certain tumors which stimulated neovascularity (folkman et al., ) . ryu and albert ( ) demonstrated a variable nonspecific neovascular response to viable or nonviable melanoma or retinoblastoma cells in a rabbit corneal model. the response was negligible in immune-deficient animals. more recently experiments by federman et al. ( ) have indicated that implanta tion of ischemie ocular tissues into the rabbit cornea can stimulate a non inflammatory neovascular response distant to the site of implantation. in diabetes capillary dropout in the retina is a well-documented phenomenon and is probably the precursor of proliferati ve retinopathy. reports of eye changes, especially capillary changes, in animals with spontaneous or induced diabetes are common (table iv) , but many of these reports represent sporadic findings. engerman ( a,b) has reviewed the subject and feels that the re tinopathy associated with alloxan-induced diabetes in dogs is the best animal model based on morphology and reproducibility of the lesions. alloxan diabetes produces a similar retinopathy in monkeys but microaneurysms require about years to develop (r. l. engerman, unpublished communication, ) . the retinopathic changes seen in these animals consists of microaneurysms and other structural capillary changes. we are not aware of any animal model of proliferative diabetic retinopathy with extraretinal neovascularization. patz and maumenee ( ) ; patz et al. ( ) sibay and hausier ( ) gepts and toussaint ( ) bloodworm ( ) engerman et al. ( ) ; engerman and bloodworth ( retrolental fibroplasia (rlf) is a result of oxygen toxicity to the immature retina. thus premature babies treated with oxygen are most susceptible, and the most immature part of the retina, usually the temporal periphery, is the most common area of involvement. the disease can be induced in newborn puppies, kittens, rats, and mice by exposure to high oxygen concentrations because these animals have an immature incompletely vascularized retina at birth. the kitten has been the most popular animal model for rlf, but the changes seen are comparable only to the early stages of rlf in humans with severe extraretinal disease complicated by retinal detachment being a rare finding in any of the animal models. there is some evidence that there are subtle differences in the pathophysiology of human rlf and that seen in animal models (ashton, ) . isolated retinal branch vein occlusion produced with the argon laser has been shown to cause a neovascular response in monkeys (hamilton et al., ) . once again, however, the new vessels are intraretinal and do not grow into the vitreous. the injection of inflammatory or toxic substances, such as blood (yamashita and cibis, ) , ammonium chloride (sanders and peyman, ), or immunogenemic substances including insulin (shabo and maxwell, ) into the vitreous may result in intravitreal vessels and fibrous membranes. these eyes show similarities to the late stages of diabetes or rlf in humans, but the pathophysiology is most likely different. the difficulty in producing intravitreal neovascular growth in animals may be due in part to an inhibitory effect of vitreous on proliferating blood vessels (brem et al., ; felton et al., ) . rabbit v- carcinoma implanted into rabbit vitreous results in a neovascular response only after the tumor has grown into contact with the retina (finkelstein et al., ) . the same tumor implanted into the corneal stroma stimulates a neovascular ingrowth toward the tumor (brem et al., ) , and, as already mentioned, federman et al. ( ) have reported that corneal implantation of ischemie retina stimulates a similar response. rubeosis, or neovascularization of the iris, may accompany diseases which cause retinal neovascularization and often produces glaucoma which is difficult to treat. there is apparently no reproducible way to induce true rubeosis in animals at present. attempts to produce the condition experimentally have been reviewed by gartner and henkind ( ) . occlusive vascular disease of the eye includes not only the relatively spectacu lar central retinal artery and vein occlusions but also a number of disorders with more subtle or insidious signs and symptoms. ischemie optic neuropathy, geo-graphic choroiditis, some forms of persistent uveitis (knox, ) , and possibly acute posterior multifocal placoid pigment epitheliopathy (ueno et al., ) are all examples of ocular disorders which can result from vasoocclusive events. experimental occlusion of the larger ocular vessels can be accomplished by occlusion of the vessel with a ligature or clamp. this includes the long and short posterior ciliary arteries (hayreh, ; hayreh and baines, a,b; anderson and davis, ) and the central retinal artery and vein (hayreh et al., (hayreh et al., , . lesseil and miller ( ) reported effects on the optic nerve and retina in surviving monkeys following complete circulatory arrest for to minutes. experimental occlusion of the smaller vessels has been produced by embolization of the arterial system with plastic beads or various other particulate matter. most of the early embolization experiments were limited by the fact that the particulate material was injected into the carotid artery, thus indiscriminately embolizing small vessels throughout the eye (reinecke et al., ; hollenhorst et al., ; ashton and henkind, ) . more recent reports describe tech niques for selective embolization of the choroidal and retinal circulations based on injection site (kloti, ) or temporary occlusion of the retinal circulation during injection (stern and ernest, ) . the most selective occlusion of retinal vessels has been accomplished with photocoagulation using the argon laser (hamilton et al., ) or xenon photocoagulator (ernest and archer, ) . the concepts of using the globe as an in vivo tissue culture medium has been discussed and exemplified in a previous section; the technique is applicable to a variety of autologous, homologous, and heterologous tumors of a variety of cell line origins. specifics are more within the realm of the oncologist rather than the ophthalmologist and as such will not be dwelt on. several experimental tumor models have been well defined and have been utilized to eludiate spontaneous tumorogenic processes and to study the biological behavior of the induced tumors. intraocular injection of viruses will induce tumors in a number of laboratory animals. injection of human adenovirus type , a dna virus, into newborn rat vitreous produced retinoblastoma-like tumors in of animals between and days postinjection . albert and his associates ( ) had earlier described similar observations in hamsters injected subcutaneously with tissue-cultured ocular cells lines exposed to the virus. muaki and kobayashi ( ) produced extraocular orbital tumors in of newborn hamsters to days following intravitreal injection of the same virus; the tumors resembled a neuroepithelioma, suggesting that they were derived from neurogenic primordia in the retrobulbar space. further investigations demon strated the retinoblastoma in mice (muaki et al., ) . jc polyoma virus was injected into the eyes of newborn hamsters by ohashi and his associates ( ); % of the animals developed retinoblastoma-or ependymoblastoma-like intraocular tumors vi-\?> months postinjection. eleven percent developed extraocular tumors, including schwannomas, fibrosarcomas, lacrimai gland carcinomas, and ependymal tumors. feline leukemia virus, an oncornavirus, was injected systemically and intraocularly into fetal and newborn kittens; a tumor of apparent retinal origin developed in of subjects (albert et al., a) . the same virus injected into the anterior chamber of cats results in the development of iris and ciliary body melanomas or, less commonly, fibrosarcomas, in a high percentage of injected animals (shadduck et al., ; albert et al., b) . in taylor and his associates ( ) utilized intravenous ra to induce ciliary body melanomas in dogs; the tumors appeared to originate from the pigmented epithelium. albert et al. ( ) transplanted human choriodal melanoma into the vitreous of the "nude" mouse, a homozygous mutant (nu/nu) with a defect in cellular immunity. serial passage transplantation was possible. the fact that the animal is immunodeficient limits the value of this model to study biologic behavior, but this in vivo system provides abundant tissue for morphologic, biochemical, immunologie, and therapeutic studies. ophthalmic res. , . baum the eye manual of tonography system of ophthalmology. the eye in evolution the biology of the laboratory rabbit the eye tonography and the glaucomas immunopathology of uveitis ocular pharmacology proc. am. coll /« "physiology of the human eye and visual system cataract and abnormalities of the lens becker-schaffer's diagnosis and therapy of the glaucomas anterior segment fluorescein angiography drugs and ocular tissues cells and tissues in culture retinal circulation in man and animals nuclear ophthalmology cell and tissue culture the vertebrate visual system comparative anatomy of the eye anatomy and histology of the eye and orbit in domestic animals textbook of veterinary internal medicine techniques in autoradiography atlas of veterinary ophthalmoscopy the rabbit in eye research immunopathology of uveitis the eye a treatise on gonioscopy the eye the vertebrate eye and its adaptive radiation transplantation of tissues and organs proc. am. coll key: cord- - men dfk authors: gupta, varsha; sengupta, manjistha; prakash, jaya; tripathy, baishnab charan title: biosafety and bioethics date: - - journal: basic and applied aspects of biotechnology doi: . / - - - - _ sha: doc_id: cord_uid: men dfk the advancement in technology is likely to tame several life forms present on earth. microorganisms are posing a big challenge due to difficulties encountered to control the diseases caused by them. working with deadly disease-causing microorganisms for their characterization, diagnostics or therapeutics and vaccine development purposes are posing increasingly potential biosafety problems for laboratory workers. thus, an appropriate biosafe working environment may protect workers from laboratory-induced infections. biotechnology has the ability to solve the upcoming problems of the world’s increasing population. however, there is often reluctance among the public to accept and support biotechnological products in medicine, industry, or agriculture. there are many safety and ethical issues raised for gm crops and human cloning. raising transgenic animals and plants has fueled ethical concerns, and the scientists have faced a lot of resistance where genetically modified crop plants or reproductive cloning research of human beings is involved. thus, biosafety and bioethics are continuously being expanded to combine the rationale of ever-increasing scientific knowledge in biotechnology that is often in conflict with the long-standing social and moral value system of our society. the advancement in technology is likely to tame several life forms present on earth. microorganisms are posing a big challenge due to diffi culties encountered to control the diseases caused by them. working with deadly diseasecausing microorganisms for their characterization, diagnostics or therapeutics and vaccine development purposes are posing increasingly potential biosafety problems for laboratory workers. thus, an appropriate biosafe working environment may protect workers from laboratory-induced infections. biotechnology has the ability to solve the upcoming problems of the world's increasing population. however, there is often reluctance among the public to accept and support biotechnological products in medicine, industry, or agriculture. there are many safety and ethical issues raised for gm crops and human cloning. raising transgenic animals and plants has fueled ethical concerns, and the scientists have faced a lot of resistance where genetically modifi ed crop plants or reproductive cloning research of human beings is involved. thus, biosafety and bioethics are continuously being expanded to combine the rationale of ever-increasing scientifi c knowledge in biotechnology that are often in confl ict with the long-standing social and moral value system of our society. however, biotechnological tools have resulted in high-yielding crop plants, more nutritive values of food grains, longer shelf life, and resistance to insects and pests, but the public acceptance to these biotechnological products is rather low. for example, gm foods have low support in europe and india. with the passing years, there is further decline in public support, probably due to media focus and the visible debates on gm crops for fear of their long-term effects and unknown risks and environmental safety issues. the controversies from nongovernment organizations (ngos), scientists, and media have led to a negative impact on the gm crops. gm crop usage has been controversial with fears being expressed on flavr savr tomato and many others. gm food again went into controversy due to labeling issues. the bt crops were challenged with the development of resistance in insects. so-called terminator technology and a gene use restriction technology (gurt) met with a lot of resistance due to introduction of negative traits of sterility in the seeds and never came to the market. the development of cloned animals and its impact on other wild-type animals and the environment also triggered many safety and ethical concerns. whether or not animals should be used in research, their welfare, sufferings, and wellbeing were debated throughout the world. in india many plants and animals are linked to religious beliefs and are respected and worshipped for their contribution in the life of human beings. likewise usage of embryonic stem cells faced controversies and concerns. protestants agree to have stem cell research under strict public regulations. many however are opposed to embryonic stem cell research as it destroys the embryo. the debates and issues surrounding the stem cell are all centered on the potential source of these cells. embryonic stem cell usage either is prohibited or is under strict public regulation. somatic stem cells and dedifferentiated somatic cells can be used for therapy. the public support for xenotransplantation was low due to safety issues with fear of spread of unknown contaminants. however, some xenogeneic tissue-engineered products are now available to treat dermal wounds and burn patients. the usage of biological weapons (use of living organism or its product for human killing) in war is presently banned. the biotechnology has tremendously evolved including the technical aspects of medical sciences like diagnostics and therapeutics. with all these, we are also witnessing rapid and dangerous adaptations in microorganisms especially for developing resistance to antibiotics. microbial pathogens are causative agents of many diseases and due to mutation in their genes, they have developed multiple drug resistance. it is gradually becoming diffi cult to control these multidrug-resistant infectious pathogenic microorganisms. to search for solutions to overcome mdr of these pathogenic agents, several scientists and medical workers are handling these pathogens . this poses enormous biohazards and raises serious "biosafety" issues, i.e., scientifi c practices, methods, and use of appropriate equipment in a biosafe environment. the biosafety aspects have become very important in various conditions and require many precautions in health-care systems as hospitals, diagnostic laboratories, animal care systems, biological laboratories, and so on. the precautions, which can be taken to reduce or nullify the risk, associated with samples by continuously monitoring and recognizing potential hazards, their risk assessment, and preventive measures to avoid exposure which might result in infection. individual worker should be appropriately trained and must understand the conditions like containment (conditions where infectious agent can be safely manipulated) and good laboratory practices which can minimize exposure to pathogens . [ ] biorisk risk is the likelihood of the occurrence of an adverse event, thus biorisk is the likelihood of the occurrence of serious infection due to exposure to pathogenic microorganism or biohazards. upon exposure there may be mild to severe infections, allergies, or other clinical problems associated with the pathogen . the biorisk can be managed by risk assessment, effective biosafety measures, and biocontainment . the fi rst report on laboratory-associated infections was published at the beginning of the twentieth century. however, , infections associated with laboratory were reported with deaths somewhere between and . the major pathogenic agents responsible for these infections were brucella spp., blastomyces dermatitidis , chlamydia psittaci , coccidioides immitis , coxiella burnetii , hepatitis b virus ( hbv ), salmonella typhi , francisella tularensis , mycobacterium tuberculosis , and venezuelan equine encephalitis virus [ , , , ] . after this report, many more laboratories associated infections and deaths were reported [ , ] . in these cases also the infectious agents were arboviruses , brucella spp., coxiella burnetii , cryptosporidium spp., hantavirus , mycobacterium tuberculosis , hbv , salmonella spp., shigella spp., and hepatitis c virus . the processes used for risk assessment are ( ) identifi cation of the hazardous properties of a familiar infectious agent or material, ( ) the activities responsible for pathogen exposure to the person, ( ) the chances of the exposure becoming a laboratory-associated infection, and ( ) the ultimate consequences of infection. risk assessment needs very alert judgments. if risks are underestimated, they might result in seri-ous and adverse consequences. overestimation of risks can result in undue protective measures, which may be a burden for the laboratory. assessment of risk also needs to evaluate the factors responsible for infections i.e., whether it is due to hazardous agents or due to hazards of laboratory procedure, or the working attitude of the laboratory staff. the staff's capability can be improved by appropriate training and inculcating good laboratory practices with training of accidental spillage/inoculations. biohazards may be the microbial infectious agent or other biological materials posing a risk for human health, parasites, viruses, prions , or biologically derived toxins, allergens, venoms, or recombinant dna that can adversely affect human or animal health and environment [ ] . the properties of an agent which make it potentially hazardous are [ ] ( ) infection capability, ( ) ability to cause diseases in human or animal host, ( ) its minimum infective dose, ( ) severity of the disease, ( ) availability of vaccine, ( ) availability of therapeutic modality to control it, ( ) the probable route followed by it for transmission, ( ) stability in the environment, and ( ) host (animal-human or only human). the probable routes of pathogen transmission may be: . through exposed part of the body such as the skin, eye, or mucous membrane . through inhalation . through needle or other sharp objects . through accidental ingestion . through bites of mosquito the world health organization (who) has recommended the classifi cation based upon the risks and route of transmission of the pathogenic agents in a laboratory environment. according to their disease-causing capacity and preventive measures available, the human pathogenic agents have been divided into four groups (according to who and nih) [ , ] . national biosafety guidelines came into being because of efforts of microbiological and biomedical communities. these involved the recognition of hazards, assessment of risk, and appropriate use of measures including biocontainment to prevent hazards. they promoted safe laboratory practices and usage of safety equipments along with occupational health programs to reduce laboratory-induced infections while handling microorganisms [ , ] . preventive measures also require personal protective equipment like gloves, mask, eye protection, laboratory coat, shoe covers, and respiratory protective measures for prevention of exposure. the biosafety level (bsl) indicates the kinds of precautions of biocontainment for handling dangerous microbial pathogens . the bsl- indicates lowest safety measures while bsl- indicates highest possible safety measures. the containment of laboratory refers to: current biosafety and biocontainment practices and procedures are designed to reduce the exposure of laboratory personnel, the public, agriculture, and the environment to potentially hazardous biological agents [ ] . biosafety levels (bsl) are laboratory designations, which are based upon the degree of risk. they are designated as bsl- , bsl- , bsl- , and bsl- . these safety levels ensure different levels of protection when working with virulent microbial strain. these laboratories are very sophisticated and have very good design, engineering control, and safe work practices. their different levels are used depending upon the pathogen properties like vaccine preventability, infectivity and its contagious nature, severity of the diseases, and risk assessment in case of infection. every level of containment has its own safety aspects: any kind of activity that includes usage of potential hazardous human pathogens , zoonotic agents ( rabies virus, infl uenza virus , trypanosomes (sleeping sickness)), toxins, and agricultural threats which may pose danger to human civilization is recommended for use only in high biosafety levels . the various biosafety levels appropriate for these works are biosafety levels and , animal facility/vivarium (absl- and absl- ), and biosafety level agricultural facilities (bsl- -ag). high containment refers to the highest level of biosafety. good biosafety and biocontainment practices can help in effective laboratory biosecurity. there is an urgent requirement to provide adequate safety for all life forms in case of natural emergency. natural emergency can be the outbreak of any disease or other conditions requiring attention due to attempt for spreading bioterrorism. thus federally funded research programs are being developed to protect all life forms. in the measures the products were designed and developed which can protect public health, are called medical countermeasures. these include public health and hygiene, diagnostics, vaccines, therapeutics, and development of biosafety level laboratories. for the purpose of biosafety, the general laboratory workers (students, scientists, and laboratory staff), along with non-laboratory workers as electricians, plumbers, and sweepers, should also be given appropriate training. the trainings are required in the areas of: every scientifi c revolution brings with it a host of ethical and social questions. the so-called genetics revolution is no exception, giving rise to a broad international debate on how the undoubted benefi ts of progress in this area can be reconciled with certain core human values. (unesco a : ) present-day biotechnology opens many opportunities in research and development, addressing medical issues and new ways to explore things; improving human health conditions, fi ght food, and feed problems; and so on. it has greatly infl uenced medicine and agriculture. it has tremendously affected the thinking process also like consuming vegetables and fruits without pesticides, using biofertilizer in place of chemical fertilizer, using renewable sources of energy, and switching to products which are biodegradable. it has also advocated the sustainability of systems (agricultural, environmental), knowledge sharing, and brought many products in the market. as the techniques involved gene manipulations in many life forms (plants, animals, microbes), thus with these advancements came the concerns in the form of ethical issues giving rise to bioethics. in our routine life also, we encounter ethical questions in terms of right or wrong or ethical or unethical; thus, in science also things are judged as being ethical or unethical. bioethics addresses policy and ethical issues arising by researches and products targeted for human applications. bioethics addresses the ethical issues in all the streams of life sciences like health care, genetics, and medical research by applying the principles of morality and philosophy [ ] . bioethics has evolved from medical ethics and moral philosophy. the ethical concerns for patient well-being were in use in the form of the hippocratic oath. it starts right from the laboratory to industry and government and affects a very wider society. the study of the social and moral responses arising due to scientifi c invention or experimentation is "bioethics." thus it led to granting of ethical clearance for any proposed research projects requiring animal or human experimenta-tions. the project is fi rst reviewed by research/ institutional ethics committee (rec/iec). the rec evaluates the risks, benefi ts, animal sufferings, utility of work and then only grants approval for conducting research projects and proposals. nuremberg code ( ) this code surfaced in the response of human rights abuses that had taken place under the nazis during world war ii. the war captives were subjected to abuses through experiments on human subjects. this propounded the use of informed consent and non-malefi cence, benefi cence, justice, and autonomy. these provided normative framework used by researchers and medical practitioners. the ethical principles consist of certain virtues such as autonomy, non-malefi cence, beneficence, and justice. now the area has gained so much of momentum that before even small experimental work, one needs to justify its rightness or wrongness. analyzing the ethical issues before starting any new experimental work [ ] would depend upon the assessment of these points: • consequences : what would be effect of this experiment? who is going to be benefi ted? what would be harms associated with this work? thus weighing the possible outcomes and their effects. • rights and responsibilities : are we exploiting somebody else's rights? do those rights need protection? who would protect these? these are questions whose justifi cation would have to be weighted. • autonomy : if one feels it is right, should things move on? or else someone feels it is wrong, should things stop? alternatively, who would decide things should be or should not be? these questions would again need to be addressed. • virtue ethics : is this the best thing to do? what is good about it? • animal rights : one big question is "are we authorized to use animals the way we want?" knowing that they too can think, they are aware of family members, they feel pain (at different levels), and they are alive. animals like primates and whales resembling human brain features, family behavior, and some sensitivity like humans should not be manipulated but others should be, why? animals have been in use since long. they are used for manipulation of their genes, as model system for study of human diseases, and as transgenics for production of pharmaceuticals. is inflicting pain in any form, which may be by creating a disease model or studying mutations in animals, ethical or not? • morality : it is a system of moral values and conduct, which govern our decision of right and wrong or good and bad. • empathy : the ability of one to understand and share the feelings of the other. • euthanasia : it refers to painless killing of any terminally ill patient suffering from painful and incurable disease on demand of the patient and the court. removing all life support system from a patient in irreversible coma is also termed as euthanasia. • autonomy is freedom from external control or infl uences, independence. • justice : it is the treatment or behavior, which is genuine, right, or just according to the prevailing laws. • equality : state of being at par or equal in status or ideology or opportunities. • benefi cence : it is related to kindness, good, or charity for good. • non-malefi cence : an act done to avoid harm or any act which would not harm or violate the trust of others. in the case of physicians, it is their act which will not harm the patient. • accountability : the condition where somebody is held responsible, answerable, or accountable for an act. philosophical ethics can be divided into descriptive ethics and normative ethics . it explains the actual moral viewpoints of the people; thus, it gives an accurate estimation of people's ethics. opinion polls or questionnaire evaluation are used to build a consensus of the view of the people. however, these are not affected by facts and reasons so they may be right or wrong [ , , ] . it explains the viewpoint, which people should have, and accordingly the actions, which people should undertake (according to what ought to be). cult situations about what should be done [ , ] . some of the important ethical theories are discussed: . ethical egoism : this theory states that morality is mere fulfi lling our self-interest. it criticizes the views associated with morality that are in the way of personal self-interest. it advocates that the actions are entirely dependent on desire and self-interest of the people. this theory advocates the selection of moral action, which in its consequence brings greatest happiness for the large number of people. it works to decide the result or consequences of good action, which would maximize happiness. thus it deals with consequences of action, in that happiness is judged because of some decision. however, greatest good for an individual or society, human race, or animals is debated. their supporters say that the theory aims to do good for the human race as a whole including nonhuman animals. for example, terminally ill patients should be allowed for physician-assisted mercy killing to avoid pain and sufferings. utilitarians also believe that abortion and infanticide may be possible in case the baby has severe disability. research on human embryo and genetic enhancement, which can benefi t humans, may be made possible. which states that morality arises from inner sense of duty, was infl uenced by the german philosopher immanuel kant ( - ). according to this all people have rights and duties and they should follow these. it states that the decision should come from inner sense of duty without considering the consequences of action. thus according to kant any act that follows categorical imperative is moral. kantian theory requires the use of morality based on rationality with respect to persons and human dignity. kantian views refrain from hiding the truth from patients who have terminal cancer. kant's categorical imperative-"act in such a way that you treat humanity, whether in your own person or in the person of any other, never merely as a means to an end, but always at the same time as an end" (kant / ) [ ] . it has been used to avoid abuses in research experiments on human subjects. this theory has been propounded and supported by a number of philosophers [ ] . main features of his theory are as follows: (a) respect : dignity of each and every human being should be respected, and one person should not use the other person as a means to fulfi ll one's own desire. (b) universality : if something ethically right is applicable to us as an individual, then it applies to others also, making it a universal right. it should be respected and followed by everyone. (c) the formula of ends : rational agents should be treated as ends. virtue ethics there are certain ethical virtues and any decision or action is considered morally correct when taken considering the virtues. these promote fl ourishing and well-being. thus, any action with a right motive based upon good character or virtue is considered morally right, for example, an action where treatment of a patient is sponsored to gain publicity and honor. the action is right but morally it is not good. jansen ( ) had highlighted ethical issues with virtue ethics: ( ) problem of content, vague virtues are unable to give proper guidance, and ( ) problem of pluralism, competing conceptions of the good life complicate a sound solution. the social and political background of feminist bioethics is feminism and feminist theory with its major social and political goal to end the oppression of women and to empower them to become an equal gender. the use of transgenic animals has sparked signifi cant controversy in many areas. some groups see the generation of genetically modifi ed organisms as interfering with natural biological states or processes. they feel that these natural biological states have evolved over long periods which should not be altered. a few other groups are concerned with the limitation of modern science to fully comprehend the potential negative ramifi cation and unforeseen possible effects of genetic manipulation . despite the importance of transgenic animals in biomedical research, there are some concerns raised about their use in research. transgenic animals suffer more abnormalities than regular research animals. the introduction of dna into an animal can be very complex and the possible side effects can be diffi cult to predict. possible harm might arise from surgical techniques used to harvest and reimplant embryos, the collection of tissue from the tip of the tail for genotyping, and nonspecifi c effects caused by damage to genes adjoining the altered area. in addition, reduced fertility and/or oversize fetuses may result from this technology. in most cases the mutations highly affect specifi c metabolic processes or cell receptors without actually resulting in disease but causing discomfort, pain, or malfunctioning in the animals. transgenic animals not expressing foreign dna or not containing a particular gene modification are destroyed. because transgenesis is a complex science, it is not % effi cient. however, new methods are being developed to increase the accuracy in transgenesis. again, it should be remembered that such genetic alteration could only be attempted if the authorities are persuaded that there is no other way to pursue important research. the potential risks of transgenics to animals, humans, and the environment are too great to justify their use. the genetic modifi cation of organisms regulations and the environmental protection act ( ) address the risks to those working with animals and the impact on the environment of accidental or planned releases . the intrinsic worth of animals may be devalued and their integrity violated by genetic modification. transgenic animals have not chosen to have foreign dna or other genetic modifi cations. however, this potential "cost" to the animals is routinely assessed under the ethical review of proposed procedures and weighed against the potential benefi ts. medical researchers only employ this technology when no alternative research avenue exists but with appropriate breeding facility of animals. as the royal society concluded in its report "the use of genetically modifi ed animals", the use of transgenic animals is fundamentally little different from the use of other animals in biomedical research. however, the technology has opened new frontiers in biomedical applications and has provided new opportunities for exploring the organization, biological pathway, regulation, and pathological function of molecular processes. contractarianism : it says that the animals are important as many people feel their importance. humanity is the virtue of morality. thus, humans have obligations toward other animals. utilitarianism : it says that as animals can also suffer; thus, they are morally relevant. it chooses the action, which aims to give happiness to all. thus, its goal is to maximize the total sum of happiness in the world and is impartial, that is, the pain and happiness of everyone count. animal activists view : according to these whatever is the motif there is no justifi cation of harming animals for human purposes. ruthless view : this has no concern about animals, whichever way they are being used. however, principles governing animal rights are the rs, which mean replace , reduce , and refi ne . it involves ethical and humane approach while using animals. the alternatives of animals in the research were also explored. the rs are replacement of conscious living animals with nonsentient animals or materials, reduction of the number of animals used in an experiment or procedure, and refi nement of the techniques used in order to decrease the incidence or amount of animal pain and distress [ ] . genetically modifi ed crops or gm crops have given new dimensions to agriculture. with the advancements in the technology, the agricultural productivity is increased, the food's nutritive value is enhanced, the requirement of pesticides and insecticides is greatly reduced, and pharmaceutical substances (vaccines) are being produced in the plants. however, gm foods have become the target of public concern due to unknown and unseen fears of their effects on the ecosystem and risks to human health. the gm foods are also not labeled leading to distrust and fear in the customers regarding the safety of food. the ethical issues raised are: • what about evolution? • each living being has evolved to grow in one condition, and if we put human gene or fi sh's gene in a plant or animal, what would be its effects? • what would happen if the gene can fi nd an escape route to introduce itself in other members of that species or other species? • what would happen if the herbicide resistance gene could transfer itself in weeds, generating superweeds? • if we have all gm crops of any grain or pulses, then occurrence of any disaster would lead to complete loss of the crop, as there is no biodiversity where one is sensitive and the other one is resistant. this would lead to disaster and damage of whole of the crop. • would the monogenetic crops, if not able to react appropriately to environmental stress , again face a situation like "ireland's potato famine"? • what if gm crops breed with other crops, would the biodiversity be lost? • what are their risks to other animals like birds, insects, and other higher animals (like monarch butterfl y and bt crops)? • there is tremendous fear of gmos on human health. • if the gm can induce allergy due to insertion of unrelated gene what would happen, because gm foods are not labeled. for example, a brazil nut gene was transferred to a soybean variety; this nut gene product was a source of allergy for many individuals. as gm products are not labeled, therefore, the consumers may consume soybean without being aware of nut gene product in them. this might lead to allergy causing major allergic reactions in the consumers. however resultant modifi ed crop was never released to the public . • human food supply may have gm products as evidenced by the settlement between syngenta and the us government over the accidental sale of unapproved gm (bt ) corn seed to farmers. • these seeds can only be afforded by big farmers and landowners, thus negatively affecting small-scale farmers. • the farmers need to buy seeds every year, as they cannot collect, store, and replant these seeds. • there would be huge differences in the farming practices in one country only, and that gap would be widened much between developed and developing nations [ ] . • as gm food is not labeled, thus, public trust and acceptance of gm food would be a big challenge . • the gm crops have higher productivity as they are resistant to pests; thus, loss due to diseases is prevented. • they are engineered to survive in high salt and frost, thus utilizing the land, which was unused for agricultural purposes. • they also help to reduce contaminants as insecticides and pesticides, thus preventing their entry in soil, water, and organic matter. • all these effects would ultimately affect the environment and consumers. • biotechnology can fulfi ll the increasing requirements of food, with maximum utilization of land. • biotechnology can help reduce the environmental contaminants as insecticides and pesticides preventing their entry in food chain. • it can help maintain clean environment. • it can enhance nutritive values of the food, for example, golden rice can help prevent blindness due to nutritional defi ciency of vitamin a. • the longer shelf life of fruits and vegetables can decrease their wastage and increase their availability with simple storage conditions. • the genes for allergins can also be engineered so that the allergins can be effectively removed from the food crops. • thus they are benefi cial for humans and animals. • it can also eliminate trans fats, producing healthier foods. • nowadays, plants are also being explored and used for production of biopharmaceuticals like vaccines, scfv, antibodies, and so on. there is reduced risk of adverse reactions in terms of animal pathogen transmission, and the protein undergoes posttranslational modifi cations. these genetically modifi ed varieties faced tremendous resistance in europe and india. their public acceptance has also met with resistance in terms of fears, controversies, and acceptance. the controversy from ngos, media, and scientists . genetically modifi ed crops and bioethics has led to tremendous negative impacts on the gm crops. the environmental council of the european union further augmented the controversy by halting regulatory approval of gm crops. the ethical issues and safety issues in gm cops can be addressed by evaluating their safety and other benefi ts and risks. the risks associated with humans, animals, and plants should be well considered along with the intention to do good. the consideration of well-being of all (humans, animals, plants, and environment) is very important for the sustainability of life, along with moving forward with positive aspects of technology. the ultimate aim of the technology should be improvement and sustainability in the future of all life forms with minimal adverse effects on our surroundings and other plants and animals. assisted reproductive technology (art) techniques are used in the case of male or female infertility . the technique is detailed in chap. . the process of art involves ovulation, artifi cial . aren't we imparting sufferings for the plants and animals by using these tools and technology on them? . rights about the well-being of other plants and animals. thus, there is a requirement of welldefi ned guidelines when using gene manipulations [ ] as well as labeling of the products produced by gene transfer technologies. there is no doubt that apart from genetically modifi ed plants and animals, the gene manipulation techniques are well accepted in production of recombinant pharmaceuticals for production of life-saving drugs. the technique has enormous potential for solving food problems and sustainable agricultur al practices and preventing and treating diseases. there are many concerns regarding reproductive cloning of humans. thus, the concerns should be there whether the technology is being used for what is worth or we simply want to see the extremes of what technology can do. each achievement of science has both negative and positive aspects; nuclear power when used judiciously can help us solve energy problems but can also result in disaster like what happened in hiroshima and nagasaki during world war ii. the gene manipulation technology is with tremendous potential but should be used just for benefi t of mankind, the environment, and animals. the experiments being done to create transgenics have left the people with many concerns regarding the health and life. the changes which unnatural gene transfer might be doing to the environment and the society? there have been concerns regarding the transfer of animal genes into plants (for vegetarians) and food animals. this has raised not only safety issues but also ethical issues: although many people are in favor of ivf and surrogacy , a survey shows that people are against posthumous sperm procurement or reproductive cloning. the technology is surrounded by many issues of creation and destruction of embryos [ , ] . bioethical issues are for seeking justice, non-malefi cence, and beneficence of all involved in any issue. adequate ethical guidelines and moral evaluations of new technologies are essential so that laws can be framed for national regulations and restrictions of unacceptable practices [ ] . stem cells for use as therapy have given new dimensions to medicine. due to capacity of these cells to differentiate into almost all different kinds of cells, they are being looked as potential alternative for the cure of many diseases. probably sometime in the future, scientists would be able to generate whole organs by stem cells and tissue engineering to fulfi ll the increasing demand for organs. depending upon their potency and differentiation capabilities, they can be: • adult stem cells: these are present in each tissue of the human body, where they help in replenishing cells for regular wear and tear of the cells. • embryonic stem cells are located in the human embryo at the blastocyst stage ( - days of age). leftover embryos at this age are often unwanted in assisted reproductive technology, and some parents can donate them for research. in many countries very strict laws are there for their usage. some of the nations recommend their use when no other option is available. • cord blood stem cells are derived from the umbilical cord, which is often still routinely discarded at birth. the ethical issues raised for stem cell research is the usage and destruction of human embryos for obtaining embryonic stem cell [ ] . as the life begins with the embryo, thus destruction of life is immoral; therefore, its usage and the extraction of embryonic stem cells are unethical. however, using adult stem cells or umbilical cord blood stem cells has been considered ethically superior alternative to the use of embryonic stem cells. their rejection and uncontrolled growth would be big challenges to fully explore their therapeutic potential. human cloning is a very controversial issue [ , ] to discuss and its cloning can be "reproductive" cloning and "therapeutic" or "research" cloning. though both the terms are not scientifically accurate, they are widely used. in somatic cell nuclear transfer (scnt) , the nucleus from the somatic cell is transferred into the enucleated egg. the resulting embryo can either be implanted into a mother or surrogate mother for development (the cloned sheep dolly was the outcome of scnt ) or the embryo can be used for research purposes [ ] . implantation of embryo for gestation is referred as reproductive cloning, whereas in therapeutic cloning embryo is harvested for embryonic stem cells [ ] . the stem cells obtained from embryo are embryonic stem cells , which can be induced to form different cells [ , ] . • people and scientifi c community in favor of human cloning say that it can combat infertility and very useful for therapeutic purposes. • many people are against the creation and usage of embryo for research purposes; some are concerned about the risks like ovarian hyperstimulation syndrome in egg donors. • many more object to the destruction of embryos as they represent the initial phase of human life thus considered morally equivalent to humans; therefore, they consider therapeutic cloning at par to reproductive cloning. • human reproductive cloning is unacceptable and unethical for many scientists and philosophers [ ] . most of the countries have banned all kinds of human cloning. • in the universal declaration on the human genome and human rights (udhghr) by unesco in , it says that the "practices which are contrary to human dignity, such as reproductive cloning of human beings, shall not be permitted." • the resolution in this line was also passed by the who saying that human reproductive cloning is against human dignity and urged member states to prohibit it. biotechnology has had a tremendous impact on human race [ ] . the technology has potential to change what humans are [ ] . humans may come up as transhumans. transhumanism is the international movement which is aimed to transform humans by the use of technology to augment brain function and enhance human intellect, physical potential, and psychological capabilities [ ] . their goal is to extend human life, enhance the brain and its capacities, delay aging, and improve physical conditions (socalled superhumans). attempting technological augmentation on the normal human function [ , ] can take us far away from our own species to "superhuman" function. thus, technological interventions can shift us from what we called as homo sapiens to "superhuman" homo sapiens technologicus -a species that uses, fuses, and integrates technology to enhance its own function [ , ] . we are living in the best of times as exhibited by the fast-paced economic growth of many countries around the world. various initiatives that were taken in the fi elds of molecular biology, genetics, and recombinant dna technology are beginning to show fruits, strengthening the basis of research. the technological advances have made our life easier. the problems encountered in the form of ethical dilemma need appropriate attention with a view of the interests of society. our ultimate aim should be to generate thorough knowledge on all aspects of modern biology and garner excellence in the making of an economically, socially, and morally sustainable society [ ] . . which of the following procedures is com- kant and applied ethics: the uses and limits of kant's practical philosophy control of communicable diseases manual science and society: different bioethical approaches towards animal experimentation. presentation at a symposium "use of animals in research: a science-society controversy? fatal cercopithecine herpesvirus (b virus) infection following a mucocutaneous exposure and interim recommendations for worker protection studies on release and survival of biological substances used in recombinant dna laboratory procedures health care rights and responsibilities: a review of the european charter of patients' rights goodbye dolly?" the ethics of human cloning after-birth abortion: why should the baby live what exactly is an exact copy? and why it matters when trying to ban human reproductive cloning in australia applying the four-principle approach the future of human nature conventional vs unconventional assisted reproductive technologies: opinions of young physicians human genetic data: preliminary study of the ibc on their collection, processing, storage and use, rumball s and smith am legal and ethical approaches to stem cell and cloning research: a comparative analysis of policies in latin america, asia, and africa the virtues in their place: virtue ethics in medicine human reproductive cloning and reasons for deprivation the imperative of responsibility: in search of an ethics for the technological age ethics and biogenetic art grundlegung zur metaphysik der sitten (groundwork for the metaphysics of morals) [ ] akademie-ausgabe kant werke iv life, liberty & the defense of dignity: the challenge for bioethics a companion to bioethics negotiating bioethics: the governance of unesco's bioethics programme human infection with venezuelan equine encephalomyelitis virus common sense in the laboratory: recommendations and priorities proceedings of a conference held at the asilomar conference center cloning mice and men: prohibiting the use of ips cells for human reproductive cloning transhumanism, medical technology and slippery slopes your destiny, from day one laboratory-associated infections: summary and analysis of cases past and present hazards of working with infectious agents laboratory-associated infections: incidence, fatalities, causes, and prevention continuing importance of laboratory acquired infections monitoring stem cell research the principles of humane experimental technique. methuen london, altex (alternatives to animal experimentation) offi cial publication of the johns hopkins center for alternatives to animal the technological world picture and an ethics of responsibility the paradox of choice: why more is less survey of laboratoryacquired infections nih guidelines for research involving recombinant dna molecules airborne q fever universal declaration on the human genome and human rights human genetic data: preliminary study of the ibc on their collection, processing, storage and use who, fifty-fi rst world health assembly agenda item : ethical, scientifi c and social implications of cloning in human health world health report, world health organization laboratory biosafety manual, rd edn. world health organization inventing iron man: the possibility of a human machine the potential transformation of our species by neural enhancement guidelines for biosafety laboratory competence european union, charter of fundamental human rights • biotechnology has made tremendous progress in providing therapeutics, new ways to diagnose diseases, regenerative medicines, food and feed solutions, food with nutritive values, and other countless achievements. • biosafety is a very important aspect as the health-care and laboratory workers are continuously exposed to highly contagious and infectious pathogenic agents. they work with these agents either for the purposes of diagnostics or research. • many laboratory-induced infections were reported among the workers and several of them were serious and resulted in death of workers. these laboratory-induced infections were due to organisms in biorisk- and biorisk- groups. • thus, it becomes very important to do appropriate risk assessment, monitoring, and installation of appropriate preventive measure like biosafety levels and biocontainment facility and training to the laboratory workers for prevention. • bioethical issues are there with all the major achievements of biotechnology . the issues were being raised because of rightness or wrongness of the experiment, to ensure experiments are conducted in a way that it ensures safety for all, the humans, animals, and the environment. • bioethical issues related with genetically modifi ed crops are related to development of resistance in insect-resistant crops and their impact on the environment, animals, and humans. many ethical issues were raised for genetically modifi ed animals and their sufferings. • embryonic stem cell therapy and assisted reproductive technologies along with genetically modifi ed crops faced too much of ethical discussions and resistance. human reproductive cloning created tremendous furor; thus, human cloning in all forms was banned in most of the countries. • as with any other technological advancements, each technology is faced with ethical and social issues and so is biotechnology . however, it is important that before we keep on proceeding, we should ask some basic questions to ourselves: ( ) why am i doing this? ( ) would it be benefi cial for anyone? key: cord- -pb p vrd authors: stalheim, o.h.v. title: major infectious diseases date: - - journal: handbook of animal science doi: . /b - - - - . - sha: doc_id: cord_uid: pb p vrd nan into an animal and multiplies, the animal has an infectious disease. if it spreads to other animals, it is considered a contagious disease. con sumers spend about $ billion annually for veterinary services (hayes, ) , and a significant number of people (< ) become ill each year with a zoonotic disease (i.e., one that is transmissible from animals to man) (u.s. public health service, ) . depending upon the nature of the microbial agent, the route by which it enters an animal, the animal's innate or artificially induced immunity, and many other factors, the outcome of the infection can vary from inapparent to rapidly fatal. some agents invade the skin, for example, and never penetrate deeper, while some proliferate and kill their host in days or hours (siegmund, ) . apparently, certain viruses have mutated so that they can attack a new host, and do so vigorously. the virus of avian influenza exists in many strains; some are highly virulent-the disease is called fowl plague and is an eradicable disease-while others are quite innocuous. the methods used to control animal diseases have evolved from the early ideas that the diseases of man and beast represented divine re tribution for sins and had to be endured with patience. but in germany during the seventeenth century, the desperate and heroic measures to control rinderpest (cattle plague), together with changes in attitude brought about by the reformation, convinced the people that animal plagues could and must be ruthlessly stamped out. that policy was carried to england and then to the united states, where it was instituted and carried out by the bureau of animal industry, usda, in a series of brilliant programs, and the health of our animals was greatly improved (stalheim, ) . at the present time, surveillance is emphasized to gether with a readiness to cope with outbreaks of infectious diseases. on the level of an infected animal, control and cure is often accom plished by the veterinarian and the owner using an appropriate com bination of testing and quarantine, chemotherapy and chemoprophylaxis, serum therapy and immunization, and other specialized procedures. the following tabular lists of animal diseases are quite limited. many minor diseases have been omitted as well as immunologic dis eases, diseases of laboratory animals, nutritional deficiencies, toxicoses, behavioral problems, and diseases due to stress. however, the diseases of fish (snieszko, ) , marine mammals (fowler, ) , and fur-bearing animals (lybashenko, ) now command much attention by a variety of biologists. in most cases, the names of the diseases conform to those selected for the animal diseases thesaurus (veterinary services, usda, ) , which was based on the veterinary subject headings of the commonwealth agricultural bureau in england. diseases that afflict more than one species have been listed under the most common host. for a fuller description of the geographic distribution of diseases, the reader is directed to odend'hal ( ) and trevino and hyde ( ) . animal health. yearbook of agriculture diseases of fur-bearing animals the geographical distribution of animal viral diseases the merck veterinary manual a symposium on diseases of fishes and shellfishes contributions of the bureau of animal industry to the veterinary profession foreign animal diseases: their prevention, diag nosis, and control morbidity mortality annual summary key: cord- -jdu h e authors: dubourdieu, dan title: colostrum antibodies, egg antibodies and monoclonal antibodies providing passive immunity for animals date: - - journal: nutraceuticals in veterinary medicine doi: . / - - - - _ sha: doc_id: cord_uid: jdu h e passive immunity can be provided to animals by several sources of antibodies including from colostrum, avian eggs, and monoclonal sources. these antibodies have been shown protect production and companion animals from a number of pathogens. this chapter reviews the immune system for the principles of immune response to antigens and the synthesis of immunoglobulins of the five classes of antibodies in the body. colostrum antibodies are described for passive immunity protection in animals such as calves. chicken egg antibodies are another source of antibodies for passive immunity. therapeutic monoclonal antibodies are also used to provide passive immunity in the veterinary field. the use of antibodies by veterinarians to maintain the health of animals has a long history. fundamentally, when it comes to the immune system health of production and companion animals, there are little absolute differences in the intended purpose of the immune system. mammals have the same basic immune system with minor differences between the species. even the differences between birds and mammals are not so great since the purpose of the immune system is to keep infectious microorganisms, such as certain bacteria, viruses, and fungi, out of the body and to destroy any infectious microorganisms that do invade the body. how veterinarians take advantage of the immune system to maintain health can roughly be defined as taking advantage of the body's inherent method of maintaining health through adaptive immunity provided by vaccinations to generate antibodies inside the animal or by administering preformed antibodies to an animal through a process called passive immunity. evolution has produced an amazing immune system in animals that utilizes various cell types and proteins to protect them from invasive organisms. this system has two broad categories: nonadaptive and adaptive. the nonadaptive immune system is mediated by cells that respond in a nonspecific manner to foreign substances. this response includes phagocytosis by macrophages, secretion of lysozymes by lacrimal cells, and cell lysis by natural killer cells. the adaptive immune response is mediated by lymphocytes that produce a set of proteins called antibodies that are either secreted by or found on the surface of the lymphocyte. when the antibodies themselves are created within the animal following vaccination or from exposure to pathogens, the process is called adaptive immunity. when preformed antibodies from a host animal are given to another recipient animal, such as it offspring or even a completely different species animal, the process is called passive immunity. scientists have long taken advantage of the adaptive immune system by using vaccines. vaccines for animal diseases were the first to result from laboratory-based scientific investigation. french chemist louis pasteur developed a vaccine for chicken cholera in , and one for anthrax of sheep and cattle in . the major goals of veterinary vaccines are to improve the health and welfare of companion animals, increase production of livestock in a cost-effective manner, and prevent animal-to-human transmission from both domestic animals and wildlife. these diverse aims have led to different approaches in the development of veterinary vaccines from crude but effective whole-pathogen preparations to molecularly defined subunit vaccines, genetically engineered organisms or chimeras, vectored antigen formulations, and naked dna injections (meeusen et al. ). it has also resulted in various guidelines for vaccinations of companion animals such as for dogs (ford et al. ) and production animals such as swine (alabama and auburn ) , in poultry (stewart-brown ), cow/calf (missouri ) , and other production animals. successful veterinary vaccines have been produced against viral, bacterial, protozoal, and multicellular pathogens, which in many ways have led the field in the application and adaptation of novel technologies. passive immunity whereas active immunity refers to the process of exposing the individual to an antigen to generate an adaptive immune response, passive immunity refers to the transfer of antibodies from one individual to another (marcotte and hammarström ) . passive immunity provides only short-lived protection, lasting from several weeks to up to - months, but is immediate. nature intended passive immunity to occur when maternal antibodies are transferred to the fetus through the placenta or from breast milk to the gut of the infant. however, it can also be produced artificially when antibody preparations are derived from sera or secretions of immunized donors and are delivered via oral or systemic routes to nonimmune individuals. passive immunization is a new approach to providing protection to animals against pathogens because of the emergence of new and drug-resistant microorganisms, diseases that are unresponsive to drug therapy and individuals with an impaired immune system who are unable to respond to conventional vaccines. the immune system can respond specifically to millions of different molecules and is constantly challenged by huge numbers of antigens. a major feature of the immune system is that it can synthesize a vast number of antibodies. each of these antibodies can bind to a different antigen. this binding is the basis for the molecular specificity of the immune response. antibodies are proteins produced by a type of terminally differentiated b lymphocytes. b cells take the b name from chicken bursa cells where they were first discovered (gitlin and nussenzweig ) . antibodies are produced in response to the presence of foreign molecules in the body. the antibodies circulate throughout the blood and lymph where they bind to the foreign antigens. once bound, these antibody-antigen complexes are removed from circulation, primarily through phagocytosis by macrophages. this is the basis for antibodies protecting the animal against pathogens. antibodies are a large family of glycoproteins that share key structural and functional features. from a structure standpoint, antibodies look like a y-shaped molecule (fig. ) . each y contains four polypeptides. two of the polypeptides are identical and called heavy chain. the other two, also identical, are called light chain and are connected by disulfide bonds. there are five classes of antibodies, igg, igm, iga, ige, and igd, that are classified based on the number of y-like units and the type of heavy-chain polypeptide they contain (fig. ). igm is the largest antibody, and it is the first to appear in response to initial antigen exposure. the spleen, where plasma cells responsible for antibody production reside, is the major site of specific igm production (capolunghi et al. ; marchalonis et al. ) . igg is the main type of antibody found in blood and extracellular fluid allowing it fig. antibody structure as drawn, by protein model and by electron microscopy to control infection within body tissues. approximately % of all antibodies in humans and companion animals are of the igg class. immunoglobulin a (iga) plays a crucial role in the immune function of mucous membranes. the amount of iga produced in association with mucosal membranes is greater than all other types of antibodies combined (fagarasan and honjo ; holmgren and czerkinsky ; snoeck et al. ) . igd was initially thought to be a recently evolved antibody class because it was only detected in primates, mice, rats, and dogs and not guinea pigs, swine, cattle, sheep, and frogs (preud'homme et al. ) . however, recent discoveries of igd in ancient vertebrates suggest that igd has been preserved in evolution from fish to humans for important immunological functions (chen and cerutt ) . immunoglobulin e (ige) has only been identified in mammals. ige's main function is immunity against parasites such as helminths (erb ) like schistosoma mansoni, trichinella spiralis, and fasciola hepatica (watanabe et al. ; pfister et al. ) . ige also has an essential role in type i hypersensitivity (gould et al. ) which manifests in various allergic diseases, such as allergic asthma, most types of sinusitis, allergic rhinitis, food allergies, and specific types of chronic urticaria and atopic dermatitis (mueller et al. ) . antibodies bind antigens at the upper tips of the y molecule. the region of antigen where binding occurs is called the epitope (fig. ). antibodies can bind to a wide range of chemical structures and can discriminate among related compounds. how well the antibody binds to an antigen is known as affinity. this affinity can range from low to high. colostrum and passive immunity mammals are born without a fully functional adaptive immune system even though the basic elements are present. when a mammal is born, it emerges from the sterile uterus into an environment where it is immediately exposed to a host of microorganisms. the gastrointestinal tract (git) acquires a complex microbial flora within hours. if it is to survive, the newborn animal must be able to control this microbial invasion. in practice, the adaptive immune system takes some time to become fully functional, and innate mechanisms are responsible for the initial resistance to infection. in some species with a short gestation period, such as mice, the adaptive immune system may not even be fully developed at birth. in animals with a long gestation period, such as domestic mammals, the adaptive immune system is fully developed at birth but cannot function at adult levels for several weeks. the complete development of active immunity depends on antigenic stimulation. the proper development of b cells and b-cell receptor diversity requires clonal selection and antigen-driven cell multiplication. thus, newborn mammals are vulnerable to infection for the first few weeks of life. they need assistance in defending themselves at this juncture. temporary help is provided by the mother in the form of colostrum, which contains antibodies. the passive transfer of immunity from mother to newborn is essential for survival. calves are born without an active immune system and rely on the consumption of antibodies for protection from disease such as scours and pneumonia. the cow provides its calf with nutrients for growth and development during gestation, but the cow cannot directly provide the calf with antibodies to protect it from diseases. fortunately, immunoglobulins form an important component of the immunological activity found in milk and colostrum. while humans have a large amount of iga in their colostrum, the colostrum from most other animals contains a high percentage of igg (hurley and theil ) (fig. ) . immunoglobulins found in mammary secretions arise from systemic and local sources. in the case igm iga ige igd of igg in milk, the major portion comes from the serum (mayer et al. ) . while plasma cells producing igg may occur within the mammary tissue, their contribution to the igg in colostrum is minor compared with the igg derived from serum. the other major classes of immunoglobulins transported into colostrum and milk are iga and igm. immunoglobulin a (iga) is the major immunoglobulin in human colostrum and milk; however, it is also present in milk of most other species. colostrum and milk iga and igm are found in the form of secretory iga, or siga, and sigm. much of this is produced by plasma cells in the mammary tissue. the plasma cells are part of the gut-associated lymphoid tissue (galt), the largest immune organ of an organism, which includes the peyer's patches, lymphoid and myeloid cells in the lamina propria, and intraepithelial lymphocytes (ishikawa et al. ) . interestingly enough, more than % of the immune system is located in the gastro intestinal tract, the site where many oral pathogens first interact with an animal (vighi et al. ) . galt is a part of the mucosa-associated lymphoid tissue and works in the immune system to protect animals from invasion of pathogens in the gut. one of the physiological functions of the mucosa in the gut is for food absorption. however, of equal importance of the galt is in the body's defense, due to its large population of plasma cells whose number exceeds the number of plasma cells in the spleen, lymph nodes, and bone marrow combined (nagler-anderson ) . lymphocytes from the galt system will move to the mammary gland and provide a direct link between the antigen exposure response in the mother's mucosa system and the secretory immunoglobulins of the mammary gland (brandtzaeg ) . as such, this means that maternal colostrum and milk will contain antibodies specific for pathogens that may be encountered by the neonate's intestine and other mucosal tissues. this provides a rationale for the observations that bovine colostrum from nonimmunized cows may also afford passive immune protection against human pathogens in both humans and animals (li-chan et al. ; yolken et al. ) and opens the door to new technology to provide veterinarians another way to protect animals from pathogens that does not involve antibiotics. antibodies must be obtained by drinking colostrum within the first couple of hours after birth as part of the passive immunization system in order to maximize antibody absorption (pakkanen and aalto ) . like other animals, antibodies are generated by healthy cows as a result of every day exposure to infectious agents. antibodies can also be the result of specific vaccination programs. the cow's natural antibodies to these infectious agents are passed from the cow to the calf through colostrum. the level of antibodies transmitted from the cow through the colostrum can be elevated by a pre-calving vaccination program (thomas ) . when the calf drinks colostrum, the maternal derived antibodies are absorbed from the calf's git into the blood stream. some of the immunoglobulins also remain in the gut where they can neutralize pathogenic bacteria and help prevent the development of diarrhea. the absorption of antibodies from the git into the bloodstream is called passive transfer. failure of passive transfer (fpt) in dairy calves is defined by a blood igg level of < mg/ml - h after birth (stilwell and carvalho ) . calves that experience fpt are more likely to become sick or die in the first months of life than calves with adequate immunity. many factors can contribute to fpt, but colostrum and the management of colostrum feeding are often involved. to successfully obtain passive transfer and provide the calf with protection from diseases, it is thought that the calf needs to consume a minimum of - g of immunoglobulins (meganck et al. ) . the pathway between the gastrointestinal tract and the bloodstream is only open for a short window of time. research shows that this pathway starts to close shortly after birth, and after - h, approximately % of the calf's ability to absorb colostrum antibodies is gone. it has therefore been recommended to feed calves as much colostrum as they want by bottle within - h after birth and at h of age to substantially reduce the probability of fpt (chigerwe et al. ; trotz-williams et al. ) . colostrum also provides the calf with protein, energy in the form of fat and sugar, and vitamins (quigley and drewry ) . some vitamins do not cross the placental barrier, and colostrum is the primary source of these nutrients for the calf after birth. energy is required for all metabolic functions including maintenance of body temperature. one of the leading causes of death in dairy calves is failure to initiate breathing and metabolic processes in the first hours of life. the newborn calf only has a few hours of energy reserves in stored fat and therefore needs the energy from colostrum. research also confirms that the sooner a calf consumes colostrum, the more maternal antibodies it can utilize. the quality of colostrum is a major issue that the dairy industry faces on a regular basis. generally speaking, quality of colostrum is related to the amount of antibody that is present. colostral igg concentration is an important factor that affects whether calves receive sufficient passive immunity (godden et al. ) . unfortunately, the amount of igg in maternal colostrum varies dramatically among cows (< - g/l) with . - . % of samples that do not reach the desired amount of g igg/l (gulliksen et al. ) . colostral quality is difficult to estimate by the farmer based on produced volume or appearance of the colostrum. there are many variables that impact colostrum quality, including nutrition, the time the cow is milked, heat stress, and stage of lactation. a study from iowa state university suggests that a minimum % of dairy calves in the usa are currently being fed colostrum classified below industry standards for igg content and are at a greater risk of fpt, mortality, and morbidity (morrill et al. ) . other production mammals such as piglets face the same issues as calves for colostrum quality. studies have shown that on average % of pigs with a colostrum intake below g usually die. in pigs with a colostrum intake below g, mortality was as high as % (devillers et al. ) . giving spray-dried bovine colostrum to other animals such piglets has been shown (sty et al. ) to help protect against gut dysfunction and inflammation. it may be possible that using bovine colostrum for piglets could help supplement sow colostrum. research has also been done in foals with an enhanced bovine colostrum supplementation (fenger et al. ). since up to % of cows have colostrum with an igg level below mg/ml, which will not prevent fpt, a variety of programs and protocols have been implemented by dairy producers. high-quality maternal colostrum is still the gold standard for feeding newborn calves. however, colostrum supplement and replacer products can be valuable tools to increase calf immunity when colostrum supplies are limited or disease eradication is desired. colostrum products that contain igg are regulated by the usda center for veterinary biologics and are available in bolus, gel, and powder formats. supplement products are unable to raise the blood concentration of igg above the species standard, which is mg/ml for calves. any product that is able to raise serum igg concentration above mg/ml may be called a colostrum replacer (penn state ). supplements do not contain sufficient quantities of antibodies to raise the blood igg level in calves beyond what average quality colostrum will do. colostrum replacer contains greater levels of igg and other nutrients and provides an effective, convenient method of providing passive immunity to calves when maternal colostrum is not available. colostrum supplements available today are made from dried bovine colostrum or serum and contain - g of igg per dose ( - % globulin protein). the fat content of these products ranges from . to %. spray-dried colostrum with high concentrations of immunoglobulin may be produced economically and used as an effective and convenient colostrum replacer in newborn calves (chelack et al. ) . numerous products designed to replace colostrum are now on the market. these products are made from bovine colostrum or serum and contain - g of igg per dose. these products also provide fat, protein, vitamins, and minerals needed by the newborn calf, although the amount varies between products. a summary (pennsylvania state university ) of treatment means from published studies investigating colostrum products indicated that replacer products provided an average of g of igg, with an absorption efficiency of %, and serum igg of mg/ml. supplement products (fed in addition to colostrum) provided g of igg with % absorption efficiency and resulted in serum igg of mg/ml. it has been recognized that while antibodies found in colostrum can certainly reduce diseases in animals via passive immunity when given to a newborn, they will only work if the colostrum donor animal has been exposed either naturally to the disease or given a vaccine to the disease, in order to have specific antibodies produced. bovine colostrum that is typically spray-dried for supplements or replacer will contain only the antibodies that the cow may have encountered naturally. therefore, the colostrum used may not have specific antibodies against particular diseases that a producer might be interested in. the animal industry has recognized this issue and has developed methods to produce specific antibodies in high titer against specific diseases that can be delivered in colostrum products. this is achieved by hyperimmunizing animals such as cows against specific animal diseases, collecting colostrum and processing it into powders such as by freeze-drying methods and then giving to a newborn animal in gels or boluses. a number of diseases including bacterial diseases like e. coli (selim et al. ) and viral diseases such as rotavirus and coronavirus (combs et al. ) or coccidial diseases such as giardia or cryptosporidia (graczyk et al. ; fayer et al. ; naciri et al. ) have been researched for hyperimmunized colostrum efficacy. the hyperimmunized colostrum is collected, processed, and given to newborn calves. to varying degrees of success, these hyperimmunized colostrum antibodies have been proven to be successful in providing passive immunization. besides calves, research has been done in a variety of production animals using bovine or other sources of colostrum. for example, lambs have been supplemented with ewe colostrum as well as hyperimmunized serum from sheep against e. coli (pommer ) . other research has examined vaccination of cows with clostridial antigens and passive transfer of clostridial antibodies from bovine colostrum to lambs (clarkson et al. ) . piglets have also been given passive protection against porcine epidemic diarrhea by hyperimmune bovine colostrum (shibata et al. ) . specific antibodies in a hyperimmunized colostrumderived product can be used while complementing early colostrum feeding and can be delivered at the same time as colostrum. there are some advantages to this strategy as specific immunoglobulins immediately fight at the gut level to protect against diseases that destroy the intestinal lining while also allowing for antibodies to be absorbed into the bloodstream. it's important to protect the intestinal lining because if the cells that line the digestive tract become damaged, milk cannot be digested or absorbed by the calf. besides the quality of the colostrum itself, researchers first believed that calves could not be vaccinated effectively while they had circulating maternal antibodies from the colostrum in their system. preweaning calves can respond to vaccination stimulation as early as month of age. the maternal antibodies absorbed from colostrum, however, cannot distinguish between the antigens of a natural challenge and the antigens in a vaccine. therefore, colostrum antibodies can interfere with the immune response to a vaccination (niewiesk ) . work continues to be done to develop ways to circumvent maternal antibody interference. vaccination of calves in the face of maternal antibodies (ifoma) often does not result in seroconversion as maternally derived immunity interferes with the activation of adequate antibody responses to vaccination. however, it can prime t-and b-cell memory responses that protect calves against clinical disease when maternal immunity has decayed. the activation of b-and t-cell memory responses in calves vaccinated ifoma varies and is affected by several factors, including age, level of maternal immunity, type of vaccine, and route of administration. these factors influence the adequate priming of humoral and cell-mediated immune responses and the outcome of vaccination. failure to adequately prime immune memory after vaccination ifoma could result in lack of clinical protection and an increased risk of viremia and/or virus shedding (chamorro et al. ). there is obviously some controversy about whether newborn calves should be vaccinated (cook et al. ) . it is thought that the process of the calf mounting an immune response to a vaccine requires energy that could better be used to fight off disease and gain weight and the response could actually be detrimental to the early health of that calf. on the other hand, while maternal antibodies can block response to vaccination, sometimes they do not (woolums ) . the exact immunologic outcome in calves vaccinated ifoma can vary, and this variation likely depends on many factors. these factors are not well characterized but likely include the nature of the vaccine administered, number of doses administered, age of the calf and level of maternal antibody present in the calf, and the means by which a protective response is defined. guidelines for vaccinating newborn animals such as calves require additional research to clarify the ifoma vaccination reactions. fortunately, producers also have the option of using antibody products in order to generate immediate protection in situations where colostrum quality is poor. antibody products complement colostrum feeding because they can be fed at the same time. these products are available in bolus, gel, and powder form. they also are included in some colostrum replacer and supplement formulas for added value. typically, the antibody products are from hyperimmunized cows, and the specific antibody is found in the colostrum. the colostrum is processed and typically fractionated into a semi-purified preparation of antibody that is freeze-dried and put into capsules or boluses. because antibody boluses can be fed in conjunction with colostrum, they can be a tool to help the calf not only achieve adequate passive transfer but also provide enough specific antibodies to protect against the most common early calf hood diseases. antibody products do not require the calf to react to a vaccine in order to develop antibodies. rather specific antibodies against various diseases are already present, measured, and verified to be at a high enough level to protect the calf from scour-related diseases, and they can be fed as close to birth as possible. united states department of agriculture (usda)-approved antibody products are available on the market that can be fed in conjunction with colostrum and provide the calf with immediate immunity. these antibodies go to the gut to immediately bind and neutralize diarrhea antigens while also being absorbed into the blood stream for extended protection (combs et al. ; chamorro et al. ) . another advantage of this approach is that providing specific antibody can potentially avoid vaccine stimulation. avoiding vaccine stimulation can allow a calf to conserve its minimal supply of fat and nutrients that are critical to get the calf through its first few days of life. passive immunity by egg antibodies cows pass their immunity to their offspring by colostrum, and various colostrum products are on the market to achieve passive immunity. additional sources of antibody products can be utilized in a similar manner to achieve passive immunity. these are from avian sources such as chickens. in birds, passive transfer of immunity occurs through the egg. by hyperimmunizing chickens over a period of time with inactivated multivalent bacterial or viral vaccines, this procedure results in the production of polyclonal immunoglobulins of the igy class (specific to avians) directed against the stimulating organisms. oral consumption of the "immune" eggs containing specific igy antibodies protects the animal against the specific organism(s) with which the hen was stimulated. unfortunately, the eggs really cannot be cooked since heat dentures the antibodies found in the eggs. other physical parameters such as an acidic ph will also destroy the antibodies to a certain extent. however, enough orally administrated igy may survive passage through the git and, after excretion, still retain a great deal of its antigen-binding ability indicating that orally administrated igys are useful for passive immunity. eggs as a natural source of immunoregulatory factors just as immune protection is transferred in utero in mammals or passively by a lactating mother via colostrum, hens passively transfer protection to their young by secreting immunoglobulin and other immune factors into their eggs for use by the hatching chick. the transfer of chicken immunoglobulins from the hen's serum to the yolk and from the yolk to the chick is analogous to cross-placental transfer of igg from the mammalian mother to its offspring. while igm and iga are found in chicken eggs, the principle immunoglobulin is igy which is found in the yolk of the egg (hamal et al. ) . igy (y stands for yolk) is an immunoglobulin class specific to avians and analogous in function to that of mammalian immunoglobulins. igy has a similar structure as mammalian igg with some minor differences in the heavy chains (fig. ) . both eggs and milk (including breast milk) contain naturally occurring antibodies, and there are reports of immunomodulatory factors in milk as well (li et al. ) . however, immunoglobulin levels in eggs can be significantly higher than levels found in serum or milk (woolley and landon ) . this may not be surprising since mammals have a considerably longer time of weeks or months during which they may passively transfer immunoglobulin and immune factors, while the hen has a single opportunity (the egg) to transfer all necessary survival components to its offspring. all the aspects that the chick needs to survive must be in the egg. because egg products are a common source of protein in human diets and eggs contain antibodies and immune factors, it was obvious to utilize egg antibodies to provide passive immunity to animals. as such, this has resulted in a number of commercial egg antibody products on the market for production animals as well as companion animals to prevent various diseases. with few exceptions, oral consumption of specific antibodies has been reported (diraviyam et al. ) to protect both humans and animals. furthermore, in vitro studies with specific igy antibodies have been found to inhibit processes associated with bacterial growth, adhesion to intestinal cells, and toxin production (sugita-konishi et al. ) . meta-analysis has demonstrated the beneficial effects of igy (diraviyam et al. ) for a variety of animals. this fig. structure of mammalian igg compared to avian igy analysis supports the opinion that igy is useful for prophylaxis and treatment. currently, the oral passive immunization using chicken igy has been focused as an alternative to antibiotics for the treatment and control of diarrhea. igy has been demonstrated to be effective in controlling and preventing diarrhea in humans and in animals including piglets (cui et al. ; vega et al. ) , mice (buragohain et al. ) , poultry (el-ghany ; farooq et al. ) , and calves (cook et al. ; germine et al. ; vega et al. ) . most commercially available chickens are immunized at birth to protect them from avian diseases. the only difference between such supermarket eggs and "immune" eggs is that the latter are from chickens that have received additional proprietary vaccinations with other inactivated pathogens known to be the etiologic agents of animal infections. a wide range of bacteria, viruses, and coccidia have been used in vaccines for producing commercial igy products. a partial list includes shigella dysenteriae, staphylococcus epidermidis, escherichia coli, e. coli k , salmonella enteritidis, and s. typhimurium, pseudomonas aeruginosa, klebsiella pneumoniae, haemophilus influenzae, species of streptococcus, salmonella dublin, salmonella anatum, clostridium perfringens type a and type c toxoids, rotavirus types and , coronavirus, reovirus, parvovirus, and cryptosporidium parvum (schade et al. ) . only the chicken (not the egg) is exposed to inactivated pathogens. immunoglobulins and other immune factors are passively transferred to the egg from the serum for use by the chick and, more importantly, for passive immunity for other animals. after appropriate times following vaccination, eggs are collected from the specially designated chicken flocks, washed and broken, and the yolk and egg white are typically dried to a fine proteinaceous powder. various processes have been developed to help minimize heat damage to egg antibodies and immunoregulatory factors during the spraydrying procedure. however, spray-drying eggs can denature the igy to an extent, due to at least some heating during the process. keeping the egg away from heat is an optimal way of maintaining igy titer levels and efficacy. while freezedrying is an expensive option for maintaining antibody titer levels, simply feeding fresh immunized eggs is another method which exists. this is to keep the immunized eggs in a stabilized liquid (dubourdieu ). this stabilized liquid format is a practical and inexpensive delivery method that allows the specific igy to be delivered in watering systems to production animals or incorporation into other delivery formats such as soft chews that can be readily given to companion animals (fig. ) . the effective mechanism by which avian igy's work is effective is by the same mechanism that mammalian igg antibodies work in the animal to provide passive immunity. for example, bacteria e. coli strain k typically causes problems in production animals that result in diarrhea and possible death. therefore, a vaccination program is used in chickens to create specific anti-e. coli k igy antibodies. these specific antibodies found in the yolk of the egg are given orally to animals to prevent e. coli k from causing disease. this occurs when specific anti-e. coli k igy antibodies bind to the pathogenic bacteria. this binding blocks the ability of the pathogen to bind to the mucin layer obtain egg yolk containing specific igy against antigen spray dry into powder containing specific igy stabilized into liquid containing specific igy fig. vaccination for specific antibodies and processing egg yolk strategies in the gi tract of the animal. the pathogens are flushed out of the gi tract with the feces since they have been rendered unable to bind. the total immunoglobulin content of eggs from hyperimmunized hens is identical to the total level of immunoglobulins found in conventional table eggs. however, the quantities of immunoglobulins to selected antigens are different in the two varieties of eggs. additionally, both the table egg and the "immune" egg contain immunoregulatory factors, but eggs from hyperimmunized chickens may contain many times greater concentrations of individual factors as compared to regular eggs. besides immunoglobulins, eggs contain a number of bioactive components, including phospholipids, cholesterol, lutein, zeaxanthin, and proteins, that possess a variety of proand/or anti-inflammatory properties. two major categories of immune components are found in "hyperimmune" egg: ( ) the immunoglobulins with neutralizing specificities against the stimulating pathogens and ( ) the immunoregulatory factors that modulate cellular functions. the immunoglobulins provide local protection against gastrointestinal intoxication. the immunomodulatory mediators act directly on gastrointestinal surfaces and circulate systemically, affecting every immune, physical, metabolic, and neuroendocrine pathway in the body. these may have important implications for the pathophysiology of numerous chronic diseases and immune responses to acute injury (andersen ) . given the essentiality of pro-inflammatory responses in normal immune defense against pathogens, further research into the role of egg intake on immunity is warranted and may lead to further commercial uses of eggs and igy technology. biological medicine in humans, an intervention pioneered in the last years, is now on the horizon for companion animals. this strategy includes the use of monoclonal antibodies (mabs) to selectively target proteins such as cellular receptors or soluble molecules involved in disease pathogenesis. such treatment holds the potential for targeted therapies of chronic diseases such as osteoarthritis, atopic dermatitis, or lymphomas in dogs and cats. monoclonal antibodies are antibodies that are made by identical immune cells that are all clones of a unique parent cell. monoclonal antibodies have monovalent affinity, in that they bind to the same epitope. in contrast, polyclonal antibodies bind to multiple epitopes and are usually made by several different plasma cell lineages (fig. ) . given almost any substance, it is possible to produce monoclonal antibodies that specifically bind to that substance; they can then serve to detect or purify that substance. this has become an important tool in biochemistry, molecular biology, and medicine. therapeutic mabs can be used medically to block disease-relevant proteins (e.g., cytokines or receptors on cells) and cancer and have gained significant use in humans. they can also be used to target viruses or bacteria and aid in their destruction and elimination. veterinary medicine is only now incorporating this tool. despite the success of mabs in human medicine, there are considerably fewer in the pipeline for veterinary medicine. this may be due to uncertain regulatory guidance in both europe and the usa. these regulatory documents are written from the perspective of human medicine-based risk assessment and development. it is recognized that this may present a challenge to veterinary mabs manufacturers to achieve the extent of characterization/quality control testing typically required for human mabs. (ema ) . regardless, the pet medicine industry is making strides to put mabs into the market through basic research. early human therapeutic mabs contained a high proportion of mouse-derived sequences (fully mouse or mouse/ human chimeric mabs) that were recognized by the human immune system as foreign. this immune response triggered production of anti-mabs, leading to reduced therapeutic efficacy. subsequently, the design of humanized and fully human mabs has resulted in a vast reduction in their immunogenicity, although most therapeutic mabs may have some remaining immunogenicity that is not followed by apparent adverse clinical manifestations. these same issues hold true antigen y y polyclonal antibody antigen monoclonal antibody fig. polyclonal antibody compared to monoclonal antibody binding of antigens for making veterinary mabs and utilizing canine mabs for dogs that helps efficacy. it has been found that caninized anti-ige mabs reduce ige hypersensitivity in mite-sensitized beagles (gearing et al. ) . caninized anti-nerve growth factor mab (webster ) significantly reduced pain scores (webster et al. ) in dogs, and mabs that neutralize the pruritogenic cytokine il- in dogs reduced the pruritic response for weeks after injection (dunham et al. ) . one of the aspects regarding commercializing therapeutic antibodies involves regulation from the usda. in , two usda-approved monoclonal antibody treatments for b-cell and t-cell lymphomas in dogs were granted. these mabs fight lymphoma by targeting the protein cd , which is commonly expressed in b-cell lymphoma (ogilvie et al. ; bulman-fleming et al. ) . while these particular mabs were not commercially successful, they are part of a new approach for using therapeutic antibodies in veterinary medicine. other companies are developing veterinary mabs for cancer, allergies, and chronic inflammatory disease such as atopic dermatitis and for the control of pain associated with osteoarthritis in dogs and cats (webster et al. ). the first mab approved for veterinary use was in the european union (bmj ). it treats the clinical signs of atopic dermatitis in dogs, including itch and inflammation, for up to month. the mab treatment works by mimicking the activity of natural antibodies to selectively bind to and neutralize interleukin- (il- ), a key protein involved in cell communication which triggers itching associated with atopic dermatitis in dogs. because it neutralizes il- , it has been demonstrated not to interfere with the immune response, meaning that it does not induce unintended immunosuppression or enhancement. most therapeutic mabs are delivered via intravenous, intramuscular, or subcutaneous injection. this is because antibodies can't be delivered orally because of breakdown in the stomach by acids or other factors found there. various encapsulation methodologies are required in order to overcome this basic issue, and these methods should be forthcoming. once injected, most therapeutic mabs, like natural antibodies, have a long half-life (about days). the absolute half-life for each is unique, depending upon its concentration, distribution of its target, and, if the mab is directed to a cellsurface receptor, clearance and elimination of the target receptor. therapeutic mabs have two main safety advantages: ( ) they have very specific targets, and ( ) they don't have intracellular activity. as a result, there are few anticipated side effects and reactions although they can occur (catapanoab and papadopoulosc ) . mabs are eventually eliminated via intracellular catabolism in the lysosome, where they are broken down into peptides or amino acids that can be either reused for synthesis of new proteins or excreted via the urine. concluding remarks and future directions antibodies for veterinary use have great potential for the future. passive antibody therapy in the treatment of infectious diseases is a concept which dates back more than years, to the s, when the use of serum from immunized animals provided the first effective treatment options against infections with clostridium tetani and corynebacterium diphtheriae (hey ) . however, due to the discovery of penicillin by fleming in , and the subsequent introduction of the much cheaper and safer antibiotics in the s, serum therapy was largely abandoned. but in more recent times, the broad and general use of antibiotics in human and veterinary medicine has resulted in the development of multiresistant strains of bacteria with limited or no response to existing treatments and thus a need for alternative treatment options. this situation can be partially attributed to the overuse of antibiotics as growth promoters for production animals and other indiscriminate use. the combined specificity and flexibility of antibody-based treatments in providing passive immunity makes them very valuable tools for designing specific antibody treatments to infectious agents. these attributes have already caused a revolution in new antibody-based treatments in oncology and inflammatory diseases, with many approved products for human use. however, only very few mabs are approved for veterinary use. mabs therapies are expensive, and this has been a barrier for their development in the presence of inexpensive antibiotics. the use of antibiotics as growth promoters came to an end in in the usa with the rest of the world already limiting their use for this purpose manner (fda ) . this opens the door to new technologies and antibodies from monoclonal sources, chicken eggs, cow colostrum, or other sources to be among the chief contenders for limiting diseases in a safe manner and potentially to act as growth promoters. for that purpose, antibodies and antibodyderived treatments offer very attractive tools and attributes to neutralize infectious agents or modulate the immune system to enable effector cells to escape immunosuppressed conditions and contribute to the elimination of infections. the ability to raise antibodies to any target, and the ability to modulate effector functions, half-life, and size of the treatment units, makes antibodies ideal for tailoring treatments for specific infectious agents. however, more research into the use of antibodies as growth promoters will need to occur. one area that researchers are looking to make better use of antibody treatments for veterinary use includes more use of mabs. it has been predicted that minimal amino acid changes are needed to adapt an antibody from one species to another to avoid immune rejection. using libraries of genetic information and algorithms to make sure that key amino acid sequences are recognized as "self" or "native" by the target species' immune system can reduce the chance of undesirable immune reactions. this will increase the advantages typical of mabs for potency, safety, and a prolonged elimination half-life. therefore, these second-and third-generation mabs will be at the forefront of veterinary antibody technology along with igy technology. molecular targets for therapeutic mabs, igy, and colostrum igg in animals should ( ) be involved in clinical signs or disease mechanism and ( ) not have redundant pathways compensating for blockade of the intended target. the validity of blocking a molecule or eliminating a cell type must also be weighed against the importance of this protein or cell for desirable normal body functions. it is possible to speculate on uses of these antibodies in companion animals. these might include immune-mediated hemolytic anemias/ thrombopenias, myasthenia gravis, and autoimmune blistering diseases such as pemphigus, among other conditions. for cancer, the use of mab or igy therapy targeting b-lymphocytes will be valuable for b-cell lymphomas in dogs and cats. in allergic diseases, the use of mabs to inhibit production of ige via its promoting cytokines such as interleukins il- /il- , their cytokine receptors, or ige itself might be beneficial in dogs and cats with ige-mediated atopic dermatitis or food allergies. the itch sensation itself could be altered, at least theoretically, by antibodies targeting itch-promoting cytokines such as il- , nerve growth factor, thymic stromal lymphopoietin, or neuromediators involved in itch transmission. in arthritis therapeutic mabs that inhibit pro-inflammatory cytokines (tnf-alpha, il , etc.) or their receptors are likely to be of benefit in treating dogs and cats with arthritis. the usefulness of anti-ngf mabs as an analgesic must be confirmed. in autoimmune diseases, the use of mabs specific for b-lymphocyte surface proteins could theoretically lead to reduced production of autoantibodies. veterinary vaccines have had, and continue to have, a major impact not only on animal health and production but also on human health, through increasing safe food supplies and preventing animal-to-human transmission of infectious diseases. the continued interaction between animals and human researchers and health professionals will be of major importance for adapting new technologies, providing animal models of disease, and confronting new and emerging infectious diseases. one area of research where more information is needed is on factors that limit efficacy of vaccination ifoma, particularly in calves < month old. this is a complex issue. however, research continues to evolve in the area of newborn calf vaccinations. passive immunity provided by chicken egg antibodies will gain increasing use in production animals. there is no doubt that chicken abs can be produced and used, with minor modifications, in similar ways to mammalian abs. it can also be said that, depending on the circumstances, the use of igy abs often has significant advantage over the use of mammalian abs. however, from a realistic point of view, igy abs probably will not be able to completely replace the use of igg abs in diagnostic systems in the near future (schade et al. ) . chickens have the potential to be used to complete the spectrum of animals that have been used for ab production. however, a prerequisite is to make igy technology more popular and to convince the scientific community of its significant advantages. an interesting possibility for the future is the production of chicken mabs. these would combine the advantages of mabs with the advantages of chicken abs. it is to be expected that studies on the therapeutic or prophylactic use of igy abs will be intensified in the future. in particular, because of the increasing resistance of microorganisms to antibiotics, research on all aspects related to the development of specific igy against pathogenic microorganisms will have to be intensified. in conclusion, the next decade will see continued development of therapeutic mabs, igys, and colostrum antibodies for production and companion animals in both treatment and prevention. these highly specific molecules are likely to prove beneficial to uniquely target disease mechanisms without the side effects associated with broad-spectrum pharmacotherapy. while vaccines will continue to play a very important role in maintaining the health of animals by active immunity, antibodies that provide passive immunity will be an increasing part of the arsenal available to veterinarians to promote growth in production animals in a safe manner and to maintain health in companion animals. vaccinations for the swine herd bioactive egg components and inflammation first antibody therapy in veterinary medicine launched for dogs in the uk the mucosal immune system and its integration with the mammary glands treatment of canine b-cell lymphoma with doxorubicin with or without an anti-cd monoclonal antibody: an open-label pilot study evaluation of hyperimmune hen egg yolk derived anti-human rotavirus antibodies (antihrvigy) against rotavirus infection why do we need igm memory b cells? the safety of therapeutic monoclonal antibodies: implications for cardiovascular disease and targeting the pcsk pathway comparison of levels and duration of detection of antibodies to bovine viral diarrhea virus , bovine viral diarrhea virus , bovine respiratory syncytial virus, bovine herpesvirus , and bovine parainfluenza virus in calves fed maternal colostrum or a colostrum-replacement product vaccination of calves against common respiratory viruses in the face of maternally derived antibodies (ifoma) evaluation of methods for dehydration of bovine colostrum for total replacement of normal colostrum in calves the function and regulation of immunoglobulin d evaluation of factors affecting serum igg concentrations in bottle-fed calves vaccination of cows with clostridial antigens and passive transfer of clostridial antibodies from bovine colostrum to lambs protection of neonatal calves against k -e. coli and corona virus using a colostrumderived immunoglobulin preparation conjugated linoleic acid enhances immune responses but protects against the collateral damage of immune events orally administered anti-escherichia coli o : h chicken egg yolk antibodies reduce fecal shedding of the pathogen by ruminants study and application of the hyperimmunized yolk antibodies against tgev and pedv in piglets influence of colostrum intake on piglet survival and immunity effect of chicken egg yolk antibodies (igy) against diarrhea in domesticated animals: a systematic review and meta-analysis stabilized liquid egg material for extended shelf life. united states patent evaluation of anti-il- monoclonal antibodies in a model of il- -induced pruritus in beagle dogs comparison between immunoglobulins igy and the vaccine for prevention of infectious bursal disease in chickens committee for medicinal products for veterinary use (cvmp). questions and answers on monoclonal antibodies for veterinary use european medicines agency allergic disorders and ige-mediated immune responses: where do we stand? intestinal iga synthesis: regulation of front-line body defenses passive immunization in infectious bursal disease virus infected birds using chemically purified immune yolk immunoglobulins (igy) efficacy of hyperimmune bovine colostrum for prophylaxis of cryptosporidiosis in neonatal calves enhanced bovine colostrum supplementation shortens the duration of respiratory disease in thoroughbred yearlings food and drug administration ( ) food and drug administration center for veterinary medicine veterinary feed directive guidance for industry # american animal hospital association canine vaccination guidelines a fully caninised anti-ngf monoclonal antibody for pain relief in dogs field evaluation of egg yolk antibodies in prevention and treatment of enteric colibacillosis in calves immunology: fifty years of b lymphocytes heat-treated colostrum and reduced morbidity in preweaned dairy calves: results of a randomized trial and examination of mechanisms of effectiveness the biology of ige and the basis of allergic disease hyperimmune bovine colostrum treatment of moribund leopard geckos (eublepharis macularius) infected with cryptosporidium spp risk factors associated with colostrum quality in norwegian dairy cows maternal antibody transfer from dams to their egg yolks, egg whites, and chicks in meat lines of chickens history and practice: antibodies in infectious diseases mucosal immunity and vaccines perspectives on immunoglobulins in colostrum and milk development and function of organized gut-associated lymphoid tissues immunomodulatory constituents of human breast milk and immunity from bronchiolitis survey of immunoglobulin g content and antibody specificity in cow's milk from british columbia structural, antigenic and evolutionary analyses of immunoglobulins and t cell receptors chapter -passive immunization: toward magic bullets expression of the neonatal fc receptor (fcrn) in the bovine mammary gland current status of veterinary vaccines advances in prevention and therapy of neonatal dairy calf diarrhoea: a systematical review with emphasis on colostrum management and fluid therapy programs for the cow/calf operation nation-wide evaluation of quality and composition of colostrum fed to dairy calves in the united states allergens in veterinary medicine treatment of experimental ovine cryptosporidiosis with ovine or bovine hyperimmune colostrum man the barrier! strategic defences in the intestinal mucosa maternal antibodies: clinical significance, mechanism of interference with immune responses, and possible vaccination strategies treatment of canine b-cell lymphoma with chemotherapy and a canine anti-cd monoclonal antibody: a prospective double-blind, randomized, placebocontrolled study growth factors and antimicrobial factors of bovine colostrum colostrum and replacer ige production in rat fascioliasis sheep antiserum as an antibody supplement in newborn lambs structural and functional properties of membrane and secreted igd nutrient and immunity transfer from cow to calf pre-and post-calving chicken egg yolk antibodies (igy-technology): a review of progress in production and use in research and human and veterinary medicine passive immunotherapy in neonatal calves -i. safety and potency of a j escherichia coli hyperimmune plasma in neonatal calves passive protection against porcine epidemic diarrhea (ped) virus in piglets by colostrum from immunized cows the iga system: a comparison of structure and function in different species vaccination programs in poultry clinical outcome of calves with failure of passive transfer as diagnosed by a commercially available igg quick test kit spray dried, pasteurized bovine colostrum protects against gut dysfunction and inflammation in preterm pigs immune functions of immunoglobulin y isolated from egg yolk of hens immunized with various infectious bacteria precalving vaccination programs for cows passive immunity in ontario dairy calves and investigation of its association with calf management practices egg yolk igy: protection against rotavirus induced diarrhea and modulatory effect on the systemic and mucosal antibody responses in newborn calves igy antibodies protect against human rotavirus induced diarrhea in the neonatal gnotobiotic piglet disease model allergy and the gastrointestinal system ige: a question of protective immunity in trichinella spiralis infection canine brief pain inventory scores for dogs with osteoarthritis before and after administration of a monoclonal antibody against nerve growth factor comparison of antibody production to human interleukin- (il- ) by sheep and chickens vaccinating calves: new information on the effects of maternal iimmunity antibody to human rotavirus in cow's milk key: cord- -gykw nvt authors: yadav, mahendra pal; singh, raj kumar; malik, yashpal singh title: emerging and transboundary animal viral diseases: perspectives and preparedness date: - - journal: emerging and transboundary animal viruses doi: . / - - - - _ sha: doc_id: cord_uid: gykw nvt the epidemics and pandemics of a few infectious diseases during the past couple of decades have accentuated the significance of emerging infectious diseases (eids) due to their influence on public health. although asia region has been identified as the epicentre of many eids and upcoming infections, several new pathogens have also emerged in the past in other parts of the world. furthermore, the emergence of new viral diseases/infections, such as rift valley fever, west nile fever, sars coronavirus, hendra virus, avian influenza a (h n ), nipah virus, zika virus and swine influenza a (h n ) virus, from time to time is a glaring example threatening adversely both animal and public health globally. infectious diseases are dynamic and concerning due to their epidemiology and aetiological agents, which is manifested within a host, pathogen and environment continuum involving domestic animals, wildlife and human populations. the complex relationship among host populations and other environmental factors creates conditions for the emergence of diseases. the factors driving the emergence of different emerging infectious disease (eid) interfaces include global travel, urbanisation and biomedical manipulations for human eids; agricultural intensification for domestic animal eids; translocation for wildlife eids; human encroachment, ex situ contact and ecological manipulation for wildlife–human eids; encroachment, new introductions and ‘spill-over’ and ‘spill-back’; and technology and industry for domestic animal–human eids. the concepts of sanitary and phytosanitary (sps) measures and biosecurity have gained recognition globally in almost all the realms of human activities, including livestock health and production management. this chapter provides the experience gained in the control and management of a few important tads and eids along with the successes, constraints, limitations and future research needs for developing better control approaches. for wildlife-human eids; encroachment, new introductions and 'spill-over ' and 'spill-back'; and technology and industry for domestic animal-human eids. the term 'emerging disease' is used to refer to changes in the disease dynamics in the population. emerging infectious diseases (eids) are those which have moved recently into a new host or have enhanced incidences or geographic range or are caused by evolving pathogens (lederberg et al. ; daszak et al. ) . this general definition covers a range of infectious diseases of man and animals which pose a significant threat to both medical and veterinary public health. among the oie-listed diseases of viral aetiology, major changes have been experienced in the occurrence of rinderpest, peste-des-petits ruminants (ppr), foot-and-mouth disease (fmd), african swine fever (asf), lumpy skin disease and rift valley fever (rvf). of these, rinderpest presents a success story from the s to as a result of fao, oie, eu and iaea (international atomic energy agency) guided and coordinated programmes including the pan african rinderpest campaign (parc), npre and nrep in india (yadav ) , global rinderpest eradication program (grep) of the fao and other national governments where the disease was endemic. these exemplary efforts led to the historic declaration of global rinderpest eradication by the fao on june , . the terms 'exotic disease' and transboundary animal diseases (tads) are often used interchangeably. though all transboundary diseases are of exotic origin, all exotic diseases are not included in tad listing. many eids are also transboundary diseases. the tads are defined as highly contagious and transmissible epidemic diseases of livestock which have the capability for rapid spread to new areas and regions regardless of national borders and have serious socio-economic and public health consequences. nearly all diseases affect livestock, poultry, fishes and other animals and adversely impact the quality and quantity of food and other products, such as hides and skins, bones, fibres, wool and animal draft power for tilling, transport and traction. the reduction in animal production, productivity and profitability due to tads affect the human livelihood. in the present scenario of fast-increasing globalisation, tads represent a serious threat to the economy and welfare of the public and affected nations as they drastically reduce production and productivity; disrupt trade and travel and local and national economies; and also threaten human health through inferior food quality and zoonotic diseases/infections. as such, consequences of tads could have a significant detrimental effect on the economy and public health of not only the affected nations but also the whole of the world. possibly the infectious agents which cause emerging and transboundary diseases are already present in the environment and get the opportunity to cause disease under certain altered circumstances. the transmission of the infectious agent could occur between animal and human; between wildlife, human and domestic animals; or between wildlife, domestic animal(s) and human. however, the main source for maintenance and transmission of the infectious agents in nature is determined by the zoonotic pool and spill-over and spill-back mechanisms. tads have become of great concern due to the risk for national security on account of their economic significance, zoonotic nature and ever-growing threat of newer tads in future. among the tads having zoonotic manifestations, a number of infectious diseases, such as highly pathogenic avian influenza (hpai), bse (mad cow disease caused by prion), west nile fever, rift valley fever, sars coronavirus, hendra virus, nipah virus, ebola virus, zika virus and cchf, to name a few, adversely affecting animal and human health have been in the news in recent times (malik and dhama ; munjal et al. ; singh et al. singh et al. , . the direct and indirect costs due to the fmd outbreak in the uk in were assessed to be over us$ billion. over million chicken died or were destroyed in southeast asia in to control hpai (h n ). the netherlands suffered an economic loss of $ . billion due to classical swine fever in [ ] [ ] . as per the estimates of fao nearly one-third of the world meat trade was facing import bans on account of bse, hpai and other animal diseases. there is evidence to suggest that threats from tads have increased over the years. the risk of animal disease outbreaks is likely to further grow in future as the higher incomes of people in developing countries will generate more demand for animal protein and products (milk, meat, egg, chicken and fish). the number of animals raised for meat is growing rapidly. during s poultry production in east asia has increased by about % per year to double every - years. similar to tads, new human viral diseases have emerged like ebola, sars, zika, cchf, nipah and bse as well as there is the emergence of new antigenic forms or new biotypes of the existing infectious diseases, such as a hypervirulent strain of ibd in poultry in europe and highly virulent strain of newcastle disease in the usa (riemenschneider ; singh et al. ) . vector-borne pathogens, namely, bluetongue, african horse sickness, rift valley fever and west nile fever, have the potential to spread in epidemic forms. riemenschneider ( ) has deliberated over several issues relevant in the control of tads as proposed in the institute of medicine (iom) report (anonymous ) . some of the points which could be responsible for the increased threat of tads are briefly discussed below. in the present-day world, higher quantitative levels of animal origin foods, as well as faster trade, new trade routes and air travel, have led to higher risks for contracting new infections and diseases. as it is now possible to reach any part of the world within h which is less than the incubation period of most of the infectious diseases, animals or people carrying the infectious agents go undetected in want of clinical disease/symptoms. fresh commodities vis-à-vis processed foods that have witnessed an increased trade are more likely to carry the pathogens to distant parts of the world-countries and continents. recent decades are witnessing higher demands for animal protein and other nutrients through meat and meat products, milk and milk products, eggs, and fish and fish products as a result of rising incomes in the developing countries and elsewhere which leads to the intensification of production systems and overcrowding of animals. this increased production is often required in peri-urban areas, having large human populations, under suboptimal husbandry practices. in such high-production areas, disease outbreaks affect a greater number of animals at a faster rate and speed, leading to heavy economic losses. drastic control measures are taken, such as the slaughter of infected and in-contact animals followed by burning or burial is not acceptable to the society at large. for example, the mass slaughter of pigs in the netherlands in - for the control of csf virus led to objection from the non-farm population which might influence the application of the stamping-out policy as a disease control approach in future. exposure of the domestic animals to forest niches due to deforestation and transformation of tropical rainforests for livestock grazing exposes the domestic livestock to a completely new range of pathogens and vectors which previously circulated in wildlife reservoir niches only. with the domestic livestock being fully susceptible and naïve to these infectious agents, the disease spreads more rapidly and severely in want of lack of diagnostic tests and vaccines against these new pathogens resulting in heavy morbidity, mortality, trade restrictions and economic losses. nowadays many countries face prolonged civil unrests besides inter-and intracountry conflicts, which may lead to enhanced threat of tads. civil disorders are known to disrupt enforcement of quarantine and other control measures due to refugee and army deployments/movements. breakdown in the institutional support for quarantine and difficulty in gaining access to border area due to landmines make disease surveillance more difficult. inflows of more food aids for such areas also pose additional risks as the food items may have contaminants. climate change and global warming seem to be altering rainfall and weather patterns. rising temperatures in the northern hemisphere are likely to shift the distribution of insect vectors of bluetongue, african horse sickness, rift valley fever and similar vector-borne diseases. the bluetongue virus (btv) having serotypes occurs in many parts of the world. however, until recently it was never reported from europe. the sudden incursions of some serotypes into spain, italy, greece, portugal and the balkan countries since , followed by germany, and the recent incursion of btv serotype in several farms in the netherlands, germany and belgium since august as well as serotype are also believed to be due to climate change as european weather has become hotter in recent decades. the btv serotype revealed that this serotype is closest to the nigerian strain. the incursion is believed to have been caused by the importation of an infected zoo animal or an infected midge. an upsurge of rift valley fever was observed in east and west africa due to climatic changes. many factors discussed above make the tads as a serious threat to national and international security. the developing countries are usually the worst sufferers. among other factors, veterinary public health services in developing countries are usually much behind than the medical public health services. moreover, unlike human disease reporting, animal disease reporting systems are usually based on passive reporting rather than active disease surveillance. a few other factors are also responsible for greater threat due to tads, namely ( ) lack of awareness of the farmers about the high-threat epizootic animal diseases; ( ) lack of diagnostic facilities for exotic diseases, and under-reporting of animal diseases like hpai due to the fear of loss of internal and export market till the country gets infection-free status as per oie-laid-down criteria; and ( ) poor and faulty compensation schemes. in the technological advances made in today's world, there is always a real risk of deliberate misuse of certain infectious agents/pathogens by terrorists as a means of biowarfare between nations to harm the people and/or livestock, poultry and other animals. potential for pathogenic disease agents not reported previously in a country and being misused or mishandled for bioterrorism is likely to threaten the ecosystem on a large scale. even new pathogens can be engineered as novel infectious agents. the animal diseases could even be a greater threat than human diseases as these may result in significant economic disruptions, besides causing food poisoning and deterioration, and zoonotic diseases in human beings. as animal diseases get less priority than human infections/diseases in undertaking immediate disease control measures, the threat scenario with the use of animal pathogens for bioterrorism or biowarfare will have many serious consequences. some of the viruses having significant bioterror potential for humans and or animals include hpai (h n ), when an exotic viral disease strikes a country for the first time, it may initially affect one animal, few animals or a large number of animals. the strategy to be adopted for containing the outbreak will depend on the nature of the virus, speed of its spread, role of vectors, risk assessment, communication and management, response time and country legislation on disease control and prevention. thus, there is a need to develop strategic plans for the prevention and control of exotic and tad on a case-to-case basis. examples of such viral diseases from indian perspective include african swine fever (asf)‚ transmissible gastroenteritis (tge)‚ and swine vesicular disease in pigs, rift valley fever, african horse sickness (ahs), west nile fever, eastern equine encephalomyelitis (eee), western equine encephalomyelitis (wee), and venezuelan equine encephalomyelitis (vee), fmd virus types 'c', 'sat i', 'sat ii' and 'sat iii', nipah virus, hendra virus, sars coronavirus, and prion diseases-bovine spongiform encephalopathy (bse), and scrapie. institution of appropriate and timely biosecurity measures is an important instrument for the protection and improvement of animal health. breach in biosecurity due to ignorance and avoidable lapses in the adoption of timely biosecurity and biosafety measures in the management of livestock, poultry and fish minimise the risks from infectious diseases including eids and tads. breach in biosecurity in livestock management is often an important reason for the high incidence of zoonotic and other infectious diseases of animals. this is more so in case of the viral diseases of livestock and poultry. closer contact between wildlife, animals and humans and rearing of livestock and poultry in close association with people promote spread of viral and other infectious diseases which have the potential for threatening health, economies and food security around the world. the emergence of new viral diseases/infections, such as rift valley fever, west nile fever, sars coronavirus, hendra virus, avian influenza a (h n ), nipah virus, zika virus and swine influenza a (h n ) virus, from time to time is a glaring example of zoonotic disease threats adversely affecting both animal health and public health, national economies and food and nutrition security globally. due to a lacuna in the biosecurity, viral diseases like the fmd had reoccurred in countries where these had not been reported for many decades, including the uk, a developed country. biosafety and biosecurity are interrelated terms but used in different contexts. the guidelines are developed by who, fao and oie. biosafety aims at the protection of person(s) at work and the facilities which are dealing with the biological agents, against their exposure to a disease agent, and prevents unintentional exposure to pathogens/toxins or their accidental release. thus, biosafety is the application of knowledge, techniques and equipment to prevent personal, laboratory and environmental exposure to potentially infectious agents or biohazards. biosecurity, unlike biosafety, has divergent meanings in different contexts in which it is used. it deals with the protection of microbiological assets from spill-over, theft, loss, diversion or intentional release from laboratories, preventing the import of certain organisms/ toxins. biosecurity is a set of preventive measures designed to reduce the risk of intentional transmission of infectious diseases to safeguard the facilities containing sensitive biological materials with the potential of a biological weapon. in brief, biosecurity means bio-risk management. once a disease is eradicated globally, the policy for keeping the wild and vaccine strains of the virus along with vaccine stocks for emergency use and their subsequent destruction is decided by international agencies like fao, who and oie based on the recommendations of experts in the area. these include risk assessment; communication and management; quarantine of imported animals at seaports, dry ports and farm; establishment of check posts and vaccination stations at international and interstate borders for clinical surveillance; creation of immune belts at international borders; and planning and conducting structured disease surveillance including clinical surveillance and serosurveillance. biosafety and biosecurity need to be observed at all levels beginning from farm to national and international levels. for handling the most dangerous transboundary disease pathogens, bsl iii and bsl iv laboratories are required to ensure biosafety, biosecurity and biocontainment. proper zoo sanitary measures, such as quarantine; rodent and vector control; disinfection of animal sheds and premises; proper disposal of dung, urine, feed and fodder wastes; and proper carcass disposal, need to be adopted religiously for effective management of eids and tads. every country needs strict and foolproof biosecurity mechanism at its international borders as a safeguard against the entry of exotic infectious agents/diseases from abroad along with the import of livestock and other animals and their products. for example, india has contiguous and porous borders with countries like nepal, bhutan, pakistan and bangladesh, besides free trade with nepal and bhutan. since all these countries are vulnerable to tads, there is a need for regional biosecurity plan to ensure a biosecure region. it would never be possible to have a biosecure country if the bordering countries do not have effective biosecurity in place. different countries are at risk for a number of tads like anthrax, plague, glanders, lyme disease, contagious equine metritis, salmonella abortus equi, hpai virus, fmd virus (sat - ), lyssavirus, rabies, hendra and nipah viruses, west nile virus, highly pathogenic nd virus, rabbit haemorrhagic disease virus, bovine spongiform encephalopathy (bse), african horse sickness (ahs), equine encephalomyelitis (eee, vee, wee), equine infectious anaemia, chicken infectious anaemia, equine influenza, vesicular stomatitis, rift valley fever, malignant catarrhal fever (mcf) and other tses of sheep, goat and deer. biosecurity measures are required for preventing and containing the ingress of these diseases through international trade. the oie has facilitated safe trade in animals and animal products by developing effective standards to prevent the spread of animal diseases across the globe. prevention of transmission of pathogens across intra-and inter-country borders warrants devising of biosecurity measures at par with international standards. adequate infrastructure comprising check posts and quarantine facilities at seaports, airports and porous international land border are must to check the ingress of viral and other pathogens from across the borders. diagnostic facilities with trained human resource, and well equipped with instruments and pen-side diagnostic tests/kits, should be in place for ensuring the pathogen-free status of imported livestock and livestock products. biosecurity measures at national level incorporate the components of 'external biosecurity' preventing the ingress of exotic and transboundary animal diseases and 'internal biosecurity' within the country encompassing zonal, compartmental and farm-level biosecurity. regulations for animal movement through interstate borders in india are in place but need strict implementation. modern detection systems can be used for identification and tracking of animals and animal products to provide information regarding the origin of the animal, and environmental practices used in production and food safety. for effective disease prevention and control, integration of biosecurity into every operation at the farm is essential. farm biosecurity should be inclusive of both 'bioexclusion' (measures for preventing a pathogen from being introduced to a herd/ flock) and 'biocontainment'. the latter addresses the events after the introduction of the pathogen and its ability to spread among susceptible groups of animals at the farm or further spill-over to other farms. strict implementation of biosecurity at farm level has played a crucial role in preventing the spread of diseases. a suitable plan addressing important issues, such as location and layout of the farm, animal health practices in place and general management on the farm, needs to be chalked out. it should be flexible to include new knowledge, concepts and technology. a wide range of biosecurity practices have been recommended for different livestock species and production systems, both for specific infection risks or for disease prevention in general. biosecurity practices have been recommended for cattle, sheep, pig, poultry and fish production systems. general biosecurity practices and interventions that can be applicable across species and farms include: . maintaining a closed herd procurement/purchase of animals from known sources . minimising the number of animals purchased/transferred/exchanged and the number of herds from which the animals are introduced . avoiding purchases from markets or dealers . appropriate quarantine and testing of animals upon introduction or reintroduction in farm premises . discouraging farming practices such as hiring a bull or stallion and returning it after the breeding season . avoiding the introduction of biological material of uncertain health status . health and vaccination records should be obtained for all the newly introduced animals isolation/quarantine of such animals for - weeks in a separate quarantine facility should be practiced and the animals during this period should be observed for illness/symptoms and screened for important diseases before mixing with other stock at the farm. laboratory testing of appropriate samples collected during quarantine against important infectious diseases is recommended. the incoming stock can also be given vaccine against the endemic disease prevalent in the area at least weeks before release from quarantine to boost their protective immunity. animal diseases can spread from farm to farm resulting in animal sickness, death and economic losses. visitors to the livestock farm, disease laboratory, birds, rodents, vehicles, feed and fodder and other inanimate objects are often a source of infection. in addition to adverse effects on the economy, there can be negative effects on the environment and human health. the best designs are to implement effective biosecurity practices. baths by the laboratory workers after and before visiting animal farm or laboratory and putting on gum boots, disposable overall, head gear and gloves should be a mandatory requirement. all effluent from the laboratory should be pre-treated to ensure freedom from pathogens before their disposal to the environment. disinfectant foot bath for the workers and vehicles entering the farm at the gate, exclusive separate dress and shoes for laboratory and farm workers, minimum movement of the people and animals within the farm during the outbreak period, and personal health and hygiene of the staff are some of the minimum guidelines to strengthen farm biosecurity. timely, rapid and accurate disease reporting based on oie-approved diagnostic tests is a must for effective detection of the pathogen and instituting early response without giving much time for the disease to spread further. to face the new exotic diseases, it is recommended to have a standard sop in place along with technical guidelines, decision-taking levels along with adequate provision for funds and legal backup. it has been observed that lack or inadequate compensation for culling the diseased and in-contact animals and poultry and negative effect on the sale, sale price and exports deter the farmers from reporting animal diseases in time which eventually leads to the spread of tads. for example, due to bse cattle producers in the usa lost over $ billion in exports to japan. similarly, hpai resulted in over $ billion loss in poultry exports for thailand. trade concerns also discourage the use of preventive vaccinations for some diseases such as hpai and fmd. disease-free countries are generally reluctant to import animals or animal products from the countries practicing preventive vaccinations. with these adverse trade considerations, stamping-out policy was adopted by the uk over the vaccination for fmd in outbreak. from a public health point of view, a vaccination programme might reduce the viral load circulating in a country and thus reduce the risk of hpai spreading to humans. however, stamping out rather than vaccination is preferred by most of the countries to declare themselves free from disease/infection at the earliest possible to regain access to exports. to ensure the cooperation of livestock farmers, it is essential to provide adequate and timely compensations to reduce the losses suffered by them on account of culling and closing the units for a few months. the failure of timely disease reporting hastens the spread of tads within the country as well as between countries. it is believed that the hpai (h n ) avian influenza virus might have been circulating in the poultry for months in the affected region before it was reported to the international authorities in leading to the wide spread of the disease/infection. an ex ante study of the fmd outbreak of the uk suggested that the fmd virus was probably introduced - weeks before it was reported and followed by a ban on livestock movements. earlier reporting and ban on animal movements would have cut the spread of the disease by about %. it is believed that the eids and tads will continue to remain an ever-growing threat to animal and human health, economic sustenance of the world and global environment well-being. however, it is difficult to predict the number of these diseases which could rapidly escalate in a country or region threatening the animal and human life as well as the economy of that region or nation. the rising global demands for meat, particularly in east and south asia, have put humans and animals together in numbers never seen before in the world. the fact is that the farm biosecurity in these countries where meat production is growing most rapidly is often poor. this scenario creates a great scope for animal diseases to jump species to create human health problems. some of these issues were thoroughly described in the iom report, , and further debated and discussed by riemenschneider ( ) . the steps suggested include early detection and early response, preparedness plans, decentralisation of government structure, international coordination, understanding of ecology, microbial evolution and viral traffic, expanded surveillance system, disease intelligence, preparedness, collaboration and cooperation among government agencies and cross-field partnerships. in developing countries, the preparedness plans for animal diseases are often unsatisfactory. incentives such as adequate compensation should be provided to the affected farmers as an impetus for reporting animal diseases. the level of preparedness should be assessed by conducting mock drills. this will help in confidence building for rapid detection and response to both eids and tads that appear suddenly and are capable of spreading to large areas in a short time. import bans in response to an animal disease outbreak must be based on sound scientific evidence to ensure that the concerned countries also have the incentive to report the disease to international agencies, namely oie, fao and who. deficiencies in national veterinary services have been attributed for inability in early detection of the disease and response as investigation, and diagnostic services have deteriorated in many regions. a continuing structural upgradation programme for national veterinary services will have to be taken into account for their transformation from providers of services, such as diagnosis, vaccinations and treatment of sick animals to inspection and quality assurance services. disease surveillance, early warning and emergency preparedness need to be pursued more vigorously in africa, the middle east and southeast asia as vital components of national veterinary services. though public health and national security are under the perspective of national governments, the decentralised government structure and improved international coordination are essential to address the threat of tads effectively as they do not respect local, regional or national boundaries. nevertheless, government support at the administrative level is essential to assure sufficient and timely response to avoid the spread of disease through livestock movement controls, closing of live markets, sharing of diagnostic services, expertise, funding, etc. technical support and guidance of international agencies, such as fao, who and oie, are key in the formulation and timely implementation of the plans and modalities for the control and management of eids and tads. the fao in established the global framework for the control of tads (gf-tad) through the emergency centre for transboundary animal disease (ectad) operations and emergency prevention system (empres) for transboundary animal and plant pests and disease initiatives for early warning and response to disease threats, following a collaborative approach to investigation at animal-human-ecosystem interface. these mechanisms have proved to be of immense help and use in the control, prevention and eradication of disease(s). microbial evolution, particularly viral evolution, is a continuous process. it is, therefore, necessary to conduct basic research on emerging infectious diseases, both viral and another microbial origin, for providing new insights about the factors responsible for the emergence of new microbes. for understanding the ecology of disease, social factors, viral and microbial traffic and spread, ecological and demographic changes in human and animal populations due to migration and other factors work in tandem leading to precipitation of emerging infections. these signals for viral and microbial traffic should be seen as warning signs. biodiversity should include microbes and viruses, and environmental impact assessment should include health aspects into account in development planning. enhancing surveillance systems by establishing laboratory response network at national, regional and international level is important for which adequate funds should be provided. by linking the laboratories in public and private domains, such networks are expected to enhance the capabilities at all levels to detect and prevent the spread of eids, transmitted naturally or intentionally (anonymous ) . a network of more than laboratories world over by who for a constant survey of influenza viruses is one of the best examples of networking of laboratories for eids and tads. these laboratories should have multidisciplinary teams involving veterinarians, physicians, ecologists, entomologists, vaccinologists, epidemiologists, molecular biologists, immunologists and possibly other specialists. state-of-the art disease surveillance is required having the capability to forecast when and where a particular disease is likely to occur for more targeted surveillance. such actionable intelligence may derive from the analysis of changes in climatic conditions, vegetation, wildlife demographics, trade pattern or vector demographics and distribution (anonymous ). the disease-producing microbes, particularly viruses and bacterial agents, often change their antigenic make-up as a result of spontaneous mutations, and immune pressure when the wild strains of the infectious agent persist in the host in the presence of vaccinal antibodies. rna viruses having segmented genome are more prone to such antigenic changes as a result of recombination, gene deletion, etc. influenza a viruses of human and animals continuously evolve new virulent variants by exchanging haemagglutinin (h) and neuraminidase (n) genes of various h and n types circulating in human, birds, pigs and other species including equines. with the change in antigenic make-up, the current vaccine strains do not provide protection against the new types of the virus. similar situations occur in fmd virus having seven types and further subtypes, clades and genotypes: ppr virus and newcastle disease virus, to name a few. new antigenic types of a virus or pathogen may also be introduced from abroad through imported livestock and poultry. hence, there is a need to have a plan in place to upgrade the vaccine by incorporating the current strains of the pathogen which induce strong and lasting immunity. this will require the setting of repositories of field strains isolated from disease outbreaks, particularly the ones from vaccine failure cases. such updates of vaccines are routinely followed for influenza vaccines for poultry, equines and human, and fmd and csf vaccine for livestock. vaccination is a valuable and well-tested method in preventive veterinary medicine for promoting animal health and welfare and reducing the risk of human exposure to several zoonotic pathogens. prophylactic immunisation practices, principles and vaccination protocols have helped in significantly reducing the prevalence of many life-threatening viral and bacterial diseases. the risks of not vaccinating their stock on account of lack of awareness among the stakeholders, non-availability of costeffective diagnostics and vaccines, and poor delivery of veterinary services to the livestock farmers can have serious consequences on livelihoods of rural livestock producers. effective vaccination programmes if implemented properly with a broader perspective are likely to reduce the need for antimicrobials, which in turn can help reduce the risk of emergent antimicrobial resistance. the world veterinary association (wva) and health for animals believe that it is essential for the global veterinary profession to educate the public, particularly animal keepers and producers, about the benefits of vaccination for animals and humans. the major objectives and motive of veterinary vaccines are to protect, improve and promote the health and welfare of companion and food animals; increase the production of livestock in a cost-effective manner; and prevent animal-to-human transmission of infectious diseases from domestic animals and wildlife to humans through animal-origin food, close contact and other mechanisms. these diverse aims have led to different approaches to the development of veterinary vaccines from crude but effective whole-pathogen preparations to molecularly defined subunit vaccines, genetically engineered organisms or chimeras, vectored antigen formulations and naked dna injections for immunisation of animals. the final successful outcome of vaccine research and development is the generation of a product that will be available in the marketplace on demand and suitable to be used in the field to achieve desired outcomes. successful veterinary vaccines have been produced against major bacterial, viral, protozoan and multicellular pathogens, which led to successful field application and adaptation of novel technologies. these veterinary vaccines have had, and continue to have, a major impact not only on animal health and production but also on human health through increasing safe food supplies, namely milk, meat, eggs and fish, and preventing animal-to-human transmission of infectious diseases. the continued interaction between the researchers from veterinary and medical streams and health professionals will be a major impetus for adapting new technologies, providing animal models of human diseases and confronting new and emerging infectious diseases. over different veterinary vaccines are currently commercially available (meeusen et al. ). multivalent (bivalent, trivalent and polyvalent) vaccines should be given preference over monovalent vaccines to cover more than one disease prevalent during control programmes to save money, time and other expenses and also to reduce the burden on implementing agencies, such as veterinarians and para-health livestock workers. there should be a system in place to conduct post-vaccination sero-monitoring in the field by appropriate agencies for finding evidence for adequate seroconversion in the randomly collected samples as per standard procedure preferably using diva tests to differentiate between vaccine-induced immune response and the one induced by the virulent virus. the application of risk analysis concerning the spread of disease on account of international trade in live animals and their products, namely, import risk analysis (ira), has been largely driven by the sanitary and phytosanitary (sps) agreement of the world trade organization (wto). the ira standard established by the world organisation for animal health (oie), and associated guidance, meets the needs of the sps agreement. the use of scenario trees is the core modelling approach adopted to represent the steps necessary for the hazard to occur. there is scope to elaborate scenario trees for commodity ira so that the quantity of hazard at each step is assessed (peeler et al. ) . the dependence between exposure and establishment of the hazard suggests that they should fall within the same subcomponent. ira undertaken for trade reasons must include an assessment of consequences to meet sps criteria. the integration of epidemiological and economic modelling may open a path for better methods. matrices have been used in qualitative ira to combine estimates of entry and exposure, and consequences with likelihood, but this approach has flaws, and better methods are needed. ira standards and guidance provided by oie indicate that the volume of trade should be taken into account. some published qualitative iras have assumed current levels and patterns of trade without specifying the volume of trade, which constrains the use of ira to determine mitigation measures (to reduce risk to an acceptable level) and whether the principle of equivalence, fundamental to the sps agreement, has been observed. it is questionable whether qualitative ira can meet all the criteria set out in the sps agreement. nevertheless, scope exists to elaborate the current standards and guidance, so that they better serve the principle of science-based decision-making. options for trade from disease-free zones and disease-free compartments and trading in safe commodities are now available to have a positive mechanism for facilitating international trade. in india, fmd-control program (fmd-cp) is already in operation intending to create fmd-free zones. similar zones can be created for other diseases like hs, bluetongue, sheep pox, goat pox, ppr and other important diseases. compartmental biosecurity is the new concept for the management of biosecurity in a compartment through a single set of biosecurity measures. creation of zones/compartments will ensure a boost in international trade of livestock and poultry products. in india, legislation regarding the movement of animals across these zones and compartments are required by the central and state governments. the tads are a threat to animal health and production and cause huge losses to the economy of nations. recent outbreaks of bovine spongiform encephalopathy (bse), foot-and-mouth disease (fmd) and highly pathogenic avian influenza (hpai) have unfolded the real and growing global threat that animal diseases pose to livestock systems and to human health and welfare. the tads adversely affect the trade in live animals and their products. the detection of one bse-positive animal in in the usa led to an % drop in beef exports during [ ] [ ] . similarly, the losses in the uk were estimated to be over us$ billion during the ill-fated fmd outbreak. the economic losses due to hpai (h n ) avian influenza have been estimated from . % to . % of gdp in thailand and vietnam by rushton et al. ( ) . the outbreak of avian influenza due to h n strain in the netherlands destroyed as many as million birds. direct losses due to fmd in india have been estimated to the extent of inr , million per annum (anonymous - ) . ppr has been estimated to cause global losses between us$ . billion and $ . availability of adequate financial support for animal health r&d, especially in developing countries, is not always readily ensured. as the livestock keepers in these countries are mostly socio-economically poor, the local and national governments should come forward to support these programmes, particularly for the landless and marginal farmers keeping pigs, sheep, goats, backyard poultry and low-producing bovine stocks by providing incentives or subsidies for diagnostics and vaccines. raising venture fund for emergency disease control through public and private partnership could be considered to meet the urgent requirements, besides farmer-friendly insurance policies for livestock health protection. for important tads, such as avian influenza, ppr and fmd, multinational, regional or global programmes under the supervision of fao, who and oie under 'one health' concept are suggested for better coordination and results. the 'one world-one health' (owoh) concept steered by fao, who and oie has its roots in the interaction between living beings including humans, animals and pathogens, and the environment is considered as a unique dynamic system in which the health of each component is interconnected and dependent with other components. nowadays, a newly integrated 'one health one medicine' approach reflects this interdependence with a holistic view of the ecological system. the owoh can be defined as a collaborative and a multidisciplinary effort at the local, national and global level to guarantee an optimal healthy status for humans, animals and environment. the control of infectious diseases, which have influenced the course of human history, is to be considered strictly related to the one health concept. after its first occurrence in in china, the highly pathogenic avian influenza (hpai) a virus (h n ) has affected more than countries in asia, europe, africa and north america. the virus affected wild birds as well as domestic poultry. sporadic cases of transmission to humans in close contact of infected birds with sizeable mortality raised the pandemic concern of 'bird flu'. after the first report of the h n virus from india and bangladesh in and , respectively, both these countries are experiencing outbreaks almost every year. between february-march and february , india incurred an expenditure of more than inr . million, including inr . million for compensation and inr million on the culling of . million birds (anonymous - ) . avian influenza viruses (aivs) have become a continued threat to global health and economy. after its first outbreak in , the h n hpai serotype disseminated very fast from korea to other parts of asia, europe and north america, a feature not observed in case of other highly pathogenic aivs. however, the pathobiological features of the virus that favoured its global translocation are not known. results of simulation studies undertaken in migratory birds to identify pathobiological features supporting aiv intercontinental dissemination risk suggest that characteristic differences exist among h n and other aiv subtypes, e.g. h n and h n that have not spread as rapidly. lower infection recovery and mortality rates and migration recovery rates also favour translocation in migratory bird populations. although india has been reporting h n aiv since , the h n virus was first time reported in from migratory birds and poultry in the states of delhi, madhya pradesh, kerala, karnataka, punjab and haryana. studies undertaken on comparative epidemiology of influenza viruses h n and h n among human and bird populations to find out similarities and differences between the two viruses in their genetic characteristics, distribution patterns in human and bird populations and postulated mechanisms of global spread (bui et al. ) indicated that h n viruses are diversifying at a much greater rate than h n viruses. analyses of certain h n strains demonstrated similarities with engineered transmissible h n viruses, which make it more adaptable to the human respiratory tract. these differences in the epidemiology of h n and h n viruses in human and birds raise further questions as to how h n has spread at a greater rate than the h n virus. african swine fever (asf) is a highly contagious, deadly emerging disease of pigs in many countries. although first described in and it affected more than countries in africa, europe and south america, several key issues about its pathogenesis, immune evasion and epidemiology remain uncertain (arias et al. ). in the absence of a vaccine, the disease causes greater sanitary, social and economic impacts on swine herds compared to many other swine diseases. currently, asf is present in sub-saharan africa, sardinia, the trans-caucasus, the russian federation and central and eastern states of the european union. the disease continues to spread, with first reports in china (august ), bulgaria (august ), belgium (september ) and vietnam (february ) highlighting the increasing threat of asf to the global pig industry (netherton et al. ) . ongoing outbreaks have also been reported in hungary, latvia, moldova, poland, romania, russia, south africa, ukraine, cambodia, north korea, vietnam and laos. the disease was rampant in china during , and about half of china's breeding pigs died or were slaughtered. the threat of asf looms large as presently no licensed vaccine is available against this disease, and further research is desired in this area for the development of live attenuated vaccines for asfv. it has been possible to generate pigs resistant to classical swine fever virus and prrs virus (burkard et al. ) by using genetic modification of the host species. genetic modification can be attempted as a viable solution to increase the host resistance to asfv. wild suids, namely warthog or bush pig, sequences could be engineered into the domestic pig genome to produce animals in which replication of asf virus and/or disease burden after asfv infection is reduced. however, to generate pigs fully resistant to asfv infection, a more effective strategy such as targeting the virus receptors on the host cell to block the entry of virus and viral replication may be attempted. different clinical courses of asfv infection in pigs have been described based on the virulence of the virus isolates, and sequencing the genomes of isolates of reduced virulence has identified virus genes associated with this phenotype. targeted gene modifications and deletions and testing of the genetically modified viruses in macrophages and pigs have contributed to an understanding of virulence factors and how the virus modulates host responses. in the absence of a vaccine and rapid spread of asf in europe and asia, the main emphasis should be on strict customs and border protection to keep the negative countries free from asf virus infection/ disease. research is required on priority to explore the virulence genes and genes related to host protection and immune evasion, role of multigene families in antigenic variability, mechanism of evasion of the immune response, factors determining viral persistence and infection outcomes, and interactions between asfv and wild african suids, which are tolerant to asfv infection. such studies will provide a complete understanding of the pathogenesis of asf. the specific role of different hosts including wild suids, vectors and environmental factors in disease propagation needs to be elucidated for understanding different epidemiological scenarios. in this regard, the northern european scenario in which infected wild boars drive disease transmission and maintenance needs to be investigated further. presently, asf has become of great significance in china and a real threat to the pig and pork production. the affected countries are planning to compensate for the losses in pork production by increasing broiler poultry production. gaps in sanitary control of wild boar populations make asf control difficult. raising awareness among veterinarians, hunters and farmers should be the priorities for asf control. advances in non-invasive sampling are required to facilitate surveillance in affected areas. current and future tests need to be optimised for noninvasive matrices. the availability of a confirmatory serological test and cell lines for replacing primary cell cultures should be the priorities for future work. availability of safe and potent vaccine against asf could benefit disease control and prevention substantially, but despite some advances such vaccine is still lacking (arias et al. ). after the successful eradication of rinderpest from the globe in , foot-andmouth disease (fmd) of cloven-footed animals is another oie-listed important viral disease inflicting heavy economic losses and adversely affecting the trade of livestock and livestock products from endemic countries to fmd-free nations/ regions. knight-jones et al. ( ) have given a detailed account of global fmd research update along with gaps and an overview of global status and research needs. the conclusions are drawn to highlight that currently available vaccines and control tools have enabled fmd eradication from many countries of the developed world. however, in many developing countries, fmd remains uncontrolled. the main reason given is that biosecurity measures that have been fundamental to successful fmd control in the developed country are difficult to be implemented effectively in developing countries due to obvious reasons. in the present scenario, improved vaccines, with longer lasting protection against a wider range of fmdv strains and lower production costs, could be the single most important development to enhance our ability to control fmd. although encouraging progress has been made with several novel vaccine candidates, addressing key limitations of the current inactivated vaccines, a commercial vaccine is yet awaited. while new discoveries are crucial, current vaccines have been used to effectively control fmd on numerous occasions. however, for imparting better immunity, fmd vaccines should be subjected to adequate quality assurance and be made available in sufficient quantity to provide desired coverage following appropriate strategy. there is also a need for better training and support in the design and execution of vaccine-based fmd control programmes. another area of research is genetic and molecular studies on the virus to elucidate host-virus interactions. more powerful tools and analyses are increasing our understanding of various aspects of fmdv evolution, ecology and epidemiology. this, in turn, should benefit many areas of fmd research, from basic virology to the vaccine and diagnostic development. furthermore, improved genetic technologies have the potential to reveal information crucial for control, such as transmission chains, vaccine match and level of virus circulation. fmd control has been prioritised by many governments around the world. besides traditional bastions of established research institutes in europe and north and south america, notable work is being conducted in china, india and africa. experiences in south america and europe have shown that through decades of sustained investment fmd can be controlled, even in regions where once it was rampant and control was seemingly impossible. however, if improved and more widespread fmd control is to be achieved, continued investment in fmd research at the local and international level is a must. improved diva diagnostics increase our ability to detect infected animals in vaccinated populations. greater confidence in the ascertainment of fmd status of animals and products has, in turn, opened the way for international standards for trade and disease control that are more efficient and less restrictive. rigorous licensing procedures increase the time taken for new technologies for diagnostic kits and vaccines to reach the market. however, if authorisation is less rigorous, substandard products may be released onto the market. hence, there is a need to balance these two requirements. relaxation should be provided for necessary changes to the existing technologies, such as changing vaccine strains, particularly when the need is urgent (knight-jones et al. ). rinderpest, also known as 'cattle plague', was once a deadly serious threat to the livestock industry and agriculture economy in several regions of the globe, particularly in asia, africa, europe and the americas. it periodically swept through old world, resulting in devastating epizootics and huge economic losses. the disease could be successfully eliminated from the globe with mass vaccination programmes, zoo sanitary measures, policy support, international cooperation and political will. the morbidity and mortality rates in newly exposed naïve populations could be as high as - % leading to enormous economic losses. in india mortality rate of about , animals were recorded among the affected bovine population of , per annum during the first half of the s, indicating average mortality of %. throughout the history of humankind, the social, economic and ecological consequences due to rinderpest had been more catastrophic, even changing the history of nations and empires. in india, the presence of rinderpest was confirmed by the cattle plague commission (hallen et al. ) . this disease has been conquered successfully by following mass vaccination along with zoo sanitary measures. the fao declared the global eradication of rinderpest on june , marking it the first ever viral disease of animals eradicated globally about three decades after the eradication of smallpox, a viral disease of humans in (yadav et al. ) . constraints of availability of quality vaccine in sufficient quality, freeze-drying of vaccines and maintenance of cold chain for a vaccine in tropical countries, lack of infrastructure for structured clinical surveillance and sero-surveillance were some of the limitations in executing the mass vaccination programmes. in india, dividing the country into four zones based on the epidemiological picture of the disease and adopting strategic and focused vaccinations at interstate and international borders and migration routes of bovines and caprine for creating immune belts, coupled with rigorous clinical and sero-surveillance, were of great help in achieving freedom from the infection. the financial support and/or technical guidance from fao, oie, eu and iaea were the driving forces in achieving infection-free status for india in the year . with the successful eradication of rinderpest, the livestock sector across the globe became safer, and consequently the living standard of livestock farmers improved. the success of rinderpest control and eradication proved a rewarding experience and landmark for the veterinary services in india, providing capacity building and confidence among field veterinarians, researchers, policy planners and donor agencies and other stakeholders to undertake a successful control programme of livestock diseases at the national level. the freedom of the country from rinderpest not only enabled the growth of the dairy industry in india but has also boosted the export of meat and other dairy products in the recent decade. today india tops not only in milk production in the world but is also the largest exporter of buffalo meat. cost-benefit analyses indicated that every dollar spent on rinderpest control programme gained about $ to the indian dairy industry through more milk, meat and draft power for better agricultural productivity (uppal ). in the fate of rampant threat due to eids and tads, a diverse, dynamic and wellplanned structured disease surveillance and monitoring approach would be the key for the sustainability and welfare of healthy livestock production systems of any country. preparedness for combating the prevailing, emerging, re-emerging eids and tads requires robust monitoring and precision detection systems that are flexible, feasible and adaptable under field conditions. in this regard, pen-side diagnostic tests/lab-on-the chip tools are the need of the hour. the hurdles of sampling need to be curtailed opting non-invasive methods for sample collection from different animal species and wildlife. transparency in disease reporting needs to be adhered to and reported to oie. because of trading in animals and animal products, the international obligation for oie reportable diseases of high importance must be followed by all member countries of wto. it is high time to apply developed diagnostics and molecular detection tools in the field to ensure fast detection and confirmation of pathogens capable of causing diseases in humans and animals. this must be accompanied by national-level disease surveillance, monitoring and networking to enable an early warning system for infectious diseases based on forecasting (saminathan et al. ) . due priority is also required for development and application of new potent, safe and affordable vaccines and vaccine delivery systems and adopting innovative vaccination programmes and immunomodulatory and effective therapeutic modalities, which would help in devising timely prevention and control strategies against viral and other infectious diseases. besides these, good manage-ment and standard biosecurity and biosafety measures/practices and appropriate hygienic and zoo sanitary and quarantine measures should be observed. moreover, on-the-spot control and checking of the spread of pathogens and adequate trade restrictions as envisaged under the sps agreement of wto also need to be followed. a holistic vision and approaches are required for timely implementation of these concepts and strategies along with the strengthening of various multidimensional research and development programmes supported by appropriate funding resources. these measures will greatly help to minimise disease incidences and outbreaks, and lessen economic burdens due to infectious animal diseases and boost livestock and poultry health, reproduction and production to strengthen sustainable growth of livestock and poultry industry. reduction in pandemic threats and public health concerns eventually lead to an improvement in the socioeconomic status and welfare of the society at large under 'one health' umbrella. application of artificial intelligence (ai), gps, remote sensing and traceability in disease detection and management needs priority attention in developing countries. similarly, the latest techniques of gene editing, base editing, nanotechnologies, electronic nose, etc. should be applied for efficient disease diagnosis and drug delivery. while planning the breeding policies for livestock and poultry, both higher production performance and health of the progeny should be given equal weightage. modern techniques should be used for developing disease resistance (absolute or partial) in livestock and poultry using indigenous germplasm. institute of medicine (iom) report - ) annual report, icar-directorate of foot-and-mouth disease - ) annual report, department of animal husbandry, dairying and fisheries, ministry of agriculture and farmers welfare, govt. of india gaps in african swine fever: analysis and priorities a systematic review of the comparative epidemiology of avian and human influenza a h n and h n -lessons and unanswered questions pigs lacking the scavenger receptor cysteine-rich domain of cd are resistant to porcine reproductive and respiratory syndrome virus infection emerging infectious diseases of wildlife-threats to biodiversity and human health allijan mm ( ) the cattle plague commission report to government of india global foot-and-mouth disease research update and gap analysis: -overview of global status and research needs emerging infections: microbial threats to health in the united states zika virus-an imminent risk to the world current status of veterinary vaccines advances in developing therapies to combat zika virus: current knowledge and future perspectives the genetics of life and death: virus-host interactions underpinning resistance to african swine fever, a viral hemorrhagic disease animal disease import risk analysis-a review of current methods and practice: open access article avian influenza and other transboundary animal diseases, director, liaison office for north america, food and agriculture organization of the united nations. presentation at "health in foreign policy forum impact of avian influenza outbreaks in the poultry sectors of five south east asian countries prevalence, diagnosis, management and control of important diseases of ruminants with special reference to indian scenario ebola virus-epidemiology, diagnosis and control: threat to humans, lessons learnt and preparedness plans-an update on its year's journey nipah virus: epidemiology, pathology, immunobiology and advances in diagnosis, vaccine designing and control strategies-a comprehensive review peste des petits ruminants: sheep and goat plague. today and tomorrow's printers and publishers fao sponsored final project report "national testimonies" under the global rinderpest eradication programme (grep)-(gcp/glo/ /ec) fao sponsored final project report on "laboratory contributions for rinderpest eradication in india" under the global rinderpest eradication programme (grep)-(gcp/ glo/ /ec) animal sciences. in: singh rb (ed) years of agricultural sciences in india acknowledgements all the authors of the manuscript thank and acknowledge their respective universities and institutes. there is no conflict of interest. key: cord- -bb lyvhy authors: nan title: monoclonal antiprothrombinase ( d . ) prevents mortality from murine hepatitis virus (mhv- ) infection date: - - journal: j exp med doi: nan sha: doc_id: cord_uid: bb lyvhy the induction of monocyte/macrophage procoagulant activity (pca) has been implicated in the pathogenesis of murine hepatitis virus strain (mhv- ) infection and disease. previously, we have shown that induction of pca by mhv- correlated with resistance/susceptibility to infection in different mouse strains. in this study, all balb/cj mice that were infected with ( ) plaque-forming units of mhv- developed severe liver disease and died within - h. examination of the livers of these animals showed marked hepatic necrosis, deposition of fibrin, and cellular expression of pca by direct immunofluorescence staining in areas of necrosis as well as in hepatic sinusoids. splenic mononuclear cells recovered from these mice expressed high concentrations of pca with time after infection. infusion into mice of a high-titered monoclonal antibody that neutralized pca ( d . ) attenuated the development of hepatic necrosis and enhanced survival in a dose- dependent manner. all of the animals receiving micrograms, and % and % of the animals that received and micrograms per day, respectively, survived for d and made a full recovery. administration of the antibody resulted in a dose-dependent reduction in fibrin deposition, pca expression as detected by direct immunofluorescence staining and by a functional assay. in animals treated with high concentrations of antibody, titers of antibody to pca fell from +/- micrograms/ml to +/- ng/ml during the active phase of the disease, consistent with sequestration due to binding of the immunoglobulin to cells expressing pca. surviving animals, when rechallenged with mhv- , had a % mortality, consistent with the known rates of metabolism of immunoglobulin. this further suggested that protection was by a passive mechanism. the results reported here demonstrate that a neutralizing antibody to pca protects animals from fulminant hepatitis and death associated with mhv- infection, and supports the notion that pca is a potent inflammatory mediator that plays a pivotal role in the pathogenesis of liver injury resulting from mhv- infection. . furthermore, using recombinant inbred strains of mice, we showed that genetic linkage between resistance/susceptibility to mhv- infection and induction of pca was controlled by two non-h - inked recessive genes ( ) . after infection with mhv- , disturbances of the hepatic microcirculation associated with sinusoidal thrombosis occurred coincident with the rise in pca ( , ) . intravenous infusion of macrophages, induced to express high amounts of pca by mhv- , resulted in rapid ( - min) death from disseminated intravascular coagulation in both susceptible and resistant mice (our unpublished observations). together, these observations suggest that coagulative necrosis occurring as a result of induction of pca may be a crucial feature of mhv- -induced hepatic injury. we have recently produced a panel of mabs to mhvinduced macrophage pca ( ) . the antibodies did not react with purified viral proteins nor did they inhibit viral replication. one of these mabs ( d . ) , an igg ak, strongly inhibited pca expression in a one-stage dotting assay and inhibited conversion of prothrombin to thrombin ( ) . this antibody had no reactivity with murine, rabbit, or human tissue factor. the monoclonal bound to a -kd protein that is distinct from murine and human tissue factor ( kd) ( , ) . the purpose of this study was to determine whether treatment with this specific murine antiprothrombinase would modify the morbidity and/or mortality associated with murine hepatitis virus infection. virus. the origin and growth of mhv- have been previously described ( ) . mhv- , obtained from the american type culture collection (kockville, md) (atcc-vr ), was plaque purified on monolayers of dbt cells. stock virus was grown to a titer of . x pfu/ml in cl cells. the virus was harvested by one cycle of freeze-thawing and clarified by centrifugation at , g for h at ~ virus was then assayed on monolayers of l cells in a standard plaque assay as previously described ( ) . to induce mhv- infection, each mouse received pfu of mhv- by intraperitoneal injection. mice. balb/cj mice - wk of age, were obtained from charles river laboratories, (st. constant, quebec). animals were housed in the d class facility at the university of toronto fed a diet of standard chow and water ad libitum. mice were killed on days , , , , , and after infection. blood was obtained by axillary bleeding. splenic mononuclear cells were harvested and assayed for pca both by immunofluorescence staining and in a onestage clotting assay. livers were harvested for viral titers, histopathology, and pca by immunofluorescence staining as described below. all mice used were screened by an elisa for exposure to mhv, and were found to be negative ( ) . anti-pca mat~ the preparation of mab d . has been previously described ( ) . after injection of hybridoma cells into pristane-primed caf mice, ascites was harvested. animals were treated with ascitic fluid containing , , or #g of mab daily for d preinfection and d postinfection (p.i.) by intraperitoneal injection. as a standard control, ~ of ascites from animals that were infected with sp myeloma cells was injected into six separate mice. all ascitic fluid whether from sp -or d . injected mice had no reactivity with mhv- either in the elisa or plaque reduction assays ( ) . to ensure that the protective effect of ascites containing mab to pca was specific, mab to pca ( d . ) was isolated from hybridoma supematants by affinity chromatography using goat anti-mouse igg immobilized on sepharose b (pharmacia, montreal, quebec). immunoblot analysis of the column eluate confirmed that igg was the only ig present. subclass analysis by elisa, as previously described ( ) , confirmed that igg ak was the only subclass present. #g of the purified mab was injected into mice for d preinfection and for d p.i. as described above. histology. histology was assessed by a blinded observer as previously described ( ) . briefly, li~r were cut into . x . -cm blocks and fixed by immersion into % formalin in . molar phosphate buffer, ph . . after fixation, the tissue was dehydrated in graded alcohols and xylene, then embedded in paraffin. -#m sections were cut, stained with harris' hematoxylin for min, and counterstained with eosin y for s. the sections were then washed with distilled water, dehydrated in graded alcohols and xylene, and mounted with parmount. for each group five animals were used. to quantitate the effect of the mab on liver histology, a digitalized image analysis system (hp- ; hewlett packard co., ltd., mississauga, ontario) with customized software was used. this constitutes a modification of a technique described previously ( ) . the areas of necrosis were encircled as well as the entire section yielding a percentage figure representing the proportion of diseased liver present in that particular section. for each animal, three random sections were assayed in this fashion, and the mean +_ sd was calcuhted. hbrin deposition was assessed by the morris-lendrum hcro-mallory stain as previously described ( ) . viral titers. livers that had been snap frozen at - ~ were homogenized in dmem supplemented with % fcs and mm glutamine as a % homogenate at ~ as previously described ( ) . viral titers of liver homogenates were then determined on monolayers of l cells in a standard plaque assay ( , ) . spleens were harvested aseptically and cells teased from splenic tissue and suspended in ml of dmem as previously described ( ) . smnc were isolated over ficoll-hypaque gradients (density, . ) (pharmcia) by centrifugation at , g for rain at ~ cells at the interface were collected. viability was > % as assessed by trypan blue exclusion. cells were washed three times and resuspended in dmem at a concentration of x smnc/ml. procoagutant activity. samples of frozen thawed smnc were assayed for the capacity to shorten the spontaneous clotting time of human citrated platelet-poor plasma in a one-stage clotting assay as previously described ( ) . equal volumes ( # ) of the cellular homogenate were admixed with citrated normal human plateletpoor plasma, and then # of mm caclz was added at ~ to start the reaction. the time in seconds for the appearance of a fibrin gel was then recorded. to establish arbitrary units, a rabbit brain thromboplastin standard at mg dry mass/ml (dade division, american hospital supply, miami, fl) was assigned a value of , mu. the assay was used over the range of - , mu, and the results were linear with normal human plasma substrate. data were converted to pca per splenic macrophages and expressed as the mean and standard deviation from six mice done in triplicate. media and buffers were all without activity in this assay. inhibition of viral replication. mab purified from hybridoma cell cultures containing d . and ascites were assayed for their ability to inhibit replication of mhv- in a standard plaque reduction assay as previously described ( ) . briefly, pfu of virus was admixed with dilutions of purified antibody or ascites, media as a negative control, or a high titered anti-mhv- -neutralizing antibody as a positive control, for min at ~ the mixture was then added to a monolayer of l cells in culture medium, overlayed with % agarose, and incubated at ~ in a % co environment for an additional h. the effect of antibody on viral replication was assessed by reduction of viral plaques ( ) . immunofluorescence. blocks of liver tissue were snap frozen in liquid nitrogen. cryostat sections ('~ #m thick) were fixed for rain in acetone and air dried for h as previously described ( ) . unoccupied sites were then blocked with % horse serum in pbs, ph . , for h. mab d . was conjugated with fitc (sigma chemical co., st. louis, mo) according to the method of thi and feltkamp ( ) . the fluoresceinated antibody did not react with normal liver or uninduced normal peritoneal macrophages. tissues were then stained with fitc-conjugated mab d . for h at room temperature, washed three times, mounted in % glycerol in pbs, and viewed on a phase-epifluorecence microscope equipped with a x fluotar objective (e. leitz, inc., rockleigh, nj). elisa for anti-pca antibody. titres of antibody to pca were determined in a standard elisa as previously described ( ) . well enzyme immunoabsorbant assay (eia) plates (dynatech, mclean, va) were coated with /~l/well of mhv- -stimulated pca-positive macrophage membranes or unstimuhted pcanegative membranes ( x ~ macrophage/ml) at ~ overnight. the plates were then washed three times with pbs, ph . , conraining . % tween (washing buffer), and the unoccupied sites were blocked with /zl of % ig-free horse serum (flow laboratories, mississauga, ontario) that had been dissolved in washing buffer for h at room temperature. -/~l/well dilutions of sera from treated animals were then added to the plates and incubated for h at ~ after washing three times, #l/well of alkaline phosphatase-conjugated goat anti-mouse ig in pbs containing . % bsa and . % tween was added for i h at ~ after three washings, p-nitrophenyl phosphate in . m -amino- -methyl- , -proponediol buffer, ph . (zymed laboratories, san francisco, ca), substrate was then added. the plates were then incubated at ~ for h and read at nm with a plate reader (titertek mcc/ ; icn/flow, mississauga, ontario). antibody levels were expressed (/~g/ml) by comparison to a standard curve. statisticalanalysis. statistical analysis was carried out using analysis of variance and the wilcoxon ranked sum test. a p value of . % or less was considered statistically significant. suwival. mice infected with , pfu ofmhv- (n = ) all succumbed to the infection within d (fig. ) . this was consistent with previous data reported by our group as well as others ( , ) . in contrast, there was survival in some animals that were treated with ascites containing antibody to pca (fig. ). animals treated with ascites containing /~g/d had a % survival; % of those treated with ascites containing #g/d and % of animals treated with /~g/d survived. all animals survived when treated with /~g of monodonal anti-pca (igg ak), which had been purified from hybridoma superuatant, confirming that the beneficial effect of ascites was due to the monoclonal anti-pca. mice immunized with /~ of ascites from sp injected animals and infected with mhv- all died within d (data not shown). liver histology. mhv- -infected mice that did not receive antibody developed histologic evidence of severe liver disease. by h p.i., small, discrete loci of necrosis with a sparse pmn infiltrate could be seen. at h, these lesions became both more pronounced and more numerous (fig. a) , and by - h, confluent liver necrosis was apparent (fig. b) . in contrast, mice infected with mhv- but treated with antibody to pca showed a marked reduction in liver disease in all groups ( , , and /.r ( were a few small loci of inflammatory cells with no necrosis (fig. d) . morphometric image analysis showed that the proportion of the liver that was necrotic was significantly different between the mhv- -infected, anti-pca-treated, and untreated groups at , and h (fig. ) . the histolog of livers from all survivors at and d postinfection appeared normal. fibrin deposits were seen in hepatic sinusoids as well as in areas of necrosis of untreated and mhv- -infected mice (fig. ) . in animals treated with and /~g of anti-pca, a marked reduction in fibrin was noted. no fibrin was seen in the livers of infected mice treated with /~g of mab. treatment with anti-pca alone resulted in no detectable histological evidence of liver disease in nonirlf~ed animals. viral titers. by h p.i., large amounts of infectious virus were recovered from liver homogenates of mhv- -infected and untreated animals, and these persisted until the death of the animals (fig. ) . in animals treated with and /~g of antibody to pca, there was no significant difference in viral titers from those observed in untreated mice at days and . viral titers remained high at day , however, viral titers decreased by day and no virus could be detected after day . in animals treated with the highest dose of antibody to pca ( #g/d), viral titers were markedly reduced (p < . ) and approached those seen in resistant a/j mice ( , ) . using the method of reed and meunch, the mhv- recovered from infected and antibody-treated animals was as pathogenic as stock mhv- or virus recovered from infected and untreated mice (data not shown) ( ) . pca. in untreated, but mhv- -infected animals, a sharp increase in splenic macrophage pca was noted at h p.i. (fig. ) . maximal pca was seen at h and pca remained elevated until the animals' death on day . animals treated with low concentrations of antibody to pca ( and /zg/d) expressed high amounts of pca at early time points, but by day , pca levels fell and only basal levels of pca were detected in smnc by day . in contrast, no expression of pca could be detected in animals treated with /zg of antibody to pca during the course of infection (fig. ). immunofluorescence. by direct immunofluorescence staining, pca could be detected at h in livers from mhv- - infected and untreated mice. pca was seen in areas of inflammation and necrosis, and also in hepatic sinusoids, localized primarily in endothelial cells and kupffer cells, but not expressed by hepatocytes (fig. ) . livers from animals treated with antibody to pca had significantly less pca expression, although small amounts of pca could be seen in macrophages and endothelial cells in hepatic sinusoids in animals treated with high-dose antibody to pca ( ~tg/d), even as late as day (fig. ) . antibody to pca. sera were collected from animals at all time points and analyzed in an elisa for the presence of antibody to pca as previously described ( ) . no antibody to pca could be detected in sera from normal control animals or in animals infected with mhv- who had not received antibody to pca (data not shown). sera from animals treated with ascites containing - /zg of antibody to pca contained large amounts of anti-pca before mhv- infection. after mhv- infection, the concentration of circulating antibody fell, but remained at > ng/ml in animals treated with /~g/d. in animals that were treated with and /zg of antibody, by day , antibody was undetectable (< ng/ml) (fig. ) . antibody to pca either in ascites or purified from supernatants did not neutralize mhv- in a standard plaque reduction assay as previously described ( ) to determine whether treated animals that had survived the acute infection developed long-term resistance to mhv- infection, mice previously infected with mhv- and treated with /~g of antibody (n = ) were infected with , pfu of mhv- , d after their last exposure to virus. pca antibody titers in this group were ~ % of that present during antibody therapy, consistent with known rates of disappearance of igg. the mhv-rechallenged mice experienced a % mortality rate, suggesting that protection was by passive immunization of antibody, not an acquired, active immune process (fig. ). in addition, no antibody to mhv- was detected in these mice either before or after rechallenge with virus. mhv- infection produces fulminant hepatic failure and death in balb/cj mice ( , ) . the availability of a mab to pca that neutralized acceleration of coagulation in vitro ( ) provided us an opportunity directly to examine the role of pca in vivo in murine hepatitis virus strain infection. all balb/cj mice which were infected with pfu of mhv- developed severe liver disease and died within - h. administration of the mab to pca attenuated the hepatic necrosis, fibrin deposition and enhanced survival in a dosedependent manner. all of the animals receiving /~g, and % and % of the animals that received and /~g/d, respectively, survived for d and made a full recovery. furthermore, the same protective effect was seen in mice treated with igg ak purified from hybridoma culture supernatants ( d . ). the increased survival of the treated animals was specific for anti-pca, since ascites from mice injected with sp cells alone, which contained no antibody to pca, failed to protect mice from mhv- infection. although antibodytreated, mhv- -infected animals demonstrated clinical evidence of viral hepatitis early in the course of the infection, by - d their behavior appeared normal and liver sections showed little or no apparent disease. examination of the livers of antibody-treated animals showed marked reduction in hepatic necrosis and inflammatory cells (neutrophils and mononuclear cells), each of which are prominent features of mhv- infection ( , , ) . pca expression is a feature of mhv- infection in this mouse strain ( ) ( ) ( ) , and in untreated mhv- -infected mice, pca was detected by immunofluorescence staining in areas of necrosis as well as in hepatic sinusoids. both macrophages and endothelial cells expressed detectable pca, but hepatocytes did not. it is likely that the hepatic necrosis is secondary to ischemic changes resulting from induction of pca, leading to the deposition of fibrin. administration of mab resulted in a dose-dependent reduction in the deposition of fibrin and in expression of pca as detected by immunofluorescence staining in the liver and by a direct functional assay in smnc. previously, we have shown that mhv- infection of peritoneal macrophages in vitro results in the production of pca, tnf, leukotriene b ( ) , and il- ( ) . thus, expression of pca by endothelial ceils might either be due to direct induction by mhv- or due to induction by il. and/or tnf, which have previously been shown to induce pca in endothelial cells in vitro ( , ) . treatment of mice with /~g of mab to pca not only increased survival and reduced hepatic necrosis, but also resulted one possible explanation for the decrease in viral replication observed in mice treated with antibody d . is that this antibody reacts with the mhv receptor recently described by holmes and coworkers ( , ) . we regard this explanation as unlikely, since d . does not show any neutraliza- tion of viral infectivity in a plaque reduction assay, whereas antibody to the mhv receptor does inhibit infectivity. furthermore, the mhv receptor has a molecular mass of kd, considerably different than that of the pca molecule ( kd). a second possible explanation for the effect of anti-pca antibody on mhv- growth in mice is related to the normal cleavage of the mhv s protein. it has been shown that cleavage of s by proteases is necessary to activate the membrane-fusing properties of the s protein ( ) . this fusion property facilitates the spread of virus to uninfected cells by cell-cell fusion and also increases the specific infectivity of mhv when compared with virus in which s has not been cleaved ( ) . it is possible that in infected macrophages, pca, a serine protease, mediates at least in part the proteolytic cleavage of s into $ and $ , and thereby activates the fusion properties of this molecule. consistent with this idea is the observation that infection of a/j macrophages, which do not produce pca in response to mhv infection, does not result in the appearance of syncytial giant cells (our unpublished observations). thus, antibody to pca could inhibit the spread of virus to uninfected cells by decreasing the activation of the fusion properties of s. although the mechanism by which anti-pca treatment protects the susceptible animals is not clear, the use of antibody to pca neutralizes pca, thereby preventing activation of the coagulation system and inhibiting fibrin formation. the antibody could also result in complement-mediated destruction of mhv- -infected cells that express membranebound pca or promote macrophage activation with restriction of viral growth. in animals treated with #g of antibody for d before infection and d p.i., concentrations of antibody to pca in sera fell from /~g/ml before mhv- infection to ng/ml during the active phase of disease (day ), consistent with sequestration perhaps due to binding of the ig to cells expressing pca. furthermore, in these mice, levels of pca in smnc remained at basal levels. in animals treated with lower amounts of antibody ( or #g/d) to pca, hepatic necrosis and survival were only partially attenuated, and antibody was not detected (< ng/ml) after day p.i. together, the data strongly support the notion that anti-pca antibody neutralizes pca in vivo during the infection and that this may be the basis for its protective effect. in a previous report we have demonstrated that dimethyl prostaglandin e inhibited procoagulant activity and prevented fulminant viral hepatitis, yet all animals still succumbed to the infection ( ) . in pge-treated mice, viral replication proceeded at a rate similar to that in untreated animals. recently, we have demonstrated that although pge inhibited functional pca, antigenic expression of pca was not altered as determined by western immunoblotting ( ) . we have proposed that pca may exert its effect through activation of the coagulation system with microvascular and macrovascular thrombosis ( , ), but the present results, in concert with the inhibition studies of pca by pge , suggest that pca has other sites of action as well. the protective effects of the mab to pca occurred even though the balb/cj mice failed to mount an antiviral humoral response. our ob-servations are consistent with a recent report by korner et al. ( ) and support the notion that the murine antiviral antibody response may not be required for protection from acute viral infection. cytokines can play a potent role in the course of inflammatory injuries in vivo, and interference with their action can alter the course of certain inflammatory diseases ( , ) . treatment of rats with recombinant antibody to tnf has been shown to protect animals from the hypotension, hypothermia, and mortality of gram-negative sepsis ( ) , and treatment of rabbits with an il- r antagonist reduced the mortality associated with endotoxin shock ( , ) . our studies support the notion that induction of pca during mhv- infection in mice is an integral and potentially central step in the disease. a previous report by taylor et al. ( ) demonstrated that lethal escherichia coli septic shock can be prevented by blocking tissue factor (a distinct procoagulant) with mab, demonstrating the importance of cellular coagulants in the pathophysiology of other infectious diseases. we conclude that pca is a potent inflammatory mediator that plays a pivotal role in hepatic injury resulting from mhv- infection. the biology and pathogenesis of coronaviruses induction of monocyte procoagulant activity by murine hepatitis virus type parallels disease susceptibility in mice the pathobiology of viral hepatitis and immunologic activation of the coagulation protease network activation of the immune coagulation system by murine hepatitis virus strain susceptibility/resistance to murine hepatitis virus (mhv- ) and monocyte procoagulant activity (mpca) are genetically linked and controlled by non-h- linked genes acute and chronic changes in the microcirculation of the liver in inbred strains of mice following infection with mouse hepatitis virus type the effects of mouse hepatitis virus type on the microcirulation of the liver in inbred strains of mice monoclonal antibody analysis of a unique macrophage procoagulant activity induced by mufine hepatitis virus strain infection complete sequence of the human tissue factor gene, a highly regulated cellular receptor that initiates the coagulation protease cascade molecular cloning of the cdna for tissue factor, the cellular receptor for the initiation of the coagulation protease cascade a sensitive radioimmunoassay for the detection of antibodies to mhv- induction of resistant hepatocytes as a new principle for a possible short-term in vivo test for carcinogens morris-lendrum picro-mallory stain for fibrin dimethyl prostaglandin e prevents the development of fulminant hepatitis and blocks the induction of monocyte/macrophage procoagulant activity after murine hepatitis virus strain infection lymphocyte cooperation is required for amplification of macrophage procoagulant activity, f ex f med conjugation of fluorescein isothiopropriate to antibodies. i. experiments in the conditions of conjugation a sensitive radioimmunoassay for the detection of antibody to mvh- mechanism of protective effect of prostaglandin e in murine hepatitis virus strain infection: effects on macrophage production of tumor necrosis factor, procoagulant activity and leukotriene b susceptibility to mouse hepatitis virus strain in balb/cj mice: failure of immune cell proliferation and interleukin production interleukin- activation of vascular endothelium. effects on procoagulant activity and leukocyte adhesion the role of endothelial cells in inflammation monoclonal antibody to the receptor for murine coronavirus mhv-a inhibits viral replication in vivo purification of the -kilodalton glycoprotein receptor for mouse hepatitis virus (mhv)-a from mouse liver and identification of a nonfunctional, homologous protein in mhv- resistant sjl/j mice. f virol proteolytic cleavage of the e glycoprotein of murine coronavirus: host dependent differences in proteolytic cleavage and ceu fusion effect of eicosanoids on induction of procoagulant activity by murine virus strain in vitro nucleocapsid or spike protein-specific cd + t lymphocytes protect against coronavirus-induced encephalomyelitis in the absence of cd + t cells, f immunol shcokd and tissue injury induced by recombinant human cachectin cachectin/tumor necrosis factor induces lethal shock and stress hormone responses in the dog development of partial tolerance to the gastrointestinal effects of high doses of recombinant tumor necrosis factor-alpha in rodents treatment with recombinant human tumor necrosis factor-alpha protects rats against the lethality, hypotension and hypothermia of gram-negative sepsis interleukin- receptor antagonist reduces mortality from endotoxin shock lethal e. coli septic shock is prevented by blocking tissue factor with monodonal antibody key: cord- -r usk authors: nan title: research communications of the th ecvim‐ca congress date: - - journal: j vet intern med doi: . /jvim. sha: doc_id: cord_uid: r usk nan saccharomyces boulardii (sb)is a non-pathogenic yeast used in the prevention and treatment of gastrointestinal disorders in human beings and horses. the aim of this study was to evaluate the effect of sb in healthy dogs and dogs with chronic enteropathies (ce). sb was formulated in x cfu capsules. its concentration and viability within the capsules was controlled by yeast culture in subsequent steps until expiration date. four healthy dogs (hd) and dogs with ce ( inflammatory bowel disease -ibd, protein losing enteropathy -ple) were included. in hd sb was administered for days ( x cfu/kg bid); daily clinical evaluation was performed to assess possible adverse effects and quantitative stool cultures for yeasts were performed before, during and after the administration. in dogs with ce a randomized double blind placebo-control study was performed, administering sb ( x cfu/kg bid) or placebo (pl). sb or pl administration was added to standard therapeutic protocols (diet, antibiotics and immunosuppressive drugs), to evaluate its efficacy for the treatment of ibd and ple. complete blood work, abdominal ultrasonography, gastro-duodenal and colon endoscopy and histopathological evaluation of intestinal samples were performed at diagnosis and after days of treatment. validated score system for the clinical signs (ceccai), ultrasonography, endoscopy and histopathology were applied. significance was set for p < . . results in hd showed the absence of sb in the faeces before treatment, its presence after one day, its steady state ( x cfu/g) after days and its complete elimination days after withdrawal of treatment. no adverse effects were reported. in ce dogs the clinical score improved significantly in dogs receiving sb compared to dogs receiving pl (p = . ). in ple dogs the albumin concentration increased significantly (p = . ) in the group receiving sb with respect to pl. the daily frequency of defecation in the sb group was significantly lower with respect to pl after (p = . ) and (p = . ) days of treatment. no statistical differences were found between dogs receiving sb and pl after treatment, based on the endoscopic evaluation of duodenum and colon. no statistical differences were found between the two groups on the duodenal ultrasonographic and histological evaluation after treatment. in conclusion, sb can be safely used in dogs with ce, in addition to standard treatment, to achieve a better control of clinical signs and a significant increase in albumin concentration compared to the standard therapy alone. no conflicts of interest reported. canine inflammatory bowel disease (ibd) is an immune-mediated enteropathy likely triggered by environmental and immunoregulatory factors in genetically susceptible dogs. previous studies suggest a pivotal role for gut bacteria in disease pathogenesis since luminal microbial composition is markedly altered (ie, dysbiosis) at diagnosis. probiotic bacteria appear to be therapeutically effective in some forms of human ibd. controlled studies evaluating the efficacy of probiotic therapy for canine ibd have not been previously reported. the aim of the present study was to characterize the mucosa-associated microbiota and determine the clinical, microbiological, and mucosal homeostatic effects of orally administered vsl# probiotics in dogs with ibd. twenty dogs diagnosed with moderate-to-severe ibd (cibdai score > ) were randomized to receive standard therapy (ie, elimination diet and glucocorticoids) with or without probiotic vsl# . the mucosal microbiota from endoscopic intestinal biopsies of ibd dogs and controls was evaluated by fluorescence in situ hybridization (fish) targeting the s rrna genes of total bacteria, group-specific organisms, and individual bacterial species shown to be relevant in human ibd. epithelial tight junction protein (tjp) expression was studied using immunohistochemistry. clinical signs and changes in mucosal microbiota and tjp expression were assessed before and after probiotic vsl# therapy. ibd dogs showed a reduction in gi signs following weeks of probiotic therapy compared with baseline cibdai scores (p < . ). adherent and sporadic invasive bacteria (eub) were observed in the small intestines and colon of healthy dogs. the diseased canine duodenum was nearly bacteria-free. ibd dogs given probiotic vsl # had altered spatial redistribution of most bacterial groups in the mucus and adherent compartments of the colon. subset analysis showed that lactobacilli were significantly (p < . ) increased in the lumen and mucus post-vsl # , while the number of mucus laden bifidobacteria approached significance (p = . ). expression of tjp showed that occludin was significantly lower in control intestines as compared to duodenal and colonic mucosa obtained from ibd dogs that received probiotic (p = . and p = . , respectively). in contrast, claudin- expression in the colon was significantly higher (p < . ) in control dogs versus vsl # treated ibd dogs. our data demonstrate that probiotic vsl# alters some of the mucosa-associated microbiota in dogs with ibd. these probiotic changes in bacterial composition are associated with up-regulated tjp expression indicative of enhanced epithelial barrier integrity, similar to vsl# -induced disease protection seen in human ibd. the probiotics used in the trial were supplied free of charge by the manufacturer. canine inflammatory bowel disease (ibd) is thought to be partially caused by an aberrant immune response towards the intestinal microbiome. in humans and mice, administration of probiotics can alleviate ibd severity and/or prevent relapse by induction of a more "tolerant"microenvironment. the aim of this study was to investigate the effect of probiotic enterococcus faecium ncimb e (ef) on intestinal microbiome composition. dogs were recruited to receive ef at x e cfu in a double-blinded, placebo-controlled manner in addition to an exclusion diet (hydrolysed protein). seven dogs were included in the probiotics group and dogs in the placebo group. all dogs improved clinically after treatment, however, there was no obvious effect on clinical severity in those that received probiotics. fresh naturally voided faecal samples were collected from all dogs before and after treatment, snap-frozen in liquid nitrogen and stored at - °c until further analysis. genomic dna was extracted from each faecal sample using the mobio power soil dna isolation kit (mobio laboratories), as recommended by the manufacturer. next generation sequencing was performed on the ion-torrent[trademark] (life technologies) platform based upon the v -v region (e. coli position - ) of the s rrna gene with the following primers: forward f: gag-tttgatcntggctcag and reverse r: gtnttacn gcggckgctg. raw sequence data were screened, trimmed, filtered, and chimera depleted with default settings using the qiime pipeline version . and uchime software, in which microbiome composition between treatment groups before and after treatment was compared. microbiota composition was not significantly different between probiotic and placebo treatment groups, and did not change significantly before and after treatment. however, there was large individual variability in the microbiome composition. species richness of faecal samples increased after treatment in both groups, but was only statistically significant in the probiotic treatment group. in conclusion, probiotic treatment in dogs with ibd leads to a significantly increased richness of the faecal bacterial microbiome. a possible additional effect of the change of diet cannot be excluded. further studies should investigate microbiomic changes in healthy dogs fed the same diet to assess if similar changes in the fecal microbiome occur due to dietary changes alone. this study is based on a phd supported by probiotics ltd., somerset, uk (the manufacturer of the probiotic product enterococcus faecium mentioned in this study). the aim of this study was to assess the prevalence and risk factors for faecal carriage of extended-spectrum beta-lactamases (esbl) and plasmidic ampc beta-lactamases (pampc) e. coliproducers in healthy dogs. a -month cross-sectional study was conducted at a private hospital in lisbon, portugal and rectal swabs were obtained from healthy dogs. the dogs included in the study were healthy with no history of antimicrobial consumption in the previous month. esbl and pampc genes were detected by pcr and were sequenced. potential risk factors for esbl-and pampc-producing e. coli faecal carriage were obtained through a questionnaire to the owner regarding reason for veterinary visit, hospitalisation and antimicrobial treatment within the last year, habitat (shelter, dog breeders and private owner), cohabitation with other animals, street access, kennel/hotel access, age and gender. data were analysed by sas software (version . ; sas institute inc., cary, n.c.) and logistic regression models were used. rectal swabs obtained from healthy dogs yielded positive samples for e. coli. about % of the isolates carried esbl genes (bla ctx-m- n = , bla ctx-m- n = , bla ctx-m- n = , bla ctx-m- -like n = ) and % carried pampc genes (bla cmy- n = , bla cmy- -like n = , bla dha- n = ). thirteen dogs carried an e. coli isolate with both an esbl and a pampc gene. dogs previously treated with antimicrobials within the last year were at higher risk of carrying at least one ß-lactamase (p = . ; or = . ; ci %: . - . ) or both ß-lactamases (p = . ; or = . ; ci %: . - . ) than non-treated dogs. dogs in shelters/breeders tended to show a higher incidence of esbl-producing e. coli (p = . ; or = . ; ci %: . - . ) or at least one ß-lactamase producing e. coli (p = . ; or = . ; ci %: . - . ) than dogs from private owners. males tended to be less likely to carry at least one ß-lactamase (esbl or pampc) (p = . ; or = . ; ci %: . - . ) or a pampc enzyme (p = . ; or = . ; ci %: . - . ) than females. this study suggests that dogs may act as reservoirs for resistant bacteria, namely for cephalosporin-resistant e. coli. three potential risk factors associated with the carriage of esbl-and/or pampc-producing e. coli by dogs were identified, which is important for the implementation of effective control measures and judicious antimicrobial therapy. conflicts of interest: dr pomba currently receives research funding from the government and national programmes (fundac ßão para a ciência e a tecnologia). in the past, she has occasionally received research support or honoraria for lectures from pharmaceutical companies including zoetis and atral cipan. she is vice-chair of the antimicrobial working party there are few reports in the literature reporting long-term relapse rate, owner compliance and clinical severity of dogs with chronic enteropathies. the goal of this study was to compare clinical activity index (ccecai), number of relapses and compliance rates - years after diagnosis. food-responsive disease (frd) was defined as dogs that responded to elimination diet alone within weeks after initiating therapy, whereas antibiotic-responsive disease (ard) dogs had an unsuccessful dietary trial before and responded to metronidazole within weeks after initiation of therapy, and steroidresponsive disease (srd)dogs had an unsuccessful dietary and antimicrobial trial before, and required immunosuppressive therapy to control their clinical signs. ccecai was extracted from the medical record database at - years after diagnosis. relapse rate was obtained by requesting the medical records of the referring veterinarians and defined as number of return visits to the referring practice after diagnosis. compliance data was obtained by telephone questionnaire to the owners. the frd group consisted of / dogs ( %), whereas the ard and srd groups consisted of ( %) and dogs ( %), respectively. there was a significant difference in ccecai at follow-up between frd and ard, and frd and srd (median ccecai . (range - ) for frd, (range - ) for ard, and . (range - ) for srd, p = . ). for the frd dogs, % of owners stated that they deviated from the prescription diet on a daily basis, % once a week, and % once a month, with a median ccecai at the time of deviation from the diet of . (range - ). relapse rate was highest for the ard group, when compared to frd and srd ( for ard, . for frd, and . for srd, p = . ). in the frd group, / dogs had been kept on the prescribed diets, and dogs had been changed to supermarket brands. all of the ard dogs had been given immunosuppressive treatment in addition to antibiotics at the time of follow-up, while / srd dogs were still on immunosuppressive treatments, with one dog being in remission with dietary treatment alone. in conclusion, this pilot study indicates that compliance rate for frd dogs is the lowest, with owners willing to tolerate the highest severity of clinical signs related to deviation from the prescription diet. ard dogs had the highest relapse rate in this cohort, indicating poor response to treatment in the long-term. conflicts of interest: dr allenspach has received research funding from bbsrc, american kennel club, comparative gastroenterology society, probiotics ltd uk, laboklin gmbh germany, and bioiberica sp. she has also undertaken paid consultancy work for bioiberica spain and hoffmann-laroche, switzerland. despite the high prevalence of canine pancreatitis in postmortem studies and the introduction of new diagnostic tests, it is believed that the disease, particularly in its chronic form, remains under recognised due to the non-specific nature of presenting signs. histology is considered to be the gold standard for diagnosis of canine pancreatitis, however, most clinicians are reluctant to take pancreatic biopsies due to significant risks to the patient. numerous serum markers have been reported to be elevated in canine pancreatitis, although most lack sensitivity or specificity. consequently, confirmed diagnosis requires results from a range of tests including imaging, serum biochemistry and physical examination. we and others have previously shown in other diseases that performance of individual low specificity markers can be dramatically improved by combining data from multiple markers with clinical information using analytical algorithms. we therefore applied this approach to the detection of pancreatitis in dogs. the activity of two non-specific biomarkers, amylase and lipase, was determined in serum samples from dogs suspected of having pancreatitis by their veterinarian. of these samples were positive by virtue of their pancreatic lipase (cpli) results (cpli > ug/l). the amylase and lipase data was then used to develop a series of algorithms using mathematical data mining and classification techniques. additional algorithms were developed using extra parameters including age, sex, vomiting, diarrhoea and abdominal pain in addition to the two enzyme levels. the performance of the algorithms was assessed using separate blinded serum samples taken from dogs which were scored clinically for acute pancreatitis according to the system described by mccord et al (j vet intern med ; : - ) . these cases presented for evaluation with vomiting, diarrhoea, inappetance and abdominal pain and were included if a clinical history, results of routine haematology, biochemistry, cpli assay and abdominal ultrasound were available. the results of the multifactorial analysis and cpli assay results were compared to the clinical scores. using amylase and lipase data alone, the algorithm gave a sensitivity of . % and specificity of . %, compared to cpli results for the same samples of . % and . % respectively when both methods were referred to clinical scoring. when the presence of additional clinic data was also included into the algorithm, the sensitivity increased to . % with specificity of %. the data suggests that test performance for canine pancreatitis can be dramatically improved when multiple diagnostic parameters are combined using disease specific algorithms. the author receives a salary as editor of the bsava journal companion, and has undertaken unrelated paid consultancies for bayer and merial. the author also receives a salary from avacta animal health, and duties involved working directly on this project. canine chronic enteropathy (cce) can cause significant long-term morbidity. in some cases this is due to intestinal inflammation, resulting from idiopathic inflammatory bowel disease (ibd). currently, the diagnosis of idiopathic ibd and assessment of disease severity relies on results of subjective clinical indices, laboratory data, diagnostic imaging and intestinal histopathology, whilst ruling out known causes of inflammation. in humans with ibd, a number of faecal biomarkers including lactoferrin, aid with diagnosis and determining disease activity. it may therefore be valuable to develop similar non-invasive objective methods to aid diagnosis and clinical assessment of disease severity in dogs with intestinal inflammation due to idiopathic ibd. this pilot study aimed to measure faecal lactoferrin concentration (flc) in dogs with cce and histologically confirmed intestinal inflammation (hcii) and to compare this with control dogs. in addition, the flc in dogs with hcii would be compared with the canine inflammatory bowel disease activity index (cibdai) and wsava standard histopathological criteria for intestinal inflammation to determine whether there was correlation between these methods when assessing disease severity. faecal samples were obtained from dogs with hcii (n = ) having undergone investigation for cce (serum biochemistry, complete blood count, full faecal and urinalysis, serum cobalamin, quantitative cpli, abdominal ultrasound and intestinal biopsies). the control population were dogs presented for reasons unrelated to cce (n = ). analysis was carried out using a faecal lactoferrin elisa previously validated in dogs (techlab, usa). the flc in dogs with hcii (median . lg/g -range . to . ) was significantly higher than control dogs (median . lg/g -range . - . ) (p < . ). a cut-off flc of . lg/g correctly identified / ( %) of dogs with hcii. using this cut-off, there was no overlap between non-cce dogs flc and the hcii group; giving a sensitivity of % and specificity of %. neither the presence of neutrophils nor the extent of inflammation on histopathology showed significant correlation with flc. the cibdai showed moderate correlation with flc in dogs with hcii (r = . , p = . ). the results of this pilot study suggest that flc is able to discriminate between dogs with cce due to hcii and dogs without cce. it is possible that incorporating flc into a panel of faecal biomarkers will enable non-invasive assessment of hcii and could serve as an adjunct to current measures of disease severity in dogs with idiopathic ibd. ryan bettencourt and james boone are employees of techlab, usa. they provided the elisa kits free of charge for this work. there was no other incentive provided and the results have been openly discussed between all parties. there has been no censorship placed on the results by tech-lab and they have been supportive of the work and submission of this abstract. there are no other conflicts to disclose. fecal s a and fecal calprotectin concentrations have been described as biomarkers in dogs with chronic enteropathies [ ]. however, to date there has been no direct comparison of these two markers in dogs with chronic diarrhea. the aim of this study was to evaluate the performance of these two markers in this situation. thirty one dogs presented for a history of chronic diarrhea were prospectively enrolled. the initial diagnostic workup for all patients included a serum biochemistry profile, fecal parasitology, abdominal ultrasound examination, and gastrointestinal endoscopy with collection of endoscopic biopsies. the severity of clinical signs was evaluated using the ccecai scoring index and patients were grouped by having a ccecai of < or ≥ . fecal calprotectin and s a were quantified as previously described [ ] . correlations were evaluated with the spearman rank correlation test. for both markers a receiver operating characteristics (roc) curve was used to select cut-off value that allowed the best discrimination between dogs with a ccecai< and dogs with a ccecai ≥ . sensitivity and specificity were calculated. correlation analyses revealed a significant positive correlation between s a and calprotectin (r = . ; p < . ). the optimal cut-off value for fecal calprotectin concentration was . lg/g, which was associated with a sensitivity of . % and a specificity of . % (auc= . ; p = . ). the optimal cutoff value for fecal s a concentration was . ng/g, which was associated with a sensitivity of . % and a specificity of % (auc= . ; p = . ). the sensitivity for fecal s a was higher than that for fecal calprotectin (p = . ). no significant difference was observed for the specificity of these two markers (p = . ). out of the dogs ( %) had concordant results for s a and calprotectin tests. among these dogs, presented with a ccecai < and of these dogs had both markers below their cut-off values. among the dogs with a ccecai≥ , dogs had both markers above their cutoff values. % of dogs ( / ) presented histologic signs of inflammation. sensitivities for fecal calprotectin and s a concentrations for histopathological intestinal inflammation were % and %, respectively, and specificities were % and %, respectively. at least in this group of patients fecal s a concentration was more sensitive (but less specific) to detect dogs with a cce-cai ≥ or histopathologic intestinal inflammation than fecal calprotectin concentration. weight loss and malabsorption of fat, protein, cobalamin and tocopherol in the face of normal exocrine pancreatic function have been reported in up to - % of cats older than years of age fed a variety of nutritionally balanced dry and wet foods (patil ap and cupp cj. proc. nestle-purina compan anim nutr summit, focus on gastroenterology, [ ] [ ] [ ] [ ] [ ] [ ] [ ] ) . the objectives of this study were to determine if serum cobalamin concentrations increased after oral administration of a cobalamin supplement to affected cats, and the duration of any positive response following cessation of supplementation. the study evaluated cats older than years of age with fat malabsorption demonstrated by either increased fecal fat (> %) or subnormal fat digestibility (< %), but without exocrine pancreatic insufficiency (epi) as assessed by assay of serum trypsin-like immunoreactivity (ftli). a commercially available solution of cobalamin containing mg mixed with ml of a liquid flavor enhancer was added to the food of each cat in a single dose each day for months, after which supplementation ceased. serum cobalamin (assayed by competitive binding assay performed through the gi laboratory at texas a&m university and evaluated using reference ranges derived by that laboratory) was determined immediately prior to initiation of supplementation, then weekly for weeks, then monthly for months. at the start of the study serum cobalamin was subnormal (< ng/l) in of the cats (range < to ng/l) and within the reference range ( to ng/l) in the remaining cats ( to ng/l). serum cobalamin was above the reference range in every cat ( to ng/l) after one week of supplementation and remained above the reference range in every sample collected during the supplementation period, with the exception of two cats with values within the reference range when supplementation was stopped. serum cobalamin , and months after cessation of supplementation ranged from < to , < to , and < to ng/l, respectively. at the end of the study serum cobalamin was subnormal in of the cats. it is concluded oral cobalamin supplementation can effectively increase serum cobalamin concentrations in geriatric cats with idiopathic chronic enteropathy, but that following cessation of supplementation concentrations decrease rapidly and can become subnormal again within as few as weeks. the primary author collaborated with nestle-purinaon the work reported in this abstract, and a co-authoris an employee of nestle-purina. the primary author has previously received funding from iams, mars, hills and nestle-purina. the author also acts as a paid consultant for the gi-lab, texas a&m university. left atrial measurements are crucial in assessing severity of cardiac disease in dogs with myxomatous mitral valve disease (mmvd). however, linear and area dimensions might not provide a comprehensive assessment of patient status, and cannot differentiate between severe subclinical (b ) and clinical disease (chf). estimates of left atrial function could provide additional information to help categorize these patients. we examined dogs with mmvd ( normal, b , b and c) presented for cardiac evaluations by d echocardiography. left atrial linear and area dimensions in right parasternal short and long axis views were obtained at time points -early diastole (la max ), just prior to mitral valve opening, at the onset of atrial systole (la p ) and just prior to mitral valve closure (la min ). we calculated indices of la function: total la emptying fraction (la tef ), active la emptying fraction (la act ), passive la emptying fraction (la pas ) and la reservoir function (la res ) for all sets of measurements. we examined the differences in selected la function indices between different disease stages with a kruskal wallis test with post-hoc multiple comparisons. we also examined the diagnostic accuracy of selected indices of la function in differentiating dogs in stage b and stage c (chf) using roc analysis. three functional indices consistently differed across the various stages of mmvd -la tef , la act and la res . these differences were most apparent in the rpla view for linear measurements and rpsa view for area measurements. dogs with chf had worse function than all other groups, which differed variably depending on the functional index being examined. laarea act showed the best ability to discriminate between b and chf dogs, with a % specificity, % sensitivity and an auc of . , but this was no better than use of la:ao measurements. our data suggest that la function differs between dogs with differing severities of mmvd, but does not provide a clear distinction between dogs with subclinical disease and chf. no conflicts of interest reported. materials and methods: de was used to evaluate consecutive, non-sedated dogs that weighed more than kg. the study population for the morphologic study included normal dogs, and dogs with acvim stages b or b -c mmvd. de image data were digitally recorded and then analyzed offline, using commercially available software. results: de image acquisition was feasible in / ( . %) consecutive dogs. patient anxiety ( ), arrhythmias ( ) and panting ( ) explained failure to obtain a de dataset. fortyone of ( . %) datasets were of analyzable quality. body weight and heart rate were significantly lower in dogs for which it was possible to perform de. dogs with analyzable de datasets were significantly older and weighed less than dogs in which de could not be analyzed. the mitral valve of normal dogs is saddle shaped (annulus height to commissural width ratio (ahcwr): . ae . [mean ae sd]) and has an elliptical annulus (sphericity index (si): . ae . ). the following measurements were significantly related to body surface area (bsa): antero-posterior diameter (apd) (r = . , p < . ), anterolateral-posteromedial diameter (alpmd) (r = . , p < . ), annulus area (aa) (r = . , p < . ), anterior leaflet length (all) (r = . , p < . ), anterior leaflet area (ala) (r = . , p < . ). these variables were indexed (i) to bsa for subsequent statistical analyses. dogs with mmvd had a significantly greater si, non-planar angle, apdi, alpmdi, alai and alli, while having a significantly lower posterior leaflet area (pla), posterior leaflet length (pll), annulus height (ah), tenting height (th), tenting volume (tv), tenting area (ta), and ahcwr compared to normal dogs. ah, tv and ta were significantly greater in normal dogs, compared to dogs with mmvd. si, apdi, al-pmdi, aai, alai and alli were significantly greater in dogs with stages b -c mmvd, compared to normal dogs and those in stage b . pll and pla were significantly lower in b -c dogs, compared to normal dogs. th was significantly different between the three groups; greatest in normal dogs and lowest in dogs in stages b -c, suggesting that flattening of the mv occurs with disease progression. conclusions: de assessment of the canine mv is feasible. morphologic changes associated with mmvd progression are presented. effective regurgitant orifice area (eroa), calculated from a dimensional measurement of the width of vena contracta (vc) as the narrowest portion of the proximal regurgitant jet, might be used to estimate severity of mitral regurgitation (mr). however, this simplified assumption only holds when the eroa is circular, which might not be true in dogs with myxomatous mitral valve disease (mmvd). the aim of the study was to compare measured eroa using color doppler real-time dimensional echocardiography (rt d) with calculated eroa estimated by dimensional echocardiography ( d) in chamber ( ch) and chamber ( ch) views of the left ventricle (lv) in dogs with mmvd. ninety-three privately owned dogs of breeds diagnosed with naturally acquired mmvd were examined using d and rt d. according to the acvim classification of congestive heart failure (chf), dogs were classified with chf ( in class c and in class c ) and dogs without chf ( dogs in class b and dogs in class b ). age ranged from to years (median years), and body weight ranged from . to kg (median kg). fifty-nine males ( %) and females ( %) were included, and heart rate ranged from to beats/minute (median b/min). eroa was calculated from d measurements of vc diameter, in the ch view only (assuming a circular regurgitant orifice), and from measurements of vc diameter in both ch and ch views (assuming an eliptical regurgitant orifice) of lv. bland-altman plots were used to compare eroa measured by rt d with calculated eroa obtained from d ch and ch/ ch lv views. none of the d estimations of eroa showed good agreement with the measured rt d eroa when corrected for bsa, and the difference between methods increased with increasing eroa. the difference between rt d and d methods normalized to the mean eroa value did not increase with increasing eroa, but showed a systematic underestimation of eroa by % ( ch) and % ( ch/ ch), respectively, compared to rt d. the beat-to-beat variation of eroa assessed by rt d (n = ) had a coefficient of variation ranging from . % to % (median %). in conclusion, substituting assessment of eroa with a measurement of vc in or dimensions might underestimate the mr severity in dogs with mmvd. in some dogs, the beat-tobeat variation of the eroa was large, thereby necessitating the need for several consecutive measurements. no conflicts of interest reported. micrornas (mirna) are short ( - nucleotides), singlestranded, non-coding rnas that specifically anneal with complementary sequences in multiple mrna targets, and they silence mrnas and suppress downstream protein translation. a mirna can act as a fine-tuner of gene expression or an on/off switch. these features highlight the potential of mir-nas as therapeutic targets. the role of mirnas in myocardial fibrosis and hypertrophic cardiomyopathy has been widely studied in human patients. however, there is no data available for canine and human myxomatous mitral valve disease (mmvd). the aim of this study was to investigate mirna transcriptomics in canine mmvd by using global transcriptional profiling, mirna target prediction software (diana tool, targetscan . ) and network analysis software (biolayout express d ). four myxomatous mitral valves (ckcs) and controls valves were profiled using the affymetrix canine gene . st array. in total out of mirnas were found to be statistically significantly differentially expressed (down-regulated) based on the false discovery rate, p-value, and fold-change. expression of three mirna (cfa-mir- b, cfa-mir- c, cfa-mir- ) were also validated by quantitative polymerase chain reaction (q-pcr, taqman), and the results were in agreement with the microarray findings. for network analysis and visualization, markov clustering algorithms were conducted in bio-layout express d , and major clusters of mirnas were exported and uploaded to the diana-mirpath (kegg pathway) web-server. the pathways identified in the main cluster were attributed to the biological functions of focal adhesion, cytoskeleton (actin) regulation, tgf-b signalling, glycosaminoglycan biosynthesis, osteoclast differentiation, notch signalling and vegf signalling. the most significantly down-regulated mirna in mmvd was cfa-mir- , which is an endothelial specific mirna shown to regulate endothelial migration and vessel patterning. the top predicted target of cfa-mir- is glucuronic acid epimerase (glce) which is the main enzyme controlling heparan sulphate biosynthesis. other interesting findings were down-regulation of cfa-mir- and members of the cfa-mir- family. cfa-mir- targets multiple extracellular matrix transcripts, such as collagens, elastin, integrin, laminin, mmp (matrix metalloproteinase) and adamts (a disintegrin and metalloproteinase with thrombospondin motifs), whereas cfa-mir- targets hyaluronic acid synthase (has ). since the major pathology of mmvd is aberrant turnover of extracellular matrix proteins, this may be linked to mir-na regulation. dysregulation of valve mirnas might be potential therapeutic targets in the treatment of canine mmvd. no conflicts of interest reported. mitral regurgitation (mr) progresses slowly, but dogs living long enough often develop congestive heart failure (chf). however, tools to predict onset of chf are sparse. echocardiographic examinations in dogs were performed in a longitudinal, multicenter study with a surveillance time of up to . years. client-owned dogswere enrolled at the university hospitals in finland, sweden and denmark (subset to the svep study). left ventricular end diastolic (lvidd) and systolic (lvids) diameters, fractional shortening (fs), left atrial (la) and aortic root (ao) diameters were estimated. values were normalized for body size (nlvidd, nlvids, and nla, respectively) and, for comparison, ratios to aortic root were calculated (lvidd/ao, lvids/ao and la/ao, respectively). a cox's proportional hazard analysis with a counting process approach was used. spline smoothed graphical models were constructed to evaluate linearity of hazards. curves were then used to find cut-off values for interval hazard ratios (hrs). the hr for nlvidd, nlvids and nla (per . unit, % confidence intervals), were . ( . - . , p = . ), . ( . - . , p = . ), and . ( . - . , p = . ), respectively. the hrs for lvidd/ao, lvids/ao and la/ao ( . unit increase) were . ( . - . , p = . ), . ( . - . , p = . ), and . ( . - . , p = . ), respectively. the hr for fs was . ( . - . , p = . ). the relative hazard plot presented a steep increase for fs values above %. hrs for intervals < %, < %, and ≥ % were . ( . - . , p = . ), . ( . - . , p = . ), and . ( . - . , p = . ), respectively. the hr for nlvidd increased linearly. hrs for intervals . < . , . < . , . < . and ≥ . were . ( . - . , p = . ), . ( . - . , p = . ), . ( . - . , p = . ), and . ( . - . , p = . ), respectively. in contrast, the hazard for nlvids remained stable until . , whereafter it increased. the hrs for nlvids ( < . , . < . , . < . and ≥ . ) were . ( . - . , p = . ), . ( . - . , p = . ), ( . - , p = . ), and . ( . - , p = . ), respectively. hrs for values normalized to ao diameter behaved in a parallel way. we conclude that fs, left ventricular and atrial size may be used to predict chf. however, because the value of a hr is dependent on the unit used and, more essentially, does not account for nonlinear change in hazard, interpretation of hazards is challenging. in contrast, interval hazards are only dependent on the reference interval used. therefore they are easier to implement in every day clinical work. no conflicts of interest reported. systemic arterial hypertension is not frequently recognized in dogs with mitral valve degeneration (mvd), although borderline hypertension is difficult to assess, mainly because of different measurement techniques, inter-operator variability and, most importantly, examination-related stress. the object of this study was to evaluate systolic arterial blood pressure (sbp) at initial presentation and at regular intervals in dogs with various clinical stages of mvd. fifty six dogs with mvd that had not received any heart medication prior to admission, were included in the study. based on the isachc staging system, were assigned to class i (group a), to class ii (group b) and to class iii. small-breed dogs and miniature poodles, in particular, were overrepresented. comorbidities that could affect sbp were ruled out prior to enrollment. sbp was measured using a commercially available veterinary oscillometric device, by applying the proper cuff on the cephalic artery. dogs were left to acclimate for - minutes and measurements were always taken by the same investigator, before any other examination was performed, with the dog sitting on the owner's lap. a total of readings were taken, outlier values were discarded and the mean of the remaining measurements was documented. after initial consultation, treatment was customized according to the clinical stage. sbp was then measured every months, up to months after initial admission. at presentation, all class i dogs had sbp > mm hg, with only / having spb ≥ mm hg, whereas all class ii dogs had sbp < mm hg. of class iii dogs, had sbp > mm hg, and had sbp ≥ mm hg. a linear mixed effects model was used to assess the temporal variability of the measured parameters between groups. groups were matched for gender, age and body weight. blood pressure measurements, for the duration of the study, were higher in group a dogs, compared to groups b and c (p < , ). at the same time, group c had significantly higher sbp values than group b dogs (p < , ). asymptomatic mvd dogs seem to have higher sbp measurements, compared to those with clinical evidence of heart failure. whether this difference is stress-related, a maladaptive mechanism of sympathetic and raas activation to mvd or idiopathic remains to be elucidated. no conflicts of interest reported. sarcoplasmic reticulum (sr) ca + -atpase and its regulatory proteins are pivotal determinants of myocardial active relaxation via calcium uptake against the sr-cytoplasmic gradient. the lowered density of the sr ca + -atpase has been well demonstrated in many species during chronic hemodynamic overload. the genes linked to sr calcium uptake were reported not only being expressed in peripheral blood but serving as potential cardiac biomarkers in dogs with chronic mitral regurgitation, such as sr ca + adenosine triphosphatase isoform a (ser-ca a), phospholamban (pln), and hs- associated protein x- (hax- ). the aim of this study is to determine whether the target genes expressed in the blood will be translatable to the myocardial setting as cardiac biomarkers. the mrna expression levels of the target genes (serca a, pln, hax- ) from biopsied left ventricle (lv) and peripheral white blood cells (pwbc) in surgical mitral valve repair cases were estimated with quantitative real-time pcr using comparative ct method with gapdh. the gene expression levels in lv and pwbc were compared and their clinical relationships were evaluated. the diagnostic power of the genetic expressions in pwbc was analyzed by comparing to those of normal dogs. the levels of all target genes expressed in lv and pwbc were highly correlated each other in linear regression analysis (p < . ; serca a, r = . , r = . ; pln, r = . , r = . ; hax- , r = . , r = . ), although lv and pwbc showed different expression levels in a paired comparison (p < . ). according to the severity of the heart failure (isachc), the expression levels of all genes were gradually and significantly reduced in both lv and pwbc (p < . ). especially, the serca a and pln expressed in pwbc could clearly discriminate all isachc groups from the control (p < . ). multivariate regression adjusted by age and body weight revealed that serca a and pln in lv were negatively associated with lv internal systolic dimension (p = . , adjusted r = . and p = . , adjusted r = . , respectively). pln was also negatively related with lv internal diastolic dimension (p = . , adjusted r = . ). additionally, receiver-operating characteristic analysis using pwbc showed high area under the curve (auc) values for all target genes on overall isachc groups (p < . ; serca a, auc= . ; pln, auc= . ; hax- , auc= . ). in conclusion, the transcriptional changes of the calcium uptake related genes in pwbc may be able to reflect myocardial hemodynamic stress as well as to be utilized as promising cardiac biomarkers. no conflicts of interest reported. the aim of this study was to estimate heart-rate normalized pulmonary transit times (nptt) in cardiomyopathic cats with or without congestive heart failure (chf), to assess potential associations of echocardiographic variables and nptt, and to evaluate nptt as a test for presence of chf. privately owned cats were included. nptt was measured using echocardiography and the ultrasound contrast media sonovue â in groups of cats: healthy cats (group ), cats with cardiomyopathy (cm) but without chf (group ), and cats with cm and chf (group ). receiver operating characteristic curves (roc) were created for nptt, left atrial diameter (lad) and the left atrial to aortic root ratio (la:ao) to assess and compare their usefulness as tests for presence of chf. interrelations between pulmonary blood volume (pbv), nptt, stroke volume (sv) and echocardiographic variables were investigated by means of uni-and multivariate analysis. nptt values in group , group and group were . (interquartile range (iqr) . - . ), . (iqr . - . ), and . (iqr . - . ), respectively. values were significantly different between all groups. pulmonary blood volumes in group , group , and group were . ml (iqr . ml- . ml), . ml (iqr . ml- . ml) and . ml (iqr . ml- . ml). sv, pbv and shortening fraction < % were significant predictors of nptt. nptt and la:ao ratio, not sv were the main predictors of pbv. analyzing roc for nptt as a clinical test for chf yielded an auc of . which was similar for la:ao ratio. nptt may be useful test for the presence of chf in cats with cm and as a measure of cardiac performance. nptt and la: ao ratios predict chf with equal accuracy. increased pbv is significantly associated with higher nptt and la:ao ratios. both decreased sv and increased pbv explain the increased nptt in cardiomyopathic cats. the author received a travel scholarship from zoetis to attend this congress. acute arterial thromboembolism (aate) occurs commonly in cats, and less frequently in dogs, mostly resulting in limb paresis or paralysis. diagnosis is based typically on physical examination and advanced imaging. diminished affected-limb peripheral blood flow induces changes in several analytes concentrations in affected limb venous samples, compared to their peripheral venous concentration. we hypothesized that in aate, local, affected-limb venous glucose concentration decreases below reference interval, while systemic glucose concentration remains unaffected. the study included groups for each species: paralytic aate cases, non-ambulatory controls with limb paralysis of orthopedic or neurologic disorders, and ambulatory controls diagnosed with various diseases. systemic and peripheral, affected-limb blood glucose concentrations were measured. group absolute (dglu) and relative (% dglu) differences were compared. no procedure-associated complications or pain were noted. peripheral blood glucose concentrations were decreased (p ≤ . ) only in cats and dogs with aate. dglu and %dglu were higher in the aate groups in both cats and dogs compared to their respective control groups (p < . , p < . , respectively), with no differences between the control groups. receiver operator characteristics analysis of dglu and %dglu as predictors of aate in cats had areas under the curve of . and . , respectively, and . and . , in dogs, respectively. dglu cutoffs of mg/dl and mg/dl, in cats and dogs, respectively, corresponded to sensitivity and specificity of % and % in cats, respectively, and % in dogs. dglu and %dglu are extremely accurate, readily-available, simple diagnostic markers of aate in cats and dogs. no conflicts of interest reported. glycaemia determination is usually included in routine biochemisty panels. no works are devoted to the evaluation of pheripheral glycaemia in animals suffering from arterial thrombosis. the aim of this study was to document the pheripheral glycaemia variations in hypoperfused limbs of patients affected by mriconfirmed arterial thrombosis. eleven dogs referred for monoparesis or paraparesis were recruited. inclusion criteria were a clinical examination supportive of limb hypoperfusion and availability of blood cell count, biochemical profile and urine analyses. before mri examination, peripheral glycaemia was tested. two blood samples were obtained, one from the affected limbs and one from a healty limb.plasmatic glycaemia was measured using an automated glucose analyser. all the patients underwent a total body mri (mri intera . t, philips medical systems) that provided the final diagnosis. the arterial thrombosis location was documented and the entity was scored. all the eleven patients were diagnosed with a peripheral thrombosis involving an arterial vessel and in some cases the relative branches. the thrombus was located: in the abdominal aorta ( / ), in the subclavian artery ( / ), in the axillary artery ( / ), in the iliac arteries ( / ). of the total amount of abdominal aortic thrombosis, / involved also the internal iliac arteries, / the external ones and / both internal and external. the extent of the thrombosis was classified as grade (g ), when the greatest portion of the thrombus did not reach half of the vessel lumen ( / patients); grade (g ), when the greatest portion of the thrombus was between / and / of the vessel lumen ( / ); grade (g ), when the thrombus exceded / of the lumen ( / ). a substantial decrease in pheripheral glycaemia values was found in sampling arising from the thrombosisaffected limbs. comparing thrombosis-affected limbs values with healthy limbs measurements from the same patient, the reduction was found from . % to . %. accounting only the g scored patients, the percentage of reduction was found up to the . % suggesting a proportional decrease related to the grade of occlusion. results from this study suggest that peripheral glycaemia values are affected by limb hypoperfusion disorders. if an arterial thrombosis is suspected, samples from the affected limbs and the healthy ones could be used to compare glycaemia values and to support the early stage therapy in anticipation of diagnostic imaging. further studies are needed to confirm the proportional relation of the decrease with thrombus entity. no conflicts of interest reported. canine idiopathic pulmonary fibrosis (cipf) is a progressive interstitial lung disease mainly affecting west highland white terriers (whwt). pulmonary hypertension (ph) may develop secondary to hypoxic vasoconstriction and/or pulmonary parenchymal infiltration. in the absence of measurable tricuspid regurgitation (tr), this co-morbid condition may be difficult to diagnose non-invasively. the degree of cardio-pulmonary impairment in cipf dogs can be evaluated through blood gas analysis (bga) and minute walking test ( mwt). a new echocardiographic index, the right pulmonary vein to pulmonary artery ratio (pv/pa) has been described for the detection of pulmonary venous hypertension. the aim of this study was to investigate pv/pa in cipf in order to determine its utility in the detection of ph and in the assessment of cardio-pulmonary disease severity. this prospective clinical cohort study included whwt with cipf (group a), healthy whwt (group b) and healthy dogs from other breeds (group c). diameters of right pv and pa were measured, in bi-dimensional (bd) and m-modes (mm), in a parasternal right long axis view, at the end of the t wave. other echocardiographic parameters for evaluation of ph were also measured: speed of tr, acceleration time to ejection time ratio of the pulmonary flow (at:et) and pulmonary artery to aorta ratio (pa/ao). bga was performed in dogs ( , and in groups a, b and c) and mwt in dogs ( , and ). values are given as meanaesd. in bd and mm mode, the pv/pa ratio was lower in group a (mm: . ae . , bd: . ae . ) compared to group b (mm: . ae . , bd: . ae . , p ≤ . ) and group c (mm: . ae . , bd: . ae . , p ≤ . ). the changes in pv/pa were both due to an increase of pa (p ≤ . ) and a decrease of pv (p ≤ . ). tr was found in % of dogs with cipf; mean pressure gradient was . ae . mmhg. at:et was lower in group a ( . ae . ) compared to group c ( . ae . , p = . ) and tended to be lower compared to group b ( . ae . , p = . ). pa/ao was not statistically different between groups. pv/pa was correlated with arterial po values (b mode: r = . , p = . ) and results of the mwt (b mode: r = . , p = . ). pv/pa was also correlated with at:et and the speed of tr, but not with pa/ao. in conclusion, in whwt affected by cipf, pv/pa is a useful indicator of ph and could serve in the assessment of disease severity. no conflicts of interest reported. ventricular septal defect (vsd) is the fourth most common congenital cardiac defect in dogs and the most common in cats. the aim of this study was to evaluate the long-term outcome in vsd patients. case records of animals were reviewed, of these re-evaluated echocardiographically and followed up by phone interview only. out of dogs pug was the most common breed ( %) followed by border terrier ( %). out of cats domestic short hair was most common ( %) followed by main coon ( %). isolated vsds were present in dogs and cats. complex defects (cds) were present in cases, most frequent anomalies being sub-aortic stenosis ( dogs, cat), pulmonic stenosis ( dogs, cats), tetralogy of fallot ( dogs, cats), cushion defects ( cats) and double-chambered right ventricle (dcrv) ( dogs, cats; in / dogs not present initially supporting the cause-and-effect theory). eisenmenger was observed in dog and cats. aortic insufficiency, not considered a cd, was noted in dogs. in dogs and cats ( % of isolated vsds and cat with a dcrv) the defect closed spontaneously. nine dogs and cats ( %) died of non-cardiac causes with an age at death of to (mean . ) months; dogs and cats died due to cardiac causes with an age at death of . to (mean . ) months. cardiac deaths were sudden ( dogs with cds) or euthanasia for left sided congestive heart (chf) failure associated with cds ( dogs, cat); right sided chf associated with cds ( cats); biventricular failure ( cat with cd); weakness (eisenmenger, dog and cat; fallot dog; cd cat). two cats developed chf due to unrelated hcm. only one dog with an isolated vsd was euthanized for chf. these results indicate that spontaneous vsd closure occurs more often than previously thought, most patients with isolated restrictive vsds live a normal life span without surgical intervention, but non-restrictive vsds or complex defects can be associated with significant morbidity and mortality. echocardiography early in life is crucial to identify the anomaly and cds, as well as useful to prognosticate long-term outcome and to identify patients where a surgical intervention should be considered if available. follow-up echocardiography is indicated to corroborate the prognosis, to detect complications due to the vsd and to detect unassociated acquired cardiac diseases. no conflicts of interest reported. centronuclear myopathy (cnm) is the most prevalent congenital inherited disorder affecting skeletal muscles in labrador retrievers. this disabling condition segregates worldwide and a recessive loss-of-function founder mutation was identified in the protein tyrosine phosphatase-like, member a gene (ptpla/ hacd ). the objectives of this study were ) to describe ptpla expression pattern in hearts from homozygous wild type (wt), heterozygous (het) and homozygous mutated (cnm) dogs, ) to assess and compare the left myocardial function in aging wt, het and cnm dogs. for this purpose, seven wt, four het and eleven cnm dogs were included in the study. ptpla mrna levels were assessed by rt-pcr and rt-qpcr. all dogs were examined using conventional echocardiography, d color tissue doppler imaging (tdi) and tdi-derived strain imaging. we found that the expression of the two wild type ptpla splice isoforms increased post-natally in wt dogs. their levels were halved in het dogs and drastically reduced in cnm dogs. in both het and cnm dogs, a slight left ventricular hypertrophy was detected using conventional echocardiography. tdi and strain imaging revealed that the left ventricular myocardial function was significantly altered in both het and cnm dogs compared to wt dogs. moreover, these functional defects were associated with significantly higher values of systemic arterial blood pressure, although maintained within normal ranges. in conclusion, subclinical myocardial alterations were detected in both het and cnm aging dogs from our french pedigree, suggesting a role for ptpla in long-term cardiovascular homeostasis. these findings prompt globalized confirmation in additional ptpla"'deficient dogs, which may thus be considered as a new large-size model for human left ventricular sub-clinical myocardial dysfunction. no conflicts of interest reported. mitral regurgitation (mr) secondary to degenerative mitral valve disease (dmvd) is the most common heart disease in dogs. in dogs with mr, mitral valve prolapse caused by degeneration of the mitral valve leaflet, chordae tendinae extension and/or rupture and mitral annulus dilation are observed. however, limited data are available on morphological changes in dogs with mr. currently, there are no studies confirming the anomaly of the mitral complex via direct observation in living dogs with mitral regurgitation. at our institution over the last ten years, approximately dogs have undergone mitral valve repair. during surgery, the anomaly of mitral complex can be observed macroscopically (directly visualized). to our knowledge, this is the first study evaluating the anomaly of the mitral valve leaflet and chordae tendineae in dogs undergoing mitral valve repair. animals: dogs that underwent mitral valve repair with cpb at nihon university between february and june were included in this study. methods: confirmation of chordae tendineae rupture was visually confirmed during surgery. the sites of chordae tendineae rupture were also recorded at that time. septal chordae and mural chordae were divided three division depend on the site (s , s , s and m , m , m respectively). results: ninety eight dogs were included in this study. the mean age and body weight were . ae . years and . ae . kg, respectively. of the dogs, ruptured chordae was observed in dogs ( . %). septal leaflet chordae was ruptured in dogs ( . %) and mural leaflet chordae was ruptured in dogs ( . %). chordae of both leaflets were ruptured in dogs ( . %). no chordal rupture was observed in dogs ( . %). in the dogs with ruptured septal chordae, the chordae between s and s was most often ruptured (n = , %). in this study, rupture of the septal chordae tendineae was most commonly observed. this is the first pilot study to visually evaluate the anomaly of the mitral valve leaflet and chordae tendineae in dogs undergoing mitral valve repair. future studies comparing pathological changes and molecular biological analysis to gross findings of mitral chordae tendineae in dogs undergoing mitral valve repair may be useful in advancing the understanding of the disease. no conflicts of interest reported. echocardiographic aortic valve (ao) measurements are routinely obtained during cardiac evaluation of patients. cardiologists commonly use diastolic ao measurements to obtain ratiometric weight-independent estimates of dimensions of other cardiac structures, most commonly the left atrium (la). however, no consensus exists about the point in diastole at which ao measurements should be obtained -immediately after closure of the aortic valve, when la size is largest (ao max , but often with least distinct margins), during the p-wave of the ecg (ao p ) and at the onset of ventricular electrical systole, when la size is smallest (ao min ). we examined the linear and area dimensions of the ao (aod and aoa) to determine if clinically significant differences exist at distinct diastolic time-points, or if these measurements could be interchangeable. we examined patients ( dogs and cats) presented for cardiac evaluations by d echocardiography. three replicates of each time-point linear and area measurement (ao max , ao p , ao min ) were obtained in each patient and averaged for analysis. only patients with aortic valve disease and those with atrial fibrillation were excluded from analysis. beat-to-beat variability of the ao measurements was determined. standard and normalized limits of agreement (loa) plots were generated for each pairwise comparison. the frequency of each ao measurement being the largest or smallest within-patient measurement was determined, and compared via repeated measures anova. all pairwise agreement plots of both aod and aoa demonstrated heteroscedasticity; normalized aod plots showed % loa to be % of the mean aod measurement, with a bias of approximately . % for aod max -aod min , % for aod max -aod p , and % for aod p -aod min . normalized aoa plots showed %loa to be % of the mean aoa measurement, with a bias of approximately % for aoa max -aoa min , % for aoa max -aoa p , and % for aoa p -aoa min . aod max was the largest measurement in / ( %) patients and aod min was the smallest measurement in / ( %) patients; aoa max was the largest measurement in / ( %) patients and aoa min was the smallest measurement in / ( %) patients. rmanova confirmed that ao max >ao p >ao min (p < . ). median within-patient within-measurement variability was % for aod and % for aoa measurements. our data suggest that ao measurements differ throughout diastole, with ao max >ao p >ao min . the disparity is greater for area than linear estimates. the degree of disagreement between ao max and ao p is small and similar to the within-measurement variability. thus, using either ao max or ao p measurements should result in similar ratiometric estimates of cardiac dimensions. no conflicts of interest reported. feline hypertrophic cardiomyopathy (fhcm) is the most common heart disease in cats. hcm is considered an inherited disease of the sarcomere and fhcm has been linked to mutations in one sarcomere protein i.e. mybpc . however, the pathophysiologic mechanisms behind disease development and progression are largely unknown. in this study we investigate whether mitochondrial morphological changes in the myocardium accompany mitochondrial dysfunction and enhanced oxidative stress formation that we recently found in fhcm. myocardial tissue from the left ventricle (lv) was obtained immediately after euthanasia from cats diagnosed with primary hcm on echocardiography ( maine coon, british shorthair, exotic shorthair, norwegian forest cat) and age-matched control cats ( maine coon, norwegian forest cats). ultrastructural examination was performed by the use of transmission electron microscopy. in hcm cats, marked ultrastructural changes of the cardiomyocytes were observed. the population of subsarcolemmal mitochondria (ssm) was absent in large cellular areas in cats with moderate and severe lv hypertrophy. flattening of the sarcolemma was a common finding, causing disorganization of the t-tubular system. interfibrillar mitochondria (ifm) were disorganized but not depleted. additional changes in cardiomyocytes from cats diagnosed with fhcm included remodeling of sarcomeres, disorganization of myofibrils, convolution of gap junctions, accumulation of intracellular z-disc material, perinuclear lipofuscin granula and extensive extracellular deposits of collagen. in healthy mammalian cardiomyocytes, the t-tubular system upholds cellular structure, prevents mitochondrial reticulum formation and provides calcium, oxygen and substrates, necessary for normal functioning muscle. disorganization of the sarcolemma and t-tubular system may cause the depletion of ssm. possible mechanisms are atrophy or disruption of the mitochondria or altered fusion-fission dynamics. calcium cycling and substrate supply are likely to be compromised by the observed structural changes. we propose this to be related to mitochondrial dysfunction and oxidative stress formation that occurs in fhcm, however a causative relationship remains unknown. in conclusion, morphological changes of mitochondria and extra-sarcomeric structures are common in fhcm, regardless of breed, genotype and phenotypic disease expression. moreover, mitochondrial subpopulation-specific changes occur in fhcm with depletion of ssm. ultrastructural and functional changes of cardiac muscle mitochondria are considered important molecular mechanisms, responsible for the development and progression of fhcm and may be relevant future treatment targets. no conflicts of interest reported. patent ductus arteriosus (pda) is one of the most common congenital cardiac defect in the dog. ductal patency is associated with pulmonary overcirculation, left ventricular volume overload and can rapidly determine congestive heart failure if untreated. several devices to close the pda have been used, with amplatzer canine duct occluder (acdo©) being considered the safer device with lowest complication rates. echocardiography represents the cornerstone of pda diagnosis, but its role has been recently expanded to wider field of application: device sizing and intraoperative monitoring, as well as a tool to quantify cardiac morphology and function. speckletracking echocardiography (ste) has been used to evaluate cardiac function in a wide variety of diseases in human and veterinary patients, however no study has evaluated its usefulness in dogs affected by pda both before and after percutaneous closure of pda. the aim of our study was therefore to assess standard m/bmode derived parameter of cardiac function and ste derived longitudinal, radial and circumferential strain and strain rate before and after pda closure. twenty-five dogs of different breeds, age and weight were prospectively recruited and a complete echocardiographic evaluation was performed before and hours after pda closure. end diastolic and systolic diameters indexed for body surface area (edvi/esvi) both derived by m-mode and b-mode views, allometric scaling derived allod and allos, sphericity index (si) and pulmonary to systemic flow ratio (qp/qs) were assessed both pre and postoperatively. ste derived parameters assessed were longitudinal, radial and circumferential strain and strain rate. a statistically significant difference was found in all standard parameters of cardiac function before and after pda closure (p < . ), with a general decrease in values hours postoperatively. ste derived parameters of cardiac function showed a trend toward a decrease back to normal values, which was statistically significant (p < . ) for circumferential and radial strain and strain rate, while longitudinal strain and strain rate did not reach statistically significance. based on our results, no cardiac dysfunction was identified by the use of ste derived parameters both before and after pda closure, with an increased contractility as identified by higher than normal ste values before pda closure and a decrease back to normal strain and strain rate values for both circumferential and radial immediately after percutaneous closure. longitudinal strain persists on higher than normal values, refusing the hypothesis of systolic dysfunction after pda closure and suggesting a longer reverse remodeling process after pda closure. dr bussadori receives royalties from esaote (florence, italy) related to an european patent (nr ) he developed for xstrain software. the study was not funded by a research grant. cardiac cachexia which is characterized by progressive weight loss and depletion of lean body mass, is an independent predictor of survival in human patients with congestive heart failure. chronic degenerative mitral valve disease (cdmd) is one of the most common cardiac diseases in dogs. the aims of this study were to evaluate the prevalence and the effects of cardiac cachexia in survival of dogs with cdmd. medical records of client-owned dogs with cdmd were reviewed. the mean age at entry was . ae . years; were females, and were males. data obtained from the records including breed, sex, body weight, age at diagnosis, complete blood counts, biochemical profiles, urinalysis, systemic blood pressure, thoracic radiographs, electrocardiograms, ultrasonography and echocardiographic examinations at initial visit and survival time. diagnosis of cdmd was based on echocardiographic characteristics and categorized by modified new york heart association (nyha) functional classification. cardiac cachexia was defined as presence unintentional weight loss (> % within months after diagnosis) together with anorexia and muscle weakness, anemia (red cell count < . /ll, hemoglobin < g/dl, or both), hypoalbuminemia (plasma albumin < . g/dl), and azotemia (blood urea nitrogen > g/dl, creatinine > . g/dl, or both). dogs with other cardiac disorders and other systemic disorders those would cause anemia and hypoalbuminemia were excluded from this study. prevalence of cardiac cachexia, anemia and azotemia was . %, . % and . %, respectively. these conditions were the most prevalent in nyha class , followed by nyha classes and . the prevalence of hypoalbuminemia was not significantly different among classes. the one-year body weight change was found in the nyha classes (increased . ae . %), (decreased . ae . %) and (decreased . ae . %). the difference between classes and was significant. results of the cox proportional hazard model indicated that survival time was significantly positively associated with nyha functional severity at diagnosis (p < . ), presence of cardiac cachexia, weight loss, anemia, hypoalbuminemia and azotemia (p < . , p = . , p = . , p = . and p = . , respectively). the prevalence of cardiac cachexia was common in advanced cdmd dogs, and the parameters of cardiac cachexia, namely weight loss, anemia, hypoalbuminemia and azotemia were strong prognostic factors associated with survival. no conflicts of interest reported. mitral valve disease (mvd) is the most common cardiovascular disease in dogs. it's characterized by myxomatous degeneration, which causes mitral valve prolapse (mvp), mitral regurgitation (mr) and a left apical systolic murmur (lasm). mvd affects small breed dogs with a very high prevalence in cavalier king charles spaniels (ckcs). the main goal of this study was to determine the prevalence of lasm, mvp and mr in the maltese, the most presented breed among dogs with mvd in taiwan. the correlation between these measurements and the influence of age, gender, reproductive state, and body weight were also investigated. study results were compared to other mvd prevalence studies in europe and north-america. client-owned maltese dogs ( males and females; body weight . - . kg; age - yrs) with no signs of heart failure were recruited. the intensity (grade - ) of lasm was recorded. grade of mvp (mild/severe) and mr severity (mild/moderate/ severe) were evaluated by echocardiography. logistic regression was used to determine the correlation between age and presence of lasm, mvp and mr. a chi-square test was used to evaluate whether sex and reproductive-status were related to prevalences of lasm, mvp and mr. spearman's correlation coefficient was used to assess the relationships between age, body weight, lasm intensity, grade of mvp and severity of mr. the prevalence of lasm, mvp and mr were . %, % and . %, respectively. all have positive correlation with age (p = . ). the age at which % of the dogs had lasm, mvp and mr was . , . , and . years, respectively. the lasm intensity, mvp grade and mr severity were all positively correlated to age (all p = . ) and had no correlation with bw and reproductive status. females had a significantly higher prevalence of lasm than males ( % vs. . %, p = . ). maltese dogs in taiwan have a very high prevalence and an early development of mvd as compared to other small breed dogs, similar to mvd in ckcs in other countries. since we only recruited asymptomatic dogs, this study may underestimate the prevalence of mvd in the whole maltese population. to our knowledge, this is the first report to document the high prevalence of mvd in taiwanese maltese.the maltese may be a new canine model for genetic, pathology, and natural history studies in mvd. boehringer-ingelheim sponsored the author's accommodation costs for this congress. esvc-o- cardiorenal syndrome in dogs with chronic valvular heart disease: a retrospective study. e. martinelli , p. scarpa , c. quintavalla , c. locatelli , p. brambilla . university of parma, parma, italy, university of milan, milan, italy in human medicine, primary disorders of the heart often result in secondary dysfunction or injury to the kidneys. the coexistence of the two problems in the same patient is referred as cardiorenal syndrome (crs). just little information about crs is available in veterinary medicine. the aim of this study was to define the prevalence of chronic kidney disease (ckd) complicating chronic valvular heart disease (cvhd) in dogs and to investigate the relationship between class of cardiac insufficiency (acvim) and class of renal insufficiency (iris). medical records of dogs presented at the cardiology service of the department of veterinary science and public health, university of milan, between january and december were retrospectively evaluated. dogs with a complete physical examination, thoracic radiographs, a cvhd diagnosis based on echocardiographic examination, and a serum biochemical panel, including assessment of serum creatinine (scr) and serum urea (bun), were included in the study. dogs with other heart disease, neoplasm or systemic diseases were not included in the study. one hundred eighteen dogs of both genders ( males and females), to years of age ( . ae . years), to kg of bodyweight ( . ae . kg) fulfilled the inclusion criteria. the % of males and the % of females were neutered. the most represented breeds were mongrel ( %), miniature poodle ( . %), york shire terrier ( . %), shih -tzu ( . %), pinscher ( . %) and dachshund ( . %). dogs were classified as follow: % acvim a, % acvim b , % acvim b , % acvim c and % acvim d. while the % of the dogs were normoazotemic (scr < , mg/dl), % were staged in iris , % in iris and %in iris . statistical analysis was performed using jmp . (sas institute inc.). a p value < , was considered significant. the prevalence of ckd associated with azotemia in dogs affected by cvhd was %. there was a statistically significant direct correlation between acvim and iris class (pearson test p = . ). unexpectedly, the % of dogs receiving drugs for medical management of heart failure (acvim class c and d) were normoazotemic. despite a definite conclusion about the role of cvhd on the induction and/or progression of ckd cannot be drawn from this cross-sectional study, these results suggest that there is a direct correlation between the severity of ckd and cvhd. no conflicts of interest reported. there is growing evidence of breed differences in concentrations of several blood variables in dogs. the aim of the study was to investigate breed differences in plasma concentrations of components of the renin-angiotensin-aldosterone system (raas), endothelin- (et- ) and serum cortisol concentration in healthy dogs. healthy, privately-owned dogs of nine breeds were examined at five centers as part of the european lupa-project. absence of cardiovascular or other clinically relevant organrelated or systemic disease was ensured by thorough clinical investigations. plasma concentrations of et- and aldosterone, renin activity, and serum concentration of cortisol were measured by ria or elisa assays. overall significant breed differences were found (p < . for all variables). bonferroni-corrected pair-wise significant differences between breeds were found in % of comparisons for et- , % for cortisol, % for renin and % for aldosterone. for et- , the highest median concentration was found in newfoundlands with values > times higher than most other breeds, while renin was highest in dachshunds, > times higher than in newfoundlands and boxers, which had the lowest concentrations. aldosterone was especially low in belgian shepherds with median concentration < times than the other breeds. cortisol was highest in finnish lapphunds, almost times higher than boxers with the lowest concentration. in conclusion, considerable inter-breed variation in concentrations of et- , components of raas and cortisol was found in healthy dogs. these differences are likely influenced by genetic factors and should be taken into account when designing clinical trials and tests. breed-specific reference ranges might be necessary. no conflicts of interest reported. most studies that assess weight management in obese dogs only examine the early stages of weight loss, and this may not properly reflect a complete weight management regime. the aim of the current study was to examine the kinetics of a complete weight management cycle in obese client-owned dogs. dogs referred to the royal canin weight management clinic, university of liverpool, for the management of obesity, were eligible for inclusion. all dogs were followed until they had either completed (i.e. reached target weight) or the programme was discontinued. rate of weight loss, percentage weight lost, and energy were assessed at different time points. a total of dogs were included, with a range of breeds, ages and sexes represented. rate of weight loss steadily decreased throughout the weight loss period (d : . ae . %/wk; d : . ae . %/wk; d : . ae . %/wk; d : . ae . %/wk; d . ae . %/wk; d : . ae . %/wk; p < . ). the energy intake required to maintain weight loss also progressively decreased (p < . ). by day , mean aesd weight loss was ae . %, and compliance was good, but most had not com-pleted ( % completed, % ongoing, % discontinued). thereafter, more dogs completed, but the number of discontinuing also increased (d : ae . % weight loss, % completed, % ongoing, % discontinued; d : ae . % weight loss; % completed, % ongoing, % stopped). initial weight loss is good in obese dogs but, thereafter, steadily worsens. thus, studies examining only the first few months of weight loss are not fully representative of the entire weight loss process. conflicts of interest: the following conflicts of interest apply: the diet used in this study is manufactured by royal canin.whilst vb is employed by royal canin. vb and ss are employed by royal canin. ajg's readership is funded by royal canin. obesity and obesity-related metabolic dysfunctions are increasing in humans as well as in dogs. obese dogs become affected by chronic diseases at young age, have a decreased quality of life and a shorter life-span. the aim of the study was to describe the metabolic and hormonal response to a feed-challenge test in lean and overweight dogs. twenty-eight healthy intact male labrador retrievers aged . ae . years with varying body condition score (bcs, scale - ) were included. twelve dogs were classified as lean (bcs - ), ten as slightly overweight (bcs ) and six as overweight (bcs . - ). an overnight fasting period and blood sample collection was followed by a high fat meal. after food intake, blood samples were collected hourly for four hours. a glucagon elisa was validated for use in dogs. the assigned bcs was supported by positive association with serum leptin concentrations. postprandial triglyceride concentration was significantly higher in the overweight group. a tendency to higher cholesterol concentration was seen in the overweight group but cholesterol was not affected by food intake. glucagon concentration rose after food intake and resembled the response seen in humans after a mixed meal. glucose and insulin concentrations followed the same pattern while free fatty acids had declined one hour after the meal. in this study, the metabolic and hormonal response to a high fat meal was similar between lean and slightly overweight dogs, whereas the response of overweight dogs differed. studies on the health significance of postprandial hypertriglyceridemia in dogs are warranted. conflicts of interest: the study was financially supported by the swedish veterinarian federation, the companion animal research foundation, and the foundation of thure f. & karin forsberg. feline weight-loss programs are often hindered by compliance issues and sedentary lifestyle. the purpose of this study was to assess the effectiveness of a new dietetic weight management food (ndwmf)* in achieving weight loss in overweight/obese, client-owned cats. the objectives were ) to evaluate weight loss parameters in cats fed the ndwmf* and ) to describe the owner's perception of the cat's quality of life. overweight/obese, otherwise healthy, client-owned cats (> / body condition score -bcs) were enrolled in the study (n = ). initial veterinary evaluation comprised a physical examination, nutritional assess-ment, determination of ideal body weight (ibw), and development of weight loss feeding plan. daily energy requirement (der) for weight loss was calculated as der = . x ( x ibw kg . ). initial and follow-up evaluations (monthly for months) consisted in determination of body weight (bw), bcs, body fat index (bfi), muscle condition score (mcs), and current feeding practices. quality of life assessment by owners included cat's level of energy, happiness, appetite, begging behavior, flatulence, stool volume, and fecal score. statistical analysis encompassed scatterplots, regression analysis, summary statistics as appropriate for the type of analyses (continuous or categorical variables, distribution), a mixed model anova was used to assess changes over time (statistical significance at p < . ). eighty three percent of the cats (n = ) lost weight with an average weight loss of % (sem, . %) over months and an average weekly weight loss rate of . % (sem, . %). a significant decrease in bcs from week - and in bfi from week - compared to baseline was observed. mcs did not change. average duration of weight loss was days (sem, . days) with days (sem, . days) between visits. fourteen percent of cats achieved ibw ( . , ci: . - . ). seventy nine percent of cats ate more than the recommended der (median fed above der= %), and the majority of these cats still lost weight. owners perceived a significant increase in energy and happiness (>week ) compared to baseline in the cats that lost weight without changes in appetite or begging behavior. no significant changes were seen in scores for flatulence, stool volume, and fecal score. in conclusion, this clinical study showed that feeding the ndwmf* to client-owned, overweight/obese cats resulted in weight loss. owners reported significant improvements in cat's quality of life without negative side effects. * porphyrias are a group of inborn errors of metabolism resulting from accumulation of porphyrins due to deficient activities of specific enzymes in heme biosynthesis. in humans, they are clinically classified as either erythroid with cutaneous involvement or hepatic with acute neurovisceral attacks. here we describe the clinical, biochemical, and molecular genetic studies in porphyric cats from new brunswick, canada. from to , three separately identified adult domestic shorthair cats from the city of saint john in new brunswick were found to have erythrodontia (brown discolored teeth which fluoresced pink) and pigmenturia. a mild compensated hemolytic disorder with numerous small dark blue irregularly shaped erythrocyte inclusions was noted. there was no evidence of acute lifethreatening neurovisceral attacks or cutaneous lesions. necropsy of one cat revealed massive deposition of porphyrins in all bones and teeth. urine and edta blood samples from one cat were metabolically studied, while molecular genetic studies were performed in all cats either from edta blood or a formalinized splenic tissue block. urinary d-aminolevulinic acid, porphobilinogen, uroporphyrin i, and coproporphyrin i concentrations were increased in the cat studied, suggesting an acute intermittent porphyria (aip). the erythrocytic hydroxymethylbilane synthase (hmbs) activity in erythrocytes was approximately half normal suggesting a dominant enzymopathy, while the erythrocyte uroporphyrinogen iiisynthase activity was normal. sequencing the feline hmbs gene revealed a heterozygous intronic base deletion (c. - _- del) which results in an insertion in the mrna and would predict a truncated protein. in conclusion, these three domestic shorthair cats had the same hmbs mutation causing an autosomal dominantly inherited aip. cats with discolored teeth and normal or mild hemolysis may have either acute intermittent porphyria or congenital erythroid porphyria. interestingly, seven disease-causing mutations have now been found by us in the hmbs gene -more than in any other gene in cats. the biochemical and molecular characterization facilitates clinical screening of affected cats to reach a specific diagnosis. supported in part by nih od . urs giger and raj karthik are also part of the laboratory that offers dna testing for this mutation. fibrinogen decreases when coagulation is activated to form fibrin, while fdps and d-dimers represent the products of fibrinolysis. in humans, activation of coagulation and fibrinolysis develops in all type of ascites and it is also associated with signs of systemic fibrinolysis.these results have lead to the suggestion that ascitic fluid is inherently fibrinolytic. preliminary studies showed similar results also in dogs (javma nov. , ecvim proceedings . in addition, in an old experimental study conducted in dogs, inoculation of blood or of a solution containing fibrinogen and thrombin into the pleural cavity resulted in the activation of the coagulation system followed by fibrinolysis. therefore, the objective of the present study was to determine whether the activation of coagulation and fibrinolysis (i.e. low fibrinogen and elevated fdps and ddimer) occurs not only in the ascitic fluid, as alredy been demonstrated, but also in all type of pleural effusions in dogs. thirty-three dogs referred to the san marco veterinary clinic with pleural effusion, but without ascites, were studied. fibrinogen, fdps, and d-dimer concentrations were measured and then compared in both pleural fluid and venous blood via wilcoxon signed ranks test. the dog's pleural effusions were categorized based on pathophysiology of fluid formation into dogs with transudate ( due to increased hydrostatic pressure and due to decreased osmotic pressure), with an exudate (of which due to septic causes), with a haemorrhagic pleural effusion, and with a chylous effusions. the fibrinogen concentration in the pleural effusion (median: mg/dl; range: - ) was significantly lower (p < . ) than the plasma fibrinogen concentration (median: mg/dl; range: - ). in all dogs, the fibrinogen pleural fluid concentration was lower than the plasma concentration. the fdp concentration in the pleural effusion (median: mg/dl; range: . - ) was significantly (p < . ) higher than plasma fdps concentrations (median: . mg/dl; range: . - . ). in case, the fdps pleural fluid concentration was lower than the plasma concentration and in cases the pleural fluid concentration was higher. the d-dimer concentrations were significantly(p < . ) higher in the pleural effusion (median: . lg/ml; range: . - . ) than in the plasma (median: . lg/ml; range: . - . ). in one case, the d-dimer pleural fluid concentration was lower than the plasma concentration and in cases was higher. these findings support the hypothesis that activation of coagulation followed by fibrinolysis occurs in all type of pleural effusions. no conflicts of interest reported. during primary hyperfibrinogenolysis (phf), fdps production is increased but production of d-dimer is not. therefore, elevated fdps and normal d-dimer are considered an indicator of phf. in humans and dogs, activation of coagulation and fibrinolysis develops in all type of ascites and it is associated with systemic phf, suggesting that ascitis is inherently fibrinolytic. preliminary data have shown that activation of coagulation followed by fibrinolysis occurs also in all type of pleural effusions (pe). the objective of this study was to determine if systemic phf occurs also in dogs with pe. thirty-three dogs referred to the san marco veterinary clinic with pe, but without ascites, were studied (group ). from the electronic data-base of the clinic dogs for inclusion in control groups (healthy dogs) and (sick dogs without pe or ascites) were randomly selected and individually matched to group dogs for age, sex, and breed. fibrinogen, fdps, d-dimers, c-reactive protein (crp), fibrinogen/crp ratio, and prevalence of phf (i.e., dogs with elevated plasma fdps and normal d-dimer) were determined. differences between the groups were analyzed using anova (fibrinogen), chi-square (fdps and prevalence of phf) and kruskal-wallis test (crp, fibrinogen/crp ratio, and d-dimer). post-test analysis were performed by tamhane and mann-whitney test. fibrinogen concentration in group was significantly increased compared to group (p < . ), but not compared to group (p = . ). fdps concentration in group was significantly increased compared to groups (p < . ), but not compared to group (p = . ). d-dimers concentration in group was significantly increased compared to group (p < . ), but not compared to group (p = . ). crp was significantly increased in group compared to group and (p < . for both comparison). fibrinogen/crp ratio was significantly decreased in group compared to group and (p < . for both comparison). prevalence of phf was significantly higher in group compared to groups (p = . ), but not compared to group (p = . ). these results support the hypothesis that phf occurs significantly more often in dogs with pe compared to healthy dogs. despite there was a trend of increased phf also in dogs with pe compared to sick dogs, this difference did not reach significance. nevertheless, the decreased in fibrinogen/crp ratio in group compared to group , in the face of a similar d-dimer concentration, would suggest that phf is also more prevalent in dogs with pe compared to sick control dogs. no conflicts of interest reported. the systemic inflammatory response syndrome (sirs) refers to clinical signs of systemic inflammation in response to (non-) infectious insults. current diagnosis of sirs is based on clinical and basic laboratory data and is a sensitive screening to identify patients at risk. c-reactive protein (crp) is a major canine acute phase protein with concentrations related to disease severity and underlying cause. crp rises in response to proinflammatory cytokines, mainly interleukin (il)- and tumor necrosis factor (tnf)-a, which are considered the main triggers of sirs. we therefore evaluated crp, il- and tnf-a kinetics in canine emergency sirs patients hypothesizing that crp is ( ) increased in dogs with a clinical sirs-diagnosis, ( ) correlated with il- and tnf-a concentrations, ( ) influenced by the underlying etiology, and ( ) a prognostic marker. canine emergencies with clinically diagnosed sirs were prospectively included. serum and plasma were immediately stored at - °c after sampling at presentation, after (t ), (t ), (t ) and (t ) hours, and at a control visit (t m) over one month after discharge. serum crp was measured with a caninespecific immunoturbidimetric crp assay. plasma il- and tnfa were measured using a bioassay measuring biologically active cytokine concentrations. disease categories were infection (i), neoplasia (n), trauma (t), gastric-dilation and volvulus (gdv), other gastrointestinal (gi), renal (r) and miscellaneous (m) diseases. statistical analysis was performed with sas. concentrations of inflammatory cytokines were expressed logarithmically, with univariate analysis confirming normal distribution. a correlation procedure, mixed procedure on a linear model and a logistic procedure were performed (p-value < . ). sixty seven dogs (i = , n = , t = , gdv= , gi= , r = , m = ) were included. forty-three patients survived (seven died, seventeen were euthanized). twenty patients had a control visit. crp was elevated in . % of dogs at presentation, and only remained within reference range ( - . mg/l) throughout hospitalization in four dogs ( . %). crp concentrations were significantly higher from t ( . ae . mg/l) to t ( . ae . mg/l) decreasing at t ( . ae . mg/l), and returning within reference range at t m ( . ae . mg/l) in all but one dog ( . mg/l). crp was significantly correlated with logarithmical concentrations of il- and tnf-a, however, these did not change significantly over time. none of the evaluated parameters was associated with disease category, nor outcome. crp appears useful to diagnose sirs in emergency patients, and tends to decrease during hospitalization. however, crp, neither il- nor tnf-a concentrations appear useful to predict the underlying disease and outcome in sirs patients. no conflicts of interest reported. calprotectin (s a /a complex) belongs to the s /calgranulin family, and is primarily released from activated neutrophils and macrophages. serum calprotectin concentrations (cp) were shown to be increased in dogs with inflammatory diseases such as inflammatory bowel disease, pancreatitis, systemic inflammatory response syndrome, and sepsis. canine cp thus appears to be a biomarker of inflammation. considerable day-today variation of fecal canine cp was found in both healthy dogs and dogs with chronic gastrointestinal disease. however, the biological variation of canine cp in serum has not been reported. the aim of this study was to determine the biological variation of serum canine cp and its minimum critical difference (mcd). eleven healthy dogs were used for this study. biological variation of serum canine cp was evaluated over a . -months period. tests for outliers were carried out at levels (within-run analytical variance, intra-, and inter-individual variation). a nested analysis of variance (anova) model was used to calculate analytical (cv a ), intra-individual (cv i ), inter-individual (cv g ), and total variation (cv t ), and to determine the index of individuality (ii), index of heterogeneity (ih), and mcd. a total of serial specimens were collected from dogs, serial samples from dogs, and serial samples from dogs. four within-subject outliers were detected and excluded from further analysis, yielding a total of serum samples and slightly right-skewed data. no outlying observations (cochrane test) or outliers among mean concentrations of subjects (reed's criterion) were detected. cv a was calculated as . %, cv i as . %, and cv g as . %, resulting in a cv t of . %. index of individuality (ii) was determined to be . and ih was . , yielding a one-sided mcd of . mg/l. the analytical goal of cv a ≤ ½ cv i was satisfied. although serum canine cp remained within a relatively narrow concentration range in healthy dogs, moderate individuality was detected. moderate changes in serum canine cp ( . mg/l) between sequential measurements are needed to be considered clinically relevant, and using a population-based reference interval may or may not be appropriate for serum canine cp. using the mcd with the previously determined median canine cp concentration ( . mg/l) for the reference sample group yielded a serum canine cp concentration close to the upper limit of the previously established reference interval ( . mg/l), showing that the reference interval for serum ccp ( . - . mg/l) is within reasonable limits. the assay used in the study was developed at the gi laboratory, texas a&m university. most authors also work at the gi laboratory, texa a&m university. canine leishmaniasis (canl) is a multisystemic disease that is endemic in the mediterranean region. in the past, concentrations of acute phase proteins (apps), and specifically c-reactive protein (crp), haptoglobin (hp), ceruloplasmin (cp), serum amyloid a (saa) and albumin (alb), have been reported to change in dogs with leishmaniasis, and revert to normal after successful treatment, highlighting the intrinsic inflammatory reaction of the host to the parasite. since the spectrum of clinical and laboratory derangements is broad, it is possible that apps are increased specifically because of certain clinicopathological syndromes associated with canl. a total of dogs with canl, diagnosed on the basis of cytological amastigote identification and ifat serology, were retrospectively included in the study. in all of them, crp, saa, hp and alb were measured at interlab-umu, murcia, spain, in aliquots of serum, which were stored in - °c for - years (median: years). results for each of the apps were correlated to laboratory and clinical parameters (n: ), clinical and parasitological scoring (n: ), ehrlichia and leishmania serology (n: ), and clinical staging according to leishvet (n: ), using an array of linear and ordinal regression models, as well as one-way anova, t-test and fisher's lsd test. crp and alb were by far the apps most frequently correlated with clinical and laboratory abnormalities such as nutritional status, lethargy and skin ulcers (p < , ), as well as urinary protein to creatinine ratio (upc), total serum protein, and urine specific gravity (p < , ). there were limited associations between hp, cp, saa and clinicopathological parameters. a minor linear relationship was observed between crp and clinical scoring. crp and alb were also correlated with parasitological scoring in bone marrow, but not lymph node cytology (p < . ). dogs with ehrlichia titers had higher crp, cp and lower alb concentrations. finally, crp concentrations were higher in later compared to earlier stages of the infection, as defined by the leishvet criteria. the inflammatory component to leishmania infection doesn't seem to be exemplified by the reaction of a particular tissue, with the possible exception of glomerulonephritis. the magnitude of increase in crp and decrease in albumin is correlated with clinical staging and bone marrow parasitological scoring. no conflicts of interest reported. the consequences of abnormal platelet function in dogs and cats can be devastating and the use of anti-thrombotic therapy to prevent thrombotic events is increasingly common. the ability to measure platelet function and the efficacy of anti-thrombotic therapy is difficult due to limited availability of equipment and inability to delay platelet function analysis. the aim of this study was to adapt and validate test procedures and protocols previously developed for humans for use in dogs and cats. residual samples of citrate anticoagulated blood were used from dogs and cats presented to a specialist referral centre for various reasons unrelated to clotting abnormalities. initially the blood was stimulated using specific combinations of either arachidonic acid/epinephrine (aa/epi) or adp/u , designed to assess the effects of the anti-thrombotic agents aspirin and clopidogrel respectively. after minutes stimulation, the blood samples were fixed using a patented platelet fixative solution developed for human platelets, which allows the delayed analysis of p-selectin an established marker of platelet activation. all analysis was performed by flow cytometry. in order to do this, specific antibodies were selected for the recognition of both canine and feline platelets. cd was used as a platelet identifier antibody while appropriate cd p (p-selectin) antibodies for each species were chosen. fixed samples were repeatedly analysed at time points between to days following fixation to establish the stability of the fixed samples. thirteen dogs and three cats were analysed. high p-selectin expression was detected following stimulation with aa/epi and adp/u in both dogs and cats following fixation. this was significantly different to unstimulated blood (p < . ). there was no significant difference in detectable pselectin expression following storage of the fixed samples at any time-point up to days. this confirmed the fixative was suitable as a preservative of canine and feline platelets. a limited number of dogs were evaluated whilst receiving antithrombotic medication. there was a significant difference in the activation of platelets in the dogs treated with either aspirin (p < . ) or clopidogrel (p < . ) compared with untreated dogs following stimulation with aa/epi (aspirin group) or adp/ u (clopidogrel group). our results show that fixation and delayed analysis of platelet function in dogs and cats is possible for up to days. this demonstrates an exciting opportunity to analyse platelet function remotely and to determine the efficacy of thromboprophylaxis in animals presenting to clinics that do not have on-site platelet analysers. no conflicts of interest reported. several authors consider thyroid hormone supplementation as a valid initial treatment option for dogs with aggression related problems. indeed, mood and behaviour modulating properties of thyroid hormones may, in part, be mediated through the interaction of thyroid hormones with neurohormones such as serotonin and prolactin. at present, prospective trials evaluating neurohormonal status or behaviour in hypothyroid dogs before and after thyroid supplementation are lacking. therefore, the aims of this study were to assess behaviour and measure serum serotonin and prolactin concentrations in dogs with spontaneous hypothyroidism before and after treatment.twenty three client-owned dogs diagnosed with spontaneous primary hypothyroidism were prospectively included in our study. after diagnosis all dogs were treated with levothyroxine ( micrograms/kg bid). behaviour of dogs was screened at initial presentation, at weeks and months after initiation of therapy. owners had to fill in a hard copy of the standardized canine behavioural assessment and research questionnaire (c-barq) consisting of scored questions evaluating seven behavioural categories. the average score on all questions was calculated for each dog at each of the three time periods and a paired t-test was used for comparison. serum serotonin and prolactin concentrations were evaluated at each time period using a commercially validated elisa kit and heterologous ria, respectively.results of the c-barq after six weeks of thyroid hormone supplementation when compared with the time zero demonstrated a significant increase (p < . ) in excitability, activity and aggression, which most likely became unmasked owing to improved overall activity of dogs. conversely, at six months period when compared with the time zero no significant changes in any of the behavioural symptoms were observed. serum serotonin was measured in / dogs colorimetrically at nm. at time zero, weeks and months serum serotonine was . (range, . - . ), . (range, , - . ) and . (range, . - . ). no significant difference was noted between week and month period comparing to time zero (p = . and p = . ). serum prolactin concentration measured in / dogs at time zero, weeks and months was . ng/ml (range, . - . ), . ng/ml (range, . - . ) and . ng/ml (range, . - . ) and did not differ significantly in either time period when compared with time zero (p = . and p = . ).altogether, results of this study failed to demonstrate a significant role of thyroid supplementation on the majority of evaluated behavioural symptoms as well as neurohormonal status of hypothyroid dogs during months of therapy. no conflicts of interest reported. iatrogenic hypothyroidism is a recognized complication of radioiodine treatment of hyperthyroidism in cats, but no prospective studies of the prevalence, clinical features, routine laboratory findings, or results of thyroid function tests have been reported in a series of hypothyroid cats. in this study, we describe the features of hypothyroidism in cats treated with radioiodine over a -month period (october -march ). during this same period, we treated % hyperthyroid cats with radioiodine, providing a prevalence rate of %. hypothyroidism was diagnosed - days (median, days) after i treatment, with doses ranging from - mbq (median, mbq; median pretreatment t , nmol/l). the hypothyroid cats ranged in age from - years (median, years). all were dsh/dlh; ( %) were female and were males (p = . ). clinical signs in these cats included overweight/obesity in ( %), lethargy/dullness in ( %), poor appetite in ( . %), and polyuria/polydipsia in ( %). abnormalities on physical examination included dermatologic signs (dry coat, seborrhea, matting) in ( %) and bradycardia (< bpm) in . twenty-two cats ( %) had no noticeable clinical features of hypothyroidism. routine laboratory abnormalities included hypercholesterolemia (> mmol/l) in ( %) and new or worsening azotemia (> lmol/l) in ( %) and ( %) cats, respectively. median serum concentrations of total t ( . nmol/l; reference interval [ri], - nmol/l), t ( . nmol/l; ri, . - . nmol/l), and ft ( pmol/l; ri, - pmol/l) were all in the low end of the ri. normal ri values for t and ft were maintained in ( %) and ( %) of the cats, respectively. serum ctsh values were high in all cats (median, . ng/dl; range, . - . ng/dl; ri, . - . ng/ml). thyroid scintigraphy showed less-than-normal amounts of residual tissue, as well as low values for thyroid-to-salivary ratio and %-uptake of pertechnetate, in ( %). of those cats with normal scintiscans, serum ctsh decreased into the ri without treatment when retested - months later. in conclusion, this study confirms that i-induced hypothyroidism is not uncommon, with an apparent female sex predilection. serum t and ft remain normal in most cats, but high serum ctsh values and thyroid scintigraphy aid in diagnosis. unless cats have overt, long-standing hypothyroidism, most cats with subclinical disease are relatively asymptomatic, other than worsening azotemia. subclinical hypothyroidism will be transient in some cats, with normalization of ctsh values within a few months. no conflicts of interest reported. iatrogenic hypothyroidism is a recognized complication of radioiodine treatment for hyperthyroidism in cats. at our clinic where we use a variable -i dosing protocol (based on tumor volume and severity of hyperthyroidism), the prevalence of overt or subclinical hypothyroidism is at least %. during the -month period from october to march , we treated cats with iatrogenic hypothyroidism, which had developed - days (median, days) after treatment with radioiodine (median dose, mbq). these cats ranged in age from - years (median, years); all were dsh/dlh; ( %) were female and were males. new or worsening azotemia (> lmol/l) was documented in ( %) and ( . %) cats, respectively. diagnosis of hypothyroidism was based on the following: ) low to low-normal serum concentrations of t , ft , and t ; ) high serum tsh concentration (> . ng/dl); and ) less-than-normal amounts of residual tissue on thyroid scintigraphy. all cats were given thyroid hormone replacement as a liquid l-t preparation (leventa; merck animal health). cats were monitored at - month intervals by repeating serum t and tsh concentrations - hours after the morning l-t dose. ten of the cats were started on a once-daily l-t regimen ( lg); of these, only ( %) had suppression of high serum tsh values into the reference interval (ri). of the cats that had persistently high tsh values, were switched to twice-daily administration ( - lg, bid), which successfully lowered high tsh concentrations in cats. the remaining cats were started on twice daily l-t ( lg, bid); of these, normalization of tsh occurred in cats. overall, l-t treatment was successful in normalizing tsh concentrations in ( %) cats, with once-daily and with twice-daily dosing. peak serum t concentrations of ≥ nmol/l were needed in most cats to normalize tsh values. higher serum t and lower tsh concentrations were achieved when l-t was administered on an empty stomach rather than given with food. a significant decrease (p < . ) in serum creatinine occurred after treatment with l-t . in conclusion, our results indicate that twice-daily administration of l-t is needed in most cats with iatrogenic hypothyroidism to normalize high serum tsh concentrations. many cats appear to absorb l-t rather poorly, which can be enhanced by giving the drug on an empty stomach. the azotemia that commonly develops in cats with hypothyroidism improved or stabilized with adequate l-t supplementation. no conflicts of interest reported. congenital hypothyroidism (ch) has been reported in many species; the hereditary forms can be divided into thyroid dysmorphogenesis and dyshormonogenesis. while thyroid hypoplasia has been described in dogs and cats, the molecular basis remains unknown. in contrast few breeds of dogs with goiterous ch were found to have deficient thyroid peroxidase (tpo) activity. the purpose of our study was to characterize a family of domestic shorthair cats with goiterous ch and disease-causing tpo gene mutations. clinical features included dwarfism and dullness, known as cretinism and seen with ch in all species, but also constipation and megacolon which are unique to cats with ch. pedigree analysis documented an autosomal recessive mode of inheritance. affected kittens developed a goiter and had low serum thyroxine (t ) and triiodothyronine (t ) when compared to controls, but high thyroid stimulating (tsh) hormone levels indicating thyroid dyshormonogenesis. oral thyroid supplementation corrected the progression of clinical signs and prevented further constipation and reversed the megacolon. the tpo enzyme activity was extremely low in hypothyroid cats when compared to that of normal cats. genomic dna and cdna from affected, carrier, and normal cats were extracted and sequenced based upon primers developed from the feline genome database. a homozygous missense point mutation (c. g>a) in tpo, which results in an amino acid change (p.ala thr), was discovered in affected cats and the mutant allele segregated within the family with goiterous ch. this is the first report of a tpo deficiency in cats. other unrelated domestic shorthair cats with goiterous ch did not have this same tpo mutation. the prevalence of this tpo mutation in the domestic cat population seems low, but ch is likely underreported in cats. supported in part by nih od . some of the authors are members of diagnostic laboratories (penngen). supported in part by the nih od # . glucagon-like peptide- (glp- )is a gastrointestinal hormone released in response to food intake that increases insulin secretion, inhibits glucagon secretion, slows gastric emptying and induces satiation. it is also assumed to stimulate beta-cell proliferation. glp- agonists are successfully used in humans with type diabetes mellitus usually either in combination with insulin or other anti-diabetic drugs. in healthy cats twice daily (exenatide) as well as once weekly (exenatide extended-release (er)) application of glp- agonists induced pronounced insulin secretion. benefits of exenatide er are the regimen of once weekly injection and less side effects. the objective of the study was to assess whether administration of exenatide er in addition to standard treatment leads to improved glycemic control and higher remission rates in cats with newly diagnosed diabetes. the study was designed as a prospective, placebo-controlled clinical trial. cats were randomly assigned to two groups receiving exenatide er (group : bydureon â , mg/kg, q d, sc) or . % saline solution (group : q d, sc). both groups additionally received insulin glargine (lantus â , initial dose: ≤ kg: . iu, q h; > kg . - . iu, q h) and diet (purina dm â ). exenatide er was applied over weeks or, in case of remission, for additional weeks after cessation of insulin application. cats were rechecked , , , and weeks after starting therapy. remission of diabetes was defined as absence of clinical signs of diabetes and normal blood glucose and fructosamine concentrations for at least weeks after discontinuing insulin injections. so far cats have completed the study. mild and transient side effects in group (n = ) were reduced appetite (n = ), nausea (n = ), vomitus (n = ), tiredness (n = ) and hiding in dark spots of the house (n = ). in group remission was achieved in / ( %) cats and good metabolic control in / ( %) nonremission cats. in group remission was achieved in / ( %) cats and good metabolic control in / ( %) non-remission cats. median insulin dose given during the study period was . iu/ kg/day in group and . iu/kg/day in group . the preliminary results suggest that exenatide er can be used safely in diabetic cats. a tendency for higher remission rate, better metabolic control and lower insulin requirement was seen when exenatide er was added to the standard treatment regimen. further cases need to be evaluated to verify the potential beneficial role of exenatide er. no conflicts of interest reported. feline diabetes mellitus shares many similarities with human type diabetes mellitus (t dm), including clinical, physiological and pathological features of the disease. domestic cats spontaneously develop diabetes associated with insulin resistance in their middle age or later, with residual but declining insulin secretion. humans and cats share the same environment and risk factors for diabetes, such as obesity and physical inactivity. moreover, amyloid formation and loss of beta cells are found in the diabetic cat pancreas, as in humans. subsequently, studying the molecular mechanisms in the failing beta cells may contribute to a better understanding of the pathophysiology of t dm in both cats and humans. the aim of the present study was to develop a method to study mrna expression of islet-specific genes in healthy and diabetic cats. previous attempts in isolating feline islets with different collagenase-based protocols have led to damaged islets or islets coated with exocrine acinar cells, which either way compromise the results obtained from gene expression studies. by using the laser microdissection technique, we were able to sample islets that were not contaminated with exocrine tissue, from both healthy and diabetic cats. high rna quality was confirmed with gel electrophoresis. by quantitative real-time pcr (qrt-pcr), mrna levels of the islet-specific genes insulin, pdx- , iapp, chga and ia- were detected in both healthy and diabetic cats. we used actin b, gapdh and rps as internal reference genes for normalizations of our qrt-pcr data. the laser microdissection technique allows studies of islets without contamination of acinar cells, as shown in this study, and is of great advantage since it is difficult to get pure feline islets from collagenase-based isolation. differences in gene expression in healthy and diabetic cats may reveal underlying mechanisms for beta cell dysfunction and decreased beta cell mass in human and feline type diabetes. conflicts of interest: the study was financially supported by the swedish juvenile diabetes foundation, the fredrik and ingrid thuring foundation, the magnus bergvall foundation, the lars hierta memorial foundation, and the foundation for research, agria insurance company. feline acromegaly is an increasingly recognised endocrinopathy among diabetic cats, caused by chronic excessive growth hormone secretion by a functional somatotrophinoma in the pars distalis of the anterior pituitary gland. the majority of human somatotrophinomas are sporadic, however up to % of familial isolated pituitary adenomas are caused by germline mutations of the aryl-hydrocarbon-receptor interacting protein (aip). feline acromegaly has phenotypic and biochemical similarities to human familial acromegaly with aip mutations, such as male predominance, somatotroph macroadenoma and resistance to octreotide therapy. the objective of this study was to identify the feline aip gene, identify single nucleotide polymorphisms (snps) within this gene and compare any snps with reported human aip snps. stored pituitary tissue from an acromegalic cat was used to create feline aip cdna using feline specific aip primers. stored edta blood from acromegalic cats (diagnosis of insulin resistant diabetes mellitus, serum igf- > ng/ml and pituitary mass > mm identified using pituitary computed tomography or necropsy) and control cats (no history of diabetes mellitus and greater than years of age) were selected, dna extracted and genotyped using pcr, agarose gel electrophoresis and sanger sequencing. the feline aip gene was identified, encoding a amino acid protein with % homology to the human aip protein. a blast search revealed this gene contained exons and exon specific primers were created to enable sequencing. a single nonconservative snp was identified in exon (aip:c. g>t), encoding for an amino acid change from aspartic acid to glutamic acid in / acromegalic patients and / control cats. two additional conservative snps were also identified (aip:c. t>c and aip:c. t>c). exon encodes for a region of the aip protein considered essential for aip-aip receptor interaction. although different human aip mutations have been identified to date, a human aip:c g>t mutation has not yet been identified. the aip n-terminal is required for the stability of the aip protein-aip-receptor complex, and essential for the regulation of translocation into the nucleus, where it binds to aryl hydrocarbon receptor nuclear translocator leading to activation of genes thought to act as tumor suppressors. loss of normal aip activity is thought to promote somatotrophinoma development. it is therefore possible that the detected aip:c. g>t mutation predisposed to somatotrophinoma tumorigenesis in the two affected patients, and a study containing a larger number of cases is indicated. no conflicts of interest reported. hypersomatotrophism (hs) is an important cause of feline diabetes mellitus (dm). in humans surgical removal of the somatotrophinoma is generally recommended, though hypophysectomy programs have suffered from significant initial morbidity and mortality given a documented steep learning curve in newly established programs. hypophysectomy as treatment for feline hs has thus far only been described in a handful of cases, all having been treated by one single experienced hypophysectomy team. this study's aim was to evaluate the learning curve of a de novo established hypophysectomy program, through analysis of peri-and post-operative morbidity and mortality, and endocrine outcomes in the first cohort of cats with hs treated. from owners of diabetic cats with confirmed hs (igf- > ng/ml, pituitary mass) presented at the royal veterinary college were offered hypophysectomy. all cats undergoing surgery were operated by one neurosurgeon with previously only cadaveric experience of the procedure, through an adapted transsphenoidal approach referencing bony landmarks to computed tomographic scans reconstructed on neuronavigation software. the somatotrophinoma was extirpated using fine surgical tools. all cats received intense electrolyte and blood pressure monitoring, peri-and post-operative ddavp and intravenous insulin and hydrocortisone infusion, transitioning to subcutaneous glargine, conjunctival ddavp, oral hydrocortisone and levothyroxine. between april -february , cats underwent hypophysectomy (median + range age: . years, . - . ; igf- : ng/ml, -> ; pituitary height . mm, . - . ). all displayed uncontrolled dm due to hs (median fructosamine: umol/l); none displayed overt central neurological deficits. two cats ( %, cats and ; pituitary height (mm): . and . ) required mechanical ventilation post-operatively and both were euthanized. post-mortem magnetic resonance imaging revealed brain herniation and cerebral ischaemia was suspected. one cat suffered cardiac arrest post-operatively at time of jugular catheter placement, though made an uneventful recovery. four other cats developed congestive heart failure within days, which was successfully treated not necessitating ongoing therapy. temporarily diminished tear production was seen in cats. seven of the ten surviving cats went into diabetic remission within a median of . days ( - ); others saw reduction of insulin needs by %. serum igf- normalised rapidly and significantly in all but one cat (median serum igf- ng/ml within days). persistent neurological deficits or palatal wound breakdown were not encountered. starting a hypophysectomy program to treat feline hs was associated with some risk of mortality, though surviving cases benefited from the procedure with a high incidence of diabetic remission. no conflicts of interest reported. pituitary dependent hypercortisolism (pdh) in dogs is frequently associated with high serum phosphate and parathormone concentrations. the pathogenesis of such abnormalities remains unknown and the evaluation of the urinary fractional excretion of phosphate and calcium in pdh dogs might be helpful in enhancing the knowledge regarding this issue. the aim of the present study was to evaluate the serum and urinary concentrations and the urinary fractional excretion of phosphate and calcium in dogs with pdh. medical records from one referral center were retrospectively evaluated between and . the diagnosis of pdh was confirmed using the cortisol to creatinine ratio, the ldds test and/or acth stimulation test, the plasma acth concentration, ultrasonography of the adrenal glands and computer tomography (ct) of the pituitary and the adrenal glands in dogs with consistent clinical signs. only newly diagnosed dogs, before treatment for pdh, were evaluated. two control groups were included: one healthy and one sick control dog (without pdh) for each dog with pdh were included. healthy control dogs (hcd) and sick control dogs (scd) were matched for age (ae months), breed, sex and sexual status. data were analysed using non-parametric tests and expressed as median and ranges. significance was set at p < . . one-hundredsixty-seven dogs with pdh were eligible for inclusion in the study. the median age at diagnosis was years (range: - ) and the median body weight was . kg (range: . - . ). there were female ( spayed) and male ( castrated). serum phosphate concentration ( . mg/dl, . - . ) was significantly (p < . ) higher compared to hcd ( . mg/dl, . - . ) and scd ( . mg/dl, . - . ). serum calcium concentration ( . mg/dl, . - . ) was significantly higher compared to scd ( . mg/dl, . - . ) but not different compared to hcd ( . mg/dl, . - . ). urinary fractional excretion of phosphate ( . %, . - . ) was significantly lower compared to hcd ( . %, . - . ) and scd ( . %, - . ). urinary fractional excretion of calcium ( . %, - . ) was significantly higher compared to hcd ( . %, . - . ) and scd ( . %, - . ). urinary calcium to creatinine ratio ( . , - . ) was significantly higher compared to hcd ( . . - . ) and scd ( . , - . ), while urinary phosphate to creatinine ratio were not significantly different in pdh dogs, hcd and scd. in conclusion pdh dogs have lower phosphaturia and higher calciuria compared to control dogs. this findings suggest that, at least in part, the high serum phosphate concentrations are related to the renal retention of phosphate. no conflicts of interest reported. four cortisol-based methods of monitoring trilostane treatment of canine hyperadrenocorticism were compared to the results of a clinical scoring scheme based on an owner questionnaire. cases of canine hyperadrenocorticism that had received a consistent dose of trilostane for more than one month were recruited from first opinion and referral practice. each dog was used only once. owners were asked to complete a questionnaire that assessed clinical control. the dogs were then categorised as being over-controlled, well-controlled, moderately-controlled and poorly-controlled. cortisol was measured in serum samples taken pre-trilostane (peak), hours post-trilostane (trough) and hour post-acth injection. dogs that had an increase in cortisol after trilostane administration were excluded. a scoring system was developed for each of these measurements. a fourth scoring system was developed using a novel algorithm that combined the peak and trough cortisol (peak-trough). the results of each of the scoring systems categorised the dogs into those that would be expected to be over-controlled, well-controlled, moderately-controlled and poorly-controlled. weighted kappa was calculated to assess the agreement between the categorisation according to each of the methods compared to the categorisation using the owners score. the pearson correlation coefficient was calculated to assess relationships between the various parameters. in total tests were analysed. when compared to the results of the owner's questionnaire , , and dogs were correctly categorised using the peak-trough, peak alone, post-acth and trough alone respectively. amongst the miscategorised results , , and dogs were incorrect by category and , , and dogs by categories using the peak-trough, peak alone, post-acth and trough alone respectively. all methods correctly recognised the over-controlled dog that had been identified by the owner's score. the weighted kappas for post-acth and trough cortisol categories compared to the owner score categories were . and . respectively (defined as slight agreement). in contrast the weighted kappas for the peak and peak-trough categories were . and . respectively (defined as fair agreement). there were no significant correlations between the absolute clinical scores and cortisol concentrations. there were significant correlations between the cortisol measurements. the novel methods of peak-trough and peak cortisol better reflected the level of clinical control of hyperadrenocorticism identified by the owners' questionnaire than either post-acth stimulation or trough cortisol. peak-trough and peak cortisol concentrations should be further investigated as monitoring methods for trilostane. financial support from dechra pharmaceuticals. the prognosis of canine adrenocortical insufficiency is generally regarded to be excellent. however, there is paucity of sur-vival analyses in the literature. the aim of the present study was to evaluate the survival of dogs with the diagnosis adrenocortical insufficiency based on data from a cohort of , swedish client-owned dogs insured in one insurance company (agria pet insurance, stockholm, sweden) during the time period - . dogs were identified by search for insurance claims with the register code for adrenocortical insufficiency. dogs were excluded from analysis if they had a previous history of hypercortisolism, and if they were born before begin of the study period. kaplan-meier survival analysis was performed. dogs were regarded as censored when the registered cause of death was other than adrenocortical insufficiency or hypercortisolism that was registered after the first claim for adrenocortical insufficiency. data from dogs was included. one hundred twenty-four dogs were registered to be dead. in dogs the cause of death was related to the adrenocortical insufficiency. the -year estimated survival-rate was % ( % ci, - %). the -year estimated survival-rate was % ( % ci, - %). the -year estimated survival-rate was % ( % ci, - %). twelve dogs ( . %) were still alive after years. in conclusion, the long-term survival of dogs with adrenocortical insufficiency was reasonably good. however, the diseases-related mortality was higher than expected, and occurred mainly during the first years after diagnosis. conflicts of interest: this study was supported by grants from the swedish research council and the foundation for research, agria insurance company. the concomitant occurrence of two or more endocrine tumors and/or hyperplasias, known as multiple endocrine neoplasia (men) is a well-known entity in humans. multiple gene mutations have been identified. the two major forms are men and men . in men , the main affected organs are parathyroid, pancreas and pituitary gland. men occurs in clinical variants: men a, characterized by medullary thyroid carcinoma (mtc), pheochromocytoma and primary hyperparathyroidism; men b, characterized by mtc, pheochromocytoma and additionally abnormalities; familial medullary thyroid carcinoma. in dogs and cats only a few cases have been reported and it is unknown whether hereditary men-like syndromes exist in these species. the aim of this study was to evaluate the prevalence of multiple endocrine tumors in dogs and cats at our institution, to identify possible breed and sex predispositions and to investigate similarities with the human men syndromes. autopsy reports of dogs and cats from until were reviewed. animals with at least two endocrine tumors/hyperplasias (eth) were included. autopsy reports of dogs and cats were examined. dogs had eth affecting a single organ, had multiple eth; cats had single eth, had multiple eth. in dogs with multiple eth, the most common breeds were west highland white terrier (whwt, / ), poodle, golden retriever, mixed-breed dogs (each / ). / were male ( intact); / were female ( neutered). median age was years (range - ). the most common combination was multiple testicle tumors of various types ( / ). the most common affected organs were the adrenals ( / ). adrenal cortical adenomas/carcinomas/hyperplasias were mainly associated with pheochromocytomas ( / ), testicle tumors ( / ) and insulinomas ( / ). all whwts had adrenal adenomas. both poodles had pheochromocytoma associated with pituitary adenoma or adrenal hyperplasia. dogs showed tumor combinations similar to the human men syndrome: pituitary adenoma and insulinoma; pituitary adenoma and parathyroid hyperplasia. / cats were domestic short/long hair, / were persians. / were male ( castrated); / were female ( neutered). the median age was . years (range - ). the most common affected organs were thyroid glands ( / ), combined mostly with lesions of parathyroid ( / ) and adrenal glands ( / ). none of the cats had combinations similar to the human men syndromes. the prevalence of multiple eth in dogs and cats was . % and . %. men-like syndromes were extremely rare in dogs and non-existing in cats. no sex predisposition was observed. possible breed predispositions need further investigations. no conflicts of interest reported. canine angiostrongylosis is an increasingly reported disease worldwilde, including many european countries, possibly due to climatic factors, presence of foxes (acting as reservoir) or more simply, to the availability of more accurate diagnostic methods. although detection of the first-stage larvae (l ) using the baermann technique on faecal samples (preferably collected over three consecutive days) remains the gold standard, recently developed serological and molecular tests (quantitative polymerase chain reaction, qpcr) are now available. until now, the prevalence of canine angiostrongylosis among healthy and coughing dogs in belgium was unknown. the aims of the present study were ( ) to describe a clinical series of recent autochtonous cases and ( ) to retrospectively assess angiostrongylus vasorum qpcr in bronchoalveolar lavage fluid (balf) samples, collected over the last years from a larger series of dogs, healthy or with other respiratory conditions, in order to investigate the past prevalence of the disease in belgium. seven dogs, living in southern or eastern belgium, were recently diagnosed as having angiostrongylosis (mean age= . y, mean body weight= . kg). they all presented with respiratory signs of variable severity. in dogs, balf was obtained and qpcr was positive in all of them, at moderate or high level (ct from , to , ) while larvae were detected in the faeces of only animals. in the remaining two dogs, no balf was obtained, but coproscopy was positive. all dogs responded to medical treatment, consisting in a -week course of fenbendazole and/or two spot-on application of moxidectin at -month interval. balf samples were collected between and from asymptomatic client-owned dogs and dogs with various respiratory conditions, including dogs with confirmed bordetellosis, dogs with eosinophilic bronchopneumopathy (ebp), dogs with chronic bronchitis and dogs with bacterial bronchopneumonia, and were retrospectively assessed with a a. vasorum qpcr assay. amongst those dogs, only one balf, from a dog with ebp, yielded a positive qpcr result. in this dog, faecal analysis was negative. the present data show that, based on balf qpcr and coproscopy, presence of angiostrongylosis in healthy and coughing dogs was negligible in belgium until the last months. it is now considered as an emerging condition and must be included in the differential diagnosis in coughing dogs. the present results also support that qpcr detection of a. vasorum in balf, when available, is an adequate and reliable detection technique. no conflicts of interest reported. ligneous membranitis is a rare chronic inflammatory disease associated with congenital plasminogen deficiency. it has only been described in six unrelated dogs. the objective of this study is to report the presentation, clinicopathological and post mortem findings in three related scottish terrier puppies with ligneous membranitis. ligneous membranitis is well described in humans, where it is inherited in an autosomal recessive manner. patients commonly present as infants. ocular, oral and genital lesions are most common, but other organs are occasionally involved and congenital obstructive hydrocephalus is reported in some individuals. numerous mutations and polymorphisms in the plasminogen gene have been identified in affected individuals. the affected scottish terriers (two male and one female) presented at months of age with severe proliferative and ulcerative conjunctivitis and gingivitis/stomatitis; biopsy confirmed ligneous membranitis. other clinical signs included increased upper respiratory tract noise, nasal discharge and lymphadenopathy. one male was cryptorchid. clinical pathological findings included neutrophilia, proteinuria and hypoalbuinaemia. serum plasminogen activity was measured in two dogs, and was low in one. the dam and sire of the affected dogs had normal serum plasminogen activity and no history or clinical signs consistent with ligneous membranitis. no significant clinical improvement was evident following treatment with antibiotics, glucocorticoids, topical ciclosporin or heparin. one dog died of cardiopulmonary arrest in the hospital and the two other dogs were euthanized due to progressive clinical signs. post-mortem evaluation of the affected dogs revealed multiple abnormalities including severe proliferative fibrinous lesions affecting the trachea, larynx and epicardium, and multiple fibrous adhesions throughout the thoracic and abdominal cavities. the male dog had internal hydrocephalus and lacked a cerebellar vermis. this is the first report of ligneous membranitis in related dogs and the first report in scottish terriers. sequencing the plasminogen gene in the affected dogs, their parents and unrelated control dogs to identify polymorphisms or mutations that may be associated with ligneous membranitis in dogs is ongoing. the author received a travel scholarship from zoetis to attend this congress. health screening of elderly dogs is often recommended, but scientific information on clinical and laboratory abnormalities in senior and geriatric dogs is scarce. this study was undertaken to describe blood pressure measurement, physical examination (pe) abnormalities and routine laboratory test results in senior and geriatric dogs that were apparently healthy for the owner. because life expectancy in dogs is related to body size, the inclusion of dogs was based on a human/pet analogy chart to determine whether a dog was senior (n = ) or geriatric (n = ). to verify health status, owners were asked to complete an extensive questionnaire. systolic blood pressure (sbp) was measured using the doppler technique according to the acvim guidelines. subsequently a thorough pe was performed, including body and muscle condition scoring, orthopedic examination, neurologic evaluation, indirect fundoscopy and bilateral schirmer tear test. complete blood count, serum biochemistry and urinalysis (including urinary sediment, urinary protein:creatinine ratio (upc) and bacterial culture) were evaluated. in of dogs sbp exceeded mmhg, none of the dogs had fundoscopic lesions secondary to hypertension. body condition score was abnormal in animals, were overweight or obese. physical examination revealed a heart murmur in , submandibular lymphadenopathy in , moderate to severe dental plaque in and one or more (sub)cutaneous masses in dogs. twenty-three dogs were leukopenic, had a decreased phosphorus, an increased serum creatinine and one dog a decreased total thyroxine (with concurrent increased thyroid stimulating hormone). crystalluria was commonly detected ( / ) and mostly due to low numbers (< /high power field) of amorphous crystals ( %). struvite crystals were present in % of the crystalluric dogs. overt and borderline proteinuria were detected in and of dogs, respectively. four dogs had a positive urinary culture. sbp was not significantly different between the senior and geriatric group. there was no significant effect of obesity or gender on sbp. the platelet count (p = . ), total thyroxine concentration (p = . ) and the frequency of orthopedic problems (p = . ) and cutaneous masses (p = . ) were significantly higher in the geriatric compared to the senior dogs. hematocrit (p = . ) and body temperature (p = . ) were significantly lower in the geriatric group. these findings indicate that physical and laboratory abnormalities are common in apparently healthy senior and geriatric dogs. this underlines the necessity for regular health screening in elderly dogs and the urgent need for reliable and maybe age specific reference intervals in veterinary medicine. the cost of examinations reported in this study were covered by hill's pet nutrition belgium. systemic lupus erythematosus, sle, is a chronic autoimmune disorder with varying clinical manifestations and diagnosis is based on both clinical signs and laboratory findings. other systemic rheumatic diseases, referred to as sle-related diseases or immune-mediated rheumatic disease (imrd), are also described. the most common clinical signs in dogs are stiffness and pain from varying joints. one hallmark of sle and sle-related diseases in both dogs and humans is high titres of circulating antinuclear antibodies (ana), which can be demonstrated by the indirect immunofluorescence (iif) ana test. earlier studies have shown that canine iif ana positive samples may be divided into two main subgroups: homogenous (ana h ) and speckled (ana s ) iif ana fluorescence pattern. in humans, further determination of the specificity of ana positive sera is frequently employed to characterize the ana reactivity. some of these ana specificities have been demonstrated in man to strongly associate with different systemic autoimmune diseases and also with different iif ana staining patterns. presence and character of antinuclear antibodies in canine sle-related diseases are not well described. the aim of this work was to further characterize the ana specificity in dogs with sle-related disease/imrd. sera from anapositive dogs, including different breeds, were analyzed with elisa and line blot techniques (elisa and euroline ana profile, euroimmun, germany). the five most prevalent breeds were german shepherd dog, nova scotia duck tolling retriever, cocker spaniel, crossbreed and golden retriever. sera displayed a homogenous and a speckled iif ana fluorescence pattern. several specific ana-reactivities earlier characterized in human patients were identified. the majority of ana h , n = , %, showed reactivity against nucleosomal antigens and ( %) against dsdna when conducted on line blot. these sera also reacted against nucleosomes and dsdna on the elisa. there were some additional positive with the elisa, so in total the elisa identified % with nucleosomal and % with dsdna reactivity. in few cases, other reactivities identified were against histones, pcna, jo- and rnp. in the ana s subgroup, the sm+rnp antigen evoked the most frequent reactivity, n = , % with both line blot and elisa. in few cases, reactivity against dsdna, pcna, jo- , pmscl kd, scl- , ssa and ssb were identified. in several dogs no specific antigen was identified. further studies are in progress in order to in more detail characterize and identify subtypes of already known and unknown antigens with clinical importance in canine autoimmunity. one of the authors, erik lattwein, is employed by euroimmun where the analyses were performed. chronic kidney disease (ckd) has a high prevalence in cats. routine renal markers, serum creatinine (scr) and urea are not sensitive or specific enough to detect early ckd. serum cystatin c (scysc) has advantages over scr for the detection of early kidney dysfunction, both in humans and dogs. a significant higher scysc concentration in ckd cats has been demonstrated. the objective of this study was to determine the effect of age, gender and breed on feline scysc and to establish a reference interval for feline scysc. in total, healthy cats between one and years were included. serum cysc was determined with a validated particleenhanced nephelometric immunoassay (penia). serum cr, urea, urine specific gravity (usg), urinary protein: creatinine ratio (upc) and systolic blood pressure (sbp) were also measured. to test for difference between the groups, the f-test was used. the lower and upper value of the % reference interval were obtained as the . % and . % quantiles of the scysc observations. no significant differences in scysc concentration were observed between young, middle-aged and old cats; between female, female neutered, male and male neutered cats; and between purebred and domestic short-or longhaired cats. the % reference interval for feline scysc was determined as [ . - . mg/l]. there was a significant difference in scr concentration between domestic short-or longhaired cats and purebred cats. the sbp was significantly influenced by gender as well as age, while urea was influenced by both age, gender and breed. this study showed that the biological factors age, gender and breed have little or no impact on feline scysc, in contrast to scr and serum urea, making it an interesting marker. therefore, further studies are warranted to evaluate the diagnostic value of scysc as a renal marker in cats. this study recieved support from the institute for the promotion of innovation by science and technology in flanders (iwt) through a bursary to l. ghys. assessment of renal function is often needed, however existing methods including urine and plasma clearances are invasive, cumbersome and time consuming. in this pilot study the feasibility of a transcutaneous glomerular filtration rate measurement was investigated. the transcutaneous disappearance rate (expressed as half-life) of fluorescein-isothiocyanatelabelled sinistrin (fitc-s) was measured in three healthy research dogs and three healthy research cats. plasma clearance of sinistrin ( data points) was performed in both species as previously described (res vet sci ; : - and j fel med surg ; : - ) and half-life was calculated using a -compartment model with a freely available pharmacokinetic calculator (comput meth prog bio ; : - ) . renal elimination of fitc-s was measured transcutaneously for hours ( - data points) using a miniaturized device as described previously for the same purpose in rats (kidney int : - ). the procedures were performed in awake, freely moving animals using escalating doses of fitc-s ( mg/kg, mg/kg, mg/kg) with a wash-out period of at least h in each animal. to find the best position for the device, multiple devices were placed on each animal. the resulting fitc-s disappearance curves were visually assessed to determine the most suitable location and the appropriate dose to reach an adequate transcutaneous peak signal for kinetic analysis. in both species mg/kg were adequate for kinetic calculation. the most suitable place for the device was the lateral thoracic wall in dogs and the ventral abdominal wall in cats, respectively. transcutaneous fitc-s clearance was then repeated using the optimal dose and location and in parallel with the plasma sinistrin clearance. plasma sinistrin clearances [ml/kg/min] were . , . and . in the three dogs, respectively. corresponding plasma elimination half-lives [min] were , and , and corresponding transcutaneous elimination half-lives [min] were , and , respectively. plasma sinistrin clearances [ml/kg/min] were . , . and . in the three cats, respectively. corresponding plasma elimination half-lives [min] were , and , and corresponding transcutaneous elimination half-lives [min] were , and , respectively. in conclusion, transcutaneous fitc-s clearance is a feasible method for assessment of gfr in awake dogs and cats. it is noninvasive, well tolerated and easy to perform even in a clinical setting with results being readily available. a dose of mg/kg of fitc-s seems adequate for kinetic assessment. further studies are now needed to establish reference values and evaluate transcutaneous renal clearance in various conditions. conflicts of interest: zhp and sg are supported by the ec fp marie-curie programme: nephrotools. the device development was supported by the fp activity: place-it.ng is owner of a patent covering fitc-sinistrin and the technology for its measurement. excretion of urinary biomarkers of renal damage should occur at an early stage of chronic kidney disease (ckd), thus facilitating earlier diagnosis of renal disease. albumin and cystatin c in the renal ultrafiltrate are mostly reabsorbed by the proximal tubular cells, therefore increased urinary excretion of albumin and cystatin c (uac and ucysc) would be expected to correlate with the presence of renal tubular damage and ckd. the aim of this study was to establish biological validity of two particle enhanced turbidimetric assays (petias) for the measurement of albumin and cystatin c (previously validated for use in feline urine) by comparing the uac and ucysc between non-azotaemic cats and cats with azotaemic ckd. blood and urine samples were obtained from cats at three uk first opinion practices as part of a geriatric screening programme. haematology, serum biochemistry (including total thyroxine concentration (tt )) and urinalysis (including urine protein:creati-nine ratio (upc)) were performed. dental disease score (calculus and gingivitis) and body condition score (bcs) were recorded. cats with tt > nmol/l, evidence of pyuria or bacteruria, or significant systemic disease were excluded. uac and ucysc were determined in non-azotaemic cats (n = ) and cats with azotaemic ckd (n = , defined as a serum creatinine concentration > lmol/l and concurrent urine specific gravity < . ). comparisons between the non-azotaemic and azotaemic ckd groups were made using the mann whitney u test. correlations were assessed by spearman's correlation coefficient. data are presented as median [ th , th percentile] and statistical significance was defined as p < . . uac was significantly higher in the azotaemic group than the non-azotaemic group ( . [ . , . ]x - vs. . [ . , . ]x - ; p = . ), whereas upc was not significantly different between the groups (p = . ). unexpectedly, ucysc tended to be lower in azotaemic cats than non-azotaemic cats ( . [ . , . ]x - vs. . [ . , . ]x - ; p = . ). uac was weakly positively correlated with serum urea concentration (r s = . , p = . ), but was not correlated with serum creatinine concentration. ucysc was not significantly correlated with serum concentrations of urea, creatinine or tt . uac was also weakly negatively correlated with dental calculus score (r s = - . ; p = . ) and bcs (r s = - . ; p = . ). uac appears to be a more sensitive test for azotaemic ckd than upc, however the apparent low specificity may limit the utility of uac as a urinary screening test for ckd. increased ucysc (determined by petia) would not appear to be a marker of azotaemic ckd in cats. no conflicts of interest reported. cystinuria is an inherited metabolic disorder that causes defective tubular reabsorption of the aminoacids cystine, ornithine, lysine and arginine (cola). the low solubility of cystine in acidic urine promotes formation of cystine crystals and uroliths in the urinary tract resulting in the clinical signs of stranguria, urinary obstruction and renal failure in affected individuals. cystinuria occurs in > breeds of dog and has been classified into types ia (newfoundland, landseer, labrador), iia (australian cattle dog), ib (miniature pinscher) and iii (androgen-dependent; e.g. mastiff, irish terrier). the kromfohrl€ ander is a medium-sized companion dog, bred initially as a cross between a wire fox terrier and a grand griffon vend een, first recognised internationally in . cystinuria has been suspected in this breed but no cases have been reported in the literature to date. we determined urinary cola concentrations in adult kro-mfohrl€ ander dogs aged - years comprising intact and castrated males, and intact and spayed females. a total of ( %) intact males aged . to . years had cola values > lmol/g creatinine and several developed cystic calculi. furthermore, intact male dogs had increased cola but normal cystine levels. all castrated males had normal cola concentrations. no females had increased cola and cystine concentrations or formed any cystine calculi. we conclude that cystinuria with cystine calculi occurs frequently in adult intact male kromfohrl€ ander dogs but neither is seen in females. this appears to be an androgen dependent type iii cystinuria, as seen in mastiff-type dogs and irish terriers. thus, castration may resolve the increased urinary cola excretion and risk for cystine calculi formation and obstruction. the precise mode of inheritance is still unclear. all adult intact male kromfohrl€ ander dogs should be screened by urinary cola testing. work carried out at the author's previous place of employment (university children's hospital, frankfurt, germany). acs, rk, em, md and ug provide a diagnostic service for cystinuria and other inborn errors of metabolism in companion animals. ureteral urolithiasis is an emerging medical concern in cats. there are few reports on epidemiology, diagnosis or medical management of ureteral calculi in cats, particularly in europe. cats diagnosed with ureteral urolithiasis in the teaching hospital of the veterinary school of alfort from to were included in this study. diagnosis was confirmed with radiographs, ultrasound scan and/or laparotomy. signalment, clinical signs, clinicopathologic and diagnostic imaging findings, medical treatment and outcome were recorded. epidemiological data were compared to a reference population of cats. eighty three cats were included in the study. the occurrence of ureteral urolithiasis was significantly higher in birman to the author knowledge, it is the first time that a higher prevalence of ureteral calculi in birman cats is reported in europe. spontaneous elimination of calculus is associated with a small size (< . mm). if the size of calculi tends to be bigger in cats with no improvement of renal function after medical treatment, prospective studies are still needed to determine the best medical treatment. no conflicts of interest reported. feline immunodeficiency virus (fiv) infection has been associated with kidney disease, mainly characterised by an increased prevalence of proteinuria in fiv-infected cats. however, studies evaluating renal variables in fiv-positive cats are scarce. recently, a higher systolic blood pressure (sbp) was reported in a small number of fiv-infected cats. hypertension is an important cause of proteinuria and a frequent cause of renal disease in human immunodeficiency virus (hiv) positive patients. therefore, our main objective was to describe sbp in clinically ill fivpositive cats. secondly we aimed to evaluate routine renal variables in this population. naturally infected clinically ill fiv-positive cats were prospectively included. the doppler ultrasonic technique was used to measure sbp according to acvim guidelines. serum creatinine (screat) and urea (surea) concentrations, urine specific gravity (usg) and urinary protein:creatinine ratio (upc) were determined. the study included cats, with a mean age of . ae . years and a mean body condition score of . ae . on a nine-point scale. the sbp ranged from to mmhg, with a mean of ae mmhg. only two cats were hypertensive (sbp > mmhg). both had isosthenuric urine, were borderline proteinuric (upc . - . ) and one of them was mildly azotemic. mean screat was . ae . lmol/l (reference interval (ri) . - . lmol/l) and mean surea concentration . ae . mmol/l (ri . - . mmol/l). thirteen cats showed increased screat levels, with decreased usg (< . ) in eight, proteinuria (upc > . ) in seven and increased surea concentrations in ten of them. five out of ten azotemic cats were proteinuric with a decreased usg. mean upc was . ae . , with a wide range from . to . . borderline proteinuria was present in / ( . %) and proteinuria in / ( . %). half of the proteinuric cats had a decreased usg. mean usg was . ae . . one third of all cats had a decreased usg, with isosthenuria in seven of them. these results demonstrate that proteinuria and poorly concentrated urine are common in naturally infected clinically ill fivpositive cats, confirming previous reports in cats and humans. however, longitudinal studies of (borderline) proteinuric patients are needed to elucidate the clinical relevance. the low number of hypertensive patients and low mean sbp in our study indicate that hypertension is uncommon and unlikely to be the cause of renal damage in clinically ill fiv-infected cats. aratana therapeutics nv financially supports a clinical trial on the use of antivirals in fiv cats at our university. screeninng examinations repoorted in this trial were part of the required pre-trial investigations for that study. the presenting author is also funded by a scholarship from aratana therapeutics av. the objective of this study was to identify the prevalence of bacterial species and the susceptibility of isolates to doxycycline, trimethoprim-sulfamethoxazole (tms), ampicillin, amoxicillinclavulanic acid (amc), cephalothin, and enrofloxacin in cats with urinary tract infections (uti) with and without predisposing comorbidities. a retrospective analysis of case records between and was performed and resulted in inclusion cats into the study: cats with a systemic comorbidity, cats with indwelling urinary catheters, cats with other local comorbidities, and cats with no comorbidity. the most commonly isolated bacteria were escherichia coli (e. coli), streptococcus species (spp.), staphylococcus spp., and enterococcus spp.. the proportion of gram-negative isolates was significantly higher in the cats with systemic comorbidities than in cats with indwelling urinary catheters (p < . ) and cats with other local abnormalities (p < . ), whereas gram-positive isolates were significantly more commonly isolated from cats with indwelling urinary catheters and other local comorbidities than in cats with systemic comorbidities (p < . ). the proportions of isolates susceptible to amc, enrofloxacin, and tms and the antimicrobial impact factors (if) were higher than the proportions of isolates susceptible to doxycycline, ampicillin, and cephalothin and the respective if. based on these findings, amc and tms would be the firstchoice antimicrobial agents for empiric treatment of bacterial the increasing rates of resistance exhibited by uropathogens represent a serious problem for the selection of an appropriate antibiotic. the aim of this study was to determine secular trends of companion animal urinary tract infection (uti) that involve extended-spectrum b-lactamase (esbl)-and carbapemenase-producing gram negative bacteria (namely, escherichia coli, klebsiella pneumoniae, proteus mirabilis, acinetobacter baumannii), methicillin-resistant-staphylococci (mrs) and ampicillin and high-level-gentamicin-resistance (hlgr) enterococci. nine hundred and twenty two uropathogenic bacteria were isolated from dogs and cats, between january and march , at the veterinary teaching hospital of the faculty of veterinary medicine and at veterinary private practices in the lisbon area. isolates were identified using standard commercial systems. susceptibility testing was performed using the disk diffusion and broth microdilution methods. clsi breakpoints were applied. extended-spectrum b-lactamases (esbl) production was screened by double-disk synergy test. the esbl, plasmid-mediated ampc, carbapemenases, meca and aac( ')-ieaph( '')-ia genes were detected by pcr and gene enzymes were sequenced. among enterobacteriaceae . % were dhaproducers, . % were esbl-producers and . % were cmyproducers. all isolates were also multidrug-resistant. cefalosporinases-producer enterobacteriaceae were detected in , the first being a cmy- -producer e. coli. all the esbl-producers were e. coli or k. pneumoniae producing ctx-m-group enzymes. ampicillin-resistance in enterococci was present throughout the years ( , %, n = ). hlgr appeared in enterococci in and was confirmed by the detection of the bifunctional enzyme that confers high level resistance to aminoglycosides ( out of isolates). in this study we showed that in the last decade the emergence of resistance to critically important antimicrobials among uropathogens from companion animals is a concerning fact. the multidrug-resistant enterobacteriaceae may compromise effective therapeutic options, namely third and fourth generation cephalosporins, fluoroquinolones, trimethoprim/sulpha combinations. the emergence of mrsa/mrsp and hlgr among uropathogens is also a therapeutic challenge. the detection of uropathogens with antimicrobial resistance is not only an animal health issue but also a matter of public health, since companion animals may act as reservoirs of antimicrobial resistant bacteria or resistance genes for humans. the author currently receives a phd grant funded by the portuguese foundation for science and technology. in the past, the author received once research support and honoraries from portuguese merial for a project on canine vector borne diseases. the aim of the present study was to use insurance data to estimate morbidity and mortality related to kidney disease in the swedish dog population. insurance company data from veterinary care-insured and lifeinsured dogs during the years - were studied retrospectively. incidence-and mortality rates were calculated for the whole group of dogs as well as divided by sex and breed. for the breeds with the highest incidence-and mortality rates, respectively, the reasons for kidney disease were investigated by dividing the diagnoses into ethiology groups. the total number of veterinary care-insured dogs was , . the total incidence rate of kidney disease in this group of dogs was . ( . - . ) cases/ , dog-years at risk. the number of dogs in the life insurance was , and in this group the total kidney-related mortality rate was . ( . - . ) deaths/ , dogyears at risk. the most commonly reported ethiologies of kidney disease were "ethiology not determined"and "infectious/ inflammatory". the breeds with the highest incidence rate of kidney disease were the bernese mountain dog, miniature schnauzer and boxer. the breeds with the highest mortality caused by kidney disease were the bernese mountain dog, shetland sheepdog and flatcoated retriever. in conclusion, the epidemiological information provided in this study concerning kidney disease in dogs can assist clinicians in establishing diagnoses, and can assist breeders in defining priorities for preventative measures. it can also provide valuable information for future research. the senior author has received money from the insurance company we have used data from to write our study, for another project. jens h€ aggstr€ om and ingrid ljungvall have received financial support for research from sante animale, agria insurance ltd, sveland insurance ltd, forsgren research foundation. both of these authors have also undertaken paid consulatcny work for boehringer-ingelheim, ceva sante animale. mast cell tumours represent the most common cutaneous tumour in the dog. diagnosis of a mast cell tumour can be achieved through cytological examination of fine needle aspirate. however the grade of the tumour is an important prognostic marker and requires so far histologic assessment. a -tier histologic grading system based on number of mitoses, multinucleated cells, bizarre nuclei and karyomegaly was recently proposed by kiupel et al. the aim of this study was to assess if the cytomorphological criteria proposed in the -tier histologic grading system are applicable on cytology specimens. ninety-three mast cell tumour specimens of grade i or grade iii according to patnaik with both histological specimens and fine needle aspirates were retrospectively taken from a data set and histologically and cytologically re-evaluated. according to the kiupel grading system thirty-six were diagnosed histologically as high grades and fifty-seven were considered low-grade mast cell tumours. the cytologic examination of the corresponding specimens revealed thirty-one high grade and fifty-five low-grade tumours. an agreement between histologic and cytologic diagnosis based on the kiupel grading system was achieved in eighty-six cases (accuracy . %, specificity . %, sensitivity . %). five high-grade tumours ( . %) were considered as low grade on cytology. cytologic grading of mast cell tumours in the dog has satisfactory accuracy, sensitivity, and specificity. histologic grading of canine mast cell tumours still remains the gold standard, but cytology already gives reliable information. no conflicts of interest reported. in canines mastocytomas are among the most frequently diagnosed neoplasms of the skin. high grade mastocytomas (grade iii, patnaik classification) are characterized by an uncontrolled growth of neoplastic mast cells (mc) and a poor prognosis. recently, the kit-targeting tyrosine kinase inhibitors masitinib and toceranib have been approved for the treatment of canine mc tumors. these drugs are able to induce responses in mastocytoma patients. however, in many patients, relapses are seen. therefore, research is focusing on new drug targets. recently, the transcription factor stat has been reported to play an important role in the proliferation and survival of human neoplastic mc. the aim of the present study was to evaluate the jak -stat pathway in canine mastocytomas. to address this aim, the canine mastocytoma cell lines c and ni- as well as inhibitors directed against jak or stat were employed. as assessed by immunocytochemistry, c cells and ni- cells were found to express pstat in their cytoplasm and nuclei. intracellular expression of pstat was confirmed by flow cytometry. interestingly, c cells were found to express higher levels of pstat compared to ni- cells. next, we treated c cells and ni- cells with various concentrations of the stat inhibitors piceatannol and pimozide and the jak inhibitors azd and tg . as assessed by h-thymidine uptake, all compounds were found to inhibit the proliferation of canine mc in a dose-dependent manner. drug effects were found to vary in different cell lines, with the following rank-order of potency (ic values): tg : . - . lm; pimozide: . - . lm; azd : - lm; piceatannol: - lm. to further explore the mechanism of drug-induced inhibition of proliferation, we examined cell cycle progression and apoptosis in drug-exposed cells. whereas all drugs tested induced only moderate cell cycle arrests in the g phase, these drugs were found to induce substantial apoptosis in c cells and ni- cells as evidenced by microscopy and annexin-v/pi staining. together, our data show that jak -and stat -targeting drugs exert anti-proliferative and apoptosis-inducing effects in canine mastocytoma cells suggesting that this signaling pathway may be a promising new therapeutic target in canine mastocytomas. the clinical relevance of this observation remains to be determined. no conflicts of interest reported. subcutaneous mast cell tumours (sqmct) in dogs are relatively uncommon compared to their cutaneous counterparts. the veterinary literature describes these tumours as a specific pathological entity with, in general, a low probability of aggressive progression. surgery is considered the main treatment modality, while medical treatment has not been described. the purpose of this study was to determine progression free survival (pfs) for a chemo na€ ıve cohort of dogs presented with non-resectable and/ or metastasized sqmct, which all underwent masitinib-based therapy. data were collected for patients with sqmct presented to participating centres in the netherlands and the uk from / / to / / , which received masitinib-based therapy. treatment protocols employed, included masitinib alone (m), masitinib and prednisolone (mp), masitinib plus vinblastine and/ or lomustine and prednisolone (mpc). response to therapy was measured conforming to recist . . adverse events were graded using vcog-ctcae . . patients were grouped according to presence or absence of metastasis, treatment protocol used, previous surgery, and remission status achieved; simple comparisons were made to evaluate possible significance. twenty-five cases were identified. / were female. median age of occurrence was years ( - ). diagnosis was made by histology in / ; proliferation indices were defined in only dogs. fourteen cases exhibited metastasis at initiation of therapy. pfs for all cases ranged from - days. median/mean pfs (days) according to treatment was m: / d (n = ), mp: / d (n = ), mpc: / d (n = ). median/mean pfs according to metastasis status was m : / d (n = ) and m : / d (n = ). dogs who underwent previous surgery (n = ) had a median/mean pfs of / d compared to those who had no surgery / d. looking at remission status, median pfs of patients who achieved a complete remission was not reached, with a mean pfs of d (n = ). median/mean pfs of patients with partial remission was / d (n = ), stable disease / d (n = ), and progressive disease / d (n = ). / cases experienced suspected adverse events. three dogs, two of which ultimately died, had seven grade - adverse events (anaemia (n = ), hepatotoxicity (n = ), gastrointestinal toxicity (n = )). masitinib-based treatment is effective in the management of sqmct perceived to be aggressive. patients do not appear to benefit from prior surgery. metastatic status did not influence outcome. adding chemotherapy negatively influenced pfs. complete remission is a very favourable prognostic development. the authors have received financial support from ab science to help with the costs of statistical analyses in an unrelated project advances in distinction between morphological subtypes of canine non-hodgkin's lymphomas (nhl) have provided a better understanding of this cancer in dogs. diffuse large b-cell lymphomas (dlbcl) are the most frequent form of nhl in dogs including some distinguished morphological subtypes (mainly centroblastic polymorphic and immunoblastic) according to the who classification. few clinical studies reported dlbcl clinical outcomes under treatment while survival times of the centroblastic polymorphic subgroup were reported. the aim of this retrospective study was to evaluate the response of dlbcl to a standardized multi-agent chemotherapy protocol. medical records from dogs with a diagnosis of dlbcl between and were retrospectively reviewed. inclusion criteria were the availability of complete initial and follow-up information and the application of a standardized multi-agent l-cop chemotherapy protocol as previously described. dogs which received corticosteroids before the initiation of treatment and dogs which died for other reasons than their related disease before the end of the induction period ( d) were excluded. response to chemotherapy was evaluated every week during the induction of treatment, then every to weeks. statistical analysis was performed using kaplan-meier analysis. thirty cases of dlbcl meeting all inclusion criteria were included from the initial population. seven dogs were in clinical stage iii according to the who classification, in stage iv and in stage v. nineteen dogs were in substage a, and in substage b. on dogs which have an adequate response evaluation, dogs ( . %) achieved a complete remission. the median and mean duration of first remission were d and d, respectively (range - d). the median and mean durations of survival time were respectively d and d (range - d). one year and -years survival rates were % and % respectively. according to the statistical analysis, neither clinical stage (p = . ) nor substage at presentation (p = . ) or morphological subtype (immunoblastic vs centroblastic polymorphic, p = . ) were considered as a significant prognostic factor regarding the duration of the first remission and the overall survival time. a complete response was significantly associated with longer survival times (p = . ). to conclude, the dlbcl displayed a good clinical response to l-cop protocol, with a median survival time of days. the only significant prognostic criterion identified was a complete clinical response to the treatment. a prospective controlled study on a larger population is warranted to confirm these results. no conflicts of interest reported. canine histiocytic sarcoma (hs) is an aggressive round cell neoplasm with a poor prognosis. both lomustine and doxorubicin have been evaluated as first line chemotherapy agents with response rates of up to % and median survival times around - months. the aim of this study was to evaluate the response to epirubicin in a population of dogs with hs pre-treated with lomustine. medical records of dogs with a diagnosis of hs that were treated with lomustine and subsequently epirubicin were retrospectively evaluated. fifteen dogs received lomustine followed by epirubicin. there was a measureable response to lomustine in seven of dogs with evident disease, % ( cr & pr). an additional dogs achieved stable disease for an overall biological effective response of %. median ttp following lomustine could be assessed in dogs and was days (range - ). all fifteen dogs received epirubicin as a rescue agent: nine following progressive disease and three with stable disease on lomustine. one dog received epirubicin after completing six doses of lomustine in complete remission and two dogs in partial remission changed to epirubicin due to hepatotoxicity associated with lomustine. response rate to epirubicin was % and biologic effective response was % ( cr, pr, sd, pd) in dogs. one dog was euthanized due to epirubicin associated gastro-intestinal toxicity and dog stopped treatment with no assessment of response. median duration of response to epirubicin was days (range: - ) and dog is still alive and in remission ( days). overall median survival time for dogs receiving epirubicin following lomustine was days (range: - ). single agent epirubicin is a valid rescue therapy after lomustine for canine histiocytic sarcoma and results in modestly improved overall survival times in responding patients. the author received a travel scholarship from zoetis to attend this congress. the incidence of melanocytic lesions is increasing among canine population. canine malignant melanoma could have an aggressive behavior, metastasize early in the course of the disease and be resistant to most current therapeutic regimens leading to the need of finding markers with potential as therapeutic targets. the overexpression of cox- seems to play a key role in melanocytic tumours, having being described an association between high cox- immunoexpression and the malignant behavior. in order to contribute to the understanding of the role of cox- in melanocytic tumours, three main pathways were investigated: angiogenesis, tumour cell proliferation and inflammatory microenvironment (t-lymphocytes and macrophages). fifty one ( ) melanocytic tumours [ cutaneous ( malignant melanomas and melanocytomas) and oral malignant melanomas] were included. all the samples were submitted to immunohistochemical staining carried out by the streptavidinbiotin-peroxidase method, with a commercial detection system with or without melanin blanching, for detection of the following markers (cox- , ki- , factor viii, vegf, cd and mac ). in melanocytic tumours (n = ), both cox- labelling extension and intensity revealed a statistically significant association with angiogenesis by factor viii (p < , ), vegf (p < , ); ki- (p < , ), cd + t-lymphocytes (p < , ) and mac (p < , ). considering only malignant melanomas (n = cases), cox- labelling extension revealed a statistically significant association with angiogenesis (p = , ) and cd + t-lymphocytes (p = , ). cox- intensity was also positively associated with angiogenesis (p = , ) and with mac (p = , ). present study demonstrated a link between high cox- immunoexpression and increased angiogenesis and tumoural t-lymphocyte and macrophage infiltration in malignant melanomas. these findings reinforce the usefulness of using selective cox- inhibitors as a valuable therapeutic tool in malignant melanocytic tumours. this study received financial support from a company (merial). neuter status and risk of malignant neoplasia is not well evaluated in the canine population, when excluding neoplasia not normally believed to be sex-hormone dependent. denmark and the scandinavian countries have a high proportion of intact dogs compared to populations from other parts of the world. in the present study it was hypothesized that there would be no difference in gender and neuter status between the population of canine patients with a non-sex-hormone dependent malignant neoplasia reported to the danish veterinary cancer registry and a general population. from august to march , canine neoplasms were reported to the danish veterinary cancer registry. the total number of malignant ( ) and benign ( ) were comparable ( % and %). malignant neoplasms totalled , when tumors from areas of distribution with known sex-hormone dependency (reproductive organs, mammary gland, perineal), and cases with unknown area of distribution were excluded. the overall distribution of malignant neoplasia was ( %) intact male dogs, ( . %) neutered male dogs, ( %) intact female dogs and ( . %) neutered female dogs. the distribution was even between male and female dogs ( . % and . %). compared to a known standard population of dogs, there was an overall statistically significant association of malignant neoplasia with neuter status in both sexes. for both genders this was significant for lymphoma, mast cell tumors and osteosarcomas,. for neutered females, but not males, there was increased risk of hemangiosarcoma, squamous cell carcinoma and malignant melanoma. these findings indicate that there might be an association between neuter status and development of malignant neoplasia but larger prospective studies are needed to evaluate the risk of non-sex hormone dependent cancers in neutered dogs. no conflicts of interest reported. serum acute phase proteins (apps) are considered biomarkers of the acute phase reaction, and are being increasingly used in human and veterinary medicine in diagnosis and monitoring of neoplastic diseases. in the cat, serum amyloid a (saa) is considered a positive major app, haptoglobin (hp) a moderate app, and albumin and insulin-like growth factor- (igf- ) negative apps. the aim of the present study was to characterize the apps response in cats with mammary tumours. for that purpose, saa, hp, igf- and albumin serum concentrations were determined in female cats with malignant mammary tumours. cats with history of previous tumours or with concomitant tumours or other diseases were excluded. information on cats age, gender, breed, tumour type, histological grade, tumour size and location, skin ulceration, vascular neoplastic infiltration, necrosis, metastasis to regional lymph nodes, thoracic or abdominal organs, and survival time from diagnosis was assessed. blood samples were collected before surgery in all cats, and whenever possible, serial samples collected on control visits. owners gave informed consent. studied population included domestic short-haired cats with ages ranging from eight to years ( , + /- , ). all had carcinomas, including solid carcinomas (n = ), tubulopapillary carcinomas (n = ), one cribiform carcinoma and one carcinosarcoma. at the time of diagnosis, % of cats had an increase in serum concentration of hp and % of saa, and % had a decrease in concentration of albumin. mean and standard deviation values were of , + /- , g/dl for albumin (reference range , - , g/ dl), , + /- , lg/dl for igf- , , + /- , lg/ml for saa (reference value ˂ lg/ml), and , + /- , g/l for hp (reference value ˂ g/l). a positive correlation (r = , ) was detected between increases in serum concentrations of saa and hp. the increase in the size of tumour was significantly associated with the concentration of saa (p˂ , ). serum hp concentrations were significantly increased in tubulopapillary carcinomas (p˂ , ), and igf- decreased in solid carcinomas (p˂ , ). in the cats where serial determinations were performed, development of thoracic metastasis was significantly associated with a decrease of serum concentration of albumin (p˂ , ), and with an increase of saa (p˂ , ). this study suggests that feline mammary tumours are associated with an acute phase response. according with the results obtained, saa, hp, albumin and igf- might be important serum biomarkers in diagnosis and monitoring of the evolution of feline malignant mammary neoplasias. no conflicts of interest reported. histiocytic sarcoma (hs) is a neoplastic proliferation of interstitial dendritic cells or tissue macrophages. dogs with hs can present with local disease or with multifocal (disseminated) involvement. disseminated hs is poorly responsive to therapy and almost always fatal. little is established regarding the aetio-pathology of histiocytic sarcoma in dogs. the purpose of this study was to establish and characterise a hs cell line from fresh tumour samples obtained from a dog with disseminated hs in order to further clarify disease pathogenesis and behaviour. with owner consent, treatment-na€ ıve tumour sections were collected from a dog with disseminated hs that was euthanased. tumour tissue was assessed with immunohistochemistry (ihc) using antibodies against canine cd , cd , and pax- to support the diagnosis of histiocytic sarcoma. primary cell cultures (hscs), established from the tumour were cultured and maintained in modified eagle's medium with % fetal bovine serum, l-glutamine, penicillin and streptomycin, in standard conditions. hscs were characterised by alpha naphthyl acetate esterase (anae) and lysozyme staining while pcr was used to detect cell markers cd a, cd c, mhc ii, cd , ccr , e-cadherin, and cd . cell surface markers were compared to an established canine hs cell line (dh ). phagocytic activity of hsc cells was assessed using cellular uptake of carboxylated fluorescent beads and documented using flow cytometry and fluorescent microscopy. tumour tissue was strongly cd positive and negative for cd and pax- . cultured cells exhibited morphological characteristics consist with dendritic cells, such as projections and pleomorphism. hsc cells stained positively for non-specific esterase (anae) and lysozyme, and pcr indicated cells were positive for cd a, cd c, mhc ii and cd and negative for cd and e-cadherin. hsc cells were positive for mhc ii and ccr while dh cells were negative. phagocytic activity was evident. a novel hs cell line (hsc) was established and characterized from primary tumour tissue collected from a dog with disseminated disease. hsc cells were most consistent with interstitial dendritic cell origin based on cd a, cd c, and mhc ii staining as well as demonstrable phagocytic activity. hsc cells also displayed expression of ccr , unlike the established dh line, supporting a notion that hs consists of a variety of subtypes. ccr has been linked to hs growth and metastasis, suggesting it may represent a possible therapeutic target. further studies establishing and characterising canine hs cells may contribute to the elucidation of mechanisms of tumourigenesis. no conflicts of interest reported. virulent-systemic (vs)-fcv that induce cutaneous edema, ulcerations of the head and feet, and occasionally jaundice have been described in the usa and europe. here we characterize for the first time vs-fcv outbreaks in cats in switzerland and liechtenstein. the four outbreaks occurred in three geographically separated locations: schaan (liechtenstein, shelter ), zurich (switzerland) and lausanne (switzerland, shelter ) between november and january . pcr (fcv and feline herpesvirus- , fhv- ), virus isolation and felv/fiv testing were performed on saliva and blood samples collected from clinically affected cats. furthermore, saliva for pcr was collected from additional cats in shelter . phylogenetic analyses were performed based on the capsid (vp ) gene sequence of fcv. vs-fcv isolates were tested for virus neutralization with sera raised against common fcv vaccine strains. outbreak occurred in a cattery in liechtenstein and involved five non-vaccinated, -months old siblings with fever, edema, skin and tongue ulcerations. outbreak occurred in a small animal clinic in zurich. a -year old cat presented with severe paw edema, fever, tongue and skin ulcerations, progressive hypoproteinemia and hyperbilirubinemia. outbreaks and happened in a cattery in lausanne five months apart and involved two litters of non-vaccinated, -to -months old kittens. the cats presented with fever, nasal discharge, edema and skin and oral ulcerations. all affected cats tested fcv-positive but negative for fhv- , felv and fiv, except for one kitten from outbreak (fiv-positive). all cats in outbreaks and recovered, whereas all cats in outbreaks and died or were euthanized because of clinical deterioration. each outbreak was caused by a phylogenetically distinct vs-fcv strain. in shelter , the queen and three in contact cats remained asymptomatic although infected with the same vs-fcv strain. furthermore, / other cats of shelter were infected with closely related, but distinct fcv strains. the vs-fcv isolates from the two outbreaks in shelter were distinct but phylogenetically related. all vs-fcv isolates from cats from the same outbreak showed a similar virus neutralization pattern, but neutralization differed between different outbreaks. in conclusion, all vs-fcv outbreaks involved multi-cat environments. the same vs-fcv strains with similar virus neutralization patterns were isolated from cats from the same outbreak. not all cats infected with a vs-fcv strain developed disease and mortality varied between the outbreaks. the sera for virus neutralization were provided by merial, france. defined herein as presence of sneezing, nasal-and/or ocular discharge, conjunctivitis and/or keratitis), but also oral cavity lesions, chronic stomatitis, limping syndrome and, rarely, virulent systemic disease. the aims of the present study were to compare cats suspected of fcv (fcv-sc) based on clinical symptoms and healthy controls (controls) and to investigate potential risk and protective factors, such as co-infection with feline herpesvirus- (fhv- ), mycoplasma felis, chlamydophila felis, bordetella bronchiseptica and feline retroviruses, vaccination, gender, age, breed, housing and corticosteroid and antibiotic treatment. oropharyngeal, nasal and conjunctival swabs from fcv-sc and controls were collected into transport medium, processed within hours after collection and analyzed for fcv by virus isolation and for all tested pathogens using molecular assays. the samples were collected by randomly selected veterinary practices in different areas of switzerland ( fcv-sc and controls/ area). to record clinical data, retroviral status and vaccination history of the cats, a questionnaire was filled out by the private veterinarian. the seven tested pathogens were found in the investigated population. the prevalence (fcv-sc vs. controls) was: fcv % vs. %; fhv- % vs. %, c. felis % vs. %, b. bronchiseptica % vs. %, m. felis % vs. %, feline leukemia virus % vs. % and feline immunodeficiency virus % vs. %. fcv-sc were positive for fcv significantly more often compared with controls (or . ) and shed more fcv. co-infections with up to four pathogens were detected; fcv-sc were significantly more frequently co-infected ( %) compared with controls ( %). gingivostomatitis and oral ulceration but not urtd were highly associated with fcv infection. in contrast, c. felis was associated with urtd; fhv- was associated with nasal and ocular discharge and m. felis with conjunctivitis and ocular discharge. risk factors for fcv infection were housing in groups (especially ≥ cats), an intact gender, maine coon breed and corticosteroid therapy. fcv-positive cats with gingivostomatitis were older and more commonly vaccinated than fcv-positive cats without gingivostomatitis. moreover they shed more fcv than cats with urtd. vaccination and primary immunization defined as two vaccinations - weeks apart with the same vaccine brand were protective factors against fcv but not fhv- infection. vaccination was associated with a decreased incidence of urtd in fcv-infected cats (or . ). further analyses will investigate cross-neutralization patterns of the prevailing fcv isolates. conflicts of interest: the study was partially funded by merial, france, and biokema, switzerland. antibody preparations are commonly used for the treatment of feline upper respiratory tract disease (furtd), although their efficacy has not been proven. the aim of this study was to evaluate efficacy of a commercial serum containing antibodies against feline herpesvirus- (fhv- ) and feline calicivirus (fcv) in cats with acute viral furtd. this prospective, randomized, placebo-controlled, double-blind study included cats with acute (< days) clinical signs of fhv- and/or fcv infection (confirmed by quantitative pcr). all cats received symptomatic treatment and either hyperimmune serum (n = ) (≤ weeks ml, > weeks ml, subcutaneously q h, topically into eyes, nostrils, and mouth q h) or saline (n = ) for three days. clinical signs, including a 'furtd score' and general health status, were recorded daily (day to and on day ). fcv shedding was determined on day and . statistical analyses included one-way analysis of variance, mann-whitney u, and student's t-test (improvement of clinical signs), fisher's exact test (fcv shedding), and spearman analysis (correlation clinical signs with virus load). clinical signs and general health status improved significantly in both groups. however, while placebo-treated cats had only improved significantly by day , cats receiving antibodies already significantly improved in their 'furtd score' (p = . ) and general health status (p = . ) by day . there was no significant difference in the number of cats shedding fcv and no correlation between viral load and clinical manifestation. administration of antibodies lead to faster improvement of clinical signs in cats with acute viral furtd, but did not influence fcv shedding. no conflicts of interest reported. different viral and bacterial pathogens can be involved in feline upper respiratory tract disease (furtd). although some clinical signs have been associated with certain pathogens, clinical signs can be variable and non-specific. aim of the study was to compare detection rates of feline herpesvirus- (fhv- ), feline calicivirus (fcv),and chlamydophila felis (c. felis) in cats with furtd on different sampling sites, and to correlate test results and clinical signs. swabs of nose, oropharynx, tongue, and conjunctiva were taken from cats with signs of furtd. on all samples, reverse transcription polymerase chain reaction (rt-pcr) was performed for detection of fcv, and polymerase chain reaction (pcr) for detection of fhv- and c. felis. fisher's exact test was used for all comparisons. the level of significance was p < . . pathogens were detected in . % of cats. of these, . % were positive for fhv- , . % for fcv, and . % for c. felis. fcv was isolated significantly more often from oropharynx ( . % of fcv-positive cats) and tongue ( . %) compared to conjunctiva ( . %) (p < . ). there was no significant difference between the sampling sites for detection of fhv- and c. felis. in addition, there was no preferred sampling site in cats with respective clinical signs, including oral ulceration, conjunctivitis, and keratitis. in cats with furtd, the oropharynx can be recommended as the preferred sampling site for detection of fcv, fhv- , and c. felis. based upon clinical signs it cannot be determined which sampling site should be selected for detection of the pathogens. no conflicts of interest reported. pulmonary haemorrhage syndrome (lphs). s. schuller , s. callanan , s. worrall , t. francey , a. schweighauser , j.e. nally . bern university, bern, switzerland, university college dublin, dublin, ireland leptospiral pulmonary haemorrhage syndrome (lphs) is a severe form of leptospirosis, which has been increasingly recognised in humans and many animal species in the past years. patients with lphs may develop rapidly progressive intra-alveolar haemorrhage, leading to high mortality. the pathogenic mechanisms of lphs are poorly understood hampering the application of effective treatment strategies. studies in humans and experimentally infected guinea pigs have demonstrated deposition of immunoglobulin and complement c in lphs lung tissue in the absence of significant numbers of leptospires, suggesting that lphs is, in part, caused by autoimmunity. the aim of this project was to describe the histopathologic features of lphs in dogs and to investigate whether igg and igm deposition is present in affected canine lung tissue. single-step immunohistochemistry (ihc) for dog igg, igm and leptospiral outer membrane vesicles was performed on lung tissues from dogs with lphs, dogs with pulmonary haemorrhage due to other causes and healthy dog lungs. acute intra-alveolar haemorrhage and oedema in the absence of significant inflammatory infiltrates were present in all lphs lung tissues. three ihc staining patterns were observed in lphs lung tissue: alveolar septal wall staining with (igg n = /igm n = ) and without intra-alveolar staining (igg n = /igm n = ) and staining of intra-alveolar fluid only (igg n = /igm n = ). intra-alveolar staining appeared to favour alveolar surfaces in some cases (igg n = /igm n = ). healthy control lungs showed no staining, whereas haemorrhagic lung showed staining of intraalveolar fluid (igg/igm n = )) and occasional, mild and discontinuous staining of alveolar septa (n = ). leptospiral antigens were not detected in any of the tissues. results indicate that histopathologic features of canine lphs are similar to what has been described in other species. ihc demonstrated that alveolar septal deposition of igg/igm is present in most dogs with naturally occurring lphs. while these findings support a role of the humoral immune response in the development of lphs, our findings do not indicate whether autoimmunity is a primary or secondary event in the pathogenesis of lphs. no conflicts of interest reported. leptospirosis, a zoonotic bacterial disease with a worldwide distribution, is a re-emerging disease in humans and dogs. acute renal and hepatic failure are the most frequently reported clinical manifestations of canine leptospirosis. the aim of this study was to describe clinical, laboratory and radiological features, the outcome as well as the distribution of leptospira serogroups in dogs with leptospirosis ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . medical records of dogs diagnosed with leptospirosis were evaluated retrospectively. diagnoses were based on microscopic agglutination testing (mat), blood/urine pcr, and histopathology (levaditi staining). mat-titers ≥ : against non-vaccine and ≥ : against vaccine serovars or a -fold rise of titers within - weeks were considered diagnostic. dogs met the inclusion criteria. in dogs diagnostic mat-titers were present (mainly against serogroups grippotyphosa ( %), australis ( %), and pomona ( %). at initial presentation, the most common clinical signs were lethargy ( %), anorexia ( %), vomitus ( %), a painful abdomen ( %), diarrhea ( %), oliguria ( %), tachypnea ( %), delayed capillary refill time ( %), pale mucous membranes ( %), fever ( %), hypothermia ( %), and icteric mucous membranes ( %). abnormal findings of the cbc included anemia ( %), thrombocytopenia ( %) and leukocytosis ( %). biochemistry abnormalities included increased creatinine concentrations ( %), increased liver enzyme activities ( %), hyperbilirubinemia ( %), hyperphosphatemia ( %), hyponatremia ( %), and hypoalbuminemia ( %). urinalysis often revealed glucosuria ( %) and an elevated urine-protein/creatinine-ratio ( %). radiological pulmonary changes were detected in % of the dogs initially or during the course of disease. dogs died or were euthanized, of them due to "leptospiral pulmonary hemorrhage syndrome". in this study, non-vaccine serogroups were the most common serogroups detected by mat. in the majority of patients renal ( %) and/or hepatic ( %) disease was detected. a pulmonary form of leptospirosis was present in % of the dogs. lung involvement represented a severe complication causing increased mortality depending on the severity of respiratory signs. no conflicts of interest reported. leptospirosis is a zoonotic disease that can affect multiple organs with renal and hepatic involvement being considered to be the most common. the aim of this study was to evaluate a large number of dogs with leptospirosis for cardiac and/or exocrine pancreatic involvement. a total of dogs were diagnosed with leptospirosis based on clinical signs and either microscopic agglutination test, blood/ urine polymerase chain reaction, and/or histopathology. at the time of admission and, in most patients, after an average of two weeks canine pancreatic lipase immunoreactivity (cpli, as measured by spec cpl â ), ultrasensitive cardiac troponin i (ctni), and c-reactive protein (crp) were analyzed. data were analyzed with non-parametric statistics. the level of significance was set at p < . . upon admission, common clinical signs reported included lethargy (n = ), vomiting (n = ), abdominal pain (n = ), dyspnea (n = ), pale mucous membranes (n = ), oliguria (n = ), hypothermia (n = ), and fever (n = ). anemia (n = ), thrombocytopenia (n = ), leukocytosis (n = ), were frequently reported hematology findings. increased concentrations of creatinine (n = / ), phosphorus (n = / ), alt (n = / ), sap (n = / ) and bilirubin (n = / ) were also frequently recorded. crp (median: . mg/l; range: . - . mg/l, reference interval (ri): . - . mg/l), ctni (median: . ng/l; range: . - . ng/l, ri: - . ng/l), and cpli (median: lg/l; range: - lg/l, ri: - lg/l) concentrations were above the upper limit of the reference intervals in / ( %), / ( %), and / ( %) dogs, respectively and serum cpli concentration was above the suggested cut-off value for a diagnosis of pancreatitis in / ( %) dogs. crp and ctni, but not cpli were higher upon admission compared to the re-check measurement (p = . and . , respectively). dogs with increased serum cpli concentrations also showed a higher proportion of dogs with increased serum ctni concentrations (p = . ). there was no statistically significant correlation of cpli concentrations with a history of abdominal pain and/or vomiting. biochemical results were compatible with multiple organ impairment with involvement of kidneys, liver, heart, and exocrine pancreas where at least two organs were affected in / ( %) dogs. forty ( %) of dogs recovered, ( %) died, and ( %) were euthanized. ctni and cpli were higher in non-survivors, but these differences did not reach statistical significance. however, the number of organs affected and outcome were significantly correlated (p = . ). our data suggest that infection with leptospira is characterized by a systemic inflammation with variable multiple organ involvement and damage, often including the heart and also the exocrine pancreas. the study was funded by texas a&m university. the primary author and two co-authors work at the gi laboratory, texa a&m university. canine bartonellosis is increasingly recognized worldwide and may be associated with diverse clinical manifestations. recent evidence suggests that bartonellosis also causes lameness and polyarthritis in dogs. however, pcr amplification of bartonella dna and isolation of bartonella species from canine synovial fluid (sf) samples have rarely been reported. canine leishmaniosis (canl) due to leishmania infantum is a multisystemic disease commonly associated with polyarthritis. based on the hypothesis that concurrent bartonella infection may be a contributing factor for the development of arthritis in dogs with canl, the main objective of this study was to investigate the microbiological and molecular prevalence of bartonella spp. in dogs with naturally-occurring canl, with or without cytologically documented arthritis. from a previous study, dogs with canl were retrospectively studied for bartonella spp. infection. diagnosis of canl was based on compatible clinical and clinicopathological abnormalities, positive serology, and lymph node or bone marrow (bm) cytology. dogs with serological evidence of other vector-borne infections (anaplasmosis, borelliosis, dirofilariosis and ehrlichiosis) and dogs recently vaccinated or medicated were excluded from the study. arthritis defined as a neutrophil percentage in excess of % of nucleated cells in sf cytology was documented in / ( . %) of dogs. a total of archived specimens from dogs, including edta-anticoagulated blood samples, bm and sf aspirates were tested for bartonella spp. dna using a bartonella alpha proteobacteria growth medium (bap-gm) diagnostic platform. eight ( . %) dogs were infected with one or two bartonella species, including candidatus bartonella merieuxii(n = ), b. henselae sa (n = ) and b. rochalimae (n = ). bartonella spp. dna was amplified from bm in dogs and from blood in dogs but was not amplified from any sf sample. overall, ( . %) dogs with and ( . %) dogs without arthritis were infected with a bartonella species. the prevalence of bartonella spp. dna in the dogs with or without arthritis did not differ (v test for independence, p = . the prevalence of giardia in dogs ranges between . % and . %, with a higher prevalence in puppies. however, the risk factors for giardia infection around weaning have been poorly described. the aim of the study was to evaluate risk factors for giardia infection in puppies during the first weeks of life and to determine an impact of this parasite on feces quality. puppies from litters living in a breeding kennel were followed between and weeks of age. each puppy was treated with fenbendazole (panacur â , msd, france, mg/kg, per os, q h) for consecutive days at , , and weeks of age. for each puppy, fecal consistency was evaluated using a -point scale. excretion of enteropathogens was evaluated by qpcr for canine parvovirus type (cpv ), qrt-pcr for canine coronavirus (ccv), coproantigens quantification for giardia (prospect-giardia, remel), and mcmaster flotation technique for any eggs and oocysts. a generalized linear mixed model (proc glimmix) with giardia infection as a binary outcome was used to assess the following effects: breed size, age, and cpv , ccv and isospora ohioensis infections. a linear mixed model (proc mixed) with fecal score as outcome was used to determine the following effects: breed size, age, and giardia, cpv , ccv and i. ohioensis infections. a total of fecal samples were collected; cpv , giardia, i. ohioensis and ccvwere detected in respectively . %, %, . % and . % of the samples. the risk of giardia infection increased with age (odd ratio= . ; %ci= . - . ; p = . ). neither breed, nor cpv , ccv and i. ohioensis infections influenced risk of giardia infection (p = . ; p = . ; p = . ; p = . respectively). giardia infection did not impact feces quality (p = . ), whereas a significant influence of cpv (p < . ), ccv infection (p = . ) and breed size (p < . ) was evidenced. this study underlines that even with an adapted deworming program the eradication of giardia is difficult to obtain in large dog packs. the higher prevalence of giardia in puppies of weeks and older could be linked with the immunity gap during this period. giardia was not associated in our study with an increased risk of diarrhea. the lack of pathogenicity of the parasite per se could be hypothesized, but also an efficacy of the treatment for the prevention of the clinical signs or a local and systemic immunity limiting clinical signs. financial support from royal canin. hepatic encephalopathy (he) can occur in dogs as a complication of primary liver disease or as consequence of congenital portosystemic shunts (pss). the aim of this study was to assess magnetic resonace spectroscopy (mrs) usefulness in he diagnosis. twenty dogs with a presumptive diagnosis of he were enrolled. inclusion criteria were: a clinical examination, a blood cell count and biochemical panel, at least one plasmatic ammonia determination higher than lmol/l, optional hystopatology of the liver, requirement of magnetic resonance imaging (mri) investigation by the clinician. even though mrs represents a non-invasive procedure, informed consent was obtained from dogs'owners. he diagnosis was confirmed in / : / had a pss, / showed hepatic microvascular dysplasia and / had hepatic cirrhosis histologically confirmed. the control-group was made of patients retrieved from our database that underwent mri without showing any abnormalities on brain scans. the mr protocol included t -weighted fast spin-echo, t -weighted sequences, proton density, and radio-frequency pulses gradient-echo. on mri examination cerebral atrophy was evident in all the patients ( / ), ranged from mild to severe. in / patients symmetrical bilateral hyperintensities in the globus pallidus on t -weighted images were evident. mrs was performed using a short te ( ms) and it was defined a volume of interest in the parieto-occipital region by -mm side cube ( cc of volume). glutamine-glutamate (gln), n-acetylaspartate and n-acetyl aspartyl glutamate (naa), choline derivatives (cho), and myo-inositol (ins) were analyzed. comparing spectra from the he affected dogs with those from the control-group decreased values of ins, cho, naa as well as high field mri combined with brain mrs provide accurate and non-invasive diagnosis of canine he. in accordance with human medicine publications, it could be state that mrs has a role in he diagnosis and follow-up with particular mention monitoring. no conflicts of interest reported. diagnosis of hepatobiliary diseases often requires hepatic tissue sampling for histologic evaluation. the laparoscopic technique is a safe method and allows acquisition of tissue by wedge and needle biopsies from different liver lobes in a minimally invasive way.specimens obtained with an -gauge biopsy needle must be interpreted with caution due to considerable variability in tissue involvement with certain disease processes. we hypothesized that needle biopsy specimens would produce findings divergent from those produced by wedge biopsy specimens. the goal of the study was to compare histological findings from two laparoscopic biopsy methods (wedge and needle) and assess which sampling technic can represent the overall disease process. procedure: all dogs included in this prospective study study were suspected diffuse hepatic disease and underwent laparo-scopic hepatic biopsy (wedge and needle - g) between and . all biopsy specimens were examined on the basis of morphologic criteria and a comparison was made between the two types of biopsies procedures according to wsava liver standardization group morphologic criteria. results: twenty-two dogs were included. no complications were reported during the laparoscopic procedure. the median number of portal triads per needle biopsy specimen was (range, to ) compared to (range ; ) with wedge biopsy specimen. the median length of needle biopsy specimens was mm; (range, to mm) and mm for all wedge biopsies. on the basis of biopsy interpretation, the diagnosis was overall similar with the two methods: dogs had vacuolar hepatopathy, acute cholangitis, non-specific acute form in dogs. chronic hepatitis with cirrhosis was found in cases, dogs had diffuse neoplasia and miscellaneous hepatic disorders. the fibrosis was considered to be severe in dogs, moderate in dogs and mild in dogs. no quantitative and qualitative difference was observed between the two types of biopsies specimen. this study demonstrates that the biopsies with a needle length of at least mm brings satisfactory information for the evaluation of most of the inflammatory, vacuolar hepatopathies, fibrosis and diffuse tumoral infiltrations. wedge biopsies allow to examine the largest number of portal triad, more contributory for certain forms of cholangitis affecting larger canals and for a single case, images of peri-hepatitis were counted at the level of the capsule. fibrosis does not seem to be more important in the sub-capsular zone contrary to what is observed in human pathology. no conflicts of interest reported. indocyanine green (icg), a fluorescence dye, is excreted solely by the liver without enterohepatic re-circulation. hence it has been used for decades as an ideal albeit invasive marker of hepatic function and blood flow in cats and dogs. here we evaluated the feasibility of a minimally invasive transcutaneous icg clearance to assess hepatic function instantaneously. transcutaneous icg clearance was performed in healthy research cats and healthy research dogs with normal liver function (bile acid stimulation test, ammonia tolerance test) using a modified device (kidney int : with an excitation wave length of nm and an emission wave length of nm. the devices were placed on different locations (lateral thoracic wall, ventrolateral abdomen, metatarsus and antebrachium) and fixed with a light bandage. to find a suitable dose to reach adequate transcutaneous peak concentrations escalating doses of icg ( . , . and . mg/kg) were injected intravenously with a wash out period of at least h in-between. measurement was continued for hour after injection in awake animals moving freely. the resulting icg disappearance curves were visually inspected to find best location (minimal artifacts, acceptable background noise) and dose. in all animals a dose of . mg/kg was deemed ideal and the thoracic and abdominal wall gave consistent results. half-life of icg clearance was calculated using a one-compartment model. half-lives in cats were . , . and . minutes, respectively; in dogs: . , . and . minutes, respectively. in conclusion, the transcutaneous assessment of icg clearance is feasible in a clinical setting. results are obtained within one hour and can be assessed instantaneously. the procedures are minimally invasive and well tolerated by the animals. given that most patients with a presumed liver problem undergo abdominal ultrasound no further clipping of hair is necessary as the device might be placed in this area. further studies are necessary to obtain reference values in healthy pets and those with various conditions leading to impaired hepatic function. no conflicts of interest reported. gallbladder diseases like gallbladder mucocele and cholecystitis can reduce gallbladder motility and may lead to cholestasis. since impaired gallbladder emptying contributes to sludge and gallstone formation, the evaluation of gallbladder motility requires accurate and appropriate methodology. three-dimensional ( d) ultrasonography has been shown to be accurate and appropriate tool for measurement of gallbladder volume in humans. therefore, we applied this novel technique for the first time to study preprandial and postprandial gallbladder volume in healthy mixed-breed dogs and compared the results to twodimensional ( d) ultrasonography. the dogs were placed in dorsal recumbency to obtain ultrasonographic measurements of the gallbladder. measurements by both d and d ultrasonography were recorded in preprandial state and after ingestion of full-fat milk. the preprandial and postprandial gallbladder volumes determined by d ultrasonography were significantly higher than corresponding volumes by d ultrasonography ( . ae . vs . ae . and . vs . ml/kg, respectively, p < . ). in d ultrasonography, most dogs ( / [ %]) had a preprandial gallbladder volume ≤ . ml/kg. however, in d ultrasonography, / ( %) of dogs had a preprandial gallbladder volume ≥ . ml/kg. gallbladder contraction index was higher in d ultrasonography than d ultrasonography, however, it did not reach statistical significance (p = . ). in conclusion, d ultrasonography showed larger gallbladder volumes than d ultrasonography in healthy dogs. it seems that d ultrasonography is appropriate adjunct device to d ultrasonography to estimate gallbladder volume when d ultrasonography could not detect whole gallbladder volume. more research is needed to determine clinical value of d ultrasonography in canine gallbladder imaging. no conflicts of interest reported. the aim of our study was to compare high-definition oscillometry (hdo) and doppler ultrasonographic measurements with direct blood pressure measurements in conscious dogs. the doppler study was performed by three investigators and by using different sphygmomanometers with different sized cuffs. devices and measurement sites were changed randomly among the investigators. cuffs were wrapped around the antebrachium in the forelimb or around the mid-metatarsus in the hind limb. in addition to the limb sites, cuffs were also placed around the base of the tail in case of the hdo method. cuff sizes were - % of the measured limb circumferences during the doppler measurement. for the hdo method all measurements were performed by the same investigator and the cuffs provided by the manufacturer were used: the smallest cuff was used for the hind limb and tail, and the medium cuff was used for the forelimb measurements. dogs were gently held in lateral recumbent position, measurement were performed on the nondependent limbs. radio-telemetry transducers were implanted to the right femoral artery some months to a year preceding the blood pressure measurements for reasons unrelated to our study. direct blood pressures varied - mmhg and - mmhg during the doppler and hdo measurements, respectively. two-hundred paired simultaneous doppler and direct measurements from dogs and paired simultaneous hdo and direct telemetric measurements from dogs were obtained. at least successful consecutive measurements could be obtained by the same investigator at the same site during the doppler or hdo measurement in and cases, respectively. thus, the mean of these measurements could be calculated similarly to the established everyday clinical practice. bias (mean difference), precision (standard deviation) and limits of agreements were calculated both from the individual paired measurements and from the means of the consecutive measurements using bland-altman spot analysis. systolic measurement performed on the tail with the hdomethod yielded the smallest bias and deviation and the best limits of agreement during this study. both doppler and hdo-measurements performed on the forelimb overestimated, while hind limb measurement underestimated the direct telemetric pressures. results of all three measurement sites by hdo performed better than forelimb or hind limb doppler-measurements, however hdo-measurements were more difficult to obtain and more often resulted with measurement failure compared to the doppler technique. cuff size above % of the measured limb circumference showed better results than smaller cuff sizes during the doppler measurements. no conflicts of interest reported. the doppler technique is considered the most repeatable indirect method to measure systolic arterial pressure (sap) in dogs. however, recent studies emphasized the effect of body position and used limb on sap measurement. the aim of this study was to determine whether a difference existed in sap measured simultaneously in dogs using different limbs, with two doppler units by two different operators. sixty clientowned dogs, admitted to the veterinary hospital for different reason, were enrolled. they were divided in groups based on body size: small breed dogs (< kg); medium breed ( - kg); large breed (> kg). for each dog the anxiety status was recorded. sap was measured via doppler technique when dogs were in right lateral recumbency in a quite environment. right and left forelimb sap and left forelimb and left hindlimb sap were recorded simultaneously, with two identical doppler units equipped with headphones, by two operators. measurement was performed based on the acvim guidelines. five measurements were recorded, the higher and lower values were discarded from the analysis. the relationship of mean sap for each limb with body weight, sex, anxiety status and sap value was evaluated. mean ae sd sap was significantly higher for the right forelimb ( . ae . ) compare to the left forelimb ( . ae . ) on overall population. the difference was significant for large breed dogs, males and dogs with sap ³ mmhg. sap was higher for the left forelimb ( . ae . ) compare to the left hindlimb ( . ae . ) on overall population. the difference was significant for medium and large breed dogs, females, calm animals and dogs with sap ³ mmhg. the mean sap from the left forelimb recorded by two different operators at two different moments, were compared and no difference was evident. in conclusion, sap measurement from different limbs, in dogs in right lateral recumbency, is poorly correlated. measurement of sap from the left forelimb is more repeatable during time and between different operators. sap trend monitoring should be done using the same measurement site for any animal. no conflicts of interest reported. amlodipine has been considered the treatmenf of choice for hypertension in cats for more than a decade. there is, however, an unmet need for a cat-specific formulation. the aim of the study was to assess the efficacy of chewable amlodipine tablets in reducing systolic blood pressure (sbp) in cats diagnosed with hypertension. seventy-seven client-owned cats were included in the study (mean age years). the study was randomised, double-blind, placebo controlled, and consisted of two phases. in the blinded phase, cats received . mg/kg amlodipine once daily for days. if they responded the dose remained the same to day . for non-responders, the dose was increased to . mg/kg. thirty-five cats received placebo following the same protocol. arterial blood pressure was measured using a high definition oscillometry method. at day a responder was defined as a cat showing a decrease of sbp to ≤ mmhg or a decrease from baseline of at least %. after days all cats continued with amlodipine for - months in an open phase with the placebo cats repeating the same dose escalation protocol as in the blinded phase. the responder rate was % in the amlodipine group and % in the placebo group following the dose escalation from day being applied to % and % of cats receiving amlodipine and placebo respectively. cats receiving amlodipine were . ( % ci . to . ) times more likely to be classified as responders when compared to those receiving placebo (logistic regression model, p = . ). from a baseline value of . ae . and . ae . mmhg the mean sbp decreased to . ae . mmhg with amlodipine and to . ae . mmhg with placebo (repeated measures analysis of covariance model, p < . ) by day . the responder rate was not influenced by factors other than amlodipine treatment (e.g. baseline blood pressure, concomitant ace inhibitor therapy, renal disease). there were no differences between the amlodipine and placebo groups in the frequency of adverse events reported during the -day blinded phase. likewise, there were very few changes in the laboratory values over time in either group. the present study is the first large clinical trial to show that amlodipine is clearly superior to placebo in the treatment of cats with hypertension. the chewable amlodipine formulation effectively reduced sbp, had a good palatability and was well tolerated. it can be used concomitantly with ace inhibitors and in cats with renal disease. conflicts of interest: m. huhtinen and j. aspegr en are employees of the sponsor j. elliott has the following information to disclose: amlodipine is the treatment of choice for feline hypertension. limited published data exist on serum concentrations achieved in hypertensive cats. the aim of the study was to assess serum amlodipine concentrations in cats treated with a new formulation of amlodipine and relate these to the blood pressure reduction achieved. seventy-seven client-owned hypertensive cats were enrolled into a randomized, double-blind and placebo controlled study consisting of two phases. in phase one, cats (group a) received . mg/kg amlodipine once daily for days. if they were deemed to have responded (see below) the dose remained the same to day . for non-responders, the dose was increased to . mg/kg. thirty-five cats (group b) received placebo following the same protocol. blood pressure was measured using high definition oscillometry. a responder was defined as a cat showing a decrease of systolic blood pressure (sbp) to ≤ mmhg or a decrease from baseline of ≥ %. following day (phase ), group a continued on amlodipine and group b switched to amlodipine and the dose was adjusted as per phase . both groups were followed for days on amlodipine. blood was collected at days (group a) and (both groups) and serum [amlodipine] measured by liquid chromatography mass spectrometry. the sbp measured on treatment was calculated as percentage of the baseline sbp and plotted against serum [amlodipine] using a sigmoidal emax model (winnonlin software). data are expressed as mean ae se. the serum concentrations of group a cats that remained on . mg/kg were . ae . ng/ml whereas those switched to . mg/kg were . ae . ng/ml. when data from groups a and b were pooled, a sigmoidal relationship between percentage baseline sbp and serum [amlodipine] was found. estimated values of lowest percentage baseline blood pressure on treatment (emax) was . ae . %, with an ec value of . ae . ng/ ml and a slope function of . ae . . the serum concentration required to reduce blood pressure by % was estimated to be ng/ml. the present study related blood pressure reduction to serum [amlodipine] in feline clinical hypertension. the limitations of this study were the limited number of blood samples collected and lack of information relating to the exact timing of blood sampling relative to dosing in some cats. however, these data could be used to define appropriate therapeutic serum [amlodipine] in hypertensive cats. m huhtinen is an employee of the sponsor; j. elliott has the following information to disclose: consultancy: pfizer animal health / zoetis, ceva animal health, boehringer ingelheim, vetoquinol ltd, orion ltd., elanco ltd, idexx ltd, niche generics ltd. triveristas ltd., virbac ltd., advisory board membership: international renal interest society (supported by novartis) european emesis council (sponsored by pfizer animal health -now zoetis) cardiorenal board -vetoquinol ltd. idexx renal advisory board research grants or contracts: vetoquinol ltd, novartis ltd, pfizer animal health ltd (now zoetis), royal canin ltd, boeringher ingelheim ltd, waltham centre for pet nutrition, ceva animal health orion ltd.; l pelligand has the following information to disclose: in receipt of research grant / contract funding from orion ltd., novartis animal health, transpharmation ltd, deltadot ltd; acted as a consultant for: triveritas ltd. and novartis animal health. commercial assays for the measurement of canine and feline pancreatic lipase immunoreactivity (spec cpl â and spec fpl â , respectively; idexx laboratories, westbrook, me, usa) have been available for a few years and have previously been analytically and clinically validated. recently, new commercial assays for the measurement of these parameters have become available, though neither one of these assays have been analytically or clinically validated in the literature. thus, the goal of this study was to compare these newly available assays to the established assays. leftover serum samples from diagnostic submissions to the gi laboratory were collected based on certain parameters (e.g., results throughout the working range of the assay, good quality sample or hemolytic, lipemic, or icteric sample) and were assigned random sample id numbers. the samples were evaluated by spec cpl â or spec fpl â , sent on dry ice to the klinik am hochberg, and one aliquot of each sample was blindly submitted to laboklin for measurement of cpli and fpli by their newly released in-house assay and also to the gi lab at texas a&m university for repeated analysis by spec cpl â and spec fpl â to exclude any effect of shipping. there was no significant difference between serum cpli or fpli concentrations before or after shipping at the gi lab (pvalues for wilcoxon matched-pairs signed rank tests: . and . , respectively). in contrast, there was a significant difference between serum cpli or fpli concentrations between the newly released assays and the previously established assays (pvalues < . and . , respectively). while there was a significant correlation between the newly released and the previously released assays (spearman r: . and . , respectively), this correlation was very poor for assays that supposedly measure the same analyte. also, the interpretation for serum cpli and fpli results between the previously developed assays and the new assays did not agree for many of the samples. finally, both newly developed assays showed some erratic results. in conclusion, the newly released assays for the measurement of cpli and fpli do not agree with previously established and validated assays, provide different interpretations, and show erratic results. thus, further research is needed before these newly released assays could be recommended for clinical use. dr. steiner serves as director and dr. suchodolski serves as associate director of the gastrointestinal laboratory at texas a&m university. dr. steiner also serves as a paid consultant to idexx laboratories, westbrook, me, usa. both the gastrointestinal laboratory and idexx laboratories offer cpli and fpli testing on a fee-for-service basis. digestive health is a main concern for growth, morbidity and mortality in weaning puppies. fecal immunoglobulin a (iga) has been suggested as a useful noninvasive biomarker for mucosal immunity. the purpose of this study was to evaluate the effect of infection with enteropathogens on fecal iga concentrations in puppies and that of physiological factors such as age and breed size. puppies from breeding kennels were included in the study. puppies were between and weeks of age (meanaestandard deviation (sd): . ae . weeks). depending on the mean adult body weight of their respective breed, the puppies were divided into small (if mean adult body weight < kg) or large (> kg) breed puppies. for each puppy, fecal consistency was evaluated using a -point scale and feces were collected for the evaluation of presence of fecal enteropathogens and fecal iga concentrations. the presence of enteropathogens in fecal samples was evaluated by qpcr for canine parvovirus type (cpv ), qrt-pcr for canine coronavirus (ccv), coproantigen quantification for giardia (prospect-giardia, remel), and mcmaster flotation technique for other parasite eggs and oocysts. fecal iga concentrations were measured by an elisa test. statistical analyses were performed using sas software. a linear mixed model (proc mixed) with fecal iga concentration as outcome was used to determine the following effects: enteropathogen infection, breed size, age, and fecal score. the respective influence of litter and breeding kennel as random effects was also determined. data is presented as mean ae sd. small breed dogs represented . % ( / ) of the total number of dogs included. at least one enteropathogen was identified in . % of puppies ( / ). fecal iga concentration was significantly influenced by fecal enteropathogens (p = . ). puppies infected with at least one enteropathogen had significantly lower fecal iga concentrations than puppies without any enteropathogens ( . ae . lg/g vs. . ae . lg/g). breed (p = . ), but not age (p = . ), influenced iga concentration. small breed puppies had significantly higher fecal iga concentrations than large breed puppies ( . ae . lg/g vs. . ae . lg/g). no significant relationship between fecal iga concentration and feces quality was evidenced (p = . ). this study suggests that fecal iga concentration is a promising marker for subclinical infection by at least one enteropathogen and confirms that digestive physiology varies with the breed size. a link between lower digestive immunity and higher susceptibility to enteropathogen infection needs further investigation. conflicts of interest: financial support of royal canin. canine chronic enteropathies (cce) include diet-responsive, antibiotic-responsive, and immunosuppressive-responsive enteropathies (ire). this prospective study was designed to evaluate a commercial hypoallergenic dry diet a containing oligopeptides as the only protein source for the management of dogs with ire and as an alternative to immunosuppressive therapy over a week period. nineteen dogs across france and quebec entered the study. dogs with food or antibiotic-responsive chronic enteropathy, hypoproteinemia, or treated with immunomodulating drugs were excluded from the study. dogs were included in the study after complete clinical, ultrasonographic, endoscopic evaluation and histopathological evaluation of intestinal biopsies showing signs of intestinal inflammation. the owners were instructed to feed exclusively the study diet a . canine inflammatory bowel disease activity index (cibdai) scores, fecal scores as observed by the dog-owners, and body weight were evaluated at baseline, , and weeks after inclusion. dietary treatment was regarded successful if the cibdai score was reduced by at least %. the protocol has been reviewed and accepted by royal canin ethics committee and owners completed an informed consent. results are presented as meanaesd (range). statistical comparisons were performed with a wilcoxon test. thirteen dogs ( intact males, neutered and intact females) completed the trial. seven dogs were excluded ( diagnosed with giardia, s with no histological evidence of inflammation, with hypoadrenocorticism). mean age was . years ae . ( . - . ), mean body weight . kg ae . ( . - . ). cibdai score was . ae . ( - ) at inclusion, was . ae . ( - ) after weeks and was . ae . ( - ) after weeks (p = . and p = . vs inclusion, respectively). fecal scores after [ . ae . ( - )] and weeks [ . ae . ( - ) ] were improved compared to inclusion scores . ae . ( - ) (p = . and p = . , respectively). the low molecular weight poultry feather hydrolyzed proteinbased dry extruded diet a appears to be effective in the management of idiopathic ibd without any concurrent immunosuppressive drug over the week period of this pilot study. these preliminary findings should be confirmed by a prospective, randomized double blind study. feline pancreatitis is the most common exocrine pancreatic disorder with varied mortality. however, there is no available and reliable method to evaluate the severity and prognosis of the disease. ninety-two cats diagnosed as pancreatitis with acute onset of compatible clinical signs and a positive snap â fpl tm test between october and september were enrolled in this study. all cats were divided into survival (n = ) and nonsurvival (n = ) groups. fifty-two parameters including signalments, clinical signs, physical examinations, clinicopathological examinations, diagnostic images, complications and concurrent diseases were analyzed and compared between the two groups. parameters with p ≤ . were considered for further analyses. the mortality in this study was . %. hematocrit, albumin, bun, creatinine, total bilirubin, calcium, phosphorous, body temperature, systolic blood pressure, the body cavity fluids, complications, e.g. systemic inflammatory response syndrome (sirs) and acute renal failure (arf) were found to be significantly associated with disease severity and prognosis, and were selected for constructing the scores. continuous variables outside the reference interval were separated into quartiles to yield quartile-specific odds ratios (ors) for survival. based on the integer value of the or, the scoring system was then developed by incorporating weighting factors assigned to each quartile. a predictive total score was calculated for each cat by summing all weighting factors. the total scores of each cat ranged from to . the severity scores in this study achieved an area under receiver operating characteristic (auroc) of . . the optimal cut-off point for discriminating outcome was . with the sensitivity of . % and specificity of . %, respectively. the mortality was . % with a score ≥ , whereas . % with a score ≤ . there was significant difference (p < . ) between the two groups of the cut-off point. furthermore, the mortality reached to % when the score more than . the severity scoring system of this study provides a reliable and clinical applicable method to predict clinical outcome in cats with pancreatitis. no conflicts of interest reported. convincing evidence for the role of clostridium (c.) perfringens as a primary pathogen in acute haemorrhagic diarrhoea syndrome (ahds) in dogs was recently found. it is suspected that clostridial toxins, especially c. perfringens enterotoxin, play a relevant role in the disease process. however, to date enterotoxigenic c. perfringens strains have only been described in single case reports. thus, the aim of this study was to indentify the specific c. perfringens genotype involved in adhs. small intestinal biopsies were collected with a sterile single-use biopsy forceps from ten dogs with ahds and immediately cultured. in / dogs, clostridial strains were isolated and identified as c. perfringens by mass spectrometry using maldi-tof ms. c. perfringens colonies from each dog were submitted for specific detection of the four major toxin genes (alpha, beta, epsilon, and iota), the enterotoxin gene, and the beta toxin by multiplex pcr. every clostridial isolate was typed as c. perfringens type a based on the detection of the alpha toxin encoding gene. in / isolates, additionally the beta toxin gene was identified, however, none of clostridial strains encoded for the c. perfringens enterotoxin gene. the results of this study suggest that c. perfringens type a is the most important c. perfingens genotype involved in the disease process of dogs with ahds. although c. perfringens enterotoxinhas been associated with intestinal diseases in humans, dogs, horses, pigs, and other animal species, this enterotoxin is most likely not responsible for the intestinal lesions in dogs with ahds. no conflicts of interest reported. p < . ). lack of treatment response was significantly associated with il (il: / , sre: / , fre: / , are: / ; p < . ). anemia, thrombocytopenia, and increased plasma urea were significantly associated with il (anemia: il: / , sre: / , fre: / , are: / , p < . ; thrombocytopenia: il: / , sre: / , fre: / , are: / , p = . ; increased urea: il: / , sre: / , fre: / , are: / , p = . ). hypoalbuminemia (< g/l) and hypocobalaminemia (< pg/ml) occurred significantly more frequently in dogs with sre (hypoalbuminemia: il: / , sre: / , fre: / , are: / , p < . ; hypocobalaminemia: il: / , sre: / , fre: / , are: / , p = . ). results of this study show that elderly and large breed dogs were more frequently affected with il and sre compared to other etiologies and both il and sre were associated with greater disease severity and/or a negative outcome. in comparison, anemia, thrombocytopenia, and increased plasma urea were most frequently detected in il whereas severe hypoalbuminemia and hypocobalaminemia were significantly associated with sre. no conflicts of interest reported. alpha -proteinase inhibitor (a -pi) is a proteinase-resistent protein that can be quantified in fecal, urine, and serum samples from dogs. recently, increased fecal and urinary canine a -pi (ca -pi) concentrations have been described in dogs with gastrointestinal diseases (e.g., inflammatory bowel disease [ibd] , but also in dogs with exocrine pancreatic insufficiency) and in dogs with chronic hepatitis or chronic kidney disease, respectively. decreased serum ca -pi concentrations have been reported in dogs with ibd, protein-losing enteropathy (ple), and hypocobalaminemia. treatment protocols for dogs with ibd and/or ple commonly include corticosteroids, but the effect of corticosteroid therapy on serum ca -pi concentrations have not yet been reported. the aim of this study was to evaluate the effect of hydro-cortisone on serum ca -pi concentrations in healthy dogs. twelve healthy beagle dogs were randomly allocated to a placebo-group (n = ) and to a treatment group (n = ; hydrocortisone-group). the placebo-group received an empty gelatin capsule po q h, whereas the hydrocortisone-group was treated with hydrocortisone at a dose of . mg/kg po q h. serum samples were obtained at baseline and on day , , , , and during treatment as well as day , , , , and post-treatment for all dogs. serum ca -pi concentrations were measured at all time points using an in-house radioimmunoassay. a mann-whitney u test was used to compare the baseline measurements of both groups. the effect of hydrocortisone-treatment on serum ca -pi concentrations was evaluated by comparing ca -pi at baseline and during treatment and between baseline and posttreatment period using a manova. baseline serum ca -pi concentrations did not differ between the hydrocortisone-and the placebo-group (p > . ). serum ca -pi concentrations increased significantly (p = . ) during the treatment period in the hydrocortisone-group (baseline [median in mg/l: , ] ). in contrast, no difference was observed between both groups when comparing serum ca -pi concentrations at baseline and during the post-treatment period (p > . ). this study showed that hydrocortisone-treatment over weeks did affect serum ca -pi concentrations in healthy dogs. whether corticosteroid therapy has any effects on fecal or urine ca -pi concentrations in healthy dogs remains to be determined. the author works at texas a&m university, whose gi lab currently offer a commercial assay for faecal alpha -proteinase inhibitor. canine chronic enteropathy (ce) is a common, but poorly understood syndrome, with variable response to therapy and prognosis. there is a need for novel biomarkers that are specific for intestinal disease and that provide objective measures of disease severity, progression, and prognosis. serum citrulline is a useful biomarker in human intestinal disease as it is specific to the small intestine and indicates globally reduced enterocyte mass and absorptive function in various disease states. it is used to determine, quantitatively, intestinal integrity at the enterocyte level and is not influenced by nutritional or inflammatory status. the aim of this study was to determine whether serum citrulline can be used as a biomarker for ce in dogs. in this retrospective study, computer records from the university of liverpool small animal teaching hospital were used to identify dogs with ce. disease severity was quantified by cibdai. controls were age-and breed-matched dogs without gastrointestinal disease. serum citrulline was measured by ultra-high performance liquid chromatography with tandem mass spectrometry. in dogs with ce, serum citrulline concentration was measured at presentation and at various time points after starting treatment. serum citrulline was measured in dogs with ce and controls. dogs responded to dietary manipulation (food-responsive enteropathy, fre) and responded to antibacterials (antibiotic-responsive diarrhoea, ard), with a further having invasive mucosal bacteria, of which one responded to antibacterials and one was refractory. dogs were diagnosed with idiopathic ibd (on the basis of exclusion of known causes and failure to respond to therapeutic dietary and antibiotic trials), of which responded to immunosuppressive therapy, were refractory, and were lost to follow-up. serum citrulline concentration did not differ between dogs with ce (median . lg/ml, range . - . ) and controls (median . lg/ml, range, . - . , p = . ). there was also no difference in serum citrulline concentration amongst dogs with fre, ard, ibd, and controls (p = . ). serum citrulline did not differ between dogs that responded well, or were refractory to treatment (p = . ), between dogs with and without protein-losing enteropathy (p = . ), or between dogs that survived and that were euthanased because of ce (p = . ). serum citrulline did not correlate with cibdai (r = . ). these findings do not support the use of serum citrulline as a biomarker in determining diagnosis, prognosis, or quantifying severity in dogs with ce. one of the co-authors (marco caldin) has a diagnostic laboratory offering citrulline assays. chronic enteropathy (ce) is a multi-factorial disease, which involves aberrant immune responses to commensal bacteria or dietary antigens. macrophages have an important role in human disease but little information is available in canine intestine. data to date have relied solely on macrophage identification using mac , an antibody directed against calprotectin, which recognizes both macrophages and neutrophils. in this study an alternative antibody for macrophages, am- k, directed against a scavenger receptor (cd ) was used and distribution of both markers was compared. this antigen is of interest as positive cells accumulate in intestine of humans with ce. endoscopic duodenal biopsies were obtained from seven crossbreed dogs. serial histologic sections were stained with mac or am- k. positively-stained cells were counted from random areas from both villous and crypt regions. stained cell localisation was subjectively evaluated and the percentage of positively stained cells from the total nucleated cells per , lm in the villus or crypt was compared between both antibodies using a wilcoxon signed-rank test. mac and am- k did not co-localize on serial sections. there were significantly more am- k positive cells than mac in the crypts ( . % [ - . ] versus . % [ - . ], p = . ). in contrast there was no difference in expression of either markers in the villi ( . % [ - . ] versus . % [ - . ], p = . ). this study reports for the first time the existence of two populations of macrophages in canine intestine. these results in normal dogs will be used to explore further the distribution and function of macrophages in dogs with ce. no conflicts of interest reported. chronic diarrhea and vomiting are common clinical signs in dogs. primary (e.g., inflammatory, infectious, neoplastic, mechanical, or other) and secondary gastrointestinal diseases (e.g., exocrine pancreatic, hepatic, renal, or endocrine disease) are possible underlying causes. the aim of this study was to evaluate the final diagnoses in dogs with chronic diarrhea and/or vomiting and to determine the prevalence of various primary and secondary gastrointestinal diseases in dogs with these gastrointestinal signs. medical records of dogs presented between july and august with chronic diarrhea (d), vomiting (v) or both (diarrhea and vomiting [vd]) were retrospectively reviewed. dogs were included if a minimum work-up (hematology, plasma biochemistry profile, and fecal parasitology) had been performed and if a final diagnosis was recorded ( / ). a primary gastrointestinal disease was recorded in % of the cases ( / ) and included inflammatory diseases ( / , exocrine pancreatic insufficiency, hypoadrenocorticism, polyendocrinopathy, dilated cardiomyopathy, and leukemia in one dog each). in total, % of the dogs were presented with d ( / ) followed by % with vd ( / ), and % with v ( / ). d and vd were significantly more frequent in dogs with primary gastrointestinal disease (d: / , vd: / ), compared to dogs with secondary gastrointestinal disease (d: / ; vd: / ; p = . , chi square test). v was significantly more common in dogs with secondary gastrointestinal disease ( / ) as compared to dogs with primary gastrointestinal disease ( / ; p = . ). in this study, food responsive enteropathy ( %) was the most commonly diagnosed cause of chronic gastrointestinal signs. chronic pancreatitis was the most frequent cause of secondary gastrointestinal disease ( %). diarrhea was significantly associated with primary and vomiting with secondary gastrointestinal disease. no conflicts of interest reported. matrix metalloproteinases (mmps) and are zinc-dependent endopeptidases that contribute to the control of breakdown and reconstitution of extracellular matrix under both normal and pathological conditions. intestinal mucosal levels of mmp- and - have been shown to be increased in animal models and human ibd. to our knowledge, the presense of mmp- and - has not been studied in the intestinal mucosal samples of healthy dogs as well as in canine spontaneous ibd. thus, the main aim of this study was to identify the presence of mmp- and - in the mucosa of the small and large intestines of clinically healthy beagle dogs using gelatin zymography technique. for the study, historical intestinal tissue samples from four different parts of the intestine (duodenum, jejunum, ileum and colon) were used. the samples were taken and snap frozen in liquid nitrogen during necropsy from healthy laboratory beagle dogs after being euthanized when finishing unrelated long-term trials studying canine intestinal microbiota. based on wsava histology standards, recorded findings of all samples were considered insignificant. pro-mmp- and - activities were found in / ( %) and / ( %) of the samples, respectively. among four different parts of the intestine of dogs, the ileum had the highest positivity rates of / ( . %) and / ( . %) for pro-mmp- and - activities, respectively. however, statistical analysis showed no significant difference of pro-mmp- and - activities between the separate parts of the intestine (p > . ). the enzyme activities ranged for pro-mmp- between . and . arbitrary units (au) and for pro-mmp- between . and . au. none of the intestinal samples showed gelatinolytic activity corresponding to the control bands of active mmp- and mmp- . this study showed that pro-mmp- and - could be detected in the intestinal mucosa of healthy dogs using zymography, which seems to be a useful tool to evaluate the role of mmp- and - in the pathogenesis of canine chronic enteropathies, including inflammatory bowel diseases. no conflicts of interest reported. digestive perforation is called spontaneous when it arises in the absence of foreign body ingestion, gastric dilatation and volvulus, external trauma, or previous digestive surgery. in dogs many predisposing factors have been identified, including antiinflammatory administration, severe hepatic or renal disease, stress, shock, gastric hyperacidity, neoplasia and idiopathic inflammatory bowel disease. spontaneous digestive perforation has been uncommonly reported in cats. the objectives of this study were to describe the clinical characteristics of spontaneous digestive perforation in cats, and to compare the frequency of malignant versus non-malignant causes for these perforations. to be included in this study, the perforation had to be spontaneous and confirmed by exploratory surgery. the medical records of cats diagnosed as having spontaneous digestive perforation between and were reviewed. the mean age of cats was . years ( months to years). five cats had concurrent illnesses including viral upper respiratory tract disease, pancreatitis and chronic kidney disease. the most frequently reported signs included anorexia ( %), vomiting ( %), and lethargy ( %). histological examination was performed in cats and diagnosed alimentary lymphoma in % and inflammatory lesions in the other % of them. six cats had received anti-inflammatory within the previous months. half of them were finally diagnosed with lymphoma. five cats with lymphoma received chemotherapy.three cats died early in the postoperative recovery period, cats were euthanized to days after surgery, and cats were still alive at the end of this study. in the absence of pneumoperitoneum, clinical signs and clinicopathological abnormalities are not specific enough to allow differentiation between cats with gastrointestinal ulceration and those with perforation. in most cases, there is no diagnostic test that individually determine whether perforation has occurred or is impending, and clinicians should use of multimodality diagnostic procedures such as radiography, ultrasonography, endoscopy, and abdominal fluid cytopathology to avoid delay in diagnosis of digestive perforation. histological examination of ulceration is essential as lymphoma should be suspected in all cats presented with spontaneous perforation. the link between anti-inflammatory administration and spontaneous perforation in cats is not established. no conflicts of interest reported. campylobacter species are commonly isolated from faeces of dogs and cats with c. upsaliensis (cu) and c. helveticus (ch) being the most frequently isolated. these two species are usually not considered pathogenic in dogs and cats and are closely related to each other and to c. jejuni, the most common cause of bacterial gastroenteritis in humans in the developed world. interestingly, despite their close genetic relationship, in humans cu is considered a pathogen while ch is not. this study aimed to describe whole genomes of cu and ch isolated from dogs and cats and to in silico investigate their pathogenic potential with comparison to several published genomes of c. jejuni and c. coli. genomic dna was extracted from three isolates of each of cu and ch recovered from the faeces of healthy dogs and cats. sequencing was performed using an illumina miseq to generate base paired reads. reads were trimmed for both length and quality. contigs were assembled using the velvet assembler. the concatenated contigs generated for each assembly were quality ranked (by number, size, maximum length and n ) and the three top ranked assemblies were annotated using the prokka annotation tool. ribosomal mlst nucleotide sequences were used as a proxy for the core genome to compare the phylogeny of cu and ch with other species in the campylobacter genus and visualised as a neighbornet using splitstree. annotated draft genomes were clustered using orthomcl and pathogenic traits were investigated in silico using pathogenfinder and viru-lentpred software. the cu and ch draft genomes were~ . mb and . mb in size, and comprised on average and contigs, and on average and predicted genes, respectively. of these cu had on average and ch hypothetical proteins. using orthomcl, a core genome of and genes resulted for cu and ch, respectively. neighbornet trees based on ribosomal mlst nucleotide sequences and the core genome confirmed the close phylogenetic relationship of ch and cu within the campylobacter genus. pathogenfinder predicted all isolates as human pathogens with probabilities of . - . %. both pathogenfinder and virulentpred identified many pathogenic proteins in cu and ch of different functions (e.g. chemotaxis, transporter and motility systems) but considerably fewer than in c. jejuni and c. coli. this study provides many insights into the pathogenic potential of pet-associated emerging campylobacter pathogens and is to our knowledge, the first to report a draft genome of ch. no conflicts of interest reported. there are only few laboratory markers being evaluated for diagnosing and/or monitoring canine chronic enteropathies, including inflammatory bowel disease (ibd). s a belongs to the s /calgranulin-protein family and has been proposed to play a central role in both innate and acquired immune responses. it has been reported to be increased in stool samples, serum and/or intestinal mucosa in human patients with ibd. myeloperoxidase (mpo) is an enzyme found mostly in granulocytes. intestinal mucosal levels of mpo have been shown to be increased in animal models and human ibd. to date, s a and mpo levels in intestinal mucosal samples have been reported neither from healthy dogs nor from dogs suffering from ibd. to start investigating this aspect in dogs, the objective of this study was to evaluate mucosal s a and mpo levels in the small and large intestines by using enzyme-linked immunoassay (elisa) and spectrophotometric methods, respectively. for the study, historical intestinal tissue samples from four different parts of the intestine (duodenum, jejunum, ileum and colon) were used. the samples were taken and snap frozen in liquid nitrogen during necropsy from healthy laboratory beagle dogs after being euthanized when finishing unrelated long-term trials studying canine intestinal microbiota. based on wsava standards the histologic findings of all samples were considered insignificant. s a concentrations were from the highest to the lowest: ileum, . ( . - . ) lg/l; colon, . ( . - . ) lg/l; duodenum, . ( . - . ) lg/l; and jejunum, . ( . - . ) lg/l. the concentration in the ileum was significantly higher than in all other segments (p < . ), and the colonic mucosal concentration was higher than the jejunal (p < . ). the highest mpo activity was found in the ileum ( . [ . - . ] da/min), followed by jejunum ( . [ . - . ] da/min), duodenum ( . [ . - . ] da/min), and colon ( . [ . - . ] da/min). mpo activity was significantly higher in ileal and duodenal than in colonic mucosal samples (p < . ). the jejunal mpo activity was higher than the colonic and duodenal activity (p < . ). this study showed that using elisa and spectrophotometry allow the detection of canine intestinal mucosal s a and mpo, respectively. the levels on s a and mpo seem to differ between certain parts of the intestinal mucosa of healthy dogs. both assays appear to be useful to further evaluate the role of s a and mpo in the pathogenesis of canine chronic enteropathies, including ibd. dr. heilmann, dr. suchodolski, and dr. steiner have a patent pending that includes the canine s a assay used in this study. the authors declare that they have no further conflicts of interest. the aim of the present study was to establish the incidence of innocent cardiac murmurs in a fairly large number of clinically healthy puppies. a second aim was to evaluate a possible correlation between the presence of an innocent cardiac murmur and a lower hematocrit value. puppies of certain breeds are routinely screened for the presence of congenital porto-systemic shunts in the netherlands. breeders bring their nests to our clinic for individual measurement of blood ammonia concentration. in one year time (from february until january ) dogs of different breeds were examined, with of them being cairn terriers. the age of the dogs varied from to days (mean days). while the breeders were waiting for the blood results, the cardiac auscultation was performed by a single board-certified cardiologist (vsz). hematocrit was measured with an automatized hematology analyzer system from the surplus blood sample. cardiac murmur was found in dogs ( %). in all cases this was a soft ( - out of ) systolic murmur, most of the time with a musical character and with the point of maximal intensity on the left hemithorax, compatible with the description of an innocent cardiac murmur. no murmurs were found that could be compatible with a congenital cardiac anomaly. the hematocrit was significantly (p = . ) lower in the group of dogs with a murmur (mean . %, standard deviation . %) compared to the group without a murmur (mean . %, standard deviation . %). multivariate analysis shows that the presence of murmur is correlated with the hematocrit, but not with the age of the dogs. physiologic anemia has been long suspected to be one of the possible causes of innocent cardiac murmurs in young animals and children. however, according to the authors knowledge, no published reports exist that looked for a possible correlation. which other factors contribute to the presence of an innocent murmur is largely unknown. because of a large overlap between the hematocrit values of dogs with and without a cardiac murmur, measuring hematocrit in a particular pup would not help a less experienced first line veterinary practitioner to decide whether a murmur is innocent or the result of a congenital cardiac anomaly. limitation of this study is that no echocardiography was performed to rule out congenital cardiac anomalies as the cause of the murmurs. neither were the dogs re-checked for spontaneously disappearance of the murmur. no conflicts of interest reported. low intensity systolic murmurs, with point of maximal intensity over the outflow tract on the left side of thorax, are not uncommonly heard during cardiac auscultation of apparently healthy siberian husky dogs, often with excellent exercise tolerance. the origin of these murmurs in athletic dogs such as huskies is unlikely to be due to heart disease but more likely due to turbulent blood flow in the outflow tract caused by a large stroke volume and forceful cardiac contractility in early systole. the differentiation of these murmurs from "pathological"significant murmurs can however be problematic in general practice. the aim of the present study was to investigate the prevalence of murmurs in a sample of successfully racing siberian husky dogs and furthermore to study the phonocardiographic characteristics of these murmurs. phonocardiograms and ecgs were recorded in actively racing siberian husky dogs, with normal or excellent exercise tolerance. normal stamina was confirmed by successful racing. phonocardiograms were easy and rapid to record on a pc laptop connected to the meditron stethoscope in ambulant "field"practice. systole was measured as the duration measured from the onset of the first heart sound to the onset of the second heart sound and the murmur duration from the onset the first heart sound to the end of the murmur. the duration of the first heart sound plus the murmur was measured and calculated as a percentage of the duration of systole. cardiac murmurs of grade - were heard in % of dogs examined. phonocardiogram from these dogs revealed early systolic crescendo-decrescendo or decrescendo murmurs with a duration of maximally % into systole. all dogs with murmurs had a silent pause at the end of systole. ecg was normal in all dogs. murmurs in adult athletic dogs should not be regarded as a definite sign of heart disease. physiological flow murmurs of up to % of systole is a common finding in active siberian husky dogs (prevalence % in the examined sample). phonocardiography is a rapid and practical method for differential diagnoses between pathological murmurs and physiological flow murmurs. no conflicts of interest reported. nt-probnp has a degree of overlap with clinically normal animals, particularly those with mild or subclinical heart disease. prior studies have evaluated the sensitivity and specificity of a point-of-care second generation elisa that utilizes snap technology. the snap feline probnp test uses the same biological reagents as the cardiopet probnp test but provides results in minutes. we sought to prospectively validate the assay in a population of clinically normal cats. cats were recruited based upon the absence of a heart murmur, gallop, and/or arrhythmia. all cats received physical examination, non-invasive blood pressure measurement, complete biochemical analysis including a t , urinalysis and echocardiogram. only cats considered free of underlying cardiac or systemic disease were enrolled. sixteen adult cats were enrolled and blood samples were obtained for nt-probnp concentrations at , hr, hr, hr, hr, hr. samples were placed in edta tubes and centrifuged within one hour and split into two tubes for duplicate samples at each time point and stored at - °c. once all samples were collected, they were shipped on dry ice overnight and run in one batch (idexx laboratories) for measurement of nt-probnp concentrations. snap tests were visually evaluated by one blinded reader. comparison of snap assay vs. quantitative elisa revealed a . (auc) degree of correlation between assays, and that a positive snap test result was associated with a nt-probnp concentration of . pmol/l or greater. the average bnp concentration of abnormal cats ( . ae . ) determined by the snap assay was significantly greater than the normal ( . ae . ). this study was funded through idexx and the university of florida college of veterinary medicine resident grant competition. we acquired d echocardiographic cineloops from the left apical -chamber view optimized for the la, and analyzed atrial longitudinal strain (st) and strain rate (sr) in dogs ( healthy dogs and dogs with mmvd - acvim stage b , stage b and stage c). endocardial la ste curves were obtained and peak atrial longitudinal strain (pals), peak atrial contraction strain (pacs), conduit atrial longitudinal strain (cals); pals-pacs) and contraction strain index (csi -pacs/pals* ) were calculated. la sr curves were similarly obtained to determine the peak positive strain rate (srs) during left ventricle systole, the first negative peak strain rate (sre) during early diastole and the second negative peak strain rate (sra) during atrial contraction. for all variables, a mean of measures was used for the statistical analysis. we compared each of these variables between each acvim stage by kruskal-wallis tests and post-hoc pairwise comparisons, with comparison-wise a= . . normal dogs had higher pals and cals than dogs with mmvd (p < . and p = . ); stage c dogs had lower pals, pacs and cals than all other dogs (p < . , p = . and p = . ), but csi did not differ between groups (p = . ). stage c dogs had lower srs (p = . ), higher sre (p = . ) and sra (p = . ) than other dogs. normal dogs had lower sre and sra than dogs with mmvd (p < . ). our data suggest that ste might be useful in assessing la function in dogs with mmvd, and might potentially differentiate dogs with severe subclinical disease from dogs with congestive heart failure. no conflicts of interest reported. sighthounds are athletic dogs and they have been claimed to have larger hearts compared to similar sized breeds. the left ventricle (lv) may enlarge in response to cardiac disease, but also in response to training, so called athlete's heart syndrome, which is a benign condition. to distinguish abnormal echocardiographic measurements from normal, breed-specific reference values are needed. the aim of this study is to establish normal reference ranges for echocardiographic measurements in the saluki breed. the study comprised clinically healthy salukis ( males and females), mean age months (ae sd months), bodyweight (bw) , kg (ae , kg). case history was ascertained and dogs underwent physical examination, complete blood count, serum biochemistry profile, thyroid profile, blood pressure measurement and -min ecg. standard m-mode and d echocardiographic measurements were obtained. dogs with systolic murmur / , and dogs with mitral valve regurgitation (mr) < % (mr color flow jet area/left atrium areax % in apical view) were considered normal. linear regression models were used to establish reference ranges. heart rate (hr) varied from to bpm ( ae bpm). bw was a significant predictor for lv dimensions, i.e. m-mode lv diameter and d volume in diastole (lvidd and lvedv) and systole (lvids and lvesv), and mitral valve end point septal separation (epss). hr was a significant predictor for fs % (fractional shortening). predicted values ( % prediction intervals) were calculated from regression models where mean bw ( , kg) and age ( months), and median hr ( bpm) were used. normal reference ranges were: lvidd , mm ( , - , ), lvids , mm ( , ) , lvedv , ml ( , - , ), lvesv , ml ( , - , ), fs%: , % ( , - , ), ejection fraction ef%: , % ( , - , ), epss , mm ( , - , ), sphericity index , ( , - , ), interventricular septum in diastole , mm ( , - , ) and systole , mm ( , - , ), lv free wall in diastole , mm ( , - , ) and systole , mm ( , - , ), left atrial (la) diameter , mm ( , ) , aortic (ao) diameter , mm ( , - , ) , la/ao , ( , - , ), and aortic and pulmonic flow velocity , m/s ( , ) and , m/s ( , - , ), respectively. this study provides echocardiographic values for normal salukis which can be used as a reference values. no conflicts of interest reported. in mitral valve disease, atrial remodeling is an indicator of evolution and prognosis, the duration of the p wave being considered suggestive of the dilatation of the left atrium. in humans' studies, neurological conditions have a significant impact on cardiac electrophysiology by altering the electrical impulse conductibility. the aim of this study is to examine the duration of the p wave in dogs suffering from mitral valve disease in comparison to dogs diagnosed with different neuropathies without cardiac abnormalities. we analyzed standard electrocardiograms ( min of ecg, on peripheral leads) performed on three polymorphic groups of dogs (different age, weight and breed): group (n = ) healthy dogs, group (n = ) dogs diagnosed with mitral valve disease and group (n = ) dogs suffering from different neuropathies (without any associated or previously diagnosed cardiovascular disease). the duration of the p wave was measured for all dogs (five consecutive p waves without anomalies or artifacts) and reported to the degree of atrial remodeling, assessed by left atrium/ aorta ratio on echocardiography. the interpretation of the ecg and echocardiography was made by the same examiner (md). the results were statistically evaluated in a specialized program (ibm spss vs. ). the p wave recorded average values was . ae . seconds for the mvd group with significant differences between the stages of heart failure (p = . ). no correlations were found between its increase and the dilation of the left atrium (r = . ). there was no statistically significant difference regarding p wave duration when compared dogs of the neuropathy group and those of the mitral valve disease group (the p wave recorded average values = . ae . sec.). both, atrial tissue lesions (as in mitral valve disease) and autonomic nervous system anomalies (secondary to a neurological condition), may change the conductibility of the electrical impulse in the left atrium. the conductibility of the electrical impulse at this level does not seem to be influenced by its actual dilation, but by the impairment of the intra-atrial and inter-atrial conduction pathways. caution must be given when p wave is analyzed in dogs with concurrent cardiologic and neurologic condition. no conflicts of interest reported. newfoundland dogs were prospectively recruited among those undergoing screening for congenital and acquired heart disease. screening includes patient history, physical examination, and systemic arterial pressure measurement by doppler flow meter and transthoracic echocardiography (m-mode, d and echo-doppler). screening is performed on conscious dogs of at least year of age. dogs without historical, clinical, electrocardiographic and echocardiographic signs of cardiovascular disease were included in the study. unpaired, two-tailed student's t-test and linear regression were performed to evaluate the influence of gender, age and body weight (bw) on echocardiographic parameters. echocardiographic measurements were compared to previously reported reference values. the reference limits of echocardiographic parameters in the newfoundland dogs were calculated. forty-six healthy adult newfoundland dogs of both genders ( males and females), to years of age (mean . ae . years), to kg (mean . ae . kg) fulfilled the inclusion criteria. significant but weak correlations were detected between aortic diameter (ao) and age (p = . , r = . ), left atrial to aortic ratio (la/ao) and age (p = . , r = . ), e-point to septum separation (epss) and bw (p = . , r = . ), m-mode left ventricular internal diameter (lvid) in diastole (d) and systole (s) and bw (respectively p = . , r = . and p = . , r = . ), and between ao and bw (p = . , r = . ). none of the echocardiographic measurements was statistically different between males and females. left ventricular internal diameter in diastole, lvids, ao, epss increased with bw, as expected. the aorta appears to become wider with advancing age. a proportion of the studied population had m-mode parameters below the allometric scaling reference range, suggesting that this method can over-estimates m-mode parameters in this breed. these findings stress the importance to report newfoundland breed specific normal ranges for echocardiographic parameters. no conflicts of interest reported. canine idiopathic pulmonary fibrosis (cipf) is a progressive interstitial lung disease usually diagnosed by thoracic ct-scan that mainly affects west highland white terriers (whwt). pulmonary hypertension (ph), a severe co-morbid condition with a challenging diagnosis, may develop in cipf dogs. the ratio between the right pulmonary vein and pulmonary artery (pv/ pa) has been described as an echocardiographic indicator of ph in cipfdogs. this study was intended to investigate whether ct-scan angiography cardiac findings are ) altered in dogs with cipf compared to healthy control dogs and ) correlated with pv/pa measured by echocardiography (pv/pa us ). thoracic cta images from whwt with cipf (group a) and healthy controls from various breeds (group b) were retrospectively reviewed by one observer. all measurements were obtained in transverse post-contrast images displayed in a soft tissue window. pv and pa were measured dorsal to the right atrium, perpendicular to the long axis of these vessels. in addition, pulmonary trunk (pt) was assessed just ventral to the division of pulmonary arteries, perpendicular to its long axis. ascending aorta (ao) was also measured perpendicular to its long axis. transverse reformatted images were obtained to have a view equivalent to the standard chambers-echocardiographic view where right ventricle (rv) and left ventricle (lv) were measured. three ratios were calculated pv/pa ct , pt/ao and rv/lv, compared between groups and correlated with pv/pa us in both bi-dimensional (bd) and m-modes (mm). statistical analyses were performed with xlstat â software. values are given as mean ae sd. statistical significance was set at a p ≤ . . pv/pa ct was lower in group a ( . ae . ) in comparison to group b ( . ae . , p = . ) and correlated with pv/pa us (bd: r = . , p = . ; mm: r = . , p = . ). pt/ao was higher in group a ( . ae . ) compared to group b ( . ae . , p = . ) and correlated only with pv/pa us measured in bd mode (r = - . , p = . ). the rv/lv ratio was increased in group a ( . ae . ) in comparison to group b ( . ae . , p = . ) and a correlation between rv/lv and pv/pa us was found (bd: r = - . , p = . ; mm: r = - . , p = . ). in conclusion, in whwt with cipf, pv/pa ct , pt/ao and rv/lv ratios measured on thoracic cta images are correlated with pv/pa us and may serve in the assessment of ph. no conflicts of interest reported. companion animals presenting to the emergency room in distress need to be assessed rapidly and accurately to implement life-saving therapies. focused cardiac ultrasound (focus) can be a useful adjunct to the physical examination in assessing dyspneic animals in the emergency room. rapid bedside ultrasound evaluations performed by ec are commonly used in human medicine, however feasibility and utility of focus by ec in veterinary medicine has not been fully evaluated. the purpose of this study is to determine the baseline accuracy of focus performed by ec and whether or not a basic training session could improve accuracy compared to evaluation by a cardiology specialist. fifteen ec including boarded emergency-critical care specialists and emergency res-idents performed focus on four animals; a normal cat and dog, and a cat and dog with severe valvular and myocardial heart disease, respectively. ec semi-quantitatively assessed thoracic and echocardiographic parameters including left atrial dimension, left ventricular systolic function and wall thickness, right heart dimension, and presence or absence of pleural or pericardial effusion before and after a structured didactic lecture and hands-on practical session. primary outcome was the level of agreement with examination performed by a cardiologist. level of agreement regarding ec assessment of all parameters improved from . to . after training (p < . ). level of agreement concerning left atrial diameter improved from . to . (p < . ). ec confidence in their overall focus evaluation and findings improved from % to % (p < . ). in summary, ec accuracy and confidence in semi-quantitatively assessing basic cardiac parameters using focus were improved following a simple structured training session. focus might be a valuable tool to rapidly assess simple thoracic and cardiac parameters in the emergency setting. no conflicts of interest reported. obesity is an increasing health problem in dogs. success of weight-loss programs is often limited by compliance issues. the purpose of this study was to determine the effectiveness of a new dietetic weight management food (ndwmf)* in achieving weight loss in overweight/obese, client-owned dogs, under typical household conditions. the objectives were ) to evaluate weight loss parameters in dogs fed a ndwmf* and ) to assess the owner's perception of the dog's quality of life. overweight/obese, otherwise healthy, client-owned dogs (> / body condition score -bcs) were enrolled in the study (n = ). initial veterinary evaluation included physical examination, nutritional assessment, determination of ideal body weight (ibw), and development of weightloss feeding guidelines. daily energy requirement (der) for weight loss was calculated as der = x ibw kg . . initial and follow-up evaluations (monthly for months) encompassed determination of body weight, bcs, body fat index (bfi), muscle condition score (mcs), and feeding practices. quality of life assessment by owners included dog's level of energy, happiness, appetite, begging behavior, flatulence, stool volume, and fecal score. statistical analysis comprised scatterplots, regression analysis, summary statistics as appropriate for the type of analyses performed (continuous or categorical variables, distribution), and a mixed model anova to assess changes over time (with statistical significance at p < . ). ninety four percent of the dogs lost weight (n = ) with an average weight loss of . % (sem, . %) over months and an average weekly weight-loss rate of . % (sem, . %). the mean duration of weight loss was days (sem, . days) with an average of days (sem, . days) between rechecks. thirty nine percent of dogs achieved ibw ( . , ci: . - . ). fifty five percent of dogs ate more calories from ndwmf* than the recommended der for weight loss (median fed above der= %) and % of these dogs ( . , ci: . - . ) still lost weight. thirty six percent of dogs received treats. bcs and bfi decreased significantly over time compared to baseline. owners perceived a significant increase in energy and happiness in the dogs that lost weight without changes in appetite or begging behavior. in conclusion, this clinical study confirmed the effectiveness of the ndwmf* in achieving weight loss in overweight/obese client-owned dogs in spite of higher than recommended caloric intake. owners reported significant improvements in dog's quality of life without negative side effects. * haptoglobin is a moderate acute phase protein in cats. as a part of the innate immune system its concentration rises within - hours after tissue damage. aim of the study was to validate an elisa which was recently developed for the measurement of feline haptoglobin and to compare it with a commonly used spectrophotometric assay. the concentration of haptoglobin was measured in healthy and sick cats using a sandwich-elisa (tecomedical group, rheinbach, germany). the validation included the detection of intra-assay and inter-assay variation, dilution linearity, spike recovery and lower detection limit. a spectrophotometric assay (tridelta development ltd, maynooth, ireland) was used as a reference method. all samples were measured in duplicate. statistical analysis was performed using ibm â spss â statistics (ibm corporation â ) and included descriptive statistics, spearman correlation (rs) and coefficients of variation (cv). the coefficients of variation were . %, . % and . % for intra-assay variability and . %, . % and . % for inter-assay variability. the ratio of observed to expected dilutional parallelism of serum samples diluted times ranged from to %. the ratio of observed to expected spike recovery of serum samples ranged from % to %. the lower detection limit was . mg/ml. the correlation between the assays was significantly strong (rs = . , p < . ). the recently available sandwich-elisa provides a high accuracy and precision and can therefore be used for the measurement of feline haptoglobin. the rd and th author (m. hennies and c. wienen) work for the company tecomedical group that developed the elisa which was evaluated in the study. they provided the kits and they helped with performing the tests, but they did not have any influence on the results and the interpretation of the data. canine idiopathic pulmonary fibrosis (cipf) is a progressive interstitial lung disease that mainly occurs in the west highland white terrier (whwt) breed. the cipf diagnosis commonly relies on thoracic high-resolution computed tomography (hrct) findings and ultimately on histopathology. as those tests are not easily performed in practice, identification of measurable markers of fibrosis, that might help to diagnose and/or monitor the course of cipf, is helpful. vegf is an angiogenic regulator involved in a variety of physiological and pathological processes. in human ipf, serum vegf concentration has been shown to be higher in ipf patients compared to healthy volunteers and may reflect the severity of the lung disease. the aims of the present study were ( ) to investigate the potential role of vegf as a peripheral blood biomarker in cipf; and ( ) to investigate possible breed-related differences in basal vegf concentration, that might explain the high predisposition of the whwt breed for cipf.therefore, vegf was determined by elisa (canine vegf quantikine elisa kit, r&d systems) in the serum of whwt with cipf confirmed by hrct and/or histopathology (median age years, range - ), healthy whwt ( , - ), and healthy dogs of other breeds, including: scottish terrier (st) ( , - ), jack russell terrier (jrt) ( , - ), maltese ( , - ), king charles spaniel (kcs) ( , - ), labrador retriever (lr) ( , - ) and malinois belgian shepherd ( , - ). health status was based on clinical examination, serum biochemistry and haematology in all healthy dogs and a thoracic hrct was performed in / healthy whwt. the khi² test with the threshold % was used for the statistical analysis (xlstat â software). eight cipf whwt ( %) have serum vegf concentrations above the kit detection limit ( . pg/ml) compared to whwt ( . %) in the group of healthy dogs (p = . ). concerning inter-breed differences in healthy dogs, most values obtained were below the kit detection limit with only kcs ( %), jrt ( %), lr ( %) and st ( %) having vegf serum levels above . pg/ml (p = . ). results of the present study show that ( ) vegf might be an interesting blood biomarker for cipf; ( ) canine vegf quantikine elisa kit is not appropriate for measurement of serum vegf levels in healthy canine populations. no conflicts of interest reported. the total protein (tp) concentration and cell count of pleural and abdominal fluid is used to differentiate a transudate from an exudate. tp can be measured by automated wet chemistry analyser or more easily using a refractometer. the aim of this study was to assess if refractometer values of tp are useful for this purpose. retrospectively samples from canine pleural and abdominal effusions in which tp concentration was measured both with a refractometer as well using pentra (abx horiba, montpellier) were included. samples were collected into heparinized tubes and analysed within hours. bland-altman diagrams were created and correlation between both measurements was calculated by spearman s nonparametric correlation. over a -months period, pleural and abdominal effusion samples were analysed with both techniques. median (range) tp concentrations in pleural effusion measured by refractormeter or by pentra was ( - ) g/l and ( - ) g/l, respectively. median (range) tp concentrations in abdominal effusions measured by refractometer or pentra was ( - ) g/l and ( - ) g/l, respectively. tp measurement between refractometer and pentra values were significantly correlated in pleural (r = . , p < . ) and abdominal (r = . , p < . ) effusion. the bland-altman graph showed a bias in the thorax and abdomen of . and . . the refractometer is an acceptable, rapid and efficient method for determination of total protein concentration in pleural and abdominal effusions in dogs to differentiate transudates from exudates. no conflicts of interest reported. coagulation factor vii (fvii) deficiency has been reported in beagles since the 's. deficient dogs show a mild hemorrhagic tendency, but often remain asymptomatic and are incidentally discovered by an isolated prolonged prothrombin time due to < % plasma fvii activity. factor vii deficiency occurs commonly in beagles, alaskan klee kais and scottish deerhounds. in these breeds it is caused by a single missense mutation (c. g>a, p.gly glu) in the second epidermal growth factorlike domain of fvii, which drastically reduces the secretion and activation of fvii. research beagles were also commonly affected which may have pharmaco-toxicologically affected studies but specific dna screening programs have been established. we report here on the discovery of fvii deficiency in welsh springer spaniels (wss) in finland based upon a novel screening panel for~ known mutations underlying inherited disorders in different canine breeds (www.mydogdna.com). among wss initially tested, were heterozygously ( %), and homozygously affected for the same fvii mutation, which was confirmed by sequencing in all dogs. in order to determine whether the mutation causes fvii deficiency also in this breed, we recruited littermates and their mother. none of these wss had shown an increased hemorrhagic tendency, but affecteds bled excessively following blood collection. we found that the homozygous affected dogs of the litter exhibited markedly prolonged prothrombin time but normal partial thromboplastin time. they also had drastically reduced fvii activities but normal to high fviii and fix activities compared to their littermate controls. the heterozygous carriers tested did not show any prolongations in their prothrombin time, but had half normal fvii activity. in conclusion, we document here the presence of fvii deficiency in wss based upon dna and coagulation activity testing. the common gly glu mutation must have arisen prior to the separation of the very different fvii deficient breeds. there is no knowledge of an advantage of the heterozygote state. while there is only a mild hemorrhagic tendency, bleeding dogs could be treated with fresh frozen or cryo-poor plasma or human recombinant fviia. this preliminary study indicates a high carrier frequency in wss. screening by new platform dna methods for this and other ancestral defects is helpful to detect additional hereditary diseases and genetic predispositions in different breeds, while other mutations are new and restricted to one or related breeds. authors are affiliated with genetic disease screening test laboratory. remarkably little has been published on haematological and serum biochemical phenotypes of the domestic dog. information on the signalment and complete blood cell count of all dogs with normal red and white blood cell parameters judged by existing reference intervals was extracted from a veterinary database; similar information was collected from all dogs with normal serum biochemical profiles, considering all parameters other than glucose as inclusion criteria. normal haematological profiles were available for dogs, of which also had machine platelet concentrations within the reference interval; normal serum biochemical profiles were available from dogs, of which also had accompanying normal serum glucose concentrations. for the haematological data, pure breeds plus a mixed breed control group were represented by or more dogs, while for the serum biochemical data, pure breeds plus a mixed breed control group were represented by or more individuals. all measured haematological parameters except mean corpuscular haemoglobin concentration (mchc), and all serum biochemical analytes except sodium, chloride and glucose, varied with age. concentrations of white blood cells (wbcs), neutrophils, monocytes, lymphocytes, eosinophils and platelets, but not red blood cell parameters, all varied with sex, as did total protein, globulin, potassium, chloride, creatinine, cholesterol, total bilirubin, and activities of alanine aminotransferase (alt), creatine kinase (ck), amylase and lipase. neutering status had an impact on haemoglobin concentration, mean corpuscular haemoglobin (mch), mchc, and concentrations of wbcs, neutrophils, monocytes, lymphocytes and platelets, as well as all serum biochemical analytes except albumin, sodium, calcium, urea and glucose. principal component analysis (pca) of haematological data revealed pure breeds with distinctive phenotypes, while pca of serum biochemical data revealed over pure breeds with distinctive phenotypes. furthermore, all haematological parameters except mchc and all serum biochemical analytes except urea and glucose showed significant differences between specific individual breeds and the mixed breed group. twenty-nine breeds had distinctive haematological phenotypes and breeds had distinctive serum biochemical phenotypes when assessed in this way. tentative breed-specific reference intervals were generated for breeds with a distinctive phenotype identified by comparative analysis. this study represents the first large-scale analysis of haematological and serum biochemical phenotypes in the dog and underlines the important potential of this species in the elucidation of genetic determinants of haematological and biochemical traits, triangulating phenotype, breed and genetic predisposition, as well as the urgent need for breed-specific reference intervals in clinical practice. the author has received funding from bbsrc, petplan charitable trust, and cruk), but none of thesegrants were for this study. for each cat aged years or older, irrespective of their suspected thyroid status, presented to eight veterinary practices in portugal, the veterinarian and the pet owner had to complete a questionnaire and the veterinarian had to take a venous blood sample (into a plain tube) from the cat, after obtaining signed owner consent. the veterinary questionnaire included history, attitude, activity, heart rate and thyroid palpation. cats aged < years and those diagnosed previously with hyperthyroidism were excluded. blood samples were centrifuged and the serum harvested and stored frozen until collection by the laboratory within days of sampling. total t was measured using a chemiluminescent method (immulite , siemens). cats were classified as hyperthyroid, equivocal or euthyroid based on a total t concentration of > nmol/l, - nmol/l or < nmol/l, respectively. repeat measurement of total t after - weeks was recommended for all equivocal cases. the individual cat was the statistical unit. descriptive statistics was used to summarise the data and associations between different clinical signs analysed using chi-square, fisher's exact test or the mann-whitney u test. the level of significance was set at . . thirty cats were excluded from the prevalence analysis because they were aged < years (between and years, n = ) or their age was not stated (n = , four of these cats were hyperthyroid). by the end of february , samples had been submitted from cats that met the inclusion criteria. based on the thyroid hormone analysis, there were / ( %) hyperthyroid, ( %) equivocal and ( %) euthyroid cats. very few follow-up blood samples were taken. hyperthyroidism appears to be not uncommon in portuguese cats. getting owners to return for follow-up blood sampling appears to be problematic. under-reporting of hyperthyroidism appears to be a significant problem in portugal, as has been reported for some other countries. thyroid palpation should form part of routine physical examinations, especially of middle aged and older cats. older cats in portugal should be screened for hyperthyroidism even in the absence of a detectable thyroid nodule. both authors are employees of msd animal health. msd animal health funded the study. msd animal health has an approved veterinary medicinal product for the treatment of feline hyperthyroidism. this product is available commercially in some eu markets but not in portugal. diabetes mellitus is one of the most commonly encountered endocrinopathies in cats and its prevalence has increased in the past. similar to human type diabetes, feline diabetes is associated with comparable lesions occurring in the pancreatic islets, namely islet amyloidosis and beta-cell loss. studying the pathophysiology of feline diabetes and the molecular mechanisms through which glucose metabolism is disturbed is largely hampered by the lack of a method for the isolation of pure pancreatic islets. the aim of this project was to improve a previously established method for the isolation of pancreatic islets; in particular enhancing the purity of isolated islets in this species. cats that died or were euthanized due to severe illness other than pancreatitis or other pancreatic disease were enrolled. pancreata were perfused post-mortem with ml collagenase type iv ( . mg/ml) through the pancreatic duct. the perfused organ was then digested for ', ' and ' at °c in a water-bath and purified using a filtration method. islet cell viability and purity were determined by thiazolyl blue tetrazolium bromide (mtt assay) and dithizone staining, respectively. perfusing the pancreas through the pancreatic duct allowed collagenase to access the islets using anatomical structures and to improve islet yield compared to previously established protocols in this species. the digestion time of ' provided the best islet yield. after digestion, feline pancreatic islets remained satisfactorily viable for days in the culture system following regular media changes. the current study has successfully optimized the isolation, purification and culture maintenance of feline islets. the successful yield and viability of islets isolated through the suggested protocol may provide promising potential as a source of islets for diabetes research in cats. no conflicts of interest reported. nesidioblastosis describes a syndrome of acquired hyperinsulinaemia and associated hypoglycaemia secondary to focal or diffuse (non-neoplastic) beta cell hyperplasia within the pancreas. beta cell dysregulation is thought to occur secondary to pancreatic injury. this syndrome has been reported in humans with increasing frequency, but it has not previously been described in domestic pets. a six year old, de-sexed female british shorthair cat presented with acute onset weakness and mental dullness. upon initial presentation the cat was mildly hyperglycaemic ( . mmol/l; . - . mmol/l). over the following hours the cat developed central blindness, tremors, intermittent seizures and opisthotonus. repeat blood sampling revealed a marked hypoglycaemia ( . mmol/l). an insulin level (performed on serum obtained while the cat was hypoglycaemic) was inappropriately elevated ( pmol/l; reference range - pmol/l). an intravenous bolus of % glucose resulted in rapid resolution of all clinical signs and mild transient hyperglycaemia ( . mmol/l). despite frequent feeding, the hypoglycaemia ( . mmol/l) recurred, so an intravenous glucose continuous rate infusion was commenced. an abdominal ultrasound was unremarkable, although three cranial mesenteric lymph nodes were noted to be prominent ( mm in width). an exploratory laparotomy revealed a firm and erythematous left limb of the pancreas. the body and right limb of the pancreas appeared grossly normal. following surgical resection of the left limb of the pancreas, the cat returned to a euglycaemic state after a brief rebound hyperglycaemia. histopathology revealed pancreatic fibrosis with marked multifocal micronodular hyperplasia of exocrine and endocrine cells, mild lymphoplasmacytic inflammation and ductular ectasia. synaptophysin immunohistochemistry confirmed nodular beta cell hyperplasia. mild granulomatous lymphadenitis and hydropic change within hepatocytes was also noted. the cat recovered uneventfully without any further intervention. it gained weight and remained euglycaemic over the following six months. while beta cell hyperplasia has been reported as an incidental histopathological finding in euglycaemic young beagles, this is the first reported case of clinically significant hypoglycaemia secondary to nesidioblastosis in a domestic pet. while this condition is rare, nesidioblastosis is being increasingly recognised in the human field and it is an important differential to consider when investigating hypoglycaemia as it cannot be differentiated from insulinoma without histopathological evaluation. age of onset may provide a clue to this non-neoplastic disease, as this cat was much younger than all previously reported cases of feline insulinoma (all > years of age at diagnosis). while recurrence has been reported in humans, a favourable outcome is anticipated following partial pancreatectomy. no conflicts of interest reported. hyperthyroidism is the most common feline endocrinopathy of geriatric cats worldwide. nonetheless, data concerning the accurate prevalence of feline hyperthyroidism (fh) is scarce; and apparently exhibit geographical variation, which can be an important instrument in investigating risk factors through the analysis of exposure to different factors in areas of high and low prevalence. in europe, fh is considered more frequent in the northern than in the southern countries. the aims of this study were to determine the occurrence of fh in a region of portugal, to characterize clinical presentation and potential risk factors. during an -month period, geriatric cats ≥ eight years from aveiro (central region of portugal) were selected. cats were excluded if presented in shock or moribund, or in treatment with drugs that might affect total t (tt ) serum concentration. the tt concentration was determined through chemiluminescence (immulite â , siemens), and diagnosis of fh established if tt serum concentration ≥ . lg/dl (reference values . - . lg/dl) associated with compatible clinical signs. information on age, gender, breed, weight, housing conditions (indoor vs outdoor), use of external parasiticides, food (dry vs canned food and flavor), use of litter box, environment, clinical signs and laboratory data was collected. all owners gave informed consent. population studied included males ( . %) and females ( . %), mainly domestic short-haired cats ( . %). ages ranged from eight to years old ( . + /- . ). eight ( . %) cats were diagnosed with hyperthyroidism. if only cats ≥ years of age were considered (n = ), prevalence raised to . %. hyperthyroid population comprised four males and four females, ranging from to years old ( . + /- . ). increasing age (p = . ), polyphagia (p˂ . ), weight loss associated with increased (p˂ . ) or normal appetite (p˂ . ), presence of thyroid uni or bilateral nodules (p˂ . ), vomiting (p = . ) and hyperactivity (p = . ) were significantly associated with hyperthyroidism in the geriatric population studied. environment was also significantly associated with development of fh (p˂ . ), with cats from urban or semi-rural areas at higher risk of developing the disease than cats living in a rural environment. no other significant associations were found between hyperthyroidism and other factors analyzed. in our knowledge, no epidemiologic studies on fh have been performed in portugal, a country where the occurrence is believed to be low, but in which the population of pet cats, the feline geriatric population and the clinical cases diagnosed have been increasing. conflicts of interest: the study was partially supported by laborat orio segalab s.a. and dechra veterinary products. canine diabetes mellitus (cdm) has been proposed to be a spontaneous animal model of human autoimmune diabetes, and comparative research can be undertaken to investigate the interaction between genetic and environmental factors. most epidemiological studies of cdm have been performed in northern european and north american populations. our aim was to evaluate the epidemiology and clinical features of the diabetic dog population from the canary islands, with special focus on immune-mediated disease. dogs attending our veterinary teaching hospital were included from january to january . previously diagnosed and new cases were considered. prevalence was calculated as number of cdm/total number of dogs attending the hospital and incidence as newly diagnosed cases divided by the same value per year. anti-insulin antibodies were assessed by elisa. genotyping for dog leukocyte antigen (dla) and measurement of canine anti-gad and anti-ia antibodies by radio-immunoprecipitation assay were performed in dogs with suspected immune-mediated diabetes. twenty-nine dogs with cdm were identified from a mean population of ( - ) dogs per year (mean prevalence . % and mean incidence cases per year per , ). age at diagnosis was . years (range: . - y). most dogs were not neutered ( % females; % males). nine breeds were represented, including poodle ( %) and andalusian wine-cellar rathunting dog ( %). seasonality was observed in the diagnosis with peaks in december and march-april. diabetes was classified as dioestrus diabetes ( %), idiopathic/immune-mediated ( %), iatrogenic ( %) and secondary to pancreatitis ( %) or other endocrine disorders ( %). insulin-treated dogs were negative for anti-insulin antibodies (n = ). from the suspected immune-mediated cases (n = ), autoantibody reactivity was shown in two cases (anti-gad , n = ; anti-ia , n = ). no previously described, diabetes-risk dla-types were identified. although age, prevalence and incidence did not differ from previous studies, the high proportion of entire females likely explained the high frequency of dioestrus diabetes. the andalusian wine-cellar rat-hunting dog was identified as a high-risk breed for cdm. most of the dla-types seen have not been previously described, but at least two have been associated with increased risk of autoimmunity in dogs. further population-based studies are needed in different regions, to assess the heterogeneous nature of this disease. no conflicts of interest reported. the cortisol-dehydroepiandrosterone (dhea)-ratio is widely used in human medicine as a marker for stress however it is not clear whether it could also help in distinguishing hyperadrenocorticism (hac) from other diseases which might have a negative impact on the outcome of a dexamethasone low dose test. therefore the aim of the study was to evaluate the cortisol-dhea-ratio as an additional diagnostic marker for hac in dogs. to achieve this aim, a reference range of this ratio depending on the sex should be evaluated in healthy dogs and compared with dogs having a hac. in healthy dogs (age: - . years) and in dogs with hac (age: . - . years) of different breeds the plasma concentration of cortisol (immulite system, siemens healthcare diagnostics) and dhea (beckman coulter) was measured and the ratio was calculated. all dogs were patients of the small animal clinic except five of the healthy dogs which were recruited from the institute of pharmacology, toxicology and pharmacy of the university. with these data the cortisol-dhea-ratio was calculated for male dogs (healthy dogs n = ; dogs with hac n = ), neutered males (healthy dogs n = ; dogs with hac n = ), female dogs (healthy dogs n = ; dogs with hac n = ) and spayed females (healthy dogs n = ; dogs with hac n = ). the statistical analysis was performed with sigma stat. the plasma cortisol-dhea-ratio of healthy male dogs was the lowest ratio of all sexual categories (mean average . ae ) and it differed significantly to all other sexes (neutered males = ae , p = . ; females = ae , p < . and spayed females ( ae . , p = . ). the cortisol-dhea-ratio showed no significant difference between male and female dogs with hac. spayed females with hac had significantly higher cortisol-dhea-ratios ( ae ) than healthy spayed females (p = . ) but no significant differences were found in other sexual categories. this preliminary data indicates that the cortisol-dhea-ratio might not be a very promising tool for the diagnosis of hac. in addition, the significant gender-dependency of this parameter has to be considered and may generally limit its clinical usefulness. this study is financially supported by the bruns-stiftung. no conflicts of interest reported. hyperthyroidism is common in older cats. the aim of this study was to assess the prevalence of feline hyperthyroidism and potential intrinsic risk factors in a hospital population in southern germany. total thyroxine (t ) was prospectively measured by enzyme immunoassay (eia) in sera of cats older than years that were presented to the clinic of small animal medicine. a standardized physical examination was performed, and body condition score (bcs) and thyroid palpation score (tps) were assessed. association between signalement, bcs and tps was analyzed by student s unpaired t-test, chi-square, and mann-whitney test. level of significance was set at . . fifty nine cats were diagnosed with hyperthyroidism leading to a prevalence of . % (ci . - . ). hyperthyroid cats were older than non-hyperthyroid cats (p < . ) and more often female (p = . , odds ratio . ). domestic short or long hair cats were more often affected than pedigrees (p = . ). hyperthyroid cats had higher tps (p < . ) and lower weight than non-hyperthyroid cats (p < . ) although bcs was not different (p = . ). in ( . %) cats, the elevated t was an incidental finding. in of those, the disease was confirmed later (the others were dead due to unrelated diseases). in patients, hyperthyroidism was considered a differential diagnosis and was confirmed in ( . %) cats although in cats additional diagnostic means were necessary. older female domestic cats are predisposed to hyperthyroidism which is frequently diagnosed after the initial clinical suspect. in a few affected cats an elevated t is not present or can precede clinical signs. conflicts of interest: the study was partially funded by msd intervet. the main endocrinopathy affecting both humans and pet felines is diabetes mellitus. accurate diagnosis is the most important aspect in the future outcome of the disease. a computer based decision support system (dss) is targeted on assisting clinicians with one or more steps of the diagnostic process. the novelty of our dss emerges from the possibility of assisting both clinical and paraclinical diagnosis stages of diabetes mellitus and all common combination of disorders associated with this endocrinopathy. the motivation behind the development of such system is the desire to maximize the reliability of clinical decisions. the design of our feline diabetes mellitus dss emerges from the syndrome of polyuria-polydipsia, with the possibility of spotting the accompanying pathologies. fuzzy logic is used for dealing with knowledge representation and uncertainty. the fuzzy rules proposed to represent this knowledge emerge from anamnesis, clinician's input, clinical and paraclinical description, and confirmation diagnostic tests. clinical signs such as polyuriapolydipsia, persistent hyperglycemia, polyphagia, weight fluctuations, administration of drugs with a diabetogenic potential, were considered decisive in the pattern of diagnosis establishment. registered medical records of cats, males and females, whit ages from to years old, were analyzed in order to validate the dss. using matlab software, the dss was implemented and tested. for any case with polyuria-polydipsia the system provides, via a friendly graphical user interface, the diagnosis with the highest probability. the set of diagnoses which can be generated by the dss consists in: a) diabetes mellitus; b) diabetes mellitus induced by (b. ) hypersomathotropism, (b. ) hyperthyroidism, (b. ) hyperadrenocorticism and (b. ) diabetogenic medication; c) diabetes mellitus in association with (c. ) chronic kidney failure and (c. ) heart failure; d) ketoacidodic diabetes mellitus; e) pancreatitis. the dss was applied with success on all cases, revealing the following diagnoses / no of cases: (a) - , (b. ) - , (b. ) - , (b. ) - , (c. ) - , (c. ) - , (d) - . an adequate treatment protocol requires an accurate and complete diagnosis. advanced computational systems accompany clinicians in their decision making, leaving a reduced space for medical errors and superfluous, expensive and time consuming tests. future work will be targeted on exploring the possibilities of combining the dss with an artificial neural network model for diabetes mellitus. this can be the foundation of a complete case oriented management system for feline diabetes mellitus and associated disorders. no conflicts of interest reported. activins are cytokines belonging to the transforming growth factor (tgf)-b superfamily. it is thought that activins may be the key intermediary in tgf-b mediated fibrotic response. activin a has been suggested to participate in the pathogenesis of human idiopathic pulmonary fibrosis (ipf), but studies regarding the role of activin b are still spares. canine ipf (cipf) is a chronic, incurable interstitial lung disease occurring particularly in west highland white terriers (whwts). during the disease course, acute exacerbations (aes), with poor prognosis, can occur. histopathologically aes of cipf are featured by diffuse alveolar damage, which is also a key feature in acute respiratory distress syndrome (ards). our objective was to study the expression of activin a and b by immunohistochemistry in the lung tissue of cipf whwts (n = ), cipf whwts with concurrent ae (n = ), and dogs of various breeds with ards (n = ), and to compare these findings to healthy whwts (n = ). in addition, western blot analysis of activin b from bronchoalveolar lavage fluid (balf) of cipf whwts (n = ) and healthy whwts (n = ) was conducted. we demonstrated that activin b, but not activin a, is strongly expressed in the altered alveolar epithelium in lungs of diseased whwts as well as in ards lungs. furthermore, activin b was detected in balf of cipf whwts, most notably in samples from dogs with ae, but not in balf of healthy whwts. this novel finding suggests that activin b participates in the pathophysiology of cipf and might act as a potential marker of alveolar epithelial damage. no conflicts of interest reported. dogs of the breed nova scotia duck tolling retriever (nsdtr) are affected by several immune-mediated diseases, in particular steroid-responsive meningitis-arteritis (srma) and an immune-mediated rheumatic disease (imrd). imrd is a systemic lupus erythematosus-related disease characterized by chronic stiffness and pain in several joints. the aim of this study was to investigate the morbidity in nsdtrs and to test the hypothesis that nsdtrs are predisposed to srma and imrd. insurance data from a swedish insurance company (agria insurance company, stockholm, sweden) from - was used for the study. approximately one third of swedish dogs are insured by agria and the insurance database is a validated tool for epidemiological studies. assessment of morbidity was based on veterinary care events. disease diagnoses were grouped in both general and specific disease categories. individual diagnoses that were likely to represent imrd were combined. morbidity was defined as incidence rates and presented as number of cases per dog years at risk (dyar). relative risk (rr) for nsdtrs compared to other breeds combined was calculated. the study included dogs, were nsdtrs. the most common general causes of veterinary care for nsdtrs were injuries followed by gastrointestinal and musculoskeletal disorders with significant increased risk (rrs between . and . ) for nsdtrs compared to other breeds. the highest relative risk for nsdtrs was for systemic lupus erythematosus (rr . ). compared to other breeds, nsdtrs had an increased risk for srma (rr . ) and imrd (rr . ) with an incidence rate of . cases per dyar for srma and . cases per dyar for imrd. the incidence rate for srma and imrd in nsdtrs were also compared to dogs of other retriever breeds. the comparison revealed that nsdtrs also had a significant increased risk for both srma (rr . ) and imrd (rr . ) when compared to other retrievers only. this study is the first to investigate the morbidity for imrd in nsdtrs, which is important for further research and breeding practice. for several reasons the incidence rates might be underestimated and exact numbers should be interpreted with caution. however underestimation of incidence rates should not differ between dogs of different breeds, therefore not affecting the risk calculations. it can be concluded that nsdtrs are predisposed to the diseases srma and imrd with an increased risk compared to other breeds and to other retrievers. brenda n. bonnett consults with agria insurance company on various projects. agria insurance company has also funded work leading to the development of the insurance data base that my study was based on. canine infectious respiratory disease (cird) is a multifactorial contagious disease caused by respiratory viruses and selected bacterial pathogens. cird has been shown to be a predisposing factor in the development of bacterial pneumonia (bp) in dogs housed in dense populations such as kennels and rehoming centers. the aim of this study was to determine the prevalence of viral co-infection and to assess its effects on disease severity in household dogs diagnosed with bp. a prospective cross-sectional observational study was conducted and dogs diagnosed with bp caused by opportunistic bacteria were included. dogs with chronic (> days) tracheobronchitis caused by bordetella bronchiseptica were included as controls for virus analysis. diagnosis was confirmed by thorough clinical examinations as well as with cytological and bacterial analysis of bronchoalveolar lavage (bal) or transtracheal wash (ttw) samples. canine parainfluenssavirus (cpiv), canine adenovirus, canine herpesvirus, canine distempervirus, canine respiratory coronavirus (crcov) and canine pneumovirus were analysed in bal or ttw samples using rt-pcr assay. cpiv was detected in / ( %) and crcov in / ( %) respiratory samples in dogs with bp. respiratory viruses were not detected in dogs with chronic tracheobronchitis. there were no significant differences in the duration of hospitalization (p = . ) or arterial pao at presentation (p = . ) between bp dogs with and without a viral co-infection. these results indicate that co-infections with respiratory viruses are common also in household dogs with bp. additionally, viral co-infections did not cause a more severe course of bp. the author's researcjh is financially supported by the finnish foundation of veterinary r and the finnish veterinary foundation. esvim-p- causes of canine anemia in taiwan: a five-year retrospective survey. e.c.y. lin , p.c. liu , l.l. chueh , b.l. su . graduate institute of veterinary medicine, national taiwan university, taipei, taiwan, institute of veterinary clinical sciences, national taiwan university, taipei, taiwan anemia is a common hematologic disorder in dogs, however, few data are available regarding epidemiology and causes in taiwan. to investigate the causes of anemia, anemic cases (pcv< %) collected between january and december at national taiwan university veterinary hospital (ntuvh) were analyzed. most dogs ( . %, n = ) presented with a mild form ( %≦pcv< %), which was followed by a moderate form ( %≦pcv< %; . %, n = ) and a severe form (pcv< %; . %, n = ). among the dogs with identifiable causes, . % ( dogs) were induced by single cause, whereas . % ( dogs) by multiple causes. neoplasia-related anemia (n = ), infectious pathogens-related anemia (n = ), renal disease-related anemia (n = ) and post-surgery/ traumarelated anemia (n = ) account for . , . , . and . % of single-cause cases, respectively. furthermore, of them ( . %) presented with severe anemia. severe anemia primarily resulted from infectious disease-related anemia ( . %), followed by imha ( . %), and tumor-related anemia ( . %). of the infectious disease-related severe anemic dogs, the most common diagnosed pathogen was babesia gibsoni ( . %, n = ), followed by ehrlichia canis ( . %, n = )and babesia canis ( . %, n = ). taken together, tumors, infectious diseases, and renal failure are the most frequently causes of canine anemia in taiwan, furthermore, b. gibsoni appeared to be the most important infectious pathogen causing severe anemia which may be associated with the climate in this geographical area. no conflicts of interest reported. bordetella bronchiseptica (bb) is one of the primary causative agents of canine infectious respiratory disease (cird). this contagious disease, commonly seen in young dogs, is often self-limiting, although a wide range of respiratory signs can be found, from mild illness to severe pneumonia leading to death. although mycoplasma cynos (m. cynos) was recently identified as an emerging and possibly lethal pathogen in cird , the role of m. canis and m. cynos as primary respiratory pathogens still remains unclear. detection of these bacteria is now improved by quantitative polymerase chain reaction (qpcr). in dogs with cird due to bb, the frequency of co-infection with mycoplasma spp, in par-ticularm. cynos, and their possible role in the severity of the clinical signs are unknown. the aim of the present study was to investigate the presence of m. canis and m. cynos in a population of dog infected with bb, compared with other populations: healthy dogs and dogs with bacterial bronchopneumonia where bb was not involved (bbp). therefore, bb, m. canis and m. cynos were detected by qpcr in the bronchoalveolar lavage fluid (balf) sample in dogs with bb (mean age = . y, mean bw = . kg), dogs with bbp ( . y, . kg), and healthy dogs ( . y, . kg). bordetellosis was diagnosed based on clinical findings together with demonstration of pleiomorphic cocci/coccobacilli adhering to the cilia of the epithelial cells on cytospin balf preparations, and positive qpcr on balf. a clinical severity index (csi to ) was assigned based on clinical signs (cough - , dyspnea - , lethargy - , fever - ), thoracic radiographic pattern ( - ), and balf score ( - ). bbp was diagnosed based on clinical findings, balf cytology and culture. m. canis was indifferently detected in healthy ( / , %), bbp ( / , %) and bb dogs ( / , %) while m. cynos tended to be more frequently detected in bb group ( / , %) than in healthy ( / , %) and bbp dogs ( / , %) (khi² test, p = , ). in bb dogs, no correlation could be detected between csi and presence of m. cynos (khi² test p = , ). in conclusion, the present data suggest that, in cird, coinfection with bb and m. cynos is frequent, but is not correlated with clinical disease severity. further studies are required to investigate whether coinfection of bb and m. cynos deserves specific therapeutic considerations. no conflicts of interest reported. canine idiopathic eosinophilic bronchopneumopathy (ebp) is a disease characterized by eosinophilic infiltration of the lung and bronchial mucosa in young adults. aetiology remains unclear although immunologic hypersensitivity is clearly suspected, while inciting antigens are generally unidentified. in humans as in cats, infections with mycoplasma spp. have been discussed as potential triggers in inflammatory bronchial disease , . bordetella bronchiseptica (bb) is a recognized pathogen agent of canine infectious tracheobronchitis. detection of bb and mycoplasma spp, especially mycoplasma cynos (m. cynos), and their potential role of in canine inflammatory bronchitis, have not been investigated. the aim of the present study was to investigate the frequency of bb, mycoplasma canis (m. canis) and m. cynos in canine ebp. therefore, presence of bb, m. canis and m. cynos were retrospectively assessed by quantitative polymerase chain reaction (qpcr) in bronchoalveolar lavage fluid (balf) samples from dogs with ebp (mean age = . y, mean body weight = . kg) as well as in dogs with aspecific chronic bronchitis ( . y, . kg). based on clinical signs, a clinical severity score (css, - ) was assigned each ebp dog. although all balf culture and cytology were negative for this bacteria, bb was more frequently detected by qpcr in ebp dogs ( / , %) than in cb dogs ( %) (khi² test, p = , ). presence of bb in ebp dogs was independant of age but significantly associated with css (khi² test, p = , ). results of qpcrwere positive for m. canis and m. cynos in ( %) and ( %) ebp dogs and in ( %) and ( %) cb dogs, respectively. there was no difference between the groups for any of the organisms. any relation between age or css and presence of m. spp in ebp dogs was observed. in conclusion, m. canis and m. cynos do not seem to be predominantly involved in the pathogenesis of canine ebp. however, bb is more frequently detected in balf from ebp dogs than from dogs with aspecific cb and its presence is associated with clinical severity. whether bb is able to trigger eosinophilic inflammation or is only more easily collected in an inflamed environment is unclear. but ebp dogs could potentially act as bb carriers and source of infection. therefore, bb should be systematically searched for in canine ebp cases and treated accordingly. no conflicts of interest reported. distal renal distal renal tubular acidosis (drta) was recently reported in three dogs with imha. the purpose of this study was to explore the hypothesis that drta is an underdiagnosed concurrent disorder in dogs with imha. we report the clinical presentation and outcome of three dogs where the combination of imha and drta was strongly suspected. the medical records of dogs diagnosed with imha at the university of edinburgh hospital for small animals between january and may were reviewed to identify cases where venous blood gas analysis and urinalysis had also been carried out. for the purpose of this retrospective study imha was defined by the presence of anaemia with pcv < %, and one or more of the following criteria; a positive slide agglutination test, positive coombs' test or moderate to marked spherocytosis. the criteria for diagnosis of drta included moderate to marked hyperchloremic metabolic acidosis with a normal anion gap; urine ph (> . ) in the face of metabolic acidosis; hypokalaemia (< . mmol/l). fifty-seven records were evaluated, with cases being excluded due to insufficient clinical information, including inability to determine urinary ph due to the severity of pigmenturia in four cases. of the cases where there was sufficient clinical data to assess the likelihood of drta only one case fulfilled all the criteria; two cases fulfilled all but one of the criteria and drta was strongly suspected based on clinical progression and persistence of urine ph > in the face of severe metabolic acidosis. of the three cases where concurrent imha and drta was suspected, two survived to discharge; one was still alive at the time of writing ( months after discharge) and the other was euthanased months after discharge following the development of multiple joint effusions and skin lesions suggestive of sle. venous blood gas analysis and assessment of urine ph should be considered in all cases of imha to exclude the possibility of concurrent drta, particularly where persistent hypokalaemia is detected. prospective evaluation of a larger cohort of imha cases is required to determine the actual incidence of concurrent drta. no conflicts of interest reported. persistent renal proteinuria is considered an early marker of chronic kidney disease (ckd) and it is listed among the initiation factors and progression factors according to kdoqi guidelines. nevertheless, few data are available about the prevalence of proteinuria in cats affected with ckd, in which it is assumed that nephropathy is mainly characterized by tubulointerstitial damage. the aim of this study is to determine the prevalence of proteinuria in cats affected with ckd and to valuate the relations between urine protein to creatinine ratio (upc) or iris substaging by proteinuria, towards purebred, sex, age, haematology, biochemistry and urinalysis. wilcoxon test, linear regression and chi-square test were used for the statistical analysis. data from cats were considered. non-renal proteinuria was an exclusion criterion. proteinuric cats (upc> . ) were . % in ckd cats, while . % could be substaged as bordeline proteinuric ( . ) in cats, proteinuria tends to increase with aging (p < . ) and with worsening of the nephropathy (p = . ). proteinuria was related to the anaemic state in ckd cats: upc significantly increases with rbc count, hb, ht and mch decreasing (p < . and p = . respectively). proteinuria tends to increase with wbc count (p = . ) and neutrophils increasing (p = . ), while tends to decrease with lymphocytes increasing (p = . ). furthermore, upc significantly increases in presence of an inflammatory serum protein electrophoretic pattern. upc tends to increase with phosphorus and alp increasing (p < . and p = . respectively); while the role of phosphorus in ckd is well known, the increase of alp is questionable: it has been hypothesized that higher alp levels in ckd could be related to b-alp increase due to bone remodelling in secondary renal hyperparathyroidism. considering urine parameters, upc increases when urinary specific gravity and ph decrease (probably related to worsening of ckd and development of a metabolic acidosis) and when glicosuria is present, regardless of the cause. furthermore, proteinuria increases in presence of rbc in urinary sediment and in samples where casts were observed, in particularly when rbc casts (considered always pathological and indicative of glomerular damage) were present. upc values assessed in proteinuric cats and data analysis suggest the need of deepen the analytical variability of upc and the opportunity to reconsider the intervals of substaging by proteinuria in cats. no conflicts of interest reported. the aim of this retrospective study was to evaluate: a) the relations between urine culture results and urinalysis parameters; b) the results of the antimicrobial susceptibility tests. urine samples were collected by cystocentesis from dogs and cats, whose diagnostic workup included a differential diagnosis of uti: all samples underwent a complete urinalysis, upc ratio assessment and urine culture. infected vs sterile results were related to urine physical, chemical parameters and observations from urinary sediment analysis. statistical analysis was performed using jmp . (sas institute inc.). a p value < , was considered significant. urine culture resulted positive in dogs ( %) and cats ( %). the presence of uti was significantly related to urine physical properties (color and turbidity), usg and leukocyturia: infections tended to be more frequent in urine samples characterized by a light yellow color, cloudy or sub-limpid aspect and low usg. nevertheless, urine was limpid in % of infected samples, and a normal usg was found in . % of dog's uti but only in . % of cats. although leukocyturia tends to become higher in infected samples both in dogs and cats (p < . ), in . % of infected sediments wbc count was normal. haematuria detected by dipstick was significantly related to uti in dogs but not in cats, nevertheless the rbc count in sediment was not related to infection in both species: rbc count was normal in . % of infected feline samples and in . % of canine samples. no significant relation between presence or absence of uti and albuminuria, bilirubinuria, glycosuria was detected, while upc tends to become significantly higher in dogs. although the chi-square test showed a significant relation between infection and the detection of bacteria in urinary sediment, a pseudobacteriuria was found in . % of samples; furthermore bacteria weren't observed in . % of infected samples (usg< . ). e. coli was isolated in the majority of samples ( , %), compared to other species: staphylococcus( . %), proteus ( . %) and streptococcus. ( . %). the urinalysis pitfalls and the high antibiotic resistance verified towards the most widely used molecules (penicillins, cephalosporins, quinolones) strongly indicates the importance to perform antimicrobials susceptibility tests to avoid the risk of failure associated with the use or abuse of empiric therapies in utis. no conflicts of interest reported. azotemia in dogs with chronic heart failure may reflect impaired renal function not only because of inadequate renal perfusion, but also due to organic renal injury. impaired renal function is observed in % of dogs with heart failure. altered renal hemodynamics due to decreased cardiac output results in renal hypoperfusion, and resultant elevation of blood urea nitrogen and creatinine, defined as azotemia. azotemia is a prognostic factor in dogs with mitral regurgitation, therefore, preservation and/or restoration of renal function is thought to improve prognosis. medical treatment for heart failure, however, includes angiotensin converting enzyme inhibitors and loop diuretics, which has been shown to increase the risk of developing azotemia. we hypothesized that mitral valve repair surgery ameliorates renal function by improvement of systemic hemodynamics. the change in renal function in dogs with mitral regurgitation was assessed by evaluating time-dependent changes in glomelular filtration rate by inulin clearance before and after cardiac surgery. eighteen dogs with severe mitral regurgitation with azotemia (plasma urea nitrogen level > mg/dl, plasma creatinine level > . mg/dl) were included in this study. the glomerular filtration rate in all dogs were evaluated by determining inulin clearance before and months after surgery. serum atrial natriuretic peptide level, plasma nt-pro brain natriuretic peptide level, plasma urea nitrogen concentration, and plasma creatinine concentration were measured at each time point as well as during the initial staging of heart failure based on the international small animal cardiac health council (isachc). left atrial/aorta ratio by echocardiography and vertebral heart size by thoracic radiographs were also measured. glomerular filtration rate significantly increased months after surgery ( . ml/min/m [ . - . ], . ml/min/kg [ . - . ]) compared to before surgery ( . ml/min/m [ . - . ], . ml/min/kg [ . - . ]) (p < . ). the isachc stage of heart failure was improved at months after surgery compared to before surgery. in addition, serum atrial natriuretic peptide level, plasma nt-pro brain natriuretic peptide level, plasma urea nitrogen concentration, la/ao and vhs significantly decreased after surgery (p < . ). the use of diuretics decreased after mitral valve repair surgery and consequently, a decrease in plasma urea nitrogen and creatinine levels were observed. therefore, this suggests that the main cause of azotemia in dogs with mitral regurgitation may be due to inadequate renal blood flow and exacerbation by the use of diuretics. no conflicts of interest reported. glomerular filtration rate (gfr) is generally considered to be the gold standard measurement of kidney function. gfr can be calculated by measuring serum iohexol clearance using concentrations at , and hours following a bolus injection. for validation, serum samples were spiked at low ( . mg/ ml), medium ( . mg/ml) and high ( . mg/ml) iohexol concentrations. they were analysed, along with standard calibration curves ( concentrations ranging from . to . mg/ml), using deltadot's label-free high performance capillary electrophoresis (hpce) system. data were analysed using deltadot's general separation transform (gst). clinical and spiked serum samples were also sent for analysis by mass spectrometry (ms) at a reference laboratory ( samples for comparison). concentrations obtained by hpce and ms were compared in a bland altman plot. gfr for clinical samples was calculated from the measured iohexol concentrations using the method reported by bexfield ( ) . a validated method was produced, with a lower limit of detection of . mg/ml and an lower limit of quantification of . mg/ml. the upper limit of quantification was . mg/ml. the standard curve had excellent linearity (r = . ). maximum inaccuracy was less than . % of the true value, except at lloq, where it was within . %. average within day variability was less than . % at all levels, while between day variability was less than . %, except at lloq, where it was less than . %. agreement between the results obtained by measurement with hpce and ms was good (bias . %, lower and upper limits of agreement of - . and . %, respectively). method specificity was confirmed by the absence of matrix effect in six serum specimen obtained from clinical dogs. clinical samples were analysed and gfr reported with a day turnaround time. in conclusion, hpce provides an accurate and precise method for measuring iohexol in canine serum. conflicts of interest: l pelligand has the following information to disclose: in receipt of research grant / contract funding from orion ltd., novartis animal health, transpharmation ltd, deltadot ltd; acted as a consultant for: triveritas ltd. and novartis animal health; s williams is an employee of the rvc and works in collaboration with deltadot ltd; j. elliott has the following information to disclose: consultancy: pfizer animal health / zoetis, ceva animal health, boehringer ingelheim, vetoquinol ltd, orion ltd., elanco ltd, idexx ltd, niche generics ltd. triveristas ltd., virbac ltd., advisory board membership: international renal interest society (supported by novartis) european emesis council (sponsored by pfizer animal health -now zoetis) cardiorenal board -vetoquinol ltd. idexx renal advisory board research grants or contracts: vetoquinol ltd, novartis ltd, pfizer animal health ltd (now zoetis), royal canin ltd, boeringher ingelheim ltd, waltham centre for pet nutrition, ceva animal health orion ltd. esvonc-p- cystic pancreatic neoplasia in cats. c.m. borschensky , k. steiger , a. staudacher , m. schlitter , i. esposito , h. aupperle . laboklin gmbh&co. kg, bad kissingen, germany, tu m€ unchen, institute of pathology, m€ unchen, germany, veterinary clinic dr. staudacher, aachen, germany pancreatic neoplasms in the cat mostly exhibit a solid growth pattern and are diagnosed as carcinomas. in contrast, only few reports about cystic pancreatic lesions exist. until now, only benign cystic pancreatic lesions are described in the literature. according to the histological pattern, they have been termed as cysts, (acinar) cystadenoma or pseudocysts. in man, cystic pancreatic neoplasms are classified according to the localisation (intra-/extraductal), growth pattern and differentiation (mucinous, (tubulo)papillary, serous, acinar). the aim of this study was to characterise feline pancreatic neoplasms in more detail, based on the human classification system with a special view on cystic lesions. pancreatic masses sent to laboklin from domestic cats ( - years) were investigated routinely macroscopically and by histological methods (h.&e. stain). the neoplasms showed a cystic (n = ) or solid (n = ) pattern. cystic pancreatic tumors were up to cm in diameter and were classified as benign variants in five and malignant variants in three cases. based on the human classification system, they were classified as tubulopapillary (n = ), acinar (n = ) and mixed (n = ) adenomas and mixed carcinomas (n = ), respectively. solid pancreatic nodules were diagnosed as carcinomas with a tubular (n = ) or acinar (n = ) differentiation pattern. in summary, the gross structure (solid versus cystic) seems to be of prognostic relevance. in contrast to solid tumors, cystic pancreatic lesions in the cat behave benign in a higher percentage of cases, resulting in a better prognosis. therefore, surgical excision of these cystic masses can be recommended. with respect to the human classification system, three different subtypes of cystic pancreatic neoplasms were detected in the cat that have not been described before in veterinary medicine: tubulopapillary, acinar and mixed. to best of our knowledge, this is the first report of cystic adenocarcinomas in feline pancreas. further corresponding clinical and histological investigations are needed for a better diagnostic (ultrasound, mri) and prognostic characterisation of cystic lesions in feline pancreas. no conflicts of interest reported. the immunohistochemical detection of cyclooxygenase- (cox- ) expression in canine mast cell tumours was recently described by our team (prada et al., ) . however its prognostic value needs to be established. the aim of the present work was study the prognostic value of cox- expression by investigating the relationship with several clinical and pathological variables including the overall survival (os) time. we included dogs with mast cell tumours ( grade i; grade ii and grade iii). cox- immunohistochemical expression was carried out by a streptavidin-biotin method. for the cox- immunoreactivity evaluation were considered the number of positive cells (cox- extension), the intensity and the score of cox- . the following clinical and pathological features were considered: animal age, sex, tumour anatomical location, tumour size, skin ulceration, histological grade, histological safety margins and number of mitosis. cox- expression was correlated with the clinical and pathological data and with the overall survival. cox- intensity was statistical significantly associated with skin ulceration (p = , ); histological grade (p = , ) and absence of histological safety margins (p = , ), high mitotic number (p = , ) and with overall survival (p = , ). both cox- extension and cox- immunohistochemical score present no statistical relationship with the variables considered neither with the overall survival. our results suggest that cox- have an important role in dog mast cell tumours progression and could constitute a promising therapeutic target in this neoplasia. however, our study also demonstrated that in mcts, is the cox- intensity that has the prognostic value, not the number of cox- positive cells (cox- extension) and not the cox- immunohistochemical score. consequently, cox- intensity should be elected for evaluating the cox- positivity in mcts immunohistochemical studies. merial provided financial support for immunohistochemical analayis. the research centres has also received financial suppoprt from cecav, ceca and citab. dogs which were radiated for a subcutaneous sarcoma between and were included. medical records were reviewed and patient characteristics, treatment protocols, adjuvant therapies and outcome were analysed. follow-up information was obtained from medical records and by phone conversations with veterinarians or pet owners. thirty-two dogs were included into this study. mean age was years and mean body-weight was kg. male dogs were slightly overrepresented ( . %). curative intent radiotherapy was applied in dogs and palliative intent in dogs with a mean total dose of and . gray, respectively. in dogs microscopic disease was radiated. five dogs received liposomal doxorubicin concomitantly with radiotherapy, two received adjuvant doxorubicin and one intralesional cisplatin. overall median survival time was days with curative and days with palliative treatment. overall median survival time in dogs with macroscopic disease was days and in patients with microscopic disease it was not reached. radiotherapy was generally accepted as new treatment modality by pet owners and referring veterinarians. comparable to the literature, best outcome was achieved for dogs radiated with microscopic disease conflicts of interest: no conflicts of interest reported. oncept â , is indicated for the treatment of stage ii or iii oral melanoma after local control with survival times significantly increased following vaccination. a similar improvement in survival times has also been reported with digit melanoma. medical records of dogs diagnosed with melanoma between march and december were retrospectively evaluated. inclusion criteria were a histopathological diagnosis of melanoma, surgical excision of the tumour, and vaccination using the oncept â vaccine. dogs met the inclusion criteria. nificant renal involvement in dogs with cvl. this result indicates staffordshire terrier ( ), bouvier, giant schnauzer, maltese, irish setter, kerry blue terrier, golden retriever, scottish terrier, and great dane ( of each). dogs had stage ii digit melanoma with an equal sex distribution and a median age of . years (range - ). currently still alive and one dead, the latter following surgery for resection of a rib osteosarcoma the median survival time of the dogs still alive is . months (range - ) versus . months (range - ) for the dogs that have died. none of the dogs showed any adverse effect to the vaccine infected macrophages can cause injury in different organs, including the kidney. cvl is known as a common cause of glomerulonephritis. thus, this study aimed to investigate and characterize the renal lesions in dogs seropositive for leishmania sp. in brazil. this project was approved by the animal ethics committee of uece, brazil. twenty adult dogs seropositive for cvl from center for zoonosis control were randomly selected for this experiment. cvl was diagnosed by immunofluorescence and elisa. urine and blood sampling and kidney harvesting were performed immediately after euthanasia that the glomerulonephritis is a common sequelae related to leishmaniasis infection. even dogs in stage of ckd showed significant renal histopathological changes. animals infected with leishmania sp. may have severe renal damage and risk of progressive chronic kidney disease even when no increase of creatinine levels or proteinuria is detected.conflicts of interest:the authors received funding to pay the phd scholarship of one student (conselho nacional de pesquisa e desenvolvimento -cnpq-brazil) and received a research grant from fundac ßão cearense de apoio ao desenvolvimento cient ıfico e tecnol ogico -funcap. anaplasma phagocytophilum, the causative agent of canine granulocytic anaplasmosis, is an obligatory intracellular bacterium transmitted by ixodes ticks. transmission via blood transfusion has rarely been described in human medicine and once in a dog. in the berlin/brandenburg area the seroprevalence rate in dogs was % regardless of health status.the aim of this study was to evaluate pcr screening results for a. phagocytophilum in canine blood donors between - in order to estimate the risk of transfusion-transmitted infection. edta blood samples from dogs were submitted for a. phagocytophilum real-time pcr testing (targeting the msp gene). altogether dogs were tested up to times. clinical and laboratory data were examined before each donation. statistical analysis was performed using spss . .the pcr test was positive for of the samples. none of the dogs tested pcr positive more than once. positive results were most often detected in june ( ), may ( ), and july ( ), but also in five other months. in three dogs a mild increased in rectal temperature (≥ , °c) was documented. mild laboratory abnormalities were noted in dogs: thrombocytopenia ( ), leukocytosis ( ), leukopenia ( ), anemia ( ) and hyperproteinemia ( / ); four dogs had more than one abnormality. there was no significant difference between the pcr negative and positive blood samples in regard to laboratory abnormalities.altogether, . % of blood samples from healthy canine blood donors were pcr positive for a. phagocytophilum. therefore, blood donors should be screened by pcr in endemic areas all year round. no conflicts of interest reported. the study population consisted of cats, including control cats recruited from veterinary practices across the country. among the disease cats, cats presented urtd, cats had conjunctivitis and cats suffered chronic gingivostomatitis, many of them presenting more than one clinical sign. pcr for the above-mentioned pathogens was performed from pooled conjunctival and oropharyngeal swabs for each cat. a questionnaire regarding signalment (age, breed, sex, neuter status), environment (indoor, number of cats in household) and vaccination history was obtained. data was analysed by multivariable logistic regression with alpha equal to < . .the prevalence for the four pathogens has been previously reported in detail. briefly, the prevalence among the four groups (including controls) ranged from to % for fhv- , - % for fcv, - % for chlamydophila felis and - % for mycoplasma felis.in the univariate analysis, age, neutering status, being purebred, indoor keeping, number of cats in the household and body weight were variably associated with the different groups of disease and the presence of the pathogens. in the multivariable analysis, only the following factors remained significant. in the multivariable analysis, only the following factors remained significant: urtd was significantly associated with positive results for fhv- , chlamydophila felis and mycoplasma felis (in addition to being male and not castrated); conjunctivitis was significantly associated to positive results for fhv- and chlamydophila felis (in addition to being young, not castrated and purebred) and cgs was significantly associated to positive results for fcv (in addition to being young, male and purebred). not being properly vaccinated was a significant risk factor only when all three groups were analyzed together. the number of cats in the household was an independent risk factor for detecting each of the pathogens studied. the age was also a significant factor in cats with fcv and chlamydophila felis, being older cats predisposed to fcv and younger cats predisposed to chlamydophila felis.the present study describes important epidemiological data for cats presenting urtd, conjunctivitis and/or cgs, and emphasizes the complex interrelationships occurring among the different pathogens. our results also support the role of fcv in cats with chronic gingivostomatitis.conflicts of interest:the study was funded and designed by merial laboratories. reports of methicillin-resistant staphylococci (mrs) in animals have become more frequent in last years. various studies have demonstrated the transmission of mrsa between animals and humans in daily contact with animals, however there is only limited data so far available on the transmission of methicillinresistant coagulase-negative staphylococci between animals and humans. the objective of this study was to investigate the frequency of methicillin-resistant staphylococci (mrs) carriage in healthy veterinarians, veterinary nurses, veterinary assistants, veterinary students and farm workers from several veterinary hospitals, clinics and farms.nasal swabs were collected from veterinarians ( small animal veterinarians and pig veterinarians), veterinary nurses, veterinary students, veterinary assistants and farm workers. mrs were screened on brilliance tm mrsa agar (oxoid) or chromid tm mrsa (biom erieux). after - h of incubation at °c, suspected colonies on both media were subcultured onto blood agar plates. species identification was obtained by species-specific pcr. methicillin-resistance was confirmed by pcr amplification of the meca gene. mrsa isolates were characterized by mlst.thirty-nine mrs were identified in humans ( veterinarians, veterinary nurses, veterinary students, veterinary assistants and farm workers). the mrs isolates were identified as staphylococcus aureus (mrsa, n = ), s. epidermidis (mrse, n = ), s. pseudintermedius (mrsp, n = ), s. haemolyticus (mrsh, n = ) and mrs coagulase-negative staphylococci. the frequency of colonization by mrs was similar in both small animals and pigs veterinarians (ae %). one veterinary student was colonized simultaneously with an mrse and an mrsh. the predominant st in humans in contact with small animals was st and in humans in contact with pigs was st .in our study the frequency of colonization by mrsa was high, but the frequency of mrse should not be underestimated. mrsa isolates in this work belonged mainly to the st lineage which is the most frequent in small animals and humans in europe. humans in daily contact with animals can become colonized by mrs of animal origin and thus are important keys for infection control programs in veterinary hospitals and farms.conflicts of interest: dr pomba currently receives research funding from the government and national programmes (fundac ßão para a ciência e a tecnologia). in the past, she has occasionally received research support or honoraria for lectures from pharmaceutical companies including zoetis and atral cipan. she is vice-chair of the antimicrobial working party (awp) and member of the antimicrobial advice ad hoc expert group (ameg) of the european medicines agency (ema). , species not determined [ ]) did not. all cats underwent antibiotic treatment (doxycycline or a fluoroquinolone); cats received blood transfusions and/or oxyglobin â . three cats were euthanatized within days due to concurrent disease (fiv, pancreatitis/cholangitis) or financial constraints, one cat due to persistent anemia after weeks. four cats were lost to follow-up. the remaining cats underwent follow-up for a period of - weeks (median ). hemoplasma pcr analysis was conducted - times on blood samples at variable time points from of the follow-up cats. the first negative pcr in cases occurred after (cmhm, during antibiotic treatment), (cmhm, during antibiotic treatment), (cmhm, during antibiotic treatment) and (mhf, after completion of antibiotics) weeks. one cat remained pcr positive (cmhm) at , , and (all during antibiotic treatment) weeks, and another cat (cmhm) was pcr positive at weeks. reactivation of the hemoplasma species (documented by hemolysis and positive pcr) occurred in cats (both cmhm) and times, respectively, up to weeks after initial presentation. reactivation was suspected (no pcr testing available) in additional cases (cmhm [ ], mhf [ ]). four of the follow-up cats were euthanatized after - weeks (median ) due to concurrent disease (cardiomyopathy, immune-mediated thrombocytopenia, postoperative complications, diabetes mellitus). infection with hemoplasmas is often chronic, can reactivate months later and is rarely the reason for euthanasia. no conflicts of interest reported. canine distemper (cd) is a worldwide occurring infectious disease caused by a morbillivirus of the family paramyxoviridae. cdv infection can result in a systemic infection. dogs presented with neurologic signs revealed the terminal stage of the disease and usually failed to therapy. additional passive immunotherapy is hypothesized to be beneficial in the early stage of cdv infection. porcine anti-cdv antibody subunit f (ab') [f(ab') ] was produced by animal technology laboratories, agriculture technology research institute. eighteen cdvnaturally infected dogs showing respiratory signs but no neurological signs were treated with the combination of f(ab') and supportive therapy (group ). group included dogs in a similar clinical signs (without neurological signs) that received only supportive therapy. the survival rate was . % ( / ) in group and . % ( / ) in group , respectively, with a significant difference between the two groups (p < . ). the progressive rates of developing neurological signs during therapy of group and group were . and . %, respectively. there was no significant difference between the two groups. the survival rates of dogs developing neurological signs during therapy were % ( / ) in group and . % ( / ) in group , respectively, with a significant difference between the two groups. in conclusion, additional administration of porcine anti-cdv antibody subunit f(ab') before developing of neurological signs could decrease the mortality and furthermore reduce the rate of developing neurological signs. no conflicts of interest reported. key: cord- -rr xua authors: nan title: news date: - - journal: aust vet j doi: . /avj. sha: doc_id: cord_uid: rr xua nan photo: istock adelaide vet speaks out against dog anti-vaxxers an adelaide vet has taken to the air to address a growing number of pet owners who believe that vaccinations cause autism in dogs. speaking to the fiveaa radio station, dr derek mcnair expressed concern that certain areas of adelaide had low vaccination rates as a new trend of using herbal remedies instead of vaccines grows in popularity. "i'm not anti-holistic treatments and helping for some things, but to use it in place of vaccines is totally ridiculous and stupid." the trend comes as "anti-vax" material floods the internet, with the british veterinary association recently forced to publicly deny that dogs can get autism. similar discussions have been across the media throughout australia. the avas president, dr paula parker, has been responding to the media with a clear message that it is dangerous to forgo pet vaccinations. shipments of australian and new zealand dairy cows to sri lanka have been halted after a large number of the imported livestock have died on-farm. the sri lankan government has ordered an inquiry into the high mortality rates, along with reports of poor milk yields, low conception rate, stillbirths, and unrecognised diseases. queensland country live reports that , holstein-jersey heifers have been shipped to sri lanka under a government program to increase the country's fresh milk production. local farmers say the cows are unsuited to sri lanka's hot and humid tropical conditions, despite the same australian-bred cows have been performing well in indonesia. the ava is monitoring the story, particularly additional disclosures on the compliance with the nutritional, veterinary and animal husbandry recommendations made. woolworths over price rise news report a number of pet food brands have recently gone missing from supermarket shelves following a pricing dispute with manufacturer mars petcare. coles and woolworths' customers will have to go elsewhere for their pet's favourite brand after mars pulled the plug on distribution to the two supermarket giants following a price rise disagreement. mars produce different pet food brands globally, including royal canin, pedigree, whiskas, dine and my dog. representatives from the supermarkets said they expected a deal to be brokered soon. you can't be what you can't see i spent much of january and february of this year buried in research and thinking about women, leadership and the future of our profession. social media is littered with quotes and catchphrases about leadership. one that stuck with me through the data and the papers is 'you can't be what you can't see'. if you can't see someone that seems similar to you in a position of leadership, then it can be difficult to believe that that role, or one similar to it, is possible for you, or although it might be possible, the cost of forging a scarcely trodden path may be too great. there is some veterinary specific data that outlines this well. in the november report 'motivation, satisfaction and retention: understanding the importance of vets' day to day work experiences' from the bva and the university of exeter, two of the key findings included 'feeling like one fits in with those who have been successful before you, and having role models' as being important to motivating veterinarians, facilitating their professional satisfaction and retaining them in the profession. of the myriad of issues that our profession faces and cares deeply about, the financial security of veterinarians, and particularly of women, is the one closet to my heart. i have seen several members of our profession profess that as a natural consequence of more women in the profession, there will be increasing gender representation in leadership positions overtime. this is an alluring proposition, but not one supported by the data. vertical segregation describes the phenomenon where there is predominance of men in more senior positions, and a predominance of women in more junior positions. vertical segregation occurs across every aspect of the australian economy, and even in female dominated professions, women are under-represented in leadership roles. the reasons for this are a complex interaction of unconscious bias, structural and cultural factors that require more discussion than can be accommodated in this column. one of the recommended interventions however, is to shed light on diverse role models. one of the greatest pleasures of being the ava president, is the opportunity to work with leaders across our profession, in all of the contexts where we contribute to the community. so, in this column, i wanted to introduce you to some of the women i have had the privilege of seeing in action and whom i admire for their leadership, their integrity and their service. i hope that you can see them too and know that regardless of the leadership role you would like to play, that there is someone who has forged a path. • if you want to be a pillar of your community, meet melissa rogers. • if you want to steward the standards of our education and be proud to promote them to the world, meet julie strous. • if you want to be a young veterinary business owner, meet kat lovell. • if you want to be a part of our leadership of animal health in the world, meet jennifer davis. • if you want to lead a global animal health company, meet stephanie armstrong. • if you want to be excited about nutrition and life, meet penny dobson. • if you want to champion collaboration across our sector, meet janet murray. • if you want to know how to exemplify excellence, compassion and grace, meet liisa ahlstrom. • if you want to be a pioneer in our sector and do so with ethics, charisma and a smile, meet rachel chay. • if you want to meet a leader who understands how looking after your team makes good business sense and puts it into practice, meet jacqui von hoff. • if you want to change the face of dairy medicine, meet gemma chuck. • if you want to feel excited about being a veterinarian and putting that out there into the world, meet zoe vogels. • if you want to lead the education of the next generation of veterinarians, meet cristy secombe. corey snell, ceo achieving better health outcomes for pets as you read this column our flagship ava annual conference in perth will have concluded. this year the theme was "the profession: better working together". here we introduced an innovation symposium and our food security day. the food security day focused on biodiversity and welfare, bringing experts from around the world together to discuss the challenges and opportunities faced to sustainably feed more than . billion people by . both of these broke new ground for the ava and our aim is to continue these inaugural events. breaking new ground is not limited to our conference events. at the ava we have been asking ourselves how the ava could assist you in contributing to the optimum health and welfare outcomes for australian pets. most importantly how could we assist our members in providing better care to ensure happier clients? as i have learned more about the challenges our veterinarians face, i have been particularly concerned by the pressure experienced by veterinarians resulting from the inherent tension between cost of veterinary care and our clients propensity to pay for services. i understand that this is one of the biggest causes of stress for the profession. i imagine it must be heartbreaking when a pet's owner is unable to pay for veterinary treatment and they are forced to make a decision that is not in the best interests of the animal. one of the tools which can help the financial preparedness of our clients is pet insurance. at the ava annual conference, we launched our partnership with guild insurance in the development of an ava endorsed pet insurance offering called 'vets choice'. guild insurance and the ava have collaborated to develop a pet insurance solution that that has been designed to meet the needs of our profession and our customers. it is my hope that by increasing the proportion of pet insurance in australia will lead to a more sustainable veterinary practices and i am particularly excited at the opportunity this provides not only the broader public but our profession and our members as well. we've got something exciting coming… vets choice is a modern pet insurance product, packed with market leading features and benefits, brought to you by the ava and guild insurance. we'd love to tell you what the product is, but it's different now than it was three weeks ago, and we're still working to refine and respond to the needs of what both you as the vet and your patients need most. send an email to vetschoice@guildinsurance.com.au if you want to be among the first to be part of it… is the dingo really just another type of dog? the dingo has been present in australia for at least the past few thousand years, with the earliest dated fossil being around years old. they are recognised native animals, but are also classified as a pest species in many jurisdictions and often defined as 'wild dogs'. since the commencement of livestock farming across australia over the past years, the dingo has had a negative relationship with graziers. early on, many efforts were made to eliminate populations and this has resulted in the dingo being absent from many parts of its historical range. the control of wild dogs is an ongoing issue for graziers because they can cause significant financial losses through predation of sheep, goats and calves. by sight alone, it is often difficult to differentiate dingoes and their hybrids from wild dogs and it is this dilemma that lead to the western australian government to recently consider removing the dingo's status as native wildlife and classify them as 'non-fauna'. this would make them no different to any other dog and allow them to be killed throughout the state. the canidae is a diverse group that includes the domestic dog, wolves, coyotes and dingoes. individuals can interbreed and produce fertile offspring and therefore technically could be classified as one species using the 'biological species' concept. istock photo news n a paper published in the veterinary record last month investigated the presence of bacterial contamination in commercially available raw meat pet food products in sweden. samples were taken from raw meat pet food products sold in supermarkets in sweden and produced by different pet food manufacturers. results showed all samples were contaminated with enterobacteriaceae bacteria, suggesting faecal contamination because these bacteria are found in the gastrointestinal tracts of animals. the presence of e. coli and other enterobacteriaceae bacteria is often used as an indicator of hygiene during processing. in addition to the presence of enterobacteriaceae bacteria being found on the samples, salmonella species were found in four of the samples and campylobacter species in three samples. in two samples, the level of clostridium perfringens bacteria exceeded bacteria/g, which is above the maximum limit for anaerobic bacteria permitted by swedish national guidelines. the authors concluded that raw pet meat food sold commercially in sweden should be handled carefully, with owners needing to implement strict hygiene protocols when storing, handling and feeding raw meat pet food products. they also discussed the potential health risks that raw meat pet food products present to both humans and animals, particularly geriatric, neonatal and immunocompromised individuals. these findings come at a time when raw meat-based diets for pets are becoming increasingly popular. the proliferation of both commercially available raw diets such as 'biologically appropriate raw food (barf)', as well as homemade diets, is leading to growth in the raw pet meat sector. although commercial dry and canned pet foods still dominate the pet food market, the introduction of raw diets and homemade diets is leading to a small but passionate group of owners who promote raw diets for health and philosophical reasons. the pet food industry in australia is currently under review. after a senate inquiry examined regulatory approaches to ensure the safety of pet food in late , the resulting inquiry's report recommended widespread changes to both oversight and safety standards of the sector. the federal department of agriculture and water resources is currently leading a working group comprising representatives from state governments, the australian veterinary association, rspca australia and food standards australia and new zealand. this working group is examining how best to regulate and improve safety standards in the pet food industry. commercially available raw pet meat products will be included in this review, as well as treats and manufactured pet food. it is currently unknown if the results of this swedish study are replicated in the raw pet meat market here in australia. similar results were found when raw pet meat products were tested in the netherlands in , with high levels of bacterial contamination as well as several parasites including sarcocystis species and toxoplasma gondii detected. if owners insist on feeding raw pet meat products to their pets, the authors of the veterinary record paper recommend the following risk reduction tips: keep raw pet meat frozen until use, always handle raw pet meat with separate utensils as well as wash hands thoroughly after contact and do not feed raw pet meat products in households with immunocompromised individuals. in early april the animal health quadrilateral group (quads), senior animal health officials from australia, canada, new zealand and the united states met for their annual meeting. the issue of attracting, developing and retaining government veterinarians was an item of keen interest on the agenda. government veterinarians play critical, unique and diverse roles in a country's veterinary services, which underpin a country's animal health status and trade. the risks and opportunities in animal health will continue to evolve, as will the expectations, interests and demographics of the veterinary profession. the future will bring new information and diagnostic technologies; changes to risk and community expectations; and evolving communication needs. therefore, we need to be proactive, and ensure the skills of future veterinarians reflect this changing context. in the field of government veterinary services there may be a need for increased veterinary public health specialist expertise, such as epidemiology, risk management, and diagnostics. it's also expected that we will need to increase our collaboration with experts from other disciplines such as information technology and data analysis; laboratory, public health, social and environmental scientists; communications professionals; and economists. the world organisation for animal health (oie) is currently developing their th strategic plan ( - ) and they too are looking at their future role, particularly on global issues such as food security, climate change, species conservation and the future of the veterinary profession. as president of the oie, i have been leading discussions in the development of the oie's th strategic plan. in reviewing the external forces that will impact the organisation's future we see the issues of big data, centrality of social media, changing public perceptions and declining trust in governments, and we need to take these issues into account. at home, the australasian veterinary boards council (avbc) has discussed the future of the veterinary profession, and therefore how to educate the veterinarians of the future. similar to the quads group and the oie, the avbc also recognised that the profession's future is inherently linked to broader global issues. veterinarians will be managers of expert knowledge and will need to work in the one health space, where environmental, human and animal health intersect. technology is playing an increasing and positive role in veterinary science education. assisted and virtual reality enhance practical learning and learning is expected to become increasingly collaborative with students study virtually across a range of institutions. it reminds me of the plenary presentation by jordan nguyen at last year's ava conference on 'the boundaries between human and technological evolution and intelligent technologies'. he certainly opened my eyes to what the future might look like! and speaking of the future, i have recently really enjoyed getting around to a few of our veterinary schools during my travels and speaking with the veterinarians of the future. i recently dropped in to the campus in gatton and met with university of queensland staff and students. i really value the perspectives gained from these visits and hearing what's happening at the forefront of our profession. this month, following the ava conference in perth, i'll return to my alma mater, murdoch university, to speak with and meet staff and students about the importance of one health and the broad range of opportunities available to us as veterinarians. vets really do play such an important role in animal and human health and we have the opportunity to truly contribute to the important issues facing the globe. following the ava conference and trip to perth, i will be flying to paris for oie council meetings in mid-may and the general session at the end of the month, my first as president of the oie assembly. i look forward to introducing you to a couple of members of the australian delegation to the oie general session so they can tell you about their veterinary career paths. assisted and virtual reality enhance practical learning and learning is expected to become increasingly collaborative with students study virtually across a range of institutions. veterinarians have been thinking about preventive care protocols in their clinics for some time now offering annual wellness checks. these annual checks may include vaccination based on the patient's individual needs, and may or may not include diagnostics. for some, the decision not to include diagnostics in the wellness check has been due to consideration of whether there is value in adding diagnostic testing when a patient has an unremarkable history and clinical examination, but the evidence to do so is mounting. several peak bodies have produced guidelines on care of dogs and cats at different life stages, including the american association of feline practitioners (aafp) , , the american animal hospital association (aaha) , , and the australian veterinary association . all of these recommend regular health checks (annual for adults, and every months for seniors and older), with diagnostic testing included, especially as animals age. add to this, a series of published studies evaluating the results of diagnostic testing in apparently healthy adult and senior pets, and we can start to draw some evidence-based conclusions around the value of diagnostics in this preventive care setting. in a study of cats and one involving dogs , there were significant numbers of patients with changes on blood and urine screens, and a more recent study undertaken in australia involving dogs and cats showed . % of dogs and . % of cats with significant changes that warranted further investigation . aaha however, were interested in leveraging the huge volumes of data produced by the industry each year to understand the issue on a much larger scale, and took a somewhat different approach to data collection . in their 'big data' study -the aaha research team evaluated over , patients, but not necessarily in the same detail as the previous studies. here are some details from the study that all veterinarians should be aware of: • data was gathered from , consenting practices and harvested from their practice management software • investigators evaluated profiles on patients that were classified as 'wellness examinations' in the practice information management system • the patient visit needed to include a cbc, full chemistry profile including idexx sdma®, and ideally a urinalysis. • for some parameters, the reference interval was widened slightly to a critical threshold before a result was abnormal (for example for liver enzymes the lower range was set to zero, and in dogs the upper end of phosphorus was increased by %). • for the majority of biochemical parameters, the requirement was that at least three or more results had to be abnormal before the findings were considered significant. • a single abnormal sdma result was considered significant. • for haematology, only numerical values were assessed (changes on blood film reviews were not included), and abnormalities identified included: • anaemia • reticulocytosis • leucocytosis • stress leucogram (lymphopaenia and eosinopaenia) • eosinophilia in many regards the approach taken was a conservative one and means that potentially significant changes preventive care were under-reported. this in-built 'conservativism' is because not all the haematological parameters were evaluated, and there was no comparison to previous results. where we now recognise the benefits of evaluating the health status of the pet by using an individual reference interval (comparing current results to previous results obtained for that patient in health) for a number of routine parameters, creatinine being a good example. in the aaha study the results were as follows, with respect to results that warranted further investigation. in this study the addition of sdma made a significant difference to the number of patients warranting further evaluation. in the adult and senior populations there were an extra per patients who required further investigation, whereas it was per for geriatric patients. in conclusion, and as we seek to enhance the health and wellbeing of pets, people and livestock, i would suggest that studies such as the ones discussed here provide supporting evidence that the inclusion of diagnostic testing in discussions on wellness and preventive care is critical. moreover, observation suggests that it is also something that clients have come to expect. the animal medicines australia pet ownership report showed nearly as many clients presented their pets to the vet to keep them well as those presenting for illness . dr graham swinney bvsc, fanzcvs (canine medicine) the eastern bettong is a small marsupial that was once common across the east coast of australia. shortly after the introduction of predators, particularly the fox, their populations crashed and they became extinct on the australian mainland around years ago. fortunately for the eastern bettong, tasmania is free of foxes and as a result is a remaining stronghold for the species. breeding programs for the bettong exist in predator-proof areas in the act and the animals in these protected areas are continuing to flourish. in , eastern bettongs were introduced to the wild near the lower cotter river catchment, kilometres southwest of canberra in an area that had been heavily baited to reduce fox population numbers. eight months after their initial release and through intense monitoring, ecologists involved with the program discovered that every female bettong found showed signs of breeding success. at the time this was a huge success for the program as it was the first time that wild-bred eastern bettongs were present on the mainland in the past years. some concerns were raised at this time, because survival rates of released bettong were found to be around %, but there was hope that the new joeys being born would help boost future populations. act parks and conservation was responsible for controlling feral pests in the release area and at the peak of the project were servicing bait stations on a weekly basis as well as monitoring between and wildlife cameras to identify the animals present in the area. unfortunately, abc news has recently reported that all eastern bettongs released into the wild as part of the project have now died and foxes are being held responsible. although the project was not as great a success as had initially been hoped for, director of act parks and conservation services, daniel iglesias said that the team has still gained valuable information through the project because during the early months the bettongs were coexisting and reproducing in the presence of foxes. however, this only occurred when the fox population was low enough to allow the bettongs to survive, and the methods used to achieve this low level of foxes in the wild are not practical on an ongoing or widespread basis. the introduction of predators to australia has been a contributing factor to the demise and extinction of our native mammals and given us the world's worst rate of mammal extinction. it is not possible to ever eliminate the threat of introduced predators from the wild, but finding ways to successfully manage their populations on a large scale is something that will need to be focussed on and invested in by the government, if we wish to slow down or reverse our rapid extinction rates of native wildlife. if this ever becomes a reality, we may one day find eastern bettongs scurrying around in areas they've been absent from for over years. the complexity of chf means a multitude of treatment approaches are required to ensure a good quality of life and longevity in our pets. in her thesis, mariko examines current treatments of chf, posing vital questions on how they can be improved. a new focus in congestive heart failure? it is well known that pimobendan, in conjunction with other drugs, is key to the treatment of chf. it is currently most commonly used and prescribed in tablet form; however, mariko assesses the use of a pimobendan oral solution in dogs. the pimobendan solution was seen to enhance echocardiographic measures of cardiac systolic function, reaching a maximal effect at - hours following administration. this study is notably the first to use non-invasive imaging of the acute cardiovascular effects of pimobendan. in addition to validating the efficacy of this solution, it raises the possibility of therapeutic monitoring in the future. mariko then examined the use of pimobendan in cats and her findings clearly showed reduced metabolism of pimobendan to its active metabolite in cats compared with dogs. interestingly, the cardiovascular effects of pimobendan could not be readily identified on echocardiography. these interspecies differences highlight the necessity for further research into the role and optimal dosing regimen of this drug in feline cardiovascular disease. in addition to examining existing treatment options for chf, mariko also studied the potential of the natriuretic peptide system as a therapeutic target in dogs with chf. in a recently published article, she identified that the cardiorenal effects of bnp - (a type of natriuretic peptide) were diminished in dogs with chf caused by myxomatous mitral valve disease. this suggested defects in what is essentially a protective mechanism in the context of chf and is a finding that prompts further investigations into therapeutics targeting this system. a recent case involving a fashion industry start up being penalised around $ , for underpaying staff has brought the issue of unpaid work into the spotlight. this came only weeks after a prominent australian company director was slammed for making comments around millennials "not wanting to work for free". these situations -while very different -both raise the question: is unpaid work permissible? the case. it's important to first outline exactly how and why this particular fashion start up fell foul of the fair work commission. over a -year period, the business was reported to have underpaid separate employees around $ , . one particular worker was said to be on an "unpaid internship" for a -month period -even though the nature of their engagement was that of an employee. the commission found that the business had failed to pay the workers their minimum entitlements under relevant employment legislation including minimum hourly rates, penalty rates and leave entitlements. as a result of the underpayment, the company was penalised around $ , and the sole director was individually penalised a further $ , . back pay was also ordered in full to the three workers. the severity of the penalty was largely due to the fact the company were said to have known they were under paying staff, with the commission labelling the underpayments "significant and deliberate". in short, yes, but be careful. as highlighted by the fair work ombudsman, sandra parker in the wake of the case, "unpaid placements are lawful where they are part of a vocational placement related to a course of study. however, the law prohibits the exploitation of workers when they are fulfilling the role of an actual employee." if you have been approached by a student looking for work experience, ensure it is part of a genuine placement through their learning institution. it might also be prudent to contact the institution (tafe, university etc.) directly to discuss the finer details including insurance and whether it can genuinely be unpaid. other than student placements -are there any other situations where unpaid work is permissible? there may be other situations where work can be unpaid, but this is assessed on a case by case basis and employers should check with the ava hr advisory service before providing any unpaid work. when the work is not part of a vocational placement, some of the major factors when considering whether work can be unpaid include; • will the work be for the benefit of the individual, or the business? • if the work primarily benefits the business, it is likely to require payment. • how long did the arrangement last? • the longer the arrangement, the more likely it is to requirement payment. • what was the nature of the work? productive work that is meaningful to the business will almost certainly requirement payment. observing, learning and performing very rudimentary tasks that give the individual a general "feel" for a role or industry may form part of an unpaid working arrangement. considering the above, unpaid work should almost solely benefit the individual (not the business), should only be for a brief period and should be largely observational. an unpaid trial can be permissible if it is purely to demonstrate a skill or skills associated with a particular role. the engagement should be brief (maximum one shift) and the individual should be under direct supervision the entire time. let it be clear -this is unrelated to a probation period which is sometimes referred to as a trial period. a probation period is generally between to months and must be paid as an employment relationship has already commenced. in summary. if you are considering engaging someone to perform unpaid work, ensure what you are doing is lawful and permissible. ask yourself the following; • is it part of a vocational placement related to a course of study? • if so, check with the learning institute to ensure it is unpaid. • -is it an individual wanting to gain experience in the industry (no vocational placement)? • ensure they do not perform any productive work, and their engagement is brief and observational. • is it a trial? it must be purely about the individual demonstrating skill/s relating to the role, it must be brief and the individual must be directly supervised for the entire engagement. if your plan to engage someone doesn't match any of the above, it is likely they will be an employee and will require payment. if you are unsure about anything in this article, contact the ava hr advisory service on or email hrhotline@ava.com.au. the material contained in this publication is general comment and is not intended as advice on any particular matter. no reader should act or fail to act on the basis of any material contained herein. the material contained in this publication should not be relied on as a substitute for legal or professional advice on any particular matter. wentworth advantage pty ltd, expressly disclaim all and any liability to any persons whatsoever in respect of anything done or omitted to be done by any such person in reliance whether in whole or in part upon any of the contents of this publication. without limiting the generality of this disclaimer, no author or editor shall have any responsibility for any other author or editor. for further information please contact wentworth advantage pty ltd. results from the survey revealed there were more than , volunteer wildlife rehabilitators in nsw, with almost half of those belonging to the largest wildlife rehabilitation group wires. on average, , animals go through wildlife rehabilitator hands every year and more than , calls for assistance and education are provided to the public annually. it was estimated it would cost the government, conservatively, more than a$ million to replace the time and resources spent by volunteer wildlife carers every year in nsw alone. most wildlife rehabilitation is conducted in the homes of volunteers, although a small number of wildlife hospitals and centrally based facilities supplement this home care. stemming from surveys and consultation work with peak bodies, the work of private veterinary practices was recognised as an essential element in the wildlife rehabilitation sector. it was estimated around , wild animals are seen by nsw private veterinary practices annually at a cost of more than a$ . million of pro bono services and products. this differs significantly from a previously published survey by orr and tribe, which estimated around , wild animals were seen annually by nsw private practices. it is currently unknown just how many wild animals pass through veterinary practices every year, although the strategy did recognise that "balancing the running of a private practice with the lack of time, facilities and resources for treating free-living wildlife is very challenging." through the findings of the two surveys and stakeholder consultation, several challenges facing the sector were identified. wildlife rehabilitators overwhelmingly identified the finding and keeping of new volunteers as a significant issue, with social demographics, time, financial demands, group politics, burnout and conflict all affecting increased participation rates. other challenges identified in this process were the lack of succession planning in many groups, particularly because of the older demographic of wildlife rehabilitators. this may lead to a loss of skills, less community support and fragmented and inefficient use of volunteer time if unmitigated. three-quarters of wildlife rehabilitators who responded to the survey indicated their support for stronger standards of care for wildlife as well as improved access to mentoring. one of the aims of this draft strategy is to standardise training and introduce a minimum standard of care for the sector. this would include a standard induction for all volunteers, as well as specialised species training to improve volunteers' depth of knowledge. it's important to note that the strategy acknowledges this is an ambitious goal. other challenges identified by volunteers as affecting the sector included a lack of strategic support from a strong, unified peak body. even though more than half of all wildlife carers belong to the group wires, most carers wanted improved advocacy and leadership for the sector and greater access to funding opportunities. less than % of all respondents were satisfied with the current level of support provided by the state government. unsurprisingly, a lack of funds was another key challenge facing volunteers. around % of wildlife carers surveyed reported incurring out-of-pocket expenses in the year prior. specific issues facing private veterinary practices many of the veterinarians and nurses surveyed reported their formal education wasn't very useful for dealing with injured free-living wildlife. additionally, the most common complaint from veterinary respondents was in relation to the behaviour of volunteers and their group leaders, as well as response times for collecting injured wildlife. the role of wildlife rehabilitation in environmental management was recognised as a key strength of the sector. most volunteers considered their efforts as greatly benefiting the environment. increasing the collection and use of data captured by the wildlife rehabilitation sector would improve wildlife and threatened species management, which was identified as an important outcome of this strategic document. additionally, much-needed research into post-release outcomes could be facilitated by better engaging with the wildlife rehabilitation community to improve treatment options and management decisions. several of the changes proposed by this draft strategy could have important outcomes for improving wildlife health and welfare in nsw. the strategy aims to: • develop a peak body for wildlife rehabilitation in nsw by either encouraging the two main bodies to unite or create an advisory board comprised of government, wildlife rehabilitation, animal welfare, veterinary and natural resource representatives • develop standards to be adopted by the sector • develop a charter for volunteer engagement with veterinary practices • commit a$ . million to implement the strategy • introduce minimum standards for volunteer training, with learning outcomes and performance criteria to demonstrate proficiency • commit a$ . million (of the $ . million total) to deliver standard resources for veterinarians and veterinary nurses in the handling, triage and treatment of wildlife; this will be delivered by taronga zoo in partnership with the university of sydney and the office of environment and heritage • streamline data capture via online reporting of wildlife rehabilitation statistics and prepare annual reports for the sector and community • fund post-release monitoring of rehabilitated koalas • fund a single wildlife rescue telephone number for the public. nsw is not the only state that will see changes to the wildlife rehabilitation sector in . at the start of the year, western australia introduced licensing for all wildlife carers to keep track of volunteers and the relevant department in tasmania is currently finalising resources for veterinarians and nurses to aid in the triage and treatment of wildlife. it is expected the final nsw strategy will be implemented from july . news n i've recently been involved in the development of a business plan as part of my role as the ava student president. given my 'young grasshopper' status i found myself thinking, what does it take to be an entrepreneur in the veterinary industry? do people just find themselves taking over the mantle of head vet/ business owner as a rite of passage? is it a matter of luck, just serendipitously having the opportunity present itself, or can a fresh out of school veterinarian manifest it through sheer will power and effort? personally, i'm still holding out for a small loan of $ million from my parents. to find out more i had a chat with zachary lederhose, a recent graduate who has stepped into the world of business ownership. it might be more accurate to describe his current success in the field as 'hitting the ground running' but he won't claim as much. what motivated you to start your own business, zach? well, like just about every new graduate, i started out working as a general practitioner. loved the job and was a fantastic learning experience. that said, the main thing that stood out for me was not being able to have as much say in the direction we took as a team that i wanted. it wasn't that anything was wrong, it was a great atmosphere and we all got along really well. the sense of autonomy was what i sought. did it change much for your income? i wouldn't say it was the main driver behind my ambition to start a business. i was doing well in my previous role and could have very happily continued as such in that space. it all depends on what you, as a professional, value and where you get satisfaction in your job. for me, it felt like my earning potential was capped as an associate and that limited my opportunities. being my own boss makes it easier to pursue some of the things that interest me. for example, being able to purchase certain medical equipment to provide niche services or the flexibility to attend conferences that interest me. how do you find being responsible for a team? i think i was very fortunate that in my first job i had a great mentor in mike mesley at snowy vets. mike taught me a lot and we share similar opinions on practice management. with that behind me, i was confident in my abilities and i think that's a big part of the equation, believing you can actually do it. i also invested a lot of effort into developing my communication skills and bring the same focus to my current clinic. it's such an important skill -not just for interacting with clients and achieving the best outcome but to make sure we're working well together. the feedback i get from my team is important to me. as great as it is to have the constant loop of positives and 'yes men', sometimes i just need to be told i'm being a bit of an idiot. keeping my team comfortable to be that voice of reason helps me take stock of what i'm doing and means they are engaged in the decisions we make for the clinic. do you find your age and experience level makes it harder? to be honest, not a great deal. sure, i've faced some challenges along the way but nothing that anyone else starting out wouldn't come across. i would probably put it down to having a fantastic support network, including people from my private life. i can acknowledge i've been fortunate in that regard and it probably isn't the case for many of my peers. in a lot of ways, it comes down to having someone you can relate to who's been through the process. with a shifting demographic in the profession, i can imagine that's not always easy. that said, i did have a client ask how work experience was going for me the other day… i've certainly drawn some inspiration from my conversation with zach. hopefully for some of you reading it does the same, giving you that extra bit of encouragement to be brave and take a leap. similarly, if you're already a business owner keep an eye out; there are a fair few sharp minds coming through the ranks that could use your help. i'm fascinated by the prospect of more people like zach taking steps forward in business and the impact their innovation has on the profession, even if it's just one workplace at a time. all things considered, i should probably pick up that copy of barefoot investor that's been sitting on my coffee surprisingly, cats of all ages, and with both effusive (wet) and non-effusive (dry) forms, responded equally well to the treatment. the response was dramatic, with fever typically resolving within - hours of commencement and concurrent daily improvements in appetite, activity levels and weight gain were seen. cats with the effusive form had a reduction in abdominal effusion over a - -week period within - days of commencing treatment. after weeks of treatment, all cats that had responded to treatment appeared normal, or near normal, to their owners. the safety profile of gs- was also impressive, with no systemic signs of toxicity based on complete blood counts and serum chemistry values monitored over the treatment periods of - weeks in almost all individuals, with only one possible exception. a single cat showed a mild increase in renal parameters, but month later these abnormalities were not detected and the cat was also in remission. the study suggests using gs- at a dose of . mg/kg by subcutaneous injection every hours for weeks is a feasible way to treat fip. although the number of animals in this field trial study was limited, there were very promising results for commercialisation of this or similar nucleoside analogues in the future and someday fip may no longer be considered an untreatable disease. the studies examined the effects of two manufactured chemicals; diethylhexyl phthalate (dehp) and polychlorinated biphenyl (pcb ). dehp is a common plasticiser found in many household items (e.g. containers, toys, upholstery) and is used as a fragrance carrier in cosmetics, laundry detergents and air fresheners. pcb was used in coolant liquid but is now banned globally. both chemicals are widely detectable in the environment and one of the most common routes of exposure is ingestion of food. the researchers exposed sperm samples from both species to these two chemicals at concentrations that have been previously detected in the male reproductive tract and at levels commonly found in the environment. in both species, the exposure to these chemicals at these levels resulted in reduced sperm motility and increased the fragmentation of dna, which negatively affects overall fertility. as we increasingly share our homes with our pets, they are exposed to the same environmental and household contaminants as us and it is this exposure that researcher associate professor richard lea believes may be responsible for the fall in sperm quality in both dogs and humans in recent years. this finding also highlights the opportunity to use dogs as an effective model for future research into the effects of pollutants on human fertility, as other external influences, such as diet, are much easier to control in dogs than humans. stephen reinisch even more surprisingly, when used to inseminate ewes, both samples had almost the same pregnancy rate, with a rate of % for the -year-old sperm compared with % for the recently frozen sperm. this finding of the long-term viability of frozen semen is significant because it shows that genetics from individuals can be stored and used long after the original male has passed away, which could prove useful for breeding programs for many species in the future. the lambs produced from the study will also provide a useful baseline for the researchers to examine the genetic progress made by the wool industry through selective breeding over the past years. these lambs displayed characteristics that were common in merinos at that time, with large body wrinkles to maximise skin surface area and therefore wool yields. that particular characteristic has since gone out of favour, having been found to increase the risk of fly strike and create difficulties with shearing. stephen reinisch veterinary writer stephen reinisch veterinary writer delivery terms and legal title prices include delivery of print journals to the recipient's address. delivery terms are delivered at place (dap); the recipient is responsible for paying any import duty or taxes. title to all issues transfers free of board (fob) our shipping point, freight prepaid. we will endeavour to fulfil claims for missing or damaged copies within six months of publication, within our reasonable discretion and subject to availability. printing and despatch printed in australia by ligare pty ltd. all journals are normally despatched direct from the 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creating new collective works or for resale. special requests should be addressed to permissions@wiley.com offprints printed offprints may be ordered online for a fee. please click on the following link and fill in the necessary details and ensure that you type information in all of the required fields: www.sheridan.com/wiley/eoc. policy predicaments or precedents i write with concern that the current ava live export policy has serious implications and potentially sets a precedent that is detrimental to the advancement of the profession. through its constitution , the ava seeks to speak on behalf of the profession (members and non-members) to government, authorities, any person, or company. policy and position statements are drafted by the policy council or the board and once approved by the board become official statements of the association. the ava uses policy and position statements to outline the position of the association when talking on behalf of the profession and these form the basis of advice given to government, other authorities, media, etc. the ava live export policy states: "where live export occurs, an australian-registered shipboard veterinarian must accompany each shipment and this veterinarian must be independent and thus not employed by either the exporting company or the shipping company." background information is provided in policy statements and is supposed to explain the rationale and scientific evidence supporting the policy position. the background information in the ava's live export policy does not provide any rationale or evidence why australian-registered shipboard veterinarian must not be employed by the exporter or shipping company. the exporter, in many cases, is the owner of the animals during the export voyage. if ownership has been transferred to another party, the exporter is the owner's representative and responsible for the animals during the export voyage. the rest of the policy is concerned about animal welfare, it is assumed the reason for the non-employment recommendation is to ensure animal welfare. this is concerning for a number of reasons. firstly, it implies that the legislative system regulating veterinarians in australia is broken, as the peak professional body is recommending the only way to ensure animal welfare is for veterinarians to be independent and not employed by the animal owner or owner's representative. it seems, the ava does not believe that the state and territory veterinary surgeons boards or the federal government have effective legislation or are able to effectively regulate against the current legislation. secondly, it implies there is a problem with fee-for-service arrangements between veterinarians and animal owners and achieving acceptable animal welfare outcomes. nearly all veterinarians providing clinical services to any species, either as an owner of a veterinary business or employee veterinarians, operate under this fee-forservice arrangement with the animal owner. how does the ava systematically review and assess policies for possible unintended consequences, implications, misrepresentation of the profession, or the possibility of setting a precedents that is not in the interest of the profession as whole? should the board review the live export policy, will the ava stand by its recommendations to the moss review and ensure that relevant people with leadership skills, deep knowledge and understanding of the industry, animal health and welfare, and epidemiology are sought? dr andrew way face the facts: gender equality corporate-site/documents/ animals-and-plants/native-animals/volunteerwildlife-rehabilitation-sector-strategyconsultation-draft- animal welfare implications of treating wildlife in australian veterinary practices tel: + ( ) japan: for japanese speaking support, email: cs-japan@wiley.com. visit our online customer help available in languages at www.wileycustomerhelp.com/ask production editor sylvia cheong. email: avj@wiley.com information for subscribers australian veterinary journal is published in issues per year. institutional subscription prices for are: print & online: us$ (australia & new zealand), us$ (us), € (europe), £ (uk), us$ (rest of world). prices are exclusive of tax. asia-pacific gst, canadian gst/hst and european vat will be applied at the appropriate rates. for more information on current tax rates, please go to www.wileyonlinelibrary.com/tax-vat. the price includes online access to the current content and all online back files to ava members communication live export update # this will be going to policy council and the chair of the policy council will provide a response following this meeting.access it on the go across any device -where you want, when you want.the avj will be a tap and swipe away from july www.ava.com.au key: cord- -f o owg authors: navarre, christine b.; pugh, d.g. title: diseases of the gastrointestinal system date: - - journal: sheep & goat medicine doi: . /b - - - / - sha: doc_id: cord_uid: f o owg nan the gastrointestinal system is, arguably, more prone to disease than any other part of the sheep or goat. gastrointestinal parasitism alone is the most significant cause of production and animal losses in much of north america. , there is no substitute for a thorough physical examination when trying to determine the affected body systems of a sick animal; this is especially true in diseases of the gastrointestinal system. a complete physical examination should include palpation for body condition, assessment of abdominal shape and rumen motility, observation of the consistency of the stool, and evaluation for the presence of bloat. however, because rectal palpation cannot be performed in sheep and goats, diagnosis of disease in a particular segment of the gastrointestinal system can be difficult. therefore, the clinician may have to perform ancillary diagnostic procedures to characterize gastrointestinal diseases properly. clinicopathologic data consisting of a complete blood count (cbc), serum biochemical evaluation (sbe), and urinalysis can be helpful in differentiating gastrointestinal diseases, developing a prognosis and plan for treatment, and monitoring treatment. a cbc rarely identifies a specific disease, but it can be helpful in evaluating the severity of dehydration, anemia, and hypoproteinemia. the clinician must take care to interpret the packed cell volume (pcv) and total protein in light of the hydration status of the animal as noted on physical examination. an anemic or dehydrated hypoproteinemic animal may have normal pcv and total protein values. both the cbc and sbe can be helpful in characterizing the presence and severity of an inflammatory disease process. changes in the total and differential white blood cell count indicate acute or chronic inflammation; increases in globulins or fibrinogen suggest a chronic inflammatory disease. low protein levels, especially albumin, can point to chronic blood loss from intestinal parasitism or infiltrative bowel disease. liver disease should be suspected if liver enzymes or bilirubin are elevated. however, liver enzymes can be normal in chronic liver disease. also, albumin levels rarely drop in ruminants with liver disease, as they do in other species. changes in electrolytes can occur with gastrointestinal disease, especially if the animals are anorexic. electrolyte measurements also are helpful in formulating a treatment plan. hypochloremia and metabolic alkalosis occasionally occur in abomasal disease. a mild hypocalcemia may be encountered in some small ruminants with gastrointestinal atony. because many animals with gastrointestinal disease are dehydrated and therefore azotemic and possibly hypoproteinemic, urinalysis is helpful to eliminate urinary disease as a cause of these pathologies. normal ranges for clinicopathologic values are included in this textbook (see appendix iii) and also have been published in several other textbooks. [ ] [ ] [ ] [ ] however, clinicians would do well to learn the normal values, especially serum biochemistry values, established by the laboratory most commonly used for analysis. analysis of rumen fluid can help differentiate diseases of the forestomachs. an appropriately sized orogastric tube can be passed through the oral cavity for fluid collection (figure - ) . the clinician must properly restrain the animal, using a mouth speculum (figure - ) to prevent tube chewing. if the tube is chewed, its roughened surface may damage the esophagus; parts of a broken tube can be swallowed. rumen fluid also can be collected using percutaneous rumenocentesis , [ ] [ ] [ ] [ ] [ ] (figure the clinician then aspirates fluid with a syringe. local anesthesia and sedation of the animal may be necessary. this technique avoids the saliva contamination that can occur during collection with an orogastric tube, and it appears to be less stressful. rumenocentesis presents a slight risk of peritonitis, but this risk can be minimized with proper restraint. percutaneous rumenocentesis should not be performed on pregnant females. after the fluid is collected, it can be analyzed for color, odor, ph, protozoal species and motility, methylene blue reduction time (mbr), gram's staining characteristics, and chloride levels. normal values are listed in table - . anorexia may cause the fluid to appear darker, the ph to increase, and the number and motility of protozoa to decrease. a gray color, low ph, and dead or no protozoa are seen in rumen acidosis from grain overload. the mbr is prolonged with any type of indigestion. large numbers of gram-positive rods (lactobacillus species) also may be seen in rumen acidosis. elevated rumen chloride indicates an abomasal or proximal small intestinal obstruction (either functional or mechanical). the most important reason for examining feces in sheep and goats is to determine the presence and relative number of nematode parasites infesting an animal or flock. the quantitative technique for determining eggs per gram of feces (epg) is shown in box - . fecal epg values of more than to indicate serious infestation and the need for intervention. fecal occult blood testing and acid-fast staining of fecal smears also can be performed. fecal occult blood tests can detect microscopic amounts of blood in the feces. however, they cannot indicate which part of the gastrointestinal tract is bleeding. acid-fast stains of fecal smears that reveal clumps of acid-fast rods usually indicate infection with mycobacterium paratuberculosis ( johne's disease). generally, individual acid-fast rods found on fecal examination are nonpathogenic. abdominocentesis is useful in discerning the causes of fluid distention in the abdomen. two methods can be used. the first technique involves tapping the lowest point of the abdomen slightly to the right of midline; it is useful in ruling out a ruptured bladder as the cause of general ascites (figure - ) . , the clinician should take care to avoid the prepuce in males. the second technique is useful if peritonitis is suspected. because localized peritonitis is more common than generalized peritonitis, four sites are tapped. the two cranial sites are slightly caudal to the xyphoid and medial to the milk veins on the left and right sides. the two caudal sites are slightly cranial to the mammary gland and to the left and right of midline. for either technique, manual restraint with sedation is recommended; the use of real-time ultrasonography may help locate fluid pockets. a -gauge needle or teat cannula can be used for fluid collection. the clinician should prepare the site using sterile technique and provide local anesthesia when employing a teat cannula. fluid should be collected in a small ethylenediamine tetra-acetic acid (edta) tube for analysis and a sterile tube for culture. abdominal fluid can be difficult to obtain because of the small amounts normally present in both sheep and goats. the clinician should minimize the ratio of edta to fluid because edta can falsely elevate protein levels. using edta tubes made for small animals or shaking excess edta out of large tubes resolves this problem. normal culture values are similar to those for cattle (clear, colorless to slightly yellow, to g/dl protein, less than , cells). cytologic examination . weigh g of feces and thoroughly mix with ml of water. this is the preferred method. however, if a gram scale is not available, feces can be added to the ml of water until the water level indicates ml. this approximates g of feces. . remove ml of well-mixed fecal-water suspension, add to ml of sheather's solution,* and mix well. is needed to characterize the cell population and assess for the presence of phagocytized bacteria. radiography of the abdomen can be performed in small ruminants using small animal techniques. in adults, the rumen normally fills the entire abdomen. radiography can detect gas distention of the small intestine, abdominal fluid, and foreign bodies. , contrast techniques are useful for diagnosing atresia of the rectum or colon. unlike in other small animals, contrast techniques are not practical for characterizing small intestinal problems in sheep and goats because the rumen dilutes and slows passage of the contrast media. ultrasonography ultrasonography can be used to provide better characterization of abdominal distention, internal and external abdominal masses, and gross lesions of the liver. ascites may be differentiated from fluid in the intestinal tract, and gas distention of the intestines can be differentiated from fluid distention. normal ultrasonographic examination of the liver in sheep has been described. the liver can be viewed on the right side from the seventh or eighth rib caudally to the thirteenth rib ( . ultrasonography can be used to perform biopsies of organs or masses and to locate pockets of fluid. laparoscopy is more commonly used as a reproductive tool, but it also can be used diagnostically as an alternative to exploratory laparotomy in small ruminants. , general anesthesia is recommended. the technique for laparoscopic exploration of the abdomen used for cattle can be modified for use in sheep and goats. the clinician inserts a cannula in the caudal abdomen and carefully inflates the abdomen with carbon dioxide (co ). with the animal restrained in dorsal recumbency and either sedated or anesthetized, the clinician places the cannula in the inguinal area as described for laparoscopic insemination in chapter . entrance on the right side allows visualization of most of the abdominal organs. the clinician should avoid the rumen when introducing the laparoscope into the abdomen. this procedure may be enhanced by lowering the head or rear of the animal, allowing better visualization of the entire abdomen. animals should be properly ventilated during this procedure because inflation of the abdomen and lowering of the head can put pressure on the diaphragm. exploratory laparotomy can be a valuable diagnostic tool in evaluating gastrointestinal diseases when other tests indicate abdominal disease. in some cases, therapeutic surgical techniques can be performed at the same time. the technique of exploratory laparotomy used in cattle can be adopted for sheep and goats as long as the clinician keeps in mind that these animals are more likely to lie down during surgery and standing surgery should only rarely be attempted. small ruminants should be heavily sedated or placed under general anesthesia during this procedure. they may show signs of postoperative pain, anorexia, and depression and should be treated accordingly with a nonsteroidal antiinflammatory drug (nsaid) (flunixin meglumine . to . mg/kg intravenously [iv] ). the decision to use perioperative and postoperative antimicrobial agents should be based on the conditions under which the surgery is performed and the diagnosis made at surgery. sheep the area should be clipped, but in goats alcohol can be applied to the overlying hair and skin. if the area is clipped, the clinician should apply a bland coupling material (e.g., methyl cellulose, vegetable oil) between the skin and the transducer. liver biopsy in sheep and goats is performed using the same technique and instruments as in cattle. however, sedation and ultrasound guidance are recommended. the biopsy can be performed in the ninth to tenth intercostal space slightly above an imaginary line from the tuber coxae to the point of the elbow (figure - ) . the site should be surgically prepared, and a local anesthetic ( % lidocaine hydrochloride) infused subcutaneously. a small scalpel blade is used to make a stab incision through the skin. a -gauge, . -cm liver biopsy instrument is inserted through the incision and the intercostal muscles and into the liver. the biopsy instrument should be directed toward the opposite elbow in most cases, but the use of real-time ultrasonography can help determine the direction and depth needed ( to cm). the clinician should avoid the vessels along the caudal border of the ribs. on reaching the liver, the clinician will note a slight increase in resistance. samples can be submitted for culture (in a sterile plastic or glass vial or tube), histopathology (in formalin at a Ϻ ratio of formalin to tissue); and/or mineral analysis (in a plastic tube). when performing a liver biopsy for mineral analysis, the clinician should rinse the biopsy site with distilled and deionized water after sterile preparation to minimize sample contamination. samples for mineral analysis should not be placed in formalin. the skin incision can be sutured, and aseptically prepared, the surgeon makes a stab incision in the skin and introduces a -gauge biopsy needle. bloat is less common in small ruminants than in cattle, with goats having the condition less commonly than sheep. bloat is the accumulation of either free gas or froth in the rumen, which causes rumen distention. the causes of bloat can be divided into three categories , : . frothy bloat-caused by diets that promote the formation of stable froth . free gas bloat-caused by diets that promote excessive free gas production . free gas bloat-caused by failure to eructate pathogenesis. frothy bloat is usually associated with the ingestion of legume forages or hay (particularly alfalfa) and with grazing on lush cereal grain pastures, but it also may occur with high-grain diets. in the case of frothy bloat from a finely ground diet (usually corn), mucoprotein released from rumen protozoa stabilizes the foam at a low ph. in legume-associated frothy bloat, plant chloroplasts released into the rumen trap gas bubbles. regardless of the form of frothy bloat, the small bubbles fill much of the rumen, preventing clearance of the rumen's cardia and resulting in a cessation of eructation. free gas bloat also occurs with grain diets, especially if the animals are not adapted to the diet. failure to eructate has a variety of causes. physical obstructions of the esophagus such as choke or swollen mediastinal lymph nodes can cause free gas bloat. any disease of the rumen wall can interfere with rumen contractions and eructation. hypocalcemia, endotoxemia, pain, peritonitis, and some pharmaceutical agents (especially xylazine) can all interfere with rumen function and eructation. , , , clinical signs. clinical signs of frothy bloat and free gas bloat from either food intake or physical obstruction of the esophagus are usually more severe and immediately life-threatening than bloat seen from rumen wall diseases and systemic influences. abdominal enlargement occurs, particularly in the dorsal left paralumbar fossa. this may be subtle in sheep or angora goats with full fleece. signs of colic and anxiety are common. the rumen may be either hypomotile or hypermotile. respiratory distress is evident, with some animals breathing through their mouths; death can ensue if the bloat is not treated. this condition is a medical emergency, and therefore diagnosis and treatment should occur almost simultaneously. if the animal is not in immediate danger of dying, an orogastric tube can be passed. most cases of free gas bloat are relieved with passage of the tube. a clinician should then take a thorough history and perform a complete physical examination to find the cause of the bloat. if the bloat is not relieved with an orogastric tube, the tube should be removed and examined for evidence of froth. frothy bloat can be treated with poloxalene ( mg/kg) or dioctyl sodium sulfosuccinate (dss) ( cc [ oz]) delivered by orogastric tube. the froth encountered in frothy bloat caused by the ingestion of finely ground grain has a ph of less than . . if frothy bloat occurs while animals are being fed concentrates, mineral oil ( ml) may work better. peanut oil ( to mg/kg), vegetable oil ( to ml), and hand soap ( ml) also have been recommended in emergency situations. if the animal is in severe respiratory distress, the clinician should insert a trocar or large needle into the rumen at the paralumbar fossa. if gas does not escape, or froth is seen coming out of the trocar, an emergency rumenotomy should be performed (see the rumenotomy section of this chapter). if several cases of bloat are encountered in a group of pastured animals, the entire group should be removed from the pasture and reintroduced slowly after gradual acclimation. if only one or two cases of bloat are encountered, the healthy animals can remain on the offending pasture, but grazing should be limited to ensure gradual acclimation. prevention. prevention of frothy bloat involves limiting access to offending pastures or feedstuffs; providing supplemental feed and providing poloxalene in mineral supplements; and adding ionophores to the ration or supplement. when grazing or consuming legumes as "greenchop," animals should be introduced to the feed or pasture slowly, preferably over to weeks. animals should be closely monitored after a frost and during the rapid growth phase of plants because legumes, particularly alfalfa, may be more likely to cause bloat at this time. certain varieties of legumes that are designed for intensive grazing systems (e.g., alfagraze) should be planted and managed in a manner that decreases the incidence of bloat (limited or creep grazing). feeding dry, stemmy hay for to hours before allowing access to the legume pasture also may help minimize bloat. grass-legume pastures in which legumes are limited to less than % of the forage are safer but can still pose a problem for animals that are selective grazers. grazing legumes with high leaf tannin concentrations (e.g., arrowleaf clover, kudzu) is usually safer because tannins help break down rumen foam. the inclusion of poloxalene ( to mg/kg daily) in the feed or mineral supplement is useful in preventing frothy bloat. if poloxalene supplements are used, keepers should feed them for to weeks before moving animals onto a problem pasture. free gas bloat from concentrate feeds can be controlled by slow introduction to these feeds to allow for rumen adaptation and by the inclusion of ionophores in the diet. monensin ( mg/head/day in ewes, mg/kg/day in goats) and lasalocid ( . to mg/kg/day in sheep and goats) both decrease the formation of free ruminal gas. by enhancing propionic acid formation, these drugs not only reduce the amount of methane produced in the rumen, they also improve the efficiency of nutrient assimilation from feedstuffs. bloat in lambs and kids can have the same causes as in adults but also can be caused by improper milk feeding. overfeeding, feeding of large infrequent meals, and feeding spoiled or cold milk have all been associated with bloat in lambs and kids. rapid overdistention of the abomasum and improper chemical or physical composition of milk replacers inhibit rumen motility, leading to bloat. even though the feeding of cold milk has been associated with bloat, the practice can be used effectively in orphan feeding programs. lambs and kids tend to limit their intake of cold milk after they have become accustomed to cold milk in a free-choice feeding system. milk is usually placed in the rumen when animals are tube-fed; this may result in milk spoilage. , simple indigestion is a mild form of upset of the reticulorumen caused by a change in feeding routine. it can be caused by an alteration in the type of feed or in the amount of feed offered. the most common causes of simple indigestion are the addition of grain to the diet, an increase in the amount of grain fed, and an increase in the energy density of the diet. examples of such dietary changes are replacing oats with corn or changing from whole to ground corn. if the changes are drastic, rumen acidosis can occur (see the following section). other common causes are changes in hay or pasture, consumption of moldy hay, and ingestion of weeds and toxic plants after overgrazing or droughts. clinical signs include mild anorexia that lasts for to days. mild diarrhea and bloat also may occur. rumen fluid ph can be unchanged, increased, or decreased depending on the inciting cause. most animals improve with no treatment. pathogenesis. rumen acidosis is caused by the rapid rumen fermentation of highly digestible carbohydrates that are ingested in excessive amounts. although corn is commonly implicated, other cereal grains (oats, wheat, barley) may be involved, particularly if they are finely ground. the smaller the particle size, the more quickly rumen bacteria are able to ferment the carbohydrates contained in the feed. the common name of this condition is "grain overload," but breads, candy, apples and other fruits, beets, and potatoes also can cause this condi-tion. rumen acidosis usually occurs in animals that have been fed predominantly forage-based rations and are suddenly given access to large amounts of highly fermentable concentrates or concentrated forms of energy. it also can occur in animals that have been receiving concentrates previously, if the amount is suddenly and drastically increased; if access is denied for a time, then suddenly returned (e.g., during weather changes and alterations in water availability); or if ration mixing errors occur (e.g., leaving out monensin and rumen buffers) as highly digestible carbohydrates are fermented, rumen ph drops. lactobacillus species, which are lactic acid producers, proliferate in the acidic rumen environment and further lower rumen ph. as the rumen ph drops, rumen protozoa and many of the lactate users begin to die. lactic acid production causes the osmotic pressure in the rumen to increase. fluid is drawn from the systemic circulation into the rumen, resulting in dehydration and possibly hypovolemic shock. lactate concentrations increase in the blood and may cause systemic lactic acidosis. the lactic acid in the rumen also is toxic to the rumen epithelium. damage to the epithelium can result in leakage of bacteria and toxins into the portal and systemic circulation. chronic sequelae to rumen acidosis include fungal rumenitis and occasionally liver abscesses. , liver abscesses are less commonly encountered in sheep and goats than in cattle. laminitis also can occur, but may be more of a problem in sheep than in goats. the severity of the disease depends on the composition of the feed, particle size, amount of feed consumed, and the period of adaptation to the diet. clinical signs. clinical signs vary with the amount and type of feed ingested and the time since ingestion. signs first appear to hours after ingestion of the offending feed; they vary from anorexia, depression, and weakness to a down animal suffering from severe circulatory shock. dehydration is usually severe and evidence of toxemia is present (e.g., injected mucous membranes, increased scleral injection). colic, bilateral ventral abdominal distention, rumen stasis, and a "splashy" feel to the rumen also may be present. diarrhea can develop, adding to dehydration. , , the diarrhea can range from a pastelike feces to very watery droppings with foam and occasionally pieces of grain easily recognized. dehydration, lactic acidosis, and toxemia result in neurologic signs, including ataxia, head pressing, opisthotonos, and seizures. the body temperature is initially elevated but may drop as the condition worsens or the animal becomes toxic. some animals develop polioencephalomalacia and appear blind. diagnosis. the rumen fluid ph may fall below . . the fluid itself is milky gray and particles of the inciting feed may be noticed. protozoa are usually reduced in number or absent, and large gram-positive rods (lactobacillus species) may be seen on gram's stain. clinicopathology is consistent with dehydration (increased pcv and total protein, prerenal azotemia) and metabolic acidosis. liver enzymes (gamma-glutamyl transpeptidase [ggt], aspartate aminotransferase [ast], lactate dehydrogenase [ldh]) may be elevated on serum biochemical analysis. , the leukogram can vary from normal to a degenerative left shift, depending on the severity of the case. urinalysis reveals an increased specific gravity. treatment. treatment is aimed at correcting cardiovascular shock, dehydration, acidosis, and toxemia and removing or neutralizing the offending feedstuffs. iv fluids containing % sodium bicarbonate should be administered. , oral fluids are contraindicated because they cannot be absorbed and may increase the rumen distention and discomfort of the animal. nsaids are indicated for toxemia (flunixin meglumine, . to . mg/kg iv). , oral administration of magnesium hydroxide and magnesium oxide ( g/kg) may neutralize the acidic ph and is sufficient in mild cases. however, if much of the feed is still in the rumen, these two alkalinizing agents will only work temporarily. oral antibiotics have been recommended to kill rumen microflora and stop fermentation. however, the authors of this chapter feel they are contraindicated because the gram-negative anaerobes that need to flourish to reestablish normal rumen microflora are susceptible to most antimicrobials effective against lactobacillus species. removing the substrate for the lactobacillus species is more effective. because orogastric tubes with large enough bores to reflux feedstuffs are too large for sheep and goats, rumenotomy is indicated in severe cases to remove the feed (see the section on rumenotomy in this chapter). after the rumen ph is cor-rected, transfaunation of the rumen microflora with about qt of rumen fluid from a small ruminant is beneficial (box - ). thiamine supplementation (vitamin b , mg/lb subcutaneously [sc] three times a day [tid] to four times a day [qid]) is indicated until rumen function returns. in certain instances, calcium may be indicated and can be included in the iv fluids (calcium gluconate). the clinician should avoid mixing calcium salts and sodium bicarbonate. bacterial leakage into the rumen wall, liver, and systemic circulation makes antimicrobial therapy necessary. the systemic antimicrobial agent of choice is penicillin (procaine penicillin g, , iu/kg im bid) because anaerobes are the most likely offending organisms. if treated aggressively, the prognosis for immediate survival is good. feed (grass hay only) and water should be limited until rumen contractions return to prevent overdistention of the rumen. the chronic sequelae discussed previously influence long-term survival. prevention. prevention involves introducing concentrate feeds slowly to allow rumen microflora adaptation. dietary change from a lower to a higher fermentable energy concentration should occur slowly and preferably over a -to -week period. in the case of animals being fed high-grain rations (e.g., club lambs, feedlot lambs, dairy goats), buffering agents can be added to the diet. rumen buffers may improve milk production, increase feed intake, and increase rate of gain. the crude fiber content should comprise a minimum of % of the diet's total digestible nutrients (tdn). for example, the tdn is %, the minimum acceptable crude fiber is %. crude fiber levels lower than this can be fed for short periods if the rumen is properly adapted, but problems may nevertheless occur. sodium bicarbonate is probably the most commonly used buffer; it can be offered free choice or included in the diet as % of dry matter intake. calcium carbonate or limestone (which both have low can be collected through a weighted orogastric tube. alternatively, fluid can be collected from any normal ruminant at slaughter. handling rumen contents collected from a fistulated cow or at slaughter can be strained through gauze or cheesecloth to separate the fluid from the fibrous contents. fluid collected through a weighted tube should be ready for storage. rumen fluid should ideally be administered immediately. however, it can be stored for to hours. the surface of the fluid should be covered with a layer of mineral oil to maintain an anaerobic environment and stored at refrigerator temperature. caution: do not store rumen fluid in a closed container because it may explode. rumen solubility) and magnesium oxide (which has poor palatability) also can be included in the feed. magnesium oxide should be limited to . % to . % of the dry matter intake. pathogenesis. reticulitis and rumenitis can result from chemical or mechanical damage to the mucosal lining of the reticulorumen. the most common cause of chemical damage is rumen acidosis. however, ingestion of caustic toxins also can damage the mucosa. mechanical damage can occur from ingested foreign bodies or the formation of rumen bezoars. in cattle, some viruses such as the ones that cause bovine virus diarrhea and infectious bovine rhinotracheitis can infect the rumen wall. similar viruses have yet to be identified in sheep and goats. after the mucosa has been damaged, secondary infection by bacteria or fungi can occur. previous treatment with oral antibiotics may predispose to fungal infections of the rumen wall, especially if the mucosa is already damaged. actinobacillosis, actinomycosis, and tuberculosis rarely affect the rumen wall. tumors of the rumen wall also have been reported. , not all of these causes of reticulitis and rumenitis have been reported in sheep and goats, but all are potential problems. clinical signs. the clinical signs of these diseases are vague. anorexia and forestomach hypomotility may be the only clinical signs. confirming a diagnosis also may prove difficult. samples of rumen fluid may only show changes associated with anorexia (alkaline ph, decreased numbers and motility of protozoa, prolonged mbr time; see table - for normal values). occasionally fungal organisms may be seen on diff quik stained slides of rumen fluid. in these cases a diagnosis of fungal rumenitis should be made. an exploratory laparotomy and rumenotomy may be required to diagnose foreign bodies or masses. rumen parakeratosis is characterized by dark, thickened, and clumped rumen papillae. it is seen mainly in feedlot lambs that consume finely ground or pelleted rations. the parakeratotic rumen papillae are fragile and predisposed to damage, which can increase the chances of rumenitis. treatment and prevention. treatment depends on the inciting cause. dietary changes should be made to decrease energy density and increase fiber intake. mild rumenitis may improve with time and supportive care (transfaunation, fluid support, high-quality feed). fungal rumenitis can be treated with thiabendazole ( mg/kg orally). severe changes may lead to scarring and permanent impairment of rumen function. traumatic reticuloperitonitis is not as common in small ruminants as in cattle, but it has been reported. goats are affected more commonly than sheep. this is probably because of the dietary habits of small ruminants; they tend to be selective grazers and do not "vacuum" the ground as cattle do. offending foreign bodies that cause traumatic reticuloperitonitis include pieces of wire and needles. , the clinical signs are identical to those in cattle and may include anorexia, depression, colic, signs of heart failure, and evidence of draining tracts from the chest cavity. treatment is usually difficult. rumen impaction can occur after dehydration, blockage of the omasal orifice by a foreign body, sand ingestion, or consumption of diets high in fiber and low in digestibility. clinical signs are nonspecific, but the firm rumen can usually be palpated in the left flank. the feces may be scant and dry. oral fluids containing magnesium sulfate ( g) may loosen impactions, but a rumenotomy is required in severe cases. to reduce rumen fill, sheep or goats should ideally have feed withheld for hours before rumenotomy. however, this is usually impossible because in most cases rumenotomy is an emergency procedure. the perioperative administration of antimicrobial agents is essential because even with meticulous technique some contamination of the incision site and possibly the peritoneal cavity is inevitable. because the rumen microflora is predominantly composed of anaerobic bacteria, penicillin ( , iu/kg) is the antimicrobial agent of choice and should be administered to hours before surgery. if the rumenotomy is being performed in an emergency situation, penicillin salts (potassium or sodium) that can be given iv provide therapeutic concentrations more rapidly than procaine penicillin. nsaids (flunixin meglumine, . to . mg/kg iv) also are recommended before surgery. if necessary, treatment of cardiovascular shock and dehydration with iv fluids also should begin before surgery and continue until the animal is rehydrated and in stable condition (see appendix ii). general anesthesia is recommended, but heavy sedation and local anesthetic infiltration of the incision site can be efficiently used (see chapter ) . the clinician should clip and surgically prepare a square area from cm in front of the last rib to the tuber coxae, and from the dorsal midline to the lower abdomen, encompassing the entire left paralumbar fossa. the surgeon makes a skin incision approximately cm longer than the width of the hand cm caudal and parallel to the last rib. the incision is continued through the muscle layers into the abdomen. because the abdominal wall is relatively thin, the surgeon should take care not to enter the rumen or bowel. the surgeon grasps the rumen wall and pulls it through the incision; suturing it to the skin with a simple continuous circular pattern around the entire incision. this forms a seal that minimizes rumen content contamination of the deep layers of the incision and peritoneal cavity. the rumen wall is then incised inside the circle of sutures. the incision in the rumen wall should be large enough for the surgeon to put his or her hand inside the rumen without traumatizing the rumen wall. after the rumen has been explored and emptied and the primary reason for doing the procedure has been completed, the surgeon closes the rumen wall in a continuous inverting pattern (cushing, lembert, or guard's rumen stitch) with absorbable suture ( catgut). the area should be rinsed with copious amounts of sterile isotonic fluids, and a new set of sterile instruments, sterile gloves, and surgical attire should be used for the remainder of the surgery. the surgeon then removes the suture securing the rumen to the skin and rinses the area again before performing routine closure of the abdominal muscles and subcutaneous layers with absorbable suture ( catgut) in simple continuous patterns, taking care to close dead space between layers. the skin is closed with a continuous pattern (ford interlocking) using a nonabsorbable suture material. the sheep or goat should be observed closely by the clinician for signs of complications, including peritonitis, incisional dehiscence, incisional hematoma, abscess, and hernia formation. penicillin therapy (procaine penicillin g, , iu/kg bid) should continue for at least days. the skin sutures can be removed to days after surgery. abomasitis and abomasal ulcers in adult sheep and goats are associated with rumen acidosis or chronic rumenitis but also can be caused by infections. [ ] [ ] [ ] [ ] finely ground feeds, pelleted rations, systemic stress, and feeding lush forages have all been implicated. anecdotal associations with mineral deficiency (copper) have gone unproved. clinical signs and diagnosis. this disease often goes unnoticed in mild cases, and the most common signs are anorexia and colic. no definitive antemortem diagnostic tests are available. fecal occult blood is often absent. occasionally dark stool, altered appetite (wood chewing), and bruxism are seen. therefore other causes of colic should be eliminated. diagnosis is based on clinical signs. effective therapy can be difficult. oral medications such as coating agents must first pass through the rumen, and therefore arrive at the abomasum diluted. iv (not oral) ranitidine ( mg/kg once a day [sid]) may be beneficial. herd problems of rumen acidosis may be addressed with buffers in the feed. a syndrome of abomasal hemorrhage, bloat, and ulceration is seen in lambs and kids to weeks of age. sarcina-like bacteria, clostridium falax, clostridium sordelli, and clostridium septicum have been isolated from many of these cases. [ ] [ ] [ ] [ ] c. septicum infections of the abomasum are commonly called braxy. the feeding of milk replacer free choice, iron deficiency, and bezoars have been implicated as predisposing factors. , clinical signs. the signs of this syndrome are severe, acute abdominal distention; colic; and death. [ ] [ ] [ ] [ ] diagnosis and treatment. the diagnosis of this condition is by postmortem examination. treatment in suspected antemortem cases is unsuccessful. prevention. adding formalin to milk replacers and vaccinating for clostridial diseases may decrease the occurrence of this disease. , lambs or kids on problem farms can be vaccinated for clostridium species during the first week of life with multivalent bacterins. similar to rumen impaction, abomasal impaction usually occurs when poor-quality roughage is fed, but it also can be seen with foreign body obstruction of the pylorus. , , goats appear to be more commonly affected than sheep, and boer goats are more commonly affected than angora goats. pregnant animals may be more prone to this condition. clinical signs and diagnosis. affected animals are usually anorexic. they have mild distention of the ventral abdomen, and in some cases the firm abomasum can be palpated through the abdominal wall on the right side. weight loss may be apparent. clinicopathologic evaluation may be normal, or mild hypochloremic metabolic alkalosis may be present, with elevated rumen chloride concentrations (more than meq/l). treatment. diet changes and mineral oil by mouth (po) are the most commonly employed treatments. abomasotomy can be attempted, but it has rarely been reported in small ruminants and does not usually improve the animals' long-term prognosis. when attempting abomasotomy, the clinician should perform the procedure with the animal in dorsal recumbency and under general anesthesia. the abomasum can best be visualized through an incision parallel and to the right of midline, caudal to the xyphoid process. the prognosis is poor. prevention. dietary manipulation to improve feed or forage quality is the best mode of prevention. abomasal emptying defect is a disease that presents similarly to abomasal impaction but is recognized only in suffolk sheep. the underlying cause is unknown. unlike abomasal impaction, this disease is associated with concentrate feeding and often occurs around lambing time. the clinical signs are chronic weight loss, abdominal distention, and anorexia. clinical pathology and rumen chloride levels are the same as described for abomasal impaction. on necropsy the abomasum is greatly distended, and the contents may be liquid or dry. treatment with laxatives, cathartics, motility modifiers, and abomasotomy has been mostly unsuccessful. [ ] [ ] [ ] azalea, laurel, and rhododendron toxicity members of the azalea, laurel, and rhododendron plant group produce andromedotoxins that alter sodium metabolism, resulting in prolonged nerve depolarization. these plants are cardiotoxic, but affected animals generally exhibit acute gastrointestinal upset. these evergreen shrubs produce thick, dark green leaves. they also have five-lobed, white to pink, saucer-shaped flowers that bloom around july. some of these plants are grown as ornamental shrubbery around homes, whereas others grow wild along streams, cliffs, and rocky slopes. they can be short or tall (as large as m) and can form thickets. all parts of these plants are toxic. clinical signs. animals browsing a new area, those fed clippings from trimmed azalea hedges, and underfed, hungry animals given access to these plants are likely candidates for intoxication. animals that ingest as few as two or three leaves may show signs of salivation, grinding teeth, nasal discharge, colic, epiphora, and acute digestive upset within hours of ingestion. as the intoxication progresses, animals become depressed and exhibit projectile vomiting, frequent defecation, and a slowed pulse. terminally intoxicated animals become paralyzed and comatose. some sheep and goats develop aspiration pneumonia secondary to intoxication. diagnosis. the diagnosis of this condition is usually based on clinical signs coupled with a history of ingestion of one of these plants and/or the discovery of these plants in the gastrointestinal tract. treatment. intoxicated animals may recover in to days without any therapy if the offending plants are removed from the diet. however, the administration of charcoal ( to g/kg po), atropine ( . to . mg/kg iv), other antiarrhythmic drugs, and iv fluids all may be indicated. to manage the aspiration pneumonia, the administration of antibiotics (penicillin , units/kg bid im) and oral magnesium hydroxide also may be beneficial. obviously, any existing dehydration should be corrected (see appendix ii). mountainous or hilly areas should be fenced. feeding shrubbery clippings is discouraged. diarrhea in lambs and kids is a complex, multifactorial disease involving the animal, the environment, nutrition, and infectious agents. decades of research have been devoted to the study of the pathophysiology of infectious diarrhea of calves; the pathology in lambs and kids is quite similar. despite improvements in management practices and prevention and treatment strategies, diarrhea is still the most common and costly disease affecting neonatal ruminants. [ ] [ ] [ ] [ ] some general preventive measures (e.g., improved sanitation) decrease disease no matter the cause. however, specific control measures such as vaccination require the definition of a specific cause of diarrhea. table - lists the agents most likely to cause diarrhea in lambs and kids, tissues or other samples required for diagnosis, and commonly employed test methods. the color and consistency of the feces and any gross lesions can appear similar no matter the cause. therefore laboratory identification of infectious agents and tissue histopathology are key to establishing a diagnosis. because autolysis and secondary bacterial invasion of the gut begins within minutes of death, necropsy samples taken immediately from euthanized lambs and kids yield the most reliable diagnostic material. mixed infections with two or more pathogens are common, and pathogens that are a problem on a farm change from year to year. , , in some cases an underlying nutritional deficiency or excess may occur concurrently with an infectious agent. therefore the clinician should be careful to take a variety of samples to ensure that all pathogens and predisposing factors involved are recognized; continued reevaluation of the causes of diarrhea is crucial. examination of several cases, with a focus on those in the acute phases, is important. although examination of antemortem fecal samples can be diagnostic, laboratory testing of tissue samples may yield better results. treatment and preventive measures specific to a particular disease are discussed with that disease in the following paragraphs. general supportive treatment and control measures are covered at the end of this section. four major pathogens cause diarrhea in lambs and kids during the first month of life: enterotoxigenic escherichia coli (etec), rotavirus, cryptosporidium species, and salmonella species. the relative prevalence of these infectious agents varies greatly among studies. this variance most likely results from differences in location, season, diagnostic techniques, and the occurrence of mixed infections. other, less common causes of diarrhea in neonates are giardia infections and nutritional diarrhea. pathogenesis. etec employs two virulence factors to cause disease. the first is the ability to attach and colonize the intestinal villi, which is accomplished via fimbria or pili. the most important fimbria in lambs are k and f . , the fimbrial antigens can be recognized from samples sent to most diagnostic laboratories and are im- portant in diagnosing this agent as a cause of diarrhea. after the organism attaches to the villi, it produces the second virulence factor, enterotoxin. enterotoxin interferes with the normal physiology of the gut, with resultant diarrhea. calves have an age-associated resistance, most likely related to the blocking of fimbrial attachment to the gut, so etec occurs mainly in calves less than a week old. , the mode of infection is fecal-oral. clinical signs. etec is seen in lambs and kids less than days of age but is most common at to days of age, so age-related resistance also may occur in these animals. , it usually presents as an outbreak in lambs and kids between and hours of age. because etec causes a "secretory" diarrhea, bicarbonate loss in the diarrhea leads to severe acidosis, with lambs and kids quickly becoming dehydrated and recumbent. however, many infected animals die before developing diarrhea. affected neonates are depressed, stop nursing, and may show excessive salivation. fluid sequestration in the abomasum causes a "splashing" sound on movement.this condition results in high mortality if animals are not treated promptly. diagnosis. fecal culture and serotyping for the k and f fimbrial antigens are the basis for diagnosis. because many nonpathogenic e. coli are normal gut inhabitants, simply culturing this organism is usually insignificant. occasionally the bacteria do not express the fimbrial antigens in culture, so etec cannot be ruled out if the culture is negative for k and f . histologic evidence of colonization of the small intestine can support a diagnosis. treatment. supportive care consisting of fluid therapy with either oral, iv, or sc administration of a polyionic solution is the mainstay of therapy. the use of oral antimicrobial agents is controversial. although antibiotics may kill the etec, they also may interfere with normal gut flora. if fluid support is provided, the diarrhea usually subsides without antibiotic treatment. still, oral neomycin ( to mg/kg bid) or trimethoprim sulfa ( mg/kg po) and systemic ampicillin ( to mg/kg im bid) or amoxicillin ( to mg/kg im tid) may be beneficial. nsaids are indicated to decrease inflammation of the gut and provide some analgesia. the use of flunixin meglumine ( to mg/kg im) has been shown to decrease fecal output in etec infections in calves and appears to be beneficial in lambs. prevention. it is recommended that clinicians vaccinate ewes and does with bovine etec vaccine before they give birth to increase passive immunity. , , monoclonal and polyclonal antibody products for calves may be beneficial during an outbreak if it can be given to lambs or kids within the first hours of life. the use of neomycin ( to mg/kg po bid) in lambs that appear normal may help stop the progression of an outbreak. shearing ewes prepartum to minimize fecal ingestion by neonates and ensuring that newborns ingest adequate colostrum both help decrease the incidence of this disease. making sure that ewes and does give birth at a . to . body condition score increases the chance of adequate colostrum manufacture by the dam. pathogenesis. lambs and kids are infected with group b rotaviruses, whereas most other animals and human beings are infected with group a rotaviruses. rotaviruses infect villus tip cells of the small intestine, which results in villus atrophy and malabsorptive diarrhea. clinical signs. rotavirus generally causes diarrhea in lambs and kids to days old, but older animals also can be affected. young animals can become very depressed and dehydrated. , , , diagnosis. detection of the organism by electron microscopy of fecal or colonic samples or by immunologic techniques on feces or tissue sections is the basis of diagnosis. , because these organisms are sloughed with the villus tip cells they infect, and viral antigens are complexed with the lambs' and kids' antibodies, tissue samples from acutely infected animals are best. rotavirus has been detected in animals without diarrhea, so other causes of diarrhea should be investigated as well. , treatment and prevention. rotavirus is treated with supportive care. prevention by vaccination of ewes and does with bovine rotavirus vaccines before they give birth is recommended to increase passive immunity. clinical signs. cryptosporidia can cause diarrhea in lambs and kids to days of age. , , affected animals are often active, alert, and nursing. the diarrhea is usually very liquid and yellow. diarrhea can vary from mild and self-limiting to severe, especially with mixed infections. , , , relapses are quite common, and this organism usually occurs as a component of mixed infections. diagnosis. acid-fast staining of air-dried fecal smears is a quick and easy method of diagnosis. examination under ϫ to ϫ magnification reveals round protozoa that have taken up the red color of the carbol fuchsin portions of the stain on a green background (figure - ). although they can be diagnosed by fecal flotation, their very small size ( - mm) makes this method difficult and subject to false negative results. , both immunologic and polymerase chain reaction (pcr) techniques have been developed to improve detection limits. , cryptosporidia also can be identified with histology. cryptosporidiosis is a zoonotic disease, and people can easily become infected from handling infected animals or feces. prevention. no consistently effective treatment for cryptosporidiosis in ruminants has been identified. anecdotal reports suggest that decoquinate and monensin sodium may be useful in control of cryptosporosis. decoquinate ( . mg/kg po) may be very useful in prevention of cryptospirosis in goats and possibly kids. during an outbreak affected animals should be isolated from the rest of the flock. no new animals should be added to a pen in which the disease has been diagnosed. keepers should depopulate pens in which the disease has been diagnosed and attempt to clean the environment. cryptosporidiosis can be particularly difficult to control because of the organisms' persistence in the environment and resistance to most chemical disinfectants. however, ammonia ( % to %) and formalin ( %) seem to be most effective. , feeders should be constructed to minimize fecal contamination. studies are currently underway to develop a vaccine for cryptosporidiosis in cattle. early results are favorable, and this may prove the best way to control the disease in the future. this is potentially a zoonotic disease, and therefore clinicians and keepers should exercise great caution when handling affected animals. pathogenesis. the bacterial genus salmonella has thousands of serotypes, and all can potentially cause diarrhea in animals. salmonella can cause diarrhea in lambs and kids of any age. , the microbes produce enterotoxins, are invasive, and cause severe inflammatory disease and necrosis of the lining of the small and large intestines. clinical signs. animals less than week old are more likely to die acutely without clinical signs, whereas animals older than week are more likely to have diarrhea. , , an acute onset of fever, depression, tenesmus, and shock is occasionally observed. salmonella-induced diarrhea is more likely to contain blood. this also is a zoonotic disease that warrants protective measures. a diagnosis of this condition is based on culture of the organism in feces or tissues and histologic examination of the small and large intestine. more sensitive pcr techniques for identifying salmonella species in feces are being developed. the diarrhea may occa-sionally contain fibrin, but many animals die before this is observed. clinicians may note leukopenia or leukocytosis in the cbc results. treatment. therapy for salmonella-induced diarrhea involves supportive care and possibly parenteral antimicrobial therapy. the use of antimicrobial agents is controversial and probably does not influence the gastrointestinal infection. however, because this is an invasive organism, parenteral use of antimicrobial agents may be beneficial in preventing septicemia. antimicrobial susceptibility patterns are difficult to predict for salmonella species, so antimicrobial therapy should be based on culture and sensitivity results. ceftiofur sodium ( . to . mg/kg im bid) or trimethoprim sulfadiazine ( mg/kg sc sid) can be administered until antimicrobial sensitivity results are known. prevention. latent carriers of salmonella can potentially shed organisms to other animals, particularly when they are stressed. newly introduced animals should be isolated for month, and fecal culture should be considered. bleach is an effective disinfectant to use during an outbreak. identification of carrier animals by fecal culture is recommended for herd problems. vaccine efficacy is questionable, and to date its effects have not been thoroughly evaluated in sheep and goats. giardia: giardia-induced diarrhea is more commonly seen in but not limited to -to -week-old lambs and kids. , the diarrhea is usually transient, but infected animals can continue to shed cysts for many weeks, even when they are clinically normal. , , therefore simply finding the agent in feces does not mean it is the cause of diarrhea, especially in older animals. however, these animals may be a source of infection for other animals and possibly humans. , iodine-stained wet mounts of feces or tissue is the classic method of diagnosing giardiasis, but more sensitive immunologic techniques are now available. , infected animals can be treated effectively with fenbendazole ( to mg/kg bid for days or sid for days). giardia has historically been treated with metronidazole ( mg/kg po sid for days). however, use of this drug class in food animals is currently illegal in the united states.this is potentially a zoonotic condition. infectious agents are not the only cause of diarrhea in neonates. nutritional problems can result in diarrhea, but these causes are overshadowed in the literature because the resulting diarrhea is usually mild and subsides without treatment. nutritional diarrhea is most common in orphaned animals as a result of keepers offering poorquality milk replacers, making mixing errors, or feeding large amounts infrequently (see chapter ) . diarrhea re- sulting from consumption of lush pasture or high-energy rations is a common occurrence. in most cases such diarrhea is self-limiting. the incidence of this form of gastric upset can be minimized by a slow introduction (over to weeks) to energy-dense diets. calves with infectious diarrhea that develop maldigestion or malabsorption can have secondary nutritional diarrhea from an inability to digest carbohydrates (lactose, xylose). , this has been reported in goats, and also is probably a cause of diarrhea in lambs. diarrhea resulting from primary lactose deficiency also has been reported in calves. calves on poor-quality milk replacers can develop an overgrowth of normal enteric e. coli, resulting in diarrhea. if lactose intolerance is suspected, decreasing the amount of lactose fed and using commercially available lactose enzymes may alleviate signs. the most common cause of diarrhea in older lambs and kids is nematode infestation. this condition is discussed later in this chapter in the section on causes of adult diarrhea. other major causes of diarrhea in older lambs and kids are c. perfringens and coccidiosis. c. perfringens types a, b, c, and d can all cause diarrhea in lambs and kids, but type d is the most common agent. , , pathogenesis. the disease occurs in peracute, acute, and chronic forms and is commonly called enterotoxemia or overeating disease. in the case of type c infection, a beta-toxin can cause acute hemorrhagic enteritis. type c infection is seen mostly in lambs or kids younger than weeks of age. an epsilon-toxin is responsible for pathology in type d infections. enterotoxemia is usually seen in rapidly growing feedlot lambs on high concentrate rations. it also is associated with other feeding changes, including changes in type of pasture. however, it occasionally occurs with no reported dietary changes, particularly in goats. , , this disease usually occurs in the fastest-growing and most well-conditioned animals. it can occur in vaccinated herds (again, more commonly in goats) so it should not be ruled out if a history of previous vaccination is present. clinical signs. the peracute form of clostridial infection is characterized by the rapid onset of severe depression; abdominal pain; profuse, bloody diarrhea; and neurologic signs. death occurs within hours of the onset of signs. sudden death may occur without signs of diarrhea. the onset of neurologic signs followed by sudden death is more common in sheep, whereas goats are more likely to show signs of diarrhea before death. similar but less severe signs are seen in the acute form of the disease. the chronic form occurs more commonly in goats. , diagnosis. antemortem diagnosis is based on clinical signs. at necropsy, c. perfringens can be cultured from intestinal tissue samples. however, the significance of a positive culture can be difficult to interpret because these organisms can be present in the gut normally and then proliferate after death. histologic examination of sections of the gut can be helpful. identification of the toxins (namely the epsilon-toxin) in intestinal contents is required for a definitive diagnosis. , because the toxin degrades within several hours of death, not finding the toxin does not preclude enterotoxemia as a diagnosis. treatment. treatment is rarely effective but consists mainly of aggressive supportive care. c. perfringens type d antitoxins ( to ml sc) can be administered to animals during an outbreak of enterotoxemia if clinical signs are noted before death. the antitoxin may be more effectively used as a preventive in the face of an outbreak. during an outbreak any animals that have not been vaccinated should be given the antitoxin and vaccinated with the toxoid simultaneously; those previously vaccinated should receive a booster vaccination. prevention. routine vaccination should start at to weeks of age and be followed by a booster to weeks later. however, on farms where the disease has become endemic, lambs or kids can be vaccinated and given antitoxin during the first week of life. yearly vaccination, preferably a few weeks before the ewes and dams give birth increases colostral immunity in neonates and improves prevention programs. goats may not respond as well to vaccination as sheep, so biannual or triannual vaccination is recommended, especially in problem herds. , vaccination with only c. perfringens types c and d and tetanus is superior to the use of more polyvalent clostridial vaccines. reducing the energy density of the diet and avoiding sudden dietary changes or alterations of the feeding routine are crucial to prevention. reducing internal parasites, particularly tapeworms, may further reduce the incidence of these disorders. pathogenesis. coccidiosis is a protozoan parasitic disease that is a common cause of diarrhea in lambs and kids. it also may cause subclinical production losses. clinical disease is often seen when some form of stress (e.g., dietary change, weather changes, parturition, weaning) is occurring on the farm or in the flock. eimeria species cause the disease in sheep and goats; each is infested with its own host-specific species. unlike cryptosporidium, which can be shed in feces in the infective stage, coccidia must sporulate outside the host to become infective. sporulation occurs under moderate temperatures and high moisture conditions. the nonsporulated and sporulated oocysts can survive a wide range of temperatures and may survive for years under certain conditions. clinical signs. lambs and kids are most susceptible to the problem at approximately to months of age, although younger animals may become infected. clinical disease is common after the stress of weaning, feed changes, or shipping. crowded conditions result in excessive manure and urine contamination, which is ideal for the buildup and sporulation of the oocysts. under these conditions, animals may be exposed to high numbers of infective organisms and develop diarrhea.the diarrhea in lambs and kids is usually not bloody, but it can contain blood or mucus and be very watery. anorexia, dehydration, weakness, rough hair coat, and death all may occur. weight loss is common, and constant straining can result in rectal prolapse. in severe cases the disease becomes protracted because of necrosis of the mucosal lining. even if these animals are treated appropriately, the diarrhea continues until the intestinal mucosa heals, which can take several days to weeks. permanent scarring can result in chronic poor development, even if the diarrhea subsides. , [ ] [ ] [ ] diagnosis. acute coccidiosis can be easily diagnosed from a direct smear or flotation of feces (figure - ) . in the chronic stages, most of the organisms have been shed and very low numbers are seen on fecal examination. because normal animals can shed small numbers of pathogenic species or large numbers of nonpathogenic species, interpretation of fecal examinations in the chronic stages of coccidiosis or in animals with diarrhea from other causes can be difficult. [ ] [ ] [ ] in these cases the clinician should rule out other diseases before making a diagnosis of coccidiosis. blood analysis may show both anemia and hypoproteinemia. treatment. treatment of affected animals with clinical signs includes supportive care and administration of coccidiostats. all animals in the group should be treated during an outbreak. the use of coccidiostats has little effect on the existing infection, but it does prevent the spread of the disease from continued exposure to infective organisms. many coccidiostats inhibit coccidia development and prevent disease if given prophylactically. they are of little value if they are given after the onset of clinical disease. sulfa drugs appear to be clinically beneficial, but they may simply decrease secondary or concurrent bacteria-induced diarrhea. because coccidia develop some resistance to coccidiostats, these drugs should be administered only before stressful events (e.g., shipping, weaning, parturition). the drugs listed in table - and trimethoprim sulfa ( mg/kg orally sid for days) are approved for use in the united states. , prevention. control involves improved sanitation and possibly the use of coccidiostats. preventing overcrowding decreases the buildup of manure and infective oocysts. exposure to sunlight and desiccation are two of the most effective means of killing the organisms. minimizing stress and optimizing nutritional intake also are important. coccidiostats available in the united states are shown in table - and appendix i. to avoid toxicity in growing animals, the clinician or keeper must carefully adjust dosages to the changing levels of feed intake as animals grow. all agents except amprolium should be fed for at least weeks. , this allows exposure and subsequent development of immunity to occur while preventing the detrimental effects of clinical disease. however, coccidia can become resistant to coccidiostats; fecal samples should be periodically evaluated after prolonged use of a particular product. anecdotal reports suggest amprolium resistance may occur on some farms. moreover, if amprolium is offered with a creep feed rich in thiamine, its ability to act as a thiamine antagonist may be compromised. year-round use of coccidiostats increases the potential for resistance. therefore they should be fed only during times of expected risk. the inclusion of lasalocid ( kg of % premix) or decoquinate ( kg of % premix) in kg of trace mineralized salt fed as the only source of salt for days prepartum can reduce the number of oocysts shed in ewe or dam feces. this practice can reduce the coccidia contamination of pasture and thereby remove a source of infection for kids and lambs. the benefits of administering lasalocid and monensin beyond coccidia control include increased feed efficiency, enhanced growth rate, and decreased incidence of free gas bloat. however, if coccidiostats are included in either mineral or feed supplements, inconsistent or depressed intake may result in subtherapeutic drug dosing. lambs are resistant to infection in the first few weeks of life. exposure to the protozoa during this time confers immunity and resistance to later infections. , adenovirus, caprine herpesvirus, coronavirus, campylobacter jejuni, yersinia species, and strongyloides papillosus can cause diarrhea in lambs and kids of various ages. , , enterohemorrhagic e. coli (ehec) and enteropathogenic e. coli (epec) also have been isolated in the feces of kids with diarrhea. , these e. coli types are k and f -negative. culture and serotyping of these organisms from feces and tissue samples with typical histopathologic lesions is diagnostic. although etec is not zoonotic, ehec and epec can potentially affect humans. although some causes of diarrhea have specific treatments, many animals need to be treated for dehydration and metabolic acidosis regardless of the inciting cause. animals with only mild diarrhea, especially mild nutritional diarrhea, may not require therapy unless they become dehydrated. if kids or lambs become less than % dehydrated and are only mildly depressed but still willing to nurse, they can be treated with oral electrolytes designed for calves. fluids can be administered by bottle or by tube if the animal will not nurse. the keeper or clinician should carefully adjust the amount of fluids for lambs and kids ( to ml, or to oz, as opposed to l in a calf ). because most electrolyte solutions designed for calves contain glucose, after they have been mixed they should be refrigerated and any leftovers discarded within hours. iv fluids may be needed to treat more severe dehydration. if the lamb or kid is too weak to stand, iv fluids are indicated. isotonic fluids containing electrolytes should be given to replenish losses. glucose can be added to fluids to make a % to . % solution. sodium bicarbonate also may be administered, especially if the dehydration is severe. a rule of thumb is to give one fourth of the calculated fluid need (see appendix ii) as isotonic bicarbonate ( . %). extra potassium ( to meq/l) can be added to fluids because most animals are severely dehydrated from diarrhea and depleted in potassium, even though their blood potassium levels may be elevated. if extra potassium is added, acidosis must be corrected concurrently. after correcting the dehydration, the keeper or clinician can offer oral electrolyte-enriched fluids to replace ongoing losses caused by continued diarrhea. removing milk or milk replacer from the diet is not recommended. young animals need nutrients, and even high-energy, glucose-containing electrolyte solutions are no substitute for milk. animals should continue to receive milk replacer in normal amounts or be allowed to nurse; they can be supplemented with oral electrolytes if necessary. animals being hand fed should be offered small amounts frequently to help minimize problems. electrolytes should never be mixed with milk, but should instead be given in separate feedings. if lactose deficiency is suspected, lactase drops or capsules (available in health food stores) can be added to milk or milk replacer. nsaids (flunixin meglumine, . to . mg/kg iv; ketoprofen, . to . mg/kg iv) are beneficial, especially if toxemia is involved, as in etec, enterotoxemia, and salmonellosis. it is the authors' opinion that antimicrobial agents should be reserved for proven outbreaks of salmonellosis and for animals with other causes of diarrhea that do not respond to fluid therapy and nsaids; these drugs should only be administered parenterally. the use of oral coating agents and antacids is popular, but it has not been shown to be beneficial and is not therapeutically logical in light of the pathogenesis of these diseases. probiotics may be beneficial in reestablishing the normal flora of the small intestine. the authors' rule of thumb is that nothing should be given orally except milk, oral electrolytes, and probiotics. ensuring adequate intake of high-quality colostrum and minimizing stress are important for prevention of all neonatal diseases. a normal lamb or kid will stand and nurse within minutes to hour of birth. the ingestion of colostrum within to hours is essential in preventing hypothermia and hypoglycemia and decreasing the incidence of various diseases. lambs or kids born as twins or triplets, weak or injured neonates, those born during severe weather, those born from a dam with dystocia, and those delivered by cesarean section are all candidates for colostrum supplementation. if supplemental colostrum is provided, it should be good-quality colostrum from females that have tested negative for johne's disease, ovine progressive pneumonia (opp), and caprine arthritis-encephalitis (cae). mixing colostrum from several cows decreases the incidence of the "cow colostrum-associated" hemolytic disease sometimes seen in lambs. if the lamb or kid is unable to nurse, it should be tube fed ml/kg of colostrum. the veterinarian or animal handler can sit comfortably holding the lamb or kid in sternal recumbency in the lap. a to french soft feeding tube is then lubricated, inserted into the side of the mouth, and passed slowly. if the tube is placed in the trachea, the lamb or kid will become uncomfortable and may shake and cough. the tube may be palpated on the left side of the throat. after the tube has been slowly passed to the thoracic inlet, colostrum can be administered by gravity flow (see chapter ) . prepartum shearing of the dam may decrease the ingestion of feces by lambs. good sanitation of lambing and kidding areas is paramount in management programs that stress prevention. the presence of organic matter interferes with the effectiveness of many disinfectants, so removal and proper disposal of feces, carcasses, and placentas are essential. when disposing of waste material containing either cryptosporidium or giardia, the keeper should be careful to avoid contaminating water sources. infected animals should be isolated to prevent spread of the infection throughout the flock. in general, infected animals should remain in the environment where the infection was first diagnosed, because it is already contaminated. removing pregnant ewes or dams to a clean area before lambing or kidding helps minimize the continued spread of disease. if possible, lambs and kids already born but not showing clinical signs should be removed to a third area. if "safe" pastures are maintained for internal nematode control, they are ideal for use in an emergency situation to control these diseases. although some animals may appear normal, they may be incubating and possibly shedding the infective agents of a disease. if such animals are moved with pregnant females, they can be a source of contamination in a clean area. if healthy lambs and kids cannot be moved to a third, relatively safe area, they should be left with the clinically infected animals because they have already been exposed. . schultheiss p: diarrheal disease in calves, large anim vet ( ) the differential diagnosis list for acute and chronic diarrhea in small ruminants is very long. the most common cause of diarrhea in adult sheep and goats is parasitism; another major cause is johne's disease. both of these diseases are discussed in the following sections. other causes of acute diarrhea include rumen acidosis, peritonitis, endotoxemia, and ingestion of toxins. the list of toxins that cause diarrhea also is very long, and often the diarrhea is not the primary clinical sign. some of the more common toxins that produce diarrhea are arsenic, toxic amounts of salt, levamisole, copper, oak, selenium, and pyrrolizidine alkaloids. salmonella species and chronic enterotoxemia can cause diarrhea in adult animals. coccidiosis can occur in adults under severe stress or in animals that possess limited immunity because of lack of exposure. hepatic and renal disease and copper deficiency are sometimes accompanied by chronic diarrhea, but weight loss is a more common sign in adults. etiology and pathogenesis. sheep and goats are infested with many of the same gastrointestinal nematode parasites as cattle, but these parasites tend to either be species-specific or have some amount of host specificity. sheep and goats are susceptible to the same nematodes and tend to share resistance to those that infect cattle and horses. the major gastrointestinal nematodes that parasitize pastured sheep and goats are haemonchus, ostertagia, trichostrongylus, cooperia, nematodirus, oesophagostomum, and bunostomum species. the acronym hotc comes from the first letter of each of the first four genera of parasites listed. the specific parasites that produce disease vary from flock to flock. climate usually determines which parasites are of clinical significance on a farm, and the weather determines when the parasites will be transmitted and infective. in much of the united states, haemonchus is the most significant parasite with respect to both clinical disease and anthelmintic resistance. most of these parasites affect the abomasum or small intestine of young, recently weaned animals and occasionally adult animals. sheep (and, to a lesser extent, goats) that are older than months may be less susceptible. overcrowding and overgrazing with concurrent pasture mismanagement and malnutrition usually increase susceptibility to these parasites. inadequate nutrient or protein intake may result in greater susceptibility. the life cycle appears to be similar in most of these parasite species. adults lay eggs that are passed in the feces; except for nematodirus species, the eggs hatch under favorable environmental conditions. the larvae go through several free-living developmental stages becoming infective. when the infective larvae are ingested by the host, the parasite completes its life cycle as an adult. trichuris eggs are the infective stage and can survive for extended periods in dry lots or barns. however, trichuris is associated with minimal pathology. during dry environmental conditions, fecal pellets tend to trap the nematode larvae, whereas in wet conditions, larvae are released onto the pasture. therefore drought conditions followed by rain can result in devastatingly high rates of pasture contamination as larvae that have remained in fecal pellets are released. very high environmental temperatures result in shorter survivability of some stages of infective larvae. most of the larvae have adapted the ability to over-winter, but can survive only for short periods outside the host during spring. nematodirus is an exception in that the developmental stages leading to infective larvae occur while the microbe is still encapsulated in the egg. however, compared with other species of parasite, nematodirus is of minor importance. nematodirus battus may pose a threat to young, newly weaned and therefore immunologically naive grazing lambs. the hookworm bunostomum also is different, as it may infect the host by either oral ingestion or percutaneous penetration. with the exception of the small intestinal parasite strongyloides, lambs or kids fed indoors or in dry pens tend to be free of parasites. clinical signs. all intestinal nematode infections produce similar signs, although infection with the more rarely encountered bunostomum may perhaps result in more profound anemia. if they infest the animal in sufficient numbers, all nematodes may cause poor growth, decreased feed conversion, decreased milk production, weight loss, diarrhea, anemia, ventral edema (bottle jaw), midline edema, and death. again, all these parasites can potentially result in disease, but haemonchus is the most devastating, particularly in more temperate regions. diagnosis. antemortem diagnosis of nematode infestation is made by examining the feces for nematode eggs. although a direct fecal smear can be examined, the mere presence of parasite eggs is not helpful in determining the parasite load of an animal or animals. quantifying of the epg of feces is the best way of estimating parasite loads. the quantitative mcmaster's technique for determining the epg of feces is shown in box - . common nematode eggs are shown in figure - . treatment and control program. after taking a thorough history of the previous parasite control program used on a farm and determining its effectiveness, the clinician can design and implement a new control program. , however, before deciding to implement a deworming program, the clinician should decide which parasites are in need of control and whether control of these parasites is cost effective in a particular flock. whenever possible, a dewormer that can reduce epg counts on the farm by % should be identified and used for at least a year. of all the parasite prevention programs, strategic deworming or a combination of strategic and tactical programs appear to produce the best results. , , strategic deworming is used when most of the parasites are inside the animals and not on the pasture. in northern climates, strategic deworming can best be carried out during the winter, when the nematode parasites are in a hypobiotic state. when environmental conditions are inhospitable for the survival of the infective larvae, some of the most pathogenic nematodes (e.g., haemonchus) may become hypobiotic; that is, they assume a state of arrested development. they may then mature to the adult stage when environmental conditions become conducive for the survival of their eggs or larvae. preventing or decreasing the numbers of maturing adults by killing the larvae before the periparturient rise in parasite egg production and pasture contamination is an excellent management tool. , , unfortunately, in warmer, more temperate to subtropical environments, this method is less effective because larvae can survive the environment for longer periods. the addition of a protein supplement overlapping the expected periparturient rise has been shown to decrease the number of parasite eggs shed around the time of parturition. however, the cost of the protein supplement may outweigh its benefits. a strategic program entails the use of an anthelmintic agent that is capable of killing encysted larvae. animals are then moved to parasite-free or safe pastures-areas in which the level of parasite contamination is too low to result in infection of grazing animals. examples of safe pastures include pastures where sheep or goats have not grazed for to months in the spring or fall, respectively (and depending on the climate); pastures used for hay production; new pastures (i.e., those used for corn, cotton, or other crops); and pastures grazed by horses or cattle. the use of safe pastures is paramount in any de-worming program. rotating pastures after less than months during the warm part of the year or less than months during cooler months is ineffective. however, if pastures are tilled and replanted, by the time new grazeable forage is available, infective parasite larvae will be dead or significantly decreased. an alternative to pasture rotation is to perform an initial strategic deworming before lambing or kidding and follow it with two to four more dewormings at week intervals throughout the lambing and kidding period. [ ] [ ] [ ] treating lambs or kids at weaning and moving them to a safe pasture is a form of strategic deworming. in lambs or kids to be sold at an early age, the administration of a single anthelmintic treatment followed by a move to a safe pasture may be all that is required. a "double treat and move" system is required for lambs kept for to weeks after weaning, particularly during the summer. , this form of strategic deworming requires two treatments to weeks apart as well as two safe pastures. in northern climates where animals are moved to a dry lot or barn for the winter, a strategic anthelmintic administration as animals are moved off pasture can help reduce the parasite burden through the winter. if this deworming is followed by minimal or no exposure to grazing areas and another dewormer is administered before the spring rise in fecal egg counts, the total parasite burden on spring pasture can be drastically reduced, effectively controlling parasites until summer or fall. tactical deworming programs are used to remove parasites from their hosts before they enter their reproductive phase and can contaminate the pasture. an example of tactical deworming is treating animals to days after a rain, particularly if the rain has followed a drought. parasite transmission is worse in most flocks during this time as pastures become heavily contaminated. mcmaster's counts of more than epg in the spring or more than epg in the fall warrant tactical deworming. , , opportunistic deworming and salvage deworming are usually less effective in long-term flock management. many times salvage deworming programs are used to save the lives of heavily parasitized animals. if animals are dewormed only after showing signs of parasitism (e.g., bottle jaw, anemia), animal and flock productivity have already been depressed. deworming during handling for other procedures (e.g., castration, vaccination, shearing) is an example of an opportunistic program. it is convenient but is not conducive to long-term flock health. flock work should be scheduled around parasite management programs, not vice versa. , suppressive deworming programs entail the use of anthelmintics at regular intervals, usually every to weeks. suppressive programs are labor-intensive, tend to be very expensive, fail to identify animals with superior immunity to parasites, and ultimately result in anthelmintic resistance despite initial effectiveness. , as a general rule, the more frequently deworming occurs, the more quickly resistance is attained to anthelmintics. after deworming, only resistant parasites remain to infect the animal and they are able to reproduce freely, resulting in proliferation of resistant strains. , using drugs that remain in tissues at inappropriately low concentrations and treating and retaining immunocompromised animals encourage the development of anthelmintic resistance. practices that ensure adequate dosages, proper treatment techniques, and appropriate types of anthelmintics should be emphasized. , the clinician should do everything in his or her power to minimize the incidence of anthelmintic resistance, both through their own actions and by counseling owners in proper use of deworming drugs. the product development market for anthelmintics is the cattle industry. the small sheep and goat markets simply use drugs made available for cattle. because most available anthelmintics are highly effective in controlling parasites, anthelmintic resistance in sheep and goats must be avoided. the anthelmintics that have been used previously on a flock, the route of administration (e.g., po, sc, im, pour-on), and the length of use should be determined. few dewormers are approved for use in sheep and goats, but many approved for use in cattle and horses may be effective. , if sheep graze with goats that harbor anthelmintic-resistant parasites, the sheep also may become infected. however, if sheep are allowed to graze while the goats browse and the two groups rarely mingle, less parasite movement will occur between these species. resistance to macrolides (e.g., ivermectin, doramectin, moxidectin) does occur. resistant worms are generally not very tolerant of cold temperatures and therefore resistance to this drug class in northern environments is not as large a problem as it is in more temperate or subtropical zones. although moxidectin is not approved for use in sheep and goats in the united states, it has been shown to be effective in cases where ivermectin resistance is encountered. still, this drug should be avoided until all other anthelmintics have failed. craig , has suggested that clinicians refrain from injecting or using pour-on macrolide preparations designed for cattle in small ruminants. this practice may enhance the development of resistant strains of some internal parasites because of inappropriately low drug absorption (with pour-on use) or long-term subtherapeutic levels (with injection). if resistance to tetrahydropyrimidines (e.g., morantel, pyrantel) occurs in a flock, levamisole also may be ineffective. morantel and levamisole resistance in parasites appears to be sex-linked. therefore if animals are not exposed to these drugs for several years, reversion to susceptibility can occur. , if resistance to one of the benzimidazole dewormers has been documented in a flock, some resistance to all members of that class is likely. benzimidazole-resistant haemonchus species appear to be more virulent, produce more eggs, cause greater environmental contamination, and survive in the environment as free-living larvae for longer periods. benzimidazole-resistant parasites apparently do not revert to susceptible forms, even over long periods. therefore the clinician or keeper should exercise caution to minimize resistance. benzimidazole efficacy can be improved by increasing dosages, dividing dosages into two treatments administered at -hour intervals, and instituting pretreatment fasting. if resistance to numerous classes of anthelmintics occurs on a farm, combining two of the resistant classes of dewormers (fenbendazole and levamisole) has proven effective. when using combined dewormers, the clinician should administer the full therapeutic dosage of each. anthelmintics are metabolized at different rates by sheep and goats. goats may require larger dosages of some dewormers than sheep. craig has suggested that if no dose rate is known for a particular anthelmintic for sheep or goats, the animals should be treated at twice the suggested cow dosage. pour-on anthelmintics designed for cattle tend to be of limited value when used topically on either goats or sheep. table - . to maximize a parasite control program, anthelmintics that appear effective should be used for only year before a new class of deworming drug is used. more frequent rotation (after less than year) of anthelmintic agents hastens resistance and should be avoided whenever possible. whenever a flock is dewormed, animals should be treated based on the heaviest animal in the group and not on the group's average weight. underdosing can hasten the formation of parasitic resistance and therefore should be avoided. holding the sheep or goats in a dry lot overnight or feeding only dry hay for to hours before and hours after deworming appears to improve the efficacy of some orally administered anthelmintic agents (benzimidazole). limiting feed intake before deworming slows the rate of passage of ingesta through the bowel, enhancing drug effectiveness. , , feed should never be withheld from sick or debilitated animals or late-term females. , , most dewormers may effectively control adult or larval parasites but are ineffective against eggs. therefore animals should be kept on a dry lot for as long as days after deworming, then moved to a safe pasture. use of this procedure minimizes parasite egg contamination of the new pasture because most of the egg-contaminated feces is voided within hours of deworming. if more than one dosage appears on the drug label, the larvacidal dose should be used (fenbendazole at mg/kg rather than mg/kg). anthelmintic effectiveness can be determined by comparing a mcmaster's fecal epg on the day of deworming with one taken to days later. if less than a % drop in epg is found, anthelmintic resistance exists and the animals should be switched to another class of dewormer. although it is a controversial method, the authors have used this technique to identify anthelmintic effectiveness for many years and on many farms and ranches. , the authors randomly collect feces from % to % (or a minimum of animals) of the sheep or goats on the farm. a composite sample is prepared by combining equal amounts of stool from all animals. craig has suggested that combining stool samples from many animals alters the accuracy of the tests because great individual variation in fecal egg counts occurs among animals. composite egg counts more accurately reflect parasite burdens in groups of young animals, and individual fecal examinations are more accurate in adults. , still, the authors prefer to use composite samples unless obvious differences in stool character or body condition score exist among the sampled animals. anthelmintic resistance can be minimized by using drugs that reduce fecal egg counts by %. pre-and postdeworming changes in epg should be evaluated yearly or whenever resistance is suspected. in vitro methods of assessing flock parasite resistance also are available at some diagnostic laboratories. in most in vitro tests, larvae are hatched from collected feces and the sensitivity of different anthelmintics is determined by larval exposure. these tests are very accurate but tend to be quite expensive. the most effective method to prevent anthelmintic resistance is to not use deworming drugs at all. one of the most overlooked management procedures is the identification and selection of parasite-resistant sheep and goats. some breeds or familial lines within breeds have excellent parasite resistance (e.g., gulf coast native and barbados sheep, some strains of spanish, pygmy, and tennessee myotonic-fainting goats). one study comparing boercrossed goats with non-boer crosses found that the boer crosses had significantly more parasite infestations. only a small number of flock members contribute the greatest amount of environmental parasite contamination because susceptible animals shed the most eggs in their feces. animals with the lowest epg in a flock may be those that possess the most parasite resistance. salvage deworming programs should generally be avoided, but they may be used as aggressive selection criteria. that is, animals that do well with little or no deworming, particularly those grazing heavily contaminated pastures, should be identified and retained in the breeding flock. those that become infected should be dewormed to salvage them or save their lives and then sold when possible. proper record keeping and identification of all animals is paramount in selecting for parasite resistance. , this aggressive approach can yield excellent results if it is carefully implemented, but devastating losses can occur if it is poorly managed. when introducing new animals to a flock, keepers should have biosecurity programs in place to limit the introduction of new or potentially anthelmintic-resistant parasites. new flock additions should be kept in a dry lot for weeks and dewormed at least twice with two different classes of dewormers during this period. the effectiveness of the anthelmintic agent used should be deter-mined by fecal examination before the animal is allowed contact with the rest of the flock. other nontraditional chemical methods of parasite control are used by some owners. some appear to be worthless (e.g., diatomaceous earth), but others (e.g., nematophagous fungi, herbal dewormers) may prove effective in some situations. pathogenesis. the most common gastrointestinal tapeworm of sheep and goats seen in north america belongs to the genus moniezia. cestodes (tapeworms) are usually of more concern to owners than clinicians, who generally consider them only incidental low-grade pathogens, particularly in adult animals. still, several to -foot-long tapeworms can compete with the host for nutrients, hinder normal gut motility, and excrete some toxic wastes into the host's gastrointestinal tract. mature tapeworm eggs are passed in the feces individually or protected in proglottides, which are usually visible to the owner. the eggs embryonate and infect a mite, a small pasture-living arthropod that serves as the intermediate host. a sheep or goat ingests the mite while grazing, allowing the tapeworm to complete its life cycle. clinical signs. tapeworms may rarely cause disease in lambs and kids less than months of age. anecdotal reports suggest a cause-effect relationship between heavy tapeworm infestation and an increased incidence of c. perfringens enteritis, digestive disturbances (e.g., diarrhea, constipation), poor condition, and anemia. ulceration at the site of attachment may be seen on necropsy. rarely species of trypanosoma, the fringed tapeworm, may cause liver condemnation. a presumptive diagnosis can be made by finding proglottides in the stool, eggs on direct smears, or eggs on fecal flotations (see figure - ).treatment with albendazole ( mg/kg), fenbendazole ( to mg/kg), or praziquantel ( to mg/kg) may be effective either with a single treatment or with daily therapy (e.g., fenbendazole daily for to days). because of the free-living nature of the arthropod intermediate host, animals are readily reinfected after treatment, which may give rise to the false assumption that the therapy was ineffective. again, tapeworm infestation may result in disease, but often it is easier to blame the tapeworm segment seen in the stool as a cause of disease than to implicate the unseen thousands of hotc complex parasites in the abomasum and small intestine of the animal. , johne's disease johne's disease (also called paratuberculosis) is a chronic wasting and diarrheal disease caused by the bacteria my-cobacterium avium subspecies paratuberculosis. transmission of the organism is primarily by the fecal-oral route. young animals are more susceptible to infection than adults. it can be transmitted through milk and placenta. pathogenesis. bacterial shedding in feces and milk and transplacental transmission is more common in animals showing clinical signs. [ ] [ ] [ ] therefore the offspring of infected animals and especially the offspring of animals showing clinical signs are most likely to acquire the infection. after an animal is exposed, it will either clear the organism or develop a chronic, persistent infection. the infection is most commonly isolated to the ileal regions of the small intestine, where it causes granulomatous thickening of the intestine and subsequent malabsorptive diarrhea. infected animals may be asymptomatic for years. clinical signs. morbidity rates are low (approximately %), but for every animal with clinical signs, several exist in the subclinical state, and may be a source of both horizontal and vertical transmission. both sheep and goats appear to remain asymptomatic until to years of age. the most consistent clinical sign in sheep and goats is chronic weight loss. chronic diarrhea occurs in approximately % of cases. signs may appear with or be exacerbated by stress, especially after parturition. , hypoproteinemia and chronic mild anemia are the only consistent clinicopathologic findings. because of their low protein levels, infected animals can develop submandibular edema. diagnosis is by culture of the organism from feces. unfortunately, this testing takes between and weeks, but it can detect % to % of clinically infected goats. sheep strains of johne's disease and some goat variant strains seem to be more difficult to culture in media used to identify cattle strains of the disease.therefore fecal culture in sheep and goats appears to be of limited benefit. , a relatively inexpensive and easily performed method of identifying approximately % of all clinically infected animals is acid-fast staining of fecal smears. , a pcr test of feces also is available, but its sensitivity is lower than that of fecal culture. good diagnostic results can be obtained with serologic testing for antibodies (e.g., agar gel immunodiffusion [agid], enzyme-linked immunospecific assay [elisa], complement fixation) in animals showing clinical signs. the specificity of all the serologic tests is greater than % in sheep and goats with signs of clinical disease, although the sensitivity is not as high. [ ] [ ] [ ] therefore a positive serologic test in an animal showing clinical signs indicates that the animal has johne's disease. however, the disease cannot be ruled out with a negative test. sheep and goats appear to respond differently in regard to the formation of antibodies. sheep tend to develop antibodies in the later stages of the disease, whereas antibodies may be detected much earlier in the goat. the agid test appears to be the best serologic test currently available. , the elisa and complement fixation tests can cross-react with corynebacterium pseudotuberculosis, making them of limited value in flocks with caseous lymphadenitis infections. , necropsy diagnosis is based on the finding of thickened, corrugated intestines, especially in the area of the ileum. acid-fast staining of impression smears (taken from the ileum and ileocecal lymph nodes) can help yield a quick diagnosis. the staining of numerous clumps of acid-fast rods is highly suggestive of johne's disease. prevention. johne's disease has no effective treatment, so prevention and control are imperative. however, preventing the introduction of johne's disease into a herd can be difficult. because animals with subclinical infection may not shed the organism or may shed only small quantities of it, fecal culture is helpful only if a positive culture is obtained. the sensitivity of serologic tests of animals with subclinical disease is low and variable among flocks. , negative test results in subclinically infected animals are common. however, the specificity of serologic tests remains high, and therefore a positive test is a valid reason to not purchase an animal. because johne's disease also occurs in cattle, supplemental colostrum supplies should come only from dairy herds with no history of johne's disease. after johne's disease is diagnosed in a herd, several control measures can be taken. sanitation is important because the organism is highly resistant in the environment (able to survive more than year under most conditions). reduced stocking rates, frequent cleaning of pens, and use of automatic waterers decrease fecal transmission. keepers should cull the offspring of infected animals. culling animals based on the results of agid tests or fecal culture of the flock is recommended. animals should be tested at least once a year. more frequent testing as resources allow speeds the identification of infected animals. a vaccine for cattle is only available in some locales and clinicians or keepers may require official permission to use it. vaccine use does not eliminate infection, but it can decrease herd prevalence, delay the onset of clinical signs, and decrease cross-transmission by infective bacterial shedding in the feces. any cause of intestinal obstruction that occurs in other ruminants may occur in sheep and goats. most of these diseases produce abdominal discomfort and occasionally abdominal distention. diagnosis can be difficult because rectal palpation cannot be performed on small ruminants. abdominal radiographs and ultrasonography may help differentiate these diseases, but exploratory surgery may be required to obtain a definitive diagnosis and select appropriate treatment. intussusception is more common in young animals, but it can occur in adults. it occurs when one segment of the intestine telescopes into an adjacent segment. any portion of the intestine can intussuscept, but the ileum and ileocecal junction are the most common areas involved. when intussusception occurs, the lumen of the intestine narrows to the point of obstruction. the initiating cause is not always known. , it is associated with an intestinal mass in adults and enteritis in young animals. oesophagostomum infestations have been implicated as a cause in sheep. clinical signs. the initial complaint is colic (manifested as kicking at the abdomen, repeated rising and lying down, and vocalization) followed by low-grade pain. true colic signs are variable in lambs and kids. in some cases, after the initial colic episode subsides, animals show no evidence of pain until the abdomen becomes distended. the time between the initial intussusception and abdominal distention depends on where the blockage occurs. intussusception of the ileal area may take several days to cause bilaterally symmetric abdominal distention. fecal output is scant, and what little there is may be dark or tarry, or may contain mucus. dehydration becomes evident, hypochloremic metabolic alkalosis may develop and rumen chloride levels may increase with obstructions of the duodenum. diagnosis. abdominocentesis may yield fluid compatible with a transudate (increased protein concentration and leukocyte numbers). radiography and ultrasonography reveal fluid-distended intestinal loops. occasionally the intussusception itself can be visualized with ultrasonography or palpable through the abdominal wall. if the disease is not treated, intestinal rupture and peritonitis can occur. treatment. surgical correction is required. if the intussusception is corrected early, the prognosis is good in the absence of peritonitis. fluid support is needed to correct dehydration and metabolic abnormalities. fluids should be administered iv until rumen function returns. ringer's solution with added calcium (approximately ml calcium borogluconate per liter) and potassium ( to meq/l) is a good choice for fluid therapy. ingested foreign bodies or bezoars can obstruct portions of the intestines. , the signs are similar to those of obstruction caused by intussusception and depend on the part of the intestine that is blocked. in some cases the obstructing body can be seen with radiography or ultrasonography. surgical removal is required for treatment. cecal volvulus and torsion of the root of the mesentery occur sporadically in sheep and goats. , extreme abdominal pain, rapid abdominal distention, and circulatory collapse are typical signs. immediate surgical correction and circulatory support are needed. atresia of the colon, rectum, and anus can all occur as congenital problems. the clinical sign of progressive abdominal distention usually is noted in the first week of life. atresia of the anus can be diagnosed on physical examination, but atresia of the colon and rectum may require contrast radiography for a definitive diagnosis. surgical establishment of anal patency can be performed for atresia ani. a permanent colostomy may be required for atresia of the colon and rectum. atresia of the anus and rectum are considered heritable in cattle. in the authors' experience, atresia ani is more common in sheep than in goats. if surgical correction of atresia ani is attempted, the animal should be neutered or kept out of the breeding program because of the potential genetic basis for this condition. occasionally a slight bulge in the skin may occur where the anus should be located, especially in male lambs. ultrasonography can be used to locate a fecesfilled rectum. for surgical correction, the clinician should locate the area where the anus should be, prepare it with sterile technique, and infiltrate it with a local anesthetic. the surgeon then makes a circumferential incision to remove the overlying skin covering the rectum. an alternative is to make an x-shaped incision into the rectum. treated animals should be given mineral oil, dss, or stool softeners as needed. ileus of the small intestine is a pseudo-obstruction that occurs when there is an absence of intestinal motility. the animal's failure to pass ingesta leads to signs similar to intussusception. the cause of ileus is usually unclear, but it often occurs secondary to systemic diseases. the same elements that cause rumen stasis may potentially result in intestinal stasis and ileus. symptomatic treatment with nsaids for pain and inflammation and fluids for dehydration is usually curative. however, if signs persist, surgical exploration is indicated to rule out true obstructive diseases. pathogenesis. infection of the peritoneal lining of the abdominal cavity may lead to septic peritonitis. common causes include uterine tears; rupture of the rumen or abomasum secondary to rumenitis, abomasitis, or abomasal ulcers; trocarization of the rumen for bloat; and rupture of the intestine secondary to obstruction. clinical signs. signs depend on the severity of the condition. abdominal discomfort and distention, dehydration, injected mucous membranes, depression, and death can all occur in cases of peritonitis. the presence of a fever is variable, both heart rate and respiration rate are usually elevated, and respiratory effort may be guarded. animals may be febrile early, but have a normal to low body temperature as the condition progresses. abdominal ultrasound can be useful in locating pockets of fluid for abdominocentesis, which usually yields fluid with increased protein concentration and leukocyte numbers. on occasion, intracellular bacteria are observed on cytologic examination. the presence of extracellular bacteria is not diagnostic because accidental enterocentesis can occur. culture of abdominal fluid and subsequent antimicrobial sensitivity tests are indicated for the implementation of proper treatment. the causative organisms vary depending on the source of the bacteria. rumen bacteria are typically gram-negative anaerobes, and e. coli and other enteric bacteria are common if the intestine is the source of infection. exploratory surgery may be required to diagnose a gastrointestinal rupture. the cbc can be normal but often shows an inflammatory leukogram and, in severe cases, a degenerative left shift. treatment. treatment includes the prescription of appropriate antimicrobial agents, the administration of nsaids for pain and endotoxemia, and fluid support for dehydration. the prognosis is guarded, especially if an intestinal rupture has occurred. pathogenesis, clinical signs, and diagnosis. rectal prolapse is more common in sheep than in goats. this evagination of the rectal mucosa and rectal structures (and possibly the descending colon) is usually associated with excessive straining. straining is seen in lambs with diarrhea caused by coccidiosis, salmonella, or dietary imbalances, in ewes or ewe lambs with vaginal prolapse, in males with urolithiasis, and in animals grazing lush forage (particularly legumes such as alfalfa and clover). it also can occur secondary to chronic coughing, short tail docking, and the use of growth implants. [ ] [ ] [ ] rabies also can cause chronic straining and rectal prolapse. [ ] [ ] [ ] [ ] [ ] regardless of the cause, after the rectal mucosa becomes everted and exposed, irritation of the mucosa causes further straining, which exacerbates the problem. venous drainage of the prolapse may be compromised, but the arterial supply usually remains intact and contributes to the swelling. rectal prolapses are graded as type i to iv, based on the portion of rectum and distal colon that is everted. a description of these grades in shown in table - . treatment. correction may be cost prohibitive for feedlot lambs, and immediate slaughter is recommended. in more valuable animals, very mild, early cases can be treated with frequent application of hemorrhoidal ointment designed for humans and manual replacement of the prolapsed mucosa into the anus. the authors try to avoid applying purse-string sutures in the anus because they tend to serve as a nidus and result in further straining. however, if less aggressive therapies do not relieve the problem in hours, a purse-string suture may become necessary, particularly in type i and ii prolapse. in all cases and modes of treatment, restricting feed for to hours and administering mineral oil is recommended. dusty feedstuffs (concentrates, pellets, hay) should be avoided because they may contribute to coughing, which exacerbates this condition. adding molasses to feeds and lightly wetting hay may help reduce problems with dust. purse-string suture is easily performed. the prolapsed tissue and perineal area are washed with mild soap and lubricated with petroleum jelly or hemorrhoidal ointment before the prolapse is replaced. , after replacing the prolapsed mucosa, the clinician inserts a tubular object (syringe case, wooden dowel, gloved finger) into the rectum. he or she then places a purse-string suture of nonabsorbable suture material ( - nylon) in the skin around the anus, tightens it around the tubular object, and ties it off. the suture should be placed around the anus using a cutting needle, and entering and exiting at the o'clock position. tying the knot above the anus ensures that less fecal soiling of the suture will occur. the clinician should tie the suture in a bow knot to allow easy identification over the next few days and then remove the tubular object. the suture should be tight enough to prevent prolapse but loose enough to allow feces to pass. the clinician should regularly reevaluate the animal and if possible loosen the purse-string suture at -hour intervals until no tension exists. after a full day of no tension, the suture can be removed. if animals continue to strain, an epidural anesthetic can be administered. petroleum jelly and hemorrhoid gels should be placed on the anus daily. , the injection of counterirritants around the rectum ( ml or less of lugol's iodine) either alone or in conjunction with anal purse-string suturing is a quick and inexpensive treatment. , , the clinician inserts an -gauge needle ( cm) deeply into the skin around the anus at , , and o'clock. an injection at the o'clock position should be avoided because swelling around the urethra can result in obstruction. for more severe cases, submucosal resection or rectal amputation of tissue may be necessary. , rectal amputation can be performed with a prolapse ring or suture technique. prolapse ring usage is a salvage technique. the clinician inserts the prolapse ring into the rectum and places an elastrator band or suture around the area to be amputated to induce vascular compromise and necrosis of tissue. if a ligature is used, it should be tightened to allow purchase on the tube or ring. a fibrosis is induced just proximal to the band or suture, and mucosa subsequently grow across the areas. strictures, peritonitis, and abscesses are possible complications, but this technique may be useful as a field procedure. submucosal resection can be performed under epidural analgesia after the prolapse and the perineal area have been surgically prepared. the clinician places two spinal needles ( to cm) at -degree angles to each other to mm distal to the anal sphincter and through the entire prolapse. a circular incision is made to mm distal to the spinal needles through the mucosa and around the outside of the anus. another circular incision is made just distal to the caudal extent of the prolapse into the point where the mucosa reflects on itself on the innerside of the prolapse. the clinician connects these two incisions with a longitudinal incision parallel to the prolapse and dissects the mucosa between the circumferential incisions. the mucosal edges are then sutured with a simple interrupted pattern using a suitable absorbable suture material. after completely suturing the mucosal surfaces, the clinician removes the two spinal needles and places a purse-string suture in the anal sphincter. placement of the suture and follow-up care are the same as described for the purse-string suture technique. submucosal resection decreases the incidence of both peritonitis and stricture formation compared with other surgical techniques, but it is expensive. in all of these techniques, a caudal epidural anesthetic ( % lidocaine, . ml per kg) is recommended to decrease straining and ease pain from the procedures. , a xylazine epidural ( . to . mg/kg as sufficient [qs] to ml with % lidocaine) may give longer relief (approximately to hours) from straining than lidocaine. an alcohol epidural also may prevent straining for extended periods. either isopropyl alcohol or ethanol can be used to demyelinate the motor and sensory nerves. this type type i small, circular amount of submucosal swelling good prognosis if there is no damage to mucosa; protrudes through anus; probing reveals a pocket purse-string suture, iodine injection, submucosal or fornix just inside anus resection type ii slightly more circular submucosal and mucosal good prognosis if treated quickly and no mucosal swelling, possibly containing retroperitoneal rectal damage; purse-string suture, iodine injection, tissue from anus; probing reveals a pocket just submucosal resection, rectal amputation inside anus type iii complete prolapse containing part of the if there is vascular injury to the descending colon, retroperitoneal structures of the rectum and the prognosis is guarded to poor; submucosal descending colon; probing reveals a fornix just resection or rectal amputation are the methods of inside anus; the affected portion of the descending choice colon does not prolapse through the anus type iv the descending colon appears as a tube, and has if there is vascular injury to the descending colon, intussuscepted through the rectum and anus; prognosis is poor; abdominal exploration may unlike the previous types, in this case a probe be required to determine the extent of damage to or finger can be inserted into the prolapse through the descending colon the anal sphincter for a distance of to cm of anesthesia can be permanent and therefore should be used only for animals intended for slaughter. because of the potential for some loss of sciatic nerve function, the clinician should perform a test injection of a local anesthetic ( % lidocaine) before using alcohol. if the epidural appears effective and no ataxia or muscle weakness of the rear limbs occurs, the clinician can inject a mixture of equal parts of lidocaine and alcohol into the sites where the test epidural was performed. possible problems with alcohol epidural anesthesia include injection site necrosis, sciatic nerve dysfunction, and the inability to void feces. regardless of the type of epidural used, the clinician clips, washes, and dries the area before placing a small needle ( -to -gauge [ . cm]) in the most cranial yet moveable intracaudal vertebral space-usually c to c or c to c . the needle is placed on the dorsal midline, with the needle degrees to the skin and the hub moved slightly caudal, and then slowly advanced. liver abscesses usually occur as a result of chronic rumenitis in cattle, but they are rare in sheep and goats. they can occur in feedlot lambs and kids and other animals fed rations high in grain. in lambs and kids, septicemia or extension of an umbilical vein infection can cause liver abscesses. in most cases, however, liver abscesses are an incidental finding. weight loss, anorexia, depression, and decreased production (growth, milk) may occur. in adults, corynebacterium pseudotuberculosis is the most common cause. actinomyces pyogenes and fusobacterium necrophorum also are cultured from abscesses. [ ] [ ] [ ] liver enzymes may or may not be elevated. diagnostic ultrasonography of the liver may help detect abscesses, especially if they are numerous and widespread. however, no specific treatment or control measure is available. many of the preventive protocols used for feeder cattle apply to the control of abscesses in sheep and goats. these include slowly introducing concentrates into the diet, offering long-stemmed hay free choice, and including rumen buffers (alkalinizing agents) and antimicrobial agents in the feed. pathogenesis. fatty liver occurs in conjunction with pregnancy toxemia in ewes and does during the last month of gestation. it is most common in thin or obese ewes or does with a single large fetus, twins, or triplets. during late gestation, particularly in obese females, the abdominal space is filled with accumulated fat and an ever-expanding uterus. because of the lack of rumen space, these females have difficulty consuming enough feedstuffs to satisfy energy requirements. in most management systems, late gestation occurs during the winter months, when less pasture is available and poorer-quality feedstuffs are offered. energy requirements for ewes and does carrying twins or triplets is greatly increased during the final months of gestation because % to % of fetal growth occurs during this time. ewes with twins require % more energy, and those with triplets need % to % more dietary energy. pregnancy toxemia also occurs in association with anorexia caused by other diseases (foot rot, opp, cae) or sudden stresses (feed or weather changes, predator attacks, hauling). whatever the initiating cause, a period of anorexia and lack of sufficient energy intake result in a negative energy balance. these animals begin to mobilize body stores of fat and transport them to the liver. in the liver, fat is catabolized to glycerol and free fatty acids (ffas). ffas can be used in the citric acid cycle (krebs cycle) as an energy source, but not in the direct formation of glucose. anorexic animals have less ruminal substrate available for production of the glucose precursor propionic acid. however, oxaloacetate, which is an integral part of the citric acid cycle, is removed from the cycle and converted into glucose. depletion of oxaloacetate inhibits the normal citric acid cycle's function, inhibiting the use of ffas. as the pool of ffas increases, they are converted to ketone bodies or repackaged into lipoproteins. because ruminants are not efficient at transporting lipoproteins out of the liver and back to the adipose stores, the lipoproteins overwhelm the liver's ability to handle this massive buildup, resulting in a fatty liver. because less substrate is available for glucose formation, more oxaloacetate is "cannibalized" from the citric acid cycle, further inhibiting the body's ability to use ffas. this in turn causes the continued accumulation of ketone bodies. hypoglycemia, hy-perketonemia, and potentially uremia and death can occur. clinical signs. animals suffering from fatty liver or pregnancy toxemia become anorexic and depressed, display altered behavior, and become recumbent. some are constipated, grind their teeth, have a ketone smell to their breath, and suffer from dystocia. neurologic signs include blindness, circling, incoordination, star-gazing, tremors, and convulsions. , death can occur if the condition is left untreated. in the case of in-utero fetal death, maternal septicemia-endotoxemia and death are common sequelae. diagnosis. diagnosis is based on clinical signs, the presence of multiple fetuses, and typical clinicopathologic findings. cbc results may be normal or show an eosinophilia, neutropenia, and lymphocytosis. these animals may or may not be hypoglycemic, but ketoacidosis, hypocalcemia, and hypokalemia are common. , liver enzymes are usually within normal limits but occasionally may be increased. azotemia, both from dehydration and secondary renal disease, is a common finding, and a fatal uremia may occur. blood concentrations of ß-hydroxybutyric acid greater than mmol/l are consistent with pregnancy toxemia. urinalysis will be positive for both ketones and protein. urine is collected from sheep by holding the nares and from does by frightening them and then allowing them a perceived escape when they stop, squat, and void. although not commonly performed, liver biopsy can help determine the extent of fatty infiltration. this syndrome must be differentiated from hypocalcemia, hypomagnesemia, polioencephalomalacia, encephalitis, lead toxicity, and cerebral abscesses. treatment. very early cases (before the animal exhibits recumbency) may be treated with oral or iv glucose. a balanced electrolyte solution with extra calcium ( ml of a % calcium borogluconate per liter), potassium ( to meq/l), and % dextrose is needed. in some cases, sodium bicarbonate is valuable in treating acidosis (see appendix ii). energy intake must be increased, and propylene glycol can be administered ( to ml every hours) as a glucose precursor. rumen transfaunation and supplementation with vitamin b complex (including vitamin b , biotin, niacin, and thiamine) also are recommended. after females become recumbent, treatment must be very aggressive. removal of the fetuses is crucial in these cases. chemically inducing parturition (by administering . to mg of prostaglandin f a or . mg/ kg of cloprostenol in does and to mg of dexamethasone in ewes) and giving the ewe or doe medical support (fluids, b vitamins, glucose) while waiting is a useful protocol in some cases. unfortunately, during the time before parturition, endotoxemia from dead fetuses further compromises the female. for this reason, the authors recommend immediate cesarean section on depressed moribund animals (see chapters and ) . the owner should be forewarned of the poor prognosis for animals already in a moribund state. fluid support during and after surgery is crucial. regardless of the therapeutic plan, the animal should be offered a palatable, energy-rich, highly digestible feedstuff. the keeper and clinician should take care to minimize the risk of a confounding disease during convalescence (e.g., lactic acidosis, polioencephalomalacia). prevention. fatty liver and pregnancy toxemia can be prevented through proper nutrition. maintaining animals in proper body condition throughout the year and making sure energy and protein levels are adequate in late gestation (see chapter ) are two key preventive measures. , the owner/manager should be taught to assess body condition in individual animals and should maintain emergency stores of feed in case of severe weather or natural disasters. the requirement for energy may be one and a half to two times maintenance for single fetuses and two to three times maintenance for multiple fetuses. prevention of concurrent disease that may further increase energy demands or cause anorexia (e.g., intestinal parasitism, foot rot) is crucial. the keeper should take care to increase the grain portion of the diet slowly because anorexia from rumen upset can lead to this disease. ewes should be offered . to kg of a cereal grain (corn, oats, barley, or a combination) every day during the final months of gestation; does can be offered ⁄ to kg of grain. keepers should maintain ewes and does at a body condition score of . to (see chapter ) throughout gestation and evaluate the animals' energy every to weeks. ultrasonography can help identify females with multiple fetuses. these animals should be separated into groups and fed accordingly. ultrasonographic determination of fetal numbers is best accomplished between days and after breeding with a . mhz transducer; a mhz transducer produces better results between days to . either type of transducer may be of value and these windows of time may be expanded by the ability of the operator (see chapter ) . determination of fetal numbers may be enhanced by shearing the hair or fiber in front of the udder, applying a coupling substance to the skin, and viewing as much of the abdomen as possible, building a mental image of its structures and the number of fetuses while systematically moving from one side of the posterior abdomen to the other. keepers and clinicians should ensure that ewes are healthy and free of chronic diseases (e.g., opp, cae, foot rot, chronic parasitism) and that a good-quality trace mineral salt mixture is available free choice. the addition of lasalocid ( . to mg/kg/day) or monensin ( mg/kg/day) to the feed or mineral mixture enhances the formation of the glucose precursor propionic acid and improves the efficiency of feed use. however, monensin should be used with caution because toxicity may occur; the agent should comprise no more than ppm of the complete diet. the inclusion of niacin ( g/head/day) in a feed supplement or mineral mixture helps prevent pregnancy toxemia. supplementation with lasalocid, monensin, or niacin should begin to weeks before the females give birth. shearing in the last trimester also is recommended in ewes. many sheep producers routinely clip the wool around the vulva. if complete body shearing is performed, the incidence of fatty liver or pregnancy toxemia may be decreased. sheared sheep require less energy to walk and graze. sheared ewes also tend to shiver on cold days, exercising the enzyme systems that promote the more efficient use of ffas as energy substrate. these ewes tend to seek shelter during cold weather, which may decrease lamb losses resulting from hypothermia. obviously, if ewes are to be shorn, keepers should make adequate shelter available. keepers should avoid hauling or moving females during late gestation. proper predator control measures should be maintained. good hoof care programs should be in place on farms or ranches where grazing is the predominant form of nutrient intake. sheep and goats should have their teeth checked to ensure good dentition before the breeding season. animals with poor teeth should be culled. measuring serum b-hydroxybutyric acid concentrations is useful in assessing energy status in ewes. values of . to . mmol/l suggest a negative energy balance. keepers should take steps to correct the problem by feeding better-quality, more digestible feedstuffs. white liver disease is a form of fatty liver disease reported only in angora and angora-cross goats and sheep. it is associated with cobalt deficiency. pathogenesis. cobalt is needed by rumen microflora to produce cyanocobalamin, or vitamin b , which is a coenzyme for methylmalonyl-coa mutase. in turn, this enzyme is needed to convert propionate to glucose through the krebs cycle. cobalt deficiency leads to the accumulation of methylmalonyl-coa, or methylmalonic acid, which is converted to branched chain fatty acids that accumulate in the liver. , high-grain diets that are fermented to propionate coupled with deficient or marginal cobalt intake may predispose to this condition. , white liver disease has not been reported in the united states, but ill thrift from cobalt deficiency has been observed. it is therefore possible that the disease goes unrecognized. clinical signs. signs are most commonly seen in young animals, and include ill thrift, anorexia, and diar-rhea; sheep may exhibit photosensitization. clinicopathologic findings include a macrocytic, normochromic anemia and hypoproteinemia. , diagnosis. abnormal serum or liver concentrations of vitamin b or liver cobalt are the basis of diagnosis. liver cobalt concentrations on a dry matter basis of . Ϯ . ppm were reported in goats with white liver disease, compared with . Ϯ . ppm in controls. treatment and prevention. sheep can be treated with oral cobalt ( mg/head/day) or vitamin b injections. the condition can be prevented by including cobalt in the ration by feeding a good-quality trace mineral salt. both fasciola hepatica and fascioloides magna can infest sheep and goats. the disease occurs along the gulf coast and in the pacific northwest and great lakes areas. clinical signs. f. hepatica infestation usually causes acute disease in sheep and goats but can present as a chronic condition. chronic disease is the result of the mature flukes in the bile ducts and is manifested in depressed growth and milk production. acute disease occurs when large numbers of immature flukes migrate at once, particularly in animals with limited immunity to flukes. signs include anorexia, depression, weakness, dyspnea, anemia, ascites, colic-like signs, dry feces, and sudden death. the clinical signs are identical to those of nematode infestations (i.e., chronic weight loss, ill thrift, diarrhea, anemia, hypoproteinemia). similar but more severe signs occur with f. magna infection, which is usually fatal. , , diagnosis. antemortem diagnosis of fluke infestation can be difficult. finding eggs in feces is diagnostic for f. hepatica. eggs are only produced by adults and not in great numbers, so a negative fecal test cannot preclude acute or chronic fascioliasis. fluke eggs do not float in routine fecal flotation methods used for nematode diagnosis; a sedimentation technique should be used for suspected fluke infestations. to perform a sedimentation test, the clinician mixes to g of feces with ml of tap water and strains the mixture through a tea strainer into a beaker. the sediment can be examined minutes later under a dissecting microscope. eggs are light yellow to golden and have an operculum at one end (see figure - ). f. magna does not mature, so eggs are not produced and fecal examination is of no value. most fluke infestations are discovered by finding the flukes at necropsy or slaughter. an elisa test may be available in the future. , , cbcs of affected animals may indicate eosinophilia and anemia. increased liver enzymes and hypoalbuminemia also are occasional findings. because antemortem diagnosis is difficult, the clinician should institute fluke treatment after ruling out other differential diagnoses if the possibility of fascioliasis exists. if fascioliasis is diagnosed at necropsy, the remaining animals in the herd should be treated. because flukicides available in the united states are highly effective only against mature flukes, the timing of treatment is important. in the southern portions of north america the snails are ingested in the spring and the flukes migrate in the summer and mature in the fall. in cooler, northern climates, snails may remain active during summer, so flukes can mature in the fall and into the winter. clinicians should begin treatment in the southern united states in the late summer or early fall. a single treatment in late winter or early spring is commonly used in the northern climates of north america. albendazole ( - mg/kg orally) and clorsulon ( mg/kg orally, mg/kg sc) are very effective against adult f. hepatica. , , clorsulon has no efficacy against nematode parasites but is highly efficacious against both adult and late-stage immature flukes. albendazole ( mg/kg orally) is somewhat useful in controlling f. magna at weeks after infestation, and clorsulon is effective only at very high dosages. [ ] [ ] [ ] unfortunately, neither agent can kill % of f. magna, and only a few remaining flukes can be fatal. control of fluke infestations is difficult, although timely treatment of animals can decrease infec-tions in successive years. decreasing exposure is the key to control. eliminating the snail is impractical, but fencing off low-lying areas may prevent ingestion. depending on local fluke life cycles, keepers should avoid grazing animals on areas with high fluke populations during peak infection times. areas where water stands or flows over grazing pastures, streams, and irrigation ditches (particularly those with clay soil) are high-risk zones. cysticercus tenuicollis is the larval stage of the dog tapeworm taenia hydatigena, of which sheep and goats are intermediate hosts. the larval stage migrates through the liver, then attaches to the liver or other abdominal organs and causes black, winding tracts and cysts in the liver. acute disease occurs only with large numbers of cysticerci and is characterized by depression and weakness resulting from liver damage. the chronic cystic stage is usually asymptomatic. no treatment is available and control is problematic because it requires treating infestation in dogs and preventing contact with dogs. , , pathogenesis. copper (cu) toxicosis is more common in sheep than in goats. goats appear closer to cattle than sheep in their ability to store and handle cu and resist toxicosis. toxicity results from chronic accumulation in the liver from the ingestion of excess cu in relation to molybdenum (mo) or sulfate in the diet. in sheep, a cuto-mo ratio greater than Ϻ leads to the accumulation of excess cu. the most common sources of excess cu in sheep and goats are trace mineral mixtures and feeds formulated for cattle or horses. clinical signs are often absent during the chronic accumulation phase. acute disease is seen when cu is suddenly released from the liver in large amounts. stress usually precipitates this acute phase. acute release and subsequent high blood cu concentrations cause an acute hemolytic crisis, resulting in anemia, hemoglobinuria, and acute renal failure. existing hepatic disease (such as that caused by liver flukes) may predispose animals to this condition. some breeds seem to be prone to cu absorption and storage problems (merino sheep), whereas others tend to be more resistant and prone to deficiency (pygmy goats) (see chapters and ). clinical signs. anorexia, depression, diarrhea, and weakness are all signs of cu toxicity. many affected animals are found dead with hemolysis and icterus. signs of abdominal pain and diarrhea are sometimes present. port wine-colored urine is evidence of hemoglobinuria. hemoglobinemia produces icterus of the mucosal membranes and fever. diagnosis. on clinicopathologic examination, anemia, hemoglobinemia, hyperbilirubinemia, increased liver enzymes, and azotemia are present. urinalysis reveals hemoglobinuria and isosthenuria. the combination of azotemia and isosthenuria indicates acute renal failure. definitive diagnosis of acute disease requires measurement of cu concentrations in serum. normal blood cu concentrations are approximately to mg/dl in sheep and goats. , , these concentrations increase to -fold with an acute hemolytic crisis. on necropsy, kidney cu concentrations are the most diagnostic because liver concentrations may be normal from release into the bloodstream. generally kidney concentrations greater than ppm and liver concentrations greater than ppm on a dry matter basis are diagnostic. , if tissue copper is reported in wet weight, the conversion to dry tissue weight can be estimated by multiplying the tissue concentration by a factor of . treatment. treatment of acutely affected animals is often futile. it consists of supportive therapy for the acute renal failure and anemia and attempts to lower liver cu stores. fluid therapy for the acute renal failure (see appendix ii) is of therapeutic value, and a blood transfusion may be needed if the pcv drops precipitously. ammonium tetrathiomolybdate ( . mg/kg iv or . mg/kg sc on alternate days for three treatments) is the most economical agent for treatment for acute cases. in valuable animals, d-penicillamine ( to mg/kg bid or mg/kg sid po for days) increases urinary cu excretion. trientine is used in human beings, but has shown variable results in sheep. treatment of the remainder of the flock should include the administration of ammonium molybdate ( to mg/head/day po) and sodium thiosulfate ( to mg/head/day po) for weeks. stress should be minimized, so keepers and clinicians should delay routine maintenance procedures such as deworming and hoof trimming until after treatment. the offending source of cu should be eliminated. prevention. avoiding high dietary cu (more than ppm), a high cu-to-mo ratio (greater than Ϻ ) in the feed, cu-containing foot baths, and other sources of cu is crucial. including supplemental mo in the diet to lower the cu-to-mo ratio to Ϻ to Ϻ is beneficial. this requires to ppm of mo in many instances. often too much emphasis is placed on the trace mineral component of the diet. the clinician should be aware that even if no cu is added to the trace mineral mixture and the element does not appear on the product label, the mineral mixture may still contain cu. many components of mineral mixes are contaminated with cu (zinc sulfate may contain ppm of cu, dicalcium phosphate may contain more than ppm of cu). therefore the clinician needs to perform a dietary analysis to find and correct the problem. pathogenesis. the liver is vulnerable to toxic insult because one of its major functions is detoxification. the most common plants that are gastrointestinal and liver toxins are shown in table - . clinical signs depend on the cause. acute, severe toxicity is more common with chemical toxicosis, whereas plant toxins usually cause chronic disease. a thorough history is important and in many cases inspection of the animals' environment is required. clinical signs. the clinical signs of toxic hepatitis can be vague. animals may only show anorexia and depression. icterus is more common with hemolytic diseases and is not always seen with liver disease. photosensitivity is a common clinical feature in ruminants and hepatoencephalopathy also can occur. clinicopathologic data are more helpful in diagnosing acute toxicity. serum ast and ldh levels can increase with hepatocellular necrosis but are not liverspecific, so muscle injury and disease must be ruled out. these enzymes also increase if serum is not separated from a blood clot in a timely fashion. increased levels of alkaline phosphatase (ap) and ggt indicate biliary stasis. ap also is not liver-specific, but increased serum levels of ggt are very specific for liver disease. ggt also increases in some hepatocellular diseases, so testing for its normal concentrations is important. unfortunately, all of these enzymes can be normal with liver disease, especially if it is chronic. hyperbilirubinemia, hypoglycemia, low blood urea nitrogen (bun), and hypoalbuminemia are not always evident as classically taught. if hepatoencephalopathy is suspected, blood ammonia concentrations may be elevated. blood ammonia analysis may be impractical in the field because the blood should be kept on ice, and the test should be performed within minutes of collection. to enhance the accuracy of blood ammonia analysis, the clinician should collect blood from a normal control animal for comparison. ammonia concentrations three times those of the control animal are diagnostic. liver biopsy remains the most valuable tool in diagnosing liver disease. although clotting dysfunction may occur in liver disease, it is an uncommon complication in ruminants and should not discourage the clinician from performing a liver biopsy. treatment. if the intoxication is caught in the acute stage, activated charcoal ( g per adult animal) can be given. supportive care, especially fluid support with dextrose solutions, is the mainstay of therapy. low-protein diets may suppress ammonia production temporarily, but they can be detrimental over time depending on the production status of the animal. if photosensitization occurs, animals should be housed indoors if possible, and broad-spectrum (systemic or topical) antibiotics may be necessary to control secondary bacterial dermatitis. corticosteroids (dexamethasone . to mg/kg iv or im) may be indicated in early cases of photosensitization to decrease inflammation. neurologic signs can be controlled with phenobarbital (initial dose: to mg/kg iv diluted in saline and administered over minutes; subsequent doses: to mg/kg iv diluted in saline, as needed up to tid). diazepam (valium) is contraindicated in hepatoencephalopathy because it may worsen signs. congenital hyperbilirubinemia, or black liver disease, occurs in mutant corriedale sheep (dublin-johnson syn-drome). this is a genetically recessive condition. it is characterized by an abnormality in the excretion of conjugated bilirubin and phylloerythrin and is often seen in animals consuming green forage. clinical signs include anorexia, photodermatitis, and icterus. liver biopsy of affected animals reveals dark to black granules in otherwise normal hepatocytes. the syndrome first manifests itself in lambs around months of age. a similar condition occurs in southdown lambs around months of age (gilbert's syndrome). this too is a recessive condition that causes decreased hepatic uptake of phylloerythrin and bilirubin, with concurrent renal failure. signs include icterus, photodermatitis, and ulceration around the ears and mouth. a liver biopsy reveals normal hepatic tissue. in both of these conditions, animals should be kept out of sunlight and fed minimal various tumors of the liver, including fibrosarcoma, lymphosarcoma, and cholangiocellular carcinoma, have been reported. , the use of ultrasonography and ultrasound-guided liver biopsy may aid in diagnosis. the umbilicus is an opening in the ventral abdominal wall that allows passage of the umbilical vessels and allantoic stalks. this opening should close within a few day of birth. the failure of this opening to close properly is termed umbilical hernia. the hernial sac has an inner peritoneal layer and an outer layer of skin. these hernias are probably of genetic origin but may occur as sequelae to umbilical remnant infection. the opening in the abdominal wall is perceived as a ring on palpation. if the clinician can insert more than one finger into the hernial ring or if the hernia persists for more than to weeks, surgical intervention is indicated. penning. clamps or rubber bands may be of value for closing small hernias (those less than cm in diameter). the clinician should either lightly sedate the animal or infiltrate the skin around the hernia with a local anesthetic ( % lidocaine). the animal should be placed on its back and held by a technician-helper. any viscera prolapsing into the hernial sac should be replaced into the abdomen. the clinician then inserts two metal pins (baby diaper pins can be used) through the skin and on opposite sides of the hernial ring, just on the edge of the linea alba. the pins should be placed deep enough to sit next to the abdominal wall. slight tension is placed on the skin in the center of the umbilical sac, pulling it away from the abdomen. when the clinician is confident that all viscera have been cleared from the hernial sac, he or she places an elastrator band between the pins and the abdominal wall. this results in ischemic necrosis of the skin. the skin will slough and the abdominal defect will heal in to days. lambs should be given tetanus prophylaxis. this procedure and other clamping techniques are useful in females and some males. however, urine scalding of the skin may occur in some males. clinicians should closely monitor animals that have undergone clamping. surgical resection. in cases in which the hernial ring is larger than cm, surgical intervention should be carried out. animals can be sedated and then infiltrated with a local anesthetic or placed under general anesthesia. the area around the hernia is clipped and surgically prepared. the clinician opens the hernial sac and introduces a finger into the abdomen to ensure that no viscera have adhered to the inner lining of the ring and that no enlarged or infected umbilical remnants are present. he or she then carefully excises the ring and closes the defect in the abdominal wall. this closure can be made by simply opposing the abdominal wall with a horizontal mattress pattern stitch (absorbable suture). an alternate closure of the abdominal wall is to suture the peritoneal lining in a separate pattern and close the abdominal wall defect so one side of the defect is pulled to overlap the other side. the upper free edge is sutured to the opposite wall with a near-far-far-near pattern. the authors choose not to employ surgical techniques that slow this procedure. the subcutaneous tissue can be closed with simple interrupted pattern using absorbable suture and the skin should be closed with whatever pattern the clinician prefers. animals should be given tetanus prophylaxis and antibiotics. they should be closely monitored for signs of sepsis and surgical failure. exercise should be limited for to days after surgery. infections of the umbilical arteries (omphaloarteritis) and veins (omphalophlebitis) and urachal disease can occur because of failure or partial failure of passive transfer of colostral antibodies and subsequent sepsis. contamination of the umbilicus, retracting of these structures after stretching and breaking, and chemical damage (from strong tincture of iodine) to the amniotic remnants are other possible causes. , , dipping the umbilicus with iodine or iodine-chloriodine substances is a common practice. aggressive use of these chemicals may precipitate serious inflammation of the cord. excessive torsion of the umbilical cord, distention of the proximal urachus, and some genetic factors may all be associated with patent urachus, which also may occur as a sequela to omphaloarteritis or omphalophlebitis. clinical signs and diagnosis. the clinical signs include umbilical swelling, pain, and occasionally drainage or discharge of the umbilical stump. palpation and transabdominal ultrasonographic evaluation reveal an enlarged cord-like structure ascending from the umbilicus cranially (umbilical vein) or caudally (urachus or um-bilical artery). ultrasonographic evaluation may indicate an abscess or thickened tissue. patent urachus is associated with dermatitis, urine scalding of the ventral abdomen, and urine dribbling. if the urachus becomes infected it may leak urine intraperitoneally or subcutaneously. both of these developments may be identified with abdominal palpation, ballottement, ultrasonographic evaluation, and, when indicated, paracentesis. the cbc may indicate neutrophilia. blood culture is indicated if sepsis occurs simultaneously. occasionally infection of the internal structures may occur with no outward umbilical swelling. deep abdominal palpation and/or the use of real-time ultrasound are necessary to attain a diagnosis. animals with umbilical infections also may have signs of septicemia, anorexia, depression, joint distention, and fever. treatment. if a patent urachus occurs without inflammation of the associated tissues, it can be cauterized daily with iodine or silver nitrate. however, if it remains patent for more than days, it should be surgically closed. the animal should be placed under general anesthesia (see chapter ) . the area around the umbilicus should be clipped and surgically prepared, and the animal should be placed on a broad-spectrum antimicrobial agent to hours before surgery. the clinician opens the abdomen lateral to the umbilicus and digitally explores the adjacent area for adhesion formation. the urachus should be identified and followed to the urinary bladder. after this, the clinician should amputate the urachal attachment to the bladder and close the bladder with a double-layered inverting pattern (cushing). the abdominal wall, subcutaneous tissue, and skin are closed as described for umbilical hernia repair. on occasion some cases of omphalophlebitis-omphaloarteritis can be treated medically. prolonged antibiotic therapy with a broad-spectrum antimicrobial agent (ceftiofur . mg/kg sid or oxytetracycline mg/kg sc every hours) may be attempted. however, if medical therapy is ineffective, the infected umbilical remnants should be marsupialized or excised. the authors prefer more aggressive, surgical removal of the umbilical rem-nants. as with urachal surgery, the abdomen should be opened lateral to the umbilicus. depending on the severity of infection and the amount of tissue involved, the clinician may need to perform extensive dissection of necrotic tissue and possibly intestinal resection. if the infection of the umbilical vein extends to and involves the liver, marsupialization of the umbilical vein is an effective method of therapy. , the clinician can pull the vein to the most cranial portion of the abdominal incision and suture it to the muscle layers and skin before closing the abdomen as described for umbilical hernia repair. however, a preferable method is to close the abdominal wall, pull the transected umbilical vein through, and suture it to a separate stab incision. this may help minimize the incidence of abdominal wall herniation. only monofilament, absorbable, non-gut suture material should be used. the venous stump should be flushed daily with antiseptic solution ( % chlorhexidine, . % povidone iodine), and the animal should be maintained on antibiotics for more than days. the venous stump usually closes within a month. prevention. umbilical infections can be prevented or drastically reduced by ensuring adequate intake of goodquality colostrum. lambs and kids also should be only minimally stressed (particularly during the first to days of life) to enhance colostral absorption. in some management scenarios, proper dipping of the navel with non-caustic materials also helps reduce the incidence of this disease. indigestion in ruminants bloat or ruminal tympany diseases of the goat on the effect of xylazine on forestomach motility in sheep anorexia during febrile conditions in dwarf goats: the effect of diazepam, flurbiprofen and naloxone bloat in kids experimentally induced lactic acidosis in nubian goats: clinical, biochemical, and pathological investigations diagnosis of enteric disease in small ruminants ruminal lactic acidosis in sheep and goats biochemical alterations in serum and cerebrospinal fluid in experimental acidosis in goats commonly encountered diseases of goats generalized aspergillosis in dairy sheep rumen papillomas in sheep effect of monensin on development of ruminal parakeratosis in fattening lambs, zentralblatt fur veterinarmedizin lactic acidosis foreign body syndrome in goats-a report of five cases traumatic gastritis in sheep and goats goat medicine references . sherman dm: causes of kid morbidity and mortality: an overview, proceedings of the fourth international conference on goats enteric infections in young goats and their control enteritis and diarrhea goat medicine infectious gastrointestinal diseases of young goats occurrence of cryptosporidia, rotaviruses, coronavirus-like particles and k ϩ escherichia coli in goat kids and lambs jensen and swift's diseases of sheep escherichia coli in domestic animals and humans development of resistance with host age to adhesion of k ϩ escherichia coli to isolated intestinal epithelial cells references . smith bp: alterations in alimentary and hepatic function control programs for gastrointestinal nematodes in sheep and goats epidemiology of internal parasites: effects of climate and host reproductive cycles on parasite survival anthelmintic resistance: the selection and successful breeding of superior parasites control and prevention of specific diseases of sheep and goats herd rp: control of periparturient rise in worm egg counts of lambing ewes production medicine and health programs for goats the effects of dietary protein on establishment and maturation of nematode populations in adult sheep nematode infections-cattle, sheep, goats, swine parasites affecting goats in the southeast, proceedings of goat production and marketing opportunities in the south grazing management strategies for the control of parasitic diseases in intensive sheep production systems goat medicine helminth parasites of the gastrointestinal tract. nematode infections in cattle, sheep, goats, and swine parasite control programs in sheep and goats epidemiology and control of trematodes in small ruminants use of anthelmintic combinations against multiple resistant haemonchus contortus in angora goats preliminary investigation of anthelmintic efficacy against gi nematodes of goats and susceptibility of goat kids to gastrointestinal nematode infection johne's disease in sheep and goats paratuberculosis in small ruminants, proceedings of the small ruminants for the mixed animal practitioner, western veterinary conference paratuberculosis in small ruminants, deer, and south american camelids comparison of the absorbed elisa and agar gel immunodiffusion test with clinicopathologic findings in ovine clinical paratuberculosis paratuberculosis in a large goat herd serodiagnosis of paratuberculosis in sheep by use of agar gel immunodiffusion corynebacterium pseudotuberculosis infection in sheep and the complement fixation test for paratuberculosis obstructive intestinal diseases intussusception in goats goat medicine duodenal obstruction by a phytobezoar in a goat general surgical techniques for small ruminants: part ii, proceedings of the small ruminants for the mixed animal practitioner, western veterinary conference rectal prolapse in ruminants and horses jensen and swift's diseases of sheep rectal prolapse in food animals. part : cause and conservative management rectal prolapse in food animals. part ii: surgical options, comp cont ed pract vet : , . references . fetcher a: liver diseases of sheep and goats goat medicine a retrospective study of hepatic abscesses in goats: pathological and microbiological findings hepatic lipidosis jensen and swift's diseases of sheep hepatic lipidosis associated with cobalt deficiency in omani goats epidemiology of internal parasites: effects of climate and host reproductive cycle on parasite survival epidemiology and control of trematodes in small ruminants efficacy and safety of albendazole against experimentally induced fasciola hepatica infections in goats efficacy of an injectable ivermectin/clorsulon combination against fasciola hepatica in sheep albendazole treatment of experimentally induced fascioloides magna infection in goats efficacy of clorsulon against fascioloides magna infection in sheep evaluation of clorsulon against fascioloides magna in cattle and sheep clinical biochemistry of domestic animals metals and other inorganic compounds interpreting a bovine serum chemistry profile: part i large animal internal medicine large animal internal medicine neonatal conditions, with emphasis on equine neonate umbilical hernia, umbilical abscess, and auricle fistula general surgical techniques for small ruminants: part ii, proceedings of the small ruminants for the mixed animal practitioner, western veterinary conference key: cord- -dd gw t authors: armbruster, walter j.; roberts, tanya title: the political economy of us antibiotic use in animal feed date: - - journal: food safety economics doi: . / - - - - _ sha: doc_id: cord_uid: dd gw t this chapter examines the evidence for antibiotic resistance in the united states and globally, the public health implications, and the impact of—and related industry and political responses to—antibiotic use in animal feed. in , the swann report in the united kingdom noted a dramatic increase in antibiotic-resistant bacteria in food animals receiving low levels of antibiotics in their feed. while the food and drug administration of the united states sought to control antibiotics in animal feed as far back as , only in were such regulations fully implemented. the farm-level costs of such controls are estimated by the us department of agriculture’s economic research service to be minimal, while the centers for disease control and prevention’s estimates of the public health costs of antibiotic resistance without implementing controls are $ billion annually. the complex interactions which exist between economic interests, regulatory policy, and human and animal health are explored in this chapter. antibiotic resistance has been widely recognized as a serious public health problem. hence, there is a major public good to be realized in safeguarding the effectiveness of existing antibiotics and creating new ones. antibiotics are used to treat human infections and used in animal agriculture. while many drugs are dual-use, others are animal-or human-use specific. the production benefits of sub-therapeutic levels of antibiotics in animal agriculture have been recognized since the late s (cast ) . in animal agricultural production, antibiotics are used at therapeutic levels to treat infections and at sub-therapeutic levels to prevent infections and promote animal growth (sneeringer et al. ; van boeckel et al. ; who ) . as the organizational complexity of the animal agricultural supply chain increased, the number of economic stakeholders in on-farm antibiotic use has also increased. the major stakeholders include pharmaceutical companies, production integrators, feed suppliers, farm groups, producers, restaurants, food retailers, the public, the medical community, the scientific community, government regulators and policy makers. each of these stakeholders faces a different set of incentives and disincentives related to on-farm use of antibiotics in animal agriculture. knowledge of these incentives and disincentives has evolved with the accumulation of scientific and economic research. to understand the regulatory outcomes governing antibiotic use in agriculture, it is important to recognize the political economy context in which they are developed. the various stakeholders are driven by the relative benefits they receive under policies as they affect their industry segment (zilberman et al. ). alexander fleming, who discovered penicillin, warned that "…misuse of the drug could result in selection for resistant bacteria" (rosenblatt-farrell ) . antibiotic resistance (ar), a term sometimes used interchangeably with antimicrobial resistance, occurs when bacteria change in ways that make antibiotics less effective in treating infections, thereby allowing the bacteria to survive, multiply, and cause additional harm. ar has been recognized as a serious public health problem among the medical and scientific communities. antibiotics are used to treat human infections and used in animal agriculture. particularly concerning is resistance for those antibiotics that are of value in treating human health issues, the so-called medically important antibiotics. the use of antibiotics along with other advances in agricultural technology has facilitated the concentration of animal production on farms in the united states (us) and elsewhere. for example, in , % of all us sales of hogs and pigs were by the % of farms with , or more head, and % of all layers were produced on the less than % of farms that sold , or more to egg producers (nass ) . the majority of the production of hogs, broilers, and eggs occurred under contractual arrangements between growers and integrators, with the integrators prescribing certain production practices, including the use of antibiotics for treating infections, for disease prevention and for promoting growth. many of the antimicrobial drugs administered to food-producing animals are also important in treating humans, worldwide. domestic sales of medically important antimicrobial drugs for use in food-producing animals in the united states accounted for % of the domestic sales of all antimicrobials approved for use in food-producing animals. and, % of domestic sales of all medically important antimicrobials approved for use in food-producing animals are labeled for therapeutic use only . importantly, animal drug sales data represent products sold or distributed by manufacturers through various outlets for intended sale to the user. since veterinarians and others in the supply chain may have substantial inventory on hand for possible use, these numbers do not accurately reflect the amount of product ultimately administered to animals. given the number of humans versus a much larger number of animals in each of the species, as well as other confounding factors, no definitive conclusions from any direct comparisons between the quantities of antimicrobial drugs sold for use in humans versus animals can be drawn (fda a) . there are obvious situations where antibiotics are required to treat sick animals in agriculture, but the proper therapeutic use versus prophylactic use remains in question among stakeholders. farm groups and others in the food animal supply chain recognize that antibiotics in animal feed keep animals healthy and meat costs down. but over medical doctors and other healthcare providers signed petitions to congress asking for new legislation to reduce non-therapeutic antibiotic use in food animals (miller ) . the animal health industry is very concerned that needed preventative use will be threatened by the recent fda ban on use of medically important antibiotics for growth. fda classifies as therapeutic those antimicrobials targeted for treatment, control, and prevention of bacteria or disease identified on the product label. fda explicitly states that the use of antibiotics in animal feed for growth promotion is not allowed. those who characterize preventative use as routine overlook the difference between treating animals versus humans. if preventative measures are not taken and a disease outbreak occurs and spreads rapidly within a flock or herd, it risks large numbers of animals developing a deadly, high mortality disease. waiting until a disease is clearly evident makes successfully treating the active infections very difficult due to the large number of animals involved. by contrast, a human patient can generally be quickly diagnosed and treated. while some are concerned that producers will continue to use antibiotics for growth under the guise of prevention, the fda-approved label is specific about dose and duration for a specified bacterium or disease. off-label use of antibiotics in animal production is illegal, and fda only allows a veterinarian to decide whether to use or not to use a preventative treatment based on their judgment of a disease threat (carnevale ) . in an economic framework, antimicrobial resistance can be considered as an unwanted side effect, or externality, associated with the use of antibiotics. the efficacy of antibiotics can be considered as a public good that must be managed with government involvement. this is because the costs of overuse by any single individual are borne by society and, in the case of antibiotics, globally. hence, not only is there a role for government involvement with the animal agriculture industry in managing the stock and use of antibiotics as an important public good, but it must be done cooperatively across countries. in , the united kingdom's (uk) parliament received the swann report, which concluded that using antimicrobials at sub-therapeutic levels in food-producing animals created risks to human and animal health (joint committee on the use of antibiotics in animal husbandry and veterinary medicine ). it noted a dramatic increase in numbers of animal-origin bacteria strains which showed resistance to one or more antibiotics and that these strains could transmit resistance to other bacteria. it recommended that only antimicrobials that are not medically important for humans should be used without prescription in animal feed and that antimicrobials should only be used for therapeutic purposes under veterinary supervision. the primary reason that producers were using these sub-therapeutic doses of antibiotics was to promote faster weight gain in the animals. in , a us food and drug administration (fda) task force was charged to do a comprehensive review of antibiotic use in animal feed (fda ). its report found that sub-therapeutic use of antimicrobials in food-producing animals was associated with development of resistant bacteria and that treated animals might provide a reservoir of antimicrobial-resistant pathogens capable of causing human disease. the task force recommended that medically important antimicrobial drugs meet certain guidelines they identified or be prohibited from growth promotion or other sub-therapeutic use by certain dates. further, antimicrobials not meeting the guidelines should be limited to short-term therapeutic use only under veterinarian control. in the s, the animal health institute (ahi), a us trade association for the animal drug industry, funded an on-farm study to determine the impact of adding low-dose antibiotics to chicken feed. within week of adding tetracycline, the intestinal flora in the chickens "…contained almost entirely tetracycline-resistant organisms" (levy et al. ). the antibiotic resistance was not located in the dna of the bacteria which is hard to transfer among bacteria but in plasmids located on the outside surface of the bacteria. plasmids are easily exchanged among bacteria living in the intestine. importantly, the tetracycline-resistant bacteria in the chicken's intestines were resistant to multiple antibiotics. furthermore, some members of the farm families began to harbor these same antibiotic-resistant bacteria in their intestines within months. in , the fda proposed withdrawing the new animal drug approvals for the sub-therapeutic uses of human medically important penicillin and tetracycline in animal feed based on lack of evidence to show they were safe. however, the us congress intervened and asked for more research first. the ahi was one of the groups advocating in congress to delay regulation pending additional research, then and now. in congressional testimony, richard carnevale, vice-president at ahi, testified that while it is possible for human antibiotic resistance to be caused by antibiotic use in farm animals, "…it does not happen enough that we can find it and measure it" (carnevale ) . this statement contradicted the data produced by the ahi-funded study by levy (levy et al. ) that was published in the prestigious new england journal of medicine in . richard carnevale also mentioned in his testimony that prior to joining ahi he was deputy director of new animal drug evaluation in fda and had worked at usda in the food safety and inspection service (fsis). his testimony illustrates two points in the political economy of food production: ( ) how industry has an opportunity to influence regulators' decision-making via the revolving door of employment and ( ) how industry carefully selects its facts to present a point of view that bolsters their profits, namely, for drug companies in this case (oreskes and conway ) . another example of the political economy in action involved usda prohibiting an agency research microbiologist from talking about the significant levels of antibiotic-resistant bacteria detected in the air near midwest hog confinement operations (union of concerned scientists ). a third element of the political economy is shown by industry efforts to influence policy makers through campaign contributions and strong lobbying of proposed legislation which may affect their bottom line. pharmaceutical companies spent at least $ million and agribusiness companies another $ million during , in large part to fight possible limits on antibiotic use in animal feed (mason and mendoza ) . in response to congressional pressure in the late s, fda withdrew its proposal and instead funded three studies to determine the impact of using low levels of antibiotics in animal feed (industry won this round, obtained a delay in regulations, and funded more reports): . in , the national academy of sciences reported that there was limited epidemiological research on the topic. available evidence at that time did not prove nor disprove dangers of seven therapeutic antimicrobials in animal feed, but that did not preclude the existence of hazards (national academy of sciences ). . in , the fda funded the seattle-king county department of public health to analyze salmonella and campylobacter, which were chosen as models to estimate the flow of potentially pathogenic bacteria from animals to humans through the food chain. their report was based on sampling retail meat and poultry and investigating salmonella and campylobacter enteritis cases in humans. isolates from human illness cases and retail foods were analyzed for antibiotic resistance of these pathogens, using plasmid analysis and serotyping. the report found that campylobacter was a more common cause of enteritis than salmonella and appeared to flow from chickens to humans through consumption of poultry products, with tetracycline resistance being plasmid-mediated (seattle-king county department of public health ). . in , the institute of medicine (iom) undertook a fda-requested independent quantitative risk assessment of human health impacts from sub-therapeutic use of penicillin and tetracycline in animal feed. based on a risk-analysis model of salmonella infections that resulted in human death, the iom did not find substantial direct evidence that sub-therapeutic use in animal feed posed a human health hazard. however, they found a considerable body of indirect evidence implicating both sub-therapeutic and therapeutic use of antimicrobials as a potential health hazard and strongly recommended additional study of the issue (institute of medicine ). numerous research results quantifying the extent of the antimicrobial resistance problem have been published in the scientific literature and indicate a growing and serious threat to human health. the many channels for ar to affect humans are shown in fig. . . the two main channels for food animals are ( ) ar bacteria in the food animal's gut can contaminate the meat or poultry eaten and ( ) environmental contamination, such as manure used to fertilize fields that contain ar bacteria, may contaminate the environment and some of the food crops grown on these fields. consumer reports (cr) tested products sold in us supermarkets and found resistance to multiple antibiotics in the following percent of samples: beef %, shrimp %, turkey %, and chicken % (consumer reports ). cr also found that ground beef from conventionally raised cows was twice as likely to contain antibiotic-resistant pathogens as ground beef from cows raised without antibiotics. like other threats to human health, ar is best managed across national boundaries. increasing international trade may spread antibiotic resistance through imported food products as more trade agreements are approved. this scenario could be exacerbated to the extent fsis approves additional international facilities, local regulations, and inspections as "equivalent to the united states." future trade agreements will need to include provisions which address reduced use of medically important antibiotics in producing food animals. numerous trusted institutions from the united states (us) and the united kingdom (uk) as well as international organizations such as the world health organization (who), the united nations' food and agriculture organization (fao), and the world organization for animal health (oie) have acknowledged the threat of antibiotic resistance related to use in producing food animals. the fol- lowing excerpts from a few recent reports highlight the role that low-dose antibiotic use in animal feed plays in spreading ar. the centers for disease control and prevention ( b) reported that: each year in the united states, at least million people acquire serious infections with bacteria that are resistant to one or more of the antibiotics designed to treat those infections. at least , people die each year as a direct result of these antibiotic-resistant infections. many more die from other conditions that are complicated by an antibiotic-resistant infection. antibiotic-resistant infections add considerable and avoidable costs to the already overburdened u.s. healthcare system. in most cases, antibiotic resistant infections require prolonged and/or costlier treatments, extend hospital stays, necessitate additional doctor visits and healthcare use, and result in greater disability and death compared with infections that are easily treatable with antibiotics. the total economic costs of antibiotic resistance to the u.s. economy has been difficult to calculate. estimates vary but have ranged as high as $ billion in excess direct healthcare costs. adding on the costs for lost productivity brings the total societal costs (sic) for ar to $ billion a year ( dollars). (cdc a, p. ) this cdc report also indicates that foodborne cases are responsible for % of human ar infections ( fig. . ). thus, societal costs of these ar foodborne illnesses could total $ billion annually of the $ billion/year total costs to the us economy. these societal costs could be prevented if the foods were free of contamination with ar pathogens. there may be additional costs associated with environmental pathways of human contamination from use of antibiotics in meat production, such as exposure to contaminated water. in , who stated: "antimicrobial resistance (ar) is an increasingly serious threat to global public health. ar develops when a microorganism (bacteria, fungus, virus or parasite) no longer responds to a drug to which it was originally sensitive. this means that standard treatments no longer work; infections are harder or impossible to control; the risk of the spread of infection to others is increased; illness and hospital stays are prolonged, with added economic and social costs; and the risk of death is greater-in some cases, twice that of patients who have infections caused by non-resistant bacteria. the problem is so serious that it threatens the achievements of modern medicine. a post-antibiotic era-in which common infections and minor injuries can kill-is a very real possibility for the st century" (who , p. ) . in , oie noted: "today, in many countries, including developed countries, antimicrobial agents are widely available, directly or indirectly, practically without restriction. of countries recently evaluated by the oie, more than do not yet have relevant legislation on the appropriate conditions for the import, manufacture, distribution and use of veterinary products, including antimicrobial agents. consequently, these products circulate uncontrolled like ordinary goods and are often falsified." to date, there is no harmonized system of surveillance on the worldwide use and circulation of antimicrobial agents. that information is necessary, however, to monitor and control the origin of medicines, obtain reliable data on imports, trace their circulation, and evaluate the quality of the products in circulation. it is in this context that the oie was mandated by its member countries to gather that missing information and create a global database for monitoring the use of antimicrobial agents, linked to the oie's world animal health information system (wahis). that mandate is also supported by fao and the who within the framework of the who's global action plan on antimicrobial resistance. the database will form a solid basis for the three organizations' work to combat antimicrobial resistance (oie , p. ) . in , a uk evaluation of academic, peer-reviewed research articles addressing antibiotic use in agriculture determined that only % found no link and % found a positive link between antibiotic use in animals and antibiotic resistance (ar) in humans (o'neill ) . taken to evaluate proposals to ban the use of growth-promoting or sub-therapeutic levels of antibiotics in food animals, usda's economic research service (ers) economists added questions on antibiotic use to the agricultural and resource management survey (arms). arms is a nationally representative survey of farms administered jointly by ers and usda's national agricultural statistics service (nass). hog producers were surveyed in and , and broiler producers were surveyed in and . ers also drew upon their research using data in the national animal health monitoring system (nahms) to develop a model to estimate the impacts of withdrawing antibiotics for other than therapeutic use in food animals. using monte carlo simulations, ers estimated the impacts of eliminating antibiotic use for growth promotion of poultry and pork, not just the fda-specified "medically important" antibiotics (table . ). simulation results showed less than . % reduction in output and an approximate . % increase in wholesale prices, netting pork producers greater total revenue of . % and poultry producers . %. ers concluded that these small effects were not statistically significant (sneeringer et al. ) . these ers results are consistent with research studies post- indicating that productivity gains from using antibiotics for growth promotion were lower than earlier research had found (teillant and laxminarayan ) . another report suggested that phase out of growth promotion use in food animals over a -year period would avoid most of the % projected global growth in such use and cost agricultural sectors a small portion of the costs of ar in each country. further, reduced infection risk and costs of medications would cover most farm-level costs of improving animal husbandry practices to offset loss of antimicrobials for production purposes (laxminarayan and chaudhury ) . presuming that any new antibiotic classes probably will not be made available for veterinary medicine, it is important to preserve the effectiveness of existing antibiotics which are necessary for treatment of infectious diseases to maintain animal health (teillant and laxminarayan ) . an alternative to encourage development of new antibiotics would be to delay or not approve drugs which mimic others, but for which the applicant company has not performed antibiotic research (amábile-cuevas ). even better, several production practices may be used to enhance animal health in the absence of using antimicrobials for growth or for prophylactic disease prevention (sneeringer et al. ; who ; macdonald and wang ) . these include: • improved management practices, such as more space per animal and better control of the housing environment • tightened biosecurity to prevent diseases and improve productivity by avoiding introduction of infectious agents by wild animals, domestic pets, and nonessential workers or other humans; through increased cleanliness of production facilities; and from timely removal of dead animals • optimized nutrition to increase growth and mitigate stress-related factors and provide vitamin and mineral supplements to reduce disease susceptibility • improved gut microflora to improve feed efficiency by providing enzymes, organic acids, prebiotics, probiotics, and immune modulators • vaccinations to prevent some diseases • hazard analysis critical control point plans to improve productivity in the absence of using sub-therapeutic antibiotics in animal production generally, these practices may raise production costs modestly at the farm level because of the need for more resources required to successfully manage them. since ers found no statistically significant evidence that antibiotics reduce the costs of producing pork or broilers, we conclude that there are small or no costs to producers from withdrawing growth-promoting or prophylactic uses of antibiotics in production of food animals. in contrast, the public health benefits of withdrawing these antibiotics from agriculture are significant. as reported above, cdc estimates that the medical costs and productivity losses of ar illnesses attributed to agriculture are $ billion us dollars annually. the benefit/cost analysis becomes $ billion in public health protection benefits vs. the very small costs to animal production from withdrawing antibiotics from non-therapeutic use. in other words, the protection of the public health will come at little or no cost to agriculture. furthermore, this benefit/cost analysis provides a conservative estimate of public health protection benefits. the cdc public health protection benefits do not include estimates for protection from an increasing number of "superbugs" that would be created if low-level antibiotics would continue to be used. and cdc does not include the costs of long-term health outcomes caused by foodborne pathogens (see chap. ). aside from costs to agricultural producers, there are also other societal costs related to ar and connected to antimicrobial use in animal production, both in their production and use/misuse, affecting human and environmental health. in economic terminology, these costs are considered negative externalities to society from the individual use of antibiotics. moreover, since the science of ar is unfolding, there may be additional unknown human health and environmental risks associated with the use of antibiotics in food animal production. pharmaceutical production. a major issue involved with manufacturing of active ingredients for antibiotics and the effluent from factories producing them is the potential to contaminate nearby water systems. pharmaceutical factories often contaminate the environment, since guidelines for pharmaceutical waste discharge focus on chemicals used in manufacturing, rather than active pharmaceutical ingredients. this is a primary concern in countries outside the united states, but international trade makes it a worldwide problem. use and misuse. worldwide, antibiotics are used heavily in animal agriculture. this practice has created resistance problems transmissible from animals to humans. for example, china has mrc- colicin resistance in pork and salmonella resistance to cephalosporins at higher levels than in the united states (zhang et al. ) . their practice of applying human waste on fields and the closeness of population centers to agriculture contribute to cross-mixing of pathogens in china. parasites are common in chinese soil and can contaminate pork. and low levels of chlorine in chinese water supplies allow accumulation of biofilms containing antibiotic-resistant pathogens in water pipes. in india, manufacturing of pharmaceuticals and waste disposal practices lead to contamination of water and soil. further, over-the-counter antibiotics are available and heavily used there. farm antibiotic use is of concern in india and china in poultry and pigs (apua newsletter ). the threat of superbugs via food is worldwide, due to the distribution of animal food products from china (zhang et al. ; zhu et al. ) . rosenblatt-farrell ( ) drew upon existing literature to identify additional environmental paths to exposure to antibiotic resistance. veterinary antibiotics are frequently excreted intact from food animals (table . ). for the widely used tetracycline, - % of the antibiotic is excreted in the feces or urine and not metabolized by the food animal. the transfer of this animal waste to croplands may transfer antibiotics and possibly ar pathogens. in one study, ar genes in soil increased fourfold after manure from hog and dairy farms was applied to the soil (moyer ) . runoff from farms, feedlots, or cropland can lead to antimicrobial resistance problems in soil, surface water runoff, and groundwater. animal waste held in lagoons allows birds and insects to become contaminated with antibioticresistant bacteria, and flies around food animal facilities can carry antibioticresistant enteric bacteria which increases potential human exposure. migratory birds and seagulls which become infected with antibiotic-resistant bacteria or viruses can widely transmit resistance to other birds as well as marine life. others note that antibiotics should never be used to compensate for poor hygiene and husbandry practices or conditions in livestock production (van boeckel et al. ) . veterinary medicine should only use antibiotics to treat diagnostically determined bacterial infections not otherwise treatable and only those antibiotics authorized for the diagnosed pathogenic indication and the specific bacteria involved. further, given the potential for acute diseases that require immediate treatment, it is important that routine testing (surveillance) be carried out for farm-specific pathogens for all relevant antibiotic classes (silley and stephan ) . who also emphasizes the need for surveillance and monitoring antimicrobial use in food-producing animals to evaluate the extent to which their guidelines are implemented. fda has increased regulation of antibiotic use in food animals. as noted in sect. . above, fda attempted to withdraw new animal drug approvals for subtherapeutic uses of human medically important penicillins and tetracyclines in animal feed based on lack of evidence to show they were safe. after industry opposition and congressional intervention to require further study, this early policy response was withdrawn. subsequently, the us congress gave something to each group when it enacted the animal drug availability act (adaa) in . this act both table . , fda in recent years issued three core documents to implement a policy framework for judicious use of medically important antimicrobial drugs in food animals: on january , , fda announced that it had completed implementation of the guidance for industry # . this means that medically important antimicrobials provided to food-producing animals may no longer be used for growth promotion purposes and may be used to treat, prevent, or control animal illnesses only under direction of a veterinarian. fda worked with industry participants to implement this voluntary compliance to slow development of antimicrobial resistance and preserve effectiveness of medically important antibiotics. more than percent of new drug applications subject to gfi # were converted from over-the-counter to prescription status, applications were withdrawn, and all applications indicating production use withdrew that specified use. fda also indicated plans to work with industry stakeholders to support antimicrobial stewardship in food animal production and to evaluate the effectiveness of strategies to reduce antimicrobial resistance development under the allowed uses (fda ). some industry stakeholders in the supply chain are actively engaged in responding to consumer and general public health concerns about ar in the food supply chain amidst mounting scientific evidence, but responses vary considerably by country, place in the supply chain, and individual company. aside from farm groups, stakeholders include feed companies, pharmaceutical companies, integrators or meat processors, restaurant chains and other retail outlets, and consumer and other interest groups. pharmaceutical companies. in the case of pharmaceutical companies, little evidence exists that they are responding to the ar problem yet. as described earlier, most antibiotics are produced in india and china, and their production has resulted in significant risk, especially environmental risk. regulators need to set minimum standards for the treatment of manufacturing waste before it is released into the environment. other industries which purchase these pharmaceuticals need to establish higher standards through their supply chains to help correct this environmental pollution (o'neill ). furthermore, the drug companies are not required to compensate victims who become ill or die from either the environmental or food exposure. the drug companies and their trade associations have resisted more regulation to prevent misuse of antibiotics. the companies therefore have been getting a "free ride" at the expense of the ill consumers and the general public. integrators and meat processors. some chains and food retailers have recently responded to customer concerns by restricting the use of antibiotics in their food supply chains. large meat processors committing to judicious use of antibiotics have already led many producers to eliminate the use of antibiotics for production enhancement purposes. in a case study of voluntary labeling in the broiler industry, "raised without antibiotics" (rwa) label claims by tyson foods and by perdue farms in , respectively, numbers one and three in total broiler production, resulted in mixed outcomes. at that time, usda fsis had not published a standard for such claims, nor was a clear definition established. perdue and tyson developed their own standards and submitted the label claims to fsis for approval along with supporting documentation. after initially approving both firms' label claims, fsis determined in september that tyson's claim was false and misleading and gave them the opportunity to submit a revised label claim. however, tyson continued their advertising of the rwa claims. the diverse label claims in which tyson and their competitors were using different standards for their claim resulted in consumer confusion, and eventually court challenges were filed jointly by sanderson farms, the fourth largest producer, and perdue against tyson. the suit was upheld in court in april . tyson was found not to have delivered the rwa attribute promised to the marketplace and to thereby have harmed competitors, while tyson profited from introducing a false and misleading claim. in june , fsis rescinded tyson's qualified rwa label claim and required its removal within weeks, after the claims and advertising had continued for more than a year. the authors found no evidence that the events had any impact on tyson's brand, suggesting that companies may have incentives to introduce misleading label claims since the size of penalties is uncertain (bowman et al. ) . perdue farms inc. was the only major chicken producer to eliminate all medically important and animal-specific antibiotics from use in its chicken production as of . by replacing antibiotics with vaccines and improving its production facilities and practices, it has been able to produce chicken at virtually the same cost as when using antibiotics. perdue estimates that its conventional chicken sales are increasing by not more than % annually, while sales for product raised without antibiotics are growing - % annually (bunge ) . gnp company, a leading provider of premium natural chicken products, is adopting antibiotics-free production of chicken products. its gold'n plump brand will feature a "no antibiotics-ever" claim. this will go well beyond what many companies are currently focusing on-eliminating the use of medically important antibiotics, rather than all antibiotics. usda regulations allow this label claim only for chicken never having received antibiotics, even inside the egg. the company will continue to treat flocks for illness as necessary, but not market them under their premier gold'n plump brand. the company plans extensive media and in-store support to educate consumers about the transition to its chicken products raised totally without antibiotics (gnp company ). tyson foods, a leading producer of chicken, pork, and beef and products thereof, adopted a position to eliminate the use of human-use antibiotics in broiler production by september . they stopped the human antibiotic use in their hatcheries and reduced usage in producing broilers by % since . they also have worked with farmers and others in the beef, pork, and turkey supply chains to explore ways to reduce human antibiotic use at the farm level. tyson is employing alternative husbandry strategies such as use of probiotics and essential oils, improved housing, and selective breeding to offset the potential impact of eliminating the use of the antibiotics. they are also interacting with the food industry and other involved supply chain participants, as well as academics, to increase research on disease prevention and alternatives to replace antibiotics (tyson foods ). feed companies. the feed companies are also getting into the discussion to address public health concerns about antibiotic resistance and the relationship to livestock production uses of antibiotics. phibro animal health corporation recently launched a website animalantibiotics.org to "…provide accurate and credible information while still creating open dialogue about animal agriculture in the use of antibiotics." it will address all issues involving animal antibiotics and changes underway within the industry to promote responsible use of antibiotics in livestock (johansen ) . this is very consistent with the historical pattern of the animal agriculture industry making its case in the political economy in reaction to the strong push to limit use of antibiotics to help quell rising antibiotic resistance of medically important drugs. restaurant chains. an interesting example of restaurant chains and poultry producers working together is provided by panera bread co. and perdue farms inc. panera is one of the restaurant companies for which perdue supplies chickens raised without antibiotics. when panera pioneered antibiotic-free chicken in its restaurant products over years ago, they paid a % premium versus chicken produced using antibiotics. with improved production practices, the cost differential has virtually disappeared (bunge ) and is thus consistent with the ers estimates cited earlier. consumer and other interest groups. in the process of developing these new fda regulations, activist groups petitioned the federal courts. for example, in may , the natural resources defense council (nrdc), center for science in the public interest (cspi), food animal concerns trust (fact), public citizen, and union of concerned scientists (ucs) filed a case against the fda. they charged that fda failed to ban penicillins and tetracyclines used at low doses in animal feed for growth promotion, despite evidence fda put forth in that penicillin and most tetracyclines were not shown to be safe and may pose a risk to human health (apua ). in , the federal court ruled in favor of these petitioners. in a later ruling in , the federal court directed fda to reexamine its decision on five other classes of "medically important drugs" used as growth promoters addressed in two citizen petitions (filed in and ) to ensure the safety and effectiveness of all drugs sold in interstate commerce (ibid). given that most governments have neglected to acknowledge and address the problem of increasing antibiotic resistance, international organizations with a role in health issues have been stymied from doing so. it will take more concerted action by societies around the globe to successfully address this cross-border issue (amábile-cuevas ). us consumers, in general, have much less information about the product than does the seller (chaps. and ). this asymmetric information can offer opportunity for the selling firm behavior that is detrimental to the interests of the consumer, as when a product is labeled as containing or not containing certain desirable or undesirable attributes. in the case of many products known as credence goods, it is impossible to determine whether the attributes are as stated, even when the product is used or consumed. this market failure can be addressed either through government regulations or through voluntary steps by the sellers to assure that the stated attributes are factual. the latter could be accomplished through advertising to build and maintain the firm's brand and reputation, and competition with other sellers could result in consumers having increased variety of product choices. however, some consumers may not trust private companies' word about product attributes and prefer certification programs which monitor products against some standard established either by the private sector or by government agencies. lusk (lusk ) argues that voluntary labels are dynamically efficient in responding to changes in market conditions and encouraging innovation more than mandatory labels implemented through regulations, since the latter are more subject to manipulation by those with vested interests. further, usda's agricultural marketing service (ams) process-verified and certification programs are very effective in helping to assure the credibility of voluntary labeling, while accommodating innovation from the private sector. gnp's adoption of antibiotics-free production discussed in . is an example of dynamic market efficiency through use of voluntary labeling to innovate in response to changing consumer demand usda's food safety inspection service (fsis) currently employs an animal production claims protocol for evaluating and allowing or denying labeling claims. labeling applications must provide supporting documentation such as operational protocols detailing production practices and affidavits or testimonials about production practices. fsis then evaluates whether protocols support the accuracy of the proposed label. also, feed formulations must be provided and reviewed to ensure they do not include substances not permitted by the claim. commonly approved claims relevant to the use of antibiotics include "raised without added hormones" (only allowed for use in beef cattle and lamb production) and "raised without antibiotics." claims not allowed include that animal products are antibiotic-, hormone-, or residue-"free" (fsis ) . given the current trend among meat producers, restaurants, and retail livestock product marketers, it can be anticipated that there will be increasing attention to labeling the lack of antibiotic use for other than therapeutic purposes. this will likely result in animals that have been raised with antibiotics to promote growth and uniformity of size consistent with processor contract agreements being diverted to marketing outlets where such promises do not exist. the impact of labeling in this manner will vary according to how much consumers know about the use of antibiotics in livestock production and their ability to currently purchase antibiotic-free livestock products (lusk et al. ) . o'neill and his british colleagues emphasize improving transparency as a major step in addressing antimicrobial resistance related to the livestock production. recent attention by companies such as food retailers, wholesale producers, and fastfood chains, as well as investors, for reducing antibiotic use in their supply chains, has been in response to consumer pressure. providing greater transparency through voluntary approaches is helpful in the short term, but it may be necessary to mandate transparency requirements about how antibiotics are used in the supply chains to have longer-term impacts. labeling that refers to antibiotic use could improve consumer knowledge to allow them to make better informed decisions. they also argued that third-party validation of support from independent institutions to monitor progress may be beneficial (o'neill ). improved transparency by food producers about antibiotics used in producing meat could help consumers make better informed purchasing decisions. but there are large gaps in data needed to allow monitoring of types and quantities of antibiotics used in animal agriculture and their impacts (cfi ), as well as on emergence and spread of resistance in animals. the who also identified major gaps in surveillance and data sharing on emergence of antibiotic resistance in bacteria and its impact on animal and human health. who called for integrated surveillance systems harmonized across countries to enable better comparison of data from foodproducing animals, food products, and humans (who ). in the united states, fda requires drug companies to voluntarily submit data on drugs sold for use in food animals. the publicly available data are not detailed, and % of the sales of medically important antimicrobials are over-the-counter (otc). tetracyclines are primarily added to feed and accounted for % of domestic sales of animal drugs that are "medically important" to human medicine in (fda b) . from to , domestic sales and distribution of tetracycline products approved for use in food-producing animals increased by %. while levy et al. ( ) discovered how rapidly tetracycline created antibiotic resistance in the gut of chickens, years later, the public does not have access to information on what antibiotics are used in which food animals at what stage of life. this will change somewhat in fda implementation of gfi # (fda ) that will identify whether the sales are intended for use in cattle, sheep, hog, or poultry. fda ( b) issued a final rule amending an existing requirement that sponsors of drug products containing antimicrobial active ingredients report annually the amount of each such ingredient in the drug products sold or distributed for use in food-producing animals. effective july , , drug sponsors were required to submit species-specific estimates of product sales as a percent of their total sales. additional reporting requirements are expected to facilitate better understanding of antimicrobial drug sales for specific food-producing animal species and the relationship between such sales and antimicrobial resistance. as reported above, drug sponsors have all adopted voluntary revision of fda-approved labels for use of new medically important antimicrobial animal drugs administered through feed or water. under this rule, sponsors all voluntarily removed the growth promotion and feed efficiency uses and brought the remaining therapeutic uses under veterinarian oversight by the end of december . the rule makes it illegal to use medically important antibiotics for production purposes. despite the scientific and economic evidence, many comments to the proposed final regulation reflected ongoing resistance to the elimination of food animal production use of medically important antibiotics. data available on antibiotics used in the us livestock industry is derived primarily from two nationally representative surveys of farms conducted by the usda's economic research service (ers) and national agricultural statistics service (nass). the agricultural and resource management survey (arms) is designed to collect information on farm finances, production practices, and resource use focuses on three commodities annually, livestock included. different types of livestock are resurveyed every - years and represent commercial producers in states producing % of production for that livestock type. some questions have been included in these surveys on antibiotic use for hogs and broilers. the hog surveys ask about use of antibiotics in feed or water for growth promotion, disease preven-tion, and/or disease treatment in breeding, nursery, and finishing hogs. given the widespread use of hog production contracts under which farm operators may receive feed from integrators, the surveyed operators may not know if antibiotics are included in it. for broilers, there is only a single question about whether they were raised without antibiotics in feed or water other than for therapeutic treatment of illness. production contracts dominate the broiler industry, so surveyed farm operators are in a similar situation as hog producers in not necessarily knowing whether antibiotics are included in the feed provided. a further complication is that arms does not separate traditional antibiotics and ionophores, which are not used in human medicine (sneeringer et al. ) . the national animal health monitoring system (nahms) consists of national studies to provide essential information on livestock and poultry health and management. major food livestock species are surveyed about every years to provide current and trend information important to industry participants, researchers, and policy makers. each study includes states that represent at least % of the targeted animal population and at least % of the farm operations involved and provides statistically sound information for decision-making. a nahms study is a collaborative, voluntary, confidential, scientifically sound product. descriptive reports are prepared along with information sheets which briefly address very specific topics, such as biosecurity practices (aphis ). the nahms focuses on animal health and management, providing information on disease occurrence and disease prevention practices, as well as more detailed information on antibiotics used in production, including by specific purpose. however, the information collected on antibiotics varies greatly across commodities, as well as over time with the same commodities. further, arms focuses on hog production operations with or more head versus nahms focus on or more head. this complicates comparison of statistics across surveys, assuming smaller operations may have different characteristics than larger ones (sneeringer et al. ) . to track antimicrobial resistance changes over time, the national antimicrobial resistance monitoring system-enteric bacteria (narms) was established by cdc in . the program is a collaboration between state and local public health departments and three federal agencies to monitor changes in antimicrobial susceptibility for certain enteric bacteria from ill people (cdc), retail meats (fda), and food animals (usda) in the united states. it provides information about emergent bacterial resistance, the ways resistance is spread, and how resistant infections differ from susceptible ones (narms ). the world organization for animal health (oie) plans to address antimicrobial resistance as a major risk to the international community, in the face of concern about agriculture's role in increased antimicrobial resistance. the goal is to preserve effectiveness of antimicrobials used in animal medicine, protect animal welfare, and help maintain important antimicrobials for use in human medicine. oie has already developed international standards, most recently revised in . the new strategy introduced at the th oie general session in may outlines plans to help nations improve legal frameworks to preserve antibiotics, communicate about the ar problem, train animal health workers, and monitor antibiotic use in animals. they are currently working to create a database of information on the use of antimicrobial agents in animals and develop performance indicators to assist countries by increasing information flow and transparency in their use of antimicrobials. further, the oie expert network is working to reinforce scientific knowledge about new technologies and replacement solutions for current antimicrobials (mitchell ) . the eu has banned the use of antimicrobials in food animal production, other than by veterinarian prescription for specific therapeutic use. some other countries have adopted similar bans, and, as discussed above, the united states fully implemented voluntary guidelines in requiring current drug sponsors to withdraw antibiotics for growth promotion. however, the animal health institute's carnevale has said that the new fda guidance on antibiotics may not decrease the total quantities of antibiotics used in animal food production (moyer ) . generally, variations among countries in implementing regulations have resulted in the spread of resistance. there is ample evidence to support the need for global coordination to prevent continued spread of antimicrobial resistance, and elements of a framework to make such global coordination effective have been posited (so et al. ) . the uk review on antimicrobial resistance final report proposes three broad steps to deal with reducing unnecessary use of antibiotics in animals. first, establish -year targets for reduction in use, with milestones to support progress consistent with countries' economic development. this could encourage farmers to reduce non-therapeutic use to be able to allocate the resulting reduced amounts of antibiotics to treating sick animals. second, implement restrictions or bans on certain types of highly critical last-line antibiotics for humans from being used in agriculture. this would require a harmonized approach to identify the most important human health antimicrobials across countries and good systems of veterinarian oversight to assure compliance. third, improve transparency from food producers on antibiotic use in meat production to allow consumers to make better informed buying decisions. voluntary industry efforts may be one of the most practical approaches to reduce antibiotic use in the near term, but third-party validation to monitor progress would be beneficial (o'neill ) . generally, voluntary industry approaches require monitoring by an outside party to assure both industry participants and consumers that standards are being met as required. the uk medical research council (mrc) recently made three large grants focused on antimicrobial resistance through an initiative established in to address the growing ar issue. the projects will use new technology to exploit natural compounds, develop a better and faster diagnostics tool, and study how the body's immune system can be harnessed to better fight infections. the goal is not only to develop antibiotics but also explore alternatives to antibiotic use, working with other uk research councils to bring to bear a wide range of disciplines to tackle ar (mrc ). the need to focus increased attention to developing new antibiotics is supported by cdc data which shows that many of the most widely used drugs have developed resistance. the number of years to develop resistance varies greatly but never extends more than a couple of decades, and more recent antibiotic introductions have been resistant for only a year or two. for example, the widely used tetracycline was introduced in and developed resistance to shigella by . this is near the midrange of years to resistance reported (cdc a). given this scientific fact, the slow pace of adopting policies to proscribe use of human-use antibiotics in animals and to encourage greater investment in developing newer antibiotics or alternatives is unacceptable. increasing detection of bacteria resistant to last-resort drugs has driven stakeholders to countenance accelerating government efforts to increase surveillance of drug use and to develop new antibiotics (fda week ). promising approaches which provide more rapid assessment utilizing newer technologies such as genomics are now being utilized by scientists. microbiologists are embracing high-throughput genomics to quickly examine individual organisms or entire microbial communities. a project underway at the university of california, davis, the k foodborne pathogen genome sequencing project, will sequence , foodborne isolates for the most important worldwide foodborne illness outbreak organisms. it involves a consortium of academic, government, and industry to create a massive database of genome signatures for the most significant foodborne disease-causing microbes. the goal is to allow public health agencies and the food industry supply chain to trace any foodborne illness outbreaks to their source. by comparing the pathogen genome to the database which includes millions of pieces of information on previously detected strains, including their exact location, the contamination source will be positively identified. bioinformatics and the analytics involved can be used to turn the vast amount of genomic information into actionable knowledge. these event sequencing approaches will enable new diagnostic and public health approaches to manage foodborne disease to facilitate improved public health. the database will increase ability to detect and mitigate pathogenic organisms in food, the environment, and livestock. that capacity is now constrained by continual genetic evolution of pathogens which hinders the ability to defend the food supply. this project will facilitate speedy testing of raw ingredients and finished products from outbreak investigations with precision and accuracy unparalleled using existing methods of analysis. genomics enabled diagnostics with molecular tools will allow surveillance, risk assessment, and diagnosis of foodborne pathogens directly throughout the global food chain. the result will be a genetic catalog for some of the most important outbreak organisms impacting human health. the database will provide insights into molecular methods of infection and drug resistance for use in creating new vaccines and therapies. and importantly, it will assist in systematic definition of biomarker gene sets associated with antibiotic resistance (weimer ). a recent innovative metagenomics study also provides new insights on possible impacts of antibiotic use in food animal production and ar in humans. the research investigated antimicrobial resistance potential-the resistome-by tracking specific pens of intensively managed cattle from feedlot through slaughter to market-ready beef products. study results found no antibiotic-resistant determinants (ards) in the beef products beyond the slaughter facility. this suggests that intervention during slaughter minimizes potential for antibiotic-resistant determinants passing through the food chain. the results also highlight potential risks through indirect environmental exposures to the feedlot resistome through wastewater runoff, manure application on cropland, and wind-borne particulate matter. the insights provided can be used to better inform future agricultural and public health policy. however, this first of its kind study suggests the scientific community must develop a better understanding of the risk of different resistomes and resistance genes. it also identifies a pressing need to standardize ard nomenclature so that databases and analyses are comparable across studies (noyes et al. ) . the world health organization has recently developed guidelines to mitigate human health consequences from use of medically important antimicrobials in food-producing animals (who ). the guidelines are evidence-based recommendations and include best practices for use of medically important antimicrobials in food-producing animals, especially antimicrobials deemed critically important to human medicine. they also can help preserve effectiveness of antimicrobials for veterinary medicine. the recommendations include: • an overall reduction in use of all classes of medically important antimicrobials in food-producing animals. • complete restriction for use in growth promotion. • complete restriction of use to prevent infectious diseases that have not yet been clinically diagnosed. • antimicrobials designated as critically important for human medicine should not be used to control spread of clinically diagnosed infectious disease identified within a group of food-producing animals, nor for treatment of food-producing animals with a clinically diagnosed infectious disease. • for best practices, any new class of antimicrobials for use in humans will be considered critically important for human medicine unless otherwise categorized by who. further, medically important antimicrobials not currently used in food production should not be so used in the future. these guidelines apply universally, and improved animal health management can be used to reduce the need for antimicrobials including improvements in disease prevention strategies, housing, and husbandry practices as noted in sect . above. economic incentives in regulations were addressed in a recent article. in some european countries, capping total antimicrobial use per animal through regulations has been successful in reducing use by more than half while maintaining competitive livestock sectors. the second option was to impose user fees on veterinary antimicrobials, applied at the point of manufacture or wholesale purchases for imported products, which could also reduce use significantly. as a policy option, some combination of these two strategies would significantly reduce antimicrobial use in food animal production (van boeckel et al. ) . finally, as discussed in chap. , sweden does not allow use of antibiotics in broiler production. if there is the political will, strong regulations can provide strong economic incentives to control antibiotic use. to promote the understanding and implementation of the fda's new veterinary feed directive, the farm foundation and the pew charitable trusts sponsored a series of meetings with livestock and farming communities throughout the united states. twelve educational workshops provided livestock producers, feed suppliers, veterinarians, and support service organizations information and insights on the new policies. the workshops also provided opportunity for participants to interact with fda and usda's animal and plant health inspection service (aphis) personnel about implementation challenges. among livestock producers attending the workshops, small-and medium-sized operators, as well as many veterinarians, were unaware of the pending requirements. lack of understanding about responsibilities under the revised vfd rule means that producers and veterinarians need education. some land grant university extension services are now offering balanced education programs to inform these audiences about their obligations going forward, rather than having interested parties in the food animal industry be the primary source of information to producers and veterinarians about the requirements. while seeing positives of improved public perception and livestock management as result of the new rules, workshop participants were concerned about increased costs in animal health due to restrictions on access to antibiotics and lack of veterinary services. perhaps the biggest challenge is that many small producers do not have established relationships with veterinarians needed to establish a veterinarianclient-patient relationship (vcpr). this may be particularly challenging in remote rural and urban fringe areas where fewer veterinarians are available to treat foodproducing animals. in sum, workshop participants saw a need for education and outreach; continuing dialogue between industry representatives, consumers, and state or federal regulators; and the need to provide better access to veterinary services for food animals. there is widespread agreement that the scientific evidence indicates a global human health and environmental crisis due to antibiotic resistance, in part resulting from production practices in animal agriculture. government action in regulating the animal agriculture industry, to date, has done little to slow the advance of ar. most countries still need to pass legislation to establish appropriate conditions for the import, manufacture, distribution, and use of veterinary products, including antimicrobial agents. continued easy access to antimicrobial drugs for use on the farm is not acceptable. important stakeholders in the animal production industry include pharmaceutical companies, feed companies, livestock production integrators, and some farm groups, each with their own set of incentives and supporters. they must be engaged in the effort to reduce agriculture's role in contributing to development of ar, which cdc estimates at % (fig. . ) . even so, other major industry groups must be engaged to significantly reduce their % contribution to resistance development. in the united states, some progress was made with the passage of the animal drug availability act in and its very gradual implementation through various regulations over the past two decades. however, there are serious gaps in these regulations. given the gridlock that has prevailed in the us congress and the power of the pharmaceutical lobby at the national level, state actions are leading the way to responsive regulation in the public interest. for example, california is the first us state to prohibit all human antibiotic use in food animal production. in contrast to the halting actions of governments and industry, consumer and interest group actions are being at least partially successful in getting fast-food and retail establishments to not market animals fed human-use antibiotics for growthpromoting purposes. this suggests that a productive approach may be finding ways to provide information to and educate consumers about the risks of antibiotic resistance to enable them to make better informed decisions. there is an important role for educators to extend scientific information in a nontechnical way to the lay public. the drive to use antibiotics more responsibly and in the public interest may be facilitated by recent economic results that show that reducing antibiotic use in animal production need not come at a significant economic cost to producers or consumers. since the benefits of using antibiotics for livestock growth promotion appear to have resulted in increasingly smaller productivity gains, independent producers where input mix is a farmer-driven choice based on farm-level economics may be better off to substitute good management practices rather than using antibiotics for prophylactic disease prevention. however, much of meat animal production on us farms is produced under contract, where the integrator provides inputs, often including antibiotics, that the grower is required by contract to use. recent actions by integrators and meat processors to reduce antibiotic use and substitute alternative strategies to protect animals from diseases and maintain productivity are an important development, especially since production-purpose use of antibiotics is now prohibited. presuming that any new human-use antibiotic classes will probably not be made available for veterinary medicine, it is important to preserve the effectiveness of existing antibiotics. some policy makers and industry now recognize the urgency to identify new antibiotics. this will require increased antibiotic research funding and judicious use of existing antibiotics. the ban of human-use antibiotics in animals for production purposes is expected to help slow the growth of antimicrobial resistance, giving more time to discover new antibiotics for animal uses and for human health uses. to the extent they can be developed and used separately, the potential for animal antibiotic use leading to antimicrobial resistance for important human antibiotics will be mitigated. the ban on antibiotics used for humans also being used for animal production purposes will necessitate adopting improved cultural practices to reduce the poten-tial for disease and to increase feed efficiency. this calls for research on best management practices to accommodate today's supply chain requirements for food safety, production efficiency, and attribute verification. moreover, there is a need to educate producers-for example, through the usda-state cooperative extension service-about safe production practices for managing ar. this will allow producers to maintain efficiency in their operations and assure that they comply with current regulations to address the growing concern about antimicrobial resistance in the food supply chain. improved data collection and analysis to allow tracking of potential antimicrobial resistance development are essential to facilitate the food animal industry implementation of cultural practices to reduce the potential for contributing to antimicrobial resistance. it would also allow policy makers to better understand the need for any necessary interventions. these investments in the public good can be very cost-effective, though not without additional public investment or internal agency budget reallocation. increasing international trade may spread antibiotic resistance through imported food products as more trade agreements are approved. this scenario could be exacerbated to the extent fsis approves additional international facilities, local regulations, and inspections as "equivalent to the united states." in many developed and developing countries, antimicrobial agents are readily available. policies need to be implemented establishing appropriate conditions for use of veterinary products, including antimicrobial agents. future trade agreements will need to include provisions which address reduced use of medically important antibiotics in producing food animals. to date, there is no harmonized system of surveillance on the worldwide use and circulation of antimicrobial agents. that information is necessary to monitor and control the origin of medicines, obtain reliable data on imports, trace their circulation, and evaluate the quality of the products in circulation. the oie initiative to create a global database for monitoring the use of antimicrobial agents is an important step that can provide valuable information for private sector and public policy leaders worldwide. the serious implications of growing antibiotic resistance require a concerted effort across all stakeholders and society generally. increased attention to this issue is emerging in the medical community where overuse of existing drugs and inadequate sanitary precautions account for % of the resistance. lack of development of new antibiotics exacerbates the problem, and industry focus and perhaps government policy are needed to improve this situation. animal agriculture stakeholders need to improve production practices to reverse the other % of resistance attributable to foodborne sources. government policy and agencies have been slow to acknowledge the seriousness of antibiotic resistance and appropriately address it. public and private sector collaboration internationally is necessary to successfully deal with this critical societal issue. changes in the measurement of antibiotics and in evaluating their impacts in agroecosystems: a critical review society must seize control of the antibiotics crisis animal and plant health inspection service, united states department of agriculture apua. major developments in u.s. policy on antibiotic use in food animals. alliance for the prudent use of antibiotics farm antibiotic use remains worrisome in india. alliance for the prudent use of antibiotics raised without antibiotics: lessons from voluntary labeling of antibiotic use practices in the broiler industry perdue chickens now free of antibiotics antibiotic resistance and the use of antibiotics in animal agriculture: hearing before the subcommittee on health of the house committee on energy & commerce across the divide: the importance of antibiotics for animal health antibiotic resistance threats in the united states estimates of foodborne illness in the united states. centers for disease control and prevention tracking animal antibiotic use in food animals. the center for foodborne illness research and prevention america's antibiotic crisis council for agricultural science and technology (cast). . antibiotics in animal feeds the judicious use of medically important antimicrobial drugs in food-producing animals. guidance for industry # summary report on antimicrobials sold or distributed for use in foodproducing animals anti-microbial animal drug sales and distribution reporting. fda, department of health and human services fda announces implementation of gfi # , outlines continuing efforts to address antimicrobial resistance stakeholders: colistin-resistance shows need for congressional action animal production claims: outline of current process. fsis, u.s. department of agriculture gnp company. gold'n plump to add 'no antibiotics-ever' and humane certified attributes. . perishable news.com report of a study: human health risks with the subtherapeutic use of penicillin or tetracyclines in animal feed. committee on human health accurate, credible info on animal antibiotics. phibro animal health report presented to parliament by the secretary of state for social services, the secretary of state for scotland, the minister of agriculture, fisheries and food and the secretary of state for wales by command of her majesty antibiotic resistance in india: drivers and opportunities for action changes in intestinal flora of farm personnel after introduction of a tetracycline-supplemented feed on a farm consumer information and labeling consumer demand for a ban on antibiotic drug use in pork production foregoing sub-therapeutic antibiotics: the impact on broiler grow-out operations pressure rises to stop antibiotics agriculture antibiotic discussions intensify in wdc. pork network world animal health organization sets out action on antibiotic resistance. poultry news the looming threat of factory-farm superbugs mrc announces cross-council awards worth nearly £ m to tackle antibiotic resistance department of health and human services, cdc united states summary and state data the effects on human health of subtherapeutic use of antimicrobial drugs in animal feeds. committee to study the human health effects of subtherapeutic antibiotic use in animal feeds resistome diversity in cattle and the environment decreases during beef production tackling drug-resistant infections globally: final report and recommendations. the review on antimicrobial resistance world organization for animal health merchants of doubt: how a handful of scientists obscured the truth on issues from tobacco smoke to global warming the landscape of antibiotic resistance prepared for united states department of health and human services, public health service, food and drug administration, bureau of veterinary medicine prudent use and regulatory guidelines for veterinary antibiotics-politics or science? economics of antibiotic use in u.s. livestock production an integrated systems approach is needed to ensure the sustainability of antibiotic effectiveness for both humans and animals restricting the use of antibiotics in food-producing animals and its associations with antibiotic resistance in food-producing animals and human beings: a systematic review and meta-analysis economics of antibiotic use in u.s. swine and poultry production position statements: antibiotic use scientific integrity in policymaking: an investigation into the bush administration's misuse of science insights: reducing antimicrobial use in food animals veterinary feed directive a rule by the food and drug administration on / / . illustrative examples of probable transfer of resistance determinants from food animals to humans: streptothricins, glycopeptides, and colistin weimer bc k genome project . veterinary medicine, uc davis antimicrobial resistance: global report on surveillance. summary. world health organization who guidelines on use of medically important antimicrobials in food-producing animals. geneva: world health organization ctx-m- producing salmonella enterica serotypes typhimurium and indiana are prevalent among food-producing animals in china diverse and abundant antibiotic resistance genes in chinese swine farms political economy of biofuel acknowledgments we deeply appreciated the conversations with and review comments from mary ahearn in developing and writing this chapter. any remaining errors, mistakes, or omissions are of course ours. w. j. armbruster and t. roberts key: cord- -kssjdn y authors: niemann, heiner; kues, wilfried; carnwath, joseph w. title: transgenic farm animals: current status and perspectives for agriculture and biomedicine date: journal: genetic engineering in livestock doi: . / - - - - _ sha: doc_id: cord_uid: kssjdn y the first transgenic livestock were produced in by microinjection of foreign dna into zygotic pronuclei. this was the method of choice for more than years, but more efficient protocols are now available, based on somatic cell nuclear transfer (scnt) which permits targeted genetic modifications. although the efficiency of transgenic animal production by microinjection technology is low, many animals with agriculturally important transgenic traits were produced. typical applications included improved carcass composition, lactational performance, and wool production as well as enhanced disease resistance and reduced environmental impact. transgenic animal production for biomedical applications has found broad acceptance. in the european medicines agency (emea) approved the commercialization of the first recombinant protein drug produced by transgenic animals. recombinant antithrombin iii, produced in the mammary gland of transgenic goats, was launched as atryn® for prophylactic treatment of patients with congenital antithrombin deficiency. pigs expressing human immunomodulatory genes have contributed to significant progress in xenotransplantation research with survival periods of non-human primates receiving transgenic porcine hearts or kidneys approaching six months. lentiviral vectors and small interfering ribonucleic acid (sirna) technology are also emerging as important tools for transgenesis. as the genome sequencing projects for various farm animal species progress, it has become increasingly practical to target the removal or modification of individual genes. we anticipate that this approach to animal breeding will be instrumental in meeting global challenges in agricultural production in the future and will open new horizons in biomedicine. the first transgenic livestock were produced in by microinjection of foreign dna into zygotic pronuclei. this was the method of choice for more than years, but more efficient protocols are now available, based on somatic cell nuclear transfer (scnt) which permits targeted genetic modifications. although the efficiency of transgenic animal production by microinjection technology is low, many animals with agriculturally important transgenic traits were produced. ty pical applications included improved carcass composition, lactational performance, and wool production as well as enhanced disease resistance and reduced environmental impact. transgenic animal production for biomedical applications has found broad acceptance. in the european medicines agency (emea) approved the commercialization of the first recombinant protein drug produced by transgenic animals. recombinant antithrombin iii, produced in the mammary gland of transgenic goats, was launched as atryn ® forp rophylactic treatment of patients with congenital antithrombin deficiency. pigs expressing human immunomodulatory genes have contributed to significant progress in xenotransplantation research with survival periods of non-human primates receiving transgenic porcine hearts or kidneys approaching six months. lentiviral vectors and small interfering ribonucleic acid (sirna) technology are also emerging as important tools for transgenesis. as the genome sequencing projects for various farm animal species progress, it has become increasingly practical to target the removal or modification of individual genes. we anticipate that this approach to animal breeding will be instrumental in meeting global challenges in agricultural production in the future and will open new horizons in biomedicine. the production of transgenic farm animals is extraordinarily labor and cost intensive and depends upon advanced techniques in molecular biology, this contribution is mainly based on the following reviews of the authors: niemann et al. ; cell culture, reproductive biology and biochemistry. the transfer of the foreign dna is only one step in this process. critical steps involved in the production of transgenic farm animals are: milestones (live offspring) in transgenesis and somatic cloning in farm animals. modified from niemann et al. . milestones trategyr eference first transgenic sheep and pigs microinjection of dna into one pronucleus of a zygote hammer et al. hammer et al. embryonic cloning of sheep nuclear transfer using embryonic cells as donor cells willadsen willadsen cloning of sheep with somatic donor cells nuclear transfer using adult somatic donor cells wilmut et al. wilmut et al. transgenic sheep produced by nuclear transfer random integration of the construct schnieke et al. schnieke et al. transgenic cattle produced from fetal fibroblasts and nuclear transfer random integration of the construct cibelli et al. cibelli et al. generation of transgenic cattle by mmlv injection infection of oocytes with helper viruses chan et al. gene targeting in sheepg ene replacement and nuclear transfer mccreath et al. mccreath et al. trans-chromosomal cattle artificial chromosome kuroiwa et al. kuroiwa et al. knockout in pigs heterozygous knock-out dai et al. ; lai et al. lai et al. homozygous gene knockout in pigs homozygous knock-out phelps et al. the first successful gene transfer method in animals (mouse) was based on the microinjection of foreign dna into zygotic pronuclei. this was used to produce the first transgenic livestock more than years ago (hammer et al. ) . despite the inherent inefficiency of microinjection technology, a broad spectrum of genetically modified large animals has been generated since then for applications in agriculture and biomedicine, with the use of transgenic livestock for 'gene pharming' already at the level of commercial exploitation . however, microinjection has several major shortcomings including low efficiency, random integration and variable expression patterns which mainly reflect the site of integration. research has focused on the development of alternate methodologies for improving the efficiency and reducing the cost of generating transgenic livestock. these include sperm mediated dna transfer (lavitrano et al. ; lavitrano et al. ; chang et al. ) , intracytoplasmic injection (icsi) of sperm heads carrying foreign dna (perry et al. ; perry et al. ) , injection or infection of oocytes and/or embryos by different types of viral vectors (haskell and bowen ; chan et al. ; hofmann et al. ) , rna interference technology (rnai) (clark and whitelaw ) and the use of somatic cell nuclear transfer (scnt) cibelli et al. ; baguisi et al. ; dai et al. ; lai et al. ; table ) . to date, somatic cell nuclear transfer, which has been successful in species, holds the greatest promise for significant improvements in the generation of transgenic livestock (figure ). the typical success rate (live births) of mammalian somatic nuclear transfer is low and usually is only - % of the transferred embryos. cattle seem to be an exception to this rule as levels of - % can be reached . recently, we have also obtained a significant improvement of porcine cloning efficiency by better selection and optimized treatment of the recipients, specifically by providing a h asynchrony between the pre-ovulatory oviducts of the recipients and the reconstructed embryos. presumably, this gives the embryos additional time to achieve the necessary level of nuclear reprogramming. the improved protocol has resulted in pregnancy rates of~ % and only a slightly reduced mean litter size ). these results show that the efficiency of scnt is likely to be improved in the near future with significant impact on transgenic animal production. further qualitative improvements may be derived from technologies that allow precise modifications of the genome including targeted chromosomal integration by site-specific dna recombinases, such as cre or flp, or methods that allow temporally and/or spatially controlled transgene expression (capecchi ; kilby et al. ) . the genomes of farm animals (cattle, chicken, horse, dog, bee) have been sequenced and annotated in http://www.ensembl.org and http://www.ncbi.org (both july ). they, thus, provide new opportunities for selective breeding and transgenic animal production. after , years of domestic animal selection (copley et al. ) based on the random mutations resulting from environmental factors such as radiation and oxidative injury, technology is now available to introduce or remove genes with known functions. here, we provide a comprehensive overview on the current status of transgenic animal production and look at future implications. we focus on large domestic species and do not cover recent developments in poultry breeding or in aquaculture. gene 'pharming' entails the producti on of recombinant pharmaceutically active human proteins in the mammary gland or blood of transgenic animals. this technology overcomes the limitations of conventional and recombinant dna based production systems rudolph ) and has advanced to the stage of commercial application (ziomek ; dyck et al. ; schnieke this proceedings and walsh this proceedings) . the mammary gland is the preferred production site mainly because of the quantities of protein that can be produced in this organ using mammary gland specific promoter elements and established methods for extraction and purification of the respective protein rudolph ) . guidelines developed by the food and drug administration (fda) of the usa require monitoring the animals' health in a specific pathogen free (spf) facility, sequence validation of the gene construct, characterization of the isolated recombinant protein, and monitoring the genetic stability of the transgenic animals over several generations. this has necessitated, for example, the use of animals from scrapie free countries (new zealand) and maintenance of production animals under strict hygienic conditions. several products derived from the mammary glands of transgenic goats and sheep have progressed to advanced clinical trials (echelard et al. ) . phase iii trials for antithrombin iii (atiii) (atryn ® from gtc-biotherapeutics, usa), produced in the mammary gland of transgenic goats, have been completed and the recombinant product was approved as drug by the european medicines agency (emea) in august . this protein is the first product from a transgenic farm animal to be accepted as a fully registered drug. atryn ® is registered for treatment of heparin resistant patients undergoing cardiopulmonary bypass procedures. gtc-biotherapeutics has also expressed at least eleven other transgenic proteins in the mammary gland of transgenic goats at concentrations of more than one gram per liter. the enzyme α-glucosidase (pharming bv) from the milk of transgenic rabbits has orphan drug status and has been successfully used for the treatment of pompe's disease (van den hout et al. ) . similarly, recombinant c inhibitor (pharming bv), produced in the milk of transgenic rabbits, has completed phase iii trials and is expected to receive registration in the near future. the overall global market for recombinant proteins from domestic animals is expected to exceed $ b illion in and to reach $ . billion in . an interesting new development is the production of recombinant proteins in the mammary gland of transgenic animals for use as antidotes against organophosphorus compounds used as insecticides in agriculture and chemical warfare. butyrylcholinesterase is a potent prophylactic agent against these compounds. recombinant butyrylcholinesterase has been produced at a concentration of g/liter in the mammary gland of transgenic mice and goats (huang et al. ). the recombinant product was biologically active and had a half life in vitro which was sufficient to provide protection against organophophorus intoxication. transgenic goats can produce sufficient butyrylcholinesterase to protect all humans at risk of organophosphorus poisoning. some gene constructs have failed to produce economically significant amounts of protein in the milk of transgenic animals indicating that the technology needs further refinement to insure consistent high-level expression. this is particularly true for genes having complex regulation, such as those coding for erythropoietin (epo) or human clotting factor viii (hfviii) (hyttinen et al. ; massoud et al. ; niemann et al. ) . with the advent of transgenic plants that also produce pharmacologically active proteins, there is now an array of recombinant technologies that will allow selection of an appropriate production system for each required protein . the use of somatic nuclear transfer will accelerate production of transgenic animals for mammary gland specific synthesis of recombinant proteins. numerous monoclonal antibodies are being produced in the mammary gland of transgenic goats ) and cloned transgenic cattle have been created which produce a recombinant bi-specific antibody in their blood (grosse-hovest et al. ). when purified from serum, this antibody is stable and mediates target-cell restricted t cell stimulation and tumor cell killing. an interesting new development is the generation of trans-chromosomal animals. a human artificial chromosome (hac), containing the complete sequences of the human immunoglobulin heavy and light chain loci, has been introduced into bovine fibroblasts, which were then used in nuclear transfer. trans-chromosomal bovine offspring were obtained that expressed human immunoglobulin in their blood. this system is a significant step forward in the production of therapeutic human polyclonal antibodies (kuroiwa et al. ) . follow-up studies showed that the hac was maintained in most animals for several years in first generation cattle (robl et al. ). how the hacs behave during meiotic cell divisions remains to be shown. functional human hemoglobin has been produced in transgenic swine. the transgenic protein could be purified from the porcine blood and showed oxygen bindingcharacteristics similartonatural humanhemoglobin. the main obstacle was that only a small proportion of porcine red blood cells contained the human form of hemoglobin (swanson ) . alternate approaches to produce human blood substitutes have focused on linking hemoglobin to the superoxide-dismutase system (d'a gnillo and chang ). today more than , people are alive only because of a successful human organ transplantation (allotransplantation). ironically, the success of organ transplantation technology has led to an acute shortage of appropriate organs, because cadaveric and live organ donation falls far short of meeting the demand in western societies. to close the growing gap between demand and availability of appropriate organs, transplant surgeons are now considering the use of xenografts from domesticated pigs (platt and lin ; kues and niemann ; yang and sykes ) . prerequisites for successful xenotransplantation are: (i) overcoming the immunological hurdles, (ii) preventing the transmission of pathogens from the donoranimal to the human recipient, and (iii) compatibility of donor organs with human physiology. with a discordant donor species such as the pig, it is necessary to overcome both hyperacute rejection (har) and acute vascular rejection (avr). the two strategies that have been successfully explored for long term suppression of the har of porcine xenografts are: i) transgenic synthesis of human proteins regulating complement activity (rcas) in the donor organ (cozzi and white ; bach ; platt and lin ) and ii) inactivation of the genes producing antigenic structures on the surface of the donor organ, e.g. the α-gal-epitope (dai et al. ; lai et al. ; phelps et al. ) . prolonged survival of xenotransplanted porcine organs where the , -α-galactosyltransferase (α-gal) gene has been knocked out has been demonstrated. survival rates of up to six months have been achieved with transplanted porcine hearts (kuwaki et al. ) and survival of up to three months has been obtained with kidneys transplanted from α-gal knockout pigs to baboons (yamada et al. ) . the current approach to increasing survival time beyond six months is to create donor pigs with multiple transgenes that block a range of additional immunological barriers. to this end, we have recently produced triple transgenic pigs expressing either human thrombomodulin (htm) or human heme oxygenase- (hho- ) on top of one or two rcas to suppress both har and the later stage coagulatory disorders observed in experimental porcine-to-primate xenotransplantation . reproducible survival of porcine xenografts for more than six months in non-human primate recipients is considered to be a necessary precondition to starting clinical trials with human patients. a particularly promising strategy for achieving long-term xenograft survival is to induce tolerance by creating permanent chimerism in the recipient by intraportal injection of embryonic stem cells (fändrich et al. ) or by co-transplantation of vascularized thymic tissue (yamada et al. ) . long term tolerance of hla-mismatched kidneys has recently been demonstrated in humans (kawai et al. ) . extensive research has revealed that the risk of porcine endogenous retrovirus (perv) transmission to human patients is low, opening the door for preclinical testing of xenografts (switzer et al. ; irgang et al. ) . rna interference (rnai) is a promising method for knocking down the already low level of perv expression in porcine somatic cells. using rnai mediated knockdown, perv expression has been further reduced in porcine somatic cells for - months, these cells were successfully used in scnt and gave normal piglets (dieckhoff et al. ; dieckhoff et al. ) . rnai mediated perv expression knockdown provides an additional level of safety for porcine-to-human xenotransplantation. although additional refinements will always be possible, it is expected that appropriatelines of transgenic pigs will be availableasorgan donors within thenextfivetoten years. transplantationofpancreaticisletsfrom (transgenic) pigs may take place even earlier. guidelines for the clinical application of porcine xenotransplants already exist in the usa and are currently being developed in other countries. the worldwide consensus is that thet echnologyi se thically acceptable provided that thei ndividual's well-being does not compromise public health (e.g., the risk of perv recombination).t he improvement in qualityo fl ifef or patients receivingc onventionala llotransplants is dramatic,b ut xenotransplantation is also economically attractive because thecostofmaintaining patients with severe kidney diseaseondialysisorlongtermtreatment of patients with chronich eart diseasec an be greater than thec osto fas uccessfult ransplantation.preliminary functional data on porcinekidneys andheartsin non-humanp rimatesi sp romising although thel ongt erme ffecto fp orcine organs on humanp hysiologyi st oag reat extent unexplored (ibrahim et al. ). the physiology, anatomy, and life span of mice differ significantly from humans, making the rodent model inappropriate for many human diseases farm animals, such as pigs, sheep or cattle, may be more appropriate models in which to study the treatment of human diseases such as artherosclerosis, non-insulin-dependent diabetes, cystic fibrosis, cancer and neuro-degenerative disorders, which require longer periods of observation than is possible with mice (theuringe ta l. ;p almarini and fan ; li and engelhardt ; hansen and khanna ) . cardiovascular disease is increasing in ageing western societies where coronary artery diseases already account for the majority of deaths. because genetically modified mice do not manifest myocardial infarction or stroke as ar esulto fa therosclerosis,n ew animal models, sucha sp igsthate xhibit similar pathologies, are needed to develop effective therapeutic strategies (rapacz and hasler-rapacz ; grunwald et al. ). an important porcine model has been developed for the rare human eye disease retinitis pigmentosa (pr) (pettersetal. ). patientswithp rsufferfromnight blindness earlyinlifedue to loss of photoreceptors.t ransgenicpigswith am utated rhodopsing eneh aveaphenotypeq uite similart othe human patients and effective treatments are being developed (mahmoud et al. ). an important aspect of scnt derived large animal models of human diseases (and the development of regenerative therapies using these models) is that somatic cloning per se does not necessarily result in shortened telomeres as once feared and thus does not necessarily lead to premature ageing (schätzlein and rudolph ) . telomeres are the repetitive dna sequences at the ends of the chromosomes and are crucial for their structural integrity and function and are thought to be related to lifespan. telomere shortening is correlated with severe limitation of the regenerative capacity of cells, the onset of cancer, ageing and chronic dis-ease with significant impact on human lifespan (schätzlein and rudolph ) . expression of telomerase, which is the enzyme primarily responsiblefor theformation andrebuildingoftelomeres,issuppressed in most somatic tissues postnatally. however, recent studies have revealed that telomerel engthi s( re-)establishede arly in preimplantationd evelopment at themorula-blastocyst transition duetotelomeraseactivity ( schätzlein et al. ). agricultural exploitation of transgenic animal technology lags behind applications in biomedicine . nevertheless, table gives an overview of work in the production of animals transgenic with improved agricultural traits. transgenic pigs bearing a hmt-pgh construct (human metallothionein promoter driving the porcine growth hormone gene) showed significant improvement in economically important traits including growth rate, feed conversion and body composition (muscle/fat ratio) without the pathological phenotype seen with earlier gh constructs nottle et al. ) . similarly, transgenic pigs carrying the human insulin-like growth factor-i gene (higf-i) had~ % larger loin mass,~ % more carcass lean tissue and~ % less total carcass fat (pursel et al. ) . unfortunately, commercialization of these pigs has been postponed due to the current lack of public acceptance of genetically modified foods. an important step towards the production of more healthful pork products was made by creating pigs with a desaturase gene, derived either from spinach or from caenorhabditis elegans, which increases the non-saturated fatty acid content in the skeletal muscles of these animals. the higher ratio of unsaturated to saturated fatty acids means more healthful pork, since it is well known that a diet rich in non-saturated fatty acids is associated with a reduced risk of stroke and coronary diseases in humans (niemann ; saeki et al. ; lai et al. ) . the physicochemical properties of milk are mainly due to the ratio of casein variants, making these a prime target for the improvement of milk composition. dairy production is an attractive field for targeted genetic modification (yom andb remel ; karatzas andt urner ) andi t is possible to produce milk with a modified lipid composition by modulation of the enzymes involved in lipid metabolism and to increase curd and cheese yield by enhancing expression of the casein gene family in the mammary gland. the bovine casein ratio has already been altered by the over-expression of beta-and kappa-casein, demonstrating the potential of transgenic technology for improving the economic value of bovine milk (brophye tal. ). it should also be possible to create 'hypoallergenic' milk by knocking out or knocking down the β-lactoglobulin gene. one could envision the production of enhanced 'infant milk' containingh uman lactoferrino rt he production of milk whichr esists bacterial contamination by expressing lysozyme, the antibacterial component of egg white and human tears. to generate lactose-free milk, a knockout or knockdown at the α-lactalbumin locus would suppress this key step in milk sugar synthesis. lactose reduced or lactose-free milk would render dairy products suitable forconsumption by thelarge proportion of theworld's adult population who do not produce an active intestinal lactase. lactose is themajor osmoticallyactivesubstance in milk andits absencemight be expected to interfere with milk secretion. however, a lactase construct has been tested in the mammary gland of transgenic mice and in hemizygous mice; this reduced lactose content by - % without altering milk secretion (jost et al. ). on the other hand, experimental transgenic mice with a homozygous knockout for α-lactalbumin could not nurse their offspring because of the high viscosity of their milk (stinnakre et al. ). in the pig, increased transgenic expression of a bovine lactalbumin construct in the mammary gland resulted in increased lactose content and increased milk production which resulted in improved survival and development of the piglets (wheeler et al. ) . increased survival of piglets at weaning would provide significant commercial benefits to the producer and improved animal welfare. these findings demonstrate the feasibility of producing significant alterations in milk composition by application of an appropriate transgenic strategy. transgenic sheep carrying a keratin-igf-i construct expressed in their skin produced . % more clear fleece than non-transgenic controls and no adverse effects on health or reproduction were observed (damak et al. a, b) . similar efforts to alter wool production by transgenic modification of the cystein pathway have met with more limited success, although it is known that cystein is the rate limiting biochemical factor for wool growth (ward ) . phytase transgenic pigs have been developed to address the problem of manure-related environmental pollution. these pigs carry a bacterial phytase gene under transcriptional control of a salivary gland specific promoter, which allows the pigs to digest plant phytate. without the bacterial enzyme, phytate passes through the animal undigested and pollutes the environment with phosphorus if uncontained. with the bacterial enzyme, fecal phosphorus output was reduced up to % (golovan et al. ) . these environmentally friendly pigs may be used for commercial production in canada within the next few years. in most cases, susceptibility to pathogens represent the interplay of numerous genes, i.e. the trait is polygenic in nature. however, some genetic loci are known to confer resistance against specific diseases. transgenic strategies to enhance disease resistance include the transfer of major histocompatibility-complex (mhc) genes, t cell receptor genes, immunoglobulin genes, genes that affect lymphokines, or specific disease resistance genes (müller and brem ) . a prominent example for a specific disease resistance gene is the murine mx-gene. production of the mx -protein is induced by interferon. this was discovered in inbred mouse strains that were resistant to influenza viruses (staeheli ) . microinjection of an interferonand virus-inducible mx-construct into porcine zygotes resulted in two transgenic pig lines which expressed the mx-mrna; but no mx protein was detected (müller et al. ). the bovine mxi gene was identified and shown to confer antiviral activity when transfected into in vero cells (baise et al. ). transgenic constructs bearing the immunoglobulin-a (iga) gene have been successfully introduced into pigs, sheep and mice in an attempt to increase resistance against infections (lo et al. ) . expression of the murine iga gene was successful in two transgenic pig lines but only the light chains could be detected and the iga-molecules showed only marginal binding to phosphorylcholine (lo et al. ). on the other hand, high levels of monoclonal murine antibodies with a high binding affinity for their specific antigen have been produced in transgenic pigs (weidle et al. ) . attempts to increase ovine resistance to visna virus infection by transgenic production of visna envelope protein have been reported (clements et al. ). the transgenic sheep developed normally and expressed the viral gene without pathological side effects. however, the transgene was not expressed in monocytes, the target cells of the viral infection, and antibodies were detected after artificial infection of the transgenic animals (clements et al. ) . passive immunity has been induced against an economically important porcine disease in a transgenic mouse model (castilla et al. ). these transgenic mice secrete a recombinant antibody in their milk that neutralized the corona virus responsible for transmissible gastroenteritis (tgev) and this conferred resistance to tgev. strong mammary gland specific expression was achieved over the entire period of lactation. extension of this work to pigs is promising. knockout of the prion protein is the only secure way to prevent infection and transmission of spongiform encephalopathies including scrapie and bse (weissmann et al. ) . it was possible to knock out the ovine prion locus; however, the cloned lambs carrying the knockout locus died shortly after birth (denning et al. ). on the other hand, cloned cattle with a knockout for the prion locus have been successfully produced and indeed show clear evidence of resistance to bse infection (richt et al. ). transgenic animals with modified prion genes will be an appropriate model for studying the development of spongiform encephalopathies in humans and are crucial for developing strategies for the elimination of prion carriers from the farm animal population. this work is a prerequisite for the future production of recombinant proteins for human medicine in the blood or the mammary glands of transgenic cattle. the level of anti-microbial peptides (lysozyme and lactoferrin) in human milk is many times higher than in bovine milk and transgenic expression of the human lysozyme gene in mice causes a significant reduction in bacterial contamination and a reduced frequency of mammary gland infections . lactoferrin has bactericidal and bacteriostatic effects in addition to being the main source of iron in milk. these properties make an increase in lactoferrin levels in the bovine mammary gland and are a practical way to improve milk quality. human lactoferrin has, in fact, been expressed at high levels in the milk of transgenic mice and cattle (krimpenfort et al. ; platenburg et al. ) and was associated with an increased resistance against mammary gland diseases (van berkel et al. ) . similarly, lysostaphin was shown to confer specific resistance against mastitis caused by staphylococcus aureus. mastitis resistant cows have been produced by expressing a lysostaphin gene construct in the mammary gland (wall et al. ) . as discussed above, most work in transgenic technology has focussed on livestock species either for biomedical or agricultural purposes. however, the methodology is becoming routine and recent applications include the development of new varieties of ornamental fish. for example, fluorescent green transgenic medaka (oryzias latipes, rice fish) have been produced and approved for sale in taiwan (chou et al. ) . the fluorescent medaka is currently marketed by the taiwanese company taikong. the "glofish ® " is a trademarked transgenic zebra fish (danio rerio) expressing red fluorescent protein from a sea anemone under the transcriptional control of a muscle-specific promoter (gong et al. ) . green and yellow fluores-cent proteins have also been introduced into the zebra fish to give different fluorescence colors. yorktown technologies (www.glofish.com, july ) initiated commercial sales of the transgenic zebrafish in the united states with retail prices of approximately $ , each. the glofish ® thus became the first transgenic animal freely distributed throughout the usa. a recent report of the fda contained no evidence that glofish ® represents a risk (us fda, ) . commercialization of fluorescent fish has gone forward in several countries other than the usa, including taiwan, malaysia, and hongkong, whereas marketing in australia, canada and european union is currently prohibited. transgenic applications will undoubtedly become more widespread if even more efficient gene transfer methods will be developed and specific genetic traits can be targeted. some of the emerging technologies are described below. lentiviruses have proven to be efficient vectors for the delivery of genes into oocytes and zygotes. for example, transgenic cattle have been produced by injecting lentiviral vectors into the perivitelline space of oocytes (hofmann et al. ) and injection of lentiviruses into the perivitelline space of porcine zygotes resulted in a high proportion of transgene expressing founder animals from which several lines of transgenic pigs were established (hofmann et al. ; whitelaw et al. ) . lentiviral mediated gene transfer in livestock has generated unprecedented high yields of transgenic animals due to multiple integration events. unfortunately, multiple integration brings the disadvantage that here is an increased probability of unwanted side effects caused by oncogene activation or insertional mutagenesis. additional problems which have been identified are gene silencing due to the presence of viral sequences (hofmann et al. ) and a high frequency of mosaicism in founder animals. transgenic mice and farm animals harbouring the first-generation of conditional promoter elements showed expression in response to heavy metals or steroid hormones but suffered from high basal expression levels and pleiotropic effects (lee et al. ; mayo et al. ; miller et al. ; pursel et al. ) . newer, binary expression systems based on prokaryotic control elements are responsive to exogenous iptg (isopropyl-β-dthiogalactopyranosid), ru- , ecdysone or tetracycline derivatives and give more tightly controlled expression (lewandowski ; corbel and rossi ) . the first tetracycline system that was successfully used in mice required two dna constructs. one was for doxycycline controlled expression of a transactivator and the other contained regulatory elements which conferred transactivator dependent expression of the target gene. these dna constructs were typically integrated into two different lines of transgenic mice. after crossbreeding the two lines of transgenic mice, their offspring expressed the target gene only after stimulation with doxycycline (furth et al. ) . unfortunately, the long generation intervals make this approach unfeasible in livestock species (niemann and kues ) . recently, we reported on a tetracycline-responsive bicistronic expression cassette (nta) in which expression was amplified by transactivator mediated positive feedback . this was used to produce the first tetracycline controlled transgenic expression in a farm animal. the auto-regulatory cassette was integrated at a single chromosomal site in the pig genome after pronuclear dna injection and was designed to give ubiquitous expression of a human regulator of complement activation (rca) independent of cellular transcription cofactors. expression from this construct could be inhibited reversibly by feeding the animals doxycycline (tet-off system). in ten transgenic pig lines in which only one copy of the nta cassette was integrated, the transgene was silenced in all tissues with the exception of skeletal muscle where expression was limited to a small number of discrete muscle fibers (niemann and kues ) . however, crossbreeding of lines to produce animals with two nta cassettes resulted in reactivation of the cassettes and strong, tissueindependent, tetracycline-sensitive rca expression. it seems that crossing the transgenic pig lines, which doubled the level of transactivator, was able to overcome epigenetic silencing. transgene expression in the double transgenic pigs was inversely correlated with the level of nta cassette dna methylation . this approach highlights the importance of understanding epigenetic (trans)-gene regulation in the pig. pluripotent embryonic stem (es) cells have the ability to participate in organ and even germ cell development following injection into blastocysts or aggregation with morulae (rossant ) . true es cells (i.e. those able to contribute to the germ line) are currently only available from inbred mouse strains (kues et al. a ). these murine es cells have become an important tool for gene knock-out and knock-in experiments and to study large chromosomal rearrangements (downing and battey ) . es-like cells and primordial germ cell cultures have been reported for several farm animal species, and es-like cells which can produce chimeric animals albeit without germ line contribution have been reported in swine (anderson ; shim et al. ; wheeler ) and cattle (cibelli et al. ) . recent data indicate that somatic stem cells have a much broader developmental potential than previously assumed kues et al. b ). whether these cells will improve the efficiency of chimera generation or somatic nuclear transfer in farm animals has yet to be shown conclusively (kues et al. b; hornen et al ) . pluripotent cells are a valuable tool for improved production of animals with targeted genetic modification. a revolutionary breakthrough in direct nuclear reprogramming of mouse somatic cells was recently reported (takahashi and yamanaka ; okita et al. ; wernig et al. ). cells transfected with constructs expressing oct , sox , myc and klf , carried in retroviral vectors, were reprogrammed to a totipotent state and were indistinguishable from es cells generated from fertilized embryos with regard to differentiation potential and morphology. these induced pluripotent stem cells (ips), derived from somatic cells, were able to populate the germ line upon injection into blastocysts and after transfer into recipient mice, clearly indicating complete reprogramming (okita et al. ; wernig et al. ; maherali et al. ). the same genes have recently been found to be effective in reprogramming human fibroblasts and other human somatic cells into cells with pluripotent properties (takahashi et al. ). this affords a new approach to the generation of pluripotent cells from farm animal species. transplantation of transgenic primordial germ cells into the testes is potentially an alternative approach to the generation of transgenic animals. initial experiments in mice showed that the depletion of endogenous spermatogonial stem cells by treatment with busulfan prior to transplantation is effective and permits re-colonization of the testes by donor cells. transmission of the donor haplotype to the next generation after germ-cell transplantation has been achieved in goats (honaramooz et al. ) . current major obstacles of this strategy are the lack of efficient in vitro culture methods for primordial germ/prospermatogonial cells and the lack of efficient gene transfer techniques for these cells. recently adeno-associated virus (aav ) was found to be suitable for delivering transgenes to infect a mal germ cell and germline tansmission was reported in goats and mice (honaramooz et al. ). the efficiency of this approach and putative silencing of the aav introduced transgenes requires further investigation. rna interference (rnai) is a conserved post-transcriptional gene regulatory process found in most biological systems including fungi, plants and animals. the common element is double stranded rna which is cleaved to form small interfering rna (sirna) molecules - base pairs in length. a single strand of these small rna molecules is incorporated in an rnainduced silencing complex (risc) which specifically binds to the complementary sequence of its target mrna causing endonuclease mediated degradation. the result is that no protein is produced from that mrna transcript (plasterk ) . natural rna interference is involved in gene regulation, specifically to suppress the translation of mrnas from endogenous and exogenous viral elements, so this can be exploited for therapeutic purposes (dallas and vlassow ) . for transient gene knockdown, synthetic sirnas can be transfected into cells or early embryos (clark and whitelaw ; iqbal et al. ) . for stable gene repression, the sirna sequences must be incorporated into a gene construct and constitutively expressed. the combination of sirna with lentiviral vector technology is now a highly effective tool in this respect. rnai knockdown of porcine endogenous retrovirus (perv) has been demonstrated in porcine primary cells (dieckhoff et al. ) and in cloned piglets (dieckhoff et al. ) . sirna mediated knockdown of the prion protein (prnp) gene has been accomplished in bovine embryos (golding et al. ) . the modification appears to be permanent as lentiviral delivered sirna has been shown to persist for three generations in rats (tenenhaus dann et al. ) . the combination of sirna and lentiviral vector technology provides a method for highly efficient targeted gene knockdown for functional genetic analysis in farm animals and could easily be integrated into existing breeding programs. concerns have been raised about the health of transgenic farm animals because it is known that insertional mutagenesis and other undesirable side effects can be caused by the integration and expression of recombinant gene constructs (van reenen et al. , van reenen this proceedings) . the health of all transgenic animals is closely monitored because of the time and money invested in their creation and because all work is basic research. a small number of studies have systematically investigated the health effects of transgenesis. a study of the effects of human growth hormone expression in pigs and sheep identified specific pathological phenotypes related to their accelerated growth rate. these problems were eliminated in subsequent transgenic animals by modifications to the gene constructs (nottle et al. ) . in pigs, transgenic for human daf and maintained under qualified pathogen free conditions, haematological parameters and blood chemistry were similar to non-transgenic controls (tucker et al. ) . with the exception of slightly accelerated growth rates, no deviations were found. a detailed pathomorphological examination of nine lines of hemizygous pigs expressing human rcas revealed no adverse effects related to transgene expression (deppenmeier et al. ) , providing clear evidence that transgenesis per se does not compromise animal health and welfare. investigation of animals carrying the nta bicistronic expression cassette, driving hcd and a tetracycline regulated transactivator revealed that multi-transgenic animals display a normal health status (deppenmeier et al. ) . the hemizygous lines were fertile and produced normal litter sizes. transgenesis based on scnt is increasingly used forf armanimals.i n cloned animals, both pre-and postnataldevelopment can be compromised and a proportion of scnt offspring in both ruminants and mice exhibit increased perinatal mortality. the list of developmental abnormalities includes: extended gestation length, oversized offspring, aberrant placental function, cardiovascular problems, respiratory defects, immunological deficiencies, problems with tendons, adult obesity, kidney or hepatic malfunctions, behavioral changesand higher susceptibilitytoneonataldiseases, all of which are aspects of what has been called the "large offspring syndrome" (los) (renard et al. ; tamashiro et al. ; ogonuki et al. ; rhind et al. ) . the incidence of los is stochastic and has not been linked to aberrant expression of any single genes or to any specific pathophysiology. the general assumption is that the underlying cause of los is faulty epigenetic reprogramming of the transferred somatic cell nucleus. despite these problems, critical surveys of the published literature have revealed that most cloned animals are healthy and develop normally (cibelli et al. ; panarace et al. ). this demonstrates that mammalian development can tolerate minor epigenetic aberrations and subtle variations in gene expression without affecting survival of cloned animals (humpherys et al. ) . six months old cloned cattle do not differ from age-matched controls with regard to biochemical blood and urine parameters (lanza et al. ; chavatte-palmer et al. ) , immune status (lanza et al. ) , body score (lanza et al. ) , somatotrophic axis (govoni et al. ) , reproductive parameters , or milk yield and composition (pace et al. ) . no differences were found in the meat or milk composition of bovine clones compared to age matched counterparts (tian et al. ; miller ) . similar findings were reported for cloned pigs archer et al. ) . regulatory agencies around the world have agreed that food derived from cloned animals is safe and there is no scientific basis for questioning this (c.f. national academy of sciences, committee on defining science-based concerns associated with products of animal biotechnology, (national academy of sciences ). expert committee from the japanese ministry of agriculture, forestry and fisheries (maff; kumugai ) , the fda (rudenko et al. ; food and drug administration ) and efsa (european food safety agency, ) . since somatic cloning has only been used since and the lifespan of domestic animals is relatively long, the specific effects of cloning on longevity and senescence have not yet been fully assessed; however, preliminary data indicate no cumulative pathology even after serial cloning of mice and cattle kubota et al. ). there are still insufficient numbers of transgenic farm animals produced by the newest technologies including viral vectors and spermatogonial transgenesis to reveal subtle effects on animal health and welfare. biological products from any animal source are unique and must be handled differently than chemically synthesized drugs to assure their safety, purity and potency. proteins are heat labile, subject to microbial contamination, can be damaged by shear forces and can be immunogenic and allergenic. in the united states, the fda has developed guidelines to assure safe commercial exploitation of recombinant biological products. a crucial consideration with animal derived products is the prevention of transmission of pathogens from animals to humans . this requires sensitive and reliable diagnostic and screening methods for various pathogenic organisms. furthermore, transgenic farm animal based applications require strict standards of quality control. maldi-tof-spectometry is an important tool in this context (hughes et al. ; templin et al. ) . meanwhile, improvements in rna isolation and in unbiased global amplification of picogram amounts of mrna enable researchers to analyse rna from single embryos (brambrink et al. ; ). one can now monitor the entire transcriptome of a transgenic organ or organism to ensure the absence of unwanted effects (hughes et al. ; templin et al. ) . detailed genomic information and new genetic engineering tools will accelerate and improve transgenic animal production in the future. genetic technology presents not only a major opportunity to improve agricultural production but also offers exciting prospects for medical research by exploiting large animals as models of human health and disease. progress in animal genomics has broadly followed the route pioneered by the human genome project in terms of the assembly, publication and utilization of the data. this is evident in the advanced drafts of the bovine, porcine, horse, canine, chicken, and honeybee genomic maps. the ability to engineer the genome is new and the advent of new molecular tools and breeding technologies is benefiting this field. however, full realization of this exciting potential is handicapped by our currently limited understanding of epigenetic controls and the role of natural sirna and microrna in regulating gene expression. the convergence of recent advances in reproductive technology with the tools of molecular biology opens a new dimension for animal breeding. major goals are the continued refinement of reproductive biotechnology and a rapid completion of the various genome sequencing and annotation projects. induced pluripotent stem cell (ips) (takahashi and yamanaka ) research will play a critical role in understanding epigenetic controls. despite continued efforts, no es-cell lines with germ line potential have been established from mammals other than the mouse although es-like cells have been reported in several species and have been maintained in culture from weeks to three years (gjorret and maddox-hyttel ) . true germ line competent es cell lines from farm animal species will permit exploitation of the full power of recombinant dna technology in animal breeding. this is critical for the development of sustainable and diversified animal production systems for the future. we anticipate that in the near future genetically modified animals will play a significant role in the biomedical field but that agricultural applications will develop more slowly due to the complexity of many economically important traits and to current resistance to the concept of engineered farm animals. embryonic stem cells in agricultural species hierarchical phenotype and epigenetic variation in cloned swine production of goats by somatic cell nuclear transfer conditional expression of type i interferon-induced bovine mx gt-pase in a stable transgenic vero cell line interferes with replication of vesicular stomatitis virus application of cdna arrays to monitor mrna profiles in single preimplantation mouse embryos cloned transgenic cattle produce milk with higher levels of b-casein and k-casein the new mouse genetics: altering the genome by gene targeting phenotyping of transgenic cloned pigs engineering passive immunity in transgenic mice secreting virus-neutralizing antibodies in milk transgenic cattle produced by reverse-transcribed gene transfer in oocytes effective generation of transgenic pigs and mice by linker based sperm-mediated gene transfer clinical, hormonal, and hematologic characteristics of bovine calves derived from nuclei from somatic cells uniform gfp-expression in transgenic medaka (oryzias latipes) at the f generation transgenic bovine chimeric offspring produced from somatic cell-derived stem like cells the health profile of cloned animals a future for transgenic livestock development of transgenic sheep that express the visna virus envelope gene direct chemical evidence for widespread dairying in prehistoric britain latest developments and in vivo use of the tet system: ex vivo and in vivo delivery of tetracycline-regulated genes the generation of transgenic pigs as potential organ donors for humans polyhemoglobin-superoxide dismutase-catalase as a blood substitute with antioxidant properties targeted disruption of the α , -galactosyltransferase gene in cloned pigs rnai: a novel antisense technology and its therapeutic potential improved wool production in transgenic sheep expressing insulin-like growth factor targeting gene expression to the wool follicle in transgenic sheep deletion of the alpha( , )galactosyltransferase (ggta ) and the prion protein (prp) gene in sheep health status of transgenic pig lines expressing human complement regulator protein cd inhibition of porcine endogenous retroviruses (perv) in primary porcine cells by rna interference using lentiviral vectors knockdown of porcine endogenous retrovirus (perv) expression by perv-specific shrna in transgenic pigs technical assessment of the first years of research using mouse embryonic stem cell lines making recombinant proteins in animals -different systems, different applications production of recombinant therapeutic proteins in the milk of transgenic animals reproductive characteristics of cloned heifers derived from adult somatic cells preimplantation-stage stem cells induce longterm allogeneic graft acceptance without supplementary host conditioning fda statement regarding glofish animal cloning: a risk assessment -final temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter attemptstowards derivationand establishment of bovine embryonic stem cell-like cultures suppression of prion protein in livestock by rna interference pigs expressing salivary phytase produce lowphosphorus manure development of transgenic fish for ornamental and bioreactor by strong expression of fluorescent proteins in the skeletal muscle age-related changes of the somatotropic axis in cloned holstein calves cloned transgenic farm animals produce a bispecific antibody for t cell-mediated tumor cell killing identification of a novel arg->cys mutation in the ldl receptor that contributes to spontaneous hypercholesterolemia in pigs production of transgenic rabbits, sheep and pigs by microinjection spontaneous and genetically engineered animal models; use in preclinical cancer drug development efficient production of transgenic cattle by retroviral infection of early embryos efficient transgenesis in farm animals by lentiviral vectors generation of transgenic cattle by lentiviral gene transfer into oocytes epigenetic regulation of lentiviral transgene vectors in a large animal model fertility and germline transmission of donor haplotype following germ cell transplantation in immunocompetent goats adeno-associated virus (aav )-mediated transduction of male germ line stem cells results in transgene transmission after germ cell transplantation production of viable pigs from fetal somatic stem cells recombinant human butyrylcholinesterase from milk of transgenic animals to protect against organophosphate poisoning widespread aneuploidy revealed by dna microarray expression profiling epigenetic instability in es cells and cloned mice generation of transgenic dairy cattle from transgene-analyzed and sexed embryos produced in vitro selected physiologic compatibilities and incompatibilities between human and porcine organ systems parent-of-origin dependent gene-specific knock down in mouse embryos porcine endogenous retroviruses: no infection in patients treated with a bioreactor based on porcine liver cells pluripotency of mesenchymal stem cells derived from adult marrow production of low-lactose milk by ectopic expression of intestinal lactase in the mouse mammary gland toward altering milk composition by genetic manipulation: current status and challenges hla-mismatched renal transplantation without maintenance immunosuppression site-specific recombinases: tools for genome engineering generation of transgenic dairy cattle using in vitro embryo production serial bull cloning by somatic cell nuclear transfer the contribution of farm animals to human health from fibroblasts to stem cells: implications for cell therapies and somatic cloning isolation of murine and porcine fetal stem cells from somatic tissue epigenetic silencing and tissue independent expression of a novel tetracycline inducible system in doubletransgenic pigs safety of animal foods that utilize cloning technology cloned transchromosomic calves producing human immunoglobulin heart transplantation in baboons usion a , -glactosyltransferase knock out pigs as donors: initial experiments generation of cloned transgenic pigs rich in omega- fatty acids production of α , -galactosyltransferase knockout pigs by nuclear transfer cloning cloned cattle can be healthy and normal sperm cells as vectors for introducing foreign dna into eggs: genetic transformation of mice efficient production by sperm-mediated gene transfer of human decay accelerating factor (hdaf) transgenic pigs for xenotransplantation glucocorticoids regulate expression of dihydrofolate reductase cdna in mouse mammary tumour virus chimaeric plasmids conditional control of gene expression in the mouse progress toward generating a ferret model of cystic fibrosis by somatic cell nuclear transfer expression of mouse iga transgenic mice, pigs and sheep the production of recombinant pharmaceutical proteins in plants the effects of mammary gland expression of human lysozyme on the properties of milk from transgenic mice mammary gland expression of transgenes and the potential for altering the properties of milk directly reprogrammed fibroblasts show epigenetic remodeling and widespread tissue contribution lensectomy and vitrectomy decrease the rate of photoreceptor loss in rhodopsin p l transgenic pigs the deleterious effects of human erythropoietin gene driven by the rabbit whey acidic protein gene promoter in transgenic rabbits the mouse metallothionein-i gene is transcriptionally regulated by cadmium following transfection into human or mouse cells expression of recombinant proteins in the milk of transgenic animals food from cloned animals is part of our brave old world expression of human or bovine growth hormone gene with a mouse metallothionein- promoter in transgenic swine alters the secretion of porcine growth hormone and insulin-like growth factor-i disease resistance in farm animals transgenic pigs carrying cdna copies encoding the murine mx protein which confers resistance to influenza virus infection safety of genetically engineered foods: approaches to assessing unintended health effects. sub-report on methods and mechanisms of genetic manipulation and cloning of animals. the national transgenic pigs expressing plant genes expression of human blood clotting factor viii in the mammary gland of transgenic sheep application of transgenesis in livestock for agriculture and biomedicine transgenic farm animals: present and future transgenic farm animals: an update application of dna array technology to mammalian embryos production and analysis of transgenic pigs containing a metallothionein porcine growth hormone gene construct early death of mice cloned from somatic cells generation of germline-competent induced pluripotent stem cells ontogeny of cloned cattle to lactation retrovirus-induced ovine pulmonary adenocarcinoma, an animal model for lung cancer how healthy are clones and their progeny: years of field experience mammalian transgenesis by intracytoplasmic sperm injection efficient metaphase ii transgenesis with different transgene archetypes preovulatory embryo transfer increases cloning efficiencies in pigs production of pigs transgenic for human hemeoxygenase-i by somatic nuclear transfer genetically engineered large animal model for studying cone photoreceptor survival and degeneration in retinitis pigmentosa production of alpha , -galactosyltransferasedeficient pigs rna silencing: the genomes immune system expression of human lactoferrin in milk of transgenic mice the future promises of xenotransplantation expression of insulin-like growth factor-i in skeletal muscle of transgenic pigs animal models: the pig hot topic: using a stearoyl-coa desaturase transgene to alter milk fatty acid composition lymphoid hypoplasia and somatic cloning cloned lambs--lessons from pathology production of cattle lacking prion protein transgenic animal production and animal biotechnology animal cloning and the fda: the risk assessment paradigm under public scrutiny stem cells from the mammalian blastocyst biopharmaceutical production in transgenic livestock functional expression of a delta fatty acid desaturase gene from spinach in transgenic pigs telomere length is reset during early mammalian embryogenesis telomere length regulation during cloning, embryogenesis and aging human factor ix transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts isolation of pluripotent stem cells from cultured porcine primordial germ cells intracellular immunization: a new strategy for producing diseaseresistant transgenic livestock? creation and phenotypic analysis of α-lactalbumin-deficient mice production of functional human hemoglobin in transgenic swine lack of cross-species transmission of porcine endogenous retrovirus infection to nonhuman primate recipients of porcine cells, tissues and organs induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors induction of pluripotent stem cells from adult human fibroblasts by defined factors postnatal growth and behavioral development of mice cloned from adult cumulus cells protein microarray technology heritable and stable gene knockdown in rats transgenic animals as models of neurodegenerative disease in humans meat and milk compositions of bovine clones the production of transgenic pigs for potential use in clinical xenotransplantation: baseline clinical pathology and organ size studies large scale production of recombinant human lactoferrin in the mik of trangenic cows enzyme therapy for pompe disease with recombinant human α-glucosidase from rabbit milk transgenesis may affect farm animal welfare: a case for systemic risk assessment cloning of mice to six generations genetically enhanced cows resist intramammary staphylococcus aureus infection transgene-mediated modifications to animal biochemistry genes encoding a mouse monoclonal antibody are expressed in transgenic mice, rabbits and pigs transmission of prions in vitro reprogramming of fibroblasts into a pluripotent es-cell-like state development and validation of swine embryonic stem cells: a review transgenic alteration of sow milk to improve piglet growth and health efficient generation of transgenic pigs using equine infectious anaemia virus (eiav) derived vector nuclear transplantation in sheep embryos viable offspring derived from fetal and adult mammalian cells xenotransplantation: current status and a perspective on the future risk assessment of meat and milk from cloned animals marked prolongation of porcine renal xenograft survival in baboons through the use of α , -galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue genetic engineering of milk composition: modification of milk components in lactating transgenic animals commercialization of proteins produced in the mammary gland key: cord- - oew e authors: aurigemma, rosemarie; tomaszewski, joseph e.; ruppel, sheryl; creekmore, stephen; sausville, edward a. title: regulatory aspects in the development of gene therapies date: journal: cancer gene therapy doi: . / - - - - _ sha: doc_id: cord_uid: oew e preclinical therapeutics development research is directed toward fulfilling two overlapping sets of goals. a set of scientific goals includes defining the best molecule or biologic construct for the task at hand, and proving the case for its development. the second set of goals addresses regulatory requirements necessary to introduce the agent into human subjects. in the case of “small molecule” drugs, in most cases the identity of the molecule and appropriate safety studies are straightforward. in contrast, the development of biologic agents, including gene therapies discussed here, presents distinct challenges. the nature of the “drug” may be an organism subject to mutation or selection of variants through recombination. its properties may vary depending on the scale and method of its preparation, purification, and storage. how to test adequately for its safety prior to first introduction in humans may not be straightforward owing to intrinsic differences in response to the agent expected in humans as compared to animals. preclinical therapeutics development research is directed toward fulfilling two overlapping sets of goals. a set of scientific goals includes defining the best molecule or biologic construct for the task at hand, and proving the case for its development. the second set of goals addresses regulatory requirements necessary to introduce the agent into human subjects. in the case of "small molecule" drugs, in most cases the identity of the molecule and appropriate safety studies are straightforward. in contrast, the development of biologic agents, including gene therapies discussed here, presents distinct challenges. the nature of the "drug" may be an organism subject to mutation or selection of variants through recombination. its properties may vary depending on the scale and method of its preparation, purification, and storage. how to test adequately for its safety prior to first introduction in humans may not be straightforward owing to intrinsic differences in response to the agent expected in humans as compared to animals. the general principles, however, in allowing "first-in-human" experiences are similar for both small molecules and biologics. the ethical conduct of clinical trials in patients with a dire or lifethreatening disease demands an understanding of the identity and dose of an agent that has the possibility of causing clinical benefit with adverse events expected at worst to be easily reversible and well predicted by the preclinical experience. in normal volunteers or patients who are otherwise well, evidence should be gathered that would support an initial range of doses of the test agent expected to be without substantial toxicity or long-term effects. thus, the successful clinical introduction of a novel therapeutic concept requires an organized approach to integrate scientific, technical, and regulatory requirements. this integration should begin in the research laboratory, as the concept becomes a candidate for the clinic, to prevent avoidable and expensive delays in clinical development. for example, if a product is created using a mammalian cell line for which viral or other contamination has not been ruled out, costly rederivation will be required before that product can be manufactured for clinical trials using current good manufacturing practices (cgmp). on the other hand, during the discovery phase, an excessive and premature concern over cgmp compliance can impede research. therefore, a clear strategic understanding of the principles underlying regulatory issues is desirable and is the goal of this chapter. we proceed from the experience of the developmental therapeutics program (dtp) of the national cancer institute in the manufacture of biologicals, including gene therapy constructs for preclinical and clinical use. we outline the basis for our approach to safety testing studies to be included in an investigational new drug (ind) application to the food and drug administration (fda). we focus on studies that would allow phase i and perhaps early phase ii clinical trials. in , dtp contributed to over different cgmp biological projects. most of these activities were selected competitively from applications received from academic researchers or from the intramural laboratories of the national institutes of health (nih). dtp products include viruses for vaccines or gene therapy, plasmids, monoclonal antibodies, recombinant proteins, synthetic peptides, natural product fermentations, and oligonucleotides. during the fiscal year, over different lots were manufactured and released under cgmp for clinical use or further cgmp manufacturing. most products are destined for phase i or phase ii clinical trials in cancer. beyond early (phase i/ii) clinical trials, technology transfer for some products has occurred, with further development through phase iii now addressed by pharmaceutical companies. based on experience accumulated over several years, we abstracted the initial profiles of the more successful concepts (table ) , as well as some early project characteristics that can impede clinical development ( table ) . we note correlations between the thoroughness of the early research, attention to "the rules," outlined in the references cited here, and the development of commercial interest in the product. gene therapy and other biologic therapeutics are regulated within the fda by the center for biologics evaluation and research (cber), which was created in to address products emerging from the new biotechnology. reorganization at the fda is currently under way that will result in regulation of many biotherapeutics by the center for drug evaluation and research, which has oversight of small molecule drugs. it is anticipated, however, that blood products, vaccines, and gene therapy products will remain with cber. the biological response modifiers advisory committee is a chartered advisory group with the role of advising the fda to ensure the safety and effectiveness of biological products, including gene therapy. the recombinant dna advisory committee (rac) also oversees gene therapy research through the nih office of biotechnology activities. the rac was established in in response to public concerns regarding the safety of recombinant deoxyribonucleic acid (dna) technology. human gene transfer trials in which nih funding is involved (either directly or indirectly) are to be submitted to the rac for review. table beyond a good idea: what the successful investigator has already done with a project leading to commercial development defined candidate biologic (or molecule) made comparisons with similar products characteristics of product are consistent with pharmaceutical requirements production scale is adequate product characterization is adequate laboratory reference standard exists in vitro potency assay has been developed stability studies develop confidence product is a "drug" reproducible model systems have confirmed in vivo activity with clinical product early animal work includes some toxicology scale-up requirements practical for initial clinical trials in general, reflects experience and scientific maturity of investigator in addition to the us agencies that develop the regulations that govern drug development and licensing, the international conference on harmonization (ich) was formed in april involving the united states, the european union, and japan to address the issue of globalizing such regulations. the ich steering committee meets at least twice a year to continue their agenda of updating and harmonizing regulations for medicinal products; they emphasize safety, quality, and efficacy. expert working groups were formed within ich to address specific topics related to these basic areas. although the fda has not formally adopted all of the ich guidelines, these guidelines should be followed when they exist in preliminary form. for investigators planning to conduct investigational drug trials in foreign countries, it is imperative that they be familiar with, and adhere to, the regulations set forth by ich. in - , a series of fda and ich guidance documents on characterization and preclinical safety evaluation of biotechnology-derived pharmaceuticals was developed ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . these guidances represent the fda's current thinking on preclinical safety evaluation of biotechnology-derived pharmaceuticals. these are defined as products derived from characterized cells using a variety of expression systems, including bacteria, yeast, insect, plant, and mammalian cells. the active substances may include proteins and peptides, their derivatives, and products of which they are components. these materials could be derived from cell cultures or produced using recombinant dna technology, including production by transgenic plants and animals. examples include cytokines, enzymes, fusion proteins, growth factors, hormones, monoclonal antibodies, plasminogen activators, recombinant plasma factors, and receptors. the intended indications for use in humans may include in vivo diagnostic, therapeutic, or prophylactic uses. the principles outlined in these guidance documents may also be applicable to recombinant dna protein vaccines, chemically synthesized peptides, plasmaderived products, endogenous proteins extracted from human tissue, and oligonucleotide drugs. the fda defines gene therapy as "a medical intervention based on modification of the genetic material of living cells" ( ) . cells may be modified ex vivo for subsequent administration to humans or may be altered in vivo by gene therapy given directly to the patient. when the genetic manipulation is performed ex vivo on cells that are then administered to the patient, this is also considered a form of somatic cell therapy ( ) . "the genetic manipulation may be intended to have a therapeutic or prophylactic table issues requiring attention at the outset of a project inappropriate antibiotic selection markers (e.g., ampicillin for recombinant proteins) lab-scale affinity purification solubility problems low yield errors in genetic sequence extraneous genetic material poorly defined production systems inadequate purification schemes unvalidated or nonexistent in vitro potency assay(s) lack of key reagents (e.g., antibodies to desired product) poor biochemical characterization inappropriate raw materials raw material qualification problems inappropriate cell banks difficult or unidentified toxicology systems failed vendor qualification intellectual property concerns effect or may provide a way of marking cells for later identification. recombinant dna materials used to transfer genetic material for such therapy are considered components of gene therapy and as such are subject to regulatory oversight". specific information related to gene therapy issues is contained in the "fda guidance for industry: guidance for human somatic cell therapy and gene therapy" ( ) . this guidance document updates and replaces the fda "points to consider" on this subject ( a). new information was intended to provide manufacturers with current information regarding regulatory concerns for production, quality control testing, and administration of recombinant vectors for gene therapy and of preclinical testing of both cellular therapies and vectors. the fda defines somatic cell therapy as "the administration to humans of autologous, allogeneic, or xenogeneic living non-germline cells, other than transfusable blood products, for therapeutic, diagnostic, or preventive purposes." the evaluation of the safety of gene therapy products is perhaps one of the more difficult tasks that faces toxicologists in the drug development arena today. because many of the agents, like other biologicals, are species specific and because these agents integrate into the host genome, the choice of animal models and study designs is fraught with uncertainty, and each product frequently breaks new regulatory ground. until recently, many investigators working in this field were probably lulled into a false sense of security because of the close scrutiny that preclinical studies and clinical protocols received from the nih rac and the fda. with the death of jesse gelsinger, a patient enrolled in a gene therapy clinical trial to correct a metabolic disease, in and the recent reports of a leukemialike disease produced in children who received gene therapy treatments to correct severe combined immunodeficiency disease (scid) ( ) ( ) ( ) ( ) , the safety of these agents is called into question more than ever. as a result, the toxicologist is under even more pressure to design more rigorous safety evaluation programs. there have been a number of reviews in this area in recent years by toxicologists from the fda ( ) , industry ( , ) , and international workshops ( ) that cover many of the fundamentals regarding safety evaluation of gene therapy products. these resources, in conjunction with this chapter and the various guidance documents from the fda and the ich ( , , ) , can be used by toxicologists to develop sound safety programs. these issues are discussed in greater detail in the latter half of this chapter. the basic foundation of regulations for drug development can be found in the code of federal regulations, title food and drugs ( cfr; ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . in addition to title , fda maintains an extensive number of web sites containing regulatory information that should be consulted during the development of a novel biotherapeutic. the collection of available regulatory information includes points to consider, guidance documents, drafts, and reports from public forums and symposia as well as information on the meetings of the biological response modifiers advisory committee. ich also sponsors a web site for obtaining the most recent guidelines. a free subscription to an e-mail advisory update service is also available ( table ) . in addition to the regulatory guidelines provided by the fda, the nih has published, and frequently updates, the nih guidelines for research involving recombinant dna molecules, which can also be found electronically ( table ) . although published documents disseminated by the fda and nih are essential starting points for planning a cgmp development strategy, it is important to realize that, in this rapidly evolving field, some requirements may be reflected in public comments or a growing consensus among industry long before they are formally adopted. furthermore, it is not unusual during the development of a new biologic to have also developed alternatives to conventional practices that are based on sound scientific data and are then implemented after discussions with the fda. product-specific factors can influence the regulatory requirements for an investigational agent. these issues should be explored in detail with the fda in a pre-ind meeting at which the ind sponsor presents relevant preclinical data and manufacturing and animal safety testing to support the proposed approach to clinical development. the types of further studies pertinent to the particular agent can then be proposed, and input from the agency help shape the final development plan. interactions should take place with regulatory authorities at intervals that will facilitate the development of a product (table ) . a key issue frequently not understood is that regulatory demands become more stringent as a product moves from phase i (safety), through phase ii (activity), to phase iii (comparative efficacy) trials and licensure ( , ( ) ( ) ( ) ( ) ( ) . this philosophy reflects the conscious effort not to stifle innovation in early phase clinical testing, but to ensure that, by the time registrationoriented late-stage trials are contemplated, issues related to production variability, assay, and assurance of safety are mature and well-substantiated because the results of such trials could be the basis for sale of the agent to the public. another factor that affects the level of regulatory compliance is the nature of the study population. products manufactured for phase i trials in healthy normal volunteers typically must meet much stricter requirements than those studied in patients with dire, life-threatening conditions (e.g., cancer or end-stage acquired immunodeficiency syndrome). as improved technology becomes available, requirements also tend to increase ( - ). the level of regulatory compliance to be followed during different stages of development is dependent on the type of biologic product and the technology available for supporting its development. assays, methods, and technologies for monoclonal antibody development ( ) , for instance, are better defined than the techniques available for some of the new virus vectors that are emerging. furthermore, new technologies to support product development are also constantly evolving. the number of specific viral contaminant tests required of cgmp human cell lines, for example, has increased steadily as new pathogens are identified and assays become available. as new scientific knowledge accumulates, novel regulatory challenges can appear. the issue of transmissible spongiform encephalopathy, for example, has resulted in stringent requirements in raw material qualifications and traceability ( ) . to minimize the impact of regulatory changes, careful record keeping of all processes and materials involved in deriving the product is highly recommended. finally, because of the availability of improved techniques for characterizing certain biologicals, the fda is reorganizing its regulatory approach in ways that are analogous to the regulation of small molecule drugs. technical demands will rise as regulatory requirements become more standardized. beyond identification and confirmation of an interesting novel concept, a major challenge in the preclinical development of biologicals is the optimal allocation of research and development resources. key to this is proper assessment of a candidate concept's readiness for clinical development. all applicants for the national cancer institute's biologicals production resources now receive a list of "generic questions" corresponding to the appropriate product type. at the beginning of a project, it is not always reasonable to expect all issues to be resolved, but the assumption is that, for a successful candidate, these issues should be in hand by the time the project is completing phase i clinical testing. table lists the generic questions for cgmp production of recombinant virus vectors. because it is not possible to provide a complete guide to cgmp development in a few pages, we highlight some concerns common to many projects arising from academic laboratories. these are based on dtp's experience (both successes and failures) with projects making the transition from the preclinical research phase to pilot clinical studies. our discussion is organized primarily around the concepts of identity, purity, potency, and safety that underlie development, manufacture, and release. from the viewpoint of regulatory compliance, it is essential to establish the identity of the product and the components used to generate it during manufacturing ( , ) . we have noted that, frequently in proposed gene therapy or recombinant dna-derived products submitted to us, dna sequencing shows some deviation from the sequence published and/or submitted by the investigator, sometimes with major consequences for the project. when the dna product, such as plasmid vaccines, will be administered to the patient, full plasmid sequencing has occasionally revealed unacceptable genetic sequences outside the open reading frame, as passengers from previous experiments, spurious promoters, frame shifts resulting in translation of nonsense sequences beyond the intended termination, and so on. dna sequencing and repair are available at relatively low cost compared to the cost of repeating critical preclinical experiments. the fda now requires complete sequencing of vectors of sizes up to kilobases (kb) ( ) . for viral vectors, genetic stability is a major concern, particularly with respect to the possible issues of recombination with generation of replication-competent viruses. specific guidelines are provided for adenovirus, retrovirus, and lentivirus vectors ( , , ) . for other virus vectors, specific assays (e.g., neurovirulence testing of recombinant poliovirus and herpes virus vectors) are required to ensure that an attenuated phenotype is preserved after scale-up. if possible, the investigator should attempt to assess the genetic stability of the vector during preclinical studies, after administration in vivo or propagation in vitro. in addition to the gene therapy product itself, the cells used to produce the product must be similarly identified and qualified for cgmp manufacturing. excellent guidance documents are available for the production of master cell banks, working cell banks, and master virus banks ( , , ) . at minimum, the complete cell history should be known and documented, and the cells should be tested to verify their origin. peptide sequencing or mapping employing liquid chromatography-mass spectrometry is typically used to provide critical information for synthetic peptides and recombinant proteins. for recombinant vectors containing transgenes, the expression of the desired gene product should be verified, for example, by immunoassay using a specific antibody against the product. a. non-gmp for additional preclinical development b. cgmp (clinical grade) . provide details of molecular construct(s), including starting materials (e.g., plasmid, relevant vector maps, detailed vector construction scheme, and so on) . does the construct contain an antibiotic resistance gene or other selectable marker? are alternative methods of selection available? why was the proposed selection chosen? . is the vector replication competent or replication defective? (for replication-selective vectors, what is the molecular basis of the selectivity and the conditions under which the vector would replicate?) . does the vector have an altered cell tropism? define. what is the effect of altered tropism on anticipated host toxicity? . has this construct been sequenced? provide a sequence in an electronic format. . are data available evaluating the genetic stability of the recombinant vector? have mutation rates been established and/or rates of reversion to either wild-type or alternate viral genomes? . are data available evaluating the potential for genetic recombination with other organisms in the patient or in the environment? . is the organism currently grown in a qualified cgmp cell line? if not, is there a qualified cell line available for propagation of this vector? was the cell line genetically modified to support this vector? provide details of its construction and any information regarding the stability of the genetic alteration in the cell line. . is there a cgmp-qualified virus seed bank? . provide details of the proposed production method. . has this material ever been produced for laboratory or clinical studies using this production system? . has this material been produced in a related or other production system? if so, please provide the details. . please provide details of existing purification methods. . what is the average yield of the production system before and after purification? what is the largest amount of material that you have produced in your laboratory in a single production batch? please provide average ratios obtained by this production method for virus particle/infectious unit and infectious units/cell. how does this scale to anticipated quantities for clinical trial? . how much material is available as a reference standard? . is material available as bulk biological substance for preliminary pharmacology and toxicology studies? . are there reproducible assays for the product? please provide the following assays, if available: a. identity b. purity c. potency . what are to be the release criteria for the product? how does one know that a lot of product is qualified for use? . in what form (lyophilized, formulated product, and so on) and fill size is the desired final product? what is the desired final product formulation? . are there issues of formulation that must be resolved? . what is known about the product stability with respect to physical integrity and activity? . do you have any information regarding the estimated costs of this production project? . have you identified any possible sources of production with any commercial firms? please provide details. . are there any safety issues connected with the production, purification, and/or handling of the product? . what is the status of the product(s) regarding intellectual property issues? . sometimes, proposed projects are an improvement or modification of an existing approach. in these cases, this information may significantly affect the analysis of feasibility, cost, and other production issues. please provide a brief summary of the nature of any such antecedents or other approaches that appear closely related to the proposed project. . have there been any meetings scheduled with regulatory agencies, such as a pre-ind meeting with the fda or a presentation to the nih rac? if so, please indicate the type of meeting, the regulatory agency, and the date or proposed date. . if you have had a pre-ind or rac meeting, were any issues concerning manufacturing, safety, or stability raised that will have an impact on producing your product? . who will sponsor the ind for the proposed study? . has a source of funding been identified for performing the clinical trial with this product? purification strategies depend on the nature of the biologic agent and expected impurities. these approaches are guided by the early development of reliable analytical techniques appropriate to the product and to the manufacturing approach. for example, purification of recombinant proteins and monoclonal antibodies for cgmp manufacture typically involve large-scale chromatography based on multiple isolation principles (e.g., charge, hydrophobicity, size, and so on). specific contaminants, such as dna, endotoxin, viruses from mammalian cell production systems, contaminants introduced in the manufacturing process, and the like must be quantified and may require additional specific purification measures to remove or inactivate them. problems in refolding or solubility, tendencies to aggregate, and product stability at intermediate holding points can be significant issues in process development for scale-up. these represent key challenges in scale-up from investigator laboratory-generated lots to a potentially suitable scale to allow clinical testing. additional concerns include subtle degradations of proteins that can lead to undesirable immunogenicity. a major concern is the impact of each additional step on the downstream product, which should be reassessed using in vitro potency assays as well as physicochemical characterization. at major development milestones, selected in vivo models should be reexamined using purified product. production cells must be cgmp qualified and tested for adventitious agents and other contaminants, before initiation of production as well as at end of production. a number of cgmp-qualified cell lines and starting vectors are available commercially at relatively low cost and should be considered for use as raw materials to initiate cgmp seed banks in preference to shared materials of uncertain provenance despite the good intentions of the original provider. in the handling of cell lines, care should also be taken to avoid contamination (e.g., from media components, trypsin, or activities taking place in nearby laboratory space). postproduction cells can be tested for specific contaminants in the presence of a viral product (e.g., using polymerase chain reaction [pcr]). in the presence of virus product, however, it is unlikely that the full set of cgmp tests (e.g., cocultivation) for adventitious agents can be performed on postproduction cells. before initiating cgmp production, therefore, investigators should consider the parallel propagation of a mock-infected control to provide a surrogate postproduction test article. in addition to the usual tests for sterility and purity of purified investigational product ( , , ) , it is important to have an assay for residual host cell dna. assays for host cell proteins are not always required for all phase i products, but are required for phase ii and beyond. this consideration is another reason to start with cgmp-qualified cell lines from a commercial source because host cell dna and protein assays may already be available. the general requirement for adventitious agent testing is given in guidance documents (e.g., ref. ). it is important to note that some specific assays are not yet described in published fda documents, but can enter widespread cgmp practice by sponsor-based industry consensus, liability considerations, or other factors. endotoxin assays are available as kits, which are useful to guide laboratory process development. a qualified good laboratory practice (glp) laboratory, however, should perform endotoxin assays for clinical product release. specific assays may also be required to quantify process residuals from production and purification components (cscl, antibiotics, and so on). in production facilities, particularly those where different types of gene therapy products may be produced, assays are necessary to support decontamination and cleaning, product changeover, environmental monitoring, and raw material qualification ( , ) . in general, all equipment that has contact with the investigational product should be verified free of contaminants before use. special consideration must also be given to assays to qualify virus seeds and end product for the presence of defective particles, replication-competent viruses, or defective genomes. in addition to monitoring for replication-competent and/or pathogenic vectors during manufacture, suitable assays may also be required to monitor patients receiving the therapeutic agent. in this case, levels of sensitivity for detection must be suitable for different types of patient specimens (serum, urine, sputum, and so on). evolving requirements for long-term follow-up of gene therapy patients ( ) should be consulted to ensure that proper assay support is maintained beyond the duration of the planned clinical trial. the fda regulations governing the performance of assays that support the production of biologics for human use can be found in cfr , subpart i, "laboratory controls" ( ) . glp regulations only per-tain to the performance of preclinical animal and in vitro studies ( ). the measured activity of a biological candidate depends on the hypothesized molecular mechanism of therapeutic action ( cfr . ; , ) . although it is most reassuring to see in vivo demonstration of efficacy in appropriate animal models, the efficient development of a cgmp process will strongly benefit from the availability of rapid, reliable, and reproducible in vitro assays relating to the mechanism of action in addition to assays for purity and identity. assays based on the basic therapeutic mechanism, therefore, are critical goals of early research and development. formulation development should begin as early as possible as suitable assays become available and experience with real-time stability accumulates. it is preferable to choose formulations from candidates likely to be acceptable to the fda, such as those whose components are already used for licensed products. as production reaches larger scales, handling and storage considerations become increasingly important. stability studies incorporate assays for identity, purity (including aggregation), and potency. although they can provide some useful information, accelerated stability studies are typically not reliable for predicting real-time stability of biologics. therefore, there is a need for real-time stability studies to be initiated as soon as possible. suitability of formulated product should also be assessed in the identical administration and handling conditions expected in the clinic. this may include transient exposure to conditions expected during transit to the study site and storage in an environment that closely mimics study site conditions. these may result in markedly different product behavior at the study site from that expected from the behavior of vouchered specimens at a central repository site. the results of ongoing stability studies are useful to support process development; to evaluate product at intermediate hold points in scaleup production and at product release; and to support formulation development, product storage, shipment, and handling during toxicology studies and clinical applications. as development proceeds, master specifications for release of intermediate and final product should be established and refined. the ind must indicate a schedule for real-time stability studies to be performed throughout the duration of the clinical trial. some key early milestones common to all product areas include the attainment of an adequate scale of high-quality, single-batch production, the availability of adequate amounts of high-quality laboratory reference standard, and the development of reliable assays for identity, purity, and potency of the product. these milestones are necessary in addition to the exploration of animal models showing safety and promising evidence of efficacy. at early stages in a project, investigators should expect substantial variability in product quality, assays, and animal models. ideally, therefore, a single high-quality batch should be used to establish laboratory standards, support multiple assay qualification runs, and perform replicate animal model experiments. multiple production runs could then be performed to explore process development issues, including scale-up. in this way, fundamental issues could be explored at the research stage to prepare for development required for cgmp manufacture. following this reasoning, our facility often manufactures high-quality glp lots to provide a uniform supply of material for additional preclinical research and development for selected biologics of interest before making the decision to undertake cgmp production. the early establishment of certain aspects of glp ( cfr ) is crucial to the advancement of a drug development project. by following simple rules of laboratory cleanliness, documentation, and segregation of materials and activities at the start of development, time can be saved by avoiding the necessity to duplicate results that were not properly performed or documented from the outset. at the discovery phase, development of reliable assays to explore basic therapeutic mechanisms of action are just as important as the performance of animal models in laying the groundwork for future cgmp product development. laboratory facilities and staff should be adequate to perform necessary studies. assay protocols should be specific and reproducible. research documentation should be kept at a glp level with complete and secure laboratory notebooks. records of all reagents (i.e., manufacturer, catalog number, lot number, certificates of analysis [coas] , and expiration dates) should be routinely archived. even if cgmp production or testing is not contemplated in the development laboratory, fluctuations in product activity are not unusual during later scale-up, and these materials and information may be useful in resolving such issues. key assays for product or reagent identity should be repeated at appropriate intervals. access to critical raw materials and reagents, reference standards, and cell banks should be limited. staff should avoid comingling of research-grade, glp, and gmp activities or reagents by labeling reagent containers and sequestering them as much as possible. similarly, signs should be posted on dedicated equipment, and access should be limited as appropriate. if common equipment must be used, standard operating procedures should be developed to define the use and control of such equipment, to clean equipment before and after use to avoid cross-contamination, and to document the use, cleaning, and calibration of the equipment. critical raw materials (e.g., those used in seed development or pilot product manufacture) must be traceable to their source and obtained from reliable vendors. it is beneficial when possible to use vendors subjected to commercial audits. animal-derived reagents should be avoided; reagents such as glycerol, detergents, proteins, amino acids, and the like should preferably come from vegetable sources. if this is not possible, animal-derived reagents should come from acceptable herds in countries without endemic or questionable transmissible spongiform encephalopathy ( ) . it is important to ensure that raw materials are stored under appropriate conditions and not used beyond their expiration date. inventories and logbooks should be used to track use of important reagents. critical materials requiring special storage conditions should be stored at more than one location to prevent loss in the event of equipment failure. cgmp-qualified cell lines should be purchased from vendors if possible, but if cgmp-qualified cell lines do not exist, cell lines should be obtained directly from a reliable repository source such as the american type culture collection (atcc) and documentation should be archived. incoming cell lines must be tested for sterility and mycoplasma contamination. thorough records should be kept on cell passages, observations, frozen storage, and the coas from media and other components used to grow, freeze, and otherwise manipulate the cells. critical cell lines should be segregated to prevent cross-contamination. stock cells should not be cultured in incubators containing virus-infected cell lines. vectors should be purchased from a reliable vendor, and documentation should be kept on the propagation, storage, and use, including coas, lot numbers, and so on of the reagents used to propagate the vector. if the vector is acquired from another laboratory (i.e., is unavailable for purchase from a vendor), a detailed history should be obtained of the generation of the vector, and detailed records should be kept from that time. all genetic manipulations of the vectors should be well documented and verified by sequencing. a lab-generated reference standard is a critical raw material for a biologic. it is ideal to have a large enough stock of this reference standard to use for the duration of the development work. it is often not possible, however, to produce sufficient quantities or material of sufficient stability at the early development stages. for this reason, it may be necessary to produce fresh batches and to test them thoroughly against independent standards or the current standard before that standard is depleted or loses potency. the same consideration should be given to other critical reagents, such as cell lines and compounds obtained from outside sources. reference standards and key reagents should be made or obtained in adequate amounts, characterized as well as possible, and stored under conditions that will maintain stability for at least the duration of the development process. as improved manufacturing and assay processes are developed, improved reference standards will be required, but quantities of the original standards should also be preserved to provide material for later comparisons as required ( ) . in some cases, such as for retrovirus and adenovirus vector development, the fda has made available reference material against which all sponsors can standardize their own reference reagents. to avoid future questions about data reliability, investigators should consider outsourcing of difficult but common technologies, such as transmission electron microscopy, tandem liquid chromatography mass spectroscopy, peptide mapping, or dna sequencing, if these are not adequate in their facility. product-specific assays, such as potency studies and pilot animal efficacy and toxicity studies, are likely to be performed best at the researcher's own facility, early in development. preclinical assay protocol development and record keeping must ensure that data are useful for later ind submission. at some point in cgmp process development, consideration should be given to technology transfer of critical assays to a glp-compliant laboratory prepared to support the repetitive studies required during cgmp process development, manufacture, release, and postrelease stability studies for the duration of the clinical trial. toxicology material should ideally be manufactured using the same process used to manufacture the cgmp clinical material. therefore, a toxicology lot is produced late in process development. if there are concerns over batch-to-batch variability, production of a single lot for both toxicology and the initial phase i trial is recommended. typically, a toxicology lot can be available several months before the clinical lot is ready for release. for studies involving autologous cells, the handling of cells must be under gmp conditions to preserve sterility and prevent cross-contamination with other cells. for allogeneic cells, it is important to use a cgmp-tested cell line with adequate traceability, including its origin, passage history, and exposure to media products that may have been derived from animal sources. starting material must be routinely checked for sequence accuracy; therefore, the complete plasmid should be sequenced. it is also preferable to examine genetic stability that can lead to the introduction of coding errors and changes in protein expression. the use of penicillin-like antibiotics (b-lactams) for selection is unacceptable because of the possibility of allergic reactions in patients administered products produced using this selection system. other antibiotics, such as kanamycin, are substituted, or alternative methods of selection are employed. measures of dna quality include supercoiled content, as well as assays for endotoxin, genomic dna, and other contaminants from the production system. more in-depth guidance is available through guidance documents ( ). it is generally recommended by the fda that a vector smaller than kb must be completely sequenced ( ) . as technology improves, this criterion may well be expanded to include larger vector genomes. those vectors with genomes larger than kb (e.g., herpes viruses, poxviruses) must have the transgene sequenced along with ' and ' flanking regions and any significant modifications to the vector backbone or sites vulnerable to alteration during molecular manipulation. when qualified vaccine strains exist for the vector of interest (e.g., vaccinia, poliovirus) it is preferable for cgmp manufacture to derive investigational constructs using the vaccine strain if available from the nih, atcc, or commercial sources. for adenovirus vectors, the recent availability of an adenovirus reference standard allows for the normalization of dosing based on virus particle concentration and infectious unit (iu) titer. current recommendations by the fda are for a ratio of viral particle to infectious unit of less than or equal to : ( ) . for replication-defective adenoviruses, generation of replication-competent adenoviruses (rca) must be measured in lots produced for clinical use. the current target requirement is fewer than rca per ´ virus particles as measured by a cell culture/cytopathic effect method ( ) . for viruses that are replication selective, different testing strategies (e.g., quantitative pcr) may be called for and should be discussed with the fda. similarly, the agency may have special considerations for viruses with altered tropism to ensure appropriate containment and prevent the generation of a replicationcompetent adenovirus with an expanded cell tropism. it should be noted that rca assays must be optimized regarding the presence of defective particles and other factors that may affect the sensitivity of the assay. retroviruses are of special concern because of the possibility of insertional carcinogenesis. this potential safety problem is amplified if replication-competent retroviruses (rcrs) are generated ( , ) . the general guideline is to test at least % of the total virus vector supernatant produced by amplifying any rcr on a permissive cell line. in addition, % of the producer cells or (whichever is less) must also be tested at the end of production by the method of coculturing on permissive cells ( ) . as with adenovirus vectors, retrovirus vectors with tropism modifications are of special concern and may require more stringent containment and patient follow-up ( ) . promoter modifications may also affect the safety profile of these virus vectors. lentiviruses generally have the same safety concerns as retroviruses, particularly because they can replicate in a broader variety of cells (dormant as well as actively dividing cells). although there is a retrovirus standard available through atcc to investigators who are developing retrovirus vectors, there is no lentivirus standard currently offered. a lentivirus standard is not planned for the future primarily because of the great variability in lentivirus backbones currently under development for clinical investigations (e.g., equine, murine, human). herpes viruses under development for clinical use either must be replication defective or, if replication competent, must be shown to be nonneurovirulent. neurovirulence is an issue for poliovirus as well. adeno-associated virus (aav) vectors are of concern because, although these vectors are designed to be maintained episomally, there can be reversion to wild type, resulting in integration into the host chromosome, or the vector could be rescued in a patient with a concurrent adenovirus infection ( ) . several interesting concepts seek to employ modified bacteria as the therapeutic agent. as with recombinant viruses, general issues of safety as well as specific issues of genetic stability and exchange should be considered. stabilization of the new genetic material may be required by incorporation into the bacterial genome rather than through a plasmid that can be lost or exchanged. strategies to incorporate new genetic material into bacterial dna will depend on confirming the sequence accuracy of the target bacterial sequences as well as the novel genetic material. introduction of an antibiotic resis-tance gene through a manufacturing step raises special concerns and can be avoided using alternative selection approaches. whether evaluating small molecules or biologically derived materials such as gene therapy products, the basic intent of nonclinical toxicity studies is to define the pharmacological and toxicological effects predictive of the human response, not only prior to initiation of phase i clinical trials in humans, but also throughout the entire drug development process leading ultimately to biologics license application (bla). the goals of these studies include, first, to define an initial safe starting dose and dose escalation schemes for first-in-human clinical trials; second, to identify potential target organs for toxicity, biomarkers or other parameters that can be monitored in patients receiving these therapies, and to determine if this toxicity is reversible; and finally, to determine which patient populations may be at greater risk for developing toxicity to a given cellular or gene therapeutic product ( ) . these nonclinical studies should be designed with the following points in mind: whether the product is transduced cells, the population of cells to be administered, or the class of vector used; the most appropriate animal species and physiological state of that model most relevant for the clinical indication and product class; and the intended doses, route of administration, and treatment regimens that will be used in the clinic. many of the questions that need to be taken into consideration and addressed during the design phase for safety studies include what is already known about the most likely toxicities related to the agent's biodistribution, local as well as systemic toxicity, immune responses (immunogenicity and immunotoxicity), the potential for insertional mutagenesis, and biological activities of the transgene product. then specific questions that arise with the new product or use are addressed. for example, are the safety issues primarily related to the vector, the transgene product, the method of administration, the formulation/excipient, or some combination of the above? how might existing published or unpublished nonclinical or clinical data address the questions mentioned above? safety issues that should be addressed in these studies include evaluation of the toxicity of the vector alone (irrespective of the transgene), including its potential toxicity and/or tumorigenicity (in some cases, this is apparent from previous evaluations with the same vector); toxicity of transgene expression in vivo that may not be evident from in vitro studies; occurrence and consequences of ectopic transgene expression in nontargeted tissues; occurrence and consequences of immune responses to transgene or vector proteins such as autoimmunity; and finally the possibility of germline transduction ( ) . because conventional pharmacology and toxicity testing as typically used for the evaluation of small molecules may not always be appropriate to determine the safety and biologic activity of cellular and gene therapy products, issues such as species specificity of the transduced gene, permissiveness for infection by viral vectors, and comparative animal to human physiology should be considered in the design of these studies. available animal models mimicking the disease indication may be useful in obtaining both sufficient safety and efficacy data prior to entry of these agents into clinical trials. early pre-ind discussions with the fda during development of a toxicology plan may prevent delays and added expenses because of inadequate data or the use of inappropriate species. some of the questions that should be answered by preclinical pharmacology/toxicology studies are the following ( ) : what is the relationship of the dose to the biologic activity? what is the relationship of the dose to the toxicity? does the route and/or schedule affect activity and/or toxicity? what risks can be identified for the clinical trial? for ex vivo gene transfer, the product is considered to be the transduced cells. the general safety test ( cfr . ) must be performed on the final product. when appropriate, modified procedures may be developed according to cfr . . the fda is considering proposed rule making to amend the general safety test rules and scope of applicability, especially for cell therapy products ( ) . finally, it is expected that these nonclinical toxicity studies will be conducted in compliance with glp regulations. however, there will be situations in which highly specialized assays will be required because of the nature of biotechnology-derived products, and it will not be possible to conduct these assays in full compliance with glps (e.g., in university or other discovery laboratories). it will be important that these areas be identified for any impact that they may have on the interpretation of toxicity data. in most cases, carefully performed studies such as this can be used to support inds and blas ( ). when selecting the animal model that will be used in the various preclinical biodistribution, pharmacology, and toxicology studies, consideration should be given to the scientific rationale for the animal species used. for example, would there be an advantage to performing the studies in rodents when larger numbers of animals might be more practical, or is there a necessity for a large animal model, such as a canine or nonhuman primate? if nonhuman primate studies are proposed, is it clear that another large animal or rodent model would not provide the same information? would there be any utility in a genetically deficient model, and would this deficient model be more relevant to the proposed study either because of the potential for adverse immunologic consequences or because of the biological effects in the deficient condition? animal models of disease may not be available for every cellular or gene therapy system proposed for development. this makes species selection an even more difficult process. preclinical pharmacological and safety testing of these agents should employ the most appropriate, pharmacologically relevant animal model available. a relevant animal species might be one in which the biological response to the therapy mimics the human response. this entails some knowledge of the pathophysiology of the disease in humans and of how faithfully it is reproduced in the animal model. the species of animal chosen for preclinical toxicity evaluations of viral preparations should be selected for its sensitivity to infection and production of pathologic sequelae induced by the wild-type virus related to the chosen vector, as well as its utility as a model of biologic activity of the vector construct. there should be a reasonable expectation of a similar distribution of receptors or permissivity in the animal model as there is in humans. thus, the species utilized may vary with the vector administered, the transgene expressed, the route of administration, the patient population treated, and the disease studied. rodent models rather than nonhuman primates may be useful if they are susceptible to pathology induced by the virus class (e.g., cotton rats are semipermissive hosts for adenovirus infections) ( ) ; the use of the scid mouse ( ) or the cotton rat ( ) may be suitable for the evaluation of herpes simplex virus (hsv) rather than the aotus monkey. some investigators have also suggested the use of miniature swine for evaluation of adenoviral vectors ( , ) . when evaluating the activity of a vector in an animal model for the clinical indication, safety data can be gathered from the same model to assess the contribution of disease-related changes in physiology or underlying pathology to the response to the vector. some specialized circumstances illustrating these points follow. the inbred cotton rat (sigmodon hispidus) has been used extensively as an animal model for research since the s, when it was first used in poliomyelitis research. since that time, it has been shown to be a semipermissive host for adenoviral infection ( , ) . in those studies, it was shown that the pulmonary lesions and replication pattern of the virus seen in the cotton rat paralleled those seen in humans. virus persisted in the nasal mucosa and lung for up to and days, respectively, after inoculation. this was even in the presence of high-titer neutralizing antibody that was detected by day . although cotton rats have readily adapted to the laboratory environment, they have retained a number of the characteristics of their wild counterparts. these animals have a tendency to bite, panic when handled, jump out of their cages, and have a large fight-or-flight zone. care and handling of these animals have been described by other investigators ( , ) . the cotton rat has been used for the evaluation of numerous adenovirus vectors by many routes of administration, and some of these studies are described here. when early e -deleted adenoviral vectors were evaluated in the cotton rat, it was discovered that the e region was not required for replication, but that this region plays a critical role in the pathogenesis of the disease in that these mutants induced a markedly greater lymphocyte and macrophage/monocyte inflammatory response in the lungs ( ) . e replacement recombinants were significantly less pathogenic than e -deleted viruses after intranasal administration ( ) . this study also demonstrated that adenovirus replicated in balb/c and cba mice and produced results that were similar to those seen in cotton rats. the intracranial administration of a replication-defective adenovirus expressing the herpes simplex virus thymidine kinase (hsvtk) gene at a dose of . ´ pfu into both adenoviral immune and adenoviral naïve cotton rats resulted in only mild gliosis and trace meningitis along the injection tract and approximated a "no toxic effect" dose ( ) . when this same vector was administered to either wistar rats or rhesus monkeys, direct neuronal injury or a dose-related inflammatory response was seen at the injection site and in the surrounding parenchyma. there was no apparent injury to tissues not of the central nervous system in any of the three models, and all cerebral spinal fluid, blood, urine, and stool samples failed to culture for adenovirus. in a study with a similar hsvtk adenovirus inoculated into cotton rats via intracardiac injection at doses up to ´ viral particles per animal with and without ganciclovir (gcv), the only significant microscopic lesions observed were epicardial inflammation and splenic hemosiderosis ( ) . vector sequences persisted throughout the -day assay period in the heart, lung, and lymphoid organs. infectious virions were detected for hours, but these virions were only detected at the site of injection of two animals in the highest dose group. when a similar vector was administered as either one or two subcutaneous injection cycles with . ´ viral particles/kg each or as a single course with . ´ viral particles/kg, the only significant treatment-related histopathological finding was dermatitis with mild acanthosis at the site of vector injection ( ) . in addition to these local effects, mild hyperamylasemia, lymphocytosis, and granulocytosis were seen clinically, but no other clinical signs of toxicity or death were observed. vector sequences were detected in the skin at the injection site and to a lesser extent in the liver, spleen, and lungs, and small amounts of vector dna were detected in the ovaries. these were cleared rapidly, and the absence of viral sequences in the excreta and swabs of the majority of animals suggested that there was no significant replication of this adenovirus vector in this host and little shedding. the owl monkey (aotus trivirgatus or nancymae) has been an excellent model for oncogenic and nononcogenic viruses such as hsv type (hsv- ) and others ( ) , and the herpes virus that infects these animals is a strain of hsv- ( ). these animals have been routinely used to test vaccines against hsv- and found to mimic the course of the disease in humans ( , ). as a result, it was only natural that these animals be used to evaluate the safety of gene therapy vectors produced from hsv- . however, these animals tend to be more fragile to use than other species and as a result must be handled with greater care. g , an attenuated, replication-competent hsv- recombinant, was tested for safety after intracerebral inoculation in the aotus ( ). these animals received doses of either or pfu of g or pfu of the wild-type hsv- strain f. wild-type hsv- caused rapid mortality and symptoms consistent with hsv encephalitis, including fever, hemiparesis, meningitis, and hemorrhage in the basal ganglia. for up to year after g inoculation, seven of the treated animals were alive and exhibited no evidence of clinical complications, indicating that this form of hsv was considerably attenuated in comparison to wild-type virus. two animals were reinoculated with pfu of g at the same stereotactic coordinates year after the initial dose. these animals were alive and healthy years after the second inoculation. as a further, more comprehensive clinical evaluation, animals were subjected to cerebral magnetic resonance imaging (mri) studies both before and after g inoculation. these studies failed to reveal radiographic evidence of the typical hsv-related sequelae in the brain seen in the animals treated with the wild-type virus. microscopic examination of multiple tissues found no evidence of hsv-induced histopathology or dissemination in spite of the fact that measurable increases in serum anti-hsv titers were detected. viral shedding and biodistribution in the aotus were also evaluated using pcr analyses and viral cultures of tear, saliva, or vaginal secretion samples ( ) . neither infectious virus nor viral dna was detected at any time-point up to month postinoculation. analyses of tissues obtained at necropsy at month or years after inoculation showed the distribution of g dna was restricted to the brain, although infectious virus was not isolated in these samples. the safety of this construct was also evaluated in the aotus after intraprostatic injections ( ) . safety was assessed on the basis of clinical observations, viral biodistribution, virus shedding, and histopathology. none of the injected monkeys displayed evidence of clinical disease, shedding of infectious virus, or spread of the virus into other organs. no significant microscopic abnormalities were observed in the organs evaluated. the results of these studies demonstrated that g can be safely inoculated into either the brain or the prostate, and that the aotus monkey could be successfully used in preclinical toxicological evaluations. in addition to the studies performed with this vector in aotus monkeys, balb/c mice were also used to evaluate the safety of g . mice were inoculated in the same manner as the aotus either intracerebrally or intraventricularly with pfu of g and survived for over weeks with no apparent symptoms of disease. in contrast, over % of animals inoculated intracerebrally with . pfu of hsv- wild-type strain kos and % of animals inoculated intraventricularly with pfu of wild-type strain f died within days. when mice were inoculated intrahepatically with g ( ´ pfu), all animals survived for over weeks, whereas no animals survived for even week after inoculation with pfu of wild-type kos ( ) . mice were also injected in the prostate with either g or wild-type hsv- strain f and observed for months ( ) . none of the g -injected animals exhibited any clinical signs of disease or died. however, % of mice injected with strain f displayed sluggishness and hunched behavior and were dead by day . on microscopic examination, the prostates injected with g were normal, whereas those injected with strain f showed epithelial flattening, sloughing, and stromal edema. these studies and those described by whitley with the scid mouse ( ) point to the fact that rodents can be used in place of the owl monkey and produce adequate safety data for the evaluation of hsv- vectors for gene therapy. finally, safety data can also be obtained in well-designed efficacy studies. in many cases, mouse studies can provide similar information as studies conducted in nonhuman primates, so smaller species should not be automatically rejected. the nonhuman primate should not be relied on for use as a model simply because of the comfort of going into studies in humans only after evaluation of the toxicity of the agent in nonhuman primates. experience has repeatedly shown for numerous classes of agents, both small molecules as well as biologicals, that no one species may be predictive of all human toxicities, and that not all human toxicities may be seen in other animal species ( ) . finally, certain human populations may not be predictive of all other human populations. this last fact makes predicting each and every toxicity almost impossible. the doses of vectors used in nonclinical studies should be selected based on preliminary efficacy/ activity data from both in vitro and in vivo studies. a no-effect dose level, an overtly toxic dose, and several intermediate doses should be evaluated, along with appropriate controls, such as naïve or vehicle-treated animals. for new formulations, it is very important to include this last group to distinguish formulation-related effects from those of the agent of interest. when products are difficult to produce in large quantities and as a result are in limited supply or for products with an inherently low toxicity, a maximum tolerated dose may not be achievable; as a result, a maximum feasible dose may be administered as the highest level tested in the preclinical studies and should be so designated in appropriate reports. although this may not be intellectually or scientifically satisfying, the data derived from such a study should at least establish the safety of the clinical starting dose. preclinical safety/ toxicity studies should include at least one dose that is equivalent to and at least one dose escalation level exceeding those proposed for the clinical trial. the multiples of the human dose required to determine adequate safety margins may vary with each class of vector employed, and the relevance of the animal model to humans and the rationale for dose selection should be provided. scaling of doses based on either body weight or total body surface area as appropriate facilitates comparisons across the animal species used and humans. although most small molecule cancer therapeutics are scaled based on body surface area ( ), body surface area may not be appropriate for gene therapeutics. information generated in the preclinical studies can be used to determine the margin of safety of the vector for use in the clinical trial, as well as gage an acceptable dose escalation scheme depending on the steepness of the toxicity curve. in a cross comparison of doses for an adenoviral vector for cystic fibrosis ( ), very similar toxicities were seen in cotton rats, mice, hamsters, rhesus monkeys, and baboons when the agent was directly instilled into the lungs of the animals. when the doses were scaled for body surface area, the no observable adverse effect levels for the various species were remarkably similar to one another and to the first human dose at which toxicity was observed, . - . ´ iu/m vs . ´ iu/m for humans. the only notable exception was the rhesus monkey at . ´ iu/m . studies like this enable other investigators to make wiser choices in the selection of doses and species to evaluate. the route of administration of vectors can have an obvious influence on toxicity in vivo because of the distribution and concentrations of the agent that are produced. for example, intravenous bolus doses can produce very high concentrations for short durations; other routes of administration, such as subcutaneous, may produce much lower concentrations and more prolonged exposure. current practice recommends that safety evaluations in preclinical studies should be conducted by the identical route and method of administration as that proposed for the phase i clinical trial whenever possible. when this is difficult to achieve in a small animal species, a method of administration similar to that planned for use in the clinic is advised. for example, intrapulmonary instillation of adenoviral vectors by intranasal administration in cotton rats or mice is an acceptable alternative to direct intrapulmonary administration through a bronchoscope because the latter procedure is simply not feasible in rodents. if the proposed clinical route is a nonintravenous (e.g., intratumoral), it may be wise also to conduct an intravenous study to provide perhaps "worse-case" data for what may happen in the event of an accidental injection directly into a patient's blood vessel. when possible, the schedule of administration in the animal studies should also be identical to that intended in the phase i clinical trial. this may not be feasible in certain instances because of the production of neutralizing antibodies in the animal model that might preclude repeated administration; that may not be a factor in humans. in studies in which additional agents will be administered in combination with the gene therapy agent (e.g., in suicide therapy using hsvtk and gcv or hsv cytosine deaminase and -fluorocytosine), the route and schedule should also be identical to that planned for the clinic. evaluating the vector alone in animal models would not provide sufficient data for predicting additional toxicity that may be produced by the combination, but should be at least one arm of the planned preclinical animal studies. at a minimum, treated animals should be monitored for general health status (clinical observations, body weight and temperature changes, changes in food and water consumption), serum biochemistry, and hematological profiles. target organs and other critical tissues should be examined for gross and microscopic changes. the addition of other parameters to be evaluated will depend on the nature of the product studied, the species used, and the route of administration. there is no set of all-inclusive parameters that is sufficient for each and every new agent. studies should be designed specifically for each agent, utilizing the most appropriate tests to capture as much relevant data as possible. because many biotechnology-derived pharmaceuticals intended for human use will be immunogenic in animals, the use of animal-derived proteins/products, if available, should be considered to define the intrinsic toxicity of the new agent. this may entail parallel development processes in which a construct relevant to the species in the safety test is developed to a point to allow a most relevant safety test to proceed. the analogous human construct then may actually be brought into the clinic supported by these results. if human material is used, measurement of antibodies associated with administration of products should always be performed when conducting repeated dose toxicity studies. these data will assist the investigator in the interpretation of the results of these studies. antibody responses produced in animals should be fully characterized (e.g., titer, number of responding animals, neutralizing or nonneutralizing), and their appearance should be correlated with any pharmacological and/or toxicological changes observed. more specifically, the effects of antibody formation on pharmacokinetics and/or pharmacodynamics, incidence and/or severity of adverse effects, complement activation, or the emergence of new toxic effects should be considered when interpreting the data. attention should also be paid to the evaluation of possible pathological changes related to immune complex formation and deposition, especially in the kidney of treated animals. the detection of antibodies in animals should not be the sole criterion for the early termination of a preclinical safety study or modification of the duration of the study unless the immune response neutralizes the pharmacological and/or toxicological effects of the biopharmaceutical in a large proportion of the animals. in most cases, the immune response to biopharmaceuticals in animals will be variable, similar to such responses in humans. if these issues do not compromise the interpretation of the data from the safety study, then no special significance should be ascribed to the antibody response. the induction of antibody formation in animals is not necessarily predictive of a potential for antibody formation in humans. by the same token, humans may also develop serum antibodies against humanized proteins, and frequently the therapeutic response persists in their presence. the same may happen in animals if a purified protein is administered via a gene therapy viral vector. in the case of human factor ix, when the purified protein was administered to rhesus macaques, the monkeys did not make antibodies ( ) . however, when factor ix was administered in a first-generation adenoviral vector, the animals mounted an acute phase response that produced neutralizing antibody that eliminated factor ix from the circulation ( ) . finally, the occurrence of severe anaphylactic responses to recombinant proteins is rare in humans. the results of guinea pig anaphylaxis tests, which are generally positive for protein products, are not considered predictive for reactions in humans; therefore, studies such as this are considered of little value for the routine evaluation of these types of products even though they are frequently performed. inflammatory, immune, or autoimmune responses induced by the gene product may be of concern. animal studies should be conducted over a sufficient duration of time to allow development of such responses. host immune responses against viral or transgene proteins may limit their usefulness for repeated administration in the clinic. the immune status of the intended recipients of a gene therapy should be considered in the risk-benefit analysis of a product, particularly for viral vectors. if exclusion of immunocompromised patients would unduly restrict a clinical protocol, immune-suppressed, genetically immunodeficient, or newborn animals may be used in preclinical studies to evaluate any potential safety risks. it is extremely important to investigate the potential for undesirable pharmacological activity in appropriate animal models and, when necessary, to incorporate particular monitoring for these activ-ities in nonclinical toxicity studies and/or clinical studies. safety pharmacology studies are designed to measure functional indices of potential toxicity. these indices may be investigated in separate studies or may be carefully incorporated into the design of nonclinical toxicity studies. the aim of these studies should be to reveal any functional effects on the major physiological systems of the body (e.g., cardiovascular, respiratory, renal, and central nervous systems) that will have a major impact on whether or how the agent is administered in the clinic. some of these investigations may include the use of isolated organs or other test systems that do not involve the use of intact animals, such as the use of a perfused rabbit heart model for the evaluation of torsade de pointes and qt prolongation ( , ) . the results from all of these safety pharmacology studies may allow a mechanistically based explanation of specific organ toxicities, which should be considered carefully with respect to human use and intended indications. the use of additional biomarkers, exemplified by cardiac troponin t or i ( , ) for agents with potential cardiac toxicity, may be warranted in additional nonclinical animal studies and/or in clinical studies in humans. pharmacology studies can be divided into three main categories, depending on the nature of the effect: primary and secondary pharmacodynamic studies and safety pharmacology studies. safety pharmacology studies are defined in the ich guidance document (s a) on this subject ( ) "as those studies that investigate the potential undesirable pharmacodynamic effects of a substance on physiological functions in relation to exposure in the therapeutic range and above." this last point is particularly important in that these studies should be conducted at dose levels or serum concentrations that are therapeutic targets based on prior efficacy/activity studies. simply conducting these studies at low doses does not provide much useful information or adequately assess the safety of the agent. the objectives of these studies are to identify undesirable pharmacodynamic properties of a drug substance that may have relevance to its human safety and toxicity; to evaluate more fully adverse pharmacodynamic and/or pathophysiological effects of a drug substance that were previously observed in nonclinical toxicology and/or clinical studies; and to investigate the mechanism of action of the adverse pharmacodynamic effects that were either previously observed or suspected. the investigational plan developed to meet these objectives should be clearly identified and delineated by the drug development team. for biotechnology-derived products that achieve highly specific receptor targeting, it is often sufficient to evaluate safety pharmacology end-points as a part of well-designed toxicology and/or pharmacodynamic studies; therefore, the need for separate safety pharmacology studies can be reduced or eliminated. for those bioproducts that represent a new therapeutic class and/or those products that do not achieve highly specific receptor targeting, a more extensive evaluation in separate safety pharmacology studies should be considered. biodistribution studies are generally performed for gene therapy products, and typical pharmacokinetic studies used for most types of drugs that measure serum or plasma levels, half-life, clearance, and the like are generally not performed. these preclinical animal biodistribution studies are designed to determine the distribution of the vector to sites other than the intended therapeutic site as an indicator of potential toxicity. the goals of these studies are generally twofold: determination of dissemination of the vector to the germline and distribution of vector to nontarget tissues. the first has been routinely accomplished by assaying total gonadal tissue. the second provides information on potential target organs of toxicity. both may be addressed in the same preclinical study. studies may use normal, intact animals or animal models of disease. the latter study may be more representative of the clinical setting. whenever possible, the intended route of administration should be employed, again with the consideration that groups of animals might also be treated intravenously as a worst-case scenario. transfer of the gene to normal, surrounding, and distal tissues as well as the target site should be evaluated using the most sensitive detection methods possible, such as reverse transcriptase pcr, and should include evaluation of gene persistence. when aberrant or unexpected localization is observed, additional studies should be conducted to determine whether the gene is expressed and whether its presence is associated with any pathologic effects. biodistribution studies may not be necessary for all new agents ( ) . with "previously defined" vectors, if there is previous experience with a similar vector, route of administration, formulation, and schedule (e.g., adenovirus type vectors), if the transgene product is considered "innocuous" if expressed ectopically, and when the size of the new vector is not essentially different, biodistribution aspects of the prior agent may be referenced. on the other hand, studies may not be postponed if a new class of vector is used (i.e., there is little or no previous experience; e.g., aav, lentivirus, others); if there is a change in the formulation (i.e., lipid carrier instead of an aqueous formulation); if the route of administration is changed to an intentional systemic route from local administration of the "established" vector; and finally, if the transgene has the potential to induce toxicity if it is aberrantly expressed in nontarget organs. as with toxicity studies, there are a number of factors that should be taken into consideration when designing vector biodistribution studies. regarding species selection, nonhuman primates are not always needed. rodents may be perfectly acceptable. the animal gender should reflect the intended patient population. at least - animals per sex and group should be used as a minimum. the use of smaller animals (i.e., mice or rats) allows the inclusion of larger numbers of animals and the easy evaluation of more time points. as in other studies, the following dose groups should be included when practical: controls, the maximally feasible/clinically relevant dose, and a lower dose for establishment of the no observable adverse effect level. the route of administration should mimic intended clinical route to the greatest extent possible. regarding animal sacrifice and/or sampling time points, an early point that reflects peak vector transduction/expression should be included, as should a later timepoint determined by intended clinical use and a time-point that should reflect clearance from the gonads and nontarget organs to determine persistence. the following tissues are generally recommended: peripheral blood; gonads; injection site; highly perfused organs (to assist in determination of toxicity) such as brain, liver, lung, kidneys, heart, spleen; other tissues based on toxicity/pathology as determined by transgene (e.g., bone marrow); and those based on the route of administration, such as draining, contralateral lymph nodes. the methodology used to detect the agent should detect a sequence of the vector dna (or ribonucleic acid) that is unique to that product and should be appropriate to detect the vector sequence adequately in tissue samples from both preclinical animal studies and samples obtained during the initial clinical trials. many of the points presented and discussed in this section are elaborated in publicly accessible fda documents ( , ). shedding of viral vectors through the skin or in excreta is of obvious concern with highly infectious viruses. to measure the dissemination, persistence, and shedding of these vectors, multiple tissues (e.g., brain, heart, lungs, spleen, liver, kidneys, ovaries, and skin) as well as bodily fluids such as urine, feces, tears, saliva, vaginal secretions, and skin swabs are taken at multiple time-points throughout the study and analyzed by real-time quantitative pcr for the presence of vector sequences. if the vector sequences are rapidly cleared and viral sequences are absent in the excreta and swabs of the majority of animals, this suggests that there was no significant replication of the vector in the host ( , , ). even if the intended clinical schedule involves repeated doses, single-dose studies may generate useful data to describe the relationship of dose to systemic and/or local toxicity and the steepness of the dose/toxicity curve. data from these studies can be used to select doses for repeated-dose toxicity studies. information on dose-response relationships may be gathered through the conduct of a single-dose toxicity study or as a component of pharmacology or animal model efficacy studies. the incorporation of safety pharmacology parameters in the design of these studies should be considered, which will reduce the number of animals used, the amount of product required, and the number of studies that must be performed. the route and dosing regimen for these studies (e.g., daily vs intermittent dosing) should reflect the intended clinical use or exposure (e.g., once a week for weeks, every other day, etc.). a recovery period should be included to determine the reversal or potential worsening of pharmacological/ toxicological effects and/or the potential for delayed toxic effects. for biopharmaceuticals that induce prolonged pharmacological/toxicological effects, recovery group animals should be monitored until reversibility is demonstrated. this may not be fundamentally obvious at the outset of the study. the duration of repeated dose studies should be based on the intended duration of clinical exposure and disease indication. this duration of animal dosing has generally been - months for most biotechnology-derived pharmaceuticals, but this probably will not be the case for most gene therapy studies. however, in the case of life-threatening diseases such as cancer, longer term studies are generally not required to support phase i trials. one aspect of immunotoxicological evaluation includes the assessment of potential immunogenicity as described in section . . many biotechnology-derived pharmaceuticals are intended to stimulate or suppress the immune system and therefore may affect not only humoral, but also cell-mediated immunity. inflammatory reactions at the injection site may be indicative of a stimulatory response. it is important, however, to recognize that simple injection trauma or specific toxic effects caused by the formulation vehicle may also result in toxic changes at the injection site. in addition, the expression of surface antigens on target cells may be altered, which has implications for autoimmune potential. for conventional small molecule drugs, reproductive toxicity is usually assessed in rats and rabbits. the species specificity and potential immunogenicity of biologicals has led to the increased use of nonhuman primates for this purpose. the need for reproductive and developmental toxicity studies will depend on the product, clinical indication, and intended patient population. the specific study design and dosing schedule may be modified based on issues related to species specificity, immunogenicity, biological activity, and/or a long elimination half-life. the issue of germline integration has prompted considerable public discussion ( ) . for gene therapy products directly administered to patients, the risk of vector transfer to germ cells should be seriously considered. animal testicular or ovarian samples should be analyzed for vector sequences by the most sensitive method available. if a signal is detected in the gonads, further studies should be conducted to determine if the sequences are present in germ cells as opposed to stromal tissues; techniques used may include, but are not limited to, cell separations, in situ pcr, or other techniques. semen samples for analysis can be collected from mature animals, including mice, by well-established methods ( , ) for determination of vector incorporation into germ cells. evaluation of biodistribution to the gonads may not be needed prior to all phase i clinical trials, and this issue should be considered carefully in pre-ind meetings with the fda. the informed consent form should address the lack of data and the unknown risks. genotoxicity studies, such as the ames salmonella assay, the micronucleus test, and the mouse lymphoma assay, which are routinely conducted for small molecule pharmaceuticals, are not appli-cable to biotechnology-derived pharmaceuticals, especially gene therapy products, and therefore are not needed. the administration of large quantities of peptides, proteins, or viruses may yield uninterpretable results. when there is cause for concern about the product, genotoxicity studies should be performed in available and relevant systems, including newly developed systems. the use of standard genotoxicity studies as indicated for assessing the genotoxic potential of process contaminants is not considered appropriate. if standard assays are performed for this purpose, the rationale should be provided. standard -year carcinogenicity bioassays in normal mice and rats are generally inappropriate for biotechnology-derived pharmaceuticals and probably more so for gene therapy products. this issue has received additional attention owing to the emergence of a lymphoproliferative syndrome in a potentially significant fraction of recipients of a vector designed to treat scid syndrome ( , ) . this clinical result actually recapitulates to a certain degree toxicities anticipated from experience in animal models ( ) . thus, product-specific assessment of carcinogenic potential will still be needed for biopharmaceuticals, and studies utilized must be refined after consideration of the duration of anticipated clinical dosing, the patient population, or the biological activity of the product (e.g., retrovirus vectors, growth factors, immunosuppressive agents, etc.). when there is a concern about carcinogenic potential, a variety of new approaches may be considered to evaluate this risk. when the potential to support or induce proliferation of transformed cells and clonal expansion leading to neoplasia is considered possible, the product should be evaluated with respect to receptor expression for the biopharmaceutical or for the transgene's expressed form in various malignant and normal human cells, especially those potentially relevant to the patient population under study. the ability of the biopharmaceutical to stimulate growth of normal and/or malignant cells expressing the relevant receptors should be determined. when in vitro studies such as this give cause for concern about carcinogenic potential, further studies in relevant animal models may be needed if these are available and relevant. as stated in this section, when gene transfer agents must be evaluated, the standard rodent models (mice and rats) and the -year carcinogenicity bioassay are probably not appropriate. daily administration of vector as is usually performed in these studies is not feasible; however, several of these vectors, including aav, continue to express over the lifetime of the animal. the other factor that may be limiting is that the host immune response to the vector or to the transgene may either limit the toxicity, perhaps because of the development of neutralizing antibodies, or may have effects on tumor development. it will be necessary to consult with the fda to develop product-specific studies on an individualized basis or to determine whether and which carcinogenicity studies are needed. local tolerance to administration of the new agent should be evaluated. the formulation intended for the clinical trial should be tested unless there is a cogent reason why this would not be feasible or biologically meaningful. in most cases, the potential adverse effects of the product at the site of administration can be evaluated in the single-or repeated-dose toxicity studies that are usually conducted in the normal course of development, thus eliminating the need for separate studies. adenoviral vectors can efficiently deliver genes to a wide variety of dividing and nondividing cell types both in vitro and in vivo, resulting in a high level of transient gene expression. considerable modifications have been made in the wild-type virus to reduce infectivity and toxicity in normal tissues or to improve transduction or tropism for tumor cells. the death of jesse gelsinger because of several complications, including liver failure, coupled with the fact that adenovirus infections in immunocompromised oncology patients can lead to fatal hepatotoxicity ( , ) , and reports of serious hepatotoxicity and death in nonhuman primates treated with different adenoviral vectors make the safety evaluation of these vectors for cancer treatment paramount. when a first-generation adenovirus vector expressing human factor ix was intravenously injected into rhesus macaques at doses from ´ to ´ pfu/kg, no toxicity was seen at the lower dose level, but substantial, dose-limiting liver toxicity was observed at the higher dose ( ) . this hepatotoxicity was manifested as elevated serum transaminase levels, hyperbilirubinemia, hypoalbuminemia, and prolongation of clotting times. all evidence of liver toxicity resolved except for persistent hypofibrinogenemia in the high-dose recipient, indicating possible permanent liver damage. these data suggested a very narrow therapeutic window for this first-generation adenovirus-mediated gene transfer vector. in follow-up studies ( ) , it was concluded that these abnormalities may be caused by direct toxic effects of the adenovirus vector itself, or may result indirectly from the accompanying acute inflammatory response marked by elevations in interleukin . when another first-generation adenoviral vector expressing b-galactosidase was intravenously injected into two baboons at doses of . ´ or . ´ particles/kg, the baboon receiving the high dose developed acute symptoms, decreased platelet counts, and increased liver enzymes and became moribund at hours after injection; the baboon receiving the lower dose developed no symptoms ( ) . again, a very narrow therapeutic index was demonstrated. recombinant adenoviruses infused into the portal vein of adult rhesus monkeys at a dose of particles/kg resulted in the formation of neutralizing antibody, severe liver toxicity, and death. readministration of a second vector was associated with the same degree of toxicity as the first vector, but prompted a much more vigorous neutralizing antibody response ( ) . the administration of several gene transfer vehicles and routes was studied in rhesus monkeys to develop a model for adenovirusmediated gene transfer for liver. vectors administered via the portal vein or saphenous vein were efficient, but this resulted in transient gene expression and was accompanied by an immune response to both vector and transgene products and acute hepatitis ( ) . turning to models of intracerebral administration, baboons received intracerebral injections of either a high dose of a replication-defective adenoviral vector expressing hsvtk ( . ´ pfu) with or without gcv or a low dose of adv/rsvtk ( . ´ pfu) with gcv to evaluate the safety of this regimen. animals receiving the high-dose vector and gcv either died or became moribund and were sacrificed during the first days of treatment. necropsy of these animals revealed cavities of coagulative necrosis at the injection sites. animals that received only the high-dose vector were clinically normal; however, lesions were detected with mri at the injection sites corresponding to cystic cavities at necropsy. animals that received the low-dose vector and gcv were clinically normal and exhibited small mri abnormalities, and although no gross lesions were present at necropsy, microscopic foci of necrosis were present. neutralizing antibodies were produced in the animals, but no shedding of the vector was found in urine, feces, or serum days after intracerebral injection ( ) . intrapulmonary administration uses are exemplified through the use of recombinant adenovirus vectors containing expression cassettes for human cystic fibrosis transmembrane conductance regulator, which were instilled through a bronchoscope into limited regions of lung in baboons. the only adverse effect noted was a mononuclear cell inflammatory response within the alveolar compartment of animals receiving doses of virus that were required to induce detectable gene expression. minimal inflammation was seen at and pfu/ml, but at and, more prominently, at pfu/ml, a perivascular lymphocytic and histiocytic infiltrate was seen ( ). host immune elimination of infected cells often limits gene expression in vivo to - weeks after infection ( , ) . in addition to a cell-mediated immune response to the adenovirus infection, a humoral response to the injected virus is often generated ( ) . although this humoral response may prevent the use of adenoviral vectors for repeated dosing, it may be blocked or reduced by coadministration of immunosuppressive agents or cytokines. alternatively, the use of adenoviruses of different serotypes may allow for repeated administration, even in the presence of neutralizing antibodies ( ) . harvey et al. ( ) reported on years of experience with the local administration of low (< particle units) and intermediate ( to particle units) doses of e -/e adenovirus vectors to six different sites. with a group incidence of only . % for major adverse events and no deaths related to administration of the adenovirus vectors, local administration of low and intermediate doses of adenovirus vectors was well tolerated. second-generation adenoviral vectors, mutated in e a, have been proposed to decrease host immune responses against transduced cells, reduce toxicity, and increase duration of expression as compared with first-generation vectors deleted only in e . the safety of and e -, e a-, e -deleted adenoviral vector (av h ) encoding an epitope-tagged b-domain-deleted human factor viii complementary dna was evaluated in cynomolgus monkeys. animals received intravenous administration of either ´ or ´ particles/kg. vector distribution was widespread, with the highest levels observed in liver and spleen. histopathology, hematology, and serum chemistry analysis identified the liver and blood as major sites of toxicity. transient mild serum elevations of liver enzymes were observed, along with a dose-dependent inflammatory response in the liver. in addition, mild lymphoid hyperplasia was observed in the spleen. mild anemia and a transient decrease in platelet count were observed, as was marrow hyperplasia and extramedullary hematopoiesis ( ) . when vectors deleted in e and containing either a temperature-sensitive mutation in the e a gene or a deletion of the e region were infused into the hepatic artery of nonhuman primates, minimal toxicity was seen. histopathology showed that portal inflammation was present throughout both livers in the animals receiving the high dose. no differences were seen in the level of portal inflammation in targeted and untargeted lobes. pcr analysis detected viral dna sequence in gonads and brain as well as many other tissues in baboons treated with high-dose vector. in baboons treated with lower doses of an e -e -deleted vector expressing the human ornithine transcarbamylase gene, dna was detectable by nested pcr in liver, but not gonads, at days and . the data suggested that intraarterial administration of recombinant adenoviral e -e -deleted vector is feasible and safe. ( ) . toxicity of first-generation and e a-deleted vectors expressing human a -antitrypsin was evaluated in c h mice after administration of increasing doses starting at ´ particles/kg. both vectors induced dose-dependent toxicity, including transient thrombocytopenia, elevated alanine aminotransferase, and increased hepatocyte proliferation, followed by inflammation and then hypertrophy. there were no differences in toxicity between the two vectors when measured at matching levels of human a -antitrypsin expression. however, the e a-deleted vector had slightly reduced hepatocyte toxicity at an intermediate particle dose ( ) . although these vectors are purported to be less toxic, the fact remains that the human fatality that occurred in the ornithine transcarbamylase deficiency trial at the university of pennsylvania was an e , e -deleted construct ( ). the current e -deleted adenoviruses can infect a wide variety of cells through a specific interaction between the viral fiber protein and at least one cell surface receptor. entry of the virus into the cell is further enhanced through a specific interaction of the fiber with an integrin "coreceptor." the host's range of tissue susceptibilities to the virus can therefore be altered by various strategies so that it can bind more efficiently to the target cell surface ( ) ( ) ( ) . antibodies against tissue-specific cell surface proteins can also be coupled to the fiber protein to facilitate partial targeting of the virus ( ) . another approach to achieve "targeting" of the virus is the use of cell-specific promoters to drive expression of a therapeutic gene in the context of the recombinant virus ( ) . enhanced uptake strategies through fiber modification may present special concerns for toxicity, especially regarding hepatotoxicity when administered by an intravenous or direct hepatic artery injection. careful comparison of a tropism-modified adenoviral vector to the nontropism-modified vector in mouse toxicity and biodistribution studies as well as nonhuman primate and toxicity studies might be desirable. members of the family parvoviridae aavs are among the smallest of the dna viruses ( ) . unlike autonomous parvoviruses, aavs or dependo-viruses require coinfection with unrelated helper viruses for a productive infection to occur ( , ) . as recombinant vectors for gene therapy, they seem to have several advantages compared to other vectors, such as the transduction of terminally differentiated and nondividing cells ( , ) , relatively high stability of transgene expression ( ) , and the potential for targeted integration ( , ) . from a safety point of view, aav vectors show a lack of pathogenicity ( - ), low immunogenicity ( , ) , and low risk of insertional mutagenesis ( ) . also, there did not appear to be any evidence of transduction in the gonads of rhesus monkeys ( ) . however, aav has a limited dna capacity. hsv vectors can deliver large amounts of exogenous dna; however, cytotoxicity and maintenance of transgene expression are obvious obstacles to their use. they also have the advantages of the abilities to infect nondividing cells and to establish latency in some cell types. the ability to establish latency in neuronal cells makes hsv an attractive vector for treating neurological disorders such as parkinson's and alzheimer's diseases. in addition, the ability of hsv to infect efficiently a number of different cell types, such as muscle and liver, may make it an excellent vector for treating nonneurological diseases. one problem associated with hsv-based vectors has been the toxicity of the vector in many different cell types. the generation of hsv vectors with deletions in many of the immediate early gene products, which is similar to the strategy used for adenovirus, has resulted in vectors with reduced toxicity and antigenicity as well as prolonged expression in vivo ( ) ( ) ( ) ( ) ( ) ( ) . no clinical study has been reported in detail with these vectors. section . . . details a summary of preclinical safety considerations pertaining to use of the aotus monkey in comparison to rodent species. retrovirus vectors are replication-defective and are primarily based on the moloney murine leukemia virus (mmlv), which is a well-studied and well-characterized retrovirus ( , ) with numerous advantages. they have been extensively studied, produce stable integration into the host genome, and are very efficient at gene transfer. disadvantages include an infection that is limited to dividing cells, which makes gene transfer into nondividing cells such as hematopoietic stem cells, hepatocytes, myoblasts, and neurons an impossibility, and low titer of products. there are four theoretical concerns that exist for retroviral-mediated gene transfer that relate to two potential delayed toxicities. these are insertional mutagenesis, recombination with endogenous retroviral sequences, transfer of exogenous genetic material, and accidental exposure to replicationcompetent murine retroviruses ( ) . because retroviral vectors can permanently integrate into the genome of the infected cell, there is a serious concern regarding insertional mutagenesis causing the development of a secondary malignancy. the presence of rcrs is of major concern because of the fact that rcrs have produced lethal malignant t-cell lymphomas in / rhesus monkeys ( ) . these concerns resulted in a publication concerning the fda considerations on these issues ( ) and the issuance of a new fda guidance on this subject in october ( ) . some of these con-cerns are no longer theoretical. the elation that this type of retroviral-mediated therapy was successful in curing a number of children with scid ( ) has been severely dampened by the reports of a leukemialike disease produced in two of these children ( - ). unlike oncoretrovirusus such as moloney murine leukemia virus, one subclass of retroviruses, the lentiviruses, can infect nondividing cells. this makes these viruses attractive for gene transfer. one of these viruses, human immunodeficiency virus (hiv), has been the subject of investigation by a number of groups. the most obvious concerns with using hiv for gene therapy is safety and the possible generation of replication-competent virus during vector production. this involves engineering the vector so that it is replication defective. this has been done in a number of cases by eliminating all accessory genes, such as tat, vif, vpr, vpu, and nef, from a packaging construct that still has the ability to transduce cells ( ) . concern about the possibility of insertional activation of cellular oncogenes by a random integration of the vector provirus into the host genome has led to the development of self-inactivating vectors ( ) ( ) ( ) ( ) . the use of self-inactivating viruses significantly improves the biosafety of hiv-derived vectors because it reduces the likelihood that rcrs will originate during vector production and target cells and hampers recombination with wild-type hiv in an infected host. in an attempt to make even safer constructs, other groups are working on the development of lentiviral vectors from hiv- ( ), simian immunodeficiency virus ( ), bovine immunodeficiency virus ( , ) , and feline immunodeficiency virus ( ) . these last vectors may be inherently more acceptable because they are not based on hiv- . none of these newer constructs has moved toward the clinic, so there is little animal safety data and no human data on these vectors yet. this chapter presents a range of issues that might be considered in contemplating the development of a gene therapy agent to the point of an early phase clinical trial. as no gene therapy product has yet been recognized as "safe and effective," the standard approach to these issues should be regarded very much as a "work in progress." indeed, the nature of these agents would suggest that each new opportunity would call for its own unique set of requirements, so that a single approach will probably never "standardly" exist. rather, the principles that underlie regulatory policy should be woven into the approach to each new agent. in broad strokes, these involve approaches to answering the following questions: is the identity of the agent clearly defined? can successive batches of the material be made reproducibly in the quantity to support clinical development, and how is this known? are the biological features of the vector, and its transgene when applicable, clearly similar in the animal species used for safety studies and in the human, at least as far as this can be ascertained? what dose is likely to be required for therapeutic effect? what level of gene expression or replication is necessary to attain a therapeutic effect? when toxicity occurs because of the agent, what is the evidence the toxicity will be reversible? is toxicity after repeated doses of agent likely to be attenuated or magnified by immunological response to the agent? what are the consequences of long-term presence of the therapeutic agent in the recipient? is there a danger of producing directly (as the therapeutic agent itself) or indirectly (through recombination and/or replication) an infectious agent that acts horizontally in the population or vertically across generations? how will the presence and distribution of the gene therapy agent be followed in the patient? sponsors are above all encouraged to see the regulatory process as a collaborative interaction with the regulatory agencies with the end not only of protecting the patient, but also of advancing the most scientifically defensible and rigorous questions to clinical trial. far more costly than the conduct of experiments designed to be compliant with regulatory requirements is a failed or overtly injurious clinical trial. a clear understanding and a proactive approach in addressing regulatory issues outlined here will maximally ensure the likelihood of an interpretable clinical outcome. the regulatory issues outlined here must be approached with continuing appreciation of the evolving science associated with the gene therapy field. as such, requirements may evolve with the state of the science, and careful sustained contact with the regulatory agencies is important in incorporating the best and most current science into the design, conduct, and interpretation of regulatory studies. ich guidance on quality of biotechnology/biological products: derivation and characterization of cell substrates used for production of biotechnical/biological products ich: final guideline on quality of biotechnological products: analysis of the expression construct in cells used for production of r-dna derived protein products ich guidance on specifications: test procedures and acceptance criteria for biotechnological/biological products ich guidance for industry q a (r ), stability testing of new drug substances and products ich guidance on viral safety evaluation of biotechnology products derived from cell lines of human or animal origin ich guidance: q a good manufacturing practice guidance for active pharmaceutical ingredients ich guidance for industry: s preclinical safety evaluation of biotechnology-derived pharmaceuticals ich: final guidance on stability testing of biotechnological/biological products fda guidance for industry: guidance for human somatic cell therapy and gene therapy gene therapy: a tragic setback a serious adverse event after successful gene therapy for x-linked severe combined immunodeficiency regulators split on gene therapy as patient shows signs of cancer second cancer case halts gene-therapy trials preclinical development strategies for novel gene therapy products the role of the toxicologic pathologist in the preclinical safety evaluation of biotechnologyderived pharmaceuticals nonclinical safety evaluation of biotechnologically derived pharmaceuticals non-clinical safety studies for biotechnology-derived pharmaceuticals: conclusions from an international workshop ich guidance to industry: s a safety pharmacology studies for human pharmaceuticals code of federal regulations, title , food and drugs, part . , subpart b, general biological products standards; general provisions; potency code of federal regulations, title , food and drugs, part . , subpart b, general biological products standards code of federal regulations, title , food and drugs, part . , subpart b, general biological products standards code of federal regulations, title , food and drugs, part . , subpart b, general biological products standards code of federal regulations, title , food and drugs, part , good laboratory practice for nonclinical laboratory studies current good manufacturing practice in manufacturing, processing, packing, or holding of drugs; general code of federal regulations, title , food and drugs, part , current good manufacturing practice for finished pharmaceuticals code of federal regulations, title , food and drugs, part , investigational new drug application inds) for phase i studies of drugs, including well-characterized, therapeutic, biotechnology-derived products fda guidance for industry: ind's for phases and studies of drugs, including specified therapeutic biotechnology-derived products, chemistry, manufacturing and controls content and format fda guidance for industry: content and format of chemistry, manufacturing, and controls information and establishment description information for a vaccine or related product fda guidance for industry for the submission of chemistry, manufacturing, and controls information for a therapeutic recombinant dna-derived product or a monoclonal antibody product for in vivo use fda guidance for industry: formal meetings with sponsors and applicants for pdufa products fda points to consider in the manufacturing and testing of monoclonal antibody products for human use fda letter to manufacturers of biological products: recommendations regarding bovine spongiform encephalopathy (bse) fda biological response modifiers advisory committee: current policy on sequence characterization of gene transfer products fda biological response modifiers advisory committee: adenovirus titer measurements and rca levels fda guidance for industry: supplemental guidance on testing for replication competent retrovirus in retroviral vector based gene therapy products and during follow-up of patients in clinical trials using retroviral vectors fda points to consider in the characterization of cell lines used to produce biologicals fda gene therapy patient tracking system final document fda guidance concerning demonstration of comparability of human biological products, including therapeutic biotechnology-derived products third national nih gene transfer safety symposium: safety considerations in the use of aav vectors in gene transfer clinical trials basic principles of gene therapy: basic principles and safety considerations preclinical animal models in gene therapy research a new animal model for human respiratory tract disease due to adenovirus use of aotus monkey to assess neurovirulence of replication-selective herpes vectors herpes simplex type infects and establishes latency in the brain and trigeminal ganglia during primary infection of the lip in cotton rats and mice tropism of human adenovirus type -based vectors in swine and their ability to protect against transmissible gastroenteritis coronavirus porcine toxicology studies of sch , an adenoviral vector for the p gene pathogenesis of adenovirus type pneumonia in cotton rats (sigmodon hispidus) the cotton rat in biomedical research handling the cotton rat for research role of early region (e ) in pathogenesis of adenovirus disease early region -replacement adenovirus recombinants are less pathogenic in cotton rats and mice than early region -deleted viruses intracranial administration of adenovirus expressing hsv-tk in combination with ganciclovir produces a dose-dependent, self limiting inflammatory response distribution, persistency, toxicity, and lack of replication of an e a-deficient adenoviral vector after intracardiac delivery in the cotton rat subcutaneous administration of a replication-competent adenovirus expressing hsv-tk to cotton rats: dissemination, dersistence, shedding, and pathogenicity. hum the owl monkey (aotus trivirgatus) as an animal model for viral diseases and oncologic studies characterization of four herpesviruses isolated from owl monkeys and their comparison with herpesvirus saimiri type (herpesvirus tamarinus) and herpes simplex virus type immunization of experimental animals with reconstituted glycoprotein mixtures of herpes simplex virus and : protection against challenge with virulent virus in vivo behavior of genetically engineered herpes simplex viruses r and r . ii. studies in immunocompetent and immunosuppressed owl monkeys (aotus trivirgatus) attenuated, replication-competent herpes simplex virus type mutant g : safety evaluation of intracerebral injection in nonhuman primates viral shedding and biodistribution of g , a multimutated, conditionally replicating herpes simplex virus type , after intracerebral inoculation in aotus preclinical safety evaluation of g , a replication-competent herpes simplex virus type , inoculated intraprostatically in mice and nonhuman primates attenuated, replication-competent herpes simplex virus type mutant g : safety evaluation in mice concordance of the toxicity of pharmaceuticals in humans and in animals quantitative comparison of toxicity of anticancer agents in mouse, rat, hamster, dog, monkey, and man the rhesus macaque as an animal model for hemophilia b gene therapy adenovirus-mediated expression of human coagulation factor ix in the rhesus macaque is associated with dose-limiting toxicity experimental models of torsade de pointes drug-related torsade de pointes in the isolated rabbit heart: comparison of clofilium, d,l-sotalol and erythromycin elevations in cardiac troponin measurements: false false-positives: the real truth predicting cancer therapy-induced cardiotoxicity: the role of troponins and other markers preclinical considerations for gene transfer clinical trials: vector biodistribution adenoviral-mediated thymidine kinase gene transfer into the primate brain followed by systemic ganciclovir: pathologic, radiologic, and molecular studies meeting summary (available on-line at: www .od.nih.gov/oba/rac/summaries/ - sum.htm.) and meeting minutes rac minutes- / - / . available on-line at: www .od a technique for the artificial insemination of mice an improved method for the artificial insemination of mice helper virus induced t cell lymphoma in nonhuman primates after retroviral mediated gene transfer adenovirus infection in the immunocompromised patient fulminant hepatic failure due to disseminated adenovirus infection in a patient with chronic lymphocytic leukemia toxicity of a first-generation adenoviral vector in rhesus macaques lethal toxicity, severe endothelial injury, and a threshold effect with high doses of an adenoviral vector in baboons gene transfer into the liver of nonhuman primates with e -deleted recombinant adenoviral vectors: safety of readministration liver-directed gene transfer in non-human primates adenovirus-mediated transfer of the cftr gene to lung of nonhuman primates: toxicity study role of viral antigens in destructive cellular immune responses to adenovirus vector-transduced cells in mouse lungs clearance of adenovirus-infected hepatocytes by mhc class i-restricted cd + ctls in vivo circumvention of anti-adenovirus neutralizing immunity by administration of an adenoviral vector of an alternate serotype safety of local delivery of low-and intermediate-dose adenovirus gene transfer vectors to individuals with a spectrum of morbid conditions adenoviral vector-mediated expression of physiologic levels of human factor viii in nonhuman primates selective gene transfer into the liver of non-human primates with e -deleted, e a-defective, or e -e deleted recombinant adenoviruses toxicological comparison of e a-deleted and first-generation adenoviral vectors expressing alpha -antitrypsin after systemic delivery recombinant adenovirus gene transfer in adults with partial ornithine transcarbamylase deficiency (otcd). hum targeting of adenovirus penton base to new receptors through replacement of its rgd motif with other receptor-specific peptide motifs generation of recombinant adenovirus vectors with modified fibers for altering viral tropism towards the use of replicative adenoviral vectors for cancer gene therapy targeted adenovirus gene transfer to endothelial and smooth muscle cells by using bispecific antibodies a new adenoviral vector: replacement of all viral coding sequences with kb of dna independently expressing both full-length dystrophin and b-galactosidase characteristics and taxonomy of parvoviridae adenovirus-associated defective virus particles herpes simplex virus types and completely help adenovirus-associated virus replication prospects for the use of adeno-associated virus as a vector for human gene therapy adeno-associated virus vectors for gene therapy stable in vivo expression of the cystic fibrosis transmembrane conductance regulator with an adeno-associated virus vector site-specific integration by adeno-associated virus mapping and direct visualisation of regionspecific viral dna integration site on chromosome q -qter epidemiology of adenoassociated virus infection in a nursery population a seroepidemiologic study of adenovirus-associated virus infection in infants and children adeno-associated viruses of humans high efficiency transfer of the t cell co-stimulatory molecule b - to lymphoid cells using high-titer recombinant adeno-associated virus vectors. hum regulated high level expression of a human g-globin gene introduced into erythroid cells by an adeno-associated virus vector preclinical evaluation of aav vectors expressing the human ctfr cdna retroviral vectors for use in human gene therapy for cancer, gaucher disease, and arthritis effects of retroviral vector design on expression of human adenosine deaminase in murine bone marrow transplant recipients engrafted with genetically modified cells safety aspects of gene therapy evaluation of recommendations for replication competent retrovirus testing associated with use of retroviral vectors sustained correction of x-linked severe combined immunodeficiency by ex vivo gene therapy minimal requirement for a lentivirus vector based on human immunodeficiency virus type development of a self-inactivating lentivirus vector advanced modular self-inactivating lentiviral expression vectors for multigene interventions in mammalian cells and in vivo transduction transduction of acute myeloid leukemia cells with third generation self-inactivating lentiviral vectors expressing cd and gm-csf: effects on proliferation, differentiation, and stimulation of allogeneic and autologous anti-leukemia immune responses self-inactivating lentivirus vector for safe and efficient in vivo gene delivery human immunodeficiency virus type (hiv- ) vectormediated in vivo gene transfer into adult rabbit retina pseudotyped lentivirus vectors derived from simian immunodeficiency virus sivagm with envelope glycoproteins from paramyxovirus construction and molecular analysis of gene transfer systems derived from bovine immunodeficiency virus mapping of the bovine immunodeficiency virus packaging signal and rre and incorporation into a minimal gene transfer vector gene transfer to the nonhuman primate retina with recombinant feline immunodeficiency virus vectors key: cord- -szb jm authors: reza khorramizadeh, m.; saadat, farshid title: animal models for human disease date: - - journal: animal biotechnology doi: . /b - - - - . - sha: doc_id: cord_uid: szb jm this chapter introduces some types of animal models which are used for better understanding the disease mechanisms and its treatment. these experimental models fall into two categories: spontaneous models and induced models. among the diseases, rheumatoid arthritis (ra) as an autoimmune disease was considered. to study the pathogenesis of ra, we explained collagen-induced arthritis as an animal model that reflects a characteristic feature of ra patients. in addition, experimental allergic encephalomyelitis (eae) as an experimental model for multiple sclerosis (ms) was explained in detail to represent a standard method to investigate in its mechanism, finding the way for the amelioration of this incurable neurological disorder. the architecture of human body is comprised in such a manner that cells cannot be considered as a separate entity. physiologically, homeostasis is the reason that these components live and perform their functions within that environment. disruption of this process leads to fatal conditions and is considered a disease. to investigate the mechanism of disease and to find the means to reverse adverse conditions, various strategies are used including cell-based assays and tissue culture studies. although these models can provide useful information, they fail to address various physiological conditions and the complex interactions among different cell types of tissues and organs. ideally a useful animal model for any disease has to have pathology similar to the disease conditions in humans. use of animals in research has a long history that dates back to the fourth century b.c. in the s, william harvey used animals to describe the blood circulatory system. many scientists, such as louis pasteur and emil von behring, have used animal models for experimental purposes to prove their hypotheses. animal models are good for understanding disease mechanisms and treatment and for overcoming the limitations of clinical trials that use human subjects. for example, experimental animal models for diseases like rheumatoid arthritis or multiple sclerosis have been successfully employed to screen new bioengineered, chemical, or herbal therapeutics that might have the potential for the treatment of human patients. so far, more than , studies have been reported in the ncbi database; they use animal models for different diseases. animal model studies have been the main reason for a better understanding of disease mechanisms. animal models of disease can be divided into two categories (kurkó et al., ) : spontaneous disease models and ( van heemst et al., ) induced disease models. in the case of induced disease models, induction can occur by various agents, both chemical and biological. this chapter discusses some of the most important animal models. rheumatoid arthritis (ra) is an autoimmune disorder with progressive occurrence that preferentially affects peripheral joints. in spite of the fact that ra is severe and crippling and affects large numbers of people, very little knowledge about its etiology and pathogenesis is available in the literature. rheumatoid arthritis affects about % of the population. the ratio of the prevalence of ra in males and females is À . . ra can occur at any age, but it is mainly reported to affect the -to -year-old age group. no doubt the incidence has been reported to increase with age. the etiology of ra is unknown, but it has been predicted that genetic and environmental factors play an important role in the onset of ra. recent advances have identified genetic susceptibility markers both within and outside of the major histocompatibility complex (mhc). human leukocyte antigen (hla) genes located on chromosome p have been found to have a strong association with rheumatoid arthritis. the contribution of hla to heritability of ra has been estimated to be %À %. individuals carrying hla-dr and hla-dr alleles have been shown to have a higher risk of ra. apart from the known shared epitope alleles (hla-drb à , drb à ), other hla alleles, such as hla-drb à and drb à , have been linked to ra susceptibility (kurkó et al., ) . the hla class ii locus is the most important risk factor for anticitrullinated protein antibodies (acpa)-positive ra (acpa ra) (van heemst et al., ) . a positive correlation has been suggested for the role of hla in terms of the severity of ra rather than the onset of the disease. the most relevant non-hla gene single nucleotide polymorphisms (snps) associated with ra include ptpn , il r, traf , ctla , irf , stat , ccr , and padi (kurkó et al., ; suzuki and yamamoto, ; stanford and bottini, ) . although the data regarding this conclusion are inconsistent, some of the studies have shown associations between tumor necrosis factor (tnf) alleles and rheumatoid arthritis. other genes like those for corticotrophin-releasing hormone, interferon (ifn)-γ, and interleukin- (il- ) have also been implied for ra. it can be concluded that the role of genetic components in ra is modest at the best (viatte et al., ) . epigenetics is another important factor that contributes to ra. in the case of identical twins, ra has not been shown to have % concordance; therefore, the role of nongenetic factors has also been implicated in the etiology of ra (meda et al., ) . throughout the world, rheumatoid arthritis is more common in women than in men. this indicates that hormones may play an important role in the development of the disease. pregnancy has also been considered as a risk factor for rheumatoid arthritis. studies show that the onset of ra is rare during pregnancy, but the risk increases after delivery. smoking is associated with increased incidences of ra, especially in men. on the contrary, populations that consume a diet high in omega- fatty acids have been reported to be protected from rheumatoid arthritis. from experimental models in animals, a large number of infectious agents such as viruses and bacteria have also been suggested to trigger or contribute to the development of rheumatoid arthritis. however, no relationship between infectious agents and the development of ra has been found. tissue edema and fibrin deposition are prominent and can manifest clinically as joint swelling and pain. within a short period, the synovial lining becomes hyperplastic, commonly becoming ten or more cells deep and consisting of type a (macrophage-like) and type b (fibroblast-like) synoviocytes that produce glycosaminoglycans (e.g., hyaluronan, as reported to be present in synovial tissue and synovial fluid). the sublining also undergoes alterations for its cellularity, both in cell type and in cell numbers, with prominent infiltration of mononuclear cells, including t cells, b cells, macrophages, and plasma cells. the abundance and activation of macrophages at the inflamed synovial membrane correlates significantly with the severity of the disease. activated macrophages over-express major histocompatibility complex (mhc) class ii molecules and produce pro-inflammatory or regulatory cytokines and growth factors [il- , il- , il- , il- , il- , il- , tnf-α, and granulocyte macrophage colony stimulating factor (gm-csf)], chemokines [il- , macrophage inflammatory protein (mip- ), monocyte chemoattractant protein (mcp- )], metalloproteinases, and neopterin. these biomolecules are routinely detected in inflamed joints. most of the t cells infiltrating the rheumatoid synovium express cd ro and cd , which is an indication that the t-cell subset present in the synovium is memory helper t cells. surprisingly, %À % of the t cells present in the case of the synovium have granzymes a and perforins. this %À % of cells present in the synovium represents cytotoxic t-cell subsets. therefore, it can be concluded that cd -expressing cells are infrequent in the synovium. in the synovial fluid of rheumatoid arthritis patients, cd and cd t cells are equally represented. tcrα/tcrß is expressed on most of the t cells while only a minority of cells show tcrγ/tcrδ expression. it has, however, been found that the expression of tcrγ/tcrδ is increased in the synovium of patients with active ra. synovial-vessel endothelial cells transform into high endothelial venules early during the course of disease. high endothelial venules are specialized post-capillary venules usually present in secondary lymphoid tissue or inflamed nonlymphoid tissues; these venules facilitate the transit of leukocytes from the bloodstream into tissues. the cytokine-mediated events have conventionally been viewed in the milieu of the cd th /th paradigm. nowadays, newer cytokines of the il- /il- axis and others have changed investigations into the immunopathogenesis of arthritis. both the cd th and γδ-t cells secreted il- which is a chemotactic for neutrophils and its response inhibited by il- due to ifn-γ induction. the roles of other cytokines such as il- and il- in arthritis have been clarified further with inhibitors of them (veenbergen et al., ; palmer et al., ) . recent studies in arthritis models have revealed new aspects toward regulatory t cell (treg) activity. in the cia model, treatment with il- induced the regression of arthritis via expansion of regulatory t cells (kochetkova et al., ) . the formation of locally invasive synovial tissue (i.e. pannus) is a characteristic feature of rheumatoid arthritis. pannus is involved in the erosion of joints in rheumatoid arthritis. pannus is histologically distinct from other regions of the synovium and shows phases of progression. initially, there is penetration of cartilage by synovial pannus, which is composed of mononuclear cells and fibroblasts, with a high-level expression of matrix metalloproteinases (mmps) by synovial lining cells. in later phases of the disease, cellular pannus can be replaced by fibrous pannus comprised of a minimally vascularized layer of pannus cells and collagen overlying cartilage. the tissue derivation of pannus cells has not been fully elucidated, although they are thought to arise from fibroblast-like cells (type-b synoviocytes). in vitro work shows that these fibroblast-like synoviocytes have anchorageindependent proliferation and loss of contact inhibition, which a phenotype is usually found in transformed cells. however, the molecular pathogenic mechanisms driving pannus formation still remains poorly understood. the range of presentations of rheumatoid arthritis is broad, but the disease onset is insidious in most cases, and several months can elapse before a firm diagnosis can be ascertained. the predominant symptoms are pain, stiffness, and swelling of peripheral joints. although articular symptoms are often dominant, rheumatoid arthritis is a systemic disease. active rheumatoid arthritis is associated with a number of extraarticular manifestations, including fever, weight loss, malaise, anemia, osteoporosis, and lymphadenopathy. the clinical course of the disorder is extremely variable, ranging from mild, self-limiting arthritis to rapidly progressive multisystem inflammation with a profound morbidity and mortality. analyses of clinical course and laboratory and radiological abnormalities have been defined as negative prognostic factors for progressive joint destruction; unfortunately, none of these are reliable enough to allow therapeutic decisionmaking. frequent assessment of disease symptoms and responses to therapy is crucial for a successful and long-term management of rheumatoid arthritis. joint destruction from synovitis can occur rapidly and early in the course of the disorder; radiographic evidence is present in more than % of patients within the initial years. more sensitive techniques such as magnetic resonance imaging (mri) can identify substantial synovial hypertrophy, bone edema, and early erosive changes as early as months after the onset of disease. these radiographic changes predate misalignment and functional disability by years; by the time physical deformity is evident, substantial irreversible articular damage has commonly occurred. furthermore, the biopsy analysis of clinically symptomless knee joints in patients with early rheumatoid arthritis shows active synovitis, highlighting the poor correlation between clinical assessment and disease progression, and the rapid development of polyarticular synovitis. the main goal of ra treatment is to stop inflammation, relieve symptoms, prevent joint damage, and reduce long-term complications. the past decade has seen a major transformation in the treatment of rheumatoid arthritis in terms of approach and choice of drugs. the previous therapeutic approach generally involved initial conservative management with nonsteroidal antiinflammatory drugs (nsaids) for several years; disease-modifying antirheumatic drugs (dmards) were withheld until a clear evidence of erosion was seen. dmards were then added individually in slow succession as the disease progressed. this form of treatment has been supplanted by early initiation of dmards and combination dmard therapy in patients with the potential for progressive disease. the idea of early intervention with the conventional disease-modifying antirheumatic drugs (cdmard) has been validated in several randomized trials. cdmards contain medications from different classes of drugs including methotrexate, gold salts, hydroxychloroquine, sulfasalazine, ciclosporin, and azathioprine. dmards are often partly effective and poorly tolerated for long-term therapy. in metaanalyses of dropout rates from clinical trials, %À % of patients discontinued the use of dmards assessed as monotherapy during the duration of the trial; even in clinical practice, the median duration of dmard monotherapy was less than years for nonmethotrexate agents. although there are many reasons for the lack of long-term adherence to treatment, poor efficacy, delayed onset of action, and toxic effects are major limitations. additionally, dmards therapy requires patients to undergo frequent monitoring of blood and physical examinations for toxic effects of treatment protocol. results from clinical trials showed that dmard therapy decreased markers of inflammation such as erythrocyte sedimentation rate and swollen joint counts, and that improved symptoms in a selected subset of patients; however, most patients continued to show progression of irreversible joint destruction on radiography. cdmards is increasingly burdened by side effects or clinical inefficacy, so other immunosuppressive drugs such as tacrolimus that blocks t-cell activation by specifically inhibiting calcineurin pathway and leflunomide have been developed. a new synthetic dmard, iguratimod, which exerts its action by the inhibition of the inflammatory cytokines (tnf-α, interleukin (il)- β, il- , il- , and il- ), is recently developed. the findings illustrate the consequences of progressive disease and have shown the need for the development of new and more effective therapies based on the therapeutic principles used for oncology; it means that treatment protocols for ra patients require the use of several therapeutic agents from different classes to be used in combination. recent studies have shown that combination therapy of biological dmards like tnfα inhibitors with methotrexate has clear-cut benefits with tolerable toxic effects. treatment with agents that can block tnf-α function has proved to be highly effective against ra. further studies reported downregulation of synovial gm-csf, il- , and il- , suggesting that tnf-α supports the production of other pro-inflammatory cytokines. however, the mechanisms behind the clinical effect of the tnf-α-blocking treatment are not fully understood. in an animal model, tnf-α-blocking agents such as etanercept (a soluble tnf-α receptor) and infliximab (a monoclonal antibody) reduce the expression of vascular adhesion molecules and inhibit the spontaneous production of il- and il- . patients with a new onset of symptoms and those with diseases of several years' duration and who had failed previous dmard therapy all benefited. these results suggest that patients in many stages of disease progression can benefit from combination therapy (chiu et al., ) . with the approval of tnf-α inhibitors (infliximab, etanercept, adalimumab, certolizumab, and golimumab), non-tnf biologic agents (rituximab, abatacept, tocilizumab, and anakinra), and other biologic agents, determining advances in treatment options of ra were made. rituximab (chimeric monoclonal antibody targeted against cd ) is a selective b-cell depleting agent for treating refractory rheumatoid arthritis. abatacept selectively modulates t-cell co-stimulation and has shown efficacy in several clinical trials. tocilizumab, a humanized monoclonal antiinterleukin- receptor antibody, has proven to be efficacious in patients who did not respond to methotrexate or other synthetic dmards. recently, several clinical trials have focused on a new class of drug: the janus kinase (jak) inhibitors. jaks are a family of nonreceptor tyrosine kinases . animal models for human disease (jak , jak , jak , and tyk ) involved in the intracellular signal transduction of many cytokines. tofacitinib is a pan-jak inhibitor that primarily inhibits jak and jak . in addition to tofacitinib, other jak inhibitor molecules including baricitinib, peficitinib, and decernotinib have also been studied in rcts. finally, filgotinib is a selective jak inhibitor which is currently in clinical development for the treatment of ra (calabrò et al., ) . as mentioned before, pro-inflammatory/regulatory cytokines and growth factors play important roles in the pathogenesis of ra. therefore, each of them or their pathway represents an attractive therapeutic target for ra. tocilizumab, a humanized monoclonal antibody targeting il- receptor, has already been approved for the treatment of ra in patients who failed to achieve remission with cdmards. another cytokine that plays an important role in the pathogenesis of ra is il- in which ixekizumab and brodalumab as humanized monoclonal antibody were developed against il a and its receptor. a new possible therapeutic target for the treatment of ra is the gm-csf pathway. the efficacy and safety of mavrilimumab (an anti-gm-csf receptor monoclonal antibody) in patients with moderate-to-severe ra has been investigated (takeuchi et al., ) . as an increased activation of osteoclasts contributes to bone erosions in ra, the inhibition of rankl that is essential for the osteoclast activation by denosumab (human monoclonal antibody against rankl) can reduce joint destruction in ra patients. takeuchi et al. ( ) , finally, do not forget that certain nutritional components interfere in the pathological inflammatory process, so that they should be considered as coadjuvant in the treatment of ra. it has been mentioned that flavonoids reduce cytokine expression and secretion. in this regard, flavonoids may have a therapeutical potential in the treatment of inflammationrelated diseases as cytokine modulators (rosillo et al., ; leyva-lópez et al., ) . in order to study the pathogenesis of ra, one can use different animal models. there are many experimental models that resemble ra in different respects. since ra is a heterogeneous disease, there is probably a need for different animal models that each reflect a characteristic feature of a particular subgroup of ra patients or illustrate a particular aspect of the disease. despite the fact that ra is not a spontaneously developing disease, spontaneously developing models for arthritis may be useful to study the role of genetics in the development of the disease. an activated immune response was reported in models such as the human tumor necrosis factor-α transgenic (htnftg), interleukin receptor-α (il- ra) knockout, il- ractivating mutation knockin, or skg mouse which bears the primary inflammatory response in joints (keffer et al., ) . in addition, arthritis can be rapidly induced with an adoptive transfer of t-helper cells in the il- r knockin mouse. transgenic mice expressing a tcr specific for bovine pancreas ribonuclease develop spontaneous arthritis that is mediated by antibodies (korganow et al., ) . this model is particularly interesting because it demonstrates that t cells specific for a ubiquitous antigen may induce an organspecific autoimmune disease. the expression of the gene product causes an upregulation of several cytokines (il- , il- , tgf-β , ifn-γ, and il- ) and subsequent development of arthritis (iwakura et al., ) . there are some other spontaneous models for arthritis in nontransgenic mice (bouvet et al., ) . arthritis can be induced by complete freunds' adjuvant (cfa). pearson (pearson, ) described this model for the first time. subsequently, it was demonstrated that other adjuvants, such as ifa, pristane, or squalene, could also induce arthritis (carlson et al., ) . microbially derived products such as lipopolysaccharide (lps), muramyle dipeptide (mdp), and trehalosedimycolate (tdm) can also induce arthritis when given with mineral oil (lorentzen, ; kohashi et al., ) . collagen-induced arthritis (cia) is normally induced by the immunization of susceptible mouse (e.g., dba/ ) or rat (da, lewis) strains at the base of the tail. the inoculum used for immunization contains both adjuvant and collagen type ii. the adjuvant has to be sufficiently strong to cause tissue destruction as well as induction of a strong pro-inflammatory immune response (holmdahl and kvick, ; kleinau et al., ) . susceptibility to cia is dependent on both mhc (class ii region) and non-mhc genes (lorentzen and klareskog, ) . antibodies against collagen ii are essential for the development of cia. this fact has been demonstrated by the passive transfer of anti-cii antibodies, which results in synovitis (svensson et al., ) . t cells are also important for cia development during early stages of disease progression. the dependence of both t-and b-cell responses has also been demonstrated in the same model (seki et al., ) . in cia, an immune response is being directed against a joint collagen type ii (cii) antigen. inflamed joints in cia are infiltrated by inflammatory cells that accumulate in the synovial membrane and fluid, similar to ra. the most frequent cell type in the synovial fluid is granulocyte. there is also a great infiltration of leukocytes into the synovial membrane. these cells have signs of an activated phenotype of ra since mhc class ii molecules are expressed (klareskog and johnell, ). in addition, there is an intense production of macrophage-derived cytokines in inflamed joints (e.g., tnf-α and il- β) (mussener et al., ; ulfgren et al., ) . a small number of t cells are encountered, and some of these t cells have il- receptor α chain upregulated. the disease shows a thickened synovial membrane that subsequently forms a pannus on the cartilage surface (holmdahl et al., ; holmdahl et al., ) . in both cia and ra, cartilage and bone destruction occurs mainly at the carti-lageÀpannus junction. there are some features of the pathology of cia that differ from what is usually observed in ra (e.g., extra-articular manifestation). although the compatibility of the cia model to human ra has been argued, many pathological features of cia are similar to those of rheumatoid arthritis. currently, collagen type ii-induced arthritis in mice and rats is one of the most widely used arthritis models in academia and industry. experimental collagen-induced arthritis was initiated by injecting bovine collagen type ii at the base portion of the tail of the animal (saadat et al., ) . male lewis rats weighing about À g were used. after the induction of cia, animals were divided randomly into four or more groups based on the experimental design. at least four different groups were needed, including a control group without arthritis, animals with collagen-induced arthritis, cia animals with treatment, and cia animals treated with methotrexate as a positive control. sample preparation: bovine collagen type ii (cii) was dissolved in . m acetic acid at a concentration of mg/ml by stirring overnight at c (the dissolved cii can be stored at c if it has to be used at a later time). before injecting the animals, cii was emulsified with an equal volume of complete freund's adjuvant (cfa). for the induction of cia, on day rats were injected intradermally at the base of the tail with μl of emulsion (containing μg of cii). after À days, animals showed the development of inflammation at peripheral joints ( fig. . ). on day , a booster injection of cii in cfa was administered. this model was used to evaluate the anti-ra effect by giving intraperitoneally injections of test materials (e.g., chemical or herbal extracts). methotrexate was a control used to evaluate the effect of the test compound and to compare the efficacy of the new compound with methotrexate. in this model, the test compound was given from day , where the frequency, route of administration, and dose could be selected as needed. the end point and days for the evaluation of different parameters were selected; one of the most common points was day (flow chart . ). the paws and knees were then removed for the histopathological assay. the visual observation can be done by using the macroscopic system as given in table . . moreover, rats immunized with cfa should be checked for weight gain from the first to the end of experiment at least every other day. the decline in body weight that followed on the onset of arthritis was proportional to the disease severity and, hence, can be used as a measure of disease activity. the scaling to record the observation should be from to for each paw (szabó et al., ) . on day , animals were anesthetized with sodium pentobarbital ( mg/kg intraperitoneally) and euthanized. blood was collected by intracardiac puncture, and paws and knees were removed, trimmed, and fixed in % buffered formalin, decalcified, and then embedded in paraffin, sectioned at μm, and stained with hematoxylin and eosin for the histological examination. joint damage was assessed based on synovial hypertrophy, pannus formation, inflammatory cell infiltration, and cartilage and subchondral bone destruction. joint erosion was graded on a scale of À for each limb (table . ), according to the severity of damage ( fig. . ). radiological scoring was performed by an investigator who was blind to the treatment protocol (on day ). radiographical analysis of affected joints in control rats typically showed soft tissue swelling, joint space narrowing, reduced lucency due to demineralization, and areas of recalcification indicative of new bone formation. a score was assigned to each joint on the basis of the information as listed in table . . scores were À per joint ( , normal; , maximum joint destruction). multiple sclerosis (ms) is a chronic inflammatory demyelinating disorder of the central nervous system (cns) that affects over . million individuals worldwide. similar to the affected population in other autoimmune diseases, twice as many women as men have ms. multiple sclerosis, like many other diseases, has existed as long as human life. in the s, the first report by dr. jean-martin charcot certified ms as a disease. a patient of him who suffered an unusual symptom died. after dissection, brain lesions were discovered. he called the disease scleroseen plaques. myelin was subsequently discovered, although its exact role was not recognized. about one century of research resulted in the discovery multiple sclerosis is the most common inflammatory demyelinating disease of the cns in europe and north america. the prevalence of ms in north america and europe is b À per , people. however, the prevalence is not globally uniform, geographically decreases in latitudes, and has been observed in only b À per , individuals in africa and asia. the etiology of ms is unknown. both genetic involvement and environmental factors have been indicated in ms. the only consistent correlation of involvement of the mhc locus is the mhc class ii allele hla-dr , which reflects a linkage with ms. in addition to associations within the major histocompatibility complex (mhc) region, other non-mhc loci reached a genome-wide significance. they map to the genes l mbtl , maz, erg, and shmt . products of the genes l mbtl , maz, and erg play important roles in immune cell regulation. shmt encodes a serine hydroxymethyl transferase catalyzing the transfer of a carbon unit to the folate cycle, which is important for establishment and maintenance of epigenetic signatures (andlauer et al., ) . some other factors like dietary components (e.g., milk), pathogens like human herpes virus (hhv- ), measles virus, epsteinÀbarr virus, and chlamydia have been implied as etiological factors. however, the association between any of these agents with ms is debatable. the presence of cns inflammation is a hallmark of ms. this inflammatory process greatly increases in the cns by the activation and deregulation of different cell types of the immune system. activation and entry of myelin-specific lymphocytes into the cns cause damage to oligodendrocytes, leading to demyelination. most of the cells from the immune system can contribute toward demyelination, but the main process of demylation is mediated by antibody and complement. so far, it has been noticed that antibody and complement are responsible for lesions in %À % of ms patients. myeloid cells may cause axonal damage by releasing molecules, such as glutamate, reactive oxy-gen species, and reactive nitrogen species. besides, these cells decreased the expression of glutamate clearance. as a result of increased glutamate in the cerebrospinal fluid, ms patients would be vulnerable to degeneration (yandamuri and lane, ) . in addition, cd t cells play an important role in ms pathogenesis. cd t cells make up the largest percentage of lymphocytes found in the brain of ms patients. all neuroectodermal cells in ms lesions express mhc class i molecules, making them an excellent target for cd t cells. in addition to their proinflammatory properties, cd t cells can also suppress the immune system and down-regulate inflammation. however, experiments with perforin, an important regulator of cytotoxic damage to immune cells, have made it clear that cd t cells present at ms lesions cause cytotoxicity, which could be the main source for demyelination and axonal damage (sinha et al., ) . bystander cd t cells do not contribute to the demyelinating process, but once cd t cells move into the cns and become activated against myelin antigen, these cd t cells could be contributing directly toward the demyelination of cns (basdeo et al., ) . moreover, cd th effector t cells are postulated to play a crucial role in the pathogenesis of ms (bettelli et al., ) . in addition to autoreactive immune cells against myelin and nerves, a progressive loss of the structure and function of neurons occurs. it has been reported that the alterations in the expression of mirnas may play a crucial role in ms pathogenesis (huang et al., ) . after demyelination, remyelination is possible, which could further damage the cns. the ratio of demyelination to remyelination determines whether a patient will develop secondary progressive ms (spms) or relapse remitting ms (rrms). if remyelination occurs before axonal damage, irreversible physiological damage can be prevented. none of the fda-approved therapies target oligodendrocytes to stimulate remyelination, but it is a very interesting possibility for future therapeutic intervention (huang et al., ) . the majority of symptoms associated with ms can be directly attributed to inflammation, edema, demyelination, and/or axonal damage within the brain, spinal cord, and optic nerves. clinical motor manifestations include weakness, stiffness, and/or pain in arms or legs, abnormal reflex activity, and spasticity. often, the earliest symptoms of ms are somatosensory, including numbness and tingling. in ms, cerebral involvement is often accompanied by symptoms such as ataxia and intention tremor. many individuals with ms complain of increased urinary frequency, urgency, and incontinence. bladder and bowel disturbances remain among the most disabling and embarrassing symptoms experienced by ms patients. sexual symptoms are also very common among both men and women. fatigue, sleep disturbances, depression, and deficits in cognitive functioning are also common. the clinical course of ms is often highly variable and is generally characterized by relapses or exacerbations and deterioration of neurologic function, which entitle relapsing remitting ms. the features of relapsing remitting ms are defined as "episodes of acute worsening of neurologic function followed by a variable degree of recovery, with a stable course between attacks." approximately %À % of patients are initially diagnosed with rrms that evolves from an isolated demyelinating attack, which is characterized by multifocal inflammation along with varying degrees of axonal injury. a patient may experience disease progression with or without relapses and minor remissions; that clinical condition is defined as secondary progressive ms. it has been seen that % of rrma patients will eventually develop the spms state. primary progressive ms (ppms) affects b %À % of ms patients. ppms is defined as "disease progression from onset, with occasional plateaus and temporary minor improvements in clinical condition." the duration of ms varies significantly among ms patients. some patients will live with ms for several decades, while about % will develop an acute, fulminant form of ms. patients with an acute and fulminant form of ms show a rapid deterioration in their clinical signs and symptoms that have fatal consequences; these patients usually die within À years after the onset of disease. in general, the clinical spectrums among ms patients represent a benign disease and a low relapse rate, and these may never develop into secondary progressive disease. the heterogeneity of the clinical course of ms is shown to have a similar variation in its pathology. multiple sclerosis lesions were recently segregated into four distinct subtypes. the general pathology of ms, the formation of demyelinating lesions in the cns associated with infiltrating cd t cells, activated macrophages, and microglia-containing myelin debris, and infiltrating b cells, is common to all forms of the disease. it is thought that ms lesions are mediated by soluble factors such as tnf-α and immunoglobulin deposition on the myelin sheath, and the local activation of the complement cascade. the diagnostic criteria for clinically definite ms (cdms) include factors such as clinical history, mri imaging, and csf abnormalities. at present, there are no identifiable biomarkers that can predict the clinical subtype of ms. similarly, there are no factors that can assist in predicting whether a patient diagnosed with ms will develop either a progressive or a benign version of the disease. the clinical and pathological heterogeneity in ms has made it important to either develop or identify reliable biomarkers. several cytokines, immunoglobulins, mmps, markers of axonal/neuronal injury, and apoptotic markers have been suggested to have potential as biomarkers, but these biomarkers need validation by rigorous durability trials. in ms, treatment strategies can be either acute or long term. during a relapse, the goal of acute treatment is to reverse neurological disability as well as to delay further neurological dysfunction, so that the normal function can be restored. this type of treatment for ms patients is in contrast to the goals of long-term treatments. the main objective of long-term treatment for ms patients is to decrease relapses (both severity and frequency), which could lend support to stopping the progression of disability. patients experiencing a relapse, such as optic neuritis or transverse myelitis, are often administered high-dose corticosteroid firstline therapy. during progressive phases of the disease, patients may be prescribed immunosuppressive agents such as cyclophosphamide or mitoxantrone because the progressive phase is often accompanied by worsening inflammatory demyelination and axonal degeneration (rommer et al., ) . in , ifn-β was the first agent to demonstrate the significant clinical efficacy among patients suffering with rrms. although the exact disease-modifying effects of ifn-β in ms are unknown, several immunomodulatory mechanisms have been suggested. presently, two forms of ifn-β, including ifn-βla (avonex and rebif) and ifn-βlb (betaseron and extavia), have been prescribed. glatiramer acetate (copaxone) is a synthetic mixture of polypeptides that has been approved to treat rrms. similar to ifn-β, glatiramer acetate is found to be not effective for progressive forms of ms. natalizumab (tysabri) is an alpha- integrin antagonist and is the first drug of an entirely new class of immune-directed therapies that has been approved by the fda to treat relapsing ms. natalizumab is a humanized recombinant monoclonal antibody that blocks leukocyte migration into the cns by binding to α- integrins; these are components of the very late antigen- (vla- ) complex constitutively expressed on the leukocyte surface. in monotherapy trials, natalizumab has been reported to reduce the risk for sustained progression of disability as well as decrease the frequency of relapses. based on the current literature, natalizumab appears to be one of the most effective agents to prevent relapses as well as to stop disease progression. other monoclonal antibodies administered by intravenous (iv) infusion include lemtrada (alemtuzumab) and novantrone (mitoxantrone). currently, numerous other monoclonal antibodies are under investigation as potential therapies for ms; for example, anti-cd (daclizumab), anti-cd (rituximab), and so on. a number of other agents are under investigation for possible future use in ms including secukinumab (a humanized monoclonal antibody to il- ), rtl (inhibitor of the activation of myelin-reactive t cells), firategrast (affect on the vla- system) and aimspro (neuropeptide stabilizer). moreover, stem cell-based therapy might be considered as another approach for attenuating ms through regulating the immune system, although several challenges should be resolved. more investigations and clinical trials should be designed to assess the effectiveness of several drugs and approaches that can target both inflammatory and degenerative components of ms. these kinds of approaches may offer hope for individuals who are suffering from this debilitating disease (mansoor et al., ; agrawal and yong, ; hart and bainbridge, ) . to gain ideas about ms mechanisms, a number of models have been developed. these experimental models fall into two categories: spontaneous models and induced models. each model reflects characteristic features of ms patients and has its own merits and demerits. myelin basic protein mutant (taiep rat), proteolipid protein mutants (rumpshaker and jimpy mice), as well as gene-knockout animals (the myelin-associated glycoprotein (mag) knockout, thy -eb -yfp mice, and thy -xfp mice) show dysmyelination, altered neurotransmission and, in some instances, clinical disease. these models have frequently been used to study myelination. with chemically induced lesions, viral and autoimmune models are developed to show some evidence of demyelination, which is considered a pathological hallmark of ms. direct injection of ethidium bromide or lysolecithin into the cns produces demyelination. these induced models are usually effectively repaired once macrophages clear the myelin debris. for this reason, these models are rarely used at the present time. besides, local administration of glutamate or nitric oxide donors induces axonopathy in mice and have also been used to understand mechanisms of axonal degeneration and regeneration (luchtman et al., ) . a number of viruses, including semliki forest virus, theiler's murine encephalomyelitis virus, and a murine coronavirus have been found to induce disease by neurotrophic infection of the cns, specifically oligodendrocytes (lane and hosking, ) . moreover, studies using immunodeficient rag / mice have indicated that cd and cd t lymphocytes as well as macrophages are key contributors to demyelination in coronavirus-infected mice (dandekar et al., ) . finally, experimental allergic encephalomyelitis (eae) has received the most attention as a model for ms; this animal model is routinely used for testing different therapeutic strategies. today, eae as the most commonly used preclinical murine model of ms induced actively by the injection of defined encephalitogenic myelin protein epitopes plus cfa, or passively by the transfer of encephalitogenic myelin-sensitized t lymphocytes. some of these eae models also require the administration of the microbial-based immunologic adjuvant pertussis toxin (pt) (yandamuri and lane, ) . eae exhibits many clinical and histological features of ms and is caused by autoimmunity induced against antigens that are expressed either naturally or artificially in cns (denic et al., ) . the method for eae induction and preparation of antigens to induce eae in c bl/ mice was adapted from the method described by kafami et al. ( ) . it is important for the successful induction of eae to follow standard precautions for the use of animals. female c bl/ mice that are -to -week old are used for the induction of eae. animals must adhere to the normal laboratory animal maintenance guide. animals were immunized with the hooke kits (hooke labs, ek- , lawrence, ma, usa). it is recommended to follow the manufacturer's instructions. a mesh was dampened in ether and put in a desiccator. the mouse was kept in the desiccator and observed until breathing slowed down to ascertain whether the mouse had been anesthetized. the mouse was removed from the anesthetic chamber and laid on its side. two syringes were filled with ml of myelin oligodendrocyte glycoprotein (mog) emulsion with complete freund's adjuvant. each animal was given an injection of μl. the needle was gently inserted into the subcutaneous space at the base of the tail, and μl of emulsion was injected into the site. since it was difficult to give the mouse a -μl injection, every mouse was given a -μl injection at two different sites on the same day. immediately, and after hours from the first injection, each mouse was given an intraperitoneal injection of pertussis toxin ( μl/ animal). the animal was observed until complete recovery, and it could move without a floppy gate. this procedure was repeated for all animals. after À days, the flanks were bulging in response to the subcutaneous injection (flow chart . ). one day before immunization, and from the th to the th day post-immunization, the animals were evaluated on a daily basis for signs of eae following the -point score system (table . three different clinical parameters were analyzed to compare the course of eae (fig. . ) : ( ) severity of disease as the cumulative disease index (cdi) was the mean of the clinical scores of the animals; ( ) disease onset, calculated as the mean of the first-day animals showed the signs of the disease in experimental animals; and ( ) peak of disease score, which represented the mean of the highest clinical score of disease for all animals in each group. tonicity of the tail and the distal part of the tail was ascertained by touching the tip of the tail. if the distal part of the tail was flaccid, the animal was removed from the base and observed to see if its tail remained erect or fell down (examined with the touch of the finger). after ascertaining tonicity of the tail, the gate of the animal was observed by keeping it in an open area (like a tabletop) and allowing it to walk. after checking the gate, the hind limb was observed by grabbing its tail. after that, the paralysis score was recorded for unilateral paralysis. by holding the animal in the palm of the hand, it was easy to evaluate the type of paralysis (unilateral or bilateral). it was noted whether the mouse rolled spontaneously in its cage or was dead with complete paralysis. after days, animals that had an eae score of and did not change for more days were euthanized by chloral hydrate injection ( . ml, ip). for histopathlogical evaluations, different tissues were harvested after dissecting the animals. animals were placed appropriately in the dissection tray. a midline incision was made on the abdomen; the diaphragm was opened while ribbons were cut to expose the beating heart. the needle was inserted into the left ventricle of the heart while a phosphate-buffered saline (pbs) tap was allowed to fill the heart for seconds. the right aorta was cut with small scissors to allow the pbs and pfa to circulate to exit. pbs allowed perfusion until the liver turned from red to yellow (b À minutes). the best sign was when the liquid flowed out of the incised left aorta and turned from red to clear. another indicator was when pbs entered the pulmonary system and emerged through the nose of the animal. then, the pbs tap was closed and the tap was turned on for % paraformaldehyde (pfa, ph . at c) to allow pfa to flow and perfuse the circulatory system for minutes. perfusion was evaluated by involuntary hind limb movement and tail shivering. when the mouse became stiff, it was time to stop pfa perfusion. after the perfusion was complete with pfa, the system was washed with pbs to remove residual pfa. after perfusion, the various tissues of interest were harvested and stored in fresh % pfa for days at c. then, these tissues were washed with pbs and the pfa-fixed tissue could be stored in pbs for a few months. these tissues were then available for sectioning and staining (fig. . ) . for immunohistochemistry, the three sections showing the highest infiltrations were studied. an area $ . μm from the brain/spinal cord was selected and analyzed under magnification to assess the average number of positive cells per millimeter square and to quantify it on a computerized imaging system [bx microscope (olympus, hamburg, germany) with analysis software (special sis docu; soft imagingsystem)] by planimetry. the inflammatory index had to be calculated as a percentage determined by dividing the number of visual fields with . cd t cells by the total number of visual fields examined. detection of amyloid precursor protein (app) was performed for acute axonal damage. to assess the content of circulating proinflammatory cytokines like il- , il- , il- , il- , tnf-α, and ifn-γ, enzyme-linked immunosorbent assay (elisa) was employed. to evaluate the levels of different cytokines, blood was collected into tubes by a retro-orbital plexus method. the collected blood was kept in the tube to clot. after the clotting of the blood serum, it was separated and stored at c. these serum samples were then used for the evaluation of different cytokines using the elisa. in order to quantify the mrna of different proinflammatory cytokines such as tnf-α and ifn-γ, antiinflammatory cytokines like il- , myelin-deteriorating matrix metalloproteinase mmp- , and the content of . animal models for human disease myelin basic protein (mbp À ), samples from animals had to be analyzed by real-time pcr. animals were sacrificed with lethal injection and perfused with cold pbs. then, the limbs and muscles were removed with scissors and the skin removed from these organs. a transverse cut was made at the base of the skull and vertebral column to separate them. the nasal bridge was broken with a small scalpel and the eyeballs removed. very thin forceps were used under the skull bones to break it into pieces from the frontal to occipital lobes. the bony connection under the cerebellum was broken to expose the cerebellum. the broken bones of the skull needed to be removed. the nerve root connection with the brain was cut. the brain was removed and stored in liquid nitrogen. for the removal of the spinal cord, an oblique cut was made from the lateral side of the spinal cord (started from the cervical part) to the furthest part of the vertebral column (both sides). the spinal cord was then exposed by cutting the boney flap. the steps above were repeated to get to the coda aquina. the spinal cord was taken out by cutting its adhesion to the base. it was then stored in liquid nitrogen. the frozen tissue sample was used for rna extraction. first, the sample was homogenized by pushing and rotating it with a sterile glass homogenizer. next, the homogenate sample was left on the bench top at room temperature ( cÀ c) for minutes to promote the dissociation of nucleoprotein complexes. then, μl of chloroform was added to the tube and the tube was shaken vigorously for seconds. the tube containing the homogenate was placed on the bench top at room temperature for À minutes and then centrifuged again at , rpm for minutes at c. after centrifugation, the sample separated into three phases: an upper, colorless, aqueous phase containing rna; a white interphase; and a lower, red, organic phase. the upper, aqueous phase was transferred to a new sterile eppendorf tube. one volume (usually μl) of % ethanol was added to the tube containing the aqueous phase and mixed thoroughly by vortexing. visible precipitates after the addition of ethanol could then be noticed. up to μl of the sample was processed for total rna extraction by using an rneasy mini spin column (roche germany) according to the kit instructions. after rna extraction, rna was quantified spectrophotometrically and the purity of rna was ascertained by taking out a ration between the od at and nm. a quantitative real-time reverse transcriptase pcr was performed to analyze the levels of mrna of different cytokines using cytokine-specific primers. the first step was to perform cdna synthesis by using a cdna synthesis kit (takara, japan), which was followed by a syber green i real-time pcr master mix kit (takara, japan). a house-keeping gene (like the β-actin gene) was included in the study to compare the results. the use of laboratory animals in research is of major ethical concern. much of the argument revolves around moral values. today, there is a wide spectrum of views on animal rights. this has prompted the establishment of guidelines on the care and use of experimental animal models. the guidelines endorse some essential principles for the care and use of animals for scientific projects. the basis of these principles is to replace animals with other methods such as mathematical models, computer simulations, and in vitro biological systems, thus reducing the number of animals used in order to obtain valid results without unnecessary duplication, and finally, refining projects by selecting appropriate species and techniques to minimize pain or distress to animals using appropriate sedation or anesthesia. as a researcher, one must always assume that procedures that cause pain to humans will cause pain in such situations in animals. surgical procedures should be performed on anaesthetized animals. it should be kept in mind that if the animal would suffer severe pain during a procedure, or if at the end point cannot be alleviated swiftly, the animals must be killed humanely. the transportation, housing, feeding, and handling of animals are also important. housing facilities should be compatible with the needs of the species and equipped to achieve a high standard of animal care. the place should be designed to facilitate control of environmental factors. cages should be comfortable and should fulfill behavioral requirements such as free movement and activity, bedding, contact with others of the same species, lighting, temperature, air quality, appropriate day/night cycles, and protection from excessive noise. the population density of animals within cages should also be considered from an ethical standpoint. this statement refers to the need for the reader to operate in accordance with the guidelines at her/his academy. the concept of translational research is to try to convert the results derived in animal models into a new understanding of disease mechanisms and therapeutics in human beings. it is a bridge from experimental models to clinical medicine. over recent years, the importance of this kind or research has progressively increased. consequently, translational research is considered a key component to finding practical applications, especially within medicine. with the improvement of technologies, significant progress has been made in producing various types of engineered experimental animal models based on a better understanding of the molecular and genetic principles of disease. as a result, any interventions in experimental models are more practical and repeatable when compared to patient-oriented research. various risk factors that are linked to, or even responsible for, differences in clinical results should also be considered as significant for the development of experimental models; this will enhance the translational value of experimental models. these risk factors can be categorized into genetic factors, acquired factors, and health conditions, which can be studied in models in a controlled manner. in medicine, the performance of successful translational research requires data from hospitals. as we mentioned before, rheumatoid arthritis as a progressive debilitating disease is characterized by hyperplasia of synoviocytes leading to joint destruction and permanent deformity. although the definite pathophysiology of ra is ambiguous, some evidence suggests that telomerase is also involved in the pathogenesis of this disease. nobel laureates in physiology/ medicine in , elizabeth blackburn, jack szostak and carol greider, have solved a major problem of the chromosomal protection against degradation during cell divisions. they identified telomerase and a unique dna sequence in the telomeres. telomerase is a ribonucleoprotein enzyme that adds repeated units of ttaggg to the ends of chromosomes. this enzyme is composed of an rna component, called htert which serves as a template for addition of telomeric repeats. although it is now known that the dna sequence in the telomere attracts proteins that form a protective cap around the fragile ends of the dna strands, a number of reports have mentioned a link between the increased telomerase activity of human tumor samples and degree of invasiveness. in patients with ra, an impaired telomerase enzyme and premature cellular ageing (senescence) of thymic naïve and memory t cells was reported. moreover, transfection of rheumatoid arthritis synovial fibroblasts with vectors expressing antisense oligonucleotide against the htert component of telomerase enzyme has led to cytolysis of these cells that exhibit high telomerase activity. taken together, their discoveries have shed light on disease mechanisms and stimulated the development of potential new therapies in experimental models. there are many methods to evaluate telomerase activity, but we measured it by telomere repeat amplification protocol using trapeze telomerase detection kit (intergen, inc., usa) in animals treated with camellia sinensis stew. in detailed, biopsies of synovial tissue were obtained aseptically from the knee joints of rat after the induction of cia. synovial tissue specimens were rinsed, minced, and digested with . % collagenase in high-glucose dmem containing % fbs and antibiotics. following overnight incubation at c, cells were collected, plated in culture flask, and allowed to reach confluency at c in a humidified atmosphere of % co . after the lysis of equal number of cells which harvested from synovial tissue with the chaps lysis buffer, the telomerase was first extended for minutes at % oc and then amplified by cycles of pcr. the products of pcr were detected by polyacrylamide gels and revealed by silver nitrate staining. telomerase activity was calculated as the ratio of the intensity of telomerase ladders to the intensity of the -bp internal standard. in conclusion, we show that c. sinensis stew effectively suppresses collagen arthritis and a potent inhibitory effect on telomerase activity. so, natural products should continue to provide innovative lead compounds currently entering clinical trials. recently, the circular plant peptide kalata b (cyclotide) was investigated by thell et al. using the ms mouse model experimental autoimmune encephalomyelitis. according to their findings, treatment of mice with the cyclotide resulted in a significant delay and diminished symptoms of eae by oral administration. taken together, natural product should be considered as a candidate for the future investigations to possible implication for human health. using these above-mentioned models associated with other experimental models gives us such an opportunity to accomplish many findings in human medicine. until now, many progresses in medical sciences have been achieved. the discovery of numerous types of antibiotics for controlling infectious disease and elimination some viral disease like smallpox might be considered as one of researcher and indeed experimental animals honor. also, blood transfusions, open heart surgery, and other life-saving techniques have all been developed. nevertheless, there are many unsolved subjects included cancer, aging, alzheimer's disease, and acquired immunodeficiency syndrome in front of the society. without no doubt, until to find another means for answering human beings dilemma, the use of living animals in scientific research would be the best and applicable procedure. with all those valuable function, it is pivotal to consider ethical concerns over the quality of life of animals when you as a young researcher start to write a proposal. medlineplus is the national institutes of health (nih) site for patients and their families and friends. produced by the national library of medicine, it brings you information about diseases, conditions, and wellness issues in easy-to-understand language. medlineplus offers reliable, up-to-date health information, anytime, anywhere, for free. http://www.ebi.ac.uk/ipd/imgt/hla/ the imgt/hla database provides a specialist database for sequences of the human major histocompatibility complex (hla) and includes the official sequences for the who nomenclature committee for factors of the hla system. the imgt/hla database is part of the international immunogenetics project. adjuvant a substance such as complete freund's adjuvant (cfa) that enhances t-and b-cell activation, mainly by promoting the accumulation and activation of antigen-presenting cells at the site of antigen exposure. adjuvants stimulate the expression of t-cellactivating co-stimulators and cytokines by antigen-presenting cells and may also prolong the expression of peptideÀmhc complexes on the surface of these cells. autoimmune disease a disease caused by a breakdown of selftolerance such that the adaptive immune system responds to selfantigens and mediates cell and tissue damage. autoimmune diseases can be organ specific (e.g., thyroiditis or diabetes) or systemic (e.g., systemic lupus erythematosus). cd molecules cell surface molecules expressed on various cell types in the immune system that are designated by the "cluster of differentiation (cd) number." disease-modifying antirheumatic drugs (dmards) they contain medications from different classes including methotrexate, gold salts, hydroxychloroquine, sulfasalazine, ciclosporin, and azathioprine. dmards were often only partly effective and poorly tolerated in long-term therapy of autoimmune diseases. enzyme-linked immunosorbent assay (elisa) a method of quantifying an antigen immobilized on a solid surface by use of a specific antibody with a covalently coupled enzyme. the amount of antibody that binds the antigen is proportional to the amount of antigen present and is determined by spectrophotometrically measuring the conversion of a clear substrate to a colored product by the coupled enzyme. experimental autoimmune encephalomyelitis (eae) this is an animal model of multiple sclerosis, an autoimmune demyelinating disease of the central nervous system. eae is induced in rodents by immunization with components of the myelin sheath (e.g., myelin basic protein) of nerves, mixed with an adjuvant. the disease is mediated in large part by cytokine-secreting cd t cells specific for the myelin sheath proteins. granulocyte-monocyte colony-stimulating factor (gm-csf) a cytokine made by activated t cells, macrophages, endothelial cells, and stromal fibroblasts that acts on bone marrow to increase the production of neutrophils and monocytes. gm-csf is also a macrophage-activating factor and promotes the differentiation of langerhans cells into mature dendritic cells. granuloma a nodule of inflammatory tissue composed of clusters of activated macrophages and t lymphocytes, often associated with necrosis and fibrosis. granulomatous inflammation is a form of chronic delayed-type hypersensitivity, often in response to persistent microbes or to particulate antigens that are not readily phagocytosed. granzyme a serine protease enzyme found in the granules of ctls and nk cells is released by exocytosis, enters target cells, and proteolytically cleaves and activates caspases and induces target cell apoptosis. homeostasis in the adaptive immune system, the maintenance of a constant number and diverse repertoire of lymphocytes, despite the emergence of new lymphocytes and the tremendous expansion of individual clones that may occur during responses to immunogenic antigens. homeostasis is achieved by several regulated pathways of lymphocyte death and inactivation. human leukocyte antigens (hla) mhc molecules expressed on the surface of human cells. human mhc molecules were first identified as alloantigens on the surface of white blood cells (leukocytes) that bound serum antibodies from individuals previously exposed to other individuals' cells. interferons a subgroup of cytokines originally named for their ability to interfere with viral infections, but that have other important immunomodulatory functions. type i interferons include interferon-α and interferon-β, whose main functions are antiviral; type ii interferon, also called interferon-γ, activates macrophages and various other cell types. interleukins any of a large number of cytokines named with a numerical suffix roughly sequentially in order of discovery or molecular characterization (e.g., interleukin- and interleukin- ). some cytokines were originally named for their biological activities and do not have an interleukin designation. lipopolysaccharide (lps) a component of the cell wall of gramnegative bacteria that is released from dying bacteria and stimulates many innate immune responses, including the secretion of cytokines, induction of microbicidal activities of macrophages, and expression of leukocyte adhesion molecules on endothelium. lps contains both lipid components and carbohydrate moieties. major histocompatibility complex (mhc) molecule a heterodimeric membrane protein encoded in the mhc locus that serves as a peptide display molecule for recognition by t lymphocytes. two structurally distinct types of mhc molecules exist. class i mhc molecules are present on most nucleated cells, bind peptides derived from cytosolic proteins, and are recognized by cd t cells. class ii mhc molecules are restricted largely to dendritic cells, macrophages, and b lymphocytes, bind peptides derived from endocytosed proteins, and are recognized by cd t cells. matrix metalloproteinase (mmp) mmps are a family of highly conserved endopeptidases dependent on zn ions for activity. mmps can collectively cleave most extracellular matrix. at present, vertebrate mmps and human homologs have been identified and characterized. mmps participate in many physiological processes, such as embryonic development, organ morphogenesis, blastocyst implantation, ovulation, nerve growth, cervical dilatation, postpartum uterine involution, mammary development, endometrial cycling, hair follicle cycling, angiogenesis, inflammatory cell function, apoptosis, tooth eruption, bone remodeling, and wound healing. myelin oligodendrocyte glycoprotein (mog) mog is a cnsspecific type i membrane glycoprotein of the immunoglobulin superfamily expressed mainly on the outermost layer of the myelin sheath, making it an ideal target for antibody-mediated demyelination. it is highly immunogenic, and unlike other myelin proteins used to induce eae, is unique in inducing both an encephalitogenic t-cell response and a demyelinating response in eae. multiple sclerosis (ms) a chronic inflammatory demyelinating disorder of the central nervous system. the majority of symptoms associated with ms can be directly attributed to inflammation, edema, demyelination, and/or axonal damage within the brain, spinal cord, and optic nerves. nitric oxide (no) a biologic effector molecule with a broad range of activities that in macrophages functions as a potent microbicidal agent to kill ingested organisms. pannus formation of locally invasive synovial tissue is a characteristic feature of rheumatoid arthritis. perforin a protein that is homologous to the c complement protein and is present in the granules of ctls and nk cells. when perforin is released from the granules of activated ctls or nk cells, it promotes the entry of granzymes into the target cell, leading to apoptotic death of the cell. rheumatoid arthritis (ra) an autoimmune disease characterized primarily by inflammatory damage to joints and sometimes inflammation of blood vessels, lungs, and other tissues. cd t cells, activated b lymphocytes, and plasma cells are found in the inflamed joint lining (synovium), and numerous proinflammatory cytokines, including il- and tnf, are present in the synovial (joint) fluid. reverse transcriptase (rt) an enzyme encoded by retroviruses, such as hiv, that synthesizes a dna copy of the viral genome from the rna genomic template. purified reverse transcriptase is used widely in molecular biology research for purposes of cloning complementary dnas encoding a gene of interest from messenger rna. th cells subset of cd helper t cells whose principal function is to stimulate phagocyte-mediated defense against infections via secretion of a group of cytokines, including ifn-γ. th cells subset of cd helper t cells whose principal functions are to stimulate ige and eosinophil/mast cell-mediated immune reactions via a particular set of cytokines, including il- and il- . th cells subset of cd helper t cells that are protective against certain bacterial infections and also mediate pathogenic responses in autoimmune diseases. tumor necrosis factor (tnf) a cytokine produced mainly by activated mononuclear phagocytes that stimulates the recruitment of neutrophils to sites of inflammation. tnf-α blocking agents a group of biological disease-modifying antirheumatic drugs such as etanercept (a soluble tnf-α receptor) and infliximab (a monoclonal antibody). very late antigen (vla) the set of integrins that shares a common beta- chain. long-answer questions immunopathogenesis of multiple sclerosis novel multiple sclerosis susceptibility loci implicated in epigenetic regulation immunology increased expression of tbet in cd t cells from clinically isolated syndrome patients at high risk of conversion to clinically definite ms reciprocal developmental pathways for the generation of pathogenic effector th and regulatory t cells spontaneous rheumatoid-like arthritis in a line of mice sensitive to collagen-induced arthritis one year in review : novelties in the treatment of rheumatoid arthritis the endogenous adjuvant squalene can induce a chronic t-cell-mediated arthritis in rats access to the next wave of biologic therapies (abatacept and tocilizumab) for the treatment of rheumatoid arthritis in england and wales axonal damage is t cell mediated and occurs concomitantly with demyelination in mice infected with a neurotropic coronavirus the relevance of animal models in multiple sclerosis research current and emerging treatment of multiple sclerosis hla and rheumatoid arthritis: how do they connect? vaccination and genetic experiments demonstrate that adjuvant-oil-induced arthritis and homologous type ii collagen-induced arthritis in the same rat strain are different diseases early appearance of activated cd t lymphocytes and class ii antigen-expressing cells in joints of dba/ mice immunized with type ii collagen involvement of macrophages and dendritic cells in synovial inflammation of collagen induced arthritis in dba/ mice and spontaneous arthritis in mrl/lpr mice micrornas associated with the pathogenesis of multiple sclerosis autoimmunity induction by human t cell leukemia virus type in transgenic mice that develop chronic inflammatory arthropathy resembling rheumatoid arthritis in humans intermittent feeding attenuates clinical course of experimental autoimmune encephalomyelitis in c bl/ mice transgenic mice expressing human tumour necrosis factor: a predictive genetic model of arthritis induced expression of class ii transplantation antigens in the cartilage-pannus junction in ra: chronic synovitis as a model system for aberrant t-lymphocyte activation role of adjuvants in turning autoimmunity into autoimmune disease il- stimulation of cd regulatory t cells confers protection against collagen ii-induced arthritis via the production ofil- arthritis-inducing ability of a synthetic adjuvant, n-acetylmuramyl peptides, and bacterial disaccharide peptides related to different oil vehicles and their composition from systemic t cell self-reactivity to organ-specific autoimmune disease via immunoglobulins genetics of rheumatoid arthritis -a comprehensive review the pathogenesis of murine coronavirus infection of the central nervous system flavonoids as cytokine modulators: a possible therapy for inflammation-related diseases identification of arthritogenic adjuvants of self and foreign origin susceptibility of da rats to arthritis induced with adjuvant oil or rat collagen is determined by genes both within and outside the major histocompatibility complex in vivo and in vitro effects of multiple sclerosis immunomodulatory ther-apeutics on glutamatergic excitotoxicity the potential use of mesenchymal stem cells for the treatment of multiple sclerosis the epigenetics of autoimmunity cytokine production in synovial tissue of mice with collagen-induced arthritis (cia) inhibition of interleukin- signaling attenuates the severity of experimental arthritis development of arthritis, periarthritis and periostitis in rats given adjuvants an update on dietary phenolic compounds in the prevention and management of rheumatoid arthritis effect of pyrimethamine in experimental rheumatoid arthritis type ii collagen-induced murine arthritis. i. induction and perpetuation of arthritis require synergy between humoral and cell-mediated immunity cd ( ) t-cells as immune regulators of multiple sclerosis ptpn : the archetypal non-hla autoimmunity gene from genetics to functional insights into rheumatoid arthritis b celldeficient mice do not develop type ii collagen-induced arthritis (cia) protection against peroxynitrite-induced fibroblast injury and arthritis development by inhibition of poly (adpribose) synthetase efficacy and safety of mavrilimumab in japanese subjects with rheumatoid arthritis: findings from a phase iia study effect of denosumab on japanese patients with rheumatoid arthritis: a doseresponse study of amg (denosumab) in patients with rheumatoid arthritis on methotrexate to validate inhibitory effect on bone erosion (drive)-a -month, multicentre, randomized, double-blind, placebo-controlled, phase ii clinical trial interindividual and intra-articular variation of proinflammatory cytokines in patients with rheumatoid arthritis: potential implications for treatment the natural soluble form of il- receptor beta exacerbates collagen-induced arthritis via modulation of t-cell immune responses genetics and epigenetics of rheumatoid arthritis imaging axonal degeneration and repair in preclinical animal models of multiple sclerosis use of animals in scientific research. indian council of medical research ministry of health & family welfare new delhi animal models of rheumatoid arthritis and their relevance to human disease animal models of multiple sclerosis-potentials and limitations the rights of animals animal models of multiple sclerosis. neuroinflammation À . glossary adhesion molecule a cell surface molecule (e.g., selectin, integrin, and member of the ig superfamily) whose function is to promote adhesive interactions with other cells or the extracellular matrix describe the significance of animal modeling in biotechnology? how cia is induced and how the ability of medications is evaluated in mice? discuss about different types of animal model to study the pathogenesis of rheumatoid arthritis? why the presence of inflammation in the cns is considered as a hallmark of multiple sclerosis? explain the various methods for evaluation of experimental models of multiple sclerosis? short answer questions what is the reason of reportedly experiencing different animal models for studying the pathogenesis of rheumatoid arthritis? give an example which shows the impact of the epigenetics in the initiation of ra? which types of evaluation should be performed after "collagen-induced arthritis" aroused? . what are the "intervening factors after the activation of the immune system, which type of lymphocytes enters into central nervous system answers to short answer questions . there are many experimental models that resemble ra in different respects. since ra is a heterogeneous disease there is probably a need for different animal models that each reflect a characteristic feature of a particular subgroup of ra patients or illustrate particular aspect of the disease. . the epigenetics of ra have also been responsible in the initiation of ra. since the concordance of rheumatoid arthritis in identical twins is not % other nongenetic factors also play a role in the disease etiology. . daily clinical assessment according to a macroscopic scoring system, histological processing and assessment of arthritis damage, radiographic evaluation by an investigator blinded to the treatment protocol on day . . age, weight, and possible infectious disease in animals should be considered as the intervening factors. . following activation, myelin-specific lymphocytes enter into the cns and oligodendrocytes are damaged.yes/no type questions . natalizumab blocks leukocyte migration into the cns by binding to icam. . eae induced actively by injection of the microbialbased immunologic adjuvant pertussis toxin. . housing facilities should be compatible with the needs of the species and equipped to achieve a high standard of animal care.answers to yes/no type questions . yes-the most important risk factor for acpa ra is the hla class ii locus. key: cord- -fkddo n authors: griffin, brenda title: population wellness: keeping cats physically and behaviorally healthy date: - - journal: the cat doi: . /b - - - - . - sha: doc_id: cord_uid: fkddo n nan o u t l i n e whereas feline practitioners are usually well versed in the creation of wellness programs tailored to individual cats, optimizing the health of a population of cats requires additional knowledge and poses unique challenges. these challenges will vary depending on many factors, including the nature and purpose of the population itself. indeed, veterinarians may be tasked with developing health care programs for cat populations in a wide spectrum of settings-from facilities housing laboratory animals, to animal shelters, home-based rescue and foster providers, care-for-life cat sanctuaries, breeding catteries, or large multicat households. regardless of the setting, a systematic approach to the health of the clowder is crucial for success. merriam-webster's dictionary defines wellness as "the quality or state of being in good health especially as an actively sought goal." ensuring population health requires careful planning and active implementation of comprehensive wellness protocols that address both animal health and environmental conditions ( figure - ). addressing physical health alone is not sufficient to ensure wellness. for example, a cat may be in proper physical condition and free from infectious or other physical disease, yet suffering from severe stress and anxiety. in this case, the patient cannot be assessed as healthy, because its behavioral (emotional) state is compromising its health and well-being. thus physical health and behavioral health are both essential components of wellness, and preventive health care must actively address each of these. addressing the environment of the population is also critically important when considering wellness. even the best-designed facilities cannot favor good health in a multicat environment without thoughtful implementation of environmental wellness protocols. in small animal practice, environmental wellness is frequently not emphasized simply because many owners are accustomed to providing a reasonably healthy environment for their pets. in contrast, a structured program to address environmental wellness is essential in the more specific goals will vary depending upon the given population and its purpose. for example, in an animal shelter, specific goals of the wellness program might include decreasing the incidence and prevalence of infectious diseases in the shelter and following adoption, decreasing the incidence of problem behaviors in the shelter, decreasing the rate of return of cats to the shelter for problem behaviors, increasing the adoption rate, and so forth. in the context of a breeding colony, the goals might include increasing kitten birth weights, decreasing neonatal mortality, or improving socialization of kittens. by identifying and tracking measurable factors (often called performance targets in large animal medicine), it is possible to measure progress toward these goals. once baseline data (such as disease rates) are established, it is possible to measure the impact of protocol changes on population health by evaluating these performance targets. both medical records and a system for regular surveillance and reporting are required to accurately track and access trends in animal health. early recognition is crucial for effective control of infectious disease and problem behavior in a group. therefore a regular system of health surveillance must be in place to monitor every individual. in a population setting, daily "walk-through rounds" represent the foundation of an effective animal health care program. rounds should be conducted at least once daily (preferably twice a day or more often, depending upon the needs of individual cats) for the purpose of monitoring and evaluating both physical and behavioral health. medically trained caregivers should visually observe every animal and its environment, taking note of food and water consumption, urination, defecation, attitude, behavior, ambulation, and signs of illness, pain or other problems. monitoring should take place before cleaning so that food intake and the condition of the enclosure, including the presence of feces, urine, or vomit can be noted. alternatively, observation logs can be completed by caregivers at the time of cleaning and reviewed during walk-though rounds. any cat that is observed to be experiencing a problem, whether it be signs of respiratory infection, diarrhea, anxiety, or obvious pain, suffering, or distress must be assessed and treated in a timely manner. regardless of length of stay, regular daily assessment is imperative to identify new problems (medical or behavioral) that may develop so that they can be identified and addressed in a timely fashion to ensure the welfare of the individual animal as well as that of the population. context of a population, regardless of the actual physical facility. proactive measures to maintain clean, sanitary environments that are not overcrowded-where cats are segregated by age and health status and provided with regular daily schedules of care by well-trained dedicated caregivers-are essential. simply stated, the overarching goals of a population wellness program are to optimize both the physical and behavioral health of the cats as well as preventing transmission of zoonotic diseases. in other words, a population wellness program should be designed to keep animals "healthy and happy" while keeping human caregivers safe. it is not difficult to identify a healthy population of cats: when wellness protocols are successful, cats "look healthy" and "act like normal cats." in other words, they appear in good physical condition and display a wide variety of normal feline behaviors, including eating, stretching, grooming, scratching, playing, rubbing, resting, and if allowed, courtship and breeding. just as changes in a cat's physical appearance should alert the clinician to potential problems, so should the absence of such normal feline activities and behaviors by members of the group. wellness goals must include maintaining the health of individual animals as well as that of the population as a whole. in the context of the population, the individuals that are physically or behaviorally ill serve as indicators or "barometers" of the health care and conditions of the population. when individuals are ill, their health and well-being is always a priority; however, it should also immediately trigger the clinician to ask, "why is this individual sick? what is the cause of its illness, and how can i prevent this from affecting others?" to optimize feline health, wellness programs must be carefully structured to address both the physical and behavioral health of the animals, which are intimately linked to their environment, making it crucial to systematically address environmental conditions as well. behavioral health . freedom from thirst, hunger, and malnutrition by providing ready access to fresh water and a diet that maintains full health and vigor . freedom from discomfort by providing a suitable environment, including shelter and a comfortable resting area . freedom from pain, injury, and disease by prevention or rapid diagnosis and treatment . freedom to express normal behavior by providing sufficient space, proper facilities, and company of the animals' own kind . freedom from fear and distress by ensuring conditions that avoid mental suffering in addition to early recognition of health problems, timely action is crucial to effectively limit their morbidity. ideally, all facilities that house multiple cats should have written policies and protocols in place that detail how medical and behavioral problems will be handled. , , a committee or team of individuals composed of medical staff, managers, and caregivers can establish and oversee these policies and protocols. such protocols serve as guidelines for systematic triage and care of animals and help to prevent delays in care that may otherwise arise if such plans were not in place. policies and protocols should be based on medical facts, taking into account the entity's purpose or mission and the availability of resources for care. they should include a definition or description of the disease or condition in question, a description of the methods that will be used for diagnosis, and a general policy regarding the handling and disposition of affected cats. in addition, protocols should include details on notification, housing, decontamination, treatment, and documentation (box - ). just as quality-of-life assessment is the responsibility of every veterinarian as they guide the medical care of individual animals, quality-of-life assessment is also a critical part of population health care and monitoring. the factors that affect physical and mental well-being are broad, complex, and often vary substantially among individuals. exacting criteria are lacking for the objective measurement of quality of life of cats. however, subjective assessments can and should be made by medical and behavioral personnel at regular intervals (weekly or even daily, as indicated) considering the most information possible. , the "five freedoms," which were originally described by the farm animal welfare council in the s, represent a benchmark for ensuring quality of life or animal welfare (box - ). these principles provide a useful framework that is applicable across varying situations and species and have been widely accepted and endorsed by animal care experts. many agencies have used the five freedoms as the basis of recommendations for minimum standards of care for many species, including cats housed in catteries, shelters, and research facilities. , , , the tenets of the five freedoms define essential outcomes and imply criteria for assessment but do not prescribe the methods by which to achieve those outcomes. regardless of the setting, population wellness programs should ensure the five freedoms for all cats. wellness always starts with prevention: it is far more time and cost efficient than treatment, and it is kinder to the animals and their caregivers. with this in mind, population wellness programs should provide broad-based, holistic approaches to preventive care, rather than being based on the control of a single disease or problem, regardless of the setting. maintenance of good health or wellness is especially challenging in populations with high turnover and interchange of cats of varying ages and susceptibilities, such as animal shelters. infectious diseases can become endemic in facilities where populations of animals are housed. even in closed populations, certain pathogens can be difficult to exclude or to eliminate once introduced. notably, upper respiratory viruses, dermatophytes, and coccidia are among the most difficult pathogenic agents to control because of their persistence in the environment through carrier states and/or resistance to environmental disinfection. in particular, upper respiratory disease is the most common endemic disease in cat populations and is impossible to completely prevent in an open population. feline herpes virus type (fhv- ) and feline calicivirus (fcv) have been implicated as the causes of most infections: both viruses induce persistent carrier states and are widespread in the cat population. cats that recover from fhv- remain latently infected and shed virus intermittently, especially following periods of stress. fcv carriers shed continuously for months to years following infection. a variety of other viral and bacterial pathogens may also contribute to feline upper respiratory disease, and bordetella, chlamydophila, and mycoplasma are problematic in some populations. feline infectious peritonitis (fip) is another disease that is nearly impossible to eradicate from a multicat environment, and sporadic cases can be expected to occur, especially in young cats. fortunately, proper wellness programs can greatly limit the incidence and severity of diseases, even for pathogens that are difficult to control. the multicat environment also presents enormous opportunities for inducing stress. because of their unique biology, cats are particularly prone to experiencing acute stress and fear in novel environments. anything unfamiliar to a cat can trigger apprehension, activating the stress response. confinement in a novel environment can result in a wide variety of behavioral indicators of stress including hypervigilance, feigned sleep, constant hiding, activity depression, and loss of appetite, among others. in the long term, if cats are unable to acclimate or cope in their environments, chronic stress, fear, frustration, or learned helplessness may result. in group settings, signs of social stress may also manifest with medical decisions must be weighed in the context of the health of the population as well that of the individual, while considering animal welfare and the availability of resources for care. when large numbers of animals are involved, situations may arise in which animal health and welfare cannot be managed in the case of every individual animal. this may be due to physical or behavioral illness, or environmental conditions that negatively impact animal health, such as crowding. regardless of the cause, it may be necessary to euthanize affected individuals if no other remedies exist to relieve animal suffering or to protect population health. these decisions can be difficult and emotionally challenging, especially in instances where the individual could easily be treated or otherwise accommodated if adequate resources were available. however, such decisions may be crucial for disease control, animal welfare, and population health. that being said, euthanasia should never be used as a substitute for providing proper husbandry and care. indeed, a critical need for a comprehensive wellness program exists in every multicat setting. it is unacceptable to house animals under conditions likely to induce illness and poor welfare, and such conditions can be expected when wellness programs are not in place and carefully monitored. when facilities elect to house cats with medical or behavioral problems, appropriate veterinary care must be provided. it is imperative that a humane plan for diagnosis, treatment/management, monitoring, and housing be implemented in a timely fashion. when determining if cats with special needs can be humanely cared for in a population setting, the following goals and considerations should be addressed: what measures must be implemented to prevent transmission of disease to other cats or people? can appropriate care realistically be delivered? will the care provided result in a cure or adequate management of the disease or problem behavior? can the facility afford the cost and time for care? how will it impact resources available for other cats? in the case of animal shelters, additional considerations should include will the cat be adoptable? what steps can be taken to minimize the holding time required for treatment? if the cat is not adopted, do humane long-term care options exist in the shelter? what welfare assessment will be used to measure quality of life in the shelter? disease control efforts when disease is present. however, the best method of disease control is always prevention. when creating preventive medicine programs for a population, consideration must be given to all components of wellness: physical, behavioral, and environmental health. with regard to promoting physical health, wellness programs should address the following essential elements: implementing population wellness protocols and ensuring quality and timely care require reliable systems for medical record keeping and animal identification. regardless of the system used, medical record keeping procedures should comply with state and local practice acts, guidelines provided by state and national veterinary medical associations, and, in the case of laboratory animals, regulations as prescribed by federal law, the increases in problem behaviors, including urine marking, spraying, or other inappropriate elimination; constant hiding; and/or aggression. stress not only has the potential to negatively impact behavioral health but also physical health as well. the intimate link between stress and immunity has been well described. in fact, stress is a leading factor in the development of infectious disease and is particularly important in the pathogenesis of feline upper respiratory infections. , wellness programs that reduce stress will also serve to minimize the morbidity of infectious disease. despite the fact that infectious agents can never be completely eliminated from the environment, it is still possible to maintain good health. this is because the development of disease is determined by a complex interaction of many factors surrounding the host, the infectious agent, and the environment. keeping these factors in mind provides a rational context for many of the recommendations in this chapter. some of the host factors that influence health and the development of disease include age, sex and reproductive status, immune status, body condition, stress, and genetics. the amount and duration of exposure to an infectious agent (i.e., the "dose effect"), as well as its virulence and route of inoculation, also influence the likelihood and severity of disease. in addition, environmental conditions contribute to the development of infectious disease, including such factors as housing density, sanitation, and fluctuations in temperature or air quality. the fact that disease results from such a large combination of factors underscores the importance of a holistic and broad-based approach to population wellness. when infectious disease does occur in a population, general principles of infectious disease control should guide the response. these include . some facilities prefer to use safety collars that are designed to break away should the collar become caught on something. even for kittens, collars can be used and may be especially beneficial, because they will learn to wear them from an early age. microchips may also be used for identification and are safe and simple to implant (figure - ) . the procedure is well tolerated by the vast majority of cats without the need for sedation. unlike visual means of identification, a scanner is necessary for positive identification of a microchipped animal. for this reason, microchips are often used in conjunction with a visual means of identification and serve as important permanent means of backup identification. box - describes the proper technique for scanning for a microchip. during the last decades, microchips of varying radiofrequencies ( , , and khz) have been introduced in the united states. the -khz chips have historically been the most common, whereas the accepted standard in the rest of the world is the -khz chip. because some scanners read only certain radiofrequencies, it is possible to miss detecting a microchip that is present, depending on the scanner being used. currently, there are efforts to standardize microchipping in the united states, including widespread distribution of universal (global) scanners to ensure that all implanted microchips can be reliably identified. once global scanners are widely available, the american veterinary medical association (avma) recommends adoption of the -khz (iso) microchip as the american standard, because this frequency is recognized as the international standard for microchips institute for laboratory animal research and institutional animal care and use committees. computerized records are preferred; however, written records may also be used. computerized records offer the advantage of mechanized reporting, which facilitates detection and monitoring of health trends in the population. a medical record should be prepared for each cat and should include the cat's entry date, identification (id) number, date of birth, gender, breed, and physical description, as well as historical and physical/behavioral examination findings. in addition, it should contain the dosages of all drugs administered and their routes of administration, including vaccines, parasite control products, other treatments, and anesthetic agents; the results of any diagnostic tests performed; any surgical procedure(s) performed; and other pertinent information regarding the animal's condition. standardized examination and operative reports may be used, but should allow for additions when necessary. identification of cats in the form of a neckband, collar and tag, tattoo, earband, and/or a microchip is also essential for preventive health care and ongoing surveillance of individuals. whenever possible, some form of identification should be physically affixed to every individual cat. in addition, enclosures should be labeled with the cats' unique identification number and/or name. contrary to popular belief, most cats can reliably wear collars safely and comfortably. many facilities use disposable collars, including commercially available plastic or paper neckbands made for animals or hospital-type wristbands made for human patients (figure - ). commercially available cat collars with an id tag affixed in the rest of the world. efforts have also focused on improving, updating, and centralizing microchip registries. this is extremely important in the context of animal shelters. box - contains information on the use of collars and microchips as tools for improving cat-owner reunification. in laboratory settings, tattoos may be used as a means of permanent identification of cats ( figure - ). tattoos are most commonly applied to the inner pinna of the ear using a tattoo machine with multiple needles. care must be taken to properly disinfect the needles between patients. a significant disadvantage of tattooing is that tattoos can sometimes be difficult to read because of the presence of hair, fading, or distortion that may occur as the cat grows. in addition, their application requires anesthesia or heavy sedation. small stainless steel ear tags manufactured for wing banding of birds are especially useful for identifying newborn kittens in some settings and are highly economical (figure - ). they can be placed without the need for anesthesia or sedation when kittens are less than to days old. placing earbands requires skill and experience. they must be positioned in such a way as to provide adequate space for growth of the ear, while seating them deeply enough in the ear margin to ensure a secure piercing far enough away from the edge. if placed too close to the ear margin, the ear flap may tear, resulting in loss of the band. other complications include local inflammation or infection at the site of the piercing. ear tags are a practical method for identifying individual kittens in institutional or commercial breeding colonies, because when applied skillfully, they are seldom lost and provide reliable, long-lasting visual identification. in contrast, private breeding catteries and animal shelters generally prefer to use methods that will not alter the cat's cosmetic appearance long term. colored ribbon, nail polish, or clipping of hair in various areas of the body can all be useful means of temporary kitten identification in the neonatal stage, especially when coat color or patterns do not easily allow individuals to be distinguished. every cat, including those surrendered by their owners, should be systematically scanned for the presence of a microchip at the time of intake, as well as prior to being made available for adoption or being euthanized. proper technique and scanning more than once are crucial to avoid missing microchips. , a universal (global) scanner (e.g., one that will read all microchip frequencies that are currently in use) should be used to ensure that all microchip frequencies are detected. at this time, the only universal scanners available in the united states are the new home again global world scanner (schering plough, whitehouse station, ny) and the imax black label resq scanner (bayer animal health, shawnee mission, kans.). one of the most common causes of scanner failure is weak batteries; therefore it is imperative that batteries be checked and replaced regularly. to ensure a thorough scan and avoid missing chips, cats must be removed from carriers or cages prior to scanning. metal and fluorescent lighting may interfere with chip detection. metal exam surfaces should be covered with a towel or other material prior to scanning to minimize interference. the entire animal should be scanned using a consistent speed, scanner orientation, scanning pattern, and distance. • scanner orientation: the scanner should be held parallel to the animal. rocking the scanner slightly from side to side will maximize the potential for optimal chip orientation and successful detection. the button on the scanner should be depressed continuously during the entire scanning procedure. • scanning distance: the scanner should be held in contact with the animal during scanning such that it is lightly touching the hair coat. • scanner speed: the scanner should not be advanced any faster than . m/second ( . ft/second). scanning slowly is crucial, because universal scanners must cycle through various modes to read all possible chip frequencies. • areas of animal to scan: the standard implant site is midway between the shoulder blades, and scanning should begin over this area. if a microchip is not detected here, scanning should proceed systematically down the back, on the sides, neck, and shoulders-all the way to the elbows in the front and the hindquarters in the rear. • scanning pattern: the scanner should be moved over the scanning areas in an "s"-shaped pattern in a transverse direction (from side to side). if no microchip is detected, the scanner should be rotated degrees, and then the "s"-shaped pattern should be repeated in a longitudinal direction (e.g., the long way) on both sides of the animal. this pattern of scanning will maximize the ability of the scanner to detect the microchip, regardless of its orientation. • less than % of cats are reunited with their owners, compared to as many as % to % of lost dogs. • the use of collars and tags as visually obvious forms of identification is extremely valuable, although overlooked by many cat owners. • cats wearing collars are more likely to be identified as owned and not mistaken for strays. • even indoor cats require identification in case they escape, and studies clearly demonstrate that visual identification improves the odds of pet-owner reunification. • the provision of permanent identification in the form of a microchip represents an important backup, further improving the odds of pet-owner reunification because collars and tags can be lost. • because owners and shelter staff often describe cat coat color and patterns differently, photographs that can be posted online are a useful method of improving lost-pet matching and enabling owners to look for their pet, even if they are physically unable to come to the shelter. • adopted animals should be sent home with id collars and microchips. • shelter staff should always register microchips before the cat leaves the shelter, because many owners will neglect to do so following adoption, making the microchip an ineffective means of identification. • web-based search engines for pet microchip identification numbers (http://www.checkthechip.com and http://www.petmicrochiplookup.org) have been established in an effort to functionally centralize microchip registries by linking existing national databases. facility that houses cats establish a formal relationship with one or more veterinarians who have direct knowledge of their animal population. this is essential to ensure that medical protocols are established with the proper professional oversight, and helps to ensure compliance with local veterinary practice acts that restrict the practice of veterinary medicine to licensed veterinarians. in facilities such as animal shelters, trained shelter staff can carry out preventive health care under the instructions of a veterinarian. the success or failure of a population wellness program hinges in large part on its implementation and oversight. a knowledgeable, cohesive, and dedicated team, where accountability, responsibility, and lines of authority are well defined, is crucial for management success. as a part of the management structure and plan, veterinarians must be involved in the oversight of all aspects of animal care and must be given direct authority for the oversight of medical decisions. this requires that every physical examination is the clinician's single most important tool for evaluating health. following a standardized physical examination form will ensure a complete and systematic review of all body systems. a veterinarian should carefully examine any new cat entering a closed population prior to admittance. in the context of animal shelters, every cat that is safe to handle should receive a physical examination at or as close to the time of admission to the shelter as possible. in many shelters, a veterinarian may not be available to examine incoming animals. however, staff can and should be trained to perform basic evaluations including sexing, aging, body condition scoring, and looking for evidence of fleas, ear mites, dental disease, overgrown claws, advanced pregnancy, or other obvious physical conditions. of particular importance in the shelter physical examination are an accurate physical description of the animal and careful inspection for the presence of identification, both of which may aid in pet-owner reunification. the gold standard for maintaining the health of a population is through exclusion of pathogens in combination with implementation of comprehensive wellness protocols. this requires that members of a population be free from specific pathogens when the group is established and that the colony be closed to any new individuals that do not meet the health standards of the group. this is the foundation of disease control procedures in a laboratory animal setting, and these concepts should be applied to other population settings whenever possible. consideration should be given to testing for the following: feline leukemia virus (felv), feline immunodeficiency virus (fiv), dermatophytosis, intestinal parasites and infections (e.g., campylobacter, giardia, coccidia), as well as other endoparasites and ectoparasites. the setting and resources available, as well as the individual's history and physical examination findings, should guide the clinician's decisions regarding selection of testing for cats entering a specific population. when new stock is added to a closed colony, disease testing is imperative. the american association of feline practitioners (aafp) maintains detailed professional guidelines for the management of felv and fiv infections. identification and exclusion of infected cats is the most effective method of preventing new infections. cats and kittens should always be tested prior to entry to a closed population. those that test negative should be retested, because it the clinician should develop a program for physical health for the population that addresses all of the essential elements as noted. none of these should be considered as optional, but their implementation will depend on the setting, purposes, and resources of the group. the value of obtaining an accurate medical history on any cat entering a population is immeasurable, because it will often alert the clinician to the presence of potential problems. in a laboratory setting, obtaining cats from commercial purpose-bred colonies or institutional breeding colonies ensures that an accurate history will be available, maximizing the odds that only healthy cats will be added to the population. likewise, private breeding catteries should always strive to obtain an accurate medical history on any cat that may be accepted into the cattery. the introduction of cats from random sources to closed populations of cats risks the health of the population and should be avoided whenever possible. in contrast, by their very nature, animal shelters must frequently receive cats from multiple random sources, and it will not always be possible to obtain accurate histories. in some cases, cats are brought in by animal control officers or good samaritans who have little if any information about them. furthermore, some shelters provide a location (e.g., drop-off cages) where cats can be relinquished after business hours. this practice should be discouraged; however, if facilities elect to do this, every effort must be made to obtain a history through questionnaires that can be completed when the cat is left. the presence of staff to directly accept cats and obtain a history at the time of relinquishment is greatly preferred. even so, surrendering owners may or may not provide complete or accurate information, fearing that if they are honest about a pet's problems, the pet may be euthanized. nonetheless, when available, a history can be extremely valuable, saving time and money as well as preventing unnecessary stress for cats and staff alike. intake procedures should be in place to capture basic patient information, including both physical and behavioral data as well as the reason(s) for relinquishment. the importance of obtaining historical information cannot be overemphasized. in many cases, historical information may be used to expedite the disposition of the cat in the shelter. can be problematic. in relation to population health, testing is of little value, because infected cats pose no risk to other cats. nonetheless, a clinician may elect testing as part of an initial database for individual cats, especially if they will be used for breeding. with heartworm tests readily available in combination with pointof-care felv/fiv tests, many animal shelters have been faced with determining whether or not to perform routine screening of cats in their care. to answer this question, it is helpful to consider the following: in consideration of these facts, the author does not recommend routine screening of cats for heartworm disease in shelters. monthly chemoprophylaxis, however, is a safe and effective option for cats sheltered in areas where heartworm infection is considered endemic. dermatophytosis or ringworm, the most common skin infection of cats, is a known zoonosis. it is caused by infection of the skin, hair, and nails with microscopic fungal organisms that cause varying degrees of hair loss and dermatitis. the dermatophyte that causes the majority of cases in felines is microsporum canis, which is responsible for greater than % of all cases. if left untreated, most infections will spontaneously resolve within to weeks postinfection. however, during this time, the infected cat will infect the surrounding environment and other animals or humans in the area. not all cats infected with dermatophytosis develop lesions, and some may become chronic carriers. control of dermatophytosis is difficult, because the spores formed by m. canis can survive in the environment for up to months or longer and are extremely resistant to disinfectants and detergents. in addition, the presence of asymptomatic carriers makes it difficult to readily recognize all infected cats. for this reason, consideration should be given to culturing all cats prior to entry to a closed colony. in particular, persian cats may be predisposed to dermatophyte infection and can be particularly difficult to clear once infected. in closed colony settings, dermatophyte testing by culture is highly recommended unless the source of the cat excludes the possibility of infection (e.g., specific pathogen-free [spf] cats, purposebred laboratory cats). to screen cats using cultures, may take as long as days following exposure for a cat to test positive. , in the context of animal shelters, testing decisions are often influenced by the availability of resources. the aafp's guidelines include recommendations specifically for shelters. they state that all cats should ideally be tested at the time of entry and again in days in case of recent exposure. when cats test positive on screening tests (e.g., point-of-care enzyme-linked immunosorbent assay [elisa] tests), the aafp recommends that the results be confirmed by additional testing, including testing over an interval of time, because false positives can occur. however, such confirmatory testing requires substantial time and monetary investment and may not be feasible in many shelters. in recognition of this, the association of shelter veterinarians established a policy statement on "management of cats who test positive for felv and fiv in an animal shelter," which states that the logistics and cost of holding and retesting unowned cats may be an ineffective use of resources. in addition, it can be difficult to find homes for retroviruspositive cats, which in many instances translates into stressful, prolonged shelter stays. such long-term confinement may compromise quality of life and may compound the emotional stress of caregivers who may later be faced with euthanizing cats that have been held for long periods awaiting confirmatory testing or adoption opportunities. for all of these reasons, many shelters elect to euthanize cats that test positive on retrovirus screening tests. although it may be ideal for shelters to test cats on entry, it is not always feasible because of financial constraints. the next best practice might be to test cats prior to adoption as well as those that are housed in the shelter long term. in addition, cats should be tested prior to placement in group housing with unfamiliar cats and prior to investment, such as foster care, treatment, or spay/neuter surgery. however, given the limited resources of many shelters, the relatively low prevalence in healthy cats and the fact that transmission can be prevented by housing cats separately, it may not be cost effective for all shelters to screen every cat before selection for adoption. each shelter should evaluate its own resources and determine their best use. when testing is performed, samples must never be pooled, and the negative results of one cat (such as a mother cat) should not be extrapolated to other cats (such as her kittens). these practices are invalid and can falsely lead to misidentification of a cat's true infection status. , if testing is not performed prior to adoption, adopters should be advised to have their new pet tested and to keep them separate from any other cats they may own prior to doing so. point-of-care heartworm tests for cats have recently become more widely available, but interpreting results vaccination protocols are typically applied uniformly to all of the individuals comprising the population. this simplifies their application and helps to afford the best possible protection for the group. detailed vaccination records should be maintained for each cat, including vaccine name, manufacturer and serial number, date, the initials of the person who administered it, and any adverse reactions. proper vaccination can substantially reduce disease in cat populations, and serious adverse reactions are relatively rare. for this reason, vaccination against certain core diseases is recommended in all population settings. although exclusion of infectious disease is always a goal of health management, certain pathogens are so widespread that even with careful biosecurity in a closed population, an infection may be introduced to susceptible cats. only in the case of specific pathogen-free colonies, where there may be a compelling reason not to vaccinate as dictated by the purposes of the research, should vaccination be foregone. the aafp maintains published guidelines for vaccination of cats in a variety of settings and includes detailed recommendations for cats in animal shelters. although many vaccines are commercially available for cats, only a few are recommended for routine use in populations. unnecessary use of vaccines should be avoided to minimize the incidence of adverse reactions and reduce cost. core vaccines involve diseases that represent significant morbidity and mortality and for which vaccination has been demonstrated to provide relatively good protection against disease. core vaccines for cats in a population setting include feline parvovirus (fpv or panleukopenia), fhv- (feline herpes virus type or feline rhinotracheitis virus), and feline calicivirus (fcv). these vaccines are usually given in a combination product commonly referred to as an fvrcp vaccine (feline viral rhinotracheitis, calicivirus, panleukopenia). in most cases, timely vaccination against panleukopenia will prevent the development of clinical disease. in contrast, vaccination against the respiratory viruses (fhv- and fcv) does not always prevent disease. in many instances, it affords only partial protection, lessening the severity of clinical signs but not preventing infection. to optimize response, modified live vaccines (mlv) should be used in most cases, because they evoke a more rapid and robust immune response and are better at overcoming maternal antibody interference than killed products. this is especially important in multicat environments in which the risk of infection is high, such as animal shelters, foster homes, as well as any population setting where upper respiratory disease is endemic. a samples should be collected using the mckenzie toothbrush method, where a new toothbrush is used to brush the cat's entire body, giving special attention to the face, ears, and limbs. in addition, if skin lesions are present, hair should be plucked around these areas for culture as well. campylobacter, salmonella, giardia, coccidia, tritrichomonas, and other gastrointestinal parasites and pathogens are common in some cattery situations and can be very difficult to eliminate once they are introduced. in fact, in some settings, these pathogens may become endemic and nearly impossible to eliminate. treatment of coccidia in shelter kittens is described in although clinical signs, such as diarrhea, may be associated with infection, some cats remain asymptomatic. these pathogens have the potential for high morbidity in a population (especially in young kittens), and some possess zoonotic potential. therefore routine fecal examinations, cultures, and/or empirical treatments should be considered prior to the introduction of new cats. it is well recognized that vaccination plays a vital role in the prevention and control of infectious diseases. protocols should be established in the context of the population's exposure risk, which will vary depending upon the setting. in the context of population medicine, ponazuril is a metabolite of toltrazuril that has proven activity against coccidia.* because there is no approved product for use in cats, the equine product marquis oral paste ( % w/w ponazuril; bayer healthcare) may be dosed at mg/kg, po, once daily for to days. prophylactic treatment may be instituted in high-risk situations, such as young kittens in environments with documented infection. proper hygiene, including the use of disposable litter boxes and frequent removal of feces, is also necessary. oocysts survive in the environment and are not treated by routine disinfectants, such as bleach and quaternary ammonium compounds. with a history of upper respiratory infection) may benefit from vaccination prior to breeding to maximize passage of maternal antibody to their kittens. for pregnant cats in such environments, administration of mlv should be avoided, because the potential risk of injury to the developing kittens may outweigh the risk of infection in this case. vaccination of lactating queens should also be avoided in a low-risk environment. a series of vaccinations should be administered to kittens less than months of age to minimize the window of susceptibility to infection and ensure that a vaccine is received as soon as possible after maternal antibodies have decreased sufficiently to allow vaccine response. for kittens, vaccines should be administered every to weeks until they are weeks (e.g., months) of age or their permanent incisors have erupted. the minimum interval of weeks is recommended in high-risk settings to narrow the window of susceptibility as maternal antibody wanes. a vaccination interval of less than weeks is not recommended, because it may actually blunt the immune response from previous vaccination. in the case of an outbreak of panleukopenia, extending vaccination to months of age may be warranted to ensure than no animal remains susceptible. although the vast majority will respond by months of age, a few may fail to respond, while others are provided with a boost to enhance the immune response. just as in owned pets, booster vaccines are generally not required until year later for modified live vaccines but should ideally be administered once in to weeks whenever resources permit. this may be especially important for cats that were ill at the time of initial vaccination, as may be the case in an animal shelter. revaccination in long-term shelter facilities should follow the guidelines set forth for pets: boost at one year, then every years for fvrcp. vaccination against rabies virus is regarded as a core requirement for pet cats and is required by law in some jurisdictions. thus vaccination against rabies is recommended in the context of private catteries. in contrast, rabies vaccination may be considered optional in most closed laboratory settings, because the risk of exposure should be absent and legal requirements may not apply. in animal shelters, vaccination against rabies is not generally recommended at the time of admission, simply because there is no benefit in terms of disease prevention or public health. vaccination on admission will not provide protection against an infection acquired prior to entry, nor will it limit concern if a cat with an unknown health history bites someone soon after admission. rabies vaccination is recommended for cats prior to adoption when a veterinarian is available to administer it (or as otherwise legally prescribed by state laws). alternatively, rabies vaccination may be administered as single modified live fvrcp vaccine will usually afford protection to cats that are at least months of age. in contrast, killed products require a booster in to weeks to confer immunity, making their use largely ineffective in such environments. to ensure rapid protection against panleukopenia, injectable fvrcp vaccines are preferred, but intranasal vaccines may offer advantages for feline respiratory disease, because they have been shown to rapidly induce local immunity at the site of exposure. furthermore, intranasal vaccines may be better at overriding maternal antibody in young kittens. for this reason, they are often used to reduce the morbidity and severity of upper respiratory infection (uri) in preweaningage kittens. when intranasal vaccines are used in animal shelters, they should be used in combination with injectable fvrcp vaccines to ensure and optimize response against panleukopenia as well as the respiratory infections. ideally, all cats should receive a mlv fvrcp vaccine at least week prior to entering a population. in the context of an animal shelter setting, this is seldom feasible. vaccination immediately upon entry is the next best practice and can provide clinically significant protection for the majority of cats. if neither maternal antibody nor another cause of vaccine failure interferes, modified live vaccinations against panleukopenia will often confer protection against disease in only days. intranasal vaccines against respiratory infections, including fhv and fcv, typically provide partial protection within to days. , in animal shelters, all incoming cats and kittens weeks of age and older that can be safely handled should receive an injectable mlv fvrcp vaccine immediately upon entry. a delay of even a day or two significantly compromises the vaccine's ability to provide timely protection. even injured cats, those with medical conditions, and those that are pregnant or lactating should be vaccinated on entry, because vaccination will likely be effective and the small risk of adverse effects is outweighed by the high risk of disease exposure and infection in the shelter. when vaccination of all cats on entry is not financially feasible, the next best practice is to vaccinate all those that are deemed adoptable at the time of entry or that are likely to be in the shelter long term. whenever possible, vaccinated cats should be separated from those that will remain unvaccinated (e.g., those that will be euthanized following a brief holding period) as soon as that determination can be made. in contrast, in lower-risk settings, ensuring that cats are in good health prior to vaccination should be a priority. vaccination of kittens with injectable fvrcp vaccinations may be delayed to to weeks of age. however, when respiratory disease is endemic, administration of intranasal vaccines beginning at weeks of age may be beneficial. in breeding catteries, queens (especially those control and prevention of internal and external parasites represent another important component of a population wellness program. common products used for their management are described elsewhere in this book. of particular importance are roundworms and hookworms, common intestinal parasites with zoonotic potential (see chapter ) . although uncommon, the risk of human infection from contaminated environments is real and can result in organ damage, blindness, and skin infections. for this reason, the centers for disease control and prevention and the companion animal parasite council strongly advise routine administration of broad-spectrum anthelminthics for their control. , pyrantel pamoate is one of the most costeffective and efficacious drugs for treatment and control of roundworms and hookworms. in both shelter and cattery settings, the author recommends administration of pyrantel pamoate at a dosage of mg/kg to all cats with re-treatment in weeks and then at monthly intervals. in shelters, if it is not possible to treat all cats at the time of entry, at a minimum, all cats that are deemed adoptable should be treated as soon as possible. in addition, kittens should be treated at -week intervals until months of age. for cats with diarrhea, fecal examination (e.g., flotation or centrifugation, direct fecal smear and cytology) should be performed with treatment according to results. even if results are negative, the administration of broad-spectrum anthelminthics should be strongly considered. in animal shelters, ectoparasites, particularly ear mites and fleas, are also very common in cats and kittens. shelter staff should be trained to recognize infestation and protocols should be established for treatment. in terms of shelter treatment protocols, the author recommends treating ear mites with ivermectin, because it is highly efficacious and costs only pennies per dose. the recommended dosage is . mg/kg subcutaneously. for fleas, the author recommends topical treatment with fipronil (frontline, merial, duluth, ga.) as a spray or top spot. in particular, the spray is very cost effective. it is safe for use in cats of all ages, including pregnant and nursing mothers and neonatal kittens. in addition, fipronil also has activity against ear mites, cheyetiella, chewing lice, and ticks. , spaying and neutering is another important consideration in the context of population wellness. reproductive stress from estrous cycling in queens and sex drive in tomcats can decrease appetite, increase urine spraying/ marking and intermale fighting, and profoundly increase social and emotional stress in the group. for these reasons, spaying and neutering cats that will not be used soon as possible following adoption. the latter may encourage new owners to establish a relationship with a private veterinarian. rabies vaccination is warranted when cats are housed long term in shelter facilities. in addition, if individual cats must be held for bite quarantines, they should be vaccinated against rabies in accordance with the current compendium of animal rabies prevention and control. noncore vaccines include those that may offer protection against disease, but because the disease in question is not widespread or only poses a risk of exposure in certain circumstances, vaccination is only recommended based on the individual risk assessment of a population of animals. noncore vaccines include felv, fiv, chlamydophila, and bordetella. vaccination against felv is not warranted in a closed population of cats in which there is no risk of exposure (e.g., most laboratory animal settings). in private catteries, a risk assessment should be done to determine if vaccination is warranted (e.g., cats permitted in outdoor enclosures, frequent introduction of cats from external sources, other opportunities for exposure). special consideration should be given to vaccinating kittens because of their high susceptibility to felv infection and the high likelihood that they will become persistently infected if exposed. in general, felv vaccination is not recommended in animal shelters when cats are housed short term. however, its use is warranted when cats are group housed when resources permit. fiv vaccination is not generally recommended in population environments. a confounding feature of fiv vaccination is that vaccinated cats develop false-positive test results on most commercially available tests (see chapter ) . if fiv vaccination is elected, vaccinated cats should be permanently identified (e.g., by use of a microchip) to help clarify their status. chlamydophila felis (c. psittaci) and bordetella bronchiseptica vaccines may be of benefit when clinical signs of these diseases are present in the population and diagnosis is confirmed by laboratory evaluation. their efficacy is moderate, and reactions are more common than with most other feline vaccines; therefore ongoing use should be periodically reassessed. some vaccines are not generally recommended for use because of undemonstrated efficacy, such as the feline infectious peritonitis (fip) vaccine. wants whenever he or she chooses. dry food is used for this method of feeding, because canned products left at room temperature are prone to spoiling. the major advantage of free choice feeding is that it is quick and easy: caregivers simply need to ensure that fresh dry food is always available. major disadvantages include the fact that cats that are not eating may remain unrecognized for several days, especially when more than one animal is fed together, and some cats may choose to continually overeat and become obese. free choice feeding is an excellent method for cats that require frequent food consumption. these include kittens up to to months of age, queens in late gestation, and those that are nursing. unlike dogs, who are competitive eaters by nature, free choice feeding may benefit cats that are group housed, because it ensures that there will be ample time for all members to eat, provided that dominant members of the colony do not block the access of subordinate cats. meal feeding using controlled portions of dry and/or canned food may be done as an alternative to or in conjunction with free choice feeding. when used alone, a minimum of two meals should be fed per day. meal feeding is ideal for any cat that requires controlled food intake and facilitates monitoring of appetite. meal feeding also has the benefit of enhancing caregiver-cat bonding and provides a pleasant and predictable experience for cats when done on a regular daily schedule. using a combination of free choice plus once daily meal feeding takes advantage of the positive aspects of both methods and works well for most cats in a population setting. typically, dry food is available free choice, and a small meal of canned food is offered once daily. this combination approach accommodates the normal feeding behavior of cats by allowing them to eat several smaller meals throughout the day while allowing caregivers to monitor the cat's appetite at least for the canned food meal. as necessary for the individual cat, some may be fed additional meals of canned food to ensure adequate nutritional support. good body weight and condition and a healthy hair coat are evidence of an adequate nutritional plane and proper nutritional management. both appetite and stool quality should be monitored daily. normal stools should be well-formed and medium to dark brown. adult cats typically defecate once daily, although healthy adults may defecate anywhere between twice a day and twice a week. kittens tend to produce a larger volume of stool more frequently, which is often lighter in color and softer in form than that of adults. simple scales can be used for monitoring appetite (e.g., good, some, none), and fecal scoring charts are available. the author recommends the purina fecal scoring system chart available from nestlé purina petcare company (figure - ) . for breeding is recommended. in animal shelters, spaying and neutering cats prior to adoption will ensure that they do not reproduce and contribute to the surplus of community cats. this will also serve to enhance husbandry, because the procedures rapidly decrease spraying, marking, and fighting; eliminate heat behavior and pregnancy; and greatly mitigate stress. in addition to reducing stress and odor, spaying and neutering sexually mature cats will facilitate group housing, which is often beneficial for cats, especially when housed longer term (see below). the medical benefits of spay/neuter have also been well described, including dramatic reductions in the risk of mammary carcinoma, elimination of cystic endometrial hyperplasia, pyometra and ovarian cancer in queens, and decreased risk of prostate disease in toms. thus spaying and neutering favors both individual as well as population health. proper nutrition has a profound impact on wellness. not only is it essential for management of healthy body weight and condition, good nutrition is also known to support immune function. a regular diet of palatable commercial food consistent with life stage should be offered, and fresh water must always be available. although some cats tolerate changes in food without apparent problems, it is important to recognize that for others, changing from one diet to another can cause loss of appetite and/or gastrointestinal upset. for this reason, it is generally best to provide the most consistent diet possible. whereas this may be relatively easy to do in a laboratory or cattery setting, it can be more challenging in a shelter environment. some pet food companies offer feeding programs for animal shelters, providing a consistent food for purchase at a special rate for shelters. however, some shelters rely heavily on donations of food. in this case, by requesting donation of certain brands of food, shelters are able to provide a consistent diet whenever possible. it is also feasible to mix donated foods with the shelter's usual diet to minimize problems caused by abrupt diet changes while taking advantage of other donated products. the wild ancestors of domestic cats hunted to eat, feeding up to times in a -hour period. this style of feeding behavior is preferred by many domestic cats that would nibble throughout the day and night, consuming many small meals if left to their own devices. although this is true, most cats are capable of adapting to either free choice or meal feeding as their daily feeding pattern. , there are advantages and disadvantages to each in a population setting. with free choice or ad libitum feeding, food is always available such that a cat can eat as much as he or she trends in body weight, because both weight loss and gain can compromise health and well-being. appropriate grooming is also essential to ensure wellness and must never be considered as optional or purely cosmetic. most cats require minimal grooming because of their fastidious nature. however, long-haired cats are notable exceptions, often experiencing matting of the hair coat without regular grooming sessions. matted hair coats are not only uncomfortable for the animal, but may lead to skin infection. overgrown nails can also be a problem for some cats, particularly those that are geriatric or polydactyl. the provision of appropriate surfaces for scratching will encourage cats to condition their own claws; and a system for regular inspection of the hair coat and nails should be established. in addition to ensuring proper coat and nail maintenance, regular grooming sessions provide an excellent opportunity to monitor body condition; and some cats enjoy the physical contact and attention. in high-risk settings, the use of stainless steel combs or undercoat rakes that can be readily disinfected are generally preferable to the use of in addition to appetite and stool quality, it is essential to monitor body weight and condition. body condition can be subjectively assessed by a process called body condition scoring, which involves assessing fat stores and, to a lesser extent, muscle mass. fat cover is evaluated over the ribs, down the top line, tail base, and along the ventral abdomen and inguinal (groin) areas. body condition score charts have been established on scales of to and to . the author recommends use of the purina body condition score chart which is based on a scale of to with being emaciated and being severely obese (see figure - ). cats should be weighed and their body condition scored at routine intervals. ideally, body weight should be recorded at entry to the population and then weekly during the initial month of care, after which it could be recorded once a month or more often as indicated based on the individual's condition. this is especially important for cats, because significant or even dramatic weight loss may be associated with stress or illness during the first few weeks of confinement in a new setting. on the other hand, in long-term-housed cats, excessive weight gain may occur in some individuals. therefore protocols must be in place to identify and manage unhealthy score -very moist (soggy); distinct log shape visible; leaves residue and loses form when picked up. score -very moist but has distinct shape; present in piles rather than as distinct logs; leaves residue and loses form when picked up. score -has texture, but no defined shape; occurs as piles or as spots; leaves residue when picked up. score -watery, no texture, flat; occurs as puddles. score -firm, but not hard; should be pliable; segmented appearance; little or no residue left on ground when picked up. score -log-like; little or no segmentation visible; moist surface; leaves residue, but holds form when picked up. fecal scoring system bristle brushes because the latter are impossible to disinfect and have the potential to spread common skin infections such as ringworm. dental health is another component of wellness. in the context of population wellness, it may not be the highest priority; however, it should always be a consideration in terms of individual health care and well-being. this is important because periodontal disease will occur unless it is actively prevented, and plaque and tartar buildup may contribute to serious health concerns, ranging from oral pain to chronic intermittent bacteremia and organ failure. feline tooth resorption and gingivostomatitis are also common conditions of the feline oral cavity that can lead to chronic pain, affecting the cat's appetite and ability to self-groom, and negatively impacting quality of life. when painful dental disease is present, a plan for timely treatment should be identified and implemented. preventive dental care may include tooth brushing, dental-friendly diets, and treats and chew toys in combination with periodic professional dental care. these should be tailored to meet the needs of individuals in the population to optimize dental health. cats with stomatitis should be removed from breeding programs. wellness protocols may also be dictated by the specific needs of certain breeds of cats. for example, persian, himalayan, and other brachycephalic cats are predisposed to respiratory disease and tend to be more severely affected than other cats because of their poor airway conformation. because of the high likelihood of exposure in a shelter setting, these cats should be housed in highly biosecure areas that are well ventilated and should be prioritized for immediate adoption or transfer to foster care or rescue. in the author's experience, even intranasal vaccination of these breeds can result in severe clinical signs of respiratory disease and is best avoided. just as a physical wellness program must be tailored to the population in question, a behavioral wellness program, composed of all of the essential elements, should be created to meet its specific needs as well. even when animals will only be housed for short periods, considerations for behavioral care are essential to ensure humane care. short-term confinement can induce severe stress and anxiety, and when confined long term, cats may suffer from social isolation, inadequate mental stimulation, and lack of exercise. a behavioral wellness program should strive to decrease stress from the moment cats arrive at a facility until the moment that their stay ends. as previously described, a thorough behavioral history will provide an important baseline for action and follow-up. understanding the importance of minimizing stress in cats and possessing the ability to recognize and respond to it are essential to facilitate a cat's transition into a population. , staff should be trained to evaluate cats beginning at intake and to recognize and respond to indicators of stress. active daily monitoring of cats for signs of stress or adjustment should be performed, and staff should record their findings daily, noting trends and making adjustments in the care of individual cats and the population as indicated. in animal shelter environments, proper behavioral care of cats also requires an understanding of the wide spectrum of feline lifestyles and an approach tailored to the individual needs of each group. domestic cat lifestyles and levels of tractability range from the most docile, sociable housecat, to free-roaming strays and truly unsocialized feral cats that will not allow handling. stray cats include those that may have been previously owned or are "loosely owned" neighborhood or barn cats. because of their lack of socialization, capture, handling, and confinement are especially stressful for feral cats. however, fearful cats may resort to overt aggressive or may "teeter on the edge" of defensive aggression regardless of their socialization status. in fact, even the tamest house cats may exhibit the same behaviors as feral cats when they are highly stressed (figure - ) . , these responses can compromise cat welfare and staff safety and hinder adaptation to a new environment. regardless of their demeanor, all cats and kittens should be provided with a hiding box in their enclosure at the time of entry, because the ability to hide has been shown to substantially reduce feline stress. for those cats that are severely stressed or reactive, covering the cage front, in addition to providing a hiding box, and posting signage to allow the cat "chill out" time for several hours or even a few days can facilitate adaptation. this is important because, once highly stressed or provoked, cats often remain reactive for a prolonged time and may become more reactive if they are stimulated again before they have been allowed a period of time to calm down. soft bedding should be available for comfort and so that cats may establish a familiar scent, which aides in acclimation to a new environment. care should be taken during cleaning procedures to minimize stress and noise, behavioral evaluation may be useful, especially for cats that will be re-homed. several evaluations have been recommended, but none are scientifically validated for predicting future behavior with certainty. , , nonetheless, this form of evaluation may be useful for determining behavioral needs while cats remain in a facility, as well as guiding appropriate placement. box - describes common components of a feline behavioral evaluation (figure - ) . housing design and operation can literally make or break the health of a population. regardless of the species in question, housing should always include a comfortable resting area and allow animals to engage in species-typical behaviors while ensuring freedom from fear and distress. it is not sufficient for the design to address only an animal's physical needs (e.g., shelter, warmth). it must meet their behavioral needs as well, and both the structural and social environment are essential considerations for housing arrangements. furthermore, the environment must provide opportunities for both physical and mental stimulation, which become increasingly important as length of stay increases. a sense of control over conditions is well recognized as one of the most critical needs for behavioral health. thus housing design must provide cats with a variety of satisfying behavioral options. specifically, housing arrangements must take into account the following feline behavioral needs : • opportunities for social interactions with humans and/or other compatible cats and cats should be allowed to hide while their cage is quietly tidied and replenished around them as needed. commercially available "cat dens" are ideal for this purpose, because they can be secured from a safe distance such that the cat is closed inside a secure, familiar hiding place during cleaning procedures (figure - ) . cats should be returned to the same cage and only spot cleaning should be performed to preserve their scent, which is necessary for stress reduction. if it becomes necessary to house the cat in another location, the den and towel should accompany the cat to ease the transition. finally, the use of commercially available synthetic analogues of naturally occurring feline facial pheromones (feliway, veterinary product laboratories, phoenix, ariz.) have been shown to be useful for stress reduction in cats during acclimation to new environments and can be sprayed onto bedding and allowed to dry prior to use or dispersed in the room using plug-in diffusers. the way in which cats are handled at intake has a profound impact on their behavior, health, and wellbeing and will impact the cat's ability to adapt to its new environment. when stress is successfully mitigated, cats are more likely to adapt and to "show their true colors" rather than reacting defensively. during a period of a few days, many cats that did not appear to be "friendly" at intake will become tractable and responsive to their human caregivers, facilitating care. aside from informally "getting to know" cats during their initial acclimation period in a facility, a systematic • the ability to create different functional areas in the living environments for elimination, resting, and eating • the ability to hide in a secure place • the ability to rest/sleep without being disturbed • the ability to change locations within the environment, including using vertical space for perching • the ability to regulate body temperature by moving to warmer or cooler surfaces in the environment • the ability to scratch (which is necessary for claw health and stretching, as well as visual and scent marking) • the ability to play and exercise at will • the ability to acquire mental stimulation because these needs will vary depending upon such factors as life stage, personality, and prior socialization and experience, facilities should maintain a variety of housing styles in order to meet the individual needs of different cats in the population (figure - ) . managing housing arrangements for a population of cats of varying ages, genders, personality types, social experiences, and stress levels requires knowledge of normal feline social behavior and communication. during the past decades, knowledge of feline social structure has evolved from the widespread belief that cats are generally an asocial and solitary species to the realization that they are social creatures. , with the exception of solitary hunting, free-roaming cats perform responses are observed and recorded for each of the following: • the tester approaches cage, stands quietly for seconds, then offers verbal encouragement. • if deemed safe to proceed, tester opens the cage door and calmly extends an open hand towards the cat, then attempts to gently touch the cat's head. • if the caregiver is unsure if this is safe to do, a plastic hand may be used to gauge the cat's receptiveness to touch (see figure - ). • if the cat allows handling, the cat is gently lifted and carried to a secure, quiet room for further observation. • the tester sits quietly on a chair and/or the floor; the tester calls and solicits the cat's attention. • the tester pets the cat on the head. • the tester strokes the cat down the back several times. • the tester picks up the cat and hugs it for seconds. • with the cat standing on the floor, the tester strokes the cat down the back and firmly but gently grasps the base of the tail and lifts the cat off of its hind feet for second. the tester repeats this a second time. • the tester engages the cat in play with an interactive toy. in some instances, it is difficult to determine if a cat will accept handling. to prevent injury to staff, a plastic hand (assess-a-hands; great dog productions, accord, ny) is used to approach this cat. as the hand approaches, the cat appears tense (a) but begins to relax and accepts petting (b and c) . the absence of normal behaviors (e.g., grooming, eating, sleeping, eliminating, stretching, greeting people). defensive behavior may involve characteristic postural and/or vocal responses, and is usually motivated by fear. disruptive behavior involves destruction of cage contents and creation of a hiding place. stereotypic behaviors (e.g., repetitive pacing, pawing, and circling) may also develop as a result of stress but generally occur less commonly. as an illustration of these feline behaviors, consider the responses of a typical social domestic cat when caged in a novel environment (box - and figures - to - ) . behavioral signs of stress may be further classified as active communication signals or passive behaviors. , signals of anxiety, fear, aggression, and submission may be subtle or obvious and include vocalization (growling, hissing), visual cues (facial expression, posturing of the body, ears, and tail), and scent marking (urine, feces, various glands of the skin). passive signs of stress include the inability to rest/ sleep, feigned sleep, poor appetite, constant hiding, absence of grooming, activity depression (decreased play and exploratory behavior), and social withdrawal. high-density housing exacerbates these signs. lowsocial-order cats in such an environment may exhibit decreased grooming, poor appetite, and silent estrus. cats that are consistently fearful or anxious may hide, most of their activities within stable social groups where cooperative defense, cooperative care of young, and a variety of affiliative behaviors are practiced. affiliative behaviors are those that facilitate close proximity or contact. cats within groups commonly practice mutual grooming and allorubbing (e.g., rubbing heads and faces together). this may serve as a greeting or as an exchange of odor for recognition, familiarization, marking, or development of a communal scent. cats of both genders and all ages may exhibit affiliative behaviors, and bonded housemates often spend a large proportion of their time in close proximity to one another. maternal behavior is the primary social pattern of the female cat, and cooperative nursing and kitten care are common. if allowed, queens form social groups along with their kittens and juvenile offspring. , tomcats typically reside within one group or roam between a few established groups. within groups of cats, a social hierarchy or "pecking order" forms. , once established, this hierarchy helps to support peaceful co-existence of cats within a stable group, minimizing agonistic behaviors between members. social hierarchy formation occurs within groups of cats that are sexually intact, as well as in those that are neutered. knowledge of behavioral signaling is critical for successful management of housing arrangements. manifestations of both normal and abnormal behavior indicate how successfully an animal is coping with its environment. common behavioral expressions of feline anxiety may manifest with inhibited or withdrawal behavior, defensive behavior or disruptive behavior. , inhibited or withdrawal behavior refers to activity depression or • fear is typically the initial response, and if threatened by the proximity of unfamiliar caregivers, defensive aggression may be displayed. alternatively, the cat may freeze or appear catatonic. • if provided with a box for concealment, the cat will hide or otherwise slink against the back of the enclosure, behind the litter box, or disrupt the cage and hide under the paper. • given time, most cats become more active and engage in greeting behaviors, coming to the front of the cage and pawing or mewing as caregivers approach. • if the cat remains confined with time without adequate periods of exercise, mental stimulation, and social companionship, stress and frustration will manifest with activity depression and withdrawal (lying in the litter box, failure to groom, failure to greet caregivers, and, in some cases, displaying aggression towards caregivers). • displays of stereotypic behavior (such as pacing) may occur; however, inhibited or withdrawal behaviors are much more common (see unfamiliar or new cats entering the group. within an established group, however, most social conflicts are not characterized by overt aggression. instead, the main mode of conflict resolution is avoidance or deference (figure - ) . , , deference behaviors include looking away, lowering the ears slightly, turning the head away, and leaning backward. large numbers of cats peacefully co-exist together, using such strategies for avoidance provided ample space and resources are available for all members of the group. signs of social stress within groups of cats may manifest with overt aggression, increased spraying and turn their back, huddle, and avert their eyes from the gaze of other cats. hiding is a normal and important coping behavior; however, when hiding is occurring with increased frequency or in response to stimuli that did not previously cause hiding, it should be recognized as a sign of stress. , in group settings, the complexity of the social structure cannot be overestimated. the internal structure of social groups rarely represents a straightforward linear hierarchy, except in very small groups of less than four to five animals. in larger groups of cats, there are usually one or two top-ranking individuals and one or two obvious subordinates, while the remaining cats share the middle space. , most cats within the group form affiliative or friendly relationships; however, some may fail to form such relationships and remain solitary. colony members commonly display aggression toward c marking, or constant hiding. , lower-ranking cats may spend little time on the floor, remaining isolated on single perches or other locations where they may even eliminate, while higher-ranking cats remain more mobile, controlling access to food, water, and litter resources. high-density housing conditions frequently result in such abnormal behaviors and are associated with increases in transmission of infectious diseases and reproductive failure as well. cats are commonly housed in three basic arrangements: cage or condo units, multiple runs within a room, or free ranging in a room. cage housing of cats should be avoided unless necessary for short periods for intake observation, legal holding periods in shelters as required by local ordinances, medical treatment or recovery, or to permit sample collection. although space recommendations vary substantially in the literature, common sense dictates that a determination of necessary housing space should take into account the cat's length of stay. in the author's opinion, it is neither appropriate nor humane to house cats in traditional cage housing long term (e.g., more than to weeks). the design of short-term housing should include provisions for housing individual animals, litters, families, or bonded housemates for intake evaluation and triage. housing must be easy to clean and sanitize, well ventilated, and safe for animals and caregivers. short-term housing should provide sufficient space to comfortably stand, stretch, and walk several steps; sit or lay at full body length; and separate elimination, feeding, and resting areas. litter boxes should be of appropriate size to comfortably accommodate the cats for which they are intended ( figure - ) . resting areas should include comfortable surfaces, soft bedding, and a secure hiding place to provide a safe refuge. a hiding place is essential, because it reduces stress by allowing cats to "escape," facilitating adaptation to a new environment. the addition of a sturdy box to a cage will provide a hiding place as well as a perch (figures - and - ) . in addition, cages should be elevated off of the floor by at least . m ( . feet), because this serves to reduce stress as well. in most instances, cage or condo style housing is used in most facilities for short-term holding at intake for observation, acclimation, and/or triage. runs or small rooms are also appropriate for intake housing, and offer cats the obvious benefit of additional space to meet their behavioral needs (figure - ) . regardless of their configuration, enclosures for short-term housing of cats should be large enough to allow them to stretch, groom, and move about while maintaining separate functional areas, at least . m ( feet) apart, for sleeping, eating, and elimination. , , laboratory guidelines in the united states call for a minimum floor area of . m ( ft ) for cats weighing less than kg and . m ( ft ) for cats weighing kg or more, with a minimum height . m ( ft). a resting upper respiratory infections. double-sided enclosures (e.g., cat condos) are ideal for meeting these specifications and have the benefit of easily allowing cats to remain securely in one side of the enclosure while the opposite side is cleaned (figure - ) . this helps to minimize stress, prevent exposure to infectious disease, perch is also required. current guidelines (european convention for the protection of vertebrate animals used for experimental and other scientific purposes, ets ) promulgated by the council of europe (http:// www.coe.int) for laboratory cats are similar, but proposed revisions call for substantially more floor space for cats, at . m ( . ft ) per adult cat with a height of at least m ( . ft). , the revisions, which have not been approved to date, also call for the provision of shelves, a box-style bed, and a vertical scratching surface. animal shelter facilities in the united states have traditionally been equipped with small perchless cages (e.g., . to . m or . to . ft long) that are poorly designed for housing cats. the association of shelter veterinarians (http://www.sheltervet.org) recommends a minimum enclosure size of m ( ft ) for adult cats. commercially available cages are typically approximately . m ( . ft) deep (e.g., an arm-length deep so that they can be readily accessed); therefore a cage with a length of . m ( ft) is required to provide this approximate square footage, and it will also allow for adequate separation of food, water, and litter ( figure - ) . similarly, the cat fanciers' association (http:// www.cfa.org) recommends a minimum of . m ( ft ) of space per cat for those weighing kg or more. cubic measurements take into account the use of vertical space in addition to floor space, which is crucial for improving the quality of the environment. for example, a . -m ( - larger enclosures also allow for better air circulation, which is an important consideration for control of feline housing for a single cat. note the large -ft long cage, provision of a secure hiding place and perch with bed, separation of litter from resting and feeding areas, and appropriately sized litter box for this large cat. housing units are available for cats and serve to separate functional living areas and provide improved opportunities for exercise and exploration. this unit (tristar metals, boyd, tex.) is constructed of powder-coated stainless steel, which is highly durable and easy to disinfect but less noisy than uncoated stainless steel. note the elevation from the floor and the grills on both the front and back, which allows flow-through ventilation. is both mentally and physically stimulating for cats and preferably that which is esthetically pleasing to humans. the latter is an important consideration to facilitate adoption in animal shelters. and, even in other types of facilities, it is important to create a pleasant environment not only for the animals, but also for their caregivers. studies indicate that employee satisfaction improves animal care and staff retention, both of which may positively impact population health and well-being. for long-term housing of cats alternatives to traditional cage housing should be afforded. , , at an absolute minimum, cats that are cage housed must be released each day and allowed an opportunity to exercise and explore in a secure enriched setting. for long-term housing, most cats will benefit from colony-style housing, provided there is sufficient space, easy access to feeding and elimination areas, an adequate number of comfortable hiding, and resting places and careful grouping and monitoring to ensure social compatibility among cats. not every cat, however, will thrive in a group setting, and certain individuals will require enriched single housing, depending on their unique physical or behavioral needs. these may include cats that bully other cats or are otherwise incompatible and those with special medical needs. it is important to recognize that such singly housed cats will require more regular contact with their human caregivers and higher levels of mental and physical stimulation in order to maintain behavioral health during long-term confinement. whenever possible, long-term housing of such individuals should be avoided. when cats are housed in amicable groups, it is easier to maintain proper behavioral welfare in the long term, because many of their social and emotional needs can be met by conspecifics. group housing affords cats with opportunities for healthy social contact with others, which, in turn, provides additional mental and physical stimulation. when properly managed, this housing arrangement enhances welfare.* insufficient space and crowding or poor compatibility matching of cats serves to increase stress and negates the benefits of the colony environment. group housing should never be used as a means of simply expanding the holding capacity of a facility. in animal shelters, the high turnover rate of cats contributes substantially to feline stress levels, especially in the context of groups of unfamiliar animals. because it may take days to weeks to acclimate to a group environment, enriched individual housing may be preferable when a brief stay is anticipated. however, the benefits of enriched social group housing become evident when stays extend beyond a few weeks. and preserve staff safety, which are especially crucial for newly arrived cats. traditional cages can be modified into condo-style enclosures by creating portals to adjoin two or three smaller cages (figure - ) . regardless of the precise specifications of the enclosures, the importance of the overall quality of the living environment cannot be overemphasized. this includes a holistic approach to husbandry, with careful attention to the way in which cats are handled, noise levels, the provision of creature comforts, positive contact with caregivers, and strict avoidance of overcrowding, as well as good sanitation, medical protocols, and careful monitoring to ensure health and welfare. for long-term housing (e.g., greater than weeks), consideration should also be given to providing space that a b *references , , , , , , , , , , . breeding age should be avoided whenever possible. at a minimum, mature tomcats should be neutered to prevent intermale aggression, urine spraying, and breeding. reproductively intact females may be co-housed with other intact females or with neutered males. in contrast, in breeding colonies, harem-style housing may be used to facilitate breeding (e.g., a few queens with a tomcat). it is also advantageous to house compatible pregnant queens together before delivery, because they will usually share nursing and neonatal care ( figure - ) . after delivery, pairing of queens becomes more difficult. when tomcats are not breeding, they can usually be co-housed with a spayed female, a neutered male, or a compatible juvenile for companionship. other recommended groupings in the context of a breeding colony include postweaning family groups, prepubertal juveniles, or compatible single-sex adults. personality type there are two basic feline personality types: cats that are outgoing, confident, and sociable and those that are relatively timid and shy. cats with bold, friendly temperaments tend to cope and adapt more readily than shy, timid cats. a subset of the bold, friendly personality type is the "assertive" or "bully" cat. bully cats constantly threaten other cats in a group setting in order to control access to food, litter, perches, or the attention of human caregivers. to maintain harmony, removing cats of this personality type from a colony is usually necessary. reassignment is possible, but may prove difficult, necessitating single housing. shy, timid cats sometimes have difficulty interacting successfully with more dominant members of a group or may fall victim to a bully, resulting in chronic stress and increased hiding. placement of shy cats in smaller groups or with calm juvenile cats, where they will not be intimidated or harassed, is generally rewarding and often helps them to "come out of their shells." , similarly, dominant cats will often accept calm, younger cats, as opposed to other adults by whom they may feel threatened. and finally, in the case of some dominant males, the introduction of a female cat will be more likely to be successful. , , the precise space requirements for long-term housing of cats will vary, because it is dependent on many factors (box - ). , of paramount importance is that group size must be small enough to prevent negative interactions among cats and to permit daily monitoring of individuals. cats typically prevent social conflict through avoidance, and adequate space must be available so that cats can maintain social distance as needed. crowding can make it impossible for animals to maintain healthy behavioral distance, creating situations where individuals may not be able to freely access feeding, resting, or elimination space because of social conflicts over colony careful attention to groupings of cats is essential for success. family groups and previously bonded housemates are natural choices for co-housing, , but unfamiliar cats may also be grouped using careful selection criteria. many cats do have preferences for housemates, necessitating conscientious compatibility matching combined with the provision of a high-quality environment. groupings of unfamiliar cats should always be given priority for the largest available enclosures. in addition, cats should always receive appropriate health clearances prior to admission to a group. these should be determined by the specific protocols of the facility; but in most cases, minimum requirements would include that cats be free of signs of contagious disease, tested for felv and fiv, vaccinated against fvrcp, and treated for parasites. in addition to prior relationships, selection criteria for groupings should include age, reproductive status, and personality. age age is an important consideration regarding housing arrangements. to ensure proper social and emotional development, kittens should be housed with their mother at least until they are weaned. because it can be behaviorally beneficial, it is desirable for them to remain with her for a longer period of time when this is feasible. in fact, queens frequently do not fully wean their kittens until to weeks of age if left to their own devices. if older kittens are housed with their mother, it is important to provide a perch that allows her the option of periodically resting away from them if desired. most queens will accept the kittens of another cat; therefore young orphan or singleton kittens should be housed with other lactating queens and/or kittens of similar age/size. in a shelter setting where there is a high turnover of cats, it may be beneficial to house young kittens up to to months of age in large cages or condos for biosecurity purposes. juveniles and adults can be housed in colony rooms or runs but should be segregated by age (e.g., juveniles to months old, young adults, mature adults, geriatrics). well-socialized juveniles tend to adapt quickly in a group setting with other cats of similar ages and exhibit healthy activity and play behavior. in contrast, mature adults and geriatric cats often have little tolerance for the high energy and playful antics of many younger cats, which can cause them substantial stress. for this reason, adult cats should be kept separate from juvenile cats, and aging or geriatric cats separate from other age groups. in animal shelters, compatible cats that enter the shelter together should be housed together regardless of age, whenever possible. unless cats will be used for breeding, group housing of sexually intact cats of all of these reasons, housing cats in small groups is preferred. , , in most instances, the author recommends housing cats in compatible pairs or small groups of not more than three to four individuals. housing cats in runs is ideal for this purpose (figure - ) . a well-equipped, . -× . -m ( -× -ft) run can comfortably house two to three adult cats depending on their familiarity and compatibility, or up to four juveniles (e.g., to months old). juveniles tend to accept a slightly higher housing density than adults. likewise, previously bonded housemates and families will generally peacefully co-exist at a higher density than will unfamiliar cats. when runs are used, they must have a top panel and should be at least . m ( ft) high to allow caregivers easy access for cleaning and care. if chain-link is used, . -cm ( -inch) mesh is ideal, but larger mesh can be used. existing dog kennel runs can be converted into areas for cat housing. this is an important and practical consideration in animal shelters, because many shelters have experienced a decrease in dog intake, while the need for improved cat housing is great. cats and dogs should never be co-housed in the same area; thus conversion should result in an exclusive cat housing area. for colony rooms, the author recommends a minimum enclosure size of approximately to . m × to . m ( to ft × to ft) for colonies of up to a maximum of eight adult cats, or in the case of juveniles, a few more. doubling the size of an enclosure does not necessarily allow a twofold increase in the number of cats that can be properly housed. another author recommends . m ( ft ) per cat as a general guideline for group housing, resources. both crowding and constant introduction of new cats induce stress and must be avoided to ensure proper welfare. the addition of new cats always results in a period of stress for the group, and if there is constant turnover within the group, cats may remain stressed indefinitely. high turnover also increases the risk of infectious disease. if cat group numbers are small, disease exposure will be limited, facilitating control. for • length of stay • overall quality of the environment, including use of vertical space • overall quality of behavioral care • physical and behavioral characteristics of the cat (e.g., age, personality type, prior experience, and socialization) • individual relationships between cats (e.g., family groupings, previously bonded housemates, versus unfamiliar groupings and degree of social compatibility among cats) • turnover of cats (e.g., frequency of introduction of new members) • total room size • absolute number of cats • individual needs and levels of enrichment being used to meet these needs enabling caregivers to better monitor individual appetites and litter box results while allowing cats a period of rest away from one another. alternatively, individual enclosures may only be used for brief periods for meal feedings of canned food, with dry food available free choice in the colony. this sort of arrangement can also be used to facilitate introduction of new cats to the group and represents a desirable option. if design and biosecurity procedures permit, portable intake enclosures could even be transferred to group rooms to smooth the transition of new cats from intake to long-term housing areas. tremendous individual variation exists among cats in the context of social relations with other cats. although introduction of some previously unfamiliar cats will seem effortless and uneventful, introduction of others will result in considerable stress, not only for the new cat but for the entire group as well. for this reason, introductions should always be done under supervision, and whenever possible, they should be gradual. to accomplish this, a new cat can be kept in a separate cage within or adjacent to the group enclosure equipped with food, water, litter, and a hiding box. usually, within a few days, it will be evident by the behaviors of the cats whether or not the new cat can be transferred into the group enclosure without risk of fighting. wellsocialized kittens and juvenile cats frequently adapt readily to group accommodations, and prolonged introductions may not be necessary unless they are shy or undersocialized. in established groups of cats, the introduction or removal of individuals will require a period of adjustment and may result in signs of social stress for members of the colony. these signs usually subside once a new social hierarchy and territorial limits (usually favored resting places) are established. in some cases, arrangement of incompatible cats, even within visible distance of one another, may create substantial anxiety, necessitating rearrangement (figure - ) . in the case of animal shelters, where population interchange is high, it is generally not feasible to maintain consistent groupings of cats. this underscores the absolute necessity of careful selection and compatibility matching, as well as maintaining a variety of housing styles. even in modestly populated, carefully introduced, environmentally enriched colonies, behavior problems may occur. for this reason, some facilities elect to use an "all in-all out" approach to avoid repeated introductions of new cats into stable groups. in animal shelters, bonded pairs and family groupings of cats frequently enter the shelter together and are usually perfect choices for co-housing. because cats do have strong preferences for new roommates, caregivers must expect to find many that are incompatible as roommates. if only one or two cats are responsible for social destabilization of a group, they can acknowledging that many factors influence the spatial needs of cats, including the overall quality of the environment as well as the relationships of the individual animals. in sanctuary and laboratory situations where cats are housed for months to years in stable colonies, larger groupings of cats may be feasible, provided ample space is available. housing arrangements can also be created in which individual enclosures are maintained within a colony room. in this case, cats could be allowed to wander and interact freely in the colony room by day but be confined to their respective enclosures at night, for pair-housing of two adult cats. note the multiple separate areas for resting, perching, hiding, feeding, eliminating, scratching, and playing. b, cats enjoy the increased behavioral options provided by run-style housing. should exceed the number of cats and should be arranged in as many locations within the enclosure as possible. open single perches should be separated by at least . m ( ft) or staggered at different heights to ensure adequate separation, while larger perches should be available for cats who choose to rest together in close proximity. many cats enjoy hammock-style perches or semienclosed box-style perches where they can hide. if there are not enough comfortable, desirable resting and hiding places, cats may choose to lie in litter boxes. comfortable bedding (that is either disposable or can be easily laundered) should be provided. not only do cats demonstrate preferences for resting on soft surfaces, they experience longer periods of normal deep sleep with soft bedding. the environmental temperature should be kept comfortable and constant, and living quarters should be well ventilated, without drafts. by changing location within the colony (e.g., from the cooler surface of the floor to a sunny window), cats should be able to choose the environmental condition they prefer (figure - ) . in colony rooms, installation of stairs, shelves, and walkways are ideal for increasing the use of vertical space (figure - ) . in larger rooms, installation of freestanding towers provides additional living and activity space and contributes to functionally reducing overcrowding (figure - ) . depending on the setting, it may not be desirable for cats to access areas above the level of an arm's reach so that cleaning is easy and cats can be easily retrieved from the highest perches if needed. colony room design should also ensure that cats cannot easily escape. in some cases, constructing a foyer at the entrance to the room will be necessary to minimize the risk of escape when the room is entered (figure - ) . in addition, ceilings should be constructed of solid surfaces, because cats can easily dislodge the usually be reassigned to another colony, because it is often the social grouping, not the individual, that is the problem. if a cat shows persistent incompatibility with other cats, he or she should be housed singly. studies indicate that cats that fight at the time of initial introduction are nearly times more likely to continue fighting in the following weeks and months. if overt fighting occurs, cats should be permanently separated. cohousing of incompatible cats or cats that fight is unacceptable. the success of group housing depends not only on selection of compatible cats and the size of the enclosure but also on the quality of the environment.* a variety of elevated resting perches and hiding boxes should be provided to increase the size and complexity of the enclosure and to separate it into different functional areas, allowing a variety of behavioral choices. the physical environment should include opportunities for hiding, playing, scratching, climbing, resting, feeding, and eliminating. whenever possible, a minimum of litter box and food and water bowl should be provided per to cats and arranged in different locations of the colony space, taking care to separate food and water from litter by at least . m ( ft). in addition, placement should allow cats to access each resource from more than one side, whenever possible, without blocking access to doorways. litter boxes should not be covered, to allow easy access and to prevent entrapment or ambush by other cats. the number of resting boards and perches the importance of a cat-savvy staff that enjoys working with cats cannot be overemphasized. animal care staff must be willing to spend quality time interacting with cats to assure socialization and tractability. , whenever possible, caregivers should be assigned to care for the same cats on a regular basis so that they become aware of the personality of each individual cat, which is necessary for detection of health problems, incompatibilities between cats, and, in the case of breeding colonies, estrous cycling. this is also important, because not all cats uniformly enjoy human companionship and will be more likely to be stressed by the presence of different caregivers, rather than becoming familiar and more at ease with one. in general, regular daily contact and socialization is essential to ensure that cats are docile, easy to work with, and have no fear of humans. caregivers should schedule time each day to interact with "their" cats aside from the activities of feeding and cleaning. some cats may prefer to be petted and handled, while others prefer to interact with caregivers by playing with toys (figure - ) . in particular, human contact is essential for proper socialization of young kittens. a sensitive period of socialization occurs during the development of all infant animals, during which social attachments to members of the same species and other species form easily and rapidly. in kittens, the sensitive period of socialization occurs between and weeks of age, and cats not properly socialized to humans during this time may never permit handling. , beginning shortly after birth, kittens should be handled daily, talked to in a soothing panels typically used for dropped ceilings, and escape into the rafters (figure - ) . in addition to contact with conspecifics, cats must be afforded time for pleasant daily contact with human caregivers. as previously discussed, daily social contact and exercise sessions with humans are especially important for individually caged cats. although social contact is usually highly desirable, it is not invariably pleasant for all cats. personality, socialization, previous experience, and familiarity contribute to whether or not social interactions are perceived as pleasurable, stressful, or somewhere in between. , a b voice, gently petted, and held. interactions should include play (stimulated with toys) as the kittens become ambulatory. for kittens housed in a shelter, socialization must always be balanced with infectious disease control, and caregivers should take precautions accordingly. other forms of stimulation, including those that engage the various senses, are important methods of enriching the living environment by promoting healthy mental and physical activity. for singly housed cats and longterm residents, appropriate levels of additional enrichment should be provided on a daily basis. the provision of birdfeeders, gardens, or other interesting stimuli in the external environment can enhance the internal environment of the colony. resting perches in view of windows or other pleasant areas of the facility are especially desirable. other novel and enriching visual stimuli include cat-proof aquariums with fish, water fountains, bubbles, perpetual motion devices, and videos especially designed for cats (figures - to . a radio playing soft, low music in the room provides a welcome distraction and important source of play items that stimulate prey drive and physical activity, such as plastic balls, rings, hanging ropes, springmounted toys, plastic wands, and catnip toys, should also be provided but must be either sanitizable or disposable. empty cardboard boxes and paper bags are inexpensive, disposable, and stimulate exploration and play behavior as well as scratching. cats tend to be most stimulated by active toys, including wiggling ropes, wands with feathers, kitty fishing poles, and toys that can be slid or rolled to chase. many cats enjoy chasing stimulation. in addition, it may help to habituate cats to human voices and prevent them from being startled by loud noises. most caregivers also enjoy listening to the radio, and happy caregivers create a relaxed environment. the provision of scratching boards is especially important, and a variety of sturdy surfaces, both horizontal and vertical, should be provided for scratching. sisal rope, the backs of carpet squares, and corrugated cardboard are all useful (figure - ) . many cats like to smell and chew grass, and containers of cat grass or catnip can be introduced for brief periods to stimulate activity (figure - ) . providing novel sources of food is another important source of stimulation and can be easily accomplished by hiding food in commercially available food-puzzle toys or in cardboard boxes or similar items with holes such that the cat has to work to extract pieces of food ( figure - ) . , positive reinforcement-based training obedience training using clickers with food rewards is an excellent form of enrichment, combining social contact with caregivers together with both mental and physical stimulation. positive reinforcement training using a target stick is a powerful tool for teaching shy cats to approach the front of an enclosure. teaching cats awaiting adoption to perform tricks is not only stimulating for them, but it often makes them more attractive to potential adopters (figure - ) . a cardboard tube, and a plastic container with holes). treats are hidden inside, and they will have to work to extract pieces of food. novel feeding is an excellent source of enrichment for cats that are housed long term. (e.g., feeding, cleaning, enrichment activities), and unpredictable caregiving has been shown to dramatically increase stress. if events that are perceived as stressful (such as cleaning time) occur on a predictable schedule, cats learn that a predictable period of calm and comfort will always occur in between. cats also respond to positive experiences in their daily routines. for example, feeding and playtime may be greatly anticipated; thus scheduling positive daily events (e.g., a treat at : pm every day) should also be a priority. erratic periods of light and darkness are also known to be significant sources of stress for cats. animals possess natural circadian rhythms and irregular or continuous patterns of light or darkness are inherently stressful. lighting should be maintained on a regular the beams of laser pointers, small flashlights, or suspended rotating disco balls. commercially available electronic toys that stimulate play are especially useful in long-term settings (figure - ) . varied toys should be substituted regularly to ensure continued interest. in some climates, cats may be housed comfortably in outdoor enclosures where fresh air, sunshine, and other stimuli can help to create a healthy environment ( figures - and - ) . , , when indoor group enclosures connect to outdoor enclosures, it is important to have ample space for passage between them (e.g., more than one doorway) so that cats can pass freely. cats will also benefit greatly from consistent daily routines of care. they become entrained to schedules of care adoption, or euthanasia (when no other options exist) may be necessary to ensure cat welfare. achieving population wellness requires a healthy environment. thus the clinician's final task in creating a population wellness program is to develop tailored schedule, with lights on by day and off by night. whenever possible, full-spectrum and/or natural lighting is ideal. housed cats require active daily monitoring by staff trained to recognize signs of stress and social conflict. to the inexperienced observer, such signs may appear subtle (figure - ) . it is often the absence of normal behaviors (such as engaging in grooming or exercise) or subtle social signals (such as covert guarding of resources or dominant staring) that signify problems. careful observers will note these behaviors and respond accordingly to ensure that stress or conflicts do not persist. when cats are well adjusted and housing arrangements meet their behavioral needs, they display a wide variety of normal behaviors, including a good appetite and activity level, sociability, grooming, appropriate play behavior, and restful sleeping (figure - ) . , ultimately, the success of adaptation of cats to a new environment will depend on both the quality of the environment and the adaptive capacity of the individual. although most adapt to new environments with time, some never adjust and remain stressed indefinitely, ultimately resulting in decline of physical as well as emotional health. when cats fail to adjust to their environment and remain markedly stressed and fearful despite appropriate behavioral care, every effort must be made to prevent long-term stays. depending on the circumstances, transfer to another colony room, foster care, a shelter will result in saving more lives. to the contrary, euthanasia rates are highly correlated to intake rates, regardless of the number of animals that a facility houses. in many instances, keeping more animals in the shelter may actually reduce the organization's ability to help animals, because time and resources are tied up caring for a crowded, stressed population, rather than focusing on adoption or other positive outcomes. in shelter medicine, the term population management is used to refer to an active process of planning, ongoing daily evaluation, and responding to changing conditions as an organization cares for multiple animals. , the major goal of population management is to minimize the amount of time any individual animal spends confined in the shelter, while maximizing the organization's lifesaving capacity. moving animals through the system efficiently is the foundation of effective population management. to move animals through the shelter more quickly, delays in decision making and the completion of procedures (e.g., intake processing, transfer from holding to adoption areas, spay/neuter surgery) must be eliminated or minimized whenever possible. in openadmission shelters, even delays of to days can have a dramatic effect on the shelter's daily census, particularly for shelters handling thousands of animals per year. this, in turn, affects the ability to provide adequate care. it is important to recognize that effective population management does not change the final disposition of an animal. it does mean that determinations are made as soon as possible, which serves both the individual animal as well as the population as a whole. for wellness programs to be effective, a clean and sanitary environment must be maintained. not only does this promote cat and human health, but it also promotes staff pride as well as public support. in addition to protocols for routine daily cleaning and disinfection procedures, protocols should be in place for periodic deep cleaning and disinfection as well as procedures to be used in the event of disease outbreaks. when crafting protocols, it is important to recognize that cleaning and disinfection are two separate processes. the cleaning process involves the removal of gross wastes and organic debris (including nonvisible films) through the use of detergents, degreasers, and physical action. although this process should result in a visibly clean surface, it does not necessarily remove all of the potentially harmful infectious agents that may be present. disinfection is the process that will destroy most of these agents, but it cannot be accomplished until surfaces have been adequately cleaned. disinfection is usually accomplished through the application of chemical compounds or disinfectants. the most commonly used of these are reviewed in protocols focused on optimizing environmental conditions that favor cat health. once again, all essential elements as noted should be addressed. perhaps the most critical aspect of environmental management is to ensure a modest population density. high population density increases opportunities for introduction of infectious disease while increasing the contact rate among members of a group. both the number of asymptomatic carriers of disease, such as those with upper respiratory infection, as well as susceptible cats in a given group are likely to increase, enhancing the odds of disease transmission among group members through both direct contact as well as fomites. in addition, crowding also increases the magnitude of many environmental stressors (e.g., noise levels, air contaminants) and compromises animal husbandry, all of which induce unnecessary stress and further inflate the risk of disease in the population. indeed, crowding is one of the most potent stressors recognized in housed animals. although adequate space for animals is essential, it is crucial to recognize that crowding is not solely dependent on the amount of available space. it is also a function of the organization's ability to provide proper care that maintains animal health and well-being. every organization has a limit to the number of animals for which it can provide proper care. when more animals are housed than can be properly cared for within the organization's capacity, caregivers become overwhelmed, and animal care is further compromised. , , in animal shelters, crowding may also negatively impact adoption rates, because potential adopters often find crowded environments to be overwhelming and uninviting. if disease spread results as a consequence of the environmental conditions, animal adoptions may be further disrupted. although unexpected shelter intake may occasionally result in temporary crowding, a good wellness program dictates that protocols must be in place to alleviate crowding and maintain a modestly populated environment for the health and protection of the animals and staff. regardless of the setting, facilities must limit the number of animals housed to the number for which they can provide proper space and care. there are three basic methods of reducing crowding: ( ) limiting the admission (or births) of new animals into a population, ( ) increasing release of animals from a population, and ( ) euthanasia. in animal shelters, management practices that minimize each animal's length of stay and programs that speed or increase adoption, owner reunification, or transfer (e.g., to rescue or foster care) help to minimize crowding and maximize the number of animals that an organization can serve. it is a common misperception that housing more animals in • although commonly used, they must be applied to clean hands and allowed seconds of contact time to be effective. • they are highly effective against bacteria, but have only moderate activity against viral agents, including feline calicivirus (fcv). • they should not be used as a substitute for hand washing or the use of gloves. • chlorhexidine is the most commonly used biguanide and is relatively expensive. its major use is as a surgical preparation agent. • although biguanide compounds have broad antibacterial activity, they have limited efficacy against viruses and are ineffective against nonenveloped viruses, such as panleukopenia and fcv. therefore they are not recommended as general-purpose environmental disinfectants. • household bleach ( . % sodium hypochlorite) is the most commonly used chlorine compound and is an excellent, safe, and highly cost-effective disinfectant when used correctly. • at a dilution of : , bleach is highly effective against bacteria and viruses, including nonenveloped viruses, such as panleukopenia and fcv. • solutions must be made fresh daily and stored in opaque containers, because bleach is highly unstable once mixed with water and degrades in the presence of ultraviolet light. • surfaces must be thoroughly cleaned with a detergent, rinsed, and dried prior to the application of bleach, because it is ineffective in the presence of detergents and organic material. • proper disinfection requires minutes of contact time with a bleach solution. • although bleach is not effective when mixed with detergents, it can be safely and effectively mixed with quaternary ammonium compounds, which do provide some cleaning activity. therefore this combination can be used for cleaning and disinfection, provided gross organic material is first removed and adequate contact time is allowed. the addition of bleach improves the disinfection properties of the solution, making it effective against nonenveloped viruses, including panleukopenia and fcv. • concentrations stronger than a : dilution can result in respiratory irritation for both animals and people, as well as increased facility corrosion, and are therefore not recommended for routine use. • at a dilution of : , bleach will destroy dermatophyte spores. however, cats must be removed from the environment prior to application of this concentration. • the use of calcium hypochlorite (wysiwash, st. cloud, fla.) is becoming more common and offers the potential advantages of reduced contact time and a neutral ph, which prevents corrosion. oxidizing agents quarantine involves the holding of healthy-appearing animals. it is most useful when animals enter a closed population to ensure that they are not incubating disease when they are introduced into the general population. quarantine areas, with rigid biosecurity procedures in place, should be used to segregate healthy animals for observation. the use of such areas not only allows apparently healthy animals to be observed for developing signs of infectious disease, but it also allows time for response to vaccination in a highly biosecure environment where exposure risks are minimized. the use of quarantine is a mainstay of effective infectious disease control programs and is intended to prevent the introduction of disease into a population. it should be used whenever it is feasible to implement effectively, such as in a laboratory setting, a private cattery, or a low-volume, limited-admission sanctuary setting. however, quarantine practices are not effective in most animal shelters, because the high volume and turnover of animals precludes proper implementation of a true quarantine where an "all in-all out" system is used. instead, incoming animals are usually added to the "quarantine group" on a daily basis, effectively defeating the purpose of true quarantine and simply prolonging the animal's stay. this is especially concerning given the fact that a cat's length of stay in a shelter is a major risk factor for development of upper respiratory infection. for this reason, the use of quarantine is not recommended in most shelter settings. instead, high biosecurity areas are recommended for housing the most susceptible animals (e.g., kittens less than to months of age). on the other hand, quarantine is warranted when a serious disease is discovered in a shelter population. if healthyappearing animals are exposed during a serious outbreak, quarantine procedures should be used to stop the movement of animals and prevent further spread of disease. if possible, temporary closure to admittance is also recommended. quarantine may also be required in bite cases to ensure compliance with state rabies laws. the particular population setting will guide the clinician's determination regarding implementation and length of quarantine, if any. a -day quarantine is sufficient to determine that cats are not incubating many common infectious diseases, including feline panleukopenia. however, other diseases, including feline leukemia virus and dermatophytosis, can have longer incubation periods and will therefore require a longer quarantine period. , in breeding colonies, early weaning and quarantine have been advocated to prevent infection of kittens with feline coronavirus. pregnant queens are isolated, and product selection should take into consideration the conditions present in a given environment (e.g., the type of surface and the presence of organic matter) and the compound's activity against the pathogens for which the animals are at greatest risk. the nonenveloped viruses, panleukopenia and feline calicivirus, are of particular concern. it is important to note that despite product label claims to the contrary, multiple independent studies have consistently shown that quaternary ammonium disinfectants do not reliably inactivate these important feline pathogens. , , in addition to selecting effective agents, ensuring adequate contact time followed by thorough drying of surfaces is essential for achieving proper disinfection. protocols should include detailed methods for achieving both cleaning and disinfection. when performed properly and regularly, these practices decrease both the dose and duration of exposure to infectious agents. box - outlines essential considerations for the development of cleaning and disinfection protocols. segregation refers to the separation of animals from the main group or into subpopulations as necessary to promote health. segregation of cats by physical and behavioral health status is essential for infectious disease control, stress reduction, and safety. in animal shelters, segregation may also be required to ensure compliance with animal control procedures as prescribed by state or local ordinances. depending on the setting, consideration should be given to separating cats by gender and reproductive status (e.g., intact, neutered, in heat, pregnant, nursing), physical and behavioral health status (e.g., apparently healthy, signs of contagious disease, reactive, feral), and life stage. a variety of separate areas will be necessary, depending on the needs of the given population and the context of the setting. the wellness program should define these areas in order to optimize cat health, while providing for the necessary functions of the facility. depending on the setting, consideration should be given to establishing areas for quarantine, isolation, general holding, adoption, and long-term housing, as well as to tailoring these by life stage. for example, the very young and very old typically require more specialized care than healthy juveniles and adults. kittens less than to months of age are particularly susceptible to infectious disease, and extra care must be taken to heighten biosecurity and limit their exposure. in particular, geriatric cats require comfortable, quiet quarters with careful attention to stress reduction (e.g., the provision of a secure hiding place and a dedicated caregiver to enhance bonding and comfort). if cats are used for breeding, functional areas will be required to facilitate mating, queening, and kitten care. and, as previously • staff must wear personal protective equipment, as necessary, to prevent exposure to chemicals and/or pathogens. • thorough cleaning and disinfection of enclosures should occur between occupants and as part of periodic deep cleaning procedures. • cats must be removed from enclosures during these procedures. • "spot cleaning" is generally sufficient for apparently healthy cats that will continue to occupy the same enclosure. • the cat remains in the enclosure while it is cleaned and soiled material is removed. • this method is often less stressful for cats (see figure - ). • separate staff should clean and care for animals in areas with highly susceptible or sick animals, whenever possible. • at minimum, attention should be given to the order of cleaning. • the least contaminated areas should be cleaned before those that are the most contaminated. kittens are weaned as early as possible (e.g., to weeks of age) and placed in strict quarantine. in this manner, as maternal antibody wanes and kittens become susceptible to the virus, exposure and infection are prevented. however, it is important to note that the level of biosecurity required for success is difficult to achieve. furthermore, eventual exposure and infection are highly likely because of the ubiquitous nature of coronaviruses. in addition, because of the importance of the mother-kitten relationship to normal social and emotional development, this management practice may not always be desirable. healthy environmental conditions in isolation areas promote recovery, and their importance cannot be overemphasized. crowding, noise, and stress must be avoided, and facilities must be easy to clean and disinfect. room temperature should be warm and comfortable with good air quality. windows are ideal, because natural sunlight is always beneficial to animal health and healing. strict biosecurity in quarantine and isolation areas, with attention to traffic patterns and the use of protective clothing, such as gowns and shoe covers, is essential. footbaths are insufficient to prevent transfer of infectious agents on shoes. this is because disinfectants typically require minutes of contact time and may be poorly effective in the presence of organic debris. in fact, footbaths may even contribute to the spread of disease. dedicated boots or shoe covers should be used when entering contaminated areas. , in addition, separate, designated staff should care for animals in high biosecurity areas whenever possible. by design, traffic patterns should move from the healthiest and most susceptible groupings to the least susceptible, and finally to isolation areas housing sick animals. observation windows and signage are useful to reduce traffic flow into high-risk areas. staff hygiene is extremely important, and the importance of diligent hand washing cannot be overemphasized. where space or facilities are not available, foster care may represent a viable and medically sound option for quarantine or isolation in some settings. for instance, in animal shelters, foster care is particularly useful for the care of preweaning-age kittens. foster homes must be monitored to ensure that cats receive proper care and that resident animals are protected from disease exposure. in addition to ensuring proper population density, segregation, and sanitation procedures, there are several other essential aspects of facility operations that must be incorporated into a population wellness program. these include heating, ventilation, and air conditioning (hvac) considerations, noise and pest control, general facility maintenance, and staff training. extremes or fluctuations in temperature and humidity, as well as poor ventilation and air quality, can compromise animal health. poor ventilation and high humidity favor the accumulation of infectious agents, while dust and fumes may be irritating to the respiratory tract. many cats are particularly sensitive to drafts and chilling, both of which can predispose to upper respiratory general holding, housing, adoption, and other areas in many settings, general holding areas are used for housing healthy juvenile and adult cats at intake. in animal shelters, it is important to consider that length of stay is associated with an increased risk of feline upper respiratory disease and that vaccination against core diseases often rapidly confers immunity. for these reasons, holding periods should be minimized whenever possible. in some cases, holding times will be influenced by legally required holding periods prescribed by state laws that allow owners a chance to reclaim lost pets. legal holding periods are usually not required for owner-relinquished pets and preweaning-age animals, but a brief medical hold (e.g., to days) for evaluation and triage is usually warranted. regardless, management practices that reduce length of stay are generally best for population health in a shelter setting. immunity is often strengthened with time through a combination of both active and passive immunity resulting from vaccination and exposure. upper respiratory disease is often endemic in cat populations, and in open populations, constant introduction of large numbers of carriers and susceptible cats make exposure likely. as length of stay increases, many cats develop and recover from respiratory disease. as animals acclimate to their environment and gain immunity, less stringent biosecurity requirements may be required for long-term housing areas, depending on the particular setting. in animal shelters, the public is usually allowed to interact with cats in adoption areas, which is another management consideration. isolation areas are used to segregate sick animals from the general population. immediate isolation of animals with signs of infectious disease is critical to effective control. isolation should be targeted by age and disease. for example, separate isolation areas should be available for cats and kittens with respiratory disease and those with gastrointestinal disease, whenever possible. in populations where upper respiratory infection is problematic, having two isolation areas for cats with respiratory infections is ideal: one area for those cats with moderate to severe signs that will require more intensive monitoring and treatment, and a separate area for those cats with only mild clinical signs and those that have been treated and are nearly recovered. when mildly symptomatic cats can be housed separately from those that are very ill, staff compliance with isolation procedures are often improved. cats with non-infectious conditions should also be housed in separate areas for treatment, and, in some cases, housing in the general population is appropriate. prevents exchange of air among them and is recommended, because air pressure gradients that recirculate or cause exchange of air between rooms have been associated with the spread of disease by aerosols. when applying these standard recommendations to a particular setting, there are some practical considerations that should be taken into account. first, even when ventilation systems provide to room air changes per hour, airflow may be restricted inside of cages or other enclosures within the room. in other words, the body of the room may be well ventilated, yet inside the cages, the air may remain relatively stagnant. in this case, ventilation may be improved by altering the housing design or arrangement; for example, the use of flow-through cages, runs, free-range rooms, or outdoor access may result in improved air quality. when considering the recommendations for % fresh, nonrecycled air with separate ventilation systems in various areas of the facility, consideration should be given to the fact that respiratory pathogens in cats are not aerosolized because of the cats' small tidal volume. although droplet transmission is possible, droplet spray does not extend more than feet, and most transmission of respiratory disease in cats is through direct contact with infected cats, carriers, or fomites. although this recommendation seems prudent to consider, especially for isolation areas, it is very expensive to install and operate this type of ventilation system throughout a facility, especially in very cold or very hot climates. if air quality remains good and the facility maintains effective comprehensive wellness protocols, it may not be necessary for animal health. more research is needed on the impact of such air exchange, but in the meantime, the author recommends consulting with an hvac specialist to establish effective and efficient settings to suit the specific needs of the given population. in addition to ensuring good ventilation, it is imperative to use other strategies for maintaining good air quality, including regular maintenance of filters, control of dust and dander through routine vacuuming and periodic deep cleaning, and the use of dust-free litter. noise control is another important consideration. it is crucial to keep cats out of auditory range of dogs, because many are profoundly stressed by the sounds of barking. also, staff should be trained to reduce or avoid other sources of noise whenever possible. the installation of sound-proofing systems may be necessary for noise abatement and stress reduction. routine pest control may also be required, depending on the setting. it may be necessary to treat the environment for fleas, ticks, or other insects or ectoparasites. products used to treat the environment must be selected carefully, because cats are extremely sensitive to the toxic effects of many insecticides. in many instances, it will infection. heating, ventilation, and air conditioning (hvac) specialists are uniquely qualified to help establish and maintain the environmental conditions required for animal health. when facilities are designed specifically for housing animals, these specialists should be consulted beforehand to ensure installation of the most effective and efficient systems possible. in reality, many facilities that house cats, including private catteries and shelters, among others, were not originally built for this purpose. retrofitting existing facilities with the ideal hvac system is often neither logistically nor financially feasible. regardless, consultation with hvac specialists is recommended in order to maximize the potential of the facility's existing system. the recommended temperature range for cats is between ° c and ° c ( ° f and ° f) with a temperature setting in the low-to mid- s celsius ( s fahrenheit) being typical. the temperature setting should be determined according to the specific animals' needs. for example, neonatal kittens are more susceptible to hypothermia and generally require warmer temperatures than healthy adult cats. the location of the cats may also be a consideration. for example, enclosures located closer to floor level are often a few degrees cooler than those at higher levels. the exact temperature setting may also vary somewhat based on the season of the year. for instance, power companies typically recommend keeping the temperature between ° c and ° c ( ° f and ° f) during hot weather to conserve electricity and reduce power bills. laboratory guidelines recommend % to % humidity for cats. higher humidity (e.g., %) may be advantageous in areas housing cats with respiratory disease because moist air may be beneficial to the respiratory passages, whereas lower humidity (e.g., % to %) may be beneficial in other areas in order to reduce survival of infectious agents in the environment. although the range considered acceptable is large, a given room should have a relatively constant humidity (i.e., it should not have large fluctuations). hosing or even mopping a room usually results in temporary spikes in humidity, but these will be short lived in a well-ventilated room. adequate ventilation is crucial for good air quality. this is especially important for cats, because good air quality is essential for control of upper respiratory disease. ten to fifteen air changes per hour is the standard recommendation for an animal room, but more or less airflow may be acceptable or necessary depending on the housing density. theoretically, the best case scenario, and what is typical in laboratory animal settings, is for the hvac system to allow for % fresh (e.g., nonrecycled) air in each room so that the air entering a given room is exhausted out of the building and not recirculated to another room. maintaining separate ventilation systems for various rooms or areas of a facility ensure both the physical and behavioral health of cats, as well as a healthy environment. a proactive, holistic approach coupled with compassion is required. when these are combined with careful attention to the unique needs and stress responses of cats, the result will be "healthy, happy cats." be necessary to remove cats during their application and only return them to the environment once it is thoroughly dried and ventilated. if rodent control is necessary, the use of rodenticide baits should be avoided, because cats can be exposed even if the bait is not within their reach. rodents that have ingested the poisonous bait may enter an animal enclosure and, if the animal ingests the rodent, the poison will affect that animal. humane live traps can be used to capture rodents for removal from a facility. food containers should be kept tightly sealed, and clutter should be minimized to discourage pests in the environment. general building maintenance procedures (e.g., regular inspection and servicing with repairs as needed) are also important considerations for the maintenance of a healthy environment. for example, periodic resealing of floors may be required as well as maintenance of plumbing fixtures to repair leaks or other problems. developing and following written standard operating procedures and daily, weekly, monthly, and quarterly checklists will ensure that systematic schedules of maintenance are carried out in a timely fashion. regular staff training is essential for implementing effective population wellness programs. simply stated, staff caring for animals must be qualified to do so. to a large extent, their knowledge and skill will determine the success or failure of the wellness program. embracing a culture of training promotes high-quality animal care as well as human safety. both formal and on-thejob training should be provided to ensure that a staff has the knowledge and skills required to perform their assigned tasks. protocols should be established for all levels of training, and a system should be in place to ensure proficiency. staff training should be documented, and continuing education should be provided to maintain and improve skills. finally, training must include the provision of information about zoonoses and other occupational health and safety considerations. regardless of the setting, maintaining population health is essential for animal welfare as well as to meet the goals of the particular population. population health depends on implementation of comprehensive wellness protocols, systematic surveillance, and excellent management. facilities must establish goals for animal health, and wellness protocols must be regularly evaluated and revised to ensure that these goals are met. the bulk of efforts must focus on preventive strategies to control of feline coronavirus in breeding catteries by serotesting, isolation, and early weaning guidelines for the diagnosis, treatment and prevention of heartworm (dirofilaria immitis) infection in cats american society for the prevention of cruelty to animals (aspca): mission possible, comfy cats. shelter temperament evaluations for cats microchipping of animals association of shelter veterinarians (asv): board position statement on cats who test positive for felv and fiv association of shelter veterinarians (asv): board position statement on veterinary supervision in animal shelters association of shelter veterinarians (asv): standards of care for animal shelters gender differences in the social behavior of the neutered indoor-only domestic cat beaver bv: fractious cats and feline aggression a game of cat and house: spatial patterns and behavior of domestic cats (felis catus) in the home affiliative behaviours of related and unrelated pairs of cats in catteries: a preliminary report immunisation against panleukopenia: early development of immunity cvma): a code of practice for canadian cattery operations behavioral and physiological correlates of stress in laboratory cats the cat: its behavior, nutrition and health cfa cattery standard minimum requirements feline heartworm (dirofilaria immitis) infection: a statistical elaboration of the duration of the infection and life expectancy in asymptomatic cats environmental factors in infectious disease immunoprophylaxis and immunotherapy infectious disease management in animal shelters scaredy cat or feral cat? animal sheltering the lowdown on upper respiratory infections in cats in griffin b: a model of non-invasive monitoring of feline ovarian function in the domestic cat domestic cats as laboratory animals recognition and management of stress in housed cats stress and immunity: a unifying concept effects of a synthetic facial pheromone on behavior of cats small animal clinical nutrition housing design and husbandry management to minimize transmission of disease in multi-cat facilities implementing a population health plan in an animal shelter: goal setting, data collection and monitoring, and policy development feline infectious disease control in shelters commission on life sciences, national research council: guide for care and use of laboratory animals questions and answers on the effects of surgically neutering dogs and cats virucidal efficacy of the newer quaternary ammonium compounds stress and adaptation of cats (felis silvestris catus) housed singly, in pairs, and in groups in boarding catteries effects of density and cage size on stress in domestic cats (felis silvestris catus) housed in animal shelters and boarding catteries socialization and stress in cats (felis silvestris catus) housed singly and in groups in animal shelters centers for disease control: healthy pets, healthy people responses of cats to nasal vaccination with a live, modified feline herpesvirus type use of fipronil to treat ear mites in cats counsel of europe: guidelines for accommodation and care of animals. proposed revision to appendix a to the european convention for the protection of vertebrate animals used for experimental and other scientific purposes. minimum cage floor area for cats soft surfaces: a factor in feline psychological well-being social organization in the cat: a modern understanding social behavior and aggressive problems of cats descriptive epidemiology of feline upper respiratory tract disease in an animal shelter sanitation and disinfection all together now: group-housing cats behavioral differences between owner surrender and stray domestic cats after entering an animal shelter a clinical trial of intranasal and subcutaneous vaccines to prevent upper respiratory infection in cats at an animal shelter virucidal efficacy of four new disinfectants influence of visual stimulation on the behaviour of cats housed in a rescue shelter feline dental prophylaxis and homecare disinfection efficacy against parvoviruses compared with reference viruses farm animal welfare council: five freedoms feline respiratory disease experimental induction of feline viral rhinotracheitis in fvr-recovered cats evaluation of the efficacy of disinfectant footbaths as used in veterinary hospitals national council on pet population study and policy: the shelter statistics survey animal welfare advisory committee: companion cats code of welfare five key population management factors affecting shelter animal health cat housing in rescue shelters: a welfare comparison between communal and discrete-unit housing recognizing and managing problem behavior in breeding catteries quality of life in long-term confinement maddie's infection control manual for animal shelters a review of feline infectious peritonitis virus observations on the epidemiology and control of viral respiratory disease in cats employee reactions and adjustment to euthanasia related work: identifying turning points through retrospective narratives the aafp feline vaccine advisory panel report welfare of cats in a quarantine cattery recommendations for the housing of cats in the home, in catteries and animal shelters, in laboratories and in veterinary surgeries comfortable environmentally enriched housing for domestic cats efficacy of fipronil in the treatment of feline cheyletiellosis virucidal disinfectants and feline viruses the influence of food presentation on the behavior of small cats in confined environments validation of a temperament test for domestic cats evaluation of the effects of footwear hygiene protocols on nonspecific bacterial contamination of floor surfaces in an equine hospital the human-cat relationship editor: cattery design: the essential guide to creating your perfect cattery intercat aggression in households following the introduction of a new cat feline leukemia virus and feline immunodeficiency virus american association of feline practitioners feline retrovirus management guidelines intercat aggression: a retrospective study examining types of aggression, sexes of fighting pairs and effectiveness of treatment evaluation of collars and microchips for visual and permanent identification of pet cats characterization of animal with microchips entering shelters in vitro sensitivity of commercial scanners to microchips of various frequencies sensitivity of commercial scanners to microchips of various frequencies implanted in dogs and cats search and identification methods that owners use to find a lost cat provision of environmentally enriched housing for cats environmentally enriched housing for cats when singly housed managing oral health in breeding catteries assessment of stress levels among cats in four shelters enriching the environment of the laboratory cat the impact of paternity and early socialisation on the development of cats' behaviour to people and novel objects quality of life in animals development of a mental wellness program for animals toxicology brief: the most common toxicoses in cats the domestic cat. perspective on the nature and diversity of cats definition of wellness behavioral effects of cage enrichment in single-caged adult cats treatment of dermatophytosis in dogs and cats: review of published studies development of an in vitro, isolated, infected spore testing model for disinfectant testing of microsporum canis isolates quality-of-life assessment in pet dogs aafp-aaha. feline life stage guidelines meet your match and feline-ality adoption program key: cord- - swjzic authors: nan title: scientific opinion on the public health hazards to be covered by inspection of meat from sheep and goats date: - - journal: efsa j doi: . /j.efsa. . sha: doc_id: cord_uid: swjzic a risk ranking process identified toxoplasma gondii and pathogenic verocytotoxin‐producing escherichia coli (vtec) as the most relevant biological hazards for meat inspection of sheep and goats. as these are not detected by traditional meat inspection, a meat safety assurance system using risk‐based interventions was proposed. further studies are required on t. gondii and pathogenic vtec. if new information confirms these hazards as a high risk to public health from meat from sheep or goats, setting targets at carcass level should be considered. other elements of the system are risk‐categorisation of flocks/herds based on improved food chain information (fci), classification of abattoirs according to their capability to reduce faecal contamination, and use of improved process hygiene criteria. it is proposed to omit palpation and incision from post‐mortem inspection in animals subjected to routine slaughter. for chemical hazards, dioxins and dioxin‐like polychlorinated biphenyls were ranked as being of high potential concern. monitoring programmes for chemical hazards should be more flexible and based on the risk of occurrence, taking into account fci, which should be expanded to reflect the extensive production systems used, and the ranking of chemical substances, which should be regularly updated and include new hazards. control programmes across the food chain, national residue control plans, feed control and monitoring of environmental contaminants should be better integrated. meat inspection is a valuable tool for surveillance and monitoring of animal health and welfare conditions. omission of palpation and incision would reduce detection effectiveness for tuberculosis and fasciolosis at animal level. surveillance of tuberculosis at the slaughterhouse in small ruminants should be improved and encouraged, as this is in practice the only surveillance system available. extended use of fci could compensate for some, but not all, the information on animal health and welfare lost if only visual post‐mortem inspection is applied. following a request from the european commission to the european food safety authority (efsa), the panel on biological hazards (biohaz) was asked to deliver a scientific opinion on the public health hazards to be covered by the inspection of meat from sheep and goats. the panel was supported by the efsa panels on contaminants in the food chain (contam) and animal health and welfare (ahaw) in the preparation of this opinion. briefly, the main risks for public health that should be addressed by meat inspection were identified and ranked; the strengths and weaknesses of the current meat inspection system were evaluated; recommendations were made for inspection methods fit for the purpose of meeting the overall objectives of meat inspection for hazards currently not covered by the meat inspection system; and recommendations for adaptations of inspection methods and/or frequencies of inspections that provide an equivalent level of protection were made. in addition, the implications for animal health and animal welfare of any changes proposed to current inspection methods were assessed. sheep and goats were considered together, unless otherwise stated. decision trees were developed and used for priority ranking of biological and chemical hazards present in meat from sheep and goats. for biological hazards the ranking was based on the magnitude of the human health impact, the severity of the disease in humans and the evidence supporting the role of meat from sheep and goats as a risk factor for disease in humans. the assessment was focused on the public health risks that may occur through the handling, preparation for consumption and/or consumption of meat from these species. the term 'priority' was considered more appropriate than 'risk' for categorizing the biological hazards associated with meat from small ruminants, given that a significant amount of data on both the occurrence of the hazards and on the attributable fraction of human cases to meat from small ruminants were not available. risk ranking of chemical hazards into categories of potential concern was based on the outcomes of the national residue control plans (nrcps), as defined in council directive / /ec for the period - , and of other testing programmes, as well as on substance-specific parameters such as the toxicological profile and the likelihood of the occurrence of residues and contaminants in sheep and goats. based on the ranking for biological hazards, toxoplasma gondii and pathogenic verocytotoxinproducing escherichia coli (vtec) were classified as high priority for public health regarding meat inspection of small ruminants. the remaining hazards were classified as low public health relevance, based on available data, and were therefore not considered further. for chemical hazards, dioxins and dioxin-like polychlorinated biphenyls (dl-pcbs) were ranked as being of high potential concern owing to their known bioaccumulation in the food chain, their frequent findings above maximum levels (mls), particularly in sheep liver, and in consideration of their toxicological profile; all other substances were ranked as of medium or lower concern. it should be noted that the ranking into specific-risk categories of hazards is based on current knowledge and available data and therefore ranking should be updated regularly, taking account of new information and data and including 'new hazards'. the main elements of the current meat inspection system include analysis of food chain information (fci), ante-mortem examination of animals and post-mortem examination of carcasses and organs. the assessment of the strengths and weaknesses of the current meat inspection was based on its contribution to the control of the meat-borne human health hazards identified in sheep and goats. a number of strengths and weaknesses of the current inspection system were identified. currently, the use of fci for food safety purposes is limited for small ruminants because the data that it contains is very general and doesn't address specific hazards of public health importance. however, fci could serve as a valuable tool for risk management decisions and could be used for risk categorisation of farms or batches of animals. to achieve this, the system needs further development to include additional information important for food safety, including definition of appropriate and standardized indicators for the main public health hazards identified above. ante-mortem and post-mortem inspections of sheep and goats enable the detection of observable abnormalities and provide a general assessment of animal/herd health, which if compromised may lead to a greater public health risk. visual inspection of live animals and carcasses can detect animals heavily contaminated with faeces, which increase the risk for cross-contamination during slaughter and may constitute a food safety risk if the animals are carrying hazards of public health importance. if such animals or carcasses are dealt with adequately, this risk can be reduced. visual detection of faecal contamination on carcasses can also be an indicator of slaughter hygiene, but other approaches to verify this should be considered. post-mortem inspection can also detect non meat-borne hazards of public health significance, such as echinococcus granulosus, that can be present in carcasses or offal from small ruminants. ante-mortem and post-mortem inspection also have the potential to detect new diseases, which may be of direct public health significance. with regard to chemical hazards, it was noted that current procedures for sampling and testing are, in general, well established and coordinated, including follow-up actions subsequent to the identification of non-compliant samples. the regular sampling and testing for chemical residues and contaminants is an important disincentive for the development of undesirable practices and the prescriptive sampling system allows for equivalence in the control of eu-produced sheep and goat meat. the current combination of animal traceability, ante-mortem inspection and gross tissue examination can support the collection of appropriate samples for residue monitoring. the main weakness of ante-mortem and post-mortem inspection is that they are not able to detect any of the public health hazards identified as the main concerns for food safety. in addition, given that the current post-mortem procedures involve palpation and incision of some organs, the potential for crosscontamination of carcasses exists. for chemical hazards, a major weakness is that, with very few exceptions, presence of chemical hazards cannot be identified by current ante-/post-mortem meat inspection procedures at the slaughterhouse level and there is a lack of sufficient cost-effective and reliable screening methods. in addition, sampling is mostly prescriptive rather than risk or information based. there is limited ongoing adaptation of the sampling and testing programmes to the results of the residue monitoring programmes, with poor integration between the testing of feed materials for undesirable substances and the nrcps and sampling under the nrcps reflecting only a part of testing done by a number of mss, the results of which should be taken into consideration. as neither of the main public health hazards associated with meat from small ruminants can be detected by traditional visual meat inspection, other approaches are necessary to identify and control these microbiological hazards. a comprehensive meat safety assurance system for small ruminants, combining a range of preventive measures and controls applied both on the farm and at the slaughterhouse in a longitudinally integrated way, is the most effective approach to control the main hazards in the context of meat inspection. information on the biological risks associated with the consumption of meat from sheep or goats is sometimes scant and unreliable. in order to facilitate decision making, harmonised surveys are required to establish values for the prevalence of the main hazards t. gondii and pathogenic vtec at flock/herd, live animal and carcass level in individual mss. epidemiological and risk assessment studies are also required to determine the specific risk to public health associated with the consumption of meat from small ruminants. if these studies confirm a high risk to public health through the consumption of meat from sheep or goats, consideration should be given to the setting of clear and measurable eu targets at the carcass level. to meet these targets and criteria, a variety of control options for the main hazards are available, at both farm and abattoir level. flock/herd categorisation according to the risk posed by the main hazards is considered an important element of an integrated meat safety assurance system. this should be based on the use of farm descriptors and historical data in addition to batch-specific information. farm-related data could be provided through farm audits using harmonised epidemiological indicators (heis) to assess the risk and protective factors for the flocks/herds related to the given hazards. in addition, classification of abattoirs according to their capability to prevent or reduce faecal contamination of carcasses can be based on two elements: ( ) the process hygiene as measured by the level of indicator organisms on the carcasses (i.e. process hygiene criteria); and ( ) the use of operational procedures and equipment that reduce faecal contamination, as well as industry led quality systems. there are a variety of husbandry measures that can be used to control t. gondii on sheep and goat farms but at present these are impractical to implement in most farms. a number of post-processing interventions are effective in inactivating t. gondii such as cooking, freezing, curing, high pressure and irradiation treatments, although further research is required to validate these treatments in meat from small ruminants. there are also a variety of husbandry measures that can be used to reduce the levels of vtec on farms, but their efficacy is not clear in small ruminants. there are also a number of challenges that need to be overcome regarding the setting of targets for pathogenic vtec, including the difficulties in identifying husbandry factors that can be used to classify farms according to pathogenic vtec risk, the intermittent nature of shedding, and the problems with the interpretation of monitoring results for pathogenic vtec due to the difficulty to correctly identify pathogenic vtec. the main sources of vtec on sheep and goat carcasses are the fleece/hide and the viscera. to control incoming faecal contamination only clean animals should be accepted for slaughter. there are also a number of measures that can help reducing the spillage or leakage of digestive contents onto the carcass, as well as post-processing interventions to control pathogenic vtec are also available. these include hot water and steam carcass surface treatments. risk categorisation of slaughterhouses should be based on trends of data derived from process hygiene assessments and from hazard analysis critical control point programmes. improvement of slaughter hygiene through technological and managerial interventions should be sought in slaughterhouses with repeatedly unsatisfactory performance. fci can be improved by including information on participation in quality assurance schemes and by greater feedback to the primary producer, as this would likely result in the production of healthier animals. ante-mortem inspection assesses the general health status of the animals and helps to detect animals heavily contaminated with faeces on arrival at the slaughterhouse, so no adaptations for the existing visual ante-mortem inspection are required. routine post-mortem examination cannot detect the meat-borne pathogens of public health importance. palpation of the lungs, the liver, the umbilical region and the joints, and incision of the liver could contribute to the spread of bacterial hazards through cross contamination. for these reasons, palpation and incision should be omitted in animals subjected to routine slaughter. sheep and goat production in the eu is marked by being largely extensive in nature, involving frequent trading of animals and involving nomadic flocks. these differences in husbandry systems and feeding regimes result in different risks for the occurrence of chemical residues and contaminants. extensive periods on pasture or/as nomadic flocks and the use of slaughter collection dealerships may preclude detailed lifetime fci. it is recommended regarding chemical hazards, that fci should be expanded for sheep and goats produced in extensive systems to provide more information on the specific environmental conditions where the animals are produced and that future monitoring programmes should be based on the risk of occurrence of chemical residues and contaminants, taking into account the completeness and quality of the fci supplied, and the ranking of chemical substances into categories of potential concern, which ranking needs to be regularly updated. control programmes for chemical residues and contaminants should be less prescriptive, with sufficient flexibility to adapt to results of testing, should include 'new hazards', and the test results for sheep and goats should be separately presented. 'new' chemical hazards identified are largely persistent organic pollutants that have not been comprehensively covered by the sampling plans of the current meat inspection or which have not been included in such sampling plans. there is a need for an improved integration of sampling, testing and intervention protocols across the food chain, nrcps, feed control and monitoring of environmental contaminants. a series of further recommendations are made in relation to chemical hazards dealing with control measures, testing and analytical techniques and also on data collection and source attribution studies for biological hazards, as well as on methods of detection of viable t. gondii in meat and on assessing the effect of the omission of palpation and incision on the risk posed by non-meat-borne zoonoses. the implications for surveillance of animal health and welfare of the changes proposed to the current meat inspection system were evaluated quantitatively and qualitatively. the proposed changes related to biological hazards included shorter transport and lairage time, improved collection of food chain information, and omission of palpation and incision in animals subjected to routine slaughter at postmortem inspection. recommendations on chemical hazards included the ranking system for chemical substances of potential concern and its updating, the use of food chain information to help facilitate risk based sampling strategies, and the inclusion of 'new hazards' in control programmes for chemical residues and contaminants. from the quantitative assessment, a change to visual only inspection caused a significant reduction of the probability of detection of detectable cases of fasciolosis and tuberculosis in goats. with regard to exotic diseases, clinical surveillance had a greater sensitivity for detecting foot and mouth disease than slaughterhouse surveillance. a change in post-mortem protocol to a visual only system did not significantly reduce the detection of any welfare conditions. following the qualitative analysis, it was concluded that a change to visual inspection (which implies no palpation) would reduce detection effectiveness for tuberculosis. surveillance of tuberculosis at the slaughterhouse in small ruminants should be improved and encouraged, as this is in practice the only surveillance system available in these species. the detection of tuberculosis in small ruminants should be adequately recorded and followed at the farm level. moving to a visual only meat inspection system would decrease the sensitivity of inspection of fasciolosis at animal level, however it would be sensitive enough to identify most if not all affected herds. therefore the consequences of the change would be of low relevance. the feedback to farmers of fasciola hepatica detected at meat inspection should be improved, to allow farmer information to support rational on-farm fluke management programmes. qualitative analysis suggested that the proposal for shortened transport and lairage time would be beneficial to improving the welfare of small ruminants. food chain information should include animal welfare status in order to complement the slaughterhouse surveillance systems (ante-mortem and postmortem inspection) and the latter could be used to identify on farm welfare status. regulation (ec) no / of the european parliament and of the council lays down specific rules for the organisation of official controls on products of animal origin intended for human consumption . inspection tasks within this regulation include: checks and analysis of food chain information animal welfare specified risk material and other by-products laboratory testing the scope of the inspection includes monitoring of zoonotic infections and the detection or confirmation of certain animal diseases without necessarily having consequences for the placing on the market of meat. the purpose of the inspection is to assess if the meat is fit for human consumption in general and to address a number of specific hazards: in particular the following issues: transmissible spongiform encephalopathies (only ruminants), cysticercosis, trichinosis, glanders (only solipeds), tuberculosis, brucellosis, contaminants (e.g. heavy metals), residues of veterinary drugs and unauthorised substances or products. during their meeting on november , chief veterinary officers (cvo) of the member states agreed on conclusions on modernisation of sanitary inspection in slaughterhouses based on the recommendations issued during a seminar organised by the french presidency from to july . the cvo conclusions have been considered in the commission report on the experience gained from the application of the hygiene regulations, adopted on july . council conclusions on the commission report were adopted on november inviting the commission to prepare concrete proposals allowing the effective implementation of modernised sanitary inspection in slaughterhouses while making full use of the principle of the 'risk-based approach'. in accordance with article of regulation (ec) no / , the commission shall consult efsa on certain matters falling within the scope of the regulation whenever necessary. efsa and the commission's former scientific committee on veterinary measures relating to public health have issued in the past a number of opinions on meat inspection considering specific hazards or production systems separately. in order to guarantee a more risk-based approach, an assessment of the risk caused by specific hazards is needed, taking into account the evolving epidemiological situation in member states. in addition, methodologies may need to be reviewed taking into account risks of possible cross-contamination, trends in slaughter techniques and possible new inspection methods. the scope of this mandate is to evaluate meat inspection in order to assess the fitness of the meat for human consumption and to monitor food-borne zoonotic infections (public health) without jeopardising the detection of certain animal diseases nor the verification of compliance with rules on animal welfare at slaughter. if and when the current methodology for this purpose would be considered not to be the most satisfactory to monitor major hazards for public health, additional methods should be recommended as explained in detail under points and of the terms of reference. the objectives of the current legal provisions aimed at carrying out meat inspection on a risk-based analysis should be maintained. in order to ensure a risk-based approach, efsa is requested to provide scientific opinions on meat inspection in slaughterhouses and, if considered appropriate, at any other stages of the production chain, taking into account implications for animal health and animal welfare in its risk analysis. in addition, relevant international guidance should be considered, such as the codex code of hygienic practice for meat (cac/rcp - ) , and chapter . on control of biological hazards of animal health and public health importance through ante-and post-mortem meat inspection, as well as chapter . on slaughter of animals of the terrestrial animal health code of the world organisation for animal health (oie). the following species or groups of species should be considered, taking into account the following order of priority identified in consultation with the member states: domestic swine, poultry, bovine animals over six weeks old, bovine animals under six weeks old, domestic sheep and goats, farmed game and domestic solipeds. in particular, efsa, in consultation with the european centre for disease prevention and control (ecdc), is requested within the scope described above to: . identify and rank the main risks for public health that should be addressed by meat inspection at eu level. general (e.g. sepsis, abscesses) and specific biological risks as well as chemical risks (e.g. residues of veterinary drugs and contaminants) should be considered. differentiation may be made according to production systems and age of animals (e.g. breeding compared to fattening animals). . assess the strengths and weaknesses of the current meat inspection methodology and recommend possible alternative methods (at ante-mortem or post-mortem inspection, or validated laboratory testing within the frame of traditional meat inspection or elsewhere in the production chain) at eu level, providing an equivalent achievement of overall objectives; the implications far animal health and animal welfare of any changes suggested in the light of public health risks to current inspection methods should be considered. . if new hazards currently not covered by the meat inspection system (e.g. salmonella, campylobacter) are identified under tor , then recommend inspection methods fit for the purpose of meeting the overall objectives of meat inspection. when appropriate, food chain information should be taken into account. . recommend adaptations of inspection methods and/or frequencies of inspections that provide an equivalent level of protection within the scope of meat inspection or elsewhere in the production chain that may be used by risk managers in case they consider the current methods disproportionate to the risk, e.g. based on the ranking as an outcome of terms of reference or on data obtained using harmonised epidemiological criteria (see annex ) . when appropriate, food chain information should be taken into account. the scope of the mandate is to evaluate meat inspection in a public health context; animal health and welfare issues are covered with respect to the possible implications of adaptations/alterations to current inspection methods, or the introduction of novel inspection methods proposed by this mandate. issues other than those of public health significance but that still compromise the fitness of the meat for human consumption (regulation (ec) no / , annex i, section ii, chapter v) are outside the scope of the mandate. examples of these include sexual odour or meat decolouration. transmissible spongiform encephalopathies (tses) are also outside the scope of the mandate. the impact of changes to meat inspection procedures on the occupational health of abattoir workers, inspectors, etc. is outside the scope of the mandate. additionally, hazards representing primarily occupational health risks, the controls related to any hazard at any meat chain stage beyond the abattoir, and the implications for environmental protection are not dealt with in this document. in line with article of regulation (ec) no / the european commission has recently submitted a mandate to efsa (m- - ) to cover different aspects of meat inspection. the mandate comprises two requests: one for scientific opinions and one for technical assistance. the european food safety authority (efsa) is requested to issue scientific opinions related to inspection of meat in different species. in addition, technical assistance has been requested on harmonised epidemiological criteria for specific hazards for public health that can be used by risk managers to consider adaptation of the meat inspection methodology. meat inspection is defined by regulation / . the species or groups of species to be considered are: domestic swine, poultry, bovine animals over six weeks old, bovine animals under six weeks old, domestic sheep and goats, farmed game and domestic solipeds. taking into account the complexity of the subject and that consideration has to be given to zoonotic hazards, animal health and welfare issues and chemical hazards (e.g. residues of veterinary drugs and chemical contaminants), the involvement of several efsa units was necessary. more specifically, the mandate was allocated to the biological hazards panel (biohaz), which prepared this scientific opinion with the support of the animal health and welfare (ahaw) and contaminants in the food chain (contam) panels. in addition, the delivery of the technical assistance was allocated to the biological monitoring (biomo), scientific assessment support (sas), and dietary and chemical monitoring (dcm) units of the risk assessment and scientific assistance directorate. this scientific opinion therefore concerns the assessment of meat inspection in sheep and goats, and it includes the answer to the terms of reference proposed by the european commission. owing to the complexity of the mandate, the presentation of the outcome does not follow the usual layout. for ease of reading, main outputs from the three working groups (biohaz, contam and ahaw) are presented at the beginning of the document. the scientific justifications for these outputs are found in the various appendices as endorsed by their respective panels, namely biological hazards (appendix a), chemical hazards (appendix b), and the potential impact that the proposed changes envisaged by these two could have on animal health and welfare (appendix c). differentiation may be made according to production systems and age of animals (e.g. breeding compared to fattening animals). based on the priority ranking, the hazards were classified as follows: -toxoplasma gondii and pathogenic verocytotoxin-producing escherichia coli (vtec) were classified as high priority for sheep/goat meat inspection. -the remaining identified hazards, bacillus anthracis, campylobacter spp. (thermophilic) and salmonella spp. were classified as low priority, based on available data. as new hazards might emerge and/or hazards that presently are not a priority might become more relevant over time or in some regions, both hazard identification and the risk ranking should be revisited regularly to reflect this dynamic epidemiological situation. particular attention should be given to potential emerging hazards of public health importance. a multi-step approach was used for the identification and ranking of chemical hazards. evaluation of the - national residue control plans (nrcps) outcome for sheep and goats indicated that only . % of the total number of results was non-compliant for one or more substances listed in council directive / /ec. potentially higher exposure of consumers to these substances from sheep and goat meat takes place only incidentally, as a result of mistakes or non-compliance with known and regulated procedures. available data however, do not allow for a reliable assessment of consumer exposure. ranking of chemical residues and contaminants in domestic sheep and goats based on predefined criteria, relating to bioaccumulation, toxicological profile and likelihood of occurrence, and taking into account the findings from the nrcps for the period - was as follows: -dioxins and dioxin-like polychlorinated biphenyls (dl-pcbs) were ranked as being of high potential owing to their known bioaccumulation in the food chain, their frequent findings above mls, particularly in sheep liver, and in consideration of their toxicological profile. -stilbenes, thyreostats, gonadal (sex) steroids, resorcylic acid lactones and beta-agonists, especially clenbuterol, chloramphenicol and nitrofurans were ranked as being of medium potential concern, as they have proven toxicity for humans, are effective as antibacterial treatments for sheep/goats and as non-compliant samples are found in most years of the nrcps. -chloramphenicol and nitrofurans were ranked as being of medium potential concern, as they have proven toxicity for humans, they are effective as antibacterial treatments for sheep/goats and as non-compliant samples are found in most years of the nrcps. -non dioxin-like polychlorinated biphenyls (ndl-pcbs) bioaccumulate, and there is a risk of exceeding the mls, but they were ranked in the category of medium potential concern, because they are less toxic than dioxins and dl-pcbs. -the chemical elements cadmium, lead and mercury were allocated to the medium potential concern category taking into account the number of non-compliant results reported under the nrcps and their toxicological profile. -all other substances listed in council directive / /ec were ranked as of low or negligible potential concern owing to the toxicological profile of these substances at residue levels in edible tissues or to the very low or non-occurrence of non-compliant results in the nrcps - , and/or to the natural occurrence in sheep and goats of some of these substances. strengths -ante-mortem and post-mortem inspection of sheep and goats enable the detection of observable abnormalities. in that context, they are an important activity for monitoring animal health and welfare. they provide a general assessment of animal/herd health, which if compromised may lead to a greater public health risk. visual inspection of live animals and carcasses can also detect animals heavily contaminated with faeces. such animals increase the risk for cross-contamination during slaughter and may consequently constitute a food safety risk if carrying hazards of public health importance. if such animals or carcasses are dealt with adequately, this risk can be reduced. visual detection of faecal contamination on carcasses can also be an indicator of slaughter hygiene, but other approaches to verify slaughter hygiene should be considered. -post-mortem inspection can also detect non meat-borne hazards of public health significance that can be present in carcasses or offal from small ruminants. ante-mortem and post-mortem inspection also have the potential to detect new diseases if these have clinical signs, which may be of direct public health significance. weaknesses -currently, the use of food chain information (fci) for food safety purposes is limited for small ruminants, mainly because the data that it contains is very general and doesn't address specific hazards of public health importance. however, fci could serve as a valuable tool for risk management decisions and could be used for risk categorisations of farms or batches of animals. to achieve this, the system needs further development to include additional information important for food safety, including definition of appropriate and standardized indicators for the main public health hazards identified in section of appendix a. -ante-and post-mortem inspection is not able to detect any of the public health hazards identified as the main concerns for food safety. it would therefore be expected that more efficient procedures might be implemented to monitor the occurrence of non-visible hazards. in addition, given that the current post-mortem inspection procedures involve palpation and incision of some organs, the potential for cross-contamination of carcasses exists. strengths of the current meat inspection methodology for chemical hazards are as follows: -the current procedures for sampling and testing are a mature system, in general well established and coordinated including follow-up actions subsequent to the identification of non-compliant samples. -the regular sampling and testing for chemical residues and contaminants in the system is an important disincentive to the development of undesirable practices. -the prescriptive sampling system allows for equivalence in the control of eu-produced sheep and goat meat. any forthcoming measures have to ensure that the control of imports from third countries remains equivalent to the controls within the domestic market. -the current combination of animal traceability, ante-mortem inspection and gross tissue examination can support the collection of appropriate samples for residue monitoring. weaknesses of the current meat inspection methodology for chemical hazards are as follows: -a weakness of the system is that presence of chemical hazards cannot be identified by current ante-/post-mortem meat inspection procedures at the slaughterhouse level, indicating the need for further harmonization of the risk reduction strategies along the entire food chain. -integration between testing of feed materials for undesirable contaminants and the nrcps in terms of communication and follow-up testing strategies or interventions is currently limited. moreover, a routine environmental data flow is not established and keeping habits for sheep and goats provides opportunities for feed coming in without a clear feed chain history. -under the current system, sampling is mostly prescriptive rather than risk-or informationbased. it appears that individual samples taken under the nrcp testing programme may not always be taken as targeted samples, as specified under council directive / / ec, but sometimes may be taken as random samples. -there is a lack of sufficient cost-effective and reliable screening methods and/or the range of substances prescribed/covered by the testing is sometimes limited. -there is limited flexibility to adopt emerging chemical substances into the nrcps and limited ongoing adaptation of the sampling and testing programme to the results of the residue monitoring programmes. in addition, sampling under the nrcps reflects only a part of testing done by a number of mss, the results of which should be taken into consideration. -sheep and goats may not be subject to surveillance over their lifetime at the same level as is the case for other food animal categories such as pigs, poultry and, to a large extent, bovine animals due to the traditional nomadic/outdoor farming systems. as shown in the comisurv assessment, a change to visual only inspection would cause a significant reduction in the probability of detection (i.e. non-overlapping % probability intervals) of detectable cases of fasciolosis and of tuberculosis in goats. small ruminants are usually not subjected to official tuberculosis eradication campaigns, and farm controls are only performed on premises where cattle and goats are kept together, or in flocks/herds that commercialise raw milk. surveillance for small ruminant tuberculosis at present relies on meat inspection of sheep and goats slaughtered for human consumption, or other limited diagnostic surveillance activities. as is the case with tuberculosis in bovines, the contribution of meat inspection surveillance of tuberculosis in small ruminants is to support the detection of flocks/herds with tuberculosis. detection of tuberculosis in individual animals is merely the first step in improving the effectiveness of flock/herd surveillance, and for any given flock/herd, the flock/herd sensitivity will increase with the number of animals slaughtered. in recent years tuberculosis has been reported in small ruminants in several eu countries and most information derives from recognition of tuberculous lesions at the slaughterhouse and from laboratory reports. although small ruminants are not considered to represent a significant reservoir of the disease for the persistence of bovine tuberculosis in cattle, it is still possible that infected sheep and goat herds could act as vectors of infection for other domestic and wild animals. therefore, surveillance and control of tuberculosis in domestic small ruminants does have consequences for the overall surveillance and control of tuberculosis. the feedback to farmers of fasciola hepatica detected at meat inspection is low at present and the real risk to animal health/welfare for this disease, caused by a change to a visual only meat inspection method, is probably low. implementation of welfare assessment protocols using appropriate animal based indicators during clinical and slaughterhouse (ami + pmi) surveillance system would improve the welfare of small ruminants. extended use of food chain information has the potential to compensate for some, but not all, of the information on animal health and welfare that would be lost if visual only post-mortem inspection is applied. food chain information is a potentially effective tool to perform more targeted ante-mortem and post-mortem inspection tasks in the slaughterhouse which may increase the effectiveness of those tasks in detecting conditions of animal health and animal welfare significance. the existing ineffective flow of information from primary production to the slaughterhouses and vice versa reduces the ability of detection of animal diseases and animal welfare conditions at the slaughterhouse and as a result it limits possible improvements on animal health and welfare standards at the farm as farmers will not be aware of the slaughterhouse findings. the conclusions and recommendations on chemical hazards were reviewed by the ahaw working group and none of them were considered to have impact on animal health and welfare surveillance and monitoring. as neither of the main public health hazards associated with meat from small ruminants can be detected by traditional meat inspection, other approaches are necessary to identify and control these microbiological hazards. a comprehensive meat safety assurance system for meat from small ruminants, combining a range of preventive measures and controls applied both on the farm and at the slaughterhouse in a longitudinally integrated way, is the most effective approach to control the main hazards in the context of meat inspection. information on the biological risks associated with the consumption of meat from sheep or goats is sometimes scant and unreliable. in order to facilitate decision making, harmonised surveys are required to establish values for the prevalence of the main hazards t. gondii and pathogenic vtec at flock/herd, live animal and carcass level in individual member states. epidemiological and risk assessment studies are also required to determine the specific risk to public health associated with the consumption of meat from small ruminants. in the event that these studies confirm a high risk to public health through the consumption of meat from sheep or goats, consideration should be given to the setting of clear and measurable eu targets at the carcass level. to meet these targets and criteria, a variety of control options for the main hazards are available, at both farm and abattoir level. flock/herd categorisation according to the risk posed by the main hazards is considered an important element of an integrated meat safety assurance system. this should be based on the use of farm descriptors and historical data in addition to batch-specific information. farmrelated data could be provided through farm audits using harmonised epidemiological indicators (heis) to assess the risk and protective factors for the flocks/herds related to the given hazards. classification of abattoirs according to their capability to prevent or reduce faecal contamination of carcasses can be based on two elements: ( ) the process hygiene as measured by the level of indicator organisms on the carcasses (i.e. process hygiene criteria); and ( ) the use of operational procedures and equipment that reduce faecal contamination, as well as industry-led quality systems. as mentioned in section . of appendix a, further studies are necessary to determine with more certainty the risk of acquiring t. gondii through consumption of meat from small ruminants. in addition, the lack of tests that can easily identify viable cysts in meat is a significant drawback. further, if there is a high prevalence in the animal population, this will hamper the development of systems based on risk categorisation of animals. for these reasons, the setting of targets for t. gondii is not recommended at the moment. there are a variety of animal husbandry measures that can be used to control t. gondii on sheep and goat farms but at present these are impractical to implement in most farms. a number of post-processing interventions might be effective in inactivating t. gondii such as cooking, freezing, curing and high-pressure and -irradiation treatments. however, most of the information available for these treatments originates from research in pigs, so further research is required to validate these treatments in meat from small ruminants. there are also a variety of animal husbandry measures that can be used to reduce the levels of vtec on infected farms, but their efficacy is not clear in small ruminants. in addition, there are a number of challenges that need to be overcome regarding the setting of targets for pathogenic vtec, including the difficulties in identifying husbandry factors that can be used to classify farms according to pathogenic vtec risk, the intermittent nature of shedding, and the problems with the interpretation of monitoring results for pathogenic vtec due to the difficulty to correctly identify pathogenic vtec. the two main sources of vtec on sheep and goat carcasses are the fleece/hide and the viscera. to control faecal contamination from the fleece or hide only clean animals should be accepted for slaughter, as currently required by eu legislation. there are also a number of measures that can help reducing the spillage or leakage of digestive contents onto the carcass, particularly rodding of the oesophagus and bagging of the rectum. post-processing interventions to control vtec are also available. these include hot water and steam pasteurization. risk categorisation of slaughterhouses should be based on trends of data derived from process hygiene assessments and from hazard analysis critical control point programmes. improvement of slaughter hygiene through technological and managerial interventions should be sought in slaughterhouses with repeatedly unsatisfactory performance. dioxins and dl-pcbs which accumulate in food-producing animals have been ranked as being of high potential concern. as these substances have not yet been comprehensively covered by the sampling plans of the current meat inspection (nrcps), they should be considered as 'new' hazards. in addition, for a number of chemical elements used as feed supplements and for organic contaminants that may accumulate in food-producing animals only limited data regarding residues in sheep and goats are available. this is the case, in particular, for brominated flame retardants, including polybrominated diphenylethers (pbdes) and hexabromocyclododecanes (hbcdds) and perfluorinated compounds (pfcs) including (but not limited to) perfluorooctane sulphonate (pfos) and perfluorooctanoic acid (pfoa). fci can be improved by including information on participation in quality assurance schemes and by giving greater feedback to the primary producer, as this would probably result in the production of healthier animals. ante-mortem inspection assesses the general health status of the animals and helps to detect animals heavily contaminated with faeces on arrival at the slaughterhouse. taking these factors into consideration, and given that current methods do not increase the microbiological risk to public health, no adaptations to the existing visual ante-mortem inspection procedure are required. although visual examination contributes by detecting visible faecal contamination, routine post-mortem examination cannot detect the meat-borne pathogens of public health importance. palpation of the lungs, the livers, the umbilical region and the joints and incision of the liver could contribute to the spread of bacterial hazards through cross-contamination. for these reasons, palpation and incision should be omitted in animals subjected to routine slaughter. sheep and goat production in the eu is marked by being largely extensive in nature, involving frequent trading of animals and nomadic flocks. this involves differences in husbandry systems and feeding regimes resulting in different risks for chemical substances and contaminants. extensive periods on pasture or/as nomadic flocks and the use of slaughter collection dealerships may preclude detailed lifetime fci. similarly, in these situations, the level of feedback from the slaughterhouse and authorities to farmers regarding the results of residue testing may be suboptimal. there is less concern about fci from dairy sheep and goats as they are reared under more intensive and controlled conditions. better integration of results from official feed control with residue monitoring seems essential to indicate whether monitoring of residues in slaughter animals needs to be directed to particular substances. therefore, there is a need for an improved integration of sampling, testing and intervention protocols across the food chain, nrcps, feed control and environmental monitoring. to provide a better evidence base for future risk ranking of hazards, initiatives should be instigated to: improve and harmonise data collection of incidence and severity of human diseases caused by relevant hazards; systematically collect data for source attribution; collect data to identify and risk rank emerging hazards that could be transmitted through handling, preparation and consumption of sheep and goat meat. source attribution studies are needed to determine the relative importance of meat and to ascertain the role of the different livestock species as sources of t. gondii and pathogenic vtec for humans. methods should be developed to estimate the amount of viable t. gondii tissue cysts in meat, especially in meat cuts that are commonly consumed. the effect of the omission of palpation and incision on the risk posed by non-meat-borne zoonoses such as echinococcus granulosus and fasciola hepatica should be assessed, particularly in those regions where these hazards are endemic. fci should be expanded for sheep and goats produced in extensive systems to provide more information on the specific environmental conditions where the animals are produced. it is recommended that sampling of sheep and goats should be based on the risk of occurrence of chemical residues and contaminants and on the completeness and quality of the fci supplied. regular updating of the ranking of chemical substances in sheep and goats as well as of the sampling plans should occur taking into account any new information regarding the toxicological profile of chemical residues and contaminants, usage in sheep and goat production, and actual occurrence of individual substances in sheep and goats. control programmes for chemical residues and contaminants should be less prescriptive, with sufficient flexibility to adapt to results of testing, should include 'new hazards', and the test results for sheep and goats should be separately presented. there is a need for an improved integration of sampling, testing and intervention protocols across the food chain, nrcps, feed control and monitoring of environmental contaminants. the development of analytical techniques covering multiple analytes and of new biologically based testing approaches should be encouraged and incorporated into the residue control programmes for prohibited substances, testing should be directed where appropriate towards the farm level and, in the case of substances that might be used illicitly for growth promotion, control measures, including testing, need to be refocused to better identify the extent of abuse in the eu. in addition, control measures for prohibited substances should not rely exclusively on nrcp testing, but should include veterinary inspection during the production phase and the use of biological methods and biomarkers suitable for the identification of abuse of such substances in sheep and goat production in the eu. data collected during clinical and slaughterhouse (ante-mortem and post mortem inspection) surveillance systems should be utilised more effectively to improve animal welfare at farm level. slaughterhouse surveillance of tuberculosis in small ruminants should be improved and encouraged, as this is in practice the only surveillance system available. the detection of tuberculosis in small ruminants should be adequately recorded and notified, followed by control measures at the farm level. lack of feedback of post-mortem inspection results to the farmer prevents instigation of a fluke management programme, which could be detrimental to animal health and welfare. an improvement in this feedback of information is recommended. welfare surveillance systems should become an integral part of the food chain information. an integrated system should be developed whereby food chain information for public health and for animal health and welfare can be used in parallel, more effectively provide farmers with background information on the animal diseases and welfare conditions of key concern that may affect their livestock and why it is important to provide this information to the slaughterhouse through the use of food chain information. following a request from the european commission, the panel on biological hazards (biohaz) was asked to deliver a scientific opinion on the public health hazards to be covered by inspection of meat for several animal species, with the contribution of the panel on contaminants in the food chain (contam) and the panel on animal health and welfare (ahaw). briefly, the main risks for public health that should be addressed by meat inspection were identified and ranked; the strengths and weaknesses of the current meat inspection were evaluated; and recommendations were made for inspection methods capable of meeting the overall objectives of meat inspection for hazards currently not covered by the meat inspection system, as well as recommendations for adaptations of inspection methods and/or frequencies of inspections that provide an equivalent level of protection. in addition, the implications for animal health and animal welfare of any changes proposed to current inspection methods were assessed. this opinion covers the inspection of meat from sheep and goats. the biohaz panel considered sheep and goats together . a decision tree was used for priority ranking of meat-borne hazards present in meat from sheep and goats. the ranking was based on the magnitude of the human health impact, the severity of the disease in humans and the evidence supporting the role of meat from sheep and goats as a risk factor for disease in humans. the assessment was focused on the public health risks that may occur through the handling, preparation and/or consumption of meat from these species. the term 'priority' was considered more appropriate than 'risk' for categorizing the hazards associated with meat from small ruminants, given that a significant amount of data on both the occurrence of the hazards and on the attributable fraction of human cases to meat from small ruminants were not available. based on the priority ranking, the hazards were classified as follows: toxoplasma gondii and pathogenic vtec were classified as high priority for sheep/goat meat inspection. the remaining identified hazards, bacillus anthracis, campylobacter spp. (thermophilic) and salmonella spp., were classified as low priority, based on available data. as new hazards might emerge and/or hazards that presently are not a priority might become more relevant over time or in some regions, both hazard identification and the risk ranking should be revisited regularly to reflect this dynamic epidemiological situation. particular attention should be given to potential emerging hazards of public health importance. the main elements of the current meat inspection system include analysis of fci, ante-mortem examination of animals and post-mortem examination of carcasses and organs. the assessment of the strengths and weaknesses of the current meat inspection was based on its contribution to the control of the meat-borne human health hazards identified in sheep and goats. a number of strengths and weaknesses of the current system were identified. currently, the use of food chain information (fci) for food safety purposes is limited for small ruminants because the data that it contains is very general and doesn't address specific hazards of public health importance. however, fci could serve as a valuable tool for risk management decisions and could be used for risk categorisation of farms or batches of animals. to achieve this, the system needs further development to include additional information important for food safety, including definition of appropriate and standardized indicators for the main public health hazards identified above. ante-mortem and post-mortem inspections of sheep and goats enable the detection of observable abnormalities and provide a general assessment of animal/herd health, which if compromised may lead to a greater public health risk. visual inspection of live animals and carcasses can detect animals heavily contaminated with faeces, which increase the risk for cross-contamination during slaughter and may constitute a food safety risk if the animals are carrying hazards of public health importance. if such animals or carcasses are dealt with adequately, this risk can be reduced. visual detection of faecal contamination on carcasses can also be an indicator of slaughter hygiene, but other approaches to verify this should be considered. post-mortem inspection can also detect non meat-borne hazards of public health significance, such as echinococcus granulosus, that can be present in carcasses or offal from small ruminants. ante-mortem and post-mortem inspection also have the potential to detect new diseases, which may be of direct public health significance. the main weakness of ante-mortem and post-mortem inspection is that they are not able to detect any of the public health hazards identified as the main concerns for food safety. in addition, given that the current post-mortem procedures involve palpation and incision of some organs, the potential for crosscontamination of carcasses exists. as neither of the main public health hazards associated with meat from small ruminants can be detected by traditional visual meat inspection, other approaches are necessary to identify and control these microbiological hazards. a comprehensive meat safety assurance system for small ruminants, combining a range of preventive measures and controls applied both on the farm and at the slaughterhouse in a longitudinally integrated way, is the most effective approach to control the main hazards in the context of meat inspection. information on the biological risks associated with the consumption of meat from sheep or goats is sometimes scant and unreliable. in order to facilitate decision making, harmonised surveys are required to establish values for the prevalence of the main hazards t. gondii and pathogenic vtec at flock/herd, live animal and carcass level in individual mss. epidemiological and risk assessment studies are also required to determine the specific risk to public health associated with the consumption of meat from small ruminants. if these studies confirm a high risk to public health through the consumption of meat from sheep or goats, consideration should be given to the setting of clear and measurable eu targets at the carcass level. to meet these targets and criteria, a variety of control options for the main hazards are available, at both farm and abattoir level. flock/herd categorisation according to the risk posed by the main hazards is considered an important element of an integrated meat safety assurance system. this should be based on the use of farm descriptors and historical data in addition to batch-specific information. farm-related data could be provided through farm audits using harmonised epidemiological indicators (heis) to assess the risk and protective factors for the flocks/herds related to the given hazards. in addition, classification of abattoirs according to their capability to prevent or reduce faecal contamination of carcasses can be based on two elements: ( ) the process hygiene as measured by the level of indicator organisms on the carcasses (i.e. process hygiene criteria); and ( ) the use of operational procedures and equipment that reduce faecal contamination, as well as industry led quality systems. there are a variety of husbandry measures that can be used to control t. gondii on sheep and goat farms but at present these are impractical to implement in most farms. a number of post-processing interventions are effective in inactivating t. gondii such as cooking, freezing, curing, high pressure and irradiation treatments, although further research is required to validate these treatments in meat from small ruminants. there are also a variety of husbandry measures that can be used to reduce the levels of vtec on farms, but their efficacy is not clear in small ruminants. there are also a number of challenges that need to be overcome regarding the setting of targets for pathogenic vtec, including the difficulties in identifying husbandry factors that can be used to classify farms according to pathogenic vtec risk, the intermittent nature of shedding, and the problems with the interpretation of monitoring results for vtec due to the difficulty to correctly identify pathogenic vtec. the main sources of vtec on sheep and goat carcasses are the fleece/hide and the viscera. to control incoming faecal contamination only clean animals should be accepted for slaughter. there are also a number of measures that can help reducing the spillage or leakage of digestive contents onto the carcass, as well as post-processing interventions to control vtec are also available. these include hot water and steam pasteurization. risk categorisation of slaughterhouses should be based on trends of data derived from process hygiene assessments and from hazard analysis critical control point programmes. improvement of slaughter hygiene through technological and managerial interventions should be sought in slaughterhouses with repeatedly unsatisfactory performance. fci can be improved by including information on participation in quality assurance schemes and by greater feedback to the primary producer, as this would likely result in the production of healthier animals. ante-mortem inspection assesses the general health status of the animals and helps to detect animals heavily contaminated with faeces on arrival at the slaughterhouse, so no adaptations for the existing visual ante-mortem inspection are required. routine post-mortem examination cannot detect the meat-borne pathogens of public health importance. palpation of the lungs, the liver, the umbilical region and the joints, and incision of the liver could contribute to the spread of bacterial hazards through cross contamination. for these reasons, palpation and incision should be omitted in animals subjected to routine slaughter. a series of recommendations were made on data collection, source attribution studies, methods of detection of viable t. gondii in meat and on assessing the effect of the omission of palpation and incision on the risk posed by non-meat-borne zoonoses. assessing current meat inspection systems for sheep and goats with the aim of introducing improvements requires a common understanding of the term "meat inspection". however, as discussed previously (efsa, (efsa, , , it seems that there is no precise, universally agreed definition of meat inspection. the term meat inspection is not described specifically in current european union (eu) legislation (regulation (ec) no / ) or in the codex alimentarius's code of hygienic practice for meat (cac/rcp - ) ; rather, there are references to elements of the inspection process for meat such as ante-and post-mortem inspections and food chain information. consequently, the current understanding of the term meat inspection is probably based more on its practical application, and somewhat intuitive, than on a specific, formal definition. the biohaz panel defined the main scope of this scientific opinion as identifying and ranking the most relevant public health risks associated with meat from sheep and goats, assessing the strengths and weaknesses of the current meat inspection system, proposing alternative approaches for addressing current meat safety risks, and outlining a generic framework for inspection, prevention and control for important hazards that are not sufficiently covered by the current system. outside of the scope of the opinion were: microbiological hazards representing only occupational health risks transmissible spongiform encephalopathies (tses) issues other than those of public health significance, but which still compromise fitness of meat for human consumption (for example quality issues such as dark firm and dry (dfd) meat). as the eu regulations do not include different inspection requirements for sheep and goats, both species are considered together, but any important differences between these species are considered when necessary. in this document, the term small ruminant is used to refer to a combination of sheep and goats. in order to evaluate any important differences in meat inspection procedures between countries and/or regions as well as between species, the biohaz panel was supported by input provided during a technical hearing on meat inspection of small ruminants, during which experts from several stakeholder organisations presented information that had previously been requested by means of a questionnaire. following the hearing, an event report was compiled (efsa, ). the conclusions from this report are referred to when relevant. chemical hazards and associated meat safety risks in small ruminants are considered in a separate part of this opinion (see appendix b). although highest priority is given to the public health aims of the improvements of the biological/chemical meat safety system, any implications for animal health and welfare of the proposed changes were assessed (see appendix c). furthermore, issues related to epidemiological indicators and associated sampling/testing methodologies for hazards dealt with in this opinion were addressed by the biological monitoring unit in a separate document (efsa, ). the structure of the eu small ruminants farming industry has already been described in an efsa opinion (efsa, ) . briefly, sheep farming takes place in many areas of europe because sheep are able to live in a wide range of environments, even those hostile for other animals. goats are generally reared in extensive systems, traditionally in less developed areas, such as mountains or arid regions, and are often reared with sheep, especially in southern europe. milk sheep and goats are reared in similar systems, either grazed near the farm or kept housed, with the milk used in most cases for cheese production. meat production in europe reflects the diverse farming systems. lamb meat production originates from sheep milk farms or from farms raising meat breeds. in the mediterranean countries, the lambs from milk farms are slaughtered at approximately one month of age (suckling lambs, the same applies to goat kids). in some of these countries, lambs from meat breeds are generally slaughtered at - days of age and represent the majority of total national lamb meat production. in northern countries, the rearing systems usually produce heavier lambs that may be slaughtered at six or more months of age. the proportion of sheep raised for wool production has steadily decreased over time, but it is still significant in parts of the eu. sheep and goats at the end of their productive life can also be destined for meat production, with the resulting meat usually processed into meat products or exported. although the production and consumption of lambs have decreased in recent years, lamb meat continues to be a traditional product consumed in some countries of the eu such as the united kingdom, ireland and the mediterranean countries (spain, france, greece and italy). these countries have the largest populations of sheep in the eu. in general, the southern countries produce lighter carcasses (about kg) than the northern ones ( - kg). sheep are relatively small animals, with a lower yield of meat per carcass and higher slaughter and processing costs per unit of meat produced. as a result, sheep meat is relatively expensive in the market compared with other protein sources. the co-products (e.g. hides, wool, offal, feet, tails, etc.) have a major effect on the prices received by producers, and the impact on the profitability of the enterprise is profound (byrne et al., ) . eurostat statistics show that sheep meat production in the eu was over tonnes in , with the united kingdom and spain as the greatest producers ( figure ). goat meat production in the eu is concentrated in the southern european countries, especially greece and spain ( there are many forces instigating change in sheep and goat meat production. legislative forces present in the hygiene package and microbiological regulation have increased meat hygiene service costs through structural and food safety requirements as well as mandating the provision of traceability and food chain information (palmer c.m., ) . commercial considerations, such as lower coproduct returns, higher costs of by-product disposal and the sourcing policies of the multiple retailers (using their market power to control margins) have also put pressure on slaughterhouse profitability (palmer, ) . in spite of the eu being only about % self-sufficient in sheep meat, the predictions are that eu sheep numbers are expected to continue to decline over the next years. this problem of falling sheep supplies has led to an overcapacity in the processing sector (byrne et al., ) . the effect of this decline is most acute for large slaughterhouses, which can only be run profitably at certain levels of throughput. given the energy market expectations, greater environmental controls and the pressure on enforcement costs, relief from falling costs looks unlikely (palmer, ) . , ) . these variations, individually and their combinations, lead to between-slaughterhouse differences in process hygiene performance and, consequently, in the hygienic status of the final carcass. at the end of the slaughter line prior to chilling, process hygiene microbiological criteria, as defined in regulation (ec) no / , verify the effectiveness of each plant's food safety management system (which includes ghp and good manufacturing practices (gmp) prerequisite programmes), based on the principles of hazard analysis and critical control points (haccp) systems. generally, smaller slaughterhouses process much smaller quantities of meat for localised markets and operate at a slower line speed. operators in such establishments tend to have a wider skill base than their counterparts in large establishments owing to the many varied roles they perform. however, small slaughterhouses have reduced investment capital for expenditure on premises, equipment and staff food safety management training. disposal of animal by-products and compliance with the microbiological testing regulation (ec) no / places further financial pressure on these lowthroughput businesses. to ameliorate the financial impact of this testing, article in this regulation states that the frequency of this microbiological sampling may be adapted to the nature and size of the food business, based on a standardised risk assessment and authorised by the competent authority. larger slaughterhouses operate more efficiently, with greater separation of duties and better sampling and food safety oversight. these larger units have larger co-product/by-product markets and therefore produce less waste per animal processed. however, the requirement for high-volume throughput with increased slaughter line speed can impinge on operational hygiene and therefore food safety (food standards agency, a; palmer, ) . such differences in structure and operational practices in the varying sized slaughterhouses can determine the effectiveness of the food safety management system (motarjemi, ). hazard identification and risk ranking . . a hazard is defined by the codex alimentarius commission (cac) as a "biological, chemical or physical agent or property of food with the potential to cause an adverse health effect". the first step in the hazard identification carried out in this assessment focused on identifying biological hazards occurring in small ruminants and small ruminant meat that can be transmitted to humans, where they may cause disease. hazards were identified based on evidence found in peer-reviewed literature, textbooks, through reported data (e.g. eu summary reports on zoonoses), previous assessments and efsa opinions, and the biohaz panel's and working group's expert knowledge. from this "long" list of identified hazards, the panel excluded those hazards: for which no causal relationship between human infections and the handling, preparation and consumption of meat from small ruminants could be documented through targeted literature reviews. not presently found in the small ruminant population in the eu. the final "short" list of identified hazards to be included in the priority ranking consisted of hazards occurring in the eu and for which evidence could be found of foodborne transmission through the handling, preparation and/or consumption of sheep and goat meat. in the context of this opinion, when referring to handling and preparation this should be interpreted as handling of sheep and goat meat that occurs immediately prior to consumption, when these activities are carried out by consumers or professional food handlers. based on a review of the scientific literature, a wide range of biological hazards were identified as potential zoonotic hazards related to small ruminants (see table ). from these, the majority were considered not to be small ruminant meat-borne pathogens, as no evidence could be found in the literature to support transmission through handling, preparation or consumption of small ruminant meat (for further information on hazards not included see annex , and section . . in this appendix for those for which evidence for meat-borne transmission was documented). other potential pathogenic microorganisms were found not to be relevant as they are not considered to be currently present in small ruminants in europe (chandipura virus, cryptococcus neoformans var. neoformans and hepatitis e virus), or, if they are, consumption of meat is not considered a significant source of infection. the latter situation applies in particular to linguatula serrata, for which contact with the final host (canids) is the source for the human cases described in europe. for some of these hazards (e.g. extended-spectrum β-lactamase-(esbl-)/ampc-carrying escherichia coli), despite their presence in the animal reservoir, no studies have been conducted to establish whether there is a link between consumption of meat from small ruminants and disease in humans. the presence of mycobacteria has been previously reported in the small ruminant population in the eu (domenis et al., ; malone et al., ; marianelli et al., ; . despite these reports, evidence of meat-borne transmission of these pathogens to humans from small ruminants is lacking, so this potential pathway of infection remains unproven in the context of livestock processed through the eu meat inspection system. a more detailed discussion on the potential for meat-borne transmission of mycobacteria can be found in the scientific opinion dealing with bovines (efsa biohaz panel, ). the remaining hazards were considered eligible for further assessment and risk ranking ( table ). the panel developed a decision tree that was used for the risk ranking of the small ruminant meatborne hazards according to their risk of causing infection in humans following the handling, preparation and/or consumption of sheep or goat meat ( figure ). the cac defines risk as "a function of the probability of an adverse health effect and the severity of that effect, consequential to one or more hazards in a food". in other words, a foodborne risk is a product of the likelihood of occurrence of the hazard and the magnitude and severity of the consequences of the illness it causes on human health. this decision tree was adapted from that presented in the scientific opinion on poultry meat inspection (efsa panel on biological hazards (biohaz), efsa panel on contaminants in the food chain (contam) and efsa panel on animal health and welfare (ahaw), ). however, there are key differences as follows: carcass pathogen prevalence and source attribution are not considered as separate questions, or ranking steps, but these two questions are addressed together in a single step. this modification was considered appropriate as there was insufficient data at eu level for qualifying carcass prevalence and source attribution for the given hazards. furthermore, consumption of meat from small ruminants is both lower and unevenly distributed in the eu relative to that of meat from other animal species such as pigs or poultry. attribution at the population level, as applied in the previous scientific opinions on meat inspection (efsa panel on biological hazards (biohaz), efsa panel on contaminants in the food chain (contam) and efsa panel on animal health and welfare (ahaw), , ), may not provide a sufficiently detailed perspective on the relative risk of different hazards in meat from small ruminants. the risk to consumers of meat from these species, rather than to the population as a whole, was therefore assessed. an added consequence is that the categorisation has been reduced from three to two categories (i.e. the medium category is not used in this opinion). the term "priority" has replaced the term "risk" used in the pork and poultry opinions. risk ranking requires a significant amount of data on both the occurrence of the relevant hazards and the fraction of cases of human disease caused by the different hazard-meat species combinations (i.e. source attribution). while there were sufficient data to perform a risk ranking of the hazards associated with pork and poultry, this was not the case for all potential hazards in small ruminants, for which eu-wide baseline surveys and harmonised monitoring do not exist and relevant studies published in the scientific and technical literature are often limited. the term "priority" was therefore considered more appropriate than "risk" for categorising the hazards associated with meat from small ruminants. based on this, the panel identified the following criteria to be important for determining the final priority category: step : identifying and excluding those hazards that are introduced and/or for which the risk for public health requires microbial growth during steps that take place after carcass chilling. the reasons for excluding such hazards from further assessment were that: ( ) the scope and target of meat inspection are focused on the food safety risks of the carcasses at the end of slaughter when they are chilled but before they are further processed; and ( ) hazards introduced and/or for which the risk relates exclusively to growth during post-chilling processes are better controlled later in the food production chain through, for instance, haccp programmes. step : to assess the magnitude of the human health impact, as measured by the reported incidence (notification rate) or number of cases. where data allowed, the estimated total number of cases was presented, i.e. adjusting for under-reporting. incidence was considered high if the notification rate in humans at eu level, as reported to ecdc, was equal to or higher than cases in population in any given year. step : to assess the severity of the disease in humans based on mortality. if necessary, severity was also evaluated by comparing disease burden estimates, expressed for example in disability-adjusted life-years (dalys) per cases. the daly metric quantifies the impact of disease on the health-related quality of life of acute diseases and sequelae, as well as the impact of premature deaths. severity was considered high if mortality in humans at eu level, as reported to ecdc, was higher or equal to . % in more than one year. step : evidence supporting the role of meat from small ruminants as a risk factor for disease in humans. for this, the following sources of information were considered: . epidemiological link, based on a significant likelihood that the consumption of meat from the given species is a risk factor for human cases, or on outbreak data . carcass prevalence /farm level prevalence (prevalence studies) . comparative considerations for meat from related species . expert opinion that meat consumption is a risk factor. the final outcome of this process involved categorising each hazard as high or low priority, as follows: the priority was characterized as 'high' when a hazard was identified as causing a high incidence and/or severity of illness in humans, and when strong evidence existed for meat from sheep or goats being an important risk factor for human disease. considering the limitations of the data available for the priority ranking, this risk category could be regarded as combining both the medium and high risk categories of the risk ranking carried out in the poultry meat inspection opinion (efsa panel on biological hazards (biohaz), efsa panel on contaminants in the food chain (contam) and efsa panel on animal health and welfare (ahaw), ); the priority was characterized as 'low' when a hazard was identified as not associated with a high incidence and a high severity of human disease or if, despite the hazard causing a high incidence and/or severity in humans, the evidence available did not identify meat from sheep or goats as an important risk factor for human disease; all hazards placed in the low priority category were further evaluated to determine if this was low due to currently controls applied (i.e. any hazard specific control measure implemented at farm and/or slaughter level before chilling of the carcass, including meat inspection procedures). if this was not the case, the hazard was not considered further. however, if this was the case then the hazard was further considered and the effect of any recommendations regarding the removal of specific control measures or meat inspection activities on these hazards was assessed and the categorisation of the hazard was reconsidered. figure : flowchart for priority ranking different public health hazards. risk of infection by handling, preparation or consumption of sheep and/or goat meat. current controls: any hazard-specific control measures implemented at farm and/or slaughterhouse level before chilling of the carcasses. for the hazards shortlisted (table ) , data on the incidence and severity in humans and the prevalence of the pathogens in the carcasses of small ruminants were sought to allow the risk from these microbiological hazards to be ranked, based on the decision tree in figure . see tables , , and for details. the data in table were obtained from the european surveillance system (tessy), covering the years , , and . the data are officially reported to the european centre for disease prevention and control (ecdc) by eu mss; however, some countries do not report on certain diseases; these were specified. the data were supplied as aggregates from all reporting mss. data show notification rates of confirmed human disease cases as per persons, and severity of illness in humans. cases include all reported confirmed occurrences of the disease, regardless of the origin of the infection. in fact, establishing the food-related origin of infection is often not possible and seldom reported. the data on severity include as a proxy the proportion of confirmed human cases that died. this information is usually only available in a small proportion of cases. finally, it has to be kept in mind that the surveillance systems are set up differently in the various eu mss, with different case definitions, national or restricted coverage, voluntary or compulsory reporting, focus, target groups, etc., in addition to the fact that only a small fraction of diseased patients is sampled and the casual organism typed and reported to the respective national health institutes. because of all these caveats, the incidence and severity figures quoted here are only approximate and must be considered with caution. n.a. h a eu population data based on individual ms population sizes reported in eurostat (data extracted: september ). when the given hazard was not reported by a ms to tessy, the population size reported by that ms was also taken out of the calculation of the overall eu population size. b calculated as the percentage of cases with fatal outcome over all cases of disease with known outcome, for a given hazard. c portugal, greece not reporting. d portugal not reporting. for a more detailed review of vtec (including serotype o ) incidence and severity in the eu see the recently published efsa opinion on vtec-seropathotype and scientific criteria regarding pathogenicity assessment (efsa panel on biological hazards, ). e portugal not reporting. f s. enterica subsp. enterica serovar typhi and s. paratyphi serovars not included; netherlands not reporting. g seroprevalence. belgium, denmark, greece, italy, netherlands, portugal and sweden not reporting; spain reporting through the sentinel system and thus not taken into account. france not reported in at the time of extraction of these data. h n.a. = not available. data presented in tables - are related to flock/herd and carcass prevalence of the hazards identified in sheep and goats. they were obtained from monitoring data as reported by the eu mss in the frame of the zoonosis directive ( / /ec), when available. data reported in the period from to were considered. no information was available at carcass level for goats. in these tables, data described as originating from suspect or selective sampling and from clinical investigations were excluded as they do not, in most cases, represent the actual epidemiological situation. food samples described as collected for haccp and own-check purposes were also excluded because the sampling scheme may be biased. samples included are described as originating from control and eradication plans and monitoring and surveillance; consequently they are supposed to represent the occurrence of the zoonotic agent in the reporting country over the years, based on objective sampling. however, it has to be noted that monitoring and surveillance systems for most of zoonotic agents are not fully harmonised between mss. furthermore, data may not necessarily be derived from sampling plans that have a sound statistical design, and may therefore not accurately represent the national situation regarding the true prevalence of zoonoses. data in tables and originate from samples taken from either farms or slaughterhouses, while for table . ( - ) a n.a., no data available. b includes those reported as human pathogenic and non-human pathogenic (i.e. no harmonised scheme to discriminate between both, and data available does not preclude that they are not human pathogenic). c seroprevalence. includes those reported as human pathogenic and non-human pathogenic (i.e. no harmonised scheme to discriminate between both, and data available does not preclude that they are not human pathogenic). c seroprevalence. includes those reported as human pathogenic and non-human pathogenic (i.e. no harmonised scheme to discriminate between both, and data available does not preclude that they are not human pathogenic). c seroprevalence. listeria monocytogenes and toxins of bacillus cereus, clostridium botulinum, clostridium perfringens and staphylococcus aureus were all considered to fall within the category of risk related to growth or introduction post-chilling, for different reasons: b. cereus, c. botulinum and c. perfringens and their spores and s. aureus are considered ubiquitous bacteria, and can be found in a variety of foods. their vegetative forms need temperatures above those used for refrigeration to grow in raw meat to concentration levels of relevance for public health and thus the risk of disease seems not to be correlated with occurrence in raw meat but rather with improper hygiene and storage that allows the production of toxins. illness caused by l. monocytogenes is usually associated with ready-to-eat products, in which contamination has occurred before or during processing followed by growth during storage at refrigeration temperatures. based on incidence and severity in humans (table ) , flock/herd, animal and carcass prevalence (tables , and ) and other epidemiological evidence, the hazards in table were ranked and categorised according to the flowchart in figure , as described in section . . above. a summary of the outcome is provided in table at the end of this section. none of the hazards identified as low priority were found to be such owing to currently applied controls. this organism has a worldwide distribution, persisting in the soil in the form of extremely resistant spores for many years. infection is initiated with the introduction of the spore through a break in the skin or entry through the mucosa. after ingestion by macrophages at the site of entry, germination to the vegetative form occurs, followed by extracellular multiplication and capsule and toxin production. humans can acquire anthrax by exposure to infected animals, animal products or spores in the soil and, depending on the mode of transmission, can develop one of four distinct clinical forms: respiratory, cutaneous, gastrointestinal and oropharyngeal. human cases of pulmonary anthrax have been linked to the large-scale processing of hides and wool in enclosed factory spaces, where aerosolised anthrax spores may be inhaled. humans also acquire the cutaneous form of anthrax from handling contaminated animal products, such as hides, wool and hair. cases of gastrointestinal anthrax have resulted from the ingestion of raw or undercooked meat (spickler, ) and well-cooked beef from infected animals (centers for disease control and prevention, ) . recently, a case of anthrax possibly acquired through handling or consumption of contaminated beef in a household in romania has been reported (popescu et al., ) . consumption of meat (including sheep and goat meat) from carcasses of animals showing clinical signs of anthrax, or animals that have died from the disease, is the most reported common route of foodborne infection resulting in gastrointestinal anthrax. human incidence: based on eu data, low. anthrax has a low human prevalence in the eu (see table for details). between and , the number of anthrax cases reported to the ecdc ranged from two confirmed cases ( ) severity of disease: based on eu data, high. the severity of these infections is considered high, and this is supported by the mortality figures in table . evidence for meat from small ruminants as an important risk factor: no. the organism causes a highly infectious notifiable disease in farmed and wild animals that have grazed on contaminated land or ingested contaminated feed (swartz, ) . the livestock species most susceptible, in descending order, are cattle, sheep, horses, pigs, goats and camels (fasanella et al., a) . the disease is endemic in most countries in africa and asia (thurnbull, ) and in defined regions of other countries. flooding may often concentrate spores of b. anthracis in particular locations. in sheep and goats, the disease is usually peracute, or acute and rapidly fatal, with death occurring in some cases within hours and affected animals showing multiple haemorrhages from natural orifices. although most cases are found dead without showing premonitory signs, pyrexia with temperatures up to c along with depression, congested mucosae and petechiae may be observed ante-mortem. post-mortem findings are characterised by incomplete rigor mortis, widespread ecchymotic haemorrhages and oedema, dark, unclotted blood and blood-stained fluid in body cavities and severe splenomegaly (quinn et al., ) . handling, or direct contact with such animals and carcasses is highly dangerous. anthrax is now rare in livestock in the eu. the major enzootic areas are greece, spain, france and southern italy (fasanella et al., ; fouet et al., ) . a severe outbreak of anthrax occurred in southern italy in (fasanella et al., b . over days, cattle, sheep, goats, horses and deer died. also in italy, an outbreak of anthrax of similar magnitude was reported among cattle, sheep and horses in . given the low number of cases of anthrax in the small ruminant population in the eu, the risk of acquiring this disease through consumption of meat from these species can be considered very low. based on the data presented and on the above discussions, the biohaz panel concluded that b. anthracis was a low priority hazard with regard to meat inspection of small ruminants. this result is not due to current controls (i.e. any hazard-specific control measures implemented at farm and/or slaughter level before chilling of the carcasses, including current meat inspection procedures). human incidence: based on eu data, high. campylobacteriosis is the most frequently reported zoonotic illness in the eu with a reported incidence of . confirmed cases per in (table ) , and it is estimated that there are nine million cases of illness annually in the eu- (efsa panel on biological hazards, ). severity of disease: as the incidence is high, the severity does not need to be considered. campylobacter jejuni is common in the intestines of ruminants of sheep and lambs. the reported prevalence of campylobacter spp. in sheep and goats can be found in tables , and . for sheep and at flock level, the prevalence was . %, while for goats it was . % (at individual animal level there were . % and . %). with regards to carcasses, no data were available for goats. for sheep, the batch prevalence was %, and at individual sample level . %. information from the scientific literature also suggests that campylobacter spp. is often found in small ruminants, with a wide range of prevalences reported. in a study of lambs in the united kingdom campylobacter spp. was isolated from % of the samples taken from the small intestines (stanley et al., ) . on the other hand, sproston et al. ( ) found this bacterium in just % of fresh faecal samples from sheep on a farm in scotland. other studies have reported prevalences somewhere in between these two figures (milnes et al., ; ogden et al., ; oporto et al., ; schilling et al., ) . a seasonal variation in prevalence and the number of campylobacter spp. has also been reported in some studies (milnes et al., ; sproston et al., ) . several studies have investigated the presence of campylobacter spp. in carcasses or meat from small ruminants. garcia et al. ( ) investigated the presence of campylobacter spp. on sheep carcasses, with a resulting prevalence of %. the authors concluded that the prevalence on carcasses reflected the occurrence of campylobacter spp. in both wool and faeces. however, there is a significant reduction in detection following chilling, probably owing to both the low temperature and drying of the carcass (norwegian scientific committee for food safety, ). after swabbing of cm around the circum-anal incision of lamb carcasses before chilling campylobacter spp. was isolated from eight ( . %) of the carcasses. after a relatively slow chilling process (the air temperature was never below °c) campylobacter spp. was recovered from only one carcass ( for contact with sheep as a risk factor for human campylobacteriosis, but consumption of meat from these species was not considered a risk factor. an earlier case-control study in households with primary campylobacter spp. infection in the netherlands also failed to identify consumption of mutton as risk factor (oosterom et al., ) finally, people that had consumed mutton were less likely to become ill with campylobacter spp. infection in a prospective case-control study of campylobacteriosis carried out in norway (kapperud et al., ) . like their sensitive counterparts, antimicrobial-resistant campylobacter spp. involved in human disease are mostly spread through foods, especially poultry meat. as stated in a previous efsa opinion (efsa, ) , "a major source of human exposure to fluoroquinolone resistance via food appears to be poultry, whereas for cephalosporin resistance it is poultry, pork and beef that are important, these food production systems require particular attention to prevent spread of such resistance from these sources". there are no indications that resistant strains behave differently in the food chain compared with their sensitive counterparts, hence there is no need to consider these strains separately in the context of meat inspection. based on the presented data, it is concluded that campylobacter spp. are a low public health priority with regard to meat inspection of small ruminants. this ranking is not the result of current controls. verocytotoxin/shiga toxin (vt/stx)-producing e. coli (vtec/stec) are characterised by the ability to produce potent cytotoxins. pathogenic vtec usually also harbour additional virulence factors that are important for the development of the disease in human (efsa and ecdc, , b) . not all vtec strains have been associated with human disease and there is no single marker or combination of markers that defines a "pathogenic" vtec (efsa panel on biological hazards, ). while stx and eae gene-positive strains are associated with a high risk of more serious illness, other virulence gene combinations and/or serotypes may also be associated with serious disease in humans. for the purposes of this opinion, pathogenic vtec are defined as vtec capable of causing disease in humans. human incidence: based on eu data, low. most reported meat-borne human vtec infections are sporadic cases. in (efsa and ecdc, ), the total number of confirmed vtec cases in the eu was , representing a . % increase compared with , with a fatality rate of . %. table includes data from tessy from to inclusive. in that period the incidence (all vtec serotypes) per population varied between . and . . the data are not easily comparable between eu countries, owing to underlying differences in the national surveillance systems. the concentration of laboratory testing on the o serogroup means that the proportion of non-o strains is largely under-reported (ecdc and efsa, ) . data for have to be interpreted with caution, as vtec o :h caused a major outbreak which resulted in confirmed cases, including cases of vtec infection and of acute renal failure, known as haemolytic-uraemic syndrome (hus), with deaths reported in eu countries, the united states and canada when the epidemic was declared to be over at the end of july (karch et al., ) . it has to be noted, however, that the source of the outbreak was sprouted seeds and not meat. severity of disease: based on eu data, high. pathogenic vtec infections can be severe, and are often associated with bloody diarrhoea, but there is a wide clinical spectrum in the association between specific subtypes of pathogenic vtec and the clinical outcome. bloody diarrhoea has been shown to be associated with an increased risk of developing hus and neurological injury, such as paralysis. hus develops in up to % of patients infected with vtec o and is the leading cause of acute renal failure in young children (efsa and ecdc, ) . this is reflected in the severity figures in table and the corresponding classification in table , which are also supported by high daly (havelaar et al., a) and quality-adjusted lifeyear (qaly) estimates (hoffmann et al., ) published in the literature. evidence for meat from small ruminants as an important risk factor: yes. pathogenic vtec can be found in the gut of numerous animal species, but ruminants have been identified as a major reservoir of vtec that are highly virulent to humans, in particular vtec o . although cattle are considered to be the most important source of human infections caused by vtec o , they have also been isolated from the intestinal contents of sheep and goats. food of small ruminant origin has been reported as a source for human vtec infections (kosmider et al., ; schimmer et al., ; werber et al., ) . transmission occurs through consumption of undercooked meat, unpasteurised dairy products, or water and vegetables contaminated by faeces of carriers. person-to-person transmission has also been documented (rey et al., ) . data reported in the frame of the zoonoses directive ( / /ec) from to can be found in tables - . for all vtec serotypes, the reported prevalence was . % and . % for sheep at flock and individual animal level, respectively. for goats, the figures were . % and . %. prevalences for vtec o were much lower across the board. isolation of e. coli o from goats has been reported in studies from several countries, with isolation rates ranging between % and % (cortes et al., ; keen et al., ; orden et al., ; orden et al., ; schilling et al., ) . vtec strains have also been detected in sheep, with a similarly wide range of prevalence figures (milnes et al., ; oporto et al., ; prendergast et al., ; pritchard et al., ; schilling et al., ; sekse et al., ) . thus it is clear that small ruminants can play an important role by shedding these pathogens in the faeces (blanco et al., ; la ragione et al., ) . the prevalence can be influenced by the sampling and testing methodology, but these studies nevertheless clearly indicate that pathogenic vtec is present in the small ruminant population in the eu. table includes data from official monitoring of sheep carcasses. the reported prevalence was % at batch level and . % at individual carcass level ( . % for vtec o ). the scientific literature also indicates that sheep and goat carcasses or meat can be contaminated with vtec, albeit generally at lower levels compared with those in the animal reservoir. at the higher end of the range, barlow et al. ( ) in australia and zweifel et al. ( ) in switzerland reported prevalences around % in carcasses and lamb cuts. brooks et al. ( ) reported a prevalence of % in lamb cuts in new zealand and other, less recent, studies reported much lower prevalence-between % and % (doyle and schoeni, ; heuvelink et al., ; pierard et al., ; samadpour et al., ) . it has to be noted that this variation in prevalence could be a result of the different testing methodologies used (e.g. use of polymerase chain reaction (pcr) testing), and the fact that not all these vtec isolates would necessarily be pathogenic to humans. a case-control study on risk factors for human vtec in germany identified lamb as an important risk factor for human infection (werber et al., ) . consumption of dry cured sausages made with sheep meat was identified as the cause of an outbreak of vtec o :h infection in humans (schimmer et al., ; sekse et al., ). in the latter study, bacteria with the same properties, including identical dna profiles, were found in five dry cured sausage products and sheep meat used as raw material in sausage production and were identical to the isolates from patients. e. coli with the same virulence genes, serotypes, biochemical characteristics and dna profiles as those found in patients from the e. coli o :h outbreak, were detected in sheep from of farms in norway (brandal et al., ) . more recent research in norway and spain comparing virulence characteristics between strains isolated from humans and sheep has suggested that the latter can be an important reservoir for pathogenic vtec (brandal et al., ; sanchez et al., ) . the evidence arising from epidemiological or source attribution studies points to a minor role for meat from small ruminants as a source of human cases of vtec, although the model used in this study was found to underestimate the observed prevalence of vtec in lamb, so this attribution estimate should be interpreted with caution (kosmider et al., ) . based on the data (see table ) and the assessment presented above, the biohaz panel concluded that pathogenic vtec can be considered to be of high priority for meat inspection of small ruminants given the relatively high prevalence of this hazard in the small ruminant population, the epidemiological links to outbreaks in humans and the severity of the disease in humans. human incidence: based on eu data, high. in the eu, s. enterica subsp. enterica serovar enteritidis and s. typhimurium are the serovars most frequently associated with human illness, although the number of reported cases of s. enteritidis has more than halved since . human s. enteritidis cases are most commonly associated with the consumption of contaminated eggs and poultry meat, while s. typhimurium cases are mostly associated with the consumption of contaminated pig, poultry and bovine meat. human salmonellosis is the second-ranking foodborne disease reported in the eu and most european countries, exceeded only by campylobacteriosis (efsa, ; efsa and ecdc, b) . a total of confirmed cases were reported from eu mss in through tessy, corresponding to a notification rate of . confirmed cases per (table , which also includes data on the severity of human disease). accounting for under-reporting, it is estimated that there are six million cases of this illness annually in the eu- (efsa, ; havelaar et al., b) . severity of disease: as the incidence is high, the severity does not need to be considered. evidence for meat from small ruminants as an important risk factor: no. the common reservoir of salmonella spp. is the intestinal tract of a wide range of domestic and wild animals, which results in a variety of foodstuffs, of both animal and plant origin, as sources of human infections. the organism may be transmitted through direct contact with infected animals or between humans or from faecally contaminated environments. in animals, subclinical infections are common. the organism may easily spread between animals in a herd or flock without detection, and animals may become intermittent or persistent carriers. the variant, s. enterica subsp. diarizonae iiib .k: , , ( ), which might be referred to as "the sheep variant" owing to its adaption to sheep, is endemic in sheep in several regions of the world such as the united kingdom (hall and rowe, ) and norway (norwegian scientific committee for food safety, ) in europe and canada (greenfield et al., ; pritchard, ) and the united states (weiss et al., ). however, the overall conclusion is that s. enterica subsp. diarizonae iiib .k: , , ( ) is very rarely demonstrated as a cause of human infections, including in those areas in which the endemic prevalence in sheep is high such as the united kingdom and norway ((norwegian scientific committee for food safety, ). another salmonella spp. variant well adapted to sheep, causing abortion and death of ewes, is s. brandenburg, which is endemic in the south island of new zealand (sabirovic, ) , but its human health relevance seems to be limited. eu monitoring data for sheep and goats are presented in tables - , which contain data collected by mss from to . the prevalence reported in both herds and individual animals is . % and . %, respectively, for sheep and . % and . % for goats. although salmonella spp. is commonly found in live sheep or goats at variable prevalence levels (bonke et al., ; duffy et al., ; duffy et al., ; hjartardottir et al., ; moriarty et al., ; zweifel et al., ) , there is a more limited number of studies looking at the occurrence of salmonella spp. in sheep and goats carcasses. a prevalence of . % was reported in individual sheep carcasses in the eu monitoring (see table ). some outbreaks linked to meat from small ruminants can be found in the scientific literature (evans et al., ; hess et al., ; synnott et al., ) . these involved unusual consumption patterns (e.g. raw lamb liver) or cross-contamination of raw food ingredients (e.g. yoghurt relish contaminated with carcass blood), therefore it is unclear how significant these events are when assessing the role of sheep or goat meat as a source of salmonella spp. infection. data from epidemiological or source attribution studies suggest that the role of meat from small ruminants as a vehicle for salmonella spp. the occurrence of antimicrobial resistance among zoonotic salmonella spp. is an increasing problem. antimicrobial-resistant salmonella spp. involved in human disease are, like salmonella spp. in general, mostly spread through foods, predominantly poultry meat, eggs, pork and beef (hald et al., ) . as there are no indications that resistant strains behave differently from their sensitive counterparts in the food chain, there is no need to consider these strains separately in the context of meat inspection. fluoroquinolone and cephalosporin resistance are currently considered to be those of most public health concern. meat, particularly poultry meat and pork, is recognised as an important source of human exposure to fluoroquinolone-resistant salmonella spp., and high levels of esbl-/ampc-producing salmonella spp. have also been reported in poultry in some eu mss (efsa and ecdc, a). such resistant strains may or may not be associated with a significant level of human infection, depending on the pathogenicity of the strains involved and the opportunity for them to contaminate the food chain (butaye et al., ; de jong et al., ; efsa panel on biological hazards, c; rodriguez et al., ) . the control of antimicrobial-resistant bacteria in food including poultry meat is further complicated by the fact that resistance mechanisms can be located on mobile genetic elements such as plasmids and thereby be transferred between different bacterial species, for instance between generally apathogenic e. coli and salmonella spp. based on the data (see table ) and the assessment presented above, the biohaz panel concluded that the risk arising from consumption of meat from small ruminants with regards to salmonella spp. is of low priority for meat inspection of small ruminants. this ranking is not the result of current controls. human incidence: based on eu data on congenital toxoplasmosis, low. toxoplasmosis can be contracted by the oral ingestion of oocysts present in cat faeces and the environment, or tissue cysts present in the meat of infected animals (tenter et al., ) . in pregnant women, the parasite can cause congenital infections (abortion, stillbirth, mortality and hydrocephalus in newborns or retinochoroidal lesions leading to chronic ocular disease) and complications (lymphadenopathy, retinitis or encephalitis) in immunocompromised individuals such as organ graft recipients and individuals with acquired immune deficiency syndrome (aids) or cancer (efsa, b) . in immune-competent individuals, - % of cases of toxoplasma gondii infection are asymptomatic, and the majority of the remainder have only mild, self-limiting symptoms. thus, reports of acute symptomatic t. gondii infection (toxoplasmosis) do not provide a reliable basis for assessing overall disease incidence. given these limitations, the incidence of human disease caused by toxoplasmosis is rare ( table ). the prevalence of antibodies to t. gondii in the general population provides an alternative for estimating the number of cases and disease burden (food standards agency, ). t. gondii seroprevalence is known to vary geographically and with age (montoya and liesenfeld, ). although antibodies are found in - % of adults in the united kingdom, seroprevalence is higher in central europe, and similar or lower in scandinavia ( - %). climate and consumption of raw meat, meat from animals farmed outdoors or frozen meat may be factors that contribute to these variations (kijlstra and jongert, ) . seropositivity also varies within countries, being highest in those from rural or small town backgrounds and lowest in those from urban or suburban areas (food standards agency, ). data showing the variation in seropositivity with age are available from a number of countries. for example, in the netherlands, it was found to range from % at years of age to % at years (efsa, b; hofhuis et al., ) . there is evidence of a sharp decrease in seroprevalence over the last years in many populations. for example, in there was a reported seroprevalence of % in france, falling to % in (afssa, ) . this decrease is in part attributable to a decrease in infection in childhood, probably associated with increased standards of living, and has also been linked to changes in meat husbandry and consumption. severity of disease: based on eu data for congenital toxoplasmosis, high. owing to the lifelong impact of symptoms related to toxoplasmosis, the burden of disease is high. mead et al. ( ) showed that t. gondii ranked fourth in hospitalisations and third concerning deaths when compared with other foodborne pathogens. more recent research ranked t. gondii among the highest in population burden estimates (daly or qaly) among foodborne pathogens from both an individual and a population perspective (havelaar et al., a; hoffmann et al., ) . evidence for meat from small ruminants as an important risk factor: yes. the relative role of t. gondii oocysts in the environment versus tissues cysts in meat and meat products as a source of infection for humans could not be determined by laboratory tests until recently. hill et al. ( ) have developed a test to identify a sporozite specific antigen which will be a useful tool in providing information on the relative importance of oocysts as the agent of infection. until this recent development, source attribution information came from epidemiological studies. in europe, three large case-control studies have pinpointed uncooked meat as the most important risk factor for pregnant women (baril et al., ; cook et al., ; kapperud et al., ) . with regard to the prevalence in the animal population, despite t. gondii infection being a major cause of abortion and stillbirth in sheep and goats in the eu, most infections exist subclinically in flocks/herds (dumetre et al., ) . in response to natural infection, seropositive sheep have been shown to harbour infectious parasites as tissue cysts (dubey et al., ; kijlstra and jongert, ; opsteegh et al., ) . antibodies to t. gondii and tissue cysts persist in infected sheep (dubey, ). this implies that serological tests can be used to estimate the number of animals carrying t. gondii tissue cysts in the meat and thereby indicate the risk for public health (opsteegh et al., b) . seroprevalence increases with increasing age (dubey, ; halos et al., ) , and sheep and goats are identified as the main source of infected meat in southern european countries (berger et al., ; dumetre et al., ) . seroprevalence of t. gondii in sheep can range from % to % in certain european countries (efsa, b) . limited data available in slaughtered sheep report seropositive rates of - % in europe (dumetre et al., ; tenter et al., ) . seroprevalence in farmed goats in europe ranges from % to % (efsa, b) . no data have been published about seroprevalence in slaughtered goats in europe, but findings in goats in non-european countries range from % to % (efsa, b; tenter et al., ) . data reported by eu member states under the zoonoses directive ( / /ec), showing a relatively high seroprevalence for this hazard in flocks/herds and individual animals, can be found in tables - . notwithstanding this, significant uncertainty remains regarding this hazard. the prevalence of toxoplasmosis in humans and its importance in terms of overall disease burden still requires research. despite the development of recent laboratory procedures, the proportion of human toxoplasmosis attributable to the consumption of sheep meat is unknown. furthermore, the relationship between seropositivity in sheep and the number of viable tissue cysts in edible tissue has yet to be established (food standards agency, ). these uncertainties hinder the development of control procedures for this hazard. with regard to the role of meat from small ruminants as a risk factor for human toxoplasmosis, a prospective case-control study designed to identify preventable risk factors for t. gondii infection in pregnancy, conducted in norway (kapperud et al., ) found eating raw or undercooked mutton to be independently associated with an increased risk of maternal infection (or = . , p = . ). in the case-control study carried out by baril et al. ( ) , an odds ratio of . was estimated for the consumption of undercooked or raw mutton/lamb. the same odds ratio was obtained for the consumption of undercooked or raw mutton/lamb in the study carried out in (cook et al.) . in addition, raw or undercooked lamb meat is considered a delicacy in certain countries, such as france, and is therefore considered an important source of infection in that country (afssa, ) . this has been recently corroborated by a report of an outbreak of toxoplasmosis linked to the consumption of undercooked lamb (ginsbourger et al., ) . given its high seroprevalence in sheep and goat meat and the correlation of human infection to animal incidence, t. gondii in sheep and goat meat was considered by the panel to be of high priority for meat inspection of small ruminants within the eu (see table ). based on the priority ranking, the hazards were classified as follows: t. gondii and pathogenic vtec were classified as high priority for sheep/goat meat inspection. the remaining identified hazards, b. anthracis, campylobacter spp. (thermophilic) and salmonella spp., were classified as low priority, based on the available data. as new hazards might emerge and/or hazards that presently are not a priority might become more relevant over time or in some regions, both hazard identification and the risk ranking should be revisited regularly to reflect this dynamic epidemiological situation. particular attention should be given to potential emerging hazards of public health importance that arise only in small ruminants. to provide a better evidence base for future risk ranking of hazards, initiatives should be instigated to: improve and harmonise data collection of incidence and severity of human diseases caused by relevant hazards. systematically collect data for source attribution. collect data to identify and risk rank emerging hazards that could be transmitted through handling, preparation and consumption of sheep and goat meat. protection of public health is the top priority objective of meat inspection. the origin of western european meat inspection goes back to the end of the th century, when it became obvious that meat could play a role in the transmission of disease, particularly tuberculosis, and that the trade in animals, meat and meat products needed some sort of safety and quality assurance (johnson, ; theves, ; von ostertag, ) . the first meat inspection act was drawn up in by professor ostertag at the university of berlin. there is no doubt that the meat inspection procedures were highly risk based at that time. ever since, an ante-mortem and post-mortem inspection has been carried out at individual animal level in cattle and it has been extended to other species. the ante-mortem inspection is a simple clinical examination aimed at identifying sick or abnormal animals, as well as assessing the level of cleanliness of the animals entering the slaughtering process. the post-mortem inspection is a pathological-anatomical examination aiming at detecting and eliminating macroscopic abnormalities that could affect the fitness of meat for human consumption. it is based on visual inspection, palpation, incision and, when required, laboratory examination. postmortem inspection is laborious and expensive. the previous situation of slaughtering a few animals originating from a farm has evolved into large numbers of uniform, relatively young and healthy animals presented for slaughter, which have a common genetic background and prior history. at the same time, it is common to find mixed batches of animals arriving at the slaughterhouse, having been assembled at markets and where several farms have each contributed a few animals. transport can also increase the level and/or duration of shedding of pathogens, as well as the surface contamination of animals with pathogens via animal-animal or animal environment-animal contacts in the vehicle, at the market or in the lairage. therefore, it can be assumed that the food/meat safety risks increase as the number/frequency of movements of animals between farm and slaughter increases (scientific committee on veterinary measures relating to public health (scvmph), ). the current state of meat inspection in the eu and six selected exporting countries outside the eu has been reviewed and summarised recently in an external report. for further, more detailed information on the current eu meat inspection system, the reader is referred to that report. still, irrespective of the meat inspection procedures in place, it is well recognised that small ruminants presented at slaughter can be carriers of zoonotic microorganisms (see section . . above), which cannot be detected during ante-and post-mortem inspections. in the following section, an assessment of the strength and weaknesses of the current practices for protection of public health will be given. the food chain information (fci) principle includes a flow of information from farm to slaughterhouse in order to help classify the batch of animals according to its expected food safety risk, so that slaughter procedures and/or decisions on fitness for consumption can be adapted to the health status and food safety risk presented by the batch of sheep or goats. regulation (ec) no / also requires the feedback of information from slaughterhouse to farmers, describing also the information that has to be provided (appendix to annex i of the regulation). fci is recorded at the flock/batch level and its minimum content is described in regulation (ec) no / . fci related to primary production of flocks or herds is based on a farmer's declaration. most mss have made available a standardised fci declaration form to farmers (e.g. ireland, the united kingdom, italy, france). fci must be checked for completeness and content as part of antemortem inspection. in theory, fci may be used to adapt ante-and/or post-mortem inspections. fci serves as a channel of communication between the primary producer and the inspectors at the slaughterhouse. this, theoretically, facilitates the process of evaluating the health of incoming batches and prevents sick or abnormal animals entering the slaughterhouse, by providing early data on probable disease conditions that may be present in the flock or herd. this is based on information related to the on-farm health status of the animals (occurrence of disease, veterinary treatments, specific laboratory testing). the main benefit of the food chain information is that it may create an awareness among primary producers of the need for high standards of animal health and welfare, proper identification of animals and appropriate use of medicines. by contributing to the overall health of the animals sent to slaughter, such a system should have a positive impact on public health by ensuring that the animals are less likely to carry hazards of public health importance. in practice, ante-or post-mortem inspections of sheep and goats are rarely adapted to take account of fci. the main reason that current fci is insufficiently utilised is because of the lack of adequate and harmonised indicators that could help in classifying the animals according to the risk to public health they may pose. the food safety relevance of fci is often limited because the data that it contains is very general and does not address specific hazards of public health importance. also, farmers might not be in a position to properly assess the presence of relevant hazards. feedback of the results of the meat inspection process to farmers is not implemented in all mss to the full extent foreseen in the legislation. the flow of information back to the farm is not straightforward in the absence of a fast and reliable animal movement tracing system, e.g. through the use of electronic individual animal identification linked to a database containing information on the movements of animals (e.g. change of farm, last farm). for example, in ireland between % and % of small ruminants come to the slaughterhouse from assembly centres or markets (efsa, ) . in this case, it is difficult to consider a batch of small ruminants as an epidemiological unit. good feedback to farmers also requires harmonisation of the reasons for condemnation and the systematic use of the same terminology for each reason for condemnation. the ante-mortem clinical examination is carried out to evaluate the health and welfare of the animals, and to prevent sick or abnormal animals entering the slaughterhouse. this is a visual-only inspection, consisting of the identification of clinical signs of a disease and an assessment of the cleanliness of the incoming animals. it is performed at the individual level in sheep and goats. the public health-related strengths of ante-mortem inspection include inspection of individual animals for signs of disease and animal identification. ante-mortem inspection also helps in identifying dirty animals, as required by current legislation. regulation (ec) no / , annex i requires primary producers to ensure the cleanliness of animals going to slaughter. regulation (ec) no / annex ii, section ii states that food business operators, operating slaughterhouses, must have haccp-based intake procedures to guarantee that each animal or, where appropriate, each lot of animals accepted on to the slaughterhouse premises are clean. regulation (ec) no / , annex i, section ii, chapter iii states that animals with hides, skins or fleeces posing an unacceptable risk of contamination to meat during slaughter cannot be slaughtered for human consumption unless they are cleaned beforehand. adjustments can be made to the slaughter process depending on how dirty the batch of sheep or goats is. current pre-slaughter control procedures include: rejection of dirty lots, washing of animals, fleece/hide trimming or clipping (at the farm or at the slaughterhouse, either pre-or post-slaughter), and slaughter of dirty animals at the end of the day (byrne et al., ) . dirty animals that are presented for slaughter are rejected at ante-mortem inspection until their fleece/hide condition improves. suppliers are sometimes penalised financially through reduced price and the cost imposed by remedial actions required to improve fleece/hide condition. certain countries have adopted such measures as part of a "clean livestock policy". these policies were adopted to meet the requirements of the hygiene package and have proved to be effective in reducing the risk posed by dirty sheep (see section . . below). from a public health perspective, ante-mortem examination is of no value in the detection of toxoplasmosis in small ruminants, as animals infected with this previously identified hazard do not show clinical signs. despite the haccp-based intake procedures guaranteeing the health, welfare and cleanliness of animals going for slaughter, it is difficult to identify animals infected with pathogenic vtec and other enteric pathogens. supplying clean animals reduces, but does not prevent, the possibility of introducing this invisible hazard as infected animals are asymptomatic transient shedders (duffy, ) . post-mortem inspection of carcasses is designed to detect and withdraw from the food chain any carcass or part thereof that has grossly identifiable abnormalities that could affect its meat safety or wholesomeness. the meat inspector examines external and internal surfaces of the carcass and internal organs after evisceration for disease conditions and contamination that could make all or part of the carcass unfit for human consumption. generally, inspection procedures include mainly visual examination of the carcass and offal. these procedures are described in annex i, section iv, chapter ii of regulation (ec) no / . palpation is compulsory for liver, lungs and their lymph nodes. in addition, palpation is mandatory for the umbilical region and joints in young animals. incision is currently required only for the gastric surface of the liver. this procedure can be reduced to a visual-only inspection for sheep less than a year old or goats less than six months of age, if certain conditions are met, as stated in regulation (ec) no / , amending regulation (ec) no / , in the spirit of a risk-based inspection. it is unclear to what extent this derogation is currently used as intended. a more thorough examination, involving palpation and incision of other organs, is performed if abnormalities are detected during visual inspection. table in annex summarises these requirements for post-mortem inspection. ultimately, the production of safe food is the responsibility of the food business operator (fbo) as defined by regulation ( post-mortem inspection enables, to a certain extent, detection of lesions related to animal health and welfare, which are not dealt with in this part of the document (see appendix c). for food safety concerns, post-mortem examination can detect visibly contaminated carcasses and offal, which might present an increased food safety risk and is an indication of a hygienically inefficient slaughter process. post-mortem inspection allows for an assessment of the general health status of the animal to be carried out, which could influence the likelihood of important meat-borne hazards to be present in the carcass. classic zoonotic diseases, such as tuberculosis, which can be detected by post-mortem examination, are now controlled in many areas where modern systems of animal husbandry, disease control and animal health care were introduced. hence, the ability of current post-mortem inspection to detect lesions caused by mycobacteria is only relevant in regions where they are present. post-mortem inspection can also detect other non meat-borne hazards of public health significance that can be present in carcasses or offal from small ruminants. examples of these hazards are echinococcus granulosus and trematode parasites such as fasciola hepatica. acquisition of these parasites by humans occurs when subjects inadvertently swallow eggs or cysts attached to tainted vegetation or by drinking contaminated water containing free-floating eggs (e. granulosus) or cysts (f. hepatica) (fried and abruzzi, ) . from the public health standpoint, only e. granulosus is still relatively important in some mss (efsa and ecdc, b), while trematode parasites are less commonly reported in humans in the eu. meat inspection contributes to the monitoring of these parasites as they are routinely detected during post-mortem examination of sheep and goats. this also allows for appropriate disposal of infected organs, thus breaking the life cycle of the parasites. the extent to which meat inspection contributes to reducing the risk to human health posed by these parasites, compared with control measures elsewhere (e.g. anti-parasitic treatments of the final hosts) is not known, so it is difficult to assess the relative importance or effectiveness of this activity in protecting public health. nevertheless, the importance of meat inspection as a monitoring tool has been stressed previously (efsa, ). the slaughter of sheep involves greater challenges than the slaughter of cattle and pigs since the animal is relatively small and has a wool fleece increasing the risk of surface contamination at dehiding (buncic, ) . as mentioned in section . of this appendix, many challenges are posed by the processing procedure at the slaughterhouse, which has a direct effect on the final microbial disposition of the carcass (e.g. line speed, operational hygiene, equipment and training) (hansson, ; palmer, ) . in this context, the mandatory bacteriological analysis of carcasses to evaluate slaughter process hygiene is important. the maximum acceptable microbiological values are set in the phc for the indicators mentioned in regulation (ec) no / . some risks determined at postmortem examination are under the direct influence of the processor and can be ameliorated by corrective action procedures. in the case of the identified hazard, pathogenic vtec, post-mortem corrective actions may include clipping after stunning and bleeding, adjustments to operational hygiene practices, slowing the slaughter line down and/or adding extra personnel at certain carcass dressing stations, with feedback to producers (see section . . below). the competent authority also verifies the fbo's responsibility to produce safe food, as mandated by regulation (ec) no / , through audit and inspection of the slaughterhouse's food safety management system. in terms of the slaughter process, phc are end-product criteria. compliance with these criteria, in regulation (ec) no / , is one aspect of system compliance verification carried out by the competent authority. more details about phc can be found in section . . . . potential threats to public health associated with slaughtered sheep or goats including agents such as pathogenic vtec and t. gondii are carried by animals without signs or lesions. current meat inspection is not designed to detect or eliminate these agents. sometimes, cysts of t. gondii can be macroscopically visible but it is impossible to distinguish them from sarcocystis cysts, except cysts of s. ovifelis. visible meat quality-related abnormalities are detectable at post-mortem inspection, but these are not important for human health (see table ). sometimes, septicaemia and conditions associated with foci of infection in tissue such as arthritis, bronchopneumonia, mastitis, pleuritis or abscesses can be detectable at post-mortem inspection. some of these are caused by pathogens that might have zoonotic implications (e.g. erysipelothrix rhusiopathiae, s. aureus), but, as explained in section of this appendix, the risk to public health arising from these hazards is not considered to be important and is mostly related to occupational exposure or the way the meat is handled after it leaves the slaughterhouse. other conditions that result in condemnation of offal or carcasses are parasitic lesions. these parasites (c. tenuicollis, sarcocystis, fasciola, dicrocoelium, etc.) are not transmissible via meat consumption. in cases where these abnormalities are observed, the meat must be removed as unfit for human consumption on aesthetic or meat quality grounds. the potential for cross-contamination of carcasses exists whenever palpation and/or incision methods are used in the inspection process. palpation of the liver, the lungs, the umbilical region and the joints, and the incision of the gastric surface of the liver during the post-mortem examination of sheep and goats could contribute to the spread of the bacterial hazards of public health importance in small ruminants through cross-contamination. the importance of cross-contamination is not clear in small ruminants, although it has been considered important in other species (walker et al., ) . however, it should be borne in mind that incision is compulsory only for the liver, whereas in cattle and pigs incision of muscle is also required, so the level of contamination is likely to be smaller in small ruminants than in these species. current legislation foresees more detailed palpation and incision if abnormalities are detected during visual inspection. this could also facilitate cross-contamination of normal carcasses with microbiological hazards of public health importance. judgement of the fitness of meat for human consumption in current post-mortem inspection is based on the identification of "conditions making meat unfit for human consumption" but does not make a clear foodborne risk distinction between different subcategories i.e. between non-zoonotic conditions making meat unfit (inedible) on aesthetic/meat quality grounds (e.g. repulsive/unpleasant appearance or odour), non-zoonotic conditions making meat unfit in order to prevent spreading of animal diseases (e.g. foot and mouth disease), zoonotic conditions making meat unfit due to transmissibility to humans via the foodborne route (e.g. toxoplasmosis) and zoonotic conditions making meat unfit due to transmissibility via routes other than meat borne (e.g. brucellosis). the legislation on official controls on fresh meat from (regulation (ec) / , annex i) has a more horizontal approach than the former one (council directive no / , amended by council directives no / and no / and has also in theory a risk-based approach. however, the main experiences are that alternative control regimes, such as visual control of young animals (sheep of less than a year old and goats less than six months old) are not implemented due to the fact that the gains are limited due to: the threshold in terms of implementation of quality assurance systems and extra procedures at herd level is too high logistical challenges connected to the post-mortem meat inspection procedures as some flocks/herds are certified for visual control while others are not due to the fact that alternative control methods are not accepted by some importing countries outside the eu. ante-mortem and post-mortem inspections of sheep and goats enable the detection of observable abnormalities. in that context, they are an important activity for monitoring animal health and welfare. they provide a general assessment of animal/herd health, which if compromised may lead to a greater public health risk. visual inspection of live animals and carcasses can also detect animals heavily contaminated with faeces. such animals increase the risk for cross-contamination during slaughter and may consequently constitute a food safety risk if carrying hazards of public health importance. if such animals or carcasses are dealt with adequately, this risk can be reduced. visual detection of faecal contamination on carcasses can also be an indicator of slaughter hygiene, but other approaches to verify slaughter hygiene should be considered. post-mortem inspection can also detect non meat-borne hazards of public health significance that can be present in carcasses or offal from small ruminants. ante-mortem and post-mortem inspection also have the potential to detect new diseases if these have clinical signs, which may be of direct public health significance. currently, the use of food chain information for food safety purposes is limited for small ruminants, mainly because the data that it contains is very general and doesn't address specific hazards of public health importance. however, fci could serve as a valuable tool for risk management decisions and could be used for risk categorisation of farms or batches of animals. to achieve this, the system needs further development to include additional information important for food safety, including definition of appropriate and standardised indicators for the main public health hazards identified above (section of this appendix). ante-and post-mortem inspection is not able to detect any of the public health hazards identified as the main concerns for food safety. it would therefore be expected that more efficient procedures might be implemented to monitor the occurrence of non-visible hazards. in addition, given that the current postmortem procedures involve palpation and incision of some organs, there is potential for crosscontamination of carcasses. as identified by priority ranking earlier in this opinion, the principal biological hazards associated with meat from small ruminants are t. gondii and pathogenic vtec. the ranking presented in section of this appendix classified all other hazards in the low-risk category. this ranking is provisional because of the limited information available for some of the hazards. neither of the principal hazards identified can be detected by traditional meat inspection, which is focused on identification of visible abnormalities and issues relating to the health and welfare of the animals on the farm, in transit and at the slaughterhouse before slaughter. detection and quantification of those hazards in/on sheep or goats and their carcasses is possible only through laboratory testing. the occurrence and levels of t. gondii and pathogenic vtec on carcasses are highly variable depending on various factors, including particularly: (i) their occurrence in the sheep and goat population before slaughter and the application and the effectiveness of related pre-slaughter controls strategies; (ii) the extent of direct and/or indirect faecal cross-contamination during slaughter line operation (this does not apply to t. gondii); and (iii) the application and the effectiveness of possible interventions to eliminate/reduce them on carcasses (e.g. decontamination). therefore, as far as the presence of these pathogens in/on carcass meat is concerned, the risk reduction strategies, and related controls, are focused on these three aspects. changes are therefore necessary to identify and control microbiological hazards, and this can be most readily achieved by improved use of fci and interventions based on risk. control measures for pathogenic vtec at the slaughterhouse are also likely to be effective against other enteric pathogens, as they would all be controlled by addressing faecal contamination of carcasses. a comprehensive meat safety assurance system for meat from small ruminants, combining a range of preventive measures and controls applied both on the farm and at the slaughterhouse in a longitudinally integrated way, is the most effective approach to control the main hazards in the context of meat inspection. the main responsibility for such a system should be allocated to fbos, whereby compliance is to be verified by the competent authority. the setting up of such a comprehensive meat safety assurance system at eu level is dependent on the availability of reliable information on the biological risks associated with the consumption of meat from these species. as indicated in the priority ranking section of this opinion (section of this appendix), information on the biological risks associated with the consumption of meat from sheep or goats is sometimes scant and unreliable. consequently, in order to facilitate decision making, harmonised surveys are required to establish values for the prevalence of the main hazards t. gondii and pathogenic vtec at flock/herd, live animal and carcass level in individual mss. epidemiological and risk assessment studies are also required to determine the specific risk to public health associated with the consumption of meat from small ruminants. in the event that these studies confirm a high risk to public health through the consumption of meat from sheep or goats, consideration should be given to the setting of clear and measurable eu targets at the carcass level. eu targets to be reached at the national level are already in place for salmonella spp. in breeding flocks of gallus gallus and turkeys and production flocks of broilers, turkeys and laying hens. similar targets in primary production could also be considered for the main hazards of other species, including small ruminants. the use of specific hazard-based targets (i.e. pathogenic vtec or t. gondii related) for chilled carcasses provides: . measurable and transparent focus for the abattoir meat safety assurance system. ) on what has to be achieved at earlier steps in the food production chain. . information for the purpose of consumer exposure assessment for each hazard. . measurable aim for the meat industry in the context of global pathogen reduction programmes. for all these reasons, the chilled carcass targets have to be specific hazard based. this, however, may not be always practical (e.g. in very low hazard prevalence situations). therefore, proper functioning of meat safety quality assurance systems may not rely exclusively on hazard-based testing of the final carcass but on the general hygiene of the slaughter process. this issue is discussed further in the following sections. ). in addition, information on harmonised epidemiological indicators (heis) and related methodologies for the main hazards that can be used in studies to establish prevalence of the main pathogens to establish targets for carcasses and performance criteria for slaughterhouses, as well as targets for incoming small ruminant animals, is provided in the efsa report (efsa, ) . therefore, this opinion should be used in combination with that report. at farm level, the primary goal is reduction of risk for the main hazards, which can be achieved through preventive measures such as flock/herd health programmes, including biosecurity and good farming practices (gfps) that specifically address the hazards identified in section of this appendix. husbandry practices will vary considerably for small ruminants, particularly, the intensity of the rearing system. so, although it is not possible to detect any of the main foodborne zoonotic infections visually at the farm, there are known risk factors that are likely to increase the risk of infection with the main hazards. an important element of an integrated meat safety assurance system is considered to be risk categorisation of flocks or herds based on the use of farm descriptors and data on clinical disease and use of antimicrobials, in addition to data provided by ongoing monitoring of high-risk hazards that constitute the fci. such data could be provided through farm audits using heis to assess the risk and preventive factors for the flocks or herds related to each of the prioritised microbiological hazards (see efsa report, ( )). ongoing monitoring could be put in place for particular pathogens at eu level if, following the completion of the prevalence studies described earlier, these pathogens are identified as presenting a high risk. an assessment of the historical data over a time period could also be used for adjusting the sampling frequency of the main hazards in order to focus control efforts where the risk is highest. a structured approach to gathering more detailed farm information should become an additional farmrelated element of the fci that, in combination with the monitoring results for the main hazards, should form the basis for the risk categorisation of the farms. the frequency of monitoring in higher risk farms could be adapted in a cost-effective manner, e.g. there would be no need to sample every batch of animals to be slaughtered if the result is very likely to be "high risk" or "very low risk". thus, animals from higher risk farms could be systematically directed to, for example, logistic slaughter, or specific treatments such as decontamination at the slaughterhouse, until these high-risk farms demonstrated a decreased risk following the implementation of adequate on-farm measures. this system could act as an incentive for the primary producer to improve farm standards by means of reduced monitoring costs associated with low-risk status. at slaughterhouse level, the primary goal is risk reduction for the main hazards that can be achieved through integrated programmes based on gmp, ghp and haccp, including the use of phc: logistic slaughter based on the risk categorisation of the slaughtered batches; this could be slaughter of higher risk animals at the end of the day. hygienic practices and technology-based measures aimed at avoiding direct and indirect cross-contamination with the main hazards. interventions such as the scheduling of higher risk animals for carcass decontamination or for risk-reducing processes such as heat treatment to reduce pathogenic microorganisms or freezing-based treatments to eliminate parasites such as t. gondii. enteric pathogens are carried in the gastrointestinal tract and/or on the fleece of sheep and goats presented for slaughter, and carcass meat becomes contaminated as a result of direct or indirect crosscontamination that is highly dependent on slaughterhouse technology and the skills of the operators. technical aspects of individual steps of the slaughter process for small ruminants may vary considerably. the order of the processing steps at the slaughterhouse is generally as follows: transport/lairaging-stunning-bleeding-skinning-evisceration-chilling. each of these steps contributes differently to the final microbial load of the carcass. crosscontamination between animals can occur from transport and lairaging and during the slaughter process. contamination occurs particularly during skinning and evisceration. the slaughter of sheep involves greater challenges than the slaughter of cattle and pigs since the animal is relatively small and has wool. "during sheep de-pelting, it is difficult to achieve the low contamination rates capable of being achieved during cattle de-hiding, as the animal is smaller, the fleece is longer and there is a much greater chance of fleece inrolling and contacting the carcass. therefore, overall, de-skinning is a 'dirtier' procedure in small ruminants than in larger ones" (buncic, ) . chilling can help to control the numbers of pathogenic and spoilage microorganisms on carcasses. decontamination treatments for carcasses might be used to reduce the levels of enteric pathogens and can be divided into physical and chemical treatments. physical interventions include water-based treatments, irradiation, ultrasound or freezing. hot water, steam and irradiation effectively reduce the bacterial load. chemical interventions such as treatments with acetic and lactic acid reduce the bacterial load, as observed in poultry (loretz et al., ) . some combinations of treatments further enhance the reductions (loretz et al., ) . freezing and -irradiation can also be effective in eliminating t. gondii in carcasses. however, some of these methods are limited by their practicability, regulatory requirements or acceptability to consumers (acmsf, ) . thus, the best way to achieve reductions in carcass contamination is likely to come either from physical decontamination treatments or from technological developments in the process that are designed to improve hygiene, as long as they are acceptable to the industry and the consumer. each slaughterhouse can be viewed as unique, owing to differences in species slaughtered, logistics, processing practices, plant layout, equipment design and performance, standardised and documented procedures, personnel motivation and management, and other factors. these variations individually and in combination lead to between-slaughterhouse differences in risk-reduction capacity and, consequently, in the microbiological status of the final carcass. hansson ( ) indicated that there was a significantly greater amount of aerobic bacteria in ruminant carcasses slaughtered at lowcapacity slaughterhouses than in high-capacity slaughterhouses. this difference in carcass microbiological status can be accounted for by better separation of low-and high-risk areas, less variation in evisceration techniques, uniformity of the animals slaughtered, increased specialisation of labour and equipment, and improved measures taken to prevent contamination through effective operational hygiene practices in high-volume slaughter establishments (hogue et al., ; rahkio and korkeala, ) . consequently, a risk categorisation of slaughterhouses is also possible, based on the assessment of individual hygiene process performance. for that, a standardised methodology and criteria for the assessment of process hygiene is a prerequisite. in respect to process hygiene, differentiation of abattoirs in current eu regulation is based on the use of process hygiene criteria providing two categories: "acceptably" and "unacceptably" performing abattoirs. however, this differentiation is based solely on carcass testing, and so does not differentiate the abattoirs in terms of the processes but only the end products. more in-depth differentiation, even within each of the two global categories of abattoirs, would have been possible if improved process hygiene assessment methodology and indicators were used. the main guiding principle (koutsoumanis and sofos, ) in abattoir process hygiene differentiation is that abattoir phc need to address the initial level of a hazard and the reduction of that hazard during the production process. in the process of creating phc for abattoirs, the possibilities that need to be considered are whether they should be linked to individual stages of the process (e.g. reduction of occurrence/level of indicator organisms or hazards at a selected one or more specific steps along the slaughter line) or only related to the starting and the end point of the process (e.g. reduction of the occurrence/level in/on the final carcass meat compared with that in/on incoming animals). establishing the frequency of the official controls on the basis of pre-defined and objective criteria. carrying out the official controls using homogeneous criteria for plants with a comparable risk profile. these criteria take six parameters into account: food safety management systems can combine official control and supervision based on compulsory requirements prescribed by law (haccp, traceability, fci, etc.) , and private quality assurance schemes. besides those aspects included in the legislation, abattoirs can voluntarily implement their own quality requirements in the form of certification schemes. certification of production processes is based on the auditing and approval by accredited third-party organisations on an accredited standard. these schemes include official requirements but also pay attention to additional, more stringent, quality and safety aspects of the processes and products. at the slaughterhouse, standards are implemented for animal welfare and hygiene, slaughtering, dressing and evisceration, hygiene control, carcass quality and grading, storage conditions, carcass cutting and processing, etc. the adherence to certification schemes reassures stakeholders (suppliers, clients), government and consumers of the quality and safety of their products, with a view to meeting market demands and consumer satisfaction. retailers and manufacturers are increasingly demanding that their suppliers hold an approved certification. in this sense slaughterhouses are becoming increasingly important throughout supply chains in integrated food safety management systems. some examples of quality assurance schemes are the international standards organisation (iso) , food safety system certification (fssc) , international food standard (ifs), british retail consortium (brc) and globalgap. in summary, classification of abattoirs according to their capability to prevent or reduce faecal contamination of carcasses can be based on two elements: (i) the process hygiene as measured by the level of indicator organisms on the carcasses (i.e. process hygiene criteria); and (ii) the use of operational procedures and equipment that reduce faecal contamination (as described in section . . . above), as well as industry-led quality systems. information about the risk categorisation of slaughterhouses could be then considered with the fci when assessing the risk arising from incoming animals. herbivorous animals most likely contract t. gondii infection via ingestion of pasture, hay, forage, feed or surface water contaminated with oocysts shed by infected cats (skjerve et al., ; tenter et al., ) . oocysts are very resistant and can survive a range of temperatures in the environment. a continuous input of sporulated oocysts, originating from young infected cats, must be present to sustain the oocyst reservoir in the environment (kijlstra and jongert, ). the risk of environmental oocyst contamination can be addressed by using sterilised feed and bedding, and not allowing sheep and goats outdoor access; however, such husbandry practices are not economically viable in most eu commercial sheep and goat enterprises (kijlstra and jongert, ). removing cats from the farm surroundings, or vaccinating cats, could theoretically lead to a reduction of the oocyst load in the neighbourhood of the farm. in reality, most of these measures would not be practical to implement in most situations at the moment. (dubey, ; kijlstra and jongert, ) . moreover, the vaccine may revert to a pathogenic strain and is, therefore, not suitable for human use (hiszczynska-sawicka et al., ). an oral vaccine composed of live bradyzoites from an oocyst-negative mutant strain (t- ) has been effective in preventing oocyst shedding by cats in experimental trials but a vaccine for cats is not yet commercially available (innes et al., ) . while the s strain vaccine remains the only one commercially available, there has been significant progress over the last years in the development of vaccines against toxoplasmosis due to technological advances in molecular biology (kur et al., ) . a cocktail dna vaccine has been shown to prime the immune system of animals against toxoplasmosis with increased immune responses being observed after experimental challenge (hoseinian khosroshahi et al., ) . in principle, an effective recombinant vaccine against both sexual and asexual stages of the parasite should be able to address all three targets listed above, but this is hampered by stage-specific expression of t. gondii proteins (jongert et al., ) . for this reason, the development of vaccines that prevent t. gondii infection in ruminants and/or cats is recommended. surveillance and monitoring of t. gondii in animals preharvest is essential in the control of this parasite, something that is currently not addressed effectively within the eu (efsa, b; opsteegh et al., b) . the most feasible surveillance method is the use of indirect serological tests (e.g. enzyme-linked immunosorbent assay, elisa) on live sheep and goats for the detection of t. gondii antibodies, as seropositivity has been correlated with tissue cyst presence in non-vaccinated animals (buxton, ; conde de felipe et al., ; dubey, ; opsteegh et al., b) . however, a more practical solution is taking a blood sample during bleeding of the animal at the slaughterhouse, or even freezing a piece of meat and collecting the meat juice during thawing. studies have indicated regional differences in seroprevalence in small ruminants which can be accounted for by differences in environmental contamination or by factors that influence the level of exposure of sheep to the environment, such as age and farm management (alvarado-esquivel et al., ; opsteegh et al., b) . monitoring programmes could help in the risk assessment and categorisation of small ruminants with regard to t. gondii at the slaughterhouse as part of the fci provided. for more details on the different options for indicators of the presence of t. gondii we refer the reader to the technical specifications on harmonised epidemiological indicators for biological hazards to be covered by meat inspection of small ruminants (efsa, ). with this background of high t. gondii prevalence in the national flocks and herds of small ruminants, a more realistic approach could be to focus the efforts in setting up a system to identify negative flocks/herds instead. for example, animals raised exclusively indoors and under controlled husbandry conditions (which would need to include for example the exclusion of cats from the farms and the absence of contamination of feed, bedding and water with t. gondii oocysts) would present a much lower risk with regards to t. gondii. when these husbandry conditions are combined with serological testing and the selection of young animals for slaughter, the production of t. gondii-free meat should be a feasible goal. this meat could be then used for either subpopulations at greater risk (e.g. pregnant women or immunocompromised people), or for the elaboration of particular dishes that require little cooking of the meat (e.g. agneau rosé in france). at the moment, this system might be practical to implement only for some intensive farms dedicated to milk or cheese production in some mss. a more detailed explanation about how harmonised epidemiological indicators could help setting up this system is provided in the accompanying report on these indicators mentioned above (efsa, ). there is no way to distinguish t. gondii-infected meat carcasses from uninfected carcasses during meat inspection (dubey et al., ) . similarly, current process hygiene criteria do not address the risk arising from this hazard (or any non-enteric hazard). the presence of t. gondii tissue cysts can be determined only by laboratory methods, particularly by using serological methods. this can be done on farm or at the slaughterhouse. studies on pcr methods to detect and quantify t. gondii in meat samples have shown promise with detection sensitivities comparable to those of bioassay (opsteegh et al., a) . studies using such laboratory techniques allow epidemiological studies to be conducted to determine the seroprevalence of toxoplasmosis in ovine meat and the risks such meat poses to human health (efsa, b; opsteegh et al., b) . additional information on sampling and testing methodologies to detect t. gondii can be found in the efsa report on harmonised epidemiological indicators for sheep and goats ( ). given that t. gondii cannot be horizontally transmitted between ruminants, there is no issue of between-animal cross-contamination with t. gondii at slaughter, and therefore separating sheep and goats from negative and positive flocks or herds during transport, lairage and on the slaughter line would not have any impact on the levels of t. gondii. studies have indicated that t. gondii tissue cysts in meat are susceptible to various physical procedures that can take place at the abattoir or beyond. these include heat treatment, freezing, irradiation, high pressure treatment and curing (addition of salt combined with drying) (table ). heat treatment is the most secure method to inactivate the parasite; however, freezing would probably be the most practical risk management option to control t. gondii for the meat industry (kijlstra and jongert, ). most of the information available for these treatments originates from research in pigs, so further research is required to validate these treatments in meat from small ruminants. these treatments would be particularly appropriate for meat cuts that are intended to be consumed rare. the ranking presented in section of this appendix classified pathogenic vtec as high risk. however, it is important to note that measures aimed at controlling this hazard will also probably be effective in reducing the level of other enteric pathogens such as salmonella spp. or campylobacter spp. control of pathogenic vtec at farm level is complicated by the fact that animals are asymptomatic carriers of these organisms, thus without an active monitoring programme there is no way of knowing which animals are infected and/or shedding at any given time. control activities must therefore be directed at the flock or herd. good management practices such as maintaining stable rearing groups, keeping a closed herd and preventing young animals from having contact with older animals all decrease the spread of vtec on and between farms. a number of studies have reported reductions in bacterial contamination and, in particular, in e. coli o :h levels on carcasses by reducing the level of fleece/hide contamination (hadley et al., ; longstreeth and udall, ) . in this context, the provision of a dry lying area for sheep improves hygiene. in outdoor rearing systems, this is achieved by access to sheltered free-draining land, avoiding access to wet or boggy areas. the housed rearing environment is more easily controlled by the producer. the shelter provided, in addition to the effect of good-quality bedding and the ability to influence access to food/water in the housed system, result in pre-slaughter housing being recommended as a clean fleece policy control measure (food standards agency, b) . other husbandry practices such as internal parasite control, effective mineral supplementation, regular dagging/crutching and the planned pre-slaughter preparation by the producer can have an impact on the on-farm clean sheep policy (food standards agency, b; pugh and baird, ) . although no such information is available for goats, it is probably safe to assume that these principles would also work in this species. controlling diet and feeding before slaughter to minimise digestive upset is essential in ensuring that animals are clean prior to slaughter. the provision of a high-fibre, nutritionally balanced diet, with easily digestible protein, helps develop good faecal consistency (collis et al., ; pugh and baird, ) . lush grass, contaminated water sources, overfertilised grassland, excessive concentrate supplementation and root/forage crop consumption prior to slaughter are dietary causes of fleece contamination (food standards agency, b ). in addition, in a recent review, pointon et al., ( ), considered the impact of pre-slaughter feed curfews of cattle, sheep and goats on food safety and carcass hygiene in australia. the authors examined the ecology of salmonella spp. and e. coli and the efficacy of on-farm withholding of feed, carried out to reduce soiling during transport, in terms of microbial reduction. they suggested that, to minimise carcass contamination with salmonella spp. and generic e. coli, the animals should be fasted before transport only for a period sufficient to complete faecal expulsion, i.e. hours, but not exceeding hours, and they concluded that the implementation of these practices as good agricultural practice is likely to improve the effectiveness in terms of reducing pathogens on the carcasses. good management of animal waste is also essential to prevent spread and cross-infection of other animals. animal waste from animals housed indoors generally accumulates as slurry or farmyard manure. vtec survive for extended periods in faecal, slurry, soil and water environments (besser et al., ; bolton et al., ; bolton et al., ; fremaux et al., ; himathongkham et al., ; hutchison et al., a; hutchison et al., b; islam et al., ; mcgee et al., ; o'neill et al., ) . current control measures to reduce the pathogen risk in animal waste can be applied before, during or after spreading manure. pre-spreading controls include the provision of proper storage facilities for animal waste to prevent leakage of waste into ground water, and keeping animals away from slurry pits or dung heaps. spreading should not take place in conditions where contamination of a water course is more likely to occur. after manure is spread, the land should not be used for grazing for a certain amount of time (at least one month or until all visible signs of animal waste have disappeared in the case of grazing (hutchison et al., ) . despite there being a range of on-farm measures to control vtec at farm level, the efficacy of such measures in reducing the prevalence (or load) of pathogenic vtec in small ruminants is not clear. transport has also been identified as a risk factor for hide cleanliness (animalia, ; byrne et al., ; food standards agency, b) . in compliance with council regulation (ec) no / on the protection of animals during transport and related operations, livestock should be carried in wellventilated, clean vehicles, at the correct stocking density, with the provision of shelter, bedding and access to food and water where appropriate. these measures, particularly relating to vehicle facilities, design and journey distances directly affect fleece/pelt cleanliness. industry standards on stocking densities during transport and lairage also facilitate the requirements of clean livestock policies (anonymous, ; minihan et al., ) . section . . above indicated that categorisation of flocks or herds according to risk can be an important element of an integrated meat safety assurance system. however, for pathogenic vtec there are a number of challenges that need to be overcome for this approach to be feasible, including the difficulties in identifying husbandry factors that can be used to classify farms according to pathogenic vtec risk, the intermittent nature of shedding, and the problems with the interpretation of monitoring results for vtec due to the difficulty of correctly identifying pathogenic vtec. the two main sources of enteric bacteria on sheep and goat carcasses are the fleece/hide and the viscera, but contamination from the former is more common. a number of studies have established a relationship between the dirtiness of sheep at the time of slaughter and the amount of contamination, and therefore the amount of pathogenic bacteria transferred to the carcass during skinning (duffy et al., ; gerrand, ; hauge et al., a; longstreeth and udall, ) . this relationship is addressed by legislation at the production and processing level, within the hygiene package, as previously mentioned in section . . . to meet this requirement for clean fleece/hides, some mss have adopted formalised "clean livestock policies" to categorise livestock including sheep and goats at ante-mortem examination, thereby placing the responsibility of presenting clean animals for slaughter with the producer and the processor (byrne et al., ; hauge et al., a) . pre-slaughter washing of sheep is widely used in new zealand (biss and hathaway, ) , together with routine shearing at high-risk sites. the clean livestock policy adopted by the food standards agency in the united kingdom has had considerable success in meeting the requirements of the hygiene package. it is based on a visual inspection during unloading or lairaging and the categorisation of the animals as acceptable for slaughter, acceptable for slaughter following shearing or clipping (conducted at the primary producers expense), and unsuitable for slaughter. extra time spent in lairage, clipping, subsequent reduction in slaughter line speed, separate processing or excessive trimming and rejection of animals all incur additional costs to producers and processors (food standards agency, b) . similarly, in , the norwegian meat industry also adopted national guidelines for good hygiene slaughter practices regarding the categorisation of fleece cleanliness for sheep (hauge et al., a) . the policy coordinators, in both the united kingdom and norway, communicate the risk of contaminated sheep to the sheep producer, suggesting various husbandry practices, handling methods and pre-slaughter preparation to limit contamination prior to slaughter (animalia, ; food standards agency, b) . as part of the norwegian clean sheep policy, developed by the associations of producers and slaughterers, sheep are shorn in the slaughterhouses. if they do not become visually clean after shearing or they are already shorn on farm and are contaminated after shearing, the carcasses of these animals are processed in a separate line. this separate processing may include heat treatment of meat products and processing into a restricted range of products, with the farmers receiving a lower price (a reduction of - % in the carcass value). hauge et al. ( a) demonstrated that the measures taken as part of the norwegian policy decreased the risks posed by carcasses and thereby validated the use of such clean sheep policies. the influence of animal cleanliness on small ruminant carcass contamination was investigated by several authors in australia, canada, new zealand, ireland and norway (biss and hathaway, a , b , c duffy et al., ; gill, ; hauge et al., a; sumner et al., ) , but there is contradictory evidence on the impact of measures to improve fleece cleanliness on microbiological contamination of lamb carcasses. roberts ( ) and field et al. ( ) found no effect of shearing of sheep on carcass contamination. some more recent studies have reported that shearing of sheep decreased carcass surface bacterial counts (biss and hathaway, a; collis et al., ; schroder and ring, ) . in a study carried out in norway by hauge et al. ( a) a significantly lower level of aerobic plate count (apc) and e. coli was found on carcass surfaces from shorn lambs when compared with unshorn lambs at skinning. at this sampling point, shearing proved to be effective for reducing microbial loads on carcasses. results in this study showed a trend of increasing contamination of carcasses with increasing duration of the time between shearing and slaughter. sheep shorn immediately before slaughter yielded carcasses with the lowest microbial loads with respect to apc. the e. coli results were less definitive, but a similar trend was demonstrated. biss and hathaway ( b), investigating the effect of pre-slaughter washing of lambs on the microbiological and visible contamination of the carcasses at four slaughterhouses in new zealand, showed that total aerobic bacteria and e. coli contamination was greater on carcasses that had been washed before slaughter, irrespective of wool length, and it was generally higher on carcasses derived from woolly lambs than those derived from shorn lambs. other researchers have found that pre-slaughter washing of sheep will only have positive effects if the washed animals are allowed sufficient time to dry before they are slaughtered (newton et al., ; patterson and gibbs, ) . many studies have reported difficulties in making valid microbiological comparisons associated with differences in slaughter hygiene due to individual operators, uneven distribution of microorganisms on carcasses, variations between groups of animals, day-to-day variations, and seasonal variations (biss and hathaway, b; hauge et al., a; ingram and roberts, ) . this may explain the conflicting results obtained in relation to the effect of shearing or washing on carcass contamination in such studies. irrespective of this conflicting evidence about how to best ensure that incoming animals are clean, it seems necessary to continue accepting only clean animals for slaughter as currently required by eu legislation, as it can be assumed that the dirtier the animals are in terms of faecal material, the higher the risk of cross-contamination of the slaughterline environment, including the carcasses. as mentioned above, a second source of enteric bacteria on carcasses are the viscera. during carcass dressing, bacteria are transferred from the gastrointestinal tract to the carcass directly by contact with gut spillage or indirectly via contaminated hands, knives, other equipment and the air. in general, prerequisite gmp and ghp implemented to reduce bacterial contamination will also prevent or reduce carcass contamination with pathogenic vtec, salmonella spp. and other pathogens. during evisceration, the abdominal cavity is opened using a knife and the connective tissue joining the bung and the viscera to the carcass is cut. rodding (sealing the oesophagus with a crocodile clip, plastic ring or starch cone) may be performed to prevent leakage. the spread of faecal material from the rectum can be prevented or reduced by bagging and tying the bung. the current throat sticking practice in halal slaughter (cutting of blood vessels, oesophagus and trachea) limits the effect of rodding as the leakage from the oesophagus occurs before rodding can be applied. if a sticking method such as chest sticking is applied, the effect of rodding will be greater as the oesophagus remains intact, with reduced leakage from the oesophagus until rodding is performed as one of the first steps after bleeding. using this method, contamination from the oesophagus of wool, skinned surfaces and the abdominal and chest cavity, in addition to the operator's hands, equipment, walls and floor, will be avoided to a high degree. the effect of bagging on the level of e. coli on sheep carcasses has been previously investigated (norwegian scientific committee for food safety, ). although the numbers of carcasses were limited, based on relevant -cm sampling sites (circum-anal incision and pelvic duct), it could be concluded that the use of the plastic bag technique during circum-anal incision and removal of the rectum results in a to log reduction in e. coli (figure ). if a plastic bag is not used and the rectum is inserted in the abdomen, the chances of contamination are larger. the hygienic effect of rodding and bagging will depend on the operator's experience at these critical hygienic positions. skinning and evisceration may also be designated as critical control points (ccps) as part of the haccp programme, the critical limit for both being zero visible faecal contamination on the carcasses. monitoring occurs at the trimming stand where every carcass is visually inspected. this inspection may be facilitated using the online monitoring system described by tergney and bolton ( ) . when faeces or faecal stains are detected they are immediately removed by trimming. the cause of the breach in hygiene should also be investigated, and secondary corrective actions require retraining of personnel, replacement of knives, etc. setting and using indicators/criteria for "process hygiene" of slaughterhouses is an integral part of the meat safety assurance system, which targets specifically contamination of the carcasses with enteric pathogens. according to the regulation on microbiological criteria, a microbiological criterion means a criterion defining the acceptability of a product, a batch of foodstuffs or a process, based on the absence, presence or number of microorganisms, and/or on the quantity of their toxins/metabolites, per unit(s) of mass, volume, area or batch. phc included in regulation (ec) no / are defined as criteria indicating the acceptable functioning of the production process. they give guidance on the acceptable implementation of pre-requisite programmes (gmp/ghp) and haccp-based systems to ensure hygienic functioning of slaughterhouse processes and are applicable only to the product at the end of the manufacturing process (final carcass after dressing but before chilling), and not to products placed on the market. in eu countries, phc involve the evaluation of indicators of overall contamination (total viable count of bacteria), indicators of contamination of enteric origin (enterobacteriaceae) and salmonella spp. prevalence. bacteriological analysis of carcasses, as outlined in this regulation, is carried out by the fbo. it involves pooled samples from four risk-assessed sampling sites on each of five sampled carcasses. this must be carried out weekly or, depending on the previous results, once a fortnight. phc set an indicative microbial contamination value above which corrective actions are required by the fbo in order to maintain the hygiene of the process in compliance with eu food law. these corrective actions should include the improvement of slaughter hygiene and the review of process controls. the phc communicate the expected outcome of a process, but they neither characterise nor differentiate between the processes themselves. process compliance must be verified by audits of haccp plans and inspections of processing procedures. the competent authority carries out this role on behalf of the member state as defined by regulation (ec) no / . as phc verifies the hygienic functioning of the process rather than the safety of the product, it does not require validation by independent sampling on behalf of the competent authority. microbiological testing alone may convey a false sense of security due to the statistical limitations of sampling plans, particularly in the cases where the hazard presents an unacceptable risk at low concentrations and/or low and variable prevalences. in addition, for pathogens other than enteric hazards (e.g. t. gondii), phc does not provide any information about risk. sampling and testing, as required by regulation (ec) no / , is only part of the verification process of systems in place. these criteria should not be considered without other aspects of eu food legislation, in particular haccp principles and official controls to fbos' compliance (efsa, c) . with current eu legislation, one element of the phc indicates the maximum acceptable prevalence of salmonella spp. on carcasses at the end of the slaughter line. the inclusion of this pathogen as a process hygiene criterion for carcasses highlights the importance of salmonella spp. as a foodborne pathogen in the eu and the need for good hygiene measures for controlling it in the abattoir. however, the use of this hazard presents some problems, because the salmonella spp. occurrence on carcasses depends not only on process hygiene performance of a given abattoir, but also on the salmonella spp. carriage by incoming animals (or lack of it). hence, when slaughtering batches that are salmonella spp. free or that have a low prevalence, such phc will be satisfied even if the actual process hygiene is inadequate-and vice versa. on the other hand, the current eu salmonella-based process hygiene criterion partly has the nature of a salmonella-related target to be achieved by abattoirs. the important difference is that with the current eu phc the hazard is measured on the carcass before chilling, while with the target-based concept the hazard is measured on the chilled carcass (i.e. just before dispatch onwards to the meat chain). however, the chilled carcass is better suited for assessing consumer exposure, and for the hazard-related target concept, as the prevalence/levels of microbial hazards on the carcass may change during chilling. furthermore, these current salmonella-related eu criteria for chilled carcasses are not clearly linked to other salmonellarelated criteria/targets at preceding and/or consecutive steps of the meat chain. in addition, current eu-legislated phc for abattoirs actually do not provide information on ratios between initial contamination associated with incoming animals versus final contamination associated with carcasses, i.e. on the actual capacity of the process to reduce the incoming contamination, but only on the process outcomes. when the main purpose is to microbiologically characterise the abattoir process itself, which is the subject of this subsection and a prerequisite for related differentiation of abattoirs, this is a significant weakness of the current eu-legislated phc. these shortcomings could be addressed by the setting of specific targets for pathogenic vtec, as described in section . above, instead of using salmonella spp. in phc. in addition, to measure the performance of the slaughter line, phc based on indicator organisms should be implemented, measuring microbial loads in at least two stages of the processing line. this would allow determination of the ratio between indicator organisms on pre-chill carcasses and those found in incoming animals, for a given batch. the phc is considered to be a key component of the proposed meat safety assurance system so, in that context, careful consideration would need to be given to issues such as the number of samples taken per week, the areas where those samples are taken from (both in carcasses and incoming animals) and the need for regulatory auditing of the process hygiene assessment (which may include microbial testing, as well as record verification). this more accurate information based on trends of data derived from process hygiene assessments and from haccp programmes would enable differentiation ("risk categorisation") of abattoirs with respect to pathogenic vtec which, in turn, would enable different risk management options for different risk categories of abattoirs to be used, including: optimisation of balancing pathogenic vtec risk categories of small ruminants with risk categories of abattoirs where they are to be slaughtered. optimisation of the decision whether/where additional pathogenic vtec risk-reducing interventions are to be applied (e.g. carcass decontamination step). more stringent requirements for monitoring/verification/auditing programmes for higher risk abattoirs. more reliable feedback to the farm of origin on the root of problems with pathogenic vtec on carcasses of small ruminants. clearer identification of slaughterhouses where improvement of the slaughtering practices and/or technology is needed. small ruminants represent a reservoir of enteric pathogens. in that context, the slaughtering of sheep involves greater challenges because the animal is relatively small and has a wool fleece, thus increasing the risk of surface contamination at dehiding (buncic, ) , which might result in suboptimal hygiene during slaughtering compared with the slaughtering of cattle. technological and operational shortcomings, such as a too high line speed, no rodding and bagging and the use of seasonal workers not sufficiently trained for the purpose, are reported as additional challenges in some abattoirs (norwegian scientific committee for food safety, ). accordingly, interventions such as surface pasteurisation using hot water might be considered as one of several options to reduce the bacterial contamination on carcasses. hot water at - °c achieves a . to . log reduction in colony-forming units (cfus)/cm in salmonella spp. on beef carcasses (arthur et al., ; cutter and rivera-betancourt, ) . in a study by hauge et al. ( b) lamb carcasses were subjected to hot water pasteurisation at °c for eight seconds. the reduction in e. coli just after pasteurisation was . %, corresponding to . log cfus/cm , and after hours' storage . log cfus/cm . accordingly, surface pasteurisation of sheep carcasses might represent an important and efficient step (ccp) to reduce vtec on the carcasses and the risk of disease among consumers. an automatic surface pasteurisation step is easy to control by measurement of time/temperature, and these results, together with the quality of the process water, might be documented on display and/or on hard copy. the pasteurisation step might be recognised as a ccp in a haccp concept. steam treatment is also allowed in the eu and has been found to reduce bacterial contamination in sheep carcasses by a log cfus/cm , both in enterobacteriaceae (milios et al., ) and aerobic plate counts (james et al., ) . greater reductions of up to . log cfus/cm have been described when using a combination of steam and a hot water wash in sheep carcasses (dorsa et al., ) . similar effects have been observed with salmonella spp. counts in beef carcasses, achieving reductions of < . to . log cfus/cm (phebus et al., ; retzlaff et al., ) . surface pasteurisation can also be achieved by manual steam vacuum technology. however, the use of this technology depends on skilled and responsible operators, and will require close supervision in order to ensure the pasteurisation procedure is correctly applied to the whole carcass. the use of manual steam vacuum was evaluated in a norwegian slaughterhouse, showing a real reduction (median of . log cfus/cm )(hassan, ). surface pasteurisation of ruminant carcasses is an option that allows dealing with carcasses presenting greater risk, such as emergency slaughtered carcasses or unclean carcasses, without the need, for example, to apply a heat treatment on these carcasses. although not permitted in the eu, a range of specific interventions are applied in us slaughter plants targeting enteric pathogens such as pathogenic vtec and salmonella spp. these include the application of organic acids. the application of acetic acid to beef carcasses will reduce e. coli counts by . - . log cfus/cm (sofos and smith, ) . significant reductions achieved with lactic acid have also been described (efsa panel on biological hazards, ). as neither of the main public health hazards associated with meat from small ruminants can be detected by traditional meat inspection, other approaches are necessary to identify and control these microbiological hazards. a comprehensive meat safety assurance system for meat from small ruminants, combining a range of preventive measures and controls applied both on the farm and at the slaughterhouse in a longitudinally integrated way, is the most effective approach to control the main hazards in the context of meat inspection. information on the biological risks associated with the consumption of meat from sheep or goats is sometimes scant and unreliable. in order to facilitate decision making, harmonised surveys are required to establish values for the prevalence of the main hazards t. gondii and pathogenic vtec at flock/herd, live animal and carcass level in individual mss. epidemiological and risk assessment studies are also required to determine the specific risk to public health associated with the consumption of meat from small ruminants. in the event that these studies confirm a high risk to public health through the consumption of meat from sheep or goats, consideration should be given to the setting of clear and measurable eu targets at the carcass level. to meet these targets and criteria, a variety of control options for the main hazards are available, at both farm and abattoir level. flock/herd categorisation according to the risk posed by the main hazards is considered an important element of an integrated meat safety assurance system. this should be based on the use of farm descriptors and historical data in addition to batch-specific information. farm-related data could be provided through farm audits using heis to assess the risk and protective factors for the flocks/herds related to the given hazards. classification of abattoirs according to their capability to prevent or reduce faecal contamination of carcasses can be based on two elements: (i) the process hygiene as measured by the level of indicator organisms on the carcasses (i.e. phc); and (ii) the use of operational procedures and equipment that reduce faecal contamination, as well as industry-led quality assurance systems. as mentioned in section . , further studies are necessary to determine with more certainty the risk of acquiring t. gondii through consumption of meat from small ruminants. in addition, the lack of tests that can easily identify viable cysts in meat is a significant drawback. furthermore, if there is a high prevalence in the animal population, this will hamper the development of systems based on risk categorisation of animals. for these reasons, the setting of targets for t. gondii is not recommended at the moment. there are a variety of animal husbandry measures that can be used to control t. gondii on sheep and goat farms but at present it would not be practical to implement them on most farms. a number of post-processing interventions might be effective in inactivating t. gondii such as cooking, freezing, curing and high-pressure and -irradiation treatments. however, most of the information available for these treatments originates from research in pigs, so further research is required to validate these treatments in meat from small ruminants. there are also a variety of animal husbandry measures that can be used to reduce the levels of vtec on infected farms, but their efficacy is not clear in small ruminants. in addition, there are a number of challenges that need to be overcome regarding the setting of targets for pathogenic vtec, including the difficulties in identifying husbandry factors that can be used to classify farms according to pathogenic vtec risk, the intermittent nature of shedding, and the problems with the interpretation of monitoring results for vtec due to the difficulty of correctly identifying pathogenic vtec. the two main sources of vtec on sheep and goat carcasses are the fleece/hide and the viscera. to control faecal contamination from the fleece or hide only clean animals should be accepted for slaughter, as currently required by eu legislation. there are also a number of measures that can help to reduce the spillage or leakage of digestive contents onto the carcass, particularly rodding of the oesophagus and bagging of the rectum. post-processing interventions to control pathogenic vtec are also available. these include hot water and steam pasteurisation. risk categorisation of slaughterhouses should be based on trends of data derived from process hygiene assessments and from hazard analysis critical control point programmes. improvement of slaughter hygiene through technological and managerial interventions should be sought in slaughterhouses with repeatedly unsatisfactory performance. source attribution studies are needed to determine the relative importance of meat, as well as to ascertain the role of the different livestock species as a source of t. gondii and pathogenic vtec. methods should be developed to estimate the amount of viable t. gondii tissue cysts in meat, especially in meat cuts that are commonly consumed. recommend adaptation of inspection methods that provide an equivalent protection for current hazards the main rationale behind the concept of fci is that animals sent for slaughter can be categorised into different risk groups based on relevant information from the flock/herd of origin. this enables appropriate measures to be put in place during slaughter to deal with the level of risk identified. although regulation (ec) no / mentions the basic requirements for fci, these are very general and as a consequence the information reported in fci is rarely used as described above (section . . of this appendix). there are a number of ways in which fci could be improved. as explained in section . . above, more specific information about the main hazards could be used for assessing the risks associated with batches of animals arriving at the slaughterhouse, resulting in a classification according to these risks. to achieve this, the system needs further development to include additional information important for food safety, including the definition of appropriate and standardised indicators for the main public health hazards identified in section of this appendix. in addition, membership of quality assurance schemes and certification systems can have a positive impact on public health by contributing to the overall health of the animals sent to slaughter. certification procedures at farm level are voluntary tools to ensure compliance with given standards and regulations in the quality assurance system. they are aimed at achieving continuous improvement in production standards by monitoring quality assurance standards or criteria. audits or inspections of farms ensure that the animal (final product) is being raised and handled in accordance with the standards or guidelines, which producers should adhere to. the main areas covered by the standards include usually animal health, welfare and hygiene, identification and traceability, adequate and prudent use of medicines and chemicals at farm level, safety of feed and water, environmental guidelines, hygiene of personnel, processes and infrastructure, and preparation of animals for slaughter. the standards should be regularly updated in line with changes in legislation and with scientific developments. certifications are issued by independent agencies or bodies which confirm that an auditing process has been passed. farmers can also adopt other schemes such as the guides to good farming practices, recommendations of best practice published by international bodies (i.e. world organisation for animal health (oie) and the food and agriculture organization of the united nations (fao)). adherence to such quality schemes and guidelines at farm level has multiple benefits, providing slaughterhouse operators with useful information about animals intended to be slaughtered and could be integrated in the fci. it also increases farmers' responsibilities and has a beneficial influence on meat safety and quality. schemes such as the beef and lamb quality assurance scheme in ireland cover a broad area, relating to animal identification and animal health and welfare, and contribute to ensuring that healthy animals enter the slaughterhouse. farmers should be encouraged to participate in these schemes, and information on whether or not a primary producer is a member should be included in the fci. eu regulations (ec) no / and (ec) no / already require that information gathered during meat inspection is fed back to the primary producer. the main value of such feedback relates to animal health and welfare and production-related diseases, such as liver fluke and pneumonia. however, as mentioned previously, use of this information to produce healthier animals would have indirect benefits for public health. from discussions with stakeholders, it is clear that feedback to the producers is very limited in most mss and that there is considerable room for improvement in that area (see the report from the technical hearing on meat inspection of small ruminants ). ante-mortem inspection assesses the general health status of the animals on arrival at the slaughterhouse. meat for human consumption should be derived from the slaughter of healthy animals. inspection of animals on arrival at the slaughterhouse will help to enforce acceptable standards of transport and handling. this might indirectly contribute to the maintenance of operating standards that minimise the general risk associated with unhygienic and stressful management of food-producing animals. stress has been shown to be an important factor in the excretion of enteric pathogens such as pathogenic vtec, salmonella spp. and campylobacter spp., so inspection procedures that prevent stress are likely to be beneficial (efsa panel on biological hazards, b). measures to keep the transport-lairaging period as short as possible may be beneficial in terms of reducing crosscontamination with these enteric pathogens. the ante-mortem procedure will help to detect animals heavily contaminated with faeces and other material. measures to prevent excessively dirty animals from entering the slaughter line will help to prevent contamination of the carcasses and may reduce the level of enteric pathogens. taking these factors into consideration, and given that current methods do not increase the microbiological risk to public health and have considerable benefits in relation to the monitoring of animal health and welfare, no adaptations for the existing visual ante-mortem inspection are proposed. in the inspection procedure for sheep and goats, as set out in eu regulation (ec) no / , carcasses are subject to visual inspection only. incision is mandatory for the gastric surface of the liver. palpation is mandatory for the lungs, bronchial and mediastinal lymph nodes, the liver and its lymph nodes. in addition, palpation is mandatory for the umbilical region and joints of young animals. palpation of lungs, liver, umbilical region and joints, and incision of the liver could contribute to the spread of bacterial hazards through cross-contamination. although the importance of such crosscontamination has not been studied in small ruminants, it has been considered important in other species ( the pathogens of importance for public health cannot be detected by routine post-mortem examination. consequently, palpation of liver, lungs, the umbilical region and joints and incision of the gastric surface of the liver do not contribute to preventing the risk to public health arising from the meat-borne hazards identified in this opinion. for these reasons, palpation and incision should be omitted in animals subjected to routine slaughter. visual examination contributes by detecting visible faecal contamination and/or spilled intestinal contents, although it is unclear how sensitive the current system is or what contribution this detection makes towards preventing public health risk. the current legislation foresees palpation and incision if abnormalities are detected during visual inspection. it is recommended that these procedures, if necessary, are carried out separately from the routine inspection of carcasses to prevent cross-contamination. elimination of abnormalities on aesthetic/meat quality grounds can be ensured through a meat quality assurance system and not through the official meat safety assurance system including meat inspection. any handling should be performed on a separate line and accompanied by laboratory testing as required. palpation and incision currently assist in the identification of zoonootic pathogens that are not meat borne, such as echinococcus granulosus, fasciola hepatica (although cysts are usually visible before incisions are made) and mycobacterium bovis. the removal of palpation and incision as a requirement in the post-mortem procedure in small ruminants could have a significant effect on monitoring echinococcus, in particular, as meat inspection is the principal method of detection of this pathogen (efsa, ) . in countries where hazards such as echinococcus are present it might be appropriate to conduct a risk assessment to evaluate the benefits to public health of stopping cross-contamination through palpation and incision of viscera with those obtained through monitoring of these non-meatborne zoonotic hazards. fci can be improved by including information on participation in quality assurance schemes and by giving greater feedback to the primary producer, as this would probably result in the production of healthier animals. ante-mortem inspection assesses the general health status of the animals and helps to detect animals heavily contaminated with faeces on arrival at the slaughterhouse. taking these factors into consideration, and given that current methods do not increase the microbiological risk to public health, no adaptations to the existing visual ante-mortem inspection procedure are required. although visual examination contributes by detecting visible faecal contamination, routine postmortem examination cannot detect the meat-borne pathogens of public health importance. palpation of the lungs, the liver, the umbilical region and the joints and incision of the liver could contribute to the spread of bacterial hazards through cross-contamination. for these reasons, palpation and incision should be omitted in animals subjected to routine slaughter. the effect of this omission on the risk posed by non-meat-borne zoonoses such as e. granulosus, f. hepatica and m. bovis should be assessed, particularly in those countries where these hazards are prevalent. conclusions tor identify and rank the main risks for public health that should be addressed by meat inspection at eu level. general (e.g. sepsis, abscesses) and specific biological risks as well as chemical risks (e.g. residues of veterinary drugs and contaminants) should be considered. differentiation may be made according to production system and age of animals (e.g. breeding compared to fattening animals). based on the priority ranking, the hazards were classified as follows: -t. gondii and pathogenic verocytotoxin-producing escherichia coli (vtec) were classified as high priority for sheep/goat meat inspection. -the remaining identified hazards, b. anthracis, campylobacter spp. (thermophilic) and salmonella spp., were classified as low priority, based on the available data. as new hazards might emerge and/or hazards that presently are not a priority might become more relevant over time or in some regions, both hazard identification and the risk ranking should be revisited regularly to reflect this dynamic epidemiological situation. particular attention should be given to potential emerging hazards of public health importance. assess the strengths and weaknesses of the current meat inspection methodology and recommend possible alternative methods (at ante-mortem or post-mortem inspection, or validated laboratory testing within the frame of traditional meat inspection or elsewhere in the production chain) at eu level, providing an equivalent achievement of overall objectives; the implications for animal health and animal welfare of any changes suggested in the light of public health risks to current inspection methods should be considered. ante-mortem and post-mortem inspection of sheep and goats enable the detection of observable abnormalities. in that context, they are an important activity for monitoring animal health and welfare. they provide a general assessment of animal/herd health, which if compromised may lead to a greater public health risk. visual inspection of live animals and carcasses can also detect animals heavily contaminated with faeces. such animals increase the risk for cross-contamination during slaughter and may consequently constitute a food safety risk if carrying hazards of public health importance. if such animals or carcasses are dealt with adequately, this risk can be reduced. visual detection of faecal contamination on carcasses can also be an indicator of slaughter hygiene, but other approaches to verify slaughter hygiene should be considered. post-mortem inspection can also detect non-meat-borne hazards of public health significance that can be present in carcasses or offal from small ruminants. ante-mortem and post-mortem inspection also have the potential to detect new diseases if these have clinical signs, which may be of direct public health significance. currently, the use of food chain information (fci) for food safety purposes is limited for small ruminants, mainly because the data that it contains is very general and doesn't address specific hazards of public health importance. however, fci could serve as a valuable tool for risk management decisions and could be used for risk categorisation of farms or batches of animals. to achieve this, the system needs further development to include additional information important for food safety, including definition of appropriate and standardised indicators for the main public health hazards identified in section of this appendix. ante-and post-mortem inspection is not able to detect any of the public health hazards identified as the main concerns for food safety. it would therefore be expected that more efficient procedures might be implemented to monitor the occurrence of non-visible hazards. in addition, given that the current post-mortem procedures involve palpation and incision of some organs, there is potential for cross-contamination of carcasses. if new hazards currently not covered by the meat inspection system (e.g. salmonella, campylobacter) are identified under tor , then recommend inspection methods fit for the purpose of meeting the overall objectives of meat inspection. when appropriate, food chain information should be taken into account. as neither of the main public health hazards associated with meat from small ruminants can be detected by traditional meat inspection, other approaches are necessary to identify and control these microbiological hazards. a comprehensive meat safety assurance system for meat from small ruminants, combining a range of preventive measures and controls applied both on the farm and at the slaughterhouse in a longitudinally integrated way, is the most effective approach to control the main hazards in the context of meat inspection. information on the biological risks associated with the consumption of meat from sheep or goats is sometimes scant and unreliable. in order to facilitate decision making, harmonised surveys are required to establish values for the prevalence of the main hazards t. gondii and pathogenic vtec at flock/herd, live animal and carcass level in individual member states. epidemiological and risk assessment studies are also required to determine the specific risk to public health associated with the consumption of meat from small ruminants. in the event that these studies confirm a high risk to public health through the consumption of meat from sheep or goats, consideration should be given to the setting of clear and measurable eu targets at the carcass level. to meet these targets and criteria, a variety of control options for the main hazards are available, at both farm and abattoir level. flock/herd categorisation according to the risk posed by the main hazards is considered an important element of an integrated meat safety assurance system. this should be based on the use of farm descriptors and historical data in addition to batch-specific information. farmrelated data could be provided through farm audits using harmonised epidemiological indicators (heis) to assess the risk and protective factors for the flocks/herds related to the given hazards. classification of abattoirs according to their capability to prevent or reduce faecal contamination of carcasses can be based on two elements: (i) the process hygiene as measured by the level of indicator organisms on the carcasses (i.e. process hygiene criteria); and (ii) the use of operational procedures and equipment that reduce faecal contamination, as well as industry-led quality assurance systems. as mentioned in section . of appendix a, further studies are necessary to determine with more certainty the risk of acquiring t. gondii through consumption of meat from small ruminants. in addition, the lack of tests that can easily identify viable cysts in meat is a significant drawback. furthermore, if there is a high prevalence in the animal population, this will hamper the development of systems based on risk categorisation of animals. for these reasons, the setting of targets for t. gondii is not recommended at the moment. there are a variety of animal husbandry measures that can be used to control t. gondii on sheep and goat farms, but at present these would not be practical to implement on most farms. a number of post-processing interventions might be effective in inactivating t. gondii such as cooking, freezing, curing and high-pressure and -irradiation treatments. however, most of the information available for these treatments originates from research in pigs, so further research is required to validate these treatments in meat from small ruminants. there are also a variety of animal husbandry measures that can be used to reduce the levels of vtec on infected farms, but their efficacy is not clear in small ruminants. in addition, there are a number of challenges that need to be overcome regarding the setting of targets for pathogenic vtec, including the difficulties in identifying husbandry factors that can be used to classify farms according to pathogenic vtec risk, the intermittent nature of shedding, and the problems with the interpretation of monitoring results for vtec due to the difficulty of correctly identifying pathogenic vtec. the two main sources of vtec on sheep and goat carcasses are the fleece/hide and the viscera. to control faecal contamination from the fleece or hide only clean animals should be accepted for slaughter, as currently required by eu legislation. there are also a number of measures that can help to reduce the spillage or leakage of digestive contents onto the carcass, particularly rodding of the oesophagus and bagging of the rectum. post-processing interventions to control vtec are also available. these include hot water and steam pasteurisation. risk categorisation of slaughterhouses should be based on trends of data derived from process hygiene assessments and from hazard analysis critical control point programmes. improvement of slaughter hygiene through technological and managerial interventions should be sought in slaughterhouses with repeatedly unsatisfactory performance. tor recommend adaptations of inspection methods and/or frequencies of inspections that provide an equivalent level of protection within the scope of meat inspection or elsewhere in the production chain that may be used by risk managers in case they consider the current methods disproportionate to the risk, e.g. based on the ranking as an outcome of terms of reference or on data obtained using harmonised epidemiological criteria (see annex ). when appropriate, food chain information should be taken into account. fci can be improved by including information on participation in quality assurance schemes and by giving greater feedback to the primary producer, as this would probably result in the production of healthier animals. ante-mortem inspection assesses the general health status of the animals and helps to detect animals heavily contaminated with faeces on arrival at the slaughterhouse. taking these factors into consideration, and given that current methods do not increase the microbiological risk to public health, no adaptations for the existing visual ante-mortem inspection are required. although visual examination contributes by detecting visible faecal contamination, routine post-mortem examination cannot detect the meat-borne pathogens of public health importance. palpation of the lungs, the liver, the umbilical region and the joints, and incision of the liver could contribute to the spread of bacterial hazards through cross-contamination. for these reasons, palpation and incision should be omitted in animals subjected to routine slaughter. to provide a better evidence base for future risk ranking of hazards, initiatives should be instigated to: improve and harmonise data collection of incidence and severity of human diseases caused by relevant hazards systematically collect data for source attribution collect data to identify and risk rank emerging hazards that could be transmitted through handling, preparation and consumption of sheep and goat meat. source attribution studies are needed to determine the relative importance of meat, as well as to ascertain the role of the different livestock species, as a source of t. gondii and pathogenic vtec for humans. methods should be developed to estimate the amount of viable t. gondii tissue cysts in meat, especially in meat cuts that are commonly consumed. assessment of the importance of the hazards in table with regard to their potential as zoonotic agents that can be transmitted via consumption of meat from small ruminants. these bacteria are considered zoonotic, although this characteristic has only been documented in fish. transmission via consumption of meat from small ruminants has not been reported, despite aeromonas being detected in lamb and meat products and having the potential to be a foodborne pathogen (daskalov, ) . these obligate intracellular bacteria are found in sheep, cattle, horses and dogs, as well as deer and rodents in europe (kalinova et al., ), and although they cause human disease, this illness is rare (scharf et al., ) . they are transmitted by ticks of the genus ixodes, therefore they do not present a risk to humans via consumption of sheep meat. arcobacter spp. the genus arcobacter includes species that can be defined as aerotolerant campylobacter-like organisms. they were first isolated from aborted bovine foetuses. information on the real prevalence and clinical importance of arcobacter is limited because of the absence of routine testing protocols and the fact that most laboratories do not use appropriate culture conditions or do not identify isolates to species level. small ruminants have been found to be carriers of these bacteria (de smet et al., ) in europe. recent reports suggest that arcobacters, especially a. butzleri, may be involved in human enteric disease, although the evidence is not conclusive (houf, ). there are no specific epidemiological data establishing a link between arcobacter infection with consumption of meat from small ruminants. in addition, the public health significance of arcobacter remains unclear. borrelia are transmitted by ticks of the genus ixodes, and infect a wide range of hosts including sheep, although their contribution to the maintenance of b. burgdorferi is still not clear (mannelli et al., ) . although present throughout europe, currently there is no evidence that borrelia can be transmitted via consumption of meat. sheep and goat brucellosis is a zoonotic infection. brucellosis is caused by some bacterial species belonging to the genus brucella. of the six species known to cause disease in humans b. melitensis affects goats and sheep, their specific animal reservoir. humans are usually infected from direct contact with infected animals or via contaminated food, typically raw milk, cheese made thereof or other milk products such as cream and ice cream. meat is not considered a source of infection since muscle tissue contains low concentrations of brucella organisms and the survival time in meat seems extremely short. the number of organisms per gram of muscle is small and rapidly decreases as the ph of the meat drops. brucella spp. die off rapidly when incubated at º c in a medium at ph < (icmsf (international commission on microbial specifications for food), ). an exception in survival behaviour seems to be frozen carcasses, in which the organism can survive for years. c. abortus is known to be transmissible from animals to humans, causing significant zoonotic infections. c. abortus causes the enzootic abortion of ewes (ovine enzootic abortion), which has become recognised as a major cause of loss in sheep (and goats) in europe, north america and africa. (pospischil, ) . most cases of c. abortus infection are directly associated with exposure to infected sheep or goats, with transmission most probably occurring by mouth following the handling of an infected ewe or lamb or of contaminated clothing (longbottom and coulter, ) . the role of meat from small ruminants in the epidemiology of human infection with c. abortus is nevertheless unclear. c. difficile is a species of anaerobic, spore-forming gram-positive bacteria that causes severe diarrhoea and other intestinal disease when competing bacteria in the gut flora have been eliminated by antibiotic treatment. there are reports of c. difficile being isolated from small ruminants (hunter et al., ; koene et al., ; rieu-lesme and fonty, ; saif and brazier, ) . however, there is to date no indication of meat-borne transmission to humans. c. pseudotuberculosis is the causative agent of caseous lymphadenitis in small ruminants. these bacteria are commonly found in europe in the ruminant population. cases of human lymphadenitis have been described previously (peel et al., ) , although transmitted via occupational exposure and not through consumption of meat. c. burnetti has been isolated from a large range of animals including farm animals (e.g. cattle, sheep and goats), wildlife and arthropods. it has a near-worldwide distribution. c. burnetti causes q fever in humans, in whom it was traditionally considered an occupational disease in farm and abattoir workers. airborne transmission is also important, and has played a major role in recent outbreaks. the meatborne transmission route has so far not been identified as a possibility (georgiev et al., ) . erysipelothrix rhusiopathiae e. rhusiopathiae is a ubiquitous bacterium which can cause polyarthritis in sheep and lambs. it can also infect humans, in whom it causes either cutaneous (localised or general) or septicaemic disease (wang et al., ) . humans usually acquire the infection through contact with infected animals, i.e. erysipelas is considered an occupational disease. meat from small ruminants has not been identified as a vehicle for human infection. esbls may be defined as plasmid-encoded enzymes found in the enterobacteriaceae that confer resistance to a variety of ß-lactam antibiotics, including penicillins, and second-, third-and fourthgeneration cephalosporins. in contrast, ampc β-lactamases are intrinsic cephalosporinases found on the chromosomal dna of many gram-negative bacteria, which confer resistance to penicillins, second-and third-generation cephalosporins, including β-lactam/inhibitor combinations, and cefamycins (cefoxitin), but usually not to fourth-generation cephalosporins. a growing number of these ampc enzymes are now plasmid-borne (efsa panel on biological hazards, c). a targeted literature search found references that reported the presence of esbl/ampc-gene carrying enterobacteriaceae (geser et al., ; snow et al., ) in small ruminants but none indicated transmission of these enzymes to humans via consumption of meat from sheep or goats. helicobacter pylori h. pylori was previously known as campylobacter pylori (it is taxonomically related to campylobacter spp. and belongs to the family helicobacteraceae). infection of the stomach by h. pylori is associated with several alterations in gastric mucosal cell proliferation, and disorders such as chronic gastritis, gastric ulcers, duodenal ulcers and stomach cancer. colonisation of the stomach by h. pylori is well established and the bacterium is able to withstand digestive enzymes and concentrated hydrochloric acid. h. pylori is believed to be transmitted orally but no food has been as yet identified as a source. no reservoir other than the human gastric mucosa has been identified for h. pylori. plonka et al. ( ) suggest a zoonotic link to sheep, but no evidence of meat-borne transmission is presented. although the isolation of k. pneumoniae from small ruminants' meat has been described (brahmbhatt, ; sharma et al., ) , no evidence for meat-borne transmission of this pathogen to humans could be found. l. spiralis has been reported in the small ruminant population in europe and elsewhere (bisias et al., ; savalia et al., ; seixas melo et al., ) ; however, although it has been considered a potential occupational hazard (heuer et al., ) , there is no current evidence that it can be transmitted to humans via consumption of meat from small ruminants. mycobacterium avium subsp. paratuberculosis m. avium subsp. paratuberculosis (map), which causes chronic enteritis in all ruminants, is the most prevalent mycobacterium found in small ruminants within the m. avium complex (mac). mac includes eight mycobacteria species and several subspecies with different degrees of pathogenicity, a broad host range and environmental distribution in numerous biotopes including the soil, water, aerosols, etc. (alvarez et al., ; biet et al., ) . a link between map and the human chronic enteritis, crohn's disease, has been speculated and supported by several lines of evidence, such as the demonstration of map-specific sequences in crohn's disease-affected tissues. however, at present, there is no agreed consensus on any aetiological role for map in crohn's disease (chiodini, ; waddell et al., ; wagner et al., ) and no evidence that it presents a risk via consumption of meat or meat products. the presence of mycobacteria has been previously reported in the small ruminant population in the eu (domenis et al., ; malone et al., ; marianelli et al., ; . despite these reports, evidence of meat-borne transmission of these pathogens to humans from small ruminants is lacking, so this potential pathway of infection remains unproven in the context of livestock processed through the eu meat inspection system. mrsa has been isolated from most food-producing animals and from most meats, as well as from milk including sheep and lamb meat. where mrsa cc prevalence is high in food-producing animals, people in contact with these live animals (especially farmers and veterinarians, and their families) are at greater risk of colonisation and infection than the general population. food may be contaminated by mrsa (including cc ): eating and handling contaminated food is a potential vehicle for transmission. there is currently no evidence for increased risk of human colonisation or infection following contact or consumption of food contaminated by cc both in the community and in hospital (efsa, ). streptococcus spp. have been isolated in small ruminants, most commonly in milk or mastitis samples (pisoni et al., ; zdragas et al., ) . zoonotic transmission has been described (poulin and boivin, ), but there is no evidence to date that it can cause meat-borne disease in humans. foodborne yersiniosis is caused primarily by yersinia enterocolitica, with y. pseudotuberculosis representing a low fraction of isolates (less than %) from human cases reported (efsa and ecdc, b). the majority of isolates of y. enterocolitica isolated from food and environmental sources are non-pathogenic types, and therefore discrimination between pathogenic and non-pathogenic strains for humans is necessary. no reports of y. pseudotuberculosis have been published of isolates in food items tested during - (efsa and ecdc, , b . pigs are considered to be the primary reservoir for the human pathogenic types of y. enterocolitica, and they can be isolated from the oral cavity, the submaxillary lymph nodes, the intestine and faeces (nesbakken, ) . y. enterocolitica is found in small ruminants, and is considered to be responsible for certain infections in sheep and goats such as enteritis (arnold et al., ; fearnley et al., ; fredriksson-ahomaa et al., ; fukushima et al., ; gourdon et al., ; krogstad, ; mcnally et al., ; milnes et al., ; philbey et al., ; slee and button, ; slee and skilbeck, ; soderqvist et al., ; wojciech et al., ) . mcnally et al. ( ) investigated the relationship between livestock (sheep, cattle and pigs) carriage of y. enterocolitica and human disease with inconclusive results. the majority of the strains isolated from animal reservoirs differ from clinical strains found in humans, biochemically and serologically. so far pigs are the only species pinpointed as being significant reservoirs for pathogenic y. enterocolitica. there is no evidence that sheep and goats are important animal reservoirs for strains involved in human cases, although slee and button ( ) reported the infection of an animal attendant in connection with an outbreak of y. enterocolitica infection in a goat herd in norway. no evidence that yersinia spp. present a risk via consumption of meat or meat products from sheep or goats is currently available. c. albicans is a fungus that is the causal agent of opportunistic oral and genital infections in humans and has also been isolated from sheep and goats, for example in milk samples of goats suffering from mastitis (langoni et al., ) or from sheep droppings (nardoni et al., ) . no evidence to date could indicate transmission of this fungus to humans via consumption of meat. cryptococcosis is a rare disease in animals in europe. a few cases have been described in sheep and goats (lung and mammary gland) in australia. the source of microorganisms is largely environmental. no cases of transmission from animal to animal or from animal to man or from man to man (except corneal transplant) have been described (acha and szyfres, ). c. neoformans is therefore currently considered not relevant in the eu sheep and goat population and not transmissible via meat. species of microsporidia infecting humans have been identified in water sources as well as in wild, domestic, and food-producing farm animals, raising concerns for waterborne, foodborne, and zoonotic transmission (didier, ) . no evidence could be found in the literature of meat-borne transmission of this hazard from small ruminants to humans. enterocytozoon bieneusi e. bieneusi, the species now known to be the most frequent in microsporidial infections of humans, was not discovered until . e. bieneusi has recently been found in the faeces of animals, including pigs, rhesus macaques, cats and cattle. however, the potential reservoirs and the mode of transmission of this pathogen are still unknown (dengjel et al., ) . phylogenetic analysis revealed the lack of a transmission barrier between e. bieneusi from humans and animals (cats, pigs and cattle). thus, e. bieneusi appears to be a zoonotic pathogen." (dengjel et al., ). however, no evidence could be found in the literature for meat-borne transmission of this hazard from small ruminants to humans. parasites of the genus ascaris have very occasionally reported in sheep. however, the transmission of these parasites to humans is via ingestion of eggs that are excreted in faeces of the definite hosts (e.g. in pigs a. sum and in and humans a. lumbricoides), therefore there is currently no evidence of a link between human ascariasis and the consumption of ruminant meat. babesia spp. are vector-mediated parasites, and are transmitted by hard ticks (e.g. ixodes, dermacentor, rhipicephalus and hyaloma spp.). in europe, they are found in cattle and rodents, although they have also been reported in sheep ( (sreter et al., ) . human babesiosis is rare in europe, and only transmitted via tick bites, i.e. there have been no reports of meat-borne transmission to humans from animals. cerebral coenurosis is caused by the metacestode stage of the cestode t. multiceps, which has canids as the final host. both humans and sheep are intermediate hosts in the life cycle of this parasite, which is present in parts of europe (scala and varcasia, ) . infection occurs by ingestion of vegetables or water contaminated with the tapeworm eggs shed by the final host. meat has not been recorded as being involved in transmission of this parasite. cryptosporidiosis in humans is usually linked to consumption of contaminated water or contact with infected animals, mainly cattle but also young sheep and goats. although its presence in meat is considered possible, a quick review of the literature did not reveal any evidence describing the isolation in meat or any outbreaks caused by consumption of meat from small ruminants. c. ovis and c. tenuicollis are the larval stages of taenia ovis and taenia hydatigena respectively, found in the intestines of canids. humans can act as intermediate hosts for these cysticerci, but cases are very rare. consumption of meat is not associated with the transmission of these parasites, but they are targeted during meat inspection because cysticerci are visible and render the meat unfit for human consumption on quality grounds. ) . humans are a dead-end host and may become infected through accidental ingestion of the eggs, shed in the faeces of infected dogs or other canids. this usually occurs via the ingestion of contaminated food (especially vegetables) or water, and also through accidental soil ingestion or by acquiring the eggs directly from the coat of the definitive host. meat, however, has not been identified as a vehicle for transmission of e. granulosus. the trematode fasciola is a parasite of herbivores that can infect humans accidentally, and is commonly found in europe. humans can become infected by ingesting freshwater plants or water containing metacercariae (fried and abruzzi, ). there is currently no evidence of meat-borne transmission of this parasite to humans. g. intestinalis is a ubiquitous protozoan parasite with global distribution, which infects humans as well as a wide range of other mammals. g. intestinalis is excreted in faeces, and it is transmitted to humans via contaminated water or fresh vegetables. no evidence is available for a role for meat from small ruminants in transmitting this parasite to humans. virus of the family astroviridae are associated with gastroenteritis in birds and mammals, including small ruminants and humans. although a potential zoonotic link has been suggested (jonassen et al., ) , information available in the scientific literature does not point at potential transmission of astroviruses to humans via consumption of meat. bdv infections can result in neurological disease that mainly affects horses and sheep in certain areas of germany (bilzer et al., ; durrwald, ; grabner and fischer, ; ludwig et al., ) . the endemic area also includes areas of switzerland, austria and the principality of liechtenstein (caplazi et al., ; weissenbock et al., ) . bdv received worldwide attention when it was reported that sera and/or cerebrospinal fluids from neuropsychiatric patients can contain bdv-specific antibodies. as infected animals produce bdv-specific antibodies only after virus replication, it was assumed that the broad spectrum of bdv-susceptible species also includes man. however, reports describing the presence of other bdv markers, i.e. bdv-rna or bdv-antigen, in peripheral blood leucocytes or brain tissue of neuropsychiatric patients are highly controversial and, therefore, the role of bdv in human neuropsychiatric disorders is questionable (richt and rott, ) . in any case, no evidence of meat-borne transmission has been found. there is a lack of clarity in relation to the taxonomy of bovine enterovirus (bev). while it appears that bev may be zoonootic, based on a serological survey in turkey, and that sheep and goats in europe are infected, it is likely that the main source of infection for humans is contact with infected animals and/or material contaminated with faeces of infected animals. on the basis of the information obtained from the scientific literature, it is proposed that bev should not be considered for risk ranking. chandipura virus, a member of the rhabdoviridae family and vesiculovirus genus, has recently emerged as a human pathogen associated with a number of outbreaks of acute encephalitis in different parts of india (basak et al., ) . the virus closely resembles the vesicular stomatitis virus, and there are reports of antibody detection in small ruminants, also in india (joshi et al., ) . there are no reports of this virus being present in the eu or being able to be transmitted to humans via food. the information available in the scientific literature for this virus in the small ruminant reservoir is very limited. cchf is a tick-borne disease that can also be transmitted to humans through contact with infected tissues or blood from affected (viraemic) livestock, including sheep. cases of cchf have been reported in butchers and abattoir workers (ergonul, ) as well as health care workers, therefore it can be considered an occupational disease. currently, there is no evidence of meat-borne transmission, and it has been reported that "meat itself is not a risk because the virus is inactivated by post slaughter acidification of the tissues and would not survive cooking in any case." (ergonul, ) . hev has been found in both livestock, especially pigs, and humans in europe. the epidemiology of hev is complex, and a foodborne transmission of hev from animal products (e.g. pork and pork products) to humans is an emerging concern. however, only very few systematic studies have been performed so far, therefore the importance of specific food items has not been sufficiently substantiated. although the presence of hev antibodies in sheep has been previously reported in europe (peralta et al., ), there is no evidence that meat from small ruminants has played a role in transmitting the virus to humans (efsa panel on biological hazards, a). the presence of influenza virus has been occasionally reported in small ruminants (abubakar et al., ; shukla and negi, ; zupancic et al., ) . although no information is available for small ruminants, the safety of meat from pigs infected with influenza has been previously assessed, and it was found that these viruses are not known to be transmissible to humans through the consumption of meat (fao/who/oie, ). orf, also known as contagious ecthyma, is caused by a parapoxvirus and is commonly found in the small ruminant population in europe. this virus is transmitted to humans through direct contact with infected animals and thus is considered an occupational disease (uzel et al., ) . meat-borne transmission has not been reported to date. small ruminants are susceptible to infection with rabies virus, which is present in the wild animal reservoir in europe (mainly in bats and wild canids). cases of rabies in sheep and goats have been occasionally reported in europe (maciulskis et al., ; mudura et al., ) , and although experimental oral transmission has been described (bell and moore, ; fischman and ward, ) , transmission of this virus to humans through the consumption of meat from small ruminants has not been reported to date. this rna virus of the family bunyaviridae causes disease in cattle, sheep and goats, and is transmitted to humans by a wide range of mosquitoes, as well as by handling diseased animals (davies and martin, ) . contact with and consumption of meat, as well as other animal products, has been identified as a risk factor for human infection (anyangu et al., ; mohamed et al., ) . the presence of this virus has not been reported in europe so far (efsa panel on animal health and welfare (ahaw), ). rotaviruses are responsible for causing enteritis and diarrhoea in young livestock, including sheep and goats, as well as in humans. some studies that used gene sequencing point to a common evolutionary origin for rotavirus strains found in small ruminants and those found in humans (ghosh et al., ; matthijnssens et al., ). this could suggest that there is potential for zoonotic transmission between livestock and humans, or at least that some exchange of viruses has occurred in the past (nakagomi et al., ) . it is, however, unclear if meat-borne transmission is possible, as there are no data in the literature reporting this possibility. tbe is an infection caused by flavivirus found in both wild and domestic animals in europe, including small ruminants. humans acquire the infection following the bite of an infected tick. transmission via aerosol and direct contact is also possible, as well as by consuming fresh milk from infected animals. however, transmission via consumption of meat has not been described (krauss et al., ) . objective of meat inspection is to ensure that meat is fit for human consumption. historically, meat inspection procedures have been designed to control slaughter animals for the absence of infectious diseases, with special emphasis on zoonoses and notifiable diseases. the mandate that meat needs to be fit for human consumption, however, also includes the control of chemical residues and contaminants that could be potentially harmful for consumers. this aspect is not fully addressed by the current procedures. the efsa panel on contaminants in the food chain (contam panel) was asked to identify and rank undesirable or harmful chemical residues and contaminants in meat from sheep and goats. such substances may occur as residues in edible tissues from the exposure of the animals to contaminants in feed materials as well as following the possible application of non-authorised substances and the application of authorised veterinary medicinal products and feed additives. a multi-step approach was used for the ranking of these substances into categories of potential concern. as a first step, the contam panel considered substances listed in council directive / /ec and evaluated the outcome of the national residue control plans (nrcps) for the period - . the contam panel noted that only . % of the total number of results was non-compliant for one or more substances listed in council directive / /ec. potentially higher exposure of consumers to these substances from sheep and goat meat takes place only incidentally, as a result of mistakes or noncompliance with known and regulated procedures. the available aggregated data indicate a low number of samples that were non-compliant with the current legislation. however, in the absence of substance-and/or species-specific information, such as the tissues used for residue analysis and the actual concentration of a residue or contaminant measured, these data do not allow for a reliable assessment of consumer exposure. independently from the occurrence data as reported from the nrcps, other criteria used for the identification and ranking of chemical substances of potential concern included the identification of substances that are found in other testing programmes and that bio-accumulate in the food chain, substances with a toxicological profile of concern, and the likelihood that a substance under consideration will occur in sheep and goat carcasses. taking into account these criteria, the individual compounds were ranked into four categories denoted as being of high, medium, low and negligible potential concern. dioxins and dioxin-like polychlorinated biphenyls (dl-pcbs) were ranked as being of high potential concern owing to their known bioaccumulation in the food chain, the frequent findings above maximum levels (mls), particularly in sheep liver, and in consideration of their toxicological profile. the following substances were ranked in the category of medium potential concern: stilbenes, thyreostats, gonadal (sex) steroids, resorcylic acid lactones and beta-agonists (especially clenbuterol) because of their toxicity for humans, their efficacy as growth promoters in sheep and goats and the incidence of non-compliant results; chloramphenicol and nitrofurans because they have proven toxicity for humans, are effective as antibacterial treatments for sheep/goats and non-compliant samples are found in most years of the nrcps; non-dioxin-like polychlorinated biphenyls (ndl-pcbs) because, while they bioaccumulate and there is a risk of exceeding the mls, they are less toxic than dioxins and dl-pcbs; and the chemical elements cadmium, lead and mercury because of the number of non-compliant results reported under the nrcps and their toxicological profile. residues originating from other substances listed in council directive / /ec were ranked as of low or negligible potential concern. the contam panel emphasises that this ranking into specific categories of potential concern is based on the current knowledge regarding toxicological profiles, usage in sheep and goat production and occurrence as chemical residues and contaminants. where changes in any of these factors occur, the ranking might need amendment. the contam panel was also asked to assess the main strengths and weaknesses of current meat inspection protocols within the context of chemical hazards. it was noted that current procedures for sampling and testing are, in general, well established and coordinated, including follow-up actions subsequent to the identification of non-compliant samples. the regular sampling and testing for chemical residues and contaminants is an important disincentive for the development of undesirable practices and the prescriptive sampling system allows for equivalence in the control of eu-produced sheep and goat meat. the current combination of animal traceability, ante-mortem inspection and gross tissue examination can support the collection of appropriate samples for residue monitoring. nevertheless, a major weakness is that, with very few exceptions, presence of chemical hazards cannot be identified by current ante-/post-mortem meat inspection procedures at the slaughterhouse level and there is a lack of sufficient cost-effective and reliable screening methods. in addition, sampling is mostly prescriptive rather than risk or information based. there is limited ongoing adaptation of the sampling and testing programmes to the results of the residue monitoring programmes, with poor integration between the testing of feed materials for undesirable substances and the nrcps and sampling under the nrcps reflecting only a part of testing done by a number of mss, the results of which should be taken into consideration. the contam panel was also asked to identify and recommend inspection methods for new hazards. as dioxins and dl-pcbs have not yet been comprehensively covered by the sampling plans of the current meat inspection, they should be considered as 'new' hazards as they have been ranked as being of high potential concern. moreover, for other organic contaminants that may accumulate in foodproducing animals and for a number of chemical elements used as feed supplements, only limited data regarding residues in sheep and goats are available. this is the case, in particular, for brominated flame retardants, including polybrominated diphenylethers (pbdes) and hexabromocyclododecanes (hbcdds) and perfluorinated compounds (pfcs) including (but not limited to) perfluorooctane sulphonate (pfos) and perfluorooctanoic acid (pfoa). the contam panel concludes that sheep and goat production in the eu is marked by being largely extensive in nature, involving frequent trading of animals and nomadic flocks. these differences in husbandry systems and feeding regimes result in different risks for the occurrence of chemical residues and contaminants. extensive periods on pasture or/as nomadic flocks and the use of slaughter collection dealerships may preclude detailed lifetime food chain information (fci). the contam panel recommends that fci should be expanded for sheep and goats produced in extensive systems to provide more information on the specific environmental conditions where the animals are produced and that future monitoring programmes should be based on the risk of occurrence of chemical residues and contaminants, taking into account the completeness and quality of the fci supplied and the ranking of chemical substance into categories of potential concern, which needs to be regularly updated. control programmes for chemical residues and contaminants should be less prescriptive, with sufficient flexibility to adapt to results of testing, should include 'new hazards', and the test results for sheep and goats should be separately presented. there is a need for an improved integration of sampling, testing and intervention protocols across the food chain, nrcps, feed control and monitoring of environmental contaminants. the development of analytical techniques covering multiple analytes and of new biologically based testing approaches should be encouraged too and incorporated into the residue control programmes. for prohibited substances, testing should be directed where appropriate towards the farm level and, in the case of substances that might be used illicitly for growth promotion, control measures, including testing, need to be refocused to better identify the extent of abuse in the eu. meat inspection in the eu is specified in regulation (ec) no / . the main objective of meat inspection is to ensure that meat is fit for human consumption. historically, meat inspection procedures have been designed to control slaughter animals for the absence of infectious diseases, with special emphasis on zoonoses and notifiable diseases. the mandate that meat needs to be fit for human consumption, however, also includes the control of chemical residues and contaminants in meat that could be potentially harmful for consumers. this aspect is not fully addressed by the current procedures. for the purposes of this document 'chemical residues' refer to chemical compounds which result from the intentional administration of legal or illegal pharmacologically active substances while 'chemical contaminants' refer to chemical compounds originating from the environment. this document aims to identify undesirable or harmful chemical residues and contaminants that may occur in meat from sheep and goats taking into account the current legislation and the results from the national residue control plans (nrcps) implemented in line with council directive / /ec. these findings, together with the characteristics of the individual substances and the likelihood that a substance will occur in meat from sheep or goats were used to rank chemical residues and contaminants into categories of potential concern. four categories were established constituting a high, medium, low or negligible potential concern. in the second part, the main strengths and weaknesses of current meat inspection protocols were assessed within the context of chemical hazards. the ultimate aim is an overall evaluation of the current strategies for sampling and analytical testing, resulting in recommendations for possible amendments to the current meat inspection protocols. in this opinion, where reference is made to european legislation (regulations, directives, decisions), the reference should be understood as relating to the most current amendment, unless otherwise stated. sheep (ovis aries) were domesticated from ancestral subspecies of wild mouflon approximately years ago in south-west asia, and by years ago, sheep had been transported throughout europe. today, over sheep breeds are recognised worldwide, and europe supports a greater number of breeds than any other continent. sheep are raised for three main purposes: meat, milk and wool. therefore, a range of different breeds have been developed over centuries to suit the land and weather and husbandry conditions in different areas of the eu. in mountain and arid areas, for example, sheep are bred for hardiness and self-reliance (e.g. scottish blackface). they must be able to survive poor weather and thrive on poor grazing. lowland or grassland breeds, on the other hand (e.g. suffolk and texel), usually do not cope as well with bad weather or poor-quality feed, but produce higher numbers of lambs that are often better suited for meat production. most lambs are born in late winter or spring. many lambs are born outside, particularly those in mountain flocks. indoor lambing is also common, particularly for lowland flocks. good housing facilities and management are important in order to prevent disease and heat stress problems. most meat-breed sheep are slaughtered and presented for meat inspection as younger stock "lambs" from ten weeks up to one year. in accordance with commission regulation (ec) no / , a "young ovine animal" means an ovine animal of either gender, not having any permanent incisor teeth erupted and not older than months. sheep have also been raised for milk production for thousands of years. the east friesian type is one of the most common and productive breeds of dairy sheep. europe's commercial dairy sheep industry is concentrated in the countries on or near the mediterranean sea. most of the sheep milk is used to produce cheese, such as feta, ricotta, manchego and pecorino romano. in france, the lacaune is the breed of choice for making roquefort cheese. dairy sheep kept on small farms are milked seasonally by hand but more modern sheep dairies use sophisticated machinery for milking. ewes are milked once or twice per day. sheep are also widely kept for wool production, particularly in the united kingdom and spain. wool may range from fine or medium fibre diameter to specialised breeds producing wool for carpets. some flocks may be kept for both meat and wool production purposes. meat from older sheep carcasses (mutton), derived from cull adult sheep from the dairy or wool industries, is tougher and is not as widely consumed as fresh meat, but may also be used in sausage production. goats have been associated with man in a symbiotic relationship for up to years. the goat eats little, occupies a small area and each produces enough milk to sustain a family. in europe, goat farming is strongly oriented towards milk production, which is mostly used for cheese production. it has been estimated that the eu has . % of the world's goat population, but it produces . % of goat milk and . % of goat meat generated in the world annually (casey, ) . during the last ten years, the overall eu goat count has diminished. in france, greece and spain, annual goat milk production is , , and million litres, respectively, which comprises % of the total goat milk produced in the eu. france produces a great number of goat's milk cheeses, especially in the loire valley, with examples of french chèvre including bucheron. like sheep, dairy goats kept on small farms are milked seasonally by hand but modern goat dairies use more sophisticated machinery for milking. does are milked once or twice per day. goats produced for fibre are not common in europe, but small local flocks occur in many member states (mss). the fibre taken from an angora goat breed is called mohair. a single goat produces between four and five kilograms of hair per year, shorn twice a year. cashmere is the valuable fine undercoat found to varying degrees and qualities on all goats, except the angora. it grows as a winter down which is shed in early spring, when it is harvested by either shearing or combing. goat meat or chevron is not widely consumed in the eu. specialised larger goat meat breeds such as the boer goat are currently only held in small local herds, but crosses of a boer sire and a cashmeretype breed dam can also be used to provide a suitable carcass. these meat-line goats can grow to slaughter weight ( - kg) in approximately six to nine months on low-quality feeding. again, the meat from older goat carcasses derived from the cull goats from dairy or fibre industries tends to be very tough. meat from older male goats ('billy' goats) can have an offensive odour. the extensive farming practices and generally low economic value of sheep and goats mean that veterinary treatment of individual animals is often limited. sheep and goats are often exposed to parasites, which explain the necessary use of anti-parasitic programmes for the flocks. other veterinary interventions follow normal clinical practice, such as the use of registered mastitis treatments for milking animals, with appropriate withdrawal periods and residue monitoring. it is important to note that, despite recent developments towards large milking goat holdings, sheep and goat production in the eu largely remains extensive in nature, involving frequent trading of animals and nomadic flocks. this involves varied husbandry systems and feeding regimes resulting in different risks for chemical substances and contaminants. sheep and goat populations in the eu as reported by eurostat are presented in table . in accordance with annex i of regulation (ec) no / all animals should be inspected prior to slaughter (ante-mortem inspection) as well as after slaughter and evisceration (post-mortem inspection). there are concerns about slaughter outside licensed premises where animals are not subject to appropriate meat inspection. since january , a mandatory identification of small ruminants has been implemented in the eu by regulation (ec) no / . domestic sheep and goats may be presented for slaughter in small numbers or even as individuals. visual ante-mortem inspection is carried out at the level of the individual animal. extensive periods on pasture or as nomadic flocks, sale at open markets of many sheep and goats, and the presence of slaughter collection dealerships that may combine small numbers of animals purchased from several farmers, means that there is a level of concern that food chain information (fci) shared between farmers and the slaughterhouse (where residue data are managed), may be suboptimal. similarly, in these situations, the level of feedback from the slaughterhouse and authorities to farmers regarding the results of residue testing may be suboptimal. here the individual identification of animals, which has now become mandatory, may contribute to more transparency in the future. there is less concern about fci from dairy sheep and goats as they are reared under more intensive and controlled conditions. fci is the animal's life history data from birth, through all stages of rearing, up to the day of slaughter. in particular, the food business operator (fbo) at the slaughterhouse should receive information related to the veterinary medicinal products (vmps) or other treatments administered to the animals within a relevant period prior to slaughter, together with their administration dates and their withdrawal periods. moreover, any test results for samples taken from the animals within the framework of monitoring and control of residues should also be communicated to the slaughterhouse operators before the arrival of the animals. based on regulation (ec) no / , post-mortem inspection was, and still is, directed primarily at the detection of lesions due to infections, based on observation, palpation, and incision. an exception is the mandatory sampling of adult animals for transmissible spongiform encephalopathies (tses). in contrast to bovine animals, tse testing is not directed at individual animals, but is based on a region and animal stock related monitoring system. visual inspection of the carcass (and offals) may allow, in some cases, for the identification of gross alterations in carcass conformation (e.g. abscesses or deposits) and organ-specific lesions in kidneys, liver, lungs or other organs that might be indicative of recent use of vmps (with the possibility of noncompliance with withdrawal periods) or acute or chronic exposure to toxic substances. in most cases, exposure to chemical compounds does not result in typical organ lesions. hence it needs to be considered that evidence for the presence of chemical residues and contaminants will in most cases not be apparent during the visual inspection of ovine and caprine carcasses. therefore, the meat inspection approach based on "detect and immediately eliminate", used for biotic (microbiological) hazards in slaughterhouses, is generally not applicable to abiotic hazards. while monitoring programmes (council directive / /ec, described in section . ) may provide a gross indication of the prevalence of undesirable chemical residues and contaminants in ovine and caprine carcasses, the sole intervention at abattoir level is the isolation of a suspect carcass as potentially unfit for human consumption, pending results of residue testing. council directive / /ec prescribes the measures to monitor certain substances and residues thereof in live animals and animal products. it requires that mss adopt and implement a national residue monitoring programme, also referred to as the national residue control plan (nrcp, for defined groups of substances. mss must assign the task of coordinating the implementation of the controls to a central public body. this public body is responsible for drawing up the national plan, coordinating the activities of the central and regional bodies responsible for monitoring the various residues, collecting the data and sending the results of the surveys undertaken to the commission each year. the nrcp should be targeted; samples should be taken on-farm and at abattoir level with the aim of detecting illegal treatment or controlling compliance with the maximum residue limits (mrls) for vmps according to commission regulation (eu) no / , with the maximum residue levels (mrls) for pesticides as set out in regulation (ec) no / , or with the maximum levels (mls) for contaminants as laid down in commission regulation (ec) no this means that in the national monitoring plans, the mss should target those groups of animals/gender/age combinations in which the probability of finding residues is highest. this approach differs from random sampling, in which the objective is to gather statistically representative data, for instance to evaluate consumer exposure to a specific substance. the minimum number of animals to be checked for all kind of residues and substances must be at least equal to . % of sheep and goats over three months of age slaughtered the previous year, with the following breakdown (further details on group a and b compounds is presented in section . group a: . % of total samples -each sub-group a must be checked each year using a minimum of % of the total number of samples to be collected for group a. the balance is allocated according to the experience and background information of the ms. group b: . % of the total samples - % must be checked for group b substances - % must be checked for group b substances - % must be checked for group b substances. the balance must be allocated according to the situation of the ms. in the case of imports from third countries, chapter vi of council directive / /ec describes the system to be followed to ensure an equivalent level of control on such imports. in particular it specifies (i) that each third country must provide a plan setting out the guarantees which it offers as regards the monitoring of the groups of residues and substances referred to in annex i to the council directive, (ii) that such guarantees must have an effect at least equivalent to those provided for in council directive / /ec, (iii) that compliance with the requirements of and adherence to the guarantees offered by the plans submitted by third countries shall be verified by means of the checks referred to in article of directive / /eec and the checks provided for in directives / /eec and / /eec, and (iv) that mss are required to inform the commission each year of the results of residue checks carried out on animals and animal products imported from third countries, in accordance with directives / /eec and / /eec. in accordance with article of council directive / /ec, the mss are requested, as a follow-up, to provide information on actions taken at regional and national level as a consequence of non-compliant results. the commission sends a questionnaire to the ms to obtain an overview of these actions, for example when residues of non-authorised substances are detected or when the maximum residue limits/maximum levels established in eu legislation are exceeded. in summary, this means that the term 'suspect sample' applies to a sample taken as a consequence of: non-compliant results, and/or suspicion of an illegal treatment, and/or suspicion of non-compliance with the withdrawal periods. non-compliant results for a specific substance or group of substances or a specific food commodity should result in intensified controls for this substance/group or food commodity in the plan for the following year. article it should be noted that targeted sampling as defined by council directive / /ec aims at monitoring certain substances and residues thereof in live animals and animal products across eu mss. in contrast to monitoring, under suspect sampling, a 'suspect' carcass has to be detained at the abattoir until laboratory results confirm or deny conformity with legislative limits for chemical residues. based on the test results, the carcass can be declared fit or unfit for human consumption. in the first scenario, the carcass is released into the human food chain whereas in the second case the carcass is disposed of. in addition to the minimum testing requirements which form part of the nrcps, council directive / /ec also establishes the requisites for self-monitoring and co-responsibility on the part of operators. in accordance with article , chapter iii of council directive / /ec, mss shall ensure that the owners or persons in charge of the establishment of initial processing of primary products of animal origin (slaughterhouses) take all necessary measures, in particular by carrying out their own checks, to: accept only those animals for which the producer is able to guarantee that withdrawal times have been observed satisfy themselves that the farm animals or products brought into the slaughterhouse do not contain residue levels which exceed maximum permitted limits and that they do not contain any trace of prohibited substances or products. farmers and food processors (including slaughterhouses) must place on the market only: animals to which no unauthorised substances or products have been administered or which have not undergone illegal treatment animals for which, where authorised products or substances have been administered, the withdrawal periods prescribed for these products or substances have been observed. tor : identification, classification and ranking of substances of potential concern . . in the current eu legislation, chemical residues and contaminants in live animals and animal products intended for human consumption are addressed in as one of the objectives of this assessment of current meat inspection protocols is the identification of chemical substances of potential concern that may occur as residues or contaminants in sheep and goats, but have not been specifically addressed in council directive / /ec, a more general grouping of chemical substances was chosen, resulting in the following three major groups: substances that are prohibited for use in food-producing animals, corresponding to group a substances in council directive / /ec veterinary drugs, also denoted vmps, corresponding to groups b and b substances in council directive / /ec and contaminants, corresponding to group b substances in council directive / /ec. the first group of chemicals that may occur in edible tissues as residues are those substances prohibited for use in food-producing animals; these substances correspond largely with group a substances in council directive / /ec. there were different rationales for banning these substances for application to animals and the list of group a substances comprises compounds that are of toxicological concern (including vmps for which an acceptable daily intake (adi) could not be established), as well as substances having anabolic effects and pharmacologically active compounds that may alter meat quality and/or affect animal health and welfare. a second group of chemicals that may be a source of residues in animal-derived foods are vmps (including antibiotics, anti-parasitic agents and other pharmacologically active substances) and authorised feed additives used in the health care of domestic animals; these substances correspond largely with group b and b substances in council directive / /ec. these substances have been subjected to assessment and pre-marketing approval by the with regard to antibacterial agents, it is important to state that the ranking of substances of concern in this part of the document considers only toxicological concerns related to the presence of residues. other aspects, such as the emergence of antimicrobial resistance is considered by the efsa panel on biological hazards (biohaz panel) in a separate part of this opinion (see appendix a of the biohaz panel). a third group of chemical substances that may occur in edible tissues of sheep and goats are contaminants that may enter the animal's body mainly via feed, ingested soil, drinking water, inhalation or direct (skin) contact; these substances include the group b substances in council directive / /ec. feed materials can contain a broad variety of undesirable substances comprising persistent environmental pollutants, toxic metals and other elements as well as natural toxins, including toxic secondary plant metabolites and fungal toxins (mycotoxins). feed producers have to act in compliance with commission directive / /ec, listing the undesirable substances in feed and feed materials and presenting maximum content in feed materials or complete feedingstuffs. in a recent re-assessment of these undesirable substances in animal feeds, the contam panel reevaluated the risk related to exposure to these substances for animals. special attention was given to toxic compounds that accumulate or persist in edible tissues, including meat, or that are directly excreted into milk and eggs. . . , pp. - . the term "dioxins" used in this opinion refers to the sum of polychlorinated dibenzo-p-dioxins (pcdds) and polychlorinated dibenzofurans (pcdfs). as an early warning tool, the european commission has set action levels for dioxins and dl-pcbs in food through commission recommendation / /ec. owing to the fact that their sources are generally different, separate action levels for dioxins and dl-pcbs were established. the action levels for meat and meat products of sheep are . pg who-teq/g fat for dioxins and . pg who-teq/g fat for dl-pcbs. in cases where levels of dioxins and/or dl-pcbs in excess of the action levels are found, it is recommended that mss, in cooperation with fbos, initiate investigations to identify the source of contamination, take measures to reduce or eliminate the source of contamination and check for the presence of ndl-pcbs. maximum residue levels for certain elements in sheep and goats are also laid down in regulation (ec) no / of the european parliament and of the council on maximum residue levels of pesticides in or on food and feed of plant and animal origin, related to the use of copper-containing and mercury-containing compounds as pesticides. for copper, the maximum residue levels are each mg/kg for meat and fat and mg/kg each for liver, kidney and edible offal. for mercury compounds (sum of mercury compounds expressed as mercury), the maximum residue levels are . mg/kg each for meat, fat, liver, kidney and edible offal. a multi-step approach was used for ranking the potential concern of the three groups of substances that are presented in sections . and . . the steps are: evaluation of the outcomes of the nrcps indicating the number of results that are noncompliant with the current legislation evaluation of the likelihood that specific residues or contaminants, including 'new hazards', may be present in sheep and goat carcasses consideration of the toxicological profile for chemical substances. data from the nrcps are published annually and these data were considered as the first step for hazard ranking. aggregated data for the outcome of the nrcps for targeted sampling of sheep and goats from to are presented in tables - results from suspect sampling are not included, as these results are considered not to be representative of the actual occurrence of chemical residues and contaminants. as stated above, suspect sampling arises as (i) a follow-up to the occurrence of a non-compliant result, and/or (ii) on suspicion of illegal treatment at any stage of the food chain, and/or (iii) on suspicion of non-compliance with the withdrawal periods for authorised vmps (articles , and of council directive / /ec, respectively). a non-compliant result refers to an analytical result exceeding the permitted limits or, in the case of prohibited substances, any measured level with sufficient statistical certainty that it can be used for legal purposes. it should be noted that information on the number of total analyses performed for an individual substance is only transmitted by those mss that were reporting at least one non-compliant result for that substance. therefore, it is not possible to extract from the data supplied complete information on the individual substances from each sub-group tested or the number of samples tested for an individual substance where no non-compliant result is reported. in addition, in some cases the same samples were analysed for different substance groups/sub-groups and therefore the number of substance groups/sub-groups tested is higher than the total number of samples collected from sheep and goats. it is to be noted that there is a lack of harmonisation regarding details provided on non-compliant results for the nrcps from mss. this hampers the interpretation and the evaluation of these data. moreover, in some cases, no information is available on the nature of the positive samples (i.e. whether this refers to muscle, liver, kidney, skin/fat or other samples) and these results often give no indication of the actual measured concentrations of residues or contaminants. as a result, in the absence of substance-specific information and the actual concentration of a residue or contaminant measured, these data do not allow for an assessment of consumer exposure. in addition, particularly in the case of prohibited substances, much of the testing may be done in matrices such as urine, faeces and hair and so no data on residue levels in edible tissues are available. another problem with interpreting the data provided arises from the failure to clearly identify in all cases (i) the proportion of total samples tested that are of sheep and that are of goat and (ii) whether a particular non-compliant result refers to a sample from a sheep or from a goat. in spite of the limitations highlighted above, an overall assessment of these data indicates that the percentage of non-compliant results is of a low order of magnitude compared with the total number of samples tested. ( ( ) ( ) ( ) antibacterials (unspecified) c published at http://ec.europa.eu/food/food/chemicalsafety/residues/control_en.htm a summary of the data presented in the previous tables (tables [ ] [ ] [ ] shows that of the ( . %) samples analysed in the eu nrcps during the period - were non-compliant for one or more substances listed in annex i of council directive / /ec. further details are presented in table . as mentioned above, one sample can be non-compliant for multiple substances, so that the number of non-compliant results is higher than the number of non-compliant samples. for example, for b substances, there were non-compliant results in non-compliant samples. one sample can be non-compliant for more than one substance. b published at http://ec.europa.eu/food/food/chemicalsafety/residues/control_en.htm c some of the samples were analysed for several substances in different subgroups (e.g. same sample analysed for b a, b b and b c), this total represents the total number of samples analysed for at least one substance in the group. it should be noted that the data in tables - provide the results for sampling and testing carried out by mss under the terms of council directive / /ec within the nrcps. however, there may be other chemical substances of relevance for control in sheep and goats, particularly in the case of contaminants, which are not included in the nrcps at all or which are not systematically covered by the nrcps. some of these substances are addressed further under tor of this opinion ('new hazards'). of the total number of samples taken for analysis during the period - , . % were taken at farm level while the remaining . % were taken at slaughterhouse level. no information on the types of animals sampled is readily available. results indicate that: . % of the total samples were non-compliant for one or more substances, with . %, . % and . % being non-compliant for group a, group b /b and group b substances, respectively. . % of all samples taken at farm level were non-compliant for one or more substances, with . %, . % and . % being non-compliant for group a, group b /b and group b substances, respectively. . % of all samples taken at slaughterhouse level were non-compliant for one or more substances, with . %, . % and . % being non-compliant for group a, group b /b and group b substances, respectively. the highest proportion of non-compliant results overall ( . %) was for group b substances, contaminants, representing largely exceedances of the mls/mrls specified for these substances. the proportions of non-compliant results overall for group a, prohibited substances ( . %), and for group b /b substances, vmps ( . %) represent largely illicit use of prohibited substances and exceedances of the mrls specified for vmps, respectively. an analysis of the results for sampling at farm level compared with slaughterhouse level indicates that for prohibited substances (group a) the rate of non-compliant results determined for sampling at farm level is considerably lower than that for sampling at slaughterhouse level. the majority ( %) of samples found to be non-compliant for prohibited substances relate to those having anabolic effects (thyreostats, steroids, zeranol, beta-agonists) and only a minority ( %) were non-compliant for substances such as chloramphenicol, nitrofurans and nitroimidazoles. while the incidence of noncompliant results from farm level sampling is low, such sampling is an integral component of the system for controlling illicit use of prohibited substances in food-producing animals, particularly in the case of substances having anabolic effects. in the case of vmps (group b /b ) the rate of non-compliant results determined at farm level is markedly higher than for sampling at slaughterhouse level. however, slaughterhouse-level sampling is more appropriate for identifying non-compliant samples for vmps, based on compliance with or exceedance of the specified mrls in edible tissues. in the case of contaminants (group b ) the rate of non-compliant results determined for sampling at slaughterhouse level is almost twofold higher than for sampling at farm level. indeed, sampling for group b substances is more appropriate, generally, at slaughterhouse level where identification of non-compliant results, based on compliance with or exceedance of specified mrls/mls in edible tissues, can be made. it should be noted also that a direct comparison of data from the nrcp over the years is not entirely appropriate as the test methods used and the number of samples tested for an individual residue varied between mss, and the specified mrls/mls for some substances may change over time. in addition, there are ongoing improvements in analytical methods, in terms of method sensitivity, accuracy and scope (i.e. number of substances covered by the method), which affects inter-year and inter-country comparisons. therefore, the cumulative data from the nrcps provide only a broad indication of the prevalence and nature of non-compliant samples. in conclusion, this compilation of data clearly indicates the low prevalence of abiotic hazards (residues and contaminants) in edible tissues of sheep and goats. only . % of the total number of analysed samples was non-compliant for one or more substances listed in annex i of council directive / /ec. based on these results, it can be concluded that potentially higher exposure of consumers to these substances from edible tissues of sheep and goats takes place only incidentally, as a result of mistakes or non-compliance with known and regulated procedures. the available aggregated data indicate the number of samples that were non-compliant with the current legislation. however, in the absence of species-and substance-specific information, such as the tissues used for residue analysis and the actual concentration of a residue or contaminant measured, these data do not allow for a reliable assessment of consumer exposure. while the data from the annual nrcp testing by mss indicate a relatively low incidence of noncompliant results for sheep and goats, there may be human health concerns regarding certain contaminants. for example, an evaluation undertaken by efsa (efsa contam panel, b) on the risk to public health related to the presence of high levels of dioxins and dioxin-like pcbs in liver from sheep (and deer) concluded that regular consumption of sheep liver would result, on average, in an approximate % increase of the median background exposure to dioxins and dioxin-like pcbs (dl-pcbs) for adults. the study also concluded that on individual occasions, consumption of sheep liver could result in high intakes exceeding the tolerable weekly intake (twi), and that the frequent consumption of sheep liver, particularly by women of child-bearing age and children, may be a potential health concern. independent from the occurrence data as reported from the nrcps, each substance or group of chemical substances that may enter the food chain was also evaluated for the likelihood that potentially toxic or undesirable substances might occur in sheep and goat carcasses. for prohibited substances and vmps/feed additives, the following criteria were used: the likelihood of the substance(s) being used in an illicit or non-compliant way in sheep and goats (suitability for sheep and goat production; commercial advantages) the potential availability of the substance(s) for illicit or non-compliant usage in sheep and goat production (allowed usage in third countries; availability in suitable form for use in sheep and goats; non-authorised supply chain availability ('black market'); common or rare usage as a commercial licensed product) the likelihood of the substance(s) occurring as residue(s) in edible tissues of sheep and goats based on the kinetic data (pharmacokinetic and withdrawal period data; persistence characteristics; special residue issues, e.g. bound residues of nitrofurans) toxicological profile and nature of hazard and the relative contribution of residues in sheep and goats to dietary human exposure. for contaminants, the following criteria were considered: the prevalence (where available) of occurrence of the substances in animal feeds/forages and pastures, and of the specific environmental conditions in which the animals are raised the level and duration of exposure, tissue distribution and deposition including accumulation in edible tissues of sheep and goats toxicological profile and nature of hazard and the relative contribution of residues in sheep and goats to dietary human exposure. considering the above mentioned criteria, a flow-chart approach was used for ranking of the chemical residues and contaminants of potential concern. the outcome of the nrcps (indicating the number of non-compliant results), the evaluation of the likelihood that residues of substances of potential concern can occur in sheep and goats and the toxicological profile of the substances were considered in the development of the general flow chart, presented in figure . ml, maximum level; mrl, maximum residue limits; nrcp: national residue control plan. a contaminants from the soil and the environment, associated with feed material, are considered to be part of the total feed for the purposes of this opinion. b potential concern was based on the toxicological profile and nature of hazard for the substances. c the contam panel notes that the ranking of vmps/feed additives was carried out in the general context of authorised usage of these substances in terms of doses, route of treatment, animal species and withdrawal periods. therefore, this ranking is made within the framework of the current regulations and control and within the context of a low rate of exceedances in the nrcps. d see definitions as provided in the next section . . . figure : general flow chart used for the ranking of residues and contaminants of potential concern that can be detected in sheep and goats. outcome of the ranking of residues and contaminants of potential concern that can occur in sheep/goat carcasses four categories were established resulting from the application of the general flow chart: substance irrelevant in sheep/goat production (no known use at any stage of production); no evidence for illicit use or abuse in sheep/goats; not or very seldom associated with exceedances in mrls in nrcps; no evidence of occurrence as a contaminant in feed for sheep/goats. vmps/feed additives which have an application in sheep/goat production, residues above mrls are found in control plans, but substances are of low toxicological concern; contaminants and prohibited substances with a toxicological profile that does not include specific hazards following accidental exposure of consumers and which are generally not found or are not found above mls in sheep/goats. contaminants and prohibited substances to which sheep/goats are known to be exposed and/or with a history of misuse, with a toxicological profile that does not entirely exclude specific hazards following accidental exposure of consumers; evidence for residues of prohibited substances being found in sheep/goats; contaminants generally not found in concentrations above the mrl/mls in edible tissues of sheep/goats. contaminants and prohibited substances to which sheep/goats are known to be exposed and with a history of misuse, with a distinct toxicological profile comprising a potential concern to consumers; evidence for ongoing occurrence of residues of prohibited substances in sheep/goats; evidence for ongoing occurrence and exposure of sheep/goats to feed contaminants. . . . . substances classified in the category of high potential concern . . . . . contaminants: dioxins, dioxin-like polychlorinated biphenyls (dl-pcbs) in the high potential concern category are dioxins and dioxin-like polychlorinated biphenyls (dl-pcbs) as the occurrence data from the monitoring programmes show a number of incidents due to contamination of feed, such as illegal disposal of dioxin-and dl-pcb-containing waste materials into feed components, or open drying of feed components with dioxin-containing fuel materials. dioxins are persistent organochlorine contaminants that are not produced intentionally and have no targeted use, but are formed as unwanted and often unavoidable by-products in a number of thermal and industrial processes. because of their low water solubility and high lipophilic properties, they bioaccumulate in the food chain and are stored in fatty tissues of animals and humans. the major pathway of human dioxin exposure is via consumption of food of animal origin which generally contributes more than % of the total daily dioxin intake (efsa, ) . a number of incidents in the past years were caused by contamination of feed with dioxins. examples are feeding of contaminated citrus pulp pellets, kaolinitic clay containing potato peel or mixing of compound feed with contaminated fatty acids. all these incidents were caused by grossly negligent or criminal actions and led to widespread contamination of feed and subsequently to elevated dioxin levels in the animals and the foodstuffs produced from them. monitoring programmes also demonstrated that certain food commodities, such as sheep liver can have high dioxin levels even when not affected by specific contamination sources. in , the contam panel delivered a scientific opinion on the risk to public health related to the presence of high levels of dioxins and dl-pcbs in liver from sheep and deer (efsa contam panel, b). efsa evaluated, inter alia, the dioxin and pcb results from sheep liver and sheep meat samples submitted by eight european countries. almost all sheep meat samples were below the relevant mls set by regulation (ec) no / . however, the corresponding liver samples from the same sheep in more than half of the cases exceeded the relevant maximum levels considerably. this finding is likely to be associated with differences in the level of biotransformation enzymes in sheep compared with bovine animals. dioxins have a long half-life and are accumulated in various tissues. the findings of elevated levels in food are of public health concern owing to their potential effects on liver, thyroid, immune function, reproduction and neurodevelopment (efsa, a (efsa, , . the available data indicate that a substantial part of the european population is in the range of or already exceeding the twi for dioxins and dl-pcbs. a report on "monitoring of dioxins and pcbs in food and feed" (efsa, ) estimated that between . % and . % of individuals were exposed above the twi of pg teq/kg body weight (b.w.) for the sum of dioxins and dl-pcbs. in addition to milk and dairy products and fish and seafood, meat and meat products also contributed significantly to total exposure. owing to the high toxic potential of dioxins and the incidence of samples of sheep meat and sheep liver exceeding the maximum limits, efforts need to be undertaken to reduce exposure where possible. in summary, based on the high toxicity and the low maximum levels set for meat and fat of sheep (see table ) and considering that food of animal origin contributes significantly (> %) to human exposure, dioxins have been ranked in the category of substances of high potential concern. (b) dioxin-like polychlorinated biphenyls (dl-pcbs) in contrast to dioxins, pcbs had widespread use in numerous industrial applications, generally in the form of complex technical mixtures. due to their physicochemical properties, such as nonflammability, chemical stability, high boiling point, low heat conductivity and high dielectric constants, pcbs were widely used in industrial and commercial closed and open applications. they were produced for over four decades, from onwards until they were banned, with an estimated total world production of . - . million tonnes. according to council directive / /ec, mss were required to take the necessary measures to ensure that used pcbs are disposed off and equipment containing pcbs is decontaminated or disposed of at the latest by the end of . earlier experience has shown that illegal practices of pcb disposal may occur resulting in considerable contamination of animals and foodstuffs of animal origin. on the other hand, monitoring programmes also demonstrated that certain food commodities, such as sheep liver can have high pcb levels even when not affected by specific contamination sources. this was demonstrated by efsa in its scientific opinion on the risk to public health related to the presence of high levels of dioxins and pcbs in liver from sheep and deer (efsa contam panel, b). efsa evaluated, inter alia, the dioxin and pcb results from sheep liver and sheep meat samples submitted by eight european countries. for sheep liver, the mean upper bound concentration for dl-pcbs (expressed as who-teq ) was . (range: . - . ) pg who-teq/g fat. the corresponding levels in sheep meat were considerably lower: . (range: . - . ) pg who-teq/g fat (efsa contam panel, b). based on structural characteristics and toxicological effects, pcbs can be divided into two groups. one group consists of congeners that can easily adopt a coplanar structure and have the ability to bind to the aryl hydrocarbon (ah) receptor, thus showing toxicological properties similar to dioxins (effects on liver, thyroid, immune function, reproduction and neurodevelopment). this group of pcbs is therefore called "dioxin-like pcbs". the other pcbs do not show dioxin-like toxicity but have a different toxicological profile, in particular with respect to effects on the developing nervous system and neurotransmitter function. this group of pcbs is called "non dioxin-like pcbs" (see below). as dl-pcbs, in general, show a comparable lipophilicity, bioaccumulation, toxicity and mode of action as dioxins (efsa, a) , these two groups of environmental contaminants are regulated together in european legislation and are considered together in risk assessments. based on the high toxicity, widespread use and potential for improper disposal practices of technical pcb mixtures, dl-pcbs have been ranked in the category of substances of high potential concern. . . . . substances classified in the category of medium potential concern . . . . . prohibited substances: stilbenes, thyreostats, gonadal (sex) steroids, resorcylic acid lactones, beta-agonists, chloramphenicol and nitrofurans (a) stilbenes the toxicity of stilbenes is well established (for review see waltner-toews and mcewen, ) and this has led to their prohibition for use as growth promoters in animals in most countries, based also on their involvement in the baby food scandal in the late s (loizzo et al., ) . in particular, diethylstilbestrol is a proven human genotoxic carcinogen (group i iarc (international agency for research on cancer)) (iarc, ), while sufficient evidence for hexestrol and limited evidence for dienestrol for carcinogenicity in animals were found (iarc, ) . diethylstilbestrol is associated with cancer of the breast in women who were exposed while pregnant, and also causes adenocarcinoma in the vagina and cervix of women who were exposed in utero; finally, a positive association has been observed between exposure to diethylstilbestrol and cancer of the endometrium, and between in utero exposure to diethylstilbestrol and squamous cell carcinoma of the cervix and cancer of the testis. in , the use of stilbenes in all species of food-producing animals was prohibited in the european community by directive / /eec. diethylstilbestrol, and other stilbenes such as hexestrol and dienestrol, are likely to be available on the black market and, therefore, might be available for illicit use in sheep and goats. no non-compliant results for stilbenes in sheep and goat samples have been reported from the european nrcps - , indicating that abuse of stilbenes in sheep and goat production in the eu is unlikely. considering that stilbenes have proven toxicity for humans, these substances are ranked as of medium potential concern. however, considering that there is no evidence for current use of stilbenes in sheep and goat production and that no non-compliant results have been found over a number of years of nrcp testing, control measures for stilbenes might be focused on identifying any potential future abuse of these substances in sheep and goat production in the eu. thyreostats are a group of substances that inhibit the thyroid function, resulting in decreased production of the thyroid hormones triiodothyronine (t ) and thyroxine (t ). enlargement of the thyroid gland has been proposed as a criterion to identify illicit use of these compounds (vos et al., ; vanden bussche et al., ) . they are used in human and in non-food-producing animal medicine to deal with hyperthyroidism. the use of thyreostats for animal fattening is based on weight gain caused by filling of the gastrointestinal tract and retention of water in muscle tissues (courtheyn et al., ) . synthetic thyreostats include thiouracil, methylthiouracil, propylthiouracil, methimazole, tapazol (methylmercaptoimidazole) and mercaptobenzimidazole (mbi). use of synthetic thyreostats in food-producing animals has been prohibited in the eu since (council directive / /ec). naturally occurring thyreostats include thiocyanates and oxazolidine- -thiones, which are present as glucosinolates in plant material such as in the seeds of cruciferae, like rapeseed (efsa, b; vanden bussche et al., ) . evidence for the occurrence of thiouracil in urine of cattle fed on a cruciferous-based diet has been demonstrated (pinel et al., ) . thyreostats are very widely available on the black market so there is the possibility for illicit use in sheep/goat production. the results from the european nrcps - show that sheep/goat samples were found to be non-compliant for thyreostats ( non-compliant results out of the total samples analysed for thyreostats). however, it has been shown that the source of the generally low levels of thiouracil determined in urine samples may be from exposure of animals through their diet (le bizec et al., ) . some mss reporting the highest numbers of non-compliant samples for thiouracil state that "the presence of thiouracil in low concentrations may be due to the animals eating cruciferous plant material" and "in line with scientific evidence, the competent authority has concluded that the residues resulted from dietary factors". thyreostats have been considered to be carcinogenic and teratogenic. while the in utero exposure to methimazole or propylthiouracil has been associated with aplasia cutis and a number of other congenital defects (löllgen et al., ; rodriguez-garcia et al., ) , an iarc evaluation found inadequate evidence in humans, but limited evidence (in the case of methimazole) and sufficient evidence (in the case of thiouracil, methylthiouracil and propylthiouracil) in experimental animals for carcinogenicity (iarc, ; efsa b) . thyreostats are prohibited substances owing to their potential toxicity to humans and their efficacy as growth promoters in sheep/goats, but considering that the non-compliant results that have been found in most years of nrcp testing have been attributed largely to a dietary source, these substances are ranked as of medium potential concern. control measures for thyreostats might focus on identifying potential abuse of these substances in sheep and goat production in the eu. (c) gonadal (sex) steroids a broad range of steroids derived from oestrogens, androgens and progestagens are available and have been used as growth-promoting agents in food-producing animals. there is an extensive body of animal production research demonstrating the efficacy of anabolic steroids, often in combination treatments of an oestrogen and an androgen (or progestagen), as growth promoters. all use of steroids as growth-promoting agents in food-producing animals is banned according to council directive / /ec, as amended by directives / /ec and / /ec. the latter included βoestradiol in the list of prohibited substances owing to its demonstrated tumour-promoting (epigenetic) and tumour initiating (genotoxic) properties (russo et al., ) . certain uses of β-oestradiol, progesterone and medroxyprogesterone acetate in sheep and/or goats are allowed for therapeutic or zootechnical purposes only (commission regulation (eu) no / ). there is evidence that anabolic steroids are of economic value for farmers as animals respond to their application with increased growth rate and feed conversion efficiency. anabolic steroids are widely available on the black market so there is the possibility for illicit use in sheep and goat production. , . . , p. - . an accurate estimate for the level of abuse of anabolic steroids in european sheep and goat production from these data. there are divergent views on the potential adverse effects for the consumer from residues of anabolic steroids in edible tissues of treated animals. there is concern regarding the carcinogenic effects of oestrogenic substances, and the long-term effects of exposure of prepubescent children to oestrogenic substances. in the scientific committee on veterinary measures relating to public health (scvph) performed an assessment of the potential risks to human health from hormone residues in bovine meat and meat products (scvph, (scvph, , (scvph, , , particularly as regards the three natural hormones ( β-oestradiol, testosterone, progesterone) and the three synthetic analogues (zeranol, trenbolone acetate, melengestrol acetate) that may be legally used as growth promoters in third countries. it was concluded that, taking into account both the hormonal and nonhormonal toxicological effects, the issues of concern include neurobiological, developmental, reproductive and immunological effects, as well as immunotoxicity, genotoxicity and carcinogenicity. in consideration of concerns relating to the lack of understanding of critical developmental periods in human life as well as uncertainties in the estimates of endogenous hormone production rates and metabolic clearance capacity, particularly in prepubertal children, no threshold level and therefore no adi could be established for any of the six hormones. according to iarc, β-oestradiol and steroidal oestrogens are classified as proven human carcinogens (group ), androgenic (anabolic) steroids as probably carcinogenic to humans (group a); for most progestagens, evidence for human carcinogenicity is inadequate while that for animals varies from sufficient to inadequate (iarc, ) . notwithstanding the toxicological profile of gonadal (sex) steroids, owing to the low prevalence of non-compliant samples from confirmed illicit use in the nrcps, these substances are ranked as of medium potential concern. (d) resorcylic acid lactones (rals) in the eu, zeranol was evaluated together with other hormonal growth promoters by the scvph (scvph (scvph , (scvph , . in these scientific opinions it was concluded that, taking into account both the hormonal and non-hormonal toxicological effects, no adi could be established for any of the six hormones, including zeranol. use of zeranol as a growth promoter in cattle production was widespread in some mss prior to its prohibition in europe in . zeranol is widely available as a commercial product and is used extensively in third countries. hence it is readily available on the market and there is the possibility for its illicit use in cattle production in the eu. zeranol is derived from, and can also occur as, a metabolite of the mycotoxin zearalenone, produced by fusarium spp. the results from the european nrcps - show sheep/goat samples non-compliant for resorcylic acid lactones (a total of seven non-compliant results out of the total samples analysed). however, it has been shown that the source of the generally low levels of zeranol and its metabolites determined in these samples may be from exposure of sheep/goats to the mycotoxin zearalenone through their diet (efsa, a) . some mss reporting non-compliant results for zeranol and its metabolites state that "the residue was found to be as a result of feed contamination on the farm" and it was "probably attributable to mycotoxin contamination of feed". rals are prohibited substances owing to their potential toxicity to humans and their efficacy as growth promoters in sheep and goats, but considering that the non-compliant results that have been found in some years of nrcp testing have been attributed largely to a dietary source, these substances are ranked as of medium potential concern. control measures for rals might focus on identifying potential abuse of these substances in sheep and goat production in the eu. (e) beta-agonists beta-agonists, or β-adrenergic agonists, have therapeutic uses as bronchodilatory and tocolytic agents. a wide range of beta-agonists have been developed, such as clenbuterol, salbutamol, cimaterol, terbutaline, ractopamine, etc., and all of these are prohibited for use as growth-promoting agents in food-producing animals in the eu. salbutamol and terbutaline are licensed human medicines indicated for treatment of asthma and bronchospasm conditions and for prevention of premature labour, respectively. one of the beta-agonists, clenbuterol, is licensed for therapeutic use in cattle (as a tocolytic agent) and in the treatment of obstructive airway conditions in horses (commission regulation (eu) no / ). other beta-agonists, such as ractopamine, have been approved for use in food-producing animals in a number of third countries. treatment of sheep with beta-agonists, such as clenbuterol, results in increased muscle mass and increased carcass leanness (baker et al., ) . the commercial benefits of using beta-agonists in sheep and goat production, particularly lambs, combined with the availability of these substances, indicates that illicit use of beta-agonists as growth promoters cannot be excluded. an outbreak of collective food poisoning from the ingestion of lamb meat containing residues of clenbuterol has been reported in portugal; symptoms shown by the intoxicated people may be generally described as gross tremors of the extremities, tachycardia, nausea, headaches and dizziness (barbosa et al., ) . in the light of the known adverse biological effects of beta-agonists in humans, particularly clenbuterol, and the efficacy of such drugs as repartitioning agents in sheep/goats, but considering that no non-compliant results for sheep/goats have not been found in the nrcps since , these substances currently are ranked as of medium potential concern. (f) chloramphenicol chloramphenicol is an antibiotic substance, first used for the treatment of typhoid in the late s. chloramphenicol may produce blood dyscrasias in humans, particularly bone marrow aplasia, or aplastic anaemia, which may be fatal. there is no clear correlation between dose and the development of aplastic anaemia and the mechanism of induction of aplastic anaemia is not fully understood (watson, ) . although the incidence of aplastic anaemia associated with exposure to chloramphenicol is apparently very low, no threshold level could be defined (emea, ). in addition, several studies suggest that chloramphenicol and some of its metabolites are genotoxic (fao/who, emea, ) . considering the available evidence from in vitro experiments and from animal studies, as well as from a case-control study conducted in china, in which there was evidence for the induction of leukaemia in patients receiving a long-term treatment with chloramphenicol, iarc classified chloramphenicol as group a (probably carcinogenic to humans) substance (iarc, ) . based on these evaluations, the use of chloramphenicol in food-producing animals is prohibited within the eu to avoid the exposure of consumers to potential residues in animal tissues, milk and eggs. consequently, chloramphenicol is included in table until its prohibition, chloramphenicol was used on food-producing animals, including sheep and goats, for treatment of salmonella infections and for prevention of secondary bacterial infections. currently, chloramphenicol, which is licensed for use as a broad-spectrum bacteriostatic antibacterial in pets and non-food-producing animals in the eu, is used also in some third countries for foodproducing animals. hence, chloramphenicol may be available on the black market for illicit use in sheep/goat production. however, the availability for use on food-producing animals of related substances with similar antibacterial properties, thiamphenicol and florfenicol (with no toxicological concern), should mitigate the illicit use of chloramphenicol in sheep/goat production as these alternative drugs are available as prescription medicines. non-compliant results for chloramphenicol in sheep/goats have been reported in most years' results from the european nrcps - ( non-compliant results), indicating that abuse of chloramphenicol in sheep/goat production in europe may be a continuing occurrence. chloramphenicol has proven toxicity for humans and is effective as an antibacterial treatment for sheep/goats but, considering that lower numbers of non-compliant results have been found in recent years of the nrcp testing, chloramphenicol currently is ranked as of medium potential concern. (g) nitrofurans nitrofurans, including furazolidone, furaltadone, nitrofurantoin and nitrofurazone, are very effective antimicrobial agents that, prior to their prohibition for use on food-producing animals in the eu in , were widely used on livestock (cattle, sheep/goats, pigs, sheep and goats), in aquaculture and in bees. various nitrofuran antimicrobials are still applied in human medicine particularly for the treatment of urinary tract infections. a characteristic of nitrofurans is the short half-life of the parent compounds and the formation of covalently bound metabolites which, under the acidic conditions of the human stomach, may be released as active agents (hoogenboom et al., ) . these covalently bound metabolites are used as marker residues for detecting the illicit use of nitrofurans in animal production. it should be noted that the metabolite semicarbazide (sem) has been shown not to be an unambiguous marker for abuse of the nitrofuran drug nitrofurazone because the sem molecule may occur from other sources (hoenicke, et al., ; sarnsonova et al., ; bendall, ). nitrofurans are effective in treatment of bacterial and protozoal infections, including coccidiosis, in food-producing animals. although prohibited for use on food-producing animals in many countries, nitrofurans are likely to be available on the black market for illicit use in sheep/goat production. noncompliant results for nitrofurans in sheep/goats have been reported in most years' results from the european nrcps - , indicating that abuse of nitrofurans in sheep/goat production in europe is a continuing occurrence. a metabolite of furazolidone that can be released from covalently bound residues in tissues has been shown to be mutagenic and may be involved in the carcinogenic properties of the parent compound (emea, a). nitrofurans have proven toxicity for humans and are effective as antibacterials for sheep and goats but, considering that non-compliant results, other than for the marker residue sem, are found only sporadically in the nrcp testing, these substances currently are ranked as of medium potential concern. . . . . . contaminants: non dioxin-like pcbs (ndl-pcbs), chemical elements and mycotoxins (a) non dioxin-like pcbs (ndl-pcbs) the non dioxin-like pcbs (ndl-pcbs) show a different toxicological profile to the dl-pcbs. in , the contam panel performed a risk assessment on ndl-pcbs in food (efsa, a) . in the final conclusion, the contam panel stated that no health-based guidance value for humans can be established for ndl-pcbs because simultaneous exposure to dioxin-like compounds hampers the interpretation of the results of the toxicological and epidemiological studies, and the database on effects of individual ndl-pcb congeners is rather limited. there are, however, indications that subtle developmental effects, caused by ndl-pcbs, dl-pcbs, or polychlorinated dibenzo-pdioxins/polychlorinated dibenzofurans alone, or in combination, may occur at maternal body burdens that are only slightly higher than those expected from the average daily intake in european countries. in its risk assessment the contam panel decided to use the sum of the six pcb congeners (- , - , - , - , - and - ) as the basis for their evaluation, because these congeners are appropriate indicators for different pcb patterns in various sample matrices and are most suitable for a potential concern assessment of ndl-pcbs on the basis of the available data. moreover, the panel noted that the sum of these six indicator pcbs represents about % of total ndl-pcbs in food (efsa, a because of their somewhat lower toxicity than that of dl-pcbs, ndl-pcbs are classified in the medium potential concern category. (b) chemical elements (heavy metals: cadmium, mercury and lead) among the chemical elements, heavy metals traditionally have gained attention as contaminants in animal tissues, as they may accumulate in certain organs, particularly in kidneys over the lifespan of an animal. kidney tissue from sheep forms a specific dietary component in many european cultures. exposure of animals is commonly related to contaminated feed materials, despite older reports of accidental intoxication of animals from other sources (paints, batteries). the contam panel has issued within the framework of the re-evaluation of undesirable substances in animal feeds according to directive / /ec several opinions addressing heavy metals and arsenic in feed materials and the transfer of these elements from feed to edible tissues, milk and eggs. cadmium (efsa, a) is a heavy metal found as an environmental contaminant, both through natural occurrence and from industrial and agricultural sources. cadmium accumulates in humans and animals, causing concentration-dependent renal tubular damage. older animals are expected to have higher concentrations of cadmium accumulated in the kidneys. most of the non-compliant results were for kidney samples with some non-compliant results for muscle and liver being reported. mercury (efsa, a , efsa contam panel, a exists in the environment as elemental mercury, inorganic mercury and organic mercury (primarily methylmercury). methylmercury bioaccumulates and biomagnifies along the aquatic food chain. the toxicity and toxicokinetics of mercury in animals and humans depends on its chemical form. elemental mercury is volatile and mainly absorbed through the respiratory tract, whereas its absorption through the gastrointestinal tract is limited ( - %). following absorption, inorganic mercury distributes mainly to the kidneys and, to a lesser extent, to the liver. the critical effect of inorganic mercury is renal damage. in contrast, in animals, as in humans, methylmercury and its salts are readily absorbed in the gastrointestinal tract (> %) and rapidly distributed to all tissues, although the highest concentrations are also found in the kidneys. data from mss indicated the presence of mercury in animal feeds, but the measured concentrations remained below the maximum content for feed materials ( lead (efsa contam panel, ) is an environmental contaminant that occurs naturally and, to a greater extent, from anthropogenic activities such as mining and smelting and battery manufacturing. lead is a metal that occurs in organic and inorganic forms; the latter predominate in the environment. human exposure is associated particularly with the consumption of cereal grains (except rice), cereal and cereal-based products, potatoes, leafy vegetables and tap water. the contribution of sheep and goat meat and offal to human exposure is limited. given the toxicological profile of these elements and the fact that cadmium accumulates in animals and humans, these three elements have been allocated to the group of substances of medium potential health concern. . . . . substances classified in the category of low potential concern . . . . . prohibited substances: nitroimidazoles, chlorpromazine (a) nitroimidazoles the -nitroimidazoles, dimetridazole, metronidazole and ronidazole, are a group of drugs having antibacterial, antiprotozoal and anticoccidial properties. owing to the potential harmful effects of these drugs on human health (emea, b)-carcinogenicity, mutagenicity, genotoxicity and the occurrence of covalent binding to macromolecules of metabolites with an intact imidazole structuretheir use in food-producing animals is prohibited in the eu, united states, china, and other countries. nitroimidazoles had been used as veterinary drugs for the treatment of cattle, pigs and sheep and goats. although prohibited for use on food-producing animals, not only in the eu but also in many third countries, nitroimidazoles are likely to be available on the black market for illicit use in animal production, particularly as drugs such as metronidazole are readily available as human medicines. however, there are no clinical conditions in sheep/goats for which nitroimidazoles are particularly appropriate. non-compliant results (two) for nitroimidazoles in sheep/goats have been reported only in one year and from one ms from the european nrcps - , suggesting that abuse of nitroimidazoles in sheep/goat production in europe is not widespread. considering that nitroimidazoles have proven toxicity for humans and that they may be effective as antibacterial/antiprotozoal treatments for sheep/goats, these substances might be ranked as of medium potential concern. however, as only occasional non-compliant results have been found over a number of years of nrcp testing, nitroimidazoles currently are ranked as of low potential concern. (b) chlorpromazine chlorpromazine is a sedative and is also used against motion sickness and as an anti-emetic in pets. its use is banned in food-producing animals, including sheep/goats. chlorpromazine is likely to be available as a black market substance for illicit use in sheep/goat production. no non-compliant results for chlorpromazine were reported from the nrcp for the period - , indicating that the substance may not be rarely used illicitly in sheep/goat production in the eu. chlorpromazine is used as an antipsychotic drug in human therapy and has long-term persistence in humans and numerous side effects, including the more common ones of agitation, constipation, dizziness, drowsiness, etc. (emea, ) . chlorpromazine may be effective as a tranquilliser for sheep/goats but, since no non-compliant results have been found over a number of years of nrcp testing, chlorapromazine currently is ranked as of low potential concern. . . . . . contaminants: organochlorine pesticides, organophosphorus compounds, and natural toxins (a) organochlorine compounds organochlorine pesticides, such as dichlorodiphenyltrichloroethane (ddt) and its metabolites, hexachlorocyclohexanes (hchs), dieldrin, toxaphene and others have been assigned to the category of contaminants of low potential concern. occurrence of residues of these substances has declined over the years, because of their long-standing ban, and relatively low levels in animal products can be expected, as shown by results from the nrcps - , which indicate that results out of the total of samples tested for the category of organochlorine compounds were non-compliant for organochlorine pesticides. organophosphorus compounds are classified in council directive / /ec as group b b contaminants, although they may be used also as vmps for the therapy of parasitic infestations of sheep and goats. however, their probably infrequent use and short half-life results in these compounds being assigned to the category of low potential concern, or even negligible potential concern where mrls are not exceeded. results from the nrcps from - indicate that results out of the total of samples tested for the category of organophosphorus compounds were non-compliant. (c) natural toxins: mycotoxins and toxic plant secondary metabolites (c. ) mycotoxins mycotoxins comprise a chemically diverse group of secondary metabolites of moulds which may induce intoxication in humans and animals following ingestion of contaminated food or feed materials. mycotoxins evaluated by the contam panel as undesirable contaminants in animal feeds, including aflatoxins (efsa, b ), deoxynivalenol (efsa, c , fumonisins (efsa, b) and zearalenone (efsa, a) , t- toxin (efsa contam panel, c), ergot alkaloids (efsa contam panel, b) may pose a risk for animal health and productivity when present in feed materials that are used for sheep and goat animals over an extended period of time. however, most of the known mycotoxins are efficiently degraded by the rumen microflora and have a short biological half-life. hence, even if residues of mycotoxins are occasionally detected in animal tissues (monogastric animal species) they do not contribute significantly to human exposure, which is mainly related to the consumption of cereal products, nuts and spices. considering that some mycotoxins like aflatoxins have proven toxicity for humans, some of these substances might be ranked as of medium potential concern. however, since non-compliant results have been found incidentally (two out of samples) over a number of years of nrcp testing, these substances currently are ranked as of low potential concern. (c. (efsa, f) . although for several of these substances potential concerns for animal health could be identified following ingestion with feed, none of these natural toxins appeared to accumulate in edible tissues. the limited data on the kinetics of these metabolites does not preclude in all cases a transfer from the feed into animal tissues under certain circumstances of exposure. for example, residues of gossypol in meat of cattle (and sheep) were demonstrated under experimental conditions (feeding of cotton meal as the main feed component), but such residues are not expected under the conditions of european farming, where cotton seeds or cotton seed by-products are infrequently used and only with limited inclusion rates in feed (efsa, e) . other natural substances, such as the fungal metabolite (mycotoxin) zearalenone, are intensively metabolised in the rumen and following absorption in the liver and other animal tissues, and this may explain certain noncompliant analytical results. zearalanol (zeranol) is one of these metabolites and which is used in certain third countries as a growth-promoting agent owing to its oestrogenic activity (see section . . . . (d) ). this applies also to certain thiocyanates and oxazolidinethiones, originating from glucosinolates produced by a broad variety of plants of the brassicaceae family. they target different steps in the synthesis of thyroid hormones, leading eventually to hypothyroidism and enlargement of the thyroid gland (goitre) (efsa, b) . again, these natural products may explain some of the noncompliant results found in nrcp testing where treatment of animals with antithyroid agents (thyreostats) has been suspected. recently, an increasing use of herbal remedies, given as so-called alternatives to antibiotics for animals, has been reported also in ruminants. many of the herbal products contain biologically active substances that are also addressed in the list of undesirable plant metabolites. however, the remedies are given in low concentrations (lower than the larger amount that could be ingested with feed), and for a limited period. although specific data are lacking, it seems unlikely that residues of these compounds may be found in edible tissues of slaughtered animals. such substances, therefore, are placed in the category of low potential concern within the current classification. vmps, such as antimicrobials, anti-coccidials and anti-parasitics, are used commonly on sheep and goats for prophylactic purposes, particularly prior to turning animals out to grazing (anti-parasitic treatments). therapeutic use of vmps, particularly antimicrobials, may occur in response to diagnosis of infection in individual animals or in the flock. in general, vmps, except the substances allocated to annex table of regulation (ec) no / , are categorised as being of low potential concern because they have all been subjected to premarketing approval which specifies adis, and mrls, with the aim of guaranteeing a high level of safety to the consumer. where exceedances of mrls are found in the residue monitoring programmes (i.e. non-compliant results out of the samples analysed for antibacterials, non-compliant results for anthelmintics out of the samples analysed, and eight non-compliant results out of the samples analysed for anti-coccidials), these are typically of an occasional nature that is not likely to constitute a concern to public health. despite only two non-compliant results being reported out of the samples analysed for corticosteroids, there is concern about their potential illicit use, particularly in fattening lambs. in the negligible potential concern category are the dyes and the prohibited substances, colchicine, dapsone, chloroform and aristolochia spp. . . . . . prohibited substances: colchicine, dapsone, chloroform and aristolochia spp. colchicine is a plant alkaloid that has been used in veterinary medicine to treat papillomas and warts in cattle and horses by injection at the affected area. a possible contamination of food with colchicine has been identified through consumption of colchicum autumnale in forage by animals such as cattle or sheep and, in this context, colchicine has been determined in milk of sheep after exposure to c. autumnale (hamscher et al., ) . colchicine is genotoxic and teratogenic and may have toxic effects on reproduction. no non-compliant results for colchicine in sheep/goats have been reported from the european nrcps - ; however, it is probable that testing for this substance may not be included in monitoring programmes in many countries. in the absence of the absence of evidence for use of colchicine in sheep/goats colchicine currently is ranked as of negligible potential concern. (b) dapsone dapsone is a drug used in humans and formerly in veterinary medicine: in human medicine it is used for treatment of leprosy, malaria, tuberculosis and dermatitis; and in veterinary medicine it is used as an intramammary treatment for mastitis, for oral treatment of coccidiosis and for intra-uterine treatment of endometriosis. following scientific assessment by the committee for medicinal products for veterinary use (cvmp), a provisional mrl of µg/kg parent drug was established for muscle, kidney, liver, fat and milk for all food-producing animals (emea, ) . further information on teratogenicity and reproductive effects for dapsone was required but, when this was not provided, the substance was recommended for inclusion in annex iv to council regulation (eec) no / (now annex, table , of commission regulation (ec) no / ). more recently, the cvmp has reviewed the alleged mutagenicity of dapsone in the context of its occurrence as an impurity in vmps containing sulphonamides and concluded that it is not genotoxic (cvmp, ) , and efsa has issued a scientific opinion on the product as a food-packaging material (compound ), proposing an acceptable level of mg/kg food (efsa, c) . in the absence of evidence for use of dapsone in sheep and goats, dapsone currently is ranked as of negligible potential concern. (c) chloroform and aristolochia spp. in the negligible potential concern category are the prohibited substances, chloroform and plant remedies containing aristolochia spp., as these are not relevant to sheep/goat production and there is no evidence for use of these substances in sheep/goat production. vmps used in sheep and goat production but with no evidence for residues above mrls being found in monitoring programmes and vmps irrelevant for sheep and goat production are ranked as of negligible potential concern. (a) carbamates and pyrethroids carbamates and pyrethroids are used in animal houses and occasionally in animals including sheep for control of environmental infections, such as lice eggs in buildings. there are no recent incidents of non-compliant results reported in nrcp testing in sheep and goats during the period - , resulting in these substances being assigned to the category of negligible potential concern. (b) sedatives a range of sedative substances including barbiturates, promazines, xylazine and ketamine, are licensed for use in sheep, goats and other animal species for sedation and analgesia during surgical procedures or for euthanasia. they are rarely used in sheep and goats. no non-compliant results were found in the nrcp testing for the period - . due to their rapid excretion, these substances generally do not have detectable residues in muscle and so do not have mrls registered in the eu. animals euthanised with these substances are not allowed to enter the food chain. however, it should be noted that testing for this category of substances is not required under the provisions of council directive / /ec. there are no indications for use of dyes such as (leuco-)malachite green in sheep and goat animals. testing of sheep and goat animals for this group of substances is not required under council directive / /ec . a summary of the outcome of the ranking is presented in table . dioxins dl-pcbs mrl, maximum residue limit; nrcp, national residue control plan; psm, plant secondary metabolite; vmp, veterinary medicinal product. the ranking into specific categories of potential concern of prohibited substances, vmps and contaminants presented in this section applies exclusively to sheep and goats and is based on current knowledge regarding the toxicological profiles, usage in ovine animal production, and occurrence as residues or contaminants, as demonstrated by the data from the nrcps for the - period. where changes in any of these factors occur, the ranking might need amendment. another element of future aspects is the issue of 'new hazards'. in this context, new hazards are defined as compounds that have been identified as anthropogenic chemicals in food-producing animals and derived products and in humans and for which occurrence data are scarce. it does not imply that there is evidence for an increasing trend in the concentration of these compounds in food or in human samples. examples are brominated flame retardants, such as polybrominated diphenyl ethers (pbde) and hexabromocyclododecanes (hbcdds) or perfluorinated compounds (pfc), such as perfluorooctane sulphonate (pfos) and perfluorooctanoic acid (pfoa). in , efsa performed a risk assessment on polybrominated diphenyl ethers (pbdes) in food (efsa contam panel, e) . pbdes are additive flame retardants which are applied in plastics, textiles, electronic castings and circuitry. pbdes are ubiquitously present in the environment and likewise in biota and in food and feed. eight congeners were considered by the contam panel to be of primary interest: . the highest dietary exposure is to . toxicity studies were carried out with technical pbde mixtures or individual congeners. the main targets were the liver, thyroid hormone homeostasis and the reproductive and nervous system. pbdes are not genotoxic. the contam panel identified effects on neurodevelopment as the critical endpoint, and derived benchmark doses (bmds) and their corresponding lower % confidence limit for a benchmark response of %, the bmdl , for a number of pbde congeners: bde- , μg/kg b.w.; bde- , μg/kg b.w.; bde- , μg/kg b.w.; bde- , μg/kg b.w. owing to the limitations and uncertainties in the current database, the panel concluded that it was inappropriate to use these bmdls to establish health based guidance values, and instead used a margin of exposure (moe) approach for the health risk assessment. as the elimination characteristics of pbde congeners in animals and humans differ considerably, the panel used the body burden as the starting point for the moe approach. the contam panel concluded that for bde- , - and - current dietary exposure in the eu does not raise a health concern. for bde- there is a potential health concern with respect to current dietary exposure. the contribution of ovine meat and ovine-derived products to total human exposure is currently not known. as these compounds bioaccumulate in the food chain, they deserve attention and should be considered for inclusion in the nrcps. in , efsa delivered a risk assessment on hbcdds in food (efsa contam panel, f) . hbcdds are additive flame retardants, primarily used in expanded and extruded polystyrene used as construction and packing materials, and in textiles. technical hbcdd consists predominantly of three stereoisomers (α-, βand γ-hbcdd). also δ-and ε-hbcdd may be present but at very low concentrations. hbcdds are present in the environment and likewise in biota and in food and feed. data from the analysis of hbcdds in food samples were provided to efsa by seven european countries, covering the period from to . the contam panel selected α-, β-and γ-hbcdd as of primary interest. as all toxicity studies were carried out with technical hbcdd, a risk assessment of individual stereoisomers was not possible. main targets were the liver, thyroid hormone homeostasis and the reproductive, nervous and immune systems. hbcdds are not genotoxic. the contam panel identified neurodevelopmental effects on behaviour as the critical endpoint, and derived a bmdl of . mg/kg b.w. owing to the limitations and uncertainties in the current data base, the contam panel concluded that it was inappropriate to use this bmdl to establish a healthbased guidance value, and instead used an moe approach for the health risk assessment of hbcdds. as the elimination characteristics of hbcdds in animals and humans differ, the panel used the body burden as the starting point for the moe approach. the contam panel concluded that current dietary exposure to hbcdds in the eu does not raise a health concern. the occurrence data reported to efsa have shown that hbcdds could be detected in a limited number of meat samples. as the total number of sheep and goat meat samples analysed for hbcdds are sparse and thus the current knowledge about the prevalence and their levels in edible tissues of ovine animals is limited, their inclusion into nrcps even as a temporary measure should be considered. perfluorinated compounds (pfcs), such as pfos, pfoa and others have been widely used in industrial and consumer applications including stain-and water-resistant coatings for fabrics and carpets, oil-resistant coatings for paper products approved for food contact, fire-fighting foams, mining and oil well surfactants, floor polishes, and insecticide formulations. a number of different perfluorinated organic compounds have been widely found in the environment. in , efsa delivered a risk assessment on pfos and pfoa in food (efsa, g) . the contam panel established a tdi for pfos of ng/kg b.w. per day, and a tdi for pfoa of . μg/kg b.w. per day. a few data indicated the occurrence of pfos and pfoa in meat samples. however, owing to the low number of data, it has not been possible to perform an assessment of the relative contribution from different foodstuffs to human exposure to pfos and pfoa. a recent study in which contaminated feed was fed to sheep demonstrated the transfer of pfos, pfoa and various other pfcs with different chain lengths into milk and meat of the sheep (kowalczyk et al., ) . as pfcs have found widespread use and ubiquitous distribution in the environment, but representative data on their occurrence in meat are still limited, an intensified monitoring of these compounds in tissues as well as feed should be considered. besides the heavy metals discussed in section . . . . , attention should be given also to those compounds that may be used as feed supplements (e.g. copper, selenium, zinc). the correct use of these supplements cannot be guaranteed. although supplementary feeding to sheep and goats at pasture with trace elements is practised, supplements for sheep are not permitted to contain copper. however, the risk of copper supplementation cannot be ruled out on mixed livestock farms where supplements containing copper for other livestock, e.g. pigs or calves, may be given in error to sheep, resulting in undesirable residues in animal organs, such as the liver. sheep are particularly susceptible to copper toxicity; goats appear to be able to tolerate higher intakes (underwood and suttle, ) . in the absence of supplementation, the main source of copper is the pasture, the uptake of which is a complex interaction between the copper, molybdenum and sulphate levels in the plants and the grass plants themselves. for example, sheep that consume excess subterranean clover (trifolium spp.) will develop chronic copper accumulation in their tissues as a result of the copper/molybdenum balance in the clover (radostits et al., ) . there are also large differences between breeds in susceptibility to copper toxicity (underwood and suttle, ) . a closer communication of results from official feed control seems essential to decide whether or not analytical monitoring of residues in slaughter animals needs to be directed to these substances that might be overused or mistakenly used in sheep or goat feeds. in light of the existing regulations and the daily practice of the control of residues/chemical substances in sheep/goat carcasses, the strengths and weaknesses of the current meat inspection methodology can be summarised as follows: the strengths of the current meat inspection methodology for chemical hazards are as follows: the current procedures for sampling and testing are a mature system, well established and coordinated, and subject to regular evaluation that is in place across eu mss, with residue testing that is based on common standards for method performance and interpretation of results (commission decision / /ec), laboratory accreditation (iso/iec ) and quality assurance schemes. the residue monitoring programmes are supported by a network of eu and national reference laboratories and by research in the science of residue analysis that serves to provide state-of-the-art testing systems for control of residues (see annex ). there are well-developed systems and follow-up actions subsequent to the identification of non-compliant samples. as indicated in section . , follow-up on non-compliant samples is typically through intensified sampling (suspect sampling), withholding of slaughter and/or of carcasses subject to positive clearance as compliant, and on-farm investigations potentially leading to penalties and/or criminal prosecutions. the regular sampling and testing for chemical residues is a disincentive for the development of bad practices. there is constant development of new approaches in sampling and testing methodologies, particularly in the area of prohibited substances, directed at identifying illicit use of such substances in animal production; for example, use of samples other than edible tissues, such as excreta, eyes, fibre, etc. that demonstrate enhanced residue persistence characteristics, and use of indirect testing procedures, such as genomics, proteomics and metabolomics, to identify treated animals. the prescriptive sampling system allows for equivalence in the control of eu-produced sheep/goat meat. any forthcoming measures have to ensure that the control of imports from third countries remains equivalent to the controls within the domestic market (this issue is addressed further in tor ). the current combination of animal traceability, ante-mortem inspection and gross tissue examination can support the collection of appropriate samples for residue monitoring. however, any indication of misuse or abuse of pharmacologically active substances through visual assessment needs to be confirmed by chemical analysis for potential residues. the weaknesses of the current meat inspection methodology for chemical hazards are as follows: presence of chemical hazards cannot be identified by current ante-/post-mortem meat inspection procedures at the slaughterhouse level, indicating the need for further harmonisation of the risk reduction strategies along the entire food chain. at present, there is poor integration between the testing of feed materials for undesirable contaminants and the nrcps in terms of communication and follow-up testing strategies or interventions. moreover, a routine environmental data flow is not established and keeping habits for sheep and goats provide opportunities for feed coming in without a clear feed chain history. under the current system, sampling is mostly prescriptive rather than risk or information based. it appears that individual samples taken under the nrcp testing programme may not always be taken as targeted samples, as specified under council directive / /ec, but sometimes may be taken as random samples. there is a lack of sufficient cost-effective and reliable screening methods and/or the range of substances prescribed/covered by the testing is sometimes limited. there is limited flexibility to adopt new chemical substances into the nrcps and limited ongoing adaptation of the sampling and testing programme to the results of the residue monitoring programmes. the sampling under the nrcps reflects only a part of testing done by a number of mss and, therefore, data from the nrcps may not provide the most complete information for certain categories of substances. sheep and goats may not be subject to surveillance over their lifetime at the same level as is the case for other food animal categories such as pigs, poultry and, to a large extent, bovine animals owing to their traditional nomadic/outdoor farming systems. current monitoring of residues and contaminants in edible tissues of slaughter sheep/goats is based on council directive / /ec. in turn, risk ranking as presented under tor is also based largely on the chemical substances listed in council directive / /ec. the outcome of the ranking showed that only a small number of compounds are considered to constitute a high potential concern for consumers. however, considering the recent information available from the re-assessment of undesirable substances in the food chain, covered by more recent efsa opinions from the contam panel, additional compounds have been identified that require attention. prominent examples of such substances are and dl-pcbs, which were identified as compounds of high potential concern as they bioaccumulate in the food chain, are likely to be found in sheep/goat carcasses and have a toxicological profile that points towards public health concerns even at low (residue) concentrations. in addition, it has been shown that these substances are found in edible tissues of sheep, particularly in sheep liver. other halogenated substances such as brominated flame retardants, including polybrominated diphenylethers (pbdes) as well as hexabromocyclododecanes (hbcdds) and perfluorinated compounds (pfcs), such as pfos and pfoa have a different toxicological profile. these compounds bioaccumulate in the food chain and deserve attention, as currently the knowledge about the prevalence and level of residues of these compounds in edible tissues of sheep and goats is limited. chemical elements, such as copper, selenium and zinc, given as feed supplements may be mistakenly provided to sheep and goats resulting in undesirable residues in animal organs, such as the liver. inclusion of these various substances in the nrcps (even as a temporary measure) should be considered together with an intensified monitoring of feed materials for the presence of these compounds, to support forthcoming decisions on whether or not these substances require continued monitoring either in feed materials and/or in slaughter animals. due to the nature of the husbandry systems applied, sheep and goats are more likely to be exposed to environmental contaminants than other livestock. therefore, any incident giving rise to contamination of the environment may be noted primarily in animals kept outdoors, i.e. in sheep and goats. it is important to note that sheep and goat production in the eu is marked by being largely extensive in nature, involving frequent trading of animals and nomadic flocks. this involves differences in husbandry systems and feeding regimes resulting in different risks from chemical substances and contaminants. extensive periods on pasture or/as nomadic flocks, sale at open markets of many sheep and goats, and the presence of slaughter collection dealerships that may combine small numbers of animals purchased from several farmers, means that there is a level of concern that fci shared between farmers and the slaughterhouse (where residue data is managed), may be suboptimal. similarly, in these situations, the level of feedback from the slaughterhouse and authorities to farmers regarding the results of residue testing may be suboptimal. here the individual identification of animals, which has now become mandatory, may contribute to more transparency in the future. there is less concern about fci from dairy sheep and goats if they are reared under more intensive and controlled conditions. fci should be expanded for sheep and goats produced in extensive systems to provide more information on the specific environmental conditions where the animals are produced. it is recommended that sampling of sheep and goats should be based on the risk of occurrence of chemical residues and contaminants and on the completeness and quality of the fci supplied. to achieve this, better integration of results from official feed control with residue monitoring seems essential to indicate whether monitoring of residues in slaughter animals needs to be directed to particular substances. it should be noted that for the small ruminant chains more environmental information should be provided. therefore, there is a need for an improved integration of sampling, testing and intervention protocols across the food chain, nrcps, feed control and monitoring of environmental contaminants. moreover, the combination of data from both sheep and goats into one data set is based on the assumption that both food chains are identical. in many cases such an assumption is not justified. a separation of records for both species is recommended. in addition, there is a need to develop new approaches to chemical residues and contaminants testing. recent developments in chemical analytical techniques allow the simultaneous measurement of a broad range of substances. analytical techniques covering multiple analytes should be encouraged too and incorporated into feed quality control and national residue control programmes. application of such validated methods for multi-residue analyses comprising veterinary drugs, pesticides and natural and environmental contaminants should be encouraged. for prohibited substances, testing should be directed towards the farm level. one of the limitations of the currently applied analytical strategies is the generally poor sensitivity of some screening methods, resulting in the potential failure to detect residues in the low µg/kg range and, therefore, to identify non-compliant samples. new approaches including molecular biological techniques for the identification of indirect biomarkers of exposure in animals, as well as the development of reliable in vitro assays based on the biological action(s) of the compounds under analysis, are considered to be of additional value. such approaches may help in detecting molecules of unknown structure or that are not included in the nrcps but share a common mechanism of action, thereby better orienting and rationalising the subsequent chemical analysis. in the case of many of the substances that might be used illicitly for growth-promoting purposes in sheep and goat production, the results of nrcp testing show no non-compliant results (e.g. stilbenes) or indicate that reported non-compliant results may be attributable to dietary sources (e.g. thyreostats, zeranol) or are the result of endogenous production (e.g. gonadal (sex) steroids). therefore, future nrcp testing relating to such substances needs to be reduced and/or refocused, in terms of the range of analytes tested and the appropriateness of samples taken for testing, to better identify the extent of abuse of growth-promoting substances in sheep and goat production in the eu. in addition, control measures for such substances must not rely exclusively on nrcp testing, but should include veterinary inspection/police activities along the food chain directed at identifying abuse of such substances in sheep and goat production in the eu. finally, it should be noted that any measures taken to improve the efficacy of meat inspection protocols also need to address the compliance of imports to the eu with these strategies. where eu meat inspection would move to a risk-based approach, particular attention to the achievement of equivalent standards of food safety for imported food from third countries will be required. currently, within the prescriptive system for meat inspection and residue monitoring applying in the eu, third countries exporting food products of animal origin to the eu need to demonstrate that they have the legal controls and residue monitoring programmes capable of providing equivalent standards of food safety as pertains within the eu. if eu meat inspection moves to a risk-based approach, particular attention will need to be paid to the achievement of equivalent standards of food safety for imported food from third countries. the risk-ranking appropriate within the eu in relation to veterinary drugs and contaminants might not be appropriate in third countries to achieve equivalent standards of food safety. rather than requiring that a risk-based monitoring programme applying within eu mss should be applied similarly in the third country, an individual risk assessment for each animal product(s)/third country situation may be required, which should be updated on a regular basis. this section contains conclusions derived from the material discussed in the document, together with recommendations for improvements to meat inspection with regard to chemical hazards within the eu. tor to identify and rank the main risks for public health that should be addressed by meat inspection at eu level. general (e.g. sepsis, abscesses) and specific biological risks as well as chemical risks (e.g. residues of veterinary drugs and contaminants) should be considered. differentiation may be made according to production systems and age of animals (e.g. breeding compared to fattening animals) as a first step in the identification and ranking of chemical substances of potential concern, the contam panel considered the substances listed in council directive / /ec and evaluated the outcome of the national residue control plans (nrcps) - . the contam panel noted that only . % of the total number of results was non-compliant for one or more substances listed in council directive / /ec. potentially higher exposure of consumers to these substances from sheep and goat meat takes place only incidentally, as a result of mistakes or non-compliance with known and regulated procedures. the available aggregated data indicate a low number of samples that were non-compliant with the current legislation. however, in the absence of substance-and/or species-specific information, such as the tissues used for residue analysis and the actual concentration of a residue or contaminant measured, these data do not allow for a reliable assessment of consumer exposure. other criteria used for the identification and ranking of chemical substances of potential concern included the identification of substances that that are found in other testing programmes and that bioaccumulate in the food chain, substances with a toxicological profile of concern, and the likelihood that a substance under consideration will occur in sheep and goat carcasses. taking into account these criteria the individual compounds were ranked into four categories denoted as being of high, medium, low and negligible potential concern. the highest overall proportion of non-compliant results under the nrcps were for group b substances, contaminants ( . %) representing largely exceedances of the maximum residue limits/maximum levels (mrls/mls) specified for these substances. the proportion of noncompliant results overall for group a substances, prohibited substances ( . %) and for group b /b substances, veterinary medicinal products (vmps) ( . %) represent largely illicit use and exceedances of the mrls, respectively. dioxins and dioxin-like polychlorinated biphenyls (dl-pcbs) were ranked as being of high potential concern owing to their known bioaccumulation in the food chain, their frequent findings above mls, particularly in sheep liver, and in consideration of their toxicological profile. stilbenes, thyreostats, gonadal (sex) steroids, resorcylic acid lactones and beta-agonists, especially clenbuterol, were ranked as being of medium potential concern because of their toxicity for humans, their efficacy as growth promoters in sheep and goats and the incidence of non-compliant results. chloramphenicol and nitrofurans were ranked as being of medium potential concern, as they have proven toxicity for humans, they are effective as antibacterial treatments for sheep/goats and as non-compliant samples are found in most years of the nrcps. non-dioxin-like polychlorinated biphenyls (ndl-pcbs) bioaccumulate, and there is a risk of exceeding of the mls, but they were ranked in the category of medium potential concern, because they are less toxic than dioxins and dl-pcbs. the chemical elements cadmium, lead and mercury were allocated to the medium potential concern category taking into account the number of non-compliant results reported under the nrcps and their toxicological profile. residues originating from other substances listed in council directive / /ec were ranked as of low or negligible potential owing to the toxicological profile of these substances at residue levels in edible tissues or to the very low or non-occurrence of non-compliant results in the nrcps - , and/or to the natural occurrence in sheep and goats of some of these substances. the low potential concern category includes nitroimidazoles chlorpromazine, organochlorine pesticides, organophosphorus compounds, natural toxins, as well as and vmps exceeding mrls. in the negligible potential concern category are the prohibited substances colchicine, dapsone, chloroform and aristolochia spp., the dyes, as well as vmps occurring below mrls. the contam panel emphasises that this ranking into specific categories of potential concern is based on the current knowledge regarding toxicological profiles, usage in sheep and goat production and occurrence as contaminants or chemical residues, as demonstrated by the data from the nrcps for the - period. future monitoring programmes should be based on the system for the ranking of chemical compounds into potential concern categories as presented in this document. regular updating of the ranking of chemical compounds in sheep and goats as well as of the sampling plans should occur, taking into account any new information regarding the toxicological profile of chemical residues and contaminants, usage in sheep and goat production, and actual occurrence of individual substances in sheep and goats. tor to assess the strengths and weaknesses of the current meat inspection methodology and recommend possible alternative methods (at ante-mortem or post-mortem inspection, or validated laboratory testing within the frame of traditional meat inspection or elsewhere in the production chain) at eu level, providing an equivalent achievement of overall objectives; the implications for animal health and animal welfare of any changes suggested in the light of public health risks to current inspection methods should be considered strengths of the current meat inspection methodology for chemical hazards are as follows: the current procedures for sampling and testing are a mature system, in general well established and coordinated including follow-up actions subsequent to the identification of non-compliant samples. the regular sampling and testing for chemical residues and contaminants in the system is an important disincentive to the development of undesirable practices. the prescriptive sampling system allows for equivalence in the control of eu-produced sheep and goat meat. any forthcoming measures have to ensure that the control of imports from third countries remains equivalent to the controls within the domestic market. the current combination of animal traceability, ante-mortem inspection and gross tissue examination can support the collection of appropriate samples for residue monitoring. weaknesses of the current meat inspection methodology for chemical hazards are as follows: a weakness of the system is that presence of chemical hazards cannot be identified by current ante-/post-mortem meat inspection procedures at the slaughterhouse level, indicating the need for further harmonisation of the risk reduction strategies along the entire food chain. integration between testing of feed materials for undesirable contaminants and the nrcps in terms of communication and follow-up testing strategies or interventions is currently limited. moreover, a routine environmental data flow is not established and keeping habits for sheep and goats provides opportunities for feed coming in without a clear feed chain history. under the current system, sampling is mostly prescriptive rather than risk or information based. it appears that individual samples taken under the nrcp testing programme may not always be taken as targeted samples, as specified under council directive / / ec, but sometimes may be taken as random samples. there is a lack of sufficient cost-effective and reliable screening methods and/or the range of substances prescribed/covered by the testing is sometimes limited. there is limited flexibility to adopt emerging chemical substances into the nrcps and limited ongoing adaptation of the sampling and testing programme to the results of the residue monitoring programmes. in addition, sampling under the nrcps reflects only a part of testing done by a number of ms, the results of which should be taken into consideration. sheep and goats may not be subject to surveillance over their lifetime at the same level as is the case for other food animal categories such as pigs, poultry and, to a large extent, bovine animals owing to their traditional nomadic/outdoor farming systems. meat inspection systems for chemical residues and contaminants should be less prescriptive and should be more risk and information based, with sufficient flexibility to adapt the residue monitoring programmes to results of testing. if new hazards currently not covered by the meat inspection system (e.g. salmonella, campylobacter) are identified under tor , then recommend inspection methods fit for the purpose of meeting the overall objectives of meat inspection. when appropriate, food chain information should be taken into account dioxins and dl-pcbs which accumulate in food-producing animals have been ranked as being of high potential concern. as these compounds have not yet been comprehensively covered by the sampling plans of the current meat inspection (nrcps), they should be considered as 'new' hazards. in addition, for a number of chemical elements used as feed supplements and for organic contaminants that may accumulate in food-producing animals only limited data regarding residues in sheep and goats are available. this is the case, in particular, for brominated flame retardants, including polybrominated diphenylethers (pbdes) and hexabromocyclododecanes (hbcdds) and perfluorinated compounds (pfcs) including (but not limited to) pfos and pfoa. control programmes for residues and contaminants should include 'new hazards' and take into account information from environmental monitoring programmes which identify chemical hazards to which animals may be exposed. provide an equivalent level of protection within the scope of meat inspection or elsewhere in the production chain that may be used by risk managers in case they consider the current methods disproportionate to the risk, e.g. based on the ranking as an outcome of tor or on data obtained using harmonised epidemiological criteria. when appropriate, food chain information should be taken into account sheep and goat production in the eu is marked by being largely extensive in nature, involving frequent trading of animals and nomadic flocks. this involves differences in husbandry systems and feeding regimes resulting in different risks for chemical substances and contaminants. extensive periods on pasture or/as nomadic flocks and the use of slaughter collection dealerships may preclude detailed lifetime fci. similarly, in these situations, the level of feedback from the slaughterhouse and authorities to farmers regarding the results of residue testing may be suboptimal. there is less concern about fci from dairy sheep and goats as they are reared under more intensive and controlled conditions. better integration of results from official feed control with residue monitoring seems essential to indicate whether monitoring of residues in slaughter animals needs to be directed to particular substances. therefore, there is a need for an improved integration of sampling, testing and intervention protocols across the food chain, nrcps, feed control and environmental monitoring. fci should be expanded for sheep and goats produced in extensive systems to provide more information on the specific environmental conditions where the animals are produced. it is recommended that sampling of sheep and goats should be based on the risk of occurrence of chemical residues and contaminants and on the completeness and quality of the fci supplied. there is a need for an improved integration of sampling, testing and intervention protocols for domestic sheep and goats across the food chain, nrcps, feed control and environmental monitoring. the development of analytical techniques covering multiple analytes and of new biologically based testing approaches should be encouraged and incorporated into feed quality control and chemical residue/contaminants testing in the nrcps. the combination of data from both sheep and goats into one data set assumes that both food chains are identical, which is not the case. a separation of test records for both species is recommended. for prohibited substances, testing should be directed where appropriate towards the farm level. future nrcp testing relating to substances that might be used illicitly for growth promoting purposes needs to be refocused to better identify the extent of abuse in the eu. in addition, control measures for prohibited substances should not rely exclusively on nrcp testing, but should include veterinary inspection during the production phase and the use of biological methods and biomarkers suitable for the identification of abuse of such substances in sheep and goat production in the eu. commission decision / /ec specifies the performance criteria for methods, including recovery and accuracy, trueness and precision. the decision specifies, also, the validation required to demonstrate that each analytical method is fit for purpose. in the case of screening methods, validation requires determination of the performance characteristics of detection limit, precision, selectivity/specificity and applicability/ruggedness/stability. for confirmatory methods, in addition to determination of those performance characteristics, validation requires, also, determination of decision limit and trueness/recovery. the analytical requirements for the determination of dioxins, dl-pcbs and ndl-pcbs are laid down in commission regulation (ec) no / . following a criteria approach analyses can be performed with any appropriate method, provided the analytical performance criteria are fulfilled. while methods, such as gc-ms and cell-and kit-based bioassays are allowed for screening purposes, the application of gc/high-resolution ms is mandatory for confirmation of positive results. screening methods include a broad range of methods, such as elisa, biosensor methods, receptor assays, bioassays and biomarkers for the presence of residues of concern. these screening methods generally use specific binding of the molecular structure of the residue(s) by antibodies or other receptors to isolate and measure the presence of the residues in biological fluids (urine, plasma) or sample extracts. more recently, biomarkers for the use of prohibited substances such as hormonal growth promoters have been identified as potential screening methods for these substances. physicochemical methods, such as lc or gc with various detectors, may be used, also, as screening methods. in the particular case of antimicrobials, microbiological or inhibitory substance tests are widely used for screening. in such tests, using multiple plates/organisms or kit formats, the sample or sample extract is tested for inhibition of bacterial growth. if, after a specific period of incubation, the sample inhibits the growth of the bacteria, it is considered that an antibacterial substance is present in the sample, but the specific substance is not identified. given that this is a qualitative analytical method, a misinterpretation of the results cannot be ruled out, and some false-positives can occur. microbiological methods are screening methods that allow a high sample throughput but limited information is obtained about the substance identification and its concentration in the sample. when residues are found in a screening test, a confirmatory test may be carried out, which normally involves a more sophisticated testing method providing full or complementary information enabling the substance to be identified precisely and confirming that the maximum residue limit has been exceeded. with the significant developments in liquid chromatography and in mass spectrometry over the last decade, confirmatory methods are largely ms-based, using triple quadrupole, ion trap, and other ms techniques. indeed, with current methodology in a modern residue laboratory with good ms capability, much of the two-step approach of screening followed by confirmatory testing has been replaced by single confirmatory testing. this has been made possible by the greatly-enhanced separation capability of ultra-high-performance liquid chromatography (uplc), coupled with sophisticated ms detection systems. the parallel growth in more efficient sample extraction/clean-up methods is an integral part of these advances in confirmatory methods and such chemistries produce rapid, sometimes (semi)-automated procedures providing multi-residue capability. techniques based on highly efficient sorbent chemistries for solid-phase extraction and techniques such as quechers are examples of these advances. such combinations of uplc-ms/ms methods with appropriate sample extraction/cleanup technologies allows for unequivocal, quantitative determination of a broad spectrum of substances in a single analytical method. particularly in the area of prohibited substances, the power of ms techniques is being applied to identify hitherto unknown compounds and to identify exogenous from endogenous substances. for example, time-of-flight ms provides accurate mass capability and may allow for retrospective analysis capability from the ms data. the technique of gc-combustion-isotope ratio ms has been utilised to study the c/ c ratio of substances in urine samples, where, for example, such c/ c ratio differs significantly between endogenous (or natural) testosterone and exogenous (or synthetic) testosterone. liver examination at slaughter is the most direct, reliable, and cost-effective technique for the diagnosis of fasciolosis. moving to a visual only meat inspection system would decrease the sensitivity of inspection of fasciolosis at the animal level; however, it would be sensitive enough to identify most if not all affected herds. therefore the consequences of the change would be of low relevance. the feedback to farmers of fasciola hepatica detected at meat inspection should be improved, to allow farmer information to support rational on-farm fluke management programmes. quantitative analysis indicated that the proposed changes to the meat inspection system would not affect detection of welfare conditions; however, for leg and foot disorders and sheep scab a combination of the two surveillance components (clinical surveillance and meat inspection) were found to be more effective than either one of the surveillance component on its own. qualitative analysis suggested that the proposal for shortened transport and lairage time would be beneficial to improving the welfare of small ruminants. food chain information should include animal welfare status in order to complement the slaughterhouse surveillance systems (ante-mortem and post-mortem inspection) and the latter could be used to identify on-farm welfare status. other recommendations on biological and chemical hazards would not have a negative impact on surveillance of animal diseases and welfare conditions. in this mandate, the ahaw panel and the ad hoc working group (wg) are focusing on the implications for animal health and welfare of any changes to the current meat inspection (mi) system, as proposed by biological hazards (biohaz) and contaminants in the (contam) panels. "implications for animal health and welfare" relates specifically to monitoring and surveillance of animal diseases and welfare conditions during mi (that is, inspection at the slaughterhouse before and after slaughter, in this document referred to as ante-mortem (ami) and post-mortem (pmi) inspection, respectively). therefore, the objective of this work was to identify possible effects and to assess the possible consequences on surveillance and monitoring of animal diseases and welfare conditions if the proposed changes in the mi system were applied. apart from its contribution to assuring public health, current mi also contributes to surveillance and monitoring of animal diseases and welfare conditions (efsa, ) , and may be an important component of the overall monitoring and surveillance system. further, mi offers the only opportunity for monitoring some diseases and welfare conditions at certain stages of a control and eradication programme. therefore, any change in mi system that could lead to a loss of sensitivity (reduced probability of detection) may compromise the surveillance efficacy. in the case of animal welfare, ami and pmi also play a role in surveillance and monitoring of the welfare of farmed animals, and, moreover, it is the only place to assess poor welfare during the transport of animals to the slaughterhouse. small ruminants are subjected to different periods of feed and water restriction, handling and transport prior to arrival at the slaughterhouse. ami begins with the observation of animals at the time of unloading from the transport vehicle and the purpose is to determine whether animal welfare has been compromised in any way on the farm and during handling and transport. welfare conditions such as fitness to travel, prevalence of injury, lameness and exhaustion, and the cleanliness of the animals are ascertained during ami. certain other welfare conditions such as bruising may not always be detectable during ami, but become visible during routine pmi. welfare conditions related to foot and leg disorders would be detectable only if the animals are observed during walking, e.g. unloading or moving to lairage pens, and are also less likely to be detected by visual examination during pmi. when mi detects apparent defects or abnormalities, incision of the relevant joints, tendons and/or muscles could be necessary to determine the presence as well as the severity of foot and leg disorders. implications for surveillance and monitoring for small ruminant health and welfare of changes to meat inspection as proposed by the biohaz panel the proposed modifications to the mi system that may have implications for animal health and welfare (see biohaz appendix a for full details), include: shorter transport and lairaging, which may be beneficial in terms of reducing crosscontamination of pathogens salmonella spp. and human pathogenic escherichia coli (see biohaz appendix a, section . ). the changes to address prioritised hazards not currently detected by mi will focus on improved collection and use of relevant food chain information (fci), including the use of harmonised epidemiological indicators, to provide information for categorisation of farms, which can be used for, for example, risk-based ami, logistic slaughter and/or decontamination (see biohaz appendix a, sections and . ). omission of palpation and incision in animals subjected to routine slaughter at pmi. if abnormalities are detected during visual inspection, palpation and incision should be carried out separately from the routine inspection of carcasses to prevent cross-contamination) (see biohaz appendix a, section . ). to assess the impact of proposed changes to the current mi on the overall sensitivity for surveillance and control of animal diseases and welfare conditions, a quantitative assessment was performed based on expert opinion and modelling. an external consortium (comisurv), under the provision of an efsa procurement, performed this work. the detailed methodology, as well as results and conclusions, together with assumptions and limitations of the modelling, can be found in the comisurv report for small ruminants mi (hardstaff et al., ) . these limitations include: the parameters for the probability of detection were based on expert opinion and therefore there is uncertainty as to the true range of these values. limited number of experts to cover the different subjects needed for the assessment. variations in the epidemiological situation of the disease and welfare conditions between countries. a brief description of the methodology that was applied is given below. an initial long list of small ruminant diseases and welfare conditions relevant to the eu was established, based on general textbooks, references, and expert opinion. wg experts filtered this list using a decision tree, following previous methodology and criteria developed for previous opinions (efsa biohaz, contam and ahaw panels, , ) . a disease or condition was retained on the list by the wg experts using the following criteria: a high likelihood of detection of a disease or welfare condition at mi, at the age that animals are presented at the slaughterhouse (if likelihood was medium, low, or the condition was undetectable, it was excluded from the list). the disease or welfare condition is considered relevant to the eu (conditions not occurring in eu member states (ms) were omitted). the condition is relevant to animal health and welfare (conditions mainly relevant to public health were not retained, as they should be dealt with by the biohaz panel). the slaughterhouse surveillance component (ami + pmi) provided by mi is significant for the overall surveillance of the disease or welfare condition (if there are other surveillance or detection systems much more effective and highly preferable to mi, the conditions were removed from the list). the final list of conditions established by the wg experts to be assessed by the comisurv consortium is shown in table . a total of twenty conditions (eleven diseases and nine welfare conditions) were included in this list. a stochastic model to quantify the monitoring and surveillance effectiveness of mi in small ruminants was developed. a definition of a typical and a mild case for each of the diseases and welfare conditions listed in table was provided by the comisurv experts. typical cases were by definition detectable cases and express more developed clinical signs than mild cases. typical cases were defined as the clinical signs and/or lesions that are expected to be observed in more than % of affected or infected small ruminants arriving at slaughter. the mild case of a disease or welfare condition is the form that could be seen at the early stages of the disease or at some point between the subclinical (and without pathological lesions that are observable through the meat inspection process) and the fully developed form (i.e. "typical" form). a mild case is neither typical nor non-detectable. the animal will probably present more subtle signs than in the typical case. as an example, a typical case of echinococcosis would show hydatid cysts in the liver and in the lungs, and a mild case would have a low number of small cysts in liver and lungs. the proportion of affected animals presenting as typical or mild cases, as well as the non-detectable fraction was estimated (see comisurv report for details). the most likely detection probability, as well as th and th percentiles (the probability intervals) of the output distribution of ami, pmi, and ami and pmi combined were derived for each of the conditions in table , both prior to and following suggested changes to the mi system as proposed by the biohaz panel. the inspection protocols in the current and visual only systems are compared in table . the probability of detection was calculated for both detectable cases (mild and typical), and for all cases (referred to as stage in the comisurv report). mastitis x a stage -all diseases and welfare conditions listed were evaluated with regards to their probability of being detected at mi. b stage -for selected diseases and welfare conditions, surveillance by mi was to be compared with clinical surveillance. as inspection tasks aimed to detect orf do not change in a visual-only system, orf was not further discussed. in addition, for three of the selected diseases and two welfare conditions, considered to be more adversely affected in terms of probability of detection following the proposed changes to the mi system, further modelling was implemented to quantify the effectiveness of monitoring and surveillance in the overall monitoring and surveillance system, both prior to and following suggested changes to the mi system (referred to as stage in the comisurv report). the objective for exotic diseases (i.e. foot and mouth disease (fmd), was to evaluate the probability of detecting at least one infected case of infected small ruminants by slaughterhouse inspection relative to other surveillance system components (component sensitivity), which for the purpose of this opinion was clinical surveillance. for endemic diseases (fasciolosis, lower respiratory tract infection) and welfare conditions (leg and feet disorders including foot rot, sheep scab) the objective was to calculate the case-finding capacity i.e. the proportion of infected or affected animals detected by the surveillance components (detection fraction) during both slaughterhouse and clinical surveillance. note that the word surveillance as used in this opinion does not imply that any action is taken to capture, or act upon, the information that is collected. it merely points to the potential of these systems to be used for such purposes. the detection probability for each disease and condition using the current mi system and the visual only system is shown in table (detectable cases) and table a of annex (all cases, including subclinical cases not detectable at slaughterhouse). a change to visual only inspection caused a significant reduction in the probability of detection (i.e. non-overlapping % probability intervals, stage ) during mi of detectable cases of fasciolosis (with a % reduction in detection probability) and tuberculosis (tb) in goats ( %) ( table ). when all cases were considered (see annex , table a) , the change to a visual only pmi protocol resulted in a clear reduction in the detection probability of three diseases, tb in goats (with a % reduction in detection fraction), fasciolosis ( %) and pulmonary adenomatosis/maedi-visna ( %)), although none of these reductions was significant when the overlap of probability intervals was considered. values for the probability of detection at ami and for the two proposed pmi scenarios for all cases (detectable and non-detectable cases combined) were also determined for welfare conditions (table and annex , table a , respectively). the probability of detection was significantly higher for ami than pmi for broken bones, diarrhoea, leg and foot disorders, partial prolapses/hernias and sheep scab. a change in pmi protocol to a visual only system did not significantly reduce the detection of any welfare conditions. pmi had a significantly higher probability of detection than ami for mastitis. combined slaughterhouse probabilities of detection were higher for detecting cases of many welfare conditions than when the slaughterhouse inspection components were considered separately (table and annex , table a ). where this was not the case, i.e. the detection probability of the combined mi process yielded equal values as either ami or pmi on its own. this was due to the fact that the experts had agreed that the respective welfare condition could not be detected at all with the one of the two mi steps. therefore the results of the combined mi are solely based on the results of either ami or pmi. for three welfare conditions (arthritis, broken bones and poor body condition), the pmi of detectable cases with visual only protocol also reduced the detection probability, although this was not significant. when considering all cases (annex , table a ), the probability of detection for the combined inspection was lower than for detectable cases. the change in pmi protocols led to a slight reduction in the detection probability of two welfare conditions (arthritis and poor body condition), yet none of these reductions were significant when the overlap of probability intervals was considered. the probability of detection for all detectable cases of diseases and welfare conditions at ami, pmi (two proposed scenarios-current and visual) inspection scenarios with the most likely (ml), th and th percentiles. shaded rows indicate diseases identified as having a significant reduction in detection probability in the visual-only scenario. for the two welfare conditions (leg and foot disorders and sheep scab) included in the overall surveillance analysis (stage ), a combination of the two surveillance components (clinical surveillance and mi) was found to be more effective (detecting a higher fraction of affected animals) than either one of the surveillance component on its own. however, the change in pmi protocol did not greatly affect the detection fraction of these welfare conditions (table ) . with regard to epizootic diseases, clinical surveillance (detection of clinical signs) had a greater sensitivity for detecting fmd than slaughterhouse surveillance, and the sensitivity increased with an increase in population size (table , stage ). a change to a visual only system would not have a negative impact on sensitivity of detection. a qualitative assessment was conducted, based on a literature review and expert opinion from the wg members, for the diseases identified as having a significant reduction in detection probability of detectable cases in the quantitative assessment of the comisurv report (tb in goats and fasciolosis) and welfare conditions. therefore routine inspection, unlike inspection for btb in the bovine, does not differ substantially from the visual only mi being proposed. information regarding the presence of tb is not specifically recorded at pmi. the comisurv report relating to the contribution of meat inspection to animal health surveillance in sheep and goats investigated the probability of detection of specific diseases and welfare conditions for three scenarios: one for inspection tasks as currently required by the legislation; one with visual inspection only; and one in which risk categorisation based on a hypothetical public health risk formed the basis for subsequent inspections. according to the comisurv report, the most likely values for the proportion of non-detectable, mild and typical cases elicited by experts for tb in goats were . , . and . , respectively. the pmi had a significantly higher probability of detection of tb in goats than ami for detectable cases and all cases, and the reduction in the probability of detection of tb in goats was significant for visual only pmi. the probability of detection (most likely values) of tb in goats (table for detectable cases and annex , table a for all cases) for combined ami and pmi was . ( . for all cases) changing to . ( . for all cases) for visual only, which represents a % reduction. as is the case with btb in bovines, the contribution of mi surveillance of tb in small ruminants is to support the detection of flocks/herds with tb, and the detection of individual animals with tb is merely the first step in improving herd surveillance. since more than one sheep or goat per flock/herd is likely to be slaughtered per time period (e.g. per year), the flock/herd probability of detection is a function of the individual animal sensitivity, the number of animals slaughtered from the herd and the within-herd prevalence of tb. for any given flock/herd, the flock/herd sensitivity will increase with the number of animals slaughtered. officially tuberculosis free (otf) status, however, is not available for small ruminants as it is for bovine herds, so the herd status is important in controlling tb in small ruminants, but not in substantiating freedom from tb. for tb in goats, the results from the comisurv report suggest that a change from the current inspection to visual only will reduce the probability of detection for detectable cases. a qualitative risk and benefit assessment for visual only pmi of cattle, sheep, goats and farmed/wild deer, commissioned by the uk food standards agency (fsa) (fsa, a), considered the absolute and relative animal health risk of tb in small ruminants as negligible when moving to a visual only pmi system when compared with the current legal requirements of inspection for sheep and goats (regulation (ec) / ). the main reason to reach this conclusion is that the current legal pmi requirements for small ruminants are mainly visual and do not require the incision of the lungs. incision of lymph nodes are required if in doubt after the initial visual inspection. considering that the majority of positive submissions to government labs in the united kingdom are associated with lesions in the mediastinal and bronchial lymph nodes, it is likely that the most frequent tb-like lesions in small ruminants (as described above) are not detected under the current traditional pmi requirements, which are initially visual, and therefore nor would be by visual only inspection. this lack of sensitivity is aggravated by the current commercial speed of slaughtering lines and the limited time available to carry out the inspection of carcases and offal. in the united kingdom, tb in non-bovine farmed animals is rare. small ruminants are not considered to represent a significant reservoir of the disease for other animals or to be of any significance in the persistence of btb in cattle. although small ruminants are considered as spillover hosts, it is still possible that severely infected sheep and goats could act as vectors of infection for other domestic and wild animals. in these circumstances, on-farm identification of possible sick small ruminants by farmers and a differential diagnosis from other respiratory disease and necropsy examination of lungs and relevant lymph nodes by farm veterinarians are the most effective control activities. fasciolosis (liver fluke) in small ruminants has a world wide distribution and is caused by the trematode parasite, fasciola hepatica. the direct losses due to fasciolosis are mortality, liver condemnation and reduced growth rate. disease results from the migration of large numbers of immature flukes through the liver, from the presence of adult flukes in the bile ducts, or both. liver fluke can infect all grazing animals, but is most pathogenic in sheep (armour, ) . the incidence of liver fluke is inextricably linked to high rainfall and is particularly prevalent in years when summer rainfall is high, which facilitates the survival and proliferation of the snail intermediate host and infective parasite stages present in the environment (ollerenshaw, ) . changes in recent epidemiological patterns, due to climate change, have resulted with increasing prevalence in northern european countries and the survival of fluke on pasture over winter, exposing sheep to infection for long periods (daniel and mitchell, ) . there have been increasing reports of liver fluke disease over the last decade in countries such as the united kingdom and ireland, most likely due to higher than average rainfall and temperatures through the seasons, and greater stock movements (taylor, ) . in southern european regions, for example in spain, the infection of snails could occur throughout the year, with a higher infection rate at the end of summer-autumn and at the end of the winter, and sheep eliminating eggs throughout the year (manga et al., ) . prevalence studies in the north-west of spain have indicated a liver fluke infection rate of approximately % of sheep flocks (ferre et al., ) . regulation (ec) / requires that domestic sheep and goats going for human consumption must have visual inspection of the liver and the hepatic lymph nodes, palpation of the liver and its lymph nodes, and incision of the gastric surface of the liver to examine the bile ducts. liver examination at slaughter is the most direct, reliable, and cost-effective technique for diagnosis of fasciolosis (urquhart et al., ) . reliance upon clinical signs to diagnose fasciolosis may result in low detection rates (rojo-vázquez et al., ) . mi is a convenient means of confirming a suspected herd or flock infestation, assessing the extent of infestation or determining the effectiveness of anthelmintic treatment (kissling and petrey, ) . pmi can confirm acute and sub-acute liver damage with liver enlargement, caused by the presence of immature flukes. animals suffering from chronic fasciolosis show a deterioration of the carcass, cholangitis, biliar occlusion and hepatic fibrosis with adult fluke present in bile ducts. besides the liver, other organs and structures can be found damaged, such as periportal and mesenteric lymph nodes that are enlarged and exhibit a brownish colour (rojo-vázquez et al., ) . mckenzie's study ( ) compared the new zealand inspection procedure (observation and palpation of livers) with the european community procedure (observation and incision through the gastric surface of liver to examine the bile ducts) and found that the new zealand method detected fewer truly infected livers, but misdiagnosis by inspectors gave more false-positives. the gastric surface incision procedure has a specificity of % and sensitivity of . % to . % (kissling and petrey, ) . this underlines their importance in animal disease surveillance and the importance of the present mi technique in liver fluke surveillance. effective disease monitoring systems are essential to the provision of reliable information on diseases to producers, thereby protecting a nation's agricultural system and its potential for production (glosser, ) . information on fluke infestation at herd level allows farmers to develop and implement control programmes that can attempt to reduce risk factors and recommend the use of drugs in a more strategic fashion (fairweather, ) . edwards et al., ( ) demonstrated that one-third of farmers would improve their animal husbandry if informed of the mi findings for their lambs. the comisurv report on the contribution of mi to animal health surveillance determined that there would be a significant difference in detection rates between the current and the visual only mi techniques (a probability of . of all detectable cases by current method compared with . by the visual only method). a reduction in liver fluke surveillance by the use of a less sensitive mi procedure will reduce the quality of information available for producers and thereby directly impact animal health and welfare. the quantitative analysis (see comisurv report) of detection levels for welfare conditions indicated that none of them will be significantly affected by the proposed changes to mi. however, the results also revealed that when both ami and pmi were considered, the probability of detection was high for most welfare conditions. it was also evident that detection of two welfare conditions, i.e. leg and foot disorders (including foot rot) and sheep scab, would be more effective when a combination of clinical and slaughterhouse surveillance systems are used. leg and foot disorders in sheep are caused by either infectious conditions, i.e. interdigital dermatitis (also known as scald), foot rot, contagious ovine digital dermatitis, or non-infectious conditions such as white line disease (shelly hoof), granulomas, foot abscesses, interdigital fibromas, and foreign bodies such as thorns, wire or soil balls (kaler and green, ; conington, et al., a conington, et al., , b fawc, ) . overgrown and misshapen hooves are also attributed to lameness in sheep and erysipelas can cause outbreaks of lameness in lambs. the importance of routine feet examination in sheep health management is well documented (hodgkinson, ) . the farm animal welfare council (fawc) suggested that there is adequate legal protection for sheep suffering from lameness as the european transport regulation ec/ / prohibits the transport of unfit animals, and specifically includes those that are "injured or present physiological weaknesses or pathological processes" and, in particular, are "unable to move independently without pain or to walk unassisted". the fawc also recommended that the surveillance of lameness in sheep should be undertaken by the uk government, in conjunction with farm assurance schemes, to determine trends in lameness over time, which would also apply to other mss where the prevalence of lameness is high (e.g. more than % of flocks being affected at national level). lameness in dairy goats is also a common welfare problem and abnormalities detected in the united kingdom were horn separation, white line lesions, slippering, abscess of the sole, foreign bodies and granulomatous lesions (hill et al., ) . interdigital dermatitis has also been reported to be the cause of lameness in goats kept indoors in greece (christodoulopoulos, ). sheep scab is a skin disease caused by the mite psoroptes ovis and has been widely prevalent in europe. it is a major animal welfare, husbandry and economic problem (bisdorff et al., ; bisdorff and wall, ) . the objectives of the ami in the current hygiene legislation, regulation (ec) / , are to determine: conditions that may might adversely affect human or animal health, paying particular attention to the detection of zoonotic diseases and animal diseases for which animal health rules are laid down in eu legislation, and whether there is any sign that welfare has been compromised. council regulation ( implementation of welfare assessment protocols using appropriate animal based indicators during clinical and slaughterhouse (ami + pmi) surveillance systems would improve the welfare of small ruminants. these welfare surveillance systems should become an integral part of the food chain information (fci). sheep are thought to be tolerant of being transported and deprived of food and water for long periods (knowles et. al., ) . it is a common practice by farmers to withdraw food on the farm for several hours prior to transport of sheep / lambs to auction markets or slaughterhouses, primarily to reduce soiling. however, dehydration can be a welfare problem during long transport distances/times, especially in high ambient temperatures (knowles, ) . recovery from the effects of food and water deprivation is a very slow process and therefore lairage appears to be of very little benefit. in this regard, full recovery from hours of transport has been shown to take up to hours (knowles, ) . owing to these, the biohaz panel's proposal for shortened transport and lairage time would be beneficial to animal welfare. the eu regulation (ec) no / on the hygiene of foodstuffs requires slaughterhouse operators to request fci declarations to ensure animals entering the food chain are safe for human consumption. fci is also a good source of information to facilitate the detection in the slaughterhouse of abnormalities indicative of animal health and welfare conditions. fci is recorded at the flock/herd level, and its minimum content is described in regulation (ec) no / . fci related to primary production of small ruminant herds/flocks is based on a farmer's declaration. most mss have made available to farmers a standardised fci declaration form. a whole-chain approach to food safety, animal health and animal welfare requires slaughterhouse operators to be provided by livestock producers with information about their animals consigned to slaughter. based on the fci provided food business operators (fbos) can assess potential hazards presented by the animals and are required to act upon any information recorded on the fci declaration as part of their hazard analysis and critical control point (haccp) plan. this helps the slaughterhouse operator to organise slaughter operations and to ensure that no animals affected by disease or certain veterinary medicines enter the food chain. quality assurance schemes at primary producer level are voluntary tools operated by independent agencies or bodies to ensure compliance with given standards and regulations. these schemes increase farmers' responsibilities with regard to animal health and welfare and have potential for integration within the fci provided (oie, ) . the fci also assists risk management to determine the required inspection procedures and should be analysed by risk management and used as an integral part of the inspection procedures. the value of the fci in guiding risk management to discriminate between animals subsequently going through different types of inspection procedures should be evaluated. as for any evaluation of (pre-) screening procedures, the sensitivity and specificity of the classification should be estimated. priority should be given to improving test sensitivity, noting that (pre-) screening tests should preferably produce few false negative classifications for the sake of animal disease detection and surveillance. test specificity will largely be an economical parameter, since the subsequent inspection of all "fcipositive" animals or groups should detect any false positives not correctly identified during the fci pre-screening. regulation (ec) no / requires that data from the ami and pmi at the slaughterhouse is delivered back to the farmer/producer when the inspections reveal the presence of any disease or condition that might affect public or animal health, or compromise animal welfare. currently this feedback of information to primary producers is not fully implemented in all mss (efsa biohaz, contam and ahaw panels, concludes that the effective and efficient flow of information provides valuable information to both the farmer and the fbo and allows more targeted and effective inspection procedures in the slaughterhouse and effective interventions on the farm that should contribute to a cycle of continuous improvement with positive implications for animal health and welfare. the effectiveness of this information cycle depends on a reliable animal identification and recording system at the slaughterhouse and an information transfer system to the primary producer. the collection and communication of slaughterhouse inspection results is an opportunity to collect and use data and knowledge applicable to disease control and the effectiveness of interventions, animal production systems, food safety and animal health/welfare (garcia, ) . at national and eu level such data can contribute to disease surveillance (for the detection of exotic diseases, monitoring of endemic diseases and identification of emerging diseases) and targeted animal health and welfare interventions. therefore fci, if consistently and effectively implemented as enshrined within the hygiene package, will form an integral part of a risk-based mi system. extended use of fci has the potential to compensate for some, but not all, of the information on animal health and welfare that would be lost if visual only pmi is applied. for the fci to be effective it should include species-specific indicators for the occurrence of disease and welfare conditions. fci for public health purposes may not have an optimal design for the surveillance and monitoring of disease and welfare conditions; therefore, an integrated system should be developed whereby fci for public health and for animal health and welfare can be used in parallel, more effective. the conclusions and recommendations from the contam panel refer to areas such as the ranking system for chemical substances of potential concern and its updating, the use of fci to help facilitate risk-based sampling strategies; the inclusion of new hazards in control programmes for chemical residues and contaminants (see contam appendix b, for full details). none of these were considered to have an impact on animal health and welfare surveillance and monitoring. as shown in the comisurv assessment, a change to visual only inspection would cause a significant reduction in the probability of detection (i.e. non-overlapping % probability intervals) of detectable cases of fasciolosis and of tuberculosis in goats. clinical surveillance had a greater sensitivity for detecting fmd than slaughterhouse surveillance following the assessment by comisurv, although the sensitivity of meat inspection increased with an increase in population size. a change to a visual only system would not have a negative impact on sensitivity of detection. as shown in the comisurv assessment, the proposed changes to meat inspection would not greatly affect the probability of detection of any of the welfare conditions analysed. from the comisurv assessment, for two welfare conditions (leg and foot disorders and sheep scab), a combination of the two surveillance components (clinical surveillance and meat inspection) were shown to be more effective (detecting a higher fraction of affected animals) than either one of the surveillance components on its own. according to regulation (ec) / , current inspection in small ruminants includes visual inspection and palpation of the lungs and respiratory lymph nodes. a change to visual inspection would imply that palpation is abandoned. small ruminants are usually not subjected to official tuberculosis eradication campaigns, and farm controls are only performed on premises where cattle and goats are kept together, or in flocks/herds that commercialise raw milk. surveillance for small ruminant tuberculosis at present relies on meat inspection of sheep and goats slaughtered for human consumption, or other limited diagnostic surveillance activities. as is the case with tuberculosis in bovines, the contribution of meat inspection surveillance of tuberculosis in small ruminants is to support the detection of flocks/herds with tuberculosis. detection of tuberculosis in individual animals is merely the first step in improving the effectiveness of flock/herd surveillance, and for any given flock/herd, the flock/herd sensitivity will increase with the number of animals slaughtered. results of two recent risk assessments (comisurv report; fsa, a) show that a change from the current inspection to visual only will reduce the probability of detection of tuberculosis in small ruminants. however, the consequences for animal health were considered as negligible in the fsa assessment, due to the fact that current meat inspection does not prescribe routine incision of lymph nodes, and the only inspection task omitted will be palpation of lungs and respiratory lymph nodes. in recent years tuberculosis has been reported in small ruminants in several eu countries and most information derives from recognition of tuberculosus lesions at the slaughterhouse and from laboratory reports. although small ruminants are not considered to represent a significant reservoir of the disease for the persistence of bovine tuberculosis in cattle, it is still possible that infected sheep and goat herds could act as vectors of infection for other domestic and wild animals. therefore, surveillance and control of tuberculosis in domestic small ruminants does have consequences for the overall surveillance and control of tuberculosis. liver examination at slaughter is the most direct, reliable, and cost-effective technique for diagnosis of fasciolosis. moving to a visual only meat inspection system would decrease the sensitivity of inspection at animal level for fasciolosis, however it would be sensitive enough to identify most, if not all, affected herds. therefore the consequences of change are low (charleston et al., ) . the feedback to farmers regarding fasciola hepatica detected at meat inspection is low at present and the real risk to animal health/welfare for this disease, caused by a change to a visual only meat inspection method, is probably low. implementation of welfare assessment protocols using appropriate animal based indicators during clinical and slaughterhouse (ami + pmi) surveillance systems would improve the welfare of small ruminants. extended use of food chain information has the potential to compensate for some, but not all, of the information on animal health and welfare that would be lost if visual only post-mortem inspection is applied. food chain information is a potentially effective tool to perform more targeted ante-mortem and post-mortem inspection tasks in the slaughterhouse which may increase the effectiveness of those tasks in detecting conditions of animal health and animal welfare significance. the existing ineffective flow of information from primary production to the slaughterhouses and vice versa reduces the ability of detection of animal diseases and animal welfare conditions at the slaughterhouse and as a result it limits possible improvements on animal health and welfare standards at the farm as farmers will not be aware of the slaughterhouse findings. the conclusions and recommendations on chemical hazards were reviewed by the ahaw working group and none of them were considered to have impact on animal health and welfare surveillance and monitoring. data collected during clinical and slaughterhouse (ante-mortem and post mortem inspection) surveillance systems should be utilised more effectively to improve animal welfare at farm level. slaughterhouse surveillance of tuberculosis in small ruminants should be improved and encouraged, as this is in practice the only surveillance system available. the detection of tuberculosis in small ruminants should be adequately recorded and notified, followed by control measures at the farm level. lack of feedback of post-mortem inspection results to the farmer prevents instigation of a fluke management programme, which could be detrimental to animal health and welfare. an improvement in this feedback of information is recommended. welfare surveillance systems should become an integral part of the food chain information. an integrated system should be developed whereby food chain information for public health and for animal health and welfare can be used in parallel, more effectively provide farmers with background information on the animal diseases and welfare conditions of key concern that may affect their livestock and why it is important to provide this information to the slaughterhouse through the use of food chain information. all cases: the combination of detectable cases (mild and typical) and non-detectable cases. case-finding capacity: characteristic of a surveillance system for endemic disease, describing the ability of the system to identify infected or affected herds or individuals, so that a control action can (potentially) be taken. the detection fraction is a measure of the case-finding capacity. case type: includes detectable (mild or typical cases) and non-detectable cases. clinical surveillance: surveillance based on clinical observations in the field. combined inspection: taking into account ante-mortem and post-mortem inspection. component sensitivity: the probability that one or more infected animals will be detected by the surveillance component during a specified time period, given that the disease is present at a level defined by the design prevalence. a risk assessment of shiga toxin-producing escherichia coli (stec) in the norwegian meat chain with emphasis on dry-cured sausages. panel on biological hazards, norwegian scientific committee for food safety norwegian scientific committee for food safety konsekvenser for dyr og mennesker norwegian scientific committee for food safety great hygienic effects from more strict requirements for the sheep slaughter campylobacter excreted into the environment by animal sources: prevalence, concentration shed, and host association comparative studies on the survival of verocytotoxigenic escherichia coli and salmonella in different farm environments epidemiological investigations on campylobacter-jejuni in households with a primary infection escherichia coli o : h and non-o shiga toxin-producing e-coli in healthy cattle, sheep and swine herds in northern spain genetic diversity among campylobacter jejuni isolates from healthy livestock and their links to human isolates in spain direct detection and genotyping of toxoplasma gondii in meat samples using magnetic capture and pcr a quantitative microbial risk assessment for meatborne toxoplasma gondii infection in the netherlands evaluation of elisa test characteristics and estimation of toxoplasma gondii seroprevalence in dutch sheep using mixture models a longitudinal study of verotoxinproducing escherichia coli in two dairy goat herds prevalence and characterization of vero cytotoxin-producing escherichia coli isolated from diarrhoeic and healthy sheep and goats liver distomatosis in cattle, sheep and goats of northeastern iran subtilase cytotoxin encoding genes are present in human, sheep and deer intimin-negative, shiga toxin-producing escherichia coli o :h studies on the reservoir status of leptospirosis in gujarat updates on morphobiology, epidemiology and molecular characterization of coenurosis in sheep distinct host species correlate with anaplasma phagocytophilum anka gene clusters sheep and goat medicine zoonotic agents in small ruminants kept on city farms in southern germany outbreak of haemolytic uraemic syndrome in norway caused by stx -positive escherichia coli o :h traced to cured mutton sausages minimising the bacterial count on lamb carcases by means of slaughtering methods scvmph (scientific committee on veterinary measures relating to public health) . opinion on revision of meat inspection in veal calves. european commission, health and consumer protection directorate-general. directorate c-scientific opininos main aspects of leptospira sp infection in sheep an outbreak of escherichia coli o :h -bacteriological investigations and genotyping of isolates from food potentially human-pathogenic escherichia coli o in norwegian sheep flocks tuberculosis in goats on a farm in ireland: epidemiological investigation and control study on prevalence of klebsiella pneumoniae in meat and meat products and its enterotoxigenicity a study of the sero-prevalence of influenze-a viruses in sheep and goats risk factors for the presence of antibodies to toxoplasma gondii in norwegian slaughter lambs enteritis in sheep and goats due to yersinia enterocolitica infection epidemiology of yersinia pseudotuberculosis and y. enterocolitica infections in sheep in australia investigation of the presence of esbl-producing escherichia coli in the north wales and west midlands areas of the uk in to using scanning surveillance yersinia enterocolitica in sheep -a high frequency of biotype a nonacid meat decontamination technologies: model studies and commercial applications temporal variation and host association in the campylobacter population in a longitudinal ruminant farm study babesia microti and anaplasma phagocytophilum: two emerging zoonotic pathogens in europe and hungary seasonal variation of thermophilic campylobacters in lambs at slaughter attribution of campylobacter infections in northeast scotland to specific sources by use of multilocus sequence typing microbial contamination on beef and sheep carcases in south australia current concepts -recognition and management of anthrax -an update an outbreak of salmonella mikawasima associated with doner kebabs diagnosis of human visceral pentastomiasis pathogenicity of yersinia enterocolitica biotype a procedures in the current meat inspection of domestic sheep and goats . . criteria used for the evaluation of the likelihood of the occurrence of residues or contaminants in sheep and goats . weaknesses of the current meat inspection methodology for chemical hazards tor : adaptation of inspection methods use of a β-adrenergic agonist to alter muscle and fat deposition in lambs food poisoning by clenbuterol in portugal semicarbazide is non-specific as a marker metabolite to reveal itrofurazone abuse as it can form under hofmann conditions special issue plenary papers of the th international conference on goats recent developments in the use and abuse of growth promoters regulation (ec) no / for dapsone as an impurity in veterinary medicinal products containing sulphamethoxazole or other sulphonamides scientific opinion of the panel of contaminants in the food chain (contam panel) on request from the european commission related to zearalenone as undesirable substance in animal feed scientific opinion of the panel of contaminants in the food chain (contam panel) on request from the european commission related to aflatoxin b as undesirable substance in animal feed scientific opinion of the panel of contaminants in the food chain (contam panel) on request from the european commission related to deoxynivalenol (don) as undesirable substance in animal feed scientific opinion of the panel of contaminants in the food chain (contam panel) on request from the european commission related to the presence of non dioxin-like polychlorinated biphenyls (pcb) in feed and food scientific opinion of the panel of contaminants in the food chain (contam panel) on request from the european commission related to fumonisins as undesirable substances in animal feed opinion of the scientific panel on food additives, flavourings, processing aids and materials in contact with food (afc) on a request related to a th list of substances for food contact materials scientific opinion of the panel of contaminants in the food chain (contam panel) on request from the european commission related to pyrrolizidine alkaloids as undesirable substaces in animal feed scientific opinion of the panel of contaminants in the food chain (contam panel) on request from the european commission related to cyanogenic compounds as undesirable substances in animal feed mercury as undesirable substance in animals feed-scientific opinion of the panel of contaminants in the food chain glucosinolates as undesirable substances in animal feed-scientific opinion of the panel of contaminants in the food chain scientific opinion of the panel of contaminants in the food chain (contam panel) on request from the european commission on tropane alkaloids (from datura spp.) as undesirable substances in animal feed scientific opinion of the panel of contaminants in the food chain (contam panel) on request from the european commission on theobromine as undesirable substances in animal feed scientific opinion of the panel of contaminants in the food chain (contam panel) on request from the european commission on gossypol as undesirable substances in animal feed scientific opinion of the panel of contaminants in the food chain (contam panel) on request from the european commission on ricin (from ricinus communis) as undesirable substances in animal feed opinion of the scientific panel on contaminants in the food chain (contam panel) on perfluorooctane sulfonate (pfos), perfluorooctanoic acid (pfoa) and their salts cadmium in food -scientific opinion of the panel of contaminants in the food chain (contam panel) scientific opinion of the panel of contaminants in the food chain (contam panel) on request from the european commission on saponins in madhuca longifolia l. as undesirable substances in animal feed results of the monitoring of dioxin levels in food and feed update of the monitoring of dioxins and pcbs levels in food and feed scientific opinion on lead in food efsa panel on contaminants in the food chain (contam), a. statement on tolerable weekly intake for cadmium scientific opinion on the risk to public health related to the presence of high levels of dioxins and dioxin-like pcbs in liver from sheep and deer scientific opinion on the risk for animal and public health related to the presence of t- and ht- toxin in food and feed scientific opinion on pyrrolizidine alkaloids in food and feed scientific opinion on polybrominated diphenyl ethers (pbdes) in food scientific opinion on hexabromocyclododecanes (hbcdds) in food scientific opinion on the risk for public health related to the presence of mercury and methylmercury in food scientific opinion on ergot alkaloids in food and feed emea (the european agency for the evaluation of medicinal products) emea (the european agency for the evaluation of medicinal products), a. committee for veterinary medicinal products emea (the european agency for the evaluation of medicinal products), b. committee for veterinary medicinal products emea (the european agency for the evaluation of medicinal products), . committee for veterinary medicinal products. dapsone ( ) committee for veterinary medicinal products expert committee on food additives. chloramphenicol -toxicological evaluation of certain veterinary drug residues in food evaluaion of certain veterinary drug residues in food agriculture organization of the united nations/world health organization expert committee on food additives), . safety evaluation of certain contaminants in food who food additives series, . who/jecfa monographs , who determination of colchicine residues in sheep serum and milk using high-performance liquid chromatography combined with electrospray ionization ion trap tandem mass spectrometry depletion of protein-bound furazolidone metabolites containing the -amino- -oxazolidinone side-chain from liver, kidney and muscle tissues from pigs formation of semicarbazide (sem) in food by hypochlorite treatment: is sem a specific marker for nitrofurazone abuse? monographs on the evaluation of carcinogenic risks to humans. sex hormones (ii) iarc monographs on the evaluation of carcinogenic risks to humans. pharmaceutical drugs iarc monographs on the evaluation of carcinogenic risks to humans. some thyrotropic agents iarc monographs on the evaluation of carcinogenic risks to humans transfer of perfluoroctanoic acid (pfoa) and perfluoroctane sulfonate (pfos) from contaminated feed into milk and meat of sheep: pilot study towards a criterion for suspect thiouracil administration in animal husbandry italian babyfood containing diethylstilbestrol - years later aplasia cutis congenita in surviving co-twin after propylthiouracil exposure in utero evidence that urinary excretion of thiouracil in adult bovine submitted to a cruciferous diet can give erroneous indications of the possible illegal use of thyreostats in meat production diseases associated with inorganic and farm chemicals aplasia cutis congenita and other anomalies associated with methimazole exposure during pregnancy estrogen and its metabolites are carcinogenic agents in human breast epithelial cells the identification of potential alternative biomarkers of nitrofurazone abuse in animal derived food products opinion on the risk assessment of dioxins and dioxins-like pcbs in food. cs/cntm/dioxin/ final opinion of the scientific committee on veterinary measures relating to public health on assessment of potential risks to human health from hormone residues in bovine meat and meat products, adopted on review of specific documents relating to the scvph opinion on april on the potential risks to human health from hormone residues in bovine meat and meat products opinion of the scientific committee on veterinary measures relating to public health on review of previous scvph opinions of the mineral nutrition of livestock analysis of thyreostats: a history of years the world health organization reevaluation of human and mammalian toxic equivalency factors for dioxins and dioxin-like compounds weight increase of the thyroid gland as a tentative screening parameter to detect the illegal use of thyreostatic compounds in slaughter cattle residues of hormonal substances in foods of animal origin: a risk assessment pesticide, veterinary and other residues in food quantitative assessment of the impact of changes on meat inspection on the effectiveness of the detection of animal diseases and welfare conditions (comisurv report) implications for surveillance and monitoring for small ruminant health and welfare of changes to meat inspection as proposed by the contam panel and has also zoonotic implications. the pathological and histological findings in sheep and goats are similar to those seen in cattle these reports highlight the possible role of domestic goats and sheep as reservoirs of tb, and the need to re-evaluate the evidence for m. bovis (and m. caprae) transmission among cattle and small ruminants described the main pathological lesions in goats in the lungs in the form of abscesses ( - cm in size) with yellowish white, caseous or caseocalcareous lesions. lesions are also seen in the retropharyngeal, mediastinal or mesenteric lymph nodes and in liver, spleen and udder. lesions is sheep are very similar to those in goats, ranging from encapsulated soft, caseous lesions and encapsulated, calcified tubercles are also present in the lungs and liver in general, small ruminants are not subjected to official tb eradication campaigns; however, sheep and goats may undergo a bovine tuberculin skin test for detecting tb infection if located on premises where btb has been confirmed in cattle (subject to findings of a veterinary risk assessment), or if m. bovis infection has been confirmed in the sheep/goat flock/herd itself. if goats are kept together with cows, such goats must be inspected and tested for tb. furthermore, given that milk has it involves a visual inspection of the lungs, trachea and oesophagus, with palpation of the lungs and the bronchial and mediastinal lymph nodes both m. bovis and m. caprae cause tuberculosis in bovines and other species, including humans. further in the text, only m. bovis is mentioned regulation (ec) no / of the european parliament and of the council of april laying down specific hygiene rules for food of animal origin oj l interference of paratuberculosis with the diagnosis of tuberculosis in a goat flock with a natural mixed infection elevation of mycobacterium tuberculosis subsp. caprae aranaz et al. to species rank as mycobacterium caprae comb. nov., sp. nov liver fluke morphopathology of caprine tuberculosis. i. pulmonary tuberculosis. anales de veterinaria de murcia prevalence and regional distribution of scab, lice and blowfly strike in great britain control and management of sheep mange and pediculosis in great britain liver fluke (fasciola hepatica) in slaughtered sheep and cattle in new zealand, - foot lameness in dairy goats contribution of meat inspection to animal health surveillance in sheep and goats characterisation of white line degeneration in sheep and evidence for genetic influences on its occurrence foot health in sheepprevalence of hoof lesions in uk and irish sheep tb in goats caused by mycobacterium bovis fasciolosis in cattle and sheep outbreak of tuberculosis caused by mycobacterium bovis in golden guernsey goats in great britain scientific opinion of the panel on biological hazards (biohaz) on a request from the commission on tuberculosis and control in bovine animals: risks for human health strategies scientific opinion on the public health hazards to be covered by inspection of meat (swine) scientific opinion on the public health hazards to be covered by inspection of meat (poultry) reducing the future threat from (liver) fluke: realistic prospect or quixotic fantasy? veterinary parasitology seroprevalence of fasciola hepatica infection in sheep in northwestern spain a qualitative risk and benefit assessment for visual-only postmortem inspection of cattle, sheep, goats and farmed/wild deer an evaluation of food chain information (fci) and collection and communication of inspection results (ccir) for all species the use of data mining techniques to discover knowledge from animal and food data: examples related to the cattle industry back to the future: the animal health monitoring system -a political necessity being addressed in the united states differentiation by molecular typing of mycobacterium bovis strains causing tuberculosis in cattle and goats lameness and foot lesions in adult british dairy goats the importance of feet examination in sheep health management naming and recognition of six foot lesions of sheep using written and pictorial information: a study of english sheep farmers comparison of new zealand and european community ovine liver inspection procedures a review of the road transport of slaughter sheep effects of stocking density on lambs being transported by road evaluation of the gammainterferon assay for eradicaiton of tuberculosis in a goat herd cost-effective meat inspection: the scientific basis. surveillance kinetics of fasciola hepatica egg passage in the faeces of sheep in the porma basin a case of generalized bovine tuberculosis in a sheep tuberculosis due to mycobacterium bovis and mycobacterium caprae in sheep guide to good farming practices for animal production food safety the ecology of the liver fluke (fasciola hepatica) tuberculosis in goats aranaz a and spanish network on surveillance monitoring of animal tuberculosis, . a database for animal tuberculosis (mycodb.es) within the context of the spanish national programme for eradication of bovine tuberculosis update on trematode infections in sheep tuberculosis in goats on a farm in ireland: epidemiological investigation and control concurrent outbreak of tuberculosis and caseous lymphadenitis in a goat herd emerging parasitic diseases in sheep . fasciolidae. in: veterinary parasitology mycobacterium bovis causing clinical disease in adult sheep meat inspection, comprising both ante-mortem and post-mortem inspection, is recognised as a valuable tool for surveillance and monitoring of animal diseases and welfare conditions, and helps in the recognition of outbreaks of existing or new disorders or disease syndromes, in situations where clinical signs are not detected on-farm. meat inspection represents a practical way to evaluate the welfare of small ruminants on-farm, and the only way to evaluate their welfare during transport and associated handling. changes in the meat inspection system may negatively affect the efficiency of the surveillance and monitoring of animal diseases and welfare conditions. the focus of the animal health and welfare (ahaw) panel was to assess the implications for surveillance of animal health and welfare of the changes proposed to the current small ruminants meat inspection system by the biological hazards (biohaz) and contaminants in the food chain (contam) panels. briefly, the recommendations of the biohaz panel were related to (i) shorter transport and lairaging, (ii) improved collection of food chain information to provide information for categorisation of farms, which can be used for e.g. risk-based ante-mortem inspection, logistic slaughter and/or decontamination, (iii) omission of palpation and incision in animals subjected to routine slaughter at post-mortem inspection (if necessary, detailed inspection with potential use of palpation and incision should be carried out separately). the contam panel recommendations included (i) the ranking system for chemical substances of potential concern and its updating, (ii) the use of fci to help facilitate risk-based sampling strategies, and (iii) the inclusion of new hazards in control programmes for chemical residues and contaminants.to assess the impact of proposed changes to the current meat inspection on the overall sensitivity for surveillance and control of animal diseases and welfare conditions, the results and conclusions of a quantitative assessment, carried out by an external consortium (comisurv) under an efsa procurement, were analysed. this report assessed the impact of a change from the current small ruminant meat inspection to a visual only system in terms of detection efficiency of a list of twenty selected diseases and welfare conditions of sheep and goats. additional information from scientific literature and other recent assessments were also taken into account by experts to assess the impact of proposed changes on the detection probability and overall surveillance of animal diseases and welfare conditions.a change to visual only inspection caused a significant reduction in the probability of detection (i.e. non-overlapping % probability intervals) of detectable cases of fasciolosis and tuberculosis in goats (stage ).with regard to exotic diseases, clinical surveillance (stage ) had a greater sensitivity for detecting foot and mouth disease than slaughterhouse surveillance, and the sensitivity increased with an increase in population size. this indicates that for those countries in europe with a large sheep population, clinical surveillance is highly effective for detecting at least one case of foot and mouth disease in an infected sheep. for countries with high slaughter numbers of sheep, slaughterhouse surveillance would be almost equally efficient in detecting the disease. a change in post-mortem protocol to a visual only system did not significantly reduce the detection of any welfare conditions.in recent years tuberculosis has been reported in small ruminants in several eu countries and most information derives from recognition of tuberculosus lesions at the slaughterhouse and from laboratory reports. according to regulation (ec) / , current inspection in small ruminants aimed at detecting tuberculosis includes visual inspection and palpation of the lungs and respiratory lymph nodes. a change to visual inspection would imply abandoning palpation, which is the reason for this reduced detection. surveillance of tuberculosis at the slaughterhouse for small ruminants should be improved and encouraged, as this is in practice the only surveillance system available. the detection of tuberculosis in small ruminants should be adequately recorded and followed at the farm level. detectable cases: cases that are detectable by routine meat inspection procedures. they will express a range of combinations of clinical and pathological signs. a proportion of detectable cases will fit the definition of the typical case and a proportion will be milder cases.detection effectiveness: the proportion of animals with lesions (i.e. detectable by visual inspection, palpation and/or incision) that are actually detected.detection fraction: the proportion of infected or affected units that are successfully detected by the surveillance system.mild cases: the mild case of a disease or condition is the form that could be seen at the early stages of the disease or at some point between the subclinical and the fully developed (i.e. "typical") form. a mild case is neither typical nor subclinical. the animal will probably present more subtle signs than in a typical case. mild cases fit the mild case definition validated by experts.monitoring: investigating samples or animals in order to obtain information about the frequency of disease or infection as it varies in time and/or space.non-detectable cases: cases that are beyond the detection capacity of current meat inspection protocols. these will often be early cases at a stage where distinct clinical signs have not yet developed, but they can be cases with mild infection that leads to only subclinical conditions, without pathological lesions detectable by meat inspection.non-overlapping probability intervals: indicates that scenarios differ significantly from each other.overall surveillance system: includes several components, such as slaughterhouse surveillance and clinical surveillance. stage : assessment of the probability of detection at meat inspection. the objective of stage modelling was to estimate case type-specific (for typical and mild cases) as well as overall probabilities of detection at meat inspection.stage : assessment of the relative effectiveness of meat inspection within the overall surveillance system by comparing meat inspection with other available surveillance methods.typical cases: cases that are, by definition, detectable cases and express more developed clinical signs than mild cases. they fit the typical case definition provided by the experts, which is defined as signs and/or lesions that are expected to be observed in more than % of affected or infected of animals seen at the slaughterhouse. key: cord- -jqgervt authors: fenner, frank; bachmann, peter a.; gibbs, e. paul j.; murphy, frederick a.; studdert, michael j.; white, david o. title: laboratory diagnosis of viral diseases date: - - journal: veterinary virology doi: . /b - - - - . - sha: doc_id: cord_uid: jqgervt tests for the specific diagnosis of a viral infection in an animal are of two general types: ( ) those that demonstrate the presence of the virus and ( ) those that demonstrate the presence of specific viral antibody. the provision, by a single laboratory, of a comprehensive service for the diagnosis of viral infections of domestic animals is a formidable undertaking. there are about individual viral species in some different viral families that infect the eight major domestic animal species. if antigenic types within an individual viral species are considered and the number of animal species is broadened to include turkey, duck, and zoo and laboratory animals, then the number of individual viruses exceeds . it is, therefore, not surprising that few single laboratories could have available the necessary specific reagents, skills, and experience for the diagnosis of such a large number of infections. tests for the specific diagnosis of a viral infection in an animal are of two general types: ( ) those that demonstrate the presence of the virus, and ( ) those that demonstrate the presence of specific viral antibody. the provision, by a single laboratory, of a comprehensive service for the diagnosis of viral infections of domestic animals is a formidable undertaking. there are about individual viral species, in some different viral families, that infect the eight major domestic animal species (cattle, sheep, goat, swine, horse, dog, cat, and chicken) . if antigenic types within an individual viral species are considered, and the number of animal species is broadened to include turkey, duck, and zoo and laboratory animals, then the number of individual viruses exceeds . it is therefore not surprising that few single laboratories could have available the necessary specific reagents, skills, and experience for the diagnosis of such a large number of infections. one consequence of this great variety of viruses is that veterinary diagnostic laboratories tend to specialize, e.g., in diseases of food ani- mais, or companion animals, or poultry, or in "exotic" viruses. within these specialized laboratories there is considerable scope for the development of rapid diagnostic methods that short-circuit the need for the isolation of viruses, which is expensive, time-consuming, and rarely necessary. many viral diseases can be diagnosed clinically, others with the assistance of the pathologist; but there are several circumstances under which laboratory confirmation of the specific virus involved is desirable or, indeed, essential. the industrialized countries of europe, north america, australasia, and japan are free of many devastating diseases of livestock that are still enzootic in other parts of the world, such as foot-and-mouth disease, african swine fever, rinderpest, and fowl plague. all industrialized countries maintain or share the use of specialized biocontainment laboratories (such as those at plum island in the united states and pirbright in the united kingdom) devoted to diagnosis and research on such "exotic" viruses. clearly it is of the utmost importance that the clinical diagnosis of a suspected exotic virus should be confirmed quickly and accurately (see chapter ). several animal viral diseases such as rabies, rift valley fever, and eastern, western, and venezuelan encephalomyelitis are zoonotic and are of sufficient human public health significance to require the maintenance of specialized diagnostic laboratories. for example, confirmation of the diagnosis of rabies in a skunk that has bitten a child provides the basis for postexposure treatment of the human patient (see chapter ). confirmation and early warning of an equine encephalomyelitis virus epizootic allows implementation of mosquito control and other measures such as restriction of the movement of horses. for diseases in which there is lifelong infection, such as bovine and feline leukemia, equine infectious anemia, and herpesvirus infections, a negative test certificate is often required as a condition of sale, particu-larly export sale, for exhibition at a state fair, or show, or for competition, as at race meetings. males used for semen collection and females used in embryo transfer programs, especially in cattle, and blood donors of all species, are usually screened for a range of viral infections to minimize the risk of transmission to recipient animals. for retrovirus infections, marek's disease, pseudorabies, and certain other diseases, it is possible to reduce substantially the incidence of disease or eradicate the causative virus from the herd or flock by test and removal programs. laboratory diagnosis is essential for the effective implementation of such operations. provision of a sound veterinary service in any state or country depends on a knowledge of prevailing viral diseases; hence, epidemiologica! studies to determine the prevalence and distribution of particular viral infections are frequently undertaken, usually based on the detection of specific antibody. many relatively nonspecific disease syndromes, such as respiratory disease (e.g., kennel cough and shipping fever), diarrhea, and some skin diseases, may be caused by a variety of agents, viral and bacterial. proper management of individual cases or infected herds or flocks may require specific viral diagnosis. specific diagnosis of the kind outlined above can be achieved by one of three methods: ( ) isolation and characterization of the causative virus, ( ) direct demonstration of virions, viral antigens, or viral nucleic acids in tissues, secretions, or excretions, and ( ) detection and measurement of antibodies (table - ). each group of methods has its place. viral isolation remains the benchmark against which other methods are measured, and is essential when decisions of major economic importance depend on the diagnosis, e.g., with suspected exotic diseases such as foot-and-mouth disease or fowl plague. on the other hand, the direct demonstration of virions or viral components may provide a much more rapid and cheaper method of specific diagnosis than viral isolation, particularly when large numbers of samples must be tested. epidemiologica! surveys, eradication programs, and the provision of certificates of freedom from specific infections are often based on serological methods or rapid tests for viral antigen. it requires at least as much effort, and often more, to process a negative specimen as it does one from which virus is isolated. the chance of isolating a virus depends critically on the knowledge, care, and attention of the veterinarian who collects the specimen (see plate - ). obviously such a specimen must be taken from the right place and at the right time. the right time is always as soon as possible after the onset of clinical signs; virus is usually present in maximum amount at about this time and diminishes, sometimes quite rapidly, in the ensuing few days. specimens taken as a last resort when days or weeks of empirically chosen antibiotic therapy have failed are almost invariably a waste of effort. the site from which the specimen is collected will be influenced by the clinical signs and a knowledge of the pathogenesis of the suspected disease (table - ). having collected the appropriate specimen(s), it should be properly labeled and sent to the laboratory, with a history, including the provisional diagnosis. where ambient temperatures are moderate and transit time to the laboratory is less than day, ice or cold packs (< °c) in a styrofoam box are frequently used. if the environmen tal temperature is high and transit times longer than a day, dry ice (- °c) may be used, although wet ice with provision to replenish it in transit is better. if exotic or zoonotic viruses are suspected, the styrofoam boxes must be replaced by or enclosed within sturdier, dou ble-walled containers with absorbent padding. appropriate permits must be obtained for interstate and international transportation, and in "blood: refers to clotted sample for serology and sample with anticoagulant added for other tests. large animals, - ml; small animals, - ml; others as appropriate. if possible remove clot before dispatch. such circumstances the collection and transport arrangements need to be discussed with the laboratory and/or the appropriate government regulatory agency. for particularly labile viruses such as respiratory syncytial virus, herpesviruses, or coronaviruses, it may be an advantage to take the cell culture to the animal. isolation and identification requires at least a week, usually longer, and it is expensive. however, it is probably the most sensitive available method, if properly collected material is used, and it provides material for further study. the sooner the specimen is processed and inoculated after arrival at the laboratory, the better. if delays of more than day are anticipated, the specimen may be frozen to - °c. swabs are processed by twirling them in the transport medium and expressing the fluid by pushing the swab firmly against the side of the container. feces are dispersed on a vortex mixer. tissue specimens are finely minced with scissors and ho mogenized in a glass or mechanical homogenizer. prior to inoculation, contaminating microorganisms are removed by filtering through membrane filters of average pore diameter . μιη, although such filters allow the passage of mycoplasmas. once virus is successfully isolated and grown to a high titer, the suspension can be refiltered through . μιη filters to exclude mycoplasmas. feces and tissue homogenates should be diluted at least : and centrifuged at g for minutes to obtain a supernate that can be filtered. if the concentration of virus is suspected to be very low, high concentrations of antibiotics may be preferable to filtration. whatever the origin of the specimen, some of the original sample and some of the filtrate should be retained at °c or frozen until the isolation attempt is finalized. virus can be grown from the suitably prepared specimen by inocula tion into cell cultures, laboratory animals, or the species of host animal from which the specimen was obtained. by far the most widely used substrate is cultured cells. choice of cultured cells. the choice of the optimal cell culture for the primary isolation of a virus of unknown nature from clinical specimens is largely empirical. primary or low-passage, homologous, monolayer cell cultures derived from fetal tissues probably provide the most sen sitive substrate for isolation of the greatest variety of different viruses. continuous cell lines derived from the homologous species are almost as good. often the nature of the disease from which the material was obtained will suggest what species of virus may be found, and in such cases the optimum cell culture for that virus can be chosen, in parallel, perhaps, with a second type of culture with a wide spectrum. cell lines offer some advantages and are available for most domestic animals ex cept avian species (table - ) . monolayer cell cultures for virus isolation should be grown in sealed containers, such as plastic flasks or glass tubes with screw caps. openculture systems such as petri dishes or microtiter trays should not be used because of the risks of cross-contamination. for some viruses, rolling the cultures on a drum improves isolation rates. special types of cultures are utilized for particular viruses. for exam ple, betaherpesviruses and gammaherpesviruses may be recovered from monolayer cultures of tissue taken directly from the diseased animal, whereas inoculation of established monolayer cell cultures with cell-free material may be negative. for some corona viruses and rhinoviruses that do not grow well in monlayer cultures, growth may occur in expiant cultures (i.e., small cubes of tissue from the trachea or gut), probably because these do not dedifferentiate in culture (see chapter ). recognition of viral growth. cultures are usually incubated at °c, despite the fact that the normal body temperatures of all domestic animal species are somewhat higher. cultures are observed daily for cytopathic effects. the speed and nature of the cytopathic effect caused by different viruses varies considerably. cytopathic effect must always be based on comparison with uninoculated cell cultures; this is particularly important for viruses requiring incubation periods of longer than a week. where none or a doubtful cytopathic effect is observed, it is usual to make a second or even a third ("blind") passage. when cytopathic effect is observed, there is a range of options: . the speed and appearance of the cytopathic effect, coupled with the case history, may immediately suggest the diagnosis. . after suitable manipulation, material from the cell culture may be examined by electron microscopy. . infected monolayers on glass coverslips or special slide/culture chambers may be fixed and appropriately stained, and the cells examined for inclusion bodies, syncytia or other characteristic cell changes. better, they may be stained with fluorescent antibody, which may provide an immediate definitive diagnosis. where prior experience and knowledge suggest it, such slide cultures may be included at the time of primary inoculation, with a consequent saving in time. table - ). their growth in monolayer culture may sometimes be recognized by means of hemadsorption. most viruses that hemagglutinate will also hemadsorb; the growth of paramyxoviruses, orthomyxoviruses, and, to a lesser extent, the flaviviruses and toga viruses, is routinely recognized in this way (see plate - ). nowadays laboratory animals play a minor role in the virus diagnostic laboratory. however, some virologists still regard intracerebral inoculation of baby mice as the method of choice for the isolation of rabies virus, flaviviruses, and toga viruses. the developing chick embryo occupies a special place. intraamniotic inoculation provides the most sensitive method for influenza viruses and for several avian viruses, and species diagnosis of orthopoxviruses can be made directly from the type of pock produced on the chorioallantoic membrane. in addition, chick embryos are extensively used as a source of primary monolayer cultures (fibroblasts, kidney cells) for the isolation of avian viruses. in veterinary medicine it is feasible to consider using the natural host species, especially susceptible young animals (e.g., calves, pigs, chicks), for the recovery of a virus from suspect material. such animals, if free of antibodies, must be considered a highly sensitive substrate. however, their use would now be contemplated only for viruses not yet cultivable, or where cell culture procedures were negative in circumstances that strongly indicated a viral etiology, and/or where there might be serious repercussions if the diagnosis were missed. a newly isolated virus can usually be provisionally allocated to a particular family, and sometimes to a genus or species, on the basis of the clinical findings, the host cell used for virus isolation, and the visible result of viral growth (cytopathic effect, hemadsorption, hemagglutination, electron microscopy of the cytopathogenic agent, etc.). definitive identification, however, usually depends on serological procedures. by using the new isolate as antigen against known antisera, e.g., in a complement fixation test, the virus can often be placed into its correct family or genus. having allocated it to a particular family (e.g., adenoviridae), one can then go on to determine the species or serotype (e.g., canine immunodiffusion antibody neutralizes infectivity of virion; inhibits cytopathology, reduces plaques, or protects animals antibody inhibits viral hemagglutination antigen-antibody complex binds complement, which is thereafter unavailable for the lysis of hemolysissensitized sheep red blood cells antibody-aggregated virions are visible by electron microscopy antibody labeled with fluorochrome binds to intracellular antigen; fluoresces by uv microscopy peroxidase-labeled antibody binds to intracellular antigen; colored precipitate forms on adding substrate enzyme-labeled antibody (or antigen) binds to antigen (or antibody); substrate changes color radiolabeled antibody (or antigen) binds to antigen (or antibody), e.g., attached to solid phase antibodies and soluble antigens produce visible lines of precipitate in a gel adenovirus ) by more discriminating serological procedures. this sequential approach is applicable only to families with a common family antigen. the range of available serological techniques is now extremely wide (table - ). some are best suited to particular families of viruses. each laboratory makes its own choice of favored procedures, based on considerations such as sensitivity, specificity, reproducibility, speed, convenience, and cost. currently most serological procedures are carried out with "hyperimmune" sera comprising a polyclonal mixture of antibodies, sometimes after they have been absorbed to eliminate antibodies of certain specificities. monoclonal antibodies with defined specificity are now becoming available. these make it possible to proceed quickly to very specific diagnosis even to the level of subtypes, strains,or variants, e.g., rabies viruses from different geographical areas. family-, genus-, and typespecific monoclonal antibodies are also being developed. as their properties are defined and they become commercially available, we can expect monoclonal antibodies to be widely used for all methods of serological identification. . immunofluorescence, used here for determining the site of assembly of components of influenza virus. antibody against the nudeoprotein antigen shows nuclear accumulation at hours after infection of chick cells. procedure: guinea pig antiserum to nudeoprotein antigen is added to a monolayer of infected chick cells, then fluoresceinconjugated rabbit anti-guinea pig igg. (courtesy dr. n. j. dimmock.) immunofluorescence. the simplest way of identifying a newly iso lated virus is by fluorescent-antibody staining of the infected cell monolayer itself (plate - ). this can provide definitive diagnosis within an hour or so of recognizing the earliest suggestion of cytopathic effect. immunofluorescence is best suited to the identification of monotypic genera, or genera of which only a single species affects that particular species of animal, or to epidemic situations when a particular virus is suspected; otherwise, replicate cultures must be screened with a range of antisera. the advantages and disadvantages of monoclonal anti bodies, in comparison with polyclonal or "absorbed" sera, discussed below in the context of radioimmunoassays, apply equally to other serological procedures, including immunofluorescence and neutrali zation. electron microscopy and immunoelectron microscopy. these pro cedures (see plate - ) are most useful in the rapid identification of cell culture virus isolates, as well as directly on specimens (see below). elec tron microscopy allows identification only to the level of family, whereas immunoelectron microscopy using suitable specific antibody may per mit finer distinctions to be made. complement fixation. for the complement fixation test, the acute and convalescent sera are heated ( °c for minutes) to inactivate complement, then serially diluted in a plastic tray. two to four units of antigen (e.g., a crude preparation of live or inactivated virus) are then added to each serum dilution together with units of complement, derived from a guinea pig. the reagents are allowed to interact at °c overnight, to allow the complement to become "fixed/' sheep erythrocytes, "sensitized" by the addition of rabbit antiserum to them ("hemolysin") are then added and the trays are incubated at °c for minutes. in those cups where the complement has been fixed by the virusantibody complex, the hemolysin fails to lyse the sheep erythrocytes; where complement is still present, they are lysed. crude cell culture supernatants often used for complement fixation tests contain not only mature virions but a range of soluble antigens, both structural and nonstructural. since many of these are shared by many or all viruses within a particular genus or family, e.g., mastadenovirus, they will cross-react with antibodies raised against any other member of the genus or family. this property makes complement fixation a useful method for preliminary screening of an isolate-to place it within the correct family or genus. immune-adherence hemagglutination is basically a somewhat simplified version of the complement fixation test; currently it is applied more often to the detection of antibody than that of antigen. hemagglutination and hemagglutination inhibition. virions of several viral families bind to red blood cells and cause hemagglutination. if antibody and virus are mixed prior to the addition of red blood cells, hemagglutination is inhibited (table - ; plate - ). the hemagglutination titer of certain viruses, e.g., canine distemper virus, may be increased by dissociation of the virions with detergents. antisera may have to be pretreated to remove nonspecific inhibitors of hemagglutination (see chapter ). the hemagglutination inhibition test is sensitive and, except in the case of the togaviruses, highly specific, since it measures antibodies binding to the surface protein most subject to antigenic change. moreover, it is simple, inexpensive, and rapid, and is therefore the serological procedure of choice for identifying isolates of hemagglutinating viruses. virus neutralization. the infectivity of a virus may be neutralized by specific antibody by a variety of mechanisms (see chapter ). serum must first be "inactivated" by heating at °c for minutes to remove nonspecific virus inhibitors. serum-virus mixtures are inoculated into appropriate cell cultures, which are then incubated until the "virus titer only" controls develop cytopathic effects (plate - ). antibody, by neutralizing the infectivity of the virus, protects the cells against viral destruction. in keeping with the general trend toward miniaturization, most neutralization tests are now conducted in disposable nontoxic sterile plastic trays with, say, flat-bottomed wells in each of which a cell monolayer can be established. virus-antiserum mixtures can be added to established monolayers, or, more usually, serum dilutions are made in the wells, a standard amount of virus is added, and the mixture incubated, after which cells are added. in the standard neutralization test the end point is read by cytopathic effect, the titer of the serum being defined as the highest dilution that inhibits the cytopathic effect. in a version of the neutralization test known as the plaque reduction assay, cell monolayers inoculated with virus-serum mixtures are overlaid with agar or methylcellulose and incubated until plaques develop (see plate - ); the end point is usually taken to be the highest dilution of serum reducing the number of plaques by at least %. if a newly isolated virus proves to be "untypeable," i.e., not neutralizable by antisera against any of the known serotypes, it may be a novel serotype, or it may indicate a mixed infection with two distinct viruses, or aggregation of virions in the specimen. aggregates can be removed by vigorous agitation, filtration, or, in the case of some nonenveloped viruses, dispersed with sodium deoxycholate, prior to repeating the neutralization test. for most routine diagnostic purposes it is usually not necessary to "type" the isolate antigenically, even to the degree just described. sometimes, however, important epidemiological information can be obtained by going even further, to identify differences between "variants" or subtypes within a given serotype (see table - ). short of determining the complete nucleotide sequence of viral nucleic acid, the most useful methods of doing this are by oligonucleotide fingerprinting of viral rna or the determination of restriction endonuclease fragment patterns of viral dna. with rna viruses, viral rna is labeled with p during replication of the virus in culture; the labeled rna is phenol-extracted from purified virions, digested with ribonuclease tl, and the resulting oligonucleotide fragments separated by two-dimensional polyacrylamide gel electrophoresis, or by cellulose acetate electrophoresis followed by deae-cellulose chromatography. autoradiography reveals a // fingerprint ,, unique to that particular viral strain. an example of the epidemiological use of this technique to trace the origin and spread of foot-and-mouth disease virus in europe in is described in chapter . similarly, viral dna prepared from virions or infected cells can be cut with appropriately chosen restriction endonucleases and the fragments separated by agarose gel electrophoresis. when stained with ethidium bromide or silver, restriction endonuclease fragment patterns (also called fingerprints) are obtained. the method has found application with all dsdna virus families, particularly in epidemiological studies, but also in understanding pathogenesis. depending on the viral family, the resolution of these methods is such that different isolates of the same viral species may be distinguishable, unless they come from the same epizootic. minor degrees of genetic drift, often not reflected in serological differences, can sometimes be detected in this way. the isolation and identification of a particular virus from an animal with a given disease is not necessarily meaningful in itself. fortuitous subclinical infection with a virus unrelated to the illness in question is not uncommon. koch-henle postulates (see chapter ) are as apposite here as in any other microbiological context, but are not always easy to fulfill. in attempting to interpret the significance of any virus isolation, one must be guided by the following considerations: . the site from which the virus was isolated is important; e.g., one would be quite confident about the etiological significance of equine herpesvirus isolated from the tissues of a -month-old aborted equine fetus with typical gross and microscopic lesions, or of distemper virus isolated from the cerebrospinal fluid of a dog with encephalitis, because these sites are usually sterile, i.e., they have no normal bacterial or viral flora. on the other hand, recovery of an enterovirus from the feces, or a herpesvirus from a nasal or throat swab may not necessarily be significant, because such viruses are often associated with inapparent infections at these sites. . interpretation of the significance of the isolation in such instances will be facilitated by recovery of the same virus from several cases of the same illness during an epizootic. . knowledge that the virus and the disease in question are often causally associated provides confidence that the isolate is significant. it is appropriate to conclude this section with some remarks about safety precautions in virus diagnostic laboratories in general and regulations about exotic viruses in particular. diagnostic virology is one of the less hazardous human occupations, but over the years a number of deaths have been caused by laboratory-associated infections. some of the commoner hazards are listed in table - . it is important to note that many of the procedures that may be dangerous for laboratory workers, particularly aerosolization, may also be sources of laboratory contamination-something that may give rise to mistaken diagnoses and sometimes a great deal of misdirected administrative action. precautions to avoid these hazards consist essentially of good laboratory technique, but special measures may be needed. mouth-pipetting is banned. gowns must be worn at all times, and gloves for anything besides personal hazard, exotic animal viruses pose special community risks such that major developed countries with large livestock indus- tries support special laboratories for their investigation. these are the so-called maximum containment laboratories, popularly designated by their location, e.g., plum island in the united states, pirbright in the united kingdom, and geelong in australia. "restricted" animal viruses in the united states and australia, the importation, possession, or use of which is prohibited or restricted by law or regulation, are listed in table - . we use the term direct identification, in contrast to virus isolation, to encompass a variety of methods that can be used to detect and often identify the etiological agent by the direct demonstration of virions or viral constituents in the tissues, secretions, or excretions of infected animals. although they do not provide the laboratory worker with a culture of the causative virus for further study, these direct methods have great advantages in terms of speed, cost, and the number of samples that can be examined. they can be subdivided into methods used to detect virions, viral antigens, or viral nucleic acids. the introduction of negative staining procedures, together with a realization that in many clinical situations the concentration of virions frequently exceeds the critical lower limit of per milliliter required for visualization in the electron microscope, has led to the use of this instrument for rapid viral diagnosis (plate - ). the procedure is particularly suited to enteric infections, in which a crude fecal suspension can be clarified by low-speed centrifugation, followed by high-speed centrifuga tion to yield a pellet for negative staining. in addition to its value in the recognition of known viruses, this technique has led to the discovery of new viruses of etiological importance in diarrheal diseases which were, and in some cases remain, uncultivable (e.g., some adenoviruses, astroviruses, caliciviruses, coronaviruses, parvoviruses, and rotaviruses). the procedure is also suited to viral infections of the skin and mucous membranes, the appropriate specimen being scabs, vesicular fluid, or scrapings made with a scalpel. also, as described earlier, electron microscopy can be used for the rapid identification of viruses isolated in cell culture, allowing immediate and definitive diagnosis to the family or sometimes the genus or species level. the sensitivity of electron microscopic methods can be enhanced by the use of immune serum, by a procedure known as immunoelectron microscopy. the sample, usually clarified by low-speed centrifugation, is mixed with antibody, and after overnight interaction, the immune complexes are pelleted by low-speed centrifugation and the pellet negatively stained. the antibody used may be serum from an old animal hyperimmune to a large number of viruses, or it may be type-specific polyclonal or monoclonal antibody, or such antibodies may be used sequentially. solid-phase immunoelectron microscopy procedures have also been developed, in which virus-specific antibody is first bound to the plastic supporting film on the copper grid. sensitivity is enhanced by a double-layering procedure, in which staphylococcal protein a (which binds the fc moiety of igg) is bound to the film, then virus-specific antibody, to which the sample is then added. . negative staining for electron microscopy. (a) direct staining: virions of bovine papular stomatitis virus. an electron-opaque stain ( % phosphotungstic acid, ph . ) was mixed with scrapings from the lesion and applied to plastic film supported by the copper electron microscope grid (xl , ). (b) immunoelectron microscopy: an isolate of foot-and-mouth disease virus type o was incubated with homotypic antiserum and stained with phosphotungstic acid (χΙΟΟ,ΟΟΟ). note aggregation of virions by antibody. [from e. p. ] . gibbs et al., vet. ree. , ( ) .] these methods are based on direct interaction between virions or viral antigens, in situ in tissues or in excretions or secretions, and specific antibodies which are prelabeled in some way so as to permit the ready recognition of the interaction. the methods are specified by the method of labeling used: immunofluorescence, immunoperoxidase staining, radioimmunoassay, or enzyme-linked immunosorbent assay (elisa). vir- al antigens can also be detected by such time-honored serological procedures as precipitation and complement fixation. immunofluorescence. its specificity, sensitivity, rapidity, and relative simplicity make immunofluorescence a procedure of singular importance in the rapid diagnosis of viral infections. the prototypic example of immunofluorescence is the diagnosis of rabies, for which it has been the standard test for more than years (see chapter ). it is now being used for a wide range of viruses. immunofluorescence can be applied to smears and frozen sections of tissues or organs. two alternative staining procedures are used: ( ) direct immunofluorescence, in which the antiviral antibody is conjugated to the fluorescent dye, fluorescein, and ( ) indirect ("sandwich") immunofluorescence, in which an antiimmunoglobin specific for the animal species providing the antiviral antibody is conjugated to fluorescein ( fig. - ) . for instance, an acetone-fixed smear or frozen tissue section is treated with virus-specific antibody (prepared, say, in rabbits), then rinsed before the second antibody, a fluorescein-conjugated anti-rabbit immunoglobulin made in goats, is added. indirect procedures have two significant advantages over direct-staining procedures: . if antibodies to different viruses are raised in a single animal species, e.g., rabbits, then only a single conjugated antibody is required. . the amount of bound labeled antibody is greatly augmented, hence the method is much more sensitive. although simple in principle, the effective use of immunofluorescence demands careful attention to many technical details if false positive results are to be avoided. in addition to immunofluorescent staining of specimens taken directly from clinical cases, the method is an important adjunct in the identification of viruses isolated in cell cultures. it may also be used in reverse, for the detection of antibody in serum. slides containing viral antigen, either smears, sections, or, more usually, cell cultures, are prepared in large numbers and stored at - °c. for use, they are flooded with the serum under test and a second fluorescein-conjugated antispecies antibody is used to detect the bound antibody. special care needs to be exercised in the application of immunofluorescence to herpesviruses, in that some herpesvirus-infected cells are known to express fc receptors on their plasma membrane; such receptors bind all igg molecules, not only those with herpesvirus specificity, hence additional controls are required. staining. an alternative method for locating and identifying viral antigen in infected cells employs an enzyme-labeled antibody. the procedure requires less expensive equipment than immunofluorescence-an ordinary light microscope is used-and produces a morphologically clearer, nonfading, permanent preparation. the procedures and principles are similar to those of immunofluorescence. the conjugated antibody, bound to antigen by a direct or indirect procedure, is detected by adding a substrate appropriate to the particular enzyme; in the case of peroxidase this is h mixed with a benzidine derivative which forms a colored, insoluble precipitate in the presence of enzyme. a disadvantage of the technique is that endogenous peroxidase present in the cells of many tissues, particularly leukocytes, produces false positive results. this problem can be circumvented by meticulous technique and adequate controls. in radioimmunoassay the label is a radioactive element, commonly i. the method is exquisitely sensitive, enabling viral antigens to be detected at concentrations as low as ~ m. many protocols for radioimmunoassay s have been devised. both direct and indirect methods can be used, the principles being the same as for immunofluorescent staining (fig. - which the "capture" antibody (or antigen) is bound to a solid substrate, typically a polystyrene tube or bead, or to the wells of a plastic microtiter plate. in the simplest format ( fig. - , left) the sample suspected to contain virus or viral antigen is allowed to bind to the bound antibody, then after washing, i-labeled antiviral antibody ("detector" antibody) is added. after a further washing, the bound labeled antibody is mea sured in a gamma counter. a more commonly used protocol is the indirect radioimmunoassay, in which the detector antibody is unlabeled but a further layer, i-labeled anti-igg, is added as "indicator" anti body. (the antiviral antibodies constituting the capture and detector antibodies must be raised in different animal species; see fig. - , right.) enzyme-linked immunosorbent assays (elisa). elisa (also known as enzyme immunoassay, eia) offers the same sensitivity as radioim munoassay without the inherent disadvantages of expensive isotopes of short half-life and the need for safe handling and disposal and a costly gamma counter. the basic principles are similar to those of radioim munoassay ( fig. - ) . antibody is bound to a solid phase, usually the wells of a microtiter tray. samples suspected to contain antigen are added to the wells. after an appropriate reaction time, the wells are rinsed and a second virus-specific antibody that has been conjugated to an enzyme is added. after allowing this to bind, the contents of the well are rinsed and a substrate for the enzyme is added. the assay is read by a color change in the substrate and can be made quantitative by serially diluting the antigen to obtain an end point or by photometrically reading the amount of color change, a reflection of the amount of enzymeconjugated antibody bound. as in radioimmunoassay, there are many variations in protocol, e.g., exploiting the very high affinity of avidin for biotin ( fig. - , right) . moreover, if antigen is bound to the plate first, the procedure is equally suitable for the detection and quantitation of viral antibody. elisa procedures have been developed for a wide variety of applications in veterinary medicine. at one level, kits have been marketed for the rapid diagnosis of a number of important viral diseases by veterinary practitioners themselves. at another level elisa procedures have been automated by the introduction of automatic dispensing, washing, and spectrophotometric reading and recording instruments, that permit hundreds of samples to be processed in a day, e.g., in the testing of swine for pseudorabies antibodies. immunodiffusion (precipitation-in-gel). if wells are cut in agar and antibody and antigen are placed in separate wells, the two diffuse toward each other (immunodiffusion) and form visible bands of precipitate (plate - ). several ingenious applications of the procedure have been developed and the test is widely used for the diagnosis of some diseases of domestic animals (e.g., bovine leukemia, equine infectious anemia; see chapter ). although now considered too cumbersome a procedure for general use in the rapid detection of viral antigen, the complement fixation procedure is still employed for the rapid and specific detection of foot-and-mouth disease viral antigen in vesicular fluid, providing both rapid diagnosis and specific typing of the virus involved. if dsdna is separated ("melted") into single strands by heat or alkali treatment, the single strands will, under appropriate conditions, reanneal to each other, or competitively to an identical or related complementary strand. if the original dna is labeled with either p or s, it may be used as a probe for the detection of related dna in infected cells. this procedure, known as in situ hybridization, can be made even more specific by using probes that are shorter than the full-length genome. it can also be made more sensitive by maximizing the amount of label incorporated into the probe. hybridization is detected by autoradiography. recently, nonradioactive hybridization procedures, based on the incorporation of biotin-conjugated nucleotides into the dna probe, have been developed; avidin, which binds strongly to biotin, is subsequently added. the avidin is detected by elisa, immunofluorescence, or immunoperoxidase staining. in situ hybridization procedures are particularly useful when viral dna is present in cells but is not expressed, as with integrated retroviral dna, or episomal dna in some papo va virus-infected cells. probes can be made highly specific by selection from a collection of cloned fragments of the whole viral genome. the probes are labeled by in vitro nick translation procedures, and are then applied to nitrocellulose blot transfers of the animal tissue (active hybridization) or to nitrocellulose blots taken from gels on which viral nucleic acid has been separated (southern blotting), or from nitrocellulose onto which viral nucleic acid-containing samples have been spotted (dot-blot hybridization). these procedures have proved of great value in virus research, but it remains to be seen to what extent nucleic acid probes and hybridization procedures displace other methods for rapid diagnosis of viral infections. it may have advantages over virus isolation in the case of viruses that are noncultivable, slow growing, dangerous, or nonviable as a result of suboptimal conditions of transport or storage. detection of viral antibody can be used for the diagnosis of viral infections, either in individual animals or in populations. the method is particularly useful in the latter context, since serum samples are readily obtained with simple equipment, in contrast to special requirements, time, and effort needed for collecting samples for virus isolation. furthermore, tests for antibody such as elisa lend themselves to automation, so that large numbers of samples can be tested. they form the basis of epidemiological surveys and of control and eradication programs, but have major limitations in diagnosis. for diagnosis in the individual animal, paired sera are tested for specific viral antibody, the first sample being taken when the animal is first examined (acute-phase serum), and the second sample - weeks later (convalescent-phase serum). a rise in antibody titer between the first and second samples is a basis, albeit in retrospect, for a specific viral diagnosis. sometimes the demonstration of antibody in a single serum sample is diagnostic of current infection, e.g., with retroviruses and herpesviruses, since these viruses establish lifelong infections. however, in such circumstances there is no assurance that the persistent virus was responsible for the disease under consideration. detection of antiviral antibody in presuckle newborn cord or venous blood provides a basis for specific diagnosis of in utero infections. it was used, for example, in showing that akabane virus was the cause of arthrogryposis-hydranencephaly in calves (see chapter ). since transplacental transfer of immunoglobulins is rare in domestic animals (see table - ), the presence of either igg or igm is indicative of exposure of the fetus to antigen. serological methods based on the detection of virus-specific igm may also be used for the specific diagnosis of recent viral infection, since antibodies of the igm class appear first after primary infection and de-clines to relatively low levels, compared to igg, by about months after infection. however, the method has not yet been much exploited in veterinary medicine. technical advances such as miniaturization (microtiter plates), automation for large numbers of samples, monoclonal antibodies, and the development of diagnostic kits such as latex agglutination assays for detecting specific igm, have resulted in a revolution in the approach to diagnostic serology in human medicine. the costs, coupled with the technical problems associated with the large number of animal hosts and their many viruses, have delayed the development of these procedures in veterinary medicine, but their use can be expected to expand considerably in the future. however, screening programs to establish regional or national prevalence rates for particular viruses, based on detection of specific antibody in single serum samples, are an essential feature in defining the epidemiology of viruses of domestic animals. biosafety in microbiological and biomedicai laboratories oligonucleotide fingerprinting of viral genomes diagnostic virology using electron microscopic techniques rapid virus diagnosis radioimmunoassay in diagnostic virology new developments in practical virology laboratory diagnosis of viral infections diagnostic procedures for viral and rickettsial infections manual of clinical microbiology recent advances in virus diagnosis guide to the collection and transport of virological specimens laboratory diagnosis of viral diseases laboratory safety: principles and practices manual of clinical laboratory immunology automated systems in viral diagnosis immunochemistry of viruses. the basis for serodiagnosis and vaccines enzyme immunoassays for the detection of infectious antigens in body fluids: current limitations and future prospects key: cord- -cz ruqxx authors: scott, danny w.; miller, william h.; griffin, craig e. title: dermatoses of pet rodents, rabbits, and ferrets date: - - journal: muller & kirk's small animal dermatology doi: . /b - - - - . - sha: doc_id: cord_uid: cz ruqxx nan subcutaneously and repeated every weeks until the disorder being treated is cured. it has also been reported that ivermectin is effective topically (one or two drops applied between the scapulael.fv " special precautions must also be observed when using topical medications. first, these animals routinely remove topical agents through their grooming behaviors. second, these small creatures are prone to hypothermia after shampooing and dipping, thus, aqueous solutions should be kept at body temperature, and the animals must be kept warm and dry and away from drafts after treatment. avoid alcohol-based sprays. in addition, inhalation/aspiration pneumonia is a potential problem. thus, the face is best avoided when dips are administered. the structure and function of small exotic mammal skin are similar to those described in chapter , hair growth in rodents (not guinea pigs) and rabbits occurs in periodic orderly waves originating on the ventrum between the front legs and spreading dorsally and caudally. in some rabbits, thickened patches of skin can be associated with the variation in hair growth cycles. this is more common in young rabbits and becomes less obvious with increasing age. the mean thickness in these patches is mm as compared with mm in normal skin. histologic examination of the patches reveals increased size of hair follicles and increased vascularity. notable histologic differences include ( ) the absence of epitrichial sweat glands in rodents and ( ) the structure of the tail epithelium of the mouse and rat, which features orthokeratosis with a stratum granulosum at the follicular osia and parakeratosis without a stratum granulosum in the interfollicular areas. hamsters have large glands on either flank, which are visible as dark brown patches that are more prominent in male animals. these flank or hip glands are sebaceous and are used for marking territory. in sexually aroused males, the haircoat over the glands becomes matted from secretions and readily visible, and the animals scratch the areas as if they are pruritic. these findings may be interpreted as signs of skin disease by some owners. gerbils have a yellowish tan, midventral scent gland, which is also sebaceous, more prominent in male animals, and occasionally mistaken as a cutaneous abnormality. rabbits have a mental or chin gland (which is sebaceous in nature) with which they mark their territory, normal ferrets typically have visible accumulations of brownish cerumen around the external auditory meatus. in addition, normal ferrets can have several comedones on the tail. most rodents are burrowing animals that spend most of their time in the wild seeking food and escaping predators. when they are placed in sterile environments with ad libitum feeding and no danger of predators, they are left with little to do except to chew on themselves or on others. in addition, male rodents tend to be territorial and aggressive. self-inflicted trauma or that inflicted on cagemates can be triggered or amplified by crowding. some other normal behaviors may be misinterpreted as pruritus. a rabbit rubbing its mental, or chin, gland on a cage or furniture is only marking its territory. likewise, a guinea pig scooting or dragging its perineal area on the ground is usually scent-marking, although in some male animals, the glandular secretions can become impacted and cause irritation. male hamsters may clean and fuss with their flank glands. staphylococci, especially staphylococcus aureus , are frequently isolated from the skin, the ears, the nostrils, and the haircoat of rodents and rabbits." , not surprisingly, s. aureus is a common opportunist and cause of skin infections in these species. finally, these small creatures, especially mice, rats, guinea pigs, and rabbits, are frequently used for studying models of human diseases (e.g., hereditary hypotrichoses and ichthyoses in mice and rats), for examining the pathogenesis of various dermatoses also seen in humans (e.g., contact hypersensitivity and candidiasis in guinea pigs), for evaluating therapeutic agents used in various human dermatoses (e.g., treatment of malassezia dermatitis in guinea pigs and the use of retinoids in rhino mice), for studying percutaneous absorption and various aspects of dermatopharmacology (e.g., the mouse tail assay for studying epidermal drug effects), and for screening the potential irritancy or sensitization of topical agents (e.g., the guinea pig draize test for contact allergens and the rabbit skin test for topical irritants)." in the wild, chinchillas keep their haircoats clean and healthy by bathing in fine volcanic dust.!? a similar dust is commercially available (chinchilla dust, or fuller's earth) and should be provided daily." , , the dust is poured to ern deep in a metal pan and left in the animal's cage for about minutes. chinchillas deprived of their dust baths are prone to abnormalities of the haircoat and skin. chinchillas kept in a warm (warmer than °f) humid environment develop matted fur. ferrets have active sebaceous glands that contribute to their distinctive odor and somewhat greasy feeling coat. occasional small, red-brown waxy deposits can be found on normal skin. during breeding season, intact males have increased sebaceous secretions, to the point of having a yellowish discoloration of the undercoat, a very oily fur, and quite a musky odor. frequent bathing may strip essential oils from the skin, resulting in keratinization disorders and pruritus.!? owners should be encouraged to use mild shampoo no more frequently than once monthly, if possible. bacterial skin infections are uncommon in the ferret and are usually caused by s. aureus or streptococcus spp." infections are usually secondary to bite wounds (especially on the neck of female ferrets during breeding season, perpetrated by aggressive male animals as a prelude to coitus) or the pruritus associated with ectoparasites. there may be superficial and follicular (fig. - ) lesions or deep abscesses and fistulae, staphylococcal or streptococcal cellulitis of the neck may be associated with dental disease and mandibular osteomyelitis.f' diagnosis is based on cytologic examination. treatment consists of a regimen of various combinations of topical antimicrobials ( % hydrogen peroxide or . % to % chlorhexidine), surgical drainage, and the administration of systemic antibiotics (table - ) . actinomycosis ("lumpy jaw") is rarely reported in ferrets.!" affected animals have nodules or abscesses in the neck, and fistulae and discharge of thick green-yellow pus may be seen. dermatophytosis appears to be rare in ferrets." m. canis and t. mentagrophytes are the most common causes, and young animals are most frequently affected. lesions consist of annular areas of alopecia, broken hairs, scale, and varying degrees of erythema and crusting. pruritus is usually absent. diagnosis is confirmed by microscopic examination of affected hairs and fungal culture. therapy consists of topical application of antifungal agents and environmental clean-up as described for cats. griseofulvin is usually not needed. dermatophytosis in ferrets is a potential zoonosis. blastomycosis was diagnosed in a ferret with chronic cutaneous plaques and ulcers. i? histoplasmosis was diagnosed in a "see references , , , , , , , . • ferret with multiple subcutaneous nodules." coccidioidomycosis was diagnosed in a ferret with a persistent draining tract of the stifle. an outbreak of otitis extema and pinnal necrosis in association with mites (otodectes?) and yeast (malassezia?) infection was reported." a painful, rapidly progressing crusting and necrosis of the pinnae spread, if untreated, onto the face. mites and yeast were found in smears. histopathologic findings included suppurative epidermitis, numerous surface yeast, and hemorrhagic necrosis with thrombosis. treatment with ketoconazole ( mg/ferret, q h, per os) and a polypharmaceutical otic preparation was rapidly effective. otodectic mange (ear mites) is common in ferrets. , , , affected ferrets may manifest no clinical signs or variable degrees of excessive cerumen production. pruritus, inflammation, and secondary bacterial infection are uncommon. diagnosis is confirmed by finding otodectes cynotis in ear swabs. treatment is accomplished with topical acaricides or topical ivermectin ( jlg/kg divided between the two ears);'? injectable ivermectin is the treatment of choice. in one study, a topical treatment with either tresaderm ( drops in ears q h for days, sequence repeated after a week of no treatment) or ivermectin (ivomec diluted : in propylene glycol; jlg/kg divided equally in both ears, repeated in weeks) was more effective than the subcutaneous administration of ivermectin ( jlglkg, repeated in weeks). ivermectin should be used with caution in pregnant jills.l" . when ivermectin was administered at to weeks of gestation, an increased incidence of congenital defects, such as cleft palates, was seen. however, when ivermectin was administered after weeks of gestation, no problems were noted. fleas (especially ctenocephalides felis felis) are commonly found on ferrets.'? animals may be asymptomatic or have cutaneous reaction patterns similar to flea bite hypersensitivity in cats. ferrets manifesting presumed flea bite hypersensitivity have a pruritic papulocrustous dermatitis over the rump, ventral abdomen, and caudomedial thighs, or a self-induced, symmetric alopecia over the rump, flanks, ventral abdomen, or medial thighs (fur-mowing) in which the skin appears normal. treatment strategies must include the ferret; in-contact ferrets, cats, and dogs; and the environment, as described for cats. fipronil spray has been found to be safe and effective for ferrets." sarcoptic mange is uncommon in ferrets and has two clinical presentations: ( ) intense pruritus and dermatitis over the face, the pinnae ( fig. - a) , and the ventrum and ( ) pruritic pododermatitis. . in the pododermatitis form, the feet are swollen, erythematous, and crusted, and the claws may be dystrophic. affected ferrets may actually slough claws or digits. diagnosis is confirmed by finding sarcoptes scabiei mites in skin scrapings. however, mites can be extremely difficult to find, so response to miticidal therapy is often used as a diagnostic procedure. treatment includes % lime sulfur dips (weekly until weeks after clinical cure) or ivermectin injections. demodicosis was reported in two unrelated ferrets that were living in the same household and receiving long-term treatment with a glucocorticoid-containing otic ointment.p' the ferrets exhibited excessive cerumen in the ears and pruritus, alopecia, comedones and orange discoloration of the skin behind the ears and on the ventrum. skin scrapings and ear smears revealed numerous short mites that resembled demodex gatoi. amitraz dips were curative, and no side effects were reported. ferrets housed outdoors may occasionally have cysts, abscesses, or fistulae in the neck associated with infestation by hypoderma sp. or cuterebra sp. larvae. , treatment includes careful surgical removal of the larva and routine wound care. flystrike (especially wohlfahrtia vigil) can be a problem for commercial ferret ranchers and outdoor ferrets." eggs are commonly laid on the face, neck, and flanks of young ferrets, causing irritation and subcutaneous abscesses. ticks may occasionally be found on ferrets, especially around the head and the ears.!? treatment is as described for cats. ferrets can be experimentally infected with dracunculus insignis and have been used as an animal model for studying dracunculiasis.t? lesions consist of tender swellings, which abscess and develop fistulae. lesions occur most commonly on the legs. the ferret is susceptible to canine distemper virus." , , typical cutaneous findings include an erythematous rash under the chin and in the inguinal region; swelling and brownish crusts on the chin, lips, nose, and the periocular area; and swollen, hyperkeratotic nose and footpads ( fig. - ) . some ferrets develop an orange-tinged dermatosis on the anus and inguinal region. adrenocortical neoplasia and hyperplasia are the most common causes of progressive bilaterally symmetric alopecia in the ferret. , , a, , two authors indicated that this disorder accounted for about % of all the ferrets examined in their practice.v the condition was initially diagnosed as cushing's syndrome. however, other classic clinical signs (polyuria, polydipsia, and polyphagia) and hematologic, biochemical, or urologic abnormalities associated with cushing's syndrome are rarely present. in addition, basal plasma cortisol levels are usually within the normal range and adrenal function tests (adrenocorticotropic hormone [acth] stimulation, dexamethasone suppression) have not been useful in separating normal from diseased ferrets.l" , , , the contralateral adrenal gland is not atrophied. these considerations in concert with the common occurrence of vulvar enlargement in affected female ferrets and return to male sexual behavior in castrated male ferrets suggested that the endocrine abnormality was in the adrenocortical production of sex hormones." median plasma concentrations of -hydroxyprogesterone, androstenedione, and estradiol are significantly higher in ferrets with hyperadrenocorticism than in normal ferrets." ninety-six percent of affected ferrets had a high concentration of at least one of these three hormones, but only % had high concentrations of all three hormones. these concentrations decreased after adrenalectomy. the etiology of hyperadrenocorticism in ferrets is unknown, but several hypotheses have been put forth. , early neutering may lead to metaplasia of undifferentiated gonadal cells in the adrenal capsule, in a dutch study, a significant linear correlation was found between the age at neutering and the age at the time of diagnosis, a hyperadrenocorticism in ferrets is common in the united states, but apparently rare in the united kingdom, suggesting that diet (prepared foods in the united states, whole prey in the united kingdom), photoperiod (housed indoors in the united states, outdoors in the united kingdom), and genetics (u,s, population more inbred) may also playa role, clinical signs are seen between and years (average ) of age and with equal frequency in females and males. in most cases, clinical signs are first noted in the spring. the initial abnormality is usually a bilaterally symmetric, noninflammatory alopecia that usually begins on the tail and tail base ( fig. - ) and progresses to the ventral abdomen, caudomedial thighs, dorsal surface of hind paws, lumbosacral region, and shoulder blades. in some animals, the alopecia may come and go. in such cases, clinical signs appear in the spring and spontaneously regress in the autumn. however, clinical signs recur with increasing severity each spring and eventually persist.l? , most affected males and females have a strong musky odor. vulvar enlargement is common in female animals , as is return to male sexual behavior in castrated males. in more chronic cases, the back of the neck, top of the head, and trunk become alopecic, and the body skin becomes thin and hypotonic ( fig. - ). about % to % of affected ferrets are reported to have varying degrees of pruritus, mostly over the dorsum between the shoulder blades. , , a when present, pruritus is often severe and unresponsive to glucocorticoids and antihistamines. numerous comedones are occasionally found on the tail, and phlebectasias may be seen. lethargy, muscle atrophy, splenomegaly, and return to male behavior (in neutered males) occur in about %, %, %, and %, respectively, of the cases.f mammary gland hyperplasia has been reported, and stump pyometras are not uncommon. stranguria may be present in up to % of affected males. a the enlarged adrenal gland can be palpated in about % of cases. diagnosis is usually confirmed by ultrasonographic examination or exploratory laparotomy.lo· . ultrasonography may fail to diagnose about % of the cases and may not be cost-effective in a practice situation." in rare instances, the adrenal glands appear normal on both ultrasonographic examination and exploratory laparotomy.t- urinary cortisol: creatinine ratios were significantly higher in ferrets with hyperadrenocorticism than in normal ferrets.p however, this is not specific for hyperadrenocorticism, and affected ferrets can have normal ratios.v' eighty-four percent of affected ferrets have unilateral adrenal tumors, whereas % have bilateral adrenal tumors." of the unilateral tumors, over % are in the left adrenal gland. . . a histologically, % of the tumors are nodular hyperplasia, % are adenocarcinomas, % are adenomas, and % are "normal." all tumors larger than ern diameter were adenocarcinornas.v in % of the patients with bilateral tumors, each gland had a different histologic type of tumor.s- a all male ferrets with a return of male sexual behavior had adenocarcinomas." in one study, % of the ferrets with hyperadrenocorticism had concurrent insulinomas. this emphasizes the importance of performing a complete exploratory. the "adrenal panel" (clinical endocrinology laboratory, department of comparative medicine, university of tennessee, knoxville, tn, - - ) may give diagnostic results in % of the cases.s" however, results are not always diagnostic, and the test may not be cost-effective in a practice situation.f- a in most animals, the treatment of choice is unilateral adrenalectomy. . , clinical improvement is evident within to weeks, and complete recovery is usually seen within months. hair regrowth usually occurs in the opposite direction to which it was lost. in two cases in which a unilateral adrenalectomy was performed and no hair growth had occurred for to months, a secondary exploratory revealed tumors on the remaining adrenal.p seventeen percent of the animals with unilateral tumors develop tumors on the remaining adrenal, resulting in a recurrence of clinical signs within months." ferrets with bilateral adrenal tumors should have the largest adrenal gland completely removed, along with % to % of the other one (subtotal bilateral adrenalectomyi.v- a even following subtotal bilateral adrenalectomy, % of the ferrets had recurrence of clinical signs within to months.f> medical treatment has been used, usually unsuccessfully, when surgery could not be performed, or when, after surgical removal of a neoplastic adrenal, the remaining adrenal gland also became neoplastic and clinical signs recurred.!? mitotane (o,p'-ddd) has been used at mg orally, once daily for days, then every days until clinical cure. administration of the drug is then stopped. if clinical signs recur, o,p'-ddd is given at a weekly maintenance dose of mg. to facilitate administration, the o,p'-ddd is mixed with com starch and -mg doses are put in gelatin capsules. however, mitotane is rarely effective.!" a ketoconazole has been ineffective when given orally at mglkg every hours. o • a anecdotal reports indicate that leuprolide (lupron, tap pharmaceuticals), when given at jlglkg subcutaneously, every days, is very effective for the treatment of hyperadrenocorticism in ferrets. response is usually seen by the third injection and is complete by months. once maximum response is achieved, the interval between injections is extended. ferrets with aggressive adrenal carcinomas may not respond. hyperestrogenism is well recognized in the female ferret.l" . . however, this disorder is rare because large-volume ferret breeders are neutering and descenting the animals at weeks of age. an ovarian remnant may be suspected in a neutered female with signs of estrus. jills allowed to remain in estrus during the breeding season are susceptible to the toxic effects of estrogen on bone marrow. affected jills develop pancytopenia and varying degrees of alopecia. clinical signs accompanying the pancytopenia include pale mucous membranes, petechial or ecchymotic hemorrhages, anorexia, depression, and weight loss. the alopecia is bilaterally symmetric, beginning on the tail, the perineum, the abdomen, the medial thighs, and the rump and progressing cranially ( fig. - ) . vulvar enlargement is a constant finding. untreated animals die of infectious or hemorrhagic complications. treatment is often unrewarding. ovariohysterectomy; intravenous blood transfusions; administration of dexamethasone, anabolic steroids, and systemic antibiotics; and supportive care have rarely been reported to result in recovery, but transfusions may need to be repeated several times for to months.!" . . testicular neoplasia is rare, because most large-volume breeders neuter male ferrets at weeks of age. a sparse haircoat and a bald tail were reported in association with an interstitial cell carcinoma of the testicle in one ferret.!? total body alopecia and pruritus were reported in association with a testicular sertoli's cell neoplasm in another ferret.'? breeding season alopecia is commonly seen, especially in the female and less frequently in the male ferret, during the period of sexual activity from march through august. , . . , photoperiod plays an important role in this condition, because even neutered ferrets are affected. bilaterally symmetric alopecia affects the tail, the perineum, the ventral abdomen, the rump, and occasionally, the periocular region and the paws. affected ferrets are otherwise healthy, and spontaneous hair regrowth occurs in the fall. seasonal shedding is seen in spring and early summer.f- , , variable degrees of hypotrichosis or alopecia may be seen over the trunk and resolve spontaneously within a month or two. telogen defluxion is occasionally seen to months after a stressful circumstance (high fever, severe illness, surgery, and anesthesial.l"' bilaterally symmetric hypotrichosis or alopecia is most prominent on the trunk ( fig. - ) . although anecdotal reports suggest that hypothyroidism is a common cause of endocrinelike alopecia in ferrets," the authors and others , l have never made such a diagnosis, and know of no documented cases. some data have been published on basal serum thyroxine and triiodothyronine levels and thyroid function tests in normal ferrets. l • some authors believe that the most common cause of alopecia and dull, dry haircoat in ferrets is poor dietary practices." food passage averages to hours in ferrets; thus, diets high in protein and fat but low in fiber are important. a low-fat diet may result in a dry, dull haircoat. biotin deficiency (from excessive feeding of raw eggs) can result in bilaterally symmetric alopecia in ferrets." , , severe intestinal parasitism (especially toxascaris leonina) can produce variable degrees of hair loss and scaling in ferrets." contact dermatitis can occur with frequent use of shampoos or insecticide sprays.p focal areas of alopecia have been seen at the site of previous injections.p the authors have seen an occasional ferret with presumptive atopy. affected animals manifested symmetric, nonlesional pruritus over the trunk, the rump ( fig. - ), and the paws. fleas were not present, hypoallergenic diets were ineffective, and response to glucocorticoids or chlorpheniramine was good. one of the authors (whm) has seen one food-hypersensitive ferret with the same clinical signs. the ferret was normal when fed a commercial hypoallergenic diet for cats (innovative veterinary diets [ivd] venison). erythema annulare centrifugum was reported in a ferret with hyperadrenccorticism." parallel linear bands of erythema were present over the dorsolateral lumbosacral area and encircling the tail (fig. - ) . the dermatitis disappeared after days of treatment with a commercial omega /omega fatty acid-containing product (derm caps). the blue ferret syndrome is an unusual idiopathic condition affecting ferrets of either sex, neutered or intact." the abdominal skin shows bilaterally symmetric bluish discoloration. affected ferrets are asymptomatic. the condition regresses spontaneously during a few weeks. in the authors' experience, this condition is most commonly seen in ferrets that have been clipped for surgery or to provide access to veins during the resting phase of the hair cycle. the clipped area remains hairless for a long time, then suddenly begins to tum blue. it appears that hair follicles are making melanin, which will be incorporated dermatoses of pet rodents, rabbits, and ferrets • figure - . a, self-induced alopecia over the rump of a ferret with a pruritic dermatosis resembling atopy. b, same ferret. self-induced hypotrichosis of hind paw. into growing hairs. soon after the ferret's skin turns blue, hair regrowth begins (within to weeks). self-inflicted facial excoriations (burrowing) may be seen in ferrets that have inadequate bedding, nesting material, or hiding spots.'? intact females may pull out hair to use as bedding.'? skin neoplasms are fairly common in the ferret, and the majority are benign. lo, in one large survey." the skin was the third most common site for primary neoplasms in ferrets (about % of all neoplasms). one of the most frequent of these is the mast cell tu-mollo, , lesions may be solitary or multiple and may come and go over time. mast cell tumors present as papules or nodules (see fig. - b), which vary from skin colored to yellow, brown, or red. they may be firm, soft, or cystic. some lesions are pruritic. lesions may occur anywhere, but are most commonly reported on the neck and dorsal trunk. similar to the situation in cats, most cutaneous mast cell tumors in the ferret are benign.p: basal cell tumors and sebaceous gland neoplasms (especially on the head, neck, limbs, tail and the shoulder [see fig. - c)) are also common in ferrets.!" , most previously diagnosed "basal cell tumors" were probably sebaceous epitheliomas.l? epitrichial sweat gland carcinomas (especially on the tail and the groin), chondromas (especially on the tail), chondrosarcomas (especially on the tail), and squamous cell carcinomas (especially on the digit and the lip [see fig. - d) ) have also been reported on numerous occasions.fv'" cutaneous epitheliotropic lymphoma was reported in an -year-old ferret with a month history of progressive alopecia and pruritic dermatitis." the ferret had generalized alopecia and erythema, excoriations, erosions, crusts, and ulcerated plaques on the head, trunk, limbs, paws, footpads, and tail. the nasal planum and footpads were depigmented, and the claws were onychogryphotic. isotretinoin ( mglkglq h, per os) produced a marked improvement after days, but the ferret was euthanized due to renal failure associated with pyelonephritis. other cutaneous neoplasms reported in ferrets include papilloma, fibroma, fibrosarcoma, malignant fibrous histiocytoma, histiocytoma, hemangioma, hemangiosarcoma, neurofibroma, neurofibrosarcoma, myxoma, myxosarcoma, ceruminous gland adenocarcinoma, lymphoma, and rhabdomyosarcoma. the treatment of choice for cutaneous neoplasms is surgical excision. bacterial skin infections, usually associated with s. aureus, are common in gerbils. , , these infections are virtually always secondary to other perhaps less obvious conditions, especially trauma (cage-related injuries and bite wounds), ectoparasite infestations, and accumulated harderian gland secretions.v infections resulting from cage-related injury are typically seen on the nose and the muzzle (from rubbing on the cage and equipment or burrowing in abrasive litter), whereas those caused by bite wounds typically occur around the head, the tail, the rump, and the perineal area. those secondary to accumulated harderian gland secretion typically begin on the nose and the periocular area." staphylococcal infections may be superficial (alopecia, erythema, oozing, crust, and scale) or deep (abscess, fistula, and ulcer), and are usually nonpruritic. treatment of bacterial dermatitis includes some combination of eliminating predisposing causes, daily topical cleaning with a % hydrogen peroxide or . % to % chlorhexidine, and administration of systemic antibiotics (see table - ). demodicosis has rarely been reported in gerbils. , lesions occurred on the face, the thorax, the abdomen, and the limbs and were characterized by alopecia, oozing, crusts, scales, and secondary bacterial infection. demodex merioni was isolated in skin scrapings. details concerning pathogenesis and treatment are presently unpublished. a dermatosis associated with acarus farris was reported.v' alopecia, scaling, and thickening of the skin began on the tail, spread to the hind paws, then to the head. pruritus and excoriation were seen with chronicity. ivermectin injections were ineffective, but environmental changes (decreased humidity, new litter, new food) and a single application of fipronil spray were curative. although gerbils tolerate crowding better than do most rodents, they chew or "barber" the hair of cagemates," the affected areas appear clipped or shaved, and rarely are any actual skin lesions present. the dorsum of the tail and the top of the head are most frequently involved. bald nose describes a clinical condition common to the gerbil, which is characterized by alopecia on the muzzle and the dorsum of the nose." , , , there are usually no skin lesions. the alopecia has been attributed to mechanical trauma associated with rubbing against cages and cage equipment, and burrowing in abrasive bedding. placing animals in a smooth-sided enclosure or aquarium with soft bedding such as shredded paper may be curative. bald nose may also be an early stage of the nasal dermatitis (sore nose [see fig. - e]) associated with accumulated harderian gland secretion." , , , these secretions are rich in porphyrins and accumulate about the nasal and facial areas, and apparently lead to the development of an irritant contact dermatitis and secondary staphylococcal infection. the animal's failure to groom the areas adequately leads to irritation, which can then lead to self-inflicted trauma and secondary infection. the stress of overcrowding and high humidity may contribute to the development or the severity of the condition. this condition is common in research colonies and commercial breeding colonies. the earliest clinical sign is the accumulation of a reddish brown discharge and crust around the nose, the lips, and less frequently, the eyes. this porphyrin-rich secretion exhibits an orange fluorescence when viewed under ultraviolet light (wood's light). there is frequently protrusion of the nictitans. this is followed by alopecia and, if not treated, dermatitis, pruritus, and secondary staphylococcal infections. lesions can then spread to the paws, the legs, and the ventrum. therapy consists of topical or systemic antibiotic therapy for secondary staphylococcal infection, if present, and housing with access to sand. surgical removal of the harderian gland is effective but less practical.s an occasional litter of gerbils is born with abnormalities of hair growth and pigmentation," typically, the back is completely bald, the surrounding haircoat is thinned or patchily alopecic, and the haircoat shows profound leukotrichia. the majority of affected animals fail to thrive and die at the time of weaning. surviving gerbils develop a normal haircoat as they mature. the etiopathogenesis of this condition is unknown. the ventral scent gland in gerbils can become inflamed from being rubbed against wood chips or other abrasive bedding.' in addition, impaction of these glands can lead to self-mutilation. when relative humidity is greater than %, the normally sleek and smooth gerbil haircoat often appears greasy and stands out." pine shavings can also cause this appearance." a gerbil's tail skin is very thin, and easily peels off. if the tail skin is lost, the exposed tail becomes necrotic and sloughs off. , alternatively, the bare tail can be surgically removed where the skin stops. the skin is the second most common site of neoplasms in gerbils. , skin neoplasms are typically seen in aged animals ( to years of age). the most commonly reported skin neoplasms in the gerbil are melanocytomas and melanomas (especially of the paw and the pinna), , sebaceous gland adenomas (especially of the ventral scent gland), , and squamous cell carcinoma (especially of the ventral scent gland [see fig. - f] and the pinna)." . other reported skin neoplasms in gerbils include papilloma, fibrosarcoma, and neurofibroma." diagnosis of skin neoplasms is based on exfoliative cytologic study or biopsy, and the treatment of choice is surgical excision. bacterial skin infections are common in guinea pigs. - . these infections are virtually always secondary to other factors, especially trauma (cage-related injuries and bite wounds) and ectoparasites. those secondary to bite wounds are typically found around the head, the tail, the rump, and the genital area and are associated with s. aureus. abscesses are occasionally associated with corynebacterium kutscheri, streptococcus zooepidemicus (especially abscesses on the neck), streptobacillus moniliformis, or yersinia pseudotuberculosis,": , . a staphylococcal cellulitis characterized by thickening and hyperkeratosis of the lips was associated with feeding of tough, fibrous hay.!" treatment of these infections includes elimination of predisposing factors, surgical drainage, daily topical applications of % hydrogen peroxide or . % chlorhexidine, and systemic antibiotic administration (see table - ). an exfoliative dermatitis resembling staphylococcal scalded skin syndrome was reported in a guinea pig colony, chiefly among female animals in the late stages of gestation.!? bacterial contamination and the abrasive effects of rusty cage floors were suggested as initiating factors. alopecia was first noted on the ventral abdomen. after a few days, the skin became acutely erythematous and painful. affected skin subsequently fissured and large thick flakes were desquamated. the condition spontaneously resolved after a course of to days. s. aureus was isolated from the skin, the pharynx, the trachea, and nasal washings of affected animals. skin biopsies revealed intragranular acantholysis and cleavage within the epidermis with minimal inflammation. an exfoliative toxin produced by the staphylococci was reported to cause the skin lesions. the most common skin disease associated with s. aureus infection (occasionally corynebacterium pyogenes) in the guinea pig is pododermatitis (bumble foot). - . predisposing factors include trauma to the footpad, poor sanitation, obesity, aging, and vitamin c deficiency, affected animals react vigorously when the feet are palpated. the footpad is markedly enlarged, edematous, and erythematous (see fig. - g) . crusts, ulcers, and hemorrhages may be present on the volar surfaces. in chronic or severe cases, the disease process extends to phalangeal and metatarsal or metacarpal bones and joints. most guinea pigs with pododermatitis have a poor prognosis, because treatment is difficult and systemic amyloidosis is a frequent consequence of chronic infection. pododermatitis can be prevented by frequently cleaning cages and changing bedding, using cages with smooth surfaces, instituting individual weight control, and providing routine foot care. early lesions may respond to management changes and daily topical therapy (povidoneiodine or chlorhexidine scrubs, soaks, and ointments under a bandage). extensive infections also necessitate systemic antibiotics (see table - ). dermatophytosis is common in guinea pigs and is almost always caused by t. mentagrophytes. lo lesions typically begin as scaling, broken hairs, and alopecia on the nose, which spread to the periocular, forehead, and pinnal areas. in severe cases, the dorsal lumbosacral area is also affected (fig. - ), but the limbs and the ventrum are usually spared. pruritus is usually minimal or absent. some animals have more inflammatory lesions characterized by erythema, follicular papules, pustules, crusts, and pruritus. diagnosis is confirmed by microscopic examination of affected hairs and fungal culture. treatment is usually accomplished with the topical application of antifungal agents ( % lime sulfur, % chlorhexidine, or . % enilconazole dips weekly until the animal is cured). - . griseofulvin is not usually needed, but can be given at mg/kg every hours orally until the animal is cured. - o • the administration of griseofulvin should be avoided in pregnant animals .. s, dermatophytosis in guinea pigs is an important zoonosis.!? cryptococcosis was reported in a single guinea pigy the animal had a plaque on the dorsum of the nose, which became crusted and ulcerated, and spread into the nostrils. skin biopsy was diagnostic, and the animal was euthanized. guinea pigs have been used as an experimental model for studying the pathogenesis of cutaneous candidiasis., skin lesions are readily produced by the application of candida albicans under occlusion and consist of erythema, pustules, oozing, and crusts. guinea pigs have also been used as an experimental model for studying the pathogenesis and treatment of malassezia dermatitis.p skin lesions are readily produced by the application of inocula of malassezia (pityrosporum) ovale and consist of erythema, edema, crusts, and scales. hairless guinea pigs have been used as an experimental model for studying the pathogenesis of recurrent herpes simplex infection." intracutaneous inoculation of herpes simplex virus produces pustules and crusts, which spontaneously resolve and can be reactivated by local trauma. poxvirus-like virions were demonstrated during the electron microscopic examination of specimens from two guinea pigs with a chronic, crusting cheilitis."? trixacarus (caviacoptes) caviae is a burrowing sarcoptiform mite and, arguably, the most common cause of ectoparasitic skin disease of guinea pigs. . . . trixacariasis is always the first differential diagnostic suspect in intense pruritus in guinea pigs. the mite is similar to s. scabiei in appearance but is smaller in size (average, ilm in length), and the female mite has a dorsal rather than terminal anus. t. caviae is similar in size and appearance to notoedres spp. but differs by lacking prominent, sharp, dorsal cuticular spines. the areas most commonly affected include the dorsal neck and the thorax (see fig. - h ), but in severe cases, the entire body may be involved (fig. - a) . early clinical signs include pruritus, erythema, and traumatic alopecia. chronic lesions include lichenification, hyperplgmentation, crusts, thick, whitish to yellowish scales (see fig. - b) , and brittle, easily epilated hair. the extreme irritation and self-mutilation cause lethargy, anorexia, progressive emaciation, and death associated with bacterial infection or immune-mediated renal disease. hyperesthesia, behavior such as furiously running in circles and blindly walking into objects, and seizures can be seen and may be triggered by examining the affected animal. resorption of fetuses and abortion may be seen in breeding animals. diagnosis may be confirmed by skin scrapings (fig. - a ), but it is not unusual for these to be negative. treatment consists of % lime sulfur dips (once weekly until cure is achieved) or ivermectin injections. orally administered ivermectin is not effective in guinea pigs. • t. caviae can temporarily infest humans, producing a pruritic papular dermatitis in areas contacted by the guinea pig (arms, thighs, and abdomen). chirodiscoides caviae is the guinea pig fur mite.l" , infestation is uncommon, and clinical disease is probably rare. heavy infestations can cause pruritus, alopecia, erythema, scaling, and crusting, especially on the dorsolateral lumbosacral area (see fig. - c ) and the perineum. diagnosis is confirmed by the microscopic identification of the mites (see fig. - b) , which are often found attached to hair shafts. treatment is as described for lice. although injections of ivermectin have been reported to eliminate c. caoiae.» , others found this form of therapy to be ineffectlve.v other mites rarely reported to affect guinea pigs and produce clinical syndromes characterized by pruritus and dermatitis, which is most prominent on the face, the pinnae, and the dorsum, include s. scabiei, notoedres muris, and myocoptes musculinus.p diagnosis and treatment are as described for t. caviae infestation. lice are commonly encountered in guinea pigs. , the guinea pig may be parasitized by two different biting lice, with gliricola porcelli (the slender guinea pig louse) (see fig. - c ) being much more frequently encountered than gyropus ovalis (the oval guinea pig louse). trimenopon hispidum is a biting louse rarely isolated from guinea pigs. most infestations occur without clinical signs, but heavy infestations may produce a roughened, disheveled haircoat, scaling, crusting, alopecia, and pruritus, especially around the ears and over the dorsum (see fig. - d) . heavy infestations are more commonly encountered in young animals and those with decreased resistance and under poor management. diagnosis is confirmed by gross or microscopic visualization of lice or nits. treatment can be accomplished with pyrethrin-or pyrethroid-containing flea powders or sprays approved for cats, % lime sulfur dips, or ivermectin lnjections.s" , cage sanitation is important. pelodera dermatitis is rarely reported in guinea pigsp affected animals have ventral dermatitis consisting of erythema, papules, oozing, crusts, and alopecia. skin scrapings and skin biopsy reveal the presence of the nematode pelodera strongyloides. removing contaminated bedding and maintaining a clean, dry cage environment are curative. cheyletiellosis is rarely reported in guinea pigs. affected animals have scaling and variable degrees of hypotrichosis and pruritus on the dorsum. skin scrapings reveal the mite cheyletiella parasitivorax. treatment is as described for rabbits. demodicosis is rare in guinea pigsp lesions occur most commonly on the trunk (fig. figure - - ) and consist of alopecia, erythema, papules, and crusts. pruritus is variable. diagnosis is confirmed by finding numerous demodex caviae mites in skin scrapings (see fig. - d) . amitraz dips ( ppm, every week until weeks after skin scrapings are negative) are reported to be an effective treatment. fleas (c. felis) and ticks may be rarely encountered on guinea pigs. . treatment is the same as that described for cats. nutritional deficiencies, other than vitamin c deficiency, are unlikely to be encountered in pet guinea pigs. experimental production of nutritional deficiencies with resultant cutaneous abnormalities have been reported and are briefly mentioned here. protein deficiency produces generalized alopecia.v fatty acid deficiency results in generalized alopecia, scaling, and dermatitis.s pyridoxine deficiency produces alopecia, scaling, and dermatitis, which is most prominent on the limbs, the face, and the pinnae.v vitamin c deficiency is associated with cutaneous petechiae, ecchymoses, hematomas, generalized scaling, and a rough, unkempt hatrcoat.v . , vitamin c deficiency is common and is most commonly seen in animals being fed a commercial rabbit chow or an outdated commercial guinea pig chow as the sale diet or in anorectic animals, telogen defluxion is frequently seen in the last trimester of pregnancy or during lactation. , , the alopecia is most prominent on the lumbosacral area and the flanks. marked shedding during stress is common in guinea pigs. when guinea pigs are sick, it is often easy to epilate an entire area of haircoat when tenting the skin. guinea pigs establish male-dominated social hierarchies, and animals of low social ranking or young animals may lose considerable amounts of hair, especially on the head, the rump, the perineum, and the prepuce owing to barbering or receiving bite wounds.v " . ear chewing can be a problem, resulting in ear margin notches or actual cropping close to the head. guinea pigs may also self-barber, producing hair loss in only those areas that they can reach with their mouth (the fur on the head, the neck, and the anterior shoulders is intact). hair loss due to barbering appears irregular in length and clipped. the underlying skin is usually normal in appearance. in some cases, the addition of long-stemmed hay resolves the barbering, suggesting that the cause was boredom or a need for fiber." , , necrosis of digits and paws is occasionally seen when owners put objects such as cloths and socks in cages with guinea pigsy segments of these materials become wrapped around distal extremities and serve as constricting bands or tourniquets. tissues distal to the constriction become swollen and painful, then necrotic, and then they slough ( fig. - ). apparent ergot poisoning was characterized by the sudden onset of anorexia, lethargy, and lameness." the feet became discolored, desiccated, insensitive to touch, and woodlike. thinning of the haircoat is common near the time of weaning in neonates and spontaneously resolves." hereditary hairlessness has been reported in guinea pigs (fig. - ) , bilaterally symmetric alopecia over the flanks and trunk has been seen in guinea pigs with cystic ovaries (fig. - ) . , , affected females usually present with progressive enlargement of the abdomen, and ovarian cysts may be palpable as discrete, large, rounded masses in the dorsal middle abdomen. ultrasonographic examination may be diagnostic." ovariohysterectomy is curative. sebaceous glands are especially abundant around the anus, in the folds of the perianal and genital regions, and in the dorsal sacral skin, especially in sexually mature male animals." . excessive accumulations of sebaceous debris, and occasionally bedding material and feces, may become entrapped in these folds or mat down the dorsal sacral haircoat. intertrigo, secondary bacterial infections, and unpleasant odors may intervene in this situation. these areas should be cleaned as needed with % hydrogen peroxide, . % chlorhexidine, or a mild astringent (aluminum subacetate) to prevent the aforementioned sequelae. heavy guinea pigs, especially those maintained in wire-bottom cages, frequently develop hyperkeratosis and cutaneous horns of the footpads." these horny growths are most commonly observed on the ventral aspect of the front paws. they can be removed with scissors and an emery file. replacement of cage surfaces with smoother materials retards or prevents further hyperkeratoses. overgrown claws are a frequent problem in pet guinea pigs. , frequent examination and trimming prevents traumatic and infectious complications. the skin is the second most common site of neoplasia in the guinea pig. , , , , the most common cutaneous neoplasm is the trichofolliculomay this tumor is most commonly encountered as a benign, solitary lesion over the dorsal lumbar area (see fig. - £). the overlying skin is usually alopecic and crusted. frequently, a central pore is seen, through which keratinous material or dark, hemorrhagic exudate is discharged. other cutaneous neoplasms reported in guinea pigs include sebaceous adenoma, fibroma, fibrosarcoma, lipoma, liposarcoma, schwannoma, and lymphoma.v bacterial skin infections are uncommonly reported in hamsters. s, aureus and pasteurella pneurrwtropica have been recovered from isolated cases of skin abscesses and bite wounds." hamsters are prone to periodontal disease and dental caries, and any facial abscess located ventral or cranial to the eye may be a tooth root abscess.'? treatment of bacterial dermatitis includes elimination of predisposing causes, surgical drainage, daily topical cleaning with % hydrogen peroxide or . % chlorhexidine, and occasionally systemic antibiotic administration (see table - ) . experimentally, the hamster is susceptible to infection with treponema pallidum subsp. endemicum, the agent of endemic syphilis of humans." intradermal injection of the spirochete results in erythematous papules and ulcers, which eventually heal but are followed by perioral ulcers and an erythematous maculopapular rash on the paws and the trunk and death. this form of hamster syphilis has been proposed as a useful model for the study of the immune response, antibiotic therapy, and vaccination techniques in human venereal and congenital syphilis. dermatophytosis is rare in hamsters and is caused by t. mentagrophytes. , , . , , - diagnosis is confirmed by microscopic examination of affected hairs and fungal culture. treatment is as described for the other rodents. demodicosis is the most common ectoparasitism of the hamster. , demodex criceti and d. aurati are both normal residents of hamster skin.!? d. aurati is long and tapered (average, /lm long) and inhabits hair follicles, whereas d. criceti is short and stubby (average, /lm long) and inhabits the keratin and pits of the epidermal surface. clinical demodicosis is seen in aged hamsters, is usually associated with d. aurati, and is usually associated with conditions that suppress immune responses (e.g., malnutrition, concurrent disease, cancer, and exposure to carcinogensj.v b, . lesions are most commonly seen over the dorsal lumbosacral area but may be generalized (fig. - ). moderate to severe alopecia is accompanied by variable degrees of scaling, erythema, and small hemorrhagic crusts. pruritus is usually absent. diagnosis is confirmed by skin scrapings. treatment of demodicosis in hamsters has not been extensively evaluated or reported. the authors and others , were successful with ppm of amitraz applied as a whole-body dip once weekly until weeks after skin scrapings are negative. notoedric mange is rarely reported in hamsters.f- , lesions are characterized by thick yellowish crusts, erythema, and alopecia on the pinnae, the muzzle, the tail, the genitalia, and the paws. pruritus is severe, and self-mutilation can be extreme. diagnosis is confirmed by finding notoedres sp. mites in skin scrapings. treatment with % lime sulfur dips (whole-body application weekly until two treatments after clinical cure) or ivermectin injections is effective. s. scabiei, t. caviae, and ornithonyssus bacoti are reported to be rare causes of pruritus and dermatitis in hamsters.'? clinical signs, diagnosis, and treatment are the same as that described for notoedric mange. fleas (c. felis felis) are rarely encountered on hamsters." treatment is the same as that described for cats. female hamsters are generally more aggressive than are males.v !" aggressive behavior in male hamsters is testosterone dependent and is markedly reduced by castration.v the establishment of social dominance among male hamsters is positively correlated with the weight, size, and degree of pigmentation of the flank glands." aggression-related bite wounds are most commonly seen around the head, the tail, and the perineal area. aggression can produce severe wounding, such as the complete removal of the flank glands of the victim.!" nutritional deficiencies are unlikely to be seen in pet hamsters. however, one author" indicates that hair loss in hamsters is associated most often with continuous feeding of low protein (less than %) feed, such as is commonly found in pet stores. experimental production of nutritional deficiencies with resultant cutaneous abnormalities have been reported and are briefly mentioned here. pantothenic acid deficiency produces exfoliative dermatitis, depigmentation of the haircoat, and the accumulation of porphyrin-rich secretions around the nose, the mouth, and the eyes. , riboflavin deficiency produces alopecia, scaling, and dermatitis, which are most evident on the extremities.s pyridoxine deficiency results in generalized alopecia and depigmentation of the haircoat.v niacin deficiency produces a generalized alopecia.v fatty acid deficiency results in generalized alopecia, scaling, and the production of profuse amounts of cerumen.v copper deficiency results in alopecia and depigmentation.f foreign body granulomas associated with bedding consisting of wood shavings and sawdust were reported on the paws and the shoulders of hamsters.!? the problem was eliminated by using shredded paper as bedding. swelling and pruritus of the face and the paws has been reported in several hamsters." in all instances, owners had recently purchased a fresh bag of cedar or pine shavings produced by a leading pet company or bulk shavings from landscaping or nursery outlets. when affected animals were housed on plain newspaper, the lesions spontaneously regressed. presumably, the shavings had been treated with some chemical that produced a contact dermatitis. hereditary hairlessness has been reported in hamsters.!? hyperadrenocorticism is rarely reported in hamsters.s clinical signs include bilaterally symmetric alopecia, hyperpigmentation, and thinning of the skin. the baseline plasma cortisol value in one affected hamster was elevated (approximately twofold increase) compared with that in one normal hamster. one hamster was treated with metyrapone ( mg orally q h for month), and hair regrowth was complete after weeks. a second hamster was treated with o,p'-ddd ( mg orally q h for month), then metyrapone as described previously, and responded to neither drug. this hamster was euthanized, and a chromophobe adenoma of the hypophysis and bilateral adrenocortical hyperplasia were found at necropsy. the flank scent glands of the hamster can become inflamed from being rubbed against wood chips or other abrasive cage equipment.' in addition, impaction of these glands can lead to self-mutilation. ' ringtail is occasionally seen in the hamster (see rat in this chapter)." skin neoplasms are rare in hamsters.v , , the most frequently reported cutaneous neoplasms are melanomas and melanocytomas.s , these tumors occur much more frequently in male hamsters and most commonly on the back, the head, the neck, and the flank gland. epitheliotropic lymphoma (mycosis fungoides) is the second most common cutaneous neoplasm of the hamster.s , affected animals have an exfoliative erythroderma (see fig. - f) , which is generally pruritic, and go on to manifest peripherallymphadenopathy, lethargy, anorexia, emaciation, and death. some animals also have cutaneous plaques and nodules (fig. - ) , which may become ulcerated and crusted. light and electron microscopic examinations demonstrated an epitheliotropic lymphoma in which many cells show the typical features of sezary cells." immunohistochemical studies showed that the neoplastic cells are t lymphocytes." therapeutic trials have not been reported. other cutaneous neoplasms reported in hamsters include basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, papilloma, epitrichial sweat gland adenoma, fibrosarcoma, and plasmacytoma.v , bacterial skin infections are uncommon in pet mice, are usually caused by s. aureus, and are secondary to trauma (cage-related injuries and bite wounds) or self-inflicted (the pruritus associated with ectoparasites)." , , , infections resulting from cage-related injury are typically seen on the nose and the muzzle, whereas those associated with bite wounds typically occur around the head, the tail, the rump, and the perineal area. staphylococcal infections may be superficial (alopecia, erythema, oozing, and crust) or deep (abscess, fistula, necrosis, and ulcer) and are usually nonpruritic. submandibular and periorbital granulomas were associated with bacterial pseudomycetuna (botryomycosis) due to s. aureusl? other bacteria occasionally isolated from cutaneous abscesses and pyogranulomas in mice include streptococcus sp., p. pneumotropica, actinobacillus sp., actinomyces sp., and klebsiella. , , treatment of bacterial dermatitis includes some combination of elimination of predisposing causes, surgical drainage, daily topical cleaning with % hydrogen peroxide or . % to % chlorhexidine, and systemic antibiotic administration (see table - ). surgical excision may be the best treatment for abscesses." s. rrwniliformis is a rare cause of epizootics of edema and cyanosis of the extremities. , , c. kutscheri (murium) is a rare cause of epizootics of furunculosis and cutaneous pyogranulomas, which may progress to necrosis and sloughing of extremities.v , the mouse has been used as an experimental model for staphylococcal scalded skin syndrome. , mycobacterium chelonae infection caused nodular, granulomatous lesions on the tails of immunocompromised mice. a dermatophytosis is uncommon in mice and is usually caused by t. mentagrophytes. i - , , - this dermatophyte can be isolated from the haircoat of up to % of clinically normal mice in pet shops and represents an important zoonosis. lesions are most commonly seen on the face, the head, the tail, and the trunk and consist of annular areas of alopecia, broken hairs, scales, and variable degrees of erythema and crusting. pruritus is usually minimal to absent. diagnosis and treatment are the same as that described for guinea pigs. mouse pox (infectious ectromelia) causes epizootics in research colonies but is rarely, if ever, seen in practice.t- , - , , skin lesions include a generalized papular dermatitis with eventual swelling, necrosis, ulceration, and even sloughing of digits, pinnae, and tail. diagnosis is confirmed by skin biopsy, electron microscopic examination of crusts, viral isolation of the orthopoxvirus, and polymerase chain reactiony· reovirus type infection of suckling mice causes severe illness and an oily haircoat.v , animals that survive past weaning experience alopecia. sialodacryoadenitis virus infection causes eye rubbing and scratching, periorbital swelling, and red tears (chromodacryorrheal.vv myobia musculi is a mite commonly found on mice. - , , - some animals are asymptomatic carriers, whereas other animals show varying degrees of skin disease and pruritus. severely inflammatory and pruritic forms of the infestation are associated with genetic susceptibility and mite-related hypersensitivity reactions.!" immature mice or those that are immunocompromised may be more susceptible to severe forms of the disease. clinical signs may be mild and include patchy alopecia, slight erythema, and minor scaling on the head and the muzzle. other mice may show intense pruritus and self-mutilation of the face, the head, the pinnae, the neck, and the shoulders (see fig. - g) . severely affected animals can become debilitated and die. diagnosis is confirmed by skin scrapings (see fig. - e) . the treatment of choice is subcutaneous injections of ivermectin.v , , , ivermectin administered topically or in drinking water is less effective : . , , myocoptes musculinus is a mite commonly found on mice. - , , - some animals are asymptomatic carriers, whereas others manifest varying degrees of skin disease and pruritus. lesions are most severe on the back and the ventrum. unlike the case with m, musculi infestations, severe ulceration is not seen with m. musculinus infestations. diagnosis is confirmed by skin scrapings (see fig. - f) ; however, the mites are often difficult to find. the treatment of choice is subcutaneous injections of ivermectin every weeks until the animal is cured." , , , ivermectin administered topically or in drinking water is less effective.p: , other mites that are rarely found on mice include radfordia affinis, psorergates simplex, . bacoti, s. scabiei, n. muris, and trichoecius romboutsi.i:" , , , fleas (especially c. felis felis) may be recovered from pet mice maintained in households frequented by dogs and cats. - , , the sucking louse polyplax serrata is occasionally found on pet mice. - , , , , some animals may be asymptomatic carriers, but others manifest varying degrees of dermatitis and pruritus. young animals, debilitated animals, and animals in poor management situations are more likely to be affected. lice and related dermatoses are most commonly found on the neck and back. treatment can be accomplished with topical insecticides or ivermectin injections. nutritional deficiencies are unlikely to be encountered in pet mice. experimental production of nutritional deficiencies with resultant cutaneous abnormalities have been reported and are briefly mentioned here. zinc deficiency produces exfoliative dermatitis, alopecia, and depigmentation of the haircoat." pantothenic acid deficiency results in exfoliative dermatitis and depigmentation of the haircoat.v riboflavin deficiency produces alopecia, scaling, and dermatitis, especially on the extremities.v pyridoxine deficiency results in exfoliative dermatitis, especially on the face, the ears, the limbs, and the taij. · biotin deficiency causes exfoliative dermatitis." fatty acid deficiency produces an exfoliative dermatitis.v male mice are aggressive. - • . . barbering and bite wounds are frequently seen, especially on the muzzle, the whiskers, the face, the head, the rump, the tail, and the perineum. these behaviors can be exacerbated by crowding, stress, and boredom. mice frequently rub the hair off the muzzle as they stick their face through slotted feeders or wire bars. mice develop numerous types of hereditary hairlessness and keratinization defects, which are probably never seen by the practitioner.v , . . some of these conditions have been used as laboratory models for the study of various aspects of cutaneous pathophysiology and pharmacology, such as ichthyosis, asebia, rhino, and blotchy (similar to the menkes kinky hair syndrome in humans) and flaky skin (similar to psoriasis in humansi.i" . , . ringtail is rarely reported in mice (see rat in this chapter)." idiopathic dry gangrene of the pinna is sporadically seen in young mice." the incidence appears to increase when the mice are exposed to cold temperatures and when the ears are traumatized by excessive grooming in attempts to remove lice. the condition progresses rapidly from initial erythema of the distal one third of the pinna to necrosis and slough. rarely, the distal one third of the tail is also involved. perianal pruritus is seen in association with pinworms (syphacia obvelata). . , , , infected mice often mutilate the base of the tail. diagnosis is confirmed by microscopic examination of strips of cellophane (scotch) tape that have been applied to the perineum. the eggs of s. obvelata are banana shaped and about /lm by /lill. treatment with ivermectin injections is curative. a spontaneous immune complex vasculitis was reported to affect up to % of certain strains of aged mice." mice developed multiple crusts between the scapulae or on the dorsal neck. these lesions rapidly evolved into irregular ulcers and spread laterally and caudally on the body. pruritus was intense. histologic examination revealed leukocytoclastic vasculitis and igg, igm, and fibrinogen were demonstrated in dermal blood vessel walls. alopecia areata was reported in the c hihej mouse and proposed as a model of the human disease." although mice are sensitive to the induction of various skin neoplasms by the topical or systemic administration of chemical carcinogens or ultraviolet light exposure, spontaneous skin neoplasms are rare.s , , , the most commonly reported cutaneous neoplasms are papilloma, squamous cell carcinoma, and fibrosarcoma. , other reported cutaneous neoplasms include hair follicle tumors, sebaceous gland tumors, mast cell tumors, hemangiomas, hemangiosarcomas, melanomas, lymphomas, and a solitary epitheliotropic lymphoma resembling pagetoid reticulosis in humans.!": , pasteurellosis (snuffles) is the most common bacterial disease of the rabbit." most rabbits carry pasteurella multocida asymptomatically in the nasal cavity, and under conditions of stress, the bacteria multiply and cause disease. subcutaneous abscesses develop as a result of septicemia, external wound contamination, or direct extension from deeper sties. the abscesses are variable in size, are usually firm on palpation, and are filled with a thick, white to tan exudate. diagnosis is confirmed by microscopic examination of direct smears of exudate and culture. in the rabbit, subcutaneous abscesses are due to pasteurellosis until proven otherwise. other causes of abscesses include s. aureus, fusobacterium, pseudomonas aeruginosa, streptococcus sp., and c. pyogenes." abscesses on the head may be secondary to dental disease, a tooth root abscess, or an oral foreign body. abscesses in rabbits are typically filled with thick, caseated pus and are surrounded by a thick capsule. these two attributes make the use of drainage, drains, and topical and systemic antibiotics unsuccessful in most cases.v- , the treatment of choice is surgical excision and at least weeks of antibiotic treatment (see table - ). , , if this is impossible, radical debridement, flushing, and antibiotics for several weeks may be effective.?" in some rabbits, abscesses continue to recur in the same spot or at some other sites in an apparently healthy individual.'? some of these rabbits may require lifetime antibiotic therapy to prevent recurrences. necrobacillosis (schmorl's disease) is a sporadic bacterial infection of rabbits caused by fusobacterium necrophorum. t it is characterized by inflammation, necrosis, ulceration, and abscessation, especially on the face, the head, and the neck. diagnosis is confirmed by culture. treatment is accomplished with surgical debridement, topical antimicrobial applications, and systemic penicillin or tetracycline administration. p. aeruginosa causes a localized moist dermatitis (sore dewlap) and, occasionally, subcutaneous abscess in areas of skin that are continuously wet. t the muzzle, the dewlap (see fig. - h) , the flank, and the haunches are most commonly involved. the affected skin is moist, erythematous, edematous, alopecic, and often ulcerated. the fur is often clumped, creating a spiked appearance. the most striking clinical feature is the blue-green color of the fur in animals with white fur, which is caused by a water-soluble pigment (pyocyanin) produced by the bacteria. diagnosis is confirmed by microscopic examination of direct smears from oozing areas and culture. treatment includes clipping, gentle cleaning, and application of astringents (aluminum acetate) and topical gentamicin sulfate ointment. prevention is directed at removing the cause of continued wetness of the fur. the most common cause is the constant drooling ("slobbers") associated with dental disease.l" leaking water valves or water bottles should be replaced. water bowls or pans should be replaced by water bottles with sipper tubes. malocclusion of the teeth should be corrected to prevent drooling. wet bedding should be changed more frequently. ulcerative pododermatitis (sore hocks) is a common disorder in rabbits. § genetic predilection is important, as large body size and thinner plantar fur pads are important predtsposing factors. unsanitary cage conditions, rough cage surfaces, and obesity also contribute to pressure necrosis and secondary bacterial infection with s. aureus. lesions commonly occur unilaterally or bilaterally on the plantar aspect of the metatarsal region or, less commonly, the volar surface of the metacarpal area. focal inflammation, oozing, crusts, and alopecia progress to ulcers, hemorrhage, and abscesses. in severe infections, the disease may extend to the bony structures of the foot and result in septicemia. treatment includes correction of predisposing conditions, surgical drainage, topical antimicrobial applications, and systemic antibiotic administration (see table - ). severe cases usually do not respond. venereal spirochetosis (treponematosis, rabbit syphilis, or vent disease) is uncommon. frequently seen on the nose (fig. - a) , the lips, the chin, the face, the eyelids, the ears, and the paws as well as the genitalia. lesions consist of vesicles, papules, erythema, edema, oozing, erosions, and brownish crusts. focal ulcers and hemorrhage may be seen. diagnosis is confirmed by skin biopsies, the venereal disease research laboratory (vdrl) slide test, and the rapid plasma reagin (rpr) card test. treatment with penicillin g benzathine or penicillin g procaine is curative ( , iulkg, subcutaneously, once a week for three treatments). tetracycline or chloramphenicol is also effective.". an outbreak of s. aureus infection in a rabbitry was associated with a pustular, exudative dermatitis in the young and mastitis in lactating does.p" a cellulitis due to s. aureus infection is occasionally seen and is characterized by the acute onset of fever, and painful edematous swelling, especially over the head, neck, and thorax." necrosis and slough may occur. dermatophytosis is common in rabbits. t. mentagrophytes is the most common dermatophyte isolated, but m. canis, m. gypseum, m. audouinii, t. verrucosum, and t. schoenleinii have been reported." t. mentagrophytes can be isolated from the haircoat and skin of approximately % of clinically normal rabbits, representing an important potential zoonosis. the disease is most common in young animals and where husbandry and management are suboptimal. lesions are characterized by patchy alopecia, broken hairs, erythema, and yellowish crusting, and typically first appear on the bridge of the nose (see fig. - b) , the eyelids, the pinnae, and the paws, and occasionally, on many body sites. the condition is usually pruritic. diagnosis and treatment are the same as that described for the guinea pig. griseofulvin is teratogenic, and should not be used in breeding does. a modified live t. mentagrophytes vaccine may prove useful in prophylaxis.!? aspergillosis of the lungs and skin was reported in a whole litter of -week-old rabbits.!" multiple -to -mm papules were present all over the body. histologically, the papules were cystic follicles distended with necrotic debris and dichotomously branching hyphae. aspergillus sp. was isolated in culture. the animals were raised on moldy grass hay bedding material. a change in nesting materials prevented further occurrences. myxomatosis is occasionally observed in domestic rabbits. t the myxoma virus (a poxvirus) is transmitted from reservoir wild rabbit hosts by mosquitoes. there are several strains of virus with variable virulence. in domestic rabbits, severe disease and high mortality are frequently produced. affected rabbits are febrile, lethargic, and depressed. in the acute form of the disease, there is edema and erythema of the anus, the genitalia, the lips, the nares, and the eyelids. less virulent strains of the virus produce numerous skin tumors (see fig. - c ). myxomatosis appeared in the depilated skin of angora rabbits.'? lesions were a few millimeters to em in diameter, erythematous, and plaquelike, and became hemorrhagic and necrotic. morbidity was low and mortality infrequent. diagnosis is based on distinctive clinical signs, biopsy, and virus isolation. there is no effective treatment, and control of insect vectors and screening of enclosures are paramount in endemic areas. heterologous vaccine may be useful. rabbit pox is infrequently reported in domestic rabbits. t the causative poxvirus is closely related to vaccinia virus. initial clinical signs of profuse nasal discharge, depression, and fever are followed in to days by a generalized, erythematous, macular to papular to nodular eruption. the rabbits have extensive edema of the face and perineum. diagnosis is confirmed by biopsy and virus isolation. shope fibroma virus and shope papilloma virus are oncogenic (see neoplasia). psoroptes cuniculi, a nonburrowing mite, is the most common ectoparasite of the rabbit, and all rabbits should be considered infected until proven otherwise." rabbits are also susceptible to p. ovis (cattle and sheep).'? p. cuniculi is transmitted by direct contact with infected rabbits, fomites, and contaminated environment. starving mites survive for approximately days off the host over the usual range of temperatures ( to °c [ to °f]) and relative humidities ( % to %), crusts dislodged into the environment contain many mites. the mites pierce skin to feed, and hypersensitivity to mite-related antigens may be important in the pathogenesis of the dermatitis and pruritus.!" p. cuniculi typically produces otitis extema (otoacariasis, ear canker, and ear mites) (see fig. - d) . affected rabbits shake their heads and scratch at the head and ears. alopecia, excoriations, and secondary bacterial infection may be present around the head, neck, and ventrum.f: in early stages, a dry, whitish gray to tan crusty exudate forms inside the vertical ear canal. later, a dry, crusty material with a layered appearance accumulates in the ear and the lateral surface of the pinnae. a secondary bacterial infection may complicate the parasitic otitis extema, contributing to the foul odor and pain. occasionally, mites may produce lesions on the face, the head, the neck, the limbs, the abdomen, and the back.n, , diagnosis is confirmed by finding the mites in ear swabs or skin scrapings (see fig. - g) , in one report in which natural infections were studied and mite numbers were quantitated, affected animals harbored to , mites per rabbit. the treatment of choice is the subcutaneous injection of ivermectin.l" , , do not attempt to clean out the crusts and debris, because this causes pain and bleeding. the cage and environment should be sanitized, and reducing the relative humidity to less than % while increasing the temperature to °c ( °f) is of benefit in this regard.n, in one report.v? an incontact guinea pig also developed psoroptic mange. s. scabiei var. cuniculi, a burrowing mite, is a rare ectoparasite on rabbits in north america but is commonly found in some other parts of the world, such as africa and india. t typical lesions include tan to yellow, often powdery crusts, alopecia, erythema, and excoriation on the muzzle, the lips, the bridge of the nose, the eyelids, the head, the margins of the pinna, the paws, and the external genitalia. pruritus is intense. severe infestations can lead to anorexia, lethargy, emaciation, and death. these mites can transiently produce lesions in humans. diagnosis is confirmed by finding s. scabiei mites in skin scrapings. however, mites are often difficult to demonstrate, and response to therapy is a frequently used diagnostic test. the treatment of choice is iverrnectin." , , cheyletiella parasitovorax, a nonburrowing mite, is a common ectoparasite on rabbits. - , , . - most rabbits harbor the mites without overt signs of skin disease. with heavy infestations or in hypersensitive hosts, a variably pruritic dermatosis is seen. lesions consist of scaling, crusting, and variable degrees of erythema, alopecia, and greasiness over the withers, the back (see fig. - e) , and the ventral abdomen. occasionally, lesions are limited to the face. these mites can produce skin lesions in humans. diagnosis is confirmed by finding c. parasitovorax in skin scrapings or acetate tape preparations. the treatment of choice is ivermectin. listrophorus (leporacarus) gibbus is a common fur mite of rabbits, which is rarely associated with clinical skin disease.t" , , - , ., . most affected rabbits are asymptomatic. the mite is usually found attached to hair shafts, especially on the back, the groin, and the ventral abdomen. occasional rabbits may manifest a variably pruritic, scaly, erythematous, alopecic dermatitis in the aforementioned sites. some animals only manifest pruritus and traumatic alopecia with no skin lesions. diagnosis is confirmed by finding l. gibbus in skin scrapings and acetate tape preparations (see fig. sulfur dips is curative.i" a, a ivermectin may also be effective.'? fipronil spray was reported to be effective.?' one author reported treating over rabbits with fipronil spray ( mllkg) with no adverse effects.?? however, the therapeutic index for this product is fairly narrow in rabbits, possibly due to the isopropyl alcohol content." company representatives received a number of reports of suspected adverse reactions to fipronil spray in rabbits, and recommended that the product not be used in this species." notoedres cati, a burrowing mite, is a rare ectoparasite on rabbits in north america, but is commonly found in other parts of the world, such as india." clinical signs, diagnosis, and treatment are identical to those described for s, scabiei. fleas (especially c, felis felis) are occasionally found on rabbits, especially those in households with dogs and cats. - , , , - , a in the united states, rabbits may also be infested with cediopsylla simplex (common eastern rabbit flea), especially around the head and the neck, and odontopsyllus multispinosis (giant eastern rabbit flea), especially over the rump, clinical signs, diagnosis, and therapy are the same as that described for cats. the rabbit sucking louse haemodipsus ventricosus is uncommon in the united states, - , , , - pediculosis is usually associated with poor management. lice are most commonly found on the dorsum and may produce intense pruritus, severe infestations in debilitated animals may produce anemia, weakness, emaciation, and death, h. ventricosus is a vector of tularemia, therapy is the same as that described for cats, demodex cuniculi mites have been isolated from rabbits with generalized pruritus and scaling, but their pathogenic significance is in doubt.!? members of the fly genus cuterebra occasionally produce myiasis in domestic rabbits reared outdoors or in nonscreened enclosures, t among those fly species reported in the united states are cuterebra cuniculi, c, buccata, and c, horripilum. larvae and, therefore, lesions appear in the summer and early fall. the incidence of infestation decreases with age, which correlates with the development of immediate and delayed-type hypersensitivity reactions to larval antigens, c, horripilum prefers the ventral cervical region, whereas c. buccata larvae localize in the interscapular, axillary, inguinal, or rump area, initial lesions include subcutaneous cystlike structures, as the larvae (warbles) enlarge, a "breathing hole," or fistula, is produced. the surrounding haircoat is moist and matted, secondary bacterial infection is common, and the lesions are often painful. treatment consists of surgical removal of the larvae (one should not crush or otherwise damage the larvae), routine wound care, and occasionally, administration of systemic antibiotics, prevention and control are aimed at eliminating contact with the warble fly, flystrike (maggots) is most commonly seen in the perineal region, and may spread dorsally onto the rump.!? moist dermatitis and fur matting are present, rabbits that are sedentary, overweight, or that have perineal dermatitis (urine scald) may be predisposed, treatment includes cleansing and one injection of ivermectin.l'' nutritional deficiencies are unlikely to be encountered in pet rabbits, experimental production of nutritional deficiencies with resultant cutaneous abnormalities have been reported and are briefly presented here, copper deficiency results in alopecia and a depigmented haircoat.v zinc deficiency produces alopecia, scaling, and a depigmented haircoat. , several days before parturition, the female rabbit undergoes a generalized loosening of the fur, , , is especially prominent on the abdomen, chest, forelegs, and hips. some rabbits pull out fur as a behavioral vice.v other rabbits rub fur off against the cage surface or feeders." does in heat and rabbits on low-fiber diets may barber their own hair.'? the barbered rabbit typically has patches of broken-off hairs over the head and back.'? seasonal molts can result in haircoat irregularities and thinning." compulsive self-mutilating behavior was encountered in % to % of the rabbits in a colony of checkered crosses." extensive auto mutilation of digits and pads of the front feet was observed. the behavior could be interrupted by giving the rabbits haloperidol ( . mglkg m qi h), a dopamine antagonist. because the condition was never seen in animals of other breeding lines kept in the same building under identical conditions, and the affected animals came from highly inbred stock, it was hypothesized that the disorder was genetically determined. hereditary alopecias (fig. - ) are rarely described in rabbits'? but are unlikely to be seen by practitioners. cutaneous asthenia has been reported in rabbits (fig. _ ). . . the animals had a history of skin fragility and repeated spontaneous skin tears, and were covered with scars. the skin extensibility index (see chap. ) in two rabbits was % to % in the affected rabbits as compared with a mean of % in normal rabbits. light microscopic examination was unremarkable, but electron microscopic examination revealed distorted and tangled collagen bundles with collagen fibrils being of different diameters and having a loose, frayed appearance. hutch bum is a contact dermatitis caused by urine scalding of the perineal region because of an unclean environment or an inability of the rabbit to void urine without soiling itself, such as after an orthopedic or neurologic injury, or with obesity." washing the area frequently with antimicrobial agents and applying a protectant cream, such as zinc oxide, are helpful. frostbite may be seen in rabbits that are suddenly exposed to cold climates without a period of acclimatization.!" erythema, acrocyanosis, necrosis, and sloughing are typically seen on the pinna (see fig. - f) . both male and female rabbits possess two sebaceous scent glands on either side of the vulvar or testicular area that secrete a brown waxy debris." this secretion can build up and can be easily removed by gentle traction or soap and water. the authors have seen a condition resembling alopecia areata in rabbits. affected animals presented with one or more areas of noninflammatory annular alopecia, especially on the black-furred areas of the pinnae. spontaneous recovery was accompanied by the regrowth of white fur (fig. - ) . sebaceous adenitis was reported in domestic rabbits. • a the animals varied from lf to years of age. lesions began around the neck or face, remained localized for several months, then became generalized. all rabbits eventually had a generalized, nonpruritic exfoliative dermatosis with patchy to coalescing areas of alopecia. skin biopsies were diagnostic. no response was seen to antibiotics, glucocorticoids, ivermectin, griseofulvin, fatty acids, azathioprine, or oral retinoids. facial eczema has been reported in young suckling rabbits.'! the condition is sporadic and of unknown etiology. areas of alopecia and slight erythema occur on the bridge of the nose and the periocular region. affected animals are otherwise healthy. the condition responds rapidly to topical glucocorticoid therapy. spontaneous nonviral cutaneous neoplasms are rare in rabbits." papilloma, basal cell carcinoma, squamous cell carcinoma, sebaceous carcinoma, melanoma, osteosarcoma, and lymphoma have been reported. shope papillomas are uncommon in domestic rabbits. t in the united states, the disease occurs in the southwest and along the mississippi river. shope papilloma virus (a papovavirus) commonly infects wild rabbits, with insects serving as vectors. lesions are characterized by multiple hornlike growths from a single site, especially about the eyelids and the pinnae. removal of the papillomas usually results in healing, and recovered rabbits are resistant to reinfection. spontaneous regression of lesions occurs within months. experimental infection of domestic rabbits resulted in malignant transformation to squamous cell carcinoma within to months in a high percentage of the inoculation sites. a program of screening animal enclosures and vector control should be instituted in endemic areas. shope fibromas are uncommon in domestic rabbits.] shope fibroma virus (a poxvirus) commonly infects wild rabbits in north and south america and is transmitted via insect vectors. lesions consist of single or multiple flat, firm, subcutaneous nodules, especially on the genitals, the perineum, the ventral abdomen, the paw, the nose, the pinna, and the eyelid. newborn rabbits are more susceptible than are older animals and have more extensive lesions. experimentally infected adult rabbits often show spontaneous involution of their fibromas within months through necrosis and sloughing. mosquito eradication and enclosure screening is indicated to prevent infection in endemic areas. cutaneous lymphoma was reported in domestic rabbits from weeks to / years old. b most had early internal organ involvement and systemic disease. lesions consisted of multifocal areas of alopecia, scale, erythema, and plaques. histologically, the lymphomas were epitheliotropic and cd +. one rabbit was unsuccessfully treated with oral isotretinoin and interferon-a. bacterial skin infections are uncommon in pet rats, are usually caused by s. aureus, and are secondary to trauma (cage-related injuries and bite wounds) or self-inflicted (the pruritus associated with ectoparasitesl.p" , [ ] [ ] [ ] [ ] rats are more resistant to experimental wound infection with s. aureus than are mice or hamsters.!? infections resulting from cage-related injury are typically seen on the nose and the muzzle, whereas those associated with bite wounds typically occur around the head, the tail, the rump, and the perineal area. staphylococcal dermatitis may be superficial (alopecia, erythema, oozing, and crust) or deep (abscess, fistula, necrosis, and ulcer) and is usually nonpruritic. granulomas occurred on the trunk and mammary gland in association with infection by an atypical slow-growing s. aureus . other bacteria occasionally isolated from cutaneous abscesses and pyogranulomas in rats include streptococcus sp., p. pneumotropica, klebsiella pneumoniae, p, aeruginosa, and mycobacterium lepraemurium (rat leprosy). , , [ ] [ ] [ ] [ ] treatment of bacterial dermatitis includes some combination of elimination of predisposing causes, surgical drainage, daily topical cleaning with % hydrogen peroxide or . % to % chlorhexidine, and systemic antibiotic administration (see table - ). s. moniliformis is a rare cause of epizootics of edema and cyanosis of the extremities. , , c. kutscheri (murium) is a rare cause of epizootics of furunculosis and cutaneous pyogranulomas, which may progress to necrosis and sloughing of extremities.v dermatophytosis is rare in rats and is usually associated with t. mentagrophytes. - , , , - this dermatophyte can be isolated from the haircoat of clinically normal rats and is a potential zoonotic agent. lesions are most commonly seen on the neck, the back, and the base of the tail and consist of annular areas of alopecia, broken hairs, scales, and variable degrees of erythema and crusting. pruritus is usually minimal to absent, diagnosis and treatment are as described for guinea pigs. sialodacryoadenitis virus (a coronavirus) infection causes eye rubbing and scratching, periorbital swelling, and red tears (chromodacryorrhea) (see fig. - g) . , , poxvirus infection has been described in laboratory white rats.!? skin lesions consisted of erythematous papules, which became crusted and occurred mainly on the glabrous areas of the body (tail, paws, and muzzle). sometimes, the affected portions of paws and tail underwent necrosis and sloughing. diagnosis was confirmed by biopsy, electron microscopy, and viral isolation. n. muris occasionally causes a severely pruritic dermatitis in rats. - , , , - lesions are most commonly present on the pinnae, the nose, the paws, and the ventrum and consist of erythema, papules, yellowish hyperkeratotic crusts, and excoriations, diagnosis is confirmed by skin scrapings. treatment is accomplished with topical % lime sulfur dips (once weekly until weeks after cure) or subcutaneous injections of ivermectin. other mites that are rarely found on rats include radfordia ensifera, . bacoti (tropical rat mite), s. scabiei, trixacarus diversus ( fig. - h) , t. caviae, m. musculi, and demodex sp." fleas (especially c. felis felis) may be recovered from pet rats maintained in households frequented by cats and dogs. - , , the sucking louse polyplax spinulosa is occasionally found on pet rats. t some animals may be asymptomatic carriers, and other animals manifest varying degrees of dermatitis and pruritus. young animals, debilitated animals, and animals in poor management situations are more likely to be affected. lice and related dermatoses are most commonly found on the neck and back. treatment can be accomplished with topical insecticides or ivermectin injections. nutritional deficiencies are unlikely to be encountered in pet rats. experimental production of nutritional deficiencies with resultant cutaneous abnormalities has been reported, and these are briefly mentioned here. zinc deficiency produces exfoliative dermatitis, alopecia, and depigmentation of the hairco at, , pantothenic acid deficiency results in exfoliative dermatitis, depigmentation of the haircoat, and excessive harderian gland activity with increased porphyrin secretion resulting in red tears and blood-caked whiskers.v riboflavin deficiency produces alopecia, scaling, and dermatitis, especially on the extremities." pyridoxine deficiency results in exfoliative dermatitis, especially on the face, the ears, the limbs, and the tail. ' biotin deficiency causes exfoliative dermatitis and periocular alopecia." niacin deficiency causes alopecia and excessive harderian gland activity, increased porphyrin secretion, and blood-caked whiskers.v essential fatty acid deficiency produces an exfoliative dermatitis and, occasionally, necrosis of the tail. , protein deficiency causes alopecia, exfoliative dermatitis, and depigmentation of the haircoat." barbering and bite wounds are frequently seen when rats are housed together. - , , these behaviors can be exacerbated by crowding, stress, and boredom. areas most commonly affected include the muzzle, the whiskers, the face, the head, the rump, the tail, and the perineum. rats may also rub the hair off the muzzle as they stick their face through slotted feeders or wire bars.!? rats have numerous types of hereditary hairlessness that are probably never seen by the practitioner (fig. - ) . ringtail is a poorly understood condition seen in rats. - , the incidence of the disorder increases as the relative humidity falls below % and is especially common in young unweaned animals housed in cages with wire mesh bottoms, on hygroscopic bedding, and in rooms with excessive ventilation. in the northern hemisphere, most cases are seen from november to may, when heating systems often cause marked reductions in relative humidity. some strains of rats seem more susceptible than others. the condition usually occurs after months of reduced relative humidity. one or more annular constrictions develop in the tail, which becomes edematous, inflamed, and necrosed distal to the constrictions (fig. - ). ringtail is prevented by maintaining a relative humidity of at least %. perianal pruritus is seen in association with pinworms (syphacia muris). , , , infected rats occasionally mutilate the base of the tail. diagnosis is confirmed by microscopic examination of strips of cellophane (scotch) tape that have been applied to the perineum. the eggs of s, muris are banana shaped and about p.,m by us«. ivermectin is effective treatment. auricular chondritis has been described in rats.!" the condition has occurred spontaneously and in association with the placement of metal ear tags or immunization with type ii collagen. typically, both ears are affected, although one ear may be affected days to weeks before the other. the pinnae are swollen, erythematous, and nodular, and they become thickened and deformed. pain and pruritus are rare. histologically, there is a multifocal granulomatous chondritis with progressive destruction of cartilage. systemic hair embolism has been reported subsequent to intravenous injections in rats.!? cutaneous lesions consist of focal areas of necrosis and ulceration on the ventral aspect of the body. histologic examination reveals granulomatous and necrotizing dermatitis and panniculitis, and intravascular hair shaft fragments. the fur of the aged rat frequently turns yellow and becomes more coarse.f the cause is unknown. alopecia areata was reported in debr rats and proposed as a useful model of the human disease."? brownish scales were observed to occur on the skin of rats, mainly on the dorsum and tail and were more numerous in males and with increasing age. the scales also occurred in strain-dependent patterns. gonadectomy produced a fading of the scales in males, whereas the administration of androgen to gonadectomized males and to females produced a darkening of the scales. spontaneous cutaneous neoplasms are uncommon in rats. - . . . mesenchymal neoplasms are more common than are epithelial neoplasms. the most common are fibromas, fibrosarcomas, and lipomas. the face, the shoulder, the flank, the tail, and the paws are typically affected. other reported skin neoplasms in rats include papilloma, keratoacanthoma, sebaceous gland tumors, squamous cell carcinoma, basal cell carcinoma, hair follicle tumors, hemangiosarcoma, melanoma, and malignant fibrous histiocytoma. dermatophytes in clinically healthy laboratory animals pathology of laboratory animals dermatology for the small animal practitioner. veterinary learning systems co skin disorders of rodents, rabbits, and ferrets practical treatment and control of common ectoparasites in exotic pets dermatologic disorders of common small nondomestic animals pathology of laboratory rats and mice bacterial diseases and antimicrobial therapy in small mammals the biology and medicine of rabbits and rodents ill. lea & febiger, philadelphia small rodents ferrets, rabbits, and rodents handbook of diseases of laboratory animals. heinemann veterinary books exotic animals dermatophytes isolated from laboratory animals antibiotic therapeutics in laboratory animals bacterial infections and antibiotic therapy in small mammals individual care and treatment of rabbits, mice, rats, guinea pigs, hamsters, and gerbils muller & kirk's small animal dermatology v handbook of rabbit and rodent medicine pruritus in rabbits, rodents, and ferrets husbandry and medicine of small rodents chinchilla . hoefer hl: chinchillas. vet clin north am ferret . ackerman j: ultrasonographic detection of adrenal gland tumors in two ferrets biology and medicine of the ferret skin diseases of ferrets yeast infection in ferrets coccidioidomycosis in three european ferrets biology and diseases of the ferret evaluation of urinary cortisol: creatinine ratios for the diagnosis of hyperadrenocorticism associated with adrenal gland tumors in ferrets superficial spreading pyoderma and ulcerative dermatitis in a ferret university sydney postgraduate foundation veterinary cutaneous lymphoma in a ferret (mustela putorius furo) neoplastic diseases in ferrets: cases - ) demodicosis in ferrets (mustela putonus fum) guide du furet domestique comparison of three treatments for control of ear mites in ferrets cutaneous epitheliotropic lymphoma in a ferret hyperadrenocorticism associated with adrenocortical tumor or nodular hyperplasia of the adrenal gland in ferrets: cases ( - ) evaluation of plasma androgen and estrogen concentrations in ferrets with hyperadrenocorticism adrenal gland diseases in ferrets complex ceruminous gland adenocarcinoma in a brown-footed ferret (mustela putonus [uro) bilaterally symmetric alopecia associated with an adrenocortical adenoma in a pet ferret figurate erythema resembling erythema annulare centrifugum in a ferret with adrenocortical adenocarcinoma-associated alopecia correlation between age at neutering and age at onset of hyperadrenocorticism in ferrets clinical aspects and surgical treatment of hyperadrenocorticism in the domestic ferret: cases ( - ) surgical treatment and long-term outcome of ferrets with bilateral adrenal tumors or adrenal hyperplasia: cases ( - ) jacklin mr: dermatosis associated with acarus farris in gerbils guinea pig . bobrowski pj, et al: latent herpes simplex virus reactivation in the guinea pig. an animal model for recurrent disease apparent spontaneous ergot-induced necrotiting dermatitis in a guinea pig acariose a chirodiscoides caviae et dermatophytie a microsporum canis chez un cobaye ivermectin treatment of a colony of guinea pigs infested with fur mite (chirodiscoides caviae) guinea pigs haematological and pathological responses to experimental trixacarus caviae infection in guinea pigs trixacarus caviae infestation in a guinea pig: failure to respond to ivermectin administration mechanisms involved in elimination of organisms from experimental cutaneous candida albicans infections in guinea pigs the in vitro antifungal activity of ketoconazole, zinc pyrithione, and selenium sulfide against pityrosporum and their efficacy as a shampoo in the treatment of experimental pityrosporosis in guinea pigs prevalence of dermatophytes in asymptomatic guinea pigs and rabbits hyperadrenocorticism in three teddy bear hamsters epidermotropic cutaneous t-cell lymphoma (mycosis fungoides) in syrian hamsters (mesocricetus auratusi. a report of six cases and the demonstration of t-cell specificity mouse . abbott dp, et al: a condition resembling pagetoid reticulosis in a laboratory mouse immune complex vasculitis with secondary ulcerative dermatitis in aged c bu nnla mice the effectiveness of ivomec and neguvon in the control of murine mites the use of repeated treatment with ivomec and neguvon in the control of murine mites and oxurid worms hemangiomas and hemangiosarcomas in inbred laboratory mice the mouse in biomedical research the mouse epidermis as a model in skin pharmacology: influence of age and sex on epidermal metabolic reactions and their circadian rhythms spontaneous vulvar carcinomas in j mice granulomatous inflammation in the tails of mice associated with mycobacterium chelonae infection specific detection of mousepox virus by polymerase chain reaction the inflammatory and immune response to mousepox (infectious ectomelia) virus treatment with ivermectin in drinking water against myobias musculi and myocoptes musculinis mange in naturally infected laboratory mice inherited mouse mutations as models of human adnexal, cornification, and papulosquamous dermatoses inherited mouse mutations: models for the study of alopecia alopecia areata in humans and other mammalian species l'utilisation d'ivermectin pour ie traitement des acariens, myobia musculi et myocoptes musculinus, dans une colonie de souris transgeniques diagnostic exercise: head and neck swelling in aljcr mice rabbit . arlian lg, et al: sarcoptes scabiei: the circulating antibody response and induced immunity to scabies sarcoptes scabiei: histopathological changes associated with acquisition and expression of host immunity to scabies abnormalities of collagen fibrils in a rabbit with connective tissue defect similar to ehlers-danlos syndrome caring for rabbits: an overview and formulary use of frontline spray on rabbits ectopic psoroptes cuniculi infestation in a pet rabbit rabbit husbandry and medicine how to handle respiratory, ophthalmic, neurological and dermatologic problems in rabbits experimental staphylococcal dermatitis in rabbits hillyer ev: pet rabbits malignant melanoma in two rabbits hereditary compulsive self-mutilating behavior in laboratory rabbits skin disorders of the rabbit diagnosis and prevalence of leporacarus gibbus in the fur of domestic rabbits in the uk use of levamisole for the treatment of mange due to notoedres cati in rabbits clinicotherapeutic efficacy of amitraz against notoedres cati infestation in rabbits. indian generalized exfoliative dermatosis with sebaceous adenitis in three domestic rabbits an evaluation of ivermectin oral preparation in the treatment of sarcoptic mange in rabbits use of frontline spray in rabbits the biology of the laboratory rabbit ii cutaneous staphylococcosis in rabbits leporacarus gibbus and spilopsyllus cuniculi infestation in a pet rabbit a case of ehlers-danlos-like syndrome in a rabbit with review of the disease in other species therapeutic efficacy of ivermectin in rabbits (orydolagus cuniculus) experimentally infected with psoroptes cuniculi the biology of the laboratory rabbit sebaceous adenitis in four domestic rabbits (orydalagus cuniculus) lymphoma with cutaneous involvement in three domestic rabbits (onjdolagus cuniculus) rat . binhazim aa, et al: spontaneous hemangiosarcoma in the tail of a long-evans rat carrying the elcer mutation behandlung med ivermectin mot springmask och piilskvalster hos laboratorieratta: strategisk program i en produktion-sanhet granulomatous dermatitis and mastitis in two spf rats associated with a slowly growing staphylococcus aureus-a case report mange in domesticated rats development of pigmented scales on rat skin: relation to age, sex, strain, and hormonal effect key: cord- -vf xbaug authors: dysko, robert c.; nemzek, jean a.; levin, stephen i.; demarco, george j.; moalli, maria r. title: biology and diseases of dogs date: - - journal: laboratory animal medicine doi: . /b - - / - sha: doc_id: cord_uid: vf xbaug nan status as a cooperative companion animal of reasonable size. dogs were used in the mid- s by william harvey to study cardiac movement, by marcello malpighi to understand basic lung anatomy and function, and by sir christopher wren to demonstrate the feasibility of intravenous delivery of medications (gay, ) . the use of dogs continued as biomedical research advanced, and they were featured in many noteworthy studies, including those by pavlov to observe and document the conditioned reflex response and by banting and best to identify the role of insulin in diabetes mellitus. for a comprehensive but concise review of the use of the dog as a research subject, the readers are directed to the manuscript by gay ( ) . the breed of dog most commonly bred for use in biomedical research is the beagle. some commercial facilities also breed foxhounds or other larger-breed dogs for use in surgical research studies. some specific breeds with congenital or spontaneous disorders are also maintained by research institutions (see specific examples below). random-source dogs used in research are most frequently mongrels or larger-breed dogs (e.g., german shepherd, doberman pinscher, labrador and golden retrievers) that are used for surgical research and/or training. according to a computerized literature search for beagle for the years - , approximately % of the biomedical scientific publications identified were in the fields of pharmacology or toxicology. especially common were studies focusing on pharmacokinetics, alternative drug delivery systems, and cardiovascular pharmacology. the next most common areas of research using beagles were dental and periodontal disease and surgery ( % of publications), orthopedic surgery and skeletal physiology ( %), and radiation oncology ( %). other research areas that utilized beagles included canine infectious disease, surgery, imaging, prostatic urology, and ophthalmology. most large-sized dogs (either purpose-bred or randomsource) are used in biomedical research because of their suitability for surgical procedures. anesthetic protocols and systems for dogs are well established, and the organs of larger-breed dogs are often an appropriate size for trials of potential pediatric surgical procedures. surgical canine models have been used extensively in cardiovascular, orthopedic, and transplantation research. there are also some unique spontaneous conditions for which dogs have proven to be valuable animal models. a colony of gray collies is maintained at the university of washington (seattle) for the study of cyclic hematopoiesis. this condition is manifested by periodic fluctuations of the cellular components of blood, most notably the neutrophil population. these dogs are used to study the basic regulatory mechanisms involved with hematopoiesis, as well as possible treatments for both the human and the canine conditions (brabb et al., ) . golden retrievers affected with muscular dystrophy have been used as models of duchenne muscular dystrophy in human children. duchenne muscular dystrophy is caused by an absence of the muscle protein dystrophin, inherited in an x-linked recessive manner. the dystrophy in golden retrievers is caused by absence of the same protein and is inherited in the same way. the clinical signs (such as debilitating limb contracture) are also similar between the canine and human conditions (kornegay et al., ) . bedlington terriers have been used to study copper storage diseases (such as wilson's disease), and the development of spontaneous diabetes mellitus and hypothyroidism in a variety of dogs has also been studied for comparisons with the human conditions. although historically the dog has been a common laboratory animal, the use of dogs in research has been waning over the past few years. according to the u.s. department of agriculture ( ) , the number of dogs used in research has declined from a high use of , in , in to only , in . this decrease was caused by a variety of factors, including (but not limited to) increased cost, decreased availability, local restrictive regulations, conversion to other animal models (such as livestock or rodents), and shift in scientific interest from pathophysiology to molecular biology and genetics. dogs used for research are generally segregated into two classes: purpose-bred and random-source. purpose-bred dogs are those produced specifically for use in biomedical research; they are intended for use in long-term research projects and/or pharmacologic studies in which illness or medication would require removal from the study. usually these dogs are either beagles or mongrel foxhounds, although other breeds may be available. purpose-bred dogs typically receive veterinary care throughout their stay at the breeding facility. they are usually vaccinated against canine distemper virus, parvovirus, adenovirus type , parainfluenza virus, leptospira serovars canicola and icterohemorrhagiae, and bordetella bronchiseptica. rabies virus vaccination may also be included. purpose-bred dogs are also usually treated prophylactically for helminths and ectoparasites, intestinal coccidia, and bacterial ear infections (r. scipioni and j. ball, personal communication, ) . random-source dogs are not bred specifically for use in research. they may be dogs bred for another purpose (e.g., hunting), retired racing dogs, or stray dogs collected at pounds or shelters. the health status of these dogs can be the same quality as purpose-bred dogs, or it can be an unknown entity. randomsource dogs that have been treated and vaccinated in preparation for use in research are termed conditioned dogs. these dogs are then suitable for long-term studies or terminal preparations that require unperturbed physiologic parameters. conditioned dogs are often tested for heartworm antigen because of the implications that infestations can have on cardiovascular status and surgical risk. nonconditioned random-source dogs are useful only in a limited number of research studies, such as nonsurvival surgical training preparations. options for procurement of dogs for biomedical research typically include purchase from a u.s. department of agriculturedesignated class a or class b licensed dealer or directly from a municipal pound. the requirements for usda licensure are detailed in code of federal regulations (cfr), title , chapter ( - - edition), subchapter a, animal welfare, . , definitions, and . , requirements and application. briefly, class a licensees are breeders who raise all animals on their premises from a closed colony (suppliers of purpose-bred dogs are typically class a dealers). class b licensees purchase the dogs from other individuals (including unadopted animals from municipal pounds) and then resell them to research facilities. there are additional regulations that apply to class b dealers (such as holding periods and recordkeeping documentation) because of the public concern that stolen pets could enter biomedical research facilities in this manner. regulations regarding the sale of pound dogs to research facilities or class b dealers vary from state to state and include some bans on this practice. the best resource for identification of possible vendors is the "buyer's guide" issue of the periodical lab animal. typically the last issue of each year, the "buyer's guide" lists sources for both purpose-bred and random-source dogs and denotes such features as pathogen-free status, documentation of health status, and availability of specific breeds and timed pregnant females. some suppliers also have separate advertisements within that issue of the journal. welfare act ( cfr . , . , and . [g] ) are described in cfr chapter ( - - edition), subchapter a, animal welfare. regulations pertaining specifically to the care of dogs used in research are found in subpart a, specifications for the humane handling, care, treatment, and transportation of dogs and cats, of part (standards) of subchapter a. particular attention should be paid to section . c (primary enclosures--additional requirements for dogs), because the space required for housing dogs is calculated using the length of the dog rather than the body weight (which is used for other species and also for dogs, according to national research council (nrc) guidelines). section . (exercise for dogs) describes the requirements that dealers, exhibitors, and facilities must follow in order to provide dogs with sufficient exercise. the institute of laboratory animal research (ilar) has written the "guide for the care and use of laboratory animals" (seventh edition, ) . the "guide" is the primary document used by institutional animal research programs to develop and design their programs, as well as by the association for assessment and accreditation of laboratory animal care international (aaalac international) and other animal care evaluation groups to facilitate site visits and inspections. the ilar committee on dogs has also written "dogs: laboratory animal management " ( ) . this publication describes "features of housing, management, and care that are related to the expanded use of dogs as models of human diseases" and includes "an interpretive summary of the animal welfare regulations and the requirements of the public health service policy on humane care and use of laboratory animals." the reader is encouraged to use these publications to obtain further information on care and husbandry of dogs in the biomedical research setting. growth data for beagles from a purpose-bred dog breeding facility are provided in table i . table ii features hematology data from beagles from the same commercial facility. table iii lists serum and urine chemical data for beagles. normal physiologic data for dogs (no breed specified) are provided in table iv . the information presented in the tables represents a range of normal values that can vary, depending on the analytical method and equipment used as well as the age, breed, gender, and reproductive status of the animal. federal regulations promulgated by the animal and plant health inspection service, usda, in response to the animal good nutrition and a sound, balanced diet are essential to the health, performance, and well-being of the animal. the basic nutrient requirements for dogs have been compiled by the nrc and represent the average amounts of nutrients that a group of animals should consume over time to maintain growth and prevent deficiencies (national research council, ) . the reader is referred to these guidelines for useful reference points for management of an animal's diet during various physiologic states (e.g., gestation, lactation, maturational age). most commercially available balanced dog diets are "closedformula" diets, in which the labeled specific minimum requirements for protein and fat, and the maximum values for ash and fiber, are met. these diets do not necessarily provide the identical composition of ingredients from batch to batch. ingredient composition varies, depending on the cost relationships of the various ingredients as the manufacturer attempts to achieve the label requirements at the lowest ingredient cost. an "openformula" (or "fixed-formula") diet provides more precise dietary control. in these diets the ingredients are specified, and the percentage of each ingredient is kept constant from batch to batch. "semipurified" diets provide for the strictest control of ingredients and are formulated from the purified components: amino acids, lipids, carbohydrates, vitamins, and minerals. the animal care provider should be aware of the manufacture date of the diet, which should be clearly visible on the bag. as a general rule, diets are generally safe for consumption up to months following the manufacture date when stored at room temperature. refrigeration may prolong the shelf life, but the best strategy is to use each lot based on the date of manufacture in order to prevent food from expiring and to ensure that only fresh diets are fed. specifications for feeding and watering of dogs are provided in the regulations of the animal welfare act. recommendations for feeding the appropriate amount of diet are determined by the dog's metabolic requirements. the basal metabolic rate, or basal energy requirement (ber), refers to the amount of energy expended following sleep, - hours after food consumption, and during thermoneutral conditions (kleiber, ; lewis et al., ) . the maintenance energy requirement (mer) is the amount of energy used by a moderately active adult animal in a thermoneutral environment, which in the dog is approximately twice the ber (lewis et al., ) . for dogs weighing greater than kg, the mer may be calculated using this simplified linear equation: mer (metabolizable kcal/day) = ( weightkg + ) (national research council, ; lewis et al., ) . the quantity of a correctly balanced diet to be fed to each dog can then be determined by dividing the mer by the energy density of the diet. fat provides three major dietary functions, including absorption of fat-soluble vitamins (a, d, e, and k), enhancement of palatability, and provision of essential (unsaturated) fatty acids. dietary fat is an excellent, highly digestible energy source, providing . times more energy on a per weight basis than either soluble carbohydrates or proteins (lewis et al., ) . however, fats are not needed for this purpose when adequate carbohydrate and protein are present. consumption of fat in excess of an animal's ability to metabolize it results in steatorrhea and has been related to the development of acute pancreatitis, whereas lack of dietary fat may lead to a fatty acid/energy deficiency. fatty acid deficiency is associated with poor growth, poor physical performance, reduced reproductive performance, and weight loss. dogs are considered to be "easy keepers," because they do not have as many absolute nutritional requirements as their domestic counterpart, the cat. however, they do possess a unique requirement for certain polyunsaturated fatty acids, a deficiency of which may predispose them to decreased growth rates and dermatologic abnormalities, such as "hot spots." dogs require linoleic (f - ) acid, an essential fatty acid (national research council, ) , and more recently it has been demonstrated that the f - fatty acids may play a role in maintaining healthy skin (logas and kunkle, ) . supplementation with a balanced essential fatty acid product (e.g., derm caps) may alleviate allergy-related dermatoses such as flea-bite dermatitis and pyoderma (logas and kunkle, ; miller, ) . essential fatty acid deficiency can occur in dogs receiving low-fat dry dog food that has been stored too long, particularly under warm, humid conditions (lewis et al., ) . there are a-amino acids, of which cannot be synthesized in sufficient quantity to meet a dog's normal metabolic demands for growth and maintenance. hence, as their name implies, these essential amino acids are required by all dogs and must be provided in the diet. the essential amino acids and the minimal requirements for growth are listed elsewhere (lewis et al., ) . chronic excessive protein intake may be detrimental to the kidney by contributing to accelerated renal aging and subsequent glomerulosclerosis (lewis et al., ) . conversely, inadequate protein intake results in retardation of growth and adata graciously provided by r. scipioni and j. ball of marshall farms usa, inc., north rose, new york. beagles tested for period / / - / / . b s.d., standard deviation; wbc, white blood cells; rbc, red blood cells; hgb, hemoglobin; hct, hematocrit; mcv, mean corpuscular volume; mch, mean corpuscular hemoglobin; mchc, mean corpuscular hemoglobin concentration; rdw, red cell distribution width; hdw, hemoglobin distribution width; plt, platelets; mpv, mean platelet volume; neut, neutrophils; lymp, lymphocytes; mono, monocytes; eos, eosinophils; baso, basophils; luc, large unstained cells; li, lobularity index; mpxi, mean peroxidase activity index reduction in production and/or performance. protein deficiency, a potential consequence of decreased food intake, results in decreased energy intake. as a compensatory mechanism for a lack of fat or carbohydrate, body protein catabolism ensues in order to meet energy demands, thus exacerbating the negative protein balance and contributing to the clinical signs of edema/ascites, unkempt appearance, lethargy, and weight loss. thus, caloric needs must be met before protein needs (lewis et al., ) , an important concept to bear in mind in the event of research experiments that may predispose to anorexia. in general, providing a good quality commercial diet that supplies the required amount of amino acids and caloric requirements of the animal, while avoiding excess protein, will ensure nutritional stability and promote longevity. appropriate mineral balance in the diet is very important. the best approach in the laboratory setting is to feed a commercial diet that has been formulated with the proper amount and balance of minerals for normal growth. the recommended amount of dietary minerals and the major causes and clinical signs of deficiencies are published elsewhere (lewis, ) . determining the specific mineral involved in an imbalance can be a diagnostic challenge, because the clinical signs for several excesses/ . (basal) . (anestrus) . c < . c - c - r (continues) deficiencies are similar and nonspecific. a definitive diagnosis is often made only after the diet has undergone analysis of the mineral components. once the imbalance has been identified, the safest resolution to the problem is to discard the entire lot of misformulated diet. attempting to correct the imbalance through oral supplementation is likely to be more harmful than beneficial, and it risks intensifying the problem by creating additional mineral imbalances. vitamins function as enzymes that regulate a wide variety of physiologic processes. they are divided into two groups based on their solubility. the fat-soluble vitamins include a, d, e, and k, whereas the rest are water-soluble. a list of the vitamins, their requirements, and clinical signs associated with deficiencies and toxicities is published elsewhere (lewis et al., ) . cases of dietary deficiency are rarely encountered in the research setting, because laboratory dog chows are fortified with vitamins. additional vitamin supplementation may occasionally be required during prolonged clinical illnesses, such as polyuria or diarrhea, which predispose to loss of water-soluble vitamins (b complex and c) (lewis et al., ) . however, as with minerals, routine supplementation of vitamins may induce inadvertent toxicity and exacerbation of an imbalance. management of a breeding colony requires broad knowledge of the dog's anatomy, reproductive physiology, and behavioral needs during breeding, gestation, and parturition. although a comprehensive discussion of the biology of canine reproduction is beyond the scope of this chapter, essential features of the broad topics noted above are presented. this section is largely based on information assimilated from texts such as "miller's anatomy of the dog" (evans and christensen, ) , "veterinary reproduction and obstetrics" (arthur et al., ) , and an issue of veterinary clinics of north america: small animal practice devoted to pediatrics of puppies and kittens (hoskins, ) . the ovaries of the bitch are attached to the dorsolateral walls of the abdominal cavity caudal to the kidneys by the broad ligaments and are not palpable abdominally. the uterus consists of the cervix, uterine body, and uterine horns. the cervix is an abdominal organ, located approximately halfway between the birchard and sherding ( ) . ovaries and the vulva. when the bitch is in proestrus and estrus, the cervix can be distinguished during abdominal palpation as an enlarged, turgid, walnut-shaped structure. catheterization of the cervix is usually not possible in the normal bitch at any stage of the reproductive cycle, except during or immediately following parturition. thus, semen is deposited at the external cervical os during natural or artificial insemination. the vagina is a long musculomembranous canal that extends from the uterus to the vulva. when the vagina is examined, the gloved finger or examination instrument should be introduced through the dorsal commissure of the vulva so as to avoid the deep ventral clitoral fossa. examination should proceed at an angle of approximately ~ until the instrument or fingertip has passed over the ischial arch, after which it can be directed further craniad toward the cervix. the bitch has a monoestrous cycle, with clinical estrus occurring predominantly in january or february and again in july or august (although it can occur at any time of year). the estrous cycle consists of four stages: proestrus, estrus, diestrus, and anestrus. the average duration of proestrus is days. during this stage the vulva is enlarged, turgid, and firm, and a sanguinous vaginal discharge is present. endocrinologically, proestrus is the follicular stage of the cycle, and estrogen levels peak at this time. estrus generally lasts days, and the vulva is softer and smaller than in proestrus. a vaginal discharge persists during estrus and may remain serosanguinous or become straw-colored. the endocrine feature of estrus is the luteinizing hormone (lh) surge, followed by ovulation within - hours. diestrus begins approximately days after the onset of standing heat. the end of this stage is days later, which would be coincident with whelping if the bitch had become pregnant. serum progesterone levels peak during diestrus. the duration of anestrus is approximately months. anestrus is the stage of reproductive quiescence, characterized by an absence of ovarian activity and serum progesterone levels of less than ng/ml. components of the canine spermatic cord include the ductus deferens, the testicular artery and vein, the lymphatics and nerves, and the cremaster muscle. the cremaster muscle and pampiniform plexus aid in thermoregulation of the testicles, which are maintained at ~ lower than basal body temperature. sweat glands in the scrotum assist in lowering the scrotal temperature through evaporation. the penis is a continuation of the muscular pelvic urethra and is attached to the ischiatic arch by two fibrous crura. it is composed of fibrous tissue and three cavernous sinuses: corpus cavernosum, corpus spongiosum penis, and corpus spongiosum glandis. the accessory sex glands of the dog consist of only a well-encapsulated prostate gland that surrounds the pelvic urethra, and ampullary glands at the termination of the vas deferens in the urethra. the dog does not have seminal vesicles or bulbourethral glands. the onset of puberty ranges from to months of age and is affected by breed, season, and nutritional and disease status. testicular growth is rapid at this time, and the seminiferous tubules begin to differentiate. the sertoli cells form the bloodtestis barrier, the tubules become hollow, and spermatogenesis commences. this process is initiated by the secretion of lh from the anterior pituitary, which stimulates the production of testosterone by the interstitial, or leydig's, cells. secretion of follicle-stimulating hormone (fsh) by the anterior pituitary stimulates the production of other key hormones by the sertoli cells, including inhibin, androgen binding protein, and estrogen. fsh stimulates spermatogenesis in the presence of testosterone, while inhibin and estrogen play a role in a feedback loop on the pituitary gland to decrease fsh production. spermatogenesis in the dog is completed in days, with subsequent maturation of sperm occurring in the epididymis for approximately days. thus, the entire process from initiation of spermatogonial mitosis to delivery of mature sperm to the ejaculate is days. a breeding soundness exam should be conducted to assess the probability of a male dog's successful production of offspring. factors affecting male fertility include libido, ability to copulate, testicular size, and quality and number of sperm produced. problems with libido may occur in dogs due to early weaning, isolation, or inherited abnormalities that suppress sexual behavior. animals with poor hindlimb conformation or with trauma to the back or hindlimbs may be unable to properly mount the female. there is a positive correlation with the size of the testicles as measured by scrotal circumference and the number of sperm produced. finally, parameters used to assess the quality of sperm include motility, morphology, volume, and concentration. an ejaculate ( ml) that contains approximately million progressively motile sperm without significant morphological abnormalities (such as a kinked tail) is a good indicator of normal male fertility. in general, erection, which involves muscular contractions and increased arterial blood flow to the penis, is controlled by the parasympathetic nervous system, whereas ejaculation is under sympathetic control. on mounting, the initial thrusting and ejaculation of semen last about minute. the bulbus glandis becomes enlarged, which lodges the penis in the female reproductive tract. the male then dismounts and brings one hindleg over the female, and the two continue to be joined "rear to rear," a position classically termed "the tie." ejaculation of the accessory gland fluid continues for - minutes. the continued expulsion of prostatic fluid during the "tie" may serve to propel the semen from the vagina through the cervix into the uterus. fertilization occurs in the oviduct and may occur as late as days after coitus, because of the long life span of sperm in the dog. however, once ovulated, oocytes generally remain viable for only - hours. therefore, the bitch should be bred prior to ovulation to ensure the presence of sperm for fertilization of live oocytes. cells of the vaginal epithelium mature to keratinized squamous epithelium under the influence of estrogen. because of the rise in estrogen throughout proestrus, with peak levels occurring just prior to the onset of standing heat, the vaginal smear can be used as an indicator of the bitch's readiness for breeding. the smear will not confirm the presence of ovulation, nor is it of prognostic value in normal bitches during anestrus. the percentage of vaginal epithelial cell cornification is an index of estrogen secretion by the ovarian follicles. as cornification of vaginal epithelial cells proceeds, the cells become larger, with more angular borders. the nuclear-cytoplasmic ratio decreases until the nuclei reach a point where they no longer take up stain (coincident with the onset of estrus). the cells appear "anuclear" and are classified as "cornified" or "anuclear squames." cornification occurs approximately days prior to the estrogen peak and days prior to standing heat. the percentage of cornified cells (of the total number of epithelial cells) decreases gradually to zero after the onset of diestrus. the vaginal cytology smear of the bitch changes from predominantly cornified to noncornified days after ovulation. the day of this change is the first day of diestrus. other epithelial cell types noted on vaginal cytology include superficial cells (large, angular cells with small nuclei); intermediate cells (round or oval cells with abundant cytoplasm and large, vesicular nuclei); and parabasal cells (small round or elongated cells with large, well-stained nuclei, and a high nuclear-cytoplasmic ratio). based on vaginal cytology, the estrous cycle is classified as follows: although vaginal cytology is a useful tool, it is not a substitute for observation of behavioral estrus, which is the best criterion to use in breeding management. during proestrus the male is attracted to the bitch and will investigate her hindquarters, but she will not accept breeding. the behavioral hallmark of estrus is standing receptivity toward the male. during this stage the bitch will exhibit "flagging," or elevation of her tail with muscular elevation of the vulva to facilitate penetration by the male. in order to maximize the conception rate, and the number of pups whelped per egg ovulated, it is recommended to breed the bitch on days , , and of the standing heat. fertilization is completed in the mid-to distal oviduct. implantation is evident by areas of local endometrial edema - days after breeding. there is no correlation between the number of corpora lutea and the number of fetuses in the corresponding uterine horn, suggesting transuterine migration of embryos. the dog has endotheliochorial placentation. the endothelium of uterine vessels lies adjacent to the fetal chorion, mesenchymal, and endothelial tissues, so that maternal and fetal blood are separated by four layers. the canine placenta is also classified as zonary and deciduate, indicating that the placental villi are arranged in a belt and that maternal decidual cells are shed with fetal placentas at parturition. the length of gestation is - days. luteal progesterone is responsible for maintaining pregnancy, and canine corpora lutea retain their structural development throughout gestation. serum progesterone rises from less than ng/ml in late proestrus to a peak of - ng/ml during gestation, then declines to - ng/ml just prior to parturition. progesterone is essential for endometrial gland growth, secretion of uterine milk, attachment of the placentas, and inhibition of uterine motility. pregnancy detection can be performed by abdominal palpation of the uterus days after breeding. the embryos and chorioallantoic vesicles form a series of ovoid swellings in the early gravid uterus. they are approximately inches in length at - days, the time at which pregnancy is most easily and accurately diagnosed. by day the uterus begins to enlarge diffusely, so that the vesicles (and, therefore, pregnancy) are difficult to identify by palpation. fetal skeletons become calcified and are radiographically evident by day . bitches in which a difficult whelping is anticipated should be radiographed in late pregnancy to determine the litter size and to evaluate the size of the fetal skulls in relation to the bony maternal birth canal. real-time ultrasound can be utilized for pregnancy detection of vesicles as early as - days. an abrupt drop in body temperature to less than ~ indicates impending parturition within - hours. the process of parturition has been divided into three stages, stage of labor lasts - hours and is characterized by uterine contractions and cervical dilation. during this stage, the bitch may appear restless, nervous, and anorexic. other common clinical signs include hard panting and increased pulse and respiration rates. fetal expulsion occurs during stage , which lasts approximately - hours. as the fetus engages the cervix, the neuroendocrine system induces the release of oxytocin; this is referred to as the ferguson reflex. oxytocin strengthens the uterine contractions and may elicit voluntary abdominal contractions as well. the bitch is usually recumbent during stage but is able to inhibit this stage if labor if disturbed. the chorioallantois ruptures either during passage of each neonate through the birth canal or by the bitch's teeth at birth. interestingly, posterior presentation is common in dogs but does not predispose to dystocia. the time interval between delivery of each pup is irregular, but the average time lapse is less than hour between pups until parturition is complete. veterinary assistance is necessary if the bitch remains in stage for more than hours without delivering the first pup, or for more than hours before delivering subsequent pups. the placentas are expelled during stage of labor, immediately following delivery of a pup, or up to minutes thereafter. if two pups are delivered from alternate uterine horns, then the birth of both puppies may precede expulsion of the respective placentas. the bitch will lick the newborn vigorously to remove the membranes from its head and to promote respiration. she will also sever the umbilical cord. the bitch may ingest the placentas, although they confer no known nutritional benefit and may induce a transient diarrhea. thermal support should be provided prior to parturition. dogs housed on grated flooring should be provided with mats, and those on solid floors would benefit from blankets placed in a corner of the primary enclosure. shavings are discouraged as they have the potential to coat the umbilical cord, which may predispose to ascending infections. heat lamps may be placed hours prior to parturition and remain until all neonates dem-onstrate vigorous and successful suckling behavior. however, the use of heat lamps necessitates strict supervision in order to prevent thermal burns. if possible, whelping bitches should be housed in a quiet corridor in order to decrease periparturient stress, especially in primiparous or young mothers. thus, monitoring of parturition is important, but human intervention should be minimal in order to prevent stress-induced cannibalism. weak or debilitated puppies may be cannibalized by the bitch before the research staff recognizes the need for veterinary attention. the postpartum use of oxytocin is required only in the event of uterine inertia, stillbirths, or agalactia. in these cases, - units of oxytocin may be administered intramuscularly. uterine involution occurs during anestrus within - weeks of parturition. during this time a greenish to red-brown vaginal discharge, or lochia, may be noted. although lochia is normal, the presence of an odiferous, purulent discharge, accompanied by systemic signs of illness, indicates metritis or pyometra. desquamation of the endometrium begins by the sixth postpartum week, with complete repair by months. newborn puppies are easily sexed by examination of the anogenital distance. in female puppies the vulva is evident a short distance from the anus, whereas the prepuce of male puppies is nearly adjacent to the umbilicus. eyes are open at approximately days, and ears are patent at approximately - days. solid food can be introduced between . and weeks of age, and puppies can be weaned at - weeks. artificial insemination (ai) is indicated when the male is physically incapable of mounting or penetrating the bitch, when there are vaginal abnormalities such as strictures, or when the bitch refuses to stand for breeding. semen for ai is collected using a plastic centrifuge tube and rubber latex artificial vagina. the male is introduced to the bitch's scent and manually stimulated. after collection of the first two fractions, a sufficient amount of the third fraction, which consists predominantly of prostatic fluid, is collected to bring the total semen volume to - ml. the semen is then drawn into a sterile or ml syringe attached to a sterile disposable insemination pipette. the bitch is inseminated either standing or with raised hindquarters. a gloved index finger is inserted into the dorsal commissure of the vulva and directed craniodorsally until it is over the ischial arch. the tip of the insemination pipette is introduced and guided by the gloved finger toward the external cervical os. the semen is injected, and - ml of air are then flushed through the syringe and pipette. the pipette is withdrawn, and the gloved finger is used to feather the ceiling of the vagina until contractions of the vaginal musculature are palpable. the bitch's hindquarters are subsequently elevated to promote pooling of semen around the external cervical os. as with natural breeding, ai should be performed on days , , and of standing heat, or on the days of maximal vaginal cornification. the bitch should be palpated for pregnancy approximately weeks after the first insemination. false pregnancy (pseudocyesis), a stage of mammary gland development and lactation associated with nesting or mothering behavior, is common in the bitch. the condition occurs after the decline in serum progesterone toward the end of diestrus. there is no age or breed predisposition. pseudopregnancy does not predispose the bitch to reproductive disease or infertility. however, in the event of extreme discomfort due to mammary gland enlargement, bitches may be treated with mibolerone (cheque drops) at an oral dose of ~tg/kg q hr for - days (brown, ) . reproductive performance in the bitch is optimal prior to years of age. although normal cycle lengths are reported to occur up to the ages of - years, the interestrous interval tends to increase by years of age. cycling does not completely cease; however, after years of age, bitches demonstrate significant decreases in conception rate and number of live pups whelped. by - years of age, pathologic conditions of the uterus, such as cysts, hyperplasia, atrophy, and neoplasia are extremely common. beagles have been a popular animal model because of their docile nature. they are easily handled and for the most part respond favorably to repetitive manipulations such as body weight measurements, physical examination, electrocardiogram (ecg) recordings, oral gavage, and venipuncture. dogs are sexually mature by - months of age, but they are not socially mature until - months of age. the socialization process should begin early during development, when puppies are receptive to conspecific and human contact. for example, from - weeks of age, puppies are most capable of learning about how to interact with other dogs. between weeks and , puppies are most capable of learning how to interact with people. by - weeks of age dogs voluntarily wander and explore new environments. thus, early handling and mild stress (such as vaccination) appear to be extremely beneficial components of a dog's social exposure. the extent to which breed affects behavior has been the subject of popular speculation but is difficult to prove. in general, breed-specific patterns do tend to emerge. for example, it appears that beagle pups are very motivated by food reward (overall, ). this is not surprising, because the breed was selected to work with its nose, and this may be a useful attribute for laboratory investigations that are predicated on food restriction. canid social systems use signals and displays that minimize the probability of outright aggression. these behavior patterns are most likely elicited during distressful situations, such as strange environments, being handled by strange people, or encountering new animals. an excellent, illustrated discussion of normal canine behavior patterns can be found in the third chapter of "clinical behavioral medicine for small animals" (overall, ). by virtue of the dog's status as a companion animal, there are many veterinary publications and reference texts on the diagnosis, medical management, pathology, and epidemiology of the disorders that can affect this species. the authors of this chapter have chosen to emphasize those diseases that are more frequently encountered in the research setting. especially noted in this chapter are infectious diseases associated with the use of random-source dogs that have unknown vaccination history and have had intensive contact with other similar animals at pounds and/or shelters, or conditions seen frequently in the beagle, the most common breed used in biomedical research. for more thorough and detailed discussion of these diseases, as well as those not discussed in this chapter, the reader should consult standard veterinary textbooks, such as the "current veterinary therapy" series (j. d. bonagura and r. w. kirk, eds.), "veterinary internal medicine" (s. j. ettinger and e. c. feldman, eds.), and "infectious diseases of the dog and cat" (c. e. greene, ed.) . full citations of some chapters from these texts are listed in the references (w. b. saunders co. of philadelphia publishes all three texts.) canine infectious tracheobronchitis (kennel cough complex) etiology. infectious tracheobronchitis (itb) is a highly contagious illness of the canine respiratory tract that usually manifests as an acute but self-limiting disease. several organisms have been incriminated as causative for this condition: bordetella bronchiseptica; canine parainfluenza virus (cpiv); canine adenovirus types and (cav- , cav- ); canine herpesvirus; canine reovirus types , , and ; and mycoplasms and ureaplasms. clinical signs. clinical infectious tracheobronchitis can be subdivided into mild or severe forms. the mild form is the more common presentation and is characterized by an acute onset of a loud, dry, hacking cough. increased formation of mucus sometimes results in a productive cough, followed by gagging or retching motions. cough is easily elicited by tracheal palpation and may be more frequent with excitement or exercise. otherwise the dog is typically asymptomatic, with normal body temperature, attitude, and appetite. mild tracheobronchitis usually lasts - days, even if left untreated. the severe form of tracheobronchitis generally results from mixed infections complicated by poor general health, immunosuppression, or lack of vaccination. secondary bronchopneumonia can occur and may be the determinant of severity (sherding, ) . animals are clinically ill and may be febrile, anorexic, and depressed. productive cough and mucopurulent naso-ocular discharge are more common than in the mild form. these cases require more aggressive treatment and may be fatal. bordetella bronchiseptica is considered to be the respiratory tract of infected animals (bemis, ) . this bacterium is very easily spread by aerosol and direct contact, and fomite transmission is also possible (bemis, ) . transmission is favored by confined housing of multiple animals. in experimental studies, b. bronchiseptica transmission to susceptible individuals was % (thompson et al., ; mccandlish et al., ) . the incubation period is - days. cpiv and cav- are also spread by aerosols. of these two viruses, cav- is the most persistent, lasting for up to several months in the environment, whereas cpiv is fairly labile (hoskins, a) . both viruses can be destroyed by quaternary ammonium disenfectants. pathogenesis. the most common clinical isolates are cpiv and bordetella bronchiseptica. however, b. bronchiseptica may be a commensal organism, and it is often recovered from asymptomatic animals. in cases of clinical infection, b. bronchiseptica attaches to the cilia on the mucosal surface of the upper airway epithelium, causing suppurative tracheobronchitis and bronchiolitis. infections with cpiv or cav- alone are usually subclinical; coinfections with b. bronchiseptica or other microbes may result in clinical itb (keil and fenwick, ; wagener et al., ) . the characteristic lesion from cpiv or cav- infection is necrotizing tracheobronchiolitis (dungworth, ) . pathogenic infection of the upper airways typically results in inflammation and ciliary dysfunction. diagnosis and differential diagnosis. diagnosis of infectious tracheobronchitis is often based on clinical signs. isolation of bordetella bronchiseptica or mycoplasma by nasal swabs allows only a presumptive diagnosis. viral isolation or paired serology can be done but is often impractical and expensive. if cough persists for more than days, other disease conditions should be considered. canine distemper virus infection, pneumonia, heartworm disease, tracheal collapse, and mycotic infections are differential diagnoses for dogs with similar signs. bronchial compression as a result of left atrial enlargement, hilar lymphadenopathy, or neoplasia may also elicit a nonproductive cough (johnson, ) and should be considered as a differential for itb. prevention. prevention is best achieved by avoiding exposure to infected animals, but this is oftentimes not practical. dogs should be vaccinated prior to, or at the time of, admission to the animal research facility. intranasal vaccine combinations for bordetella bronchiseptica and cpiv are preferred. intranasal vaccines protect against both infection and disease, can be given to dogs as young as weeks of age, and can produce immunity within days. control. sanitation and ventilation are critical for control. the animal care staff must practice proper hygiene to prevent fomite transmission. symptomatic animals should be isolated, and animal-to-animal contact avoided. kennels should be disinfected with agents such as bleach, chlorhexidine (nolvasan) or quaternary ammonium chloride (roccal-d). proper ventilation and humidity are important in controlling spread of these infectious agents; - air changes per hour at % relative humidity are recommended (sherding, ) . no specific treatment is available for viral infections. bordetella bronchiseptica is typically sensitive to potentiated sulfas, chloramphenicol, quinolones, tetracyclines, gentamicin, and kanamycin. use of antibiotics is indicated when severe or persistent clinical signs occur, and it should be continued for days. use of empirical antibiotic treatment in mild cases may hasten the resolution of clinical signs. for severe or unresponsive infection, treatment should be based on bacterial culture sensitivity patterns; nebulized gentamicin may be helpful. cough suppressants (e.g., dextromethorphan) should be avoided if the cough is bringing up mucus (productive); however, their use is indicated if coughing is causing discomfort or interfering with sleep. bronchodilators such as aminophylline, theophylline, or terbutaline can be helpful in reducing reflex bronchoconstriction and minimizing discomfort. tis results in altered respiratory tract histology and impaired mucociliary clearance, infected animals should not be used for pulmonary studies. animals with clinical disease would also be poor surgical candidates. etiology. [ -hemolytic lancefield's group c streptococcus (streptococcus zooepidemicus) is a gram-positive non-spore-forming coccus and an etiologic agent for pneumonia and septicemia in dogs. clinical signs. clinical signs vary based on the organ system affected. pneumonic disease is typically associated with coughing, weakness, fever, dyspnea, and hematemesis. peracute death without clinical signs has been reported in a previously healthy research dog (bergdall et al., ) , and conjunctivitis can also be caused by this organism (murphy et al., ) . epizootiology and transmission. lancefield's group c streptococci have been isolated as commensal flora in the upper respiratory tract and the vagina of clinically normal dogs (olson et al., ) . epizootics have been reported in both racing greyhounds and research colonies (sundberg et al., ; garnett et al., ) . in these epizootics, and in the reported case of peracute death (bergdall et al., ) , recent transportation (within days) was associated with the disease. as such, lancefield's group c streptococcus may be an opportunistic pathogen in dogs. pathologic findings. in the peracute case reported (bergdall et al., ) , hemorrhage from the mouth and nose and within the pleural cavity was the most striking lesion. ecchymotic and petechial hemorrhages were seen on other organ surfaces. the lungs were heavy and wet, and blood oozed from cut surfaces. "bull's-eye" lesions were observed on the pleural surface of affected lung lobes, similar to ischemic lesions seen with fungal infections (fig. ) . histologically, the lungs were characterized by areas of hemorrhage surrounding foci of degenerative neutrophils, blood, and necrotic debris. gram-positive cocci were seen in both the lung and the tonsils. pathogenesis. the pathogenesis for disease caused by lancefield's group c streptococcus is unclear. strain variation with respect to virulence and host immune factors is probably significant. diagnosis and differential diagnosis. definitive diagnosis is made based on bacterial culture and identification. any cause of pneumonia and/or peracute death in dogs needs to be considered as a differential diagnosis. bacterial pneumonias or septicemias can be caused by other pathogenic streptococcus spp., staphylococcus spp., escherichia coli, pasteurella multocida, pseudomonas spp., klebsiella pneumoniae, and bordetella bronchiseptica. nonbacterial causes include rodenticide intoxication, coagulopathies, heartworm disease, pulmonary thromboembolism, ruptured aneurysm, and left-sided congestive heart failure. prevention and control. too little is known about the pathogenesis of lancefield's group c streptococcus to make any recommendations about prevention and control. treatment. antibiotic therapy should be provided, based on culture and sensitivity. intravenous fluids are indicated for febrile or systemically ill patients. for dyspneic patients, oxygen therapy and strict activity restriction are required. research complications. clearly, dogs with severe hemorrhagic pneumonia or septicemia are not appropriate for any research study. the association between epizootics of this disease and transportation shipment supports the philosophy of providing acclimation periods to animals upon arrival at research facilities to evaluate health status and enable the animals to normalize physiologically. etiology. serovars of the spirochete leptospira interrogans sensu lato cause canine leptospirosis. disease in dogs is primarily due to serovars canicola, icterohemorrhagiae, grippotyphosa, pomona, and bratislava. clinical signs. leptospirosis may present as either an acute or a chronic problem. clinical signs are nonspecific and include lethargy, depression, abdominal discomfort, stiffness, anorexia, and vomiting. animals may be febrile and may be reluctant to move, because of muscle or renal pain or meningitis. icterus, congested mucous membranes, or signs referable to disseminated intravascular coagulation (petechial/ecchymotic hemorrhages, melena, epistaxis, or hematemesis) are also possible. animals with peracute leptospirosis are characterized by septicemia, shock, vascular collapse, andrapid death. uveitis, abortions, and stillbirths have also been associated with leptospirosis. epizootiology and transmission. vaccination and reduced exposure to reservoir hosts have markedly decreased the prevalence of leptospirosis over the past years. wild animals, cattle, and rodents are reservoirs for leptospira. the epidemiology of the disease is not static, and recent changes have been observed. serovars pomona, grippotyphosa, and bratislava are becoming more common causes of canine disease, with canicola and icterohemorrhagiae becoming less common. this may be due to vaccination practices and increased movement of wildlife reservoirs (raccoons, skunks, and opossums) into urban/suburban areas. rats have been implicated as important in the transmission of serovars canicola and icterohemorrhagiae (rentko et al., ; brown et al., ; kalin et al., ) . transmission occurs primarily by environmental contact, and not directly from animal to animal. infected hosts shed leptospires in urine, thereby contaminating the environment; naive animals are infected when the organisms contact mucous membranes or abraded skin. recovered animals may shed organisms in their urine for months to years. the organisms are actually labile in the environment; moisture, moderate temperatures, and alkaline soil favor survival and subsequent transmission. close contact, bites, ingestion of infected meat, and transplacental and venereal transmission are also possible. leptospirosis is a zoonotic disease. pathologic findings. the kidneys consistently have gross and microscopic lesions. in the acute phase of the infection, kidneys are swollen and have subcapsular and cortical ecchymotic hemorrhages. petechial or ecchymotic hemorrhages and swelling of the lungs and liver may also be noted. hepatic lesions during the acute phase consist of diffuse hemorrhage and focal areas of necrosis (searcy, ) . in chronic stages of leptospirosis the kidneys become small and fibrotic. endothelial cell degeneration and focal to diffuse lymphocytic-plasmacytic interstitial nephritis are the characteristic histopathological findings. pathogenesis. infection occurs after the leptospires penetrate a mucous membrane or abraded skin. the organisms then invade the vascular space and multiply rapidly. several days postinfection the renal tubular epithelium (and, to a variable extent, the liver) is colonized. the hematogenous phase lasts - days. acute renal failure or progressive renal failure leading to oliguria or anuria may occur. the most common clinical syndrome is chronic or subclinical infections after recovery from the acute phase (greene, ) . the nephritis may or may not be accompanied by hepatitis, uveitis, and meningitis. icterus, if it develops, is most common in the acute phase. the combination of azotemia and icterus should alert the clinician to the possibility of leptospirosis. disseminated intravascular coagulation is often a secondary complication. the severity and course of leptospirosis depend on the causative serovar and the age and immune status of the patient. diagnosis and differential diagnosis. zinc toxicity in dogs most closely mimics the clinical syndrome of leptospirosis. other causes of acute and chronic renal failure, icterus, and acute hepatic failure must also be considered. paired serology is the most reliable means of definitive diagnosis; however, seroconversion may not occur until after the first week of infection. prevention and control. vaccination for leptospirosis is standard veterinary practice. bivalent inactivated bacterins for serovars of l. interrogans canicola and serovars of l. interrogans icterohemorrhagiae are commercially available. however, immunization does not prevent development of the carrier state or protect against other serovars. for outdoor-housed dogs, an effective program to prevent contact with wildlife reservoirs is important. control requires identification and either treatment or elimination of carrier animals. treatment. penicillins are the drugs of choice for treating leptospiremia, and prompt use reduces fatal complications. aggressive fluid therapy and supportive care may also be needed. elimination of renal colonization and the carrier state can be accomplished with dihydrostreptomycin or doxycycline administration. should not be used in research studies because of the effects of the disease on renal and hepatic function. etiology. campylobacteriosis in dogs is caused by campylobacter jejuni, a thin, curved or spiral, microaerophilic, thermophilic motile gram-negative rod. clinical signs. most adult animals infected with c. jejuni are asymptomatic carriers; clinical signs are most commonly noted in dogs that are less than months of age (greene, ; burnens et al., ) . in cases of clinical illness, small volumes of mucoid or watery diarrhea, with or without frank blood, are most commonly noted. these signs are usually mild, may be intermittent, and typically last - days. tenesmus, inappetance, vomiting, and a mild fever may accompany the diarrhea. epizootiology and transmission. the role of c. jejuni as a primary pathogen has been questioned; it may require a coenteropathy to produce disease (sherding and johnson, ) . clinical signs of disease most often occur in dogs less than months of age, although any age may be affected. stress or immunosuppression may make animals more susceptible to clinical disease. pound and shelter populations have the highest rates of fecal excretion of c. jejuni (sherding and johnson, ) . transmission is via the fecal-oral route, mostly through fecally contaminated food or water. unpasteurized milk, poultry, and meat are other sources of infection. campylobacter jejuni can be zoonotic; children and immunocompromised individuals are at the greatest risk. pathologic findings. the actual lesions observed depend upon the mechanism of the enteropathy (van kruiningen, ) . enterotoxin production results in dilated fluid-filled bowel loops, with little or no histopathologic alteration. in cytotoxin-mediated disease, hyperemia and a friable, hemorrhagic mucosal surface are noted. on histopathology the mucosal surface is irregular and ulcerated, and a lymphocytic-plasmacytic ileitis or colitis may be seen. when translocation occurs, the lamina propria becomes edematous and congested, with focal accumulation of granulocytes in the crypts and lamina propria. focal areas of epithelial hyperplasia and decreased numbers of goblet cells are also noted. with warthin-starry silver staining, c. jejuni may be seen between enterocytes but only rarely inside them. pathogenesis. clinical disease may be produced by several different mechanisms after the campylobacter has populated the intestinal tract (van kruiningen, ) . after colonization of the enterocyte surface, c. jejuni can produce an enterotoxin that causes a secretory diarrhea. campylobacterjejuni can also cause an erosive enterocolitis by invasion of the ileal and colonic epithelium along with production of a cytotoxic agent; this may be the mechanism that causes hematochezia. in addition, c. jejuni can produce illness by translocation, i.e., multiplication in the lamina propria and transportation to regional lymph nodes by macrophages. this causes mesenteric lymphadenitis. diagnosis and differential diagnosis. fresh feces (per rectum) are best for ensuring an adequate diagnostic sample. presumptive diagnosis may be made by demonstration of highly motile curved or spiral organisms with dark-field or phase-contrast microscopy. gram-stained c. jejuni appear as gull-winged rods. definitive diagnosis requires isolation of the organism (sherding and . culture requires selective isolation media, and growth is favored by reduced oxygen tension and a temperature of ~ any disorder that can cause diarrhea in dogs should be considered as a differential diagnosis, including canine parvovirus, coronavirus, distemper virus, giardia, and salmonella infections; helminth infestations; and hemorrhagic gastroenteritis. clinical signs. based on experimental infections in dogs, three phases to the disease have been described: acute, subclinical, and chronic. clinical signs observed vary with the phase of the disease, and the acute and subclinical phases are often missed or misdiagnosed (c. g. couto, personal communication, ; waddle and littman, ; woody and mcdonald, ) . a history of tick exposure may be noted prior to onset of signs. in the acute phase, clinical signs range from mild to severe and may last - weeks. they include inappetance, lethargy, fever, generalized lymphadenopathy, hepatosplenomegaly, exercise intolerance or dyspnea, petechial or ecchymotic hemorrhages, and peripheral edema. central nervous system (cns) signs may also be present such as hyperaesthesia, myoclonus, and cranial nerve deficits. clinical laboratory abnormalities noted during the acute phase include thrombocytopenia, anemia, neutropenia or neutrophilia, and bicytopenia or pancytopenia. hyperplastic bone marrow, mild hyperglobulinemia, and elevated hepatic enzymes may be noted during this phase (kuehn and gaunt, ) . clinical signs are generally absent during the subclinical phase. mild thrombocytopenia, anemia, or leukopenia may be seen. the chronic phase develops - months after the initial infection, and signs may be subclinical to severe. an extremely varied clinical picture can emerge during this time and can mimic several other clinical syndromes. the following constellation of clinical signs may be observed: chronic lethargy, weight loss, inappetance or anorexia, fever, generalized lymphadenopathy, hepatosplenomegaly, petechial or ecchymotic hemorrhages, epistaxis, hematuria, melena, pallor, anterior or posterior uveitis, chorioretinitis, peripheral edema, ataxia, upper and lower motor neuron deficits, altered mentation, cranial nerve deficits, and seizures. persistent thrombocytopenia is the most consistent laboratory abnormality noted for all three stages. many other hematologic abnormalites may be found, such as regenerative or nonregenerative anemia (more frequently the latter), positive coombs' test, bicytopenia or pancytopenia, and splenic plasmacytosis or lymphocytosis. on bone marrow evaluation, plasmacytosis along with hypoplasia of erythroid, myeloid, and/or megakaryocyte lines may be seen. hyperglobulinemia as a result of polyclonal or occasionally monoclonal gammopathy has been noted in - % of e. canis seropositive or infected dogs (kuehn and gaunt, ; breitschwerdt et al., ; shimon et al., ) . proteinuria and/or hypoalbuminemia have also been seen. epizootiology and transmission. ehrlichia canis is an obligate intracellular parasite that infects mononuclear cells. the definitive hosts are arthropods; domestic and wild canids are parasitized secondarily. the primary vector and reservoir is the brown dog tick, rhipicephalus sanguineus. ehrlichia canis is found worldwide and follows the distribution of the vector. infection in dogs is most prevalent in tropical and subtropical areas (greene, ) . in the united states, cases are concentrated in the southeastern and southwestern states but have been reported in almost every state (breitschwerdt, ) . transmission is primarily by tick bites, but it can also occur via blood transfusions from dogs infected for as long as years. ticks become infected by feeding on an infected dog that is in the first - days of an acute infection (lewis et al., ) , and ticks can shed the organisms for up to months. within the tick population, e. canis is transmitted transstadially (within developmental stages) but not transovarially (from female to offspring) (groves et al., ) . pathogenesis. in experimental infections, the incubation period prior to the onset of the acute phase is - days. during the acute phase, which can last from - weeks, the bacteria replicate within circulating and tissue monocytes, resulting in lymphoreticular hyperplasia in affected tissues. infected monocytes then spread hematogenously to other organs in the body, in particular the lungs, kidney, and meninges. infected cells adhere to the vascular endothelium and induce vasculitis, which is the primary mechanism whereby the organism causes disease. the thrombocytopenia during the acute phase is due to both sequestration and destruction, and the development of anemia is a result of red blood cell destruction and suppression of erythrocyte production. the subclinical phase of the disease occurs - weeks after initial infection. during this stage, dogs that can mount an effective immune response clear the infection. those that cannot mount such a response progress to the chronic stage. infection does not confer protective immunity in dogs that recover. german shepherds and doberman pinschers seem to be more severely affected than other breeds. pathologic findings. gross lesions are varied and change, depending on the phase of the disease. the most common findings are petechial and ecchymotic hemorrhages and edema of dependent tissues (woody and hoskins, ) . the most common histologic abnormality noted is lymphocytic-plasmacytic inflammation of numerous organs. mononuclear phagocytic system hyperplasia, extramedullary hematopoiesis, and splenic erythrophagocytosis may also be seen. diagnosis and differential diagnosis. the most sensitive, specific, and commonly employed method for diagnosing e. canis infections is the indirect fluorescent antibody (ifa) test. antibodies can be detected as early as days postinfection, although some dogs may not seroconvert until days postinfection (buhles et al., ) . cross-reaction may occur between e. canis, e. chaffeensis, and e. ewingii. titers greater than : are considered positive and indicative of infection and may persist for up to year. effective treatment typically produces seronegative results in - months. in some cases, asymptomatic dogs may remain seropositive for years after treatment or may be seropositive with a persistent hematologic abnormality (bartsch and greene, ) . the exact mechanism for this finding has not been elucidated. ehrlichia canis morulae can be demonstrated in circulating monocytes of giemsa-stained blood smears. however, this method is labor-intensive and has low sensitivity, as morulae are present transiently and in low numbers. using buffy coat smears from capillary blood may increase the diagnostic yield. polymerase chain reaction (pcr) assays are also available to identify e. canis. differential diagnoses include immune-mediated hemolytic anemia/thrombocytopenia, multiple myeloma, chronic lymphocytic leukemia, and lymphoma. prevention. preventing laboratory animals from contacting ticks is the primary means to avoid monocytic ehrlichiosis in research dogs. avoid exercising dogs in areas infested with ticks. use topical acaricides to prevent tick infestations. keep kennel areas tick-free. dogs used as blood donors and dogs from unproven sources should be tested for e. canis. treatment. doxycycline is the drug of choice for treating monocytic ehrlichiosis. oral doses of either . - mg/kg q hr or mg/kg q hr for days are very effective at eliminating the organism. tetracycline, chloramphenicol, and enrofloxacin are also effective antibiotics; however, chloramphenicol should not be used in animals with cytopenias. in chronic cases, antibiotic treatment should be extended for an additional - weeks. research complications. the most significant research complication is the thrombocytopenia that persists for all stages of the disease. additionally, there is probable alteration in immune function and increased susceptibility to infectious agents. for these reasons, dogs positive for antibodies to e. canis should not be used in research. etiology. this disease, caused by ehrlichia platys, was first described as cyclic thrombocytopenia by harvey et al. in . clinical signs. in most cases, infection with e. platys results in subclinical disease. a generalized lymphadenopathy may be noted. epizootiology and transmission. the vector for e. platys is assumed to be a tick; however, this mode of transmission has not been established. experimental studies by simpson et al. ( ) failed to demonstrate rhipicephalus sanguineus as a vector for e. platys. coinfection with e. canis has been reported, which suggests a common vector for both organisms (french and harvey, ; kordick et al., ) . dogs have been experimentally infected by inoculation with infected blood or infected platelets from other dogs (harvey et al., ; gaunt et al., ) . the geographic distribution of thrombocytic ehrlichiosis is assumed to follow that of other ehrlichia organisms. the highest concentration of cases seems to be in southeastern states, but isolated cases have been reported as far north as michigan and as far west as oklahoma (wilson, ; mathew et al, ) . the prevalence of seropositive dogs can be high in some parts of the country. a study by bradfield et al. ( ) reported that % of the dogs entering a research institute's quarantine facility from sources in eastern north carolina were seropositive for e. platys. hoskins et al. ( ) reported a . % seropositive prevalence in healthy dogs from kennels in louisiana. pathologic findings. gross and histopathologic findings during experimental e. platys infection in dogs have been described by baker et al. ( ) . generalized lymphadenopathy was the only gross lesion noted. follicular hyperplasia and plasmacytosis were the predominate findings in lymphoreticular tissues. all dogs also had extramedullary hematopoiesis, erythrophagocytosis, and crescent-shaped hemorrhages in the spleen. multifocal kupffer's cell hyperplasia was noted in the liver, and mild multifocal lymphocytic-plasmacytic interstitial inflammation was seen in the kidneys. pathogenesis. the pathogenesis of e. platys in dogs has primarily been determined through experimental infection (harvey et al., ) . after inoculation the organism directly infects platelets. thrombocytopenia occurs by day - and fluctu-ates, along with parasitemia, at to day intervals. in some cases the rebound may be within the normal range for thrombocyte counts. the nadir can be lower than , platelets/~d. concurrent with low platelet counts is the development of megakaryocytic hyperplasia in the bone marrow. interestingly, despite extremely low platelet counts, spontaneous bleeding has not been reported in cases of e. platys infection. the mechanism responsible for the cyclic nature of the infection has not been elucidated. diagnosis and differential diagnosis. ehrlichia platys infection may be diagnosed on stained blood smears by visualization of the organisms within platelets. however, this method is very unreliable due to the cyclic nature of the parasitemia and the low numbers of infected thrombocytes. available ifa assays are much more sensitive and specific, and there is reportedly no serologic cross-reaction with other ehrlichia species. dogs usually develop detectable titers - weeks postinfection. pcr assays for e. platys have now been developed as well (chang and pan, ; mathew et al., ) . differential diagnoses for thrombocytic ehrlichiosis include e. canis infection, immunemediated thrombocytopenia, and disseminated intravascular coagulation (dic). platys is the same as described for e. canis, above. research complications. ehrlichia platys infection may increase the risk of bleeding during surgical or traumatic procedures. coinfection with e. platys may potentiate the pathogenicity of other infectious agents, in particular e. canis (breitschwerdt, ) . etiology. lyme disease is caused by borrelia burgdorferi sensu lato, a microaerophilic spirochete that is primarily an extracellular pathogen. clinical signs. clinical signs may be highly variable; lameness due to polyarthritis has been reported as the most common sign. the onset of lameness may be acute or chronic, shift from limb to limb, and be accompanied by swelling and joint pain. synovial fluid analysis from affected joints is consistent with a diagnosis of suppurative arthritis. other clinical signs include fever, anorexia, lethargy, lymphadenopathy, and weight loss. over the course of the disease, signs may wax and wane over a period of weeks to months. dogs rarely develop erythema chronicum migrans (the characteristic rash seen in infected people) and do not exhibit the severe arthritis and neurologic sequelae seen in human beings (greene, ; manley, ) . hematologic and biochemical profiles are generally unremarkable. lyme disease is thought to be the most common arthropod-borne disease of human beings (and possibly of dogs) in the united states. it affects humans and dogs worldwide. the geographic distribution of canine borreliosis is assumed to follow that of the human disease and is related to the range of the arthropod vectors. three major endemic foci that have been identified in the united states account for % of reported human cases (appel and jacobson, ). the distribution of these cases is as follows: northeast/mid-atlantic focus, %; midwestern focus (michigan, wisconsin, minnesota, iowa, illinois, and missouri), %; and california and oregon, %. for the most part, dogs in the remainder of the country are not at risk for contracting lyme disease. borrelia burgdorferi is transmitted exclusively by ixodes ticks. other arthropod hosts may carry the organism but have not as yet been implicated in the transmission of disease. ixodes scapularis, a three-host tick with a to year life cycle, is the prototypical vector for north america. the spirochetes are spread by tick bites from both nymphs and adults. ticks become infected by feeding on an infected mammal and by transstadial transmission (transovarial passage is rare). in endemic areas, - % of adult ticks may be infected (appel and jacobson, ) . the primary reservoir for the organism is the whitefooted deer mouse, peromyscus ieucopus, which can carry spirochetes for its life span without becoming ill. evidence also indicates that the eastern chipmunk, tamias striatus, is an important reservoir (slajchert et al., ) , and birds may also be a significant reservoir. deer, however, serve only as hosts for the tick vectors and not as a reservoir for the spirochete. pathogenesis. the pathogenesis of lyme disease is poorly understood, primarily because of a lack of good animal models and the chronic nature of the disease. infection can be induced experimentally by the bite of a single infected tick. clinical signs develop - days postinfection. some evidence points to the host's inflammatory response to the organism as etiologic for disease (pershing et al, ; greene, ) . seroconversion in dogs occurs - weeks after infection with b. burgdorferi. antibody titers may remain extremely elevated for at least months. igm titers also remain elevated for several months and are indicative of neither acute nor active infection (appel and jacobson, ) . because antibiotic treatment may not eliminate the organism, persistent infections in dogs (treated for days with antibiotics) can be reactivated by steroid treatment up to days postinfection (straubinger et al., ) . diagnosis and differential diagnosis. appel and jacobson ( ) recommend that three of the following four criteria be met to establish a diagnosis of lyme disease in dogs: ( ) history of exposure to ixodes ticks in an endemic area, ( ) characteristic clinical signs, ( ) positive serology, and ( ) rapid resolution of clinical signs with antibiotic therapy. ifa or elisa tests for borrelia antibodies are the assays of choice. it should be re-membered, however, that a positive titer in an endemic area indicates exposure and not necessarily disease and that vaccinated dogs will also have a positive titer. responses to vaccine versus infection may be distinguished by western blot. culture or identification of the organism provides a definitive diagnosis but is very difficult to perform. differential diagnoses include immune-mediated polyarthritis and septic arthritis from other etiologic agents. prevention and control. prevention and control are the same as for the other tick-borne diseases (see discussion of monocytic ehrlichiosis, section iii,a,l,e above). a vaccine against b. burgdorferi is available but should not be necessary in a research setting. treatment. doxycycline is the drug of choice for treating lyme borelliosis. a typical dosing regimen is mg/kg q hr for - weeks. amoxicillin, tetracycline, and the quinolones are also effective. of significant note is that antibiotic treatment results in resolution of clinical signs but may not result in elimination of the organism. (fox and lee, ) . "helicobacter heilmannii" and h. bizzozeronii are thought be the same species, with the latter being the updated nomenclature. this species, as well as h. rappini and h. canis, is considered to be zoonotic (fox and lee, ) . clinical infections may present with vomiting, diarrhea, fever, and anorexia, pica, or polyphagia. epizootiology and transmission. the epizootiology and transmission of helicobacter spp. in the dog remains to be elucidated. the prevalence of canine helicobacter infections in colony or shelter situations has been reported to range from % to almost % (fox, ; hermanns et al., ) . both oral-oral and fecal-oral routes for transmission have been suggested. pathologic findings. no gross lesions are noted; the primary lesion is that of histologic gastritis. this is typically characterized by reduced mucus content of the surface epithelium; vacu-olation, swelling, karyolysis, and karyorrhexis of parietal cells; and multifocal infiltrates of plasma cells and neutrophils into the subepithelium, primarily around blood vessels and between the gastric pits (hermanns et al., ) . focal areas of lymphocytic inflammation and lymphoid follicles may also be seen. pathogenesis. some helicobacter spp. colonize the gastric epithelium exclusively and other species colonize lower parts of the gastrointestinal tract. helicobacter felis and "h. heilmannii" infections have been linked to gastric lesions in laboratoryraised beagles (fox and lee, ) . the mechanism by which these organisms cause disease may be related to the host's inflammatory response to colonization and the helicobacter's ability to produce urease. urease splits urea into ammonia and bicarbonate; ammonia is toxic for the epithelial cells, and bicarbonate may help the organism survive the acidic environment (marshall et al., ; shimoyama and crabtree, ). diagnosis and differential diagnosis. any of the numerous causes of acute or chronic vomiting and diarrhea in the dog (including canine distemper, viral or bacterial gastroenteritis, and ingested toxicants) should be considered as differential diagnoses. definitive diagnosis for dogs requires either endoscopic or surgical biopsy. confirmation of infection with helicobacter spp. requires demonstration of the organism in biopsy samples by histopathology, culture, or recognition by pcr. a positive urease test on a biopsy sample may give a presumptive diagnosis, but only for those species that produce urease. the use of warthin-starry silver stain may increase the sensitivity for histopathologic diagnosis. prevention and control. until more is known about the epizootiology and transmission of helicobacter spp. in the dog, specific recommendations cannot be made about prevention and control in this species. treatment. combination therapy has proven to be the most effective method for treating helicobacter spp. infections in dogs. combination therapy of amoxicillin ( mg/kg q hr), metronidazole ( mg/kg q hr), and sucralfate ( . - . mg/kg q hr) for days has been suggested for dogs (hall and simpson, ) . replacing the sucralfate with famotidine ( . mg/kg q hr), omeprazole ( . mg/kg q hr), or bismuth subsalicylate ( . ml/kg q - hr) may also be effective (marks, ; jenkins and bassett, ; denovo and magne, ) . the benefits of antimicrobial therapy in dogs still need to be established by controlled therapeutic studies. research complications. helicobacter spp. infections could result in altered gastrointestinal responses to drugs and toxic or carcinogenic compounds. therefore, dogs used in gastric physiology or oral pharmacology studies should be free from helicobacteriosis. clinical signs. clinical signs of canine parvovirus usually appear days after inoculation by the fecal-oral route and are characterized by anorexia, fever, depression, and vomiting. profuse, intractable diarrhea ensues, which may become hemorrhagic. approximately % of affected dogs develop severe leukopenia, with a total granulocyte/lymphocyte count ranging from - wbc/~d or less. repeated hemograms may provide prognostic value, because rebounds in leukocyte counts are indicative of impending recovery. terminally ill dogs may develop hypothermia, icterus, or disseminated intravascular coagulation due to endotoxemia. parvovirus can infect dogs of any age, but puppies between and weeks of age appear to be particularly susceptible. puppies less than weeks of age are generally protected from infection by passive maternal antibody. adult dogs probably incur mild or inapparent infections that result in seroconversion. pathogenesis. canine parvovirus has an affinity for rapidly dividing cells of the intestine and causes an acute, highly contagious enteritis with intestinal crypt necrosis and villus atrophy. the virus also has tropism for the bone marrow and lymphoid tissues; thus leukopenia and lymphoid depletion accompany the intestinal destruction. diagnosis and differential diagnosis. parvovirus can be detected in fecal samples with a commercially available elisa from cite. at necropsy, diagnosis is based on gross and histopathologic evidence of necrosis and dilatation of intestinal crypt cells with secondary villous collapse. other lesions include myeloid degeneration and widespread lymphoid depletion. parvovirus can also be demonstrated in frozen sections by fluorescent antibody techniques. differential diagnoses should include other viral enteritides, salmonellosis, and small intestinal obstruction. prevention and control. prevention of transmission begins with isolation of affected animals and quarantine for week after full recovery. disinfection of potentially infected kennel and diagnostic areas with diluted bleach ( : ) or commercially prepared disinfectant (such as kennesol, available from alphatech, lexington, massachusetts) is essential for elimination of the virus. six-week-old puppies should be vaccinated every - weeks with a commercially available modified live vaccine until - weeks of age. young rottweilers and doberman pinschers appear to be predisposed to parvoviral enteritis and should be vaccinated every weeks ( times) from - weeks of age. treatment. treatment is largely supportive and is aimed primarily at restoring fluid and electrolyte balance. research complications. infection with parvovirus obviously precludes the use of a particular dog in an experimental protocol. given the potential for significant discomfort of the affected animal, and the cost of therapy, humane euthanasia is usually the option chosen in a research setting. canine coronavirus infection is usually inapparent or causes minimal illness. this epitheliotropic virus preferentially invades the enterocytes of the villous tips, resulting in destruction, atrophy, and fusion and subsequent diarrhea of varying severity. subclinical infections are most common, but abrupt gastrointestinal upset accompanied by soft to watery, yelloworange feces is possible. definitive diagnosis by virus isolation or paired sera is usually not made, because supportive therapy generally results in rapid resolution of the diarrhea. inactivated coronavirus is present in commercially available combination vaccines, which are administered immunoprophylactically at - , - , and - weeks of age and then annually thereafter. the role of these vaccines in protection from coronaviral infection is unknown, because the virus typically causes inapparent or mild illness (hoskins, ) . etiology. canine distemper virus (cdv) belongs to the family paramyxoviridae, within the genus morbillivirus, which includes human measles virus and rinderpest virus of ruminants. although there is only one serotype of cdv, there is a wide difference in strain virulence and tissue tropism. some strains produce mild clinical signs that are similar to tracheobronchitis, whereas other strains cause generalized infections of the gastrointestinal tract, integument, and central nervous system, resulting in enteritis, digital hyperkeratosis, and encephalitis, respectively. other factors contributing to the severity and progression of clinical signs include environmental conditions, immune status, and age of the host. a transient subclinical fever and leukopenia occur - days after exposure, with a subsequent fever spike - days later, accompanied by conjunctivitis and rhinitis. other clinical signs associated with acute distemper include coughing, diarrhea, vomiting, anorexia, dehydration, and weight loss. secondary bacterial infections can cause progression to mucopurulent oculonasal discharge and pneumonia. an immune-mediated pustular dermatitis may develop on the abdomen; this is usually a favorable prognostic sign (greene and appel, ) , because dogs that develop skin lesions often recover. neurologic complications of distemper infection may occur weeks to months after recovery from an acute infection. dogs that develop late-onset disease are usually immunocompetent hosts, suggesting that the virus may have escaped complete elimination by the immune system, possibly because of protective effects by the blood-brain barrier. classic neurologic signs that may occur in acute or chronic cdv infection include ataxia, incoordination, vocalization, "chewing gum" seizures, and myoclonus with or without paresis of the affected limb. canine distemper is the most common cause of seizures in dogs less than months of age. dogs with extensive neurologic involvement often have residual clinical deficits, including flexor spasm and olfactory dysfunction. cdv has also been associated with two forms of chronic encephalitis in mature dogs: multifocal encephalitis and "old dog encephalitis." epizootiology and transmission. the virus is highly prevalent and contagious to dogs and other carnivores, especially at the age of - months, coincident with the waning of maternal antibody. transmission is primarily by aerosolization of infective droplets from body secretions of infected animals. pathologic findings. the predominant histopathologic lesion in neurologic forms of distemper is demyelination, which may .. be accompanied by gliosis, necrosis, edema, and macrophage infiltration. acidophilic cytoplasmic inclusions can be found in epithelial cells of mucous membranes, reticulum cells, leukocytes, glia, and neurons, while intranuclear inclusions are often present in lining or glandular epithelium and ganglion cells. diagnosis and differential diagnosis. diagnosis of cdv is based on history of exposure and clinical signs. young dogs who have not received routine immunoprophylaxis (or similarly, mature dogs with a questionable vaccination history) and present with rhinitis, mucopurulent oculonasal discharge, plus or minus hyperkeratosis of the footpads and neurologic signs, are highly likely to have cdv. ophthalmologic examination may reveal chorioretinitis with acute disease or retinal atrophy in chronic cases. definitive diagnosis of acute infection can be made by fluorescent antibody testing of intact epithelial cells from conjunctival and mucous membranes. attenuated strains of cdv, found in modified live vaccines, are not disseminated from lymphoid tissue to epithelial cells and thus are not detected by the fluorescent antibody. serologic testing is usually not useful, because dogs frequently fail to mount a measurable immunologic response. because of the variety of clinical signs, there are many differential diagnoses for canine distemper. an important differential diagnosis for respiratory illness is infectious tracheobronchitis (kennel cough). bacterial, viral, and protozoal causes of gastroenteritis must be considered for cases presenting with vomiting and diarrhea, and rabies, pseudorabies, bacterial meningitis, and poisonings are differential diagnoses for dogs with central nervous system disorder. prevention and treatment. a series of three immunizations from to weeks of age, followed by yearly boosters, is a recommended preventative. treatment is largely supportive, but because of the profound immunologic effects and significant morbidity of cdv, humane euthanasia is usually undertaken in the research setting. etiology. canine herpesvirus (chv) infection causes a generalized hemorrhagic disease with a high mortality rate in newborn puppies less than weeks of age. in adult dogs, chv causes a persistent, latent infection of the reproductive tract with recrudescence and shedding during periods of physiologic stress. clinical signs. clinically affected puppies do not suckle, cry persistently, become depressed and weak, and fail to thrive. petechial hemorrhages of the mucous membranes and erythema of sparsely haired regions such as the caudal abdomen and inguinal area are evident. older puppies, aged - weeks, develop less severe clinical signs and are likely to survive with neurologic sequelae such as ataxia and blindness resulting from reactivation of latent infection. infection in adult dogs may result in stillbirths, abortions, and infertility. lesions in adult bitches include raised vesicular foci in the vaginal mucosa, accompanied by mild vaginitis. adult males have preputial discharge due to vesicular lesions at the base of the penis and on the preputial mucosa. passage of puppies through the birth canal or venereally in adult dogs. puppies can also be horizontally infected by littermates. entire primiparous litters may be lost, with subsequent litters protected by colostral antibody. pathologic findings. pathologic findings include multifocal ecchymotic hemorrhages of the kidneys, liver, lungs, and gastrointestinal tract. basophilic intranuclear inclusions in necrotic areas of parenchymal organs are characteristic findings. diagnosis and differential diagnosis. diagnosis of canine herpesvirus infection in adult dogs is based on a history of reproductive infertility and the presence of genital vesicular lesions. differential diagnoses for stillbirths, abortions, and infertility include canine brucellosis, canine distemper virus and parvovirus infections, and pyometra. the diagnosis in infected puppies is usually made based on clinical history and characteristic lesions (multifocal systemic hemorrhages) (carmichael and greene, ) . differential diagnoses for the disease in neonates would include canine ehrlichiosis and causes of disseminated intravascular coagulation, including bacterial endotoxemia. there is no effective curative treatment. supportive therapy is unrewarding, and death usually ensues within hours in in-fected neonates. in general, adult bitches that have multiple abortions, stillbirths, or persistent infertility should be culled from the breeding colony. examination of these animals may reveal raised vesicular lesions on the vaginal mucosa. adult male dogs that have vesicular lesions on the base of the penis and preputial mucosa should be similarly culled. adult dogs would obviously interfere with production operations, and affected animals should be culled based on the criteria noted above in the discussion of prevention and treatment. because of the severity of clinical illness in puppies, such animals should be humanely euthanatized. etiology. rabies virus is a member of the rhabdovirus family and is essentially contagious to all species of warm-blooded animals. clinical signs. clinical progression of neurologic disease occurs in three stages. the first, or prodromal, stage is characterized by a change in species-typical behavior. the loss of the instinctive fear of humans by a wild animal is a classic sign of impending rabies. in the second, or furious, stage animals are easily excited or hyperreactive to external stimuli and will readily snap at inanimate objects. the third, or paralytic, stage is characterized by incoordination and ascending ataxia of the hindlimbs due to viral-induced damage of motor neurons. death usually occurs within - days of the onset of clinical signs, due to respiratory failure. epizootiology and transmission. wild animals such as raccoons, skunks, and bats are common reservoirs of infection for domestic animals, which in turn are the principal source of infection for humans. transmission occurs primarily by contact of infected saliva from a rabid to a naive animal (or human), usually via bite wounds. pathogenesis. the incubation period for rabies is generally - weeks from the time of exposure to the onset of clinical signs but can range from week to year. bites of the head and neck typically result in shorter incubation periods because of the proximity to the brain. following infection, the virus migrates centripetally via peripheral nerve fibers to the central nervous system and eventually to neurons within the brain, resuiting in neurologic dysfunction. on reaching the brain, the virus migrates centrifugally to the salivary glands, thus enabling shedding and subsequent transmission. diagnosis and differential diagnosis. diagnosis of rabies is based on clinical signs; differential diagnoses include pseudorabies, canine distemper, bacterial meningitis, and toxicants that affect neurologic function. definitive diagnosis is based on fluorescent antibody demonstration of the virus in negri bodies of hippocampal cells. prevention and treatment. puppies should be vaccinated at - months of age, "boostered" in year, then vaccinated annually or triennially, depending on state and local laws and which vaccine product is used. treatment of rabies is not recommended, because of the risk of human exposure. research complications. in a research setting, dogs are often not vaccinated for rabies, because of the low incidence of exposure to wild-animal reservoirs. a healthy, purpose-bred dog that bites a human in a research facility should be quarantined for days and observed for signs of rabies. this quarantine interval is based on the knowledge that dogs do not shed rabies in the saliva for more than a few days before the onset of neurologic disease. a random-source dog with an unknown vaccination history that bites a human should be immediately euthanized. the brain should be examined for rabies virus to determine if the dog was infected, and if the test is positive, postexposure immunization should be initiated for the human patient. a rabies vaccine licensed for use in humans is available, and immunoprophylaxis is recommended for animal care and research personnel who may have high work-related risks of exposure. a. protozoa i. giardiasis etiology. giardiasis is a small-intestinal disease of the dog caused by giardia duodenalis (lamblia), a binucleate flagellate protozoan. clinical signs. most giardia infections are subclinical. when dogs are clinically affected, diarrhea is the most prominent sign. the diarrhea is a result of intestinal malabsorption and is often characterized as voluminous, light-colored, foul-smelling, and soft to watery. weight loss has also been associated with clinical infection. clinical illness is more often seen in young animals. epizootiology and transmission. giardia has a direct life cycle. dogs (and people) typically become infected when they consume water (or food) contaminated with giardia cysts. the ph change from the stomach (acid) to duodenum (neutral) causes excystation. trophozoites migrate to the distal duodenum and proximal jejunum and attach to the villus surface. eventually the trophozoites encyst and pass in the feces to perpetuate the life cycle. pathologic findings. giardiasis is rarely fatal. on histopathology of duodenal or jejunal specimens, giardia trophozoites can be seen attached to enterocytes. mucosal inflammation and ulceration, and villous atrophy, have been observed. pathogenesis. the exact pathogenesis of giardia-induced illness is unknown. it is thought that tissue invasion, although occasionally observed, is unimportant for pathogenesis. it is suspected that illness is caused by physical obstruction of enteric absorption, enterotoxicity, competition for nutrients, excess mucus production, and/or secondary bacterial overgrowth. diagnosis and differential diagnosis. definitive diagnosis requires observation of the organism in fecal or intestinal samples. direct fecal smears are considered best for observing trophozoites, and zinc sulfate flotation is preferred for detection of cysts. commercial elisa kits and direct immunofluorescent tests are available to detect fecal giardia antigens, but the diagnostic specificity and/or sensitivity of these tests may not be sufficient to warrant substitution for the less expensive direct fecal examination or zinc sulfate preparation (barr, ) . differential diagnoses for giardiasis include bacterial and protozoal enteritis, coccidiosis, and whipworm infestation. prevention. high-quality water sources will eliminate the possibility of infection developing within an animal research facility. use of dogs with a known husbandry and medical background will minimize the chances of giardiasis developing in a research colony. control. once giardiasis has been diagnosed in a canine population, segregation of infected animals will help to reduce further infection (provided other dogs were not preinfected at the same source location as the signal case). disinfection with quaternary ammonium compounds, bleach, or steam is usually successful in eradication of giardia cysts. treatment. the most common treatment for giardiasis is metronidazole (flagyl) at - mg/kg per os twice per day for - days. quinacrine hydrochloride (atabrine) at mg/kg per os once per day for days, furazolidone (furoxone) at mg/kg per os twice per day for - days, and the anthelmintics albendazole and fenbendazole have been proposed for use against metronidazole-resistant strains of giardia. a bendazole is recommended at mg/kg per os q hr for days, and fenbendazole at mg/kg per os q hr for days. fenbendazole was thought to be safer for both puppies and pregnant females (nonteratogenic) (barr, ) . research complications. typical asymptomatic infections probably have no consequence on research protocols, with the exception of intestinal physiology or immunology studies. clinical diarrhea would clearly need to be treated before a dog could be used as a research subject. ii. coccidiosis etiology. intestinal coccidia that have been associated with enteropathy in dogs include cystoisospora canis, c. ohioensis, c. burrowsi, and c. neorivolta. clinical signs. dogs are typically asymptomatic when infected with intestinal coccidia, and oocysts are an incidental finding on fecal flotation or direct smear. dogs that are clinically infected usually develop diarrhea, which can vary from soft to watery and may contain blood or mucus. vomiting, dehydration, lethargy, and weight loss can also be seen. epizootiology and transmission. cystoisospora oocysts are typically spread by fecal-oral transmission, usually by ingestion of fecal-contaminated food or other objects in the environment. an indirect form of transmission is also possible, whereby the dog consumes a rodent or other animal that is serving as a transport host. once inside the small intestine, the cyst releases sporozoites that infect enteric epithelium. several generations of asexual reproduction can occur in the enterocyte before sexual reproduction produces gamonts. the gamonts fuse to become a zygote, which encysts, ruptures the enterocyte, and passes in the feces. once in the environment the cyst sporulates and is now an infective stage for ingestion by another host. pathologic findings. dogs with coccidiosis may have hyperemia or fluid retention at affected intestinal segments. the mucosa may appear normal, raised, or ulcerated. histologically, there may be necrosis of enterocytes, hyperemia, and submucosal inflammation. the oocysts are usually readily apparent within the epithelial cells (van kruiningen, ) . pathogenesis. intestinal coccidia are opportunistic organisms; they do not typically cause illness unless other predisposing factors are present. such factors include immunodeficiency, malnutrition, and/or concurrent disease. overcrowding and unsanitary conditions can also promote clinical coccidiosis by providing a high population of infective oocysts to stressed animals. diagnosis and differential diagnosis. diagnosis is somewhat difficult, as coccidian oocysts (of both cystoisospora and non-cystoisospora spp.) can be seen on fecal examinations of clinically healthy dogs, as well as animals with diarrhea. other causes for diarrhea (e.g., parvovirus, roundworms, giardia spp., campylobacter jejuni, and inflammatory bowel disease) should be excluded before a coccidial etiology is implicated. prevention. clinical coccidiosis can be readily prevented by adhering to proper sanitation guidelines, reducing any over-crowding, and providing as stress-free an environment as possible. treatment. treatment for the presence of coccidial oocysts may often not be necessary, because cystoisospora infections are typically self-limiting and clinically insignificant. treatment may, however, help to limit the number of oocysts shed in a kennel housing situation and may be necessary in cases of protracted clinical illness. possible choices for treatment include daily administration of sulfadimethoxine ( - mg/lb per os for days), trimethoprim sulfa ( mg/lb per os for days), or quinacrine ( mg/lb per os for days). amprolium, which is not labeled for dogs, can also be used as a coccidiostat. it can be given in gelatin capsules for - days at a daily dose of mg for small-breed pups and mg for larger breeds. research complications. as with any enteric disease, the presence of clinical coccidiosis can cause aberrations in gastrointestinal physiological parameters. dogs used in intestinal pharmacokinetic studies should be confirmed to be free of cystoisospora infections. b. nematodes i. ascarids etiology. the most common ascarid of dogs is toxocara canis. toxascaris leonina can also infect both dogs and cats. clinical signs. ascarid infestations are most commonly subclinical. however, large worm burdens can cause diarrhea, vomiting, dehydration, and abdominal discomfort with vocalization. puppies may have a classical "potbellied" appearance and dull hair coat. heavy infestations can cause intussusception and/or intestinal obstruction, in which case the young dogs may be found dead. visceral larval migrans caused by toxocara canis can cause pneumonia. epizootiology and transmission. toxocara canis typically infects puppies. in fact, a unique characteristic of t. canis is its ability to infect prenatal puppies by transplacental migration, and neonatal puppies by transmammary migration. ingestion of infective eggs that have been shed in the feces is another common route of transmission, and infection by ingestion of a transport or intermediate host is also possible. pathologic findings. puppies that die from ascarid infestations typically have large worm populations in the lumen of the small intestine. such populations can cause intestinal obstruction and may also result in intussusception or intestinal perforation. puppies that experience lung migrations of large larval worm populations can have severe pulmonary parenchymal damage and develop fatal pneumonia. pathogenesis. the infective stage of t. canis is the third-stage larva (l ). infections initiated by ingestion of infective eggs have three possibilities for larval migration: liver-lung migration (which leads to intestinal infection), somatic tissue migration, and intestinal wall migration. older dogs that become infected typically have an age-related resistance to liver-lung migration and instead experience the other two migratory patterns. these larval migrations are often asymptomatic, and progression of the l larvae is arrested in the tissues. it is these larvae that become reactivated in a pregnant bitch, thus establishing the transplacental and transmammary routes of transmission. if the source of infection is transplacental, puppies may be born with l larvae in their lungs, because larval migration is already in progress (sherding, ). diagnosis and differential diagnosis. the characteristic large ( - ~tm in diameter) and relatively round ascarid eggs can be readily diagnosed by standard fecal flotation methods. prevention and control. monthly administration of milbemycin or ivermectin plus pyrantel pamoate (heartgard plus) is recommended for prevention and control of canine ascarid infestation (hall and simpson, ) . treatment. most anthelmintics are effective for treatment of ascariasis. pyrantel pamoate (nemex) and fenbendazole (panacur) are commonly used. treatment should be started early in puppies ( , , , and weeks) because of the possibility of prenatal or neonatal infection. pyrantel pamoate, dosed at mg/kg per os, is safe for puppies and is also effective in treatment of hookworms (see section iii,a, ,b,ii). in breeding colonies in which ascarid infestation is a known problem, treatment of the pregnant and nursing bitch may be advantageous. extended fenbendazole therapy ( mg/kg per os twice per day for days or once per day from day of gestation through day of lactation) has been shown to be experimentally safe and effective in decreasing ascarid burdens in puppies. research complications. puppies with large worm burdens make poor research subjects and should be treated aggressively before placement on an experimental study. ii. hookworms etiology. the most common and most pathogenic hookworm of dogs is ancylostoma caninum. other, less pathogenic canine hookworms found in north america are a. braziliense, which can be found in the american tropics and southern united states, and uncinaria stenocephala, which is distributed in the northern united states and canada. clinical signs. only a. caninum infestation typically results in clinical illness, because of the amount of blood that it con-sumes. puppies with a. caninum infestations are typically pale and weak (from anemia), with bloody diarrhea or melena. other clinical signs include lethargy, anorexia, dehydration, vomiting, and poor weight gain. epizootiology and transmission. infective larvae (l ) are typically ingested by puppies and develop directly in the intestinal tract. ingestion can be from the bitch's milk (transmammary migration occurs with a. caninum), from food or objects contaminated with infective larvae, or from ingestion of a paratenic host. transplacental migration does occur with a. caninum, but to a much lesser extent than is seen with toxocara canis. larvae can also penetrate intact skin, migrate to the lung via somatic or circulatory routes, and be coughed and swallowed to reach the intestine. the prepatent period is weeks. pathologic findings. infected puppies often have severe anemia and eosinophilia. the anemia can be from acute blood loss or can also be an iron-deficiency anemia caused by chronic blood loss coupled with limited iron reserves. on gross necropsy, the small-intestinal tract contains worms admixed with intestinal contents containing fresh or digested blood (fig. a) . ulcerative enteritis caused by hookworm attachment is evident on histopathologic examination, and worms with mouthparts embedded in the mucosa can be identified in some sections (fig. b) . pathogenesis. the severe pathogenicity of a. caninum is a direct result of its voracious consumption of blood and body fluids. each adult hookworm can consume . - . ml of blood; thus an extensive infection could deplete a puppy of ml of blood per day, which is approximately % of the blood volume of a . kg animal. in contrast, a. braziliense and u. stenocephala consume . and . ml per worm, respectively. diagnosis and differential diagnosis. diagnosis of ancylostomiasis is made by identification of eggs or larvae from fecal samples by either flotation or direct smear. parvovirus should be considered for puppies with bloody diarrhea, and autoimmune hemolytic anemia should be considered in the diagnosis of a young dog with anemia. prevention and control. purchase of purpose-bred animals will limit the exposure to hookworm larvae, and effective sanitation programs will easily eradicate the infective larvae. unlike ascarid eggs, hookworm eggs are readily killed by drying, sunlight, or cold; however, they do survive readily in warm, moist environments. monthly administration of milbemycin or ivermectin plus pyrantel pamoate (heartgard plus) is recommended for prevention and control of canine ascarid infestation (hall and simpson, ) . treatment. pyrantel pamoate (nemex) is the anthelmintic of choice because it is safest in young ill animals and is also effective against ascarids and other enteric helminths. because of the possibility of transplacental or milk-borne infection, puppies should be treated every weeks from weeks - . a follow-up treatment at weeks is recommended to kill any larvae that have migrated and matured since the initial therapy. severely ill puppies may require supportive fluid therapy and possibly whole blood transfusions and iron supplementation. research complications. anemic puppies with large worm burdens make poor research subjects and should be treated aggressively before placement on an experimental study. iii. strongyloides etiology. strongyloides stercoralis is a small strongyle that can cause hemorrhagic enteritis in puppies. it is found in warm, humid climates such as the southeastern united states. fects dogs and other animals by third-stage larval penetration of the skin or mucous membranes. larvae migrate via the circulatory system to the lung and then are coughed and swallowed to initiate the intestinal parasitism. the eggs of s. stercoralis hatch within the gut lumen, and so it is the first-stage larvae that pass in the feces and need to be identified by diagnostic examination. once passed, the larvae can either develop into the infectious third-stage larvae or mature into free-living, nonparasitic adults. diagnosis and differential diagnosis. the baermann procedure is usually performed on fresh feces in order to detect the motile first-stage larva ( - ~tm x - ~tm). the larvae must be distinguished from larva of filaroides hirthi and hatched ancylostoma caninum. treatment. the usual treatment for s. stercoralis is fenbendazole (panacur) at mg/kg per day for days. iv. whipworms etiology. trichuris vulpis, the canine whipworm, can cause acute or chronic large-intestinal diarrhea. the adult whipworm typically resides in the cecum or ascending colon. clinical signs. most whipworm infections are subclinical. in symptomatic cases, the typical clinical sign is diarrhea with blood and/or mucus. abdominal pain, anorexia, and weight loss are also seen. dogs may have eosinophilia, anemia, and/or hypoproteinemia on clinical hematology. severe dehydration with electrolyte imbalance has occurred occasionally as an acute crisis episode. life cycle. adult worms residing in the canine large intestine intermittently release eggs that pass in the feces. the eggs are very hardy and can persist for years. in optimal conditions, the eggs develop into an infective embryo within days. after ingestion by a dog, the larvae hatch in the small intestine, burrow into the small-intestinal mucosa, and then reemerge several days later to travel and burrow into the cecal and colonic mucosa. the prepatent period is typically - months long. pathologic findings. dogs do not typically die from whipworm infestations. lesions seen as incidental findings feature adult worms embedded into the colonic and cecal mucosae, causing local granulomatous inflammatory reactions and mucosal hyperplasia. pathogenesis. the penetration of the adult worm into the enteric mucosa, and the associated inflammation, can lead to the clinical development of diarrhea. factors that influence the possible.development of clinical symptoms are the number and location of adult whipworms; the severity of inflammation, anemia, or hypoproteinemia in the host; and the overall condition of the host. diagnosis and differential diagnosis. whipworm infestation is diagnosed by the presence of characteristic trichurid eggs on fecal flotation. these eggs are barrel-shaped, with thick walls and bipolar plugs. because of the intermittent release of eggs by the adult female worms, negative fecal flotation does not exclude the possibility of clinical whipworm infection. adult worms can be seen on colonoscopy (jergens and willard, ) . differential diagnoses for whipworm infestation include giardiasis, coccidiosis, and bacterial enteritis. prevention and control. trichuris eggs are resistant to disinfection, making control difficult. dessication or incineration is the only completely effective means to eradicate whipworm eggs from the environment. treatment. fenbendazole, oxibendazole, and milbemycin have all been recommended for treatment of whipworms. treatment for whipworm infestation should be at monthly intervals for months (jergens and willard, ) . treatment is also suggested in cases wherein whipworm infestation is suspected but not confirmed by multiple fecal flotation. rapid response to treatment would be indicative of a correct diagnosis; lack of response should prompt further diagnostic efforts. research complications. whipworm infestation has not been documented to interfere with research protocols, although one would anticpate that aberrations in local enteric immune function and absorptive functions of the large intestine could result from trichuriasis. etiology. heartworm disease of dogs is caused by the filarial worm, dirofilaria immitis. adult heartworms reside in the pulmonary artery; severe infestations can result in the presence of worms in the right ventricle and atrium. microfilariae, the immature worms produced by the adults, circulate in the bloodstream until a mosquito (intermediate host) ingests them. clinical signs. most heartworm infestations are asymptomatic. the most common clinical signs observed are coughing and dyspnea. clinical signs of exercise intolerance and rightsided heart failure can be seen in severe infestations. epizootiology and transmission. successful heartworm transmission requires the presence of mosquitoes. for this reason, random-source dogs or dogs housed in outdoor kennels are much more likely to have heartworm infestations than indoor, purpose-bred dogs. mosquitoes become infested with heartworm microfilariae when they take a blood meal from the dog. the microfilaria progress through several larval stages within the mosquito, eventually terminating at the third stage. this stage is then returned to the canine bloodstream during feeding. this stage matures within the dog's circulatory system, and the adults reside in the pulmonary artery. male and female heartworms will then sexually reproduce to create more microfilariae and propagate the parasitic life cycle. in the united states, transmission of heartworm by mosquitoes occurs over a month or shorter period, except for the southeastern and gulf coast states. here, climatic conditions enable longer survival of the mosquitoes (possibly year-round), thus resulting in the highest prevalence of heartworm infestation (knight, ) . pathologic findings. on necropsy, the small, slender worms can be seen in the pulmonary artery, right ventricle, and/or right atrium (fig. a ). there may be no histologic abnormalities associated with a minor worm burden, although typically the arterial endothelium in these areas is hyperplastic (fig. b) . endothelial cell hyperplasia, vascular smooth muscle hyperplasia, inflammation, and thrombosis of the pulmonary arteries and arterioles characterize more significant infestations. severe infestations can lead to right-sided heart failure and its pathologic sequelae of ascites, pleural effusion, hepatomegaly, and right heart and pulmonary artery enlargement. verminous pulmonary embolism can result from treatment of dogs with anthelmintics when a worm burden is present. immune responses to circulating microfilariae can cause pathologic lesions, most commonly glomerulonephritis. pathogenesis. the physical presence of the worms in the pulmonary artery is partially responsible for clinical signs observed in severe cases. however, the host immunologic response to this infestation, coupled with secretion by the heart-worms of physiomodulative factors, contributes significantly to the complications seen with this disease. endothelial cell proliferation, damage, and sloughing stimulates periarteritis and proliferation of the vascular media of pulmonary arteries and arterioles. these changes lead to thrombosis of these vessels and the arterial truncation that can be seen radiographically in severe infestations. the heartworms also release circulating factors that affect vascular tone and can promote bronchoconstriction (dillon, ) . these factors are discussed in more detail below, under "research complications." diagnosis and differential diagnosis. for dogs used in biomedical research, diagnosis of asymptomatic heartworm disease is important, especially if the dogs are used in cardiovascular, pulmonary, or long-term studies. a diagnosis of dirofilariasis is typically made by detection of adult heartworm antigens in a blood sample. use of adult heartworm antigen tests has virtually eliminated the historical status of "occult" heartworm disease, which was caused by infestation of adult worms without corresponding microfilarial circulation. commercial test kits that assay for the presence of adult heartworm antigens, and designed for use by veterinary practitioners, are readily available. false-negative results can occur during the prepatent period after initial infection (first - months), and when the adult worm burden is light or predominantly male. infections consisting of more than three mature female worms are usually detected by antigenic serology (knight, ) . a significant feature of these tests for circulating antigen is that they have a very high specificity (low rate of false-positive resuits). if a dog were negative on initial testing because of prepatency or small worm burden, it will more than likely be detected on a follow-up test months later. examination for circulating microfilariae could be used to confirm an antigenic diagnosis of dirofilariasis or to establish that microfilarial production had occurred. microfilarial detection can be done by microscopic examination of the buffy coat of a microhematocrit tube or by concentration techniques, such as the modified knott test and filter tests. tests that examine for microfilariae have the inherent problem of false positives caused by microfilariae of dipetalonema reconditum, a nonpathogenic filarial worm. other serologic diagnostic tests that were more common historically, and that may still be useful, include detection of antibodies to either adult heartworm antigens or microfilarial antigens. these same techniques can be used to diagnose clinical heartworm disease. additional diagnostic tests that can augment a diagnosis of clinical heartworm disease include thoracic radiography (pulmonary artery and right-heart enlargement), electrocardiography (right-heart enlargement), and hematology (eosinophilia). differential diagnoses for symptomatic heartworm disease (coughing, dyspnea, and exercise intolerance) include canine distemper, canine infectious tracheobronchitis (complicated), streptococcal or other bacterial pneumonia, nocardiosis, and congestive heart failure. prevention and control. for dogs used in biomedical research, prevention is primarily via insect control and housing of the dogs in a controlled, indoor environment. purpose-bred dogs reared in such an environment are usually free from dirofilariasis. however, any dog (random-source or purposebred) exposed to mosquitoes could become inoculated with infective larvae and, if untreated, could develop adult heartworm disease. there are many commercial anthelmintic preparations used to prevent heartworm infestation by killing the larval stages in the canine bloodstream before they become adult worms (e.g., ivermectin, milbemycin, and diethylcarbamazine). these could be used in a research setting in which heartwormnegative dogs are housed outdoors and thus could potentially be infected through mosquito bites. if a research facility is conditioning random-source dogs for long-term use, the presence of circulating adult heartworm antigen should disqualify an animal from the conditioning program. treatment. treatment for eradication of heartworms (adults, juveniles, and microfilaria) is a long process that can pose a significant risk to the patient with regard to both drug side effects (hoskins, ) and immunologic reactions to dead worms lodged in the pulmonary vasculature. for this reason, medical treatment of heartworm disease is not usually attempted in research dogs. in a rare instance when such treatment was in the best interest of a long-term canine experiment, thiacetarsamide (caparsolate) and ivermectin (ivomec) were used to eradicate adults and microfilariae, respectively (authors' personal experience). alternative choices include melarsomine (immiticide) as an adulticide and milbemycin (interceptor), levamisole (levasol), or fenthion (spotton) as microfilaricidal agents. dosing regimens for these agents are detailed in dillon ( ) . research complications. the physiomodulative properties of heartworm infection have been studied. such studies have looked at factors released by adult heartworms, as well as changes in the function of host tissues in response to the worm presence. probably the most consistent finding is that endothelial cell-dependent relaxation of pulmonary arterial smooth muscle is depressed in heartworm-infected dogs as compared with control dogs, indicative of alterations in local endothelial cell behavior (maksimowich et al., ; matsukura et al., ; mupanomunda et al., ) . the extension of this effect on peripheral arteries (in vivo and in vitro) has been supported in some studies (kaiser et al., ) but refuted in others (tithof et al., ) . it is thought that the endothelium is perturbed by a factor released from the adult dirofilaria, possibly a cyclooxygenase product such as prostaglandin d (kaiser et al., (kaiser et al., , . these products have also been demonstrated to cause constriction in in vitro rat tracheal ring preparations (collins et al., ) , suggesting that bronchoconstriction could be an aspect of the pathogenesis of the infestation. platelet reactivity was also been found to be enhanced in dogs naturally infected with dirofilaria, when compared with uninfected controis (boudreaux and dillon, ) . based on these data, dogs that are positive for adult heartworm antigen should be considered inappropriate for use as research subjects and, if used, should be restricted to nonsurvival preparations that do not require physiological measurements. etiology. several species of cestodes (tapeworms) parasitize the small intestine of dogs. the most common is dipylidium caninum. other species include taenia pisiformis and, more rarely, echinococcus granulosus, multiceps spp., mesocestoides spp., and spirometra spp. clinical signs. most cestode infestations are subclinical. severe infestations with dipylidium can be associated with diarrhea, weight loss, and poor growth. epizootiology and transmission. the cestode life cycle requires an intermediate host. for dipylidium caninum, the intermediate hosts are fleas and lice. thus this species of tapeworm can be readily transmitted by ingestion of arthropods that are canine parasites in and of themselves. taenia pisiformis requires small ruminants, rabbits, or rodents for intermediate hosts, so spread is less likely, especially in a research setting. echinococcus granulosus uses not only sheep as an intermediate host but also human beings, and thus the zoonotic potential of this cestode must be considered. pathologic findings. adult cestodes in the small intestine are usually an incidental finding at necropsy. diagnosis and differential diagnosis. definitive diagnosis is usually made by the identification of egg capsules or proglottids (tapeworm segments) on the surface of the feces or around the anus. dipylidium egg packets are large ( x bm) and contain - eggs per packet (hall and simpson, ) . prevention and control. the most significant means to limit cestode infestation is to control the population of fleas and/or lice infesting the colony. see the sections on these ectoparasites for effective means to treat infested dogs and kennels. treatment. praziquantel at - . mg/kg orally or subcutaneously is the standard treatment for cestodiasis, especially taenia or echinococcus species. fenbendazole, mebendazole, or oxfendazole may also be effective against dipylidium caninum (hall and simpson, ) . clinical signs. most lung fluke infestations are inapparent, but coughing can develop in cases that prompt a strong inflammatory response. pneumothorax has been a sequela of cyst rupture, in which case dyspnea with reduced lung sounds would be the typical presentation. epizootiology and transmission. the lung fluke life cycle requires two intermediate hosts: a snail and then a crayfish. dogs become infested after eating crayfish, which essentially limits this disease to random-source dogs. on ingestion, the immature flukes (metacercariae) migrate to the lungs and encyst in the pulmonary parenchyma. eggs produced by adult flukes are passed into the bronchioles, coughed up, swallowed, and passed in the feces to complete the life cycle. pathologic findings. grossly, the trematode cysts containing adult flukes can be seen in the lung parenchyma. areas of eosinophilic inflammation surround the cysts, and eosinophilic granulomas can also be seen encircling released eggs. pleural hemorrhages may also be caused by the migrating metacercariae (lopez, ) . pathogenesis. clinical illness is usually a result of a severe eosinophilic inflammatory response, pneumothorax caused by cyst rupture, or secondary bacterial pneumonia. diagnosis and differential diagnosis. definitive diagnosis of paragonimus infestation requires identification of the characteristic ovoid eggs ( - ~tm long) with a single operculum in either the feces or a transtracheal wash. identification from fecal samples requires sedimentation techniques. other causes of coughing in dogs (e.g., infectious tracheobronchitis, dirofilariasis, congestive heart failure) need to be considered. radiographically, the appearance of (multi)focal densities within the air-filled lung field needs to be differentiated from pulmonary neoplasia (primary or metastatic) or systemic fungal pneumonias. prevention. use of purpose-bred dogs virtually eliminates the chance of pulmonary trematodiasis in a research animal. treatment. praziquantel (at mg/kg q hr x days) or fenbendazole ( - mg/kg q hr x - days) are recommended for treatment of canine paragonimus infestation (hawkins, ) . effectiveness is monitored by fecal sedimentation tests for eggs and resolution of radiographic lesions (which may never resolve entirely). early diagnosis of pulmonary trematodiasis should warrant discontinuation of a dog from a long-term study because of the possibility of more serious clinical sequelae, such as pneumothorax. research complications. experimental studies involving the immune system, especially eosinophilic or local pulmonary responses, would be significantly affected by even minor infestations. clinical illness would complicate almost any research project and makes dogs poor anesthetic risks. radiographic lesions may confound diagnostic evaluation for pulmonary metastasis of tumors. e. mites i. demodicosis etiology. canine demodicosis is caused by demodex canis, a commensal mite that lives in the hair follicles. it is considered to be normal fauna of dog skin, but certain conditions (i.e., immunosuppression) cause development of clinical illness. clinical signs. demodex canis infestation is typically asymptomatic. clinical demodicosis presents with variable and nonspecific clinical signs, such as alopecia, erythema, pruritus, crusts, and hyperpigmentation. it can occur anywhere on the body but is often seen on the feet and the face and around the ears (demanuelle, a). secondary bacterial pyoderma is a common complication. epizootiology and transmission. demodex canis mites pass to nursing pups from the dam. they live their entire lives on one dog and are not considered contagious to other dogs or humans. certain breeds are predisposed to the generalized form of demodex dermatitis (see "pathogenesis," below). beagles are among the predisposed breeds, as are german shepherds, doberman pinschers, old english sheepdogs, collies, boxers, and shorthair brachycephalic breeds (muller et al., ) . pathologic findings. histologically, demodex infections are characterized by perifolliculitis and folliculitis with mites and keratin debris visible in the hair follicles. cases with generalized demodicosis (see "pathogenesis," below) may have a minimal cellular response with no eosinophils, indicative of severe immunosuppression . pathogenesis. when clinical demodicosis develops, it is classified into "localized" or "generalized" (e.g., more than one foot affected, or five or more small areas, or one large body area). localized demodicosis is typically seen in juvenile dogs (< months) and usually resolves without treatment as natural immunological control develops. generalized demodicosis can develop in juvenile or adult populations. juvenile-onset generalized demodicosis occurs in dogs with a genetic predisposition, thought to be an inherited t-lymphocyte dysfunction. adult-onset generalized demodicosis is usually indicative of an underlying endocrine (hyperadrenocorticism, diabetes mellitus, hypothyroidism) or neoplastic disorder or can develop as a result of immunosuppressive therapy (such as corticosteroid administration). diagnosis and differential diagnosis. demodex is readily identified from deep skin scrapings of lesioned areas (campbell, ; noli, ) . demodex canis has a characteristic "cigar shape," with short, stubby legs on a body - ~tm long. differential diagnoses for local demodicosis include dermatophytosis, allergic contact dermatitis, and seborrheic dermatitis. the primary differential diagnosis for generalized demodicosis is primary bacterial pyoderma; remember, however, that bacterial pyoderma is a common secondary complication of the generalized form of this parasitism. prevention and control. dogs with generalized demodicosis should not be maintained in a breeding colony. treatment: ivermectin (ivomec) at - ~tg/kg and oral milbemycin (interceptor) at - mg/kg/day have been found to be effective treatments. these parasiticides are probably the most practical to use in a research setting, although they are not labeled for treatment of demodex canis. amitraz (mitaban) dips ( ppm every days) can be used for more problematic cases. treatment duration can be extensive and must be accompanied by repeated skin scrapings. research complications. dogs with generalized demodicosis should not be used in research studies, because this disease is indicative of another underlying disorder (endocrine or immunological). dogs that receive immunosuppressive agents or paradigms could develop generalized demodicosis as an unexpected consequence of the experimentation. ii. sarcoptic mange etiology. canine sarcoptic mange is caused by sarcoptes scabiei var. canis. clinical signs. the most common clinical sign is an intense pruritus, usually beginning at sparsely furred areas such as the ear pinnae, elbows, and ventral thorax and abdomen. lesions are characterized by alopecia and yellowish dry crusts with a macular papular eruption. these lesions may be exacerbated by excoriation due to the pruritic nature of the condition. epizootiology and transmission. sarcoptes mites live their entire lives in the stratum corneum of the host animal; however, they can survive for - weeks away from the host, and it is this ability that enables them to spread from dog to dog. sarcoptes scabiei var. canis can also infect cats and humans. pathologic findings. histologic examination can be unrewarding because mites are rarely seen on tissue sections, and the associated dermatitis is nondiagnostic: perivascular and interstitial dermatitis with hyperkeratosis, with or without eosinophilic infiltration. suggestive histopathologic lesions are epidermal "nibbles," small foci of edema, exocytosis, degeneration, and necrosis . pathogenesis. lesions and illness are a result of the female mites burrowing through the epidermal layers to deposit eggs, and the larvae migrating back to the surface. the typical locations of mange lesions are a result of the mite's preference for relatively hairless areas. diagnosis and differential diagnosis. sarcoptic mange can be difficult to diagnose because multiple skin scrapings can yield negative results with this parasitic disorder. hopefully, adult mites, mite eggs, or mite feces can be observed on superficial skin scrapings. even if scrapings are negative, however, a therapeutic trial should be initiated if the clinical signs and history suggest a sarcoptes etiology. demonstration of anti-mite ige in either the serum or via an intradermal antigen test can be used as a diagnostic aid (campbell, ). an important differential diagnosis is flea allergy dermatitis; in contrast, mange is nonseasonal and contagious. prevention and control. use of purpose-bred dogs limits the possibility of having research animals with sarcoptic mange. for random-source dogs, an ectoparasite control program should be in place to limit possible infestations. many institutions use ivermectin as a means to control both endoparasites and ectoparasites. treatment. unless treatment would interfere with research objectives, all dogs with sarcoptic mange (no matter how minor the lesions) and their kennel mates should be treated because of the contagious nature of the disease and its zoonotic potential. in research colonies, the usual means of treatment is either ivermectin (ivomec) at - ~tg/kg q days or milbemycin (interceptor) at oral doses of mg/kg q days . neither of these agents is approved for treatment of sarcoptic mange, but they are considered to be effective. acaricidal dips (e.g., lime sulfur, organophosphates, amitraz) can also be used. research complications. the local skin inflammation and systemic immune response to sarcoptic mange probably make infected dogs poor subjects for dermatologic and immunologic studies. f lice and ticks i. lice etiology. dogs can be infested by one species of sucking louse (linognathus setosus) and two species of biting lice (trichodectes canis and heterodoxus spiniger). clinical signs. mild cases of pediculosis may be asymptomatic or may cause pruritic areas of dry skin. more severe infestations can cause significant pruritus and produce alopecia, papules, and crusts. these lesions lead to excoriation and secondary bacterial dermatitis. severe linognathus infestations could cause anemia, because this species feeds on blood. epizootiology and transmission. louse infestations are uncommon in both pet animal practice and the research setting. they would most likely be seen in random-source dogs that were obtained from a pound or shelter. transmission is usually by direct contact, for lice spend their entire lives on the host species. lice are host-specific and not zoonotic. pathogenesis. the biting lice usually cause more local irritation than the sucking louse and therefore are more apt to induce clinical dermatologic signs. trichodectes canis can serve as vector for the canine tapeworm dipylidium caninum. the most severe complication of infestations by the sucking louse is the potential anemia. diagnosis and differential diagnosis. pediculosis is diagnosed by direct observation of the lice or nits (eggs) on the dog's skin. cellophane tape can be used to pick up surface debris from skin lesions, which may include nits or immobilized lice (muller et al., ) . differential diagnoses include dermal acariasis, flea allergy dermatitis, and seborrhea. prevention. use of high-quality conditioned dogs for research should prevent pediculosis from ever being seen within a research facility. random-source dogs should be shampooed or treated prophylactically with topical insecticide before being permitted to enter the research colony. treatment. most commercially available insecticide shampoos and dips readily treat louse infestations. treatment should be repeated in - days, because any nits that were not killed would have hatched by that time (muller et al., ) . there is probably minimal interference with research, unless severe linognathus infestations cause anemia. ii. ticks etiology. ticks are obligate arachnid parasites that require vertebrate blood as their sole food source. except for the brown dog tick (rhipicephalus sanguineus), ticks have a wide host range and are not especially host-specific; so any number of tick genera and species can be found on dogs. genera that more commonly infest dogs in the united states include species of rhipicephalus, dermacentor, and ixodes. the primary significance of tick infestation is the tick's ability to be a vector for many other infectious diseases, including rocky mountain spotted fever (caused by rickettsia rickettsii), lyme disease (borrelia burgdorferi), and the canine forms of ehrlichiosis (ehrlichia canis and e. platys), babesiosis (babesia canis), haemobartonellosis (haemobartonella canis), and hepatozoonosis (hepatozoon canis). clinical signs. as an entity unto itself, tick infestation causes minimal clinical signs. most infestations are subclinical, although some dogs may lick and bite at the site, aggravating the local lesion. some dogs can develop a hypersensitivity reaction after several tick bites; these dogs develop a more granulomatous response at the location of the bite (merchant and taboada, ) . some species of ticks (primarily dermacentor andersoni and d. variabilis) produce a salivary neurotoxin that can cause an ascending flaccid paralysis (malik and farrow, ) . the paralysis develops within - days of tick attachment and can result from a single tick. this paralysis is fatal once the respiratory musculature is affected. epizootiology and transmission. in dogs used for biomedical research, tick infestation may occasionally be seen in randomsource dogs, because these dogs are more likely to have been in tick habitats than purpose-bred dogs. ticks commonly reside in wooded areas until they contact a suitable host for a blood meal. the brown dog tick may reside within kennels (attics, bedding, wall insulation) (garris, ) . pathologic findings. under most circumstances, tick infestation will be an incidental finding on necropsy (unless tick paralysis was the cause of death). pathogenesis. tick-bite paralysis is caused by the presence of a salivary neurotoxin released by female ticks of certain genera (e.g., dermacentor) while consuming a blood meal (malik and farrow, ) . interestingly, dogs seem to be most affected by this condition, whereas cats appear to be resistant. the primary dysfunction appears to be at the neuromuscular junction, as stimulation of the motor nerves fails to elicit a response, but direct stimulation of the muscle tissue results in contractions. tick bites can also transmit pathogen microorganisms to the dog, because ticks serve as vectors for several infectious diseases, including lyme borreliosis, ehrlichiosis, babesiosis, and rocky mountain spotted fever. diagnosis and differential diagnosis. for uncomplicated tick bites and tick-bite paralysis, definitive diagnosis is made by identification of the offending arachnid (and improvement of paralysis after removal). differential diagnoses for tick-bite paralysis include botulism, snakebite, polyradiculoneuritis, and idiopathic polyneuropathy (malik and farrow, ) . prevention. purpose-bred dogs should be free from all ectoparasites, but ticks can occasionally be seen on randomsource animals. research dogs should not be exercised in outdoor areas infested with ticks, and kennels must be cleaned properly and regularly so as to remain free of ticks and other parasites. treatment. removal of the offending tick is the primary treatment for both local inflammation as well as tick-bite paralysis. dogs with tick-bite paralysis usually show improvement within hr, with complete recovery within hr (malik and farrow, ) to remove an attached tick from a dog, forceps should be used to grasp the tick as close to the dog's skin as possible. the tick should not be grabbed by the body, as this may cause the parasite to either rupture or inject its body contents into the dog. the tick should be pulled away from the dog with steady pressure. many of the diseases transmitted by ticks are zoonotic so precautions, such as wearing gloves, should be taken. use of topical acaricide/insecticides on newly arrived random-source dogs should help to limit infestations. probably have minimal impact on research variables. the significant concern for tick infestation is the possible development of tick-bite paralysis or of any one of a number of systemic diseases spread by ticks (see sections iii,a,l,e-g). g. other i. flea infestation etiology. fleas are laterally flattened wingless insects that feed on animal blood. the most common flea to infest dogs is ctenocephalides felis, the cat flea. other fleas that can affect dogs are ctenocephalides canis, pulex irritans, and echidnophaga gallinacea. the fleas are speciated by the shape of their head and by the presence or absence of ctenidae (spiny combs on or behind the head) (campbell, ) . clinical signs. flea infestations usually cause foci of alopecia and pruritus. dogs that are hypersensitive to antigenic proteins in flea saliva develop the more severe "flea allergy dermatitis," which features papules and crusting. acute moist dermatitis ("hot spots") can also be seen in these cases, and secondary pyoderma or seborrhea can develop. lesions from flea allergy dermatitis generally appear in the dorsal lumbosacral region, as well as the flanks, thighs, and abdomen (muller et al., ) . the lesions are typically worse in the summer and autumn months and are progressively more severe as the dog ages. epizootiology and transmission. fleas are readily transmitted between animals and even between host species. they move readily between the host and the environment, making transmission easy and control difficult. because fleas require host blood for food, they can survive off of a host for only - months (muller et al., ) . pathologic findings. biopsy samples are usually nondiagnostic in cases of flea allergy dermatitis. lesions are typically characterized by perivascular eosinophilic inflammation and may feature pustules and folliculitis if secondary pyoderma develops (muller et al., ) . pathogenesis. fleas are parasites that require animal blood for their meals. when they bite host animals, they inject some saliva into the host's skin. if the host develops an allergic response to the flea saliva, it will develop the more pruritic flea allergy dermatitis. fleas can also transmit or serve as vectors for other pathogens (e.g., dipylidium tapeworms). flea allergy dermatitis are definitively diagnosed by observing the fleas on the host's skin. given that this may be difficult because of the mobility of the flea and the majority of the time it spends off of the host, diagnosis is often based on clinical signs, history, and lesion distribution. sometimes the presence of flea excrement ("flea dirt") on the dog's skin can support a presumptive diagnosis (demanuelle, b) . circulating eosinophilia is seen in some dogs with flea allergy dermatitis. differential diagnoses include mite and louse infestations, bacterial folliculitis, and allergic or atopic conditions that present with skin lesions in dogs (e.g., food, drug, or contact hypersensitivity). prevention. most dogs obtained from high-quality purposebred facilities should be free from flea infestations. dogs received from pounds, shelters, or licensed dealers would be more likely to be affected by fleas (or any ectoparasitism). thorough knowledge of prevention, control, and treatment measures at these facilities should be obtained, and dogs from sources where proper prevention and/or therapy are not practiced should be evaluated and/or empirically treated upon arrival at the facility. control. thorough cleaning of the dog's housing environment should remove the risk of perpetuating or transmitting flea infestation in the colony. treatment. treatment for fleas needs to address treatment of both the dog and the environment. many insecticide formulations such as shampoos, sprays, dips, powders, and oral systemics can be used for initial treatment of the individual dog. the active ingredients include pyrethrins, pyrethroids, carbamates, and organophosphates. flea control in the kennel may need to include outdoor areas in warm climates. typically combinations of adult insecticides and juvenile growth regulators are used for environmental treatment. directed sprays are the most effective means of treating housing areas, because flea "bombs" or foggers do not penetrate adequately into tight areas where fleas might hide (demanuelle, b) . in addition to insecticide therapy, dogs with flea allergy dermatitis may also require anti-inflammatory medication to relieve clinical signs. oral prednisone or prednisolone at . mg/kg q hr for - days has been proposed as a starting therapy (muller et al., ) . the use of hyposensitization with flea-bite antigens is controversial and not practical for the research setting. research complications. mild flea infestation probably has minimal impact on most research protocols, and treatment measures may in fact be more detrimental to the experimental objective than the actual ectoparasitism. in a research setting, the residual effects of insecticides may preclude their use in experimental animals. such treatments should be used judiciously to ensure that experimental results are not more seriously affected by the therapy rather than the infestation. dogs with flea-allergy dermatitis are more severely affected by the flea infestation and should be treated apigropriately; however, systemic corticosteroids may also interfere with experimental objectives, especially in studies involving functions of the immune system. the ability of fleas to transmit other parasitic diseases must also be considered. etiology. dermatophytoses ("ringworm") are fungal skin infections, which in dogs in the united states are usually caused by either microsporum canis, m. gypseum, or trichophyton mentagrophytes (muller et al., ) . clinical signs. uncomplicated superficial dermatophytoses are characterized by circumscribed circular areas of alopecia, usually with minimal to no inflammation. these skin lesions are usually seen around the face, neck, and forelimbs but can be found anywhere on the body. secondary bacterial infections can develop; these lesions are called kerions and are selflimiting, for the fungus cannot survive in inflamed skin (muller et al., ) . ep&ootiology and transmission. the fungi that cause skin infections are very contagious and readily transmissible between dogs and other species (including human beings), but they can also be obtained from the soil. pathologic findings. on close inspection of skin samples, broken hair shafts (and not complete hair loss) would be seen with uncomplicated dermatophytosis. histologically, fungal elements can be seen within the stratum corneum or in and around the hair and hair follicles (muller et al., ) . stains that facilitate visualization of fungal elements include periodic acid-schiff (pas) or gomori methenamine-silver. the pattern of inflammation in the affected foci is very variable and can feature folliculitis, perivascular dermatitis, hyperkeratosis, and/or vesicular dermatitis. pathogenesis. the dermatophytes typically infect the hair shaft itself, the hair follicle, and possibly the skin around the affected hair. the hair follicle is not destroyed (unless by secondary bacterial infection), but the hair itself becomes brittle and breaks. this causes short stubbly hair to be seen within the lesion. as the lesion progresses, the hairs in the center recover from the infection, thus leading to the classic "ringworm" appearance of the alopecic areas. it is postulated that the inflammatory process produces an environment that is unfavorable for dermatophyte survival, whereas the periphery of the lesion still enables continued fungal growth (muller et al., ) . diagnosis and differential diagnosis. diagnosis of dermal fungal infection is typically made by scraping the affected area to obtain hair and superficial epidermal cells. these scrapings are then digested with potassium hydroxide to facilitate observation of fungal elements. fungal elements can also be seen on skin biopsy samples. for speciation of a fungus, skin scrapings can also be inoculated onto agars that promote fungal growth, such as sabouraud's medium or dermatophyte test medium (dtm). incubation should be at ~ with % humidity for - days. lesions caused by m. canis may fluoresce when inspected using a wood's ( . nm ultraviolet) light. unfortunately, some strains of m. canis do not fluoresce, and neither does m. gypseum or t. mentagrophytes. differential diagnoses for dermatomycosis include seborrhea, localized demodecosis, folliculitis, histiocytoma, and acral lick dermatitis (muller et al., ) . prevention. purpose-bred dogs are typically free of infectious dermatophytes, but ringworm may be diagnosed on randomsource animals. control. in cases of dermatophytosis, isolation of the affected animal(s) is prudent, because the fungi are easily spread to other dogs, as well as to people. treatment, if acceptable, should be started immediately. treatment. topical antifungal therapy is most commonly used. shampoos, rinses, and creams containing miconazole, ketoconazole, enilconazole, or chlorhexidine are commercially available to treat ringworm (stannard et al., ) . severe cases may require systemic therapy with griseofulvin, ketoconazole, itraconazole, or fluconazole. however, these systemic antifungal agents may have considerable side effects (such as vomiting and teratogenicity with griseofulvin). many of the newer agents are also expensive and not labeled for use in dogs. impact on most research applications for dogs. unfortunately, the zoonotic implications of dermatophytoses force the issue of aggressive treatment, and many antifungal agents may not be compatible with biomedical research studies. systemic fungal infections disseminate to multiple organ systems from a single mode of entry (usually through the respiratory tract). dogs are susceptible to several fungi that characteristically cause systemic mycosis, including blastomyces dermatitidis, histoplasma capsulatum, coccidioides immitis, and cryptococcus neoformans var. neoformans. these diseases are not typically seen in the research setting, because of the low overall incidence and noncontagious nature of these disorders and because of the use of purpose-bred animals. these conditions could, however, present in the rare random-source dog that was subclinical at its point of origin, especially if the animal becomes immunosuppressed (either naturally or by virtue of experimental manipulation). typical clinical signs include weight loss, fever, lymphadenopathy, and cough and dyspnea (if the lungs are affected). the reader is advised to read veterinary medical text chapters (e.g., taboada, ) for more complete information on these disorders and their possible treatments. although the incidence of hypothyroidism in the canine population is not high (kemppainen and clark, ) , deficiency in thyroid hormone can significantly affect basal metabolism and immune function. because these factors are important in many biomedical research studies, it is imperative that laboratory animal veterinarians be able to recognize, diagnose, and treat this problem. etiology. the majority of cases of canine hypothyroidism are due to lymphocytic thyroiditis, an autoimmune disorder, or idiopathic atrophy of the thyroid gland. both of these causes result in a gradual loss of functional thyroid tissue (kemppainen and clark, ) . lymphocytic thyroiditis is the major cause of hypothyroidism in laboratory beagles and appears to be familial in that breed (tucker, ; beierwaltes and nishiyama, ; manning ) . rarely, congenital defects or nonfunctional tumors may cause hypothyroidism (peterson and ferguson, ; kemppainen and clark, ) . clinical signs. because it affects metabolism in general, hypothyroidism can produce a large number of clinical signs referable to many organ systems. an individual dog with hypothyroidism may have one or any combination of clinical signs. hypothyroidism reduces the dog's metabolic rate, which then produces such signs as obesity, lethargy, cold intolerance, and constipation. additionally, hypothyroidism can produce several dermatologic abnormalities, including alopecia, hyperpigmentation, seborrhea, and pyoderma (peterson and ferguson, ; panciera, ) . several clinicopathologic abnormalities have also been reported in a large percentage of hypothyroid dogs. these aberrations include increased serum cholesterol and triglycerides due to a decrease in lipolysis and decreased numbers of low-density lipopolysaccharide receptors (peterson and ferguson, ; panciera, ) . normocytic normochromic nonregenerative anemia and increased serum alkaline phosphatase and creatine kinase have also been reported in a significant number of hypothyroid dogs (peterson and ferguson, ; panciera ) . neurologic signs of hypothyroidism, which include lameness, foot dragging, and paresis, may be caused by several mechanisms such as segmental nerve demyelination or nerve entrapment secondary to myxedema (peterson and ferguson, ) . mental impairment and dullness have also been reported in hypothyroid dogs, secondary to atherosclerosis and cerebral myxedema (peterson and ferguson, ) . hypothyroidism has been implicated in other neurological abnormalities such as horner's syndrome, facial nerve paralysis, megaesophagus, and laryngeal paralysis; however, these conditions do not always resolve with treatment (bischel et al., ; panciera, ) , and so the relationship between hypothyroidism and these problems has not been completely defined (panciera, ) . myopathies associated with hypothyroidism are caused by metabolic dysfunction and atrophy of type ii muscle fibers and can present with signs similar to neurological disease (peterson and ferguson, ) . hypothyroidism can also cause bradycardia as a result of decreased myocardial conductivity. abnormalities that may be detected by ecg include a decrease in p and r wave amplitude (peterson and ferguson, ) and inverted t waves (panciera, ) . these electrocardiographic abnormalities are caused by lowered activity of atpases and calcium channel function. several reports have suggested that hypothyroidism is associated with von willebrand's disease and bleeding abnormalities. however, the relationship is probably one of shared breed predilection and not a true correlation. it has been demonstrated that dogs with hypothyroidism are not deficient in von willebrand's factor when compared with other dogs. in addition, the replacement of thyroid hormone in dogs did not increase the levels of vwf:ag in naturally occurring (panciera and johnson, ) or experimentally induced (panciera and johnson, ) hypothyroidism. epizootiology. the prevalence of hypothyroidism in the general canine population has been reported to be less than % (panciera, ) . the disorder occurs most often in large-breed dogs but has been reported in several other breeds as well as mongrels. doberman pinschers and golden retrievers appear to have a higher incidence of hypothyroidism when compared with other breeds (panciera, ; peterson and ferguson, ; scarlett, ) . there have been several reports about hypothyroidism in laboratory colonies of beagles (manning, ; tucker, ; beierwaltes and nishiyama, ) . in general, the problem is usually recognized in middle-aged animals, and some reports state that there is a higher incidence of hypothyroidism in spayed female dogs (panciera, ; peterson and ferguson, ). diagnosis and differential diagnosis. because of the large number of clinical manifestations in dogs, the recognition of hypothyroidism is not always straightforward. likewise, the diagnosis of hypothyroidism can be difficult because of the lack of definitive diagnostic tests available for the dog. the tests currently available and in popular use will be discussed further. however, a complete understanding of the diagnosis of hypothyroidism requires a familiarity with thyroid hormone metabolism and function that is beyond the scope of this writing. for additional information, the reader is referred to one of several manuscripts available (peterson and ferguson, ; ferguson, ) . currently, the ability to diagnose hypothyroidism relies heavily on the measurement of serum total t (thyroxine) and free t (peterson and ferguson, ; ferguson, ) . t serves primarily as a precursor for t in the body and is heavily proteinbound. free t represents the unbound fraction that is available to the tissues (peterson and ferguson, ) . using the measurement of serum total t and free t , hypothyroidism can usually be ruled out if the values are within the normal range or higher. if both hormone concentrations are low, it is highly likely that the patient has hypothyroidism, and a therapeutic trial is in order (peterson and ferguson, ) . however, it must be noted that nonthyroidal illnesses and some drugs (e.g., glucocorticoids, anticonvulsants, phenylbutazone, salicylates) can falsely lower these values (peterson and ferguson, ; ferguson, ) . therefore, low values do not always indicate that hypothyroidism is present, and animals should not be treated solely on the basis of serum hormone levels if clinical signs are absent. if the clinical signs are equivocal or if only total t or free t is decreased, further diagnostic testing is warranted (peterson and ferguson, ) . although t is the most biologically active form of thyroid hormone in the body, the measurement of serum t levels is an unreliable indicator of hypothy-roidism (peterson and ferguson, ; ferguson, ) . like t , serum t can be falsely lowered by many nonthyroidal illnesses and many drugs (see above). in addition, t may be preferentially released, and conversion of t to t may be enhanced in the hypothyroid dog (peterson and ferguson, ; ferguson, ) . t was within normal limits in % of the hypothyroid dogs in one study (panciera, ) . autoantibodies can be responsible for false elevations in the concentrations of t and t found in these respective assays. it has been recommended that free t , measured by equilibrium dialysis, be assayed in dogs that are suspected of hypothyroidism and have autoantibodies with normal or high t and t . autoantibodies have been found in less than % of the samples submitted to one laboratory (kemppainen and behrend, ) . other means of diagnosing hypothyroidism have been described. in humans, endogenous tsh (thyroid-stimulating hormone) levels provide reliable information on thyroid status, and an assay for endogenous tsh is now available in dogs. however, tsh levels can be normal in some dogs with hypothyroidism, and high tsh levels have been noted in normal dogs. therefore, it is recommended that tsh levels be considered along with other information (clinical signs, t ) prior to diagnosis and treatment (kemppainen and behrend, ) . tsh stimulation testing using exogenous bovine tsh provides a good and reliable method for establishing a diagnosis. unfortunately, the availability and expense of tsh limit the use of this diagnostic tool (peterson and ferguson, ; ferguson, ) . another drawback of tsh testing is that the test must be postponed for weeks if thyroid supplementation has been given (peterson and ferguson, ) . when tsh is available for testing, there are several recommendations for dosage, routes of administration, and sampling times. one recommendation is . u of tsh per pound of body weight (up to a maximum of u) to be administered iv. for this protocol, blood samples are taken prior to administration of tsh and hours after. a normal response to the administration of tsh should create an increase of t levels at least ktg/dl above the baseline levels or an absolute level that exceeds ~tg/dl (peterson and ferguson, ; wheeler et al., ) . treatment. the treatment of choice for hypothyroidism in the dog is l-thyroxine (sodium levothyroxine). a recommended dosing regimen is . mg/kg once a day or . mg/m (body surface area)/day for very small or very large dogs. if drugs that decrease thyroxine levels are being administered concurrently, it may be necessary to divide the thyroxine dose for twice daily administration. after the supplementation has begun, the thyroid hormone level should be rechecked in - weeks, and blood samples should be drawn - hours after the morning pill. a clinical response is usually seen in - weeks and would include weight loss, hair regrowth, and resolution of other signs (panciera, ) . ecg abnormalities also return to normal (peterson and ferguson, ) . for dogs with neurologic signs, the prognosis is guarded, because the signs do not always resolve with supplementation (panciera, ) . weight gain and eventual obesity are also frequent findings in dogs in the research environment. because obesity can adversely affect several body systems as well as general metabolism, the laboratory animal veterinarian must be aware of the development of obesity and the potential effect that it can have on research. etiology. obesity is defined as a body weight - % over the ideal. in general, obesity occurs when the intake of calories exceeds the expenditure of energy. excessive caloric intake resuits from overeating or eating an unbalanced diet. overeating is a common cause of obesity in pet dogs and may be triggered by boredom, nervousness, or conditioning (macewen, ) . in addition, pet animals are often subjected to unbalanced diets supplemented with high-fat treats. in the laboratory animal setting, overeating is less likely than in a household, because access to food is more restricted and diets are usually a commercially prepared balanced ration. however, obesity can still be a problem if specific guidelines for energy requirements are not followed. in addition, the necessary caging of dogs in the research environment and thus the limitation to exercise reduces energy expenditure and predisposes dogs to weight gain. it is also important to realize that other factors may predispose dogs to obesity, even when guidelines for caloric intake and energy expenditure are followed (butterwick and hawthorne,. ). as in humans, genetics plays an important role in the development of obesity in dogs. it has been established that certain breeds are more predisposed toward obesity. in a study of dogs visiting veterinary clinics in the united kingdom, labrador retrievers were most likely to be obese. other breeds affected included cairn terriers, dachshunds, basset hounds, golden retrievers, and cocker spaniels. the beagle was also listed as a breed predisposed to obesity in the household environment (edney and smith, ) . in addition to genetics, several metabolic or hormonal changes are associated with obesity. it has been well established that neutering promotes weight gain. in one study, spayed female dogs were twice as likely to be obese when compared with intact females (macewen, ) . the authors proposed that the absence of estrogen promotes an increase in food consumption. a similar trend toward obesity was found in castrated male dogs (edney and smith, ) . in addition, hypothyroidism and hyperadrenocorticism may present with obesity as one of the clinical signs (macewen, ) . epizootiology. ewen, ) . obesity affects up to % of pet dogs (mac-diagnosis and differential diagnosis. the diagnosis of obesity is somewhat subjective and relies on an estimate of ideal body weight. the ideal body condition for dogs is considered to be achieved when the ribs are barely visible but easily palpated beneath the skin surface. when the ribs are not easily palpated and/or the dog's normal function is impaired by its weight, the animal is considered obese. there are few objective, quantifiable methods for establishing this diagnosis. ultrasound has been evaluated for measurement of subcutaneous fat in dogs, and measurements taken from the lumbar area can be used to reliably predict total body fat (wilkinson and mcewan, ) . after a diagnosis of obesity has been made, additional diagnostic tests should be performed to determine if there is an underlying cause for the problem. a complete physical exam should be performed to look for signs of concurrent disease and to establish if obesity has adversely affected the individual. serum thyroid hormones should be evaluated (see section iii,b,l,a), and serum chemistry may reveal an increased alkaline phosphatase associated with hyperadrenocorticism. treatment. restricting food intake readily treats obesity, and this is easily done in the research setting. it has been suggested that a good weight loss program involves restriction of intake to % of the calculated energy requirement to maintain ideal body weight. it has been shown that restriction of calories down to % produces no adverse health effects. however, t levels will decrease in direct proportion with caloric intake. ideally, weight loss will occur at a rate of - % of body weight per week (laflamme et al., ) . with more severe calorie restriction and more rapid weight loss, the individual is more likely to rebound and gain weight after restrictions are relaxed. there has been agreat deal of attention in humans as to the correct diet to be fed to encourage weight loss. likewise, the type of diet fed to dogs has been examined. as mentioned above, the restriction of calories is most important, and feeding less of an existing diet can do this. alternatively, several diet dog foods are available, and there is some evidence that these diets are superior to simple volume restriction (macewen, ) . there has been much concern about the addition of fiber to the diet in both humans and animals as a method for reducing caloric intake while maintaining the volume fed. studies in dogs have examined the addition of both soluble and insoluble fibers to calorierestricted diets. these studies have shown that the addition of fiber does not have an effect on satiety in dogs and therefore does not have a beneficial effect in weight loss protocols (butterwick and thorne, ; butterwick and thorne, ) . it is important to control weight gain in research animals, because of the association of obesity and several metabolic changes. although an association between obesity and reproductive, dermatologic, and neoplastic problems has been reported (macewen, ) , this relationship is not consistently apparent (edney and smith, ) . obesity in dogs over years of age appears to be related to an increase in cardiovascular problems (edney and smith, ) , and obesity has been linked to hypertension. joint problems including osteoarthritis and hip dysplasia have also been related to obesity (macewen, ; kealy et al., ) . in addition, diabetes mellitus has been linked to obesity, and obesity induces hyperinsulinism in several experimental models (macewen, ) . in the laboratory setting, the majority of traumatic wounds will be small in size. in facilities with good husbandry practices and a diligent staff, traumatic wounds will generally be observed quickly and attended to promptly. under these conditions, proper initial treatment will lead to uncomplicated wound healing. complications such as infection and delayed healing arise when wounds are not noticed immediately or. when the basic principles of wound management are not followed. to aid in the description of wounds and in decision making about wound therapy, several classification systems have been developed for traumatic injuries. at one time, decisions about wound therapy were largely based upon the length of time since wounding, or the concept of a "golden period." it is now recognized that several factors must be considered prior to initiating wound care, including (but not limited to) the type and size of the wound, the degree of wound contamination, and the capability of the host's defense systems (swaim, ; waldron and trevor, ) . one of the most widely used classification systems is based upon wound contamination and categorizes wounds as either clean, clean-contaminated, contaminated, or dirty (see table v ). the vast majority of the wounds seen in the laboratory setting will fall into the clean and clean-contaminated categories. these wounds may be treated with the basic wound care described below and primary closure of the wound. contaminated and dirty wounds, which are seen infrequently in the laboratory setting, require more aggressive therapy. dirty wounds can occur as postsurgical infections or complications of initial wound therapy. when one is in doubt as to the classification of a wound, the worst category should be presumed in order to provide optimal therapy and reduce the chance for complications. the initial treatment of a wound is the same regardless of the wound's classification. when first recognized, the wound should be covered' with a sterile dressing until definitive treatment is rendered. bleeding should be controlled with direct waldron and trevor ( ) . pressure; tourniquets are discouraged because of the complications that may arise with inappropriate placement (swaim, ) . it is best to avoid using topical disinfectants in the wound until further wound treatment (culture, debridement, lavage) has been performed (swaim, ) . when the treatment of a wound begins, anesthesia or analgesia may be necessary, and the choice of anesthetic regimen will depend on the size and location of the wound as well as the preference of the clinician. if the wound is contaminated or dirty, bacterial cultures, both aerobic and anaerobic, should be performed at this time. then a water-soluble lubricant gel may be applied directly to the wound. a wide margin of hair should then be clipped from around the wound, using a # blade. after the clipping, a surgical scrub is performed around the edges of the wound. povidone-iodine alternating with alcohol or chlorhexidine gluconate scrub alternating with water is most often recommended for surgical preparation of the skin surface (osuna et al., a,b) . simple abrasions that involve only a partial thickness of the skin do not generally require further treatment. full-thickness wounds require further attention, including irrigation with large quantities of a solution delivered under pressure. two solutions, . % chlorhexidine diacetate in water (lozier et al., ) and % povidone-iodine in saline, are most often recommended for wound lavage (waldron and trevor, ) . the chlorhexidine solution may offer the advantage of greater bactericidal activity but does not significantly alter wound healing when compared with povidone-iodine (sanchez et al., ) . actually, the type of solution chosen may not be as important to wound care as the volume and pressure at which the solution is delivered. it has been suggested that psi is required to obtain adequate tissue irrigation, and this may be achieved by using a ml syringe and an -or gauge needle (waldron and trevor, ) . for wounds that are contaminated or dirty, debridement is an important part of initial therapy. debridement usually proceeds from superficial to deeper layers. skin that is obviously necrotic should be removed. although it is often recommended to remove skin back to the point at which it bleeds, this may not be feasible with large wounds on the limbs. in addition, other factors such as edema or hypovolemia may reduce bleeding in otherwise viable skin (waldron and trevor, ) . if one is unsure about tissue viability in areas that are devoid of extra skin, the tissue may be left (swaim, ; waldron and trevor, ) , and nonviable areas will demarcate within - days (waldron and trevor, ) . necrotic fat should be resected liberally, because it does not have a large blood supply and will provide an environment for infection. often, resection of subcutaneous fat is necessary to remove debris and hair that could not be removed during wound irrigation. damaged muscle should also be liberally resected (swaim, ) . the wound should be irrigated several times during debridement and again after completion. after initial wound treatment, the options concerning wound closure must be weighed. the principles of basic surgery are discussed in several good texts, and readers are encouraged to pursue additional information. primary wound closure is defined as closure of the wound at the time of initial wound therapy and is the treatment of choice for clean and clean-contaminated wounds. closure is performed in two or more layers, carefully apposing tissues and obliterating dead space. if dead space will remain in the wound, a drain should be place d. subcutaneous closure should be performed with absorbable suture such as polydioxanone (pds), polyglactin (vicryl), or polyglycolic acid (dexon). it is best to use interrupted sutures and avoid leaving excess suture material in the wound. it may be necessary to choose tension-relieving suture patterns, such as horizontal mattress. skin closure is generally performed with nylon ( - or - ). in situations where gross contamination cannot be completely removed, closure of the wound should be delayed or avoided. after debridement and irrigation, the wound should be bandaged. initially, the wound can be covered by gauze sponges soaked in saline or chlorhexidine to create a wet-to-dry bandage. when the sponges are later pulled from the wound, dried exudates will also be removed. when the wound appears clean, the layer in contact with the wound may be changed to a nonadherent dressing such as vaseline-impregnated gauze (swaim, ) . the contact layer is covered by cotton padding, and the entire bandage is covered by a supportive and protective layer. the bandages should be changed once or twice daily, depending upon the amount of discharge coming from the wound. wound closure within - days of wounding (prior to the formation of granulation tissue) is considered delayed primary closure. when the wound is closed after days, this is considered secondary closure (waldron and trevor, ) . secondintention healing involves allowing the wound to heal without surgical intervention. this type of healing is often used on limbs when there is an insufficient amount of skin to allow complete closure (swaim, ) . it is important to note that second-intention healing will take longer than surgical repair of a wound, and in the case of large wounds it will be more expensive because of the cost of bandaging materials. several factors must be weighed concerning the use of antibiotics in traumatic wounds, including the classification and site of the wound, host defenses, and concurrent research use of the animal. when wounds are clean or clean-contaminated, antibiotics are seldom necessary unless the individual is at high risk for infection. when wounds have been severely contaminated or are dirty, antibiotics are indicated, and the type of antibiotic will ultimately depend on culture and sensitivity results. until such results are available, the choice of antibiotic is based on the most likely organism to be encountered. in skin wounds, staphylococcus spp. are generally of concern, whereas pasteurella multocida should be considered in bite wounds. cephalosporins, amoxicillin-clavulanate, and trimethoprim sulfas are often recommended for initial antibiotic therapy (waldron and trevor, ) . etiology. pressure sores (decubital ulcers) can be a problem in long-term studies that require extended periods of recumbency. decubital ulcers usually develop due to continuous pressure from a hard surface contacting a bony prominence such as the elbow, the tuber ischii, tarsus, or carpus. the compression of the soft tissues between the hard surfaces results in vascular occlusion, ischemia, and ultimately tissue death (swaim and angarano, ) . several factors that increase pressure at the site and/or affect the integrity of the skin will predispose an individual to develop pressure sores. these factors include poor hygiene, self-trauma, low-protein diet, preexisting tissue damage, muscle wasting, inadequate bedding, and ill-fitting casts or bandages (swaim and angarano, ) . clinical signs. at first, the skin at the developing site will appear red and irritated. over time, constant trauma can result in full-thickness skin wounds and can progress to necrosis of underlying structures such as bone. the severity of the sores may be graded from i to iv, according to the depth of the wound and the tissues involved, from superficial skin irritation to bone necrosis. epizootiology. the problem usually occurs in large-breed dogs, but any type of dog can be affected. prevention and control. minimizing or eliminating those factors that can predispose to decubital ulcers is important to both the prevention and the control of this condition. if the dogs are going to experience long periods of recumbency, adequate bedding or padding must be provided. skin hygiene is of the utmost importance when trying to prevent or treat pressure sores. the skin should be kept clean and dry at all times. if urine scalding is a problem, the affected area should be clipped, bathed, and dried thoroughly at least once or twice daily. finally, an appropriate diet to maintain good flesh and adequate healing is also important (swaim and angarano, ) . treatment. the treatment of pressure sores must involve care of the wound and attention to the factors causing the wound. the extent of initial wound management will largely depend on the depth of the wound. for simple abrasions and small wounds involving the skin only, simple wound cleansing and openwound management provide adequate treatment. when wounds involve deeper tissues, including fat, fascia, or bone, more aggressive therapy must be performed. the affected area should be radiographed to assess bone involvement, and the wound should be cultured. all of the damaged tissue should be debrided, and wound management guidelines should be followed (see section iii,c, ). when a healthy granulation bed has formed over the entire wound, a delayed closure over a drain may be performed (swaim and angarano, ) . with extensive lesions, reconstruction with skin flaps may be necessary. bandaging should be performed on all full-thickness wounds; however, it is important to remember that ill-fitting or inadequately padded bandages or casts may worsen the problem. the area over the wound itself should not be heavily padded, because this will increase the pressure over the wound. the wounded area should be lightly covered and then a doughnut, created from rolled gauze or towel, should be fitted around the wound. this will displace the forces acting on the wound over a larger area and over healthier tissue. then the doughnut is incorporated into the bandage. if a cast has been applied to the area for treatment or for research purposes, a hole can be cut over the wound to reduce pressure in that area and allow treatment of the wound (swaim and angarano, ). bandages should be removed at least once or twice a day to allow wound care. after wound care has been initiated the causative factors for the pressure sore must be addressed (see "prevention and control," above). recumbent animals should be moved frequently to prevent continuous compression on the wound. if the dog tends to favor a position that aggravates the problem, splinting the body part to reduce contact with hard surfaces may be necessary. etiology. acral lick granuloma is a psychodermatosis, a skin lesion caused by self-trauma. in a few cases, self-trauma begins because of identifiable neurologic or orthopedic causes (tarvin and prata, ) . however, the majority of the cases begin because of repetitive licking by dogs that are confined and lack external stimuli (swaim and angarano, ) . it has been theorized that the self-trauma promotes the release of endogenous endorphins, which act as a reward for the abnormal behavior (dodman et al., ) . the laboratory setting is an environment that could promote this abnormal behavior and lead to acral lick granuloma. epizootiology. the lesions associated with acral lick granuloma are seen most often in large-breed dogs, but any type of dog can be affected (walton, ) . clinical signs. at first, lesions appear as irritated, hairless areas usually found on the distal extremities (swaim and angarano, ). the predilection for the limbs may be due to accessibility or possibly may be caused by a lower threshold for pruritus in these areas. as the lesions progress, the skin becomes ulcerated, and the wound has a hyperpigmented edge. the wounds may partially heal and then be aggravated again when licking resumes. diagnosis and differential diagnosis. acral lick granulomas must be differentiated from several other conditions, including bacterial or fungal infection, foreign bodies, and pressure sores. in addition, mast-cell tumors and other forms of neoplasia can mimic the appearance of acral lick granuloma. many of these problems can be ruled out by the history of the animal. when in doubt, a biopsy should be taken. an uncomplicated acral lick granuloma would feature hyperplasia, ulceration, and fibrosis without evidence of infection or neoplasia (walton, ) . prevention and control. behavior modification and relief of boredom are important aspects of preventing (and treating) acral lick granuloma. the environment of a dog with this problem can be enriched with exercise and the introduction of toys. in addition, the relief of boredom or anxiety can be attempted through the use of drugs such as phenobarbital, megestrol acetate, and progestins. these drugs may produce side effects, however (swaim and angarano, ) , and may interfere with experimental results. treatment. several treatments have been reported for acral lick granuloma, and none of them have been proven to be successful in ah cases. one of the most important aspects of treatment is to break the cycle of self-trauma. mechanical restraint with an elizabethan collar is one of the easiest methods to accomplish this goal. several direct treatments have been examined, including intralesional and topical steroids, perilesional cobra venom, acupuncture, radiation, and surgery (swaim and angarano, ; walton, ) . opioid antagonists have been used in an attempt to treat acral lick granuloma by blocking endogenous opioids. in one study, either naltrexone ( mg/kg sq) or nalmefene ( - mg/kg sq) successfully reduced the excessive licking behavior in of dogs; however, lesions returned after the drug was discontinued (dodman et al., ) . the use of a mixture of flunixin meglumine, steroid, and dimethyl sulfoxide ( ml of banamine [schering] mixed with ml of synotic [diamond laboratories]) applied topically twice daily has also been shown to be effective (walton, ) . the prognosis for acral lick granuloma should be considered guarded, because the lesions often recur or new lesions develop when treatment is discontinued. etiology. hygromas are fluid-filled sacs that develop as a result of repeated trauma over a bony prominence. the area over the olecranon is most frequently affected, but hygromas have been reported in association with the tuber calcis, greater trochanter, and stifle (newton et al., ) . epizootiology. elbow hygromas are most frequently reported in large and giant breeds of dogs around - months of age (johnston, ; bellah, ) . elbow hygromas are seen infrequently in the laboratory animal setting because the commonly affected breeds are seldom used in research. however, the housing environment for research dogs predisposes them to hygromas, because these animals spend a large amount of time on hard surfaces such as cage bottoms or cement runs. for this reason, laboratory animal veterinary and husbandry staff should be familiar with this condition. clinical signs. a dog with an elbow hygroma presents with a unilateral or bilateral, painless, fluctuant swelling over the point of the elbow. the animals are not usually lame. over a long period of time, elbow hygromas may become inflamed and ulcerated. if the hygroma is secondarily infected, the animal may exhibit pain and fever (johnston, ; bellah, ) . pathology. the fluid-filled cavity in the hygroma is lined by granulation and fibrous tissue. hygromas lack an epithelial lining and therefore are not true cysts. the fluid within the cavity is yellow or red and is a serous transudate. this fluid is less viscous than joint fluid, and elbow hygromas do not communicate with the joint (johnston, ) . treatment. the treatment of elbow hygromas should be conservative whenever possible, and surgical options should be reserved for complicated or refractory cases. conservative management of the elbow hygroma is aimed at relieving pressure at the point of the elbow by providing a padded cage surface and/or bandaging the elbow in a manner similar to that used to treat pressure sores (see section iii,c, ). more aggressive therapy, including needle drainage and the injection of corticosteroid into the hygroma, has been described but is not recommended, because infection is a serious complication of this treatment (johnston, ) . likewise, simple surgical excision of elbow hygromas can be associated with complications such as wound dehiscence and ulceration (johnston, ) . a technique that has been used successfully involves placement of multiple penrose drains. the drains are kept in place for - weeks, and the limb remains bandaged for weeks with this technique (bellah, ) . another technique has been described that involves the removal of a crescent-shaped piece of the skin and capsule. the remaining dead space is closed with mattress sutures over stents, and then the wound is closed in a routine fashion. the stents are removed in - days, and the wound is bandaged until suture removal in - days (newton et al., ) . regardless of the method used to treat an elbow hygroma, recurrence of the problem is likely unless the predisposing factors are identified and relieved. etiology. in the research environment, corneal ulcers are most often associated with either direct trauma, contact with irritating chemicals, or exposure to the drying effects of air during long periods of anesthesia. chronic or recurrent corneal ulcers may also be associated with infection or hereditary causes in some breeds of dogs; however, these cases would be rare in the laboratory setting. clinical signs. the signs of corneal ulceration are blepharospasm, epiphora, and photophobia. the eye may appear irritated and inflamed. in minor cases, the cornea may not appear abnormal; however, in cases of deeper ulceration, the cornea may appear roughened or may have an obvious defect. in addition, the periocular tissues may be swollen and inflamed because of self-inflicted trauma from rubbing at the eye. a tentative diagnosis of corneal ulcer or abrasion may be based on the clinical signs. a definitive diagnosis of corneal ulcers may be made by the green appearance of the cornea when stained with fluorescein dye. when a corneal ulcer has been diagnosed, the eye should be inspected for underlying causes such as foreign bodies or abnormal eyelids or cilia. treatment. the treatment of corneal ulcers will depend on the depth and size of the affected area. deep ulcers may require debridement and primary repair. in such cases, a third eyelid or conjunctival flap may be applied to the eye until experienced help can be obtained. superficial abrasions are generally treated with topical application of antibiotics. a triple antibiotic ointment that does not contain steroids given times a day for - days usually provides adequate treatment. ointments are preferred over drops, because use of the former requires less frequent. simple corneal ulcers are restained with fluorescein after days and should show complete healing at that time. if the ulcer is not healed, this may indicate that the ulcer has an undermined edge impeding proper healing. topical anesthetic should be applied to the eye, and a cotton-tipped applicator can be rolled over the surface of the ulcer toward its edge. this will remove the unattached edge of the cornea and healing should progress normally after debridement. in all cases, an elizabethan collar or other restraint may be necessary to prevent additional trauma to the eye. indwelling intravascular catheters, including intracaths and vascular access ports, often play a vital role in research protocols. the catheters are most often placed in a central vein or artery where they may be used for repeated blood sampling, administration of anesthetics and experimental compounds, or measurement of hemodynamic parameters. although catheters vary in composition, number of ports, and port placement, the basic principles of their implantation and maintenance are similar. it is important that the laboratory animal veterinarian be familiar with these principles and the potential complications of catheter use. when appropriately maintained, indwelling catheters may remain functional for months without serious complication. the actual incidence of complications associated with indwelling vascular catheters in dogs is unknown. this is due largely to the fact that many of the problems may be incidental findings or related to a particular research protocol. one study (hysell and abrams, ) examined the lesions found at necropsy in animals with chronic indwelling catheters (exact vascular locations not specified). the lesions found were categorized as traumatic cardiac lesions, visceral infarcts, and fatal hemorrhages. the traumatic cardiac lesions consisted primarily of masses of fibrin and inflammatory cells on the heart valves. the visceral infarcts were noted in the spleen, kidney (fig. ) , and brain and resulted from fibrin embolization from either the valvular lesions or the catheter tip. fatal hemorrhages were most often found in animals with experimentally induced hypertension. these animals developed clinical signs of sepsis and later ruptured a major vessel associated with mycotic infection and aneurysm. etiology. the leading complication associated with the use of indwelling vascular catheters is infection, either systemic or local at the point of entry through the skin. septicemia may develop from bacterial colonization of either the tract around the catheter or the catheter lumen. clinical signs. the signs and treatment of systemic infection are covered in section iii,d, . problems with the skin defect associated with the catheter port vary from mild skin irritation to obvious infection. the signs may include redness and swelling of the skin around the external port, discharge from the skin wound, or even abscess formation. prevention. because indwelling catheters play an important role in many research protocols, it is highly desirable to prevent catheter complications that may result in loss of the device. the catheter should be made of nonthrombogenic material. in addition, it is recommended that catheters be as simple as possible. a catheter with extra ports or multiple lumens requires addi- tional management and supplies more routes for infection. the use of vascular access ports that lie entirely under the skin eliminates many problems with infection. it has also been found that a long extension of tubing connected to the port may actually reduce the potential for infection of the catheter (ringler and peter, ) . the initial placement of an indwelling catheter must be done under aseptic conditions by individuals who are familiar with the procedure. the placement of the catheter should be verified by radiography. catheters that are used for delivery of drugs or blood sampling should be positioned in the vena cava and not in the right atrium, thereby minimizing trauma to the tricuspid valve. after catheter placement, the animals should be observed daily for signs of either local or systemic infection. the catheter entry site should be disinfected, coated with antibiotic ointment, and rebandaged every other day. once a month, the catheter line may be disinfected with chlorine dioxide, as described below (see "treatment"). throughout the life of the catheter, injections into and withdrawals from the catheter should be done in a sterile manner, and the number of breaks in the line should be kept to a minimum. treatment. the treatment of catheter infections almost invariably involves removal of the catheter, as demonstrated in both dogs and monkeys (ringler and peter, ; darif and rush, ) . superficial wound irritation or infection may be treated locally with antibiotic ointment, sterile dressing changes and efforts to minimize catheter movement; however, more extensive problems require aggressive therapy. systemic antibiotic therapy should be initiated for a -day period. the choice of drug will ultimately be based on previous experience and culture results. aerobic and anaerobic cultures of blood and locally infected sites should be performed (ringler and peter, ) . localized abscesses or sinus tracts may be managed by establishing drainage and flushing with chlorhexidine. again, the catheter should be removed. if retention of a catheter is important, the catheter lumen may be disinfected by filling with chlorine dioxide solution. it has been shown that there are no adverse effects from the use of chlorine dioxide in catheters (dennis et al., ) . the solution is removed after min and replaced with heparinized saline. all of the extension lines and fluids used in the catheter should be discarded. the blood cultures should be repeated days after the antibiotic therapy has ceased. if bacteria are still cultured, the catheter must be removed. intestinal access ports have been used to study the pharmacokinetics of drugs at various levels in the intestinal tract. these catheters are usually vascular access ports with several modifications to allow secure placement in bowel (meunier et al., ) . when placed and managed correctly, these ports may remain in place for months without complications. the most frequently reported complication associated with these catheters is infection around the port site (meunier et al., , kwei et al., . these infections lead to removal of the catheters despite treatment with local lavage and systemic antibiotics. there have also been reports of catheters dislodging from the intestinal tract and resulting in peritonitis. this complication has largely been eliminated with the improved security afforded by a synthetic cuff added to the end of the catheter (meunier et al., ) . the chapter authors have also seen migration of the catheter end within the lumen of the intestine (caused by peristaltic motion to egest the catheter), extensive intra-abdominal adhesions, and intestinal torsion (figs. a,b) as complications of intestinal access ports. the procedures for placement and maintenance of the catheters are similar to those outlined previously for indwelling vascular catheters. it is important that the catheters be firmly secured to the intestine to prevent migration or dislodgment. an omental patch placed over the site of entry may help form a firm adhesion. in addition, it is important to place the proper length of catheter within the peritoneal cavity; excess catheter length can promote adhesion formation, whereas insufficient catheter length to account for visceral organ motion can result in detachment. the placement and patency of the catheters can be verified periodically by contrast radiography using iodinated contrast material or by fecal occult blood testing after a small amount of blood has been injected through the catheter (meunier et al., ) . etiology. sepsis is defined as the systemic response to infection. most often, sepsis is a result of infection with gramnegative bacteria; however, sepsis may also be associated with gram-positive bacteria and fungi. in laboratory animals, sepsis is seen as a complication of surgical procedures or associated with chronic implants. sepsis may also be seen as a complication of infectious diseases such as parvovirus. clinical signs. the signs of sepsis can vary, depending on the source of the infection and the stage of the disease. early in the course of sepsis, dogs will present with signs of a hyperdynamic response, including an increased heart rate, increased respiratory rate, red mucous membranes, and a normal to increased capillary refill time. systemic blood pressure and cardiac output will be increased or within the normal range. the animals will often be febrile. later in the course of the syndrome, the animals will show the classic signs of septic shock, including decreased temperature, pale mucous membranes, and a prolonged capillary refill time. cardiac output and blood pressure are decreased as shock progresses. peripheral edema and mental confusion have also been reported (hauptman and chaudry, ) . pathogenesis. the pathophysiology of sepsis is complex and is mediated by immune responses involving mediators such ~ as cytokines, eicosinoids, complement, superoxide radicals, and nitric oxide. the body responds to overwhelming infection with an attempt to optimize metabolic processes and maximize oxygen delivery to tissues. however, if inflammation is left unchecked, the system may be unable to compensate, and the result is cardiovascular collapse. diagnosis. in general, a presumptive diagnosis of sepsis is made based on the occurrence of several in a group of signs, including altered body temperature, increased respiratory and/or heart rate, increased or decreased white blood cell count, increased number of immature neutrophils, decreased platelet count, decreased blood pressure, hypoxemia, and altered cardiac output. however, extreme inflammation without infection (e.g., pancreatitis, trauma) may create similar signs. one study examined the diagnosis of sepsis in canine patients at a veterinary hospital based on easily obtainable physical and laboratory findings. that study found that septic individuals had higher temperatures, wbc counts, and percentage of bands than nonseptic individuals, whereas platelet counts were lower in the septic dogs. there were no differences in respiratory rate or glucose levels between the groups. using these criteria, the results had a high sensitivity and a tendency to overdiagnose sepsis (hauptman et al., ) . ultimately, the presence of a septic focus simplifies diagnosis greatly; however, the focus may not be obvious. if the signs of sepsis are evident but the focus is not, several systems should be evaluated for infection, including urinary tract, reproductive tract, abdominal cavity, respiratory tract, teeth, and heart valves (kirby, ) . treatment. the treatment of sepsis has three aims. the first aim is to support the cardiovascular system. all septic animals should be treated with fluids to replace deficits and to maximize cardiac output. crystalloids are most frequently used to maintain vascular volume, primarily because of their low cost. colloids offer the advantage of maintaining volume without fluid overload and may have other positive effects on the cardiovascular system. acid-base and electrolyte imbalances should also be addressed. after the animal has stabilized, the treatment of sepsis should be aimed at removing the septic focus. obvious sources of infection should be drained or surgically removed. if an implant is associated with the source of infection, the implant should be removed. antibiotic therapy should also be instituted. the choice of antibiotic will ultimately depend upon the results of culture; however, the initial choice of antibiotics is based on previous experience, source of infection, and gram stains. the organisms associated with sepsis are often gram-negative bacteria of gastrointestinal origin or are previously encountered nosocomial infections. ideally, the antibiotic chosen for initial therapy should be a broad-spectrum, bactericidal drug that can be administered intravenously. second-or third-generation cephalosporins provide good coverage, as does combination therapy with enrofloxacin plus metronidazole or penicillin. finally, the treatment of sepsis is aimed at blocking the mediators of the systemic response. several studies have examined the effects of steroids, nonsteroidal anti-inflammatory drugs, and antibodies directed against endotoxin, cytokines, or other mediators of the inflammatory response; however, none of these treatments have proven greatly effective in clinical trials. consequently, there is no "magic bullet" for the treatment of sepsis at this time. successful therapy remains dependent on aggressive supportive care coupled with identification and elimination of the inciting infection. etiology. in research animals, aspiration into the lungs may occur accidentally during the oral administration of various substances or by the misplacement of gastric tubes. aspiration of gastric contents may also occur as a complication of anesthesia. in pet animals, aspiration is often seen as a result of metabolic and anatomical abnormalities; however, such occurrence would be rare in the research setting. clinical signs. the signs of aspiration lung injury may include cough, increased respiratory rate, pronounced respiratory effort, and fever. when respiration is severely affected, the oxygen saturation of blood will be decreased. the diagnosis of this problem is based on a history consistent with aspiration and the physical findings. classically, radiographs of the thorax demonstrate a bronchoalveolar pattern in the cranioventral lung fields. however, these lesions may not appear for several hours after the incident of aspiration. in addition, the location of the lesions may be variable, depending on the orientation of the animal at the time of aspiration. pathogenesis. aspiration of gastric contents or other compounds can create lung injury of variable severity, depending upon the ph, osmolality, and volume of the substance. the compounds aspirated can produce direct injury to lung tissue, but more importantly, the aspiration provokes an inflammatory response probably mediated by cytokines. the result is a rapid influx of neutrophils into the lung parenchyma and alveolar spaces. the inflammation leads to increased vascular permeability with leakage of fluid into the alveolar spaces and can eventually lead to alveolar collapse. if the condition is severe, it may result in adult respiratory distress syndrome and respiratory failure. it should be noted that infection is not present in the early stages of this condition but may complicate the problem after - hr. treatment. the treatment of aspiration lung injury is largely supportive and depends upon the severity of the inflammation and the clinical signs. in cases in which a small amount of a relatively innocuous substance (e.g., barium) has been aspirated, treatment may not be necessary. when severe inflammation is present, systemic fluid therapy should be instituted. support of the cardiovascular system should be performed judiciously; fluid overload could lead to an increase in pulmonary edema. the use of colloids is controversial because of the increase in vascular permeability that occurs in the lungs. oxygen therapy is also controversial, because it may increase lung injury if administered at high concentrations for long periods of time (nader-djahal et al., ) . several studies have addressed the use of anti-inflammatory agents to reduce lung injury associated with aspiration; however, none are used clinically in human or veterinary medicine at this time. in humans, antibiotics are reserved for use in cases with confirmed infection, in order to prevent the development of antibiotic-resistant pneumonia. it has been suggested that dogs should be treated with antibiotics immediately when the aspirated material is either not acidic or has potentially been contaminated by oral bacteria associated with severe dental disease. amoxicillin-clavulanate has been recommended as a first line of defense, reserving enrofloxacin for resistant cases (hawkins, ) . the presence of pneumonia should be verified by tracheal wash and cultures. etiology. in laboratory animals, accidental burns usually result from thermal injury (heating pads, water bottles) or harsh chemicals (strong alkalis, acids, disinfectants). the insult to the skin results in desiccation of the tissue and coagulation of proteins. in addition, the severely injured area is surrounded by a zone of vascular stasis, which promotes additional tissue damage. even small burns can result in significant inflammation that could affect the outcome of some research investigations and cause considerable discomfort to the animal. the proper and immediate treatment of burn wounds can reduce the effects of the injury on both the individual and the research. clinical signs. the clinical signs vary with the type and degree of burn injury. initially, the injury may not be noticed. the first signs may be oozing from the skin and matting of the overlying hair. within a couple of days, progressive hair and skin loss may be observed (johnston, ) . the wounds may vary in severity from very superficial (involving only the epidermis) to those in which the epidermis and dermis are completely destroyed. superficial wounds appear as red, inflamed skin similar to sunburn in humans. the pain associated with these injuries usually subsides in - days, and the wound reepithelializes without complications in - days. deeper burns develop a thick covering, or eschar, composed of the coagulated proteins and desiccated tissue fluid. the wound heals by granulation under the eschar, which eventually sloughs or is removed to allow further healing by contraction and reepithelialization. within - days of injury, the burn wound will be colonized by grampositive bacteria that rapidly cover the entire wound. several days later, gram-negative organisms can appear in the burn wound (johnston, ) . at this point, signs of wound infection and sepsis may occur (see section iii,d, ). treatment. appropriate and timely treatment of a burn wound will reduce the extent of the injury. thermal injuries should be immediately cooled to reduce edema and pain (demling and lalonde, ) . chemical burns should be thoroughly lavaged for min after wounding. the damaged tissues may be unable to mount appropriate responses to changes in temperature; therefore, the lavage should be performed with warm water to prevent hypothermia. after the initial treatment, all burn wounds should be gently cleansed - times a day (demling and lalonde, ) . burns involving the epidermis and part of the dermis can be extremely painful, and analgesia should be addressed throughout the treatment period. systemic antibiotics are unable to penetrate eschar and are not adequately distributed through the abnormal blood supply of burned tissues. therefore, topical wound dressings are recommended in the early stages of treatment. a thin film of a water-soluble broad-spectrum antibiotic ointment should be applied to the wound surface after each cleaning. silver sulfadiazine has a broad spectrum, penetrates eschar well, and is often the preparation of choice for burn wound therapy. povidone-iodine ointment will also penetrate thin eschar and provides a broad spectrum. mafenide has a good spectrum that covers gram-negative organisms well and is often used to treat infected wounds, although it is associated with pain upon application (demling and lalonde, ) . when signs of wound or systemic infection are present, systemic antibiotics should be employed, and their ultimate selection should be based on culture and sensitivity results. after the topical antibiotic has been applied, a nonadherent dressing should be placed on the wound. burn wounds covered in such a manner tend to epithelialize more rapidly and are less painful than uncovered wounds. when the eschar over a burn wound has formed and become fully defined, a small or moderately sized wound may be completely resected. prevention. obviously, prevention of burn wounds is preferable to a long course of treatment. care should be taken to prevent direct exposure to harsh chemicals. tables, floors, and other surfaces should be rinsed thoroughly after chemical use, prior to allowing any animal contact. electric heating pads should be avoided, and only heated water blankets or circulating warm-air devices should be used to provide warmth to the animals. in rare instances, heated water blankets have also caused burns; therefore these devices should be carefully monitored. as a precaution, a thin towel may be placed between the animal and the water blanket. etiology. research and/or anesthetic protocols may require the intravenous injection of various solutions. when these substances have a ph or osmolarity significantly different from that of the surrounding tissues, the accidental perivascular extravasation of the solutions may result in tissue damage. several drugs have been shown to cause problems when injected perivascularly, including pentobarbital, thiamylal, thiopental, thiacetarsemide, vincristine, vinblastine, and doxorubicin (swaim and angarano, ; waldron and trevor, ) . clinical signs. the immediate signs of perivascular injection are swelling at the injection site and withdrawal of the limb or other signs of discomfort. later, the area may appear red, swollen, and painful as inflammation progresses. often there will be eventual necrosis of the skin around the injection site. in cases of doxorubicin extravasation, signs may develop up to a week after the injection, and the affected area may progressively enlarge over a to month period. this is because the drug is released over time from the dying cells (swaim and angarano, ) . prevention. because the degree of injury and extensive treatment associated with perivascular extravasation of a drug can be detrimental to research protocols and can cause severe discomfort to the dog, prevention of these injuries is preferred. prior to the use of any substance, the investigator should be aware of its chemical composition and the potential for problems. if a potentially caustic compound is to be used in a fractious subject, sedation of the dog is warranted if this will not interfere with the research protocol. whenever possible, insertion of an indwelling catheter is extremely important. access to a central vessel such as the cranial or caudal vena cava is preferred over the use of peripheral vessels. when peripheral catheters are used, the injection should be followed by a vigorous amount of flushing with saline or other physiological solution and removal of the catheter. additional injections are best given through newly placed catheters in previously unused vessels. the repeated use of an indwelling peripheral catheter should be approached cautiously and done only out of necessity. prior to use, the catheter should be checked repeatedly for patency by withdrawal of blood and injection of saline. any swelling at the catheter site or discomfort by the subject indicates that the catheter should not be used. treatment. the treatment of perivascular injections will depend on the amount and type of substance injected. in most cases, dilution of the drug with subcutaneous injections of saline is recommended. in addition, steroids may be infiltrated locally to reduce inflammation. topical application of dimethyl sulfoxide (dmso) may also be helpful in reducing the immediate inflammation and avoiding the development of chronic lesions (swaim and angarano, ). the addition of lidocaine to subcutaneous injections of saline has been used in cases of thiacetarsemide injection (hoskins, ) , and local infiltration of hyaluronidase accompanied by warm compresses has been suggested for use in cases of vinblastine injection (waldron and trevor, ) . despite these treatments, necrosis of skin may be observed and would require serial debridement of tissues with secondary wound closure or skin grafting. in cases of doxorubicin extravasation, early excision of affected tissues is advocated to prevent the progressive sloughing caused by sustained release of the drug from dying tissues (swaim and angarano, ) . in all cases, the condition can be painful, and analgesia should be addressed. etiology. hepatic encephalopathy is the result of the derangements in metabolism associated with abnormal liver function. this condition may be seen in young dogs with congenital portosystemic shunting of blood flow. however, in the research setting, encephalopathy occurs more often in canine models of hepatic disease that lead to liver failure. a well-developed knowledge of the pathophysiology of liver disease is necessary for the initial treatment and long-term management of hepatic encephalopathy. pathogenesis. when the liver function is severely impaired because of either portosystemic shunting of blood flow or loss of metabolically active hepatic tissue, the result is an accumulation of ammonia, toxic amines, aromatic amino acids, and short-chain fatty acids (hardy, ; center, ) . these compounds have several toxic effects that result in a decrease in cerebral energy metabolism and a decrease in excitatory neurotransmitter synthesis. concurrently, there is an increase in the concentration of false neurotransmitters and the inhibitory substance -aminobutyric acid (gaba). clinical signs. the signs of hepatic encephalopathy include lethargy, depression, muscle tremors, and convulsions. diagnosis and differential diagnosis. a presumptive diagnosis of hepatic encephalopathy may be based on the appearance of clinical signs following experimental manipulation of the liver. additional diagnostic tests to verify the loss of liver function can be performed to confirm the diagnosis. serum glucose and protein levels may be low if hepatic function is severely impaired. a low serum urea nitrogen level suggests that the normal hepatic metabolism of ammonia into urea has been impaired. elevated levels of serum bile acids and blood ammonia also verify the loss of liver function (hardy, ) . measurement of serum hepatic leakage enzymes are nondiagnostic, because they can be low, high, or normal. treatment. because of the severity of hepatic encephalopathy, treatment may be initiated based on a presumptive diagnosis. during initial treatment, supportive care with fluids and electrolytes should be instituted, based on the results of serum chemistry and blood gas analysis. the majority of animals with hepatic dysfunction will be hypokalemic, alkalotic, and hypernatremic; therefore, either . % sodium chloride or . % sodium chloride with . % dextrose, supplemented with potassium chloride, is recommended (hardy, ) . the type of drug to be used for seizure control is controversial. the short halflife of diazepam makes it an attractive choice compared with barbiturates, which have prolonged metabolism when hepatic function is impaired (maddison, ) . however, endogenous benzodiazepines mediate some of the cns signs seen with hepatic encephalopathy. therefore, the use of diazepam has been discouraged in favor of phenobarbital (johnson, ) . the drug selected for seizure control should be titrated carefully, given the altered liver metabolism. most importantly, the treatment of dogs with hepatic encephalopathy must be aimed at reducing the levels of toxic metabolites in the bloodstream. because protein metabolism is a major source of ammonia, all oral food intake should cease until the signs of hepatic encephalopathy have abated. because gastrointestinal bleeding may occur in individuals with liver failure and this is also a source of protein, the use of h blockers such as cimetidine or ranitidine is suggested (swalec, ) . in addition, lactulose retention enemas should be performed ( - ml/lb of a % solution in water, retained for - min) (hardy, ) . lactulose is an indigestible semisynthetic sugar that is metabolized in the gut to lactic and other acids. the decrease in colonic ph reduces ammonia levels in the bloodstream by converting intestinal ammonia into less diffusible ammonium ions. lactulose will also cause an osmotic diarrhea. antibiotics such as neomycin ( mg/lb, - times/ day) or metronidazole ( mg/lb, times/day) should also be used to reduce the intestinal load of urease-producing bacteria responsible for splitting urea into ammonia (hardy, ) . when the signs of hepatic encephalopathy have resolved, the dog may be fed a low-protein diet. diets suitable for dogs with renal insufficiency are recommended initially. this type of diet is not suitable for long-term use, however, because it appears that individuals with some types of hepatic disease actually have increased protein requirements. these requirements may be met by slowly increasing protein in the diet as long as signs of hepatic encephalopathy do not recur. to maintain the appropriate balance of aromatic and branched-chain amino acids, the diet should be based on vegetable and dairy protein instead of meat or fish protein (center, ) . in addition, the antibiotics suggested above should be continued to reduce the effects of increasing dietary protein levels. the prevalence of cancer in the general canine population has increased over the years (dorn, ) . this can be attributed to the longer life spans resulting from improvements in nutrition, disease control, and therapeutic medicine. because of these changes, cancer has become a major cause of death in dogs (bronson, ) . in a lifetime cancer mortality study of intact beagles of both sexes, albert et al. ( ) found death rates similar to the death rate of the at-large dog population (bronson, ) . approximately % of the male beagles died of cancer. the majority of the tumors were lymphomas ( %) and sarcomas ( %), including hemangiosarcomas of the skin and fibrosarcomas. of the female beagles dying of cancer ( % of the population studied), three-quarters had either mammary cancer ( %), lymphomas ( %), or sarcomas ( %). of the sarcomas in females, one-third were mast cell tumors. in addition to these tumors that cause mortality, the beagle is also at risk for thyroid neoplasia (hayes and fraumeni, ; benjamin et al., ) . because of the popularity of the beagle as a laboratory animal, discussion of specific neoplasms will focus on the tumors for which this breed is at risk, as well as tumors that are common in the general canine population. fine-needle aspirates are generally the first diagnostic option for palpable masses, because they can easily be performed in awake, cooperative patients. this technique allows for rapid differentiation of benign and neoplastic processes. in cases where cytologic results from fine-needle aspirates are not definitive, more invasive techniques must be used. needle-punch or core biopsies can also be performed in awake patients but typically require local anesthesia. an instrument such as a tru-cut needle (travenol laboratories, inc., deerfield, illinois) is used to obtain a mm x to . cm biopsy of a solid mass. a definitive diagnosis may be limited by the size of the sample acquired using this technique. incisional and excisional biopsies are utilized when less invasive techniques fail to yield diagnostic results. excisional biopsies are the treatment of choice when surgery is necessary, because the entire mass is removed. surgical margins should extend at least cm around the tumor, and cm if mast cell tumors are suspected (morrison et al., ) . incisional biopsies are performed when large soft-tissue tumors are encountered and/or when complete excision would be surgically difficult or life-threatening. when performing an incisional biopsy, always select tissue from the margin of the lesion and include normal tissue in the submission. etiology. lymphomas are a diverse group of neoplasms that originate from lymphoreticular cells. whereas retroviral etiologies have been demonstrated in a number of species (e.g., cat, mouse, chicken), conclusive evidence of a viral etiology has not been established in the dog. in humans, data implicate the herbicide , -dichlorophenoxyacetic acid ( , -d) as a cause of non-hodgkin's lymphoma, but studies in dogs with similar conclusions have come under scrutiny (macewen and young, ) . clinical signs. multicentric and alimentary lymphomas account for most cases of canine lymphoma. in multicentric lymphoma, animals usually present with enlarged lymph nodes and nonspecific signs such as anorexia, weight loss, polyuria, polydypsia, and lethargy. when the liver and spleen are involved, generalized organomegaly may be felt on abdominal palpation. alimentary lymphoma is associated with vomiting and diarrhea, in addition to previous clinical signs. less commonly, dogs develop mediastinal, cutaneous, and extranodal lymphomas. dogs with mediastinal lymphoma often present with respiratory signs secondary to pleural effusion. hypercalcemia is most frequently associated with this form of lymphoma and may result in weakness. cutaneous lymphoma varies in presentation from solitary to generalized and may mimic any of a number of other skin disorders. the tumors may occur as nodules, plaques, ulcers, or dermatitis. approximately half of the cases are pruritic. a number of extranodal forms of lymphoma have been reported, including tumors affecting the eyes, central nervous system, kidneys, or nasal cavity. clinical presentation varies, depending on the site of involvement. epizootiology. the incidence of lymphoma is highest in dogs - years old, accounting for % of cases. although the neoplasm generally affects dogs older than year, cases in puppies as young as months have been reported (dorn et al., ) . pathologic findings. enlarged neoplastic lymph nodes vary in diameter from to cm and are moderately firm. some may have areas of central necrosis and are soft to partially liquefied. the demarcation between cortex and medulla is generally lost, and on cut section, the surface is homogenous. the spleen may have multiple small nodular masses or diffuse involvement with generalized enlargement. the enlarged liver may have disseminated pale foci or multiple large, pale nodules. in the gastrointestinal tract, both nodular and diffuse growths are observed. these masses may invade through the stomach and intestinal walls. histologically, the most common lymphomas are classified as intermediate to high grade and of large-cell (histiocytic) origin. the neoplastic lymphocytes typically obliterate the normal architecture of the lymph nodes and may involve the capsule and perinodal areas. pathogenesis. all lymphomas regardless of location should be considered malignant. a system for staging lymphoma has been established by the world health organization. the average survival time for dogs without treatment is - weeks. survival of animals undergoing chemotherapy is dependent on the treatment regimen as well as the form and stage of lymphoma (macewen and young, ) . hypercalcemia is a paraneoplastic syndrome frequently associated with lymphoma. the pathogenesis of this phenomenon is not fully understood but may be a result of a parathormone-like substance produced by the neoplastic lymphocytes. diagnosis and differential diagnosis. differential diagnoses for multicentric lymphoma include systemic mycosis; salmonpoisoning and other rickettsial infections; lymph node hyperplasia from viral, bacterial, and/or immunologic causes; and dermatopathic lymphadenopathy. alimentary lymphoma must be distinguished from other gastrointestinal tumors, foreign bodies, and lymphocytic-plasmacytic enteritis. in order to make a definitive diagnosis, whole lymph node biopsies and full-thickness intestinal sections are frequently needed. treatment. therapy for lymphoma typically consists of one or a combination of several chemotherapeutic agents. the treatment regimen is based on the staging of the disease, the presence of paraneoplastic syndromes, and the overall condition of the patient. macewen and young ( ) provide a thorough discussion of therapeutic options for the treatment of lymphomas in the dog. research complications. given the grave prognosis for lymphoma with or without treatment, euthanasia should be considered for research animals with signifcant clinical illness. etiology. the fibrosarcoma group of tumors encompasses not only malignant tumors of fibroblasts but also a number of indistinguishable tumors, all of which are capable of collagen production (pulley and stannard, ) . frequently classified in this group are undifferentiated leiomyosarcomas, liposarcomas, malignant melanomas, and malignant schwannomas. clinical signs. although these neoplasms can arise throughout the body, they are most commonly found in the skin, subcutaneous tissues, and oral cavity. fibrosarcomas are extremely variable in size and can grow to be quite large. in general, they are irregular and nodular, poorly demarcated, and nonencapsulated, and they frequently invade deeper tissues. epizootiology. most fibrosarcomas develop in adult and aged animals but can affect dogs as young as months or less. pathogenesis. fibrosarcomas exhibit rapid, invasive growth, recurring frequently after excision. metastasis occurs in only one-fourth of cases, usually by the bloodstream to the lungs. less frequently, spread to local lymph nodes is observed. diagnosis and differential diagnosis. differential diagnoses for fibrosarcomas vary with the location of the tumor. histopathologic exam should be used to distinguish these tumors from round cell tumors (mast cell tumors, histiocytomas, transmissible venereal tumors), papillomas, and other neoplasms. treatment. treatment of any soft-tissue sarcoma would begin with wide surgical excision. if the tissue margins indicate incomplete resection, radiotherapy could be used. for any highgrade tumors, adjuvant chemotherapy would be recommended (see macewen and withrow, a , for a complete discussion). research complications. because fibrosarcomas are locally invasive and often recur, dogs with these neoplasms should not be considered good subjects for long-term studies. etiology. neoplasms of lipocytes and lipoblasts are welldifferentiated tumors referred to as lipomas. clinical signs. these growths can be found as single or multiple round, ovoid, or discoid masses in the subcutaneous tissues of the lateral and ventral thorax, abdomen, and upper limbs. generally they are well circumscribed, encapsulated, and soft on palpation. further, the skin is freely movable over the tumor. epizootiology. lipomas occur principally in aged animals (average years), and the incidence increases with age (pulley and stannard, ) . the tumors are most commonly seen in overweight female dogs, but no breed predisposition is observed. pathologic findings. histologically, lipomas are indistinguishable from normal adipose tissue except when a fibrous capsule is present. pathogenesis: lipomas are typically slow-growing and do not recur after complete surgical excision. diagnosis and differential diagnosis. lipomas are not frequently confused with other tumors but can sometimes be difficult to distinguish from normal adipose tissue. generally, the distinction can be made from the clinical history. treatment. treatment for lipomas is not usually necessary unless the mass is causing problems with normal ambulation. in such cases, surgical excision is usually curative. research complications. lipomas usually do not complicate research studies unless they are interfering with other systemic functions or ambulation. etiology. histiocytomas are benign skin growths that arise from the monocyte-macrophage cells in the skin. some debate exists as to whether this growth is actually a neoplasm or a focal inflammatory lesion (pulley and stannard, ) . clinical signs. the most frequent sites for histiocytomas are the head (especially the pinna) and the skin of the distal forelegs and feet. the masses are usually domelike or buttonlike (often referred to as "button tumors") and usually measure - cm in diameter. epizootiology. histiocytomas are the most common tumors of young dogs, mostly occurring in dogs less than years of age. pathologic findings. histologically, these tumors contain round to ovoid cells with pale cytoplasm and large nuclei. the cells infiltrate the dermis and subcutis, displacing collagen fibers and skin adnexa. despite being benign lesions, histiocytomas characteristically have a high mitotic index. pathogenesis. this tumor typically exhibits rapid growth ( - weeks) but does not spread. most histiocytomas will spontaneously regress in less than months. diagnosis and differential diagnosis. histiocytomas must be distinguished from potentially metastatic mast cell tumors. this is accomplished by staining with toluidine blue, which would stain the cytoplasmic granules of mast cells red or purple. treatment. although most histiocytomas will spontaneously resolve, conservative surgery or cryosurgery will provide an expeditious resolution. research complications. histiocytomas should not interfere with most studies. etiology. neoplastic proliferations of mast cells are the most commonly observed skin tumor of the dog (bostock, ) . mast cells are normally found in the connective tissue beneath serous surfaces and mucous membranes, and within the skin. clinical signs. well-differentiated mast cell tumors are typically solitary, well-circumscribed, slow-growing, to cm nodules in the skin. alopecia may be observed, but ulceration is not usual. poorly differentiated tumors grow rapidly, may ulcerate, and may cause irritation, inflammation, and edema. mast cell tumors can be found on any portion of the dog's skin but frequently affect the hindquarters, especially the thigh and in-guinal and scrotal areas. mast cell tumors usually appear to be discrete masses, but they frequently extend deep into surrounding tissues. epizootiology. these tumors tend to affect middle-aged dogs but have been observed in dogs ranging from months to years (pulley and stannard, ). pathologic findings. because of the substantial variation in histologic appearance of mast cell tumors, a classification and grading system described by patnaik et al. ( ) has become widely accepted. in this system, grade i has the best prognosis, and grade iii the worst prognosis. grade i tumors are well differentiated, with round to ovoid uniform cells. the nuclei are regular, the cytoplasm is packed with large granules that stain deeply, and mitotic figures are rare to absent. grade ii (intermediately differentiated) mast cell tumors have indistinct cytoplasmic boundaries with higher nuclear-cytoplasmic ratios, fewer granules, and occasional mitotic figures. grade iii (anaplastic or undifferentiated) mast cell tumors have large, irregular nuclei with multiple prominent nucleoli. the cytoplasmic granules are few, but mitotic figures are much more frequent. in addition to skin lesions, mast cell tumors have been associated with gastric ulcers. these lesions are most likely secondary to tumor production of histamine. histamine stimulates the h receptors of the gastric parietal cells, causing increased acid secretion. gastric ulcers have been observed in large numbers (> %) of dogs with mast cell tumors (howard et al., ) . the ulcers can be found in the fundus, pylorus, and/or proximal duodenum. although all mast cell tumors should be considered potentially malignant, the outcome in individual cases can be correlated with the histologic grading of the tumor. grade iii tumors are most likely to disseminate internally. this spread is usually to regional lymph nodes, spleen, and liver and less frequently to the kidneys, lungs, and heart. diagnosis and differential diagnosis. mast cell tumors can be distinguished histologically from other round cell tumors (such as histiocytomas and cutaneous lymphomas) by using toluidine blue, which metachromatically stains the cytoplasmic granules of the mast cells red or purple. treatment. initial treatment for mast cell tumors is generally wide surgical excision ( to cm margins). even with wide surgical margins, approximately % of mast cell tumors may recur. if the site is not amenable to wide surgical excision, debulking surgery and radiation therapy may be used. other alternatives include amputation (if on a limb) or radiation therapy alone. as an adjunct to surgery, grier et al. ( grier et al. ( , found that deionized water injected into surgical margins reduced tumor recurrence by hypo-osmotically lysing any mast cells left behind. this technique has recently been refuted by jaffe et al. ( ) . for systemic mastocytosis, and nonresectable or incompletely excised mast cell tumors, chemotherapy can be used. treatment options would include oral prednisolone, intralesional triamcinalone, and the combination of cyclophosphamide, vincristine, and prednisolone (graham and o'keefe, ) . research complications. because of the possibility of systemic histamine release and tumor recurrence, dogs with mast cell tumors are not good candidates for research studies. grade i mast cell tumors may be excised, allowing dogs to continue on study; however, monitoring for local recurrence should be performed on a regular basis (monthly). grade ii tumors are variable; animals that undergo treatment should be monitored for recurrence monthly, and evaluation of the buffy coat should be performed every - months for detection of systemic mastocytosis. because of the poor prognosis for grade iii tumors, treatment is unwarranted in the research setting. etiology. hemangiosarcomas are malignant tumors that originate from endothelial cells. clinical signs. these tumors may arise in the subcutis but are more commonly found in the spleen and the right atrium. clinical signs are associated with the site of involvement. vascular collapse is frequently observed secondary to rupture and hemorrhage from splenic masses. heart failure can be observed secondary to tumor burden or hemopericardium. when found in the skin, hemangiosarcomas are poorly circumscribed, reddish black masses that range in size from to cm in diameter. the most common cutaneous sites are the ventral abdomen, the prepuce, and the scrotum. epizootiology: hemangiosarcomas occur most frequently in -to -year-old dogs. the german shepherd dog is most commonly affected. pathologic findings. grossly, splenic hemangiosarcomas resemble nodular hyperplasia or hematomas (fig. ) . the masses are spherical and reddish black and can range in size up to - cm in diameter. on cut section the masses may appear reddish gray or black and have cavernous areas of clotted blood. when the masses are found in the heart, the endocardium may be covered by a thrombus, giving the to cm tumors a reddish gray or yellow appearance. histologically, hemangiosarcomas are composed of immature endothelial cells that form vascular channels or clefts. these spaces may be filled with blood or thrombi. the neoplastic cells are elongated with round to ovoid, hyperchromatic nuclei and frequent mitotic figures. pathogenesis. hemangiosarcomas can be found in one or many sites. in cases where multiple sites are involved, it may be impossible to identify the primary tumor. this neoplasia is highly malignant and spreads easily. metastasis occurs most frequently to the lungs but can be found in any tissue. diagnosis and differential diagnosis. splenic hemangiosarcoma may resemble nodular hyperplasia or some manifestations of lymphoma. when the heart is affected, other causes of heart failure must be ruled out. echocardiography is a valuable tool for identifying the primary lesion. histopathology should be used to differentiate dermal hemangiosarcoma from hemangiomas and other well-vascularized tumors. treatment. surgery is generally the first choice of treatment for hemangiosarcoma. dermal tumors are treated with radical resection, splenic tumors by total splenectomy, and heart tumors by debulking and pericardiectomy. because of the high likelihood of metastasis, adjunct chemotherapy should always be considered. research complications. dogs with dermal hemangiosarcoma may be cured after complete resection with margins, but monitoring should be done regularly for recurrence. the other forms of hemangiosarcoma have a much poorer long-term prognosis, and treatment is typically unwarranted in the research setting. etiology. also known as infectious or venereal granuloma, sticker tumor, transmissible sarcoma, and contagious venereal tumor, the transmissible venereal tumor is transmitted to the genitals by coitus (nielsen and kennedy, ) . the origin of this tumor is still unknown but has been described as a tumor of lymphocytes, histiocytes, and reticuloendothelial cells. although this tumor has been reported in most parts of the world, it is most prevalent in temperate climates (macewen, ) . clinical signs. the tumors are usually cauliflower-like masses on the external genitalia, but they can also be pedunculated, nodular, papillary, or multilobulated. these friable masses vary in size up to cm, and hemorrhage is frequently observed. in male dogs, the lesions are found on the caudal part of the penis from the crura to the bulbus glandis or on the glans penis (fig. ) . less frequently, the tumor is found on the prepuce. females typically have lesions in the posterior vagina at the junction of the vestibule and vagina. when located around the urethral orifice, the mass may protrude from the vulva. these tumors have also been reported in the oral cavity, skin, and eyes. epizootiology and transmission. transmissible venereal tumors are most commonly observed in young, sexually active dogs. transmission takes place during coitus when injury to the genitalia allows for transplantation of the tumor. genital to oral to genital transmission has also been documented (nielsen and kennedy, ) . extragenital lesions are believed to be a result of trauma prior to exposure to the tumor. pathogenesis. tumor growth is rapid after implantation but later slows. metastasis is rare (< % of cases) but may involve the superficial inguinal and external iliac lymph nodes as well as distant sites. diagnosis and differential diagnosis. transmissible venereal tumors have been confused with lymphomas, histiocytomas, mast cell tumors, and amelanotic melanomas. cytology may be of benefit in making a definitive diagnosis, so impression smears should be made prior to processing for histopathology. prevention. thorough physical examinations prior to bringing new animals into a breeding program should prevent introduction of this tumor into a colony. control. removing affected individuals from a breeding program should stop further spread through the colony. treatment. surgery and radiation can be used for treatment, but chemotherapy is the most effective. vincristine ( . - . mg/m ) iv once weekly for - treatments will induce remission and cure in greater than % of the cases (macewen, ). research complications. experimental implantation of transmissible venereal tumors has been shown to elicit formation of tumor-specific igg (cohen, ) . this response may occur in natural infections and could possibly interfere with immunologic studies. etiology. dogs are susceptible to a wide variety of mammary gland neoplasms, most of which are influenced by circulating reproductive steroidal hormones. clinical signs. single nodules are found in approximately % of the cases of canine mammary tumors. the nodules can be found in the glandular tissue or associated with the nipple. masses in the two most caudal glands (fourth and fifth) account for a majority of the tumors. benign tumors tend to be small, well circumscribed, and firm, whereas malignant tumors are larger and invasive and coalesce with adjacent tissues. epizootiology. mammary tumors are uncommon in dogs under years of age with the incidence rising sharply after that. median age at diagnosis is - years. mammary tumors occur almost exclusively in female dogs, with most reports in male dogs being associated with endocrine abnormalities, such as estrogen-secreting sertoli cell tumors. pathologic findings. based on histologic classification of mammary gland tumors, approximately half of the reported tumors are benign (fibroadenomas, simple adenomas, and benign mesenchymal tumors), and half are malignant (solid carcinomas, tubular adenocarcinomas, papillary adenocarcinomas, anaplastic carcinomas, sarcomas, and carcinosarcomas) (bostock, ) . extensive discussions of classification, staging, and histopathologic correlations can be found in macewen and withrow ( lb) and moulton ( ) . pathogenesis. mammary tumors of the dog develop under the influence of hormones. receptors for both estrogen and progesterone can be found in - % of tumors. futher, schneider et al. ( ) showed that the risk of developing mammary tumors increased greatly after the first and second estrus cycles. dogs spayed prior to the first estrus had a risk of . %, whereas dogs spayed after the first and second estrus had risks of % and %, respectively. malignant mammary tumors typically spread through the lymphatic vessels. metastasis from the first, second, and third mammary glands is to the ipsilateral axillary or anterior sternal lymph nodes. the fourth and fifth mammary glands drain to the superficial inguinal lymph nodes where metastasis can be found. many mammary carcinomas will eventually metastasize to the lungs. diagnosis and differential diagnosis. both benign and malignant mammary tumors must be distinguished from mammary hyperplasia and mastitis. prevention. mammary tumors can effectively be prevented by spaying bitches prior to the first estrus. this is commonly done in the general pet population at months of age. recently, the topic of spaying sexually immature dogs ( - weeks of age) has received much attention for the control of the pet population. kustritz ( ) reviewed the techniques for anesthesia and surgery, as well as possible pros and cons of spaying at this young age. treatment. surgery is the treatment of choice for mammary tumors, because chemotherapy and radiation therapy have not been reported to be effective. the extent of the surgery is dependent on the area involved. lumpectomy or nodulectomy should be elected in the case of small discrete masses, while mammectomy and regional or total mastectomies are reserved for more aggressive tumors. at the time of surgery, axillary lymph nodes are removed only if enlarged or positive on cytology for metastasis. inguinal lymph nodes should be removed any time the fourth and fifth glands are excised (macewen and withrow, lb). research complications. because % of mammary tumors are benign, treatment may be rewarding, allowing dogs to con-tinue on study. if removed early enough, malignant masses could yield the same results. all dogs should be monitored regularly for recurrence and new mammary tumors. etiology. beagles are among the breeds with the highest prevalence of thyroid carcinomas. benjamin et al. ( ) reported a correlation between lymphocytic thyroiditis, hypothyroidism, and thyroid neoplasia in the beagle. clinical signs. thyroid carcinomas generally present as palpable cervical masses. affected animals may experience dysphagia, dyspnea, and vocalization changes. precaval syndrome resulting in facial edema is also observed in some cases. epizootiology and transmission. the mean age of dogs presented with thyroid carcinomas is years, with equal distribution of cases between the sexes. pathologic findings. grossly, thyroid carcinomas are multinodular masses, frequently with large areas of hemorrhage and necrosis. they tend to be poorly encapsulated and invade local structures such as the trachea, esophagus, larynx, nerves, and vessels. the masses are unilateral twice as often as bilateral (capen, ) . histologically, thyroid carcinomasare divided into follicular, papillary, and compact cellular (solid) types (see capen, , for complete discussion). pathogenesis. thyroid carcinomas tend to grow rapidly and invade local structures. early metastasis is common and occurs to the lungs by invasion of branches of the thyroid vein. diagnosis and differential diagnosis. nonpainful cervical swellings such as seen with thyroid tumors are also consistent with abscesses, granulomas, salivary mucoceles, and lymphomas. often a preliminary diagnosis can be made by fineneedle aspirate. treatment. surgery is the treatment of choice for thyroid carcinomas that have not metastasized. when the tumor is freely movable, surgery may be curative. surgical excision may be difficult for tumors that adhere to local structures, requiring excision of the jugular vein, carotid artery, and associated nerves. when bilateral tumors are observed, preservation of the parathyroid glands may not be possible. in such cases, treatment for hypoparathyroidism will be necessary. both chemotherapy and radiation therapy have been suggested for extensive bilateral tumors and/or after incomplete excision, but no controlled trials have been performed (ogilvie, ) . in the research setting, treatment of this tumor may not be rewarding. only freely movable tumors can be practically treated without seriously affecting research efforts. euthanasia is warranted in the more advanced cases when clinical illness is apparent. beagles are subject to many of the inherited and/or congenital disorders that affect dogs in general. in a reference table on the congenital defects of dogs (hoskins, b) , disorders for which beagles are specifically mentioned include brachyury (short tail), spina bifida, pulmonic stenosis, cleft palate-cleft lip complex, deafness, cataracts, glaucoma, microphthalmos, optic nerve hypoplasia, retinal dysplasia, tapetal hypoplasia, factor vii deficiency, pyruvate kinase deficiency, pancreatic hypoplasia, epilepsy, gm gangliosidosis, globoid cell leukodystrophy, xx sex reversal, and cutaneous asthenia (ehlers-danlos syndrome). in addition, there are defects that affect so many breeds that the author simply lists "many breeds" for the breeds affected by those disorders. thus these defects could also affect beagles and include pectus excavatum, polydactyly, radial and ulnar dysplasia, hypoadrenocorticism, entropion, lens coloboma, factor viii deficiency (von willebrand's disease), renal agenesis or ectopia, and developmental defects of the reproductive and lower urinary tracts. at a commercial breeder of purpose-bred beagles, the most common birth defects were umbilical hernia ( . % of births) and open fontanelle ( . % of births) (r. scipioni and j. ball, personal communication, ) . other defects observed include cleft palate and cleft lip, cryptorchidism, monorchidism, limb deformity, inguinal hernia, diaphragmatic hernia, hydrocephaly, and fetal anasarca. each of these other congenital defects occurred at less than . % incidence. etiology. cataract is an opacification of the lens or the lens capsule. it is the pathologic response of the lens to illness or injury, because the lens has no blood supply. cataracts can be caused by metabolic, inflammatory, infectious, or toxic causes and can be congenital, juvenile, or degenerative. nuclear sclerosis is an apparent opacification of the lens caused by the compression of older lens fibers in the center of the lens (nucleus) as a consequence of the production of new fibers. because the nucleus increases in size as the animal ages, the sclerosis is more apparent in older animals and may be mistaken as a senile cataract. the ability to see the fundus during ophthalmoscopy persists with nuclear sclerosis but is obstructed by a true cataract. clinical signs. the first clinical sign is typically the ability to visualize the opaque lens through the pupil of the dog's eye. dogs have an impressive ability to tolerate bilateral lens opacity (especially when development is gradual), and often visual impairment is detected late in the development of the condition (helper, ) . moderate vision loss may cause the dog to be hesitant in moving in new surroundings or unable to locate movable objects (such as a toy). rapid cataract development can result in a sudden vision loss, such as can occur with diabetic cataracts. epizootiology and transmission. certain dog breeds can be predisposed to the development of juvenile or senile cataracts or to metabolic disorders that result in cataract development, such as diabetes mellitus. dogs in studies for diabetes mellitus should be observed regularly for cataract development. toxicological studies may also induce formation of cataracts. pathogenesis. lens fibers respond to all biological or chemical insults by necrosis and liquefaction (render and carlton, ) , because they have no blood supply with which to recruit an inflammatory and repair process. disruption of these fibers by any means, therefore, leads to opacification. the exact processes by which the varieties of congenital and juvenile cataracts are produced have not been determined. in diabetic cataracts, the excess glucose is metabolized to sorbitol and fructose. as these alcohols and sugars accumulate in the lenticular cells, they produce an osmotic imbalance, which brings fluid into the cells, causing swelling and degeneration of lens fibers and resultant opacity (capen, ) . diagnosis and differential diagnosis. the ability to visualize the retina and fundus during ophthalmoscopy differentiates true cataracts from nuclear sclerosis. dogs with cataracts should be evaluated for possible causes, especially diabetes mellitus. diabetes mellitus will typically affect middle-aged dogs and feature rapid cataract formation, whereas juvenile and senile cataracts are slow to develop and affect younger and older dogs, respectively. progressive retinal atrophy can also cause secondary cataract formation; pupillary light response is maintained with primary cataracts (even if the lens is completely opaque), whereas this reflex is obtunded by retinopathy. prevention. most forms of cataracts cannot be prevented, for their exact etiologic pathogenesis is unknown. diabetic cataracts, however, can be prevented by proper regulation of blood glucose concentrations with insulin therapy and proper diet. treatment. because dogs do not need to focus visual images as accurately as human beings, proper lens clarity and function are not necessary for an adequate quality of life. many dogs adjust quite well to the visual impairment caused by persistent cataracts. lens removal can be performed for dogs seriously affected by cataracts, but this would not be anticipated for dogs in the research setting. information on surgical lens extraction procedures can be found in helper ( ) or other veterinary ophthalmology textbooks. research complications. research complications would be minimal with cataracts, unless the dogs were intended for use in ophthalmologic or visual acuity-based studies. etiology. hip dysplasia is a degenerative disease of the coxofemoral joint. a specific etiology is unknown, but the development of hip dysplasia has a strong genetic component (pedersen et al., ) , modified by age, weight, size, gender, conformation, rate of growth, muscle mass, and nutrition (smith et al., ) . clinical signs. the initial clinical abnormality caused by hip dysplasia is laxity of the coxofemoral joint. this may present as a gait abnormality without any indication of lameness or stiffness. eventually, affected dogs will have periods of lameness and, in protracted cases, will be rendered immobile by severe pain. epizootiology and transmission. hip dysplasia has been seen in most dog breeds, but it typically affects larger breeds of dogs. in the research setting, it is primarily a condition of randomsource large-breed dogs used for surgical research. diagnosis and differential diagnosis. hip dysplasia is classically diagnosed by radiography of the pelvis and hip joints. radiographic abnormalities consistent with hip dysplasia include shallow acetabula with remodeling of the acetabular rim, flattening of the femoral head, subchondral bone sclerosis (caused by erosion of articular cartilage and exposure of underlying bone), and osteophyte production around the joint (pedersen et al., ) . hip dysplasia needs to be differentiated from other musculoskeletal or neurological conditions that can cause unusual gaits and/or lameness. this may be somewhat difficult, because clinical signs of hip dysplasia may develop before radiographic abnormalities. radiographic calculation of the distraction index (di) to measure joint laxity has proven to be a good means to predict future hip dysplasia before other radiographic changes are evident (smith et al., ) . prevention. because of the genetic component, dogs with hip dysplasia should not be used in breeding colonies. dogs should be provided a good plane of nutrition but not be allowed to become overweight. dogs that were limit-fed at % of the food amount eaten by ad libitum-fed dogs had lower body weights and decreased severity of radiographic lesions of hip dysplasia (kealy et al, ) . treatment. in the stages when clinical signs are episodic, cage rest and analgesics for several days can be used to treat the symptoms. more advanced cases may require continuous analgesia. sectioning of the pectineus muscle or tendon may provide some pain relief but does not affect the progression of the disease (pedersen et al., ) . surgical treatments for hip dysplasia include femoral head ostectomy and total hip replacement. neither surgical treatment is likely in a research setting. research complications. long-term studies using large-breed dogs may be affected by the eventual development of hip dysplasia. in studies where hip dysplasia would be a serious complication or confounding variable (e.g., orthopedic research), dogs should be radiographed upon arrival to assess possibility of early coxofemoral joint degeneration and suitability for use in the study. etiology. benign prostatic hyperplasia (bph) is an agerelated condition in intact male dogs. the hyperplasia of prostatic glandular tissue is a response to the presence of both testosterone and estrogen. clinical signs. bph is often subclinical. straining to defecate (tenesmus) may be seen because the enlarged gland impinges on the rectum as it passes through the pelvic canal. urethral discharge (yellow to red) and hematuria can also be presenting clinical signs for bph. epizootiology and transmission. bph typically affects older dogs (> years), although it has been seen as early as years of age. pathologic findings. in its early stages, canine b ph is hyperplasia of the prostatic glandular tissue. this is in contrast to human bph, which is primarily stromal in origin. eventually, the hyperplasia tends to be cystic, with the cysts containing a clear to yellow fluid. the prostate becomes more vascular (resulting in hematuria or hemorrhagic urethral discharge), and bph may be accompanied by mild chronic inflammation. pathogenesis. bph occurs in older intact male dogs because increased production of estrogens (estrone and estradiol), combined with decreased secretion of androgens, sensitizes prostatic androgen receptors to dihydrotestosterone. the presence of estrogens may also increase the number of androgen receptors, and hyperplastic prostate glands also have an increased ability to metabolize testosterone to a-dihydrotestosterone (kustritz and klausner, ) . diagnosis and differential diagnosis. bph is diagnosed in cases of nonpainful symmetrical swelling of the prostate gland in intact male dogs, with normal hematologic profiles and urinalysis characterized by hemorrhage, at most. differential diagnoses include squamous metaplasia of the prostate, paraprostatic cysts, bacterial prostatitis, prostatic abscessation, and prostatic neoplasia (primarily adenocarcinoma). these differential diagnoses also increase in frequency with age and, except for squamous metaplasia, can also occur in castrated dogs. as such, these conditions do not necessarily abate or resolve when castration is used for treatment of prostatic enlargement. prevention. castration is the primary means for prevention of benign prostatic hyperplasia. treatment. the first and foremost treatment for b ph is castration. in pure cases of b ph, castration results in involution of the prostate gland detectable by rectal palpation within - days. for most dogs in research studies this is a viable option to rapidly improve the animal's condition. the alternative to castration is hormonal therapy, primarily with estrogens. this may be applicable in cases in which the dog is a valuable breeding male (e.g., genetic diseases), and semen collection is necessary. if the research study concerns steroidal hormone functions, then neither the condition nor the treatment is compatible. newer drugs marketed for human males have also shown promise in treating canine bph. finasteride (proscar) is a a-reductase inhibitor that limits metabolism of testosterone to a-dihydrotestosterone. treatment at daily doses of - mg/kg has been effective in causing prostatic atrophy without affecting testicular spermatogenesis (kustritz and klausner, ) . dogs given . mg/kg were proven to still be fertile. there are also indications that lower doses may be effective in relieving b ph. androgen receptor antagonists (flutamide and hydroxyflutamide) have also been studied in the dog and found to be effective for treatment of bph while maintaining libido and fertility (kustritz and klausner, ) . unfortunately, both the areductase inhibitors and the androgen receptor antagonists are not presently labeled for use in male dogs in the united states. research complications. bph can cause complications to steroidal hormone studies, in that the condition may be indicative of abnormal steroidal hormone metabolism, and neither castration nor estrogen therapy is compatible with study continuation. it is presently unknown whether the use of the newer antihyperplastic agents systemically alters physiologic parameters outside of the prostate itself. the development of tenesmus as a clinical sign may also affect studies of colorectal or anal function. etiology. juvenile polyarteritis syndrome (jps) is a painful disorder seen in young beagles (occasionally reported in other breeds). the lesion consistent with the syndrome is systemic necrotizing vasculitis. the cause of the vasculitis has not been established, but it appears to have an autoimmune-mediated component and may have a hereditary predisposition. clinical signs. clinical signs of jps include fever, anorexia, lethargy, and reluctance to move the head and neck. the dogs tend to extend the neck ventrally. most dogs seem to be in pain when touched, especially in the neck region. the syndrome typically has a course of remissions and relapses characterized by - days of illness and - weeks of remission (scott-moncrieff et al., ) . there may be a component of this condition that is subclinical, given that a vasculitis has been diagnosed postmortem in beagles that had no presenting signs. epizootiology and transmission. jps typically affects young beagles ( - months), with no sex predilection. pathologic findings. on gross necropsy, foci of hemorrhage can be seen in the coronary grooves of the heart, cranial mediastinum, and cervical spinal cord meninges (snyder et al., ) . local lymph nodes may be enlarged and hemorrhagic. histologically, necrotizing vasculitis and perivasculitis of small to medium-sized arteries are seen. these lesions are most noticeable in the three locations where gross lesions are observed, but they may be seen in other visceral locations. the perivasculitis often results in nodules of inflammatory cells that eccentrically surround the arteries (fig. a) . the cellular composition of these nodules is predominantly neutrophils, but it can also consist of lymphocytes, plasma cells, or macrophages (snyder et al., ) . fibrinous thrombosis of the affected arteries is also seen (fig. b) . a subclinical vasculitis has also been diagnosed in beagles postmortem; it is not known whether this subclinical condition is a different disorder or part of a jps continuum. this subclinical vasculitis often affects the coronary arteries (with or without other sites). pathogenesis. the initiating factors for jps are unknown. it was once presumed to be a reaction to test compounds by laboratory beagles, but this may have been coincident to the fact that the beagle is the breed most often affected with jps. immune mediation of jps is strongly suspected, because the clinical signs have a cyclical nature and respond to treatment with corticosteroids, and the affected dogs have elevated a -globulin fractions and abnormal immunologic responses. there may be hereditary predisposition, given that pedigree analysis of some affected dogs has indicated that the offspring of certain sires are more likely to be affected, and breeding of two affected dogs resuited in / affected pups (scott-moncrieff et al., ) . diagnosis and differential diagnosis. differential diagnoses include encephalitis, meningitis, injury or degeneration of the cervical vertebrae or disks, and arthritis. in the research facility, the disorder may be readily confused with complications secondary to the experimental procedure, or with postsurgical pain. beagles with jps that were in an orthopedic research study were evaluated for postsurgical complications and skeletal abnormalities prior to the postmortem diagnosis of systemic vasculitis (authors' personal experience). are known at this time. no prevention and control measures brane (third eyelid). this is not considered a congenital anomaly, but there is breed disposition for this condition, including beagles. a specific etiology is not known. clinical signs. the glandular tissue of the nictitating membrane protrudes beyond the membrane's edge and appears as a reddish mass in the ventromedial aspect of the orbit (fig. ) . excessive tearing to mucoid discharge can result, and severe cases can be associated with corneal erosion. treatment. clinical signs can be abated by administration of corticosteroids. prednisone administered orally at . mg/kg, q hr, was associated with rapid relief of clinical symptoms. maintenance of treatment at an alternate-day regimen of . - . mg/kg was shown to relieve symptoms for several months. however, withdrawal of corticosteroid therapy led to the return of clinical illness within weeks. pathologic findings. typically the glandular tissue is hyperplastic, possibly with inflammation. rarely is the tissue neoplastic. pathogenesis. prolapse of the gland may be a result of a congenital weakness of the connective tissue band between the gland and the cartilage of the third eyelid (helper, ) . research complications. because of the potentially severe clinical signs and the need for immunosuppressive treatment, jps is often incompatible with use of the animal as a research subject. it is unknown whether subclinical necrotizing vasculitis causes sufficient aberrations to measurably alter immunologic responses. etiology. "cherry eye" is a commonly used slang term for hyperplasia and/or prolapse of the gland of the nictitating mem-prevention. hyperplasia of the third eyelid cannot be prevented, but dogs that develop this condition unilaterally should have the other eye evaluated for potential glandular prolapse. preventative surgical measures might be warranted. treatment. corticosteroid treatment (topical or systemic) can be used to try to reduce the glandular swelling. however, surgical reduction or excision of the affected gland is typically required to resolve the condition. in the reduction procedure, the prolapsed gland is sutured to fibrous tissue deep to the fornix of the conjunctiva (helper, ). if reduction is not possible (as with deformed nictitating cartilage) or is unsuccessful, removal of the gland can be performed. such excision is fairly straightforward and can be done without removal of the nictitating membrane itself. the gland of the third eyelid is important in tear production; although the rest of the lacrimal glands should be sufficient for adequate tear production, keratoconjunctivitis sicca is a possible consequence after removal of the gland of the nictitating membrane. research complications. in most cases, research complications would be minimal, especially if treated adequately. either the presence of the hyperplastic gland, or its removal, might compromise ophthalmologic studies. etiology. interdigital cysts are chronic inflammatory lesions (not true cysts) that develop in the webbing between the toes (fig. ). the cause for most interdigital cysts is usually not identified unless a foreign body is present. bacteria may be isolated from the site, but the lesions may also be sterile (hence the synonym "sterile pyogranuloma complex"). clinical signs. dogs with interdigital cysts are usually lame on the affected foot, with licking and chewing at the interdigital space. exudation may be noticed at the site of the lesion. the lesion appears as a cutaneous ulcer, usually beneath matted hair, with possible development of sinus tracts and purulent exudate. epizootiology and transmission. interdigital cysts are common in a variety of canine breeds, including german shepherds. beagles have been affected in the research setting. interdigital cysts usually occur in the third and fourth interdigital spaces (bellah, ) . pathologic findings. histopathologically, interdigital cysts are sites of chronic inflammation, typically described as pyogranulomatous. pathogenesis. initial development of the cysts is unknown, except for those cases in which a foreign body can be identified. diagnosis and differential diagnosis. bacterial culture swabs and radiographs should be taken of the cysts to rule out bacterial infection, and radiopaque foreign bodies or bony lesions, respectively. a biopsy should be taken if neoplasia is suspected. treatment. if a foreign body is associated with the lesion, then removal is the first order of treatment. if biopsy of the site provides a diagnosis of sterile pyogranuloma complex, then systemic corticosteroid therapy (e.g., prednisolone at mg/kg ql h) can be initiated and then tapered once the lesion heals. interdigital cysts that are refractory to medical therapy require fig. . interdigital cyst between the third and fourth digits of the forelimb of a research beagle. surgical removal. excision includes removal of the lesion and the interdigital web, and a two-layer closure of the adjacent skin and soft tissues is recommended (bellah, ) . the foot should be put in a padded bandage and a tape hobble placed around the toes to reduce tension when the foot is weightbearing. the prognosis for idiopathic interdigital cysts is guarded, because the cysts tend to recur (bellah, ) . research complications from the cysts are minimal, unless the dogs need to be weight-bearing for biomechanic or orthopedic studies. treatment with systemic steroids could be contraindicated with some experimental designs. post-therapy antibody titers in dogs with ehrlichiosis: follow-up study on patients treated primarily with tetracycline and/or doxycycline dog thyroiditis: occurrence and similarity to hashimoto's struma surgical management of specific skin disorders bordetella and mycoplasma infections in dogs and cats associations between lymphocytic thyroiditis, hypothyroidism, and thyroid neoplasia in beagles diagnostic exercise: peracute death in a research dog saunders manual of small animal practice neurologic manifestations associated with hypothyroidism in four dogs neoplasms of the skin and subcutaneous tissues in dogs and cats neoplasia of the skin and mammary glands of dogs and cats platelet function, antithrombin-iii activity, and fibrinogen concentration in heartworm-infected and heartworm-negative dogs treated with thiacetarsamide pancreatic adenocarcinoma in two grey collie dogs with cyclic hematopoiesis ehrlichia platys infection in dogs the rickettsioses monoclonal gammopathy associated with naturally occurring canine ehrlichiosis variation in age at death of dogs of different sexes and breeds leptospira interrogans serovar grippotyphosa infection in dogs efficacy and dose titration study of mibolerone for treatment of pseudopregnancy in the bitch tropical canine pancytopenia: clinical, hematologic, and serologic response of dogs to ehrlichia canis infection, tetracycline therapy, and challenge inoculation comparison of campylobacter carriage rates in diarrheic and healthy pet animals. zentralbl advances in dietary management of obesity in dogs and cats effect of level and source of dietary fiber on food intake in the dog effect of amount and type of dietary fiber on food intake in energy-restricted dogs external parasites: identification and control tumors of the endocrine glands thomson's special veterinary pathology infectious diseases of the dog and cat nutritional support for dogs and cats with hepatobiliary disease specific amplification of ehrlichia platys dna from blood specimens by two step pcr detection of humoral antibody to the transmissible venereal tumor of the dog dirofilaria immitis: heartworm products contract rat trachea in vitro dogs: laboratory animal management management of septicemia in rhesus monkeys with chronic indwelling catheters client information series: canine demodicosis client information series: fleas and flea allergy dermatitis management of the burn wound chlorine dioxide sterilization of implanted right atrial catheters in rabbits current concepts in the management of helicobacter associated gastritis dirofilariasis in dogs and cats use of narcotic antagonists to modify stereotypic self-licking, self-chewing, and scratching behavior in dogs epidemiology of canine and feline tumors epizootiologic characteristics of canine and feline leukemia and lymphoma study of obesity in dogs visiting veterinary practices in the united kingdom miller's anatomy of the dog update on diagnosis of canine hypothyroidism helicobacter-associated gastric disease in ferrets, dogs, and cats the role of helicobacter species in newly recognized gastrointestinal tract disease of animals serologic diagnosis of infectious cyclic thrombocytopenia in dogs using an indirect fluorescent antibody test hemorrhagic streptococcal pneumonia in newly procured research dogs control of ticks platelet aggregation studies in dogs with acute ehrlichia platys infection health benefits of animal research: the dog as a research subject soft tissue sarcomas and mast cell tumors textbook of veterinary internal medicine infectious diseases of the dog and cat canine lyme borreliosis mast cell tumor destruction by deionized water mast cell tumour destruction in dogs by hypotonic solution transmission of ehrlichia canis to dogs by ticks (rhipicephalus sanguineus) textbook of veterinary internal medicine diseases of the liver and their treatment cyclic thrombocytopenia induced by a rickettsia-like agent in dogs shock evaluation of the sensitivity and specificity of diagnostic criteria for sepsis in dogs textbook of veterinary internal medicine canine thyroid neoplasms: epidemiologic features magrane's canine ophthalmology helicobacter-like organisms: histopathological examination of gastric biopsies from dogs and cats thiacetarsamide and its adverse effects infectious diseases of the dog and cat pediatrics: puppies and kittens canine viral diseases textbook of veterinary internal medicine antibodies to ehrlichia canis, ehrlichia platys, and spotted fever group rickettsia in louisiana dogs mastocytoma and gastroduodenal ulceration complications in the use of indwelling vascular catheters in laboratory animals deionised water as an adjunct to surgery for the treatment of canine cutaneous mast cell tumours helicobacter infection textbook of veterinary internal medicine textbook of veterinary internal medicine hygroma of the elbow in dogs thermal injuries dirofilaria immitis: do filarial cyclooxygenase products depress endothelium-dependent relaxation in the in vitro rat aorta? depression of endotheliumdependent relaxation by filarial parasite products three cases of canine leptospirosis in quebec cvt update: interpretation of endocrine diagnostic test results for adrenal and thyroid disease etiopathogenesis of canine hypothyroidism five-year longitudinal study on limited food consumption and development of osteoarthritis in coxofemoral joints of dogs role of bordetella bronchiseptica in infectious tracheobronchitis in dogs kirk's current veterinary therapy : small animal practice the fire of life kirk's current veterinary therapy : small animal practice coinfection with multiple tick-borne pathogens in a walker hound kennel in north carolina tarsal joint contracture in dogs with golden retriever muscular dystrophy clinical and hematological findings in canine ehrlichiosis early spay-neuter in the dog and cat textbook of veterinary internal medicine chronic catheterization of the intestines and portal vein for absorption experimentation in beagle dogs evaluation of weight loss protocols for dogs the brown dog tick rhipicephalus sanguineus and the dog as experimental hosts of ehrlicha canis the clinical chemistry of laboratory animals double-blinded crossover study with marine-oil supplementation containing high-dose eicosapentaenoic acid for the treatment of canine pruritic skin disease thomson's special veterinary pathology effects of four preparations of . % chlorhexidine diacetate on wound healing in dogs transmissible venereal tumors kirk's current veterinary therapy : small animal practice soft tissue sarcomas tumors of the mammary gland canine lymphoma and lymphoid leukemias kirk's current veterinary therapy : small animal practice effect of heartworm infection on in vitro contractile responses of canine pulmonary artery and vein tick paralysis in north america and australia saunders manual of small animal practice thyroid gland and arterial lesions of beagles with familial hypothyroidism and hypedipoproteinemia bacterial gastroenteritis in dogs and cats: more common than you think urea protects helicobacter (campylobacter) pylori from the bactericidal effect of acid characterization of a new isolate of ehrlichia platys using electron microscopy and polymerase chain reaction decreased pulmonary arterial endothelium-dependent relaxation in heartworm-infected dogs with pulmonary hypertension vaccination against canine bordetellosis: protection from contact challenge dermatologic aspects of tick bites and tick-transmitted diseases a chronic access port model for direct delivery of drugs into the intestine of conscious dogs clinical trial of dvm derm caps in the treatment of allergic diseases in dogs: a nonblinded study diagnosis of neoplasia tumors of the mammary gland dirofilaria immitis: heartworm infection alters pulmonary artery endothelial cell behavior survey of conjunctival flora in dogs with clinical signs of external eye disease hyperoxia exacerbates microvascular injury following acid aspiration nutrient requirements of dogs surgical closure of elbow hygroma in the dog tumors of the genital system practical laboratory methods for the diagnosis of dermatologic diseases walker's mammals of the world tumors of the endocrine system beta hemolytic streptococcus isolated from the canine vagina comparison of three skin preparation techniques in the dog comparison of three skin preparation techniques in the dog. part : clinical trial in dogs clinical behavioral medicine for small animals hypothyroidism in dogs: cases ( - ) plasma von willebrand factor antigen concentration in dogs with hypothyroidism plasma von willebrand factor antigen concentration and bleeding time in dogs with experimental hypothyroidism canine cutaneous mast cell tumor: morphologic grading and survival time in dogs joint diseases of dogs and cats target imbalance: disparity of borrelia burgdorferi genetic material in synovial fluid from lyme arthritis patients textbook of veterinary internal medicine tumors of the skin and soft tissue thomson's special veterinary pathology canine leptospirosis: a retrospective study of cases dogs and cats as laboratory animals effects of chlorhexidine diacetate and povidoneiodine on wound healing in dogs epidemiology of thyroid diseases of dogs and cats factors influencing canine mammary cancer development and postsurgical survival muller and kirk's small animal dermatology systemic necrotizing vasculitis in nine young beagles thomson's special veterinary pathology textbook of veterinary internal medicine canine infectious tracheobronchitis (kennel cough complex) saunders manual of small animal practice serum protein alterations in canine erhlichiosis bacterial factors and immune pathogenesis in helicobacter pylori evaluation of rhipicephalus sanguineus as a potential biologic vector of ehrlichia platys role of the eastern chipmunk (tamias striatus) in the epizootiology of lyme borreliosis in northwestern illinois evaluation of risk factors for degenerative joint disease associated with hip dysplasia in dogs pathologic features of naturally occurring juvenile polyarteritis in beagle dogs textbook of veterinary internal medicine clinical manifestations, pathogenesis, and effect of antibiotic treatment on lyme borreliosis in dogs streptococcus zooepidemicus as the cause of septicemia in racing greyhounds trauma to the skin and subcutaneous tissues of dogs and cats chronic problem wounds of dog limbs portosystemic shunts textbook of veterinary internal medicine lumbosacral stenosis in dogs experimental respiratory disease in dogs due to bordetella bronchiseptica dirofilaria immitis: depression of endothelium-dependent relaxation of canine femoral artery seen in vivo does not persist in vitro thyroiditis in a group of laboratory dogs: a study of beagles of agriculture, animal and plant health inspection service thomson's special veterinary pathology a retrospective study of cases of naturally occurring canine ehrlichiosis role of canine parainfluenza virus and bortedella bronchiseptica in kennel cough management of superficial skin wounds serum concentrations of thyroxine and , , '-triiodothyronine before and after intravenous or intramuscular thyrotropin administration in dogs use of ultrasound in the measurement of subcutaneous fat and prediction of total body fat in dogs ehrlichia platys in a michigan dog ehrlichial diseases of dogs canine ehrlichiosis. miss albert, r. e., benjamin, s. a., and shukla, r. ( ). life span and cancer mortality in the beagle dog and human. key: cord- -jkug jpz authors: schaefer, a. l.; cook, n. j.; church, j. s.; basarab, j.; perry, b.; miller, c.; tong, a.k.w. title: the use of infrared thermography as an early indicator of bovine respiratory disease complex in calves date: - - journal: research in veterinary science doi: . /j.rvsc. . . sha: doc_id: cord_uid: jkug jpz abstract bovine respiratory disease (brd) complex causes considerable distress to domestic livestock and economic hardship to the beef industry. furthermore, the resulting extensive use of antimicrobial treatments is a growing concern from the perspective of facilitating antibiotic resistant microbes. the earlier detection of brd would enable an earlier, more targeted treatment regime and earlier isolation of infected individuals. the objective of the present study was to investigate the use of non-invasive infrared thermography in the early detection of brd in cattle. studies were conducted on head of weaned calves. data demonstrated that infrared thermography was able to identify animals at early stages of illness, often several days to over one week before clinical signs were manifest. data indicated that – days prior to the onset of clinical symptoms of brd, greater positive and negative predictive values and test efficiency for infrared thermography ( %, % and %, respectively) compared to the industry standard practice of clinical scoring ( %, % and %, respectively). the use of infrared thermography as an early indicator of bovine respiratory disease complex in calves infectious diseases such as bovine respiratory disease (brd) are known to have a significant economic impact on the cattle industry with respect to treatment costs and a negative impact on animal performance and welfare (basarab et al., ; wittum et al., ) . although the term brd can refer to a host of complex diseases this term is generally used to refer to an animal displaying an undifferentiated fever as well as some number of clinical signs. as discussed by jericho and kozub ( ) the presence of brd is common in intensively raised calves and the industry dependence on antibiotic treatment is high. such management practices are becoming increasingly scruti-nized in many countries due to concern regarding antibiotic use and the potential promotion of antibiotic resistant microbes (fleming, ; jericho and kozub, ) . this situation is in fact also becoming a growing issue in the popular press (zimmerman and zimmernan, ; mellon et al., ) . the ability to treat bovine respiratory disease (brd) especially in multiple sourced and co-mingled animals, is becoming more difficult. shahriar et al. ( ) for example report the presence of antibiotic resistant pneumonia in feedlot cattle. as a result, a more targeted and selective use of antimicrobials in the animal industries is sought. as discussed by cusack et al. ( ) the effectiveness of treating brd depends primarily on the early recognition and treatment. unfortunately, traditional clinical signs of brd are known to often occur late into the course of the disease. by contrast, humblet et al. ( ) demonstrated the utility of acute phase proteins, particularly haptoglobin and fibrinogen, as early indicators of bronchopneumonia in calves. however, measurements of acute phase proteins requires collection of biological samples and complicated laboratory-based analysis that cannot be performed in situ. other early indicators of disease onset have been observed such as the actual appearance of antibodies in the blood, cytokines, acute phase proteins and fever. in a review of the behaviour of sick animals, hart ( ) describes and references the observation that the acute phase fever response is initiated by endogenous pyrogens such as interleukin- following infection from many known microorganisms. the value or function of such a fever is thought to be in enhancing the animal's ability to combat infection. the mechanisms involved are shown to include the increase of neutrophils, an enhanced lymphocyte proliferation as well as an enhanced antibody synthesis. recent research (schaefer et al., ) has demonstrated the diagnostic potential of infrared thermography (irt) under controlled conditions for an induction model of bovine viral diarrhea (bvd). considering up to % of the heat loss from an animal can occur in the infrared range (kleiber, ) the observation that radiated heat loss can serve as an early indicator of fever conditions is perhaps logical. in terms of treating animals displaying symptoms of brd, the industry standard practice has been to use one or more antibiotics. in fact it is considered by some researchers to be difficult to even place and raise calves in feedlots without using antibiotics (jericho and kozub, ) . again, this is a practice that is increasingly considered undesirable. the european community's scientific steering committee for example has recommended against the abundant use of antibiotics in the livestock industries (jericho and kozub, ) . the primary objective of the present study was to investigate the use of infrared thermography in the early identification of spontaneously occurring brd. perceived advantages of the infrared thermography technology were that diagnosis could be made in real time and could be conducted non-invasively. however, a comparative test of the irt technology under spontaneous conditions was considered prudent. research studies are reported on multiple sourced, commingled commercial weaned calves. these studies were conducted at the lacombe research centre beef research facility and all management practices followed canadian council of animal care guidelines (canadian council on animal care, ) and canadian beef cattle code of practice guidelines (agriculture canada, ) . in addition, the research protocols were reviewed and approved by the lacombe research centre animal care committee. the cattle demographics for the initial procurement of animals involved; ninety three ( ) these calves were chosen in order to provide study groups displaying a bovine respiratory disease (brd) incidence range of - % which is typical of the beef industry in canada for these types of cattle. data used in the present study for true positive and true negative animals is corrected for any health aberrations due to non-respiratory disease (example pink eye, foot rot) and for any data that could not be verified. for example, a calf received at the lacombe beef research unit displaying keratoconjunctivitis (pink eye), pododermatitis (foot rot) or an infected wound of any other kind would be examined by the research centre veterinarian and placed on a treatment program. while it is recognized that such animals are also of interest and importance from a herd health perspective, these animals were also not ill due to brd nor, for the purpose of a gold standard, was the time of the onset of their condition known. thus, such animals would risk biasing the brd population data and were not considered part of the brd data set. this verification process left calves in the study from an initial . for the cattle under study at the lacombe research centre, the weaned calves were typically received in the afternoon having undergone transport for approximately h, handling and co-mingling. these calves were offloaded into clean holding pens, given access to water and allowed to rest for a few hours prior to being processed. processing consisted of measurements for haematology, clinical scores, salivary cortisol, infrared thermal measurements and body weights. blood samples for haematology and serology assessment were collected by venous puncture of the jugular vein. for this purpose animals were briefly restrained in a conventional hydraulic cattle handling catch. repeated measures were made on all animals at approximately weekly intervals. animals were maintained on cereal grain silage rations which met or exceeded nrc ( ) recommendations. any animals deemed ill either by direct veterinary assessment or under veterinary guidance were provided with routine antimicrobial therapy and/or other treatment and support until recovered. all calves were vaccinated for common brd agents including clostridia, bovine viral diarrhoea and infectious bovine rhinitis at the end of the research study. the cattle in the present study were used to develop a ''gold standard'' for defining a true positive population of the presence of brd. the approach of subjective ''judging ''who might be ill and who might be well, on the basis of pen checking, has been found wanting in the cattle industry. hence, the use of a gold standard has been deemed useful in both human (american college, ) and animal clinical studies (galen and gambino, ; thrusfield, ) . several research groups have developed gold standards for the presence of brd and there tends to be common aspects to all standards. in the current study we based our gold standards around parameters for normal and non-normal parameters as per the approach followed by humblet et al. ( ) . in the current study, the criteria for a gold standard for true positive disease was defined as any animal displaying two or more of the following symptoms: a core temperature of °c or higher, a white blood cell count of less than or greater than · ll À , a clinical score of or higher and a neutrophil/lymphocyte ratio of less than . or greater than . . a true negative animal was defined as any animal displaying one or fewer of the above signs or symptoms. these parameters are consistent with ranges for normal or abnormal parameters suggested by several clinical texts (blood et al., ; kaneko, ) as well as historical data from our own lab. using the above criteria, true positive and true negative populations of animals were established. a second objective of the study was to develop a predictive index for the infrared thermal data by establishing cut off values for thermal parameters useful in the early prediction of respiratory disease. this again would be possible once and only once true positive and true negative populations of animals could be established. in the present study, the methods described by galen and gambino ( ) and humblet et al. ( ) were utilized, whereby, the positive predictive value (ppv) was defined as the proportion of all positive test results that were true positive. the negative predictive value (npv) was the proportion of all negative test results that were true negative and test efficiency was defined as the proportion of all test results that were true results, whether positive or negative. data for true positive and true negative animals was used only for those animals in which the day of illness could be evaluated. for example, some commercial calves would arrive at the research facility already displaying signs of illness. hence, for those animals an exact day for the onset of symptoms could not be established in terms of monitoring days prior to illness. for infrared scans (irt) the data was representative of orbital maximum temperatures collected with a flir s camera (flir comp. boston, ma) from approximately meter distance. digitized infrared images were collected and saved in greyscale. each greyscale pixel corresponded to a specific temperature. pseudo-colours (fig. ) were added or generated by computer enhancement. an orbital image was obtained by auto tracing a circle over the orbital area including the eyeball and approximately cm of surrounding skin of the eye socket. this area including the lachrymal gland is, in the author's observation, very sensitive to thermoregulatory changes associated with stress and disease conditions. clinical score were based on those used in other studies and reported previously (schaefer et al., ) . a clinical score was additive of the four components. the scoring system was as follows: respiratory insult: ( - ): = no insult, normal breath sounds (nbs); = very fine crackle (rale) (vfcr) on auscultation and/or a moderate cough; = fine crackle (subcrepitant) (fcr) on auscultation and/or a moderate nasal discharge and moderate cough; = medium crackle (crepitant) (mcr) on auscultation and/or a moderate to severe viscous nasal discharge with cough; = course crackles (ccr), tachypnea (> % of the norm) and/or a severe discharge with respiratory distress and obtunded lung sounds; = ccr with dyspnea, tachypnea, marked respiratory distress and/or lung consolidation. digestive insult: ( - ): = no insult, normal, eating and drinking; = mild or slight diarrhoea with slight dehydration (< %) and reduced eating; = moderate diarrhoea with % dehydration and reduced feed intake (< %); = moderate to severe diarrhoea with % or less of feed intake and more than % dehydration; = severe diarrhoea, and less than % of normal feed intake. = severe diarrhoea and not eating, not drinking and dehydrated. temperature score: core temperature (rectal) ( - ): = < . ; = . - . ; = . - . °c; = . - . °c; = . - . °c; = > °c. disposition or lethargy score: ( - ): = no lethargy, normal posture; = mild anorexia or listlessness, depressed appearance; = moderate lethargy and depression, slow to rise, anorectic; = recumbent or abnormal posture, largely depressed; = prostrate, recumbent or abnormal posture; = death. salivary cortisol was measured by collecting saliva onto cotton swab and determining the cortisol levels in the saliva by an enzyme immunoassay modification of an existing method reported by cook et al. ( ) . hematology values were measured on a cell-dyn hematology analyser (sequoia -turner corp. mountain view, ca). differential blood cell counts were determined via stained blood smears (geisma-wright quick stain) and direct microscope examination of cells. animals displaying overt clinical symptoms of brd in the present study received immediate treatment as recommended by the lacombe research centre veterinarian followed by continued monitoring and re-treatment if required. to investigate the qualitative and quantitative presence of typical brd viruses in this class of cattle a representative subgroup of animals underwent serology assessment for five common viral causes of brd. the selection criteria for these animals was as follows: the calves had to originate from a minimum of two farm herds or sources; the calves had to have been weaned within h of auction sale; the calves had to have been recently transported on a commercial carrier (in the present case the transport time was approximately h); the calves had to have been exposed to handling and managed through a commercial auction facility and finally, the calves had to have been exposed to a time off feed and water since weaning of between and h. the viruses assessed were infectious bovine rhinotracheitis (ibr), bovine virus diarrhea (bvd), corona virus, pi (bovine para-influenza) and brsv (bovine respiratory syncytial virus). assessment of viral presence and titres was done by either elisa methods (pi , corona and brsv) or serum neutralization methods (ibr, bvd) and was performed by prairie diagnostic services (saskatoon, saskatchewan). the titre values for these viruses are shown in table . for samples analysed by elisa methods (brsv, pi and corona), antigen and tissue control well plates were used. antibody concentrations are expressed by measuring the optical density as a percentage of a positive control serum whereby: (mean net optical density of test serum À mean net optical density of fetal bovine serum)/(mean net optical density of positive standard À mean net optical density of fetal bovine serum) · = units. calculations for positive predictive value (ppv), negative predictive value (npv) and test efficiency were conducted as per the methods of humblet et al. ( ) . response operant characteristic curves (roc, fig. ) were based on the methods described by zweig and campbell ( ) . medcalc software (medcalc, ) was used in the calculation of roc (fig. t ) curves and in developing the predictive values. roc curves were used to calculate optimal efficiency cut off values as shown in tables and . for infrared temperatures, the irt absolute temperature was the direct temperature measured from the flir camera and calculated using the flir''researcher'' Ò software. the irt mean ratios values represent the mean orbital maximum temperature value for an individual animal divided by the mean orbital maximum temperature value for the contemporary group of calves as they arrived at the research centre. in addition to the above, a statistical comparison of test performance for tables and was conducted with fishers exact test using medcalc software (galen and gambino, ) . this study illustrates how one aspect of the thermal information, the orbital max temperature and the use of mean ratios have utility as early predictors of illness. tables and illustrated how these predictors compare to other clinical standards commonly used in the animal industries both at the time a positive diagnosis is made with the calves or the day of illness and for a subset of animals measured - days prior to the positive identification of the disease. of the sixty five animals meeting the criteria for a true positive diagnosis, it was possible to also collect prior data from - days earlier on thirty seven of these calves. bovine respiratory disease predictive values, cut off values as determined by roc curves and efficiency calculations were completed for all calves in the true positive and true negative populations. these calculations were made for both clinical (table ) and - days pre clinical (table ) time frames. both absolute and mean ratio infrared values are displayed compared to several other commonly used predictors of disease in cattle including core temperature, white blood cell count, neutrophile/lymphocyte ratios, clinical scores and salivary cortisol values. examples of response operant characteristic (roc) curves are shown for both clinical scores and infrared absolute values calculated at both clinical and pre clinical times. for illustrative purposes, the progression and onset of bovine respiratory disease as detected by infrared thermography is shown in one animal (fig. ) . in terms of test comparisons, fishers exact test results indicated that on the day of illness there were no significant differences in the ability of the tests to distinguish diseased animals (p > . ). in other words, when an animal was clinically ill then clinical scores, core temperature, haematology values or infrared thermography would suggest that animals was ill with equivalent accuracy. however, for data - days prior to the appearance of clinical illness, fisher exact test scores indicated that infrared thermography was significantly better (p < . ) at identifying true positive animals than either clinical scores or core temperature. as illustrated by the serology data in table collected from a representative group of contemporary multiple sourced, co-mingled auction cattle, the calves used in the present study would appear to display significant titres to many of the viruses thought to be commonly responsible for brd. while the presence of these viruses in serology samples would be expected as a normal profile especially in any calves that had been vaccinated, the high titres for many of the animals are suggestive of significant viral infectivity and were likely responsible for the onset of brd in the calves. the identification of true positive and true negative populations of calves was attempted in the present study. the authors appreciate the fact that there are often debates regarding the optimal composition of parameters to define a gold standard. predictably, some debate would likely also arise regarding the parameters and values used in the present study. however, as seen in table , the development of a gold standard set of values as used in the present study was nonetheless seen to be efficacious as a decision tool for brd onset and in establishing true positive and true negative subsets in the calf population. this arguably is more accurate than simply trying to utilize a single subjective score or opinion for illness as is a common industry standard practice in such situations. the development of the gold standards for true positive and true negative criteria enabled an objective comparison of several common tests of disease presence. while none of the tests are perfect, as evident in table , when an animal is actually verifiably ill a number of tests including clinical scores, core temperatures and hematology will perform comparably well at identifying ill animals. likewise, the predictive values of many tests (table ) will perform comparably well when the animals are actually ill. the challenge, however, as discussed earlier is how to develop and test predictive methods that are diagnostically relevant prior to the onset of the clinical signs of disease. in this respect, candidate methods such as infrared thermography are appealing both from the prospect of being non-invasive and, in contrast to laboratory based analysis, irt technology can operate in real time. apparent in the present study was the observation that infrared values, especially the mean ratio values, were as or more efficient than clinical scores, core temperatures or haematology in identifying ill animals prior to the clinical appearance of brd. of particular greater significance is that prior to the onset of clinical brd infrared data was also seen to show predictive capability. of interest was the observation that the predictive value of a comparatively non-invasive technique, salivary cortisol, likewise demonstrated a strong positive and negative predictive value as well as test efficiency compared to conventional measures such as core temperature and clinical scores. table illustrates how, with the use of a response operant characteristic curve calculation, such data could be used to optimise the prediction of positive and negative values for animals at risk of brd and how different measurements perform. data are used for - days prior to illness in a subset of calves subsequently determined as true positive using the gold standard values. a please see methods section in text for description of clinical score system. b irt = infrared thermography value for the orbital (eye) maximum temperature. c probability determined by least squares analysis using microsoft excel two tailed t-test. ( ) software. g,h the absolute irt or infrared thermography value is equal to the orbital (eye) maximum value in°c, the mean ratio value is the mean infrared maximum temperature for the individual animal divided by the mean infrared maximum temperature value for the contemporary group of calves. data are given for the day of illness or day clinical assessment was true positive using the ''gold standard'' values. a please see methods section in text for description of clinical score system. b irt = infrared thermography value for the orbital (eye) maximum temperature. c probability determined by least squares analysis using microsoft excel two tailed t-test. in addition to the above, the authors would, however, point out that the incidence of brd in the animal populations used in the current study was comparatively high. a valid question therefore would remain regarding how the infrared thermography technology might perform as an early indicator of disease in animal populations displaying a lower incidence of brd. one must also keep in mind that as discussed by galen and gambino ( ) the use of parallel and/or series testing can be used to increase all measures of diagnostic test per-formance. in the case of infrared values just as an example, using both infrared absolute values and infrared mean ratio values in a parallel test would improve the negative prediction values and test efficiency by - %. there are many additional ways infrared data can be utilized and only two such possibilities (absolute and mean ratio data) are presented in the present manuscript. optimising the use of such values will be the next step in this research. furthermore, the use of infrared thermography lends itself to a non-invasive automation of data collection. in concurrent research ( ) software. g,h the absolute irt or infrared thermography value is equal to the orbital (eye) maximum value in°c, the mean ratio value is the mean infrared maximum temperature for the individual animal divided by the mean infrared maximum temperature value for the contemporary group of calves. fig. . retrospective facial infrared thermography images of the same animal during the onset of bovine respiratory disease. clinical signs were positive (> ) on days - . (stewart et al., ) , individual animal identification using radio frequency identification tags (rfid) and an irt camera located at a water station have been employed to automatically collect thermal data. this situation is impossible to achieve with more invasive measures such as haematology or core temperatures. of interest from the point of speculation would be the question that if an early disease detection system, perhaps employing an infrared parameter, were possible, what then would be the optimal way to utilize such data? animals identified early might be re-vaccinated, or simply isolated from the contemporary group to avoid viral shedding and infecting other individuals. in this regard, recent trials at lacombe research centre have demonstrated that animals identified early as at risk of developing brd benefited from the prophylactic application of respired nitric oxide (schaefer et al., ) . as discussed by mcmullin et al. ( ) and ghaffari et al. ( ) nitric oxide is a known microcidal agent in vitro and thus may well function effectively in vivo also. data collected in the present study demonstrated that infrared thermography scans of the orbital area in calves was efficacious as an early identifier of bovine respiratory disease onset. such information would enable the earlier and more targeted treatment of affected animals thereby reducing animal suffering, improving the animal industry economics and reducing the likelihood of promoting antibiotic resistant microbes. recommended code of practice for the care and handling of farm animals. beef cattle. publication /e. communications branch definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis automatic monitoring of watering behaviour in feedlot steers: potential use in early detection of respiratory disease and in predicting growth performance. can veterinary medicine guide to the care and use of experimental animals a radioimmunoassay for cortisol in pig saliva and serum the medicine and epidemiology of bovine respiratory disease complex in feedlot antibiotics vs the super bugs. ideas, cbc potential application of gaseous nitric oxide as a topical antimicrobial agent biological basis of the behaviour of sick animals. neurosci acute phase proteins assessment for an early selection of treatments in growing calves suffering from bronchopneumonia under field conditions experimental infectious respiratory disease in groups of calves: lobular distribution, variance, and sample size requirements for vaccine evaluation clinical biochemistry of domestic animals the fire of life the antimicrobial effect of nitric oxide on bacteria that cause nosocomial pneumonia in mechanically ventilated patients in the intensive care unit hogging it. estimates of antimicrobial abuse in livestock. union of concerned scientists nutrient requirements of beef cattle early detection and prediction of infection using infrared thermography. can infrared detection and nitric oxide treatment of bovine respiratory disease coinfection with bovine viral diarrhea virus and mycoplasma bovis in feedlot cattle with chronic pneumonia infrared thermography as a non-invasive tool to study animal welfare diagnostic testing p - . veterinary epidemiology, second ed relationships among treatment for respiratory tract disease, pulmonary lesions evident at slaughter and rate of weight gain in feedlot cattle receiver operating characteristic (roc) plots: a fundamental evaluation tool in clinical medicine the authors wish to acknowledge the alberta livestock industry development fund, alberta agriculture food and rural development, agriculture and agri-food canada, the alberta farm animal care organization, animal diseases research institute (lethbridge, alberta), penridge feeders (acme, alberta), veterinary agri-health services ltd. (airdrie, alberta), olds college (olds, alberta), the lacombe veterinary clinic and pulmonox medical comp. (edmonton, alberta) for their support during this study. we also wish to thank the following persons for their technical expertise without which this study could not have been done; b. chabot, j. colyn, d. froehlich, l. holt-klimec, p. lepage, s. lohmann and t. lui. the assistance of the lacombe research centre beef unit staff and the animal staff at the animal diseases research institute is also gratefully acknowledged. key: cord- -rrverrsj authors: delano, margaret l.; mischler, scott a.; underwood, wendy j. title: biology and diseases of ruminants: sheep, goats, and cattle date: - - journal: laboratory animal medicine doi: . /b - - / -x sha: doc_id: cord_uid: rrverrsj nan since the first edition of this book, the use of ruminants as research subjects has changed dramatically. formerly, large animals were primarily used for agricultural research or as models of human diseases. over the past decade, ruminants have continued in their traditional agricultural research role but are now extensively used for studies in molecular biology, genetic engi-british stock with egyptian and indian goats. this breed is relatively heat tolerant and produces milk with the highest butterfat (about - %). fiber breeds include the angora and the cashmere. the angora, the source of mohair, originated in turkey. the cashmere breed is found primarily in mountainous areas of central asia. the la mancha, a newer breed of dairy goat first registered in the united states in , has rudimentary ears that are a genetically dominant distinguishing characteristic of the breed. the meat breeds include the boer, sapel, ma tou, kambling, and pygmy. the pygmy goat is small and is sometimes used for both meat and milk. the mubend of uganda and the red sokoto of west africa produce quality skins for fine leather (smith and sherman, ) . most breeds of cattle are classified as "dairy" or "beef"; a few breeds are considered "dual-purpose." common dairy breeds in the united states include holstein-friesian, brown swiss, jersey, ayrshire, guernsey, and milking shorthorn. holsteins have the largest body size, whereas jerseys have the smallest. of breeds in temperate regions, jerseys have been considered to be the most heat tolerant, but holsteins have been found to adapt to warmer climates. there are many beef breeds. the more common in the united states include angus (also called aberdeen-angus), hereford (both polled and horned), and simmental (briggs and briggs, ; schmidt et al., ) . breeds indigenous to other continents, such as the cape buffalo, have been found to have unique innate immune characteristics that protect them from endemic trypanosomiasis (muranjan et al., ) . more detailed information regarding these and other ruminant breeds is available in briggs and briggs ( ) . "rare" or "minor" breeds of sheep, goats, and cattle are studied for their genetic and production characteristics. discussions of these and efforts at conservation are described in detail elsewhere (national research council, ) . several terms are unique to ruminants. in relation to sheep, a ewe is the female, and a ram is the adult intact male. a lamb is the young animal, and ram lamb and ewe lamb are commonly used terms. a wether is a castrated male. the birthing process is referred to as lambing. with respect to goats, a doe or nanny is the female. a buck or billy is the adult intact male. a kid or goatling is a young goat. a young male may be referred to as a buckling, and a young female may be referred to as a doeling. a castrated male in this species is also called a wether. the birthing process is called kidding. with respect to cattle, an adult female is a cow, and an adult male is a bull. a calf is a young animal. a heifer is a female who has not had her first calf. a steer is a castrated male. calving refers to the act of giving birth. ruminants have been used as research models since the inception of the land grant college system, first in production agriculture and now also in basic and applied studies for the anatomic and physiologic sciences and in biomedical research for a variety of purposes. healthy, normal young ruminants serve as models of cardiac transplantation and as preclinical models for evaluation of cardiac assist or prosthetic devices, such as vascular stents and cardiac valves (salerno et al., ) . for many years, ruminants have been useful research subjects for reproductive research, such as research on embryo transfer, artificial insemination, and control of the reproductive cycle (wall et al., ) . several important milestones in gene transfer, cloning, nuclear transfer, and genetic engineering techniques have been developed or demonstrated using these species (ebert et al., ; schnieke, ; cibelli et al., a,b) (see fig. ). one of many proposed uses of genetically engineered ruminants is the production of proteins that will be secreted in the milk and later isolated (ebert et al, ; memon and ebert, ) . healthy sheep and goats are also often used for antibody production (hanly et al., ) . genome mapping developed rapidly during the s; extensive information is available and is increasing for sheep and cattle (broad et al., ; womack, ) . sheep are often selected for studying areas such as ruminant physiology and nutrition. these animals provide obvious bene-fits over the use of cattle in research from the standpoint of size, ease of handling, cost of maintenance, and docile behavior. sheep are also widely used models for basic and applied fetal and reproductive research (buttar, ; rees et al., ; ross and nijland, ) . the species is used for investigating circadian rhythms related to day length (lehman et al., ) , and the interaction between olfactory cues and behavior (kendrick et al., ) . the number and diversity of natural-and induceddisease research models in sheep are great and increasing. natural models include congenital hyperbilirubinemia/hepatic organic anion excretory defect (dubin-johnson syndrome) in the corriedale breed, congenital hyperbilirubinemia/hepatic organic anion uptake defect (gilbert syndrome) in the southdown breed, glucose- -phosphate dehydrogenase deficiency in the dorset breed, gm~ gangliosidosis in the suffolk breed, and pulmonary adenomatosis (jaagsiekte) in many breeds (hegreberg, l a) . induced models include arteriosclerosis, hemorrhagic shock, copper poisoning (wilson's disease), and metabolic toxocosis (hegreberg, lb) . goats are used in a wide variety of agricultural and biomedical disciplines such as immunology, mastitis, nutrition, and parasitology research. vascular researchers select the goat because of the large, readily accessible jugular veins. goats with inherited caprine myotonia congenita ("fainting goats") have been used as a model for human myotonia congenita (thomsen's disease) (kuhn, ) . a line of inbred nubians serves as models for the genetic disease [ -mannosidosis and prenatal therapeutic cell transplantation strategies (lovell et al., ) . (these disorders are discussed in more detail in section iii,b, .) goats are used as a model for osteoporosis research (welch et al., ) . cattle are often used as a source of ruminal fluid for research, teaching, or treatment of other cattle, by placing a permanent fistula in the left abdominal wall to allow sampling of ruminal fluid (dougherty, ) . cattle also serve as models of many infectious diseases, including zoonoses, and several inherited metabolic diseases. this species is useful for the basic and comparative research on the pathogenesis and immunology of inherited and infectious diseases. bovine trichomoniasis, caused by tritrichomonas (trichomonas)fetus, has been identified as a useful model for the human infection by trichomonas vaginalis (corbeil, ) . inherited cardiomyopathies have been found in the holstein-friesian, simmental-red holstein, black spotted friesian, and polled hereford with woolly coat (weil et al., ) . lipofuscinosis has been identified in ayrshires and friesians, and glycogenesis in shorthorns and brahmans. metabolic diseases such as hereditary orotic aciduria and hereditary zinc deficiency have been characterized in holstein-friesian or friesian cattle. holstein cattle also serve as a model for leukocyte adhesion deficiency syndrome (afip, ) . common breeds of normal, healthy ruminants are usually readily available, although seasonality may play a role, as noted below. agricultural sources and reputable farms may be located through land-grant universities or agricultural schools, cooperative extension and -h networks, regional ruminant breeders' associations, and farm bureaus. commercial sources of purposebred animals are found in technical publications and annual listings of research animal vendors. breeds carrying genetic traits of interest, either as animal models or as valuable production characteristics, may be located through literature or internet searches, animal science societies, breed or livestock conservation associations, and information resources such as the armed forces institute of pathology. organizations such as the institute for laboratory animal research (ilar), national center for research resources (ncrr), or the animal welfare information center (awic) may also serve as information sources about the animals needed. purpose-bred research sheep and goats are available from commercial vendors and are usually maintained in registered facilities under federal standards that are also acceptable to research animal accrediting agencies. these commercial animals are frequently described as specific pathogen-free (spf) and housed as biosecure or closed flocks. animal health programs are in place, and health reports or other quality assurance reports are usually available on request. agricultural sources of either small ruminant may be acceptable, but specific research needs may not have been addressed or may not be understood. lambs, kids, and milking goats may be difficult to locate in fall and winter months because most breeds of sheep and goats are seasonal breeders. management practices exist, however, to extend the breeding and milking seasons. most cattle used as animal models in research in the united states are from one of the dairy breeds, usually holstein, because this breed is now the most common. purpose-bred, specific pathogen-free research cattle are not typically available. because of selection and the management of dairy production units, calves and young stock are available year-round. availability of young beef cattle is more seasonal, according to production cycles typically followed by that industry. auction barns or sales are not appropriate sources for research ruminants. many of these animals are culls and will be poor-quality research subjects. they may be in poor body condition and stressed, may be sources of disease, and may contaminate other healthy animals, as well as the research facility. selection of the suppliers should be made only after research needs have been carefully considered. consistently working with and buying directly from as few sources as possible are best. certain types of research (i.e., agricultural nutrition studies) may better be served by selecting animals from local agricultural suppliers rather than commercial vendors located in a different geographical area. the selection of sources for research ruminants includes scrutiny of flock or herd record keeping; health monitoring, vaccination, and preventive medicine programs (including hoof care); production standards and management practices consistent with the industry; management of the breeding flock or herd; sanitation and waste handling programs; vermin and insect control measures (especially for flies and other flying insects); rearing programs for and condition of young stock; the location, health, and condition of the other animals on the premises; intensity of housing; and animal housing facilities. preliminary and periodic visits to the source farms should be conducted. it is important to establish a good relationship with the local attending large-animal veterinarians, who will be valuable resources for current approved therapies and practices. they may need to be oriented on the specific requirements of animal research. creative ways can be used to initiate and foster a good working relationship between the agricultural supplier and the research facility. supplying the vaccines or dewormers required for flock health programs, providing services such as quarterly serological testing or fecal examinations for the herd or flock, and paying a premium (rather than market price) for animals that meet the quality criteria established for the research animals are often helpful. a set of testing standards can be developed based on one high-quality supplier, and then flocks or herds can be "qualified" based on those standards. qualifying entails evaluations utilizing the facility and management aspects mentioned above and testing either a percentage of the herd or flock or the entire herd or flock for a number of infectious agents. the testing regimen itself should be carefully developed and evaluated. once qualified, each source farm should be reevaluated periodically to maintain its status. slaughter checks may be appropriate; otherwise necropsy of sentinel animals may be required. selected animals undergoing screening tests should be quarantined from the rest of flock or herd while awaiting test results. vaccination and deworming regimens can be instituted during these quarantine periods. a second quarantine should occur when animals arrive at the research facility. the animal screening process also depends on the origin of the animal (state, country) and the scientific program. federal and state regulations must be followed. socialization of the animals at the source facility should also be considered in terms of ease of handling and safety for personnel in the confinement of the research lab, barn, or farm. for example, frequently handled calves will be easier to manage, and adult dairy goats that have been acclimated to human contact are preferable. several texts provide information on industry standards for flock and herd management and preventive medicine strategies that can provide helpful orientation to those unfamiliar with these aspects. these references also provide information regarding vaccination products licensed for use in ruminants and typical herd and flock vaccination parasite control schedules ("current veterinary therapy," , "council report," ; "large animal internal medicine," ; smith and sherman, ) when designing a vaccination program during qualification of a source or at the research facility, it is important to evaluate the local disease incidence and the potential for exposure. vaccination programs should be conducted with an awareness of duration of passive immunity and stresses in ruminants' lives (e.g., weaning, grouping, management changes, and shipping) that may impair immunity or increase susceptibility to infectious diseases. it is also prudent to evaluate the cost-effectiveness of vaccination; labor and vaccine expenses may be much higher than the potential animal morbidity or mortality for diseases in a particular locality. not all of the vaccines mentioned subsequently will be necessary in all herds or flocks. vaccination needs for research animals will also depend on the local disease history, intent of the research, the age of the animals needed for research, and the length of time the animals will be housed. typical health screening programs for sheep include q fever (coxiella burnetii); contagious ecthyma; caseous lymphadenitis (corynebacterium pseudotuberculosis); johne's disease (mycobacterium paratuberculosis); ovine progressive pneumonia; internal parasitism such as nasal bots, lungworms, and intes-tinal worms; and external parasitism such as sheep keds. each supplier should be queried about vaccination programs for bluetongue, brucella ovis, campylobacter spp., chlamydia (enzootic abortion of ewes), clostridial diseases, pneumonia complex (parainfluenza , pasteurella haemolytica, and p. multocida), ovine ecthyma, rabies, dichelobacter (bacteroides) nodosus, arcanobacterium pseudotuberculosis, bacillus anthracis, and fusobacterium necrophorum. because of the limited number of biologics approved for small ruminants, products licensed for cattle have been used with success in sheep, and some licensed for sheep are used in goats ("council report," javma, ) . in some cases, approved feed additives, such as coccidiostats, are fed to sheep. the basic screening profile for goats should include q fever (coxiella burnettii), caprine arthritis encephalitis (cae), brucellosis, tuberculosis, and johne's disease (mycobacterium paratuberculosis) . goats may also be tested for caseous lymphadenitis, contagious ecthyma, or mycoplasma as needed. herd vaccination programs may include immunizations against tetanus and other clostridial diseases, chlamydia, campylobacter, contagious ecthyma, caseous lymphadenitis, corynebacterium pseudotuberculosis, and escherichia coli. cattle herds should be screened for johne's disease, brucellosis, tuberculosis, respiratory diseases, internal and external parasitism, and foot conditions such as hairy heel warts and foot rot. determination of the status of the herd with respect to bovine leukemia virus (blv) may be worthwhile. herd programs may include essential or highly recommended vaccines against bovine viral diarrhea virus (bvdv), infectious bovine rhinotracheitis virus (ibrv), bovine respiratory syncytial virus (brsv), parainfluenza (pi- ), leptospira pomona, tritrichomonas fetus, rotavirus, coronavirus, campylobacter (vibrio) , pasteurella haemolytica and p. multocida, and brucella abortus. other vaccination programs, dependent on herd status, endemic diseases, or geographic location, may include immunizations against the clostridial diseases, moraxella bovis (pinkeye), fusobacterium necrophorum (foot rot), staphylococcus aureus (mastitis), haemophilus somnus, rabies, tetanus, bacillus anthracis, enterotoxigenic e. coll anaplasma, and other leptospira species. some products considered to have limited efficacy include vaccines against salmonella dublin and s. typhimurium. some autogenous vaccines may be more effective than the commercially available products--for example, the bovine papillomavirus (warts) vaccines. rearing programs for dairy calves differ from those for the smaller ruminants, including the withdrawal of calves from their dams immediately or by hours after birth. in the cattle industry, antibiotics, ionophores (antibiotics that control selected populations of ruminal organisms), coccidiostats, probiotics, and other approved additives may be part of the milk replacers, grain and concentrate formulations, and/or creep feeding regimens. use varies by the segment of the industry, and regulations vary by country. subcutaneous hormonal implants (such as estradiol benzoate and progesterone combined, zeranol, or [~-estradiol) are administered, especially to beef calves destined for market rather than breeding, to promote growth. transportation of the animals from the source to the research facility must be carefully planned, and all applicable livestock travel regulations followed. it is best to have the animals transported in vehicles regularly utilized by the source farm. if commercial haulers are used, then disinfecting trucks, trailers, and associated equipment, such as ramps and chutes, beforehand is particularly important. the loading, footing, and distribution of the animals in the trailers and trucks, as well as environmental conditions during shipping, are important to consider to minimize stress and injury to the animals. sufficient time for acclimation to the facility, pens, handlers, feed, and water must be allowed once at the destination ("livestock handling and transport," ). recent publications address many general considerations as well as specifics about the facilities, husbandry, space requirements, and standard practices for research and production ruminants. institutions, private entities, researchers, and facility staff must also be aware of the recent adoption by the u.s. department of agriculture (usda) of specific guidelines for regulation of farm animals, such as ruminants, that are used in biomedical and other nonagricultural research. the usda animal care policy notes that the "guide for the care and use of agricultural animals in agricultural research and teaching" and the "guide for the care and use of laboratory animals" provide additional information to supplement the existing animal welfare act regulations (cfr, ; fass, ; hays et al., ; nrc, a; usda, ) . in all cases, stress should be considered and minimized in the husbandry and handling of ruminants. animals need to be provided adequate time to adapt to new surroundings. stress decreases feed intake, and the resulting energy, vitamin, and mineral deficiencies will affect the growth and development in younger animals. reproductive soundness and rumen function are affected by transport and similar stresses. standard practices such as weaning, castration, dehorning, vaccinations, deworming and treatments for external parasites, shipping and the associated feed and water deprivation, introduction to a new housing environment and new personnel, and intercurrent disease are all stressors (houpt, ) . animals should be acclimated to the use of halters and leads, temporary restraint devices, and other handling equipment associated with the research program. personnel in the research facility who are unfamiliar with ruminants should be trained in appropriate handling techniques. ap-preciation for ruminant behaviors has grown in recent years, and refined ruminant handling techniques have been published (houpt, ; grandin, ) . when ruminants are confinement-housed, care should be taken to provide adequate but draft-free ventilation. ammonia buildup and other waste gases may induce respiratory problems. in cold weather, if the ceiling, walls, or water pipes condense water, then the ventilation should be increased even at the expense of lower temperatures. even adult goats and younger cattle are quite comfortable in cold, even subfreezing temperatures, if provided with adequate amounts of dry dust-free bedding and draft protection. sheep, because of their wool, are remarkably tolerant to both hot and cold extremes. newborn lambs and recently shorn adults are susceptible to hypothermia, hyperthermia, and sunburn. therefore, in outside housing areas, sheep should be provided with shelters to minimize exposure to sun and inclement weather. animals housed under intensive confinement should be kept clean, and excreta should be removed from the pens or enclosures daily. feed and water equipment should be maintained in sound, clean condition and should be constructed to prevent fecal contamination. waterers should not create a muddy environment in paddocks or pens. there should be sufficient continuous-access waterers placed around the area to prevent competition or fighting. feeders should be constructed to conform to species size and feeding characteristics and to prevent entrapment of head and limbs. pens, other enclosures, passageways, chutes, and floors must be very sturdy to withstand such factors as the frequent cleaning; the strength, weight, and curiosity of all ages of animals; and the investigative and climbing behaviors of goats. chain-link fences are dangerous because goats (as well as some breeds and ages of sheep) are curious and tend to stand on their hind legs against fencing or walls. forelimbs may be caught easily in the mesh. floors in any areas where animals will be housed, led, or herded must ensure secure footing at all times to prevent slipping injuries. all ruminants are social and herding animals. therefore, they should be housed in groups or at least within eyesight and hearing of other animals. singly housed animals should have regular human contact. environmental enrichment should be governed by the experimental protocol or standard operating procedures, and durable play objects should be supplied to those animals that are housed in confinement. calves, in particular, that must be singly housed or that have been recently weaned, need play objects (morrow-tesch, ) . because sheep and goats are sensitive to changes in light cycle (especially reproductive parameters), photoperiod must be taken into account. normally, sheep and goats should be maintained on a cycle comparable to natural conditions. light intensity should be maintained at about lux (ilar, ; fass, ) . light cycles can be manipulated for experimental reasons. the development of the digestive system and the unique function of the rumen are among the most notable comparative anatomic and physiologic characteristics of ruminants. there is a three-compartment forestomach (rumen, reticulum, and omasum) and a true stomach (abomasum). the mature rumen functions as an anaerobic fermentation chamber in which the enzymes, such as cellulase, of the resident bacteria allow the animals to prosper as herbivores. digestion is also aided by other microorganisms, such as protozoa ( - /ml) and bacteria ( - ~ that contribute to rumen fermentation. the result is the production of volatile fatty acids (acetic, propionic, and butyric) . unlike in the monogastrics, fermentative digestion and volatile fatty acid absorption also occur in the large intestines. the main sources of energy for ruminants are volatile fatty acids (vfas) rather than glucose. glucose is formed from propionic acid (or from amino acids) for metabolism in the central nervous system (cns), uterus, and mammary glands. plasma glucose in ruminants is much lower than and is regulated differently from that in nonruminants. the rumen microorganisms also synthesize vitamins, such as b and k, and provide protein that is used by the animals' systems. large amounts of fermentation gases such as co and methane, and small amounts of nitrogen, are naturally eructed (hecker, ; schimdt et al., ) . intestinal immunoglobulin absorption by pinocytosis in the neonates is crucial to the success of passive transfer. this transfer mechanism is functional for approximately the first hr after birth. neonatal ruminants are immunocompetent, however, and this condition is used to advantage for vaccinations against some common diseases of the neonatal and later juvenile periods, such as infectious bovine rhinotracheitis (ibr) vaccine (using modified live virus vaccines) to calves when their dams' colostrum is lacking antibody against this virus. unlike hepatic lipogenesis in humans, lipogenesis in sheep primarily occurs in adipose tissue and the mamrnary gland (hecker, ) . in addition to normal lymph node chains, and as in other ruminants, sheep have small red "nodes" associated with blood vessels. inadvertently named hemal "lymph nodes," they contain numerous red blood cells. sheep have a relatively large pituitary gland, and accessory adrenal medullary tissue may be interspersed throughout the abdominal cavity. three major ovine histocompatability classes have been identified and designated as ovar (ovis aries) classes i, ii, and iii (franz-werner et al., ) . bovines are recognized as having several unique aspects involving their immune systems. the bovine lymphocyte antigen (bola) system ranks after the hu-man (hla) and murine (h- ) systems in terms of depth of knowledge (lewin, ) . cattle are considered free of autoimmune diseases (schook and lamont, ) . the complexity of the immunobiology of the bovine mammary gland is being studied extensively because mastitis is the most prevalent disease in the dairy industry. several innate immune mechanisms and cellular defenses, and their variation throughout lactation, have been described (sordillo et al., ) . hematology and clinical reference texts are available for the ruminant species and include overviews of normal values for age, sex, and breed-specific ranges, as well as discussions regarding the influences on the hemogram of many management, nutritional, geographic, metabolic, physiologic (including lactation), medication, and iatrogenic variables (duncan and prasse, ; jain, ; kaneko et al., ) . these references should be consulted when preparing to include blood collection data in research protocols and when reviewing hematologic findings. in addition, most veterinary diagnostic laboratories have also developed databases for normal ranges for hematologic and clinical chemistry values based on subjects from their service areas, and these may be useful as local and breed references. appropriate control groups must be incorporated into each research plan, however, to establish the normal values (see table i ) for the particular locale, diagnostic facilities, breed, age, sex, and research circumstances. normal hematologic and clinical biochemistry data are presented in tables ii and iii. some general statements apply to most ruminants. most ruminants have fewer neutrophils than lymphocytes. the blood urea nitrogen (bun) values cannot be used as an indicator of renal function because of the metabolism of urea nitrogen by rumen microflora. because of the large volume of rumen water, ruminants can generally go several days without drinking before significant dehydration occurs. erythrocytes may become more fragile during rehydration, resulting in some degree of hemolysis and hemoglobinuria. severe dehydration can occur quickly, however, in animals that are ill. urine ph is generally alkaline in adult ruminants. ruminant erythrocytes are smaller than those in other mammals, and hematocrits tend to be overestimated unless blood samples are centrifuged for longer amounts of time for packing of the cell pellet. increased red-cell fragility is also associated with the smaller erythrocyte. rouleau formation does not occur in cattle but does to a limited extent in sheep and goats. in addition to fetal hemoglobin, sheep are reported to have at least six different hemoglobins (hecker, ) . blood coagulation in sheep is similar to that in humans. (di / , dc / , dp / ) = (di / , dc / , dp / ) = (di / , dc / , dp / ) = permanent dental formula ( / , c / , m / ) = ( / , c / , m / ) = ( / , c / , m / ) = avital sign data for goats are from "large animal internal medicine" ( ) . sheep weight data represent weights of feeder lamb and adult dry ewe (federation of animal science societies [fass], ) . goat weight data are for a large-breed male goat. cattle weight data represent weights of female holstein or guernsey dairy cattle (fass, ) . life span data for sheep and cattle are from brooks et al. ( ) . erythrocytes in pygmy and toggenburg goats tend to be more fragile than erythrocytes from other goat breeds. normal caprine erythrocytes lack central pallor because they are fiat and lack biconcavity. normal caprine erythrocytes may exhibit poikilocytosis. at least five blood groups have been reported in goats: b, c, m, r-o, and x. because transfusion reaction rates may be as high as - %, cross-matching is advisable although not always practical (smith and sherman, ) . blood loss of up to % of the red cell mass at a single time point can be tolerated by healthy goats. blood may safely be obtained in volumes of ml/kg body weight and given in volumes of - ml/kg. in general, aspartate aminotransferase (ast) and lactate dehydrogenase (ldh) are not liverspecific in goats, and alanine aminotransferase (alt; formally serum glutamic-pyruvic transaminase, or sgpt) cannot be used to evaluate hepatic disease in goats. ~,-glutamyltransferase (ggt) and alkaline phosphatase (ap) are associated with biliary stasis, and elevations in ggt are generally associated with hepatic damage. the nutritional needs of ruminants vary considerably according to the species, breed type, different phases of development, the use of the animals, location, and different stresses in their lives. for example, mineral requirements and other nutritional requirements vary even among breeds of cattle. several references are available that describe the varying requirements and are useful for determining the requirements of ruminants consistent with the parameters noted above and the type of feeds available (jurgens, ; "large animal clinical nutrition," ; nrc, nrc, , nrc, , nrc, , b "large animal internal medicine," ) . preformulated commercial feeds, concentrates, and supplements are available specifically for the different species of ruminants. some of these provide complete energy and protein requirements or may be used as supplements for what cannot be provided entirely by pasture, forage, hay, or silage. concentrate mixtures contain salt, minerals, and other elements. concentrates should contain a protein source such as soybean meal, cottonseed meal, or linseed meal. computer programs are also readily available for those who may need to formulate and balance rations. the palatability of feeds should be taken into account. mineral deficiencies and supplementation have been shown to influence several physiologic parameters such as immune function. introduction of young stock should include continuation of the feeding program of the source or gradual transition to appropriate feed for the animals available in the region of the research facility (nrc, ) . good-quality pasture can support ruminants under certain circumstances. lush spring pastures, especially pastures containing alfalfa, can induce bloat, diarrhea, grass tetany, or nitrate poisoning. ruminants not acclimated to lush pasture should be fed good-quality hay and slowly introduced to pasture environments. when ruminants have access to pasture, it is important to be aware of different eating habits. sheep and cattle are grazers. goats are browsers and will readily eat grasses, as well as seeds, nuts, fruit, and woody-stemmed plants. goats, however, can also be selective eaters and will only eat the leafy, more nutritious parts of the plant. therefore, goats have a tendency to "waste" hay. other eating habits should also be considered. finely ground concentrates are not tolerated well by goats; pelleted concentrates are preferred because the goat will pick out large particles in mixes. generally, goats do not prefer "sweet" feeds that contain molasses and do not need supplemental concentrates if a good-quality pasture or hay is fed. when given access to a salt block, goats generally are self-regulating. grass-fed goats and lactating goats may need supplementation with calcium and phosphorus, whereas alfalfa-fed goats do not (bretzlaff et al., ) . horse and sheep feeds may be fed to goats provided that the feed does not contain much molasses (bretzlaff et al., ) . the copper content of horse feed is not excessive for goats, as it is for sheep. pelleted horse feeds with - % fiber and - % protein are good goat rations. goats will consume - % of body weight in dry-matter intake (whereas cattle will usually consume only % of body weight). goats enjoy human contact, and small alfalfa cubes make tasty treats for the goat. rations that have excessive calcium-phosphorus ratios or elevated magnesium levels may induce urinary calculi in male ruminants. these may also occur when forage grasses are high in silicates and oxalates. to increase ovulation rate in does, some producers "flush" females by feeding . - lb concentrate per head per day for several weeks before and after the initiation of the breeding season. thin pregnant dairy goats should be fed lb concentrate per ) . - . ( . ) . - . potassium (k; mmol/l) hp . - . ( . ) . - . ( . ___ . ) . - . ( . adata presented as ranges with mean and standard deviation in parentheses, s, serum; p, plasma; hp, heparinized plasma. clinical biochemistry data from kaneko et al. ( ) . day, with the amount increasing to . lb per head per day during the last weeks of gestation. forage should be fed ad libitum during this time. all newborn ruminants must receive passive immunity from colostrum, the first postpartum milk of a dam that contains concentrated protective maternal antibodies (most as igg ), functional leukocytes, cytokines, vitamins, minerals, and protein. colostrum also has laxative properties. trypsin inhibitors in the colostrum allow the passage of intact antibody molecules, by pinocytosis, through the neonate's gut wall and into the bloodstream during the first few days after birth. the quality of the colostrum is directly related to herd or flock management, vaccination programs, and the dam's overall condition and nutrition throughout gestation and at the time of parturition. ensuring effective colostrum transfer is also dependent on the timing and amount taken by the neonate. most neonatal ruminants can suckle well within hr of birth. those that do so have been shown to have significantly less diarrhea (naylor, ) . neonates weakened by dystocia or hypothermia, for example, should be hand-fed or tube-fed colostrum. if necessary, the dam should be hand-milked and the newborn fed colostrum (for example, - ml for kids) every - hr for the first - days. in typical management situations, dairy calves either are separated from their dams immediately after birth and bottle-fed colostrum, or they remain with their dams for only about hr and suckle fresh colostrum during this time. dairy producers then refrigerate and/or freeze the colostrum that cannot be consumed by the calf during that time and then feed this diluted : with warm water times a day to the calves during the next - days. extra frozen colostrum for emergencies may be obtained from dairy farmers; it is advantageous to obtain colostrum from well-managed herds and from the multiparous cows in the herd (not heifers) in the same geographic locale. holstein calves, for example, should receive a minimum of - liters within hr of birth and then be fed about - % of body weight in colostrum by hr of age. after days, calves are then placed on milk replacers. although young ruminants generally do well receiving their dams' milk, commercially available milk replacers are available and should generally be prepared and fed according to the manufacturer's recommendations. containers used to prepare and feed these replacers should be sanitized daily. the fat content of both calf and lamb milk replacers is excessive; however, calf milk replacers can be used for kids if care is taken not to overfeed. young ruminants can be offered good-quality hay (such as second cutting) to nibble on by week of age. calves may be provided with calf starter, a commercially available concentrate with appropriate levels of energy and protein, fed according to the manufacturer's recommendations at - weeks of age. they can be weaned off milk replacer by - weeks of age. young ruminants ( - months of age) need good-quality forage as well as grain and concentrate supplementation to promote development of the rumen. in farm management situations, forage can be silage, pasture, and hay. in a confinement situation like a research unit, good-quality hay, such as second cutting, is desirable. animals should not be overfed and should be offered a mineral mix free-choice. in contrast to dairy calves, beef calves remain with their mother cows until weaning at months of age. calves tend to suckle many times per day. as they mature, calves are creepfed, with the energy and protein content of the ration determined by the milk production of the dams and by the available forage, such as pasture. several useful references addressing ruminant reproduction in detail are available ("current veterinary therapy: food animal practice," practice," , practice," , "large animal internal medicine," ; "current therapy in large animal theriogenology," ; hafez, ) . sheep are seasonally polyestrous; most breeds will express estrus in the fall (northern hemisphere) and subsequently lamb in the spring. some breeds of sheep may cycle in both the fall and the spring. between seasonal periods of receptivity, the females undergo a long period of sexual quiescence called anestrus. in a research environment, ewes can be artificially stimulated to progress from anestrous to estrous cyclicity by maintaining the females in hr of light and hr of dark for - weeks. puberty is reached at about - months (or earlier) in both rams and ewes; rams will typically reach puberty before their female counterparts. ewes will display signs of estrus for about - hr and will ovulate spontaneously at the end of estrus. the estrous cycle length is - days, with an average of about days. following breeding, the average length of gestation is - days. slightly longer gestations are observed in animals carrying single lambs (singlets), in animals carrying rams, and in certain breeds such as those derived from merinos. prolificacy, or the number of lambs produced per gestation, tends to be dependent on the maturity of the dam (older dams tend to have multiple lambs) and on breed characteristics (some fine-wool breeds have fewer multiple births). the finn and dorset breeds are especially prolific. lambs vary in size at birth from about - lb up to lb. factors that affect birthweight include parental size, number of lambs in the litter (fewer lambs or singlets tend to be larger), age of the ewe (younger ewes have smaller lambs), lamb gender (males tend to be heavier), nutrition, and season or temperature (spring lambs tend to be larger than fall lambs). goats are seasonally polyestrous in temperate regions, so that young are born in favorable times of the year. they are shortday breeders, in that estrus (heat) is brought about by the decreasing light of shorter days. in temperate climates of the northern hemisphere, goats are normally anestrous during the summer and begin cycling in the fall. the actual length of the sexual cycle depends on day length, breed, and nutrition. most dairy goats cycle between august and february or march. nubians often have extended breeding cycles, and the sexual season of some breeds, including the alpine, can be extended by artificial means. the caprine gestation length averages days with a variation of - days. does bear singletons, twins, and triplets, with slightly shorter gestation when the doe is carrying triplets. cows are polyestrous. domestication of cattle has included selection against seasonality of the breeding season, particularly in dairy breeds but to some extent also in the beef breeds. in spite of this, cattle have been found to be still sensitive, in varying manifestations, to photoperiodicity. reproductive physiology in cattle is influenced by many factors. the reproductive programs in source herds and at well-managed facilities will be production-related. extensive coverage of both physiologic basics and specific industry-related criteriamfor retention of a cow as a breeder, for examplenare addressed in detail in texts and references oriented toward herd and production management ("current veterinary therapy," ). gestation in cattle is approximately days, with a range of - days. the length of gestation in cattle is influenced by fetal sex; fetal numbers; age and parity of the cow; breed; genotype of cow, bull, or fetus; nutrition; and local environmental factors. as noted, these factors are also important in sheep and goats. cows usually bear single calves, although twin births do occur. when twins are combinations of male and female calves, the female should be evaluated for freemartinism. ovine estrus detection is usually accomplished by the ram. nonetheless, because artificial insemination is achievable in ewes, clinical signs of estrus are important. typically, ewes in heat will show a mild enlargement of the vulva, with slight increases of mucus secretion. ewes may isolate from the flock and appear anxious. it is often better and clearly more reliable to employ the help of a sterile ram to mark females when they are in standing heat. two mating systems commonly employed include hand mating and group mating. with hand mating, ewes are placed either singly or in small groups with the ram of choice. ewes are removed as serviced. group mating involves placement of a mature ram with approximately - ewes for the entire -week breeding season. in either mating system, it is best to attach a marking harness to the male so that individual ewes can be identified as serviced. this is important so that parturition dates can be calculated. an easy, natural way to estimate pregnancy is by placing sterile teaser rams with the ewes at the end of the breeding season. any animal marked by the ram probably has not conceived. ultrasound scanners are also used for pregnancy detection. the ultrasound transducer is placed against the right abdomen; presence of a fetus is indicated on the machine. claims of % accuracy at weeks postbreeding have been made, although accuracy is generally best beyond days of gestation. interrectal doppler ultrasound probes detect fetal pulses. fetal heart rate is in the range of - beats per minute, whereas maternal heart rates tend to be - beats per minute. accuracy is best beyond days of pregnancy. rectal-abdominal palpation is an inexpensive alternative. a plastic probe is introduced intrarectally into the ewe, which is restrained on her back in a cradle. the plastic probe is then manipulated toward the abdomen while palpating for the fetus with the opposite hand. the age of the doe when she first expresses heat varies with breed. some does will express signs of heat between and months old. however, does should be - months old or at least - lb in weight before being bred. the caprine estrous cycle lasts - days. the duration of estrus is - hr but averages about hr. the estrous cycle can be more erratic in the beginning than in the end of the breeding season (smith, season (with winter delaying), and the level of nutrition (with higher levels hastening puberty). in some cases, the presence of mature cycling cows influences heifer puberty. with adequate nutrition, dairy breeds will reach puberty at - months and beef breeds at - months, and estrous cycles will occur regularly after the pubertal (first) estrus, maturing heifers will often have one or more ovulations before showing overt signs of estrus. only one follicle usually ovulates per estrous cycle (hafez, ) estrus, or standing heat, in cattle averages - hr in length, with a range of - hr ("large animal internal medicine," ) . detection of standing heat is important because it is closely related to the time of ovulation. ovulation occurs approximately - hr after estrus. detection of estrus is usually accomplished by visual observation of vaginal mucous discharge, mounting behavior by other females (i.e., the cow standing to be mounted is the individual in estrus), and receptivity to a bull (willingness to stand). successful visual detection of standing heat is dependent on observation skills of handlers, knowledge of the herd, stresses (e.g., detection decreased in bos taurus during heat stress), barn and yard surfaces (estrus detected better on dirt than on concrete), and maintaining a consistent observation schedule. teaser animals outfitted with marking devices are also used. other methods of detecting estrus include monitoring progesterone levels; glass slide and other evaluations of cervical mucus; change in vaginal ph; and body temperature changes (hafez, ) . estrous cycles are usually days in length, with a range of - days. it is recommended that a heifer deliver her first calf by years of age. after successful conception, progesterone levels in the cow remain elevated for most of the pregnancy, as the result of the ). "standing heat" is usually - hr but can be as short corpus luteum of pregnancy, and they decline only during the as a few hours. signs of estrus in goats include uneasiness, tail switching or "flagging," redness and swelling of the vulva, clear vaginal discharge that becomes white by the end of estrus, vocalization such as continuous bleating, and occasionally riding and standing with other does. a doe that is not in heat will not stand to back pressure or for attempts to hold her tail. does can be induced to show signs of heat by buck exposure and will ovulate within - days after introduction of the buck. goats ovulate during the later part of the estrous cycle, most between - hr after the onset of estrus. nevertheless, goats should be mated once signs of estrus are recognized and every hr until the end of estrus. most goats kid only once a year, although some goats near the equator may kid twice. once bred successfully, a goat will only rarely show signs of heat again. in fact, the first sign of pregnancy is usually a failure to return to heat, so animals should be carefully watched. pregnancy can be affirmed by a variety of means. goats will generally decrease milk production with pregnancy and should have at least a -to -week dry period for the udder to fully involute and prepare for the next milking period. in cattle, age of first estrus is dependent on the breed, the final month. conceptus implantation occurs beginning at about day . if the pregnancy fails before this time, the cow will begin to cycle again between days - , but if the pregnancy ends after day , there may be a delayed return to estrus. realtime ultrasonography can be used to determine pregnancy as early as days after insemination, with embyros seen by days - . fetal gender can also be determined by experienced personnel by this method by about day . detection of pregnancy can be successful by - days after conception by observation of failure to return to estrus or by palpation per rectum (detecting fetal membrane slip by days - and/or amniotic vesicle by days - ). palpation of the fetus is possible by day and placentomes by approximately days - . palpation later in presumed pregnancy will provide information based on differences in size of the two uterine horns, changes in the uterine wall, and fremitus in the miduterine artery. pregnancy can also be determined with reasonable success rates by determining if progesterone levels are elevated at days - after insemination. levels of bovine pregnancy-specific protein b may also be measured; this is produced by trophoblastic cells and is detectable by days - and elevated throughout pregnancy. placentation in sheep, goats, and cattle is epitheliochorial and ft. evaluation of a cow's udder prior to breeding and especotyledonary, in contrast to the diffuse or microcotyledonary cially as parturition approaches is important in order to assure placentas of horses and pigs. the placentomes, the infolded adequate nutrition and success of passive transfer by the functional units of the placenta, are formed as the result of fu-neonate. if the udder is edematous or if mastitis is present, for sion of the villi of the fetal cotyledons projecting into the crypts example, an alternate source of colostrum (such as frozen reof the maternal caruncles (specialized projections of uterine " serves) must made be available. poor udder conformation may mucosa). caruncles of sheep and goats are concave in shape, whereas those of cows are convex. the placentomes are distributed between the pregnant and nonpregant horns of the uterus in sheep, and there are - . in cattle, although the placentomes initially develop around the fetus, they will eventually be distributed to the limit of the chorioallantoic membrane even in the nongravid horn. the placentomes in the nongravid horn will be smaller than in the gravid horn. the total number will be - . the best birthing preparation for all dams is to ensure a proper plane of nutrition (not overnutrition) and adequate exercise. if possible, the dam should be confined to a birthing pasture or sanitized maternity pen a few days prior to parturition. the birthing environment will be very important in the overall health of the dam and offspring; stress minimization and a clean environment will benefit the immune health of both in the short and long term. outdoor parturition in a small birthing pasture has advantages. there is less stress and less intensity of pathogens. indoor maternity pens should be clean, dry, warm, well bedded, well ventilated but draft-free, and well lighted. adequate space per pen minimizes losses of neonates from being stepped and sat on by the dam. management of these pens, especially if concentrated in an area, is important to minimize pathogens to which dam and young are exposed. water troughs or buckets should be elevated or placed outside the pen, because lambs and kids have a tendency to fall or be pushed into them. soiled bedding should be removed from the birthing pen between dams, the area sanitized and allowed to dry, and fresh bedding installed for the next occupant. moving the female immediately before or during parturition may delay the birthing process. in goats, furthermore, in utero death may occur if parturition is unduly delayed. dams should be monitored closely during parturition for dystocias; these may result in loss of young or in young severely weakened from the prolonged birthing process. prior to parturition, ewes should be sheared or crutched. crutching refers to removing wool around the perineal and mammary areas; this minimizes fetal contamination during the birth process. foot trimming can be done at this time as well. the tail and perineal area of the doe should be clipped and cleaned to improve postbirth sanitation. in general, the pregnant doe needs a ft ( . m x . m) area for the birthing process, and area needs to be increased after birthing to allow spacing for kids. each cow should have a minimum pen area of ft x also be problematic; contingency plans should be made to ensure adequate support for the young if they cannot suckle from those udders. inexperienced heifers may react indifferently or aggressively to their offspring and should be monitored more closely than older, multiparous cows with uneventful calving histories. ewes approaching parturition generally isolate themselves from the flock, become restless, stamp their feet, blat, and periodically turn and look at their abdomen. the pelvic region will appear relaxed, and milk will be present in the udder. once hard labor contractions begin, lambs will usually be born quickly. animals that do not appear to be progressing correctly should be examined for dystocia. most cases of fetal malpresentation or malpositioning can be corrected via vagino-uterine manipulation. occasionally cesarean sections will be necessary. sanitation, cleanliness, and adequate lubrication are of utmost importance when performing obstetrical procedures. for about a week before parturition, rectal temperature of the doe will be above normal, or about ~ depending on environmental temperatures. approximately hr prior to birth, rectal temperature will fall to slightly below normal. many large dairy-goat facilities attempt to control the onset of parturition in order to assist birthing. the drug of choice to induce parturition in the goat is prostaglandin f ~ (pgf ~) (ott, ) . on day of gestation, goats given pgf ~ ( . - mg) will deliver kids within - hr. most goats prefer to kid alone and do so unaided. human interaction can actually interfere with normal birthing, especially in young or nervous does. some does may reject kids if extensive human interference occurs. does nearing parturition have an obviously swollen udder and a red, swollen vulva. pelvic ligaments at the base of tail relax. the doe may circle to make a bed, get up and down, look at her tail or sides, push other goats away, and bleat softly. signs of impending parturition include restlessness; vocalization (bleating softly); uneasiness, including getting up and down, pawing, and bedding; and a mucous discharge, leading to a moist tail. eight to hr prior to parturition, the cervix will dilate and the cervical mucous plug will be evident as a tan, smeared substance on the tail and perineum of the dam. kids should present within - hr in either anterior or posterior position. a posterior presentation can be recognized by the presence of upward-pointing feet. most does will rest between fetuses and are best left alone. however, if labor is prolonged more than hr, a vaginal exam is indicated. if the pregnant goat is housed with other goats, then herdmates will express great interest in the dam. unless moved prior to parturition, it is best to leave the dam with the group until after parturition, because removal may delay parturition. goats are not prone to retained placenta. normal kids will be quite active and will quickly attempt to stand and nurse. weak kids should be towel-dried, warmed (via heat lamp, heat pad, or warm water bottle), and assisted to nurse or fed colostrum. the goat is one of the few ungulate species that will exhibit "false pregnancy," or pseudopregnancy. this is a fairly common condition. does may have characteristically distended abdomens and may develop hydrometra and "deliver" large volumes of cloudy fluid at expected due dates. subsequent pregnancies can be normal. goats should be tested for pregnancy by days of age. veterinary use of prostaglandins has been successful in treating this condition. as in other species, parturition in cattle results from a combination of hormonal changes associated with the maturity of the fetus, notably acth (adrenocorticotropic hormone) and subsequent increases in fetal corticosteriods within days of birth. administration of acth to a fetus, or administration to the dam, results in premature birth. pregnancy is extended if fetal pituitary or adrenal glands are removed surgically. the fetal cortisol probably affects placental steroid production, accounting for sharp increases in the estrogens and estrogen precursors. coincident with this, maternal progesterone levels fall. the rising levels of estrogen cause release of maternal pgf ~ and induction of oxytocin receptors. most cows will separate themselves from the rest of the herd. a cow will lift her tail and arch her back when she is within a few hours of delivering the calf, and most cows are recumbent when delivering the calf. typically, the whole birthing process takes about min. the length of labor of cows carrying larger calves also will be longer. nervous heifers will take longer to deliver, and if they are disturbed, their labor may cease. all postparturient animals should be monitored for successful passage of these fetal membranes within hr of birth. veterinary intervention is required if not. cows occasionally eat placentas, which may subsequently obstruct rumen outflow and require surgical correction. for cattle, it is now recommended practice to remove membranes that have passed, in order to prevent ingestion. following lambing, it is critical that the newborns be "processed" so that they will have greatest survival chances. in a well-managed flock, many lambs and ewes will not need much assistance. when assistance is given, the newborn lamb's nose and mouth should be wiped free of secretions; gently swinging the lambs, head down, aids in removal of these fluids. the lamb should be dried off and stimulated through rubbing to aid its breathing. the lamb's navel should be dipped in an iodine solution to prevent subsequent navel infections. and the lamb should be identified by the application of an ear tag or ear notch. it is extremely important that the lamb be supplied with highquality colostrum within the first hr of birth. lambs that are not nursing on their own should be tube-fed with colostrum that has been collected and saved previously (i.e., frozen in ice cube trays) or collected from the mother after parturition. passive transfer can be assessed by measuring serum y-glutamyltransferase (ggt) levels (tessman et al., ) . after the first few days, colostrum changes over to milk. nursing lambs will ingest increasing amounts of milk as they grow. if the ewe cannot produce sufficient milk, the lamb should be "grafted" onto another ewe or fed artificially with a baby bottle. powdered milk replacers are commercially available; the content of ewe milk is much different from that of cow's milk; thus lamb milk replacer should specifically be used. one report notes that - % of lamb deaths occur during the first week of life and up to % occur within the first month. good management of ewes during gestation, care of the lamb at parturition, application of an appropriate vaccination program, and observation and intervention within the first several weeks of a lamb's life will minimize losses (ross, ) . immediately after birth, the placenta and any birthing materials should be removed from the doe's pen. kids do not usually need assistance. if kids are to be raised by the dam, they can be left alone; otherwise, kids should be towel-dried and removed from the dam. kids are cold-sensitive and may require a heat lamp or other source of added warmth in cold weather. navel cords should be dipped in tincture of iodine, and kids should be dehorned and castrated within the first several days of life. to control caprine arthritis encephalitis (cae), kids should be immediately removed from the dam and hand-fed heattreated colostrum. colostrum should be heat-treated for hr at ~ e the first feeding can be up to ml of colostrum. kids should receive a total of ml colostrum within the first - hr of birth. after day , kids can be placed on milk replacer. milk replacers should contain - % fat and - % milkbased protein. by days of age, kids should be consuming approximately . - . liters of milk per day. kids should be introduced to forages as soon as possible and may be weaned by - weeks or - lb body weight. milk that is fed can be reduced by weeks of age by decreasing either the volume fed or the number of feedings. as with other dams, a cow is usually very attentive to her newborn calf, cleaning and softly vocalizing to the neonate. calves typically are standing by hr after birth and are suckling within hr. as noted previously, dairy calves may be removed from the cow even before suckling, and the colostrum milked from the dam and given to the calf. assistance may be required for nervous heifers, after dystocias and in extreme circumstances such as severe cold. cleaning the newborn's nose and mouth, rubbing down the neonate, assuring that the calf does not get chilled, and assuring that it receives adequate colostrum are all important under any of these circumstances. a stressed calf's umbilical may be treated with an iodine or chlorhexidine solution, although some authors note no benefit of navel treatment, specifying that successful transfer of passive immunity and sound sanitary management of birthing area are the most crucial factors in preventing omphalitis (navel ill) (house, ; kersting, ; kasari and roussel, ) . because newborn calves can be deficient in vitamin a and iron, these may be injected to improve disease resistance (wikse and baker, ) . in cases in which the dams' colostrum is known to be deficient in antibodies against common diseases, vaccinations may be administered at day old and followed with boosters at regular intervals. dehorning is performed when horn buds appear. castration is performed between and weeks of age or later. sexing the young in any of the ruminant species is straightforward. the vulva of the female young is located just ventral to the anus. the genitalia of the male include a penis, located along the ventral midline, and a scrotum, located in the inguinal region. the phenomenon of the freemartin, a genetic female born as a twin to a male, is the result of anastomoses between placental circulations of the twin fetuses; the mixing of bloodforming cells and germ cells results in the xx/xy chimeras. this occurs in - % of phenotypic bovine females born as co-twins with males. the female will often have abnormal vulva and clitoris, and the vagina will be a blind end because of the lack of a cervix. sometimes singleton freemartins are born if the male fetus is lost after days' gestation. multiple births are selected for and are common in sheep; the freemartin phenomenon is regarded as rare. twinning is common in goats, and freemartinism occurs in about % of male-female pairs of twins. intersexes are seen in some goat breeds and when polled goats are mated. proof is usually based on evidence of abnormal genital development and reports of abnormal sexual behavior. prior to weaning, it must be established that lambs can nutritionally survive without mother's milk. thus, grain, and later roughage, should be offered to lambs well in advance of the day of weaning so that they can adjust to the feedstuff. to prevent the ewes from ingesting the lamb ration, a "creep" should be set up by building an area adjacent to the ewe-lamb pen and devising a slatted entry for the lambs to enter but not the ewes. therefore, the lambs will be accustomed to the new ration through this creep-feeding process. if lambs and ewes will be pastured later in the spring, it is still beneficial to creep-feed lambs until pasture growth is adequate enough to fulfill the requirements of the growing lambs. lambs that are consuming . - lb of creep feed per day may be weaned. depending on the individual program, lambs may be weaned as early as weeks of age, although - weeks of age is more common. if ewes are of a breed that will cycle twice a year, and if it is expected that they will be rebred, then the lambs must be weaned as early as possible so that lactational anestrus will resolve and ewes will recycle. another factor is the cost of lactation rations for the ewes; if lamb grain is more economical than ewe grain, then lambs should be weaned. about - days prior to weaning, feeding of the lactation ration to the ewes should be discontinued, and only roughage fed. at weaning, the lambs should be removed in the creep, and the ewes removed to an area that is not within sight (and preferably sound) of the lambs. the ewes should be monitored for postweaning mastitis and treated as necessary. ewes that have physical or disease problems or that have not been productive at lambing or feeding their lambs should be culled. the lambs should be monitored to assure that they continue to gain weight and are eating the new ration. kids should be introduced to forages within the first week of life because the natural curiosity of these animals will cause them to investigate sources of feed. kids can be weaned by - weeks or - lb. hand-fed milk should be reduced by weeks of age by reducing the volume fed or by decreasing the number of feedings. dairy calves are now usually removed from their dams immediately after birth. it is less common now to allow the calves to remain with their dams for about hr and suckle fresh colostrum during this time, because their intake will be inadequate. dairy producers refrigerate and/or freeze the colostrum produced during the first hr and feed this, diluted : with warm water, twice a day to the calves during the next - days. holstein calves, for example, should receive a minimum of - liters within hr of birth and then be fed about - % of body weight in colostrum by hr of age. after days, calves are then placed on milk replacers, preformulated powders reconstituted with water that provide complete nutrition. milk replacers are commercially available and should be fed according to manufacturer's recommendations vaccination programs for calves vary with the preventive medicine program for the overall herd. passive immunity provided by colostrum from cows on sound management programs will last until a calf is about - months old; normally vaccinations are not necessary and are contraindicated during those first months. the duration of passive immunity varies considerably among calves, however; some producers choose to begin vaccinating calves at - months of age and continue with monthly booster immunizations until the animals are months old, when passive immunity is no longer a possibility. artificial insemination (ai) in sheep is more difficult than in cattle because sheep are smaller and cannot be reproductively manipulated via the rectum and because the cervix of sheep is more difficult to traverse with the insemination pipette. breeding animals artificially with fresh semen produces pregnancy rates averaging % (not unlike that of cattle); artificial insemination with frozen semen is less successful. several artificial insemination techniques have been used. laparoscopic ai involves the surgical instillation of semen into the uterus through a small abdominal opening. the procedure is successful but is technically involved and costly. cervical ai involves the transvaginal introduction of semen into the cervix. a modification of this technique (transcervical ai) allows for penetration through the cervix into the uterus. this method (called the guelph system for transcervical ai) leads to successful penetration into the uterus in up to % of ewes when performed by an experienced inseminator. artificial insemination is now an integral part of dairy herding; natural insemination as a management practice is relatively rare. technicians performing the ai technique are available through commercial enterprises. dairy production employees are also trained. information regarding the management of the donors and recipients, the storage and handling of the semen, and the skills and record keeping required is covered extensively elsewhere (nebel, ) . because sheep are hormonally similar to other ruminants, estrous synchronization techniques are comparable. progesterone suppresses follicle-stimulating hormone (fsh) secretion, preventing animals from developing follicles and exhibiting estrus. artificial or natural progesterone can be administered in the feed, through parenteral injection, subcuticular implants, and vaginal pessaries. the progesterone is withdrawn in about - days, after which the fsh secretion will initiate the process of follicle development (trower, ) . estrus usually will occur in - hr (average is hr). a natural method of synchronization, often applied to promote flock breeding within a short period of time (and thus parturition will be within a narrow window as well), is the introduction of sterile rams with the ewes before the beginning of the normal fall mating period. pheromones released from males naturally stimulate the females to cycle and to synchronize their heats. it should be noted that introduction of a male during late anestrus will often stimulate ovulation in about days; however, this cycle will generally be without clinical signs of estrus (silent heat). vasectomy of rams is one method of producing sterile "teaser rams." introduction of the buck to a group of does will induce ovulation and may even synchronize does. does that are kept separate from the buck will show signs of estrus, will ovulate within - days, and will have normal pregnancies when introduced to a buck. bucks with horns and intact scent glands are better able to induce ovulation than dehorned bucks, whose scent glands often been removed. control of breeding in the goat has been studied mostly in dairy breeds in order to produce milk throughout the year and to reduce kidding labor. goats in the luteal phase of the estrous cycle, days - , are sensitive to pgf ~ ( . - mg im) and will show estrus in - hr postinjection (bretzlaff, ) . dosing cycling animals twice days apart will synchronize goats, and artificial insemination using this method has resulted in - % conception rates (bretzlaff, ; greyling and van niekerk, ) . programs for timed breeding have been described and involve administering progestogens (bretzlaff, ) . vaginal pessaries of fluorogestone acetate left in place for days in the doe followed by an injection of pregnant mare serum gonadotropin (pmsg) at the time of pessary removal have proven successful. also, when primed by pgf ~, an day regimen of fluorogestone acetate with pmsg given on day has been successful. synchronization of cattle estrous cycles and superovulation are used as management techniques in certain commercial cattle and dairy production settings where estrus synchronization or embryo transfer is advantageous to production and management. the methodology is also used in the research setting for coordinating donors and recipients of embryos or other genetically manipulated tissues for implantation. the options and dosing regimens are described in detail in veterinary clinical texts (wenzel, ; vanderboom et al., ) . in synchronization, the principle is lysis of the existing corpus luteum. the more common practices involve the use of products approved for use in cattle such as pgf ~, one of its analogs, or products containing estradiol valerate. progestogens are also used in conjunction with estradiol valerate. other approaches, involving management techniques combined with pharmacologic interventions, are considered less successful. superovulation regimens involve injections of fsh either alone or with pgf ~ at timed internals. estrus is expected hr after the final injection, and two inseminations are performed at hr intervals after estrus detection. preparation of recipients involves injection of pgf ~ or progestogens with gonadotropins such as pmsg. for greatest success as management tools, these must be combined with a consistent program that provides appropriate nutrition for all cattle involved. synchronization of animals is also influenced by several other factors, however, such as time in the cycle when hormones are administered, response by each individual animal, whether the cow is a dairy or beef animal, parity and maturity of the cows, success of heat detection after the luteolysis, and accurate record keeping. embryo transfer involves the removal of multiple embryos from a superovulated embryo donor and transferring them to synchronized recipients. this method maximizes the genetic potential of the donor animal. the donor animal is hormonally superovulated and inseminated. in sheep, about week after breeding, the embryos are surgically removed from the donor's uterus. in cattle, the procedure is nonsurgical. about % of expected embryos (determined by counting corpora lutea) can be recovered; successful recovery is affected by factors such as age of the donor, reproductive health, and experience of the surgeon or technician. furthermore, not all collected embryos are of transferable quality. recipients are hormonally synchronized with the donor animals. on the day of embryo collection, transferable embryos are implanted into the uterus of the recipient; laparoscopy has been used in the past and is now being replaced by nonsurgical methods. pregnancy rates average about %. if recipients are not available, embryos, like sperm, can be frozen and kept for later transfer. embryo transfer is commonly practiced in cattle as a herd improvement technique and as a research technique for engineered embyros. disease screening programs for all animals involved are important because several pathogens can be transmitted directly or indirectly, such as bovine viral diarrhea virus, bluetongue virus, infectious bovine rhinotracheitis virus, and mycoplasmal species. in sheep flocks and goat herds, as noted, male young are usually castrated by month of age. the elastrator method is the more popular for animals less than week of age. other methods include the emasculatome (crushing) and surgical removal ("knife method"). the distress associated with castration and tail docking in lambs is the subject of debate and has been researched recently (kent et al., ) . as noted, male calves are usually castrated as early as possible and no later than month of age. in some production situations, however, where maximum hormone responsive muscle development and grouping animals together for procedures dictate scheduling, the procedure may be performed on older males. open and closed techniques are used, depending on the age of animals and on veterinary or farm practice. breeding and vasectomized rams and bucks are usually maintained by medium to large production farms. smaller farms often borrow breeding males. breeding males are typically selected by production record, pedigree, and/or breed. vasectomized males are often retired breeders and should be tattooed or identified clearly to avoid any wasted breeding time. the vasectomy technique for both species is comparable (smith and sherman, ) . rams may be housed together for most of the year, whereas bucks are penned separately. because ewes will exhibit only a limited number of estrous cycles before becoming reproductively quiescent, it is critical that the male be capable of successfully breeding the female in an expeditious manner. any defects in the external genitalia, reproductive diseases, or musculoskeletal abnormalities may prevent successful copulatory behaviors. furthermore, it is impor-tant to know the semen quality of the ram as one indicator of fertility. semen can be collected via electroejaculation or by use of a teaser mount. once semen is collected, it should be handled carefully and kept warm to prevent sperm death, leading to improper conclusions about the male. typically, the characteristics usually evaluated as a determinate of sperm quality are volume (normal between . and . ml); motility (% of sperm moving in a forward wave; high quality is associated with motility of approximately %); concentration (sperm count per unit of volume as measured by a hemocytometer; high-quality semen should contain . x sperm per ml); morphology (live versus dead cells, as determined by special stains and the percentage of abnormal-appearing sperm; neither the abnormalities nor the dead sperm should exceed % in high-quality semen). the extensive use of artificial insemination in the dairy cattle industry has minimized the use of bulls on many farms, although a farm may maintain a few bulls for heat detection and for "cleanup" breeding. breeding bulls are maintained in beef production establishments. breeding bulls must be part of the herd vaccination program, with special attention to appropriate timing of immunizations for the commonly transmitted venereal diseases campylobacteriosis and trichomoniasis. tail docking is a relatively recent development in dairy herd management and is practiced in the belief that it will minimize bacterial contamination of the udder and therefore the milk. tails are typically docked to about inches in length. the practice is more popular in certain regions in the united states. to date, there is no published study indicating that this technique provides any distinctive advantage over keeping the tail switch hair clipped short. healthy ruminants have good appetites, chew cud, are alert and curious, have healthy intact coats, move without hindrance, and have clear, bright, clean eyes and cool dry noses. even adult animals, when provided sufficient space, will play. sheep and goats have tidy "pelleted" dark green feces. cattle have pasty, moist, dark green-brown feces. ruminants normally vocalize, and handlers will learn to recognize normal communication among the group or directed at caregivers in contrast to that when animals are stressed. excessive, strained vocalizations are often a sign of stress in cattle. "bruxism," or grinding of the teeth by a ruminant, is usually associated with discomfort or pain. other signs of discomfort, stress, or illness include decreased time spent eating and cud chewing, restlessness, prolonged recumbency with outstretched neck and head, and hunched back when standing. unhealthy ruminants may be thin, may arch their backs or favor a limb, or may have external lumps or swollen joints, an unusual abdominal profile, or rough or dull coats. all ruminants are herd animals to some extent and social individuals; therefore, every effort should be made to allow contact among animals, in terms either of direct contact or of sound, smell, or sight. human contact and handling should be initiated promptly and maintained regularly and consistently throughout the animal's stay in the research facilities. animals should be provided sufficient time to acclimate to handlers and research staff. cattle and sheep can hear at higher frequencies than humans can and may react to sounds not perceived by handlers. knowledge of the peculiarities of sheep behavior will increase the ease of handling and decrease stress-related effects in research. generally, fine-wooled breeds, such as rambouillet, are the most gregarious and are best handled in groups. the meat, or "downs," breeds tend to be less gregarious, and the long-wooled breeds tend to be solitary (ross, ; asia, ) . nonetheless, movement of animals is simplified by proper facility design. sheep have a wide-angle visual field and are easily scared by activities that are taking place behind them. sheep should be moved slowly and gently. to capture individuals within a flock, it is best to confine the flock to a smaller space and use a shepherd's crook or to gently catch the animal in front of the neck/thorax. grabbing the wool can injure the animals, as well as damage the wool and the underlying tissues. sheep move best in chutes that have solid walls, and individual animals will generally follow a lead animal. any escape route will be challenged and, if successfully breached, will disrupt the entire flock movement. sheep movement is also disrupted by contrasts such as light and shadows that impinge on a chute or corral. finally, like most animals, sheep have a flight zone (minimum zone of comfort), the penetration of which will result in sheep scattering. this minimal flight distance can be modified by increasing handling of the animals and working at the edge of the zone, but it should always be considered when working with animals in chutes, pens, or other confined areas. goats exhibit behavioral characteristics that make them quite distinct from other ruminants. their browsing activity makes them quite orally investigative. goats will readily nibble or chew just about anything they come in contact with, so researchers should keep all paperwork and equipment out of reach. a herd of goats will readily chew through wood gates and fencing, especially when confined in areas without alternatives for chewing behavior. goats are also inquisitive, restless, agile jumpers and climbers, and quite mischievous. if maintained in paddocks, strong high fences are essential, as are adequate spaces for exercise or boulders or rock piles for hoof maintenance and recreational climbing. goats are more tolerant of isolation and are more easily acclimated to human contact than sheep are, but goats will confront unfamiliar intruders and make sneezing noises. goats with horns will use them to advantage, and horns may also become entangled in fencing. although less strongly affected by flock behavior, goats are social animals. most goats raised in close human contact are personable and cooperative and can easily be taught to stand for various procedures, including blood collection. an understanding of breed behaviors, sources of stress in cattle, play behaviors, calf behaviors, and dominance determinants will contribute to prevention of injuries to handlers and better health and welfare of the animals. ruminants of all ages, especially cattle of all ages, should be handled with an appreciation of the serious injury to human handlers that may result (houpt, ) . cattle have a wide visual field, as sheep do, and a flight zone that varies in size, according to previous handling experiences (gentle handling and animal tameness make the flight zone smaller) and the circumstances of the moment (grandin, ) . groups of cattle are moved effectively around a facility by utilizing chute systems, with sequences of gates, that minimize chances of animals turning around. dairy cattle have been bred and selected over centuries for their docile, tractable characters and production characteristics. in contrast, beef breeds have not been selected for docility and are generally more difficult to handle and restrain. beef breeds, such as angus, are known for their independent natures and protective maternal instincts. all cattle respond well to feed as a reward for desired behavior. healthy cattle typically are very curious and watchful and are alert to sounds and smells. when not grazing or eating, they hold their heads up. when sleeping, the head and neck may be tucked back. because of ruminant digestive and metabolic needs, much of the day is spent eating or cud chewing. occasionally, adult cows sit upright like dogs. cattle maintained inside tend to be more docile. in addition to forced isolation from other cattle, sources of stress include rough attitudes of handlers and unfamiliar visual patterns, routines, or environments. these stressors may exacerbate signs of systemic illnesses. calves are known for non-nutritive suckling, bar licking, and tongue rolling. non-nutritive suckling behavior is greater in hungry calves and also right after a milk meal. it is best to provide nipples and other clean noninjurious materials for the animals to suck. non-nutritive suckling can be detrimental in group-housed calves because it can result in disease transmission and hair ball formation. environmental enrichment devices have been developed to cope with this behavior. the behavior diminishes as the animals are weaned onto solid food (morrow-tesch, ) . play activity and vocalizations of calves mimic adult dominance behaviors. play activity by young adult cattle is more common in males, can be quite rough, and is often triggered by a change in the environment. dominance behaviors are dependent on direct physical contact among the cattle, and dominance hierarchies are established within a herd. horns, age, and weight have been reported to be the most important determi-nants. aggressive behaviors in cattle may be triggered by newly introduced animals or unfamiliar visual patterns and by feeding when animals are very hungry. aggression is more common among intact adult males. this section focuses primarily on the more common diseases affecting sheep, goats, and cattle in the united states and elsewhere in north america and those that are reportable. for detailed information not included in this limited overview and for diseases of importance internationally, the authors recommend several excellent comprehensive and focused veterinary clinical texts and periodicals that address ruminant diseases, preventive medicine, and individual and flock or herd management. these are listed under "major references" in the reference list at the end of this chapter. recommendations for current drug therapies, both approved and off-label use in ruminants, including withholding prior to slaughter, formularies, and related information can be found in the references noted above and in formularies (hawk and leary, ; plumb, ) . in addition, the food animal residue avoidance databank (farad), accessible on the internet <http://www.farad.org>, should be used as a resource. farad is a food safety project of the u.s. department of agriculture and is an information resource to prevent drug and pesticide residues in food animals and animal products. food; may be anorexic, weak, unthrifty and depressed; and may salivate excessively. diagnosis is made based on clinical signs and is confirmed by culture. epizootiology and transmission. the organism penetrates wounds of the skin, mouth, nose, gastrointestinal tract, testicles, and mammary gland. rough feed material and foreign bodies may play a role in causing abrasions. actino bacillus lignieresii then enters into deeper tissues, where it causes chronic inflammation and abscess formation. lymphatic spread may occur, leading to abscessation of lymph nodes or infection of other organs. necropsy findings. purulent discharges of white-green exudate drain from the tracts that often extend from the area of colonization to the skin surface. exudates will also contain characteristic small white-gray (sulfurlike) granules. the pus is usually nonodorous. differential diagnosis. contagious ecthyma and caseous lymphadenitis are the primary differentials. diseases or injuries causing oral pain and discomfort, such as dental infections, foreign bodies, and trauma, should be considered. treatment. animals should be fed softer feeds. antibiotics such as sulfonamides, tetracyclines, and ampicillin are effective, although high doses and long durations of therapy are required. penicillin is not effective. weekly systemic administration of sodium iodide for several weeks is not as effective as antibiotic therapy. surgical excision and drainage are not recommended. etiology. actinobacillus lignieresii is an aerobic, nonmotile, non-spore-forming, gram-negative rod that is widespread in soil and manure and is found as normal flora of the respiratory, gastrointestinal, and reproductive tracts of ruminants. in sheep and cattle, a. lignieresii causes sporadic, noncontagious, and potentially chronic disease characterized by diffuse abscess and granuloma formation in tissues of the head and occasionally other body organs. this disease, called wooden tongue, has not been documented in goats. clinical signs. skin lesions are common. tongue lesions are more common in cattle than in sheep. lip lesions are more common in sheep. soft-tissue or lymph node swelling accompanied by draining tracts is observed in the head and neck regions, as well as other areas. animals may have difficulty prehending prevention and control. because the organism enters through tissue wounds, especially those associated with oral trauma, feedstuffs should be closely monitored for coarse material and foreign bodies. b. arcanobacterium infection (formerly actinomycosis, or "lumpy jaw") etiology. arcanobacterium (formerly known as actinomyces or corynebacterium) pyogenes and a. bovis are anaerobic, nonmotile, non-spore-forming, gram-positive, pleomorphic rods to coccobacilli. arcanobacterium bovis is a normal part of the ruminant oral microflora and is the organism associated with "lumpy jaw" in cattle; this syndrome is rarely seen in sheep and goats. this organism has also been associated with pharyngitis and mastitis in cattle. clinical signs and diagnosis. arcanobacterium bovis causes mandibular lesions primarily. the mass will be firm, nonpainful, and immovable. draining tracts may develop over time. if teeth roots become involved, painful eating and weight loss are evident. radiographic studies are helpful for determining fistulas. diagnosis is based on clinical signs, and culture is required to confirm arcanobacterium. the prognosis is poor for lumpy jaw. epizootiology and transmission. these organisms are normal flora of the gastrointestinal tracts of ruminants and gain entrance into the tissues through abrasions and penetrating wounds. necropsy. draining lesions with sulfurlike granules (as with actinobacillosis) are frequently observed. ious degrees of depression and anorexia, and purulent discharges may be seen draining from the umbilicus. involvement of the urachus is usually followed by cystitis and associated signs of dysuria, stranguria, hematuria, and so on. severe sequelae may include septicemia, peritonitis, septic arthritis (joint ill), meningitis, osteomyelitis, and endocarditis. research complications. young stock affected by omphalophlebitis may be inappropriate subjects because of growth setbacks and physiologic stresses from the infection. affected adult animals will not thrive and, even with therapy, may not be appropriate research subjects. pathogenesis. arcanobacterium pyogenes is known to produce an exotoxin, which may be involved in the pathogenesis. differential diagnosis. actinobacillus lignieresii and caseous lymphadenitis are important differentials for draining tracts. a major differential for omphalophlebitis is an umbilical hernia, which will typically not be painful or infected. there are many differentials for septic joints and polyarthritis: chlamydia spp., mycoplasma spp., streptococci, coliforms, erysipelothrix rhusiopathiae, fusobacterium necrophorum, and salmonella spp. tumors, trauma to the affected area, such as the mandible, and dental disease or oral foreign body should also be considered. prevention and control. arcanobacterium bovis lesions can be prevented or minimized by feeds without coarse or sharp materials. treatment. penicillin or derivatives such as ampicillin or amoxicillin are treatments of choice. sodium iodides (intravenous) and potassium iodides (orally) have been utilized also. extended antibiotic therapy may be necessary. surgical excision is an option. in addition to medications noted above, isoniazid is somewhat effective for a. bovis infections in nonpregnant cattle. research complications. the possibility of long-term infection and long therapy are factors that will diminish the value of affected research animals. omphalophlebitis, omphaloarteritis, omphalitis, and navel ill are terms referring to infection of the umbilicus in young animals. arcanobacterium pyogenes is the most common organism causing omphalophlebitis, an acute localized inflammation and infection of the external umbilicus. most cases occur within the first months of age, and animals are presented with a painful enlargement of the umbilicus. animals may exhibit var- etiology. bacillus anthracis is a nonmotile, capsulated, sporeforming, aerobic, gram-positive bacillus that is found in alkaline soil, contaminated feeds (such as bonemeal), and water. common names for the disease anthrax include woolsorters' disease, splenic fever, charbon, and milzbrand. clinical signs and diagnosis. anthrax is a sporadic but very serious infectious disease of cattle, sheep, and goats characterized by septicemia, hyperthermia, anorexia, depression, listlessness, depression, and tremors. subacute and chronic cases may occur also and are characterized by swelling around the shoulders, ventral neck, and thorax. the incubation period is day to weeks. bloody secretions such as hematuria and bloody diarrhea often occur. abortion and blood-tinged milk may also be noted. the disease is usually fatal, especially in sheep and goats, after - days. death is the result of shock, renal failure, and anoxia. diagnosis is based on the clinical signs of peracute deaths and hemorrhage. stained blood smears may show short, single to chained bacilli. blood may be collected from a superficial vein and submitted for culture. epizootiology and transmission. cattle and sheep tend to be affected more commonly than goats, because of grazing habits. older animals are more vulnerable than younger, and bulls are more vulnerable than cows. although the disease occurs worldwide, and even in cold climates, most cases in the united states occur in the central and western states, and outbreaks usually occur as the result of spore release after abrupt climatic changes such as heavy rainfall after droughts or during warmer, dryer months. spores survive very well in the environment. the anthrax organisms (primarily spores) are generally ingested, sporulate, and replicate in the local tissues. abrasive forages may play a role in infection. transmission via insect bites or through skin abrasions rarely occurs. necropsy. necropsies should not be done around animal pens or pastures, and definitive diagnoses may be made without opening the animals. incomplete rigor mortis, rapid putrefaction, and dark, uncoagulated blood exuding from all body orifices are common findings. blood collected carefully and promptly from peripheral veins of freshly dead animals can be used diagnostically. splenomegaly, cyanosis, epicardial and subcutaneous hemorrhages, and lymphadenopathy are characterisitic of the disease. pathogenesis. the rapidly multiplying organisms enter the lymphatics and bloodstream and result in a severe septicemia and neurotoxicosis. encapsulation protects the organisms from phagocytosis. liberated toxins cause local edema. differential diagnosis. although anthrax should always be considered when an animal healthy the previous day dies acutely, other causes of acute death in ruminants should be considered, e.g., bloat, poisoning, enterotoxemia, malignant edema, blackleg, and black disease. prevention and control. outbreaks must bereported to state officials. anthrax is of particular concern as a bioterrorism agent. any vaccination programs should also be reviewed with regulatory personnel. herds in endemic areas and along waterways are usually vaccinated routinely with the sterne-strain spore vaccine (virulent, nonencapsulated, live). careful hygiene and quarantine practices are crucial during outbreaks. dead animals and contaminated materials should be incinerated or buried deeply. biting insects should be controlled. the disease is zoonotic and a serious public health risk. treatment. treatment of animals in early stages with penicillin and anthrax antitoxin (hyperimmune serum, if available) may be helpful. amoxicillin, erythromycin, oxytetracycline, gentamicin, and fluoroquinolones are also good therapeutic agents. during epidemics, animals should be vaccinated with the sterne vaccine. research complications. natural and experimental anthrax infections are a risk to research personnel; the pathogen may be present in many body fluids and can penetrate intact skin. the organism sporulates when exposed to air, and spores may be inhaled during postmortem examinations. etiology. brucella is a nonmotile, non-spore-forming, nonencapsulated, gram-negative coccobacillus. brucella abortus is one of several brucella species that infects domestic animals but cross-species infections occur rarely. brucella abortus or b. melitensis may cause brucellosis in sheep, cattle, and goats. brucella melitensis (biovar , , or ) is the primary cause of sheep disease (garin-bastuji et al., ) . brucella ovis is more commonly associated with ovine epididymitis or orchitis than abortion. in the united states, clusters of brucellosis are still found in western areas contiguous to yellowstone national park. bang's disease is the common name given to the disease in ruminants. clinical signs and diagnosis. brucella melitensis in the adult ewe is generally asymptomatic and self-limiting within about months. however, because the organism may enter and cause necrosis of the chorionic villi and fetal organs, abortion or stillbirths may occur. abortion usually occurs in the third trimester, after which the ewe will appear to recover. it has been reported that up to % of infected ewes may abort more than once. rams will also be infected and may develop orchitis or pneumonia. the disease caused by b. ovis is manifested by clinical or subclinical infection of the epididymis, leading to epididymal enlargement and testicular atrophy. brucella ovis causes decreased fertility. brucella melitensis is the more common cause of brucellosis in goats. brucella abortus has been shown to infect goats in natural and experimental infections, and b. ovis has also been shown to infect goats experimentally. does infected with b. melitensis will also abort during the third trimester. infections with b. abortus in cattle produce few clinical signs. there may be a brief septicemia during which organisms are phagocytosed by neutrophils and fixed macrophages in lymph nodes. in cows, the organism localizes in supramammary lymph nodes and udders and in the endometrium and placenta of pregnant cows. infection may cause abortions after the fifth month, with resulting retained placentas. permanent infection of the udder is common and results in shedding of organisms in milk. in bulls, the organism may cause unilateral orchitis and epidydimitis and involvement of the secondary sex organs. organisms may be in the semen. in infected herds, lameness may also be a clinical sign. diagnosis of brucellosis can be made by bacterial isolation of the brucella organism from necropsy samples (especially the fetal stomach contents), as well as by supportive serological evidence. many serological tests are available, such as the tube and plate agglutination tests, the card or rose bengal test, the rivanol precipitation test, complement fixation, enzyme-linked immunosorbent assay (elisa), polymerase chain reaction (pcr), and others. test selection is often dependent on state requirements in the united states. epizootiology and transmission. the primary route of transmission of b. abortus is ingestion of the organism from infected tissues and fluids (milk, vaginal and uterine discharges) during and for a few weeks after abortion or parturition; contaminated semen is considered to be a minor source of infection. exposure to the organism may occur via the gastrointestinal tract (contaminated feed or water), the respiratory tract (droplet infection), or the reproductive tract (contaminated semen) and through other mucous membranes such as the conjunctiva. brucella ovis is transmitted in the semen, as well as orally or nasally through contaminated feed and bedding. necropsy findings. a sheep fetus aborted due to brucella will exhibit generalized edema. the liver and spleen will be swollen, and serosal surfaces will be covered with petecchial hemorrhages. peritoneal and pleural cavities often contain serofibrinous exudates. the placenta will be leathery. pathogenesis. ruminants are considered especially susceptible to brucella infection, because of higher levels of erythritol (a sugar alcohol), which is a growth stimulant for the organism. brucella utilizes erythritol preferentially over glucose as an energy source. placentas and male genitalia also contain high levels of erythritol. brucella organisms also evade lysis when phagocytosed by macrophages and neutrophils and survive intracellularly in phagosomes. abortion is the result of placentitis, typically during the third trimester of gestation. brucella ovis enters the host through the mucous membranes, then passes into the lymphatics, causes hyperplasia of reticuloendothelial cells, and is spread to various organs via the blood. the organism localizes in the epididymides, the seminal vesicles, the bulbourethral glands, and the ampullae. orchitis may be a sequelae of the disease. epididymitis can be diagnosed by identifying gross lesions by palpation of the epididymides, by serological evidence of antibodies to b. ovis, and by semen cultures. differential diagnosis. differential diagnoses include all other abortion-causing diseases. many other agents, such as actinobacillus spp., arcanobacterium (actinomyces) pyogenes, eschericia coli, pseudomonas spp., proteus mirabilis, chlamydia, mycoplasma, and others may be associated with ovine epididymitis and orchitis. a clinically and pathologically similar agent, actinobacillus seminis, has been isolated from virgin rams. this organism has morphological and staining characteristics similar to those of b. ovis and complicates the diagnosis (genetzky, ) . prevention and control. the rev vaccine has been recommended for vaccination of ewe lambs in endemic areas, but this vaccine is not used in the united states. separating young rams from potentially infected older males, sanitizing facilities, and vaccinating them with b. ovis bacterin can prevent the disease. over the past years, aggressive federal and state regulatory and cattle herd health programs in the united states have provided control and prevention mechanisms for this pathogen through a combination of serological monitoring of herds, slaughter of diseased animals, herd management, vaccination programs, and monitoring of transported animals. most states are considered brucellosis-free in the cattle populations; thus, procurement of ruminants that have been exposed to this infectious agent will be unlikely. cattle vaccination programs can be very successful when conducted on a herd basis to reduce likelihood of exposure. strain and the recently validated attentuated strain rb are live vaccines and can be used in healthy heifer calves - months old. vaccination for older animals may be done under certain circumstances. vaccination of bull calves is not recommended, because of low likelihood of spread through semen and possibility of vaccination-induced orchitis. the strain vaccine induces long-term cell-mediated immunity, protects a herd from abortions, and protects the majority of a herd from reactors during a screening and culling program. the vaccine will not, however, protect the animals from becoming infected with b. abortus. strain vaccine induces an antibody response in cattle. the rb vaccine does not result in antibody titers and therefore is advantageous because infection with brucella can be determined serologically. the rb vaccine has been designated as the official calfhood bovine brucellosis vaccine in the united states by the u.s. department of agriculture's animal and plant health inspection service (aphis) (stevens et al., ) . brucella vaccine should be administered to unstressed, healthy cattle, with attention to particular side effects of the vaccination material and to prevention of compounding stresses associated with weaning, regrouping, other management changes, and shipping. the rb is regarded as less pathogenic and abortigenic in cattle. clinical signs and diagnosis. ovine vibriosis is a contagious disease that causes abortion, stillbirths, and weak lambs. the organism inhabits the intestines and gallbladder in subclinical carriers. abortion generally occurs in the last trimester, and abortion storms may occur as more susceptible animals, such as maiden ewes, become exposed to the infectious tissues. it is reported that - % of the flock may become infected and up to % of the ewes will die (jensen and swift, ) . some lambs may be born alive but will be weak, and dams will not be able to produce milk. diagnosis is achieved by microscopic identification or isolation of the organism from placenta, fetal abomasal contents, and maternal vaginal discharges. tentative identification of the organism can be made by observing curved ("gull-wing") rods in giemsa-stained or ziehl-neelsen-stained smears from fetal stomach contents, placentomes, or maternal uterine fluids. epizootiology and transmission. campylobacteriosis occurs worldwide. campylobacter spp., such as c. jejuni, normally inhabit ovine gastrointestinal tracts. transmission of the disease occurs through the gastrointestinal tract, followed by shedding, especially associated with aborted tissues and fluids. in abortion storms, considerable contamination of the environment will occur due to placenta, fetuses, and uterine fluids. ewes may have active campylobacter organisms in uterine discharges for several months after abortion. the bacteria will also be shed in feces, and feed and water contamination serve as another source. there is no venereal transmission in the ovine. necropsy. aborted fetuses will be edematous, with accumulation of serosanguinous fluids within the subcutis and muscle tissue fascia. the liver may contain - cm pale foci. placental tissues will be thickened and edematous and will contain serous fluids similar to those of the fetus. the placental cotyledons may appear gray. pathogenesis. the organism enters the bloodstream and causes a short-term bacteremia ( - weeks) prior to the localizing of the bacteria in the chorionic epithelial cells and finally passing into the fetus. should be considered in late gestation ovine abortions. a bacterin is available to prevent the disease. carrier states have been cleared by treating with a combination of antibiotics, including penicillin and oral chlortetra-cycline. aborting ewes should be isolated immediately from the rest of the flock. after an outbreak, ewes will develop immunity lasting - years. treatment. infected animals should be isolated and provided with supportive therapy. prompt decontamination of the area and disposal of the aborted tissues and discharges are important. research complications. losses from abortion may be considerable. campylobacter ssp. are zoonotic agents, and c. fetus subsp, intestinalis may be the cause of "shepherd's scours." ii. clinical signs and diagnosis. preliminary signs of a problem in the herd will be a high percentage of cows returning to estrus after breeding and temporary infertility. this will be particularly apparent in virgin heifers that may return to estrus by days after breeding. long interestrous intervals also serve an indication of a problem. spontaneous abortions will occur in some cases, typically during the fourth to eighth months of gestation. severe endometritis may lead to salpingitis and permanent infertility. demonstration or isolation of the organism, a curved rod with corkscrew motility, is the basis for diagnosis. the vaginal mucous agglutination test is used to survey herds for campylobacteriosis. serology will not be worthwhile, because the infection does not trigger a sufficient antibody response. culture from breeding animals may be difficult because campylobacter will be overgrown by faster-growing species also present in the specimens. epizootiology and transmission. the bacteria is an obligate, ubiquitous organism of the genital tract. transmission is from infected bulls to heifers. older cows develop effective immunity. necropsy findings. necrotizing placentitis, dehydration, and fibrinous serositis will be found grossly. in addition, bronchopneumonia and hepatitis will be seen histologically. pathogenesis. campylobacter organisms grow readily in the genital tract, and infection is established within days of exposure. the resulting endometritis prevents conception or causes embyronic death. differential diagnosis. the primary differential diagnosis for campylobacteriosis is trichomoniasis. other venereal diseases should be considered when infertility problems are noted in a herd. these include brucellosis, mycoplasmosis, ureaplasmosis, infectious bovine rhinotracheitis-infectious pustular vulvovaginitis (ibr-ipv), and bovine virus diarrhea (bvd). leptospirosis should also be considered. in addition, management factors such as nutrition and age of heifers at introduction to the herd should be considered. prevention and control. killed bacterin vaccines are available, either as oil adjuvant or as aluminum hydroxide adsorbed. the former is preferred because of duration of immunity but causes granulomas. that vaccine also has specific recommendations regarding administration several months before the breeding season. the latter product is administered closer to the breeding season, and the duration of immunity is not as prolonged. in both cases, boosters should be given after the initial immunization and as part of the regular prebreeding regimen. only one bacterin product is approved for use in bulls. many combination vaccine products contain only the aluminum hydroxide adsorbed product. artificial insemination (ai) is particularly useful at controlling the disease, but bulls used for ai must be part of a screening program for this and other venereal diseases such as trichomoniasis. treatment. cows will usually recover from the infection, and treatment with antibiotics such as penicillin, administered as an intrauterine infusion, improve the chances of returning to breeding condition. etiology. the most common caprine bacterial skin infection is caused by staphylococcus intermedius or s. aureus and is known as staphylococcal dermatitis (smith and sherman, ) . the staphylococcus organisms are cocci and are categorized as primary pathogens or ubiquitous skin commensals of humans and animals. staphylococcus aureus and s. intermedius are classified as primary pathogens and produce coagulase, a virulence factor. clinical signs and diagnosis. small pustular lesions, caused by bacterial infection and inflammation of the hair follicle, occur around the teats and perineum. occasionally, the infection may involve the flanks, underbelly, axilla, inner thigh, and neck. staphylococcal dermatitis may occur secondary to other skin lesions. diagnosis is based on lesions. culture will distinguish s. aureus. pathogenesis. simple boredom may cause rubbing, followed by staphylococcal infection of damaged epidermis. differential diagnosis. the presence of scabs makes contagious ecthyma a differential diagnosis, along with fungal skin infections and nutritional causes of skin disease. treatment. severe infections should be treated with antibiotics based on culture and sensitivity. severe lesions and lesions localized to the underbelly, thighs, and udder benefit by periodic cleaning with an iodophor shampoo and spraying with an antibiotic and an astringent (smith and sherman, ) . h. clostridial diseases i. clostridium perfringens type c infection (enterotoxemia and struck) etiology. clostridium perfringens is an anaerobic, grampositive, nonmotile, spore-forming bacterium that lives in the soil, in contaminated feed, and in gastrointestinal tracts of ruminants. the bacteria is categorized by toxin production. toxins include alpha (hemolytic), beta (necrotizing), delta (cytotoxic and hemoltyic), epsilon, and iota. types of c. perfingens are a, b, c, d, and e. this is a common and economically significant disease of sheep, goats, and cattle. clinical signs and diagnosis. the beta toxin associated with overgrowth of this bacterium results in a fatal hemorrhagic enterocolitis within the first hr of a young ruminant's life. many animals may be found dead, with no clinical presentation. affected animals are acutely anemic, dehydrated, anorexic, restless, and depressed and may display tremors or convulsions as well as abdominal pain. feces may range from loose gray-brown to dark red and malodorous. morbidity and mortality may be nearly %. a similar noncontagious but acutely fatal form of enterotoxemia in adult sheep, called struck, occurs in yearlings and adults. struck is rare in the united states. the disease is also caused by the beta toxin of c. perfringens type c and is often associated with rapid dietary changes or shearing stresses in sheep. although affected animals are usually found dead, clinical signs include uneasiness, depression, and convulsions. mortality is usually less than %. diagnosis is usually based on necropsy findings, although confirmation can be made by culture of the organism. identification of the beta toxin in intestinal contents may be difficult because of instability of the toxin. necropsy findings. necropsy findings include a milk-filled abomasum, and hemorrhage in the distal small intestine and throughout the large intestine. petechial hemorrhages of the serosal surfaces of many organs, especially the thymus, heart, and gastrointestinal tract, will be visible. hydropericardium, hydroperitoneum, and hemorrhagic mesenteric lymph nodes will also be present. pulmonary and brain edema may also be seen. histologically, the gram-positive c. perfringens organisms may be visible in excess numbers along the mucosal surface of the swollen, congested, necrotic intestines. in cases of struck, necropsy findings include congestion and erosions of the mucosa of the gastrointestinal tract, serosal hemorrhages, and serous peritoneal and pericardial fluids. in late stages of the disease and especially if prompt necropsy is not performed, the organism will infiltrate the muscle fascial layers and produce serohemorrhagic and gaseous infiltration of perimysial and epimysial spaces. pathogenesis. hemorrhagic enterotoxemia is an acute, sporadic disease caused by the beta toxin of clostridium perfringens type c. neonates ingest the organism, which then proliferates and attaches to the gastrointestinal microvilli and elaborates primarily the beta toxins. the trypsin inhibitors present in colostrum prevent inactivation of the beta toxin. the toxins injure intestinal epithelial cells and then enter the blood, leading to acute toxemia. the intestinal injury may result in diarrhea, with small amounts of hemorrhage. associated electrolyte and water loss result in dehydration, acidosis, and shock. differential diagnosis. differential diagnoses include other clostridial diseases such as blackleg and black disease, as well as coccidiosis, salmonellosis, anthrax, and acute poisoning. clinical signs in chronic cases in older animals, such as adult goats, include soft stools, weight loss, anorexia, depression, and severe diarrhea, sometimes with mucus and blood. mature affected sheep may be blind and anorectic and may head-press. necropsyfindings. necropsy findings are similar to those seen with c. perfringens type c. additionally, extremely necrotic, soft kidneys ("pulpy kidneys") are usually observed immediately following death. (this phenomenon is in contrast to what is normally associated with later stages of postmortem autolysis.) focal encephalomalacia, and petechial hemorrhages on serosal surfaces of the brain, diaphragm, gastrointestinal tract, and heart are common findings. diagnosis can be made from the typical clinical signs and necropsy findings as well as the observation of glucose in the urine at necropsy. shock, probably through vascular damage. the noncontagious, peracute form of enterotoxemia occurs in suckling, fast-growing animals, either nursing from their dams or on high-protein, high-energy concentrates. the largest, fastest-growing animals generally are predisposed to this condition; for example, lambs, fat ewe lambs, and usually singleton lambs tend to be most susceptible. the hyperglycemia and glucosuria seen in acute cases are due to epsilon toxin effects on liver glycogen metabolism. should be administered to the pregnant animals prior to parturition. an alternative includes administration of an antitoxin to the newborn lambs. the disease may become endemic once it is on the premises. treatment. treatment is difficult and usually unsuccessful. antitoxin may be useful in milder cases, and the antitoxin and toxoid can also be administered during an outbreak. differential diagnosis. tetanus, enterotoxigenic e. coli, botulism, polioencephalomalacia, grain overload, and listeriosis are differentials. prevention and control. vaccination prevents the disease. maternal antibodies last approximately weeks postpartum; thus young animals should be vaccinated at about this time. feeding regimens to young, fast-growing animals and feeding of concentrates to adults should be evaluated carefully. research complications. this disease can be costly in losses of neonates and younger animals. treatment. treatment consists of support (fluids, warmth), antitoxin administration, oral antibiotics, and diet adjustment. toxin that is proteolytically activated by trypsin. this disease caused by c. perfringens tends to be associated with sheep and is of less importance in goats and cattle. clinical signs. the peracute condition in younger animals is characterized by sudden deaths, which are occasionally preceded by neurological signs such as incoordination, opisthotonus, and convulsions. because the disease progresses so rapidly to death (within - hr), clinical signs are rarely observed. hypersalivation, rapid respirations, hyperthermia, convulsions, and opisthotonus have been noted. in acute cases, hyperglycemia and glucosuria are considered almost pathognomonic. etiology. clostridium tetani is a strictly anaerobic, motile, spore-forming, gram-positive rod that persists in soils and manure and within the gastrointestinal tract. at least serotypes of c. tetani exist. clinical signs. infection by c. tetani is characterized by a sporadic, acute, and fatal neuropathy. after an incubation period of days to weeks, the animal exhibits bloat; muscular spasticity; prolapse of the third eyelid; rigidity and extension of the limbs, leading to a stiff gate; an inability to chew; and hyperthermia. erect or drooped ears, retracted lips, drooling, hypersensitivity to external stimuli, and a "sawhorse" stance are frequent signs. the animal may convulse. death occurs within - days, and mortality is nearly %, primarily from respiratory failure. diagnosis is based on clinical signs. musclerelated serum enzymes such as aspartate aminotransferase (ast), creatinine kinase (ck), and lactate dehydrogenase (ldh) might be elevated. (jensen and swift, ) . serum cortisol may also be elevated, and stress hyperglycemia may be evident. permanent lameness may result in survivors. contaminant and is often found as part of the gut microflora of herbivores. the organisms sporulate and persist in the environment. all species of livestock are susceptible, but sheep and goats are more susceptible than cattle. individual cases may occur, or herd outbreaks may follow castration, tail docking, ear tagging, or dehorning. mouth wounds may also be sites of entry. pathogenesis. tetanus, or lockjaw, is caused by the toxins of c. tetani. all serovars produce the same exotoxin, which is a multiunit protein composed of tetanospasmin, which is neurotoxic, and tetanolysin, which is hemolytic. a nonspasmogenic toxin is also produced. contamination of wounds results in anaerobic proliferation of the bacterium and liberation of the tetanospasmin, which diffuses through motor neurons in a retrograde direction to the spinal cord. the toxin inhibits the release of glycine and y-aminobutyric acid from renshaw cells; this resuits in hypertonia and muscular spasms. proliferation of c. tetani in the gut of affected animals may also serve as a source and may produce clinical signs. the uterus is the most common site of infection in postparturient dairy cattle with retained placentas. differential diagnoses. early in the course of the infection, differential diagnoses include bloat, rabies, hypomagnesemic tetany, polioencephalomalacia, white muscle disease, enterotoxemia in lambs, and lead poisoning. polyarthritis of cattle is a differential for the gait changes in that species. necropsy findings. findings are nonspecific except for the inflammatory reaction associated with the wound. because of the low number of organisms necessary to cause neurotoxicosis, isolation of c. tetani from the wound may be difficult. administering tetanus antitoxin (e.g., at least iu in an adult sheep or goat); vaccinating with tetanus toxoid; administering of antibiotics (penicillin, both parenterally [potassium penicillin intravenously and procaine penicillin intramuscularly] and flushed into the cleaned wound), a sedative or tranquilizer (e.g., acepromazine or chlorpromazine) and a muscle relaxant; and keeping the animal in a dark, quiet environment. supportive fluids and glucose must be administered until the animal is capable of feeding. if the animal survives, revaccination should be done days after the previous dose. prevention and control like other ubiquitous clostridial diseases, tetanus is impossible to eradicate. the disease can be controlled and prevented by following good sanitation measures, aseptic surgical procedures, and vaccination programs. tetanus toxoid vaccine is available and very effective for stimulating long-term immunity. tetanus antitoxin can be administered ( iu in lambs) as a preventive or in the face of disease as an adjunct to therapy. both the toxoid and the antitoxin can be administered to an animal at the same time, but they should not be mixed in the syringe, and each should be administered at different sites, with a second toxoid dose administered weeks later. animals should be vaccinated or times during the first year of life. does and ewes should receive booster vaccinations within months of parturition to ensure colostral antibodies. research complications. unprotected, younger ruminants may be affected following routine flock or herd management procedures. contaminated or inadequately managed open wounds or lesions in older animals may provide anaerobic incubation sites. etiology. clostridium novyi, an anaerobic, motile, sporeforming, gram-positive bacteria, is the agent of bighead and black disease. clostridium novyi type d (c. hemolyticum) is the cause of bacillary hemoglobinuria, or "red water." clostridium chauvoei is the causative agent of blackleg. clinical signs. bighead is a disease of rams characterized by edema of the head and neck. the edema may migrate to ventral regions such as the throat. additional clinical signs include swelling of the eyelids and nostrils. most animals will die within - hours. black disease, or infectious necrotic hepatitis, is a peracute, fatal disease associated with c. novyi. it is more common in cattle and sheep but may be seen in goats. the clinical course is - days in cattle and slightly shorter in sheep. otherwise healthy-appearing adult animals are often affected. clinical signs are rarely seen, because of the peracute nature of the disease. occasionally, hyperthermia, tachypnea, inability to keep up with other animals, and recumbency are observed prior to death. bacillary hemoglobinuria is an acute disease seen primarily in cattle and characterized by fever and anorexia, in addition to the hemoglobinemia and hemoglobinuria indicated by the name. animals that survive a few days will develop icterus. mortality may be high. blackleg, a disease similar to bighead, causes necrosis and emphysema of muscle masses, serohemorrhagic fluid accumulation around the infected area, and edema (jackson et al., ) . blackleg is more common in cattle than in sheep. the incubation period is - days and is followed by hyperthermia, muscular stiffness and pain, anorexia, and gangrenous myositis. the clinical course is short, - hr, and untreated animals invariably die. blackleg in cattle can be associated with subcutaneous edema or crepitation; these do not usually occur in sheep. most lesions are associated with muscles of the face, neck, perineum, thigh, and back. epizootiology and transmission. bighead is caused by the toxins of c. novyi, which enters through wounds often associated with horn injuries during fighting. the c. novyi type b organisms produce alpha and beta toxins, and the alpha toxins are mostly responsible for toxemia, tissue necrosis, and subsequent death. clostridium novyi type d is endemic in the western united states. it is hypothesized that the c. chauvoei organisms enter through the gastrointestinal tract. black disease and bacillary hemoglobinuria are associated with concurrent liver disease, often associated with fasciola infections (liver flukes); it is sometimes seen as a sequela to liver biopsies. the diseases are more common in summer months, and fecal contamination of pastures, flooding, and infected carcasses are sources of the organism. birds and wild animals may be vectors of the pathogen. ingested spores are believed to develop in hepatic tissue damaged and anoxic from the fluke migrations. necropsy. diagnosis of black disease is usually based on postmortem lesions. subcutaneous vessels will be engorged with blood, resulting in dried skin with a dark appearance. carcasses putrefy quickly. in addition, hepatomegaly and endocardial hemorrhages are common, and hepatic damage from flukes may be so severe that diagnosis is difficult. blood coagulates slowly in affected animals. pathogenesis. the propagation of the clostridial organisms is self-promoted by the damage caused by the toxins and the increased local anaerobic environment created. clostridium novyi proliferates in the soft tissues of the head and neck, and the resultant clostridial toxin causes increased capillary permeability and the liberation of serous fluids into the tissues. mixed infections with related clostridial organisms may lead to increasing hemorrhage and necrosis in the affected tissues. diagnosis is based on clinical signs. in black disease and bacillary hemoglobinuria disease, the ingested clostridial spores are absorbed, enter the liver, and cause hepatic necrosis. associated toxemia causes subcutaneous vascular dilatation; increased pericardial, pleural, and peritoneal fluid; and endocardial hemorrhages. the toxins produced by c. novyi, identified as beta, eta, and theta, and each having enzymatic or lytic properties or both, also contribute to the hemolytic disease. clostridium chauvoei spores proliferate in traumatized muscle areas damaged by transportation, rough handling, or injury. differential diagnosis. differential diagnoses include other clostridial diseases as well as photosensitization. hemolytic diseases such as babesiosis, leptospirosis, and hemobartonellosis should be included as differentials. treatment. for c. chauvoei infection (blackleg), early treatment with penicillin or tetracycline may be helpful. treatment for black disease is not rewarding even if the animal is found before death. carcasses from bacillary hemoglobinuria losses should be burned, buried deeply, or removed from the premises. prevention and control. vaccinating animals with multivalent clostridial vaccines can prevent these diseases. subcutaneous administration of vaccine material is recommended over intramuscular. vaccinations may be useful in an outbreak. careful handling of ruminants during shipping and transfers will contribute to fewer muscular injuries. for bighead, mature rams penned together should be monitored for lesions, especially during breeding season. control of fascioliasis is very important in prevention and control of black disease and in the optimal timing of vaccinations. etiology. clostridium septicum is the species usually associated with malignant edema, but mixed infections involving other clostridial species such as c. chauvoei, c. novyi, c. sordellii, and c. perfringens may occur. clostridium spp. are motile (c. chauvoei, c. septicum) or nonmotile, anaerobic, spore-forming, gram-positive rods. clinicial signs. malignant edema, or gas gangrene, is an acute and often fatal bacterial disease caused by clostridium spp. the incubation period is approximately - days. the affected area will be warm and will contain gaseous accumulations that can be palpated as crepitation of the subcutaneous tissue around the infected area. regional lymphadenopathy and fever may occur. the animal becomes anorexic, severely depressed, and possibly hyperthermic. edema and crepitation may be noted around the wound; death occurs within hr to days. epizootiology and transmission. the organisms are ubiquitous in the environment and may survive in the soil for years. the disease is especially prevalent in animals that have had recent wounds such as those that have undergone castration, docking, ear notching, shearing, or dystocia. necropsy findings. the tissue necrosis and hemorrhagic serous fluid accumulations resemble those of other clostridial diseases. pathogenesis. in most cases, the clostridial organisms cause a spreading infection through the fascial planes around the area of the injury; vegetative organisms then produce potent exotoxins, which result in necrosis (alpha toxin) and/or hemolysis (beta toxin). furthermore, the toxins enter the bloodstream and central nervous system, resulting in systemic collapse and high mortality. necropsy. spreading, crepitant lesions around wounds are suggestive of malignant edema. affected tissues are inflamed and necrotic. gas and serosanguineous fluids with foul odors infiltrate the tissue planes. large rod-shaped bacteria may be observed on histopathology; confirmation is made through culture and identification. intramuscular inoculation of guinea pigs causes a necrotizing myositis and death. organisms can be cultured from guinea pig tissues. treatment. infected animals can be treated with large doses of penicillin and fenestration of the wound is recommended. prevention and control. proper preparation of surgical sites, correct sanitation of instruments and the housing environment, and attention to postoperative wounds will help prevent this disease. multivalent clostridial vaccines are available. research complications. morbidity or loss of animals from lack of or unsuccessful vaccination and from contaminated surgical sites or wounds may be consequences of this disease. etiology. escherichia coli is a motile, aerobic, gram-negative, non-spore-forming coccobacillus commonly found in the environment and gastrointestinal tracts of ruminants. escherichia coli organisms have three areas of surface antigenic complexes (o, somatic; k, envelope or pili; and h, flagellar), which are used to "group" or classify the serotypes. colibacillosis is the common term for infections in younger animals caused by this bacteria. clinical signs. presentation of e. coli infections vary with the animal's age and the type of e. coli involved. enterotoxigenic e. coli infection causes gastroenteritis and/or septicemia in lambs and calves. colibacillosis generally develops within the first hr of life when newborn animals are exposed to the organism. the enteric infection causes a semifluid, yellow to gray diarrhea. occasionally blood streaking of the feces may be observed. the animal may demonstrate abdominal pain, evidenced by arching of the back and extension of the tail, classically described as "tucked up." hyperthermia is rare. severe acidosis, depression, and recumbancy ensue, and mortality may be as high as %. the septicemic form generally occurs between and weeks of age. animals display an elevated body temperature and show signs suggestive of nervous system involvement such as incoordination, head pressing, circling, and the appearance of blindness. opisthotonos, depression, and death follow. occasionally, swollen, painful joints may be observed with septicemic colibacillosis. blood cultures may be helpful in identifying the septicemic form. in ruminants, e. coli is is a less common cause of cystitis and pyelonephritis. the cystitis is characterized by dysuria and pollakiuria; gross hematuria and pyuria may be present. the infection may or may not be restricted to the bladder; in the later presentation, and in cases of pyelonephritis, a cow will be acutely depressed, have a fever and ruminal stasis, and be anorexic. in chronic cases, animals will be polyuric and undergo weight loss. escherichia coli may also cause in utero disease in cattle, resulting in abortion or weakened offspring. epizootiology and transmission. escherichia coli is one of the most common gram-negative pathogens isolated from ruminant neonates. zeman et al. ( ) classify e. coli infections into four groups: enterotoxigenic, enterohemorrhagic, enteropathogenic, and enteroinvasive. enterotoxigenic e. coli (etec) attach to the enterocytes via pili, produce enterotoxins, and are the primary cause of colibacillosis in animals and humans. fimbrial (pili) antigens associated with ovine disease include k and f . enterohemorrhagic e. coli (ehec) attach and efface the microviuus, produce verotoxins, and occasionally cause disease in humans and animals. enteropathogenic e. coli (epec) colonize and efface the microvillus but do not produce verotoxins. epec are associated with disease in humans and rabbits and cause a secretory diarrhea. enteroinvasive e. coli (eiec) invade the enterocytes of humans and cause a shigella-like disease. overcrowding and poor sanitation contribute significantly to the development of this disease in young animals. the organism will be endemic in a contaminated environment and present on dams' udders. the bacteria rapidly proliferate in the neonates' small intestines. the bacteria and associated toxins cause a secretory diarrhea, resulting in the loss of water and electrolytes. if the bacteria infiltrate the intestinal barrier and enter the blood, septicemia results. diagnosis of the enteric form can be made by observation of clinical signs, including diarrhea and staining of the tail and wool. necropsy findings. swollen, yellow to gray, fluid-filled small and large intestines, swollen and hemorrhagic mesenteric lymph nodes, and generalized tissue dehydration are common. septicemic lambs may have serofibrinous fluid in the peritoneal, thoracic, and pericardial cavities; enlarged joints containing fibrinopurulent exudates; and congested and inflamed meninges. isolation and serotyping of e. coli confirm the diagnosis. elisa and latex agglutination tests are available diagnostic tools. differential diagnosis. differential diagnoses include the enterotoxemias caused by c. perfringens type a, b, or c; campylobacter jejuni; coccidia, rotavirus, coronavirus, salmonella, and cryptosporidia. other contributing causes of abomasal tympany in young ruminants, such as dietary changes, copper deficiency, excessive intervals between feedings of milk replacer, or feeding large volumes should be considered. prevention and control. the best preventive measures are maintenance of proper housing conditions, limiting overcrowding, and frequently sanitizing lambing areas. attention to colostrum feeding techniques and colostral quality are important means of preventing disease. treatment must include intravenous fluid hydration and reestablishment of acid-base and electrolyte abnormalities. treatment. antibiotics such as trimethoprim-sulfadiazine, enrofloxacin, cephalothin, amikacin, and apramycin may be helpful; oral antibiotics are not recommended. vaccines are available for prevention of colibacillosis in cattle. etiology. corynebacterium pseudotuberculosis (previously c. ovis) are nonmotile, non-spore-forming, aerobic, short and curved, gram-positive coccobacilli. caseous lymphadenitis (cla) is such a common, chronic contagious disease of sheep and goats that any presentation of abscessing and draining lymph nodes should be presumed to be this disease until proven otherwise. the disease has been reported occasionally in cattle. clinical signs and diagnosis. abscessation of superficial lymph nodes, such as the superficial cervical, retropharyngeal, subiliacs (prefemoral), mammary, superficial inguinals, and popliteal nodes, and of deep nodes, such as mediastinal and mesenteric lymph nodes, is typical. radiographs may be helpful in identifying affected central nodes. peripheral lymph nodes may erode and drain caseous, "cheesy," yellow-green-tan secretions. the incubation period may be weeks to months. over time, an infected animal may become exercise-intolerant, anorexic, and debilitated. fever, increased respiratory rates, and pneumonia may also be common signs. exotoxin-induced hemolytic crises may occur occasionally. morbidity up to % is common, and morbid animals will often eventually succumb to the disease. diagnosis is based on clinical lesions; elisa serological testing is also available. smears of the exudate or lymph nodes aspirates can be gram-stained. lymph node aspirates may also be sent for culturing. epizootiology and transmission. the organism can survive for months or more in the environment and enters via skin wounds, shearing, fighting, castration, and docking. ingestion and aerosolization (leading to pulmonary abscesses) have been reported as alternative routes of entry. necropsy findings. disseminated superficial abscesses as well as lesions of the mediastinal and mesenteric lymph nodes will be identified. cut surfaces of the affected lymph nodes may appear lamellated. lungs, liver, spleen, and kidneys may also be affected. cranioventral lung consolidation with hemorrhage, fibrin, and edema are seen histologically. pathogenesis. corynebacterium pseudotuberculosis produces an exotoxin (phospholipase d) that damages endothelial and blood cell membranes. this process enhances the organisms' ability to withstand phagocytosis. the infection spreads through the lymphatics to local lymph nodes. the necrotic lymph nodes seed local capillaries and hematogenously and lymphatically spread the organisms to other areas, especially the lungs. differential diagnosis. differentials include pathogens causing lymphadenopathy and abscessation. treatment. antibiotic therapy is not usually helpful. abscesses can be surgically lanced and flushed with iodinecontaining and/or hydrogen peroxide solutions. abscessing lymph nodes can be removed entirely from valuable animals. during warmer months, an insect repellent should be applied to and around healing lesions. all materials used to treat animals should be disposed of properly. because of the contagious nature of the disease, animals with draining and lanced lesions should be isolated from cla-negative animals at least until healed. commercial vaccines are available (piontkowski and shivvers, ) . minimizing contamination of the environment, using proper sanitation methods for facilities and instruments, segregating affected animals, and taking precautions to prevent injuries are all important. research complications. this pathogen is a risk for animals undergoing routine management procedures or invasive research procedures, because of its persistence in the environment, its long clinical incubation period, and its poor response to antibiotics. etiology. corynebacterium renale, c. cystitidis, and c. pilosum are sometimes referred to as the c. renale group. these are piliated and nonmotile gram-positive rods and are distinguished biochemically. corynebacterium renale causes pyelonephritis in cattle, and c. pilosum and c. cystitidis cause posthitis, also known as pizzle rot or sheath rot, in sheep and goats. in many references, all these clinical presentations are attributed to c. renale. clinical signs and diagnosis. acute pyelonephritis is characterized by fever, anorexia, polyuria, hematuria, pyuria, and arched back posture. untreated infections usually become chronic, with weight loss, anorexia, and loss of production in dairy animals. relapses are common, and some infections are severe and fatal. diagnosis of pyelonephritis is based on urinalysis (proteinuria and hematuria) and rectal or vaginal palpation (assessing ureteral enlargement). urine culturing may not be productive. in chronic cases, e. coli and other gram-negatives may be present. posthitis and vulvovaginitis are characteriazed by ulcers, crusting, swelling and pain. the area may have a distinct malodor. necrosis and scarring may be sequelae of more severe infections. fly-strike may also be a complication. diagnosis is based on clinical signs and on investigation of feeding regimens. epizootiology and transmission. ascending urinary tract infections with cystitis, ureteritis, and pyelonephritis are widespread problems, but incidence is relatively low. the vaginitis and posthitis contribute to the venereal transmission, but indirect transmission is possible because the organisms are stable in the environment and present on the wool or scabs shed from affected animals. posthitis occurs in intact and castrated sheep and goats. necropsy findings. pyelonephritis, multifocal kidney abscessation, dilated and thickened ureters, cystitis, and purulent exudate in many sections of the urinary tract are common finding at gross necropsy. of bovine genitourinary tracts. the pilus mediates colonization. conditions such as trauma, urinary tract obstruction, and anatomic anomalies may predispose to infection. in addition, more basic ph urine levels may block some immune defenses. infections ascend through the urinary tract. the bacteria are urease-positive when tested in vitro, and the ammonia produced in vivo during an infection damages mucosal linings, with subsequent inflammation. corynebacterium cystitidis and c. pilosum are normally found around the prepuce of sheep and goats. high-protein diets, resulting in higher urea excretion and more basic urine, are contributing factors. posthitis and vulvovaginitis may develop within a week of change to the more concentrated or richer diet, such as pasture or the addition of high-protein forage. the ammonia produced irritates the preputial and vulvar skin, increasing the vulnerability to infection. differential diagnosis. urolithiasis is a primary consideration for these diseases. contagious ecthyma should be considered for the crusting that is seen with posthitis and vulvovaginitis, although the lesions of contagious ecthyma are more likely to develop around the mouth. ovine viral ulcerative dermatosis is also a differential for the lesions of posthitis and vulvovaginitis. prevention and treatment. because high-protein feed is often associated with posthitis and vulvovaginitis, feeding prac-tices must be reconsidered. clipping long wool and hair also is helpful. treatment. long-term ( weeks) penicillin treatment is effective for pyelonephritis. reduction of dietary protein, clipping and cleaning skin lesions, treating for or preventing fly-strike, and topical antibacterial treatments are effective for posthitis and vulvovaginitis; systemic therapy may be necessary for severe cases. surgical debridement or correction of scarring may also be indicated in severe cases. etiology. erysipelothrix rhusiopathiae is a nonmotile, nonspore-forming, gram-positive rod that resides in alkaline soils. clinical signs. erysipelothrix causes sporadic but chronic polyarthritis in lambs less than months of age. in older goats, erysipelas has been associated with joint infections. epizootiology and transmission. the disease may follow wound inoculation associated with castration, docking, or improper disinfection of the umbilicus. following wound contamination and a -to -day incubation period, the lamb exhibits a fever and stiffness and lameness in one or more limbs. joints, especially the stifle, hock, elbow, and carpus, are tender but not greatly enlarged. necropsy findings. thickened articular capsules, mild increases in normal-appearing joint fluid and erosions of the articular cartilage are usually found. the joint capsule is infiltrated with mononuclear cells, but bacteria are difficult to find. diagnosis is based on clinical signs of polyarthritis, and confirmation is made by culturing the organism from the joints. differential diagnosis. differential diagnoses include polyarthritis caused by chlamydia or other bacteria and stiffness caused by white muscle disease. other bacteria causing septic joints include areanobacterium pyogenes and fusobacterium necrophorum. caprine arthritis encephalitis (cae) should also be considered. prevention and control. proper sanitation and prevention of wound contamination are important in preventing the infection in lambs. screening of goat herds for cae is recommended. therapy. erysipelas is sensitive to penicillin antibiotic m. etiology. dermatophilus congolensis is an aerobic, grampositive, filamentous bacterium with branching hyphae. dermatophilosis is a chronic bacterial skin disease characterized by crustiness and exudates accumulating at the base of the hair or wool fibers (scanlan et al., ) . clinical signs. animals will be painful but will not be pruritic. two forms of the disease exist in sheep: mycotic dermatitis (also known as lumpy wool) and strawberry foot rot. mycotic dermatitis is characterized by crusts and wool matting, with exudates over the back and sides of adult animals and about the face of lambs. strawberry foot rot is rare in the united states but is characterized by crusts and inflammation between the carpi and/or tarsi and the coronary bands. animals will be lame. in goats and cattle, similar clinical signs of crusty, suppurative dermatitis are seen; the disease is often referred to as cutaneous streptothricosis in these species. lesions in younger goats are seen along the tips of the ears and under the tail. diagnosis is based on clinical signs as well as the typical microscopic appearance on stained skin scrapings, cultures, and serology. epizootiology and transmission. the disease occurs worldwide, and the dermatophilus organism is believed to be a saprophyte. transmission occurs by direct or indirect contact and is aggravated by prolonged wet wool or hair associated with inclement weather. biting insects may aid in transmission. necropsy findings. lymphadenopathy as well as liver and splenic changes may be observed. histopathologically, superficial epidermal layers are necrotic and crusted with serum, white blood cells, and wool or hair. dermal layers are hyperemic and edematous and may be infiltrated with mononuclear cells. pathogenesis. lesions typically begin around the muzzle and hooves and the dorsal midline. prevention and control. potash alum and aluminum sulfate have been used as wool dusts in sheep to prevent dermatophilosis. minimizing moist conditions is helpful in controlling and preventing the disease. in addition, controlling external parasites or other factors that cause skin lesions is important. lesions will resolve during dry periods. treatment. animals can be treated with antibiotics such as penicillin and oxytetracycline. treating the animals with povidone-iodine shampoos or chlorhexidine solutions is also useful in clearing the disease. n. etiology. two bacteria, dichelobacter (bacteroides) nodosus and fusobacterium necrophorum, work synergistically in caus-ing contagious foot rot in sheep and goats. other organisms may be involved as secondary invaders. both dichelobacter and fusobacterium are nonmotile, non-spore-forming, anaerobic, gram-negative bacilli. foot rot is a contagious, acute or chronic dermatitis involving the hoof and underlying tissues (bulgin, ) . it is the leading cause of lameness in sheep. at least serotypes of dichelobacter are known. arcanobacterium pyogenes may also contribute to the pathogenicity or to foot abscesses in goats. foot scald, an interdigital dermatitis, is caused primarily by d. nodosus alone. clinical signs. varying degrees of lameness are observed in all ages of animals within - weeks of exposure to the organisms. severely infected animals will show generalized signs of weight loss, decreased productivity, and anorexia associated with an inability to move. the interdigital skin and hooves will be moist, with a distinct necrotic odor. morbidity may reach % in susceptible animals. diagnosis is based on clinical signs. smears and cultures confirm the definitive agents. clinical signs of the milder disease, foot scald, include mild lameness, redness and swelling, and little to no odor. epizootiology and transmission. fusobacterium necrophorum is ubiquitous in soil and manure, in the gastrointestinal tract, and on the skin and hooves of domestic animals. in contrast, dichelobacter contaminates the soil and manure but rarely remains in the environment for more than about weeks. some animals may be chronic carriers. overcrowded, warm, and moist environments are key elements in transmission. outbreaks are likely in the spring season. shipping trailers and contaminated pens or yards should be considered also as likely sources of the bacteria. pathogenesis. both organisms are transmitted to the susceptible animal by direct or indirect contact. the organisms enter the hoof through injuries or through sites where strongyloides papillosus larvae have penetrated. fusobacterium necrophorum initiates the colonization and is followed by d. nodosus. the latter attaches and releases proteases; these cause necrosis of the epidermal layers and separation of the hoof from the underlying dermis. the pathogenicity of the serotypes of d. nodosus is correlated with the production of these proteases and numbers of pili. additionally, f. necrophorum causes a severe, damaging inflammatory reaction. differential diagnosis. foot abscesses, tetanus, selenium/ vitamin e deficiencies, copper deficiency, strawberry foot rot, bluetongue virus infection (manifested with myopathy and coronitis), and trauma are among the many differentials that must be considered. treatment. affected animals are best treated by manually trimming the necrotic debris from the hooves, followed by application of local antibiotics and foot wraps. systemic antibiotics such as penicillin, oxytetracycline, and erythromycin may be used. goats have improved dramatically when given a single dose of penicillin ( , u/kg) (smith and sherman, ) . footbaths containing % zinc sulfate, % copper sulfate, or % formalin (not legal in all states) can be used for treatment as well as for prevention of the disease. affected animals should be separated from the flock. vaccination has been shown to be effective as part of the treatment regimen. some breeds of sheep and some breeds and lines of goats are resistant to infection. individual sheep may recover without treatment or are resistant to infection. epizootiology and transmission. cases may be sporadic, or epizootics may occur. bos taurus dairy breeds and animals with wide interdigital spaces are more commonly affected. the factors here are comparable to those present in foot rot of smaller ruminants. necropsy findings. findings at necropsy include dermatitis and necrosis of the skin and subcutaneous tissues. although necropsy would rarely be performed, secondary osteomyelitis may be noted in severe cases by sectioning limbs. prevention and control. prevention and control programs involve scrutiny of herd and flock management; quarantine of incoming animals; vaccination; segregation of affected animals; careful and regular hoof trimming; discarding trimmings from known or suspected infected hooves; maintaining animals in good body condition; avoiding muddy pens and holding areas; and culling individuals with chronic and nonresponsive infections. dichelobacter nodosus bacterins are commercially available; cross protection between serotypes varies. biannual vaccinination in wet areas may be essential. some breeds may develop vaccination site lumps. footbaths of % zinc sulfate, % formalin (where allowed by state regulations), or % copper sulfate are also considered very effective preventive measures. goats are less sensitive than sheep to the copper in the footbaths. treating and controlling foot rot is costly in terms of time, initial handling and treatments and their follow-up, housing space, and medications. etiology. interdigital necrobacillosis of cattle is caused by the synergistic infection of traumatized interdigital tissues by fusobacterium necrophorum and bacteroides melaninogenicus. like f. necrophorum, b. melaninogenicus is a nonmotile, anaerobic, gram-negative bacterium. dichelobacter nodosus, the agent of interdigital dermatitis, may be present in some cases. this is a common cause of lameness in cattle. clinical signs. clinical signs include mild to moderate lameness of sudden onset. hindlimbs are more commonly affected, and cattle will often flex the pastern and bear weight only on the toe. the interdigital space will be swollen, as will be the coronet and bulb areas. characteristic malodors will be noted, but there will be little purulent discharge. in more severe cases, animals will have elevated body temperature and loss of appetite. the les~ons progress to fissures with necrosis until healing occurs. the diagnosis is by the odor and appearance. anaerobic culturing confirms the organisms involved. pathogenesis. the bacteria enter through the skin of the interdigital area after trauma to the interdigital skin, from hardened mud, or from softening of the skin due to, for example, constant wet conditions in pens. colonization leads to cellulitis. in addition, f. necrophorum releases a leukocidal exotoxin that reduces phagocytosis and causes the necrosis, whereas the tissues and tendons are damaged by the proteases and collagenases produced by b. melaninogenicus. zinc deficiency may play a role in the pathogenesis in some situations. differential diagnoses. the most common differentials for sudden lameness include hairy heel warts and subsolar abcesses. bluetongue virus should also be considered. grain engorgement and secondary infection from cracks caused by selenium toxicosis should also be considered. the exotic footand-mouth disease virus would be considered in areas where that pathogen is found. prevention and control. as with foot rot in smaller ruminants, management of the area and herd are important. paddocks and pens should be kept dry, well drained, and free of material that will damage feet. footbaths and chlortetracycline in the feed have been shown to control incidence. affected animals should be segregated during treatment. chronically affected or severely lame animals should be culled. new cattle should be quarantined and evaluated. ing within a week include cleaning the feet and trimming necrotic tissue; parenteral antimicrobials, such as oxytetracycline or procaine penicillin, or sulfonomethazine in the drinking water or tetracyclines in feed; and footbaths (such as % zinc sulfate, . % formalin, or % copper sulfate) twice a day. in severe cases, more aggressive therapy such as bandaging the feet or wiring the digits together may be needed. animals can recover without treatment but will be lame for several weeks. acquired immunity is reported to be poor. research complications are comparable to those noted for foot rot in smaller ruminants. fusobacterium necrophorum is also associated with foot abscesses, the infection of the deeper structures of the foot, in sheep and goats. only one claw of the affected hoof may be involved. the animals will be three-legged lame, and the affected hoof will be hot. pockets of purulent material may be in the heel or toe. etiology. bacteria such as fusobacterium spp., bacteroides spp., and dichelobacter nodosus have been isolated from bovine heel lesions. spirochete-like organisms have also been shown in the lesions of cows with papillomatous digital dermatitis (pdd), in the united states and europe; these have culturing requirements similar to those of treponema species. treatment. antibiotic and antiseptic regimens have been used successfully for this problem. antibiotics include parenteral cephalosporins and pencillins, as well as topical tetracyclines with bandaging. antiseptic or antibiotic solutions in footbaths include tetracyclines, zinc sulfate, lincomycin, spectinomycin, copper sulfate, and formalin. the footbaths must be well maintained, minimizing contamination by feces and other materials. tandem arrangements, such as the cleaning footbaths and then the medicated footbaths, and preventing dilution from precipitation are useful. other treatments such as surgical debridement, cryotherapy, and caustic topical solutions have been successful. research complications. infectious, contagious ppd is one of the major causes of lameness among heifers and dairy cattle and is a costly problem to treat. the outbreaks are generally worse in younger animals in chronically infected herds. the immune response is not well understood, and it may be temporary in older animals. clinical signs. all lesions occur on the haired, digital skin. one or all feet may be affected. most lesions occur on the plantar surface of the hindfoot (near the heel bulbs and/or extending from the interdigital space), but the palmar and dorsal aspect of the interdigital spaces may also be involved. progression of lesions, typically over - weeks, includes erect hairs, loss of hair, and thickening skin. moist plaques begin as red and remain red or turn gray or black. exudate or blood may be present on the plaque. plaques enlarge and "hairs" protrude from the roughened surface. lesioned areas are painful when touched. the lesions may or may not be malodorous. epizootiology and transmission. facility conditions and herd management are considered contributing factors. the following have been examined as contributing factors: nutrition, particularly zinc deficiency; poorly drained, low-oxygen, organic material underfoot; poor ventilation; rough flooring; damp and dirty bedding areas; and overcrowding. these interdigital lesions occur commonly in young stock and in dairy facilities throughout the world. the disease is seen only in cattle. pathogenesis. the organisms noted above, combined with poor facility and herd management, are critical in the pathogenesis. differential diagnosis. differentials for lameness will include sole abscesses, laminitis, and trauma. prevention and control. each facility and management condition noted above should be addressed in conjunction with appropriate antibiotic and/or antiseptic treatment regimens. all equipment used for hoof trimming must be cleaned and disinfected after every use. trucks and trailers should also be sanitized between groups of animals. etiology. haemophilus somnus is a pleomorphic, nonencapsulated, gram-negative bacterium. diseases caused by this organism include thromboembolic meningoencephalitis (teme), septicemia, arthritis, and reproductive failures due to genital tract infections in males and females. haemophilus somnus is a also major contributor to the bovine respiratory disease complex. haemophilus spp. have been associated with respiratory disease in sheep and goats. clinical signs. the neurologic presentation may be preceded by - weeks of dry, harsh coughing. neurologic signs include depression, ataxia, falling, conscious proprioceptive deficits; signs such as head tilt from otitis interna or otitis media, opisthotonus, and convulsions may be seen as the brain stem is affected. high fever, extreme morbidity, and death within hr may occur. respiratory tract infections are usually part of the complex with infectious bovine rhinotracheitis virus, bovine respiratory syncytial virus, bovine viral diarrhea virus, parainfluenza , mycoplasma, and pasteurella, and the synergism among these contributes to the signs of bovine respiratory disease complex (brdc). in acute neurologic as well as chronic pneumonic infections, polyarthritis may develop. abortion, vulvitis, vaginitis, endometritis, placentitis, and failure to conceive are manifestations of reproductive tract disease. in all cases, asymptomatic infections may also occur. diagnosis based on culture findings is difficult because h. somnus is part of the normal nasopharyngeal flora. paired serum samples are recommended; single titers in some animals seem to be high because of passive immunity, previous vaccination, or previous exposure. in cases of abortion, other causes should be eliminated from consideration. because the organism is considered part of the normal flora of cattle and can be isolated from numerous tissues, the distinction between the normal flora and the status of chronic carrier is not clear. outbreaks are associated with younger cattle in feedlots in western united states, but stresses of travel and coinfection with other respiratory pathogens are involved in some cases. adult cattle have also been affected. vaccination for viral respiratory pathogens may increase susceptibility. transmission is by respiratory and genital tract secretions. the organism does not persist in the environment. times of stress to the cattle is worthwhile. killed whole-cell bacterins are commercially available; these have been shown to be effective in controlling the respiratory disease presentation. control of other clinical aspects of the h. somnus disease by these bacterins has not been well described. treatment. rapid treatment at the first signs of neurologic disease is important in an outbreak. haemophilus somnus is susceptible to several antibiotics, such as oxytetracycline and penicillin, and these are often used in sequence until the cattle are recovered. necropsy findings. pathognomonic central nervous system lesions include multifocal red-brown foci of necrosis and inflammation on and within the brain and the meninges. many thrombi with bacterial colonies will be seen in these affected areas. ocular lesions may also be seen, including conjunctivitis, retinal hemorrhages, and edema. usually animals with neurological disease will not have respiratory tract lesions. the respiratory tract lesions include bronchopneumonia and suppurative pleuritis. when combined with pasteurella infection, the pathology becomes more severe. aborted fetuses will not show lesions, but necrotizing placentitis will be evident histologically. pathogenesis. inhalation of contaminated respiratory secretions from carrier animals is the primary means of transmission. the anatomical location of bacterial residence within the carriers has not been identified. after gaining access by way of the respiratory tract, the bacteria proliferate, and a bacteremia develops. the bacteria are phagocytosed by neutrophils but are not killed. the thrombosis formation is due to the adherence by the nonphagocytosed organisms to vascular endothelial cells, degeneration and desquamation of these cells, and exposure of subendothelial collagen, with subsequent initiation of the intrinsic coagulation pathway. antigen-antibody complex formation, resulting in vasculitis, is also correlated with high levels of agglutinating antibodies. other pathogens associated with neurological disease and respiratory disease such as pasteurella hemolytica, p. multocida, and p. aeruginosa. in smaller ruminants, corynebacterium pseudotuberculosis should be considered. prevention and control. stressed animals or those exposed to known carriers can be treated prophylactically with tetracycline administered parenterally or orally (in the feed or water). the late-stage polyarthritis is resistant to antibiotic therapy, because of failure of the antibiotic to reach the site of infection. planning vaccination programs carefully will decrease chances of outbreaks. for example, avoiding vaccinating animals for infectious bovine rhinotrachetitis and bovine viral diarrhea during clinical signs. leptospirosis is a contagious but uncommon disease in sheep and goats. the disease may cause abortion, anemia, hemoglobinuria, and icterus and is often associated with a concurrent fever. after a -to -day incubation period, the organism enters the bloodstream and causes bacteremia, fever, and red-cell hemolysis. leptospiremia may last up to days. immune stimulation is apparently rapid, and antibodies are detectable at the end of the first week of infection; crossserovar protection does not occur. during active bacteremia, hemolysis may result in hemoglobin levels of % below normal. hyperthermia, hemoglobinuria, icterus, and anemia may be observed during this phase, and ewes in late gestation may abort. abortion usually occurs only once. mortality rates of above % have been reported in infected ewes and lambs (jensen and swift, ) . subclinical infection is more common in nonpregnant and nonlactating animals. sheep infected with leptospirosis may display a hemolytic crisis associated with igm acting as a cold-reacting hemagglutinin. acute and chronic infections in cattle are more common than infections in sheep and goats. acute forms in cattle display signs similar to those in sheep. acute infection in calves may progress to meningitis and death. lactating cows will have severe drops in production. chronic cases may lead to abortion, with retained placenta, and weakened calves or animals that carry the infection. infertility may also be a sequela. epizootiology and transmission. leptospires are a large genus, and leptospirosis is a complicated disease to prevent, treat, and control. the organism survives well in the environment, especially in moist, warm, stagnant water. cattle, swine, and other domestic and wild animals are potential carriers of serovars common to particular regions. wild animals often serve as maintenance hosts, but domestic livestock may be reservoirs also. organisms are shed in urine, in uterine discharges, and through milk. animals become carriers when they are infected with a host-adapted serovar; sporadic clinical disease is more commonly associated with exposure to a non-hostadapted serovar (heath and johnson, ) . infection may occur via oral ingestion of contaminated feed and water, via placental fluids, or through the mucous membranes of the susceptible animal. placental or venereal transmission may occur. as the organisms are cleared from the bloodstream, they chronically infect the renal convoluted tubules and the reproductive tract (and occasionally the cerebrospinal fluid or vitreous humor). chronically infected animals may shed the organism in the urine for days or longer. necropsy. diagnosis is confirmed by identification of leptospires in fetal tissues. the leptospires are visible in silver-or fluorescent antibody-stained sections of liver or kidney. leptospires may also be seen under dark-field or phase-contrast microscopy of fetal stomach contents. fetal and maternal serology, and diagnostic tests such as the microscopic agglutination test, are useful; interpretation is complicated because of cross reaction of antibodies to many serovars. differential diagnosis. more than one serovar may cause infection in one animal, and each serovar should be considered as a separate pathogen. because of the associated anemia, differential diagnoses should include copper toxicity and parasites, in addition to other abortifacient diseases. prevention and control. polyvalent vaccines, tailored to common serovars regionally, are available and effective for preventing leptospirosis in cattle. immunity is serovar specific. because serological titers tend to diminish rapidly ( - days in sheep [jensen and swift, ] ), frequent vaccination may be necessary. other prevention measures such as species-specific housing, control of wild rodents, and proper sanitation should be instituted. treatment. antibiotic treatment is aimed at treating ill animals and trying to clear the carrier state. treatment methods for acute leptospirosis include oxytetracycline for - days. addition of oxytetracycline or chlortetracycline to the feed for week may be helpful. these antibiotics are considered best for removal of the carrier state of some serovars. vaccination and antibiotic therapy can be combined in an outbreak. research complications. leptospirosis is zoonotic and may be associated with flulike symptoms, meningitis, or hepatorenal failure in humans. etiology. listeria monocytogenes is a pleomorphic, motile, non-spore-forming, [ -hemolytic, gram-positive bacillus that inhabits the soil for long periods of time and has been often found in fermented feedstuffs such as spoiled silage. of the known serovars, several produce clinical signs in ruminants. listeria ivanovii (associated with abortions in sheep) is serovar . clinical signs. listeriosis is an acute, sporadic, noncontagious disease associated with neurological signs or abortions in sheep and other ruminants. the overall case rate is low. the disease may present as an isolated case or with multiple animals affected. three forms of disease are described: encephalitis, placentitis with abortion, and septicemia with hepatitis and pneumonia. the encephalitic form is most common in sheep; septicemic forms may occur in neonatal lambs (scarratt, ) . clinically, the encephalitic form begins with depression, anorexia, and mild hyperthermia after an incubation period of - weeks. as the disease progresses, animals exhibit nasal discharges and conjunctivitis and begin to walk in circles, as if disoriented. facial paralytic lesions, including drooping of an ear or eyelid, dilation of a nostril, or strabismus occur unilaterally on the affected side as the result of dysfunction of some or all the cranial nerves v-xii. the neck will by flexed away from the affected side. facial muscle twitching, protrusion of the tongue, dysphagia, hypersalivation, and nasal discharges may be noted. the hypersalivation may lead to metabolic acidosis in advanced cases in cattle. anorexia, prostration, coma, and death follow. the placental form usually results in last-trimester abortions in ewes and does, which typically survive this form of the disease. the affected females may be asymptomatic or may show severe clinical signs such as fever and depression, with subsequent retained placenta or endometritis. abortion usually occurs within weeks of listeria infection. in cattle, abortion occurs during the last months of gestation and has been induced experimentally - days after exposure. cows present with the range of clinical signs seen in smaller-ruminant dams. there is no long-term effect on the fertility of affected dams. epizootiology and transmission. the organism is transmitted by oral ingestion of contaminated feeds and water or possibly by inhalation. by the oral route, the organism enters through breaks in the oral cavity and ascends to the brain stem by way of nerves. when severe outbreaks occur, feedstuffs should be assessed for spoilage. listeria organisms can be shed by asymptomatic carriers, especially at the end of pregnancy and at lambing. diagnosis and necropsy findings. diagnosis is usually made from clinical signs. culture confirms the diagnosis (cold enrichment at ~ is preferable but not essential for isolation). impression smears will show the pleomorphic gram-positive characterisitics of the pathogen. tissue fluorescent antibody techniques may also be utilized. gross lesions are not observed with the encephalitic form. microscopic lesions include thrombosis, neutrophilic or mononuclear foci in areas of inflammation, and neuritis. the pons, medulla, and anterior spinal cord are primarily affected in the encephalitic form. microabscesses of the midbrain are characteristic of listeria encephalitis in sheep. aborted fetuses that are intact may show fibrinous polyserositis, with excessive serous fluids; small, necrotic foci of the liver; and small abomasal erosions. necrotic lesions of the fetal spleen and lungs may also be seen. in goats, listeria-induced neurological lesions occur only in the brain stem. placentitis, focal bronchopneumonia, hepatitis, splenitis, and nephritis may be seen with other forms. pathogenesis. with the encephalitic form, the organism penetrates mucosal abrasions and enters the trigeminal or hypoglossal nerves. the listeria organisms then migrate along the nerves and associated lymphatics to the brain stem (medulla and pons). in the septicemic form, the organism penetrates tissues of the gastrointestinal tract and enters the bloodstream, to be distributed to the liver, spleen, lungs, kidneys, and placenta. after infection, organisms are shed in all body secretions (infected milk is an important risk factor for zoonosis). a toxin produced by listeria monocytogenes is correlated with pathogenicity, but the mechanism of the pathogenesis of this molecule has not been elucidated. differential diagnoses. rabies, bacterial meningitis, brain abscess, lead toxicity, and otitis media must be considered as differentials. in sheep, the differentials include organisms that cause abortion, and neurological signs, such as enterotoxemia due to clostridium perfringens type d. in goats, the major differentials include caprine arthritis encephalitis viral infection and chlamydial and mycoplasmal infections. in both species, scrapie is a differential. in cattle, aberrant parasite migration or hemophilus somnus infection must also be considered. prevention and control. affected dams should be segregated and treated. other animals in the group may be treated with oxytetracycline as needed. aborted tissues should be removed immediately. proper storage of fermented feeds minimizes this source of contamination. when silage spoils, the ph increases, producing a suitable growth environment for the organism. commercial vaccines are not available in the united states. treatment. affected animals can be treated aggressively with penicillin, ampicillin, oxytetracycline, or erythromycin. exceptionally high levels of penicillin are required for treating affected cattle. severely affected animals should receive appropriate fluid support and other nursing care. treatment is less successful, and mortality is especially high in sheep. recovered animals tend to resist reinfection. research complications. in addition to the loss of fetal animals, stress to the dams, and risks to other animals, any aborted tissue by a ruminant should be regarded as a potential zoonotic risk. listeria can cause mild to severe flulike symptoms in humans and may be a particular risk for pregnant women and for older or immune-compromised individuals. listeriosis in humans is a reportable disease. etiology. lyme disease is caused by the spirochete borrelia burgdorferi. clinical signs and diagnosis. reports in ruminants indicate seroconversion to b. burgdorferi, but there are few definitive correlations to the arthritis that is present. diagnosis requires culturing from the affected joints and diagnostic elimination of other causes of lameness and arthritis. epizootiology and transmission. the organism is present throughout much of the northern hemisphere and has been reported in many mammals and also in birds. ticks of the ixodes ricinus complex are the major vectors of the spirochete and must be attached for hr for successful transmission. pathogenesis. the ixodes ticks have three life stages: larval, nymphal, and adult. feeding occurs once during each stage, and wild animals are the source of blood meals. the larval stages feed from rodents, such as the white-footed deer mouse, peromyscus leucopus, from which they acquire the spirochete. the nymphal stage is that which usually infects other animals. the adult ticks are usually found on deer. differential diagnosis. seroconversion to b. burgdorferi does not necessarily confirm the cause of arthritis. other causes of arthritis and lameness in ruminants include trauma, caprine arthritis encephalitis virus, mycoplasma spp., chlamydia psittaci, erysipelothrix spp., arcanobacterium pyogenes, brucella spp., and rickets. prevention and control. control of the tick vector is the most important factor in preventing the possibility of exposure or disease. treatment. antibiotic therapy, with tetracycline, penicillin, amoxicillin, and cephalosporins, is used for diagnosed or suspected lyme arthritis. research complications. lyme disease is zoonotic, and the lxodes ticks transmit the disease to humans. v. mastitis i. ovine mastitis mastitis in ewes may be acute, subclinical, or chronic. acute mastitis often results in anorexia, fever, abnormal milk, and swelling of the mammary gland. pasteurella haemolytica is the most common cause of acute mastitis. additional isolates may include, in order of prevalence, staphylococcus aureus, actinomyces (corynebacterium) spp., and histophilus ovis. escherichia coli and pseudomonas aeruginosa have also been found to cause acute mastitis. as many as six serotypes of pasteurella haemolytica have been isolated from the mammary glands of mastitic ewes. furthermore, intramammary inoculation of these organisms isolated from ovine and bovine pulmonary lesions has resulted in clinical mastitis in ewes (watkins.and jones, ) . subclinical mastitis is detected only indirectly, by counting somatic cells. the most common isolate from ewes with subclinical mastitis is coagulase-negative staphylococci. other isolates include actinomyces bovis, streptococcus uberis, s. dysgalactiae, micrococcus spp., bacillus spp., and fecal streptococci. most of these organisms are commonly found in the environment. diffuse chronic mastitis, or hardbag, results from interstitial accumulations of lymphocytes in the udder. both glands are usually affected, but no inflammation is present. serological evidence suggests that diffuse chronic mastitis is caused by the retrovirus that causes ovine progressive pneumonia (opp or maedi/visna virus). other bacterial agents or mycoplasma have not usually been isolated from udders with this type of mastitis. acute mastitis occurs in approximately % of lactating ewes annually, and it usually occurs either soon after lambing or when lambs are - months old (lasgard and vaabenoe, ) . subclinical mastitis occurs in - % of lactating ewes (kirk and glenn, ) . subclinical mastitis is more common in ewes from high-milk-producing breeds. skin or teat lesions and dermatitis increase the prevalence of disease. acute mastitis can be diagnosed in ewes with associated systemic signs of disease by physical examination of the udder and inspection of the milk. subclinical mastitis is often suggested by somatic cell counts elevated above x cells/ml. when high somatic cell counts are identified, subclinical mastitis can be diagnosed by milk culture. the california mastitis test may also be helpful as an indicator of mastitis. manual palpation of a hard, indurated udder as well as serological testing for the maedi/visna virus is helpful in confirming the diagnosis of diffuse chronic mastitis. treatment for acute bacterial mastitis should include aggressive application of broad-spectrum antibiotics (intramammary and systemic) and supportive therapy such as fluids and anti-inflammatory drugs. it is may be helpful to milk out the infected ud-der frequently; oxytocin injections preceding milking will improve gland evacuation. because somatic cell counting is often not routinely performed, treatment of subclinical mastitis is seldom done. there is currently no treatment available for diffuse chronic mastitis. ii. caprine mastitis lactating goats are subject to inflammation of mammary gland, or mastitis. the primary causative organisms are staphylococcus epidermidis and other coagulasenegative staphylococcus spp. clinical signs of mastitis include abnormal coloration or composition of milk, mammary gland redness, heat and pain, enlargement of the mammary gland, discoloration of the mammary gland, and systemic signs of septicemia. large abscesses may be present in the affected gland. staphylococcus aureus is also associated with caprine mastitis, and toxemia may be part of the clinical picture. this organism produces a necrotizing alpha toxin that can result in gangrenous mastitis. caprine mastitis may be clinical or subclinical, and the first indication of mastitis may be weak, depressed, or thin kids. diagnosis is based on careful culture of mastitic milk. treatment includes frequent stripping, intramammary antibiotics, and nonsteroidal anti-inflammatory drugs. oxytocin ( - u) may help milk letdown for frequent strippings. bovine mastitis products can be used in the goat; however, care should be taken not to insert the mastitis tube tip fully, because damage to the protective keratin layer lining the teat canal may occur. in severe acute systemic cases, steroids, fluids, and systemic antibiotics may be necessary. other less common causes of mastitis in goats include streptococcus spp. (s. agalactiae, s. dysgalactiae, s. uberis, and zooepidemicus). gram-negative causes of caprine mastitis include escherichia coli, klebsiella pneumoniae, pasteurella spp., pseudomonas, and proteus mirabilis. corynebacterium pseudotuberculosis can cause mammary gland abscessation, whereas mycoplasma mycoides may cause agalactia and systemic disease. "hard udder" can be caused by caprine arthritis encephalitis virus (caev). brucellosis and listeriosis can cause a subclinical interstitial mastitis (smith and sherman, ) . iii. bovine mastitis mastitis is the disease of greatest economic importance for the dairy cattle industry. the majority of the impact will be on the production and overall health of the cows, but low-incidence herds also diminish the risk of calves' ingesting or being exposed to pathogens. the most common bovine mastitis pathogens include staphylococcus aureus and streptococcus agalactiae, s. dysgalactiae, and s. uberis; coliform agents such as escherichia coli, enterobacter aerogenes, serratia marcescens, and klebsiella pneumoniae; mycoplasmal species such as mycoplasma bovis, m. bovigenitalium, m. californicum, m. canadensis, and m. alkalescens; and salmonella spp. such as s. typhimurium, s. newport, s. enteritidis, s. dublin, and s. muenster. many of these agents such as staphylococcus spp., salmonella spp., and the coliforms can cause both acute and chronic mastitis, as well as severe systemic disease, including fever and anorexia. these must be regarded as herd and environmental pathogens in terms of treatment and prevention. the pathogenesis of staphylococcal infections is comparable to that in goats. staphylococcus agalactiae can be cleared from udders because it does not invade other tissues, is an obligate resident of the glands, and is susceptible to penicillin. in contrast, s. uberis and s. dysgalactiae are environmental organisms and can be highly resistant to pencillin. mycoplasma bovis is the more common of the mycoplasmal pathogens and can cause severe infections. transmission of the mycoplasmas is not well defined but may be related to their presence in other organ systems. treatments for mycoplasmal mastitis are not successful; culling is recommended. there are many interrelated factors associated with prevention and control of mastitis in a herd, including herd health and dry cow management, order of animals milked, milking procedures, milking equipment, condition of the teats, and the condition of the environment. management of the overall herd includes aspects such as vaccination programs, nutrition, isolation of incoming animals, and quarantine and treatment of or culling diseased individuals. culturing or testing newly freshened cows and monitoring the bulk milk tank serve as indicators of subclinical mastitis. herd management will diminish teat lesions. bacterin vaccines are available for preventing and controlling coliform mastitis and s. aureus mastitis. at the time of dry-off, all cows must be treated by intramammary route. some infections can be successfully cleared during this time. younger, disease-free animals should be milked first; any animals with diagnosed problems should be milked after the rest of the herd and/or segregated during treatment. milkers' hands easily serve as a means of pathogen transmission, and wearing rubber gloves is recommended. teat and udder cleaning practices include washing and drying with single-service paper or cloth towels or pre-and postmilking dipping. milking equipment must be maintained to provide proper vacuum levels and pumping rates, and liners should be the appropriate size. facilities that provide clean and dry areas for the animals to rest, feed, and move will diminish teat injuries and reduce exposures to mastitis pathogens. in that regard, inorganic bedding such as clean sand harbors few pathogens in contrast to shavings and sawdust. w. etiology. moraxella bovis, a gram-negative coccobacillus, is the most common cause of infectious bovine keratoconjunctivitis (ibk) in cattle. this organism is not a cause of keratoconjunctivitis in sheep and goats. the disease includes conjunctivitis and ulcerative keratitis. the pathogenic m. bovis strain is piliated, and at least seven serotypes exist. clinical signs. lacrimation, photophobia, and blepharospasm are seen initially. conjunctival injection and chemosis develop within a day of exposure, and then keratitis with corneal edema and ulcers. anterior uveitis may be a sequela within a few days, and thicker mucopurulent ocular discharge may be seen. corneal vascularization begins by days after onset. reepithelialization of the corneal ulcers occurs by - weeks after onset. diagnosis is usually based on clinical signs, but culturing is helpful and fluoroscein staining is useful for demonstrating corneal ulceration. epizootiology and transmission. the disease is more severe in younger cattle. the clinical signs of ibk tend to be more severe in cattle that are also infected with infectious bovine rhinotracheitis (ibr) virus or those that have been vaccinated recently with modified live ibr vaccine. the bacteria are shed in nasal secretions and cattle with no clinical symptoms may be carriers. transmission is by fomites, flies, aerosols, and direct contact. incidence in winter months is very low. nonhemolytic strains are associated with the winter epidemics, and hemolytic strains are associated with summer epidemics. necropsy findings. necropsy is not typically performed on these cases. corneal edema, ulceration, hypopyon, and uveitis would be noted, depending on the stage of infection. pathogenesis. the pili ofm. bovis bind to receptors of corneal epithelium. the virulent strains of the bacteria then release the enzymes that damage the corneal epithelial cells. other factors contributing to infection include ultraviolet light and trauma from dust and plant materials. differential diagnoses. infectious bovine rhinotrachetitis virus causes conjunctivitis, but the central corneal ulceration that is characteristic of ibk is not seen with m. bovis infections. mycoplasma, listeria, branhamella (neisseria) , and adenovirus may be cultured from affected bovine eyes but none has been shown to produce the corneal lesions when inoculated into susceptible animals. prevention and control. cattle should not be immunized intranasally with modified live infectious bovine rhinotracheitis vaccine during ibk outbreaks; this will likely exacerbate the infection. new animals should be quarantined and treated prophylactically before introduction to herds. the available vaccines, containing. m. bovis pili or killed m. bovis, help decrease incidence and severity of disease; these preparations are not completely effective, because the m. bovis strain may not be homologous to that used for the vaccine preparation. other preventive measures include % permethrin-impregnated bilateral ear tags, pour-on avermectins, or dust bags or face rubbers containing insecticide (such as % coumaphos) to control flies throughout the season and premises; mowing of high pasture grass to minimize ocular trauma; provision of shade; control of dust and sources of other mechanical trauma; and segregation of animals by age. treatment. cattle can recover without treatment, but younger animals should be treated as soon as the infection is detected. antibiotic treatments include topical, subconjunctival administration and intramuscular dosing. several standard topical antibiotics have been shown to be effective, including oxytetracycline, gentamicin, and triple antibiotic combinations. these should be administered twice per day. subconjunctival injections of antibiotics, such as penicillin g, provide higher corneal levels of drug; these are typically administered only once or twice in severe cases. intramuscular doses of long-acting oxytetracycline, given on alternate days, are effective in larger herds, and doses hr apart eliminate carriers. third-eyelid flaps, temporary tarsorrhaphy, or eye patches may be useful in certain cases. epizootiology and transmission. although m. bovis can be killed by sunlight, it otherwise survives a long time in the environment and in cattle feces. animals acquire the infection from the environment or from other animals via aerosols, from contaminated feed and water, and from secretions such as milk, semen, genital discharges, urine, and feces. clinically normal animals may serve as carriers. the bacilli stimulate an initial neutrophilic tissue response. neutrophils become necrotic and are phagocytosed by macrophages, forming giant epithelioid cells called langhans' giant cells. an outer lymphocytic zone is formed, and fibrotic encapsulation creates the classical caseous nodules. vascular erosion and hematogenous migration of the organisms may lead to lesions throughout the body. necropsy findings. yellow primary tubercles (granulomas) with central areas of caseous necrosis and calcification are present in the lungs. caseous nodules are also associated with gastrointestinal organs and mesenteric lymph nodes. research complications. this pathogen does present a complication due to the carrier status of some animals, the likelihood of herd outbreaks, the severity of disease in younger animals, and the morbidity, possible progression to uveitis, and time and treatment costs associated with infections. the overall condition of the cattle will be affected for several weeks, and permanent visual impairment or loss, as well as ocular disfigurement, may occur. mycobacterium bovis infection (tuberculosis) etiology. mycobacteria are aerobic, nonmotile, non-sporeforming, acid-fast pleomorphic bacteria. most cases of tuberculosis in sheep are related to mycobacterium bovis or m. avium. cases in goats have been attributed to m. bovis, m. avium, or m. tuberculosis. mycobacterium bovis, or the bovine tubercle bacillus, is the cause in cattle but has been isolated from many domestic and wild mammals. other agents of mammalian tuberculosis include m. microti and m. africanum. clinical signs. tuberculosis is a sporadic, chronic, contagious disease of ruminants and is zoonotic. the infection is often asymptomatic later in the illness, and it may be diagnosed only at necropsy. the respiratory system (m. bovis) or the digestive system (m. avium) is the primary site of infection; other tissues such as mammary tissue and reproductive tract may be infrequently involved. locations of the characteristic tubercles will determine whether clinical signs are seen. respiratory signs may include dyspnea, coughing, and pneumonia. digestive tract signs include diarrhea, bloat, or constipation; diarrhea is most common. lymphadenopathy occurs in advanced cases. fever and generalized disease may be seen after calving. infected goats lose weight and develop a persistent cough. prevention and control. significant progress has been made in eradication programs in the united states during the past several decades, but during the s, infected animals continued to be found in domestic cattle herds and particularly in captive deer herds in hunting preserves. the intradermal tuberculin test, using purified protein derivative (ppd), is usually used as a diagnostic indicator in live animals. this test should be performed annually on bovine and caprine dairy herds (and bison herds); the official tests are the caudal fold, comparative cervical, and single cervical tests. notification to state officials is required following identification of intradermal-positive animals. great care must be exercised in any handling of tissue or necropsies of reactors, and state animal health officials should be consulted regarding disposal of materials and cleaning of premises following depopulation of positive animals. no treatment is recommended, and treatment is usually not allowed, because of the zoonotic potential, chronicity of the disease, and the treatment costs. slaughter is preferred, to prevent potential transmission to humans. paratuberculosis, or johne 's disease (mycobacterium paratube rculo sis) etiology. mycobacterium paratuberculosis, the causative agent of johne's disease, is a fastidious, non-spore-forming, acid-fast, gram-positive rod. the organism is actually a subspecies of m. avium, but m. paratuberculosis does not produce the siderophore mycobactin (an iron-binding molecule) of m. avium. clinical signs and diagnosis. johne's disease is a chronic, contagious, granulomatous disease of adult ruminants and is characterized by unthriftiness, weight loss, and intermittent diarrhea. in sheep and goats, chronic wasting is usually seen, occasionally with pasty feces or diarrhea. in cattle, chronic diarrhea and rapid weight loss are the most common clinical signs of the disease. usually older adult animals are infected, but over time in an infected herd, younger animals will become infected when sufficient doses of organisms are ingested. although clinical signs are nonspecific, johne's disease should be considered if the affected diarrheic animals have a good appetite and are on a good anthelmintic program. the disease is diagnosed based on clinical signs and laboratory analyses, although none of the tests is more than % sensitive. in addition, the sensitivity of the serological tests differs between species. the standard is the fecal culture that takes - weeks. theenzyme-linked immunosorbent assay (elisa) is now considered the most reliable serological test, but false negatives do occur. other serological tests such as agar gel immunodiffusion (agid) and complement fixation are useful. herd screening may be done using the agid or elisa serological tests. identification of the organism on culture, or the presence of acid-fast organisms on mucosal or mesenteric lymph node smears or from rectal biopsies, helps confirm the diagnosis. some animals serologically negative for johne's disease, however, have been found to be positive on fecal culture. commercial agid tests approved for use in cattle may be useful in diagnosing johne's disease in sheep (dubash et al., ) . serological tests cross-react with other species of mycobacterium, especially m. avium. epizootiology and transmission. the organism is prevalent in the environment and is transmitted to young animals by direct or indirect contact. although vertical transmission has been reported, the organism more commonly enters the gastrointestinal tract and penetrates the mucosa of the distal small intestine, primarily the ileum. chronic carriers may intermittently shed the organisms. parasite that grows only in macrophages of infected animals. nursing infected dams are a primary source of infection of neonates. if the organism is not cleared, it proliferates slowly in the tissue, leading to inflammatory reactions that progress through neutrophilic to mononuclear stages. the organism may penetrate the lymphatics and proliferate in mesenteric lymph nodes. after an incubation period of a year or more, some of the carriers will progress to clinical disease manifested by fibrotic and hyperplastic changes in the ileum, leading to the classic thickening in the region. gut changes result in intermittent diarrhea, with subsequent dehydration, electrolyte imbalances, and malnutrition, although this clinical sign is more common in cattle than in sheep or goats. necropsy and diagnosis. the ileum from infected cattle is grossly thickened; this is not seen in sheep and goats. ileal and ileocecal lymph nodes provide the best samples for histology and acid-fast staining. differential diagnosis. diseases causing chronic wasting and poor coat and body condition of all ruminants should be considered. these include chronic salmonellosis, peritonitis, severe parasitism, winter dysentery, and pyelonephritis. deer can be infected, and the lesions can be confused with those of tuberculosis. prevention and control. prevention is the most effective method to manage this pathogen. efforts should be focused on eliminating the disease through test and slaughter. neonates should not be reared by infected dams. some states have johne's disease eradication programs. facilities and pastures where animals testing positive for johne' disease were maintained should be thoroughly cleaned and kept vacant for a year after culling. other considerations. mycobacterium paratuberculosis is being investigated as a factor in the development of crohn's disease in humans. etiology. the most common organism causing infection of the umbilicus is arcanobacterium (formerly actinomyces, corynebacterium) pyogenes; other bacteria may be present. arcanobacterium spp. are anaerobic, nonmotile, non-sporeforming, gram-positive, pleomorphic rods to coccobacilli. other environmental contaminants are also associated with this disease, such as escherichia coli, enterococcus spp., proteus, streptococcus spp., and staplylococcus spp. clinical signs and diagnosis. navel ill is an acute localized inflammation and infection of the external umbilicus. animals present with fever and painful enlargement of the umbilicus. animals may exhibit various degrees of depression and anorexia, and purulent discharges may be seen draining from the umbilicus. involvement of the urachus is usually followed by cystitis and associated signs of dysuria, stranguria, and hematuria. other common severe sequelae include septicemia, pneumonia, peritonitis, septic arthritis (joint ill), meningitis, osteomyelitis, uveitis, endocarditis, and diarrhea. neonates, and most cases occur within the first months of age. cleanliness of the birthing and housing environment and successful transfer of passive immunity are important factors in the occurrence of the disease. dystocia resulting in weak neonates can be a factor predisposing to the development of the disease. navel ill is diagnosed by typical clinical signs. the presence of microabscesses and palpation of the umbilical area for firm intra-abdominal structures extending from the umbilicus are abnormal. assessment of colostral immunoglobulin transfer may contribute to determination of the prognosis. navel ill should always be considered for young ruminants with fever of unknown origin during the first week of life and for slightly older lambs, kids, or calves that are not thriving. arthrocentesis of affected joints and culture of the fluid for identification of the pathogen are also diagnostic options and essential for effective antimicrobial selection. differential diagnosis. the major differential is an umbilical hernia, which will typically not be painful or infected and can often be reduced. mycoplasmal arthritis is a differential in kids. in the past, erysipelothrix rhusopathiae was a common navel ill pathogen in sheep. treatment. omphalitis can be treated with a to day course of broad-spectrum antibiotics such as ampicillin, amoxicillin, penicillin, ceftiofur, florfenicol, and erythromycin. if an isolated abscess is palpable, it should be surgically opened and repeatedly flushed with iodine solutions. surgical reduction of the infected umbilicus is indicated if intra-abdominal structures are involved. the prognosis for recovery is good if systemic involvement has not occurred. prevention and control. the disease is best prevented and controlled by providing clean birthing environments, ensuring adequate colostral immunity, thoroughly dipping the umbilicus of newborns in tincture of iodine or strong iodine solution (lugol's), monitoring for dystocias, and maintaining young growing animals in noncontaminated environments. may invade the bloodstream, causing disseminated septicemia. clinically, the lambs may exhibit nasal discharge of mucopurulent to hemorrhagic exudate, hyperthermia, coughing, dyspnea, anorexia, and depression. with the respiratory form, auscultation of the thorax suggests dullness and consolidation of anteroventral lobes; this will be confirmed by radiographs. the disease is diagnosed by clinical signs, blood cultures from septicemic animals, blood smears showing bipolar organisms, and history of predisposing stressors. in cultures, p. hemolytica is distinguished from p. multocida by hemolysis on blood agar; only p. multocida produces indole. epizootiology and transmission. the organism is ubiquitous in the environment and in the respiratory tracts of these animals. younger ruminants, between and months of age, are especially prone to infection during times of stress, such as weaning, transportation, dietary changes, weather changes, and overcrowding. the pneumonic form appears as a complex associated with concurrent infections such as parainfluenza , adenovirus type , respiratory syncytial virus, mycoplasmas, chlamydia, pasteurella multocida and bordetella parapertussis (martin, ; brogden et al., ) . the organism is transmitted between animals by direct and indirect contact, through inhalation or ingestion. necropsy findings. necropsy lesions include areas of necrosis and hemorrhage in the small intestines and multifocal mm lesions distributed on the surfaces of the lungs and liver. with the pneumonic form, serofibrinous exudates fill the alveoli; ventral lung lobes are consolidated and are congested and purple-gray in color. fibrinous pleuritis, pericarditis, and hematogenously induced arthritis also may be evident.. the disease can be costly to treat, and the toll taken on young animals due to the consequences of systemic infection may detract from their research value. etiology. pasteurella hemolytica and p. multocida are aerobic, nonmotile, non-spore-forming, bipolar, gram-negative rods. biotype a serotypes are associated with pneumonia and septicemia in all ruminants (ellis, ) . serotype of p. hemolytica is considered a major cause of pulmonary lesions of bovine bronchopneumonia and fibrinous bronchopneumonia. clinical signs. pasteurellosis is an acute bacterial disease characterized by bronchopneumonia, septicemia, and sudden death. the organism invades the mucosa of the gastrointestinal tract or respiratory tract and causes localized areas of necrosis, hemorrhage, and thrombosis. the lungs and liver are frequent areas of formation of microabscesses. acute rhinitis or pharyngitis often precedes the respiratory form. the organism also pathogenesis. a leukotoxin is considered to be a key factor in the pathogenesis of the p. hemolytica infection. macrophages and neutrophils are lysed by the toxin as they arrive at the lung, and the enzymes released by the neutrophils cause additional damage to the tissue. treatment. treatment may include the use of antibiotics such as penicillin, ampicillin, tylosin, sulfonamides, or oxytetracycline. newer antibiotics, such as ceftiofur, tilmicosin, spectinomycin, and florfenicol, are very effective and approved for use in cattle. in outbreaks, cultures from fresh necropsies are helpful for determining sensitivities useful for the remaining group. prevention and control. the incidence of disease can be decreased by minimizing the degree of stress; by improving management, such as nutrition and control of parasitism; and, in cattle and sheep, by vaccinating for viral respiratory infections such as parainfluenza. early pasteurella hemolytica bacterin vaccines for use in cattle are not considered effective, but newer products based on immunizing against the leukotoxin and some bacterial capsule surface antigens are effective. pasteurella multocida bacterins and live streptomycin-dependent mutant vaccines are available. in young animals, passive immunity is protective. preventive measures also include maintaining good ventilation in enclosures and barns. new animals to the flock or herds should be quarantined for at least weeks before introduction. etiology. salmonella typhimurium is a motile, aerobic to facultatively anaerobic, non-spore-forming, gram-negative bacillus and is the organism associated with enteric disease and some abortions in ruminants. it is a common inhabitant of the gastrointestinal tract of ruminants. current nomenclature categorizes s. typhimurium as a serovar within the species s. enteritidis (the other two species are s. typhi and s. choleraesuis). salmonella typhimurium, s. dublin, and s. newport are the common species seen in bovine cases. salmonella typhimurium, s. dublin, s. anatum, and s. montevideo are seen in ovine and caprine cases, although a host-adapted species has not been identified in the goat. ovine abortions due to various salmonella species are not reported in the united states but are enzootic in other countries. salmonella serotypes have been associated with aborted fetuses in all ruminant species. clinical signs and diagnosis. salmonellosis causes acute gastroenteritis, dysentery, and septicemia (anderson and blanchard, ) . clinically, the animals become anorexic and hyperthermic. diarrhea or dysentery develops; feces may contain mucus and/or blood and have a putrid odor. animals become severely depressed and weak, losing a high percentage of their body weight. animals may die in - days because of dehydration associated with dysenteric fluid loss, septicemia, shock, and acidosis. morbidity may be %, and mortality may be high. septicemia may result in subsequent meningitis, polyarthritis, and pneumonia. chronically infected animals may have intermittent diarrhea. in goats, salmonellosis may be recognized as diarrhea and septicemia in neonates, as enteritis in preweaned kids and mature goats, and, rarely, as abortion. adult cases may be sporadic, with intermittent bouts of diarrhea, subacute or even chronic. morbidity and mortality will be highest in neonates, and some may simply be found dead. the older animals generally tend to fare better during the disease. abdominal distension with profuse yellow feces is common. kids become severely depressed, anorexic, febrile (with temperatures as high as ~ dehydrated, acidotic, recumbent, and comatose. salmonella abortions may occur throughout gestation. there may not be any other clinical signs, or abortion may be seen with diarrhea, fever, and vulvar discharges. hemorrhage, placental necrosis, and edema will be present. metritis and placental retention may occur. some mortality of dams may occur. diagnosis is based on clinical signs and can be confirmed by culturing fresh feces or at necropsy. because of intermittent shedding of organisms, culture may be difficult; repeated cultures are recommended. leukopenia and a degenerative shift to the left are not uncommon hematological findings. epizootiology and transmission. stresses associated with recent shipping, overcrowding, and inclement weather may predispose the animal to enteric infection. birds and rodents may be natural reservoirs of salmonella in external housing environments. transmission is fecal-oral. after ingestion, the organisms may proliferate throughout the gastrointestinal tract and may penetrate the mucosa of the intestines, invade the peyer's patches and lymphatics, and migrate to the spleen, liver, and other organs. animals that survive may become chronic carriers and shedders of the organisms, and this has been demonstrated experimentally (arora, ) . fecal-oral transmission is also associated with salmonella abortion; veneral transmission has not been reported. necropsy findings and diagnosis. animals will have noticeable perineal staining. intestines (particularly the ileum, cecum, and colon) may contain mucoid feces with or without hemorrhages. petechial hemorrhages and areas of necrosis may be noticed on the surface of the liver, heart, and mesenteric lymph nodes. the wall of the intestines, gallbladder, and mesenteric lymph nodes will be edematous, and a pseudodiphtheritic membrane lining the distal small intestines and colon may be observed. this membrane is not normally seen in the goat (smith and sherman, ) . splenomegaly may be present. aborted fetuses will often be autolysed. placentitis, placental necrosis, and hemorrhage are commonly seen. serologic evidence of recent infection can be demonstrated in the dam. salmonella can be isolated from the aborted tissues. pathogenesis. after ingestion, the organism proliferates in the intestine. damage to the intestines and the resulting diarrhea are due to the bacterial production of cytoxin and endotoxin. although the salmonella organisms will be taken up by phagocytic cells involved in the inflammatory response, they survive and multiply further. septicemia is a common sequela, with the bacteria localizing throughout the body. in latently infected animals, it is often shed from the gallbladder and mesenteric lymph nodes. younger animals may be susceptible because of immature immunity and intestinal flora and higher intestinal ph. carriers may develop clinical disease when stressed. differential diagnoses. in young animals, differentials include other enteropathogens: escherichia coli, rotavirus and coronavirus, clostridia, cryptosporidia, and other coccidial forms. these pathogens may also be present in the affected animals. differentials in adults include bovine viral diarrheas and winter dysentery in cattle and parasitemia and enterotoxemia in all ruminants. prevention and control. affected animals should be isolated during herd outbreaks. samples for culture should include herdmates, water and feed sources, recently arrived livestock (other species), and area wildlife, including birds and rodents. repeated cultures, culling of animals, intensive cleaning, and disinfection of facilities are all important during outbreaks. the bacteria survive for about a week in moist cow manure. vaccination using the commercially available killed bacterin or autologous bacterins may be useful in outbreaks involving pregnant cattle, although the j- bacterin is now considered better. treatment. nursing care includes rehydration and correction of acid-base abnormalities. antibiotic therapy may be useful in cases with septicemia, but it is controversial because it may induce carrier animals. gentamicin, trimethoprim-sulfadiazine, ampicillin, enrofloxacin, and amikacin antibiotics may be successful. negative, rod-shaped bacterium. type a is more virulent than type b. clinical signs. although tularemia is a disease of livestock, pets, and wild animals, sheep are most commonly affected. the disease is characterized by hyperthermia, muscular stiffness, and lymphadenopathy. infected animals move stiffly, are depressed, and are hyperthermic. anemia and diarrhea may develop, and infected lymph nodes enlarge and may ulcerate. mortality may reach %. animals that recover will have immunity of long duration. epizootiology and transmission. the disease is most commonly transmitted by ticks or biting flies. the wood tick, dermacentor andersoni, is an important vector in transmitting the disease in the western united states, and, as natural hosts, wild rodents and rabbits tend to be reservoirs of the pathogen. research complications. salmonellosis is zoonotic, and some serotypes of the organism have caused fatalities even in immunocompetent humans. attempts should be made to identify and cull carrier animals. pathogenesis. the organisms, entering the tick bite wound, move via lymphatics to lymph nodes and subsequently to the bloodstream, where they cause septicemia. the organisms can also be transmitted orally through contaminated water. etiology. spirochete-like organisms are associated with this disease; it is now recognized that the agent is not a chlamydial organism. the disease has been reported only in the foothills bordering the central valley of california. necropsy findings. ticks may also be present on the carcasses. suppurative, necrotic lymph nodes are typical. lungs will be congested and edematous. diagnosis is confirmed by prompt culturing of the organism from lymph nodes, spleen, or liver where granulomatous lesions form; p. tularensis does not survive for long periods in carcasses. serological findings may also be helpful. clinical signs. cows that become infected with the causative agent before months of gestation abort or give birth to weak calves without any clinical sign of infection. cows infected after months of gestation give birth to normal calves. affected cows rarely abort in subsequent pregnancies. the tick vector is ornitho- necropsy. fetuses show several pathological changes, including enlargement of the cervical lymph nodes, spleen, and liver. the calf's thymus will be small, and histologically there will be losses of thymic cortical lymphocytes. histologic changes in lymph nodes and spleen include vasculitis, necrosis, and histiocytosis. treatment. chlortetracycline treatment has been effective in controlling this disease. etiology. tularemia is caused by pasteurella (francisella) tularensis a nonmotile, non-spore-forming, aerobic, gram-control and prevention. eliminating the tick vectors can prevent tularemia. animals should be provided with fresh water frequently. the organism can survive in freezing conditions and in water and mud for long periods of time. caretakers, veterinarians, and researchers should take special precautions before handling the tissues of infected sheep, because this is a method of zoonotic spread. research complications. the disease is zoonotic, and transmission to people may result from tick bites or from handling contaminated tissues. although not a major disease of concern in sheep, researchers using potentially infected animals from western range states of the united states should be aware of it. the organism is antigenically related to brucella spp. etiology. yersiniosis is caused by infections with yersinia enterocolitica, a gram-negative, aerobic, and facultative anaerobe of the family enterobacteriaceae. there are serotypes reported for y. enterocolitica. yersinia pseudotuberculosis infections have also been seen in ruminants. enteric infections predominate in the diseases caused by these bacteria. clinical signs and diagnosis. clinical disease may be seen rarely in many groups of ruminants. goats of - months old suffer from the enteric form of the disease, which is characterized by sudden death or the acute onset of watery diarrhea lasting or more days. spontaneous abortions and weak neonates are also clinical manifestations of infection. lactating does may have mastitis that becomes chronically hemorrhagic. bacteremia results in internal abscesses, abortion, and acute deaths. yersinia pseudotuberculosis has been associated with laboratory goat epizootics (obwolo, ) . diarrhea in pastured sheep, stressed by other factors, has also been reported. diagnosis is based on culture and serology. epizootiology and transmission. the bacteria are carried by wild birds and rodents, and transmission is by ingestion of contaminated feed and water. research complications. yersinia is zoonotic. prevention and control. control measure are not well defined, because the epidemiology of the disease is poorly understood (smith and sherman, ) . tissues from affected goats must be handled and disposed of properly. areas housing affected goats must be thoroughly sanitized. treatment. in case of an abortion storm, treatment of goats with tetracycline has been useful. other broad-spectrum antibiotics may also be useful. clinical signs. contagious caprine pleuropneumonia is characterized by severe dyspnea, nasal discharge, cough, and fever (mcmartin et al., ) . infections with other mycoplasma species also have similar clinical signs. septicemia without respiratory involvement may also be a presentation. epizootiology and transmission. this disease is highly contagious, with high morbidity and mortality. transmission is by aerosols. mycoplasma mycoides subsp, mycoides has become a serious cause of morbidity and mortality of goat kids in the united states. necropsy. large amounts of pale straw-colored fluid and fibrinous pneumonia and pleurisy are typical. some lung consolidation may be present. meningitis, fibrinous pericarditis, and fibrinopurulent arthritis may also be found. diagnosis is usually made at necropsy by culture of the organism from lungs and other internal organs. differential dagnosis. in the united states, the principal differential for m. mycoides subsp, mycoides is caprine arthritis encephalitis. treatment. tylosin and oxytetracycline are effective. some infections are slow to resolve. prevention and control. vaccines are available in some areas. infected herds are quarantined. new goats should be quarantined before introduction to the herd. research complications. the worldwide distribution of the f biotype, as well as the aerosol transmission and high mor-bidity and mortality characteristics of mycoplasmal infectious, make these infections economically important diseases. considerable attention is presently given to this genus as a source of morbidity and mortality in goats. iv. mycoplasma conjunctivae (mycoplasmal keratoconjunctivitis) etiology. mycoplasma conjunctivae causes infectious conjunctivitis, or pinkeye, in sheep and goats with associated hyperemia, edema, lacrimation, and corneal lesions. mycoplasma mycoides subsp, mycoides, m. agalactiae, m. arginini, and acholeplasma oculusi have also been associated with keratoconjunctivitis in these species. respiratory disease and other infections, such as mastitis, may also be observed. clinical signs and diagnosis. all ages of animals may be affected. initially, lacrimation, conjunctival vessel injection, and then keratitis and neovascularization are seen. sometimes uveitis is evident. although the presentation is usually unilateral, bilateral involvement is possible. recurring infections are common. culturing provides the better diagnostic information, and cultures will be positive even after clinical signs have diminished. ily between animals by direct contact. animals can become reinfected, and carrier animals may be a factor in outbreaks. necropsy. it is unlikely that animals would die or be euthanized and undergo necropsy for this problem. conjunctival scrapings would include neutrophils during earlier stages and lymphocytes during later stages. epithelial cell cytoplasm should be examined for organisms. differential diagnosis. the primary differential in sheep and goats is chlamydia, as well as branhamella, rickettsia (colesiota) conjunctivae, and infectious bovine rhinotracheitis in goats only. it is important to consider these differentials if arthritis, pneumonia, or mastitis is present in the group or the individual. treatment. animals do recover spontaneously within about weeks. tetracycline ointments and powders are also used. third-eyelid flaps may be necessary if corneal ulceration develops. prevention and control. new animals should be quarantined and, if necessary treated, before introduction to the flock or herd. etiology. eperythrozoonosis is a rare, sporadic, noncontagious, blood-borne disease in ruminants worldwide caused by the rickettsial agent eperythrozoon. host-specific species of importance are e. ovis, the causative species in sheep and goats, and e. wenyoni, e. tegnodes, and e. tuomii, the causative agents in cattle. although the disease is of minor importance, it can cause severe anemia and debilitation in affected animals. haemobartonella bovis is also rare, and is usually found only in association with other rickettsial diseases. clinical signs and diagnosis. the disease is more severe in sheep. following an incubation period of - weeks, infected animals exhibit episodic hyperthermia, weakness, and anemia. losses may be greater in younger lambs. cattle are usually latently infected but may have swollen and tender teats and legs. fever, anemia, and depression will be present if the cattle are stressed by another systemic disease. diagnosis is based on clinical evidence of anemia and is confirmed by observing the rickettsiae on the surface of red blood cells in a blood smear. epizootiology and transmission. the rickettsial organisms are transmitted typically to young sheep by biting insects, ticks, contaminated needles or blood-contaminated surgical instruments. necropsyfindings. necropsy findings include splenic enlargement and tissue icterus. has resulted in transient hyperthermia, mild respiratory disease, and mastitis. abortions, stillbirths, and births of weak lambs are also seen. epizootiology and transmission. coxiella burnetii is extremely resistant to environmental changes as well as to disinfectants; persistence in the environment for a year or longer is possible. the organism is associated with either a free-living or an arthropod-borne cycle. coxiella burnetii is found in a variety of tick species, such as ixodid or argasid, where it replicates and is excreted in the feces. once introduced into a mammal, coxiella may be maintained without a tick intermediate. the organism is especially concentrated in placental tissues, replicates in trophoblasts, and will be in reproductive fluids. additionally, the organism is shed in milk, urine, feces, and oronasal secretions. necropsy findings. no specific lesion will be seen in aborted or stillborn fetuses, but necrotizing placentitis will be a finding in cases of abortion. the placenta will contain white chalky plaques and a red-brown exudate. the disease can be diagnosed by identifying the rickettsial organisms in smears of placental secretions. the organism has been found in the placentas of clinically normal animals. the organism stains red with modified ziehl-neelsen and macchiavello stains and purple with giemsa stain. pathogenesis. the organism invades and destroys red blood cells. it is believed that intravascular hemolysis and erythrophagocytosis contribute to the macrocytic anemia. as with other red blood cell parasites, splenectomy aggravates the disease. differential diagnosis. because of the organisms' similarity to chlamydia, confirmation must be made by culture techniques, immunofluorescent procedures, elisa, and complement fixation tests. differential diagnosis. clontridium novyi type d, babesiosis, and leptospirosis are the primary differentials. prevention and control. following strict sanitation practices for surgical procedures and controlling external parasites prevent the disease. treatment. treatment is not usually recommended, but oxytetracycline has been used. sheep will develop immunity if supported nutritionally during the disease. research complications. splenectomized animals are the experimental models used to study these diseases. ii. q fever, or query fever (coxiella burnetii) etiology. coxiella burnetii is a small, gram-negative, obligate intracellular rickettsial organism that causes query fever and is regarded as a major cause of late abortion in sheep. clinical signs. infection of ruminants with c. burnetii is usually asymptomatic. experimental inoculation in other mammals treatment. coxiella can be treated with oxytetracyclines. a vaccine is not commercially available. prevention and control. any aborting animals should be segregated from other animals, and other pregnant animals should be treated prophylactically with tetracycline. serologic screening of ruminant sources should be performed routinely. barrier housing, a review of ventilation exhaust, and defined handling procedures are often required. all placentas and all aborted tissues should be handled and disposed of carefully. q fever has been reported in many mammalian species, including cats. research complications. coxiella burnetii-free animals are particularly important in studies involving fetuses and placentation. because of its zoonotic potential, c. burnetii presents a unique problem in the animal research facility environment. a single organism has been shown to cause disease. some of the greatest concerns are the risk to immunocompromised individuals, pregnant women, and other animals, and the presence of carrier animals or those that may shed the organism in placentas, for example. etiology. the ruminant adenoviruses are dna viruses that cause respiratory and reproductive tract diseases. nine antigenic types of the bovine adenovirus have been identified, with type associated with respiratory disease. two of the ovine and two of the caprine antigenic types have been identified. clinical signs. signs of infection range from subclinical to severe, including pneumonia, enteritis, conjunctivitis, keratoconjunctivitis, weak calf syndrome, and abortion. respiratory tract and intestinal tract diseases may be concurrent. infections caused by this virus are often found associated with other viral and bacterial infections. epizootiology and transmission. the virus is believed to be widespread, but prevalence and characteristics of infection have not been characterized. transmission of adenoviruses in other species (e.g., canine) is by aerosols or fecal-oral routes. necropsy findings. lesions found after experimental infections include atelectasis, edema, and consolidation of the lungs. etiology. the bluetongue virus is an rna virus in the orbivirus genus and reoviridae family. five serotypes ( , , , , and ) have been identified in the united states, where it is seen mostly in western states. bluetongue is an acute arthropodborne viral disease of ruminants, characterized by stomatitis, depression, coronary band lesions, and congenital abnormalities (bulgin, ) . clinical signs and diagnosis. sheep are the most likely to show clinical signs. clinical disease is less common in goats and cattle. early in the infection, animals will spike a fever and will develop hyperemia and congestion of tissues of the mouth, lips, and ears. the virus name, bluetongue, is associated with the typical cyanotic membranes. the fever may subside, but tissue lesions erode, causing ulcers. increased salivary discharges and anorexia are often related to ulcers of the dental pad, lips, gums, and tongue, although salivation and lacrimation may precede apparent ulceration. chorioretinitis and conjunctivitis are also common signs in cattle and sheep. lameness may be observed associated with coronitis and is evident in the rear legs. skin lesions such as drying and cracking of the nose, alopecia, and mammary glands are also observed. secondary bacterial pneumonia may also occur. animals may also develop severe diarrhea and become recumbent. sudden deaths due to cardiomyopathy may occur at any time during the disease. hematologically, animals will be leukopenic. the course of the disease is about weeks, and mortality may reach %. if animals are pregnant, the virus crosses the placenta and causes central nervous system lesions. abortions may occur at any stage of gestation in cattle. prolonged gestation may result from cerebellar hypoplasia and lack of normal sequence to induce parturition. cerebellar hypoplasia will also be present in young born of the infected dams, as well as hydrocephalus, cataracts, gingival hyperplasia, or arthrogryposis. diagnosis is suspected with the characteristic clinical signs and exposure to viral vectors. virus isolation is the best diagnostic approach if blood is collected during the febrile stage of the disease or brains from aborted fetuses. fluorescent antibody tests, elisa, virus neutralization tests, pcr, and agar gel immunodiffusion (agid) tests are also used to confirm the diagnosis. necropsy findings. at necropsy, erosive lesions may be observed around the mouth, tongue, palate, esophagus, and pillars of the rumen. ulceration or hyperemia of the coronary bands may also be seen. many of the internal organs will contain petechial and ecchymotic hemorrhages of the surfaces, and hemorrhage may be seen at the base of the pulmonary artery. pathogenesis. the virus multiplies in the hemocoel and salivary glands of the fly and is excreted in transmissible form in the insect's saliva. after entering the host, the virus causes prolonged viremia. the incubation period is - days. the virus migrates to and attacks the vascular endothelium. the resulting vasculitis accounts for the lesions of the skin, mouth, tongue, esophagus, and rumen and the edema often found in many tissues. ballooning degeneration of affected tissues, followed by necrosis and ulceration, occurs. the effects on fetuses appear to be due to generalized infections of developing organs. differential diagnosis. differentials include other infectious vesicular diseases such as foot-and-mouth disease, contagious ecthyma, bovine viral diarrhea virus-mucosal disease, infectious bovine rhinotracheitis, bovine papular stomatitis, and malignant catarrhal fever. rinderpest is a differential in countries where it is endemic. photosensitization should be considered. foot rot is a differential for the lameness and coronitis. differentials for the manifestations such as arthrogryposis include border disease virus and genetic predispositions of some breeds such as charolais cattle and merino sheep. prevention and control. cellular and humoral immunity are necessary for protection from infection. the bluetongue virus is insidious because the genome is capable of reassortment, and some vaccines will not have the antigenic components represented in the local infection. in addition, there is little to no cross protection between strains. modified live vaccines are available in some parts of the united states but should not be used in pregnant animals. vaccinating lambs and rams in an outbreak is worthwhile, for example, but vaccinating lategestation ewes may cause birth defects or abortions. congenital defects are more common from vaccine use than from naturally occurring infection. minimizing exposure to the vector in endemic areas will decrease the incidence of the disease. treatment. supportive care and nursing care are helpful, including gruels or softer feeds, easily accessed water, and shaded resting places. nonsteroidal anti-inflammatory drugs are often administered. for the cases of secondary bacterial pneumonia and some cases of bluetongue conjunctivitis, antibiotics may be administered. research complications. this is a reportable disease because clinical signs resemble foot-and-mouth disease and other exotic vesicular diseases. etiology. bovine lymphosarcoma refers to lymphoproliferative diseases in young cattle that are not associated with bovine leukemia virus (blv) infection, and those in older cattle that are associated with b lv. b lv is a b lymphocyte-associated retrovirus (johnson and kaneene, a,b,c) . clinical signs. forms of bovine lymphosarcoma that are not associated with blv infection are calf, or juvenile; thymic, or adolescent (animals months to years old); and cutaneous (any age). the calf form is rare and characterized by generalized lymphadenopathy. onset may be sudden, and the disease is usually fatal within a few weeks. signs include lymphadenopathy, anemia, weight loss, and weakness. some animals may be paralyzed because of spinal cord compression from subperiosteal infiltration of neoplastic cells. the adolescent form is also rare, the course rapid, and the prognosis poor. the disease is seen most often in beef breeds such as hereford cattle and is characterized by space-occupying masses in the neck or thorax. these masses are also often present in the brisket. secondary effects of the masses are loss of condition, dysphagia, rumen tympany, and fatal bloat. the cutaneous presentation has a longer course and may wax and wane. the masses are found at the anus, vulva, escutcheon, shoulder, and flank; they are painful when palpated, raised, and often ulcerated. the animals are anemic, and neoplastic involvement may affect cardiac function. generalized or limited lymphadenopathy may be apparent. only the adult, or enzootic, form of bovine lymphosarcoma is associated with blv infection. many animals do not develop any malignancies or clinical signs of infection and simply remain permanently infected. some cows manifest disease only during the periparturient period. malignant lymphoma is the more common, whereas leukosis, due to b-lymphocyte proliferation, is rare. clinical signs are loss of condition and a drop in production of dairy cattle, anorexia, diarrhea, ataxia, paresis, and other signs dependent on the location of the neoplastic tissue. tumors are associated with lymphoid tissues. common sites also include the abomasum, spinal canal, and uterus. cardiac tumors develop at the right atrial or left ventricular myocardium, and associated beat and rate abnormalities may be auscultated. the common ocular manifestation of the disease is exophthalmos due to retrobulbar masses. many internal organs may be involved, and tumors may be palpable per rectum. secondary infections will be due to immunosuppression and the weakened state of the animal. sheep have acquired blv infection naturally and have been used as experimental models; in both situations, this species is susceptible to tumor and leukemia development. goats seroconvert but do not develop the clinical syndromes. diagnosis is based on the animal's age, clinical signs, serology, hematology findings according to the form, aspirates or biopsies of masses, and necropsy findings. kits are available for running agid, for which the blv antigens gp- and gp- are used; antibodies may be detected within weeks after exposure and may also help in predicting disease in clinically normal cattle. elisa and pcr diagnostic aids will also be helpful. worldwide. it is estimated that at least % of the cattle in the united states are infected with blv. as few as % of these animals develop lymphosarcoma, but the adult form of the disease described here is the most common bovine neoplastic disease in the united states. larger herds tend to have higher rates. genetic predisposition may be involved; in addition to the presence of blv, the type of bovine lymphocyte antigen (bola) may be correlated to resistance or susceptibility and to the course of the disease. transmission is believed to be by inhalation of blv in secretions; in colostrum; horizontally by contaminated equipment not sanitized between cattle; and by rectum (e.g., mucosal irritation during per-rectum exams or procedures). natural-service bulls may transmit the infection to cows. cows infected with blv may transmit the infection to their calves in utero. tabanid and other flies also serve as vectors, but these represent a minor means of transmission. necropsy findings. neoplastic infiltration of many organs and tissues are found in the calf form and the cutaneous forms. tumors may be local or widely distributed in the enzootic form. definitive diagnosis of neoplastic tissue specimens is by histology. pathogenesis. as with other retroviruses, the blv integrates viral dna into host target cell dna by means of the reverse transcriptase enzyme, creating a provirus. epizootiology and transmission. the virus is reported to be widespread. occurrence is often seasonal, and biting insects may be vectors. transmission with successful infection requires deep penetration of the skin. transmission may be by contaminated milkers' hands, contaminated equipment, and other fomites. differential diagnosis. differential diagnoses include other diseases that cause lesions on teats such as pseudocowpox, papillomatosis, and vesicular stomatitis. other vesicular diseases may be considered, but other more severe clinical signs might be associated with those. there is no vaccine for this disease. development and maintenance of a blv-free herd, or controlling infection within a herd, requires financial and programmatic commitments: blv-positive and blv-negative animals maintained separately; serologic testing (such as at least every months) and separating positive animals; and washing and then disinfecting instruments, needles (or using sterile singleuse products), and equipment for ear tagging and dehorning and other such equipment between animals. a fresh rectal exam sleeve and lubricant should be used for each animal examined. otherwise serologically positive cows may have undetectable antibodies during the periparturient period. embryo transfer recipients should be negative, and the virus will not be transferred by the embryonic stage. calves should be fed colostrum from serologically negative cows. treatment. treatment regimens of corticosteroids and cancer chemotherapeutic agents provide only short-term improvement. in cases where ova, embryos, or semen need to be collected, supportive care for the affected animals is essential. research complications. the united states and several countries, some in europe, have official programs for eradication of enzootic bovine leukosis. prevention and control. established milking hygiene practices are important control measures: having milkers wash their hands with germicidal solutions or wear gloves, cleaning equipment between animals, and separating affected animals. treatment. there is no treatment, and affected animals should be separated from the herd and milked last. lesions can be cleaned and treated with topical antibacterials. etiology. the bovine viral diarrhea virus (bvdv) is a pestivirus of the flaviviridae family. the flaviviridae include hog cholera virus and border disease virus of sheep. the virus contains a single strand of positive-sense rna. a broad range of disease and immune effects is produced by b vdv only in cattle. in addition, this virus is important in the etiology of bovine pneumonias. bovine viral diarrhea/mucosal disease (bvd/md) is one of the most important viral diseases and one of the most complex diseases of cattle. strains of bvdv are characterized as cytopathic (cp) and noncytopathic (ncp), based on cell-culture growth characteristics. the virus has also been categorized as type and type isolates. heterologous strains exist that may confound even sound vaccination programs. etiology. bovine herpesvirus causes bovine herpes mammillitis, a widespread disease characterized by teat and udder lesions, as well as oral and skin lesions. clinical signs and diagnosis. lesions begin suddenly with teat swelling; the tissue will be edematous and tender when touched. the udder lesions may extend to the perineum. the lesions progress to vesicles, then to ulcers; these may take weeks to heal. lesions rarely may also develop focally around the mouth and generally on the skin of the udder. secondary mastitis may occur, because of bacteria associated with the scabs. diagnosis is by clinical signs and serologically. clinical signs and diagnosis. signs of bvdv infections may be subclinical but also include abortions, congenital abnormalities, reduced fertility, persistent infection (pi) with gradual debilitation, and acute and fatal disease. the presence of antibodies, whether from passive transfer or immunizations, does not necessarily guarantee protection from the various forms of the disease. an acute form of the disease, caused by type bvdv, occurs in cattle without sufficient immunity. after an incubation period of - days, clinical signs include fever, anorexia, oculonasal discharge, oral erosions (including on the hard palate), diarrhea, and decreased milk production. the disease course may be shorter with hemorrhagic syndrome and death within days. clinical signs of b vdv in calves also include severe enteritis and pneumonia. when susceptible cows are infected in utero from gestational be found extending throughout the gastrointestinal tract to the days - , or gestational cows are vaccinated with a modi-cecum. the respiratory tract lesions will often be complicated fled live vaccine, abortion or stillbirth result. congenital defects caused by bvdv during gestational days - include impaired immunity (thymic atrophy), cerebellar hypoplasia, ocular defects, alopecia or hypotrichosis, dysmyelinogenesis, hydranencephaly, hydrocephalus, and intrauterine growth retardation. typical signs of cerebellar dysfunction will be evident in calves, such as wide-based stance, weakness, opisthotonus, hyperflexion, hypermetria, nystagmus, or strabismus. some severely affected calves will not be able to stand. ophthalmic effects include retinal degeneration and microphthalmia. fetuses can also be infected in utero, normal at birth, immunotolerant to the virus, and persistently infected (pi). the term mucosal disease is commonly associated with this form of the infection. many pi animals do not survive to maturity, however, and many have weakened immune systems. the pi animals are important because they shed virus and will probably show the clinical signs of mucosal disease (md) caused by a cp b vdv strain derived from an ncp b vdv strain. these md clinical signs include fever, anorexia, and profuse diarrhea that may include blood and fibrin casts, and oral and pharyngeal erosions, as well as erosion at the interdigital spaces and on the teats and vulva. many other associated clinical signs include anemia, bloat, lameness, or corneal opacities and discharges. secondary effects of hemorrhage and dehydration also contribute to the morbidity and mortality. animals that do not succumb to the disease will be chronically unthrifty, debilitated, and infection-prone. diagnosis in affected calves is based on herd health history, clinical signs, and antibodies to b vdv in precolostral serum. viral culturing from blood may be useful. in older animals, oral lesions, serology, detection of viral antigen, and virus isolation contribute to the diagnosis. leukopenia, and especially lymphopenia, are seen. serology must be interpreted with the awareness of the possibility of pi immunotolerant animals. vaccination against the disease carries its own set of side effects and potential problems, especially when using modified live vaccines, whether against cp or ncp strains. the condition of the animals is also a variable. epizootiology and transmission. bvdv is present throughout the world. transmission occurs easily by direct contact between cattle, from feed contaminated with secretions or feces, and by aborted fetuses and placentas. pi females transmit the virus to their fetuses. semen also is a source of virus. necropsy findings. in affected calves, histopathologic findings include necrosis of external germinal cells, focal hemorrhages, and folial edema. later in the disease, large cavities develop in the cerebellum, and atrophy of the cerebellar folia and thin neuropil are evident. older calves may have areas of intestinal necrosis. in cases where oral erosions occur, erosions will by secondary bacterial pneumonia. when the hemorrhagic syndrome develops, petechiation and mucosal bleeding will be present. pathogenesis. the cp and ncp strains are thought to be related mutations of the bvdv; the cp short-lived isolates are believed to arise from the ncp strains. the ncp strains are those present in the pi animals, and the strains are maintained in cattle populations. cp and ncp isolates vary in virulence, and classification of these types is based on viral surface proteins. considerable antigenic variation also exists between strains and types. other viral infections, such as bovine respiratory syncytial virus and infectious bovine rhinotracheitis, may also be present in the same animals. the pathology caused by b vdv is due to its ability to infect epithelial cells and impair the functioning of immune cell populations through out the bovine system. in type bvdv hemorrhagic syndrome, death results from viral-induced thrombocytopenia. in fetuses, the virus infects developing germinal cells of the cerebellum. the purkinje's cells in the granular layer are killed, and necrosis and inflammation follow. the immune effects are the result of the virus's interfering with neutrophil and macrophage functions and of lymphocyte blastogenesis. all of these predispose the affected animals to bacterial infections with pasteurella haemolytica. b vdv damages dividing cells in fetal organ systems, resulting in abortions and congenital effects. differential diagnosis. many differentials must be considered for the clinical manifestations of b vdv infections. differentials for enteritis of calves include viral infections, cryptosporidia, escherichia coli, salmonella, and coccidia. salmonella, winter dysentery, johne's disease, intestinal parasites, malignant catarrhal fever (mcf), and copper deficiency are differentials for the diarrhea seen in the disease in adult animals. respiratory tract pathogens such as bovine respiratory syncytial virus, pasteurella, haemophilus, and mycoplasma must be considered for the respiratory tract manifestations. oral lesions are also produced by mcf, vesicular stomatitis, bluetongue, and papular stomatitis. infectious bovine herpesvirus , leptospirosis, brucellosis, trichomoniasis, and mycosis should be considered in cases of abortion. prevention and control. combined with sound management in a typical cattle herd, vaccination is the best way to prevent b vdv and should be integrated into the herd health program, timed appropriately preceding breeding, gestation, or stressful events. vaccine preparations for b vdv are modified live virus (mlv) or killed virus. each has advantages and disadvantages. the former induces rapid immunity (within week) after a single dose, provides longer duration of immunity against sev-eral strains, and induces serum neutralizing antibodies. mlv vaccines are not recommended for use in pregnant cattle, may induce mucosal disease, and may be immunosuppressive at the time of vaccination. the immunosuppression is detrimental if cattle are concurrently exposed to field-strain virus because it will facilitate infection and possible clinical disease. the mlv strains may cross the placenta, resulting in fetal infections. the killed vaccines are safer in pregnant animals but require booster doses after the initial immunization, may need to be given - times per year, and do not induce cell-mediated immunity. passive immunity may protect most calves for up to - months of age. subsequent vaccination with mlv may provide lifelong immunity, but this is not guaranteed. annual boosters are recommended to protect against vaccine breaks. the virus persists in the environment for weeks and is susceptible to the disfectants chlorhexidine, hypochlorite, iodophors, and aldehydes. maintenance of a closed herd to prevent any possibility of the introduction of the virus is difficult. isolation of new animals, avoidance of the purchase of pregnant cows, scrutiny of records from source farms, use of semen tested bulls, minimization of stress, testing of embryo-recipient cows, and maintainenance of populations of ruminants (smaller or wild species) separately on the premises will minimize viral exposure. other management strategies may require a program for testing and culling pi cattle. this can be expensive but may be a worthwhile investment to remove the virus shedders from a herd. no specific treatment is available. supportive care and treatment with antibiotics to prevent secondary infection are recommended. animals that survive the infection should be evaluated a month after recovery to determine their status as pi or virus-free. etiology. cache valley virus (cvv), of the arbovirus genus of the bunyaviridae family, is a cause of congenital defects in lambs. cvv infection in fetal and newborn lambs include arthrogryposis, microencephaly, hydranencephaly, porencephaly, cerebellar hypoplasia, and micromyelia. stillbirths and mummified fetuses are seen. lambs will be born weak and will act abnormally. diagnosis is by evidence of seroconversion in precolostral blood samples or fetal fluids, as the result of in utero infection. western united states, although it has been isolated in a few midwestern states. although considered a disease of sheep, virus has been isolated from cattle and from wild ruminants and antibodies found in white-tailed deer. transmission is by arthropods during the first trimester of pregnancy. etiology. caprine arthritis encephalitis virus (caev) occurs worldwide, with a high prevalence in the united states. caprine arthritis encephalitis (cae) is considered the most important viral disease of goats. the caev is in the lentivirus genus of the retroviridae family. it causes chronic arthritis in adults and encephalitis in young. caev is in the same viral genus as the ovine progressive pneumonia virus (oppv). clinical signs and diagnosis. the most common presentation in goats is an insidious, progressive arthritis in animals months of age and older. animals become stiff, have difficulty getting up, and may be clinically lame in one or both forelimbs. carpal joints are so swollen and painful that the animal prefers to eat, drink, and walk on its "knees." in dairy goats, milk production decreases, and udders may become firmer. this retrovirus also causes neurological clinical signs in young kids - months old. kids may be bright and alert, afebrile, and able to eat normally even when recumbent. some kids may initially show unilateral weakness in a rear limb, which progresses to hemiplegia or tetraplegia. mild to severe lower motor neuron deficits may be noted, but spinal reflexes are intact. clinical signs may also include head tilt, blindness, ataxia, and facial nerve paralysis. older animals in the group may experience interstitial pneumonia or chronic arthritis. the pneumonia is similar to the pneumonia in sheep caused by oppv; the course is gradual but progressive, and animals will eventually lose weight and have respiratory distress. some animals in a herd may not develop any clinical signs. diagnosis is based on clinical signs, postmortem lesions, and positive serology for viral antibodies to caev. an agar gel immunodiffusion (agid) test identifies antibodies to the virus and is used for diagnosis. kids acquire an anti-caev antibody in colostrum, and this passive immunity may be interpreted as indicative of infection with the virus. the antibody does not prevent viral transmission. ep&ootiology and transmission. the virus is prevalent in most industrialized countries. the common means of transmission, from adults to kids, is in the colostrum and milk in spite of the presence of anti-caev antibody in the colostrum. transmission may occur among adult goats by contact. intrauterine transmission is believed to be rare. transmission to sheep has occurred only experimentally; there is no documented case of natural transmission. necropsy findings. necropsy and histopathology reveal a striking synovial hyperplasia of the joints with infiltrates of lymphocytes, macrophages, and plasma cells. other histologic lesions include demyelination in the brain and spinal cord, with multifocal invasion of lymphocytes, macrophages, and plasma cells. in severe cases of mastitis, the udder may appear to be composed of lymphoid tissue. tem, resulting in the formation of non-neutralizing antibody to viral core proteins and envelope proteins. immune complex formation in synovial, mammary gland, and neurological tissue is thought to result in the clinical changes observed. most commonly, the carpal joint is affected, followed by the stifle, hock, and hip. the infection is lifelong. differential diagnosis. the differential diagnosis for the neurologic form of caev should include copper deficiency, enzootic pneumonia, white muscle disease, listeriosis, and spinal cord disease or injury. the differential diagnosis for caev arthritis should include chlamydia and mycoplasma. prevention and control. herds can be screened for cae by testing serologically, using an agid or an enzyme-linked immunosorbent assay (elisa) test. the elisa is purported to be more sensitive, whereas the agid is more specific. individual animals show great variation in development of antibody. because cae is highly prevalent in the united states, and because seronegative animals can shed organisms in the milk, retesting herds at least annually may be necessary. recently, an immunoprecipitation test for cae has been developed that has high sensitivity and specificity. control measures include management practices such as test and cull, prevention of milk transmission, and isolation of affected animals. parturition must be monitored, and kids must be removed immediately and fed heat-treated colostrum ( ~ for hr). caev-negative goats should be separated from caevpositive goats. treatment. there is no treatment for caev. is also referred to as bovine herpesvirus (bhv- ) and is an alphaherpesvirus. ibrv causes or contributes to several bovine syndromes, including respiratory and reproductive tract diseases. it is one of the primary pathogens in the bovine respiratory disease complex. strains include bhv-i. (associated with respiratory disease), bhv . (associated with respiratory and genital diseases), and bhv . (associated with neurological diseases), which has been reclassified as bovine herpesvirus . clude conjunctivitis, rhinotracheitis, pustular vulvovaginitis, balanoposthitis, abortion, encephalomyelitis, and mastitis. the respiratory form is known as infectious bovine rhinotracheitis, and clinical signs may range from mild to severe, the latter particularly when there are additional respiratory viral infections or secondary bacterial infections. the mortality rate in more mature cattle is low, however, unless there is secondary bacterial pneumonia. fever, anorexia, restlessness, hyperemia of the muzzle, gray pustules on the muzzle (that later form plaques), nasal discharge (that may progress from serous to mucopurulent), hyperpnea, coughing, salivation, conjunctivitis with excessive epiphora, and decreased production in dairy animals are typical signs. open-mouth breathing may be seen if the larynx or nasopharygneal areas are blocked by mucopurulent discharges. neonatal calves may develop respiratory as well as general systemic disease. in these cases, in addition to the symptoms already noted, the soft palate may become necrotic, and gastrointestinal tract ulceration occurs. young calves are most susceptible to the encephalitic form; signs include dull attitude, head pressing, vocalizations, nystagmus, head tilt, blindness, convulsions, and coma, as well as some signs, such as discharges, seen with respiratory tract presentations. this form is usually fatal within days. abortion may occur simultaneously with the conjunctival or respiratory tract diseases, when the respiratory infection appears to be mild, or may be delayed by as much as months after the respiratory tract disease signs. infectious pustular vulvovaginitis is most commonly seen in dairy cows, and clinical signs may be mild and not noticed. otherwise, signs are fever, depression, anorexia, swelling of the vulvar labia, vulvar discharge, and vestibular mucosa reddened by pustules. the cow will often carry her tail elevated away from these lesions. these soon coalesce, and a fibrous membrane covers the ulcerated area. if uncomplicated, the infection lasts about - days, and lesions heal in weeks. younger infected bulls may develop balanoposthitis with edema, swelling, and pain such that the animals will not service cows. epizootiology and transmission. ibrv is widely distributed throughout the world, and adult animals are the reservoirs of infection. the disease is more common in intensive calf-rearing situations and in grouped or stressed cattle. transmission is primarily by secretions, such as nasal, during and after clinical signs of disease. modified live vaccines are capable of causing latent infections. necropsy findings. fibrinonecrotic rhinotracheitis is considered pathognomic for ibrv respiratory tract infections. there will be adherent necrotic lesions in the respiratory, ocular, and reproductive mucosa. when there are secondary bacterial infections, such as pasteurella bronchopneumonia, findings will include congested tracheal mucosa and petechial and ecchymotic hemorrhages in that tissue. lesions from the encephalitic form include lymphocytic meningoencephalitis and will be found throughout the gray matter (neuronal degeneration, perivascular cuffing) and white matter (myelitis, demyelination). intranuclear inclusion bodies are not a common finding with this herpesvirus. pathogenesis. in the encephalitic form, the virus first grows in nasal mucosa and produces plaques. these resolve within days, and the encephalitis develops after the virus spreads centripetally to the brain stem by the trigeminal nerve dendrites. latent infections are also established in neural tissue. differential diagnosis. the severe oral erosions seen with bvdv infections are rare with infectious bovine rhinotracheitis-infectious pustular vulvovaginitis (ibr-ipv). the conjunctivitis of ibr may initially be mistaken for that of a moraxella bovis (pinkeye) infection; the ibr will be peripheral, and there will not be corneal ulceration. bovine viral diarrhea virus and ibrv are the most common viral causes of bovine abortion. differentials for balanoposthitis include trauma from service. vated, attenuated, modified live, and genetically altered preparations. some are in combination with parainfluenza (pi- ) virus. the mlv preparations are administered intranasally; these are advantageous in calves for inducing mucosal immunity even when serologic passive immunity is already present and adequate. some newer vaccines, with gene deletion, allow for serologic differentiation between antibody responses from infection or immunization. bulls with the venereal form of the infection will transmit the virus in semen; intranasal vaccine may be used to provide some immunity. treatment. uncomplicated mild infections will resolve over a few weeks; palliative treatments, such as cleaning ocular discharges and supplying softened food, are helpful in recovery. antibiotics are usually administered because of the high likelihood of secondary bacterial pneumonia. the encephalitic animals may need to be treated with anticonvulsants. etiology. parainfluenza , an rna virus of the family paramyxoviridae, causes mild respiratory disease of ruminants when it is the sole pathogen. the viral infection often predisposes the respiratory system to severe disease associated with concurrent viral or bacterial pathogens. viral strains are reported to vary in virulence. serotypes seen in the smaller ruminants are distinct from those isolated from cattle. clinical signs and diagnosis. infections ranging from asymptomatic to mild signs of upper respiratory tract disease are associated with this virus by itself; infections are almost never fatal. clinical signs include ocular and nasal discharges, cough, fever, and increased respiratory rate and breath sounds. in pregnant animals, exposure to pi- can result in abortions. clinical signs become apparent or more severe when additional viral pathogens are present, such as bovine viral diarrhea virus, or a secondary bacterial infection, such as pasteurella haemolytica infection, is involved. greater morbidity and mortality will be sequelae of the bacterial infections. viral isolation or direct immunofluorescence antibody (ifa) from nasal swabs can be used for definitive diagnosis. presently it is assumed that the virus is widespread in goats, but firm evidence is lacking. for an infection of pi- only, findings will be negligible. some congestion of respiratory mucosa, swelling of respiratory tract-associated lymph nodes, and mild pneumonitis may be noted grossly and histologically. intranuclear and intracytoplasmic inclusion bodies may be present in the mucosal epithelial cells. findings will be similar but not as severe as those caused by bovine respiratory syncytial virus. immunohistochemistry may also be used. pathogenesis. pi- infects the epithelial mucosa of the respiratory tract; however, the disease is often asymptomatic when uncomplicated. differential diagnosis. differentials, particularly in cattle, include infections with other respiratory tract viruses of ruminants: ibrv, bvdv, bovine respiratory syncytial virus, and type bovine adenovirus. prevention and control. immunization, management, and nutrition are important for this respiratory pathogen, as for others. in cattle, modified live vaccines for intramuscular (im), subcutaneous (sc), or intranasal (in) administration are available. the im and sc routes provide immune protection within week after administration but will not provide protection in the presence of passively acquired antibodies. it is contraindicated for pregnant animals because it will cause abortion. the in route immunizes in the presence of passively acquired antibodies, provides immunity within days of administration, and stimulates the production of interferon. other vaccine formulations, about which less information is reported, include inactivated or chemically altered live-virus preparations; both are administered im, and followup immunizations are needed within weeks. booster vaccinations are recommended for all preparations within - months after the initial immunization. all presently marketed vaccine products come in combination with other bovine respiratory viruses as multivaccine products. the humoral immunity protects against pi- abortions. there is no approved pi- vaccine for sheep and goats. the use of the cattle formulation in these smaller ruminants is not recommended. sound management of housing, sanitation, nutrition, and preventive medicine programs are all equally important components in prevention and control. treatment. uncomplicated disease is not treated. etiology. the respiratory syncytial viruses are pneumoviruses of the paramyxoviridae family and are common causes of severe disease in ruminants, especially calves and yearling cattle. two serotypes of the bovine respiratory syncytial virus (brsv) have been described for cattle; these may be similar or identical to the virus seen in sheep and goats. clinicalfindings and diagnosis. infections may be subclinical or develop into severe illness. clinical signs include fever, hyperpnea, spontaneous or easily induced cough, nasal discharge, and conjunctivitis. interstitial pneumonia usually develops, and harsh respiratory sounds are evident on auscultation. development of emphysema indicates a poor prognosis, and death may occur in the severe cases of the viral infection. secondary bacterial pneumonia, especially with pasteurella haemolytica, with morbidity and mortality, is also a common sequela. abortions have been assciated with brsv outbreaks. diagnosis is based on virus isolation and serology (acute and convalescent). nasal swabs for virus isolation should be taken when animals have fever and before onset of respiratory disease. prevention and control. vaccination should be part of the standard health program, and all animals should be vaccinated regularly. vaccinations should be administered within - months of stressful events, such as weaning, shipping, and introduction to new surroundings. currently available vaccines include an inactivated preparation and a modified live virus preparation administered intramuscularly or subcutaneously; immunity develops well in yearling animals, and colostral antibodies develop when cows are vaccinated during late gestation. passive immunity from colostrum provides at least partial protection to calves in herds where disease is prevalent. but this immunity suppresses the mucosal iga response and serum antibody responses. the basis for successful immune protection is the mucosal memory iga, but this is difficult to achieve with present vaccine formulations. the virus is easily inactivated in the environment. preventive measures in preweaning animals should include preconditioning to minimize weaning stress. treatment. recovery can be spontaneous; however, antibiotics and supportive therapy are useful to prevent or control secondary bacterial pneumonia. in severe cases, antihistamines and corticosteriods may also be necessary. use of vaccine during natural infection is not productive and may result in severe disease. etiology. ulcerative dermatosis is a contagious disease of sheep only. it is caused by a poxvirus similar to but distinct from the causative agent of contagious ecthyma ("current veterinary therapy," ). epizootiology and transmission. these viruses are considered ubiquitous in domestic cattle and are transmitted by aerosols. teroventral lung lobes. edema and emphysema are present. as the name indicates, syncytia, which may have inclusions, form in areas of the lungs infected with the virus. necrotizing bronchiolitis, bronchiolitis obliterans, and hyaline membrane formation will be evident microscopically. crusts associated with the skin and mucous membranes of the genitalia, face, and feet (bulgin, ) . genital lesions are much more common than the facial or coronal lesions. discomfort may be associated with the lesions. paraphimosis occasionally occurs. these lesions are painful; during breeding season, animals will avoid coitus. morbidity is low to moderate, and mortality negligible if the flock is otherwise healthy. diagnosis is based on clinical signs. pathogenesis. the severe form of the disease, which often follows a mild preliminary infection, is thought to be caused by immune-mediated factors during the process of infection in the lung. virulence may vary greatly among viral strains. united states, ulcerative dermatosis is transmitted through direct contact with abraded skin of the prepuce, vulva, face, and feet. necropsy findings. necropsy would rarely be necessary to diagnose an outbreak in a healthy flock. findings will be similar to those described for contagious ecthyma. when no contact with cattle has occurred. persistently infected animals, such as lambs, are shedding reservoirs of the virus in urine, feces, and saliva throughout their lives. pathogenesis. following an incubation period of - days, the virus replicates in the epidermal cells and leads to necrosis and pustule formation. pustules rapidly break, forming weeping ulcers. the ulcers scab over and eventually form a fibrotic scar. the disease usually resolves in - weeks. rarely, the disease will persist for many months to more than a year. differential diagnosis. the main differential is contagious ecthyma, which is grossly and histopathologically associated with epithelial hyperplasia. this is also a feature of ulcerative dermatosis. imals, especially males, should not be used for breeding. treatment. affected animals should be separated from the rest of the flock. treatment is supportive, including antiseptic ointments and astringents. research complications. breeding and maintenance of the flocks' condition, because of the pain associated with eating, will be compromised during an outbreak. etiology. border disease, also known as hairy shaker disease (or "fuzzies" in the southwestern united states), is a disease of sheep caused by a virus closely related to the bovine viral diarrhea virus (bvdv), a pestivirus of the togaviridae family. goats are also affected. the virus causes few pathogenic effects in cattle. clinical signs and diagnosis. border disease in ewes causes early embryonic death, abortion of macerated or mummified fetuses, or birth of lambs with developmental abnormalities. lambs infected in utero that survive until parturition may be born weak and often exhibit a number of congenital defects such as tremor, hirsutism (sometimes darkly pigmented over the shoulders and head), hypothyroidism, central nervous system defects, and joint abnormalities, including arthrogryposis. later, survivors may be more susceptible to diseases and may develop persistent, sometimes fatal, diarrhea. the virus infection produces similar clinical manifestations in goats, except that the hair changes are not seen. diagnosis includes the typical signs described above, as well as serological evidence of viral infection. virus isolation confirms the diagnosis. wide, and reports of disease are sporadic. disease has occurred necropsy findings. lesions include placentitis, and characteristic joint and hair-coat changes in the fetus. histologically, axonal swelling, neuronal vacuolation, dysmyelination, and focal microgliosis are observed in central nervous system structures. pathogenesis. the virus entering the ewe via the gastrointestinal or respiratory tracts penetrates the mucous membranes and causes maternal and fetal viremia. infection during the first days of gestation causes embryonic death. in lambs infected between and days, the virus activates follicular development, diminishes the myelination of neurons, and causes dysfunction of the thyroid gland. infection after days of gestation results in lambs that are born persistently infected. infected lambs have high perinatal mortality; survivors have diminished signs over time but, as noted, continue to shed the virus. prevention and control. border disease can be prevented by vaccinating breeding ewes with killed-bvdv vaccine. congenitally affected lambs should be maintained separately and disposed of as soon as humanely possible. new animals to the flock should be screened serologically. if cattle are housed nearby, vaccination programs for bvdv should be maintained. treatment. there is no treatment other than supportive care for affected animals. etiology. contagious ecthyma, also known as contagious pustular dermatitis, sore mouth, or off, is an acute dermatitis of sheep and goats caused by a parapoxvirus. this disease occurs worldwide and is zoonotic. naturally occurring disease has also been reported in other species such as musk ox and reindeer. other parapoxviruses infect the mucous membranes and skin of cattle, causing the diseases bovine pustular dermatitis and pseudocowpox. clinical signs and diagnosis. the disease is characterized by the presence of papules, vesicles, or pustules and subsequently scabs of the skin of the face, genitals of both sexes, and coronary bands of the feet. lesions develop most frequently at mucocutaneous junctions and are found most commonly at the commissures of the mouth. off is usually found in young animals less than year of age. younger lambs and kids will have difficulty nursing and become weak. lesions may also develop on udders of nursing dams, which may resist suckling by offspring to nurse, leading to secondary mastitis. the scabs may appear nodular and raised above the surface of the surrounding skin. morbidity in a susceptible group of animals may exceed %. mortality is low, but the course of the disease may last up to weeks. diagnosis is based on characteristic lesions. biopsies may reveal eosinophilic cytoplasmic inclusions and proliferative lesions under the skin. electron microscopy will reveal the virus itself. disease is confirmed by virus isolation. epizootiology and transmission. all ages of sheep and goats are susceptible. seasonal occurrences immediately after lambing and after entry into a feedlot are common; stress likely plays a role in susceptibility to this viral disease. older animals develop immunity that usually prevents reinfection for at least or more years. resistant animals may be present in some flocks or herds. the virus is very resistant to environmental conditions and may contaminate small-ruminant facilities, pens, feedlots, and the like for many years as the result of scabs that have been shed from infected animals. transmission occurs through superficial lesions such as punctures from grass awns, scrapes, shearing, and other common injuries. necropsy findings. necropsy findings include ballooning degeneration of epidermal and dermal layers, edema, granulomatous inflammation, vesiculation, and cellular hyperplasia. secondary bacterial infection may also be evident. pathogenesis. the virus is typical of the poxviridae, resembling sheep poxvirus (not found in the united states) and vaccinia virus and replicating in the cytoplasm of epithelial cells. following an incubation period of - days, papules and vesicles develop around the margins of the lips, nostrils, eyelids, gums, tongue, or teats; skin of the genitalia; or coronary band of the feet. the vesicles form pustules that rupture and finally scab over. virus should be considered in both sheep and goats. an important differential in goats is staphylococcal dermatitis. prevention and control. individuals handling infected animals should be advised of precautions beforehand, should wear gloves, and should separate work clothing and other personal protective equipment. clippers, ear tagging devices, and other similar equipment should always be cleaned and disinfected after each use. colostral antibodies may not be protective. vaccinating lambs and kids with commercial vaccine best prevents the disease. dried scabs from previous outbreaks may also be used by rubbing the material into scarified skin on the inner thigh or axilla. animals newly introduced to infected premises should be vaccinated upon arrival. precautions must be taken when vaccinating animals, because the vaccine may induce orf in the animal handlers; it is not recommended to vaccinate animals in flocks already free of the disease. affected dairy goats should be milked last, using disposable towels for cleaning teat ends. treatment. affected animals should be isolated and provided supportive care, especially tube feeding for young animals whose mouths are too sore to nurse. treatment should also address secondary bacterial infections of the orf lesions, including systemic antibiotics for more severe infections. treatment for myiasis may also be necessary. the viral infection is self-limiting, with recovery in about weeks. research complications. carrier animals may be a factor in flock or herd outbreaks. contagious ecthyma is a zoonotic disease, and human-to-human transmission can also occur. the virus typically enters through abrasions on the hands and results in a large (several centimeters) nodule that is described as being extremely painful and lasting for as many as weeks. lesions heal without scarring. etiology. foot-and-mouth disease (fmd) is caused by the foot-and-mouth disease virus, a picornavirus in the aphthovirus genus. the disease is also referred to as aftosa or aphthous fever. seven immunologically distinct types of the virus have been identified, with subtypes within those . epidemics of the disease have occurred worldwide. north and central america have been free of the virus since the mid- s. this is a reportable disease in the united states; clinical signs are very similar to other vesicular diseases. cattle (and swine) are primarily affected, but disease can occur in sheep and is usually subclinical in goats. clinical signs and diagnosis. in addition to vesicle formation around and in the mouth, hooves, and teats, fever, anorexia, weakness, and salivation occur. vesicles may be as large as cm, rupture after days, and subsequently erode. secondary bacterial infections often occur at the erosions. anorexia is likely due to the pain associated with the oral lesions. high morbidity and low mortality, except for the high mortality in young cattle, are typical. diagnosis must be based on elisa, virus neutralization, fluorescent antibody tests, and complement fixation. epizootiology and transmission. domestic and wild ruminants and several other species, such as swine, rats, bears, and llamas are hosts. asymptomatic goats can serve as virus reservoirs for more susceptible cohoused species such as cattle. greater mortality occurs in younger animals. the united states, great britain, canada, japan, new zealand, and australia are fmd-free, whereas the disease is endemic in most of south america, parts of europe, and throughout asia and africa. the virus is very contagious and is spread primarily by the inhalation of aerosols, which can be carried over long distances. transmission may also occur by fomites, such as shoes, clothing, and equipment. human hands, soiled bedding, and animal products such as frozen or partially cooked meat and meat products, hides, semen, and pasteurized milk also serve as sources of virus. necropsy findings. vesicles, erosions, and ulcers are present in the oral cavity as well as on the rumen pillars and mammary alveolar epithelium. myocardial and skeletal muscle degeneration (zenker's) is most common (and accounts for the greater mortality) in younger animals. histological findings include lack of inclusion bodies. vesicular lesions include intracellular and extracellular edema, cellular degeneration, and separation of the basal epithelium. replicates in the pharynx and digestive tract in the cells of the stratum spinosum, and viremia and spread of virus to many tissues occur before clinical signs develop. virus shedding begins about hr before clinical signs are apparent. vesicles result from the separation of the superficial epithelium from the basal epithelium. fluid fills the basal epithelium, and erosions develop when the epithelium sloughs. persistent infection also occurs, and virus can be found for months or years in the pharnyx; the mechanisms for the persistence are not known. differential diagnosis. vesicular stomatitis is the principal differential. other differentials include contagious ecthyma (orf), rinderpest, bluetongue, malignant catarrhal fever, bovine papular stomatitis, bovine herpes mammillitis, and infectious bovine rhinotracheitis virus infection. products from endemic areas is regulated. quarantine and slaughter are practiced in outbreaks in endemic areas. quarantine and vaccination are also used in endemic areas, but vaccines must be type-specific and repeated or times per year to be effective and will provide only partial protection. autogenous vaccines are best in an outbreak. passive immunity protects calves for up to months after birth. the virus is inactivated by extremes of ph, sunlight, high temperatures, sodium hydroxide, sodium carbonate, and acetic acid. treatment. nursing care and antibiotic therapy to minimize secondary reactions help with recovery. humoral immunity is considered the more important immune mechanism, with cellmediated immunity of less importance. research complications. rare cases in humans have been reported. importation into the united states of animal products from endemic areas is prohibited. etiology. malignant catarrhal fever (mcf) is a severe disease primarily of cattle. the agents of mcf are viruses of the gammaherpesvirinae subfamily. alcelaphine herpesvirus and and ovine herpesvirus are known strains. the alcelaphine strains are seen in africa. the ovine strain is seen in north america. the alcelaphine and ovine strains differ in incubation times and duration of illness. disease may occur sporadically or as outbreaks. clinical signs and diagnosis. signs range from subclinical to recrudescing latent infections to the lethal disease seen in susceptible species, such as cattle. sudden death may also occur in cattle. presentations of the disease may be categorized as alimentary, encephalitis, or skin forms; all three may occur in an animal. corneal edema starting at the limbus and progressing centripetally is a nearly pathognomonic sign; photophobia, severe keratoconjunctivitis, and ocular involvement may follow. other signs include prolonged fever, oral mucosal erosions, salivation, lacrimation, purulent nasal discharge, encephalitis, and pronounced lymphadenopathy. as the disease progresses, cattle may shed horns and hooves. in north america, cattle will also have severe diarrhea. the course of the disease may extend to week. recovery is usually prolonged, and some permanent debilitation may occur. the disease is fatal in severely affected individuals. history of exposure, as well as the clinical signs and lesions, contributes to the diagnosis. serology, pcr-based assays, viral isolation, and cell-culture assays, such as cytopathic effects on thyroid cell cultures, are also used. because of the susceptibility of rabbits, inoculation of this species may be used. in less severe outbreaks or individual animal disease, definitive diagnosis may never be made. necropsy. gross findings at necropsy include necrotic and ulcerated nasal and oral mucosa; thickened, edematous, ulcerated, and hemorrhagic areas of the intestinal tract; swollen, friable, and hemorrhagic lymph nodes and other lymphatic tissues; and erosion of affected mucosal surfaces. lymph nodes should be submitted for histological examination. histological findings include nonsuppurative vasculitis and encephalitis; large numbers of lymphocytes and lymphoblasts will be present without evidence of virus. pathogenesis. the incubation period may be up to months. vascular endothelium and all epithelial surfaces will be affected. the virus is believed to cause proliferation of cytotoxic t lymphocytes with natural killer cell activities, and the resulting lesions are due to an autoimmune type of phenomenon. differential diagnoses. the differentials for this disease are bovine viral diarrhea/mucosal disease, bovine respiratory disease complex, infectious bovine rhinotracheitis, bluetongue, vesicular stomatitis, and foot-and-mouth disease. causes of encephalitis, such as bovine spongiform encephalopathy and rabies, should be considered. in africa, rinderpest is also a differential. other differentials are arsenic toxicity and chlorinated naphthalene toxicity. in north america, sheep, as well as cattle that have been either exposed or that have survived the disease, are reservoirs for outbreaks in other cattle. if there is concern regarding presence of the virus, animals should be screened serologically; once an animal has been infected, it remains infected indefinitely. lambs can be free of the infection if removed from the flock at weaning. the virus is very fragile outside of host's cells and will not survive in the environment for more than a few hours. lobes; and hematological findings indicate anemia and leukocytosis. the rare neurological signs include flexion of fetlock and pastern joints, tremors of facial muscles, progressive paresis and paralysis, depression, and prostration. death occurs in weeks to months. the disease can be serologically diagnosed with agar gel immunodiffusion (agid) tests, virus isolation, serum neutralization, complement fixation, and enzyme-linked immunosorbent assay (elisa) tests. sixty-eight percent of sheep in some states have been infected with the virus (radostits et al., ) . it is transmitted horizontally via inhalation of aerosolized virus particles and vertically between the infected dam and fetus. in addition, transmission through the milk or colostrum is considered common (knowles, ) . necropsy findings. lesions are observed in lungs, mammary glands, joints, and the brain. pulmonary adhesions, ventral lung lobe consolidation, bronchial lymph node enlargement, mastitis, and degenerative arthritis are visualized grossly. meningeal edema, thickening of the choroid plexus, and foci of leukoencephalomalacia are seen in the central nervous system (cns). histologically, interalveolar septal thickening, lymphoid hyperplasia, histiocyte and fibrocyte proliferation, and squamous epithelial changes are seen in the lungs. meningitis, lymphoid hyperplasia, demyelination, and glial fibrosis are seen in the cns. affected and any exposed animals should be isolated from healthy animals. there is no specific treatment for mcf; supportive treatment may improve recovery rates. corticosteroids may be useful. etiology. an rna virus in the lentivirus group of the retroviridae family causes ovine progressive pneumonia (opp), or maedi/visna. maedi refers to the progressive pneumonia presentation of the disease; visna refers to the central nervous system disease, which is reported predominantly in iceland. visna has been reported in goats but may have been due to caprine arthritis encephalitis infection. clinical signs and diagnosis. opp is a viral disease of adult sheep characterized by weakness, unthriftiness, weight loss, and pneumonia (pepin et al., ; de la concha bermejillo, ) . clinically, animals exhibit signs of progressive pulmonary disease after an extremely long incubation period of up to years. respiratory rate and dyspnea gradually increase as the disease progresses. the animal continues to eat throughout the disease; however, animals progressively lose weight and become weak. additionally, mastitis is a common clinical feature. thoracic auscultation reveals consolidation of ventral lung pathogenesis. the virus has a predilection for the lungs, mediastinal lymph nodes, udder, spleen, joints, and rarely the brain. after initial infection, the virus integrates into the dna of mature monocytes and persists as a provirus. later in the animal's life, infected monocytes mature as lung (and other tissue) macrophages and establish active infection. the virus induces lymphoproliferative disease, histiocyte and fibrocyte proliferation in the alveolar septa, and squamous metaplasia. pulmonary alveolar and vascular changes impinge on oxygen and carbon dioxide exchange and lead to serious hypoxia and pulmonary hypertension. secondary bacterial pneumonia may contribute to the animal's death. pulmonary adenomatosis is the differ-prevention and control. isolating or removing infected animals can prevent the disease. facilities and equipment should also be disinfected. ii. proliferative stomatitis (bovine papular stomatitis) etiology. a parapoxvirus is the causative agent of bovine papular stomatitis. this virus is considered to be closely related to the parapoxvirus that causes contagious ecthyma and pseudocowpox. it is also a zoonotic disease. the disease is not considered of major consequence, but high morbidity and mortality may be seen in severe outbreaks. in addition, lesions are comparable in appearance to those seen with vesicular stomatitis, bovine viral diarrhea virus, and foot-and-mouth disease. the disease occurs worldwide. clinical signs and diagnosis. raised red papules or erosions or shallow ulcers on the muzzle, nose, oral mucosa (including the hard palate), esophagus, and rumen of younger cattle are the most common findings. in some outbreaks, the papules will be associated with ulcerative esophagitis, salivation, diarrhea, and subsequent weight loss. lesions persist or may come and go over a span of several months. morbidity among herds may be %. mortalities are rare. bovine papular stomatitis is associated with "rat tail" in feedlot cattle. animals continue to eat and usually do not show a fever. no lesion is seen on the feet. the infection may also be asymptomatic. diagnosis is based on clinical signs, histological findings, and viral isolation. epizootiology and transmission. cattle less than year of age are most commonly affected, and disease is rare in older cattle. transmission is by animal-to-animal contact. necropsy findings. raised papules may be found around the muzzle and mouth and involve the mucosa of the esophagus and rumen. histologically, epithelial cells will show hydropic degeneration and hyperplasia of the lamina propria. eosinophilic inclusions will be in the cytoplasm of infected epithelial cells. pathogenesis. following exposure to the virus, erythematous macules most commonly appear on the nares, followed by the mouth. these become raised papules within a day, regressing after days to weeks; the lesions that remain will be persistent yellow, red, or brown spots. some infections may recur or persist, with animals showing lesions intermittently or continuously over several months. differential diagnosis. pseudocowpox, vesicular stomatitis, foot-and-mouth disease, and bovine viral diarrhea virus infection are the differentials for this disease. the differential for the "rat tail" clinical sign is sarcocystis infection. there is no vaccine available for bovine papular stomatitis. because of the similarity of this virus to the parapoxvirus of contagious ecthyma, it is important to be aware of the persistence in the environment and susceptibility of younger cattle. vaccination using the local strain, and the skin scarification technique for off, have been protective. handlers should wear gloves and protective clothing. treatment. cattle usually will not require extensive nursing care, but lesions with secondary bacterial infections should be treated with antibiotics. their hands at sites of contact with lesions of cattle. iii. pseudocowpox etiology. pseudocowpox is a worldwide cattle disease caused by a parapoxvirus related to the causative agents of contagious ecthyma and bovine papular stomatitis (see sections iii,a, ,m and iii,a, ,q,ii). lesions are confined to the teats. this is also a zoonotic disease. clinical signs and diagnosis. minor lesions are usually confined to the teats. these are distinctive because of the ring-or horseshoe-shaped scab that develops after days. additional lesions sometimes develop on the udder, the medial aspect of the thighs, and the scrotum. the teat lesions may predispose to mastitis. etiology. pulmonary adenomatosis is a rare but progressive wasting disease of sheep, with worldwide distribution. pulmonary adenomatosis is caused by a type d retrovirus antigenically related to the mason-pfizer monkey virus. jaagsiekte was the designation when the disease was described originally in south africa. progressive respiratory signs such as dyspnea, rapid respiration, and wasting. the disease is diagnosed by these chronic clinical signs and histology. epizootiology and transmission. the disease is transmitted by aerosols. body fluids of viremic animals, such as milk, blood, saliva, tears, semen, and bronchial secretions, will contain the virus or cells carrying the virus. necropsy. the adenomas and adenocarcinomas will be small firm lesions distributed throughout the lungs. the adenocarcinomas metastasize to regional lymph nodes. pathogenesis. as with ovine progressive pneumonia (opp), the incubation period is up to years long. adenocarcinomatous lesions arising from type ii alveolar epithelial cells may be discrete or confluent and involve all lung lobes. with or is a differential diagnosis for opp. etiology. cutaneous papillomatosis is a very common disease in cattle and is much less common among sheep and goats. the disease is a viral-induced proliferation of the epithelium of the neck, face, back, and legs. these tumors are caused by a papillomavirus (dna virus) of the papovaviridae family, and the viruses are host-specific and often body site-specific. most are benign, although some forms in cattle and one form in goats can become malignant. in cattle, the site specificity of the papillomavirus strains are particularly well recognized. designations of the currently recognized bovine papillomavirus (bpv) types are bpv- through bpv- . clinical signs and diagnosis. the papillomas may last up to months and are seen more frequently in younger animals. lesions have typical wart appearances and may be single or multiple, small ( mm) or very large ( mm). the infections will generally be benign, but pain will be evident when warts develop on occlusal surfaces or within the gastrointestinal tract. in addition, when infections are severe, weight loss may occur. when warts occur on teats, secondary mastitis may develop. in cattle, bpv- and bpv- cause fibropapillomas on teats and penises or on head, neck, and dewlap, respectively. bpv- causes flat warts that occur in all body locations, b pv- causes warts in the gastrointestinal tract, and b pv- causes small white warts (called rice-grain warts) on teats. warts caused by bpv- and bpv- do not regress spontaneously. prognosis in cattle is poor only when papillomatosis involves more than % of the body surface. in sheep, warts are the verrucous type. the disease is of little consequence unless the warts develop in an area that causes dis-comfort or incapacitation such as between the digits, on the lips, or over the joints. in adult sheep, warts may transform to squamous cell carcinoma. in goats, the disease is rare, and the warts are also of the verrucous type and occasionally may develop into squamous cell carcinoma. warts on goat udders tend to be persistent. diagnosis is made by observing the typical proliferative lesions. epizootiology and transmission. older animals are less sensitive to papillomatosis than young animals, although immunosupressed animals of any age may develop warts as the result of harbored latent infections. the virus is transmitted by direct and indirect (fomite) contact, entering through surface wounds and sites such as tattoos. pathogenesis. the incubation period ranges from to months. the virus induces epidermal and fibrous tissue proliferation, often described as cauliflower-like skin tumors. the disease is generally self-limiting. differential diagnosis. in sheep and goats, differentials include contagious ecthyma, ulcerative dermatosis, strawberry foot rot, and sheep and goat pox. for cattle) or autogenous vaccines must be used with a recognition that papovavirus strains are host-specific and that immunity from infection or vaccination is viral-type-specific. autogenous vaccines are generally considered more effective. some vaccine preparations are effective at prevention but not treatment of outbreaks. viricidal products are recommended for disinfection of contaminated environments. minimizing cutaneous injuries and sanitizing equipment (tattoo devices, dehorners, ear taggers, etc.) in a virucidal solution between uses are also recommended preventive and control measures. halters, brushes, and other items may also be sources of virus. treatment. warts will often spontaneously resolve as immunity develops. in severe cases or with flockwide or herdwide problems, affected animals should be isolated from nonaffected animals, and premises disinfected. warts can be surgically excised and autogenous vaccines can be made and administered to help prevent disease spread. cryosurgery with liquid nitrogen or dry ice has also proven to be successful for wart removal. topical agents such as podophyllin (various formulations) and dimethyl sulfoxide may be applied to individual lesions once daily until regression. etiology. pseudorabies is an acute encephalitic disease caused by a neurotropic alphaherpesvirus, the porcine herpesvirus . one serotype is recognized, but strain differences exist. the disease has worldwide distribution. it is a primarily a clinical dis-ease of cattle, with less frequent reports (but no less severe clinical manifestations) in sheep and goats. during the rapid course of this usually fatal disease. at the site of virus inoculation or in other locations, abrasions, swelling, intense pruritus, and alopecia are seen. pruritus will not be asymmetric. animals will also become hyperthermic and will vocalize frantically. other neurological signs range from hoof stamping, kicking at the pruritic area, salivation, tongue chewing, head pressing and circling, to paresthesia or hyperesthesia, ataxia, and conscious proprioceptive deficits. nystagmus and strabismus are also seen. animals will be fearful or depressed, and aggression is sometimes seen. recumbency and coma precede death. diagnostic evidence includes clinical findings; virus isolation from nasal or pharyngeal secretions or postmortem tissues; and histological findings at necropsy. serology of affected animals is not productive, because of the rapid course. if swine are housed nearby, or if swine were transported in the same vehicles as affected animals, serological evaluations are worthwhile from those animals. epizootiology and transmission. swine are the primary hosts for pseudorabies virus, but they are usually asymptomatic and serve as reservoirs for the virus. the infection can remain latent in the trigeminal ganglion of pigs and recrudesce during stressful conditions. other animals are dead-end hosts. the unprotected virus will survive only a few weeks in the environment but may remain viable in meat (including carcasses) or saliva and will survive outside the host, in favorable conditions, in the summer for several weeks and the winter for several months. transmission is by oral, intranasal, intradermal, or subcutaneous introduction of the virus. when the virus is inhaled, the clinical signs of pruritus are less likely to be seen. transmission can also be by inadvertent exposure (e.g., contaminated syringes) of ruminants to the modified live vaccines developed for use in swine. spread between infected ruminants is a less likely means of transmission, because of the relatively short period of virus shedding. transport vehicles used for swine may also be sources of the virus. raccoons are believed to be vectors of the virus. horses are resistant to infection. there is no pathognomonic gross lesion. definitive histologic findings include severe, focal, nonsuppurative encephalitis and myelitis. eosinophilic intranuclear inclusion bodies (cowdry type a) may be present in some affected neurons. methods such as immunofluorescence and immunoperoxidase staining can be used to show presence of the porcine herpesvirus . pathogenesis. the incubation period is - hr and duration of the illness is - hr. the longest duration is seen in animals with pruritus around the head. differential diagnoses. differentials for the neurologic signs of pseudorabies infection include rabies, polioencephalomalacia, salt poisoning, meningitis, lead poisoning, hypomagnesemia, and enterotoxemia. those for the intense pruritus include psoroptic mange and scrapie in sheep, sarcoptic mange, and pediculosis. prevention and control. pseudorabies is a reportable disease in the united states, where a nationwide eradication program exists; states are rated regarding status. effective disinfectants include sodium hypochlorite ( % solution), formalin, peracetic acid, tamed iodines, and quaternary ammonium compounds. five minutes of contact time is required, and then surfaces must be rinsed. other disinfectant methods for viral killing include hr of formaldehyde fumigation, or min of ultraviolet light. transport vehicles should be cleaned and disinfected between species. serological screening for pseudorabies of swine housed near ruminants is essential. there is no treatment, and most affected ani-research complications. swine housed close to research ruminants should be serologically screened prior to purchase, and all transport vehicles should be cleaned and disinfected between loads of large animals. humans have been reported to seroconvert. the porcine herpesvirus shares antigens with the infectious bovine rhinotracheitis virus. etiology. rabies is a sporadic but fatal, acute viral disease affecting the central nervous system. the rabies virus is a neurotropic rna virus of the lyssavirus genus and the rhabdoviridae family. sheep, goats, and cattle are susceptible. the zoonotic potential of this virus must be kept in mind at all times when handling moribund animals with neurological signs characteristic of the disease. rabies is endemic in many areas of the world and within areas of the unites states. this is a reportable disease in north america. clinical findings and diagnosis. animals generally progress through three phases: prodromal, excitatory, and paralytic. many signs in the different species during these stages are nonspecific, and forms of the disease are also referred to as dumb or furious. during the short prodromal phase, animals are hyperthermic and apprehensive. animals progress to the excitatory phase, during which they refuse to eat or drink and are active and aggressive. repeated vocalizations, tenesmus, sexual excitement, and salivation occur during this phase. the final paralytic stage, with recumbency and death, occurs over several hours to days. this paralytic stage is common in cattle, and animals may simply be found dead. the clinical course is usually - days. diagnosis is based on clinical signs, with a progressive and fatal course. confirmation presently is made with the fluorescent antibody technique on brain tissue. epizootiology and transmission. the rabies virus is transmitted via a bite wound inflicted by a rabid animal. cats, dogs, raccoons, skunks, foxes, wild canids, and bats are the common disease vectors in north america. virus is also transmitted in milk and aerosols. necropsy findings. few lesions are seen at necropsy. many secondary lesions from manic behaviors during the course of disease may be evident. histological findings will include nonsuppurative encephalitis. negri bodies in the cytoplasm of neurons of the hippocampus and in purkinje's cells are pathognomonic histologic findings. pathogenesis. after exposure, the incubation period is variable, from weeks to several months, depending on the distance that the virus has to travel to reach the central nervous system. the rabies virus proliferates locally, gains access to neurons by attaching to acetylcholine receptors, via a viral surface glycoprotein, migrates along sensory nerves to the spinal cord and brain, and then descends via cranial nerves (trigeminal, facial, olfactory, glossopharyngeal) to oral and nasal cavity structures (i.e., salivary glands). the fatal outcome is currently believed to be multifactorial, related to anorexia, respiratory paralysis, and effects on the pituitary. differential diagnosis. rabies should be included on the differential list when clinical signs of neurologic disease are evident. other differentials for ruminants include herpesvirus encephalitis, thromboemobolic meningoencephalitis, nervous ketosis, grass tetany, and nervous cocciodiosis. prevention and control. vaccines approved for use cattle and sheep are commercially available and contain inactivated virus; there is not one available in the united states for goats. ruminants in endemic areas, such as the east coast of the united states, should be routinely vaccinated. any animals housed outside that may be exposed to rabid animals should be vaccinated. vaccination programs generally begin at months of age, with a booster at year of age and then annual or triennial boosters. awareness of the current rabies case reports for the region and wildlife reservoirs, however, is important. monitoring for and exclusion of wildlife from large-animal facilities are worthwhile preventive measures. the virus is fragile and unstable outside of a host animal. research complications. aerosolized virus is infective. personal protective equipment, including gloves, face mask, and eye shields, must be worn by individuals handling animals that are manifesting neurological disease signs. bovine spongiform encephalopathy, a transmissible spongiform encephalopathy (tse), is not known to occur in the united states, where since it has been listed as a reportable disease. the profound impact of this disease on the cattle industry in great britain during the past two decades is well known. the disease may be caused by a scrapielike (prion) agent. it is believed that the source of infection for cattle was feedstuff derived from sheep meat and bonemeal that had been inadequately treated during processing. the incubation period of years, the lack of detectable host immune response, the debilitating and progressive neurological illness, and the pathology localized to the central nervous system are characteristics of the disease, and are is comparable to the characteristics of other tse diseases such as scrapie, which affects sheep and goats. in addition, the infectious agent is extremely resistant to dessication and disinfectants. confirmation of disease is by histological examination of brain tissue collected at necropsy; the vacuolation that occurs during the disease will be symmetrical and in the gray matter of the brain stem. molecular biology techniques, such as western blots and immunohistochemistry, may also be used to identify the presence of the prion protein. differentials include many infectious or toxic agents that affect the bovine nervous and musculoskeletal systems, such as rabies, listeriosis, and lead poisoning. metabolic disorders such as ketosis, milk fever, and grass tetany are also differentials. there is no vaccine or treatment. prevention focuses on import regulations and not feeding ruminant protein to ruminants; recent usda regulations prohibit feeding any mammalian proteins to ruminants. etiology. scrapie is a sporadic, slow, neurodegenerative disease caused by a prion. scrapie is a reportable disease. it is much more common in sheep than in goats. the disease is similar to transmissible mink encephalopathy, kuru, creutzfeldt-jakob disease, and bovine spongiform encephalopathy (mad cow disease). prions are nonantigenic, replicating protein agents. clinical signs and diagnosis. during early clinical stages, animals are excitable and hard to control. tremors of head and neck muscles, as well as uncoordinated movements and unusual "bunny-hopping" gaits are observed. in advanced stages of the disease, animals experience severe pruritus and will self-mutilate while rubbing on fences, trees, and other objects. blindness and abortion may also be seen. morbidity may reach % within a flock. most animals invariably die within - weeks; some animals may survive months. in goats, the disease is also fatal. pruritus is generally less severe but may be localized. a wide range of clinical signs have also been noted in goats, including listlessness, stiffness or restlessness, or behavioral changes such as irritability, hunched posture, twitching, and erect tail and ears. as with sheep, the disease gradually progresses to anorexia and debilitation. diagnosis can be made by clinical signs and histopathological lesions. a newer diagnostic test in live animals is based on sampling from the third eyelid. tests for genetic resistance or susceptibility require a tube of edta blood and are reasonably priced. epizootiology and transmission. the suffolk breed of sheep tends to be especially susceptible. scrapie has also been reported in several other breeds, including cheviot, dorset, hampshire, corriedale, shropshire, merino, and rambouillet. it is believed that there is hereditary susceptibility in these breeds. targhees tend to be resistant. genomic research indicates there are two chromosomsal sites governing this trait; these sites are referred to codons (q, r, or h genes can be present) and (a or v genes can be present). of the five genes, r genes appear to confer immunity to clinical scrapie in suffolks in the united states. affected suffolks in the united states that have been tested have been aa qq. the disease is also enzootic is many other countries. the disease tends to affect newborns and young animals; however, because the incubation period tends to range from to years, adult animals display signs of the disease. scrapie is transmitted horizontally by direct or indirect contact; nasal secretions or placentas serve as sources of the infectious agent. vertical transmission is questioned, and transplacental transmission is considered unlikely. necropsy findings. at necropsy, no gross lesion is observed. histopathologically, neuronal vacuolization, astrogliosis, and spongiform degeneration are visualized in the brain stem, the spinal cord, and especially the thalamus. inflammatory lesions are not seen. pathogenesis. replication of the prions probably occurs first in lymphoid tissues throughout the host's body and then progresses to neural tissue. differential diagnosis. in sheep and goats, depending on the speed of onset, differentials for the pruritus include ectoparasites, pseudorabies, and photosensitization. prevention and control. if the disease diagnosed in a flock, quarantine and slaughter, followed by strict sanitation, are usually required. the u.s. department of agriculture has approved the use of % sodium hydroxide as the only disinfectant for sanitation of scrapie-infected premises. prions are highly resistant to physicochemical means of disinfection. artificial insemination or embryo transfer has been shown to decrease the spread of scrapie (linnabary et al., ) . research complications. as noted, this is a reportable disease. stringent regulations exist in the united states regarding importation of small ruminants from scrapie-infected countries. etiology. vesicular stomatitis (vs) is caused by the vesicular stomatitis virus (vsv), a member of the rhabdoviridae. three serotypes are recognized: new jersey, indiana, and isfahan. the new jersey and indiana strains cause sporadic disease in cattle in the united states. the disease is rare in sheep. clinical signs and diagnosis. adult cattle are most likely to develop vs. fever and development of vesicles on the oral mucous membranes are the initial clinical signs. lesions on the teats and interdigital spaces also develop. the vesicles progress quickly to ulcers and erosions. the animal's tongue may be severely involved. anorexia and salivation are common. weight loss and decreased milk production are noticeable. morbidity will be high in an outbreak, but mortality will be low to nonexistent. diagnostic work should be initiated as soon as possible to distinguish this from foot-and-mouth disease. diagnosis is based on analysis of fluid, serum, or membranes associated with the vesicles. virus isolation, enzyme-linked immunosorbent assay (elisa), competitive elisa (celisa), complement fixation, and serum neutralization are used for diagnosis. epizootiology and transmission. this disease occurs in several other mammalian species, including swine, horses, and wild ruminants. vsv is an enveloped virus and survives well in different environmental conditions, including in soil, extremes of ph, and low temperatures. outbreaks of vs occur sporadically in the united states, but it is not understood how or in what species the virus survives between these outbreaks. incidence of disease decreases during colder seasons. equipment, such as milking machines, contaminated by secretions is a mechanical vector, as are human hands. transmission may also be from contaminated water and feed. transmission is also believed to occur by insects (blackflies, sand flies, and culicoides) that may simply be mechanical vectors. it is believed that carrier animals do not occur in this disease. necropsy. it is rare for animals to be necropsied as the result of this disease. typical vesicular lesion histology is seen, with ballooning degeneration and edema. there is no inclusion body formation. pathogenesis. lesions often begin within hr after exposure. the virus invades oral epithelium. injuries or trauma in any area typically affected, such as mouth, teats, or interdigital areas, will increase the likelihood of lesions developing there. animals will develop a long-term immunity; this immunity can be overwhelmed, however, by a large dose of the virus. differential diagnosis. foot-and-mouth disease lesions are identical to vs lesions. other differentials in cattle include bovine viral diarrhea, malignant catarrhal fever, contagious ecthyma, photosensitization, trauma, and caustic agents. prevention and control. quarantine and restrictions on shipping infected animals or animals from the premises housing affected animals are required in an outbreak. vaccines are available for use in outbreaks and have decreased the severity of lesions. phenolics, quaternaries, and halogens are effective for inactivating and disinfecting equipment and facilities. treatment. affected animals should be segregated from the rest of the herd and provided with separate water and softened feed. these animals should be cared for after unaffected animals. any feed or water contaminated by these animals should not be used for other animals; contaminated equipment should be disinfected. topical or systemic antibiotics control secondary bacterial infections. cases of mastitis secondary to teat lesions must be treated as necessary. any abrasive materials that could cause further trauma to the animals should be removed. research complications. animals developing vesicular lesions must be reported promptly to eliminate the possibility of an outbreak of foot-and-mouth disease. personal protective equipment, especially gloves, should be worn when handling any animals with vesicular lesions. vsv causes a flulike illness in humans. x. viral diarrhea diseases i. ovine. rotavirus, of the family reoviridae, induces an acute, transient diarrhea in lambs within the first few weeks of life. four antigenic groups (a-d) have been identified by differences in capsid antigens vp and vp . primarily group a, but also groups b and c, have been isolated from sheep. the disease is characterized by yellow, semifluid to watery diarrhea occurring - days after infection. the disease can progress to dehydration, anorexia and weight loss, acidosis, depression, and occasionally death. the virus is ingested with contaminated feed and water and selectively infects and destroys the enterocytes at the tips of the small intestinal villi. the villi are replaced with immature cells that lack sufficient digestive enzymes; osmotic diarrhea results. virus may remain in the environment for several months. the disease is diagnosed by virus isolation, electron microscopy of feces, fecal fluorescent antibody, fecal elisa tests (marketed tests generally detect group a rotavirus), and fecal latex agglutination tests. rotavirus diarrhea is treated by supportive therapy, including maintaining hydration, electrolyte, and acid-base balance. a rotavirus vaccine is available for cattle; because of cross-species immunity, oral administration of high-quality bovine colostrum from vaccinated cows to infected sheep may be helpful ("current veterinary therapy," ). coronavirus, of the family coronaviridae, produces a more severe, long-lasting disease when compared with rotavirus. clinical signs are similar to above, although the incubation period tends to be shorter ( - hr), and animals exhibit less anorexia than those with rotavirus. additionally, mild respiratory disease may be noted (janke, ) . like rotavirus, coronavirus also destroys enterocytes of the villus tips. the virus can be visualized with electron microscopy. treatment is supportive; close consideration of hydration and acid-base status is essential. bovine vaccines are available. ii. caprine. rotavirus, coronavirus, and adenoviruses affect neonatal goats; however, little has been documented on the pathology and significance of these agents in this age group. it appears that bacteria play a more important role in neonatal kid diarrheal diseases then in neonatal calf diarrheas. iii. bovine. rotaviruses, coronaviruses, parvoviruses, and bovine viral diarrhea virus (bvdv) are associated with diarrheal disease in calves. each pathogen multiplies within and destroys the intestinal epithelial cells, resulting in villous atrophy and clinical signs of diarrhea (soft to watery feces), dehydration, and abdominal pain. these viral infections may be complicated by parasitic infections (e.g., cryptosporidium, eimeria) or bacterial infections (e.g., escherichia coli, salmonella, campylobacter). treatment is aimed at correcting dehydration, electrolyte imbalances, and acidosis; cessation of milk replacers and administration of fluid therapy intravenously and by stomach tube may be necessary, depending on the presence of suckle reflex and the condition of the animals. diagnosis is by immunoassays available for some viruses, viral culture, exclusion or identification of presence of other pathogens (by culture or fecal exams), and microscopic examination of necropsy specimens. prevention focuses on calves suckling good-quality colostrum; other recommendations for calf care are in section ii,b, . combination vaccine products are available for immunizing dams against rotavirus, coronavirus, and enterotoxigenic e. coli. additional supportive care for calves includes providing calves with sufficient energy and vitamins until milk intake can resume. rotaviruses of serogroup a are the most common type in neonatal calves; -to -day old calves are typically affected, but younger and older animals may also be affected. the small intestine is the site of infection. antirotavirus antibody is present in colostrum, and onset of rotavirus diarrhea coincides with the decline of this local protection. transmission is likely from other affected calves and asymptomatic adult carriers. the diarrhea is typically a distinctive yellow. colitis with tenesmus, mucus, and blood may be seen. this virus may be zoonotic. coronaviruses are commonly associated with disease in calves during the first month of life, and they infect small-and large-intestinal epithelial cells. the virus infection may extend to mild pneumonia. transmission is by infected calves and also by asymptomatic adult cattle, including dams excreting virus at the time of parturition. calves that appear to have recovered continue to shed virus for several weeks. parvovirus infections are usually associated with neonatal calves. b vdv infections also are seen in neonates and also affect many systems and produce other clinical signs and syndromes that are described in section iii,a, ,e. iv. winter dysentery. winter dysentery is an acute, winterseasonal, epizootic diarrheal disease of adult cattle, although it has been reported in -month-old calves. the etiology has not yet been defined, but a viral pathogen is suspected. coronavirus-like viral particles have been isolated from cattle feces, either the same as or similar to the coronavirus of calf diarrhea. outbreaks typically last a few weeks, and first-lactation or younger cattle are affected first, with waves of illness moving through a herd. individual cows are ill for only a few days. the incubation period is estimated at - days. the outbreaks of disease are often seen in herds throughout the local area. clinical signs include explosive diarrhea, anorexia, depression, and decreased production. the diarrhea has a distinctive musty, sweet odor and is light brown and bubbly, but some blood streaks or clots may be mixed in with the feces. animals will become dehydrated quickly but are thirsty. respiratory symptoms such as nasolacrimal discharges and coughing may develop. recovery is generally spontaneous. mortalities are rare. diagnosis is based on characteristic patterns of clinical signs, and elimination of diarrheas caused by parasites such as coccidia, bacterial organisms such as salmonella or mycobacterium paratuberculosis, and viruses such as b vdv. pathology is present in the colonic mucosa, and necrosis is present in the crypts. etiology. chlamydia psittaci is a nonmotile, obligate, intracytoplasmic, gram-negative bacterium. clinical signs. enzootic abortion in sheep and goats is a contagious disease characterized by hyperthermia and late abortion or by birth of stillborn or weak lambs or kids (rodolakis et al., ) . the only presenting clinical sign may be serosanguineous vulvar discharges. other animals may present with arthritis or pneumonia. infection of animals prior to about days of gestation results in abortion, stillbirths, or birth of weak lambs. infection after days results in potentially normal births, but the dams or offspring may be latently infected. latently infected animals that were infected during their dry period may abort during the next pregnancy. ewes or does generally only abort once, and thus recovered animals will be immune to future infections. and specific antigens associated with the cell surface. the group antigen is common among all chlamydia; the specific antigen is common to related subgroups. two subgroups are recognized, one that causes eae and one that causes polyarthritis and conjunctivitis. the disease is transmitted by direct contact with infectious secretions such as placental, fetal, and uterine fluids or by indirect contact with contaminated feed and water. necropsy. placental lesions include intercotyledonary plaques and necrosis and cotyledonary hemorrhages. histopathological evidence of leukocytic infiltration, edema, and necrosis is found throughout the placentome. fetal lesions include giant-cell accumulation in mesenteric lymph nodes and lymphohistiocytic proliferations around the blood vessels within the liver. diagnosis is based on clinical signs and laboratory (serological or histopathological) identification of the organism. impression smears in placental tissues stained with giemsa, gimenez, or modified ziehl-neelsen can provide preliminary indications of the causative agent. immunofluorescence, enzyme-linked immunosorbent assay (elisa), and polymerase chain reaction (pcr) methods also aid in diagnosis. differential diagnosis. q fever will be the major differential for late-term abortion and necrotizing placentitis. campylobacter and toxoplasma should also be considered for late-term abortion. treatment. animals may respond to treatment with oxytetracycline. abortions are prevented through administration of a commercial vaccine, but the vaccine will not eliminate infections. this is a sheep vaccine and should be administered before breeding and annually to at least the young females entering the breeding herd or flock. research complications. in addition to losses or compromise of research animals, pregnant women should not handle aborted tissues. etiology. chlamydia psittaci is a nonmotile, obligate intracellular, gram-negative bacterium. chlamydial polyarthritis is an acute, contagious disease characterized by fever, lameness (bulgin, ) , and conjunctivitis (see section iii,a, ,c) in growing and nursing lambs. clinical signs. clinically, animals will appear lame on one or all legs and in major joints, including the scapulohumeral, humeroradioulnar, coxofemoral, femorotibial, and tibiotarsal joints. lambs may be anorexic and febrile. animals frequently also exhibit concurrent conjunctivitis. the disease usually resolves in approximately weeks. joint inflammation usually resolves without causing chronic articular changes. epizootiology and transmission. the disease is transmitted to susceptible animals by direct contact as well as by contaminated feed and water. the organism penetrates the gastrointestinal tract and migrates to joints and synovial membranes as well as to the conjunctiva. the organism causes acute inflammation and associated fibrinopurulent exudates. necropsy findings. lesions are found in joints, tendon sheaths, conjunctiva, and lungs. pathological sites will be edematous and hyperemic, with fibrinous exudates but without articular changes. lesions will be infiltrated with mononuclear cells. lung lesions include atelectasis and alveolar inspissation. diagnosis is based on clinical signs. synovial taps and subsequent smears may allow the identification of chlamydial inclusion bodies. treatment. animals respond to treatment with parenteral oxytetracycline. etiology. chlamydia psittaci, a nonmotile, obligate intracellular, gram-negative bacterium, is the most common cause of infectious keratoconjunctivitis in sheep. chlamydia and mycoplasma are considered to be the most common causes of this disease in goats. chlamydial conjunctivitis is not a disease of cattle. clinical signs. infectious keratoconjunctivitis is an acute, contagious disease characterized in earlier stages by conjunctival hyperemia, epiphora, and edema and in later stages by, corneal edema, ulceration, and opacity. perforation may result from the ulceration. animals will be photophobic. in less severe cases, corneal healing associated with fibrosis and neovascularization occurs in - days. lymphoid tissues associated with the conjunctiva and nictitating membrane may enlarge and prolapse the eyelids. morbidity may reach - %. bilateral and symmetrical infections characterize most outbreaks. relapses may occur. other concurrent systemic infections may be seen, such as polyarthritis or abortion in sheep and polyarthritis, mastitis, and uterine infections in goats. epizootiology and transmission. direct contact, and mechanical vectors such as flies easily spread the organism. necropsy. if the chlamydial or mycoplasmal agents are suspected, diagnostic laboratories should be contacted for recommendations regarding sampling. conjunctival smears are also useful. pathogenesis. the pathogen penetrates the conjunctival epithelium and replicates in the cytoplasm by forming initial and elementary bodies. the infection moves from cell to cell and causes an acute inflammation and resultant purulent exudate. the chlamydial organism may penetrate the bloodstream and migrate to the opposite eye or joints, leading to arthritis. diagnosis is suggested by the clinical signs. cytoplasmic inclusions observed on conjunctival scrapings and immunofluorescent techniques help confirm the diagnosis. differential diagnosis. nonchlamydial keratoconjunctivitis also occurs in sheep and goats. the primary agents involved include mycoplasma conjunctiva, m. agalactiae in goats, and branhamella (neisseria) ovis. a less common differential for sheep and cattle is listeria monocytogenes. other differentials include eye worms, trauma, and foreign bodies such as windblown materials (pollen, dust) and poor-quality hay; these latter irritants and stress may predispose the animals' eyes to the infectious agents. should be minimized whenever possible. quarantine of new animals and treatment, if necessary, before introduction into the flock or herd are important measures. shade should be provided for all animals. treatment. the infections can be self-limiting in - weeks without treatment. treatment consists of topical application of tetracycline ophthalmic ointments. systemic or oral oxytetracycline treatments have been used with the topical treatment. atropine may be added to the treatment regimen when uveitis is present. shade should be provided. a. protozoa i. anaplasmosis etiology. anaplasmosis is an infectious, hemolytic, noncontagious, transmissible disease of cattle caused by the protozoan anaplasma marginale. anaplasma is a member of the anaplasmatacae family within the order rickettsiales. in sheep and goats, the disease is caused by a. ovis and is an uncommon cause of hemolytic disease. anaplasmosis has not been reported in goats in the united states. some controversy exists regarding the classification. most recently it is classified as a protozoal disease because of similarities to babesiosis. it has also been classified as a rickettsial pathogen. this summary addresses the disease in cattle with limited reference to a. ovis infections, but there are many similarities to the disease in cattle. clinical signs and diagnosis. acute anemia is the predominant sign in anaplasmosis, and fever coincides with parasitemia. weakness, pallor, lethargy, dehydration, and anorexia are the result of the anemia. four disease stagesnincubation, developmental, convalescent, and carriermare recognized. the incubation stage may be long, - weeks, and is characterized by a rise in body temperature as the infection moves to the next stage. most clinical signs occur during the -to -day developmental stage, with hemolytic anemia being common. death is most likely to occur at this stage or at the beginning of the convalescent stage. death may also occur from anoxia, because of the animal's inability to handle any exertion or stress, especially if treatment is initiated when severe anemia exists. reticulocytosis characterizes the convalescent stage, which may continue for many weeks. morbidity is high, and mortality is low. the carrier stage is defined as the time in the convalescent stage when the animal host becomes a reservoir of the disease, and anaplasma organisms and any parasitemia are not discernible. common serologic tests are the complement fixation test and the rapid card test. these become positive after the incubation phase and do not distinguish between the later three stages of disease. definitive diagnosis is made by clinical and necropsy findings. staining of thin blood smears with wright's or giemsa stain allows detection of basophilic, spherical a. marginale bodies near the red blood cell peripheries. evidence will most likely be found before a hemolytic episode. a negative finding should not eliminate the pathogen from consideration. epizootiology and transmission. the disease is common in cattle in the southern and western united states. anaplasma organisms are spread biologically or mechanically. mechanical transmission occurs when infected red blood cells are passed from one host to another on the mouthparts of seasonal biting flies. sometimes mosquitoes or instruments such as dehorners or hypodermic needles may facilitate transfer of infected red cells from one animal to another. biological transmission occurs when the tick stage of the organism is passed by dermacentor andersoni and d. occidentalis ticks. the carrier stage covers the time when discernible anaplasma organisms can be found on host blood smears. recovered animals serve as immune carriers and disease reservoirs. necropsy. pale tissues and watery, thin blood are typical findings. splenomegaly, hepatomegaly, and gallbladder distension are common findings. pathogenesis. the parasites infect the host's red blood cells, and acute hemolysis occurs during the parasites' developmental stage. the four stages of the parasite's life cycle are described above because these are closely linked to the clinical stages. differential diagnosis. the clinical disease closely resembles the protozoal disease babesiosis. whole organism) programs are not entirely effective, and vaccine should not be administered to pregnant cows. neonatal isoerythrolysis may occur because of the antierythrocyte antibodies stimulated by one vaccine product. vaccinated animals can still become infected and become carriers. the cattle vaccine has shown no efficacy in smaller ruminants, and there is no a. ovis vaccine. identifying carriers serologically and treating with tetracycline during and/or after vector seasons may be an option. removing carriers to a separate herd is also an approach. interstate movement of infected animals is regulated. treatment. oxytetracycline, administered once, helps reduce the severity of the infection during the developmental stage. other tetracycline treatment programs have been described to help control carriers. ii. babesiosis (red water, texas cattle fever, cattle tick fever) etiology. babesia bovis and ba. bigemina are protozoa that cause subclinical infections or disease in cattle. these are intraerythrocytic parasites. babesia bovis is regarded as the more virulent of the two organisms. this disease is not seen in the smaller ruminants in the united states. clinical signs and diagnosis. the more common presentation is liver and kidney failure due to hemolysis with icterus, hemoglobinuria, and fever. hemoglobinuria indicates a poor prognosis. acute encephalitis is a less common presentation and begins acutely with fever, ataxia, depression, deficits in conscious proprioception, mania, convulsions, and coma. the encephalitic form generally also has a poor prognosis. sudden death may occur. thin blood smears stained with giemsa will show babesia trophozoites at some stages of the disease, but lack of these cannot be interpreted as a negative. the trophozoites occur in a variety of shapes, such as piriform, round, or rod. complement fixation, immunofluorescent antibody, and enzyme immunoassay are the most favored of the available serologic tests. babesiosis is present on several continents, including the americas. in addition to domestic cattle, some wild ruminants, such as white-tailed deer and american buffalo, are also susceptible. bos indicus breeds have resistance to the disease and the tick vectors. innate resistance factors have been found in all calves. if infected, these animals will not show many signs of disease during the first year of life and will become carriers. stress can cause disease development. prevention and control. offspring of immune carriers resist infection up to months of age because of passive immunity. vector control and attention to hygiene are essential, such as between-animal rinsing in disinfectant of mechanical vectors such as dehorners. there is no entirely effective means, however, to prevent and control the disease. vaccination (killed necropsy findings. signs of acute hemolytic crisis are the most common findings, including hepatomegaly, splenomegaly, dark and distended gallbladder, pale tissues, thin blood, scattered hemorrhages, and petechiation. animals dying after a longer course of disease will be emaciated and icteric, with thin blood, pale kidneys, and enlarged liver. pathogenesis. the protozoon is transmitted by the cattle fever ticks boophilus annulatus, b. microplus, and b. decoloratus; these one-host ticks acquire the protozoon from infected animals. it is passed transovarially, and both nymph and adult ticks may transmit to other cattle. only b. ovis is transmitted by the larval stage. clinical signs develop about weeks after tick infestations or mechanical transmission but may develop sooner with the mechanical transmission. hemolysis is due to intracellular reproduction of the parasites and occurs intra-and extravascularly. in addition to the release of merozoites, proteolytic enzymes are also released, and these contribute to the clinical metabolic acidosis and anoxia. the development of the encephalitis form is believed to be the result of direct invasion of the central nervous system, disseminated intravascular coagulation, capillary thrombosis by the parasites and infarction, and/or tissue anoxia. differential diagnosis. in addition to anaplasmosis, other differentials for the hemolytic form of the disease are leptospirosis, chronic copper toxicity, and bacillary hemoglobinuria. several differentials in the united states for the encephalitic presentation include rabies, nervous system coccidiosis, polioencephalomalacia, lead poisoning, infectious bovine rhinotracheitis, salt poisoning, and chlorinated hydrocarbon toxicity. prevention and control. control or eradication of ticks and cleaning of equipment to prevent mechanical transmission, as noted in section iii,a, ,a,i, are important preventive measures. some vaccination approaches have been effective, but a commercial product is not available. treatment. supportive care is indicated, including blood transfusions, fluids, and antibiotics. medications such as diminazene diaceturate, phenamidine diisethionate, imidocarb diprionate, or amicarbalide diisethionate are most commonly used. treatment outcomes will be either elimination of the parasite or development of a chronic carrier state immune to further disease. research complications. this is a reportable disease in the united states. iii. coccidiosis etiology. coccidiosis is an important acute and chronic protozoal disease of ruminants. in young ruminants, it is characterized primarily by hemorrhagic diarrhea. adult ruminants may carry and shed the protozoa, but they rarely display clinical signs. intensive rearing and housing conditions and stress increase the severity of the disease in all age groups. coccidia are protozoal organisms of the phylum apicomplexa, members of which are obligatory intracellular parasites. there are at least reported species of coccidia in sheep, of which several are considered pathogenic: eimeria ashata, e. crandallis, and e. ovinoidalis (schillhorn van veen, ). at least species of eimeria have been recognized in the goat (foreyt, ) . eimeria ninakohlyakimovae, e. arloingi, and e. christenseni are regarded as the most pathogenic. eimeria bovis and e. zuernii (highly pathogenic), and e. auburnensis and e. alabamensis (moderately pathogenic), are among the species known to infect cattle. eimeria zuernii is more commonly seen in older cattle and is the agent of "winter coccidiosis." clinical signs and diagnosis. hemorrhagic diarrhea develops days to weeks after infection. fecal staining of the tail and perineum will be present. animals will frequently display tenesmus; rectal prolapses may also develop. anorexia, weight loss, dehydration, anemia, fever (infrequently), depression, and weakness may also be seen in all ruminants. the diarrhea is watery and malodorous and will contain variable amounts of blood and fibrinous, necrotic tissues. the intestinal hemorrhage may subsequently lead to anemia and hypoproteinemia. depending on the predilection of the coccidial species for small and/or large intestines, malabsorption of nutrients or water may occur, and electrolyte imbalances may be severe. concurrent disease with other enteropathogens may also be part of the clinical picture. in sheep, secondary bacterial infection with organisms such as fusobacterium necrophorum may ensue. young goats may die peracutely or suffer severe anemia from blood loss into the bowel. older goats may lose the pelleted form of feces. cattle may have explosive diarrhea and develop anal paralysis. the disease is usually diagnosed by history and clinical signs. numerous oocysts will frequently be observed in fresh fecal flotation (salt or sugar solution) samples as the diarrhea begins. laboratory results are usually reported as number of oocysts per gram of feces. coccidia seen on routine fecal evaluations reflect shedding, possibly of nonpathogenic species, without necessarily being indicative of impending or resolving mild disease. epkzootiology and transmission. as noted, coccidiosis is a common disease in young ruminants. in goats, young animals aged weeks to months are primarily affected, but isolated outbreaks in adults may occur after stressful conditions such as transportation or diet changes. coccidia are host-specific and also host cell-specific. the disease is transmitted via ingestion of sporulated oocysts. coccidial oocysts remain viable for long periods of time when in moist, shady conditions. necropsy. necropsies provide information on specific locations and severity of lesions that correlate with the species involved. ileitis, typhlitis, and colitis with associated necrosis and hemorrhage will be observed. mucosal scrapings will frequently yield oocysts. various coccidial stages associated with schizogony or gametogony may be observed in histopathological sections of the intestines. fibrin and cellular infiltrates will be found in the lamina propria. pathogenesis. this parasite has a complex life cycle in which sexual and asexual reproduction occurs in gastrointestinal enterocytes (speer, ) . the severity of the disease is correlated primarily with the number of ingested oocysts. specifics of life cycles vary with the species, and those characteristics contribute to the pathogenicity. in most cases, the disease is well established by the time clinical signs are seen. oocysts must undergo sporulation over a -to -day period in the environment. after ingestion of the sporulated oocysts, sporozoites are released and penetrate the intestinal mucosa and form schizonts. schizonts initially undergo replication by fission to form merozoites and eventually undergo sexual reproduction, forming new oocysts. the organisms cause edema and hyperemia; penetration into the lamina propria may lead to necrosis of capillaries and hemorrhage. differential diagnosis. differential diagnoses include the many enteropathogens associated with acute diarrhea in young ruminants: cryptosporidia, colibacilli, salmonella, enterotoxins, yersinia, viruses, and other intestinal parasites such as helminths. in cattle, for example, bovine viral diarrhea virus and helminthiasis caused by ostergia must be considered. management factors, such as dietary-induced diarrheas, are also differentials. in older animals, differentials in addition to stress are malnutrition, grain engorgement, and other intestinal parasitisms. prevention and control. good management practices will help prevent the disease. oocysts are resistant to disinfectants but are susceptible to dry or freezing conditions. proper sanitation of animal housing and minimizing overcrowding are essential. coccidiostats added to the feed and water are helpful in preventing the disease in areas of high exposure. treatment. affected animals should be isolated. on an individual basis, treatment should also include provision of a dry, warm environment, fluids, electrolytes (orally or intravenously), antibiotics (to prevent bacterial invasion and septicemia), and administration of coccidiostats. coccidiostats are preferred to coccidiocidals because the former allow immunity to develop. although many coccidial infections tend to be self-limiting, sulfonamides and amprolium may be used to aid in the treatment of disease. other anticoccidial drugs include decoquinate, lasalocid, and monensin; labels should be checked for specific approval in a species or specific indications. animals treated with amprolium should be monitored for development of secondary polioencephalomalacia. pen mates of affected animals should be considered exposed and should be treated to control early stages of infection. mechanisms of immunity have not been well defined but appear to be correlated with the particular coccidial species and their characteristics (for example, the extent of intracellular penetration). immunity may result when low numbers are ingested and there is only mild disease. immunity also may develop after more severe infections. iv. cryptosporidiosis etiology. cryptosporidium organisms are a very common cause of diarrhea in young ruminants. four cryptosporidium species have been described in vertebrates: c. baileyi and c. meleagridis in birds and c. parvum and c. muris in mammals. cryptosporidium parvum is the species affecting sheep (rings and rings, ) . debate continues regarding whether there are definite host-specific variants. clinical signs and diagnosis. cryptosporidiosis is characterized by protracted, watery diarrhea and debilitation. the diarrhea may last only - days or may be persistent and fatal. the diarrhea is watery and yellow, and blood, mucus, bile, and undigested milk may also be present. infected animals will display tenesmus, anorexia and weight loss, dehydration, and depression. in relapsing cases, animals become cachectic. overall, morbidity will be high, and mortality variable. mucosal scrapings or fixed stained tissue sections may be useful in diagnosis. the disease is also diagnosed by detecting the oocysts in iodine-stained feces or in tissues stained with periodic acid-schiff stain or methenamine silver. cryptosporidium also stains red on acid-fast stains such as kinyoun or ziehl-neelsen. fecal flotations should be performed without sugar solutions or with sugar solutions at specific gravity of . (foryet, ) . fecal immunofluorescent antibody (ifa) techniques have also been described. epizootiology and transmission. younger ruminants are commonly affected: lambs, kids (especially kids between the ages of and days old), and calves less than days old. like other coccidians, cryptosporidium is transmitted via the fecal-oral route. in addition to local contamination, water supplies have also been sources of the infecting oocysts. the oocysts are extremely resistant to desiccation in the environment and may survive in the soil and manure for many months. necropsy findings. the lesions caused by cryptosporidium are nonspecific. animals will be emaciated. moderate enteritis and hyperplasia of the crypt epithelial cells with villous atrophy as well as villous fusion, primarily in the lower small intestines, will be present. cecal and colonic mucosae may sometimes be involved. gastrointestinal smears may be made at necropsy and stained as described above. pathogenesis. although cryptosporidium infections are clinically similar to eimeria infections (moore, ) , cryptosporidium, in contrast to eimeria, invades just under the surface but does not invade the cytoplasm of enterocytes. there is no intermediate host. the oocysts are half the size of eimeria oocysts and are shed sporulated; they are, therefore, immediately infective. within - days of exposure, diarrhea and oocyst shedding occur. the diarrhea is the result of malabsorption and, in younger animals, intraluminal milk fermentation. autoinfection within the lumen of the intestines may also occur and result in persistent infections. in addition, several other pathogens may be involved, such as concurrent coronavirus and rotavirus infections in calves. environmental stressors such as cold weather increase mortality. intensive housing arrangements increase morbidity and mortality. differential diagnosis. other causes of diarrhea in younger ruminants include rotavirus, coronavirus, and other enteric viral infections; enterotoxigenic escherichia coli; clostridium; other coccidial pathogens; and dietary causes (inappropriate use of milk replacers). in addition, these other agents may also be causing illness in the affected animals and may complicate the diagnosis and the treatment picture. eimeria is more likely to cause diarrhea in calves and lambs at - weeks of age. giardia organisms may be seen in fecal preparations from young ruminants but are not considered to play a significant role in enteric disease. blood. animals exhibit fever, dehydration, and depression. chronic cases may result in a "poor doer" syndrome with weight loss and unthriftiness. giardia can be diagnosed by identifying the motile piriform trophozoites in fresh fecal mounts. oval cysts can be floated with zinc sulfate solution ( %). standard solutions tend to be too hyperosmotic and to distort the cysts. newer enzyme-linked immunosorbent assay (elisa) and ifa tests are sensitive and specific. epizootiology and transmission. giardia infection may occur at any age, but young animals are predisposed. chronic oocyst shedding is common. transmission of the cyst stage is fecaloral. wild animals may serve as reservoirs. necropsy findings. gross lesions may not be evident. villous atrophy and cuboidal enterocytes may be evident histologically. prevention and control. precautions should be taken when handling infected animals. affected animals must be removed and isolated as soon as possible. animal housing areas should be disinfected with undiluted commercial bleach or % ammonia. formalin ( %) fumigation has proven successful (foryet, ) . after being cleaned, areas should be allowed to dry thoroughly and should remain unpopulated for a period of time. because enteric disease often is multifactorial, other pathogens should also be considered, and management and husbandry should be examined. no known drug treatment is available. the disease is generally self-limiting, so symptomatic, supportive therapy aimed at rehydrating, correcting electrolyte and acid-base balance, and providing energy is often effective. supplementation with vitamin a may be helpful. age resistance begins to develop when the animals are about month old. research complications. cryptosporidiosis is a zoonotic disease. it is easily spread from calves to humans, for example, even as the result of simply handling clothing soiled by calf diarrhea. adult immunocompetent humans are reported to experience watery diarrhea, cramping, flatulence, and headache. the disease can be life-threatening in immunocompromised individuals. v. giardiasis etiology. giardia lamblia (also called g. intestinalis and g. duodenalis) is a flagellate protozoon. giardiasis is a worldwide protozoal-induced diarrheal disease of mammals and some birds (kirkpatrick, ), but it not considered to be a significant pathogen in ruminants. clinical signs and diagnosis. diarrhea may be continuous or intermittent, is pasty to watery, is yellow, and may contain pathogenesis. following ingestion, each giardia cyst releases four trophozoites, which attach to the enterocytes of the duodenum and proximal jejunum and subsequently divide by binary fission or encyst. the organism causes little intestinal pathology, and the cause of diarrhea is unknown but is thought to be related to disruption of digestive enzyme function, leading to malabsorption. disturbances in intestinal motility may also occur (rings and rings, ) . prevention and control. intensive housing and warm environments should be minimized. cysts can survive in the environment for long periods of time but are susceptible to desiccation. effective disinfectants include quaternary ammonium compounds, bleach-water solution ( : or : ), steam, or boiling water. after cleaning, areas should be left empty and allowed to dry completely. treatment. giardia has been successfully treated with oral metronidazole. benzimidazole anthelmintics are also effective, but these are not approved for use in animals for this purpose. should be taken when handling infected animals. etiology. neosporosis is a common, worldwide cause of bovine abortion caused by the protozoal species neospora caninum. abortions have also been reported in sheep and goats. neonatal disease is seen in lambs, kids, and calves. until , these infections were misdiagnosed as caused by toxoplasma gondii. some similarities exist between the life cycles and pathogeneses of both organisms. clinical signs and diagnosis. abortion is the only clinical sign seen in adult cattle and occurs sporadically, endemically, or as abortion storms. bovine abortions occur between the third and seventh month of gestation; fetal age at abortion correlates with the parity of the dam as well as with pattern of abortion in the herd. although cows that abort tend to be culled after the first or second abortion, repeated n. caninum-caused abortions will occur progressively later in gestation (up to about months) and within a shorter time frame in the same cow (thurmond and hietala, ) . although infections in adults are asymptomatic other than the abortions, decreased milk production has been noted in congenitally infected cows. many neospora-infected calves will be born asymptomatic. weakness will be evident in some infected calves, but this resolves. rare clinical signs include exophthalmos or asymmetric eyes, weight loss, ataxia, hyperflexion or hyperextension of all limbs, decreased patellar reflexes, and loss of conscious proprioception. some fetal deaths will occur, and resorption, mummification, autolysis, or stillbirth will follow. immunohistochemistry and histopathology of fetal tissue are the most efficient and reliable means of establishing a postmortem diagnosis. serology (ifa and elisa) is useful, including precolostral levels in weak neonates, but this indicates only exposure. titers of dams will not be elevated at the time of abortion; fetal serology is influenced by the stage of gestation and course of infection. earlier and rapid infections are less likely to yield antibodies against neospora. none of the currently available tests is predictive of disease. epizootiology and transmission. the parasite is now acknowledged to be widespread in dairy and cattle herds. the life cycle of n. caninum is complex, and many aspects remain to be clarified. the definitive host is the dog (mcallister et al., ) . placental or aborted tissues are the most likely sources of infection for the definitive host and play a minor role in transmission to the intermediate hosts. the many intermediate hosts include ruminants, deer, and horses. transplacental transmission is the major mode of transmission in dairy cattle and is the means by which a herd's infection is perpetuated. a less significant mode of transmission is by ingestion of oocysts, which sporulate in the environment or in the intermediate host's body. reactivation in a chronically infected animal's body is the result of rupture of tissue cysts in neural tissue. seropositive immunity does not protect a cow from future abortions. many seropositive cows and calves will never abort or show clinical signs, respectively. some immunological cross-reactivity may exist among neospora, cryptosporidia, and coccidium. necropsy findings. aborted fetuses will usually be autolysed. in those from which tissue can be recovered, tissue cysts are most commonly found in the brain. spinal cord is also useful. histological lesions include mild to moderate gliosis, nonsuppurative encephalitis, and perivascular infiltration by mixed mononuclear cells. pathogenesis. as with toxoplasma, cell death is the result of intracellular multiplication of neospora tachyzoites. neospora undergoes sexual replication in the dog's intestinal tract, and oocysts are shed in the feces. the intermediate hosts develop nonclinical systemic infections, with tachyzoites in several organs, and parasites then localize and become encysted in particular tissues, especially the brain. infections of this type are latent and lifelong. except when immunocompromised, most cattle do not usually develop clinical signs and do not have fetal loss. fetuses become infected, leading to fetal death, mid-gestation abortions, or live calves with latent infections or congenital brain disease. it usually takes - weeks for a fetus to die and to be expelled. many aspects of the role of the maternal immune response and pregnancy-associated immunodeficiency in the patterns of neospora abortions remain to be elucidated. differential diagnosis. even when there is a herd history of confirmed neospora abortions, leptospirosis, bovine viral diarrhea virus (bvdv), infectious bovine rhinotracheitis virus (ibrv), salmonellosis, and campylobacteriosis should be considered. bvdv in particular should be considered for abortion storms. differentials for weak calves are b vdv, perinatal hypoxia following dystocia (immediate postpartum time), bluetongue virus, toxoplasma, exposure to teratogens, or congenital defects. prevention and control. the primary preventive measure is preventing contact with contaminated feces. oocysts will not survive dry environments or extremes of temperature. dog populations should be controlled, and dogs and other canids should not have access to placentas or aborted fetuses. dogs should also be restricted from feed bunks and other feed storage areas. preventive culling is not economically practical for most producers. a vaccine recently became available. if embryo transfer is practiced, recipients should be screened serologically before use. laxis. there is no known treatment or immunoprophy- clinical signs and diagnosis. clinical signs of sarcocystosis infection are seen in cattle during the stage when the parasite encysts in soft tissues. often the infections are asymptomatic. fever, anemia, ataxia, symmetric lameness, tremors, tail-switch hair loss, excessive salivation, diarrhea, and weight loss are clinical signs. abortions in cattle occur during the second trimester and in smaller ruminants days after ingestion of the sporulated oocysts. definitive diagnosis is based on finding merozoites and meronts in neural tissue lesions. clinical hematology results include decreased hematocrit, decreased serum protein, and prolonged prothrombin times. sarcocystis-specific igg will increase dramatically by - weeks after infection. there is no cross-reaction between sarcocystis and toxoplasma. epizootiology and transmission. infection rates among cattle in the united states are estimated to be very high. transmission is by ingestion of feed and water contaminated by feces of the definitive hosts. dogs are the definitive hosts for the species that infect the smaller ruminants. cats, dogs, and primates (including humans when s. hominis is involved) are the definitive hosts for the species that infect cattle. necropsy. aborted fetuses may be autolysed. lesions in neural tissues, including meningoencephalomyelitis, focal malacia, perivascular cuffing, neuronal degeneration, and gliosis, are most marked in the cerebellum and midbrain. lesions may be found in other tissues, such as lymphadenopathy, and hemorrhages may be found in muscles and on serous surfaces. cysts in cardiac and skeletal muscles are common incidental findings during necropsies. pathogenesis. ingestion of muscle flesh from an infected ruminant results in sarcocystis cysts' being broken down in the carnivore's digestive system, release of bradyzoites, infection of intestinal mucosal cells by the bradyzoites, differentiation into sexual stages, fusion of the male and female gametes to form oocysts, and shedding as sporocysts by the definitive hosts. the sporocysts are eaten by the ruminant and penetrate the bowel walls; several stages of development occur in endothelial cells of arteries. merozoites are the form that enters soft tissues, such as muscle, and subsequently encysts. prevention and control. feed supplies of ruminants must be protected from fecal contamination by domestic and wild carnivores. these animals should be controlled and must also not have access to carcasses. in larger production situations, monensin may be fed as a prophylactic measure. treatment. monensin fed during incubation is prophylactic, but the efficacy in clinically affected cattle is not known. etiology. toxoplasmosis is caused by the obligate intracellular protozoon toxoplasma gondii, a coccidial parasite of the family eimeridae. cats are the only definitive hosts, and several warm-blooded animals, including ruminants, have been shown to be intermediate hosts. the disease is a major cause of abortion in sheep and goats and less common in cattle. clinical signs and diagnosis. clinical signs depend on the organ or tissue parasitized. toxoplasmosis is typically associated with placentitis, abortion, stillbirths, or birth of weak young (underwood and rook, ; buxton, ) . it has also been shown to cause pneumonia and nonsuppurative encephalitis. the enteritis at the early stage of infection may be fatal in some hosts. hydrocephalus does not occur in animals as it does in human fetal toxoplasma infections. rare clinical presentations in ruminants include retinitis and chorioretinitis; these are usually asymptomatic. infection of the ewe during the first trimester usually leads to fetal resorption, during the second trimester leads to abortion, and during the third trimester leads to birth of weak to normal lambs with subsequent high perinatal mortality. congenitally infected lambs may display encephalitic signs of circling, incoordination, muscular paresis, and prostration. in sheep, weak young will develop normally if they survive the first week after birth. infected adult sheep show no systemic illness. infected adult goats, however, may die. diagnosis may be difficult, and biological, serological, and histological methods are helpful. serological tests are the most readily available. complement fixation and the sabin-feldman antibody test may assist in diagnosis. antibodies found in fetuses are indicative of congenital infection and are typically detectable days after infection; fetal thoracic fluid is especially useful in demonstrating serological evidence of exposure. biological methods, such as tissue culture or inoculation of mice with maternal body fluids, or with postmortem or necropsy tissues, are more time-consuming and expensive. epizootiology and transmission. this protozoon is considered ubiquitous. fifty percent ( %) of adult western sheep and % of feedlot lambs have positive hemagglutination titers ( : or higher) (jensen and swift, ) . transmission among the definitive host is by ingestion of tissue cysts. necropsy findings. at necropsy, placental cotyledons contain multiple small white areas that are sites of necrosis, edema, and calcification. fetal brains may show nonspecific lesions such as coagulative necrosis, nonsuppurative encephalomyelitis, pneumonia, myocarditis, and hepatitis. histologically, granulomas with toxoplasma organisms may be seen in the retina, myocardium, liver, kidney, brain, and other tissues. impression smears of these tissues, stained appropriately (e.g., with giemsa), provide a rapid means of diagnosis. identification of the organism in tissue sections (especially of the heart and the brain) also confirms the findings. toxoplasma gondii is crescent-shaped, with a clearly visible nuclei, and will be found within macrophages. pathogenesis. the protozoon has three infectious stages: the tachyzoite, the bradyzoite, and the sporozoite within the oocyst. the definitive hosts, felids, become infected by ingesting cyst stages in mammalian tissues, by ingesting oocysts in feces, and by transplacental transfer. ingested zoites invade epithelial cells and eventually undergo sexual reproduction, resulting in new oocysts, which the cats will shed in the feces. cats rarely show clinical signs of infection. one cat can shed millions of oocysts in gm of feces, but the asymptomatic shedding takes place for only a few weeks in its life. oocysts sporulate in cat feces after day. ruminants are intermediate hosts of toxoplasmosis and become infected by ingesting sporulated oocyst-contaminated water or feed. as in the definitive host, the ingested sporozoite invades epithelial cells within the intestine but also further invades the bloodstream and is transported throughout the host. the organism migrates to tissues such as the brain, liver, muscles, and placenta. placental infection develops about days after ingestion of the oocysts. the damage caused by an infection is due to multiplication within cells. toxoplasma does not produce any toxin. campylobacter, chlamydia, and q fever. prevention and control. feline populations on source farms should be controlled. eliminating contamination of feed and water with cat feces is the best preventive measure. sporulated oocysts can survive in soil and other places for long periods of time and are resistant to desiccation and freezing. vaccines for abortion prevention in sheep are available in new zealand and europe. treatment. toxoplasmosis treatment is ineffective, although feeding monensin during pregnancy may be helpful (underwood and rook, ) . (monensin is not approved for this use in the unites states.) weak lambs that survive the first week after birth will mature normally and will not deliver toxoplasmainfected young. research complications. because toxoplasmosis is zoonotic, precautions must be taken when handling tissues from any abortions or neurological cases. infections in immunocompromised humans have been fatal. etiology. trichomoniasis is an insidious venereal disease of cattle caused by tritrichomonas (also referred to as trichomonas) fetus, a large, pear-shaped, flagellated protozoon. the organism is an obligate parasite of the reproductive tract, and it requires a microaerophilic environment to establish chronic infections. in the united states, it is now primarily a disease seen in western beef herds. there are many similarities between trichomoniasis and campylobacteriosis; both diseases cause herd infertility problems. clinical signs and diagnosis. clinical signs include infertility manifested by high nonpregnancy rates as well as periodic py-ometras and abortions during the first half of gestation. often the problem is not recognized until herd pregnancy checks indicate many "open," delayed-estrus, late-bred cows, or cows with postcoital pyometras. the abortion rate varies from % to %, and placentas will be expelled or retained. tritrichomonas fetus also causes mild salpingitis but this does not result in permanent damage. other than these manifestations, infection with t. fetus causes no systemic signs. diagnosis is based on patterns of infertility and pyometras. for example, pyometras in postcoital heifers or cows are suggestive of this pathogen. diagnostic methods include identifying or culturing the trichomonads from preputial smegma, cervicovaginal mucus, uterine exudates, placental fluids, or abomasal contents of aborted fetuses. other nonpathogenic protozoa from fecal contamination may be present in the sample. the trichomonad has three anterior flagellae, one posterior flagella, and an undulating membrane; it travels in fluids with a characteristic jerky movement. culturing must be done on specific media, such as diamond's or modified pastridge. real exposure from breeding bulls or cows or, in some cases, contaminated breeding equipment. necropsy findings. nonspecific lesions, such as pyogranulomatous bronchopneumonia of fetuses and placentitis, may be seen in aborted material; some cases will have no gross lesions. histologically, trichomonads may be visible in the fetal lung lesions and the placenta; those tissues are also the most useful for culturing. pathogenesis. tritrichomonas fetus colonizes the female reproductive tract, and subsequent clinical manifestations may be related to the size of the initial infecting dose. tritrichomonas fetus does not interfere with conception. embryonic death occurs within the first months of infection. affected cows will clear the infection over a span of months and maintain immunity for about months. infections in younger bulls are transient; apparently organisms are cleared by the bulls' immune systems and are dependent on exposure to infected females. older bulls become chronic carriers, probably because of the ability of t. fetus to colonize deeper epithelial crypts of the prepuce and penis. differential diagnosis. campylobacteriosis is the other primary differential for reduced reproductive efficiency of a herd. other venereal diseases should be considered when infertility problems are noted in a herd: brucellosis, mycoplasmosis, ureaplasmosis, and infectious pustular vulvovaginitis. in addition, management factors such as nutrition and age of heifers at introduction to the herd should be considered. heifers, cows, and breeding bulls are vaccinated subcutaneously twice at to week intervals, with the booster dose administered weeks before breeding season starts. similar timing is recommended for administration of the annual booster; a long, anamnestic response does not occur. bulls used for artificial insemination (ai) are screened routinely for t. fetus (and campylobacter) . ai reduces but does not eliminate the disease. the use of younger, vaccinated bulls is recommcmded in all circumstances. new animals should be tested before introduction to the herd. control measures also include culling affected cows or else removing them from the breeding herd for months to rest and clear the infection. culling chronically infected bulls is strongly recommended. treatment. imidazole compounds have been effective, but the use of these is not permitted in food animals in the united states. therapeutic immunizations are worthwhile when a positive diagnosis has been made. these will not curtail fetal losses but will shorten the convalescence of the affected cows and improve immunity of breeding bulls. research complications. trichomoniasis should be considered whenever natural service is used and fertility problems are encountered. nematodes are important ruminant pathogens that cause acute, chronic, subclinical, and clinical disease in adults and adolescents. the major helminths may cause gastroenteritis associated with intestinal hemorrhage and malnutrition. nematodiasis is associated with grazing exposure to infective larvae; animals procured for research may have had exposure to these helminths. mixed infections of these parasites are common. generally, older animals develop resistance to some of the species; thus, animals between about months and years of age are most susceptible to infection. because of the parasites' effects on the animals' physiology, infection in these younger animals is a major contributor to a cycle of poor nutrition and digestion, compromised immune responses, and impaired growth and development. diagnosis is primarily based on fecal flotation techniques; however, because many of these nematodes have similar-appearing ova, hatching the ova and identifying the larvae are often required (baermann technique). a number of anthelmintics can be used to interrupt nematode life cycles. see zajac and moore ( ) and pugh et al. ( ) for comprehensive reviews of treatment and control of nematodiasis. i. haemonchus contortus, h. placei (barber's pole worm, large stomach worm) . haemonchus contortus is the most important internal parasite of sheep and goats, and the brief description here focuses on the disease in the smaller ruminants. haemonchus contortus and h. placei infections do occur in younger cattle and are similar to the disease in sheep. haemonchus is extremely pathogenic, and the adults feed by sucking blood from the mucosa of the abomasum. severe anemia may lead to death. weight loss, decreased milk production, poor wool growth, and intermandibular and cervical edema due to hypoproteinemia ("bottle jaw") are also common clinical signs. diarrhea is not seen in all cases but may sometimes be severe or chronic. the life cycle is direct. under optimal conditions, a complete life cycle, from ingestion of larvae to eggs passed in the feces, occurs in weeks. embryonated eggs may develop into infective larvae within a week. hypobiotic (arrested) larvae may exist for several months in animal tissues, serving as a reservoir for future pasture contamination. periparturient increases in egg shedding by ewes contribute to large numbers of eggs spread on spring pastures ("spring rise"). resistance to common anthelmintics has developed; currently ivermectin or benzimidazole products are used, with a minimum of dosings given - weeks apart. levamisole is also used. in severe cases, animals may benefit from blood transfusions and iron supplementation. because animals may easily acquire infective larvae from ingestion of contaminated feed and from contaminated pastures, general facility sanitation and pasture management and rotation are important preventive and control measures. haemonchus contortus is susceptible to destruction by freezing temperatures and dry conditions. ii. ostertagia (teladorsagia) circumcincta (medium stomach worm). ostertagia circumcincta is also highly pathogenic for sheep and goats and, like haemonchus, attaches to the abomasal mucosa and ingests blood. the life cycle is comparable to that of haemonchus, including the phenomenon of hypobiosis. larvae are especially resistant to cool temperatures, however, and will overwinter on pastures. larvae-induced hyperplasia of abomasal epithelial glands results in a change of gastric ph from about . to near . , leading to decreased digestive enzyme activity and malnutrition. clinical syndromes are categorized as type or type . the former type is associated with infections acquired in fall or spring and is seen in younger animals. the latter type is associated with emergence of the arrested larvae during spring or fall. clinical signs include anemia, weight loss, decreased milk production, and unthriftiness. diarrhea is usually seen in type only; the symptoms of type are comparable to those of haemonchus infections. anthelmintic drug therapy is comparable to that for haemonchus, and drug resistance is also a problem with ostertagia. iii. ostertagia ostertagi (cattle stomach worm). ostertagia ostertagi is the most pathogenic and most costly of the cattle nematodes. ostertagia leptospicularis and o. bisonis also cause disease. the life cycle is direct, and egg shedding by the cattle may occur within - weeks of ingestion of infective larvae. hypobiosis is also a characteristic of o. ostertagi. in the initial steps of infection, the normal processes of the abomasum are profoundly disrupted and cells are destroyed as the larvae develop within and emerge from the glands. moroccan leather appearance is the term to describe the result of cellular hyperplasia and loss of cell differentiation. cycles of infection and morbidity depend on geographic location, climate, and production cycles. type cattle ostertagiasis is associated with ingestion of large numbers of infective larvae, occurs in animals less than years old, and causes diarrhea and anorexia. type ostertagiasis occurs in cattle - years old and older adults, is the result of the emergence and development of hypobiotic larvae, and in addition to signs seen with type , hypoproteinemia with development of submandibular edema, fever, and anemia is a clinical sign. treatment options include ivermectin, fenbendazole, and levamisole; all are effective against the arrested larvae. ostertagia is susceptible to desiccation but is resistant to freezing. iv. trichostrongylus vitrinus, t. axei, t. colubriformis (hair worms) . trichostrongylus species favor cooler conditions, and some larvae may overwinter. although the different species may affect different segments of the gastrointestinal tract, the nematode attaches to the mucosa and affects secretion and/or absorption. trichostrongylus vitrinus and t. colubriformis infect the small intestine of sheep and goats. trichostrongylus axei infects the abomasum of cattle, sheep, and goats and causes increases in abomasal ph similar to those seen with ostertagia. mucosal hyperplasia is not seen. the prepatent period is about weeks. affected animals display unthriftiness, anorexia, decreased milk production, weight loss, diarrhea, and dehydration. these worms show intermediate resistance to freezing temperatures and dry conditions. v. nematodirus spathiger, n. battus (thread-necked worms vii. strongyloides papillosus. strongyloides papillosus is a small-intestinal parasite of sheep and cattle. strongyloides has a different life cycle from that of many nematodes. the eggs, expelled in the feces, are larvated, and when they hatch, they form both free-living males and females or parasitic females only. the parasitic females may enter the gastrointestinal tract through oral ingestion, such as in milk during nursing, or through direct penetration of the skin. penetrating larvae enter the bloodstream and are transported to the lungs, where they penetrate the alveoli, are coughed up, and then swallowed to ultimately enter the gastrointestinal tract. adult females may reproduce in the small intestines by parthenogenesis. clinical signs associated with strongyloides include weight loss, diarrhea, unthriftiness, and dermatitis in cases where large numbers migrate through the skin. the current broad-spectrum anthelmintics are effective against strongyloides. strongyloides, bunostomum infection may involve oral ingestion or direct penetration of the skin (followed by tracheal migration and swallowing). the larvae mature in the small intestines and suck blood. larvae are susceptible to desiccation and freezing. heavy infection with bunostomum may result in anemia, diarrhea, intestinal hemorrhage, edema, and weight loss. ix. oesophagostomum columbianum, o. venulosum (nodule worms) . oesophagostomum spp. primarily infect the large intestine and occasionally the distal small intestine, causing nodule worm disease, or simply gut. oesophagostomum columbianum and o. venulosum infect sheep and cattle. these nematodes may affect sheep from months to years of age, and the prepatent period is about weeks. larvae are highly sensitive to freezing and desiccation and rarely overwinter. larvae penetrate the large-intestinal mucosa but occasionally move into the deeper areas of the intestinal wall near the serosa. the resultant inflammatory reaction may lead to the formation of a caseous nodule that may mineralize over time. intestinal lesions may accelerate peristalsis, leading to diarrhea, or may inhibit peristalsis (later stages), resulting in constipation. clinical signs include weakness, unthriftiness, alternating episodes of diarrhea and constipation, and severe weight loss. nodular lesions are typical at necropsy. x. chabertia ovis (large-mouth bowel worm). chabertia ovis is a minor colon parasite of sheep, goats, and cattle and is seen primarily in sheep. signs of infection are not usually seen in cattle. prepatent periods are up to days. heavy infection, which may result from as few as worms located at the proximal end of the colon, may lead to hemorrhagic mucoid diarrhea, weight loss, weakness, colitis, and mild anemia. xi. trichuris (whipworms). trichuris spp. are mildly pathogenic nematodes and are usually attached to the cecal mucosa. trichuris has a rather long prepatent period, extending from to months. the oval eggs are double-operculated and survive well in pasture environmental extremes. the adult worms also have a characterisitic morphology, with one thicker end appearing as a whip handle. the nematodes cause a minor cecitis and will feed on blood. clinical infection is rare and results in diarrhea with mucus and blood. treatment and prevention methods are similar to those for other nematodes. xii. dictyocaulus (lungworms). dictyocaulus spp., or lungworms, are nematodes that cause varying clinical signs in ruminants. in sheep, dictyocaulus filaria, protostrongylus rufescens, and muellerius capillaris cause disease; dictyocaulus is the most pathogenic. goats are infected by the same species as sheep, but infections are uncommon. dictyocaulus viviparus is the only lungworm found in cattle, causing "fog fever." infections with these parasites in the united states tend to be associated with cooler, moister climates. lungworms induce a severe parasitic bronchitis (known as husk, or verminous pneumonia) in sheep between approximately and months of age. sheep infected with any of the lungworm species may display coughing, dyspnea, nasal discharge, weight loss, unthriftiness, and occasionally fever. coughing and dyspnea are symptoms in goats. diagnosis is suggested by persistent coughing and nasal discharge and is confirmed by identifying larvae in the feces or adults in pathological samples. the baermann technique, involving prompt examination of room-temperature feces, is usually used; zinc sulfate flotation is also used. dictyocaulus has a direct life cycle. the adult worms reside in the large bronchi. dictyocaulus produces embryonated eggs that are coughed up and swallowed; the eggs then hatch in the intestines, and larvae are expelled in the feces. the expelled larvae are infectious in about - days and, after ingestion, penetrate the intestinal mucosa and move through the lymphatics and blood into the lungs, where they develop into adults in about weeks. dictyocaulus filaria causes an especially severe bronchitis in sheep. protostrongylus inhabits smaller bronchioles. muellerius is of minor pathogenicity. protostrongylus and muellerius require the snail or slug as an intermediate host. infection occurs through ingestion of infected snails; infections are less likely than those caused by the direct ingestion of dictyocaulus larvae. immunity wanes over a year. viral and bacterial respiratory tract infections may be associated with the parasitic infection. more severe illness is seen after infections with cooperia and ostertagia, because of a synergism between the nematodes even if the cattle are not currently infected with those parasites. hypobiosis (arrested development of immature worms in lung tissue) is associated with dictyocaulus infections; cattle will be silent carriers, showing no clinical signs and serving as a means for the infection to survive over winter or a dry season. pastures can be heavily contaminated during the next grazing season. necropsy lesions include bronchiolitis and bronchitis, atelectasis, and hyperplasia of peribronchiolar lymphoid tissue. nematodes frequently reside in the bronchi of the diaphragmatic lung lobes and are frequently enmeshed with frothy exudate. prevention and control of the disease involve appropriate pasture management. elimination of intermediate hosts is important in sheep and goat pastures. in a laboratory setting, animals may be procured that are already harboring the disease. infected animals can be treated with anthelmintics such as ivermectin or levamisole. muellerius tends to be resistant to levamisole. there is no anthelmintic currently approved for goats, but fenbendazole, administered weeks apart, has been effective for all three tapeworms are rarely of clinical or economic importance. in younger animals, heavy infections result in potbellies, constipation or mild diarrhea, poor growth, rough coat, and anemia. moniezia expansa, and less commonly moniezia benedini, inhabit the small intestines of grazing ruminants. moniezia expansa has the widest distribution of the tapeworm species in north america. soil mites (galumna spp. and oribatula spp.) contribute to the life cycle as intermediate hosts, a period that lasts up to weeks. cysticercoids released from the mites are grazed, pass into the small intestines, and mature. no clinical or pathological sign is usually observed with moniezia infection; diagnosis is made by observing the characteristic triangularshaped eggs in fecal flotation examinations. infection is treated with cestocides. thysanosoma actinoides, or the fringed tapeworm, is a cestode that resides in the duodenum, bile duct, and pancreatic duct of sheep and cattle raised primarily west of the mississippi river in the united states. thysanosoma is of the family anoplocephalidae. the life cycle is indirect, and the intermediate host is the psocid louse. larval forms, or cysticercoids, are ingested by grazing animals, and the prepatent period is several months. typically, no clinical signs are observed with thysanosoma infection; nonetheless, liver damage, resulting in liver condemnation at slaughter, occurs. necropsy lesions include bile and/or ductal hyperplasia and fibrosis. thysanosoma is diagnosed premortem by identifying the gravid segments in the feces. ii. abdominal or visceral cysticercosis. abdominal or visceral cysticercosis is an occasional finding at slaughter. the socalled bladder worms typically affect the liver or peritoneal cavity and are the larval form of taenia hydatigena, the common tapeworm of the dog family. taenia hydatigena resides in the small intestines of canids, and its gravid segments, oncospheres, contaminate feed and water sources. after ingestion, the larvae penetrate the intestinal mucosa, are transported via the bloodstream to the liver, and cause migration tracts throughout the liver parenchyma. the larvae may leave the liver and migrate into the peritoneal cavity, where they attach and develop over the next - months into small fluid-filled bladders. the life cycle is completed only after these bladders are ingested by a carnivore, thus completing the maturation of the adult tapeworms. although larval migration may cause nonspecific signs such as anorexia, hyperthermia, and weight loss, affected animals are usually asymptomatic. at necropsy, the bladder worms will be observed attached to the peritoneal or organ surfaces. migration tracts may result in fibrosis and inflammation. diagnosis is usually made at necropsy. because of the migration through the liver, fasciola hepatica is a differential diagnosis. minimizing exposure to canine feces-contaminated feeds and water effectively interrupts the life cycle. research animals may have been exposed prior to purchase. echinococcosis, like cysticercosis, is an occasional finding at slaughter or necropsy. the hydatid cyst is the larval intermediate of the adult tapeworm echinococcus granulosus, which resides in the small intestines of dogs and wild canids. embryonated ova are expelled in the feces of the primary host and are ingested by herbivores, swine, and potentially humans. the eggs hatch in the gastrointestinal tract, and the oncospheres penetrate the mucosal lining, enter the bloodstream, and are transported to various organs such as the liver and lungs. the cystic structure develops and potentially ruptures, forming new cystic structures. clinically, echinococcosis presents minimal clinical signs; unthriftiness or pneumonic lesions may be associated with infected organs. cysts are typically observed at necropsy. prevention should be aimed at decreasing fecal contamination of feed and water by canids. additionally, tapeworm-infected dogs can be treated with standard tapeworm therapies. treatment of infected ruminants is uncommon. iv. gid. coenuris cerebralis, the larval form of the canid tapeworm taenia (multiceps) multiceps, is the causative agent of the rare condition called gid. the disease occurs in ruminants as well as many other mammalian species. the larval parasite, ingested from fecal-contaminated food and water, invades the brain and spinal cord and develops as a bladder worm that causes pressure necrosis of the nervous tissues. the resultant signs of hyperesthesia, meningitis, paresis, paralysis, ataxia, and convulsions are observed. diagnosis is usually made at necropsy. eliminating transfer from the canid hosts prevents the disease. the cercariae leave the intermediate host, swim to grassy vegetation, lose their tail, and become a cystlike metacercaria. the metacercariae may remain in a dormant stage on the grass for months or longer until ingested by a ruminant. the ingested metacercariae penetrate the small-intestinal wall and migrate through the abdominal cavity to the liver. there they locate in a bile duct, mature, and remain for up to years. acute liver fluke disease is related to the damage caused by the migration of immature flukes. migratory flukes may lead to liver inflammation, hemorrhage, necrosis, and fibrosis. fascioloides magna infections in sheep and goats can be fatal as the result of just one fluke tunneling through hepatic tissue. in cattle, infections are often asymptomatic because of the host's encapsulation of the parasite. liver fluke damage may predispose to invasion by anaerobic clostridium species such as c. novyi that could lead to fatal black disease or bacillary hemoglobinuria. chronic disease may result from fluke-induced physical damage to the bile ducts and cholangiohepatitis. blood loss into the bile may lead to anemia and hypoproteinemia. liver damage also is evidenced by increases in liver enzymes such as y-glutamyl transpeptidase (ggt). persistent eosinophilia is also seen with liver fluke disease. other clinical signs of liver fluke disease include anorexia, weight loss, unthriftiness, edema, and ascites. at necropsy, livers will be pale and friable and may have distinct migration tunnels along the serosal surfaces. bile ducts will be enlarged, and areas of fibrosis will be evident. diagnosis can be made from clinical signs and postmortem mites cause a chronic dermatitis. the principal symptom of these infections is intense pruritus. in addition, papules, crusts, alopecia, and secondary dermatitis are seen. anemia, disruption of reproductive cycles, and increased susceptibility to other diseases may also occur. mites are rare in ruminants in the united states, but infections of sarcoptes and psorergates mange must be reported to animal health officials. ruminants in poorly managed facilities are generally the most susceptible to infection, and infections are more frequent during winter months. diagnosis is based on signs, examination of skin scrapings, and response to therapy. no effective treatment for demodectic mange in large animals has been found. the differential for mite infestations is pediculosis. several genera of mites may affect sheep. these have been eradicated from flocks in the united states or are very rare and include psoroptes ovis (common scabies), sarcoptes scabiei (head scabies, barn itch), psorergates ovis (sheep itch mite), chorioptes ovis (foot scabies, tail mange), and demodex ovis (follicular mange). goats can also be infected by sarcoptic, chorioptic, and psoroptic mange. the scabies mite sarcoptes rupicaprae invades epidermal tissue and causes focal pruritic areas around the head and neck. the chorioptic mite, either chorioptes bovis or c. caprae, does not invade epidermal tissue but rather feeds on dead skin tissue. the chorioptic mite prefers distal limbs, the udder, and the scrotum and can be a significant cause of pruri-tus. the psoroptic mite psoroptes cuniculi commonly occurs in the ear canal and causes head shaking and scratching. repeated treatments of lime sulfur, amitraz, or ivermectin may be effective (smith and sherman, ) . goats are also susceptible to demodectic mange caused by demodex caprae. adult mites invade hair follicles and sebaceous glands. pustules may develop with secondary bacterial infection. psoroptes bovis continues to be present in cattle in the united states, although it has been eradicated from sheep. chorioptes bovis typically infects lower hindlimbs, perineum, tail, and scrotum but can become generalized. the sarcoptic mange mite s. scabei can survive off the host, so fomite transmission is a factor. the mange usually begins around the head but then spreads. this parasite can be transmitted to humans. demodex bovis infects cattle; nodules on the face and neck are typical. demodex bovis infections may resolve without treatment. lindane, coumaphos, malathion, and lime sulfur are used to treat psoroptes and psorergates. ivermectin is effective against sarcoptes and is approved for use in cattle. lice that infect ruminants are of the orders mallophaga, biting or chewing lice, and anoplura, sucking lice. these are wingless insects. members of the mallophaga are colored yellow to red; members of the anoplura are blue gray. lice produce a seasonal (winter-to-spring), chronic dermatitis. in sheep, biting lice include damalinia (bovicola) ovis (sheep body louse). sucking lice that infect sheep include linognathus ovillus (blue body louse) and l. pedalis (sheep foot louse). in goats, biting lice infection are caused by d. caprae (goat biting louse), d. limbatus (angora goat biting louse), and d. crassipes. suckir/g louse infections in goats are caused by l. stenopis and l. africanus. damalinia bovis is the cattle biting louse. sucking lice include l. vituli, solenopotes capillatus, haematopinus eurysternus, and h. quadripertusus. pruritus is the most common sign and often results in alopecia and excoriation. the host's rubbing and grooming may not correlate with the extent of infestation. hairballs can result from overgrooming in cattle. in severe cases, the organisms can lead to anemia, weight loss, and damaged wool in sheep and damaged pelts in other ruminants. young animals with severe infestations of sucking lice may become anemic or even die. pregnant animals with heavy infestations may abort. in sheep infected with the foot louse, lameness may result. lice are generally species-specific. those infecting ruminants are usually smaller than mm. goats may serve as a source of infection for sheep by harboring damalinia ovis. transmission is primarily by direct contact between animals. transmission can also occur by attachment to flies or by fomites. some animals are identified as carriers and seem to be particularly susceptible to infestations. biting or chewing lice inhabit the host's face, lower legs, and flanks and feed on epidermal debris and sebaceous secretions. sucking lice inhabit the host's neck, back, and body region and feed on blood. lice eggs or nits are attached to hairs near the skin. three nymphal stages, or instars, occur between egg and adult, and the growth cycle takes about month for all species. lice cannot survive for more than a few days off the host. all ruminant mite infestations are differentials for the clinical signs seen with pediculosis. animals that are carriers should be culled, because these individuals may perpetuate the infection in the group. lice are effectively treated with a variety of insecticides, including coumaphos, dichlorvos, crotoxyphos, avermectin, and pyrethroids. label directions should be read and adhered to, including withdrawal times. products should not be used on female dairy animals. treatments must be repeated at least twice at intervals appropriate for nit hatches (about every days) because nits will not be killed. fall treatments are useful in managing the infections. systemic treatments in cattle are contraindicated when there may be concurrent larvae of cattle grubs (hypoderma lineatum and h. bovis). back rubbers with insecticides, capitalizing on self-treatment, are useful for cattle. sustained-release insecticide-containing ear tags are approved for use in cattle. etiology. ruminants are susceptible to many species of ixodidae (hard-shell ticks) and argasidae (softshell ticks). many diseases, including anaplasmosis, babesiosis, and q fever are transmitted by ticks. clinical signs and diagnosis. tick infestations are associated with decreased productivity, loss of blood and blood proteins, transmission of diseases, debilitation, and even death. feeding sites on the host vary with the tick species. ticks are associated with an acute paralytic syndrome called tick paralysis. this disease is characterized by ascending paralysis and may lead to death if the tick is not removed before the paralysis reaches the respiratory muscles. diagnosis is based on identification of the species. epizootiology and transmission. ticks are not as host-specific as lice. ticks are classified as one-host, two-host, or three-host; this refers to whether they drop off the host between larval and nymphal stages to molt. pathogenesis of tick infestations. patterns of feeding on the host differ between argasidae and ixodidae. the former feed repeatedly, whereas the latter feed once during each life stage. pathogenesis of tick paralysis. following a tick-feeding period of - days, the tick salivary toxin travels hematogenously to the myoneural junctions and spinal cord and inhibits nerve transmission. removal of the ticks reverses the syndrome unless paralysis has migrated anteriorly to the respiratory centers of the medulla. in these cases, death due to respiratory failure occurs. insecticides. ticks can be treated using systemic or topical h. other parasites i. nasal bots (nasal myiasis, head grubs). nasal myiasis causes a chronic rhinitis and sinusitis. the disease is caused by the larval forms of the botfly oestrus ovis. the botfly deposits eggs around the nostrils of sheep. the ova hatch, and the larvae migrate throughout the nasal cavity and sinuses, feeding on mucus and debris. in - months, the larvae complete their growing phase, migrate back to the nasal cavity, and are sneezed out. the mature larvae penetrate the soil and pupate for - . months and emerge as botflies. clinically, early in the disease course, animals display unique behaviors such as stamping, snorting, sneezing, and rubbing their noses against each other or objects. hypersensitivity to the larvae occurs (dorchies et al., ) . later, mucopurulent nasal discharges associated with the larval-induced inflammation of mucosal linings will be observed. at necropsy, larvae will be observed in the nasal cavity or sinuses. mild inflammatory reactions, mucosal thickening, and exudates will accompany the larvae. the disease is diagnosed by observing the behaviors or identifying organisms at necropsy. up to % of a flock will potentially be infected; treatment should be employed on the rest of the flock. ivermectins and other insecticides will eliminate the larvae; but treatment should be done in the early fall, when larvae are small. fly repellents may be helpful at preventing additional infections. ii. screwworm flies. cochliomyia hominivorax (callitroga americana) is the the screwworm that causes occasional disease in the southwestern united states along the mexico border. eradication programs have been pursued, and the disease is reportable. large greenish flies lay large numbers of white eggs as shinglelike layers at the edges of open wounds (including docking and castration sites), soiled skin, or abrasions. eggs hatch within hr. larvae are obligate parasites of living tissue, and the cycle is perpetuated because the increasingly large wound continues to be attractive to the next generation of flies. larvae eventually drop off, pupate best in hot climates, and hatch in weeks. large cavities in parasitized tissue are formed, and lesions are characterized by malodor, large volumes of brown exudate, and necrosis. single animals or entire herds may be affected. treatment is intensive, with dressings and larvicidal applications. if there is no intervention, the host succumbs to secondary infections and fluid loss. effective current control regimens include subcutaneous injection of ivermectin and programs that release sterile male flies. iii. sheep keds ("sheep ticks"). in sheep and goats, sheep keds produce a chronic irritation and dermatitis with associated pruritus. the disease is caused by melophagus ovinus, which is a fiat, brown, blood-sucking, wingless fly; the term sheep tick is incorrectly used. the adult fly lives entirely on the skin of sheep. females mate and produce - larvae following a gestation of about - days. the larvae attach to the wool or hair and then pupate for about weeks. the adult female feeds on blood and lives for - months; the life cycle is completed in about - weeks. infection is highest in fall and winter. pruritus develops around the neck, sides, abdomen, and rump. in severe cases, anemia may occur. keds can transmit bluetongue virus. keds are diagnosed by gross or microscopic identification. ivermectin or other insecticides are useful treatment agents. portant, other immune mechanisms are not well understood. immunity may not be of long duration. recovery is enhanced by correcting nutritional deficiencies and improving housing and ventilation problems. a number of topical treatments, such as - % lime-sulfur solution, % captan, iodophors, thiabendazole, and . % sodium hypochlorite, can be used. in severe cases, systemic therapy with griseofulvin may be successful. prevention and control. the animals' environment and overall physical condition should be reassessed with particular attention to ventilation, crowding, sanitation, and nutrition. pens should be thoroughly cleaned and disinfected. research complications. ringworm is a zoonotic disease. etiology. dermatophytosis, or infection of the keratinized layers of skin, is caused mostly by species of the genera trichophyton and microsporum. the primary causes in sheep are t. mentagrophytes and t. verrucosum. in goats, the agents are t. mentagrophytes, m. canis, m. gypseum, t. verrucosum, t. schoenleinii, and epidermophyton floccosum. in cattle, t. verrucosum is the primary causative agent. dermatophytosis is a common fungal infection of the epidermis of cattle and is less common in sheep and goats. clinical signs and diagnosis. multiple, gray, crusty, circumscribed, hyperkeratotic lesions are characteristic of infection. lesions will vary in size. in all ruminants, lesions will be around the head, neck, and ears. in goats and cattle, lesions will extend down the neck, and in cattle, lesions develop particularly around the eyes and on the thorax. cattle lesions are unique in the marked crustiness, which progressively appears wartlike. hair shafts become brittle and break off. intense pruritus is often associated with the alopecic lesions. the disease can be diagnosed by microscopic identification of hyphae and conidia on the hairs following skin scraping and % potassium hydroxide digestion. dermatophyte test media (dtm) cultures are the most reliable means to diagnose the fungus. broken hairs from the periphery of the lesion are the best sources of the fungus. epizootiology and transmission. younger animals are more susceptible, and factors such as crowding, indoor housing, warm and humid conditions, and poor nutrition are also important. transmission is by direct contact or by contact with contaminated fomites, such as equipment, fencing, or feed bunks. pathogenesis. incubation can be as long as weeks. the organisms invade and multiply in hair shafts. treatment. spontaneous recovery occurs in all species in - months. although cell-mediated immunity is considered im- inverted eyelids are a common inherited disorder of lambs and kids of most breeds. generally, the lower eyelid is affected and turns inward, causing various degrees of trauma to the conjunctiva and cornea. young animals will display tearing, blepharospasm, and photophobia initially. if the disorder is left uncorrected, corneal ulcers, perforating ulcers, uveitis, and blindness may occur. placing a suture or a surgical staple in the lower eyelid and the cheek, effectively anchoring the lid in an everted position, successfully treats the condition. the procedure likely results in the formation of some degree of scar tissue within the lower lid, because when the suture eventually is removed, the condition rarely returns. other treatments include the injection of a "bleb" of penicillin in the lid, regular manual correction over a -day period early in the animal's life, and application of ophthalmic ointments, powders, and solutions. boric acid or % argyrol solutions have been used as treatments. because of the genetic predisposition, prevention of the condition requires removal of maternal or paternal carriers. [ -mannosidosis is an autosomal recessive lysosomal storage disease of goats. the disease affects kids of the nubian breed and is identified by intention tremors and difficulty or inability of newborns to stand. cells of affected animals are vacuolated because of a lack of lysosomal hydroxylase, which results in accumulation of oligosaccharides. newborn kids are unable to rise, and they have characteristic flexion of the carpal joint and hyperextension of the pastern joint. kids are born deaf and with musculoskeletal deformities such as domed skull, small narrow muzzle, small palpebral fissures, enophthalmos, and depressed nasal bridge (smith and sherman, ) . carrier adults can be identified by plasma measurements of [ -mannosidase activity. caprine congenital myotonia is an inherited autosomal dominant disease that affects voluntary striated skeletal muscles. goats with this disease are commonly known as fainting goats. "fainting" is actually transient spasms of skeletal musculature brought about by visual, tactile, or auditory stimuli (smith and sherman, ) . muscle fiber membranes appear to have fewer chloride channels than normal, resulting in decreased chloride conduction across the membrane, with subsequent increased membrane excitability and repetitive firing (smith and sherman, ) . contractions of skeletal muscle are sustained for up to min. kids exhibit the condition by weeks of age, and males appear to exhibit more severe clinical signs than females (smith and sherman, ) . electromyographic studies produce an audible "dive-bomber" sound characteristic of hyperexcitable cell membranes (smith and sherman, ) . i. congenital erythropoietic porphyria. congenital erythropoietic porphyria (cep) is an autosomal recessive disease of cattle seen primarily in holsteins, herefords, and shorthorns. the disease also occurs in limousin cattle, humans, and some other species. in the homozygous recessive animal, symptoms of the disease may vary from mild to severe and occur at different times of the year and in different ages of animals. a reddish brown discoloration of teeth and bones is a characteristic of the disease, as is discolored urine, general weakness and failure to thrive, photosensitization, and photophobia. bones are more fragile compared with bones of normal animals. a regenerative anemia occurs as the result of the shortened life span of erythrocytes, due to accumulations of porphyrins. the genetic defect is associated with low activity of an essential enzyme, uroporphyrinogen iii synthase, in the porphyrin-heme synthesis pathway in erythrocytic tissue. the ranges in the presentation of the disease are believed to be related to varying cycles of porphyrin synthesis. porphyrins are excreted in varying amounts in the urine and the discoloration fluoresces under a wood's lamp. diagnosis is based on these clinical and visible signs of porphyria; skin biopsy provides definitive diagnosis. heterozygotes may have milder symptoms. many other genetic defects, in all major organ systems, have been described in numerous breeds of cattle and are described in detail elsewhere ("large animal internal medicine," ) . in many cases, the genetic basis has been clarified, and associated defects also noted. many defects are reported in particular breeds, but as crossbreeding increases and new breeds are developed, these traits are appearing in these animals. the bovine genome continues to be further characterized, and more linkage maps and gene locations are forthcoming (womack, ) . some bovine genetic defects are also regarded as models of genetic disease, such as leukocyte adhesion deficiency of holstein cattle. some of the more commonly reported defects include syndactyly in holsteins and other breeds and polydactyly in simmentals; lysosomal storage diseases such as a-mannosidosis in some beef breeds; enzyme deficiencies such as citrullinemia in holsteins; and progressive degenerative myeloencephalopathy ("weaver") in brown swiss. ii. goiter of sheep. a defect in the synthesis of thyroid hormone has been identified in merino sheep (radostits et al., ) . lambs born with the defect have enlargement of the thyroid gland, a silky appearance to the wool, and a high degree of mortality. edema, bowing of the legs, and facial abnormalities have also been noted in animals with this disorder. immaturity of the lungs at birth causes neonatal respiratory distress and resuits in dyspnea and respiratory failure. spider lamb syndrome is an inherited, often lethal, musculoskeletal disorder primarily occurring in suffolk and hampshire breeds. severely affected lambs die shortly after birth. animals that survive the perinatal period develop angular limb deformities, scoliosis, and facial deformities. with time, affected animals become debilitated, exhibit joint pain, and develop neurological problems associated with the spinal abnormalities. radiologically, secondary ossification centers--especially the physis, subchondral areas, and cuboidal bonesmare affected. abnormal endochondral ossification leads to excess cartilage formation, notably apparent in the elbows. lambs will typically display abnormally long limbs, medial deviation of the carpus and tarsus, flattening of the sternum, scoliosis/kyphosis of the vertebrae, and a rounded nose. muscle atrophy is common. diagnosis can be based on typical clinical signs, which are similar to those seen with marfan syndrome in humans (rook et al., ) . long-term survival is rare; treatment is unsuccessful. i. abomasal and duodenal ulcers. abomasal and duodenal ulcers occur more frequently in calves and adult cattle than in sheep and goats. like rumenitis, abomasal and duodenal ulcers may be associated with lactic acidosis. concurrent disease, such as salmonellosis, bluetongue, or overuse of anti-inflammatory drugs, or recent shipping or environmental stresses may also lead to ulcer formation. copper deficiency, dietary changes, mycotic infections, clostridium perfringens abomasitis, and abomasal bezoars are associated with this disease in calves. in older adult cattle, abomasal lymphosarcoma may be the underlying condition. gastric acid hypersecretion in conjunction with insufficient gastric mucous secretion will physically destroy the gastric epithelium. deep ulceration may cause serious hemorrhage and/or perforation with peritonitis. chronic hemorrhage may lead to anemia. although ulcers are often asymptomatic in calves, perforation with peritonitis is more common than hemorrhage. dark feces or melena and abdominal pain may be observed. arched back, restlessness, kicking at the abdomen, bruxism, and anorexia are common signs of abdominal pain. fecal occult blood is as an easy diagnostic test. treatment includes gastrointestinal protectants and histamine antagonists. anemia may be symptomatically treated with parenteral iron injections and anabolic steroids. preventive measures in cattle herds include ensuring optimal passive immunity for calves, minimizing stress to calves, and striving for a herd free of bovine leukosis virus. ii. abomasal emptying defect. abomasal emptying defect of sheep is a sporadic syndrome associated with abomasal distension and weight loss. suffolks tend to be especially predisposed, although the disease has been diagnosed in hampshires, columbias, and corriedales. the mechanism of the disease is unknown. affected animals will exhibit a gradual weight loss with a history of normal appetites. feces will continue to be normal. ventral abdominal distension associated with abomasal accumulation of feedstuffs will be apparent in many of the animals. diagnosis is primarily based on history and clinical signs. elevations in rumen chloride concentrations (> meq/liter) are commonly found. radiography or ultrasonography may be helpful at identifying the distended abomasum. abomasal emptying defect is usually eventually fatal. medical treatment with metoclopramide and mineral oil may be helpful in early disease. iii. abomasal displacement. displaced abomasum (da) is a sporadic disorder usually associated with multiparous -to year-old dairy cows in early lactation, but the condition can occur even in young calves. displacement to the right (rda) may be further complicated by torsion (rta), a surgical emergency. left displacement (lda) is more common than rda. clinical signs include anorexia, lack of cud chewing, decreased frequency of ruminal contractions, shallow respirations, increased heart rate, treading, and decreased milk production. diagnosis is based on characteristic areas of tympanic resonance during auscultation-percussion of the lateral to lateral-ventral abdomen ("pings"), ruminal displacement palpated per rectum, and clinical signs. cow-side clinical chemistry findings include hypoglycemia and ketonuria; more extensive evaluations will often indicate moderate to severe electrolyte and acid-base abnormalities. da occurs because of gas accumulation within the viscus, and the abomasum "floats" up from its normal ventral location to the lateral abdominal wall. no exact cause of da has been identified, but it is commonly associated with stress; high levels of concentrate in the diet, leading to forestomach atony; and many disorders, including lack of regular exercise, mastitis, hypocalcemia, retained placenta, metritis, or twins. factors such as body size and conformation indicate the possibility of genetic predisposition. treatments include surgical and nonsurgical techniques for lda; the former has a better chance of per-manent correction. emergency surgery is necessary for rta; the disorder is fatal within hr. recurrence is rare after surgical correction. electrolyte and acid-base imbalances are likely in severe cases and especially with rta. prevention includes reducing stress, taking greater care in the introduction and feeding of concentrates, and reducing incidence of predisposing diseases noted above (rohrbach et al., ) . fat cow syndrome is seen in peri-or postparturient overconditioned or obese multiparous dairy cows. factors in the development of the condition include negative energy balance related to the normal decreased dry matter intake as parturition approaches; hormonal changes associated with parturition; and concurrent diseases of parturition that decrease feed intake and increase energy needs. the possible concurrent diseases include metritis, retained fetal membranes, mastitis, parturient paresis, and displaced abomasum. signs are nonspecific and include depression, anorexia, and weakness. prognosis is usually guarded. diagnosis is based on herd management, the animal's condition, ketonuria, and clinical signs. in prepartum cattle and in lactating cows, blood levels of nonesterified fatty acids (nefa) greater than ~teq/liter and - ~teq/liter, respectively, are abnormal (gerloff and herdt, ) . triglyceride analysis of liver biposy specimens are useful. in affected cows, body fat is mobilized, in the form of nefa in response to the energy demands. hepatic lipidosis occurs rapidly as the nefa are converted into hepatic triglycerides. the ability of the liver to extract the albumin-bound nefa from the blood is better than that of other tissues that need and can also use nefa as an energy source. treatment for any concurrent diseases must be pursued aggressively, as well as measures to increase and stabilize blood glucose, decrease nefa production, and increase forestomach digestion to improve production of normally metabolized volatile fatty acids. therapeutic measures include intravenous glucose drips, insulin (nph or lente) injections every hr, and transfaunation of ruminal fluid from a normal cow. prevention includes minimizing stress to lategestation cows. dry and lactating cows should be maintained separately; their energy, protein, and dry matter requirements are very different. cows with prolonged lactation or delayed breeding should be managed to prevent weight gain. i. bloat. bloat or tympanites refers to an excessive accumulation of gas in the rumen. the condition most frequently occurs in animals that have been recently fed abundant quantities of succulent forages or grains. bloat is classified into two broad categories: frothy bloat and free-gas bloat. frothy bloat is associated with ingestion of feeds that produce a stable froth that is not easily expelled from the rumen. fermentation gases such as co , ch , and minor gases such as n , , h , and h s incorporate into the froth, overdistend the rumen, and eventu-ally compromise respiration by limiting diaphragm movement. the froth is often derived from a combination of salivary mucoproteins, protozoal or bacterial proteins, and proteins, pectins, saponins, or hemicellulose associated with ingested leaves or grain. typical foodstuffs that cause frothy bloat include green legumes, leguminous hay (alfalfa, clover), or grain (especially barley, corn, and soybean meal). free-gas bloat is less related to feeds ingested; rather, it is caused by rumen atony or by physical or pathological problems that prevent normal gas eructation. some examples of causes of free-gas bloat are esophageal obstructions (foreign bodies, tumors, abscesses, and enlarged cervical or thoracic lymph nodes), vagal nerve paralysis or injury, and central nervous system conditions that affect eructation reflexes. clinically, the animal will exhibit rumen distension, and tympany will be observed in the left paralumbar fossa. additional signs may include colic-like pain of the abdomen and dyspnea. passage of a stomach tube helps to differentiate between free-gas bloat and frothy bloat; and with free-gas bloat, expulsion of gas through the stomach tube aids in treatment of the disorder. once rumen distension is alleviated with free-gas bloat, the underlying cause must be investigated to prevent recurrence. frothy bloat is more difficult to treat, because the foam blocks the stomach tube. addition of mineral oil, household detergents, or antifermentative compounds via the tube may help break down the surface tension, allowing the gas to be expelled. in acute, life-threatening cases of bloat, treatment should be aimed at alleviating rumen distension by placing a trocar or surgical rumenotomy into the rumen via the paralumbar fossa. limiting the consumption of feedstuffs prone to induce bloat can prevent the disease. additionally, poloxalene or monensin will decrease the incidence of frothy bloat. ii. lactic acidosis. lactic acidosis, or rumen acidosis, is an acute metabolic disease caused by engorgement of grains or other highly fermentable carbohydrate sources. the disease is most frequently related to a rapid change in diet from one containing high roughage to one containing excessive carbohydrates. diet components that predispose to acidosis include common feed grains; feedstuffs such as sugar beets, molasses, and potatoes; by-products such as brewer's grains; and bakery products. biochemically, ingestion of large amounts of the carbohydrate-rich diet causes the normally gram-negative rumen bacterial populations to shift to gram-positive streptococcus and lactobacillus species. the gram-positive organisms efficiently convert the starches to lactic acid. the lactic acid acidifies the rumen contents, leading to rumen mucosal inflammation, and increases the osmolality of rumen fluids, leading to sequestration of fluids and osmotic attraction of plasma and tissue fluid to the rumen. lactic acid-induced rumenitis predisposes the animal to ulcers, to liver abscesses from "absorbed" bacterial pathogens, to laminitis from absorbed toxins, and to polioencephalomalacia from the inability of the new rumen bacterial populations to produce sufficient thiamine needed to maintain normal nervous system function. clinically, animals will become anorexic, depressed, and weak within - days after the initial insult. incoordination, ataxia, dehydration, hemoconcentration, rapid pulse and respiration, diarrhea, abdominal pain, and lameness will also be noted rumen distension and an acetone-like odor to the breath, milk, or urine may also be observed. diagnosis is based on history and clinical signs. blood, urine, or milk ketones can be detected (moore and ishler, ) . additionally, rumen ph, which is normally above . , will drop to less than . and in severe cases may achieve levels as low as . . similarly, urine ph will become acidic, blood ph will drop below . , and hematocrit will appear to increase due to the relative hemoconcentration. necropsy findings will be determined by secondary conditions. the primary lactic acidosis will cause swelling and necrosis of rumen papillae and abomasal hemorrhages and ulcers. treatment must be applied early in the syndrome. in early hours of severe carbohydrate engorgement, rumenotomy and evacuation of the contents are appropriate. the t patient should be given mineral oil and antlfermentatlves to prevent the continued conversion of starches to acids and the absorption of metabolic products. bicarbonate or other antacids like magnesium carbonate or magnesium hydroxide introduced into the rumen will aid in adjusting rumen ph. furthermore, animals can be given oral tetracycline or penicillin, which will decrease the gram-positive bacterial population. iii. rumen parakeratosis. parakeratosis is a degenerative condition of the rumen mucosa that leads to keratinization of the papillary epithelium excessive and continuous feeding of diets low in roughage causes the mucosal changes generally, this condition is seen in feedlot lambs and steers that are fed an all-grain diet. clinically, animals may exhibit only poor rates of gain, due to changes in the absorptive capacity of the injured mucosa. at necropsy, papillae will be thickened and rough. they will frequently be dark in color, and multiple papillae will clump together. abscessation may be observed. histopathologically, papilla surfaces will have hyperkeratinization of the squamous epithelium. chronic laminitis may be observed. however, diagnosis of parakeratosis is generally made at necropsy. feeding adequate roughage, such as stemmy hay, will prevent the disease. antibiotics may be administered to prevent secondary liver abscess formation. iv. rumenitis. rumenitis is an acute or chronic inflammation of the rumen, which occurs most commonly as a sequela to lactic acidosis in addition to concentrate feeding, inadequate roughage in the diet is also associated with this disorder rumenitis may occur with contagious ecthyma infection or following ingestion of poisons or other irritants. because rumenitis is often associated with lactic acidosis, it tends to occur in feedlot animals. the inflamed ruminal epithelium becomes necrotic and sloughs, creating ulcers. endogenous rumen bacteria such as fusobacterium necrophorum may invade the ulcers, penetrate the circulatory system, and induce abscesses of the liver. clinically, the animals will appear depressed and anorexic. rumen motility will be decreased, and animals will lose weight. the disease may resolve in a week to days; mortality may reach %. necropsy lesions include rumen inflammation and ulcers in the anteroventral sac. granulation tissue and scarring may be observed following healing. rumenitis is not typically diagnosed clinically; thus, specific treatment is not commonly done. the disease can be prevented by minimizing the incidence of lactic acidosis. etiology. traumatic reticulitis-reticuloperitonitis is a disease of cattle related to their exploratory tendencies and ingestion of many different, nonvegetative materials. the disease is rarely seen in smaller ruminants. clinical signs. clinical signs range from asymptomatic to severe, depending on the penetration and damage by the foreign object after settling in the animal's forestomach. many signs during the early, acute stages will be nonspecific, ranging from arched back, listlessness, anorexia, fever, decrease in production, ketosis, regurgitation, decrease or cessation of ruminal contractions, bloat, tachypnea, tachycardia, and grunts when urinating, defecating, or being forced to move. the prognosis is poor when peritonitis becomes diffuse. sudden death can occur if the heart, coronary vessels, or other large vessels are punctured by the migrating object. epizootiology and transmission. this is a noncontagious disease. the occurrence is directly related to sharp or metallic indigestible items in the feed or environment that the cattle mouth and swallow. necropsy findings. in severe cases, necropsy findings include extensive inflammation throughout the cranial abdomen, malodorous peritoneal fluid accumulations, and lesions at the reticular sites of migration of the foreign objects. cardiac puncture will be present in those animals succumbing to sudden death. pathogenesis. consumed objects initially settle in the rumen but are dumped into the reticulum during the digestive process, and normal contraction may eventually lead to puncture of the reticular wall. this sets off a localized inflammation or a localized or more generalized peritonitis. the inflammation may also temporarily or permanently affect innervation of local tissues and organs. further damage may result from migration and penetration of the diaphragm, pericardium, and heart. diagnosis is based on clinical signs, knowledge of herd management techniques in terms of placement of forestomach magnets, and reflection of acute or chronic infection on the hemogram. radiographs and abdominocentesis may be useful. differential diagnosis. differentials include abomasal ulcers, hepatic ulcers, neoplasia (such as lymphosarcoma, usually in older animals, or intestinal carcinoma), laminitis, and cor pulmonale. infectious diseases that are differentials include systemic leptospirosis and internal parasitism. diseases causing sudden death may need to be considered. prevention and control. this problem can be prevented entirely by elimination of sharp objects in cattle feed and in the housing and pasture environments. adequately sized magnets placed in feed handling equipment and forestomach magnets (placed per os with a bailing gun in young stock at - months of age) are also significant prevention measures. treatment. provision of a forestomach magnet, confinement, and nursing care, including antibiotics, are the initial treatments. in severe cases, rumenotomy may be considered. etiology. pregnancy toxemia is a primary metabolic disease of ewes and does in advanced pregnancy. beef heifers are susceptible to protein energy malnutrition (pem) syndrome, which is also referred to as pregnancy toxemia. clinical signs. in sheep, this disease is characterized by hypoglycemia, ketonemia, ketonuria, weakness, and blindness. hypoglycemic and ketotic ewes begin to wander aimlessly and to move away from the flock. they become anorexic and act uncoordinated, frequently leaning against objects. advanced signs may include blindness, muscle tremors, teeth grinding, convulsions, and coma. body temperature, heart rate, respiratory rate, and rumen motility continue normally. up to % of infected ewes may die from the disease. the course of the disease may last up to a week. in goats, the disease usually occurs in the last weeks of gestation, especially in does carrying triplets. pregnancy toxemia should be considered with any goat showing signs of illness in late gestation. the doe may separate herself from the herd, stagger, or circle and may appear blind. appetite is poor, and tremors may be evident. a rapid metabolic acidosis results in subsequent recumbency. urinalysis will readily reveal ketonuria. if fetal death occurs, acute toxemia and death of the doe may result. in beef heifers, weight loss and thin body condition, weakness and inability to stand, and depression are clinical signs. some cows develop diarrhea. because the catabolic state is often so advanced, most affected heifers die even if treated. pregnancy toxemia is diagnosed by evidence of typical clinical signs. sodium nitroprusside tablets or ketosis dipsticks may be used to identify ketones in the urine or plasma of ewes and does. blood glucose levels found to be below mg/dl and ketonuria are good diagnostic indicators. in cattle, ketonuria is not a typical finding; hypocalcemia and anemia may be present. that are obese or bearing twins or triplets. the disease develops during the last weeks of pregnancy. pem most frequently occurs in heifers during the final trimester of pregnancy. necropsy findings. at necropsy, affected ewes will often have multiple fetuses, which may have died and decomposed. the liver will be enlarged, yellow, and friable, with fatty degeneration. the adrenal gland may also be enlarged. in cattle, heifers will be very thin, and in addition to a fatty liver, signs of concurrent diseases may be present. pathogenesis. rapid fetal growth, a decline in maternal nutrition, and a voluntary decrease in food intake in overfat ewes result in an inadequate supply of glucose needed for both maternal and fetal tissues. the ewe develops a severe hypoglycemia in early stages of the disease. the ruminant absorbs little dietary glucose; rather, it produces and absorbs volatile fatty acids (acetic, propionic, and butyric acids) from consumed feedstuffs. propionic acid is absorbed and selectively converted to glucose through gluconeogenesis. when the animal is in a state of negative energy balance, it hydrolyzes fats to glycerol and fatty acids. glycerol is converted to glucose while the fatty acids are metabolized for energy. the oxidation of fatty acids in the face of declining oxaloacetate levels (required for normal krebs cycle function) results in the formation of ketone bodies (acetone, acetoacetic acid, and [ -hydroxybutyric acid), thus causing the condition ketoacidosis. heifer cattle have high energy requirements for completing normal body growth and supporting a pregnancy. additional energy requirements are needed during pregnancy for winter conditions and during concurrent diseases. marginal diets and poor-quality forage will place the cows in a negative energy balance. differential diagnosis. hypocalcemia is a common differential diagnosis. in cattle, differentials include chronic or untreated diseases such as johne's disease, lymphosarcoma, parasitism, and chronic respiratory diseases. prevention and control. pregnancy toxemia can be prevented by providing adequate nutrition during late gestation and by maintaining animals in appropriate nonfat condition during pregnancy. in late pregnancy, the dietary energy and protein should be increased . - times the maintenance level. pem can be prevented by maintaining appropriate body condition earlier in pregnancy and supplying good-quality forage for the last trimester. treatment. in sheep, because the morbidity may be as high as %, treatment should be directed at the flock rather than the in-dividual. treating the individual is usually unsuccessful. oral administration of ml of propylene glycol or % glucose twice a day, anabolic steroids, and high doses of adrenocorticosteroids may be helpful. if ewes are still responsive and not severely acidotic or in renal failure, cesarean section may be successful by rapidly removing the fetus, which is the dietary drain for the ewe. in goats, pregnancy toxemia is best treated by removal of the fetuses either by cesarean section or induction of parturition. parturition can be induced in does by either dexamethasone ( mg) or pgf a ( ~tg). in addition, goats may be treated with % dextrose ( to ml iv) or propylene glycol ( ml per os or times a day). adjunctive therapy includes normalizing acid base and hydration status, administration of vitamin b and transfaunation. heifers may be force-fed alfalfa gruels, given propylene glycol per os, placed on iv % glucose drips, and treated for concurrent disease. research complications. in research requiring pregnant ewes in late stages of gestation, for example, this disease should be considered if the animals are likely to bear twins and will be transported or stressed in other ways during that time. f hypocalcemia (parturient paresis, milk fever) etiology. hypocalcemia is an acute metabolic disease of ruminants that requires emergency treatment; the presentation is slightly different in ewes, does, and cows. clinical signs and diagnosis. in sheep, the disease is seen in ewes during the last weeks of pregnancy and is characterized by muscle tetany, incoordination, paralysis, and finally coma. as calcium levels drop, ewes begin to show early signs such as stiffness and incoordination of movements, especially in the hindlimbs. later, muscular tremors, muscular weakness, and recumbency will ensue. animals will frequently be found breathing rapidly despite a normal body temperature. morbidity may approach %, and mortality may reach as high as % in untreated animals. affected does become bloated, weak, unsteady, and eventually recumbent. cows are affected within - hr before or after parturition. cows initially are weak and show evidence of muscle tremors, then deteriorate to sternal recumbency, with the head usually tucked to the abdomen, and an inability to stand. tachycardia, dilated pupils, anorexia, hypothermia, depression, ruminal stasis, bloat, uterine inertia, and loss of anal tone are also seen at this stage. the terminal stage of disease is a rapid progression from coma to death. heart rates will be high, but pulse may not be detectable. hypocalcemia is diagnosed based on the pregnancy stage of the female and on clinical signs. it is later confirmed by laboratory findings of low serum calcium. with hypocalcemia in ewes, the plasma concentrations of calcium drop from normal values of - mg/dl to values of - mg/dl. in cattle, plasma levels below . mg/dl are hypocalcemic; at the terminal stages levels may be mg/dl. ewes during the last weeks of pregnancy or during the first few weeks of lactation. the disease is not as common in the dairy goat as in the dairy cow. high-producing, older, multiparous dairy cows are the most susceptible, and the jersey breed is considered susceptible. cows that have survived one episode are prone to recurrence. in addition, dry cows must be managed carefully regarding limiting dietary calcium. the disease is not common in beef cattle unless there is an overall poor nutrition program. ing at necropsy. there is no pathognomonic or typical find-pathogenesis. during the periparturient period, calcium requirements for fetal skeletal growth exceed calcium absorbed from the diet and from bone metabolism. additionally, dietary calcium intake is thought to be compromised because, in advanced pregnancy, animals may not be able to eat enough to sustain adequate nutrient levels, and intestinal absorption capabilities do not respond as quickly as needed. after parturition, calcium needs increase dramatically because of calcium levels in colostrum and milk. recent information suggests that legume and grass forages, high in potassium and low in magnesium, create a slight physiological alkalosis (at least in cattle), which antagonizes normal calcium regulation (rings et al., ) . thus, bone resorption, renal resorption, and gastrointestinal absorption of calcium are less than maximal. prevention and control. maintaining appropriate nutrition during the last trimester is helpful in preventing the disease. in cows and does, for example, limiting calcium intake by removing alfalfa from the diet is helpful. treatment. hypocalcemia must be treated quickly based on clinical signs; pretreatment blood samples can be saved for later confirmation. twenty percent calcium borogluconate solution should be administered by slow intravenous infusion. response will often be rapid, with the resolution of the animal's dull mentation. less severely affected animals will often try to stand in a short time. relapses are common, however, in sheep and cattle. hypermagnesemia and hypophosphatemia often coincide with hypocalcemia. these imbalances should be considered when animals appear to be unresponsive to treatment. hypocalcemia in the goat can be treated with - ml of calcium borogluconate. heart rate should be monitored closely throughout calcium administration. if an irregular or rapid heart rate is detected, then calcium treatment should be slowed or discontinued. calcium gels and boluses are also available for treatment (rings et al, ) . prognosis is generally good if the animal is treated early in the disease, but the prognosis will often be poor when treatment is initiated in later stages of the disease. etiology. urolithiasis is a metabolic disease of intact and castrated male sheep, goats, and cattle that is characterized by the formation of bladder and urethral crystals, urethral blockage, and anuria (murray, ) . the disease occurs rarely in female ruminants. clinical signs and diagnosis. affected animals will vocalize and begin to show signs of uneasiness, such as treading, straining postures, arched backs, raised tails, and squatting while attempting to urinate. these postures may be mistaken for tenesmus. male cattle may develop swelling along the ventral perineal area. affected animals will not stay with the herd or flock. small amounts of urine may be discharged, and crystal deposits may be visible attached to the preputial hairs. additionally, in smaller ruminants, the filiform urethral appendage (pizzle) often becomes dark purple to black in color. the pulsing pelvic urethra may be detected by manual or digital rectal palpation, and bladder distention may be noticeable in cattle by the same means. as the disease progresses to complete urethral blockage, the animal will become anorexic and show signs of abdominal pain, such as kicking at the belly. the abdomen will swell as the bladder enlarges, and rupture can occur within hr after development of clinical signs. bladder or urethral rupture may cause a short-lived period of apparent pain relief; subsequent development of uremia will eventually lead to death. the disease may progress over a period of - weeks, and the mortality is high unless the blockages are reversed. diagnosis is made by the typical clinical signs. abdominal taps may yield urine. calculi are usually composed of calcium phosphate or ammonium phosphate matrices. clinical disease is usually seen in growing intact or castrated males. the disease may be sporadic or there may be clusters of cases in the flock or herd. necropsy findings. necropsy findings include urine in the abdomen with or without bladder or urethral rupture. renal hydronephrosis may be evident. calculi or struvite crystal sediment will be observed in the bladder and urethra. histologically, trauma to the urethra and ureters will be present. etary, anatomical, hormonal, and environmental factors. male sheep and goats have a urethral process that predisposes them to entrapment of calculi. in cattle, the urethra narrows at the sigmoid flexure, and calculi lodge there most frequently. additionally, the removal of testosterone by early castration is thought to result in hypoplasia of the urethra and penis. this physical reduction in the size of the excretory tube may predispose to the precipitation of and blockage by the struvite minerals. grains fed to growing animals tend to be high in phosphorus and magnesium content. these calculogenic diets lead to the formation of struvite (magnesium ammonium phosphate) crystals. other minerals associated with urolithiasis include silica (range grasses), carbonates (some grasses and clover pastures), calcium (exclusively alfalfa hay), and oxalates (fescue grasses). differential diagnosis. grain engorgement colic, gastrointestinal blockage, and causes of tenemus, such as enteritis or trauma, are differentials. trauma to the urethral process should be considered. urinary tract infections are uncommon in ruminants. prevention and control. one case often is indicative of a potential problem in the group. urolithiasis can be minimized by monitoring the calcium:phosphorus ratio in the diet. the normal ratio should be : . additionally, increasing the amount of dietary roughage will help balance the mineral intake. increasing the amount of salt (sodium chloride, - %) in the diet to increase water consumption, or adding ammonium chloride to the diet, at gm/head/day or % of the ration, to acidify the urine, will aid in the prevention of this disease. palatability of and accessibility to water should be assessed as well as functioning of automatic watering equipment. treatment. treatment is primarily surgical (van metre et al. ) . initially, amputation of the filiform urethral appendage may alleviate the disease since urethral blockage often begins here. as the disease progresses, urethral blockage in the sigmoid flexure as well as throughout the urethra may occur. in more advanced stages, perineal urethrostomy may yield good results. the prognosis is poor when the condition becomes chronic, reoccurs, or surgery is required. research complications. young castrated and intact male ruminants used in the laboratory setting will be the susceptible age group for this disorder. rickets is a disease of young, growing animals but rarely occurs in goats. it is a metabolic disease characterized by a failure of bone matrix mineralization at the epiphysis of long bones due to lack of phosphorus. the condition can occur as an absolute deficiency in vitamin d , an inadequate dietary supply of phosphorus, or a long-term dietary imbalance of calcium and phosphorus. the syndrome must be differentiated from epiphisitis (unequal growth of the epiphyses of long bones in young, rapidly growing kids fed diets with excess calcium). clinical signs include poor growth, enlarged costochondral junctions, narrow chests, painful joints, and reluctance to move. spontaneous fractures of long bones may occur. animals will recover when dietary phosphorus is provided and if joint damage is not severe. a. copper deficiency (enzootic ataxia, swayback) etiology. chronic copper deficiency in pregnant ewes and does may produce a metabolic disorder in their lambs and kids called enzootic ataxia. in goats, this deficiency also causes swayback in the fetuses. clinical signs and diagnosis. this disease results in a progressive hindlimb ataxia and apparent blindness in lambs up to about months of age. additionally, because copper is essential for osteogenesis, hematopoiesis, myelination, and pigmentation of wool and hair, ewes may appear unthrifty, may be anemic, and may have poor, depigmented wool with a decrease in wool crimp. affected kids are born weak, tremble, and have a characteristic concavity to the spinal cord, leading to the name swayback. when the deficiency occurs later during gestation, demyelination is limited to the spinal cord and brain stem. kids are born normally but develop a progressive ataxia, leading to paralysis, muscle atrophy, and depressed spinal reflexes with lower motor neuron signs. diagnosis is based on low copper levels found in feedstuffs and tissues at necropsy. diagnosis is based on clinical signs, feed analysis, and pathological findings. epizootiology and transmission. enzootic ataxia is rarely seen in western states; most north american diets have sufficient copper levels to prevent this disease. copper antagonists in the feed or forage at sufficient levels, such as molybdenum, sulfate, and cadmium, however, may predispose to copper deficiencies. pathogenesis. the maternal copper deficiency leads to a disturbance early in the embryonic development of myelination in the central nervous system and the spinal cord. copper is part of the cytochrome oxidase system and other enzyme complexes and is important in myelination, osteogenesis, hematopoiesis (iron absorption and hemoglobin formation), immune system development, and maintenance and normal growth (smith and sherman, ) . differential diagnosis. the differential diagnosis for newborns includes [ -mannosidosis, hypoglycemia, and hypothermia. for older animals the differential should include caprine arthritis encephalitis (goats), enzootic muscular dystrophy, listeriosis, spinal trauma or abscessation, and cerebrospinal nematodiasis. prevention and control. copper deficiency can be prevented by providing balanced nutrition for pregnant animals. necropsy findings. gross encephalomalacia has been noted. histopathologically, white matter of the brain and spinal cord displays gelatinization and cavitation. extensive nerve demyelination and necrosis are evident. postmortem lesions include extensive demyelination and neuronal degeneration. treatment. because the condition is developmental, supplemental copper may improve clinical signs but not eliminate them. necropsy findings. common findings at necropsy include icterus; a soft, dark, friable, enlarged spleen; an enlarged, yellow-brown friable liver; and "gun-barrel" black kidneys. hemoglobin-stained urine will be visible in the bladder. copper accumulations in the liver reaching - ppm are toxic. pathogenesis. hemolysis occurs when sufficient amounts of copper are ingested or released suddenly from the liver and is believed to be due direct interaction of the copper with red-cell surface molecules. stresses such as transportation, lactation, and poor nutrition or exercise may precipitate the hemolysis. etiology: acute or chronic copper ingestion or liver injury often causes a severe, acute hemolytic anemia in weanling to adult sheep and in calves and adult dairy cattle. growing lambs may be the most susceptible. copper toxicosis is rare in goats. differential diagnosis. other causes of hemolytic disease include babesiosis, trypanosomiasis, and plant poisonings such as kale. arsenic ingestion, organophosphate toxicity, and cyanide or nitrate poisoning should also be considered as the source of poisoning. urethral obstruction and gastrointestinal emergencies should be considered for the abdominal pain. clinical signs and diagnosis. the clinical course in sheep can be as short as - days, and mortality may reach %. hemolysis, anemia, hemoglobinuria, and icterus characterize the acute hemolytic crisis, associated with copper released from the overloaded liver. some clinical signs are related to direct irritation to the gastrointestinal tract mucosa. weakness, vomiting, abdominal pain, bruxism, diarrhea, respiratory difficulty, and circulatory collapse are followed by recumbency and death. hepatic biopsy is currently considered the best diagnostic approach; serum or plasma levels of copper and hepatic enzymes such as aspartate aminotransferase (ast) and y-glutamyltransferase (ggt) may provide some information, but it is generally believed that these will not accurately reflect total copper load or hepatic damage. and goats is the range of - mg/kg, and for cattle it is - mg/kg. chronic poisoning in sheep may occur when . mg/kg is ingested. copper-containing pesticides, soil additives, therapeutics, and improperly formulated feeds may potentially lead to copper toxicity. phytogenous sources include certain pastures such as subterranean clover. feed low in molybdenum, zinc, or calcium may lead to increased uptake of copper from properly balanced rations. a common cause of the disease in sheep is feeding concentrates balanced for cattle; cattle feeds and mineral blocks contain much higher quantities of copper than are required for sheep. chronic ingestion of these feedstuffs leads to copper accumulation and toxicity. copper toxicosis has been reported in calves given regular oral or parenteral copper supplements, and in adult dairy cattle given copper supplements to compensate for copper-deficient pasture. pregnant dairy cattle may be more susceptible to copper toxicity. rare sources of copper ingestion may include copper sulfate footbaths. control and prevention. the disease is prevented by carefully monitoring copper access in sheep and copper supplementation in cattle. sheep and goats should not be fed feedstuffs formulated for cattle, and dairy calf milk replacer should not be used for lambs and kids. molybdenum may be administered to animals considered at high risk. molybdenum-deficient pastures may be treated with molybdenum superphosphate. herd copper supplementation should be undertaken with the knowledge of existing hepatic copper levels, and existing copper and molybdenum levels, in the feedstuffs. treatment. oral treatment for sheep consists of ammonium or sodium molybdenate ( - mg/day), and sodium thiosulfate ( . - . mg/day) for weeks aids in excretion of copper. oral d-penicillamine daily for days ( mg/kg) has also been shown to increase copper excretion in sheep. ammonium molybdenate has been administered intravenously to goats at . mg/kg for treatments on alternate days. cattle have been treated orally with sodium molybdenate ( gm/day) or sodium thiosulfate ( gm/day). treatment for anemia and nephrosis may be necessary in severe cases. merino crosses and the british breeds, may be more susceptible to copper toxicosis caused by phytogenous sources. (nutritional muscular dystrophy, nutritional myodegeneration, white muscle disease, stiff lamb disease) etiology. white muscle disease, also known as stiff lamb disease, is a nutritional muscular dystrophy caused by a deficiency of selenium or vitamin e. clinical signs and diagnosis. clinically two forms of the disease have been identified: cardiac and skeletal. the cardiac form occurs most commonly in neonates. in these, respiratory difficulty will be a manifestation of damage to cardiac, diaphragmatic, and intercostal muscles. young will be able to nurse when assisted. in slightly older animals, the disease is characterized by locomotor disturbances and/or circulatory failure. clinically, animals may display paresis, stiffness or inability to stand, rapid but weak pulse, and acute death. mortality may reach % (jensen and swift, ) . paresis and sudden death in neonates with associated pathological signs are frequently diagnostic. with the skeletal form, affected animals are stiff and reluctant to move, and muscles of affected animals are painful. young will be reluctant to get up but will readily nurse when assisted. peracute to acute myocardial degeneration may occur in the cardiac form, and animals may simply be found dead. serum selenium levels are usually below ppb (normal is - ppb) (nelson, ) . diagnosis may also include determination of antemortem whole blood levels of selenium and plasma levels of vitamin e. glutathione peroxidase levels in red blood cells can be measured as an indirect test. clinical biochemistry findings of significant elevations of aspartate aminotransferase (ast) in creatinine kinase (ck) are also supportive of the diagnosis. epizootiology and transmission. selenium deficiency has been associated with formulated diets deficient in selenium, forages grown on selenium-deficient soils in certain geographic regions, and forages such as alfalfa and clover that have an inability to efficiently extract available selenium from the soils. rumen bacterial reduction of selenium compounds to unavailable elemental selenium may also contribute to the disease. necropsy findings. necropsy lesions include petechial hemorrhages and muscle edema. hallmarks are pale white streaking of affected skeletal and cardiac muscle. these are due to coagulation necrosis. pale striated muscles of the limb, diaphragm, and tongue are also seen. antioxidants that protect lipid membranes from oxidative destruction. selenium is a cofactor for glutathione peroxidase, which converts hydrogen peroxide to water and other nontoxic compounds. lack of one or both results in loss of membrane integrity. differential diagnosis. in neonatal ruminants presenting with respiratory and cardiac dysfunction, differentials include congenital cardiac anomalies. differentials generally for weak neonates or sudden or peracute neonatal deaths should include septicemia, pneumonia, toxicity, diarrhea, and dehydration. prevention and control. awareness of regional selenium deficiencies is important. control involves providing good-quality roughage, vitamin e and selenium supplementation, and parenteral injections prior to parturition and weaning. treatment. affected animals may be treated by administering vitamin e or selenium injections. administering vitamin e or selenium to ewes in late pregnancy can prevent white muscle disease (kott et al., ) . the label dose for selenium is . - mg/ kg of body weight. combination products are available and can be used in goats at the sheep dose (smith and sherman, ) . proper mineral balance in the diet is critical. selenium toxicity occurs most frequently as the result of excessive dosing to prevent or correct selenium deficiency or as the result of ingestion of selenium-converting plants. the main preventive measure for the former is the use of the appropriate product for the species. secondarily, the concentration of the available product should be double-checked. in the united states, ruminants in the midwest and western areas may be subject to selenium toxicity when pastured in areas containing selenium-converting plants. signs of overdosing include weakness, dyspnea, bloating, and diarrhea. shock, paresis, and death may occur. initial clinical signs of excessive selenium intake from plants are observed in the distal limb, with cracked hoof walls and subsequent infection and irregular hoof growth. etiology. polioencephalomalacia (pem) is a noninfectious, noncontagious disease characterized by neurological signs. growing and adult ruminants on high-concentrate diets are typically affected. animals exposed to toxic plants or moldy feed containing thiaminases, feed high in sulfates, or unusually high doses of some medications are also at risk. clinical signs and diagnosis. an early sign may be mild diarrhea. acute clinical signs include bruxism, hyperesthesia, involuntary muscle contractions, depression, partial or complete opisthotonus, nystagmus, dorsomedial strabismus, seizures, and death. in subacute cases of the disease, animals may appear to walk aimlessly as if blind or may display head-pressing postures. hypersalivation may be present, but body temperatures and ocular reflexes are normal. morbidity and mortality may be high, especially in younger animals. diagnosis is suggestive from clinical signs and from response to intensive parental thiamine hydrochloride. epizootiology and transmission. pem is caused by a thiamin deficiency. the disease tends to be seen more frequently in cattle and sheep feedlots where the concentrates fed are high in fermentable carbohydrates. pastured animals are also vulnerable if grain is feed. thiaminase-containing plants, such as bracken fern, are often unpalatable so will less likely be a contributing factor. recent studies have also indicated that high levels of sulfate in the diet, such as in the fermentable, low-fiber concentrates, may play an important role. medications such as as amprolium, levamisole, and thiabendazole have thiaminantagonizing activity when given in excessive doses. sherman, ) . vitamin a deficiencies associated with hyperkeratosis have been reported, as well as vitamin e-responsive and selenium-responsive dermatitis. necropsy signs. cerebral lesions characterized by softening and discoloration are grossly observed in the gray matter. microscopically, neurons will exhibit edema, chromatolysis, and shrinkage. gliosis and cerebral capillary proliferation may be observed. a lack of thiamin results in inappropriate carbohydrate metabolism and accumulation of pyruvate and other intermediaries that lead to cerebral edema and neuronal degeneration. differential diagnosis. several important differentials include acute lead poisoning, nitrofuran toxicity, hypomagnesemia, vitamin a deficiency, listeriosis, pregnancy toxemia, infectious thromboembolic meningoencephalitis, and type d clostridial enterotoxemia. prevention and control. the disease can be prevented by monitoring the diet and by providing adequate roughage necessary to prevent overgrowth of thiaminase-producing ruminal flora and to maximize ruminal production of b vitamins. if excess sulfur is the primary factor, immediate removal of the source is critical. neonatal ruminants are born without immunoglobulins and must receive colostrum by hr after birth. the morbidity and mortality associated with failure of or inadequate passive transfer, such as enteric and respiratory illnesses, can be severe. measures to assure passive immunity for neonatal ruminants are covered in section ii,b, , and clinical signs of illness associated with lack of immunity are addressed in the discussions of bacterial diseases (e.g., escherichia coli infections) and, of viral diseases (e.g., diarrheas) in section iii,a, and iii,a, . generally, transfer of less than mg/dl of immunoglobulins in the serum is classified as failure of transfer, - mg/dl is partial, and above mg/dl is complete transfer. methods to determine success of transfer should be performed within a week of birth and include single radial immunodiffusion (quantitates immunogloblin classes); zinc sulfate turbidity (semiquantitative); sodium sulfite precipitation (semiquantitative); glutaraldehyde coagulation (coagulates above specific level); and, y-glutamyltransferase (assays enzyme in high concentration in colostrum and absorbed simultaneously with colostrum). treatment. early aggressive treatment is essential to save animals. the disease is treated by frequent parenteral administration of thiamine hydrochloride, the first dose being administered intravenously. dexamethasone, b vitamins, and diazepam may also be required. treatment is less successful when sulfur plays a prominent role in the etiology. research complications. this disease is preventable. although the disease is less likely to occur in smaller groups of confined ruminants, the risks of feeding concentrates or moldy feed, for example, with minimal good-quality roughage, should be kept in mind. vitamin d toxicity can result either from iatrogenic overadministration or ingestion of the plant trisetum flavescens. serum calcium levels may be high enough that blood in edta tubes will clot. laminitis is common in ruminants and can be caused by sudden changes in diet, excess dietary energy, and grain overload (or overeating). laminitis is also associated with mastitis and metritis. facility conditions, such as concrete flooring, poor manure management, and inadequate resting areas may also contribute to the pathogenesis of the disease. the complete pathogenesis of laminitis is poorly understood; however, it is thought that changes in the diet cause changes in rumen microbial populations, resulting in acidosis and endotoxemia. dramatic changes in the vascular endothelium result in chronic inflammation of the sensitive laminae of the hoof, separation of corium and hoof wall, and rotation of the third phalanx. affected animals may be reluctant to get up or walk, will shift their weight frequently, and will grind teeth or walk on carpi. chronically, the hoof wall takes on a "slipper" appearance. treatment consists of identifying the underlying cause, administering antiinflammatories (phenylbutazone, flunixin meglumin), feeding good-quality forages only, and regular foot trimming. in goats, nutritional deficiencies often manifest as a generalized poor coat that is dry, scaly, thin, and erectile. zincresponsive dermatitis has been reported in goats (smith and otherwise normal, well-managed lambs, kids, and calves can develop loose, pasty feces due to a nutritional imbalance caused by overfeeding and/or improper mixing of milk replacers. only milk replacer formulated for the particular species should be used. once nutritional imbalances are corrected, the feces readily return to normal. sudden changes in diet can also result in loose feces. photosensitization is an acute dermatitis associated with an interaction between photosensitive chemicals and sunlight. the photosensitive chemicals are usually ingested, but in some cases exposure may be by contact. animals with a lack of pigment are more susceptible to the disease. three types of photosensitization occur: primary; secondary, or hepatogenous; and aberrant. primary photosensitization is related to uncommon plant pigments or to drugs such as phenothiazine, sulfonamides, or tetracyclines. secondary photosensitization is more common in large animals and is specifically related to the plant pigment phylloerythrin. phylloerythrin, a porphyrin compound, is a degradation product of chlorophyll released by rumen microbial digestion. liver disease or injury, which prevents normal conjugation of phylloerythrin and excretion through the biliary system, predisposes to photosensitization. the only example of aberrant photosensitization is congenital porphyria of cattle (see section iii,b, ). pathologically, the photosensitive chemical is deposited in the skin and is activated by absorbed sunlight. the activated pigments transfer their energy to local proteins and amino acids, which, in the presence of oxygen, are converted to vasoactive substances. the vasoactive substances increase the permeability of capillaries, leading to fluid and plasma protein losses and eventually to local tissue necrosis. photosensitization can occur within hours to days after sun exposure and produces lesions of the face, vulva, and coronary bands; lesions are most likely to occur on white-haired areas. initially, edema of the lips, corneas, eyelids, nasal planum, face, vulva, or coronary bands occurs. the facial edema, nostril constriction, and swollen lips potentially lead to difficulty in breathing. with secondary photosensitization, icterus is also common. necrosis and gangrene may occur. diagnosis is based on clinical lesions and exposure to the photosensitive chemi-cals and sunlight. treatment is symptomatic. the prognosis for hepatogenous type may be guarded if hepatic disease is severe. from excessive straining associated with dysuria from the pressure of the fetuses and/or abdominal contents on the bladder. if the prolapse obstructs subsequent urination, rupture of the bladder may occur. the vaginal prolapse can be reduced and repaired if discovered early, and techniques in small and large ruminants are comparable. the animal should be restrained, and the prolapsed tissue should be cleansed with disinfectants. best done under epidural anesthesia, the vagina is replaced into the pelvic canal and the vulvar or vestibular opening is sutured closed (buhner suture). alternatively, a commercial device called a bearing retainer (or truss) can be placed into the reduced vagina and tied to the wool, thereby holding the vagina in proper orientation without interfering with subsequent lambing. vaginal prolapses may have a hereditary basis in ewes and cows and may prolapse the following year. these animals should be culled. vaginal prolapses may occur in nonpregnant animals that graze estrogenic plants or as a sequela to docking the tail too close to the body (ross, ) . uterine prolapses occur sporadically in postpartum ewes and cattle. the gravid horn invaginates after delivery and protrudes from the vulva. the cause is unknown, but excessive traction utilized to correct dystocia or retained placenta, uterine atony, hypocalcemia, and overconditioning or lack of exercise have been implicated. in cattle, the uterine prolapses usually develop within week of calving, are more common in dairy cows than in beef cows, and are often associated with dystocia or hypocalcemia. cows may also have concurrent parturient paresis. initially, the tissue will appear normal, but edema and environmental contamination or injuries of the tissue develop quickly. clinical signs will include increased pulse and respiratory rates, straining, restlessness, and anorexia. if identified early, the uterus can be replaced as for vaginal prolapses. electrolyte imbalances should be corrected if present. additional supportive therapy, including the use of antibiotics should always be considered. tetanus prophylaxis should be included. oxytocin should be administered to induce uterine reduction. vaginal closures are less successful at retaining uterine prolapses. preventive and control measures include regular exercise for breeding animals, and management of prepartum nutrition and body condition. vaginal and uterine prolapses occur in ewes, does, and cows. the conditions are not common in does. vaginal prolapses usually occur during late gestation and may be related to relaxation of the pelvic ligaments in response to hormone levels. in sheep, these are most common in overconditioned ewes that are also carrying twins or triplets. overconsumption of roughages, which distends the rumen, and lack of exercise leading to intraabdominal fat may predispose an animal to vaginal prolapse by increasing intra-abdominal pressure. the condition may result f rectal prolapse rectal prolapse is common in growing, weaned lambs and in cattle from months to years old. the physical eversion of the rectum through the anal sphincter is usually secondary to other diseases or management-related circumstances. rectal prolapses may occur secondary to gastrointestinal infection or inflammation, especially when the colon is involved. diseases that cause tenesmus, such as coccidiosis, salmonellosis, and intestinal worms, may result in prolapse. urolithiasis may result in prolapses as the animal strains to urinate. any form of cystitis or urethritis, vaginal irritation, or vaginal prolapse and some forms of hepatic disease may lead to rectal prolapse. abdominal enlargement related to advanced stages of pregnancy, excessive rumen filling or bloat, and overconditioning may cause prolapse. finally, excessive coughing during respiratory tract infections, improper tail docking (too short), growth implants, prolonged recumbency, or overcrowded housing with animal piling may lead to prolapses. diagnosis is based on clinical signs. early prolapses may be corrected by holding the animal with the head down, while a colleague places a pursestring suture around the anus. the mucosa and underlying tissue of prolapses that have been present for longer periods of time will often become necrotic, dry, friable, and devitalized and will require surgical amputation or the placement of prolapse rings to remove the tissue. rectal prolapse may also be accompanied by intestinal intussusceptions that will further complicate the treatment and increase mortality. occasionally, acute rectal prolapse with evisceration will result in shock and prompt death of the animal. prognosis depends on the cause and extent of the prolapse as well as the timeliness of intervention. in all cases of treatment, determination and elimination of the underlying cause are essential. gastrointestinal accumulations or obstructions of hair (and/ or sometimes very coarse roughage, forming bezoars) occur in cattle and sheep. cattle that are maintained on a low-roughage diet, that lick their coats frequently, that have long hair coats from outdoor housing, or that have heavy lice or mite infestations and associated pruritus will often develop bezoars. in addition, younger calves with abomasal ulcers have been found to be more likely to have abomasal tric. hobezoars as well. clinical signs may be mild or severe according to size, number, and location. ruminal trichobezoars rarely result in clinical signs. obstruction will be accompanied by signs of pain, development of bloat, and decreased fecal production. serum profiles will show hypochloridemia; other imbalances depend on the duration of the problem. diagnosis is also based on abdominal auscultation, rectal palpation, and ultrasound (useful in calves and smaller ruminants). treatment is surgical, such as paracostal laparotomy (for abomasal), paralumbar celiotomy with manual breakdown, or enterotomy. supportive care should be administered as necessary to correct electrolyte imbalances and to prevent inflammation and sepsis. prognosis is generally good if the condition is diagnosed and treated before dehydration and imbalances become severe and peritonitis develops. prevention includes providing good-quality roughage and treating lice and mange infestations. wounds may be sustained from poorly constructed pens or fences, or from skirmishes among animals. predators will usu-ally be sources of bite wounds. standard veterinary wound assessment and care are essential for wounds or bites. tetanus antitoxin may be indicated. use of approved antibiotics may be appropriate. the lesion should be cleaned with disinfectants and repaired with primary closure if it is clean and uncontaminated. thorough cleaning, regular monitoring, and healing by second intention are recommended for older wounds. abscesses may also occur in the soft tissues of the hooves (sole abscesses; see section iii,c, ) because of entrapped foreign bodies or hoof cracks that fill with dirt. preventive measures include improvement of housing facilities, pens, and pastures; monitoring hierarchies among animals penned together; and implementing predator control measures, such as sound fencing, flock guard dogs, or donkeys, in pasture situations. acute anaphylatic reactions in sheep, goats, and cattle are often clinically referable to the respiratory system. anaphylactic vaccine reactions cause acute lung edema; lungs are the primary site of lesions if collapse and death are sequelae. the animals will also be anxious and shivering and will become hyperthermic. salivation, diarrhea, and bloat also occur. immediate therapy must include epinephrine by intravenous infusion at ( ml of : per kg of body weight for goats and : , ( . mg/ml) or . mg/kg (about ml) for adult cows.) furosemide ( mg/kg) may be beneficial to reduce edema. prognosis is usually guarded. recovery can occur within hr. in a research environment, catheter sites or experimental surgeries may be sources of iatrogenic infection. traumatic injuries to peripheral nerves can cause acute lameness. improper administration of therapeutics can easily cause this type of lameness. injections given in gluteals or between the semimembranosus and semitendinosus can cause irritation to the sciatic nerve and subsequent lameness. contraction of the quadriceps results in the limb being pulled forward. injections in the caudal thigh can damage the peroneal nerve and cause knuckling at the fetlock. traumatic injury to the radial nerve can result in a "dropped elbow" (nelson, ) . husbandry procedures such as tail docking, castration, dehorning, dosing with a bailing gun, and shearing may result in superficial lesions, dermal infections, or cases of tetanus. bailing-gun injuries to the pharynx may lead to cellulitis with coughing, decreased appetite, and sensitivity to palpation. standard veterinary assessment and care are essential for these cases. local and systemic antibiotics with supportive care may be indicated. swelling around peripheral nerves caused by inoculations may be reduced by diuretics and anti-inflammato-ries. mild cases of peripheral nerve damage may recover in - days. personnel training, including review of relevant anatomy, preprocedure preparation, appropriate technique, careful surgical site preparation, rigorous instrument sanitation, and sterile technique will minimize the incidence of potential complications from surgical procedures. albumin values and foaming urine. the proteinuria also distinguishes amyloidosis (and glomerulonephritis) from other causes of weight loss and diarrhea in cattle such as johne's disease, parasitism, copper deficiency, salmonellosis, and bovine viral diarrhea virus infection. prognosis is poor, and no treatment is reported. neoplasia and tumors are relatively rare in ruminants. lymphosarcoma/leukemia in sheep has been shown to result from infection by a virus related (or identical) to the bovine leukemia virus. pulmonary carcinoma (pulmonary adenomatosis) and hepatic tumors are found in sheep. virus-induced papillomatosis (warts), discussed in section iii,a, ,s, and squamous cell carcinomas have also been reported in sheep. in goats, thymoma is one of the two most common neoplasias reported, although no distinct clinical syndrome has been described. cutaneous papillomas are the most common skin and udder tumor of goats, and although outbreaks involve multiple animals, no wart virus has been identified. persistent udder papillomas may progress to squamous cell carcinoma. lymphosarcoma is reported rarely in goats. although adrenocortical adenomas have been reported frequently and almost exclusively in older wethers, no clinical condition has been described. lymphosarcoma of various organ systems and "cancer eye" (bovine ocular squamous cell carcinoma, or oscc) are the most commonly reported cancers in cattle. lymphosarcoma is described in section iii,a, ,c. lack of periocular pigmentation and the amount and intensity of exposure to solar ultraviolet light are considered important factors in oscc. genetic factors may also play a role. many cases occur in herefords. this is a disease of older cattle; no case has been reported in animals less than years of age. the cancer metastasizes through the lymph system to major organs. treatment in either lymphosarcoma or oscc is recommended only as a palliative measure. the extent of ocular neoplastic involvement is a significant criterion for carcass condemnation. papillomatosis (warts) are common in cattle (see section iii,a, ,s). dental wear is seen most commonly in sheep. as sheep age, excessive dental wear may lead to an inability to properly masticate feed, manifesting as weight loss and unthriftiness. several factors predisposing to dental wear should be considered. the diet should be properly balanced for minerals, especially calcium and phosphorus, because primary or secondary calcium deficiency during teeth development results in softening of the enamel and dentin. dietary contamination with silica (i.e., hays and grains harvested in sandy regions) will lead to mechanical wear on the teeth. likewise, animals grazing or being fed in sandy environments will have excessive tooth wear. sheep older than about years of age are especially prone to tooth wear and should be checked frequently, especially if signs of weight loss or malnutrition are evident. managing the content and consistency of the diets can best prevent the disease. of the ruminants, cows are the most frequently affected by subsolar absesses. dirt becomes packed into cracks in the horny layer of the sole of the hoof, and contamination eventually extends into the sensitive areas of the hoof, with lameness and infection resulting. animals maintained in very soiled or muddy conditions, combined with poor hoof care, are more likely affected. fusobacterium necrophorum is often the pathogen involved. separation of the animal, supportive care, surgical drainage, and antibiotic treatment are indicated. amyloidosis amyloidosis in adult cattle is due to accumulations of amyloid protein in the kidney, liver, adrenal glands, and gastrointestinal tract. the disease has been classified as aa type, or associated with chronic inflammatory disease, although other unknown factors are believed to be involved in some cases. clinical signs include chronic diarrhea, weight loss, decreased production, nonpainful renomegaly, and generalized edema. the loss of protein in the urine contributes to abnormal plasma advances in sheep and goat medicine animals and animal products, subchapter a, animal welfare formulary for laboratory animals domestic animal behavior for veterinarians and animal scientists schlam's veterinary hematology diseases of sheep animal feeding and nutrition guide for the care and use of laboratory animals veterinary drug handbook veterinary medicine: a textbook of the diseases of cattle, sheep, pigs, goats, and horses sheep production and management animal and plant health inspection service (aphis), policy # , farm animals used for nonagricultural purposes goats the clinical syndromes caused by salmonella infection armed forces institute of pathology (afip) ( ) the effect of stress on the carrier state of salmonella typhimurium in goats bibliography of naturally occurring models of human disease clinical signs, treatment, and postmortem lesions in dairy goats with enterotoxemia: cases control of the estrous cycle the goat industry: feeding for optimal production neurologic disease in sheep and goats modern breeds of livestock the sheep gene map pasteurella haemolytica complicated respiratory infections in sheep and goats ungulates as laboratory animals diagnosis of lameness in sheep an overview of the influence of ace inhibitors on fetalplacental circulation and perinatal development protozoan infections (toxoplasma gondii, neospora caninum, and sarcocystis spp.) in sheep and goats: recent advances cloned transgenic calves produced from nonquiescent fibroblasts transgenic bovine chimeric offspring produced from somatic cell-derived stem-like cells use of an animal model of trichomoniasis as a basis for understanding this disease in women council report: vaccination guidelines for small ruminants (sheep, goats, llamas, domestic deer, and wapiti) ( ) maedi-visna and ovine progressive pneumonia pathophysiology of oestrus ovis infection in sheep and goats: a review experimental surgery in farm animals evaluation of an agar gel immunodiffusion test kit for detection of antibodies to mycobacterium paratuberculosis in sheep veterinary laboratory medicine induction of human tissue plasminogen activator in the mammary gland of transgenic goats pasteurella haemolytica infections in sheep coccidiosis and cryptosporidiosis in sheep and goats the major histocompatibility complex region of domestic animal species brucella melitensis infection in sheep: present and future hemoglobin switching epididymitis in rams current veterinary therapy: food animal practice livestock handling guide: management practices that reduce livestock bruises and injuries, and improve handling efficiency. livestock conservation institute synchronization of oestrus in the boer goat doe: dose effect of prostaglandin in the double injection scheme. south afr guide to the dissection of domestic ruminants reproduction in farm animals review of polyclonal antibody production procedures in mammal and poultry considerations in the design and construction of facilities for farm species clinical update: leptospirosis the sheep as an experimental animal bibliography oflnduced animal models of human disease bibliography of naturally occurring models of human disease postpartum assessment and care of the newborn ruminant animal genetics guide for the care and use of laboratory animals blackleg: a new perspective on an old disease protecting calves from viral diarrhea bovine leukemia virus. part : descriptive epidemiology, clinical manifestations, and diagnostic tests bovine leukemia virus. part : risk factors of transmission bovine leukemia virus. part : zoonotic potential, molecular epidemiology, and an animal model. in "infectious disease in food animal practice bovine leukemia virus. part : economic impact and control measures brucella abortus strain rb vaccine: its advantages and risks current veterinary therapy: food animal practice neural control of maternal behavior and olfactory recognition of offspring comparison of the burdizzo and rubber ring methods for castrating and tail docking lambs postpartum care of the cow and calf advances in the control of foot rot in sheep mastitis in ewes giardiasis in large animals laboratory diagnostic tests for retrovirus infections of small ruminants effects of dietary vitamin e supplementation during late pregnancy on lamb mortality and ewe productivity myotonia congenita (thomsen) and recessive myotonia genetic and environmental causes of variation in mastitis in sheep the gnrh system of seasonal breeders: anatomy and plasticity genetic organization, polymorphism, and function of the bovine major histocampaticulity complex scrapie in sheep biochemical and morphological expression of early prenatal caprine beta-mannosidosis respiratory infections of sheep dogs are the definitive hosts of neospora caninum a century of classical contagious caprine pleuropneumonia from original description to aetiology sheep and goat practice gene manipulation in goats through biotechnology minimizing morbidity and mortality from cryptosporidiosis managing dairy cows during the transition period: focus on ketosis environmental enrichment for dairy calves and pigs the trypanocidal cape buffalo serum protein is xanthine oxidase oral rehydration therapy for diarrheic calves neonatal ruminant diarrhea techniques for artificial insemination of cattle with frozenthawed semen noninfectious causes of lameness neosporosis: its prevalence and economic impact a review of yersinosis (yersinia pseudotuberculosis infection) dairy goat reproduction maedivisna virus in sheep: a review evaluation of a commercially available vaccine against corynebacterium pseudotuberculosis for use in sheep ruminant production management: control programs for gastrointestinal nematodes in sheep and goats fetal brain injury following prolonged hypoxemia and placental insufficiency: a review managing cryptosporidium and giardia infections in domestic ruminants milk fever: seeking new solutions to an old problem recent advances on ovine chlamydial abortion risk factors for abomasal displacement in dairy cows the spider syndrome: a report on one purebred flock development of ingestive behavior current state of in vivo preclinical heart valve evaluation dermatophilus congolensis infections in cattle and sheep ovine listeric encephalitis coccidiosis in ruminants principles of dairy science human factor ix transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts the major histocompatibility complex region of domestic animal species clinical reproductive anatomy and physiology of the doe immunobiology of the mammary gland coccidiosis brucella abortus strain rb : a new brucellosis vaccine for cattle use of age and serum gamma-glutamyltransferase activity to assess passive transfer status in lambs effect of congenitally acquired neospora caninum infection on risk of abortion and subsequent abortions in dairy cattle artificial control of breeding in ewes toxoplasmosis infection in sheep bovine reproductive biotechnology transgenic dairy cattle. genetic engineering on a large scale the effect of intra-mammary inoculation of lactating ewes with pasteurella haemolytica isolates from different sources bovine surgery and lameness reduction of myocardial myoglobin in bovine dilated cardiomyopathy intraosseous infusion of prostaglandin e prevents disuse-induced bone loss in the tibia estrous cycle synchronization the bronchopneumonias (respiratory disease complex of cattle, sheep, and goats) the cattle gene map treatment and control of gastrointestinal nematodes in sheep diagnosis, treatment, and management of enteric colibacillosis key: cord- - kgfwjzd authors: neo, jacqueline pei shan; tan, boon huan title: the use of animals as a surveillance tool for monitoring environmental health hazards, human health hazards and bioterrorism date: - - journal: veterinary microbiology doi: . /j.vetmic. . . sha: doc_id: cord_uid: kgfwjzd abstract this review discusses the utilization of wild or domestic animals as surveillance tools for monitoring naturally occurring environmental and human health hazards. besides providing early warning to natural hazards, animals can also provide early warning to societal hazards like bioterrorism. animals are ideal surveillance tools to humans because they share the same environment as humans and spend more time outdoors than humans, increasing their exposure risk. furthermore, the biologically compressed lifespans of some animals may allow them to develop clinical signs more rapidly after exposure to specific pathogens. animals are an excellent channel for monitoring novel and known pathogens with outbreak potential given that more than % of emerging infectious diseases in humans originate as zoonoses. this review attempts to highlight animal illnesses, deaths, biomarkers or sentinel events, to remind human and veterinary public health programs that animal health can be used to discover, monitor or predict environmental health hazards, human health hazards, or bioterrorism. lastly, we hope that this review will encourage the implementation of animals as a surveillance tool by clinicians, veterinarians, ecosystem health professionals, researchers and governments. zoonosis is derived from the greek words "zoon" (animals) and "nosos" (disease), referring to any infectious diseases transmitted from animals to humans, either directly or indirectly (world health organization, ) . as the global human population increases, so will anthropogenic pressures on wildlife and the environment, augmenting the likelihood of zoonotic pathogen spillover from animal to human populations. the world health organization (who) identifies zoonoses as emerging threats and describe them as previously occurring phenomena that have an increasing trend and expansion in geographical, host or vector range. more than % of all emerging infectious diseases are from zoonoses (mackenzie and jeggo, ) . despite acting as the main reservoir for only % of the known zoonoses, humans are the main source of identification for disease outbreaks (frank, ) . as such, epidemiological relationships agents in an analogous manner to humans and manifest similar disease symptoms. some animals have biologically compressed lifespans, consequently developing clinical signs more rapidly after exposure to specific pathogens. furthermore, they may be more susceptible to contaminants than humans and they do not share some human behaviors that may confound investigation results (e.g. smoking). table provides a list of websites containing information related to the use of animals for surveillance. proper utilization of animals for surveillance may allow the early identification of epidemics, which facilitates mitigation of its magnitude, or prevention of its occurrence (chomel, ; kahn, ) . this is due to the ability of animals to: ) exhibit changes in the occurrence or prevalence of a pathogen or disease with time, ) serve as markers for on-going exposure risk, ) allow examination of hypotheses on the ecology of pathogens, and ) provide information on the efficiency of disease control measures (mccluskey, ) . this review attempts to highlight animal illnesses, deaths, biomarkers or sentinel events, to remind human and veterinary public health programs that animal health can be used to discover, monitor or predict environmental and human health hazards, or bioterrorism. animals useful for surveillance mostly exist in the environment as hosts of naturally cycling pathogens. they can be utilized in passive, active or sentinel surveillance programs. passive (reactive) surveillance involves the spontaneous reporting of disease data from the animal sector to veterinary authorities (hoinville et al., ) . data reported can include illnesses or deaths in animals, or notifiable diseases that must be reported by law. the data is then analyzed to observe disease trends and identify potential outbreaks. active (proactive) surveillance on the other hand involves calling on animal facilities to interview workers and to review animal health records to identify diseases under surveillance. it also involves actively monitoring domestic or wild animals for biomarkers. the choice of surveillance type depends on the characteristics of a pathogen and the objective of the program. passive surveillance is best employed when the objective of the program is targeted towards early detection of outbreaks or monitoring the extent of disease for making decisions on control strategies; whereas, active surveillance is best employed when a disease is targeted for elimination. passive surveillance is the least time consuming, labor intensive and expensive of the three forms of surveillance, and covers an extensive range. however, because it relies heavily on reports from veterinarians who receive little incentive for reporting, the data reported is frequently incomplete and delayed. underreporting of disease suspicions is also known to be a major cause of disease control failure (fao, ) and multiple studies have been conducted to better comprehend the decisionmaking processes behind underreporting so as to develop recommendations for improved passive surveillance (bronner et al., ; delabouglise et al., ; paul et al., ; sawford et al., ; thompson et al., ) . in contrast, active surveillance demands more time and resources and is thus less commonly employed. however, it provides more complete and accurate data than passive surveillance. a study comparing active and passive animal surveillance in chad concluded that for monitoring of existing diseases, the less expensive passive surveillance is better (marr and calisher, ) . historian plutarch mentioned that flock of ravens displayed unusual behavior and died subsequently as alexander entered babylon. several hundred livestock deaths death of several hundred livestock in lake alexandrina, australia allowed the identification of cyanobacteria nodularia spumigena in water. despite warnings issued, there was undescribed illness was reported in one individual after consuming contaminated water (codd et al., ) . th century canaries in coal mines coal miners in the u.k. and the u.s. brought canaries into coalmines as an early-warning signal for toxic gases including methane and carbon monoxide. the birds, being more sensitive, would become sick before the miners, who would then have a chance to escape or put on protective respirators (burrell and seibert, ). minamata disease cats from a fishing village, minamata developed a neurological disease. people of minamata later displayed similar symptoms. investigations later found that effluent from a factory had polluted surrounding waters resulting in accumulation of mercury in fish (takeuchi et al., (takeuchi et al., ). chicken sentinels chickens as sentinels for surveillance of arboviruses like wnv, wee and sle viruses (rainey et al., ) . rabbits warn of nerve gases during transportation rabbits were placed in small cages in railcars during transportation of nerve gases and sudden animal mortality would warn of gas release (brankowitz, ) . april sverdlovsky anthrax release anthrax was accidentally released from a soviet military microbiology facility. livestock died at a greater distance of km from the plant, compared to human cases which occurred within a narrow km zone downwind of the facility (meselson et al., ) . west nile virus wnv was reintroduced into the u.s., where it caused the ongoing epizootic in birds with a spillover of infections to humans and equines (chancey et al., ) . chickens on alert in kuwait u.s. marine corps employed chickens for the detection of nerve and blister agents. they were meant to act as a backup to false alarms the automated detectors were notorious for (ember, (ember, ). dog, livestock, wildlife deaths death of dogs, livestock and wildlife in the buccaneer bay lake, eastern nebraska, u.s. allowed identification of cyanobacteria anabaena, microcystis, oscillatoria and aphanizo-menon in water. > incidences of skin lesions, rash, gastroenteritis and/or headache were reported in humans. warnings were issued (walker et al., (walker et al., ). plague cases in yulong county of the yunnan province, china serologic survey found antibodies against the f antigen from domestic dogs around the affected county, demonstrating that domestic dogs could serve as animals for plague surveillance (li et al., ) . windblown lead carbonate in esperance, western australia windblown lead carbonate causing huge number of bird deaths in esperance, western australia, prevents lead exposure to esperance community (gulson et al., ). suited for chad's conditions. whereas, for monitoring of rare diseases, active surveillance of animal herds is required to complete passive surveillance (ouagal et al., ) . occasionally, when high-quality data about a specific disease is required, animal sentinels are intentionally deployed for surveillance. these animals receive greater attention than would be possible with active or passive surveillance. sentinel surveillance is less extensive in terms of range and personnel involved compared to passive surveillance, but often yields more detailed data. it is best employed when thorough investigation of each animal or site is necessary, however it may not be as effective for detecting diseases outside the demarcated limits of the sentinel sites. examples of events involving the various forms of animal surveillance spanning bce and the st century can be found in table . the review concludes with the one health approach. a sentinel is a naïve animal which is intentionally placed in an environment of potential infection that is monitored at short time intervals to detect infection. if the sentinel is deployed close to human populations, the sentinel should react to the infectious agent (but not become infectious), thereby providing early warning of human health hazards in the environment (van der schalie et al., ) . a classic example of an animal sentinel system is the well-known canary in the coalmine (burrell and seibert, ) . canaries are sensitive to the effects of poisonous gases, particularly carbon monoxide, and were routinely taken into the mines to warn of dangers. its inclination to sing much of the time, coupled with its brightly colored plumage offered both "audio and visual" cues to the miners. if the canary stopped singing and/or fell from its perch, this was the signal for the miners to don their respirators or evacuate. many miners owe their existence and livelihood to this historic animal sentinel. besides canaries, other animal species have also been used as sentinels of toxic chemical exposure. for example, birds, horses, cats, guinea pigs, rats, mice and rabbits were employed as sentinels for chemical agent exposure during world war i (wwi) and wwii. until , rabbits were placed in small cages in railcars during the transportation of nerve gases and sudden animal mortality would warn of gas release (brankowitz, ) . although technological advancements have since resulted in the more widespread use of electronic detectors for detecting toxic chemicals, animal sentinels are still superior because of the complexity and likeness of the animal and human physiology. this is evidenced by more recent uses of animals, in particular avian species, as sentinels of toxic chemical exposure. for example, on march , , the tokyo underground trains were hit by synchronized chemical terrorist attacks (national research council of the national academies, ). the aum shinrikyo religious sect dispensed a concoction of military nerve agent, sarin, killing twelve and injuring thousands. as a precaution, the japanese policemen carried canaries À the very primitive animal sentinel À in cages with them to warn of the presence of toxic gases during raids. another recent example of the use of avian species as sentinels of toxic chemical exposures was in , when u.s. marine corps employed chicken sentinels at the kuwaiti staging area despite the deployment of automated detectors (ember, ) . the chickens were employed to complement the m ionmobility spectrometer, which was used to tag nerve and blister agents. they were meant to act as a backup to false alarms the automated detectors were notorious for. other than as sentinels of toxic chemical exposure, the avian species has also proven itself to be a valuable sentinel of disease outbreaks. for example, chickens have been used as sentinels for the surveillance of arboviruses like west nile virus (wnv), western equine encephalomyelitis (wee) and st. louis encephalitis (sle) viruses (moore et al., ) . they are amenable to and can tolerate arboviral infections with little or no symptoms, developing antibodies within a week of being bitten by an infected mosquito. they produce low tittered viremia, are cheap to purchase, robust and easily bled (biweekly or monthly during the peak season from june to october), making them excellent sentinel animals of arboviruses. despite providing accurate spatiotemporal information on virus transmission, the relationship between mosquito transmission and percentage of bird and mosquito infections in a particular region still needs to be determined in order to precisely evaluate human risk. in order to improve their use as sentinels, chicken interferon-a can be administered perorally in drinking water, where it acts as an adjuvant, inducing rapid seroconversion in chickens after infection by low pathogenic avian influenza (lpai). these chickens are called 'super-sentinels' since they are able to detect clinically inapparent lpai (marcus et al., ) . lpai strains are the most widespread, and can mutate into highly pathogenic avian influenza (hpai) strains, which can lead to human transmission and potential fatalities. thus, by placing super-sentinel chickens in locations prone to bird flu outbreaks, for example live-bird markets, this would allow early detection of lpai, thereby buying time for its control. in spite of the value of animal sentinels in monitoring the presence of pathogens or chemicals in the surroundings, there are ethical concerns regarding the deliberate exposure of animals to danger by placing them at sites of suspected contamination. consequently, the surveillance of extant animals in their natural habitats could act as an alternative means to warn of human, veterinary or environmental health hazards. animals in many habitats can be studied to monitor health hazards in the environment (reif, ) . environmental health hazards refer to both natural and unnatural contaminants in air, water, soil or food, which can potentially lead to acute or chronic health issues in humans (national research council (u.s.) and committee on animals as monitors of environmental hazards, ) . a variety of marine species are excellent surveillance tools of environmental stress and potential health threats for humans. for example, the mussel watch program actively analyzes sediment and bivalve tissue chemistry for a suite of organic contaminants and trace metals to identify trends at over selected u.s. coastal sites from to today. it is designed to identify deleterious changes in the marine habitat and indicate potential human health concerns (kim et al., ) . anomalocardia brasiliana is also a good surveillance tool for actively monitoring contamination levels of coliforms in shellfish harvesting regions in brazil's northeast coast (lima-filho et al., ) . mussels, clams and oysters are particularly suitable for us as surveillance tools because they are able to bioaccumulate many chemicals (o'connor and lauenstein, ) , as well as concentrate microbial organisms and pathogens (kueh and chan, ) to concentrations in excess of fold (grodzki et al., ) . thus, the high concentrations of chemical and pathogens make it easier to detect environmental and health threats in these organisms. moreover, improved sequencing technologies have led to the monitoring of bivalves via genomics, epigenomics, transcriptomics, proteomics and metabolomics (suarez-ulloa et al., ) . such integrative omic studies will make powerful tools in the biomonitoring of marine pollution. besides marine species, cats can be potentially used as a passive surveillance tool for monitoring toxic chemicals in the aquatic ecosystem. in , japanese veterinarians discovered a neurological disease in cats in the minamata fishing village (takeuchi et al., ) . it was called "dancing cat fever" because the cats displayed convulsions and involuntary jumping movements. however, this disease was not investigated rigorously until similar symptoms also manifested in the people of minamata. as a result of subsequent epidemiologic studies in minamata, researchers realised that effluent from a factory had polluted surrounding waters resulting in the accumulation of mercury in fish. subsequent consumption of contaminated fish by fishermen and their families resulted in high mercury concentrations in their brains, kidneys and livers. had the authorities paid more attention to the cats' disease symptoms, this could have been prevented. nevertheless, this episode raised the awareness of cats as a surveillance tool for monitoring mercury poisoning, food safety and public health throughout the world. besides mercury poisoning, there have been increasing numbers of reports of human or animal illnesses or deaths associated with harmful cyanobacteria blooms in freshwater systems. hence, in a similar way, the surveillance of fish, dogs or livestock can provide important early warnings of cyanobacteria-associated environmental hazards (hilborn and beasley, ) . more recently in , the "birds dropping from the sky" phenomenon demonstrated how passive monitoring of bird dieoffs alerted the community of esperance, western australia to a case of lead poisoning in the environment (gulson et al., ) . during that period, the community was alarmed by the sudden death of more than birds. this sparked an urgent investigation which eventually revealed that the birds had died of lead poisoning. the lead ore concentrate had originated from the handling of lead carbonate concentrate at the megallan mine km north of esperance. the western australia department of health and local shire council measured lead concentrations in rainwater from household tanks (the main source of drinking water) and discovered that % of households had lead concentrations exceeding who guidelines of mg/l. although the death of numerous native bird species was tragic, it triggered the investigation, which ultimately prevented the catastrophic exposure of lead to the community. the clear and present dangers of emerging infectious diseases have propelled world governments to enhance animal surveillance activities (gubernot et al., ) . due to the zoonotic origin of most human health hazards, it is thought that animals may have a greater susceptibility to zoonotic pathogens, thereby justifying their use as surveillance tools for monitoring human health hazards. for example, in , death and illness in multiple avian species aided investigators in identifying wnv as the root of the encephalitis outbreak in humans in new york. during those periods, the unusually high numbers of encephalitis cases in humans was concurrent with a surge in dying crows with neurological symptoms similar to encephalitis patients (eidson et al., ) . this prompted investigations, which identified wnv as the cause of the outbreak, and demarcated its geographical limits since the crows were amplification hosts for viral transmission. the cdc, u.s. geological survey national wildlife health centre and u.s. department of agriculture have since been involved in the battle against wnv. strategies are currently in place to consolidate data on human, mosquito, bird, chicken and veterinary cases of west nile infection in the arbonet system (u.s. geological survey and cdc, ). other than birds, dogs could also act as surveillance tools for monitoring wnv circulation. seroconversion was detected in juvenile dogs weeks before wnv appeared in humans in houston, texas (resnick et al., ) . hence, active surveillance of wnv seroprevalence in birds and dogs can be used for monitoring wnv activity. additionally, the active surveillance of swine and live bird markets or supply abattoirs at the human-animal interface could be used to monitor the risk of hpai to human and animal populations. in and , the h n swine flu pandemic claimed more than , lives. this new strain resulted when a triple reassortment of northern american swine, bird and human flu viruses further combined with a eurasian pig flu virus (trifonov et al., ) . also in was the h n avian influenza outbreak which led to the intense surveillance of wild ducks for avian flu viruses in europe (globig et al., ) . in eastern asia, wild swans are an ideal surveillance species as there is vast geographical overlap between whooper swan distributions and h n outbreak areas (newman et al., ) . other hosts of hpai include cats and dogs (cleaveland et al., ; kuiken et al., ) . these studies show that active surveillance of suitable animal species through serosurveys could provide early warnings of hpai foci, accelerating public health investigation and action. bats are also important animals from a surveillance perspective. they have long life spans, are highly mobile and are increasingly well adapted to human environments due to habitat loss as a result of land use changes. they live in close proximity to humans, and interact with livestock and domestic animals that are potential intermediate hosts for pathogens. bats are the natural reservoir of severe acute respiratory syndrome coronavirus (sars-cov) (lau et al., ; li et al., ) , which was responsible for the sars-cov outbreak in , with known infected cases and confirmed human deaths worldwide (who, ) . besides sars-cov, bats are also reservoir hosts to filoviruses like ebola (leroy et al., ) and marburg (towner et al., ) viruses; paramyxoviruses like hendra (halpin et al., ) and nipah (yob et al., ) viruses; rubulaviruses like tioman (chua et al., ) and menangle (philbey et al., ) viruses; and the australian fruit bat lyssavirus (fraser et al., ) . there are however various challenges associated with the active surveillance of bats. firstly, the collection of blood and fluid samples from bats is dangerous given their highly infectious nature. secondly, the collection of bat specimens is difficult in remote areas. thirdly, bats are sensitive to disturbances and may migrate as a consequence of investigations, making it difficult to locate bat colonies. hotspots with high human and bat population density have thus been identified to focus bat surveillance efforts on areas with the highest probability of the emergence of zoonoses (jones et al., ) . dromedaries may also be potentially useful as surveillance tools. in particular, active surveillance of dromedaries in herds and large abattoirs could potentially reveal the prevalence of middle east respiratory syndrome coronavirus (mers-cov) infection. in , mers-cov was first detected in humans in saudi arabia. sera from dromedary camels across and beyond the arabian peninsula were found to harbor high levels of antibodies against mers-cov (reusken et al., ) . indeed, viral sequencing revealed nucleotide polymorphism signatures, indicative of cross-species transmission (chu et al., ; memish et al., ) . this suggests that human mers-cov infections could have been zoonotically acquired from camels and that their surveillance could reveal mers-cov foci. mosquitoes, aedes aegypti and potentially aedes albopictus, transmit brazilian zika virus (zikv) among humans. in , the first zikv infection was confirmed, and within a few months it was declared by the who to be a public health emergency of international concern (aziz et al., ) . health authorities have found zikv disease to be associated with auto-immune and neurological complications, and microcephaly in babies. transmission has been rampant in various regions and zikv is expected to spread to new territories. hence, the active surveillance of mosquitoes could enable the evaluation of vector control measures to determine the efficacy of zikv outbreak interventions. bioterrorism is the intentional release of microorganisms or biological agents to cause disease or death in humans, animals or plants to influence government conduct or threaten civilian population (cdc, ) . since more than % of bioweapons are zoonoses, animals are likely to be at high risk (ryan, ) and thus the surveillance of animals may provide early warning of a bioterrorist attack (rabinowitz et al., ) . farm animals like sheep and cows are potentially valuable surveillance tools for passively monitoring the production or release of bioterrorism weapons in rural areas. bacillus anthracis, the causative agent of anthrax, which has fatality rates of near % in both humans and animals, has been identified by cdc as one of the most likely biological agents to be used (cdc, ) . moreover, anthrax can form resilient spores that persist for decades in soil. during wwii, the british government was experimenting the use of anthrax on gruinard island. despite efforts to decontaminate the island, the long-lasting contamination of the soil by anthrax spores put the island under quarantine for years before it was considered safe for human use ("britain's anthrax island," ) . this highlights the importance of passively monitoring random cases in animals to identify anthrax hot spots. in , b. anthracis spores were inadvertently released from a soviet military microbiology facility in sverdlovsky (meselson et al., ) . livestock km away from the plant died, whereas human cases occurred within km downwind of the facility. analysis showed that the dosage of b. anthracis at which sheep and cows became ill was more than an order of magnitude lower than the dosage required to affect humans. this analysis therefore suggests that livestock are much more susceptible than humans to b. anthracis and would be ideal for use as surveillance tools for b. anthracis since lower dosages at greater distances from the source were sufficient to affect the animals. furthermore, animals like domestic dogs and rodents spend more time outdoors and have greater exposure to the environment than humans, making them great surveillance tools for monitoring plague. yersinia pestis, the etiological agent of plague, has also been identified by cdc as a bioterrorism agent (cdc, ) . it appeared in humans in the u.s. in the s (link, ; lipson, ) , and became established enzootically in wild rodents by the mid- s. it is hypothesized that plague is maintained by reservoir species like rodents, and that carnivores become ill following the ingestion of plague-infected rodents. in , plague cases were confirmed in the yulong county of the yunnan province, china (li et al., ) . a survey of serum samples of domestic dogs in and around the affected county confirmed that they could be used for active plague surveillance. the dynamics of infectious diseases are highly variable. they are determined by infecting dose, pathogen characteristics, host susceptibility and transmission routes. it is therefore important to have a framework for the proper evaluation of animals to determine if they are suitable for use as sentinels for surveillance. to this end, the halliday et al. ( ) conceptual framework effectively evaluates animals as sentinel populations for various surveillance aims and ecological settings (halliday et al., ) . there are three fundamental components of the sentinel framework: the pathogen under surveillance, the target population, and the sentinel population. the sentinel framework produces a sentinel response which could take the form of seroconversion, current infection, morbidity, mortality and changes in morphology or behavior. in addition to the sentinel response, other sentinel practical factors and host factors also influence the detectability of the sentinel response, which eventually determines the utility of the sentinel. as alluded to earlier, although there are numerous benefits in using animal sentinels, its use is associated with ethical concerns which might be alleviated by the surveillance of existing animals in their natural habitats. in spite of the obvious potential of animals as a surveillance tool for monitoring environmental damage, risks to human health and bioterrorism, animals currently appear to be underutilized as surveillance tools. a likely reason is that human and animal health surveillance efforts mostly stem from disparate initiatives, resulting in data being kept in entirely separate databases (scotch et al., ) . there is thus an increasing need for interdisciplinary integration between human and veterinary medicine, and communication before we can completely exploit the benefits of animals for surveillance (bisdorff et al., ; wendt et al., ) . to this end, committees have been established to increase awareness of the mutual reliance between human, animal, plant, microbial, and ecosystem health (rabinowitz et al., ) . this includes the 'one medicine' or 'one health' initiatives like the one health commission, the one health initiative and the comparative clinical science foundation (zinsstag et al., ) . the term 'one medicine' was coined in by calvin w. schwabe in recognition of the mutualism of human and animal health, nutrition and livelihood (schwabe, ) . this mutualism is further supported by the close genomic relationship of humans and animals (peters et al., ) . today, the appreciation of the interdependence of the well-being and health of humans, animals and the ecosystems, evolved the term 'one medicine' towards 'one health', to include public health, ecology and broader societal dimensions (zinsstag et al., ) . in support of one health, predict was launched in (usaid, a). the predict project is part of united states agency for international development's (usaid's) emerging pandemic threats (ept) program, designed to identify zoonotic viral threats with pandemic potential at wildlife-human viral transmission interfaces (kelly et al., ) . it has successfully improved surveillance and laboratory capabilities for monitoring humans (that have had animal contact) and wildlife for new and known pathogens with outbreak potential; defined ecological and human causes of zoonosis; and reinforced and perfected models for predicting outbreaks. it became the largest zoonotic virus surveillance project worldwide, successfully identifying and predicting the emergence of pathogens from wildlife. it also established infrastructure and expertise required for the operation of pandemic threat surveillance and diagnostics to support the one health workforce (ohw). the huge success of ept led to the launch of ept which aims to discover diseases of known and unknown origin; minimize the possibility of disease outbreaks by reducing human activities that promote disease spillover; boost national readiness; and ultimately to reduce the repercussions of novel zoonotic pathogen emergence (usaid, b). in southeast asia, there is the one health network south east asia platform supported by the european union to promote collaboration, networking and sharing between southeast asian one health programs (massey university new zealand, ). it presently hosts two programs, lacanet and comacross. lacanet is a cambodia and laos effort aimed at improving detection of zoonotic diseases, developing capabilities for surveillance, promoting regional and national collaborations, and researching into land-use change and wildlife trade À the two main causes of disease emergence. on the other hand, comacross is a thailand, cambodia and laos effort aimed at assembling a multidisciplinary framework to address complex one health problems and to improve integration between public health, animal health, agriculture and livestock, ecology, environmental science, social science and engineering. besides the one health network south east asia platform, in southeast asia, there is also biodivhealthsea supported by the french anr cp&es. it investigates the impact of global changes and global governance on zoonotic diseases, biodiversity and health (morand et al., ) . together with comacross, biodivhealthsea and a few other bodies have proposed in a southeast asian interdisciplinary conference that ecosystems could reveal potentially harmful developments for human health (walther et al., ) . they made recommendations for the implementation of the one health approach; future research direction; education, training, and capacity building; potential science-policy interactions; and ethical and legal considerations. these recommendations should influence legislation and enforcement, thereby strengthening the health and resilience of southeast asian societies. as typical with large programs, ept, ept , lacanet, comacross and biodivhealthsea will need to apportion sufficient funds and resources for conducting surveillance, laboratory diagnosis, consumables, equipment and infrastructure. it is therefore critical that they continue to collaborate with partners like the cdc, who, ohw, food and agriculture of the united nations, institut pasteur du cambodge and lao-oxford-mahosot hospital-wellcome trust research unit, to strengthen their capacity in surveillance and laboratory capabilities, and to ensure that efforts are not replicated. it is also important that one health programs continue to receive the support and funds that they need to sustain their work. the extent of support for animal disease surveillance in communities is largely built upon its understanding of the dangers of zoonoses to human health, trade and the economy, rather than out of interest in wildlife health. accordingly, it is crucial to boost public awareness of the importance of wildlife health to societies. lastly, governments are occasionally unwilling to announce potential disease outbreaks for various reasons, including preventing the disruption of trade. hence, it is important that reporting of wildlife diseases be standardized and made necessary, and for a reporting global clearinghouse to be established. altogether, 'one health' is a unifying paradigm encouraging integration and leverage of existing capabilities among clinicians, veterinarians, ecosystem health professionals, researchers and governments. ultimately, this will hopefully lead to the development and application of sustainable and effective community health interventions, thereby reducing zoonotic disease emergence, outbreaks and repercussions, and addressing some of the biggest multidisciplinary challenges of the st century. none. zika virus: global health challenge, threat and current situation active animal health surveillance in european union member states: gaps and opportunities chemical weapons movement history compilation. office of the program manager for chemical munitions (demilitarization and binary britain's anthrax island why do farmers and veterinarians not report all bovine abortions, as requested by the clinical brucellosis surveillance system in france? experiments with small animals and carbon monoxide bioterrorism overview [www document bioterrorism agents/diseases [www document the global ecology and epidemiology of west nile virus control and prevention of emerging zoonoses mers coronaviruses in dromedary camels tioman virus, a novel paramyxovirus isolated from fruit bats in malaysia dogs can play useful role as sentinel hosts for disease toxic blooms of cyanobacteria in lake alexandrina, south australia -learning from history the perceived value of passive animal health surveillance: the case of highly pathogenic avian influenza in vietnam crow deaths as a sentinel surveillance system for west nile virus in the northeastern united states chickens on alert in kuwait challenges of animal health information systems and surveillance for animal diseases and zoonoses one world, one health, one medicine encephalitis caused by a lyssavirus in fruit bats in australia ducks as sentinels for avian influenza in wild birds bioaccumulation efficiency, tissue distribution, and environmental occurrence of hepatitis e virus in bivalve shellfish from france animals as early detectors of bioevents: veterinary tools and a framework for animal-human integrated zoonotic disease surveillance windblown lead carbonate as the main source of lead in blood of children from a seaside community: an example of local birds as canaries in the mine a framework for evaluating animals as sentinels for infectious disease surveillance isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus one health and cyanobacteria in freshwater systems: animal illnesses and deaths are sentinel events for human health risks proposed terms and concepts for describing and evaluating animal-health surveillance systems global trends in emerging infectious diseases confronting zoonoses, linking human and veterinary medicine one health proof of concept: bringing a transdisciplinary approach to surveillance for zoonotic viruses at the humanwild animal interface relationship of parasites and pathologies to contaminant body burden in sentinel bivalves: noaa status and trends mussel watch program bacteria in bivalve shellfish with special reference to the oyster public health. pathogen surveillance in animals severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats human ebola outbreak resulting from direct exposure to fruit bats in luebo, democratic republic of congo bats are natural reservoirs of sars-like coronaviruses serologic survey of the sentinel animals for plague surveillance and screening for complementary diagnostic markers to f antigen by protein microarray coliform risk assessment through use of the clam anomalocardia brasiliana as animal sentinel for shellfish harvesting areas in brazil's northeast a history of plague in united states of plague in san francisco in : the united states marine hospital service commission to study the existence of plague in san francisco reservoirs and vectors of emerging viruses super-sentinel chickens and detection of low-pathogenicity influenza virus alexander the great and west nile virus encephalitis one health network south east asia use of sentinel herds in monitoring and surveillance systems human infection with mers coronavirus after exposure to infected camels the sverdlovsk anthrax outbreak of guidelines for arbovirus surveillance programs in the united states infectious diseases and their outbreaks in asia-pacific: biodiversity and its regulation loss matter migration of whooper swans and outbreaks of highly pathogenic avian influenza h n virus in eastern asia trends in chemical concentrations in mussels and oysters collected along the us coast: update to comparison between active and passive surveillance within the network of epidemiological surveillance of animal diseases in chad practices associated with highly pathogenic avian influenza spread in traditional poultry marketing chains: social and economic perspectives the mouse as a model for human biology: a resource guide for complex trait analysis an apparently new virus (family paramyxoviridae) infectious for pigs, humans, and fruit bats animals as sentinels of bioterrorism agents animals as sentinels: using comparative medicine to move beyond the laboratory toward proof of concept of a one health approach to disease prediction and control a sentinel chicken shed and mosquito trap for use in encephalitis field studies animal sentinels for environmental and public health juvenile dogs as potential sentinels for west nile virus surveillance middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan zoonoses likely to be used in bioterrorism a focused ethnographic study of sri lankan government field veterinarians' decision making about diagnostic laboratory submissions and perceptions of surveillance veterinary medicine and human health, sub edit linkages between animal and human health sentinel data meeting report: panel on the potential utility and strategies for design and implementation of a national companion animal infectious disease surveillance system bivalve omics: state of the art and potential applications for the biomonitoring of harmful marine compounds the outbreak of minamata disease (methyl mercury poisoning) in cats on northwestern ontario reserves improving animal disease detection through an enhanced passive surveillance platform marburg virus infection detected in a common african bat geographic dependence, surveillance, and origins of the influenza a (h n ) virus west nile virus human provisional emerging pandemic threats [www document emerging pandemic threats program [www document nebraska experience summary of probable sars cases with onset of illness from biodiversity and health: lessons and recommendations from an interdisciplinary conference to advise southeast asian research, society and policy zoonotic disease surveillance?inventory of systems integrating human and animal disease information nipah virus infection in bats (order chiroptera) in peninsular malaysia from one medicine to one health and systemic approaches to health and well-being the writing of this review was supported by dso national laboratories. key: cord- - s l kua authors: kunstyr, ivo; nicklas, werner title: control of spf conditions: felasa standards date: - - journal: the laboratory rat doi: . /b - - . - sha: doc_id: cord_uid: s l kua only experimental animals of a good microbiological quality will give any kind of guarantee of an experiment undisturbed by health hazards. it is for this reason that so-called (specific pathogen free) spf animals are used for animal experiments. certain requirements are necessary to maintain the desired spf organism. physical barriers together with appropriate operating methods aim at preventing contamination with pathogens and penetration by wild rodents. as a consequence, barrier units are not easily accessible for personnel, which is sometimes considered a disadvantage by experimenters. finally, monitoring programs help to detect and control potential sources of contamination and may therefore be of crucial importance for the management of a facility housing animals of a good microbiological quality. the main purpose of health monitoring is to detect or prevent infections, which might influence physiological characteristics of animals or their health. appropriate health monitoring helps to avoid imprecise results and allows all the experiments necessary to be carried out with a minimum number of animal. it is found that sufficient number of animals have to be monitored to obtain relevant information on a given population. it is important that the monitoring must be performed on a regular basis to detect unwanted microorganisms in good time. the recommended frequency is every week. only experimental animals of a good microbiological quality will give any kind of guarantee of an experiment undisturbed by health hazards. it is for this reason that so-called 'spf' (or specific pathogen free) animals are used for animal experiments. here we focus on 'spf' rats, although experimental rats of conventional and possibly even germ-free hygienic status are also used in research and testing. most infectious agents can severely influence experimental results. therefore the detection and subsequent elimination of infectious agents is essential if improved and more reliable results from animal experiments are to be obtained. at the same time, the use of such animals reduces the number of animals needed and therefore makes an important contribution to animal welfare. the term 'spf' means that the absence of individually listed microorganisms has been demonstrated for a population by regular monitoring of a sufficient number of animals at appropriate ages by appropriate and accepted methods. 'spf' animals originate from germ-free animals. these are usually associated with a defined microflora and subsequently lose their gnotobiotic status by contact with environmental and human microorganisms. such animals are bred and housed under conditions that prevent the introduction of unwanted microorganisms, i.e. organisms that have the potential to induce disease in animals (or humans) or which are known to influence the physiological properties of their host and thus the outcome of experiments (table . ). 'spf' animals are morphologically and physiologically 'normal', well suited for modelling the situation of a human population. it has to be stressed that most infections in experimental rodents are subclinical. the absence of clinical manifestations therefore has very limited diagnostic value. however, modifications of research results due to natural infections often occur in tlhe absence of clinical disease. such modifications may be devastating for experiments because they often remain undetected (table . ). the types of interference of an agent with exper!imental results may be diverse. as an example, a detailed list of the potential influences of kilham rat virus (krv), a frequently occurring rat pathogen, on research results is given in table . (see also mossmann et al., ) . more information about the considerable effects on research due to infectious agents can be found in various review articles (bhatt et al., ; lussier, ; national research council, ; hansen, ; mossmann et al., ; baker, ; nicklas et a/., ) . most infectious diseases are multifactorial. an infectious agent alone or in insufficient quantities is usually not able to elicit the disease. support by other factors is necessary. some factors that can lead to an overt disease are listed in table certain requirements are necessary to maintain the desired hygienic quality. physical barriers together with appropriate operating methods aim at preventing contamination with pathogens and penetration by wild rodents. as a consequence, barrier units are not easily accessible for personnel, which is sometimes considered a disadvantage by experimenters. finally, monitoring programmes help to detect and control potential sources of contamination and may therefore be of crucial importance for the management of a facility housing animals of a good microbiological quality. o a% m g keeping rodents free of pathogens is a much more complex problem in research facilities than in breeding units. it is necessary that all potential sources of infections are considered and evaluated. they have been discussed in more detail by nicklas ( ) . unwanted microorganisms may be introduced into a barrier unit by various routes and materials. the most important sources of infections are infected animals of the same or closely related species (e.g. mice). in addition, biological materials (e.g. cell lines, sera, monoclonal antibodies, transplantable tumours, isolated organs, virus strains or parasites after animal-to-animal passages) may be contaminated (collins and parker, ; nicklas et al., a) . the contaminating agents may survive for years or decades when contaminated samples are stored frozen or freeze-dried. therefore, such materials must be included in regular health monitoring programmes to avoid transmission of unwanted monitoring is usually done by the rat antibody production test (rap test). this test is based on the immune response to rat viruses which is stimulated in pathogen-and antibody-free animals if the material injected is contaminated. a short protocol is given in table . ; for more details see nicklas eta/. ( a) . the polymerase chain reaction (pcr) can also be used to demonstrate the presence or absence of microorganisms in such materials but is more expensive and time consuming to perform. all additional materials that have been in contacz with infected animals may be contaminated and may act as potential vectors. however, many of them (e.g. cages, feeders, bottles, etc.) can easily be decontaminated by hygienic procedures or appropriate disinfection. another important factor is human contact. although the risk of transmitting rat pathogens by humans is very low if all personnel (caretakers, technicians, researchers) are properly educated and motivated, in practice pathogens are often transmitted as a consequence of a lack of discipline or thoughtlessness. the: main purpose of health monitoring is to detect or prevent infections which might influence physiological characteristics of animals or their health. appropriate health monitoring helps to avoid imprecise results and allows all the experiments necessary to be carried out with a minimum number of animals. in contrast to troubleshooting, which means an ad hoc search and identification of unknown causes of abnormalities in an experiment, health monitoring describes a scheduled programme for monitoring the microbiological status of an animal population. the health monitoring programme aims at determining the microbiological status of a population before and during an experiment through regular and repeated examination and monitoring for previously defined, known infectious agents. another aim of health monitoring is prevention of the introduction of unwanted organisms. as the major risk factor, the animal remains the main target of the monitoring laboratory. we must emphasize that all diseased or dead animals should be examined in addition to regular and scheduled monitoring of clinically healthy animals. they are a valuable source of information about the hygienic status of the colony. the federation of the european laboratory animal science associations (felasa) publish recommendations dealing with health monitoring of either breeding colonies or experimental units (kraft et al., ; rehbinder et al., ) . in experimental units in particular, the monitoring programme will differ between institutions or between different units of the same facility in its dependence on (a) research objectives, (b) physical conditions and the layout of the animal house, (c) husbandry methods, (d) sources of animals, (e) staff quantity and qualification, (f) diagnostic laboratory support, (g) finances. an overview on monitoring of experimental rodent colonies has been given by nicklas ( ) . in most experimental units, animals of appropriate ages will not always be available for random sampling to monitor the microbiological status. furthermore, diverse special experimental animals -transgenic, immunodeficient, pretreated-which are only available in small quantities, have been used increasingly during recent years. the use of sentinel animals is therefore advisable. sentinels are animals from a breeding colony of known hygienic status (negative for all known pathogens) which aid in the evaluation of the microbiological status of the colony. they must be housed in the population to be monitored for a sufficiently long time (minimum of - weeks) in order to develop detectable antibody titres or parasitic stages. sentinels should be kept in such a way that they receive maximum exposure to potential infections (on bottom shelves of different racks within an animal room, open cages, use of 'dirty bedding') (national research council, ) . a sufficient number of animals has to be monitored to obtain relevant information on a given population. clearly, infections with an attack rate of % or more (sendai virus, rat coronavirus/sialodacryoadenitis virus, rcv/sdav) require far fewer animals to detect their presence than infections with low attack rates. it has been recommended by the ilar committee on long-term holding of laboratory rodents ( ) that at least eight randomly sampled animals should be monitored, which is (theoretically) sufficient to detect an infection with a % probability if at least % of a population is infected. the formula which can be used to calculate the number of animals for an estimated prevalence rate is given in table . . in breeding units these animals should be at least weeks old, which ensures that they have reached immunological maturity and had sufficient time to develop detectable antibody titres or parasitic stages (e.g. worm eggs). for experimental units, the time animals have been housed in the unit to be monitored may be more important than their age. as already mentioned for the sentinel animals, they should have been housed in the respective population for a minimum of - weeks before serological monitoring is conducted. according to the felasa recommendations two additional weanlings should be monitored because they may be better suited for the detection of specific parasites or bacterial pathogens than older animals. monitoring must be performed on a regular basis to detect unwanted microorganisms in good time. the recommended frequency is every weeks. most commercial breeders test more frequently (e.g. every weeks). in most multipurpose experimental units animals are regularly bought and introduced into a facility. it may, in such cases, be reasonable to tesl-with a higher frequency (e.g. - animals every -- weeks instead of every three months) as this will result in the earlier detection of an infection (kunstyr, ) . for each facility or even for every single unit within a facility, the agents that are acceptable must be defined. besides felasa (kraft et al., ) , various other organizations (kunstyr, ; national research council, ; waggie et al., ) have published similar lists of microorganisms which should be monitored for in routine programmes. the list will usually be restricted to organisms that pose a threat to animals (or humans) or organisms which are known to affect experiments and that can be eliminated. however, infections in immunodeficient animals frequently result in increased mortality due to reduced or lack of resistance to weakly pathogenic or even saprophytic microorganisms. it may therefore be necessary to include organisms with low pathogenicity in a monitoring protocol for immunodeficient animals. on the other hand, some pathogens of laboratory rats have disappeared during domestication or gnotobiotic derivation (e.g. francisella tularensis, leptospira sp., rickettsia sp., spmllum minus) and are less likely to infect laboratory animals housed behind barriers. some parasites (e.g. most cestodes) need an intermediate host not found in barrier units. monitoring for these agents may therefore be less urgent or even unnecessary and may be performed less frequently. felasa recommends testing once a year for such agents, i.e. agents of lower priority (kraft et al., ) . some of the most important bacteria, fungi and parasites for which rats should be monitored are given in table . iji @ a number of new organisms have emerged during recent years and are not included in existing lists. a number of pasteurellaceae that have not yet been definitely classified seem to infect rats, in addition to the only known species, pasteurella pneumotropica (nicklas et al., b) . several helicobacter species have been isolated recently from rats, such as /-/. muridarum (lee et al., ) ,/-/. hepaticus (fox et al., ; riley et al., ) ,/-/. bilis (fox et al., ; riley et al., ) ,/-/. trogontum (mendes et al., ) . a rat parvovirus has also been detected (ueno et al., (ueno et al., , jacoby et al., ) in addition to those parvoviruses already known (io'lham rat virus, . other organisms, such as closmdium piliforme, have been renamed recently (duncan et al., ) , which leads to some confusion in those scientists who are not sufficiently familiar with health monitoring of laboratory rats. in general, the examination methods are: (a) necropsy-following after sacrifice, (b) serology, (c) bacteriology and (d) parasitology. most of these methods are described in special publications (feldman and seely, ; kunstyr, ; owen, ; kraft et al., ) and in various textbooks. reliable results are only obtained if appropriate and sufficiently sensitive methods are used for health monitoring. it is therefore evident that the methods must be adapted to the actual 'state of art', i.e. to introduce new proven methods as they become available. microscopic methods such as stereomicroscopy are commonly used for monitoring for ectoparasites. adhesive tape, flotation or direct microscopy of wet mounts taken from the intestinal tract are used for detection of endoparasites. monitoring for bacteria is usually done by culture methods. however, serology or pcr may in some cases be superior or the only reliable approaches (e.g. for streptobacillus moniliformis, clostridium piliforme or mycoplasma pulmonis) (van kuppeveld et al., ; goto and itoh, ) . monitoring for viral infections is primarily done by serological methods. pcr, as an example of a new method, might be applicable in the case of acute infections (clinical disease) or for agents causing persistent infections (e.g. parvoviruses under specific conditions; gaertner et al., ; besselsen et al., ) . however, the lack of macroscopical changes during necropsy or lack of histopathological changes are still commonly used as the sole basis for declaring a population negative for a specific organism. this must be considered insufficient and unacceptable. serological methods must be selected properly as they may differ in their sensitivity and specificity (smith, ; lussier, ) . unexpected serological results should always be confirmed by an independent method or, preferably, by virus isolation or antigen detection in order to avoid false-positive results. some acceptable serological methods for the most common viral and some bacterial pathogens are given in table . . a health status report is usually requested and necessary when animals are shipped from breeders or between scientific institutions. it must contain sufficient data to provide reliable information on the quality of a population. usually, each animal facility or breeder has its own style of report sheets which are sometimes difficult to read and to interpret. the felasa (kraft et al., ; rehbinder et al., ) recommends using a uniform health report for breeding and for experimental colonies. some additional information might be reasonable (e.g. housing conditions, treatment) and should be included. table . gives a checklist of the basic information that should be included in a health status report. table . information which should be included in a health report when animals are shipped to external colonies viral and mycoplasma infections of laboratory rodents: effects on biomedical research necropsy guide: rodents and the rabbit handbook of laboratory animal science long-term holding of laboratory rodents list of pathogens for xpeci~cation in si~ v laboratory animals diagnostic microbiology for laboratory animals infectious diseases of mice and rats mycoplasmal infections of i~aboratory rodents manual of microbiologic monitoring of s tory animals key: cord- - o utlfx authors: gray, carol; fordyce, peter title: legal and ethical aspects of ‘best interests’ decision-making for medical treatment of companion animals in the uk date: - - journal: animals (basel) doi: . /ani sha: doc_id: cord_uid: o utlfx simple summary: making decisions about medical treatment for animal patients involves two key decision-makers, the animal owner and the veterinary surgeon. we aim to show that these decisions should and can be based on the ‘best interests’ of the animal, with both human decision-makers acting as advocates for the animal requiring treatment. we suggest that the role of the animal owner is similar to that of a parent in making decisions for a child, drawing on legal cases to demonstrate the limits of parental (and owner) decision-making. to provide a firmer basis for ‘best interests’ decision-making, we adapt the factors included in the united nations convention on the rights of the child and demonstrate how these could be used with a typical clinical situation. finally, we analyse the decisions from an ethical point of view. abstract: medical decisions for young children are made by those with parental responsibility, with legal involvement only if the decision is potentially detrimental to the child’s welfare. while legally classified as property, some argue that animals are in a similar position to children; treatment decisions are made by their owners, posing a legal challenge only if the proposed treatment has the potential to cause harm or unnecessary suffering, as defined by animal protection legislation. this paper formulates the approach to a ‘best interests’ calculation, utilising the factors included in the united nations convention on the rights of the child and relying on exchange of information between the human parties involved. although this form of decision-making must primarily protect the animal from unnecessary suffering, it recognises that the information provided by the owner is critical in articulating the animal’s non-medical interests, and hence in formulating what is in the animal’s best overall welfare interests. while statute law does not mandate consideration of ‘best interests’ for animals, this approach might reasonably be expected as a professional imperative for veterinary surgeons. importantly, this version of a ‘best interests’ calculation can be incorporated into existing ethical frameworks for medical decision-making and the humane treatment of animals. according to coggon, for medical treatment to be lawful, it requires that treatment is deemed to be in the patient's best interests (in the opinion of the medical professionals involved); that it aligns with the patient's values (demonstrated, for example, by the patient giving informed consent); and that the necessary resources (including finances) are available to provide the treatment [ ] . consequently, for adult patients with capacity to consent, the legality of treatment is unequivocal. however, for patients without capacity, the picture is somewhat obscured by attempts to make decisions in of their 'best interests,' when the latter term is regarded as being vague, subjective, and too difficult to determine [ ] [ ] [ ] . for adult patients without capacity, whether the loss of capacity is permanent or temporary, defining a 'best interests' basis for treatment decisions involves, at some stage, incorporating their values and wishes in the calculations, as demonstrated in several high-profile cases in the united kingdom (uk), for example, in 'aintree university hospitals nhs foundation trust v james' [ ] and in 'briggs v briggs' [ ] , where the patients' previously expressed wishes were pivotal to the eventual 'best interests' decisions. for those adult patients lacking capacity, for example, due to severe learning disabilities, the uk mental capacity act requires that they should be supported to make decisions wherever possible; they may have capacity for some decisions and not for others. thus, adult patients without capacity may not be suitable as a comparison for animal patients. in view of the problem with finding out what animals want, particularly if relying on human interpretation of their values and preferences [ ] , it is more usual to compare the situation of the animal patient with that of the infant child, whose values and preferences are not yet known [ , ] . this comparison, although enticingly straightforward, must incorporate an acknowledgement that, eventually, animal emotions and desires will be more accurately interpreted by humans, perhaps allowing determination of the individual animal's preferences with regards to the life circumstances in which it finds itself. such interpretations will also depend on the developing knowledge from animal welfare science that underpins the concept of 'critical anthropomorphism' [ ] . importantly, the difference in legal status between children and animals must also be appreciated, notably the animal's position as property. for both paediatric and veterinary patients, the decision-maker is also the carer. by limiting the comparison to companion animals, we focus on animals that are frequently regarded as family members [ ] , with decisions made for them accordingly. unlike decision-making for farm animals or laboratory animals, where interventions are mainly for the benefit of (human) individuals or society, the authors argue that, ideally, decision-making in companion animals should be made on the basis of the 'best interests' of the individual animal [ ] . for children, decisions are made by an adult with parental responsibility. for animals, decisions are made by the person legally regarded as the owner, or the owner's agent. although many scholars are critical of the use of the term 'ownership' with regards to animals, we will use this term to denote the person with primary caring responsibility for an animal. we undertook the comparison by researching uk case law regarding medical decision-making for children, focusing on judgments containing key references to the best interests of the child. we scrutinised uk child and animal welfare legislation and utilised the united nations (un) convention on the rights of the child as a potential source of a 'best interests' calculation. direct comparisons between paediatric and veterinary decision-making require specific assumptions; first, that the owner regards the animal as having intrinsic value, and second, that there is an ultimate ethical acceptance of the subservience of the owner's potential 'selfish interests' in the relationship with the animal to the best interests of that animal. because of the different legislation and funding arrangements for treatment that protect children and animals from harm, this 'selfish interest' concept may include resolution of the dilemmas surrounding the ability or desire of the owner to fund any potential treatment. ultimately the outcome of what is in the animal's best interests is likely to depend on the dialogue between the veterinary surgeon, who has the technical and legal knowledge relating to the animal's health and welfare, and the owner/carer, who is able to inform the discussion regarding aspects of the animal's wider welfare, its individual circumstances and temperament. bridgeman observes that decision-making for children involves two dependencies: first, the parent's dependence on the healthcare professional for information and professional advice and second, the child's dependence on the parent for care and responsible decision-making [ ] . while recognising that similar dependencies may apply to decision-making in veterinary medicine in the uk, given the ethical imperative explicit in the oath sworn by members of the royal college of veterinary surgeons (mrcvs) "that, above all, my constant endeavour will be to ensure the health and welfare of the animals committed to my care" [ ] , the authors argue that the role of the two human parties to the veterinary treatment decision should be for both to advocate for the animal patient, enabling a decision that is in the animal's best interests in its individual medical situation and practical circumstances. nevertheless, any theoretical advocacy must be considered in the practical context of the legal protection constraining the level of harm the animal has to endure. both paediatric and veterinary decision-making are constrained by baseline protection for the patient. for children in the uk, protection against cruelty and unnecessary suffering is still provided under the auspices of the children and young persons act , although in other areas, much of this legislation has been replaced by the provisions of the children acts and . protection for animals in the uk is provided by the animal welfare act (awa) (as devolved), which replaced the protection of animals act . both sets of legislation prohibit anyone (not just the parent or owner) from treating the child or animal cruelly. for medical scenarios, there are two likely applications of these welfare protection laws: ( ) to ensure that the patient is given any necessary treatment, i.e., to rule out the option of no treatment (while acknowledging that refusal of treatment would be accepted as a decision for adult patients with capacity to make such a decision). ( ) to prevent the carrying out of any treatment that may cause harm or unnecessary suffering (for animals, this would include prohibited mutilations, and experimental medical treatments not considered to be 'recognised veterinary practice' by the rcvs [ ] ). in the case of the awa , the requirement to provide treatment is encompassed in section , where a person commits an offence "if he does not take such steps as are reasonable in all the circumstances to ensure that the needs of an animal for which he is responsible are met to the extent required by good practice", one such need being: "its need to be protected from pain, suffering, injury and disease" (albeit with caveats in subsection ). the legal liabilities incumbent on both owner and veterinary surgeon under sections , and of the awa act as constraints to decision-making, not least the fact that killing or euthanasia of the patient are legal options open to the animal's owner under section , and that due to liabilities in section , coggon's issue relating to resource provision may have a significant impact on the practical outcome of where 'best interests' lie in a specific situation. an egregious breach of section may lead to a prosecution under section for 'unnecessary suffering.' while what constitutes 'suffering' under the act is vaguely defined [ , ] , there is guidance in section ( ) on what a court might consider 'unnecessary.' such considerations include whether the suffering was for the longer-term benefit of the animal. as pointed out by grimm and others, while any clinical treatment can cause harm, if it can be justified in the animal's health-related interests, it can be justified under the act [ ] . however, other caveats also apply to this principle; any suffering caused by the treatment must reasonably be avoided, or reduced, and be proportionate to the purpose of the conduct. importantly, animal welfare protection legislation in the uk, despite having recently progressed from a resolutely negative (what an owner must not do to an animal) to a more positive (what an owner must provide for an animal) stance, still fails to promote the best interests of the animal (as exemplified by subsections and of section , which allow for lawful use and humane destruction). as robertson notes, animal protection legislation focuses on minimum standards rather than mandating 'best practice' [ ] . therefore, consideration of decision-making that prioritises the best interests of the animal under veterinary care requires a substantial input from the field of veterinary ethics, as outlined in the development of the veterinary ethical tool by grimm and others [ ] . while the latter authors centre 'best interests' as their moral foundation for decision making in companion animal practice, based on restoration of the animal's health while aiming for a positive balance of quality of life, factors other than bridgeman's first dependency (the parent/carer's reliance on the healthcare professional for information and professional advice) are clearly key to establishing what an animal's 'best interests' are in its particular circumstances. if grimm and others' second imperative (to respect the animal's quality of life experience) is to be upheld, information regarding the animal's temperament and character, the (owner-provided) physical environment, and owner-related factors such as knowledge, ability, and financial and time resources may all impact on the animal's 'quality of life' experience. in adding the preservation of the human-animal relationship to this list, schnobel proposes that the primary role of the veterinary professional may be to provide the owner with the information about proposed treatment(s), with a view to maintaining the ideal human-animal relationship, enabling the animal to "participate within a companion relationship between owner and animal where both derive a significant benefit" [ ] . in common with many other lists, the preceding examples maintain a degree of flexibility and individual interpretation that can frustrate attempts to base decisions on 'best interests.' therefore, in seeking to identify specific factors that may constitute the calculation of the 'best interests' of an animal patient, we now turn to examine the lessons that can be learned from the field of children's rights and best interests. for children, the best interests test originated as a legal requirement in child custody cases at common law and was later applied to paediatric healthcare [ ] . a major problem with an objective 'best interests' standard is the difficulty of defining 'best interests, which, according to baines, may be ontological (there may be no such thing as objective best interests), or epistemological (best interests may exist, but there is no way of discovering what they are) [ ] . section . of the uk children act proposes a list of factors which courts should take into account in determining the best interests of the child, including the risk of any harm, and assessing the emotional as well as physical needs of the child. however, it seems that in many uk cases involving children, the avoidance of harm and the 'reasonableness' of the decision is not enough. some early cases took the approach that a procedure may be performed provided it would not harm the child [ ] , but this has been firmly rejected, first by the court of appeal in the charlie gard case, where the child's parents, seeking to take him overseas for treatment with an unproven therapy, appealed on the grounds that the judge had erred in preventing such treatment when there was no risk of the treatment causing significant harm to the child. dismissing the appeal, mcfarlane lj reinforced the 'best interests' standard and refused to create a "subset of cases based upon establishing significant harm" [ ] . this ruling was endorsed by the supreme court in the alfie evans case [ ] , where the court unanimously dismissed an argument made by the parents of a terminally ill child that the appropriate test for their request to take their child to italy for treatment should be that such an intervention would not cause significant harm, even if it were not in the child's best interests. thus, the 'gold standard' now applies a positive version of best interests (a decision should positively promote the interests of the child) rather than a negative version where the focus is on the avoidance of harm. the 'gold standard' should also extend to more than just medical interests, as illustrated in the 're t' case, where a child's best medical interests would have been served by undergoing a proposed liver transplant. however, the court of appeal overturned the decision in the lower courts, which had found for the medical professionals based on the 'unreasonableness' of the mother's refusal to consent. in her judgment, butler-sloss lj stated that "to prolong life . . . ( . . . ) . . . is not the sole objective of the court and to require it at the expense of other considerations may not be in a child's best interests" [ ] . therefore, a 'best interests' decision for children cannot be based solely on the preservation of life, meaning that extension of life at any cost to the child's welfare is of itself not a justification for medical intervention, nor can it be based solely on the avoidance of harm. turning to the animal patient, the authors are concerned that many veterinary decisions are based on precisely these two parameters, by considering 'extension of life' and 'avoidance of harm' to the exclusion of other factors that should inform a "best interests" decision. moreover, in animals, there is considerable debate over the place of death in these calculations. authors such as webster [ ] and broom [ ] do not consider death to be a harm to an animal's welfare, as once the animal's brain has ceased to function, it clearly will have no awareness of any feelings that could be described as aversive mental states, i.e., suffering. however, others would argue that suffering is not the only harm that can be caused to an animal; death is also potentially a harm, by depriving the animal of the possibility of experiencing positive mental states (pleasure) through continuing to live [ , ] . harms can involve suffering or deprivation. rollin regards pain as the worst harm that can be inflicted on an animal, due to the perceived inability of animals to anticipate the end of their suffering [ ] . thus, although death can be regarded as the ultimate deprivation, it may not be the worst harm [ ] . expanding on this argument, if "the presence of a life" has positive value to the animal then death is a harm, but conversely if that life has negative value then death is a benefit [ ] . end-of-life decision-making in veterinary medicine prioritises the prevention of suffering, requiring that in cases of poor welfare, the decision is made for euthanasia [ ] . in agreeing with the priority given to avoidance of pain in these situations, linzey proposes that, faced with a choice between the duty to preserve life and the duty to prevent suffering, the second duty should take precedence [ ] . therefore, decision-making in animals tends to prioritise the avoidance of harm. although it has been rejected as the basis for decision-making for babies with life-limiting conditions, where the intention to preserve life must take priority, input from a 'harm avoidance' perspective helps to define the limits to 'best interests' decision-making for both children and animals. grimm and others' ethical framework for decision-making for veterinary patients incorporates the idea that short-term harm for long-term best interests can be justified utilising a similar approach to that used to justify research on animals [ ] . in this framework, the veterinary surgeon is placed at the centre of the process, first deciding whether the proposed treatment is in the best interests of the animal. this decision is made using criteria such as improving health, improving quality of life, minimising harm and performing a harm-benefit analysis. only after evaluating these parameters is there a discussion of client-associated (secondary) factors such as effect on client quality of life and the owner-animal relationship. although this tool could prove useful when considering cutting-edge or novel therapies, we consider that it neglects the contribution of the owner to the 'best interests' discussion for more conventional treatments. we therefore propose that basing the decision on calculations of 'best interests' for children may lead to a more universal approach for veterinary treatments. we now attempt to utilise an existing framework, developed for children, to define the contents of a 'best interests' discussion for an animal patient. while positing an approach that leaves the determination of 'best interests' open to interpretation, and lauding the flexibility of such an approach, the un convention on the rights of the child (crc) proposes several factors for inclusion in any 'best interests' calculation. in its accompanying document, general comment [ ] , the crc includes a (non-exhaustive) list of factors to be considered when compiling a 'best interests' calculation, some of which may be suitable for inclusion in a similar list for animals. these are included in table . table . the components suggested for a "best interests' calculation for children with interpretation for companion animal patients. although, at first glance, "the child's views" and "the child's identity" seem uniquely applicable to children rather than animals, they are in fact worthy of closer examination. in light of expanding knowledge of animal preferences, there may be a place for inclusion of the animal's views in the list. in suggesting that the owner may be best placed to report on the animal's personality and preferences, we are highlighting that, in a similar role to those with parental responsibility, animal owners have caring responsibilities that give them unique insight to the animal's world. nevertheless, assigning the role of interpreter of animal preferences to the animal owner may add to the 'caregiver burden' felt by many owners [ ] . additionally, a number of caveats need to be applied to the owner's perception of the state of their animal's welfare [ , ] . these caveats include assessing whether the owner has failed to observe and report physical signs in the pet that the veterinary surgeon would perceive as indicators of poor welfare, for example, obesity [ ] , or whether having noticed these signs, the owner does not consider them to constitute a welfare problem, for example, breathing difficulties in brachycephalic dogs [ ] . hence, while the owner's intent towards their animal's welfare may be beneficent, their interpretation of the state of its welfare should be caveated with a professional assessment of welfare, followed by an appropriate discussion with the owner. nevertheless, the authors would argue that consideration of the owner's interpretation of what is in the animal's best interests should stand as a 'prima facie' principle in the decision-making process, and only then should each owner-animal dyad be scrutinised on an individual basis. subsequent factors appear more straightforward to relate to the veterinary context. "preservation of the family environment and maintaining relations" foregrounds the importance of the relationship between the patient and their carer. as bridgeman [ ] asserts, most parents seek to 'do their best' for their children, therefore, in most cases, parents are the most appropriate decision-makers, as was observed by baker j in 're ashya king (a child)': " . . . the parents are the best people to make decisions about a child and the state . . . ( . . . ) . . . has no business interfering with the exercise of parental responsibility unless the child is suffering or is likely to suffer significant harm as a result of the care given to the child not being what it would be reasonable to expect a parent to give." [ ] in applying the same test to animal owners, we propose that they are the best people to make decisions about their charges, unless there is clear evidence that the animal is suffering or is likely to suffer harm. of course, there are some owners who do not prioritise the animal's interests, however we maintain that such owners are in the minority [ , ] . importantly, some owners may be unable to prioritise the animal's interests due to financial constraints that restrict the choices available to them. the fundamental requirement to preserve the "care, protection and safety" of the child or animal patient engages the main principles of welfare legislation, with more positive responsibilities to provide for the patient's basic welfare needs. it could be stated that a "situation of vulnerability" applies to all animal patients, although its application in the crc is limited to children with disabilities, those who are refugees, belong to a minority group or are victims of abuse. certainly, rescued and abused animals would fit this category, and the suggestion that their interests should be specifically analysed seems appropriate. this situation would prioritise 'protection from harm' as a key area for discussion regarding such patients. two basic rights, to health and education, complete the crc's list of factors that must be considered for a 'best interests' calculation in children. while education seems less relevant for animals (unless translated as training, but that may be stretching the comparison), the right to health could be given prominence, although such an assignment may unfairly prioritise health-based interests above all others. additionally, switching to a rights-based narrative may invoke protest. conversely, in light of the perceived problems with assigning rights to animals, a 'best interests' approach may achieve what a rights-based agenda could not. even children's rights are not universally accepted, and as kilkelly observes, "without rights language, the child's best interests appears as a conciliatory gesture with its softer language reaching out to those to whom rights are not acceptable, whatever the basis of that position" [ ] . assigning a right to health to the animal through a 'best interests' calculation could perhaps move the argument further along the road for animal rights. indeed, the veterinary profession's "self-imposed ethical ideal of advocating and defending its patients' interests in health" [ ] already gives the animal patient status as a subject. any 'best interests' calculation, whether for children or animals, necessarily involves two key aspects. the first is the medical knowledge of the healthcare professional, translated into accessible information for the caregiver. the second, which invokes the relational responsibility to which bridgeman [ ] alludes, is the unique personality of the patient, interpreted by the caregiver as the patient's preferences and values. in the authors' opinion, these two (human) agents should have equal input to the discussion surrounding 'best interests,' which could be achieved by incorporating the ideals of collaborative decision-making [ ] . such collaboration between veterinary healthcare professionals and animal owners requires discussion of the financial burdens of treatment, which may impact on the choices available. where an owner is unable to afford the treatment required, the private enterprise veterinary healthcare market dictates that the options available may include severance of the ties between owner and animal. to illustrate the stark choices involved, we now consider the hypothetical case of a nine-month-old neutered female cat who has been involved in a road traffic accident, sustaining several fractures that will require surgical repair. the patient has been made comfortable, while the owner and veterinary surgeon discuss the options. the owner has stated that he cannot afford the proposed surgery and is not eligible for referral to a veterinary charity for treatment. the options under consideration are: ( ) the practice will perform the surgery at a vastly reduced rate, or offer a payment plan that fits with the owner's financial situation. ( ) the owner will relinquish ownership of the animal to the practice, with an agreement that the cat will be rehomed once the surgery has been successfully carried out. ( ) the owner will agree to euthanasia of the cat. these options can be evaluated in more detail with a principal focus on the 'best interests' of the cat, relating these to similar provisions in the crc. we propose that option may prevent harm by ending suffering, but it would also deprive the cat of the ability to experience a 'life worth living' for a member of her species. we would argue that euthanasia in these circumstances cannot be said to be in the cat's best interests, as it neglects her "care, protection and safety," her "right to health," and, from previous ethical discussion, her ability to experience pleasure in the future. additionally, this option would sever the unique owner-companion animal relationship. option would also entail the severance of the human-animal relationship. in the hypothetical scenario, the owner rescued the cat as a six-week-old kitten, and they have a bonded relationship that seems to give the cat (and owner) pleasure. moreover, the cat enjoys exploring her owner's garden and the local neighbourhood, where there are few other cats. therefore, we propose that a "best interests"-based decision would need to "preserve the family environment and maintain relations" and would not contemplate removal of the cat from her current living arrangements. thus, we consider option as unique in preserving the current relationship, which seems to be of value to the cat, thus respecting her "views and identity." following a period of recovery from surgery, the cat will return to her home territory, which is the ideal outcome from the perspective of her 'best interests'. note that in selecting the option that prioritises the cat's best interests, we have ignored the interests of the veterinary practice as a business entity, and have assumed that the veterinary staff involved will also prioritise the interests of the cat over personal financial interests, such as practice profit or receiving bonus payments on turnover. however, both options and involve financial loss to the practice on a similar scale, so perhaps the real choice is between performing the surgery, regardless of ownership, and euthanasia. the discussion of 'best interests' in the context of financing veterinary treatment illustrates how ethical dilemmas resulting from multiple competing interests require consideration and resolution. such dilemmas can be viewed through the perspective of differing normative ethical systems such as utilitarianism (looking at what would be the greatest good for the greatest number of moral agents and patients involved); deontology (examining the duties owed by the parties to each other); virtue ethics (assessing the actions of the moral agents involved in relation to a vocabulary of virtues and vices such as 'compassionate' or 'cruel'); rights discourse (examining what legal and moral rights or entitlements the parties have, including the right of an individual involved to not have their interests overridden by utilitarian claims); and relational and care ethics (arguably a branch of virtue ethics, which places the nature of the interpersonal relationships between the various parties at the centre of the moral argument). however, no one normative ethical perspective is likely to provide a calculus resulting in a morally acceptable conclusion for all parties; for example, utilitarian perspectives may violate deontological imperatives for individuals not to be treated 'as a means to an end', while 'rights discourse' may infringe on concepts from virtue ethics such as fairness, when considering distribution of resources such as financing the surgery discussed above. perhaps unsurprisingly, two of the most relevant contemporary ethical frameworks are associated with human patient care, and the use of animals as a food source by humans. these frameworks incorporate multiple elements from normative ethics, to allow not only consideration of the interests of the moral agents and patients involved, but also the consideration of these interests from the perspective of different ethical approaches. while not universally accepted, the framework of 'principlism' is a dominant ethical approach used in human medicine to highlight, and help resolve, ethical dilemmas in medical decision making [ ] . principlism asks the clinician to consider four 'prima facie' principles (principles that stand, until found to be in conflict with another principle) within the wider context of the patient's situation (scope). the principle of 'patient autonomy' provides a deontological perspective, 'beneficence and non-maleficence' a utilitarian perspective, while 'justice' incorporates concepts from virtue ethics, consideration of legal and moral justice, fairness in relation to resource distribution, and concepts from 'care ethics.' scope provides a perspective of the potential effect of the decisions made on all parties likely to be affected and might be considered to incorporate aspects of communitarian ethics [ ] . the second framework, mepham's matrix, was developed to consider the competing interests of those involved in production of animals for food, including the animals themselves [ ] . this matrix provides a format to consider the impact of different decisions on various stakeholders, thus approximating scope, and examines three areas of ethical concern: the impact on the stakeholders' wellbeing, including health and welfare (utilitarian perspective); autonomy, including freedom and choice (deontological perspective); and whether the decision would be seen as just and fair to them (virtue ethics perspective). as has been discussed elsewhere by both authors [ , ] , the issue of using 'autonomy' as a prima facie principle in the context of animals is problematic, not least given the anthropocentric weight that tips the scales in favour of human benefit when discussing the moral value and utility of animals, a point acknowledged by the food ethics council. such inconsistency between the moral value of humans and animals is reflected in statute legislation, as previously discussed. however, replacing the principle of 'autonomy' with 'best interests' in the principlism paradigm avoids many of the issues with interpretation of autonomy for animals. specifically, it avoids the debate over whose autonomy should be respected; the owner's autonomy, which is already constrained by animal welfare legislation and financial limitations, or the animal patient's autonomy, which is difficult to interpret with current knowledge about animal cognition and behaviour. at the same time, it highlights the importance of the moral standing of the veterinary patient that might be expected in the light of the veterinary surgeon's professional ethical obligations. grimm and others' veterinary ethical tool [ ] adds much to the debate in terms of examining beneficence and non-maleficence with regard to treatment options, in particular the avoidance of 'futile' treatment. however, the authors would suggest that while much of the focus of their approach is on the animal's health, which is a major contributor, welfare encompasses much more than just health. the concept of 'best interests' enables this wider perspective to be considered. such analysis requires considerable input from the animal's primary caregiver, both in relation to issues such as the animal's nature and temperament, its likes and dislikes, pleasures and fears, as well as the nature of the resources the caregiver is able (or willing) to bring to bear to achieve those best interests. in considering the clinical scenario described above for the cat involved in the road accident, substituting the concept of 'best interests' for 'autonomy' as a prima facie principle in the principlism paradigm makes this central to any debate in relation to the other principles, should these clash at a practical level. such an approach upholds the veterinary surgeon's professional responsibilities to the oath sworn on admission to the profession. additionally, it allows the wider welfare issues surrounding the animal's wellbeing to be discussed outside the purely technical issues of the veterinary surgeon's area of health and medicine. issues such as the good or harm a treatment (or lack of treatment) may cause, and particularly discussions about futile treatment can be raised within the 'beneficence/non-maleficence' principles. similarly, issues relating to legal and moral rights and duties, as well as fairness with regard to issues such as funding treatment and breaking of relationships would fall under the principle of justice. clearly, any ethical framework is unlikely to function as an algorithm to provide a 'morally correct output' that is accepted by all parties affected by the decision; a point highlighted by the food ethics council. it should, however, highlight the interests of all involved, including the responsibilities of the moral agents to the law, professional ethical obligations, and ethical duties as an owner. where moral dilemmas arise due to a clash of prima facie principles, rational discussion can then take place about the way forward. the 'best interests' principle ensures that the wider wellbeing of the veterinary patient is at the centre of the discussion. in the case of the cat discussed in the clinical scenario above, option (performing the surgery at a reduced rate or offering a suitable payment plan) appears to be in the 'best interests' of the animal. while option (relinquishing ownership) may meet the test for beneficence and non-maleficence in the narrower context of the animal's health, it clashes with the principle of 'best interests' for the animal for the reasons discussed above and would likely have a negative psychological impact on the owner. similarly, while option (euthanasia) may address the welfare issue of the cat's current suffering, as once dead, she can no longer suffer, it deprives the cat of the potential of a favourable balance of pleasure over suffering during her life if treated successfully. when the decision is considered in terms of 'scope', it would also have a negative psychological impact on the humans involved. the ethical dilemma arising from this scenario is the tension between what is in the patient's 'best interests' and the principle of 'justice' with regard to paying for the treatment. such dilemmas may well depend on the individual circumstances of the case, and disagreement may arise over what is considered 'ethically acceptable'. the farm animal welfare council (fawc) provides some guidance on ethical disagreements, suggesting that confidence in the validity and worth of the conclusion depends on three criteria: ( ) the arguments that lead to the particular conclusion are convincingly supported by facts, scientific deductions, and reason; ( ) the arguments are conducted within a well-established ethical framework; ( ) a considerable degree of consensus exists, arising from a process of genuine discussion and debate, about the validity of the conclusions [ ] . in relation to fawc's first point, the concept of 'best interests' allows facts relating to the individual animal and its circumstances to be incorporated into the decision-making process by the owner and discussed in conjunction with the medical knowledge of the clinician. inclusion of such information is therefore more likely to produce an ethically valid outcome for the parties involved. as discussed, evaluating exactly what is in an animal's wider best interests (not just its immediate health interests) must involve significant input from the owner, all be it caveated by the individual circumstances of the owner's knowledge and understanding of the wider concept of welfare, and assessment of its translation to the individual pet at the time. we propose that the framework used in the un crc provides useful insights into how questions relating to the 'best interests' of a veterinary patient might be addressed in the conversation between the veterinary professional and the patient's owner. the use of a 'best interests' calculation has been prioritised in decision-making about the medical treatment of young children. in considering whether a similar calculation would be appropriate for animals, we have questioned the basis for such a calculation, drawing on the parameters advocated by the convention on the rights of the child. the multiple difficulties inherent in such an approach, not least the disparity in legal status between children and animals, make such a comparison problematic. moreover, the calculation of best interests for animal patients is heavily reliant on the human interpretation of animal preferences and values, and the willingness and ability of the human carer to pay for any required treatment. in producing a draft set of criteria for consideration when attempting to calculate the 'best interests' of an animal patient, we have deliberately left some criteria open to interpretation, resisting the temptation to produce a 'step-by-step' guide. such an approach fits with the intention of the un crc, which, in drawing up a 'non-exhaustive' and 'non-hierarchical' list of factors to be taken into account, intended the guidelines to be flexible. a key factor to the success of this approach is the importance of good communication skills. the veterinary surgeon must be able to communicate appropriately with the client to facilitate a 'best interests' discussion. importantly, with the involvement of at least two human advocates, a 'best interests' approach to veterinary decision-making gives the animal patient the priority that has perhaps been absent from veterinary medicine until relatively recently. the acknowledgement that we are discussing 'best interests' as a basis for veterinary decision-making, not to mention comparing it to medical decision-making for young children, requires elevation of the status of the animal patient to more than just 'property' or legal 'object'. furthermore, as our knowledge of animal cognition and behaviour increases, the possibility of incorporation of the animal's views in such calculations may become a reality. the underpinning of 'best interests' discussions with previously posited ethical frameworks lends more weight to their use, with the term 'best interests' used in place of 'autonomy' when invoking human medical frameworks. the animal patient is thus given a position of moral relevance in veterinary science that fits with emerging views on how we decide on appropriate and ethical veterinary treatment. finally, through these endeavours, we propose that the animal patient, although currently lacking the status of legal subject, deserves to be considered a moral subject whose interests are worthy of primary consideration by the humans involved in making decisions on her behalf. author contributions: conceptualization, c.g.; writing-original draft preparation, c.g. and p.f.; writing-review and editing, c.g. and p.f. all authors have read and agreed to the published version of the manuscript. acknowledgments: this paper is based on one that was accepted for the sociolegal studies association annual conference in the animal law stream. when the conference had to be cancelled due to the covid- restrictions, the authors revised the paper for publication. the authors declare no conflict of interest. the funders had no role in the design of the study, in the writing of the manuscript, or in the decision to publish. mental capacity law, autonomy, and best interests: an argument for conceptual and practical clarity in the court of protection the best interest standard and children: clarifying a concept and responding to its critics clarifying the best interests standard: the elaborative and enumerative strategies in public policy-making making healthcare decisions in a person's best interests when theylack capacity: clinical guidance based on a review of evidence aintree university hospitals nhs foundation trust v james uksc what do animals want? the use and abuse of aesculapian authority in veterinary medicine informed consent in veterinary medicine: ethical implications for the profession and the animal critical anthropomorphism, animal suffering, and the ecological perspective post-human families? dog-human relations in the domestic sphere meeting the patient's interest in veterinary clinics. ethical dimensions of the st century animal patient parental responsibility, young children and healthcare law royal college of veterinary surgeons. declaration on admission to the profession code of professional conduct: supporting guidance : recognised veterinary practice assessment of unnecessary suffering in animals by veterinary experts suffering in non-human animals: perspectives from animal welfare science and animal welfare law drawing the line in clinical treatment of companion animals: recommendations from an ethics working party welfare and the law: fundamental principles for critical assessment regulating the veterinary profession: taking seriously the best interests of the animal deciding together? best interests and shared decision-making in paediatric intensive care. health care anal death and best interests: a response to the legal challenge great ormond street hospital for children nhs foundation trust ewca civ children's nhs foundation trust and evans supreme court animal welfare: a cool eye towards eden a history of animal welfare science death is a welfare issue how should death be taken into account in welfare assessments? animal pain the case for animal rights veterinarians' role in clients' decision-making regarding seriously ill companion animal patients why animal suffering matters: philosophy, theology and practical ethics united nations convention on the rights of the child, general comment caregiver burden in owners of a sick companion animal: a cross-sectional observational study how happy is your pet? the problem of subjectivity in the assessment of companion animal welfare not all dogs are equal: perception of canine welfare varies with context mapping discussion of canine obesity between veterinary surgeons and dog owners: a provisional study do dog owners perceive the clinical signs related to conformational inherited disorders as 'normal' for the breed? a potential constraint to improving canine welfare ewhc (fam) guardians and owner-guardians: differing relationships with pets my family and other animals: pets as kin the best interests of the child: a gateway to children's rights? the importance of communication in collaborative decision making: facilitating shared mind and the management of uncertainty principles of biomedical ethics center for the study of language and information (csli) reconciling autonomy and beneficence in treatment decision-making for companion animal patients a discussion of teaching clinical veterinary ethics to undergraduates: personal thoughts from the front line appendix iv ethical principles: how can we decide what is right and what is wrong in the treatment of farm animals? in farm key: cord- - nk je authors: mätz‐rensing, k.; winkelmann, j.; becker, t.; burckhardt, i.; van der linden, m.; köndgen, s.; leendertz, f.; kaup, f.‐j. title: outbreak of streptococcus equi subsp. zooepidemicus infection in a group of rhesus monkeys (macaca mulatta) date: - - journal: j med primatol doi: . /j. - . . .x sha: doc_id: cord_uid: nk je background a severe upper respiratory tract infection occurred in a breeding group of rhesus monkeys housed together in one of six indoor/outdoor corals of the german primate center. the clinical signs of the disease included severe purulent conjunctivitis, rhinitis, pharyngitis, respiratory distress and lethargy. six of animals died within a few days after developing signs of infection. methods and results histopathologic and microbiologic examinations of the dead animals were consistent with a severe fibrinopurulent bronchopneumonia. microbiology revealed a lancefield group c streptococcus identified as streptococcus equi subsp. zooepidemicus as the causative agent of infection. conclusions the infection was passed on from animal to animal but did not spread to the other five breeding groups nearby. extensive diagnostic testing failed to reveal the consisting presence of copathogens in individual cases. a visitor with upper respiratory disease was suspected as source of infection. streptococcus (s.) equi subspecies zooepidemicus belongs to the b-hemolytic group c streptococci. it is able to cause disease both in animals and humans. streptococcus equi subsp. zooepidemicus primarily causes equine infections. the agent may be found in the nasopharynx, on the tonsils, in the respiratory tract and on the genital mucous membranes of healthy horses and cattle. it is an important cause of respiratory tract infections in foals and young horses and it is involved in uterine infections in mares. the agent has also been associated with a wide variety of infections including mastitis in pigs, sheep, cows, goats and several other mammalian species [ ] . these hosts can be a reservoir for human infections. the clinical manifestation includes pharyngitis, septicemia, meningitis, purulent arthritis and endocarditis. the source of human infection is often traced back to contact with domestic animals, especially horses, or ingestion of unpasteurized milk or milk products. streptococci are colonizers of mucous membranes and are transmitted through droplets or direct contact. streptococcus equi subsp. zooepidemicus has sporadically been described in non-human primates. one outbreak occurred in the national zoological park, washington d.c., leading to the death of several callithrichids after contact to infected horse meat fed to armadillos kept in the same exhibition area [ , ] . another outbreak of group c streptococcal infection with a high mortality rate occurred in a group of wanderoos (macaca silenus) of the zoological garden of rheine/germany. the source was suspected to be a human being [ ] . the russian primate center in sochi background a severe upper respiratory tract infection occurred in a breeding group of rhesus monkeys housed together in one of six indoor/outdoor corals of the german primate center. the clinical signs of the disease included severe purulent conjunctivitis, rhinitis, pharyngitis, respiratory distress and lethargy. six of animals died within a few days after developing signs of infection. methods and results histopathologic and microbiologic examinations of the dead animals were consistent with a severe fibrinopurulent bronchopneumonia. microbiology revealed a lancefield group c streptococcus identified as streptococcus equi subsp. zooepidemicus as the causative agent of infection. conclusions the infection was passed on from animal to animal but did not spread to the other five breeding groups nearby. extensive diagnostic testing failed to reveal the consisting presence of copathogens in individual cases. a visitor with upper respiratory disease was suspected as source of infection. reported an outbreak of septicaemia caused by s. equi subsp. zooepidemicus in representatives of five species of lower monkeys [ ] . in , an outbreak among the pig and monkey population was reported from the island of bali, indonesia. the infection spread from pigs to monkeys and infected animals died within a few days with signs of bronchopneumonia, pleuritis, epicarditis, endocarditis and meningitis [ ] . however, most of the reported disease outbreaks were characterized by symptoms of an enteric infection. the present case report describes an outbreak of respiratory diseases among rhesus monkeys induced by s. equi subsp. zooepidemicus. the outbreak involved one of six breeding colonies housed in an indoor-outdoor facility. each unit was composed of an indoor area, a heated and roofed outdoor room and a large henced outdoor enclosure. contact to any other animal species except wild birds was not possible. the animals could move freely between the different compartments of the unit. they were fed twice a day with a primate specific diet, composed of standard commercial monkey pellets (ssniff, spezialdia¨ten gmbh, soest, germany), fresh fruits and vegetables. water was available ad libitum. the animals are kept in accordance with the guidelines of the european union for the accommodation and care of animals used for experimental and other scientific purposes ( / /eg). the primate husbandry is controlled by local and regional veterinary authorities in accordance with the german animal protection law. all procedures are supervised by an animal welfare officer and the ethical committee for experiments using animals in the federal state of lower saxony. a total of animals died in one of these breeding colonies in a short time period. at the moment of the outbreak, the group was composed of animals; most of them were adult females with their offspring and one adult male. necropsies were performed on all carcasses and samples for histology, parasitology, microbiology and molecular diagnostics were taken. for histopathology, samples were taken from all thoracic, abdominal and pelvic organs, fixed in % neutrally buffered formalin, embedded in paraffin and stained with hematoxylin and eosin. for parasitological diagnosis fresh samples were examined microscopically. for microbiological, investigations swabs from liver, spleen, kidney, heart and lung were streaked immediately onto columbia blood agar plates. swabs recovered from the intestine were swabbed onto columbia agar, emb, mcconkey and skirrows campylobacter agar plates and inoculated into rapaport and skirrow's enrichment broth. biochemical characterization of the isolated bacterial colonies was achieved using the crystel-system from becton dickinson. bacterial isolates were sent to the german national reference center for streptococci for further characterization. pulsed field gel electophoresis (pfge) of bacterial dna was carried out on a bio-rad chef-dr iii system using the bio-rad (bio-rad laboratories gmbh, munich, germany) gene path gel kit and the restriction enzyme smai. the outbreak started at the end of october . two adult females were found seriously ill with signs of severe upper respiratory tract infection and severe purulent conjunctivitis (fig. ). antibiotic therapy with baytril Ò (dosage . mg/kg) and aviapen Ò (dosage . - . ie/kg) was started but both animals died within days after onset of the first symptoms (animals no. , ). one month later, four more female animals developed severe upper respiratory tract infection. two died suddenly without obvious clinical findings (animals no. , ), one of these was a pregnant one (animal no. ). the other two died after days on antibiotic therapy with the antibiotics mentioned above (animals no. , ). necropsy findings were similar for all six animals. the main findings were severe inflammatory alterations of the upper respiratory tract and the lung ( table ) . all six animals showed a severe subacute tonsillitis and pharyngitis and a severe subacute fibrinopurulent pleuritis and pneumonia. the inflammation spread to the heart and developed into a fibrinopurulent epi-and pericarditis with exception of animal no. (figs and ). variably sized and poorly demarcated atelectatic areas were present in all lung lobes. histologically, there was a fibrinosuppurative pneumonia with extensive to lobular obliteration of the airways and alveolar spaces with neutrophils (fig. a) . large and medium sized airways were unaffected but terminal airways were intensely altered and showed signs of a purulent bronchopneumonia (fig. b) . the inflammatory process extended from the pleura to the diaphragm leading to severe purulent myositis (fig. c ). one pregnant animal additionally developed a severe purulent endometritis. the aborted fetus was nearly fully developed and showed signs of a purulent hepatitis and splenitis (animal no. a). splenitis and hepatitis were mild side effects in four more animals. only one animal developed a purulent encephalomeningitis (animal no. ). clusters and chains of gram-positive cocci were present within the altered organs. they were found within the cytoplasm of macrophages and free within the extracellular spaces laying in pairs or chains (fig. ) . septic thromboemboli were less consistently observed (fig. d) . septicemia was attributed as cause of death in all cases. septicemia and bacteriemia were confirmed by microbiological investigations on samples taken during necropsy. pure cultures of large colonies of b-hemo- mä tz-rensing et al. lytic streptococci were grown not only from the respiratory tract, respectively tonsils and lung, but also from nearly all internal organs including the central nervous system. bacterial characterization was based on the observation of gram-positive, catalase-negative chain forming cocci which fermented lactose and trehalose. agglutination tests showed that they belong to lancefield group c. pulsed field gel electophoresis patterns were identical for all seven isolates analyzed, showing that the infections were caused by the same strain in all cases (fig. ) . polymerase chain reaction based on amplification of common primate respiratory pathogens was performed on the lung tissue of the necropsied animals to exclude the presence of other important viral pathogens. the methods used are summarized in ko¨ndgen et al. [ ] . all samples were negative for influenza a and b, parainfluenza, rhinovirus, coronavirus, adenovirus, human metapneumovirus, respiratory syncytial virus and enterovirus. the six dead animals were high ranked closely related animals. their loss induced severe ranking fights, a problem which is well known in rhesus monkey colonies. as a consequence of these heavy fights six more animals (animal no. - ) died due to severe injuries, stress and shock-symptomatic. streptococcus equi subsp. zooepidemicus was isolated from the tonsils of three of these animals (animal no. - ). a fatal outbreak of streptococcus infection occurred in one of six breeding colonies housed in an indoor/outdoor facility. the source of infection with s. equi subsp. zooepidemicus in the present outbreak remains unknown. contact to horsemeat, equines or domestic animals could reliably be excluded. instead a human to animal transmission was suspected. generally s. equi subsp. zooepidemicus can be transmitted by aerosols, via the oral route or through wound contamination. aerosol transmission is most likely in the present case. it was assumed that contact to a visitor with upper respiratory disease led to the initial infection of two elderly and closely related animals. streptococci of lancefield group c can be recovered from the pharynx of . % of normal humans. infections of the upper respiratory tract in humans are rare but may occur after contact with infected horses [ ] . it was assumed that the infection was subsequently transmitted from animal to animal because ill and dead animals were closely related to each other. there was a high degree of relationship and close contact among them. close contact seems to be necessary for the transmission of this agent. none of the animals in adjacent corrals situated at a distance of - m became infected. none of the staff members was ill before or during the time of the outbreak. in horses s. equi subsp. zooepidemicus is a commonly isolated mucosal commensal that opportunistically invades after virus infections, transport stress or other immunosuppressive conditions. in the described monkey group the situation seems to be different. bacteriologic investigation of tonsillar swabs indicates that there was no colonization or asymptomatic infection with s. equi subsp. zooepidemicus among the group members which could have been activated by a stressor. the only exceptions were three animals (animal no. - ) that died in the consequence of heavy position fights. in these animals streptococci were found in the tonsils but they developed no clinical findings. the three animals belonged to the same family and had been in close contact to the dead animals. they developed a purulent tonsillitis and it is speculative if these animals may have established a latent infection comparable to horses if they survived the attacks. the streptococcus outbreak among rhesus monkeys clearly demonstrates the high susceptibility of these animals. this is confirmed by data from the literature describing outbreaks caused by s. equi subsp. zooepidemicus in non-human primates marked by sudden, explosive appearance and high fatality rate [ , , ] . this observation should lead to a critical discussion of housing primates and other animal species in close vicinity of each other as is often the case in zoological gardens. particularly equines should not be kept in the vicinity of primate facilities and there should be a strict separation between the different units regarding animal keepers and other staff members. the problem of the described outbreak for the breeding colony was not only the initial fatal infection but also the death of the first six animals. the loss of these animals caused ranking fights, injuries and death of six more animals. as a consequence the group had to be split up and newly composed. the outbreak ended after the dramatic depopulation of the group and the intense cleaning and disinfection of the facility. the disease caused heavy losses; not only the loss of the animals, but also a reduced reproductive rate and high costs for therapy. the experience from this outbreak leads to the conclusion that contact of important and valuable breeding colonies to visitors should be strictly avoided. an outbreak of streptococcus equi ssp. zooepidemicus infection of probable human origin in wanderoos (macaca silenus) -case report outbreak of infection caused by streptococcus zooepidemicus among laboratory primates pandemic human viruses cause decline of endangered great apes diseases of zoo marmosets, tamarins and goeldi's monkeys streptococcus zooepidemicus (group c) pneumonia in a human persistent occurrence of a single streptococcus equi subsp. zooepidemicus clone in the pig and monkey population in indonesia streptococcus zooepidemicus infections of possible horsemeat source in red-bellied tamarins and goeldie's monkeys identification and molecular characterization of serological group c streptococci isolated from diseased pigs and monkeys in indonesia key: cord- -rv j authors: boes, katie m.; durham, amy c. title: bone marrow, blood cells, and the lymphoid/lymphatic system date: - - journal: pathologic basis of veterinary disease doi: . /b - - - - . - sha: doc_id: cord_uid: rv j nan within the marrow spaces, a network of stromal cells and extracellular matrix provides metabolic and structural support to hematopoietic cells. these stromal cells consist of adipocytes and specialized fibroblasts, called reticular cells. the latter provides structural support by producing a fine network of a type of collagen, called reticulin, and by extending long cytoplasmic processes around other cells and structures. both reticulin and cytoplasmic processes are not normally visible with light microscopy but are visible with silver reticulin stains (e.g., gordon and sweet's and sometimes with periodic acid-schiff). bone marrow is highly vascularized but does not have lymphatic drainage. marrow of long bones receives part of its blood supply from the nutrient artery, which enters the bone via the nutrient canal at midshaft. the remaining arterial supply enters the marrow through an anastomosing array of vessels that arise from the periosteal arteries and penetrate the cortical bone. vessels from the nutrient and periosteal arteries converge and form an interweaving network of venous sinusoids that permeates the marrow. these sinusoids not only deliver nutrients and remove cellular waste but also act as the entry point for hematopoietic cells into blood circulation. sinusoidal endothelial cells function as a barrier and regulate traffic of chemicals and particles between the intravascular and extravascular spaces. venous drainage parallels that of the nutrient artery and its extensions. • bleeding time (template bleeding time, buccal mucosal bleeding time). this assay assesses primary hemostasis (platelet plug formation) by measuring the time interval between inflicting of standardized wound and cessation of bleeding. sedation may be required. in small animals the test is usually performed on the buccal mucosa; in large animals it may be performed on the distal limb. prolonged bleeding time may be because of a platelet function defect, von willebrand disease, or a vascular defect. the sensitivity of this test is low; reference intervals are species and site dependent (can perform test on a normal animal as a control). this test is contraindicated in cases of thrombocytopenia because significant thrombocytopenia can cause a prolonged bleeding time (invalidates interpretation of test results). • clot retraction test. this assay assesses retraction of a clot, in which platelets play an essential role. this is a crude test that is rarely performed. different protocols are described. significant thrombocytopenia invalidates interpretation of test results. • tests to characterize platelet function abnormalities more specifically are available through specialized laboratories. • aggregometry-to assess platelet aggregation in response to different physiologic agonists. • adhesion assays-to assess the ability of platelets to adhere to a substrate (e.g., collagen). • flow cytometry-to assay for expression of surface molecules. • pfa- -an instrument that simulates a damaged blood vessel, by measuring time for a platelet plug to occlude an aperture; to date, this instrument has mainly been used in research applications. • thromboelastography (teg)-global assessment of hemostasis (platelets, coagulation, and fibrinolysis) based on viscoelastic analysis of whole blood. • tests for immune-mediated thrombocytopenia (imt). • flow cytometry-to detect immunoglobulin bound to the platelet surface, using a fluorescent-labeled antibody. • bone marrow immunofluorescent antibody (ifa) test-to detect bound immunoglobulin. sometimes referred to as the "antimegakaryocyte antibody test," this assay actually detects the presence of immunoglobulin nonspecifically: a smear of a bone marrow aspirate is incubated with a fluorescent-labeled antibody to species-specific immunoglobulin. other components of the marrow include myelinated and nonmyelinated nerves, as well as low numbers of resident macrophages, lymphocytes, and plasma cells. of note, the macrophages play an important role in iron storage and erythrocyte maturation. the following basic concepts provide a framework for understanding the mechanisms of injury and diseases presented later in the chapter. • hematopoietic tissue is highly proliferative. billions of cells per kilogram of body weight are produced each day. • pluripotent hematopoietic stem cells are a self-renewing population, giving rise to cells with committed differentiation programs, and are common ancestors of all blood cells. the process of hematopoietic differentiation is shown in fig. - . • hematopoietic cells undergo sequential divisions as they develop, so there are progressively higher numbers of cells as they mature. cells also continue to mature after they have stopped dividing. conceptually, it is helpful to consider cells in the bone marrow as belonging to mitotic and postmitotic compartments. examples of developing hematopoietic cells are shown in fig. - . • mature cells released into the blood circulation have different normal life spans, varying from hours (neutrophils), to days (platelets), to months (erythrocytes), and to years (some lymphocytes). • the hematopoietic system is under exquisite local and systemic control and responds rapidly and predictably to various stimuli. • production and turnover of blood cells are balanced so that numbers are maintained within normal ranges (steady-state kinetics) in healthy individuals. • normally the bone marrow releases mostly mature cell types (and very low numbers of cells that are almost fully mature) into the circulation. in response to certain physiologic or pathologic stimuli, however, the bone marrow releases immature cells that are further back in the supply "pipeline." the composition of the marrow changes with age. the general pattern is that hematopoietic tissue (red marrow) regresses and is replaced with nonhematopoietic tissue, mainly fat (yellow marrow). thus in newborns and very young animals the bone marrow consists largely of hematopoietically active tissue, with relatively little fat, whereas in geriatric individuals the marrow consists largely of fat. in adults, hematopoiesis occurs primarily in the pelvis, sternum, ribs, vertebrae, and the proximal ends of humeri and femora. even within these areas of active hematopoiesis, fat may constitute a significant proportion of the marrow volume. immature hematopoietic cells can be divided into three stages: stem cells, progenitor cells, and precursor cells. hematopoietic stem cells (hscs) have the capacity to self-renew, differentiate into mature cells, and repopulate the bone marrow after it is obliterated. progenitor cells and precursor cells cannot self-renew; with each cell division, they evolve into more differentiated cells. later-stage precursors cannot divide. stem cells and progenitor cells require immunochemical stains for identification, but precursor cells can be identified by their characteristic morphologic features (see fig. - ). control of hematopoiesis is complex, with many redundancies, feedback mechanisms, and pathways that overlap with other physiologic and pathologic processes. many cytokines influence cells of different lineages and stages of differentiation. primary growth factors for primitive cells are interleukin (il) , produced by t lymphocytes, and stem cell factor, produced by monocytes, macrophages, fibroblasts, endothelial cells, and lymphocytes. interleukin is an early lymphoid growth factor. lineage-specific growth factors are discussed in their corresponding sections. hematopoiesis occurs in the interstitium between the venous sinusoids in the so-called hematopoietic spaces. there is a complex functional interplay among hematopoietic cells with the supporting connective tissue cells, extracellular matrix, and soluble factors, which form the hematopoietic microenvironment. behavior of hematopoietic cells is influenced by direct cell-to-cell and cellmatrix interactions and by soluble mediators, such as cytokines and hormones that interact with cells and with matrix proteins. cells localize to specific niches within the hematopoietic microenvironment via adhesion molecules, such as integrins, immunoglobulins, lectins, and other receptors, which recognize ligands on other cells or matrix components. cells also express receptors for soluble molecules such as chemokines (chemoattractant cytokines) and hormones that influence cell trafficking and metabolism. iron is essential to hemoglobin synthesis and function. it is acquired through the diet and is transported to the bone marrow via the iron transport protein, transferrin. central macrophages either store iron as ferritin or hemosiderin, or transfer the iron to erythroid precursors for hemoglobin synthesis. hemosiderin is identifiable in routinely stained marrow preparations as an intracellular brown pigment. however, perls's prussian blue stain is more sensitive and specific for iron detection. the earliest erythroid precursor identifiable by routine light microscopy is the rubriblast, which undergoes maturational division to produce to progeny cells. late-stage erythroid precursors, known as metarubricytes, extrude their nuclei and become inhibiting apoptosis of developing erythroid cells. the stimulus for increased epo production is hypoxia. within the bone marrow, erythroid precursors surround a central macrophage in specialized niches, termed erythroblastic islands . the central macrophage, also known as a nurse cell, anchors the precursors within the island niche, regulates erythroid proliferation and differentiation, transfers iron to the erythroid progenitors for hemoglobin synthesis, and phagocytizes extruded metarubricyte nuclei. although erythroblastic islands occur throughout the marrow, those with more differentiated erythroid cells neighbor sinusoids, whereas nonadjacent islands contain mostly undifferentiated precursors. as erythroid cells mature from a rubriblast to a mature erythrocyte, their nuclei become smaller and more condensed. the nucleus is eventually extruded to form a polychromatophil. erythroid cells also become less basophilic and more eosinophilic as more hemoglobin is produced and as rna-rich organelles are lost during maturation. (hemoglobin stains eosinophilic, and rna stains basophilic with routine romanowsky's stains.) as granulocytes (e.g., neutrophils, eosinophils, and basophils) mature from a myeloblast to their mature forms, their nuclei become dense and segmented. granulocytes acquire their secondary or specific granules during the myelocyte stage and can be morphologically differentiated starting at this stage. neutrophils have neutral-staining secondary granules, eosinophil secondary granules have an affinity for acidic or eosin dyes, and basophil secondary granules have an affinity for basic dyes. monoblasts differentiate into promonocytes with ruffled nuclear boarders and then into monocytes. in most mammals, mature erythrocytes have a biconcave disk shape, called a discocyte. microscopically, these cells are round and eosinophilic with a central area of pallor. however, the central concavity may not be microscopically apparent in species other than the dog. camelids normally have oval erythrocytes, termed ovalocytes or elliptocytes, which facilitate better gas exchange at high altitudes. the erythrocytes of some animals are prone to in vitro shape change, including those of cervids, pigs, and some goat breeds (e.g., angora). erythrocyte size during health depends on the species, breed, and age of the animal. in dogs, some breeds have relatively smaller (e.g., akitas and shibas) or larger (e.g., some poodles) erythrocytes. akitas and shibas also have a high concentration of potassium, unlike erythrocytes in other dogs. juvenile animals may have larger erythrocytes because of the persistence of fetal erythrocytes, which is followed by a period of relatively smaller cells before reaching adult reference intervals. mature mammalian erythrocytes lack nuclei and organelles and are thus incapable of transcription, translation, and oxidative metabolism. however, they do require energy for various functions, including maintenance of shape and deformability, active transport, and prevention of oxidative damage. red blood cells generate this energy entirely through glycolysis (also known as the embden-meyerhof pathway). except in pigs, glucose enters erythrocytes from the plasma through an insulin-independent, integral membrane glucose transporter. within circulation the erythrocyte mean life span varies between species and is related to body weight and metabolic rate: approximately days in horses and cattle, days in dogs, and days in cats. when erythrocytes reach the end of their life span, they are destroyed in a process termed hemolysis. hemolysis may occur within blood vessels (intravascular hemolysis) or by sinusoidal macrophages (extravascular hemolysis). during intravascular hemolysis, erythrocytes release their contents, mostly hemoglobin, directly into blood. however, during extravascular hemolysis, macrophages phagocytize entire erythrocytes, leaving little or no hemoglobin in the blood. normal turnover of erythrocytes occurs mainly by extravascular hemolysis within the spleen, and to a lesser extent in other organs such as the liver and bone marrow. the exact controls are not clear, but factors that likely play a role in physiologic hemolysis include the following: • exposure of membrane components normally sequestered on the inner leaflet of the erythrocyte membrane, particularly phosphatidylserine. reticulocytes, and subsequently mature erythrocytes. the normal transit time from rubriblast to mature erythrocyte is approximately week. reticulocytes start maturing in the bone marrow but finish their maturation in the blood circulation and spleen. horses are an exception in that they do not release reticulocytes into circulation, even in situations of increased demand. unlike mature erythrocytes, which lack organelles, reticulocytes still contain ribosomes and mitochondria, mainly to support completion of hemoglobin synthesis. these remaining organelles impart a bluish-purple cast (polychromasia) to reticulocytes on routine blood smear examination. the resultant cells are termed polychromatophils. because older reticulocytes do not exhibit polychromasia, more sensitive laboratory techniques must be used for accurate reticulocyte quantification. when a blood sample is incubated with new methylene blue stain, the reticulocytes' ribosomal rna precipitates to form irregular, dark aggregates . cats also have a more mature form of reticulocyte, termed punctate reticulocyte, which is stippled when stained with new methylene blue. punctate reticulocytes indicate prior, not active, regeneration and do not appear polychromatophilic on routine blood smear evaluation. storage pool, which consists of a reserve of fully mature neutrophils. the size of the storage pool varies by species; it is large in the dog, but small in ruminants. in homeostasis mostly mature segmented granulocytes are released from the marrow into the blood. the first monocytic precursor identifiable by morphologic features is the monoblast, which develops into promonocytes and subsequently monocytes (see fig. - ). unlike granulocytes, monocytes do not have a marrow storage pool; they immediately enter venous sinusoids upon maturation. after migrating into the tissues, monocytes undergo morphologic and immunophenotypic maturation into macrophages. within blood vessels there are two pools of leukocytes: the circulating pool and the marginating pool. circulating cells are free flowing in blood, whereas marginating cells are temporarily adhered to endothelial cells by selectins. in most healthy mammals there are typically equal numbers of neutrophils in the circulating and marginal pools. however, there are threefold more marginal neutrophils relative to circulating neutrophils in cats. only the circulating leukocyte pool is sampled during phlebotomy. the concentration of myeloid cells in blood depends on the rate of production and release from the bone marrow, the proportions of cells in the circulating and marginating pools, and the rate of migration from the vasculature into tissues. the fate of neutrophils after they leave the bloodstream in normal conditions (i.e., not in the context of inflammation) is poorly understood. they migrate into the gastrointestinal and respiratory tracts, liver, and spleen and may be lost through mucosal surfaces or undergo apoptosis and be phagocytized by macrophages. lymphopoiesis. lymphopoiesis-from lympha (latin, water)refers to the production of new lymphocytes, including b lymphocytes, t lymphocytes, and natural killer (nk) cells. b lymphocytes primarily produce immunoglobulins, also known as antibodies, and are key effectors of humoral immunity. they are distinguished by the presence of an immunoglobulin receptor complex, termed the b lymphocyte receptor. plasma cells are terminally differentiated b lymphocytes that produce abundant immunoglobulin. t lymphocytes, effectors of cell-mediated immunity, possess t lymphocyte receptors that bind antigens prepared by antigen-presenting cells. a component of innate immunity, nk cells kill a variety of infected and tumor cells in the absence of prior exposure or priming. main growth factors for b lymphocytes, t lymphocytes, and nk cells are il- , il- , and il- , respectively. lymphocytes are derived from hscs within the bone marrow. b lymphocyte development occurs in two phases, first in an antigenindependent phase in the bone marrow and ileal peyer's patches (the site of b lymphocyte development in ruminants), then in an antigendependent phase in peripheral lymphoid tissues (such as spleen, lymph nodes, and mucosa-associated lymphoid tissue [malt] ). t lymphocyte progenitors migrate from the bone marrow to the thymus, where they undergo differentiation, selection, and maturation processes before migrating to the peripheral lymphoid tissue as effector cells. unlike granulocytes, which circulate only in blood vessels and migrate unidirectionally into target tissues, lymphocytes travel in both blood and lymphatic vessels and continually circulate between blood, tissues, and lymphatic vessels. also in contrast to nonlymphoid hematopoietic cells, blood lymphocyte concentrations in adult animals are primarily dependent upon extramedullary lymphocyte production and kinetics, and not lymphopoiesis by the marrow. in healthy nonruminant mammals, lymphocytes are the second most numerous blood leukocyte. according to conventional wisdom, • decreased erythrocyte deformability. • binding of immunoglobulin g (igg) and/or complement to erythrocyte membranes. complement binding may be secondary clustering of the membrane anion exchange protein, band . • oxidative damage to erythrocytes. macrophages degrade erythrocytes into reusable components, such as iron and amino acids, and the waste product bilirubin. bilirubin is then exported into circulation, where it is transported to the liver by albumin. the liver conjugates and subsequently excretes bilirubin into bile for elimination from the body. intravascular hemolysis normally occurs at only extremely low levels. hemoglobin is a tetramer that, when released from the erythrocyte into the blood, splits into dimers that bind to a plasma protein called haptoglobin. the hemoglobin-haptoglobin complex is taken up by hepatocytes and macrophages. this is the major pathway for handling free hemoglobin. however, free hemoglobin may also oxidize to form methemoglobin, which dissociates to form metheme and globin. metheme binds to a plasma protein called hemopexin, which is taken up by hepatocytes and macrophages in a similar manner to hemoglobin-haptoglobin complexes. free heme in the reduced form binds to albumin, from which it is taken up in the liver and converted into bilirubin. the concentration of circulating erythrocytes typically decreases postnatally and remains below normal adult levels during the period of rapid body growth. the age at which erythrocyte numbers begin to increase and the age at which adult levels are reached vary among species. in dogs, adult values are usually reached between and months of age; in horses, this occurs at approximately year of age. granulopoiesis is the production of neutrophils, eosinophils, and basophils, whereas monocyte production is termed monocytopoiesis. granulocytic and monocytic cells are sometimes collectively referenced as myeloid cells. however, the term myeloid and the prefix myelo-can be confusing because they have other meanings; they may reference the bone marrow, all nonlymphoid hemic cells (erythrocytes, leukocytes, and megakaryocytes), only granulocytes, or the spinal cord. the main purpose of granulocytes and monocytes is to migrate to sites of tissue inflammation and function in host defense (see chapters and ). briefly, these cells have key immunologic functions, including phagocytosis and microbicidal activity (neutrophils and monocyte-derived macrophages), parasiticidal activity and participation in allergic reactions (eosinophils and basophils), antigen processing and presentation, and cytokine production (macrophages). neutrophils are the predominant leukocyte type in blood of most domestic species. primary stimulators of granulopoiesis and monocytopoiesis are granulocyte-macrophage colony-stimulating factor and il- , il- , and il- (granulocytes and monocytes), granulocyte colonystimulating factor (granulocytes), and macrophage colonystimulating factor (monocytes). in general, these cytokines are produced by various inflammatory cells, with or without contribution from stromal cells. the earliest granulocytic precursor identifiable by routine light microscopy is the myeloblast, which undergoes maturational division over days to produce to progeny cells (see fig. - ). these granulocytic precursors are conceptually divided into those stages that can divide, including myeloblasts, promyelocytes, and myelocytes (proliferation pool), and those that cannot, including metamyelocytes, and band and segmented forms (maturation pool). within the neutrophil maturation pool is a subpool, termed the platelet aggregation and adherence to subendothelial collagen. expansion of surface area and release of granule contents is aided by a network of membrane invaginations known as the open canalicular system. this system is not present in horses, cattle, and camelids. information on this topic is available at www.expertconsult.com. information on this topic is available at www.expertconsult.com. information on this topic is available at www.expertconsult.com. information on this topic is available at www.expertconsult.com. mechanisms of bone marrow disease are summarized in box - . hematopoietic cells' response to injury is dependent upon whether the insult is on the marrow or within extramarrow tissues. in general, marrow-directed injury or disturbances result in production of abnormal hematopoietic cells (dysplasia), fewer hematopoietic cells (hypoplasia), or a failure of hematopoietic cell development (aplasia). dysplasia, hypoplasia, and aplasia may be specific for one cell line, such as pure red cell aplasia, or affect multiple lineages, as seen with aplastic anemia. accordingly, decreased blood concentrations of the involved cell types are expected with hypoplasia or aplasia. erythroid, myeloid, and megakaryocytic hypoplasia or aplasia causes nonregenerative anemia, neutropenia, and thrombocytopenia, respectively. bicytopenia is used to describe decreased blood concentrations of two cell lines, whereas pancytopenia indicates decreased blood concentrations of all three cell types. bicytopenia or pancytopenia may indicate generalized marrow disease, such as occurs with aplastic anemia or marrow malignancies (leukemia), necrosis, fibrosis (myelofibrosis), or inflammation (myelitis). replacement of hematopoietic tissue within the bone marrow by abnormal tissue, including neoplastic cells, fibrosis, or inflammatory cells, is termed myelophthisis. cattle normally have higher numbers of lymphocytes than neutrophils in circulation. however, recent studies suggest that is no longer the case, most likely due to changes in genetics and husbandry. in most species the majority of lymphocytes in blood circulation are t lymphocytes. the concentration of blood lymphocytes decreases with age. thrombopoiesis. thrombopoiesis-from thrombos (gr., clot)refers to the production of platelets, which are small ( to µm), round to ovoid, anucleate cells within blood vessels. platelets have a central role in primary hemostasis but also participate in secondary hemostasis (coagulation) and inflammatory pathways (see chapters and ). thrombopoietin (tpo) is the primary regulator of thrombopoiesis. the liver and renal tubular epithelial cells constantly produce tpo, which is then cleared and destroyed by platelets and their precursors. therefore plasma tpo concentration is inversely proportional to platelet and platelet precursor mass. if the platelet mass is decreased, less tpo is cleared, and there is subsequently more free plasma tpo to stimulate thrombopoiesis. the earliest morphologically identifiable platelet precursor is the megakaryoblast, which undergoes nuclear reduplications without cell division, termed endomitosis, to form a megakaryocyte with to nuclei. as the name suggests, megakaryocytes are very large cells, much larger than any other hematopoietic cell ; also see fig. - ). megakaryocytes neighbor venous sinusoids, extend their cytoplasmic processes into vascular lumens, and shed membranebound cytoplasmic fragments (platelets) into blood circulation. orderly platelet shedding is partially facilitated by β -tubulin microtubules within megakaryocytes. platelets circulate in a quiescent form and become activated by binding platelet agonists, including thrombin, adenosine diphosphate (adp), and thromboxane. platelet activation causes shape change, granule release, and relocation of procoagulant phospholipids and glycoproteins (gps) to the outer cell membrane. specific procoagulant actions include release of calcium, von willebrand factor (vwf), factor v, and fibrinogen, as well as providing phosphatidylserine-rich binding sites for the extrinsic tenase (factors iii, vii, and x), intrinsic tenase (factors ix, viii, and x), and prothrombinase (factors x, v, and ii) coagulation complexes. platelet gp surface receptors include those for binding vwf (gpib-ix-v), collagen (gpvi), and fibrinogen (gpiib-iiia), which facilitate increased destruction hemorrhage (especially erythrocytes) consumption (platelets) neoplasia altered distribution abnormal function bone marrow is not routinely sampled during postmortem examinations. however, indications for bone marrow evaluation include suspected leukemia, metastatic neoplasia within bone marrow, or infectious myelitis, as well as cytopenia(s) or hematopoietic dysplasia of unknown cause. multimodal evaluation is ideal, including a recent (< hours) complete blood count with bone marrow cytologic and histopathologic examination. however, antemortem blood analyses are not always available, and interpretation of hematopoietic cytomorphologic examination results becomes difficult to impossible shortly after death. postmortem bone marrow should be collected as soon as possible after death or euthanasia, preferably within minutes. samples may be collected from the proximal femur, rib, sternum or vertebrae. when collecting from the femur, the femoral neck is removed with a bone saw, or a fragment of the shaft is removed with bone-cutting shears. cytologic samples are first collected using the paintbrush technique: gently sample the red marrow with a clean, dry, naturalbristle brush, and then carefully brush the material onto a clean glass microscope slide in two to four parallel wavy lines. the brush should be cleaned and dried before its use on a different animal. the slide is then air dried, stored away from formalin fumes, and then stained with a routine (romanowsky) stain. for histologic evaluation the entire femoral head or femoral shaft or rib fragment with exposed red marrow is submersed in % neutral buffered formalin. for cosmetic necropsies, samples may be obtained by antemortem techniques, such as needle biopsies for cytologic examination and core biopsies for histopathologic examination. the complete blood count (cbc) is the cornerstone for diagnosis of hematologic disturbances and is often part of a minimum database in sick patients. the cbc includes numeric data indicating the concentration of different cell types, as well as other estimations of red blood cell mass (hemoglobin concentration, packed-cell volume, and hematocrit), red blood cell volume (mean cell volume), and red blood cell hemoglobin content (mean cell hemoglobin and mean cell hemoglobin concentration). cell morphologic features and the presence or absence of hemic parasites are assessed upon microscopic review of a blood smear and are also included in a cbc report. (note: some parasites may infect blood cells, such as hepatozoon organisms within circulating neutrophils or monocytes or bartonella organisms within erythrocytes, but mainly cause disease in other body systems and are therefore not discussed in this chapter.) learning to evaluate blood smears is a valuable skill for any practicing veterinarian. the cbc also may include the plasma protein concentration, as measured with a refractometer. it is important to remember that changes in hydration status and in the distribution of body fluids between the vascular and extravascular compartments affect the concentration of both cells and proteins in the blood. other tests that may help with evaluation of the hematopoietic system include cell or tissue biopsies, the direct antiglobulin test, flow cytometry, immunophenotyping, and polymerase chain reaction (pcr). aspiration cytology and/or histopathology of organs other than the bone marrow can be pursued to assess for the presence of emh, increased destruction of erythrocytes, neoplasia, or infection. the coombs test, or direct antiglobulin test, detects excessive antibody or complement bound to red blood cells' surfaces and is the standard assay for immune-mediated hemolytic anemia. flow cytometry and immunofluorescent antibody tests may also be used to detect autoantibody bound to erythrocytes or other hematopoietic cells. immunophenotyping and pcr are further discussed in the section on hematopoietic neoplasia. structural or functional abnormalities of blood vessels, platelets, or coagulation factors may result in a tendency toward hypocoagulability (bleeding), hypercoagulability (inappropriate thrombosis), or both. in veterinary medicine there has been a great deal of work on specific mechanisms of hypocoagulability, whereas mechanisms of hypercoagulability are less fully characterized. disorders of primary hemostasis typically result in "small bleeds" (e.g., petechiation, mild ecchymosis, bleeding from mucous membranes, bleeding immediately after venipuncture), whereas disorders of secondary hemostasis typically result in "big bleeds" (e.g., hemorrhage into body cavities/ joints, marked ecchymosis, large hematomas, delayed bleeding after venipuncture). this chapter concentrates on primary disorders of hemostasis and also covers disseminated intravascular coagulation, which is the secondary condition. however, it is important to note that coagulation disorders can also result from other underlying disease processes. for example, advanced liver disease can lead to abnormal hemostasis through decreased or defective synthesis of coagulation factors or impaired clearance of fibrinolytic products that inhibit coagulation reactions and platelet function. vascular disorders may also result in a bleeding tendency because of abnormalities of endothelial function or collagen-platelet interactions. specific diseases involving abnormal structure or function of hematopoietic or hemostatic elements are discussed later in this chapter. the cbc provides basic information about platelets, including numeric values for platelet concentration and mean platelet volume (mpv), subjective assessment of platelet morphologic features (size, shape, and granularity), and a rough estimation of platelet numbers based on examination of a blood smear. some laboratories measure reticulated platelets (platelets recently released from the bone marrow), although this test is mostly used in the research setting at present. increased mpv and increased numbers of reticulated platelets tend to indicate increased thrombopoiesis. bone marrow examination is indicated with any unexplained cytopenia, including thrombocytopenia, to evaluate production. tests to evaluate the components of the hemostatic process are described and listed in e-appendix - . secondary myelofibrosis is the enhanced deposition of collagen within the marrow by nonneoplastic fibroblasts and reticular cells. disease pathogenesis is unclear, but there are two leading theories. first, it may represent scar formation after marrow necrosis, as previously presented. and second, high concentrations of growth factors present during times of marrow injury or activation may stimulate fibroblast proliferation. in particular, stimulated megakaryocytes and macrophages produce fibrogenic cytokines, including plateletderived growth factor, transforming growth factor-β, and epidermal growth factor. early in disease there is reticulin deposition without reduction of hematopoietic elements. however, fibrous collagen replaces hematopoietic cells with disease progression. histologic identification of reticulin and collagen fibers can be aided with reticulin silver and masson's trichrome stains, respectively. in animals, secondary myelofibrosis occurs most commonly with leukemias, extramarrow malignancies, and chronic hemolytic anemias, but many cases are idiopathic. experimental whole-body gamma irradiation, dietary strontium- exposure, and certain drugs and toxins can also induce myelofibrosis. the responses of marrow adipocytes to systemic and localized disease are under current investigation, especially in relation to energy metabolism, inflammation, and bone trauma. during times of severe energy imbalance, such as cachexia, the marrow may undergo serous atrophy of fat, also known as gelatinous marrow transformation (e- fig. - ). the pathogenesis of this phenomenon is unknown, but it is characterized by adipocyte atrophy, hematopoietic cell hypoplasia with subsequent cytopenias, and replacement of the marrow with extracellular hyaluronic acid-rich mucopolysaccharides. positive alcian blue staining identifies the extracellular material as mucin. marrow adipocytes secrete adipose-derived hormones, termed adipokines, including leptin and adiponectin. in general, leptin is proinflammatory, prothrombotic, and mitogenic for various cell types, including lymphocytes, hematopoietic progenitors, and leukemic cells. conversely, adiponectin has antiinflammatory and growth inhibitory properties. during times of inflammation and infection, leptin production is increased. in response to marrow trauma, such as orthopedic surgery, fat may enter the vasculature, embolize to various tissues, and cause tissue ischemia. the severity of tissue injury caused by fat embolism is dependent upon the quantity of fat entering circulation and the tissue's susceptibility to ischemia (see chapter ). responses of circulating blood cells to injury include decreased survival (destruction, consumption, or loss), altered distribution, and altered structure or function (see box - ). these responses are not mutually exclusive-for example, altered erythrocyte structure may lead to decreased survival. often, but not always, these responses result in decreased concentrations of blood cells in circulation. abnormal concentrations of blood cells. the concentration of blood cells may be decreased, termed cytopenia (from kytos [gr., hollow vessel] and penia [gr., poverty]) or increased, designated cytosis (from osis [gr., condition]). a specific blood cell type is denoted as being decreased by using the suffix -penia (table - ) . a decreased concentration of erythrocytes is the exception and is termed anemia (from a [gr., without] and haima [gr., blood]). decreased concentrations of blood basophils are not recognized in domestic animals because the lower reference interval is typically zero. an increased blood cell type is denoted with the suffix -osis or -philia (see table - ). postmortem quantification of blood cell insults to extramarrow tissues and cells tend to cause increased production of the involved cell types (hyperplasia) with or without dysplasia. loss of erythrocytes from blood vessels (hemorrhage), or premature destruction of erythrocytes (hemolysis) causes erythroid hyperplasia. tissue inflammation may cause neutrophilic, eosinophilic, basophilic, and/or monocytic hyperplasia, depending on the type of inflammation. megakaryocytic hyperplasia may occur with increased platelet use during hemorrhage or disseminated intravascular coagulation (dic) or with immune-mediated platelet destruction. exceptions to these generalizations, such as anemia of chronic disease, iron deficiency anemia, and anemia of renal failure, are discussed in more detail later. endothelial cell response to injury specifically within the marrow is poorly characterized, but it is likely similar to that of endothelial cells elsewhere, playing active roles in coagulation and inflammation (see chapters and ). however, one potential sign of marrow sinusoidal injury is the presence of circulating nucleated erythrocytes in the absence of erythrocyte regeneration, termed inappropriate metarubricytosis. it is proposed that injured marrow endothelial cells allow premature passage of metarubricytes into blood circulation during times of stress. however, a conflicting theory proposes that marrow stress causes decreased metarubricyte attachment to central macrophages, and subsequent release into circulation. specific causes of marrow injury-induced metarubricytosis include sepsis, hyperthermia, malignancies, hypoxia, and certain drugs and toxins. inappropriate metarubricytosis may also occur with erythroid dysplasia and splenic disorders. in addition to a suspected role in inappropriate metarubricytosis, marrow macrophages are integral to altered iron metabolism, including anemia of chronic disease and hemosiderosis. anemia of chronic disease is a mild to moderate nonregenerative anemia observed in animals with a variety of inflammatory and metabolic disorders. this anemia is discussed in more detail later, but briefly, it is primarily a result of iron sequestration within macrophages. hemosiderosis is the excessive accumulation of iron in tissues, typically macrophages. accumulation of iron in parenchymal organs, leading to organ toxicity, is termed hemochromatosis. in animals, iron overload due to blood transfusions or chronic hemolytic anemias may cause marrow hemosiderosis and hemochromatosis. myelitis can take different forms. granulomatous myelitis occurs with systemic fungal infections (e.g., histoplasmosis) or mycobacteriosis. acute or neutrophilic myelitis may occur with lower-order bacterial infections or those with an immune-mediated component. dogs and cats with nonregenerative immune-mediated hemolytic anemia (imha) often have myelitis, in addition to myelofibrosis and necrosis. the inflammation is evident as fibrin deposition, edema, and multifocal neutrophilic infiltrates; immune-mediated cytopenias may also concurrently occur with bone marrow lymphocytic and/or plasma cell hyperplasia. bone marrow necrosis is the necrosis of medullary hematopoietic cells, stromal cells, and stroma in large areas of bone marrow. potential causes include leukemias, extramarrow malignancies, infection (bovine viral diarrhea virus [bvdv] , ehrlichia canis, and feline leukemia virus [felv]), sepsis, drugs or toxins (carprofen, chemotherapeutic agents, estrogen, metronidazole, mitotane, and phenobarbital), and irradiation. direct hematopoietic or stromal cytotoxicity and altered marrow microvasculature (disseminated intravascular coagulation) are proposed pathogeneses. extensive marrow necrosis results in decreased hematopoiesis and subsequent blood cytopenias, including anemia, neutropenia, and thrombocytopenia. if the animal survives the initial insult, the marrow may recover and resume normal hematopoiesis, or it may undergo scar formation, termed myelofibrosis. concentrations is not possible due to perimortem coagulation. however, a complete blood count (cbc) with microscopic blood smear evaluation is the foundation for antemortem assessment of blood cells. anemia. anemia causes clinical signs referable to decreased red hemoglobin pigment (e.g., pale mucous membranes), decreased oxygen-carrying capacity (e.g., depression, lethargy, weakness, and exercise tolerance), and decreased blood viscosity (e.g., heart murmur). recumbency, seizures, syncope, or coma may occur with severe anemia. anemia is confirmed by identifying a decreased hemoglobin concentration or reduced erythrocyte mass, as measured by the packed-cell volume, hematocrit, or red blood cell concentration. the three general causes of anemia are blood loss (hemorrhage), red blood cell destruction or lysis (hemolysis), and decreased red blood cell production (erythroid hypoplasia). classifying anemia as regenerative or nonregenerative is clinically useful because it provides information about the mechanism of disease; regenerative anemia indicates hemorrhage or hemolysis, whereas erythroid hypoplasia or aplasia causes nonregenerative anemia (table - ). the hallmark of regenerative anemias, except in horses, is reticulocytosis (i.e., increased numbers of circulating reticulocytes [immature erythrocytes]), which is evident as polychromasia on a routinely stained blood smear (see fig. - ). reticulocytosis indicates increased bone marrow erythropoiesis ( fig. - ) and release of erythrocytes before they are fully mature. reticulocytosis is an appropriate marrow response to anemia and is often seen with hemorrhage or hemolysis. on a cbc a strong regenerative response may produce an increased mean cell volume (mcv) and decreased mean cell hemoglobin concentration (mchc) because reticulocytes are larger and have a lower hemoglobin concentration than mature erythrocytes. horses are an exception to this classification scheme because they do not release reticulocytes into circulation, even with erythroid hyperplasia. horses with a regenerative response may have an increased mcv and red cell distribution width (an index of variation in cell size). but definitive determination of regeneration in a horse requires demonstration of erythroid hyperplasia via bone marrow examination or an increasing red cell mass over sequential cbcs. in addition to reticulocytosis there may be increased numbers of nucleated red blood cells (nrbcs) in circulation with erythrocyte regeneration, termed appropriate metarubricytosis. when nrbcs are present as part of a regenerative response, they should be in low numbers relative to the numbers of reticulocytes. however, the presence of circulating nrbcs is not in itself definitive evidence of regeneration and may signify dyserythropoiesis (e.g., lead poisoning or bone marrow disease) or splenic dysfunction. these processes should be suspected when nrbcs are increased without reticulocy- such as into the peritoneal cavity, because iron is not lost from the body and can be reused for erythropoiesis. in hemolytic anemia, erythrocytes are destroyed at an increased rate. whether the mechanism is intravascular or extravascular, or a combination, depends on the specific disease process (specific diseases are discussed later in this chapter). some clinical indicators of hemolytic anemia and their pathogeneses are summarized in fig. - and are further described in the following discussion. a classic sequela of hemolytic anemias in general is hyperbilirubinemia, which is an increase in the plasma bilirubin concentration. bilirubin is a yellow pigment, which explains why hyperbilirubinemia, if severe enough, causes icterus-the grossly visible yellowing of fluid or tissues ( fig. - ) . icterus, also known as jaundice, is usually detectable when the plasma bilirubin concentration exceeds mg/dl. however, it is important to note that hyperbilirubinemia and icterus are not pathognomonic for hemolysis and may also occur with conditions of impaired bile flow (cholestasis), such as hepatopathy or cholangiopathy. in addition to icterus, hemolytic anemia often results in splenomegaly , which is secondary to extravascular hemolysis and macrophagic hyperplasia within the spleen, as well as splenic emh. splenomegaly may also occur in other conditions, as discussed elsewhere in this chapter. intravascular hemolysis is grossly evident as pink-tinged plasma or serum, termed hemolysis or hemoglobinemia. hemolysis is not apparent until the concentration of extracellular hemoglobin is greater than approximately mg/dl. cell-free hemoglobin is scavenged by haptoglobin until haptoglobin becomes saturated with hemoglobin at a concentration of approximately mg/dl. when haptoglobin is saturated, any remaining free hemoglobin has a low enough molecular weight to pass through the renal glomerular filter into the urine. this imparts a pink or red discoloration to the urine, called hemoglobinuria. thus extracellular hemoglobin can cause gross discoloration of the plasma, where it is bound to haptoglobin, before becoming grossly visible in urine. the half-life of haptoglobin is markedly decreased when bound to hemoglobin, so when large amounts of haptoglobin-hemoglobin complex are formed, the concentration of haptoglobin in the blood decreases and hemoglobin can pass through the glomerulus at even lower concentrations. hemoglobinuria is a contributing factor in the renal tubular necrosis (hemoglobinuric nephrosis) that often occurs in cases of acute intravascular hemolysis (see chapter ). a similar lesion occurs in the kidneys of individuals with marked muscle damage and resulting myoglobinuria (see chapters and ). hemoglobinuria cannot be distinguished grossly from hematuria (erythrocytes in the urine) or myoglobinuria (myoglobin in the urine), and all three processes cause a positive reaction for "blood protein" on urine test strips. comparing the colors of the plasma and the urine may be informative. in contrast to hemoglobin, myoglobin causes gross discoloration of the urine before the plasma is discolored. this is because myoglobin is a low-molecular-weight monomer, freely filtered by the glomerulus, and does not bind plasma proteins to a significant degree. hematuria can be distinguished from hemoglobinuria on the basis of microscopic examination of urine sediment (i.e., erythrocytes are present in cases of hematuria). in addition to red plasma and urine, hemoglobinemia may also be identified by increased mch or mchc values on a cbc. this is because the hemoglobin concentration is measured by lysing all erythrocytes in the sample and then measuring the total hemoglobin via spectrophotometry. by this method, hemoglobin that originated within or outside of erythrocytes is measured together. however, calculations for mch and mchc, which include results for the hemoglobin and red blood cell concentrations, assume that all of tosis, or their numbers are high relative to the degree of reticulocytosis, termed inappropriate metarubricytosis. in ruminants, reticulocytosis is often accompanied by basophilic stippling (fig. - ) . however, like metarubricytosis, basophilic stippling without reticulocytosis is concerning for lead poisoning or other causes of dyserythropoiesis. recall that the stimulus for increased erythropoiesis is increased secretion of epo in response to tissue hypoxia. although the action of epo on erythropoiesis is rapid, evidence of a regenerative response is not immediately apparent in a blood sample. one of the main effects of epo is to expand the pool of early-stage erythroid precursors, and it takes time for these cells to differentiate to the point where they are released into circulation. in a case of acute hemorrhage or hemolysis, for example, it typically takes to days until reticulocytosis is evident on the cbc and several more days until the regenerative response peaks. the term preregenerative anemia is sometimes used to describe anemia with a regenerative response that is impending but not yet apparent on the cbc. confirming a regenerative response in such cases requires either evidence of erythroid hyperplasia in the bone marrow or emergence of a reticulocytosis on subsequent days. hemorrhage results in escape of erythrocytes and other blood components, such as protein, from the vasculature. as a result, a decreased plasma or serum protein concentration, termed hypoproteinemia, may be evident on a cbc or chemistry panel. if the hemorrhage is into the gastrointestinal lumen, some of the protein may be resorbed and converted to urea, resulting in an increased urea nitrogen concentration relative to creatinine in plasma. hemorrhage within the urinary tract may cause red urine with erythrocytes observed in the urine sediment. causes of hemorrhage include trauma, abnormal hemostasis, certain parasitisms, ulceration, and neoplasia. hemorrhage may be acute or chronic, or internal or external. during acute hemorrhage, there are ample iron stores within the body for hemoglobin synthesis and erythrocyte regeneration. however, with chronic external hemorrhage, continued loss of iron may deplete the body's iron stores. as iron stores diminish, so does erythrocyte regeneration, eventually leading to iron deficiency anemia. iron deficiency anemia is either poorly regenerative or nonregenerative and is discussed in more detail later in the chapter. iron deficiency anemia does not occur with chronic internal hemorrhage, spherocytes form when macrophages (mainly in the spleen) phagocytize part of an erythrocyte plasma membrane bound with autoantibody ( fig. - ) . the remaining portion of the erythrocyte assumes a spherical shape, thus preserving maximal volume. this change in shape results in decreased deformability of the cells. erythrocytes must be extremely pliable to traverse the splenic red pulp and sinusoidal walls; spherocytes therefore tend to be retained in the spleen in close association with macrophages with risk for further injury and eventual destruction. in the dog, spherocytes appear smaller than normal and have uniform staining ( fig. - , a), in contrast to normal erythrocytes, which have a region of central pallor imparted by their biconcave shape. this difference in staining between spherocytes and normal erythrocytes is not consistently discernible in many other domestic animals (including horses, the hemoglobin originated within erythrocytes. in the case of hemoglobinemia, the excess extracellular hemoglobin may cause an artifactual increase in the calculated mch and mchc. it is important to remember that similar artifactual increases may also occur with lipemia. once hemolytic anemia has been identified, the specific cause for hemolysis should be investigated based on signalment, clinical history, and microscopic blood smear evaluation. the most common causes of hemolytic anemia in domestic animals are immunemediated, infectious, oxidative, and mechanical fragmentation (i.e., microangiopathic) disorders (table - ) . spherocytosis and autoagglutination are hallmarks of immunemediated hemolytic anemia, either primary (also known as idiopathic) or secondary to infectious disease, drugs/toxins, or neoplasms. several initiating processes can cause intravascular hemolysis; formation of the complement membrane attack complex is pictured. with intravascular hemolysis, free hemoglobin is release directly into the plasma, where it is scavenged by haptoglobin and hemopexin. when haptoglobin and hemopexin are saturated, the cell-free hemoglobin causes red discoloration of the plasma (hemolysis) and is excreted in the urine (hemoglobinuria; dark red urine). the liver clears haptoglobinhemoglobin and hemopexin-methemoglobin complexes from plasma and converts hemoglobin to unconjugated bilirubin and then conjugated bilirubin. conjugated bilirubin is normally excreted in the bile and then converted to urobilinogen (yellow) and subsequently stercobilinogen (dark brown). however, excessive bilirubin will spill over into the plasma, resulting in hyperbilirubinemia, icteric plasma (if severe enough), and urinary excretion of bilirubin (bilirubinuria; icteric urine). extravascular hemolysis: during extravascular hemolysis, erythrocytes are phagocytized by macrophages, which digest erythrocytes, and convert hemoglobin to unconjugated bilirubin. excessive bilirubin in plasma causes hyperbilirubinemia with or without icteric plasma. unconjugated bilirubin is processed and excreted by the liver (as previously described) and in dogs, the kidney. kidney u-bilirubin cattle, and cats), whose erythrocytes differ from those of the dog in that they are smaller and have less pronounced biconcavity and therefore less pronounced central pallor. autoagglutination occurs because of cross-linking of antibodies bound to erythrocytes (see . autoagglutination is evident macroscopically as blood with a grainy consistency (see fig. - , b) , and microscopically as clusters of erythrocytes (see fig. - , c). autoagglutination may also result in a falsely increased mcv and decreased red blood cell concentration when clustered cells are mistakenly counted as single cells by automated hematology analyzers. when autoagglutination is present, the packed-cell volume is the most reliable measurement of red blood cell mass. ghost cells are ruptured red blood cell membranes devoid of cytoplasmic contents (see a) . they indicate intravascular hemolysis and may be seen with a variety of hemolytic disorders, including those with immune-mediated, infectious, oxidative, or fragmentation causes. in the case of immune-mediated hemolytic anemia, antibody or complement binds to red blood cell membranes and activates the complement membrane attack complex (see fig. - ). this causes pore formation in the red blood cell membrane and release of cytoplasmic contents into the plasma. ghost cells are eventually cleared from circulation by phagocytic macrophages, mainly within the spleen. oxidative damage to erythrocytes occurs when normal antioxidative pathways that generate reducing agents (such as reduced nicotinamide adenine dinucleotide [nadh] , reduced nicotinamide adenine dinucleotide phosphate [nadph] , and reduced glutathione [gsh]) are compromised or overwhelmed, resulting in hemolytic anemia, abnormal hemoglobin function, or both. hemolysis caused by oxidative damage may be extravascular or intravascular, or a combination. evidence of oxidative damage to erythrocytes may be apparent on blood smear examination as heinz bodies or eccentrocytes or on gross examination as methemoglobinemia. heinz bodies are foci of denatured globin that interact with the erythrocyte membrane. they are usually subtly evident on routine wright-stained blood smears as pale circular inclusions or blunt, rounded protrusions of the cell margin but are readily discernible on smears stained with new methylene blue . cats are particularly susceptible to heinz body formation and may have low numbers of heinz bodies normally. there is no unanimity of opinion, but some clinical pathologists believe that the presence of heinz bodies in up to % of all erythrocytes in cats is within normal limits. this predisposition is believed to reflect unique features of the feline erythrocyte, whose hemoglobin has more sulfhydryl groups (preferential sites for oxidative damage) than do erythrocytes of other species and may also have lower intrinsic reducing capacity. it is also possible that the feline spleen does not have as efficient a "pitting" function (splenic structure and function are discussed in more detail later in this chapter). eccentrocytes, evident as erythrocytes in which one side of the cell has increased pallor ( fig. - , a) , are another manifestation of oxidative damage. they form because of cross-linking of total hemoglobin), methemoglobin imparts a grossly discernible chocolate color to the blood. by itself, mechanical fragmentation hemolysis tends to cause mild or no anemia. mechanical fragmentation results from trauma or shearing of erythrocytes within blood vessels. normal erythrocytes may be flowing through abnormal vasculature, such as with heart valve defects, intravascular fibrin deposition (e.g., disseminated intravascular coagulation), vasculitis, or hemangiosarcoma. alternatively, the red blood cells may be particularly fragile within normal blood vasculature, as occurs with iron deficiency. in either instance, microscopic evidence of mechanical fragmentation includes the presence of erythrocyte fragments (schistocytes [see fig. - , c]), erythrocytes with irregular cytoplasmic projections (acanthocytes), erythrocytes with blister-like projections (keratocytes), or ghost cells (see figs. - , a, - , d, and - , e). membrane proteins, with adhesion of opposing areas of the cell's inner membrane leaflet, and displacement of most of the hemoglobin toward the other side. the fused membranes may fragment off of the eccentrocyte, leaving a slightly ruffled border; this cellular morphologic abnormality is called a pyknocyte (see fig. - , b) . oxidative insult may also result in conversion of hemoglobin (iron in the fe + state) to methemoglobin (iron in the fe + state), which is incapable of binding oxygen. methemoglobin is produced normally in small amounts but reduced back to oxyhemoglobin by the enzyme cytochrome-b reductase (also known as methemoglobin reductase). methemoglobinemia results when methemoglobin is produced in excessive amounts (because of oxidative insult) or when the normal pathways for maintaining hemoglobin in the fe + state are impaired (as in cytochrome-b reductase deficiency). when present in sufficiently high concentration (approximately % of degradation. antierythrocyte antibodies bind rbc surface antigens, resulting in rbc opsonization by immunoglobulins (mainly immunoglobulin g [igg] ) and complement (primarily c b). immunoglobulin-or c b-bound rbcs are phagocytized and digested by sinusoidal macrophages. , spherocytes. spherocytes form when the membrane of immunoglobulin-or c b-bound rbcs are phagocytized by macrophages, without removing the entire rbc from circulation. compared to normal erythrocytes, spherocytes appear smaller, more eosinophilic, and lack central pallor. , rbc aggregation (agglutination). rbc aggregation occurs when antierythrocyte immunoglobulins (immunoglobulin m [igm] or high concentrations of igg) bind multiple erythrocytes simultaneously. , ghost cells. antierythrocyte antibodies bind rbc surface antigens, resulting in complement activation and formation of the membrane attack complex (mac). macs form membrane pores, resulting in rupture of rbcs, and the release of hemoglobin into the circulation. ghost cells are rbc membrane remnants that lack cytoplasm (hemoglobin normocytic, normochromic anemia). it has long been known that patients with inflammatory or other chronic disease often become anemic, and that this condition results in increased iron stores in the bone marrow. sequestration of iron may be a bacteriostatic evolutionary adaptation because many bacteria require iron as a cofactor for growth. in recent years, investigators have begun to elucidate the molecular mechanisms underlying anemia of inflammation. hepcidin, an acute phase protein and antimicrobial peptide synthesized in the liver, is a key mediator that limits iron availability. hepcidin expression increases with inflammation, infection, or iron overload and decreases with anemia or hypoxia. hepcidin exerts its effects by causing functional iron deficiency. it binds to and causes the degradation of the cell surface iron efflux molecule, ferroportin, thus inhibiting both absorption of dietary iron from the intestinal epithelium and export of iron from macrophages and hepatocytes into the plasma ( fig. - ). anemia of inflammation involves factors besides decreased iron availability. inflammatory cytokines are likely to inhibit erythropoiesis by oxidative damage to and triggering apoptosis of developing erythroid cells, by decreasing expression of epo and stem cell factor, and by decreasing expression of epo receptors. in addition, experimentally induced sterile inflammation in cats resulted in shortened erythrocyte survival, indicating that anemia of inflammation is likely also a function of increased erythrocyte destruction. other causes of decreased erythropoiesis are listed in table - . specific examples of diseases causing nonregenerative anemia by these mechanisms are discussed later in this chapter. neutropenia. neutropenia refers to a decrease in the concentration of neutrophils in circulating blood. neutropenia may be caused by decreased production, increased destruction, altered distribution, or a demand for neutrophils in tissues that exceeds the rate of granulopoiesis. decreased production is evident on bone marrow examination as granulocytic hypoplasia. this usually results from an insult that affects multiple hematopoietic lineages, such as chemical insult, radiation, neoplasia, infection, or fibrosis, but may also be caused by a process that preferentially targets granulopoiesis. in marked contrast to erythrocytes, neutrophils have a very short life span in circulation. once released from the bone marrow, a neutrophil is in the bloodstream only for hours before migrating into the tissues. when neutrophil production ceases, a reserve of mature neutrophils in the bone marrow storage pool may be adequate to maintain normal numbers of circulating neutrophils for a few days; however, after the bone marrow storage pool is depleted, neutropenia rapidly ensues. immune-mediated neutropenia is a rare but recognized condition in domestic animals. bone marrow findings range from granulocytic hypoplasia to hyperplasia, depending on where the cells under immune attack are in their differentiation programs. neutropenia with no evidence of decreased production and in which other causes of neutropenia have been excluded may be a result of destruction of neutrophils before they leave the bone marrow, a condition known as ineffective granulopoiesis. like other forms of ineffective hematopoiesis, this condition is often presumed to be immune mediated; in cats this condition may occur as a result of infection of hematopoietic cells with felv. as presented in the earlier section on granulopoiesis and monocytopoiesis (myelopoiesis), neutrophils within the blood vasculature are in two compartments: a circulating pool, consisting of those cells flowing freely in the blood, and a marginating pool, consisting of those cells transiently interacting with the endothelial surface. (in reality, neutrophils are constantly shifting between these two pools, but the proportion of cells in either pool normally remains fairly schistocytes are the only red blood cell morphologic abnormality specific for mechanical fragmentation because all other morphologic abnormalities can be seen with other disease processes. for example, ghost cells may be observed with other types of hemolysis. nonregenerative anemia is characterized by a lack of reticulocytosis on the cbc; however, reticulocytosis does not occur in horses even in the context of regeneration. most often this is a result of decreased production in the marrow (i.e., erythroid hypoplasia). erythrocytes circulate for a long time, so anemias caused by decreased production tend to develop slowly. the most common form of nonregenerative anemia is known as anemia of inflammation or anemia of chronic disease. in this form of anemia, erythrocytes are decreased in number but are typically normal in size and hemoglobin concentration (so-called c or because of one specifically depressing thrombopoiesis. in either case, decreased thrombopoiesis is evident as megakaryocytic hypoplasia upon bone marrow examination. general causes of decreased hematopoiesis outlined earlier in the sections on anemia and neutropenia also apply to thrombocytopenia. increased platelet destruction due to immune-mediated thrombocytopenia (imtp) is a fairly common disease in dogs and may also occur in other species. thrombocytopenia with immune-mediated thrombocytopenia is often severe (e.g., < , platelets/µl), resulting in spontaneous multisystemic hemorrhage. increased use of platelets occurs with hemorrhage and disseminated intravascular coagulation. thrombocytopenia secondary to hemorrhage is often mild to moderate, whereas disseminated intravascular coagulation may cause mild to severe thrombocytopenia, often with evidence of mechanical fragmentation hemolysis (e.g., schistocytes). disseminated intravascular coagulation is a syndrome in which hypercoagulability leads to increased consumption of both platelets and coagulation factors in the plasma, with subsequent hypocoagulability and susceptibility to bleeding. risk factors for developing disseminated intravascular coagulation include severe inflammation, such as sepsis or pancreatitis, neoplasia, and organ failure. the spleen normally contains a significant proportion of total platelet mass (up to one-third in some species), and abnormalities involving the spleen may result in changes in the number of circulating platelets. for example, splenic congestion may result in platelet sequestration and thrombocytopenia, and splenic contraction may cause thrombocytosis. lymphopenia. lymphopenia refers to a decreased concentration of lymphocytes in blood. it is a common hematologic finding in sick animals. usually the precise mechanism of lymphopenia is not clear but is often presumed secondary to endogenous glucocorticoid excess that occurs with stress. excess glucocorticoids, either endogenous or exogenous, cause an altered distribution of lymphocytes; there is increased trafficking of lymphocytes from blood to lymphoid tissue, and decreased egress of lymphocytes from lymphoid tissue to blood. at higher concentrations of glucocorticoids, lymphocytes are destroyed. other causes of lymphotoxicity include chemotherapeutic agents, radiation therapy, and some infectious agents. lymphopenia may occur with various mechanisms, including loss of lymphocyte-rich lymphatic fluid (e.g., gastrointestinal disease, repeated drainage of chylous effusions), and disruption of the normal lymphoid tissue architecture because constant in any given species.) circulating neutrophils are part of the blood sample collected during routine venipuncture and are thus counted in the cbc, whereas marginating neutrophils are not. pseudoneutropenia refers to the situation in which there is an increased proportion of neutrophils in the marginating pool. this may occur because of decreased blood flow or in response to stimuli, such as endotoxemia, that increase expression of molecules promoting interaction between neutrophils and endothelial cells. this mechanism of neutropenia is rarely observed in clinical practice. neutropenia may also result from increased demand for neutrophils in the tissue. how rapidly such a situation develops depends not only on the magnitude of the inflammatory stimulus but also on the reserve of postmitotic neutrophils in the bone marrow. the size of this reserve, or storage pool, is species dependent. in dogs this pool contains the equivalent of days' normal production of neutrophils. cattle represent the other extreme in that they have a small storage pool and thus are predisposed to becoming neutropenic during times of acute inflammation. horses and cats are somewhere between the two extremes, closer to cattle and dogs, respectively. it stands to reason that the clinical significance of neutropenia because of a supply and demand imbalance is also species dependent. in dogs, neutropenia as a result of inflammation is an alarming finding because it is evidence of a massive tissue demand for neutrophils that has exhausted the patient's storage pool and is exceeding the rate of granulopoiesis in the bone marrow. however in cows, neutropenia is commonly noted in a wide range of conditions involving acute inflammation and does not necessarily indicate an overwhelming demand. eosinopenia/basopenia. eosinopenia and basopenia are decreased concentrations of blood eosinophils and basophils, respectively. in many laboratories, cbc reference values for eosinophils and basophils are as low as zero cells per microliter, precluding detection of eosinopenia or basopenia. when detectable, eosinopenia is often a result of stress (i.e., glucocorticoid mediated). monocytopenia. monocytopenia denotes a decreased concentration of monocytes in blood; it is of little to no pathologic significance by itself. thrombocytopenia. thrombocytopenia refers to a decrease in the concentration of circulating platelets. mechanisms of thrombocytopenia include decreased production, increased destruction, increased consumption, and altered distribution. decreased production may occur because of a condition affecting cells of multiple hematopoietic lineages, including megakaryocytes, is often used interchangeably with erythrocytosis, but technically and for the purposes of this chapter, polycythemia refers to a specific type of leukemia called primary erythrocytosis or polycythemia vera. causes of erythrocytosis are either relative or absolute. relative erythrocytosis results from a fluid deficit or an altered distribution of erythrocytes within the body (i.e., the body's total erythrocyte mass of generalized lymphadenopathy (e.g., lymphoma, blastomycosis). some hereditary immunodeficiencies, such as severe combined immunodeficiency or thymic aplasia, can cause lymphopenia due to lymphoid aplasia. erythrocytosis. an increase in the measured red cell mass above the normal range is known as erythrocytosis. the term polycythemia rarely, an epo-secreting tumor may cause inappropriately elevated levels of epo in the absence of hypoxia. absolute erythrocytosis, whether primary or secondary, causes increased viscosity of the blood, resulting in impaired blood flow and microvasculature distention. affected individuals are at increased risk for tissue hypoxia, thrombosis, and hemorrhage. clinical signs of hyperviscosity syndrome may include erythematous mucous membranes ( fig. - ) , prolonged capillary refill time, prominent scleral vessels, evidence of thrombosis or hemorrhage, and secondary signs related to specific organ systems affected (e.g., neurologic and cardiovascular signs). neutrophilia. neutrophilia, an increased blood concentration of neutrophils, occurs in response to a number of different stimuli, which are not mutually exclusive. major mechanisms of neutrophilia are shown in fig. - . understanding the cbc findings characteristic of these responses is an important part of clinical veterinary medicine. inflammation can result in neutropenia, as discussed earlier, or neutrophilia, as discussed next. however, before moving on to a discussion of inflammatory neutrophilia and the so-called left shift, it is important to mention two other common is not increased). it occurs most frequently with dehydration, when the decreased proportion of water in the blood results in hemoconcentration. it is observed less frequently with epinephrine-mediated splenic contraction, wherein erythrocytes move from the spleen into peripheral circulation. erythrocytosis from splenic contraction occurs to the most pronounced degree in horses and cats, especially in young, healthy animals. absolute erythrocytosis is a true increase in red blood cell mass due to erythroid neoplasia or hyperplasia and includes causes of primary and secondary erythrocytosis. primary erythrocytosis, or polycythemia vera, is a neoplastic proliferation of erythroid cells with a predominance of mature erythrocytes. diagnosis is based on a marked increase in red cell mass (hematocrit in normally hydrated dogs ranges from % to > %), an absence of hypoxemia, an absence of other tumors, and a normal or decreased plasma epo concentration. secondary erythrocytosis refers to epo-mediated erythroid hyperplasia causing an increased red blood cell mass. the erythroid hyperplasia may be an appropriate response to chronic hypoxia, such as occurs with right-to-left cardiac shunts or chronic pulmonary inflammatory mediators, including interleukin- (il- ), interleukin- (il- ), interferon (inf), and tumor necrosis factor (tnf), cause anemia of inflammatory disease due to oxidative hemolysis, iron sequestration within enterocytes and macrophages, and impaired erythroid responsiveness to erythropoietin (epo). during homeostasis the membrane transport molecule, ferroportin, transports iron from the cytosol to the extracellular space. the iron is then used for various physiologic processes, including hemoglobin production within bone marrow erythroid precursors. during times of inflammation the liver increases production of hepcidin, which binds ferroportin and causes its internalization and lysosomal degradation. with fewer membrane ferroportin molecules, less iron is absorbed from the diet and mobilized from macrophages. rbc, red blood cell. ( of course, neutrophilia may also indicate inflammation, and inflammatory stimuli of varying magnitude and duration produce different patterns of neutrophilia. a classic hematologic finding in patients with increased demand for neutrophils is the presence of immature forms in the blood, known as a left shift. not all inflammatory responses have a left shift, but the presence of a left shift almost always signifies active demand for neutrophils in the tissue. the magnitude of a left shift is assessed by the number of immature cells and their degree of immaturity. the mildest form is characterized by increased numbers of band neutrophils, the immediate predecessor to the segmented neutrophil normally found in circulation. progressively immature predecessors are seen with increasingly severe inflammation. a left shift is considered orderly if the number of immature neutrophils in circulation decreases as they become progressively immature. the term degenerative left shift is sometimes used to describe cases in which the number of immature forms exceeds the number of segmented neutrophils. as with glucocorticoidmediated neutrophilia, the typical magnitude of neutrophilia caused by inflammation varies by species, with dogs having the most pronounced response. it might be useful to think of neutrophil kinetics in terms of a producer-consumer model in which the bone marrow is the factory, and the tissues (where the neutrophils eventually go) are the customers. the bone marrow storage pool is the factory inventory, and the neutrophils in the bloodstream are in delivery to the customer. within the blood vessels, circulating neutrophils are on the highway, and marginating neutrophils are temporarily pulled off to the side of the road. during health, there is an even flow of neutrophils from the factory to the customer. thus the system is in steady state, and neutrophil numbers remain relatively constant and within the normal range. however, disease states may perturb this system at multiple levels. decreased granulopoiesis is analogous to a factory working below normal production level. ineffective granulopoiesis is analogous to goods that are produced at a normal to increased rate but are damaged during manufacturing and never leave the factory. a left shift is analogous to the factory meeting increased customer demand by shipping out unfinished goods. cases of persistent, established inflammation are characterized by bone marrow granulocytic hyperplasia and mature neutrophilia, analogous to a factory that has had time to adjust to increased demand and is meeting it more efficiently by increasing its output. eosinophilia/basophilia. eosinophilia and basophilia are increased concentrations of blood eosinophils and basophils, respectively. they may occur with parasitism, hypersensitivity reactions, paraneoplastic responses (e.g., lymphoma, mast cell neoplasia, or leukemia), and nonparasitic infectious disease. eosinophilia has also been documented with hypoadrenocorticism and rare idiopathic conditions (e.g., hypereosinophilic syndrome). most cases of eosinophilia and basophilia are due to eosinophilic and basophilic hyperplasia within the bone marrow in response to inflammatory growth factors. however, cortisol deficiency is thought to cause eosinophilia in dogs with hypoadrenocorticism. monocytosis. monocytosis is an increased concentration of monocytes in blood. it most commonly occurs with excessive glucocorticoids or inflammation and uncommonly to rarely with monocytic leukemia, immune-mediated neutropenia, and cyclic hematopoiesis. with excessive endogenous or exogenous glucocorticoids, monocytes shift from the marginating pool to the circulating pool. this stress monocytosis is most common in dogs, less frequent in cats, and rare in horses and cattle. inflammatory diseases cause monocytosis by cytokine-mediated monocytic hyperplasia in the bone marrow. causes of neutrophilia: glucocorticoid excess and epinephrine excess. less common causes of neutrophilia, such as leukocyte adhesion deficiency and neoplasia, are discussed later in the chapter. glucocorticoid excess, either because of endogenous production or exogenous administration, results in a cbc pattern known as the stress leukogram, characterized by mature neutrophilia (i.e., increased concentration of segmented neutrophils without immature neutrophils) and lymphopenia, with or without monocytosis and eosinopenia. mechanisms contributing to glucocorticoid-mediated neutrophilia include the following: • increased release of mature neutrophils from the bone marrow storage pool • decreased margination of neutrophils within the vasculature, with a resulting increase in the circulating pool • decreased migration of neutrophils from the bloodstream into tissues the magnitude of neutrophilia tends to be species dependent, with dogs having the most pronounced response (up to , cells/µl) and in decreasing order of responsiveness, cats ( , cells/µl), horses ( , cells/µl), and cattle ( , cells/µl) having less marked responses. with long-term glucocorticoid excess, neutrophil numbers tend to normalize, whereas lymphopenia persists. epinephrine release results in a different pattern, known as physiologic leukocytosis or excitement leukocytosis, characterized by mature neutrophilia (like the glucocorticoid response) and lymphocytosis (unlike the glucocorticoid response). this phenomenon is short lived (i.e., < hour). neutrophilia occurs primarily because of a shift of cells from the marginating to the circulating pool. physiologic leukocytosis is common in cats (especially when they are highly stressed during blood collection) and horses, less common in cattle, and uncommon in dogs. of a regenerative response in patients recovering from thrombocytopenia, as a result of redistribution after splenic contraction, or within the several weeks after splenectomy. in these cases, thrombocytosis is transient. in the case of splenectomy, thrombocytosis may be marked but normalizes after several weeks. because the body's total platelet mass regulates thrombopoiesis, and a significant portion of the platelet mass is normally in the spleen, it makes sense that splenectomized animals develop thrombocytosis. however, the reason that the number of circulating platelets normalizes in these individuals in the weeks after splenectomy is not thrombocytosis. thrombocytosis, or an increased concentration of platelets in the blood, is a relatively common, nonspecific finding in veterinary patients. in the vast majority of cases, thrombocytosis is reactive-a response to another, often apparently unrelated, disease process. examples of conditions having reactive thrombocytosis include inflammatory and infectious diseases, iron deficiency, hemorrhage, endocrinopathies, and neoplasia. factors that may contribute to reactive thrombocytosis include increased plasma concentration of thrombopoietin, inflammatory cytokines (e.g., il- ), or catecholamines. thrombocytosis may also occur as part , neutrophils and their precursors are distributed in five pools: a bone marrow precursor pool, which includes mitotically active and inactive immature cells; a bone marrow storage pool, consisting of mitotically inactive mature neutrophils; a peripheral blood marginating pool; a peripheral blood circulating pool; and a tissue pool. the relative size of each pool is represented by the size of its corresponding wedge. the peripheral blood neutrophil count measures only neutrophils within the circulating peripheral blood pool, which can be enlarged by ( ) increased demargination, ( ) diminished extravasation into tissue, ( ) increased release of cells from the marrow storage pool, and ( ) cells. the preceding section focused on abnormalities in the number of blood cells. there are also various acquired and congenital conditions involving abnormal structure or function of blood cells. this section briefly discusses abnormal blood cell structure or function occurring secondary to other underlying disease. primary disorders of blood cells are discussed later in the chapter in the section on specific diseases. morphologic abnormalities detected on routine microscopic examination of blood smears may provide important clues about underlying disease processes. poikilocytosis is a broad term referring to the presence of abnormally shaped erythrocytes in circulation. e- table - lists conditions with and mechanisms involved in the formation of a number of specific types of erythrocyte morphologic abnormalities, and fig. - shows some examples. the acquired neutrophil morphologic abnormality known as toxic change (fig. - ) reflects accelerated production of neutrophils as part of the inflammatory response. features of toxic change include increased cytoplasmic basophilia, the presence of small bluegray cytoplasmic inclusions known as döhle bodies (often noted incidentally in cats), and in more severe cases, cytoplasmic vacuolation. although not causing impaired neutrophil function, toxic change occurs during granulopoiesis and thus is technically a form of dysplasia (e.g., döhle bodies are foci of aggregated endoplasmic reticulum). toxic change may accompany any inflammatory response, but in general the more marked the toxic change, the higher the index of suspicion for infection or endotoxemia. other secondary changes to neutrophils may not be evident morphologically. for example, studies in human beings and dogs have shown that individuals with cancer have abnormal neutrophil function (including phagocytic activity, killing capacity, and oxidative burst activity) before initiation of therapy. the clinical significance of this finding is not clear. platelet function disorders, also known as thrombopathies or thrombopathias, may be primary or secondary. many conditions are known or suspected to cause secondary platelet dysfunction (hypofunction or hyperfunction) by altering platelet adhesion or aggregation or by mechanisms that are not fully understood. box - shows underlying conditions having secondary platelet dysfunction. clear. there is also a rare form of megakaryocytic leukemia known as essential thrombocythemia, which is characterized by marked thrombocytosis. lymphocytosis. lymphocytosis refers to an increase in the concentration of lymphocytes in blood circulation. there are several causes of lymphocytosis, including age, excessive epinephrine, chronic inflammation, hypoadrenocorticism, and lymphoid neoplasia; lymphoid neoplasms are presented later in the chapter. young animals normally have higher concentrations of lymphocytes than older animals, and normal healthy young animals may have counts that exceed adult reference values. because this is not pathologic lymphocytosis, but normal physiologic variation, it is often termed pseudolymphocytosis of young animals. as discussed earlier in the section on neutrophilia, lymphocytosis is also a feature of epinephrine-mediated physiologic leukocytosis, resulting from redistribution of lymphocytes from the blood marginating pool into the blood circulating pool. epinephrine-mediated lymphocytosis may be more marked than neutrophilia, particularly in cats (lymphocyte counts of > , /µl are not uncommon). antigenic stimulation may result in lymphocytosis, which may be marked in rare cases (up to approximately , /µl in dogs and , /µl in cats); however, this is not usually the case, even when there is clear evidence of increased immunologic activity in lymphoid tissues. in cases of antigenic stimulation, it is common for a minority of lymphocytes to have "reactive" morphologic features-larger lymphocytes with more abundant, deeply basophilic cytoplasm and more open chromatin ( fig. - ). just as glucocorticoid excess can cause lymphopenia, glucocorticoid deficiency (hypoadrenocorticism) can cause lymphocytosis, or lack of lymphopenia during conditions of stress that typically result in glucocorticoid-mediated lymphopenia. a condition known as persistent lymphocytosis (pl) occurs in approximately % of cattle infected with the bovine leukemia virus (blv). the condition is defined as an increase in the blood concentration of lymphocytes above the reference interval for at least months. this form of lymphocytosis is a nonneoplastic proliferation (i.e., hyperplasia) of b lymphocytes. in the absence of other disease, cattle with persistent lymphocytosis are asymptomatic. however, cattle infected with blv, especially those animals with persistent lymphocytosis, are at increased risk for developing b lymphocyte lymphoma. aplastic anemia (aplastic pancytopenia) aplastic anemia, or more accurately aplastic pancytopenia, is a rare condition characterized by aplasia or severe hypoplasia of all hematopoietic lineages in the bone marrow with resulting cytopenias. the term aplastic anemia is a misnomer because affected cells are not limited to the erythroid lineage. many of the conditions reported to cause aplastic anemia do so only rarely or idiosyncratically; more frequently, they cause other hematologic or nonhematologic abnormalities. a partial list of reported causes of aplastic anemia in domestic animals includes the following: most of these causes, especially the chemical agents, are directly cytotoxic to hscs or progenitor cells, resulting in their destruction. however, another proposed mechanism is disruption of normal stem cell function because of mutation or perturbation of hematopoietic cells and/or their microenvironment. this pathogenesis is mostly recognized in retroviral infections. aplastic anemia occurs in both acute and chronic forms. most of the chemical causes result in acute disease. grossly, affected animals may show signs of multisystemic infection and hemorrhage due to severe neutropenia and thrombocytopenia, respectively. severe neutropenia typically develops within week of an acute insult to the bone marrow, and severe thrombocytopenia occurs in the second week. this sequence is a result of the circulating life spans of each cell type; in health, neutrophils have a blood half-life of to hours, whereas platelets circulate for to days. the development of signs of anemia, such as pale mucous membranes, is more variable. the presence and severity of anemia depends on how rapidly the marrow recovers from the insult and the erythrocyte life span of the particular species. microscopically, bone marrow is hypocellular with markedly reduced hematopoietic cells. hematopoietic cells are replaced with adipose tissue, and there is a variable inflammatory infiltrate of lymphocytes, plasma cells, and macrophages. in addition, there may be necrosis, hematopoietic cell apoptosis, and an increase in phagocytic macrophages. fig. - shows bone marrow aspirates from a dog with pancytopenia from acute -fluorouracil toxicosis, before and during recovery. many inherited or presumably inherited disorders of blood cells have been recognized in domestic animals, including rare or sporadic cases and conditions that are of questionable clinical relevance. this section and the later sections covering species-specific disorders are invading cells or microorganism gain access to the bone marrow or blood circulation either hematogenously or by trauma. trauma may be as obvious as a gaping wound or as subtle as the bite of an insect. portals of entry for the bone marrow are summarized in box - . diseases that arise from the bone marrow, such as leukemia, typically spread to other tissues hematogenously. the bone marrow is encased by a protective shell of cortical bone, and blood supply to the marrow provides access to systemic humoral and cellular defenses. of course, leukocytes themselves function as an essential part of inflammation and immune function, as discussed briefly in the section on granulopoiesis and monocytopoiesis (myelopoiesis) and in greater detail in chapters and . biochemical steps in the glycolytic pathway or linked to it generate antioxidant molecules that enable erythrocytes to withstand oxidative insults throughout their many days in circulation. in addition to producing energy in the form of adenosine triphosphate (atp), glycolysis generates nadh, which helps convert the oxidized, nonfunctional form of hemoglobin, known as methemoglobin, back to its active, reduced state. another antioxidant erythrocyte metabolic pathway, the pentose shunt or hexose uremia antiplatelet antibodies (also cause immune-mediated thrombocytopenia) infection (bvdv, felv) hyperglobulinemia increased fibrinolytic products hypoammonemia snake envenomation platelet inhibitors nsaids-irreversible (aspirin) or reversible inhibition of cyclooxygenase colloidal plasma expanders (e.g., hydroxyethyl starch) other drugs and exogenous agents (many) hematogenously direct penetration (trauma) including hypercellular glomeruli, thickened glomerular and tubular basement membranes, and tubular epithelial lipidosis, degeneration, and necrosis. other porphyrias have been diagnosed in cattle, pigs, and cats but are not known to cause hemolytic anemia. pyruvate kinase deficiency. pyruvate kinase (pk) deficiency is an inherited autosomal recessive condition due to a defective r-type pk isoenzyme that is normally present in high concentrations in mature erythrocytes. to compensate for this deficiency, there is persistence of the m -type pk isoenzyme, which is less stable than the r-type isoenzyme. the disease is reported in many dog breeds and fewer cat breeds (e.g., abyssinian, somali, and domestic shorthair). erythrocyte pk deficiency results in decreased atp production and shortened erythrocyte life spans. in dogs the hemolytic anemia is typically chronic, moderate to severe, extravascular, and strongly regenerative. with chronicity, hemolytic anemia causes enhanced intestinal absorption of iron and subsequent hemosiderosis, especially of the liver and bone marrow. dogs typically die at to years of age of hemochromatosis-induced liver and bone marrow failure. however, cats with pk deficiency typically show no clinical signs, have milder anemia, and do not develop organ failure. grossly, affected animals have lesions attributed to hemolytic anemia, including splenomegaly, pale mucous membranes, and rarely icterus. dogs with end-stage disease have cirrhosis, myelofibrosis, and osteosclerosis. dogs with pk deficiency do not necessarily have the same genetic defect, so mutation-specific dna-based assays are required. in contrast, a single dna-based test is available to detect the common mutation affecting abyssinian, somali, and domestic shorthair cats. cytochrome-b reductase deficiency. deficiency of cytochrome-b reductase (cb r, also known as methemoglobin reductase), the enzyme that catalyzes the reduction of methemoglobin (fe + ) to hemoglobin (fe + ), has been recognized in many dog breeds and in domestic shorthair cats. it is probably an autosomal recessive trait. affected animals may have cyanotic mucous membranes or exercise intolerance but usually lack anemia and clinical signs of disease. life expectancies are normal. glucose- -phosphate dehydrogenase deficiency. deficiency of glucose- -phosphate dehydrogenase (g pd), the ratecontrolling enzyme of the pentose phosphate pathway (ppp), has been reported in an american saddlebred colt, its dam, and one male dog. the ppp is an antioxidative pathway that generates nadph, which maintains glutathione in its reduced form (gsh). therefore in animals with g pd deficiency, oxidants are not scavenged, and erythrocyte oxidative injury occurs. the colt with g pd deficiency had severe oxidative hemolytic anemia with eccentrocytes on blood smear evaluation. however, the colt's dam only had eccentrocytes, and showed no hematologic signs of disease. leukocyte adhesion deficiency. leukocyte adhesion deficiency (lad) is a fatal autosomal recessive defect of leukocyte integrins, in particular the β chain (also known as cluster of differentiation [cd] [cd ]). disease has been recognized in holstein cattle (known as bovine leukocyte adhesion deficiency [blad] ) and irish setter dogs (known as canine leukocyte adhesion deficiency [clad]) (see chapter ). without normal expression of this adhesion molecule, leukocytes have severely impaired abilities to migrate from the blood into tissues. as a result, animals with leukocyte adhesion deficiency have marked neutrophilia with nonsuppurative multisystemic infections. blood neutrophils often have nuclei with greater than five nuclear segments, termed not comprehensive but instead focus on the more common, wellcharacterized, or recently reported conditions. erythropoietic porphyrias. porphyrias are a group of hereditary disorders in which porphyrins accumulate in the body because of defective heme synthesis. inherited enzyme defects in hemoglobin synthesis have been identified in holstein cattle, siamese cats, and other cattle and cat breeds, resulting in bovine congenital erythropoietic porphyria and feline erythropoietic porphyria, respectively. accumulation of toxic porphyrins in erythrocytes causes hemolytic anemia, whereas accumulation of porphyrins in tissues and fluids produces discoloration, including red-brown teeth, bones, and urine (see fig. - ). because of the circulation of the photodynamic porphyrins in blood, these animals have lesions of photosensitization of the nonpigmented skin. all affected tissues, including erythrocytes, exhibit fluorescence with ultraviolet light. histologically, animals may exhibit perivascular dermatitis, as well as multisystemic porphyrin deposition, hemosiderosis, emh, and marrow erythroid hyperplasia. cats may show evidence of renal disease, a b syndrome exhibit oculocutaneous albinism (due to altered distribution of melanin granules) and are prone to infection and bleeding. blood smear evaluation reveals granulocytes with large cytoplasmic granules. glanzmann thrombasthenia. glanzmann thrombasthenia (gt) is an inherited platelet function defect caused by a mutated α iib subunit of the integrin α iib β (also known as glycoprotein iib-iiia [gpiib-iiia]). the disorder has been recognized in great pyrenees and otterhound dogs and several horse breeds, including a quarter horse, a standardbred, a thoroughbred-cross, a peruvian paso mare, and an oldenburg filly. the α iib β molecule has multiple functions but is best known as a fibrinogen receptor that is essential for normal platelet aggregation. bleeding tendencies vary widely between affected individuals but mainly occur on mucosal surfaces. the condition is characterized by an in vitro lack of response to all platelet agonists and severely impaired clot retraction (i.e., whole blood samples without anticoagulant often fail to clot). molecular testing is available to detect diseased or carrier states in dogs and horses. thrombopathia. calcium diacylglycerol guanine nucleotide exchange factor i (caldag-gefi) is a molecule within the signaling pathway that results in platelet activation in response to platelet agonists. mutated caldag-gefi has been documented in basset hound, eskimo spitz, and landseer dogs, and simmental cattle. all reported mutations have a bleeding tendency. in vitro platelet aggregation responses to platelet agonists, such as adp, collagen, and thrombin, are absent or impaired. information on this topic is available at www.expertconsult.com. information on this topic is available at www.expertconsult.com. information on this topic is available at www.expertconsult.com. oxidative agents. a variety of oxidative toxins cause hemolytic anemia and/or methemoglobinemia in domestic species. more common or well-characterized oxidants are listed here: • horses-acer rubrum (red maple) • ruminants-brassica spp. (cabbage, kale, and rape), copper hypersegmented neutrophils, due to neutrophil aging within blood vessels ( fig. - ). these animals are highly susceptible to infections and usually die at a young age. pelger-huët anomaly. pelger-huët anomaly (pha) is a condition of hyposegmented granulocytes due to a lamin b receptor mutation. it has been described in dogs, cats, horses, and rabbits, especially in certain breeds. in australian shepherd dogs the mode of inheritance is autosomal dominant with incomplete penetrance. most cases of pelger-huët anomaly are the heterozygous form and of no clinical significance. however, skeletal abnormalities, stillbirths, and/or early mortality may accompany pelger-huët anomaly in rabbits and cats, especially homozygotes. in pelger-huët anomaly the nuclei of neutrophils, eosinophils, and basophils fail to segment, resulting in band-shaped, bean-shaped, or round nuclei. although the nuclear shape is similar to that of an inflammatory left shift, healthy animals with pelger-huët anomaly do not have clinical signs or other laboratory findings indicating inflammation. for example, neutrophils in healthy animals with pelger-huët anomaly have mature (clumped) chromatin and do not show signs of toxicity ( fig. - ). an acquired, reversible condition mimicking pelger-huët anomaly, known as pseudo-pelger-huët anomaly, is occasionally noted in animals with infectious disease, neoplasia, or drug administration. chédiak-higashi syndrome. chédiak-higashi syndrome (chs) is a rare autosomal recessive defect in the lysosomal trafficking regulator (lyst) protein. the syndrome has been identified in hereford, brangus, and japanese black cattle, persian cats, and several nondomestic species. the defective lyst protein results in granule fusion in multiple cell types, including granulocytes, platelets, and melanocytes, as well as abnormal cell function. individuals with chédiak-higashi syndrome have severely impaired cellular innate immunity because of neutropenia, impaired leukocyte chemotaxis, and impaired killing by granulocytes and cytotoxic lymphocytes. platelets lack the dense granules that normally contain key bioactive molecules involved in hemostasis, including platelet agonists, such as adp and serotonin. in vitro platelet aggregation is severely impaired. as a result, animals with chédiak-higashi .e chapter bone marrow, blood cells, and the lymphoid/lymphatic system von willebrand disease (vwd) is the most common canine hereditary bleeding disorder and has also been described in many other domestic species. the disease actually refers to a group of inherited conditions characterized by a quantitative or qualitative deficiency of vwf. this factor is a multimeric glycoprotein that is stored in platelet α-granules and endothelial cells and circulates as a complex with coagulation factor viii. its primary functions are to stabilize factor viii and mediate platelet binding to other platelets and subendothelial collagen. although not technically a platelet disorder, von willebrand disease is often classified as such because it results in a loss of normal platelet function. different types of von willebrand disease vary in terms of mode of inheritance and severity of clinical disease. type i von willebrand disease is characterized by low plasma vwf concentration but normal multimeric proportions and a mild to moderate clinical bleeding tendency; it has been reported in many dog breeds. type ii von willebrand disease is characterized by low vwf concentration, absence of large multimers, and a moderate to severe bleeding tendency; it has been reported in german short-haired pointer and german wirehaired pointer dogs. type iii von willebrand disease is characterized by absence of vwf and a severe bleeding tendency; familial and sporadic cases have been reported in numerous dog breeds. the buccal mucosal bleeding time is prolonged with von willebrand disease, often with adequate concentrations of platelets and normal prothrombin time and partial thromboplastin time (ptt). however, ptt may be mildly prolonged because vwf stabilizes factor viii, and deficiency of vwf results in enhanced factor viii degradation. grossly, affected animals exhibit bleeding tendencies, especially in the form of inherited coagulation factor deficiencies have been documented in most domestic species, including deficiencies of prekallikrein and factors i, ii, vii, viii, ix, x, xi, and xii. of these disorders, hereditary coagulation factor viii (hemophilia a) and factor ix (hemophilia b) deficiencies are most common. hemophilia a has been recognized in horses, cattle, dogs, and cats, and hemophilia b occurs in dogs and cats. both disorders have an x-linked recessive mode of inheritance, meaning that clinical disease is more common in males. affected males have variable tendencies to bleed, depending on the severity of the deficiency, exposure to trauma, and size and activity level of the affected individual. carrier females are usually asymptomatic. laboratory tests often reveal adequate platelets, normal prothrombin times, and prolonged partial thromboplastin times. hereditary defects in γ-glutamyl carboxylase, the enzyme required for normal carboxylation of vitamin k-dependent coagulation factors, have been recognized in a flock of rambouillet sheep and two devon rex cats from the same litter. the genetic defect is not known in cats, but in sheep it is an autosomal recessive trait that results in a premature stop codon and truncated γ-glutamyl carboxylase. in sheep there is increased lamb mortality with excessive bleeding during parturition, especially through the umbilicus or into subcutaneous tissues. gingival bleeding, epistaxis, and hematuria or at sites of injections, venipuncture, or surgery. of loss is through the gastrointestinal tract (e.g., neoplasia in older animals or hookworm infection in puppies). chronic blood loss may also be caused by marked ectoparasitism (e.g., pediculosis in cattle or massive flea burden in kittens and puppies), neoplasia in locations other than the gastrointestinal tract (e.g., cutaneous hemangiosarcoma), coagulation disorders, and repeated phlebotomy of blood donor animals. rapidly growing nursing animals may be iron deficient when compared with adults because milk is an iron-poor diet. in most cases this has little clinical significance (and in fact is normal). an important exception is piglets with no access to iron, which may cause anemia, failure to thrive, and increased mortality. neonatal piglets are routinely given parenteral iron for this reason. copper deficiency can cause iron deficiency in ruminants and may occur because of copper-deficient forage or impaired usage of copper by high dietary molybdenum or sulfate. it is believed that copper deficiency impairs production of ceruloplasmin, a copper-containing enzyme involved in gastrointestinal iron absorption. iron deficiency causes anemia by impaired hemoglobin synthesis. iron is an essential component of hemoglobin, and when it is absent, hemoglobin synthesis is depressed. because erythrocyte maturation is dependent upon obtaining a critical hemoglobin concentration, maturing erythroid precursors undergo additional cell divisions during iron-deficient states. these additional cell divisions result in small erythrocytes, termed microcytes (see fig. - , g). however, erythrocytes with low hemoglobin concentrations are produced when microcyte formation can no longer compensate for iron deficiency. the classic hematologic picture with iron deficiency anemia is microcytic (i.e., decreased mcv), hypochromic (i.e., decreased mchc) anemia. microcytes and hypochromasia (see fig. - , g) may also be discernible on blood smear examination as erythrocytes that are abnormally small and paler-staining, respectively. early iron deficiency anemia is poorly regenerative, whereas continued hemorrhage and iron loss cause nonregenerative anemia. additional hematologic changes may include evidence of erythrocyte mechanical fragmentation (e.g., schistocytes) and reactive thrombocytosis. hypophosphatemic hemolytic anemia. marked hypophosphatemia is recognized as a cause of intravascular hemolytic anemia in postparturient dairy cows and diabetic animals receiving insulin therapy. in postparturient cows, hypophosphatemia results from increased loss of phosphorus in their milk. insulin therapy may cause hypophosphatemia by shifting phosphorus from the extracellular space to the intracellular space. in either case, marked hypophosphatemia (e.g., mg/dl in cows, or ≤ . mg/dl in cats) is thought to decrease erythrocyte production of atp, leading to inadequate energy required for maintenance of membrane and cytoskeletal integrity. an accompanying decrease in reducing capacity and increase in methemoglobin concentration have also been noted in experimental studies of hypophosphatemic hemolytic anemia in dairy cattle, suggesting that oxidative mechanisms may also contribute to anemia. affected animals are anemic and hemoglobinuric. gross postmortem findings include pallor, decreased viscosity of the blood, and lesions arising from the underlying metabolic derangement (e.g., discolored pale yellow and swollen liver due to hepatic lipidosis). renal tubular necrosis and hemoglobin pigment within the tubules is evident microscopically. this section covers infectious agents within the same genus that are recognized to cause disease in multiple species. other infectious agents with more limited host specificity (e.g., cytauxzoonosis in cats, feline and equine retroviruses) are covered in later sections on species-specific diseases. throughout both sections, diseases are • dogs-acetaminophen, propofol, zinc • cats-acetaminophen, propofol, propylene glycol • all species-allium spp. (chives, garlic, and onions) in horses, red maple leaves and bark are toxic, especially wilted or dried leaves. the toxic principle is believed to be gallic acid. plants that contain high concentrations of nitrates, such as cabbage, kale, and rape, may cause oxidative injury to erythrocytes; cattle are more susceptible than sheep and goats. however, sheep are more prone to copper toxicosis relative to other ruminants. the condition occurs in animals that have chronically accumulated large amounts of copper in the liver through the diet. the copper is then acutely released during conditions of stress, such as shipping or starvation. continuous rate infusions of the anesthetic propofol may cause oxidative hemolytic anemia in dogs and cats, but single or multiple single doses are not expected to cause clinical hemolysis. zinc toxicosis has been identified in a wide range of animals; however, it is most common in dogs due to their indiscriminate eating habits. common sources include pennies, batteries, paints, creams, automotive parts, screws, nuts, and coating on galvanized metals. propylene glycol is an odorless, slightly sweet solvent and moistening agent in many foods, drugs, and tobacco products. although it is "generally recognized as safe" for animal foods other than for cats by the food and drug administration, it has been banned from cat food since . grossly and microscopically, animals show varying signs of oxidative hemolysis and/or methemoglobinemia, as previously presented in the section discussing anemias (see bone marrow and blood cells, dysfunction/responses to injury, blood cells, abnormal concentrations of blood cells, anemia). in sheep with copper toxicosis, hemoglobinuric nephrosis, frequently described as gunmetal-colored kidneys with port wine-colored urine, is a classic postmortem lesion. snake envenomation. hemolytic anemia from snake envenomation has been reported in horses, dogs, and cats. it is most commonly reported with viper and pit viper envenomations, including those from rattlesnakes. hemolysins within viper venom directly injure erythrocytes, causing intravascular hemolysis. other mechanisms of hemolysis include the action of phospholipase a on erythrocyte membranes and erythrocyte mechanical fragmentation due to intravascular coagulation and vasculitis. nonhemolytic lesions depend on the venom's additional components and may include hemorrhage, paralysis, and/or tissue edema, inflammation, and necrosis. on blood smear evaluation, animals with snake envenomation may have ghost cells, spherocytes, and/or echinocytes (see figs. - and - ). information on this topic is available at www.expertconsult.com. severe malnutrition is probably a cause of nonregenerative anemia in all species attributable to combined deficiencies of molecular building blocks, energy, and essential cofactors. by far the most commonly recognized specific deficiency that results in anemia is iron deficiency. other specific nutritional deficiencies causing anemia in animals are uncommon or rare. acquired cobalamin (vitamin b ) and folate deficiencies are recognized as causes of anemia in human beings but are rare in animals. iron deficiency anemia. iron deficiency is usually not a primary nutritional deficiency but rather occurs secondary to depletion of iron stores via chronic blood loss. the most common route .e chapter bone marrow, blood cells, and the lymphoid/lymphatic system antagonism of vitamin k leads to production of a nonfunctional form of some coagulation factors and resulting coagulopathy; a similar condition results from vitamin k deficiency. conditions with avitaminosis k include poisoning with coumarin-related molecules, fat malabsorption (vitamin k is a fat-soluble vitamin) caused by primary intestinal disease or impaired biliary outflow (uncommon), dietary deficiency (rare), and antibiotics that interfere with vitamin k absorption or usage. a number of coagulation factors-factors ii, vii, ix, and x (collectively known as the vitamin k-dependent factors), as well as the regulatory molecules protein c and protein s-must undergo carboxylation to be functional. this posttranslational modification is catalyzed by the enzyme γ-glutamyl carboxylase, and requires vitamin k as a cofactor. vitamin k is oxidized during the carboxylation reaction and is converted back into its active reduced form by the enzyme vitamin k epoxide reductase. coumarin-related rodenticides, such as warfarin, act by inhibiting vitamin k epoxide reductase, resulting in an absence of vitamin k in its active reduced form (e- fig. - ). this inhibition lasts until the rodenticide is metabolized and cleared. how long this takes depends on the half-life of the rodenticide and dose, but it may take many weeks. secondgeneration rodenticides, such as bromadoline and brodifacoum, are more potent than warfarin, with longer half-lives. spoiled sweet clover contains dicumarol, which causes coagulopathy by the same mechanism. e- figure - mechanism of anticoagulant rodenticide toxicity. anticoagulant rodenticides inhibit the enzyme that converts vitamin k back to its active reduced form. active factors (carboxylated) inactive factors (ii, vii, ix, x) = inactivation of vitamin k epoxide reductase, the enzyme that maintains vitamin k in the active form vitamin k epoxide (inactive) x x laboratory findings include prolonged coagulation times (prothrombin time [pt] , ptt, and activated clotting time [act] ). early in the course of rodenticide and related toxicoses, pt may be the only one of these tests that is prolonged because factor vii has the shortest half-life of the vitamin k-dependent factors. however, the other tests become prolonged as nonfunctional forms of the other factors accumulate. in uncomplicated cases, patients are not thrombocytopenic. a wide range of hemorrhagic lesions may occur in affected individuals, including ecchymoses, epistaxis, gingival bleeding, hematomas, hemoptysis, melena or hematochezia, hematuria, and other forms of hemorrhage. there are also lesions with regenerative anemia, such as pale mucous membranes and splenomegaly. histologically, there is hemorrhage, emh, and marrow erythroid hyperplasia. the treatment of cases of rodenticide and related toxicoses is regular administration of exogenous vitamin k until the toxin is cleared (determined by repeat coagulation testing after withholding treatment). babesiosis may cause intravascular and extravascular hemolytic anemia via direct red blood cell injury, the innocent bystander effect, and secondary immune-mediated hemolytic anemia. infection with highly virulent strains may cause severe multisystemic disease. in these cases, massive immunostimulation and cytokine release cause circulatory disturbances, which may result in shock, induction of the systemic inflammatory response, and multiple organ dysfunction syndromes. babesia organisms can usually be detected on a routine blood smear in animals with acute disease. infected erythrocytes may be more prevalent in capillary blood, so blood smears made from samples taken from the pinna of the ear or the nail bed may increase the likelihood of detecting organisms microscopically. buffy coat smears also have an enriched population of infected erythrocytes. pcr-based tests are the most sensitive assay for detecting infection in animals with very low levels of parasitemia. at necropsy, gross lesions are mainly related to hemolysis and include pale mucous membranes, icterus, splenomegaly, dark red or black kidneys, and reddish-brown urine. the cut surface of the congested spleen oozes blood. the gallbladder is usually distended with thick bile. less common lesions include pulmonary edema, ascites, and congestion, petechiae, and ecchymoses of organs, including the heart and brain. parasitized erythrocytes are best visualized on impression smears of the kidney, brain, and skeletal muscle. microscopic findings in the liver and kidney are typical of a hemolytic crisis and include anemia-induced degeneration, necrosis of periacinar hepatocytes and cholestasis, and hemoglobinuric nephrosis with degeneration of tubular epithelium. erythroid hyperplasia is present in the bone marrow. in animals that survive the acute disease, there is hemosiderin accumulation in the liver, kidney, spleen, and bone marrow. in chronic cases there is hyperplasia of macrophages in the red pulp of the spleen. theileriosis (piroplasmosis). theileria spp. are tick-borne protozoal organisms that infect many domestic and wild animals worldwide. numerous theileria spp. have been documented, but only the more economically or regionally important species are mentioned here. diseases with the greatest economic impact in ruminants are east coast fever (theileria parva infection) and tropical theileriosis (theileria annulata infection). • horses-theileria equi (formerly babesia equi) • cattle-theileria annulata, theileria buffeli, t. parva • sheep and goats-theileria lestoquardi (formerly theileria hirci) like babesiosis, theileriosis is generally restricted to tropical and subtropical regions, including parts of africa, asia, the middle east, and europe. except for t. buffeli, all previously listed species are exotic to the united states. infection is characterized by schizonts within lymphocytes or monocytes, and pleomorphic intraerythrocytic piroplasms (merozoites and trophozoites). within host leukocytes the parasite induces leukocyte cellular division, which expands the parasitized cell population. infected cells disseminate throughout the lymphoid system via the lymphatic and blood vessels. the infected leukocyte may block capillaries, causing tissue ischemia. later in infection some schizonts cause leukocyte lysis and release of merozoites. merozoites then invade and parasitize erythrocytes, causing hemolytic anemia. possible mechanisms of anemia in theileriosis include invasion of erythroid precursors by merozoite stages and associated erythroid hypoplasia (as occurs with t. parva infection), immune-mediated hemolysis, mechanical fragmentation because of vasculitis or microthrombi, enzymatic destruction by proteases, and oxidative damage. gross and microscopic lesions are similar to those of babesiosis, except that cattle with east coast fever tend not to develop organized by taxonomy (protozoal, bacterial and rickettsial, and viral). babesiosis (piroplasmosis). babesia spp. and theileria spp., presented in the next section, are members of the order piroplasmida, and are generally referenced as piroplasms. these organisms are morphologically similar but have different life cycles; babesia spp. are primarily erythrocytic parasites, whereas theileria spp. sequentially parasitize leukocytes and then erythrocytes. both are protozoan parasites spread by ticks, but other modes of transmission are possible (e.g., biting flies, transplacental, and blood transfusions). evidence is accumulating that dog fighting also transmits babesia gibsoni infection. babesia organisms are typically classified as large ( to µm) or small (< µm) with routine light microscopy ( fig. - ). over babesia species have been identified, some of which are listed here, along with their relative microscopic size in parentheses: geographic distributions vary with the species, but most have higher prevalences in tropical and subtropical regions. for example, equine and bovine babesiosis are endemic in parts of africa, the middle east, asia, central and south america, the caribbean, and europe. both were eradicated from the united states and are now considered exotic diseases in that country. of the previously mentioned species, only agents of canine babesiosis are thought to be endemic in the united states. fever. anaplasmosis, ehrlichiosis, heartwater, and tick-borne fever are tick-borne diseases caused by small, pleomorphic, gramnegative, obligate intracellular bacteria within the order rickettsiaceae, also colloquially known as rickettsias. as a group, rickettsias primarily infect hematopoietic cells and endothelial cells. rickettsias that predominantly infect endothelial cells (e.g., rickettsia rickettsii [rocky mountain spotted fever]), or cause gastrointestinal disease (e.g., neorickettsia helminthoeca [salmon poisoning disease] and neorickettsia risticii [potomac horse fever]) are discussed elsewhere (see chapters and ). less commonly, transmission may occur via blood transfusions or blood-contaminated medical supplies. rickettsias that infect erythrocytes include the following species (the disease name follows in parentheses): • cattle-anaplasma marginale, anaplasma centrale (bovine anaplasmosis) • sheep and goats-anaplasma ovis (ovine and caprine anaplasmosis, respectively) anaplasma marginale and a. ovis have worldwide distributions, but a. centrale is mostly restricted to south america, africa, and the middle east. hemolytic anemia. in acute east coast fever, lymph nodes are enlarged, edematous, and hemorrhagic. but with chronic cases they may be shrunken. there is often splenomegaly, hepatomegaly, and hemorrhagic enteritis with white foci of lymphoid infiltrates (pseudoinfarcts) in the liver and kidney. microscopically, infected leukocytes may block capillaries. african trypanosomiasis. trypanosomes are flagellated protozoa that can infect all domesticated animals. the most important species that cause disease are trypanosoma congolense, trypanosoma vivax, and trypanosoma brucei ssp. brucei. disease is most common in parts of africa where the biologic vector, the tsetse fly, exists. however, t. vivax has spread to central and south america and the caribbean, where other biting flies transmit the parasite mechanically. in africa, cattle are mainly affected due to the feeding preferences of the tsetse fly. african trypanosomiasis must be distinguished from nonpathogenic trypanosomiasis, such as trypanosoma theileri infection in cattle. animals become infected when feeding tsetse flies inoculate metacyclic trypanosomes into the skin of animals. the trypanosomes grow for a few days, causing a localized chancre sore, and then sequentially enter the lymph nodes and bloodstream. trypanosomal organisms do not infect erythrocytes but rather exist as free trypomastigotes (i.e., flagellated protozoa with a characteristic undulating membrane) in the blood ( fig. - , a) or as amastigotes in tissue. the mechanism of anemia is believed to be immune mediated. cattle with acute trypanosomiasis have significant anemia, which initially is regenerative, but less so with time. the extent of parasitemia is readily apparent with t. vivax and t. theileri infections because the organisms are present in large numbers in the blood. this is in contrast to t. congolense, which localizes within the vasculature of the brain and skeletal muscle. chronically infected animals often die secondary to poor body condition, immunosuppression, and concurrent infections. gross examination of animals with acute disease often reveals generalized lymphadenomegaly, splenomegaly, and petechiae on serosal membranes. an acute hemorrhagic syndrome may occur in cattle, resulting in lesions of severe anemia (e.g., pale mucous membranes) and widespread mucosal and visceral hemorrhages. main lesions of chronic infections include signs of anemia, lymphadenopathy (e.g., enlarged or atrophied lymph nodes), emaciation, subcutaneous edema, pulmonary edema, increased fluid in body cavities, and serous atrophy of fat. trypanosoma cruzi is the flagellated protozoal agent of american trypanosomiasis. infections have been reported in more than mammal species in south america, central america, and the southern united states, but dogs and cats are among the more common domestic hosts. infected triatomine insects, or "kissing bugs," defecate as they feed on their mammalian host, releasing infective t. cruzi organisms. the parasite then enters the body through mucous membranes or breaks in the skin. like the other trypanosomes described previously, t. cruzi lives in the blood as extracellular trypomastigotes (see fig. - , b) and in the tissues as intracellular amastigotes. trypanosoma cruzi primarily causes heart disease. lesions of acute disease include a pale myocardium, subendocardial and subepicardial hemorrhages, and yellowish-white spots and streaks. there may also be secondary lesions, such as pulmonary edema, ascites, and congestion of the liver, spleen, and kidneys. in chronic disease the heart may be enlarged and flaccid with thin walls. microscopically, there is often myocarditis and amastigotes within cardiomyocytes. most of these rickettsias have worldwide distributions. however, e. ewingii has been reported only in the united states, and e. ruminantium is endemic only in parts of africa and the caribbean. although a. phagocytophilum has a wide geographic distribution, strain variants are regionally restricted. for example, a. phagocytophilum causes disease in ruminants in europe, but it has not been documented in ruminants in the united states. reservoirs of disease vary, depending upon the rickettsial species. cattle are the reservoir host for e. ruminantium, canids are the reservoir host for a. platys and e. canis, and the other rickettsias have wildlife reservoirs. pathogenesis of disease involves endothelial cell, platelet, and leukocyte dysfunction. those agents that infect endothelial cells cause vasculitis and increased vascular permeability of small blood vessels. if only plasma is lost, then there is hypotension and tissue edema. however, more severe vasculitis causes microvascular hemorrhage with the potential for platelet consumption thrombocytopenia, disseminated intravascular coagulation, and hypotension. infection of platelets may cause thrombocytopenia by direct platelet lysis, immune-mediated mechanisms, or platelet sequestration within the spleen. pathogenesis of leukocyte dysfunction is unclear, but may involve sepsis, inhibited leukocyte function, endothelial cell activation, and platelet consumption. chronic e. canis infection may cause aplastic anemia with pancytopenia by an unknown mechanism. some studies indicate that german shepherd dogs with ehrlichiosis are predisposed to have particularly severe clinical disease. some breeds of cattle (bos taurus), sheep (merino), and goats (angora and saanen) are more susceptible to heartwater. upon blood smear evaluation, thrombocytopenia is the most common hematologic abnormality; anemia and neutropenia occur less frequently. in early stages of infection, blood cells may contain morulae, which are clusters of rickettsial organisms within cytoplasmic, membrane-bound vacuoles ( fig. - ). examination of buffy coat smears increases the probability of detecting the organism. chronic infection may cause lymphocytosis, particularly of granular lymphocytes. anaplasma platys causes recurrent marked thrombocytopenia. in general, more common gross lesions are splenomegaly, lymphadenomegaly, and pulmonary edema and hemorrhage. more severe cases may also exhibit multisystemic petechiae, ecchymoses, and bovine anaplasmosis causes anemia mainly by immune-mediated extravascular hemolysis. the severity of disease in infected animals varies with age. infected calves under year of age rarely develop clinical disease, whereas cattle years of age or older are more likely to develop severe, potentially fatal, illness. the reason for this discrepancy is not clear. indian cattle (bos indicus) are more resistant to disease than european cattle (bos taurus). surviving cattle become chronic carriers (and thus reservoirs for infection of other animals) and develop cyclic bacteremia, which is typically not detectable on blood smears. splenectomy of carrier animals results in marked bacteremia and acute hemolysis. pcr testing is the most sensitive means of identifying animals with low levels of bacteremia. grossly, acute disease causes lesions of acute hemolytic anemia, including pale mucous membranes, low blood viscosity, icterus, splenomegaly, hepatomegaly, and a distended gallbladder. in animals with acute disease it is usually easy to detect a. marginale organisms on routine blood smear evaluation ( fig. - ) or impression smears from cut sections of the spleen. however, in recovering animals, the organisms may be difficult to find. rickettsias that infect leukocytes are broadly divided into those that preferentially infect granulocytes ( in addition to anemia, common findings in animals with leptospirosis-induced hemolysis include hemoglobinuria and icterus. on necropsy, renal tubular necrosis, which occurs in part because of hemoglobinuria (hemoglobinuric nephrosis), may also be present. hemotropic mycoplasmosis (hemoplasmosis). the term hemotropic mycoplasmas, or hemoplasmas, encompasses a group of bacteria, formerly known as haemobartonella or eperythrozoon spp., that infect erythrocytes of many domestic, laboratory, and wild animals. hemotropic mycoplasmas affecting common domestic species are as follows: • cattle-mycoplasma wenyonii • camelids-"candidatus mycoplasma haemolamae" • sheep and goats-mycoplasma ovis • pigs-mycoplasma suis (e- fig. - ) • dogs-mycoplasma haemocanis, "candidatus mycoplasma haematoparvum" • cats-mycoplasma haemofelis, "candidatus mycoplasma haemominutum," "candidatus mycoplasma turicensus" like other mycoplasmas, hemoplasmas are small ( . to µm in diameter) and lack a cell wall. they are epicellular parasites, residing in indentations and invaginations of red blood cell surfaces. the mode of transmission is poorly understood, but blood-sucking arthropods are believed to play a role; transmission in utero, through biting or fighting, and transfusion of infected blood products are also suspected. effects of infection vary from subclinical to fatal anemia, depending on the specific organism, dose, and host susceptibility. most hemoplasmas are more likely to cause acute illness in individuals that are immunocompromised or have concurrent disease. however, m. haemofelis is an exception and tends to cause acute hemolytic anemia in immunocompetent cats. anemia occurs mainly because of extravascular hemolysis, but intravascular hemolysis also occurs. although the pathogenic mechanisms are not completely understood, an immune-mediated component is highly probable, as well as direct red blood cell injury by the bacteria and the innocent bystander effect. hemotropic mycoplasmas induce cold agglutinins in infected individuals, although it is not clear whether these particular antibodies are important in the development of hemolytic anemia. when detected on routine blood smear evaluation, the organisms are variably shaped (cocci, small rods, or ring forms) and sometimes arranged in short, branching chains ( fig. - ) . the organisms may also be noted extracellularly, in the background of the blood smear, especially if the smear is made after prolonged storage of the blood in an anticoagulant tube. in animals dying of acute hemoplasma infection, the gross findings are typical of extravascular hemolysis, with pallor, icterus, splenomegaly, and distended gallbladder ( fig. - ). additional lesions documented in cattle include scrotal and hind limb edema and swelling of the teats. microscopic lesions in the red pulp of the spleen include congestion, erythrophagocytosis, macrophage hyperplasia, emh, and increased numbers of plasma cells. bone marrow has varying degrees of erythroid hyperplasia, depending on the duration of hemolysis. immune-mediated hemolytic anemia. immune-mediated hemolytic anemia is a condition characterized by increased destruction of erythrocytes because of binding of immunoglobulin to red blood cell surface antigens. it is a common, life-threatening condition in dogs but also has been described in horses, cattle, and cats. immune-mediated hemolytic anemia may be idiopathic (also called edema, cavitary effusions, and effusive polyarthropathy. hydropericardium gives heartwater its name but is more consistently found in small ruminants than in cattle. chronically infected dogs are emaciated. the bone marrow is hyperplastic and red in the acute disease but becomes hypoplastic and pale in dogs with chronic e. canis infection. equine anaplasmosis is often mild but may cause edema and hemorrhages. disease in cats is rare and poorly documented. histologic findings include generalized perivascular plasma cell infiltration, which is most pronounced in animals with chronic disease. multifocal, nonsuppurative meningoencephalitis, interstitial pneumonia, and glomerulonephritis are present in most dogs with the disease. rickettsial organisms are difficult to detect histologically; examination of wright-giemsa-stained impression smears of lung, liver, lymph nodes, and spleen is a more effective method for detecting the morulae within leukocytes. heartwater is often diagnosed by observing morulae in endothelial cells of giemsastained squash preparations of brain. rickettsial diseases are often diagnosed on the basis of serologic testing, but pcr testing is more sensitive. clostridial diseases. certain clostridium spp. may cause potentially fatal hemolytic anemias in animals; nonhemolytic lesions are presented elsewhere (see chapters , , , and ) . clostridium haemolyticum and clostridium novyi type d cause the disease in cattle known as bacillary hemoglobinuria. (the phrase "red water" has also been used for this disease and for hemolytic anemias in cattle caused by babesia spp.) similar naturally occurring disease has been reported in sheep. in cattle the disease is caused by liver fluke (fasciola hepatica) migration in susceptible animals. ingested clostridial spores may live in kupffer cells for a long time without causing disease. however, when migrating flukes cause hepatic necrosis, the resulting anaerobic environment stimulates the clostridial organisms to proliferate and elaborate their hemolytic toxins, causing additional hepatic necrosis. the mechanism of hemolysis involves a bacterial β-toxin (phospholipase c or lecithinase), which enzymatically degrades cell membranes, causing acute intravascular hemolysis. bacillary hemoglobinuria also occurs with liver biopsies in calves. clostridium perfringens type a causes intravascular hemolytic anemia in lambs and calves-a condition known as yellow lamb disease, yellows, or enterotoxemic jaundice because of the characteristic icterus. the organism is a normal inhabitant of the gastrointestinal tract in these animals but may proliferate abnormally in response to some diets. c. perfringens causes intravascular hemolytic anemia in horses with clostridial abscesses, and clostridial mastitis in ewes. c. perfringens type a produces hemolytic α-toxin, which also has phospholipase c activity. leptospirosis. leptospirosis is recognized as a cause of hemolytic anemia in calves, lambs, and pigs. specific leptospiral organisms that cause hemolytic disease include leptospira interrogans serovars pomona and ictohaemorrhagiae. leptospira organisms are ubiquitous in the environment. infection occurs percutaneously and via mucosal surfaces and is followed by leptospiremia; organisms then localize preferentially in certain tissues (e.g., kidney, liver, and pregnant uterus). proposed mechanisms of hemolytic disease include immune-mediated (immunoglobulin m [igm] cold agglutinin) extravascular hemolysis and enzymatic (phospholipase produced by the organism) intravascular hemolysis. leptospirosis can also cause many disease manifestations besides hemolysis (e.g., renal failure, liver failure, abortion, and other conditions) that are not discussed here. primary immune-mediated hemolytic anemia or autoimmune hemolytic anemia) or secondary to a known initiator, termed secondary immune-mediated hemolytic anemia. although the cause of idiopathic immune-mediated hemolytic anemia is unknown, certain dog breeds (e.g., cocker spaniels) are predisposed to developing disease, suggesting the possibility of a genetic component. causes of secondary immune-mediated hemolytic anemia include certain infections (e.g., hemoplasmosis, babesiosis, and theileriosis), drugs (e.g., cephalosporins, penicillin, and sulfonamides), vaccines, and envenomations (e.g., bee stings). immune-mediated hemolysis directed at nonself antigens, such as in neonatal isoerythrolysis, is presented later. in most cases of idiopathic immune-mediated hemolytic anemia, the reactive antibody is igg, and the hemolysis is extravascular (i.e., erythrocytes with surface-bound antibody are phagocytized by macrophages, mainly in the spleen). igm and/or complement proteins may also contribute to idiopathic immune-mediated hemolytic anemia. complement factor c b usually acts as an opsonin that promotes phagocytosis and extravascular hemolysis. however, formation of the complement membrane attack complex on red blood cell surfaces causes intravascular hemolysis; this mechanism more commonly occurs with igm autoantibodies. most immunoglobulins implicated in immune-mediated hemolytic anemia are reactive at body temperature (warm hemagglutinins). a smaller portion, usually igm, are more reactive at lower temperatures, young (hours to days old) with typical gross and microscopic changes of immune-mediated hemolytic anemia. pure red cell aplasia. pure red cell aplasia (prca) is a rare bone marrow disorder characterized by absence of erythropoiesis and severe nonregenerative anemia. primary and secondary forms of pure red cell aplasia have been described in dogs and cats. primary pure red cell aplasia is apparently caused by immune-mediated destruction of early erythroid progenitor cells, a presumption supported by the response of some patients to immunosuppressive therapy and by the detection of antibodies inhibiting erythroid colony formation in vitro in some dogs. administration of recombinant human erythropoietin (rhepo) has been identified as a cause of secondary pure red cell aplasia in dogs, cats, and horses, presumably caused by induction of antibodies against rhepo that cross-react with endogenous epo. experimentation with the use of speciesspecific recombinant epo has produced mixed results. dogs treated with recombinant canine epo have not developed pure red cell aplasia. however, in experiments reported thus far involving cats treated with recombinant feline epo, at least some animals have developed pure red cell aplasia. parvoviral infection has been suggested as a possible cause of secondary pure red cell aplasia in dogs. infection with felv subgroup c causes secondary erythroid aplasia in cats, probably because of infection of early-stage erythroid precursors. grossly, animals with pure red cell aplasia have pale mucous membranes without indicators of hemolysis (e.g., icterus). microscopic examination of the bone marrow shows an absence or near absence of erythroid precursors with or without lymphocytosis, plasmacytosis, and myelofibrosis; production of other cell lines (e.g., neutrophils and platelets) is normal or hyperplastic. immune-mediated neutropenia. immune-mediated neutropenia is a rare condition that has been reported in horses, dogs, and cats. this disease is characterized by severe neutropenia from immune-mediated destruction of neutrophils or their precursors. the range of causes is presumably similar to that of other immunemediated cytopenias (e.g., immune-mediated hemolytic anemia, pure red cell aplasia, and immune-mediated thrombocytopenia). affected animals may have infections, such as dermatitis, conjunctivitis, or vaginitis, which are secondary to marked neutropenia and a compromised innate immune system. microscopically, there may be neutrophil hyperplasia, maturation arrest, or aplasia in the bone marrow, depending on which neutrophil maturation stage is targeted causing a condition known as cold hemagglutinin disease. this results in ischemic necrosis at anatomic extremities (e.g., tips of the ears), where cooling of the circulation causes autoagglutination of erythrocytes and occlusion of the microvasculature. typically immunemediated hemolytic anemia targets mature erythrocytes, causing a marked regenerative response. however, as discussed earlier in the chapter, immune-mediated destruction of immature erythroid cells in the bone marrow may also occur, resulting in nonregenerative anemia. pathogenesis of secondary immune-mediated hemolytic anemia is dependent upon the cause. erythrocytic parasites may cause immune-mediated hemolysis by altering the red blood cell surface and exposing "hidden antigens" that are not recognized as selfantigens by the host's immune system. alternatively, the immune attack may be directed at the infectious agent, but erythrocytes are nonspecifically destroyed because of their close proximity-this is called the innocent bystander mechanism. certain drugs, such as penicillin, may cause immune-mediated hemolytic anemia by binding to erythrocyte membranes and forming drug-autoantigen complexes that induce antibody formation, termed hapten-dependent antibodies. other proposed mechanisms include binding of drugantibody immune complexes to the erythrocyte membrane, or induction of a true autoantibody directed against an erythrocyte antigen. hematologic, gross, and histopathologic abnormalities are typical of those of hemolytic anemia, as presented in the earlier section on bone marrow and blood cells, dysfunction/responses to injury, blood cells, abnormal concentrations of blood cells, anemia). in brief, there may be spherocytes and autoagglutination on blood smear evaluation, icterus and splenomegaly on gross examination, and emh, erythrophagocytic macrophages, and hypoxia-induced or thromboemboli-induced tissue necrosis on histopathologic examination. dogs with immune-mediated hemolytic anemia also frequently develop an inflammatory leukocytosis and coagulation abnormalities (prolonged coagulation times, decreased plasma antithrombin concentration, increased plasma concentration of fibrin degradation products, thrombocytopenia, and disseminated intravascular coagulation). intravascular hemolysis plays a relatively insignificant role in most cases of immune-mediated hemolytic anemia, but evidence of intravascular hemolysis (e.g., ghost cells, red plasma and urine, dark red kidneys) is noted occasionally, presumably in those cases in which igm and complement are major mediators of hemolysis. neonatal isoerythrolysis. neonatal isoerythrolysis (ni) is a form of immune-mediated hemolytic anemia in which colostrumderived maternal antibodies react against the newborn's erythrocytes. it is common in horses ( fig. - ) and has been reported in cattle, cats, and some other domestic and wildlife species. in horses, neonatal isoerythrolysis occurs as a result of immunosensitization of the dam from exposure to an incompatible blood type inherited from the stallion (e.g., transplacental exposure to fetal blood during pregnancy or mixing of maternal and fetal blood during parturition). a previously mismatched blood transfusion produces the same results. some equine blood groups are more antigenic than others; in particular, types aa and qa are very immunogenic in mares. in cattle, neonatal isoerythrolysis has been caused by vaccination with whole blood products or products containing erythrocyte membrane fragments. neonatal isoerythrolysis has been produced experimentally in dogs, but there are no reports of naturally occurring disease. in cats the recognized form of neonatal isoerythrolysis does not depend on prior maternal immunosensitization but on naturally occurring anti-a antibodies in queens with type b blood. affected animals are s l rather a secondary complication of many types of underlying disease, including severe inflammation, organ failure, and neoplasia. it is included in the section on inflammatory disorders because the coagulation cascade is closely linked to inflammatory pathways. information on this topic, including e- fig. - , is available at www.expertconsult.com. as well as in chapter . the term hematopoietic neoplasia encompasses a large and diverse group of clonal proliferative disorders of hematopoietic cells. historically, numerous systems have been used to classify hematopoietic neoplasms in human medicine, some of which have been applied inconsistently to veterinary species (examples include the kiel classification and national cancer institute working formulation). the world health organization (who) classification of hematopoietic neoplasia was first published in (updated in ) and is based on the principles defined in the revised european-american classification of lymphoid neoplasms (real) from the international lymphoma study group. the who classification system is considered the first true worldwide consensus on the classification of hematopoietic malignancies and integrates information on tumor topography, cell morphology, immunophenotype, genetic features, and clinical presentation and course. a veterinary reference of the who classification system, published in , was later validated in using the canine model of lymphoma. this project, modeled after the study to validate the system in human beings, yielded an overall accuracy (i.e., agreement on a diagnosis) among pathologists of %. currently this classification system is accepted as the method of choice in both human and veterinary medicine. the who classification broadly categorizes neoplasms primarily according to cell lineage: myeloid, lymphoid, and histiocytic. this distinction is based on the fact that the earliest commitment of a pluripotent hsc is to either a lymphoid or nonlymphoid lineage. many pathologists and clinicians distinguish leukemias from other hematopoietic neoplasms. leukemia refers to a group of hematopoietic neoplasms that arise from the bone marrow and are present within the blood. leukemia may be difficult to differentiate from other forms of hematopoietic neoplasms that originate outside of the bone marrow but infiltrate the bone marrow and blood. for simplicity, cases of secondary bone marrow or blood involvement may not be considered leukemia but rather the "leukemic phase" of another primary neoplasm. it is now recognized that certain lymphomas and leukemias are different manifestations of the same disease (e.g., chronic lymphocytic leukemia and small lymphocytic lymphoma), and the designation of lymphoma or leukemia is placed on the tissue with the largest tumor burden. based on their degree of differentiation, leukemias are classified as acute or chronic. acute leukemias are poorly differentiated or undifferentiated, meaning that there are high percentages of early progenitor and precursor cells, including lymphoblasts, myeloblasts, monoblasts, erythroblasts, and/or megakaryoblasts. in contrast, well-differentiated cells predominate in chronic leukemias. because well-differentiated cells also predominate with nonneoplastic proliferations, chronic leukemias must be differentiated from reactive processes, such as those cells that occur in chronic and/or granulomatous inflammation. diagnosis of chronic leukemia is often made by excluding all other causes for the proliferating cell type. for example, causes of relative and secondary erythrocytosis are excluded to be able to diagnose polycythemia vera. furthermore, the designation of acute or chronic also refers to the disease's clinical course. acute leukemias tend to have an acute onset of severe and rapidly progressive clinical signs, whereas animals with chronic leukemia for destruction. marrow lymphocytosis and plasmacytosis may be marked (e.g., > % of nucleated cells). the diagnosis may be supported by flow cytometric detection of immunoglobulin bound to neutrophils but is most often made on the basis of exclusion of other causes of neutropenia and response to immunosuppressive therapy. immune-mediated thrombocytopenia. immune-mediated thrombocytopenia (imtp) is a condition characterized by immunemediated destruction of platelets. it is a fairly common condition in dogs and is less frequent in horses and cats. the disease is usually idiopathic but may be secondary to infection (e.g., equine infectious anemia and ehrlichiosis), drug administration (e.g., cephalosporins and sulfonamides), neoplasia, and other immunemediated diseases. when immune-mediated thrombocytopenia occurs together with immune-mediated hemolytic anemia, the condition is called evans's syndrome. the thrombocytopenia is often severe (e.g., < , platelets/µl), resulting in varying degrees of bleeding tendencies, mainly in skin and mucous membranes. microscopically, there are multifocal perivascular hemorrhages in multiple tissues, and the bone marrow exhibits megakaryocytic and erythroid hyperplasia. rarely, immune-mediated destruction of megakaryocytes may cause megakaryocytic hypoplasia, termed amegakaryocytic thrombocytopenia. neonatal alloimmune thrombocytopenia. a form of immune-mediated thrombocytopenia, known as neonatal alloimmune thrombocytopenia, is recognized in neonatal pigs and foals. the pathogenesis of this disease is virtually identical to that of neonatal isoerythrolysis as a cause of anemia: a neonate inheriting paternal platelet antigens absorbs maternal antibodies against these antigens through the colostrum. in principle, a similar situation may occur after platelet-incompatible transfusion of blood or blood products containing platelets. gross and microscopic changes are similar to those of immune-mediated thrombocytopenia except that the animal is young (e.g., to days). hemophagocytic syndrome. hemophagocytic syndrome is a term used to describe the proliferation of nonneoplastic (i.e., polyclonal), well-differentiated but highly erythrophagic macrophages. the condition is rare but has been recognized in dogs and cats. unlike hemophagocytic histiocytic sarcoma, which is a neoplastic proliferation of phagocytic macrophages, hemophagocytic syndrome is secondary to an underlying disease, such as neoplasia, infection, or an immune-mediated disorder. the primary disease process causes increased production of stimulatory cytokines, which results in macrophage proliferation and hyperactivation. these activated macrophages phagocytize mature hematopoietic cells and hematopoietic precursors at an enhanced rate, resulting in one or more cytopenias. affected animals usually have lesions of the primary disease, as well as signs of the anemia (e.g., pale mucous membranes), neutropenia (e.g., bacterial infections), and thrombocytopenia (e.g., petechiae and ecchymoses). microscopically, phagocytic macrophages are found in high numbers in the bone marrow and commonly in other tissues, including lymph nodes, spleen, and liver. additional bone marrow findings reported in animals with hemophagocytic syndrome vary widely, ranging from hypoplasia to hyperplasia of cell lines with peripheral cytopenias. disseminated intravascular coagulation. disseminated intravascular coagulation is a syndrome characterized by continuous activation of both coagulation and fibrinolytic pathways and is also known as consumptive coagulopathy. it is not a primary disease, but .e chapter bone marrow, blood cells, and the lymphoid/lymphatic system disseminated intravascular coagulation is a consumptive coagulopathy resulting from activation of both coagulation and fibrinolytic pathways. it is a secondary complication of many types of underlying disease, including many infectious diseases, trauma, burns, heat stroke, immune-mediated disease, hemolysis, shock, neoplasia, organ failure, obstetric complications, and noninfectious inflammatory disease, such as pancreatitis. it is common in critically ill domestic animals. disseminated intravascular coagulation involves an initial hypercoagulable phase, resulting in thrombosis and ischemic tissue damage, and a subsequent hypocoagulable phase as a result of consumption of coagulation factors and platelets, resulting in hemorrhage (e- fig. - ) . the pathogenesis of disseminated intravascular coagulation typically involves the release of tissue factor (thromboplastin) and subsequent activation of coagulation pathways and platelets but may also involve defective normal inhibition of coagulation or defective fibrinolysis. classically diagnosis of disseminated intravascular coagulation is based on clinical evidence of hemorrhage and/or thromboembolic disease and a triad of laboratory findings: thrombocytopenia, usually moderate (below the lower reference value but above , /µl); prolonged coagulation times (prothrombin time and/or partial thromboplastin time); and decreased fibrinogen or increased concentration of plasma fibrin degradation products or d-dimer. milder forms of disseminated intravascular coagulation that do not meet all of the diagnostic criteria also occur. decreased plasma antithrombin (antithrombin iii) concentration and schistocytosis are other laboratory abnormalities often found in patients with disseminated intravascular coagulation. dysplasia of myeloid cells, and fewer than % myeloblasts and "blast equivalents." acute myeloid leukemia. acute myeloid leukemia (aml) is uncommon in domestic animals but most frequently occurs in dogs and cats. in veterinary species, acute myeloid leukemia is most commonly of neutrophil, monocyte, and/or erythroid origin, with rare reports of eosinophil, basophil, or megakaryocytic lineages. it is caused by felv infection in cats. evaluations of blood smears show many early myeloid precursors, including myeloblasts and blast equivalents ( fig. - , a) . in dogs the total leukocyte concentration averages approximately , /µl; anemia, neutropenia, and thrombocytopenia commonly occur. grossly, animals show lesions attributed to anemia, neutropenia, and thrombocytopenia, such as pale mucous membranes, secondary infections, and multisystemic bleeding, respectively. neoplastic cells often infiltrate tissues, resulting in splenomegaly, hepatomegaly, and lymphadenomegaly. microscopically, myeloid cells efface (replace) the bone marrow and infiltrate extramedullary tissues, especially lymphoid tissue. chronic myeloid leukemia. chronic myeloid leukemia (cml), also called chronic myelogenous leukemia or myeloproliferative neoplasia, is rare in animals. most reported cases occur in dogs and cats. there are various subclassifications of chronic myeloid leukemia, including excessive production of erythrocytes (polycythemia vera), platelets (essential thrombocythemia), neutrophils (chronic neutrophilic leukemia), monocytes (chronic monocytic leukemia), neutrophils and monocytes (chronic myelomonocytic leukemia), eosinophils (chronic eosinophilic leukemia), or basophils (chronic basophilic leukemia). complete peripheral blood count analysis often reveals very high concentrations of the neoplastic cells, such as greater than , to , leukocytes/µl (see fig. - , b) or , , platelets/µl. cellular morphologic features are often normal, but slight dysplasia may be observed. later in the disease there may be cytopenias of nonneoplastic cell types. animals with polycythemia vera often have red mucous membranes and lesions of hyperviscosity syndrome, such as bleeding and dilated, tortuous retinal vessels. essential thrombocythemia results in multisystemic bleeding due to dysfunctional platelets, or multisystemic infarcts from hyperaggregability and excessive platelets. chronic myeloid leukemias of leukocytes often result in splenomegaly, hepatomegaly, and lymphadenomegaly because of infiltration by the neoplastic cells. histologically, the bone marrow shows proliferation of the neoplastic cell type characterized by dysplasia and low numbers (e.g., < %) of myeloblasts and blast equivalents. mast cell neoplasia. mast cell tumors (mcts) of the skin and other sites are common in animals (see chapters , , and ), but mast cell leukemia is rare. in cats, mcts are the most common neoplasm in the spleen (e- fig. - ) . mast cells normally are not present in the blood vascular system, but the finding of mast cells in the blood (mastocytemia) is highly suggestive of disseminated mast cell neoplasia (systemic mastocytosis) in cats. however, mastocytemia does not necessarily indicate myeloid neoplasia in dogs. in fact, one study found that the severity of mastocytemia in dogs was frequently higher in animals without mcts than those with mcts and that random detection of mast cells in blood smears usually is not the result of underlying mct. granulocytic sarcoma. granulocytic sarcoma is a poorly characterized extramedullary proliferation of myeloid precursors, most typically have indolent, slowly progressive disease. this classification scheme is summarized in table - . subcategories exist within each of these groups, as discussed further later. information on this topic is available at www.expertconsult.com. this section discusses examples of myeloid neoplasms, including myelodysplastic syndrome, myeloid leukemias, and mast cell neoplasms (technically a form of myeloid neoplasia), and lymphoid neoplasms, including lymphoid leukemias and multiple myeloma. other lymphoid neoplasms, such as the numerous subtypes of lymphoma and extramedullary plasmacytomas (emps), as well as histiocytic disorders are described in the section on lymphoid/lymphatic system, disorders of domestic animals, neoplasia. additional discussion of hematopoietic neoplasia occurs in the species-specific sections at the end of this chapter. myelodysplastic syndrome. myelodysplastic syndrome (mds) most commonly occurs in dogs and cats and may be caused by felv infection in cats. the disease refers to a group of clonal myeloid proliferative disorders with ineffective hematopoiesis in the bone marrow, resulting in cytopenias of more than one cell line. hematopoietic proliferation in bone marrow with concurrent peripheral blood cytopenias is likely a result of increased apoptosis of neoplastic cells within the bone marrow, before their release into circulation. clinical illness and death often result from secondary manifestations, such as secondary infections or cachexia, attributable to the effects of cytopenias and/or transformation of the neoplasm into acute myeloid leukemia. gross lesions are dependent upon the type and severity of the cytopenias. however, essential microscopic findings within the bone marrow are normal or increased cellularity, "blast equivalents" include other stages of immature myeloid cells, such as abnormal promyelocytes, monoblasts, promonocytes, erythroblasts, and megakaryoblasts. before the discussion of specific diseases, it is worthwhile to describe the diagnostic techniques required to classify hematopoietic neoplasms that are becoming increasingly available for routine use in veterinary medicine. immunophenotyping refers to the use of antibodies recognizing specific molecules expressed on different cell types to determine the identity of a cell population of interest. immunophenotyping on the basis of these lineage-specific or lineage-associated markers can be performed on histologic sections (immunohistochemistry [see fig. - ]), air-dried cytologic examination smears (immunocytochemistry), or by laser analysis of cells in suspension in blood or buffer solutions (flow cytometry). in cases of lymphoid neoplasia, immunophenotyping most routinely refers to determination of b or t lymphocyte origin. clonality assays, pcr for antigen receptor rearrangement (parr), can help identify neoplastic lymphoid proliferations on the basis of clonal rearrangements of genes encoding lymphocyte antigen receptors. in terms of practical application the parr assay is most useful in helping to distinguish lymphoid neoplasms from those nonneoplastic lymphoid proliferations mimicking neoplasia. cytogenetic testing has not been routinely used in veterinary medicine, though several genetic mutations have been identified in dogs. for example, breakpoint cluster region-abelson (bcr-abl) translocations have been identified in some canine leukemias, including acute myeloblastic leukemia and chronic monocytic leukemia. dogs with burkitt-like lymphoma have a translocation leading to constitutive c-myc expression. a b thrombocytopenia commonly occur. gross and microscopic lesions also are similar to those that occur in cases of acute myeloid leukemia, except that neoplastic cells may differentiate into morphologically identifiable lymphoid cells. chronic lymphocytic leukemia. chronic lymphocytic leukemia (cll) is uncommon in veterinary medicine. it is predominantly a disease of middle-aged to older dogs but is also documented in horses, cattle, and cats. most canine chronic lymphocytic leukemia cases are of t lymphocyte origin, typically cytotoxic t lymphocytes expressing cd . in cats the majority of chronic lymphocytic leukemia cases have a t helper lymphocyte immunophenotype. a cbc often shows very high numbers of small lymphocytes with clumped chromatin and scant cytoplasm. proliferating cytotoxic t lymphocytes frequently contain a few pink cytoplasmic granules when stained with most methanol-based romanowsky stains (e.g., wright-giemsa). however, these granules may not be appreciated with some aqueous-based romanowsky stains (e.g., diff-quik). although the number of total blood lymphocytes is often greater than , /µl, relatively mild lymphocytosis (e.g., , /µl) has been reported. seventy-five percent of affected dogs also have often of eosinophilic or neutrophilic cell lines. although rare, there are reports of granulocytic sarcoma in dogs, cats, cattle, and pigs, and it may arise in a number of sites, such as lung, intestine, lymph nodes, liver, kidney, skin, and muscle. acute lymphoblastic leukemia. acute lymphoblastic leukemia (all) is uncommon in dogs and cats, and rare in horses and cattle. in a recent immunophenotype study of cases of acute lymphoblastic leukemia in dogs, arose from b lymphocytes and arose from double-negative t lymphocytes that were immunonegative for cd and cd markers. in the blood of animals with acute lymphoblastic leukemia, there are typically many medium to large lymphoid cells with deeply basophilic cytoplasm, reticular to coarse chromatin, and prominent, multiple nucleoli (see fig. - , c) . in affected dogs the mean blood lymphoid concentration is approximately , /µl, but cats with acute lymphoblastic leukemia often have low numbers of neoplastic cells in the circulation. as with animals with acute myeloid leukemia, anemia, neutropenia, and c d m the light chains deposit as nonamyloid granules, it is termed light chain deposition disease. light chains are low-molecular-weight proteins that pass through the glomerular filter into the urine, wherein they are also known as bence jones proteins. they tend to not react with urine dipstick protein indicators and are most specifically detected by electrophoresis and immunoprecipitation. in addition to aiding in the diagnosis of multiple myeloma, paraproteins have an important role in pathogenesis of disease. these proteins may inhibit platelet function, increase blood viscosity, deposit in glomerular basement membranes (see chapter ; see figs. - and - , or precipitate at cool temperatures, which results in bleeding tendencies, hyperviscosity syndrome, glomerulopathies, and cryoglobulinemia, respectively. hyperviscosity syndrome refers to the clinical sequelae of pathologically increased blood viscosity, which are slowed blood flow and loss of laminar flow. clinical signs include mucosal hemorrhages, visual impairment due to retinopathy, and neurologic signs, such as tremors and abnormal aggressive behavior. cryoglobulinemia is the condition in which proteins, typically igm, precipitate at temperatures below normal body temperature (cold agglutinins). precipitation often occurs in blood vessels of the skin and extremities, such as the ears and digits, and results in ischemic necrosis. in multiple myeloma the neoplastic proliferation of plasma cells results in osteolysis. work with human cell cultures has shown that anemia, and % have thrombocytopenia. autopsy findings depend on the stage of disease. in advanced cases with marked infiltration of organs with neoplastic cells, there is often uniform splenomegaly, hepatomegaly, and lymphadenomegaly, and the bone marrow is highly cellular (e- fig. - ; see fig. - , d) . other lesions depend on whether there are concurrent cytopenias, such as anemia, neutropenia, and thrombocytopenia, and if the neoplastic cells produce excessive immunoglobulin. lesions caused by excessive immunoglobulin are further discussed in the section on multiple myeloma. histologically, the bone marrow is densely cellular with welldifferentiated lymphocytes. small lymphocytes infiltrate and often efface in the architecture of the lymph nodes and spleen. the liver may have dense accumulations of neoplastic cells in the connective tissue around the portal triad. plasma cell neoplasia. plasma cell neoplasms are most easily categorized as myeloma or multiple myeloma, which arises in the bone marrow, and extramedullary plasmacytoma, which as the name implies involves sites other than bone; the latter is discussed in the section on lymphoid/lymphatic system, disorders of domestic animals: lymph nodes, neoplasia, plasma cell neoplasia. multiple myeloma. multiple myeloma (mm) is a rare, malignant tumor of plasma cells that arises in the bone marrow and usually secretes large amounts of immunoglobulin. the finding of neoplastic plasma cells in blood samples or smears is rare. dogs are affected more frequently than other species, but multiple myeloma has also been reported in horses, cattle, cats, and pigs. diagnosis of multiple myeloma is based on finding a minimum of two or three (opinions vary) of the following abnormalities: • markedly increased numbers of plasma cells in the bone marrow ( fig. - , a) • monoclonal gammopathy • radiographic evidence of osteolysis • light chain proteinuria the classic laboratory finding in patients with multiple myeloma is hyperglobulinemia, which results from the excessive production of immunoglobulin or an immunoglobulin subunit by the neoplastic cells. this homogeneous protein fraction is often called paraprotein or m protein. paraproteins produced from the same clone of plasma cells have the same molecular weight and electric charge. therefore they have the same migration pattern using serum protein electrophoresis, which results in a tall, narrow spike in the globulins region, termed monoclonal gammopathy (see fig. - , b) . the term gammopathy is used because most immunoglobulins migrate in the γ-region of an electrophoresis gel. however, some immunoglobulins, especially immunoglobulin a (iga) and igm, migrate to the β-region. occasionally, biclonal or other atypical electrophoretic patterns may be seen with multiple myeloma as a result of protein degradation, protein complex formation, binding to other proteins, or when the tumor includes more than one clonal population. it is important to note that monoclonal gammopathy is not specific to multiple myeloma but has also been reported with lymphoma, chronic lymphocytic leukemia, canine ehrlichiosis, and canine leishmaniasis. definitively distinguishing monoclonal from polyclonal gammopathy requires immunoelectrophoresis or immunofixation using species-specific antibodies recognizing different immunoglobulin subclasses and subunits. occasionally, multiple myeloma cells produce only the immunoglobulin light chain. an immunoglobulin monomer consists of two heavy chains and two light chains connected by disulfide bonds. these light chains may deposit in tissues and cause organ dysfunction, especially renal failure. when the light chains form amyloid deposits, the disease is called amyloid light chain amyloidosis. but if marrow is dark red as a result of replacement of fat by hematopoietic tissue; the extent of replacement is an indication of the duration of the anemia. the severity of microscopic lesions is dependent on the chronicity of the disease, and they are most significant in the spleen, liver, and bone marrow. as would be anticipated, microscopic findings of the spleen are predominantly influenced by the number and activity of macrophages, which is a reflection of the duration of the disease and the frequency of hemolytic episodes. hemosiderin-laden macrophages persist for months to years; therefore large numbers are consistent with chronicity. kupffer cell hyperplasia with hemosiderin stores and periportal infiltrates of lymphocytes are the most significant changes in the liver. bone marrow histologic findings vary depending on the duration of the disease. in most animals the marrow is cellular because of the replacement of fat by intense, orderly erythropoiesis. granulocytes are relatively less numerous, and plasma cells are increased. as in the spleen, hemosiderin-laden macrophages are present in large numbers in chronic cases. emaciated animals with chronic disease have serous atrophy of fat (see e- fig. - ) . clinical findings with viremic episodes include fever, depression, icterus, petechial hemorrhages, lymph node enlargement, and dependent edema. equine infectious anemia infection is diagnosed on the basis of the coggins test, an agarose gel immunodiffusion test for the presence of the antibody against the virus. congenital dyserythropoiesis in polled herefords. a syndrome of congenital dyserythropoiesis and alopecia occurs in polled hereford calves. the cause and pathogenesis of this often fatal disease are unknown. early in disease there is hyperkeratosis and alopecia of the muzzle and ears, which progresses to generalized alopecia and hyperkeratotic dermatitis. histologically, there is orthokeratotic hyperkeratosis with dyskeratosis, as well as erythroid hyperplasia, dysplasia, and maturation arrest in the bone marrow. ineffective erythropoiesis results in nonregenerative to poorly regenerative anemia. erythrocyte band is integral membrane protein that connects to the cytoskeleton and aids in erythrocyte stability. a hereditary deficiency of this protein has been identified in japanese black cattle, resulting in increased erythrocyte fragility, spherocytosis, intravascular hemolytic anemia, and retarded growth. affected calves show lesions consistent with hemolytic anemia, including pale mucous membranes, icterus, and splenomegaly. histologically, there are bilirubin accumulations in the liver, and hemosiderin in renal tubules. bovine leukemia virus. bovine leukemia virus is discussed in the later section on lymphoma (see lymphoid/lymphatic system, disorders of domestic animals: lymph nodes, neoplasia, lymphoma). bovine viral diarrhea virus. bvdv infection may cause thrombocytopenia in cattle, and a thrombocytopenic hemorrhagic syndrome has been specifically caused by type ii bvdv infection. investigations of the mechanism of bvdv-induced thrombocytopenia have resulted in varying, sometimes conflicting, conclusions. more than one study has shown viral antigen within bone marrow megakaryocytes and circulating platelets. evidence of impaired thrombopoiesis (megakaryocyte necrosis, megakaryocyte pyknosis, osteoclasts support the growth of myeloma cells, and that direct contact between the two cell types increases the myeloma cell proliferation and promotes osteoclast survival. increased osteoclast activity causes osteolysis, but the exact mechanism is not known. osteolysis often results in bone pain, lytic bone lesions on radiographs, hypercalcemia, and increased serum alkaline phosphatase activity. later in disease, osteolysis may cause pathologic fractures. morphologically, myeloma cells tend to grow in sheets that displace normal hematopoietic cells in the bone marrow. a proposed diagnostic criterion of multiple myeloma is that plasma cells constitute % or more of the nucleated cells in the marrow. welldifferentiated plasma cells are round with abundant basophilic cytoplasm (due to increased rough endoplasmic reticulum) and a perinuclear pale zone (enlarged golgi apparatus for the production of immunoglobulin); anisocytosis and anisokaryosis are often mild but may be marked. some plasma cell neoplasms have a bright eosinophilic fringe due to accumulated iga (see fig. - , a) . nuclei are round with clumped chromatin and often peripherally placed with the cytoplasm; binucleation and multinucleation are common. poorly differentiated myeloma cells may lack and/or display less characteristic features. osteolysis of bone may be present microscopically. common sites of metastasis include the spleen, liver, lymph nodes, and kidneys. flavin adenine dinucleotide deficiency. flavin adenine dinucleotide (fad) is a cofactor for cytochrome-b reductase, the enzyme that maintains hemoglobin in its functional reduced state, and for glutathione reductase, an enzyme that also protects erythrocytes from oxidative damage. reported in a spanish mustang mare and a kentucky mountain saddle horse gelding, erythrocyte fad deficiency is a result of an abnormal riboflavin kinase reaction, which is the first reaction in converting riboflavin to fad. clinicopathologic changes include persistent methemoglobinemia of % to %, eccentrocytosis, a slightly decreased or normal hematocrit, and erythroid hyperplasia in the bone marrow. equine infectious anemia virus. equine infectious anemia virus (eiav), the agent of equine infectious anemia, is a lentivirus that infects cells of the monocyte-macrophage system in horses (also ponies, donkeys, and mules). the virus is mechanically transmitted by biting flies, such as horseflies and deer flies. less common routes of transmission include blood transfusions, contaminated medical equipment, and transplacentally. disease may present in acute, subacute, and chronic forms and is potentially fatal. after an acute period of fever, depression, and thrombocytopenia that lasts to days, there is a prolonged period of recurrent fever, thrombocytopenia, and anemia. in most cases, clinical disease subsides within a year, and horses become lifelong carriers and reservoirs of eiav. eiav causes anemia by both immune-mediated hemolysis and decreased erythropoiesis. hemolysis is typically extravascular but may have an intravascular component during the acute phase. decreased erythropoiesis may result from direct suppression of earlystage erythroid cells by the virus, as well as anemia of inflammation. thrombocytopenia likely results from immune-mediated platelet destruction and suppressed platelet production. animals dying during hemolytic crises are pale with mucosal hemorrhages and dependent edema. the spleen and liver are enlarged, dark, and turgid, and they and other organs have superficial subcapsular hemorrhages. petechiae are evident beneath the renal capsule and throughout the cortex and medulla. the bone affected animals are not necessarily anemic. however, acute intravascular hemolytic episodes may occur with hyperventilationinduced alkalemia. lesions are typical of hemolytic anemia and include pale mucous membranes, icterus, hepatosplenomegaly, and dark red urine with microscopic emh and marrow erythroid hyperplasia. a single dna-based test is available to detect the common mutation. erythrocyte structural abnormalities. congenital erythrocyte structural abnormalities may occur with abnormal membrane composition or defective proteins within the membrane or cytoskeleton. some of these morphologic changes occur concurrently with clinical disease, but others do not. hereditary stomatocytosis is recognized in alaskan malamutes, drentse patrijshonds, and schnauzers. the specific defects are not known, but they are likely different in the various dog breeds. however, all affected dogs have stomatocytes on blood smear evaluation, as identified by their slit-shaped area of central pallor. erythrocytes also have increased osmotic fragility and decreased survival. schnauzers are clinically healthy and not anemic but do have reticulocytosis, suggesting that the hemolytic anemia is compensated by erythroid hyperplasia. mild to marked hemolytic anemia is documented in alaskan malamutes and drentse patrijshonds. alaskan malamutes have concurrent short-limb dwarfism, and drentse patrijshonds have hypertrophic gastritis and polycystic kidney disease. other (presumably heritable) erythrocyte abnormalities in dogs that do not have clinical signs include elliptocytosis caused by band . deficiency or β-spectrin mutation, and familial macrocytosis and dyshematopoiesis in poodles. scott's syndrome. an inherited thrombopathy resembling scott's syndrome in human beings, in which platelets lack normal procoagulant activity, has been recognized in a family of german shepherd dogs. the specific defect in these dogs has not been identified on the molecular level but involves impaired expression of phosphatidylserine on the platelet surface. affected dogs have a mild to moderate clinical bleeding tendency characterized by epistaxis, hyphema, intramuscular hematoma formation, and increased hemorrhage with surgery. macrothrombocytopenia. macrothrombocytopenia is an inherited condition in cavalier king charles spaniels in which there are lower than normal concentrations of platelets with enlarged and giant platelets. the condition is caused by defective β -tubulin, which results in impaired microtubule assembly. affected dogs are asymptomatic but may have abnormal platelet aggregation in vitro. canine distemper. canine distemper virus preferentially infects lymphoid, epithelial, and nervous cells and is presented in greater detail in the lymphoid section. canine distemper virus may also infect other hematopoietic cells, including erythrocytes, nonlymphoid leukocytes, and platelets ( fig. - ) , and can cause decreased peripheral blood concentrations of neutrophils, lymphocytes, monocytes, and platelets during viremia. the thrombocytopenia is a result of virus-antibody immune complexes on platelet membranes and direct viral infection of megakaryocytes. increased erythrocyte osmotic fragility. a condition characterized by increased erythrocyte osmotic fragility has been described in abyssinian and somali cats. the specific defect has and degeneration) and increased thrombopoiesis (megakaryocytic hyperplasia, increased numbers of immature megakaryocytes) in the bone marrow has been reported in type ii bvdv-infected animals, including concurrent megakaryocyte necrosis and hyperplasia in some experimental subjects. calves infected with type ii bvdv also have impaired platelet function. cattle with the hemorrhagic syndrome are severely thrombocytopenic and neutropenic with multisystemic hemorrhages, particularly of the digestive tract, spleen, gallbladder, urinary bladder, and lymph nodes. histologic lesions include hemorrhage, epithelial necrosis of enterocytes, intestinal erosions, crypt proliferation with microabscesses, and lymphoid depletion of the gut-associated lymphoid tissue, peyer's patches, and spleen. lesions of the bone marrow are variable, as previously described. bovine neonatal pancytopenia. bovine neonatal pancytopenia (bnp) is caused by alloantibodies absorbed from colostrum, resulting in a hemorrhagic syndrome in calves. the syndrome was first recognized in europe in the early s and has since been experimentally correlated with prior vaccination of affected calves' dams with a commercial bvdv vaccine (pregsure bvd; pfizer animal health). the vaccine has since been voluntarily recalled from the market. it is thought that vaccination induces alloantibody formation by the dam. the alloantibodies are ingested by the calf and bind to the calf's hematopoietic progenitor cells, resulting in functional compromise of those cells. acutely affected calves are less than a year of age and have peripheral thrombocytopenia and neutropenia. death results from thrombocytopenia-induced hemorrhages or neutropenia-induced secondary infections, including pneumonia, enteritis, and septicemia. within the bone marrow there is erythroid, myeloid, and megakaryocytic hypoplasia. cyclic hematopoiesis. cyclic hematopoiesis (also known as lethal gray collie disease) is an autosomal recessive disorder of pluripotent hscs in gray collie dogs. a defect in the adaptor protein complex (ap ) results in defective intracellular signaling and predictable fluctuations in concentrations of blood cells that occur in -day cycles. the pattern is cyclic marked neutropenia, and in a different phase, cyclic reticulocytosis, monocytosis, and thrombocytosis. production of key cytokines involved in regulation of hematopoiesis is also cyclic. neutropenia predisposes affected animals to infection, and many die of infectious causes. affected animals have dilute hair coats and lesions with acute or chronic infectious disease, especially of the lungs, gastrointestinal tract, and kidneys. dogs older than weeks of age have systemic amyloidosis, which occurs because of cyclic increases in concentration of acute phase proteins during phases of monocytosis. phosphofructokinase deficiency. inherited autosomal recessive deficiency of the erythrocyte glycolytic enzyme, phosphofructokinase (pfk), is described in english springer spaniel, american cocker spaniel, and mixed-breed dogs. there are three genes encoding pfk enzymes, designated m-pfk in muscle and erythrocytes, l-pfk in liver, and p-pfk in platelets. a point mutation in the gene coding for m-pfk results in an unstable, truncated molecule. erythrocytes in pfk-deficient dogs have decreased atp and , -diphosphoglycerate ( , -dpg) production and increased fragility under alkaline conditions. the disease is characterized by chronic hemolysis with marked reticulocytosis. the marked regenerative response may compensate for the ongoing hemolysis; therefore erythrophagocytosis, thrombosis, and histologic changes of ischemia are common, especially within the spleen, liver, and lungs. affected cats typically become acutely ill with fever, pallor, and icterus and usually die within to days. for many years, cytauxzoonosis was considered to be almost always fatal. however, a recent not been identified, but pk deficiency (which has been reported in these breeds) was excluded as the cause. affected cats have chronic intermittent severe hemolytic anemia and often have other lesions secondary to hemolytic anemia (e.g., splenomegaly and hyperbilirubinemia). cytauxzoonosis. cytauxzoonosis is a severe, often fatal disease of domestic cats caused by the protozoal organism, cytauxzoon felis. disease is relatively common in the south central united states, particularly during summer months. bobcats (lynx rufus) and other wild felids are thought to be wildlife reservoirs of disease. c. felis is transmitted by a tick vector, dermacentor variabilis, which is probably essential for infectivity of the organism. cytauxzoonosis has a schizogenous phase within macrophages throughout the body (especially liver, spleen, lung, lymph nodes, and bone marrow) that causes systemic illness. these schizontcontaining macrophages enlarge and accumulate within the walls of veins, eventually causing vessel occlusion, circulatory impairment, and tissue hypoxia. later in disease, merozoites released from schizonts enter erythrocytes, resulting in an erythrocytic phase of infection. infected domestic cats often have nonregenerative anemia, but the pathogenesis for the anemia is unclear. however, it likely represents preregenerative hemolytic anemia because erythrocyte phagocytosis is a prominent finding in many organs. infected cats often also develop neutropenia and thrombocytopenia, which likely result from inflammation and disseminated intravascular coagulation, respectively. on blood smear evaluation, signet ring-shaped erythrocytic inclusions (piroplasms) may be observed during the erythrocytic phase of disease ( fig. - ) . these inclusions closely resemble small-form babesia (see fig. - , a) and some theileria organisms. postmortem examination typically shows pallor, icterus, splenomegaly, enlarged and red lymph nodes, diffuse pulmonary congestion and edema, and multisystemic petechiae and ecchymoses. vascular obstruction may cause marked distention of abdominal veins. cavitary effusions are present in some cats. microscopically, large, schizont-laden macrophages accumulate within venous and sinusoidal lumens and often completely occlude the lumens (fig. - ) . which the b and t lymphocytes proliferate, differentiate, and mature. in mammals, lymphocytes arise from hscs in the bone marrow, and b lymphocytes continue to develop at this site. ruminants also have b lymphocyte proliferation and maturation within their peyer's patches. progenitor t lymphocytes migrate from bone marrow to mature and undergo selection in the thymus. the spleen, lymph nodes, and lymph nodules are secondary lymphoid organs and are responsible for the immune responses to antigens, such as the production of antibody and cell-mediated immune reactions. at these sites, lymphocytes are activated by antigens and undergo clonal selection, proliferation, and differentiation (see also chapter ). in addition, the spleen and lymph nodes contain cells of the monocyte-macrophage system and thus also participate in the phagocytosis of cells and materials. the bone marrow is described in the first section of this chapter. the remaining primary lymphoid organ, the thymus, is described first in this section, followed by the secondary lymphoid organs: spleen, lymph nodes, and diffuse and nodular lymphatic tissues. errors from selection of inappropriate sampling sites and artifacts from compression and incorrect fixation for histopathologic and immunohistochemical examinations are common in routine veterinary pathologic analysis. the identification and remedies for these problems are discussed in e-appendix - . the thymus is essential for the development and function of the immune system, specifically for the differentiation, selection, and maturation of t lymphocytes generated in the bone marrow (see also chapter ). the basic arrangement of the thymus in domestic animals consists of paired cervical lobes (left and right), an intermediate lobe at the thoracic inlet, and a thoracic lobe, which may be bilobed. the cervical lobes are positioned ventrolateral to the trachea, adjacent to the carotid arteries, and extend from the intermediate lobe at the thoracic inlet as far cranially as the larynx. the intermediate lobe bridges between the cervical and the thoracic lobe. the right thoracic lobe is usually small or completely absent. the left lobe lies in the ventral aspect of the cranial mediastinum (except in the ruminant, where it is dorsal) and extends caudally as far as the pericardium. horse-the cervical lobes in foals are small, and the thoracic lobe constitutes the bulk of the thymus. ruminant-the cervical lobes are large. the left and right thoracic lobes are fused and unlike other domestic animals, lie in the dorsal aspect of the cranial mediastinum. pig-the cervical lobes are large. dog-the cervical lobes regress very early and thus appear absent. the thoracic lobe extends caudally to the pericardium. cat-the cervical lobes are small, and the thoracic lobe, which forms the majority of the thymus, extends caudally to the pericardium and molds to its surface. the thymus is referred to as a lymphoepithelial organ and hence is composed of epithelial and lymphoid tissue. formed from the endoderm of the third pharyngeal pouch in the fetus, the thymic epithelium is infiltrated by blood vessels from the surrounding mesoderm, resulting in the development of the thymic epithelial reticulum. the lymphocyte population consists of bone marrow-derived progenitor cells, which fill spaces within the epithelial network. a connective tissue capsule surrounds the thymus, and attached thin septa subdivide the tissue into partially separated lobules. each report, in which numerous cats from a subregion of the endemic area in the united states survived infection with an organism with greater than % homology to cytauxzoon felis, suggests the emergence of a less virulent strain. felv is an oncogenic, immunosuppressive lentivirus that causes hematologic abnormalities of widely varying types and severity. manifestations of disease caused by felv infection vary depending on dose, viral genetics, and host factors, but normal hematopoiesis is probably suppressed to some degree in all cases. felv infects hematopoietic precursor cells soon after the animal is exposed and continues to replicate in hematopoietic and lymphatic tissue of animals that remain persistently viremic. the virus disrupts normal hematopoiesis by inducing genetic mutations, by other direct effects of the virus on infected hematopoietic cells, or by an altered host immune system. hematologic changes include dysmyelopoiesis with resultant cytopenias or abnormal cell morphologic features, and neoplastic transformation of hematopoietic cells (leukemia). a notable form of dysplasia is the presence of macrocytic erythrocytes (macrocytes) and metarubricytosis in the absence of erythrocyte regeneration (inappropriate metarubricytosis). the relatively uncommon subgroup c viruses cause erythroid hypoplasia, probably because of infection of early-stage erythroid precursors. felv may be detected in megakaryocytes and platelets in infected cats and may result in platelet abnormalities, including thrombocytopenia, thrombocytosis, increased platelet size, and decreased function. proposed mechanisms of felv-induced thrombocytopenia include direct cytopathic effects, myelophthisis, and immunemediated destruction. platelet life span and function have been shown to be decreased in felv-positive cats. persistently viremic cats are immunosuppressed and are prone to developing other diseases, including infectious diseases, bone marrow disorders, and lymphoma. cbc abnormalities attributed to felv infection include various cytopenias, especially nonregenerative anemia, which may be persistent or cyclical. regenerative anemia may also occur with felv infection, often because of coinfection with m. haemofelis. hematopoietic cell dysplasia or neoplasia may also be evident. grossly, infected cats are often pale, but other lesions are dependent upon the presence of other cytopenias or concurrent disease. microscopically, the bone marrow is hypocellular, normocellular, or hypercellular. there may be erythroid hypoplasia, erythroid hyperplasia with maturation arrest, or acute leukemia. feline immunodeficiency virus. feline immunodeficiency virus (fiv), another feline lentivirus, causes anemia in a minority of infected cats. immunosuppressive effects of fiv from thymic depletion are discussed elsewhere. it is generally accepted that anemia does not result directly from fiv infection but instead develops because of concurrent disease such as coinfection with felv or hemotropic mycoplasma, other infection, or malignancy. the severity and type of anemia in fiv-infected cats depends on the other specific disease processes involved. the thymus, spleen, lymph nodes, and lymph nodules, including malt, are classified as part of both the lymphoid and immune systems. the lymphoid system (also known as lymphatic system in some texts) is broadly categorized into primary and secondary lymphoid organs. the main primary lymphoid organs include thymus, bone marrow, and bursa of fabricius in birds and are the sites at .e chapter bone marrow, blood cells, and the lymphoid/lymphatic system because the thymus involutes after sexual maturity, evaluation of whether it is smaller than normal is difficult to discern unless the change is extreme or age-matched control animals are available. before sexual maturity the thymus is easily identified as a lobular white to gray organ with a thin capsule. after sexual maturity the gland is often grossly indistinguishable from adipose connective tissue within the cranial mediastinum, although microscopic remnants may remain. an extremely small thymus in a neonatal animal should be considered abnormal and may indicate a primary immunodeficiency or secondary lymphoid depletion caused by extreme stress, often due to infectious diseases. enlargement of the thymus is most often due to neoplasia. serial sectioning of the thymus allows for gross identification of neoplasms, cysts, or hematomas. spleens vary in size within the same species and among the different species of domestic animals. the spleen can be enlarged (splenomegaly), normal in size, or small (atrophy), and the surface can be smooth, wrinkled, or nodular. the appearance of the cut surface of the spleen in normal animals depends on the amount of stroma (e.g., trabeculae are prominent in ruminants); the size and visibility of the white pulp, which reflects the amount of lymphoid tissue; and whether the red pulp is congested with blood. during an autopsy (syn: necropsy), the spleen is dissected free and checked for torsion of the gastrosplenic ligament (in nonruminants). the spleen is then sliced transversely at approximately -mm intervals (serial sectioning), and the cut surfaces are checked for lesions. specimens are taken for tests that require fresh tissue (e.g., bacteriologic and virologic examinations), and the remaining cross-sections are placed in fixative ( % buffered neutral formalin). a diffusely enlarged spleen should be serially sectioned to determine if the splenomegaly is due to congestion. the cut surface of severely congested spleens is red to bluish-black and exudes blood (bloody spleens), whereas cut surfaces of noncongested enlarged spleens ooze little blood (meaty spleens) and the color depends on how much of the normal parenchyma is replaced by inflammatory cells, stored materials, or neoplastic cells (see splenomegaly and table - ). the spleen may be measured and weighed, but because of the wide variation in the dimensions and weight of normal spleens and the amount of blood stored, this information is difficult to interpret. it is essential that spleens with one or more nodules also be serially sectioned and the nodules evaluated for size, shape, and consistency. nodules may be dark red and ooze blood on cut surface, white-tan with a more firm texture, or a mixture of both. multiple wedge sections that include the interface between a nodule and the adjacent nonmass spleen should be collected, because the center of the nodules often consists only of hemorrhage and necrosis, and neoplasms may be missed. the color of the capsular surface of the spleen also varies among species of domestic animals and depends on the opacity or translucence of the splenic capsule. the degree of opacity of the capsule is a function of its thickness and the amount of collagen. the splenic capsules of horses and ruminants are thick and usually appear gray because the color of the red pulp is not visible through the capsule. in the pig, dog, and cat, the splenic capsule is thin, and thus the surface of the spleen is red. the tenseness of the capsule depends on how much the splenic parenchyma is distended; storage spleens devoid of blood usually have a wrinkled surface. irregular contraction of storage spleens is common, especially in dogs, and consists of nonuniform areas of congestion with intermingled contracted and wrinkled regions. lymph nodes should be dissected free of fat and connective tissue, and any firm attachment to adjacent tissues should be noted because these attachments may indicate neoplastic infiltration through the capsule. gross examination includes evaluating size (measurement or weight), shape, and whether the capsule is intact. the cut surface is examined for the presence of bulging tissue, edema, congestion, exudate, discoloration (see pigmentation), obscuration of the normal architecture, and masses such as abscesses, granulomas, and discrete neoplasms. cytologic evaluation of superficial lymph nodes through fine-needle aspirates provides excellent cellular detail and often yields a diagnosis. however, diagnosis of certain diseases (including lymphomas for complete world health organization [who] classification) requires architectural assessments, and therefore cytologic or small histologic samples are not sufficient. tru-cut biopsies are not ideal, but a -mm tru-cut needle may provide adequate tissue. the surgeon or pathologist must handle lymph nodes carefully to minimize artifacts. compression (e.g., squeezing with forceps) may cause crush artifacts, usually resulting in nuclear "streaming." immediately after removal, imprints/impression smears should be prepared and then kept away from formalin fumes. formalin fixation (in this case by formaldehyde fumes) destroys the differential staining seen with romanowsky stains such as wright's and giemsa and results in diffuse blue staining. prompt transfer of biopsy or postmortem specimens into fixative is crucial because delayed fixation can lead to numerous artifacts, including an artificial decrease in mitotic index (up to % reduction with a more than -hour delay in fixation); this reduction can alter tumor classification and grade. the current recommendation for the duration of formalin fixation is to hours; complete fixation of -to -mm thick tissues is likely to be achieved after to hours. both underfixation and overfixation may lead to difficulties with antigen retrieval for immunohistochemistry, though underfixation is considered the more common and serious problem. thinly slicing some nodes may be difficult, and allowing the node to fix for hour before slicing may help. some pathologists prefer not to incise very small lymph nodes to avoid compression artifacts, but instead nick the capsule to allow formalin penetration. however, fixation of unincised lymph nodes can also cause compression artifacts because the fibrous capsule contracts in the fixative. once fixed, nodes should be cut in uniformly thick cross section to include both the cortex and medulla. transverse sections are usually sufficiently small to allow the entire cross section of most lymph nodes to fit on one microscopic slide, which facilitates histologic interpretation. the longitudinal plane is preferred for porcine lymph nodes because the location and amount of cortex and medulla vary at different sites in transverse sections. molecules) but not self-antigens are permitted to mature by a process called positive selection. cells that do not recognize mhc molecules are removed by apoptosis. those t lymphocytes that recognize both mhc molecules and self-antigens are removed by macrophages at the corticomedullary junction, a process called negative selection. because of the rigid differentiation requirements attributable to mhc restriction and tolerance (positive and negative selection, respectively), only a small fraction (< %) of the developing t lymphocytes that arrive at the thymus from the bone marrow survive. mature naïve t lymphocytes exit the thymus through postcapillary venules in the corticomedullary region, enter the circulation, and recirculate through secondary lymphoid tissues, primarily located in the paracortex of lymph nodes and the periarteriolar sheaths of the spleen. in these specialized sites, the mature naïve t lymphocytes are activated upon exposure to their specific antigens and undergo additional phases of development to differentiate into effector and memory cells. the thymus attains its maximal mass relative to body weight at birth and involutes after sexual maturity; the rate of involution may vary among domestic species. the lymphoid and epithelial components are gradually replaced by loose connective tissue and fat, although remnants remain histologically, even in aged animals. lobule is composed of a central medulla and surrounding cortex (fig. - ) . the thymic cortex consists mainly of an epithelial reticulum and lymphocytes ( fig. - ) . the stellate cells of the epithelial reticulum have elongate branching cytoplasmic processes that connect to adjacent epithelial cells through desmosomes, thus forming a supportive network (cytoreticulum). the lymphoid component is composed of differentiating lymphocytes derived from progenitor (also known as precursor) t lymphocytes in the bone marrow. the medulla is composed of similar epithelial reticular cells, many of which are much larger than those in the cortex and have a more obvious epithelial structure. some of the epithelial reticular cells form thymic corpuscles, also called hassall's corpuscles, which are distinctive keratinized epithelial structures (see fig. - ). interdigitating dendritic cells (dcs) are also present within the medulla, but there are far fewer lymphocytes than in the cortex. the progenitor t lymphocytes released from the bone marrow into the blood enter the thymus in the subcapsular zone of the cortex and begin the differentiation and selection processes, developing into mature naïve t lymphocytes as they traverse the thymic cortex to the medulla. in the cortex, t lymphocytes that recognize self-molecules (major histocompatibility complex [mhc] the responses of the thymus to injury and causes are listed in boxes - and - . the most common change is lymphoid atrophy caused by physical and physiologic stresses, toxins, drugs, and viral infections. atrophy. because the thymus does not contain any lymphopoietic tissue, it depends on the bone marrow for the supply of progenitor t lymphocytes. thus thymic lymphoid atrophy can be the result of either an inadequate supply of lymphocytes from the bone marrow or lysis of lymphocytes (lymphocytolysis) in the thymus. thymic aims to define the terms used in this chapter. the term splenic sinusoid is used to describe a vascular structure present in the sinusal spleen (also known as sinusoidal spleen); dogs are the only domestic animal with true splenic sinusoids. the term red pulp vascular spaces is used (as opposed to "sinus") to describe the vascular spaces in the red pulp of both the nonsinusal and nonsinusoidal spleens of all domestic animals. the other terms used here include marginal sinus, marginal zone, periarteriolar lymphoid sheath (pals), periarteriolar macrophage sheath (pams), and splenic lymphoid follicles. the spleen is located in the left cranial hypogastric region of the abdomen, where it is typically suspended in the gastrosplenic ligament between the diaphragm, stomach, and the body wall. the exception is in domestic ruminants, where it is closely adhered to the left dorsolateral aspect of the rumen. the gross shape and size of the spleen vary markedly among domestic animals, but generally it is a flattened, elongated organ. some species, notably birds, demonstrate seasonal variation in splenic shape and size. the spleen is covered by a thick capsule composed of smooth muscle and elastic fibers, from which numerous intertwining fibromuscular trabeculae extend into the parenchyma. these trabeculae and reticular cells form a spongelike supportive matrix for the parenchyma of the mammalian spleen in all domestic species. in cattle and horses the three muscular layers of the capsule lie perpendicular to each other, forming a capsule thicker than that of carnivores. carnivores, small ruminants, and pigs have interwoven smooth muscle within the splenic capsule, and pigs also have abundant elastic fibers within the capsule. the spleen differs from many other organs in the organization of its parenchyma. instead of a cortex and medulla, the spleen is divided into two distinct structural and functional components: the red pulp and white pulp (fig. - ) . with hematoxylin and eosin (h&e) staining, red pulp appears red-pink because of the abundance of red blood cells, whereas white pulp appears blue-purple because of the heavy concentration of lymphocytes. the white pulp consists of splenic follicles, populated by b lymphocytes; the pals, inhabited by t lymphocytes; and the marginal zone at the periphery of follicles. macrophages, antigen-presenting cells, and trafficking b and t lymphocytes populate the marginal zone. the radial arteries, branches of the central artery (also known as central arteriole), and capillaries from both red and white pulp drain into the marginal sinus of the marginal zone, although the latter has not been shown to be the case in all species to the same degree (e.g., the cat has a small marginal sinus but a well-developed pams) (figs. - and - ). the red pulp consists of cells of the monocyte-macrophage system, pams, sinusoids (dogs, rats, and human beings only), red pulp vascular spaces, and associated stromal elements such as reticular cells, fibroblasts, and trabecular myocytes. the labyrinth of the splenic red pulp vascular spaces serves as both a functional and physical filter for circulating blood cells. the blood circulation of the spleen is particularly suited to enable its functions, namely, ( ) filtering and clearing the blood of atrophy must be differentiated from involution, which normally begins at sexual maturity. this distinction is difficult to make, unless the change is extreme or age-matched control animals are available for comparison. inflammation. inflammation of the thymus is rare. neutrophils and macrophages are often present within keratinized hassall's corpuscles during involution and should not be mistaken for a true thymitis. thymitis has been reported in salmon poisoning disease of dogs (see chapter ), epizootic bovine abortion (see chapter ), and in pigs infected with porcine circovirus type (pcv ). necrosis and secondary infiltrates of neutrophils and macrophages may be seen in other infectious diseases (e.g., equine herpesvirus [ehv- ]). the main portal of entry to the thymus is hematogenous. portals of entry used by microorganisms and other agents and substances to access the lymphatic system are summarized in box - . these portals include the blood vessels (hematogenous spread by microorganisms free in the plasma or within circulating leukocytes or erythrocytes), afferent lymphatic vessels (lymphatic spread), direct penetration, or through m (for "microfold") cells and dcs in malt. defense mechanisms used by the thymus to protect itself against microorganisms and other agents are the innate and adaptive immune responses, discussed in chapters , , and . viruses, bacteria, and particles arriving in the lymph and blood interact with cells of the monocyte-macrophage system through phagocytosis and antigen processing and presentation. hyperplasia of the macrophages often occurs concurrently. antigen processing and presentation are followed by an immune response resulting in proliferation of b lymphocytes, plasma cells, and the subsequent production of antibody; proliferation of t lymphocytes may also occur. the relationships between anatomic structures and the different functions of the spleen are complicated. there are also anatomic differences among domestic animal species and confusion about the correct and up-to-date terminology. the following brief discussion malt, mucosa-associated lymphoid tissue. there are numerous synonyms and misuse of terms within the literature, which have contributed to the confusion over terminology for red pulp vascular spaces. these terms include reticular space, red pulp, splenic cords, sinuses, red pulp sinuses, sinus spaces, pulp spaces, mesh space of the spleen, reticular cell-lined meshwork, interstices of the reticulum network, bloodfilled reticular meshwork of the red pulp, chordal spaces, splenic cords, and cords of billroth. the latter two terms are defined as the red pulp between the sinusoids, which most domestic animals do not have (except the dog). therefore the term red pulp vascular spaces is more appropriate. as a result of this pattern of blood flow, macrophages in the marginal sinus have the first opportunity to phagocytize antigens, bacteria, particles, and other material before macrophages in the sinusoids (in the dog) or in the pams and red pulp vascular spaces (all other domestic animals). in the dog the marginal sinus drains into the sinusoids, but in other domestic animals it drains into the red pulp vascular spaces. the central arteries leave the white pulp, enter the red pulp, and branch into smaller penicillar arterioles. each arteriole is surrounded by a sheath of macrophages known as periarteriolar macrophage sheaths (pams, previously known as ellipsoids), which are notably prominent in pigs, dogs, and cats. in horses, cattle, pigs, and cats the terminal branches of the penicillar arterioles empty into the red pulp vascular spaces lined by reticular cells. because the red pulp vascular spaces are not lined by endothelium, this type of circulation is known as an open system. this system is in contrast to the sinusoidal spleen of the dog (also of the rat and human beings), where the branches of the central artery of the white pulp and vessels from the marginal sinus enter into the sinusoids, which are lined by a discontinuous endothelium, and these empty into splenic venules. this type of circulation is known as a closed system because the blood flow is through blood vessels (arterioles, capillaries, sinusoids, and venules), all of which are lined by endothelium. although circulation in the red pulp is anatomically open in nonsinusoidal spleens, under certain conditions (e.g., during splenic contraction) the circulation is functionally closed, and the blood in the red pulp is particulate matter and senescent cells; ( ) transporting recirculating lymphocytes and naïve b and t lymphocytes to the follicle and pals, respectively, to fulfill their specific immune functions; and ( ) storage of blood in some domestic animal species (dog, cat, and horse) (fig. - ) . phagocytosis is particularly effective in the spleen because blood flows through areas within the red pulp that are populated with increased concentrations of macrophages, namely, within the marginal sinuses, in cuffs around the penicillar arteries (pams), diffusely on the reticular walls of the red pulp vascular spaces, and along the sinusoids in dogs. trafficking of naïve and recirculating lymphocytes is facilitated by the proximity of the marginal sinus to the follicular germinal centers and pals. maps of the vascular blood flow in sinusoidal and nonsinusoidal spleens are illustrated in figures - to - . the celiac artery is the major branch of the abdominal aorta from which the splenic artery arises. the splenic artery enters the splenic capsule at the hilus, where it branches and enters the fibromuscular trabeculae as trabecular arteries to supply the splenic parenchyma. trabecular arteries become the central arteries of the white pulp and are surrounded by cuffs of t lymphocytes forming the pals. the splenic follicles, populated by b lymphocytes, are eccentrically embedded within or just adjacent to the pals. the central arteries send branches-the radial arteries-to supply the marginal sinus surrounding the splenic follicles. thus the cells at the circumferences of the follicles are brought into intimate contact with blood-borne antigens and trafficking b and t lymphocytes in the marginal sinus. diverted into "channels" lined by reticular cells. because the dog has both sinusoids and red pulp vascular spaces, it has both open and closed splenic circulations, which may allow for both fast and slow flows of blood depending on the physiologic need of the animal. blood flowing through the sinusoids or red pulp vascular spaces is under the surveillance of macrophages. in dogs the pseudopodia of these perisinusoidal macrophages project into the sinusoidal lumen through the spaces in the discontinuous endothelium. in all domestic animals, blood in the red pulp vascular spaces is under surveillance of macrophages attached to the reticular walls. blood from the red pulp vascular spaces and sinusoids then drains into the splenic venules, splenic veins, and ultimately into the portal vein, which empties into the liver. the spleen filters blood and removes foreign particles, bacteria, and erythrocytes that are senescent, have structural membrane abnormalities, or are infected with hemotropic parasites. as a secondary lymphoid organ, its immunologic functions include the activation of macrophages to process and present antigen, the proliferation of b lymphocytes and production of antibody and biologic molecules, and the interaction of t lymphocytes and antigens. in some species the spleen stores significant quantities of blood (box - ). the functions of the spleen are best considered on the basis of the two main components of the spleen: the red and white pulp and the anatomic systems contained within them (monocyte-macrophage system, red pulp vascular spaces, and hematopoiesis in the red pulp, and the b and t lymphocyte systems within the white pulp). monocyte-macrophage system. within the red pulp, macrophages are located in the marginal sinus, pams, and attached to the reticular walls of the red pulp vascular spaces. in the dog, macrophages are also located perisinusoidally. the supportive reticular network of the red pulp vascular spaces is composed of a fine meshwork of reticular fibers made of type iii collagen, on which macrophages are dispersed. exactly in which of these concentrations of macrophages phagocytosis of blood-borne particles takes place depends upon ( ) the sequence in which they are exposed to the incoming blood, ( ) the concentration of macrophages in these areas (e.g., the cat marginal sinus is small and thus not a major site of clearance; there is a compensatory increase in pams for phagocytosis), and ( ) the functions of the macrophages. some of the macrophages in the marginal sinus and marginal zone are responsible for phagocytosis of particulate matter and others for the trapping and ingestion of antigens and antigen-antibody complexes. macrophages responsible for phagocytosis of blood-borne foreign material (fig. - ) , bacteria, and senescent and/or damaged erythrocytes (e.g., as seen in immune-mediated anemias and infections with hemotropic parasites) are also found in the red pulp. in the dog, sinusoidal macrophages remove entire erythrocytes (erythrophagocytosis), as well as portions of an erythrocyte's membrane and cytoplasmic inclusions, such as nuclear remnants like heinz bodies, by a process called pitting. as such, the presence of large numbers of nuclear remnants in erythrocytes in canine blood smears may indicate malfunction of the sinusoidal system. the normal rate of removal of senescent erythrocytes from the circulating blood does not cause an increase in size of the spleen; however, splenomegaly can be observed when large numbers of defective erythrocytes must be removed, as in cases of severe acute hemolytic anemia. nonsinusoidal spleens lack the fenestrated endothelium and perisinusoidal macrophages of canine sinusoids that allow for slow processing of red blood cells to determine which are to be returned to the equine, canine, and feline spleens all have considerable storage and contractile capacity because of their muscular capsule, increased numbers of trabeculae, and the relatively small amount of splenic parenchyma devoted to white pulp. the storage capacity in dogs and horses is remarkable: it has been claimed that the canine spleen can store one-third of the dog's erythrocytes while the animal sleeps and the equine spleen holds one-half of the animal's circulating red cell mass (which is considered advantageous because it reduces the viscosity of the circulating blood). storage spleens expand and contract quickly under the influence of the autonomic nervous system, via sympathetic and vagal fibers in the trabeculae and reticular walls of the red pulp vascular spaces and other circulatory disruptions, such as hypovolemic and/or cardiogenic shock. thus storage spleens may be either grossly enlarged and congested or small with a wrinkled surface and a dry parenchyma depending on whether the spleen is congested from stored blood or shrunken from contraction (see uniform splenomegaly and small spleens). hematopoietic tissue. in the developing fetus the liver is the primary site of hematopoiesis, with the spleen making a minor contribution. shortly before or after birth, hematopoiesis ceases in the liver and spleen, and the bone marrow becomes the primary hematopoietic organ. under certain conditions, such as severe demand due to prolonged anemia, splenic hematopoiesis can be reactivated; this outcome is called extramedullary hematopoiesis (emh). studies have indicated that splenic emh in dogs and cats most commonly occurs with degenerative or inflammatory circulation, pitted, or phagocytized. instead, the macrophages of the red pulp perform these functions, and phagocytized cells remain in the red pulp vascular spaces. the location of the primary sites of pitting in nonsinusoidal spleens is unclear, but it is likely that most erythrophagocytosis takes place in the red pulp vascular spaces. the cat's spleen is deficient in pitting, and removal of heinz bodies is slow; however, some erythrophagocytosis does occur in the marginal sinus. the macrophages of the sinusoids, marginal sinus, and red pulp vascular spaces are of bone marrow origin. from the bone marrow these cells circulate in the blood as monocytes and migrate into the spleen. some macrophages are replenished by local proliferation. for example, after phagocytizing large amounts of material from the blood, the macrophages of the pams migrate through the wall of the cuff into the adjacent red pulp, denuding the pams of macrophages. after hours, local residual macrophages have proliferated to repopulate the pams. the fixed macrophages elsewhere in the body, namely, those in connective tissue, lymph nodes (sinus histiocytes), liver (kupffer cells), lung (pulmonary intravascular macrophages and pulmonary alveolar macrophages), and brain (resident and perivascular microglial cells), are also derived from bone marrow (see chapters , , , and ) . storage or defense spleens. spleens are also classified as either storage or defense spleens, based on whether or not they can store significant volumes of blood. the ability to store blood in the spleen depends on the fibromuscular composition of the splenic capsule and trabeculae. splenic capsules and trabeculae with a low percentage of smooth muscle and elastic fibers cannot expand and contract and are designated as defense spleens. these are found in rabbits and human beings. the spleens of other domestic animal species have distention of the red pulp from stored blood separates the foci of white pulp (pals and lymphoid follicles), making white pulp appear sparser. splenic white pulp is organized around central arteries in the form of pals, which are populated primarily by t lymphocytes (see . primary splenic follicles are located eccentrically in pals and are primarily composed of b lymphocytes. when exposed to antigen, the splenic lymphoid follicles develop germinal centers (see lymphoid/ lymphatic system, lymph nodes, function) . macrophages in the white pulp follicles remove apoptotic b lymphocytes not selected for expansion because of low binding affinity for antigen. failure of these macrophages to phagocytize has been experimentally correlated with decreased production of growth factors like tgf-β and increased production of inflammatory cytokines that predispose the animal to autoimmune conditions. the marginal zone surrounds the marginal sinus at the interface of the white and red pulp and consists of macrophages, dcs, and t and b lymphocytes. the blood supply of the marginal sinus is from conditions (e.g., hematomas, thrombosis) and may occur without concomitant hematologic disease (see uniform splenomegaly with a firm consistency). it is also found in splenic nodular hyperplasia (see splenic nodules with a firm consistency). in some species, such as the mouse, emh is a normal function of the adult spleen and not necessarily a response to disease or hypoxic challenge. the splenic red pulp also contains large numbers of monocytes, which function as a reserve for generating tissue macrophages in response to ongoing tissue inflammation in the body. white pulp. white pulp consists of pals, each with a splenic lymphoid follicle surrounded by a marginal zone. normally these foci of white pulp are so small that they may not be visible on gross examination of a cross section of the spleen. however, if nodules are enlarged either by lymphoid hyperplasia, amyloid deposits, or a neoplastic process (e.g., lymphoma), they can become grossly visible on the cut surface, initially as . -to . -mm white circular foci scattered through the red pulp. in animals with storage spleens, the the splenic artery enters at the hilus and divides into arteries, which enter the trabeculae. when a trabecular artery emerges from a trabecula it becomes the central artery and is encased in a periarteriolar lymphoid sheath (pals), which is composed of t lymphocytes. it then enters the splenic follicle and gives off branches-the radial arteries, which supply the marginal sinus and marginal zone. the central artery emerges from the splenic follicle to enter the red pulp and branches into the penicillary arterioles, which are enclosed in a cuff of macrophages-the periarteriolar macrophage sheath (pams). the emerging penicillar arteries branch into arterioles and capillaries that supply the red pulp vascular spaces (see fig. - ) . the red pulp vascular spaces also receive blood from capillaries draining from the marginal sinus and drain into the splenic venules and then into the trabecular veins and splenic vein. b, sinusoidal spleen, dog. the blood flow is essentially the same but with the additional feature that arterioles from the marginal sinus drain into the sinusoids and some blood from the red pulp vascular space passes through slits in the sinusoidal wall to enter the sinusoid (see fig. - ) . this is the site of pitting and erythrophagocytosis. note that the major flow in a is sequentially past concentrations of macrophages in the marginal sinus, pams, and red pulp vascular spaces. in b there is the additional route from the marginal zone into the sinusoids. dog. red pulp vascular spaces macrophages in the marginal zone are phenotypically distinct from those in the red pulp. the red pulp macrophages function primarily to filter the blood by phagocytizing particles and by removing senescent or infected erythrocytes and pathogenic bacteria and fungi. marginal zone macrophages are divided into two types based on their location and the type of cell surface receptors they possess. the first group is positioned toward the periphery of the marginal zone, whereas the second group, the marginal metallophilic macrophages (so called for their silver staining positivity), is at the inner margin of the marginal zone closer to the splenic follicle and pals. it has been difficult to generate mammalian models that eliminate one of the two classes of marginal zone macrophages, so the degree to which one group specializes in a particular function is not clear. some marginal zone macrophages actively phagocytize particulate matter or bacteria (e.g., septicemias caused by streptococcus pneumoniae, listeria monocytogenes, campylobacter jejuni, or bacillus anthracis) in the blood (see fig. - ) . they also play a similar role in limiting the spread of viral infections. other marginal zone macrophages phagocytize and process antigens. thus macrophages of the marginal zone serve to bridge the innate and adaptive immune responses by secreting inflammatory cytokines to activate other immune cells and providing receptor-based activation of marginal zone lymphocytes. studies have shown that a loss of marginal zone macrophages coincides with decreased antigen trapping by resident b lymphocytes of the marginal zone and consequently a decrease in the early igm response to antigens. the responses of the spleen to injury (box - ) include acute inflammation, hyperplasia of the monocyte-macrophage system, hyperplasia of lymphoid tissues, atrophy of lymphoid tissues, storage of blood or contraction to expel reserve blood, and neoplasia. these responses are also best considered on the basis of the two main components of the spleen, the red and white pulp, and the anatomic systems associated with each. monocyte-macrophage system. the distribution and function of macrophages in the spleen is described earlier in the section on structure and function. these interactions are complex, and their relationships to both innate and adaptive immunity are areas of intense study (see also chapter ). to facilitate filtering, all of the blood in the body passes through the spleen at least once a day, and % of the cardiac output goes to the spleen. in dogs, blood flow and transit time depend on whether the spleen is contracted or distended; blood flow is slower in the distended spleen. the extent to which macrophages of the monocyte-macrophage system phagocytize particles depends to a large degree on the sequence in which they receive blood. in most species, macrophages of the marginal sinus are the first to receive blood, and consequently phagocytized particles and bacteria tend to be more concentrated here initially. however, there are differences among domestic animal species; the cat, for instance, has a comparatively small marginal sinus, and thus the pams play a larger role in phagocytosis. the spleen is able to mount a strong response to blood-borne pathogens, which has been demonstrated in several studies. the blood of immunized rabbits injected intravenously with pneumococci cleared % of those bacteria within minutes and % within an hour. the blood of dogs injected with billion pneumococci per pound of body weight into the splenic artery was cleared of all bacteria in minutes. after splenectomy, blood-borne the radial branches of the central artery, and it serves as the portal of entry into the spleen for recirculating b and t lymphocytes. from here, t lymphocytes migrate to the pals and b lymphocytes to the germinal centers. macrophages in the marginal zone capture bloodborne antigens, process them, and present them to the lymphocytes. senescent erythrocytes damaged erythrocytes (e.g., immune-mediated anemias) parasitized erythrocytes (e.g., hemotropic parasites) storage of blood (in storage spleens) extramedullary hematopoiesis severe demand (e.g., anemias) degenerative/inflammatory conditions without concomitant hematologic disease incidental (e.g., within nodules of hyperplasia) monocytes within splenic cords reserve for generating tissue macrophages in response to inflammation homing of circulating lymphocytes in the blood phagocytosis and processing of antigen macrophage activation inflammation, which may be diffuse or multifocal/focal (e.g., blastomycosis and tuberculosis, respectively). red pulp vascular spaces. the main response to injury of the red pulp vascular spaces is congestion (see uniform splenomegaly with a bloody consistency), as well as the storage of blood or contraction to expel reserve blood. white pulp. the responses to injury within the white pulp are most pronounced in the splenic lymphoid follicles. lymphoid follicular hyperplasia is a response to antigenic stimuli and results in the formation of secondary follicles; marked hyperplasia may be grossly evident. hyperplasia of splenic lymphoid follicles follows a similar sequence of events and morphologic changes as seen in other secondary lymphoid organs and is discussed in more detail in lymphoid/lymphatic system, lymph nodes, dysfunction/responses to injury. similarly, atrophy of splenic lymphoid follicles has similar causes as lymphoid atrophy in other lymphoid organs (see box - ). briefly, atrophy occurs in response to lack of antigenic stimulation (e.g., from regression after antigenic stimulation has ceased), from the effects of toxins, antineoplastic chemotherapeutic agents, microorganisms, radiation, malnutrition, wasting/cachectic diseases, or aging, or when the bone marrow and thymus fail to supply adequate numbers of b and t lymphocytes, respectively. the follicles are depleted of lymphocytes, and with time, germinal centers and follicles disappear. the amount of the total lymphoid tissue is reduced, and the spleen may be smaller. the response to injury of the monocyte-macrophage system in the marginal sinus and marginal zone is also phagocytosis and proliferation. capsule and trabeculae. lesions in the capsule and trabeculae are uncommon and include splenic capsulitis secondary to peritonitis, and complete or partial rupture of the splenic capsule, usually due to trauma. the two main portals of entry to the spleen for infectious agents are hematogenous spread and direct penetration. the splenic capsule is thick, and thus direct penetration is less common. inflammation from an adjacent peritonitis is unlikely to penetrate the capsule into the splenic parenchyma. cattle with traumatic reticulitis may have foreign objects migrate into the ventral extremity of the spleen, causing a splenic abscess. splenic abscesses also develop secondary to perforation of the gastric wall in horses, due to foreign body penetration, gastric ulcers, or gastric inflammation. portals of entry used by microorganisms and other agents and substances to access the lymphoid/lymphatic system are summarized in box - . defense mechanisms used by the spleen to protect itself against microorganisms and other agents are the innate and adaptive immune responses, discussed in chapters , , and . other defense mechanisms are structural in nature to protect against external trauma and include the thick fibrous capsule of the spleen. lymph nodes are soft, pale tan, round, oval or reniform organs with a complex three-dimensional structure. on gross examination of a cross section of lymph nodes, two main areas are visible: an outer rim of cortex and an inner medulla (fig. - ) . to understand the pathologic response of the lymph node, it is important to consider its anatomic components and their relationship with antigen processing (fig. - ) : organisms multiply rapidly and may disseminate widely in the body to cause an overwhelming postsplenectomy infection. studies have also shown that the phagocytic function of the spleen is critical in the control of plasmodium (causative agent of malaria) in human beings and babesiosis in cattle. if the number of pathogenic bacteria in the circulation exceeds the capacity of the splenic macrophages, as in cases of severe septicemia, it may result in acute splenic congestion (see uniform splenomegaly with a bloody consistency). this may be followed by inflammation with areas of necrosis, fibrin deposition, and infiltration by neutrophils in bacteremias of pyogenic bacteria. the marginal zone can be the initial site of response to blood-borne antigens and bacteria delivered by the radial branches of the central arteries to the marginal sinus. similar to the response of the red pulp vascular spaces, the marginal zone can become congested and with time (only hours with highly pathogenic organisms) may contain aggregates of neutrophils and macrophages. histologically, the congestion and inflammation form a complete or partial concentric ring around the circumference of the splenic nodule (see anthrax). hyperplasia of the red pulp macrophages is also seen in chronic hemolytic diseases, because there is a prolonged need for phagocytosis of erythrocytes. similarly, chronic splenic congestion, usually the result of portal or splenic vein hypertension, can lead to proliferation of the macrophages present on the walls of the red pulp vascular spaces and results in thickening of the reticular walls between the red pulp vascular spaces. macrophages in the red pulp also proliferate in response to fungi and facultative intracellular pathogens (e.g., mycobacterium bovis) arriving hematogenously to the spleen. the number of red pulp macrophages may be augmented by monocytes recruited from the blood to form granulomatous acute inflammation with fibrin and necrosis abscesses, microabscesses granulomatous inflammation (diffuse, multifocal, focal) fibroblastic reticular cells and fibers. besides providing structural support, this reticulum helps form a substratum for the migration of lymphocytes and antigen-presenting cells to the follicles and facilitates the interaction with b and t lymphocytes. cortex. the outer/superficial cortex contains the lymphoid follicles (also referred to as lymphoid nodules) (see . the follicles are designated as primary if they consist mainly of small lymphocytes: mature naïve b lymphocytes expressing receptors for specific antigens exit the bone marrow and circulate through the bloodstream, lymphatic vessels, and secondary lymphoid tissues. on their arrival at lymph nodes, b lymphocytes exit through hevs in the paracortex and home to a primary follicle (which also contains follicular dcs in addition to the resting b lymphocytes). lymphoid follicles with germinal centers are designated as secondary follicles: b lymphocytes that recognize the antigen for which they are expressing receptors are activated and proliferate to form the secondary lymphoid follicles characterized by prominent germinal centers. germinal centers are areas with a specialized • stroma-capsule, trabeculae, and reticulum • cortex-"superficial" or "outer" cortex (lymphoid follicles, b lymphocytes) • paracortex-"deep" or "inner" cortex (t lymphocytes) • medulla-medullary sinuses and medullary cords • blood vessels-arteries, arterioles, high endothelial venules (hevs), efferent veins • lymphatic vessels-lymphatic afferent and efferent vessels; lymphatic sinuses (subcapsular, trabecular, and medullary) • monocyte-macrophage system-sinus histiocytes stroma. the lymph node is enclosed by a fibrous capsule penetrated by multiple afferent lymphatic vessels, which empty into the subcapsular sinus (see also . at the hilus, efferent lymphatic vessels and veins exit, and arteries enter the node. fibrous trabeculae extend from the capsule into the parenchyma to provide support to the node and to house vessels and nerves. the lymph node is also supported by a meshwork of medulla. the medulla is composed of medullary cords and medullary sinuses (see . the medullary cords contain macrophages, lymphocytes, and plasma cells. in a stimulated node the cords become filled with antibody-secreting plasma cells. the medullary sinuses are lined by fibroblastic reticular cells and contain macrophages ("sinus histiocytes"), which cling to reticular fibers crossing the lumen of the sinus. these macrophages phagocytize foreign material, cellular debris, and bacteria from the incoming lymph. vasculature: blood vessels, lymphatic vessels, and lymphatic sinuses. the blood vessels of the lymph node include arteries, arterioles, veins, and postcapillary venules (hevs) lined by specialized cuboidal endothelium (see figs. - and - ) . microenvironment that support the proliferation and further development of b lymphocytes to increase their antigen and functional capacity (see lymphoid/lymphatic system, lymph nodes, function). the mantle cell zone surrounds the germinal center and consists of small inactive mature naïve b lymphocytes and a smaller population of t lymphocytes (approximately %). paracortex. the diffuse lymphoid tissue of the paracortex (also referred to as the deep or inner cortex) consists mainly of t lymphocytes, as well as macrophages and dcs (see . this region contains the hevs through which b and t lymphocytes migrate from the blood into the lymphoid follicles and paracortex, respectively. t and b lymphocytes may also enter the lymph node via the lymphatic vessels. and larger molecules, small molecules and free antigens, and antigen within dcs. it is helpful to consider the paths taken by particles, molecules, antigens, and cells arriving at a lymph node. the following account describes the journey of an antigen as it enters a lymph node to trigger an immune response. antigen in the lymph arriving in the afferent lymphatic vessels empties into the subcapsular sinus. hydrostatic pressure here is low, and reticular fibers crossing the sinus impede flow, and thus particles tend to settle, which facilitates phagocytosis by the sinus macrophages. lymph then flows down the trabecular sinuses that line the outer surface of fibrous trabeculae, to the medullary sinus, and eventually exits via efferent vessels. as antigens within the lymph travel through the sinuses, they are captured and processed by macrophages and dcs. alternatively, dcs charged with antigen can migrate within blood vessels to the node and enter the paracortex via the hevs. circulating b lymphocytes also enter across the hevs, and if they encounter antigen-bearing dcs, there is a local reaction involving the appropriate t helper lymphocytes, b lymphocytes, and dcs. this results in the migration of the activated b lymphocytes to a primary follicle, where they initiate formation of a germinal center. germinal centers, upon migration of antigen-activated b lymphocytes, develop a characteristic architecture. distinct polarity composed of a superficial or light zone and a deep dark zone is present in cases of antigenic stimulation. the light zone, orientated at the source of antigen, consists mainly of small lymphocytes, called centrocytes, which have moderate amounts of pale eosinophilic cytoplasm. the cells of the dark zone, called centroblasts, are large, densely packed lymphocytes with scant cytoplasm, giving this area a darker appearance on h&e staining. the centroblasts undergo somatic mutations of the variable regions of the immunoglobulin gene, followed by isotype class switching (from igm to igg or iga). during this process most centroblasts undergo apoptosis, and cell fragments are phagocytized by macrophages, which are then termed tingible (stainable) body macrophages. the cells that have survived the affinity maturation process are now called centrocytes and along with t lymphocytes and follicular dcs, populate the germinal center light zone. these post-germinal center b lymphocytes leave the follicle as plasma cell precursors (immunoblasts or plasmablasts) and migrate from the cortex to the medullary cords, where they mature and excrete antibody into the efferent lymph. some of these cells may colonize the region surrounding the mantle cell zone to form a marginal zone. marginal zones are apparent only in situations of prolonged and intense immune stimulation and serve as a reservoir of memory cells. the elliptical mantle cell cuff is wider over the light pole of the follicle, though in instances of strong antigenic stimulation, the cuffs can completely encircle the germinal center. responses to injury are listed in box - , and the responses are discussed on the basis of the following systems: sinus histiocytes of the monocyte-macrophage system, cortex, paracortex, and medulla (medullary sinuses and medullary cords). generally, enlarged lymph nodes can be distributed in several different patterns in the body. first, all lymph nodes throughout the body (systemic or generalized) may be enlarged (lymphadenopathy or lymphadenomegaly). this pattern is usually attributed to systemic infectious, inflammatory, or neoplastic processes. if a single lymph node or regional chain of nodes is enlarged, then the area drained by that node should be checked for lesions (e.g., evaluate the oral cavity if the mandibular lymph nodes are enlarged). thus it is important to know the area drained by specific lymph nodes. mesenteric lymph nodes are normally larger because of follicular approximately % to % of lymphocytes enter lymph nodes through the hevs, which also play an important role in lymph fluid balance. the lymphatic vasculature consists of afferent lymphatic vessels, which pierce the capsule and drain into the subcapsular sinus. lymph continues to drain through the trabecular sinuses to the medullary sinuses and finally exits at the hilus via efferent lymphatic vessels. all lymph nodes receive afferent lymphatic vessels from specific areas of the body. the term lymphocenter is often used in veterinary anatomy to describe a lymph node or a group of lymph nodes that is consistently present at the same location and drains from the same region in all species. for example, the popliteal lymph node, caudal to the stifle, drains the distal hind limb. the tracheobronchial nodes (bronchial lymphocenter), located at the tracheal bifurcation, collect lymph from the lungs and send it to the mediastinal nodes or directly to the thoracic duct. because lymph from a single afferent lymphatic vessel drains into a discrete region of a lymph node, only these regions of the node may be affected by the contents of a single draining lymph vessel (e.g., antigen, infectious organisms, or metastatic neoplasms [ fig. - ] ). the lymph node of the pig has a different structure. the afferent lymphatic vessels enter at the hilus instead of around the periphery of the node and empty lymph into the center of the node. the lymph drains to the "subcapsular" sinus (the equivalent of the medullary sinuses of other domestic animals) and then into several efferent lymphatic vessels, which pierce the outer capsule. this reversal of flow is the result of an inverted nodal architecture, with the cortex in the middle of the node surrounded by the medulla at the periphery. thus a pig lymph node that is draining an area of hemorrhage will have blood accumulate in the periphery (subcapsular) instead of in the center of the node (which may be grossly visible). the functions of the lymph node are ( ) to filter lymph of particulate matter and microorganisms, ( ) to facilitate the surveillance and processing of incoming antigens via interactions with b and t lymphocytes, and ( ) to produce b lymphocytes and plasma cells. material arriving in the lymph can be subdivided into free particles that is undergoing follicular hyperplasia is enlarged and has a taut capsule, and the cut surface may bulge. histologically, the follicles contain active germinal centers with antigenic polarity (light and dark zones) (figs. - and - ; also see fig. - ) . depending on the duration and continued exposure to the antigen, there may also be concomitant paracortical hyperplasia and medullary cord hyperplasia and sinus histiocytosis, because these nodes continuously receive and respond to barrages of antigens and bacteria from the intestinal tract. sinus histiocytes (macrophages) are part of the monocyte-macrophage system and the first line of defense against infectious and noninfectious agents in the incoming lymph. in response to these draining agents, there is hyperplasia of the macrophages ("sinus histiocytosis"), most notable in the medullary sinuses ( fig. - ) . leukocytes, often monocytes, may harbor intracellular pathogens (e.g., mycobacterium spp., cell-associated viruses such as parvovirus), arrive in the blood or lymph, infect the lymph node, and then are disseminated throughout the lymphoid tissues of the body via the efferent lymph and circulating blood. cortex (lymphoid follicles). follicular hyperplasia of the cortex is discussed in the section lymphoid/lymphatic system, lymph node, function. an antigenically stimulated lymph node the two main portals of entry to the lymph node for infectious agents and antigens are afferent lymphatic vessels (lymphatic spread) and blood vessels (hematogenous spread). portals of entry used by microorganisms and other agents and substances to access the lymphoid/lymphatic system are summarized in box - . infectious microorganisms, either free within the lymph or within lymphocytes or monocytes, are transported to regional lymph nodes through lymphatic vessels. agents may escape removal by phagocytosis in one lymph node and be transported via efferent lymphatic vessels to the next lymph node in the chain and cause an inflammatory or immunologic response there. this process can continue serially down a lymph node chain, and if the agent is not removed, it may eventually be transported via the lymphatic vessels to either the cervical or thoracic ducts and then disseminated throughout the body. although most pathogens are transported to lymph nodes via afferent lymphatic vessels, bacteria can be transported to lymph nodes hematogenously (free or within leukocytes such as monocytes) in septicemias and bacteremias. direct penetration of a lymph node is uncommon, because it is protected by a thick fibrous capsule. occasionally, inflammatory cells or neoplasms can extend directly into nodal parenchyma from adjacent tissues. defense mechanisms used by the lymphatic system to protect itself against microorganisms and other agents are the innate and adaptive immune responses, discussed in chapters , , and . other defense mechanisms are structural in nature to protect against external trauma and include the thick fibrous capsules of lymph nodes. hemal nodes are small, dark red to brown nodules found most commonly in ruminants, mainly sheep, and have also been reported in horses, primates, and some canids. their architecture resembles that of a lymph node with lymph follicles and sinuses, except that in the hemal node, sinuses are filled with blood (e- fig. - ) . because erythrophagocytosis can be present, it is presumed that hemal nodes can filter blood and remove senescent erythrocytes, but as their blood supply is small, their functional importance is not clear. malt is the initial site for mucosal immunity and is crucial in the protection of mucosal barriers. malt is composed of both diffuse lymphoid tissues and aggregated lymphoid (also known as lymphatic) nodules, which can be subcategorized based on their anatomic location: ( ) bronchus-associated lymphoid tissue (balt), which is often at the bifurcation of the bronchi and bronchioles; ( ) tonsils (pharyngeal and palatine) form a ring of lymphoid tissue at the oropharynx; ( ) nasal-, larynx-, and auditory tube-associated lymphoid tissues (nalt, lalt, and atalt, respectively) within the nasopharyngeal area; ( ) gut-associated lymphoid tissue (galt), which includes peyer's patches and diffuse lymphoid tissue in the gut wall; ( ) conjunctiva-associated lymphoid tissue (calt); ( ) other lymphoid nodules (e.g., genitourinary tract) (fig. - ) . diffuse lymphoid tissue consists of lymphocytes and dcs within the lamina propria of the mucosa of the alimentary, respiratory, and genitourinary tracts. these cells intercept and process antigens, which then travel to regional lymph nodes to initiate the immune response, leading ultimately to the secretion of iga, igg, and igm. plasmacytosis. less florid follicular reactions will have smaller separated germinal centers, whereas nodes receiving persistent high levels of antigen stimulation may have coalescing germinal centers (termed "atypical benign follicular hyperplasia"). in such cases of chronic strong antigenemia, the highly reactive nodes may also exhibit colonization of lymphocytes into perinodal fat, and germinal centers may contain irregular lakes of eosinophilic material, known as follicular hyalinosis. as the immune response declines, there is follicular lymphoid depletion and the concentration of lymphocytes in the germinal centers is reduced, allowing the underlying follicular stroma (including dcs and macrophages) to become visible. with ongoing lymphocyte depletion, the mantle cell zones are thinned, less populated, and discontinuous. eventually, residual mantle cells collapse into the follicular stroma, forming clusters of small dark cells within the bed of dcs and macrophages, referred to as fading follicles. paracortex. paracortical atrophy may result from a variety of causes, including deficiency in lymphocyte production in the bone marrow, reduced differential selection of lymphocytes in the thymus, or destruction of lymphocytes in the lymph node by viruses, radiation, and toxins directly on the lymphocytes in the lymph node (see box - ). examination of h&e-stained sections allows evaluation of follicular activity in the cortex and the concentration of plasma cells in the medullary cords, which serve as a reasonable estimate of b lymphocyte activity for comparison. paracortical hyperplasia may have a nodular or diffuse appearance depending on which and how many afferent lymphatic vessels are draining antigen. this reaction may precede or be concurrent with the germinal center reaction of follicular hyperplasia. proliferation of t lymphocytes has been reported in the paracortex (and pals of the spleen) in malignant catarrhal fever (mcf) in cattle and in pigs with porcine reproductive and respiratory syndrome. pcv can cause a diffuse proliferation of macrophages within the paracortex. responses to injury by the medullary sinuses are dilation of the sinuses and proliferation of histiocytes ("sinus histiocytosis"). sinus macrophages proliferate in response to a wide variety of particulate matter in the lymph, including bacteria and erythrocytes (erythrophagocytosis) draining from a hemorrhagic area (see lymphoid/lymphatic system, disorders of domestic animals: lymph nodes, pigmentation of lymph nodes). dilation of the sinuses due to edema occurs with many underlying conditions, including chronic cardiac failure or drainage from an acutely inflamed area. as the inflammation progresses, the sinuses become filled with neutrophils, macrophages, and occasionally fibrin, in addition to the hyperplastic resident sinus histiocytes (see fig. - ) . depending on the intensity of the inflammation, the adjacent parenchyma may become affected (see lymphoid/lymphatic system, disorders of domestic animals: lymph nodes, enlarged lymph nodes [lymphadenomegaly], acute lymphadenitis). as pointed out in the section on lymph nodes, function, after activation and proliferation of b lymphocytes in the follicle, the immunoblasts formed there move to and mature in the medullary cords, which as a result are distended with plasma cells that secrete antibody into the efferent lymphatic vessels ("medullary plasmacytosis"). the concentration of medullary plasma cells correlates with the activity of the germinal centers. as the immune response subsides, the number of plasma cells decreases and the medullary cords return to their resting state populated by few lymphocytes and scattered plasma cells. chapter bone marrow, blood cells, and the lymphoid/lymphatic system composition. for instance, m cells increase in animals transferred from pathogen-free housing to the normal environment. m cells may also be exploited as a portal for entry by some microbes (see lymphoid/lymphatic system, portals of entry/pathways of spread). table - lists the interactions of the malt with different microorganisms. the responses of malt to injury are similar to those of other lymphoid tissues: hyperplasia, atrophy, and inflammation (box - ). hyperplasia. hyperplasia of lymphoid nodules is a response to antigenic stimulation and consists of activation of germinal centers with subsequent production of plasma cells (see fig. - , b) . lymphoid nodule hyperplasia is often present in chronic disease conditions, such as balt hyperplasia in chronic dictyocaulus spp. (horses, cattle, sheep, and goats) or metastrongylus spp. (pigs) associated bronchitis or bronchiolitis. mycoplasma spp. pneumonias of sheep and pigs display marked balt hyperplasia that can encircle bronchioles and bronchi ("cuffing pneumonia"). hyperplastic lymphoid nodules can be so enlarged that they become grossly visible as discrete white plaques or nodules (see fig. - , a). they can be seen in the conjunctiva of the eyelids and the third eyelid in chronic conjunctivitis, the pharyngeal mucosa in chronic pharyngitis, the gastric mucosa in chronic gastritis, and the urinary bladder in chronic cystitis (follicular cystitis). the normal fetus has no detectable balt, though it may be present in fetuses aborted due to infectious disease. atrophy. atrophy of the diffuse lymphoid tissue and lymphoid nodules has the same causes as atrophy affecting other lymphoid tissues (see box - ) and includes lack of antigenic stimulation, cachexia, malnutrition, aging, viral infections, or failure to be repopulated by b lymphocytes from the bone marrow or t lymphocytes from the thymus. lymphocytolysis of germinal center lymphocytes of peyer's patches is a characteristic lesion in bvdv infection in ruminants and canine and feline parvovirus infections ("punchedout peyer's patches) (see chapters and ). the main portals of entry to malt for infectious agents are hematogenous spread and through migrating macrophages, dcs , and m cells. pathogenic bacteria such as escherichia coli, yersinia pestis, mycobacterium avium ssp. paratuberculosis (map), l. monocytogenes, salmonella spp., and shigella flexneri can invade the host from the lumen of the intestine through dendritic or m cells. some viruses (e.g., reovirus) may be transported by m cells. the scrapie prion protein (prp sc ) may also accumulate in peyer's patches. many viruses, such as bovine coronavirus, bvdv, rinderpest virus, malignant catarrhal fever virus, feline panleukopenia virus, and canine parvovirus, cause lymphocyte depletion within the malt. portals of entry used by microorganisms and other agents and substances to access the lymphoid system are summarized in box - . defense mechanisms used by malt to protect itself against microorganisms and other agents are the innate and adaptive immune responses, discussed in chapters , , and . congenital disorders of the thymus are discussed in detail in chapter . summaries of the gross and microscopic morphologic changes are solitary lymphoid nodules are localized concentrations of lymphocytes (mainly b lymphocytes) in the mucosa and consist of defined but unencapsulated clusters of small lymphocytes (primary lymphoid nodule). they are usually not grossly visible in the resting or antigenically unstimulated state, but upon antigenic stimulation, they proliferate and form germinal centers and surrounding mantle cell zones (secondary lymphoid nodules). aggregated lymphoid nodules consist of groups of lymph nodules, the most notable of which are the tonsils and peyer's patches. the aggregated lymphoid follicles of the peyer's patches are most obvious in the ileum. the latter are covered by a specialized epithelium, the follicle-associated epithelium (fae). the fae is the interface between the peyer's patches and the luminal microenvironment and consists of enterocytes and interdigitated m cells. m cells transport (via endocytosis, phagocytosis, pinocytosis, and micropinocytosis) antigens, particles, bacteria, and viruses from the intestinal lumen to the underlying area rich in dcs , which deliver the material to the lymphoid tissue of the peyer's patches. m cells also express iga receptors, which allows for the capture and transport of bacteria entrapped by iga. the proportion of enterocytes and m cells within the fae is modulated by the luminal bacterial a b mercury have a suppressive effect on the immune system. halogenated aromatic hydrocarbons cause dysfunction of dcs through several mechanisms that lead to atrophy of the primary and secondary lymphoid organs. heavy metals, such as lead, mercury and nickel, are immunosuppressive and generally affect the levels of b and t lymphocytes, nk cells, and inflammatory cytokines. other metals, such as selenium, zinc, and vanadium, may be immunostimulatory at low doses. the immunotoxic mechanisms may differ and include chelation of molecules and effects on protein synthesis, cell membrane integrity, and nucleic acid replication. the toxic effects of mycotoxins such as fumonisins b and b (secondary fungal metabolites produced by members of the genus fusarium) and aflatoxin (produced by aspergillus flavus) include lymphocytolysis in the thymic cortex. box - responses of mucosa-associated lymphoid tissue to injury described in the sections on disorders of horses and disorders of dogs. thymic cysts can be found within the developing and mature thymus and in thymic remnants in the cranial mediastinum. thymic cysts are often lined by ciliated epithelium and represent developmental remnants of branchial arch epithelium and are usually of no significance. thymitis is an uncommon lesion and may be seen in pcv infection (see disorders of pigs and also chapter ), enzootic bovine abortion (see chapter ), and salmon poisoning disease of dogs (see chapter ). infectious agents more commonly cause thymic atrophy. variable degrees of acquired immunodeficiency can be also be caused by toxins, chemotherapeutic agents and radiation, malnutrition, aging, and neoplasia. of infectious agents, viruses most commonly infect and injure lymphoid tissues and include the following: ehv- in aborted foals (fig. - ) , classic swine fever virus, bvdv, canine distemper virus, canine and feline parvovirus, and fiv; severe thymic lymphoid depletion is an early lesion in fiv-infected kittens. environmental toxins, such as halogenated aromatic hydrocarbons (e.g., polychlorinated biphenyls and dibenzodioxins), lead, and thymic hyperplasia. asymptomatic hyperplasia may occur in juvenile animals in association with immunizations and results in symmetrical increase in the size of the thymus. autoimmune lymphoid hyperplasia of the thymus has germinal center formation and occurs with myasthenia gravis. asplenia or the failure of a spleen to develop in utero occurs rarely in animals, and the effect on the animal's immune status is uncertain. (splenic aplasia is present in certain strains of mice, but because these are usually maintained under either germ-free or specific pathogen-free [spf] conditions, the effect of asplenia cannot be evaluated.) congenital immunodeficiency diseases are described in detail in chapter , and in the sections on disorders of horses and disorders of dogs. gross examination of the spleen involves deciding whether the spleen is enlarged (splenomegaly), normal, or small (see e-appendix - ). diffuse enlargement of the spleen may be due to congestion (termed bloody spleen) or other infiltrative disease (termed meaty spleen). the cut surface of congested spleens will exude blood, whereas meaty spleens are more firm and do not readily ooze blood. the diseases and disorders having splenomegaly are discussed using the following categories, which list the common causes of uniform splenomegaly (table - ): • uniform splenomegaly with a bloody consistency (bloody spleen) ( fig. - , a) • uniform splenomegaly with a firm consistency (meaty spleen) (see fig. - , b) • splenic nodules with a bloody consistency • splenic nodules with a firm consistency uniform splenomegaly with a bloody consistency-bloody spleen. the common causes of a bloody spleen are ( ) congestion (due to gastric volvulus with splenic entrapment, splenic volvulus chemotherapeutic drugs inhibit the cell cycle through various mechanisms, and thus all dividing cells, including lymphocytes, bone marrow cells, and enterocytes, are sensitive to their effects. as such, bone marrow suppression, immunosuppression, and gastrointestinal disturbances are common side effects of anticancer drugs. purine analogues (e.g., azathioprine) compete with purines in the synthesis of nucleic acids, whereas alkylating agents like cyclophosphamide cross-link dna and inhibit the replication and activation of lymphocytes. cyclosporin a specifically inhibits the t lymphocyte signaling pathway by interfering with the transcription of the il- gene. methotrexate, a folic acid antagonist, blocks the synthesis of thymidine and purine nucleotides. the immunosuppressive effects of some of these agents is desirable for the treatment of immune-mediated disease (e.g., immune-mediated hemolytic anemia) or to prevent allograft rejection after transplantation. corticosteroids may be given at an immunosuppressive dose, though the degree of suppression is highly variable among species. local or palliative treatment of cancer may include radiotherapy (ionizing radiation) to target and damage the dna of the neoplastic cells. although some immunosuppression may be noted, particularly if bone marrow or lymphoid tissue is within the therapeutically irradiated field, mounting evidence suggests that radiotherapy can induce a cascade of proimmunogenic effects that engage the innate and adaptive immune systems to contribute to the destruction of tumor cells. malnutrition and cachexia, which may occur with cancer, lead to secondary immunosuppression through several complex metabolic and neurohormonal aberrations. thymic function may be impaired in young malnourished animals, resulting in a decrease in circulating t lymphocytes and subsequent depletion of t lymphocyte regions of secondary lymphoid organs. lymphoid atrophy may result from physiologic and emotional stress, which can cause the release of catecholamines and glucocorticoids. as part of the general effects of aging in cells (see chapter ), all lymphoid organs decrease in size (atrophy) with advancing age. in the case of the thymus this reduction in size occurs normally after sexual maturity and is more appropriately termed thymic involution. the term involution should be reserved for normal physiologic processes in which an organ either returns to normal size after a period of enlargement (e.g., postpartum uterus) or regresses to a more primitive state (e.g., thymic involution). because the thymus has both lymphoid and epithelial components, neoplasms may arise from either component. thymic lymphoma arises from the t lymphocytes in the thymus (and very rarely b lymphocytes). it is most often seen in young cats and cattle and less frequently in dogs (fig. - ) (see hematopoietic neoplasia). thymomas arise from the epithelial component and are usually benign neoplasms that occupy the cranial mediastinum of older animals. histologically, these neoplasms consist of clustered or individualized neoplastic epithelial cells, often outnumbered by nonneoplastic small lymphocytes ("lymphocyte-rich thymoma"). thymomas are common in goats and often contain large cystic structures. immunemediated diseases, including myasthenia gravis and immunemediated polymyositis, occur with thymomas in dogs, and also rarely in cats. myasthenia gravis is caused by autoantibodies directed toward the acetylcholine receptors, which lead to destruction of postsynaptic membranes and reduction of acetylcholine receptors at neuromuscular junction. megaesophagus and aspiration pneumonia are common sequelae to this condition. (syn: necropsy) in horses and dogs that have been euthanized or anesthetized with barbiturates. grossly, the spleen is extremely enlarged (fig. - ) , and the cut surface bulges and oozes copious blood. because of the splenic distention, the splenic capsule can be fragile and easily ruptured. histologically, the red pulp is distended by erythrocytes, and the lymphoid tissues of the white pulp are small and widely separated (fig. - ). electric stunning of pigs at slaughter may result in a large congested spleen; the mechanism is unknown, but it should not be confused with a pathologically congested spleen. splenic congestion in acute cardiac failure is rarely seen in animals. acute congestion/hyperemia. acute septicemias may cause acute hyperemia and concurrent acute congestion of marginal zones and splenic red pulp. microbes are transported hematogenously to these sites, where they are rapidly phagocytized by macrophages. enormous numbers of intravenous bacteria can be cleared by the spleen from the blood in to minutes, but when this defensive mechanism is overwhelmed, the outcome is usually fatal. the response of the spleen depends on the duration of the disease. in acutely fatal cases, such as anthrax and fulminating salmonellosis, distention by blood may be the only gross finding. if the animal survives longer, as in swine erysipelas and the less virulent forms of [all of which compress the splenic vein], and barbiturate euthanasia, anesthesia, or sedation), ( ) acute hyperemia (due to septicemia), and ( ) acute hemolytic anemia (due to an autoimmune disorder or an infection with a hemotropic parasite). splenic torsion. torsion of the spleen occurs most commonly in pigs and dogs; in dogs this usually involves both spleen and stomach and is seen more often in deep-chested breeds (see chapter ). in contrast to ruminants, in which the spleen is firmly attached to the rumen, the spleens of dogs and pigs are attached loosely to the stomach by the gastrosplenic ligament. it is the twisting of the spleen around this ligament that results initially in occlusion of the veins, causing splenic congestion, and later in occlusion of the artery, causing splenic infarction. in dogs the spleen is uniformly and markedly enlarged and may be blue-black from cyanosis. it is often folded back on itself (visceral surface to visceral surface) in the shape of the letter "c." treatment for this condition is most often splenectomy. barbiturate euthanasia, anesthesia, or sedation. intravenous injection of barbiturates induces acute passive congestion in the spleen due to relaxation of smooth muscle in the capsule and trabeculae. this phenomenon is seen most dramatically at autopsy only histologic lesion may be marked congestion of the marginal sinuses and the splenic red pulp vascular spaces. at low magnification, congestion of the marginal sinus may appear as a circumferential red ring around the splenic follicle, and there is marked lymphocytolysis of follicles and pals. intravascular free bacilli are noted and may be seen in impression smears of peripheral blood, presumably because death is so rapid from the anthrax toxin that there is insufficient time for phagocytosis to take place. if the animal lives longer, scattered neutrophils are present in the marginal sinuses and red pulp vascular spaces (fig. - ). anthrax cases are not normally autopsied because exposure to air causes the bacteria to sporulate-anthrax spores are extremely resistant and readily contaminate the environment. acute hemolytic anemias. hemolytic diseases, including acute babesiosis, hemolytic crises in equine infectious anemia, and immune-mediated hemolytic anemia, can cause marked splenic congestion. the splenic congestion is due to the process of removal (phagocytosis) and storage of large numbers of sequestered parasitized and/or altered erythrocytes from the circulation. histologically, there is dilation of the red pulp vascular spaces with erythrocytes and erythrophagocytes. with chronicity there is hyperplasia of the red pulp macrophages, hemosiderosis, and reduced congestion because the number of sequestered diseased erythrocytes is diminished. spleen. the three general categories of conditions leading to uniform splenomegaly with a firm meaty consistency are ( ) marked phagocytosis of cells, debris, or foreign agents/material; ( ) proliferation or infiltration of cells as occurs in diffuse lymphoid and histiocytic hyperplasia, diffuse granulomatous disease (e -table - ), emh, and neoplasia; ( ) storage of materials in storage diseases or amyloidosis. it is important to recognize that more than one of these processes can occur in the same patient (e.g., dogs with immunemediated hemolytic anemia may have both marked erythrophagocytosis and emh). the appearance of the cut surface of a meaty spleen depends on the underlying cause. in diffuse marked lymphoid hyperplasia, large, disseminated, discrete, white, bulging nodules are visible. spleens with diffuse infiltrative neoplasms, such as lymphoma, are pink-light purple on cut surface. diffuse lymphoid hyperplasia. lymphoid hyperplasia has been described in detail in the section on dysfunction/responses to injury. in cases of prolonged antigenic stimulation the lymphoid salmonellosis, there may be sufficient time for neutrophils and macrophages to accumulate in the marginal sinuses, marginal zones, and splenic red pulp vascular spaces. anthrax. b. anthracis, the causative agent of anthrax, is a grampositive, large, endospore-forming bacillus, which grows in aerobic to facultative anaerobic environments. anthrax is primarily a disease of ruminants, especially cattle and sheep (see chapters , , , and ) . once the spores are ingested, they replicate locally in the intestinal tract, spread to regional lymph nodes, and then disseminate systemically through the bloodstream, resulting in septicemia. b. anthracis produces exotoxins, which degrade endothelial cell membranes and enzyme systems. grossly, the spleen is uniformly enlarged and dark red to bluishblack and contains abundant unclotted blood. in peracute cases the .e chapter bone marrow, blood cells, and the lymphoid/lymphatic system diffuse granulomatous disease. chronic infectious diseases may cause a uniformly firm and enlarged spleen, mostly due to macrophage hyperplasia and phagocytosis, diffuse lymphoid hyperplasia, or diffuse granulomatous disease. diffuse granulomatous diseases (see e- table - ) occur in ( ) intracellular facultative bacteria that infect macrophages (e.g., mycobacterium spp., brucella spp., and francisella tularensis); ( ) systemic mycoses (e.g., blastomyces dermatitidis, histoplasma capsulatum) (see lymphoid/lymphatic system, disorders of domestic animals: lymph nodes, enlarged lymph nodes [lymphadenomegaly]) ( fig. - , a and b) , and ( ) protozoal infections that infect macrophages (e.g., leishmania spp.). some of these organisms may also produce nodular spleens with the formation of discrete to coalescing granulomas (e.g., m. bovis) (see splenic nodules with a firm consistency). follicles throughout the splenic parenchyma can become enlarged and visible on gross examination (fig. - ), leading to diffuse splenomegaly. in contrast to b lymphocyte hyperplasia of the lymphoid follicles, certain diseases (e.g., malignant catarrhal fever in cattle) may lead to t lymphocyte hyperplasia of the pals. diffuse histiocytic hyperplasia and phagocytosis. splenomegaly from hyperplasia and increased phagocytosis of splenic macrophages is a response to the need to engulf organisms in prolonged bacteremia or parasitemia from hemotropic organisms. whereas acute hemolytic anemias cause splenomegaly with congestion (bloody spleen), with chronicity there is decreased sequestration of diseased erythrocytes and hence less congestion. therefore in cases of chronic hemolytic disease, splenomegaly is attributed to diffuse proliferation of macrophages, phagocytosis, and concurrent hyperplasia of the white pulp due to ongoing antigenic stimulation. for example, equine infectious anemia has cyclical periods of viremia, with immune-mediated damage to erythrocytes and platelets, and phagocytosis to remove altered erythrocytes and platelets. these cycles result in proliferation of red pulp macrophages, hyperplasia of hematopoietic cells (emh) to replace those lost, and hyperplasia of lymphocytes in the white pulp. in animals less than year of age. in general, these substrates are lipids and/or carbohydrates that accumulate in the cells, the result of the lack of normal processing within lysosomes. major categories of stored materials include mucopolysaccharides, sphingolipids, glycolipids, glycoproteins, glycogen, and oligosaccharides. macrophages are commonly affected by storage diseases, and thus extramedullary hematopoiesis. emh is the development of blood cells in tissues outside the medullary cavity of the bone (e- fig. - ). the formation of single or multiple lineages of hematopoietic cells is often observed in many tissues and commonly in the spleen. the ability of blood cell precursors to home, proliferate, and mature in extramedullary sites relies on the presence of hscs and pathophysiologic changes in the microenvironment (i.e., extracellular matrix, stroma, and chemokines). in the spleen, hscs have been found within vessels and adjacent to endothelial cells to form a vascular niche; thus splenic emh occurs in the red pulp, both within the red pulp vascular spaces and sinusoids (of the dog). the predilection for emh to occur varies among species (for instance, splenic emh persists throughout adulthood in mice), and the underlying mechanisms are not completely understood, but four major theories to explain the causes of emh are ( ) severe bone marrow failure; ( ) myelostimulation; ( ) tissue inflammation, injury, and repair; and ( ) abnormal chemokine production. because splenic emh is often observed in animals without obvious hematologic abnormalities, tissue inflammation, injury, and repair is the most likely mechanism of emh in this organ. in dogs and cats emh occurs most frequently with degenerative and inflammatory disorders, such as lymphoid nodular hyperplasia, hematomas, thrombi, histiocytic hyperplasia, inflammation (e.g., fungal splenitis), and neoplasia. emh in multiple tissues may be observed in chronic cardiovascular or respiratory conditions, chronic anemia, or chronic suppurative diseases in which there is an excessive tissue demand for neutrophils that exceeds the supply available from the marrow (e.g., canine pyometra). primary neoplasms. primary neoplastic diseases of the spleen arise from cell populations that normally exist in the spleen and include hematopoietic components, such as lymphocytes, mast cells, and macrophages, and stromal cells, such as fibroblasts, smooth muscle, and endothelium. the primary neoplasms that result in diffuse splenomegaly are the round cell tumors, including lymphoma ( fig. - ) , leukemia, visceral mast cell tumor, and histiocytic sarcoma. it is important to note that all of these types of neoplasms can produce nodular lesions instead of-or along with-a diffusely enlarged spleen. the different types of lymphoma in domestic animals are discussed in the section on hematopoietic neoplasia. secondary neoplasms of the spleen are due to metastatic spread and most often form nodules in the spleen, not a uniform splenomegaly. amyloid. the accumulation of amyloid in the spleen may occur with primary (al) or secondary (aa) amyloidosis (see chapters and ). rarely, severe amyloid accumulation may cause uniform splenomegaly ( fig. - ) , in which the spleen is firm, rubbery to waxy, and light brown to orange. microscopically, amyloid is usually in the splenic follicles, which if large enough, are grossly visible as approximately -mm-diameter gray nodules. amyloid deposition can also be seen within the walls of splenic veins and arterioles. plasma cells tumors within the spleen may also be associated with amyloid (al) deposits. lysosomal storage diseases. storage diseases are a heterogeneous group of inherited defects in metabolism characterized by accumulation of storage material within the cell (lysosomes). genetic defects, which result in the absence of an enzyme, the synthesis of a catalytically inactive enzyme, the lack of activator proteins, or a defect in posttranslational processing, can lead to a storage disease. acquired storage diseases are caused by exogenous toxins, most often plants that inhibit a particular lysosomal enzyme (e.g., swainsonine toxicity due to indolizidine alkaloid found in astragalus and oxytropis plant spp.). storage diseases typically occur diagnosis, it may be difficult (and futile) to determine the primary site. histologically, hemangiosarcomas are composed of plump neoplastic endothelial cells, which wrap around stroma to form haphazardly arranged and poorly defined blood-filled vascular spaces ( fig. - ). splenic nodules with a firm consistency. the most common disorders of the spleen with firm nodules are ( ) lymphoid nodular hyperplasia, ( ) complex nodular hyperplasia, ( ) primary neoplasms, ( ) secondary metastatic neoplasms, ( ) granulomas, and ( ) abscesses. lymphoid and complex nodular hyperplasia. see lymphoid/ lymphatic system, disorders of dogs. primary neoplasms. the primary neoplastic diseases of the spleen that result in firm nodules include lymphoma (multiple subtypes), histiocytic sarcoma, leiomyoma, leiomyosarcoma, fibrosarcoma, myelolipomas, liposarcomas, myxosarcomas, undifferentiated pleomorphic sarcomas, solid hemangiosarcomas, and rare reports of primary chondrosarcomas. these locally extensive neoplasms may be solitary or multiple, raised above the capsular surface, but usually confined by the capsular surface. the consistency and cut surface appearance varies depending on the type of neoplasm; spindle cell tumors like leiomyosarcomas and fibrosarcomas will be white and firm, liposarcomas and myelolipomas are soft and bulging, and myxomatous neoplasms are gelatinous. it is important to remember that many round cell neoplasms, such as lymphoma, mast cell tumors, plasma cell tumors, myeloid neoplasms, and histiocytic sarcomas, can form nodules or diffuse splenic enlargement (or both). metastatic neoplasms. neoplasms that metastasize to the spleen usually result in enlarged nodular spleens (fig. - ) and include any number of sarcomas, carcinomas, or malignant round cell tumors. metastatic sarcomas can include fibrosarcomas, leiomyosarcomas, chondrosarcomas, and osteosarcomas. mammary, prostatic, pulmonary, anal sac gland and neuroendocrine carcinomas may metastasize widely to abdominal viscera, including the spleen. granulomas and abscesses. microorganisms that cause diffuse granulomatous splenitis and uniform splenomegaly may also cause focal to multifocal nodular lesions (e.g., mycobacterium spp., fungal organisms) (see diffuse granulomatous diseases of the spleen and also enlarged lymph nodes). although there are a large number of abscesses bulge from the capsule and cut surfaces, and the exudate can vary in amount, texture, and color depending on the inciting organism and the age of the lesion. the most common diseases or conditions that have small spleens are ( ) developmental anomalies, ( ) aging changes, ( ) wasting and/or cachectic diseases, and ( ) splenic contraction. splenic hypoplasia. primary immunodeficiency diseases can result in splenic hypoplasia, as well as small thymuses and lymph nodes (which may be so small as to be grossly undetectable in some diseases). these diseases affect young animals and involve defects in t and/or b lymphocytes (fig. - ) . spleens are exceptionally small, firm, and pale red and lack lymphoid follicles and pals. these diseases and their pathologic findings are discussed in chapter and in the sections on disorders of horses and disorders of dogs. congenital accessory spleens. accessory spleens can be either congenital or acquired (see splenic rupture). congenital accessory spleens are termed splenic choristomas, which are nodules of normal splenic parenchyma in abnormal locations. these are usually small diseases and conditions commonly caused by bacteremia (e.g., navel ill, joint ill, chronic respiratory infections, bacterial endocarditis, chronic skin diseases, castration, tail docking, and ear trimming and/ or notching), these rarely result in visible splenic abscesses. pyogranulomas and abscesses in the spleen (multifocal chronic suppurative splenitis) that do develop after septicemia and/or bacteremia are usually caused by pyogenic bacteria such as streptococcus spp., rhodococcus equi (fig. - ) , trueperella pyogenes ( fig. - ) , and corynebacterium pseudotuberculosis. cats with the wet or dry form of feline infectious peritonitis virus may have nodular pyogranulomatous and lymphoplasmacytic inflammatory foci throughout the spleen. splenic abscesses due to direct penetration by a migrating foreign body are reported in cattle (from the reticulum) and less commonly in the horse (from the stomach). perforating gastric ulcers in horses due to gasterophilus and habronema spp. have also reportedly led to adjacent splenic abscesses. granulomas and a b and may be located in the gastrosplenic ligament, liver, or pancreas (see fig. - , b) . splenic fissures. fissures in the splenic capsule are elongated grooves whose axes run parallel to the borders of the spleen. this developmental defect is seen most commonly in horses but also occurs in other domestic animals and has no pathologic significance. the surface of the fissure is smooth and covered by the normal splenic capsule. aging changes. as part of the general aging change of cells as the body ages, there is reduction in the number of b lymphocytes produced by the bone marrow and decline of naïve t lymphocytes due to age-related thymic involution. consequently, there is lymphoid atrophy in secondary lymphoid organs. the spleen is small, and its capsule may be wrinkled. microscopically, the white pulp is atrophied, and splenic follicles, if present, lack germinal centers. sinuses may also collapse from a reduced amount of blood, possibly because of anemia, which makes the red pulp appear fibrous. wasting/cachectic diseases. any chronic disease, such as starvation, systemic neoplasia, and malabsorption syndrome, may produce cachexia. starvation has a marked effect on the thymus, which results in atrophy of the t lymphocyte areas in the spleen and lymph nodes, which is in part mediated by leptin. b lymphocyte development is also diminished, because b lymphocytes require accessory signals from helper t lymphocytes to undergo somatic hypermutation and immunoglobulin isotype switching. splenic contraction. contraction of the spleen is a result of contraction of the smooth muscle in the capsule and trabeculae of storage spleens. it can be induced by the activation of the sympathetic "fight-or-flight" response and is seen in patients with heart failure or shock (cardiogenic, hypovolemic, and septic shock) and also occurs in acute splenic rupture that has resulted in massive hemorrhage (hemoabdomen/hemoperitoneum). the contracted spleen is small, its surface is wrinkled, and the cut surface is dry. hemosiderosis. hemosiderin is a form of storage iron derived chiefly from the breakdown of erythrocytes, which normally takes place in the splenic red pulp. thus some splenic hemosiderosis is to be expected, and the amount varies with the species (it is most extensive in the horse). excessive amounts of splenic hemosiderin are seen when erythropoiesis is reduced (less demand for iron) or from the rapid destruction of erythrocytes in hemolytic anemias (increased stores of iron), such as those caused by immune-mediated hemolytic anemias or hemotropic parasites. excess splenic hemosiderin may also occur in conditions such as chronic heart failure or injections of iron dextran or as focal accumulations at the sites of old hematomas, infarcts, or trauma-induced hemorrhages. hemosiderin is also present in siderofibrotic plaques. siderofibrotic plaques. siderofibrotic plaques are also known as siderocalcific plaques and gamna-gandy bodies. grossly, they are gray-white to yellowish, firm, dry encrustations on the splenic capsule. usually they are most extensive along the margins of the spleen but can be elsewhere on the capsule (fig. - ) and sometimes in the parenchyma. with h&e staining these plaques are a multicolored mixture of yellow (hematoidin), golden brown (hemosiderin), purple-blue (hematoxylinophilic calcium mineral), and pink (eosinophilic fibrous tissue) (fig. - the diseases or conditions with small lymph nodes are ( ) congenital disorders, ( ) lack of antigenic stimulation, ( ) viral infections, ( ) cachexia and malnutrition, ( ) aging, and ( ) radiation. congenital disorders. primary immunodeficiency diseases are described in detail in chapter and in the sections on disorders of horses and disorders of dogs. neonatal animals with primary immunodeficiency diseases often have extremely small to undetectable lymph nodes. in dogs and horses with severe combined immunodeficiency disease (scid), lymphoid tissues, including lymph nodes from affected animals, are often grossly difficult to identify and characterized by an absence of lymphoid follicles. congenital may represent sequelae to previous hemorrhages from trauma to the spleen. splenic rupture. splenic rupture is most commonly caused by trauma, such as from an automobile accident or being kicked by other animals. thinning of the capsule from splenomegaly can render the spleen more susceptible to rupture, and this may occur at sites of infarcts, hematomas, hemangiosarcomas, and lymphoma. in acute cases of splenic capsular rupture, the spleen is contracted and dry and the surface wrinkled from the marked blood loss (fig. - ) . in more severe cases the spleen may be broken into two or more pieces, and small pieces of splenic parenchyma may be scattered throughout the omentum and peritoneum (sometimes called splenosis) (fig. - , a) . clotted blood, fibrin, and omentum may adhere to the surface at the rupture site. if the rupture is not fatal, the spleen heals by fibrosis, and there may be a capsular scar. occasionally there are two or more separate pieces of spleen adjacent to each other and sometimes joined by scar tissue in the gastrosplenic ligament. the functional capabilities of the small accessory spleens are questionable, although erythrophagocytosis, hemosiderosis, hyperplastic nodules, emh, and neoplasia can be present in these nodules. accessory spleens due to traumatic rupture should be distinguished from peritoneal seeding of hemangiosarcoma and the developmental anomaly splenic choristomas (see fig. - , b) , which are nodules of normal splenic parenchyma in abnormal locations (such as liver and pancreas). chronic splenic infarcts. in the early stage, splenic infarcts are hemorrhagic and may elevate the capsule (see splenic nodules with a bloody consistency). however, as the lesions age and fibrous connective tissue is laid down, they shrink and become contracted and often depressed below the surface of the adjacent capsule. conditions causing lymphadenomegaly include ( ) lymphoid hyperplasia (follicular or paracortical), ( ) hyperplasia of the sinus histiocytes (monocyte-macrophage system), ( ) acute or chronic lymphadenitis, ( ) lymphoma, and ( ) metastatic neoplasia. macrophage system. detailed descriptions of lymphoid follicular hyperplasia, paracortical hyperplasia, and hyperplasia of sinus histiocytes are in the sections on lymph nodes, function, and lymph node, dysfunction/responses to injury. follicular lymphoid hyperplasia can involve large numbers of lymph nodes, as in a systemic disease, or can be localized to a regional lymph node draining an inflamed or antigenically stimulated (e.g., vaccine injection) area. acute lymphadenitis. lymph nodes draining sites of infection and inflammation may develop acute lymphadenitis (e.g., retropharyngeal lymph nodes draining the nasal cavity with acute rhinitis, tracheobronchial lymph nodes in animals with pneumonia ( fig. - ), and mammary [supramammary] lymph nodes in animals with mastitis). grossly, affected lymph nodes in acute lymphadenitis are red and edematous, have taut capsules, and may have necrotic areas ( fig. - ). in some instances the afferent lymphatic vessels may also be inflamed (lymphangitis). the material draining to the regional lymph node may be microorganisms (bacteria, parasites, protozoa, and fungi), inflammatory mediators, or a sterile irritant. in septicemic diseases, such as bovine anthrax, the lymph nodes are markedly congested and the sinuses filled with blood. examination of these lymph nodes should include culturing for bacteria and the examination of smears and histologic sections for bacteria and fungi. pyogenic bacteria, such as streptococcus equi ssp. equi in horses , streptococcus porcinus in pig, and trueperella pyogenes in cattle and sheep, cause acute suppurative lymphadenitis (see disorders of horses and disorders of pigs). hereditary lymphedema has been reported in certain breeds of cattle and dogs. grossly, the most severely affected animals have generalized subcutaneous edema (see fig. - ) and effusions. in severe cases the peripheral and mesenteric lymph nodes are hypoplastic and characterized by an absence of follicles. nodes draining an edematous area may be grossly enlarged from marked sinus edema. lack of antigenic stimulation. the size of the lymph node depends on the level of phagocytosis and antigenic stimulation; lymph nodes that are not receiving antigenic stimuli (e.g., spf animals) will be small with low numbers of primary lymphoid follicles and few, if any, secondary follicles or plasma cells in the medullary cords. conversely, nodes receiving constant antigenic material (such as those draining the oral cavity or intestines) are large with active secondary lymphoid follicles. the number of follicles increases or decreases with changes in the intensity of the antigenic stimuli, and the germinal centers go through a cycle of activation, depletion, and rest, as described previously (see lymph nodes, function). as the antigenic response wanes, germinal centers become depleted of lymphocytes, and lymphoid follicles become smaller. viral infections. many viral infections of animals target lymphocytes and cause the destruction of lymphoid tissue. of infectious agents, viruses most commonly infect and injure lymphoid tissues and include the following: ehv- in aborted foals, classic swine fever virus, bvdv, canine distemper virus, and canine and feline parvovirus. although some viruses destroy lymphoid tissue, others can lead to lymph node hyperplasia (e.g., follicular b lymphocyte hyperplasia in fiv and paracortical t lymphocyte hyperplasia in malignant catarrhal fever virus) or cause neoplasia (e.g., felv, blv, and marek's disease). cachexia and malnutrition. malnutrition and cachexia, which occur with cancer, lead to secondary immunosuppression through several complex metabolic and neurohormonal aberrations. starvation has marked effect on the thymus with resultant atrophy of the t lymphocyte areas in the spleen and lymph nodes and may also affect b lymphocyte development. lymphoid atrophy may result from physiologic and emotional stress and the concurrent release of catecholamines and glucocorticoids. glucocorticoids reduce b and t lymphocytes via redistribution of these cells and glucocorticoidinduced apoptosis. t lymphocytes are more sensitive to glucocorticoid-induced apoptosis than are b lymphocytes. aging. as part of the general aging change of cells as the body ages, there is reduction in the number of lymphocytes produced by the bone marrow and regressed thymus, and consequently a reduction in the b and t lymphocytes in secondary lymphoid organs, resulting in lymphoid atrophy. consequently, lymph nodes are small, with loss of b and t lymphocytes and plasma cells in the cortical follicles, paracortex, and medullary cords, respectively. radiation. local or palliative treatment of cancer may include radiotherapy (ionizing radiation) to target and damage the dna of the neoplastic cells. although some immunosuppression may be noted, particularly if bone marrow or lymphoid tissues are within the irradiated field, mounting evidence suggests that radiotherapy can induce a cascade of proimmunogenic effects that engage the innate and adaptive immune systems to contribute to the destruction of tumor cells. fibrosis of tissues within the irradiated field also occurs as mainly a late effect of chronic radiation. bouts of chronic lymphadenitis (e.g., regional lymph node draining chronic mastitis in cows) lead to fibrosis and lymphoid hyperplasia, in addition to chronic abscesses. the classic example of chronic suppurative lymphadenitis with encapsulated abscesses is caseous lymphadenitis, a disease of sheep and goats caused by c. (also see disorders of ruminants). it is also the cause of ulcerative lymphangitis in cattle and horses and pectoral abscesses in horses. classic examples of focal to multifocal granulomatous lymphadenitis are mycobacterium tuberculosis complex, which includes m. bovis among others. members of m. avium complex cause similar lesions and have been described in a number of species, including dogs, cats, primates, pigs, cattle, sheep, horses, and human beings. infection may begin by inhalation of aerosol droplets containing the bacilli, which may spread via the lymphatic vessels to regional lymph nodes, resulting in granulomatous lymphangitis and lymphadenitis (fig. - ) . initially lesions in the lymphatic system are confined to the lymphatic vessels (granulomatous lymphangitis) and regional lymph nodes (e.g., the tracheobronchial lymph nodes in the case of pulmonary histologically, the subcapsular, trabecular, and medullary sinuses and the parenchyma of the cortex and medulla have focal to coalescing foci of neutrophilic inflammation, necrosis, and fibrin deposition ( fig. - ). if inflammation in the lymph node continues for several days or longer, the lymph node is further enlarged by follicular hyperplasia and plasmacytosis of the medullary cords from the expected immune response. chronic lymphadenitis. the types of chronic lymphadenitis include chronic suppurative lymphadenitis, diffuse granulomatous inflammation, and discrete granulomas. in chronic suppurative inflammation, abscesses range in size from small microabscesses to large abscesses that occupy and obliterate the whole node. recurrent figure - acute lymphadenitis, lymph node, dog. acute lymphadenitis usually occurs when a regional lymph node drains a site of inflammation caused by microorganisms and subsequently becomes infected. the lymph node is firm and enlarged with a tense capsule. the cut surface bulges and is wet with blood, edema, and an inflammatory cell infiltrate. histoplasmosis (see disorders in dogs). in feline cryptococcosis (most often cryptococcus neoformans), the inflammatory response may be mild due to the thick polysaccharide capsule, which has strong immunomodulatory properties and promotes immune evasion and survival within the host. therefore the nodal enlargement is due mainly to a large mass of organisms (see e- fig. - ) . pigs with pcv infection may have a multifocal to diffuse infiltrate of macrophages and multinucleated giant cells of varying severity (see disorders of pigs). secondary (metastatic) neoplasms. carcinomas typically metastasize via lymphatic vessels to the regional lymph node. other common metastatic neoplasms include mast cell tumor and malignant melanoma. although sarcomas most often metastasize hematogenously, some more aggressive sarcomas (e.g., osteosarcoma) may spread to regional lymph nodes. histologically, single cells or clusters of neoplastic cells travel via the afferent lymphatic vessels and are deposited in a sinus, usually the subcapsular sinus (see fig. - ). here the cells proliferate and can ultimately occupy the whole lymph node, as well as drain to the next lymph node in the chain. tuberculosis), but once disseminated in the lymph or blood, lymph nodes throughout the body will have lesions. well-organized granulomas consist of a central mass of macrophages with phagocytized mycobacteria, surrounded by epithelioid and foamy macrophages and occasional multinucleated giant cells (langhans type). these inflammatory nodules are surrounded by a layer of lymphocytes enclosed in a fibrous capsule. over time the center of the granuloma may undergo caseous necrosis due to the high lipid and protein content of the dead macrophages (see chapter ). in bovine johne's disease the mesenteric lymph nodes draining the infected intestine can have noncaseous granulomas (fig. - ). diffuse granulomatous lymphadenitis. coalescing to diffuse granulomatous lymphadenitis is seen in disseminated fungal infections such as blastomycosis, cryptococcosis (e- fig. - ) , and extent of the emphysema. in severe cases the lymph node is light, puffy, and filled with discrete gas bubbles, and the cut surface may be spongy. histologically, the sinuses are distended with gas and lined by macrophages and giant cells. this change has been considered a foreign body reaction to the gas bubbles. macrophages and giant cells are also seen in afferent lymphatic vessels (granulomatous lymphangitis). vascular transformation of lymph node sinus (nodal angiomatosis). vascular transformation of the sinuses is a nonneoplastic reaction to blocked efferent lymphatic vessels or veins. this pressure-induced lesion results in the formation of anastomosing vascular channels and may be confused with a nodal vascular neoplasm. these proliferative but noninvasive masses usually begin in the subcapsular sinuses and may be followed by lymphoid atrophy, erythrophagocytosis/hemosiderosis, and fibrosis. the blockage may be caused by malignant neoplasms of the tissues that the lymph node drains (e.g., thyroid carcinoma with nodal angiomatosis of the mandibular lymph node). see section on bone marrow, disorders of domestic animals, hematopoietic neoplasia for a discussion of the who classification of hematopoietic neoplasia that predominantly arise and proliferate within bone marrow. this section will cover neoplasms of lymphoid tissue(s) arising outside of bone marrow. lymphoma. the term lymphoma (also known as lymphosarcoma) encompasses a diverse group of malignancies arising in lymphoid tissue(s) outside of bone marrow. grossly, there may be diffuse to nodular enlargement of one or more lymph nodes (fig. - ) , and the cut surface is soft, white, and bulging with loss of normal corticomedullary architecture. there is great variation in the clinical manifestations and cytopathologic features of lymphoma, which underlie the importance of classification to better predict the clinical behavior and outcome. an understanding of lymphocyte maturation is crucial, because the who classification of lymphoma postulates a normal cell counterpart for each type of lymphoma (when possible). in other words, lymphoma can arise at any stage in the development/maturation of a lymphocyte-from precursor lymphocytes (b or t lymphoblasts) to mature lymphoid b and t, lymphocytes and nk cells (table - and box - ). pathologists use gross features, histomorphologic features, immunophenotype (b or t lymphocyte), and clinical characteristics to red discoloration is caused by ( ) draining erythrocytes from hemorrhagic or acutely inflamed areas, ( ) acute lymphadenitis with hyperemia and/or hemorrhage, ( ) acute septicemias with endotoxininduced vasculitis or disseminated intravascular coagulation, and ( ) dependent areas in postmortem hypostatic congestion. blood in pig lymph nodes is especially obvious due to the inverse anatomy (the equivalent of the medullary sinuses are subcapsular and thus readily visible in the unsectioned node). initially erythrocytes fill trabecular and medullary sinuses and then rapidly undergo erythrophagocytosis by proliferating sinus macrophages. hemosiderin deposition occurs within to days in these macrophages, imparting a brown discoloration of the node. black discoloration is often present in the tracheobronchial lymph nodes due to draining of carbon pigment (pulmonary anthracosis, see chapter ). black ink from skin tattoos will drain to the regional lymph node. these pigments are usually noted within the medullary sinus macrophages. brown discoloration may be due to melanin, parasitic hematin, or hemosiderin. melanin pigment is seen in animals with chronic dermatitis when melanocytes are damaged and their pigment is released into the dermis and phagocytized by melanomacrophages (pigmentary incontinence) and drained to the regional lymph node. the mandibular lymph nodes often contain numerous melanomacrophages in animals with heavily pigmented oral mucosa, presumably due to chronic low levels of inflammation. this must be distinguished from metastatic malignant melanomas. lymph nodes draining areas of congenital melanosis may have melanin deposits. parasitic hematin pigment is produced by fascioloides magna (cattle) and fasciola hepatica (sheep) in the liver and then transported via the lymphatic vessels to the hepatic lymph nodes. hemosiderin, an erythrocyte breakdown product, may form in a hemorrhagic node or arrive in hemosiderophages draining from congested, hemorrhagic, or inflamed areas. drainage of iron dextran from an intramuscular injection may also cause hemosiderin pigment accumulation within the draining lymph node. green discoloration is rare and may be caused by green tattoo ink (often used in black animals); ingestion of blue-green algae, which drain to mesenteric lymph nodes; massive eosinophilic inflammation; and in mutant corriedale sheep, which have a genetic defect that results in a deficiency in the excretion of bilirubin and phylloerythrin by the liver. the phylloerythrin or a metabolite stains all the tissues of the body a dark green, except for the brain and spinal cord, which are protected by the blood-brain barrier. miscellaneous discolorations of lymph nodes may be seen with intravenously injected dyes (e.g., methylene blue or trypan blue) or subcutaneous drug injections. lymph nodes may be yellow in severely icteric patients. the pigmented strain of map (johne's disease) may impart an orange discoloration in the mesenteric lymph nodes of sheep. inclusion bodies. many viruses produce inclusion bodies, and some of these occur in lymph nodes. these viruses include ehv- in horses, bovine adenovirus, cytomegalic virus in inclusion body rhinitis and pcv , herpesvirus of pseudorabies in pigs, and rarely parvovirus in dogs and cats. emphysema. emphysema in lymph nodes is a consequence of emphysema in their drainage fields and is seen most frequently in tracheobronchial lymph nodes in bovine interstitial emphysema and in porcine mesenteric lymph nodes in intestinal emphysema (see chapter ). the appearance of the lymph node varies with the immunohistochemistry (mum /irf is particularly sensitive and specific for plasma cell neoplasms). histiocytic disorders. histiocytic disorders are frequently diagnosed in dogs and occur less often in cats. briefly, histiocytes are categorized as macrophages and dcs, the latter of which are subdivided into langerhans cells (lcs), found in skin, gastrointestinal, respiratory, and reproductive epithelia (mucosae), and interstitial dcs (idc), located in perivascular spaces of most organs. the term interdigitating dcs describes dcs (either resident or migrating) found in t lymphocyte regions of lymph nodes (paracortex) and spleen (pals); interdigitating dcs consist of both lcs and idcs. these lineages can be differentiated using immunohistochemical stains. histiocytic disorders that are diagnosed in veterinary medicine at this time include the following: canine cutaneous histiocytoma, canine lc histiocytosis, canine cutaneous and systemic histiocytosis, feline pulmonary lc histiocytosis, feline progressive histiocytosis, dendritic cell leukemia in the dog, and histiocytic sarcoma and hemophagocytic histiocytic sarcoma in both dogs and cats. lymph node involvement is seen in many of these conditions. rare reports of regional lymph node metastasis in cases of solitary canine cutaneous histiocytoma have been published. lymphatic invasion with subsequent regional nodal involvement may be seen in dogs with lc histiocytosis, which is a poor prognostic indicator and likely reflects systemic infiltration. the normal architecture of tracheobronchial lymph nodes is often effaced in cats with pulmonary lc histiocytosis. canine reactive histiocytoses are not clonal neoplastic proliferations but likely reflect an immune dysregulation consisting of activated dermal idcs (and t lymphocytes). they are categorized as cutaneous histiocytosis (ch), involving skin and draining lymph nodes, and a more generalized systemic histiocytosis (sh), affecting skin and other sites (e.g., lung, liver, bone marrow, spleen, lymph nodes, kidneys, and orbital and nasal tissues). histiocytic sarcoma complex. histiocytic sarcomas (hss) are neoplasms of idcs and therefore can arise in almost any tissue, frequently the spleen, lung, skin, meninges, lymph nodes, bone marrow, and synovium. secondary involvement of the liver is common as the disease progresses. this neoplasm is most commonly diagnosed in dogs, and a lower incidence is seen in cats. localized histiocytic sarcoma may be a focal solitary lesion or multiple nodules within a single organ. disseminated histiocytic sarcoma describes classify lymphomas. the morphologic features used in histopathologic classification are the following: • histologic pattern-nodular or diffuse. • cell size-the nuclei of the neoplastic lymphocytes are compared to the diameter of a red blood cell (rbc ≅ µm). small is less than . times the diameter of an rbc; intermediate is . to . times the diameter of an rbc; large is more than . times the diameter of an rbc. • grade-mitotic figures are counted in a single high-power ( ×) field. indolent is to ; low is to ; mid is to ; high is more than . although there are numerous subtypes of lymphoma recognized under the who system, a detailed discussion of each subtype is outside the scope of this textbook. however, a select number of subtypes are more commonly seen in domestic animals (see table - ) and currently best described in the dog (see disorders of dogs, neoplasms, lymphomas). the most common types in dogs are large cell lymphomas and include diffuse large b cell lymphoma and peripheral t cell lymphoma. t cell-rich large b cell lymphoma is thought to be a variant of diffuse large b cell lymphoma with a distinctive reactive t lymphocyte infiltrate. intermediate cell lymphomas include b or t lymphocyte lymphoblastic lymphomas and burkitt-like lymphoma (both high grade), marginal zone lymphoma, and the intermediate cell variant of t zone lymphomas (both indolent and nodular). small cell lymphomas most commonly diagnosed in domestic animal species include enteropathy-associated t cell lymphoma, commonly seen in the cat, t zone lymphoma (small cell variant), and small cell lymphoma. cutaneous lymphomas are most often of t lymphocyte origin and may be epitheliotropic or nonepitheliotropic, and a distinct entity of inflamed t cell lymphoma has been recently described in dogs (see chapter ). plasma cell neoplasia. plasma cell neoplasms are most easily categorized as myeloma or multiple myeloma, which arises in the bone marrow, and extramedullary plasmacytoma, which as the name implies involves sites other than bone. multiple myeloma. see bone marrow and blood cells, disorders of domestic animals, types of hematopoietic neoplasia, plasma cell neoplasia. extramedullary plasmacytomas. extramedullary plasmacytomas are most commonly diagnosed in the skin of dogs (also cats and horses), where they constitute . % of all canine cutaneous tumors (see chapter ). the pinnae, lips, digits, and chin are the most commonly affected locations, and most lesions are solitary, though multiple plasmacytomas are infrequently diagnosed. other tissues affected include the oral cavity, intestine (colorectal in particular), liver, spleen, kidney, lung, and brain; of these, the oral cavity and intestine (colorectal) are involved most often. in one study, extramedullary plasmacytomas represented % of all canine oral tumors and % of all extramedullary plasmacytomas diagnosed. most cutaneous extramedullary plasmacytomas are benign, and complete excision is usually curative; oral cavity and colorectal extramedullary plasmacytomas are likely to behave in a similar manner. more aggressive forms may occur at any site. as with multiple myeloma, the neoplastic cells composing the tumor may vary from well differentiated to pleomorphic, often within the same tumor. the cells often have a characteristic perinuclear golgi clearing or "halo," and the more pleomorphic cells exhibit karyomegaly and binucleation (fig. - ) . extramedullary plasmacytomas may produce monoclonal immunoglobulins with resulting monoclonal gammopathy. amyloid deposition (which may mineralize) is also observed in a proportion of cases. differentiation from other round cell tumors may be aided by severe combined immunodeficiency disease of arabian foals is an autosomal recessive primary immunodeficiency disorder characterized by the lack of functional t and b lymphocytes caused by a genetic mutation in the gene encoding for dna-dependent protein kinase catalytic subunit (dna-pkcs). this enzyme is required for receptor gene rearrangements involved in the maturation of lymphocytes, and the resulting loss of functional t and b lymphocytes leads to a profound susceptibility to infectious diseases. though normal at birth, these foals develop diarrhea and pneumonia by approximately days of age, often due to adenovirus, cryptosporidium parvum, and pneumocystis carinii infections. affected foals often die before months of age. lymph nodes and thymus are small and often grossly undetectable, and the spleen is small and firm due to the absence of white pulp (see fig. - ) . the development of genetic tests to identify carriers of the disorder has led to a decrease in the prevalence of severe combined immunodeficiency disease. recently, severe combined immunodeficiency disease was diagnosed in a single caspian filly, though the exact genetic defect was not determined. congenital immunodeficiency diseases are also discussed in detail in chapter . streptococcus equi ssp. equi, the etiologic agent of equine strangles, is inhaled or ingested after direct contact with the discharge from infected horses or from a contaminated environment. the bacteria attach to the tonsils, penetrate into deeper tissues, enter the lymphatic vessels, drain to regional lymph nodes (mandibular, retropharyngeal, and occasionally parotid and cervical lymph nodes), and cause large abscesses (see fig. - ). retropharyngeal enlargement from abscesses may lead to compression of the pharynx and subsequent respiratory stridor and dysphagia. abscesses may rupture and discharge pus through a sinus to the skin surface or spread medially into guttural pouches, where residual pus dries and hardens to form chondroids (which serve as a nidus for live bacteria to persist in carrier animals). in up to % of these cases, ruptured abscess material may spread via blood or lymph to other organs (metastatic abscess formation, bastard strangles), including lung, liver, kidney, synovia, mesenteric and mediastinal lymph nodes, spleen, and occasionally brain. purpura hemorrhagica, a type iii hypersensitivity reaction, may result in necrotizing vasculitis in some horses with repeated natural exposure to s. equi ssp. equi or after vaccination in horses that have had strangles. the typical manifestation of r. equi infection is chronic suppurative bronchopneumonia with abscesses (see chapter ). approximately % of foals also develop intestinal lesions characterized by pyogranulomatous ulcerative enterotyphlocolitis, often over peyer's patches, and pyogranulomatous lymphadenitis of mesenteric and colonic lymph nodes (see chapter ; see fig. - ) . large abdominal abscesses may be the only lesion in the abdomen and presumably originate from an infected mesenteric lymph node. the diffuse lymphatic tissue in the lamina propria may contain granulomatous inflammation with the phagocytized bacteria. mediastinal pyogranulomatous lymphadenitis may compress the trachea, causing respiratory distress. r. equi lesions also can develop in the liver, kidney, spleen, or nervous tissue. lymphoma is the most common malignant neoplasm in horses and mostly affects adult animals (mean age to years) with no lesions that involve distant sites and has replaced the term malignant histiocytosis. breed predispositions to histiocytic sarcoma complex are seen in bernese mountain dogs, rottweilers, golden retrievers, and flat-coated retrievers, though the disease can occur in any breed. histiocytic sarcoma complex is considered to have a rapid and highly aggressive course, and the clinical signs depend on the particular organ(s) involved. grossly, affected organs may be uniformly enlarged and/or contain multiple coalescing white-tan nodules. tissue architecture is effaced by sheets of pleomorphic round to spindle-shaped cells. there is marked cellular atypia with numerous karyomegalic and multinucleated neoplastic cells (fig. - ) . hemophagocytic histiocytic sarcoma. hemophagocytic histiocytic sarcoma is seen in dogs and cats and is a neoplasm of macrophages of the spleen and bone marrow. clinically, dogs present with hemolytic regenerative anemia and thrombocytopenia, thus mimicking evans's syndrome, though they are coombs negative. this form of histiocytic sarcoma carries the worst prognosis of the histiocytic sarcomas, which is likely in part related to the severe anemia and coagulopathy. it is characterized by a non-mass forming infiltrate of histiocytes within the bone marrow and splenic red pulp, causing diffuse splenomegaly. the neoplastic cells exhibit marked erythrophagocytosis, but the severe cellular pleomorphism seen in the histiocytic sarcoma complex may be lacking. the neoplastic cells are often intermixed with emh and plasma cells. metastasis is frequently to the liver, where the cells concentrate within the sinuses. tumor emboli within the lung are often present. malt is involved in a variety of ways with bacteria and viruses, and these are summarized for large animals in table - . these interactions include being a portal of entry for pathogens (e.g., salmonella spp., yersinia pestis, map, and l. monocytogenes); a site of replication for viruses (e.g., bvdv); a site for hematogenous infection (e.g., panleukopenia virus and parvovirus); and a site of gross or microscopic lesions in some viral diseases. bovine coronavirus, bvdv, rinderpest virus, malignant catarrhal fever virus, feline panleukopenia virus, and canine parvovirus cause lymphocyte depletion within the malt. anthrax is caused by b. anthracis, a gram-positive bacillus found in spore form in soil. cattle, sheep, and goats become infected when grazing on infected soil, and infection causes fulminant septicemia. the spleen in infected animals is markedly enlarged and congested (see uniform splenomegaly with a bloody consistency; see also chapter ). bovine viral diarrhea is caused by bvdv, a pestivirus. cattle are the natural host, but other animals such as alpacas, deer, sheep, and goats are also affected. bvdv preferentially infects cells of the immune system, including macrophages, dcs, and lymphocytes. the associated lesions in lymphoid tissues are severe lymphoid depletion in mesenteric lymph nodes and peyer's patches, whose intestinal surface may be covered by a fibrinonecrotic membrane. histologically, there is marked lymphocytolysis and necrosis of germinal centers in peyer's patches and cortices of lymph nodes. there is thymic atrophy because the thymus is markedly depleted of lymphocytes and may consist of only collapsed stroma and few scattered lymphocytes. bvd is discussed in detail in chapters and . apparent breed or sex predisposition. the most frequent anatomic locations of equine lymphoma are multicentric, cutaneous, and gastrointestinal tract. multicentric lymphoma, defined as involving at least two organs (excluding the regional lymph nodes), is the most common manifestation, followed by skin and gastrointestinal tract types. solitary locations have been reported in the mediastinum, lymph nodes, ocular/orbital region, brain, spinal cord, oral cavity, and spleen. of the multicentric lymphomas, the most frequently observed type is t cell-rich large b cell lymphoma (tcrlbcl), reportedly in one study affecting % of the cases. peripheral t cell lymphoma (ptcl) was the second most common, followed by diffuse large b cell lymphoma (dlbcl). the most common lymphoma type in the gastrointestinal tract is also t cell-rich large b cell lymphoma, followed by enteropathyassociated t cell lymphoma. cutaneous lymphomas in horses account for up to % of all equine skin tumors. t cell-rich large b cell lymphoma is again the most common lymphoma subtype in the skin, representing up to % of all cutaneous lymphomas, and most frequently presents clinically as multiple skin masses. cutaneous t cell lymphoma (ctcl) is the second most common form and arises as smaller solitary nodules. thoroughbreds may have a higher incidence of cutaneous t cell lymphoma compared to other breeds. overall, horses with cutaneous t cell-rich large b cell lymphoma appear to have a longer survival time than horses with other types of lymphoma of the skin. progesterone receptor-positive lymphomas have also been identified in horses, and there is one report of subcutaneous tumor regression following removal of an ovarian granulosa-theca cell tumor. there may be an increased frequency of lymphoma in horses diagnosed with equine herpesvirus (ehv- , gammaherpesvirus), when compared to healthy horses, although the exact cause-effect role of this observation in lymphomagenesis is not yet known. histologically, the hallmark features of t cell-rich large b cell lymphoma include a majority of small (nuclei approximately the size of an rbc), reactive, mature t lymphocytes admixed with a neoplastic population of large b lymphocytes whose nuclei are two to three times the diameter of an equine rbc. these large atypical cells are often binucleated and have prominent eosinophilic nucleoli ( fig. - ). the large cells may be observed in mitosis or in necrosis as single cells with retracted cytoplasm and pyknotic nuclei. t cell-rich large b cell lymphoma is often accompanied by the presence of a dense fibrovascular network. johne's disease primarily affects domestic and wild ruminants (and rarely pigs and horses) and is due to infection by map. the characteristic lesions include granulomatous enteritis usually confined to the ileum, cecum, and proximal colon; lymphangitis; and lymphadenitis of regional lymph nodes (see fig. - ) . the bacteria are ingested, engulfed by the m cells overlying peyer's patches, and then transported to macrophages in the lamina propria and submucosa. among cattle, sheep, goats, and wild ruminants, there is wide variation in the severity, distribution of lesions, primary inflammatory cell type (lymphocytes, epithelioid macrophages, multinucleated giant cells), and numbers of bacteria within lesions (multibacillary or paucibacillary). histologically, the architecture of the ileocecal lymph nodes may be partially replaced by aggregates of epithelioid postweaning multisystem wasting syndrome pcv , a small single-stranded dna virus, is highly prevalent in the domestic pig population. several clinical syndromes are attributed to pcv infection and collectively termed pcv-associated diseases (pcvads). these include postweaning multisystemic wasting syndrome (pmws), porcine respiratory disease complex (prdc), porcine dermatitis and nephropathy syndrome, and enteric disease (see chapters and ). the major postmortem findings of postweaning multisystemic wasting syndrome are poor body condition, enlarged lymph nodes, and interstitial pneumonia. the lesions of the lymphoid system are commonly observed in the tonsil, spleen, peyer's patches, and lymph nodes. some pigs have all lymphoid tissues affected, whereas others may have only one or two affected lymph nodes. the characteristic microscopic lesions are lymphoid depletion of both follicles and paracortex with replacement by histiocytes, mild to severe granulomatous inflammation with multinucleated giant cells, and intrahistiocytic sharply demarcated, spherical, basophilic cytoplasmic inclusion bodies. necrosis of prominent lymphoid follicles (necrotizing lymphadenitis) is occasionally observed, and pcv can be detected within the necrotic regions. the loss of lymphocytes may be due to reduced production in the bone marrow, decreased proliferation in the secondary lymphoid organs, or necrosis of lymphocytes. splenic abscesses can be the result of bacteremia (see fig. - ) or direct penetration by a foreign body from the reticulum (see spleen and also portals of entry/pathways of spread). c. pseudotuberculosis is a gram-positive intracellular bacterium that causes caseous lymphadenitis, a chronic suppurative disease of sheep and goats. the bacterium may enter through skin wounds (e.g., shearing cuts in sheep, tagging, tail docking, or castration), drain to the regional lymph node, and then be disseminated in lymph and circulating blood to external and internal lymph nodes, as well as other internal organs, including lung. external abscesses are most often detected in the "jaw and neck" region, specifically in the mandibular and parotid lymph nodes. on gross examination the abscesses are encapsulated and filled with greenish semifluid pus due to an infiltrate of eosinophils (see fig. - ) . over time the abscesses lose the greenish hue, and contents become inspissated to form the characteristic concentric laminations (see fig. - ); old abscesses may reach a diameter of to cm. bovine lymphoma is broadly classified into enzootic and sporadic forms. the enzootic form, called enzootic bovine leukosis (ebl), is caused by blv, a retrovirus common in cattle. there is a higher prevalence in dairy cattle compared to beef breeds. blv is transmitted horizontally (e.g., blood, milk/colostrum, saliva) or iatrogenically (e.g., rectal sleeves, instruments/equipment). following infection, blv invades and integrates into the genome of infected b lymphocytes, resulting in a polyclonal b lymphocyte lymphocytosis in approximately % of cattle. in approximately % to % of blv-infected cattle, a single clone will emerge, leading to the development of b lymphocyte leukemia/lymphoma. the average incubation period between infection and development of lymphoma is to years, and this low conversion rate suggests that the latency period may be longer than the life span of most animals (dairy cattle seldom live to the -to -year peak incidence of lymphoma occurrence). other contributing variables, such as genetic background, coinfections, and environmental factors, may also play a role in lymphomagenesis. the exact mechanism of blv-induced tumorigenesis is poorly understood. recently blv micrornas (mirnas) were identified in preleukemic and malignant b lymphocytes, which showed repression of structural and regulatory gene expression. these findings suggested that mirnas may play a key role in tumor onset and progression. grossly, multiple tissues may be affected in cattle that develop lymphoma, including peripheral lymph nodes (cephalic, cervical, sublumbar) ( fig. - ) , abdominal lymph nodes, retrobulbar region, abomasum, liver, spleen, heart, urogenital tract, bone marrow, vertebral canal ( fig. - ) , and spinal cord. one study indicates most of these high-grade lymphomas are diffuse large cell lymphomas ( %), and approximately % are intermediate cell lymphomas (burkitt-like and lymphoblastic lymphomas). the sporadic form of bovine lymphoma is most often of t lymphocyte immunophenotype and has three subcategories: cutaneous, calf, and thymic. there is no known viral cause for the sporadic form, and each subcategory has a much smaller prevalence compared to the enzootic form. of the three sporadic forms, the cutaneous form seems to be the most common and manifests itself as multiple skin nodules in -to -year-old cattle. the calf form presents as generalized lymphadenopathy with weight loss, lethargy, and weakness in calves less than months old. the thymic form is reportedly more common in beef cattle, to months of age. lymphoma is the most frequently reported cancer of pigs based on abattoir surveys. affected pigs are typically less than year of age, and there is no reported breed predisposition, although a hereditary basis is suspected in cases arising in inbred herds. the two main forms of porcine lymphoma are thymic/mediastinal and multicentric; the latter is more common. spleen, liver, kidney, bone marrow, and lymph nodes are affected in the multicentric form, with visceral lymph nodes reportedly more commonly involved than peripheral nodes. a recent study of lymphoma in pigs found the majority to be multicentric, and subtypes included the following: b lymphoblastic leukemia/lymphoma, follicular lymphoma, diffuse and intestinal large b cell lymphoma, and peripheral t cell lymphoma. one case each of thymic b cell and t cell lymphomas were also described. several types of severe combined immunodeficiency diseases have been described in dogs. a mutation in dna-pkcs (similar to arabian horses) with an autosomal recessive mode of inheritance is seen in jack russell terriers. an x-linked form of severe combined immunodeficiency disease is well described in basset hounds and is caused by mutations in the common γ-chain (γc) subunit of the receptors for il- , il- , il- , il- , il- , and il- . a similar disease is seen in cardigan welsh corgi puppies, though it is an autosomal mode of inheritance in this breed. the mutation inhibits the signal transduction pathways initiated by any of these cytokines, which are critical for the proliferation, differentiation, survival, and function of b and t lymphocytes. affected dogs have normal numbers of circulating b lymphocytes that are unable to class switch to igg or iga and reduced numbers of t lymphocytes, which are nonfunctional due to the inability to express il receptors. affected puppies are remarkably susceptible to bacterial and viral infections and rarely survive past to months of age. the thymus of these dogs is small and consists of only small dysplastic lobules with a few of hassall's corpuscles. tonsils, lymph nodes, and peyer's patches are often grossly unidentifiable due to the severe lymphocyte hypoplasia. congenital immunodeficiency diseases are also discussed in detail in chapter . thymic hemorrhage and hematomas have been reported in dogs and are most often seen in young animals. a variety of causes are described, including ingestion of anticoagulant rodenticides (warfarin, dicumarol, diphacinone, and brodifacoum), dissecting aortic aneurysms, trauma (e.g., automobile accident), and idiopathic/ spontaneous. histologically, hemorrhage variably expands the thymic lobules and septa, and in severe cases the lobular architecture is obscured by hemorrhage. in cases of anticoagulant rodenticide toxicosis, the medulla appears to be the main site of hemorrhage. see uniform splenomegaly with a bloody consistency (also see figs. - and - ). see the section on splenic nodules with a bloody consistency for discussion on splenic hematomas (including those induced by nodular hyperplasia or occurring with hemangiosarcoma), incomplete splenic contraction, acute splenic infarcts, and hemangiosarcomas. porcine reproductive and respiratory syndrome (prrs) is caused by an arterivirus and causes two overlapping clinical syndromes: reproductive failure and respiratory disease. the virus is transmitted by contact with body fluids (saliva, mucus, serum, urine, and mammary secretions and from contact with semen during coitus), but often it first colonizes tonsils or upper respiratory tract. the virus has a predilection for lymphoid tissues (spleen, thymus, tonsils, lymph nodes, peyer's patches). viral replication takes place in macrophages of the lymphoid tissues and lungs, though porcine reproductive and respiratory syndrome virus antigen is found in resident macrophages in many tissues and may persist in tonsil and lung macrophages. the result of this infection is a reduction in the phagocytic and functional capacity of macrophages of the monocytemacrophage system. as a consequence, there is reduction in resistance to common bacterial and viral pathogens. most porcine reproductive and respiratory syndrome-infected pigs are coinfected with one or more pathogens, including streptococcus suis and salmonella choleraesuis. infection with bordetella bronchiseptica and mycoplasma hyopneumoniae appear to increase the duration and severity of the interstitial pneumonia. the major lesions are interstitial pneumonia and generalized lymphadenopathy, and tracheobronchial and mediastinal lymph nodes are most commonly affected. coinfections often complicate the gross and histopathologic changes. lymph nodes are enlarged, pale tan, occasionally cystic, and firm; some strains of virus also cause nodal hemorrhage. microscopically, the lesions in the lymph nodes, tonsils, and spleens consist of varying degrees of follicular and paracortical hyperplasia and lymphocyte depletion in follicular germinal centers. streptococcus porcinus causes jowl abscesses in pigs. the bacteria colonize the oral cavity and spread to infect tonsils and regional lymph nodes. the mandibular lymph nodes are the most often affected and have multiple, -to -cm abscesses; the retropharyngeal and parotid lymph nodes may also be involved (fig. - ). this once-prevalent disease is now rare, presumably due to improvements in husbandry, feeder design, and hygiene. it is occasionally isolated in pigs with bacteremia. disseminated histoplasmosis include wasting, emaciation, fever, respiratory distress, diarrhea with hematochezia or melena, and lameness. the clinicopathologic changes of disseminated histoplasmosis may include neutrophilia, monocytosis, nonregenerative anemia in chronic infections, changes in total serum protein level, and liver enzyme level elevations with hepatic involvement. the anemia is likely a result of chronic inflammation, histoplasma infection of the bone marrow, and/or intestinal blood loss in dogs with gi disease. cytologic examination is useful for the diagnosis of histoplasmosis (tracheal wash preparations, aspirates of bone marrow and lymph nodes), where organisms are often visible in macrophages (e- fig. - ) . grossly, there is hepatosplenomegaly, the intestines are thickened and corrugated, and the lymph nodes are uniformly enlarged (fig. - ) with loss of normal architecture (somewhat similar to siderofibrotic plaques, splenic rupture, and accessory spleens see the section on miscellaneous disorders of the spleen for discussions on siderofibrotic plaques, splenic rupture, and accessory spleens. splenic nodular hyperplasia is common in dogs and categorized based on their cellular components as lymphoid nodular hyperplasia or complex nodular hyperplasia. hematomas may arise within nodules of hyperplasia (see splenic nodules with a bloody consistency). lymphoid (or simple) nodular hyperplasia consists of a focal well-demarcated mass composed of discrete to coalescing aggregates of lymphocytes. the lymphocytes may form follicular structures with germinal centers and/or consist of a mixture of lymphocytes with mantle and marginal zone cell morphologic features. the intervening tissue is often congested and may contain plasma cells, but stroma is not observed (fig. - ; e-fig. - ) . complex nodular hyperplasia is a focal mass that contains two proliferative components: lymphoid and stroma (e- fig. - ) . the lymphoid component resembles lymphoid nodular hyperplasia described above. there is proliferation of the intervening stromal tissues with fibroplasia, smooth muscle hyperplasia, and histiocytic hyperplasia; emh and plasma cells may also be present. it has recently come to light that the entity splenic fibrohistiocytic nodule (sfhn), first described in , is not a single condition, but in fact a complex group of diseases. our better understanding of the spectrum of diseases once described under the term splenic fibrohistiocytic nodule is due to increasing knowledge of histiocytic disorders and immunochemistry. the original definition of splenic fibrohistiocytic nodule is a nodule characterized by a stromal population of histiocytoid and spindle cells intermixed with lymphocytes. grading was based on the lymphocyte percentage of the population (e.g., > % lymphocytes = grade ; < % lymphocytes = grade ); dogs with grade splenic fibrohistiocytic nodule had a much better -year survival rate, and dogs with grade nodules may develop sarcomas (often malignant fibrous histiocytoma, a now outdated term). with our increasing knowledge of histiocytic disorders and additional immunohistochemical stains, diseases that likely were encompassed by the term splenic fibrohistiocytic nodule include the following: complex and lymphoid nodular hyperplasia (see earlier), stromal sarcoma, histiocytic sarcoma, marginal zone hyperplasia, marginal zone lymphoma, and diffuse large b cell lymphoma (see lymphoid/lymphatic system, disorders of domestic animals: lymph nodes, neoplasia, lymphoma). histoplasma capsulatum can cause a disseminated fungal disease that is widely endemic, particularly in areas with major river valleys and temperate or tropical climates (e.g., midwestern and southern united states). free-living organisms in the mycelial phase produce macroconidia and microconidia that are inhaled and converted to the yeast phase in the lung. yeasts are phagocytized and harbored by macrophages of the monocyte-macrophage system. in some dogs the disease is limited to the respiratory tract and causes dyspnea and coughing. however, in most dogs, the disease is disseminated throughout the body, predominantly affecting the liver, spleen, gastrointestinal tract, bone marrow, skin, and eyes; primary gastrointestinal disease is also reported. the clinical signs in cases of and hepatosplenomegaly (e- fig. - ) . histologically, the lymph node sinuses and splenic red pulp are filled with macrophages that contain intracytoplasmic, round, -µm-diameter organisms with a small kinetoplast. though there is an initial stage of lymphoid hyperplasia in the spleen and lymph node, subsequent lymphoid atrophy occurs with chronicity. the atrophy is due to impairment of follicular dcs, b lymphocyte migration, and germinal center formation. there may be lymphoid atrophy of the spleen and lymph nodes in severe chronic infections. canine distemper virus preferentially infects lymphoid, epithelial, and nervous cells (see chapter ). dogs are exposed through contact with oronasal secretions, and the virus infects macrophages within the lymphoid tissue of the tonsil and respiratory tract (including tracheobronchial lymph nodes) and later disseminates to the spleen, lymph nodes, bone marrow, malt, and hepatic kupffer cells. the virus causes necrosis of lymphocytes (especially cd t lymphocytes) and depression of lymphopoiesis in the bone marrow, leading to severe immunosuppression. dogs are therefore susceptible to secondary infections, including bordetella bronchiseptica, toxoplasma gondii, nocardia, salmonella spp., and generalized demodicosis. canine parvovirus type (cpv- ) is a highly contagious disease of dogs spread through the fecal-oral route or oronasal exposure to contaminated fomites. the virus has tropism for rapidly dividing cells, and replication begins in the lymphoid tissues of the oropharynx, thymus, and mesenteric lymph nodes and then is disseminated to the small intestinal crypt epithelium. by infecting lymphoid tissues, canine parvovirus type causes immunosuppression directly through lymphocytolysis and indirectly though bone marrow depletion of lymphocyte precursors. there is marked lymphoid atrophy of thymus and follicles of the spleen, lymph nodes, and maltparticularly of peyer's patches to produce the classic gross lesion of depressed oval regions of the mucosa (so-called punched-out peyer's patches). thymomas. see lymphoid/lymphatic system, disorders of domestic animals: thymus, neoplasia. lymphomas. lymphoma is the most common hematologic malignancy in the dog. using the who classification scheme, several lymphoma subtypes are identified in dogs and clinically range from slow-growing indolent tumors to highly aggressive tumors. of all domestic animal species, lymphoma is the most extensively studied in dogs. the most common clinical presentation in dogs is generalized lymphadenopathy, with or without clinical signs such as lethargy and inappetence. the majority of lymphomas in dogs are large cell mid-to highgrade lymphomas, and up to half of all lymphoma cases are subtyped as diffuse large b cell lymphoma. diffuse large b cell lymphomas are further subdivided into centroblastic or immunoblastic based on nucleolar morphologic features (see table - and box - ), although it is unclear if this difference has any prognostic significance. histologically, lymph node architecture is most often completely effaced by sheets of large neoplastic cells, which may invade through the capsule and colonize the perinodal tissue. these dogs are often treated with chemotherapy and achieve remission. the overall median survival time for dogs with diffuse large b lymphocyte lymphoma is approximately months, although this number lymphoma, though the nodes tend to be more firm in histoplasmosis). histologically within the node, there is a multifocal to coalescing infiltrate of epithelioid macrophages with intracytoplasmic, small ( to µm in diameter) yeast organisms with spherical basophilic central bodies surrounded by a clear halo (fig. - ) . leishmaniasis is a disease of the monocyte-macrophage system caused by protozoa of the genus leishmania. it occurs in dogs and other animals and is endemic in parts of the united states, europe, mediterranean, middle east, africa, and central and south america. the protozoa proliferate by binary fission in the gut of the sand fly and become flagellated organisms, which are introduced into mammals by insect bites, where they are phagocytized by macrophages and assume a nonflagellated form. cutaneous and/or visceral forms of the disease are observed. in the visceral form, dogs are emaciated and have general enlargement of abdominal lymph nodes numerous other subtypes of lymphoma have been reported in dogs, including several forms of cutaneous lymphomas, most often of t lymphocyte origin and epitheliotropic (see chapter ). hepatosplenic t cell lymphoma, thought to be of γ/δ t lymphocyte origin, affects the liver and spleen without significant nodal involvement. hepatocytotropic t cell lymphoma is a distinct form of lymphoma with tropism for the hepatic cords; clusters or individual neoplastic lymphocytes invade the hepatic cords, without hepatocyte degeneration. intravascular lymphoma is a proliferation of large neoplastic lymphocytes within blood vessels of many tissues, leading to progressive occlusion and subsequent thromboses and infarcts. this neoplasm does not form an extravascular mass, and neoplastic cells are not found in peripheral blood smears or bone marrow. indolent lymphomas constitute up to % of all canine lymphomas. indolent lymphomas in dogs, in descending order of frequency, include t zone lymphoma (tzl), marginal zone lymphoma (mzl), mantle cell lymphoma (mcl), and follicular lymphoma (fl). mantle cell lymphoma and follicular lymphoma are less commonly diagnosed than t zone lymphoma and marginal zone lymphoma; therefore the reader is referred to the suggested readings to learn more on mantle cell lymphoma and follicular lymphoma. t zone lymphoma. t zone lymphoma is the most common indolent lymphoma in dogs ( fig. - ) . it presents as a solitary or varies based on the study and the grade of the tumor (as determined by mitotic figures). peripheral t cell lymphomas-not otherwise specified are the second most common subtype in dogs. this category includes all t cell lymphomas that do not fit into the other categories (e.g., t zone lymphoma, enteropathy-associated t cell lymphoma, and hepatosplenic t cell lymphoma). peripheral t cell lymphoma also effaces nodal architecture, and when compared to diffuse large b cell lymphoma, there is more variation in nuclear size and morphologic features. dogs with this subtype tend to have shorter survival times. intermediate cell size, high-grade lymphomas are less common in dogs, and the two most frequently encountered subtypes are lymphoblastic lymphoma (lbl) and burkitt-like lymphoma (bll). lymphoblastic lymphoma may be of b or t lymphocyte origin, though t cell lymphoblastic lymphoma is more common of the two. it is important to recognize a common misuse of the term "lymphoblast" in lymphoblastic lymphoma-by definition in lymphoblastic lymphoma, lymphoblasts are intermediate-sized cells with a distinct dispersed chromatin pattern, and not the large lymphocytes seen in cases of diffuse large b cell lymphoma or peripheral t cell lymphoma. t lymphocyte lymphoblastic lymphoma is an aggressive disease that is often resistant to treatment. burkitt-like lymphoma is a high-grade lymphoma of b lymphocytes. consequently there is marked lymphoid atrophy of thymus, spleen, lymph node, and malt (particularly peyer's patches). see bone marrow and blood cells, disorders of domestic animals, types of hematopoietic neoplasia, myeloid neoplasia, mast cell neoplasia and see e- fig. - . lymphoma is the most commonly diagnosed neoplasm in cats, and the incidence is reportedly the highest for any species. mediastinal or multicentric lymphomas are seen in young, felv-infected cats (see fig. - ). with the advent of felv vaccine and routine testing, the prevalence of felv-associated lymphoma is decreased. currently the alimentary tract is the most commonly affected site, and typically occurs in cats greater than years of age . other miscellaneous sites commonly affected are brain, spinal cord, eye, kidney, and nasopharynx. the retrovirus felv has long been recognized as a cause of lymphoma in cats-the risk for lymphoma is increased sixtyfold in infected cats. before the advent of a vaccine in , approximately % of cats (mainly young animals) with lymphoma were felv positive. felv infects t lymphocytes and can cause myelodysplastic syndrome, acute myeloid leukemias (see myeloid neoplasia), and t lymphocyte leukemia/lymphoma. in the latter the mediastinum (thymus, mediastinal, and sternal lymph nodes) is the site most commonly involved, although a multicentric distribution also occurs. routine felv vaccination has led to a significant decrease in the prevalence of felv infection, which has resulted in a decrease in the proportion of mediastinal lymphomas. the risk for developing lymphoma in fiv-infected cats is fivefold to sixfold higher than in uninfected cats. cats that underwent kidney transplantation and thus received immunosuppressive drug therapy had a similar risk for developing lymphoma. both fivinfected and posttransplantation cats predominantly developed extranodal, high-grade, diffuse large b lymphocyte lymphomas. this form is also the most common subtype in human immunodeficiency virus and posttransplantation patients caused by the epstein-barr virus (ebv). therefore it is reasonable to question whether these two groups of immunosuppressed cats may be more prone to infection by a gammaherpesvirus similar to ebv, leading to lymphoma. recently a novel feline gammaherpesvirus (fcaghv ) was multiple peripheral lymphadenomegaly (often mandibular lymph nodes) in otherwise healthy-appearing dogs. the characteristic histopathologic architecture is a nodular expansion of the paracortex by neoplastic cells, which push atrophied "fading" cortical follicles against the thinned capsule and trabeculae. this unique architectural feature is best highlighted with immunohistochemical stains (often cd for t lymphocytes and cd a, pax , or cd for b lymphocytes). the neoplastic cells are small to intermediate in size with pale eosinophilic cytoplasm and oval nuclei with sharp shallow indentations. mitotic figures are rare. dogs with this lymphoma subtype tend to be diagnosed with an advanced stage of the disease, likely because they present clinically healthy, without loss of appetite or activity level. even so, dogs with t zone lymphoma have a relatively long survival time compared to other lymphomas: reports on median survival time range from to months, and data suggest that dogs who do not receive chemotherapy actually have longer median survival times. marginal zone lymphoma. marginal zone lymphoma is an indolent b lymphocyte neoplasm derived from the cells of the marginal zone of lymphoid follicles. most marginal zone lymphomas (and mantle cell lymphomas) are assumed to originate in the spleen with slow spread to lymph nodes and often present as a mottled white-red smooth spherical splenic mass. histopathologic assessment of tissue architecture is needed for a diagnosis of marginal zone lymphoma and is characterized by a distinct nodular pattern in which the lighter-staining neoplastic marginal zone cells form a dense cuff around small foci of darkly stained mantle cells (fading follicles). the neoplastic marginal zone lymphocytes are intermediate in size and have a single prominent central nucleolus. mitotic figures are often rare or absent early on and increase with disease progression. differentiating between marginal zone lymphoma and marginal zone hyperplasia (which refers to a proliferation of marginal zone cells and contains a mixture of small and intermediate lymphocytes) is challenging because marginal zone lymphoma arises on the background of marginal zone hyperplasia. additionally, lymphoid and complex nodular hyperplasia are common in the dog spleen (see disorders of dogs), and it is possible that many cases of nodular hyperplasia contain areas of marginal zone lymphoma. therefore immunophenotyping and molecular clonality are ultimately required for a definitive diagnosis of marginal zone lymphoma. the overall median survival time in dogs with splenic marginal zone lymphoma after splenectomy is approximately months (even longer if it is diagnosed as an incidental finding). plasmacytomas. see disorders of domestic animals: lymph nodes, neoplasia, plasma cell neoplasia, extramedullary plasmacytomas (see fig. - ). feline panleukopenia, caused by the single-stranded dna virus feline parvovirus (fpv), is a highly contagious and often lethal disease of cats and other felidae, as well as other species (including raccoons, ring-tailed cats, foxes, and minks). fpv is transmitted by the fecal-oral route through contact with infected body fluids, feces, or fomites. following intranasal or oral infection, the virus initially replicates in the macrophages in the lamina propria of the oropharynx and regional lymph nodes, followed by viremia, which distributes the virus throughout the body. because fpv requires rapidly multiplying cells in the s phase of division for its replication, replication occurs in mitotically active tissues (lymphoid tissue, bone marrow, and intestinal mucosa). by infecting lymphoid tissues, fpv causes immunosuppression directly through lymphocytolysis and of the small intestine. the neoplastic cells are small (nuclei are equal to the diameter of a feline rbc), mitotic figures are infrequent (low grade), and mucosal and crypt epitheliotropism is common (see fig. - ) . a diagnosis of this subtype of lymphoma may be difficult (particularly in endoscopic biopsy samples), because this disease often is multifocal and concurrent with or arises within lymphoplasmacytic inflammatory bowel disease (ibd). the neoplastic lymphocytes are morphologically similar to the inflammatory lymphocytes. early small cell mucosal t cell lymphomas often require additional diagnostic testing, namely, immunohistochemistry and molecular clonality testing (pcr for antigen receptor rearrangement [parr]) to confirm a clonal neoplasm. transmural t cell lymphomas also occur focally or multifocally in the small intestine of cats (best classified as enteropathy-associated t cell lymphoma type i) and by definition must extend into the submucosa and muscularis. some tumors invade the serosa and adjacent mesentery. t cell large granular lymphocyte (lgl) lymphoma is often diagnosed, and the intestinal segments orad and aborad to the transmural mass may also have mucosal lymphoma. gastrointestinal b cell lymphomas are less prevalent in cats but occur in the stomach, jejunum, and ileocecocolic region as transmural lesions. most are diagnosed as diffuse large b cell lymphomas. lymphomas in other sites also occur less frequently in cats. the upper respiratory tract (nasal and/or nasopharyngeal region) is a relatively rare site for lymphoma. however, lymphoma is the most common primary nasal tumor, and diffuse large b cell lymphomas (of immunoblastic type) are the predominant subtype. both cutaneous (cutaneous t cell lymphoma) and subcutaneous lymphomas (usually large cell lymphomas) are rare. presumed solitary ocular lymphomas have also been reported. t cell-rich large b cell lymphoma, also referred to as feline hodgkin-like lymphoma in some studies, is composed of a mixture of reactive small lymphocytes and large neoplastic b lymphocytes, many of which may be binucleated and/or have prominent nucleoli (thus resembling the reed-sternberg cells of human hodgkin's lymphoma). this disease is typically characterized by a distinctive clinical presentation of an indolent unilateral neoplasm of the cervical lymph nodes, which spreads slowly to adjacent nodes within the chain. however, a proportion of cases may go on to develop into a more aggressive multicentric large to anaplastic b lymphocyte lymphoma that can affect peripheral and central nodes and multiple organs. suggested readings are available at www.expertconsult.com. a b discovered in domestic cats with a % prevalence in north america, and further studies to investigate its role in lymphomagenesis are needed. the overall incidence of feline lymphomas has increased, mainly due to an increase in gastrointestinal lymphomas. mucosal t cell lymphoma, also known as enteropathy-associated t cell lymphoma (eatcl type ii), is the most common and arises from diffuse malt partial thromboplastin time (aptt or ptt) • required sample: citrated plasma • measures time for fibrin clot formation after addition of a contact activator, calcium, and a substitute for platelet phospholipid • deficiencies/dysfunction in intrinsic and/or common coagulation pathway (all factors except for vii and xiii) causes prolongation of ptt • insensitive test-prolongation requires % deficiency. • other causes of prolongation include polycythemia (less plasma per unit volume, so excess amount of citrate is available to chelate calcium) and heparin therapy activated clotting time (act) • required sample: nonanticoagulated whole blood in special act tube (diatomaceous earth as contact activator) used in practice setting-performed by warming sample to body temperature, monitoring for clot formation; normal clotting times are within to seconds in dogs, seconds in cats • less sensitive version of ptt-prolongation requires % deficiency severe thrombocytopenia may cause prolongation. • one-stage prothrombin time (ospt or pt) • required sample: citrated plasma • measures time for fibrin clot formation after addition of tissue factor (tf; thromboplastin), calcium, and a substitute for platelet phospholipid • deficiencies/dysfunction in extrinsic (factor vii) and/or common coagulation pathway cause prolongation of pt • insensitive test-prolongation requires % deficiency proteins induced by vitamin k antagonism or absence (pivka) test • required sample: citrated plasma • essentially a version of the pt using an especially sensitive thromboplastin reagent • pivka are inactive (uncarboxylated) vitamin k-dependent factors; an increase in pivka is not specific for vitamin-k antagonism but may be an earlier and more sensitive detector than pt or ptt • thrombin time (tt) • required sample: citrated plasma • measures time for fibrin clot formation after thrombin (factor iia) is added • defects directly involving formation and/or polymerization of fibrin prolong this test (i.e., if the lesion is upstream of the conversion of fibrinogen to fibrin, the tt will be normal). hypofibrinogenemia or dysfibrinogenemia causes prolongation of the tt required sample: citrated plasma. • fibrinogen concentration measured based on time to clot formation after addition of thrombin; this is essentially the same as the tt mentioned earlier and is a more accurate method than the heat precipitation method • decreased fibrinogen may be because of increased consumption (disseminated intravascular coagulation) or decreased production (liver disease) increased fibrinogen is associated with inflammation, renal disease, and dehydration required sample: special fdp tube. • used in the practice setting. • performed by adding blood to a special tube containing thrombin and a trypsin inhibitor (sample clots almost instantly in normal dogs and cats) and incubating two dilutions of serum ( : and : ) with polystyrene latex particles coated with sheep anti-fdp antibodies (should be negative in normal dogs and cats required sample: citrated plasma. • latex agglutination test. • to date, only validated in dogs and horses assay detects a specific type of fdp resulting from breakdown of cross-linked fibrin; concentration of plasma d-dimer indicates the degree of fibrinolysis; often used as part of a disseminated intravascular coagulation panel • required sample: citrated plasma. • decreased because of decreased production (liver disease), loss (protein-losing nephropathy or enteropathy) • specific factor assays • required sample: citrated plasma. • performed at specialized laboratories intermediary spleen microvasculature in canis familiaris-morphological evidence of a closed and open type molecular methods to distinguish reactive and neoplastic lymphocyte expansions and their importance in transitional neoplastic states characteristics, diagnosis, and treatment of inherited platelet disorders in mammals making sense of lymphoma diagnostics in small animal patients canine granulocytic anaplasmosis: a review lymphoid tissues and organs lymph nodes and thymus diagnostic cytology and hematology of the dog and cat two hundred three cases of equine lymphoma classified according to the world health organization (who) classification criteria hepcidin: a key regulator of iron metabolism and mediator of anemia of inflammation changes to bovine hematology reference intervals from to atlas of veterinary hematology: blood and bone marrow of domestic animals pathogenesis, laboratory diagnosis, and clinical implications of erythrocyte enzyme deficiencies in dogs, cats, and horses feline leukemia virus infection and diseases tumors of the hemolymphatic system proposed criteria for classification of acute myeloid leukemia in dogs and cats immunobiology: the immune system in health and disease extramedullary hematopoiesis: a new look at the underlying stem cell niche, theories of development, and occurrence in animals lineage-specific hematopoietic growth factors hepatosplenic and hepatocytotropic t-cell lymphoma: two distinct types of t-cell lymphoma in dogs histology and cell biology: an introduction to pathology lymphoid neoplasms in swine jc: robbins & cotran pathologic basis of disease the spleen ehrlichiosis and related infections hemotropic mycoplasmas (hemoplasmas): a review and new insights into pathogenic potential clinical, laboratory, and histopathologic features of equine lymphoma classification and clinical features in cases of equine cutaneous lymphoma histologic and immunohistochemical review of splenic fibrohistiocytic nodules in dogs feline gastrointestinal lymphoma: mucosal architecture, immunophenotype, and molecular clonality current state of knowledge on porcine circovirus type -associated lesions molecular pathology of severe combined immunodeficiency in mice, horses, and dogs hematologic abnormalities associated with retroviral infections in the cat pathology of the pig: a diagnostic guide pathologic and prognostic characteristics of splenomegaly in dogs due to fibrohistiocytic nodules: cases splenic myeloid metaplasia, histiocytosis, and hypersplenism in the dog ( cases) fundamentals of veterinary clinical pathology feline parvovirus infection and associated diseases novel gammaherpesviruses in north american domestic cats, bobcats, and pumas: identification, prevalence, and risk factors palmer's pathology of domestic animals veterinary comparative hematopathology histologic classification of hematopoietic tumors of domestic animals. in world health organization international histological classification of tumors in domestic animals, second series canine lymphomas: association of classification type, disease stage, tumor subtype, mitotic rate, and treatment with survival classification of canine malignant lymphomas according to the world health organization criteria canine indolent nodular lymphoma the revision of the world health organization (who) classification of myeloid neoplasms and acute leukemia: rationale and important changes the coombs' test in veterinary medicine: past, present, future a retrospective study of the incidence and the classification of bone marrow disorders in the dog at a veterinary teaching hospital ( - ) schalm's veterinary hematology chronic lymphocytic leukemia in dogs and cats: the veterinary perspective grossly, incompletely contracted areas are characterized by multiple, variably sized and irregularly shaped, dark red to black, raised, soft, blood-filled "nodules." these areas are usually at the margins of the spleen, and the intervening tissues are depressed and pink-red, corresponding to the contracted portions of red pulp devoid of blood. incompletely contracted areas may be confused with acute splenic infarcts or hematomas on gross examination.acute splenic infarcts. splenic infarcts are wedge-shaped or triangular hemorrhagic lesions that occur primarily at the margins of the spleen. in dogs, splenic infarcts most often occur with hypercoagulable states (e.g., liver disease, renal disease, cushing's disease), neoplasia, and cardiovascular disease. splenic vein thrombi may occur in association with traumatic reticulitis, splenic abscesses, portal vein thrombosis, and arterial thrombosis in bovine theileriosis in cattle. valvular endocarditis may also lead to multiorgan infarcts, including the spleen. splenic infarcts are common in pigs with classical swine fever.acute splenic infarcts may not always be grossly visible in the early stages but develop into discrete, dark red and blood-filled, bulging, wedge-shaped foci with the base toward the splenic capsule ( fig. - , a) . with chronicity the lesion becomes gray-white and contracted due to fibrosis (see fig. - , b).hemangiosarcoma. hemangiosarcoma is a malignant neoplasm of endothelial cells and is a common primary tumor of the spleen, especially in dogs. benign splenic hemangiomas are extraordinarily rare. grossly, hemangiosarcomas may appear as single, multifocal, or coalescing dark red-purple masses and cannot be easily differentiated from a hematoma ( fig. - ). on cut surface they are bloody with varying amounts of soft red neoplastic tissue; in more solid areas the neoplasm can be slightly more firm and whitetan. metastatic spread occurs early in the disease process. seeding of the peritoneum results in numerous discrete red-black masses throughout the omentum and serosa of abdominal organs, and hematogenous spread to liver and lung are common. hemangiosarcomas in dogs also occur in the right atrium of the heart, retroperitoneal fat, and skin (dermal and/or subcutaneous) and multiorgan hemangiosarcomas are described in horses, cats, and cattle. because hemangiosarcomas have often metastasized at the time of initial accumulations of macrophages within several organs, including splenic macrophages, kupffer cells of the liver, and macrophages in the brain are often observed.splenic nodules with a bloody consistency. the most common disorders of the spleen with bloody nodules are ( ) hematomas, including those induced by nodular hyperplasia or occurring with hemangiosarcoma, ( ) incompletely contracted areas of the spleen, ( ) acute splenic infarcts, and ( ) hemangiosarcomas. the term nodule has been applied rather loosely here. in some of these conditions, such as incompletely or irregularly contracted areas of the spleen, the elevated area of the spleen is not as well defined as the term nodule would imply.hematomas. bleeding into the red pulp to form a hematoma is confined by the splenic capsule, and produces a red to dark red, soft, bulging, usually solitary mass of varying size ( to cm in diameter) (fig. - ). resolution of a splenic hematoma progresses over days to weeks, through the stages of coagulation and breakdown of the blood into a dark red-brown soft mass (fig. - , a) , infiltration by macrophages that phagocytize erythrocytes and break down hemoglobin to form hematoidin and hemosiderin (see fig. - , b), and repair leading to fibrosis. on occasion the capsule (splenic capsule and visceral peritoneum) over the hematoma can rupture, resulting in hemoperitoneum, hypovolemic shock, and death.the origin or cause of many hematomas is unknown. some are due to trauma, and others may also be induced by splenic nodular hyperplasia. it is postulated that as the splenic follicles become hyperplastic they distort the adjacent marginal zone and marginal sinus, which compromises their drainage into sinusoids and red pulp vascular spaces. the result is an accumulation of pooled blood surrounding the hyperplastic nodule, which leads to hematoma formation. splenic hematomas can also occur secondary to the rupture of hemangiosarcomas within the spleen.incompletely contracted areas of the spleen. incompletely or irregularly contracted areas of the spleen are caused by failure of the smooth muscle to contract in response to circulatory shock (hypovolemic, cardiogenic, or septic) or sympathetic "fight-or-flight" response, resulting in a lack of splenic evacuation of stored blood. key: cord- -ujflw b authors: newcomer, benjamin w.; cebra, chris; chamorro, manuel f.; reppert, emily; cebra, margaret; edmondson, misty a. title: diseases of the hematologic, immunologic, and lymphatic systems (multisystem diseases) [image: see text] date: - - journal: sheep, goat, and cervid medicine doi: . /b - - - - . - sha: doc_id: cord_uid: ujflw b nan in this chapter, multisystemic diseases are discussed in small ruminants (sheep, goats, and cervids). these include diseases of the hematologic, immunologic, and lymphatic systems. in general, species will be discussed together, but when pertinent data are available, each species will be considered separately. the terms "cervid" and "deer" have been used interchangeably in parts of this chapter by the authors. an adequate volume of blood for hematologic and biochemical analysis is best obtained from the jugular vein. a docile animal may be restrained in a standing position or tipped up (sheep only) with the head turned away from the jugular vein to be used. wilder ones, such as some cervids, may require restraint devices or chemical sedation. ideally, the animal should be restrained by someone other than the blood collector, although the same person may be able to both restrain a sheep and collect blood if the animal is tipped up or a halter is used (see chapter ) . the animal should be at rest, with minimal excitement. the collector parts or clips the wool or hair to visualize the jugular vein and then uses the hand not holding the needle to apply digital pressure proximally just above the thoracic inlet to block blood movement through the vein. the vessel may take a second or more to distend after pressure is applied. the collector may then use the needlebearing hand to "strum" the vessel and cause the blood to oscillate. if in doubt about whether the distended vessel is the jugular vein, the collector can release the hand placing pressure on the vessel and observe whether the distended vessel disappears; if it does, the distended vessel was probably the jugular vein. the collector should avoid vessels that pulsate because these are likely to be the carotid arteries. the area should be cleaned with alcohol or other disinfectant, water, or a clean, dry gauze sponge. an -or -gauge, -to . -inch needle is usually adequate to collect blood from an adult, whereas a -gauge needle may be used in a neonate. the skin of adults or males may be thicker and more difficult to penetrate with the needle. a syringe or evacuated tube attached to a vacutainer (becton dickinson inc., rutherford, nj) can be used to collect blood. the needle should be plunged through the skin into the vein at an approximate -degree angle. the blood should not come out of the vessel in pulsatile waves; this is suggestive of an arterial stick. after aseptically obtaining an adequate volume of blood, the collector removes the needle and releases the pressure on the vessel near the thoracic inlet. pressure should be applied to the site of puncture for a minute or more to prevent extravascular leakage of blood and hematoma formation. the blood should be carefully transferred to a vial containing the appropriate anticoagulant to prevent red blood cell (rbc) rupture. goat erythrocytes are small and particularly prone to hemolysis. to minimize this problem, goat blood should be collected with a needle and syringe, not a vacutainer. white blood cell (wbc) differential distribution, individual blood cell staining characteristics, and morphology may be assessed by microscopic examination of a stained blood film. the differential distribution provides more information than total wbc count because inflammatory conditions in artiodactyls often result in a shift in neutrophil populations toward more degenerate, toxic, or immature forms without changing the overall wbc count. the preferred anticoagulant for a complete blood count (cbc) is ethylenediaminetetraacetate (edta), and tubes should be filled to ensure the proper blood-to-anticoagulant ratio. blood samples should be processed as soon as possible after collection. if a delay is anticipated, the blood sample should be refrigerated ( ° c) and an air-dried blood smear should be made because prolonged contact of blood with edta causes changes in wbc morphology and the separation of some rbc parasites. blood can be refrigerated for hours and still yield an accurate cbc. a reference range for hematologic data for sheep and goats is provided in table . (see appendix , tables and ) . goats tend to have a low mean corpuscular volume (mcv) because of their small erythrocytes. sheep and goats younger than months old tend to have lower hematocrit, rbc count, hemoglobin, and plasma protein concentrations, as well as a higher total wbc count. neonates often have a high hematocrit at birth that decreases with colostral ingestion. lactating animals may have decreased hematocrits, rbc counts, and hemoglobin concentrations. animals grazing at high altitude (mountain goats and bighorn sheep) tend to have increased rbc counts, hematocrits, and hemoglobin concentrations. interpreting hematologic changes in cervids is more complex. restraint method affects a variety of parameters in non-acclimated individuals. physical restraint yields red cell counts and hematocrit and hemoglobin concentrations that are to % higher than animals immobilized chemically. , neutrophil, lymphocyte, monocyte, and total white cell counts are also to % higher in physically restrained cervids (see appendix , tables and ) . adult deer also have seasonal variations in their hemogram. red cell numbers and related values are highest during midsummer and late winter. white cells, especially neutrophils, are also highest in midsummer, and platelet counts are highest in spring and fall. these changes may relate to diet or to seasonal activities, such as antler growth and rutting conflicts, which increase the chance of trauma. red cell stickling has also been reported in a variety of deer species. this appears to relate to a mutation in hemoglobin's b-globin component, similar to the disorder in people, but no pathologic role has been described. bone marrow aspirates and core biopsy samples taken from sites of active erythropoiesis can be useful to evaluate erythrocyte production and determine the cause of anemia and other hemogram abnormalities. the sites of biopsy include the sternebrae, femur, and ileum. the procedure should be done under chemical sedation or anesthesia (see chapter ) . the area over the biopsy site is clipped and surgically prepared; the sampler should wear sterile gloves to maintain asepsis. aspirates can be obtained by inserting a sterile needle attached to a -or -cc syringe containing one or two drops of edta through the bone and into the bone marrow. drawing back on the syringe plunger several times may aid in the procurement of an acceptable sample; such a sample may consist of as little as . ml of bone marrow. if the sample is going to be processed immediately, no anticoagulant is required. core biopsies are obtained using a jamshidi or westerman-jensen biopsy needle. the skin is incised with a scalpel and the biopsy needle is inserted into the bone and turned several times to obtain a core sample. more than one site may be used. the sampler then closes the skin with sutures or staples. biopsy samples are preserved by placing them in % neutral buffered formalin solution. impression smears can be made from these samples by gently rolling them on a clean glass slide before placing them in the formalin solution. information obtained from bone marrow samples includes subjective data regarding cell density, megakaryocyte numbers, abnormal cells, maturation patterns of rbcs and wbcs, and the ratio of erythroid to myeloid cells. prussian blue stain can be used on bone marrow to demonstrate iron stores. bone marrow aspirates and biopsies are painful and invasive procedures. therefore, animals should be placed on antibiotics and antiinflammatory drugs prophylactically. blood cultures can be useful in diagnosing bacteremia in an intermittently or persistently febrile animal or one with numerous sites of organ infection. ideally, the clinician should obtain the sample before instituting antimicrobial therapy. however, if this is impossible, antimicrobial therapy should be discontinued to hours before sampling. samples should be taken before and during febrile episodes. the jugular vein is most commonly used to attain a blood culture. as described previously, the skin over the jugular vein should be clipped and surgically prepared. the person collecting the blood sample should wear sterile gloves and use a sterile needle and syringe. blood samples should be placed immediately in a blood culture flask. the chances of attaining a positive culture from bacteremic animals increase with the size of the sample up to about ml, but adding more than the recommended amount to any single culture vial may overwhelm the capacity of the specialized antibiotic-absorbing resins within the flasks. the clinician should change the needle on the sample syringe after collecting the blood and before putting the sample in the culture medium. samples should be refrigerated until they can be sent to a diagnostic laboratory, where aerobic and sometimes anaerobic cultures are made. as an alternative to hematologic testing, comparing conjunctival color to swatches on a standardized famacha chart has been used as a rapid and inexpensive assessment of anemia in whole flocks, primarily to assess the impact of haemonchus contortus and other blood-sucking parasites. , results from a number of trials have yielded fair to good sensitivity to packed cell volume and h. contortus load in both sheep and goats. similar to body condition scoring systems, it is essential to calibrate assessors to ensure consistency when using this system. also, some breeds hematocrit (%) - - hemoglobin (g/dl) - . [ ] [ ] [ ] [ ] [ ] red blood cell count ( /ml) - . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] mean corpuscular volume (fl) - - mean corpuscular hemoglobin concentration (g/dl) - [ ] [ ] [ ] [ ] [ ] [ ] [ ] platelet count ( /ml) . - . . - . total white blood cell count (/ml) - , - , segmented neutrophils (/ml) - - band neutrophils (/ml) lymphocytes (/ml) - - monocytes (/ml) - - eosinophils (/ml) - - basophils (/ml) - - total plasma protein (g/dl) . - . . - . fibrinogen (mg/dl) - read differently on the cards, and use of an electronic color analyzer, while more expensive and less field-friendly, may detect anemia earlier (see chapter , figure . a, b, and chapter ) . easy use of this technique in deer is limited by their intractability and has not been reported. the most common and significant abnormality of the hemogram is anemia. anemia occurs most commonly after blood loss, hemolysis, or chronic disease. blood loss is usually covert and commonly caused by gastrointestinal or external parasites. overt blood loss is usually caused by major trauma such as that caused by dog bites, severe lacerations, male rivalry fighting, or complications of castration or dehorning. cbc values appear normal immediately after acute blood loss. however, after a few hours of fluid redistribution, anemia and hypoproteinemia are evident. evidence of red cell regeneration (macrocytosis, reticulocytosis, and nucleated red cells) should appear within a day or two of the blood loss. hemolysis occurs most commonly after ingestion of toxic plants, rbc parasitism, intravenous (iv) injection of hypotonic or hypertonic agents, contact with bacterial toxins, water intoxication, or immune-mediated destruction of opsonized erythrocytes. ingested toxins include sulfur compounds from onions and brassica plants (kale and canola), [ ] [ ] [ ] [ ] nitrates, nitrites, and copper. [ ] [ ] [ ] [ ] except for that caused by copper, hemolysis usually occurs within a day or two after ingestion. copper toxicosis can occur after acute overingestion but more commonly is seen in animals that are chronically overfed copper and suffer some stressful event. goats are more tolerant of excess copper than sheep are, and certain breeds of sheep, particularly the suffolk, are highly sensitive to copper toxicosis (see chapters and ) . hemolytic bacterial toxins include those from clostridium perfringens type a, clostridium haemolyticum, and leptospira interrogans. , intraerythrocytic parasites include anaplasma species, mycoplasma (eperythrozoon) species, and babesia species. [ ] [ ] [ ] [ ] [ ] immune-mediated rbc destruction is very uncommon except with parasitemia, the administration of certain drugs (penicillin), or bovine colostrum to small ruminant neonates. rapid reduction of plasma osmolality can lead to osmotic lysis of erythrocytes. this can occur locally as a sequela to rapid iv injection of hypotonic substances or after ingestion of a large quantity of water following a period of water deprivation and dehydration (water intoxication). selenium and copper deficiency have also been associated with heinz body anemia. parasite infestation, opsonization, and ingestion of toxic plants typically cause extravascular hemolysis. in these cases, damaged erythrocytes are removed by cells of the reticuloendothelial system, resulting in anemia, pallor, weakness, depression, icterus, and dark urine. bacterial toxins, changes in plasma osmolality, and copper toxicosis cause intravascular hemolysis, resulting in the additional signs of hemoglobinemia and hemoglobinuria. other signs such as fever, neurologic symptoms, and sudden death may be seen with specific diseases. signs of regeneration should be seen on the hemogram to days after the onset of hemolysis. anemia that is not related to the loss or destruction of erythrocytes usually results from a lack of production and thus are nonregenerative. although mild forms may exist in pregnant sheep and goats and those deficient in vital minerals (e.g., iron, selenium, copper, and zinc), the most common cause of nonregenerative anemia is chronic disease. under these conditions, iron is sequestered in an unusable form in the bone marrow; staining a marrow sample with prussian blue stain reveals large iron stores, differentiating this disease from iron-deficiency anemia. the causes of anemia of chronic disease are numerous and include infectious conditions (e.g., pneumonia, foot rot, and caseous lymphadenitis), malnutrition, and environmental stressors. most anemia does not require treatment. unless loss of rbc mass is rapid and severe, the animal is usually able to compensate to the decreased oxygen-carrying capacity by decreasing activity. it is important to remember in this regard that anemia often first becomes apparent to the manager of a flock or herd when animals appear overly stressed or die during movement or handling. if possible, the cause of the anemia should be addressed. this can involve trying to control internal and external parasites, changing the diet, and treating infectious diseases. maintaining adequate hydration is essential in animals with intravascular hemolysis to avoid hemoglobin-induced renal tubular damage. specialty compounds such as molybdenum salts, such as ammonium molybdate, and sulfur or penicillamine for copper toxicosis and methylene blue ( mg/kg in a % solution in % dextrose or normal saline intravenously) for nitrate toxicity are usually too expensive or difficult to be used on a flock-wide basis but may be useful in valuable individual animals. veterinarians should be aware that methylene blue is no longer approved for use in food-producing animals. animals with severe acute blood loss or hemolysis may benefit from a whole blood transfusion. because transfusion reactions are rare and strong erythrocyte antigens have not been identified in small ruminants (including cervids), almost any donor of the same species is acceptable for a first transfusion. cross-matching can be done to ensure compatibility, which becomes more important if the animal receives more than one transfusion. blood should be withdrawn aseptically from the donor and collected by a bleeding trocar into an open flask or by a catheter into a special collection bag. blood should be mixed at a . : ratio with acid-citrate dextrose, or : with % sodium citrate, or another suitable anticoagulant and administered through a filtered blood administration set. if the jugular vein is not accessible, blood may be infused into the peritoneal cavity, but the slower absorption from that site makes it less effective for treating acute blood loss. the first to minutes of administration should be slow. if no reaction is seen (fever, tenesmus, tachypnea, tachycardia, and shaking), the rate may be increased. transfused erythrocytes may only survive a few days, and therefore, the original cause of the anemia must be addressed. peripheral wbcs include granulocytes (neutrophils, eosinophils, and basophils) and mononuclear cells (lymphocytes and monocytes). immature forms of neutrophils and lymphocytes may be seen during severe inflammatory diseases. abnormalities of the neutrophil line are usually the best cellular evidence of inflammation in small ruminants, and inflammation is almost always a sequela of infection. an increase in neutrophil numbers and their proportional contribution to the total wbc count is usually seen in mild gram positive, subacute, or chronic bacterial infections. animals with more severe disease may exhibit high or normal counts, but a greater proportion of the neutrophils will have toxic changes or be immature forms (band cells, metamyelocytes, or myelocytes). in severe, acute inflammation and many diseases caused by gram negative bacteria, a temporary reduction in neutrophil numbers is observed, often with a concurrent shift toward more toxic or immature forms. if the animal survives the peracute disease, neutropenia should resolve over to days, first through an increase in immature cells, and later through a mature neutrophilic response. another important cause of increased total and relative neutrophil counts is stress (or glucocorticoid administration), which inhibits neutrophil margination and extravasation and thereby increases the number of these cells in the midstream blood. increases in eosinophil counts are usually related to exposure to eukaryotic parasites. decreases are rarely of clinical significance and may be part of the stress response. idiopathic allergic-type reactions also are indicators of pathology but are very rare. increases in basophils are rarely clinically significant. increases in lymphocyte counts often reflect chronic inflammatory disease such as that seen with internal abscesses. in rare cases, lymphocytosis may consist of abnormal, blast-type cells and indicate a lymphoproliferative neoplasm. lymphopenia is an important part of the stress response; nevertheless, the clinician must keep in mind that many diseases stimulate a stress response. therefore, lymphopenia and neutrophilia may represent either stress or inflammation, and an examination of neutrophil morphology and plasma fibrinogen concentrations may be useful in distinguishing the two situations. a high fibrinogen concentration, toxic changes, and high counts of immature neutrophils indicate inflammation under those circumstances. blood monocyte counts also may indicate stress or chronic inflammation. the difficulties in interpreting individual cell count abnormalities highlight the importance of obtaining a differential wbc count and description of cellular morphology in assessing sick sheep and goats. leukogram abnormalities are rarely given specific treatment. it is far more common and useful to use the information from the leukogram to develop a plan to treat the disease responsible for the abnormality. palpation of external lymph nodes is part of the thorough physical examination. lymph nodes that can be found in normal sheep and goats include the submandibular, prescapular, and prefemoral nodes. none of these should be prominent or painful on palpation. additional nodes that may be palpated occasionally in normal animals include the parotid, retropharyngeal, supramammary, perirectal, and popliteal nodes. internal lymph nodes that may be identified during specialized diagnostic procedures include the mediastinal, mesenteric, and other abdominal nodes. enlargement of lymph nodes may be focal, multifocal, or generalized. identification of a single enlarged superficial node does not always rule out a multifocal or generalized disorder because the status of the internal nodes often cannot be determined. enlargement generally indicates either inflammation or neoplasia. inflammatory enlargement is generally related to an associated disease with an infectious component. small ruminants are particularly sensitive to lymph node-based infections (e.g., caseous lymphadenitis), so the search often does not extend beyond aspirating or draining the lymph node itself. neoplastic enlargement almost always results from lymphosarcoma. lymphosarcoma pathogenesis. neoplastic transformation of a member of the lymphocyte cell line leads to unregulated clonal expansion of that cell. the cause of transformation is usually unknown; in rare cases, especially in flock outbreaks in sheep, it can be linked to exposure to the bovine leukemia virus, which has occurred experimentally and as a result of the administration of whole blood anaplasma vaccines. whether the bovine leukemia virus can induce lymphosarcoma in goats and cervids is still unclear. multicentric lymphosarcoma has been reported sporadically in white-tailed deer (odocoileus virginiatus) and other deer, but bovine leukemia virus infection has not been diagnosed in cervids. in one study of neoplastic diseases affecting goats from to , lymphoma was identified as the most common neoplasm, accounting for . % of the assessed tumors. in contrast to other species such as cattle, sheep, and horses, lymphomas in goats are predominantly t-cell lymphomas affecting the mediastinum. a recent study attempted to classify the type of lymphoma affecting goats. using immunohistochemistry (ihc), it was determined that % (n ) of affected goats had t-cell lymphoma and only % (n ) had b-cell lymphoma. proliferation of t or b lymphocytes leads to mass lesions and infiltration of viscera. these changes cause physical obstruction (to breathing, blood flow, urination, defecation, etc.), ulceration of mucosal surfaces (blood loss, bacterial invasion), immune system dysfunction, organ failure, and generalized malaise and cachexia. tissue masses may be internal or visible on external examination. clinical signs. clinical signs in affected animals vary according to the type of lymphoma (t-or b-cell) and the location of the masses. t-cell lymphomas in goats are usually localized in the thoracic cavity and/or neck, suggesting thymic origin or homing. in contrast, b-cell lymphomas tend to have a multicentric distribution. lymphoma in small ruminants usually presents with non-specific signs that can mimic other respiratory or gastrointestinal conditions. slowly progressive weight loss is the most common finding. in some cases, generalized peripheral lymphadenopathy and expansile masses are noted ; at first, they usually are presumed to be caseous lymphadenitis abscesses. progressive chemosis and exophthalmos have been reported in a sheep and a goat with multicentric b-cell lymphoma. , most masses form at the sites of internal or external lymph nodes. in sheep, masses in the brain, skin, joint, and lymphoid tissue have been reported. leukemia is rare. the most common abnormalities are those of chronic disease and cachexia and include nonregenerative anemia and hypoalbuminemia. bone marrow examination may reveal clonal expansion of lymphoid precursor cells. in cervids, lymphadenopathy and multifocal masses affecting the heart, blood vessels, kidney, urinary bladder, and peritoneum have been reported. a more recent report described a subcutaneous maxillary mass in a -year-old captive-born, female whitetailed deer. the mass was diagnosed as focal lymphosarcoma with local metastasis. diagnosis. history and clinical signs are important in the diagnosis of lymphoma in small ruminants. age of affected animals ranges from to years and no gender or breed predisposition has been reported. final diagnosis of affected animals is achieved through necropsy, histopathology, and ihc. lesions seen at necropsy include homogeneous white to tan masses that bulge on the cut surface. they may be small or large. less commonly, diffuse paleness of the reticuloendothelial organs is noted. microscopic examination of these tissues reveals infiltrates of abnormal cells of the lymphocyte line. prevention. avoiding exposure to the bovine leukemia virus and restricting the use of instruments to one animal between cleaning procedures may help prevent the spread of lymphosarcoma. in most animals, however, this neoplasm appears to develop spontaneously. pathogenesis. lambs, kids, and fawns are born with functional lymphocytes that can produce endogenous immunoglobulin. these cells develop the ability to respond to foreign antigens in the fetus during mid to late gestation. because of a lack of in utero exposure, however, basal concentrations of immunoglobulin are very low at birth. these cells therefore are naïve to foreign antigens and unable to develop protective immunity through specific cellmediated and immunoglobulin production. additionally, as with other ruminants, no transplacental passage of maternal immunoglobulin to fetal sheep, goats, and fawns occurs. lambs, kids, and fawns depend exclusively on intestinal absorption of maternally derived colostral antibodies, immune cells (t-lymphocytes), and other immune factors to provide a ready supply of specific immunity and allow opsonization of pathogens for the first months of life. adequate passive transfer requires delivery of a sufficient quantity of good-quality colostrum (immunoglobulin g [igg] concentration in mg/ml) into the gastrointestinal tract, as well as adequate absorption of antibodies (timely) from the colostrum into the blood. however, the amount of maternal colostrum produced by the dam, and its composition, as well as the ability of the newborn to stand and nurse in a timely manner, can be affected by several factors. colostrum igg concentration and volume of production can be influenced by breed, age, nutrition, body condition score (bcs) at parturition, and vaccination status of the dam. the igg concentration in colostrum samples from ewes of different breeds can vary between and mg/ml. one study demonstrated that primiparous ewes with low bcs (, . ) at lambing produced less colostrum compared with multiparous ewes with similar bcs. additionally, ewes with higher bcs (. . ) tended to produce higher volumes of colostrum compared with ewes with lower bcs. another study suggested that undernutrition of ewes during late gestation can affect colostrum quality and immune development and function in newborn lambs. it has been suggested that at least g of total igg should be fed to newborn lambs and kids during the first hours of life to reach adequate transfer of passive immunity. adequate transfer of passive immunity in small ruminant neonates has been suggested as serum igg levels at hours of life of  mg/ml. one study indicated that lambs that nurse low-quality colostrum (igg , mg/ml) had lower serum igg concentrations compared with lambs that that nurse colostrum of higher quality (igg . mg/ml), indicating that the concentration of igg in colostrum is a determining factor for the presentation of failure in the transfer of passive immunity. other factors such as pregnancy toxemia, gastrointestinal parasitism, excess of iodine intake during pregnancy, and inadequate vaccination of the dam can result in poor colostrum synthesis and quality. timely consumption of maternal colostrum during the first hours of life is essential to achieve adequate transfer of passive immunity. in small ruminants, cells of the small intestine are able to internalize and transfer igg into the blood during the first hours of life; however, the absorption efficiency of igg is higher during the first to hours of life. , factors associated with the neonate, such as weakness, inability to stand, and congenital abnormalities, will prevent timely nursing of maternal colostrum and lead to failure of passive transfer (fpt). litter size and body weight (bw) of the kid(s) have also been correlated with inadequate absorption of igg from colostrum. one study demonstrated that litter sizes of three light goat kids (, . kg bw) or more had significantly lower mean serum igg levels at hours of life when compared with litter sizes of one or two heavier kids ( . versus . mg/ml, respectively). this suggests that special attention and monitoring should be paid to multiple fetus gestation as the risk of fpt under these circumstances at kidding is higher; however, the quality of colostrum, amount ingested, and adequacy of absorption are rarely monitored by small ruminant producers in natural or artificial rearing systems. the use of monitoring tools to evaluate colostrum quality and igg absorption is common in modern dairy cattle operations, and these tools are readily available for small ruminant production systems. recent reports have presented the use of %brix in maternal colostrum and neonate serum and its positive correlation with serum total proteins (stps) at hours as effective monitoring tools of fpt in lambs and goat kids. [ ] [ ] [ ] the use of stp has also been used to monitor colostrum deficiency intake in mule deer fawns ; however, adequate values of serum igg for cervid neonates have not been established yet. inadequate colostrum intake and low serum igg at to hours of life have been consistently associated with higher morbidity and mortality rates in lambs, goat kids, and fawns. one study reported that % of lamb mortality between hours and weeks of age can be attributed to fpt. another study suggested that colostrum deficiency and low serum igg in goat kids resulted in higher mortality rates at weeks and of life due to chronic infections with pasteurella multocida and escherichia coli. other reports demonstrated that % of lambs with a serum igg of , mg/ml at hours died before weeks of age compared with only % of the lambs with a serum igg of . mg/ml at hours. in a previous report, mule deer (odocoileus hemionus) fawns with a stp of # g/dl between days and of age developed diarrhea and died before days of age compared with fawns with stp . g/dl. in a more recent report, a -day-old formosan sambar deer (rusa unicolor swinhoei) with a history of colostrum deprivation died due to severe suppurative meningitis caused by e. coli infection. in addition to immunoglobulins, colostrum also contains large quantities of fat-soluble vitamins that do not cross the placenta. the most important of these are vitamins a, d, and e, which are important in bone development and the immune or inflammatory response. neonates that have not ingested enough colostrum are likely to be deficient in these vitamins. diagnosis. history of dam dystocia, inadequate colostrum nursing, complete colostrum deprivation, and signs of undernourishment or sepsis in the first few days after birth are usually a presumptive indication of failure in the transfer of passive immunity. a high prevalence of diarrhea and respiratory disease in neonates should prompt investigation and evaluation of passive transfer of immunity in affected herds or flocks. owners occasionally evaluate lambs or kids for adequate intake by picking up the animal and holding it at ear level, while carefully cradling the head and neck, and then shaking the abdomen to hear milk in the abomasum; however, this is not a reliable indication of adequate transfer of passive immunity. a definitive diagnosis of fpt can be made by direct laboratory measurement (single radial immunodiffusion [srid]) of igg in serum at hours of life. although some practitioners use the value of igg used in dairy calves ( mg/ml), others have suggested an igg value , mg/ml to establish the presence of fpt in small ruminants. numerous semiquantitative methods of estimating igg are available and are easy to use in sheep, goats, and cervids. the most common is the measurement of serum total solids or stp values at hours of life through an optical refractometer. the stp at hours of life in a well-hydrated animal has demonstrated correlation with serum igg in calves, lambs, and goat kids. studies in goat kids indicated that an stp between . and . g/dl was associated with adequate transfer of passive immunity. , another study demonstrated fpt in lambs with stp values , . g/dl at hours of life. a study in mule deer suggested that fawns with an stp # g/dl had inadequate colostrum intake and fpt. recently, the measurement of %brix in maternal colostrum and serum with a digital brix refractometer has become an alternative method to evaluate colostrum quality and fpt in dairy operations. colostrum %brix . % and serum %brix . . % have been associated with adequate transfer of igg in calves and goat kids. other qualitative methods to assess the transfer of passive immunity in large animals include various agglutination (glutaraldehyde), precipitate assays (sodium sulfate), and measurement of g-glutamyl transferase (ggt) in serum. these methods may be relied on to give an overall flock assessment of adequacy of passive transfer, but they are rarely accurate enough to provide definitive information on individual animals. treatment. fpt is not in itself pathologic, but it greatly increases the neonate's susceptibility to infectious diseases. the amount of colostrum absorbed across the gut decreases with time, especially in animals that have been ingesting other proteins (e.g., the casein in milk); it also decreases with illnesses that decrease gastrointestinal function. neonatal small ruminants should receive at least g of igg/kg of bw or ideally g of total mass of igg from a good-quality colostrum source (. mg/ml of igg) during the first hours of life. other authors recommend an intake of to ml of colostrum/kg during the first hours of life. in artificial rearing systems or lamb feedlots, feeding of colostrum every hours until hours of life is recommended. when same species' maternal colostrum is unavailable, goat colostrum or bovine colostrum/colostrum replacers or are a good alternative; however, hemolysis has been reported in lambs receiving cattle colostrum. one study demonstrated that there was no difference in serum igg levels of lambs that received the same volume of sheep or goat colostrum at birth. another study demonstrated that lambs that received ml of a bovine colostrum replacer at birth in addition to ml of stored sheep colostrum at hours of life had higher serum %brix values at hours and had less incidence of disease during the preweaning period compared with lambs that received the same volume of stored sheep colostrum at birth and at hours of life. since igg absorption cannot be extended more than hours after birth, administration of an oral colostrum source is the best treatment in the immediate postpartum period in still-healthy neonates. after the window for immunoglobulin absorption has closed, plasma, serum, or whole blood administered by the iv or intra-peritoneal route is the best way to raise the neonate's blood immunoglobulin concentrations. adult donor plasma contains approximately . to . g of immunoglobulin/dl, so administration of a volume equivalent to % of bw or a dose of to ml/kg has been recommended for the treatment of large animal neonates. if plasma is used instead of colostrum, administration of vitamins a, d, and e also may be beneficial. if colostrum and plasma are unavailable or cost-prohibitive, "closing" the gut as quickly as possible with milk, maintaining high standards of hygiene, and possibly administering prophylactic antibiotics offer the greatest prospects for preventing infectious disease. vaccination of the neonate or the administration of antitoxin hyperimmune serum should not be considered protective but may be of value. prevention. prevention of fpt should be based on the establishment of an adequate colostrum program managing the previously mentioned factors that affect production, quality, and absorption of maternal colostrum components in lambs, goat kids, and fawns. ensuring colostral quality is best done through good nutrition, health care, and vaccination of dam (see chapters and ) . administration of vaccines weeks before parturition, followed in weeks with a booster, provides the highest quantity of protective immunoglobulin in the colostrum. antepartum leakage is rarely the problem in small ruminants that it is in horses and cattle. however, in a flock or herd environment, still-pregnant dams may steal babies from other sheep or goats. to prevent such theft and the resultant loss of colostrum by the "adopted" neonate, owners may choose to keep pregnant animals separate from those that have already delivered. if complete separation is not possible, the dam and her offspring should be allowed to bond with each other in a private pen ("jug" or "crate") for at least hours before being placed back with the flock. clipping excessive wool or mohair from around the perineal area and udder before lambing or kidding, expressing the teats to ensure they are not plugged, and having extra colostrum available when pregnant females are placed in jugs or crates are other good preventive measures. etiology and pathogenesis. uncomplicated diarrhea in lambs, goat kids, and fawns may be caused by infectious agents such as viruses, bacteria, and protozoa. in goat kids and elk calves, metabolic causes of diarrhea have been described. , group b and a rotavirus, enterotoxigenic e. coli k , cryptosporidium parvum, and other cryptosporidium spp. have been commonly identified as causal agents of diarrhea in small ruminant neonates. [ ] [ ] [ ] [ ] with recent advances in diagnostics and metagenomics of the enteric environment of large animals, novel viruses have been identified as potential causal agents of diarrhea in lambs and goat kids. adenovirus, astrovirus, calicivirus, coronavirus, and picornavirus have been identified in feces of diarrheic lambs and goat kids ; however, their role in the pathogenesis of neonatal diarrhea is still uncertain. these organisms differ from the agents of complicated diarrhea in that they do not invade beyond the gut wall or result in systemic toxemia (see chapter ) . additional causes of diarrhea reported in goat kids and elk include lactose intolerance and hypernatremia, respectively. , less frequently, bacteria such as c. perfringens, clostridium difficile, and attaching and effacing e. coli have been associated with complicated diarrhea in small ruminant neonates. , the net result of such an infection is that a large volume of water and electrolytes are lost into the bowel due to malabsorptive, hypersecretory, or hyperosmolar processes. if enough fluid and electrolytes are lost, dehydration and metabolic acidosis arise, inducing systemic clinical signs of depression and weakness in association with diarrhea. in goats, this clinical entity is one component of the floppy kid syndrome. clinical signs. profuse, watery, yellowish-green to brown diarrhea without fever is the hallmark clinical sign. with severe dehydration and acidosis, affected lambs, kids, and fawns become weak and dull and lack appetite. [ ] [ ] [ ] excessive salivation and loss of suckle reflex have also been reported in affected lambs and kids. , mucous membranes become tacky, and skin tenting times are prolonged. shock signs may develop. physical assessment often must take the place of clinicopathologic analysis in affected neonates. mild, nonclinically complicated diarrhea is characterized by profuse diarrhea with minimal systemic signs. the affected animal is bright and alert, with minimal skin tenting, and can stand and eat readily, with a strong suckle reflex. it is less than % dehydrated, with a blood ph of . to . , and bicarbonate deficit is minimal. moderate uncomplicated diarrhea is characterized by profuse diarrhea in a dull but responsive animal. skin tenting is prolonged, but eye luster is normal. the affected animal is able to stand and eat but eats slowly and has a weak suckle reflex. the head typically is held down. it is to % dehydrated, with a blood ph of . to . and a bicarbonate deficit of to meq/l. severe uncomplicated diarrhea is characterized by profuse diarrhea. the affected animal is dull and minimally responsive, with a very long skin tent time and dull, sunken eyes. it can stand only with assistance and prefers to stay in sternal recumbency with its head up. the animal eats very slowly, if at all, and has a minimal suckle reflex. it is to % dehydrated, with a blood ph of . to . and a bicarbonate deficit of meq/l. very severe uncomplicated diarrhea is characterized by profuse diarrhea and profound weakness. the animal's skin remains tented for more than minute, and its eyes are very sunken and dull. it is nonresponsive with no suckle response. it is unable to maintain sternal recumbency, lying on its side instead. the animal is to % dehydrated, with a blood ph of . to . and a bicarbonate deficiency of to meq/l. epidemiology. morbidity and mortality of uncomplicated diarrhea in small ruminants and fawns vary depending on the cause. reports of rotaviral diarrhea in newborn lambs indicate morbidity rates between % and % and mortality rates between and % , ; however, one study reported a % case fatality rate in lambs affected with types b and a rotavirus diarrhea. another study reported mortality rates between % and % in lambs and kids affected with c. parvum diarrhea. most of infectious agents associated with uncomplicated neonatal diarrhea in small ruminants are shed by adult animals and older lambs/kids around stressful events such as lambing/kidding and extreme weather conditions. one study reported that pregnant does shed to times more oocysts during the weeks around kidding compared with other time periods. additionally, poor husbandry/hygiene of lambing/kidding sheds, fecal soiling, flock size (. animals), lambing/kidding season (winter/spring), and the presence of c. perfringens type a in feces have been suggested as potential risk factors for uncomplicated diarrhea in small ruminant neonates. [ ] [ ] [ ] clinical pathology. the leukogram should be normal or show abnormalities compatible with stress. serum biochemical or blood gas analysis may reveal evidence of intestinal malabsorption, electrolyte loss, metabolic acidosis (hypoglycemia, hyponatremia, hypochloremia, hyperkalemia, low bicarbonate, and increased anion gap), and dehydration (hyperalbuminemia and increased blood urea nitrogen [bun] and creatinine). in contrast with the common leukogram and biochemical abnormalities found in calves, lambs, and goat kids with uncomplicated diarrhea, elk calves with diarrhea develop leukocytosis, hyperchloremia, and hypernatremia (serum na . meq/l). additionally, increased anion gap, bun, creatinine, and albumin concentrations have been reported in affected elk calves. a presumptive diagnosis may be based on the characteristic history and clinical signs. response to conservative treatment also is supportive of this diagnosis. identification of the specific causative agent is less important than proper treatment of affected animals; however, feces or intestinal contents from affected animals can be submitted for electron microscopy, reverse-transcription polymerase chain reaction (pcr), and cell culture immunofluorescent assays to identify viruses. [ ] [ ] [ ] additionally, intestinal tissue can be submitted for ihc for rotavirus and c. parvum. , feces of affected animals can also be submitted for enzyme-linked immunosorbent assay (elisa), ziehl-neelsen staining technique, light or fluorescence microscopy, sugar flotation, and auramine or fluorescent antibody staining for the diagnosis of c. parvum infection. fecal culture to determine a bacterial cause is recommended. treatment. the immediate goals of treatment are rehydration, replacement of lost electrolytes, and restoration of acid-base balance as these are usually the leading causes of death in affected neonates. less immediate goals are provision of nutrition and replacement of ongoing losses. the aggressiveness of treatment is dictated by the severity of the condition, as well as economic considerations. . rehydration: calculate the percent dehydration and use to calculate fluid requirements for a -hour period. example: % dehydration in a -kg lamb: dehydration: . kg kg/l . l or ml. maintenance: ml/kg/day . l or ml. total fluids to replace in hours . l or ml fluid loss due to dehydration ( ml in this case) should be replaced during the first hours and the rest can be replaced in the next hours. . replace lost electrolytes: sodium, chloride, and bicarbonate are lost roughly in proportion to extracellular fluid (ecf) in the acute phase of diarrhea ( - days) in untreated animals. potassium tends to be increased in this phase due to the presence of metabolic acidosis and care should be taken when selecting fluids containing potassium to treat affected animals at this time. in chronic cases of diarrhea, and especially in cases where the owner/producer has given oral milk replacer or electrolyte supplements/replacements to affected animals before veterinary evaluation, the serum concentration of sodium, potassium, and bicarbonate might be variable or increased. special care should be taken in these cases when selecting fluids to treat affected animals as the risk of causing hypernatremia is higher. in cases of diarrhea in elk calves, hypernatremia is common, and fluids should be selected accordingly. in the majority of cases, initial replacement of sodium, chloride, and bicarbonate with fluids containing proper composition is recommended. example: assessment suggests a bicarbonate deficit of meq bicarbonate in a -kg, comatose lamb with prolonged skin tenting ( . is the multiplier for ecf in a neonate): . ( meq) kg meq bicarbonate. commercial iv . % sodium bicarbonate solutions contain meq of bicarbonate per milliliter and could be added directly to iv fluids in severely dehydrated and acidotic animals. therefore, the immediate goal is to provide ml of fluid and meq of bicarbonate to this lamb in a formulation that resembles normal ecf. fluids can be given by various routes. selection of route of administration of fluids depends on degree of dehydration, presence or not of a strong suckle reflex, and degree of depression. neonates with advanced degrees of dehydration, depression, and absence of suckle reflex will benefit from iv fluid therapy. in contrast, neonates with mild dehydration and active suckle reflex can be effectively treated with oral electrolytes ; however, if oral fluids have not produced an improvement within to hours, iv treatment should be strongly considered. other routes such as subcutaneous, intra-peritoneal, and intra-osseous can also be used for fluid administration to neonates. • advantages: oral fluids are inexpensive (nonsterile) and easy to give. they are less likely to cause fatal arrhythmias or neurologic disease than iv fluids. • disadvantages: an animal receives a maximum of its gastric volume ( % of bw), and good gastric motility is required. oral fluids may not be well absorbed by a damaged gut. absorption also is slow. intravenous • advantage: this method allows rapid correction of all deficits, even in moribund animals. • disadvantages: it is expensive (sterile), requires venous access, and can rapidly lead to overcorrection. subcutaneous • advantages: this method does not require venous access or good gut motility. • disadvantages: it is expensive (sterile), and the fluids may not be well absorbed in very dehydrated animals. absorption is not as quick as by iv administration. animals should be given only hypotonic or isotonic fluids. intra-peritoneal • advantages: this method does not require venous access or gut motility. fluids are absorbed quickly by this route. • disadvantages: it is expensive (sterile) and can cause peritonitis. isotonic fluids are best used in this route. only a limited volume can be given. many commercial oral electrolyte solutions for neonatal ruminants are available; however, not all of them fulfill the requirements to adequately replace fluids and electrolytes in neonatal ruminants with diarrhea. oral electrolyte solutions must contain enough sodium , provide agents that increase absorption of water (glycine, glucose, and acetate), provide an alkalinizing agent (bicarbonate, propionate, acetate, and citrate; acetate has demonstrated best results), and an energy source (glucose). the amount of carbohydrates might vary and is usually higher in "high-energy" solutions specifically used for severely affected neonates that are not eating and develop negative energy balance. less carbohydrate is needed in less severely affected animals because they are usually eating some and are less likely to have severe negative energy balance. fluids to be avoided include medicated milk replacers and unbuffered saline solutions. iv treatment should be provided with a sterile commercial product. such preparations typically contain to meq/l of base. additional sodium bicarbonate solution or sterile powder can be added to fluid therapy based on the bicarbonate deficit ( meq/ml of . % solution and meq of bicarbonate/g of powder, respectively). the bicarbonate deficit should be over the first hours. after deficits are replaced, the following continued treatments and adjuncts may be considered: . continued administration of fluids (oral rather than iv, if possible) to replace ongoing losses: • oral electrolytes at a volume equal to % of the bw per feeding can be given; the number of feedings can be increased from two (normal) to three to six per day. • iv fluids can be continued at twice the maintenance fluid rate until appetite is restored. • more bicarbonate may be necessary. . consideration of addition of milk to the treatment regimen: • milk or milk replacers should be added to the therapy of neonates with diarrhea. they provide nutrition to the affected neonate, preventing negative energy balance and promoting intestinal healing. • care should be taken to not mix oral electrolyte solutions with milk or milk replacers in the same container as the concentration of sodium and overall osmolarity of the solution can dramatically increase, leading to hypernatremia or other metabolic abnormalities. • milk or milk replacers should be given in small volumes (, % of total requirements) but at a higher frequency (every - hours) to avoid overloading the abomasum and intestine of affected animals. lambs fed milk lose less weight with scours. • free water helps prevent hypernatremia. • milk is a good potassium source (see chapter ). elk deer calves. elk deer calves commonly develop diarrhea with hypernatremia (serum na . meq/l) compared with other large animal neonates, where hyponatremia is more common. therefore, administration of oral electrolyte solutions designed for other ruminants (calves, lambs, and kids) should be avoided in these animals. a dilution ( : or : ) of commercially available bovine calf electrolyte solutions to reduce sodium content is recommended for the treatment of elk calf diarrhea. the use of lactated ringer's solution, which has a low sodium concentration in addition to a very low reduction rate of serum sodium (, . meq/l/hour) has been advocated in the fluid therapy of hypernatremic elk calves with diarrhea. additional therapy. dextrose ( . - %) solutions can be added to the fluid therapy of hypoglycemic animals. the use of nonsteroidal antiinflammatory drugs (nsaids) in neonatal ruminants with diarrhea is controversial due to the risk of renal damage and abomasal ulceration; however, in cases of diarrhea complicated by septicemia or endotoxemia, nsaids should be used to reduce the effects of systemic inflammation. flunixin meglumine at a dose of . to . mg/kg is the only nsaid approved for food animal use. similarly, the use of oral or systemic antibiotics in cases of uncomplicated diarrhea is controversial due to its potential effect on the intestinal microbiota and development of bacterial resistance; however, their use is warranted in the presence of septicemia or endotoxemia in addition to diarrhea. in these cases, b-lactams such as oral amoxicillin or systemic ceftiofur are usually good choices. the effect of mucosal protectants and probiotics in cases of diarrhea is unknown in small ruminant neonates, and their use is left to practitioners based on their own experiences (see appendix ) . prevention. prevention of uncomplicated diarrhea in small ruminant neonates is based primarily on the timely feeding of adequate amounts of good quality maternal colostrum or colostrum replacer (see "failure of passive transfer" section). vaccination of dams with antigens of common infectious agents associated with uncomplicated neonatal diarrhea before parturition has demonstrated to be effective increasing colostrum immunity and prevention of diarrhea in lambs. maintenance of adequate husbandry and hygiene conditions in lambing/kidding sheds or barns is necessary to reduce neonatal exposure to infectious agents normally shed in feces of dams during parturition such as rotavirus and c. parvum. ruling out infectious causes of depression and weakness is difficult, and clinicians often do well to assume that an infectious disease is contributing to clinical signs when making treatment decisions. however, several noninfectious systemic disturbances also can depress neurologic and muscular function. successful treatment often requires identification and correction of each of these disturbances. among the more common abnormalities leading to depression in neonates are hypoxemia, metabolic or respiratory acidosis, hypothermia, hyperthermia, hypoglycemia, dehydration, azotemia, and some electrolyte imbalances. hypothermia and hyperthermia can easily be diagnosed by measuring body temperature with a rectal thermometer. hypothermia is far more common and can result from weakness, shock, and environmental stress. cold, windy weather or tube feeding with cold milk replacer or fluids can lead to a rapid drop in core body temperature, especially in neonates that are small or weak or have been inadequately licked off or were rejected by their dams. strong, vigorous neonates usually are protected by heat produced during muscular activity and are able to seek food and shelter. clinical signs appear when the rectal temperature drops to ° f ( . ° c) or below. protection from wind and cold such as with an individual ewe jug or pen, heat lamps (positioned far enough away so as not to burn the neonate), hot water bottles, blankets, and administration of warm fluids is helpful in treating and preventing hypothermia. shearing the ewe before lambing is of value because it forces the ewe to seek shelter. if this management technique is used, care should be taken to avoid inducing severe hypothermia in the dam. environmental hyperthermia is much less common than fever in neonates. therefore, treatment for infectious diseases in young animals with high temperatures usually is warranted. providing cool shelter with good ventilation, minimizing stressful events, ensuring adequate fluid intake, and shearing the adults are the best defenses against environmental heat stress. hypoglycemia also is easy to diagnose with the aid of an inexpensive, portable glucose meter. lambs and kids typically develop hypoglycemia under the same circumstances as those leading to hypothermia. administering ml/kg of dextrose (approximately . fl oz/lb, or % of bw) in warm milk replacer or ml/kg of % dextrose, by either the iv or oral route (diluted to % dextrose), should provide ample energy to correct hypoglycemia. iv administration may be necessary if gut motility is absent. follow-up treatment may be necessary if the neonate does not regain its appetite. except during severe conditions, normal lambs and kids should be able to maintain normal body core temperature. they should therefore be examined for an underlying disorder if they exhibit signs of hypothermia or hyperthermia. clinicians and owners should not assume that warming and feeding a cold, weak neonate will always correct the problem. hypoxemia is much more difficult to diagnose. portable blood gas meters for arterial analysis and radiography units for thoracic imaging are available but are still not in common use in small ruminant practice. for those reasons, hypoxemia usually is underdiagnosed. hypoxemia can result from prematurity or dysmaturity, infection, depression or weakness (decreased ventilation), meconium aspiration, bullous emphysema, hernias, and other thoracic fluid or tissue masses. it is likely to be a contributing factor in illness and death in most weak neonates younger than days of age. such animals benefit from the provision of supplemental oxygen, either through a nasal insufflation tube or by oxygen tent. in addition to its direct effect on general wellbeing and behavior/ attitude, hypoxemia at birth leads to poor gut function and subsequent poor colostral absorption. many animals that exhibit fpt and subsequent sepsis had a previous bout of hypoxemia. azotemia, metabolic acidosis, and electrolyte imbalances are difficult to diagnose without clinicopathologic analysis. therefore, these problems are best treated in animals showing signs of dehydration with the administration of a balanced, physiologic electrolyte solution. metabolic acidosis usually is accompanied by either obvious evidence of bicarbonate loss (diarrhea) or severe dehydration. however, neither of these conditions is present with floppy kid syndrome. this descriptive title is applied to muscle weakness, anorexia, and depression in kids observed in the first weeks of life. by its strictest definition, floppy kid syndrome refers to metabolic acidosis with a high anion gap without dehydration or any known cause in young kids that were normal at birth. a variety of disorders and conditions have been proposed as the cause of metabolic acidosis without dehydration, including intestinal fermentation of milk in well-fed kids with subsequent absorption of volatile fatty acids, transient neonatal renal tubular acidosis, and lactic acidosis secondary to toxic impairment of cardiovascular function. overgrowth of c. perfringens type a often is suggested as a source of the toxin. with a high anion gap, a pathologic condition that leads to overproduction of an organic acid is more likely than one that leads to bicarbonate loss. the disease can occur in individual animals or in outbreaks; although parity of the dam and number of offspring have not been associated with this metabolic disturbance, aggressively feeding kids are more likely to suffer from milk fermentation or clostridial overgrowth. an infectious etiology appears to be more likely in herds displaying an increased incidence of this metabolic disturbances as the kidding season progresses. the disease also is reported to be more common in meat goats than in dairy goats. the prevalence can vary tremendously from year to year in a single flock or region. a similar disease has been reported in calves and llama crias, and lambs are likely to be susceptible under the right conditions. because blood gas analysis and exclusion of other diseases often are impractical, the term floppy kid syndrome frequently is used by owners to refer to any kid that is weak and does not have an overt, organ-specific sign (e.g., diarrhea). different pathologic processes are grouped together by their common clinical endpoint (as with "thin ewe syndrome"), and the veterinarian is charged with determining the etiology in a specific flock. most possible causes are found in the previous list of conditions that cause weakness and depression in neonates. among these entities, sepsis and hypoxemia are the most important items and therefore must also be considered possible causes of floppy kid syndrome. treatment and prevention of floppy kid syndrome currently follow the same lines as for treatment and prevention of neonatal sepsis or enteritis. spontaneous recovery of animals with floppy kid syndrome may occur. however, in valuable kids, quick assessment of blood chemistry and base deficits will allow requisite correction of electrolyte and blood ph abnormalities with . % sodium bicarbonate. tissue-invading clostridia are large, straight, gram positive rods that are to mm in length. c. perfringens and c. haemolyticum are smaller bacteria, and clostridium novyi, clostridium chauvoei, and clostridium septicum are larger. the bacteria grow best under anaerobic conditions and produce waste gases. clostridia bear spores, which may be the only viable form in the environment (soil and decomposed organic matter). identification of these spores within bacteria on microscopic examination is useful to identify clostridia, but it is not diagnostic of disease. spores in c. perfringens are central and do not affect the shape, whereas most other species have the spore toward one end and appear slightly club shaped. clostridia cause infectious, noncontagious disease. the bacteria inhabit the intestinal tract and are present in the feces of ruminants. small numbers of organisms in their dormant spore form also may reside in tissues such as liver and skeletal muscle. they can be isolated from soil, where most are thought to have short life spans. soil concentrations are highest in locations recently contaminated with ruminant feces, especially crowded, overused facilities such as feedlots and lambing sheds. environmental contaminations are associated with cool, damp times of the year such as late winter and spring. the concentration of organisms and their toxins found in the feces, gut contents, and internal organs of most adult ruminants usually is small. competition and peristalsis prevent overgrowth in the gut, and aerobic conditions prevent overgrowth in other tissues in live animals. however, rapid overgrowth and tissue invasion ensue after death, making rapid postmortem examination essential to ascertain whether clostridial organisms are responsible for the death. pathogenic clostridial organisms all produce heat-labile protein exotoxins. most make a variety of toxins, and the relative contribution of each toxin to the disease state is not known. c. perfringens is a normal commensal of the intestinal tract of clinically healthy large animals, including cervids; however, the number of bacteria and their toxin production within the intestine usually remain low due to peristalsis and normal homeostasis. c. perfringens is classified into five biotypes (a, b, c, d, and e) based on the production of four major exotoxins, namely alpha (cpa), beta (cpb), epsilon (etx), and iota (itx); however, the production of more than different exotoxins in various combinations has been associated with these bacteria, including perfringolysin o (pfo), enterotoxin (cpe), and beta toxin (cpb ). the different biotypes of c. perfringens cause different diseases in relation with the exotoxins they produce. the major effect of the phospholipase/ sphingomyelinase cpa, produced by all c. perfringens biotypes, is cell lysis and hemolysis, and its role on intestinal disease of large animals is not well understood. however, this toxin has been associated with hemolytic disease and hemorrhagic enteritis in large animals; cpb, produced by c. perfringens types b and c, is a trypsinlabile toxin associated with necrotizing enteritis and enterotoxemia in large animal neonates; etx, produced by c. perfringens types d and b, is a trypsin-activated necrotizing toxin associated to vasculitis, edema, and necrosis of the cns and enterotoxemia; and itx, another trypsin-activated necrotizing toxin produced by c. perfringens type e, has also been associated to intestinal disease in small ruminants. , c. perfringens types c and d are considered the most important types in veterinary medicine as they can cause disease in most farm animals. severe clinical disease due to bacteria sporulation and massive toxin production only occurs when the normal intestinal environment and microbial balance are disrupted in affected individuals. decreased peristalsis and poor ruminal and abomasal function have also been proposed as factors that contribute to disease presentation. weather and handling stresses, feed changes, and an overabundance of high-energy feeds such as milk, bakery products, and cereal grains might promote bacteria overgrowth and exotoxin synthesis and release. additionally, other enteric infections that disrupt the mucosal border may increase systemic absorption of toxins and promote severe disease. c. perfringens type a is a normal inhabitant of the intestinal tract of large animals and is ubiquitous in the environment (soil). one study reported c. perfringens type a as the most common isolate among other clostridia from healthy young lambs. c. perfringens type a has been associated with a fatal hemolytic syndrome in younger lambs and cattle but not goats ("yellow lamb disease"), , acute hemorrhagic enteritis and hemolytic enterotoxemia in cattle (hemorrhagic bowel/jejunal syndrome) and goats, , , and intestinal hemorrhage and splenomegaly in farmed deer. , risk factors for infection have not been established; however, high soluble carbohydrate diets and high bcss have been associated with clinical disease. , , this disease occurs most commonly in lambs to months old. under favorable conditions, the organisms proliferate and cause a corresponding increase in alpha toxin production. the alpha toxin (cpa), in synergy with the beta toxin (cpb ), is responsible for hemolytic crisis, vasculitis, and gastrointestinal lesions. the clinical course usually is less than hours. clinical signs. in most cases, sudden death or history of found dead is common. clinical signs observed usually include weakness, depression, fever or hypothermia, icterus, anemia, hemoglobinuria, tachypnea, colic, hemorrhagic diarrhea or absence of feces, and terminal recumbency. , , [ ] [ ] [ ] adult animals also are susceptible to hemolytic disease and vasculitis caused by c. perfringens type a infection. fatal abomasitis and rumenitis in neonates and juveniles also have been blamed on c. perfringens type a, but the rapid postmortem proliferation of the organism makes substantiation of this claim difficult. morbidity in a flock is lower than for many of the other enteric clostridial diseases, but the mortality rate is very high. diagnosis. the most characteristic clinicopathologic change is neutrophilic leukocytosis with a left shift. other evidence of systemic toxemia (metabolic acidosis, azotemia, and increases in liver and muscle enzymes) also may be seen. laboratory evaluation reveals evidence of intravascular hemolysis. necropsy in sheep, goats, and cervids usually reveals evidence of hemolysis, pallor, jaundice, hemoglobinuria, hyperemic and edematous intestines, splenomegaly, gastrointestinal serosal and mucosal hemorrhage, and multifocal internal petechial hemorrhages. , , [ ] [ ] [ ] the isolation of c. perfringens type a from necropsied animals is not itself diagnostic. definitive diagnosis can be made based on identification of the alpha toxin and the absence of other toxins by elisas or older, live animal assays. more recently, multiplex pcr techniques are replacing immunodiffusion assays for the identification of a specific toxin-producing gene isolate, typing of bacteria, and demonstration of toxins or toxin genes. gut content and intestinal samples collected from freshly dead animals make the most meaningful samples for diagnosis. treatment. administration of high doses (. , iu/kg bid) of penicillin and clostridium antitoxin ( - ml subcutaneously [sc] or orally [po] ) is the mainstay of treatment, although animals may die acutely before therapies can be instituted. prevention. a conditionally licensed toxoid against the clostridial alpha toxin is available for cattle in the united states. a recent report demonstrated that a new vaccine including recombinant cpa, cpb, and etx was effective at inducing protective antibodies to c. perfringens biotypes in cattle, sheep, and goats. this could be an alternative for the prevention of morbidity and mortality caused by c. perfringens type a. prevention efforts should focus on environmental hygiene and avoiding gut conditions favorable for proliferation of the organism (high content of soluble carbohydrates in the diet). because this type appears to survive better in soil than other types, preventing ingestion of soil may be important in preventing disease. c. perfringens types b and c occur in the soil and the animals' housing environment and can be shed by asymptomatic individuals. the reported geographic range of both diseases is limited (type b to the united kingdom and south africa and type c to the united kingdom, australia, and north america), even though infection with c. perfringens type c appears to occur worldwide. these organisms cause very similar diseases called lamb dysentery and hemorrhagic enterotoxemia, respectively. very young lambs and kids ( - days to - weeks of age) are usually affected due to the presence of trypsin inhibitors in colostrum. older animals may become susceptible as a result of overwhelming infection or trypsin inhibition by some soy and sweet potato products or temporary suppression of pancreatic trypsin production (struck in adult sheep). with both diseases, the beta toxin (cpb) is a required pathophysiologic factor, and inactivation of this toxin after maturation of pancreatic trypsinogen secretion is what commonly limits the susceptible population to neonatal animals. the cytolytic and necrotizing effects of the beta toxin (cpb), in synergy with the beta toxin (cpb ), cause necrosis and ulceration of the intestinal mucosa and are translocated into circulation, causing severe toxemia and death. the diseases initially affect lambs and kids younger than days of age, with illness occasionally occurring in older lambs. the incidence of disease in lambs and kids can be around to %, with a case fatality rate of %. high stocking density in lambing areas, cold weather, single-born lambs, and high milk production of dams have been suggested as potential risk factors for type b and c enterotoxemia. because of management practices in young animals and age-related vulnerability, fecal contamination of teats, hands, and equipment that enter the mouths of the neonates (orogastric tubes and nipples) is a major cause of infection. clinical signs. severely affected animals or those at the beginning of an outbreak usually are found dead. less acutely affected animals expel initially yellow, fluid feces that progressively become brownish and/or hemorrhagic. feces may also contain flecks of blood and show splinting of the abdomen, especially when handled, along with signs of colic and feed refusal. the clinical course usually is short, and the disease is almost always fatal. one study reported acute abdominal pain, hemorrhagic diarrhea, and death within hours of experimental oral inoculation of three goat kids with a field strain of c. perfringens type c. dehydration, anemia, and severe weakness are also common clinical signs in affected animals. terminal convulsions and coma occasionally are noted, especially in outbreaks in the united states. c. perfringens type c in older sheep causes the disease known as "struck." affected animals usually are found dead or with signs of toxemia. specific antemortem signs of gastrointestinal disease are rare. specific antemortem signs of gastrointestinal disease are rare. clinical pathology changes observed in these animals include neutrophilic leukocytosis with a left shift. additional evidence of systemic toxemia (metabolic acidosis, azotemia, and increases in liver and muscle enzymes) also may be seen. necropsy findings. postmortem examination reveals focal hemorrhagic ulcers (up to . cm in diameter) in the small intestine (mostly in the ileum) with type b infection and diffuse reddening with hemorrhage and necrosis of the abomasum and the entire segments of the intestine with type c infection. type c infections in ruminants can also present with generalized peritonitis, subendocardial and subepicardial hemorrhages, and hemorrhagic lymph nodes. animals that die very rapidly may exhibit minimal or no gross abnormalities of the intestine. a similar syndrome of type c enterotoxemia has been previously reported in a sika deer (cervus nippon). sudden death, severe hemorrhagic gastritis including forestomach and abomasum, and catarrhal enteritis was observed in the affected animal. diagnosis. diagnosis of these diseases is made by identification of characteristic history, clinical signs, postmortem lesions, and positive toxin assays. because the beta toxin is very labile, negative toxin assays are less significant than negative assays for presence of other tissue-invading clostridia. the isolation of c. perfringens type b or c from necropsied animals is not itself diagnostic. immunodiffusion assays or multiplex pcr of intestinal contents for specific isolate and beta toxin (cpb) identification are recommended to obtain final diagnosis (see "diagnosis" in "c. perfringens type a" section). treatment. if the infection is identified early in the disease course, high doses of oral and parenteral penicillin and c. perfringens c and d antitoxin may be of benefit. iv fluids and antiinflammatory agents may be indicated as well. usually, the condition is not recognized early enough, and animals are found dead or dying. prevention. a beta toxoid is available in the united states and other countries. it usually is packaged with an epsilon toxoid. the best protection is achieved by vaccinating pregnant dams twice, with the second dose administered approximately to weeks before lambing or kidding and annual booster. deer does should receive double the dose of sheep as low antibody responses to clostridia have been reported in these animals. , vaccination of pregnant dams is directed to increase specific colostrum antibodies to protect neonates. juveniles also should be vaccinated twice or three times at months, months, and months. adults, including males, should receive an annual booster. in the face of an outbreak, the lambing area should be moved to a different place. additionally, vaccination of dams and newborns with a beta toxoid and administration of c. perfringens c and d antitoxin can be carried out in the face of an outbreak to reduce morbidity and mortality. c. perfringens type d produces epsilon toxin (etx), which is responsible for causing type d enterotoxemia in sheep, goats, calves, and deer. , other common names for the disease include "overeating disease" or "pulpy kidney disease." the disease has a worldwide distribution and occurs primarily in suckling lambs of to weeks of age, although it has also been reported in weaned lambs up to months of age and adult sheep. the disease is also common in grazing goats and deer. the prevalence of disease has been reported from . to . %, with a % case fatality rate in feedlot lambs. one study on proportional distribution of goatherd mortality in the province of quebec, canada, reported a . % mortality of goats to c. perfringens type d enterotoxemia. the disease is more common in feedlot lambs after they enter the lot. tail docking, castration, and other management interventions are thought to decrease the incidence of this disease by temporarily decreasing appetite. the disease also affects unvaccinated adult sheep, even without any history of stressors or feed changes. sudden changes in the diet are the main predisposing factor in goats. the disease can occur in vaccinated goats, as vaccination has not demonstrated to be completely protective in this species. , c. perfringens type d is normally found in the gastrointestinal tract of healthy ruminants, but the acid environment of the abomasum and continuous peristalsis help to keep numbers of bacteria and levels of toxin production low. however, under specific conditions such as overingestion of high-energy feeds (milk, grain, and lush pasture), excess of fermentable starches in the intestine, and intestinal stasis, the organism proliferates rapidly, producing lethal quantities of epsilon toxin. these conditions are usually triggered in well-conditioned, fast-growing animals that are on a highly nutritious diet. the epsilon toxin, once produced, acts locally, causing increasing gut permeability and widespread tissue damage. epsilon toxin and other exotoxins are then absorbed through the intestinal tract into systemic circulation and transported to the brain, lungs, and kidneys, causing increased endothelial permeability, perivascular edema, and generalized necrosis. , the characteristic increased vascular permeability and perivascular edema in the kidney and brain are responsible for the name of "pulpy kidney disease" and "focal symmetric encephalomalacia." clinical signs. the course of the disease is usually very short ( . - hours), so sudden or spontaneous death is a common clinical sign across affected small ruminant species. , - natural disease caused by c. perfringens type d differs between sheep and goats, possibly because of a difference in relative local and systemic actions of the epsilon toxin, although experimental models have demonstrated that both species develop similar lesions. , , in sheep, systemic actions of the toxin leads to mostly neurological signs such as dullness, depression, ataxia, trembling, stiff limbs, opisthotonus, convulsions, frothy mouth, and rapid death. in goats, actions of the toxin appear to be more localized to the intestinal tract, causing enterocolitis, colic, diarrhea, dehydration, and occasional neurological signs. , necropsy findings. postmortem findings in sheep are characterized by edema of the brain, lungs, and heart in addition to hydropericardium. edema of the kidneys (pulpy kidney lesion) is inconsistent. sheep usually demonstrated minor and inconsistent intestinal changes. other lesions reported in cattle and deer include hemorrhages on the epicardium, thymus, and diaphragm and petechial hemorrhages in the jejunal mucosa. , necropsy lesions reported in goats include pseudomembranous enterocolitis with mucosal ulceration, as well as fibrin, blood clots, and watery contents in the bowel lumen. evidence of systemic toxemia, including multifocal petechial and ecchymotic hemorrhage, proteinaceous exudates in body cavities, pulmonary edema, hydropericardium, and cerebral malacia with perivascular cuffing, have also been reported in goats and affected deer. , , , , clinical pathology. characteristic clinicopathological changes include pronounced hyperglycemia and glucosuria, which are considered a hallmark of c. perfringens d enterotoxemia. additionally, neutrophilic leukocytosis with a left shift and evidence of systemic toxemia (metabolic acidosis, azotemia, and increases in liver and muscle enzymes) also may be seen. treatment. in general, the course of disease is too acute for the establishment of any treatment. however, as with infections with types b and c, if the disease is identified early in the disease course, high doses of oral and parenteral penicillin in addition to clostridium c and d antitoxin may be of benefit. iv fluids and antiinflammatory agents may be indicated as well. prevention. vaccination of pregnant ewes with two doses of toxoid, with the second dose given to weeks before lambing, and adequate ingestion of colostrum are the best methods of protecting newborn lambs. vaccination of older lambs should occur before exposure to diets rich in carbohydrates (grain-feedlot settings) or lush pastures. in these cases, lambs should be vaccinated twice or three times around , , and months of age. males and adult females that are not part of the breeding program may be vaccinated annually. vaccination has been shown to protect goats from experimental disease, but clinical evidence suggests that well-vaccinated goats are still susceptible to developing clostridial enteritis. the toxoids may not protect against local action of the toxins in the goat, which appears to play a greater role in their disease than it does in the sheep. , , more frequent vaccination (every months) in goats is suggested to increase protection. the adjuvant present in some multivalent clostridial vaccines may cause subcutaneous reactions that may lead to abscess formation. in the face of an outbreak, immediate mass administration of c and d antitoxin ( iu/kg) in addition to vaccination is recommended. nonenteric clostridial infections c. novyi, c. septicum, c. chauvoei, and c. sordelli have been identified as causal agents of severe muscle, liver, and abomasal necrosis in small ruminants and cervid species. , [ ] [ ] [ ] these organisms are usually present in the soil and environment and in the gastrointestinal tract and liver of healthy ruminants. pathogenesis is usually facilitated by trauma of affected tissues, local multiplication of the organism, local and systemic damage by exotoxin production, and ultimately death. , four types of c. novyi have been described, a, b, c, and d. c. novyi type c is considered nontoxigenic and therefore is not associated with disease. c. novyi type a produces alpha toxin and is associated with wound infections and myonecrosis in cases of "bighead" and "malignant edema." c. novyi type b produces alpha and beta toxins and is associated with infectious necrotic hepatitis or "black disease." , the temporal and geographic distributions of black disease resemble those of fascioliasis, with the highest incidence of disease in milder, moister months in many countries. black disease is less common in sheep than in cattle and is rare in goats. , c. novyi type d (c. haemolyticum) produces beta toxin and is associated with bacillary hemoglobinuria (red water disease). c. septicum produces alpha toxin and is associated with malignant edema and necrotic abomasitis (braxy). c. chauvoei produces alpha and beta toxins and is associated with severe myonecrosis observed in blackleg and c. sordelli produces a hemolytic toxin associated with myonecrosis in cases of malignant edema and blackleg. , pathogenesis. spores of the organism shed in feces of carrier animals contaminate the environment and are ingested with feed/ grass and stored within kupffer cells. , liver damage caused by migrating liver fluke larvae (fasciola hepatica, fasciola gigantica, and cysticercus tenuicollis) create perfect ischemic conditions that induce germination of c. novyi type b spores and toxin synthesis and production. , , the alpha toxin is necrotoxic and causes liver necrosis and diffuse damage of the vascular system. the beta toxin is produced in smaller amounts and contributes to vascular damage and systemic toxemia. infective organisms also may be brought into the liver by the flukes. clinical signs. the course of disease from first illness to death is short and never lasts more than a few hours in sheep. therefore, peracute or sudden death is not uncommon in this species. wellnourished adult sheep between and years are more commonly affected. the disease course is a little longer ( - days) in cattle and deer. , affected sheep are debilitated, fail to keep up with the flock, and exhibit generalized weakness, sternal recumbency, separation, and anorexia. tachypnea and tachycardia may be seen; high fever ( - ° f) occurs early in the disease. clinical signs observed in cattle, goats, and deer are similar and may include severe depression, anorexia, abdominal distention, colic, ruminal stasis, and lateral recumbency. , , , , , a report of black disease in a forest reindeer (rangifer tarandus fennicus) described serosanguinous discharge from mucocutaneous orifices (nostrils and anus), periorbital edema, and nystagmus in addition to other clinical signs. necropsy findings. necropsy might be difficult due to rapid autolysis of tissues in affected animals. severe venous congestion usually darkens the underside of the skin of affected animals, giving this disease its common name of "black disease." fluid in the pericardial sac, pleural space, and peritoneal cavity is usually present. endocardial and epicardial hemorrhages are a common finding. the liver is swollen and congested and on its diaphragmatic surface presents pale foci of coagulation necrosis; however, solid organs such as liver and kidneys could be in an advanced state of autolysis. characteristic lesions of black disease in the liver are single or multiple yellow to white areas ( - cm in diameter) of necrosis surrounded by a bright hyperemic zone. a recent report of black disease in a reindeer described moderate amounts of dark red thoracic and pericardial fluid, edema of the lungs and upper respiratory tract, swollen spleen, and several well-circumscribed areas of black discoloration in the liver. diagnosis. the most characteristic clinicopathological change is neutrophilic leukocytosis with a left shift. additional evidence of systemic toxemia (metabolic acidosis, azotemia, and increases in liver and muscle enzymes) also may be seen; however, diagnosis of black disease is based on characteristic history (endemic liver fluke areas), clinical signs, and postmortem findings and testing. an impression smear of the margins of the liver might reveal large numbers of gram positive rods, but this is not definitively diagnostic. anaerobic culture of c. novyi from typical liver lesions, in addition to demonstration of the alpha/beta toxins from peritoneal fluid or liver (fresh-refrigerated), through elisa or pcr is required to establish final diagnosis. , the use of fluorescent antibody and ihc for the identification of c. novyi on liver impression smear samples or other liver (formalin-fixed) samples have also been described. , treatment and prevention. treatment is rarely possible because of the fulminant clinical course of the disease; however, if treatment is attempted, high doses of penicillin g sodium ( , - , iu/kg) iv every hours or oxytetracycline mg/kg iv every hours should be initiated. supportive care, including iv fluids, nutritional support, and stress reduction, may be beneficial. in the face of an outbreak, vaccination of the whole herd/flock should be initiated immediately. efforts to control fluke infestation constitute the most effective approach to prevention of this disease. administration of multivalent clostridial vaccines containing c. novyi is highly effective. animals should be vaccinated every months starting around to months of age and before parturition as protective immunity is short lived. in flocks at high risk for developing this disorder, a booster vaccine given month before expected fluke exposure may provide additional protection. , deer should be vaccinated in the same fashion as sheep but double the vaccine dose for sheep should be used for these animals as they do not develop a strong antibody response to commercially available multivalent vaccines. , efforts to eliminate the organism from soil and environment are usually unrewarding but carcasses of animals dying from the disease should be burned, deeply buried, or removed from the premises. pathogenesis. c. novyi type d (c. haemolyticum) is the etiologic agent associated with red water disease. c. haemolyticum is similar to other clostridial species in its life cycle and appears to thrive on alkaline soils and pastures with standing water. the disease tends to be seasonal occurring at times of high larval fluke migration. similar to c. novyi b, c. haemolyticum colonizes the livers of healthy animals and proliferates after liver damage, including damage caused by migrating flukes (f. hepatica, fascioloides magna, dicrocoelium dendriticum, and c. tenuicollis), liver abscessation (fusobacterium necrophorum or trueperella pyogenes), or damage incurred during liver biopsy. , under ischemic conditions of the liver, spores of c. haemolyticum germinate and produce high amounts of beta toxin. the beta toxin causes localized hepatic necrosis and after reaching circulation induces severe intravascular hemolysis and damage of the capillary endothelium. intravascular hemolysis leads to rapid anemia and death due to anoxia. the disease is seen worldwide and is more commonly reported in sheep than in goats. bacillary hemoglobinuria has been reported in a free-ranging elk calf (cervus elaphus roosevelti) found dead in the southwest of the state of washington, united states. clinical signs. bacillary hemoglobinuria usually affects wellnourished animals older than year of age. , in most cases, the disease is per-acute and sudden dead or found dead is the only sign. in cases where signs are recognized antemortem, affected animals appear weak, depressed, and febrile ( - ° f); blood or blood-tinged froth may be present in the nostrils; rectal bleeding and bloody feces may be present; and severe hemoglobinuria (dark red, port wine-colored urine) is usually observed. , blood appears thin and watery and mucous membranes are pale and icteric. heart and respiratory rates are high and become much higher with any sort of effort or stress. other terminal signs include bloat and the presence of blood in the nostrils, mouth, vagina, and rectum. death occurs within hours to a few days after onset of clinical signs. necropsy findings. gross lesions include jaundice of mucous membranes and tissues and subcutaneous petechial/ecchymotic hemorrhages, edema, and emphysema. marked autolysis of internal organs might prevent identification of typical lesions. dark red urine is present in the bladder. lymph nodes and spleen are congested and hemorrhagic. hemorrhagic abomasitis and enteritis might occur, as well as the presence of hemoglobin-stained transudate in pleural and peritoneal cavities and pericardial sac. pulmonary edema is common. the pathognomonic lesion is the ischemic hepatic infarcts ranging from to cm in diameter with a hyperemic interface with healthy liver tissue. , diagnosis. clinicopathological abnormalities usually include anemia, leukocytosis with mature neutrophilia, and degenerative left shift (immature forms of neutrophils and toxic changes) often is present. , serum biochemical evaluation may reveal increased levels of liver enzymes such as sorbitol dehydrogenase, ggt, aspartate aminotransferase, and increased indirect total serum bilirubin. [ ] [ ] [ ] presumptive diagnosis can be made on history, clinical sigs, clinicopathological abnormalities, and postmortem findings; however, similar to black disease, final diagnosis should be based on anaerobic culture of c. novyi from typical liver lesions in addition to demonstration of the beta toxins from peritoneal fluid or liver (fresh-refrigerated) through elisa or pcr techniques. , , , the use of fluorescent antibody and ihc for the identification of c. novyi on liver impression smears or other liver (formalin-fixed) samples has also been described. , more recently, a pcr assay for the detection of c. novyi type d in cattle has been reported. treatment and prevention. treatment is rarely possible because of the fulminant clinical course of the disease; however, if treatment is attempted, high doses of penicillin g sodium ( , - , iu/kg) iv every hours or oxytetracycline mg/kg iv every hours should be initiated. supportive therapy should include the administration of iv fluids, blood transfusions, and antiinflammatory agents. efforts to control liver flukes and prevent other causes of liver damage are most important. administration of multivalent clostridial vaccines containing c. novyi is highly effective. animals should be vaccinated every months starting around to months of age and before parturition as protective immunity is short lived. in flocks at high risk for developing this disorder, a booster vaccine given month before expected fluke exposure may provide additional protection. deer should be vaccinated in the same fashion as sheep, but double the vaccine dose for sheep should be used as these animals as they do not develop a strong antibody response to commercially available multivalent vaccines. , efforts to eliminate the organism from soil and environment are usually unrewarding but carcasses of animals dying from the disease should be burned, deeply buried, or removed from the premises. pathogenesis and clinical signs. fecal and soil contamination of wounds received during fighting (head-butting) or dehorning (disbudding) leads to proliferation of c. novyi type a in damaged head and neck tissues. accumulation of secreted toxins leads to swelling, edema, serohemorrhagic exudates, and local tissue necrosis. wounds appear and smell gangrenous. systemic toxemia may affect internal organs, leading to the death of the animal. c. sordelli causes identical disease. diagnosis. laboratory analysis may reveal an increase in enzymes of muscle or liver origin as well as neutrophilic leukocytosis with many immature and toxic neutrophils. postmortem findings include local necrosis around the injury site. diagnosis usually is made by characteristic clinical signs and lesions. treatment. wound management (disinfection, debridement) and administration of high doses of penicillin g sodium ( , - , iu/kg) iv every hours are important treatment considerations. prevention. ram management may aid in the prevention of head-butting wounds. vaccination with multivalent clostridial toxoids starting around weaning time ( - months of age) and with annual boosters also may be helpful. in flocks with a high prevalence of this disorder, a booster vaccine given to rams month before the breeding season and to ewes/does before parturition may provide additional protection. pathogenesis. c. septicum is the most important agent in the pathogenesis of malignant edema and braxy. in the case of malignant edema, other tissue-invasive clostridia (c. chauvoei, c. sordelli, and c. perfringens a) have also been associated with this disease, and mixed infections are common. the pathogenesis of infection is often similar to that seen with bighead and blackleg: soil or fecal clostridial invasion of a contaminated wound. in sheep and goats, this disease has been reported following lambing/kidding, after shearing of tail docking. c. septicum can also invade the abomasal lining of lambs, causing severe hemorrhagic, necrotic abomasitis known as braxy. activation of dormant bacteria in previously damaged tissue (myositis/abomasitis) similar to that seen in clostridial necrotic hepatitis also occurs. in both cases (malignant edema and braxy), bacterial toxins precipitate local tissue necrosis and systemic toxemia. the alpha, beta, gamma, and delta toxins produced by c. septicum are lecithinase, deoxyribonuclease, hyaluronidase, and hemolysin, respectively. commonly affected sites of malignant edema include castration, dehorning, and injection sites; the umbilicus; and the postpartum uterus. factors that promote braxy have not been identified, although it usually affects weaned and yearling lambs in the winter after ingestion of frozen feedstuffs implicated as initial causes of abomasitis. , both forms of the disease have worldwide distribution and are described more in sheep than in goats. , clinical signs. malignant edema is characterized by local lesion (wound) or regional pain characterized by swelling and edema that progressively becomes tense and dark (skin). high fever, signs of shock/toxemia, and frothy exudation of the wound are usually present. evidence of subcutaneous gas production is less common in this infection than in blackleg. uterine infection may cause a fetid vaginal discharge. death occurs within hours to a few days after onset of clinical signs. braxy usually causes death before any abnormalities are noted. on rare occasions, signs of sudden onset of illness with high fever, abdominal distention, depression, colic, and recumbency may be seen before death. diagnosis. characteristic clinicopathologic changes include neutrophilic leukocytosis with a left shift. a decrease in wbc and rbc counts also is possible because of the leukocidal and hemolytic effects of the toxins. additional evidence of systemic toxemia (metabolic acidosis, azotemia, and increases in liver and muscle enzymes) also may be seen. examination of a gram-stained smear from the edematous swelling(s) or wound swabs could give an early diagnosis. one study reported the successful use of a pcr assay for the identification of bacteria associated with malignant edema in cattle, sheep, and other ruminants. postmortem changes with malignant edema include dark red, swollen muscle filled with hemorrhagic, proteinaceous exudate and little or no gas. with braxy, the abomasal wall is hemorrhagic and necrotic. both diseases are associated with rapid postmortem decomposition of the carcass. treatment and prevention. wound management and the rapid administration of high doses of penicillin (penicillin g sodium at , - , iu/kg iv every hours) are important in treating malignant edema. local treatment consists of surgical incision of the affected area to provide drainage and irrigation with peroxide. injection of penicillin directly into or in the periphery of the lesions may help. ancillary treatments such as iv fluids, antiinflammatory agents (e.g., flunixin meglumine, mg/kg iv), and nutritional support may be necessary. maintenance of good hygiene during procedures such as lambing, tail docking, shearing, castration, obstetric manipulation, and administering injections is helpful in preventing malignant edema. multivalent clostridial toxoids may provide some protection and should be given annually to animals at risk for the disorder. pathogenesis. several species of clostridial organisms can cause myonecrosis in small ruminants. , , the disease is acute to per-acute, has a short course of duration, and is usually fatal. c. chauvoei, c. septicum, and c. sordelli are commonly involved with clostridial myonecrosis in ruminants. [ ] [ ] [ ] blackleg can be enzootic in some areas or farms because of increased bacterial contamination and occurs more commonly in the warm months of the year. [ ] [ ] [ ] animals between months and years of age can be affected. , c. chauvoei is the most important cause of blackleg. c. sordelli tends to be involved in the myonecrosis of older feedlot animals. these organisms are found in the soil and can gain access to muscles after translocation from the gastrointestinal tract and liver into systemic circulation. additionally, direct inoculation of the organisms by penetrating wounds or intramuscular injections has been suggested. local tissue trauma, wounds, unsanitary procedures (i.e., shearing, tail docking, and castration), umbilical infection (neonates), or vaginal trauma from lambing can create perfect conditions for the germination of clostridial spores inducing rapid toxin synthesis and production. in some cases, bacterial proliferation appears to occur in a site distant from the original wound (i.e., fetal infections after shearing of a ewe and myocardial necrosis in cattle and sheep). bacterial toxins cause severe local tissue necrosis, systemic toxemia, and ultimately death. as with braxy, several other strains of tissue-invasive clostridia can cause this disease and mixed infections are common. clinical signs. clostridial myonecrosis usually progresses rapidly and sudden death or history of found dead is not uncommon. [ ] [ ] [ ] clinical signs in animals who are still alive include local to regional painful, edematous swelling most commonly in the limbs or trunk muscles. skin of the affected area can become discolored and crepitus; however, in affected sheep, subcutaneous edema and gaseous crepitation are uncommon and cannot be felt before death. other signs might include stiff gait, lameness, fever, and signs of shock. in cases where the infection occurred through a wound, there is extensive local damage and malodorous serosanguinous fluid discharge. c. chauvoei also causes uterine infection and severe gangrenous mastitis in postparturient ewes. , in these cases, uterine and mammary infections may cause fetid vaginal and mammary discharge, respectively. death often occurs within to hours after onset of clinical signs. necropsy findings. rapid tissue autolysis is not uncommon in animals that succumb to clostridial myonecrosis. bloodstained fluid and froth can be observed discharging from nostrils and anus. in small ruminants and especially sheep, affected muscle areas are more localized and deeper, brown to black discoloration is present, the subcutaneous edema is not as severe, and, although there is gas present, is not in such large amounts as in cattle. in cases of infection from skin wounds, the area demonstrates subcutaneous edema, swelling, and underlying muscle discoloration. in cases of infection through the urogenital tract, typical lesions are found in the perineal area, vagina, uterus, and fetus. lung congestion, fibrinohemorrhagic pleuritis, pericarditis, myocardial damage, and bloat are also common findings. , diagnosis. it is rarely possible to obtain samples for clinicopathological analysis due to the per-acute course of the disease. if samples can be obtained, common findings include neutrophilic leukocytosis with a left shift. a decrease in wbc and rbc counts also is possible because of the leukocidal and hemolytic effects of the toxins. additional evidence of systemic toxemia-metabolic acidosis, azotemia, and increases in liver and muscle enzymesalso may be seen. presumptive diagnosis can be made from history, characteristic clinical signs, and gross pathology findings; however, aspirates or tissue specimens from affected muscles for direct smear examination, fluorescent antibody testing, or anaerobic culture are required for definitive diagnosis. , a multiplex pcr is available for identification of pathogenic clostridia on fluid and tissue samples. treatment and prevention. aggressive antibiotic therapy (e.g., penicillin g sodium or potassium penicillin at , - , iu/kg iv every hours), in combination with surgical debridement of affected tissues (fasciotomy), and supportive care (nutritional support, iv fluids, and antiinflammatory agents) are important within the treatment plan for clostridial myositis. prognosis for treatment of all types of clostridial myositis cases is usually guarded to poor and depends on the duration and extension of the lesions. maintaining excellent hygiene during invasive procedures such as castration, obstetric manipulation, shearing, tail docking, and administering injections is helpful in preventing blackleg. multivalent clostridial toxoids may provide some protection and should be given to all animals starting at weaning time, before parturition, and annually. , both tetanus and botulism are important diseases in small ruminant medicine. these two diseases are covered elsewhere in this book (see chapters , , , , and ) . older animals are generally more resistant to sepsis than neonates because they have larger amounts of circulating antibodies. however, this resistance can be overwhelmed by aggressive bacteria, or loss of immune function can allow invasion by opportunistic bacteria. malnutrition, parasitism, transport, overcrowding, other diseases, extreme weather conditions, and other stressors are the major causes of immune suppression. sepsis may produce peracute, acute, or chronic disease signs. peracute signs include fever, injected mucous membranes (including the sclera), tachycardia, tachypnea, dyspnea, swollen joints, lameness, splinting of the abdomen, weakness, depression, anorexia, recumbency, seizures, coma, and sudden death. acute signs are similar, except that they persist for a longer period and therefore are more likely to be noticed. chronic signs usually result from the partial clearance of infection after an acute episode, which may be clinical or inapparent. pathogenesis. gram negative bacteria and their toxins gain access to the blood from a site of proliferation or destruction. the most important toxin is endotoxin, a group of lipopolysaccharide molecules that reside within the wall of the bacteria. bacteria or endotoxins incite a systemic inflammatory response, chiefly through activation of host macrophages and stimulation of host cytokine release. these cytokines cause inflammation, produce leukocyte recruitment, increase capillary permeability, induce fever through stimulation of the hypothalamus, and have regional or diffuse vasomotor effects. because the ruminant gut has a plentiful population of gram negative bacteria, it is implicated as the source of most cases of gram negative sepsis. grain overload causes a die-off of the normal gram negative ruminal flora, ulcerative enteric disease allows invasion of bacteria or absorption of their toxins, and ingestion of pathogens provides a suitable place for proliferation and route for invasion of the body. gram negative sepsis caused by opportunistic organisms is best recognized in immunocompromised neonates but also can be seen in stressed or immunocompromised animals of all ages. e. coli is commonly found in fecal material, klebsiella pneumoniae is found in feces and wood products, f. necrophorum lives in the gastrointestinal tract and in soil and invades through compromised gastric mucosa or foot-rot lesions, and pseudomonas aeruginosa is commonly found in water and wash solutions. primary pathogens are most common in adults. although some coliform bacteria may fit into this category, by far, the most important genus is salmonella. sources of salmonella infection are numerous and include carrier animals of the same species, cattle, rodents, birds, other animals, environmental contamination, and possibly feedstuffs. only one serotype of salmonella is specifically adapted to sheep (salmonella abortus ovis), and it is not found in north america. no strain is known to be host-adapted to goats or cervids. therefore, all infections in sheep, goats, and cervids have the potential to spread to and from other species, including humans. serotypes of salmonella that have caused important infections in sheep or goats include salmonella typhimurium, salmonella dublin, and salmonella montevideo. most of these infections lead to bacteremia with mild systemic signs, followed by abortion. s. dublin and s. typhimurium tend to cause more illness in adults because of fibrinonecrotic enteritis. clinical signs. affected animals can exhibit anything, from mild depression with a low-grade fever to shock. common signs include fever, tachycardia, tachypnea, depression with slow or absent eating and drinking, weakness or recumbency, and injection or cyanosis of mucous membranes. organ-specific signs may betray the source or at least the primary location of the infection. fetid discharge may be seen with metritis or abortion; dyspnea and abnormal lung sounds may be seen with pulmonary infection; and bloat, ruminal atony, abdominal distention, and diarrhea may be seen with gastrointestinal infections. diagnosis. the most common abnormality identified on a cbc with peracute gram negative sepsis is panleukopenia. over the course of several days, this condition may resolve, first through an increase in immature neutrophils and later through an increase in mature neutrophils and restoration of lymphocyte counts. very immature cells, severe toxic changes, and persistence of neutropenia suggest a poor prognosis. serum biochemical changes often reflect the severity of the condition. the greater the evidence of shock or tissue damage, the worse the prognosis. metabolic acidosis with a large anion gap and azotemia suggest advanced disease. necropsy findings include diffuse evidence of inflammation, including pulmonary congestion, and polyserositis with body cavity exudates. hemorrhagic pneumonia or fibrinonecrotic enteritis may be seen and reflect the source of bacterial invasion. in all cases, diagnosis is best confirmed by bacteriologic culture of body tissues or fluids. in the live animal, culture of blood, feces, or tracheal fluid yields the best results. when several animals are infected, environmental samples (including feed, water, and bedding) should be tested for the presence of the bacteria. bacteriologic culture of aborted fetuses or placentas frequently yields heavy growth of the organism. prevention. maintaining overall good health and hygiene is the best means of preventing gram negative sepsis. antiendotoxin bacterins are available for cattle in the united states, but their use in small ruminants has been too limited to assess their efficacy. during a flock outbreak, the use of autogenous bacterin may help prevent the spread of disease on a farm. actinobacillus seminis is a gram negative bacillus or coccobacillus that affects primarily the male and female reproductive tracts. infection causes posthitis, epididymitis, and orchitis in rams and metritis and abortion in ewes. other sites of infection, including rare occurrences of chronic sepsis, also are possible. serologic tests are much more useful for identifying infected flocks than infected individuals within flocks. definitive diagnosis depends on bacteriologic culture of the organism and differentiation of it from brucella ovis. the bacillus is common in sheep in some parts of the world but is uncommon in north american sheep and goats. t. pyogenes is best known as an abscess-forming bacterium because of the thick pus formed in response to infection by it and the fibrinous response it elicits. it occasionally also causes sepsis. its association with chronic sepsis lends credence to the belief that trueperella is often a secondary invader that colonizes tissues damaged by another bacterium (see chapter ) . bacillus anthracis is a large, gram positive, anaerobic bacillus that causes anthrax. it forms spores under aerobic conditions (such as on culture plates) but rarely does so when oxygen tensions are low, as in carcasses. the organism affects most mammals, with herbivores being most susceptible. it is usually carried from one area to another by shedding or dying animals and also can multiply in alkaline, nitrogenous soils. periods of heat and intermittent flooding promote overgrowth of the organism. b. anthracis spores may be inhaled or ingested; in rare cases, the bacillus itself may be spread by biting flies. after local replication, the organism gains access to the blood, where it multiples readily. large numbers of the organisms colonize the spleen. b. anthracis secretes a holotoxin made of edema factor), protective antigen, and lethal factor. this toxin impairs phagocytosis, increases capillary permeability, and inhibits clotting. splenic engorgement, generalized edema, circulatory shock, and bleeding diathesis are the most common lesions and signs of anthrax. generalized infection should be considered uniformly fatal. death may occur before or within hours of initial recognition that the animal is sick. prophylactic antibiotic treatment of healthy animals (oxytetracycline mg/kg iv sid) may decrease spread and mortality during outbreaks. the disease is reportable in many areas. local forms of anthrax also occur, most commonly after transmission through a skin wound or fly bite. local heat, pain, swelling, and necrosis are seen first, and the generalized syndrome often follows. bacterial organisms are rarely identified before important treatment decisions must be made. therefore, treatment should follow general principles and have a wide spectrum of efficacy. antimicrobial drugs are the cornerstone of treatment. in meat-or milkproducing animals, the veterinarian must be careful to use drugs within label directions or have a rational plan for extra-label drug use. the issue of extra-label drug use is especially important in small ruminants and cervids because very few pharmaceutical products have been licensed for them in north america. unless a specific organism is suspected (clostridiosis or anaplasmosis), a single antibiotic or combination of antimicrobial drugs to provide a broad spectrum of coverage should be selected. penicillins, macrolides, tetracyclines, and cephalosporins all provide effective coverage against gram positive pathogens. the newer third-generation cephalosporins are effective against many systemic and enteric gram negative pathogens. the gram negative pathogens of the respiratory tract are often sensitive to other classes of antibiotics. macrolides and tetracyclines also are effective against mycoplasma species and rickettsial organisms. nsaids are almost always beneficial in severe infectious conditions because of their antiinflammatory, antipyretic, and antiendotoxic effects. they are likely to be more effective than corticosteroids because they provide benefits without suppressing the immune response. all such drug use should be considered extralabel and administered accordingly with appropriate withdrawal times established. specific antisera are available for some of the clostridial diseases and may be beneficial if given before widespread tissue necrosis has occurred. severely compromised animals should be treated with fluids for shock (see chapter ). the most common zoonotic disease risk posed by exposure to small ruminants is orf, also known as contagious ecthyma in animals (see chapter ) . the disease is caused by an epitheliotropic poxvirus and is transmitted to humans by direct contact with infected animals. skin trauma is a significant risk factor for transmission in both humans and animals. in humans, erythematous macules or papules appear at the site of infection to days following exposure. the infection is generally self-limiting in immunocompetent individuals with complete healing occurring within weeks. brucella melitensis. apart from contagious ecthyma, the greatest risk of zoonotic disease from small ruminants is due to pathogens typically found in the reproductive tract that are transmitted to humans through contact with aborted fetuses, the placenta, or birthing fluids or through the consumption of raw or improperly pasteurized dairy products. b. melitensis is more common in goats than sheep (see chapter ) . swine, cattle, and other ruminants are common hosts. infection in animals usually causes inapparent mammary infection and abortions; infection in humans is characterized by undulant fever, myalgia, and fatigue. coxiella burnetii. c. burnetii is a rickettsial organism that is an important cause of abortion in sheep and goats (see chapter ) . wildlife and farm-raised deer may serve as reservoir hosts for infection in other ruminants and humans. infection is a documented cause of reproductive failure in farmed deer and prolonged shedding of the organism is an important source of environmental contamination. in addition to abortion, newly infected sheep and goats occasionally have mild, transient fevers. c. burnetii is far more important as the cause of q fever in humans, who become infected after inhaling particles, handling contaminated animals, or coming into contact with contaminated body fluids (uterine fluid, milk) from infected animals. infection in humans may be asymptomatic, present with flu-like symptoms, or, in the chronic form, present as granulomatous hepatitis, osteomyelitis, or bacterial endocarditis. chlamydophila spp. chlamydophila abortus (previously chlamydia psittaci) is an obligate intracellular parasite and the cause of enzootic abortion of small ruminants (see chapter ) . chlamydophila pecorum may cause polyarthritis and keratoconjunctivitis (see chapter ) in sheep and goats. transmission between animals and to humans most commonly occurs through direct contact with infected tissues or materials. infection in humans results in an acute febrile syndrome or respiratory symptoms. chlamydial diseases are more commonly reported in sheep than in goats. chlamydial diseases are suspected to cause disease in other species, including deer. recent serologic evaluation of wild ungulates identified multiple species of deer with antibodies against several chlamydial species. the clinical significance of serological infection in these species remains undetermined. francisella tularensis. f. tularensis is more common in sheep than goats. the organism has many hosts, of which the most important are wild rabbits and rodents. it can contaminate water sources. transmission to sheep is usually through biting arthropods that have previously fed on an infected wild mammal. acute or chronic sepsis may be seen, with more widespread and severe disease occurring in sheep with poor immune function. at necropsy, the disease is characterized by military foci of necrosis in the liver, and less commonly in the lymph nodes, spleen, and lungs. most cases in humans result in acute onset of flu-like symptoms a few days after exposure. l. interrogans. pathogenesis. leptospira spp. are spirochete bacteria that live in moist environments. their survival time outside of hosts is usually short, so their most important reservoirs are the kidneys of infected animals, especially rodents. infected animals shed the organisms through urine and most other body fluids. organisms enter new hosts through mucous membranes and skin breaks and cause bacteremia. signs of sepsis range from inapparent to severe, with more severe signs predominating in neonates. intravascular hemolysis may result. in animals that survive the acute stage, infection may localize in sites such as the kidneys, eyes, and fetoplacental unit. abortion may occur a month or more after acute signs first become evident while renal shedding may occur for several months. leptospirosis is zoonotic. in most cases, infections in humans are asymptomatic and selflimiting. however, in approximately % of cases, severe, and potentially fatal, systemic disease may develop, including jaundice, renal failure, and pulmonary hemorrhage. clinical signs acute leptospirosis causes signs of sepsis, including fever, depression, dyspnea, exercise intolerance, weakness, and death (see chapter ) . additionally, many affected animals show signs of intravascular hemolysis such as anemia, icterus, and hemoglobinuria. diagnosis evidence of intravascular hemolysis such as anemia, hyperbilirubinemia, hemoglobinuria, and hemoglobinemia is suggestive of this disease. in chronic infection, non-specific inflammatory changes and azotemia may be seen. animals dying in the acute hemolytic stage are likely to have dark, discolored urine, bladder, and kidneys. spirochetes can be identified on dark-field microscopy of fresh urine or plasma from infected animals and may be cultured with special techniques. in animals with less severe infection, a rise in antibody titers can be used to support a diagnosis of leptospirosis. prevention numerous vaccines are available for sheep. because protection is serotype specific, it is important to vaccinate against common serotypes in the area. leptospira pomona is the most consistent isolate from sheep and goats; leptospira hardjobovis is the predominate serovar in deer. vaccination immunity is thought to be short lived; boosters should be given at least twice a year in endemic areas. vaccination of deer against serovars hardjobovis and pomona has been associated with decreased urine shedding and increased growth rate in young animals. pathogenesis. l. monocytogenes causes disease with similar frequency in sheep and goats (see chapter ) . the organism is a common soil and fecal contaminant. it also proliferates in silage that is not properly acidified and in rotting, woody debris. risk of exposure depends on the feed and environment of the animals. environmental and fecal contamination is a more common source than silage in small ruminants overall because most sheep and goats throughout the world are not fed silage. infection in humans almost always results from ingestion of contaminated food products or unpasteurized milk. clinical signs. nervous system dysfunction and abortion are the most common manifestations of the disease. animals with the brainstem form of the disease display signs reflective of cranial nerve dysfunction, including drooped ears or eyelids, decreased facial sensation, and deviated nasal septum. a head tilt and circling may be present; in advanced cases of the disease, the animal is recumbent. clinical signs are mainly unilateral, occasionally bilateral, according to the nerve nuclei affected. diagnosis. antemortem diagnosis of listeriosis is difficult. a presumptive diagnosis is made based on history, clinical signs, and potential response to treatment. histopathologic identification of microabscesses in the brainstem and culture of the organism from affected tissues can be used to confirm the diagnosis. pathogenesis. p. multocida is a small, gram negative, bipolar, ovoid rod that inhabits the pharynx of healthy ruminants. it can survive in soil and water for varying amounts of time after contamination with ruminant nasal secretions. healthy ruminants shed p. multocida much more frequently than mannheimia haemolytica. disease occurs when bacteria colonize the lower respiratory tract or enter the blood. risk factors for pulmonary and systemic infection include viral or mycoplasmal respiratory diseases, temperature extremes, respiratory tract irritants, transport, overcrowding, changes to higher-energy feeds, and handling stress. these factors are thought to both increase bacterial replication in the airway and suppress mechanisms to clear the infection. pasteurellosis is a major problem in feedlot sheep but less common in small breeding or hobby flocks. pasteurellosis also is a significant disease in certain wild small ruminants such as bighorn sheep. direct spread of the organism between animals occurs with nasal contact, and indirect spread occurs after contact with infected nasal secretions. the organism persists in the environment for longer periods during warm, moist weather. p. multocida produces a polysaccharide capsule that inhibits phagocytosis and an endotoxin that contributes to clinical signs. the major disease caused by p. multocida is pneumonia (see chapter ). however, pasteurella spp. also are capable of entering the blood to cause septicemia in young lambs and hemorrhagic septicemia in adults. occasionally, focal infections such as septic arthritis and mastitis are found. clinical signs. clinical signs of pneumonic and septicemic pasteurellosis include severe depression, bilateral purulent nasal discharge, coughing, diarrhea, anorexia, high fever, and edema of the head, neck, and brisket. the disease course can be short with septicemic pasteurellosis and is usually more insidious with p. multocida pneumonia. pasteurella mastitis is characterized by the bluebag condition or gangrene of the udder. diagnosis. inflammatory changes in the leukogram and hyperfibrinogenemia are the most frequent abnormalities. with severe disease and in the septicemic form, immature neutrophils may predominate over mature cells. inflammation of the intestine and abomasum also may be seen. hemorrhage and fibrin are usually absent or less prominent than in pneumonia caused by m. haemolytica. samples for bacteriologic culture are usually obtained postmortem. blood or tracheal fluid may be obtained before death if the value of the animal warrants it. m. haemolytica is a gram negative rod that is a common commensal inhabitant of the tonsils of young animals. disease is much more frequently described in sheep than in goats and occurs when the organism gains access to the lower respiratory tract. clinical signs and diagnosis. the most common syndrome is enzootic pneumonia, which is seen in young lambs and their dams (see chapter ) . hemorrhagic bronchopneumonia is the major lesion and respiratory signs predominate. gangrenous mastitis (bluebag) is seen in some of the dams, presumably after they have been nursed by infected offspring. factors that promote respiratory disease, including viral infections, airborne irritants, high stocking density, and stress, are thought to promote invasion of the lower airway by these bacteria. b. trehalosi is a gram negative rod that is a commensal inhabitant of the upper respiratory tract (see chapter ) . disease is much more frequently described in sheep than in goats and occurs when the organism gains access to the lung or blood. replication occurs in the lung and systemic toxemia or bacteremia resulting in septicemic pasteurellosis. septicemic pasteurellosis is a significant cause of mortality in young lambs and in some farms is the leading cause of death in the age group. clinical signs. septicemic pasteurellosis occurs most commonly in weaned lambs, often following some form of stress such as transport, marketing, or weaning itself. the course of the disease is relatively rapid, and animals may be found dead within hours without showing premonitory clinical signs. when observed, clinical signs include depression, recumbency, and signs of toxemia. diagnosis. septicemic pasteurellosis should be suspected when presented with a dead, recently weaned, sheep with a recent history of stress. diagnosis is best confirmed by typical lesions at necropsy and culture of the organism from bodily tissues. demonstration of b. trehalosi in nasal swabs is of limited value due to the high prevalence of upper respiratory tract colonization in healthy lambs. at necropsy, there may be no evidence of pneumonia, but blood-stained foam can be found in the upper respiratory tract. ulceration of the pharynx and esophagus is commonly present as is subcutaneous hemorrhage of the neck and thorax. prevention. treatment is difficult due to the rapid course of disease. efforts should be made to minimize stressors, particularly during and following weaning, and to manage management factors that may contribute to the disease. vaccination with pasteurella bacterins is rarely effective at controlling natural outbreaks of disease. pathophysiology. abscess-forming bacteria are usually able to survive phagocytosis and thereby avoid destruction by cells of the immune system. alternatively, they invoke such an inflammatory response that the host body "walls off" the entire region with fibrous tissue. abscesses may occur locally, frequently after a wound infection, or at numerous or distant sites from the point of infection. for abscesses to occur at the latter sites, the organism must travel either by way of the blood or within leukocytes. disease characterized by multifocal or internal abscesses usually results from a low-grade, transient event of bacteremia. the best known and most important abscess-forming bacterium in small ruminants is corynebacterium pseudotuberculosis, the gram positive, facultative anaerobic coccobacillus that causes caseous lymphadenitis. infection is usually maintained in a flock by infected animals that spread the organism to others through purulent material draining from open abscesses. the organism is very hardy, so infection can occur through direct contact or indirect contact with contaminated common instruments and facilities. infection is usually introduced into a flock through acquisition of an infected animal, although it also can occur when a naive flock is moved into a contaminated area. horses, cattle, and humans also are minor hosts. infection is thought to occur after ingestion, inhalation, or wound contamination. except for lower respiratory tract invasion, a surface break is thought to be necessary. contaminated shears, tail-docking knives, and emasculators readily spread the organisms through a flock. abscesses can form at the site of invasion or more commonly at the site of the local lymph node. clinical signs. clinical signs of external abscesses include surface swellings and draining lesions. drainage may be intermittent and usually consists of thick, yellow-white purulent material. internal abscesses are more difficult to diagnose. thoracic masses may cause inspiratory dyspnea or occlude venous return to the heart. abdominal lesions may cause tenesmus, stranguria, and occasionally colic. the most common sign of internal abscesses is weight loss with or without intermittent fever. common external sites include the submandibular or retromandibular space and the preinguinal, prefemoral, and supramammary lymph nodes. head and neck lesions are more common in goats, whereas sheep have a more even distribution of cranial and caudal lesions, presumably as a result of shearing wounds. external infections rarely cause clinical illness beyond the draining abscess, although some degree of cachexia may be present. diagnosis. diagnosis is often made by the characteristic lesions with their thick, nonmalodorous pus. bacteriologic culture provides a definitive diagnosis, which may be important for flock management. serologic tests have been developed to identify carrier animals and may be useful if the manager wishes to eliminate infection from the flock. treatment. treatment is often unrewarding: antibiotic sensitivity profiles do not reflect the degree of protection afforded the organisms within the abscesses. long-term treatment with antibiotics and drainage of any compromising masses may lead to some degree of resolution, but internal abscesses are likely to persist. prevention. prevention through the use of vaccines has been attempted. vaccines appear to reduce the severity of the disease but do not completely prevent infection. moreover, live attenuated bacterins lead to de facto infection of all vaccinated animals and therefore should not be used in naïve flocks. other abscess-forming bacteria are most important as differential diagnoses for caseous lymphadenitis. t. pyogenes is another wound contaminant that affects focal areas or regional external lymph nodes. it also commonly colonizes damaged internal tissues such as postpneumonic lungs, postacidotic livers, and damaged feet and heart valves. it is thought to be ubiquitous and poorly invasive in ruminants and therefore does not have the same flock significance as c. pseudotuberculosis. flocks with outbreaks of this infection often have suboptimal management. f. necrophorum causes similar disease and often coinfects with t. pyogenes. it is generally more necrotizing and leads to greater systemic signs of acute illness, including death. f. necrophorum also produces fetid pus, whereas t. pyogenes usually does not. rhodococcus equi is a rare cause of pulmonary abscesses in sheep. numerous small, coalescent, nodular skin abscesses may result from pseudomonas pseudomallei infection (melioidosis). infection usually occurs after the sheep or goat is bitten by an insect that previously fed on an infected rodent. this organism is found in many subtropical regions, including the caribbean, but is not reported in north america. f. necrophorum causes or is associated with a variety of diseases in sheep and is likely to cause many similar diseases in goats. it is best known as a cause of foot rot and hepatic abscesses and appears to be important in lip-leg ulceration. it is an enteric gram negative anaerobe and as such can cause gram negative sepsis after entrance of the bacteria or its toxins into the circulation. f. necrophorum has a poor ability to invade healthy tissue. however, it readily colonizes regions damaged by trauma, persistent moisture, and infection. in addition to endotoxin, the bacterium produces leukocidal and cytolytic toxins that form zones of necrosis around bacterial colonies. this tissue necrosis and the foul-smelling waste gases produced by the bacteria are characteristic of necrobacillosis, or f. necrophorum infection. clinical signs include necrotic, fetid lesions, usually of the mouth or feet, that can cause ingestion or lameness problems. efforts to maintain good hygiene are helpful in preventing fecal contamination. additionally, preventing trauma to foot and mouth tissues through good surface choices and proper pasture drainage is important. pathogenesis. yersinia spp. are gram negative bacteria. yersinia enterocolitica and yersinia pseudotuberculosis both have many mammalian and avian hosts, including humans, and cause clostridial enteritis-like disease in goats. rodent and bird hosts may be important reservoir populations for infections in domestic animals. kids younger than months develop enteritis, bacteremia, and diarrhea that is watery but not bloody. severe toxemia and sudden death can occur. older kids and flocks with chronic exposure tend to have less severe acute disease. instead, chronic diarrhea and weight loss are seen, usually in association with gut wall and abdominal abscesses. sheep, deer, and wild ungulates are rarely affected. clinical signs. signs of enteritis or sepsis predominate in acute disease, whereas signs of wasting are more common in chronic disease. diagnosis. evidence of acute or chronic inflammation is provided by blood work. characteristic necropsy lesions include numerous microabscesses in the gut wall and mesenteric lymph nodes, as well as other evidence of enteritis or sepsis. culture of lesions and demonstration of a rising antibody titer are diagnostic. prevention. avoiding exposure to sources and maintaining overall flock health are helpful in preventing losses due to yersiniosis. pathogenesis. mycobacteria are small, aerobic, straight or curved pleomorphic rods with thick lipid cell walls. they can be stained with acid-fast stains and are usually gram positive. the bacteria live within infected animals of many mammalian species and survive for several years in warm, moist environments. infection occurs after ingestion or inhalation. an identifying characteristic of the mechanism of infection by mycobacteria is the bacteria's ability to survive within macrophages by preventing fusion of phagosomes and lysosomes. the organisms are carried to local lymphatic vessels or lymph nodes, where they form granulomas. as they enlarge, granulomas may develop necrotic or mineralized centers surrounded by macrophages and giant cells. disease can be local, regional, or generalized, depending on the distance the organism is carried from the original site of infection. granulomatous pneumonia, enterocolitis, and lymphadenitis are the most common local and regional forms of the disease. organisms from ruptured granulomas may be spread in contaminated respiratory secretions and feces. mycobacterial infections of all types are uncommon in north american sheep, goats, and cervids, and these species are considered to be relatively resistant to infection. mycobacterium bovis is the most common organism associated with ovine tuberculosis in other countries (see chapter ), but mycobacterium avium is more common in the united states. the most common mycobacterial infection is johne's disease (paratuberculosis) caused by the etiologic agent m. avium subsp. paratuberculosis (see chapter ) . mycobacterium tuberculosis is rare in the united states. mycobacterial infections are reportable in most parts of the united states. some debate is ongoing about human susceptibility to m. avium subsp. paratuberculosis; the other organisms are known to be pathogenic in people. clinical signs. the most common clinical sign is emaciation. diarrhea may be seen terminally in both tuberculosis and paratuberculosis. the disease is insidious, with signs becoming more apparent over several weeks to months. respiratory signs may be seen, especially with infection by m. bovis or m. avium subsp. diagnosis. reports of clinicopathologic abnormalities are rare. hypoalbuminemia and hypoproteinemia are likely to be common with chronic enterocolitis caused by either tuberculosis or paratuberculosis. the most common necropsy lesions seen in tuberculosis are nodular lesions of the lung, liver, lymph nodes, spleen, and intestines. histologic evaluation reveals the nodules to be granulomas with giant cells and acid-fast organisms. frequently, the center of the lesion is necrotic and mineralized. intestinal lesions appear to be more common than pulmonary lesions in goats. the lesions of paratuberculosis are centered around the ileocecocolic junction and the adjacent mesentery. the regions may appear normal or be notably thickened. thickening of bowel or nodular infiltrates of lung or liver may be detected antemortem using imaging modalities, such as ultrasonography or computed tomography. postmortem diagnosis is made by identifying characteristic lesions and culturing the organisms. antemortem diagnosis of tuberculosis is best achieved by observing the reaction to intradermal injection of tuberculin with or without comparative injection of purified protein derivatives of m. bovis and m. avium subsp. paratuberculosis. all tuberculosis testing should be done in accordance with local regulations. antemortem diagnosis of johne's disease can be achieved by fecal culture of the organism, but this test takes several weeks to months to complete and is far less reliable in sheep or goats than cattle, with a sensitivity as low as . . serologic tests (e.g., elisa) appear to be sensitive and specific for johne's disease in animals demonstrating clinical disease rather than preclinical infection. serologic detection of clinical johne's disease in cervids has been shown to be highly sensitive and specific while the sensitivity of fecal culture is low in both sheep and goats. the recommended organism detection method in both species is fecal pcr. fecal or milk pcr can be used on pooled samples for flock identification and to type the organism. prevention. tuberculosis should not be endemic in flocks in the united states because positive animals are quarantined or destroyed. preventing exposure to wild ruminants and other possible sources is crucial. except in goat flocks raised for the production of milk that is to be sold unpasteurized, testing is uncommon, so animals are usually not identified until they develop overt disease. paratuberculosis is much more common and may be maintained in flocks by carrier animals. no effective treatment is available for either disease, nor should any be encouraged because efforts should be concentrated on eliminating infection from the flock or herd. vaccination of sheep is used extensively in australia to control paratuberculosis. prolonged vaccination has been shown to decrease fecal shedding in infected animals over time. pathogenesis. mycoplasma spp. are very small, simple bacteria that parasitize cells of higher species. they are common inhabitants of mucous membranes and can have either a commensal or pathogenic relationship with the host. transmission between animals is most likely through direct or indirect contact with body fluids from infected animals, inhalation of respiratory droplets, and arthropod vectors. common sites for superficial infection include the ocular membranes, lung, mammary gland, and female reproductive tract. the organisms can also enter the blood and cause septicemia, abortion, pleuritis, and polyarthritis. flare-ups often occur during times of crowding and during parturition, when neonates can spread the organisms from the mother's mouth to her udder and in turn become infected by ingesting contaminated milk. the most important mycoplasma species in the united states are mycoplasma conjunctivae, mycoplasma capricolum, and the less pathogenic mycoplasma ovipneumoniae. they are most commonly associated with keratoconjunctivitis, acute or chronic sepsis, and pneumonia, respectively. m. conjunctivae and c. abortus are the most common causes of pinkeye in north american small ruminants. mycoplasma spp. are thought to inhibit tracheal ciliary function and thus may have a role similar to viruses in "shipping fever pneumonia" in facilitating lower respiratory tract invasion by primary bacterial pathogens. many of the major pathogenic serotypes found in other countries (some of which cause severe pleuropneumonia without the participation of another bacteria), including mycoplasma mycoides subsp. mycoides, mycoplasma mycoides subsp. capri, mycoplasma agalactiae, and strain f , are not found in or have been eradicated from north america clinical signs. keratoconjunctivitis, mastitis, exudative vulvovaginitis, fever, cough, dyspnea, exercise intolerance, abortion, lameness, swollen joints, neonatal death, and depression may all be seen with mycoplasma infections. diagnosis. no specific clinical pathologic findings occur with these diseases. mycoplasma infection should be suspected in sheep and goats with severe exudative pleuropneumonia in some parts of the world. mycoplasma can be identified by bacteriologic culture or staining of exudates. examiners must take care in interpreting positive cultures from body surfaces because nonpathogenic mycoplasma are common. prevention. vaccines against mycoplasmal infections are available in some parts of the world, but not in the united states. providing fly control, preventing stress and overcrowding, and isolating sick animals from healthy ones may help prevent the spread of disease. anaplasma ovis, mycoplasma ovis, and babesia spp. a. ovis and m. ovis are small bacteria that lack cells walls and parasitize erythrocytes. these and similar organisms have undergone recent reclassification following molecular analysis. other species of hemotropic mycoplasmas may affect sheep and cervids. the organisms are spread from animal to animal by insect or mechanical vectors. known arthropod vectors for a. ovis include ticks and horseflies; other biting flies may be more important with m. ovis infection. hypodermic needles and equipment used for tail-docking, castrating, or disbudding animals may be important in iatrogenic transmission. after being introduced into a naive host, the organisms proliferate, and the number of red cells infected increases rapidly until an effective immune response begins to weeks later. a similar proliferation of organisms may occur in chronically infected animals after temporary immune suppression. the humoral and cellular immune responses against a. ovis lead to opsonization of parasitized erythrocytes and their removal by cells of the reticuloendothelial system; m. ovis infection is thought to cause more intravascular hemolysis. the result in both cases is hemolytic anemia. the protozoon parasites babesia ovis and babesia motasi have similar life cycles and cause similar diseases, but they have been eradicated and are reportable in the united states. babesia spp. affecting small ruminants are generally less pathogenic than are their bovine counterparts. animals surviving acute hemolytic crisis reduce the parasites to low numbers but rarely clear the infection completely; they serve as sources of infection for other animals. sheep and goats are susceptible to infection by either organism; goats generally appear to be more resistant to the development of severe parasitemia and clinical signs. clinical signs. signs present during hemolytic crises include fever, weakness, pale mucous membranes, and pigmenturia. urine discoloration results from increased amounts of bilirubin in most cases, although hemoglobinuria may be seen in some sheep with m. ovis infection. icterus is usually present only after the acute hemolytic crisis. clinical signs are exacerbated during times of stress, and infection is often first noted when the animals are moved or handled. chronically infected animals may appear clinically normal, may have recrudescence of infection after stress, or may display signs of ill-thrift such as poor body condition and fleece. babesiosis occasionally causes concurrent central neurologic signs. diagnosis. the major clinical laboratory finding is regenerative anemia with detection of the intraerythrocytic bodies. chronically infected sheep often have high counts of nucleated erythrocytes. because m. ovis consumes glucose, hypoglycemia and metabolic acidosis may be detected, especially in blood samples that are not processed immediately. diagnosis is by identification of the organisms on blood smears. special stains are available to make the organisms more visible. postmortem lesions include pallor or icterus of membranes and splenomegaly. some evidence of vasculitis, including edema or exudates in body tissues or cavities, may be seen with m. ovis infection. treatment. mycoplasma spp. and anaplasma spp. are sensitive to tetracycline antibiotics. babesiosis is more difficult to treat. effective drugs include diminazene, pentamidine, and imidocarb dipropionate. supportive care for all blood parasite infections includes whole blood transfusions, nutritional support, and administration of fluids. prevention. prevention in most cases involves maintaining low levels of parasites rather than eliminating them entirely. this method ensures continual stimulation of the immune response, whereas eradication often leaves the animal susceptible to another bout of acute infection. vector control can also be important in management of the disease. pathogenesis. two organisms belonging to the anaplasmataceae family, ehrlichia ovis and anaplasma phagocytophilum, infect ovine wbcs, causing fever, immune suppression, and some organ damage. a. phagocytophilum is the causative agent of tick borne fever in sheep and granulocytic anaplasmosis in horses, dogs, and humans. the organism is transmitted by ticks (ixodes spp.) and maintained in the environment by asymptomatic carrier animals. the distribution and incidence of disease is seasonal with the life cycle of the tick. the organism infects cells of the granulocytic lineage, leading to severe persistent neutropenia and acute lymphopenia. fever occurs to weeks after infection, lasts as long as weeks, and occasionally relapses. chronic infection is common. spleen, lung, liver, and kidney tissue may show some damage because of immune destruction of infected cells, but organ-specific signs are usually the result of secondary infection. secondary bacterial joint infections in lambs infected with a. phagocytophilum develop debilitating lameness known as tick pyemia. e. ovis causes fever (benign ehrlichiosis) to weeks after infection. because of this organism's predilection for mononuclear cells, the degree of immunosuppression and subsequent importance of this disease are much less than for a. phagocytophilum infection. diagnosis. specific diagnosis is best made by identifying darkly stained bodies at the periphery of granulocytic cells, as well as occasional large bodies deep within the cytoplasm of some cells. stained bodies also can be seen on the periphery of mononuclear cells from a blood smear during the acute febrile stage or in tissues during chronic infection. serologic tests are available for detection of anaplasmosis. the available celisa is incapable of distinguishing species of anaplasma and serologic results must be interpreted appropriately, and the species confirmed by pcr. both infections affect sheep and goats (a. phagocytophilum also affects many other ruminants, including white-tailed deer), but neither has been reported in north america. a recent study demonstrated that sheep are capable of being experimentally infected with a human isolate a. phagocytophilum. interestingly, the sheep did not develop clinical disease. such findings suggest that sheep could serve as asymptomatic carriers and potential reservoirs for humans. a. phagocytophilum is widespread in northwestern europe, including the united kingdom, scandinavia, and india, and e. ovis is found mainly in countries bordering the indian ocean. in spite of documented seropositive status of animals, there have been no reports of sheep or goats naturally infected with a. phagocytophilum in the united states developing clinical disease. treatment and prevention. treatment and prevention efforts should focus on reducing vectors and bacterial counts during vector season. both organisms are susceptible to treatment with tetracycline. people and animals can become infected with trypanosome protozoa. the trypanosomes can complete their developmental cycle only in tsetse flies (glossina species). trypanosomes multiply in blood, tissues, and body fluids of their vertebrate hosts and are transmitted between vertebrate hosts in the saliva of blood-sucking flies as they feed. the trypanosome species that are known to infect goats and sheep include trypanosoma congolense, trypanosoma vivax, trypanosoma brucei subsp. brucei, trypanosoma evansi, and trypanosoma simiae. pathogenesis. after entering through the skin, trypanosomes reach the bloodstream by way of the lymphatic system. the parasites multiply, and the prepatent period lasts for to days after infection. the infection is characterized by periods of parasitemia, followed by the absence of parasites. this pattern of infection occurs because of antigenic variation: trypanosomes vary the antigenic nature of their glycoprotein surface coat to evade the host's immune system. this immune system-evasive maneuver prolongs infection and is responsible for chronic disease. some trypanosomes tend to invade extravascular spaces, such as the ocular aqueous humor and cerebrospinal fluid. the pathogenicity of trypanosomes varies with the different host species. trypanosomes may produce a hemolysin early in the course of the disease that causes anemia in the host. later, increased phagocytic activity results in massive erythrocyte destruction. clinical signs. the clinical signs are variable and non-specific and depend on the speed of onset of anemia and the degree of organ impairment. entire herds may be affected. all aspects of production are impaired-fertility, birth weight, lactation, weaning weight, growth, and survival. trypanosomiasis may predispose the animal to the development of other diseases that mask the underlying trypanosome infection. trypanosomiasis may be acute, subacute, or chronic, with chronic infection occurring most commonly. acute disease often causes abortion. dairy goats may show a sudden drop in milk production. depression, anorexia, and a stiff gait may be present. physical examination reveals tachycardia, tachypnea, and a slight fever. hyperemic mucous membranes and excessive lacrimation may be noted. affected animals often become recumbent and anorexic and die within to weeks of onset of clinical signs. if the animal survives, progression to the subacute phase, characterized by listlessness, weight loss, enlargement of superficial lymph nodes, and a dull, dry hair coat, may occur. in such cases, auscultation findings are similar to those in other forms of acute cardiac disease, as well as pale mucous membranes and a pronounced jugular pulse. the animal may linger for several weeks or months, or the chronic form of the disease may develop. affected animals show ill-thrift: dull and dry hair coat, inelastic skin, lethargy, emaciation, peripheral lymphadenopathy, pale mucous membranes, and exercise and stress intolerance. death may occur many months or even years after infection and usually results from congestive heart failure. subclinical trypanosomiasis causes acute episodes when animals are stressed by inadequate nutrition, increased production demands, or concurrent disease. diagnosis. diagnosis is difficult because the parasitemia is intermittent, clinical signs are non-specific, and infection is not always synonymous with disease. a pcr assay is gaining acceptance as the most sensitive diagnostic modality, but not all infected animals exhibit clinical disease. although a tentative diagnosis of pathologic trypanosomiasis can be made on the basis of history, clinical signs, and the presence of appropriate vectors, a definitive diagnosis requires identification of trypanosomes on a fresh blood smear, a giemsa-stained blood smear, or less commonly, a lymph smear. examination of the buffy coat of centrifuged blood with darkfield phase-contrast spore illumination is the most sensitive direct microscopic method and is useful when parasite numbers are low. pathogenic trypanosomes must be distinguished from more ubiquitous, nonpathogenic species particularly common in cattle, such as trypanosoma theileri. repeated blood sampling in individual animals often is necessary, because as noted, parasitemia is intermittent. the diagnosis is supported by evidence of anemia on a cbc. indirect diagnostic methods include an indirect fluorescent antibody test and the elisa. these tests are less helpful for diagnosis of a single clinical case but are useful in assessment for herd infection. both t. congolense and t. brucei readily infect rats and mice, and detection of these pathogens can be used to diagnose the infection indirectly. treatment. treatment consists of the use of trypanocidal agents and supportive care. animals with acute, subacute, and subclinical disease respond better to treatment than those with chronic disease because of the irreversible damage to hematopoiesis associated with chronic infection. with most trypanocides, the therapeutic index is low and varies with the host species. trypanocide efficacy also varies with the species of trypanosome present; resistance to agents is common. some trypanocides are irritating to the skin and may cause severe inflammation at the injection site. in sheep and goats with t. brucei infection, the trypanocide of choice is diminazene aceturate, which should be used at a higher dosage rate ( mg/kg given intramuscularly [im] or sc) than that recommended for cattle. protection after trypanocide use usually lasts to months, depending on the season. animals must be rested before and after treatment. supportive care consists of providing fluids, an environment conducive to rest, good nutrition, and possibly blood transfusions. prevention. vector control, stress and nutrition management, and selection of trypanosome-tolerant breeds of sheep and goats all help control or prevent trypanosomiasis. no vaccine is available. animals can be treated with insecticides (pyrethroids) to prevent bites by tsetse flies and other flies. control is accomplished by strategic use of trypanocides during the peak season. continued parasitologic and clinical surveillance is essential to determine the efficacy of control measures. pathogenesis. sarcocystis spp. are protozoon parasites that have a two-host life cycle. sexual reproduction occurs in the bowel of a carnivore (mainly dogs and wild canids) after the carnivore ingests cysts in the muscles of sheep, goats, and cervids. sporocysts are passed in the carnivore's feces and later ingested by a sheep, goat or cervid. the sporocysts hatch in the ruminant gut and invade the vascular endothelium during three phases of asexual reproduction. after the third phase (approximately to weeks after ingestion), merozoites enter the ruminant's muscle tissue and encyst. clinical signs are uncommon but can occur during the stages of reproduction and muscle invasion of the host. n. caninum has a similar life cycle and causes similar disease, except that it appears more likely to cause abortion and affect the central nervous system. clinical signs. most infections are asymptomatic. however, if a large number of sporocysts are ingested, tissue damage may occur during the intestinal, vascular, and muscle stages of the sarcocystis life cycle. fever, lameness or a stiff gait, reluctance to move, and diarrhea may be seen. central neurologic signs (blindness, changes in mentation, and seizures) may occur if the organisms invade the brain or interrupt blood flow to it. abortion can occur as early as weeks after ingestion. with severe chronic infections, emaciation and anorexia are seen. diagnosis. the most characteristic abnormality is an increase in muscle enzyme activity in the blood. anemia is common and may result from extravascular hemolysis. cerebrospinal fluid may show mild mononuclear pleocytosis or may appear normal. on necropsy, muscles may display pale streaks or macroscopic cysts throughout. other evidence of vasculitis includes hemorrhagic serosal surfaces, body cavity fluids, and lymphadenopathy. microscopic or ultrastructural examination of affected tissues should reveal the presence of organisms. specific antibody tests are available and do not cross-react with t. gondii antibodies. blood antibody titers often peak around the onset of clinical signs and should be markedly higher than baseline values. antibody preparations also are available for identification of organisms in tissue preparations. treatment. sheep infected with sarcocystis species can be treated with salinomycin ( ppm in complete feed), monensin ( . - mg/kg po), or amprolium ( - mg/kg po). drugs such as sulfadiazine or trimethoprim ( - mg/kg im sid), pyrimethamine ( . - mg/kg po sid), and clindamycin have shown some success in treating neospora infections. these treatments are off-label and thus are governed by regulations regarding extra-label drug use. prevention. preventing contamination of feedstuffs with the feces of infected carnivores and preventing ingestion of raw meat by carnivores are most important, but these measures may not be possible in flocks handled with dogs or those living on range land. anticoccidial drugs appear to decrease the chance of clinical disease. pathogenesis. t. gondii is a protozoon parasite with a life cycle very similar to sarcocystis, except that the definitive host is the cat and that a wider range of mammalian and avian species, including humans, appear to be capable of acting as intermediate hosts. sporocysts are infective a few days after passage in cat feces, and most ruminants are infected by eating feed contaminated with cat feces. people can become infected by ingesting raw meat or milk from infected animals. abortion, stillbirth, and neonatal death are the most common forms of clinical disease in sheep and goats, and toxoplasma should be considered one of the most common causes of perinatal losses in small ruminants (see chapter ) . abortion usually occurs during the final month of pregnancy. fever, vasculitis-induced disease, and neurologic disease are less common manifestations. clinical signs. beyond abortion, clinical disease is rare in adults and resembles systemic sarcocystosis. clinical signs include fever, dyspnea, depression, and anorexia. neurologic signs are more common than with sarcocystis infection, especially in lambs and kids infected in utero. diagnosis. no specific laboratory abnormalities are associated with toxoplasmosis. nodular lesions similar to sarcocysts may be seen in various tissues, including the brain. aborted or stillborn fetuses may appear normal except for histologic lesions in the brain, liver, or lung, but more commonly fetuses are macerated. the placenta is usually abnormal, with gross and microscopic evidence of necrosis of the cotyledons. microscopic identification of the organism in body tissues is the most common means of diagnosis. serologic tests also are available. treatment and prevention. drugs similar to those used to treat neospora may be effective against toxoplasma. preventing contamination of feeds with cat feces and preventing ingestion of dead animals by cats are the most important ways of stemming the spread of this organism. both methods are likely to be difficult in most flocks. direct spread from one animal to another is rare. clinical signs. bluetongue disease has two different manifestations-reproductive problems (see chapter ) and acute vasculitis of several organ systems. with vasculitis, a spiked fever often precedes depression, anorexia, and rapid weight loss. leukopenia is present. affected animals may develop edema of the lips, tongue, throat, ears, and brisket. other signs include excessive salivation and hyperemia or cyanosis of the oral mucosa, including the tongue (hence the name bluetongue). affected sheep often produce profuse serous nasal discharge that soon becomes mucopurulent and produces crusts and excoriations around the nose and muzzle. oral lesions progress to petechial hemorrhages, erosions, and ulcers. pulmonary edema is often severe, and pneumonia may develop. skin lesions can progress to localized dermatitis. affected sheep may exhibit stiffness or lameness because of muscular changes and laminitis. cyanosis or hemorrhagic changes of the skin of the coronet can extend into the horny tissue. after recovery, a definite ridge in the horn of the hoof may be present for many months. in severe cases, the hoof sloughs. mortality varies widely. in africa, the virus is much more virulent than in the united states, and mortality ranges from to %. the reproductive or teratogenic form of the disease varies greatly with strain, host, and environmental factors. teratogenic effects include abortions, stillbirths, and weak, live "dummy lambs." congenital defects may include hydranencephaly. diagnosis. in parts of the world where the disease is common, the diagnosis is usually based on clinical signs alone. the virus can be isolated from blood, semen, or tissues (spleen and brain from aborted fetuses). viral isolation from blood obtained during the viremic state is the most definitive means of diagnosis. serologic evaluation involves two types of viral antigen groups called p and p . the former is found in all bluetongue viruses, and the latter determines the serotype. sera are commonly tested with complement fixation, agar gel immunodiffusion (agid), or one of several elisa tests. a competitive elisa is considered the best serologic test for detecting group antibodies to bluetongue virus. a direct fluorescent antibody test is available. molecular tests (e.g., pcr) for bluetongue have recently become available and are extremely sensitive and specific. they can be useful for distinguishing serotypes. other clinicopathologic signs that aid in diagnosis include leukopenia during the early febrile stage of the disease and an increase in serum ck corresponding to the latter phase of muscle stiffness and lameness. treatment. treatment is non-specific and consists of nursing care. because of the reluctance of animals to eat, they should be fed a gruel of alfalfa pellets by stomach tube or encouraged to eat soft feeds and green grass. broad-spectrum antimicrobials are often used to treat secondary pneumonia and dermatitis. animals should be kept on soft bedding with good footing. water and shade should be readily available. nsaids are commonly used. prevention. the culicoides vector is difficult to eliminate, so animals should be kept indoors during periods of peak gnat activity (dusk and early evening). owners should attempt to eliminate gnat breeding grounds such as overflowing watering troughs and shallow septic systems and should limit exposure of sheep to gnats with the use of repellent sprays. modified live vaccines based on local strains and serotypes are available in some parts of the world. some cross-protection among serotypes does occur. the vaccine should be administered at least weeks before breeding season to prevent teratogenic effects. vaccinated breeding rams may have a slight risk of decreased fertility. lambs can be vaccinated in the face of an outbreak. pregnant animals cannot be vaccinated with modified live vaccines. sheep that have recovered from an attack of bluetongue are solidly resistant for months to infection by the same viral strain and to some other viral types. active immunity in sheep requires both humoral and cellular immunity. etiology. epizootic hemorrhagic disease virus (ehdv) is an orbivirus belonging to the family reoviridae. the virus is structurally related to bluetongue virus, and the pathogenesis and clinical signs of disease resulting from these two viral infections are very similar. at least seven distinct serotypes of ehdv are recognized, although formal classification of serotypes has yet to be finalized. only two serotypes (ehdv and ehdv ) have historically circulated throughout north america, and those serotypes are largely considered to be endemic in almost all areas of the united states, with the exception of the northeast and arid areas of the southwest. however, in , ehdv was isolated from surveillance efforts in dead white-tailed deer. since then, ehdv has been increasingly identified from both surveillance samples and clinical cases and is also believed to be endemic in several regions. pathogenesis. epizootic hemorrhagic disease (ehd) is a noncontagious disease that is transmitted by the culicoides biting midges. culicoides sonorensis is the primary vector of ehdv in the united states, although other species are also suspected to transmit the disease based on the geographic distribution of clinical cases, although this has yet to be formally shown. due to the vector-borne route of transmission, peak incidence of the disease is closely associated with peak vector population, namely, in the late summer and fall of the year. although capable of infecting a wide range of wild and domestic ruminants, ehdv is largely a pathogen of wild cervids, particularly white-tailed deer. episodes of clinical disease are less common in mule deer, pronghorn antelope, and bighorn sheep and have lower morbidity and mortality. sheep are only rarely infected with the virus and goats appear to be resistant to the virus. cattle are commonly infected based on seroprevalence surveys, but overt clinical disease is uncommon. as a rule, infection in livestock is usually asymptomatic except for periodic epidemics. the last major ehd epidemic in the united states occurred in and affected a variety of captive and wild ruminant species. in endemic areas, seroprevalence in cervids and other ruminants is high, but clinical disease is not commonly seen. conversely, where seroprevalence is low, introduction of the virus results in widespread infection, where morbidity and mortality can reach % and %, respectively. following transmission of the virus by biting midges, ehdv replicates in the endothelial cells of the lymphatics surrounding the site of the bite. a primary viremia allows for systemic spread of the virus and secondary replication in lymph nodes throughout the body and the spleen. viremia is important for disease propagation and generally lasts no more than weeks following infection, although the virus can occasionally be isolated from deer infected days previously. antibodies to ehdv are first detected to days following infection but are not always capable of completely neutralizing the infection. thus, it is possible to find both neutralizing antibodies and live virus in the same animal. passive antibodies in fawns can be found up to approximately months of age. as in adults, antibodies in fawns may not protect from infection but generally protect from severe clinical signs. clinical signs. clinical disease in white-tailed deer can be peracute, acute, or chronic. the course of the peracute syndrome of diseaseis relatively short, with death often occurring within hours of infection, with or without the presence of clinical signs. when present, clinical signs include severe edema of the head and neck, swelling of the tongue and conjunctiva, anorexia, fever, weakness, and respiratory distress. hemorrhagic diatheses are not present antemortem but may occur after death. in contrast, in the acute form of the disease, the clinical signs of the peracute form are accompanied with bleeding throughout body tissues (figure . a, b) . ulcers may be evident in the oral cavity and throughout the upper gastrointestinal tract, forestomachs, and abomasum. case fatality rates are high for both the peracute and acute forms. deer that recover after several weeks of illness are said to suffer from the chronic form of the disease. signs of previous illness may include breaks or rings in the hoof horn due to interrupted growth and synthesis leading to lameness, sometimes severe. ulceration and scarring of the rumen and gastrointestinal tract may result in loss of body condition despite a seemingly normal appetite and ample nutrition. widespread evidence of vasculitis may be observed histopathologically. diagnosis. the gold standard for ehdv diagnosis is virus isolation. demonstration of neutralizing antibodies to ehdv reference strains is evidence of previous infection but may be of limited value in endemic areas where seroprevalence levels are expected to be high. also, all potentially suspected serotypes must be used when testing the sample, thereby increasing the time and cost involved with the test. continued research and refinement of molecular techniques, including pcr, are ongoing and are attractive due to the short turnaround times and the potential for high throughput of samples. however, it is important to remember that a positive result using molecular techniques does not equate to the presence of infectious virus, and thus, interpretation of results must be done with caution. control. control of ehd is difficult and relies on a combination of disease surveillance, vector control, and potentially, vaccination. eradication of vector-borne diseases from endemic areas is difficult and time-consuming, and thus, disease control is likely more attainable than strict eradication. vector control is more important in the late fall and summer, when populations are at peak levels and viral transmission is more likely. midge-proofed housing and the treatment of animals with pyrethroid insecticides have been attempted but may be logistically challenging and have yet to have been demonstrated efficacious. vaccine availability in north america is limited, but inactivated autogenous vaccines have been developed from isolates obtained from ill or recently diseased animals. autogenous vaccines are tested for purity but not necessarily for efficacy. vaccine usage must be approved by the u.s. department of agriculture prior to administration. etiology. peste des petits ruminants (ppr) is an acute or peracute, febrile, often fatal disease of ruminants caused by a virus in the family paramyxoviridae and genus morbillivirus. sheep are less susceptible than goats and white-tailed deer. cattle are only subclinically infected, and some wild ungulates, as well as camels, appear to suffer the occasional epizootic. the virus (pprv) is serologically related to the virus that causes rinderpest. geographically, the virus is found throughout northern africa, the middle east, and adjacent regions of asia, with possible movement into southern africa and europe noted. pathogenesis. the main route of infection is respiratory, and ppr is spread by airborne droplets. all secretions and excretions of infected animals are contagious throughout the course of the disease, but no carrier state exists. the virus targets lymphoid tissue. lymphocytes are destroyed in germinal centers in lymph nodes, peyer's patches, tonsils, splenic corpuscles, and cecal lymphoid tissue. immunosuppression results from lymphoid destruction. lymphocytes are partially replaced by plasma cells, macrophages, an eosinophilic acellular matrix, and occasionally neutrophils. the epithelial lining of the mouth and digestive tract is highly vulnerable to the pprv. with the loss of the alimentary tract mucosa, weight loss and diarrhea become severe. the incubation period is usually to days, with up to days possible. clinical signs. the clinical disease produced by pprv in sheep and goats closely resembles that of rinderpest, but the course is much more rapid. with the acute form, sheep and goats typically display an abrupt rise in temperature to ° to ° f ( °- ° c). within a few days, infected animals develop nasal and lacrimal discharge, depression, thirst, anorexia, and leukopenia. congestion of the conjunctival and other mucous membranes occurs, followed by serous and mucopurulent exudates. sheep and goats develop oral erosions with necrotic foci, which results in excessive salivation. diarrhea that may be profuse but rarely hemorrhagic develops within to days and is accompanied by abdominal pain, tachypnea, emaciation, and severe dehydration. bronchopneumonia, particularly that caused by pasteurella spp., may be a terminal • fig. . a. the lungs of the adult pen-raised, white-tailed deer, have been retracted to reveal to ecchymoses on the ventral surface of the "ribcage." petechiae and ecchymoses can occur anywhere within the carcass in cases of epizootic hemorrhagic disease (ehd), but common locations are on the epicardium, on the pleural surface the ribs, subcutaneously, and on the surface of the spleen. b. ecchymoses over the surface of the reticulum (bottom right of photo) and the surface of the rumen (left side of photo). in addition to ehd, this deer also had bronchopneumonia (fibrin overlying consolidated lung can be seen in the far right of photo). (courtesy dr. kelley steury, auburn, al.) a b sequela. death usually occurs to days after the onset of fever. pregnant sheep or goats with ppr may abort. diagnosis. a presumptive diagnosis of ppr can be made on the basis of clinical, pathologic, and epizootiologic findings. the diagnosis can be confirmed by isolating the virus from blood or tissues, including lymph nodes, tonsils, spleen, and lung. immunocapture elisa or pcr may be used to detect infection several days before the development of clinical disease. most serologic tests (complement fixation or agid) cannot differentiate between ppr and rinderpest. characteristic postmortem findings include necrotic stomatitis that is generally confined to the inside of the lower lip and adjacent gum, the cheeks near the commissures, and the ventral surface of the free portion of the tongue. abomasal erosions are often present. in the small intestine, peyer's patches are markedly affected, particularly in the first portion of the duodenum and terminal ileum. the large intestine may be severely affected. lesions occurring near the ileocecal valve, at the cecocolic junction, and in the rectum are often described as zebra stripes that indicate areas of congestion along the folds of the mucosa. treatment and prevention. infection with pprv has no specific treatment. mortality can be reduced by supportive care, including the administration of antimicrobial and antiinflammatory agents, as well as nutritional support. in the united states, state and federal veterinarians should be notified if pprv is suspected. methods used to eradicate rinderpest are useful in the eradication and control of ppr. all sick sheep and goats and those exposed should be slaughtered and disposed of by burning, burying, or rendering. the premises should be decontaminated, and the area quarantined. sheep and goats can be protected against ppr by immunization with rinderpest vaccines or by the simultaneous administration of ppr hyperimmune bovine serum and virulent pprv. pathogenesis. louping ill is a tickborne disease caused by a flavivirus. it affects mainly lambs but occasionally also affects other livestock species and infrequently affects deer, camelids, and humans. transmission is most common during tick season, and ixodes ricinus is thought to be the most important infective host. many sheep clear the infection after a few days of fever and viremia, but others develop severe, fatal viral encephalitis. the virus is shed in many secretions, including milk, which is an important source of infection for other animals (and humans). the severity of the disease depends on herd immunity because previous exposure gives long-lasting immunity. colostrum from immune females is protective for the neonate. high antibody titers also appear to shorten the duration and level of viremia and thereby prevent invasion of the central nervous system. naïve flocks may have fatality rates as high as %. clinical signs. high biphasic fever, anorexia, and depression are seen in most infected sheep. lambs may die quickly before illness is noted. some sheep also develop central neurologic signs, including hyperexcitability, muscle tremors, and rigidity. abnormal coordination and muscle activity may cause sheep to move with a bounding gait (hence the name louping ill). diagnosis. the condition has no characteristic gross lesions. microscopic examination of animals with neurologic signs reveals evidence of viral meningoencephalitis. diagnosis is made by history (based on location, signs, and time of year), the identification of characteristic lesions, virus isolation, or fluorescent antibody staining of fresh brain tissue. a demonstrated increase in specific antibody titers in survivors strongly suggests the presence of this infection. prevention. vaccines are available in endemic areas to control infection. vector control during tick season also is important. lambing season should also be timed so that lambs have high colostral antibody protection at the time of exposure to ticks. pathogenesis. foot-and-mouth disease is caused by a highly contagious picornavirus and has been eradicated from the united states. vesicular stomatitis is caused by a rhabdovirus and is intermittently eradicated from the united states. both diseases are highly contagious, nearly indistinguishable from each other clinically, and reportable. foot-and-mouth disease has a broad host range that includes most hoof stock (including pigs but not horses) and several other mammalian species. vesicular stomatitis also affects many species of hoof stock, including both pigs and horses. sheep and goats are relatively less susceptible than cattle, particularly to vesicular stomatitis. the viruses are spread by aerosol and mechanical vectors and primarily colonize skin or mucous membranes. milking machines, flies, birds, and humans all may be important mechanical vectors. vesicular stomatitis tends to remain at the site of infection, and colonization is facilitated by damage to the skin. oral mucous membranes, coronary bands and interdigital skin, and teat-end skin are common sites of lesions. vesicular stomatitis outbreaks in the united states tend to occur in the summer or fall and end with the first killing frost. viremia plays more of a role with foot-and-mouth disease. the virus is present in most body tissues and fluids in infected animals and can be transmitted through milk, meat, bone, and hide products; semen; equipment that pierces the skin; and biting arthropods. it also tends to spread through the circulation from the site of infection to other susceptible tissues, including the sites of vesicular stomatitis, as well as to the nasal cavity, mammary glandular epithelium, and ruminal pillars. the basic lesion for both diseases are the vesicles that form in the oral cavity and on the teats and coronary band. the vesicles quickly rupture and may not be visualized before forming erosions. ruptured vesicles leave deep erosions on the skin or mucous membranes and appear to cause pain. tissue damage and inflammation are often compounded by secondary bacterial infection, which can cause greater morbidity and mortality than the original viral infection. morbidity is related to feed refusal, increased recumbency, and secondary infections of the mouth, udder, and feet. clinical signs. sheep and goats usually develop minor lesions, if any, and are more important in many outbreaks as transport or multiplying hosts than as primary clinical cases. however, identification of lesions should raise suspicion of this disorder. in the worst cases, vesicles, erosions, and ulcers are seen at target sites. they may appear mildly inflamed and erythematous; if they are infected, they may appear severely inflamed with hemorrhage and necrosis. other signs vary according to the location and severity of the lesions. lingual and buccal lesions cause salivation, dysphagia, and feed refusal. foot lesions, which are the most common clinical manifestation in small ruminants, cause lameness and recumbency. teat lesions cause reluctance to be milked or nursed and a decrease in production. fever also may be seen early in the disease, when vesicles are most apparent. the fever then usually abates, and vesicles are replaced by erosions or ulcers. abortion may occur, especially with foot-and-mouth disease, and is probably related to the fever rather than to fetal infection. the disease is usually self-limiting; most animals recover within to weeks. shedding of the virus causing vesicular stomatitis is thought to subside soon after healing of lesions. foot-and-mouth disease virus may be shed for as long as months, and all body secretions and tissues should be considered contagious, including milk, semen, meat, and offal. both viruses have zoonotic potential and cause a disease in humans that resembles mild influenza. the diseases are self-limiting, but people can shed the viruses in sufficient quantities to infect other animals. diagnosis. no characteristic clinicopathologic changes are reported for either virus. gross lesions resemble those seen before death and include vesicular, erosive, and ulcerative lesions of the mouth, feet, and teat ends; foot-and-mouth disease also causes lesions of the mammary gland and ruminal epithelium. microscopic findings include hydropic degeneration of cells of the stratum spinosum of the epidermis without inclusion bodies. secondary bacterial infection may lead to deeper ulcers and complicate identification of the viral etiology of these lesions. myocarditis lesions may be seen with some forms of foot-and-mouth disease. a presumptive diagnosis may be made by identifying characteristic lesions during a season and in an area at risk for one of these infections. in north america, bluetongue should be considered as an important differential diagnosis for ulcerative oral lesions in sheep. a confirmed diagnosis of foot-and-mouth disease is achieved by a combination of virus isolation (from vesicles), ihc, and serology by regulatory officials. identifying the source of infection also is very important. diagnosis of vesicular stomatitis is achieved by complement fixation or fluorescent antibody staining of virus in vesicular fluid or detection of a rise in antibody titers. flocks with either of these diseases in the united states are subject to quarantine and possible destruction (especially for foot-and-mouth disease). prevention. meticulous personal hygiene and avoidance of contact with new animals are important during outbreaks to prevent spread between flocks. vaccines against foot-and-mouth disease are available in many parts of the world, but not in the united states. most nations slaughter or quarantine affected animals. vaccines against vesicular stomatitis are available and are most commonly used if the risk of outbreak is high, but vaccination does not prevent infection or shedding. good hoof and teat care and soft feeds may help prevent spread of the virus by providing a healthy, intact barrier against invasion. pathogenesis. sheep and goat pox are caused by two closely related poxviruses. some strains are infective to both sheep and goats; most are species specific. they are maintained in populations by infected animals, and transmission occurs by aerosol or direct or indirect contact. flies may play an important role as mechanical vectors in some flocks. viruses remain infective in the environment for as long as months. after infection, viremia and inflammation of the oral, nasal, and ocular mucous membranes occur. erythematous papular pox lesions appear a few days later. severity varies according to strain pathogenicity, breed susceptibility, and immune status. mild infections are characterized by lesions concentrated in the non-wooled or hairless regions of the skin. severe infections produce lesions throughout the oral cavity, respiratory tract, and peritoneal cavity. secondary infection is common with the severe form and mortality is high. if the animal survives, lesions heal in to weeks. both diseases have been eradicated from the united states and are reportable. people can develop mild disease on exposure to these viruses. clinical signs. fever, inappetence, conjunctivitis, and upper respiratory signs are seen in the initial stages. pox lesions are visible shortly thereafter. secondary infection can lead to a variety of more serious signs indicative of respiratory disease, sepsis, and shock. diagnosis. characteristic pox lesions are highly suggestive of this disease. microscopic analysis reveals eosinophilic intracytoplasmic inclusion bodies, acantholysis, and pustule formation within the epidermis and occasionally the dermis. viral particles may be seen on ultrastructural examination. gross and microscopic lesions are characteristic with the severe form, but mild disease may produce mild lesions that are difficult to differentiate from other viral diseases that cause oral proliferative or ulcerative lesions. virus can be isolated from blood or tissues (mainly skin) during the acute viremic stage and identified by antibody staining of more chronic lesions. serologic tests are available to detect rising titers in convalescent animals. treatment and prevention. no specific treatment is available for sheep or goat pox. antibacterial drugs may be useful to treat secondary infection. judicious use of insecticides and confinement of affected animals may prevent spread. vaccines are available in some countries, but not in the united states. infected flocks are placed under quarantine or destroyed in regions where the diseases are not endemic. these viruses are difficult to eradicate from flocks because of their environmental persistence and the constant supply of susceptible hosts. caprine arthritis-encephalitis virus (caev) is an enveloped, singlestranded retrovirus in the lentivirus genus. like other retroviruses, caev integrates into the host chromosomal dna before replicating. the virus is able to remain latent or undergo sporadic bouts of productive viral replication. caev is closely related to ovine lentiviruses. clinical signs. clinical disease may be evident in only % of goats from a caev-infected herd at any given time. as many as % of seropositive goats may be clinically normal. caev produces four clinical syndromes: encephalomyelitis, arthritis, interstitial pneumonia, and indurative mastitis. the pattern of disease usually varies with age. arthritis is generally seen in sexually mature goats, whereas encephalomyelitis is generally seen in kids to months old. interstitial pneumonia and indurative mastitis are more common in adult goats. some goats suffer from a wasting disorder characterized by poor body condition and rough hair coat. diagnosis. a presumptive diagnosis of caev can be made on the basis of history and clinical signs suggestive of one or more of the syndromes. in general, elisa tests are better for detecting disease in an individual animal because the sensitivity of the test is higher than that of the agid, whereas the agid is better for herd screening that requires high specificity. with the agid test, false negatives may occur in goats that have not yet seroconverted to recent infection. individual goats may take months or years to seroconvert or may never do so. parturition or advanced stages of disease also may contribute to a false-negative result. false positives may occur in goats younger than days old that have colostral antibodies. for this reason, it is often suggested that kids be at least months old before they are tested. pcr testing has high specificity and sensitivity and can detect infection within a day of exposure. other less commonly used tests include a western blot to detect antibodies and a northern blot to look for mitochondrial rna. because of the limitations in interpreting serologic results, caev-induced disease can only be definitively diagnosed by identification of characteristic lesions from examination of biopsy specimens or postmortem viral isolation. treatment. no specific treatments are available for any of the syndromes associated with caev. young goats suffering from encephalomyelitis may benefit from physical therapy if they are recumbent, and bottle feeding may help maintain hydration and caloric intake. antibiotics may be beneficial to goats affected with interstitial pneumonia or mastitis if secondary bacterial infection is present. generally, the prognosis is poor for the encephalitic form and guarded for the other forms. prevention. prevention of caev is crucial because infection is lifelong. infected colostrum and milk are the most important sources of infection. newborn kids should be prevented from ingesting colostrum from infected does and should instead be fed pasteurized goat's milk or milk from caev-negative goats. all goats in a herd should undergo serologic testing twice yearly; seropositive goats should be segregated or culled to prevent direct contact between infected and uninfected animals. ovine progressive pneumonia (opp) is an ultimately fatal retroviral disease that causes chronic, progressive, debilitating inflammatory conditions of the lungs (united states) and central nervous system (other parts of the world). it also is called maedi-(maeði is icelandic for "shortness of breath") visna (meaning "wasting"). the virus is a member of the lentivirus genus of retroviruses and is closely related to caev. recombination between opp and cae viruses has been observed. the virus primarily affects sheep and rarely goats and has been identified worldwide, except in australia and new zealand. the disease has a long incubation period and protracted clinical course. pathogenesis. only sheep older than years of age are affected by opp virus (oppv). the virus is spread by direct contact, probably in respiratory and salivary secretions, and by excretion in the milk and colostrum. transplacental transfer is of minor importance. virus is excreted by animals that exhibit clinical signs and asymptomatic animals. infection is established in the monocyte and macrophage cell line and spread by these cells to the lungs, lymph nodes, choroid plexus, spleen, bone marrow, mammary gland, and kidneys. like caev, oppv evades the cellular and humoral immune system of the host by incorporation of its provirus in host dna, low-grade replication of virus only when monocytes differentiate into macrophages (restricted replication), and production of antigenic variants that are not neutralized by existing antibodies. continual antigenic stimulation of the host by low-grade replication of oppv results in chronic inflammation and resultant lymphoid proliferation in various target tissues. the virus may prevent b lymphocytes from differentiating into plasma cells in lymph nodes and may thereby impair immunoregulation. seroconversion occurs within to weeks after infection. clinical signs. in the united states, serologic surveys reveal infection rates of between and % but rarely is more than % of a flock lost to oppv. icelandic, texel, border leicester, and finnish landrace appear to be susceptible sheep breeds. more resistant sheep breeds include rambouillet, suffolk, and columbia. various clinical syndromes are associated with oppv and include wasting (thin ewe syndrome), dyspnea occasionally with a dry cough, pneumonia, mastitis ("hard bag"), posterior paresis, arthritis, and vasculitis. in north america, pneumonia and indurative aseptic mastitis are common sequelae of infection. coinfection with the jaagsiekte virus (the cause of pulmonary adenomatosis) worsens respiratory signs. visna, the neurologic form, is more common in goats. over the course of up to a year, subtle signs such as a head tilt or hindlimb weakness progress to gross incoordination, whole body tremors, and rarely more profound cranial nerve signs. diagnosis. a presumptive diagnosis can be made on the basis of clinical signs, poor response to treatment, characteristic postmortem findings, and serologic testing. definitive diagnosis requires pcr or isolation of the virus from wbcs (buffy coat of whole blood sample) or tissues. less expensive and faster serologic tests include agid, elisa, and an indirect immunofluorescence test. the agid test is frequently used as a flock screening test, but the elisa is more sensitive on an individual basis and can detect antibodies earlier in the course of the disease. as with caev, false negatives and false positives are possible. characteristic postmortem lesions include generalized wasting and firm, noncollapsing lung or firm, mottled mammary glands, both with regional lymphadenopathy. microscopic evaluation of those tissues reveals interstitial non-septic, mononuclear cell infiltrates, although these may be complicated by secondary infections. histopathology of nervous tissue reveals meningoleukoencephalitis. treatment. no effective treatment is available for oppv. supportive therapy that includes appropriate husbandry and control of secondary infection with antibiotics may prolong life for a few weeks or months but, ultimately, the disease is fatal. because of the poor prognosis and risk of exposure of naive animals to clinical disease, long-term treatment is not recommended. prevention. the only known method of preventing oppv infection in a flock is to prevent exposure to the virus. management practices that help decrease the incidence of horizontal transmission include disinfection of milking equipment, dehorning instruments, and tail docking and castration tools before use and between animals. contaminated feed and water also are potential routes of infection and should not be shared among infected and uninfected animals. serologic testing and separation or culling of seropositive animals may help reduce infection. although oppv can readily be isolated from ewe colostrum, colostral transmission of oppv has not been definitively established. however, many prevention guidelines recommend that offspring from infected dams be separated from the dam before they nurse and then be fed cow colostrum and artificially reared. quarantine and serologic testing of flock additions before placing them with the current flock and purchase of sheep only from oppv-free flocks are important to prevent the introduction of new infections. because of the potential cross-species spread, all precautions taken for sheep also apply to contact goats. serologic testing should be performed at least annually in a flock until two consecutive negative test results are obtained. border disease virus (bdv) is in the genus pestivirus and family flaviviridae, which also includes the two genotypes of bovine viral diarrhea virus (bvdv) and classical swine fever virus. it rarely causes disease in adults and is most important as a cause of in utero infection of lambs and kids. the condition gets its name from the fact that it was first reported in sheep along the welsh border of the united kingdom. other names such as "hairy shakers" and "fuzzy lamb disease" refer to some of the clinical signs seen in affected newborns. it is important to recognize that although bdv is genetically distinct from the two types of bvdv, sheep and goats also are susceptible to some strains of bvd. pathogenesis. horizontal transmission of bdv occurs through contact with secretions and excretions of body fluids and tissues from infected animals. the virus crosses intact mucous membranes and can spread rapidly through a flock. the major reservoir is the persistently infected sheep or goat. these reservoirs are usually asymptomatic, congenitally infected, and often seronegative animals that shed large quantities of virus. these may be residents of a flock with an ongoing problem or bought in as replacement animals to a naïve flock. some cross-infection from other species is possible, particularly from cattle. adult, immunocompetent sheep rarely show any signs of acute infection. however, if a pregnant ewe or doe is infected, the virus may be transmitted vertically to the embryo or fetus. depending on the stage of gestation, embryonic or fetal infection may have different outcomes ranging from embryonic reabsorption to normal birth. these infections are the most important aspect of border disease. the major organ system targeted by bdv is the fetal central nervous system. the hallmark lesion is hypomyelination, or degeneration of oligodendroglial cells. three factors contribute to this lesion. the first is direct viral damage. the second is viral-induced inhibition of the thyroid gland that causes decreased secretion of thyroid hormones. in the absence of these hormones, a resultant lowered concentration of a specific nucleotide in the central nervous system also contributes to the hypomyelination. the third factor is altered immune function. the virus causes the host to produce a virus-specific delayed hypersensitivity reaction that causes inflammation in the central nervous system. it also causes immunosuppression. death often results from opportunistic conditions such as parasitism, diarrhea, and bronchopneumonia. clinical signs. clinical signs depend on the time during gestation when the fetus or embryo is exposed to the virus. clinical signs also may vary in severity from animal to animal because different fetuses develop competent immune systems at different times. if the fetus or embryo is exposed to the virus within days of conception, it dies and is resorbed or aborted. these losses are not usually noticed by the flock manager. the principal manifestation in the flock is a large number of open ewes and a small lamb crop. infection of the fetus between days and of gestation causes damage to rapidly growing systems such as the skin and nervous, lymphoid, thyroid, and skeletal systems. congenital malformations are seen at birth. lambs have abnormal fleece characteristics (hairy rather than woolly in consistency), small stature, domed heads, shortened legs, and dark pigmentation of the skin, particularly on the dorsal aspect of the neck. the lamb may exhibit tonic-clonic tremors ("hairy shakers") when awake, which may prevent standing or suckling. most of these lambs die within a few days of birth. if they survive, the hair changes disappear in to weeks and the central nervous system signs resolve by weeks. goats infected at this time have similar symptoms except that they rarely exhibit hair coat changes. if kids are infected before day of gestation and are still viable, they may become persistently infected and immunologically compromised. they are small at birth and generally weak. typical outbreaks of border disease cause abortions and birth of weak lambs in the first year as the virus rapidly spreads throughout a susceptible flock and then insignificant losses in the succeeding years as adult sheep develop immunity. however, if new naïve ewes are introduced in the flock, substantial losses may occur in perpetuity. diagnosis. border disease viral antigens can be demonstrated in abomasum, pancreas, kidney, thyroid, skin, and testicle tissues from aborted fetuses and persistently infected animals using fluorescent antibody tests. however, ihc on ear notch samples is not considered as reliable for detecting persistently infected small ruminants as it is for cattle. the virus can be isolated, or viral antigen detected by elisa, from serum, heparinized whole blood, and tissue taken from brain, spinal cord, spleen, and bone marrow from affected lambs. whole blood is better than serum if colostral antibodies are likely to be high; serum is an adequate sample in neonates and juveniles that have not suckled. antibodies to the virus may be quantified by serum neutralization, agid, and complement fixation with hyperimmune bvd antiserum. serologic tests are useful to detect exposure in lategestation (after day ) neonates and unvaccinated animals but may be confounded by colostral antibodies in suckling neonates, previous exposure, and vaccination in older animals. any titer in a presuckling neonate indicates in utero exposure, whereas a serum neutralization titer of : to : suggests infection in adults. the presence of specific antibodies in the cerebral spinal fluid suggests bdv infection. negative presuckling serologic tests do not rule out exposure because persistently infected lambs tend to be immunotolerant to the bdv and therefore are born without an antibody titer. these animals may subsequently develop a titer that is indistinguishable from that of a normal animal. although persistently infected animals do not respond immunologically to the strain of the virus they carry, they may respond to other strains of the virus, including vaccine strains. as with bvd, pcr assays are gaining popularity for the detection of bdv in fluids and tissue samples. these assays appear to be superior to other techniques, except in autolyzed tissues. realtime pcr may also be used to differentiate bdv from bvd and to type isolates. gross postmortem findings include hydranencephaly, porencephaly, microcephaly, cerebellar hypoplasia, abnormal rib curvature, brachygnathia, doming of the frontal bones of the skull, narrowing of the distance between the orbits, shortening the crown-to-rump length, shortening of the diaphyseal length, retention of secondary hair fibers, and abnormal skin pigmentation. the major histopathologic changes include hypomyelination and hypercellularity of the white matter. glial cells appear normal. treatment. no treatment is available for border disease infection. supportive care may include assistance in nursing and standing for affected lambs, provision of good bedding and solid footing, and treatment of secondary opportunistic infection. prevention. control is primarily achieved by eliminating persistently infected carrier animals from the flock and preventing the addition of new carrier animals. this is easiest in a closed flock but especially difficult in small ruminant flocks because of the frequent desire to import new genetics. to identify carriers, virus isolation must be performed on every animal in the flock; carrier animals must be culled. additionally, all unborn animals must be considered potential carriers and should be tested at birth. an alternative solution in hobby flocks is to arrest breeding activity until all animals have been shown to be free of infection. new animals should be quarantined and tested before admission to the flock. herd screening with the ear skin biopsy test using fluorescent antibody staining to detect virus is less expensive and more convenient than the whole blood virus isolation test. the role of vaccination in preventing infection is still unclear. no vaccine against bdv is available, but some reports suggest that bvdv vaccines for cattle may be helpful for sheep at risk. however, these vaccines have proven to be more effective at preventing clinical disease in vaccinated animals than in preventing in utero infection because they do not prevent transient viremia. vaccination decreases viremia and fetal infection but does not eliminate them. therefore, vaccines play a role in decreasing economic loss but do not replace culling of carrier animals as the major method of control. another member of the slow infection group of diseases of small ruminants is scrapie. it is an afebrile, chronic, progressive degenerative disorder of the central nervous system of sheep and occasionally of goats (see chapter ) . scrapie is caused by a prion and, as such, is one of the transmissible spongiform encephalopathies. sheep (and goats and mouflon to a lesser degree) are the natural hosts for scrapie. clinical signs often do not usually appear until animals are years old, and animals as old as years may exhibit clinical disease. both vertical and horizontal transmission have been demonstrated experimentally in sheep and goats. abnormal scrapie protein has been identified in milk, urine, and seminal plasma of sheep up to months prior to the development of clinical signs. also, new evidence from deer with chronic wasting disease, a similar disorder, suggests that infective prions are excreted in the saliva and feces well before the development of clinical signs. these new revelations may help explain horizontal transmission of infection. clinical signs. the onset of scrapie is insidious. initially, sheep show subtle changes in behavior such as mild apprehension, staring or fixed gaze, failure to respond to herding dogs, and boldness around humans. several months later, the animals become intolerant of exercise and develop a clumsy, unsteady gait and floppy ears. later, the sheep develop itchy skin that causes them to rub themselves excessively against firm, immobile objects (origin of the name scrapie). this leads to excoriations and wool damage. there is a general decline in body condition and coordination. diagnosis. histologically, the only consistent lesions are degenerative changes in the central nervous system consisting of bilaterally symmetric vacuolation of the neurons in the brainstem and spinal cord with accompanying spongy degeneration. as a preclinical test, ihc may be performed in lymphoid tissue from the tonsils, third eyelid, or rectoanal mucosa, but none of these methods is foolproof. cwd is discussed in chapters , , and . testing for clinical anaemia caused by haemonchus spp. in goats farmed under resource-poor conditions in south africa using an eye colour chart developed for sheep validation of the fama-cha eye color chart for detecting clinical anemia in sheep and goats on farms in the southern united states validation of the famacha © eye colour chart using sensitivity/ specificity analysis on two south african sheep farms is the famacha chart suitable for every breed? correlations between famacha scores and different traits of mucosa colour in naturally parasite infected sheep breeds rumen bacteria are involved in the onset of onion-induced hemolytic anemia in sheep the role of free radicals in brassicainduced anaemia of sheep: an esr spin trapping study kale poisoning: the brassica anaemia factor hemolytic anemia in sheep fed wild onion (allium validum) copper toxicosis in sheep: a case report chronic copper poisoning in sheep. i. the relationship of methaemoglobinemia to heinz body formation and haemolysis during the terminal crisis chronic copper toxicity of ruminants copper poisoning in a flock of sheep. copper excretion patterns after treatment with molybdenum and sulfur or penicillamine evaluation of mechanisms of leptospiral hemolytic anemia fatal hemolytic anemia attributed to leptospirosis in lambs studies on eperythrozoon ovis-infection in sheep eperythrozoon ovis-a blood parasite of sheep experimental eperythrozoon ovis infection of sheep mycoplasma ovis comb. nov. (formerly eperythrozoon ovis), an epierythrocytic agent of haemolytic anaemia in sheep and goats molecular characterization of two different strains of haemotropic mycoplasmas from a sheep flock with fatal haemolytic anaemia and concomitant anaplasma ovis infection bovine colostrum as a cause of hemolytic anemia in a lamb heinz body anaemia in lambs with deficiencies of copper or selenium maxillary lymphosarcoma in a white-tailed deer (odocoileus virginianus) large animal internal medicine effect of physical restraint and xylazine sedation on haematological values in red deer (cervus elaphus) seasonal variations in red deer (cervus elaphus) hematology related to antler growth and biometrics measurements the genetic basis and evolution of red blood cell sickling in deer one hundred two tumors in goats lymphoma classification in goats exophthalmos due to multicentric b-cell lymphoma in a goat ocular involvement of multicentric malignant b-cell lymphoma in a ewe. a case report diseases and parasites of white-tailed deer, miscellaneous publication no. . tall timbers research station colostrum composition of santa inês sheep and passive transfer of immunity to lambs effects of maternal undernutrition during late gestation and/or lactation on colostrum synthesis and immunological parameters in the offspring failure in passive transfer of immunoglobulin g to lambs: measurement of immunoglobulin g in ewe colostrums iodine supplementation of the pregnant dam alters intestinal gene expression and immunoglobulin uptake in the newborn lamb short communication: apoptosis regulates passive immune transfer in newborn kids effects of newborn characteristics and length of colostrum feeding period on passive immune transfer in goat kids a field trial evaluating the health and performance of lambs fed a bovine colostrum replacement use of a digital brix refractometer to estimate serum immunoglobulin in goat kids field methods for estimating serum immunoglobulin concentrations in newborn kids colostrum deficiency in mule deer fawns: identification, treatment and influence on neonatal mortality passive transfer of colostral immunoglobulins from ewe to lamb and its influence on neonatal lamb mortality transfer of maternal passive immunity to kids in goat herd suppurative meningitis in a -day-old formosan sambar deer (cervus unicolor swinhoei) caused by escherichia coli factors affecting igg concentration in day-old lambs effects of maternal nutrition on udder development during late pregnancy and on colostrum production in scottish blackface ewes with twin lambs the effect of colostrum source (goat vs. sheep) and timing of the first colostrum feeding ( h vs. h after birth) on body weight and immune status of artificially reared newborn lambs bovine neonatal pancytopenia and anaemia in lambs caused by feeding cow colostrum secondary lactose intolerance in a neonatal goat hypernatremia in neonatal elk calves: cases ( - ) group b rotavirus associated with an outbreak of neonatal lamb diarrhea rotaviruses associated with neonatal lamb diarrhea in two wyoming shed-lambing operations novel group a rotavirus g p[ ] as primary cause of an ovine diarrheic syndrome outbreak in weaned lambs role of enteric pathogens in the aetiology of neonatal diarrhoea in lambs and goat kids in spain enteric viral infections in lambs or kids suspected clostridium difficile-associated hemorrhagic diarrhea in a -week-old elk calf observations and immunohistochemical detection of coronavirus, cryptosporidium parvum and giardia intestinalis in neonatal diarrhoea in lambs and kids giardia duodenalis and cryptosporidium parvum infections in adult goats and their implications for neonatal kids case control study of diarrhoea and faecal soiling in two-to six-month-old lambs comparison of two techniques for diagnosis of cryptosporidiosis in diarrhoeic goat kids and lambs in cyprus fluid therapy in calves passive immunisation of neonatal lambs against infection with enteropathogenic escherichia coli via colostrum of ewes immunised with crude and purified k pili floppy kid syndrome (metabolic acidosis without dehydration in kids clostridium perfringens toxins involved in mammalian veterinary diseases first isolation of clostridium perfringens type e from a goat with diarrhea clostridial enteric diseases of domestic animals isolation and molecular characterization of clostridium perfringens from healthy merino lambs in patagonia region lamb losses associated with clostridium perfringens type a hemorrhagic bowel syndrome in dairy cattle: cases clostridium perfringens type a and beta toxin associated with enterotoxemia in a -week-old goat investigation of a syndrome of sudden death, splenomegaly, and small intestinal hemorrhage in farmed deer gastric mucormycosis in a sika deer (cervus nippon) associated with proliferation of clostridium perfringens the relationship between the presence of helicobacter pylori, clostridium perfringens type a, campylobacter spp, or fungi and fatal abomasal ulcers in unweaned beef calves multiplex pcr method for genotyping clostridium perfringens the effect of clostridium perfringens type c strain cn and its isogenic beta toxin null mutant in goats beta toxin is essential for the intestinal virulence of clostridium perfringens type c disease isolate cn in a rabbit ileal loop model clostridial diseases vaccines for control, prevention and eradication of disease in farmed deer development and application of an oral challenge mouse model for studying clostridium perfringens type d infection enterotoxaemia caused by clostridium perfringens type d in farmed fallow deer rates of diseases and their associated costs in two colorado sheep feedlots ( - ) proportional mortality: a study of goats submitted for necropsy from goat herds in quebec, with a special focus on caseous lymphadenitis the pathology of experimental clostridium perfringens type d enterotoxemia in sheep enterotoxaemia in goats: a review diagnosis of clostridium perfringens intestinal infections in sheep and goats clinico-pathological findings of clostridium perfringens type d enterotoxaemia in goats and its hemolytic activity in different erythrocytes experimental clostridium perfringens type d enterotoxemia in goats clinical signs, treatments, and postmortem lesions in dairy goats with enterotoxemia: cases epsilon toxin is essential for the virulence of clostridium perfringens type d infection in sheep, goats, and mice clinicopathologic features of experimental clostridium perfringens type d enterotoxemia in cattle ulcerative enterocolitis in two goats associated with enterotoxin-and beta toxin-positive clostridium perfringens type d the passive protection of lambs against clostridium perfringens type d with semi-purified hyperimmune serum blackleg in deer bacterial diseases of farmed deer and bison black disease in a forest reindeer bovine vaccines and herd vaccination programs toxigenic clostridia characterization of the catalytic domain of clostridium novyi alpha-toxin first report of infectious necrotic hepatitis (black disease) among nubian goats in sudan clostridium novyi (myonecrosis, black disease, and bacillary hemoglobinuria) and clostridium septicum (braxy) infections first report of infectious necrotic hepatitis (black disease) among nubian goats in sudan liver and biliary system bacillary hemoglobinuria: induction by liver biopsy in naturally and experimentally infected animals bacillary hemoglobinuria in a free-ranging elk calf bacillary hemoglobinuria in dairy cows an outbreak of bacillary haemoglobinuria in sheep in india successful treatment of bacillary hemoglobinuria in japanese black cows acute abomasitis due to clostridium septicum infection in experimental sheep rapid identification and differentiation of pathogenic clostridia in gas gangrene by polymerase chain reaction based on the s- s rdna spacer region suppurative abomasitis associated with clostridium septicum infection clostridial myocarditis in lambs outbreak of clostridial myocarditis in calves clostridial myositis in cattle: bacteriology and gross pathology clostridial vaccination efficacy on stimulating and maintaining an immune response in beef cows and calves failure of clostridium chauvoei vaccines to protect against blackleg prevalence of coxiella burnetti infection in wild and farmed ungulates coxiella burnetii shedding by farmed red deer (cervus elaphus) high prevalence of antibodies against chlamydiaceae and chlamydophila abortus in wild ungulates using two regional seroprevalence of leptospirosis on deer farms in new zealand growth response and shedding of leptospira spp. in urine following vaccination for leptospirosis in young farmed deer corynebacterium pseudotuberculosis paratuberculosis (johne's disease) in cattle and other susceptible species efficacy of a killed vaccine for the control of paratuberculosis in australian sheep flocks detection of a novel reassortant epizootic hemorrhagic disease virus (ehdv) in the usa containing rna segments derived from both exotic (ehdv- ) and endemic (ehdv- ) serotypes the first years ( - ) of epizootic hemorrhagic disease virus serotype in the usa review of the epizootic hemorrhagic disease outbreak in domestic ruminants in the united states peste des petits ruminants demonstration of coinfection with and recombination by caprine arthritis-encephalitis virus and maedi-visna virus in naturally infected goats key: cord- -pz htccp authors: kohn, dennis f.; barthold, stephen w. title: biology and diseases of rats date: - - journal: laboratory animal medicine doi: . /b - - - - . - sha: doc_id: cord_uid: pz htccp nan the diversity of research for which the laboratory rat is used is probably greater than that associated with any other animal. the laboratory rat is a descendent of the wild rat, rattus norvégiens, which originated in asia and reached europe in the early s. wild and albino mutants were first used for ex perimental purposes in europe in the mid- s and in the united states shortly before . the wistar institute in phil adelphia was prominent in the development of the rat as a labo ratory animal, for here originated many of the rat strains now used worldwide. henry donaldson and his colleagues at the wistar institute used these early rat strains for a variety of stud ies dealing with neuroanatomy, nutrition, endocrinology, ge netics, and behavior. the history and evolution of the many rat strains used today have been recently summarized (lindsey, ) . the most commonly used outbred rat stocks in north amer ica are the wistar, sprague-dawley, long-evans, and holtzman. all are albino except the long-evans stock, which is usually marked with a black or gray hair coat over the shoul ders and is sometimes referred to as a "hooded rat." there are numerous inbred and mutant rat strains, although the number is less than that in the mouse. table i lists the more commonly used strains. there are a rather large number of commercial vendors of laboratory rats in the united states. most of the stocks and strains mentioned above can be obtained from more than one source. although the origin of an outbred stock, such as the sprague-dawley, may have been the same for a number of vendors, in many cases it has been to years since such a stock has been removed from its original breeding colony. ac cordingly, the genotype of outbred stocks and inbred strains may vary among sources and be reflected by differences in data when multiple sources of rats are used. a standardized scheme of identifying stocks and strains of rats has been devel oped and is now used by nearly all commercial vendors. more over, it is important that authors correctly identify stocks and strains that are used in their research since the success in re peating the work in another laboratory may be dependent upon the genotype (source of the rat). table ii summarizes the stan dardized nomenclature for outbred stocks as developed by the table i commonly used strains "national institutes of health ( ) . . letters preceding the colon designate the supplier/breeder code consist ing of a capital and two or three lowercase letters . capital letters following the colon are used by a breeder to identify his stock . letters in parentheses denote origin of stock . subscript symbols indicate rearing by means other than natural mother (f, fostered; fh, fostered by hand) international committee on laboratory animals (icla). table iii contains the scheme for designating inbred strains of rats (national institutes of health, ) . "animals for re search" (national academy of sciences, ), a directory of sources for laboratory animals sold in the united states and canada, lists all rodents according to standard nomenclature, and is a valuable aid in purchasing laboratory animals. commercial production of rats has markedly changed since the s due to the development of hysterectomy-derived and barrier-maintained breeding colonies. prior to the application of this technology to production colonies, infectious diseases were ubiquitous in rats from most sources. today, vendors can be selected who offer pathogen-defined animals for most stocks and strains. concomitant with changes in commercial sources of rats are the major advances made in the design and construction of institutional animal resources and husbandry practices within them. optimum housing of rats today includes provisions for quarantining and isolation of animals according to vendor subpopulations that have a similar microbial flora. there are various levels of sophistication to provide barriers to the spread of infections in rat colonies. since many rat pathogens are spread by aerosol, ventilation control is very important. nonrecirculating room air or high-efficiency panic ulate air (hepa)-filtered air has become a design standard in modern animal facilities. as discussed in chapter , clean/contaminated corridor-designed facilities aid in contain ment against the spread of pathogens by aerosol, personnel, table iii nomenclature for inbred rats . the strain designation is given in capital letters followed by a slash . the substrain designation follows the slash and is given as numbers or as individual or company codes. numbers are used to denote substrains that were derived from a common strain but separated before the completion of inbreeding . subscript symbols indicate rearing by means other than natural mother and contaminated equipment. a more complete barrier system may include an entry area in which incoming supplies and equipment are sterilized and in which personnel shower and don sterile clothing and filter masks before entering animal rooms. more recently, laminar-flow (mass air displacement) rooms and mobile units have become popular because they can be incorporated in existing buildings that lack design charac teristics mentioned above. environmental control in rat rooms is important to the com fort and health of the animals, as well as to the consistency of data derived from the rats. room temperatures between and °f are desirable, and the relative humidity should range be tween and %. daily fluctuations in temperature and hu midity act as significant stressors. these fluctuations may be associated with the environmental control system of a building or may be induced by procedures such as cleaning floors with a water hose or high pressure sprayer. twenty-four-hour tem perature/humidity recorders are useful in detecting changes in environmental conditions. light intensity should be evenly distributed to all animals within a room. seventy-five to fc have often been suggested as an optimal range for light inten sity. however, recent evidence indicates that this intensity can induce retinal degeneration in albino rats (anver and cohen, ) . light-timing devices are a convenient means to provide desired day/night cycles such as - or - hr. caution should be exercised in the use of insecticides and air-deodorizing chemicals, since some have been shown to in duce hepatic microsomal enzymes in rats. accordingly, their use in animal rooms is not usually recommended (baker et al., a) . rats can be housed in either wire-or solid-bottom cages. wire-bottom cages are more frequently used since they are less labor-intensive. frequency of cage and litter pan changing is a function of animal density. solid-bottom cages should be sani tized two to three times per week, while wire-bottom cages should be sanitized on a weekly or biweekly schedule with litter pans changed two or three times per week. feed should be contained in hoppers. either automatic systems or bottles are satisfactory for providing water to rats. some caution is necessary when using automatic systems, since weanling and newly arrived rats may not drink initially from such devices. to avoid undesirable microbial contamination, water bottles should be sanitized before they are refilled and automatic sys tems should be drained and flushed when racks are sanitized. acidification of water to a ph of . to . or chlorination at to ppm will control pseudomonas aeruginosa contamina tion of water (weisbroth, ) . however, this treatment is not necessary for immunocompetent animals. wood shavings or chips are the most commonly used contact bedding mate rials. hardwoods are preferred to softwoods, since the latter are capable of inducing hepatic microsomal enzymes (baker et al, a) . this section summarizes some of the anatomical characteris tics of the rat with emphasis on characteristics that are unique. the reader is advised to refer elsewhere in the literature for comprehensive descriptions (bivin et al., ; caster et al., ; hebel and stromberg, ; smith and calhoun, ; zeman and innés, ) . the rat dental formula is ( / , c / , pm / , m / ) = . the incisors are well developed and grow continuously. the rat lacks tonsils and water taste receptors. the major pairs of salivary glands are the parotid, submandibular (submaxillary), and sublingual. the parotid gland is a serous gland consisting of three to four lobes and is located ventrolaterally from the caudal border of the mandible to the clavicle. the submandibular glands are mixed glands located ventrally between the caudal border of the mandibles and the thoracic inlet. the sublingual glands are mucous glands and are much smaller than the parotid and submandibular glands. they are located at the rostral pole of the submandibular glands to which they are closely associated. brown fat deposits are present in the ventral cervical region. these multilocular deposits are well demarcated and can be confused with salivary glands or lymph nodes. the stomach of the rat is divided into two parts; the forestomach (nonglandular) and the corpus (glandular). the two portions are separated by a limiting ridge. the esophagus en ters at the lesser curvature of the stomach through a fold of the limiting ridge. this fold is responsible for the inability of the rat to vomit. the forestomach, which is thinner than the cor pus, is linked with an epithelium similar to that of the esopha gus and extends from the cardia to a narrow band of cardiac glands at the junction of the glandular portion. the small intestine is composed of the duodenum ( cm), jejunum ( cm), and ileum ( cm). the cecum is a thinwalled, comma-shaped pouch that has a prominent lymphoid mass in its apical portion. the colon is composed of the as cending colon, with prominent oblique mucosal ridges, trans verse and descending colons, with longitudinal mucosal folds; followed by a short rectum that is confined to the pelvic canal. the liver has four major lobes (median, right lateral, left, and caudate) and is capable of regeneration subsequent to par tial hepatectomy. the rat has no gallbladder. the bile ducts from each lobe form the common bile duct that enters the duo denum mm from the pyloric sphincter. the pancreas is a lobulated, diffuse organ that extends from the duodenal loop to the gastrosplenic omentum. it can be dif ferentiated from adjacent adipose tissue by its darker color and firmer consistency. up to excretory ducts fuse into - large ducts, which empty into the common bile duct. the nasal cavity is not markedly different from that of other mammals. the rat has a maxillary recess (sinus) located be tween the maxillary bone and the lateral lamina of the ethmoid bone. the recess contains the lateral iiasal gland (steno's gland) that secretes a watery product that is discharged at the rostral end of the nasal turbinate. it has been postulated that the nonviscous secretion contributes to the humidification of in spired air and acts to regulate the viscosity of the mucous layer overlying the nasal epithelium. the left lung has one large lobe, and the right lung is divided into four lobes (cranial, middle, accessory, and caudal). the pulmonary vein in the rat has cardiac striated muscle fibers within its wall that are contiguous with those in the heart. the rat does not have an adrenergic nerve supply to the bronchial musculature, and bronchoconstriction is controlled by vagai tone. unlike the guinea pig, the rat lung has a low concentra tion of histamine (bivin et al., ) . the heart and peripheral circulation in the rat differ little from that of other mammals. the blood supply to the heart is derived from both coronary and extracoronary arteries. the latter arise from the internal and subclavian arteries. the right kidney, which is more craniad than the left, has its cranial pole at the l, vertebra and its caudal pole at the level of l . the rat kidney is unipapillate as are kidneys of other ro dents, lagomorphs, and insectivores. having only one papillus and calyx makes the rat useful for studies in which cannulization of the kidney is done. the presence of superficial nephrons in the renal cortex has made the rat widely used as a model for studying nephron transport in an in vivo micropuncture system. the male reproductive system has a number of highly devel oped accessory sex glands. these include large seminal vesi cles, a bulbourethral gland, and a prostate gland composed of the coagulation gland (dorsocranial lobe) and ventral and dorsolateral lobes. the inguinal canal remains open throughout the life of a rat and testes descend initially by days of age. the female rat has a bicornate uterus that is classified as the duplex-type because the lumina of the uterine horns are com pletely separate with paired ossa uteri and cervices. the female urethra does not communicate with the vagina or vulva, but rather exits at the base of the clitoris. the brain of the rat has very large olfactory bulbs and a nonconvoluted cerebrum. the hypophysis is behind the optic chiasma and is attached to the base of the brain by a thin hol low stalk, the infundibulum. the blood supply to the brain is from the internal carotid and vertebral arteries. blood leaves the brain via a system of sinuses that are enclosed in the dura mater. the ventricular system is similar to that of other ani mals, but the rat lacks a foramen of magendie. it must be recognized that many of the normal values deter mined for a specific group of rats may be accurate for only that rat stock/strain, source, and conditions under which they are held. selected physiological, hematological, and clinical bio chemical parameters are listed in tables iv-vii. more com plete information on biological values is available (mitruka and rawnsley, ; ringler and dabich, ) . nutritionally adequate diets are readily available from com mercial sources. these standard rations are quite satisfactory for most applications. however, for some types of experimen tation there are factors, other than nutritional adequacy, which must be considered. the nutrient composition of diets and the contamination of feed components by mycotoxins, antibiotics, synthetic estrogens, heavy metals, and insecticides may have a profound impact on many studies. for instance, caloric intake and the percent of fat and protein in the diet of rats influence the incidence of neoplasia (altman and goodman, ) . sim ilarly, various contaminants have an adverse effect on data from toxicologie, gerontological, and reproductive studies. standard commercial diets are formulated from natural ingre dients and will vary in nutrient composition on a batch-tobatch basis due to differences in type and quality of ingredients used. commercial makers of rodent feeds take precautions to preclude the presence of contaminants in feeds, but only a few products have a defined profile of maximal levels of heavy metals, aflatoxins, chlorinated hydrocarbons, and organophosphates. for some investigative purposes, feeds formulated with re fined ingredients (purified diets) or with chemically defined compounds are useful when control of nutrient concentrations is essential (national research council, ) . these diets are, however, too expensive for general use. baker et al. ( b) and bivin et al. ( ). rats are commonly fed ad libitum, and food intake will vary according to requirements for growth, gestation, and lactation. the nutritive requirements for the rat are listed in table viii. the duration of storage and the temperature at which feeds are stored prior to use effect the nutritive quality of diets. com mercial diets are formulated to have a shelf life of up to months. however, storage in a hot or damp environment will reduce this shelf-life. to help assure that only fresh diets are used, products should be used which have milling dates identi fied on their containers (see chapter ). sexual maturity occurs between and weeks for both sexes, although the onset of first estrus in females occurs at about weeks. the vagina opens between and days, and the testes descend between and days, although they remain fully retractable in adults. rats ovulate spontaneously, but ovulation can also be induced by forced coitus during nonestrous intervals. vaginal stimulation during mating is impor tant in rat reproductive physiology. the more often a male inserts his penis into the vagina prior to ejaculation, the greater the probability of a resulting pregnancy. however, natural or artificial stimulation of the vagina within min of a first mat ing will abrogate pregnancy from the first mating by inhibition of sperm transport. a -hr estrous period recurs every to days and after parturition, without seasonal variation. estrus can be suppressed when females are housed in groups and syn chronized in the presence of a male or its excreta (whitten effect), but this effect is not as pronounced as in the mouse. female fertility wanes at to days, but estrous cycles may continue through months. male fertility is lost between and months. fertility of both sexes is generally regarded as maximal between and days of age (adler and zoluth, ; baker, ; farris, ; lane-petter, ; leathern, ) . males will mount estrous females numerous times with one or two rapid ejaculations in the course of to minutes. ejaculated semen coagulates, forming a copulatory plug that remains in the distal vagina for a few hours, after which time it dissolves or is extruded. copulation is usually nocturnal. du ration of gestation varies with strain, age, litter size, and other variables, and ranges from to days, with an average of or days. primiparous females tend to have a slightly longer gestation than multiparous females (farris, ) . estrus can be detected in a number of ways. females in es trus are hyperactive and brace themselves when touched. their ears quiver when they are stroked on the head or back, and stimulation of the pelvic region induces lordosis (farris, ) . the vulva becomes swollen, and the vagina becomes dry in contrast to the moist pink wall during metestrus or diestrus. as proestrus occurs (approximately hr), smears of va ginal cells contain nucleated epithelium, leukocytes, and occa sional cornified cells. estrus (approximately hr) begins with about % nucleated and % cornified cells, with cornified cells predominating as estrus continues. metestrus follows (ap proximately hr) with large numbers of leukocytes and corn ified cells, which form abundant caseous vaginal detritus. metestrus is characterized by the presence of large flat nucle ated (pavement) cells. diestrus persists for approximately hr (baker, ; farris, ) . breeding dates can be established by examination of vaginal swabs for spermatozoa or examining the distal vagina or cage pan for copulatory plugs. timed pregnancies are best achieved by placing the female in the male's cage in the afternoon and examining her for a plug or spermatozoa the following morning. abdominal enlargement becomes evident at about weeks. pseudopregnancy is rare (lane-petter, ) . rats reproduce successfully under a variety of conditions, but husbandry practices can significantly influence fecundity. rats can be bred as monogamous pairs, taking advantage of postpartem estrus for maximal breeding efficiency. polyg amous breeding is more economical, since only one male can be kept with to females. females are often removed to a separate cage prior to whelping, since they may not tolerate other females in the cage while nursing. they will tolerate their mates, however. females with litters do best on clean dust-free wood shavings in solid-bottom cages. due to heat regulation, pups neither thrive in overly spacious cages with wide flutuations in ambient temperature, nor in overly crowded cages where they cannot dissipate heat. the recom mended cage floor area for a female and her litter is in. . ambient room temperature and humidity should be within the acceptable range with minimal fluctuation. high ambient tem perature can cause male infertility (baker, ; baker et al., a; lane-petter, ) . the rat estrous cycle is particularly sensitive to variations in light. daily lighting at an average of fc with a spectrum approximating natural light for to hr is best for breeding. constant light for as few as days may induce persistent es trus, hyperestrogenism, polycystic ovaries and endometrial hy pertrophy or metaplasia (baker et ai, a; gralla, ) . nutrition may also affect reproductive performance. re quirements for certain components are increased during preg nancy and growth, but overfeeding is deleterious. caloric re striction may actually improve fertility and possibly reproduc tive life of the female (leathern, ) . excess dietary protein can adversely affect female sexual development. vitamin defi ciencies can cause infertility, particularly those vitamins (a, e, riboflavin, and thiamin) that are most labile to autoclaving or deterioration (baker, ) . it is not necessary to add nesting material to bedding for successful breeding, but rats will utilize it if offered. shredded paper or cotton nesting material will be readily accepted and used by prepartem and nursing dams. parturition is heralded by pronounced postural stretching and rear leg extension. a vagi nal discharge may be noted li- hr prepartum. parturition is usually complete in or hr, but can range from a few minutes to several hours depending on litter size. dystocia is exceed ingly rare. litters average between and pups, with highest fecundity through the sixth litter. inbred rats tend to produce smaller litters. although infrequent, cannibalism is most apt to occur with nervous or primiparous females subjected to stress (farris, ; lane-petter, ; leathern, ) . the neonate weighs about i gm, depending on litter size, sex, strain, and physical condition of the dam. pups are born hairless, blind, with closed ears, undeveloped limbs, and short they are fully haired between and days (baker, ; farris, ; lane-petter, ) . maternal antibody is trans ferred in utero, via the yolk sac and by intestinal absorption of colostrum by the neonate for up to days after birth (chev ille, ) . optimal weaning age is - days, although pups can be weaned as early as days. differentiation of sex in adult rats is relatively easy after the testes descend. the adult testes can be readily retracted through large inguinal canals. male neonates have a larger genital papillus and the anogenital space is greater in males than females. from national research council ( ) . h adequate to support growth, gestation, and lactation; based on % dry matter. ( linoleic acid, . %, is required. ^one-third to one-half can be supplied by l-cystine. ^one-third to one-half can be supplied by l-tyrosine. ^mixture of glycine, l-alanine, and l-serine. ^vitamin a, iu = . ìg retinol, . ìg retinyl acetate, . ìg retinyl palmitate. vitamin d, iu = . ì£ ergocalciferol. vitamin e, iu = mg dl-a-tocopheryl acetate. artificial insemination can be achieved in rats, but the major obstacle is the coagulative properties of their semen. sperm can be obtained by maceration of the epididymis and vasa or by electroejaculation, although the latter method is unreliable and the semen often rapidly coagulates. coagulation can be eliminated by prior surgical extirpation of the seminal vesicles and coagulating glands without significant effect on fertility. semen can be diluted with a number of media but frozen stor age of rodent semen has met with little success. insemination can be achieved surgically by direct injection of seminal fluid into the uterus and by nonsurgical means. successful concep tion seems to require not only insemination during estrus but also induction of pseudopregnancy by mating with a vasectomized male or mechanical stimulation within a few hours (before or after) insemination. egg harvest for transfer can be accomplished by excision of the preovulatory ovaries and teas ing from gravid follicles or recovery from the oviduct or uterus by flushing with transfer medium. superovulation by injection of gonadotropisms may enhance yield, but is usually not nec essary. eggs are generally injected directly into the uterus but the recipient uterus must be at the same stage of the uterine cycle (bennet and vickery, ) . synchronization of estrus can be achieved by vaginal inser tion of polyurethane sponges containing . mg medroxyprogesterone for days. females are then put in a cage previously occupied by male rats, sponges are removed, and the rats are injected with iu of pregnant mare's serum. within hr, % will be in estrus. this can also be attained by administer ing mg medroxyprogesterone in ml ethanol/liter drink ing water, prepared fresh daily for days, then intramuscular injection of iu of pregnant mare's serum (bennet and vick ery, ). the rat has been utilized extensively in a variety of research fields, including behavioral science. rats are docile, adapt to new surroundings, tend to explore, and are easily trained to a variety of sensory cues by positive or negative reinforcement. rats sleep during daylight hours and activity, including feed ing, is greater during the night and early morning. laboratory rats are easily handled, but strain differences exist. sprague-dawley and lew rats tend to be less fractious than long evans or f rats. docility is improved with routine and proper handling. rats become nervous and refractory to han dling when they hear others squeal. nutritional deficiency, particularly hypovitaminosis a, and mishandling can make rats vicious. rats seek entry into small openings, a trait that is utilized for coaxing them into restrait apparatus. like other rodents, rats are coprophagic, which must be taken into con sideration when administering drugs, measuring fecal output, or performing nutritional studies. unlike mice, rats are less apt to fight, and males can be housed together. in addition, rats are not gregarious like mice, and seem to tolerate single caging well. experimental studies indicate significant changes in plasma corticosteroid levels, depending on cage cohort size. levels tend to be least in rats housed singly, to increase in groups up to , to decrease in larger groups up to - , then rise again in groups up to (lane-petter, ). infectious agents constitute a significant environmental vari able that impacts on research data derived from laboratory rats. as is the case with other species, infectious agents induce a wide range of diseases in the rat that vary from inapparent to overt clinical disease. most investigations use large numbers of rats in which a specific group or colony consists of several to hundreds of rats. accordingly, emphasis on disease is one of prevention and placed at the colony level rather than on a sin gle or a few animals. curative use of antibiotics, which is important in the treatment of bacterial diseases of nonrodent species, is rarely useful in the laboratory rat. administration of drugs to obtain therapeutic blood levels is difficult to achieve in a colony; also some animals may improve clinically but re main colonized by the pathogen and serve as carriers, reinfecting other animals. rats seldom show clinical signs of disease upon arrival to the laboratory from commercial sources. however, these rats may harbor pathogens that are of low to moderate virulence and that are capable of severely compromising the health of animals when the rats are exposed to various types of experimental stress. moreover, some of these pathogens may never cause clinical disease, yet induce microscopic lesions or biochemical aberrations that can have profound effects on research data. for these reasons, investigators and clinicians should be aware of the pathogen status of the animals used in studies, both ini tially and throughout the course of the studies. this section on infectious diseases contains those agents that are of principal importance to the investigative use of the rat. a. streptococcosis. the causative organism, streptococcus pneumoniae, is a gram-positive coccus that is rather ubiq uitous among humans and animals. streptococcus pneumoniae is frequently recovered from respiratory tract lesions in guinea pigs, nonhuman primates, and some domestic animals. in hu mans, it is often present in the nasopharynx in the absence of clinical symptoms of infection. upper respiratory tract infec tion of conventionally raised rats has been reported to be com mon. however, it is seldom present in barrier-maintained, commercial rat sources. as in pneumococcal disease in hu mans, a number of serological types have been associated with respiratory disease in rats. streptococcus pneumoniae infection in rats often remains lo calized in the nasopharynx without the development of overt disease. a shift in the host-parasite balance due to stress or concurrent infection with another pathogen may result in bronchopneumonia and bacteremia. the most common signs of respiratory disease are serous to mucopurulent nasal discharge and "red tears" due to porphyrin pigments secreted from the harderian glands, dyspnea, rales, and depressed activity. ani mals will often die within a few days after the onset of pneu monic signs. the severity and prevalence of clinical disease within an infected colony are associated with environmental conditions that induce stress (e.g., experimental manipulation, overcrowding, fluctuations in ambient temperature and humid ity, and copathogens). although all age groups are susceptible to infection and clinical disease, young animals are more apt to be clinically affected. transmission between rats is by aerosol droplet. although both humans and rats can carry the same serotypes of s. pneumoniae, the authors are unaware of evi dence indicating zoonotic or human-to-animal transmission. the most characteristic gross lesions are pulmonary consol idation and fibrinopurulent pleuritis and pericarditis ( fig. ). an extensive fibrinopurulent peritonitis, orchitis, or meningitis may occur as well. if a bacteremia occurs early, the disease may be acute with few gross lesions. streptococcus pneu moniae induces an outpouring of exudate rich in fibrin, neutrophilic leukocytes, and erythrocytes into the alveoli. bron chioles are filled with neutrophilic leukocytes. embolie lesions may occur in multiple tissues which include the spleen, liver, kidneys, joints, and brain. streptococcosis is diagnosed by clinical signs, characteristic lesions, and isolation of s. pneumoniae from lesions. the per icarditis, pleuritis, and pleural effusion noted above differenti ate pneumococcal disease from pneumonia due to mycoplasma, although the two pathogens often are superimposed. this organism produces an á-hemolysis on blood agar plates similar to that of the streptococcus viridans group. streptococ cus pneumoniae isolates are most commonly differentiated from nonpathogenic s. viridans by the sensitivity of the former organism to optochin (hydrocuprein hydrochloride). optochin-impregnated discs are placed on a blood agar plate which has been inoculated with a pure culture of the clinical isolate. if the isolate is s. pneumoniae, a distinct zone of growth inhibition will be present around the disc. although typing of s. pneumoniae isolates is seldom done today, one can type an isolate by reacting known specific s. pneumoniae antisera with s. pneumoniae isolates. this serological test is the neufeld-quellung reaction and is based on the capsular swelling that is induced by specific antiserum. there is no effective means to control s. pneumoniae infec tion once it is enzootic in a colony. benzathine penicillin ( , units/ gm body weight) may be helpful in reducing the severity of the disease and as an aid in limiting infections to a subclinical mode in some animals. however, antibiotics will not eliminate the organism from rat colonies. hysterectomy rederivation of breeding stock from infected colonies is an ef fective method of initiating new stock free from pneumococcal infection (weisbroth, ) . b. pseudotuberculosis (corynebacteriosis). the causative agent of pseudotuberculosis is the gram-positive bacillus, corynebacterium kutscheri. on occasion, other corynebacterium species can cause similar syndromes in rats. typically, the or ganism causes inapparent infections in rats, with exacerbation of respiratory disease under conditions of stress. when clinically ill, the most commonly seen signs include serous oculonasal discharge, dyspnea, anorexia, and loss of weight or retarded growth. animals with severe pulmonary signs usually succumb within several weeks, while rats with less severe signs often survive much longer. most rats will have inapparent infections in which c. kutscheri cannot be isolated from internal organs. little is known concerning how c. kutscheri is carried or transmitted within a colony. it has been suggested that the organism is transmitted via aerosol droplet or direct contact. once rats are infected, a hematogenous spread may be involved, since lung lesions are initially interstitial and not bronchial. gross lesions are characterized by a variable number of grayish-yellow foci surrounded by red zones, particularly in the lung (fig. ) . in longer-standing cases, individual foci co alesce into raised lesions cm or larger in diameter. occasion ally, fibrous adhesions occur between the lungs and thoracic walls. similar lesions may be seen in other organs, including the liver, brain, and kidneys. the hepatic lesions resemble tu bercles and have caseous centers and fibrous capsules. prepucial adenitis, arthritis, and otitis media may also be caused by c. kutscheri. the lesions in various target organs appear to be due to septic emboli. pulmonary lesions initially consist of a polymorphonuclear cell and macrophage infiltrate of the bronchioles and interstitial tissue with a round cell infiltrate occurring later. bronchi become impacted with polymorphonuclear cells and necrotic leukocytes. giemsa or gram staining of infected tissues will reveal the rod-shaped c. kutscheri organisms. diagnosis of c. kutscheri infection is made on clinical signs, gross and microscopic lesions, and isolation of the bacterium from infected tissues. although the respiratory signs are simi lar to those present with mycoplasmosis, the rapidity with which c. kutscheri clinically affected rats succumb helps dif ferentiate it from mycoplasma pulmonis-'mduced disease. un like streptococcosis, fibrinopurulent pericarditis, peritonitis, and pleural effusion are not seen. whereas peribronchial lymphoid hyperplasia is a dominant lesion in mycoplasmosis, it is unremarkable in c. kutscheri infections. corynebacterium kutscheri is easily recovered from lesions and upper respiratory tract exudates by culturing on blood agar plates incubated aerobically at °c. epizootics of pseudotuberculosis may occur in conven tionally raised breeding colonies, but rarely occur in barrierraised colonies. epizootics often can be retrospectively associ ated with an environmental stress (e.g., fluctuation in ambient temperature or ventilation). culling of ill animals will not eliminate c. kutscheri from animals remaining in a colony. isolation of the organism from animals with subclinical infec tions is not usually successful. for this reason, cortisone ad ministration has been advocated as a means for surveillance of infection in colonies prior to necropsy and culturing for c. kutscheri. in the past, most serological methods have been un satisfactory in detecting antibody in animals with inapparent infections (weisbroth, ) . recently, however, enzymelinked immunoabsorbant assay (elisa) has been shown to be capable of detecting antibody in animals without clinical signs of infection (ackerman et al., ) . hysterectomy derivation is an effective means to establish a c. kutscheri-free colony. antibiotic therapy will not eliminate c. kutscheri from a colo ny, but a -day regimen of penicillin has been reported to be effective in curtailing an epidemic of c. kutscheri-'mductd pneumonia (fox et al., ) . since c. kutscheri infection is, in most cases, inapparent and manifests itself whenever the host is sufficiently stressed, it can be a significant problem in experimentally stressed rats. c. tyzzer's disease. tyzzer's disease is caused by the gram-negative, spore-forming rod, bacillus piliformis. this organism, which is not a true bacillus, is an intracellular pathogen that has not been cultivated on artificial media, and is, as yet, taxonomically undefined. in the laboratory, b. piliformis is propagated in the yolk sac of embryonated chick eggs. this disease occurs in other rodent species and appears to be widely distributed in many nonrodent species, but there ap pears to be a degree of species specificity among b. piliformis strains. it occurs occasionally in conventionally raised rat colo nies. the vegetative form of b. piliformis is unstable in the environment. however, spores of the organism are relatively stable and are believed to be the source of transmission among animals. clinical signs associated with tyzzer's disease are not partic ularly distinctive and, accordingly, only suggestive in making a diagnosis. typically, affected rats are apt to be adolescents with signs such as lethargy, weight loss, and distended abdo mens. diarrhea is not a common sign in rats with b. piliformis infection. animals displaying clinical signs generally die with in several weeks. clinically inapparent infections occur and are most probably responsible for transmission of the organism within a colony. clinically evident tyzzer's disease is usually associated with experimentation that compromises the immunocompetence of rats. the most remarkable gross lesions involve the liver, ileum, and myocardium. hepatic lesions consist of numerous small, pale foci on the surface and within the parenchyma. the intes tinal lesion has been termed "megaloileitis" due to a segmen tai dilatation and inflammation of the ileum (fig. ) (jonas et ai, ) . heal distension is not always present. in some rats, circumscribed gray foci also occur in the myocardium. the pathogenesis of the disease is believed to involve a pri mary intestinal infection with spread to the liver via the portal circulation. bacillus piliformis invades enterocytes, resulting in villus shortening, inflammation, necrosis, and hemorrhage. intracellular organisms are demonstratable in epithelium of crypts and villi. the necrotic foci in the liver are most often present near vessels. surrounding these foci are varying num bers of leukocytes, macrophages, and fibroblasts. intracytoplasmic bacteria may be seen in hepatocytes at the periph ery of the lesions, but may be present in very small numbers and thus be hard to find. organisms are also found in myocar dium around foci of necrosis (weisbroth, ) . a presumptive diagnosis can be made by the gross lesions, but a definitive diagnosis is dependent upon observation of the organism within hepatocytes, intestinal epithelium, or myocar dium. impression smears of liver taken at necropsy and stained with gram, giemsa, or méthylène blue stains may be useful for a rapid diagnosis. however, formalin-fixed specimens stained by giemsa or warthin-starry methods are usually per formed to confirm a diagnosis. the ileal distension seen in rat tyzzer's disease must be differentrated from other causes of adynamic ileus, particularly chloral hydrate-induced lesions. prevention of tyzzer's disease in a colony is dependent upon a barrier that excludes entry of the agent by contaminated cages, equipment, and infected animals. routine cage sanita tion probably is ineffective in killing the spores of b. piliformis, but exposure of spores to °c for min has been shown to inactivate them. sodium hypochlorite ( . %) is an effective disinfectant (ganaway, ) . although antibiotics have been shown to be effective under experimental conditions in mice, there is no evidence to indicate that antibiotic therapy can be of value under natural conditions within a colony of rats (weisbroth, ) . d. pasteur elio sis. pasteur ella pneumotropica frequently infects conventionally raised rats and has been recovered occa sionally in rats from barrier-and axenic-maintained colonies. it is a pathogen of very low virulence, and most infections remain clinically inapparent. only a relatively few reports doc ument p. pneumotropica as a primary pathogen in cases of penumonia, otitis media, and conjunctivitis. as a copathogen with either m'ycoplasma pulmonis or sendai virus, it has a con tributory role in the resultant respiratory lesions and otitis. its localization is not limited to the respiratory tract, since it is frequently isolated from the oral cavity, intestinal tract, and uterus. it also has been associated with mastitis and furunculosis in rats. it has been suggested that p. pneumotropica is essentially an enterotropic rather than a pneumotropic orga nism. the intestinal tract is probably the primary site for local ization of the organism in subclinical infections. horizontal transmission is by the oral-fecal route and direct contact. since p. pneumotropica is frequently carried in the uterus, vertical transmission can occur, and, accordingly, this can compromise the microbial status of axenic and gnotobiotic colonies. distinctive clinical signs and lesions do not occur with p. pneumotropica-induoed disease. accordingly, a diagnosis must be based upon its isolation as the sole pathogen or, as in many cases, as a copathogen within lesions. blood agar medi um is satisfactory for primary isolation from nonenteric sites. however, for recovery from the intestinal tract, enrichment in a medium such as gn broth is recommended before isolation is attempted on blood agar plates (weisbroth, ) . hysterectomy derivation and barrier maintenance are the only means to control infection. however, particular attention must be made to ensure that hysterectomy-derived young came from dams that had culturally negative uteri. antibiotic thera py is not effective in eliminating the organism from a colony. e. salmonellosis. salmonella species that infect rats in clude salmonella enteritidis, s. typhimurium, s. dublin, and s. meleagridis. salmonellosis, which was once a major cause of disease in laboratory rat and mouse colonies, is rarely reported in either species today. however, it still exists in wild popula tions of rodents and, therefore, remains a potential threat to laboratory rodents. infection in an immunologically naive colony typically re sults in an epizootic of clinically affected rats and a varying proportion of animals with inapparent infection. these latter animals act as subclinical carriers to render the infection as enzootic in a colony. acute outbreaks will occur intermittently whenever immunological and other host defense mechanisms are altered. signs associated with salmonellosis in the rat are anorexia, depressed activity, starry hair coats, and soft to formless feces. affected animals die in to weeks. lesions that occur in salmonellosis differ depending on the stage of the disease. salmonellae penetrate the intestinal mucosa at the level of the ileum and cecum. the earliest le sions occur in this locale and consist of a mild dilatation, thick ened intestinal walls, and a granular mucosal surface. involve ment of the reticuloendothelial system is reflected by enlarged peyer's patches, mesenteric lymph nodes, and spleen. in some infected animals, a bacteremic state occurs that results in the demise of the host before the development of further lesions. however, in animals not succumbing to septicemia, ulcération of the ileal, colonie, and cecal mucosa occurs. histologically, the villus epithelium of the ileum is markedly degenerated, and the lamina propria is infiltrated with neutrophils and mac rophages. concomitant with intestinal lesions is the develop ment of focal necrosis and granulomas in the spleen and liver due to hematogenous spread of the organism (buchbinder et al, ; maenzae/fl/., ) . in rats who are intermittent or chronic shedders of salmonel la, the most remarkable lesions are lymphadenitis of the mes enteric lymph nodes and ulcération of the cecal mucosa. rats from which salmonella is chronically shed have more ad vanced lesions than do intermittent shedders of the organism. a diagnosis of salmonellosis relies upon identification of an isolate as a salmonella sp. recovery of salmonella from the intestines, spleen, and liver is readily accomplished in rats clinically affected during an epizootic. however, this is not true for asymptomatic carriers, since some will shed the orga nism intermittently in the feces, and recovery from tissues is difficult. recovery in carrier animals is best accomplished by initial incubation of fecal pellets in an enrichment broth, such as selenite f plus cystine broth, followed by streaking onto brilliant green agar (weisbroth, ) . from this medium, possible salmonella colonies are inoculated into triple-sugariron slants. final identification is then made by biochemical tests and serotyping. prevention of this disease is based upon the exclusion of wild rodents from laboratory animal facilities and the use of only feed and bedding that has been properly processed and pack aged to ensure against salmonella contamination. /. pseudomoniasis. pseudomonas aeruginosa, a ubiq uitous gram-negative bacterium found in soil and water, colo nizes plants, insects, animals, and humans. it often colonizes the oropharynx and can be isolated from the intestinal tract of rodents. infection with this organism in immunocompetent rats is nearly always inapparent. however, when rats are immunosuppressed, p. aeruginosa invades the upper respiratory mucosa and cervical lymph nodes, becomes bacteremic and induces an acute, lethal disease. in some cases, rats develop facial edema, conjunctivitis, and nasal discharge. in genet ically thymic-deficient rats (nude), retro-orbital abscesses may occur prior to bacteremia. transmission in laboratory rodents occurs primarily by direct contact and contaminated water bottles and automatic watering systems. phenolics are usually effective disinfectants, but quaternary ammonium compounds may actually support its growth. diagnosis of pseudomoniasis is based upon a history of immunosuppression associated with an epizootic of acute disease and isolation of p. aeruginosa from the blood and organs of affected rats. facial edema in affected rats must be differenti ated from viral sialodacryoadenitis. pseudomonas aeruginosa grows well on blood agar and most other standard laboratory media. most strains are ß-hemolytic and produce a bluish-green pigment, pyocyanin, as well as fluorescein. the use of specialized media (pseudomonas p agar) enhances pigment production. the organism derives energy from carbohydrates via oxidation rather than fermentative me tabolism. identification of isolates as p. aeruginosa is easily made by the above characteristics and appropriate biochemical reactions (weisbroth, ) . in most research applications, p. aeruginosa-free rats are not necessary for the conduct of the work. it is a major problem, however, in rats used for burn research and in studies in which drugs or radiation induce immunosuppression. infection can be relatively well controlled in a colony by hyperchlorinating drinking water at ppm or by acidification of water to a ph of . - . . in a closed colony, it is also advisable to remove rats that remain culturally positive after water treatment has been instituted. in studies requiring pseudomonas-free rats, isolators are useful in which a gnotobiotic environment can be achieved. alternatively, laminar flow units may suffice if supplies and equipment are sterilized and personnel wear sterile garments. g. streptobacillosis. streptobacillus moniliformis is a commensal bacterium often present in the nasopharynx of con ventionally raised rats. although it may be involved occasion ally as a secondary invader within inflammatory lesions of the rat, the chief importance of s. moniliformis is that it is the principal agent causing rat-bite fever in humans (anderson et ai, ) . the other bacterium associated with this clinical syndrome is spirillum minus. clinical signs in humans usually occur within days of a rat bite and consist of headache, weakness, fever, a generalized rash, and arthritis. often clini cal signs subside in several days but then recur at irregular intervals for weeks or months (see chapter ). a. murine respiratory mycoplasmosis. murine respirato ry mycoplasmosis (mrm) is the term now accepted for a dis ease which, for many years, had an undefined etiology and a number of synonyms [i.e., infectious catarrh, enzootic bronchiectasis, chronic respiratory disease (crd), and chronic murine pneumonia]. since , the causal relationship of mycoplasma pulmonis with this disease has become well estab lished (kohn and kirk, ; lindsey et al., ; whittlestone et al., ) . of all the pathogens occurring in laboratory rats, m. pulmonis has had the greatest negative im pact on studies. this has been primarily due to the chronicity of the disease, which often manifests itself only after months of infection. long-term studies in areas of toxicology, carcinogenesis, nutrition, and gerontology, in particular, have been affected. prior to the use of gnotobiotic techniques and barrier maintenance in rat production colonies, m. pulmonis was enzootic in nearly all commercial and institutional colo nies. today, vendors can be selected who offer mycoplasmafree rats. my coplasma pulmonis is highly contagious and in duces a disease that frequently results in debilitation or demise of the host after a long period of time. the clinical signs associated with mrm range from negligi ble upper respiratory tract signs to systemic signs associated with pneumonia. the earliest and most common signs include snuffling and serous or mucopurulent oculonasal discharge. extension of m. pulmonis infection from the nasopharynx via the eustachian tubes to the middle ears is common. however, torticollis and circling due to involvement of the inner ear are infrequently observed, even though one or both middle ear bullae may be impacted with exudate. the onset of upper res piratory signs is variable, but often occurs within several weeks postinfection. signs of penumonia include dyspnea, rales, and systemic effects such as weight loss, starry hair coat, and hunched posture. characteristically, signs of pneumonia occur - months postinfection, but this is quite variable and is a function of environmental influences, such as intracage ammonia levels and the immune competence of the host. in a small percentage of cases, the disease will be nearly subclinical even in the presence of extensive pulmonary lesions. mycoplasma pulmonis is transmitted both horizontally and vertically from dams to their litters. in most instances, trans mission from the female occurs postpartum by direct contact, but if the genital tract of the dam is infected, antenatal infec tion can occur. horizontal transmission between postweanling rats of any age readily occurs, and there appears to be no sig nificant age-related resistance to either infection or disease. although little is known about differences in resistance among rat stocks and strains, the lew rat has been shown to be more susceptible to mrm than the f rat. there is little evidence available to indicate that transmission occurs through fomites such as caging equipment and garments worn by personnel. since aerosol droplet and direct contact appear to be the prima ry modes by which m. pulmonis infections are spread, the rapidity with which the organism is transmitted is dependent upon environmental factors, such as ventilation rates, degree of recirculation of air, and animal density within rooms. the basis for the pathogenicity of m. pulmonis is not well understood. mycoplasma pulmonis adsorbs to the cell mem brane of the ciliated, columnar or cuboidal epithelia in the res piratory tract (fig. ) . it has been suggested that adsorption is a means by which mycoplasmas damage host cells by uptake of essential cellular metabolites; release of cytotoxic products, such as h ; or cross reaction of antibody with cell mem brane components that are antigenically similar to or altered by mycoplasmas. infection severely distorts or ablates ciliary structures (fig. ) , interfering with mucociliary clearance mechanisms. the gross lesions in the upper respiratory tract include mucopurulent exudate in the nasal cavity, sinuses, and middle ear bullae. later, the exudate becomes caseous within the bul lae. lesions in the lower respiratory tract reflect those of a bronchopneumonia. the earliest lesion is a mucopurulent exu date within the trachea, bronchi, and bronchioles. this pre cedes grossly evident lesions of the lung parenchyma that ini tially consist of atelectasis due to bronchial occlusion. later, bronchiectatic lesions appear as numerous cream-colored nod ular abscesses on the surface of the lung. these lesions may be restricted to only a portion of a lobe or may involve nearly all of the parenchyma (fig. ) . microscopically, the inflammatory response is characterized by a lymphocyte and plasma cell infiltrate in the submucosa and neutrophilic leukocyte response within the lumina of the epithelium. nasal cavity, eustachian tubes, middle ears, and tracheobronchial tree. a consistent and prominent lesion in the lung is the peribronchial lymphoid hyperplasia that often be comes quite massive. within the lumina of the bronchi and bronchioles, mucin and neutrophil exudation increases during the course of the disease to the point of bronchiectasis. con comitant with the impaction of bronchi is a change in the epithelia from a ciliated, columnar type to a squamoid type. this change in epithelial architecture is likely associated with cytotoxic enzymes from autolyzed neutrophils, although a di rect cytotoxic effect from mycoplasmas could be involved. a tentative diagnosis of mrm can usually be made by obser vance of the clinical signs and gross lesions described above. clinical signs alone are not particularly helpful, since nasal exudates are present in bacterial infections such as s. pneumoniae. in addition, the reddish porphyrin deposition seen in the nares and periorbitally in sialodacryoadenitis virus infec tion and water deprivation may be confused with exudation. the gross lesions of otitis media and bronchiectasis are rather distinct. however, c. kutschen lung lesions may grossly mim ic those of mrm. histopathology and serological evidence will differentiate mrm from sendai virus infection, although the two infections are often superimposed. recently a filamen tous bacterium has been associated with bronchiectasis in wild and laboratory rats (mackenzie et al., ) . however, the causal relationship of this organism with lesions is undefined since the rats were also infected with m. pulmonis. this fila mentous bacterium has not been successfully grown on artifi cial media, and its presence is best verified by either histology, using the warthin-starry stain, or electron microscopy (fig. ) . although a definitive diagnosis of mrm is made by isola tion of m. pulmonis from involved tissues, it is evident that the existence of other agents must be evaluated to determine if copathogens are contributory to lesions. prevention of mrm in either breeding or experimental colo-nies is dependent upon barrier systems that preclude the entry of m. pulmonis into the facility. hysterectomy derivation is the only means of establishing an m. pulmonis-frtt breeding colo ny from a previously infected stock. due to the frequent local ization of this microorganism in the uterus, it is necessary to ensure that neonates taken by hysterectomy have not been in fected in utero. rats used in research animal facilities are ob tained from various commercial and institutional sources. ac cordingly, it is essential that the mycoplasma status of these sources is known and that the rats are housed by vendor or in groups with a similar microbial status. for assessment of whether a group of rats is m. pulmonisfree, the best sites for isolation in animals without gross lesions are the nasal cavity, middle ear, trachea, and uterus-oviduct. mycoplasma pulmonis is not particularly fastidious and grows well in several types of mycoplasma media (cassell et al., ; lentsch et al., ) . most formulations have a ph indi cator that is useful since m. pulmonis ferments glucose. in broth media, moderate to heavy growth is reflected by ph and color of the broth. in broth cultures in which the titer is low, a perceptable ph change may not occur. tissue and washing samples should be placed in broth rather than agar media, since recovery of the organism is more likely in those samples con taining few mycoplasmas. samples from broth cultures are transferred to agar media when a ph change is readily evident or at - days if no ph change occurs. mycoplasma colonies are evident in - days by observation with x stereoscopic microscopy. although culturing and histopathology have been the usual means to survey rat colonies, elisa testing has recently been shown to be a very sensitive serological assay and one that can be performed quickly in most clinical laboratories (cassell et al., a) . in vitro sensitivity tests show m. pulmonis to be susceptible to tetracycline and tylosin. tetracyline, given at mg/ml drinking water, may be useful in some situations (lindsey et al., ) . however, treatment with antibiotics seldom influences the disease course of mrm in a colony situation. b. murine genital mycoplasmosis. mycoplasma pulmonis recently has become recognized as an important pathogen in the female genital tract of rats, and thus is being treated here as a distinct disease rather than as a sequella to mrm. infection of the genital tract is usually inapparent. however, reduced fertility and fetal deaths can occur. infection of the oviduct and uterus occurs frequently in rats who have respiratory my coplasmosis. it is unknown whether localization in the genital tract occurs due to a hematogenous spread or to an ascending infection of the genital tract. it has been shown that subsequent to intravenous inoculation, m. pulmonis almost invariably lo calizes in the female oviduct-uterus. gross lesions, when present, consist of a purulent oophoritis, salpingitis (fig. ) , and pyometra. the lew strain is particu-Ä^sfcvvv ( Ä ß f/g. . electron micrograph of filamentous bacterium (large arrow) and m. pulmonis (small arrow) attached to epithelium of respiratory mucosa. the morphology of size of the filamentous bacterium are similar to that of the cilia. (courtesy of dr. w. f. mackenzie.) larly prone to develop gross lesions. mycoplasmapulmonis ad sorbs to the epithelial cells in the genital tract in a manner similar to that seen in the respiratory tract. salpingitis occurs most frequently and is characterized by exudation of neutrophils into the lumen, hyperplasia of oviductal epithelium, and a lymphoid response in the submucosa. the lesions in the ovarian bursa include edema and inflammation. uterine le sions can vary from a mild inflammatory change to pyometra (casselle/fl/., b). genital mycoplasmosis in the male rat has not been well doc umented. however, it is known that experimental inoculation can include an inflammatory response in the ductus efferens and epididymis. moreover, it is known that m. pulmonis is capable of adherence to spermatozoa in an in vitro system. since pasteur ella pneumotropica can also induce similar le sions in the female rat, a diagnosis of mycoplasmosis is depen dent upon isolation of m. pulmonis from the lesions. methods for culturing and identification are similar to those used for respiratory mycoplasmosis. because the rat is widely used in various types of reproduc tive biology research, m. pulmonis colonization, even without gross lesions, would probably impact on the validity of data. the grossly evident caseous lesions in the ovary and oviduct can be mistaken for neoplasia if microscopy is not done. c. mycoplasmal arthritis. the etiological agent of this disease is mycoplasma arthritidis. this mycoplasma species colonizes the pharynx, middle ears, and lungs of rats, although few studies have been done to document the relative frequency of this mycoplasma in rat sources. within the respiratory tract, m. arthritidis colonization is thought to induce negligible le sions, and it has been shown to coexist with m. pulmonis. although it is often considered to be the principal agent in volved in arthritis in rats, the disease has been rarely reported. nearly all reports of its involvement in clinically apparent ar thritis have been made prior to . it has been suggested that poor cage sanitation and abrasions of the extremities are in volved in entry of the organism to the joints by hematogenous spread or extension from surrounding tissues (ward and cole, ) . since the organism appears to be of low virulence, the immunocompetence of the host may be a major factor in the outcome of infection. arthritic animals limp and move with difficulty due to pain associated with the polyarthritis. any of the joints in the limbs and vertebrae can be affected, but the tibiotarsal and radiocarpal joints are most often involved. affected joints are hyperemic and swollen. incised joints reveal a purulent exudate in both articular and periarticular tissues. microscopically, there is exudation of neutrophils into the synovial spaces, and a lym phocyte and plasma cell infiltration in the synovial mem branes. destruction of the articular cartilage occurs subsequent to the inflammatory response. since polyarthritis can occur subsequent to septicemias asso ciated with other bacteria, particularly c. kutschen, a diag nosis of m. arthritidis-'mduced arthritis is contingent upon the demonstration of m. arthritidis by isolation or immunofluorescence techniques. this mycoplasma species grows well in me dia used to isolate m. pulmonis if arginine is added to the for mulation (cassell et al., ) . tetracyclines have been used to prevent the onset of arthritis when the organism has been inoculated intravenously, but there are no reports of its efficacy in spontaneous cases. mycoplasma arthritidis, like m. pulmonis, may contaminate transmissible tumors and caution should be exercised to ensure transplanted tissues are not contaminated. hemobartonellosis. the causative agent of this rickettsial disease is hemobartonella muris. this organism is an extra cellular parasite of erythrocytes and induces inapparent infec tions that may persist for long periods. the ability of the host to restrict the infection to a subclinical mode rests with the integrity of the reticuloendothelial system. evidence of infec tion is usually limited to splenomegaly and laboratory findings of mild parasitemia and reticulocytosis. transmission of h. muris involves the blood-sucking louse, polyplax spinulosa. transmission can occur during a blood meal or when rats crush infected lice and are inoculated via pruritis-induced abrasions. the organism can also be transmit ted inadvertantly with transplantable tumors and other biolog ical products. diagnosis of hemobartonellosis is dependent upon identifica tion of the organism in the peripheral blood of infected ani mals. the usual method of detection is by splenectomizing rats suspected of harboring the organism. in these rats, severe para sitemia and hemolytic anemia occur within weeks after sur gery. hemobartonella muris can be visualized on the surface of erythrocytes in romanowsky-stained blood smears as coc- coid bodies arranged singly, in clusters, or chains (cassell et al., ) . the rarity of reported cases would indicate h. mûris is no longer a significant problem in barrier-maintained colonies. however, conventionally maintained colonies may be exposed to infected wild rats and p. spinulosa and, accordingly, the disease still is of importance in the laboratory rat. the disease has had a negative impact on investigations of various types, but principally with those in which the host's immune compe tence has been impaired. a. parvoviral syndromes. parvoviruses that can infect rats include rat virus (rv), toolan h-l (h-l) virus, and minute virus of mice (mvm). parvoviruses are small nonenveloped viruses that resist extremes in temperature, ph, and drying. rat virus, or kilham rat virus (krv), has several antigenically related strains (rv, h- , x- , l , hb, sprv, her, hhp, kirk), all of which have been isolated as inadvertant contami nants of rat tissue or rat-passaged biological material. toolan h-l related serotypes (h-l and h-t) are antigenically distinct from rv serotypes. both rv and h-l are experimentally pathogenic, producing similar lesions, but only rv has been associated with natural disease. neonatal rats can be experi mentally infected with mvm, but the virus does not seem to cause natural infection. minute virus of mice antibody reac tivity can be present in rat serum, but this is probably non specific, since it can be found in germfree rat serum and is reduced or eliminated by receptor destroying enzyme. rat virus infection is usually subclinical or latent, but a num ber of clinical syndromes have been associated with it. infec tion of pregnant females can cause fetal résorption and birth of small litters. pups are runted, atactic, or jaundiced. neonates develop similar signs following postpartem exposure. rats in troduced to an infected colony can develop ruffled fur, de hydration, and sudden high mortality. a similar syndrome oc curs in latently infected adults subjected to immunosuppressive regimens. the rat is the only natural host for rv and h- , although experimental infection can be established in a number of other species. seroconversion to both rv and h-l virus is common, with a high prevalence of infection within an enzootically in fected colony. horizontal transmission is achieved by the oral and probably respiratory routes, with virus excretion primarily in the feces. some strains of rv can be excreted in the milk or in utero. clinical signs are manifest transiently upon introduc tion of rv into a previously uninfected population, but, there after, the virus spreads rapidly to produce subclinical or inapparent enzootic infection. rat virus can persist as a true latent infection in the presence of high circulating antibody, but dis ease can be activated by immunosuppression. it must, there- fore, be assumed that seropositive rats are persistently infected and can serve as a source of infection to other rats. pups infected in utero or as neonates develop intranuclear inclusions and necrosis in the outer germinal cell layer of the cerebellum. the recovered animal has severe depletion of the internal granular layer and disorganized purkinje cells. intra nuclear inclusions are also in hepatocytes, kupffer cells, endothelial cells, and biliary epithelial cells, resulting in necrotizing hepatitis and the sequellae thereof (bile retention, jaundice, peleosis, bile ductal hyperplasia, parenchymal collapse, nodu lar hyperplasia). in adults, infection is usually inapparent, but when acute disease is precipitated, rv injures vascular walls and hematopoietic elements, causing coagulative disorders, thrombosis, hemorrhage, and infarction within the central ner vous system (hemorrhagic encephalomyelopathy). hemorrhagic and necrotic lesions have also been noted in the per itoneum, testis, and epididymis. rat virus has broad tissue tropism and lesions or clinical signs may potentially be varied, depending on virus and host factors (coleman et al., ; jacoby et al, ) . infertility and unthrifty pups caused by rv must be differenti ated from environmental and husbandry factors or infectious agents such as mycoplasma or sendai virus. adult disease must be differentiated from toxicity, nutritional deficiency, and trau ma. diagnosis is made by the typical lesions, if present, virus isolation, and serology. seroconversion to each virus (rv or h-l) can be detected by serum neutralization, hemagglutination inhibition, complement fixation, and immunofluorescence. hemagglutination inhibition is currently the most commonly used means of antibody determination (jacoby et ai, ) . since rv infection is usually silent and persistent and can be transmitted either vertically or horizontally, effective control is best achieved by destroying the entire population, decon taminating, and repopulating with clean stock. virus-free rats can be obtained from selected commercial vendors or by caesarean rederivation. rederived progeny must be tested for vertically transmitted strains of virus. colonies can be kept virus-free by limiting entry to seronegative, virus-free rats (as well as transplantable rat neoplasms or tissues), periodic serological testing, and adequate physical containment. although parvovirus infection of rats is usually inapparent, there can be adverse effects on the research usefulness of in fected rats. immunosuppression may exacerbate illness and mortality in latent carriers. the viruses often contaminate transplantable tumors and cell lines, can modify immune re sponsiveness or cause teratological effects. a decision to work with infected animals should be made carefully. b. other dna virus infections. rats are susceptible to rat cytomegalovirus, which has a predilection for the salivary and lacrimai glands. infection is widespread among wild, but not laboratory rats (jacoby et al., ) . rats also seroconvert to mouse adenovirus, but it is not known if infection is due to a mouse or rat strain of virus. adenovirus-like inclusions have been reported in the intestine of rats treated with cancer chemotherapeutic agents (ward and young, ) . c. siaiodacryoadenitis virus and related coronaviral infections. two strains of coronavirus have been identified as pathogens of laboratory rats: siaiodacryoadenitis virus (sdav) and rat coronavirus (rcv). furthermore, rats are experimen tally susceptible to the coronavirus of mice, mouse hepatitis virus (mhv). coronaviruses are large, pleomorphic enveloped rna viruses with surface peplomers or spikes that confer a corona-like appearance to the virion. viruses of this group have complex antigenic interrelationships and cross-react ex tensively. common antigens are shared by sdav, rcv, and mhv, particularly by complement fixation, but antibody reac tivity is highest with homologous virus. siaiodacryoadenitis virus and rcv represent different strains of the same virus, but whether different strains of the same virus or separate viruses, they are both important natural pathogens in rats. the signifi cance of mhv for rats is not known, but the virus can replicate in the respiratory tract of intranasally inoculated rats (taguchi et al., ) . natural antibodies to mhv can occur in rats, but this is probably due to the closely related antigenicity of mhv to sdav and rcv rather than natural mhv infection of rats (barthold, ) . clinical signs of sdav infection vary widely in severity, but include blepharospasm, sneezing, porphyrin-pigmented nasal and ocular discharge, and cervical edema (fig. ) . some rats develop keratoconjunctivitis and other ocular lesions. signs persist approximately one week, but ocular sequellae can be permanent. acutely infected rats become anorectic, and estrus can cease temporarily. infection is subclinical in weanling or older rats, but intranasally inoculated neonates die and suck lings develop lower respiratory disease. siaiodacryoadenitis virus is highly contagious and spreads rapidly among susceptible rats by contact, aerosol, or fomite. susceptible rats of any age can be infected. when enzootic within a colony, clinical disease occurs only in sucklings, since adults are immune. infection is acute, lasting only about week, at which time rats seroconvert with no carrier state. maintenance of sdav in a colony requires continuous intro duction of susceptible stock as weanlings or newly introduced rats. the epizootiology of rcv is presumed to be similar to sdav. within days of intranasal inoculation, sdav causes rhi nitis followed by necrosis of the ductular and acinar epithelium of salivary and lacrimai glands, accompanied by intense in flammation and edema. tracheitis and peribronchial lymphoid hyperplasia can also be found. salivary glands appear swollen, pale, with interlobular and periglandular edema. harderian glands are flecked with yellow-gray foci. one, some, or all of the salivary or lacrimai glands can be affected, with the excep tion of the sublingual glands, which are spared. cervical lymph nodes become enlarged. glandular repair ensues within week, with squamous metaplasia of ductular epithelium and hyperplasia of acinar epithelium. the repair phase subsides within days with minimal residual lesions. interstitial pneu monia can occur in suckling, but not adult rats. conjunctivitis, keratitis, corneal ulcers, synechia, hypopyon, and hyphema can arise due to lacrimai dysfunction. eye lesions usually re solve, but can proceed to chronic keratitis, megaloglobus (fig. ) , and retinal degeneration. rat coronavirus infection causes rhinotracheitis and focal interstitial pneumonia. salivary but not lacrimai gland infection is rare, but when present, resem bles wild sdav lesions. infection with rcv also lasts approx imately week (barthold, ; jacoby et al., ) . nasal and ocular signs must be differentiated from those caused by mycoplasma, sendai virus, pathogenic bacteria, ex cess ammonia, or hypovitaminosis a. cervical swelling may fig. . epiphora and swelling of the ventral neck in a rat naturally infected with sdav. (from barthold, ; courtesy of hemisphere publishing corp.) fig. . megaloglobus and hyphema in a young rat naturally infected with sdav. (from barthoid, ; courtesy of hemisphere publishing corp.) also occur in immunosuppressed rats infected with p. aeruginosa. microscopic sdav lesions are characteristic. mild lower respiratory tract lesions associated with rcv must be differentiated from those of sendai virus or pneumonea virus of mice (pvm). seroconversion or rising complement fixing antibody titers following acute disease is confirmatory. how ever, antibody may be low or undetectable with this method. serum neutralization is another test that can be used, but the most sensitive antibody tests are immunofluorescence or elisa. either mouse or rat coronaviruses are used as antigen in these latter tests (smith, ) . rats can be kept free of sdav and rcv if they are isolated and if newly introduced rats are immune or unexposed. intro duction of a single subclinically infected rat can precipitate epizootic disease among naive rats. if an outbreak occurs, the infection will run its course and die out within - weeks if new rats are not introduced into the room and if breeding is temporarily ceased. routine disinfection of rooms and equip ment is sufficient to destroy environmental sources of virus. sialodacryoadenitis virus lesions can be confused with or contribute to changes induced by test compounds or nutritional deficiencies, particularly vitamin a. sialodoacyoadenitis virus disease can predispose to anesthetic death due to airway hypersecretion. eye lesions resulting from sdav infection can in terfere with eye research. both sdav and rcv can potentiate other respiratory infections. d. sendai viral infection. sendai virus commonly infects laboratory rats, but its clinical significance is less than in mice. sendai virus is a parainfluenza virus of the paramyxovirus family. paramyxoviruses are pleomorphic, enveloped, labile rna viruses. sendai virus infection in rats is usually subclinical, but can be manifested as ruffled fur, dyspnea, or anorexia. a decrease in average litter size and runted pups is common during outbreaks in breeding colonies. sendai virus is highly contagious and disseminates rapidly. outbreaks subside following development of an immune popu lation, with the potential of recurrence several months later as the susceptible population enlarges. sendai virus induces an acute respiratory infection with no natural carrier state. excre tion and transmission of virus occurs via the respiratory tract (jacoby <?r Ö/., ) . sendai viral lesions in the rat are similar to those in genet ically resistant strains of mice. following an initial necrotizing rhinitis, tracheobronchitis ensues within week of infection. lungs become consolidated and plum-colored in a patchy, pri marily anteroventral, distribution. necrotizing bronchiolitis, focal nonsuppurative interstitial pneumonia, and perivascular and peribronchial lymphoplasmacytic infiltrates are the hall marks of the lower respiratory component. in the rat, acute lower respiratory lesions last only a few days and coincide with clinical disease. repair of bronchial mucosa is effected through hyperplasia and squamous metaplasia. residual le sions can last for months in the mouse, but probably not in rats. during the acute stage of sendai virus infection, mucosal necrosis and inflammation can extend up the eustachian tubes to the middle ear. bacterial otitis media is often preceded by sendai viral infection in rodents (brownstein et al., ; d. g. brownstein, unpublished observations, ) . mild nonsuppurative pulmonary lesions can be seen with sendai, pvm, rcv and to a lesser extent, sdav. sendai virus can be superimposed upon and contribute to respiratory mycoplasmosis. squamous metaplasia of respiratory mucosa can mimic hypovitaminosis a and also occurs in my coplasmosis. virus isolation and serocon version or rising titers to sendai virus confirm a diagnosis based upon characteristic lesions and clinical signs. sendai virus antibody can be accu rately detected by complement fixation and hemagglutination inhibition, but newly developed immunofluorescence and elisa are replacing these methods (smith, ) . rats can be kept free of sendai virus in a similar manner as with sdav and rcv. recovered sendai virus antibody-posi tive stock can be utilized as breeders to reestablish a virus-free population. pups born of these parents will be virus-free and antibody negative, following decay of their maternal antibody. mice and hamsters can serve as sources of infection with paramyxoviruses. sendai virus infection, although usually subclinical, can cause significant pulmonary changes and act as a copathogen with mycoplasma or bacteria. squamous metaplasia and hy perplasia induced by these agents influence respiratory carcinogenesis and interpretation. sendai virus can cause immunosuppression for prolonged periods in rats (garlinghouse and vanhoosier, ) . e. pneumonia virus of mice infection. pneumonia virus of mice is a pneumovirus of the paramyxovirus family and thus shares many features in common with sendai virus. it has not been associated with clinical disease in the rat or mouse under natural conditions. several rodent species, including hamsters, guinea pigs, and gerbils, seroconvert to pvm under natural conditions. the virus seems to spread rapidly among suscepti ble rodents, based on serocon version. it is presumed to be transmitted in a manner analogous to sendai virus. the pathogenesis of pvm has been investigated in mice, but has not been thoroughly examined in rats, even though it pro duces more significant pulmonary lesions in rats than mice un der natural conditions. in the mouse, pvm replicates in the upper respiratory mucosa without obvious cytolysis following low-dose exposure (d. g. brownstein, unpublished observa tions, ) . viral antigen and cytolytic lesions are seen in the bronchial and bronchiolar epithelium as well as alveolar walls following apparently higher doses of virus in a pattern reminis cent of sendai virus. the ensuring lesion, like that of sendai virus, is necrotizing bronchiolitis and interstitial pneumonia (carthew and sparrow, ) . the active infection lasts about week, after which time lymphocytic infiltration of peribronchial and perivascular tissues and focal interstitial pneumonia can persist for several weeks. rat lungs develop prominent nonsup purative perivascular and mild interstitial inflammation in re sponse to both experimental and natural pvm infections (d. g. brownstein, unpublished observations, ; vogtsberger et al., ) . infection is usually diagnosed retrospectively in rats, where pulmonary lesions are observed following seroconversion to pvm in the absence of other respiratory pathogens. pulmonary lesions mimic those of sendai virus or possibly coronaviruses. antibody to pvm has usually been detected by hemagglutination inhibition or serum neutralization, but these tests are being replaced by immunofluorescence and elisa. hemagglutination inhibition is least expensive and highly accurate, however (smith, ) . pulmonary lesions induced by pvm can inter fere with the interpretation of experimental studies, or possibly pulmonary function, although this has not been established. hemorrhagic fever with renal syndrome, korean hemorrhagic fever, or muroid virus nephropathy is a human disease com plex caused by one or more serologically related bunyaviruses, including hantaan virus. this syndrome is mentioned here be cause of its emerging eminence as a major zoonotic disease in which several species of the suprafamily muroidae, including laboratory rats, can serve as carriers. the virus is transmitted to man by excretions and aerosols from the lungs, saliva, and urine of healthy rodent carriers. pathology, if any, in rats has not been described. the disease in humans includes proteinuria, azotemia, and, less often, petechiae, hemoconcentration, hypotension, and renal failure. human mortality can oc cur, but recovery with immunity to reinfection is usual. hemorrhagic fever with renal syndrome viruses cause human disease throughout much of eurasia, but on the basis of serological evidence, hfrs viruses exist world-wide in feral rodents and humans, including the united states. transmis sion of hfrs to laboratory personnel by silently infected al bino laboratory rats (rattus norvegicus) has been documented in japan and belgium. virus-infected lung and kidney cell lines provide excellent sources of antigen for sensitive immu nofluorescence tests, but serological monitoring of laboratory rats for hfrs virus antibody is not currently a common prac tice (gajdusek et al, ; anonymous, ) . g. other virus infections. rats harbor endogenous retroviruses or genomic retrovirus sequences that (in contrast to the mouse) are of minimal significance in terms of natural disease. a number of endogenous rat sequences have been artificially / / / recombined with mouse leukemia virus sequences or naturally recombined with other rat retrovirus sequences to form defec tive rat sarcoma viruses, for example kirsten and harvey rat sarcoma viruses, which are experimentally oncogenic to rats, mice, and hamsters (shih and scolnick, ) . natural seroconversion to reovirus- and mouse encephalomyelitis virus have been noted with no apparent disease. an enterovirus with neurotropic properties has been isolated from naturally infected, asymptomatic rats. although rats can be experimen tally infected with ectromelia and lymphocytic choriomeningitis virus, these agents are not of practical significance as natu ral infections of rats. testing for antibody to these agents is therefore a vacuous exercise. a number of other viruses or viruslike agents have been isolated or incriminated as causes of disease in rats, including rat submaxillary gland virus, novy virus, enzootic bronchiectasis agent, gray lung virus, and wild rat pneumonia agent. these agents, if extant at all, are of du bious importance in contemporary laboratory rat populations (jacoby et ai, ) . recently, clinically silent infection with an unidentified pox virus has been found in soviet rats. micro scopic necrotizing and inflammatory changes were restricted to the nose (kraft et ai, ) . the agent is believed to be cow pox virus. laboratory rats are host to far fewer parasites than wild rats, since husbandry practices interrupt complex life cycles and caesarean rederivation has simply eliminated many agents al together. control of these agents in infected colonies is also best achieved by these means. this section will mention only agents that have been reported in laboratory rats, although most are unlikely or rare. table ix outlines selected treatment regimens for control of common metazoan parasites, but the reader must beware that these drugs may render the treated rat useless for experimental work. decisions to treat rather than eliminate infected rodents must be made with care, in concert with the needs of the scientific investigator. a. protozoal infections. several flagellates can parasitize, rats but few are truly pathogenic. hemoflagellates (tripanosoma cruzi, t. lewisi) occur in wild rats, but these agents require anthropod vectors. nevertheless, t. lewisi can be en countered if a colony is infested with rat fleas. trypansoma lewisi is only mildly pathogenic even in heavy infections, and infection is short term. rats harboring this organism become ill when stressed or immunosuppressed. enteric flagellates (tritrichomonas sp., tetratrichomonas sp., pentatrichomas sp., trichomitis sp., hexamastix sp., enteromonas sp., retortamonas sp., chilomastix sp., monocercomonoides sp., and octomitus sp.) are very common but nonpathogenic in labora tory rats. giardia muris and spironucleus (hexamita) muris application - gm/liter drinking water for days, stop days, re peat days . gm/liter drinking water for days mg/liter drinking water or . mg/kg feed for weeks . mg/gm feed for day mg/g sc, single dose mg/kg po, single dose . - . % dust with or without pyrethrin hr/week on cage top for several weeks - gm of pellets in bedding for days, change bedding and cage, repeat at days, change and repeat at month . % dip - % dust, up to . % spray or up to % dip, repeat at days up to . % dip % spray "information kindly supplied by dr. j. w. streett, yale university. ; 'a number of these compounds have adverse effects on the experimental usefulness of treated rats and therefore should be used with discretion and full knowledge of the investigator. are also common and considered potentially pathogenic, caus ing unthriftiness, diarrhea, and intestinal lesions in severely affected animals. however, these are generalizations made from other species, since natural clinical disease has not been reported among rats. sporozoan parasites, common in wild rats, are encountered only on occasion in laboratory rats. hepatozoon muris infec tion is usually subclinical, but severely affected rats can have leucocytosis, splenomegaly, anemia, hepatic degeneration, emaciation, or death. infection requires ingestion of the vec tor, laelaps echidninus. toxoplasmosis (toxoplasma gondii) is subclinical in rats, requiring the ingestion of cat feces or contaminated tissues containing asexual stages, but can also be transmitted transplacentally. intracellular t. gondii organisms occur in several tissues, particularly lung and reticuloendothelial organs, and cysts are found in the central nervous system. sarcocystis muris, once common but now rare in labo ratory rats, has a life cycle similar to Ã. gondii. cysts are found in the muscle. frenkelia sp. has been described in a colony of laboratory rats, causing large thin-walled cysts and inflamma tion in the central nervous system, but no clinical signs. encephalitozoon cuniculi is apparently now uncommon in labora tory rats and seldom induces signs or lesions. its prevalence and significance are far greater in the laboratory rabbit. single or clusters of small spores form within cysts in a number of tissues. nonsuppurative inflammation can be found in the brain and kidneys. unlike t. gondii, e. cuniculi organisms are gram-positive. encephalitozoon cuniculi can be transmitted via contaminated transplantable tumors, tissue, or tissue fluids from infected rats. intestinal sporozoans include eimeria nieschulzi and e. miyairii, which infect the small intestine, and e. separata, which infect the large intestine of wild rats. they are exceedingly rare or nonexistent in laboratory rats. pneumocystis carinii, once considered to be a yeast but now considered a sporozoan, is an ubiquitous organism that infects the lung of a variety of species, including laboratory rats. prev alence of infection is high within infected colonies, but preva lence among various geographic areas in unknown. it seems to be very common among both conventional as well as barrierreared rats. normally p. carinii is not pathogenic, but infected rats can develop disease after several weeks of repeated steroid injections and starvation, particularly in younger rats. remark ably, rats from many commercial sources can be so treated and shown to be infected, even if free of other pathogens. under these circumstances, the lungs are expanded and rubbery. al veolar walls are thickened and hypercellular, and alveoli are distended with proteinaceous, eosinophilic foamy material containing macrophages and organisms in different stages of development. tissues can be stained with special stains, such as methanamine silver or periodic acid-schiff, to visualize the organisms, which are not visible with hematoxylin and eosin. other protozoa, including entamoeba muris and balantidium coli, are found in the large bowel of rats, but are not pathogenic. the reader is referred to more exhaustive coverage of protozoa for detailed means of diagnosis (flynn, ; hsu, ) . b. nematodiasis. syphacia muris is a common oxyurid (pinworm) of the cecum and colon. embryonated eggs are passed in the feces or deposited on the anus. rats are infected by ingestion of eggs or by larval migration into the colon from the anus. syphacia muris closely resembles s. obvelata, the mouse pinworm. both species of syphacia can patently infect rats or mice, but preference seems to be shown for the homolo gous host. another common oxyurid of rats and mice is aspicularis tetraptera, which is also found in the cecum and colon. aspicularis eggs are nonembryonated, and are not de posited near the anus. depending on parasite species, the pré paient period is - days. diagnosis is achieved by exam ination of large bowel contents (particularly the cecum and proximal colon) for nematodes, feces for ova, and, in the case of syphacia, cellophane tape impressions of the anus for ova. speciation is easily achieved by the differential morphology of ova. aspicularis ova are symmetrically ellipsoidal, while syphacia ova are flattened on one side. since eggs are ex tremely resistant to environmental factors and disinfectants, and autoinfection can occur with s. mûris, infection is difficult to eradicate. anthelminthic treatment ameliorates infection (table ix) , but all rats must be treated simultaneously and the room must be thoroughly cleaned. despite these actions, rein fection frequently occurs. caesarean rederivation and subse quent strict sanitation are the most effective means of control. pinworms are generally considered nonpathogenic, but heavy parasite loads can be deleterious to the host. concern has been raised regarding the effects of these agents on research but no effects have been documented in the rat. trichosomoides crassicauda is common in some rat colonies but is not generally encountered in commercially raised ani mals. it inhabits the transitional epithelium and lumen of the urinary tract. the small male resides within the reproductive tract of the female, whose anterior end embeds within the mucosa. embryonated ova with bipolar opercula are shed in the urine. following ingestion, eggs hatch in the stomach, lar vae penetrate the gastic wall, then migrate via the lungs, kidney, and ureter to the urinary bladder. migrating larvae can incite eosinophilia and formation of granulomata, particularly in the lung. drug treatment (table ix) is effective, as is cesarean rederivation. parasites do not incite an inflammatory response in the bladder, but can serve as a nidus for calculi or enhance natural and experimental bladder carcinogenesis. other nematodes are common in wild but rare in laboratory rats. trichinella spiralis adults occur in the intestine, and lar vae migrate extensively, encysting in muscle. transmission is effected by ingestion of contaminated meat. capii i aria hepatica eggs are ingested, hatch, and migrate through the cecum to the liver through the portal veins. adults mature and lay characteristic bipolar operculate ova in the liver, where they lie dormant until ingested by a carnivore. gongylonema neoplastium burrows in the squamous epithelium of the tongue and anterior stomach, where it incites a proliferative response or ulcération. the life cycle involves an intermediate insect host. angiostrongylus cantonesis, which requires the ingestion of a mollusk intermediate host, infects the brain and lung. the intestinal nematodes heterakis spumosa, nippostrongylus brasiliensis, strongyloides ratti, and trichuris muris lack in termediate hosts, but rarely occur naturally in laboratory rats. none is known to be very pathogenic. further details on nematode diagnosis and ova morphology are available (flynn, ; hsu, ; oldstone, ) . taenia taeniaformis, the cat tapeworm. following ingestion of ova, oncospheres migrate to the liver and form strobilocerci (cysticercus fasiolaris). host connective tissue capsules can give rise to sarcomas. this parasite has been used as a model of parasite-induced oncogenesis in the rat. cysticercus fasciolaris is on occasion encountered in laboratory rodents with contaminated food or bedding. hymenolepis nana and diminuta infect the small intestine of several species, including the rat. hymenolepis nana, the dwarf tapeworm, has a -mm wide segmented body ranging from to mm in length. the life cycle may be either direct or indirect. following ingestion of ova, oncospheres penetrate the intestinal mucosa, form cysticercoid larva, then emerge into the lumen and mature into adults that shed eggs in the feces. nonimmune hosts can be autoinfected; eggs are pro duced, hatched, and complete their life cycle within the intes tine of a single host. the indirect cycle involves intermediate hosts, such as grain beetles or fleas, in which cysticercoid lar vae form and await ingestion by the definitive host. hymenolepis diminuta is - mm wide and - mm long. the eggs are passed in the feces but must be ingested by an intermediate host (flour beetle, flea, moth), which in turn must be ingested by the definitive host to complete the life cycle. enteritis can occur in heavy infestations, which are most likely to be encountered with h. nana. both can infect primates, in cluding humans. hymenolepis nana, which does not need an intermediate host and can cause autoinfection, is a particular public health hazard. anthelmintic treatment is effective (table ix) . diagnosis of hymenolepis is made by finding ova containing hexacanth embryos in the feces or adults in the lumen of the bowel. differential features are detailed else where (hsu, ) . d. insect infestation. two anopluran (sucking) lice infest wild rats, polyplax spinulosa (the spined rat louse) and hoplopleura pacifica (the tropical rat louse). the latter has not been reported among laboratory rats. polyplax spinulosa is now rare in most laboratory rat populations. it completes its entire life cycle within the fur of the host and is transmitted by direct contact. infested rats are unthrifty, irritable, pruritic, restless, and anemic. polyplax spinulosa can transmit a num ber of infectious agents, which include hemobartonella muris and t. lewisi. diagnosis is made by identifying organisms in the fur. rat fleas (xenopsylla sp., leptopsylla sp., and nosopsyllus sp.) are rare in laboratory rats, since their life cycles are usu ally interrupted by proper sanitation. fleas can transmit other agents and can serve as the intermediate host of h. nana and diminuta (flynn, ; hsu, ; oldstone, ) . see sec tion iii,a, ,e for methods of control. e. arachnid infestation. mesostigmate mites can be en countered on occasion in laboratory rats. they are rapid blood suckers and have a nonselective host range. they are on the host only during feeding, spending much of their life cycle secreted in the environment. diagnosis must be attained by finding engorged mites on bedding, cages, and crevices. other hosts, including humans, fall prey to their painful and irritating bites. the most important mesostigmate is ornithonyssus bacon (tropical rat mite). heavily infested colonies contain de bilitated, anemic rats with reduced reproductive efficiency and occasional deaths. liponyssoides sanguineus has not been re ported in laboratory rats, but may be unrecognized because of its similarity to ornithonyssus sp. laelaps echidninus (spiny rat mite) does not adapt well to the laboratory rat environment unless husbandry is poor. it is a vector of hepatozoon. prostigmate mites have a more selective host range and spend their life cycle in the fur (or follicles) of the host. radfor dia ensifera, the rat fur mite, can be common in some rat colonies. this mite produces few ill effects, but heavy infesta tions can induce self-inflicted trauma (flynn, ; hsu, ; oldstone, ) . demodex sp. is also encountered, but its prevalence is unknown, since it lives deep within hair follicles and sebaceous glands where it produces minimal lesions (walbtxgetal., ) . astigmate mites include the mange mites, which have a se lective host range and complete their life cycle on the host. notoedres muris, the ear mange mite, infests the skin of the ear and to a lesser extent, the nonglabrous regions of the body. this mite burrows in the cornified epithelium, and elicit a pruritic dermatitis (flynn, ; hsu, ) . it is seldom seen in contemporary rat colonies. control of ectoparasites is gained by preventing the introduc tion of infected animals (including wild rodents), sanitation, treatment of rats, and, in the case of mesostigmates and fleas, treatment of the environment (table ix) . treatment seldom completely eliminates infestation and can have an adverse ef fect on the usefulness of the treated rats for research. repopulation or caesarean rederivation and subsequent preven tion by proper management is the best approach. deep mycoses are generally not considered to be significant natural diseases in laboratory rats. pulmonary aspergillosis is occasionally observed in rats and mice, particularly when immunocompromised. aspergillus sp. can be readily isolated or observed in affected lung tissue with selected histochemical techniques. pulmonary lesions consist of miliary granulomata containing langhans giant cells in areas with characteristic uniformly branched, septate hyphae. phycomycotic encepha litis has been reported as a natural disease in -to -week-old rats. phycomycotic fungi have thick, irregularly branched, nonseptate hyphae in tissue. dermatomycosis is probably more likely to be encountered than deep mycosis, but is also rare in laboratory rats. ring worm in rats is restricted largely to infection by trichophyton mentagrophytes. infected rats can be lesionless carriers or dis play patchy alopecia and erythema with scale, papule, or pus tule formation. fungal elements are visible within the stratum corneum and hair follicles. ecothrix spore formation in hair shafts can be present. the disease can be diagnosed by exam ination of skin scrapings treated with % potassium hydrox ide, histological sections of skin prepared with fungal stains, or isolation (weisbroth, ) . a. genetic anomalies. genetic anomalies in coat color and character, organ development, immune responsiveness, obesity, hypertension, metabolism, and others have been iden tified and in many cases developed as specific characteristics of various stocks and strains. the reader is referred tö several more reviews for further information (altman and katz, ; hansen et al., ; robinson, robinson, , . some strains nat urally develop disease syndromes, such as brattleboro rats with diabetes insipidus, while other strains have less obvious characteristics but react uniquely to different research vari ables. the researcher must be aware of these variations and judiciously select the appropriate stock or strain to accomplish the goals of the experiment. each stock, strain, substrain, and even group of rats that has drifted from its parental stock can develop its own unique dis eases, manifest its own incidence and rate of development of various spontaneous lesions, and react in different ways to in fectious and research variables. expression of genetic traits is further influenced by extraneous factors such as husbandry and diet. b. nutritional deficiencies. more is known about the nu tritional requirements and deficiencies of rats than perhaps any other species. natural deficiencies of single dietary compo nents are rare and seldom recognized. this is because rats ef fectively store fat-soluble vitamins (and b ), manufacture vi tamin c, and fulfill much of their requirement for b vitamins by coprophagy. furthermore, some deficiencies are influenced or compensated for by other dietary elements. the most com mon signs of nutritional deficiency are vague, such as poor hair coat, reduced weight gain or growth, diminished fertility, and susceptibility to infectious disease. commercially avail able rodent diets effectively supply balanced diets to rodents, but diets should not be utilized beyond their expiration date and ideally should be kept in cold storage. several components deteriorate with prolonged storage, heating, or sterilization, in cluding lysine, vitamins a and e, and, to a lesser extent, riboflavin and thiamin. nutritional requirements vary with the genetic strain, stage of life, and microbiological association. axenic or specific pathogen-free (spf) animals can lack gut microflora necessary for supplying certain vitamins, particu larly vitamin k. they must receive supplemented diets both because they need more and because autoclaving or pasteuriza tion reduce the levels of certain essential nutrients. antibiotic treatment, which affects intestinal microflora, can reduce the bacterial source of vitamins by coprophagy. nutritional ade quacy goes far beyond the needs of the rat, since the composi tion of nutritionally adequate diets is known to profoundly in fluence the biological responsiveness to research manipula tion, infectious disease, and expression of spontaneous lesions (national research council, ; rogers, ) . the signs of nutritional deficiency are often vague and must be differentiated from other syndromes. hemorrhage due to hypovitaminosis k can be confused with rv disease (which in itself can be precipitated by nutritional deficiency). infertility or poor production can be caused by rv, sdav, sendai virus, m. pulmonis, or nuturitional deficiency. squamous metaplasia in the salivary and lacrimai glands of rats recovering from sdav or in the respiratory tract of rats infected with sendai virus or m. pulmonis must be differentiated from hypo vitaminosis a, which in turn predisposes to respiratory infec tions. environmental factors, such as excess heat or low hu midity, contribute to infertility or skin disease which mimic hypovitaminosis e or other deficiencies. ordinarily mild infec tious diseases can become severe, such as pseudotuberculosis, due to nutritional deficiency. the diagnosis of many rat dis eases therefore requires review of nutritional practices. practices other than nutrition can also influence or cause dis ease in rats. sanitation is of obvious import. cage design, pop ulation density, and temperature influences have been men tioned in sections ii,d and e. outbreaks of opportunistic in fectious disease, such as pseudotuberculosis, can be precipi tated by sudden fluctuations in temperature and humidity. pseudomonas aeruginosa can be introduced and spread within a colony through inappropriate sanitation, particularly of water bottles. refilling water bottles at a faucet in the animal room is not advised for this reason. bottles and sipper tubes should be sanitized and water should be acidified or chlorinated. exces sive ammonia from dirty bedding, overcrowded cages, or poor ventilation predisposes to respiratory infections, particularly mycoplasmosis. a number of management-related syndromes can occur that are not related to sanitation or infectious disease. rats adapt well to automatic watering systems, but animals unfamiliar with these devices can become dehydrated. similarly, mal function of these systems and blockage of water bottle sippe tubes with metal filings or other detritus can have a similar effect. rats maintained for long periods on wire-bottom cages develop foot sores, which can result in severe lesions, discom fort, hemorrhage, and anemia. aging rats must be examined periodically for overgrowth of their incisors, a very common problem in rats held long term. teeth must be carefully clip ped, avoiding splitting or shattering. dusty bedding and food can result in foreign body pneumonia. plant, mineral, and even bone fragments can be found in sections of lung obtained from rats under these circumstances. softwood shavings used as bedding can cause intestinal impaction, particularly in young rats. high ambient light, or even light levels within the recom mended range, can cause retinal degeneration and cataracts in albino rats. rats maintained near ceiling light fixtures develop retinal degeneration, while those near the floor are spared. low relative humidity (< %) in concert with high tempera ture and other factors can cause one or more annular constric tions of the tail skin (ringtail) or toes. the segments distal to the constrictions swell or undergo dry gangrene (fig. ) . this is most apt to occur in suckling or preweanling rats and in rats kept in wire-bottom cages. increasing humidity, reducing air turnover rate, placing rats in solid-bottom cages, and providing nesting material for nursing dams and their litters are all ame liorative for this condition. axenic rats normally have enlarged ceca, which become ap proximately five times normal size. there is net efflux of fluid into the lumen in response to increased osmotic pressure of luminal content, reduced availability of ions needed for mucosal solute-coupled water résorption and vasoactive com pounds which exert their effect on smooth muscle tone. cecal enlargement can interfere with reproduction. dietary manip ulation can reduce the enlargement substantially (foster, ) . the effects on smooth muscle tone become pronounced in aging axenic rats, which can develop progressive cecal en largement due to atony. volvulus and torsion can result. the reader must be cognizant of the much larger list of en- vironmental variables that significantly alter physiological re sponsiveness of test animals (baker et al., a; gralla, ) . unlike the mouse, traumatic skin disease in the rat is more likely to be due to self-inflicted trauma than fight wounds. rats infested with ectoparasites or dermatophytes can develop excoriative dermatitis due to self-inflicted trauma. roughly sym metrical ulcerative dermatitis over the dorsal and lateral neck and shoulders has been reported (fig. ) . the etiology re mains to be determined, but coagulase-positive staphylococcus aureus is consistently isolated from the skin lesions, and colonies of gram-positive cocci are microscopically visible on the surface of the lesion. attempts to induce the disease in other rats with this agent have yielded inconsistent results (fox et al, ) . the rat is often considered "resistant" to infection and ac cordingly submitted to a variety of experimental procedures without regard to aseptic techniques. peritonitis and wound in fections are often found under such conditions. inappropriate handling of large rats by the tips of their tails will cause the skin to tear and slip off when they struggle. adynamic ileus occurs in rats given intraperitoneal injections of anesthetic preparations containing chloral hydrate or related compounds (fig. ) . for variable periods up to weeks after treatment, rats become lethargic, anorectic, and constipated with distended abdomens that may lead to death. gross nec ropsy findings reveal segmental atony and distension of the bowel, usually jejunum, ileum, and cecum. focal serosal in flammatory changes and fibrosis are seen microscopically (fleischman et al., ) . this syndrome mimics megaloileitis (tyzzer's disease), but can be differentiated on the basis of history, lack of characteristic bacteria in tissues, and lack of liver and heart lesions. in.addition, it should not be confused with the bowel dilatation observed in axenic rats. space exists in this chapter to only summarize the more fre quently reported tumors in the rat, and the topic will not in clude experimentally induced tumors. the reader is encouraged to refer to the articles and monographs referenced in the text for more complete information. there have been numerous studies done to assess the inci dence of neoplasia in various stocks and strains of rats (burek, ; coleman et al., ; international agency for research on cancer, mackenzie and garner, ; ringler and dabich, ; sher, ) . some of these have led to the development of animal models and provided baseline data for experimental carcinogenesis work for which the rat is regularly used. there is a wide range of values reported in regard to the prevalence and type of tumors found in a particular rat stock or strain. these differences are a function of genetic and environ mental influences and differences in sample selection. nutri tion is well known as a factor that influences the development of neoplastic disease. for instance, it has been shown that a high fat diet enhances tumorigenesis, that the protein-calorie ratio influences the occurrence of some tumors, and that re stricted food intake of post weanlings for or more weeks de creases tumor risk (ross and bras, ) . most of the surveys on tumor occurrence in rats either state nothing in regard to the presence of enzootic infectious disease or acknowledge that infectious diseases were present in the population. in many instances, infectious diseases can signifi cantly influence data on tumor risk because of their effect on longevity, preneoplastic changes, and masking of small tumors. with the exception of mammary fibroadenomas in many stocks and testicular tumors in f rats, the prevalence of tumors is quite low in rats under months of age. according ly, the age at which rats are surveyed is very important in de fining the incidence of neoplasia in a particular stock or strain. the f rat is used in the bioassay program of the national cancer institute and is widely used in toxicology studies. it has been selected for these types of studies because of its small size, longevity, and low incidence of most tumors. however, this strain has a moderate to high incidence for some tumors, such as those of the testis, mammary gland, uterus, and hematopoietic and endocrine systems (sher, ) . a sprague-dawley-derived stock that is commonly used in carcinogenesis studies is the crl:cd(sd)br rat. this stock has a high inci dence of mammary tumors and pituitary adenomas. papillomas and squamous cell carcinomas occur infre quently, but have been reported in many stocks of rats. squam ous cell carcinomas are usually located on the face and head, and tend to be highly invasive but not metastatic. most of these arise as sebaceosquamous tumors from the glands of zymbal in the ear. similar tumors occur on the prepuce and clitoris. in females from most stocks and strains, the mammary gland is the most frequent site of neoplasia, with incidences of - % being common. these tumors occur in males but much less frequently. benign fibroadenomas are the most common type, but adenocarcinomas may also occur. fibroadenomas have ductal epithelium and periductular connective tissue compo nents and tend to be less vascular than carcinomas. adenocar cinomas can metastasize to regional lymph nodes and the.lung. tumors of the mammary gland, which may arise at any site from the neck to the inguinal region, often attain a very large size (fig. ) . the prevalence of tumors in the alimentary tract is extremely low, with most cases involving the colon. although hepatic tumors are readily induced by chemical carcinogens, very few spontaneously occurring malignant neoplasms have been re ported. more commonly occurring are neoplastic nodular le sions. the term ò 'neoplastic nodule" is used to describe cir cumscribed nodules of proliferating hepatocytes that compress the parenchyma (altman and goodman, ) . there is some fig. . mammary fibroadenoma in an aged rat. controversy as to whether these nodules are in all cases neo plastic. however, there is evidence that some develop into carcinomas. leukemia is rare in many stocks and strains of rats. howev er, mononuclear cell leukemia is very frequently seen in f and wf rats. in one survey of male f rats, an incidence of % proved leukemia second in occurrence only to the testic ular interstitial cell tumors. the leukocyte count in leukemic rats ranged from , to , cells/ìÀ with an average of , (coleman et al., ) . other studies indicate a simi lar incidence for the wf rat. in the bn/bi rat, myelomonocytic leukemia has been reported to be the most common lymphoreticular tumor, with female and male incidences of and %, respectively (burek, ) . both types of leukemia oc cur in rats over months of age. neoplasms of the pituitary occur frequently with a higher proportion in females. surveys have shown an incidence of - % in f rats, % in om (osborne-mendel) rats, and - % in sprague-dawley rats. the most common pituitary tumor is the chromophobe adenoma. these are benign tumors that frequently become quite large, and, due to compression of the brain by the tumor, hydrocephalus and neurological signs may occur. they appear dark due to hemorrhagic areas arising from a vascular sinusoid component of the tumor. clinical manifestations may include neurological signs or cachexia, but these are often absent. pancreatic islet cell tumors are common in some stocks and strains. surveys have reported a % incidence in f rats, . % in holtzman rats, and a - . % in sprague-dawley stocks. tumors of the thyroid are usually parafollicular cell ade nomas. these benign tumors occur in many stocks with an incidence varying between and %. follicular cell car cinomas, which occur infrequently, can metastasize to the lung. adenomas of the adrenal cortex are very common in several strains. in at least three strains, buf/n, m /n, and om/n, over % of the females and % of the males have cortical tumors. pheochromocytomas are the most common medullary tumors. they are particularly common in the buf/n, f /n, m /m, and wn/n strains, and, unlike cortical tu mors, they appear more often in males. tumors of the brain have been found to occur rarely in those studies that included examination of the cns. less is known about the prevalence of tumors in the cns than in other sys tems because many studies have not included brain and spinal cord examination. the astrocytoma is the most commonly re ported tumor of the cns. less often reported tumors include oligodendroglioma, meningioma, ependymoma, and granular cell tumors. primary tumors of the lung are rare in the rat. the most fre quently reported types are bronchogenic carcinomas, car cinomas, sarcomas, and adenomas. tumors of the bladder are rare in most stocks and strains of rats. however, the bn/bi strain has an unusually high inci dence of carcinomas of the bladder and ureter. males of this strain have been reported to have a % incidence of bladder carcinoma, while % of females have carcinoma of the ureter (burek, ) . nephroblastomas, renal tubular adenomas, and adenocarcinomas have been reported in the rat kidney. tumors of the prostate are common in the axc rat. in one report, adenocarcinoma of the ventral prostate was histologically detected in % of -to -month-old axc rats. prostatic tumors are reportedly uncommon in most stocks and strains of rats. reportedly, however, some of these surveys did not include adequate sectioning to locate small adenomas and carcinomas. interstitial cell tumors are the most common testicular tumor type. nearly all aged f rats and the majority of aci/n rats develop these tumors, but the tumor is rare in most other strains. tumors of the vagina and cervix are rare, except in aged bn/bi rats. tumors of the uterus and ovary are common in several strains of rats. one om strain was reported to have a % incidence of granulosa cell tumors, and a high incidence of endometrial tumors has been reported in the f and m strains. congenital disorders in the rat are infrequently reported. this is probably a reflection of two factors; the first being that evidence indicates the incidence is extremely low in outbred stocks, and the second being that commercial/institutional sup pliers select against breeders who have produced abnormal young. the genitourinary system and the eye have been most associated with congenital disorders. one substrain of the axc rat has a greater than % inci dence of unilateral renal agenesis and hydronephrosis (fujikura, ) . a high incidence of unilateral and bilateral hy dronephrosis has been found in bn/bi rats (cohen et ai, ) . pseudohermaphroditism has been reported in rats that are phenotypically female but who have an xy karyotype. testicles are present either in the abdomen or inguinal canal. this condition is due to a sex-linked recessive gene that is expressed by an insensitivity of tissues to androgens (bardin et al., ) . a number of ocular disorders have been reported in the rat. retinal dystrophy is inherited as a single autosomal recessive gene (rdy). homozygotes have a progressive loss of the retinal photoreceptor cells and an overproduction of rhodopsin early in the disease process (lavail et al., ) . a retinal degener ation reported in the wag/rij rat appears to be an expression of an autosomal dominant gene. end-stage lesions include disap pearance of photoreceptor cells, migration of the pigment epi thelium, and disorganization of remaining retinal layers. this retinal disease appears to mimic the pathogenesis of retinitis pigmentosa in humans (lai et al., ) . other reported devel opmental disorders of the eye include colobomas and cataracts. reports of congenital heart disease are rare. among the few reports is one involving an inbred strain of long-evans rats (fox, ) . in this strain, approximately % of the neonates have interventricular septal defects. this malformation is be lieved to be of polygenic origin. unlike the mouse, there are few reported mutants that have disorders of the central nervous system. recently, a partially inbred strain has been developed in which one subline has nearly a % incidence of hydrocephalus (kohn et al., ) . the mode of inheritance appears to be polygenic. the hydrocephalus is classified as communicating and the pathogenesis may be due to poorly developed veins in the periosteal and durai layers, and to underdeveloped pia-arachnoid cells. neoplastic and nonneoplastic lesions vary in type, onset, and prevalence due to the hereditary and environmental factors as sociated with a specific colony of laboratory rat. the rat is widely used in gerontological research. those who use the rat must be clearly aware of the factors that can influence lesions and death in aged rats (anver and cohen, ; burek, ) . neoplastic disease, a cause of death in many aged rats, has been discussed previously, and this section will summarize the more commonly seen nonneoplastic lesions in aging rats. a. chronic progressive nephropathy (cpn). chronic pro gressive nephropathy is among the most commonly encoun tered lesions in the rat, particularly in the aged animal where it is a major life-limiting disease. because of its complex nature, cpn has a large number of synonyms, often with inaccurate descriptive titles. sequential development of lesions and fac tors that modify the course of cpn have been thoroughly stud ied, but the actual pathogenesis remains undetermined. chron ic progressive nephropathy occurs in most rats, but its rate of development is significantly influenced by numerous factors, including sex, genetics, age, diet, association with microflora, hormone treatment (particularly testosterone), and others. male rats have an earlier onset and more rapid progression of cpn than females, and albino strains and stocks seem to be more predisposed. diet is an extremely important predisposing factor, particularly the quality and quantity of dietary protein. significant differences in severity of cpn can be observed among rats of the same sex, strain, age, and source when fed different diets for their lifetime. axenic rats do not seem to develop significant cpn. rats with cpn develop progressively severe proteinuria, first with the selective excretion of only small molecules such as albumin. in late cpn there is nonselective excretion, and the electrophoretic pattern of urinary protein resembles that of serum. proteinuria related to cpn should not be confused with the normal urinary excretion of a-globulin in male rats, proba bly of tubular origin. the histopathology of cpn is charac terized as progressive basement membrane thickening of the glomerulus, bowman's capsule, and proximal tubule. base ment membranes split and wrinkle as tubules become atrophie and collapse. glomeruli enlarge, with mesangial deposition of protein and lipid, adhesion to bowman's capsule, and segmen tai sclerosis. tubular epithelium may contain granules of resorbed protein, which later is mixed with lipofuscin and hemosiderin. as glomerular protein leakage becomes more severe, tubules dilate and accumulate hyaline eosinophilic castes of protein within their lumina, resembling colloid. tubular epi thelium may undergo atrophy with collapse or dilatation. in terstitial fibrosis and leukocytic infiltration is frequently ob served. grossly, the kidneys become enlarged, pale with irregular surfaces and pigmented (fig. ) . cystic tubules can be observed. hypoproteinemia, parathyroid hyperplasia with serum chemical changes indicative of hyperparathyroidism, and nephrotic syndrome are observed in rats with severe cpn (barthold, ) . b. nephrocalcinosis. this disease, which occurs more fre quently in females, has been reported in numerous strains of rats. in most cases, affected rats have been fed purified diets (e.g., . renal lesions usually involve the corticomedullary junction and consist of mineral deposition in the lumina of the proximal tubules (nguyen and woodward, ) . e. polyarteritis nodosa. this disease is of unknown etiol ogy and affects muscular arteries, most commonly the mesenteric, pancreatic, and spermatic. the lesions may be either acute or chronic. in the acute stage, there is intimai and medial fibrinoid necrosis, focal thrombosis, disruption of the elastic lamellae, and an infiltration of polymorphonuclear leukocytes and mononuclear cells. in the chronic stage, the arteries are nodular, tortuous, and thick-walled. affected vessels fre quently have aneurysms and thrombi. both acute and chronic phases may be observed in the same artery in an animal. d. myocardial degeneration. degeneration is a commonly observed lesion that occurs in most stocks and strains, with an onset at - months of age. it occurs more frequently in males. the lesions are usually microscopic but, in advanced stages, can appear grossly as grayish foci. the papillary mus cles and their attachment sites in the wall of the left ventricle are the most frequent sites of the lesion (anver and cohen, ) . e. radiculoneuropathy. spinal nerve root degeneration has been reported in a number of rat stocks and strains. lesion distribution may vary among strains; however, the cauda equi na and ventral spinal nerve roots are commonly involved. pos terior paresis and paralysis have been associated with these lesions, but some suggest that these signs are associated with a separate degenerative process of the skeletal muscle (anver and cohen, ) . an enzyme linked immunosorbent assay for detection of antibodies to corynehacterium kutschen in experimentally infected rats copulatory behavior can inhibit preg nancy in female rats neoplastic diseases inbred and genetically defined strains of laboratory animals. part . mouse and rat rat-bite fever in animal research laboratory personnel muroid virus nephropathies lesions associated with aging reproduction and breeding housing to contro! research variables selected norma tive data. appendix i pseudohermaphroditic rat: end organ insensitivity to testoster one chronic progressive nephropathy in aging rats research complications and state of knowledge of rodent coronaviruses reproduction and breeding techniques for laboratory animals morphophysiology sendai virus infection in genetically resistant and susceptible mice observa tions on enzootic paratyphoid infection in a rat colony age-associated pathology a comparison in germ-free mice of the pathogenesis of sendai virus and pneumonia virus infection my coplasmal and rickettsial diseases detection of natural mycoplasma pulmonis infection in rats and mice by an enzyme-linked immunosorbent assay (elisa) adherence and colonization of mycoplasma pulmonis to genital epi thelium and spermatozoa in rats tissue weights of the rat. i. normal values determined by dissection and chemical methods immunopathology. in "cell pathology heritable hydronephrosis in a mutant strain of brown norway rats pathological changes during aging in barrier-reared fischer male rats naturally occurring lethal parvovirus infection of juvenile and young-adult rats breeding of the rat adynamic ileus in the rat induced by chloral hydrate parasites of laboratory animals gnotobiology ulcera tive dermatitis in the rat corynebacterium kutschen pneumonia in rats on long term tobacco inhalation studies evidence suggesting a multigenic origin of membranous septal defect in rats kidney malformations in fetuses of axc line rats bibli ography of hemorrhagic fever with renal syndrome (muroid virus nephropathies) effect of heat and selected chemical disinfectants upon infectivity of spores of bacillus piliformis (tyzzers disease) studies on adjuvantinduced arthritis, tumor transplantability and serologie response to bovine serum albumin in sendai virus-infected rats scientific considerations in monitoring and evaluating toxicological research nih rodents catalogue anatomy of the laboratory rat parasitic diseases viral diseases. /// "the laboratory rat tyzzer's disease in the rat pathogenicity of mycoplasma pulmonis in laboratory rats a new model of congenital hydrocephalus in the rat morphological evi dence for natural poxvirus infection in rats animal model: hereditary retinal degeneration in wag/rij rats the ufaw handbook on the care and management of laboratory animals photoreceptor pig ment epithelial cell relationships in rats with inherited retinal degenera tion comparison of techniques for primary isolation of respiratory mycoplasma pulmonis from rats historical foundations murine chronic respiratory disease. significance as a research complication and experimental production with mycoplasma pulmonis comparison of neoplasms in six sources of rats a filamentous bac terium associated with respiratory disease in wild rats salmonella enterocolitis in the rat clinical biochemical and hematological reference values in normal experimental animals intranephronic calculosis in rats helminths, ectoparasites and protozoa in rats and mice hematology and clinical biochemistry genetics of the norway rat nutrition lasting influence of early caloric re striction on prevalence of neoplasms in the rat tumors in control hamsters, rats, and mice: literature tabulation molecular biology of mammalian sarcoma viruses state of the art detection methods for rodent viruses the microscopic anatomy of the white rat asymptomatic infection of mouse hepatitis in the rat histological and serological response of b c f, mice and f rats to experimental pneumonia virus of mice infection demodicidosis in laboratory rats (rattus norvegicus) latent adenoviral infection of rats: intranuclear inclusions induced by treatment with a cancer chemotherapeutic agent the role of mycoplasmas and l forms of bacteria in disease bacterial and mycotic diseases respiratory disease in a colony of rats. ii. isolation of mycoplasma pulmonis from the natural disease, and the experimental disease induced with a cloned culture of this organism craigie's neuroanatomy of the rat key: cord- - u w r authors: verma, anju; verma, megha; singh, anchal title: animal tissue culture principles and applications date: - - journal: animal biotechnology doi: . /b - - - - . - sha: doc_id: cord_uid: u w r animal cell culture technology in today’s scenario has become indispensable in the field of life sciences, which provides a basis to study regulation, proliferation, and differentiation and to perform genetic manipulation. it requires specific technical skills to carry out successfully. this chapter describes the essential techniques of animal cell culture as well as its applications. cell culture is the process by which human, animal, or insect cells are grown in a favorable artificial environment. the cells may be derived from multicellular eukaryotes, already established cell lines or established cell strains. in the mid- s, animal cell culture became a common laboratory technique, but the concept of maintaining live cell lines separated from their original tissue source was discovered in the th century. animal cell culture is now one of the major tools used in the life sciences in areas of research that have a potential for economic value and commercialization. the development of basic culture media has enabled scientists to work with a wide variety of cells under controlled conditions; this has played an important role in advancing our understanding of cell growth and differentiation, identification of growth factors, and understanding of mechanisms underlying the normal functions of various cell types. new technologies have also been applied to investigate high cell density bioreactor and culture conditions. many products of biotechnology (such as viral vaccines) are fundamentally dependent on mass culturing of animal cell lines. although many simpler proteins are being produced using rdna in bacterial cultures, more complex proteins that are glycosylated (carbohydrate-modified) currently have to be made in animal cells. at present, cell culture research is aimed at investigating the influence of culture conditions on viability, productivity, and the constancy of posttranslational modifications such as glycosylation, which are important for the biological activity of recombinant proteins. biologicals produced by recombinant dna (rdna) technology in animal cell cultures include anticancer agents, enzymes, immunobiologicals [interleukins, lymphokines, monoclonal antibodies (mabs)], and hormones. animal cell culture has found use in diverse areas, from basic to advanced research. it has provided a model system for various research efforts: . the study of basic cell biology, cell cycle mechanisms, specialized cell function, cellÀcell and cellÀmatrix interactions. . toxicity testing to study the effects of new drugs. . gene therapy for replacing nonfunctional genes with functional gene-carrying cells. . the characterization of cancer cells, the role of various chemicals, viruses, and radiation in cancer cells. . production of vaccines, mabs, and pharmaceutical drugs. . production of viruses for use in vaccine production (e.g., chicken pox, polio, rabies, hepatitis b, and measles). today, mammalian cell culture is a prerequisite for manufacturing biological therapeutics such as hormones, antibodies, interferons, clotting factors, and vaccines. the first mammalian cell cultures date back to the early th century. the cultures were originally created to study the development of cell cultures and normal physiological events such as nerve development. ross harrison in showed the first nerve fiber growth in vitro. however, it was in the s that animal cell culture was performed at an industrial scale. it was with major epidemics of polio in the s and s and the accompanying requirement for viral vaccines that the need for cell cultures on a large scale became apparent. the polio vaccine from a de-activated virus became one of the first commercial products developed from cultured animal cells (table . ). tissue culture is in vitro maintenance and propagation of isolated cells tissues or organs in an appropriate artificial environment. many animal cells can be induced to grow outside of their organ or tissue of origin under defined conditions when supplemented with a medium containing nutrients and growth factors. for in vitro growth of cells, the culture conditions may not mimic in vivo conditions with respect to temperature, ph, co , o , osmolality, and nutrition. in addition, the cultured cells require sterile conditions along with a steady supply of nutrients for growth and sophisticated incubation conditions. an important factor influencing the growth of cells in culture medium is the medium itself. at present, animal cells are cultured in natural media or artificial media depending on the needs of the experiment. the culture medium is the most important and essential step in animal tissue culture. this depends on the type of cells that need to be cultured for the purpose of cell growth differentiation or production of designed pharmaceutical products. in addition, serum-containing and serum-free media are now available that offer a varying degree of advantage to the cell culture. sterile conditions are important in the development of cell lines. cells from a wide range of different tissues and organisms are now grown in the lab. earlier, the major purpose of cell culture was to study the growth, the requirements for growth, the cell cycle, and the cell itself. at present, homogenous cultures obtained from primary cell cultures are useful tools to study the origin and biology of the cells. organotypic and histotypic cultures that mimic the respective organs/tissues have been useful for the production of artificial tissues. there are three methods commonly used to initiate a culture from animals. whole organs from embryos or partial adult organs are used to initiate organ culture in vitro. these cells in the organ culture maintain their differentiated character, their functional activity, and also retain their in vivo architecture. they do not grow rapidly, and cell proliferation is limited to the periphery of the explant. as these cultures cannot be propagated for long periods, a fresh explanation is required for every experiment that leads to interexperimental variation in terms of reproducibility and homogeneity. organ culture is useful for studying functional properties of cells (production of hormones) and for examining the effects of external agents (such as drugs and other micro or macro molecules) and products on other organs that are anatomically placed apart in vivo. fragments exercised from animal tissue may be maintained in a number of different ways. the tissue adheres to the surface aided by an extracellular matrix (ecm) constituent, such as collagen or a plasma clot, and it can even happen spontaneously. this gives rise to cells migrating from the periphery of the explant. this culture is known as a primary explant, and migrating cells are known as outgrowth. this has been used to analyze the growth characteristics of cancer cells in comparison to their normal counterparts, especially with reference to altered growth patterns and cell morphology. cell culture this is the most commonly used method of tissue culture and is generated by collecting the cells growing out of explants or dispersed cell suspensions (floating free in culture medium). cells obtained either by enzymatic treatment or by mechanical means are cultured as adherent monolayers on solid substrate. cell culture is of three types: ( ) precursor cell culture, which is undifferentiated cells committed to differentiate; ( ) differentiated cell culture, which is completely differentiated cells that have lost the capacity to further differentiate; and ( ) stem cell culture, which is undifferentiated cells that go on to develop into any type of cell. cells with a defined cell type and characteristics are selected from a culture by cloning or by other methods; this cell line becomes a cell strain. the monolayer culture is an anchorage-dependent culture of usually one cell in thickness with a continuous layer of cells at the bottom of the culture vessel. some of the cells are nonadhesive and can be mechanically kept in suspension, unlike most cells that grow as monolayers (e.g., cells of leukemia). this offers numerous advantages in the propagation of cells. passaging is the process of subculturing cells in order to produce a large number of cells from preexisting ones. subculturing produces a more homogeneous cell line and avoids the senescence associated with prolonged high cell density. splitting cells involves transferring a small number of cells into each new vessel. after subculturing, cells may be propagated, characterized, and stored. adherent cell cultures need to be detached from the surface of the tissue culture flasks or dishes using proteins. proteins secreted by the cells form a tight bridge between the cell and the surface. a mixture of trypsin-edta is used to break proteins at specific places. trypsin is either protein-degrading or proteolytic; it hydrolyzes pepsindigested peptides by hydrolysis of peptide bonds. edta sequesters certain metal ions that can inhibit trypsin activity, and thus enhances the efficacy of trypsin. the trypsinization process and procedure to remove adherent cells is given in flowchart . . quantitation is carried out to characterize cell growth and to establish reproducible culture conditions. cell counts are important for monitoring growth rates as well as for setting up new cultures with known cell numbers. the most widely used type of counting chamber is called a hemocytometer. it is used to estimate cell number. the concentration of cells in suspension is determined by placing the cells in an optically clear chamber under a microscope. the cell number within a defined area of known depth is counted, and the cell concentration is determined from the count. for high-throughput work, electronic cell counters are used to determine the concentration of each sample. in some cases, the dna content or the protein concentration needs to be determined instead of the number of cells. cells propagated as a cell suspension or monolayer offer many advantages but lack the potential for cellto-cell interaction and cellÀmatrix interaction seen in organ cultures. for this reason, many culture methods that start with a dispersed population of cells encourage the arrangement of these cells into organ-like structures. these types of cultures can be divided into two basic types. cellÀcell interactions similar to tissue-like densities can be attained by the use of an appropriate ecm and soluble factors and by growing cell cultures to high cell densities. this can be achieved by (a) growing cells in a relatively large reservoir with adequate medium fitted with a filter where the cells are crowded; (b) growing the cells at high concentrations on agar or agarose or as stirred aggregates (spheroids); and (c) growing cells on the outer surface of hollow fibers where the cells are seeded on the outer surface and medium is pumped through the fibers from a reservoir. to simulate heterotypic cell interactions in addition to homotypic cell interactions, cells of differentiated lineages are re-combined. co-culturing of epithelial and fibroblast cell clones from the mammary gland allows the cells to differentiate functionality under the correct hormonal environment, thus producing milk proteins. primary cell culture these cells are obtained directly from tissues and organs by mechanical or chemical disintegration or by enzymatic digestion. these cells are induced to grow in suitable glass or plastic containers with complex media. these cultures usually have a low growth rate and are heterogeneous; however, they are still preferred over cell lines as these are more representative of the cell types in the tissues from which they are derived. the morphological structure of cells in culture is of various types: ( ) epithelium type, which are polygonal in shape and appear flattened as they are attached to a substrate and form a continuous thin layer (i.e., monolayer on solid surfaces); ( ) epitheloid type, which have a round outline and do not form sheets like epithelial cells and do not attach to the substrate; ( ) fibroblast type, which are angular in shape and elongated and form an open network of cells rather than tightly packed cells, are bipolar or multipolar, and attach to the substrate; and ( ) connective tissue type, which are derived from fibrous tissue, cartilage, and bone, and are characterized by a large amount of fibrous and amorphous extracellular materials. these cultures represent the best experimental models for in vivo studies. they share the same karyotype as the parent and express characteristics that are not seen in cultured cells. however, they are difficult to obtain and have limited lifespans. potential contamination by viruses and bacteria is also a major disadvantage. depending on the kind of cells in culture, the primary cell culture can also be divided into two types. these cells require a stable nontoxic and biologically inert surface for attachment and growth and are difficult to grow as cell suspensions. mouse fibroblast sto cells are anchorage cells. these cells do not require a solid surface for attachment or growth. cells can be grown continuously in liquid media. the source of cells is the governing factor for suspension cells. blood cells are vascular in nature and are suspended in plasma and these cells can be very easily established in suspension cultures. when primary cell cultures are passaged or subcultured and grown for a long period of time in fresh medium, they form secondary cultures and are longlasting (unlike cells of primary cell cultures) due to the availability of fresh nutrients at regular intervals. the passaging or subculturing is carried out by enzymatic digestion of adherent cells. this is followed by washing and re-suspending of the required amount of cells in appropriate volumes of growth media. secondary cell cultures are preferred as these are easy to grow and are readily available; they have been useful in virological, immunological, and toxicological research. this type of culture is useful for obtaining a large population of similar cells and can be transformed to grow indefinitely. these cell cultures maintain their cellular characteristics. the major disadvantage of this system is that the cells have a tendency to differentiate over a period of time in culture and generate aberrant cells. the primary culture, when subcultured, becomes a cell line or cell strain that can be finite or continuous, depending on its lifespan in culture. they are grouped into two types on the basis of the lifespan of the culture. cell lines with a limited number of cell generations and growth are called finite cell lines. the cells are slow growing ( À hours). these cells are characterized by anchorage dependence and density limitation. cell lines obtained from in vitro transformed cell lines or cancerous cells are indefinite cell lines and can be grown in monolayer or suspension form. these cells divide rapidly with a generation time of À hours and have a potential to be subcultured indefinitely. the cell lines may exhibit aneuploidy (bhat, ) or heteroploidy due to an altered chromosome number. immortalized cell lines are transformed cells with altered growth properties. hela cells are an example of an immortal cell line. these are human epithelial cells obtained from fatal cervical carcinoma transformed by human papilloma virus (hpv ). indefinite cell lines are easy to manipulate and maintain. however, these cell lines have a tendency to change over a period of time. nowadays, for the production of biologically active substances on an industrial scale, a mammalian cell culture is a prerequisite. with advancements in animal cell culture technology, a number of cell lines have evolved and are used for vaccine production, therapeutic proteins, pharmaceutical agents, and anticancerous agents. for the production of cell lines, human, animal, or insect cells may be used. cell lines that are able to grow in suspension are preferred as they have a faster growth rate. chinese hamster ovary (cho) is the most commonly used mammalian cell line. when selecting a cell line, a number of general parameters must be considered, such as growth characteristics, population doubling time, saturation density, plating efficiency, growth fraction, and the ability to grow in suspension. advantages of continuous cell lines . continuous cell lines show faster cell growth and achieve higher cell densities in culture. . serum-free and protein-free media for widely used cell lines may be available in the market. . the cell lines have a potential to be cultured in suspension in large-scale bioreactors. the major disadvantages of these cultures are chromosomal instability, phenotypic variation in relation to the donor tissue, and a change in specific and characteristic tissue markers (freshney, ) . the cells in the culture show a characteristic growth pattern, lag phase, exponential or log phase, followed by a plateau phase. the population doubling time of the cells can be calculated during the log phase and plateau phase. this is critical and can be used to quantify the response of the cells to different culture conditions for changes in nutrient concentration and effects of hormonal or toxic components. the population doubling time describes the cell division rates within the culture and is influenced by nongrowing and dying cells. the population doubling time, lag time, and saturation density of a particular cell line can be established and characterized for a particular cell type. a growth curve consists of a normal culture and can be divided into a lag phase, log phase, and plateau phase. this is the initial growth phase of the subculture and re-seeding during which the cell population takes time to recover. the cell number remains relatively constant prior to rapid growth. during this phase, the cell replaces elements of the glycocalyx lost during trypsinization, attaches to the substrate, and spreads out. during the spreading process, the cytoskeleton reappears; its reappearance is probably an integral part of the process. this is a period of exponential increase in cell number and growth of the cell population due to continuous division. the length of the log phase depends on the initial seeding density, the growth rate of the cells, and the density at which cell proliferation is inhibited by density. this phase represents the most reproducible form of the culture as the growth fraction and viability is high (usually %À %), and the population is at its most uniform. however, the cell culture may not be synchronized, and the cells can be randomly distributed in the cell cycle. the culture becomes confluent at the end of the log phase as growth rates during this phase are reduced, and cell proliferation can cease in some cases due to exhaustion. the cells are in contact with surrounding cells, and the growth surface is occupied. at this stage, the culture enters the stationary phase and the growth fraction falls to between % and %. also, the constitution and charge of the cell surface may be changed, and there may be a relative increase in the synthesis of specialized versus structural proteins. the animal cell culture can be grown for a wide variety of cell-based assays to investigate morphology, protein expression, cell growth, differentiation, apoptosis, and toxicity in different environments. product yields can be increased if monitoring of cell growth is managed properly. a number of factors affect the maximum growth of cells in a batch reactor. regular observation of cells in culture helps monitor cell health and the stage of growth; small changes in ph, temperature, humidity, o , co , dissolved nutrients, etc., could have an impact on cell growth. monitoring the rate of growth continuously also provides a record that the cells have reached their maximum density within a given time frame. animal cell cultures show specific characteristics and differ from microbial cultures. the important characteristics of the animal cell are slow growth rate, requirement of solid substrata for anchoragedependent cells, lack of a cell wall (which leads to zf and ab cells embryonic fibroblast cells zebrafish fragility), and sensitivity to physiochemical conditions such as ph, co levels, etc. some of the fundamental bioprocess variables are as follows: temperature is one of the most fundamental variables as it directly interferes with the growth and production processes. on a small scale, thermostatically controlled incubators can be used to control temperature. however, cell cultures grown on a large scale in bioreactors require more sensitive control of temperature. different bioreactors use different methods to maintain the temperature of the cell culture. temperature in a bioreactor is maintained by a heat blanket and water jacket with a temperature sensor. ph ph of the culture medium can be controlled by adding alkali (naoh, koh) or acid (hcl) solution. addition of co gas to the bioreactor, buffering with sodium bicarbonate, or use of naturally buffering solutes help maintain the ph of the culture. a silver chloride electrochemical-type ph electrode is the most commonly used electrode in the bioreactor. dissolved oxygen is the most fundamental variable that needs to be continuously supplied to the cell culture medium. it is consumed with a carbon source in aerobic cultures (moore et al., ) . diffusion through a liquid surface or membranes is one of the methods for providing dissolved oxygen to the medium. the number of viable cells in the culture provides an accurate indication of the health of the cell culture (stacey and davis, ) . trypan blue and erythrosin b determine cell viability through the loss of cellular membrane integrity. both these dyes are unable to penetrate the cell membrane when the membrane is intact, but are taken up and retained by dead cells (which lack an intact membrane). erythrosin b stain is preferred over trypan blue as it generates more accurate results with fewer false negatives and false positives. the toxic chemicals in the culture medium affect the basic functions of cells. the cytotoxicity effect can lead to the death of the cells or alterations in their metabolism. methods to access viable cell number and cell proliferation rapidly and accurately is the important requirement in many experimental situations that involve in vitro and in vivo studies. the cell number determination can be useful for determining the growth factor activity, concentration of toxic compound, drug screening, duration of exposure, change in colony size, carcinogenic effects of chemical compounds, and effects of solvents (such as ethanol, propylene, etc.). the assays to measure viable cells (viability assays) are as follows: bromide] (mtt)/mts/resazurin assay. . protease marker assay. . atp assay. the mtt assay allows simple, accurate, and reliable counting of metabolically active cells based on the conversion of pale yellow tetrazolium mtt. nicotinamide adenine dinucleotide in metabolically active viable cells reduces tetrazolium compounds into brightly colored formazan products or reduces resazurin into fluorescent resorufin (fig. . ). mtt and resazurin assays are widely used, as they are inexpensive and can be used with all cell types. the protease marker assay utilizes the cell-permeant protease substrate glycylphenylalanyl-aminofluorocoumarin (gf-afc). the substrate, which lacks an aminoterminal blocking moiety, is processed by aminopeptidases within the cytoplasm to release afc. the amount of afc released is proportional to the viable cell number. this assay has better sensitivity than resuzurin and the cells remain viable; thus, multiplexing is possible. the atp assay is the most sensitive cell viability assay. it is figure . schematic summary of biochemical events in different viability assays. measured using the beetle luciferase reaction to generate light. the mtt assay and procedure is given in flowchart . . assays to detect dead cells are as follows: the viable cells in culture have intact outer membranes. loss of membrane integrity defines a "dead" cell. the dead cells can be detected by measuring the activity of marker enzymes that leak out of dead cells into the culture medium or by staining the cytoplasmic or nuclear content by vital dyes that can only enter dead cells. ldh is an enzyme that is present in all cell types. it catalyzes the oxidation of lactate to pyruvate in the presence of co-enzyme nad . in the damaged cells, ldh is rapidly released. the amount of released ldh is used to assess cell death (fig. . ). this assay is widely used but has limited sensitivity as half-life of ldh at c is hours. the protease release assay is based on the intracellular release of proteases from the dead/compromised cell into the culture medium. the released proteases cleave the substrate to liberate aminoluciferin, which serves as a substrate for luciferase ( fig. . ) and leads to the production of a "glowtype" signal (cho et al., ). hayflick limit or hayflick's phenomena is defined as the number of times a normal cell population divides before entering the senescence phase. macfarlane burnet coined the term "c limit" in . hayflick and moorhead ( ) demonstrated that a population of normal human fetal cells divide in culture between and times before stopping. there appears to be a correlation between the maximum number of passages and aging. this phenomenon is related to telomere length. repeated mitosis leads to shortening of the telomeres on the dna of the cell. telomere shortening in humans eventually makes cell division impossible, and correlates with aging. this explains the decrease in passaging of cells harvested from older individuals. one of the most important factors in animal cell culture is the medium composition. in vitro growth and maintenance of animal cells require appropriate nutritional, hormonal, and stromal factors that resemble their milieu in vivo as closely as possible. important environmental factors are the medium in which the cells are surrounded, the substratum upon which the cells grow, temperature, oxygen and carbon dioxide concentration, ph, and osmolality. in addition, the cell requires chemical substances that cannot be synthesized by the cells themselves. any successful medium is composed of isotonic, low-molecular-weight compounds known as basal medium and provides inorganic salts, an energy source, amino acids, and various supplements. the basic components that make up most of the animal cell culture media are as follows: inorganic salts (ca , mg , na , k ), nitrogen source (amino acids), energy sources (glucose, fructose), vitamins, fat and fat soluble component (fatty acids, cholesterols), nucleic acid precursors, growth factors and hormones, antibiotics, ph and buffering systems, and oxygen and carbon dioxide concentrations. complete formulation of media that supports growth and maintenance of a mammalian cell culture is very complex. for this reason, the first culture medium used for cell culture was based on biological fluids such as plasma, lymph serum, and embryonic extracts. the nutritional requirements of cells can vary at different stages of the culture cycle. different cell types have highly specific requirements, and the most suitable medium for each cell type must be determined experimentally. media may be classified into two categories: ( ) natural media and ( ) artificial media. natural media consist of naturally occurring biological fluids sufficient for the growth and proliferation of animals cells and tissues. this media useful for promoting cell growth are of the following three types: . coagulant or clots: plasma separated from heparinized blood from chickens or other animals is commercially available in the form of liquid plasma. . biological fluids: this includes body fluids such as plasma, serum lymph, amniotic fluid, pleural fluid, insect hemolymph, and fetal calf serum. these fluids are used as cell culture media after testing for toxicity and sterility. . tissue extract: extracts of liver, spleen, bone marrow, and leucocytes are used as cell culture media. chicken embryo extract is the most common tissue extract used in some culture media. the media contains partly or fully defined components that are prepared artificially by adding several nutrients (organic and inorganic). it contains a balanced salt solution with specific ph and osmotic pressure designed for immediate survival of cells. artificial media supplemented with serum or with suitable formulations of organic compounds supports prolonged survival of the cell culture. the artificial media may be grouped into the following four classes: serum-containing media, serum-free media, chemically defined media, and protein-free media. the clear yellowish fluid obtained after fibrin and cells are removed from blood is known as serum. it is an undefined media supplement of extremely complex mixture of small and large molecules and contains amino acids, growth factors, vitamins, proteins, hormones, lipids, and minerals, among other components (table . ). advantages of serum in cell culture medium . it has basic nutrients present either in soluble or in protein-bound form. transferrin. insulin is essential for the growth of nearly all cells in culture and transferrin acts as an iron binder. . it contains numerous growth factors such as platelet-derived growth factor (pdgf), transforming growth factor beta (tgf-b), epidermal growth factor (egf), and chondronectin. these factors stimulate cell growth and support specialized functions of cells. . it supplies protein, which helps in the attachment of cells to the culture surface (e.g., fibronectin). . it provides binding proteins such as albumin and transferrin, which helps transport molecules in cells. . it provides minerals such as ca , mg , fe , k na , zn , etc., which promote cell attachment. . it increases the viscosity of the medium, which provides protection against mechanical damage during agitation and aeration of suspension cultures. . it provides appropriate osmotic pressure. disadvantages of serum-containing medium . expensive: fetal calf serum is expensive and difficult to obtain in large quantities. and there is no uniformity in composition of serum. this can affect growth and yields and can give inconsistent results. . contamination: serum medium carries a high risk of contamination with virus, fungi, and mycoplasma. the serum itself may be cytotoxic and may contain inhibiting factors, which in turn may inhibit cultured cell growth and proliferation. the enzyme polyamine oxidase in serum reacts with polyamines such as spermine and spermidine to form cytotoxic polyamino-aldehyde. . downstream processing: the presence of serum in culture media may interfere with isolation and purification of culture products. additional steps may be required to isolate cell culture products. the use of serum in culture media presents a safety hazard and source of unwanted contamination for the production of biopharmaceuticals. as a number of cell lines can be grown in serum-free media supplemented with certain components of bovine fetal serum, the development of this type of medium with a defined composition has intensified in the last few decades. eagle ( ) developed a "minimal essential medium" composed of balanced salts, glucose, amino acids, and vitamins. in the last years, considerable work has been carried out to develop more efficient culture media to meet the specific requirements of specific cell lines. advantages of serum-free culture media . serum-free media are simplified, and the composition is better defined. . they can be designed specifically for a cell type. it is possible to create different media and to switch from growth-enhancing media to differentiationinducing media by altering the combination and types of growth factors and inducers. . they decrease variability from batch to batch and improve reproduction between cultures. . downstream processing of products from cell cultures in serum-free media is easier. . they reduce the risk of microbial contamination (mycoplasma, viruses, and prions). . serum-free media are easily available and ready to use. they are also cost-effective when compared with serum-containing media. disadvantages of serum-free media . growth rate and saturation density attained are lower than those compared to serum-containing media. . serum-free media prove to be more expensive as supplementing with hormone and growth factors increases the cost enormously. . different media are required for different cell types as each species has its own characteristic requirements. . critical control of ph and temperature and ultrapurity of reagent and water are required as compared to serum-containing media. these media contain pure inorganic and organic constituents along with protein additions like egfs, insulin, vitamins, amino acids, fatty acids, and cholesterol. these media contain nonprotein constituents necessary for the cell culture. the formulations of dme, mem, rpmi- , procho tm, and cdm-hd are the characterization of cell lines is important to ensure the quality of cell-derived biopharmaceutical products. it helps in determining the cell source with regard to its identity and the presence of other cell lines, molecular contaminants, and endogenous agents. the characterization of mammalian cell lines is species-specific and can vary depending on the history of the cell line and type of media components used for culturing. mammalian cell line characterization can be done in four ways: identity testing can be carried out by isoenzyme analysis. the banding pattern of the intracellular enzyme (which is species-specific) can be determined by using agarose gels. dna fingerprinting and karyotyping, and dna and rna sequencing are alternative methods to identity testing. karyotyping is important as it determines any gross chromosomal changes in the cell line. the growth conditions and subculturing of a cell line may lead to alteration in the karyotype; for example, hela cells were the first human epithelial cancer cell line established in long-term culture, and they have a hypertriploid chromosome number ( n ). bacterial and fungal contamination of cell lines occurs due to impure techniques and source material. the occurrence of contaminants can be tested by a direct inoculation method on two different media. mycoplasma infection is the contamination of cell cultures/cell lines with mycoplasmas, and it represents a serious problem. detection by microscopy is not adequate and requires additional testing by fluorescent staining pcr, elisa assay, autoradiography, immunestaining, or microbiological assay. characterization and testing of cell substrate (cell line derived from human or animal source) is one of the most important components in the control of biological products. it helps to confirm the identity, purity, and suitability of the cell substrate for manufacturing use. the substrate stability should be examined at a minimum of two time points during cultivation for production. in addition, genetic stability can be tested by genomic or transcript sequencing, restriction map analysis, and copy number determination (fda guidelines, ). virus testing of cell substrate should be designed to detect a spectrum of viruses. appropriate screening tests should be carried out based on the cultivation history of cell lines. the development of characteristic cytopathogenic effect (cpe) provides an early indication of viral contamination. some of the viruses of special concern in cell production work are human immunodeficiency virus, human papilloma virus, hepatitis virus, human herpes virus, hantavirus, simian virus, sendai virus, and bovine viral diarrhea virus. for detection of viruses causing immunodeficiency diseases and hepatitis, detection of sequences by pcr testing is adequate. cells exposed to be serum or bovine serum albumin require a bovine virus test. some of the viral testing assays are xc plaque assays, s l-focus assay, reverse transcription assay. xc plaque assay is utilized to detect infectious ecotropic murine retroviruses. s l-focus assay is used to test cells for the presence of infectious xenotropic and amphotropic murine retroviruses that are capable of interacting with both murine and nonmurine cells. real-time (rt) assays such as real-time fluorescent product-enhanced reverse transcriptase (fpert) assay and quantitative real-time for fluorescent productenhanced reverse transcript (qpert) assay detect the conversion of an rna template to cdna due to the presence of the rt template when retrovirus infection is present in the cell line. advantages of animal cell culture . physiochemical and physiological condition: role and effect of ph, temperature, o /co concentration, and osmotic pressure of the culture media can be altered to study their effects on the cell culture (freshney, in addition, this system cannot replace the complex live animal for testing the response of chemicals or the impact of vaccines or toxins. despite considerable progress in the development of cell culture techniques, the potential biohazards of working with animal and human tissues presents a number of ethical problems, including issues of procurement, handling, and ultimate use of material. in most countries, biomedical research is strictly regulated. legislation varies considerably in different countries. research ethics committees, animal ethics committees for animal-based research, and institutional research boards for human subjects have a major role in research governance. some guidelines for the use of experimental or donor animals include assurances of proper conditions for housing animals and minimal pain or discomfort to any animal that is put to death or operated upon. these guidelines apply to higher vertebrates and not to lower vertebrates such as fish or other invertebrates. fetal bovine serum (fbs)-supplemented media are commonly used in animal cell cultures. in recent years, fbs production methods have come under scrutiny because of animal welfare concerns. fbs is harvested from bovine fetuses taken from pregnant cows during slaughter. the common method of harvesting the fetus is by cardiac puncture without any anesthesia. this practice of harvesting fbs is inhumane as it exposes the fetus to pain and/or discomfort. in addition to moral concerns, numerous scientific and technical problems exist with regard to the use of fbs in cell culture. efforts are now being made to reduce the use of fbs and replace it with synthetic alternatives. in the case of human tissues, some considerations that need to be addressed are as follows (freshney, in biomedical research, the use of animal and human cell cultures has become beneficial for diverse applications. it provides indispensable tools for producing a number of products, including biopharmaceuticals, mabs, and products for gene therapy. in addition, animal cell cultures provide adequate test systems for studying biochemical pathways, intra-and intercellular responses, pathological mechanisms, and virus production. some of the applications of animal cell culture are discussed below. animal cell culture technology has played an important role in the development of viral vaccine production. the establishment of cell culture technology in the s and the consequent replacement of live animals for the development of antigens have led to considerable progress in bioprocess technology. with the advent of dna technology, molecular manipulation of viruses has led to the development of a recombinant vaccine against hepatitis b virus (hbv) and several others potential vaccines that are in the final phase of clinical trials. viral particles production by cell culture viral particle production by cell culture differs from the production of molecules such as proteins, enzymes, and toxins by bacteria or animal cells. the product formation may not be related to the development or growth of a cell and may occur through secondary metabolic pathways, unlike virus production, which does not result from secondary metabolic pathway. virus production occurs after the viral infection directs cell machinery to perform viral particle production. two stages are involved in viral production: . cell culture system: this requires the development of an efficient system for conversion of the culture medium substrate in the cell mass. . virus production: this phase differs from the infection phase and has different nutritional and metabolic requirements. a number of immortalized cell lines are used for the industrial production of viral vaccines. table . gives the cell lines used for vaccines. most of the existing classical vaccines for viral disease are either altered or chemically inactive live viruses. however, incomplete inactivation of a virus or reversion of an attenuated strain can risk infection in vaccinated individuals. viruses with segmented genomes with a high degree of genetic exchange can undergo re-assortment or recombination of genetic material with viruses of different serotypes in the vaccinated host, which can result in the production of new variants of the virus. moreover, some live virus vaccines are teratogenic; for example, smithburn neurotropic strain (sns) (smithburn, ) and mp attenuated (caplen et al., ) vaccine strains of the rift valley fever virus. a new type of vaccine that does not present the typical side effects of an attenuated or inactivated viral vaccine has been made possible with the development of rdna technology. virus-like particles (vlps) are highly effective as they mimic the overall structure of the virus; however, these particles lack the infectious genetic material. capsid proteins can aggregate to form core-like particles in the absence of nucleic acids. these spontaneously assembled particles are structurally similar to authenticate viruses and are able to stimulate b-cell-mediated immune responses. in addition, vlps stimulate a cd proliferative response and cytotoxic t-lymphocyte response (jeoung et al., ) . vlps resemble and mimic virus structure and are able to elicit a strong immune response without causing harm. the major advantage of vlps is their simplicity and nonpathogenic nature. they are replicationdeficient as they lack any viral genetic information, thus eliminating the need for inactivation of the virus. this is important as inactivation treatments lead to epitope modifications (cruz et al., ) . as the structural morphology of vlps is similar to the virus, the conformational epitopes presented to the immune system are the same as for the native virus particles. the immune response/antibody reactivity in the case of vlps is significantly improved as vlps present conformation epitopes more similar to the native virus. vlps also induce a strong b-cell response. for broader and more efficient protection, it is possible to adapt one or more antigens to the multimeric protein structure. another advantage offered by vlps is that they significantly reduce vaccine costs as these can elicit a protective response at lower doses of antigen. the fda has approved vlp-based vaccines for hbv and hpv. the hbv vaccine was approved in and the hpv one in (justin et al., ) . to generate immunogenic vlps, the s gene is cloned and expressed in a eukaryotic expression host such as yeast or mammalian cells (e.g., cho cell line). the mammalian cell culture allows easy recovery because the cells are able to secrete the antigen hbsag. the two companies producing cho-based vaccines are the frenchbased pasteur-merieux aventis (gene hevac b) and the israeli-based scigen (sci-b-vac). the gene hevac b vaccine contains the hbsag s protein and m protein, whereas sci-b-vac contains the m and l proteins. viruses of the papillomaviridae family are known to induce lesions and warts and also cause cervical cancer. fifteen strains of papillomaviridae are known to cause cervical cancer. hpv- is considered a high-risk hpv type as the risk of cancer may be higher than for other high-risk hpv types. the two virally encoded proteins of hpv are l and l . l is the main capsid protein that forms the outer shell of the virus. l is found in the interior of the viral particle and is less abundant. the recombinant l vlp is able to induce neutralizing antibodies in animals. gardasil (the first hpv vaccine) was approved by the fda in . this vaccine is manufactured by merck and co., inc. ceravarix, another hpv vaccine (manufactured by glaxo smithkline), was approved by the fda in . it uses the trichoplusia ni (hi- ) insect cell line infected with l recombinant baculovirus (jiang et al., ; wang et al., ) . a number other vlp-based vaccines are in clinical trials. these include the anti-influenza a m -hbcag vlp vaccine (clarke et al., ) , two antimalarial vaccine nicotine-qβ vlps (maurer et al., ) , and an anti-angiiqβ vlp. the vlp production in mammalian cell lines and baculo cell lines of viruses infecting humans and other animals is summarized in table . . proteins play a major role in carrying out biochemical reactions, transporting small molecules within a cell or from one organ to another, formation of receptors and channels in membranes, and providing frameworks for scaffolding. the number of functionally distinct proteins in humans far exceeds the number of genes as a result of post-translational modifications. these modifications include glycosylation, phosphorylation, ubiquitination, nitrosylation, methylation, acetylation, and lipidation. the changes in protein structure as a result of mutation or other abnormalities often lead to a disease condition. protein therapeutics offer tremendous opportunities for alleviating disease. the first therapeutic from recombinant mammalian cells was human tissue plasminogen, which obtained market approval in . at present, %À % of all the recombinant therapeutic proteins are produced in mammalian cells. the main therapeutic proteins can be divided into seven groups (walsh, ): . cytokines . hematopoietic growth factors . growth factors . hormones . blood products . enzymes . antibodies most of the proteins have complex structures and undergo chemical modification to insure full biological activity. protein post-translation modifications (ptm) can happen in several ways. the most widely recognized form of ptm is glycosylation, which involves extensive sequence processing and trimming in the golgi apparatus and endoplasmic reticulum. eukaryotic cells are capable of carrying out this type of modification and are thus preferred in biopharmaceutical processes. hamster, baby hamster kidney (bhk), and cho cells are often the host cells of choice as glycosylation patterns generated from these cells are more similar to human patterns. table . lists various therapeutic proteins produced in animal cell lines. cytokines are proteins of the immune system that play a central role in immune response. cytokines are produced as a result of immune stimulus by various white blood cells. interferons (ifns) were the first family of cytokines to be discovered and used as biopharmaceuticals. ifnα is used for treatment of hepatitis, and more recently has been approved for leukemia and other types of cancers. ifnβ is used for treatment of multiple sclerosis and is marketed under the names avonex, belaseron, and rebif. ifnγ is used for the treatment of chronic granulomatous disease. interleukin is another kind of cytokine that helps regulate cell growth, differentiation, and motility and is used as a biopharmaceutical. the recombinant form of il- is used for the treatment of renal cell carcinoma. growth factors are proteins that bind to receptors on the surface of cells to activate the cells for proliferation and or differentiation. the different types of growth factors are tgf, insulin-like growth factor, and (egf. the primary sources of pdgf are platelets, endothelial cells, and the placenta. two isoforms of this protein are present in the human body and both of these have one glycosylation site and three disulfide bonds. examples of growth factors used as biopharmaceuticals are the following: . osigraft/eptotermin alfa (bone morphogenetic protein) is used for the treatment of tibia fractures, is grown commercially in cho cells, and was first approved in in europe. surgery; it is also commercially grown in cho cells. this product was first approved in europe in . insulin, glucagon, gonadotropins, and growth hormones are the most well-known therapeutic hormones. the first biopharmaceuticals that obtained approval by regulatory agencies were insulin and recombinant human growth hormones. these were produced in microbial cells. the commercial recombinant forms of the gonadotropin family of hormones are gonal-f, luveris, puregon, and ovitrelle. all these are produced using cho cells and are used for treating female infertility. a number of recombinant therapeutic enzymes are expressed in mammalian cells. tissue plasminogen activator (tpa) is a thrombolytic agent involved in dissolving blood clots. recombinant tpa is commercially is known as alteplase and tenectplase, which are used for the treatment of acute myocardial infraction. fabry disease, a genetic metabolic disorder, is characterized by a lack of enzyme α-galactosidase a. α-galactosidase a and is produced by genetically modified cho cells. hemophilia a is caused by the lack of bloodclotting factor viii, hemophilia b is caused by deficiency of factor ix, and hemophilia c by lack of factor xi. factor viii and ix are proteins. the first recombinant factor vii products were recombinate and kogenate, which were expressed in cho and bkh cells, respectively. recombinant factor fix is commercially sold as benefix and is produced in recombinant cho cells. therapeutic antibodies are used in the treatment of cancer, cardiovascular disease, infections, and autoimmune diseases. in , the antibody avasin (bevacizeimab) was approved for the treatment of metastatic colorectal cancer. this antibody acts as an inhibitor of vascular endothelial growth factor. zenapax, another commercially available antibody, is used during prophylaxis for preventing the rejection of transplanted organs. this is commercially grown in the nso cell line and was approved for human use in . gene therapy involves the insertion, removal, or alteration of a therapeutic or working gene copy to cure a disease or defect or to slow the progression of a disease, thereby improving the quality of life. the human genome map was the first major step toward a new way of addressing human health and illness. gene therapy holds great promise, however, the task of transferring genetic material into the cell remains an enormous technical challenge and requires ex vivo cell cultivation and adaptation from the lab to a clinically relevant state. the development of animal cell culture technology is imperative for advances in gene therapy. monogenic diseases caused by single gene defects (such as cystic fibrosis, hemophilia, muscular dystrophy, and sickle cell anemia) are the primary targets of human gene therapy. the first step in gene therapy is to identify the faulty gene. this is followed by gene isolation and generation of a construct for correct expression. integration of the gene followed by delivery of the genetic material in vivo or ex vivo is crucial to the success of gene therapy. in in vivo therapy, the genetic material is introduced directly into the individual at a specific site, and in ex vivo treatment, the target cells are treated outside the patient's body. these cells are then expanded and transferred back to the individual at a specific site. the ex vivo technique involves gene therapy in the cultured cells, which are expanded and subsequently transferred to the targeted tissue. a number of clinical studies and trials for gene therapy have already been approved and are being conducted worldwide. from up to the present, about clinical studies have been reported; % of these studies are intended for cancer treatment. the first product designed for gene therapy was gendicine, a medication produced by shenzhen sibiono genetech, china. gendicine is used for head and neck carcinoma treatment. the tumor suppressing gene p in recombinant adenovirus expresses protein p , which leads to tumor control and elimination. sbn-cel is a cell line that was subcloned from the human embryonic kidney (hek) cell line and has been used for the production of gendicine. in recent years, biopesticides have gained importance due to increased concerns about agrochemicals and their residues in the environment and food. biopesticides provide an effective means for the control of insects and plant disease, and they are environmentally safe. the biological control of insect pests by another living organism (in order to suppress the use of pesticides) is an age-old practice. presently, a number of biological controls are being used as biopesticides. with the high cost of chemical-based pesticides and the development of resistance to multiple chemical pesticides, baculoviruses are one of the most promising biocontrols for insect pests and have been increasingly used effectively against caterpillars worldwide. however, the major impediment in the development of baculoviruses as biopesticides is the high cost and small volumes of in vitro methods. development of an in vitro production process for large quantities of baculoviruses at comparable costs to chemical pesticides will help provide insect control that is safe, efficacious, cost-effective, and environmentally safe. baculovirus production in animal cell culture a number of factors are important for a successful commercial production of bioinsecticides: . large-scale production of viruses at competitive costs. . economic production of viruses (i.e., low cost for the media and running the culture). . effective cell line with high virus per cell productivity. . with passage of the virus into cells, there is a loss of virulence and an increased risk of mutant formation; this should be avoided. . the quality of the polyhedral produced in the cell culture should be comparable to those obtained from caterpillars. the insect baculovirus cell system offers a number of advantages. it produces recombinant proteins that are functional and are immunologically active, as it is able to make post-translational modifications. the recombinant system uses a powerful promoter polyhedron. the most commonly used cell lines in biopesticide production are the sf and sf cell lines, which are derived from ovarian tissues of the fall army worm (spodoptera frugiperda). sf cells show a faster growth rate and higher cell density than sf cells and are preferred. high five cell lines (designated bti-tn- bi- ) established from trichoplusia ni embryonic tissue are also being used. the continuous culturing of cells for virus production leads to virus instability and the so-called passage effect. this can result in a decrease of virulence and polyhedral production and a variety of mutations. all these changes affect commercial production in vitro. two types of mutations are commonly seen in continuous passaging of cell cultures for viral productions: ( ) defective infective particles (dips) and ( ) few polyhedral (fp) mutations. fp mutations are characterized by ( ) reduced polyhedral, ( ) enhanced production of bv, and ( ) lack of occluded virions in polyhedra. all these factors reduce the infectivity of the target pest. spontaneously generated fp mutants have been reported in acmnpv (autographa california nucleopolyhedroviruses) (wood, ) , galleria mellonella nucleopolyhedroviruses (gmmnpv) (fraser and hink, ) , and helicoverpa armigera nucleopolyhedroviruses (hasnpv) (chankraborty and reid, ) . dip mutations are the formation of dips. they occur due to serial passaging for long periods, which results in a decrease in the filtering of infectious virus. dips have been reported in a number of animal virus systems and in baculovirus systems. dip formation can be avoided by low multiplicity of infection. this minimizes the probability of the defective virus entering the cell with an intact helper virion. the majority of antibodies available on the market today are produced in animal cell cultures (van dijk and van de winkle, ) . animal cells are preferred because they are capable of glycosylation and structural conformation, which is essential for a drug to be productive. hybridoma technology has been the most widely used method for small-and large-scale production of mabs. however, these antibodies have limited therapeutic applications since they produce an adverse immune response on repeated use. a number of cell lines are now being used for the production of recombinant antibodies. the cho lines are the most commonly used. other cell lines used are marine myelomas nso, sp / , hek- , and bhk. a number of factors influence the production of mabs. for a high concentration of mab production, the cell line should have high productivity. for high protein productivity, it is important that the selected cell line be productive in order to avoid large reaction volumes and the high cost of protein purification. cell lines with the capacity to grow without anchorage offer an advantage in terms of scaling up the process; it is much simpler than with those designed for the growth culture of anchorage-dependent cells. sp / and nso cell lines can grow naturally in suspension; other cells such as cho and bhk can be easily adapted to this form of cultivation. stem cells are unspecified cells that have the potential to differentiate into other kinds of cells or tissues and become specialized cells. the two characteristics that define stem cells are their ability of self-regenerate and to differentiate into any other cells or tissues. these cells have the capability to renew themselves to form cells of more specialized function. in recent years, stem cell research has been hailed as a major breakthrough in the field of medicine. this property of turning a cell into any other specialized function cell has made researchers believe that stem cells could be utilized to make fully functional, healthy organs to replace damaged or diseased organs. human embryonic stem cells (hescs) are grown on nutrient broth. these cells are traditionally cultured on mouse embryonic fibroblast feeder layers, which allows continuous growth in an undifferentiated stage. the mouse cells at the bottom of the culture dish provide a sticky surface to which the cells can attach. in addition, the feeder cells release nutrients into the culture medium. researchers have now devised animalfree culture systems for hescs and have used human embryonic fibroblasts and adult fallopian tube epithelial cells as feeder layers (in addition to serum-free mediums). more recently, methods to subculture embryonic cells without the feeder layer have been developed. martigel from bd biosciences has been used to coat the culture plate (hassan et al., ) for effective attachment and differentiation of both normal and transformed anchorage-dependent epithelioid and other cell types. this is a gelatinous protein mixture isolated from mouse tumor cells. a major milestone in the biological sciences was the establishment of the tissue culture technique that can both maintain and propagate the growth of living cells under sterile in vitro conditions. traditional cell cultures, which are two-dimensional ( d), are grown as monolayer cultures on a flat and rigid surface. since their development, several advancements have been made to improve cell culture media as well as the biological materials used for culturing. the improvements have proven valuable for cell-based study due to their amalgamation of various modern analytical techniques, such as fluorescence, electrochemistry, and mass spectroscopy. d cell culture does not provide an adequate in vivo environment, where other cells surround the cells in a three-dimensional ( d) ecm (edmondson et al., ) . cells under in vivo conditions both produce and continuously consume oxygen nutrients and other molecules, and such dynamic distributions are not mimicked in conventional d cell cultures. moreover, d cell cultures fail to recapitulate the highly complex d environment, function, and physiology of living tissues, the multitudinous regulatory interactions from surrounding tissue cells, the ecm, and other systemic factors that lead to nonpredictive data of an in vivo response (li et al., ) . the limitations of d cell culture systems have recently become more evident. recent standard protocol advances in the fields of quantitative and system biology and imaging technology have allowed analysis of individual cells and observation of live individual cells growing in a natural physiological d environment. cells cultured in a d model system more closely mimic in vivo conditions. thus, unlike d cell cultures, which can sometimes cause misleading and nonpredictive data of in vivo responses, d systems are realistic for translating study findings. compared to the d cell culture system, the d cell culture system provides a physiologically relevant and closer biomimetic environment, promotes better cell differentiation, and improves cell function (edmondson et al., ) . the d culture system holds great promise for applications in various fields, such as cancer cell biology, stem cell research, drug discovery, and various cell-based analyses and devices. while this culturing model offers state-of-the-art technology for facilitating drug development and numerous other applications, several hurdles remain before a universal, standardized, and validated system can be established (sung et al., ) . recent developments in the transition from d to d cell cultures indicate promising applications for many industries; however, the cost of automation and easyto-use readout systems are still key concerns. the d cell culture system has provided a powerful tool that mimics a highly complex and dynamic in vivo environment, and it has gained greater momentum with the integration of microfluidic technology. microfluidics is a technology characterized by the manipulation of fluids at the micron-scale for the improvement of diagnostics and cell culture research. it uses microfluidic devices to manipulate fluids in the small capillaries or microchannels. microfluidics is a science of manipulating, mixing, monitoring, and analyzing minute volumes of fluids or gases on the surfaces of chips and microfluidic chips. this technology is ideal because it recreates the microenvironment of the vasculature and has become a powerful tool in cell culture research. it encompasses knowledge of the biological sciences, chemistry, physics, and engineering applications (xu and attinger, ) . the microfluidic d cell culture model also allows precise spatial control over the gradients and medium exchange. it not only mimics but also promotes several biologically relevant functions not seen in the d cell culture. furthermore, it has been increasingly used to generate high-throughput cell culture models and has shown considerable promise for improving diagnostics and biological research (el-ali et al., ) . notably, microfluidic cell cultures are potential candidates for next generation cell analysis systems. several d-based cell culture approaches have been created to provide a better biomimetic microenvironment for cells than those of d cultures. in addition, crucial liquid handling steps, including cell loading, nutrient supply, and waste removal-under physiologically relevant conditions-can be performed with real-time microscopy (xu et al., ) . numerous microfluidic devices have been developed to not only provide nutrients and oxygen continuously for cell proliferation but also to investigate several characteristics of a dynamic d cell culture, such as differences in concentration, temperature gradients, and shear force conditions on cell transport and cultivation. numerous microfluidic platforms for d cell culturing have been developed and based on the substrates used for microdevice fabrication, including glass/siliconbased, polymer-based, and paper-based platforms. polydimethylsiloxane (pdms)-based microdevices are the predominant form of microfluidic d cell culture systems because they are economical and allow permeability of o , which is vital in cell proliferation. to provide an in vivo-like environment that resembles living tissues, several natural polymers, such as collagen, fibrin, and agarose, have been used to fabricate microfluidic devices (li et al., ) . microfluidics technology has emerged as a viable and robust platform for tissue engineering-a multidisciplinary field aimed at replacing and repairing damaged and diseased tissues and/or organs and developing in vitro models to mimic physiological conditions. successful clinical applications include the development of organ-on-a-chip technology-a microfluidic perfusion device for regenerative medicineand a chip-based platform for the culture of cells and toxicological studies. scientists currently rely on in vitro cell culture platforms and in vivo animal models to study biological processes and develop therapeutic strategies, although informative have significant shortcomings (zió łkowska et al., ) . in vitro platforms may not simulate the intricate cellÀcell and cellÀmatrix interactions that are vital to regulating cell behavior in vivo (guillouzo & guguen-guillouzo, ) . organ-on-a-chip devices could offer biological relevance and be a requisite for high-throughput applications. an organ-on-a-chip is a microfluidic cell culture device comprising a microchip with continuously perfused chambers that are infused with living cells that are arranged to mimic the d tissue microenvironment and physiology (ghaemmaghami et al., ) . these chips have the potential to significantly impact drug discovery and toxicity testing (ghaemmaghami et al., ) . the simplest functional unit of organ-on-a-chip devices consists of a single, perfused microfluidic chamber that is composed of a single type of cultured cell. these systems are utilized for studying organ-specific responses, chemical responses, such as drugs or toxins, and physical stimuli. in a complex system, two or more independently perfused parallel microchannels are connected by porous membranes to recreate interfaces between different tissues. numerous tissue models have been developed to mimic in vivo biological processes. on-chip tissue models include those for the liver, kidney, lungs, intestines, muscle, fat, and blood vessels as well as models of tumors. various chemicals and drugs, when administered over a long period, result in adverse effects and acute liver toxicity, known as hepatotoxicity (gershell & atkins, ) . in vitro models used for identifying drug-induced liver toxicity have drastically limited utility. therefore, efficient and reliable tools for testing liver toxicity are required. microfluidics devices for liver tissue and cells that can maintain metabolic activity and can be used for drug discovery and toxicity studies have shown great potential for solving this problem. bioreactors with a perfused multiwell plate device were developed by domansky et al. ( ) to recapitulate both the physiological and mechanical microenvironments of hepatocytes that can support both growth and functional integrity for up to week. khetani and bhatia ( ) developed microscale cultures of human liver cells in a multiwell micropatterned co-culture system that can maintain phenotypic functions of liver cells for up to several weeks. a significant challenge for cancer research is the early detection and development of in vitro strategies for studying the role of drug-carrier design in tumor transport and therapies for targeting rapidly dividing cancer cells while leaving normal, healthy cells untouched. the microfluidics tumor-on-a-chip platform can be used for detecting circulating tumor cells (ctcs) in blood flow, which may lead to early diagnosis of cancer (millner et al., ) . a variety of designs for studying the microenvironment of microfluidic devices that culture solid and liquid tumors were reviewed by young ( ) . tatosian and shuler ( ) developed a novel microfluidic system to study the multidrug resistance of cancer cells to chemotherapeutic combinations. jang et al. ( ) fabricated a microfluidic device with an active injection system that produced of combinations of different chemical solutions at various concentrations and stored them in isolated chambers. to optimize system parameters for varied types of cancer cells while requiring minute amounts of reagents and cells, jedrych et al. ( ) generated a microfluidics system for photodynamic therapy-based measurements. this system allows light-induced photosensitizers to be delivered to the carcinoma cells, which-on reaction with oxygenproduce a chemical toxin that is lethal to tumor cells. http://amgenscholars.com/images/uploads/ contentimages/biotechnology-timeline.pdf amgen scholars provides hundreds of undergraduate students with the opportunity to engage in a hands-on summer research experience at some of the world's leading institutions. . http://monographs.iarc.fr http://monographs.iarc.fr/eng/monographs/ vol /mono - .pdf the iarc monographs identify environmental factors that can increase the risk of human cancer. these include chemicals, complex mixtures, occupational exposures, physical agents, biological agents, and lifestyle factors. . www.iptonline.com http://www.iptonline.com/articles/public/ iptfive np.pdf iptonline publishes "the pharmaceutical technology journal," which is designed to provide information on the latest ideas, cutting-edge technologies, and innovations shaping the future of pharmaceutical research, development, and manufacturing. . http://www.aceabio.com http://www.aceabio.com/userfiles/doc/ literature/xcell_appnotes/ rtca_appnote _acea_lores.pdf acea biosciences, inc. (acea) is a privately owned biotechnology company. acea's mission is to transform cell-based assays by providing innovative and cutting-edge products and solutions to the research and drug discovery community. the food and drug administration (fda or usfda) protects and promotes public health through the regulation of all foods (except meats and poultry), the nation's blood supply, and other biologics (such as vaccines and transplant tissues). drugs must be tested, manufactured ?la en promega manufactures enzymes and other products for biotechnology and molecular biology the world health organization (who) is a specialized agency that is concerned with international public health. it is affiliated with the united nations and headquartered in geneva, switzerland. who ensures that more people, especially those living in dire poverty virus-like particle and viral vector production using the baculovirus expression vector system/insect cell system animal cell culture concept and application. alpha science international limited mutagen-directed attenuation of rift valley fever virus as a method for vaccine development serial passage of a helicoverpa armigera nucleopolyhedrovirus in helicoverpa zea cell cultures recombinant hemagglutinin produced from chinese hamster ovary (cho) stable cell clones and a pelc/cpg combination adjuvant for h n subunit vaccine development a bioluminescent cytotoxicity assay for assessment of membrane integrity using a proteolytic biomarker improved immunogenicity of a peptide epitope after fusion to hepatitis b core protein integrated process optimisation: lessons from retrovirus and virus like production perfused multiwell plate for d liver tissue engineering amino acid metabolism in mammalian cell cultures threedimensional cell culture systems and their applications in drug discovery and cell-based biosensors cells on chips the isolation and characterization of the mp and fp plaque variants of galleria mellonella nuclear polyhedrosis virus culture of animal cells: a manual of basic technique culture of animal cells: a manual of basic technique and specialized applications culture of animal cells: a manual of basic technique and specialized applications a brief history of novel drug discovery technologies evolving concepts in liver tissue modeling and implications for in vitro toxicology role of c-jun n-terminal protein kinase / (jnk / ) in macrophage-mediated mmp- production in response to moraxella catarrhalis lipooligosaccharide (los) the serial cultivation of human diploid cell strains assembly of human severe acute respiratory syndrome coronavirus-like particles an integrated microfluidic device for two-dimensional combinatorial dilution evaluation of photodynamic therapy (pdt) procedures using microfluidic system immunogenicity and safety of the virus-like particle of the porcine encephalomyocarditis virus in pig synthesis of rotavirus-like particles in insect cells: comparative and quantitative analysis indian vaccine innovation: the case of shantha biotechnics microscale culture of human liver cells for drug development microfluidic d cell culture: potential application for tissue-based bioassays contribution of ebola virus glycoprotein, nucleoprotein, and vp to budding of vp virus-like particles a therapeutic vaccine for nicotine dependence: preclinical efficacy, and phase i safety and immunogenicity viral vaccines: concepts, principles, and bioprocesses circulating tumor cells: a review of present methods and the need to identify heterogeneous phenotypes apoptosis in cho cell batch cultures: examination by flow cytometry efficient assembly and release of sars coronavirus-like particles by a heterologous expression system the vp protein of rotavirus interacts with a large fraction of human naive b cells via surface immunoglobulins rift valley fever: the neurotropic adaption of virus and experimental use of this modified virus as a vaccine medicines from animal cell culture using physiologically-based pharmacokinetic-guided "body-on-a-chip" systems to predict mammalian response to drug and chemical exposure virus-like particles exhibit potential as a pan-filovirus vaccine for both ebola and marburg viral infections a novel system for evaluation of drug mixtures for potential efficacy in treating multidrug resistant cancers mers-cov virus-like particles produced in insect cells induce specific humoural and cellular imminity in rhesus macaques self-assembly of the infectious bursal disease virus capsid protein, rvp , expressed in insect cells and purification of immunogenic chimeric rvp h particles by immobilized metal-ion affinity chromatography isolation and replication of an occlusion bodydeficient mutant of the autographa californica nuclear polyhedrosis virus drop on demand in a microfluidic chip three-dimensional in vitro tumor models for cancer research and drug evaluation assembly of siv virus-like particles containing envelope proteins using a baculovirus expression system cells, tissues, and organs on chips: challenges and opportunities for the cancer tumor microenvironment microfluidic devices as tools for mimicking the in vivo environment from biopharmaceuticals to gene therapy culture of animal cells: a manual of basic techniques and specialized applications prospects for the use of animal cell cultures in screening of pharmaceutical substances microfluidic single-cell manipulation and analysis: methods and applications membrane protein of human coronavirus nl is responsible for interaction with the adhesion receptor towards single-cell lc-ms phosphoproteomics medicines from animal cell culture animal biotechnology: models in discovery and translation engineering microfluidic organoid-on-a-chip platforms cytotoxicity the degree to which an agent has specific destructive action on certain cells differentiation a change in a cell causing an increase in morphological or chemical heterogeneity immortalized changing a cell type with limited lifespan in vitro into a cell type with unlimited capacity to proliferate; sometimes achieved by animal cells in vitro or by tumor cells in vitro cell growth outside the body, in glass, as in a test tube what is the hayflick effect? . what is the source of cells for primary monolayer cell culture? serum is one of the basic components of cell culture media (true/false)? what was the first recombinant human protein? . what are the different phases of the growth curve? . is the vlp-based hpv vaccine approved by the fda? answers to short answer questions limited replication capacity of cells in culture medium lag phase, log phase, and plateau phase gardasil (the first hpv vaccine) was approved by the fda in yes/no type questions are cells obtained directly from organs and tissues in primary cell culture? is secondary culture used for studying transformed cells? is identity testing a way to determine purity of culture? is ifn-α used for the treatment of multiple sclerosis? is bevacizumab approved for the treatment of colorectal cancer? does passage effect leads to an increase in the virulence of cultured viruses? . do stem cells can not differentiate into other kinds of cells? microfluidic devices provide nutrients and oxygen for cell proliferation living cells are used in organ-on-a-chip microfluidic cell culture can embryonic cells be cultured without any feeder layer? answers to yes/no type questions yes-mechanical, chemical, or enzymatic disintegration of tissues and organs is required in primary cell culture. . yes-secondary cultures are used in the study of transformed cells as these cultures maintain their cellular characteristics no-for testing the purity, one should use fluorescent staining pcr or elisa no-ifn-β is used in the treatment of multiple sclerosis yes-it is an inhibitor of vascular endothelial growth factor no-passage effect leads to viral instability no-stem cells can differentiate into other kind of cell types yes-microfluidic devices also help in investigating characteristics of d cell culture yes-chambers of organ-on-a-chip devices are continuously infused with living cells yes-martigel from bd biosciences can be used to coat the culture plate short answer questions key: cord- -zcz zte authors: rethorst, david n. title: animal health equipment management date: - - journal: veterinary clinics of north america: food animal practice doi: . /j.cvfa. . . sha: doc_id: cord_uid: zcz zte proper health equipment management requires significant attention to detail. establishing and following protocols during processing (eg, cleaning and disinfecting equipment at the end of the work day) is required to ensure a safe product that is free of defects and residues. overall cleanliness of equipment and facilities is important not only from a food safety standpoint but many view these as an overall indicator of attention to detail in the entire production system. ensuring that needles are changed, implant guns are managed properly, vaccine is handled in an acceptable manner, and that proper chute operation occurs is essential. isolation is the most important step in disease control, because it prevents direct contact between diseased or potentially infected cattle and healthy cattle. preventing commingling of new cattle and existing groups of cattle is crucial to an effective biosecurity plan. facilities used to segregate cattle should be cleaned between groups and disinfected if deemed appropriate. traffic control includes all traffic on an operation, animals, people, and vehicles or equipment. animals to consider include all domesticated animals as well as rodents, birds, and other wildlife. fecal contamination of feedstuffs such as silage or ground hay can be controlled by limiting traffic to the silage pit and hay pile to the loader designated to load feed. another major concern of traffic control is limiting rendering truck access to an area of the yard that is away from all cattle and feeding activities. sanitation refers not only to the disinfection of people and equipment entering an operation but the cleanliness of people and equipment on the operation. prevention of fecal-oral contamination is the primary goal of sanitation. balling guns and drench guns are of primary concern for disinfecting between animals. before disinfection, the equipment should be cleaned with soap and hot water at the end of each day and stored in a dry area. another major biosecurity consideration is loaders that are used to load feedstuffs. if these loaders are used for handling manure, dead cattle, or other nonfeedstuff products, they should be cleaned and disinfected before loading feedstuffs. processing and treatment areas should be cleaned at the end of each day. this strategy reduces the likelihood of fecal-oral contamination not only of the cattle but of the caregivers as well. the first step in sanitation is the removal of organic matter, primarily feces. any blood or saliva present should also be removed. disinfection should follow this cleaning. physical contact between the disinfectant and proper contact time are crucial to ensure proper disinfection. the selected disinfectant should kill a broad spectrum of bacteria, viruses, protozoa, fungi, and spores. other selection considerations include safety to both humans and animal, effect on equipment (corrosiveness), effect on environment, and cost (tables and ). equipment used for processing and treating cattle should be cleaned and disinfected daily, after use. if this equipment becomes grossly contaminated with feces or other material while being used, it should be cleaned immediately. equipment such as balling guns and drench guns should be thoroughly cleaned at the end of each day and stored in a clean, dry environment. disinfecting this equipment between animals is recommended. the disinfectant solution should be changed when it becomes cloudy or visibly contaminated to maintain effectiveness. increased morbidity and mortality in feeder cattle have been associated with improper sanitation of this equipment in a feedyard hospital system having a high prevalence of salmonella infection. cohort cattle that did not go through the hospital system were found to have zero prevalence for salmonella spp. disinfecting of blood-contaminated equipment between animals is essential. this strategy reduces the risk of transmission for pathogens such as bovine virus diarrhea virus, bovine leukosis virus, and various other blood-borne pathogens. disinfection also reduces bacterial contamination and risk of disease transmitted animal to animal. this equipment includes dehorners, castration equipment, and various instruments used for minor surgical procedures. thorough cleaning is necessary, followed by storage in a clean, dry area. banding castration equipment should be kept free of feces [ ] [ ] [ ] [ ] [ ] and other gross contaminates between animals. thorough cleaning at the end of the day is appropriate. cleaning and disinfection of obstetric equipment is an often neglected area. all obstetric equipment, calf pullers, chains, straps, head snares, and so forth should be thoroughly cleaned and disinfected after each use. if straps are being used rather than chains, extra attention should be given to making sure that the strap is clean and dry before storing. chains should be allowed to dry completely before storing to prevent rusting. one of the most consistent, positive returns on investment procedures in the cattle industry is the use of growth-promoting implants. for this practice to be effective, care must be taken to ensure that it is performed properly. implant guns should be thoroughly washed with detergent and hot water after each use and allowed to dry. it is essential that the needles on these guns are sharp. if needles become dull or burred, they should be discarded rather than attempting to sharpen them. the ear should be free of feces and other gross contamination. if not, the ear should be cleaned with a disinfectant solution before placing the implant. a sponge or paint roller in a disinfectant solution should be available to clean the implant needle after each animal is implanted. if the needle skips off the ear, the needle should be disinfected before another attempt to implant is made. inadequate care and cleaning of syringes used for routine vaccination of livestock can be associated with localized swelling and infection after vaccination. swellings involving the injection site are common, particularly when vaccines such as clostridial vaccines are given subcutaneously. the subcutaneous use of oil-adjuvanted vaccines also results in swelling of the injection site. if these swellings do not subside in a short period, if they become larger than a small hen's egg, or if they become fluid filled, they may be infected and should be examined. proper cleaning of syringes begins with using hot water, soap, and a brush to clean the external surface and working mechanism of the syringe. care should be taken to keep soap away from the luer tip of the syringe. after cleaning the external components, rinse the internal components with distilled or deionized water that is warmer than . c ( f) by drawing the water into the syringe and squirting it out several times. all syringes, whether pistol grip repeaters or continuous feed syringes with vinyl tubing and vent spikes, can be cleaned in this manner. soap and disinfectant should not be used on the internal components of syringes. remove as much water as possible from the syringe and allow it to cool before use, because heat inactivates modified live viral vaccines. some syringes may need to be disassembled to adequately clean them. disassembly should be performed on a clean work surface followed by cleaning the components with hot tap water, avoiding the use of soap or disinfectant. if syringes are physically contaminated enough to require disassembly for cleaning, they should be boiled for minutes in deionized or distilled water. plastic, continuous feed syringes can be sanitized after cleaning, if necessary. the syringe and tubing should be filled with distilled water and wrapped in multiple layers of paper towels. after soaking the paper towels in water, the syringe is placed in an unclosed zip-lock bag. this bag is placed in a microwave oven for minutes using the high setting. care should be taken that the paper towels do not dry out during this process. syringes should be individually microwaved when using this method. after the syringes are removed from the microwave, any water remaining in them should be squirted out and the syringe allowed to cool before using. syringes should be reassembled while hot, using lubricant such as vegetable oil spray on the rubber plunger. others recommend using the first draw of vaccine as the lubricant, thus avoiding silicone, mineral oil, or vaseline. there does not seem to be a consensus as to what is acceptable (nonviricidal) in regard to syringe lubricant, because still others recommend the use of silicone or vaseline. this is an area that warrants further investigation. once assembled, the syringe should be rinsed several times with water warmer than f. after cooling, the syringes should be stored in a clean, dry environment, such as a new zip-lock bag that is placed in a freezer. syringes should be allowed to warm to room temperature before filling with vaccine. warming the syringes by artificial means may result in hot spots in the syringes, which have the potential to damage the vaccine. syringes should be labeled to ensure that the same product goes in each syringe every time cattle are worked. this strategy eliminates the possibility of trace amounts of a vaccine or pharmaceutical remaining in a syringe and having a detrimental effect on the next product in the syringe. it also ensures that syringes are not filled with the wrong product during processing. transfer needles should be cleaned in a manner similar to syringes, after using a stylet to remove rubber plugs from the needle, using hot water without soap or disinfectant. once cleaned, there are several methods that can be used to ensure that the needles are sterile. these methods include . boiling in distilled water for minutes. after cooling, the needles should be stored in a clean, dust-free container. . microwaving the needles in a cup filled with distilled water. use the high setting to bring the water to a boil. once the water boils, continue for minute. be sure the needles are completely covered with water during the entire process. once again, the needles should be stored in a clean, dust-free container after cooling. . wrap the needles in several layers of paper towels. soak the paper towels in water and place in a zip-lock bag. place the unclosed zip-lock bag in a microwave for minutes using the high setting. the paper towels should remain wet during the process. store appropriately when cool. . autoclaving works well if such equipment is available. the clean needles are placed in a -ml disposable syringe case or multiple needles in a -ml syringe case. the cap is held in place with autoclave tape. after autoclaving, the syringe cases work well for clean, dust-free storage. the size of needles used for processing and treatment of cattle should be adjusted according to cattle size, viscosity of product injected, route of administration, and type of restraint to be used ( table ). use the smallest needle possible to easily administer the product, yet large enough to prevent the needle from bending or breaking off in the animal. the use of disposable needles is recommended, because they are sterile and are sharp. using steel needles that are reusable is discouraged, because they are difficult to sterilize and keep sharp. improper needle management results in injection site lesions that requires trimming during harvest or fabrication. needles should be changed before they become dull, preferably every to head. in addition, they should be changed immediately if the needle bends, becomes contaminated with feces, dirt, or chemicals, or if the needle point is damaged. in herds that are known to be infected with pathogens that are blood borne, consideration should be given to changing needles after each animal. never use a needle that is bent, because this increases the likelihood that the needle will break off in the animal being injected. it is considered an emergency situation when a needle breaks off in an animal. if this occurs, great effort should be made to find the needle immediately. if the needle cannot be found, the animal should be identified to be checked later. an animal that is known to have a broken needle in it should not be marketed for human consumption. the best method for disposal of used needles is the collection of needles in a biohazard sharps container, which should be disposed of by a biomedical disposal company. an alternative disposal method is collecting the needles in a plastic jug or bucket and filling the bucket with cement powder and water before sealing it and disposing of it in a landfill. used needles should never be disposed of individually in select the needle to fit the cattle size (the smallest practical size without bending). adapted from beef quality assurance. best management practices-prevention and processing. national beef quality assurance manual. national cattlemen's beef association, centennial, co. the daily trash. state law should be checked if one is unsure of the legalities of needle disposal in one's local area. for vaccines to induce adequate immunity, they must be shipped, stored, and handled in a proper manner, including the handling that occurs during administration. although this factor is especially important for modified live vaccines, it applies to all vaccines whether modified live virus, killed virus, or a bacterin toxoid. refrigerators used to store animal health products come in all shapes and sizes, ranging from minirefrigerators to household-type refrigerators to glass-front display coolers. many of the household-type refrigerators are used refrigerators purchased for the sole purpose of storage of animal health products. these refrigerators are found in a variety of places from the barn, to the shop, to the mudroom, or even the kitchen. retailers such as feed stores and veterinary clinics are another common location for refrigerators used to store animal health products. two studies funded by state beef quality assurance programs have evaluated how well these refrigerators cool. the first study evaluated refrigerators. of these refrigerators, only ( . %) operated within the desired temperature range greater than % of the time over the -hour test period. thirty-eight ( . %) operated at the correct temperature % to % of the time, whereas ( . %) operated properly % to % of the time and ( . %) were in the correct range % to % of the time. the remaining refrigerators ( . %) operated at the desired temperature less than % of the time. nearly % of these refrigerators were greater than years old. the second study evaluated refrigerators. producer refrigerators accounted for of the , whereas were feedstore or veterinary clinic refrigerators. forty ( %) of the refrigerators operated at the correct temperature greater than % of the time, with another ( . %), operating properly % to % of the time. forty-three ( . %) were at the correct temperature less than % of the time. the retail coolers were similar, in that ( %) were at the correct temperature greater than % of the time. seven ( . %) operated properly % to % of the time and ( . %), were at the correct temperature % to % of the time. eight ( %) were found to be at the proper temperature less than % of the time. the desired temperature range as defined for these studies was c to c ( f- f), which is the temperature range commonly recommended by the pharmaceutical industry. temperatures lower than c ( f) can be more detrimental than temperatures higher than c ( f) because of antigen-adjuvant separation. temperatures higher than c ( f) are particularly detrimental to modified live vaccines. location of the refrigerators in this study seemed to account for a portion of the fluctuation of the temperature. those refrigerators that were in a controlled environment maintained proper temperature in a more acceptable manner than did those that were located in an uninsulated building. an interesting observation in these studies was the number of outdated products and open bottles of product, including modified live viral vaccine, that were found in the refrigerators evaluated. of the products found in producer refrigerators in the first survey, . % were outdated and . % were open. the second survey found nearly products in the refrigerators, of which . % were expired and . % were open. a summary of these refrigerator studies includes the following: . keep a thermometer in the refrigerator so that temperature can be monitored. . do not use minirefrigerators for long-term storage. . keep the refrigerator in a controlled environment. . regular cleaning of the coil and compressor are essential, particularly if the refrigerator is located in a barn or processing shed. . refrigerators that do not maintain temperature between c and c ( f - f) should be replaced. when vaccine is taken from the refrigerator to chute-side, it should be stored in a cooler box with freezer packs. this strategy not only keeps the vaccine cool but provides for storage of the vaccine out of direct sunlight once it has been mixed. no more modified live viral vaccine should be mixed than is anticipated will be used in hour. open vaccine, whether modified live or killed, should be stored in the cooler box unless syringes are being filled. modified live vaccine that has been mixed should be discarded when the cattle have been processed, because of the short life of the vaccine ( hour). care should be taken to ensure that a used needle never enters a bottle of vaccine. a used needle can contaminate the remainder of the vaccine, resulting in injection site blemishes. the importance of cleanliness during processing is shown by the fact that dried nasal secretions from a calf persistently infected with bovine virus diarrhea (bvd) virus on the rubber stopper of a vaccine bottle transmitted bvd virus to a seronegative calf. the storage environment of nonrefrigerated pharmaceutical products such as antibiotics, anthelmintics, and topical insecticides should be considered. these products should be protected from freezing and heat, because either can cause damage to the product. if the rubber stoppers on stored bottles of product are damaged enough to not seal the bottle, the product may deteriorate because of exposure to air. these bottles should be capped in some manner, such as a roll-on rubber stopper, to prevent exposure of the product to air. the operating pressure and thus the operating speed of a hydraulic chute are set by the manufacturer and should not be changed. although this pressure setting varies between manufacturers, the basic guideline is kg ( lb) of squeeze pressure measured at the bottom of the squeeze panel drop gates using a pressure bar between the squeeze panels (d.d. griffin, personal communication, ) . for this pressure to be accurate, the distance between the bottom of the squeeze panels should be slightly narrower than the distance between the squeeze panels at the bottom of the drop gates (simulate shoulder width vs stance width). the chute operator should always use hands on the controls. this technique allows for smoother handling of the cattle and reduces the bruising that can be associated with processing. before cattle enter the chute, the distance between the bottoms of the squeeze panels should be observed. this distance should be approximately the stance width for the class of cattle being processed. if this distance is too narrow, excessive pressure will be applied to the animal when the squeeze is applied. as the operator prepares to let cattle enter the chute, the tailgate is opened with the squeeze slightly closed and the headgate open no wider than the animal's shoulders. as the animal proceeds into the chute, it is slowed with the squeeze panels to prevent it from slamming into the headgate. the headgate is closed, the tailgate closed and then, if necessary the squeeze panels. when releasing the animal, the headgate should be opened first, then the squeeze released. when this sequence is followed, the cattle tend to move forward, without the use of an electrical prod. if the squeeze is released before opening the headgate, the cattle tend to back up, necessitating prod use in many instances. the use of low-stress handling practices before the animal enters the chute can reduce the risk of injury and problems associated with hydraulic chute use. proper health equipment management requires significant attention to detail. establishing and following protocols during processing (eg, cleaning and disinfecting equipment at the end of the work day) is required to ensure a safe product that is free of defects and residues. in this age of increasing production transparency, overall cleanliness of equipment and facilities is important not only from a food safety standpoint but many view these as an overall indicator of attention to detail in the entire production system. therefore, ensuring that needles are changed, implant guns are managed properly, vaccine is handled in an acceptable manner, and proper chute operation occurs is essential. current veterinary therapy: food animal practice care of veterinary vaccine syringes. neb-guide g - -a. lincoln (ne): cooperative extension, institute of agriculture and natural resources best management practices-prevention and processing. national beef quality assurance manual. centennial (co): national cattlemen's beef association biosecurity basics for cattle operations and good management practices (gmp) for controlling infectious diseases. neb-guide g . lincoln (ne): cooperative extension, institute of agriculture and natural resources overview of antiseptics and disinfectants antiseptics and disinfectants cleaning and disinfection of environmental surfaces. health care health and safety association of ontario manual on procedures for disease eradication by stamping out. rome (it): food and agriculture organization of the united nations florida cow-calf and stocker beef safety and quality assurance handbook: quality control points university of georgia college of veterinary medicine incidence of injection-site blemishes in beef top sirloin butts case study: the temperature variability of refrigerators storing animal health products case study: handling and management of animal health products by idaho producers and retailers key: cord- -hswef ky authors: khan, safdar a. title: differential diagnosis of common acute toxicologic versus nontoxicologic illness date: - - journal: veterinary clinics of north america: small animal practice doi: . /j.cvsm. . . sha: doc_id: cord_uid: hswef ky this article provides a display table laying out the differential diagnosis of common acute toxicologic versus nontoxicologic illnesses in small animals. major clinical abnormalities are listed, along with common toxicologic rule outs and nontoxicologic rule outs. further readings are also provided. safdar a. khan, dvm, ms, phd a,b, * upon presentation of an acutely ill animal, a veterinary professional must consider poisoning as a potential cause among the differentials. a complete and thorough case history in this regard is essential for differentiating a poisoning situation from a naturally occurring disease. obtaining a clear recent history may sometimes be quite challenging, especially in situations where the pet was unsupervised before the initiation of clinical signs. history questions must include animal signalment (breed, sex, and age) and weight, previous medical history, vaccination history, type of feed used (brand; home-made or commercial) and any medications the pet is taking. initial information about any other animals present in the household, timeline of clinical signs, types of clinical signs reported by the owner, number of affected animals, pet's environment (indoor vs. outdoor; fenced or free roaming), location (urban vs. rural), time of the year (summer vs. winter), recent renovations/ updates (construction material; lead in older farms/houses), recent visitors, availability of human medications in the pet's environment (antidepressants, pain killers, stimulants, nutritional supplements), presence or recent use of chemicals (insecticides, herbicides, rodenticides) in the house/yard, information about neighboring animals (outbreaks; illnesses; death) and information about indoor/outdoor plants may help provide clues to the clinician to narrow down the search for a possible cause for the pet's illness. a good case history can help speed up the process of narrowing down a potential cause; eliminate several unnecessary steps, save time, and money. before obtaining a complete case history, the first goal should be to stabilize the patient and preserve life of the acutely ill animal irrespective of the cause. relying too much on specific antidotal treatment may be dangerous. a majority of clinical cases on presentation are treated supportively as only a very few specific antidotes are available or needed for treating specific poisonings. therefore, on presentation, make sure the animal has a patent airway and adequate ventilation. support and maintain cardiac functions. monitor heart rate, rhythm, and blood pressure, and treat cardiac arrhythmias and blood pressure changes as needed. hydration status, fluids, electrolytes, and acid-base balance should be checked and corrected accordingly. treat central nervous system abnormalities (excitation, depression, seizures) as required, and maintain body temperature within the normal range (treat hypothermia or hyperthermia). after stabilizing the vital functions, obtain a history; then, provide other necessary treatment such as decontamination (administration of activated charcoal, gastric lavage, bathing, dilution), supportive care, and carrying out other diagnostics (complete blood count, chemistries, urinalysis, radiographs, ultrasound) as needed. collect samples for toxicologic analyses if required. toxicology testing performed in a diagnostic laboratory can be expensive and time consuming and mostly, results are not available immediately. therefore, to rule in or out a suspected cause, first perform commonly used in-house diagnostics before ordering toxicology analysis. for example, monitoring prothrombin time or clotting times can be useful in anticoagulant poisoning cases. other samples for toxicology testing in a diagnostic laboratory include whole blood for heavy metal analysis (lead), blood cholinesterases (organophosphate poisoning), and presence of pesticides (anticoagulant rodenticides). similarly serum/plasma can be used for some metal analysis (zinc), drugs, alkaloids, and electrolytes (useful in sodium chloride poisoning or water intoxication). stomach contents (vomitus; freeze upon collection) can be used for detecting pesticides, metals, baits, alkaloids, and drugs. urine (chilled or frozen) can be used for some metal analysis, drugs and their metabolites, and alkaloids (strychnine). table outlines some important toxicologic versus nontoxicologic rule-outs based on clinical abnormalities one must consider in an acutely ill animal. where necessary, with each rule-out, along with major clinical abnormality, a brief description of other clinical signs is also provided. an acutely ill animal with sudden onset of clinical effects may often have multiple major clinical signs/abnormalities present. the purpose here is to provide an initial guideline for considering toxicologic versus nontoxicologic rule-out. once a reasonable etiology has been narrowed down or established, the reader is encouraged to review a more detailed discussion on management of the particular poisoning or disease listed in this reference (see table ). toxicology of selected pesticides, drugs, and chemicals clinical veterinary advisor: dogs and cats intoxication versus acute, nontoxicologic illness: differentiating the two quick reference to veterinary medicine investigating fatal suspected poisonings diagnostic toxicology for the small animal practitioner , -d, dichlorophenoxyacetic acid; aki, acute kidney injury; alt, alanine aminotransferase; aptt, activated partial thromboplastin time; ckd, chronic kidney disease; cns, central nervous system; cv, cardiovascular; felv, feline leukemia virus; fiv, feline immunodeficiency virus; gi, gastrointestinal; npk, nitrogen, phosphorus, potassium; nsaid, nonsteroidal antiinflammatory drug; pcv, packed cell volume; pt, prothrombin time; rbc, red blood cell; slud, salivation, lacrimation, urination, defecation. key: cord- -c urbl l authors: hicks, t.m.; verbeek, c.j.r. title: protein-rich by-products: production statistics, legislative restrictions, and management options date: - - journal: protein byproducts doi: . /b - - - - . -x sha: doc_id: cord_uid: c urbl l congruent to a rapidly growing population is the generation of horticultural and agricultural by-products and waste. the modern-day food cycle includes agricultural production, postharvest handling and storage, food processing and packaging, and distribution and retail, ending with consumption and waste disposal. of these, agricultural production and postharvest handling and storage result in unintended losses and by-products whereas the other steps generally result in food waste. approximately one-third of the worldwide food production is wasted each year. although it is most desirable to prevent waste and by-product formation, followed by reuse or recycling, the formation of by-products and waste is inevitable, and management options must be innovative and also meet legislative requirements. however, management options are mostly a compromise between what is viewed acceptable based on legal requirements and local perceptions, and what is deemed technologically and financially feasible. most of the world's food is derived from agricultural, horticultural, and fishery processes. with a growing population, urbanization, and increased income, the food industry has become increasingly market driven. as a result of globalization and reduced trade barriers, it has grown to account for approximately % of the world's gross domestic product (murray ) . fortunately, environmental protection and sustainability are currently better aligned with the worlds' consumption of natural resources. over the past few decades, it has tried to adopt technologies to improve waste minimization and environmental performance. although the most valuable elements are extracted from foods during harvest and processing, what remains in both the product-specific and product-nonspecific wastes may also contain other potentially valuable components. predicting future food production and associated by-products is complicated and has to take into account not only changes in population size, dietary composition, land requirements, and primary resources, but also climate and environmental aspects (godfray et al., a) . overall, increased global demand for animal-based products requires a substantially greater increase in plant and other feed resources, which will subsequently generate a much larger volume of protein-rich materials than currently produced. the quantity of food materials wasted each year is exorbitant, and urbanization and the increasing per capita income will see this quantity rise further through increased consumption of staple foods and through diversification into animal products, such as meat, fish, and dairy. this will be most challenging for transitional countries, which are expected to undergo a much more rapid increase in per capita meat consumption compared to high-income countries (ie, china will increase by ∼ %, from kg in to kg per capita per year in compared to an increase of ∼ %, from kg to kg per capita per year, in higher income countries) (msangi and rosegrant, ) . such nutritional transitions result in a rapid increase in animal products, putting a significant amount of pressure on food supply chains within transitional countries than those in the developed world. a major facet of the problem we face, is being able to source adequate quantities of high-quality protein from which to feed both humans and animals, without intensifying the overall environmental impact (van huis, ) . obviously, increasing production of animal-based products will result in a much higher consumption of grain and protein feeds to feed livestock, which are estimated to require ∼ kg of plant protein for every kilogram of protein they produce (pimentel and pimentel, ) . however, this could be better perceived by the ∼ kg of grain required to produce kg of edible boneless meat from grainfed cattle (foley, ) . conversely, while chicken and pork are more efficient converters of plant proteins, pasture-fed cattle are able to convert nonfood material into usable protein. the technology for recovering nutrients and usable materials from industry is often feasible, but the regulations regarding what can be done with by-products of industry may not always allow for the technology to be adopted. despite a concerted effort to better use by-products of the agricultural and food industry to improve the management of resources, sensible legislative incentives also need to be implemented. this chapter identifies areas of food production and related industries generating waste and byproducts with high levels of recoverable protein, in particular, those derived from agricultural production itself. current and future management options for the transformation and/or disposal of these wastes and by-products are then considered in light of current legislation and technological restrictions. the modern food cycle is comprised of several stages, including agricultural production, postharvest handling and storage, food processing and packaging, distribution and retail, and finally, end-of-life and consumption ( fig. . ) (kummu et al., ) . agricultural production, postharvest handling, and storage of food give rise to unintended food losses and ancillary byproducts, while processing and packaging and distribution and retail result in "food waste." food loss, by-products, and food waste are formed at every stage of the food production process. while the generation of by-products, such as crop residues and animal by-products (abps) during agricultural production is considered unavoidable, food losses, owing to a lack of market or degradation during handling or transportation could be avoided with care, but when considering statistics, it is often difficult to distinguish between the two. for various reasons, approximately one-third of the food produced worldwide is wasted (godfray figure . general food production stages, starting from agricultural production and postharvest handling and storage to processing and packaging, distribution, retail, and consumption. et al., a; food and agriculture organization of the united nations, ). these wastes (and possible by-products) are created during the manufacturing processes and are often removed in order to give the product the desired sensory and nutritional qualities. although the magnitude of food losses, by-products, and food waste varies depending on the product type (table . ) and the stage of production considered (table . ), it is strongly influenced by the technology and infrastructure available to the region. it has been estimated that around million metric tons (mmt) of abps are produced worldwide every year (leoci, ), along with significantly higher quantities of crop residues (santana-méridas et al., ) . obviously, industrial processing of any food, whether it is intended for human or animal consumption (or other industrial processes, such as biofuels) leads to a vast quantity of waste and by-products, typically ranging between and % by weight (table . ). in the case of crops, only % of global production is used for human consumption, mostly in the form of grains, pulses, oil plants, fruits, and vegetables, leaving % as by-products (used for animal fodder) and the remaining % for conversion to biofuel and other industrial products (foley, ) . in high-income regions, most food waste occurs during distribution and consumption, with high losses also occurring during agricultural production of plant products and fish (table . ). harvesting of crops also results in an inedible portion of the biomass (including edible product lost during harvest) contributing to what is known as crop residues. for most common edible crops, the residue-to-crop-production ratio is between . and to (scarlat et al., ) . this mass is not accounted for in table . , however, typical quantities of some common food crops are given in table . . in lower-income regions, losses occur at every stage, particularly postharvest, to a much higher degree, but occur significantly less at the consumption stage. higher losses throughout production in low-income regions are an artefact of inadequate knowledge, skills, technologies, and infrastructure to support the food supply chain compared to the industrialized world (godfray et al., b) . globally, billions of tons of agro-industrial residues and by-products are generated annually (table . ). these include solid, liquid, and gaseous residues and can be seen as one of the most abundant, cheap, and renewable resources available (santana-méridas et al., ) . given that food waste has a typical composition of ∼ - wt.% starch, - wt.% lipids, and - wt.% protein (pleissner and lin, ) , millions of tons of protein, from plant and animal sources, could be better used. agricultural production also has other unavoidable wastes associated with it, including manure and effluent, which also contain high levels of recoverable protein. these by-products and wastes find new life, often as animal feed ingredients. waste materials generated during agricultural production, including inedible plant and animal parts, are removed during harvesting and postharvest processing. other unavoidable nutrient-rich wastes, such as manure and deadstock, are also produced. due to their high levels of recoverable protein, carbohydrate and fiber, many of the by-products and wastes of the agricultural industry currently find reuse as animal feeds or animal feed ingredients. animal feed ingredients are blended in such a way as to create a more nutritious food for livestock. plant-derived ingredients include grains, such as maize, barley, sorghum, oats, and wheat (which can also be used for bioethanol production), from which the by-products are often diverted back to feed. these grain by-products include corn gluten meal, brewers and distiller's grains, malt sprouts, brewer's yeast, and wheat mill feed (lefferts et al., ; naik et al., ) . more importantly, it has been assumed that by , up to % of transportation fuels will be derived from biofuels, generating up to mmt of additional protein . higher value applications for inedible and nonessential amino acids derived from these by-products may eventually be commercialized, providing a feedstock for protein-based plastics, milk and dairy . - - . - - . - regions were grouped (gustavsson et al. ) into medium-to high-income regions (europe, united states, canada, oceania, and industrialized asia) and low-income regions (sub-saharan africa, north africa, west and central asia, south and southeast asia, and latin america). production process biopesticides or commodity organic compounds (naik et al., ; scott et al., ) . oil production by-products (oil meals and press cakes) from processing oilseeds, such as soybean, canola, sunflower seed, linseed, palm kernel and others, are also important feed ingredients. oil meals are obtained by solvent extraction of the oil cakes, which are obtained by pressing the seed. in , mmt of various oil meals were produced globally, of which mmt was soymeal (united states department of agriculture, a). in the united states alone, mmt of soymeal is produced annually (united states department of agriculture, a), representing more than two-thirds of the proteinaceous animal feed in the country (lefferts et al., ) . other oilseed meals are lower in protein and higher in fiber and are often used for feeding ruminants. cottonseed meal is also high in protein and is mainly used as cattle feed in the united states or as aquaculture feed. unlike other seeds, the press cake obtained from castor seeds during castor oil production is inedible because of its high level of phytotoxins (ricin, a toxic protein), hydrocyanides, and other allergens, however, this too has a high level of protein, ∼ - % (table . ). other plant ingredients may include alfalfa by-products, such as alfalfa meal, pellets, and concentrated alfalfa solubles, which are typically fed to ruminants. further, various nuts, seeds, and their by-products, such as hulls and seed screenings; legume by-products, such as bean straw meal and hulls; and even dried roots and tubers, such as sweet potatoes and chipped or pelletized cassava, find use in animal feed. agricultural production-specifically the production of animal-derived goods-also results in by-products. in fact, around wt.% of an animal produced for food is not used directly for human consumption, and downed or dead animals are another waste artefact of production. these waste materials are processed by the rendering industry, producing protein-rich products (table . ). global production of abp meals from rendering is in excess of mmt per year ( fig. . ) . these products include meat meal, meat and bone meal, poultry by-product meal, poultry meal, blood meal, feather meal, hydrolyzed leather and leather meal, eggshell meal, hydrolyzed hair, unborn calf carcasses, ensiled paunch, bone marrow, and dried plasma (lefferts et al., ) . other than the preceding, about wt.% of the fish caught globally each year is not used directly for human consumption; instead it is used to produce protein-rich marine by-products, in excess of mmt per annum (table . ). typical animal feed ingredients derived from marine origin include fishmeal, dried fish solubles, crab meal, shrimp meal, fish protein concentrate, and other fish by-products (lefferts et al., ) . finally, animal waste has also been used as a feed ingredient, including dried ruminant waste (manure), dried poultry waste, dried poultry litter, dried swine waste, undried processed animal waste products, and processed animal waste derivatives (lefferts et al., ) . according to the association of american feed control officials, in the united states, these processed animal waste products must be treated appropriately to ensure that the product is free of harmful pathogens, pesticide residues, parasites, heavy metals, or drug residues (association of american feed control officials, ). although recycled animal wastes have been knowingly incorporated into animal feed for almost years, the food and drug administration does not endorse the use of recycled animal waste (lefferts et al., ) . regardless, protein content in dried manure ranges from to wt.% for cattle, to wt.% for poultry, and to wt.% for pigs (chen et al., ) , making it another source of valuable protein and nutrients. just as the sources of waste are diverse, so too are the wastes generated, each with a different chemical and physical makeup, directly affecting how they are best used (table . ). many studies focused on the valorization of these and ( ); fuller et al. ( ) castor seed meal . annongu and joseph ( ) corn gluten meal . - . national research council ( ) continued ) other waste streams in a profitable way. obviously, for protein meals that can be fed to livestock or fish, the price for which they are sold will generally cover the cost of producing them, and in the case of abps, the revenue generates a reasonable profit. however, for inedible protein meals (including meals which either have no market or limited market access), adding value through conversion into novel products is of greater necessity. the problems with imparting additional value to these products is not necessarily related to the scientific or technological feasibility or even cost, but are most commonly associated with the perceived risks and often restrictive supporting legislation. every nation strives to maintain its biosecurity to protect its ecological and economic resources from disease and invasive pests. the most effective means of governing the risks posed by the importation of dangerous or questionable materials, and the harm they may cause to animals or humans, is to impose legal restrictions. the importance of maintaining biosecurity is most apparent when considering the risks of international trading. the introduction of invasive pests and disease through international trade could lead to adverse effects, not only on plant a based on production statistics for - in animal feeds compendium ( (ash, total global production ∼ million metric tons. and animal health, but biodiversity and food production as a whole, and should be appropriately managed (maye et al., ) . these measures must consider not only the scientific evidence supporting such a restriction, but must also consider any reasonable precautions that can act to offset any deficiencies in a solely scientific approach. hence, during the development of a new policy, a risk analysis is first performed, followed by evaluation of that risk through the lens of current legal, institutional, social, and economic circumstances, all of which is undertaken by the stakeholders who represent them (mills et al., ) . as such, risk governance deals with the management of both perceived and scientifically founded risks. although risk management implemented through public policy is focused at the national level, many food and natural resource policies operate at levels both below and beyond the national level (mills et al., ) . however, as a result of the discrepancies between each state's local policy making and a lack of cohesive global regulations, the intersection between risk and commerce continues to be a major challenge facing the international trading system. a significant amount of trade conflict experienced at the world trade organization has involved the united states, canada, and/or the european union (hornsby, ) . some topics that became the focus of either formal or informal disputes have included hormone-fed beef, bovine spongiform encephalopathy (bse), raw milk cheese, genetically modified organisms, chlorine-washed chicken, and wood packing materials. such disputes imply the presence of a transatlantic divide over what constitutes a legitimate risk regulation, however, this is an oversimplification. although the risk regulations set forth by the european union take a precautionary approach, acting in light of scientific uncertainty and taking into account public concerns, the us system is based on a "sound science" approach, free from political influence, however, this has not always been the case. it has been argued that the united states used to be more precautionary than the european union (hornsby, ) , but was pressured to limit the calculation of risk in public policy. the eu's regulatory failures during food safety crises served to undermine public trust in the eu institutions, resulting in the use of a precautionary approach (hornsby, ) . overall, it has also been proposed that both regions partake in "occasional and selective application of precaution to different risks in different places and time" (wiener, ) . nevertheless, there are some consistencies around the world regarding the safe handling, distribution, and disposal of food, animal wastes, and by-products. products from byproducts dairy milk, butter, cream, yogurt, cheese, ice cream whey, wastewater whey protein adapted from de las fuentes et al. ( ) and ramachandran et al. ( ) . the degree to which protein by-products, particularly abps, can be used is limited by the customs, religions, and regulatory requirements of the region. all feedstuffs imported into a country must comply with rules regarding hygiene, traceability, contaminants, labeling requirements, and health issues given its expected use. the use of the product is then subject to more specific rules, largely limiting the use of those feedstuffs containing animal-derived products. the first diagnosis of bse in the united kingdom in and the subsequent publication in that new variant creutzfeldt-jakob disease in humans had most probably arisen from exposure to bse-infected meat, sparked a global crisis with respect to food safety and risk management. up until the outbreak of bse during the s, almost all protein by-products were used as feed supplements for livestock. in , the practice of feeding ruminant animal protein meals to other ruminants was banned, along with the use of specified bovine offal (brain, spinal cord, other organs potentially infected with bse) (ockerman and hansen, ) . more recent infectious disease outbreaks, such as avian influenza and severe acute respiratory syndrome, have further jeopardized diplomatic relations, frightened the public, and caused massive economic losses by disrupting global commerce (karesh and cook, ) . since then, concern over the risks posed by abps, including infectious diseases (such as swine fever, foot and mouth) and other contaminants (such as dioxins), to human and animal health, has resulted in strict regulations regarding their safe handling and disposal (cunningham, ; department for environment food and rural affairs, ). as such, most countries now have local regulations put in place that are typically broad in scope and directly affect any person or business that generates, uses, disposes, stores, handles, or transports food waste containing animal products and abps derived from the food processing industry. currently, most countries no longer allow animal by-product meals containing any amount of ruminant tissue to be fed to other ruminant animals, although meat and bone meals containing ruminant tissue are still able to be fed to nonruminant animals, such as poultry, swine, pets, and aquaculture species in most countries, including new zealand (garcia and phillips, ). to the contrary, throughout the european union, meat and bone meals are banned from the feed of any animal that may become human food, and as a result, in the european union, meat meal and meat and bone meal are primarily incinerated or used as an ingredient in pet food (kirchmayr et al., ) . in most countries, legislation for waste disposal and disposal of dead animals and of slaughterhouse materials (animal rendering) is already in place. in germany, the animal disease act, the meat hygiene act, the poultry meat act, and the meat hygiene ordinance also regulate the disposal of slaughterhouse offal. to protect animal and human health, the canadian food inspection agency (cfia) enforces federal regulations governing the production and use of rendered materials that may be used in animal feed. however, a policy established by the national renderers association, which prevented ovine material (sheep) from being used in meat and bone meals in the united states and canada, and has been withdrawn (malone, ) . compared to canadian and us policy, the framework of the eu regulations regarding abps and derived products is complex, resulting from ongoing reviews by the eu commission. each updated regulation is a result of the successive amendment to the initial regulation (ec) / , most recently amended with (eu) no. / . the regulation covers the safe disposal options available for all animal products, including meat, fish, milk, and eggs not intended for human consumption, and other products of animal origin, including hides, feathers, wool, bones, horns, and hooves. it also prohibits catering waste being used as livestock feed and covers disposal of fallen stock, companion animals, and wild animals if they are suspected of being diseased. the regulations also control the use of abps as feed, fertilizer, and technical products with rules for their transformation via composting and biogas operations and their disposal via rendering and incineration (department for environment food and rural affairs, ). when looking at the existing options available for management of these by-products (and/ or wastes), both legal regulations and the best ecological and economical solutions need to be considered. whether a material is deemed to be a valuable by-product (or a waste that needs to be disposed of) depends on the social, legal, and technological framework surrounding its origin. from there, the most sensible form of management becomes a compromise between what is viewed as acceptable, based on legal requirements and local perceptions, and what is technologically and financially feasible ( fig. . ) . although it is most desirable to prevent waste and by-product formation, followed by reuse or recycling into other product lines, the formation of by-products and waste is inevitable, and management options must be innovative and also meet local regulatory requirements. waste management is then possible through several media: to use it in its current form, dispose of it through incineration or landfill, or add value to it through bioprocessing or valorization technologies ( fig. . ) . the choice of media used will largely depend on the cost, customs, and regulatory environment. for example, converting the by-product to animal fodder (bioreduction) may not be feasible in all countries. excess and waste food has been used as animal fodder for centuries, and in many parts of the world, farmers still use waste food to feed their animals-primarily pigs and poultry. the practice of feeding waste material containing meat products to pigs was banned in the united kingdom in (statutory instrument , no. the animal by-products amendment) to prevent further spread of bse, and soon after, a new regulation was implemented throughout the european union (the animal by-products regulation, ec no: / ) prohibiting catering waste from being fed to farmed animals. this includes all waste food and used cooking oils, as well as waste from vegetarian restaurants and kitchens. based on these laws, only certain types of waste food can be given to livestock and must first be treated appropriately. if the by-product cannot be immediately used as it is or treated appropriately for use as an animal feed, it must be safely disposed of. because of the time and expense of treating these food wastes, most end up in landfill. currently, landfilling and incineration account for the treatment of greater than % of food waste in most european countries (melikoglu et al., ) . in general, using the biomass waste in the form it is in, either as an animal feed or fertilizer or as a fuel to generate electricity, is the most simplistic approach and generates a value of ∼us$ - per metric ton of biomass (tuck et al., ) . incineration is the simplest means of waste disposal, with its major advantage being the significant reduction in volume of the waste stream, which is up to % for waste streams with high amounts of paper, cardboard, plastics, and horticultural waste (hoornweg and bhada-tata, ) . however, most food wastes are not appropriate for incineration, owing to their high moisture content. when properly equipped, an incinerator can be used as a means of energy recovery to generate electricity. heat released from the combustion of waste can be used to produce steam, which can turn a steam turbine, generating electricity. however, because of the increased concentration of toxins in the ash, incinerators must be operated alongside landfill systems in order to dispose of them. combustion destroys chemical compounds and diseasecausing bacteria, leaving it pathogen free, but causes serious environmental problems through the production of carbon dioxide, nitrogen oxides, sulfur dioxide, and trace quantities of toxic pollutants, such as heavy metals and dioxins. the remaining residues are often landfilled, owing to their high heavy metal content. thermochemical conversion of food and industry wastes are an effective means of converting energy-rich biomass into a more easily used liquid or gaseous intermediate. high temperatures can be used with minimal (gasification) or no oxygen present (pyrolysis) to break down hydrocarbon containing wastes, resulting in combustible syngas mixtures, containing carbon monoxide and hydrogen ( %), with small amounts of carbon dioxide and methane. this syngas intermediate can be further processed to produce bio-based gasoline, diesel, or jet fuel, or be used in a fuel cell to generate electricity or steam. landfills-burying the material-are a common final disposal site for waste and the residues remaining from other treatment options. at atmospheric pressure, metric ton of organic material generates approximately - m of landfill gas over a - year timeframe (jardine et al., ) , comprised of - % methane and - % carbon dioxide, which represents around % of the anthropogenic methane (ch ) emitted worldwide (melikoglu et al., ) . methane has times the global warming potential of carbon dioxide and can be recovered and burned (with or without energy recovery) to reduce greenhouse gas emissions (hoornweg and bhada-tata, ) . other serious environmental implications of landfilling include the risk of leachate (potential toxic liquid that drains from landfills) entering surrounding soils and groundwater. although the use of landfills is common, their use has been discouraged through the implementation of landfill taxes and directives, such as the uk "landfill tax" in and eu landfill directive established in (jardine et al., ) . obviously, other disposal options are preferred to landfilling, which costs ∼us$ per metric ton. around % of the municipal waste sent to landfill is biodegradable and mostly comprised of food waste (hoornweg and bhada-tata, ) . this makes bioprocessing, such as composting and anaerobic digestion, sensible options for disposing of these organic waste streams. a common means of obtaining a safe end product is achieved through composting. this involves a combination of chemical and microbiological processes occurring throughout three stages that convert organic materials to a stable, soil-like product called compost (som et al., ; verbeek et al., ) . provided composting is carried out well, the volume and mass of the waste can be reduced by up to %. for composting to occur efficiently, the conditions of the composting process must be maintained at an optimal level to encourage microbial growth. because of changes in the composition of waste material with location and over time, the compost mixture needs optimization through regular adjustments. for example, if the system becomes anaerobic, offensive odors can be produced, and if it becomes too wet or too dry, the process will halt altogether. some of these organic waste materials require specific pretreatment before composting can occur. in the united kingdom, eu standards must be implemented over and above uk standards if the site treats category abps, which have first been pressure rendered, or category abps if they exclude catering waste. exceptions apply for some types of abps in the united kingdom, which can be composted in closed reactors at °c for more than h or in housed rows of piled green-waste (windrows) at °c for more than days under strict operating parameters with a maximum particle size of mm. although compost is of limited value, it is still a more economic option compared to landfilling. other bioprocesses can be employed that produce more valuable products. biofuels can be produced using fermentation, valued at us$ - per metric ton more than the initial biomass waste (tuck et al., ) . anaerobic digestion is another means of disposing of organic waste materials and is carried out in an enclosed vessel. the methane generated can either be flared or collected for combustion to generate heat and/or electricity, which also adds value to the waste biomass. the maximum value can be recovered from these waste materials by converting them into more purified streams and using them in the manufacture of lubricants, surfactants, plastics, fibers, and industrial solvents. theoretically, all abps in the european union could be combusted as fuel for energy, provided the eu commission formulates the appropriate rules and regulations, which as of yet has not been done. although there are many technologies currently available (or in developmental stages) that aim to valorize by-products of industry, legislation has yet to be passed that explicitly deals with higher technology outcomes. most current law deals with the safe handling and disposal of animals, their products, and by-products and animal feeding. although it is necessary to contain health and environmental risks through appropriate legislation, it is becoming apparent that the use of abps and food wastes (excluding crop residues and some agro-industrial byproducts) for animal fodder and composting is not only obsolete, but in many nations, illegal. many technologies exist that aim to valorize by-products of the agricultural industry. although the edible portion of these protein-rich by-products could be used for recovery of essential amino acids for human consumption, or as is for use in animal feeds, higher value applications for inedible and nonessential amino acids may include providing a feedstock for proteinbased materials, such as plastics, and for the production of biopesticides and commodity organic compounds (naik et al., ; scott et al., ) . along with more obvious uses of protein hydrolysates-animal feeds and biomass for energy recovery-protein-based meals from crop residues and agro-industrial by-products also find value addition through use in biological processes. an example is the use of various oilseed cakes, which have been shown to be ideal mediums for many types of bacteria and fungi responsible for producing a variety of enzymes, antibiotic and antimicrobial compounds, and bioactive metabolites (ramachandran et al., ) . protein-based raw materials can be used for the production of , -ethanediamine and , -butanediamine from the amino acids serine and arginine, respectively . furthermore, protein-based surfactants are valuable mild surfactants, because the structure and properties of the amino acids in the surfactants are similar to the amino acids that make up the tissue of skin. if valorization technologies are to be implemented on a commercial scale, they must work within current legal constructs. however, this does not deal directly with the science involved and may inhibit progress if new legislation is not developed that more closely examines the evidence and whether risk regarding human and animal health is still an issue. in light of current legislation and potential markets for value-added commodities, it is becoming apparent that the use of protein-rich agricultural by-products for lower value applications, such as animal fodder, is no longer a sensible use of such a valuable resource. energy values of feed ingredients for white pekin ducks comparative nutritive value of cassava feaf meal, soya beans, fish meal and casein in diets for growing pigs proximate analysis of castor seeds and cake official publication: association of american feed control officials marine and freshwater products handbook value-added chemicals from animal manure. pacific northwest national laboratory, united states department of energy total food: exploiting co-products-minimizing wastes: proceedings. institute of food research, norwich, england. department for environment, food and rural affairs, . controls on animal by-products: guidance on regulation (ec) / and accompanying implementing regulation (ec) / , enforced in england by the animal by-products (enforcement) (england) regulations . department for environment, food and rural affairs evaluation of cow pea seed meal, vigna sinensis, as a dietary protein replacer for nile tilapia shrimp byproduct feeding and growth performance of growing pigs kept on small holdings in central vietnam protein quality of shrimp-waste meal. bioresour protein utilisation, lysine bioavailability and nutrient digestibility of shrimp meal in growing pigs can we feed the world and sustain the planet? a five-step global plan could double food production by while greatly reducing environmental damage potential for detoxified castor meal physical distribution and characteristics of meat and bone meal protein food security: the challenge of feeding billion people the future of the global food system global food losses and food waste: extent, causes and prevention. food and agriculture organization of the united nations fish meal: historical uses, production trends and future outlook for sustainable supplies market potential and processing of blood products: some overseas observations what a waste: a global review of solid waste management risk regulation, science, and interests in transatlantic trade conflicts. international political economy series variation in ruminal degradation and intestinal digestion of animal by-product proteins methane uk, research report . environmental change institute, university of oxford world renderers organization: rendered products worldwide. global feed & food congress in situ ruminal dry matter and crude protein degradability of plant-and animal-derived protein sources in southern turkey the human-animal link. foreign affairs proximate composition of selected potential feedstuffs for smallscale aquaculture in ethiopia nutritional evaluation of various feedstuffs for livestock production using in vitro gas method. pak utilization of by-products and treatment of waste in the food industry feed for food-producing animals: a resource on ingredients, the industry, and regulationvol. bloomberg school of public health animal by-products (abps): origins, uses and european regulations. universitas studiorum, mantova nra rescinds sheep material policy. american sheep industry association, asi weekly blood and haemoglobin meal as protein sources in diets for gilthead sea bream (sparus aurata): effects on growth governing biosecurity in a neoliberal world: comparative perspectives from australia and the united kingdom. environ analysing global food waste problem: pinpointing the facts and estimating the energy content. cent. eur integrating natural and social science perspectives on plant disease risk, management and policy formulation feeding the future's changing diets: implications for agriculture markets, nutrition, and policy. paper presented at the international food policy research institute conference: leveraging agriculture for improving nutrition and health the world's biggest industry investigation to determine digestibility coefficients of various raw materials in diets for gilthead sea bream animal by-product processing and utilization evaluation of shrimp waste meal as a probable animal protein source for broiler chickens sustainability of meatbased and plant-based diets and the environment valorisation of food waste in biotechnological processes evaluation of traditional sorghum (sorghum bicolor) beer residue, shea-nut (vitellaria paradoxa) cake and cottonseed (gossypium spp) cake for poultry in burkina faso: availability and amino acid digestibility feed composition table use of the mobile nylon bag technique to determine the digestible energy content of traditional and non-traditional feeds for swine oil cakes and their biotechnological applications-a review biorefinery as the bio-inspired process to bulk chemicals agricultural residues as a source of bioactive natural products assessment of the availability of agricultural crop residues in the european union: potential and limitations for bioenergy use biomass in the manufacture of industrial products-the use of proteins and amino acids stability and maturity of a green waste and biowaste compost assessed on the basis of a molecular study using spectroscopy, thermal analysis, thermodesorption and thermochemolysis market report: us rendering: a $ billion industry comparative value of fish meal alternatives as protein sources in feeds for hybrid striped bass valorization of biomass: deriving more value from waste oil crops outlook. united states department of agriculture, washington, dc. united states department of commerce, . current industrial reports: manufacturing profiles . economics and statistics administration, bureau of the census editors, united states department of commerce biodegradation of bloodmeal-based thermoplastics in green-waste composting livestock meat processing: inventory data and methods for handling co-production for major livestock species and meat products. paper presented at the ninth international life cycle assessment of foods conference the reality of precaution: comparing risk regulation in the us and europe key: cord- -g z gq m authors: buffenstein, rochelle; park, thomas; hanes, martha; artwohl, james e. title: naked mole rat date: - - journal: the laboratory rabbit, guinea pig, hamster, and other rodents doi: . /b - - - - . - sha: doc_id: cord_uid: g z gq m naked mole rats are mouse-sized rodents that have become an important animal model in biomedical research. they play a unique mammalian role in behavioral and ecophysiological research of life underground. this chapter studies the general physiology, anatomy of organ systems, husbandry, and uses in research of the naked mole rats. naked mole rats belong to the order rodentia in that they have two incisor teeth on the upper and lower arcade that continuously grow. the skin is loose, wrinkled, and brownish pink in color. the body is for the most part absent of hairs with the exception of tactile hairs that are regularly arranged throughout the body and which are particularly prominent around the face and to a lesser extent on the tail. they are typically housed at – °c, and at – % relative humidity. because naked mole rats are social and have cooperative behaviors, the study of their conduct has more applicability to people. the chapter describes the models of experimental research on the naked mole rat such as the model of reproductive suppression, model of somatosensory processing, model of bone elongation, and model of aging. considered to be either neonates of a much larger species or mammals that had lost their fur as a consequence of disease. subsequent collections and descriptions were reported in the s through and are summarized by brett ( ) . most of those studies focused upon the unusual anatomy of this species and morphological adaptations to life underground. jennifer jarvis began studying these animals in the late s and was the first person to bring these hairless rodents into the laboratory. she observed that these animals were highly social and appeared to work together to excavate tunnels and find food. she also noted that she seldom found pregnant animals in the wild and even in captivity had little success with the formation and maintenance of breeding colonies. subsequently, a group of biologists primarily working with jarvis (brett, buffenstein, clarke, faulkes) began studying and reporting on their unique biology. in the mid s, alexander, an evolutionary biologist whose research focused on the evolution of eusociality (a colonial lifestyle with a strict division of labor culminating in the presence of a single breeding female), described the ideal hypothetical eusocial mammal. at that time no eusocial mammals had yet been identified and only the wasps, ants, and bees were known to be eusocial. he described his hypothetical eusocial mammal as strictly subterranean, living in hard soils and cooperatively foraging on large underground tubers. a colleague and friend of jarvis, tl vaughan, told alexander that he had aptly described the naked mole rat, rodents that vaughan had seen in the office of jarvis. alexander, his doctoral student sherman, and jarvis thereafter began an extensive collaborative study on eusociality and behavior in the naked mole rat. biologists bennett, clarke, faulkes, o'riain, and sherman have contributed greatly to the understanding of naked mole rat behavior. buffenstein studied with jarvis and accompanied her on her trip to kenya in to assess if naked mole rats in the wild also live in colonies with only one breeding female. jarvis published this seminal finding in science in (jarvis, ) . her team published extensively on ecophysiological responses which allow naked mole rats to survive and thrive in their dark, dank environment in the arid and semiarid regions of northeast africa. these responses include mineral metabolism in the absence of sunlight , kidney function (urison and buffenstein, ) , thermoregulation withers and jarvis, ) , gastrointestinal function , microecological aspects of the naked mole rat (bennett and jarvis, ) , and finally the biological characteristics that contribute to the exceptional longevity of this, the longest-lived rodent known. naked mole rats have been documented to live longer than years in the lab . more recently, neurophysiologists (catania, comer, crish, goldman, and park) have analyzed the nervous system of these animals and have identified many unique neurological features that allow these animals to thrive in their environment. one of the interesting aspects of the naked mole rat nervous system is their lack of ability to sense chemical pain (capsaicin and acid) and inflammatory pain (park et al., ) , although they respond normally to acute pinch and heat (kanue and hole, ; kanui et al., ; park et al., ; towett et al., ) . naked mole rats belong to the order rodentia in that they have two incisor teeth on the upper and lower arcade that continuously grow. they are in the family bathyergidae which includes six extant genera (kock et al., ) , and includes solitary (bathyergus; georychus; heliophobius), and social taxa (cryptomys, fukomys and heterocephalus) . the suborder is difficult to classify since the criteria for classification are the origination of the masseter muscle and the degree to which the muscle passes through the infraorbital foramen. the bathyergidae have a small infraorbital foramen and the masseter muscle does not enter into it. nonetheless, the lateral angle of the jaw relative to the plane of the incisor teeth appears more hystricomorph-like than myomorph or sciuromorph. in addition, the bathyergidae share several hystricognath features which include similar fetal membranes and sacculus rethralis, a fused malleus and incus, an anterior opening in the pterygoid foss, multiserial incisor enamel, the loss of the internal carotid artery, and albumin immunology . taxonomists thus classify the bathyergidae in the suborder hystricognathi. this suborder consists of three super families (the phiomorpha (e.g. mole rats, rock rats, and cane rats), caviomorpha (e.g., guinea pigs), and hystricidae (e.g. porcupines). interestingly, most hystricognath rodents are found in the western hemisphere, and there are no close relationships between members of the bathyergidae family and other hystricomorph families. molecular genetic data suggest that the naked mole rat is a sister lineage to the group comprising all other genera in the family. heterocephalus forms a monotypic genus with no morphological variation nor variation in chromosome number (n  ). earliest fossil records of this species were found in east africa and date back to the early miocene ~ million years ago. vi. other rodents arched back over lumbar and sacral regions (hamilton, ) (figure . ). the head is cone-shaped and blunted anteriorly in the area of a horseshoe-shaped region that surrounds the nares. procumbent incisor teeth extend well outside the mouth and are characteristic of the bathyergidae. naked mole rats have either two or three molar teeth. the eyes are tiny with thickened eyelids and minute eyelashes. the auditory meatus is raised slightly and pinnae are absent. the masseter muscles are prominent. the skin is loose, wrinkled, and brownish pink in color. the body is for the most part absent of hairs with the exception of tactile hairs that are regularly arranged throughout the body and which are particularly prominent around the face and to a lesser extent on the tail. the outer edges of the hind feet are fringed with stiff hairs that increase the surface area of the foot. there are five toes on each foot. the tail is about half the length of the body. naked mole rats are sexually monomorphic; the external genitalia of males and females appear very similar. males have internal testes and most females have unperforated vaginas. males and females have similar-looking external genitalia, ano-genital distance, perineal muscles, perineal motoneurons, brain, and spinal cord (holmes et al., ; lacey and sherman, ; peroulakis et al., ; seney et al., ) as well as femoral bone morphology and microarchitecture and strength (pinto et al., ). however, these traits differ among breeders and subordinates. there is no sex difference in adult body weight. animals raised in captivity have body weights that vary with age, food availability, colony composition, and social and reproductive status (jarvis, ; jarvis et al., ; lacey and sherman, ; o'riain and jarvis, ; o'riain et al., ) . most mature animals range from - grams in captivity but can get as large as grams. this large range in adult body mass is uncommon among small mammals (jarvis, ) . naked mole rats are one of the slowest-growing mammals and reach stable body mass by the end of the first year of age. individuals born in the first few litters of a newly established breeding female grow more rapidly than those of later litters (o'riain and jarvis, ) . by years of age all non-breeding animals have stopped growing. should a dominant animal die or be removed from the colony many individuals will have a growth surge, and this is most pronounced if the breeding female dies. any animal within the colony is capable of becoming a breeder and individuals as young as months or as old as years have changed their social status. should a female change her social status in the colony and become a breeder; she is capable of significant vertebral growth regardless of her age. indeed a female that became a breeder for the first time at years of age still underwent a prolonged period of lumbar vertebral growth. the members of the bathyergidae family are all african rodents and are found in distinct geographic locations from the southern-most tip of the continent to about °n of the equator. bathyergids are found in several distinct climatic zones, at different altitudes and in a wide variety of soil types. naked mole rats are found in the arid and semi-arid regions of eastern equatorial africa, commonly found in kenya, ethiopia, and somalia. their habitat gets irregular rain, but it is generally hot and dry with little seasonal or daily variation in climatic conditions, followed by intense short periods of high rainfall. the soil in which they burrow varies from sandy to hard clay soils. the only evidence of the presence of naked mole rats below the surface is small volcano-like mounds of soil excavated by the mole rats during burrow extensions. naked mole rats live in an extensive maze of underground tunnels and chambers. depending upon the size of the colony these may extend more than mile ( . km) in length and may reach depths of up to meters. there are multiple layers of interconnected tunnels and chambers. some tunnels are more permanent (highways) while others near the periphery and surface are opened and closed depending on the needs of the colony. the more superficial tunnels are used for gathering food from tuberous plants, and may be used during behavioral thermoregulaion by animals basking in warmer regions of the burrow system. these near-surface tunnels also serve as a path to eliminate soil from new burrows. the deepest tunnels generally open into football-sized nest chambers in which most of the mole rats will huddle together, often resting with many animals piled up on top of each other. the nest area is lined with tufts of grass roots, skins of tubers and bulbs, and other soft detritus encountered in figure . photograph of typical naked mole rats. their elongate tubular shape allows them to move through tunnels. teeth are exterior to the mouth, and the ears and the eyes are tiny with thickened eyelids and minute eyelashes. the auditory meatus is raised slightly and pinnae are absent. the masseter muscles are prominent. the burrow system. often near the nests are deep, blindending tunnels that are presumed to be bolt holes and which may possibly serve as drainage systems during periods of heavy rainfall. interconnecting tunnels that run at a slant are thought to play an important role in ventilation of the colony. adjacent to the nest chambers in the deeper confines of the burrow system is a blind-ending toilet chamber in which naked mole rats generally urinate and defecate. when these become full, the chamber is plugged with soil and a new toilet chamber excavated. similarly food hoards or pantry-like chambers are located near the nest; here small bulbs, corms, and roots that have been located during foraging are carried back to the nest and stored or shared among the colony. mole rats maintain their burrows all year round, but predominantly extend their burrow system shortly after rains occur when the dry sun-baked soils are moistened and no longer hard and impenetrable. a sealed subterranean burrow complex protects the inhabitants from predation and also protects against climatic extremes. while surface temperatures above ground may become unbearably hot, the thermal conductivity properties of soil result in attenuated fluctuations in burrow temperatures such that burrow temperatures in the warm confines of equatorial africa vary by less than °c seasonally (bennett et al., ) . while superficial burrows may heat up during the day and remain considerably warmer than deeper burrows, burrows at depths greater than cm are thermally stable. the gaseous atmosphere in the burrow system, and in particular in the deep nests, is extremely challenging (bennett and faulkes, ) . the burrows are usually sealed and gas exchange through soil is limited. this coupled with so many organisms respirating (naked mole rats, commensals such as insects and lizards and microfauna) result in an hypoxic ( - % oxygen) and hypercapnic (~ % co ) atmosphere almost saturated with water, even when soils are dry. air movement through the burrow is extremely limited and primarily facilitated by animals in tight burrows acting like pistons pushing air in front of them as they move through tunnels. respiration in such atmospheres, especially in animals that are metabolically active or resting in large communal groups may be especially problematic. furthermore the impact of high levels of ammonia or methane in nests and latrines could exacerbate respiration and acid-base balance. naked mole rats are eusocial, strictly subterranean mammals in the wild. the term eusocial describes species that live in colonies of overlapping litters in which one or a few individuals produce all the offspring and the rest serve as functionally sterile helpers in colony maintenance, rearing juveniles and protecting the colony (michener, ) . the only other mammal that meets these criteria of eusociality is the naked mole rat's close cousin the damaraland mole rat (fukomys damarensis). a typical naked mole rat colony has only one breeding female (queen) and a few ( - ) breeding males. the remainder of the colony is loosely categorized by body size. the larger animals tend to be the first captured in field studies and are thought to be the defenders of the colony. the smaller animals function as food gatherers and burrow diggers. with the exception of the "dispersomorphs," animals that occasionally leave their natal colony, presumably to outbreed and form new colonies, naked mole rats only come onto the surface if flooded out of a burrow system or if their burrow system has been breached. despite their evolution in a formidable dark, dank environment, naked mole rats thrive in a laboratory setting and display many of the social behaviors that make them unique and of considerable interest for biological and biomedical research. in the wild, colonies of naked mole rats may vary from a few animals to an average of animals to as many as or more animals (brett, ) . in an institutional setting, colonies may be formed from breeding pairs and despite being housed in much smaller burrow systems may reach comparable sizes to wild colonies. average colony size in captivity is ~ , with maximum colony size exceeding individuals. naked mole rats are extremely xenophobic and readily attack and kill conspecifices from foreign colonies. members of a colony may roll in the excreta within the toilet to confer a colony scent. this enables colony members to identify each other as members of the group or strangers. naked mole rats are strictly herbivorous, feeding on roots, bulbs, corms, and tubers which they locate by excavating tunnels until they stumble across a large tuber or a patch of small bulbs. this is an energetically costly and blind process. nmrs dig/forage in working groups. typically a lead member digs and passes the soil back to other members who move the soil to other areas of the tunnel system or to the surface. this effort has been described as a chain of diggers. occasionally members will move the soil to the surface and deposit it in volcano-like structures. this is the only time tunnels are open to predation. mole rats generally carry back to the nest small foods (roots, bulbs) they encounter and bites of the large tubers they find. the large tubers may weigh as much as kg and tend to be "farmed" in that animals will gnaw at parts of the tuber, carefully removing the often poisonous outer layers and then plug the regions they have consumed with soil, thereby enabling the tuber reproductive behavior vi. other rodents to regenerate. such large tubers may sustain the colony during the dry season when burrow excavations are energetically prohibitive (lovegrove and wissel, ) . given the high cost associated with random foraging in regions where food is patchily distributed, a single animal may easily starve to death before foods are found. by working in foraging groups; animals are able to search for food in multiple areas at once and the colony increases its chances of finding a patch of food or a tuber that is large enough to sustain the entire colony. new food sources are communicated to the group through an odor trail (judd and sherman, ) and cheep-like vocalizations. naked mole rats have an extensive vocal communicate repertoire. pepper et al. ( ) described different vocalizations that include alarm calls, food recruitment calls, mating calls, toilet-assembly calls, and vocalizations specific to pups. these vocalizations range from hz to . khz at db (hefner and hefner, ) , but the majority of noises are low frequency ( - khz) (judd and sherman, ) . lower-frequency noises propagate better underground and nmrs take advantage of this. the soft chirp is characteristic of individual members of the group and may be important in identifying individual members (yosida et al., ) . there are also several distinct alarm calls employed during cooperative predator avoidance and the defense of the colony. colony defense is an important behavior in the colony. if a major predator or member of a foreign colony is encountered in the tunnels, naked mole rats produce a pronounced alarm call and an obnoxious odor, and defenders are recruited in mass to attack the offender. indeed even snakes may be killed by the wall of teeth they encounter, should they not capture the first mole rat they come upon. young pups in the colony employ many different kinds of begging behavior and emit various vocalizations to induce an appropriate response from the breeders and older siblings. one of the most common types of these behaviors is the begging for feces from adults. adults respond by voiding feces which are directly consumed from the anus. captive colonies of naked mole rats normally contain one breeder female, and she can retain this position for many years (jarvis, ) . the breeding females may produce more than offspring during their breeding reign and litter size tends to increase sometime after the breeders become established. on rare occasions there may be an additional female breeding within the colony. the level of dominant female aggression appears to influence whether this occurs and in most cases the offspring of the second breeder in the colony seldom survive. reproductive suppression occurs by the queen's dominance-related behavioral aggressiveness and requires direct contact between the queen and subordinates abbott, , ; margulis et al., ) . the direct contact takes the form of pushing and shoving. once dominance is established subordinates readily display subordinate behaviors, they allow the breeders to eat first and they generally tend to be walked over when they encounter a breeder in the tunnel. aggression in a colony occurs mainly between the queen and other challenging females. often challengers attack and kill the breeding female when she is at her most gravid or during parturition. if the dominant female dies, succession appears to be rapid, but it may take months to years for colonies to stabilize. succession appears to come from animals that tend to be larger. this is usually the only time when fighting within a colony occurs. typically, when the queen identifies a colony member to attack the subordinate animal submits and may even be killed. captive colonies of naked mole rats have several breeding males. in contrast to females, there is no evidence of breeding competition in males (clarke and faulkes, ) . both dominant breeding males and females have higher levels of sex steroid hormones than the non-reproductive colony members. lordosis and mounting behavior are commonly observed in the queen and breeding male respectively during the periovulatory period as is seen in other rodent species (goldman et al., ) . the female is restless during estrus and initiates courtship behavior by thrusting her rear end into the face of the male and lordosing. mating occurs several times between a - -hour period and often occurs in the tunnels. anogenital nuzzling in breeders is also observed and occurs at all phases of the estrus cycle. in this behavior the queen and a breeding male lie side-by-side in the nasoanal position. this latter behavior is not dependent on androgen status in the male, and is most commonly observed in the nest. naso-genital stimulation of the breeding males is thought to play an important role in elevating and maintaining active hypothalamic/pituitary/gonadal axes compared to non-breeders. the latter have low levels of testosterone and extremely low levels of luteinizing hormone and furthermore are unresponsive to administration of exogenous gonadotropin releasing hormone (faulkes et al., ) . spermatogenesis occurs in both breeding and nonbreeding males. non-breeding males tend to be sterile, producing fewer and predominantly non-motile sperm (endo et al., ; faulkes et al., ) . similarly subordinate females for all intensive purposes appear to be sterile. endocrine and anatomical studies reveal that these females show no signs of ovarian cyclicity or ovulation and that their ovaries are in a functionally quiescent state. this state may change should the subordinate female be isolated from the breeding female of her colony. since usually only one female breeds in a naked mole rat colony, the majority of animals (males and females) spend their entire lives in a suspended sterile "prepubescent" reproductive state. these subordinates spend their entire lives maintaining the colony and its burrow systems and generally helping raise the young. a breeding female breeds continuously throughout the year and may have as many as four litters per year. the average litter size is , but may be as small as one pup and as large as . in captivity, the gestation period ranges from - days and there is a - -day post partum estrus, such that the inter-birth interval is approximately days. the first sign of pregnancy is a marked increase in body temperature of the breeding female (urison and buffenstein, ) and during this time the breeding female spends considerable time basking under heat lamps or in the warmer confines of the burrow. the female becomes obviously pregnant at about days gestation, when her weight begins to increase significantly. similarly in the last trimester of pregnancy the very gravid queen spends more time in the nest or basking under heat lamps, and other females and males in the colony show signs of nipple enlargement and some females even show perforate vaginas. by the end of pregnancy a female may more than double her non-pregnant body mass. both pregnancy and lactation are energetically costly processes, increasing metabolic rate . - -fold (urison and buffenstein, ) . the young weigh about . - . g. the queen nurses the pups for weeks, although they start eating solids and begging for and eating feces from older siblings at days. fecal consumption provides the appropriate microfaunal innoculum necessary to digest fiber. naked mole rats have the highest inbreeding coefficient known for wild mammals with a mean coefficient of relatedness among colony members estimated at . (reeve et al., ) and there is considerable evidence that most new colonies form by fission. there is, however, strong evidence that there is an outbreeding dispersomorph caste with a morphologically, physiologically, and behaviorally distinct phenotype (o'riain et al., ) . this disperser caste may occasionally promote outbreeding in naked mole rats, especially in those colonies that are not successfully reproducing. these dispersers are unwilling to engage in colony maintenance, are laden with fat, especially in the neck and pelvic regions, exhibit elevated levels of luteinizing hormone, and solicit mating behaviors with non-colony members and as such are readily accepted by foreign colonies. in captivity, these animals are often seen standing on their hind limbs, trying to escape from the colony and it seems that if there is a colony escapee it is usually a member of this caste. naked mole rats have low metabolic rates, approximately % of that of similar-sized rodents . outside the confines of their warm, humid equatorial burrows, naked mole rats cannot regulate their body temperature over a broad range of environmental temperatures ( - °c) and are thus considered poikilotherms (buffenstein and yahov, ; hislop and buffenstein, ) . indeed both their metabolic and body temperature profiles at temperatures below °c, closely track that expected for a similarsized lizard. at temperatures above °c, they display a typical mammalian endothermic pattern with the highest metabolic rate at the lowest temperature and a decline in metabolic rate, with the lowest rate at temperatures between - °c; temperatures considered the apparent themoneutral zone (mcnab, ) . the relative increase in metabolic rate from - °c is similar to that observed in mice housed within their thermoneutral zone and in a refrigerator at °c. hence, given their lack of insulatory pelage, naked mole rats have a much narrower window in which they can thermoregulate relative to other mammals. indeed it is only at subzero temperatures that mice show the similar hypothermic "reptilian" response of a decline in metabolism and body temperature as conditions become colder. at temperatures above °c, naked mole rats employ non-shivering thermogenesis (hislop and buffenstein, ) initiated by catecholaminergic innervation of interscapular brown fat (daly et al., ) . brown fat is also found in the neck, inguinal, axillary, and perirenal areas. this can generate heat and may maintain a constant body temperature if the animals are housed in large groups in a humid environment that prevents evaporative cooling. naked mole rats naturally have low thyroid hormone levels, in keeping with the warm temperatures they encounter in burrows or under institutional housing conditions. however, they show increased thyroid activity if housed in the cold for prolonged periods, thus conforming to general mammalian cold acclimation trends . although naked mole rats do not enter a daily or seasonal torpor, like many small mammals do, metabolic rate decreases following prolonged food restriction and may be a mechanism to cope with sporadic and restricted food supplies (goldman et al., ) . circadian locomotor activity was not observed in general for animals monitored while they were in a colony setting (riccio and goldman, a) . however, nocturnal rhythms were noted in four males in the colony that were thought to be dispersomorphs. for vi. other rodents individually housed naked mole rats, % showed robust circadian rhythms in locomotor activity, and three of four naked mole rats showed circadian rhythms of body temperature and metabolic rate (riccio and goldman, b) . although good descriptions (hill et al., ) of naked mole rat anatomy have been published, very little is known about the basic physiology of naked mole rats. the following are some salient features known about the systems biology of the naked mole rat relative to above ground dwellers. a unique identifying feature of naked mole rats is the large incisor teeth that are external to the mouth. having the teeth external enables the animals to dig without the earth coming inside the mouth. naked mole rats have either two or three molar teeth. the mandibles connect at their anterior extent by a flexible mandibular symphysis, allowing the lower incisor teeth to move independently and provide a greater range of movements and dexterity than is typical of most mammals (catania and remple, ) . the incisor teeth appear to be greatly involved in somatosensory perception (henry et al., ) . the parotid, submandibular, and sublingual salivary glands are all well developed (hill et al., ) . the esophagus of the naked mole rat has keratinized stratified squamous epithelium and smooth muscle surrounding it. the stomach is monogastric. the length of the small intestine is relatively short, and leads into a very large cecum (rechkemmer et al., ) . the intestinal mucosa does not have distinct subdivisions based on duodenal, jejunal, or ileal villar length. the cecum accounts for % of the body weight . despite the fact that members of the bathyergidae are strictly herbivorous and eat a diet high in fiber, they nevertheless exhibit the highest digestive efficiency of all mammals (bennett and jarvis, ) . this is primarily due to comparatively long retention times in the gut and highly efficient microbial fermentation processes in the cecum porter, ) coupled with coprophagy. the cecum has a mixed microbial population of ciliate and holotrich protozoa, biflagellates, and fungi as well as an assortment of rod-and cone-shaped bacteria porter, ) (figure . ). these abundant microorganisms within the hindgut, provide naked mole rats with high-energy compounds in the form of volatile fatty acids (such as acetic and butyric acid) that are rapidly absorbed into the bloodstream and provide the host with more than % of their basal energy requirements (parra, ) . to further maximize energy extracted from the diet, nmrs practice coprophagy which provides a good source of proteins in the form of digested microbes. fermentation processes in the gut of the naked mole rat function most efficiently at their low body temperature of °c. surprisingly, because fermentation processes are generally considered to be endothermic, cecal temperature in the mole rat is cooler than core temperature (yahav and buffenstein, ) . the scientific reason for this phenomenon has not yet been explained. the liver has four principal lobes and a number of minor ones, the main lobes being separated by deep fissures extending in most cases completely throughout the hepatic substance, and a gall bladder is absent (hill et al., ) . the anatomy and physiology of the respiratory system in subterranean animals is of great interest because their environment can have high levels of carbon dioxide ( - %) and low levels of oxygen ( %) (arieli, ; contreras and mcnab, ; kennerly, ; nevo, ) . naked mole rats have three turbinates (maxillo, ethmo, and sphenoidal), three lung lobes on the right and left sides, and a caudate lobe on the right side (hill et al., ) . histologically, they have a preponderant double pulmonary capillary arrangement, incomplete differentiation of alveolar pneumocytes, and extension of the cuboidal epithelium to the immediate vicinity of the alveoli (maina et al., (maina et al., , (figure . ). observations between field animals and animals maintained in the lab were similar, suggesting the naked mole rat is intrinsically intractable to environmental shifts in its habitat. consistent with their overall reduced metabolism, the respiratory rate of naked mole rats is about half the respiratory rate of mice (blass et al., ). subterranean rodents have increased capillary densities in skeletal, cardia, and pulmonary tissues, as well as increased fractional volumes of mitochondria in tissue (arieli and ar, ; widmer et al., ) . also, elevated myoglobin levels have been report in other subterranean species (ar et al., ) . in addition, lower resting heart rate and greater heart mass have been reported in ground squirrels and chipmunks (burlington et al., ; jones and wang, ) . in the naked mole rat, the left precaval vein is retained and is larger than the right precaval vein (hill et al., ) . however, little else is known about the cardiovascular system of this species. the left ventricular free wall and interventricular septum are thicker in relation to the right ventricular free wall than seen in rats and mice. naked mole rats have the typical variety of leukocytes (johansen et al., ) as seen under light microscopy, but there are no studies that define cells by markers or immunological function. erythrocytes are similar to those of other mammals. the spleen is in the typical rodent location and is more slender than typical rodent spleens. extramedullary hematopoiesis is noted in the spleen of young and pregnant animals. the hemoglobin molecule has higher affinity for oxygen compared to most other mammalian species, and this is viewed as an adaptation for living under chronic low-oxygen conditions in subterranean burrows (boggs et al., ) . naked mole rats exhibit levels similar to those in other mammals for , -diphosphoglycerate in red blood cells (johansen et al., ) . the skin of the naked mole rat has many features which are well-suited to its warm, subterranean niche. these include the lack of an insulating layer, and a loosely folded morphological arrangement, both contributing to poikilothermic responses to changing temperatures (daly and buffenstein, ) . the presence of pigment-containing cells in the dermis, rather than the epidermis, which is typical of most mammals, also reflects an environment devoid of sunlight. the lack of fur is compensated by a thicker epidermal layer and a marked reduction in sweat glands. in contrast to having fur composed of numerous short hairs, the naked mole rat has an array of sparsely distributed long vibrissa-like hairs (crish et al., ; daly and buffenstein, ) . the follicles of these body hairs are exceptionally large and well-innervated, similar to guard hair of furred species (figure . ) . the role of these sensory hairs is discussed below under somatosensory perception. the arrectores pilorum muscles are lacking, and the sebaceous glands are degenerate (hill et al., ) . water balance of naked mole rats is dependent on utilizing the water from food sources only, since pools of water are not normally available in a subterranean environment and even in captivity naked mole rats meet all their water requirements through the consumption of foods. the kidneys of the naked mole rat are unipapillate and unlike other arid-dwelling organisms are capable of only a moderate concentrating of urine ( mosm) (urison and buffenstein, ) . given the high humidity in their burrows in the wild, this may be adequate for both pulmonary and cutaneous evaporative water loss. however, if high humidity is not maintained under captive conditions, evaporative water loss reaches the highest levels recorded for homeotherms . not surprisingly, when water stressed under captive conditions, given their low figure . lung (h&e) section of diaphragmatic portion of the naked mole rat lung. in contrast to rat and mouse lungs, the terminal bronchiole is ectatic; and alveolar ducts are short or non-existent, allowing alveolar sacs to branch almost directly from the terminal bronchioles. terminal bronchioles are lined by cuboidal epithelium, the alveoli by flattened type i pneumocytes. thus the most distal alveolar spaces abut the pleura and connect with the terminal bronchioles. the lungs thus have an "emphysematous" appearance at low magnification. the relation of this design to the low oxygen needs of the naked mole rat is not understood. vi. other rodents metabolic rates, metabolic water production is too low to counteract high rates of insensible evaporative water loss and the animals cannot maintain plasma osmolality or body mass. both males and females are able to breed at months of age, should the opportunity arise. the external genitalia show no dramatic difference between the sexes. the males have abdominal testes and there may be a slightly greater ano-genital distance in non-breeding males and females. the only accessory sex gland is the vestibular gland. non-breeding females have a vaginal closure membrane that is slightly dark in color. breeding males are generally larger, thinner, and the penis is slightly more raised. breeding females (queens) are large, elongate (jarvis, ; o'riain et al., ) , with a swollen and raised genital area, patent vagina, and large mammae (sherman, ) . the uterus is bicornuate, there are no fornices, and the ovarian bursa is absent. the ovary is covered by a greatly expanded infundibulum of the uterine tube that has no fimbriae (hill et al., ) . the non-breeding males have hyperplastic testicular interstitial cells, reduced numbers of seminiferous tubules, and a simple epididymus without a tail for capacitation of sperm. reproductive suppression of non-breeding female naked mole rats is profound in that the majority of females remain in a pre-pubertal state for their entire lives. concentrations of urinary and plasma progesterone are consistently low in subordinate naked mole rat females (faulkes et al., a) yet the pituitaries of subordinate females are responsive to gnrh (faulkes et al., b) . this suggests that the primary site of reproductive hormone inhibition is at the level of the brain (holmes et al., ) . non-breeding males generally have lower levels of urinary testosterone compared to breeding males, but the difference is not as large as in the females. the rapid rise in urinary progesterone in subordinate females following removal from the colony is probably not an indication of reproductive capacity in that breeding rarely occurs in less than - months (holmes et al., ) . other anatomical features associated with reproduction that are sexually monomorphic in naked mole rats include onuf's nucleus of the spinal cord, perineal muscles, and brain regions (holmes et al., ) . the induction of ovulation has not been determined in naked mole rats, but the damaraland mole rats are capable of spontaneous ovulation and the act of coitus may advance the onset of ovulation (snyman et al., ) . average number of mammary glands in males and females averages .  . (sherman et al., ) . neither total numbers of mammae, nor fluctuation asymmetries in mammary numbers differ significantly between males and females, or between breeders and non-breeders. also, there is no relationship between litter sizes and numbers of mammae. naked mole rats have an unusual endocrine profile relative to above-ground rodents, although several subterranean-dwelling rodents show similar profiles. as mentioned above, naked mole rats have low levels of sex steroids; however they also have low levels of other steroid hormones. living in the dark, it is not surprising that naked mole rats are naturally deficient in the "sunshine hormone", vitamin d. levels of -hydroxyvitamin d are undetectable and the principal active metabolite is low . surprisingly, even when exposed to sunlight for prolonged periods, hormone levels remain low and the enzyme responsible for synthesis of the active metabolite ( -alpha hydroxylase) is rapidly down-regulated. despite these low levels, naked mole rats are able to tightly regulate serum calcium levels and adequately maintain mineral homeostasis, using passive gastrointestinal absorption and vitamin-d-independent mechanisms to retain calcium, magnesium, and phosphorus within the body (skinner et al., ) . thyroid hormones and thyroid-stimulating hormones are also maintained at low levels for mammals and may contribute to the low basal metabolic rate of this species . thyroid morphology is similar to that of most mammals and the thyroid responds positively to cold stress such that thyroid follicular cell height and free throxine levels were significantly greater in cold-acclimated individuals. insulin levels in naked mole rats cannot be detected using standard rodent antibody-based assays, suggesting that naked mole rats either have extremely low levels of insulin or have an insulin molecule with a different protein composition to that of other rodents. although we have not as yet attempted to sequence naked mole rat insulin, there is considerable evidence that the insulin structure in the hystricognaths (e.g. guinea pigs) is substantially different to that of laboratory mice. unlike laboratory mice, and in a similar morpholothe, insulinproducing cells are distributed in a mantle around the periphery of the islet, rather than in the more usual location of the central core. similarly, glucagon-producing cells are not located in the mantle, as previously reported in most vertebrates. indeed, only horses show a similar pancreatic arrangement. like most mammals, a greater number of insulin-producing cells than glucagon-producing cells are present, and somatostatin-producing and pancreatic polypeptide-producing cells are considerably more scarce and randomly scattered within the islet (kramer and buffenstein, ) (figure . ). naked mole rats have low fasting blood glucose levels, and show an impaired glucose tolerance test, maintaining elevated glucose levels for more than hours. when given a physiologically determined dose of human insulin, blood glucose rapidly drops to extremely low levels ( mg/dl) and remains there for several hours. the exquisite sensitivity to human insulin coupled with the abnormal glucose tolerance test and undetectable levels of insulin suggest that naked mole rats naturally only produce small quantities of this hormone and that this is usually sufficient for their needs, given their predominant reliance on volatile fatty acids produced during fermentation for their energy requirements. as such, they are unable to regulate blood glucose when given a supraphysiological dose of sugar. little is known about the hormones of the adrenal gland. the right adrenal is medial to the right kidney and the left adrenal is ventro-medial to the left kidney. there are no published data on the adrenal cortical axis in naked mole rats. mole rats have very low levels of corticosterone compared to mice and rats, but predominantly secrete cortisol. it is not known if these two hormones have identical functions or have differences in their regulatory mechanisms. apart from the hormones described above, little if anything is known about other hormone secretions and functions and considerable research is needed to glean a better understanding of the endocrine function of this species. the naked mole rat brain has the normal anatomy of a typical rodent brain (xiao et al., ) . what makes the naked mole rat different is the relative amount of cortex devoted to the different sensory inputs. the somatosensory system occupies one-third of the sensory cortex and appears to be a more important sensory input (henry et al., ) . unlike the majority of vertebrate species, subordinate and breeding naked mole rats lacked vassopressin innervation of the lateral septum and vassopressin-immunoreactive cells in the nucleus of stria terminalis . instead, the dorsomedial septum contained dense vassopressin-immunoreactive innervation, which did not vary with sex or breeding status. immunoreactive oxytocin fibers were found in the periaqueductal gray, locus coeruleus, parabrachail nucleus, nucleus of the solitary tract, and nucleus ambiguous (rosen et al., ) . the high levels in the nucleus accumbens and preoptic area are associated with maternal behavior in other animals (francis et al., ) . breeders, regardless of sex, had more cells than subordinates in the ventromedial nucleus of the hypothalamus and a larger volume of the bed nucleus of the stria terminalis, paraventricular nucleus, and medial amygdale (holmes et al., ) . non-breeding males and females did not differ on any measure. the sensation of touch is an important sense in the naked mole rat. the area of the sensory cortex devoted to the sensation of touch is much larger in comparison the islet distribution of beta and alpha cells which produce insulin and glucagon respectively have a distinct zonal arrangement with the mantle cells of beta cells framing a centrally located mass of glucagon producing cells. human islets are not organized in this manner, but cells of all types are rather randomly distributed within the islet. vi. other rodents to other mammals (catania and remple, ; henry et al., ) . the queen suppresses reproductive activity via touch by pushing and shoving . like most mammals, naked mole rats have welldeveloped facial vibrissae (whiskers). however, they also have a system of vibrissa-like hairs that are regularly spaced in rows over the body, and naked mole rats have a robust and accurate orientating response when these hairs are stimulated (crish et al., ) . a large number of vibrissa-like hairs are also present on the tail, consistent with the naked mole rat moving as quickly in reverse as in the forward direction. naked mole rats also naturally lack neuropeptides and behaviors associated with signaling of chemical irritants on the skin and nasal cavity (lavinka et al., ; park et al., ) which will be discussed in the models section. naked mole rats demonstrate prominent scent-marking behaviors and members of the colony are frequently seen rolling in the toilet areas of housing systems. yet, reproductive suppression does not appear to be influenced by hormones or pheromones present in the urine from the breeding animals (smith et al., ) . in most vertebrates, chemical (pheromonal) communication involves the vomeronasal organ, a structure within the nasal cavity. for rodents in general, the vomeronasal organ is proportionally large (relative to body size) compared to other mammals (smith et al., ). yet, the vomeronasal organ in naked mole rats is remarkably small, about ten-fold smaller than vomoeronasal organs in social rodents such as the meadow vole and mouse. also, the naked mole rat is the only known mammal to show no post-natal growth of the vomeronasal organ (smith et al., ) . this supports the notion that pheromones and chemical sense do not play a major role in sexual suppression in naked mole rats. with regards to traditional odor sensation involving the olfactory system (apart from the vomeronasal system), naked mole rats perform as well as mice in distinguishing odors in a behavioral laboratory test (lavinka et al., ) . naked mole rats do not have pinnae (important for sound localization), and have narrow external ear canals. the majority of subterranean rodents have ear canals filled with cerumen . definitive anatomical descriptions of the auditory system are not available for the naked mole rat with one exception (hefner and hefner, ) . they described the brainstem auditory nuclei as being relatively small but present. generally though, middle ears are vestigial and the inner ear resembles the guinea pig anatomically more than murid rodents in subterranean rodents (begal et al., ) . in contrast to other rodents, the naked mole rat has hearing and communication calls that are primarily in the low-frequency range (pepper et al., ) . this is probably because low-frequency sound propagates well through soil. however, naked mole rats perform poorly in tasks based on sound localization ability (hefner and hefner, ) . this may reflect that these tests are not appropriately designed to match the unusual (tubular) environment and behavior of this species. subterranean rodents live in a light-free environment and as a consequence have very small eyes and reduced or absent visual capacities. the naked mole rat is not an exception. they have a rod-dominated retina and have cones that are s-opsin dominated which is similar to other bathyergidae and unlike any other mammalin species (peichl et al., ) . the eye contains -cis retinal, the chromophore of visual pigment. the optic nerve cross-sectional area is % of the gerbil and had an erg response typical of other mammals but extremely attenuated and with much slower kinetics (hetling et al., ) . they display a variable degree of retinal detachment which may be associated with the less than gelatinous nature of the vitreous humor (hetling et al., ; nikitina et al., ) . the superior collicullus and lateral geniculate nucleus are very small in the naked mole rat . collectively these findings show that the retinogeniculocortical system in the naked mole rat is considerably smaller than that of rodents that rely heavily on their visual system, but is nevertheless less regressed than that of the extensively studied blind mole rat; this may facilitate limited responses to visual stimuli (xiao et al., ) . indeed all these findings suggest the naked mole rat eye functions for amorphous light detection that may serve for circadian function and/or for the simple assessment of whether or not the burrow has been breached and requires repair. magnetoreception is magnetic compass orientation. it has been reported in the african ansell's mole rat marhold et al., ) , the blind mole rat spalax ehrenbergi (middle eastern mole rat unrelated to the bathyergidae; burda et al., ) , the siberian hamster (deutschlander et al., ) , and the laboratory mouse (muheim et al., ) . there are no reports in the naked mole rat. aging, the progressive decline in functional morphology that over time leads to increased mortality risk is markedly abrogated in the naked mole rat. naked mole rats are the longest-lived rodents known, living approximately years in captivity; . times longer than predicted on the basis of body size and nine times longer than similar-sized laboratory mice. in captivity both breeders and non-breeders have similar maximum lifespan, whereas in the wild the more protected breeders live considerably longer ( years; s. braude, personal communication) than working, predationvulnerable subordinates ( years). not only do naked mole rats have an extraordinarily long lifespan but they show sustained good health for at least % of their maximum longevity and show no age-associated acceleration in mortality risk. from the age of to years naked mole rats maintain lean muscle mass (o'connor et al., ) , bone health (pinto et al., ) , mitochondrial mass, and enzyme activities (ungvari et al., ) . furthermore they sustain their metabolic rate (o'connor et al., ) , level of gastrointestinal absorption (yang and buffenstein, ) , and cardiovascular youthfulness (csiszar et al., ) in addition to their level of activity and reproductive potential well into their third decade (buffenstein, ) . naked mole rats support the evolutionary theory of aging which posits that species that have evolved in areas of low extrinsic mortality such as thermally buffered, sealed burrows that are protected from predation will evolve mechanisms better-suited for extended tissue maintenance and concomitant longevity; whereas those species living in a dangerous environment fraught with challenging climatic conditions and/or high predation risk will evolve life history traits that facilitate early reproduction and shorter lifespans. naked mole rats also support the environmentally selected lifespan theory which posits that animals that live where resource availability is unpredictable will exhibit extended longevity. the life history of naked mole rats provides some support for this theory: location of food is a random, blind, and energetically costly process and food resources are patchily distributed. the low metabolic rate of nmrs may help them survive periods of limited resources. extended longevity, like lower extrinsic mortality, is correlated with group-living, such that cave-roosting bats, social insects, and humans all have long lifespans. the cellular, molecular, and biochemical mechanisms facilitating this extended longevity, good health, and delayed aging are however poorly understood. naked mole rats do not support the telomere theory of aging. telomere length and maintenance will govern the number of times a cell may divide and thereby replace damaged cells. naked mole rats, like humans, have relatively short telomeres compared with mice and lack telomerase and as such cannot maintain telomeres indefinitely. these will shorten with each replication passage and eventually become too short for further replication events. similarly, the naked mole rat only provides equivocal support for the most widely accepted theory of aging, the oxidative stress theory of aging that posits that the characteristic traits of aging are due to accrued damage by unchecked reactive oxygen species (ros), generated during aerobic metabolism. naked mole rats produce similar amounts of ros to those produced by shorterlived rats and mice (lambert et al., ) , do not have a stellar antioxidant defense (andziak et al., ) , and accrue more oxidative damage even at a young age than do shorter-lived species (andziak et al., ) . however, accrued damage appears to be maintained at steadystate levels throughout their long lives (andziak and buffenstein, ) , suggesting that these animals have a higher tolerance threshold for damage prior to inducing mechanisms to remove and repair accrued oxidation products. currently, the mechanisms facilitating prolonged good health and lifespan remain elusive and new studies are focusing on mechanisms that may facilitate protein and genomic stability even in the face of cellular endogenous and environmental insults. naked mole rats are popular zoological attractions (petry, ) and are also studied at numerous research institutions (artwohl et al., ) . they are typically housed at - °c, and humidity levels at - % relative humidity. if housing temperatures are below °c, mole rats will stop breeding (woodley, ) . the only way to obtain this environment is to curtail the ventilation into the room significantly. the benefit of turning on the light-dark cycle has not been proven since colony-housed animals have not demonstrated a circadian rhythm. as in most facilities that house breeding rodents, noise should be minimized. wall-mounted shelves can be used or racks that do not rock and have locking wheels can be used. some facilities put the rack wheels in sand to reduce vibrations in the floor extending into the rack. various bedding material can be provided. all current types of mouse bedding have been used with naked mole rats. each of these kinds of bedding has different pros and cons. for example, wood shavings from pine and ash may have chemicals or odors that may be irritating, causing scratching of the skin. unless it is of a fine high quality it can also cause splinters in the feet. the advantage of wood shavings is that the shavings are less dusty and tend not to stick to skin. vermiculite and corn cob bedding are particularly harsh. some types of paper bedding produce fine particles of dust that may block noses and irritate eyes of naked mole rats. a highly absorbent material should be placed in the toilet chamber and paper towel, tissues, and corn husks should be given in addition for nesting. naked mole rats like to move the bedding material and typically gather materials into the nesting chamber. occasionally naked mole rats will make a ramp with bedding material that can be used to escape from their enclosure. no water is generally provided when housing naked mole rats in captivity. fresh food should thus always be available. food items such as sweet potato can be soaked in water prior to offering the food. the high humidity in the room will diminish insentient water loss. the staple diet for naked mole rats is sweet potato. some institutions soak the tuber in % bleach or weak acid (lemon/ vinegar) for a period of time since fecal organisms can regularly be cultured from them. sweet potatoes are difficult to disinfect because of their porous nature. additional fresh foods include apple, grapes, bananas, oranges, melons, squash, cucumbers, lettuce, and peppers. these can be supplemented with frozen beans and corn. mole rats are particularly fond of melons, however, these water-rich foods lead to frequent urination and the need to clean cages more frequently. a vitamin supplement such as pronutro (wadeville, south africa) is frequently offered. in a laboratory setting, caging is typically made from caging materials that are readily sanitized in a cage washer. housing systems consist of single and multiple cages that can be connected via -inch-diameter pipe. sanitizing complex caging that utilizes connecting pipes is more labor-intensive since pipes should be periodically scrubbed. enrichment for naked mole rats includes offering nesting materials for making a nest, increased cage complexity (figure . ) that would include multiple pipe connections or soils in which to burrow. one strategy is to provide un-husked corn. the animals eat the corn and make a nest out of the husk material. naked mole rats are relentless chewers and constantly gnaw at the corners of chambers. blocking tubes with whole corn or yams will stimulate burrowing behavior in the tubes and may possibly reduce repairs to the housing structure. that said, care must also be taken to check for holes and loose tubes or lids to prevent escapes from occurring. cages can be spot cleaned daily by removing soiled food and spoiled substrates, particularly in the toilet chamber. the entire system can be cleaned every - weeks with scent-free mild soaps. adult naked mole rats can be easily marked using felttipped pens which may last up to days. they do not seem to lick the markings off themselves. toe clipping and transponders can also be used (braude and ciszek, ) . subcutaneous implanted transponders however can migrate to different areas of the body and often fall out. tattoos have also worked in naked mole rats. neonatal and weanling mortality is common in naked mole rats. facilities should severely limit traffic and curtail noises in rooms with neonates. naked mole rats respond to noises and vibrations and can bury or carry neonates as part of their care-taking behavior. sometimes new litters die simply because the mother does not take notice of the neonates. problems associated with the birthing process include dystocia and endometritis. naked mole rats can have very large litters and uterine fatigue can occur. oxytocin given at mouse dosages helps this condition. queens with endometritis typically die a few days after birth. opportunistic bacteria can be isolated from the uterus. the queen's aggression is part of the eusocial life of naked mole rats. aggression is heightened when other females are trying to usurp the queen's control. fight wounds can be observed when this occurs and are one of the most commonly observed clinical problems. these often result in staphylococcal infections. wounds should be treated with organic iodines. naked mole rats have various non-infectious conditions that occur on occasion. bloating is not an uncommon sign. it is often associated with weaning and can be associated with ingestion of less-complex carbohydrate food sources. usually this sign will abate with time, but occasionally the bloat is debilitating, leading to dehydration and can cause death. clinical evaluation and treatment are necessary in these cases. a metastatic calcification condition has been observed (margulis, personnal communication (figures . and . ) . the cause for the condition was probably dietaryrelated since a higher level of rodent chow was used and naked mole rats typically eat diets low in calcium. some calcium deposits may be dystrophic since they can be seen in areas of inflammation such as around fight wounds and foot lesions. the foot lesions would be considered painful. the calcium deposits are made of a pasty white material that appears like an abscess, but are often sterile and radio-opaque. similarly london zoo reported nephrocalcinosis and mineral deposition in soft tissues in animals fed a calcium-rich diet supplemented with vitamin d. few pathogens have been reported in naked mole rats, which is noteworthy considering the preponderance of naked mole rat colonies in zoos and conventional institutional rodent facilities. zoos are environments that have less control over the spread of pathogens. naked mole rats seem to be extremely resilient to pathogens and other toxic insults. perhaps naked mole rats are less susceptible to murine pathogens because of their lower resting body temperature ( °c), or perhaps because naked mole rats are typically housed at higher room temperatures than other rodents, necrosis sets in rapidly when they die such that pathogens and cause of death cannot be easily identified. as such, incidences of pathogenic outbreaks may not be reported. surveys of zoo pathologists however support the premise that naked mole rats are extremely resilient and resistant to infectious agents. ross-gillespie et al. ( ) described an acute coronavirus epizootic that caused acute diarrhea, dehydration, and severe enteric hemorrhaging that killed of the animals ( . %). age (younger animals more susceptible), sex (males were more susceptible), and the degree of inbreeding (inbreeding increased susceptibility) influenced mortality. the source of the coronavirus was not identified. an acute death syndrome has been noted at multiple institutions and zoos. it is characterized by multiple deaths, sometimes in multiple species (naked mole rats and damaraland mole rats) in animals that are in good body condition. sometimes diarrhea is noted. available tests for clostridial enterotoxin and mycotoxins were negative. edematous ileum, cecum, and colon were noted histologically. since naked mole rats have a complex microflora in this region of the intestine, consideration of dysbiosis as described in rabbits or a pathogen that requires special culture conditions should be made for the cause. naked mole rats occasionally get localized abscesses where opportunistic pathogens such as pasteurella, pseudomonas, or staphylococcus sp. are isolated. helicobacter sp. have not been identified in nmrs. small colonies are particularly vulnerable to dehydration and cracked skin in response to low humidity. give small colonies mini-chambers to increase humidity. if skin is dry add moist cloths and/or supplement nesting with fresh corn husks, lettuce, or fresh (washed) grass. in the past we have rubbed vaseline intensive care or hypoallergic moisturizer creams onto skin with considerable success. recently however, a small proportion (up to % of a colony) of animals has gone into shock and although we were able to resuscitate them, we have replaced this moisturizing spa treatment with a rub of olive oil. ring tail has also been observed in naked mole rats (figure . ) . the immune function of naked mole rats has not been described. consideration for susceptibilities to experimental infections should be considered. artwohl this condition should be considered for any mass in a naked mole rat that appears like an abscess. neoplasia has not been reported in naked mole rats (buffenstein, ) . it is possible but not substantiated adequately by pathology that old naked mole rats may suffer from cardiovascular insults such as aneurysms and possible strokes. a couple of considerations when treating naked mole rats are that they take a long time to recover from anesthesia, and the reintroduction of animals back into the colony can cause a xenophobic response. gaseous anesthesia is preferred and dosages of analgesics are much lower than dog dosages. in addition, the greater amount of time the naked mole rat is removed from the colony, the greater the chance of queen aggression on the animal. gloves should be cleaned before removing the animal, minimal disinfectants should be used that would impart a foreign scent, and post-procedural monitoring should be performed to be the sure the animal is not killed after reintroduction. one tactic is to roll the animal in the toilet area before placing it back in the colony. animals that are mobbed by colony members after reintroduction should be removed again. some shoving by the queen is acceptable and usually does not last long. because naked mole rats are social and have cooperative behaviors, the study of their behavior has more applicability to people. functional castes within a colony have been identified (bennett and faulkes, ; sherman et al., ) , and some means of communication. no wonder they are popular attractions at zoos and in the biology class. naked mole rats are one of only two species of mammals identified as being eusocial. the advantage of studying this species in comparison to the damaraland mole rats (the other eusocial mammal) is that they are much smaller and colonies reach much higher numbers of animals. the basic mechanisms involved in reproductive suppression have not been elucidated, but there appears to be some psychological aspect to it . the sensation of touch is an important sense in the naked mole rat since they inhabit an environment that is devoid of light. the area of the sensory cortex devoted to the sensation of touch is larger in comparison to other mammals (catania and remple, ; henry et al., ) . like most mammals, naked mole rats have welldeveloped facial vibrissae (whiskers). however they also have a system of vibrissa-like hairs that are regularly spaced in rows over the body, and naked mole rats display a robust and accurate orientating response when these hairs are stimulated (crish et al., ) . the highly organized pattern of the body hairs and the decisive orienting responses to their touch suggest that the hairs convey somatosensory-based spatial information. since naked mole rats have no fur, the vibrissa-like body hairs are easily accessed for precise stimulation by investigators. the superior colliculus, a midbrain center involved in directing orientation movements to somatosensory, auditory, and visual stimuli, provides an excellent area for studying the neural basis of orientation to somatosensory stimuli in the absence of vision (which is dominant in the superior colliculi of more visually guided animals) . taken together, the tractable number of body hairs (~ ), their ease of access for stimulation, and the lack of visual inputs to the superior colliculus make the somatosensory system of the naked mole rat an excellent system for studying somatosensory processing as it relates to orientating behavior. naked mole rats show a unique and remarkable lack of cutaneous pain-related behaviors to two potent chemical irritants, capsaicin (from chili peppers) and acid (park et al., ) , and a lack of trigeminal pain-related behaviors to capsaicin and ammonia (lavinka et al., ) . furthermore, when exposed to inflammatory insults or known mediators (e.g. freunds' adjuvant), naked mole rats do not display sensitization to heat on the inflamed area (thermal hyperalgesia) as do other mammals. in contrast, naked mole rats do display normal (mouse-like) pain behaviors to mechanical stimuli (pinch) and heat. naked mole rats display several physiological and anatomical features that explain their insensitivity to noxious chemical and inflammatory stimuli. most notably, nerve fibers in the skin that normally respond to acid in other animals are completely insensitive to acid in naked mole rats (park et al., ) . also, while nerves in the skin do respond to capsaicin, those nerves lack the neurotransmitters that would usually be released onto spinal cord cells to signal pain . the neurotransmitters that are missing are substance p (sp) and calcitonin gene related peptide (cgrp). why do naked mole rats lack pain from chemical irritants? as mentioned earlier, naked mole rats have a very unusual combination of ecological and social characteristics. they are fully subterranean, extremely social, and they live in colonies with many individuals. in other words, naked mole rats live in large numbers in very tight quarters and in very poorly ventilated spaces where metabolic activity increases carbon dioxide (co ) and ammonia to extremely high levels. in other mammals, exposure to chronically high levels of co and ammonia induces pain, especially in the eyes and nose. it appears that the insensitivity to chemical irritants in naked mole rats is an adaptation to living in an otherwise painful environment. in addition to functioning in certain aspects of pain processing, sp and cgrp also play an important role in regulating blood flow, for example in thermoregulation and digestion. the absence of these neuropeptides in naked mole rats may be related to the finding that naked mole rats are the only known mammalian poikilotherms, and they may not require cutaneous vasodilation to regulate body temperature. cutaneous vasodilation in other mammals is mediated by nerve cells that release sp and cgrp onto small arteries in the skin to increase regional blood flow and heat transfer. of interest, naked mole rats have sp/cgrp innervation to mesenteric arteries, which presumably subserves regulation of blood flow related to digestion . sp and cgrp have been implicated in a direct effector role in tissue maintenance. absence of this peptidergic innervation raises questions about the capacity of the skin of naked mole rats to respond to insult and could explain the extreme susceptibility to herpes simplex i administered cutaneously (artwohl et al., ). hypogonadism, be it induced by delayed puberty, exercise-induced ammenorhea, or testicular malfunction, impacts upon bone quality, resulting in impaired bone strength with reduced bone mass and area as well as changes in trabecular architecture (chevalley et al., ) . suboptimal skeletal development during puberty affects long-term bone strength, leading to an increase in fracture risk later in life (yingling et al., ) . current animal models to study delayed puberty and hypogonadism have several drawbacks. ovariectomy models often manifest with high gnrh, since it is inhibition by sex steroids and inhibition is reduced, whereas gnrh antagonists affect not only the entire hypothalamic-pituitary-gonadal axis, but also have a tendency to increase body weight, serum igf- levels, and complete suppression of the onset of puberty is dose-dependent (roth et al., ; yingling and taylor, ) . while these studies have yielded considerable information in this regard, the short-term nature of these studies as well as the extended duration of bone remodeling and turnover, limits these studies in what they can tell us about "catch-up" growth or chronic hypogonadism and begs the question of whether or not there is a natural animal model for long-term hypogonadic effects on bone. one such novel animal model may be the naked mole rat. they are naturally suppressed from entering puberty and do not show differences in other hypothalamic pituitary hormones or serum igf- levels, a clear advantage to other models. further, "puberty" can be initiated within days once isolated from the dominant female and levels of the sex steroid hormones in these recently isolated nmrs reach concentrations comparable to those of their long-term breeding counterparts faulkes, , ) . this activation of gonadal activity and fertility is extremely rapid, especially when considering that non-breeders may have been reproductively suppressed for many years, and even -year-old females are capable of exhibiting rapid changes from reproductive suppression to ovarian cycling. further studies in which hormone status may be experimentally manipulated in young individuals both prior to and after attainment of adult mass may shed more light as to whether or not bone morphology and mechanical properties are sustained by sex steroid hormone-independent mechanisms. elucidating the mechanisms facilitating sustained bone structure and strength in hypogonadic subordinates may yield pivotal insights into potential mechanisms and therapies for sustaining bone in delayed puberty and exercise-induced hypogonadism. naked mole rat queens have significantly longer lumbar vertebrae for their body size compared with all other colony members o'riain et al., ) . this finding parallels eusocial insects in that the dominant breeder is physically different. the length of the spine is proportional to the number of pregnancies experienced and suggests that hormones related to pregnancy may have a role in bone growth (henry et al., ) . queens no longer experienced net gain in lumbar spine length after eight pregnancies, but did have some transient elongation during pregnancy after the eighth pregnancy (dengler- crish and catania, ). few animal models used in aging studies show slow rates of aging with affiliated prolonged good health-span and lifespan. rather, traditional aging models are chosen primarily because such animals have poor defenses against aging and are short-lived. for example the most common mammal used in aging is the c bl/ mouse that only lives % as long as predicted by body size. use of short-lived species enables rapid evaluation of whether experimental manipulation of environmental (e.g., caloric restriction (cr)) and/or genetic factors alter lifespan (miller and nadon, ) . although we recognize the many advances in understanding aging processes in these short-lived models, we do not know if their lifespan extension mechanisms are pertinent to long-lived mammals, such as humans, that naturally age slowly. long-lived species may possess certain traits germane to retarding aging that enable them to attain their impressive longevity (austad, ). thus, a complementary approach for studying mechanisms of aging, that we as humans would like to use in translational research, would be to exploit the untapped resource of natural variation in longevity and focus on long-lived successfully aging species such as the naked mole rat. similarly, since naked mole rats do not show any signs of spontaneous neoplasia during aging and also exhibit negligible declines in cardiovascular function, bone quality, and cognitive function; this unusually long-lived and healthy species may also prove to be useful models in many age-associated diseases. as such this species may reveal novel, potentially causal, mechanisms naturally involved in abrogated aging and age-associated diseases. such knowledge will enable therapies aimed at activating mechanisms to delay human aging, prevent degenerative disease, and extend quality of life in old age. antioxidants do not explain the disparate longevity between mice and the lonest-living rodent, the naked mole rat high oxidative damage levels in the longest-living rodent, the naked mole rat blood-gas properties and function in the fossorial mole rat under normal and hypoxic hypercapnic atmosphere conditions the atmospheric environment of fossorial mole rat (spalax ehrenbergi): effect of season, soil texture, rain, temperature and activity blood capillary density in heart and skeletal muscles of the fossorial mole rat naked mole rats: unique opportunities and husbandry challenges extreme susceptibility of african naked mole rats (heterocephalus glaber) to experimental infection with herpes simplex virus type comparative biology of aging acoustics, audition and auditory system daily and seasonal temperatures in the burrows of africn rodent moles coefficients of digestibility and nutritional values of geophytes and tubers eaten by southern african mole rats. (rodentia: bathyergidae) african mole rats: ecology and eusociality resistance to co -induced pulmonary edema in naked mole rats and free-tailed bats respiratory physiology of burrowing mammals and birds survival of naked mole rats marked by implantable transponders and toe-clipping the ecology and behavior of the naked mole rat heterocephalus glaber (ruppell) (rodentia: bethyergidae) the ecology of naked mole rat colonies: burrowing, food, and limiting factors is the naked mole rat heterocephalus glaber an endothermic yet poikilothermic mammal? the effect of diet on microfaunal population and function in the caecum of a subterranean naked mole rat, heterocephalus glaber subterranean mole rats naturally have an impoverished calciol status, yet synthesize calciol metabolites and calbindins cold-induced changes in thyroid function in a poikilothermic mammal, the naked mole rat negligible senescence in the longest living rodent, the naked mole rat: insights from a successfully aging species magnetic compass orientation in the subterranean rodent (cryptomys hottentotus bathyergidae the ear in subterranean insectivora and rodentia in comparison with ground-dwelling representatives. i. sound conducting system of the middle ear magnetic orientation in subterranean mole rats of the superspecies spalax ehrenbergi: experiments, patterns, and memory effect of hypoxia on glycolysis in perfused hearts from rats and ground squirrels (citelus lateralis) somatosensory cortex dominated by the representation of teeth in the naked mole rat brain the influence of pubertal timing on bone mass acquisition: a predetermined trajectory detectable five years before menarche dominance and queen succession in captive colonies of the eusocial naked mole rat, heterocephalus glaber hormonal and behavioral correlates of male dominance and reproductive status in captive colonies of the naked mole rat, heterocephalus glaber kin discrimination and female mate choice in the naked mole rat heterocephalus glaber evolution of subterranean mammals at the organismal and molecular levels somatosensory organization and behavior in naked mole rats i: vibrissa-like body hairs comprise a sensory array that mediates orientation to tactile stimuli central visual system of the naked mole rat (heterocephalus glaber) population coding strategies and involvement of the superior colliculus in the tactile orienting behavior of naked mole rats vascular aging in the longest-living rodent, the naked mole rat catecholaminergic innervation of interscapular brown adipose tissue in the naked mole rat (heterocephalus glaber) skin morphology and its role in thermoregulation in mole rats, heterocephalus glaber and cryptomys hottentotus cessation of reproductionrelated spine elongation after multiple breeding cycles in female naked mole rats learned magnetic compass orientation by the siberian hamster, phodopus sungorus localization of the cytochrome p side-chain cleavage enzyme in the inactive testis of the naked mole rat social suppression of ovarian cyclicity in captive and wild colonies of naked mole rats, heterocephalus glaber lh responses of female naked mole rat, heterocephalus glaber, to single and multiple doses of exogenous gnrh social control of reproduction in breeding and non-breeding naked mole rats (heterocephalus glaber) investigation of sperm numbers and motility in reproductively active and socially suppressed males of two eusocial african mole rats, the naked mole rat (heterocephalus glaber), and the damaraland mole rat (cryptomys damarensis) proximate mechanisms regulating a reproductive dictatorship: a single-dominant female controls male and female reproduction in colonies of naked mole rats micro-and macro-geographical genetic structure of colonies of naked mole rats heterocephalus glaber variations in maternal behaviour are associated with differences in oxytocin receptor levels in the rat factors influencing metabolic rate in naked mole rats (heterocephalus glaber) influence of gonadal sex hormones on behavioral components of the reproductive hierarchy in naked mole rats heterocephalus the remarkable african burrowing rodent degenerate hearing and sound localization in naked mole rats (heterocephalus glaber), with an overview of central auditory structures organization of somatosensory cortical areas in the naked mole rat (heterocephalus glaber) growing out of a caste -reproduction and the making of the queen mole rat features of visual function in the naked mole rat heterocephalus glaber field and laboratory studies of the nake mole rat noradrenaline induces non-shivering thermogenesis in both the naked mole rat (heterocephalus glaber) and the damara mole rat (crystomys damarensis) despite very different modes of thermoregulation social control of brain morphology in a eusocial mammal neuroendocrinology and sexual differentiation in eusocial mammals systematics and evolution of the family bathyergidae genetic variation within and among populations of the naked mole rat: evidence from nuclear and mitochondrial genomes eusociality in a mammal: cooperative breeding in nake mole rat colonies ecological studies on heterocephalus glaber, the naked mole rat, in kenya growth and factors affecting of size in naked mole rats the biology of the naked mole rat blood respiratory properties in the naked mole rat heterocephalus glaber, a mammal of low body temperature adaptive cardiovascular modifications in the west chipmunds, gunes naked mole rats recruit colony mates to food sources morphine induces aggression but no analgesia in the naked mole rat (heterocephalus glaber) the formalin test in the naked mole rat (heterocephallus glaber): analgesic effects of morphine, nefopamand, paracetamol microenvironmental conditions of the pocket gopher burrow on the nomenclature of bathyergidae and fukomys n. gen. (mammalia: rodentia) the pancreas of the naked mole rat (heterocephalus glaber): an ultrastructural and immunocytochemical study of the endocrine component of thermoneutral and cold acclimated animals social organization of naked mole rat colonies: evidence for divisions of labor low rates of hydrogen peroxide production by isolated heart mitochondria associated with long maximum lifespan in vertebrate homeotherms extreme tolerance to ammonia fumes in african naked mole rats animals that naturally lack neuropeptides from trigeminal chemosensory nerve fibers sociality in mole rats: metabolic scaling and the role of risk sensitivity morphology and morphometry of the lungs of two east african mole rats, tachyoryctes slendens and heterocephalus glaber (mammalia, rodentia) effects of change in environmental temperature and natural shifts in carbon dioxide and oxygen concentrations on the lungs of captive naked mole rats (heterocephalus glaber): a morphological and morphometric study behavioral and hormonal changes in female naked mole rats (heterocephalus glaber) following removal of the breeding female from a colony a magnetic polarity comjpass for direction finding in a subterranean mammal metabolism of fossorial rodents comparative social behavior of bees magnetic compass orientation in c bl/ mice principles of animal use for gerontological research mosiac evolution of subterranean mammals: regression, progression, and global convergence postnatal development of the eye in the naked mole rat (heterocephalus glaber) prolonged longevity in naked mole rats: age-related changes in the metabolism, body composition and gastrointestinal function a dispersive morph in the naked mole rat the dynamics of growth in naked mole rats: the effects of litter order and changes in social structure morphological castes in a vertebrate somatosensory organization and behavior in naked mole rats: ii. peripheral structures, innervation, and selective lack of neuropeptides associated with thermoregulation and pain selective inflammatory pain insensitivity of the african maked mole rat (heterocephalus glaber) comparison of foregut and hindgut fermentation in herbivores unusual cone and rod properties in subterranean african mole rats vocalizations of the naked mole rat perineal muscles and motoneurons are sexually monomorphic in the naked mole rat (heterocephalus glaber) zur artgerechten haltung von afrikanischen nacktmullen (heterocephalus glaber) lack of sexual dimorphism in femora of the eusocial and hypogonadic naked mole rat: a novel animal model for the study of delayed puberty on the skeletal system entozoa and endophyta of the naked mole rat fermentation of polysaccharides and absorption of short chain fatty acids in the mammalian hindgut dna 'fingerprinting' reveals high levels of inbreeding in colonies of the eusocial naked mole rat circadian rhythms locomotor activity in naked mole rats (heterocephalus glaber) circadian rhythms of body temperature and metabolic rate in naked mole rats distribution of vasopressin the brain of the eusocial naked mole rat distribution of oxytocin in the brain of a eusocial rodent viral epizootic reveals inbreeding depression in a habitually inbreeding mammal comparative analysis of different puberty inhibiting mechanisms of two gnrh agonists and the gnrh antagonist cetrorelix using a female rat model social structure predicts genital morphology in african mole rats naked mole rats litter sizes and mammary numbers of naked mole rats: breaking the one-half rule is vitamin d essential for mineral metabolism in the damara mole rat (cryptomys damarensis)? combined olfactory contact with the parent colony and direct contact with nonbreeding animals does not maintain suppression of ovulation in female naked mole rats (heterocephalus glaber) the vomeronasal organ of greater bushbabies (otolemur spp.): species, sex, and age differences growth-deficient vomeronasal organs in the naked mole rat (heterocephalus glaber) do dispersing nonreproductive female damaraland mole rats (cryptomys damarensis (rodentia: bethyergidae) exhibit spontaneous or induced ovulation? effects of pethidine, acetylsalicylic acid, and indomethacin on pain and behavior in the mole rat oxidative stress in vascular senescence: lesions from successfully aging species kidney concentrating ability of a subterranean xeric rodent, the naked mole rat (heterocephalus glaber) metabolic and body temperature changes during pregnancy and lactation in the naked mole rat (heterocephalus glaber) working underground: respiratory adaptations in the blind mole rat the effect of huddling on thermoregulation and oxygen consumption for the naked mole rat a steriotaxic atlas of the brain of the naked mole rat (heterocephalus glaber) the effect of temperature on caecal fermentation processes in a poikilothermic mammal, heterocephalus glaber caecal function provides the energy of fermentation without liberating heat in the poikilothermic mammal, heterocephalus glaber disparate age effects on gastrointestinal enzymes in naked mole rats delayed pubertal development by hypothalamic suppression causes in increase in periosteal modeling but a reduction in bone strength in growing female rats antiphonal vocalization of a subterranean rodent, the naked mole rat (heterocephalus glaber) key: cord- -s d ew authors: e. newcomer, christian; g. fox, james title: zoonoses and other human health hazards date: - - journal: the mouse in biomedical research doi: . /b - - / - sha: doc_id: cord_uid: s d ew zoonoses refers to the infectious diseases and infestations that are transmissible directly from an animal host to humans. the biomedical literature contains numerous reports of zoonotic diseases and parasitic infestations from laboratory mice and their wild counterparts. the extended maintenance of the laboratory mouse over a number of generations under controlled and increasingly sophisticated laboratory animal housing conditions with veterinary oversight and effective infection control measures has markedly reduced the likelihood that zoonotic agents would be encountered in a modem animal care and use environment. wild caught mice that are maintained in naturalistic housing environments in the laboratory, laboratory mice that have contact with wild or feral mice, and mice kept as pets in the home environment are examples of animal management conditions that would be conducive to the expression and transmission of zoonotic diseases and other mouse-associated hazards. in addition to the zoonoses, mice are capable of inflicting bites on inadequately trained personnel and are a rich source of allergens for a substantial number of persons predisposed to develop mouse-associated allergic sensitivities. this chapter discusses the mouse-associated zoonotic diseases and other health hazards and explains the strategies that are helpful for reducing or eliminating the risk of personnel exposure to these conditions. zoonoses is derived from the greek words zoon (meaning animals), and noses (meaning disease), and refers to the infectious diseases and infestations that are transmissible directly from an animal host to humans. the biomedical literature contains numerous reports of zoonotic diseases and parasitic infestations from laboratory mice and their wild counterparts. the extended maintenance of the laboratory mouse over a number of generations under controlled and increasingly sophisticated laboratory animal housing conditions with veterinary oversight and effective infection control measures has markedly reduced the likelihood that zoonotic agents would be encountered in a modem animal care and use environment. however, when these essential animal program quality measures fail or are not incorporated into the animal facility operations, zoonotic pathogens may be unwittingly introduced and perpetuated, placing personnel at increased risk of exposure. wild caught mice that are maintained in naturalistic housing environments in the laboratory, laboratory mice that have contact with wild or feral mice, and mice kept as pets in the home environment are examples of animal management conditions that would be conducive to the expression and transmission of zoonotic diseases and other mouse-associated implications in the new world serocomplex group are present among the wild rodents endemic to the united states such as neotoma spp. and peromyscus spp. buchmeier et al. ; fulhorst et al. ) . research animal programs that import wild rodents for laboratory studies should stay abreast of the developments on the identification and host-range characteristics of the new world arenaviruses of emerging importance. this section will focus only on lymphocytic choriomeningitis (lcm), a naturally occurring viral zoonosis of the laboratory mouse. many published reports of human lcm infection are associated with laboratory animal and pet contact, particularly mice and hamsters, and these studies now span many decades (armstrong and lillie ; bowen et al. ; dykewicz et al. ; jahrling and peters ; lehmann-grube et al. ; rousseau et al. ) . there seems to be a resurgent awareness among physicians that lcm should be sought as an etiology in human neurological disease and in pediatric congenital brain disorders (barton and hyndman ; barton et al. barton et al. , romero and newland ) . lcmv is widely distributed among the wild mouse population throughout most of the world presenting a zoonotic hazard (childs et al. ; childs and peters ; morita et al. ; smith et al. ). surveys conducted within the urban environment of baltimore, maryland, reported that % of house mice and . % of persons tested had measurable lcmv antibody titers (childs et al. (childs et al. , . a similar serological survey conducted in spain across urban and rural ecological settings also found a % prevalence in mice and a . % prevalence among persons by immunofluorescence assay (lledo et al. ) . the recent serological detection of lcmv in five mice on the treeless, sub-antarctic, macquarie island of australia, indicates the extent of distribution of this agent to the remotest areas of our planet (moro et al. ) . the apparent ease with which lcmv is transmitted to humans also occurs in a variety of other laboratory animal species; hamsters, guinea pigs, swine, dogs, and nonhuman primates, especially callitrichids, which readily sustain natural infections. in the case of the callitrichids, there have been numerous reports of epizootic infectious hepatitis (callitrichid hepatitis) due to lcmv, with a high mortality rate in zoological parks in both the united states and england over the past two decades (lucke and bennett ; montali et al. ; stephensen et al. stephensen et al. , stephensen et al. , . rodent (mouse) infestations of these zoos and/or the supplementation of the diets of tamarins and marmosets with suckling mice, a common practice (richter et al. ) , are potentially rich sources for lcmv. in the research animal facility environment, the laboratory mouse continues to merit attention as the species of primary concern as a reservoir for cases of human lcm (dykewicz et al. ). in the laboratory mouse, and to a lesser degree the hamster, breeding colonies can become endemically infected when the virus is transmitted to pups in utero or early in the neonatal period, producing a tolerant subclinical infection characterized by chronic viremia and viruria. when infected, athymic and other immunodeficient mouse strains may be predisposed to harboring silent, persistent infections and present a higher risk to personnel (dykewicz et al. ) . under some circumstances lcmv also produces a pantropic infection and may be copiously present in blood, cerebrospinal fluid, urine, nasopharyngeal secretions, feces, and tissues of infected natural hosts and possibly humans. bedding material and other fomites contaminated by lcmv-infected animals can also be important sources of infection for humans, as demonstrated in a recent outbreak among laboratory animal technicians and on many previous occasions (biggar et al. ; dykewicz et al. ) . the experimental passage of tumors and cell lines contaminated with lcmv has long been recognized (haas and stewart ) and represents one of the biggest threats for the introduction of lcmv into animal facilities at the present time (bhatt et al. ; dykewicz et al. ; nicklas et al. ) . reported that of rodent transplantable tumors screened were positive for lcmv and identified contamination in of hamster tumors and of mouse tumors that had been propagated in animals (nicklas et al. ) . the growth of lcmv in insect cell lines has also been demonstrated (rahacek ) , and the article by hotchin summarized the work of others indicating that numerous experimentally infected, bloodsucking ectoparasites are capable of transmitting the disease to laboratory rodents (hotchin ) . lcm virus also has been recovered from cockroaches (armstrong and lillie ) . the diagnosis and control of lcmv infection in mouse colonies has been reviewed in chapter of this volume. tumor and cell-line screening before animal passage, the control of wild rodent infestations in areas where animals are housed or used, and the early detection of colony infections through sound colony health surveillance practices are of critical importance to the prevention of infection in mouse colonies. once established in mouse breeding colonies, the high viral load characteristically shed by mice infected congenitally or neonatally represents a very serious hazard to personnel. most of the cases of human infection, whether involving exposure in the home, agricultural, or laboratory setting, have involved contact with live mice and their excreta or mouse carcasses (dykewicz et al. ; havens ; morbidity and mortality weekly report ) . several authors have emphasized the association between the actual handling of infected mice and the contraction of the disease by humans (dykewicz et al. ; havens ; smithard and macrae ) . several cases of human infection have suggested the possibility that infected rodent tissues can serve as a source of infection for laboratory personnel (baum et al. ; dykewicz et al. ; tobin ). humans may be infected by inhalation or by the contamination of mucous membranes or broken skin with infectious tissues or fluids from infected animals. the transmission of lcmv by the bite of an infected mouse can also occur (scheid et al. ) . also, hotchin reported the findings of other researchers that lcmv was transmissible experimentally through the intact skin of the guinea pig, but this finding has not been reported in humans (hotchin ) . airborne transmission is well documented and plays a very important role in human infections, especially through the ready dispersion and inhalation of viral-contaminated dust from the animal cage or room (biggar et al. ; hinman et al. ) . following an incubation period of to weeks, humans may experience asymptomatic or a mild febrile disease ranging to a serious flu-like illness characterized by anorexia, malaise, diffuse myalgia and arthralgia, fever, chills, vomiting, headache, stiff neck, and photophobia. some patients enter a second stage of the disease several days after the resolution of early mild symptoms, developing meningoencephalitis and exhibiting additional signs such as drowsiness, confusion, sensory disturbances, and motor abnormalities. patients can also develop more serious nonneurological manifestations of the disease such as maculopapular rash, lymphadenopathy, parotitis, orchitis, arthritis, and epicarditis (peters ). central nervous system involvement has resulted in death in several cases. infections during pregnancy pose a risk of infection for the human fetus (wright et al. ). wright et al. reported cases in human infants, with lcmv confirmed serologically over a two-year period in a major u.s. medical center (wright et al. ) . these infants presented with ocular abnormalities, macrocephaly, and hydrocephalus with microcephaly. fifty percent of the mothers reported having had illnesses compatible with lcmv infection, and over half reported exposures to rodents during their pregnancies. the diagnosis of lcm infection in humans is currently made by serological testing using either the immunofluorescent antibody (ifa) test or the enzyme-linked immunosorbent assay (elisa) (barton et al. ) . both of these tests are available through the centers for disease control and prevention and are superior to the complement fixation test that is widely available commercially. although there are no proven effective antiviral therapies for lcm infection, intravenous ribavirin therapy reduces mortality in patients infected with lassa fever virus (a member of the old world arenavirus serocomplex) and may be of some benefit in patients with severe lcmv infections (andrei and de clercq ; mccormick et al. ). this disease can be prevented in the laboratory through periodic serological surveillance using elisa and ifa tests of newly introduced animals with inadequate disease profiles and of resident animal colonies at risk. thorough screening of all tumors and cell lines intended for animal passage using the highly sensitive mouse antibody production assay or newer pcr-based laboratory tests for the presence of lcmv is another crucial element in the program to prevent the introduction of lcmv into established animal colonies (besselsen et al. and chapter of this volume). sound animal facility sanitation practices and the use of microbarrier caging systems with proper infection-control techniques should prevent or suppress the spread of lcmv if present in the environment. the elimination of wild rodent infestations in animal facilities is very important to prevent the introduction of lcmv into the animal facility environment. also, facilities with wild rodent infestations may encounter the relatively common, free-living, bloodsucking mite of the rodent, ornithonyssus bacoti, in abundance (personal communication). although the natural lcmv transmission to humans from bloodsucking ectoparasites is unproven, the control of potential ectoparasitic vectors of this type would be a prudent measure. rabies is an acute, almost invariably fatal disease that occurs worldwide with the exception of a few countries, generally island nations, and other regions that have excluded the disease through animal importation and control programs and the aid of geographic barriers. neither the laboratory mouse nor other small wild rodent hosts appear to be important as reservoirs of natural rabies infection. hence, the principal reason for our discussion of rabies as a zoonotic disease of the laboratory mouse is to provide information that should quickly allay the fears of uninformed research and animal facility staff who suffer the bite of a mouse. on the other hand, the experimental use of mice in the study and characterization of rabies virus and in rabies vaccine development is an important component of some animal care and use programs that deserves special attention by the institution during all phases of research planning and implementation. there are no known cases of human rabies from rodents in the united states ( ). the incidence in larger wild rodent species within the united states has increased in recent years, however. during the interval - , a total of cases of rabies in rodents were recorded, but from to approximately cases were reported in large rodents annually ( ) . the rodents involved were woodchucks and beavers, both species presumably large enough to survive the chance encounter and attack by a wild rabid carnivore such as the raccoon, skunk, fox, or feral cat. earlier literature on the federal republic of germany summarized findings from to , which indicated that three mice, one rat (species not given), nine norway rats, and three muskrats were infected with rabies and had bitten humans (scholz and weinhold ) . despite rare reports of this nature, rodents are not a proven source of rabies transmission to humans (johnson ). all mammals are generally regarded as susceptible to rabies. in humans, the course of the disease proceeds through several phases: incubation, prodromal, acute neurological, coma, and rarely, recovery (johnson ) . the incubation period varies from days to over months. during the prodromal stage lasting to days, patients experience a period of apprehension and develop headache, malaise, and fever. an abnormal, indefinite sensation at the site of a prior animal bite wound is the first specific symptom. patients also may develop intermittent periods of excitation, nervousness, or anxiety interspersed with quiet periods when the mental state appears normal. further progression of the disease involves paresis or paralysis, inability to swallow, and the related hydrophobia, delirium, convulsions, and coma. rabies produces an almost invariably fatal acute viral encephalomyelitis, with death due to respiratory paralysis. adult mice used experimentally in rabies studies usually exhibit clinical signs between and days following inoculation and die within days of the onset of clinical signs coinciding with the period of viral shedding. clinical signs in the mouse consist of muscular tremors, incoordination, excitation, or paralysis. certain rabies virus isolates from skunks produce a spastic paralysis in adult mice followed by recovery in a high percentage of infected mice. also, infant mice inoculated with certain strains of street rabies virus are capable of full recovery (johnson ). personnel working with mice experimentally infected with rabies virus should adhere to the well-established and detailed procedures that have been described in other sources for animal inoculation, husbandry, and tissue harvest procedures (johnson ) . vaccination of personnel involved in rabies studies with laboratory animals also is clearly indicated, regardless of the animal species involved. four other viruses that produce natural infections in the mouse have been implicated previously or are known to be infectious for humans. these include hantavirus, sendai virus, reovirus , and mouse hepatitis virus. for none of these viral agents is there documented evidence of zoonotic transmission of the agent from naturally infected laboratory mice to personnel in the laboratory. hantaviruses are zoonotic viruses comprising at least species that are maintained among natural rodent reservoirs, despite the presence of neutralizing antibody in the rodent host (elliott et al. ; meyer and schmaljohn ) . approximately half of the hantaviruses are known human pathogens producing virus-specific patterns of disease that include hantavirus pulmonary syndrome, hemorrhagic fever with renal syndrome, and its benign form, nephropathia endemica (mills and childs ) . although mus musculus can exhibit the pattern of persistent hantavirus infection in the presence of neutralizing antibody when induced experimentally (araki et al. ) , this does not occur in natural infections of the mouse (meyer and schmaljohn ) . this is the likely reason that the wild mouse apparently is not important as a natural reservoir for the hantaviruses. this interpretation is supported by serological studies of wild mus musculus that detected either no or a very low incidence of serological evidence to hantavirus exposure even when other wild rodent species in the vicinity had a high level of endemic infection (kantakamalakul et al. ; meyer and schmaljohn ; pacsa et al. ; zuo et al. ) . mus musculus is used in the laboratory as an animal model to study various aspects of hantavirus infection ranging from vaccine development (choi et al. ) , viral pathogenicity (ebihara et al. ; kim and mckee ) , and immunological aspects of persistence (araki et al. ) , and it is primarily in this context that hantavirus deserves mention as a zoonotic infection in the mouse. the control of wild rodent infestations in animal facilities, quarantine and testing of wild caught rodent species during importation, and proper observance of animal biosafety guidelines for hantavirus-infected animals would be expected to virtually eliminate the risk of this zoonosis in laboratory maintained mice. mouse hepatitis virus (mhv) remains a prevalent infection in many mouse colonies where it potentially impacts colony health and disrupts experimental studies (see chapter ). earlier studies have demonstrated that human sera contained complement-fixing and neutralizing antibodies to mhv (hartley et al. ) . later studies suggested that this was most likely due to cross-reactive antibodies from human cold virus infections (bradburne ; mcintosh et al. ). mouse hepatitis virus and the two prototype human cold viruses (oc and e) are members of the antigenic group coronaviruses, and it is now known that members of this group share four, and in some cases five, structural genes that could account for this crossreactivity (navas-martin and weiss ). the coronaviruses have a very narrow host range and generally replicate only in the cells of the host species (navas-martin and weiss ). however, under unique laboratory conditions involving persistent cell culture infection, the use of mixed cell cultures of murine and of a nonpermissive species, or the use of cells possessing modified receptors, mhv has been adapted to grow in human, nonhuman primate, and hamster cells. also, the many strains of mhv in combination with use of targeted rna recombinant system have also been very useful for experimental study of the molecular basis of coronavirus pathogenicity (masters ) . application of the targeted rna recombinant system to mhv for exploring of emerging coronavirus infections such as sars may delineate the molecular basis for expanding of host range. these studies may warrant the reader's future attention, but at the present, mhv can be reasonably dismissed as a zoonotic infection. sendai virus was once a prevalent agent in mouse colonies but has become a rarity in most institutions. this is due to its ease of eradication through the use of temporary cessation of breeding to eliminate a naive population that is susceptible to infection and through the use of caging systems that prevent transmission (see chapter ). sendai virus was originally isolated and described in the s during the investigation of cases of human respiratory illness (gerngoss ; kuroya et al. a kuroya et al. , b sano et al. ; zhandoff et al. ). in the original report involving the isolation of sendai virus from japanese newborn human infants suffering from fatal pneumonitis, lung suspensions from the newborns were inoculated intranasally into mice, producing lung consolidation and death in several cases (kuroya et al. b) . in later studies, sendai virus isolates were reported to produce disease in human volunteers (kuroya et al. a; yamada ) , and reports from many countries indicated that serological evidence of sendai virus infection was associated with outbreaks of human respiratory illness (demeio and walker ; gardner ; jensen et al. ). tennant et al. demonstrated that personnel working with laboratory animals had antibody titers to sendai virus, but personnel with no known exposure to laboratory animals also had significant titers to the agent (tennant et al. ) . recombinant sendai virus is widely used for gene transfer experiments, and these vectors can readily infect human airway epithelium and a variety of other human tissues under experimental conditions (nagai ; pinkenburg et al. ) . although these recent studies have clearly demonstrated that sendai virus is capable of infecting human tissues, the initial evidence for its role as a human pathogen remains doubtful. the mice used for the early isolations of the agent may have already been endemically infected with sendai and served as the source, or it may be that other serologically cross-reactive parainfluenza viruses, which were not characterized during this era, were responsible for producing false positive reactions to sendai virus (ishida and homma ) . reoviruses are generally regarded as the cause of childhood infections producing asymptomatic or very mild disease, and there are few reports linking these infections with a particular disease (tsai ) . reovirus was originally isolated from the feces of a clinically ill child (stanley et al. ) , and it continues to receive attention as a possible etiology for neonatal hepatitis and extrahepatic biliary atresia in infants (richardson et al. ; steele et al. ) . reovirus is highly infectious for infant laboratory mice and still receives some attention in the health-screening programs for the mouse and laboratory rodent species. jacoby and lindsey ( ) reported that mouse colonies in the united states continue to have a to % prevalence of reovirus infection. although there are no confirmed reports of reovirus transmission from mice (or other laboratory animal species) to humans, the laboratory mouse should be considered a possible source for this infectious agent for humans and other susceptible species. the rickettsiae are fastidious, small pleomorphic coccobacilliary organisms maintained in nature in a cycle of infection involving mammalian hosts and arthropod vectors as reservoirs (saah ) . in most rickettsial infections, humans serve only as an incidental host and do not contribute to the propagation of the organism in nature. such is the case for rickettsialpox, a nonfatal, self-limiting zoonotic disease caused by rickettsia akari, which is perpetuated in a cycle of infection involving mus musculus as the primary host and a mite vector (liponyssoides sanguineus). isolation of the organisms from rats (rattus) and voles (microtus) has also been reported. the first cases of human rickettsialpox were described in patients in new york city (huebner et al. a (huebner et al. , b , and outbreaks of the disease have generally remained clustered within large urban areas of the united states and in rural north carolina, utah, south africa, korea, and the former soviet union . according to koss et al. approximately cases of rickettsialpox have been reported in the literature, with nearly of these within the three years following the original description of the disease . prior to the report by koss et al. the largest case study included patients accumulated over a -year period. the recent report by koss et al., however, included patients in new york city reporting over only a -month period in the wake of the september , attacks, suggesting that perhaps the heightened sensitivities to possible bioterrorism events stimulated an upsurge in the reporting of cases as a byproduct of increased patient concerns . authors have commented on a variety of other social and demographic factors that may also be contributing to the noticeable increase in the incidence of rickettsialpox and murine typhus in the urban environment (comer et al. ; paddock et al. ) . there are no reported cases of rickettsialpox in personnel related to exposure to naturally infected laboratory mice. the mite vector l. sanguineus has not been reported in laboratory mouse colonies either historically or contemporarily. however, the tropical rat mite (ornithonyssus bacoti) can be experimentally infected with r. akari but has not been shown to play a role in the natural cycle of infection. in the authors' experience of ornithonyssus bacoti infestations of laboratory mouse or rat colonies are still seen with some frequency in facilities that have resident wild or feral mouse populations and should be addressed in the institution's pest control and infection control programs. rickettsialpox has an incubation period of to days following the bite of the infected mite (saah mild to severe with an abrupt onset, and it typically presents with a classic triad of an eschar, fever, and a papulovesicular rash. the papule develops at the site of the bite and later ulcerates and progresses to an eschar, . to cm in diameter, as r. akari proliferates locally in the skin and vasculitis develops saah ) . the rash begins with firm, generally nonpruritic, erythematous papules, to mm in diameter, that develop into vesicles and heal by crusting. in addition to fever, patients may experience chills and headache, and less commonly, backache, myalgia, and photophobia. the disease is mild and self-limiting within to days, and serious complications or death have rarely been reported (saah ) . patients typically respond quickly after the initiation of antirickettsial therapy with tetracycline, doxycycline, or other appropriate agent saah ) . however, following resolution of the infection, headache and lassitude can persist for to weeks. the reader should refer to koss et al. for information on other rash-producing or eschar-related diseases that should be considered in the differential diagnosis to rickettsialpox . rickettsia akari are diagnosed by complement fixation tests or the more sensitive indirect immunofluorescent antibody test. serum antibody to r. akari generally takes weeks to develop, and paired sera are needed to confirm a four-fold rise in antibody titer (saah ) . during the acute phase of the infection, immunohistochemistry or pcr analysis can be used for a rapid diagnosis on biopsy material obtained from the papulovesicular rash or eschar paddock et al. ) . laboratory mice infected with r. akari develop fatal pneumonia with intranasal inoculation and severe illness and death with intraperitoneal inoculation. mice develop anorexia, depression, and dyspnea. peritonitis, splenomegaly, and lymphadenitis are found upon necropsy examination. subcutaneous inoculation produces an active infection for month, with organisms being recovered from the spleen but not the feces or urine (bell ) . the control and eradicaton of r. akari infections depend on the prevention of wild mice and the mite vector from entering laboratory animal facilities and human dwellings. murine typhus is a rickettsial disease caused by rickettsia typhi (previously r. mooseri) that occurs worldwide with epidemics or with high prevalence in particular geographic areas (dumler and walker ) . in the united states, most human cases of the disease are concentrated in texas and southern california. the disease is now predominantly associated with the rat as the primary host species for the oriental rat flea (xenopsylla cheopis), which serves as the principal ectoparasitic vector transmitting the disease to humans. however, the mouse can also serve as a host for this flea, as well as for the northern rat flea (nasopsyllus fasciatus) and the mouse flea (leptosylla segnis), which also bite man and can be involved in the transmission of r. typhi (flynn ; pratt and wiseman ; yunker ). an early report in the literature indicated that x. cheopis was easily established in an animal facility inhabiting rooms used for housing laboratory mice (yunker ) . it is now also known that the cat and opossum and the cat flea (ctenocephalides felis) can be involved in sustaining the cycle in some geographic localities (azad et al. ) . clark and will ( ) reported on use of the laboratory mouse as an experimental host for rearing x. cheopis, but there have been no reports of natural infestations of mouse colonies with any of the flea vectors of r. typhi for several decades. also, r. typhi has not been isolated from natural infections in laboratory mice. murine typhus is a more serious disease than rickettsialpox and presents with fever, headache, chills, nausea, and vomiting. splenomegaly, hepatomegaly, central nervous system involvement, and multiorgan failure can occur as severe and potentially fatal complications. a skin rash, which is typically maculopapular in the case of murine typhus, occurs much less commonly than in rickettsialpox, and its absence should not dissuade the clinician from making a diagnosis of murine typhus and from promptly instituting therapy due to the potential severity of the disease (dumler and walker ) . the methods used for the laboratory diagnosis and treatment of the disease in humans and the principles of preventing the introduction of r. typhi into laboratory animal colonies are similar to those for r. akari. leptospirosis microorganisms were discovered in when they were isolated from jaundiced patients (inada et al. ) ; after further study they were named in (noguchi ) . leptospirosis is solely a zoonotic disease of livestock, pet and stray dogs, and wildlife, including wild rodents. rodent reservoir hosts of leptospirosis include, in addition to rats, mice, field moles, hedgehogs, gerbils, squirrels, rabbits, and hamsters (fox and lipman ; torten ) . human to human transmission is extremely rare. leptospira interrogans (comprising more than serovars) have been isolated worldwide (tappero et al. ) . l. interrogans contains serogroups with strains pathogenic for amphibians, reptiles, and mammals including humans. serovars australis, ballum, bataviae, hardjo, grippotyphosa, icterohemorrgagiae, javanica, and pomona are associated with rodent infections. leptospira serovars, including l. australis, bataviae, grippotyphosa, hebdomidis, icterohaemorrhagiae, pomona, and pyrogenes, are found in the house mouse (torten ) . leptospira ballum has also been reported from mice and is e. newcomer and james g. fox most commonly associated with zoonotic outbreaks (borst et al. ; friedmann et al. ; stoenner and maclean ) . although particular serovars usually have distinct host species, most serovars can be carried by several hosts. leptospira are well adapted to a variety of mammals, particularly wild animals and rodents. in the chronic form, the organism chronically infects the host and is shed in the urine inconspicuously for long periods of time. rodents are the only major animal species that can shed leptospires throughout their lifespan without clinical manifestations (fox and lipman ; torten ) . active shedding of leptospires by rodents can go unrecognized until personnel handling the animals become clinically infected or are infected by exposure to water or food contaminated by urine. rats and mice are common animal hosts for serotype, l. ballum, although it has been found in other wildlife as well. water can often be contaminated with infected rodent urine. the infection can persist unnoticed in laboratory rodents, though their carrier rates for laboratory-maintained rodents in the united states are unknown, but it is probably low. the organism is not routinely screened on health surveillance protocols for mouse colonies; however, there was a report of leptospirosis in in a research colony of mice in the united states being housed in a large research institution (alexander ) . l. icterohaemorrhagiaes antibody when compared to children living in the detroit suburbs. therefore, children living in rodent-infested tenements may be at increased risk of infection (demers et al. ) . in europe and more recently in north america, rodents including house mice have provided a source of leptospira infection for swine and by extension could also infect personnel working in swine production units (galton ; smith et al. ) . leptospira interrogans serovar bratislava is commonly reported in these mice. the disease may vary from unapparent infection to severe infection and death. a self-limited systemic illness is seen in approximately % of infected humans. the incubation period is usually to days. individuals infected with leptospira experience a biphasic disease (faine ; sanger and thiermann ; stoenner and maclean ) . they become suddenly ill with weakness, headache, myalgia, malaise, chills, and fever and usually exhibit leukocytosis. during the second phase of the disease, conjunctival suffusion and a rash may occur. upon examination, renal, hepatic, pulmonary, and gastrointestinal findings may be abnormal. intravenous penicillin is the drug of choice in treating early-onset and late-stage leptospirosis infection (faine ; taber and feigin ; watt et al. ). ampicillin and doxycycline also have been effective in treating people with mild to moderate forms of leptospirosis. because leptospirosis in humans is often difficult to diagnose, the low incidence of reported infection in humans may be misleading. from to , only cases were reported, for an incidence of . per , people per year (sanger and thiermann ) . leptospirosis was removed from the reportable disease category in the united states in because of the small number of cases reported. outbreaks have been documented in the united states from personnel working with laboratory mice (barkin et al. ; stoenner and maclean ) . in one study, of employees handling the infected laboratory mice ( % of breeding females were excreting l. ballum in their urine) contracted leptospirosis (stoenner and maclean ) . infection with leptospira most frequently results from handling infected animals (contaminating the hands with urine) or from aerosol exposure during cage cleaning (barkin et al. ; friedmann et al. ; stoenner and maclean ) . skin abrasions or exposure to mucous membranes may serve as the portal of entry. all secretions and excretions from infected animals should be considered infective. in one instance, a father apparently was infected after his daughter used his toothbrush to clean a contaminated pet mouse cage (boak et al. ) . rodent bites can also transmit the disease (looke ). in detroit, children from the inner city had a significantly higher because of the variability in clinical symptoms and the lack of pathognomonic pathologic findings in humans and animals, serologic diagnosis or actual isolation of leptospires is imperative (faine ) . as an aid to diagnosis, leptospires can sometimes be observed by examination or direct staining of body fluids or fresh tissue suspensions (sulzer et al. ). the definitive diagnosis in humans or animals is made by culturing the organisms from tissue or fluid samples, or by animal inoculation (particularly in -to -week-old hamsters) and subsequent culture and isolation. culture media with long-chain fatty acids with % bovine serum albumin are routinely used as a detoxicant (faine ) . serologic assessment is accomplished by indirect hemagglutination, agglutination analysis, complement fixation, microscopic agglutination, and fluorescent antibody techniques (faine ) . the serologic test most frequently used is the modified microtiter agglutination test. titers of : or greater are considered significant. molecular techniques including pcr and randomly amplified polymorphic dna fingerprinting are used for identification of serovars (tappero et al. ) . in mouse colonies infected with l. ballum, antibodies against l. ballum were detected in sera of mice of all ages, but . zoonoses and other human health hazards leptospires could be recovered only from mature mice. progeny of seropositive females had detectable serum antibodies at days of age but not at days. it was also reported that progeny of seropositive female mice, which possessed antibody at birth and acquired additional antibody from colostrums, remained free of leptospires if isolated from their mothers at days of age, despite exposure during the nursing period (stoenner ) . studies in mice experimentally infected with l. grippotyphosa demonstrated that maternal antibodies, whether passed through milk or placental transfer, conferred protection of long duration against the carrier state and shedding of leptospires. thus, serologically positive immune mothers do not transmit the disease to their offspring. however, mice born to nonimmune mothers, if infected at day postpartum, become carriers with no trace of antibodies. thus a population of carrier pregnant mice without antibody could serve as a precipitator in outbreaks among susceptible mouse populations (birnbaum et al. ) . field surveys have supported this data in that a significant percentage of carrier mice do not have antibodies. this led to the diagnostic approach, which specifies that both serologic and isolation methods must be utilized to determine the rate of leptospiral infection in rodents (galton et al. ) . leptospira ballum is frequently found in the common house mouse (m. musculus) (brown and gorman ; yager et al. ) . therefore, eradication of infected colonies, use of surgically derived and barrier-maintained mice or of conventional laboratory mice free of leptospira infection, coupled with the prevention of ingress of wild rodents, should effectively preclude introducing of the organism into research and commercial laboratories (loosli ) . leptospira ballum has been eliminated from a mouse colony by administration of feed containing gm chlorotetracycline hydrochloride per ton for days. after days of antibiotic therapy, mice were transferred to clean containers and administered clean water, both having been sterilized by steam. mouse traps and rodenticides were used to destroy escaped mice and to prevent reintroduction of l. ballum by the common house mouse . commercial animal colonies maintained in research vivarium today are not routinely screened for leptospirosis, assuming that the organism has been effectively eliminated from commercial and research-maintained mice. rat bite fever can be caused by either of two microorganisms: streptobacillus moniliformis or spirillum minus. streptobacillus moniliformis causes the diseases designated as streptobacillary fever, streptobacillary rat bite fever, or streptobacillosis. haverhill fever and epidemic arthritic erythemia are diseases associated with ingestion of water, food, or raw milk contaminated with str. moniliformis. sodoku, derived from the japanese words for rat (so) and poison (doku), spirilosis, and spirillary rat bite fever are caused by another bacterium, spirillum minus. the bite of an infected rat is the usual source of infection. in some cases, other animal bites, including mice, gerbils, squirrels, weasels, ferrets, dogs, and cats, or rare traumatic injuries unassociated with animal contact, cause the infection. in a retrospective analysis coveting three decades of s. moniliformis isolates ( % from humans) from the department of public health in berkeley, california, % of the isolates were from children < years of age (graves and janda ) . in % of the human infections where a diagnosis was made, rat bite fever (rbf) was suspected; % of those suspected cases involved either known rat bite or exposure to rodents. two cases of rbf were attributed to exposurem in one case a squirrel, and in the second a mouse (graves and janda ). interestingly, > % of the cases could not be attributed to a rat bite or scratch, indicating that close contact with infected rodents can be sufficient to become infected (graves and janda ) . other reports have indicated that the disease can occur in individuals who have no history of rat bites, but reside or work in rat-infested areas or have pet rats with whom they have close contact (fordham et al. ; holroyd et al. ; rumley et al. ) . rat scratches can also be the source of the organism (edwards and finch ; shanson et al. ) . exposure to cats and dogs that prey on wild rodents may also be the source of the organisms. these organisms are present in the oral cavity and upper respiratory passages of asymptomatic rodents, usually rats (wilkins et al. ). mice can be infected with resulting morbidity and mortality due to arthritis and pneumonia. in one study, streptobacillus moniliformis was isolated as the predominant microorganism from the upper trachea of laboratory rats (paegle et al. ) . presumably the incidence of str. moniliformis is now lower in high-quality, commercially reared specific pathogen-free rats. surveys in wild mice indicate to % infection with spirillum minus (hull ) . ( ). streptobacillus moniliformis incubation varies from a few hours to to days, whereas spirillum minus incubation ranges from to weeks (table - ) . fever is present in either form. inflammation associated with the bite and lymphadenopathy are frequently accompanied by headache, general malaise, myalgia, and chills (arkless ; cole et al. ; gilbert et al. ; mcgill et al. ) . the discrete macular rash that often appears on the extremities may generalize into pustular or petechial sequelae. arthritis occurs in % of all cases of s. moniliformis but is less common in spirillum minus. streptobacillus moniliformis may be cultured from serous to purulent effusion, which is recovered from affected larger joints. a total of cases of endocarditis due to s. moniliformis were reported from to (shvartsblat et al. ). death has occurred in cases of s. moniliformis involving preexistent valvular disease or as a result of endocarditits in a previously healthy individual. infants can also die of the infection (sens et al. ) . if antibiotic treatmentmusually penicillin at doses of , to , daily for daysmis not instituted early, complications such as pneumonia, hepatitis, pyelonephritis, enteritis, and endocarditis may develop (richter ). if endocarditis is present, the penicillin should be given parenterally at doses of to million units daily for for weeks. streptomycin and tetracyclines are also effective antibiotics for those individuals with penicillin-associated allergies. addition of streptomycin to standard therapy is also advised in cases where isolates of sir.. moniliformis are cell wall deficient (rupp ) . spirillum minus does not grow in vitro and requires inoculation of culture specimens into laboratory animals, with subsequent identification of the bacteria by dark-field microscopy. streptobacillus moniliformis grows slowly on artificial media but only in the presence of % blood and sera, usually % to % rabbit or horse serum incubated at reduced partial pressures of oxygen. sodium polyanethol sulfonate sometimes found in blood-based media because of its properties as a bacterial growth promoter should not be used due to its inhibitory effects on sir. moniliformis (lambe et al. ; shanson et al. ) . growth on agar consists of to mm gray, glistening colonies. the api zym diagnostic system can be used for rapid biochemical analysis and diagnosis. a pcr-based assay has also been described to diagnose sir. moniliformis (berger et al. ) . the genus salmonella are gram-negative bacteria with approximately serotypes. nontyphoidal salmonellosis is caused by any of these serotypes. other than salmonella typhi, the causative agent of typhoid fever, salmonellosis occurs worldwide and is important in humans and animals. s. typhi and salmonella choleraesuis have only one serotype, whereas the remaining serotypes are within the species salmonella enteritidis. references to the salmonella enteritidis serotypes are abbreviated such that "enteritidis" is dropped; for example, s. enteritidis serotype typhimurium is called salmonella typhimurium. salmonella typhimurium is the serotype most commonly associated with disease in both animals and humans. other serotypes most commonly reported from humans and animals are salmonella heidelberg, salmonella agona, salmonella montevideo, and salmonella newport. salmonellae are pathogenic to a variety of animals. salmonella are ubiquitous in nature and are routinely found in water or food contaminated with animal or human excreta. fecal-oral transmission is the primary mode for spreading infection from animal to animal or to humans. transmission is enhanced by crowding and poor sanitation. during the early s, rodenticides containing live cultures of s. enteritidis were distributed on a large-scale basis by commercial and public health organizations in an attempt to eliminate feral rats. these cultures were known as "rat viruses" and were widely used in europe, england, and the united states as "rat poisons" (weisbroth ). however, enthusiasm for their use waned when it was discovered that the spread of the organisms couldn't be limited; predictably, the baiting program was implicated in several epidemics among exposed human populations (weisbroth ) . surprisingly, as late as the s in england, s. enteritidis (serovar danzy) was isolated from adults living four miles apart. the source of infection was traced to contaminated cakes from a local bakery. mice that had acquired the (brown and parker ) . as with other fecal-oral transmitted diseases, control depends on eliminating contact with feces, food, or water contaminated with salmonella or animal reservoirs excreting the organism. salmonella survive for months in feces and are readily cultured from sediments in ponds and streams previously contaminated with sewage or animal feces. fat and moisture in food promote survival of salmonella. pasteurization of milk and proper cooking of food ( ~ for to minutes) effectively destroys salmonella. municipal water supplies should be routinely monitored for coliform contamination (pavia and tauxe ) . clinical signs of salmonellosis in humans include acute sudden gastroenteritis, abdominal pain, diarrhea, nausea, and fever. diarrhea and anorexia may persist for several days. organisms invading the intestine may create septicemia without severe intestinal involvement; most clinical signs are attributed to hematogenous spread of the organisms. as with other microbial infections, the severity of the disease relates to the organism's serotype, the number of bacteria ingested, and the host's susceptibility. in experimental studies with volunteers, several serovars induced a spectrum of clinical disease ranging from brief enteritis to serious debilitation. incubation varied from to hours. cases of asymptomatic carriers, persisting for several weeks, were common (hull ) . salmonella are flagellated, nonsporulating, aerobic gramnegative bacilli that can be readily isolated from feces on selective media designed to suppress bacterial growth of other enteric bacteria. salmonella serotyping requires antigenic analysis (fox ) . salmonella gastroenteritis is usually mild and self-limiting. with careful management of fluid and electrolyte balance, antimicrobial therapy is not necessary. in humans, antimicrobial therapy may prolong rather than shorten the period that salmonella are shed in the feces (nelson et al. ; pavia and tauxe ) . in one double-blind placebo study of infants, oral antibiotics did not significantly affect the duration of salmonella carriage. bacteriologic relapse after antibiotic treatment occurred in % of the patients, and % of these suffered a recurrence of diarrhea, whereas none of the placebo group relapsed (nelson et al. ). tick-borne relapsing fever occurs primarily in foci in the western part of the united states, as well as other parts of the world. the disease is caused by at least borrelia species and is transmitted to humans from a variety of rodents (chipmunks, squirrels, rats, mice, prairie dogs, hedgehogs) via soft ticks of the genus ornithodorus. of recent interest are the increasingly recognized enterohepatic helicobacter spp., which cause both hepatic and intestinal disease in mice (whary and fox ) . one of these, h. bilis, isolated routinely from mice, has been found using pcr-based assays in bile and gallbladder of chilean patients with chronic cholecystitis and in biliary cancers in japanese patients (fox et al. ; matsukura et al. ) . whether these new helicobacters will be linked to zoonotic transmission from wild or laboratory rodents will require further studies. pathogenic staphylococcus aureus of human phage type can cause clinical disease in mice and rats. this organism has been (davies and shewell ) lab worker % not determined, alopecia, increased scaling on head (booth ) and back, mice bacteriologist of , crusted or crustless plaques, circular with (cetin et al. ) prominent periphery; general alopecia; mortality in some mice technician % colony with alopecia and scaly skin (dolan et al. ) technician alopecia with crusting an erythema (povar ) introduced into spf barrier-maintained mouse colonies and spf rats and guinea pigs; the same phage type was isolated from their animal caretakers (davey ; shults et al. ) . colonization by normal s. aureus strains in the nasopharyngeal area of humans presumably minimizes the zoonotic potential of animal-originated s. aureus. as reviewed comprehensively in blank, reports of ringworm (favus) in the mouse began to appear in the european literature in the mid-nineteenth century and in the north american literature during the early twentieth century (blank ) . several of the early authors noted the similarities between the lesions of favus in the mouse and in humans. quincke, who is generally credited with isolating the causative agent which he named ~-pilz (now trichophyton mentagrophytes), suggested that the infection in the mouse was also a source of infection of the cat, and thereby, of humans. earlier reports of murine ringworm referred to the causative agent as t. quinckeanum, but the successful crossing of t. quinckeanum with the perfect state of t. mentagrophytes, arthroderma behamiae, indicates that t. quinckeanum is not a distinct species (ajello et al. ) . a later study of the two varieties, t. mentagrophytes var. mentagrophytes and t. m. var. quinckeanum, noted that the conidia from both produced two morphological variants on cultivation (granular and fluffy), and these variants were a. behamiae type + and pathogenic (hejtmanek and hejtmankova ) . in addition to t. mentagrophytes, epidermophyton floccosum, mircrosporum gallinae, m. gypseum, m. canis, t. erinacai, t. schoenleini, and t. (keratinomyces) ajello have been reported as zoophilic dermatophytes that can infect mice and cause ringworm in humans (dvorak ; krempl-lamprecht and bosse ; marples ; papini et al. ; refai and ali ) . the dermatophytes are distributed worldwide and can involve a variety of small animal host species in addition to the mouse. chmel et al. ( ) conducted field studies in a wooded farm setting in czechoslovakia and detected an overall prevalence rate of . % ( positive of ) for t. mentagrophytes infection in of species sampled; the prevalence in mus musculus was . %, with mice comprising . % of the infections detected. of the species that harbored the infection, all frequented the barn or granary area; the seasonal incidence was highest during the winter months when the rodent carriers were more likely to seek harborage indoors. chmel et al. ( ) also analyzed patient data and demonstrated that t. mentagrophytes was the predominant isolate from those who did agricultural work, while t. verrucosum was the main isolate from individuals who worked with farm animals. also, human t. mentagrophytes infections were most common on the hands, wrist, forearm, face, and neck, unprotected skin sites readily contaminated by fodder, litter, or other materials while working in the barns. ringworm infections associated with the handling of bags of grain in which mice had been living have also been reported (alteras ; blank ) . ringworm infection in laboratory mice is often asymptomatic, remaining unrecognized until laboratory personnel become infected. early reports in the literature indicated that the prevalence of t. mentagrophytes was to % among some laboratory mouse stocks (davies and shewell ; dolan et al. ). however, these reports predated the era of modem laboratory animal colony management marked by the commercial availability of cesarean-derived, barrier-maintained rodents. moreover, the modem production practices that have been universally adopted by the industry for decades and the use of microbarrier cages with appropriate technique have further reduced the opportunity for ringworm to become a significant problem in contemporary colonies. in recognition of this fact, none of the major commercial vendors in the united states survey their colonies for dermatophyte infections as part of routine health monitoring. sporadic cases of ringworm infections in rodents have been reported in the past three decades (hironaga et al. ; mizoguchi et al. ; papini et al. ) . programs involved in importing mice from sources that fail to meet contemporary rodent production and husbandry practices should consider screening mice for dermatophytes during the quarantine period. the ease of transmission of dermatophytes from animals to humans is well known and is a significant health hazard. laboratory mice, as well as other laboratory animal species, can harbor dermatophyte infection, with few or no visible skin lesions transmitting the infection to unsuspecting personnel (dolan et al. ) . transmission can occur through direct contact with the infected animal or through indirect contact with animal bedding or other materials in the environment of the contaminated animal room. rigorous facility and equipment sanitation has long been recognized as an essential element of an effective control program and should be undertaken in conjunction with efforts to treat valuable animals or to repopulate previously contaminated areas of a facility (davies and shewell ; dolan et al. ; mizoguchi et al. ). the importance of barrier protections by donning appropriate clothing, using gloves and other personal protective equipment, and modifying work practices to minimize skin exposure to dermatophytes has also been acknowledged for the prevention of transmission (dolan et al. ) . when prevention methods fail, allowing the introduction of dermatophyte infection into a mouse colony, and when transmission to humans occurs, clinical cases of dermatophytosis routinely respond well either to topical or systemic antifungal therapy. *found in laboratory animals that cause allergic dermatitis or from which zoonotic agents have been recovered in nature (see yunker ) . **wee, western equine encephalitis. +sle, st. louis equine encephalitis. ++rmse rocky mountain spotted fever. dermatophytosis or ringworm in humans can be asymptomatic and minor, often self-limiting and drawing little attention from the affected individual. the infection usually causes an expanding, scaly and erythematous inflammatory plaque on the skin that occasionally contains fissures or vesicles when severely eczematous. on the trunk and extremities, the lesion may consist of one or more circular lesions with a central clearing and sharply defined margins, forming a ring, and hence the name "ringworm" (fig. - ) (merlin et al. ) . other dermatophytes are named according to the sites of involvement on the body (e.g., tinea pedis for foot infections, tinea capitis for scalp infections). the dermatophyte infections of humans associated with direct or indirect contact from mice usually involve the body or extremities, especially the arms and hands. zoophilic t. mentagrophytes infection usually produces a highly inflammatory lesion and often undergoes rapid resolution. however, it can also produce furunculosis--deep infection of the hair follicles or widespread tinea corporism which is also seen in infections of e. f l o c c o s u m . in a laboratory-acquired infection with t. (keratinomyces) ajelloi, mice were the source of infection for a laboratory technican who developed small, grayish-white, scaly lesions on both hands. hand lesions yielded the organism, as did of apparently health mice (refai and ali ) . a. tapeworms a. reservoir and incidence although this parasite occurs in the mouse intestine, it is more commonly associated with rats and is especially common in wild norway (rattus norvegicus) and black (rattus rattus) rats throughout the world (faust and russell ; stone and manwell ; wardle and mcleod (voge and heyneman ) . larval development in tribolium sp. at ~ requires days. therefore, humans become infected only through ingestion of infected insects, such as flour beetles, which may contaminate rodent food or cereal marketed for human consumption. c. clinical signs the infection in humans is usually asymptomatic, but in moderate to heavy infections it may cause headaches, dizziness, abdominal discomfort, and diarrhea. the greatest length of an adult parasite removed from a patient was meter. usually, adult parasites are to cm long and as much as mm wide (markell et al. ). a. reservoir and incidence the dwarf tapeworm is a common parasite of both the wild house mouse and the laboratory mouse. as indicated earlier in the text, in most well-managed mouse colonies, r. nana incidence is low compared to earlier reports of its high incidence in rodent colonies (wescott ) . the estimate that million humans in the world are infected was made many years ago but probably is an underestimate (markell et al. ) . surveys conducted in central europe report that this tapeworm in humans is more prevalent in warm than in temperate regions. an incidence of % has been noted in some south american countries (jelliffe and stanfield ) . it is most commonly diagnosed in children. diagnosis is made by observing characteristic eggs in the feces. b. mode of transmission r. nana is unique among tapeworms in that the adult worm develops after the egg is ingested. the hooked oncosphere then invades the intestinal mucosa and develops into a cysticeroid larva. rodentolepsis nana eggs can contaminate hands, be trapped on particulate matter, or be aerosolized, and then accidentally ingested. since no intermediate host is required, the eggs are readily infective for the reciprocal hosts (faust and russell ) . precautions against infection include strict personal hygiene, appropriate laboratory uniforms, and use of disposable gloves and face masks when handling contaminated bedding and feces. c. clinical signs the clinical picture of r. nana infection is quite cosmopolitan. in well-nourished persons, essentially no symptoms occur; the infection is noted when the proglottids or ova are seen in the stool. in other persons, the symptoms include headaches, dizziness, anorexia, inanition, pruritis of the nose and anus, periodic diarrhea, and abdominal distress. a tapeworm identified as r. nana was found in a tumor removed from the chest wall (jelliffe and stanfield ) . the diagnosis is based on identification of the characteristic eggs or proglottids in the stool. d. treatment praziquantfel, given orally in a single dose of mg/kg body weight is the drug of choice. alternatively, niclosamide is given daily for days because of the tissue phase of the parasite. the dose is gm for adults and . gm for children > kg, and . gm for children between and kg (markell et al. ) . recently, a parasite known to naturally colonize mice, r. microstoma, has been identified in the feces of humans living in the northwest of western australia (macnish et al. ) . although r. nana was the most common enteric parasite based on microscopic examination of feces, r. microstoma was identified as a mixed infection in of individuals by using a molecular-based assay consisting of restriction fragment length polymorphism of tapeworm dna as well as a sequencing of the pcr product of the internal transcribed spacer region of ribosomal dna (macnish et al. ) . given that r. microstoma requires an intermediate host, tribolium confusum for its life cycle, it is understandable why it was not as common as r. nana in this study. however, given the morphological similarities of the eggs of r. nana and r. microstoma, the true prevalence of r. microstoma in humans won't be known until molecular techniques to differentiate the two species are utilized in future studies. syphacia obvelata is an ubiquitous parasite in both wild and laboratory mice. although parasitology texts report that syphacia is infectious to humans, this citation originates from a publication in , in which two s. obvelata adult worms and eggs reportedly were found in the formalin preserved feces of a filipino child whose entire family of five was infected with h. nana (riley ) . no mention is made of the method of collecting the feces, nor is it known whether the feces could have been contaminated with murine feces or with the parasite and/or eggs. the only other report is an unpublished finding of s. muris eggs in the feces of two children and two rhesus monkeys, cited in a personal letter from dr. e. e. faust of tulane university, dated january , (stone and manwell ) . both of these cases may therefore be examples of spurious parasitism, but definitive information for that conclusion is lacking. regardless, no published information indicates that laboratory personnel have been infected by working with syphacia-infected mice. contamination of food or utensils or accidental ingestion of syphacia ova (e.g., via contaminated hands) could result in infection of humans. people working with infected mice probably ingest ova occasionally, but there is no evidence that this exposure results in active infection. because syphacia infection in humans has not been described, clinical signs have not been noted. there are striking differences in size between specimens of female s. obvelata and those of enterobius vermicularis, the pinworm, in humans (markell and voge ) . syphacia is . to . mm long, whereas the enterobius sp. female reaches a length of to mm. the male syphacia sp. measures . to . mm compared to . mm for enterobius. the size difference between the eggs of the two species is also marked: syphacia eggs are more than twice as long ( gm versus /am as those of enterobius). it is unlikely therefore that syphacia sp. would be misdiagnosed as enterobius sp., assuming, of course, that the observer was aware of the size difference and measured the eggs. although many species of mites are found on laboratory mice, only ornithonyssus bacoti, the tropical rat mite, and liponysoides sanguineus, the house mouse mite, are vectors of human disease. ornithonyssus bacoti is seen in laboratory mice (fox ) ; l. sanguineus has been identified only on wild mice. bites from these mites, as well as from another mouse mite, haemalaelaps casalis, are responsible for allergic dermatitis, or local inflammation, in humans. ornithonyssus bacoti can be found on many rodents; the brown norway rat and the black roof rat are probably the primary host species (beaver and jung ) . since the time of the first report of human ornithonyssus bacoti-associated dermatitis in australia in , and a report in humans in the united states, many other cases have continued to be described throughout the world (see table - ) (charlesworth and clegern ; chung et al. ; dove and shelmire ; dowlati and maguire ; engel et al. ; fox ; haggard ; hetherington et al. ; riley ; theis et al. ; wainschel ; weber ) . ornithonyssus bacoti is an obligate bloodsucking parasite, usually tan but red when engorged with blood. both the male and female feed on a rodent as their preferred host. the female is ~tm to mm in length; the male is smaller (fig. - ) . eggs are laid in bedding or wall crevices by the female, which survives for about days and feeds about every two days during this period. the mite has five developmental stages: adult, egg, nonfeeding larva, bloodsucking protonymph, and nonfeeding deutonymph. after feeding, the adults and protonymphs leave their host and seek refuge in cracks and crevices. the life cycle from adult to egg requires to days at room temperature. unfed protonymphs have survived for days (brettman et al. ) . the mite often gains access to the premises on wild rodents and lives in crevices. if wild rodents are not readily available or are captured, the mite will seek blood elsewhere, either from the wild or laboratory rodent (if in an animal research facility) and/or humans. in some infestations, the rodent shows no clinical signs. however, in more chronic cases, dermatitis and anemia may develop. historically, this mite has been a troublesome parasite in certain laboratory animals, especially rats, mice, and hamsters (fox ; keefe et al. ). a. clinical signs tropical rat mites produce painful, pruritic lesions in humans. examination of patients often discloses papular lesions on the wrists, arms, abdomen, and chest. raised erythematous papules and nodules several millimeters to greater than cm in size occur singly or in linear configuration ( fig. - ) . epidemiologically, cases usually occur in clusters that involve a common source of exposure to the mite. experimentally, cases have been shown to be a vector of pathogens. in the laboratory, mite transmission of various rickettsial species, pasteurella tularensis, and coxsackie virus between different laboratory animals has been shown (hopla ; petrov ; philip and hughes ; schwab et al. ) . affected individuals may be treated with topical lindane or treated symptomatically, given that the mite does not reside on humans for any extended periods. papular dermatitis will regress after a period of to days post-therapy. recurrence of ornithonyssus bacoti infestations is common unless the premises have been treated with an appropriate insecticide, and any feral rodents eradicated (engel et al. ). fleas are seldom found in laboratory mice but are common parasites of feral rodents. the oriental rat flea, xenopsylla cheopis, and another flea, nasopsyllus fasciatus, both naturally infest mice and rats; they are vectors for murine typhus. apparently, x. cheopis is easily established in animal facilities. at a midwestern u.s. university, it inhabited a room housing laboratory mice where, on two separate occasions, the flea caused distress by biting students (yunker ) . leptopsylla segnis, the mouse flea, bites humans and is a vector for plague and typhus, serious diseases in humans. also, l. segnis can serve as an intermediate host for the rodent tapeworms r. nana and r. diminuta, which can infect people. the flea's bite can also be irritating and cause allergic dermatitis. epidemiological perspective on the transmission of infectious diseases, principally rabies. the bite of the rat is far more powerful and more likely to be disfiguring than that of the mouse, and rat bites are known to be associated with the transmission of bacterial zoonoses such as rat bite fever and leptospirosis (see elsewhere in this chapter). one should assume that the mouse is also capable of transmitting these agents via bite. rabies transmission from small rodents in the wild occurs but is exceedingly rare (gdalevich et al. ) ; therefore, rabies is of concern only if experimental studies with the virus are being conducted in mice. mice can also transmit hantavirus infection and lymphocytic choriomeningitis virus infection via bites. anecdotally, most animal care and use programs report that rodent bites among personnel are a reasonably common occurrence that often are unreported to an institution's occupational medical service, despite the fact that bites inflict pain, produce anxiety, and may have significant health consequences. in addition to the hazard of zoonotic disease or local wound infection with pyogenic or toxin producing bacteria such as clostridium tetani, staphylococcus spp., streptococcus spp., escherichia coli, and bacillus subtilis, rodent bites, including those of the mouse, can induce a severe local allergic reaction or anaphylaxis in individuals previously sensitized to allergen (hesford et al. ; teasdale et al. ; thewes et al. ) . thus, bite wounds from mice should be immediately cleaned thoroughly and reported to the institutional occupational medical service to permit evaluation of the person's tetanus immunization status and need for additional local wound or other medical care. the need for additional training of bitten persons in animal handling may also be indicated. authoritative information on the incidence and impact of animal bites in the general population over the past several decades is scant, and reliable data on the incidence of mouse bites among personnel who work in laboratory animal facilities or among the general populace is lacking. there have been occasional studies on the occurrence and clinical characteristics of rat bites within urban populations, including a recent investigation of bites over the period - that associated this phenomenon with urban blight, poverty, and unemployed populations (hirschhorn and hodge ) . traditionally, animal bites have received attention from the clinical perspective of wound management and complications and from the allergic skin and respiratory reactions to laboratory mice are very common in laboratory animal caretakers and technicians who work with these animals. a survey by lutsky ( ) demonstrated that three-fourths of all institutions with laboratory animals had animal care personnel with allergic symptoms. the prevalence of symptoms of laboratory animal-associated allergy (laa) among personnel working with laboratory animals has been estimated as between and % in numerous recent studies, and among these individuals, approximately % are estimated to eventually proceed to the development of asthma (chan-yeung and malo ; eggleston and wood ; hollander et al. ; hunskaar and fosse ; knysak ; renstrom et al. ) . furthermore, other sources have suggested that among atopic individuals with preexisting allergic disease, up to % of persons exposed to lab animal allergens may eventually develop laa (committee on occupational health and safety in research animal facilities/national research council allergens ). the population at risk for work-related exposure to rodents was estimated at , (newill et al. ); this population has likely grown in the intervening years to the expanding populations of genetically modified mice that are used in contemporary biomedical research programs and require care. moreover, a recent study would seem to suggest that the risk of exposure to mouse allergens is not confined to those working in the laboratory animal facility environment. data analyzed from the first national survey of lead and allergens in housing in the united states demonstrated that % of homes of diverse types and income levels across geographic locations had evidence of mouse allergen; % had detectable levels on the kitchen floors specifically; and % had allergen concentrations greater that . ~tg/g of dust collected, a level previously correlated with the significantly increased rate of sensitization to mouse allergen (cohn et al. ) . the large number of staff at risk of exposure in the workplace or already presensitized, in combination with the substantial added costs to employers for the medical management, operational disruptions, and retraining efforts related to employees who develop laa and later proceed to asthma, should provide the impetus for many institutions to pay grater attention to this element of the occupational health and safety program (schweitzer et al. ) . the major allergen of the laboratory mouse is the mus m protein, a member of the mouse major urinary proteins encoded by a multigene family consisting of approximately genes (clark et al. a (clark et al. , b . the earlier literature on the subject of mouse allergy referred to the mouse urinary proteins (mups), whereas recent literature cites the specific protein (mus m ) that is now known to be the primary offending allergen in the mup multigene family. the mus m protein is in the lipocalin family of proteins that are produced in the saliva and liver and are excreted in the urine at levels times higher than are present in mouse serum. lipocalins serve to bind small hydrophobic molecules and function biologically to transport vitamins, small volatile odorants, and pheromones conferring the characteristic odor to mouse urine (cavaggioni et al. ; flower et al. ; konieczny et al. ; santa et al. ; virtanen et al. ) . several studies have indicated that production of mus m is under hormonal control and that the urine of male mice contains four-fold higher levels than the urine of female mice (hastie et al. ; lorusso et al. ; price and longbottom ) . in addition to being present in the saliva and urine, mus m in the serum becomes incorporated into the pelt, conferring the allergenic property to mouse dander. the main allergens of many furred animals are structurally similar proteins within the lipocalin family, including those of the cow (bos d ), horse (equ c ), dog (can f ), and rat (rat n ) (virtanen et al. ) . the mus m and rat n lipocalin allergens, to which % of mouse and rat allergic individuals react, respectively, are closely related, sharing a % homology (clark et al. a) . some have proposed that personnel exposed to laboratory animal allergens can be categorized into four basic risk groups based on their history of allergic disease and sensitization to animal proteins (committee on occupational health and safety in research animal facilities/national research council allergens ). these risk groups are ( ) normal individuals, ( ) atopic individuals with preexisting allergic disease, ( ) asymptomatic individuals with ige antibodies to allergic animal proteins, and ( ) symptomatic individuals with clinical symptoms upon exposure to animal allergens. individuals in the normal risk group do not have a history of allergic disease, and % will never develop symptoms of laa. if laa develops in individuals in the normal risk group, it usually appears during the first three years of exposure. however, infrequently individuals in this risk group who have remained free of laa for or more years of exposure have developed a delayed onset of the condition (department of health and human services, national institute of occupational safety and health ). atopic individuals have a genetic predisposition for an exaggerated tendency to mount ige responses to common environmental allergens. atopic individuals have higher total levels of ige in the circulation and higher blood eosinophil counts compared to normal individuals, possibly as a result of the activation of cytokines involved in ige isotype switching, eosinophil survival, and mast cell proliferation (janeway et al. ). among atopic individuals, up to % of those exposed to allergenic animal proteins eventually develop symptoms (agrup et al. ) . in asymptomatic individuals with elevated circulating ige antibodies to animal allergens, up to % are at risk of developing allergic symptoms. of the individuals in risk groups that are already symptomatic for laa, approximately % will develop chest symptoms and % are likely to develop occupational asthma and face the prospect that continued exposure will result in permanent impairment. allergic rhinitis, allergic conjunctivitis, and contact urticaria are the most common disorders seen in laa (committee on occupational health and safety in research animal facilities/ national research council allergens ). clinically, allergic rhinitis and conjunctivitis present with the symptoms of sneezing, clear nasal discharge, nasal congestion, itchiness, and watery eyes. contact urticaria presenting as raised, circumscribed, erythematous lesions may also be present in laa patients who report an intense itchiness to the skin in the area of contact with the allergen. figs. - and - (fox and brayton ) illustrate the typical wheal and flare reaction on the skin provoked in an individual who had developed hypersensitivity to mouse urine over a period of several years and who was exposed by having a mouse with urine-contaminated feet walk over his arm (ohman ) . one large survey of laboratory animal workers summarized in the niosh alert (department of health and human services, national institute of occupational safety and health ) reported that of animal workers from animal facilities, % developed allergic symptoms related to laboratory animals. of the workers reporting symptoms, % had nasal or eye symptoms, % had skin complaints, and % had asthma. patients who develop asthma as a more serious complication of laa manifest symptoms of wheezing, intermittent dyspnea or shortness of breath, cough, often nocturnal or in the early morning, and tightness of the chest. the key clinical sign in these patients is wheezing on auscultation, and physiological abnormalities include airflow obstruction, which may vary over time, bronchodilator responsiveness, and increased airway responsiveness (airway hyperreactivity) (tang et al. ) . though quite rare, generalized anaphylactic reactions that are potentially life threatening can occur in individuals highly sensitized to animal allergens. anaphylaxis may manifest as diffuse itching, hives, and swelling of the face, lips, and tongue. in some individuals, breathing becomes difficult owing to laryngeal edema, and others develop asthma and wheezing. laboratory animal-associated allergy is an example of the type i, ige antibody-mediated, immune reaction, and the reader should refer to other sources for a detailed discussion of the molecular mechanisms involved in developing this reaction (janeway et al. ). in the case of animal allergens, the usual route of initial exposure is airborne, although bite exposures (saliva) and direct contact with the skin can also become important in later clinical symptoms. in the type i reaction, upon exposure to antigen, which is often a protein or glycoprotein, the allergen is taken up and processed by cells of the innate and adaptive immune systems and by dendritic cells located in the mucosal-associated lymphoid cells, gut-associated lymphoid cells, and/or the dermis. the cytokine profiles of these cells favor the development of na'fve cd t cells into th cells that induce b cells to produce ige specific for the allergen. once the ige response is initiated, it can be further enhanced by basophils, mast cells, and eosinophils that also drive allergen-specific ige production (janeway et al. ) . ige is normally found only in low levels in the circulation because it binds to tissue mast cells and circulating basophils. in the sensitized individual, restimulation with the sensitizing allergen results in allergen binding to ige and the release of histamine and other chemical mediators from the mast cells and basophils. these mediators produce the array of clinical signs and symptoms that are characteristic of the allergy: itchiness, nasal congestion, sneezing, nasal and ocular drainage, coughing, wheezing, and shortness of breath. to establish the diagnosis of laa related to mouse exposure, the physician should begin by considering the strength of the history, physical examination findings, the temporal relationship between the patient symptoms and the environmental exposure to mice, and possibilities of alternative explanations for the patient's problems such as exposure to other potential allergens in the workplace or allergens of a nonoccupational nature. the development of clinical symptoms concomitant with or following exposure to an environment containing mice or mus m laden mouse products should help in narrowing the number of allergens tested. the patient's family history of allergy is also very important to consider because atopy is a proven risk factor in developing laa (botham et al. ; meijer et al. ; venables et al. ). physical examination of the patient and clinical monitoring for the progression of allergic disease incorporate a number of approaches. pulmonary function tests such as bronchial hyperresponsiveness (in response to pharmacologic challenge with methacholine and not the specific allergen) and the forced expiratory volume in one second (fev ) are commonly used to evaluate the degree of airway impairment and the response to bronchodilators, glucocorticoids, and other therapeutic agents. radiographs may also be useful in patients with pulmonary involvement. routine laboratory tests may also aid in the characterization and management of the patient's condition, such as complete blood count and nasal smears for eosinophilia which is common in allergic individuals but also can be seen in those with perennial nonallergic rhinitis (dykewicz et al. ) . measurement of total serum ige has little value to the physician as an aid in distinguishing whether a particular patient has allergic disease, but it may offer some potential for the identifying of populations at risk for developing of laa as indicated in both prospective and cross-sectional studies of laboratory animal workers (hollander et al. ; renstrom et al. ) . use of the radioallergosorbent test (rast) for the detection of human ige antibodies of defined allergen specificity is also available for patient evaluation. however, the quality of the laboratory performing the in vitro rast assays, the specificity of the allergens used, and the potential for cross-reactivity are important considerations in adopting the rast as a diagnostic tool (hamilton ) . even when properly conducted, in vitro tests usually fail to detect a modest number of skin testpositive individuals, and on a per-test basis, skin tests have lower time and reagent costs (hamilton ) . clinicians agree that when properly performed, prick-puncture skin tests are generally considered the most convenient and least expensive screening method for detecting allergic reactions in most patients (demoly et al. ) . the valid interpretation of these tests relies on standardized allergens and methods, and negative prick-puncture tests may be confirmed by more sensitive intradermal techniques. even after falsepositive and false-negative tests have been eliminated, the proper interpretation of results requires thorough knowledge of the patient's history and physical findings. a positive skin test alone does not confirm a definite clinical sensitivity to an allergen in the asymptomatic patient but possibly predicts the onset of allergic symptoms. a positive skin test in conjunction with a history suggestive of clinical sensitivity strongly indicates the allergen as the cause of the disease (horak ) . strong positive skin tests along with a suggestive clinical history also correlate well with results of bronchial or nasal challenges with the antigen. the animal facility conditions and practices that contribute to mouse-associated laa as a serious and prevalent workplace hazard have received considerable study over the past several decades, enabling effective strategies for achieving control of exposures in most research animal care and use settings. in summary, these strategies involve exposure reduction through source reduction, containment of hazard through the use of modern equipment and engineering controls, and barrier protection with personal protective equipment. the mus m allergen load in the environment is markedly increased when male mice are used in studies due to the fact that they excrete -fold higher levels of allergen in the urine than do female mice (lorusso et al. ). therefore, sources have recommended, that whenever scientifically possible, use of only female mice would be a means of reducing allergen load in the environment and minimizing the exposure of personnel (department of health and human services, national institute of occupational safety and health ; renstrom et al. ). furthermore renstrom et al. ( ) reported a three-fold higher rate of allergic sensitization in technicians who worked with male rodents. although this approach may be useful in a few studies, this method of source reduction would appear to have only very limited applicability across the broad scope of contemporary studies using mouse models. source reduction of mouse allergen is also achieved through reduction of animal density within an animal room (the number of animals per room volume) and through use of frequent, effective facility sanitation practices (department of health and human services, national institute of occupational safety and health ). the risk of exposure to mouse allergen varies by the type of animal-related activities conducted by personnel and by the type of animal housing systems and equipment containment devices employed in the use and maintenance of laboratory mice (gordon et al. (gordon et al. , schweitzer et al. ; thulin et al. ) . many studies have examined the different caging systems used for mouse housing, and the ability of each cage system design to reduce environmental allergen is well known (gordon et al. ; schweitzer et al. ) . the application of just a simple filter sheet top or fitted filter bonnet to an open cage is effective in reducing ambient allergen levels (reeb-whitaker et al. ) . however, studies indicate the clear superiority of individually ventilated caging (ivc) systems run under negative pressure for the purpose of controlling room allergen levels (gordon et al. ; reeb-whitaker et al. ; schweitzer et al. ) . gordon et al. ( ) suggested that the use of efficient negative ivc in combination with other engineering controls for allergen containment during procedures and waste processing would potentially produce a virtually allergen-free work environment. when negative pressure ivc housing is not available, the placement of cages in a hepafiltered, ventilated cabinet is effective at reducing room allergen loads (thulin et al. ) . gordon et al. ( ) reported that individuals who have direct contact with mice (animal technicians) have the highest exposure, followed by those who have intermittent contact with anesthetized animals or mouse tissues (scientists and necropsy technicians), followed by those with indirect contact (office workers or histology technicians). the specific animal husbandry activities that are known to result in high exposure of personnel to mouse allergen are cage-changing activities, including animal transfer, stacking dirty cages, and manual emptying of cages; handling animals directly (particularly males); and room sweeping (gordon et al. ) . for each of these activities, use of improved containment equipment and changes in equipment handling procedures are effective in the controlling the allergen hazard and should be encouraged. for example, use of ventilated cabinets or biological safety cabinets during cage changing and animal handling is effective in conjunction with the use of microisolation cages (gordon et al. ; schweitzer et al. ; thulin et al. ) . containment equipment has also been designed for the capture of airborne allergens generated when the bottom of one dirty cage is placed into the opening of another to stack the cages for transport to the cage wash area or when dirty bedding is removed prior to cage washing (gordon et al. ; kacergis et al. ) . room cleaning with a vacuum equipped with hepa filtration followed by mopping with a damp mop also aids in reducing the environmental allergen load and personnel exposure (kacergis et al. ) . use of personal protective equipment and dedicated work clothing for personnel involved in high-exposure activities is an important asset in reducing allergen exposure. it is important for the work clothing to remain at work, as evidenced by the finding that children of laboratory animal workers had a higher incidence of clinical signs during provocative testing, positive skin tests, and ige specific to laboratory rodents than did the children of parents who worked in other occupations (krakowiak et al. ) . full sleeve protection and gloves should be worn to prevent the urticarial reactions in persons who are highly sensitive to the mus m allergen. personnel should also be provided with respiratory protection and eye or face protection when warranted. either filtering facepiece particulate respirators (n equivalent) or powered air purified respirators are effective in reducing exposure and alleviating clinical symptoms (schweitzer et al. ; thulin et al. ) . special attention must be paid to the selection and fitting of n filtering facepiece particulate respirators to ensure proper function (morbidity and mortality weekly report ). when the elimination of mouse allergen exposure in the workplace is not achieved through the use of engineering controls, work practices, and personal protective equipment, allergic reactions in persons sensitive to mus m can be managed with pharmacological agents that have a long history of use for this condition. these include antihistamines, topical ~-adrenergic agents (bronchodilators), cromolyn sodium as a nasal spray, and intranasal potent glucocorticoids (austen ) . prophylaxis in patients with mild symptoms is often provided by topical cromolyn sodium on a continuous basis, supplemented with the intermittent use of antihistamines often at bedtime. the selection of the antihistamine has been an area of considerable discussion, and the reader should refer to casale et al. ( ) for further insights into this matter. in more serious cases, potent topical glucocorticoids may be necessary for alleviating clinical signs. immunotherapy, or hyposensitization, is typically reserved for patients who are unable or unwilling to escape the allergen provoking the response. although allergy to the dog or the cat can be ameliorated by immunotherapy (norman (norman , , the infrequent reports in the literature of immunotherapy for allergy to mice and other small laboratory animals have failed to establish the usefulness of this approach for the control of allergy to these species (sorrell and gottesman ; wahn and siraganian ) . the progress made in the past two decades in the evolution of health care systems responsible for the monitoring, control, and elimination of infectious diseases in laboratory mice as well as the advancements in the facilities, equipment, and techniques used to maintain mice in contemporary animal care and use programs, has vastly reduced the likelihood that personnel will encounter zoonotic diseases or other health hazards in the laboratory under most circumstances. continued program success in the control of mouse-associated hazards relies on the use of well-designed and maintained animal facilities, exclusion of wild rodents and other vermin, and quality control in animal care and veterinary care practices. in unique experimental situations that place personnel at a high risk of exposure to mouse-associated hazards, institutional review should ensure that procedures are carefully planned and conducted using personal protective equipment for worker safety. allergy to laboratory animals in laboratory technicians and animal keepers the relationship of trichophyton quinckeanum to trichophyton mentagrophytes alteras, i. ( ) human infection from laboratory animals rat-bite fever in animal research laboratory personnel molecular approaches for the treatment of hemorrhagic fever virus infections fatal illnesses associated with a new world arenavirus---calif~ rabies in a beaver--florida hantavirus-specific cd (+)-t-cell responses in newborn mice persistently infected with hantaan virus rat bite fever at albert einstein medical center experimental lymphocytic choriomeningitis of monkeys and mice produced by a virus encountered in studies of the st. louis encephalitis epidemic allergies, anaphylaxis and systemic mastocytosis flea-borne rickettsioses: ecologic considerations infection by leptospira ballum: a laboratory-associated case lymphocytic choriomeningitis virus: reemerging central nervous system pathogen lymphocytic choriomeningitis virus: emerging fetal teratogen lymphocytic choriomeningitis virus: an unrecognized teratogenic pathogen epidemic nonmeningitic lymphocytic-choriomeningitis-virus infection. an outbreak in a population of laboratory personnel animal agents and vectors of human disease tick-borne (pasture) fever and rickettsial pox broad range polymerase chain reaction for diagnosis of rat-bite fever caused by streptobacillus moniliformis detection of lymphocytic choriomeningitis virus by use of fluorogenic nuclease reverse transcriptase-polymerase chain reaction analysis contamination of transplantable murine tumors with lymphocytic choriomeningitis virus lymphocytic choriomeningitis outbreak associated with pet hamsters. fiftyseven cases from new york state the influence of maternal antibodies on the epidemiology of leptospiral cartier state in mice favus of mice a case of leptospirosis ballum in california mouse ringworm eeen geval van leptospirosis ballum allergy to laboratory animals: a follow up study of its incidence and of the influence of atopy and pre-existing sensitisation on its development laboratory studies of a lymphocytic choriomeningitis virus outbreak in man and laboratory animals antigenic relationships amongst coronaviruses. archiv fur die gesante virusforschung rickettsialpox: report of an outbreak and a contemporary review salmonella infections in rodents in manchester, with special reference to salmonella enteritidis var. danysz the occurrence of leptospiral infections in feral rodents in southwestern georgia arenaviridae: the viruses and their replication first do no harm: managing antihistamine impairment in patients with allergic rhinitis pheromone signalling in the mouse: role of urinary proteins and vomeronasal organ epizootic of trichophyton mentagrophytes (interdigitale) in white mice aetiological agents in occupational asthma tropical rat mite dermatitis lymphocytic choriomeningitis virus infection and house mouse (mus musculus) distribution in urban baltimore human-rodent contact and infection with lymphocytic choriomeningitis and seoul viruses in an inner-city population ecology and epidemiology of arenaviruses and their hosts spread of trichophyton mentagrophytes var. gran. infection to man inactivated hantaan virus vaccine derived from suspension culture of vero cells outbreak of rat mite dermatitis in medical students structure of mouse major urinary protein genes: different splicing configurations in the '-non-coding region a -kb dna domain with two divergently orientated genes is the unit of organisation of the murine major urinary protein genes an advancement in cage design to provide environmental enrichment for mice when rearing the plague flea xenopsylla cheopis (roths) for behavioural studies national prevalence and exposure risk for mouse allergen in us households rat-bite fever. report of three cases serologic evidence of rickettsialpox (rickettsia akari) infection among intravenous drug users in inner-city occupational health and safety in the care and use of research animals the use of pathogen-free animals control of mouse ringworm sendai virus antibody in acute respiratory infections and infectious mononucleosis exposure to leptospira icterohaemorrhagiae in inner-city and suburban children: a serologic comparison department of health and human services. national institute of occupational safety and health ringworm epizootics in laboratory mice and rats: experimental and accidental transmission of infection the tropical rat mite, liponyssus bacoti hirst : the cause of a skin eruption of man, and a possible vector of endemic typhus fever rat mite dermatitis: a family affair rickettsia typhi (murine typhus) geophites zoophilic and anthropohilic dermatophytes: a review lymphocytic choriomeningitis outbreak associated with nude mice in a research institute diagnosis and management of rhinitis: complete guidelines of the joint task force on practice parameters in allergy pathogenicity of hantaan virus in newborn mice: genetic reassortant study demonstrating that a single amino acid change in glycoprotein g is related to virulence characterisation and antibiotic susceptibilities of streptobacillus moniliformis management of allergies to animals family bunyaviridae tropical rat mite dermatitis: case report and review leptospirosis craig and faust's clinical parasitology structure and sequence relationships in the lipocalins and related proteins parasites of laboratory animals rat bite fever without the bite man's worst friend (the rat) outbreak of tropical rat mite dermatitis in laboratory personnel zoonotic diseases. campylobacter infections and salmonellosis. semin zoonoses and other human health hazards hepatic helicobacter species identified in bile and gallbladder tissue from chileans with chronic cholecystitis infections transmitted from large and small laboratory animals leptospirosis ballum contracted from pet mice bear canyon virus: an arenavirus naturally associated with the california mouse (peromyscus californicus) leptospiral serotype distribution lists according to host and geographic area serological evidence of infection with sendai virus in england rabies in israel: decades of prevention and a human case peculiarities of the influenza outbreak in vladisvostok due to d virus rat-bite fever elimination of mouse allergens in the working environment: assessment of individually ventilated cage systems and ventilated cabinets in the containment of mouse allergens measurement of exposure to mouse urinary proteins in an epidemiological study rat-bite fever (streptobacillus moniliformis): a potential emerging disease lymphocytic choriomeningitis virus in mouse neoplasms rat mite dermatitis in children laboratory tests for allergic and immunodeficiency diseases antibodies to mouse hepatitis viruses in human sera multiple genes coding for the androgen-regulated major urinary proteins of the mouse lymphocytic choriomeningitis virus teleomorphs and mating types in trichophyton mentagrophytes complex anaphylaxis after laboratory rat bite: an occupational hazard rat mite dermatitis outbreak of lymphocytic choriomeningitis virus infections in medical center personnel trichophyton mentagrophytes skin infections in laboratory animals as a cause of zoonosis identification of risk factors in rat bite incidents involving humans cat and dog allergy and total ige as risk factors of laboratory animal allergy streptobacillus moniliformis polyarthritis mimicking rheumatoid arthritis: an urban case of rat bite fever experimental transmission of tularemia by the tropical rat mite manifestation of allergic rhinitis in latent-sensitized patients. a prospective study the contamination of laboratory animals with lymphocytic choriomeningitis virus rickettsialpox--a newly recognized rickettsial disease. v. recovery of rickettsia akari from a house mouse (mus musculus) rickettsialpoxma newyly recognized rickettsial disease. iv. isolation of a rickettsia apparently identical with the causative agent of rickettsialpox from allerdermanyssus sanguineus, a rodent mite rickettsialpox--a newly recognized rickettsial disease. i. isolation of the etiological agent diseases transmitted from animals to man allergy to laboratory mice and rats: a review of the pathophysiology, epidemiology and clinical aspects the etiology, mode of infection and specific therapy of weil's disease sendai virus health care for research animals is essential and affordable lymphocytic choriomeningitis virus. a neglected pathogen of man allergy and hypersensitivity diseases of children in the subtropics and tropics serologic evidence of american experience with newborn pneumonitis virus rabies virus air quality in an animal facility: particulates, ammonia, and volatile organic compounds prevalence of rabies virus and hantaan virus infections in commensal rodents and shrews trapped in bangkok ornithonyssus bacoti (hirst) infestation in mouse and hamster colonies pathogenesis of hantaan virus infection in suckling mice: clinical, virologic, and serologic observations animal aeroallergens the major dog allergens, can f and can f , are salivary lipocalin proteins: cloning and immunological characterization of the recombinant forms increased detection of rickettsialpox in a new york city hospital following the anthrax outbreak of : use of immunohistochemistry for the rapid confirmation of cases in an era of bioterrorism allergy to laboratory animals in children of parents occupationally exposed to mice, rats and hamsters epidermophyton floccosum (harz) langeron and milochevitsch als spontanin fektion bei mausen newborn virus pneumonitis (type sendai) ii. the isolation of a new virus newborn virus pneumonitis (type sendai) ii. the isolation of a new virus possessing hemagglutinin activity streptobacillus moniliformis isolated from a case of haverhill fever: biochemical characterization and inhibitory effect of sodium polyanethol sulfonate lymphocytic choriomeningitis virus infection in a province of spain: analysis of sera from the general population and wild rodents weil's syndrome in a zoologist zoonoses in common laboratory animals immunologic and biochemical properties of the major mouse urinary allergen (mus m ) an outbreak of hepatitis in marmosets in a zoological collection a worldwide survey of management practices in laboratory animal allergy trichophyton mentagrophytes in mice: infections of humans and incidence amongst laboratory animals a molecular phylogeny of nuclear and mitochondrial sequences in hymenolepis nana (cestoda) supports the existence of a cryptic species detection of the rodent tapeworm rodentolepis (=hymenolepis) microstoma in humans. a new zoonosis? medical parisitology non-domestic animals in new zealand and in rarotonga as a reservoir of the agents of ringworm reverse genetics of the largest rna viruses association between helicobacter bilis in bile and biliary tract malignancies: h. bilis in bile from japanese and thai patients with benign and malignant diseases in the biliary tract lassa fever. effective therapy with ribavirin rat-bite fever due to streptobacillus moniliformis growth in sucklingmouse brain of "ibv-like" viruses from patients with upper respiratory tract disease detection of workers sensitised to high molecular weight allergens: a diagnostic study in laboratory animal workers infectious and parasitic disease persistent hantavirus infections: characteristics and mechanisms ecologic studies of rodent reservoirs: their relevance for human health a new transmissible viral hepatitis of marmosets and tamarins epidemiologic notes and report: lymphocytic choriomeningitis virus-georgia mmwr seroepidemiological survey of lymphocytic choriomeningitis virus in wild house mice in china with particular reference to their subspecies pathogens of house mice on arid boullanger island and subantarctic macquarie island paramyxovirus replication and pathogenesis. reverse genetics transforms understanding sars: lessons learned from other coronaviruses treatment of salmonella gastroenteritis with ampicillin, amoxicillin, or placebo preemployment screening for allergy to laboratory animals: epidemiologic evaluation of its potential usefulness contamination of transplantable tumors, cell lines, and monoclonal antibodies with rodent viruses morphological characteristics and nomenclature of leptospira (spirochaeta) icterohemorrhagiae (inada and ido) immunotherapy: past and present immunotherapy: - allergy in man caused by exposure to mammals hantavirus-specific antibodies in rodents and humans living in kuwait rickettsialpox in new york city: a persistent urban zoonosis microbial flora of the larynx, trachea, and large intestine of the rat after long-term inhalation of per cent oxygen survey of dermatophytes isolated from the coats of laboratory animals in italy nontyphoidal. in bacterial infections of humans arenaviruses on the role of the mite ornithonyssus bacoti hirst as a reservoir and vector of the agent of tularemia the tropical rat mite, liponyssus bacoti, as an experimental vector of rickettsial pox recombinant sendai virus for efficient gene transfer to human airway epithelium ringworm (trichophyton mentagrophytes) infection in a colony of albino norway rats fleas of public health importance and their control allergy to mice. i. identification of two major mouse allergens (ag and ag ) and investigation of their possible origin cultivation of different viruses in tick tissue culture control strategies for aeroallergens in an animal facility laboratory acquired infection with keratinomyces ajelloi working with male rodents may increase risk of allergy to laboratory animals allergic sensitization is associated with increased bronchial responsiveness: a prospective study of allergy to laboratory animals prospective study of laboratory-animal allergy: factors predisposing to sensitization and development of allergic symptoms reovirus serotype infection in infants with extrahepatic biliary atresia or neonatal hepatitis incidence of rat bites and rat bite fever in baltimore primates. in laboratory animal medicine a mouse oxyurid, syphacia obvelata, as a parasite of man rat mite dermatitis in minnesota viral meningitis and encephalitis: traditional and emerging viral agents lymphocytic choriomeningitis virus in southern france: four case reports and a review of the literature rat-bite fever as a cause of septic arthritis: a diagnostic dilemma streptobacillus moniliformis endocarditis: case report and review rickettsia akari newborn virus pneumonitis (type sendai) i. report: clinical observation of a new virus pneumonitis of the newborn a bovine dander allergen, comparative modeling, and similarities and differences in folding with related proteins distribution of the virus lymphocytic choriomeningitis virus in west germany the tropical rat mite (liponyssus bacoti) as an experimental vector of coxsackie virus reducing exposure to laboratory animal allergens fatal streptobacillus moniliformis infection in a two-month-old infant comparison of media with and without 'panmede' for the isolation of streptobacillus moniliformis from blood cultures and observations on the inhibitory effect of sodium polyanethol sulphonate staphylococcal botryomycosis in a specific-pathogen-free mouse colony fatal rat bite fever in a pet shop employee a serologic survey for viruses and mycoplasma pulmonis among wild house mice (mus domesticus) in southeastern australia a survey of house mice from iowa swine farms for infection with leptospira interrogans serovar bratislava lcm: associated human and mouse infections mouse allergy: case report studies on the pathogenesis of a hitherto undescribed virus (hepato-encephalomyelitis) producing unusual symptoms in suckling mice reovirus not detected by reverse transcriptase-mediated polymerase chain reaction analysis of preserved tissue from infants with cholestatic liver disease isolation of an arenavirus from a marmoset with callitrichid hepatitis and its serologic association with disease identification, using sera from exposed animals, of putative viral antigens in livers of primates with callitrichid hepatitis cdna sequence analysis confirms that the etiologic agent of callitrichid hepatitis is lymphocytic choriomeningitis virus the laboratory diagnosis of leptospirosis leptospirosis (ballum) contracted from swiss albino mice elimination of leptospira ballum from a colony of swiss albino mice by use of chlortetracycline hydrochloride potential helminth infections in humans from pet or laboratory mice and hamsters comparison of diagnostic technics for the detection of leptospirosis in rats spirochetal infections in middleton's allergy: principles and practice anaphylaxis after bites by rodents incidence of murine virus antibody in humans in contact with experimental animals tropical rat mite dermatitis. report of six cases and review of mite infestations anaphylactic reaction after a mouse bite in a -year-old girl reduction of exposure to laboratory animal allergens in a research laboratory viruses transmissible from laboratory animals to man leptospirosis. in crc handbook series in zoonoses orthoreoviruses. in principles and practices of infectious diseases laboratory animal allergy in a pharmaceutical company important animal allergens are lipocalin proteins: why are they allergenic? development of hymenolepis nana and hymenolepis diminuta (cestoda: hymenolepididae) in the intermediate host tribolium confusum efficacy and specificity of immunotherapy with laboratory animal allergen extracts rat mite bite the zoology of tapeworms placebo-controlled trial of intravenous penicillin for severe and late leptospirosis bacterial and mycotic diseases helminths natural and experimental helicobacter infections rat-bite fever in a gerbil breeder congenital lymphocytic choriomeningitis virus syndrome: a disease that mimics congenital toxoplasmosis or cytomegalovirus infection natural occurrence of leptospira ballum in rural house mice and in an opossum on the biological character of hvj akitsugu strain and its pathogenicity in human beings as revealed after experimental inoculation of it in volunteers infections of laboratory animals potentially dangerous to man: ectoparasites and other arthropods, with emphasis on mites influenza d in early infancy key: cord- -rdhuc n authors: anderson, nancy l. title: pet rodents date: - - journal: saunders manual of small animal practice doi: . /b - - - / - sha: doc_id: cord_uid: rdhuc n nan many small rodents are commonly kept for companionship and enjoyment. this chapter provides information needed to diagnose and treat the most frequently encountered problems of mice, rats, gerbils, hamsters, guinea pigs, and chinchillas. • cages should be made of stainless steel, hard plastic, or glass. these materials are cleaned and sanitized easily and are resistant to gnawing or corrosion from urine and fecal matter. minimum floor space and height requirements are listed for each species in • guinea pigs can be housed in open-topped enclosures with walls higher than inches. ensure that dogs, cats, wild animals, and small children do not have unsupervised access to these cages. • clean cages as needed, usually to times per week for most rodents. a scrub brush, dish soap, and water work well. if cages are not kept clean, ammonia, other irritants, moisture, and bacteria concentrations rise to harmful levels, predisposing animals to disease. • disinfect the cage twice a month with part sodium hypochlorite (household bleach) mixed in parts water. let the bleach solution stand for at least minutes. rinse the cage well afterward. • all solid-floored cages need to be covered in bedding. shredded paper, non-resinous wood shavings, wood wool, and corn cobs are all acceptable. provide at least inches of bedding. most rodents enjoy burrowing in deeper bedding when it is provided in one corner of a cage. do not, however, fill the entire cage with deeper bedding. this usually leads to poor sanitation as a result of owners' failure to recognize buildup of hidden wastes such as moisture from leaking water bottles, cached foods, urine, and feces. • wire mesh floors can be used successfully only if the dimension of the mesh is correct. size the openings to be just large enough for an adult to retract a tarsal joint back through the mesh. larger holes make it difficult for the animals to walk and cause pressure sores. smaller openings may cause injuries such as tibial fractures and self-mutilation while struggling to free trapped appendages. bedding above the wire keeps waste from dropping through the wire and therefore is not recommended. wire bottom cages do not work well for breeding animals because neonatal rodents must be surrounded by nesting material to maintain moisture in the nest and prevent dehydration. young rodents often cannot walk correctly on mesh sized for adult feet. • all pet rodents require visual security. tubes, jars, or cans made of nontoxic, nonabrasive substances work well for this purpose. also provide objects for gnawing. rodents possess open-rooted teeth, and constant wear is necessary to maintain normal dentition. mice, rats, gerbils, and hamsters enjoy and benefit from exercise wheels. • a good room temperature range for most pocket pets is °f to °f. keep rodents with disease at °f to °f unless hyperthermia is of concern (some chinchillas). • provide to hours of darkness to to hours of light. this light cycle is essential if breeding is desired. • hamsters, guinea pigs, and chinchillas that are exposed to temperatures below °f may hibernate for a few days or until the ambient temperature rises. heart rates may be less than bpm during hibernation. m key point feed pet rodents laboratory animal chow appropriate for their species (table - ). seed diets are deficient in protein and contain excessive fat. • seeds, as well as vegetables and other foods, may be fed as treats but not to provide more than % of calories. intermittent exposure to vegetables and seeds causes mild, transient diarrhea. • supplementation of vitamin c is recommended for all guinea pigs. • adult chinchillas that are not obese should be fed high quality, fresh grass hay ad libitum. obese animals may need to have the hay rationed. chinchillas require / to / -cup of fresh pellets per animal each day. feeding pellets free choice leads to obesity, and the high protein and calcium levels in these diets may predispose animals to urinary tract disease. most pellets also do not provide sufficient fiber to maintain normal gastrointestinal (gi) motility. • store food in tightly sealed containers at less than °f; keep food refrigerated if possible. • feed all diets within days of milling to ensure the highest nutritional value. encourage owners to check dates on packages and ask pet store managers about providing dates on bulk items. • if possible, feed pet rodents except guinea pigs from overhead racks. these devices reduce wastage and eliminate fecal contamination of food. covered hoppers, heavy crocks, or stainless steel bowls that are attached to the side of the cage to eliminate spillage are acceptable and recommended for guinea pigs. • feed breeding females and their litters from the floor of their cages until the young are large enough to reach overhead feeders or crawl in and out of crocks. • cannibalism of neonates commonly occurs as the result of stress associated with cage cleaning. to minimize cannibalism, clean the cage and provide a to -day food supply to days before parturition. m key point provide fresh water in clean containers daily. • do not provide water in open crocks. these are contaminated or spilled easily and are a common cause of dehydration and poor sanitation. • sipper tubes and water bottles work well. clean with dish soap and water daily and disinfect them weekly. guinea pigs expel food from their mouths into their sipper tubes, so more frequent cleaning is needed. • some water bottles have special valves to minimize backflow. supplement guinea pigs' water daily with mg vitamin c/l. if the water is not dechlorinated, it will inactivate the vitamin c. quarantine all newly acquired animals in a different room from current pets for a minimum of days. feed and handle quarantined animals last. recommend that caretakers wash their hands and change clothes before handling current pets. avoid the introduction of adult animals because this frequently results in fighting. instead, place animals together while young and allow them to mature together. avoid keeping more than one male per cage because this also usually leads to fighting. a systematic history and physical examination are mandatory. many disease syndromes are caused by poor husbandry. pets that have been kept isolated from other rodents or acquired from a private breeder are less likely to harbor infectious disease than animals obtained from a pet store, laboratory, or wholesaler. see table - for normal physiologic data. obtain the following information: • observe the pet in its cage for mentation, activity, locomotion, dyspnea, head posture, haircoat, and any grossly observable abnormalities. • note respiratory and heart rates before restraint when possible. observe the condition of cagemates. m key point if dyspnea or severe depression is detected, warn the owner that the animal is critically ill and could die of stress brought on by an examination. • handle such animals as little as possible. initially, treat severely ill animals symptomatically, then • note the type of diet and bedding as well as the level of sanitation and compare these with what was described in the history. • observe quantity and quality of feces and urine. diarrhea, soft stools, absence of stools, copious urine, and discolored urine all can be signs of illness. • coprophagy is a normal behavior in rodents. • check the diet and water supply for freshness, quantity, source, and accessibility. • evaluate the presence and suitability of cage furniture. adequate visual security and the ability to exercise and gnaw are extremely important. • an accurate weight in grams is extremely important for evaluating an animal's body condition, calculating drug dosages, and monitoring treatment. the easiest method of weighing a pet rodent is to place it in a box and then subtract the weight of the container. • restraint of pet rodents is easy with experience. pets that have been handled frequently and gently by the owners require only minimal restraint. gentle pressure directs the animal as needed. grasp less cooperative patients (except chinchillas and guinea pigs) by the scruff over the back of the neck with thumb and forefinger ( fig. - ) . take care to pinch enough skin to prevent the animal from turning around, yet leave enough slack for respiration. on smaller specimens, hold the base of the tail, if present, between the fourth and fifth fingers to provide additional restraint. • hold docile guinea pigs with the palm of one hand supporting the chest while the other hand supports the hind quarters ( fig. - ). place the thumb and forefinger of the first hand in the axillas for additional control. • take care to minimize damage to the fur when handling chinchillas because they lose hair easily. grasp the animal by the tail and scoop it up into the palm of the same hand ( fig. - ). if necessary, grasp the thorax just behind the axillas. • calm uncooperative rodents by placing an appropriately sized towel over the head. complete the physical examination by wrapping the patient in a towel and exposing only needed areas. even oral, ophthalmic, and aural examinations can be performed with minimal effort if the animal is given the chance to relax in the towel "burrow." • remove particularly aggressive patients from their cages by scooping them up in a can or bucket; then slide them out onto a slick surface and pick them up or transfer them to a holding area or scale. m key point lift the hind quarters of mice and rats by the base of their tails to facilitate scruffing. never use the tip of the tail for restraint, or the skin of the tail may slough. once the animal is restrained properly, examine the head. assess the cranial nerves. check the nose for presence and character of discharge. examine the mouth for ptyalism, swelling, overgrown incisors, or discharges. to inspect the oral cavity, place an avian speculum across the mouth just caudal to the incisors. use a light source and a pair of hemostats as retractors to improve access. alternatively, use an otoscope with a pediatric head to examine the premolars and molars of guinea pigs and chinchillas for overgrowth. examine the cheek pouches of hamsters for swelling, impaction, or discharge. an ophthalmic examination, including a fundic examination, is important. • use a slit lamp to identify superficial pathology, especially corneal ulcers or foreign bodies. • if indicated, perform fluorescein stain and conjunctival scrapings or cultures. • note the presence of conditions such as discharge, asymmetry, and exophthalmos. m key point gerbils, rats, and mice produce red tears (chromodacryorrhea) with stress or disease. do not confuse them with hemorrhage. • guinea pigs suffering from hypovitaminosis c often produce dry, white tears. • check ears for discharge, foreign bodies, and mites. bluish discoloration of the ears is a sign of cyanosis. bright red injected coloration is associated with septicemia. sores behind the ears and on the neck are often a sequela of aural disease. • evaluate submandibular, axillary, inguinal, and popliteal lymph nodes for size and consistency. enlargements usually indicate infectious or neoplastic disease. • reevaluate respirations and heart rate after the stress of handling and compare them with the resting rate noted when the animal was in the cage. note dyspnea or respiratory sounds. auscultate animals weighing more than g. counting every third or fourth beat and multiplying by the appropriate factor allows recording of heart rates up to bpm. • palpate the abdomen. pay special attention to differentiating pregnancy from the bladder, kidneys, abdominal masses, enlarged cecum, and fecal balls in the colon. while palpating the abdomen, examine the mammary chain of all female rodents for signs of mastitis, lactation, or neoplasia. also check male mice and rats for mammary neoplasia. mammary tissue extends from the base of the neck to the base of the tail. gerbils typically have an elliptical sebaceous gland on their ventral midline. do not confuse this with neoplasia or infection. check the rectum and perineal area for signs of diarrhea, prolapse, irritation, parasites, and bite wounds. note that coprophagy is normal in rodents. • evaluate the urogenital tract for signs of inflammation, foreign bodies, urine scalding, and vaginal discharge. locate and palpate the testicles in males. the easiest method of determining sex in pet rodents is to compare the anogenital distance, which is twice as long in males as in females. other characteristics that allow the determination of sex are as follows: • visualization or palpation of testicles or extrusion of the penis from the prepuce indicates a male. • the presence of two external openings (i.e., anus and urethra) indicates a male. • the presence of three openings (i.e., anus, vagina, and urethra) indicates a female. • examine the skin and fur for conditions such as crusts, alopecia, masses, herniations, and wounds. • check tail and feet for swellings, coloration, sores, length of toenails, and condition of footpads. • evaluate the extremities for trauma or other abnormalities. apply cellophane tape to crusted areas of the skin and view under a microscope as an aid in diagnosing ectoparasites such as lice, mites, and fleas. skin scrapings are beneficial in detecting mites and dermatophytes. dermatophytes are diagnosed best through culture of broken hairs or crust on dermatophyte test medium. use small, cotton-tipped swabs to obtain ear swabs from animals weighing more than g. mix debris with mineral oil and view under low magnification to test for ear mites, or roll onto a glass slide and gram stain to look for bacterial or yeast infections. collect urine by placing the rodent in a clean meshbottomed cage with a plastic liner. after enough urine has been produced, collect it off the bottom of the cage with hematocrit tubes or a syringe and a -gauge needle. perform cystocentesis on non-pregnant animals weighing more than g with a -to -gauge needle. collect feces over several hours to provide a volume sufficient for fecal flotation. flotation allows the detection of nematodes and some trematodes and cestodes. cellophane tape applied to the perineal area and then viewed under a microscope often reveals oxyurid eggs. use a fresh saline smear or fecal sedimentation to diagnose protozoal parasites. fecal cultures are useful in diagnosing bacterial diarrhea. radiology is an extremely useful tool. machines capable of exposures as low as kvp and to mas effectively image mice. most radiograph machines are capable of generating diagnostic radiographs of guinea pigs, chinchillas, and mature rats at settings used for kittens. positioning is accomplished with masking tape or velcro straps. sedate unruly animals. techniques used in cats for contrast studies of both urinary and gi systems are modified easily for use in pocket pets. • use lateral or medial saphenous veins to obtain samples in animals heavier than g. liberally clip the area to allow exposure of the vessel before attempting venipuncture. place a -to -gauge needle in the vein and collect blood into microtainers or hematocrit tubes as it drips from the hub of the needle (fig. - ) . take extreme care not to col-lapse and lacerate the vein with overzealous aspiration if a syringe is attached to the needle. • it is also possible to use the cephalic vein in guinea pigs. • jugular veins are good alternatives in thin individuals under sedation. • an alternative technique that is useful in smaller animals is to coat the skin over the vein with a thin layer of petroleum jelly and then to puncture the vessel. blood is collected with a hematocrit tube as it exits the wound. samples up to % of the animal's weight are considered safe, even in stressed animals. m key point attempt tail bleeding only as a last resort in mice, rats, gerbils, and hamsters. these techniques often are not acceptable to owners. to bleed the tail, warm the tail with water or compresses to dilate the tail vessels. in large rats, perform venipuncture with a needle and obtain blood in the usual fashion. in smaller animals, lacerate the tip of the tail. blood from the wound is collected as described previously. see tables - and - for hematology and chemistry values. incorporate oral medications into a treat, or administer them in liquid form. if the medication is palatable, administer it by placing the tip of a dosing syringe into the diastema. m key point take care not to place the tip into the contralateral cheek pouch, or the patient may store the medication and expel it later. administer medication in small amounts. ensure that the animal swallows the medication in its mouth before more is administered. this technique is useful for force-feeding pellet gruels to anorexic pets if the caregiver is patient. medication or food that is administered too quickly will be spit out or aspirated. for rodents that are intractable or for administration of unpalatable substances, pass a stomach tube per os. • metal feeding needles, red rubber urinary catheters, or infant feeding tubes work well. selection is based on the size of the animal and individual preference. metal feeding needles with ball tips frequently are used in patients weighing less than g . the metal provides the necessary stiffness to pass a tube of small diameter. the ball at the end of the needle makes it difficult (but not impossible) to pass the tube into the trachea. these tubes have the potential to create esophageal tears with improper restraint or when excessive force is applied. • measure the length from the tip of the nose to the last rib. ventroflex the head slightly, and place the tip of the tube through the diastema and over the tongue. if the tube does not pass easily down the esophagus to the premeasured distance, check the tube size and/or reposition the tube before attempting further advancement. the needle is easily palpable percutaneously if it is placed correctly. it is usually safe to administer up to ml/ g body weight. • a flexible catheter is ideal for use as a stomach tube in larger rodents ( fig. - ). use a speculum to prevent chewing on the tube. an otoscope head, avian speculum, or piece of wood or plastic with a hole drilled in the center works well. measure and mark the tube for the distance from the tip of the nose to the last rib. place the speculum in the mouth and over the tongue. pass the tube while holding the speculum in place and slightly ventroflexing the head. resistance is encountered if the tube is malpositioned or is an inappropriate size. the tube must pass over the tongue before it can be advanced down the esophagus. this is difficult in some animals. palpate the throat to confirm the presence of the feeding tube in the esophagus. m key point because the placement of a stomach tube is a blind procedure, administer a small volume of sterile saline into the tube before administering the medication to ensure that the tube is not in the trachea. misplaced medications are fatal. • this method is also useful for administration of nutrition to anorexic patients. place a pharyngostomy tube if repeated dosing is necessary, using the technique for cats. flush pharyngostomy tubes with water at least every to hours. nasogastric tubes are not recommended because they are difficult to place and maintain patency because of their small size. securely suture all tubes to the skin. place a tube collar made of radiographic film or use rear leg hobbles to prevent removal of tubes. • nutritional support is critical in rodents because of their high metabolic rate. provide supplements in animals that are anorexic for longer than hours. if the gi tract is capable of digestion, use a slurry of pellets mixed with a high-calorie supplement. if the tube diameter is too small for this mixture, use avian hand-feeding formula or a mixture of vegetable and cereal baby foods in place of the pellets. if the ability of the gi tract to tolerate enteral feeding is questionable, first try isotonic electrolyte or dextrose solutions. parenteral nutrition is used successfully in research animals and may be feasible in select pet cases. administer sc injectable medications or fluids over the shoulder blades or in folds of skin on the flank. • avoid irritating substances in rats and mice because their mammary tissue extends into these areas. the resulting inflammatory response is thought to increase the occurrence of mammary neoplasia. m key point in general, avoid streptomycin and the carrier procaine in all pet rodents because of a high incidence of toxicity and hypersensitivity reaction. give im injections in the semimembranous and semitendinous muscles. inject only small volumes of nonirritating substances, or tissue damage with resulting self-mutilation may occur. use the epaxial or triceps muscles if repeated injections are necessary. m key point use intraperitoneal (ip) injections only as a last resort for large volumes of fluids or for irritating injections that cannot be administered via an iv or io route. express the bladder and aseptically prepare the abdomen. restrain the rodent with its head down to move the abdominal organs cranially. give the injection . to cm lateral to the midline in the caudal abdomen. aspirate before injecting to ensure that the injection is not being given into the bladder or bowel. never use this technique in pregnant animals. give iv injections into any of the veins as previously described. in addition, the penile vein may be used in hamsters and guinea pigs. placement of iv catheters is possible in animals heavier than g. for small rodents, give a bolus of fluids every - hours, followed by a diluted heparin flush. a pediatric iv pump is used for continuous infusion of fluids to mark distance from nose to last rib larger animals. maintenance of catheters in active animals is extremely difficult. for io injections, place a spinal needle into the proximal tibia or femur following the technique used for placing an intramedullary pin. once the needle is seated, remove the stylet. aspirate and check the hub of the needle for bone marrow. the tip of the needle should be in the bone marrow cavity that directly drains into the central venous system in normal bones (i.e., the cortex must be intact). administer drugs, blood, or fluids at a rate similar to that used for iv catheters. • in chinchillas and guinea pigs, withhold food for hours before anesthesia. withhold food from smaller, mature rodents for hours. withhold food from immature animals for up to / hour depending on age and condition. • use heat lamps and heating pads to prevent hypothermia. have a prewarmed incubator available for recovery. preoperative or intra-operative warmed sc or iv fluids are strongly recommended. place iv or io catheters whenever possible. • administer atropine preoperatively to reduce airway secretions. acepromazine, diazepam, or midazolam work well as premedications for other anesthetics. avoid acepromazine in gerbils because it potentiates seizures. see table - for anesthetic drugs and dosages. surgical anesthesia is reached when toe, tail, and ear pinch fail to generate a withdrawal reaction. depth of anesthesia is best monitored by pulse and respiratory rate and character. pulses drop to within normal ranges after induction. further reduction, especially to less than % of the original stabilized value, is an indication to lighten the plane of anesthesia. monitor the electrocardiogram (ecg) of small patients by clamping the alligator clips onto the hubs of all-metal -gauge needles or steel sutures placed through the skin at the usual sites. tape cables to the table to maintain placement. doppler units taped over the chest also provide accurate heart rates. pulse oximeters are easier to use, more sensitive, and more expensive than the instruments mentioned previously. these instruments are easily taped to the patient's ear, foot, or tail and provide heart rates as well as information regarding oxygenation. respirations are often shallow and rapid during induction. they become deep and regular as a surgical plane of anesthesia is reached. the corneal reflex varies markedly between individuals and anesthetic agents. if the animal has a corneal reflex after induction and then loses it, reduce the anesthetic. induce gas anesthesia using small face masks purchased from laboratory supply houses or make them from syringe cases and latex gloves ( fig. - ) . induction in an anesthetic chamber is also possible. all rodents induced and maintained on gas anesthesia require some form of non-rebreathing system. usual induction is achieved between % and % for isoflurane and % and % for halothane. maintenance for isoflurane and halothane varies from . % to %. there is marked individual variation in the amount of anesthetic required for induction and maintenance. use of % nitrous oxide in oxygen reduces anesthetic concentration requirements for other gases. m key point some chinchillas and guinea pigs hold their breath while being induced with gas anesthetics and then take deep rapid breaths. if the concentration of anesthetic gases is high enough, this behavior results in death. the risk of this behavior is reduced by premedication with tranquilizers, initial induction with nitrous oxide with later addition of primary anesthetic gas after relaxation, and low induction settings. changes in respirations, especially erratic or apneustic patterns and decreased respiratory rates, indicate deepening anesthesia. most pet rodents are not intubated for anesthesia because of their small size. when necessary, as in prolonged oral and other procedures, endotracheal intubation is accomplished with the animal in dorsal or ventral recumbency, depending on the clinician's preference. small non-cuffed or cole endotracheal tubes work well. a stylet usually is required to provide enough stiffness for the tube to pass the larynx. extend the animal's head and neck. grasp the tongue with forceps and use gentle traction. the tip of the tube then is advanced above the tongue and just past its base. the hard palate is used to deflect the tip of the tube ventrally into the glottis. this is a blind procedure that is difficult to master. use of a laryngoscope is helpful in larger rodents. another technique is to place an over-the-needle catheter in the trachea and move it up retrograde through the larynx to act as a guide. the catheter is removed after the endotracheal tube is in place. it is extremely important that the tube be checked for patency. rodents produce copious respiratory secretions, which frequently clog endotracheal tubes. the small diameter allows these tubes to collapse or kink, resulting in asphyxiation of the patient. check patency at least every minutes by applying positive pressure ventilation at to cm water and watching for excursion of the chest wall. if extending the head and neck does not result in air flow, suction the tube. if this is either not successful or impossible, remove the tube and continue anesthesia with a mask or reintubate the animal with a new tube. because of the small diameter of the trachea, endotracheal tube-induced tracheitis and subsequent swelling of the trachea may become a life-threatening situation. doses and routes for injectable anesthetics are listed in table - . needed doses for injectable anesthetics are tremendously variable among species and individuals. most injectable anesthetics provide safe sedation for minor procedures, but very few induce a safe surgical plane of anesthesia on a consistent basis. • ketamine in combination with diazepam is easily obtainable, is given intramuscularly, and has a wide margin of safety, but it does not provide good analgesia. • intraperitoneal injections of barbiturates provide surgical anesthesia but have a low margin of safety and a significant mortality rate. barbiturate anesthesia can result in fatal ileus. euthanasia is performed easily by induction of inhalant anesthetic through a mask or chamber followed by an overdose of barbiturates given intraperitoneally, iv, or intracranially. euthanasia by ip injection of barbiturates alone causes pain in some animals. • surgical techniques for pet rodents are similar to those used in cats and birds. • hemostasis is critical because of small blood volumes. • electrosurgery for incisions and cautery is highly recommended. • if necessary, give fresh blood transfusions drawn from a donor of the same species and mixed with sodium heparin ( iu/ml) at a rate of . ml/ ml of blood directly into an iv or io line. • the lack of a filter creates a potential for thrombosis. • transfusion reactions are possible. administer postoperative analgesics to all rodents undergoing surgical or dental procedures. common analgesics include buprenorphine, butorphanol, ketoprofen, carprofen and meloxicam. see table - for dosages. the most common surgeries are laceration repair and removal of dermal or sc masses. • most rodents will not gnaw on skin sutures. • if this occurs, use steel sutures, subcuticular sutures, or tissue glue. • if an animal still chews at its suture line, physical restraint, such as a tube or an elizabethan collar, is required. castration is a common procedure in guinea pigs. this usually is performed when owners want to house more than one male together or do not wish to breed their female any longer. common abdominal surgeries include cystotomy for urolith removal in guinea pigs and rats, and cesarean section (c-section) in guinea pigs and chinchillas because of dystocia. use a technique similar to those described for dogs and cats. preplaced stay sutures are recommended to define incision edges for closure. use - polyglactin or polydioxanone (pds) on a taper needle and suture in a simple continuous pattern to close the body wall. close the skin with a subcuticular suture (absorbable) or interrupted skin monofilament, nonabsorbable suture. fracture fixation is accomplished best with intramedullary pinning or kirschner apparatus. rodents gnaw on bandages until they remove them. if they are unable to remove a splint, self-mutilation often results in self-amputation. if a cast or splint is necessary, physical restraint often is required. healing usually takes to weeks. incisors can be trimmed with nail trimmers, but this technique often fractures the tooth, causing abscesses of the root. instead, use a high-speed dental burr or a flat cutting disk on a dremel hand tool. trim molars with a high-speed drill or pediatric rongeurs. a mouth speculum that deflects the tongue and other soft tissues is essential to prevent lacerations and provide working space ( fig. - ) . intubate the trachea to prevent aspiration pneumonia when working on molars. if a tooth is abscessed, extract both it and the occlusal tooth. • if necessary, approach cheek teeth via an incision through the cheek. • use a fine dental elevator to loosen the teeth. • patience and firm but gentle pressure are needed, or the root or surrounding bone may fracture. • the roots of the maxillary incisors curve dramatically back into the head. take care to follow the curve of the tooth. • packing an infected tooth socket with a calcium hydroxide paste may decrease the occurrence of persistent infection. remove the paste in to days. • administer meloxicam, carprofen or ketoprofen postoperatively to control pain. see table - for dosages. in chinchillas with dental malocclusion, the roots of the molars can become impacted, causing swelling of the mandible or exophthalmos and epiphora. these teeth are extremely difficult to extract without causing extensive bony and soft-tissue damage. discourage breeding of animals with malocclusion, unless it was acquired as a result of trauma or infection, because this trait is hereditary. most pet and laboratory mice are derived from mus musculus, which is the common house mouse. mice sold in the pet trade are randomly bred and less likely to suffer from the genetic problems associated with inbred laboratory rodents. mice possess brown fat tissues between their scapulae that also are known as hibernating glands; these are thought to provide an energy store. the spleen in male mice is % larger than that of females. ectoparasites usually are found in new acquisitions. • alopecia and pruritus, especially on the back of the head and dorsal midline, usually are associated with lice (polyplax serrata), mites, or fleas. • mite infestation (e.g., mycoptes musculinus, myobia musculini, radfordia affinis) often causes a greasy haircoat and folliculitis. transmission of lice and mites occurs via direct contact. fleas are transmitted by other household pets, such as cats and dogs. • diagnosis is based on clinical signs, history, visualization of parasite, skin scrape, and cellophane tape test. • treatment of fleas and lice is with pyrethrin powder. ivermectin is recommended for treatment of mites (see table - ). • change the bedding and thoroughly clean the cage between treatments to prevent reinfestation. occasionally, the surrounding environment needs to be treated with a premises spray used for killing fleas. • alopecia also may be the result of dermatophytes (see chapter ). lesions are often hyperkeratotic. • diagnosis is made by skin scrape or isolation on culture. most dermatophytes found in pet rodents do not fluoresce under a wood's lamp. • treatment is with lime-sulfur dip or griseofulvin (table - ). • ulcerative dermatitis is a common syndrome caused by staphylococcus aureus characterized by pododermatitis, mastitis, and abscesses in other areas. • administer antibiotics based on culture and sensitivity tests. chloramphenicol is recommended pending culture results (see table - ). the application of hot packs, local drainage, and topical medications are also beneficial in selected cases. • mastitis also may be caused by escheria coli or pasteurella, klebsiella, pseudomonas, or streptococcus species. mastitis usually is caused by poor sanitation, abrasive bedding, or overly aggressive young. • preputial gland abscesses are fairly common in males and are usually caused by e. coli or s. aureus. local flushing and topical treatment are usually adequate. • sc abscesses can be the result of the aforementioned bacteria or actinobacillus spp. or corynebacterium kutscheri. corynebacteria is associated with widespread abscesses, septic arthritis, gangrene, and ulcerated draining tracts. diagnosis is based on finding grampositive pleomorphic rods on gram stain and isolation on culture. • the bacteria are usually sensitive to ampicillin, chloramphenicol, and tetracycline (see table - ). • lymphoma and mammary neoplasia are common causes of sc masses. mammary neoplasia is usually malignant in mice, and metastasis to the lungs is common. (mice have five pairs of mammary glandsthree thoracic and two abdominal.) • obtain thoracic radiographs before surgery. • give a guarded prognosis for long-term survival. • other possibilities for sc masses are fungal granulomas, nodules from the psorergates simplex mite, hematoma, hernia, non-neoplastic lymphadenopathy, or emphysema. • otitis externa usually is caused by ear mites, although bacteria or fungi also may cause primary or secondary otitis. • clinical signs include erythema, pruritus, waxy debris, and excoriations behind the ears. • mites may be diagnosed by identification on otoscopic examination or microscopic examination of ear swabs (see "techniques"). • treatment requires cleaning debris from ears with a commercial ear cleanser followed by administration of three doses of ivermectin at -week intervals or topical acaricides used daily for to weeks (see table - ). • bacterial and fungal otitis is diagnosed by identification of organisms or gram-stained specimens and isolation on culture. • treatment is similar to that used in cats. • otitis media/interna usually are caused by hematogenous spread or local invasion of bacteria from a primary abscess. • clinical signs include head tilt, circling, facial nerve paralysis, and otitis externa. • rule out mouse hepatitis virus as the cause of the head tilt (see "gastroenterology"). • if treatment of the primary disease is successful, the otitis media usually resolves, although a residual head tilt may persist. • if a cluster of pseudomonas infections occurs in a population, evaluate the water source and produce for contamination. use sodium hypochlorite in the drinking water at ppm to control an outbreak while water quality is being restored. • damage to the pinnae can be associated with trauma, dermatitis, pox virus, hypersensitivity reactions, and vasculitis. dry gangrene is a common sequela and is usually self-limiting. i have observed a steroid-responsive pruritus in pet mice. the pruritus is severe enough to result in significant self-mutilation. this condition has been nonresponsive to treatment with ivermectin, lime-sulfur dips, griseofulvin, multiple antibiotics, oral prednisolone, and antihistamines. attempts at bacterial and fungal culture have failed to identify a pathogen. an inflammatory response is observed on histologic examination. mice with this condition respond to repository methylprednisolone injections every to weeks ( . mg/kg im). most owners have not elected to continue injections for longer than a few months. once the injections are stopped, the pruritus returns, often requiring euthanasia of the affected mouse. • bilateral alopecia found around the muzzle associated with no other abnormalities usually is caused by friction from overhead feeders. • alopecia occurring in smaller, weaker individuals is often the result of barbering. removal of the mice in best condition from the cage results in normal appearance of barbered mice in to weeks. • tailhead alopecia and scabbing are usually the result of aggression. separate affected animals, or additional trauma may occur. • other rare causes of alopecia are endocrinopathies, leprosy, and hereditary alopecia in nude mice. • epiphora is a common condition of pet mice. the most common causes in pets owned for longer than months are ammonia fumes and overgrown incisors. • ammonia causing contact irritation is diagnosed by examining an uncleaned cage and checking for odor. • treatment is improved sanitation. • overgrown incisors are diagnosed easily by oral examination. treat by trimming the affected teeth and providing opportunities for gnawing. • foreign bodies and the resultant corneal ulcers can cause epiphora. an eye examination, including fluorescein stain, is indicated. treat by removing the foreign body and administering an ophthalmic antibiotic (gentamicin, tetracycline, or chloramphenicol in affected eye, q h-q h). • in newly acquired pets, epiphora is often the first clinical sign of an upper respiratory infection. pasteurella pneumotropica is the most common pathogen, although salmonella spp., mycoplasma, sendai virus, lymphocytic choriomeningitis, and mouse pox also may cause epiphoria. the ocular discharge later appears mucopurulent (see "respiratory"). retinal degeneration can be either hereditary or (in albino mice) may be caused by exposure to highintensity lighting. the resulting blindness often goes undetected because patients adapt well and behave normally in their cages. • clinical signs include dyspnea (often described as chattering), mucopurulent oculonasal discharge, hunched posture, and anorexia. animals with a chronic history of this disease are often cachexic. • radiology aids in determination of the extent and severity of the pneumonia and the absence or presence of distant foci of infection. • treatment with tylosin is successful in controlling the disease if it is not too advanced. tetracycline, enrofloxacin, and chloramphenicol also have been used (see table - ). • many patients need nutritional support. • mice with pyometra or other abscesses require surgical debridement. • recovered animals are carriers and stress elicits clinical signs. strictly quarantine these animals. a common cause of pneumonia in newly acquired mice is sendai virus. acute fatalities are seen in suckling or weanling mice. • transmission is by aerosol or direct contact. • clinical signs in adults are caused by secondary bacterial infections and are similar to those in mrm. • diagnosis is based on clinical signs and serologic testing. • treat with antibiotics to control the secondary bacterial infection and provide supportive care as needed. • prohibit breeding for to weeks. • a killed vaccine is available. • recovered animals are resistant to new infection. common primary or secondary pathogens causing respiratory signs in mice are streptococcus pneumoniae, corynebacterium kutscheri, pasteurella pneumontropica, pseudomonas aeruginosa, and klebsiella pneumoniae. treatment is empiric or based on culture and sensitivity of a tracheal swab sample. dyspnea often is caused by metastasis to the lungs from mammary adenocarcinomas. primary lung tumors, especially pulmonary adenomas, also occur frequently. although not frequently diagnosed, cardiac disease can result in signs of respiratory disease. diagnosis is based on radiographic evidence of cardiomegaly and pulmonary edema. • tapeworms usually do not cause clinical signs. occasionally, heavy burdens may cause diarrhea or weight loss. the chief concern is the zoonotic potential of one species, hymenolepis nana, which is directly transmissible to humans. • diagnosis is made from visualization of eggs in the feces. treat with praziquantel or niclosamide (see table - ). improve sanitation, and remove indirect hosts (e.g., fleas, beetles, roaches). • pinworms (syphacia obvelata, aspiculuris tetraptera) may cause anal pruritus and, in severe cases, rectal prolapse. • diagnosis is based on clinical signs and observation of eggs on cellophane tape after application to the perineal region. treat with piperazine or mebendazole every to days for three treatments or with fenbendazole once daily for days (see table - ) and provide improved sanitation. • the protozoal parasite spironucleus muris causes diarrhea and slow growth associated with a pot-bellied appearance in young mice. • diagnosis is by fecal wet mount, although falsenegative findings are common. • treat with oxytetracycline (see table - ). supportive care to combat dehydration and hypothermia is extremely important. • control is achieved with improved sanitation. • giardia spp. and, rarely, eimeria falciformis show signs similar to spironucleus. giardiasis is zoonotic. treat with metronidazole. treat eimeria with sulfadiazine/trimethoprim (see table - ). most other protozoa are considered nonpathogenic. • cysticercus fasciolarus causes nonpathologic cysts of the liver. these cysts are the infective form of taenia taeniaformis in carnivores. viral diseases • diagnosis is based on clinical presentation, serology, and presence of syncytial giant cells in the epithelium of the small intestine. • treat supportively, and quarantine affected individuals. the prognosis is grave. • although less commonly seen in pet mice, reovirus occurs in older suckling mice. it is characterized by an oily diarrhea, which results in a greasy haircoat. other signs are conjunctivitis, stunted growth, and tremors. transmission is by ingestion. • diagnosis is based on clinical signs, histology, and serology. • treat supportively. the long-term prognosis is grave. initial survivors are weak and jaundiced, suffer from alopecia, and eventually die. • transmissible murine colonic hyperplasia (mch) caused by citrobacter freundii is characterized by diarrhea followed by rectal prolapse and stunted growth. transmission is feco-oral. • diagnosis is made by clinical signs and fecal culture. • treat with neomycin, tetracycline, or sulfamethazine until sensitivity results are available (see table - ). severe thickening of the distal half of the colon is observed at necropsy. • salmonellosis, also known as mouse typhoid, is transmitted by latent carriers or contaminated feed or bedding. incubation lasts for to days. • clinical signs are lethargy, anorexia, purulent conjunctivitis, arthritis, and diarrhea. • diagnosis is based on clinical signs and fecal culture. treat supportively. use of antibiotics is controversial. • quarantine survivors. • sanitation is extremely important because salmonella spp. are zoonotic. on gross postmortem examination, erythema of distal ileum and congestion of the spleen and liver are seen. with more chronic infections, necrotic foci are seen in the liver, spleen, and lymph nodes. prevent infection by feeding a fresh laboratory chow from a reputable source. thoroughly wash all produce and then dip it in a diluted bleach solution. rinse completely before feeding. • tyzzer disease is caused by bacillus piliformis. transmission is feco-oral. • clinical signs are precipitated by stress and consist of anorexia, diarrhea, and high mortality in weanlings. • diagnosis is made by clinical signs or isolation on culture. enteritis and multiple gray-yellow necrotic foci in the liver are seen on gross postmortem examination. • administer tetracycline for to days (see table - ) and reduce stress to control the disease. breeding systems vary; from one to six females may be placed with one male. all animals are housed together, and the young are removed after weaning. females in proestrus have swollen vulvas. vaginal plugs are present post-copulation. female mice that have been bred within days abort if a new male is placed in the cage. inappropriate light cycle, inappropriate age, crowding, and poor nutrition are the most common causes of infertility in pet mice. pyometra due to pasteurella pneumontropica, mycoplasma spp., or other bacteria is also common. desertion of litters is usually a result of stress, lack of nesting materials, agalactia, or mastitis. • urethral obstruction from proteinaceous plugs of inspissated ejaculum may develop in aged male mice. pseudomonas, e. coli, or proteus are the most frequently cultured pathogens. before complete obstruction, chronic hematuria may be noticed by the owner. • antibiotics, which are chosen based on the results of urine culture, are often curative with early presentation. complete obstruction requires surgical removal. • glomerulonephritis is very common in geriatric mice. it frequently is secondary to chronic viral infection. clinical signs are anorexia, lethargy, dehydration, and cachexia. urinalysis demonstrates proteinuria. as the disease progresses, the urine becomes isosthenuric, the blood urea nitrogen (bun) and creatinine levels rise, and other electrolyte abnormalities typical of chronic renal failure occur. treat supportively. prognosis for long-term survival is grave. • coccidia (e.g., klosseilla muris) occasionally is found in the urine. the clinical significance of its presence is unknown. • mice can be asymptomatic carriers of leptospirosis; however, this is rarely seen in pet mice. • diagnosis is based on darkfield microscopy of urine, serology, or histopathology. euthanasia of carriers is recommended. • infectious polyarthritis or mouse rheumatism is caused by streptobacillus moniliformis. in humans, it is known as rat bite or havernill fever. transmission is by direct contact. clinical signs are cachexia, keratitis, edema and ulceration of the appendages, and ankylosing arthritis. • diagnosis is based on the bacterial culture findings or the presence of caseous pericarditis and arthritis on necropsy. • treat with antibiotics chosen through the results of culture and sensitivity tests. use penicillin while awaiting results. supportive care is important. animals that recover remain arthritic. control is achieved through quarantine and sanitation. • the most frequently diagnosed neurologic disease in pet mice is head tilt resulting from bacterial otitis media (see "otitis"). the second most common cause of neurologic signs is trauma. • diagnosis is based on history and clinical signs. consider neoplasia in aged mice with slowly progressive signs. • lymphocytic choriomeningitis is a zoonotic arenavirus. transmission is airborne, transplacental, or by direct contact, fomites, or insect vectors. acute signs usually occur in mice that are to weeks old. approximately % of infected individuals show acute clinical signs, which include lethargy and photophobia followed by convulsions and paralysis. in animals that are latently infected, glomerulonephritis develops later. mice infected after weaning and before year in age lose weight, appear arthritic, and show signs of conjunctivitis and photophobia. the virus runs its course in several weeks. animals that recover show no residual signs. • diagnosis is based on clinical signs and the presence of immunofluorescent antibody (ifa). pleural effusion, splenomegaly, and hepatic lipidosis are found on necropsy. treat supportively. house survivors separately. • prevent the disease by improving sanitation, providing pest control, and cleaning produce. consider euthanasia because of the zoonotic potential of the virus. • mouse poliomyelitis/encephalomyelitis, also known as theiler disease, causes clinical signs in in , infected mice. two-thirds of healthy mice are carriers. transmission is by oral or respiratory routes. mice younger than weeks of age show signs of encephalitis. animals that are to weeks old are weak in the rear legs and progress to paralysis. the tail may remain mobile. affected mice continue to eat and be alert. albino mice are predisposed to show clinical signs. • diagnosis is based on clinical signs, serology, or histopathology that shows necrosis of the ganglionic cells of the anterior horn of the spinal cord. • treat supportively. consider euthanasia because of poor prognosis. • seizures in mice commonly result from otitis media, trauma, liver or kidney failure, toxin, bacterial meningitis, neoplasia, or viral encephalitis. • leukemia in mice is usually viral in origin. transmission is trans-mammary or trans-placental. • clinical signs are anemia, dyspnea (with thymic involvement), and those signs that are compatible with chronic disease. • diagnosis is based on complete blood count (cbc), bone marrow aspirate, or histopathology. prognosis is grave. • eperythrozoon coccoides is a rickettsial red blood cell (rbc) parasite of mice. affected mice are usually asymptomatic. occasionally, fever, anemia, and splenomegaly develop in infected animals. transmission is through the louse polyplax serrata. control is by extermination of the louse. • treat with tetracyclines. pet rats are derived from the norwegian or brown rat (rattus norvegicus), which did not originate from norway, but from asia. breeds of rats are called strains when they are inbred extensively and stocks when strains are hybridized. rats have brown fat, as discussed in the section on mice. they do not possess a gallbladder. their mandibular symphysis is articulated normally. rats are neophobic; therefore, make gradual changes in food or environment when possible. • fleas, mites (e.g., radfordia ensifera, ornithonyssus bacoti), lice (i.e., polyplax spinulosa), ear mites (i.e., notoedres muris), and dermatophytes cause similar signs in both mice and rats. treatment also is similar (see "mouse"). • sc masses in rats are similar to mice. pasteurella pneumotropica is a very common pathogen in mastitis and sc abscesses. • treat with chloramphenicol until culture results are available (see table - ). • mammary cancer develops in % to % of adult female rats and in approximately % of male rats. always submit biopsy specimens for histologic examination. most, but not all, of these tumors are fibroadenomas, which are benign. prognosis for longterm survival after surgical removal is good. other common neoplasms include interstitial cell tumors of the testes, which cause sc swellings in the inguinal region, and squamous cell carcinomas of the zymbals gland of the external ear canal. • ulcerative dermatitis occurs in rats as well as mice. staphylococcus aureus is the causative agent. c. kutscheri follows a similar course in rats and mice (see "mouse"). • ringtail is the formation of constrictive bands of fibrous tissue around the tail in nestling rats. these bands result in gangrene of the distal tail. this disease occurs when environmental humidity is less than %. • treat by making a longitudinal incision of the ring to release the stricture and apply topical dimethyl sulfoxide (dmso), steroid, and antibiotic solution ( ml dmso, ml mg/ml amikacin, ml mg/ml dexamethasone) four times daily. • to prevent ringtail, keep humidity above %, use solid-bottom cages and provide ample nesting material. prognosis for life is excellent. prognosis for retention of the distal tail is guarded. • epiphora and blepharospasm are caused mostly by ammonia fumes, overgrown incisors, or foreign bodies (see "mouse"). • sialodacryoadenitis virus is a coronavirus that is endemic in many rat populations. • clinical signs vary from mild keratoconjunctivitis to blepharospasm, chromodacryorrhea, severe uveitis, hyphema, buphthalmos, periorbital swelling, and pneumonia. the clinical course of the disease lasts to days. rats maintain normal activity levels and appetite. • treatment is not necessary for mild infections. place rats showing marked ocular disease or discomfort on the appropriate ophthalmic ointments (e.g., atropine, antibiotic, steroid) based on presentation. administer parenteral antibiotics to animals that show signs of respiratory problems. recovery is usually complete unless the eye ruptures or selfmutilation occurs. • control is achieved by not introducing new animals for weeks. • in contrast to mice, sendai virus rarely causes clinical signs in rats. • mucopurulent ocular discharge also may be caused by infection with mycoplasmosis, streptococcus pneumoniae, pseudomonas spp., and other less common bacterial or viral agents that cause pneumonia. • cataracts are primary hereditary defects or occur secondary to severe uveitis or diabetes mellitus. retinal dystrophy and colobomas are also inheritable traits in rats. retinal degeneration occurs in rats housed under intense lighting. • mrm is extremely common in pet rats. its presentation is similar to the disease in mice (see "mouse"). • streptococcus pneumoniae is normal flora for rats. however, during stressful situations, bacteremia may occur, resulting in pneumonia. clinical signs are similar to mrm. differentiation is based on culture and the presence of extensive fibrinopurulent pleural effusion on necropsy. • ampicillin controls clinical signs if treated early in the course of disease (see table - ). prevent the condition by minimizing stress. • corynebacterium kutscheri and pasteurella pneumotropica cause signs similar to mrm (see "mouse"). there is a serologic test for c. kutscheri. see the mouse section for a discussion of pseudomonas aeruginosa. • pneumocystosis carinii is an uncommon protozoa that infects the lung. cysts and trophozoites live in the alveoli. clinical signs occur only in immunocompromised or geriatric individuals. signs are cachexia, cyanosis, and dyspnea. • diagnosis is based on clinical signs, tracheal wash, response to therapy, or histologic examination. • treat with sulfadiazine/pyrinrethamine (see table - ). • myocardial degeneration and subsequent congestive heart failure are fairly common in geriatric rats. diagnosis is based on radiographs of the thorax and clinical signs. treat supportively, and use furosemide and digitalis at cat dosages to alleviate pulmonary edema. • polyarteritis nodosa is an idiopathic condition of geriatric rats that results in thickening and tortuosity of arteries, especially in the mesentery, pancreas, and testicles. affected areas are predisposed to clot formation and aneurysms. • nematode (syphacia muris), cestode, and intestinal protozoal parasite infestations are similar to those in mice. • capillaria hepatica has no clinical significance. yellow streaks on the liver are an incidental finding at necropsy. the causes and treatment of malocclusion are similar to those for mice. • epizootic diarrhea of suckling rats is a viral disorder found in rats to days old. the infection causes a mild diarrhea. most animals recover. occasionally, stunting occurs. treat supportively. • salmonellosis in rats is similar to that in mice. • if breeding is desired, take females showing signs of estrus (e.g., lordosis, hyperactivity, quivering ears, and swollen vulva) to a male rat's cage for hours, or keep one male in a cage with up to six females. check females for a post-copulatory plug to confirm breeding. remove females just before parturition, and house females individually while raising the young. a vaginal discharge is seen . to hours before labor. parturition is accompanied by stretching and extension of the rear legs. all neonates usually are delivered within to hours. • two extremely common conditions in geriatric rats are nephrocalcinosis and chronic progressive nephropathy. clinical signs are compatible with those of chronic renal failure. enlarged or small irregular kidneys may be found on physical or radiographic examination. isosthenuria and marked proteinuria are found in urinalysis. • definitive diagnosis is based on renal biopsy. • treat supportively. prognosis for long-term survival is grave. • trichasomoides crassicauda is an uncommon parasite of the urogenital tract. the adult worms usually reside in the kidney, but they occasionally may wander into the genital tract. the ova are passed in the urine. • clinical signs are hematuria and stranguria. proliferative mucosa of the bladder occasionally may be palpated as an abdominal mass. • treatment is somewhat successful with methyridene (see table - ). sanitation is critical in control of this disease. • klossiella muris is an incidental coccidia of the urinary tract. • many geriatric pet rats have chronic progressive radiculoneuropathy. • clinical signs are compatible with cauda equina syndrome, including posterior paresis progressing to paralysis, urine retention, and incontinence. prognosis is grave. • treat supportively or euthanize. • streptobacillus moniliformis, a normal bacteria found in the oral, nasal, and pharyngeal cavities of rodents, is isolated from % of middle ear infections and % of chronic pneumonias in rats. the bacteria is nonpathogenic for gerbils and guinea pigs. • clinical signs vary with the site of infection. head tilt and circling, septic arthritis, and respiratory disease commonly are seen. • diagnosis is based on isolation on culture. the clinical signs mimic many other diseases, especially mrm and pseudomonas infection (see "mouse"). • head tilt in rats also may be the result of trauma or neoplasia, especially pituitary adenomas. • hemobartonella muris is an rbc parasite of rats that is nonpathogenic unless the rat is immunocompromised or splenectomized. transmission is through the louse polyplax spinulosa. • clinical signs result from hemolytic anemia and hemoglobinuria. • treat with tetracyclines (see table - ). mesocricetus auratus, better known as the golden or syrian hamster, is a primarily nocturnal rodent that originated in the middle east. almost all hamsters in the united states are the offspring of three siblings imported in the s. many color variations are available. long-haired hamsters are called "teddy bear" hamsters. the stomach has two compartments, a non-glandular forestomach, which functions like a rumen, and a glandular stomach. hamsters are very territorial. they possess flank glands, which are larger in males, that are rubbed against objects to mark their territory. females are larger than males. except during estrus, they use this size advantage to attack males. do not allow groups to estivate together or recently awakened animals may cannibalize sleeping hamsters. m key point hamsters are extremely sensitive to antibiotics. penicillins, clindamycin, lincomycin, streptomycin, tylosin, erythromycin, and cephalosporins eliminate the normal intestinal flora, allowing overgrowth of pathogenic bacteria, particularly clostridium difficile. diarrhea, which is almost always fatal within to days, subsequently occurs. even antibiotics considered to be safe can have this effect. treat by discontinuing antibiotics, providing a lactobacillus supplement, and giving supportive therapy. • hamsters are susceptible to demodex criceti and d. aurati mites. d. criceti is limited to skin folds. d. aurati causes hyperpigmentation, alopecia, and seborrhea sicca affecting the dorsal midline. demodex is carried by many normal-appearing hamsters. • clinical signs occur in immunosuppressed animals, as would occur with stress, chronic infection, pregnancy, or malnutrition. • diagnosis is based on clinical signs and deep-skin scrapings. • treat with amitraz every weeks for two treatments past two consecutive negative skin scrapings. use the manufacturer's recommended dilution for dogs. • sarcoptes mites infrequently cause facial alopecia. diagnosis is based on skin scraping. treat with ivermectin (see table - ). do not confuse this condition with alopecia caused by contact with feeders or barbering. • notoedres mites affect only the external ear canal in female hamsters but may affect the ears, feet, geni-talia, and tail in males. diagnosis is made by observation of mites on samples from ear swabs, skin scrapings, or both. treat with ivermectin (see table - ). • other less common causes of alopecia in hamsters are dermatophytosis, endocrinopathies, and genetic defects. • dermal sc masses are usually abscesses caused by pasteurella pneumotropica, s. aureus, or streptococcus spp. treatment is based on results of culture and susceptibility testing. use chloramphenicol until culture results are available. other frequent causes of sc swellings are distended cheek pouches and testicles, mastitis, hernias, neoplasia, and lymphadenopathy. • epiphora and conjunctivitis are caused most frequently by increased environmental ammonia concentrations, incisor overgrowth, foreign body, or lymphocytic choriomeningitis (see "rat"; "mouse"). • mucopurulent discharge is caused by secondary infection by pasteurella or streptococcus spp. • hamsters are predisposed to rupture of the eye following trauma or infection. surgical enucleation is advised. electrosurgery is extremely helpful in controlling bleeding but do not apply heat to the stump of the optic nerve or vessels, or thermal injury to the brain may result. place gelfoam in the socket to enhance clot formation. • hamsters are susceptible to viral respiratory infections of humans. • clinical signs include nasal discharge, sneezing, otitis media, fever, and pneumonia. uncomplicated cases last to days. complications are usually the result of secondary bacterial infections. • treat supportively. use of antibiotics is indicated if copious nasal discharge, dyspnea, anorexia, or marked lethargy is observed. overuse of antibiotics may cause diarrhea-related death in hamsters that might have recovered uneventfully if left untreated. • most dyspnea in hamsters is caused by blunt thoracic trauma. hamsters often bite when startled. reflex actions on the part of humans, especially children, cause hamsters to be flung against hard objects. • diagnosis is by history and presence of fresh epistaxis. • treat supportively. emergency shock therapy, consisting of supplemental heat, oxygen administration, parenteral fluids, and glucocorticoids, frequently is required. • sendai virus can cause death in suckling hamsters housed with mice. adults show no clinical signs (see "mouse"). • primary bacterial pneumonia most frequently is caused by yersinia pseudotuberculosis, pasteurella pneumotropica, or streptococcus. clinical signs are compati-ble with those of pneumonia seen in other species, as well as weight loss and conjunctivitis. all three agents have a tendency to form distant abscesses, especially in the uterus. • diagnosis is based on clinical signs and isolation on culture. • treat with chloramphenicol until antibiotic susceptibility results are available. abscesses require surgical debridement; however, anesthesia in affected animals is very risky. recovered hamsters are carriers and must be quarantined from other rodents. prognosis is guarded. • cardiac thrombosis is seen in % of geriatric hamsters. most thromboses occur in the left atrium and are secondary to degenerative cardiomyopathy, cardiac amyloidosis, sepsis, or calcification of the great vessels. congenital myocardial necrosis also occurs. • clinical signs include cyanosis, dyspnea, and acute death. enlargement of the cardiac silhouette and pulmonary edema sometimes can be seen on thoracic radiographs. • furosemide and digitalis (using standard cat doses) may temporarily alleviate clinical signs. • hamsters can carry the zoonotic tapeworm h. nana (see "mouse"). • treat with niclosamide or praziquantel (see table - ) and provide improved sanitation. • pinworms (aspicularis tetraptera, syphacia muris, s. obvelata) occur in hamsters as well as in mice. • treat with fenbendazole (see table - ). • hamsters are predisposed to dental caries. a large percentage of affected teeth become abscessed, causing facial swelling, ptyalism, and anorexia. • diagnosis is based on clinical signs, oral examination, skull radiographs, and isolation on culture. • extract the tooth and administer antibiotic therapy based on results of susceptibility testing. prognosis is variable depending on the condition of the animal, tooth affected, and extent of the abscess (see "mouse"). • overgrown incisors also occur, as in mice. • the cheek pouches are very distensible. impaction of the pouches occurs on occasion. • clinical signs vary from ptyalism to swelling from abscess. in simple cases, removal of the material from the pouch with fine forceps is sufficient. sedation usually is not required. • if a fungal or bacterial infection of the pouch is present, remove the exudate, submit samples for gram staining and bacterial or fungal culture, and flush the pouch with diluted iodine solution. if cellulitis is present, administer systemic antibiotics as well. fistulas often heal spontaneously. • proliferative ileitis (i.e., wet-tail disease) is thought to be caused by a campylobacter-like organism with or without concurrent bacterial or viral infections. more than % of animals with clinical signs die. the highest morbidity and mortality rates occur in hamsters to weeks of age. teddy bear hamsters may be more susceptible to infection than shorter-haired varieties. transmission is feco-oral. • clinical signs include diarrhea, which mats on the ventrum and perineum, and results in anorexia, dehydration, and a hunched posture. the abdomen frequently seems painful on palpation. bowel loops often are distended on palpation because of ileal obstruction or intussusception. rectal prolapse usually occurs. • administer neomycin, gentamicin, metronidazole, or tetracycline (see table - ). supportive care is critical. prognosis is grave, even with treatment. gross postmortem findings include gas and yellow diarrhea in the distal intestinal tract, mucosal thickening in the ileum and distal jejunum, peritonitis, and liver abscesses. • other common causes of bacterial diarrhea include e. coli, tyzzer disease, or salmonella spp. (see "mouse"). in hamsters older than year of age, liver cysts that are derived from the biliary duct often develop. less frequently, similar cysts arise from the pancreas, epididymis, and seminal vesicles. this syndrome is called polycystic disease. no clinical signs are associated with cysts in these structures, which are an incidental finding on abdominal palpation. no treatment is recommended. • timing is critical to prevent injury to the male when breeding hamsters. transfer the female to the male's cage in the early evening days after a creamy, viscous vaginal discharge is noticed. monitor the pair carefully. remove the male immediately if the female is aggressive. remove the male after mating or after to hours even if mating has not occurred. two days after successful copulation, a gray malodorous vaginal discharge is observed. pregnancy is highly likely if there is no translucent vaginal discharge to days post-breeding. pseudopregnancies last to days. normal gestation is to days. before par-turition, a hemorrhagic vaginal discharge appears, and the female may pant. hamsters rarely suffer from pregnancy toxemia (see "guinea pig"). • infertility may be caused by pyometra (see p. pneumotropica and lymphocytic choriomeningitis). • cannibalism is most frequently a result of stress or mastitis. • in almost % of geriatric hamsters, renal amyloidosis develops. the disease tends to develop more rapidly in females. • clinical signs include edema and ascites due to protein loss in the urine, as well as the typical signs of chronic renal failure. • treat supportively. prognosis for long-term survival is grave. • head tilt is usually secondary to otitis media. also consider lymphocytic choriomeningitis or neoplasia as differential diagnosis (see "rat"). • in hamsters fed all-seed diets and deprived of exercise, cage paralysis syndrome often develops. usually pets are presented for acute posterior paresis which, in reality, was slowly progressive. the distinction is important in ruling out trauma. in mild cases, the hamster is able to move its hind limbs but unable to support weight. vitamin d and e supplementation, along with nutritional improvement and providing exercise, is curative in to weeks. in severe cases, recovery is negligible or incomplete. lymphoma and lymphosarcoma may be viral in origin. diagnosis is made by biopsy or fine-needle aspiration of affected lymph nodes. rule out lymphadenopathy caused by lymphadenitis from infection with streptobacillus moniliformis (see "rat"). although many hamsters initially respond well to chemotherapy protocols established for cats and dogs, prognosis for long-term survival is grave. • rarely, demodex spp. mites cause alopecia in gerbils. • diagnosis is based on skin scraping. • treat with rotenone ointment or amitraz dips every weeks for three to six treatments. use manufacturer's recommended dilution for dogs. • acute moist dermatitis usually is caused by s. aureus infection. infection on the face often begins with the harderian glands. the gland secretion is viscous and causes matting, with secondary staphylococcal infection occurring under the mats. attempts at grooming spread the infection to the feet and abdomen. • diagnosis is based on clinical signs and isolation on culture. • administer enrofloxacin, tetracycline, or chloramphenicol and apply warm, moist compresses to remove dried debris. remove possible irritants from cage (e.g., pine or cedar shavings, ammonia). occasionally, surgical removal of a chronically infected or inflamed gland is needed. • alopecia of the facial area, especially when it is symmetric, is usually the result of self-trauma from feeders, cage bars, or overzealous burrowing. • treat by changing cage construction or providing better visual security. • gerbils that catch their tails in crevices or are restrained inappropriately by their tails often are presented for avulsion of the skin from their tails. • treat initially by controlling hemorrhage and hypovolemic shock. • amputate the tail after patient stabilization to prevent ascending infection. in some animals, the infection is localized to the distal tail, which is sloughed in approximately to weeks. • generalized alopecia is normal in some weanling gerbils. the hair grows in as the animals mature. • melanomas are found most frequently on the ears, feet, or base of the tail. • diagnosis is based on biopsy. • treat by surgical removal. • sebaceous gland disease is usually the result of bacterial infections or neoplasia. • diagnosis is based on cytologic examination, culture, histologic examination, and response to antibiotic therapy. • treat bacterial infections with parenteral or topical antibiotics based on the severity of signs. • sebaceous gland adenomas, basal cell tumors, and squamous cell carcinomas are the most frequently encountered neoplasms. • take a radiograph of the thorax to diagnose metastases. prognosis for long-term survival is based on tumor type, stage, and character. • treat by surgical excision. chromodacryorrhea and epiphora occur as in mice. tapeworms (i.e., h. nana and h. diminuta) and pinworms (i.e., syphacia obvelata, dentostomella translucida, and aspicularis tetraptera) occur as in mice. incisor overgrowth occurs as in mice. • salmonella spp. cause transient diarrheas in gerbils. the source of infection is usually unwashed greens, contaminated feed, or carrier rodents of another species. most recover. animals that die have a fibrinosuppurative peritonitis. • treat supportively. use antibiotics in severe cases based on results of culture and susceptibility testing. • tyzzer disease, caused by bacillus piliformis, is seen most often in weanlings at to weeks of age and in post-partum females. • clinical signs include anorexia, lethargy, rough haircoat, and sometimes diarrhea. gross postmortem findings include yellow-gray nodules in the liver and hemorrhage at the ileocecal junction. • diagnosis is based on postmortem examination or response to therapy. • treat with oxytetracycline (see table - ) and supportive care. hepatic lipidosis and gallstones are frequent sequela to lipemia in gerbils fed diets with excessive fat. • breeding is most successful if animals are paired at weaning and kept in these pairs. male gerbils aid in raising the young. pairing older animals causes fighting. an average of % of neonates fail to survive to weaning. this is usually the result of agalactia and crushing. • chronic hemorrhagic discharge from the vulva is usually the result of cystic ovaries or ovarian tumors. most tumors occur in animals older than years of age and consist of granulosa cell tumors or theca cell tumors. leiomyomas of the uterus also cause similar clinical signs. • rule out urinary tract disease by performing a urinalysis via cystocentesis. large masses may be visualized on abdominal ultrasound. definitive diagnosis is based on vaginal cytology followed by exploratory laparotomy. • ovariohysterectomy is curative for cystic ovaries and tumors if they have not metastasized. • chronic renal failure develops in most gerbils older than . years of age. • clinical signs are polyuria, polydipsia, weight loss, and anorexia. urinalysis demonstrates proteinuria, hematuria, casts, and an increase in white and red blood cells. • treat supportively. prognosis for long-term survival is grave. • up to % of gerbils in certain family lines suffer spontaneous epileptiform seizures. the seizures are induced by stress and are self-limiting. seizures usually start as the gerbil reaches months of age. • treatment is unnecessary. chinchilla laniger and c. brevicaudata are nocturnal rodents from the andes mountains in south america. most animals kept in the united states are the descendants of animals. aside from pets, chinchillas are raised commercially for their pelts. the most common coat color is gray; the most valuable coat color is black. m key point chinchillas are sensitive to antibiotics (see "hamster"); therefore, avoid use of penicillins, lincomycin, erythromycin, and cephalosporins. house chinchillas in a cool environment because they are prone to overheating. if heat stroke occurs, treat with tepid water baths and supportive therapy. • chinchillas require dust baths to keep their skin in condition. use commercially available chinchilla dust only. sand substitutions do not condition the coat and occasionally cause conjunctivitis. offer dust at least once a week. • dermatophytosis occurs as in guinea pigs. • fur chewing is a serious problem in chinchillas that are farmed for pelts and often is seen in pet chinchillas that are recent culls from a ranch. the etiology of fur chewing is unknown. some cases seem to be related to chronic disease, malnutrition, poor caging, or stress. theories for undiagnosed cases include genetic abnormality; undiagnosed dermatophytosis; or adrenal, pituitary, or thyroid gland abnormalities. • diagnostics such as skin scraping, fungal culture, fecal, cbc, history profile, and biopsy are recommended. in general, if changes in diet and husbandry do not elicit a response or an underlying treatable disease condition is not discovered, prognosis for cure is grave. • one source advocates plucking all remaining underfur in chewed areas in an attempt to stimulate new hair growth. place collars after this procedure until the fur has grown in completely. • cystic sc masses may be caused by the intermediate stage of multiceps serialis. transmission is by ingestion of feed contaminated with canine feces. • diagnosis is made by histopathologic or cytologic examination of tissue samples. treat by surgical removal of the masses. • otitis caused by pseudomonas spp. occurs as in rats. • conjunctivitis occurs as in mice. • cataracts are congenital or developmental. • asteroid hyalosis occurs as a degenerative change. pneumonia occurs as in guinea pigs. parasites tapeworms (i.e., h. nana) occur as in mice. malocclusion of incisors and cheek teeth occurs as in guinea pigs. • diarrhea is caused most often by coccidia or giardia spp. or a bacterium. • clinical signs range from soft stools and weight loss to fluid diarrhea, dehydration, bloating, septicemia, and sudden death. • the protozoal parasites are best diagnosed on fresh saline smear or necropsy. • bacterial diarrhea is most often caused by contaminated feed and is diagnosed by isolation on culture. clostridium spp., pseudomonas aeruginosa, e. coli, salmonella enteritidis, and pasteurella spp. are the most common isolates. • treat supportively and use appropriate antiprotozoal or antibiotic drugs. • pasteurella pseudotuberculosis causes acute death from septicemia or a chronic weight loss with intermittent diarrhea. enlarged mesenteric lymph nodes are a hallmark of this disease. • diagnosis is based on clinical signs, histopathologic examination of tissue samples, and isolation on culture. • treat with sulfa drugs until sensitivity results are available. prognosis for recovery is poor. gross postmortem examination reveals yellow to white necrotic foci in the liver. • check male chinchillas four times per year for penile hair rings. roll back the prepuce and expose the penis. roll hair rings off the penis after application of a water-soluble lubricant. treat ulcerations topically or systemically as needed. • dystocia is fairly common in chinchillas (see "guinea pig"). • metritis is suspected when post-partum vaginal discharge, failure to return to a normal estrus cycle, anorexia, weight loss, polydipsia, polyuria, and chewing at flank and abdomen are present. • diagnosis is based on history, physical examination, abdominal radiographs, culture, ultrasound, and cbc. it usually is caused by bacteria introduced by the male or spread from an internal abscess. retained placentas, macerated fetuses, and dystocia are predisposing factors toward metritis. • treat with ovariohysterectomy after stabilization. females used only for breeding purposes may be treated with antibiotics alone, but the prognosis is poor. • female chinchillas are aggressive toward male chinchillas when not in estrus. breeding operations usually have separate cages for females and an interconnecting run for the male. females are kept out of the male's run by their larger size or collars. the young are precocious and do not need a nest. chinchillas only produce two litters per year. • clinical signs include hot, swollen mammary glands. suspect mastitis if previously healthy neonates become restless, then lethargic. • perform bacterial cultures on milk samples, and treat with antibiotics based on susceptibility testing. administer sulfa drugs until susceptibility results are available. local hot packing is also beneficial. occasionally, surgical drainage is required. foster neonates to another female if possible, or use puppy or kitten milk replacers to hand-raise babies. • chinchillas seem to be particularly sensitive to listeria monocytogenes. clinical signs can mimic p. pseudotuberculosis and include anorexia, lethargy, abortion, generalized central nervous system (cns) signs, hepatitis, mild enteritis, and mild emphysematous pneumonia. necropsy shows yellow foci in the liver. • diagnosis is based on isolation on culture. • treat with sulfa drugs (see table - ) until sensitivity results are available. the prognosis is poor. • other less common causes of neurologic disease in chinchillas include lymphocytic choriomeningitis, streptococcus spp., balisascaris procyonis (i.e., aberrant migration of raccoon roundworm), lead poisoning, and thiamine deficiency. guinea pigs (caviae porcellus) are nocturnal rodents that originated in the andes mountains. they are known for their dietary need for vitamin c. they are used as a food source in their native lands. there are three basic types: english, which have short hair; abyssinian, which have short, cowlicked hair; and peruvian, which have long hair. male guinea pigs are known as boars and the females as sows. guinea pigs become neophobic as they mature. offer a variety of foods early in life and make changes in diet or environment gradually. guinea pigs stampede when excited. square cages and strategically placed barriers on external walls prevent the trampling of small or weak animals. the smooth muscle of the bronchial tree is quite developed in guinea pigs. this places them at high risk for asthmatic-type anaphylactic reactions. both male and female guinea pigs have one pair of inguinal mammary glands; however, only the female's are well developed. m key point antibiotic toxicity (see "hamster"): guinea pigs also may be sensitive to tetracyclines. • fleas occur as in mice. • lice (i.e., gliricola porcelli, gynopus ovalis) usually cause no clinical signs except occasional alopecia, seborrhea, and trauma secondary to pruritus. • diagnosis is made by observation of lice on skin scraping. • treat with ivermectin, % malathion dust, or pyrethrin shampoo (see table - ). • the mite trixacarus caviae causes severe pruritus and is zoonotic. it mainly affects the dorsal midline and is difficult to find on skin scraping. it occurs most frequently in recently post-partum females, in which alopecia is the predominant clinical sign. treat with excellent sanitation and ivermectin (see table - ). • chirodiscoides caviae lives on the hair shaft of the perineal regions. it does not cause clinical signs. • treat with % carbaryl or lime-sulfur dip ( : ) (see table - ). sanitation is critical in preventing reinfestation. • about % to % of guinea pigs are carriers of trichophyton mentagrophytes. • clinical signs are alopecia and seborrhea sicca, usually starting on the face and spreading along the dorsum. • treat with lime-sulfur dips or griseofulvin (see table - ) combined with topical povidone iodine or chlorhexadine shampoos. • other causes for alopecia are barbering, alopecia of the flanks in late-gestation females, and generalized alopecia of young at weaning. subclinical hypovitaminosis c causes a poor hair coat and seborrhea sicca, as well as anorexia and large, malodorous stools. • "lumps" is the lay terminology for cervical lymphadenitis, which is characterized by lymphadenopathy in the ventral neck region. • pododermatitis and sore hocks are very common in guinea pigs. predisposing factors are untrimmed toe nails, poor sanitation, and wire flooring. s. aureus is the most commonly cultured pathogen. • clinical signs range from small ulcers on the soles of the feet to abscesses and gangrene. radiography is essential in determining whether bony involvement is present. untreated pododermatitis usually develops into osteomyelitis, which is very difficult to cure. • treat mild cases by improving sanitation and grooming. place affected individuals in solidfloored cages with paper bedding. use sulfa drugs (see table - ) until results of susceptibility testing are available. surgically remove or curette abscesses, and apply topical therapy and hot packing. amputation may be necessary when severe osteomyelitis exists. • conjunctivitis and epiphora occur as in mice. • inclusion body conjunctivitis is caused by chlamydia psittaci and is self-limiting in to weeks. • perform a conjunctival scraping to differentiate inflammatory conjunctivitis secondary to infection from allergy. i have observed an idiopathic, topical steroid-responsive lymphoplasmacytic conjunctivitis in guinea pigs. • white, dry ocular discharge is an early sign of hypovitaminosis c. • "pea-eyes" is the lay terminology for subscleral fatty deposits or protrusion of the lacrimal gland through the lower conjunctiva. the condition is thought to be hereditary. treatment is not required. • cataracts occur and are either congenital or developmental. • diabetes mellitus in guinea pigs also may cause cataracts. usually, no other clinical signs are present and urine glucose is greater than mg/dl whereas blood glucose remains within normal limits. • corneal or scleral calcification is usually an incidental finding. a thorough workup, including serum chemistry profile and radiographs, is recommended to ensure that generalized metastatic calcification is not present. • pneumonia in guinea pigs usually is caused by infection with s. pneumoniae, s. zooepidemicus, or bordetella bronchiseptica. s. aureus, p. aeruginosa, klebsiella pneumoniae, and pasteurella multocida also are cultured frequently. transmission is by direct contact, fomites, or aerosol. hypovitaminosis c and stress often predisposes guinea pigs to bacterial respiratory infections. weanlings are particularly susceptible. clinical signs and diagnosis are similar to other small mammals (see "mouse"). • take radiographs to rule out abscesses, pleural effusion, or pericardial effusion in refractory cases. • treat with chloramphenicol, sulfa drugs, or enrofloxacin (see table - ) and vitamin c (table - ) until results of culture and susceptibility testing are available. cats, dogs, rabbits, and rats are reservoirs for bordetella spp. as in other rodents, respiratory infections may lead to otitis interna/media. bordetella spp. also cause pyometra and abortions. • nasal discharge is most frequently a sign of upper respiratory tract infection but also may be associated with allergies or volatile irritants. • the diagnosis of allergic rhinitis is made by exclusion and through response to antihistamines or environmental changes. • bronchogenic papillary adenoma develops in approximately % of guinea pigs older than years of age. • diagnosis is often an incidental finding when thoracic radiography is performed for another problem. • occasionally, clinical signs are seen as a result of pressure on the heart or great vessels. • dyspnea most frequently is caused by heat stress or trauma. other causes are pregnancy toxemia, gastric bloat, volatile irritants, pleural effusion, pneumonia, or pulmonary edema. rhabdomyomatosis is a common necropsy finding. gross lesions appear as pale foci located on the endomyocardium and valves. histologic examination reveals myocardial cells that have stored excessive glycogen. do not confuse these areas with thrombi, abscesses, or neoplasia. their clinical significance is unknown. • paraspidodera ucinata is the cecal pinworm of guinea pigs. they are generally asymptomatic, but heavy infestations can cause diarrhea and weight loss. • diagnosis is based on fecal examination or cellophane tape test. • treat with piperazine or fenbendazole (see table - ). • coccidiosis caused by eimeria caviae is a fairly common cause of diarrhea in guinea pigs recently purchased from pet stores. • clinical signs are tenesmus, diarrhea, dehydration, and death. • diagnosis is based on fecal examination. on gross postmortem examination, petechiation and thickening of the colon are seen. • treat supportively and administer sulfa drugs (see table - ). • cryptosporidium wrairi and giardia spp. are found rarely. they cause a chronic enteritis. balantidium spp. are thought to be nonpathogenic. • malocclusion in guinea pigs is diagnosed on oral examination. • clinical signs are ptyalism and anorexia. the premolars are the most commonly affected teeth. • long-standing hypovitaminosis c predisposes guinea pigs to malocclusion. • treat malocclusion as in other rodents (see "dental procedures"). • hypovitaminosis c (i.e., scurvy) is associated with soft, malodorous feces. degeneration of the epithelium of the intestinal tract adversely affects digestion and absorption and allows secondary bacterial infections. • diagnosis of scurvy is based on clinical signs, the exclusion of other causes of diarrhea, and response to vitamin c therapy (see table - ). • salmonellosis usually is contracted through contaminated feed. • clinical signs range from sudden death to diarrhea and anorexia. the diarrhea is frequently light colored. sepsis is common and may cause conjunctivitis, shock, pneumonia, abortion, and neurologic symptoms. • diagnosis is based on isolation on culture of feces or other appropriate tissue samples. • treatment is controversial because recovered individuals remain carriers. use sulfa antibiotics or enrofloxacin (see table - ) until sensitivity testing results are available. supportive care is essential. • e. coli, arizona, and clostridium are other commonly cultured diarrhea-causing organisms. clostridium are diagnosed most easily by finding large numbers of spores on a gram stain fecal specimen. treat with metronidazole (see table - ). • yersinia pseudotuberculosis either causes an acutely fatal diarrhea or localizes into regional lymph nodes. • diagnosis is based on culture. • treat by surgical removal or drainage of abscessed lymph nodes. mesenteric lymph node involvement necessitates abdominal surgery. treat with sulfa drugs or enrofloxacin until susceptibility testing results are available (see table - ). • one male usually is housed with four to six females for breeding purposes. signs of estrus are vulvar swelling, lordosis, and opening of the vaginal closure membrane. fetuses are palpable at to weeks of gestation. parturition occurs within hours after the pubic symphysis has reached mm. neonates weighing less than g have a grave prognosis for survival even with intensive care. neonates normally do not nurse for the first to hours. litters with five or more fetuses usually result in abortion. • dystocia commonly occurs in females bred after the age of to months. after this age, the symphysis fuses and is unable to open the to cm required to allow passage of a fetus. dystocia in younger guinea pigs may be caused by obesity, large fetal size, fetal malpresentation, subclinical ketosis, or uterine inertia. on presentation, check the pelvic symphysis. if active contractions are present and the symphyseal gap is less than cm, perform a c-section. normal parturition is very rapid, with a rest of only to minutes between fetuses. • perform a c-section if active straining does not produce a fetus within to minutes. radiograph sows with a history of weak contractions to determine the stage of pregnancy and evaluate the size of the fetuses. if well-developed skeletons of appropriate size are seen and the pubis has not yet fused, give oxytocin and calcium (see table - ). if no fetuses are produced within to minutes, perform a c-section. • if poorly developed fetuses are seen radiographically, consider fetal death, ketosis, or a nonreproductive disorder as possible causes of dystocia. m key point pregnancy toxemia usually is seen in obese sows with large litters in late pregnancy. other risk factors include systemic disease or diet change causing anorexia, genetics, stress, and first litter. • clinical signs are tachypnea, depression, malodorous breath, seizures, and icterus. a urine ph of less than with marked proteinuria is compatible with pregnancy toxemia. a marked hyperkalemia and elevation of liver enzymes often occurs. thrombocytopenia may be present. • treat with iv or io saline, dextrose, glucocorticoids, and calcium. surgical abortion of the fetuses may be attempted, but the anesthesia risk is quite high. prognosis for survival is grave. do not rebreed affected females. do not breed sows heavier than g. • large litters can cause a hemorrhagic syndrome. compression of the portal vein and liver causes hepatic dysfunction, which results in vitamin k and clotting factor deficiency. • treat with vitamin k supplementation (see table - ). response is poor in severely compromised patients. affected individuals are at risk of ketosis developing. prognosis is guarded. • vaginitis in guinea pigs frequently is caused by foreign bodies, usually bedding. • diagnosis is made on vaginal examination. • treat by flushing the vagina to remove the foreign material. • vaginal discharge also can be caused by pyometra, uterine torsion, urinary tract infection, or urogenital neoplasia. • diagnosis is based on findings on abdominal palpation, vaginal cytology and culture, urinalysis, abdominal radiographs, ultrasound, and exploratory. • treatment varies with the condition and is similar to that used in cats. • ovarian teratomas and uterine tumors occasionally are diagnosed and usually resolve with ovariohysterectomy. • a symmetric alopecia with concurrent abdominal enlargement may be seen in female guinea pigs with cystic ovaries. • diagnosis is based on abdominal palpation, cytology, and ultrasound. • treat by performing an ovariohysterectomy. if the guinea pig is not a good candidate for surgery, human chorionic gonadotropin (hcg, usp units im, repeat in week) may temporarily resolve clinical signs. • male guinea pigs are prone to preputial foreign bodies. a preputial discharge is the usual presenting complaint. • diagnosis is based on physical examination. • treat by removing foreign bodies and performing local flushing. chronic problems require a change in bedding. • male guinea pigs produce sebaceous secretions in the folds around their perineal area. clean these areas with soap and water semiannually to prevent localized pyoderma. • if pyoderma occurs, treat with topical therapy and oral antibiotics. bacterial cystitis and urolithiasis are relatively common in guinea pigs. diagnosis is based on a history of stranguria, hematuria, painful abdomen, and anorexia, in addition to abdominal palpation, urinalysis, urine culture, abdominal radiographs, and ultrasonography. treatment consists of antibiotics based on results of culture and susceptibility testing and surgical removal of calculi, if present. prevention of recurrence is difficult if the calculi are not caused by a bacterial infection. addition of vitamin c to the drinking water as well as changing the brand of diet are sometimes helpful in preventing recurrence of metabolic stones. klossiella cobayae is a coccidia that lives in the renal tubules. it has no clinical significance. the most common orthopedic problem seen in guinea pigs is overgrown toenails. this leads to pododermatitis and sore hocks as well as to degenerative joint disease and a predisposition to tibial fractures. tibial fractures are the most common fracture seen in guinea pigs. they most frequently occur after foot entanglement. internal fixation with an im pin or application of a kirschner apparatus is the repair of choice. m key point signs of hypovitaminosis c or scurvy start to develop in guinea pigs as early as - days if they are placed on diets % deficient in vitamin c. early signs are soft, malodorous stools, weight loss, poor hair coat, and anorexia. later, petechia, gingivitis, cutaneous and oral sores, swollen costochondral junctions, joint pain and hemorrhage resulting in lameness, and conjunctivitis become apparent. treat supportively and administer parenteral vitamin c ( mg/day). • lymphocytic choriomeningitis occurs as in mice. • guinea pig paralysis syndrome starts with mild pyrexia and urinary incontinence, followed by weight loss and posterior paresis that progresses to paralysis. currently, the etiology is unknown, but it does not appear to be contagious. • treat with supportive care. prognosis for long-term survival is grave. • head tilt is usually the result of otitis or trauma (see "mouse"). cavian leukemia has a viral etiology. the liver, spleen, and lymph nodes are the primary organs involved. there is no current treatment. quarantine exposed individuals. death usually occurs within days after discovery of lymphoblasts in the peripheral blood. neutrophils normally have red granules. kurloff bodies are normally occurring eosinophilic intracytoplasmic inclusion bodies that are found in mononuclear cells. they are seen most frequently in females and appear to correspond positively with estrogen levels. metastatic calcification occurs in most guinea pigs older than year of age. it is more severe in females than in males. the stomach is one of the first organs affected. dysfunction in motility causes obstruction. the tendency appears to be exacerbated by high calcium and low phosphorus diets. exotic animal formulary the biology and medicine of rabbits and rodents veterinary clinics of north america key: cord- - pika authors: merck, melinda d. title: clinical management of large-scale cruelty cases date: - - journal: august's consultations in feline internal medicine, volume doi: . /b - - - - . - sha: doc_id: cord_uid: pika nan large-scale feline cruelty cases typically arise from investigations involving individual hoarders, rescue organizations, sanctuaries, or breeders. these cases involve the crime scene, animal processing at the scene and temporary shelter, animal removal and transport, sheltering, and final animal disposition. the veterinarian plays a role in the planning of the operation, at the crime scene, the temporary shelter, and the hospital receiving cats for more advanced treatment. the clinical management of these cases is different from traditional shelter medicine or feline practice. to act and respond appropriately, the veterinarian must have an understanding of all aspects of the operation. these are legal cases requiring certain types of documentation and procedures. the organization of the entire operation should be conducted similar to disaster response using the incident command systemwhere the medical management on scene and at the temporary shelter is incorporated into this reporting structure with direct communication to the lead investigator. extensive planning is needed, anticipating the number of cats, age ranges, known or likely medical issues, and socialization status. decisions must be made considering practicality, stress on the animals, costs, resources, future animal placement, and any legal impact. the goal is a successful outcome for the individual cat and the criminal case. supplies needed for on-scene and shelter medical operations need to be planned very early in the investigation. all team leaders should be involved in this planning, including those from the medical, forensic evidence, and shelter branches, with a supply list compiled for each branch (table - ) . arrangements should be made to obtain supplies during the case response for items that were not anticipated or depleted due to use. the use of color codes is important for large-scale cases to implement a visual system of communication at the scene, from scene to the shelter, within the shelter, and for forensic examinations. the colors are used to designate triage categories, initial cat temperament, and other conditions that determine placement and housing within the shelter. in addition, this system serves to establish the examination sequence. the assignment of color categories may vary with each case based on the availability of the color devices used (table - ) . colored duct tape is easily used and readily purchased at home improvement, hardware, office supply, and general merchandise stores. colored flagging tape may also be used but poses a risk of animal ingestion and can be difficult to remove from transport carriers or cages. a piece of the appropriate colored duct tape is placed on the animal identification (id) label located on the transport carrier or cage; some animals may have more than one color assigned (figure - ) . the primary use of the color system is at the scene, but some colors may continue to be used within the sheltering system. the color system and their assigned categories should be posted at the scene, within each forensic examination area, and at multiple areas within the shelter. the purpose of the critical triage team, comprised of veterinarians with or without veterinary technicians, is to identify any animals in need of urgent care. the critical triage team assesses animals after the initial scene walk-through is complete and as the physical evidence, animal id, and animal removal teams begin their work. any critically ill or injured animals should have priority for id assignment and removal from the scene. provisions for an on-site critical examination area should be made, such as a mobile veterinary clinic, trailer, tent, or one of the buildings within the scene itself. cats requiring more extensive diagnostics or treatment should be transported to a local veterinary hospital. the forensic examination teams are responsible for examination of all the animals. these teams are comprised of a veterinarian, scribe, and animal handler. it is important that all animals are examined in a timely fashion, within hours, to ensure the examination evidence is representative of the the effect of stress on cats has been well documented. in large-scale cases, stress can come from crowding, prolonged confinement, isolation, lack of environmental enrichment, an unsanitary environment, poor food and water resources, and untreated medical conditions. every consideration should be made to minimize stress during removal, transport, sheltering, and examination of the cats. physical handling of the cats and change of environment are additional but necessary stressors in these cases that carry a risk of exacerbating any pre-existing medical conditions. it is not unusual for these large groups of cats to be subclinical disease carriers that have reached a state of homeostasis that when disrupted results in new outbreaks. in addition, many of these cases involve animals that lack proper socialization and/or have been under severe emotional stress. they may exhibit aggression (due to fear or pain), or they may be completely unable to interact. some cats may exhibit subtle behavioral clues of extreme distress. it is the initial handling at the scene that can set the animal up for success or failure more so than any setbacks that may occur during examination or sheltering. resistance to handling and restraint is almost always the result of fear, anxiety, or pain, which is compounded when force is used. if possible, it is preferable to take several hours to remove a cat from the scene rather than forcefully remove it, which can reinforce the animal's fear and behavioral response. it is important to recognize that these cats may have reactions to novel stimuli different from those raised as pets in private homes. therefore, any negative behavioral designations assigned at the scene should be considered temporary and only for the purpose of cautionary handling. the key to successful handling of animals involves the accurate appraisal of behavior, an adequate number of staff, and the appropriate equipment; proper training in each of these areas being the most important aspect. transportation of cats from the scene to the sheltering site should be done in such a manner to minimize stress on the animals and to maintain compliance with applicable laws and regulations. considerations should be given to the environmental conditions and the time required to load the vehicle (which directly affects the first animals loaded). the spacing of the animal transport carriers within the transport unit should be planned to provide adequate ventilation for the cats inside. individual transport carriers should be of sturdy construction, put together properly to prevent animal escape or injury, have adequate ventilation, and consist of a design that allows stacking of carriers. special areas may need to be animal's condition at the original scene and any health issues are addressed as quickly as possible. the examination and treatment process should be complete and efficient. these teams usually work best when the scribe, who must be familiar with medical terminology, fills out the examination form as the veterinarian dictates the findings. the creation of special laboratory and treatment teams can improve efficiency and free the veterinarian to focus on examination and documentation. it is preferable to have one team designated to examine the more severe medical cases and animals that may require sedation to handle. this team should have ample medical supplies, controlled drugs, and the most secure examination area. there are several assessments that must be made at the crime scene that directly impact handling of the cats throughout the operation and have important legal ramifications. these evaluations may be conducted by veterinarians along with case investigators. it is critical that the veterinary teams, offsite hospitals, and sheltering operations take this into consideration. the assessment should include information about the cats and current housing conditions along with medications and supplies present. the most important consideration, and most overlooked, is the evaluation and continuous monitoring of stressors on the cats. it is important to document any obvious medical, husbandry, or behavioral care needs at the scene. this includes the presence of neonates, nursing queens, chronic medical conditions, infectious diseases, and potential zoonotic diseases. any records present should be assessed for pertinent information, including intake date, medical history, and individual cat names. any medications, supplies, and supplements should be documented, noting any prescriptions for individual cats, the date prescribed, expiration dates, the original amount and quantity remaining, and the prescribing veterinarian's information. the medications and supplies should be assessed for indicators of current or past medical issues. the general housing of the cats should be assessed and documented with photography and mapping. this assessment is an important consideration for housing decisions at the sheltering site. special note should be taken of the number of cats in a room or cage and natural groupings or obvious social bonds. the flow of people and animals within and between housing areas should be documented, with special focus on the potential for fomite transmission of pathogens. the type and brand of food offered should be recorded as well as the number of feeding stations and the number of cats that have access to each one. the availability and potability of water sources should be assessed. the litter boxes should be evaluated, including the type of litter, the amount of urine and feces, the type of litter box, and the number of cats with access to each litter box. samples of diarrhea should be taken for individual cats and representative samples for group population medicine whenever possible, the same persons should care for the same cats to help reduce stress, enhance opportunities for socialization, and decrease risk for injuries. assessments and monitoring may be improved when performed by personnel familiar with individual cats. creating separate sections in housing areas should be carefully considered, because they can create more stress on the cats and complicate management of the shelter. these separations are often based on infectious diseases, zoonotic diseases, critical medical observation, bite quarantine, reproductive status, and special needs (such as those cats that are difficult to handle, pregnant, neonates, or nursing). decisions for separate housing areas must be done with consideration of the crime scene assessment. most of these cases involve cats that have previously been exposed to each other either through housing design or fomite transmission; therefore, separation based on infectious disease is neither necessary nor recommended. in large open shelters, separate areas can be created by hanging tarps or clear plastic sheets that are open at the ceiling for airflow. other considerations for housing of cats include lighting, number of separate rooms, secure areas, and airflow. cats are more stressed when at ground level, so every effort should be made to elevate the housing. in the planning stages, it is important to develop medical protocols, including those for examinations, diagnostic testing, and all treatments. planning should include what tests will be conducted at the shelter and what outside laboratories will be used. because this is a legal case, the use of standard examination forms that are developed specifically for these types of cases is recommended (box - ) . these forms should be structured to prompt and guide the veterinarian through a complete examination. photographic id of the cat can be performed by one of the forensic examination team members, or a separate floating photographer may support multiple examination teams. the photographs should begin with the case information, including the case number, date, and animal id written on a card or dry erase board. this first picture with the case and animal information may be taken with or without the animal in the photo, and all photographs following this initial picture should only be of that animal. general photos should be taken showing the entire body of the cat: right and left sides, front (facial), hind (rear), dorsal, and ventral (if possible or appropriate) views. photographs, with and without a photo scale, should be taken of any obvious lesions, abnormal physical findings, and any evidence found on the body. it is important that each veterinarian use the same examination form (see box - ). it is the responsibility of the scribe to ensure that the entire form is complete, and it is the veterinarian's responsibility to review it for accuracy. in designated at the offloading area for animals requiring immediate veterinary care or assessment upon arrival to the shelter, especially neonates. planning for animal carrier placement based on infectious disease status is difficult and usually unnecessary as in most cases there has been ongoing exposure among animals due to their living conditions. for large-scale cat cruelty cases, there is rarely an existing animal shelter that can house all of the animals at one location. this requires creating a temporary shelter, which follows the same principles used for animal disaster sheltering, with the primary difference that the animals are evidence in a criminal case. protocols must be in place for monitoring, treatment, daily assessment, and progress documentation, including change of medical or behavioral status. there should be a shelter mapping system to identify and track the location of every animal, including when animals are moved. it is important to have the veterinarian's input on the initial design and any adjustments made based on the crime scene assessment or forensic examinations. the shelter should be designed to be flexible based on changing medical or behavioral conditions of the animals. shelter design should take into account the number of cats, special housing needs, ease of handling, infectious disease, temperature control, airflow, foot traffic, noise levels, animal stress, and environmental enrichment. continued assessment and reassessment of cat behavior and temperament should be conducted, with changes made to address any issues noted. any final behavior or temperament designations should only be assigned after the cats have had time to adjust to their new environment and recover from all stressors, which may take several days to weeks. consideration should be given to the original housing, grouping, and social bonds of the cats at the scene. often these cases involve group housing or free-roaming shelter settings, and the temporary shelter results in isolation of cats into individual cages. although this can be another stressor on the cats, it can be offset (at least for the short term) by the positive change to a sanitary environment and the elimination of competition for resources of space, litter, food, and water. cats should be paired or group-housed whenever possible and practical to reduce stress. shy or feral cats are often less stressed when paired with another cat. wire cages are especially amenable to alteration, creating a "double-wide" space for this purpose. environmental enrichment is important to incorporate into the sheltering design and protocols. this includes soft bedding, boxes, or small carriers to hide in, toys, treats, and catnip. it is critical to offer canned food, especially for cats with upper respiratory or dental disease, to assess and stimulate appetite, and for further enrichment. it is important to minimize changing of bedding and other items within the cages, which can create additional stress. the use of synthetic pheromones (e.g., feliway, ceva animal health) may be effective in stress reduction when sprayed on bedding and through the use of plug-in diffusers. addition, the veterinary teams should use consistent terminology and abbreviations, which may be posted in the exam areas. breed descriptions should be done carefully. unless the breed is known, it is often best to use "breed-type" or predominant "breed-mix." the body condition score (bcs) to be used should be posted in each examination area so that the veterinarians can refer to it. the age or estimated age of a cat should only be documented if supported by examination findings, such as dentition or ocular changes. it is preferable to use designations of neonatal, juvenile, adult, or geriatric with documentation of the basis for the determination. after the examination is complete, a check mark should be placed on the animal's id marker to create a visual indication that the animal has been examined. any color-coded markers should be assessed and removed or changed based on the examination; those that may impact shelter operations should be communicated to the shelter manager. the standard medical protocols enacted depend on the known existing conditions and diseases within the population, the expected length of stay in the temporary shelter, and the expected disposition of the cats; however, a variety of infectious diseases (including respiratory, enteric, and dermatologic pathogens) should be expected. depending on the legal process, the cats may be adopted directly from the shelter, placed at another animal facility within or outside the area, placed in foster homes, or euthanized. these potential scenarios and outcomes may be fluid and will dictate what protocols are most appropriate for the cats at any given time. the medical protocols should be implemented during the forensic examination process. standard vaccination protocols should follow the american association of feline practitioners (aafp) recommendations for shelter-housed cats. vaccinations should be given at the time of intake to the temporary shelter and should include feline herpesvirus type , feline calicivirus, feline panleukopenia, and rabies. vaccination for feline leukemia virus (felv) may be considered based on viral test results and group housing. a majority of intensively housed cats will harbor at least one enteropathogen, often of zoonotic significance; the presence of such pathogens is not limited to cats with clinical population medicine euthanasia protocols for euthanasia, necropsy examinations, and the storage of deceased animals should also be determined in advance. because the animals are evidence and also to remain sensitive to the case responders and public sentiment, the criteria and parameters for euthanasia decisions should be discussed with the lead investigator and prosecutor prior to the day of seizure. the decision for euthanasia should first be agreed upon by the examining veterinarian and the case lead veterinarian. the lead veterinarian should obtain permission from the lead investigator to perform euthanasia according to the agreed upon parameters. another factor to address in the planning phase is the use and storage of controlled drugs, including euthanasia solution and sedatives. a controlled drug log must be maintained according to applicable laws. many of the cats may have obvious medical needs. regardless of the presence or absence of clinical signs, it should be expected that most of the cats are likely carrying multiple different pathogens. these may or may not become clinically relevant, depending on the individual cat and the stressors placed on it. the most common medical finding is upper respiratory tract disease (urtd). this may vary from mild to severe with secondary pneumonia, ulcerations of the nasal or oral area, nasopharyngeal polyps, and ocular involvement ( figure - ) . mild cases may not need to be treated while being closely monitored for disease progression; most cases will resolve within to days. the more severe cases typically involve secondary bacterial infection, regardless of the primary pathogen, which should be treated aggressively. considerations for choices of medication should include the availability of personnel and their ability to administer signs of illness. therefore, routine deworming for roundworms, hookworms, and tapeworms should be part of the standard medical protocol. fecal testing is not necessary unless there is unresolved diarrhea after deworming, or it is otherwise dictated based on clinical illness. coccidiosis is a common finding in large-scale cat cruelty cases; it is found in up to % of cats with diarrhea and % of clinically healthy cats. based on observed diarrhea findings at the scene, within the temporary shelter, or diagnostic test results, it may be reasonable to treat all cats for coccidiosis. ponazuril is an effective treatment for coccidiosis and is easily administered to large numbers of cats, given orally once daily at to mg/kg for to days and repeated if necessary in week. , the stressors the cats have been exposed to along with dietary changes at the temporary shelter can contribute to diarrhea. in addition, many cats from these cases may harbor other enteropathogens, such as tritrichomonas, giardia, or cryptosporidium species, as well as feline enteric coronavirus, which may or may not be a cause of refractory diarrhea. , any additional testing and interpretation should be conducted as discussed later in this chapter. all diagnostic testing decisions for the cat population should be done with consideration for the goals of the operation; the legal impact; the health of the individual cat and the general population; the stress on the cat; the expected time length for housing; the future placement of the cats and the placing agency's responsibilities; the financial and personnel resources; and clinical relevance. for each test considered, both the test itself and the action to be taken based on its result must be appropriate and reasonable. in a large-scale case, tests that may be recommended or conducted in private or shelter medicine practice may not be appropriate or reasonable. the goal is not to cure all animals and eliminate all pathogens prior to the cat leaving the temporary shelter, rather it is to immediately identify and address any important medical issues, improve overall physical and mental health, and address any obvious zoonotic issues. this should be a clear and constant guidepost for decisions made. it cannot and should not be the expectation to test and treat for everything. as with any shelter situation, these cats may be asymptomatic carriers of a variety of pathogens. testing can result in information that is not currently relevant to the health of the animal yet force decisions for unnecessary and inappropriate actions for that individual animal or the general population. for large-scale cruelty cases, it is recommended to test cats for felv and feline immunodeficiency virus (fiv) based on the guidelines established by the aafp. depending on the test results and the original housing at the crime scene (i.e., pre-existing exposure), changes in the housing location within the temporary shelter may or may not be recommended. cats that appear ill without a grossly apparent cause should have routine laboratory work analyzed, including a full biochemistry profile, complete blood count, and urinalysis. such documentation is especially important in legal cases. medication, the possibility of administering medication through canned food or treats, dosing intervals, possibility of multiple pathogens, and risk of side effects. stress reduction plays the single greatest role in the remission and prevention of urtd in cats; therefore, particular attention should be given to providing an enriched environment as a component of the treatment plan. skin and fur conditions are quite common in large-scale cruelty cases. the cats often have urine and/or fecal staining of fur, especially on the feet, usually with associated dermatitis. other issues related to the feet include contact pododermatitis, plasma cell pododermatitis, and overgrown or embedded claws (figure - ) . cat bite abscesses are not uncommon. severe matting may be seen on longhaired cats with fecal soiling or impaction in the perineal area with associated dermatitis (figures - and - ). in addition to fleas and associated dermatitis, there may be other external parasites, such as cheyletiella, lice, and demodectic or sarcoptic mange. stress can cause alopecia due to overgrooming or self-mutilation (figure - ). less commonly, cases of streptococcus canis have been reported in intensively housed cats, often manifesting with skin ulceration and necrotizing fasciitis (figure - ) . dermatophytosis may be seen in some cats but rarely is a widespread issue within the population. cats may be mechanical carriers and never develop clinical lesions themselves; such cats are seldom clinically relevant to the case, the individual animals, or the general population. for these reasons, dermatophyte testing is not routinely recommended on intake of cats or for those without dermatologic lesions. rather, the focus should be on diagnosing and treating cats with suspicious lesions and enacting precautions for handling and cleaning. housing placement within the shelter for cats suspected or diagnosed with dermatophytosis should be based on the considerations previously discussed. population medicine in the distal extremities, may be found. other common findings include decubital ulcers, poor wound healing, and secondary bacterial or yeast skin infections. there are numerous other medical findings that may be seen or develop in these cases: a variety of clinical abnormalities of the skin result from nutritional deficiencies, including starvation. a common finding is a sparse, dry, dull, and brittle hair coat with hairs that epilate easily. seborrhea sicca and patchy alopecia may be seen. loss of hair occurs due to anagen and/or telogen defluxion. in the anagen or growth phase of the hair cycle, there can be loss and/or abnormal growth of hair, resulting in broken hair shafts. with telogen defluxion, there is widespread loss of hair in the quiescent phase of the hair cycle due to large numbers of hair follicles being synchronized in this phase. loss of normal hair color and hair keratinization abnormalities can occur. in cats, decreased melanin deposition due to amino acid deficiencies results in a reddish cast to the hairs, sometimes called red coat (figure - ). erythema or crusting in areas of friction or stretching, such as primary or secondary to other medical conditions, malnutrition, dehydration, stress, and/or an unsanitary environment. often, the investigation findings may provide information on pre-existing conditions through intake paperwork or hospital records. these underlying issues may only be detected with routine blood work and urinalysis; affected cats may require further diagnostic testing, imaging, or hospitalization. the clinical management of large-scale feline cruelty cases starts at the scene and continues to the temporary housing in a shelter or hospital. it is critical that veterinarians understand the importance of the role they play as part of the investigative team and the requirements for documentation in a legal case. the veterinarian may become involved in these cases directly at the scene and shelter or by receiving a patient into the hospital. by the very nature of large-scale cruelty cases, the cats will have a wide range of issues associated with high volume housing and long-term neglect. it is important to be mindful that this will be a fluid situation requiring a process of continuous assessment of the animal's health and behavior to detect early signs of developing issues and initiate treatment. the simple actions of providing appropriate housing, food, routine medical treatments on intake, and overall stress reduction will often result in rapid improvement of cat health. there is a greater chance of recovery-even for the most severely affected cats-when both physical and mental needs are addressed. • oral lesions, including ulcerations associated with urtd, stomatitis, and dental disease • urinary tract issues (e.g., urinary tract infection, idiopathic cystitis, nephroliths, uroliths, crystalluria, and vaginitis) are often the reason the cat was presented to the original shelter or sanctuary the clinician should be cognizant of the possibility for these and other underlying medical issues that may be special considerations in animal cruelty cases recognition and management of stress in housed cats feline care in the animal shelter effects of a synthetic facial pheromone on behavior of cats a synthetic fraction of feline facial pheromones calms but does not reduce struggling in cats before venous catheterization crime scene investigation prevalence of infectious diseases in large scale cat hoarding responses aafp feline vaccination advisory panel report prevalence of enteropathogens in four management models for unowned cats in the southeast united states clinical trial to assess the efficacy of ponazuril for the treatment of coccidiosis in dogs and cats enteropathogens identified in cats entering a florida animal shelter with normal feces or diarrhea american association of feline practitioners retrovirus management guidelines prevalence of upper respiratory pathogens in four models for unowned cats in the southeast united states feline infectious respiratory disease fatal streptococcus canis infections in intensively housed shelter cats veterinary forensics: animal cruelty investigations key: cord- - lyw gh authors: guzman, efrain; montoya, maria title: contributions of farm animals to immunology date: - - journal: front vet sci doi: . /fvets. . sha: doc_id: cord_uid: lyw gh by their very nature, great advances in immunology are usually underpinned by experiments carried out in animal models and inbred lines of mice. also, their corresponding knock-out or knock-in derivatives have been the most commonly used animal systems in immunological studies. with much credit to their usefulness, laboratory mice will never provide all the answers to fully understand immunological processes. large animal models offer unique biological and experimental advantages that have been and continue to be of great value to the understanding of biological and immunological processes. from the identification of b cells to the realization that γδ t cells can function as professional antigen presenting cells, farm animals have contributed significantly to a better understanding of immunity. great advances in immunology are usually supported by experiments carried out in animal models and by far, inbred lines of mice and their corresponding knock-out or knock-in derivatives, are the most commonly used animal systems in immunological studies. though with much credit to their usefulness, laboratory mice will never provide all the answers to fully understand immunological processes. also, some answers provided in mouse models are not applicable to other species of animals or humans. large animal models offer unique biological and experimental advantages that have been and continue to be of great value to the understanding of biological and immunological processes. the humble cow, the underestimated pig and the unassuming chicken have greatly influenced our current understanding of human immunology. for most immunologists dedicated to fundamental and applied research, it is easy to forget that b cells were first identified in chickens and vaccination first occurred because of a cow. although there are far too many important events to discuss in this paper, we have chosen to highlight a few of the most important contributions of farm animals to the current understanding of immunology ( table ) . edward jenner published in a booklet entitled "an inquiry into the causes and effects of the variolae vaccinae, a disease discovered in some of the western counties of england, particularly gloucestershire and known by the name of cow pox" ( , ) and although strictly speaking jenner did not discover vaccination, he was the first person to use scientific rigor to prove protection from disease through targeted intervention. the english dairy farmer benjamin jesty ( - ) was the first person known to vaccinate against smallpox ( ) protecting his family against the virus even after numerous exposures ( ) . however, the idea and indeed the term vaccination, only came into the light spot years later thanks to louis pasteur. this time the chicken takes a privileged position and the story was beautifully explained by pasteur himself ( , ) and has been romanticized in paul de kruif 's book "microbe hunters" ( ) . in pasteur inoculated chickens with "stale" cultures of pasteurella multocida. the chickens became sick but recovered so he decided to re-inoculate them with a fresh culture. the chickens that had received the "stale" culture recovered whereas chickens that had not been pre-exposed to the stale cultures died. pasteur recognized the similarities between his studies in chickens and what jenner had published with smallpox. he coined the term "vaccine" ( , , ) in honor of jenner. by the early s, william smith greenfield in the uk ( , ) and pasteur working with henri thullier, charles chamberland and pierre paul Émile roux in france ( , ) had begun developing and testing vaccines against anthrax in sheep and cattle. a decade later, the german scientists friedrich loffler and paul frosch identified the first ever filterable infectious agent in mammals: foot and mouth disease virus (fmdv) and developed a fully protective heat-inactivated vaccine against it ( , ) ; however an effective long-lasting and broadly protective vaccine against fmdv remains elusive. pigs also played an important role in early vaccinology studies. by the late s swine plague or hog cholera (later discovered to be caused by a virus now called classical swine fever virus, csfv ( ) was killing hundreds of thousands of pigs across the word and was particularly of concern to the us pig producing industry, causing an impressive us$ million a year in losses in ( ) and us$ million by . once again, pasteur and thullier developed a vaccine to what is now thought to be the first ever vaccine against a viral infectious disease ( ) and the first mass-vaccination campaign in history. in addition, it is rarely recognized that csfv was the first animal virus ever to be cultured in vitro ( ) and the techniques developed by carl tenbroek continue to be used today. horses have also contributed to the understanding of fundamental immunological mechanisms. in a series of experiments, emile roux working with alexandre yersin and followed by emil von behring and shibasaburo kitasato immunized horses to produce an "antidote" or immune sera against the diphtheria toxin that was eventually used to treat humans, an important step in understanding antibodies and humoral immunity ( ) . behring won the nobel prize for medicine in for this work. another milestone in vaccine development was the generation in the 's of vaccines to control marek's disease (md), a naturally occurring neoplastic disease in chickens caused by an oncogenic herpesvirus ( ) . md vaccines are the first examples of the use of vaccination to protect against cancer ( , ) . with the discovery of molecular biology techniques in the 's and 's, the race was on to develop recombinant vaccines against numerous infectious diseases. the first report of a biosynthesized polypeptide vaccine was published in ( ) . the structural protein vp of fmdv was cloned ( ). swine plague. science , and expressed in e. coli and the purified protein used to vaccinate six cattle and two swine, which developed neutralizing antibodies and were protected against challenge with fmdv ( ) . and new technologies have only helped to highlight the importance of farm animals in vaccine development: using a computational approach to assess protein-protein stability, kotecha and colleagues ( ) used molecular dynamic ranking to predict fmdv capsid stabilities and produced stabilized fmdv capsids based on these predictions, assessed their stability using x-ray crystallography and demonstrated their improved immunogenicity in vivo by vaccinating cattle. this demonstrates the potential value of structure-based design of vaccines to develop stabilized vaccine antigens for animals and humans alike. although the innate immune system of animals is largely conserved, there are significant variations in the pattern-recognition-receptor (prr) structures of various species ( ) . it has been suggested that laboratory mice have not been subjected to the selective pressures that other animals have and so innate immune studies carried out in laboratory animals do not accurately inform human biology ( ) . it has been demonstrated that human and farm animal prr responses to their ligands ( , ) are more similar to each other than human-mouse prr responses ( ) ( ) ( ) . because prr recognition is associated with adaptive immunity, a better understanding of these molecules in farm animals is likely to better inform on their effect in these animals as well as humans. a major contribution of the chicken to fundamental innate immunity was the description in of the first interferon. chicken embryos were exposed to influenza virus by alick issacs and jean lindenmann ( ) and they identified an immune soluble element responsible for regulating virus infection. this discovery was certainly one of the scientific landmarks in cell biology in the twentieth century and one which opened the doors of what we now know as innate immunity. perhaps the most recognizable contribution of the chicken to science, and immunology in particular, was in the definition of the two elements of adaptive immunity: the b-dependent and the t-dependent immunity. the avian bursa of fabricius, named after hieronymus fabricius of aquapendente ( ) is a sac-like structure located in the cloacal passage of the bird and its function remained elusive until well into the twentieth century. bruce glick and timothy chang working at the poultry science department at ohio state university ( , ) described how following the surgical removal of the bursa, chickens injected with salmonella typhimurium "o" antigen failed to develop bacteria-specific antibodies. glick and chang wrote a paper entitled: "the role of the bursa of fabricius in antibody production" and was rejected by leading scientific journals ( ) and eventually published in poultry science ( ) . several years later, the bone marrow in mammals was shown to be the equivalent of the bursa in birds ( ) , and so the term "b-lymphocyte" originated from "bursa-derived lymphocyte". several years later, cooper et al. published a fundamental paper on the demarcation of the thymic and bursal and systems in birds and proposed the existence of equivalent systems in mammals ( ) . the cannulation of lymphatic vessels was developed in the early twentieth century in rats to study the lymphatic system but due to the complexity of the surgical procedure, sheep and then cattle were used extensively in the s and s in lymphatic cannulation studies ( ) . in a series of adoptive transfers of lymph-migrating cells and fluid, hall and colleagues first identified in sheep that antibody-secreting cells (acs) encounter antigens and are activated in the lymph nodes ( ), then migrate via the efferent lymphatics to the circulatory system, and that the immune response depends on an intact lymphatic system. cattle have also contributed to fundamental b cell immunology and the generation of a highly diverse antibody repertoire. most vertebrates encode a large number of variable (v), diversity (d), and joining (j) gene segments and antibody diversity is achieved by recombination of these segments. in contrast, cattle only express a limited number of v genes and so it is thought that antibody diversity is achieved recombination events and endogenous mutation mechanisms in the cdr region ( ) . another unusual feature of bovine antibodies is their exceptionally long cdr regions ( ) . these long cdr and unusual mutation mechanisms result in "microfolds" within the cdr region that allow bovine antibodies to bind antigens that would normally be inaccessible ( ) . a recent report demonstrated that cows can be immunized with a single hiv env trimer and this results in potent hivspecific nabs which are dependent on the length of the cdr loops of bovine ig ( ) . it has been suggested that this could be an efficient way of producing super-antibodies against other human pathogens. transchromosomal cows have been engineered to produce large amounts of full human igg molecules with pathogen specificity: mers-cov ( ) , hanta virus ( ), veev ( ) , and ebola virus ( ) . this technology has the potential to generate prophylactic antibodies against emerging viral diseases. on the other hand, chickens have serum igm and iga both of which are homologs of their mammalian counterparts; in addition, they express igy, not found in mammals but thought to be an evolutionary ancestor for mammalian igg and ige. chickens however do not have either ige nor igd but instead use a distinct process for generating antibody diversity that is distinctly different to mammals ( ) . engineers frequently look to nature for inspiration. antibody engineers are no exception, modeling new therapeutics on molecules found in animals such as camels and cows. indeed, % of bovine antibodies have unusually long heavy-chain cdr s as part of their antigen-recognition sites. stanfield et al. ( ) have solved crystal structures of three new bovine fab fragments and analyzed the five known structures to show that their ultra-long cdr h s all adopt similar architectures composed of a knob domain containing a small conserved β-sheet connected by diverse disulfide-bonded loops that is separated from the antibody surface by a long conserved stalk. they propose that varying the length of the stalk and the positions and number of disulphide links in the knob may help drive antibody diversity. these structural insights could be leveraged to tailor antibody-based therapeutics. in contrast to all other mammals, camelids (dromedaries, camels, llamas, etc) also have an unique antibody type similar igg but with identical heavy chains lacking the ch domain and which do not pair with their corresponding light chains. these "heavy-chain antibodies" (hcabs) display antigenspecific variable domains or "vhh" which are structurally and functionally similar to an igg fv but have only three cdr loops defining the antigen biding sites. camel vhh domains, also called "nanobodies, " have been of great interest because of their stability and small size and strong affinity to their corresponding antigens. in fact, several camel vhh domain antibodies are in early preclinical development in oncology, infectious, inflammatory, and neurodegenerative diseases ( ), the most recent example being the generation of broadly neutralizing antibodies to influenza in llamas ( ) . cytotoxic and helper t cells are generally considered to be phenotypically different due to the mutually exclusive expression of the co-receptors cd αβ and cd and differences in mhcrestriction (class i vs. class ii). however, between (in normal individuals) and % (in certain pathologies) of human peripheral blood lymphocytes have been shown to be cd /cd double positive (dp) t cells ( ) . thymic and extra-thymic development of t cells has been studied mainly in mice and because the expression of cd and cd in mouse t cells for the most part mutually exclusive, cd /cd dp lymphocytes have generally been ignored. nevertheless, the presence of cd /cd dp t cells in many animals makes it impossible to ignore these cells. studies in pigs have shown that cd /cd dp are a distinct subset of activated and/or memory t helper cells ( ) and in humans the increase in circulating cd /cd dp t cell frequency has been identified in autoimmune and chronic inflammatory diseases ( ) ( ) ( ) ( ) ( ) suggesting the importance of this particular t cell population in human health. most circulating t cells in humans and mice are conventional t cells expressing the αβ t cell receptor (tcr) and either cd or cd . unlike mice, other species like cattle, pigs and chickens possess a substantial proportion of t cells expressing the γδ tcr cells in the circulation suggesting that circulating γδ tcr t cells have a more important role immunity than previously thought ( ) . the fact that the phenotype and frequency of circulating and tissue resident t cells is so vastly inconsistent in different species suggests that immune responses to (vaccine) antigens are also distinct. it is assumed that that all animal species have a similar immune response to a particular antigen, but this is a statement to be reviewed in light of each host particularities. in addition, the th /th polarization of t cells observed in response to particular antigens is a phenomenon of certain strains of laboratory mice and not of outbred mammals including farm animals and humans ( , ) . in fact, it has been shown that cytokine profiles defining th /th responses to antigens in cattle are more similar to human responses than those observed in mice ( ). dendritic cells (dc) as such, and their role in immunity were first described in the s and in ralph steinman published a series of papers describing that a cellular receptor called "dec- " (now cd ) was expressed on mouse dc, was involved in antigen processing ( , ) and was detected by the monoclonal antibody nldc- . in fact, it was years earlier in , that chris howard, a bovine immunologist working at the then called "institute for animal heath" in the uk published a series of papers identifying an important and until then uncharacterized marker expressed on pseudoafferent lymph veiled cells (also called aldc) detected by the monoclonal antibody wc (now cc ) ( - ). although steinman's identification of mouse cd helped characterize the binding of cc to bovine cd ( ), the importance of cd in identifying dc was first evident in cattle. as mentioned above, steinman's seminal work in characterizing dc using the mouse system has been one of the most important developments in cellular immunology of the twentieth century, and one which lead to his nobel laureate. however, the idea that a component of the immune system was involved in antigen processing and presentation had been proposed many times before. as mentioned above, cannulation of the lymphatic vessels is more practical in large than small animals, and this technique has been used to investigate dc biology. afferent or peripheral lymph dc were first described in sheep in ( ) as "very phagocytic dendritic macrophages that are involved in long term immunological reactions" that are very potent antigen presenting non-lymphoid cells ( ) and that their phagocytic and antigen presentation capacities differed from "classical" peritoneal macrophages ( ) , therefore indicating that dc and macrophages were different cell types ( ) several years before steinman's observations ( ) . in addition, lymphatic cannulation of sheep has revealed important ontologic, phenotypic and functional characteristics of dc subsets that are relevant in other mammals, particularly humans ( , ) . similitudes and differences between swine and human dc/macrophage populations have recently been described ( ) . in one striking example and in contrast to studies performed in mice, swine and human cdc , which are associated with th responses, both express fcεriα and are localized in or next to the tracheal and bronchial epithelia. these observations have been proposed to imply that swine and humans have similar allergen responses as opposed to mice. this theory is supported by the fact that localization of cdc helps them access antigens such as airborne allergens, and fcεriα expression on these cells might help proliferation observed in allergic responses. as mentioned before and unlike mice, horses and humans, most other animals have a large γδ t cell compartment. for example, up to % of all blood lymphocytes in young calves are t cell expressing the γδ t cell receptor (tcr). although the reasons for the enlarged t cell compartment in cattle, pigs and chickens is still unknown, their large numbers and ease of collection has resulted in great advances in γδ t cell biology knowledge not only for farm animals, but also for humans. for example, apcs were shown to influence γδ t cell proliferation ( , ) . cynthia baldwin's lab has defined antigen-specific bovine γδ t cell responses in various systems ( ) ( ) ( ) and adrian smith's lab has done similar observations in chickens ( , ) . it has also been shown that bovine γδ t are potent regulatory t cells ( ) , an observation that is also true for a subset of human γδ t cells ( , ) . these results in farm animals have and continue to enhance our understanding of human γδ t biology ( ) . perhaps the most important one was the realization that a subset of bovine γδ t cells expressed mhc class ii and co-stimulatory molecules on their surface, a characteristic normally attributed to macrophages, b cells and dc but not t cells ( ) . bovine γδ t cells were also shown to phagocyte antigens and of mhc iirestricted presentation to cd + t cells ( ) . this function of bovine γδ t cells was subsequently reported in pigs ( ) and much later in mouse ( ) and human ( ) ( ) ( ) γδ t cells. perhaps the best known contribution of any farm animal to scientific progress was the somatic cell nuclear transfer that gave origin to dolly, the sheep ( ) . although nuclear transfer itself is not a direct contribution to immunology, nuclear transfer technology has directly influenced many immunological concepts underpinned by technologies such as induced pluripotent stem (ips) cells and crispr-cas systems. clustered regularly interspaced short palindromic repeats (crispr) is a rna-guided endonuclease used both in vivo and in vitro. genetically modified animals becoming more common and their availability can be exploited in many applications such as comparative immunology, physiology and disease, to generate in vivo bioreactors to produce complex proteins, or to produce genetically modified organs for transplantation in humans ( ) . although the majority of pharmaceutical research is performed in laboratory mice models, it is clear that humans are not "large mice." by a large extent, studies in laboratory mice have been the victim of over interpretation; for example, by extrapolating successful pre-treatment in mice to therapeutic treatment in men. the weakness of the mouse model in pharmaceutical research was recently highlighted in a study showing that inflammatory responses in mouse models do not correlate with human inflammatory disease ( ) . an additional study showed a close similarity in expression profiles of immune-related genes between humans and pigs ( ) . cattle, pigs, and chickens, are useful, valid, and valuable models to study human infectious diseases and important clinical targets in their own right. both humans and cattle are the natural hosts for tuberculosis (being infected with the geneticallyrelated mycobacterium bovis and mycobacterium tuberculosis, respectively) and the bovine and human diseases share many similarities in terms of immunity and pathology ( ) , whereas the mouse model of tuberculosis does not provide a faithful representation of the disease in humans ( ) . similarly, bovine respiratory syncytial virus (brsv) is closely related to human (h) rsv and the pulmonary pathology, immune responses and epidemiology seen in young calves and children are very similar ( ) . swine have been shown to be a more faithful model for human influenza infection and immunity studies and the same strains of influenza infect both humans and pigs because the distribution of influenza virus receptors and physiopathology are similar in both species. the transfer of maternal-derived antibodies (mda) to new born pigs enables fancy vaccine study design to elucidate the role of mda in immunity ( , ) , vaccine efficacy and in enhancement of respiratory disease ( ) . gnotobiotic piglets have been used to study various human gastrointestinal pathogens. for example, human noroviruses are antigenically and genetically related to swine noroviruses and unlike mice, humans and pigs show genetic susceptibilities to noroviruses depending on their histoblood group antigen phenotypes and the virus strain. similarly, gnotobiotic pigs have been used in rotavirus research to study disease pathogenesis and identify virus-specific iga and asc as correlates for protection and vaccine efficacy in children ( ) . pigs have also been proposed to be better models than mice for many other infectious diseases including female genital infection with chlamydia trachomatis, helycobacter pylori, neisseria meningitides, and nipah virus among others because of the natural susceptibility of pigs to these pathogens ( ) . endogenous retroviruses were first discovered in pig kidney cell lines in ( ) and are now known to be present in most, if not all, mammalians. the presence and potential reactivation of endogenous retroviruses has very important consequences in both allo-and xeno-transplantation. immunotherapy is becoming more popular in clinical trials and vaccine efficacy studies. the success of immune cell therapies partially depends on the effective delivery of cells to target organs, a process that invariably involves the lymphatic system. dc migration in mice has not proven to be very informative, however, dc migration in pigs may be able to answer several question on dc migration that cannot be addressed otherwise. these studies demonstrate that using large animals to investigate immune cell trafficking will help improve immunotherapies in humans ( ) . in the french surgeon mathieu jaboulay ( - ) implanted a pig's kidney into one woman and a goat's liver into another thus starting the idea of xenotransplantation; unsurprisingly, both women died ( ) . the acceptance or rejection of a donor's organ or cells is fundamentally an immunological event. cellular rejection involves nk and t cells that recognize foreign antigens on the grafted tissue. using xenotransplantation models (pig-to-rat, pig-to-primate, and pigto-human), the main mechanism for organ and tissue rejection has been proposed to involve arteriosclerosis, or thickening of the arterial walls. this process if thought to be caused by activated and allo-reactive lymphocytes that migrate over time to the transplanted organ ( ) . arteriosclerosis is a major cause of chronic organ rejection ( ) . studies in laboratory mice have underpinned many concepts of immune tolerance and the generation of immune responses in the neonate. however, the peripheral immune system in mice remains unpopulated during pregnancy and it is only after birth that b and t cells begin to emmigrate to the periphery. in contrast, lymphoid cells circulate through the fetus in humans and large animals well-before birth and specialized lymphoid tissues are also well-developed and populated by the time of birth and are able to respond to a number of antigens ( , ) . certainly the immune system in neonate humans and large animals is not matured, but calves, lambs and piglets can be more useful than mice in understanding immune responses during pregnancy and in new borns and these studies can be used to better inform human developmental immunology. this advantage over mice has recently been used to develop extracorporeal support technologies using neonatal lambs with the ultimate objective to use these technologies in premature children ( ) . perhaps one of the most common uses of large animal models is in the development of vaccines with several advantages over mice. the serial collection of peripheral blood from animals such as pigs, cattle, chickens, and horses allows for immunokinetic studies to be possible in response to vaccination or infection at the level of the individual. these immunokinetic studies can be used to correlate immune responses generated with protection after challenge with the relevant pathogen. in vaccinology studies using mice, the typical approach would be to sacrifice groups of mice sequentially and harvest spleen and blood, so the immune response to vaccination at the individual level is not normally achieved. large animals also provide several advantages over mice when investigating mucosal immunity. when mice are vaccinated or inoculated intranasally, it is common for the inoculum to be digested because anesthetized mice can both swallow and inhale the material placed on their nose. in addition, the structure of the mucosal associated lymphoid tissue (malt) differs significantly in mice from that of large animals and humans; for example mice do not have tonsils but instead have undefined networks of malt, whereas cattle, pigs and sheep have well-defined tonsils ( ) ( ) ( ) . in the case of vaccine delivery through the skin, cattle, and pigs appear to be better suited than mice for these studies. skin thickness, structure of the epidermis and the presence and distribution of langerhan's cells are among many characteristics that humans and cattle and pigs have in common and which are practically relevant in transcutaneous immunizations ( ) . the process of selecting a relevant and appropriate animal model is a balanced and complicated exercise due to the diversity in vertebrate physiology, adaptive and innate immunity. studies in mice, for example, have shown the efficacy of vaccines against fmdv, however these efficacy studies have failed to be translated to the target species (cattle and pigs), presumably due to fundamental differences in the immune systems of model organisms and target species and the ability of the virus to mutate in these animals ( ) . it has recently been shown that because immunoglobulin subclass diversification occurred after speciation ( , ) a particular immunoglobulin subclass in one species bears no functional homology to one of the same name of another species ( ) . thus, our knowledge of the functions of igg in mice cannot be extrapolated to other mammals. characterizing generating reagents for each animal model hinders the development and usefulness of any of these models and therefore limiting the usefulness of cows, cattle or chickens as models for human immunology. mice and rats are and will probably continue to be the chosen model organisms over farm animals. mice can be readily mutated (knock in or knock out) to study immunological pathways; so far this has been proven to be very difficult-and expensive-in large animals. as mentioned above, the availability of reagents to study immune cells and processes in mice far out competes the availability of these reagents for large animals. pharmacokinetic and toxicology studies would be prohibitively expensive in pigs, horses or cattle, so small rodents and rabbits are the best organisms to use in these studies. in addition, studies in mice have been fundamental in the discovery of antibiotics, chemotherapy agents and more recently car-t cell therapies that can be directly applied to humans. genetic homogeneity, low cost, the availability of biologically-relevant mutants and reagents make the mouse the optimal animal model for many academic and industrial researchers. farm animals have historically contributed and continue to contribute to fundamental and applied immunology. the use of these animals in research is not difficult as long as the appropriate facilities and reagents are available. dedicated housing, cost, biosecurity, and genetic variability are some of the many disadvantages confronted when using farm animals in research. however, selecting an appropriate animal model should be more than just a matter of accessibility and common practice ( ) but should be based on the scientific question to be addressed and its relevance. not just a country doctor: edward jenner, scientist edward jenner and the eradication of smallpox new light in the dawn of vaccination on the germ theory on chicken cholera: study of the conditions of non-recidivation and of some other characteristics of this disease microbe hunters the life and works of louis pasteur lectures on some recent investigations into the pathology of infective and contagious diseases professor superintendent, the brown animal sanatory institution concerning the priority due to him for the production of the first vaccine against anthrax remarks on anthracic vaccination as a prophylactic of splenic fever summary report of the experiments conducted at pouilly-le-fort, near melun, on the anthrax vaccination, summarischer bericht uber die ergebnisse der untersuchungen der kommission zur erforschung der maul-und klauenseuche bei dem institut fur infektionskrankheiten in berlin. centralblatt fur bakteriologie berichte der kommission zur erforschung der maulund klauenseuche bei dem institut fur infektionskrankheiten in berlin. centralblatt fuxr bakteriologie the survival of the hog-cholera virus in laboratory animals, particularly the rat swine plague, or hog cholera correspondence of pasteur and thuillier concerning anthrax and swine fever vaccinations cultivation of the hog cholera virus remembering emil von behring: from tetanus treatment to antibody cooperation with phagocytes studies on the epidemiology of marek's disease herpesvirus in broiler flocks effect of vaccination with herpesvirus of turkeys (hvt) on horizontal spread of marek's disease herpesvirus protective efficacy of marek's disease vaccines cloned viral protein vaccine for foot-and-mouth disease: responses in cattle and swine structure-based energetics of protein interfaces guides foot-and-mouth disease virus vaccine design toll-like receptors in domestic animals variation matters: tlr structure and species-specific pathogen recognition human but not murine toll-like receptor discriminates between tripalmitoylated and tri-lauroylated peptides human toll-like receptor recognizes host-specific lps modifications identification of full length bovine tlr and functional characterization of lipopeptide recognition by bovine tlr / heterodimer virus interference. i the interferon a landmark contribution to poultry science-immunological function of the bursa of fabricius growth of the bursa of fabricius and its relationship to the adrenal gland in the white pekin duck, white leghorn, outbred new-hampshire, and inbred new-hampshire the bursa of fabricius and antibody production cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells restoration of gamma globulin production in agammaglobulinemic chickens surgical techniques for the collection of lymph from unanaesthetized sheep the ultrastructure and function of the cells in lymph following antigenic stimulation maintenance of antibody to pathogen epitopes generated by segmental gene conversion is highly dynamic during long-term persistent infection reshaping antibody diversity rapid elicitation of broadly neutralizing antibodies to hiv by immunization in cows human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo dna vaccine-derived human igg produced in transchromosomal bovines protect in lethal models of hantavirus pulmonary syndrome antibody preparations from human transchromosomic cows exhibit prophylactic and therapeutic efficacy against venezuelan equine encephalitis virus production of potent fully human polyclonal antibodies against ebola zaire virus in transchromosomal cattle structural and genetic diversity in antibody repertoires from diverse species conservation and diversity in the ultralong third heavy-chain complementarity-determining region of bovine antibodies universal protection against influenza infection by a multidomain antibody to influenza hemagglutinin coexpression of t and t on peripheral blood t cells demonstrated by two-color fluorescence flow cytometry phenotypic maturation of porcine nk-and t-cell subsets evaluation of t cell subsets in myasthenia gravis using anti-t cell monoclonal antibodies circulating cd + cd + cd + t lymphocytes in multiple sclerosis circulating cd +cd + t lymphocytes in patients with kawasaki disease cd + cd + double positive (dp) t cells in health and disease peripheral cd cd double positive t cells with a distinct helper cytokine profile are increased in rheumatoid arthritis porcine gammadelta t cells: possible roles on the innate and adaptive immune responses following virus infection the relative magnitude of transgene-specific adaptive immune responses induced by human and chimpanzee adenovirus vectors differs between laboratory animals and a target species differences in immune responses against leishmania induced by infection and by immunization with killed parasite antigen: implications for vaccine discovery type and type responses in regulation of ig isotype expression in cattle the receptor dec- expressed by dendritic cells and thymic epithelial cells is involved in antigen processing dec- , a -kda protein abundant on mouse dendritic cells and thymic epithelium that is detected by the monoclonal antibody nldc- : purification, characterization, and n-terminal amino acid sequence summary of workshop findings for cattle (tables and ) leukocyte antigens of cattle and sheep. monoclonal antibodies submitted to the second workshop studies of monoclonal antibodies identifying two novel bovine lymphocyte antigen differentiation clusters: workshop clusters (wc) and phenotypic variation and functional differences within dendritic cells isolated from afferent lymph dec- expression on migrating dendritic cells in afferent lymph the cells of lymph and their role in immunological reactions. handbuch der allgemeinen pathologie the properties and functional activity of non-lymphoid cells from bovine afferent (peripheral) lymph some properties of dendritic macrophages from peripheral lymph identification of a novel cell type in peripheral lymphoid organs of mice. morphology, i., quantitation, tissue distribution plasmacytoid dendritic cells migrate in afferent skin lymph existence of cd alpha-like dendritic cells with a conserved functional specialization and a common molecular signature in distant mammalian species the respiratory dc/macrophage network at steady-state and upon influenza infection in the swine biomedical model monocytes control gamma/delta t-cell responses by a secreted product bovine gamma/delta t-cell proliferation is associated with self-derived molecules constitutively expressed in vivo on mononuclear phagocytes protective killed leptospira borgpetersenii vaccine induces potent th immunity comprising responses by cd and gammadelta t lymphocytes the role of bovine γδ t cells and their wc co-receptor in response to bacterial pathogens and promoting vaccine efficacy: a model for cattle and humans the bovine model for elucidating the role of γδ t cells in controlling infectious diseases of importance to cattle and humans an alphabeta t-cell-independent immunoprotective response towards gut coccidia is supported by gammadelta cells γδ t cells play a protective role during infection with nippostrongylus brasiliensis by promoting goblet cell function in the small intestine bovine γδ t cells are a major regulatory t cell subset antigen-specific regulation of ige antibodies by non-antigen-specific γδ t cells a new effect of il- on human γδ t cells: promoting regulatory vδ t cells via il- production and inhibiting function of vδ t cells bovine γδ t cells: cells with multiple functions and important roles in immunity gammadelta t cells present antigen to cd + alphabeta t cells a sub-population of circulating porcine gammadelta t cells can act as professional antigen presenting cells mouse gammadelta t cells are capable of expressing mhc class ii molecules, and of functioning as antigen-presenting cells professional antigen-presentation function by human gammadelta t cells cross-presenting human gammadelta t cells induce robust cd + alphabeta t cell responses prolonged antigen survival and cytosolic export in cross-presenting human gammadelta t cells sheep cloned by nuclear transfer from a cultured cell line crispr is knocking on barn door genomic responses in mouse models poorly mimic human inflammatory diseases structural and functional annotation of the porcine immunome tuberculosis immunity: opportunities from studies with cattle animal models of tuberculosis for vaccine development animal models of respiratory syncytial virus infection influence of antibody-mediated immune suppression on clinical, viral, and immune responses to swine influenza infection live attenuated influenza virus vaccine reduces virus shedding of newborn piglets in the presence of maternal antibody. influenza other respir viruses universal vaccines and vaccine platforms to protect against influenza viruses in humans and agriculture the gnotobiotic piglet as a model for studies of disease pathogenesis and immunity to human rotaviruses the pig: a model for human infectious diseases de madrid at. c-type virus particles in pig kidney cell lines in vivo tracking and immunological properties of pulsed porcine monocyte-derived dendritic cells xeno's paradox: why pig cells are better for tissue transplants than human cells xenoislet rejection following pig-to-rat, pig-to-primate, and pig-to-man transplantation expanding the role of peyer's patches in b-cell ontogeny induction of immune responses in newborn lambs following enteric immunization with a human adenovirus vaccine vector an extra-uterine system to physiologically support the extreme premature lamb of mice and not men: differences between mouse and human immunology revisiting the b-cell compartment in mouse and humans: more than one b-cell subset exists in the marginal zone and beyond the lymphoid system: a review of species differences transcutaneous immunization of domestic animals: opportunities and challenges laboratory animal models to study foot-and-mouth disease: a review with emphasis on natural and vaccine-induced immunity antibody repertoire development in fetal and neonatal piglets. xiii hybrid vh genes and the preimmune repertoire revisited genomic organization of the immunoglobulin light chain gene loci in xenopus tropicalis: evolutionary implications therapeutic administration of broadly neutralizing fi antibody reveals lack of interaction between human igg and pig fc receptors model organisms: there's more to life than rats and flies all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. the authors were funded by the uk's bbsrc grants bbs/e/i/ and bbs/e/i/ . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © guzman and montoya. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - lp t q authors: salman, m. d. title: the role of veterinary epidemiology in combating infectious animal diseases on a global scale: the impact of training and outreach programs date: - - journal: preventive veterinary medicine doi: . /j.prevetmed. . . sha: doc_id: cord_uid: lp t q abstract the effectiveness of detection and control of highly contagious animal diseases is dependent on a solid understanding of their nature and implementation of scientifically sound methods by people who are well trained. the implementation of specific detection methods and tools requires training and application in natural as well as field conditions. the aim of this paper is to present the design and implementation of training in disease investigation and basic veterinary epidemiology in selected countries using the highly pathogenic avian influenza (hpai) h n asia strain as a disease detection model. indonesia, egypt, nigeria, turkey, and vietnam were each identified as either a priority country where ai was spreading rapidly or a country at risk for infection. in each of these countries, a training program on epidemiological concepts, field investigation methodology, and detection of h n asia strain cases was conducted. this report includes the impact of these training sessions on national animal health programs, including follow-up activities of animal health officers who went through these training sessions. during the last two decades, there have been several emerging and new health events in humans that have received heightened attention from society in general. most of these health events were linked to animal diseases or originated in animal products, including highly pathogenic avian influenza (h n ), bovine spongiform encephalopathy, west nile virus, and severe acute respiratory syndrome. due to the extensive involvement of animals and their products in these events, animal health authorities have been involved in measures to minimize the spread and impact of these diseases. however, it has been the public health sector, particularly within central government or international agencies, that led the effort to control or eradicate these diseases. nevertheless, the prevention effort requires major involvement of animal health officers and others in related industries since the roots of most of these diseases are found in animal populations, particularly food-producing animals and wildlife species. the the effectiveness of detection and control of highly contagious animal diseases is dependent on a solid understanding of their nature and implementation of scientifically sound methods by people who are well trained. the implementation of specific detection methods and tools requires training and application in natural as well as field conditions. the aim of this paper is to present the design and implementation of training in disease investigation and basic veterinary epidemiology in selected countries using the highly pathogenic avian influenza (hpai) h n asia strain as a disease detection model. indonesia, egypt, nigeria, turkey, and vietnam were each identified as either a priority country where ai was spreading rapidly or a country at risk for infection. in each of these countries, a training program on epidemiological concepts, field investigation methodology, and detection of h n asia strain cases was conducted. this report includes the impact of these training sessions on national animal health programs, including follow-up activities of animal health officers who went through these training sessions. ß elsevier b.v. all rights reserved. animal and plant life or health. the agreement advocates the use of international standards from the world organization for animal health (oie), codex alimentarius (cac) and the international plant protection organizations (ippc) as the basis for recommended standards. it has therefore become obvious that the health status of animals and their products plays a major role in import and export regulations. this type of requirement for trade has placed pressure on the animal health program both nationally and internationally. veterinary professionals throughout the world, mainly through their animal health services, are faced with having to fulfill a crucial role in protecting their country's animal health status, providing sound surveillance information on the occurrence of diseases within their territories, and conducting scientifically valid risk analyses to establish justified import requirements. the majority of these tasks and activities require sound epidemiological approaches. veterinary epidemiology as a discipline has therefore become the main focus of the scientific community for effectively combating infectious animal diseases nationally and internationally. this paper presents the value and role of veterinary epidemiology in combating infectious animal diseases on a global scale, emphasizing the importance of training and outreach programs. three main topics will be addressed: veterinary epidemiology and its relation to the general discipline of epidemiology. global aspects of infectious animal diseases and their impact on trade and public health. training in veterinary epidemiology and its impact on constraining the spread of infectious diseases. particular examples and demonstrations are also presented in this paper. in late s the discipline of veterinary epidemiology began to be recognized as an important contributing discipline for animal production and public health aspects of veterinary medicine (schwabe, ) . modern veterinary epidemiology was established in response to demands by veterinary and public health professionals to aid them in handling health events, including suboptimal production, within highly intensive livestock operations. in the s and s, quantitative veterinary epidemiology was attractive to intensive livestock producers, practitioners and, to a certain extent, central and local governments (schwabe et al., ) . the term ''herd health'' was used interchangeably with modern veterinary epidemiology. the tools and techniques were mainly borrowed and modified from several other disciplines such as clinical medicine, quantitative sociology, statistics, pathology, ecology, animal sciences, and microbiology, among others and the approaches were mainly applied at the individual herd or farm level. the term ''micro-veterinary epidemiology'' was used to reflect the unit of interest/study as individual animals (schwabe, ; dohoo et al., ; martin et al., ; meek, ; schwabe, ) . the dairy sector pioneered this effort and was later followed by the poultry, swine and, to a certain extent, small ruminant and equine sectors. epidemiologic techniques were also later adapted by animal shelters, laboratory animal colonies, and in the management of free-ranging wildlife species. as a consequence to opening trade and the signing of the gatt agreement, the world started to take a different shape, especially in the early s. animal health programs were in the spotlight since the primary issue that would facilitate or impede the trade of animals and their products was their effect on the safety and health of humans, animals, and plants. comprehensive surveillance, quantitative disease indices, and science-based risk analysis were a few of the new terms that emerged during this critical time. veterinary epidemiologists, animal scientists, and agricultural economists started to apply some of the herd health tools on a larger scale within regions, zones, or countries. the term ''macro-veterinary epidemiology'' emerged as an important discipline in shaping animal health strategies and policies in several parts of the world. this term is actually not new to scientists and policy makers since economists have used it when the unit of interest is country based. nevertheless, not all the tools and techniques used in micro-veterinary epidemiology can be applied directly to macro-veterinary epidemiology. modifications and adjustments to some of these tools are required and these modifications have evolved out of necessity. the discipline of veterinary epidemiology so far has not addressed methods and techniques to differentiate between these two categories of its function. a national public health program (nphp) oversees the well-being of the people/society in the country with particular emphasis on community health quality including issues of food security, social health, and environmental contamination. activities related to this type of national program therefore include detection, response, prevention, and treatment of specific diseases such as tuberculosis, salmonellosis, west nile virus, and others. a national animal health program (nahp) monitors the well-being of animal populations in the country with particular differentiation between those associated with food production, pleasure animals and, to a limited extent, with companion animals. activities related to this type of national program are almost the same as those associated with a nphp with the focus on targeted animal populations. in addition, there are several overlapping activities between nphp and nahp particularly when adverse health events involve animals or their products. in the usa, for example, it seems that the nahp generally responds to the aftermath of specific adverse public health events that are initially recognized by nphp. the response from animal health authorities during the e. coli o :h outbreak, for instance, occurred after several fatal human cases were reported. the role of animal contamination in the salmonellosis outbreak in california via sewage was not investigated until the media made the issue public. the investigators, however, identified properly the link to human sewage contamination as the source of salmonella bacteria in this outbreak (kinde et al., ) . in the majority of cases the nphp reacts to emerging issues brought to the public attention by the media. an exception to this is the new initiative through national surveillance in which health events are monitored and analyzed for their impact on the community. regardless, the nahp is almost considered to be a secondary resource for managing adverse public health events instead of working side by side with nphp. although the media should not be ignored, their role in identifying an emerging disease event should be evaluated carefully. historically one activity of the nahp in the usa has been in disease-specific programs that were initiated as a result of the public health impact of zoonotic diseases. in the usa, the nahp has been successful in reducing the impact of diseases such as bovine brucellosis, bovine tuberculosis, salmonellosis in poultry products, rabies, etc. nevertheless, the interaction with the nphp has been limited because of inadequate administrative connection or, as in the case of brucellosis, a lack of interest from the public health sector when there are few human cases because of the success of the eradication effort of this disease from the cattle population. therefore, there has been limited knowledge about or appreciation for the accomplishments or progress of the nahp. consequently, the majority of methods and approaches used by the nahp have not been recognized or valued by public health agencies. since its inception, the nphp has by nature been proactive and dynamic. society and public decision makers have paid more attention to the public health aspect especially in regard to community-based diseases. limited attention has been paid to positive impacts of nahp disease prevention activities on public health in usa. for instance, decision makers rarely acknowledge the value of consistently safe and high quality food of animal origin. the success of the nahp has also contributed to better and more efficient animal production systems regardless of the zoonotic implications. since most of the public attention is given to the nphp it receives the bulk of the resources, while nahp components receive little attention and few resources. furthermore the nahp has many mandates and also is woefully underfunded. a good example of the imbalance of resource allocation is evident in the current activities related to the global occurrence of highly pathogenic avian influenza (hpai). far more resources have been given to the detection and control of spread of hpai h n in human populations with relatively limited resources dedicated to the animal side, even though the spread of the infection can be prevented in human side if the focus is on the animal side. in a country such as the usa, there is the need for a parallel and sound relationship between nphp and nahp. an awareness system indicating the link between these two national systems must be shared with decision makers. this system would require animal health authorities and other interested parties such as the animal industries to have intensive and effective communications with decision makers and the public. the livestock industry, veterinary professionals, animal health authorities and other related sectors should attempt to build bridges with their public health counterparts by explaining the value of nahp for the well-being of society. the form of such collaboration requires comprehensive understanding of the role of each of these sectors in building a reliable, effective, and practical nahp. unfortunately, there is limited understanding of the role of each of these sectors with their counterparts. therefore, records of successful past and current accomplishments of nahp should be collected and presented to appropriate professional societies and authorities. the broad aspect of public health and preventive measures should also be emphasized. any focus on selected zoonotic diseases coupled with a speculative and less scientific approach should be avoided as much as possible. the discipline of veterinary epidemiology can be effective only if it is based on a solid understanding of its scientific principles and implemented by people who are well trained. understanding the concepts of veterinary epidemiology and its tools are mainly a result of research and education. the implementation of specific approaches and tools requires training and application in natural as well as field conditions. most of the graduate programs currently available require substantial on-campus residency periods which often make it difficult for public sector veterinarians to undertake them. therefore, it would seem appropriate to explore the possibility of establishing training programs which require short intensive periods with the focus on animal health events of contemporary importance that are of interest to the audience. distance education and working in the field with selected 'mentor' epidemiologists can enhance this type of training. this type of approach to training has several advantages for building a sound infrastructure for animal health programs. it allows opportunities for training in a way that more fully utilizes the talents of the veterinary epidemiologists already employed by the public sector. it will build a cadre of veterinary epidemiologists who can contribute a harmonized approach to national, regional, and global animal health programs. the training program will also be a major contributor in solving the most current emerging disease since it can deal directly with that disease. in addition, the field activities associated with this training program can be part of the regular public sector activities and thus serve ongoing needs. highly pathogenic avian influenza (hpai) h n asia strain is a growing problem in animal industries throughout the world, and there is the added threat to human health in the case of a human-adapted strain which might cause a pandemic. worldwide, countries are preparing and implementing response plans (efsa, ) . the u.s. government through the department of state, department of human health services, and department of agriculture has contributed to several initiatives and projects to combat this disease in both humans and animals. for example, usda-animal plant health inspection services-veterinary services has been engaged in the delivery of technical capacity to various countries. traditionally, aphis has supported the training of epidemiologists within the domestic service because it recognizes the value of these scientists in the development of strategies to combat diseases, to evaluate risk, and in many cases to form the basis for field veterinarians to detect, control and eradicate outbreaks of disease. to these same ends, aphis conducted a series of four regional epidemiology workshops in and for working-level epidemiologists of national veterinary services. these workshops were regionally convened: in bangkok for countries of asia; in vienna for the countries of eastern europe, the middle east and countries of the former soviet union; in cairo for the countries of north and east africa; and in dakar for the countries of west africa and southern africa. the criteria for selection of these countries were based on greatest need, greatest number of human cases, lack of veterinary infrastructure, and possibly additional criteria. indonesia, egypt, nigeria, turkey, and vietnam were identified as either a priority country, or a country at risk for infection where an opportunity existed to train epidemiologists. in each of these countries, a training program on epidemiological concepts, field investigation and detection of hpai cases was conducted. in addition, two sessions for selected participants were held in fort collins, co, usa with an emphasis on the national surveillance system and implementation of a comprehensive national plan for the detection of hpai, emergency planning, geographic information systems, and incident command system (ics) application to hpai control. the value and effectiveness of these training sessions have already been observed. animal health officers who have gone through these sessions have already been engaged in several of the following activities as a result of their training and exposure to alternative approaches: revision of a national surveillance plan to better reflect sound epidemiological approaches (fao-egypt plan). establishment of a more reliable case definition for investigating and reporting ai cases (fao report). establishment of a national professional network for the purpose of consolidating efforts to constrain the extent of ai (nigeria experience). change of the concept of biosecurity from the presence or absence to a continuum of efforts to prevent the introduction of the infection into premises or regions (indonesia experience). revitalization of epidemiological units as part of the government veterinary services (egypt experience). during the last two decades, the largest hurdle facing animal health has been the lack of resources available to combat several emerging and re-emerging infectious diseases. due to recent events, particularly those with public health implications, more resources than ever are currently being directed toward pressing animal heath challenges. the available funds, however, are mainly directed at specific high-profile infectious diseases instead of animal diseases in general. nevertheless, these resources provide an excellent opportunity to improve the infrastructure of organizations involved in national and global animal health programs. the emergence of diseases that receive the attention of the public and of policy makers requires technically reliable disease investigation and case findings. there also are requirements for a science-based approach to trade and assessment of risk (i.e., no longer zero risk). furthermore, international financial institutions have more involvement in shaping government veterinary services and have several requirements to justify plans of action. veterinary epidemiology has been a major discipline in supporting and improving national as well as global animal health programs. this discipline, however, should not be considered as an extension of the human public health sector or human epidemiology. veterinary epidemiology is unique in its approaches and anticipated issues to be solved. the discipline's contribution to reducing the impact of global infectious animal diseases is significant but has had limited recognition. veterinary epidemiologists are faced with several challenging questions in determining their role at the macro-level of animal health programs. answering these questions requires a detailed assessment of approaches for designing scientifically sound national animal health programs that include surveillance, prevention strategies, and response plans. the planning of training and outreach sessions is an essential component for the promotion and implementation of sound animal health programs. veterinary epidemiology is currently in its golden age in terms of its growth and encouragement of scientific approaches. the use of multilevel models to evaluate sources of variation in reproductive performance in cattle animal health and welfare aspects of avian influenza and the risk of its introduction into the european union poultry holdings-scientific opinion of the panel on animal health and welfare of the european food safety authority sewage effluent: likely source of salmonella entritidis phage type infection in a commercial chicken layer flock in southern california veterinary epidemiology: principles and methods veterinary epidemiology: challenges and opportunities in research epidemiology in veterinary practice the current epidemiological revolution in veterinary medicine. part i the current epidemiological revolution in veterinary medicine. part ii dr. m.d. salman does not have any conflict of interest. key: cord- -jy a al authors: von essen, erica; lindsjö, johan; berg, charlotte title: instagranimal: animal welfare and animal ethics challenges of animal-based tourism date: - - journal: animals (basel) doi: . /ani sha: doc_id: cord_uid: jy a al simple summary: animals of countless species, wild as well as tame, can now entertain tourists on their holidays. the popularity, however, of animal-based tourism comes with significant risks for the welfare of these animals. many animals are kept in small confinements, are broken down to interact obediently with tourists, or are made to perform, entertain, transport or even give their lives for human leisure. in this paper, the challenges of animal-based tourism are presented from the perspectives of interdisciplinary researchers. the challenges are discussed based on a two-day symposium with workshop sessions. we bring attention to the problem of cultural relativism and the difficulty of imposing universal standards of animal welfare. we conclude that reforms and individual travel decisions as a result of biosecurity concerns will impact animal welfare. in addition to this, we observe that technology has a dual role to play in enhancing edutainment but also potentially inviting new challenges. in the end, we declare some possibilities for compassionate animal based tourism. abstract: by animal-based tourism, a host of activities offering passive viewing or active interaction with wild, semi-wild or captive animals is included. the multibillion dollar industry is on the rise globally today, offering modes of engagement with animals that trade on increasingly embodied close encounters with non-human animals. as new modes of animal-based tourism proliferate, such as sloth selfies, visiting cat cafes, swimming with sharks and agri-tourism petting zoos, animal welfare standards risk deteriorating. in the following paper, we collate concerns over animal welfare into a discussion on the challenges facing animal-based tourism. our synthesis is the first to consider the full spectrum of such animal-based tourism: across agri-, hunting, zoo and safari tourism, to name a few, and crossing consumptive and non-consumptive boundaries. a literature review is first provided. findings are then presented thematically following workshops at an international interdisciplinary symposium of leading tourism, animal welfare, ethics and leisure sciences scholars together with practitioners of the industry. it discusses macrolevel drivers to animal-based tourism as an industry, the problem of cultural relativism and the role of technology in enhancing or promoting the experience. we indicate ways forward toward implementing a compassionate animal-based tourism. taking selfies with animals and uploading them to instagram may be considered tacky to some but is part of a growing suite of activities in late modernity that sell embodied encounters with animals [ , ] . and indulgence which should not be judged by the ethical criteria deployed in daily life" [ ] . nevertheless, it also appears that some tourists are increasingly discerning consumers, whose choices and preferences on holiday signal identity. this means that changes in visitor tastes may be used to improve animal welfare standards in the future [ ] . certainly, there is appeal for many western tourists of attaching oneself to practices with epithets such as "sustainable", "eco" and "ethical" tourism [ ] . nevertheless, there is cause also to be wary of eco-labeling and green-washing in the industry, and even words like animal "sanctuaries" [ ] as these, too, function as neutralising cognitive dissonance. in light of such profound challenges to animal lives and welfare, what is the future of animal-based tourism? can we expect to see a diversification in the sorts of animals commoditised, the destinations offered and array of interactions with animals available? parallel to this and as a result of a few notable scandals (such as cecil the lion, blackfish, and exposures of phajaan, breaking of the will of elephants to get them docile in thailand), can we also expect increased scrutiny of the industry in terms of its treatment of animal workers? a doctrine of cultural relativism prevents legislation from managing all animal-based recreational practices too uniformly, but consumers themselves may, as indicated, become more selective across practices that receive negative reviews or public naming and shaming. the landscape for animal-based tourism is shifting unpredictably, however, in light of covid- of , which saw a simultaneous drop in tolerance for practices involving interactions with wild animals and an increase in media stories on how animals in captivity were coping without visitors everyday-sometimes poorly, prompting concerns they were lonely and liked people to visit them. the lack of tourists due to the covid- pandemic has had two other contradictory impacts; on the one hand, animals seem to benefit from abandoned natural areas, e.g., national parks and beaches, where they increase their presence, prompting the atlantic to call the coronavirus "the biggest conservation action" of this time [ ] . on the other hand, fewer tourists mean fewer resources and less incentive to protect wild animals and biological diversity, which may come with its own risks. in namibia, the world travel and tourism council estimates a loss of us $ . million in annual tourism revenue following covid- , an additional us $ . million loss of staff salaries, and increased poaching by locals who have lost their livelihoods in tourism businesses [ ] . setting out to answer some of the most burning questions on the future of animal-based tourism, we arranged a two-day interdisciplinary symposium at the swedish university of agricultural sciences in august , titled: instragranimal: animal welfare and ethical challenges of animal-based tourism. the following report is a synthesis of its discussions and next steps in research, policy and practice. the symposium featured some fifty participants across disciplines and sectors, involving veterinarians, ethologists, ecologists, animal and environmental ethicists, philosophers, sociologists and tourism scholars on the one hand, and on the other hand practitioners from animal-tourism ventures and animal welfare and animal rights non-governmental organisations (ngos). the spread of invited presenters was global, calling for researchers at the forefront of tourism studies in leading hubs in australia and new zealand, as well as ethologists based at the swedish university of agricultural sciences. the symposium marked the gathering of these people for the first time, and also the first time that animal-based tourism was approached holistically and not divided across, e.g., prior consumptive/non-consumptive, captive/wild axes, geographical regions or segregated across industries such as hunting, agri-or ecotourism. the rationale for this was to identify common challenges across these dimensions. in what follows, we synthesise the discussions that were held during this symposium into four themes. the discussion was open to the public on the first day. on day two, it was organised in closed workshops for registered participants. these sessions sought to collate the topics and reflections of the two days into action points or themes for future research. the themes were chosen together with participants in plenary. they build on the ideas presented in the above introduction, discussing new arenas for animal tourism, societal drivers behind the phenomenon and compassionate animal tourism. at the end of each of these four themes, we present a short section containing next steps on three levels: directives to legislation and policy, guidelines to tourists and calls for further research. the themes reflect the main aims of the symposium, which were to look toward the future as to: . identify challenges in animal welfare and animal ethics in tourism; . identify new animal tourism developments conceptually or empirically; . explore and suggest needed regulative responses on the part of governments, international bodies or pressure from animal protection and rights ngos and consumers of tourism, to secure the development or enforcement of welfare standards; . to develop calls for future research on animal-based tourism, both intradisciplinary and across disciplines. the selection of invited presenters at the symposium started with erica von essen and johan lindsjö conducting a literature review and survey of tourism research centers globally that showcased prominent researchers writing about animal-based tourism, animal welfare and ethics. a list of fifteen researchers was generated at first stage and presented as part of the application for the grant supporting the symposium. these researchers were then reached out to and personally invited by email. a desire was to span the three contexts of animal tourism: ecotourism, hunting tourism and agritourism. media were also present during the two days and followed up on the findings afterwards, in radio, tv and a hunting journal. in the following section, we present four themes of animal-based tourism that merit more discussion, research and/or legislation. one working group at the symposium outlined the societal drivers and global processes that promote animal-based tourism on the one hand, and on the other hand the types of drivers and processes that may mediate people's tolerance to animal suffering in these settings. here, world events, paradigms and value shifts were discussed as to their impact on the industry going forward. a key premise in this theme may be said to be the alleviation of responsibility from the individual tourist or tourism operator, to consider macrolevel drivers responsible for the industry's appearance today. what will be the long-term impact of the so-called flight shame movement on animal-based tourism abroad? it has been speculated that a decline in long-distance travel, exacerbated also by covid- in , may give rise to new local forms of tourism with animals. on this argument, the proximate and the everyday in the animal context may be exoticised and commoditised in, for example, staycations and day trips. this may partly account for the popularity of local agri-tourism, where nearby farms are visited [ ] . these sites offer reconnecting also with local economies and pastoral culture-something that no doubt would have seemed absurd only a few decades ago. in sweden, "cow releases" are the new agri-tourism happenings that bring families and urban residents to see local farms put their cows out to pasture for the summer grazing period, freeing them from the confinement of the winter barn the pastiche of rural life is at once two forms of "liberation": a physical one for the cows to be enjoyed as spectacle, in terms of relieving the cows from indoor confinement and giving them the opportunity to realise themselves as bovines in the grassy field; and a spiritual one for urban visitors, who may experience the temporary alleviation of alienation from the modes of production of their dairy that they consume daily. however, the turn to local tourism need not necessarily be in the service of a rural renaissance. some suggest that given the urban is becoming a significant haven for wildlife, intuitively following greater urbanisation [ ] and its concentration of human-animal interaction [ ] . today, several animal tourism activities take place within the city: cat cafes and zoos, tours of the city, animal walks, but also less organised experiences such as spotting urban wildlife: pigeons in venice, rhesus monkeys in indian cities and wild boars in berlin. while there has traditionally been a strong focus on experiencing pristine nature, in particular in so-called marketed wilderness tourism and last-chance tourism, which includes endangered species, the future may have to adapt to a "messier" nature that is characterised by multispecies interfaces, including a strong human presence [ ] . insofar as the city may be a location for such tourism, of course, there are biosecurity concerns regarding the conditioning of wildlife to feeding, trespassing human-inhabited areas, affecting sanitation and increasing zoonotic disease risk [ ] . a future-oriented research agenda on animal-based tourism needs to consider the flows of people, the push and pull factors present in various parts of the world at any given time and may even need to attend to the long-term changes in tourism circulation, such as climate change opening up routes in the arctic following the melting of ice caps. the increase of invasive species, posing a threat to pristine tourism based on native flagship species, may need to be problematised and reconsidered as a potential tourism revenue. we recognize that tourist routes may be shifted with climate change and geopolitical processes, but tourist preferences may shift independently of the physical, in longer term value shifts. as we enter a post-industrial society, there may be less emphasis on the accumulation of wealth (as has characterised agricultural societies) and more on experiences and how they contribute to a person's identity. given this, one might ask whether travel may increase, but travel-associated accumulation (such as physical souvenirs) may decrease. as tourism becomes a ritual context for showing identity-based goods, moreover, animal tourism experiences may be valuable to establishing a person's status [ , ] . within this, last-chance tourism, danger tourism and slum tourism in relation to interacting with animals may feasibly be on the rise among some tourists [ , , ] . although our symposium took place before the outbreak of covid- , this is a world event which will likely have profound impacts on human mobility more broadly, and where and how tourists choose to engage with animals on holiday. biosecuritisation of borders will mean a changed travel landscape. the pandemic helped to shed a light on animal malpractices in parts of the world and generally opened the public's eyes about the risks of human-wildlife interfaces and the dangers of keeping several animal species in confined or shared spaces for human consumption or leisure. on a fundamental level, the spatial concentration of animals into confined areas for tourism consumption involves heightened biosecurity risks [ ] . since many enterprises in animal tourism, including zoos, keep animals in these ways, change may be afoot. this may extend also to the practices of tourists, who may be compelled to adopt more clinical, hygienic ways of interacting with animals and animal-derived products, involving the washing of hands, wearing of gloves or masks, quarantining in time and space following certain encounters and handling animals or products of animals with greater care or sanitation (such as the meat from hunting trips). research should begin to apprehend how the idea of increased biosecurity risk is impacting animal tourism, not merely from a legislative point of view, but how such risks add to, detract from or otherwise impart changes in clients' experience of the tourism activities. recognising that part of the drivers for animal-based tourism is in self-fulfillment and escape of "inauthentic" lives [ ] , a discussion also needs to be held on the various extreme forms that help to realise such self-fulfillment. within this, gender and masculinities play a role in enacting animal tourisms. indeed, animal tourism may be an arena in which ideas of gender can be played out and negotiated, given that touristic settings are a liminal space partly freed from everyday constraints [ ] . moreover, that animal interactions can inform one's gender identity seems apparent with an entertainment industry that readily commodifies macho, primeval and atavistic encounters with wild animals, where nature is marketed as a kind of antidote to the feminising influence of modern city life [ ] . the popularity of survival shows featuring wilderness rangers, bear grylls and survival and self-sufficiency guides appears to testify to a masculine domain of taming the wild. hunting packages such as the manly-titled scottish, and alaska: rampage, invite a particular clientele (see, for example, the macho "shoot-outs". often, the animals involved in these trips are dangerous-predators or game that fight back [ ] . oppositely, contexts involving care and nursing relations with animals on holiday, including bottle-feeding baby animals or volunteering at shelters, may be both a female domain and a context in which alternative notions of masculinity can be played out. bertella [ ] suggests that direct experience of wild animals may contribute to emotional and cognitive capacities that support caring attitudes, but there is a possibility these engagements may also be pastiches-exaggerated or contrived. below we present a simplified table that addresses next steps for this theme. table . summary of workshop conclusions on legislation and policy, guidelines to tourists and calls for further research on the impact of broader societal structures on animal-based tourism. develop, review and ensure implementation of animal welfare legislation and "best practice"-guidelines in animal-based tourism, nationally and internationally. go local and explore animal-friendly and ethically justifiable animal tourism venues at home before flying across the world. be a responsible tourist-inform yourself, contact travel retailers and tour operators, demand animal-friendly and ethically justifiable approaches to animals in tourism. society s view of animals' roles in animal-based tourism-how do the perceptions, values and attitudes of tourists correspond to those of tourism operators and animal welfare organizations? possibilities to stimulate local, animal-welfare-friendly and ethically justifiable animal-based tourism. how gender is performed, contested and negotiated in animal encounters in animal-based tourism. local customs and universal animal welfare standards sometimes clash. in many cases, the animal tourism industry is a significant source of income livelihood for local communities. this means that when external pressures are put on destination communities to restrict or prohibit traditional animal uses, we are presented with ethical dilemmas between human and cultural sustainability and animal welfare. oh and jackson [ ] write that these instances present a dilemma between two both dominant cultural scripts in late modernity: on the one hand, one pertaining to multiculturalism, individual choice and cultural rights, and on the other hand, one pertaining to animal rights. both, in effect, carry weight today and represent directions for policy and legislation. the latter script pertaining to animal welfare is frequently accused of cultural imperialism or ethnocentrism [ ] , as cultural rights and freedom of choice to animal leisure and cuisines may be a point of pride for many nationals. one can also see the dilemma as between two other cultural scripts: one of cultural globalisation and commercial homogenisation [ ] and one of cultural protectionism, respectively. in a world where we try to simultaneously adhere to these opposing scripts, increased market and legislative pressure is placed upon parts of the world such as china where animal tourism practices are generally more permissive [ ] . however, accommodating changes toward greater welfare is sometimes seen by critics as caving to international pressures [ , ] . oppositely, scholars observe how the presumption "in favour of right to culture" [ ] that endorses the multiculturalism script now presents " . . . a real danger of exoticising and marginalising immigrant minorities, placing them outside of the circle of moral dialogue, criticism and community" (p. ). a related human rights concern that may challenge animal welfare is the increased catering to persons with disabilities. for the most part, this poses no additional problems for animals, but when animal treks are made to transport obese persons across long distances, animal welfare problems may arise. such abuse has been documented in the case of donkey rides in seaside tourism in several parts of europe, notably greece and portugal [ ] . the dilemma raises questions about the expediency of hard versus soft regulatory mechanisms to implement animal welfare regulations globally, relating to both governmental legislation and voluntary standards [ ] . a neoliberalisation of government policy may exacerbate the establishment of regulation. in these cases, should we trust to market and, e.g., corporate social responsibility to push through improved standards of animal welfare in tourism? where market demand is too slow in bringing about change, additional questions are raised as to who exactly will regulate, who will inspect and whom this will affect (travelers, countries, operators, etc.). destination marketing organisations (dmos) and ngos may be able to operationalise locally universal codes of conduct. this "aspirational" universal code must be somewhat flexible to allow for local context. lovelock and lovelock [ ] write that this is in line with the multifunctionality of codes of conduct, which are to educate, aspire and regulate. moreover, this aspirational code needs to be dynamic also across time, allowing for constant evolution as conditions and priorities change. hultsman [ ] points to a needed separation between a paradigmatic (aspirational) code of ethics and an operational code of ethics. further, without either of these codes worked out in connection with local communities with, e.g., ngos and dmos, such regulation is likely to do more harm than good and be seen as imperialist. the above presuppose a dilemma between animal welfare and human sustainability, but ways forward may include a stronger emphasis on the interrelation of human and animal welfare, as in the "one health", "one welfare" concept [ ] . otherwise stated, we need to appreciate that animal welfare and human rights are not diametrically opposed. addressing animal welfare within the sustainable development goals [ ] specifically may be a potential way forward in acknowledging the intersectionality of human and non-human oppressive conditions in tourism. indeed, pitting these against one another is likely to undermine not only the welfare of both, but human-animal relations, as protected animals often become the subject of resentment among locals. following the killing of cecil the lion and the uproar of the western world, some zimbabweans expressed concern that people in the west cared more about lions than the predicament of poor zimbabweans. it is also important to note that western culture does not have a monopoly on animal welfare and rights, or compassionate animal practices. hence, attempts to implement a typically western model for other countries may be counterproductive insofar as cultures may come with their own existing and historically grounded repertoires of animal ethics, notions of stewardship and benevolence [ ] . following this, codes of conduct pertaining to animals would do well not only to list prohibitions, but to emphasise cultural virtues in dealing with animals. it may then be more effective to build on these than to implement top-down directives. it must also be recognised that there are substantial differences in moral values and animal practices also within societies [ ] . as covid- intimated, many chinese may already be opposed to wildlife markets [ ] . below in table , we present an overview of next steps for research and practice in regard to the cultural relativism challenge meeting animal-based tourism. table . summary of workshop conclusions on legislation and policy, guidelines to tourists and calls for further research on cultural relativism. develop, review and ensure implementation of animal welfare legislation and "best practice" guidelines in animal-based tourism among travel retailers, tour operators and animal users, emphasising the benefits from a sustainability and human perspective as well. develop, review and implement legislation and guidelines about information, certification and labeling, implementation of codes of conduct, also including benefits from a sustainability and human standpoint. be a responsible tourist-inform yourself, contact travel retailers and tour operators, demand animal-friendly and ethically justifiable approaches (compassion-do no harm) to animals, humans and the environment in tourism (one welfare). push for certification, labeling and information before and during traveling, a code of conduct, based on one welfare. attitudes and compliance of certification, labeling and information about animal-based tourism-potential cultural differences. the roles and responsibilities of humans in animal-based tourism. impact on un sustainability goals from animal-based tourism-and its interconnection. animal-based tourism from a one welfare perspective. technology can powerfully mediate distance and interactions with animals, changing the way we see and think about animals [ ] . it can bring us into heretofore unmatched proximity; new techniques such as drone-based thermal images and camera trapping invite us squarely into the everyday lives of wild animals in dens and burrows [ ] . no doubt, the success of bbc's planet earth series owes much to the advances in technology that enable us to learn more and get closer and into areas or animal behavior previously hidden from view. for this reason, it should be asked to what extent a fully or semi-virtual animal-based tourism may be on the rise today. we may also ask the extent to which this may replace or complement "real" encounters, thus taking some pressure and stress off the animals in their habitats, enclosed or wild. in times where crowding and overtourism is a real concern [ ], not just for the "victims" of tourism (animals, locals, cultural heritage and property) but also as something that denigrates the tourism experience also for the clients, more remote viewing appears promising. indeed, the use of binoculars to improve views, trail and surveillance cameras, sometimes even mounted in the nests and dens of animals, allow intimacy without getting civilians physically close to animals. the use of drones capturing footage, which can now come extremely close to many wild animals without or at least cause less disruption to their behavior, may hence allow for remote viewing close-ups. to this end, this provides only visual satisfaction for tourists, and the thrust of the embodied turn in animal-based tourism is that of advocating for a multisensory engagement that transcends mere passive viewing, involving tactile and auditory senses [ , , ] . one striking development that has effected human-wildlife relations is the democratisation of the access and scope of technology used to document animal encounters. no longer the purview of professional photographers, amateurs can capture footage of wild animals with their smartphones, camera traps and take part in apps that tell them where animals are. such technology has formed the basis for several citizen science monitoring programs [ ] . mobile phone technology can thus bring power down to the individual level, permitting decidedly personal encounters memorialised in custom photos. taken to its extreme, the technology could also be brought down to the animal level where animal-mounted go-pro cameras show animal points of view and agency. the ability of ordinary people to autonomously zoom in on cameras affords a potentially personalised, intimate view of animal lives that is not captured in edited documentaries that often focus on grand spectacle. moreover, future research needs to consider the use of technology from the animal side, in terms of using apps and programs to communicate their needs to us, or games on, e.g., ipads for stimulation in enclosures. recently, for example, virtual reality goggles fitted for cows were devised to stimulate green pastures for the cows when in reality, their habitats were confined indoors. could this technology be used to contribute to animal welfare goals in animal-based tourism or would it perhaps make it worse? technology also provides a divisive subject in mediating the human-animal experience in animal-based tourism. discussing "cheater technology" [ ] , critics have argued that too sophisticated equipment detracts from the animal encounter. hence, an app that lures birds to a site may be frowned upon by dedicated birders; heli-hunting is seen as "not real hunting" by hunting tourists, and the addition of technological "comforts" in nature-based trips, such as ipads and microwave ovens, may be criticised by tourists who value authenticity. use of and acceptance of technology will vary across animal-based activities, across demographics and between different types of technology. as contended, technology used in the service of promoting the virtues and authenticity of the animal experience may be permitted, while ones that do away with these virtues may be criticised and prohibited or phased out. this coheres with scholars' assertion that technology is neither good nor bad, instead showing an ambivalent face, being "empowering and hindering at the same time" [ ] . just as technology may alter or distort a tourism experience from some ideal type, technology may also serve to reproduce certain representations of animals as caricatures and facilitate the continued consumption of these animals in particular ways. burt [ ] writes that technology and visuals do not merely reflect but constitute animal ethics. the way we film particular species, and what we leave out for viewers, serve to shape our notions of animals. hansen et al. [ ] suggest that the public's vocabulary for communicating about the environment is predominantly visual. technology has thus meant greater scope for manipulating animals into televisual commodities " . . . packaged for the purposes of eliciting donations, membership monies, and repeat visits" which is reflected in our treatment and legislation of them. social media are an intuitive context and platform for both tourism advertising and generating expectations on animal encounters and for clients potentially disseminating critical reviews and exercising moral reflection [ ] . in a time of perpetual documentation of our experiences on holiday and our life achievements generally [ ] , animal tourism experiences are lived out again on social media and reviewed on travel and booking platforms-making animal tourism also a digitally reproduced endeavor. here, the influence of "intermediaries" between tourists and the industries, including expedia and tripadvisor, play a critical role. influencers on instagram showing close contrived encounters with wild animals, including #slothselfies and cuddling with tiger cubs at thai "sanctuaries", are now recognised as an increasingly harmful driver to animal tourism, insofar as it mischaracterises animal interactions. concepts such as "social envy", stemming from viewing others' holiday experiences online, and e-lineation, describing a myopic or outright harmful representation of the real thing, are now explored in relation to animal tourism [ ] . instagram has now taken action to delete images associated with certain hashtags associated with poor animal welfare, or advancing alerts for them [ ] . lastly, technology provides opportunities for edutainment in animal tourism: clients learning about the animals, their biology and ecology, not only through footage captured by sophisticated camera technologies, but interactively on digital platforms. they can also feature a kind of learning with animals, as through the anthropomorphising of particular animal into pedagogical figures for edutainment. many zoos today feature interactive quizzes and ways to educate and entertain visitors. indeed, verma, van der wal and fischer [ ] discuss whether technology may be part of an important bridging communication tool between policy-makers, the media, conservation practitioners and the general public. cloke and perkins [ ] suggest cetacean encounters in dolphin tourism, both to entertain and to educate, heavily rely on an assemblage of technology: sonar, telecommunications, radar, spotter planes and more, which may be obtrusive to the dolphins. here, a recurring challenge may be achieving a balance between presenting animal lives through "simulated spectacle" and the "objectivity of science" [ ] , balancing the public's investment from emotion and cognition, respectively. digital learning is not necessarily without its risks. within the context of many action-based tourism activities such as hunting and fishing, one now no longer learns skills from family mentors to the same extent as in the past, relying instead increasingly or at least in large part on influencers and guides on social media platforms such as youtube, for good and bad. as contended earlier, moreover, technology also allows for enhanced manipulation through editing and effects. grazian [ ] notes that animal tourism educators still have significant power in selecting the parts to be displayed and often mute key aspects of animal lives to suit the particular audience, something which they can easily do with technology. insofar as the representation of animals and the gaze with which they are represented can powerfully constitute ethics and human-animal relationships, care needs to be taken to not misrepresent reality. below in table , an overview is provided in a table format on suggested next steps and topics for a research agenda on the role of technology in animal-based tourism. we also outline ways forward for policy and practice. table . summary of workshop conclusions on legislation and policy, guidelines to tourists and calls for further research on the role of digital technology in animal-based tourism. outreach and education about animal welfare and ethical challenges, resulting in guidelines for web-based platforms and influencers. certification and labeling on internet-based platforms (websites, social media) informing about and promoting animal-based tourism activities. implement codes of conduct. promote development and implementation of virtual animal-based tourism (see rs in section . . ). develop and implement legislation/guidelines about using animals first when technology cannot replace use of real animals. require that web-based platforms and influencers consider the animals' situation and ethics surrounding animal use, demand that they take a standpoint (a condition for your attention, you following them, etc.). require tour operators to consider and implement technical development replacing, reducing and refining animal use. the impact of web-based platforms, including influencers, on animal-based tourism and how they can promote animal-friendly and ethically justifiable tourism. a moral dilemma that meets many forms of animal-based tourism is that the lives of individual animals are essentially "sacrificed" to benefit the species. this recurs across several tourism sectors: in hunting tourism perhaps most palpably, trophy hunting has been promoted on the basis of securing biodiversity conservation [ ] . it is now a prevalent slogan across hunting tourism campaigns to "be the savior" of a species by killing its individual specimens: the "kill it to save it" narrative [ ] , in which the revenues from hunting tourism are said to ensure the survival of endangered species. according to many hunters, therefore, the benefits outweigh the potential harms in canned hunting [ ] . although here animals individually pay the ultimate price for the survival of their wild cousins, other forms of animal tourism showcase a similar logic in which the welfare of the individual animal may suffer so that this particular individual can serve as a flagship animal for its species [ ] . thus, zoo tourism is now promoted as a necessary evil in which the lives of charismatic megafauna in particular are devoted to ambassadorships for the greater good of their species [ ] . the thrust of this dilemma is a trade-off between individual welfare of sentient beings and species level welfare, often conceptualised as a conflict between the sentientistic and ecocentric. the difference in moral patients across these two points of departure underpins a range of conservation conflicts today. animal welfarists accuse ecological managers of allowing the sacrifice or suffering of individual animals to save the whole. this is especially so when there is epistemic uncertainty about the consequences of our interventions on the ecosystem. managers, on the other hand, express frustration with what they see as the short-sightedness of the welfare perspective [ ] . the ecocentric, holistic perspective runs into objections on the difficulty of speaking on behalf of the good of a whole species or ecosystem, in which seemingly grandiose claims can be made that sanction the killing of individual members [ ] . nonetheless, the sentiensistic perspective comes with obvious limitations in prescribing no particular moral obligation to endangered species or the preservation of wild habitats for the sake of species conservation alone (only insofar as its members are happy): a cow has the same right to life, and living a good life, as a white rhino. moreover, if one white rhino were to be used for spreading knowledge and awareness about the plight of his species, by being placed in a life of captivity, this cannot be condoned on the sentientistic deontological rationale. given the ostensible incompatibility of these two points of departure, which one ought we to prioritise when there is a conflict? in animal-based tourism, which level of being should be ascribed moral priority? frustrated with the trade-off, a growing body of scholars have embraced so-called compassionate conservation, which promotes the consideration of animal welfare in conservation, benefitting both individuals, species and conservation outcomes compassionate conservation [ ] is debated, mainly because of the inherent conflict between the cost for (welfare of) the individual animal and the greater good for a population or species (i.e., is it possible or not to apply compassion in successful conservation activities). burns [ ] extracted valuable lessons from an approach that embodies compassionate conservation in wildlife tourism. we further extend the concept of compassionate conservation to compassionate animal-based tourism, i.e., the welfare of the individual animal within all kinds of tourism; eco-, hunting and agritourism. hence, we argue that individual animal welfare is compatible with responsible tourism. to this end, two approaches may be used to practice compassionate animal-based tourism: first, the rs (replacement of animals with alternative methods, reduction of the number of used animals and refinement of the methods, including housing and care, to mitigate suffering and promote animal welfare), initially developed to improve animal welfare for animals used in research [ ] , can be applied in other areas as well, including animal-based tourism. the initial question is: is there a need to use animals at all? is the interest for the tourism (and thus society) bigger than the cost of the individual animal? from a compassionate perspective, are there any activities where the use of animals can never be accepted? animals can be replaced with virtual reality or completely replaced by tourist activities without any animal theme. if animals are involved, how many animals need to be involved in a given activity? in line with reduction, zoos, amusement parks, elephant and horse riding camps and farms may exhibit fewer species or fewer individual animals within a species, fewer species or individuals have to be affected by safari or trophy hunting activities, catch and release fishing, etc. refining the treatment of animals used in tourism will ensure a good welfare and compassion for these animals. housing (captive animals), exposure, care and handling that enable natural behavior, health and positive feelings will not only benefit the individual animal, but also groups, populations and species, especially if these are small or otherwise vulnerable. compassionate animal-based tourism relies on animal protection, i.e., what we do, or ought to do, to provide a good animal welfare through legislation, but also education, policy making and, importantly, information that reaches out to tourists, industry and decision makers. the second approach emphasises the need of information to the tourists. certification and labeling of products and services, including the introduction of codes of practice, are well-known strategies to inform consumers. we believe that this is one strategy to help tourists to make animal-welfare-friendly and compassionate choices when traveling. to achieve credibility in society, this needs to be based on scientific knowledge and collaboration between ngos, academia, industry and local people involved in, or otherwise affected by, tourism. another way to reach out to tourists is to provide information about animal-based tourism at travel hubs, i.e., airports, ferry terminals, train and bus stations, car rentals, etc. to ensure effective communication, pr strategists and experts in advertising should be involved. the benefits for animals, humans and the environment from a responsible tourism need to be communicated. the final table summarises next steps for a compassionate-based animal tourism. table . summary of workshop conclusions on legislation and policy, guidelines to tourists and calls for further research on compassionate animal-based tourism. policy: develop, review and ensure implementation of animal welfare legislation and "best practice" guidelines (based on research on animal health, physiology, behavior, emotions and natural living) among travel retailers, tour operators and animal users. ban non-acceptable animal activities in tourism. develop, review and implement legislation and guidelines about information, certification, labeling and introduction of codes of conduct. be a responsible tourist-inform yourself, contact travel retailers and tour operators, demand animal-friendly and ethically justifiable approaches (compassion-do no harm) to animals in tourism. push for certification, labeling and information before and during traveling. require tour operators to include a r approach, replacing, reducing and refining animal use. attitudes and compliance of certification, labeling of and information about animal-based tourism-potential differences between activities, species and demography. health, physiology, behavior, emotions and natural living with regard to different species and if and how they are suitable in animal-based tourism. knowledge, attitudes, identification and implementation of compassion and the rs in animal-based industry. we began by highlighting current debates in the field of animal-based tourism as this industry is gaining in popularity and scope. animal welfare challenges were identified and traced to cognitive dissonance among tourists and to structural barriers of the industry. we noted that animals become laborers in a global capitalist economy when they are conscripted into the service of the tourism industry. the form of labor that they provide varies. in the above sections, animals featured in various roles, including: • a kind of spiritual commodity [ ] , including serving as totem animals; • a mode of transportation, such as donkey treks or dog-sledding in the arctic; • a laborer in the background to a more center-stage tourism activity [ ] , such as how research has found that having wolves in an area where hunting tourists hunt herbivores adds to the overall experience [ ] , or goats in the background to a farmers' market; • a culinary delight [ ] , such as alligator meat in louisiana [ ] ; • a front-stage performer, such as animals doing tricks in circuses [ ] ; • a marker of place, such as the kangaroo or koala for australia [ ] ; • a "facilitator" of leisure [ ] , such as an animal trained to serve drinks; • the face of a souvenir or toy [ , ] ; • the ultimate sacrifice as a game animal to be hunted as part of the big five. our symposium, devoted to identifying the main challenges for this industry from an ethical and welfare perspective, generated four overlapping themes worthy of further exploration: macro processes as drivers to animal-based tourism, cultural relativism as a potential challenge to implementing universal animal welfare standards, the role of technology in enhancing, promoting or even replacing animal-based tourism, and the potential for a compassionate animal-based tourism that could reconcile animals' well-being with tourists' interest. these are themes that point, above all, to a complex and changing landscape for animal-based leisure, with an uncertain future. a shift in public perception toward either greater reverence for animal welfare and condemnation of unethical practices, or increased concern toward host destinations and locals needing to make a living in an uncertain future, will make or break some animal-based tourisms. likewise, based on present trends and values, we suggested that global travel patterns and pandemics will significantly impact the future of certain tourism activities. in addition to the above cross-cutting themes, we noted also tension between needing to balance the educational and entertainment functions of animal-based tourism, respectively. "edutainment" seeks to provide both, but there are inherent challenges to finding an ethical, workable and profitable balance. in this regard, technology may aid in bringing us closer into animal lives and facts without becoming unduly obtrusive. this is in line with the rs (replacement, reduction and refinement). however, we also noted some risks related to technology with regard to tourists' experience and insights. for example, online reproduction of animal-based tourism and its tendency to convey partly selective or false impressions may hence build expectations and contribute to a culture of commodification. furthermore, in the suggested next steps for each of these themes or challenges for animal-based tourism, there are two levels to contend with: the structural level and that of individual clients. as evidenced, both literature and policy struggle to determine which of these levels is the most expedient to try to impart changes on. a third and final tension identified by the scholars of our symposium was that of reconciling sentientistic welfare perspectives-valuing the well-being of the individual animal-and ecocentric perspectives. indeed, this cut across multiple tourism sectors. there is as of yet no consensus on whether it is morally right to "sacrifice" animal lives to benefit their wild species kin. ultimately, this raises a larger discussion on the value of the wild, the loss of value of ex situ confined animals in zoos or parks. in the end, the future of animal-based tourism may be somewhat uncertain. one might anticipate that its growing popularity may paradoxically undermine its success, as it is often predicated on selling the rare and exotic. hence, if these products become too commonplace, animal engagement may lose part of its appeal in the future. nevertheless, as citizens, we have a moral obligation to travel with responsibility and use compassion for animals involved in tourism. facing the wild: ecotourism, conservation, and animal encounters staging tourism: bodies on display from waikiki to sea world tiger tourism: from shooting to petting an analysis of zoo visitors' favourite and least favourite animals consuming the king of the swamp: materiality and morality in south louisiana alligator tourism neo-darwinian leisures, the body and nature: hunting and angling in modernity the cow goes moo: farm animal and tourist interactions on long island's north fork nature-based tourism animals & modern cultures-a sociology of human-animal relations in modernity volatile ecologies: towards a material politics of human-animal relations multispecies leisure: human-animal interactions in leisure landscapes animals in the tourism and leisure experience exploring the boundaries of a new moral order for tourism's global code of ethics: an opinion piece on the position of animals in the tourism industry fear of the animal planet: the hidden history of animal resistance animal resistors: on the right of resistance and human duties of non-return and abolition tourism and animal welfare towards an ethical framework for animal-based attractions captive wildlife at a crossroads-sanctuaries, accreditation, and humane-washing asian elephant (elephas maximus), pig-tailed macaque (macaca nemestrina) and tiger (panthera tigris) populations at tourism venues in thailand and aspects of their welfare the customer isn't always right-conservation and animal welfare implications of the increasing demand for wildlife tourism animal welfare in tourism services: examples and practical tips for the well-being of animals used for tourism in lapland tourists' attitudes toward the use of animals in tourist attractions: an empirical investigation circus and zoo animal welfare in sweden: an epidemiological analysis of data from regulatory inspections by the official competent authorities farmed animal sanctuaries: the heart of the movement? politics anim biological impacts of ecotourism-tourists and nesting turtles in behavioural effects of tourism on oceanic common dolphins, delphinus sp., in new zealand: the effects of markov analysis variations and current tour operator compliance with regulations beasts and tourists: human-animal relationships in tourism ethical fading: the role of self-deception in unethical behavior the attitude-behavior gap in sustainable tourism abstracting animals through tourism unethical use of wildlife in tourism: what's the problem, who is responsible, and what can be done? animal ethics, dietary regimes and the consumption of animals in tourism wildlife tourism the importance of the aesthetic lockdowns could be the 'biggest conservation action' in a century the coronavirus threat to wildlife tourism and conservation agri-tourism: in between rural change, tourism restructuring and environmental imperatives a new urban dispositif? governing life in an age of climate change public perceptions and implications for species management. hum. dimensions wildl animal geographies i: hearing the cry and extending beyond biosecurity and the topologies of infected life: from borderlines to borderlands an eco-feminist perspective on the co-existence of different views of seals in leisure activities leisure, labor, and the complexity of culture: an anthropological perspective the competitive consumption and fetishism of wildlife trophies the reflexive tourist distribution and interspecies contact of feral swine and cattle on rangeland in south texas: implications for disease transmission not such smart tourism? the concept of e-lienation neo-colonialism and greed: africans' views on trophy hunting in social media tourism: an introduction a room of (his) own: italian and italian-american male-bonding spaces and homosociality animal rights vs. cultural rights: exploring the dog meat debate in south korea from a world polity perspective multiculturalism goes imperial: immigrants, animals, and the suppression of moral dialogue cultural variation, animal welfare and telos current status of animal welfare and animal rights in china the oppression of donkeys in seaside tourism just tourism: an ethical framework one welfare-a platform for improving human and animal welfare what we've got wrong about china's 'wet markets' and their link to covid- digital technology and the conservation of nature what do we know about wild boar in iberia? are tourists interesting? ways of knowing for 'response-ability' in more-than-human encounters: the role of anticipatory knowledges in outdoor access with dogs becoming tourist: renegotiating the visual in the tourist experience microscope and spectacle: on the complexities of using new visual technologies to communicate about wildlife conservation troubled shooting-the ethics of helicopter-assisted guided trophy hunting by tourists for tahr animals in film researching visual environmental communication exclusive: instagram fights animal abuse with new alert system cetacean performance and tourism in kaikoura seeing green: knowing and saving the environment on film where the wild things aren't selling the serengeti: the cultural politics of safari tourism the seven sins of hunting tourism killing with kindness: when hunters want to let you know they care problematic animals in the zoo: the issue of charismatic megafauna alternative facts and alternative views: scientists, managers, and animal rights activists animal liberationism, ecocentrism, and the morality of sport hunting promoting predators and compassionate conservation ethics and responsibility in wildlife tourism: lessons from compassionate conservation in the anthropocene the principles of humane experimental technique; methuen: london, uk, . © by the authors we want to sincerely thank all the participants of our symposium for contributing their insights and experiences on animal-based tourism during the two-day event. we also wish to thank some key helpers without whom the event could scarcely have proceeded: jennie persson assisted during the planning and execution of the event. adélaïde fouache kindly took notes for us during the proceedings, providing the basis for this report. lara tickle supplied the catchy title back in june. finally, anna martin headed up the graphic design for the symposium poster, which we retained as our aesthetic template. there is no conflict of interest to report. key: cord- - gyix authors: stull, jason w.; weese, j. scott title: hospital-associated infections in small animal practice date: - - journal: veterinary clinics of north america: small animal practice doi: . /j.cvsm. . . sha: doc_id: cord_uid: gyix hospital-associated infections (hais) occur in veterinary hospitals of all types and sizes, and their frequency is likely to increase. urinary tract infections, pneumonia, bloodstream infections, surgical site infections, and infectious diarrhea are the hais most frequently identified in veterinary medicine. a hospital infection control program, consisting of an infectious disease control officer, written protocols, and staff training, is critical to reducing hais and promoting patient, staff, and client health. infection control protocols (plans) should include discussion of hand hygiene and use of personal protective equipment, cleaning and disinfection, patient management, with-in hospital surveillance, and antimicrobial stewardship. compared with people. however, this may be countered with greater patient hygiene challenges, greater difficulty with patient compliance (eg, licking wounds), and a lesser "culture" of infection control. although current data are limited for the veterinary field, similar (or even higher) hai rates have been reported compared with human studies, such as hais in % of intensive care unit patients in one study. during a -year period, % of veterinary teaching hospitals in north america and europe reported at least one hai outbreak and % reported multiple outbreaks. many of these outbreaks required restricted patient admissions ( %) or closure of the hospital or section ( %). therefore, although hais are poorly quantified in veterinary medicine, they are undeniably a concern. there are many potential adverse events from hais in veterinary patients. animals suffering from hais may have an increased hospital stay (with accompanying increased cost to the client or clinic). these patients may also suffer permanent health consequences, or hais may result in death of the pet. multidrug-resistant organisms (mdros) are often involved in hais, complicating treatment and resulting in poor patient outcomes and extensive outbreaks. furthermore, some veterinary hospital-associated (ha) pathogens (eg, methicillin-resistant staphylococcus aureus [mrsa] , salmonella) can be transmitted to staff or pet owners, resulting in human illness. additionally, as veterinary medicine advances, there may be parallel increases in hai risk through the use of more invasive procedures, more use of invasive devices (eg, urinary catheters, intravenous catheters), more immunosuppressant therapies, and a greater intensity of critical care management. patients that might not have survived their underlying disease in the past may now be alive, but highly susceptible to infection. perhaps most important to this topic is the assumption in human medicine that % to % of all hais are preventable through the use of practical infection-control measures. large economic benefits are estimated to occur with the implementation of infection-control interventions ($ -$ billion cost savings in the united states alone). the proportion of hais that are preventable in veterinary medicine is unknown, but is likely to be similar, and even a % reduction in infections could constitute a major impact on patient health, owner cost, and owner and clinician satisfaction. the routine use of simple infection prevention practices can likely dramatically reduce hais. infection control is the term best suited to the goal in small animal veterinary medicine of preventing (or, more practically speaking, limiting) the introduction and/or spread of pathogens with a group of patients and caregivers. central to this goal is the establishment and refinement of an infection-control program at each animal hospital. every hospital's infection-control program will be different, reflecting the unique pathogen risks, facility and personnel characteristics, animal populations served, and level of risk tolerance of the practice. however, at a minimum, each practice's program should include the following: an infectious disease control officer (otherwise known as an infection-control practitioner); a written infection-control protocol (plan); regular training of staff about hospital infection-control protocols (and documentation of this training and assessment of comprehension); monitoring of both disease rates and infection-control protocol compliance. together, the components of the program should address the hai risks for patients and staff and recommended or required protocols to reduce these risks. the end stull & weese result will be a safer working environment for staff, optimal care for all patients, and protection of public health. although good infection-control practices are not the only feature defining excellence in patient care, it is impossible to achieve excellent patient care without them. the standard of what is "acceptable" from an infectioncontrol standpoint is changing in veterinary medicine, and it is clear that the "bar" is being raised in terms of the expected standard of care. in human medicine, urinary tract infections (utis), pneumonia, surgical site infections (ssis), and bloodstream infections (bsis) account for approximately % of all hais. in veterinary medicine, these sites along with gastrointestinal disease (infectious diarrhea) are likely to be the most common hais, although other conditions, such as upper respiratory tract infection, dermatophyte infection, iatrogenic blood-borne pathogen infection, and infections of a wide range of invasive devices can also occur. each of these main areas is discussed later, highlighting (where available) incidence, risk factors for disease, and commonly identified pathogens. because ssis are being exclusively covered in a separate article in this issue, they are not discussed here. catheter-associated utis are one of the more common hais in small animal veterinary medicine, although veterinary data are often limited by the failure to differentiate bacteriuria (a potentially benign condition) from uti (disease). studies have reported catheter-associated bacteriuria occurring in % to % of hospitalized dogs, with a subset of these exhibiting clinical signs or other evidence of infection. [ ] [ ] [ ] [ ] the interference of normal defense mechanisms by urinary catheters, such as mucosalsecreted adhesion inhibitors, along with patient comorbid factors and some catheter handling factors, facilitate bacterial colonization of the catheter and ascension of the organism or organisms into the bladder. these pathogens may be endogenous to the patient, arising from the rectum or perineum, or directly from the hospital environment or people through contamination of the drainage system or bag. if the collection system has been contaminated, bacteria can ascend into the bladder through the catheter if there is retrograde flow of urine. retrograde urine flow can occur if the collection system is elevated above the level of the patient; if collection lines are flushed, or if there is obstruction to flow in the collection system. in addition, biofilms (a complex structure of microorganisms and extracellular matrix) can be produced by bacteria on surfaces of urinary catheters. biofilm formation can be associated with poor antimicrobial penetration, antimicrobial resistance, and treatment failure. in human medicine, several factors, such as recumbent position, mechanical ventilation, and use of endotracheal or nasogastric tubes, are likely to increase the risk for ha pneumonia. , this topic has been minimally investigated in the veterinary field, in large part because of the limited use of mechanical ventilation. in one study, escherichia coli and acinetobacter spp were commonly identified in feline ha ventilatorassociated pneumonia cases. although not included in the human surveillance definition, aspiration pneumonia is not uncommon in small animal medicine and can occur in patients hospitalized for a wide range of disorders as well as otherwise healthy patients undergoing sedation or anesthesia. in addition to those factors listed for human ha pneumonia, factors that increase aspiration pneumonia, such as hospital-associated infections laryngeal or esophageal disorders and decreased mentation or recumbency, likely increase hai risk. [ ] [ ] [ ] if these patients have been hospitalized for multiple days before aspiration, it is more likely that the oropharynx is colonized with organisms from the hospital environment or hands of staff, and the pneumonia may be more likely to involve mdros, particularly if patients have been treated with antimicrobials. in the human literature, most ha bsis are associated with intravascular devices. duration of catheterization has been recognized as the most important risk factor for the development of catheter-related (cr) bsis (most developing - days after placement). despite this increased risk, studies have not documented a benefit with prophylactic catheter changes (eg, every days). in human medicine, the current recommendation is for catheters to be removed as soon as medically indicated, but for routine changes to be avoided. a similar approach is appropriate in veterinary medicine. veterinary studies have revealed that jugular and intravenous catheters are frequently contaminated with enteric or environmental pathogens. , several factors have been positively associated with intravenous catheter contamination/colonization in dogs and cats, including receipt of dextrose infusion, longer duration of catheter placement, and patient immunosuppression (presence of immunosuppressive diseases or receipt of immunosuppressive drugs). commonly isolated organisms include staphylococci, e coli, enterobacter spp, proteus spp, and klebsiella spp. [ ] [ ] [ ] contamination may occur from the hands of people placing or handling the catheter, the patient's own flora, or the hospital environment. however, there is little evidence indicating that contaminated but not infected catheters (ie, catheters from which bacteria can be isolated but where the catheter insertion site and vein are clinically normal) pose a risk for subsequent bsi. as a result, routine culture of catheters at time of removal or culture of catheter insertion sites is not recommended because skin bacteria are expected to be present. veterinary outbreaks involving cr bsis have been associated with inadequate skin preparation or contaminated materials used in skin preparation, , something that is of most concern when antiseptic solutions or wipes are prepared by refilling bottles or containers, which can become contaminated with biocide-resistant bacteria over time. ha gastrointestinal infections are usually recognized when there is a noted increase (outbreak) of infectious diarrhea in hospital patients. although identification of diarrhea is simple, determination of the cause is often difficult, even for known pathogens. in small animal veterinary facilities, salmonellosis is the most frequently reported gastrointestinal hai , ; however, it is unclear whether that is because it poses the greatest risk or (more likely) it is more readily identified and reported compared with other potential causes. in nonhospitalized small animal populations, several risk factors for salmonella colonization or infection have been identified, including animal species (eg, reptiles, amphibians, young poultry, exotics), consuming a raw animal-based diet or treats (eg, raw meat/eggs, rawhides), exposure to livestock, and recently receiving a probiotic. , these factors may substantially increase the risk of shedding salmonella, % to % shedding in dogs with one or more of these risk factors as compared with less than % typically noted in dogs without these risk factors. , however, the true scope of this issue is unclear because most outbreaks go unnoticed or testing is not performed, but, conversely, clusters of diarrhea seem to be uncommon in most facilities. pathogens involved in small animal hais often have one or more of the following characteristics: opportunistic pathogen in companion animals and/or humans, environmentally stable, or multidrug-resistant. many pathogens involved in hais are opportunistic pathogens that can be found in healthy animals, highlighting the inability to prevent entrance of all potential pathogens into a veterinary facility. the frequency of each pathogen varies for each veterinary practice (in part influenced by antimicrobial use/pressure, geography, animal species, vaccine coverage of animals in "catchment" area, level of care provided). in addition, environmentally stable pathogens (eg, parvovirus, clostridial spores, dermatophytes) have a demonstrated clear "advantage," increasing the chance of transmission. given the close interaction between veterinary staff and patients as well as the often poor hand hygiene practices documented in veterinary practices, human commensals with zoonotic potential are represented by hais in veterinary medicine. finally, increased resistance to antimicrobials is a common feature of most nosocomial bacteria. several pathogens are a concern from a small animal infection-control standpoint (box ). although a wide range of pathogens may be involved in hais, currently there is a strong focus on the emerging epidemic of multidrug-resistant bacteria because of dramatic increases in infections, limited antimicrobial options, and potential public health consequences. these mdros are not inherently more virulent than antimicrobial susceptible organisms, but treatment options are limited, something that ultimately can worsen the prognosis. the us centers for disease control and prevention has recently assessed domestic antibiotic resistance threats for people based on box pathogens of concern in a small animal clinic hospital-associated infections clinical and economic impact, incidence, transmissibility, availability of effective antimicrobials, and barriers to prevention. several pathogens of importance relative to veterinary hais were included as "serious antibiotic resistance threats," namely, acinetobacter spp, extended spectrum b-lactamase-producing enterobacteriaceae (esbls), pseudomonas aeruginosa, salmonella spp, and mrsa. as animals and people may share common infection sources or transmit these pathogens to each other, this concern is equally important in the veterinary field, and all of the abovenamed pathogens can be found in veterinary patients. given these relatively novel threats and the often limited knowledge by veterinary personnel on this group of pathogens, the attention here is focused on mdros as hais. in human medicine, hais are often captured through voluntary or mandatory hospital reporting. as such, the occurrence (and trends) of hais are fairly well-established. in the united states, recent data indicate bacteria are responsible for % of hais, with commonly identified groups including staphylococcus aureus, enterococcus spp, e coli, coagulase-negative staphylococci (cons), klebsiella spp, p aeruginosa, enterobacter spp, and acinetobacter baumannii. despite the importance of this field, current knowledge of many aspects of the epidemiology of important mdros and pathogens responsible for hais in veterinary medicine is unclear (eg, prevalence, risk factors, and transmission dynamics). unfortunately, companion animal veterinary medicine has been slow to implement surveillance systems; however, this is changing. currently, most data come from limited retrospective studies of clinical isolates, likely resulting in geographic and culturebased bias, potentially misrepresenting the frequency of these pathogens and potentially overestimating the prevalence of antimicrobial resistance if culture submissions are biased toward infections that failed to respond to empirical therapy. regardless, based on the reported veterinary ha outbreaks or supposition from the human literature, several important mdros responsible for hais are identifiable: s aureus, staphylococcus pseudintermedius, enterococci, salmonella spp, acinetobacter spp, e coli, and other enterobacteriaceae, and pseudomonas spp. the specific resistance profiles and treatment options for common multidrug-resistant (mdr) pathogens have recently been summarized. the reader is directed to the article elsewhere in this issue of veterinary clinics of north america: small animal practice by guardabassi and prescott entitled, "antimicrobial stewardship in small animal veterinary practice: from theory to practice," which expands on the topic of mdros in hais and antimicrobial stewardship. staphylococcus s pseudintermedius and to a lesser extent s aureus are common causes of veterinary hais. both are frequently carried on the skin and mucosal surfaces of dogs and people (respectively), creating the potential for both endogenous infection (infection caused by bacteria the animal was harboring at the time of hospital admission) and acquisition of the pathogen during hospitalization directly or indirectly from other patients, the environment, or human caregivers. the emergence of methicillin resistance in these species (methicillin-resistant s pseudintermedius [mrsp] and mrsa) has had important implications for hai prevention and control. methicillin resistance is mediated by the meca gene, which results in resistance to b-lactam antimicrobials (penicillins, cephalosporins, and carbapenems). in addition, resistance to other classes of antimicrobials is frequently observed: lincosamides (clindamycin), fluoroquinolones, macrolides (erythromycin), tetracyclines, trimethoprim-sulfonamides. , , mrsa is an important pathogen in human hais, being a common cause of ssis and various other types of infections. to a lesser extent, mrsa has also been noted in veterinary hais. risk factors for veterinary mrsa hais have not been well studied, but prior antimicrobial use, prior hospitalization, ownership by veterinary or human health care workers/students, and longer hospitalization (> days) have been associated with mrsa colonization or infection in dogs. [ ] [ ] [ ] [ ] furthermore, the use of fluoroquinolones and cephalosporins has been linked to the emergence of mrsa in people and may play a role in veterinary species. it is important to note that an abnormally high proportion of veterinarians are colonized with mrsa as compared with the general public. as such, they may serve as a source for hais in their patients if infection-control practices (notably hand hygiene) are substandard. this also likely indicates deficiencies in standard infection control and hygiene practices that allow for transmission of mrsa between veterinary personnel and animals. mrsp has rapidly spread in canine populations, often with high levels of antimicrobial resistance, something that is of tremendous concern because s pseudintermedius is the leading opportunistic pathogen in dogs (and, to a lesser degree, cats). it is the most common cause of ssis in some regions, and treatment may be complicated because of the high level of resistance. in one study, more than % of mrsp isolates were also resistant to additional antimicrobial classes. recent prior hospitalization and b-lactam antimicrobial administration have been associated with mrsp infections, suggesting hospital-associated transmission may be a factor in mrsp disease. the topic of cons deserves mention. veterinary diagnostic laboratories often consider these species as a group and speciation is rarely performed. cons are frequently identified as commensals in small animal species, with high methicillin resistance in healthy animals. with the exception of highlycompromised individuals, it has been generally assumed that cons, even those that are multidrug resistant, are of limited clinical concern. that assumption has been challenged to some degree and some cons species may be more clinically relevant than others; however, this group remains a less common cause for concern compared with s pseudintermedius and s aureus. however, their commonness as skin or mucous membrane commensals can complicate interpretation of culture results because differentiating infection from contamination may be challenging. e coli is a frequent component of the commensal gastrointestinal microbiota and is an important pathogen, particularly in utis. mdre coli is frequently shed in the feces of both community and hospitalized small animals. [ ] [ ] [ ] multiple factors have been associated with dogs shedding or acquiring mdr e coli during hospitalization, including duration of hospitalization (> days) and treatment with antimicrobials shortly before or while hospitalized (cephalosporins, metronidazole). , antimicrobial resistance is an important problem with e coli and other enterobacteriaceae (eg, enterobacter). although b-lactamase-producing isolates have been common for some time, there has been a recent emergence of esbls producers, which provide resistance to a broad range of b-lactam antimicrobials, including thirdgeneration cephalosporins. in addition, esbls are conferred resistance to other antimicrobial classes through genetic linkage with resistance mechanisms. extended spectrum b-lactamase-producing e coli has been identified as the source of veterinary hais, occurring as ssis and catheter-associated utis, with observed hospital contamination. , other genera in the enterobacteriaceae family (ie, klebsiella, enterobacter) are considered to be important in human hais; however, less is known of their involvement in veterinary infections. one of the most important drug classes for treatment of esbl-producing bacteria is carbapenems (eg, meropenem). unfortunately, carbapenemase-producing enterobacteriaceae (or carbapenem-resistant enterobacteriaceae; cre) (including e coli) have emerged as a significant problem in human health care. additional resistance mechanisms are often present, rendering isolates virtually pan-resistant, and ability for cres to spread rapidly in health care settings with extension into the community. high mortality (> %) has been documented for invasive human cre infections. carbapenemase-producing e coli have recently been identified in small animals, with suggested nosocomial transmission. nosocomial transmission currently seems to be a rare, albeit concerning, occurrence, and one that is likely to increase as cres increase in prevalence in the human population, with subsequent exposure of pets. enterococci are often found in the gastrointestinal tract of animals and humans. two species, enterococcus faecium and enterococcus faecalis, are most often involved in disease, including hais, although enterococci tend to be of limited virulence and typically cause infections in compromised hosts. enterococci are inherently resistant to several antimicrobial classes, including cephalosporins, some penicillins, fluoroquinolones, clindamycin, and trimethoprim. they may also acquire resistance to various other antimicrobial classes and, although they are typically of limited virulence, they may be difficult to eliminate in cases when disease develops. vancomycin-resistant enterococci (vre) are an increasing concern in human medicine, with vancomycin resistance noted in up to % of e faecium involved in hais. to date, vre appears to be rare in companion animals. however, other mdr enterococci are regularly recognized in small animals and have been identified in hais. , , enterococci are often identified as utis (including catheter-associated); however, infections at other anatomic sites occur (eg, ssis, bsis, pneumonia). the high degree of antimicrobial resistance, ability to propagate for extended periods in small animal hosts as a commensal, and environmental persistence make enterococci particularly challenging when involved in hais. it is important to note that isolation of enterococcus species (regardless of antimicrobial resistance) does not always indicate treatment is indicated. without clinical signs in an otherwise immune-competent animal, it may be warranted to withhold treatment and monitor the patient. when isolated in a patient with clinical signs (notably infections of the urinary tract, wound, or body cavity), treatment should often be directed at the organism or organisms also isolated that are thought to be primarily responsible for clinical disease. often, that involves ignoring the enterococcus and targeting therapy toward another, more convincing, pathogen, such as e coli. salmonella is most frequently a concern in equine facilities, but has been identified as a source of sporadic illness and hospital-associated outbreaks in small animal hospitals. , an important concern with salmonella hais is the occurrence of zoonotic transmission with accompanying human infections. , because most infections in dogs and cats are subclinical, there is a high risk for inadvertent hospital-wide environmental contamination and nosocomial transmission. reported factors leading to an increased risk of salmonella shedding in small animals include consumption of raw meat diets, exposure to livestock, and receiving a probiotic in the previous days. , as with e coli, esbl-producing strains are a concern for antimicrobial resistance and have been identified in small animals. given its environmental stability, potential shedding by healthy animals, and significant zoonotic health hazard to clinic staff and clients, salmonella needs to be considered an important companion animal nosocomial pathogen. acinetobacter is well-recognized as an important ha pathogen in human medicine, in part because of recently recognized high levels of antimicrobial resistance in a baumannii. more than % of a baumannii human isolates involved in hais were mdr in one study. given its role as an opportunistic pathogen in small animals, ability to persist in the environment for extended periods, and documented outbreaks in veterinary facilities, it is also a concern for veterinary medicine. , , documented hais involving a baumannii include intravenous and urinary catheters, surgical drain infections, ssis, pneumonia, and bsis. multidrug resistance is frequently encountered with pseudomonas spp. this along with their noted persistence in the hospital environment makes pseudomonas spp a concern for hais. in humans, most infections are ha and occur in immunocompromised hosts. in companion animal species, pseudomonas spp infections often involve the skin, urinary system, and ears, - along with ssis and invasive device infections. , biofilm formation by pseudomonas spp can further complicate treatment. identification of within hospital clusters of pseudomonas infections should prompt investigation of potentially contaminated environmental, equipment (eg, endoscope), or consumable (eg, catheter preparation supplies) sources. the admission of sick animals occurs daily in most if not all small animal veterinary facilities. furthermore, every animal admitted to the veterinary clinic, healthy or not, can reasonably be assumed to be shedding multiple microorganisms that could cause infection in humans or animals, given the opportunity. as such, there is always a risk for the introduction and spread of hais and for exposure to zoonotic pathogens. the level of risk will be determined, in part, by the population of animals served (eg, young, elderly, immunocompromised), pathogens circulating in the community animals, proportion of patients for which protective or increased-risk practices are taken by their owners (eg, vaccination, husbandry practices to reduce pathogen acquisition), intensity of care typically provided for patients, and clinic infection-control practices and adherence to these practices by staff and clients. veterinary clinic staff will not be able to alter many of these risks; however, infection-control practices is an area that with some planning and dedicated time, can be relatively easy to address. although complete prevention of hais is the goal, given the nature of patient care, bacterial adaptation, and complexity of many pathogens (subclinical shedding, insensitive diagnostic tests), it is inevitable they will continue to occur. methods to reduce the risk of hais are paramount. in general, methods to reduce hais can be divided into the following main categories: hand hygiene and use of personal protective equipment (ppe; ie, clothing and/or gloves to reduce contamination of staff, patients, and the environment); cleaning and disinfection (environmental surfaces and patient equipment); patient management (eg, cohorting patients based on risk, isolating high-risk patients, discontinuing the use of higher risk devices when indicated); surveillance (identification of infected or colonized patients, hais, and source/ risk factors); antimicrobial stewardship (prudent antimicrobial use); education and training (clients, staff). these methods will not only reduce overt problems such as hospital-associated outbreaks but also reduce the likelihood of patient colonization with a ha pathogen, which can become part of the patient's resident microbiota, potentially increasing disease risk at a later date and posing a risk to other animals and humans. each of these areas should be addressed in a hospital's infection-control manual. several "model" plans are widely available to use as a starting point for developing an individualized hospital plan; infection-control officers are encouraged to review these resources. , individual articles in this issue of veterinary clinics of north america: small animal practice are devoted to each of these areas, so they are only briefly discussed here. unfortunately, studies on the area indicate only a minority of small animal veterinary hospitals have written infection-control plans ( %- %). , given the relative ease of putting together an infection-control plan and potential health, legal, and financial benefits of doing so, every clinic should invest the time and effort to make this a priority. hand hygiene (washing hands with soap and water or using an alcohol-based hand rub) and use of ppe, such as nonsterile gloves and gowns, are simple techniques that can reduce the risk of hais. effective use of hand hygiene and appropriate ppe use reduces the risk of contamination of personal clothing, reduces exposure of skin and mucous membranes of veterinary staff to pathogens, and reduces transmission of pathogens between patients by veterinary personnel. unfortunately, several studies indicate that veterinarians and staff do a poor job at performing hand hygiene between patients (w %) or using ppe when indicated ( %- % depending on the situation). , cleaning and disinfection recent evidence suggests environmental contamination in human hospitals increases the risk for hais, whereas interventions that reduce environmental contamination have assisted with cessation of ha outbreaks or reduction of hais. , the same connection is assumed to occur in veterinary medicine. effective cleaning and disinfection of hospital equipment and environmental surfaces play an important role in reducing hais. in order for a disinfectant to work properly, the surface or item must first be clean (free of visible organic material) and the product must be applied at the manufacturer's suggested dilution and contact time (amount of time the disinfectant is in contact with the item before being removed). disinfectants should be selected based on several criteria, including the product's spectrum of activity, susceptibility to inactivation by organic matter, and potential pathogens in the environment. given the close contact between veterinary patients and their hospital housing, environmental contamination is inevitable. furthermore, staff caring for these patients is at increased risk for spreading the pathogen through contact with the patient or its stull & weese environment. to protect other patients and clinic staff, special attention to patient housing is important in managing infectious patients. isolation procedures, use of dedicated medical equipment, and patient cohorting are important stopgaps in the transmission of hais for animals suspected to be infectious. in addition, specific patient care procedures may be helpful in reducing hais associated with catheters, aspiration pneumonia, bsis, infectious diarrhea, and ssis. resident small animals are sometimes kept at veterinary facilities as blood donors, companionship for staff, or other reasons. because these animals may harbor mdr pathogens and be sources or propagation of hospital contamination or outbreaks, special attention should be devoted to hospital policies for these animals regarding staff-animal contact and restricted movement (not permitting direct contact with patients or patient areas, including areas for exercise and elimination). , surveillance the early identification of hais is critical for effective infection control. identification of "abnormal" (increases in disease incidence or patterns) depends on a reasonable understanding of "normal." understanding of endemic rates can be useful to allow for comparison with other facilities, to establish benchmarks for ongoing surveillance, to serve as a baseline for interventions, to allow for more accurate counseling of clients about risks (eg, ssi rates), and to provide a greater overall awareness of the importance of hais and corresponding control measures. it is not unusual for hai outbreaks to "smolder" below the radar of veterinary staff for extended periods because of the lack of centralized data reporting or communication, resulting in substantial environmental contamination, patient morbidity (and potentially mortality), and even increased zoonotic disease risk for staff and clients. key elements of early hai identification include ( ) a surveillance program tailored to the risks and needs of the veterinary practice and ( ) routine use of diagnostic culture and susceptibility data to establish practice-specific baseline levels of pathogen prevalence and antimicrobial resistance and detect changes from this baseline. careful selection and appropriate use of antimicrobials are important steps in combating patient mdro development and subsequent contamination and transmission in the hospital environment. antimicrobials should be avoided when a bacterial infection has not been confirmed. antimicrobials used in the initial treatment of an infection should be selected based on the effectiveness against the most likely organisms causing the infection (something that can be facilitated by having good passive surveillance data) as well as patient (eg, renal function, comorbidities) or drug (eg, penetration, route of administration, frequency of administration) factors. whenever possible, a culture should be submitted to determine the true susceptibility pattern of the bacteria involved. local therapy can be an important option that is often overlooked. during their careers, approximately two-thirds of veterinarians report a major animalrelated injury resulting in lost work or hospitalization. , animal bite injuries and infections are a large contributor to this hazard, but zoonotic infections (eg, mrsa, dermatophytosis [ringworm], salmonellosis) are also frequently reported. , educating staff and clients on zoonotic disease risks and enforcing in-hospital infection-control protocols to reduce these risks will be beneficial to the health of people and patients. all veterinary personnel and visitors should be familiar with the hospital's infectioncontrol plan and policies. the infection-control officer is integral to the successful development, maintenance, and enforcement of an infection-control plan. in the human health care field, infection-control practitioners are formally trained and certified, with the infectioncontrol program typically overseen by a physician with specialized training in infectious diseases, infection control, and/or microbiology. in veterinary medicine, this type of approach is only practiced in large facilities (mainly teaching hospitals), yet the basic concepts remain the same for veterinary facilities of any type and size. a functional infection-control program can be directed by a single infectioncontrol practitioner in a veterinary hospital, with minimal time requirements. this individual can be a technician or veterinarian who has an interest in infection control. the skills required (eg, general understanding of infection-control concepts) can be obtained on the job and need not be a prerequisite for the position, and the limited time requirement under normal circumstances means that a new position does not need to be added. rather, direction of the infection control can usually be undertaken by an existing staff member. of greatest importance for the individual filling this position is an interest in the topic, motivation to make improvements in the clinic's infection-control policies, and the support of clinic leaders (eg, practice owners, veterinarians). without full support by clinic leaders (eg, time to perform the required duties, financial investments, serving as a role model by following clinic infection-control policies), the infection-control officer, and resulting program, is unlikely to be successful. hais have been reported in veterinary medicine and their frequency is likely to increase with the increase in intensive care practices in many veterinary hospitals. prolonged hospitalization and the use of invasive devices and procedures increase the risk of hais. all staff members should be educated on the risks and signs associated with hais so that cases can be detected early and managed appropriately. ultimately, a multifaceted approach is necessary to address hais in small animal veterinary medicine, including prudent antimicrobial use, strengthening surveillance of hais in companion animals, improving infection-control practices (eg, hand hygiene, ppe, cleaning and disinfection, patient management), instilling an infection-control culture among veterinary staff, and improving health care and public education of antimicrobials. a hospital infection-control program, consisting of an infectious disease control officer, a written protocol, and staff training, is a key component to unifying these elements and successful reduction of hais in small animal veterinary practice. estimating health care-associated infections and deaths in u.s. hospitals the direct medical costs of healthcare-associated infections in u.s. hospitals and the benefits of prevention using syndromic surveillance to estimate baseline rates for healthcare-associated infections in critical care units of small animal referral hospitals van metre dc. characteristics of biosecurity and infection control programs at veterinary teaching hospitals the preventable proportion of nosocomial infections: an overview of published reports cdc/nhsn surveillance definition of health care-associated infection and criteria for specific types of infections in the acute care setting evaluation of catheter-associated urinary tract infections and multi-drug-resistant escherichia coli isolates from the urine of dogs with indwelling urinary catheters incidence of catheter-associated urinary tract infection among dogs in a small animal intensive care unit nosocomial infection surveillance in a small animal intensive care unit urinary tract infection resulting from catheterization in healthy adult dogs biofilms and catheter-associated urinary tract infections circulating immune parameters predicting the progression from hospital-acquired pneumonia to septic shock in surgical patients supine body position as a risk factor for nosocomial pneumonia in mechanically ventilated patients: a randomised trial indications for and outcome of positivepressure ventilation in cats: cases outcome of and postoperative complications in dogs undergoing surgical treatment of laryngeal paralysis: cases postoperative pulmonary complications in dogs undergoing laparotomy: anesthetic and perioperative factors incidence of and risk factors for postoperative pneumonia in dogs anesthetized for diagnosis or treatment of intervertebral disk disease risk of local and systemic infection with polyethylene intravenous catheters. a prospective study of catheterizations surveillance of infections associated with intravenous catheters in dogs and cats in an intensive care unit bacterial and fungal colonisation of peripheral intravenous catheters in dogs and cats inadequate skin preparation as a cause of intravenous catheterrelated infection in the dog a prospective study of intravenous catheter contamination salmonella typhimurium outbreak associated with veterinary clinic evaluation of pet-related management factors and the risk of salmonella spp. carriage in pet dogs from volunteer households in ontario perceptions, practices, and consequences associated with foodborne pathogens and the feeding of raw meat to dogs salmonella shedding in racing sled dogs antibiotic resistance threats in the united states antimicrobial-resistant pathogens associated with healthcare-associated infections: summary of data reported to the national healthcare safety network at the centers for disease control and prevention antibiotic treatment of resistant infections in small animals transmission of opportunistic pathogens in a veterinary teaching hospital inducible clindamycin-resistance in methicillin-resistant staphylococcus aureus and methicillin-resistant staphylococcus pseudintermedius isolates from dogs and cats methicillin-resistant staphylococcus pseudintermedius in a veterinary teaching hospital severe surgical site infection in community hospitals: epidemiology, key procedures, and the changing prevalence of methicillin-resistant staphylococcus aureus suspected transmission of methicillinresistant staphylococcus aureus between domestic pets and humans in veterinary clinics and in the household epidemiological profiling of methicillin-resistant staphylococcus aureus-positive dogs arriving at a veterinary teaching hospital methicillin-resistant and -susceptible staphylococcus aureus infections in dogs risk factors for methicillinresistant staphylococcus aureus (mrsa) infection in dogs and cats: a casecontrol study acquisition and persistence of antimicrobial-resistant bacteria isolated from dogs and cats admitted to a veterinary teaching hospital the effect of antibiotics on methicillin-resistant staphylococcus aureus methicillin-resistant staphylococcus aureus colonization in veterinary personnel clonal spread of methicillin-resistant staphylococcus pseudintermedius in europe and north america: an international multicentre study economic impact of tibial plateau leveling osteotomy surgical site infection in dogs evaluation of susceptibility test breakpoints used to predict meca-mediated resistance in staphylococcus pseudintermedius isolated from dogs factors associated with methicillin-resistant versus methicillin-susceptible staphylococcus pseudintermedius infection in dogs prevalence of antimicrobial-resistant escherichia coli in dogs in a cross-sectional, community-based study occurrence of antimicrobial resistant bacteria in healthy dogs and cats presented to private veterinary hospitals in southern ontario: a preliminary study risk factors for multidrug-resistant escherichia coli rectal colonization of dogs on admission to a veterinary hospital risk factors for dogs becoming rectal carriers of multidrug-resistant escherichia coli during hospitalization infections with extended-spectrum beta-lactamase-producing enterobacteriaceae: changing epidemiology and drug treatment choices characterization of multidrug-resistant escherichia coli isolates associated with nosocomial infections in dogs emergence and spread of two distinct clonal groups of multidrug-resistant escherichia coli in a veterinary teaching hospital in australia vital signs: carbapenemresistant enterobacteriaceae outcomes of carbapenem-resistant klebsiella pneumoniae infection and the impact of antimicrobial and adjunctive therapies emergence of oxa- carbapenemase-producing escherichia coli and klebsiella pneumoniae in dogs vancomycin-resistant enterococci: consequences for therapy and infection control monitoring of antimicrobial resistance in healthy dogs: first report of canine ampicillin-resistant enterococcus faecium clonal complex susceptibility to vancomycin and other antibiotics of enterococcus strains isolated from dogs in italy dogs leaving the icu carry a very large multi-drug resistant enterococcal population with capacity for biofilm formation and horizontal gene transfer multidrug-resistant salmonella typhimurium in four animal facilities comparison of antimicrobial resistance patterns of salmonella spp. and escherichia coli recovered from pet dogs from volunteer households in ontario ( - ) prevalence, distribution and characterisation of ceftiofur resistance in salmonella enterica isolated from animals in the usa from to the role of acinetobacter baumannii as a nosocomial pathogen for dogs and cats in an intensive care unit multidrug-resistant acinetobacter baumannii in veterinary clinics pseudomonas aeruginosa carriage, colonization, and infection in icu patients characterization of antimicrobial resistance of pseudomonas aeruginosa isolated from canine infections evidence-based veterinary dermatology: a systematic review of interventions for treatment of pseudomonas otitis in dogs comparison of three techniques for the diagnosis of urinary tract infections in dogs with urolithiasis cardiovascular device infections in dogs: report of cases and review of the literature an infected hip prosthesis in a dog diagnosed with a mtc-ciprofloxacin (infecton) scan infection prevention and control best practices for small animal veterinary clinics compendium of veterinary standard precautions for zoonotic disease prevention in veterinary personnel: national association of state public health veterinarians veterinary infection control committee infection control practices and zoonotic disease risks among veterinarians in the united states evaluation of specific infection control practices used by companion animal veterinarians in community veterinary practices in southern ontario evaluation of an educational campaign to increase hand hygiene at a small animal veterinary teaching hospital does improving surface cleaning and disinfection reduce health care-associated infections? a targeted strategy to wipe out clostridium difficile role of environmental cleaning in controlling an outbreak of acinetobacter baumannii on a neurosurgical intensive care unit outbreak of clostridium difficile-associated disease in a small animal veterinary teaching hospital resident cats in small animal veterinary hospitals carry multi-drug resistant enterococci and are likely involved in crosscontamination of the hospital environment a review of published reports regarding zoonotic pathogen infection in veterinarians a survey of veterinarian involvement in zoonotic disease prevention practices key: cord- -atltc d authors: arkow, phil title: human–animal relationships and social work: opportunities beyond the veterinary environment date: - - journal: child adolesc social work j doi: . /s - - -x sha: doc_id: cord_uid: atltc d a species-spanning approach that incorporates clients’ relationships with their companion animals into family genograms, schools of social work curricula, continuing education, interviews, assessments, and interventions offers increased career opportunities, professional and personal growth and development, and a more comprehensive resolution of clients’ issues, social justice concerns, and the prevention of family violence. this article identifies six reasons why social workers should be cognizant of human–animal relationships and introduces nine ways, with action steps, in which social workers can include these relationships into training and practice outside the more developed field of veterinary social work. these venues include: agencies working in child protection and child sexual abuse; children’s advocacy centers and courthouse facility dogs; animal shelters; domestic violence shelters; public policy advocacy; clinical practice; agencies working with older and disabled populations; veterinary sentinels for intimate partner violence; and pet support services for homeless populations. such attention to the human–animal bond can utilize social workers’ problem-solving skills to improve delivery of services, identify clients’ risk and resiliency factors, enhance social and environmental justice, expand academic inquiry, and increase attention to all of the vulnerable members of families and communities. system arousal, and improving physical fitness by providing an impetus to exercise (friedmann, ) . there is, conversely, substantial evidence of animal abuse as a potential precursor and indicator of interpersonal violence often linked to child maltreatment, intimate partner violence and elder abuse (arkow, a) . pets occupy central roles in many interpersonal and intrafamilial relationships (turner, ) . they may serve as significant others, confidants, attachment figures, and sources of companionship (mcnicholas & collis, ) . they can be vital members of an individual's support system and facilitators to foster social capital, trust, civic participation and a sense of safety and community (wood, martin, christian, houghton, & kawachi, ) . the relationships between humans and animals in a household may mirror the status of the health and safety of the people in that family (hoffer, hargreaves-cormany, muirhead & meloy, ) . inquiring about children's and adolescents' experiences with animals can help social workers address risk and resilience factors. pets' physical condition and behaviors can provide clues to human experiences and family functioning. human-animal relationships are contextual for learning and resilience in the wake of family violence. strengthening these relationships, and helping people better understand their animals' behaviors, can promote well-being in both species and enable people to leverage inclusion and a sense of belonging in community relationships. animal rights arguments about inherent "speciesism" in humans' relationships with other animals should resonate well with social work's commitment to social justice and fighting oppression (national link coalition, a) . this article explores six reasons why human-animal relationships can be significant to social workers' responsiveness to their clients and nine areas outside the veterinary social work environment where an understanding of clients' interactions with animals can be directed into enhanced professional and personal growth, innovative species-spanning solutions, and potential career opportunities. the american veterinary medical association ( ) estimated that % of u.s. households owned a pet, including . million dogs, . million cats, plus birds, horses and other companion animals. pets are predominantly found in households self-defined as "family" rather than "non-family" and the highest rates of dog and cat ownership continue to be among households with children. primary responsibility for pets' care continues to rest overwhelmingly with the female members of the household. pet ownership rates are higher in rural areas and lower in densely urbanized cities. there are unexplained racial and ethnic disparities in the rates of pet ownership; pets are found in . % of white households, compared with latino/hispanic ( . %), asian ( . %), and black/african american ( . %). some pet owners describe veterinarians as "the other family doctor" (national link coalition, b) . two studies reported that % (risley-curtiss et al. a; risley-curtiss, holley, & wolf, b) and % (risley-curtiss et al., a, b) of multi-ethnic pet owners agreed that their pets are members of the family. in short, social workers exploring a client's home life and family dynamics may be missing a significant piece of the puzzle if they neglect to inquire about the client's animals and the attachments, relationships, and problems with them. in addition to appreciating a client's individual and familial attachments or antipathy toward pets, social workers can achieve a fuller understanding of a client's connectivity or isolation from the community by seeing human-animal relationships in a social context. social capital (as contrasted with human capital, economic capital, cultural capital, technological capital, or other community resources) is the connectivity among people which enhances cooperation for mutual benefit. the concept was popularized by putnam ( ) describing the networks and other forces that build social cohesion, personal investment, reciprocity, civic engagement, and interpersonal trust among community residents. notably absent, however, from the work of putnam and other social capital researchers was inclusion of the influence of pets in a community (arkow, ) . this knowledge gap was addressed by wood et al. in studies in australia (wood, giles-corti, & bulsara, ; wood, giles-corti, bulsara, & bosch, and the u.s. ( ) which reported companion animal ownership to be positively associated with social capital, civic engagement, perceptions of neighborhood friendliness, and a sense of community. the ability of pets to generate interpersonal communications was greater than minor conversational exchanges among dog-walkers: the visible presence of people walking dogs and the impetus dogs provide for people to be outdoors and use park areas ameliorated negative mental health conditions and gave residents a feeling of greater collective safety and sense of community. companion animal owners were found to be more likely to participate in volunteer, school and sports activities, professional associations and environmental campaigns. they were reported to be more likely to vote and to exchange favors with neighbors. arkow ( a, ) proposed a converse, that the absence of companion animals in communities where rates of pet ownership are lower and incidence of animal problems is greater might contribute to less social connectivity and cohesion. levinthal ( ) used geospatial mapping techniques to correlate the distribution and prevalence of animal neglect, abuse and dog fighting in philadelphia with domestic violence and child maltreatment. she reported a highcrime neighborhood seemed to predict animal abuse, and that animal neglect correlated with demographic, cultural, and structural aspects of block groups, suggesting social disorganization may lead to animal neglect. campbell ( ) used geospatial mapping techniques to report direct correlations between animal control complaints and domestic violence incidents in neighborhoods in indianapolis. bruni ( ) suggested a way to push back on "the degradation of our country's civil culture" can be found in walking one's dog in public spaces. dog walks encourage "honest-to-goodness conversations with actual strangers" that leave their owners "feeling a little less isolated, a little less disconnected" and discourage americans retreating into "increasingly homogeneous enclaves." dog-walking encourages mutual courtesy and reciprocal generosity. "when you're about to bend down and scoop up your beloved's odoriferous bequest, there's no snobbery and no timidity, only solidarity," he wrote. there is something immensely powerful about animals that attracts and motivates humans, a force that is especially compelling with youths. whether discussing pet ownership, fascination with wild animals, or imaginary animals, children are particularly engaged, and asking about animalrelated experiences can provide important information and establish a caring persona and a trusting relationship (melson & fine, ; boat, ) . a pet is a communication waiting to happen. a recurring theme in the literature is that companion animals are what messent ( , p. ) first called "social lubricants," icebreakers who facilitate social support and interpersonal communications (garrity & stallones, ) . the nonjudgmental nature and unconditional positive regard of human-animal interactions can be a useful bridge for establishing rapport between therapists and clients (arkow, b). because animals slip under the radar of human defense mechanisms, clients who are fearful, traumatized or under stress may be more willing to talk about their concerns for their animals before opening up and describing their own vulnerabilities (melson & fine, ) . fawcett and gullone ( ) reported that even the mere observation of animals can result in reduced physiological responses to stressors and increased positive mood. lange, cox, benert, & jenkins ( ) reported animals can introduce a calming effect, stress-reducing humor, increased feelings of safety, experienced empathy, and motivation among adolescents attending anger management sessions. chandler ( ) wrote that discussing pets can easily segue into a discussion about the client's family support system and how well he or she is utilizing personal resources. an interaction with a therapy animal can enhance social workers' listening responses, convey empathy and help the client access feelings. she observed that it is not the mere presence of an animal, but rather the orchestrated child-dog and child-therapist interactions, that can facilitate client motivation and participation, enhance the relationship with the client, stimulate client focus and attention to task, and reinforce positive client change. margaret mead's oft-quoted adage ( ) that "one of the most dangerous things that can happen to a child is to kill or torture an animal and get away with it" has been substantiated with research. a growing body of evidence suggests that bonds formed or broken with companion animals in childhood reverberate and resonate across the lifespan (jalongo, ) . childhood acts of committing or witnessing animal cruelty: may be sentinel warnings that a child is living in a dysfunctional environment and may be exhibiting other antisocial behaviors (gullone, ) ; are a prime risk factor for perpetrating animal cruelty, bullying behaviors and violence against humans; and may lead to desensitization, decreased empathy, learned maladaptive coping mechanisms, and unresolved feelings of anger, fear and resentment, particularly if the child is also experiencing co-occurring family violence (ladny & meyer, ) . correlations between childhood exposure to animal abuse and bullying behaviors have been reported by several authors (for example, see baldry, ; gullone & robertson, ; henry & sanders, ; parkes & signal, ; sanders & henry, ; vaughn et al., ; walters, ) . currie ( ) reported that children exposed to domestic violence were three times more likely to have been cruel to animals than children not exposed to intimate partner violence (ipv). there is increasing academic and programmatic recognition of animal abuse and neglect as sentinel indicators of concurrent or future family violence, particularly child maltreatment and child sexual abuse, ipv and elder abuse. social workers may find that when animals are abused people are at risk, and when people are abused animals may be at risk (arkow, b) . emotional attachments to companion animals are often exploited by abusers in violence-prone households to control and coerce victims in domestic violence, sexual assault, child abuse, and elder abuse situations (ascione & arkow, ) and fear of leaving a pet behind is a significant barrier that keeps women and children from extricating themselves from abusive situations (roguski, ; ascione, ) . households marked by ipv have higher-than-average rates of pet ownership and are extreme high-risk environments when animal abuse is also present: nearly % of ipv survivors where suspects also had histories of animal abuse feared they would be killed, % had been strangled and % had been forced into non-consensual sex. multidisciplinary collaborations were seen as critical in prevention, detection and intervention to address substantial risk of harm for all children, adults and animals residing in the home (campbell, hicks, thompson, & wiehe, ) . grief over the loss of a pet is a broad, complex construct, which can be complicated by pet owners' perceptions that they have minimal social support sources and negative veterinary interactions (rémillard, meehan, kelton, & coe, ) . the loss of a pet through death or disappearance, and the decisions inherent in determining whether euthanasia of a beloved animal companion is necessary and appropriate, can generate significant emotional trauma for human members of families as well as staffs of veterinary facilities and animal shelters (see, e.g., barnard-nguyen, breit, anderson, & nielsen, ; carmack, ; dunn, mehler, & greenberg, ; laing & maylea, ; miller, prout, rourke, lefkowitz, & boyer, ; ross, ) . social workers who have had training in grief and loss theory are well positioned to be resources for individuals experiencing these emotions and can aid them in making difficult decisions and navigating the options available. because pets generally have shorter lifespans than humans, families are likely to witness significant life-cycle events such as birth, serious illness and death of their animal companions, who are often seen by children as peers and family members. the inevitable death of a pet can bring a profound sense of loss, with patterns of bereavement similar to those experienced with the death of a human family member or friend, and thus an opportunity for social work intervention. several studies have reported that by adolescence the majority of children have experienced pet loss through death or disappearance (melson & fine, ) . social workers can play important roles in offering grieving clients opportunities for validation of their feelings, memorialization of the animal, resolution of potential feelings of guilt, and closure. social work's interest in human-animal relationships has its origins in the practitioner-client-patient dynamics of the veterinary hospital or clinic. from modest beginnings at the veterinary hospital of the university of pennsylvania in (quackenbush, ; quackenbush & glickman, ) , the field of veterinary social work has grown dramatically in recent years. the term veterinary social work is believed to have been coined in by elizabeth strand, founding director of the veterinary social work certificate program at the university of tennessee-knoxville. today, dozens of students have been trained in the four areas of veterinary social work: the link between human and animal violence; grief and loss; animal-assisted interactions; and compassion fatigue management. this work may include: • supportive grief support and counseling with end-of-life decisions and follow-up. • advocacy and brokering of resources. • circulating reading materials and educational packets. • crisis intervention. • assessment of suicidal tendencies, mental health issues and domestic violence issues. • facilitation of a pet loss support group for hospital clientele and the community. • staff debriefing sessions. • client consultations and follow-up. • presentations to staff. • referral of mental health services for staff. • recommendations to administrators. • making improvements to client comfort on-site. (larkin, ) as the field of veterinary social work gains additional recognition both within and outside the world of social work, additional opportunities will continue to emerge whereby an understanding of human-animal relationships becomes a valuable asset in many aspects of social work practice. this process can begin with something as simple as routinely including pets in family genograms and adding relevant coursework in schools of social work and continuing education as well as field placement opportunities. by demonstrating additional opportunities for social workers to include human-animal relationships in interventions and assessments and to be aware of community resources that can resolve clients' animal-related concerns, social workers can be more holistic and effective in resolving clients' needs and challenges and preventing further abuse of vulnerable members of families and communities. social work's legacy of facilitating collaborative community change can open up many new career opportunities by incorporating human-animal relationships into social work practice. inquiring about the presence (or absence), stability (or turbulence), attachments, dangerousness, history, and status of animals within clients' lives can help social workers to obtain more comprehensive family assessments, validate important intra-familial relationships, build stronger support networks, support resiliency, gain earlier recognition of abusive behaviors, and address clients' animal care concerns with practical, appropriate and affordable solutions. these emerging opportunities include: despite the social reformer origins of child protective services established by humane societies and spcas in the latter third of the th century (hoy-gerlach, delgado, sloane, & arkow, ), there is a peculiar and unfortunate irony in that child and animal welfare agencies today operate wholly independently with little to no trans-species cross-fertilization of ideas, information or collaboration (arkow, ; walker, ; zilney & zilney, ) . this inter-agency communications gap can have tragic consequences, as with a case in brooklyn, n.y. in which a child whose family was under investigation by child protective services was killed by a dangerous dog in the home (baker & stelloh, ) . the author has heard anecdotally of three children under the aegis of child protection agencies in oxford, fla., and st. john, n.b., canada, who were killed by pet snakes. the potential impact of animals in the lives of children cannot be overstated and warrants expanding the ecological lens of child welfare work to include animals (risley-curtiss, ). companion animals are found in . % of households with children under age and . % of households with children over age (avma, ). melson ( ) reported that pets are more likely to be a part of children's growing up than are siblings or fathers, and that animals are pervasive in children's media, stories, imagination, and play. she reported that words for different animals-dog, cat, duck, horse, bear, bird and cow-are among the first words that toddlers learn; children include dog and cat in their initial productive vocabularies more than any other words except mama and daddy. an estimated to % of children first confront the loss of a loved one when a pet dies, disappears, or is abandoned (melson & fine, ) . melson ( ) reported that children, from an early age, view animals as living actors who have autonomy, intentionality and feeling. children are often animals' caregivers; since opportunities for and encouragement of nurturing others are rare in childhood, nurturing animals makes up a large proportion of childhood caregiving experiences. because caring for pets is gender-neutral, companion animals may develop innate nurturing skills in boys and feelings of mastery and self-efficacy among children who feel dependent and powerless. many children turn to their pets for reassurance and emotional support during times of stress. companion animals may assist children in feeling security and unconditional love and contribute to a child's cognitive and language development (risley-curtiss, ) . companion animals may be sentinels of unsafe environmental conditions, mirroring family tensions and serving as cues in assessments that explore family issues. as one of the sub-systems within a complex family system, the inclusion of questions and observations about the current and past presence of animals in a child's environment, the meaning those animals have for each family member, their care, and whether any of them have been killed or hurt can be important to effective family-centered practice (risley-curtiss, ) . children may feel safer talking about their pets' experiences before they disclose their own, thereby opening a friendly channel where children can provide important information (melson & fine, ; boat, ) . introducing therapy animals into the interview process can further advance this process, easing the stresses of such sessions, establishing rapport, providing the child with a sense of comfort, and creating a less threatening environment (menzies, ) , particularly in working with sexuallyabused children (reichert, ) . of particular concern is the nexus of animal sexual abuse or bestiality with child sexual abuse. edwards ( ) reported . % of animal sex offenders also had histories of sexually offending children or adults and . % had prior convictions for child pornography. her survey of offenders arrested for bestiality between and found at least children and adults had been directly sexually victimized by the offenders, and that in arrests ( . %) animal pornography had been used to groom a child for sexual behavior. the canadian centre for child protection ( ) reported significant overlaps of animal and human sexual abuse in a study of cases of bestiality. in % of cases, sexual abuse of children occurred as frequently as, if not more frequently, than coerced sexual abuse of an animal. in % of cases involving both child and animal sexual abuse, the offender was in a position of trust over the child, usually a close family member. child welfare workers can obtain more accurate and useful assessments of child safety and well-being by taking several steps: • in conducting child welfare checks and ongoing case management, look for: potentially abused, neglected or dangerous animals (e.g., aggressive dogs, poisonous reptiles, exotic species, dog-and cock-fighting paraphernalia); animals needing veterinary care; excess numbers of animals; and inadequate food, water or shelter. animal health issues such as fleas or other parasites could have a direct impact on the health of the humans in the home. include these findings in evaluations of the child's living environment, lifestyle and risk factors as potential threats to the child's well-being. such conditions may also be illegal under a jurisdiction's laws. • consider a turbulent history of frequent turnover of animals as potential indicators of a family's inability to make strong, lasting emotional attachments. • include the child's emotional attachment to pets as a key support which may help build resiliency and a protective factor that mitigates. treat the death or disappearance of animals as potentially as emotionally charged as the death of a human family member. • identify whether the child has been traumatized by witnessing or causing the abuse or death of animals. • consider animal maltreatment as a factor that supports a finding of child abuse or neglect. • report suspected animal maltreatment to the animal control/services, humane society/spca, or law enforcement agency in that jurisdiction. the reporter need not prove that animal abuse occurred, but rather introduces the case into those agencies' investigative systems to vet the information and follow through as appropriate. confidentiality restrictions may be waived in reporting to another such law enforcement agency, or when the welfare of the client and others in the household is threatened. establishing channels of communication with animal welfare agencies in advance can simplify the cross-reporting process when a case of suspected abuse occurs. • a history of animal cruelty, and a child's emotional attachments to animals, may have evidentiary importance in court trials, dispositions and hearings involving child maltreatment, custody, visitation, removal, and protection orders. by asking three simple open-ended questions, social workers can learn much about a child's experiences with the animals and humans who share his or her environment: • are there animals at home? • how are they cared for? • are you worried about their welfare? follow-up questions about their names, breeds, play activities, deaths or disappearances, recent health problems or injuries, and secrets the child shares with them may fill in details of the family dynamics, patterns of power and control, and risk and resiliency factors in the child's life. facility animals in children's advocacy centers, casa (court appointed special advocates), guardians ad litem programs, and courtrooms provide emotional support to sexual abuse survivors as they undergo forensic examinations, re-live their experiences, and confront their abusers (labahn, ) . as of may, , an estimated dogs are working in courtrooms and children's advocacy centers in states, plus others in canada, australia, chile, and europe (courthouse dogs foundation, a , b , c . elaborate precautions prevent handlers from violating client confidentiality and keep the dog's presence from adversely eliciting sympathy from a jury. judges must balance the accommodation for a vulnerable witness with the potential for prejudice which could impact the defendant's right to a fair trial. extensive guidelines on best practices protect the interests of the animal, the victim, the defendant, and the criminal justice system (courthouse dogs foundation, ) . social workers in victim services can be trained to be facility animal handlers or secondary dog handlers to allow the children to spend more time with the dogs after concluding their testimony. they can facilitate interactions between the dogs and distraught family members and stressed facility staff and be resources who connect individuals and institutions with facility animals in their community. nonprofit animal welfare and governmental animal control agencies have historically operated in isolation outside the purview of human services agencies, leading to a "silo" effect in which cross-disciplinary and trans-species collaborations rarely occur (becker & french, ) due to increased specialization, avoidance of "mission creep" and fear of violating confidentialities. consequently, interagency cooperation and cross-training is minimal, resulting in a significant barrier to change. meanwhile, animal shelter workers experience significant stressors including animal suffering and euthanasia, responsibility for life, abusive clients, negative public perceptions, and attachments to animals under their care (schneider & roberts, ) without recognizing that their counterparts in human services often experience similar stressors. this lack of knowledge and coordination among community systems constricts the potential for creative and effective collaborations and can increase the risk of harm to people and animals in situations where both human and animal abuse co-occur. social workers can facilitate bridging these segregated service delivery systems through the profession's longstanding commitment to community-level action, intervention and change. social workers can work with animal shelters to organize species-spanning community coalitions, link organizational champions, and connect consumers and professionals for the well-being of underserved and at-risk individuals and family members (long & kulkarni, ) . social workers can help animal shelters build capacity by coordinating inter-disciplinary interaction and communication, gathering data, conducting research, and building resources. increasing cross-systems knowledge and promoting individual and institutional relationships across systems, particularly vis-à-vis cross-reporting animal, child and elder abuse, can protect vulnerable unlike coordinated state-run child protection systems, animal protection is handled exclusively on the local level by a fragmented patchwork of independent government animal control/services agencies, municipal/county law enforcement, and nonprofit humane societies/spcas. a national directory of animal abuse investigation agencies in over , jurisdictions is available at https ://natio nalli nkcoa litio n.org/how-do-i-repor t-suspe cted-abuse . populations and develop stronger community services (long & kulkarni, ) . numerous animal shelters, often working with juvenile and adult detention centers, have implemented animal-assisted therapy interventions where individuals who have offended, or who are at risk, train dogs with behavior problems who are at risk of being euthanized. using positive reinforcement techniques, these programs teach teamwork, non-violent conflict resolution and collaboration skills to save animals' lives and modify the behaviors of abusive and traumatized individuals (arkow, b). animal shelters appear poised for such systemic change. the service philosophy in the animal shelter community is evolving to recognize that treating the symptoms of animal welfare problems, such as animal homelessness, abuse and neglect, is only a stopgap solution: to be truly effective, underlying community and family dysfunction and violence must be addressed (petlynx, ). hoy-gerlach et al. ( ) described promising opportunities for social work field placements in community animal shelters, including: reducing staff and volunteers' compassion fatigue in an exceedingly difficult and emotionally draining work environment; placement of shelter pets as emotional support animals; strengthening community responsiveness to violence through assessing overlaps and differences between child, elder and animal abuse investigations; creating and implementing educational programming across child and animal protection systems; and increasing community awareness of the link between violence to animals and violence to humans. animal control and humane officers frequently have access to pet owners' homes in the course of their investigations, and in the process may observe conditions detrimental to the welfare of children, youth and others. in addition, cruelty investigations which result in the removal of animals from a home could be an additional stressor on the family system and could lead to increased risk for vulnerable family members. social workers can train shelter personnel on the intersectionality of animal abuse and human violence and the procedures for making referrals to social services agencies. other untapped social work opportunities in animal shelters might include: strengthening collaborations with domestic violence shelters and mobile meals programs; directing and expanding pet visitation programs for long-term care facilities and animal-assisted interventions for at-risk populations; developing pet loss grief support groups; developing safety net supportive programming for individuals who experience a medical, economic or housing crisis that temporarily makes it difficult to keep an animal; defusing contentious confrontations with shelter clients; resolving customers' complaints and needs for services; and connecting pet owners with community resources, such as low-cost pet and veterinary services, animal behavioral counselors, pet food banks, and social services agencies. social workers provide essential services to survivors of intimate partner violence and their children in numerous ways, including advocacy, practice and public policy. they serve as advocates in women's shelters, criminal courts, protective order offices, hospital ers, police victim services units, government and nonprofit agencies, military family advocacy centers, fatality review teams, and elsewhere. their work encompasses crisis intervention, investigations, counseling, case management, legal services, public policy, and referrals to and liaison with community resources. all of these aspects of social work practice can take on an additional dimension by incorporating human-animal relationships into the perspective. research findings suggest that in working with pet-owning domestic violence victims, social workers must consider the welfare of the women's pets in order to effectively help the women achieve safety for themselves and their families (strand & faver, ) . domestic violence shelters in the s began raising awareness that significant numbers of survivors (usually, but not always, women) and their children are either turned away from safehouses that will not accept their pets or are refusing to leave abusive situations for fear of what would happen to their pets if they left. these fears range from the mundane-that no one remaining at home would provide adequate care-to the tragedies of seeing animals tortured and killed in an emotional extortion that warns partners that they themselves could be the abuser's next victims. the issue reached national awareness in when susan walsh, , told legislators in maine that her husband had retaliated against her and her children and prevented her from leaving a frightening relationship by killing her pets and farm animals: "it wasn't just the cats and the dogs i had, it was the sheep and the chickens -i was terrified for their welfare. i knew if i were to leave, he wouldn't hesitate to kill them. he had done it before." (belluck, ) . to address the problem, then-gov. john baldacci signed into law the first of what are now laws in states plus puerto rico and the district of columbia specifically allowing courts to include pets and, in some cases, livestock in domestic violence protection-from-abuse orders. these allow courts to grant petitioners exclusive care, custody and control of animals, and to forbid respondents from harming, taking or disposing of animals or even coming near them (national link coalition, c). abusers' obsessive jealousy and control can become a manipulative tool for power over partners and children by exploiting the vulnerability their emotional attachment to pets. % of battered women reported that their abusers had harmed, killed or threatened animals as coercive control (ascione, weber & wood, ) . % to % of abused women delay seeking safety in fear for the welfare of their animals (mcintosh, ) . % of ipv offenders had histories of animal cruelty (febres et al., ) , which is one of the four strongest risk factors for becoming a batterer (walton-moss, manganello, frye, & campbell, ) . animals are chosen as soft targets because abusers believe that they can get away with it because police generally don't care about animal abuse (roguski, ) . when perpetrators of ipv also have a history of animal abuse, victims experience to violent incidents before contacting police and the risk of lethality to first responders doubles (campbell, thompson, harris, & wiehe, ) . the risks encapsulated in these and similar findings are further escalated vis-à-vis the impact on children in these households: % of coercive ipv animal abuse incidents occurred in the presence of the woman; % occurred in the presence of the children (quinlisk, ) . % of domestic violence survivors in shelters reported their children had also harmed animals, repeating the intergenerational cycle of violence (ascione, ) . batterers have been reported to sexually abuse animals, threaten pets to get children to do something, or force the child to kill the pet (jury, thorburn, & burry, ) . the national link coalition ( ) modified the "power and control wheel" frequently used to graphically depict the dimensions of domestic violence to demonstrate how animal abuse is incorporated in abusers' coercive control tactics, as shown in fig. . in response to these situations, domestic violence shelters are developing collaborative foster care programs with local animal welfare agencies to provide off-site "safe havens" for the animal survivors, thereby removing one barrier that prevents families from escaping abuse (ascione, ) . more recently, a program called saf-t-sheltering animals and families together-is helping more than domestic violence shelters in the u.s. and other countries build co-sheltering facilities for pets to keep all family members together and safe (phillips, ) . grant funding is available to help shelters with capital costs and survivors with veterinary and boarding expenses (national link coalition, d). these concerns dictate bringing social workers into the planning process for innovative and collaborative intakes, assessments, responses, and referrals that incorporate human-animal relationships. serious gaps often separate domestic violence and animal shelters: although concern for the safety of pets and livestock is a barrier to individuals leaving situations of ipv in urban and rural areas, one study reported that . % of animal welfare representatives and . % of human service representatives, respectively, reported no collaboration between their agencies (saskatchewan spca & stops, ). acts of animal cruelty in mental health assessments and rehabilitation of abusers and in the specialized domestic violence assessment of risk to children. • including relocation of pets in domestic violence agencies' safety plans (national link coalition, ). • obtaining information from local animal welfare and control agencies about prior investigations at the household. • inviting animal-assisted therapy teams into shelters to help comfort survivors. • counseling children regarding incidents of animal maltreatment, death or disappearance of pets that they may have witnessed or committed. • developing community education campaigns to alert the public and cross-train professionals about how animal abuse is linked with ipv. veterinarians, in particular, whose staffs and clients are predominantly female, should begin to recognize a responsibility to serve as resources for survivors of ipv (larkin, ; newland, boller, & boller, ) . the well-established role of social workers as advocates for social justice provides additional opportunities to advance legislation that recognizes human-animal relationships, the beneficial aspects of pet ownership on individual and community health and well-being, and the adverse effects of animal abuse on human welfare and safety. as an underserved population, animals are classified as property and have long been ignored by the legal system; legislators frequently trivialize campaigns to protect their interests for the simple reasons that animals don't vote and human concerns are widely viewed as being more pressing. however, recognition of the foundation that animal abuse is linked to human violence and therefore improving animal welfare improves human society is generating a new respect for animal welfare legislation. current relevant public policy issues include legislation that would: • allow courts to include pets and/or livestock in protection-from-abuse orders (currently enacted in states, puerto rico and the district of columbia) (national link coalition, c). • allow courts to award custody of pets in divorce and marriage dissolutions based upon the animals' best interests, similar to long-standing similar provisions affecting child custody (four states). • redefine animal abuse when committed as coercive control as also being an act of domestic or dating violence ( states). • allow acts of violence against animals to be included in criteria for extreme risk protection orders that bar domestic violence abusers from obtaining firearms. • allow courts to appoint pro bono advocates to represent animals' interests in criminal cruelty cases, similar to established court appointed special advocates (casa) provisions for children (three states). • mandate or permit child welfare, adult protection and animal services agencies to cross-report incidents of suspected animal, elder and child abuse to each other, and veterinarians to report suspected animal, child and elder abuse to appropriate agencies, with immunity from civil and criminal liability and professional disciplinary sanctions. • increase penalties for bestiality (now often considered animal sexual abuse) based upon increased evidence of its co-occurrence with child sexual abuse and child pornography. as of august, , having sex with animals is still legal in hawai'i, new mexico, west virginia, and wyoming. • increase penalties for acts of animal cruelty when committed in the presence of a child or adolescent. clinical social workers may become aware of clients' human-animal interactions through recognizing a client's attachments and issues vis-à-vis the animals in his or her environment and by introducing animals for therapeutic purposes to enhance the client-practitioner relationship. in one of the earliest writings on the nexus of social work and human-animal interactions, netting, wilson, & new ( ) outlined seven ways in which social workers can contribute to human-animal bonding: • being sensitive and supportive in counseling clients who have pet-related problems. • being aware of clients' relationships with their pets and assisting in locating support services that include pet care. • being aware of policies that affect pet ownership, such as restrictive housing conditions, and advocating for clients' pet-related interests. • assessing clients to determine their readiness to accept pet-related interventions. • being critical of how pet-related programs are developed and collaborating with animal professionals. • acknowledging potential benefits and problems which may accompany pet-related programs. • linking veterinarians into the human services referral network. more recently, silverman ( ) published a brief guide to ways in which social workers can utilize animals as a bridge between a therapist and patient in private practice. animals can expedite rapport building with patients who have issues with attachment disorders and enhance the motivation to attend the session which improves retention and treatment outcomes. animals may function as a surrogate of the therapist and allow for more ethical therapeutic touch, which could be a corrective experience for those with histories of trauma. fostering the human-animal connection may help patients identify sustainable, long-term support to manage symptoms and maintain functioning after the therapeutic relationship with a clinical social worker has ended. she identified four categories of animals utilized in a helping capacity: • service animals, which are individually trained to do specific tasks for a person with a physical or sensory disability. clinical social workers may recommend that a patient consider having a service animal and identify resources to obtain one. • emotional support animals, a newer and vaguer category, that provide emotional benefits to a person diag-nosed with a mental health disorder that impairs or limits functioning in one or more life domains. • comfort dogs, introduced in disaster responses to offer a calming presence to survivors and first responders. • animal-assisted therapy animals, professionally evaluated to be introduced in treatment plans with intentional, goal-directed activities to complement traditional interventions. social workers should note that spouses and partners may be jealous of a disabled individual's dependence upon and emotional attachment to a service animal. also, the emotional support animal system has a potential for egregious abuses by individuals getting their animals so designated solely to accompany them on airplane flights; online services will provide such documentation from mental health professionals who have never examined the client. human-animal bonds may be particularly robust with older clients and present unique challenges. for individuals who are socially isolated, pets may be a significantly vital source of companionship and emotional support. caring for a pet may be an especially strong motivator for a client to get out of bed, have a daily routine, nurture another being, or go for a walk. the animal may be a last link to a deceased spouse (arkow, a). human-animal social work issues relevant to older adults include: • animal neglect: more than % of adult protective services respondents to a national survey reported animal neglect coexisting with a client's inability to care for himself/herself, indicating that reports of animal neglect may be an important warning sign for vulnerable adults' self-neglect (lockwood, ) . animals may be neglected by frail elders who lack financial resources, transportation, or physical or mental capacity to care for them adequately (peak, ascione & doney, ). • self-neglect: frail elders may neglect their own needs by spending limited financial resources on their animals' food and medications. some may refuse to go into hospitals, assisted living or long-term healthcare facilities unless provisions are made for their pets (boat & knight, ) . • coercive control: in more than two-thirds of domestic/ elder abuse cases, the perpetrators were family members who may neglect or abuse the elder's pet as a form of control or retaliation, out of frustration over their caretaking responsibilities, or as a way to extract financial assets from the victim (humane society of the u.s., ). • bereavement: isolated seniors may experience profound grief and depression upon the death of a beloved pet. some seniors are reluctant to replace departed pets in fear that the animals will outlive them (boat & knight, ) . many older adults, particularly the widowed and elderly, are at risk of emotional trauma and experience significant disruptions in eating, sleeping, job-related responsibilities and other daily routines and decreased socialization behaviors following the death of a pet (quackenbush, ) . • denied services: home health aides, social workers and other caregivers may be reluctant to enter seniors' dwellings if they fear the presence of aggressive animals or deteriorated environmental conditions linked with animal hoarding or neglect (boat & knight, ) . • animal hoarding: animal hoarders may come from any cohort but they are statistically over-represented by older women (patronek & nathanson, ). animal hoarders (and their children) often live in unhealthy environments surrounded by dozens and even hundreds of living and deceased animals in states of neglect, starvation and suffering. stereotypical hoarders, often labeled as "cat ladies," have been reported as living in a self-fulfilling cycle of social isolation: they gravitate towards animals because they are uncomfortable around people, and other people choose not to associate with them because of their excess number of animals. many are experiencing mental health issues and a collaborative, multi-agency response is invariably required (patronek, loar, & nathanson, ) . nathanson ( ) , in identifying four core barriers that limit adult protective services workers' involvement in these cases, called animal hoarding one of the most perplexing and problematic human-animal relationship and a deviant behavior associated with extremely deleterious conditions of comorbid animal and selfneglect. she identified training programs that can better prepare human services professionals to respond to these clients and engage in trans-species and interdisciplinary efforts essential for the safety, health and well-being of the hoarder, human and animal dependents, property, and community. social workers, whether in private practice, nonprofit organizations or governmental adult protective services agencies, can recognize the import of these human-animal relationships, locate support services for the animals and make appropriate referrals including temporary foster care and other pet services for owners who are in need of hospitalization, long-term care or other social services. social work input on multidisciplinary teams can help to resolve the particularly challenging psychosocial aspects of animal hoarding. and social workers should be attuned to the potential that a client's requesting a veterinarian to have all of his or her pets euthanized is a potential sentinel warning sign for suicidal behavior. social workers can help train veterinarians in recognizing this warning sign and responding with appropriate referrals. an emerging frontier is exploring veterinary medicine's response to suspected domestic violence. an incident in deland, fla. in , when a woman being held captive at gunpoint by her abusive boyfriend was able to alert veterinary staff who in turn called the police (robbins, ) , brought to national attention what was just beginning to be discussed in professional journals: how should veterinarians and their staffs, the majority of whom now are women (kelly, ) , respond to suspected domestic violence in their clientele? (newland et al., ; larkin, ; allison, satterwhite, ramaswamy, hynek, & agnew-svoboda, ) . veterinarians in the united kingdom and new zealand have taken the most proactive responses in addressing this concern. medics against violence, a scottish collaborative of human and veterinary healthcare professionals, created a domestic abuse veterinary initiative to train veterinarians to help pet owners needing to escape domestic violence; the initiative was featured in a british veterinary association guidance for responses to suspected domestic abuse (animal welfare federation and the links group, ). in , the scottish government put £ , into a national campaign to train , front-line professionals in the three fields identified as most likely to encounter domestic violence survivors: dentists, veterinarians and hairdressers (paterson, ) . the u.k.'s code of professional conduct for veterinary surgeons states, "given the links between animal, child and domestic abuse, a veterinary surgeon or veterinary nurse reporting suspected or actual animal abuse should consider whether a child or adult within that home might also be at risk" (royal college of veterinary surgeons, ). the new zealand veterinary association supported a national legislative response to family violence by describing veterinary medicine as a "three-dimensional profession" with a unique voice in issues that transcend animal life, human life and the environment. nzva called for domestic violence protection-from-abuse orders to specifically include animals, and for changing the definition of domestic violence to include "coercive control" which would cover emotional and psychological abuse to family members through threat or harm to pets or farm animals (national link coalition, ) . the veterinary council of new zealand ( ), whose code of professional conduct includes a recommendation that veterinarians confronted with situations of animal abuse should consider whether people within that home might also be at risk, published a guidance that included suggestions on preparing the practice and responding to domestic violence. social workers can help to introduce a response to intimate partner violence as a public health matter to a profession which has been reluctant to get involved, due to a lack of training and fears for personal safety, and help veterinary clinics develop protocols for response and dissemination of literature about community domestic violence resources to their clients. they can also coordinate programs linking students at colleges of veterinary medicine with local domestic violence shelters, such as has been done at texas a & m, mississippi state university, and the university of georgia. social workers can respond to the needs of pet owners who are homeless, whose attachments to their animal companions are often stronger compared with the general population (labrecque & walsh, ). an estimated % to % of the . million americans who experience homelessness every year have dogs and cats, with rates as high as % in some areas. because the vast majority of homeless shelters do not allow pets, these restrictions deter pet owners from seeking essential shelter (o'reilly-jones, ). many individuals who live on the street keep pets, primarily dogs, for emotional support, safety, a sense of responsibility, to combat loneliness (labrecque & walsh, ; williams & hogg, ; arnott, ) , and as social catalysts to attract passers-by who may offer them money (irvine, kahl, & smith, ; anderson, snow, & cress, ) . social workers can coordinate veterinary and foster care for the animals and advocate for pet-friendly co-shelters for the homeless much as has been done in domestic violence shelters (phillips, ) . social workers can participate in such programs as the street dog coalition, operating in states, in which social work, veterinary and medical school students host clinics and provide resources to help the pets of homeless pet owners. an accurate representation of the roles of animals in families, and of social work's responsiveness to these dynamics, is compromised by several factors. despite the above-cited market research data indicating a widespread population of companion animals and their over-representation among families with children, relatively little is known about the racial, ethnic, socioeconomic, age-related, or geographic demographics of pet-owning families, as such information has never been included in the u.s. census (arkow, a). meanwhile, until recently, social workers have historically ignored the central role that companion animals may play in the lives of their clients, adopting an anthropocentric view underpinned by human rights and social justice (laing & maylea, ) . this is reflected in reports (national link coalition, ) that identified only schools of social work in the u.s. and seven in canada that are believed to include the topic of human-animal relationships in either undergraduate or graduate level study, or in the absence of such courses have a faculty member known to have a specialization in the human-animal bond. it is hoped that publications such as this will begin to address these shortcomings and increase awareness of human-animal relationships in the lives of social workers' clients. the inclusion of human-animal relationships should be considered more widely in training and practice as part of social work's commitment to social and environmental justice and fighting oppression and seen as an expanding opportunity for research, practice, advocacy, and advancing public policy. in the process, additional career opportunities may open up with this species-spanning approach to resolving individual, family and community challenges. such inclusion can begin with something as simple as routinely including companion animals in genograms, ecomapping, and definitions of family support systems (risley-curtiss, ; hodgson & darling, ) . as assessing clients' needs is an important step in developing the best plan to solve clients' problems, including pet protective factors in clients' ecologies should be considered a relevant environmental factor in social work practice theory (sato, ) . collecting information about all the pets and humans in a family communicates interest and concern for the whole family and demonstrates an integrated approach to care that can help in planning appropriate interventions and preventive care. human-animal bond awareness can be further expanded by adding relevant coursework and field placements in schools of social work and training programs in continuing education. given the established links between animal cruelty and other forms of violence within the family system (arkow, b), questions about human-animal interactions and relationships and clients' committing and/or witnessing acts of animal abuse should be systemically, not just optionally, introduced in intakes and assessments. incorporating the significance of human-animal interactions can help modernize what has been an intrinsic anthropocentrism of social work's theoretical foundations. growing opportunities both within and beyond the veterinary environment will help convince educators, researchers and practitioners that this species-spanning approach is worthwhile and offers opportunities for career development, personal fulfillment and improved service delivery. veterinary social work blends the human side of veterinary medicine with the animal side of social work. as awareness of and interest in veterinary social work continues to grow, additional opportunities will emerge whereby social workers with an abiding interest in animals as well as people can help their clients, society, and the non-human members of families and communities. battered women's reports of their partners' and their children's cruelty to animals safe havens for pets: guidelines for programs sheltering pets for women who are battered emerging research on animal abuse as a risk factor for intimate partner violence child abuse, animal abuse and domestic violence: linking the circles of compassion for prevention and intervention the abuse of animals and domestic violence: a national survey of shelters for women who are battered boy, , is mauled to death by dog in brooklyn home animal abuse among preadolescents directly and indirectly victimized at school and at home pet loss and grief: identifying at-risk pet owners during the euthanasia process making the links: child abuse, animal cruelty, and domestic violence new maine law shields animals in domestic violence cases understanding the role of animals in the family: insights and strategies for clinicians experiences and needs of adult protective services case managers when assisting clients who have companion animals dogs will fix our broken democracy following the links: the critical role of animal control in improving detection and prevention of family violence characteristics of intimate partner violence incidents and the environments in which they occur: victim reports to responding law enforcement officers intimate partner violence and pet abuse: responding law enforcement officers' observations and victim reports from the scene bestiality" as reflected in canadian case law grieving the death of a pet animal assisted therapy in counseling facility dogs at children's advocacy centers and in legal proceedings: best practices courthouse facility dogs in the u appellate case law casa/gal programs animal cruelty by children exposed to domestic violence social work with a pet loss support group in a university veterinary hospital. social work in health care arrest and prosecution of animal sex abuse (bestiality) offenders in the united states cute and cuddly and a whole lot more? a call for empirical investigation into the therapeutic benefits of human-animal interaction for children adulthood animal abuse among men arrested for domestic violence the animal-human bond: health and wellness effects of pet contact on human well-being animal cruelty, antisocial behaviour, and aggression: more than a link the relationship between bullying and animal abuse in adolescents: the importance of witnessing animal abuse bullying and animal abuse: is there a connection? pets in the family: practical approaches violence in animal cruelty offenders rediscovering connections between animal welfare and human welfare: creating social work internships at a humane society helping vulnerable adults and their pets confrontations and donations: encounters between homeless pet owners and the public the world's children and their companion animals: developmental and educational significance of the child/ pet bond. washington: association for childhood education international pet abuse as part of intimate partner violence. wellington, nz: national collective of independent women's refuges veterinary medicine is a woman's world. veterinarian's money digest providing comfort, companionship, and relief: the use of courtroom dogs homeless women's voices on incorporating companion animals into shelter services traumatized witnesses: review of childhood exposure to animal cruelty they burn brightly, but only for a short time": the role of social workers in companion animal grief and loss is counseling going to the dogs? an exploratory study related to the inclusion of an animal in group counseling with adolescents for human needs, some veterinary clinics are turning to a professional: social workers see a place for themselves in veterinary practice when domestic violence arrives at the clinic door: how veterinary staff can respond to abused clients and patients the community context of animal and human maltreatment: is there a relationship between animal maltreatment and human maltreatment: does neighborhood context matter? (doctoral dissertation making the connection between animal cruelty and abuse and neglect of vulnerable adults cross-reporting of interpersonal violence and animal cruelty: the charlotte project animals as social supports: insights for understanding animal-assisted therapy the links between animal abuse and family violence, as reported by women entering shelters in calgary communities cultural factors in the cause and prevention of pathological homicide why the wild things are: animals in the lives of children children's ideas about the moral standing and social welfare of non-human species animals in the lives of children animals in the lives of children animal assisted therapy and young people: a review of selected literature social facilitation of contact with other people by pet dogs a therapist's guide to treating grief after the loss of a pet: a three-tier model animal hoarding: slipping into the darkness of comorbid animal and self-neglect three-dimensional" new zealand veterinarians respond to domestic violence the connection between animal cruelty and societal violence and vulnerability schools of social work with humananimal interactions in curriculum the human-animal bond: implications for practice considering the relationship between domestic violence and pet abuse and its significance in the veterinary clinical and educational contexts when fido is family: how landlordimposed pet bans restrict access to housing revisiting a link: animal abuse, bullying, and empathy in australian youth vets enlisted in bid to stop domestic abuse. the national animal hoarding: structuring interdisciplinary responses to help people, animals and communities at risk a theoretical perspective to inform assessment and treatment strategies for animal hoarders adult protective services and animal welfare: should animal abuse and neglect be assessed during adult protective services screening national urban animal report start-up manual bowling alone: the collapse and revival of american community the pet connection: its influence on our health and quality of life pets, owners, problems, and the veterinarian: applied social work in a veterinary teaching hospital. the compendium on continuing education for the small animal practitioner social work services for bereaved pet owners: a retrospective case study in a veterinary teaching hospital child abuse, domestic violence, and animal abuse: linking the circles of compassion for prevention and intervention individual counseling for sexually abused children: a role for animals and storytelling exploring the grief experience among callers to a pet loss support hotline social work practitioners and the human companion animal bond: a national study expanding the ecological lens in child welfare practice to include other animals she was family:" women of color and their animal human connections the animalhuman bond and ethnic diversity florida woman held captive by boyfriend slips note to vet staff pets as pawns: the co-existence of animal cruelty and family violence pet loss and children: establishing a healthy foundation code of professional conduct for veterinary surgeons, supporting guidance no. (client confidentiality). london: royal college of veterinary surgeons nonhuman animal cruelty, bullying, and behavioral difficulties among women saskatchewan society for the prevention of cruelty to animals and saskatchewan towards offering partnership solutions (stops) to violence social workers' attachments to their pets, organizational structures, and their impact on professional assessment regarding the roles pets play in clients' lives (doctoral dissertation) shelter-specific occupational stress among employees in animal shelters the role of animals as therapeutic aids in private practice battered women's concern for their pets: a closer look the role of companion animals throughout the family life cycle effects of childhood adversity on bullying and cruelty to animals in the united states: findings from a national sample guidance for veterinarians dealing with cases of suspected or actual animal abuse and family violence the link between interpersonal violence and animal abuse a study on the relationship of child abuse and pet abuse animal cruelty and bullying: behavioral markers of delinquency risk or causal antecedents of delinquent behavior? risk factors for interpersonal violence and associated injury among urban women the health and welfare of dogs belonging to homeless people wa: petcare information & advisory service and the centre for the built environment and health (school of population health) the pet connection: pets as a conduit for social capital? more than a furry companion: the ripple effect of companion animals on neighborhood interactions and sense of community social capital and pet ownership-a tale of four cities reunification of child and animal welfare agencies: cross-reporting of abuse in wellington county key: cord- -lmkfxme authors: schafrum macedo, aline; cezaretti feitosa, caroline; yoiti kitamura kawamoto, fernando; vinicius tertuliano marinho, paulo; dos santos dal‐bó, Ísis; fiuza monteiro, bianca; prado, leonardo; bregadioli, thales; antonio covino diamante, gabriel; ricardo auada ferrigno, cassio title: animal modeling in bone research—should we follow the white rabbit? date: - - journal: animal model exp med doi: . /ame . sha: doc_id: cord_uid: lmkfxme animal models are live subjects applied to translational research. they provide insights into human diseases and enhance biomedical knowledge. livestock production has favored the pace of human social development over millennia. today's society is more aware of animal welfare than past generations. the general public has marked objections to animal research and many species are falling into disuse. the search for an ideal methodology to replace animal use is on, but animal modeling still holds great importance to human health. bone research, in particular, has unmet requirements that in vitro technologies cannot yet fully address. in that sense, standardizing novel models remains necessary and rabbits are gaining in popularity as potential bone models. our aim here is to provide a broad overview of animal modeling and its ethical implications, followed by a narrower focus on bone research and the role rabbits are playing in the current scenario. rabbits have been used for decades by researchers in diverse scientific fields. however, only recently have they been targeted as potential bone models. with great importance in age-related bone loss research. , here, we first present a broad historical review and some key ethical points in animal modeling. we then take a closer look at bone research and the role rabbits play in this field. animal domestication was a significant turning point for mankind. human society developed into what it is today due to livestock production, and animals still provide us with food, clothing, transportation, protection, and companionship. , nowadays they contribute to human well-being in additional ways: by helping people with visual impairment or diabetes, by taking part in police enforcement, or even by entertaining people in animal shows, zoos, and social media. animals have also been pivotal to our medical knowledge and health status since ancient greece. , the first animal studies provided understanding of biological pathways and disease mechanisms. animal dissection proved to be a valuable substitute for human dissection -an illegal practice in ancient times. several philosophers and physicians, from aristotle to diocles and erasistratus, experimented on animals. alcmaeon of croton ( - bc) was the first physician to document and publish anatomical observations of canine dissections. , he established brain control over intelligence and sensory perceptions. centuries later, aelius galenus (also known as galen of pergamon, - ad) would make pivotal discoveries based on animal experimentation. galen served as a doctor to different roman emperors. his public demonstrations of cutting laryngeal nerves in squealing pigs made him famous. he also made important anatomic observations on cranial and spinal nerves. he included findings from more than animal species. , in the late nineteenth century, claude bernard set the foundations of experimental medicine by developing rigorous guidelines for controlled studies. , , animal-based research has been the cornerstone of health sciences ever since. it accounts for more than % ( / ) of all physiology or medicine nobel laureates' studies. research on the diphtheria vaccine-developed in guinea pigs (cavia porcellus)-received the very first prize in . other fundamental discoveries, like the insulin mechanism and pasteur's and koch's studies, are also credited to animal research. , , animal welfare has not always been a concern. proper acknowledgment of an animal's moral status as a sentient being is a recent development. , for most part of the history, animals were considered senseless to pain and were treated with little or no respect in research, teaching, and demonstrations. , for centuries, animals were perceived mainly as useful tools. , most greek philosophers excluded animals from moral judgments, especially those derived from stoic and epicurean beliefs. other philosophical strands, such as cynicism, were more empathetic to the well-being of animals. nevertheless, assumptions that animals are entitled to ethical consideration and can indeed perceive pain and negative feelings only emerged during the renaissance. , , french philosopher rené descartes ( - ) acknowledged that animals could perceive sensations, but in a purely mechanical way. based on this cartesian perspective, scientists justified the use of animals without concern for their feelings for centuries afterwards. , , when william harvey demonstrated blood circulation on conscious dogs, the attending public believed the painful screams were part of a "beast machinery," like an automatic sound. , only by the second half of the nineteenth century, in victorian europe, animal rights would be debated among the mainstream philosophers. , jeremy bentham's introduction to principles of morals and legislation ( ) was a turning point. emphatic attitudes displayed by influential thinkers like rousseau and schopenhauer helped shaping a new approach towards animal welfare. , darwin's evolutionary insights (published in ), emphasized our moral duty towards animals. [ ] [ ] [ ] the cruelty to animals act-passed in -was the first official legal document to set boundaries on animal experimentation. however, the dominant approach to animal research remained utilitarian. , in the late s, russell and burch developed the "three rs concept" to rationalize animal use by replacing, reducing, and refining resources. these guidelines aim to minimize animal distress and emphasize our duty to search for alternative technologies. bioethical principles are now mandatory for any animal experimentation. , today, the internet reflects public opinion on animal welfare. the attitude of young people towards animals is much more empathetic now than in previous generations. consequently, bioresearch elicits heated debates. some groups with radical views advocate banning animal research altogether. nevertheless, the unlimited potential and importance of animal-based discoveries cannot be denied. five key bioethical points are considered when assessing the moral status of animal subjects in research: the presence of life, the ability to feel and perceive stimuli, the level of cognitive behavior, the degree of sociability, and the ability to proliferate. scientific proof of animal consciousness and sentience is a recent achievement. however, there is no global consensus on the value people attach to particular animals. in some cultures, the western household dog is no more than a food source. the same is true for research. using animals like monkeys, dogs or cats as models will likely evoke adverse reactions nowadays. the social perception of the animal's "worthiness" is called "speciesism". at this point in time, animal research cannot be entirely replaced by in vitro testing. developing alternative methods is essential. scientists can now create and cultivate microfluid organ-on-a-chip models. but these new technologies are still under development. hopefully future studies will provide the means to replace animal experiments. until then, ethical treatment and rational use of all living forms are still necessary. , in that context, characterizing alternative models remains a goal. rabbits, for instance, may be potentially useful bone models. they are already used as laboratory subjects in several medical fields. even though they are also praised as household pets, particularly in europe, their use in laboratory is well accepted. , many species can be suitable models for different diseases. the research question will dictate what type of model should be considered. undoubtably, rodents are the most popular laboratory subjects worldwide. rats (rattus norvegicus) have been part of medical studies since the nineteenth century ( ). they reached peak importance with the development of the wistar strain, in . although mendel started studying the laws of inheritance on mice (mus musculus), he shifted his methods to peas after facing religious restrictions on his animal model. , rodents became the standard choice for genetic experimentation after watson and crick published their dna study. during the s, the first "gene knockout" mouse was developed. this study won a nobel prize. , using models is very attractive because one can easily ensure homogeneity between subjects-unachievable otherwise. then future studies can reproduce similar conditions. , for obvious reasons, the greater the model's similarity to humans, the greater are the moral implications. the planning phase is the moment to define the best model to answer the research question, avoiding unnecessary enrollments. the "ideal model" does not exist. no single animal-aside from humans-can perfectly exhibit human responses. researchers must choose the most suitable option, considering the objectives of the study. careful planning is mandatory. it should be kept in mind that sometimes more than one type of model might be necessary to answer the research question. multi-level assessment is required to identify the possible advantages and challenges of any given model and table provides a template guide. animal models have taught us much about bone disorders and have been central to developing many treatments throughout history. their contribution remains paramount for assessing bone physiology and immunology, since in vitro alternatives cannot fully reproduce whole-organism physiological behavior. they remain beneficial to the whole orthopedic field. either by mimicking diseases in arthrology and oncology studies or by allowing surgical training, animals are still essential to medicine. nonhuman primates are our best biological representation. for that reason, using them nowadays for scientific purposes elicits public. aside from moral implications, their size and ease of handling in experiments are difficulties, besides being financially demanding. working with primates also requires very well-trained staff (owing ta b l e schematic compilation of traits and possible challenges to consider when planning to use an animal model , , purpose/approach to their unpredictable aggressive behavior and zoonotic potential), which limits their research potential. our second closest model on the structure of bone is dogs. despite individual variations on macrostructure, their bone remodeling is somewhat similar, and they exhibit similar haversian structure. dogs used to be popular research subjects due to their medium size, ease of handling, and docile behavior. , today, these classical models are no longer feasible. , over recent decades, a paradigm shift regarding animal use in research has occurred. the fields of laboratory sciences, animal welfare and alternative methods for replacing animal use have expanded considerably to overcome the lack of public acceptance of the classical models. one of the most studied-and prevalent-disorders nowadays is osteoporosis. age-related osteopenia is a public health concern of growing importance. demographic aging and the urban lifestyle of western societies have led to this modern disease. the world health organization considers osteoporosis a significant age-related disease and has developed global strategies for its prevention, management, and surveillance. osteoporosis causes unbalanced bone formation/resorption and decreases bone mass. the weakened bones are more prone to suffer a fracture, even with low-impact injuries. pathological fractures occur mainly at the hip joint and vertebrae. they may even go unnoticed in elder patients. the domestic rabbit (oryctolagus cuniculus) is a small digging lagomorph of the family leporidae. in the modern age, there are only two living families: leporidae (rabbits and hares) and ochotonidae (pikas), with genera currently recognized. more than rabbit breeds exist worldwide. rabbits exhibit desirable traits for bone research. these calm and easily handled creatures have a short lifespan and breed readily in captivity. the new zealand white rabbit is the most popular research breed. furthermore, rabbits are phylogenetically closer to primates than rodents. they reach skeletal maturity between and weeks of age (females earlier). adults display some haversian remodeling and their bone metabolism is somewhat similar to humans. however, surgical castration is not enough to mimic satisfactory bone loss and other techniques must be associated. , rabbits display less cancellous bone than humans. , they have more fragile cortices. , cortical thickness and the diameter of drilled holes contribute to the high complication rate of fracture repair in this species. their functional anatomy allows their peculiar high-speed hopping to evade predators. cage confinement and exercise restriction might be harmful to their bone development and researchers should consider alternative in-house systems, as opposed to small cage confinement. in their natural habitat, rabbits are a prey species, which explains their curious but easily scared behavior and also explains some anatomic features that enable them to escape at high speed when in danger. their peculiar appendicular skeleton (figure ) must be light weight but also resistant to allow their burrowing and food-seeking behaviors. their hindlimbs have high power hip extensor muscles concentrated at the proximal part. muscle mass in the front limbs is distributed more distally and accounts for approximately % of the total body mass. their fibula fuses to the middle shaft of the tibia. their four long webbed toes on each hindlimb allow accelerated digitigrade hopping. their small clavicles resemble those of domestic cats and make them more agile. , a survey of the terms "rabbit" and "experimental model" in pubmed resulted in articles of indexed journals published between and , with almost from the past decade. rabbits were pivotal to the discovery of the atropine esterase enzyme, in the nineteenth century. since then, they have been used in several studies by nobel laureates. they helped characterizing the mechanisms involved in insulin production and diabetes. , , rabbits are appealing models for bone research. studies involving rabbits are now commonplace in orthopedics, and multi-species assessments of model suitability have rated rabbits as potential bone models after primates and dogs. biomechanical forces act during stance and walking in any living animal. measuring these forces is important to determine a model's bone strength, but biomechanical data on rabbit bones are still scarce. a study published the effects of in vivo loading of rabbit tibiae. biomechanical data on axial compression and bending moments in the rabbit tibia were given. the authors concluded that rabbit tibia can endure higher strain levels than goats can, therefore rabbits were better models. in another study describing the qualitative differences between mice, rats, dogs, nonhuman primates and rabbits, the authors concluded that the skeletal characteristics of rabbits were the least suitable for extrapolating to humans, but highlighted their lack of biomechanical data. rabbits are a standard model in periodontal research. they are part of diverse studies such as measurement of parathyroid hormone effects on osseointegration in osteoporosis, some recent studies have explored the potential of rabbits as models for cartilage and meniscal tears repair. they have also been used in other studies on arthrology and tendon healing. one study focused on intra-articular injections of chondroitin sulfate carried by hydrogel. others assessed tendon healing by reproducing biceps tenosynovitis, and anterior cruciate ligament and rotator cuff tears. rabbits have also increased in importance as pets. they are the third most popular companion animal in the uk, after dogs and cats. more than two million pet rabbits are estimated to have existed in the past decade. they are the most popular exotic animal in us private veterinary practices. in view of these trends, the demand for higher standards of rabbit medicine is increasing and thus the need to enhance veterinary knowledge also exists. more recent studies focus on clinical and surgical aspects of the pet rabbit. , , [ ] [ ] [ ] [ ] [ ] [ ] in a recent paper, the authors evaluated the effect of three different screw-hole diameters and torsional properties of rabbit femora. however, more in-depth biomechanical studies are lacking. there are scarce data on the torsional properties, [ ] [ ] [ ] but the main focus of these studies was bone healing and bone grafting. fracture repair in the pet rabbit remains a major challenge. rabbit bones are very thin and brittle, an important complicating factor that results in frequent implant failure. , another study has defined vertebral safe corridors for implant insertion using computer tomography. but rabbit research still has unexplored gaps to be addressed. the human-animal bond has sculpted medical knowledge. animal models play a significant role in enhancing our understanding of emerging pathologies. current in vitro technologies are very promising but still have some way to go before fully replicating whole-animal responses. rabbits have potential as bone models but conclusive studies are still lacking. however, the growing popularity of rabbits as pets may ultimately decrease their eligibility as laboratory models. the need for alternative methods to replace animals in research remains paramount. none. introduction to "working across species animal experiments in biomedical research: a historical perspective a brief history of animal modeling the development and application of laboratory animal science in china evaluation of bone regeneration using the rat critical size calvarial defect animal models for implant biomaterial research in bone: a review clinician's guide to prevention and treatment of osteoporosis the laboratory rabbit: an animal model of atherosclerosis research characterization of a rabbit osteoporosis model induced by ovariectomy and glucocorticoid an ovariectomy-induced rabbit osteoporotic model: a new perspective the rational use of animal models in the evaluation of novel bone regenerative therapies moral status as a matter of degree alkmaion's discovery that brain creates mind: a revolution in human knowledge comparable to that of copernicus and of darwin galen and the squealing pig historical perspective -andreas vesalius ( - ) research ethics in animal models foundation n. nobel prizes. nobel prizes animal consciousness, cognition and welfare animal models are essential to biological research: issues and perspectives the collected works of jeremy bentham: an introduction to the principles of morals and legislation common morality, coherence, and the principles of biomedical ethics the moral status of animals and their use in research: a philosophical review microfluidic organon-a-chip models of human intestine standards of care in the st century: the rabbit searching for animal models and potential target species for emerging pathogens: experience gained from middle east respiratory syndrome (mers) coronavirus. one heal preclinical models for orthopedic research and bone tissue engineering osteoporosis-bone remodeling and animal models the domestic rabbit, oryctolagus cuniculus: origins and history an interspecies comparison of bone fracture properties interspecies differences in bone composition, density, and quality: potential implications for in vivo bone research the recent prevalence of osteoporosis and low bone mass in the united states based on bone mineral density at the femoral neck or lumbar spine management of osteoporosis & who. prevention and management of osteoporosis: report of a who scientific group. genova: world health organization cortical bone porosity: what is it, why is it important, and how can we detect it? inter-trabecular angle: a parameter of trabecular bone architecture in the human proximal femur that reveals underlying topological motifs odanacatib, effects of -month treatment and discontinuation of therapy on bone mass, turnover and strength in the ovariectomized rabbit model of osteopenia animal models for fracture treatment in osteoporosis delay in estrogen commencement is associated with lower bone mineral density in turner syndrome bone mineral measurements of subchondral and trabecular bone in healthy and osteoporotic rabbits the mouse ascending: perspectives for human-disease models the domestic rabbit, oryctolagus cuniculus: origins and history animal models for glucocorticoidinduced postmenopausal osteoporosis: an updated review sex-related variations in bone microstructure of rabbits intramuscularly exposed to patulin effects of hole diameter on torsional mechanical properties of the rabbit femur comparative anatomical and biochemical studies on the main bones of the limbs in rabbit and cat as a medicolegal parameters a meta-analysis on the effects of the housing environment on the behaviour, mortality, and performance of growing rabbits bsava manual of rabbit medicine and surgery functional specialisation of the thoracic limb of the hare (lepus europeus) the biology of the laboratory rabbit anatomy, physiology, and behavior seasonal and sexual influences on rabbit atropinesterase the new zealand white rabbit as a model for preclinical studies addressing tissue repair at the level of the abdominal wall comparative bone anatomy of commonly used laboratory animals: implications for drug discovery biomechanics of bone: determinants of skeletal fragility and bone quality axial forces and bending moments in the loaded rabbit tibia in vivo effects of continual intermittent administration of parathyroid hormone on implant stability in the presence of osteoporosis : an in vivo study using resonance frequency analysis in a rabbit model physico-chemical and histomorphometric evaluation of zinc-containing hydroxyapatite in rabbits calvaria bone healing improvements using hyaluronic acid and hydroxyapatite/beta-tricalcium phosphate in combination: an animal study hemiarthroplasty of the shoulder joint using a custom-designed high-density nano-hydroxyapatite/ polyamide prosthesis with a polyvinyl alcohol hydrogel humeral head surface in rabbits bioactive scaffolds for osteochondral regeneration repair of osteochondral defects with biodegradable hydrogel composites encapsulating marrow mesenchymal stem cells in a rabbit model early in situ changes in chondrocyte biomechanical responses due to a partial meniscectomy in the lateral compartment of the mature rabbit knee joint intra-articular delivery of chondroitin sulfate for the treatment of joint defects in rabbit model comparison of bone tunnel and cortical surface tendon-to-bone healing in a rabbit model of biceps tenodesis a versatile protocol for studying anterior cruciate ligament reconstruction in a rabbit model into-tunnel repair versus ontosurface repair for rotator cuff tears in a rabbit model approach to preventive health care and welfare in rabbits pet rabbits comparison of two methods of long bone fracture repair in rabbits survey of the husbandry, health and welfare of pet rabbits welfare assessment in pet rabbits anesthesia and analgesia in rabbits and rodents case report surgical correction of patellar luxation in a rabbit analysis of mechanical symmetry in rabbit long bones comparison of cyclic loading versus constant compression in the treatment of long-bone fractures in rabbits physical properties of autoclaved bone: torsion test of rabbit diaphyseal bone bsava manual of rabbit surgery, dentistry and imaging computed tomographic study of safe implantation corridors in rabbit lumbar vertebrae key: cord- -rkqijcxn authors: menchaca, a.; dos santos-neto, p.c.; mulet, a.p.; crispo, m. title: crispr in livestock: from editing to printing date: - - journal: theriogenology doi: . /j.theriogenology. . . sha: doc_id: cord_uid: rkqijcxn precise genome editing of large animals applied to livestock and biomedicine is nowadays possible since the crispr revolution. this review summarizes the latest advances and the main technical issues that determine the success of this technology. the pathway from editing to printing, from engineering the genome to achieving the desired animals, does not always imply an easy, fast and safe journey. when applied in large animals, crispr involves time- and cost-consuming projects, and it is mandatory not only to choose the best approach for genome editing, but also for embryo production, zygote microinjection or electroporation, cryopreservation and embryo transfer. the main technical refinements and most frequent questions to improve this disruptive biotechnology in large animals are presented. in addition, we discuss some crispr applications to enhance livestock production in the context of a growing global demand of food, in terms of increasing efficiency, reducing the impact of farming on the environment, enhancing pest control, animal welfare and health. the challenge is no longer technical. controversies and consensus, opportunities and threats, benefits and risks, ethics and science should be reconsidered to enter into the crispr era. genome editing in large animals may be applied with different purposes, including biotechnology to improve food production, animal health and pest control, and the generation of animal models for biomedicine and basic research. the main question for an innovative technology is why it should be used. in livestock, the global context of food demand and production, as well as new concerns in terms of environmental sustainability and animal welfare, may explain the potential usefulness of crispr. increasing demand for food required by the world's growing population is one of the biggest challenges for the future of the human species. according to fao, un and wb, this consumption level is not only increased by the global population growth, exceeding billion by [ ] , but also by a higher per capita consumption of animal protein over vegetable carbohydrates. extreme poverty rates have significantly decreased over the past years and people have more access to better quality food [ ] . this growing demand has already pushed livestock production and this phenomenon will increase in the coming years [ ] . our challenge will be to achieve a balance to attain greater food production, ensuring global sustainability, preserving climate change and deforestation, respecting biological diversity and animal wellbeing, and guaranteeing equity to global food access. classical approaches to enhance productivity based on the improvement of animal health, nutrition, genetics, reproduction and management, will make an important contribution. however, this will not be enough to ensure the productive change required. this scenario needs novel ideas and disruptive technologies, and crispr appears as a powerful tool to contribute to global livestock transformation. how would the future of farm animals be with novel production traits, with resistance to transmissible diseases, or even with the eradication of pests that have harmed livestock throughout the history? with crispr we are closer to make these things happen. crispr-cas system is present in nature as a component of the bacteria and archaea immune system, protecting them from invading dna contaminants [ ] . in crispr-cas system type ii used in genetic engineering, the specificity of the cut is given by the crispr rna (crrna). this molecule needs to interact with a transactivating crrna (tracrrna) to form a crrna_tracrrna duplex, directing the crispr-associated (cas) nuclease to specific sites (spcas in the case of the streptococcus pyogenes type ii crispr system). the crrna and the tracrrna sequences can be combined into a single guide rna (sgrna), directing cas to the desired site and catalyzing the dna cleavage. once these components are introduced within the cell (or into the zygote), they will guide the cas to the complementary locus in the genome and will create a double-strand break (dsb) [ ] . the generated dsbs will be repaired through two mechanisms, non-homologous end joining (nhej) and homology-directed repair (hdr) (fig. ) . the nhej pathway frequently leads to small insertions or deletions (indels), or chromosomal rearrangements. these often disrupt open reading frames, effectively creating gene knockouts. this mechanism led to the generation of the first successful disruption of endogenous genes by crispr-cas system in livestock (sheep [ , ] , goats [ , ] , cows [ ] and pigs [ e ] ). on the other hand, the hdr pathway employs a homologous repair template to fix the dsb. this mechanism allows the creation of specific changes in the dna, which is mediated by the addition of the adequate repair template containing the desired insertion or modification. genome editing conducted by this mechanism can be used to insert a predefined single nucleotide or sequence, or even change or delete it in existing genes. after the first knockouts births reported by nhej, successful generation of knock-in large animals by hdr were achieved in sheep [ , ] , goats [ ] and pigs [ ] . amazing advancement of this technology does not cease to surprise us. recently, fusion of an inactive form of cas with a cytidine deaminase enzyme permitted to develop a new crispr tool called base editors that perform c/t substitutions [ ] . furthermore, a new promising crispr variant consisting of a fig. . the crispr pipeline to transform livestock: from editing to printing. different approaches for crispr-cas system, consisting of a single guide rna (sgrna) designed to direct cas to the desired dna site and induce double strand breaks (dsb). dna cleavage results in different gene repair mechanisms as nonhomologous end joining (nhej) or homologydirected repair (hdr, i.e., in the presence of a dna donor template). zygotes are obtained by in vitro embryo production (in vitro maturation and fertilization), or derived from in vivo production (insemination and oviduct flushing). delivery of crispr-cas components into the zygote is performed by direct microinjection into the cytoplasm, or alternatively, by electroporation with no need for embryo micromanipulation. embryo transfer is carried out either with fresh or cryopreserved embryos by vitrification with minimum volume method (e.g., cryotop). the wide range of crispr applications in large animals include improving productive traits, enhancing animal welfare through adaptation and resilience, conferring resistance to infectious and transmissible diseases, generating animal models for biomedical research, and suppressing other species considered as pests for livestock. catalytically impaired cas endonuclease fused to a reverse transcriptase allows performing precise targeted insertions, deletions and point mutations without requiring dsbs or donor dna templates. this approach, known as prime editing, presents lower offtargets activity and fewer byproducts than previous alternatives [ ] . . the design of the crispr components when designing an experiment or project, one of the main things to determine is which nuclease to use. the spcas nuclease is widely used but there are other options that should be considered depending on the purpose of the project. nucleases such as cas a or casx are also available and have unique structural and functional features, providing new opportunities for genome editing applications. cas proteins can be introduced as dna expression plasmid, in vitro transcript, or as a recombinant protein bound to the sgrna in a ribonucleoprotein particle (rnp). despite being the easiest system, plasmid dna can be randomly integrated into the genome and thus integration need to be carefully assessed. rna reagents are easy to generate and can be cloning-free, however, cas time-course experiments revealed that the rnp complex triggers mutations faster than cas mrna þ sgrna [ ] . moreover, the longer they last inside the cell, the bigger the probability to generate complex modifications and mosaics as well as more offtarget effects. the second element for genome editing by crispr involves target site selection and sgrna design. any dna locus containing the sequence ngg, named as protospacer adjacent motif (pam), represents a potential target site for crispr/cas system. nevertheless, not all the sequences induce cuts with the same efficiency and specificity. sgrnas sequences should be as unique as possible, to limit the possibility of unintended dsbs in partially complementary sequences. it is preferable to select crrnas whose seed sequences (first nucleotides adjacent to the pam) are unique, since those are essential for the specificity on a nucleotides crrna. in livestock, where gestation length is long and high-cost projects are required, failure to achieve edited offspring or inefficiencies in the system due to the editing design or reagents quality have frustrating impact and should be minimized. in this kind of projects, a careful design and previous validation of the sgrnas becomes essential. for in silico design, many web tools and software packages allow predicting the activity and specificity of the sgrnas. after selecting these sequences, it is highly recommended to validate them before generating the animals. this analysis can be done in vitro in different cell-free systems, in in vitro cultured cells of the same species, or directly in embryos. since the chromatin context influences the cutting efficiency of the crispr-cas system, more reliable results will be obtained if the sgrnas are analyzed in embryos. embryo sequencing may be conducted in the whole embryo and then, if efficiency is acceptable, proceed to embryo production sessions for embryo transfer. although it is feasible to perform a biopsy in the same blastocysts that will be transferred, pre-implantation analysis of biopsied blastomeres is not always representative of the genotype of the born animals due to the occurrence of mosaicism [ , ] . the incidence of hdr for dsb repair is low compared to nhej, thus designing optimal dna donor templates can increase hdr frequencies. ssdna donor templates have shown greater recombination efficiency and require shorter homology arms than dsdna. when searching for specific mutations or small sequences insertions, the most used templates are single-stranded donor oligonucleotides (ssodns). homology arms for ssodn donor should be approximately e bp each. although some reports suggest that asymmetric arms improve efficiency, there is no consensus in the convenience of this strategy in the template design [ , ] . larger insertions require dsdna templates with homology arms of e kb at either side of dsb sites or long ssdnas. the insertion efficiency of plasmid-mediated hdr with dsdna is much lower than with ssdna [ ] . nucleases may re-cut the target locus after the knock-in sequence is inserted. to avoid recurrent dsbs, a silent mutation should be inserted at the pam site or the target site. if an incompatible pam or target site sequence is introduced into the genome via the knock-in, the sgrna-cas complex can no longer bind to the target site, and no further edits can be made. the use of ssodn to induce knock-in or to improve knockout efficiency has been reported recently in ruminant species (sheep: [ , ] ; goats: [ ] ; cows [ ] ). advanced reproductive technologies play a critical role in the generation and propagation of edited animals. novel molecular strategies are available for the generation of genome engineered animals, but mainly in livestock, the success always requires mastering several reproductive technologies. in vitro embryo production (ivep) is the preferable method of choice to obtain enough quantity of excellent quality zygotes available to be microinjected with the crispr components ( fig. ). oocytes are collected after follicular aspiration from live females, or moreover from ovaries of a nearby slaughterhouse. of notice, although zygotes may be collected from the oviduct, in vivo zygote production involves several issues [ ] that were overcome with the development of ivep technology. the success of in vitro production implies that the ivep laboratory should be capable to obtain good fertilization and cleavage rates (~ e %) and should have optimum culture conditions to achieve acceptable in vitro development rates (~ e % blastocysts). for further revision of the procedure for ivep conducted in our laboratory, see previous review [ ] . standard microinjection protocols originally developed to obtain genome-edited mice have been adapted to livestock zygotes, since crispr system can be easily injected into the dark cytoplasm of livestock species with no need to visualize the pronucleus. the possibility of cytoplasmic injection (instead of pronuclear injection), represents another great advantage of crispr technology, avoiding the centrifugation of zygotes required in farm animals for pronuclear injection. thus, this technical advantage allows an easier, faster, more efficient and more innocuous microinjection in terms of embryo survival. for this reason, crispr microinjection into the cytoplasm of zygotes appears as the preferable method in mammals [ ] . on the other hand, as an alternative to direct microinjection into the zygotes, crispr cell transfection for somatic cell nuclear transfer (scnt) is also used in livestock species. the generation of a live edited animal resulting from scnt embryos is not an easy task, adding complexity to the whole procedure of genome editing. although crispr success in donor cells is acceptable, scnt procedure has low efficiency and a low proportion of transferred embryos results in live offspring. embryos produced by cloning have low development rates, are susceptible to developmental and epigenetic reprogramming anomalies, thus inducing pregnancy failure, stillborn or low newborn survival rates. when scnt is mastered by the laboratory, the low efficient rate will be compensated with most of the healthy born animals carrying the desired mutation. genome editing mediated by scnt is applied by some laboratories in some kind of projects (e.g., multiplex editing), but the high efficiency of crispr after direct microinjection into zygotes has allowed an easier approach (sheep: [ , , , ] ; goats: [ , ] ; pigs: [ , ] ). all together, these reports showed that with crispr it is possible to perform direct microinjection into the zygote of different species, with minimum effects on developmental competence and pregnancy rates, high newborn survival rate, and high editing rate with acceptable homozygous proportion. for zygote microinjection, the mixture containing the crispr components is prepared in the laboratory. cas , sgrna and ssodns purity, quality and concentrations are critical to achieve a good balance between embryo survival and editing rate. as no absolute rule exists, each laboratory should test their reagents and concentrations before launching projects, since getting negative results in the offspring will be discouraging and expensive. although reagents concentration is not a major problem and may vary among laboratories, as a reference we usually use cas rnp at e ng/ ml, sgrnas at e ng/ml, and ssodns at ng/ml. with these concentrations, acceptable editing rates of e % were achieved in lambs, with e % of them being knock-in (summarized from lambs born [ ] , and unpublished data). microinjection is usually performed under an inverted microscope connected with two micromanipulators. up to presumptive zygotes can be microinjected one by one into the cytoplasm ( e pl of injection mix) in each session. on the other hand, electroporation has recently been described as an alternative approach to deliver small crispr reagents into mouse and rat zygotes [ , ] and it is currently used in our laboratory [ , ] . this procedure avoids the technically demanding microinjection technique allowing a high throughput scheme in the laboratory. microinjection requires investment in expensive equipment for embryo micromanipulation, long-term expertise and technical skills. electroporation also requires equipment, but not as expensive as the microscope and micromanipulators, and it may be already available at the bench for other purposes. the procedure is much easier, the learning curve for the technicians is much faster, and many zygotes may be electroporated in few minutes. although this method has clear advantages compared with microinjection, little information is reported in livestock to date [ e ]. proper adaptation from mice and rats protocols to large animals' species would allow the replacement of microinjection by electroporation, as is happening in mice. after microinjection or electroporation, zygotes can be transferred immediately into the oviduct, or cultured in vitro until the blastocyst stage to be transferred into the uterine horn, or cryopreserved in different stages to be stored until embryo transfer. one of the greatest challenges in in vitro produced embryos in farm animals is to overcome the low cryotolerance compared with in vivo derived embryos. in addition, for genome editing the embryos are subjected to microinjection, and thus survival rate and cryotolerance may be further affected. for this reason, in most of the projects involving microinjection, embryos are transferred fresh. however, in long-term programs with hundreds of embryos produced every week, a large and continuous supply of synchronized recipients ready to use are required. in this context, current improvements on cryopreservation of in vitro produced embryos deserve to be considered. novel vitrification systems named as minimal volume methods have been proposed for in vitro produced embryos [ ] . we have been conducted a series of experiments to evaluate these minimum volume methods for different stage ovine embryos [ , e ] , allowing to standardize the method for cryopreservation in genome editing projects. the outcomes on embryos microinjected with crispr that were transferred in different developmental stages, associated or not to vitrification, are presented in table . vitrification by minimum volume methods (crytop and spatula mvd) are used as a routine in our laboratory with acceptable outcomes, both in livestock and murine embryos after microinjection for genome modification [ , ] . this practice simplifies embryo transfer programs to produce genome-edited animals, mainly in large-scale projects involving livestock. once presumptive genome-edited animals are born, an exhaustive genotypic analysis in addition to the phenotypic one is necessary. those animals in which crispr/cas system was effective, will carry a different combination of mutated alleles, which in some cases may be in the form of mosaic. the available methods to analyze crispr editing efficiency vary depending on the type of intended change. as nhej generates random and heterogeneous indels in the animals, the sgrna can be designed in such a way that the cut intervenes in a restriction site to facilitate subsequent genotyping, however, this approach limits the number of available loci. in addition, there are some methods that are based on the detection of heteroduplex formed when a wild type and a mutant pcr amplicon (or amplicons that carry two different mutations) bind together. these techniques allow genotyping mutations in any locus. methods based on nucleases include surveyor nuclease and t endonuclease i assay (t ei assay). both nucleases target and digest mismatched heteroduplex double-stranded dna, and as a result produces two or more smaller fragments in an enzymatic reaction. the digested dna fragments can thus be resolved and visualized by agarose gel electrophoresis. heteroduplex mobility assay (hma) is based on the differential migration of dna molecules with and without mismatches in a non-denaturing page and will show a retarded electrophoresis migration. none of the above assays can exactly reveal the nature of the mutation introduced. in the end, it is always necessary to sequence the region to determine the mutation. sanger sequencing of amplicons from founding animals can be problematic, mainly in the presence of mosaics, since it results in asynchronous and overlapping chromatograms. one way to overcome this problem is to clone pcr products into plasmids and sequence a sufficient number of independent clones to obtain a representative sample of the present alleles in the animal (usually between and clones). editing involving hdr, which introduces new dna sequences, can be assessed by a number of methods such as the restriction enzyme digest if the mutation results in a loss or gain of a restriction enzyme site. an alternative is the use of deep sequencing or digital pcr of the area of interest. this system allows detecting alleles that are in low abundance, and in addition, it can be simultaneously used to identify off-target sites. although off-target sites should always be analyzed mainly in those projects involving livestock for food production, the frequency and number of offtargets produced by the cas nuclease is low in live animals compared to in vitro cultured cells [ , ] . in addition, the possible off-targets can be removed with the subsequent selected breeding of animals. as a rule, a careful selection of target sequences to avoid predicted off-target sites is mandatory, avoiding guides with possible off-targets sites on the same chromosome. improving productive and fitness traits in large animals, conferring resistance to infectious and transmissible diseases, enhancing animal welfare through improving adaptation and resilience in animals, and suppressing other species considered as pests for livestock. these uses for crispr have been either reported as a proof of concept, for research, or proposed for commercial use. some crispr edited animals are summarized below, illustrating the main or first reported ideas to give an overview of how this technology can contribute to the livestock transformation (fig. ) . a wider list of reports is summarized in previous reviews [ , , ] . the first animals edited by crispr in our laboratory were born in [ ] . in that project superfine merino breed was used as genetic background to introduce a mstn mutation that improves meat production. superfine merino is recognized as the best breed to produce the finest and highest quality wool, but the problem for farmers is the lower growing rate and smaller size of the lambs in comparison with other breeds. on the other hand, several breeds have been improved through the years by classic genetics (i.e., selective breeding) to produce meat, which was in detriment of wool quality. a good example is texel, the most popular breed in the netherlands and distributed worldwide. the impressive growth rate and meat production of this breed is associated with a spontaneous mutation at the mstn gene encoding for myostatin (or gdf ) that is involved in muscle cell grow and differentiation. by suppressing the function of mstn, the muscle mass increases. in our study, merino embryos were edited with crispr to disrupt the mstn gene. as a result, double muscle lambs were obtained, thus achieving greater growing rate and heavier body weight than superfine merino lamb counterparts. the knockout lambs were % heavier [ ] and maintained the same wool quality traits than merino lambs (unpublished data). although this proof of concept did not include the study of the offspring, in following studies germline transmission of mstn mutation have been reported by its presence in the gonads of founders (in sheep [ ] and goats [ ] ) and offspring [ ] . amazingly, these early studies suggested that what farmers have pursued for centuries (i.e., animals producing both high-quality wool and more meat), might be achieved by crispr in few months. other similar models to improve productive or fitness traits have been reported in other species. in china, cashmere goats, which are known primarily for their high-quality hair, were simultaneously edited by crispr at the mstn and fgf gene, in order to improve both meat and hair production [ ] . in pigs also, after the first report conducted to disrupt mstn using crispr in this species [ ] , several studies have been conducted to improve carcass traits through this strategy. in addition, novel ideas have been proposed to use crispr for altering male:female ratio in livestock, particularly when the desired product is provided by only one sex (e.g., meat from males or milk from females). only male offspring strategy has been proposed in beef cattle [ ] , since males grow faster and are bigger than females, and only male production may improve efficiency and avoid female culling by farmers. another approach has been proposed in pigs, in this case to suppress testis development in the fetal gonadal ridges resulting in a female phenotype and avoiding the undesirable male-specific boar taint [ ] . both strategies still needs to be proven and require further investigation. these and other improvements have been proposed in different species and serve as examples of the opportunities of crispr to enhance livestock efficiency. disruption of single genes that have significant effects on traits of economic relevance appears as an interesting approach to improve farm animal genetics. diseases affecting livestock can have a devastating impact on production, industry and trade of live animals and derived products, and even on zoonosis and public health. not only limited to the region or country in which the disease appears, some pandemics have even global effects as is now happening with african swine fever in asia, disturbing the meat and crop market and producing global consequences. animal welfare should be also taken into account, as the first affected subject are the proper animals suffering the disease and death. according to oie publications [ ] , due to the intensification of livestock production among other factors, the world is facing an unprecedented increase of emerging and re-emerging animal diseases and zoonosis. thus, decreasing disease susceptibility in livestock has become an interesting focus of research. with crispr, the generation of disease-resistant animals may be achieved. some examples have been reported recently, and one of the clearest models was the generation of resistant pigs to porcine respiratory and reproductive syndrome virus (prrsv). this disease causes the most important economic losses in the pig industry, with more than . billion dollars per year only in us and europe. vaccines have not been able to control the disease, there is no effective treatment, and due to the high level of infection and expansion, the only effective method to eradicate the virus during a prrsv outbreak is depopulation of the herd, sterilization, and repopulation. the macrophage surface protein cd , which belongs to the scavenger receptor cysteine rich family, mediates the entry of prrsv into the host cell. based on this information, genome-edited pigs with a disruption in the cd gene were produced by crispr, conferring resistance to prrsv infection. cd edited piglets, reported in two different projects in us and scotland, were completely resistant to north american and european prrsv strains [ , ] . the authors reported that the animals table pregnancy outcomes after zygote microinjection with crispr/cas system of in vitro produced embryos transferred fresh or subjected to vitrification in sheep (unpublished data). fresh or vitrified showed no symptoms or suffered no infection to the viral in vivo challenge, demonstrating the effectiveness of this strategy. african swine fever, a transboundary animal disease for which there are no approved vaccines and euthanasia is required, is currently producing massive losses in pig industry in asia and represents a risk for other regions. although the virus affect domestic and wild pigs, the infection is generally asymptomatic in warthog (a wild pig of sub-saharan africa). this type of resistance to african swine fever virus in warthog was associated with a variation in the gene encoding rela [ ] . researchers from the roslin institute have proposed to edit the domestic pig inducing this variation in rela with the aim to confer disease resilience to this virus [ , ] . in another study, crispr has also been used to produce coronavirus resistant pigs, by editing a putative receptor of the transmissible gastroenteritis virus [ ] . in china, edited cows with increased resistance to tuberculosis were produced through crispr [ ] . these examples show the potential of crispr as a novel strategy to control infectious diseases in livestock. attempts to move towards more compassion and respect for animals have been encouraged in the last years, seeking to avoid unnecessary suffering in livestock. most of the strategies consisted in the improvement of the animal practices or the environment, but adaptation (or edition) of the animals to the productive systems had not been addressed. gene editing can also make a contribution in this regard. the concept of welfare-enhanced animals is a novel strategy to avoid animal suffering, designing genetic adaptation and resilience [ ] . many routine procedures used to mitigate some of the consequences of intensive livestock, such as calf dehorning, male castration, tail-docking in dairy cattle, mulesing and taildocking in sheep, abortion or offspring culling of not desired gender, often results in both immediate or chronic pain. some of these practices may be avoided with the application of genome editing. horn removal is a routine practice in calves, however, pain and stress for the animals is an inevitable consequence, increasingly questioned by consumers. several beef breeds like angus, are naturally horn-free due to a dominant trait referred to as polled, with two allelic variants on the bovine chromosome. genetics improvement programs by selective breeding have not been effective to introduce this polled trait into holstein cattle. using talens, carlson et al. [ ] introgressed the causative celtic mutation (pc) into the holstein cattle genome resulting in a polled phenotype of the offspring. this example represents a potential approach for reducing physical dehorning in dairy cattle without loss of productivity. as mentioned, the use of crispr to produce germline ablated male pigs has been proposed [ ] , and even thought this remains to be proven, it would offer new opportunities to finish with surgical castration in pigs [ , ] . avoiding these practices in animal husbandry may encourage public support of genome-edited animals for food chain production. although the regulatory system of crispr is still unclear for agricultural applications in some countries, genome editing will continue to advance biomedicine and basic research. large animal models can now answer basic and applied questions using novel approaches not available before (e.g., knockouts and single-base changes). in biomedicine, many human diseases including cancer, diabetes, heart diseases, and various neurological conditions, are caused by numerous variants in genes. generation of animal models that replicates human mutations are a well-established tool in mice since homologous recombination in embryonic stem cells was achieved in this species several years ago. on the contrary, this was not technically feasible in large animals until the genomeediting era arrives. since then, using farm animals as relevant preclinical models for human therapies is gaining worldwide interest. although the pig is the most used animal species in biomedicine and xenotransplantation, sheep, goats and cattle are also interesting models to be studied. recently, crispr-cas system was used to produce a sheep knockout model (assisted by ssodn) for human deafness by editing the otof gene (menchaca et al., unpublished data) . the absence of otoferlin, encoded by this gene, is involved in hearing impairment in humans, and a large animal model that mimics this disorder will be useful to test diverse therapies that could reverse the hearing disability. from lambs born, showed indel mutations ( . %), and eight of them ( . %) carried knock-in mutations by hdr (unpublished results). also in sheep, researchers from utah developed a knockout model for cystic fibrosis, a genetic disease with progressive lung affectation [ ] . they combined crispr with scnt to obtain cftr e/e lambs with a similar phenotype as in humans, providing an interesting model to advance the development of new therapies. another sheep model of a rare human bone disease, hypophosphatasia, was recently reported using crispr/cas to introduce a single point mutation in the tissue nonspecific alkaline phosphatase (tnsalp) gene, which induced the same disorder as in humans [ ] . in addition, an ovine model for infantile neuronal ceroid lipofuscinosis (cln disease), a devastating neurodegenerative pediatric disorder with no cure, was achieved more recently using crispr/cas system to introduce the same human mutation [ ] . the availability of crispr to accurately replicate the clinical phenotype of human diseases in large animals is an invaluable tool for the understanding of disease progression and the development of more effective therapeutics. several biomedical models have been reported in pigs, but one of the main contributions of this species is in xenotransplantation. animal organs and tissues are considered to be a promising solution to overcome the global shortage due to the growing demand for human transplantation. the crispr system may be used to produce edited pigs to intend preventing hyperacute rejection, acute vascular rejection mechanisms, and potentially promote tolerance in pig-to-human xenotransplantation. interesting strategies have been reported mainly including knockouts models [ ] . although the use of these pigs as donors in preclinical nonhuman primate models has been limited up to date, in vitro analysis of their cells has provided invaluable information. an interesting approach to inactivate porcine endogenous retrovirus (perv) was reported in crispr edited pigs [ ] . endogenous retrovirus is viewed as a potential infectious risk in xenotransplantation of pig organs, and this strategy opens new opportunities for research in this field. the increasing availability of crispr large animal models for human diseases and xenotransplantation will help to develop global therapies and personalized medicine to improve human health. if genetic engineering in livestock has been technically amazing and ethically controversial, its application in wild population is even much more challenging. gene drive enabled by crispr has brought an unprecedented possibility to propagate genetics through populations in wild species. since , crispr was finetuned to develop a genetic system with the ability to 'drive' themselves and nearby genes through populations, in a greater frequency than predicted by mendelian inheritance. in sexual reproduction, offspring inherit two versions of every gene, one from each parent with the same opportunity to be transmitted ( : ) . on the opposite, gene drive ensures that the genetic edition will almost always be transmitted, allowing the variant to rapidly spread through a given population. this application differs from classic gene editing by associating a sequence that expresses a crispr endonuclease to the rna guide into the target site, cutting the sister allele in the homologous chromosome. dna repair occurs by hdr, and crispr and any additional sequence included in the cassette is copied onto the homologous chromosome. the system ensures homozygosity for the edited segment. this kind of 'super-mendelian' inheritance makes possible to drive any edition through the desired population, and if the edition compromises essential traits, the entire population is suppressed. thus, when applied in pests affecting livestock, the environment (or fauna) where large animals will be raised can be engineered. after releasing relatively few edited insects in wildlife, a great impact in a given target population is expected, including eradication or extinction. for this reason, gene drive is as promising as controversial. a pest is any animal or plant detrimental for humans, affecting directly public health, livestock, agriculture or wildlife. in livestock, these species usually compete for the same resources than humans, and they can generate losses either by a direct effect on the host or by acting as a vector of other diseases. approximately million metric tons of pesticides are sprayed onto the global landscape each year [ ] . because chemical-based farming contributes negatively to sustainability, the exploration of different approaches is encouraged. gene drive holds the potential to control pests without the use of chemicals or pesticides [ ] . as preliminary advances, gene drive system has been successfully reported in proof-ofconcept studies in insects [ , ] , mainly with the focus on controlling vector-borne diseases. interesting ongoing projects and novel ideas are being explored nowadays, mainly in human vectorborne diseases; in our case we are involved in a collaborative project to suppress screwworm fly population (cochliomyia hominivorax) that produce important loses in livestock in south america. after the insects, targeting invasive vertebrate pests affecting livestock, agriculture and wildlife will be for sure the next step. application of this technology will require a global discussion and a case by case study; who decides to use gene drive and when, is a big challenge for the coming years. the development of crispr for genome editing has led to a range of novel ideas addressing challenges associated with modern livestock, including productive and fitness traits, animal health and welfare, environmental preservation, and impacts on human health. this technology allows moulding the animal kingdom and the environment as never achieved before, in order to pursue human purposes to enhance global well-being. once the technical challenge has been overcome, the focus will become political. who decides, and when and how it should be regulated, are questions under current debate. any decision that could affect the global population must be collectively discussed. in the end, probably sooner rather than later, this technology will be applied as part of the globalization we all live in. restrictive regulations in some countries will become opportunities for others; those who excessively restrict biotechnology today will pay-per-use tomorrow. we should decide on which side the future will find us. world population prospects: the revision, key findings and advance tables piecing together the poverty puzzle how to feed the world in . food and agriculture organization crispr/cas, the immune system of bacteria and archaea a programmable dual-rna-guided dna endonuclease in adaptive bacterial immunity biallelic beta-carotene oxygenase knockout results in yellow fat in sheep via crispr/cas efficient generation of myostatin knock-out sheep using crispr/cas technology and microinjection into zygotes efficient gene knockout in goats using crispr/cas system generation of gene-modified goats targeting mstn and fgf via zygote injection of crispr/cas system single cas nickase induced generation of nramp knockin cattle with reduced off-target effects one-step generation of knockout pigs by zygote injection of crispr/cas system use of the crispr/cas system to produce genetically engineered pigs from in vitroderived oocytes and embryos efficient generation of genemodified pigs via injection of zygote with cas /sgrna generation of crispr/ cas -mediated gene-targeted pigs via somatic cell nuclear transfer genetic engineering a large animal model of human hypophosphatasia in sheep crispr/ cas mediated generation of an ovine model for infantile neuronal ceroid lipofuscinosis (cln disease) efficient generation of goats with defined point mutation (i v) in gdf through crispr/cas efficient generation of orthologous point mutations in pigs via crispr-assisted ssodn-mediated homology-directed repair programmable editing of a target base in genomic dna without double-stranded dna cleavage search-and-replace genome editing without double-strand breaks or donor dna highly efficient gene knockout in mice and zebrafish with rna-guided endonucleases mosaicism diminishes the value of pre-implantation embryo biopsies for detecting crispr/cas induced mutations in sheep somatic mosaicism and allele complexity induced by crispr/cas rna injections in mouse zygotes optimization of scarless human stem cell genome editing enhancing homologydirected genome editing by catalytically active and inactive crispr-cas using asymmetric donor dna highfrequency genome editing using ssdna oligonucleotides with zinc-finger nucleases generation of cattle knockout for galactose-alpha , -galactose and n-glycolylneuraminic acid antigens new insights and current tools for genetically engineered (ge) sheep and goats advances and limitations of in vitro embryo production in sheep and goats generation of gene-edited rats by delivery of crispr/cas protein and donor dna into intact zygotes using electroporation efficient crispr/cas -mediated genome editing in mice by zygote electroporation of nuclease from reproductive technologies to genome editing in small ruminants: an embryos journey generation of pdx- mutant porcine blastocysts by introducing crispr/cas -system into porcine zygotes via electroporation generation of a tp -modified porcine cancer model by crispr/cas -mediated gene modification in porcine zygotes via electroporation genome mutation after introduction of the gene editing by electroporation of cas protein (geep) system in matured oocytes and putative zygotes simplified pipelines for genetic engineering of mammalian embryos by crispr-cas electroporationdagger cryopreservation of oocytes and embryos cryotolerance of day or day in vitro produced ovine embryos after vitrification by cryotop or spatula methods impact of delipidated estrous sheep serum suplementation on in vitro maturation, cryotolerance and endoplasmic reticulum stress gene expression of sheep oocytes embryo survival and birth rate after minimum volume vitrification or slow freezing of in vivo and in vitro produced ovine embryos minimum volume spatula mvd vitrification method improves embryo survival compared to traditional slow freezing, both for in vivo and in vitro produced mice embryos off-target mutations are rare in cas -modified mice unexpected mutations were expected and unrelated to crispr-cas activity gene targeting, genome editing: from dolly to editors sheep and goat genome engineering: from random transgenesis to the crispr era multiplex gene editing via crispr/cas exhibits desirable muscle hypertrophy without detectable off-target effects in sheep crispr/cas -mediated mstn disruption and heritable mutagenesis in goats causes increased body mass efficient generation of myostatin mutations in pigs using the crispr/cas system application of genome editing in farm animals: cattle pre-determination of sex in pigs by application of crispr/ cas system for genome editing ecological sources of zoonotic diseases gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus pigs lacking the scavenger receptor cysteine-rich domain of cd are resistant to porcine reproductive and respiratory syndrome virus infection species-specific variation in rela underlies differences in nf-kappab activity: a potential role in african swine fever pathogenesis live pigs produced from genome edited zygotes mammalian interspecies substitution of immune modulatory alleles by genome editing resistance to coronavirus infection in amino peptidase n-deficient pigs genetically modifying livestock for improved welfare: a path forward production of hornless dairy cattle from genome-edited cell lines generation of germline ablated male pigs by crispr/cas editing of the nanos gene is gene editing an acceptable alternative to castration in pigs? a sheep model of cystic fibrosis generated by crispr/cas disruption of the cftr gene xenogeneic transplantation and tolerance in the era of crispr-cas genome-wide inactivation of porcine endogenous retroviruses (pervs) synthetic chemicals as agents of global change gene drive research in non-human organisms: recommendations for responsible conduct. in: national academies of sciences e, and medicine a crispr-cas sex-ratio distortion system for genetic control a crispr-cas gene drive system targeting female reproduction in the malaria mosquito vector anopheles gambiae key: cord- -z vcgqxd authors: larson, r.l. title: epidemiology and disease control in everyday beef practice date: - - journal: theriogenology doi: . /j.theriogenology. . . sha: doc_id: cord_uid: z vcgqxd it is important for food animal veterinarians to understand the interaction among animals, pathogens, and the environment, in order to implement herd-specific biosecurity plans. animal factors such as the number of immunologically protected individuals influence the number of individuals that a potential pathogen is able to infect, as well as the speed of spread through a population. pathogens differ in their virulence and contagiousness. in addition, pathogens have various methods of transmission that impact how they interact with a host population. a cattle population's environment includes its housing type, animal density, air quality, and exposure to mud or dust and other health antagonists such as parasites and stress; these environmental factors influence the innate immunity of a herd by their impact on immunosuppression. in addition, a herd's environment also dictates the “animal flow” or contact and mixing patterns of potentially infectious and susceptible animals. biosecurity is the attempt to keep infectious agents away from a herd, state, or country, and to control the spread of infectious agents within a herd. infectious agents (bacteria, viruses, or parasites) alone are seldom able to cause disease in cattle without contributing factors from other infectious agents and/or the cattle's environment. therefore to develop biosecurity plans for infectious disease in cattle, veterinarians must consider the pathogen, as well as environmental and animal factors. one component of epidemiology is understanding the interaction among animals, potential pathogens, and the environment, in order to implement a herd-specific biosecurity plan [ , ] . biosecurity is the attempt to keep infectious agents away from a herd, a state, or a country and to control the spread of infectious agents within a herd. infectious agents (bacteria, viruses, or parasites) alone are seldom able to cause disease in cattle without contributing factors from other infectious agents and/or the cattle's environment. therefore to develop biosecurity plans for infectious disease in cattle, veterinarians must consider pathogen, environmental, and animal factors. pathogens differ in virulence, contagiousness, and modes of transmission (table ). these differences exist not only among pathogens, but for virulence and contagiousness, can also differ among strains of the same species of pathogen. a more virulent pathogen causes more severe clinical signs of disease, with a greater likelihood of death. a pathogen with greater contagiousness will infect more animals in a shorter interval when introduced into a population. these factors are not related; therefore, highly virulent www.theriojournal.com pathogen may not be very contagious, and a very contagious agent may not be highly virulent. in addition, different pathogens have various methods of transmission that impact how they interact with a host population. some pathogens are spread via inhalation or ingestion. infectious agents spread in these ways are further differentiated by the length of time the agent can survive outside the host in the environment, by the distance they can travel and remain infectious, and by the age of host that is susceptible to infection or disease. other pathogens are spread only through sexual contact and are not contagious outside the act of mating. and still other pathogens require an intermediate host or transmitting fomite such as an insect, snail, or mammal. the two primary animal factors that affect protection of cattle herds from disease are specific and innate immunity. specific immunity relates to an immune response directed at a specific infectious agent that the animal has been exposed to in the past, either via natural infection or vaccination, for which ''memory'' remains. innate immunity is strongly influenced by the overall health of the animal. nutritional status such as adequate energy, protein, vitamins, and minerals impacts an animal's overall health and immune status. stress due to crowding, inclement weather, unsanitary housing, or concurrent disease can cause varying degrees of immune suppression. in populations of animals, not only do pathogen factors such as virulence, and the length of the latent and infectious periods influence the number of animals infected, animal factors, such as the number of immunologically protected individuals (either due to specific or innate immunity) also determine the number of individuals the pathogen is able to infect and the speed of spread through a population [ ] . whenever a veterinarian is looking at a beef herd, they are observing not only a population of animals, but also an unseen population of present or potential pathogens. both populations have their own life cycle, immunology, and adaptations. when we investigate a disease, we almost always consider the animal side of the infectious disease interaction, because the incubation period (interval from infection to onset of clinical signs), the symptomatic period (interval from onset to end of clinical signs), and non-diseased state (interval after the end of clinical signs) are relatively visible and measurable. concurrently, the pathogen population and its dynamics are often given little attention. for the pathogen population, its interests are described by the latent period (interval from infection of one host to the time its offspring can infect a new host), the infectious period (duration that the pathogen's offspring can infect other hosts), and the noninfectious period (interval when the pathogen population of one host can no longer infect other hosts). the infectious period can end when the immune system clears the organism from the host's body, the infectious host animal is removed from the susceptible population (isolation), the animal is sent to slaughter, or the host animal dies. the relationship between the time line of infectiousness (pathogen's perspective) and the time line of disease (animal's perspective) differs between pathogens/diseases and is influenced by the virulence of the pathogen and the host's response to it. understanding the relationship between these two time lines for a particular disease is important when developing a biosecurity plan to deal with that disease. the biosecurity tools available are ( if an animal with a particular infectious disease becomes infectious at approximately the same time as clinical symptoms appear, diagnosis and isolation or culling will help, and may completely stop the spread of the disease (e.g., rotavirus and coronavirus scours in calves). in contrast, if a particular pathogen infects a host before the animal shows symptoms, diagnosis and isolation will not greatly affect the spread of the disease (e.g., ibr-and bvd-induced respiratory disease). if a disease has a long-term carrier state that accounts for all or most of the source of the infectious agent (anaplasmosis, blv, bvd, johne's, vibrio, lepto, etc.) and a testing system has both high sensitivity and specificity, a test and cull strategy may be appropriate (anaplasmosis, blv, bvd, and vibrio), whereas a test and isolate strategy could be considered for a disease with a short-term carrier state or a minimally pathogenic disease with a long-term carrier state (blv, anaplasmosis) [ ] [ ] [ ] . a test and treat strategy may be appropriate if the carrier state can be cleared with treatment (anaplasmosis, leptospirosis). if an effective treatment exists to clear a carrier state, but testing or diagnosis lacks sensitivity or exceeds the cost of testing, prophylactic treatment of an entire population may be an appropriate biosecurity strategy (anaplasmosis, brd). for disease with no easily defined carrier state or for which accurate tests are not available, vaccination should be considered as a primary biosecurity tool if vaccination will result in an increased percentage of animal resistant to infection or a decreased likelihood of transmission of the agent (ibr). vaccination can be an adjunct biosecurity measure for diseases that have an accurate test used for test and cull as long as vaccination does not interfere with interpretation of the test. for some diseases that lack either a defined carrier or an accurate diagnostic testing strategy, management intervention to decrease the probability of effective contact is the primary a cattle population's environment includes its housing type, animal density, air quality, weather effects, mud, dust, footing, and health antagonists such as internal parasite burden, external parasite burden, and social stress. these environmental factors influence the innate immunity of a herd by their impact on immunosuppression. in addition to effects on immunosuppression, a herd's environment also dictates the ''animal flow'' or contact and mixing patterns of potentially infectious and susceptible animals. some infectious agents preferentially infect only certain ages of cattle. mycobacterium avium subspecies paratuberculosis (johne's) is primarily infectious to young animals, whereas trichomonas fetus (trichomoniasis) is primarily infectious to older bulls. some infectious agents will infect all ages of cattle, but are only likely to cause disease in certain ages. rotavirus and coronavirus infections are likely to cause clinical disease (calf scours) in young calves, but not in adults. in contrast, initial infection with the parasite anaplasma marginale (anaplasmosis) is not likely to cause clinical disease in young animals, but will cause disease in adult animals. the future of food animal veterinary medicine will involve a greater emphasis on biosecurity to implement and monitor systems to prevent the introduction and spread of common livestock diseases. cattle farms will increase the use of testing and isolation systems, as well other biosecurity strategies to minimize the costs of disease to production. with an increased level of sophistication and knowledge necessary to implement these systems, veterinary involvement in food production will continue to increase. biosecurity of cattle operations epidemiologic tools for biosecurity and biocontainment testing and management strategies for effective beef and diary herd bvdv biosecurity programs persistent bovine viral diarrhoea virus infection in u.s. beef herds key: cord- -rjud iz authors: horzinek, marian c. title: vaccine use and disease prevalence in dogs and cats date: - - journal: veterinary microbiology doi: . /j.vetmic. . . sha: doc_id: cord_uid: rjud iz abstract a yearly revaccination of adult pets against distemper, the adenoviral and parvoviral diseases is scientifically unwarranted, professionally obsolete and ethically questionable; other vaccinal antigens, however, may need yearly or even more frequent injections. base immunisation is redefined: it is complete only after the multivalent booster in the second year of life. a yearly revaccination interview, not necessarily an injection, should become the new standard. this interview is a professional service that must be taught, expertly performed and invoiced. adult animals should be “vaccinated to measure”, taking age, breed, lifestyle, the epidemiologic situation, etc. into account. post-vaccination serology should become a guide in revaccination decisions. for a solid herd immunity, more animals of the population must be vaccinated. the profession should issue regular updates of the ‘code of vaccination practice’. to counteract vaccination antagonism, a concerted action of academia, the veterinary profession and industry is required. when lecturing about pet vaccination and vaccine use in the last decade, i used to confront veterinary audiences with the following scenario: 'a pediatrician client presents her cat to a vet and asks: ''doctor, why do i need to pay you a yearly visit for revaccination of my cat? i see my children patients no more than twice in their lifetimes!'' what do you suggest as an answer? can you perhaps be more creative than . . . this is what we always did. . . . this is in the product documentation. . . . this is what most clients want. . . . this is what representatives of our profession want us to do. . . . this does not hurt. . . . this is because the immunity conferred by veterinary vaccines is short-lived. . . . this is scientifically correct . . .'. the ensuing hilarity was somewhat laboured. the difference between the medical approach and the veterinary lore of immunisation against infectious diseases is striking. this is the current schedule for routine childhood vaccination in the netherlands: www.elsevier.com/locate/vetmic veterinary microbiology ( ) -  ( of which in the first year)-diphtheria, tetanus and polio (all inactivated).  ( of which in the first year)-pertussis, haemophilus influenzae b. Â-mumps, measles, rubella: at months and years (all live). Â-meningococcus c: at months. due to the different living and hygienic conditions, human babies can take their first live virus immunisations relatively late in life, at months, when they are immunologically mature and maternal antibody interference is no longer a concern. while about one-half of -month-old infants still have detectable levels of maternal antibodies to measles virus, none of the -month-olds show this. among those without passive immunity to measles, only about one-third of the -month-olds mustered enough antibodies upon vaccination to resist an infection, compared with % of the -and -month-olds. consequently, the human immune system continues to develop postnatally, acquiring key abilities past the age of months. the development of the canine immune system shares many similarities with that of the human. in both species immune competence, also of the mucosal immune system, is fully developed before birth although further maturation of the responses may continue into the postnatal period (felsburg, ) . thus, in man, a single booster at years of age is accepted as being sufficient for lifelong protection. this practice is based on epidemiologic evidence only, since challenge based duration of immunity (doi) studies are out of the question. amongst the live virus vaccines are those against measles-the human counterpart of canine distemper. dogs, however, may be treated to a dozen or more boosters during their lifetimes. the question arises whether this is rational. why has veterinary medicine adopted a practice that causes raised eyebrows in the biomedical environment, e.g. when talking to immunologists? the reason is largely historic: in the first years of vaccine development, the objective of maximum protection was thought to be achieved by maximum antigenic stimulation. at the time this seemed to be the right thing to do, with the newly developed, attenuated distemper and canine infectious hepatitis virus preparations. it became common practice in subsequent vaccine developments, including the parvovirus preparations, without asking why. in recent decades, however, the frequency of vaccination has become a matter of debate-first in the usa, more recently in europe. the scenario in germany ( ) turned grim, when a well-informed journalist issued a declaration of war in a nation-wide newspaper. the lay public was activated, the foe included vets and the biologicals industry. in england ( ), a similar initiative of several newspapers based on information contained in a letter from more than british vets, contained quotes like: ''unnecessary, potentially dangerous, fraud by misrepresentation, fraud by silence, theft by deception, complete overkill'' and the like. the scenario world-wide, including the medical scene is not different, and websites abound advertising pamphlets with titles like ''vaccination, social violence and criminality'', ''the hidden truth'', ''vaccines represent a medical assault on the immune system'', ''what doctors don't tell you'', to quote a few. whereas yearly revaccination is a veterinary specialty, indiscriminate vaccination antagonism with traits of paranoia, persecution mania and all kinds of conspiracy theories is not-it is also prominent in the medical environment. actually, the first cartoons depicting a ''vaccination monster', appeared in the th century, short after edward jenner's seminal discovery. from the immunologist's viewpoint, the veterinary profession should weigh the perceived risks of side effects due to overvaccination together with vaccination failures against the true risks of a decreased herd immunity with the re-emergence of epidemics as a consequence. statistically, these are minor problems, when weighing the significance of the sporadic cases of immune-mediated pathology with that of sweeping epidemics. the real and serious threat to veterinary medicine (and of course to the canine and feline populations) is the vaccination antagonism in the public with its aggressive undertones. the professional organizations should be even more concerned about their loss of credibility. the term 'overvaccination' is suggestive in that it evokes the picture of an organism swamped with antigens, its immune system paralysed by too many components in one shot. this is an erroneous conception, in view of the fact that any organism is bombarded with antigenic molecules during its lifetime. in both veterinary and human medicine, this issue has received much attention, also from manufacturers, and the consensus is that carefully selected, tested and registered combinations are neither inferior in immunogenicity nor in safety. in veterinary medicine, overvaccination rather refers to vaccinating with excessive frequency, which has been controversial for more that a decade (''are we vaccinating too much?'' (smith, ) . after a period of indifference, the profession is now faced with organised militant campaigning combined with scaremonging. the reported side effects fulfill the criteria of selective observations, and although some have achieved prominence in the scientific literaturelike the injection site fibrosarcoma in cats -they are comparatively rare. however, also some infections have become rare, and it must be anticipated that vaccination will be discontinued when the disease to protect against is no longer around. the scenario is similar to that during the final phase of smallpox vaccination, when the rare side effects (less than in a million vaccinees) exceeded the number of natural disease cases. in line with the romantic idea that natural infections are superior to vaccines, ''infection parties'' (measles and parvo) are being organized, e.g. in germany. this is both irrational and irresponsible. infections with wild viruses are always more serious than those with their attenuated laboratory counterparts -this is why field strains were attenuated in the first place. voltaire said: ''people who believe in absurdities will inevitably commit atrocities.'' the scientific arguments in favour of less frequent revaccinations are traditionally based on antibody titers. protection against most viral diseases is indeed antibody-mediated, and antibodies are easily measured. in dogs these have been found to persist for more than years, the study did not look later. the high prevalence of adequate antibody responses (cpv, . %; cdv, . %) in a large population (> animals) ''suggests that annual revaccinations against cpvand cdv may not be necessary'' was the authors' conclusion (twark and dodds, ) . in the cat, antibodies to fpv, fcv and fhv were detected for more than four years (mouzin et al., ) . the question whether the titers found are protective or not against a field virus challenge is irrelevant for this discussion. it is not the residual serum antibody that determines survival to challenge but the population of memory cells that can quickly expand. the question about the longevity of memory cells has now been answered experimentally; the question was not, if lifelong immunity exists (which is common knowledge), but whether its mechanism relies on a lifelong presence of the antigen in the animal's organism or of the cells' longevity. the latter was not found to be the case ''memory b-cell persistence is independent of persisting immunising antigen''; (maruyama et al., ) . however, it is not an individual memory b-cell, rather a population of slowly dividing clones that persists during the life of the organism. like in neurobiology, a paradigm has been shattered: neurons and memory cells can indeed divide. finally, duration of immunity (doi) experiments in dogs have now proven beyond reasonable doubt that years protection is achieved against challenge with distemper, adenovirus- and parvovirus (gore et al., ) . while this is a timely -though by no means surprising -finding, it has been achieved at a considerable cost. it is this financial aspect (statistically sufficiently large group sizes, isolation facilities, quarantine conditions) that will preclude any further study of doi, i am afraid. underpinned by the conservatism of the veterinary profession this study will become a monument in veterinary vaccinology and determine the periodicity of revaccination. few will dare to do otherwise, i.e. to vaccinate even less frequently. though it has been the financial mainstay of many a companion animal practice, vaccination is not exciting. an injection is technically not as demanding as repairing a fracture, and the client does not see much -if she does, it is the failures: the side effects and vaccination breaks. neither can the vet impress her client -she vaccinates, but cannot (and for time reasons does not want to) explain the basic principles of immunology and epidemiology to the client. nor can any effect of the immunisation be shown. what is commonplace in surgery and internal medicine is lacking here: vaccination is probably the only veterinary measure without a follow-up. it is what i called the ''shoot-and-trust principle''. post-vaccination serology (pvs) would introduce evidence, and serve the industry and the profession alike, but it meets with considerable opposition. a yearly vaccination interview, sensibly part of a yearly health check, but not necessarily followed by an injection, should become the standard. why yearly, why an interview, what should it be about, how should it be performed-these are the obvious questions. why yearly? because many owners are used to that routine, to contact their vets for the yearly shot, for a dental appointment or other health reasons. decisions about holiday travels are taken on a yearly basis, with possible vaccinological consequences (stay in a boarding kennel, cattery, visit to foreign countries with new pathogens). the vet can schedule these visits, to entertain the relationship with the client and show responsibility for the animal family member. why an interview? because it provides the practitioner with informations necessary to take vaccinological decisions and to explain them to the client. if she fails to do this, the client will obtain them from sources of doubtful reliability. however, a conversation not only informs about the measures to be taken, it also prepares, encourages, warns, reassures and comforts. its first purpose is to build a vet-client relationship of trust, which needs to be established and developed. ''customer loyalty'' is the term in commercial publications, and a practice is a business enterprise after all. this relationship is in need of improvement -only about % of the clientele return for follow-up vaccinations; the last visit to the vet obviously did not leave a lasting impression. this is hardly unexpected: an injection given in passing, to minimise the time investment, cannot achieve customer loyalty. this myopia has done much damage to the profession. after all, the client has prepared her visit to the veterinary clinic, comes with expectations, opinions (and prejudices), which need to be taken seriously. to reject the client's views as an irrelevant lay opinion is no basis for a dialogue. emotional and social intelligence will eventually be decisive factors for forging a lasting relationship with the owner. the vaccination interview will be different for each year, and a catalogue of questions and answers must be developed. for the first year it might look as follows: the owner is informed about the preventable diseases. the advantages of vaccination versus treatment. possible side effects and complications. possible lack of protection. risk/benefit considerations. how to handle the vaccinee after the shot. the basic vaccination program (with the boosters in the second year). the onset and duration of protection. the origin of the animal. the responsibilities within the family. other animals in the household. vacations and travel plans (abroad). medical (vaccination) history, previous treatments, etc. the interested dialogue is of paramount importance (professionals like to hold monologues); questions for self-assessment include: did i practice active listening? did i show empathy? did i choose the right place, time, situation, climate? did i feel pressed for time? did i take in all messages? did i use the correct query technique? did i stimulate the client to ask questions? did i properly structure the interview (introduction, aim, course, conclusion)? did i respect the listener's need for pauses? did i use killer phrases? did i use diversion strategies (stray, digress, evade, deviate, disparage, patronize, condescend)? did i ask too much from the client? did i ignore (or even induce) worries in the client? was my client's reality the same as mine? the vet is the authoritative source of healthrelevant information and sells her knowledge (exper-tise) to the client. as long as this context is ignored, the prestige of the profession remains at stake. the interview with its considerations of the patient history, of explanations of risks and contra-indications, of the reaching of agreements, and of an informed consent, will be followed by a clinical examination: only healthy animals are vaccinated. whereas the ''one-size-fits-all'' shot has been practiced as a routine in the past, ''vaccination to measure'' will have to come in its place in the future. any ad hoc vaccination must address the individual risk of infection and disease of the vaccinee-a pampered devon rex has a lifestyle different from a stray cat, when considering the risk of exposure; the former may leave the home only for a visit to the vet. customtailored vaccination schedules will differ the client's companion is not just a dog or cat -it is a dear family member, has been given a name, is an individual and requires individual attention. neither is the client just a time-consuming nuisance -she is a partner in a conversation and deserves interest, sometimes empathy. vaccines protect against infectious disease, and a precondition for their use is a risk of infection. in most cases this hazard is an assumption, an impression not based on epidemiological data. the prevalence of e.g. distemper virus in europe amongst domestic dogs and in feral carnivores is unknown. from yearly endemics amongst unvaccinated pups, however, it can be inferred that the virus is still around. infectious canine hepatitis, on the other hand, has been seen here by clinicians and pathologist only rarely, nor has it been evidenced by pcr analysis in dogs with respiratory signs in the uk (erles et al., ) , in contrast to e.g. the usa, canada, mexico and australia. it is present in wild carnivores though. parvovirus, however, is ubiquitous, both its canine and feline varieties. herpes-, calici-and coronaviruses abound amongst cats, with high prevalences in crowded communities. felv infections are phasing out in several countries in western europe, thanks to testing and vaccination, while the prevalence of fiv has hardly changed. this is an oversimplified global view of canine and feline viral epidemiology-the vaccinating veterinarian must positively know about the local situation in her area. there is no dedicated information service available, and contacts with the regional veterinary schools and diagnostic institutions will be left to personal initiatives. promed-mail -the program for monitoring emerging diseases -is an internet-based reporting system dedicated to rapid global dissemination of information on outbreaks of infectious diseases and can be queried (http://www.promedmail.org). the veterinarian is the designated expert to provide competent advice to the client concerning epidemic risk factors and their vaccinological consequences. as stated above, vaccination is about the only veterinary measure whose result is not routinely evaluated. post-vaccinal serology, however, is not new to companion animal medicine: evidence of antibodies to rabies virus decides whether a dog may travel. an assessment of the animal's immune status would provide the vet with information about the success rate of her measures, and reassure the client. the proposal is to assess the result of vaccination by asking the question: did the vaccinee's immune system recognise the antigen? no assertion of protection, only a probability can be given -similar to the results of many assays in clinical chemistry. the first serum samples should be tested in the rd year of life, to see whether the pup vaccination (first year) plus boost (second year) have resulted in immunological memory; later tests can be done if so desired by the client, e.g. before the -yearly interval, as an aid in the decision about revaccination. when the vaccination history of an animal is unknown, a prime-boost regime will usually be preferred, unless requested otherwise by the client. interpretation of the serology data will be an element of the vaccination interview. the purpose of pvs should be to show antibody against the core vaccine components distemper, hepatitis and parvovirus (dog and cat). if antibody is present, the animal has been immunised (which is not synonymous with immune or protected). forget about titers (titres) -the bad experience with fip serology is still on everybody's mind. titer values have been attributed a biological significance they intrinsically lack, and high coronavirus antibody titers have been the veterinary death sentence for many a healthy cat. there are many reasons for rejecting titers: values differ per laboratory, because of technical variations in the tests, '' '' and '' '' are not different titers ('' : '' and '' : '' are no titers at all but the serum dilutions tested), the difference is just two-fold. virologists start to think about specificity when the titer difference is four-fold. only yes/no-data should be communicated by the laboratory -which leave no room for doubt (no uncertainly of interpretation). ideally, the vaccine industry should be involved in pvs, which could corroborate their claims of the potent antigenicity of their products. in a nutshell: the laboratory (or an in-practice test) should give a robust yes/no answer with a threshold value safely in the positive range. if the potential vaccinee tests negative (false or true) vaccination is recommended. a false-negative test will result in vaccination (in spite of antibodies) -which is similar to the present situation in many cases. if an animal tests positive (false or true) vaccination is not recommended. a false-positive test is unlikely in view of the high prevalence of antibodies in the population. . we vaccinate the same animal too often, but too few animals of the population . . . most animals in an area, a province, a country should be vaccinated-rather than revaccinating the same dog or cat time and again, which neither improves its own immune status nor contributes to herd immunity. herd immunity is defined as the indirect protection of susceptible members of a population brought about by the presence of immune individuals. to prevent serious losses from epidemics (like during the distemper epidemic in finland finland - where > dogs were infected, of which $ % succumbed; (ek-kommonen et al., ) , active campaigning for vaccination should be targeted at achieving about % immune coverage. this figure has been obtained by mathematical modelling of epidemics and confirmed by observations from natural outbreaks (like the finnish epidemic), but may not be universal, i.e. for all infectious agents. in such a situation, the effective reproduction rate r would be reduced to < , which means that there will be less than one new case per infected individual, and (if r continues to be < ) the infection will locally die out. these considerations play a role in the eradication strategy for measles. distemper cannot be eradicated because of spill-over infections from feral carnivore reservoirs and re-introduction of the agent into the domestic populations, but the spread of infection can be contained. a milkmaid's calculation can show that we are far from that % goal. thus, in germany ( ) there are $ mio. registered dogs; there are $ . mio. vaccine doses reaching the market (shppilt & shplt); there are $ . mio. purebred dogs (most of which will be immunised); the remaining . mio. vaccine doses thus are applied to . mio. dogs = % (data kindly provided by dr. uwe schultheiss, nice/france; source: gfk nürnberg). estimates made by intuitive assessments vary widely and may reflect selective observation or wishful thinking. it is of course more arduous to solicit new clients than to summon old ones, but it needs to be done. this opening article of a special issue dedicated entirely to pet vaccination is intended to set the scene for the various aspects of immunisation of the dog and cat. the discussions to follow will hopefully result in a new degree of awareness amongst veterinary practitioners. if the profession wants to play a leading role in the public discussion, if the vet (and not the internet) is to stay the animal health authority for pet owners -if microbiological, immunological and vaccinological knowledge is to be conveyed to (and rewarded by) the clientele, the profession must change its attitude. vaccinological knowledge must be acquired, entertained and kept current, which should start at the university and be perpetuated by continued education. without further discussion, and as food for thought, these are my ten commandments of pet vaccinology: . the puppy schedule should be extended to include a vaccination at week of age. . base immunisation is complete only after the booster in the second year of life. . the routine of yearly revaccinations from the third year onward is questionable. . a yearly revaccination interview, not necessarily an injection, should become the standard. . the yearly revaccination interview is a professional service that must be taught, expertly performed and invoiced. . adult animals should be ''vaccinated to measure'', taking age, breed, lifestyle, the epidemiologic situation, etc., into account. . post-vaccination serology should become a guide in revaccination decisions. . for a solid herd immunity, more animals of the population must be vaccinated. . the profession should issue regular updates of the 'code of vaccination practice'. . to counteract vaccinophobia, a concerted action of academia, the veterinary profession and industry is of paramount importance. outbreak off canine distemper in vaccinated dogs in finland longitudinal study of viruses associated with canine infectious respiratory disease overview of immune system development in the dog: comparison with humans three-year duration of immunity in dogs following vaccination against canine adenovirus type- , canine parvovirus, and canine distemper virus memory b-cell persistence is independent of persisting immunising antigen duration of serologic response to three viral antigens in cats are we vaccinating too much? clinical use of serum parvovirus and distemper virus antibody titers for determining revaccination strategies in healthy dogs key: cord- -gvq uyfk authors: rosenberg, ronald title: detecting the emergence of novel, zoonotic viruses pathogenic to humans date: - - journal: cell mol life sci doi: . /s - - -y sha: doc_id: cord_uid: gvq uyfk rna viruses, with their high potential for mutation and epidemic spread, are the most common class of pathogens found as new causes of human illness. despite great advances made in diagnostic technology since the s, the annual rate at which novel virulent viruses have been found has remained at – . most emerging viruses are zoonoses; they have jumped from mammal or bird hosts to humans. an analysis of virus discovery indicates that the small number of novel viruses discovered annually is an artifact of inadequate surveillance in tropical and subtropical countries, where even established endemic pathogens are often misdiagnosed. many of the emerging viruses of the future are already infecting humans but remain to be uncovered by a strategy of disease surveillance in selected populations. in common with all organisms, pathogens evolve. every year brings reports of previously unrecognized human pathogens or of pathogens extending their geographic range, becoming less susceptible to treatment or prevention, or displaying unprecedented epidemic tendencies. as i write this an unprecedented ebola virus epidemic threatens west africa [ ] and chikungunya, a mosquito-borne virus, which first appeared in the western hemisphere in november , has already infected nearly , , people there [ ] . for those of us with responsibility for preventing or controlling infectious diseases, the speed with which new battles must be fought can be disconcerting. zaire ebola virus was first identified as a human pathogen only in and chikungunya in but neither reached pandemic magnitude until decades later. how can we be better prepared to identify emerging pathogens early? i will try to briefly examine some of the factors that influence our success in finding and characterizing previously unrecognized human viruses. the concept of emerging diseases is relatively recent [ ] , even if the phenomenon is not. the definition used by the world health organization [ ] is representative: ''an emerging disease is one that has appeared in a population for the first time or that may have existed previously but is rapidly increasing in incidence or geographic range''. in practice, determining if a disease is increasing in incidence or geographic range sometimes requires interpretation that might be considered arbitrary. for example, using this broad definition a recent paper [ ] claimed to have identified about emergent ''events'' between and , most of which were examples of antimicrobial resistance. a more limited and less ambiguous subset of emerging pathogens will be described here: virus species first recognized to cause human illness. before proceeding, it might be worth describing how three recently described pathogens were discovered and their disease characteristics. all three were first reported during the last six years and all three are generally accepted as distinct pathogenic entities causing serious human illness. in early september , a -year-old female resident of lusaka, zambia developed fulminant symptoms of an acute infection, beginning with headache and myalgia, and progressing over the next days to extensive rash, facial swelling and severe sore throat [ , ] . by the time she was airlifted to a hospital at johannesburg, south africa, she had developed cerebral edema, acute respiratory distress, and renal failure. despite intensive care, including hemodialysis, she died days after her initial symptoms. five of those who cared for her during transport to south africa or at the johannesburg hospital-a paramedic, two nurses and a cleaner -subsequently developed symptoms and four of these died. a previously undescribed arenavirus, lujo virus (a conflation of lusaka and johannesburg) was isolated from the index case and all four secondary cases [ ] . the arenaviruses, which have bisegmented, single-stranded, negative-sense rna genomes, are broadly divided phylogenetically into new world and old world groups. lujo belongs to the old world group, as does lassa virus. typically arenaviruses have rodent reservoirs but the specific host for lujo virus has yet to be determined and how the index case was infected is unknown [ ] . there have been no further cases reported. during summer, , two men, aged and years, were admitted within a few weeks of each other to heartland regional medical center, st. joseph, missouri, usa, with similar symptoms of fever, fatigue, anorexia, nausea and non-bloody diarrhea. the two men were farmers who lived approximately km distant from each other in northwestern missouri. both men had histories of frequent tick bite and were initially suspected to be infected with ehrlichia chaffeensis, a tick-borne rickettsia endemic to the area. serological and molecular testing of both, however, were negative for ehrlichia and neither responded to antibiotics. while in hospital both men developed precipitous thrombocytopenia and leukopenia. symptoms resolved with supportive care and both men were released from hospital and days after admission. culture of specimens indicated the presence of virus, which was confirmed by electron microscopy, and subsequently a unique bunyavirus, in the group phlebovirus, was sequenced from both patients [ ] . phleboviruses are singlestranded, negative-sense rna viruses with tripartite genomes, all of which appear to be transmitted by biting arthropods. heartland virus is the first pathogenic phlebovirus described from the western hemisphere and has a % nucleotide homology with the severe fever with thrombocytopenia syndrome (sfts) virus, reported from china in [ ] . heartland virus has since been isolated from ticks and antibodies to it have been found in a variety of wild animals, including white-tailed deer (odocoileus virginianus) and raccoons (procyon lotor) [ ] . there is no evidence for direct human to human transmission of heartland although a number of mostly nosocomial cases have been reported for sfts. between april, and late july, , middle east respiratory syndrome coronavirus (mers-cov) was definitively diagnosed in people, of whom died [ ] . the focus of cases has been in saudi arabia, united arab emirates and other middle eastern countries; the few cases detected in europe and north africa appear to be travelers from the middle east. the index case was a -year-old male admitted to hospital at jeddah, saudi arabia in june with a recent history of fever, cough and shortness of breath [ ] . at the time of admission his laboratory blood results were generally unexceptional but by days post-admission his white blood cell count had increased to , /cu mm and his platelets fallen to , . antibiotic-sensitive strains of klebsiella pneumoniae and staphylococcus aureus were cultured from his respiratory tract but he did not respond to antibiotic therapy. despite being on intensive support from the second day of admission, the patient died days post-admission of respiratory deterioration and renal failure. coronaviruses, which include severe acute respiratory syndrome coronavirus (sars-cov) and some agents of the common cold, have positive-sense, single-stranded rna genomes and are predominately transmitted between humans by fomites. many of the mers-cov cases have been nosocomial or appear to have been transmitted within families. neutralizing antibodies to mers-cov have been widely found in dromedary camels [camelus dromedarius] from the arabian peninsula and africa [ ] and virus has been isolated from them, strengthening the evidence that they are the immediate link to emergence in humans. despite the differences in clinical presentation and geographical location, these three pathogens share three characteristics: all were unknown before found infecting humans, all are rna viruses, and all have proven or putative non-human, animal sources. animal rna viruses are the most common source of emerging pathogens in a seminal study, woolhouse et al. [ ] tabulated pathogens first reported to be pathogenic to humans during - . two-thirds of these were viruses, % of which had single-stranded rna (ssrna) genomes. the predominance of rna viruses mostly owes to two characteristics. first, the rate of error during rna replication (* - ) is an order of magnitude greater than that of dna (* - ). rna replication does not benefit from the proofreading capabilities of dna polymerase or post-replication mismatch repair; consequently the potential for mutation per replication cycle is high [ ] and the lack of fidelity may have limited the size of rna genomes, many of which are in the range of , - , nucleotides. second, most rna viruses are zoonoses, that is, they were transmitted, at least initially, to humans from non-human mammal or avian hosts. examples of rna viruses retaining the capacity to be directly transmitted from animals to humans include influenza, nipa, and sars viruses, but even some viruses commonly transmitted exclusively between humans, such as hiv and hepatitis c, have likely animal origins [ ] . all arthropod-borne viruses (arboviruses) are zoonoses, although some, like dengue, yellow fever, and chikungunya, have adapted to efficient vectorborne transmission between humans. humans have been in contact with infectious animals since prehistory but their exposure accelerated with the development of livestock husbandry beginning about , years ago [ ] . the growing global population has not only increased the demand for domesticated meat in the st c but has increased encroachment on areas once wild, both are trends that increase human exposure to animals and animal products [ ] . by the end of , there had been, by one tabulation [ ] , virus species from virus families incriminated as causes of human disease. more than two-thirds of these viruses ( %) are known or presumed zoonoses. more than a quarter ( %) were first described from non-human mammals, birds or blood-feeding arthropods - years before being recognized as human pathogens. indeed, the first vertebrate virus described, the cause of foot and mouth disease, was isolated from a cow in [ ] but conclusively shown to cause human disease only in [ ] . the dates of discovery, regardless of host, are plotted in fig. a . the rate at which virulent viruses have been discovered has been governed by two equally important factors: the ability of existing technology to detect and discriminate between viruses, and the ability to collect specimens potentially containing novel viruses. initially, the lack of methods for the laboratory cultivation of viruses, which require cells for replication, prevented their isolation for study. early characterization as a virus depended mainly on demonstration that a filterable agent smaller than bacteria was responsible for transmissible disease. until the late- s, when embryonated chicken eggs and suckling mice began to be used commonly to culture animal viruses, only of the viruses now known to be pathogenic had been described. the rate of discovery again accelerated after the introduction of in vitro cell culture in (fig. a) . the mean annual rate of virus discovery during - was . . during this period methods for antigentically typing viruses using panels of antibodies were refined and came into wide use. there was a striking increase in the number of novel viruses described during - to . /year. this was followed, however, by a sudden deceleration in the rate of discovery to only about /year, which persisted through , despite the availability of increasingly powerful methods for genomic characterization, such as polymerase chain reaction (pcr) from the mid- s and, more recently, high-throughput, parallelized (''next-generation'') sequencing. vertebrate viruses can be sorted into two broad categories: those directly transmissible between humans or between animals and humans, and the arboviruses, which require the mediation of blood-feeding arthropod vectors, such as mosquitoes or ticks; % ( ) of pathogenic viruses are transmitted to humans only by arthropod vectors. the sudden, transitory increase in rate during - has been shown [ ] to be because the rates of discovery of these two classes differed (fig. b) . before the rates for the two were the same and each comprised about half the pathogenic viruses. after , however, the trends of the two classes of virus diverged. while about two non-arboviruses were discovered each year between and , the arboviruses dramatically increased during - , only to fall equally dramatically to nearly zero by . during - twice as many arboviruses ( ) were discovered than non-arboviruses ( ); by contrast, during - , only arboviruses were discovered compared to non-arboviruses. the difference in the rates for the two classes highlights the important role that strategies for specimen collection play in the recognition of novel pathogens [ ] . [ ] . all the field stations were located in tropical or sub-tropical countries and all carried out an integrated strategy that attempted to discover viruses from humans, vertebrate animals and biting arthropods. of the arboviruses discovered by the end of , ( %) were discovered by rf staff, of those during - . in comparison, the single most successful institutional discoverer of non-arboviruses, the united states national institutes of health, described of ( %). the rf protocol, which was the model for several other institutions, including the institut pasteur, was directly responsible for two additional characteristics differentiating arbovirus from non-arbovirus discovery. although % of all non-arboviruses were discovered in europe or the usa, % of all arboviruses were discovered in sub-saharan africa, latin america/caribbean, or egypt/india/ near east (fig. ) . second, % of arboviruses were first isolated from arthropods, a consequence of systematic vector collections. the predetermined cessation, by , of most rf support for international arbovirus researchincluding sponsorship of reference collections, conferences, new technology and reagents-was soon followed by a rapid, worldwide decline in arbovirus discovery (fig. b) . the east african virus research institute, for example, which was founded by rf in , isolated arboviruses after direct rf administration ended in , but none after the general rf program closed in . the disproportionate productivity of rf resembles that of especially effective individual discoverers of plant species [ ] . those botanical ''big hitters'' combined technical expertise and persistence over many years with concentration in a limited geographic area where they had gained deep knowledge. the rf was committed to a long-term strategy founded on five components. first, it chose study sites where it had evidence that arbovirus diversity would be high. these were mostly tropical and contained forested and rural areas. they chose countries where a professional work force could be recruited and where it was hoped the work would be sustained after the rf departed. second, the program concentrated only on one subclass of pathogens, the arboviruses. despite the wide competence of the professional staff, few reports were published dealing with local diseases other than arboviral. third, the research strategy called for long-term commitment. rf staff, including expatriates, typically lived on site and implemented projects for years, allowing for continuity of not only research but training. fourth, the program was integrative. human, animal and vector investigations were simultaneously pursued. because all scientists worked in a single unit connections between human virus isolates and those from animals or vectors were readily made. on the other hand, for many viruses isolated from animals or arthropods there has yet to be a link to human infection [ ] . and fifth, each unit was self-contained. each was capable of conducting both specimen collection and sophisticated laboratory analyses. how many pathogenic viruses remain to be discovered? mathematical methods for extrapolating from historical rates of discovery to estimate the pool of yet to be discovered organisms in a given taxon tend to be accurate only after most species have already been discovered [ ] . the fundamental weakness of these computations, which generally rely on analysis of cumulative frequency curves, is the assumption that the numbers of organisms known at a given time are the result of methods of discovery that have been consistent everyplace and throughout time. the increased rate of virus discovery during - was largely due to the temporary efforts of the rf, a ''big hitter'' [ ] , whose combination of active surveillance, geographic specialization, and integrated approach remains atypical. in contrast to sophisticated computations, a recently published prediction that a minimum of , mammalian viruses of families remain to be discovered was based on a simple arithmetical calculation using data from a single study [ ] . considering how few new, virulent viruses are found every year, the potential for any of , viruses jumping to humans and being discovered would then be very low ( . - ). the authors used degenerate, virus-family-level primers to amplify genomic segments from specimens of feces, urine, and throat swabs collected from the bat species pteropus giganteus in bangladesh. amplicons were as short as bp and no biological information was obtained. they found viruses (and statistically surmised an additional three existed), some of which might be novel, belonging to seven virus families. in calculating a number for the universe of viruses yet to be found they speculated that each of the known , mammal species will host an average unique viruses, unshared with other species. the authors concede that there is little evidence to support these presumptions. a single subtropical bat species hardly represents all mammal species and indeed many viruses are known to infect more than one species; they tested for only of the virus families pathogenic to humans. ultimately, the number of viruses remaining to be discovered is irrelevant if, as expected, there are many and they continue to rapidly evolve. the discovery of a virus can long predate its emergence as a recognized public health threat. the discovery of zika virus in a monkey in uganda preceded its first incrimination as the cause of a human epidemic- , km distant in micronesia-by years [ ] . as noted above, the availability of ever more powerful molecular techniques is substantially increasing the catalog of distinct viruses found in nature but the number found annually to be pathogenic to humans rarely exceeds a few each year. is it feasible to predict which animal viruses have the potential to cause disease in humans? steps to emergence the steps by which a virus might emerge from exclusively animal hosts are schematically depicted in fig. . at the most preliminary level (tier ) viruses circulate within mammals and birds, not necessarily causing disease, before some opportunistically infect humans (tier ). typical human-animal contact includes husbandry, capture of wild animals for food, and exposure to animal fomites or waste, as may happen in bat infested environments. indirect exposure via arthropods must be frequent, as evidenced by the large proportion of pathogenic viruses that are vectorborne [ ] . in most instances these tier , opportunistic infections are dead ends or remain rare events because the pathogen is not well adapted to transmission between humans (e.g., crimean-congo hemorrhagic fever virus) or because the type of contact between infected animals and humans is uncommon (e.g., sealpox virus). a small but fig. comparison of regions in which arboviruses and nonarboviruses were discovered fig. schematic of the emergence of zoonotic viruses as human pathogens. in tier , viruses are only transmitted among sub-human animals. in tier , viruses infect humans, but only directly from animals. some animal viruses (solid arrows), like west nile, can fuel zoonotic epidemics. others, like hantaviruses, are frequent but subepidemic causes of human illness (dashed black arrows), while many, like sealpox, are rare (dashed red arrows). in tier , zoonotic viruses have acquired the ability to be transmitted between humans without the contribution of the animal host. in some cases (w) a virus might leap directly to tier or transition through tier significant group of zoonotic viruses, including rift valley fever, nipah and west nile viruses, are capable of instigating human epidemics without ever adapting to humanhuman transmission. theoretically even sub-epidemic (r o \ ) transmission can favor mutations that will enhance future transmission [ ] . in rare instances (tier ) pathogens do evolve to allow human-human transmission (e.g., hiv) or appear to already posses that capacity (e.g., mers-cov, lujo). some viruses maintain animal-animal and animal-human cycles but are mostly propagated by human-human transmission (e.g., chikungunya, zika). the geographically limited sylvatic cycles of dengue in west africa and southeast asia account for a tiny percentage of the estimated million infections annually [ ] . human contact with an animal virus does not ensure infection. among the biological barriers for the virus are finding a route of entry, evading general immune defenses, invading host cells, replicating sufficient numbers before specific immune responses are mounted, and finding a route to the next host. aerosol delivery, for example, greatly enhances transmissibility but efficiency depends on the anatomical site within the respiratory system of the invaded cells [ , ] . arbovirus transmission, in which the vector amplifies, transports and inoculates the virus into humans, can be enhanced by viral mutations that increase the potential for successfully infecting the vector [ , ] or animal host [ ] without altering its virulence to humans. much recent research has focused on identifying determinants essential for viral invasion of host cells and how modification of the viral ligands might increase their ability for interspecies infectivity [ ] . it is not yet possible on the evidence of sequence alone to predict with confidence the probability of an animal virus transitioning to humans. in general, vertebrate specificity greatly limits the ability of viruses adapted to one species to invade similar cells in another, distant species. influenza a is the most studied and best understood of the few viruses that frequently jump from animals to humans. a number of mutations have been identified that enhance infectivity. these include substitutions in the hemagglutinin (ha) protein receptor binding sites that enable the virus to exploit sialylated glycan receptors on respiratory cells belonging to other species [ ] . for example, two nonsynonymous base changes in the ha receptor binding sites of avian h and h viruses, which converted their specificity from the avian a , -sa to human a , -sa, led to the pandemic of h n and the pandemic of h n [ ] . much current research on the determinants of influenza specificity is experimental and its extension to complex natural transmission of other virus families remains to be tested. understanding how such adaptability works could focus our attention on those virus families or species with the greatest chance of infecting humans but how this knowledge could be used more specifically to identify potential threats to humans among animal viruses, as has recently been proposed [ ] , is unclear. geographical bias, human behavior and likelihood of contact species richness of mammals and birds is greatest at the equator, thinning toward the poles [ ] ; mosquitoes [ ] and ticks [ ] appear to follow a similar latitudinal species diversity gradient. pathogen species are also richer in the tropics than in temperate zones [ ] although, as has been ruefully pointed out, ''the fact that warbler species distributions are better understood than the distribution of human pathogens is a gap that clearly deserves research attention'' [ ] . there is a strong association between mammal and pathogen richness but mammal diversity appears to be an indicator rather than the cause of pathogen diversity [ ] . pathogens that maintain external life cycles, for example vector-borne and helminthic, which are directly susceptible to variability in precipitation, tend to be more geographically restricted to the tropics than those directly transmissible between people, such as influenza [ ] . environmental barriers to dispersion can be circumvented. monkeypox virus, whose natural transmission is largely restricted to parts of equatorial africa by the range of its natural rodent hosts, has demonstrated the ability to make use of new hosts in temperate zones [ ] and a number of arboviruses, such as chikungunya, dengue and zika viruses, have widely expanded their natural ranges as their principal vectors have [ ] . the rf selection of field sites in was based on the relatively greater diversity of arboviruses found during the preceding years in tropical countries and it can be argued that their success in discovering new viruses owed as much to this factor as their choice of integrated, active surveillance. as would be expected, there appears to be a correlation between zones of species richness and frequency of reported vector-borne and zoonotic emerging disease events [ ] . the species richness of the tropics suggests that human populations there are exposed to greater risk and that they are fertile grounds for virus mutation. certain behaviors common to some regions, such as the harvesting of wild animals for food, aggravate that risk. the lack of housing barriers to rodents, bats and arthropod vectors are major vulnerabilities to pathogens carried by them. environmental and sanitation deficiencies also increase risk to enteric and vector-borne viruses; in the absence of dependable water supply, many people, for example, are forced to store containers of water that provide breeding for the virus-carrying mosquitoes. most importantly, as dunn et al. [ ] have shown, countries with the highest pathogen richness spend the least per capita on health care, and there is an inverse correlation between investment in public health and pathogen prevalence, independent of species richness. because of the obvious link between the abundance of novel viruses and the tropics, geospatial modeling could help target areas for surveillance. there is a tradition of developing and using models to identify those areas of the world whose species richness is most in need of conservation [ , ] and recently attempts have been made to use models to identify areas most liable to spawn emerging diseases [ , ] . considering the association between mammal and pathogen species richness there can be expected to be some overlap between the two sets of ''hot spots''. the predictive robustness of a model depends not only on the algorithms used but in the choice and weighting of variables, and the representativeness and validity of the data. for example, a widely cited model [ , ] , which chooses ''the original case or cluster of cases representing an infectious disease emerging (during - ) in human populations for the first time'', lists only of viruses first described as infecting humans during that period [ ] . on the other hand it credits the first occurrence of a number of viruses described years earlier to the period - ; these include measles, influenza a, and rabies, assigning to each a single, arbitrary origination location (us, hong kong, and costa rica, respectively) for modeling geospatial associations. because this model also lumps a variety of emerging disease types, including many examples of antimicrobial resistance, population density is a major factor, which might explain the higher likelihood for emergent events it assigns to india and java than to the amazon or equatorial africa, where so many novel viruses have been discovered. modeling has been more successful for single pathogens for which large amounts of specific data have been collected, such as for dengue [ ] or malaria [ ] . associating disease prevalence in a limited area with well-characterized environmental attributes, as has been done for plague bacteria (yersinia pestis) in uganda, can be used to predict areas potentially at risk that would be difficult to collect data from, such as large plague-prone tracts of the neighboring democratic republic of congo [ ] . because many of the arbovirus species are known only from places where long-term field operations were established by the rf, institut pasteur, and others, and because those sites were selected for logistical and political realities as well as scientific interest, their usefulness in modeling is still limited. surveillance for emerging pathogens: the problem of knowing the unknown successful surveillance depends on how and where one looks. ideally, an emerging virus will be detected at its source and contained before spreading. this ideal requires, however, extensive networks of alert health care providers, adequate laboratory resources, and an effective method for communicating results to an authority capable of responding. it also assumes that a zoonotic virus will not be spread by animal hosts impossible to control, as was the case with the avian arbovirus, west nile. in practice, human disease surveillance raises an alarm only after an arbitrary number of seemingly related, serious cases are reported and arouse attention. generally, number of cases and length of time to detection are least for anticipated pathogens with distinct presentations, such as poliovirus presenting with acute flaccid paralysis. in countries with rudimentary public health systems that threshold might be reached for unexpected pathogens only after the number of cases reaches epidemic proportions impossible to ignore, as the recent epidemic of ebola virus in west africa demonstrates. many viral disease cases present as clinically indistinguishable acute febrile illnesses (afi) or with symptoms so mild the patient does not seek attention. cases with neurological involvement or systemic bleeding can also be difficult to diagnose clinically without adequate laboratory support. in those tropical areas most likely to spawn emerging viruses, afi will be misdiagnosed or undiagnosed in at least % of patients [ ] [ ] [ ] [ ] . overlooking novel pathogens as the cause of nonspecific symptoms is not confined to developing countries: it is likely heartland virus was a cause of illness in the usa long before it was characterized in . nevertheless, the probability that more undescribed viruses infect humans in the tropics seems to be greater. it is likely, therefore, that many emerging diseases due to novel viruses will be overlooked, especially at tier , until they become epidemic, are transported to countries with more sensitive surveillance, or are discovered by chance. laboratory support for clinicians is critically deficient nearly everywhere in the rural tropics. the first step in determining if an illness might be caused by a rare or unknown virus is to eliminate the possibility of pathogens known to be endemic. simple, relatively accurate rapid diagnostic tests (rdts) are available for a few common causes of afi, notably malaria, but tests for most viruses require not only equipment, such as elisa readers, but modest, dependable infrastructural support-electric power, clean water, cold storage-rarely available outside major cities. poor roads often make the timely, proper transport of specimens to centers with laboratory capacity impractical. even in cities, many hospitals and government laboratories do not have the basic equipment, fresh reagents or accurate testing protocols to assay for most common endemic pathogens. should laboratory capability be available to eliminate most known etiologies for the disease observed, description of a novel pathogen typically requires both biological and molecular characterization [ ] . recovery of viable virus for culture and histological evidence of pathology remain fundamental steps establishing causality. the increasing power and availability of rapid, next-generation sequencing has made whole-genome analysis an increasingly routine and important part of describing novel viruses but because of the large number of commensal species found in the human virome [ ] , linking a novel genome with virulence will be tentative without supporting biological evidence. for example, wu and ki polyomaviruses, isolated in the mid- s from children suffering from acute respiratory infections and tentatively included in the list of human pathogens [ ] , have yet to be proven causal of illness [ ] . our ability to detect and characterize novel pathogenic viruses in hot spot locations lags behind global systems that have arisen to report and respond to unusual occurrences. the international health regulations (ihr) of the world health organization (who) binds countries to plans to improve their ability to detect and respond to outbreaks, and the global outbreak alert and response network (goarn) of who helps organize international response to public health emergencies. open source networks for reporting outbreaks include the program for monitoring emerging diseases (promed-mail), a free, internet-based system for disseminating information posted by , professional contributors in countries, and healthmap, which collects and continuously updates disease outbreak data from a variety of public sources, including news services. many of these reports seem never to be investigated or resolved. the full value of these systems can only be attained if provided with accurate information. considering the barriers to obtaining human surveillance data, it has been proposed [ ] that monitoring animal populations at sentinel locations could alert us to risk from viruses with pandemic potential. for such a plan to be feasible for emerging viruses it would be necessary to judge the potential risk posed by a virus not yet known to infect humans. epizootic disease in livestock or wild animals is used as a threat indicator for some known zoonotic viruses, such as influenza, rift valley fever virus, and west nile virus, but there is no assurance that an agent potentially pathogenic to humans will cause noticeable disease in animal hosts. coronaviruses closely related and putatively ancestral to sars virus, for example, seem not to cause disease in host bats [ ] . the extent of animal disease surveillance is also far less in the tropics than even the poorest human clinical networks so the likelihood of recognizing an unusual event is less. periodic sampling of animals can discover novel viruses but is too infrequent and limited to be surveillance. considerable attention has been given to human contact with bush meat, or animals captured for food [ ] . while harvesting and slaughtering wild animals appear to have provided the mechanism by which some important pathogens have emerged, such as hiv and sars, it has not played a role in the emergence of many others, including the three examples discussed in this paper: lujo, heartland and mers-cov. vectors obviate the need of direct human-mammal or human-bird contact and can move viruses across ecological zones. sequencing and cataloging the viruses of animals in selected areas can provide valuable insight to transmission dynamics and phylogenetics but, as discussed, cannot yet be used to predict. one must wonder if the us $ . billion proposed to catalog mammalian viruses not yet known to be pernicious [ ] would not be better spent on developing more suitable diagnostic tests for humans in remote areas most liable to emerging pathogen risk or on conducting sentinel human surveillance. ultimately, the best indication that a pathogen has the ability to jump to humans is finding it in humans [ ] . although there is not a strong rationale for conducting autonomous searches for potentially pathogenic viruses in animal populations, there is much value in animal investigations in support of surveillance for infectious diseases in selected, sentinel human populations. the discovery of lujo virus in a human, for example, should direct our attention to its epidemiology and ecology in the area where we suspect exposure occurred. had lujo been discovered in an animal instead, its significance as a pathogen would have been speculative until the detection of the first human case. the integrative, long-term approach of the rf can serve as a model but with primary focus on conducting population-based surveillance for acute illness. concomitant ecological profiling and virological studies in arthropods, mammals and birds can more quickly clarify the epidemiology of any novel viruses discovered in the human population. the zoonotic viruses pathogenic to humans represent a small but unknown proportion of those infecting mammals and birds. from this constantly evolving universe of vertebrate viruses two or three are recognized every year to have broken the species barrier, a remarkably small number considering the frequent contact between humans and animals, and the high adaptability of rna viruses. while most of these novel, emergent viruses have inconsequential public health significance, some, such as mers-cov or lujo, have obvious destructive potential. what is the best strategy for identifying and limiting the menace from novel, zoonotic viruses? identifying potential pathogens before they leap to humans (tier ) would seem ideal but is impractical. the determinants of pathogenicity are complex and poorly understood, while a system for wildlife or livestock surveillance in those areas with conditions most conducive to emergence cannot anytime soon reach a scale or effectiveness to be pragmatic. it is likely that yet to be recognized viruses already infecting humans will be sources of disease outbreaks of varying magnitude in the future. these tier infections can be uncovered as part of comprehensive, investigative surveillance in human populations at risk. in the near term this would be best accomplished in most places through specially designed sentinel surveillance sites. modeling might at some point provide guidance for site selection but too narrow a definition for target sites (e.g., bush meat markets) will be self-defeating. by identifying and eliminating poorly appreciated endemic agents, investigations can then focus on illnesses with unresolved etiologies. unlike investigations directed at animal populations there would be a tangible, immediate improvement in the health of the subject communities. complimentary studies of vectors and animals, as pioneered by the rockefeller foundation, would prepare for epidemiological investigations of those zoonoses uncovered but the primary focus must be on humans. ultimately, one hopes, surveillance for emerging zoonoses will be a part of improved health care systems throughout the world. global alert and response: ebola virus disease chikungunya virus in the americas emerging infections: microbial threats to health in the united states prediction and prevention of the next pandemic zoonosis nosocomial outbreak of novel arenavirus infection genetic detection and characterization of lujo virus, a new hemorrhagic fever-associated arenavirus from southern africa a new phlebovirus associated with severe febrile illness in missouri first detection of heartland virus (bunyaviridae: phlebovirus) from field collected arthropods middle east respiratory syndrome coronavirus (mers-cov)-update isolation of a novel coronavirus from a man with pneumonia in saudi arabia isolation of mers coronavirus from a dromedary camel ecological origins of novel human pathogens mutation rates among rna viruses origins of major human infectious diseases guns, germs, and steel: the fates of human societies environmental and social influences on emerging infectious diseases: past, present and future search strategy has influenced the discovery rate of human viruses berichte der kommission zur erforschung der maul-und klauenseuche bei dem institut für infektionskrankheiten in berlin das klinische bild der maul-undklauenseuche beim menschen, aufgestelt aus den bisher experimentell gesicherten erkrankungen the rockefeller foundation virus program: - with update to big hitting collectors make massive and disproportionate contribution to the discovery of plant species centers for disease control and prevention, international catalog of arboviruses including certain other viruses of vertebrates predicting unknown species numbers using discovery curves a strategy to estimate unknown viral diversity in mammals zika virus outbreak on yap island, federated states of micronesia the role of evolution in the emergence of infectious diseases the global distribution and burden of dengue airborne transmission of influenza a/h n virus between ferrets role of receptor binding specificity in influenza a virus transmission and pathogenesis venezuelan equine encephalitis emergence: enhanced vector infection from a single amino acid substitution in the envelope glycoprotein chikungunya virus adapts to tiger mosquito via evolutionary convergence: a sign of things to come? a single positively selected west nile viral mutation confers increased virogenesis in american crows investigating virus-glycan interactions using glycan microarrays early alterations of the receptor-binding properties of h , h , and h avian influenza virus hemagglutinins after their introduction into mammals global patterns of terrestrial vertebrate diversity and conservation insight into global mosquito biogeography from country species records using habitat models to map diversity: pan-african species richness of ticks (acari: ixodida) ecology drives the worldwide distribution of human diseases global drivers of human pathogen richness and prevalence spectrum of infection and risk factors for human monkeypox movement of chikungunya virus into the western hemisphere global trends in emerging infectious diseases threatened biotas: 'hot-spots' in tropical forests theory and data for simulating fine-scale human movement in an urban environment hot spot or not: a comparison of spatial statistical methods to predict prospective malaria infections improvement of disease prediction and modeling through the use of meteorological ensembles: human plague in uganda etiology of acute, non-malaria, febrile illnesses in jayapura, northeastern papua. indonesia etiology of severe non-malaria febrile illness in northern tanzania: a prospective cohort study undiagnosed acute viral febrile illnesses evidence of a major reservoir of non-malarial febrile diseases in malaria-endemic regions of recommendations for publication of viral genetic data and sample access for novel viruses and strains the human virome: new tools and concepts human polyomaviruses in disease and cancer bats and their virome: an important source of emerging viruses capable of infecting humans overviews of pathogen emergence: which pathogens emerge, when and why? key: cord- -vzw cl authors: ferella, alejandra; aguirreburualde, maría sol perez; sammarruco, ayelen; parreño, viviana; santos, maría josé dus; mozgovoj, marina title: dinámica de anticuerpos neutralizantes contra el virus respiratorio sincicial bovino date: - - journal: rev argent microbiol doi: . /j.ram. . . sha: doc_id: cord_uid: vzw cl abstract bovine respiratory syncytial virus (brsv) is one of the most relevant agents responsible for respiratory disease in cattle from both dairy and beef farms. brsv is spread by horizontal contact causing a constant presence of seropositive animals that favors viral circulation throughout the year. moreover, reinfections with brsv are frequent between animals regardless of their age as brsv does not confer long-lasting protective immunity. several studies have demonstrated the circulation of brsv in cattle from different regions of the world; however, little is known about the dynamics of brsv infection in cows before and after they begin lactation. the aim of this work was to study the dynamics of brsv neutralizing antibodies from birth up to months of age in a closed dairy herd of argentina specifically around the lactation period. passive maternal antibodies against brsv started to decrease monthly and became almost undetectable at months of age. we detected two potential infection points at months and after birth, in which % and % of the animals showed seroconversion, respectively. specifically, an increase in the proportion of seropositive cows after the start of lactation suggests that they became reinfected around the time they began lactating. we demonstrate the importance of understanding brsv dynamics in a closed dairy herd to review the vaccination schedule of the animals to achieve protection against brsv infection. the authors wish to thank osvaldo zabal for his technical assistance and dr amanda wooloms for reviewing and correcting this manuscript. this work was supported by the instituto nacional de tecnologia agropecuaria pnsa- , the mincyt secyt-bidpict- - and conicet. dynamics of neutralizing antibodies against bovine respiratory syncytial virus in a dairy herd from santa fe province, argentina resumen el virus respiratorio sincicial bovino (vrsb) es uno de los principales agentes responsables de enfermedad respiratoria en bovinos, tanto de tambos como de cría. el virus se transmite horizontalmente y causa la presencia constante de animales seropositivos, lo cual favorece la circulación viral a lo largo del año. a su vez, las reinfecciones por vrsb son frecuentes entre animales independientemente de su edad, dado que el virus no confiere inmunidad protectora a largo plazo. numerosos estudios han demostrado la circulación de vrsb en bovinos de diferentes regiones del mundo, sin embargo, poco se conoce acerca de la dinámica de infección en vacas antes y después del inicio de la fase de lactancia. el objetivo de este trabajo fue estudiar la dinámica de anticuerpos neutralizantes anti-vrsb en vacas lecheras desde el nacimiento hasta los meses de vida en un tambo cerrado de argentina, específicamente, en el período de lactancia. los anticuerpos pasivos específicos para vrsb comenzaron a declinar mensualmente hasta ser casi indetectables a los meses. detectamos dos potenciales puntos de infección a los meses y luego del nacimiento, momentos en los que el % y el % de los animales mostraron seroconversión, respectivamente. el incremento en la proporción de vacas seropositivas luego del comienzo de la lactancia sugiere que estas se reinfectaron en el inicio de dicha etapa. demostramos la importancia de entender la dinámica de circulación del vrsb en un tambo cerrado, a fin de revisar el esquema de vacunación de los animales para que estén protegidos frente a la posible infección por este virus. bovine respiratory syncytial virus (brsv) is one of the most relevant agents responsible for respiratory disease in cattle from both dairy and beef farms. brsv is spread by horizontal contact causing a constant presence of seropositive animals that favors viral circulation throughout the year. moreover, reinfections with brsv are frequent between animals regardless of their age as brsv does not confer long-lasting protective immunity. several studies have demonstrated the circulation of brsv in cattle from different regions of the world; however, little is known about the dynamics of brsv infection in cows before and after they begin lactation. the aim of this work was to study the dynamics of brsv neutralizing antibodies from birth up to months of age in a closed dairy herd of argentina specifically around the lactation period. passive maternal antibodies against brsv started to decrease monthly and became almost undetectable at months of age. we detected two potential infection points at months and after birth, in which % and % of the animals showed seroconversion, respectively. specifically, an increase in the proportion of seropositive cows after the start of lactation suggests that they became reinfected around the time they began lactating. we demonstrate the importance of understanding brsv dynamics in a closed dairy herd to review the vaccination schedule of the animals to achieve protection against brsv infection. palabras clave: virus respiratorio sincicial bovino, tambo, circulación viral, período de lactancia keyword: bovine respiratory syncytial virus; dairy herd; viral circulation; lactation period introduction bovine respiratory disease (brd) is a group of infectious respiratory diseases that affect cattle and is responsible for significant economic losses to the farming industry worldwide . bovine respiratory syncytial virus (brsv) plays a major role in this disease. brsv belongs to the genus orthopneumovirus, family pneumoviridae, order mononegavirales . morbidity can be as high as to % and mortality can reach up to % . after brsv infection, pneumonia outbreaks are a frequent outcome given its tropism for the lower respiratory tract and its ability to predispose to secondary bacterial infection . the virus is transmitted horizontally by direct contact between the animals, through aerosols and respiratory secretions; therefore, the infection is favored when there is close contact between the animals, as it happens during milking or in feedlots. antibodies against brsv can be detected in animals of all ages, even in those with no respiratory signs. infection with brsv does not induce long-lasting immunity and calves can be reinfected throughout their lives, even with the same strain. thus, antibody titers against brsv are higher in older animals . reinfections are usually subclinical or cause mild clinical respiratory disease. interestingly, it has been described that the presence of high titers of neutralizing antibodies (nas) against brsv significantly decreases the clinical severity of the disease . hägglund et al. reported that brsv is widely distributed in dairy farms in sweden and that there is horizontal dispersion of the virus with a constant presence of seropositive animals, which indicates viral circulation throughout the year . although several reports have demonstrated that the virus is widespread all over the world, information about brsv in argentina is limited, especially in dairy herds. odeón et al. reported the presence of antibodies against brsv mainly in cattle under months of age from buenos aires, corrientes and la rioja, with a greater spread of the virus in districts with larger movement of animals . recently, we described the presence of neutralizing antibodies and risk factors associated with brsv in feedlots from santa fe and córdoba, argentina . in addition, we demonstrated evidence of the circulation of brsv by antibody seroconversion in a longitudinal analysis of post-weaned animals from an argentinian beef herd . we have also reported the presence of antibodies against the virus in early and ultra-early weaned beef calves . even though brsv has been linked to decreased milk production in dairy cattle , there is no information about the dynamics of brsv infection in cows before and after they begin lactation. in this work, we studied the dynamics of neutralizing antibodies against brsv in a dairy herd from santa fe province, argentina for three years. potential infection points based on seropositivity were evaluated. the herd was a large holstein dairy herd from a farm in santa fe province, consisting of milking cows. the herd was closed; thus, there were no animals from other herds and no entry of animals from outside. calves stayed with their dams for - days and they were individually tethered by a chain to a stake holding two buckets to feed bulk tank milk and balanced feed for days, when they were weaned. after weaning, they were raised on pastures with animals of the same age group up to months. then, they were moved to pastures with animals of the same age and weight (end of first raising period). animals were vaccinated against foot-and-mouth disease virus semiannually starting at months of age. they also received doses every days against: bovine herpes virus subtypes i and v (bohv i and v), bovine viral diarrhea virus (bvdv), parainfluenza (pi ), pasteurella multocida, mannheimia haemolytica and histophilus somni and clostridial agents, between to months of age. regarding brucellosis, they were vaccinated once between to months. rotavirus-escherichia coli vaccine was administered at and days before parturition. animals did not receive vaccination against brsv during all the sampling period. beginning at months of age, heifers were artificially inseminated and placed in contact with other heifers and cows. pregnant heifers were kept on pastures and days before delivery were moved to a preparturition area. stages of production management in the dairy herd are shown in fig. . serum samples were kindly provided by geronimo gutierrez (laboratory of adventitious virus, inta). samples were collected from female calves born during july and august (n = ). sampling took place from birth until months of age. the first sample was collected within the first week of age and at months , , , , , , , , , and . all the experimental proceedings were carried out following international recommendations (guide for the care and use of agricultural animals in research and teaching) and the institutional manual of inta (guide for the care and use of experimental animals). the viral neutralization assay of serum samples was carried out as described by samal et al. . briefly, inactivated serum samples were four-fold diluted from : to : . serum dilutions were mixed with tcid of a brsv strain and incubated for hour at °c in a % co atmosphere. this mixture was inoculated in duplicate onto mdbk cell monolayers ( . cells/ml) in -well plates. plates were incubated as mentioned above and cpe was observed at dpi. samples were considered positive when no cpe was observed. na titers were expressed as the reciprocal of the maximum dilution in which no cpe was observed. samples with titers lower than were considered negative. this method was selected for its high sensitivity in seroprevalence studies. seroconversion was defined as an increase in a base four-fold dilution antibody titer. antibody titers to brsv determined by na were log -transformed prior to the statistical analysis. negative samples at a dilution of : were assigned an arbitrary antibody titer of for the calculation of geometric mean titers (gmts). percentage of seropositive animals since birth until months of age was tested using the fisher´s exact test for multiple comparison of proportions, p values were corrected by the holm method. group effects on the na titers to brsv were analyzed by a general linear mixed model (glmm). the model included one main fixed factor: time (with twelve levels, as withinsubjects factor). animals were included in the model as a random factor. heteroskedasticity of variance among time points was modeled using the var indent option. the variance and covariance matrix included the assumption of an autoregressive effect (ar ) among the titers of the same bovine determined at different time points. the akaike information criterion (aic) was used for choosing the best-fitting model as a minimal adequate one. thus, the model with the lowest aic value was selected. the glmm analysis was conducted by using the glmer function (lme package, r development core team, ) through infostat® statistical software connection to r. statistical significance was assessed at p < . for all comparisons. results obtained from this study showed that at birth (stage ), % of animals were seropositive for brsv with high na titers. these results are in concordance with those reported by tuncer et al., who detected the presence of maternal antibodies in % of the calves sampled at one month of age in dairy herds from turkey . since the vaccines used in this dairy herd did not contain brsv in their formulation, maternal antibodies must have derived from natural brsv infections. in this study, passive maternal na specific for brsv started to decrease monthly according to igg half-life. at two months of age a significant reduction in the mean na titers was observed (fig. ) . these na levels remained low (almost undetectable) between months and . in fact, % ( / ) of the animals were seronegative at months of age (fig. ) . these results are in agreement with those described by uttenthal a et al., who postulate that the level of brsv na significantly decreases during the first months of age . at months of age (stage ), when the first movement of the animals took place, (transference to pastures with other animals of the same age), primary infection was evidenced by a significant increase in na titers in % ( / ) of the animals. this fact is in agreement with the first movement of animals to pastures where they took contact with other animals of the same age. these management practices, as well as the absence of specific na against brsv from month to month , increases the susceptibility to infection by brsv. our results agree with what has been previously described by other authors, who stated that management practices within herds are predisposing risk factors for brsv infection and that the first infection in dairy farms occurs mostly during the first two months mainly in autumn or winter . na titers remained stable until months of age. interestingly, at month , na titers dropped slightly, while prevalence increased to % ( / ). at parturition ( months) a significant decline in the percentage of seropositive animals was observed ( %, / ). this could be associated, again, with management practices, since at that moment animals were isolated from the rest of the animals, explaining why there was no direct contact between them favoring infection with brsv. close contact between animals and crowding conditions are main risk factors that enhance the transmission of brsv . the concentration of serum antibodies in colostrum is another physiological process that must be considered, as it was documented that cows concentrate in colostrum times the amount of igg present in sera which can lead to a significant drop in serum igg . an increase in seropositive animals (reaching % values) was observed at month (after delivery, stage ), when the animals re-entered the productive cycle (lactation cycle), coming back into close contact with other animals and under stress conditions, including recent calving and separation from their calves. in addition, the mean na titers were significantly higher than those obtained during stage (at that time the animals were in pastures with other heifers and cows, prior to parturition). this is in agreement with the study report from león et al. stating that reinfection is common in herds causing a significant increase in antibody levels in seropositive animals . after stage , na titers began to decline. seroconversion percentages were analyzed at each stage. in this regard, two critical points of seroconversion were detected at months and , with seroconversion rates of % and . % respectively. given that the dairy herd analyzed in this study was a closed one it seems unlikely that the virus could have been introduced from other sources, suggesting an intra-herd viral circulation of brsv in this production system. it is not clear if brsv persists in the herd or if it is reintroduced from other sources before each outbreak . hypotheses about the way a virus remains in a herd or if it is reintroduced are controversial. it is possible that factors such as latency and reactivation, low level of circulation of the inter or intra-herd virus or hosts that act as a reservoir play a role in the maintenance of the brsv . studies of the viral genome analysis from isolates indicated that during an outbreak the virus within the herds remains identical, but varies between outbreaks, suggesting that these are probably caused by "new" viruses rather than by latency or the existence of animals that act as carriers . in our study, a possible hypothesis is that the virus was spread between animals over the years, which is accordance with previous results . nevertheless, further studies should be performed on isolates in order to assess the genetic diversity of the circulating virus. there are several inactivated vaccines available in argentina provided as part of multivalent products to prevent respiratory disease. at present, vaccination programs recommend two initial doses ( weeks apart) to induce immunity. protection induced by these vaccines and also after infection with the virus, tend to be short-lived (< months), especially in calves with colostral antibodies . in this study at month after birth, all calves were seronegative. this is a critical period since the animals are more susceptible to brsv infection because of the low level of na, and if an infection occurs, this could cause severe disease and even death. a conventional vaccination schedule recommends two doses; the first one at weaning and a booster days post-weaning. our results demonstrate the need to review the vaccination schedule of animals in dairy farms in order to protect them against infection through vaccination against brsv. in this regard, calves should be vaccinated with more effective vaccines in the face of maternally derived antibodies and also after months of age where neutralizing antibodies significantly decrease and become almost undetectable at month after birth. strategic measures must be implemented to overcome economic losses due to respiratory disease caused by brsv in dairy herds including the improvement of the vaccines used. reduced likelihood of bovine coronavirus and bovine respiratory syncytial virus infection on organic compared to conventional dairy farms phylogenetic analysis of bovine respiratory syncytial viruses from recent outbreaks in feedlot and dairy cattle herds duration of immunity to experimental infection with bovine respiratory syncytial virus following intranasal vaccination of young passively immune calves bovine respiratory syncytial virus seroprevalence and risk factors in feedlot cattle from córdoba detection of antibodies against bovine herpes virus , bovine viral diarrhea virus and bovine respiratory syncytial virus in early and ultra-early weaned beef calves dynamics of virus infections involved in the bovine respiratory disease complex in swedish dairy herds seroprevalencia de la diarrea viral bovina , herpesvirus bovino y virus sincicial respiratorio en argentina a longitudinal study of the dynamics of bovine corona virus and respiratory syncytial virus infections in dairy herds seroprevalence and risk factor associated with respiratory viral pathogens in dual-purpose cattle of rio de oro, and la gloria municipalities in cesar department reliable confirmation of antibodies to bovine respiratory syncytial virus (brsv) by enzyme-linked immunosorbent assay using brsv nucleocapsid protein expressed in insect cells bovine respiratory complex: evidence of multiple viral circulation in a breeding farm serological detection of infection dynamics for respiratory viruses among dairy calves antibody dynamics in brsv-infected danish dairy herds as determined by isotypespecific immunoglobulins bovine respiratory syncytial virus infection key: cord- - g n e authors: steele, james harlan title: veterinary public health: past success, new opportunities date: - - journal: preventive veterinary medicine doi: . /j.prevetmed. . . sha: doc_id: cord_uid: g n e abstract animal diseases are known to be the origin of many human diseases, and there are many examples from ancient civilizations of plagues that arose from animals, domesticated and wild. records of attempts to control zoonoses are almost as old. the early focus on food-borne illness evolved into veterinary medicine's support of public health efforts. key historical events, disease outbreaks, and individuals responsible for their control are reviewed and serve as a foundation for understanding the current and future efforts in veterinary public health. animal medicine and veterinary public health have been intertwined since humans first began ministrations to their families and animals. in the united states, the veterinary medical profession has effectively eliminated those major problems of animal health that had serious public health ramifications. these lessons and experiences can serve as a model for other countries. our past must also be a reminder that the battle for human and animal health is ongoing. new agents emerge to threaten human and animal populations. with knowledge of the past, coupled with new technologies and techniques, we must be vigilant and carry on. farmer keeping animals in his midst. the people who domesticated the animals were thus the first to be victims. those early humans then developed resistance to some zoonotic diseases that had emerged (diamond, ) . the relation of animal diseases to human disease was observed in the ancient civilizations of babylon, the nile valley, and china and noted by leviticus in the old testament, and later by hippocrates in greece, and virgil and galen in rome. millions of people across europe during the middle ages suffered from plague carried by rat fleas. the invasion of europe by rinderpest in the th century disrupted commerce and government so much that the papal authority created a medical commission to advise the vatican on what measures should be taken to control the animal plague/rinderpest . the movement of animal diseases into the americas is believed to have been in the support of the settlements founded by columbus in santo domingo in . these livestock were the foundation animals for spanish colonies in the americas. in the next century, de soto, the spanish explorer of florida and the southeast, brought cattle, horses, and swine, as well as dogs that thrived. farther north, the virginia colonists brought animals to roanoke island, but none survived, neither humans nor animals. later the jamestown colonists imported domestic animals that survived and became valuable foundation stock, but no zoonotic diseases are recorded in any of these earliest settlements. not until was rabies the first zoonosis recorded in the us colonies, and later as an epizootic in both the colonies and the federation of states in the late th century (smithcors, a,b) . in , the newly founded medical repository editors were the first to inquire about emerging diseases in the united states and territories. they asked for information on human diseases, diseases among domestic animals, accounts of insects, the condition of the vegetation, and even the state of the atmosphere. they hoped to put the facts together as an annual report on the status of health in the united states. surgeon general luther terry ( ) of the us public health service (usphs) in his address at the american veterinary medical association (avma) centennial called this report the first reference to veterinary medicine in support of public health. a few years after this report, benjamin rush called for the establishment of veterinary medical education at the university of pennsylvania. the united states sanitary commission, organized during the civil war by public-spirited women, was concerned largely with sanitary conditions, including food hygiene. they were the first to call attention to the putrid meat and later embalmed beef sent to the army. the commission was to be a forerunner of public health in the years following the civil war (furman, ) . by the s, there was interest in developing a national health service. yellow fever epidemics were frightening as they spread up the mississippi river from new orleans. the possibility that yellow fever involved animals brought professor john gamgee, a famous veterinarian, from england to investigate the epidemic. he recognized the seasonal occurrencethat cold weather stopped the epidemic -and even suggested river traffic be limited to the colder months. however, he failed to associate the effect of cold weather with the decline of the numbers of mosquito populations, the vector of yellow fever (furman, ) . the us board of health came into being largely because of the yellow fever epidemic and the morbidity and mortality that it caused. by the time of the board's inception in , malaria was widespread in the south, and tuberculosis was a recognized disease. typhoid fever and enteric diseases were also common. in addition, animal diseases were present, especially the spread of glanders and anthrax following the civil war (furman, ) . in the president of the us board of health, dr. j.l. cabell, asked james law, professor of veterinary medicine at cornell university, to advise the board on how they should supervise the diseases and movements of domestic animals. law's report ( ) was the first comprehensive recognition of the effects of zoonotic diseases upon public health published in the united states (steele, ) . the organization of public health in the post civil war period has been reviewed by miles ( ) , former historian of the national institutes of health (nih). his report discusses the struggle between public health and agricultural interests in the decade leading up to the inauguration of the bureau of animal industry in . the interest of the bureau was to protect animal health, and later to provide a meat inspection service for public health, international trade, and subsequently interstate commerce. the relation of animal diseases to the public health and their prevention by frank s. billings ( ) was the first book to review the problems and the state of bacteriology as well as parasitology in the s. although the book is limited to trichinosis, hog-cholera, tuberculosis, anthrax, texas fever, rabies, and glanders, his knowledge of these diseases is remarkable for the time. billings gained this knowledge through education in berlin, where he learned about the history of animal diseases in the greco-roman period and the latin origin of ''veterinarians,'' which he says first appeared in the th century writings of vegetii. he also traveled extensively in europe, where he observed veterinary activities. billings makes a strong plea for the development of veterinary public health to control the animal diseases that affect man. he stated that this could be accomplished only by having trained veterinarians who were scientifically educated. he was one of the veterinarians who was active in the early years of the american public health association (apha), during which discussions of trichinosis, tuberculosis and other animal diseases took place at the early annual meetings. a true visionary of veterinary public health, billings pointed out that milk from diseased cows is dangerous. he appealed to the government to set up laboratories to use the new science of bacteriology to find the cause of illness of milk origin. food hygiene came into being only with the new science of bacteriology (billings, ) . the frightful toll of milk borne disease is reviewed by stenn ( ) . in his report, he cites the shocking figure of deaths per births in new york city in . spoiled milk accounted for the deaths of thousands of children in the early s, and in many other cities. the records of , cited by stenn, list many milk borne outbreaks of typhoid fever and diphtheria. he goes on to state that % of the milk cans sampled contained tubercle bacilli, and in cities, % of the milk had tubercle bacilli. in a milk borne typhoid epidemic occurred in washington, dc, that caused president theodore roosevelt to order the usphs to investigate the local supply. surgeon general walter wyman ordered his staff to examine not only the washington milk problem but to examine the national milk problem. the report milk and its relation to public health by milton rosenau, issued by the usphs, brought reform to the dairy industry and support for the bureau of animal industry program to control bovine tuberculosis (myers and steele, ) . pasteur took milk safety even further, changing science and veterinary medicine by creating a new concept of the origin of disease. no longer would the myth of spontaneous origin of disease guide society, although there were as many objections to scientific advances then as now. the centennial celebration of the rabies vaccine revealed in pasteur a man of many accomplishments. he was a chemist who discovered the cause of fermentation and applied it to the beer and wine industries, a process that led to milk pasteurization. he was an artist who was known to the impressionists of the th century as the man who prepared better paint colors. he was also a genius who gave public health the science and vaccines to combat th century diseases and prepare for the th century's emerging problems (koprowski and plotkin, ) . although the concept of pasteurization of beer and wine brought fame to pasteur, the application to milk was less known, and it was accepted no more readily than the concept of evolution. it was asserted that all kinds of illness and changes in well being would ensue from pasteurization. the eradication of bovine tuberculosis and brucellosis (bang's disease) insured a safe milk supply and protected the health of farmers, dairymen, veterinarians and the handlers of milk and milk products. the case for pasteurized milk and milk products is conclusive. in the late th century, a new array of milk borne zoonoses is of concern to public health and veterinarians. some date back to the th century, such as salmonella. the salmonella were identified in by one of the most distinguished public servants of the veterinary profession, dr. daniel salmon. as the first chief of the bureau of animal industry (bai) from to , he assembled and trained a great staff. this included theobald smith, v.a. moore and e.c. schroeder, who solved the epidemiology of texas fever caused by babesia bigemina, which is carried by the tick boophilus annulatus. salmon was the leading proponent of veterinary public health in the s. he asked for, and received from congress, authority for a federal meat inspection service in to meet the demands of foreign commerce. however, his national program was circumvented by local interests citing states' rights; therefore, the meat inspection act of was ineffective nationally. salmon sought support from the apha and the american medical association for these early veterinary efforts to protect public health. unfortunately, these agencies did not support him (schwabe, a) . the federal meat inspection service act of came about only after sinclair ( ) exposed the filthy conditions of the chicago stockyards. salmon was blamed for the local hygiene failure over which he had no authority and was removed from office. however, he is remembered today by the usda's salmon award for leadership. in , the bai initiated tuberculin testing of dairy cattle in the district of columbia, a demonstration that revealed an infection rate of almost %. this was the beginning of a successful tuberculosis control campaign that led to its eradication under john r. mohler, bai director from to . the late jay arthur myers memorialized the near eradication of bovine tuberculosis in his book, entitled ''man's greatest victory over tuberculosis'' (myers, ) . at the start of the th century, pathologists were greatly interested in comparative medicine. they were led by karl f. meyer, a swiss veterinarian who was to become one of the leaders and outstanding scientists of the th century. he was among the early public health scientists to delve into virology as professor of pathology at the university of pennsylvania (penn), and in he may have been among the first to recover a virus causing equine encephalitis. as director of the pennsylvania livestock sanitary board laboratory, he published on glanders, anthrax, anaplasmosis, sporotrichosis, paratuberculosis, septicemia, and many other diseases of animals. in he left the university of pennsylvania to accept a position at the university of california's newly established tropical medical center. the following year, he accepted an appointment to the george williams hooper foundation for medical research at the university of california medical center. he remained there the rest of his life and become a legend. his lectures introduced medical students to the biologically active world, including the zoonoses, plant life, the atmosphere and all that is called the environment today. at the hooper foundation, meyer researched a wide spectrum of animal diseases of public health importance. after being active in the investigation of human influenza in - , he went to the field to define the epidemiology of malaria, dysentery, and even dental diseases. his study of the bacterial causes of abortion in animals resulted in bringing together brucella abortus, brucella melitensis, and brucella suis in a new genus honoring david bruce. another important event was his report on clostridium botulinum in nature. botulism became a national concern in the s when california canned fruit and vegetables were found to contain botulinum toxin. the industry asked meyer to resolve the problems and underwrote a laboratory to maintain surveillance. thereafter, meyer was active in food safety, but he was also concerned with humane animal care in which he maintained an interest all his life. in , meyer and his long time lab associate bernice eddie began their series of psittacosis reports in birds. these reports eventually led to control years later with tetracycline-impregnated seed. the same antibiotic is now used to prevent ornithosis in domestic fowl (meyer, ) . one of meyer's most memorable lectures was in when he called attention to the importance of the animal kingdom as a reservoir of diseases that endanger the health and welfare of people throughout the world (meyer, ) . in , he first reviewed the state of the animal reservoir of diseases, by then referred to as zoonotic diseases, before the world health organization (who) general assembly. he repeated the same theme before the who expert committees for the zoonoses, plague, food hygiene and for the pan american health organization (paho) until his th year. meyer's work on plague was reported in the special supplement of the journal of infectious diseases to commemorate his th birthday. this was underwritten by max stern, president of hartz mountain, which supported the psittacosis control investigations at the hooper foundation (steele, ) . meyer died in san francisco on may , , less than a month before his th birthday. larry altman ( ) , the medical editor of the new york times, wrote a lengthy obituary from which the following excerpt is taken. it also appears on the fore page of the journal of infectious diseases (supplement), may : ''dr. karl fredrich meyer was regarded as the most versatile microbe hunter since louis pasteur and a giant in public health [. . .] . public health leaders yesterday called his contributions to medicine 'monumental.' his scientific work had such broad implications that it touched on virtually all fields of medicine.'' the obituary was placed in the congressional record that same month. in , albert sabin ( ) wrote a biographical memoir of meyer for the national academy of science, of which meyer was a member from l to . sabin explains that as a youth in basel, switzerland, pictures of the black death so fascinated meyer that he became an outdoor scientist instead of following in the aristocratic business world in which he grew up. he told friends that in choosing to become a veterinarian he could ''be a universal man and study all diseases in all species.'' the s were memorable for public health growth and scientific advances. the viral etiology of influenza was uncovered by richard shope and thomas francis at the rockefeller institute. the use of egg embryos was a new method of growing viruses that would lead to the chick-embryo rabies vaccine and other viral vaccines. the development of the strain brucella vaccine and the stern anthrax vaccine in south africa were important to the control of brucellosis and anthrax worldwide. earlier investigation of toxoids by gaston ramon, a french military veterinarian, led to the discovery of tetanus toxoid for both horses and humans. discovery of the sulfa drugs and penicillin gave the clinician medication he had not dreamt of, a prelude to great advances in medicine. to be a veterinary student in the late s was both exciting and slightly dangerous. the brucellosis epidemic among veterinarians -both students and clinicians -raised epidemiological questions as to how brucellosis was spread. in michigan state college experienced an epidemic among veterinary students and others in the bacteriology building (holland, ) . up to then the disease was thought to be caused by direct exposure or ingestion of milk borne brucella, and airborne brucella was not given much consideration. the episode at michigan state college would change that oversight. as a student in the brucellosis testing laboratory, i heard discussion of the means of spread being water borne and back siphonage. professor i.f. huddleson, whose research laboratory was the focus of this investigation, disagreed with the state investigators, who were public health scientists and engineers focusing on the water borne theory. these investigators suggested contaminated glassware was not being autoclaved properly, and in turn, viable brucella was getting into the water system (newitt et al., ) . the discussion of the epidemic, which affected most of the people in the building, along with other public health interests of dean ward giltner, professor h.j. stafseth, professor i.f. huddleson and dr. w.t.s. thorp of the michigan state university college of veterinary medicine, led me to think about a career in public health. dr. stafseth encouraged many students to consider public health as a career (stalheim and steele, ) . when he learned of my interest, he and dean giltner worked out a program to make me eligible for a usphs fellowship. i was excused from senior clinics to pursue the fellowship. my assignment was an internship at the michigan health department. there i observed and learned from health department veterinarians, pathologists and bacteriologists how to remove and examine an animal brain for rabies and to inoculate mice to further confirm the diagnosis. vaccinia were grown on the belly of a calf that had been shaved, scrubbed and disinfected. after harvesting the scabs, the vaccinia would be tested for contaminants. it was a lengthy procedure. the same high standards were maintained for the pertussis/whooping cough vaccine, equine antiserum for tetanus and rabbit pneumococcal antiserum. it was a learning period that would serve me well. dean giltner and c.c. young, the director of the michigan public health laboratories, put together my fellowship application to the usphs and harvard school of public health. approval came the week before graduation, and my bride-to-be aina oberg and i were elated. we were married the evening after graduation, with many of the faculty and classmates in attendance two days later i took the michigan examination to practice veterinary medicine. the summer of was spent as an intern at the petoskey animal hospital. there i learned about swimmer's itch-a common affliction of man and pets caused by an avian schistosome. i was exposed to the parasite while swimming in inland lakes. at harvard it became my thesis subject. later it was the first subject i reported on at the avma convention in chicago ( ) with dean giltner in the audience. at harvard, there was talk of war. president conant addressed the incoming class with the admonition there would be important world changes during their student years. the school of public health's dean cecil drinker, the faculty, and the students were stimulating. i was the only veterinarian, which attracted some attention, and the medical school librarian was delighted to know there was a veterinarian around. she showed me a remote section of the library that contained many old books on veterinary medicine-harvard had a veterinary faculty from to . we students were delighted with our newly found classmates, and many of us would remain lifetime friends. to me, the academic work was not demanding except for statistics, which took much time. after all, in those days, we used hand-cranked machines for tabulations. my wife, aina, first worked at the harvard co-op and then with the british american ambulance volunteers. she enjoyed the students and compliments and being invited to fundraisers for the volunteers. then tragedy struck. a sudden collapse with fever hospitalized her. the diagnosis was advanced tracheal-bronchial tuberculosis that would confine her to sanitariums and hospitals for the next years, from january to april . after innumerable surgeries, the newly discovered streptomycin saved her life after months of treatment. eventually, we established a home and family with two sons, jay and david, in atlanta, georgia, for years. there aina died from the complications of arrested pulmonary tuberculosis in . the new year ( ) brought unforeseen problems, mainly medical bills, even though student health expenses were covered to a lesser extent. i sought work at the angel memorial animal hospital, where i knew some of the staff. when dean drinker heard of my after-school work plans, he called me into his office and told me to concern myself with school and taking care of my wife. a check signed by dean drinker awaited me in his secretary's office, a practice that continued until graduation. the drinker society still honors his contributions at the harvard school of public health, which we support. as graduation neared, many of us knew we were going into uniformed service job opportunities. i was deferred by the lansing michigan draft board, but i volunteered for the army veterinary corps and the navy special services, an epidemiology unit. both declined my services, and in the meantime, i found no positions of interest. i wanted to do epidemiology of the animal diseases affecting human health, but all the positions i was interested in required a medical degree. finally i brought my dilemma to dean drinker's attention. shortly thereafter, he asked me to his office to talk over my future. i was upset that it seemed i must have a medical degree to be an epidemiologist. should i get an md? dr. drinker and his wife, also a physician, heard me out. their reply was to list my attributes: good student, industrious, good appearance, good speaker, and creative ambitions. that said, they followed with memorable advice: ''jim, fly under one flag.'' before leaving harvard, i met kf meyer who was lecturing at the school of public health. some days later, i learned he had asked about me because he anticipated some research contracts with the us army epidemiology board and would need staff. i was elated when he offered me a position, and aina and i left boston with high expectations. some days later we arrived in chicago, only to find out a week later that meyer had not received the contract and had no funds to support the research position. i was depressed: no job and a sick wife. a few days later i visited the usphs chicago regional offices to seek their help in finding work. i appeared unannounced and asked a secretary to see the director. while waiting, a medical officer appeared and asked if he could help. i explained that i was a usphs fellow they had supported in getting an mph, and my objective was to find a position where i could investigate the epidemiology of animal diseases that affect the public health. dr. henry holle, the medical officer, listened and replied he never heard about such a situation. so he took me to see the medical director, mark ziegler, a tall, soft-spoken, southern gentleman. the availability of a young mph graduate led them to call washington. a week later after a review of my qualifications and evidence of my education, i was offered an internship as a civilian sanitarian in the ohio department of health. there i would spend the next year, july -october . the challenges were milk sanitation problems, food borne diseases, diarrhea, typhoid, rabies and the ohio river flood, a great learning experience. in september , the us army offered me a commission as a veterinary officer which i planned to tentatively accept. within days, medical director frank meriwether told me since the usphs had given me a fellowship, they should have a first call to commission me. i received a commission as a sanitarian on november , . afterward, i spent a short tour of duty in the midwest region with senior sanitarian william h. haskell, an authority on pasteurization methods and practice. he was one of the civil service veterinarians brought in by the usph milk specialists early in . in , other newly recruited veterinarians raymond helvig, ray fagan, and ted price were also commissioned as sanitarians. they were the only veterinarians in the usphs except for a veterinarian who was an animal control officer in world war i in and two parasitologists willard wright and maurice hall at the nih in the s. from chicago, i was ordered to report to washington, dc, for orientation. there i learned of my assignment to puerto rico and the virgin islands where i was to be responsible for coordinating milk and food sanitation and evaluating any zoonotic diseases in areas that had been isolated by the war. brucellosis and bovine tuberculosis were widespread. the diagnosis of venezuelan equine encephalitis and bat rabies in trinidad caused some concern in the islands but did not spread beyond trinidad. rabies was indigenous in the dominican republic and cuba in the s. in march , the pan american sanitary bureau asked the usphs san juan, puerto rico, office to do an assessment of the post-war veterinary public health problems in the dominican republic and haiti, neither of which had a functional veterinary service. i was directed to make a report on their problems. in the dominican republic, there were no reported diseases, but bovine tuberculosis, brucellosis and mastitis were known. no veterinary laboratory support existed, and the abattoirs kept no records. rabies had been reported in dogs, and possibly in horses, and some years later there was an epizootic of equine encephalitis. president trujillo kept some racing horses near ciudad trujillo (santo domingo), and i was asked to examine them. these old horses were brought to the dominican republic before the war in - . all were broken down and hardly fit to run. regardless, the dominican republic officials thought i could repair their ailments. when i told them i could not, they complained that i was not cooperative to the u.s. embassy, who then told me to be cooperative. later i visited the trainer, who told me, ''we will do what we can.'' thereafter, i was anxious to leave and went to haiti within a few days. port-au-prince was a rundown but hospitable capital. the country had been ravaged by tropical fevers for decades; malaria and filariasis were widespread. animal diseases were mainly fever and parasites, but an epizootic of anthrax in the early s was still present in : the disease was sporadic in the countryside. to my amazement, the dead animals were salvaged regardless of what they died from. there were no veterinarians in the government or in practice. still, the abattoir in port-au-prince was an elegant open iron structure. the cattle were immobilized by pithing, in which a small blade severs the spinal cord after which they are bled and eviscerated. the procedure was done rapidly, usually late at night, and the meat was distributed early the next day. however, a serious shortage of animal products existed, and few shops had any meat for sale. all in all, my stay at port-au-prince and the rural areas was a distressing experience. some weeks later i was in washington for further assignment as the war wound down. while there, i visited the pan american sanitary bureau to discuss my report with surgeon general hugh s. cumming who served the pan american sanitary bureau for a decade, after retiring from the usphs. at our meeting, i emphasized the need for a veterinary public health program to help in updating the animal health, preventing zoonotic diseases, and enhancing food safety. dr. cumming suggested i discuss the need for a veterinary public health program with his medical staff, where the proposal was enthusiastically accepted. the veterinary public health program was initiated with dr. aurelio malaga alba, a peruvian military veterinarian, as a consultant. dr. fred soper, the post-war director of the reorganized paho appointed dr. ben blood to organize a veterinary public health program in june . he carried on until and was followed by the outstanding public health veterinarian dr. pedro acha. the temporary duty in dc left my future uncertain. i was to be assigned to kansas city to prepare for the problems that might evolve with the invasion of japan. i took leave to spend some weeks with my hospitalized wife whose health was failing. the end of the war in europe and the pacific shortly thereafter changed my reassignment. i returned to dc to meet with assistant surgeon general joe mountin, whom i met earlier in puerto rico. dr. joe dean, his deputy, had arranged the interview. after a few inquiries about my wife's health, dr. mountin came to the point: ''what are you veterinarians going to do for the public health now that the war is over?'' the follow-up to that interview is in the appendix of ''the th anniversary of the veterinary medical corps officers of the u.s. public health service.'' after the approval of a veterinary public health section in the states relation division in december , i spent some months at the national institutes of health. i also worked to establish liaisons with the usda, bai, federal agencies, congressional interests, state relations, the avma and apha. in september after surgeon general parran's approval of the veterinary medical officer cadre, dr. mountin felt my washington activities were successful. he told me i was to be assigned to the newly created communicable diseases center, formerly the malaria control in war areas. there the veterinary public health program was established as a division, but it was a challenge to integrate. the new director of the centers for disease control (cdc) was dr. r.a. vonderlehr, previously chief of the puerto rico regional office, who i served under. he gave excellent support as did his deputy, dr. justin andrews, who succeeded dr. vonderlehr a year later. rabies was a national problem after the war. there was a great movement of people as war industries and encampments closed, and as a result, pets were lost or abandoned. the incidence of human rabies was the highest ever recorded, and unfortunately, human vaccine therapy was not always effective. canine rabies vaccine protection was short, with the vaccines being given every months. therefore, rabies became the lead program of the veterinary public health division. to head the activity, dr. ernest tierkel, a university of pennsylvania graduate who had completed his mph at columbia school of public health in , was recruited. he, dr. robert kissling and martha eidson along with a staff of animal handlers became the nucleus of the national rabies program at the rockefeller rabies investigation center in montgomery alabama (steele and tierkel, ) . the center was transferred to the cdc for $ . . they successfully demonstrated the effectiveness of a new chicken embryo rabies vaccine in the laboratory (tierkel et al., ) and in epidemic situations in memphis, tennessee (tierkel et al., ) . dr. mountin had learned from the public health authorities of indiana, michigan and others that brucellosis in man was of concern. they went so far as to say that as the sanitariums lost tuberculosis patients, brucellosis patients would take their place. the indiana health department was to be a brucellosis project site under dr. sam damen, the director of laboratories. the goal was to determine what action the health agencies should take. the federal bovine brucellosis control program was active in all states, so it became apparent that if the health authorities gave their support, the federal state brucella control program could eliminate the animal source of the human disease. late we brought the problem to the attention of dr. herman bunderson, chicago's dynamic health officer who remembered the struggle to eradicate bovine tuberculosis in the chicago milk shed, which included dairy herds in six midwestern states. in , he had required all milk coming into chicago to be from tb-free herds regardless of whether the milk was to be pasteurized. he recognized the brucella problem and shortly thereafter instituted the same standards for the elimination of bovine brucellosis in the s. the brucella eradication program was supported by the usphs milk code, which required that all grade a milk be from disease-free herds (us public health service, ) . the chicago brucella control program was soon adopted by big city health authorities, which gave impetus to the joint state federal brucella programs. as a result of these efforts, human brucellosis declined rapidly in the midwest from a high of thousands of human infections to hundreds in less than a decade. thereafter most of the human cases were of occupational origin, in travelers or in people using raw milk in rural areas. in the s there was a scare of brucellosis at dugway proving grounds, a military research center in western utah. dr. herbert stoenner investigated the alleged contaminated area and found the problem to be a rodent disease caused by brucella neotoma. this organism does not cause disease in man or domestic animals, but will cause antibody formation in cattle (stoenner and lackman, ) . after world war ii, there was great interest in the application of atomic energy for civilian use. professor s.f. gould ( ) at wayne state medical school initiated studies on the use of irradiation to destroy trichinella. he persuaded the american medical association to host a trichinosis symposium in in which the cdc participated. the evidence was conclusive that gamma radiation was effective at low doses (gould et al., ) . this was the beginning of my interest in promoting food irradiation, but it was not until that irradiation for commercial use was approved by federal agencies. the zimmerman human tissue survey - revealed the lowest rate of trichinosis ever (zimmerman et al., ) . modern pig raising, the prohibition of garbage feeding of swine, and consumer education are all contributing factors in the decline of the disease in pigs and humans (steele, ) . trichinosis has continued to decline in the states except in wild animals especially bears. other veterinary public health studies of parasitic diseases involved creeping eruption, also known as cutaneous larva migrans. this condition is due to the common dog hookworm larva ancylostoma caninum entering the skin and causing intense itching. this disease was common in the southeast states among persons exposed to damp, sandy soil; children playing in sandboxes; bathers at the beach, and utility men (cypess, b) . toxocariasis or visceral larva migrans is another parasite due to the dog, and sometimes the cat, roundworm larva migrating in the body of a foreign host, human beings (cypess, a) . dr. peter schantz confirmed these findings as a world health problem. toxoplasmosis was recognized as a human infection, and the domestic cat is recognized as a common source of human infection. infection is more likely to be caused by consumption of raw or undercooked meat. irradiation is effective in destroying this oocyst in meat (gould et al., ) . in the early s, a large equine encephalitis epizootic in central california required the assignment of all cdc veterinary officers. later another equine encephalitis epizootic occurred in new jersey in . since then there have been only occasional epizootics of the equine encephalitides. although the principal reservoir is birds, there is also survival of the virus in mosquito eggs that over winter. the cdc-fort collins laboratory has been at the forefront of these investigations. the most recent mosquito born disease is the introduction of west nile virus into north america in . wild birds and common city birds are the reservoirs, and the culex mosquito is involved in the transmission. horses may show clinical signs. control of the vector mosquito breaks the transmission cycle. plague, primarily a disease of rodents, is sporadic in the united states. the appearance of plague in domestic and feral cats and squirrels has brought the ancient scourge to households in the western states (poland and barnes, ) . however, dogs were never identified as carriers of the disease to man. an unusual epidemic of anthrax caused alarm in animal and human public health circles in the s. the anthrax was introduced by contaminated bone meal used in animal feed to improve lactation in sows. a radio announcer in cincinnati raised the question if cows' milk could be a vehicle for anthrax to be carried to humans. a search of literature found that milk was never a vehicle or cause of human or animal anthrax disease because the high fever of the disease stops lactation (steele and helvig, ) . salmonellosis was a recognized public health problem early in the s as well as during world war ii and afterwards among the civilian populations (galton et al., ) . after the war, investigators demonstrated it was widely disseminated. dr. phil edwards led the way at the university of kentucky and later at the cdc. mildred galton, chief of the veterinary public health laboratory contributed with her unusual ability to find evidence that others had overlooked. she demonstrated salmonella in many animals. her studies of transported pigs revealed how stress caused latently infected pigs to become shedders. the same reaction was found in other species. her work on raw eggs and meats led to the pasteurization of egg slurry used in baked or cooked products. she was among the first to find salmonella in raw milk years ago, and her work on the frequent presence of salmonella in poultry led to the federal poultry inspection program in the late s (steele and galton, ) . thirty years before weil described leptospirosis in humans in , animal leptospirosis was identified as its own problem. a record of an canine epidemic in stuttgart, germany, exists, but the etiologic agents were not determined. years after the canine epidemic, it was discovered that microorganisms morphologically identical caused the disease in both dogs and humans. leptospirosis proved confusing to all health professionals partly because ''isolated serovars were given names denoting the clinical signs observed in the patients from whom they were isolated'' (torten, ) . therefore, it was thought that serovar grippotyphosa would cause signs similar to catarrhal fever, and serovar icterohemorrhagia would cause hemorrhagic jaundice. it was not recognized that both serotypes are capable of causing both signs (torten, ) . in the us, there were numerous outbreaks among animal handlers, veterinarians and swimmers as well as people whose occupation exposed them to contaminated waste water in the - period. leptospirosis is now recognized as a problem associated with disasters such as flooding and earthquakes. there is wide agreement that vaccination of cattle and dogs has reduced environmental contamination (stoenner et al., ) . galton ( ) edited the ''leptospiral serotype distribution list '' through , and sulzer ( ) carried it up to . they were truly dedicated in keeping these records. listeriosis was first recorded in in sheep, and the first reported human case was in denmark in (bomer et al., ) . prevention of listeriosis is still not possible with the knowledge available, as there are no immunizing agents of proven worth. killed bacterins have been disappointing, and living attenuated vaccines have not been evaluated properly nor have they shown promise in limited experiments. good physical hygiene is essential to prevention (bomer et al., ) . groups at high risk of infection are pregnant women, neonates, diabetics, alcohol dependents, persons with neoplastic disease, or those being treated with corticosteroids or antimetabolites. among animals, ewes are at the highest risk late in the first pregnancy. sheep in late pregnancy should not be fed ensilage of doubtful quality nor be exposed to severe cold or inclement weather and crowding (bomer et al., ) . improved measures for preventing and controlling human listeriosis depend on increasing awareness of its diverse clinical manifestations and an increasing index of suspicion. because l. monocytogenes, the causative agent of listeriosis, is sensitive to most antibiotics, their early administration, once the diagnosis has been established, significantly decreases mortality. cortisone and its derivatives may, however, cause asymptomatic listeria infections to become overt (bomer et al., ) . after the end of the war in europe, the breakdown of food hygiene there allowed salvaged food to spread zoonotic diseases. at the same time there were numerous cases of listeriosis reported in france that caused abortion, stillbirths and reproductive tract disease (seeliger, ) . the disease remains prevalent in western europe to the extent that all midwives and obstetricians alert their patients to report symptoms. since there has been a steady decline of reported cases. food borne listeriosis elsewhere was virtually forgotten until when an outbreak occurred in the maritime provinces of canada and was associated with consumption of contaminated coleslaw (schleck et al., ) . then years later, a major outbreak in massachusetts between june and august of was epidemiologically linked to consumption of a particular brand of pasteurized whole and % milk (fleming et al., ) . although questions have been raised about the adequacy of the epidemiologic study (ryser and marth, ) , no other food has emerged as the vehicle that transmitted l. monocytogenes in this outbreak. in mexican-style cheese made in a factory near los angeles was definitively linked to a large outbreak listeriosis (linnan et al., ) . this was followed in by the linking of consumption of vacherin mont d'or, a variety of cheese, to an outbreak of listeriosis in the canton of vaud in switzerland (bille et al., ) . in recent years, food borne outbreaks continue to be reported in north america and europe. during the s, many more human cases and deaths were reported in the united states. the vehicles reported as contaminated were cold cuts, canned meats and frankfurter sausages. worldwide, listeriosis is a problem mostly in the temperate zones. another emerging zoonotic food borne disease is escherichia coli o :h , the enterohemorrhagic strains as well as those characterized by cytotoxins. these e. coli and others of human origin are major causes of the human enteric disease. however, they are less causative in food producing animals that may be infected but show few or no clinical signs. pasteurization of milk is effective in the control of e. coli spread. irradiation has proven effective for pasteurization of food of animal origin for the protection of the public health. recently improved inspection and hygiene have reduced reported human diseases, even though toxic e. coli is wide spread among cattle. the same can be said for newly identified emerging food borne zoonotic diseases. cryptosporidia parvum is a coccidian protozoa found worldwide. giardia are found in numerous animals, and during the late th century, the flagellate protozoan was identified worldwide as a water borne disease of humans and animals. old problems new to the states are taenia saginata and t. solium, largely found in immigrant workers. the tapeworm cysts found in meat, beef and pork are easily destroyed by irradiation, a technology that slowly is being accepted in the southern countries where tapeworm disease is recognized as both an economic as well as a public health problem. the acceptance of veterinary public health internationally by the paho has been previously discussed. the inauguration of veterinary public health as a national program in the usphs in stimulated interest worldwide, especially in the newly created international agencies. the united nations health office organizing committee chaired by surgeon general tom parran met in new york in june to further public health worldwide. the public health service officers and personnel were asked by the surgeon general's chief of staff, g.l. dunnahoo, to suggest topics. veterinary public health was new, but a few weeks before the organizing group was to meet, i was directed to make a veterinary public health presentation and answer questions at the surgeon general's staff meeting. after the meeting, i asked dr. dunnahoo if he would be interested in a recommendation for a veterinary public health program for the who organizing committee. he urged me to give him a memo recommending a veterinary public health activity. that may , , i wrote a memo paraphrased as follows: ''regarding our conversation and your encouragement, i propose that in the organization of the united nation's health office there be a veterinary public health (vph) program. the vph program would be concerned with animal diseases transmissible to man. the vph would carry on liaisons with veterinary activities in the agriculture agencies and collect information on animal health.'' some months later i asked how the vph recommendation was received. dr. dunnahoo said there were no objections or discussion: the vph item was accepted and placed in the records. years later i learned an american veterinarian, martin m. kaplan, was recruited by an english physician with whom kaplan worked with in the united nations relief and rehabilitation administration (unrra) in greece. in kaplan came to the newly established who in geneva, switzerland. he developed a vph program in the communicable disease division that is a model for a public health program in the developing world. during the next years, he organized the expert committee meetings and technical reports. the first was in l (who, ) to review tuberculosis, which was a major disease problem in humans and animals at the end of world war ii. an american tuberculosis authority, dr. franklin top, a us army consultant, had reported that % of the human cases in occupied germany were caused by mycobacteria tuberculosis bovis. the problem was referred to the who expert zoonoses committee by the who expert tuberculosis committee. there was no consensus on what recommendation to make. the danish veterinarian dr. plum spoke for the classical tuberculin test and identification. the french urged the use of bacille calmette-guérin (bcg) vaccinations. the success of bovine tuberculosis eradication in the united states was recommended as the ideal method. eventually the committee recommended test and removal, with the caveat for developing countries to try other methods, including the bcg vaccination, which had no success in field trials. a number of other diseases were reviewed with the recommendation for control. there was a consensus on the following: q fever, anthrax, psittacosis, and hydatidosis. another issue was to settle on a definition of veterinary public health. a current definition of public health is summarized as diseases that are naturally transmitted between animals and man. the following year, , who called together a panel of rabies experts, including e.s. tierkel of the cdc (who, ). tierkel and others who followed from the cdc, namely george baer, keith sikes, jerry winkler and currently charles rupprecht, contributed to rabies control and prevention. the first of the who expert committees on the zoonoses was followed by zoonoses study groups in , which meyer chaired in stockholm (who, ) . he was most effective in leading the committee, and in his closing remarks he passed the leadership to james steele. at the next meeting of the who zoonoses expert committee in geneva in , i was the chairman (who, ) . the next meeting in was chaired by calvin schwabe (who, ) , professor of epidemiology at the university of california school of veterinary medicine and the school of human medicine. schwabe ( b) summarizes the who veterinary public health in his monumental third edition of veterinary medicine and human health: ''the final objective of veterinary medicine does not lie in the animal species that the veterinarian commonly treats. it lies very definitely in man, and above all in humanity.' ' we in veterinary public health recognize the contributions of acha and szyfres ( ) for their invaluable book, zoonoses and communicable diseases common to man and animals in spanish and english. it has been the foundation of veterinary public health epidemiology and surveillance in the spanish speaking countries of the americas. at this time, dr. george beran is to be recognized as one of the consultants to paho and who, and for his work in the philippines. he has carried on in admirable style for more than years in teaching, research, health promotion and consulting, and as author and editor. he has updated the chemical rubber company (crc) handbook of zoonoses series (beran, ) and the paho zoonoses reports, and hopefully will continue to do so. he is a historian of veterinary public health. in closing we pay tribute to the american veterinarians who demonstrated and promoted veterinary public health in the united states. most of these early pioneers years ago were recruited by the cdc and assigned to states that had zoonotic disease problems, mainly rabies. among the early cdc recruits assigned to a state was ernest wine. he was sent to pennsylvania, where he remained for years, rising to the position of state epidemiologist. oscar sussman went to arizona, and later the new jersey health department recruited him, where he built an outstanding program. martin baum served colorado for many years after leaving the cdc. john mason served in new mexico. art wolff did excellent service in michigan before returning to washington, where he became a leader at the usphs in environmental health as a radiation authority and assistant surgeon general. herbert stoenner went to utah and raymond fagan to indiana as described earlier. monroe holmes followed stoenner to utah, and john scrugs went to indiana when fagan went to the harvard school of public health. john winn, francis abimanti, don mason, and lauri luoto were among the early investigators of q fever in california. stoenner, in addition to his investigation of brucellosis and leptospirosis, was also a leader in q fever studies. don mason, john richardson, and paul arnstein worked on the control of psittacosis in k.f. meyer's laboratory at ucsf. dick parke, joe held and robert huffaker kept the cdc office responsive to many inquires and provided service to the states. james glosser closed his career at the cdc in . his work coordinating the venezuelan equine encephalitis epizootic and epidemic with the u.s. department of agriculture veterinary services earned him the united states department of agriculture's outstanding service award. the veterinarians service to public health in the th century resulted in better health in all humans and animals. what are the st century challenges? animal medicine and veterinary public health have been intertwined since humans first began ministrations to their families and animals. dr. william foege, former director of the communicable disease center and professor at emory school of public health and now consultant to the bill gates foundation center, expressed this more forcefully in saying that we cannot have good public health unless we have good animal health. we can invert that and say we cannot have good animal health unless we have good public health. in the united states, the veterinary medical profession has carried on effectively in eliminating those major problems of animal health that had serious public health ramifications, namely bovine tuberculosis and brucellosis. in recent years the advances in rabies immunization have eliminated the disease from our pets, and humans have benefitted. the new human cases that occur are mainly the result of bat exposure. looking beyond that, we can see there is a sizable list of parasitic diseases, namely trichinosis and tapeworms, that have been brought under control in the united states. however, tapeworms are now being introduced by the recruitment of workers from mexico, central america and south america. these problems affect society in the united states, but it is apparent that we have an obligation to share our knowledge with our neighbors of the americas as well as africa and asia. all of these countries face the same problems the united states, solved in the past century. now as we move into the st century, the technology for controlling these diseases is available. these proven effective procedures in the united states can be used worldwide. some challenges exist, however, for methods that control bovine tuberculosis. there is a continuous demand for vaccines to prevent tuberculosis in animals, but there is little evidence there is any value in routine vaccination. these procedures are quite costly, and the best examples are in europe in the past years. after world war ii, tuberculosis was a major problem in central europe especially in germany, eastern europe, what is now russia, and western europe. there has been an uncalled-for degree of confidence in the tuberculosis vaccine, bcg, but with constant pressure from the world health organization, the world animal health organization (oie), the food and agricultural organization and united states agencies and consultants, the use of vaccines has been put aside. the old test and removal strategies have proven to be the most successful. to introduce that method into mexico, central america, south america and asia is difficult at this time because they are hopeful that a good vaccine will be developed. unfortunately we have lived with that hope for years. the major problem that remains is to compensate farmers for diseased animals that are removed. the neighboring countries of mexico, central america and south america have the opportunity to further their own disease control by employing the proven techniques used in the united states, canada and europe. the control of brucellosis in the developing world is a much bigger problem than tuberculosis. in veterinary epidemiologist george baer described the human disease in mexico and said that most rural people who had reached the age of had evidence of past infection with brucellosis. the same can be said for the countries of latin america where goats have a high rate of b. melitensis infections. to control b. melitensis is a difficult task and is a matter of the governments facing up to the issue. a new vaccine developed in the united states, the rb rough strain, had been researched for years or more before the united states department of agriculture veterinarians were able to find a solution to producing an effective vaccine for cattle. the vaccines have not proven valuable for goats and sheep. the control of widespread brucellosis in north africa and the middle east across asia has been given little attention. the who, through their consultancies and expert committees on rabies, has spread the knowledge of dog vaccination throughout the world. we can say with some degree of pride that the technology developed by veterinarians at the communicable disease center and carried to other parts of the world by authorities such as the late ernie tierkel and others who worked with him and george baer have made a great contribution to the world scene. we do see the light at the end of the tunnel for worldwide control of canine rabies. other efficient rabies vaccines have been developed in south america and europe. looking at the parasitic infections of the world, there is certainly a great deal of interest in control of trichinosis, which has been fostered through scientific congresses every few years. the world wide results are favorable today with a drastic reduction in north america and europe. unfortunately new problems have arisen in connection with the disease in wild animals, especially those found in the arctic zones of the world. taenia saginata and t. solium are receiving more attention as we face worldwide problems with the measurements of disease. in the americas the problem has been carried from one country to another by human carriers and then spread to animals. new foci have been established in north america, where there have been meetings to plan for initiating a worldwide control program. in my own way of thinking, the control of t. saginata is a measurement of good hygiene and good waste control in any country where it is present. dr. peter schantz has advocated world control of tapeworm and hydatid disease with the goal of eradication. many other new problems arising in zoonotic and parasitic diseases are constantly coming to our attention. the continuous migration of workers seeking better opportunities in industrialized countries also carries the risk of infections being brought with them. the surgeon general has spoken for the globalization of public health. the veterinary public health program of the cdc has been active in globalization of veterinary public health, namely in the control of rabies, parasitic diseases and food borne diseases. many of the veterinary officers have served on who expert zoonosis committees have carried out detailed missions for who. the cdc program has been supportive of paho veterinary activities with assignments of veterinary officers to mexico, panama, peru, argentina, and most recently david ashford and hugh mainzer to brazil for foot and mouth disease control and other problems. the number of emerging diseases increased in the latter part of the th century. infectious disease scientists have found that acquired immune deficiency syndrome (aids) is a disease that makes people more susceptible to zoonotic diseases, including bovine tuberculosis and related mycobacterial infections, toxoplasmosis, cryptosporidiosis, food borne salmonella and enteric infections including campylobacter, listeria and yersinia. it is possible that other zoonotic diseases that are dormant or infrequent may emerge in individuals with aids, human immunodeficiency virus infection, or other immune-compromised conditions. related latent or nonpathogenic viral diseases have been described in tropical cats of africa including lions as well as domesticated cats. in australia and malaysia new diseases which also affect humans have been reported in horses and swine. these diseases are caused by the morbilliviruses, a measles-like virus that causes canine distemper and rinderpest. another virus that killed the wild felids in the cairo zoo has not been identified. could this be another form of distemper? some of the emerging viral diseases that have a rodent or unknown animal host have caused fatal, devastating diseases in humans in africa and south america, namely lassa fever and south american hemorrhagic diseases in argentina and bolivia. in africa, ebola virus hemorrhagic fever and marburg hemorrhagic fever virus infection, linked to monkey disease, caused disease in medical personnel, handlers and people who had only casual exposure. an incident that surprised us many years ago was the deaths of workers in middle east abattoirs caused by crimean hemorrhagic fever carried by ectoparasites. one example of developing, emerging, or relatively unknown diseases is severe acute respiratory syndrome (sars), a disease that erupted a few years ago in china and was carried to many parts of the world. recently information has suggested that bats are a natural reservoir of a sars like coronavirus. even though sars may have been an occasional emerging disease that disappeared as rapidly as it appeared, there may have been other infections from bats that have been around the world for millenia. naturally, an infection that has been given much attention now is where we stand with the influenza virus. are wild birds the true reservoir? apparently, birds are the reservoir based on the information we have gathered showing that wild birds transmitted the virus to avian domestic flocks. all this new information is challenging. the emerging diseases of the world are reasonably covered in the table of the last chapter of merck veterinary manual's ninth edition zoonosis section, . in addition to infectious diseases, we have a new class of diseases that are caused by prionsproteinaceous infectious parties that transfer diseases without any dna or rna. transmissible diseases are not the same as infectious diseases which are characterized by replication of dna or rna. this is certainly a bewildering situation especially when we read that saliva may be a means of transfer. immediately veterinarians think of rabies which is transmitted by saliva. is it possible the prion of the diseased brain can be secreted through nerve fibers that innervate the salivary gland? the prions are of great and continuing concern as a cause of concern as new types of diseases. our associates in chemistry, physics, and physiology may offer clues to other neurological diseases. one last subject i want to mention is humaneness. it is important that we abide by sensible, humane policies, but humaneness can be carried to such an extreme that it destroys values that we hold so high for protecting our pets, farm animals, and the wild animals around us. periodically we all read about overpopulations in different areas. society calls for conservative measures for population control that applies to all pets, wild animals and domestic animals. in a broader sense, it has applications to the human race. we are aware of the collapse of earlier civilizations that have overpopulated their given area or were destroyed by natural events such as starvation. so i say all veterinarians, especially those in public health, have a responsibility in developing humane regulations for animal population control and public guidance. in the united states, - % of veterinarians treat our animal associates or pets for various diseases. it is important that veterinarians have a broad, basic knowledge of public health issues and are alert for new public health issues that can be resolved with tender loving care, new antibiotics, and new procedures. the , or more veterinarians in the avma in the united states are key to the control of zoonotic diseases by public health agencies. the health of our animal population is tied to the emotional and mental well being of those humans who are close to animals in their lives. animals are vital companions to those homebound, and animal health becomes a family concern. an area i have stressed is the need for basic veterinary science. we see in current publications that most research is based on support from nih. at the avma meeting in july , the speaker us senator hatch of utah spoke highly of the public health activities of veterinary medicine. he went on to say that there may be a nih veterinary institute in the future. it behooves us all that the agricultural interest in public health be recognized as an important issue to the american public. i think highly of the importance of animal health in providing good public health. public health should not be guided by economic interest but by the welfare of all society. i go back to my earlier statement that animal health and public health are of great importance to all, and we must have good animal health to have good public health. good public health provides a means for good animal health. as we look to the future, we have to have open minds and think in terms that anything can occur in biology. i would like to quote my dean from michigan state, ward giltner, who said the only thing about biology we can accept that remains a firm truth is there always is new information that provides exceptions. looking at it broadly, all infectious things in nature, and prions which may cause disease are always looking for a new host. i like to say they are seeking social security, as most of the world is. carry on in the st century. i wish i could continue to be a part of it, but it seems time has a way of saying, ''you have been here. you have enjoyed it.'' i especially enjoy the recognition of years of public health service, i am elated. to the audience, especially to the teachers of public health science, thank you. carry on. dr. steele does not have a financial or personal relationship with other people or organisations that could inappropriately influence or bias the paper entitled ''veterinary public health: past success, new opportunities.'' zoonoses and communicable diseases common to man and animals. pan american health organization viral scientist dies-public health giant handbook of zoonoses section a: bacterial, rickettsial, chlamydial and mycotic. section b: viral, second ed epidemic food-borne listeriosis in western switzerland. ii. epidemio ogy the relation of animal diseases to the public health and their prevention listeriosis visceral larva migrans cutaneous larva migrans guns, germs, and steel: the fates of human societies pasteurized milk as a vehicle of infection in an outbreak of listeriosis a profile of the us public health service - . pub. no. nih- - . us department of health, education and welfare. us government printing office leptospiral serotype distribution lists (through ). veterinary public health laboratory. us department of health, education, and welfare public health service the world problem of salmonellosis prevention of trichinosis by gamma irradiation of pork as a public health measure control of trichinosis by gamma irradiation of pork undulant fever outbreak at michigan state college. mich world's debt to pasteur report on diseases of domestic animals epidemic listeriosis associated with mexican-style cheese the animal kingdom-a reservoir of disease history of medical research and public health: an oral interview history of the bureau of animal industry, appendix man's greatest victory over tuberculosis. c.c. thomas attempts to control tuberculosis among cattle water borne outbreak of brucella melitensis infection plague milk and its relation to public health karl friedrich meyer - : a biographical memoir epidemic listeriosis: evidence for transmission by food food safety veterinary medicine and human health listeriosis the jungle. double, page & company the veterinarian in america - the socioeconomic responsibilities of veterinary medicine biographical notes on the occasion of karl f. meyer's th birthday trichinosis zoonoses - : an update of james law's report on diseases of animals epidemiology of food borne salmonellosis present status of anthrax rabies: problems and control. a nationwide program nurture turned to poison a new species of brucella isolated from the desert wood rat neotoma lepida (thomas) the epizootiology of bovine leptospirosis in washington leptospiral serotype distribution lists ( to ). veterinary public health laboratory. us department of health, education, and welfare public health service a century's progress in public health effective control of an outbreak of rabies in memphis and shelby county a brief survey and progress report of controlled comparative experiments in canine rabies immunization leptospirosis milk ordinance and code. public health bulletin no. . department of the treasury joint who/fao expert group on rabies. who technical report series no. . who joint who/fao expert group on zoonoses: bovine tuberculosis, q fever, anthrax who, . joint who/fao expert committee on zoonoses, report on the nd session. who technical report series no. . who, geneva. who, . joint who/fao expert committee on zoonoses trichiniasis in the u.s. population, - . prevalence and epidemiologic factors the symposium at which this paper was presented was sponsored by bayer animal health. the author also wishes to acknowledge and thank dr. cynthia hoobler for assistance with the preparation of this paper. key: cord- -cslrz yp authors: ehnert, karen; galland, g. gale title: border health: who's guarding the gate? date: - - journal: vet clin north am small anim pract doi: . /j.cvsm. . . sha: doc_id: cord_uid: cslrz yp changes in the global trade market have led to a thriving international pet trade in exotic animals, birds, and puppies. the flood of animals crossing the united states' borders satisfies the public demand for these pets but is not without risk. imported pets may be infected with diseases that put animals or the public at risk. numerous agencies work together to reduce the risk of animal disease introduction, but regulations may need to be modified to ensure compliance. with more than , dogs and , wildlife shipments being imported into the united states each year, veterinarians must remain vigilant so they can recognize potential threats quickly. the global trade market, the ease of transporting animals across continents and around the world, lower production costs in foreign countries, and market demand have resulted in a thriving pet trade of exotic animals, birds, and puppies, both purebred and small mixed breeds. the flood of animals crossing the united states' borders satisfies the public demand for these pets but is not without risk. trade barriers have been disappearing, creating a global marketplace. improved transportation networks allow travelers, trade goods, and animals to move across continents or the globe in a single day. improved communication and expanded use of the internet for commerce simplify the connection between consumers and suppliers worldwide. these changes have created an environment in which a new global pet trade thrives. between and , the uruguay round of the general agreement on tariffs and trade was held. the trade negotiations led to the creation of the world trade organization (wto) in and the reduction of tariffs, import limits, and quotas over the next years. agricultural product trade was liberalized, and guidelines on the trade of animals and animal products were created by the office international des epizooties. the wto operates under the principle that imported products be treated as favorably as domestic goods, but countries are permitted to take measures to protect humans and animals. these changes in trade regulations seem to have expanded the global market. the volume of world trade increased threefold from through , and the export value of goods from asia increased fivefold. exotic pet ownership is on the rise in the united states, resulting in an increased trade in live animals. the number of united states households owning reptiles increased from , in to . million in , and from to the numbers of pet birds, rodents, fish, turtles, and lizards have risen. importers, both legal and illegal, have stepped forward to meet this demand. in the early s, united states imports and exports accounted for % of the total world trade of approximately reptile species listed under the convention on international trade in endangered species of wild fauna and flora (cites). in the united states, the annual volume of live animal imports has roughly doubled since . there were , wildlife shipments in , with a declared value of more than $ . billion. from through , annual increases in wildlife trade ranged from % to %. from through , approximately , animals were imported into the united states each day. the number of animals being imported illegally is difficult to estimate. wildlife smuggling is very profitable and is estimated to bring in more than $ billion each year. interpol estimates that wildlife smuggling ranks third on the contraband list of items of value, behind drugs and firearms. customs officers have found animals stuffed in clothing, bags, containers, compartments in cars, and even inside artificial limbs. animal smuggling is likely to continue until the penalties outweigh the profits. starting in , the los angeles county veterinary public health and rabies control program (vph-rcp) noticed a sharp increase in puppies being imported from overseas, with an accompanying increase in public interest regarding how to import puppies for resale. individuals have reported that imported puppies could be sold for much more than their purchase price and shipment costs (vph-rcp, unpublished data). a kennel in los angeles county is selling yorkshire terrier puppies imported from south korea for $ to $ each. puppies smuggled from mexico often are sold for $ to $ cash. small purebred or crossbred puppies are very popular, and there is a lack of local breeders to meet the demand. the public's demand for small, cute puppies continues to stimulate the business and increase profits to puppy importers. requirements for importing animals into the united states can be found in the regulations of several federal agencies and reflect the mission of each agency. in , the lacey act became the first federal law protecting wildlife, by prohibiting the interstate movement and importation of wildlife species. additionally, the lacey act prohibits the importation of wildlife that has been determined to be injurious to people, agriculture, horticulture, forestry, or wildlife in the united states in , the bureau of fisheries and the bureau of biological survey was consolidated to create the united states fish and wildlife service (usfws) in the department of the interior, with the mission of conserving and protecting wildlife and plants. in , the endangered species act was passed to protect endangered or threatened species. the usfws also enforces requirements for cites, an international agreement between governments to ensure that international trade in wild animals and plants does not threaten the existence of those species. lists of endangered or threatened species covered under cites can be found in appendices i, ii, and iii of the agreement. usfws regulations require that all wildlife species imported for commercial, noncommercial, scientific, or personal use be declared at the time of import, be cleared by the usfws, and enter the united states through a designated port. in most cases, the importer must have a usfws permit. , if the species is covered under cites, the shipment also must be accompanied by a current cites certificate. the us department of agriculture (usda) animal and plant health inspection service (aphis) was established in to protect united states agriculture, consolidating the functions of previous animal and plant bureaus within the usda. the basis for aphis came from the usda's first regulatory program, the veterinary division, established in . in , the veterinary division became the bureau of animal industry, which was created by congress to promote research in livestock diseases, enforce animal import regulations, and regulate the interstate movement of animals. in , the usda's agriculture research service replaced the bureau of animal industry. in , the agriculture research service became the animal and plant health service (aphs), and in the meat and poultry inspection divisions of the consumer and marketing service were added, changing aphs to aphis. since , several changes have occurred, including the establishment of the food safety and quality service, known today as usda's food safety and inspection service, the transfer of the animal quarantine inspection activities at ports of entry from the veterinary services division to the plant protection division in , and the movement of the port inspection activities to the department of homeland security in . [ ] [ ] [ ] usda, aphis veterinary services limits the importations of animals, animal products, and plants based on the risk to agriculture. examples of these activities are controlling the importation of hoofed stock from countries in which foot and mouth disease is endemic or birds from countries that are experiencing outbreaks of highly pathogenic avian influenza (h n ) in poultry. importation of livestock or other hoofed stock, birds, dogs, or other animals may require a permit and possibly quarantine in a usda facility before the shipment is allowed to enter the united states. the animal welfare act was passed in to require minimum standards of animal care for animals that are used in research, bred for sale or exhibition, or transported commercially. aphis' animal care program enforces the provisions of the animal welfare act and the horse protection act, which was passed by congress in . the animal care program ensures that all animals are transported at the proper ages, in proper crates, and in appropriate conditions in accordance with the animal welfare act. the animal care program does not have regulations specific to importation of animals. the centers for disease control and prevention (cdc) of the department of health and human services has regulations prohibiting or controlling the importation of a variety of species of animals and animal products based on a specific threat to human health. for example, dogs entering the united states from countries reporting cases of rabies need proof of a current rabies vaccination, or the importer must sign an agreement to confine the animal until appropriate vaccinations can be obtained and then for an additional days after vaccination. the importation of nonhuman primates has been regulated since , limiting their importation specifically to purposes of science, education, or exhibition and requiring that importers be registered by the cdc. in , importation of civets was banned because these animals were considered to be an amplifying host or vector for severe acute respiratory syndrome (sars). in , the importation of african rodents was banned in response to an outbreak of monkeypox in the united states associated with imported gambian pouched rats. customs and border protection (cbp), located in the department of homeland security, is the first line of defense at the border to ensure that animals and animal products are being imported in accordance with all federal agency regulations. additionally, cbp has the authority to levy a fee on imported animals or products for commercial use, in accordance with the tariff codes. animal importation regulations change often, reflecting any new disease threats that arise, and imported animals may require permits or approvals from a variety of agencies. individuals planning to import animals should check with the usda, cdc, usfws, and cpb to make certain that all required documents are obtained before an animal is brought to the united states. in california, the number of legally documented dog imports began increasing in ( fig. ) . in , most imported dogs were single imports. some were personal pets; others were purebred dogs that had been purchased from an overseas breeder. few dogs were imported for resale. in , the number of imports of multiple puppies per shipment began to increase. the number of puppies imported into california through airports has increased from multidog imports documented in to in and in . each shipment contained as many as puppies. such large numbers of puppies are being imported for resale and not as personal pets. a similar increase was seen nationally. an estimated , dogs were imported into the united states in , with , lacking proof of valid rabies vaccinations, mostly because they were too young to be vaccinated. in california, most of the imported puppies were destined for los angeles county (fig. ) , and the most common countries of origin were mexico and canada (fig. ) . many dogs also were imported from asia, europe, south america, and russia. in los angeles county, many puppies were imported from south korea by pet stores or kennels. the most common breed imported was yorkshire terrier, followed by maltese, bulldogs, and poodles (fig. ) . as the number of shipments containing more than one dog increased, tracking puppies became increasingly more difficult in los angeles county. initially, several shipments went to local pet stores, but as los angeles county vph-rcp staff began enforcing postimportation quarantines until days after the puppies received their rabies immunization, shipments became harder to locate. puppies were sold before individual dog import multiple dog import vph-rcp visits, incorrect addresses were indicated on the cdc confinement agreement form, and individuals refused entrance onto their properties. in addition, some importers provided falsified rabies certificates, and puppies were not available for inspection. this problem was not limited to los angeles county. new york city sent out a veterinary alert in to notify veterinarians that puppies were being imported from rabies-endemic countries and that some were being sold without completing the mandated confinement. the cdc noted more than confinement agreement violations among imported dogs in . during the past few years, illegal shipments of puppies also have become a problem. the los angeles county vph-rcp and animal law enforcement agencies throughout california began receiving reports in that individuals were purchasing puppies in mexico and selling them in california. these puppies were advertised in free classified ads and were delivered to the purchaser at a public location, or they were sold directly from vehicles in shopping center parking lots. generally, the purchaser was required to pay cash and had no way of contacting the seller after purchase. many of the puppies were ill and died a short time after being sold to unsuspecting buyers (personal communication, captain aaron reyes, southeast area animal control authority, december , ) . in early , animal law enforcement agencies and three health agencies, including the los angeles county vph-rcp, formed the border puppy task force (bptf) to assess this growing and disturbing trend. in december , animal law enforcement officers worked alongside cbp agents for a -week period, examining and documenting animals entering from mexico through two california border crossings. more than puppies were examined during this operation; many were found huddled together in cardboard boxes in car trunks or wrapped in towels and stuffed under seats (fig. ) . only a few puppies were confiscated because of illness. most were allowed to enter california after a cdc confinement order was issued. these numbers indicate that , or more puppies may be imported each year through the two california-mexico border crossings investigated, and few are confined as required by federal law to protect against introduction of rabies. following the joint investigation, the bptf held a news conference and conducted media interviews to educate the public about the risks associated with illegally imported puppies. buyers were encouraged not to purchase puppies if the seller required cash and required that the puppy be delivered to its new owner in a public place, such as a restaurant or shopping center parking lot. individuals whose puppy became ill or died shortly after purchase were encouraged to report the matter to the bptf for follow-up investigation of illegal importers. in and , the bptf identified continued transport of puppies across the same border crossings. the cdc has responded to complaints about large-volume shipments of puppies intended for immediate resale and the need for additional regulations to prevent the introduction of zoonotic diseases into the united states by publishing an advance notice of proposed rulemaking on july , . public comments were solicited until december and are being evaluated. stakeholders were asked questions such as should the cdc establish a minimum age for importation of dogs, cats and ferrets? should imported animals have a unique identifier (microchip, tattoo)? should a valid international health certificate be required? should the importation of dogs, cats and ferrets be restricted to ports staffed by cdc quarantine personnel? these changes could have a major impact on the legal and illegal international puppy trade. until the regulations are revised, however, the flow of puppies into the united states is likely to continue. the worldwide movement of animals increases the potential for the spread of diseases that pose a risk to human and animal health. , animals are imported into the united states for use as pets, food and other animal products, scientific research, and exhibition in zoos. dogs and cats are allowed to enter the country without health certificates and, if the owners sign a confinement agreement as described previously, without proof of rabies immunization. even if a pet is ill on arrival, it may be allowed in, with a recommendation that the owner take the pet to a veterinarian for examination. many of the exotic animals are wild caught, and often there is no requirement that they be screened for zoonotic disease before or after arrival in the united states. global trade of animals creates circumstances in which diseases that generally are not found in the united states may be introduced. on the first world rabies day, september , , the cdc reported that the canine strain of rabies had been eliminated from the united states the importation of dogs from rabies-enzootic countries represents a risk for reintroducing canine rabies. , imported dogs have been found to be infected with rabies , , , on several occasions. in , a -month-old puppy imported from mexico into new hampshire became ill weeks after its arrival. the dog began whimpering and had tremors in one leg for days. it then developed urinary and fecal incontinence and finally excessive salivation. the owners took the puppy to a veterinarian, who suspected rabies based on the puppy's history and clinical signs. the puppy was euthanized, tested, and found to be rabid. seventeen people had been exposed, including the owner's classmates, partygoers, and a babysitter. in , a -month-old ill puppy was imported from thailand through the los angeles international airport and was allowed to enter the country. it had been evaluated by several veterinarians in thailand for a respiratory illness and had begun vomiting while in flight. the owner took the puppy to three veterinary clinics as she traveled to her home in northern california. the puppy was aggressive and seemed to have pain along its back. obvious neurologic signs did not develop until it was seen at the third veterinary clinic. at that point, the puppy was euthanized and tested positive for rabies (thai canine variant). numerous people had been exposed, and individuals required postexposure prophylaxis. more recently, in , a puppy imported from india by a washington state veterinarian developed rabies after being adopted by another veterinarian and taken to alaska. the puppy became ill days after arrival from india, with at least one episode of regurgitation. it then bit one of the veterinarians and another dog. clinic staff noticed it gnawing on its kennel, resulting in bleeding gums. even so, another veterinarian completed a health certificate for the puppy, and a third veterinarian transported it to alaska. the day after arriving in alaska, the puppy developed neurologic signs and died. the puppy was tested and found to be rabid (indian canine rabies variant); eight individuals received rabies postexposure prophylaxis. previous documented vaccination does not always negate the risk of imported rabies. in , a dog developed rabies months after being imported from cameroon. the dog had been vaccinated against rabies twice in west africa and once after arriving in the united states the owners took the dog to an animal hospital after it developed paralysis of the lower jaw. the dog was docile and ambulatory. it was discharged with a diagnosis of ''viral infection,'' and the owner was directed to force feed it. the dog was seen at two different clinics over days and finally was euthanized and tested for rabies. it was found to have a west african dog strain of rabies. thirty-seven individuals received postexposure prophylaxis after potential exposures to the dog during its illness and the weeks before the onset of clinical signs. in , an ill cat from mexico also was allowed to enter the country through los angeles international airport. the cat was seen by three veterinarians before being euthanized and testing positive for rabies. other countries have reported imported rabies cases. france has identified several cases of rabies in dogs imported illegally from morocco through portugal or spain by car. [ ] [ ] [ ] in and again in , three cases of canine rabies were reported in imported dogs. in , belgium and germany also reported rabies in dogs imported illegally from morocco. , imported dogs may carry other diseases, such as screwworm, , that pose risk to both animals and humans. screwworm infestation begins when a female fly lays eggs on a superficial wound. unlike typical maggots that feed on dead tissue, the screwworm feeds on living tissue. one female fly may lay up to eggs at a time and as many as eggs during a -day lifespan. the eggs hatch into larvae that burrow into the wound and begin feeding on living flesh. after feeding for to days, the larvae drop off and burrow into the soil, where they pupate. the adult screwworm fly emerges and then mates after to days. in the first day or two of screwworm infection, the clinical signs include a slight motion inside the wound and possibly a serosanguineous discharge and a distinctive odor. by the third day, the larvae may be seen easily. in dogs, the larvae often tunnel under the skin, and there may be a large pocket of larvae with only a small opening in the skin. the deep burrowing is distinctive of screwworms, because other types of maggots are surface feeders and feed on dead tissue. if screwworms are left untreated, animals may die of secondary infection or toxicity within to days of infection. daily wound treatment and larvicidal insecticides are necessary to control the screwworm larvae. in , astute veterinarians in mississippi and massachusetts identified screwworm larvae in imported dogs. , both new world (cochliomyia hominovorax) and old world (chyrsoma bezziana) screwworm myiasis are considered foreign animal diseases in the united states and are reportable within hours of diagnosis. new world screwworms were eradicated from the united states in . the old world screwworm had never been seen in this country until it was found in a -year-old dog imported from singapore to massachusetts in october . in september , a -year-old dog was imported from trinidad and entered the country through the miami airport. it was seen by a mississippi veterinarian days after arrival for ocular damage caused by larval infection. in both cases, the practitioners recognized that the larvae seemed unusual and submitted specimens for identification. their quick action prevented these insects from becoming established, which could have resulted in the united states livestock industry suffering $ million in production losses. imported dogs may introduce other non-native pathogens to the united states. in , a dog imported from england to canada was found to be infected with angiostrongylus vasorum, a nematode parasite of the pulmonary arteries and right heart of dogs and wild carnivores. this parasite is enzootic among dogs in areas of europe and uganda but is not considered established in north america. in , an investigation in french guiana, south america, determined that a dog imported from france in had leishmania infantum and subsequently spread the infection to a second dog. imported wild or exotic animals also pose a risk to human and animal health. bats have been associated with rabies virus and related lyssaviruses, nipah and hendra viruses, and a sars-like virus of bats. a highly pathogenic strain of the influenza virus, h n (hpai), first appeared in asia in and subsequently spread to russia, europe, and parts of africa. live bird markets, trade, wild birds, and illegal bird importation probably all contributed to the spread of the disease. , in , two crested hawk-eagles that had been smuggled into europe from thailand were seized at the brussels international airport. although neither appeared ill, they were euthanized and were found to be infected with hpai. bird smuggling continues to be a problem in the united states. from through , federal authorities intercepted individuals attempting to smuggle commercial quantities of live birds into the united states from mexico. before being arrested, one individual had illegally transported between and , exotic birds, valued at more than $ . million, across the border. smuggled birds are not quarantined, screened, or treated as required by federal law. in addition to avian influenza, smuggled birds may carry exotic newcastle disease, a foreign animal disease that is lethal to poultry, , or avian chlamydiosis, a zoonosis that people can contract through contact with pet birds. rodents, rabbits, and pocket pets also may pose a risk to human and animal health. in may and june , the first cluster of human monkeypox cases in the united states was reported. many of the patients developed a febrile vesicular rash after having contact with prairie dogs that had acquired the infection through contact with a shipment of african rodents at a wholesale pet store. the prairie dogs exhibited anorexia, wasting, sneezing, coughing, swollen eyelids, and ocular discharge. ultimately, there were confirmed and probable human cases of monkeypox during this outbreak. the traceback investigation showed that rodents imported from africa were held in the same area as prairie dogs before being shipped to other distributors and, ultimately, to many pet stores. the frequent mixing of species in the wildlife trade arena creates an opportunity for cross-species transmission and the introduction of new diseases to domesticated animals, wildlife, and humans. in addition to zoonotic threats, imported animals may pose a risk to agriculture. rabbit hemorrhagic disease (rhd) first was identified in china in . rhd is a highly contagious calicivirus that kills up to % of infected animals. infected rabbits often develop a blood-tinged foamy nasal discharge, severe respiratory distress, and/or convulsions before death. in % to % of the rabbits, clinical signs do not progress as rapidly but may include jaundice, malaise, weight loss, and eventually death in to weeks. this disease has spread to europe, asia, australia, new zealand, and cuba but still is considered a foreign animal disease in the united states. outbreaks of rhd occurred in the united states in , , and . the outbreak of rhd occurred at a rabbitry in indiana after the owner purchased rabbits from a flea market in kentucky. following the introduction of the new rabbits, nearly half of his herd died, and the remaining animals were euthanized to contain the outbreak. the source of the infection never was determined. imported exotic pets also may carry parasites that could pose a public health or agricultural health threat. in , florida animal health officials detected exotic ticks on a leopard tortoise that contained cowdria ruminantium, the cause of heartwater disease in ruminants. summary imported dogs bring the risk of the reintroduction of canine rabies, screwworm, and other diseases. exotic birds pose a risk for avian influenza, exotic newcastle disease, and psittacosis. rodents have been a source of imported monkeypox, and turtles can carry ticks that spread heartwater disease. regulations are in place to reduce the risk of diseases that pose a threat to public health and agriculture from imported animals. changes to the regulations are being proposed to define better the united states entry and follow-up requirements. veterinarians play an essential role in preventing the transmission of zoonotic disease between animals and the public and are on the front line dealing with imported animals. they should be aware of and compliant with state and local regulations and play an active role in educating and advising clients regarding the risk of importing an animal. veterinarians should be vigilant when examining new puppies. many imported dogs never are confined properly or inspected for infectious diseases, and many diseases may not be detected readily in imported dogs. with the current rabies vaccination requirements in the united states, most veterinarians have never seen a pet with rabies and do not consider rabies in the differential diagnosis. additionally, early signs of rabies may be very subtle and may not be recognized readily. it is important to keep rabies on the differential list, especially if the pet is known to have been or is suspected of having been imported. additional training in recognizing emerging infectious diseases may be helpful. veterinarians should contact their local health department immediately about any potential rabies cases or suspicious illness, especially in imported animals. a veterinarian could be the one who prevents the next outbreak. broken screens: the regulation of live animal importation in the united states the economic implications of greater global trade in livestock and livestock products the impact of diseases on the importation of animals and animal products global change and human vulnerability to vector-borne diseases results of the avma survey of companion animal ownership in us pet-owning households the reptile and amphibian communities in the united states all creatures great and small: wildlife smuggling ''not worth the risk''. us customs today akc dog registration statistics office of law enforcement about the u.s. fish and wildlife service convention on international trade in endangered species. wild fauna and flora convention on international trade in endangered species. wild fauna and flora. the cites appendices office of law enforcement. importing and exporting your commercial wildlife shipment animal and plant health inspection service. about usda, history of aphis animal and plant health inspection service. import export, animal and animal product import information, live animals animal and plant health inspection service. animal welfare centers for disease control and preventiondivision of global migration and quarantine. importation of pets, other animals, and animal products into the united states importing into the united states, a guide for commercial importers importation of dogs into the united states: risks from rabies and other zoonotic diseases. zoonoses public health veterinary alert # south east area animal control authority (seaaca) foreign quarantine regulations, proposed revision of hhs/cdc animal-import regulations rabies in a puppy imported from india to the usa impact of globalization and animal trade on infectious disease ecology notice to readers: world rabies day epidemiologic notes and reports imported dogs and cat rabies rabies in a puppy imported to california from thailand an imported case of rabies in an immunized dog canine rabies in france promed mail. rabies, canine-france ( ): investigation. promed mail an imported case of canine rabies in aquitaine: investigation and management of the contacts at risk rabies news archives germany, hamburg ex morocco is it just another worble? california veterinarian dangerous screwworm species found in u.s., poses little threat. dvm newsmagazine the center for food security & public health. screwworm myiasis screwworm found in mississippi dog angiostrongylosis with disseminated larval infection associated with signs of ocular and nervous disease in an imported dog first report of leishmania infantum in french guiana: canine visceral leishmaniasis imported from the old world bats: important reservoir hosts of emerging viruses summary of avian influenza activity in europe disease intelligence for highly pathogenic avian influenza highly pathogenic h n influenza virus in smuggled thai eagles exotic parrots confiscated from smugglers returned to mexico. press release investigation of an outbreak of velogenic viscerotropic newcastle disease in pet birds in michigan, indiana, illinois and texas phylogenetic relationships among highly virulent newcastle disease virus isolates obtained from exotic birds and poultry from to detection of chlamydiosis in a shipment of pet birds, leading to recognition of an outbreak of clinically mild psittacosis in humans monkeypox transmission and pathogenesis in prairie dogs the detection of monkeypox in humans in the western hemisphere spectrum of infection and risk factors for human monkeypox a pandemic strain of calicivirus threatens rabbit industries in the americas rabbit hemorrhagic disease evidence of cowdria ruminantium infection (heartwater) in amblyomma sparsum ticks found on tortoises imported into florida the authors thank dr. ben sun and sharon ernst who provided data on cdc confinement agreements completed for dogs imported into california. key: cord- -onghrm y authors: nevarez, javier title: chapter crocodilians date: - - journal: manual of exotic pet practice doi: . /b - - . - sha: doc_id: cord_uid: onghrm y publisher summary this chapter presents a general overview of the anatomy, physiology, and treatment methodology for crocodilians. most crocodilians grow to be larger than other reptile species and, therefore, have significant space requirements. like most animals requiring an aquatic environment, crocodilians need water that is clean and free of disease. crocodilians have a true hard palate in the roof of the mouth that ends caudally in a soft palate. this soft palate has a ventral flap which is referred to as the velum palati. the respiratory system of crocodilians consists of well-developed lungs benefiting from a very effective inspiration aided by the intercostal muscles and the septum post hepaticum. crocodilians have a four-chambered heart as opposed to the three-chambered heart found in other reptiles and amphibians. the temperature and humidity requirements for crocodilians in captivity vary with the species. an understanding of crocodilian biology and natural history is needed to try and duplicate their natural environment. an important consideration is the allowance of circadian variations in light cycle and temperatures to mimic their natural environment. this is not the case in many commercial operations, where they are maintained at a fairly constant temperature and humidity to achieve faster growth. c h a p t e r crocodilians javier nevarez modern-day crocodilians date back to the mesozoic era over million years ago, surviving to the present with relatively few evolutionary changes. their biology, physiology, and anatomy are unlike those of any other reptiles. however, some species have suffered the effects of a changing environment and human encroachment; this situation has led to the formation of international programs dedicated to the preservation of threatened or near-extinct species. of the species of crocodilians, can be found on the red list of endangered species. taxonomic identifi cation varies among all species, and there is debate over the proper classifi cation of species and subspecies (tables - and - ; box - ). in addition to having wild crocodilian populations, countries such as australia, india, mexico, papua new guinea, south africa, and the united states maintain intensive production operations for various species. in north america, the american alligator (alligator mississippiensis) is the best-known crocodilian. second to it is the american crocodile (crocodylus acutus), a vulnerable species found in small numbers in florida. the american alligator was considered a threatened species during the s, but a captive rearing program in louisiana has been successful at maintaining the estimated population at over million animals. the fi rst type of captive rearing operations consisted of alligator farming. in farming operations, breeding pairs of alligators were kept in enclosed areas where they could mate and nest (figure - ). the eggs were collected from the nests and incubated. the farming operations led to ranching operations in which eggs are harvested from the wild and incubated in private facilities ( figure - ). the alligators hatched on an "alligator ranch" are then raised for their hide and meat. to help maintain the wild populations, % of the hatched alligators are eventually returned to the wild. louisiana is the primary producer of american alligators in the world. captive rearing operations can also be found in florida, texas, georgia, and other southern states within the united states where alligators naturally inhabit. in louisiana the majority of the operations are ranches, whereas many farms still exist in florida. other crocodilian species, such as the nile crocodile (crocodylus niloticus), are primarily raised under farming operations in other countries. in those states where alligator productions are present, there are opportunities for veterinarians to get involved with the industry. some veterinarians, regardless of practice concentration, have alligator farmers or ranchers as part of their clientele and meet new challenges in their daily practice. a population management approach is necessary when working with these alligator production facilities. it is also advisable for veterinarians and farmers alike to be aware of the rules and regulations imposed by the local department of wildlife and fisheries. in addition to being found in their natural environment, crocodilians are also found in zoos. zoologic parks house different species of crocodilians for educational purposes, reproductive efforts, or both. as is the case with many other threatened species, zoologic institutions play a critical role in the captive reproduction of threatened species of crocodilians. here the animals can be observed by specialists in a more controlled environment that mimics their natural habitat, although design and maintenance of the enclosures can be diffi cult. disease prevention and control are other important considerations when it comes to oversight of threatened species. veterinarians may also encounter crocodilians as privately kept "pets." in the past, the spectacled caiman (caiman crocodilus) and even the american alligator (a. mississippiensis) have been sold to the general public. there are many reasons why crocodilian species should not be maintained as pets. in addition to the physical dangers and risks associated with keeping crocodilians, there is the misfortune of inadequate husbandry, adversely affecting the reptile's health. most crocodilians grow to be larger than other reptile species and therefore have signifi cant space requirements. like most animals requiring an aquatic environment, crocodilians need water that is clean and free of disease. another husbandry issue is an incorrect diet, which can lead to metabolic abnormalities and overall poor health. many private owners are unable or unwilling to provide these conditions for their crocodilians. in addition to these issues, most states will require special permits to own these animals. most people lack the proper ownership permits, and veterinarians could be liable if they treat these illegal patients. the biggest challenge that veterinarians face when treating illegally owned animals is that they are often the last chance for humane treatment for these animals. it is the duty of most of the material presented in this chapter specifi cally relates to the american alligator (a. mississippiensis). although most of the alligator information will apply to other crocodilian species, there are variations among groups that will be mentioned. the natural environment and geographic distribution of a species will often determine disease exposure. once in captivity, all species are susceptible to the same diseases within that environment. in addition, there are some inherent differences among the three families of crocodilians-alligatoridae, crocodylidae, and gavialidae-that have emerged from studying them in captivity. one of the most common questions a veterinarian may encounter regarding reptiles is "what is the difference between alligators and crocodiles?" the fi rst difference is that they belong to two different families: the family alligatoridae (see table - ) includes the alligators and caimans, and the family crocodylidae (see table - ) includes all crocodiles. there is a third family, the gavialidae (see box - ), which contains the gharial, or gavial. geographic location may help in the identifi cation of some species. alligators are thought to tolerate colder temperatures and live at higher latitudes, whereas crocodiles and caimans are less cold resistant and live in warmer areas. however, there are some anatomic features that will be most useful in differentiating alligators from crocodiles. the alligators and caimans have a broad, u-shaped snout, whereas crocodiles have a more narrow, v-shaped snout. this difference can be observed by looking at the dorsal aspect of the head (figure - ) . a more obvious distinction can be made when looking at their mouth from the side (figure - ) . alligators and caimans have notches in the maxilla that fi t the mandibular teeth. therefore, they have no mandibular teeth visible if observed from the side with their mouth closed. on the other hand, crocodiles have the fourth mandibular tooth exposed when looking at them from the side with their mouth closed. integumentary sensing organs, also known as dome pressure receptors (dprs), are clear to gray pits present on the skin of crocodilians. their function is not completely understood, but they may play a role as mechanoreceptors in prey detection or even as chemoreceptors aiding in detection of salinity levels. , alligators and caimans have dprs only on the lateral aspect of the mandible ( figure - ), whereas crocodiles and gharials have dprs all over the body, most noticeably on the ventral scales ( figure - ) . the presence of dprs can be used to differentiate the two main groups of crocodilian skins in the leather market. an additional feature that could be used for differentiation of alligators and crocodiles is the salt glands, which are absent from the tongue of alligators and caimans but well developed in crocodiles and gharials. an interesting anatomic feature of crocodilians is the palatal valve, also known as the gular valve. there is some discrepancy as to the name of this structure and its two components, but i will attempt to describe them based on the anatomic location. crocodilians have a true hard palate in the roof of the mouth that ends caudally in a soft palate. this soft palate has a ventral fl ap that is referred to as the velum palati. the velum palati is the dorsal component of the palatal valve, with its second and ventral component being the gular fold. this structure projects in a craniodorsal direction from the base of the tongue and has a cartilaginous base to it that is part of the larynx. together, the velum palati and the gular fold form what is known as the palatal, or gular, valve ( figure - ) . the function of this valve is to seal the pharyngeal cavity while under water to prevent aspiration. crocodilians also have control of the nares and are able to open and close them as needed to prevent aspiration of water. the respiratory system of crocodilians consists of welldeveloped lungs benefi ting from a very effective inspiration aided by the intercostal muscles and the septum post hepaticum. the septum post hepaticum is a diaphragm-like muscle that creates a partial separation of the thoracic and abdominal viscera. a number of membranous connections separate the lungs and the liver, and an intricate mesentery system encompasses the gastrointestinal tract and viscera. all of these tissue structures may be necessary for allowing the changes in pressures that occur during diving. the cardiovascular system of crocodilians also has special characteristics. crocodilians have a four-chambered heart as opposed to the three-chambered heart found in other reptiles and amphibians. the circulation of blood through the crocodilian heart is like that in the heart of mammals, but the crocodilians possess a viable foramen of panizza. this opening is located at the base of the heart between the left and right aortic arches and allows for venous admixture, which is essential during periods of diving to conserve oxygen. during diving, there is pulmonary hypertension. this in turn creates increased pressure in the pulmonary artery and the right ventricle, which forces deoxygenated blood through the foramen of panizza into the left side of the heart and the aorta to be distributed through the body. this mechanism allows for conservation of oxygen and supplies oxygenated blood to those organs that require it the most, allowing some crocodilian species to stay submerged for up to hours. a second anastomosis may be present in other crocodilian species as a vessel connecting the two aortic arches. submandibular (figure - ) and paracloacal glands and a gall bladder are present in crocodilians. the hard dorsal scales are known as osteoderms, bony plates lined by skin. crocodilians have a smaller gastric compartment distal to the stomach that is a gizzard-like structure in which rocks and other materials may be found. it does not appear as evolved as the ventriculus in birds, however. crocodilian intestines have a thick wall and can have well-developed diffuse aggregates of lymphoid tissues like peyer's patches. there is no urinary bladder, but the colon can hold large amounts of urine and water. sexing can be performed by palpation of the cloaca; males have a phallus that can be palpated and extracted from the cloaca. females have a well-developed clitoris that, depending on size, can be confused with a phallus. internally paired gonads are found near the ventral surface of the kidneys. physiologic data, including body temperature, heart rate, and respiration, vary with species, age, and environmental factors (e.g., environmental temperature, season of the year). environmental considerations are variable depending on where a crocodilian species lives or is farmed. the aim of captive rearing operations is to produce a large number of animals in the most effi cient way possible. in a zoologic or other educational institution, the goal is to exhibit the animals in an environmentally accurate artifi cial environment. the underlying policy of any aquatic enclosure should be clean water, appropriate diet, and enough space to accommodate the growth of the animals. as with most exotic animals, the challenge is to mimic a captive animal's natural environment. there are no specifi c references for the enclosure size of crocodilian species in captivity. the size of the enclosure will largely depend on the species of reptile and the purpose of their captivity. a general understanding of biology and natural behavior of the captive species is essential to designing appropriate enclosures. although a zoologic institution housing the species might provide more specifi c advice, there are some general enclosure guidelines for the commercial production of american alligators: square foot per alligator up to inches in length (snout to tip of tail), square feet per alligator for those between and inches in length, and an additional square foot of space for every inches in body length beyond inches. these are the recommendations for the maximum stocking rate for alligators in commercial operations. the recommendations for a zoo or educational facility are to make the exhibit as large as possible, taking into consideration the species being housed. a consideration for larger species is territoriality requiring an expanded enclosure. male crocodilians may become more aggressive during the reproductive season, and keeping them separated should be a consideration if space is a concern. exhibits can be outdoor, indoor, or a combination of both. outdoor exhibits can closely mimic the natural environment but also present more challenges for maintaining water and environmental quality as well as for controlling diseases. geographic location will also play a role in the creation of outdoor exhibits, as not all species of crocodilians can tolerate cold weather. finally, some species can dig considerably, and measures must be taken to prevent an escape. the temperature and humidity requirements for crocodilians in captivity vary with the species. once again, an understanding of crocodilian biology and natural history is needed to try and duplicate their natural environment. an important consideration is the allowance of circadian variations in light cycle and temperatures to mimic their natural environment. this is not the case in many commercial operations, where they are maintained at a fairly constant temperature and humidity to achieve faster growth. from a health standpoint, this may allow for cross exposure of reptiles in commercial operations to infectious organisms that typically affect mammals. as the commercial reptiles are maintained at higher temperatures, new diseases commonly associated with mammals may adapt to living inside a reptile host and lead to clinical disease. in an enclosure, the temperature can be maintained via heating elements contained within the concrete slab, in line water heaters, or both. the water temperature must also be maintained during the refi lling of the pen or enclosure to avoid signifi cant temperature variations. light requirements for reptiles are still a controversial subject. in general, a source of ultraviolet b (uvb) light for herbivorous and omnivorous reptiles is recommended. ultraviolet light is essential for the synthesis of vitamin d , specifi cally its active form , dihydroxyvitamin d, which is essential for the metabolism of calcium and phosphorus. a lack of vitamin d can lead to inappropriate calcium absorption, which in turn creates a metabolic imbalance resulting in metabolic bone disease. metabolic bone disease, specifi cally secondary nutritional hyperparathyroidism, is recognized in many reptile species housed with an inappropriate source of uvb light, fed a diet defi cient in calcium, or both. carnivorous reptiles may also benefi t from uvb light but are thought to obtain enough vitamin d and calcium from their prey. the uvb light requirement of crocodilians is unknown, but as true carnivores they may thrive with minimal exposure to uvb light. it is a common practice on alligator ranches to raise animals in darkness with no source of uvb light or a normal light cycle. most animals will grow well under these conditions, and some have reached adulthood without signs of metabolic diseases. however, i have also observed evidence of metabolic bone disease in a subset of captive american alligators being fed a commercial diet with no exposure to uvb light. in these cases veterinarians must also consider the possibility that the commercial diet may be defi cient in calcium. anecdotal stories from alligator ranches claim that weak, anorectic animals appear to improve after being exposed to sunlight over a period of time. further research is needed to determine the uvb light requirements of crocodilians and the potential benefi ts of exposure to uvb light. natural unfi ltered sunlight is the best source of uvb light, but various artifi cial sources are available (e.g., fl uorescent uvb light bulbs, mercury vapor light bulbs). the two main substrates in crocodilian exhibits are water and soil/sand. the species, age, and feeding habits must be taken into account; avoid substrates that may be ingested by accident and may lead to impactions. it is also important to prevent the public from throwing coins and trash into exhibits, as this may represent a source of toxicity and a cause of impactions. in commercial operations, a smooth covering is applied to the concrete to preserve the quality of the hide. either an epoxy coating or plastic liners are routinely used as substrate. crocodilians are true carnivores, as evidenced by their short gut and oral cavity. as such, they require a high protein diet, low in fi ber. their feeding habits in the wild will vary with age and food availability. early on, their diet will consist of small invertebrates, amphibians, and reptiles. as they grow, they will eat larger prey of the type described earlier and will incorporate fi sh and birds into their dietary regime. with time, and depending on the species, size, and food availability, crocodilians will start eating mammals. there have been few studies investigating the nutritional requirements and feeding protocols of alligators. [ ] [ ] [ ] various commercial feeds are available for alligators maintained in captivity. the commercial rations consist of dry pelleted diets that try to provide full nutritional requirements. these diets can be found with a %, %, or % protein content, less than % fat, and approximately % fi ber content. refi ned commercial alligator diets are widely used in production operations but may prove too expensive and/or inappropriate for the long term. a variety of whole prey feeds, such as chicken, nutria, and fi sh, are also recommended. if using nutria, be sure that it has not been killed using lead shot; if the lead is ingested, toxicosis can occur. when feeding frozen fi sh to crocodilians, provide a vitamin b supplement or another meat source to prevent thiamine defi ciency. to prevent dietary associated problems, purchase meat from a reputable source. all animals should be quarantined before their introduction to a production or zoologic facility. a detailed history should be obtained from the source facility, including information regarding diseases to which the animals in question may have been exposed. one should always purchase animals from a reputable individual or institution. little information will be known about wild-caught animals. a minimum quarantine period of to days is recommended in a building that is separate from the main facility. during the quarantine period, the animals can be examined for any sign of illness, and diagnostic tests (complete blood count [cbc], plasma or serum chemistry, west nile virus antibodies, etc.) can be performed to assess their overall health status. if the alligators originated from an area where wnv is endemic, it is advisable to test for previous exposure to this virus. unfortunately, a true quarantine process does not often occur. quarantine is limited by the availability of appropriate facilities, as well as by the time and production budget. quarantine is also a challenge in commercial operations where there may be a large population of animals. nonetheless, zoologic institutions and production facilities should try to establish an adequate quarantine program for their crocodilians. routine physical exams of crocodilians may be incorporated into a zoologic institution's preventive health program. as animals get larger in size, performing physical exams becomes more hazardous. chemical immobilization can be used, if needed, to perform necessary examinations, particularly on large crocodilian species. in commercial operations, the skins of a subset of the production animals will be examined at intervals before the anticipated time of slaughter. the time of hide examination presents an opportunity for veterinarians to examine the animals in more detail. other than hide examinations, routine physical exams are not commonly performed in crocodilians from commercial operations. biosecurity is an essential part of disease prevention in any animal facility. the creation of a sanitation station at the entrance of each building is recommended to prevent introduction of disease organisms. these stations should contain a foot bath and a hand-washing station to decrease the opportunity of disease transfer between exhibits or buildings. a brush should be provided at each foot bath to thoroughly clean boots and shoes. the solution for the bath can be made of bleach or other commercial disinfectants, preferably with virucidal activity, and should be changed daily. organic material will contaminate a foot bath, rendering it ineffective. the foot bath should be used before and after entering the building. a hand-washing station should consist of a water source, a sink, hand soap, and disposable hand towels. a waterless hand sanitizer product or exam gloves will suffi ce in lieu of a handwashing station. in addition to the sanitation station, separate tools for working in each building are required. separate working tools also prevent the transfer of diseases via nets, brooms, rakes, and so on. the buildings themselves must also be maintained free of pests and thoroughly cleaned whenever possible. in commercial operations, it is common practice to empty and disinfect the buildings after the slaughter period, before the introduction of new animals. water quality is one of the main issues of concern when keeping crocodilians in captivity, and many health problems can be prevented if attention is paid to acceptable water quality. manual restraint of crocodilians is essential for physical examination, administration of medications, administration of anesthetics, and relocations. the size and species of crocodilian will determine the best and safest restraint methods to be used. an experienced crocodilian handler must be available to help restrain the animal. although alligators and caimans are usually thought of as being less aggressive than crocodiles, this may not always be true. all sizes and species should be handled with the safety of the people as well as the animal in mind. crocodilians less than m in length (snout to tip of tail) may be handled by one or two individuals. those between and m in length should be handled by at least two or three indi-viduals. those longer than m in length will require at least four to fi ve individuals. various tools (e.g., pole snares, nets, squeeze cages, traps) also can be used to restrain crocodilians. the head, tail, and limbs must be immobilized and controlled. once the animal is under control, the mouth is secured with strong tape or a rope. albino and leucistic animals can have increased skin sensitivity compared to the normal pigmented individual; therefore, additional care should be employed to avoid irritation of the skin. restraint is stressful for the animal, and contact must be limited to the time it takes for the procedures being performed. (see anesthesia for more discussion on chemical restraint.) signs of illness in captive crocodilians are usually nonspecifi c. anorexia, lethargy, a change in behavior, or death may be the fi rst indication that something is wrong in a collection of animals or commercial operation. adequate observations of the animals made by the personnel in the facility should be taken seriously. a visit to the facility is best undertaken during feeding time to avoid additional stress to the animals. at this time, the feeding and water quality policies can be evaluated. a thorough history should include information about the number of animals, source, age, most recent introduction, quarantine practices, feed, frequency of feeding, water quality parameters, clinical signs, time since fi rst signs were observed, recent changes in management techniques, and any treatments such as salt, bleach, or antibiotics. within a commercial operation, a subset of animals should be collected for disease diagnostics and necropsy. in addition to the obtaining of routine samples, tissues should be frozen for possible bacterial, fungal, or viral cultures. within a zoologic institution, sacrifi cing live animals may not be possible, but veterinarians should obtain diagnostic samples from those with and without clinical signs. necropsies should be performed in all dead animals. live animals should undergo physical examination. once the animal is properly restrained, a physical examination can be performed. for safety purposes, veterinarians must be aware of the location of the head and tail at all times when performing the examination. a protocol should be followed for the examination process in crocodilians as in any other species. the oral examination can be performed if a speculum (e.g., pvc pipe, piece of wood) is inserted in the mouth before securing it with tape or a rope (figures - and - ). examine the eyes for evidence of discharge and assess their function ( figure - ). examine the skin for any evidence of trauma or dermatitis, and then palpate the extremities, joints, musculature of neck, pelvic region, and tail. joint swelling is often noted with infectious disease such as mycoplasmosis or trauma. poor body condition may be refl ected in atrophy of the muscles. in commercial operations, it is important to examine the skin on the animal's ventral aspect because this is the area where many disease manifestations will be noted. it is also common to fi nd tooth marks, scratches, and lacerations on the ventral epithelial surface. finally, examine the vent and cloaca for abnormalities. if working in a commercial operation, a veterinarian must examine multiple animals to determine if an observation is associated with disease. if working in a zoologic institution and any fi ndings are suspected to be infectious in origin, other animals in the exhibit should be examined. as with any species, an examiner must know what a normal presentation is in order to recognize clinical signs of disease. diagnostic tests used to determine crocodilian health status are no different than those used with other animal groups. cbcs, chemistry panels, and bacterial cultures can all be performed in crocodilians. however, there are limitations when it comes to the interpretation of test results because of the lack of reference ranges available for the multiple crocodilian species. in addition, other diagnostic tests, such as those based on polymerase chain reaction (pcr) technology, enzyme-linked immunosorbent assay (elisa), and other antibody or antigen serologic tests, are usually not validated for crocodilians. a clinician must inquire about the specifi cs of the tests being performed to make an accurate interpretation of the results. published literature and the experience of other colleagues are invaluable for interpreting diagnostic test results of crocodilian species. also there may be generally accepted diagnostic tests for diseases that have been recognized and studied in crocodilians, such as mycoplasmosis and wnv. there are various sites for venipuncture in crocodilians. the ventral coccygeal vein can be accessed from either the ventral or the lateral aspect of the tail ( figure - ). this vessel lies ventral to the vertebral processes and on midline with the vertebrae. a second alternative is the supravertebral sinus, located on midline at the junction of the head and the neck ( figure - ). the examiner must be careful not to go too deep at this site, or physical damage may occur. a third site is an unnamed vessel located on both the left and right sides of the neck. this vessel can be approached from the dorsal aspect of the neck and is surrounded by muscles, decreasing the risk of coming in contact with nervous tissue (figure - ) . a -ml syringe and a -gauge needle are recommended for collecting blood from crocodilians. lithium heparin and edta tubes are used for plasma chemistry analysis and cbcs, respectively. if collecting serum, the tubes may have to sit for at least to minutes before centrifugation to allow proper separation of the serum from the blood cells. as with other species, cbcs and chemistry panels are a fundamental part of diagnostic testing of crocodilians. these tests can help assess the overall health status of the animals. a cbc can show evidence of acute or chronic infl ammation that may prompt further investigation or lend direction to a defi nitive diagnosis. chemistry panels can give insight into the hydration status and the health of the liver and kidneys. also of importance are electrolyte values, which can show abnormalities related to poor diet and husbandry. a measurement of packed cell volume and total solids and/or total proteins is also an essential health indicator for crocodilian species. fine needle aspirates, impression smears, and fl uid analysis are diagnostic tools that can be useful in determining a defi nitive diagnosis. urinalysis is not a practical test in crocodilians due to the absence of a urinary bladder and the fact that urine will be highly contaminated in a voided sample. the hemocytometer with the unopette eosinophil determination for manual methods stain (becton dickinson and company, franklin lakes, nj - ) is often used to obtain a cbc of crocodilian species and other reptile and avian species. a blood smear stained with diff-quick (quik-dip stain, mercedes medical physician and laboratory products, commerce ct., sarasota, fl ) is also needed to complete the total estimated white blood cell count and obtain the cell differential count. interpretation of cbcs from crocodilians can be challenging for veterinarians unfamiliar with the morphology of their white blood cells, which can vary from that of other reptile species. this variation found in crocodilian species is typical and more pronounced among reptiles. to become familiarized with the different cell types and their appearance, veterinarians should spend time scanning blood smears. this will often help veterinarians differentiate heterophils from eosinophils before the initiation of the differential count. alternatively the samples can be submitted to a commercial laboratory that runs cbcs on blood collected from exotic animal species. most imaging modalities (radiographs, computed tomography [ct] scan, magnetic resonance imaging [mri], ultrasound, etc.) can be used on crocodilian species as long as there is a general understanding of their anatomy. knowledge of normal anatomy is crucial for the interpretation of radiographs, ct scans, and mri images. knowledge of the anatomy will also help locate organs during ultrasound examination. radiographs can be used to locate foreign bodies as well as diagnose fractures. contrast studies can also be performed if the facilities and personnel are allowed to hospitalize the animal for an extended period of time. barium sulfate (liquid e-z-paque, e-z-em inc., westbury, ny ) and iohexol (omnipaque, amersham health inc., princeton, nj ) can be administered at to ml/kg po. iohexol can be diluted with water at a : to : ratio depending on the concentration. depending on the size of the animal and the site of interest, some imaging procedures may be performed with manual restraint or, if needed, chemical restraint. neuromuscular blocking agents may be benefi cial as chemical restraint agents if used appropriately and if the animal is monitored closely. stress has been defi ned as "a physiological answer to a perceived threat that includes, but is not restricted to, increased adrenal secretion." stress can also be any event that challenges homeostasis. the response of the body to that event is complex and involves more than an adrenal response. the autonomic nervous system, the hypothalamic adrenal axis, neuropeptides, neurotransmitters, and neuroimmunologic mediators all have a role in the response of the immune system to stress. measuring stress and immunosuppression is a challenge in veterinary medicine. there are no specifi c tests available to provide a clinical measure of stress. veterinarians concentrate on identifying a combination of physiologic changes that give them an idea of what is involved in a stress response. the stress response in crocodilians has been examined in relation to restraint, long-term corticosterone implants, cold shock, and stocking densities. , - lance et al. provides an overview of the physiology and endocrinology of stress in crocodilians. catecholamines, glucocorticoids, glucose, and lactate have been implicated in the stress response of crocodilians. changes in the white blood cells have also been implicated with immunosuppression and the stress response. [ ] [ ] [ ] there is enough evidence to suggest that stress plays an important role in the physiology of crocodilians and may indeed predispose them to illness. overcrowding, handling, excessive noise, diet changes, temperature irregularities, and so forth, should be considered as predisposing or confounding factors of disease. all of these factors must be considered in the history of a clinical case and when determining treatment. an example of how stress can play a role in disease susceptibility was observed in a case at a commercial alligator facility. this facility had animals with a history of chronic dermatitis. the alligators with the most severe lesions were consistently located on one end of the building, whereas those at the other end were unaffected or only mildly affected. the location of the severely affected animals was consistent in all buildings. one building had no affected animals. based on a thorough history and observation of the operation, a possible explanation was determined for the occurrence of the dermatitis problem. all of the buildings with affected animals had pvc pipes on the inside walls that delivered water to each individual pen. a strong water stream fell from the pipes down into the water and the strength of the stream decreased from one end of the building (infl ow) to the other end (outfl ow). the strength of the water stream was considerable near the infl ow. the building with no affected animals did not have any source of falling water into the pens. water quality, temperature, and feed were the same in affected and nonaffected buildings. the most affected animals happened to be in the pens at the infl ow side of the building with the number of affected animals decreasing toward the outfl ow side of the building. this constant fl ow of water was creating a constant movement of the water surface and consequently stimulating the alligators via the dprs. once this watering system was changed, the cases of dermatitis decreased and no new cases were reported. although other factors, such as water quality, may have contributed to the dermatitis, the change in the watering system decreased the progression and occurrence of the disease. this case demonstrates the importance of addressing the environment, as well as the animals, when working in commercial operations. bacterial diseases in captive crocodilians occur primarily as opportunistic infections. poor water quality, trauma, and stress are some of the factors that contribute to bacterial infections in crocodilians. in their natural environment, crocodilians appear to have a stronger ability, compared with other species, to withstand bacterial infections. reports of antibacterial properties in serum and tissues of crocodilians , offer a possible explanation. the possibility of increased antibacterial properties in the serum and tissues of alligators should not mislead people into thinking that crocodilians are resistant to bacterial infections. it is true that they appear to tolerate trauma and other lesions that would be fatal in many species, but crocodilians are still capable of succumbing to an array of microorganisms, including bacteria. in fact, a number of bacterial infections have been reported in crocodilians, and septicemias are thought to be a frequent fi nding. septicemias can be diagnosed in postmortem examinations and are often associated with a wide number of bacteria, many of them normal gut fl ora. there is an abundance of information about bacteria recovered from the american alligator (a. mississippiensis) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (table - ) and from african dwarf crocodiles (osteolaemus tetraspis). salmonella spp. are normal inhabitants of the gut in most reptile species and crocodilians. [ ] [ ] [ ] the importance of salmonella sp. arises more from its zoonotic potential rather than its ability to cause disease in crocodilians, but it has been reported to cause deaths in nile crocodile (c. niloticus) hatchlings. in most commercial operations the meat is sold as a by-product of the hide production. various species of salmonella and other bacteria have been isolated from the meat of crocodiles in commercial operations in an attempt to address the concern of zoonoses. , , [ ] [ ] [ ] [ ] [ ] zoologic institutions that have smaller crocodilians as part of a "petting" station should exercise caution, as fecal shedding of salmonella sp. occurs. although the zoonotic potential exists, there are no well-documented cases of human infections originating from crocodilians. chlamydiosis has also been reported in crocodilian species. there is a report of an isolate, closely associated to chlamydophila psittaci, obtained from the livers of nile crocodiles (c. niloticus) in zimbabwe. this infection is thought to have an acute course characterized by a hepatitis in which hatchling mortalities are observed. a chronic form of reptilian chlamydiosis characterized by conjunctivitis has also been reported and may be more common. there have been reports of concurrent chlamydia sp. isolates in cases of mycoplasmosis and adeno viral infections. dermatophilosis or "brown spot disease" is thought to be caused by dermatophilus sp., with most of the cultures resembling dermatophilus congolensis, and affects both crocodiles and alligators. , , [ ] [ ] [ ] the characteristic brown to red lesions are located usually at the junction of the ventral abdominal scales, which may become ulcerated over time. this has occurred in both alligator and crocodile operations. this disease does not respond well to antimicrobial therapy; therefore, intensive hygiene practices are required to prevent and control outbreaks. mycoplasma alligatoris is a recognized respiratory pathogen of crocodilians. it has been documented in a. mississippiensis and in the broad-nosed caiman (caiman latirostris). , , other crocodilian species closely related to alligators also may be susceptible to this intracellular organism. clinical signs are nonspecifi c and include lethargy, weakness, anorexia, white ocular discharge, paresis, and edema (facial, periocular, cervical, limbs). virus identifi cation and diagnosis in reptiles has lagged compared to virus identifi cation and diagnosis in other species. this delay has occurred primarily because of diffi culties in developing diagnostic tests and lack of knowledge of viruses that affect reptiles and crocodilians. many conditions that go undiagnosed may be caused by viruses. these may be new viruses or just viral diseases unknown to occur in reptilian or crocodilian species. there are only a few recognized viruses that have been documented in crocodilians. jacobson et al. described an adenovirus-like infection in captive nile crocodiles (c. niloticus) characterized by nonspecifi c clinical signs, lethargy, and anorexia. conjunctivitis and blepharitis were also observed in one of two crocodiles. , histopathologic examination revealed intranuclear inclusions, primarily in the liver but also in the intestines, pancreas, and lung. both horizontal and vertical transmission have been postulated for this adenovirus-like infection. diagnosis is obtained through postmortem examination, and no treatment regimes have been established. coronavirus, infl uenza c virus, and paramyxovirus have been identifi ed by transmission electron microscope in the feces of crocodilians. herpesvirus-like particles were identifi ed by electron microscopy in the skin of a salt water crocodile (c. porosus). there is a second fi nding of herpesvirus identifi ed from the cloaca of an american alligator (a. mississippiensis) via pcr. the clinical signifi cance of these fi ndings is unknown. seroconversion to paramyxovirus and eastern equine encephalitis virus has also been reported in crocodilians. parapoxvirus or pox-like viruses have been identifi ed in fi ve different crocodilian species: spectacled caiman (caiman crocodilus fuscus), , brazilian caiman (caiman crocodilus acre), nile crocodile (crocodylus niloticus), , saltwater crocodile (crocodylus porosus), and freshwater crocodile (crocodylus johnstoni). pox lesions in caimans will be to mm in diameter, gray to white in color, and coalescing to macular. they may appear on the head, palpebra, maxilla, mandible, limbs, palate, tongue, and gingiva. [ ] [ ] [ ] palpebral and generalized edema also may be present. resolution of clinical signs has been observed with and without changes in husbandry practices. , in crocodiles the lesions are described as to mm in diameter, yellow to brown, wart-like, sometimes fi rm, and unraised to raised nodules with occasional shallow ulcers. in crocodiles pox lesions appear on the head, palpebra, nostrils, sides of the mouth, oral cavity, limbs, ventral neck and coelom, and at the base of the tail. , resolution of lesions was reported to occur as early as to weeks from the onset of clinical signs. histopathologic fi ndings include epithelial hyperplasia, acanthosis, hyperkeratosis, and necrosis, with intracytoplasmic borrel and bollinger's bodies being visible in some cases. [ ] [ ] [ ] [ ] [ ] secondary bacterial and fungal infections may occur concurrently with the viral infection. at this time there are no specifi c treatment recommendations. the use of an autogenous vaccine to treat poxvirus in nile crocodiles (c. niloticus) has had some success. mosquito control and good hygiene are essential in preventing and controlling poxvirus outbreaks. west nile virus (wnv) has been reported to affect various crocodilian species, including the american alligator (a. mis-sissippiensis), the nile crocodile (c. niloticus), and the morelet's crocodile (c. moreletii). crocodilians likely become infected, as birds and mammals do, via a mosquito bite. there is also the possibility of infection after ingestion of an animal with a high viral load of wnv, as reported by klenk et al. this last scenario is more likely to occur when crocodilians are housed outdoors and not in enclosed buildings, as in most ranching operations. it has been demonstrated that alligators can serve as amplifi ers of wnv. although there is a lot to be learned about wnv in croco dilians, it is believed that once infected, they can develop high viremias and shed the virus in the feces. fecal shedding leads to horizontal transmission of the virus. there is clinical evidence for horizontal transmission to occur in commercial operations. fecal shedding and high viremias also raise the concern of zoonosis, especially in commercial operations where animals are being slaughtered and people come in contact with blood and tissues. a strict building quarantine and hygiene strategies should be implemented to prevent the spread of wnv to other animals in the facility as well as to the personnel. in the state of louisiana there are a number of recommendations provided to help alligator producers cope with episodes of wnv and prevent spread of the disease (box - ). . buildings with affected animals should be maintained under isolation from other buildings in the farm. . feeding and cleaning of affected buildings should be performed last, after all other nonaffected buildings. . a foot bath ( part water, part bleach) should be placed at the entrance of the building to disinfect the shoes and boots of farm employees before and after entering the building. this foot bath should be changed on a daily basis. in addition, a set of shoes or boots may be kept only to be used inside affected buildings. . hands should be washed before and after entering the building. . the water should be changed more frequently in affected buildings, at least once a day. this may create additional stress on the animals but should decrease the amount of feces and therefore viral organisms possibly present in the water. any special treatment of the discharge water still remains to be determined. . affected animals should not be transported to other alligator farms for processing of hides or meat. . additional care should be exercised during the slaughter process to avoid direct contact with the blood and organs of animals known as exposed to, or affected with, west nile virus. . dead animals should be disposed of by burning the carcasses. . aggressive mosquito control is required to help in the prevention of the disease. affected animals in captive operations have ranged in age from month to over months. in younger animals infection is usually acute and severe, with as much as % mortality. the pattern of deaths is peracute, usually seen as a sudden onset of mortalities followed by a peak and subsequent decline in the number of deaths. however, sporadic mortalities may also be noted, especially in older animals. clinical signs of wnv in alligators include swimming in circles, head tilt, muscle tremors, weakness, lethargy, and anorexia. bloating and diffi culties swimming have also been observed but occur less commonly. gross fi ndings from wnv-infected alligators are nonspecifi c. light microscopy of tissues from infected animals may reveal diffuse severe heterophilic, histiocytic, and necrotizing enterocolitis; heterophilic meningoencephalitis; necrotizing and heterophilic hepatitis; heterophilic and histiocytic splenitis; generalized heterophilic and histiocytic lymphoid folliculitis; and necrotizing and heterophilic pancreatitis. veterinarians may also observe a mild multifocal heterophilic and lymphohistiocytic interstitial nephritis, gastritis, and mild pulmonary congestion and edema. strong immunopositivity results have been observed in the brain, liver, spleen, pancreas, kidney, and gastrointestinal tract after immunohistochemistry testing for wnv. proliferative enteritis has been diagnosed in a group of alligators positive for wnv. the colon lesions tested positive for wnv via reverse transcriptase polymerase chain reaction (rt-pcr), culture, and immunohistochemistry , ( figure - ). these affected alligators were bloated and unable to submerge themselves; these symptoms may have been due to a blockage caused by the fi brinous membrane in the colon. there is no known treatment for wnv-infected crocodilians. mosquito control, strict quarantine, and biosecurity are essential for the prevention of wnv. once wnv is present within an operation, it is critical to maintain infected and exposed animals in strict isolation. the ill and exposed animals must not be moved to other areas or buildings where wnv has not been observed. failure to maintain strict isolation may result in the spread of the virus throughout the facility. in addition, there should be strict biosecurity measures, especially in the affected areas or buildings. tools such as nets, feeding utensils, boots, and so forth, must be left in the affected area and not introduced to other, "clean" areas or buildings. the surviving animals will continue to thrive after the wnv infection has run its course, but it is unknown for how long thereafter they can shed the virus. antibody titers of : and : were observed in two alligators months after exposure to wnv. these antibody titer results were obtained via a plaque reduction neutralization test. defi nitive diagnosis of wnv infection should be based on history, clinical signs, and the results of diagnostic tests. postmortem diagnosis can be performed via rt-pcr and/or viral culture of brain, spinal cord, or liver, with immunohistochemistry providing additional supportive evidence of wnv infection. immunohistochemistry may prove useful as an antemortem test when applied to biopsies of the colon mucosa. fungal infections also occur with some frequency in captive crocodilians. most fungi are opportunistic invaders of the integument, respiratory system, and gastrointestinal tract. poor water quality, trauma, stress, and extreme temperatures can contribute to the occurrence of fungal disease. it is thought that most fungal infections in crocodilians are of enteric origin and occur in other tissues secondary to an immunocompromised state. it is important to remember that fungi are considered ubiquitous organisms in nature. therefore, it is not uncommon to isolate fungi from tissues (e.g., skin, intestines) that are in contact with water and soil. the presence of fungi will vary with geographic location and management techniques that affect the different environments for their growth. there are a number of fungi that have been isolated from crocodilians , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (table - ). some of these fungal organisms were associated with disease, whereas others were incidental fi ndings. diagnosis of fungal disease requires the identifi cation of a fungal organism via culture or special stains of infected tissues. positive identifi cation of genus and species may be diffi cult. treatment is expensive and may be affordable only when administering to a small number of animals. it also requires a long treatment period of at least or months. for these reasons, many fungal infections go untreated, and recommendations are made for prevention of disease. a number of parasites are known to affect crocodilians. protozoa, nematodes, trematodes, and pentastomes have all been reported in crocodilians. cestode larvae have been found in some species, but no adult tapeworms are reported from crocodilians. external parasites, such as leeches, fl ies, mosquitoes, ticks, and mites, can also affect crocodilians. the presence of scales has created a misconception about the possibilities for external parasitism in crocodilians. however, there are a number of areas in a crocodilian's body that have soft skin and allow an external parasite to attach and/or obtain a meal. the recent cases of wnv in the american alligator (a. mississippiensis) are a reminder that arthropods can play an important role in disease transmission. internal parasites are not a common problem in ranching operations where animals are maintained in concrete buildings with no organic substrate. parasites are of major concern in wild crocodilians or those kept captive outdoors on organic substrates. external parasites are more ubiquitous, affecting both captive and wild populations. to determine the best prevention and control methods to use, veterinarians must assess the environment as well as the life cycle of the parasite. with the increased knowledge of crocodilian dietary requirements, nutritional diseases are diagnosed less frequently than they were in the past. better understanding of the biology, natural history, and physiology of the different crocodilian species has allowed implementation of better feeding schemes and diets. over time, there has been a desire to develop commercial diets that will allow for high feed to weight gain ratios. these commercial diets are available for feeding american alligators (a. mississippiensis), and although the diets are not perfect, if fed appropriately, captive-hatched american alligators (a. mississippiensis) will grow up to inches in length within a -month period. for zoologic institutions the goal is not rapid growth but rather normal development and a foundation for excellent health. zoologic institutions and crocodile farms commonly provide crocodilians with fresh and/or frozen prey (e.g., poultry, swine, beef, fi sh, nutria, native prey, etc.) with bones providing a source of calcium. to prevent lead toxicity in crocodilians, veterinarians must use a reputable fresh or frozen prey source that has not been killed with lead projectiles (e.g., shotgun pellets, bullets). despite better feeding schemes and diets, veterinarians will still see crocodilians with diseases associated with nutritional defi ciencies. a common nutritional disease of reptiles is metabolic bone disease (mbd). clinical signs of mbd include weakness, lethargy, kyphosis, scoliosis, osteodystrophy, pathologic fractures, paresis, and tooth decalcifi cation. various possible causes of mbd have been proposed, but it is usually associated with a calcium-phosphorus imbalance that leads to increased bone resorption. calcium and vitamin d are the two main nutritional compounds defi cient in reptiles with mbd. vitamin d defi ciencies are associated with a lack of exposure to uvb spectrum light, which comes naturally from the sun. light bulbs and light tubes are commercially available to help provide adequate amounts of uvb light for reptiles. mbd is primarily observed in herbivorous or omnivorous reptiles. it appears that mbd is not commonly observed in commercial crocodilian operations, even in those were animals are raised in the dark with no source of uvb light. the carnivorous nature of crocodilians may allow them to obtain vitamin d from the diet without a true requirement for uvb light. however, if an appropriate calcium source is not offered, these animals will develop mbd. adult alligators without mbd have been observed growing in enclosed buildings with no light source while being fed a commercial diet supplemented with fresh prey meat with bones. however, anecdotal comments from various ranchers indicate that the animals appear to thrive better if exposed to sunlight. this is an area where more research is needed to determine the uvb and calcium requirements of crocodilians and their effects on growth and health. articular and visceral gout also have been reported in crocodilians. some predisposing factors for gout include high protein diets, dehydration, and stress. clinical signs associated with chronic gout in crocodilians are primarily nonspecifi c, but limb paresis or paralysis and joint enlargement can occur. ariel et al. reported a case of visceral and articular gout with concurrent hypovitaminosis a in crocodile hatchlings. defi ciencies of vitamins a, b, c, or e also can lead to a variety of musculoskeletal disorders. these are thought to be less common in crocodilians fed commercial diets in addition to fresh meat products. respiratory disease is one of the most common presentations of captive crocodilians in commercial operations. it is common to fi nd lesions in the lungs associated with respiratory disease when performing postmortem examinations (figure - ) . although most of the lung pathology is incidental, some animals will have a history of clinical signs associated with respiratory disease before death. clinical signs of respiratory disease in crocodilians are nonspecifi c anorexia, lethargy, and weakness but may also include dyspnea, tachypnea, nasal secretion, excessive basking, respiratory stridor, and abnormal swimming (either in circles or on one side of the body). in some cases the animals may appear neurologic as they become weak and ataxic. a clear distinction must be established between respiratory disease and neurologic disease. respiratory disease may be secondary to neurologic disease as a consequence of weakness, predisposing the animal to aspiration pneumonia. most respiratory infections are either bacterial or fungal in origin. upper respiratory diseases, including rhinitis and pharyngitis, also have been reported in crocodilians. neurologic diseases are not common in crocodilians. however, the causes implicated by clinical neurologic presenting symptoms deserve special attention. the most common neurologic clinical sign is abnormal swimming behavior (e.g., swimming in circles, swimming on one side of the body, etc.). once outside the water, these animals may show signs of lethargy, ataxia, head tilt, and muscle tremors. anorexia may be the earliest sign observed by the facility personnel. the most recent explanation of signs of neurologic disease in crocodilian species is wnv. this virus should be in the differential diagnosis list for any crocodilian that has neurologic signs and is housed in an area where wnv is endemic. hypoglycemia also has been reported to cause neurologic signs in alligators. neurologic signs and deaths of alligators (a. mississippiensis) were identifi ed from a commercial operation where a building was not cleaned according to the maintenance schedule. the water in the building had not been changed in a -hour period, and there was evidence of food debris from previous feedings. the owner found a few hundred dead alligators, and of those that remained alive, some had neurologic signs (e.g., slow refl exes, abnormal swimming patterns, ataxia, and lethargy). necropsy and histopathologic evaluation leaned toward toxicity as the cause of death. both dead and live animals tested negative for wnv. all other buildings were cleaned on schedule, and there was no evidence of disease in those buildings. mortalities decreased after the building housing the sick animals was cleaned. based on the history, clinical observations, and histopathologic fi ndings, veterinarians believe these animals developed neurologic signs as a result of either increased ammonia level in the water, oxygen depletion in the air, or both. musculoskeletal disease can be caused by changes in the incubation temperature or humidity; trauma from fi ghting, transport, or restraint; and infectious diseases ( figure - ) . deformities of the head, limbs, and tails are common malformations infl uenced by incubation rather than true genetics ( figure - ). fractures and limb amputations can occur as a result of trauma and fi ghting, with many healing without major complications. healed traumatic injuries are observed in captive and wild crocodilians. if the trauma is severe enough, nerve damage, muscle damage, paresis, and/or paralysis may result. the most common infectious disease known to have effects on the musculoskeletal system is mycoplasmosis. both m. alligatoris and m. crocodyli can cause polyarthritis in affected animals. ingestion of foreign bodies, gastric ulcers, enteritis, and trauma to the oral cavity are gastrointestinal diseases associated with crocodilian species. anorexia is a common sequelae to the gastrointestinal diseases listed above. ingestion of foreign bodies occurs in both wild and captive crocodilians. construction, malfunction of water pumps and fi lters, forgotten objects in the enclosure, and tossed objects by the public are some of the sources of foreign bodies for captive animals. sharp objects such as nails may cause severe gastrointestinal problems, while other less sharp or pointed ingested material may pass without major diffi culties. infectious enteritis is usually diagnosed post mortem based on gross and histopathologic evaluation. crocodilians appear to have an aggressive response to insult of the gastrointestinal tract. grossly one may observe accumulation of fi brinous or fi brous material and/or necrosis of the mucosa (figure - ) . in some instances, this can even lead to obstruction due to accumulation of fi brinous/fi brous material. fecal impactions and torsions are probably rare but can also occur in crocodilians, while gastric ulcerations may be associated with stress and diet. the intestinal tract of crocodilians appears to contain a signifi cant amount of gut associated lymphoid tissue like peyer's patches. this lymphoid tissue may allow for an aggressive infl ammatory response to infectious agents in the intestinal tract. integumentary disease is arguably the most signifi cant disease process affecting captive crocodilians. water quality, temperature, and stressors contribute to the occurrence of integumentary diseases. secondary bacterial and fungal dermatitis in crocodilians can have a devastating economic impact on com- mercial operations. lacerations, abscesses, and draining tracts can serve as a nidus for microorganisms. an additional predisposing factor of integumentary disease in captive operations is the accumulation of fat in the upper water column. this concentration of fat creates a slime layer on the walls and water surface, which is then transferred to the skin of the animals where it creates an ideal environment for fungal and bacterial growth. this is more of a problem when meat is the sole source of food or is provided in addition to a commercial diet. a surfactant disinfectant can be used to break down the fat accumulation in the water and on the walls of the enclosure. in most instances both bacteria and fungi will be present in the skin lesions associated with fat accumulation in the captive environment. it is important to recognize the fat accumulation problem early to institute appropriate cleaning measures and possible therapy. mixed fl ora is often found in the dermal lesions, making identifi cation of a single causal agent diffi cult. antimicrobial therapy is indicated if bacterial involvement is suspected. although the use of systemic antifungal medications is cost prohibitive in commercial operations, it can be used if only a small number of animals are affected. improved hygiene, better water quality, stable temperatures, and decreased stress are essential for managing these dermatitis cases. in most instances those animals that are already showing clinical signs may not recover. the key to preventing and decreasing the spread of disease is improving and implementing appropriate husbandry methods. various alligator (a. mississippiensis) ranches in louisiana have reported hatching animals with normal pigmentation that becomes "white" after a few weeks of life (figure - ) . physical examination of these animals does not reveal any abnormalities other than an apparent depigmentation or hypopigmentation of the skin. the white discoloration is not unlike that described as a result of fungal or bacterial dermatitis. upon palpation, the skin appears thinner and has a fl aky nature similar to that observed in leucistic alligators. affected animals do not appear to grow at different rates than normally pigmented animals. the processing of the hides is not affected by the pigment defi cit. a nutritional defi ciency or genetics are suspected in this presentation, but there are anecdotal reports of improvement of the condition after vitamin supplementation with a multivitamin formulation. lymphohistiocytic proliferative syndrome of alligators ("pix" disease) lymphohistiocytic proliferative syndrome of alligators (lpsa), also known as pix disease, has been recently studied in captivereared alligators (a. mississippiensis) from florida and louisiana. lpsa affects the quality of the hides in commercial operations, leading to decreased profi ts for alligator producers. gross lesions of lpsa are multifocal, -to -mm, gray to red foci on the ventral mandibular and abdominal scales ( have been found in the lungs, intestines, kidneys, ovaries, testicles, thyroid, gall bladder, mesentery, eyes, and nervous tissue. ongoing research has revealed a strong relationship between exposure to/infection by west nile virus and the occurrence of pix lesions. toxicities are not common in captive crocodilians. however, there have been cases of lead toxicity in alligators being fed nutria that had been killed with lead shotgun pellets. clinical signs include weakness, lethargy, anorexia, and death. alligator ranchers and farmers are now more diligent in inquiring about the source and kill method of nutria before feeding their animals. wild alligators, on the other hand, can be exposed to a number of toxic substances, mostly pesticides and heavy metals. many of these substances are thought to be endocrine dis rupters that affect the development and function of the reproductive organs and result in developmental defects of young crocodilians. [ ] [ ] [ ] [ ] [ ] there is an abundance of information to support the impact of environmental contamination on crocodilians as well as other species. this reiterates the critical state of the natural habitat of many species around the world. crocodilians have served as sentinels for anthropogenic changes that ultimately may come back to affect human health. runting is a phenomenon observed in captive crocodilian operations. it describes a general state of unthriftiness, lack of growth, and weakness. there is a visible size difference in same-age animals between the "runts" and the otherwise normally developing animals. it is not unusual to fi nd some buildings in a commercial operation that contain the runt animals for that year. it is also in this group of animals that disease problems may be observed with higher frequency. stress factors, including dominance by other animals, environment, and even incubation temperature, contribute to the presence of runts. fluid therapy of crocodilians is accomplished as in other reptile species. veterinarians must determine hydration status, percentage of fl uid defi cit, and maintenance requirements to develop a plan for fl uid therapy. a maintenance rate of to ml/kg can be administered intracoelomically (ice), orally (po), or intravenously (iv). in some instances, food prey items can be injected with fl uid and then fed to the animal. if the animal is anorexic, manual restraint will be required for fl uid administration. it is important to remember that restraint itself will be an additional stressor that may hinder the recovery of an animal. therefore, to limit the amount of stress in the animal, treatment protocols including fl uid therapy must be planned appropriately and administered quickly. antimicrobial therapy presents a challenge in crocodilians because of their extra label use. in commercial alligator facilities, antimicrobial treatment is complicated by the lack of drug withdrawal times for crocodilian species. currently the u.s. , enrofl oxacin was found to be effective when administered at mg/kg iv every hours. the longlasting oxytetracycline was found to be effective at mg/kg both iv and intramuscularly (im) every days. both of these treatment regimes may be practical in a zoologic institution or a commercial operation with a small number of animals. in a large commercial operation where a higher number of animals may need treatment, it may be time, cost, and labor prohibitive to administer medications intramuscularly or intravenously to individual animals. spectrum of coverage, method and frequency of administration, and cost should be considered when selecting an antimicrobial agent(s). consideration should be given to those drugs that can be mixed with, or injected into, the feed. one technique of antibiotic administration is making a sausage fi lled with the animal's feed and placing the drug in the sausage. manual medication by mouth or injection can be performed in smaller animals. whenever possible, culture and sensitivity test should be obtained to identify the organism in question and determine the effi cacy of the antimicrobial selected. unless a bacterial infection is suspected, use of antibiotics is strongly discouraged. most antimicrobial agents and their metabolites will be transported in the wastewater, potentially creating an avenue for future antimicrobial resistance. nutritional support of crocodilians should be approached as in other carnivorous reptiles. the main diffi culties result from the handling and restraint required to force-feed an ill crocodilian. nutritional support of an ill patient is more likely to occur at a zoologic institution than in a commercial operation. placing an esophagostomy tube should be considered as a less stressful alternative method of feeding the critical crocodilian. nutritional support through an esophagostomy tube will require restraint while feeding, but the stress level is reduced because the mouth does not have to be opened. any highprotein feed (e.g., commercial feed, fresh prey) that can be blended may be used as long as it can easily pass through the tube. in larger and aggressive animals, force-feeding is a serious challenge and must be performed carefully. various factors should be considered before performing a surgery procedure on a crocodilian, with the initial consideration being the ultimate purpose of the animal. is the animal part of a zoologic collection, is it from a commercial operation, or will it be released back to the wild? the next concern is the expected prognosis and time of recovery. ultimately the animal should be able to thrive in its future environment whether it be an exhibit or in the wild. cost of the procedure also is a major presurgical consideration. whereas cost may not be a major decision factor in some zoologic institutions, in other programs such as conservation programs, the money needs to be carefully allocated to obtain the highest return on the investment. the available facilities, especially for postoperative management, are also an important consideration. if the procedure requires placement of sutures, the animal should be maintained in a clean environment with shallow water for a few days to prevent infection and infi ltration of water into the surgical site. depending on the species, it may be possible to spray the animal with water throughout the day instead of keeping it in water. the fi nal consideration is the ability of the personnel to carry out the postoperative care, especially if force-feeding is required. it would be benefi cial to obtain a cbc and chemistry panel to assess the overall health status of the animal before surgery is performed. diagnostic test results will help determine if the animal is fi t for surgery and can be compared to postsurgical blood work to monitor recovery and healing, although in many instances diagnostic evaluation is an unrealistic expectation. to anesthetize crocodilian species, a basic understanding of their physiology and anatomy is needed to ensure safe delivery, induction, and maintenance of the anesthetic agent. there are various anesthetic agents, both injectable and inhalant, that can be used to anesthetize and sedate crocodilians. procedure type, species, and size are factors that will infl uence the choice of anesthetic agent(s). there are no established anesthetic protocols for crocodilians, and information is often derived from fi eld research as well as clinical experience of practitioners. injectable anesthetic agents can be delivered in the tail muscles via a pole syringe or hand syringe or administered intravenously after immobilization or restraint. neuromuscular blocking agents, such as gallamine and succinylcholine, are widely used in crocodilians. when transporting large, aggressive animals, veterinarians might use these agents to prevent injury to both the handlers and the animal. however, these drugs do not have analgesic properties and should not be used alone for any surgical procedures. when using neuromuscular blocking agents, veterinarians should ensure the animal is supported with a backboard to minimize injury to the spine. succinylcholine is a depolarizing neuromuscular blocker with primary action at the nerve end plate, and fl accid or rigid paralysis may occur depending on the dose. , there is no reversal agent for succinylcholine, so it must be used with caution and the animals monitored closely. effects can be observed within minutes of administration, and recovery takes up to . hours. recovery will depend on the excretion of the drug in the urine and metabolism of the crocodilian. gallamine is a nondepolarizing neuromuscular agent that competitively blocks acetylcholine at the motor end plate, resulting in fl accid paralysis. crocodilians immobilized with gallamine will open their mouths following relaxation of the muscles; this is known as the flaxidil reaction. increased respiratory rate and heart rate may be observed with gallamine immobilization. , an overdose of gallamine may lead to bradycardia, mydriasis, increased gastrointestinal motility, and paralysis of respiratory muscles. increased salivation and emesis can be prevented with presurgical use of atropine. an advantage of gallamine over succinylcholine is that it can be reversed with neostigmine methylsulfate. fatalities have been reported in american alligators (a. mississippiensis), chinese alligators (a. sinensis), and false gharials (tomistoma schlegelii) that were administered gallamine. diazepam can also be administered to minutes before succinylcholine or gallamine for added muscle relaxation. administration and recovery of animals with neuromuscular agents should be done far from the water to avoid drowning. noise and light stimuli should be minimized when animals have been sedated with gallamine or any other neuromuscular agent or anesthetic. table - shows doses for various neuromuscular agents. following administration of a neuromuscular agent, it may be possible to intubate a crocodilian for general anesthesia with an inhalant anesthetic such as isofl urane. remember to always provide analgesics if performing a painful procedure. injectable anesthestics have the added benefi t of providing analgesia and decreasing the amount of inhalant anesthetics used during surgery. dissociatives, opioids, benzodiazepines, and barbiturates have been administered to reptiles and croco-dilians with various rates of success. alpha- adrenergics are gaining popularity in reptile medicine and show promising results for their continued use as anesthetics in reptiles. a combination of medetomidine and ketamine works well in american alligators (a. mississippiensis) and has the added benefi t of being able to reverse the medetomidine with atipamezole. in a study involving juvenile alligators, the young animals had a higher requirement of medetomidine ( . ± . μg/kg im), atipamezole ( . ± . μg/kg im), and ketamine ( . ± . mg/kg im), compared to adult alligators' requirement of medetomidine ( . ± . μg/kg im), atipamezole ( ± μg/kg im), and ketamine ( . ± . mg/ kg im). these fi ndings are similar to those observed for other anesthetic agents used in reptiles. metabolic differences between age classes usually require higher doses for juvenile animals. an additional anesthetic agent that has gained popularity in reptile medicine is propofol (abbott laboratories, abbott park rd., abbott park, il - ). propofol is a lipid soluble drug with fast action and short duration of effects, which makes it an ideal anesthetic agent that allows for intubation of the animal to be followed with inhalant anesthesia. propofol can be administered at to mg/kg iv , and as a constant rate infusion. the iv route of administration of propofol presents a disadvantage in cases where manual restraint is not possible or is undesired. when choosing an anesthetic or immobilizing agent, veterinarians should take into account all the factors previously mentioned while keeping the safety of the animal and personnel in mind. fortunately, surgeries are not routinely performed in crocodilian species. the most common surgical procedure performed in zoologic institutions is probably the removal of foreign bodies through gastroscopy. this may be performed under immobilization or general anesthesia. enterotomies are rare but may be required if a foreign body lodges itself beyond the stomach, is too large to pass on its own, or both. the next most common procedure is the repair of fractures and lacerations caused by trauma. crocodilian fracture repair can present a real challenge to the veterinarian because of an animal's large size, demeanor, and need to live in an aquatic environment. therefore, it is preferred to perform fracture repairs with internal fi xation methods, such as intramedullary pins, cerclage wire, and bone plates. before attempting a surgical procedure, any veterinarian must realistically determine the prognosis for the animal. huchzermeyer fw: crocodiles: biology, husbandry and diseases an ancient sensory organ in crocodilians crocodilian anesthesia reptile medicine and surgery stocking rates of ranched american alligators (alligator mississippiensis) essential fatty acid nutrition of the american alligator (alligator mississippiensis) protein nutrition in the alligator alligator feed evaluations elevated lead levels in farmed american alligators (alligator mississippiensis) consuming nutria (myocastor coypus) meat contaminated by lead bullets unpublished description of a new site for venipuncture in the american alligator exotic animal formulary biotic and abiotic factors in crocodilian stress: the challenge of a modern environment stress-mechanisms of immunosuppression plasma catecholamines and plasma corticosterone following restraint stress in juvenile alligators effects of long-term corticosterone implants on growth and immune function in juvenile alligators (alligator mississippiensis) hormonal and metabolic response of juvenile alligators to cold shock growth rate and plasma corticosterone levels in juvenile alligators maintained at different stocking densities physiology and endocrinology of stress in crocodilians antibacterial properties of serum from the american alligator (alligator mississippiensis) naturally occurring antibacterial activities of avian and crocodile tissues isolation of aeromonas hydrophila from the american alligator (alligator mississippiensis) aeromonas-induced deaths among fi sh and reptiles in an eutrophic inland lake diseases of the eye and ocular adnexae in reptiles immobilization, blood sampling, necropsy techniques and diseases of crocodilians: a review initial antibiotic therapy for alligator bites: characterization of the oral fl ora of alligator mississippiensis gram-negative septicemia in american alligators (alligator mississippiensis) isolation of edwardsiella tarda from a sea lion and two alligators treatment of multiple cases of pasteurella multocida and staphylococcal pneumonia in alligator mississippiensis on a herd basis pathology of experimental mycoplasmosis in american alligators adenovirus-like infection in two nile crocodiles morbidity and mortality associated with a new mycoplasma species from captive american alligators (alligator mississippiensis) clinical values associated with opportunistic bacterial diseases in farm-raised alligators brown spot disease in the louisiana alligator industry: what we know about the disease and possible control protocols brown spot disease of commercially-raised alligators: a preliminary report colibacillosis in captive wild animals mycoplasma alligatoris sp nov, from american alligators aerobic intestinal fl ora of wild-caught african dwarf crocodiles (osteolaemus tetraspis) salmonella isolation from reptilian faeces: a discussion of appropriate cultural techniques recovery rates, serotypes, and antimicrobial susceptibility patterns of salmonellae isolated from cloacal swabs of wild nile crocodiles (crocodylus niloticus) in zimbabwe salmonella isolated from crocodiles and other reptiles during the period - in south africa hepatitis in farmed hatchling nile crocodiles (crocodylus niloticus) due to chlamydial infection public health risks of the fl esh of farmed crocodiles prevalence and serovar distribution of salmonella in fresh and frozen meat from captive nile crocodiles (crocodylus niloticus) microbial fl ora of frozen tail meat from captive nile crocodiles (crocodylus niloticus) salmonella in captive crocodiles (crocodylus johnstoni and c porosus) microbiological evaluation of dressing procedures for crocodile carcasses in queensland isolation of dermatophilus sp from skin lesions in farmed saltwater crocodiles (crocodylus porosus) pathology of skin diseases in crocodiles brown spot disease in the american alligator (alligator mississippiensis) experimental inoculation of broad-nosed caimans (caiman latirostris) and siamese crocodiles (crocodylus siamensis) with mycoplasma alligatoris in vitro susceptibility pattern of mycoplasma alligatoris from symptomatic american alligators (alligator mississippiensis) mycoplasma-associated polyarthritis in farmed crocodiles (crocodylus niloticus) in zimbabwe vaccination of farmed crocodiles (crocodylus niloticus) against mycoplasma crocodyli infection vaccination to control an outbreak of mycoplasma crocodyli infection a color atlas of diseases of the crocodile unpublished data from clinical and research cases, la herpes virus-like particles in the skin of a saltwater crocodile (crocodylus porosus) poxlike skin lesions in captive caimans pox virus infection in captive juvenile caimans (caiman crocodilus fuscus) in south africa poxvirus dermatitis outbreak in farmed brazilian caimans (caiman crocodilus yacare) poxvirus in farmed nile crocodiles poxvirus infection in nile crocodiles (crocodylus niloticus) poxvirus infection in two crocodile west nile virus in farmed alligators west nile virus infection in crocodiles alligators as west nile virus amplifi ers west nile virus in alligator (alligator mississippiensis) ranches from louisiana unpublished data on fungal isolates from the skin of american alligators (alligator mississippiensis) disease-husbandry associations in farmed crocodiles in queensland and the northern territory fatal mycotic pulmonary disease of captive american alligators cephalosporiosis in three caimans mycotic pneumonia caused by fusarium moniliforme in an alligator phycomycoses resulting in death of crocodiles in a common pool systemic mycotic disease of captive crocodile hatchling (crocodylus porosus) caused by paecilomyces lilacinus concurrent gout and suspected hypovitaminosis a in crocodile hatchlings hypoglycemic shock in captive alligators lymphohistiocytic proliferative syndrome of captive-reared american alligators (alligator mississippiensis) from louisiana unpublished data on lymphohistiocytic proliferative syndrome of alligators (lpsa) interaction of environmental chemicals with the estrogen and progesterone receptors from the oviduct of the american alligator abnormal bone composition in female juvenile american alligators from a pesticide-polluted lake sex reversal effects on caiman latirostris exposed to environmentally relevant doses of the xenoestrogen bisphenol a affi nity of the alligator estrogen receptor for serum pesticide contaminants achieving environmentally relevant organochlorine pesticide concentrations in eggs through maternal exposure in alligator mississippiensis incubation temperature affects body size and energy reserves of hatchling american alligators (alligator mississippiensis) pharmacokinetics of enrofl oxacin after single-dose oral and intravenous administration in the american alligator (alligator mississippiensis) pharmacokinetic disposition of a long-acting oxytetracycline formulation after single-dose intravenous and intramuscular administrations in the american alligator (alligator mississippiensis) physical and chemical restraint of crocodilians gallamine reversal in cuban crocodiles (crocodylus rhombifer) using neostigmine alone versus neostigmine with hyaluronidase crocodilian anesthesia diazepam and succinylcholine chloride for restraint of the american alligator evaluation of medetomidine-ketamine anesthesia with atipamezole reversal in american alligators (alligator mississippiensis) key: cord- -uokqn u authors: zhang, li; hua, ning; sun, shan title: wildlife trade, consumption and conservation awareness in southwest china date: - - journal: biodivers conserv doi: . /s - - - sha: doc_id: cord_uid: uokqn u commercial trade in wildlife is the major cause of species endangerment and a main threat to animal welfare in china and its neighboring countries. driven by consumptive use for food and traditional medicine, the large volume of both legal and illegal trade in wildlife has caused great destruction to ecosystems and pushed many species to the brink of extinction. data gathered from trading hubs at ports, boundary markets, city markets and stores, indicates the large amount of wildlife traded in the region of guangxi, yunnan and qinghai provinces, a direct result of the numerous wildlife markets available. in a survey distributed in various trading places, while about half of the respondents agreed that wildlife should be protected, % of them had consumed wildlife at some point in the last years. the results also indicated that law and regulation on wildlife trade control is insufficient. wildlife trade controls are very limited because of bias on the utilization of wildlife as a natural resource to be exploited by the government agencies. the survey also shows that the current situation of wildlife consumption in key cities in china is serious, especially the consumption for food. the main consumption groups in china are male and young people with high education levels and good incomes. the key in public awareness publicity and education is to give them more information on the negative impacts of wildlife consumption and knowledge of protection. the concept of modern wild animal protection was introduced to the chinese from abroad. china's trade of edible and medicinal wildlife dates back thousands of years. ''the skin can be worn, feathers can be used, meat is edible, and organs can be used for drugs''-throughout chinese history, wild animals have been viewed as an important source of food and income. from a traditional chinese perspective, as the same as many other countries, wild animals are a resource to be exploited, not something to be protected for their intrinsic value. in recent years, with the development of a consumer economy, people's demand for wild animal products has grown substantially. the markets for consumption are increasing, and using wild animals as pets, for medicine and health care, and as food has become a status symbol and a fashionable lifestyle pursued by many (zhou ; morgan ; wang et al. a; nooren and claridge ) . this robust market demand means lucrative profits for traders, providing a strong incentive for more people to join the trade. as a result, wild animal trade has expanded quickly, and illegal wild animal trafficking has increased sharply. trafficking, which involves excessive capture and non-sustainable utilization of wild species, poses a severe threat to many endangered species (li et al. ; li and li ) . large quantities of wild animals are now on the verge of extinction as a result of commercial development, such as chinese pangolin (manis pentadactyla) and tiger (panthera tigris) (traffic southeast asia ; s. wu , private communication; dinerstein et al. ) . as a conservative estimate, tens of millions of wild animals are shipped each year regionally and internationally destined to southern china for food or east and southeast asia for use in traditional medicine (wwf ) . according to the third annual report released by the biodiversity working group of the china council for international cooperation on environment and development (bwg/cciced), nearly % of mammal species in china are endangered because of hunting and habitat destruction, with the primary threat being excessive hunting (bwg/cciced ) . in the report entitled ''wildlife trade in southern china'' released in , the bwg pointed out that the increase in wildlife trade on mainland china in recent years could be attributed to four main of causes: (i) china's reform and economic growth has resulted in an increasingly prosperous population in the region who can afford expensive wild animals, increasing the incentive to trap animals. (ii) the improved infrastructure within china results in the availability of much sought after 'rarities' from distant or remote areas. (iii) the recent opening up of borders between china and its neighbors in southeast asia (e.g. vietnam and laos) has provided a new source of wild animals for the trade. (iv) the growing popularity of keeping reptiles and amphibians as pets in western countries, and more recently in japan, hong kong and taiwan, constitutes a substantial market outside china's mainland. thus reptiles and amphibians are imported to hong kong for re-export and, to a lesser extent, for local sale. wild animal trafficking worldwide is estimated to be worth more than us$ billion a year globally, second only to the trade in illegal drugs, with profit margins which are more attractive than illegal arms dealing (sain-ley-berry ) . adding to the problem, wild animal trade monitoring is very weak in china, making it difficult to assess the impact on wild animals domestically; relevant data obtained from consumers are also very scarce. meanwhile, the coexistence of legal trade and illegal trade makes it very difficult to monitor wild animal trade and distinguish which products are entering the market legally and which are being smuggled in illegally (li and zhang ) . identifying animal species traded at the sales terminals, estimating trading frequency, identifying species under protection or rare species, and assembling data on the countries and routes involved in trafficking are complex tasks that require support from a wide variety of sources (mill et al. ; zhou ; xie ; wan ) . challenges faced by organizations and government agencies working to combat illegal wildlife trade are vast: few key provinces house systematic wildlife trade management systems; enforcement authorities lack legal enforcement power; not enough data are available to sustain local biodiversity conservation; and a scientific monitoring and evaluation system has not yet been established to support conservation efforts (lee et al. ). in addition, limited data on wildlife trade are not efficiently shared and utilized between relevant protection and decision-making departments. another alarming trend is that the cross border trade in mammals, birds and reptiles between china and neighboring countries has reached a level previously unmatched in history, substantially affecting wildlife populations (li et al. ; yang et al. ; nooren and claridge ; wang et al. a, b; lee et al. ) . as a result, we are working to establish a database and monitoring platform on wildlife trade which can be used to enhance the communication among government administrative departments, decision-making departments and related organizations, improve monitoring and evaluation methods, strengthen law enforcement, and advance management and control on wildlife trade. the aim of this project is to eliminate commercial exploitation of animals by creating a sound public and democratic environment for the public to voice their opinions on wildlife consumption and protection issues. over-exploitation and cross-border wildlife trade are directly related to the fast growing chinese market which has inflated the price of wildlife products, making it more profitable to engage in the trade. many species of plants and animals are now in danger of becoming extinct because of over-exploitation, like liquorices (glycyrriza uralensis) in gansu and some other provinces in west china, birds and arethusa (dendrobium spp.) in guangxi and yunnan, matsutake (tricholoma matsutake) and other wildlife population in sichuan and yunnan. such threats may influence the administrations and individuals in some key departments of forestry, environment protection, agriculture, tourism and livestock farming into taking action. other stakeholders include local residents who depend on these natural resources, pharmaceutical manufacturers who want a sustainable medicine supply, and decision-makers who deal with local and global economies. the border area where yunnan and guangxi connect to vietnam, laos and burma is of particular concern. forests growing in the border area support % of wildlife population (li et al. ; yang et al. ) . problems exist in this area for all parties seeking to control illegal wildlife trade; transportation is inconvenient, the economy is undeveloped, law enforcement is weak, and wildlife management staff are few. traditionally, hunting was a main source of income for local residents. with the border trade opening and more frequent cross border trade, illegal wildlife trade is increasing, posing a serious threat to the species which can easily be transported across the international boundary (lau et al. ; li and li ) . poaching and smuggling in qinghai and tibet is also a serious problem. local residents heavily depend on natural resources for their livelihood, but lack awareness of the need to protect these resources. local government authorities lack efficient wildlife management and law enforcement capacity and are in need of training (wan ; lee et al. ) . over recent years, people's demand for wildlife has grown in some of china's developed cities, especially coastal cities along the border. as in other regions of china, eating wildlife has become a fashionable lifestyle and symbol of elite status. regions and routes (see fig. ( ) a public poll on wildlife consumption and protection awareness was carried out in shanghai, guangzhou, beijing, chengdu and kunming. ( ) interview the study used a structured questionnaire and face-to-face interviews in beijing, shanghai, guangzhou, chengdu and kunming, with at least successful samples per city. interviewees included local self-identified animal lovers, former hunters, high school biology teachers, rangers, herb collectors, field research staff, nature reserve and forestry center staff and urban residents. questions focused on the change in numbers and species of wildlife, as well as threats (special emphasis was given to the situation facing key species like tiger, bear, snakes and turtles etc.). the total valid sample size of the interview is , individuals. ( ) field and market investigation the investigation was carried out by: observing the animals and their tracks, including footprints, footprint traces, lying traces, feathers, hairs, feces and other marks; investigating the living condition and threats wildlife are facing; recording the quantities and species of the animals; visiting markets and talking to the dealers; and recording the species and volumes of endangered and rare wildlife in trade. ( ) information gathering information was gathered by visiting customs and entry-exit inspection and quarantine bureaus, referring to case records, collecting historical data, and investigating the historical situation of wildlife and changes in quantity by talking to local residents and specialty shops. posing as interested customers or dealers, we also collected information on overall trade, the source of wildlife for the trade and transportation routes. ( ) sampling in this survey, we used multi-phase random sampling methodology, and followed the order of ''overall neighborhood committees-neighborhood committee samplehousehold sample-individual'' to randomly filter the households. if this sampling method resulted in selection of an unqualified respondent in the target household, another qualified respondent was substituted. if there was more than one qualified respondent in a household, only one was interviewed, in accordance with the kish sampling method. ( ) urban residents ( ) shopping centers, restaurants, open markets, customs, ports, forestry bureaus, forest police, bureaus of foreign trade and border trade, administrations for industry and commerce, administrations for wildlife entry-exit inspection and quarantine etc. beijing, shanghai, guangzhou, kunming and chengdu are five representative chinese cities with high population density and high wildlife consumption levels. we requested a consulting company to conduct research by using a questionnaire to address the following issues: . the current attitude of chinese urban residents towards wildlife consumption, the kinds of wildlife people think can legally be consumed, and factors influence on the consumer's attitude. . the current situation includes four types of consumer behavior among chinese urban residents: using wild animals as food, using medicine or tonic products containing wildlife ingredients, wearing ornaments and garments made from wildlife, and keeping wildlife as pets. in understanding behavior, it is essential to consider level of consumption, consumption channels, species and motives so that we can understand the wildlife consumption better and find ways to improve the law enforcement, as well as public awareness and education. . characteristics of wildlife consumer groups among chinese urban residents. . chinese urban residents' awareness of wild animal protection, the attention they pay to wildlife protection efforts and their willingness to participate in conservation. by conducting the survey on public awareness of wildlife consumption, we hoped to be able to provide government authorities and ngos with useful information to enhance the management of the unregulated market and help combat illegal wildlife trade. we also hoped that it would assist the government in adopting methods to educate the public, promoting wildlife conservation awareness, establishing the concept of protection, and ultimately reducing and eliminating illegal commercial exploitation and trade. current situation of wildlife trade in key regions . trading sites. the main sites for wildlife trade are ports, border markets, and markets and stores in cities. there are ports of entry in yunnan, among which are road ports (foreigners are allowed to go through ruili, mohan and hekou ports by railway), are air ports (foreign flights are allowed to land in kunming international airport and xishuangbanna international airport), and are water based (foreign ships are permitted to go through jinghong and simao ports). in addition, there are border channels and border markets, including, along the border with burma, dehong prefecture with road ports (houqiao, ruili and wanding), provincial ports in longchuanzhangfeng and yingjiang, and ferries and roads and channels with border markets. along the border with laos, there are ports (of which honghe county of honghe prefecture is the largest), provincial ports, over ferries and numerous informal channels. in guangxi counties share a border with vietnam, with border ports of entry, among which dongxing, pingxiang, youyiguan, shuikou and longbang are national ports. along the border with vietnam, border markets are connected by road to these ports and other trading points. dongxing, puzhai and nongrao-nonghai are the three most important border trade ports. nanning, as the capital of guangxi, is one of the largest international wildlife trading hubs in china, also acting as a market for trade in domestic wildlife resources illegally harvested from within the province and yunnan. qinghai does not share a border with any of china's neighbors, but the province itself is rich with wildlife resources and trade evident in the numerous markets, drug stores, specialty stores and breeding farms within the cities in qinghai. xining is the biggest city within qinghai for trade transactions and consumption of wildlife resources. geermu is also a major trading spot for species traded within the province or coming from tibet and xinjiang autonomous region, especially for the skin of tibetan antelope. huangzhong county draws tourists, both foreign and domestic, and is also a major hub for trading wild animal skins and crafts. . traded species. the six taxonomic groups found in this study to be affected by trade in china include (species totals are in parentheses, in total): insects ( ), fish ( ), reptiles ( ), amphibians ( ), birds ( ) and mammals ( ). reptiles and mammals are the most prevalent species in wildlife trade ( confiscated over , wild animals, , kg of wildlife products and wildlife skins. at a pet market in kunming, investigators found more than turtles and birds as well as , pieces of wildlife products waiting for sale. in guangxi province, most wildlife trade takes place along the border area. for example, the number of specimens found in pingxiang city included , - , tokay geckos, - , asian water monitors, , - , pangolins, - tons of snakes, - tons of frogs, - tons of turtles, - tons of soft-shelled turtles, and - tons of birds of prey each year. in dongxing city, wildlife trade each year amounts to , - , snakes, , - , tons of frogs, , - , turtles, - , raptors, and , - , tokay geckos. in addition, investigators found frogs, , turtles, soft-shelled turtles, , snakes, asian water monitors, raptors, over small mammals, , wild birds and reptiles in the pet markets in nanning. in qinghai province, a wildlife trade survey was carried out in xining city, germu city and huangzhong county while customs' data would indicate that wildlife smuggling is gradually decreasing, the government's data on confiscated wildlife indicates otherwise, signaling that the majority of smuggling is occurring in underground and black markets. . trading purposes. wildlife trade is driven by a multitude of markets including: • food, such as snake and monkey, most of which can be found in the market as live animals or animal parts; • medicine and tonic products, such as tiger bone, bear bile, or deer horn, most of which can be found as animal parts in the drug store or supermarket; • crafts and souvenirs, such as ivory and antelope skull, most of which can be found as animal parts in the craft store, gift shop or open market; • garments and decoration, such as tiger skin, crocodile skin, and tibetan antelope wool, most of which can be found as animal skins in the market or port; and • pets, like turtles, lizards, and blue peacocks, most of which can be found as live animals in the market. ( ) sources wildlife is mainly coming in from border markets such as vietnam, laos and myanmar. some animals coming through this route include the hieremys annandalii, cuora amboinensis, indotestudo elongata, aspideretes hurum, varanus salvator, enhydris bocourti, manis javanica. from other provinces within china, such as yunnan, guizhou, sichuan and guangdong, species traded include elaphe taeniura, ptyas korros, erinaceus europaeus, passer montanus, and many species of pheasants, hawks and owls. wildlife traded mainly in local markets includes cervus elaphus (qinghai), trimeresures stejnegeri, rana guentheri, gallicrex cinerea, and lepus capensis (guangxi). breeding facilities also exist, the largest being in baoshan. species commonly found in farms include pavo cristatus (yunnan-for domestic sale) and pelodiscus sinensis. wild caught nycticebus intermedous, naja naja and manis javanica are also smuggled from vietnam into yunnan but declared as captive bred animals. ( ) routes ruili city of dehong prefecture is the largest trading port of entry between china and myanmar, and it represents more than half of the total import and export trade volume in yunnan. unlike guangdong, yunnan has more convenient road transportation and longer shared borders with neighboring countries. this means that many smugglers prefer to trade along the borders, instead of through customs, making it even harder to regulate and monitor the illegal trade. legal wildlife trade in yunnan mainly comes from river ports in eastern yunnan. guangxi benefits immensely from wildlife trade. as wildlife in guangdong is expensive in comparison to other regions, most of the imported wildlife in guangxi (about %) is traded to guangdong and the rest goes to nanning, then liuzhou. qinghai is rich in wildlife resources, so most of the wildlife traded there is sold locally or to nearby provinces (fig. ). . government trade control agencies. government agencies with a role in wildlife trade management include customs, the state forestry administration, the bureau of ( ) customs customs is responsible for inspecting imported and exported goods, managing and monitoring wildlife trade, overseeing wildlife smuggling cases, within the customs inspection area and the coastal area near customs, and confiscating wildlife during smuggling and illegal importation. ( ) state forestry administration in terms of import and export control, the forestry bureau has the right to monitor the implementation of the law of the people's republic of china on the protection of wildlife and other related regulations. according to the ''specific permit law,'' the forestry bureau can authorize or submit import and export applications to higher level authorities for authorization, and the bureau has the right to handle cases involving forging, cheating, or transferring import or export permits. the convention on international trade of endangered species of flora and fauna (cites) is enforced by offices around the country under the management of the china national management authority (cnma) and the forestry bureau. they are responsible for the monitoring of cites implementation on wildlife trade, authorizing permits and certificates, assisting relevant law enforcement bodies in illegal importing or exporting cases, rescuing and resettling confiscated live wild animals, charging wildlife import and export management fees based on relevant regulations, and organizing the training or publicity on cites and the related domestic laws. this ministry is responsible for the registration and management of import and export companies, foreign investment, international economic and technical cooperation, and port development and management. ( ) ministry of police the police bureau, particularly the administration of forestry police, is responsible for managing state protected wildlife trafficking occurring outside of customs inspection areas and coastal area near customs. the armed police bureau and the police bureau inspect people going through customs to check whether they are carrying prohibited goods. if prohibited wildlife is found, offenders are transferred to forestry police and customs for further legal procedures. this administration is responsible for the management of domestic markets, protection of wildlife species, monitoring the market selling of livestock, marine animals, flowers, medicine and wildlife, and jointly working with the forestry bureau to regulate the market. confiscated wildlife or smuggling cases identified by this administration are handed over to the police bureau. ( ) bureau of fisheries under the ministry of agriculture the bureau is responsible for the implementation of state laws and regulations on oceans and fishery management, as well as for related international conventions. this means they control the overall management of china's ocean space, including ocean environmental monitoring, aquatic animal conservation, fishery industry administration, the fishery industry business permit system, and other fishing activities, such as processing and distribution. current situation of wildlife consumption in the market ''should wild animal consumption be allowed?'' ''what kind of wild animals can be used for consumption?'' through urban chinese residents' answers to these questions, we can assess general attitudes towards wildlife consumption that the first group is explicitly opposed to wildlife consumption, and they hold ''pure protection'' (pp) viewpoint; the second, third and fifth groups are subject to the influence and inducement of various kinds of factors, and thus they may be divided into an interest-driven group-the ''conditional utilization''(cu); without misgivings about wildlife consumption, the fourth group holds the ''pure utilization'' (pu); those who are not sure about their attitude belong to the ''vague'' cognition group (see table ). attitudes regarding wildlife consumption. thus, our analysis reveals that at present . % of urban residents agree with the cu cognition, compared to . % with pp cognition; pu group and vague group account for . % and . % respectively. attitudes towards wildlife consumption varied between cities ( table ). the result showed that . % of residents in guangzhou agreed with the cu cognition, together with the pu group ( . %), the percentage of wildlife utilization group was significantly higher than that of the other four cities. consumption levels. we divided the wildlife consumers into three groups: light consumption group ( times or below per year), medium consumption group ( - times per year) and heavy consumption group ( times and more per year). there is distinct difference among cities in the level of consumption of wildlife. heavy consumption is by far higher in beijing ( . %), guangzhou ( . %) and shanghai ( %), than in chengdu ( . %) and kunming ( %). medium consumption is much higher in shanghai ( %) and guangzhou ( . %) than in other cities. among , respondents . % did not eat any of the species listed. animals that were consumed by more than % of respondents in the past year include quail ( . %), ring-necked pheasants ( . %), hares ( . %), frogs ( . %) and snakes ( %). where is wildlife consumed. among the places where wild animals are consumed by chinese urban residents, common and high-grade restaurants and hotels account for % and % respectively. the purchasing of wild animals at vegetable/non-staple/flea markets ( . %) and supermarkets ( %) is also high. why do people consume wildlife. more than % of wildlife consumers said they consume wildlife because they find the taste delicious. those who tried wild animals because they felt they were rare represent . % of the surveyed, while . % of people indicated they tried wildlife out of curiosity. those who tried wild animals for nutritional and nourishment purposes accounted for . %. about . % of respondents said they eat wild animals at dinner parties. about . % of consumers ate wild animals because they were served to them by others. geographical differences on wildlife consumption. there is a marked difference between consumers from different cities in the degree of wild animal consumption. the ''heavy consumption'' group is higher in beijing ( . %) than in other cities; the ''medium consumption'' is higher in chengdu ( %) and guangzhou ( . %) than in other cities; and the ''light consumption'' is highest in kunming ( . %). major wildlife products in consumption. the wildlife breeds or products selected in this survey consist of species of wild animal and seven breeds of wild plants. among the , respondents: • . % said they had not eaten any of the breeds mentioned on the list in the past year. • sinkgo ( . %), snake gall ( . %), cordyceps sinensis ( . %), musk (moschus spp.) ( . %), snow saussurea (saussurea spp.) ( . %) and snake oil ( . %) were all heavily consumed in the past year. • of the people who purchased medicine or health products containing wildlife ingredients: - . % of consumers purchased them at pharmacies, - . % purchased from supermarkets, and - . % purchased from general stores - . % used wildlife medicine or health products given to them by others (higher than the percentage of those who ate wild animals as food given by others ( . %)) - . % think medicine and health products containing wildlife ingredients promote health by increasing nutrition and nourishment, . % think such medicine and health products have special curative effects, and . % say that they have no other choice because the medicine they need contains these ingredients. in this survey, we listed wildlife products for respondents to identify. according to the results, %, or %, of the total respondents had articles made from wildlife (see table ). thirteen respondents said they were not sure about or refused to answer their consumption frequency. on average, the wildlife product owned had been made within the last years ( respondents). ninety seven percent of the respondents did not own any of the articles made from wild animals on the list in the past years. among the people who had articles, the percentage of the ownership of ivory and marten products were highest. in the survey, of those with wildlife products, . % of the ornaments and readymade products owned by the respondents were given to them by others, . % were purchased from leather and wool product stores, % were purchased from stores, . % were purchased at general ornament stores, . % at tourist stores or markets, and % were purchase in flea markets. in the survey, . % of the respondents believed that ornaments and ready-made clothes made from wildlife were beautiful. those who owned these products out of curiosity account for . %; those who regard it as a fashionable lifestyle make up . %, and those who think it shows their distinctive taste and status account for . %. on the survey, we also listed species of wild animals that can be raised in captivity, a total of respondents have raised the wild animals on the list, accounting for . % in the total (see table ). in terms of the average number of wild animals raised by residents in the cities, beijing ranks first, and shanghai comes second. among , total respondents, those who did not raise any of the species listed account for . %. however, tortoises, mynah, parrot, sparrow and thrush are the most common species raised by people. respondents who purchased wild animals from pet markets account for . %, those who adopted wild animals or accepted wild animals given by others account for . %. a percentage of . % of the respondents raise wild animals because they have fun in doing so, and over % raise wild animals for aesthetic purposes. what drove respondents to start wild animal consumption? among the respondents who have consumed wild animals, . % purchased wild animals or products recommended by their relatives and friends, . % consumed wild animals given to them by others, . % purchased wild animals after watching advertisements on the products or publicity materials on the species, . % purchased wildlife products recommended by professionals (like traditional medicine practitioner), and . % were unsure of how they started wild animal consumption. among those who have consumed wild animals, . % do not know whether they have consumed captive bred or wild animals, % know the animal they consumed was captive bred, . % know that they consumed both captive-bred wild animals and real wild animals, and . % know that what they consumed are real wild animals. among respondents who have consumed wild animals, . % prefer real wild animals, . % prefer captive-bred wild animals and . % do not have a preference. preference varies by city: the percentage of consumers in guangzhou who prefer wild animals ( . %) is slightly higher than that in other cities, followed by the percentage in kunming ( . %) and shanghai ( . %); the percentage of consumers who prefer captive-bred wild animals is the highest in beijing ( . %), and the percentage in chengdu ( . %) comes second. from this survey, of the , respondents had never consumed wild animals. among them, those who regard wild animal consumption as uncivilized behavior account for . %; . % think it is unhealthy and may cause infection of diseases; . % believe wild animals should be conserved and they are not for consumption; . % think it will destroy the ecological environment and it is not conducive to environmental protection; . % have no particular preference for wild animals; . % have not consumed wild animals because they could not afford it, and . % have not because they had no opportunity for consumption. the survey findings show that at present, . % of chinese urban residents believe ''all wild animals should be protected'' (protection group), . % think ''some wild animals should not be conserved because they carry viruses or bacteria that human beings are susceptible to and they may communicate the diseases to human beings'' (middle group ), . % think ''it is not necessary to protect wildlife, which is a kind of resource and valuable to human beings, and has strong ability to reproduce and survive'' (utilization group), and . % think ''wild animals that pose a threat to human beings' safety, like jackals and wolves, should not be protected'' (middle group ). those who are not sure about this issue account for . %, and respondents refused to answer this question, taking up . % in the total number of respondents. interestingly, the distribution of the four groups varies significantly from one city to another. the protection group represents the majority of respondents in kunming ( . %), beijing ( . %) and shanghai ( . %). the middle group and takes up a higher percentage in guangzhou ( . %) and chengdu ( . %). the utilization group accounts for a higher percentage in chengdu ( %) than in the other four cities. in this study, we listed species of heavily consumed wild animals under priority protection in china for respondents to identify. the results show that for the majority of the species, the protection group rate is below % among chinese urban residents (table ) . again, there is a marked difference in cognition levels of residents living in different cities. residents in kunming have the highest cognitive level regarding protected wildlife, beijing is second, shanghai and chengdu rank third, and the lowest is in guangzhou. how do the chinese regard the relationship between wild animals and human beings? in this survey, . % of the respondents see wild animals as serving a companion role for human beings, . % think wild animals are equal to human beings and both deserve protection and respect, % regard wild animals as good resources and believe the purpose of wild animal conservation is to allow better utilization by human beings, . % think wild animals and human beings belong to two different worlds, . % see no relationship between wild animals and human beings and think they are lower than human beings, and . % think wild animals pose a threat to human lives. at present, animal welfare is a popular topic in china. when asked about this issue, nearly % of urban respondents think improved animal welfare is related to societal development and . % say there is a close relation. meanwhile, . % of the respondents say they are not sure about this issue, and . % see no relation between the two. how many and why chinese people are concerned with wild animal protection work now will affect what methods are used for future intervention. more than % of chinese urban residents are supportive of wildlife conservation; those holding a negative attitude account for . %. residents of beijing are most supportive of conservation efforts ( . %), followed by shanghai and guangzhou. among respondents who explicitly noted that they were not concerned about wildlife conservation, . % say they have no personal connection to wildlife, . % believe conservation should only be through the government, . % believe any effort they make will have little impact, . % feel there are too few people involved in conservation for it to make a difference, . % of them regard the work a waste of time and money, . % of them think it unnecessary to protect wildlife, and . % indicated no specific reason for not being concerned with conservation. most urban residents have an accurate understanding of the severe situation faced by wildlife in china. in this survey, . % of the respondents are positive about the issue and believe that wildlife could avoid extinction through conservation measures, and . % of them are negative about this issue, believing that, considering the current situation, species extinction is unavoidable. in , china promulgated the law on wild animal protection, which stipulates a list of wild animals under priority protection in china. the list contains about species of wild animals under first-level state protection and more than species under second-level state protection (http://www.sepa.gov.cn/natu/swdyx/swwzzybh/ /t _ .htm). as its supporting statute, the implementation rules on the protection of land wild animals of the p.r.c., promulgated in , claims that the legal system for wild animal protection with the law on wild animal protection has been firmly established. yet, in this survey, as many as . % of the respondents are unaware of the two statutes, . % know about the law on wild animal protection, and . % know about the implementation rules on the protection of land wild animals. at present, despite these legal provisions, unlawful consumption of wild animals still exists in china. in this survey, . % of respondents impute it due to poor enforcement, . % think the law itself is lack of details on regulating the wildlife consumption that results in rampant unlawful consumption, and . % hold that the sanctions imposed by law are not stern enough, which is why the law does not truly play its role of prohibiting unlawful behavior. meanwhile, % believe that many people do not know the existence of relevant statutes, due to inadequate efforts made toward publicity of the statutes, . % think that money is more powerful than law, and that the rich can access any product they desire despite illegality, and . % maintain that traditional notions in the chinese society have significant influence on people's behavior, and it is difficult to change in a short time. at present, respondents indicated the following actions as urgently needing to be taken: • publicity efforts to heighten public awareness of wild animal protection ( %), • relevant statutes formulated and the legal system improved ( . %), • punishment on the persecution of wild animals should be increased ( . %), • cultivate public awareness of environmental protection ( . %), • tighten regulation and control over wild animal trade and transportation ( . %), • strengthen education on wild animal protection among children and adolescents ( %), • devote additional funds to wild animal protection ( . %), • improve the professional quality of foresters and law enforcement personnel in the market ( . %), • support more wild animal protection organizations ( . %). of information chinese urban residents have been exposed to in the last months, the top three types are wild animals in trade ( . %), photographs of wild animal hunting ( . %) and documentaries on wild animals' lives ( %). in additional, people also indicated learning about wildlife conservation ( . %) and the spread of diseases from wild animals to human beings ( . %). dissemination of information most commonly comes through tv and rarely through publicity activities or other community programs. for information on wild animals, the percentage of those who pay attention to visual information is much higher among heavy consumers than among light and medium consumers. a high percentage of medium consumers pay attention to non-visual information. among the various types of public benefit activities concerning wild animals, people are most willing to take part in publicity activities on wild animal protection ( . %), and a high percentage of people are interested in ecotourism ( . %). regarding consumer behavior, the respondents explicitly noted that they would abstain from purchasing wild animals ( %), using medicine or health products containing wildlife ingredients ( %), or acquiring cosmetics containing wildlife ingredients ( %). less than a quarter of respondents would be willing to provide monetary support for improving conditions of zoo animals or supporting wildlife conservation organizations. in this study, respondents noted that they had no consumption experiences involving wild animals in the past years, accounting for . % in the total number of respondents ( , ). among the respondents who had wild animal consumption experiences, three respondents consumed wild animals other than the species we listed in this survey; respondents consumed the species listed but were not sure about their consumption frequency; respondents consumed the species listed and were clear about their consumption frequency. among the respondents who have had wild animal consumption experiences, light consumers account for . %, medium consumers . %, and heavy consumers . %. among the five cities, the percentage of light consumers is the highest in kunming and chengdu, and the percentage of heavy consumers is the highest in beijing and guangzhou. the percentage of medium consumers is higher in beijing than in other cities. further analysis reveals some difference between these consumer groups in sex, age, income and occupations. the heavy consumption group ( samples in total) consists largely of young men ( individuals) who were under year old, many of whom are physical workers, students, self-employed and freelancers, with relatively good education and high monthly household income. the majority of them have a monthly household income of above rmb , . there is significant variability between different consumer groups in understanding the scope of wildlife under protection. the percentage of consumers with the pure protection cognition type is higher among light consumers and medium consumers than among heavy consumers; the percentage of those holding the conditional utilization cognition is the highest among heavy consumers. in summary, people's knowledge about state-protected wildlife does not directly influence their consumer behavior. consumption level is proportional to cognitive levels. the percentage of those with high cognitive level is the highest among heavy consumers. among residents with high cognitive level, the percentage of those who have consumed wild animals is equal to the percentage who has never consumed wild animals. a higher percentage of residents with low cognitive level have not consumed wildlife. the majority of light consumers began consuming animals on their own accord, while the majority of medium and heavy consumers involuntarily began consuming animals (e.g. pressure from family, coworkers or items given as gifts). heavy consumers are more concerned about the place of origin of wild animals compared to the other consumers. they generally know whether they have consumed wild or captive bred animals, and have a stronger preference towards wild animals than the other consumer groups. wildlife trade in key regions continues to be very active because of a continuous supply of wildlife resources and large market demand for wildlife goods. our survey statistics show that as time passes, however, there is a gradual trend toward a reduction in total wildlife trade for the following reasons: ( ) due to heavy cross-border poaching, wildlife resources have been greatly reduced, with some species even facing extinction. ( ) in recent years, the government has strengthened law enforcement and the wildlife trade control system. as a result, illegal trading is riskier, causing some traders to leave the trade. ( ) after sars in and bird flu in early , previously legal wildlife was banned for import, and the general public began becoming aware of the negative impact wildlife could have on their health. though smuggling still exists, the trade amount was reduced. recommendations: . establish a wildlife trade control & monitoring network. it is difficult to identify the numerous wildlife species and their products in daily trade control and legal procedures, and, due to language barriers and politics, it is hard to compile evidence for foreign crimes. the handling of confiscated live wild animals is also a problem. enforcement officers often release confiscated animals anywhere, without any consultation from wildlife experts. this improper release can directly affect the life of local people and the ability of the animals to survive. stringent measures must be taken to ensure that an effective trade control system is established to prevent the deficiencies in management of wildlife trade and ensure adequate enforcement so illegally traded animals cannot enter the market. the cites management authority should work with customs and the forestry bureau to set up a joint network and obtain greater support from international organizations to conduct long-term wildlife trade control and monitoring in the border regions, develop a wildlife import and export database, and give feedback on natural resource utilization. . enforcement capacity building. most of the villagers in the border regions are minority groups with their own customs and culture. there are very few wildlife conservation publicity events or education materials (such as video or audio) in their villages. there are no regulations for wildlife import and export control. limited by the slow economic development and the limited amount of education received, local governmental decision makers often feel there is no alternative but to consume the natural resources. it is necessary to educate the public on the relationship between global environmental conservation and country and local economic development, as well as short and long term potential economic benefits associated with wildlife protection. the improvement of statutes and law enforcement should not be neglected. the survey shows that people regard lax enforcement, poor operability of statutes and too lenient sanctions on violations as the main causes for the rampant unlawful wild animal consumption. even heavy consumers of wildlife recognize that improvement and enforcement of statutes and punitive measures would have great influence on their practices and reduce demand for wildlife consumption. for improvements to be made, it is essential that cites training and education programs focusing on techniques for trade control, cites regulations and species identification are delivered to law enforcers, including those from forestry bureau, forestry police bureau, customs, and administration of industry and commerce. in addition, government agencies and international ngos can work jointly to promote information exchange and cooperation between border countries. as was found in this investigation, local networks, government agencies and ngos have been established to mitigate trade but enforcement is lacking. considering the current heavy wild animal consumption, it is difficult to be too optimistic in this survey, we found that . % of residents had consumed wild animals. considering that people may conceal their actual consumption behaviors when answering our questions, we adopted aversion techniques in designing the questionnaire. however, techniques can only reduce the intervention of subjective factors to some extent. therefore, the percentage of wild animal consumers in real life is assumed to be higher than the data suggest. even thought the data likely underestimate the problem, the actual figures obtained still reveal a desired approach to wild animal consumption among chinese urban residents. those who consume wildlife regularly are the largest group, accounting for . % of the total. what is more worrying is that . % of residents who consume wildlife voluntarily started to buy wild animals due to word of mouth, media or the influence of professionals. in the analysis of different groups' attitudes towards wild animal consumption, we find that a high percentage of those who have never consumed wild animals hold the ''utilization value theory,'' and the percentage of those holding ''vague cognition'' is even higher among those who have never consumed wild animals than among wild animal consumers. given that chinese urban residents' wild animal consumption is affected to a great extent by community views and pressure, in the future it is possible that more people will begin wild animal consumption due to societal influences. considering the current situation, intervening now, before more people become wild animal consumers, is very important, as changing consumer behavior once it has begun is much more difficult. less than % of chinese urban residents hold the correct attitude toward wild animal consumption. consumer attitude is subject to various factors, and cognitive level does not play a decisive role people's attitudes influence their behavior. in this survey, we found that only . % of chinese urban residents think no wild animals should be consumed. consumer attitude is subject to various factors, and cognitive level does not play a decisive role. first we examined people's views regarding the scope of wild animals that should be protected. those who hold the view that ''all wild animals should be protected,'' were more likely to not be supportive of animal consumption. this would indicate that to some extent people's attitude toward wild animal consumption is directly proportional to their cognitive level. second, through a comparative study on consumer attitude of groups with different demographic background, we find that well educated groups have high cognitive level of wild animal protection. yet people's attitude towards wild animal consumption is not directly proportional to their educational level. among groups with different educational background, the percentage of those who could correctly identify the listed protected species is the highest among two-year college graduates, followed by high school and junior high school graduates. however, those who are not necessarily in disagreement with wildlife consumption are more likely to hold at least a bachelor's degree, and likewise the percentage of those following the ''utilization value theory'' is higher among those with bachelor's degree or above ( . %) than among those with less formal education. among groups with different income levels, the percentage of those holding the ''utilization value theory'' is higher among those with monthly household income above , yuan than among other groups. this indicates that, to a certain degree, financial strength increases people's propensity to consume wild animals. third, there is marked difference between groups with different consumption levels and their motives for consumption. for example, the behavior of eating wild animals: a greater proportion of heavy consumers ( . %) eat wild animals because they find them delicious. the percentage of those holding the utilization value theory is much higher among heavy consumers than among the other two groups. on the other hand, a higher percentage of light consumers tried wild animals out of curiosity. due to traditional notions, heavy consumers hold the ''utilization theory,'' and they also have fixed consumption behavior because of the preference for wild animals. this poses a considerable obstacle to intervention efforts. wild animal consumption has distinct regional differences and group features ( ) regional differences. when looking at consumer behavior, consumption degrees, consumption channels and consumption motives, different cities have different consumption features. for example, the behavior associated with eating wild animals: a high percentage of residents in shanghai and guangzhou eat wild animals at popular restaurants and purchase wild animals from markets to cook at home, and the percentage of heavy and medium consumers in guangzhou is higher among residents in these two cities than in other cities (the percentage of heavy consumers in guangzhou is second only to that in beijing). for consumption motives, taste and nourishment are the two primary motives for eating wild animals among residents in shanghai and guangzhou. this indicates that eating wild animals has become a habitual consumption behavior among residents in shanghai and guangzhou. among the five cities, the percentage of residents in beijing who eat wild animals at high-end restaurants and hotels is highest, as is the percentage of heavy consumer and light consumer residents. most people consume wild animals out of curiosity, with the idea that wild animals are rare. for initial consumption motives, a major percentage of people involuntarily started wild animal consumption (i.e. it was given to them by friends, family, or colleagues). this indicates that wild animal consumption has become part of the lifestyle of some high-income people in beijing; it also indicates the influence of peers and group pressure on consumer behavior and consumption methods. it is possible that without effective control and intervention, many consumers will become medium and heavy consumers. according to the current trend, beijing is likely to become a swing consumer city. ( ) group features. we divided the consumers into groups depending on their consumption degree. in terms of consumer attitude, the percentage of those with the correct attitude toward wild animal consumption is lower among heavy consumers than among light consumers and medium consumers. with regard to the viewpoint regarding the relationship between human beings and wild animals, the percentage of those holding the ''utilization view,'' ''unrelated view'' and ''threat view'' is higher among heavy consumers than among light and medium consumers. interestingly, we find that although the percentage of those with the correct cognition is higher among light consumers and medium consumers than among heavy consumers, heavy consumers' cognitive level of state-protected wild animals is the highest among the three groups. this indicates that in terms of intervention efforts, strengthening education among heavy consumers is not necessarily effective. for consumption preference, the percentage of those who know they have consumed wild animals (as opposed to farmed) and the percentage of those who prefer wild animals are higher among heavy consumers than among other groups. this indicates that the group has very high awareness of their consumer behavior. regarding wild animal protection work, the percentage of those not supportive is much higher among heavy consumers than among the other two groups. meanwhile, the percentage of those regarding conservation as the government's responsibility and the percentage of those who think few people are concerned about this issue are much higher among heavy consumers than among the other two groups. the percentage of those who do not know about the two statutes on wild animal protection is higher among heavy consumers than among light and medium consumers. the percentage of those who know about the two statutes is the highest among light consumers. for information on wild animals, the percentage of those who pay attention to visual information is much higher among heavy consumers than among light and medium consumers. with regard to the willingness to participate in public benefit activities for wild animal protection, a high percentage of heavy consumers are willing to take part in ecological tourism and provide monetary support, but the percentage of those willing to change their consumption behavior is lower than the other two groups. a high percentage of medium consumers are willing to take part in publicity activities, and a high percentage of light consumers are willing to abstain from wild animals in food, medicine or health products containing wildlife ingredients. this further indicates the importance in intervening in consumer behavior before it become habitual, as in the case of heavy consumers. merely underscoring the threat posed by wild animals to human beings' health (taking sars for example) does not help. it is necessary to use both hard and soft tactics in behavior intervention since the outbreak of sars in , there has been greater focus on human health in the discussion of eating wild animals. animal medical experts point out that wild animals such as primates, rodents and ungulates share more than diseases with human beings (karesh et al. ) . even with warnings from various consumer associations and animal protection organizations, diseases continue to spread. recent studies have identified the chinese horseshoe bat as the natural reservoir of the coronaviruses from which the sars viruses that infected humans and civets likely emerged (lau et al. ; li et al. ) . in july , academics from the chinese academy of sciences jointly called for efforts to be stepped up for resources supporting wildlife conservation, improving statutes on wildlife protection, and establishing healthy diet views. sudden threats of disasters like a widespread disease certainly affect people's lifestyle and behaviors quickly, but it is not feasible to expect such temporary impact to fundamentally change the deep-rooted motives and notions formed by long-term social customs and traditions. in this study, we find that merely educating people on the threat posed to their health by wild animal consumption cannot fundamentally change people's attitude towards wild animal consumption. it can only heighten, to a certain extent, people's hostility toward wild animals, and cannot engender a conservation-oriented view towards wild animals. in comparing residents in different cities in terms of their understanding of wild animal protection, we find that the percentage of guangzhou residents, who were the first to receive the impact of sars, hold the ''threat theory'' at the highest; while their cognitive level about protected wild animals is the lowest. among the four groups divided by the viewpoints regarding the scope of wild animals that should be protected, the percentage of those holding the ''utilization value theory'' of consumption is comparable to those holding the ''threat theory'' and the ''utilization value theory.'' in other words, the view that wild animals may spread diseases to human beings or pose a threat to their lives does not reduce peoples' propensity to utilize or consume wild animals. therefore, in conducting behavior intervention we not only need to consider differences between consumer groups in cognition, attitude and behavioral features, but, more importantly, various measures should be taken to produce changes in human behavior. the key to publicity and education should be increasing knowledge. in this survey, we find that currently a high percentage of chinese urban residents is still not clear about what wild animals are protected. undoubtedly, this poses a major obstacle to regulating consumer behavior. due to the pertinence of people's cognitive level and their attitude toward wild animal consumption, it is a crucial task to cultivate the correct cognition among general residents through publicity and education. for widespread change, it will be necessary to combine the strategies of publicity and education. through the analysis of the cognition of the scope of wild animal protection and consumer attitude, a high percentage of the ''threat theory'' and ''utilization theory'' groups adopt the consumption attitude of ''utilization value.'' meanwhile, the percentage of those with the incorrect attitude toward wild animal consumption is highest among the ''utilization theory'' group. this indicates the strong influence the utilization theory has on people's behavior. therefore, publicity and education should be aimed at changing this mentality and making it clear that all wild animals should be protected, which will certainly take a long time. we may also utilize other publicity strategies suited for specific stages/groups of consumption. for example, in analyzing reasons for not consuming wild animals, we find that a high percentage of people have never consumed wild animals because they regard the behavior unhealthy or they do not like it. different groups have different reasons. the percentage of those who regard it as unhealthy is higher among people aged - than among other age groups. a high percentage of people aged - and - do not like it. the percentage of those who think it is not right to consume wild animals is far lower among people with primary school education and below than among other groups. a high percentage of junior high school and high school graduates think it unhealthy, while a high percentage of those with bachelor's degree or above do not like to consume wild animals for different reasons. this indicates that we may guide the audiences by cultivating healthy consumption notions using various approaches in a short time. for example, we may educate young people not to eat wild animals and encourage them to abstain from wild animals early on. meanwhile, we may imbue middle-aged people and those with low education with the view that wild animal consumption is not healthy. of course, as stated earlier, even if we guide the audiences with the ''unhealthy'' view, it is also necessary to popularize correct knowledge and conduct education. recommendations: . one major strategy should be to prevent wildlife from reaching the markets. through this survey, it was found that villages along the border act as temporary stops for smugglers. if paid, villagers will often help the smugglers to escape, which strongly increases the difficulty in seizing criminals. a program needs to be implemented that targets key villages and simultaneously provides community development and biodiversity education projects to arouse their environment protection awareness, develop the local economy and convince villagers of the value of working together to stop smuggling. . as demonstrated by the survey results, heavy wildlife consumers are often those who are young, well-educated, and have a high income. without correct knowledge and understanding of wildlife consumption and protection, university students, as the next potential consumers, are the main target to be educated. schools are good places for knowledge input. as a starting point, education programs should be developed for university students and then move forward from there. biodiversity working group of china council for international cooperation on environment and development, bwg/cciced ( ) the rd annual report to china council for international cooperation on environment and development the fate of wild tiger wildlife trade and global disease emergence severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats wild animal trade monitoring at selected markets in guangzhou and shenzhen, south china guide book on wildlife import and export management a survey on wildlife trade in guangxi and guangdong bats are natural reservoirs of sars-like coronaviruses the bear facts: the east asian market for bear gall bladder. trafffic, cambridge morgan d ( ) chinese medicine raising wildlife concerns unpublished report on wildlife trade and enforcement in qinghai province sain-ley-berry c ( ) new campaign launched to fight illegal wildlife trade wildlife trade management and enforcement. china forestry publishing house status of live wildlife trade in yunnan border area souvenir alert highlights deadly trade in endangered species breeding techniques for turtles trade of wild animals and plants in china-laos border areas: status and suggestion for effective management captive breeding of economic wildlife acknowledgements we are grateful for the grant from critical ecosystem partnership fund (cepf) to support this project. we thank the support of cites management authority of china, and its kunming, guangxi and sichuan branch offices. we are grateful to chun li, fang zhou, and all participants in the field trade survey, and the horizonkey information and consulting co., ltd. who conducted and implemented the public awareness survey for this project. we are also grateful for xue wang, stacy vynne, clare sierawski and chantal elkin for reviewing the english language of the manuscript. key: cord- - g dk authors: aguirre, a. alonso; catherina, richard; frye, hailey; shelley, louise title: illicit wildlife trade, wet markets, and covid‐ : preventing future pandemics date: - - journal: world med health policy doi: . /wmh . sha: doc_id: cord_uid: g dk although the exact origin of sars‐cov‐ , the etiologic agent of covid‐ , is currently unknown, there is substantial evidence to suggest the source of transmission of the virus occurred within the wuhan wet market. in these markets, bats and wild animals are frequently sold and stored in close contact. during several of the world's past pandemics, bats were essential to the spread of zoonotic diseases from bat to another animal or to humans directly. live animal markets create the perfect conditions for novel viruses such as covid‐ to emerge. this paper suggests that to prevent future pandemics, the sale of exotic animals be banned at wet markets. it also advocates for the integration of the analysis of illicit trade with the study of zoonotic disease transmission and pandemics. as the world continues to struggle with the unprecedented ramifications of covid- , two central questions are being asked: where did this novel virus come from and how can we prevent future pandemics? although there is no definitive answer as to the origins of the virus, specialists in the field of immunology and microbiology have suggested that covid- , occurred naturally (choudhury, ) . there is significant evidence to suggest that live animal markets or wet markets in wuhan, china are the original source of transmission of the virus. a major indication is that some of the first patients to test positive for covid- had connections to the wet markets in wuhan (broad, , p. ) . additionally, the causative virus known as sars-cov- is a betacoronavirus that originated in bats, much like sars and mers diseases (centers for disease control and prevention [cdc] , ). an examination into these forms of contagion supports the hypothesis that covid- potentially transmitted from bats to another susceptible animal, such as a pangolin, and then to a human (choudhury, ) . transmission may have occurred at the wildlife market, or while en route to the wildlife market where illicitly traded wildlife may be sold. it is currently unknown as to precisely how the virus was transmitted from animals to humans but the threat of bats spreading disease has been a long-held concern for public health officials and biological researchers. viruses that are transmitted from animals to humans are very dangerous to human life due to the absence of herd immunity among the population. this article will explore the connections among the current pandemic, live-animal markets, the spread of animal-related diseases, and the illicit wildlife trade and will include a set of policy recommendations prescribed to prevent future outbreaks stemming from these issues. to explore the connections between bats and covid- , we reviewed academic literature and scientific journals on bat ecology and conservation, emerging zoonotic diseases, the covid- outbreak, and wet markets in china. we used websites of authoritative bodies such as the centers for disease control and prevention (cdc) and the world health organization (who). we looked at scholarship and reports addressing the illicit wildlife trade from prominent intergovernmental organizations and non-governmental organizations such as the united nations office on drugs and crime (unodc), and traffic. we also reviewed reporting from news media outlets such as the new york times and the wall street journal. although it could be several years before any final conclusions are drawn on the origins of the covid- outbreak, there is still substantial evidence that it originated in bats. biological and epidemiological research indicates that different species of bats may potentially carry multiple strands of the virus very similar to that of the current coronavirus. the los angeles times stated "scientists estimate that out of new or emerging infectious diseases in people come from animals" (su, ) . at the institute of virology in china, a horseshoe bat (rhinolophus affinis) identified as ratg , was discovered to be carrying a form of coronavirus, which shared % of its dna with sars-cov- (ridley, ) . although this information provides compelling circumstantial evidence, it does not prove that this particular species of bat contributed to or assisted in the spread of covid- . however, this does not mean that another species of bat does not carry this form of coronavirus that can then be shared with humans. in perspective, humans share % of our genome with chimpanzees (bat conservation trust, ). experts are not certain as to how sars-cov- was transmitted to humans; it may have been transmitted through another susceptible animal such as a pangolin, the most illegally traded mammal in the world (conciatore, ) . past research has shown that bats are central to the dissemination of covidlike diseases among animals and humans. although rodents bear the responsibility for spreading the bubonic plague during the fourteenth century, bats present a unique danger as reservoirs for zoonotic viruses, hosting even more bacteria and diseases than rodents (luis et al., , p. ) . therefore, bats have been much studied as transmitters of viruses to humans through close contact or direct consumption. humans cause harm to bats not only through consumption but also by contributing to their loss of habitat. as a consequence of environmental destruction, bats seek new areas to feed, sometimes causing them to come into contact with livestock that will be eventually sold in open markets (plowright et al., , pp. - ) . additionally, habitat destruction can cause bats to shed and contract more viruses, further increasing the likelihood that they will transmit disease (olival, , p. ) . bats are frequently stored and sold in wet markets and are often transported alongside large groups of uncommon and illicitly traded animals that are consumed by humans. this form of transportation and storage allows the spread of pathogens between bats and different, sometimes exotic, and rare creatures that can carry and advance diseases under severe, stressful situations. the concern over pathogens being transferred from bats to humans is widespread; not only among public health professionals but also has been much studied by academics. the american society for microbiology published a report entitled identification of a novel coronavirus in bats, which stated "the recent emergence of severe acute respiratory syndrome-associated coronavirus (sars-cov) highlights the importance of virus surveillance in wild animals" (poon et al., (poon et al., , p. . it further explains "the identification of severe acute respiratory syndrome-associated coronavirus (sars-cov) in civet cats and other wild animals in live animal markets suggests that this novel human pathogen emerged as a result of an interspecies transmission" (poon et al., (poon et al., , p. . this report illustrates how other coronavirus-like illnesses in animals have been previously identified as potential sources of transmission to humans. in fact, this is not a recent discovery and concerns existed already in that there was a serious danger to human life. a university of hong kong study at that time found that "the presence of a large reservoir of sars-cov-like viruses in horseshoe bats, together with the culture of eating exotic mammals in southern china, is a time bomb" (cheng, lau, woo, & yuen, , p. ) . indeed, this warning was unheeded and the failure to act on this insight has contributed to the most serious global pandemic in a century. this global health crisis originating from zoonotic transmission is not novel; in fact, over the last years, several deadly diseases are thought to have originated from live animal or wet markets. wet markets are where fresh meats, produce, and animals are often stored to be sold in open-air environments, in close proximity, with little to no health safety precautions or sanitation measures. these wet markets exist across the world and in china, they often contain foreign, rare, and sometimes endangered species that are sold, among other goods, by traveling suppliers. wet markets facilitate and heavily contribute to the practice of illicit wildlife trade and in turn, this practice has led to the spread of zoonotic diseases among the animals and to customers at markets. transportation and storage of animals for wildlife trade at these markets enables the spread of diseases from animals to other animals. as one science writer reports "live meat markets are perfect laboratories for creating new viruses. stressed animals shed more viruses and are more susceptible to infections, and cages are often stacked on top of each other, facilitating exposure" (nuwer, ) . exotic meats that are traded illegally, such as pangolins, are less likely to be inspected for health risks and may come into contact with bats while in transit. additionally, because these rare animals are often from remote areas, they may be more likely to carry diseases that humans have never come into contact with (hemley, ) . the conditions the live animals are subjected to in wet markets create a petri dish for the spread of diseases. in china and in some other asian countries, these markets are prevalent because consumers view wet markets as fresher, not expensive, and providing rare types of creatures that serve as status symbols or are believed to possess unique healing elements (daszak, olival, & li, , p. ). yet the spread of disease from the live and exotic animals represents the biggest threat. for instance, wild carnivores, birds, reptiles, and primates that are traded in these markets carry pathogens from every taxonomic origin. therefore, pathogens such as newcastle disease can jump from wild to domestic birds, and pathogens such as simian foamy virus and chlamydophila psittaci can be transmitted from wild animals to humans (gómez & aguirre, , p. ) . there is a well-documented history of regional and even global pandemics originating from the transmission of diseases from animals to humans due to the practice of wet markets and illegal wildlife trafficking. in april , the united nations (un) acting head of biodiversity stated, "countries should move to prevent future pandemics by banning wet markets that sell live and dead animals for human consumption but cautioned against unintended consequences" (greenfield, ) . public health officials in the united states and around the world have pointed to wet markets and illegal wildlife trading as conduits for the transfer of disease. the transference of diseases from animals to humans may transpire through the consumption, proximity, or mixing of animals or their by-products. typically, animals are susceptible to different types of diseases. once a strand is introduced into the human population, it can be spread from human to human and cause an outbreak or potential public health emergency. relatively recent epidemics such as ebola, middle east respiratory syndrome (mers), bird flu, severe acute respiratory syndrome (sars), and swine flu are all examples of diseases that have originated through the transfer of disease to humans from animals. for example, the highly pathogenic asian avian influenza a (h n ) virus or bird flu is highly contagious, and it occurs when humans come into direct or close contact with infected or already diseased poultry (cdc, ). therefore, the significant risk factors for infection include visiting or mixing animals in live poultry markets. similarly, while the swine flu is sporadic in humans, infections are typically the result of exposure to infected pigs within live markets, fairs, or the pork industry (cdc, a). unfortunately, due to the nature of infections, there is still a lot of information left unknown about how some of these outbreaks originated. in the case of ebola and based on the analyses of similar viruses, scientists believe it is animal-borne, with bats or nonhuman primates such as apes or chimpanzees being the most likely source (cdc, b). another example would be in , when sars broke out in the southern province of guangdong in china. experts believe this outbreak is the result of an unknown animal reservoir, some experts have suggested bats, and the spread occurred through other animals such as civet cats (world health organization [who], ). during its initial outbreak, sars resulted in more than , cases in over countries. similar to sars, mers began as the result of a zoonotic virus in saudi arabia. although different practices other than wet markets or animal reservoirs led to the outbreak of mers, the method of transfer is the same; animal to human (who, ). the director of the national institute of allergy and infectious diseases, dr. anthony fauci, insisted on a global closure of wet markets because the current crisis is a "direct result" of this practice (guzman, ) . after the sars outbreak, china attempted to institute a similar, more stringent ban on wildlife trade but this has eroded over time (su, ) . several conservationists and medical professionals, including members of the chinese academy of sciences, hoped this ban would be permanent, but the markets and trade routes returned after the crisis ended (who, ). it is certain that without cooperative, comprehensive, and enforced policies on wildlife trade and of endangered species, such as the pangolin, in place there will continue to be a spread of global sickness and international loss of life. the devastation resulting from the spread of covid- could potentially serve as a future warning for what is to come, if practices such as illicit wildlife trade and wet markets are allowed to continue on a global scale. a combination of factors ranging from loss of habitat to increased human-animal interactions through the illicit wildlife trade have increased the likelihood of novel zoonotic diseases emerging and spreading. this pandemic began in china, but there is no reason a similar pandemic could not begin elsewhere in southeast asia, south asia, sub-saharan africa, or latin america (daszak et al., , p. ) . significant actions must be taken to ensure this type of pandemic is prevented in the future. here, we outline policy proposals to address the issue of zoonotic diseases spread by illicitly traded animals. these policy suggestions are multifaceted and do not depend entirely on law enforcement strategies. the limits of law enforcement are clear. in , anti-smuggling officers in guangdong intercepted live pangolins and discovered many of them were sick with coronaviruses (ridley, ) . the illicit trade of pangolins, a suspected susceptible host and potential carrier of covid- , for their meat and scales represents over $ million in illegal activity each year (united nations office on drugs and crime [unodc], , p. ) . although increasing operations targeting the illicit wildlife trade is a worthwhile endeavor for weakening revenue sources for transnational criminals, conserving endangered species, and fighting corruption, there are limits to how effective it alone would be in stopping the spread of novel diseases. global law enforcement to address the illegal trading in exotic animals for consumption or possession represents a high priority in preventing future animal to human disease transmission. to address this problem, countering illicit wildlife trade must be a higher priority of interpol (the international police body) and of regional and law enforcement bodies. but this trade is highly linked to transnational crime that requires corruption to operate. therefore, there needs to be much more attention paid to dismantling cross-national illicit networks, addressing corruption, and following the money linked to this trade. moreover, the role of corporate actors as part of the supply chain for this illicit transit needs much more attention. much illicit wildlife trade flows through free-trade zones in asia, and they must be held much more responsible for the cargo that goes through their ports (shelley, , p. ) . a targeted ban could be effective and have fewer unintended consequences than an outright ban. focusing on the most likely vectors of zoonotic diseases could protect public health without the negative effects of a blanket ban. although a ban on wild animal and wet markets would lower the number of opportunities for chinese consumers to come into contact with meat infected with infectious agents, there is the chance that underground markets will pop up. therefore, allowing the sale of a limited category of meat in markets as the chinese government is now considering would preserve people's access to food. an overarching ban could destroy these people's livelihoods or even lead to a black market facilitated by corruption with even greater risks (samuel, ) . enforcement of regulations are essential and inspections must be carried out to ensure that meat from exotic animals is not disguised as meat from domestic animals. there are certain aspects of this pandemic that might make a total ban on wet markets politically viable. the markets in china were closed temporarily after the sars outbreak in , though this ban did not last long (samuel, ) . this time might be different, however. the sars outbreak resulted in around deaths worldwide, while the death toll from covid- has caused over , deaths as of june , . with the recommendations of experts and the drastic toll of covid- , shutting down the wet markets may be politically possible. even if wildlife markets are banned, outreach will be necessary to drive down demand for a black market. consumption of wild animals from wet markets is influenced by chinese and asian culture, which holds that wild animals and their by-products have healing powers. eating rare animals has also become a status symbol signifying that the consumer is wealthy (daszak et al., , p. ) . in addition, chinese culture places a great deal of emphasis on the "freshness" of the food being consumed. this concept of freshness extends to perceptions of food quality, healthiness, and taste, with fresher always being better. food from a grocery store is seen as less fresh than food from wet markets. some chinese consumers will make trips to easily accessible wet markets every day (zhong, crang, & zeng, , p. ) . dealing with the underground markets will require convincing people that they should not buy from underground wet markets. this could be done through public health outreach campaigns that inform people of the dangers of exotic meats and markets that have live wild animals. research has shown that public health outreach campaigns can work. the first anti-smoking campaign given federal funds by the united states government showed impressive results, inspiring . million smokers to attempt to quit and , to quit for good in (cdc, ). although there are crucial differences between cigarettes and meats from animal markets in terms of addiction and their places in different cultures, the concept may have applicability in convincing people not to buy food from wild animal markets. future research by the health, medical, and environmental community must be combined with research on illicit trade and corruption. a transdisciplinary approach has been taken in the one health perspective but this approach has not gone outside the scientific perspective wilcox, aguirre, de padua, siriaroonrat, & echaubard, ) . going forward, these barriers among academic disciplines must be bridged to consider all the risks to public health. addressing and preventing pandemics requires an array of transdisciplinary research. research must focus on the central causes of the spread of zoonotic diseases such as illicit wildlife trade and wet markets. public health, conservation biology, and illicit trade scholars should collaborate on addressing the facets of supply chains, corruption, and trade that may contribute to zoonotic transmission. there will need to be much more work with the business community to follow the supply chains linked to illicit trade in wildlife. governments will need to get involved as well and take action to suppress the wildlife trade in order to prevent future outbreaks. those who are part of the illicit wildlife trade have not taken off days during this crisis. lockdown policies and the changing dynamics of international travel have resulted in multiple changes to the supply chains for illicit wildlife trade. trade by air has been slowed due to quarantine regulations and transportation by road has been complicated by roadblocks and checkpoints, particularly at borders. the trade is still happening by sea, however. additionally, because governments have closed so many national parks, poachers have taken the opportunity to go after animals that are not being as closely guarded as before. the combination of transportation difficulties and unwatched national parks has led to stockpiling of products in the illicit wildlife trade. without addressing these issues now, the trade could come roaring back as lockdowns are lifted (wildlife justice commission, , p. ). this outbreak was caused by an array of factors, including the conditions of wet markets, the illicit wildlife trade, and environmental destruction. a multipronged approach will be necessary to make sure this never happens again. on the illegal trade aspect, policymakers will need to address the source of demand for these products: the wet markets. shutting down the wet markets, or at least strictly regulating them and banning the sale of live wild animals, will be necessary to address the problem of zoonotic diseases. hopefully, this pandemic will convince government officials that these markets need to be dealt with and convince scholars to study the intersection of illicit trade and public health. a. alonso aguirre, dvm, phd, is professor and chair, department of environmental science and policy at george mason university. transdisciplinary and social-ecological health frameworks-novel approaches to emerging parasitic and vector-borne diseases covid- and bats-bats and disease wildlife trade, covid- , and zoonotic disease risks impact of first federally funded anti-smoking ad campaign remains strong after three years highly pathogenic asian avian influenza a (h n ) in people key facts about human infections with variant viruses what is ebola virus disease coronavirus disease (covid- ) situation summary severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection covid- research: scientists prove that china did not create coronavirus up to . million pangolins are poached every year for scales and meat a strategy to prevent future epidemics similar to the -ncov outbreak infectious diseases and the illegal wildlife trade ban wildlife markets to avert pandemics, says un biodiversity chief fauci: it's mind-boggling that china's wet markets are still operating during coronavirus pandemic people want wildlife markets to close-governments should listen a comparison of bats and rodents as reservoirs of zoonotic viruses: are bats special? to prevent next coronavirus, stop the wildlife trade, conservationists say to cull, or not to cull, bat is the question ecological dynamics of emerging bat virus spillover identification of a novel coronavirus in bats the bats behind the pandemic the coronavirus likely came from china's wet markets. they're reopening anyway dark commerce: how a new illicit economy is threatening our future why china's wildlife ban is not enough to stop another virus outbreak transnational organized crime in east asia and the pacific operationalizing one heath employing social-ecological systems theory: lessons from the greater mekong subregion rapid assessment of the impact of covid- on wildlife trafficking sars (severe acute respiratory syndrome) middle east respiratory syndrome coronavirus (mers-cov) constructing freshness: the vitality of wet markets in urban china key: cord- -oovkoiyj authors: hickman, d.l.; johnson, j.; vemulapalli, t.h.; crisler, j.r.; shepherd, r. title: commonly used animal models date: - - journal: principles of animal research for graduate and undergraduate students doi: . /b - - - - . - sha: doc_id: cord_uid: oovkoiyj this chapter provides an introduction to animals that are commonly used for research. it presents information on basic care topics such as biology, behavior, housing, feeding, sexing, and breeding of these animals. the chapter provides some insight into the reasons why these animals are used in research. it also gives an overview of techniques that can be utilized to collect blood or to administer drugs or medicine. each section concludes with a brief description of how to recognize abnormal signs, in addition to lists of various diseases. the mouse is a small mammal that belongs to the order rodentia ( fig. . ). the house mouse of north america and europe, mus musculus, is the species most commonly used for biomedical research. it is likely that the mouse originated in eurasia and utilized its commensal relationship with humans to spread through to other continents as humans explored and colonized. mouse fanciers around the turn of the th century are the source of the majority of the laboratory mice that are in use today. a summary of the overarching categories of mouse models that are available is presented in table . . physiology; and the possibility for breeding genetically manipulated mice and mice that have spontaneous mutations. mice have been used as research subjects for studies ranging from biology to psychology to engineering. they are used to model human diseases for the purpose of finding treatments or cures. some of the diseases they model include: hypertension, diabetes, cataracts, obesity, seizures, respiratory problems, deafness, parkinson's disease, alzheimer's disease, various cancers, cystic fibrosis, and acquired immunodeficiency syndrome (aids), heart disease, muscular dystrophy, and spinal cord injuries. mice are also used in behavioral, sensory, aging, nutrition, and genetic studies. this list is in no way complete as geneticists, biologists, and other scientists are rapidly finding new uses for the domestic mouse in research. mice are mammals and their organ systems are very similar to organ systems in humans in terms of shape, structure, and physiology. basic physiologic data are presented in table . . mice have very long loops of henle in the kidneys, thus allowing for maximal concentration of their urine. as a result, urine output in mice usually consists of only a drop or two of highly concentrated urine at a time. they also excrete large amounts of protein in their urine with sexually mature male mice excreting the largest levels of protein possibly as pheromones. mice have only two types of teeth, incisors and molars. the incisors are openrooted and erupt (i.e., grow) continuously throughout their lives. this predisposes mice to malocclusion if not given feeds or objects such as nylon bones to help wear down the teeth during mastication. the molars are rooted and, thus, do not continuously erupt. the stomach has two compartments with the proximal portion completely keratinized and the distal portion entirely glandular. their intestines are simple, but the rectum is very short ( e mm) and hence is prone to prolapse, especially if the animal has colitis. the gastrointestinal flora consists of more than species of bacteria that form a complex ecosystem that aids digestion and health of the mouse. mice have no sweat glands but have a relatively large surface area per gram of body weight. this results in dramatic changes in physiology and behavior in response to fluctuations in ambient temperature. when too cold, mice will respond by nonshivering thermogenesis (i.e., metabolism of brown adipose tissue). in addition to the lack of sweat glands, they cannot pant or produce large amounts of saliva to aid in cooling their body temperature. therefore, when exposed to very hot situations, mice increase the blood flow to their ears to maximize heat loss; and in the wild, they move into their burrows which are at cooler temperatures. the thermoneutral zone, the range of ambient temperatures at which the mouse does not have to perform regulatory changes in metabolic heat production or evaporative heat loss to maintain its core temperature, is about . fe . f ( . ce . c). the female reproductive system is comprised of paired ovaries and oviducts, uterus, cervix, vagina, clitoris, and paired clitoral glands. pregnant female mice have hemochorial placentation, similar to humans (i.e., maternal blood is in direct contact with the chorion, the outermost layer of the fetal placental membranes). the female mouse also has five pairs of mammary glands. the male reproductive system consists of paired testes, penis, and associated sexual ducts and glands. the inguinal canals are open in the male mouse, and the testes can retract easily into the abdominal cavity. both sexes have well-developed preputial glands, which can become infected. males have a number of accessory sex glands, including large seminal vesicles, coagulating glands, and a prostate. secretions from these glands make up a large part of the mouse's ejaculate. when mice ejaculate, the semen forms a coagulum or copulatory plug. mice breed continuously throughout the year unless conditions are very unfavorable to them (e.g., lack of food). their reproductive potential can be affected by a number of external influences such as noise, diet, light cycles, population density, or cage environment. genotype also can affect reproductive performance as it is common knowledge that some inbred strains of mice are poor breeders, and if pups are born they may receive poor maternal care. additional reproductive physiologic data are presented in table . . mice can be bred using a one-on-one system (one male to one female; monogamous) or in a harem mating system (polygamous mating). in a monogamous system, the male and female are always left together, but at weaning the pups are removed from the cage. this system allows for maximal use of the postpartum estrus and the maximum number of litters for the females involved, and facilitates recordkeeping and monitoring of specific breeders in the colony. in a harem mating system, multiple males are placed with multiple females, usually at a ratio of one male to two to six females. usually, females are removed to separate cages just before parturition and the postpartum estrus is underutilized. mouse pups are born hairless, blind, and deaf and require extensive parental care that is provided mainly by the mother. due to the ruddy coloration of the skin of the hairless pups, they are also known as "pinkies." while mouse pups can increase their body temperature through the metabolism of brown fat stores, they are unable to adequately conserve body heat until they develop an adequate fur coat. thus, inclusion of nesting materials in the cage is highly recommended as huddling inside the nest can provide much needed warmth and safeguard against temperature-associated neonatal losses. the most reliable method for determining the sex of a mouse is by measuring the length of the anogenital distance, i.e., the distance from the anus to the genitalia. this distance can be measured with a ruler or animals assessed side by side with the rear ends held up by their tails. the anogenital distance is longer in males than females. in sexually mature animals one can also determine the sex of mice by the presence or absence of testicles in a testicular sac. mice are social creatures and can be group housed easily. their main method of communication is via pheromones. they use these olfactory cues to establish a pecking order (i.e., a hierarchical system of social organization). these chemicals are so important that when cage environments are changed, such as by simple cleaning or with bedding changes, a bout of fighting may occur until scent marking of the cage is completed as a way to reestablish the pecking order and social organization in that cage. pheromones also play a vital role in reproduction of these animals. this is demonstrated by the whitten effect and the bruce effect. the whitten effect occurs when a group of female mice that are not cycling are exposed to male urine, which contains a large quantity of pheromones. the females will all resume cycling as a group soon after the introduction of the male. in contrast, the bruce effect is characterized by abortion of litters when pregnant females are exposed to the urine of a strange male. as with most rodents, mice are nocturnal animals exhibiting peak levels of activity at night. because mice are a prey species, they display thigmotactic behavior or wall hugging. they avoid open spaces where they might be easily caught by predators. despite this, mice are very curious about any new objects in their territory and will often examine them at length. mice not only have poorly developed eyesight but they are also color blind. a number of inbred strains (e.g., fvb/n and c h/he) are functionally blind by weaning. they rely on their very sensitive hearing to escape detection and on their sense of smell and taste to detect food (and possibly avoid poisons). mice can hear over a range of frequencies between . and khz; however, normal mice are most sensitive to frequencies of e khz. it is important to note here that some inbred strains of mice, e.g., c bl/ , suffer significant hearing loss before year of age. mice can climb, swim, and jump (up to a foot), though they normally prefer to avoid swimming, if possible. under certain conditions they display stereotypies, which are obsessiveecompulsive behaviors. the behaviors may be strain-related, environment-related, or study-related and include wire gnawing, circling, jumping, and aggression. the use of environmental enrichment items such as cardboard tubes or other structures offer the animal an area for retreat from cage mates and add complexity to the environment. aggression is another important behavior that commonly occurs in group-housed male mice. it can also occur in group-housed females and mixed-sex cages. indications that there is an aggressive animal in the cage include bite wounds on the tail, rump, ears, and shoulders of mice ( fig. . ). the wounds can be so severe as to cause significant blood loss and abscess formation at bite sites. aggression has been shown to be influenced by strain, age, and prior encounters. in terms of strains, the more aggressive strains are balb/c, c bl/ , c bl/ , dba/ , and outbred swiss. methods to prevent or reduce aggression include use of properly designed enrichment devices; provision of adequate space and shelter for each animal; grouping of mice before they reach puberty; use of docile strains; and removal of dominant animals as soon as possible. a common manifestation of social organization in group-housed mice is barbering, a behavior in which a dominant mouse will trim, by chewing, the hair or whiskers of other mice in the cage. barbering is also sometimes referred to as the dalila effect. it is usually instigated by a dominant male or female mouse. the dominant animals retain their whiskers and full hair coats, while their cage mates have "shaved faces and bodies" (fig. . ) . although barbering does not generally result in any physical harm to the animal, removing the dominant mouse (the nonbarbered one) from the cage is a good approach to control. general types of housing mice in a laboratory setting include: conventional, specific pathogen free (spf), and germ free. in conventional housing, no attempt is made to keep out adventitious microbial and parasitic organisms. mice housed in this manner can be found in open-topped cages. room air, along with any airborne contaminants, is allowed to freely circulate into the mouse's cage. in addition, the food and water are not sterilized, though it should be noted that microbial contaminants may enter into the mouse population in this way. spf mice are raised in barrier conditions to ensure that they remain free of a specific list of pathogens. care is taken to ensure that adventitious microbes and parasites are excluded from the animals. spf mice are typically raised in specialized caging such as microisolator cages. these cages contain a . mm filter top that aids in the exclusion of microbes and parasites. individually ventilated caging systems include a rack of microisolator cages, each of which receives a filtered air supply ( fig. . ) . under spf conditions, everything that comes into contact with the animal should be sterilized or disinfected. this includes, but is not limited to, the water, food, bedding, and caging. special care must be taken by anyone handling the mice, including researchers, to ensure that handling and experimental procedures do not introduce potential pathogens into the colony. thus, all handling and procedures done on spf mice are often performed under hepa-filtered air conditions, such as within a biosafety cabinet. placing the mouse in an unfiltered environment ("room" air), even for a moment, is enough to potentially colonize the mouse with a whole host of adventitious microorganisms, thus destroying its spf status. once a contaminated mouse is placed back into the colony, the entire colony is at risk for infection. germ-free, or axenic, mice are raised to contain no microbes or parasites whatsoever. raising germ-free mice requires strict barrier maintenance. usually, this requires the use of flexible film isolators which provide hepa-filtered air to the mice within the isolator. additionally, any materials (e.g., food, water, and bedding) must be sterilized or thoroughly disinfected prior to being moved into the isolator unit so as not to contaminate the living space of the germ-free rodents with adventitious microorganisms. all procedures performed within the flexible film isolator must utilize strict aseptic technique for the same reason. in addition to the microbiological environment of the animal's housing systems, mice need to be housed at specific environmental parameters otherwise they may experience stress. the guide for the care and use of laboratory animals, th edition (national research council, ) is an internationally accepted document that outlines and discusses globally accepted environmental parameters for housing different species of animals including the mouse. table . outlines the specific environmental requirements listed in this document for housing mice. mice are omnivorous and coprophagic with at least one-third of their diet being the consumption of their feces. in the laboratory setting mice are fed a clean, wholesome, and nutritious pelleted rodent diet ad lib. there are many commercially formulated diets for the various stages of life and for animals with specific induced diseases such as diabetes mellitus or hypertension. these diets may be available as autoclavable or irradiated forms to prevent transmission of disease via contaminated feed. there are also a variety of "pet" treats available for mice. however, the treats should not make up more than e % of the daily diet. mice should be provided with a continuous supply of water daily. if the animals do not get enough water daily, their food consumption will decrease. the animals will also look scruffy and unhealthy. mice can be provided with water from water bottles or pouches, automatic watering systems with nipples, or water-based gel packs. some general signs of ill health include: weight loss, depression or lethargy, anorexia, obesity, diarrhea, scruffiness or ruffled coats, abnormal breathing, sneezing, weakness, dehydration, enlarged abdomen, discolorations (e.g., yellow for jaundiced animals or very pale for anemic animals), masses or swellings, and abnormal posture or gait. body condition scoring is an objective measure to truly assess how fat or thin the animal is and can be used for accurate determination of endpoints in studies where animals are expected to lose or gain weight ( fig. . ) (ullman-cullere and foltz, ) . some of the more commonly found diseases of mice are presented in table . . noninfectious disorders are presented in table . . based on their genetic and physiologic makeup, mice can be either immunocompetent or immunodeficient. immunocompetent means that the mouse has a normal functioning immune system and can stage an immune response to any insult or injury. in contrast, immunodeficient means that some component or components of the mouse's immune system is not working or functioning normally, and so they cannot stage an adequate immune response and are more susceptible to infectious disease. immunosuppressed mice are mice that have a complete immune system but because of a drug or chemical or disease state, the immunological response is attenuated. rats and humans have a long history of coexistence. the origins of the laboratory rat, also known as the norway rat, stretch back centuries to the areas of modern day china and mongolia (burt, ; song et al., ) . the dispersal of the norway rat has occurred across the centuries and its natural habitat stretches from the mediterranean across southeast asia and down into australia and new guinea . unfortunately, most people associate rats with disease and destruction. throughout history, outbreaks of bubonic plague, typhus, and hantaviruses have had an unwitting accomplice in the rat (zinsser, ; benedictow, ; firth et al., ) . over the centuries, rats have also been used for food (e.g., in imperial china), companionship, and sport (gorn and goldstein, ; hopkins et al., ; burt, ) . ratting, a vicious blood sport where people laid bets on the dog that could kill the most rats in a given period of time was especially popular in both the victorian england and american underworld (thomas and mayhew, ; gorn and goldstein, ) . at the turn of the th century, breeding rats as a hobby or for companionship (i.e., "fancy rats") was recognized by the addition of "rat" to both the name and mission of national mouse club in the united kingdom (american fancy rat and mouse association, ). however, as interest in pet rats waned over the following years, the club reorganized and dropped "rat" from its name. a similar club, the american fancy rat and mouse association, was founded in the united states in (american fancy rat and mouse association, ). the first recorded use of rats as research subjects occurred in (hedrich, ) , and the first known rat breeding experiments occurred in the late s (lindsey and baker, ) . the first major effort to perform research in the united states using laboratory rats occurred at the wistar institute of philadelphia, the oldest independent research institute in the united states, in (lindsey and baker, ) . rattus norvegicus constitutes one of the most commonly used laboratory species ( fig. . ), second only to the laboratory mouse. because rats and mice are not included under the animal welfare act regulations, the precise number of these species used per year within the united states is unavailable. however, examining the data collected within the european union can give some indication of their use relative to other common laboratory animal species. in , rats accounted for just fewer than % ( . million) of the total animals ( . million) used in research within the european union (european commission, ) . this contrasts to mice, which constituted % ( . million) of the total animals used within the european union (european commission, ). rats possess a number of qualities which make them a highly suitable and much preferred animal model. like mice, these traits include relatively small size; known genetic background; short generation time; similarities to human disease conditions; and known microbial status. their tractable nature makes them easier to handle in a laboratory setting than many other rodents. rats rarely bite their handlers unless extremely stressed or in pain. rats have been used as animal models in numerous areas of research from space exploration to answering more basic scientific questions regarding nutrition, genetics, immunology, neurology, infectious disease, metabolic disease, and behavior. perhaps their largest use is in drug discovery, efficacy, and toxicity studies. in the united states, the approval of any new drug for use in humans or animals usually necessitates that toxicity testing be done in at least one small animal species (e.g., rodents) and one large animal or target species (e.g., dog, nonhuman primate). there are known physiologic differences between the numerous outbred stocks and inbred strains of rats. the rat genome database (rgd) is an extensive, free resource filled with information regarding the different phenotypes, models, and genomic tools used in rat research (laulederkind et al., ) . vendors of commercially available rat stocks and strains are often good resources for normal physiologic data of these strains. many provide stock-and strain-specific data directly on their websites such as growth curves, complete blood count and serum biochemistry panels, and spontaneous lesions seen on histopathology. a summary table of normal physiologic references is in table . . sexual dimorphism exists between male and female rats. sexing of adult rats is most easily done by examining the perineal area of the rat and identifying the external reproductive structures such as the penis, testes, or vagina. in addition, male rats are typically larger and weigh significantly more than their age-and strain- matched female counterparts. sexing of rat pups is most easily performed by examining the distance between the anus and genital opening in the pup. males have a greater anogenital distance than females. male rats possess paired testicles that descend from the abdomen into the scrotal sac at approximately days of age (russell, ) . due to the lack of closure of the inguinal rings, the testes may be retracted into the abdominal cavity even as an adult. the male rat also possesses a number of accessory sex glands. a four-lobed prostate is present along with four other paired glands, including: the seminal vesicles, coagulating glands, ampullary glands, and bulbourethral glands (noted in some texts by the older name, cowper's gland) (popesko et al., ) . due to the unusual bihorned shape of the closely associated coagulating gland and seminal vesicles, individuals unfamiliar with rodent male anatomy may initially mistake these structures for the female uterus. however, the apices of these glands are freely mobile and easily exteriorized from the abdominal cavity unlike the uterus, which is attached to the dorsal body wall bilaterally via paired ovaries and their respective ovarian ligaments. the reproductive anatomy of the female rat contains some distinct features. the uterus of the female rat is classified as a duplex uterus, because the vagina is separated from the uterus by two individual cervices with each cervix leading to a separate uterine horn (popesko et al., ) . the placentation of the pregnant rat is hemotrichorial (three layers) rather than the hemomonochorial (single layer) placentation present in humans (wooding and burton, ) . the rest of the reproductive anatomy (e.g., ovary, oviduct) is structurally and functionally similar to other mammals. a summary of basic reproductive physiology is presented in table . . rat pups are born hairless, blind, and deaf and require extensive parental care that is provided mainly by the mother. as with mice, the skin of the hairless rat pups has a pink coloration, thus they are also referred to as "pinkies." the inclusion of nesting materials in the cage is recommended to assist the rat pups with thermal regulation until they have a full hair coat (whishaw and kolb, ). like other rodents, rattus norvegicus is a nocturnal species with the highest level of activity occurring during the dark phase. behaviors exhibited by rats include grooming, nesting, eating, and other social behaviors. nesting behavior serves several purposes among rats and mice (gaskill et al., (gaskill et al., , a . nests allow for better thermoregulatory control within a given environment as well as protection against predation (gaskill et al., c) . recent work in mice suggests that energy not diverted to thermoregulation can be shunted to other activities as seen via improved feed conversion and breeding performance (gaskill et al., c) . however, anecdotal evidence suggests that nest building in rats is largely a learned behavior, and it appears that there is a developmental period in young rats whereupon if exposed to nesting materials during this time they will begin using the materials to build at least rudimentary nests (gaskill, ) . at a minimum, rats benefit from having a structural shelter or nest box into which they may rest away from prying eyes ( fig. . ). rats emit alarm vocalizations during times of distress. however, these negatively associated vocalizations typically register in the ultrasonic wavelengths (approximately khz), well outside of the human hearing range (burman et al., ; parsana et al., ) . rodents may also emit high-pitched audible vocalizations when extremely alarmed, distressed, or in pain (jourdan et al., ; han et al., ) . as discussed in chapter , rats exhibiting abnormal behaviors and stereotypies can create variables in the research findings and should not be used in a study unless abnormal behavior is the object of the study subjects (baenninger, ; callard et al., ; garner and mason, ; cabib, ; ibi et al., ) . examples of stereotypies seen in rats include: bar-gnawing, pawing behavior, repetitive circling, and backflipping. it is critical that rats should be provided some form of environmental enrichment to stimulate positive species-typical behaviors. the housing of rats in a laboratory setting is similar to that described previously for mice: conventional, spf, and germ free. as rats are social animals, at minimum, they should be housed in pairs whenever possible. there is a preponderance of evidence that shows the differences in affiliative versus aggressive behavior, biochemical changes, and changes in learning between rats raised and housed in social isolation versus those housed in social groups (baenninger, ; einon and morgan, ; robbins et al., ) . enrichment items, such as a hut, nesting box, or similar type of shelter may be included in the cage to provide a visual barrier between the rat and the rest of the animal room. evidence suggests that rats prefer shelters made from opaque plastic (patterson-kane, ) . rats also spend a lot of time in the wild chewing either for eating or for manipulating objects for nest building. providing objects made of safe materials in the cage allows the rats to exhibit this natural behavior and encourage the normal wear of the rat's incisors, minimizing the incidence of malocclusion of the teeth. rodents can benefit from frequent gentle handling by the researcher and animal care staff. this concept is also known as "gentling" and has been demonstrated to reduce the stress experienced by rats during experimental handling and procedures (hirsjarvi et al., ; van bergeijk et al., ) . another positive interaction between humans and rats is found in the "tickling" of rodents. based on ultrasonic vocalization data, rodents find tickling a pleasurable experience (burgdorf and panksepp, ; panksepp, ; hori et al., ) . tickling may also decrease the stress response seen in rodents after experimental manipulations like intraperitoneal injections (cloutier et al., ). a multilevel cage with an intracage shelter. this style of caging provides opportunities for exercise for the rats. photo provided by melissa swan. as for mice, some general signs of ill health would include: weight loss, depression or lethargy, anorexia, obesity, diarrhea, scruffiness or ruffled coats, abnormal breathing, sneezing, weakness, dehydration, enlarged abdomen, discolorations (e.g., yellow for jaundiced animals or very pale for anemic animals), masses or swellings, and abnormal posture or gait. when assessing animals, a body condition score can be used as an objective measure or scale to truly assess how fat or thin the animal is; and it allows for the accurate determination of endpoints in studies where animals are expected to lose or gain weight (hickman and swan, ) (fig. . ) . some of the more commonly found diseases of rats are presented in table . . the ancestral home of the european rabbit (oryctolagus cuniculus) is the iberian peninsula (hardy et al., ) . the earliest archeological evidence of the coexistence of humans and rabbits can be found in excavation sites dated at approximately , years bce in nice, france (dickenson, ) . in antiquity, romans used rabbits as a food source and are thought to be responsible for their dispersal throughout europe, although there is no evidence that they attempted to actually domesticate them (dickenson, ) . domestication and selective breeding is thought to have begun in france in the middle ages where monks began to breed rabbits in their monasteries (dickenson, ) . the rabbits were kept confined in enclosures called "clapiers"(dickenson, ). they were kept largely as a source of food for the monks especially since ce when pope gregory i officially classified them as "fish" and thus eligible to being eaten during lent. however, rabbit wool production soon became a welcome by-product of these domestication efforts. european rabbits have been used in research since the middle of the th century. early work with the species was concentrated on the comparative anatomy of the rabbit with other species, such as the frog, and the unique features of the rabbit's heart and circulatory system (champneys, ; roy, ; smith, ) . louis pasteur used rabbits in a series of experiments that led to the development of the world's first rabies vaccine (rappuoli, ) . while there are numerous so-called "fancy" breeds of rabbits available in the pet trade, the list of breeds routinely used in research is much shorter. the new zealand white (nzw) rabbit is the most frequent breed of used in research (fig. . ). the california and dutch-belted rabbit breeds are also occasionally used. researchers have developed genetically inbred rabbit strains for particular research applications. for example, the watanabe heritable hyperlipidemic (whhl) and the myocardial infarction-prone whhl rabbit (whhlmi), both developed by researchers in japan, are used to explore diseases associated with dyslipidemia such as atherosclerosis (shiomi et al., ; shiomi and ito, ) . rabbits have been used as a model of human pregnancy and for the production of polyclonal antibodies for use in immunology research (hanly et al., ; ema et al., ; ito et al., ; fischer et al., ) . rabbits are routinely used in (southard et al., ; arslan et al., ; mcmahon et al., ; castaneda et al., ; habjanec et al., ; manabe et al., ; xiangdong et al., ; panda et al., ; sriram et al., ; wei et al., ; zhou et al., ) . the production of polyclonal antibodies is preferentially performed in the rabbit due to its relatively large blood volume compared to rodents (hanly et al., ) . their tractable nature and larger body size also make them suitable for surgical implantation of biomedical devices (gotfredsen et al., ; swindle et al., ; ronisz et al., ) . additionally, rabbits are a favored model in pharmacologic studies for teratogenicity testing of novel pharmaceutic compounds (gibson et al., ; lloyd et al., ; foote and carney, ; jiangbo et al., ; oi et al., ) . while much of the anatomy of the rabbit is similar to other mammalian species, it should be noted that there are a number of key differences. for example, the skin of the rabbit is quite thin and fragile. care should be taken when restraining a rabbit or shaving a rabbit's fur (e.g., in preparation for surgery) to avoid tearing the skin. unlike rodents and other laboratory animals, rabbits do not have pads on their feet; rather, the plantar surface is covered with fur (quesenberry and carpenter, ). new zealand white rabbits are commonly used in research. photo provided by kay stewart. the long ears of the rabbit serve several purposes. the most obvious is for hearing. in addition, the central ear artery and marginal ear veins are easily accessible for both intravenous administration and blood sampling (diehl et al., ; parasuraman et al., ) (fig. . ) . the ears also serve as a means of thermoregulation, as excess heat may be exchanged across the large surface area of the ears (sohn and couto, ) . the skin of rabbits lacks sweat glands and is therefore unable to sweat; panting is insufficient to dissipate the excess heat (sohn and couto, ) . thus, the ears play a vital role in maintaining proper body temperature. other unique features of the skin and adnexa are the presence of chin and inguinal glands used in scent marking. additionally, the female rabbit (doe) is noted by the presence of a large skin fold filled with subcutaneous fat just under the chin (the dewlap) (sohn and couto, ) . the skeleton of rabbits makes up only % of the body weight by mass (brewer, ) . this is in contrast to other similarly sized mammals. for example, the cat skeleton makes up e % of body weight (brewer, ) . the small skeletal mass of the rabbit coupled with strong back muscles mean that the back is prone to traumatic fracture (meredith and richardson, ) . proper holding and restraint techniques are necessary to avoid this undesirable outcome. there are several unique features of both the respiratory and cardiovascular systems of rabbits. for example, rabbits are obligate nose breathers (varga, ) . this is especially important during procedures involving anesthesia and placement of an endotracheal tube. with respect to the cardiovascular system, the rabbit heart is unique in that the right atrioventricular (av) valve has only two leaflets instead of three (brewer, ) . additionally, due to the similarity to humans with respect to the neural anatomy of the ventricles, the rabbit is the species of choice for purkinje fiber research (brewer, ) . rabbit teeth are "open-rooted" meaning that they continue to erupt and grow throughout life. this applies to all of the teeth in the rabbit dental arcade (i.e., incisors, premolars, and molars; rabbits do not have canines). this contrasts with rodents, where the incisors are the only open-rooted (or hypsodontic) teeth (sohn and couto, ) . thus, rabbit teeth are subject to overgrowth. another unique feature of rabbit dentition that sets them apart from rodents is the presence of a second set of incisors just behind the first set of upper incisors known as "peg" teeth (sohn and couto, ) . they are thought to aid in tearing off the succulent leaves of plants while grazing. as an obligate herbivore, the gastrointestinal tract of rabbits differs greatly from that of carnivores and omnivores. rabbits require a high fiber diet of between and % (sohn and couto, ) . the small intestine is divided up into three main sections: the duodenum, jejunum, and ileum. the ileum connects to the cecum via a structure called the sacculus rotundus. the presence of lymph follicles suggests that the sacculus rotundus has immunological functions. it is sometimes referred to as the ileocecal "tonsil" (jenkins, ) . in the rabbit, gastric associated lymphoid tissue is also present in the small intestine and the vermiform appendix (lanning et al., ) . the cecum, a large distensible outpouching of the large intestine, holds up to an estimated % of the total ingesta (sohn and couto, ) . rabbits are considered to be "hindgut fermenters." bacteria present in the cecum ferment the digestible fiber found within the diet. the product of this fermentative process becomes cecotrophs (also known as "night feces"). cecotrophs are excreted roughly h after the initial foodstuffs are ingested (sohn and couto, ) . they are softer and more mucoid in appearance than the hard, dry "day feces" produced just h after consuming food (sohn and couto, ) . the bulk of day feces consists of the indigestible fiber found in the diet. the sorting of foodstuffs destined to become either day feces or cecotrophs and the timing of their relative excretion is largely dependent upon the neural input of the fusus coli, also termed the "pacemaker" of the colon (sohn and couto, ) . the fusus coli is anatomically located between the ascending and transverse colons of the rabbit (popesko, ) . consumption of cecotrophs by rabbits is an important part of the digestive process in rabbits as they are rich in b vitamins, such as niacin and b , and vitamin k (hörnicke, ) . while cecotrophs are known colloquially as "night feces," rabbits produce and eat them at all hours of the day (sohn and couto, ) . rabbits are agile enough to eat these night feces directly from their anus (sohn and couto, ) . those researchers performing digestive research (e.g., fecal collection via metabolism cages) should keep this in mind. sexing of adult rabbits is aided by the sexual dimorphism present in the species. mature females are readily identified by the presence of the dewlap (sohn and couto, ) . females have e nipples, while in males these nipples are present, but rudimentary (sohn and couto, ) . nzw rabbits reach sexual maturity between and months (suckow et al., ) . reproductive data are summarized in table . . mature bucks have paired testicles enclosed in paired hairless scrotal sacs (sohn and couto, ) . like rodents, the inguinal rings do not close. accessory sex glands include several bilobed organs: the seminal vesicle, vesicular gland, prostate, and paraprostatic gland. the bulbourethral glands of bucks are paired (sohn and couto, ) . female rabbits are induced ovulators (dal bosco et al., ; sohn and couto, ) . that is, the egg does not ovulate spontaneously from the ovary, rather manual stimulation via copulation is required. ovulation occurs approximately h postcopulation (sohn and couto, ) . because they are induced ovulators, does do not have a defined estrous cycle. rather, they have periods of sexual receptivity lasting approximately e days followed by e days of nonreceptivity. nonfertile matings may result in a period of pseudopregnancy of up to e days (sohn and couto, ) . fertile matings result in pregnancy lasting e days (sohn and couto, ) . the placenta of rabbits is classified as hemodichorial; this is in contrast to humans which have a hemomonochorial placenta (furukawa et al., ) . birthing (i.e., parturition; also known as "kindling") occurs most often during the early morning hours (sohn and couto, ) . kits are born deaf and blind. by and days of age they can hear and see, respectively (quesenberry and carpenter, ) . amazingly, does suckle their young ones daily, usually during the dark phase, and for approximately only e min (sohn and couto, ) . the kits are able to drink about % of their entire body weight in that time. wild and domesticated does both follow this nursing behavior. rabbit kits may be weaned between and weeks of age (suckow et al., ) . earlier weaning should not be attempted as there may be profound detrimental effects on the functioning of the gastrointestinal system (bivolarski and vachkova, ). rabbits are very social, nocturnal creatures. scent marking is a normal and important part of their behavior repertoire. rabbits will rub the secretions from their chin scent glands against inanimate objects, other rabbits, and human handlers in a process called "chinning" (sohn and couto, ) . dominance hierarchies are established behaviorally. dominant animals may mount, "barber," or scent-mark subordinates (sohn and couto, ) . barbering is the act of chewing the hair of a subordinate animal, usually on the neck and back in the case of rabbits, very close to the skin giving the appearance of having been cut or "barbered" (bays, ). rabbits will "thump" one or both back feet on the ground when frightened or as an alarm call to other rabbits (bays, ) . highly stressed rabbits may actually emit a loud, piercing scream, especially when roughly caught by an untrained individual (bays, ) . it is important to approach rabbits calmly and quietly. relaxed, content rabbits may be heard making a purring sound (bays, ) . rabbits benefit by repeated, positive interactions with people similar to the concept of "gentling" in rats (see section . ). changes in behavior are often first indication that an animal is in pain. given that rabbits are a prey species, it is evolutionarily speaking not in a rabbit's best interest to display signs of pain. thus, these behaviors are most often quite subtle in nature and may be easily missed if particular attention is not paid. the first sign often seen in a rabbit experiencing pain is a decreased appetite resulting in little to no food intake (sohn and couto, ) . rabbits will often grind their teeth (i.e., bruxism) when experiencing pain (sohn and couto, ) . other rabbits may simply appear very dull and inactive. as with rodents, rabbits can develop stereotypies. due to the sensitivity of the rabbit's nose and lips many stereotypies involve chewing behaviors. bar chewing, chewing on the water bottle, and self-barbering are all stereotypical behaviors seen in rabbits (gunn and morton, ; chu et al., ) . in addition, rabbits may engage in "nose sliding" against solid surfaces like the cage walls and head swaying (sohn and couto, ) . animals that exhibit stereotypies do not make good research animals. efforts should be made, where possible, to prevent these behaviors through the use of environmental enrichment. enrichment may be in the form of chew-resistant objects (such as plastic dumbbells and stainless steel rattles) and food treats (poggiagliolmi et al., ). as a prey species, rabbits benefit from the inclusion of a hut in the cage or at least a visual barrier into which they may retreat when psychologically stressed (baumans, ) . breeding females should always have access to a nest box to allow for the necessary expression of normal nesting behavior (baumans, ) . being social creatures, ideally rabbits should be housed in compatible pairs or trios unless contraindicated by the research objectives or by incompatibility of the animals (sohn and couto, ) . stable social groups formed shortly after weaning, where animals are not added or removed, is most beneficial (boers et al., ) . structurally, rabbits benefit from housing that has both adequate vertical and horizontal space (boers et al., ) . one recommendation on the space requirements of laboratory rabbits stipulates in. as the minimum vertical cage height (national research council, ) . at a minimum, rabbits must be able to comfortably sit upright in the cage without their ears bending over (national research council, ) . laboratory rabbits are typically housed in easily sanitized stainless steel cage racks ( fig. . ). slatted flooring allows for urine and feces to fall through the slats onto special pans fixed below the cage, thus providing for easier sanitation of the cages. however, care must be taken that the slats are of sufficient width so as to prevent a condition known as bumblefoot (see section . . ). dog runs with elevated, slatted flooring or a solid floor with bedding have also been used by some investigators in group-housed rabbits with success (personal observations). again, attention should be paid to the flooring and its effect on foot health. commercially available research diets specifically formulated for rabbits are available. these diets are preferred to so-called "natural diets" and feeding individual vegetables. this is because rabbits tend to be very selective eaters which can lead to nutritional imbalances (fraser and girling, ). additionally, the use of fresh vegetables may lead to the introduction of unwanted pathogens like salmonella (varga, ) . commercial diets are available in maintenance and reproductiveperformance dietary formulations as well as presterilized diets for rabbits housed under spf conditions. rabbits are very easily heat stressed and thus must be kept at significantly lower temperatures than other laboratory animals like rats and mice. noise is another significant stressor to rabbits (verga et al., ) . sudden, high-pitched, sharp noises are most disruptive. however, in general, noise within the animal rooms should be avoided as much as possible. for this reason, rabbits should not be housed, even temporarily for short procedures, near areas of high noise. problems in rabbits related to the gastrointestinal system are relatively common. these problems can become serious very quickly. therefore, it is critical that abnormalities seen (e.g., rabbit not eating or abnormal feces) be reported to the veterinary care staff immediately. even if a researcher is unsure if there is a problem, it is best to report suspicions because without prompt intervention, seemingly minor problems can escalate to potentially life-threatening conditions. some of the most commonly seen clinical conditions in rabbits are summarized in table . . the zebrafish, danio rerio of the cyprinidae family, is a small, dark blue and yellow striped, shoaling, teleost fish, popular among aquarium enthusiasts, and increasingly among the research community (fig. . ) . the adult fish are e cm in length, with an incomplete lateral line and two pairs of barbels (laale, ) . males have larger anal fins and more yellow coloration; females have a small genital papilla just rostral to the anal fin (laale, ; creaser, ) . zebrafish are hardy, fresh water fish originating from a tropical region with an annual monsoon season. the fish are generally found among slow moving waters of rivers, streams, and wetlands, across the south asia region of india, bangladesh, example of rabbit caging for a laboratory setting. photo provided by deb hickman. and nepal (engeszer et al., ; spence et al., ) . the waters tend to be shallow, relatively clear with substrates of clay, silt, or stone of varying size (mcclure et al., ; engeszer et al., ) . the fish feed mostly on insects and plankton, with evidence of feeding along the water column as well as water surface (mcclure et al., ; engeszer et al., ; spence et al., ) . gastrointestinal upset generally secondary to the use of broad spectrum antibiotics, such as penicillin. misalignment and subsequent overgrowth of the continuously growing teeth pododermatitis ("bumblefoot") infection of the underside of the feet pasteurella multocida common cause of respiratory infections and abscesses male zebrafish have a more stream-lined body with darker blue strips while the females have a white protruding belly. photo provided by kay stewart. the small size of zebrafish, the ease of keeping large numbers, frequent spawning, large egg clutches, translucent nonadherent eggs, rapid development and complex sequencing of the zebrafish genome are all key components that make the zebrafish an attractive research model. interestingly, approximately % of zebrafish genes have at least one orthologous human gene (howe et al., ) . publications on the use of zebrafish in research are cited as early as the s (creaser, ) . until the early s, the use of zebrafish stayed fairly low, with the number of articles published staying below per year. in the mid- s, publications increased to about per year, doubling again in the s, increasing to almost articles per year in the early s, and rapidly expanding to publications by . developmental biology was the initial focus of zebrafish research use. however, in recent years, use of the zebrafish in research related to biochemistry and molecular biology, cell biology, neurological sciences, and genetics has been rapidly increasing. zebrafish are known to live for only a year in the wild (spence et al., ) . for most of the year, the fish reside in shallow streams. with the onset of monsoon rains, they move to flooded, highly vegetated shallow wetlands and floodplains, including rice paddies, with little to no current and often silt bottoms for spawning (engeszer et al., ) . the offspring then develop in these waters until the seasonal waters diminish (engeszer et al., ) . zebrafish rapidly mature, reaching sexual maturity as early as months postfertilization . the zebrafish continues to grow throughout life, which is much longer in captivity, with a mean lifespan of . years in captivity (gerhard et al., ) . in nature, spawning behavior occurs within small groups of three to seven fish. males within the group pursue females, with spawning occurring along the substrate (spence et al., ) . similar behaviors are noted in laboratory zebrafish, with spawning often occurring with the first light of day. courtship behavior involves a rapid chase of the female, the male swimming around the female, nudging her, or swimming back and forth working the female to the spawning site. interestingly, zebrafish prefer spawning near artificial plants. once there, the male remains close to the female, extending his fins to bring his genital pore in line with the female. the male may also rapidly undulate his tail against the side of the female to initiate egg release by the female, coinciding with sperm discharge by the male. the female produces eggs in batches of e over several encounters with the male for up to an hour. most eggs are released within the first min, with a peak in production during the first min (darrow and harris, ; spence et al., ) . zebrafish produce large clutches of eggs, from to eggs per clutch (laale, ) . the eggs, approximately . mn in diameter, are transparent and protected within a chorionic membrane (kimmel et al., ) . first body movements and beginning stages of organ development occur e h postfertilization (kimmel et al., ) . as development continues, the larva hatches from the egg two to three days postfertilization (kimmel et al., ) . the early larva has special secretory cells within multicellular regions of the head epidermis that allow the larvae to attach to various hard surfaces and plants until the swim bladder inflates or days postfertilization (laale, ; kimmel et al., ) . once the air bladder inflates, the fish can maneuver through the water column. in captivity, zebrafish can breed year round. the presence of males, or even just the male pheromones, is needed to induce ovulation (gerlach, ) . if females are housed away from males for an extended period, they can retain the eggs resulting in egg-associated inflammation, which can be lethal (kent et al., a) . to accommodate the fish life cycle, zebrafish are typically housed in static spawning cages to allow for fertilized egg production. spawning cages include a housing tank containing a clear slotted bottom insert, and a plastic plant. the insert is often placed in the holding tank at an angle to create a shallow region for spawning and the slotted bottom of the insert allows for ease of egg collection (lawrence and mason, ; nasiadka and clark, ) (fig. . ). the embryos are then incubated at around . c in a petri dish for at least e days postfertilization (wilson, ) . the fish are then kept in static or slow water flow containment and can be fed paramecium, rotifer, a powdered food, or a combination of these feed types. unfortunately, other than the need for essential fatty acids in their diet, little is yet known on the nutritional requirements of zebrafish. zebrafish in research settings are typically fed live feed like artemia (brine shrimp), rotifers, bloodworms example of a zebrafish spawning system. the system is designed to allow eggs to fall beneath a slotted insert to the bottom of the tank as a way to prevent the adult fish from consuming the eggs. photo provided by robin crisler. (chironomid larvae), commercial feed, or a combination of all (lawrence, ) . the size of the feed is necessary to suit the gape size of the larvae, approximately mm (lawrence, ; wilson, ) . water flow and feed size increase with development, with transition of the feed to artemia (brine shrimp), and/or use of a larger particle commercial feed during the e days postfertilization (wilson, ) . once the juvenile stage is reached, around days postfertilization, the fish are housed more like adult fish, with more frequent feeding and slower water flow to accommodate their remaining development and smaller size, respectively (wilson, ). as early as months of age the fish are sexually mature . adult zebrafish can be housed in traditional glass aquaria or elaborate computerized and automated systems that monitor and control water quality parameters such as temperature (typically . c), ph, water hardness, salinity, dissolved oxygen, and nitrogenous wastes (lawrence, ; lawrence and mason, ) . whether maintained manually or computerized, these parameters are important to monitor and maintain at appropriate levels to maximize the health of the fish. poor water quality can lead to disease in the fish (kent et al., b) . many of the organisms that cause disease in zebrafish are opportunists in the environment and remain subclinical until the fish is stressed, often due to problems with husbandry. appropriately maintained housing, combined with a healthy water quality, avoidance of overcrowding, and a functional quarantine and health surveillance program are key components to avoiding stress and disease. to date, there are currently no known viruses documented in zebrafish as naturally occurring disease concerns (kent et al., b) . mycobacterium infections are the most frequently documented bacterial infections (kent et al., a,b) . class reptilia is made up of four orders classified as chelonia, rhynchocephalia, squamata, and crocodilia (frye, ) . in class amphibia, animals more commonly encountered in research setting are in the order anura, containing frogs and toads (such as xenopus, bufo, rana, hyla, and dendrobates spp.); and in the order caudata, containing salamanders such as the tiger salamander, ambystoma tigrinum and the axolotl, ambystoma mexicanum (national research council, ) (figs. . and . ). snakes and lizards are in class reptilia, order squamata; chelonians (turtles, tortoises, terrapins) are in order chelonia; and alligators, caimans, and crocodiles are in order crocodilia. in contrast to research in mammals, there is a tendency for reptile and amphibian research to be more oriented to studying evolution and ecology as opposed to basic science evaluating models of human disease (pough, ) . salamanders and frogs are important for studying embryonic development, metamorphosis, regeneration, figure . a commonly used amphibians in research is the axolotl, ambystoma mexicanum. provided by chris konz. african clawed frogs, xenopus laevis, a commonly used amphibian. provided by randalyn shepherd. physiology, and climate change (burggren and warburton, ; hopkins, ; pough, ) . reptiles are often studied because of their more simple cardiovascular systems as well as for evaluating mechanisms of immune responses, hormonal controls, and unique reproduction methods such as parthenogenesis (frye, ) . of the amphibians, xenopus laevis (south african clawed frog) and xenopus tropicalis (western clawed frog) are commonly studied in the research setting. x. laevis is a prominent research model in comparative medicine and developmental studies, and is the most commonly studied species in the genus xenopus (denardo, ; schultz and dawson, ; o'rourke, ) . advantages include large-sized eggs for ease of observing embryo development, as well as the wealth of published literature in areas of research such as evolution, neurobiology, regeneration, endocrinology, and toxicology (koustubhan et al., ; gibbs et al., ) . rana catesbeiana (bullfrogs) have been used for developmental and toxicological studies, and for infectious disease study of the chytrid fungus batrachochytrium dendrobatidis (alworth and vazquez, ) . a. mexicanum, in particular, is studied to understand the regenerative ability of the blastema of amputated limbs at the molecular level (gresens, ; rao et al., ) . ambystoma tigrarium has been studied in regard to general amphibian decline in north america, environmental contaminants such as pesticides and effects of infection with a. tigrarium virus (sheafor et al., ; kerby and storfer, ; chen and robert, ; kerby et al., ) . a variety of snakes, crocodiles, lizards, and turtles have been studied in research. for example, anolis carolinensis (the green anole) has been used for the study of reproduction biology (lovern et al., ) . caiman crocodilus and alligator mississippiensis (crocodiles), trachemys scripta elegans (red-eared sliders) represent a few other examples of reptiles used in research (o'rourke and schumacher, ). amphibians and reptiles are considered to be ectotherms (greene, ) . unlike mammals and birds, ectotherms are unable to internally regulate body temperatures above that of the ambient environment through metabolism and require complex behavioral and thermoregulatory adaptations to regulate temperature (pough, ; seebacher and franklin, ) . in captivity, ectotherms typically require supplemental sources of heat to mimic the thermoregulatory effects of basking in the sun. some amphibians and reptiles are aquatic (xenopus frog spp.) whereas others are semiaquatic or terrestrial. x. laevis and x. tropicalis are from geographically distinct areas and have different temperature requirement depending on life stage, with x. tropicalis adults generally around c in their natural habitat versus about c for x. laevis (tinsley et al., ) . the skin of amphibians is permeable to water and some adults (semiterrestrial tree frogs in family hylidae, arboreal and terrestrial toads in bufonidae) may receive a significant portion of their daily water requirement via absorption through a vascular-rich area on the pelvic area termed the pelvic patch (pough, ; ogushi et al., ) . the skin of some amphibians contains toxins which can cause arrhythmias in human handlers, for example, alkaloids from dendrobatid frogs, and bufotoxins from toads of the genus bufo (denardo, ) . the toxins serve to keep predators away but, as with xenopus, may harm the animals themselves by continued direct contact or diffusion through the water (tinsley et al., ; chum et al., ) . the skin of amphibians is easily damaged thus to protect the animal during handling powder-free gloves should be worn (gentz, ) . researchers and animal care providers should investigate the natural environment of each species within their care and critically evaluate what features are required for normal behavior and physiology to provide the essential elements in the research setting (pough, ) . in the wild, amphibians and reptiles live in ecological environments that span a range of diversity from topical forest areas to dry desert. they may be arboreal, aquatic, or terrestrial. they are often secretive and hide when in natural habitats, preferring to hide under vegetation or in crevices. parameters from the natural habitat to evaluate include temperature, humidity, nutritional requirements, natural diet, nocturnal versus diurnal behavior, and housing density. temperature and lighting gradients should be established so animals can choose to move toward or away from the heat source as a way to avoid overheating. most amphibian species in the wild are nocturnal (pough, ; tinsley et al., ) . amphibians and reptiles are sensitive to chemicals in the environment. water quality parameters (such as ph, hardness, ammonia, nitrate/nitrite, salinity, conductivity) should be regularly monitored. chloramine and chloramines are often present in municipal water supplies and are toxic to aquatic species. water should be treated prior to use for aquatic species with an agent like sodium thiosulfate, since chloramine does not readily dissipate (browne et al., ) . ammonia is a breakdown product between the chloramine and sodium thiosfulfate reaction and is a concern for aquatic animals (browne et al., ; koustubhan et al., ; o'rourke and schultz, ) . a wide variety of caging materials may be used for housing such as glass, plastic, stainless steel, or fiberglass but should be free of contaminants or harmful chemicals like bisphenol a that could leach from the caging into the water (levy et al., ; browne et al., ; bhandari et al., ) . agents used to sanitize caging should be chosen to minimize likelihood of harmful residues. environmental enrichment should be provided to encourage natural behaviors and can include providing cage mates for social interaction, cage accessories that serve as hiding spots or shelters (fig. . ) as well as providing a variety of food treats in changing locations for foraging opportunities (hurme et al., ) . scents, sounds, and color choices may also be incorporated into enrichment strategies provided that they are carefully evaluated to ensure that they are beneficial and do not cause stress. for example, the tortoise, chelonoidis denticulata, may show a color preference for red-colored enrichment items (passos et al., ) . pvc tubes are another example of enrichment that has been provided to x. laevis for use as hiding cover (koustubhan et al., ) . some species may require haul-out ramps, areas for sun basking, floating rest areas, or enrichment devices along the water's surface to help prevent drowning. one should consider the possibility of ingestion, as reptiles and amphibians may attempt to consume the substrates provided to them. the degree to which amphibians are social varies significantly depending on the species and is not always well understood. they use visual and olfactory discrimination to help them find food, forage, and avoid predators (vitt and caldwell, ) . both in the wild and in captivity, reptiles and amphibians may exhibit excitatory behavior when fed (sometimes described as a "feeding frenzy") which may result in animal injury where animals are in close proximity (divers and mader, ; tinsley et al., ) . overcrowded tanks can result in competition for food and subsequent trauma. thus, when placed together for the first time, animals should always be observed for compatibility; and only members of the same species should be housed together. many reptiles and amphibians are escape artists and prevention of escape and injury is a critical factor when considering housing design. species that are prone to jumping must have secured lids on their enclosures. the diets of amphibians and reptiles are highly variable in the wild and are species dependent. commercially prepared pelleted diets may be available and accepted by reptiles and aquatic amphibians, however, terrestrial amphibians and many use of a rabbit feeder for xenopus enrichment. photo by randalyn shepherd. reptiles may prefer live diets (pough, ) . it is not unusual for some species to go several days of fasting between meals in nature (pough, ) . consultation with those experienced at successful housing and feeding the species in question (zoos, nutritionists, herpetologists) is recommended. there are many different types of infectious agents such as bacteria, viruses, fungi, and parasites that can cause health problems in amphibians and reptiles in addition to noninfectious conditions such as those resulting from nutritional imbalances, metabolic disease, neoplasia, trauma, and other spontaneous maladies. although significant advances in knowledge have been made over the past years regarding disease in these species, much still remains unknown. it is not possible to go into detail here, but there are excellent reference texts for diseases in amphibians and reptiles that can be consulted (jacobson, ; frye, ; wright and whitaker, ) . from a taxonomic standpoint, birds are placed into class aves which includes multiple orders based on anatomical, physiological, and genetic characteristics. passeriformes is the largest order and contains songbirds and perching birds such as the finch, canary, and cardinal ( fig. . ). order columbiformes contains pigeons and doves; order psittaciformes contains budgies and parrots such as the african gray; and order galliformes contains domestic fowl such as the chicken and quail (proctor and lynch, ; ritchie et al., ) (fig. . ). birds have been used as research models of human disease and are important in evaluation of aging, memory, parasitology, atherosclerosis, reproduction, and infectious disease among other topics (austad, (austad, , maekawa et al., ) . the genomes of several avian species have now been sequenced (jarvis et al., ) . historically, chickens (gallus domesticus) are the most common bird species studied in biomedical and agricultural research and are a classic model in areas such as immunology, virology, infectious disease, embryology, and toxicology (scanes and mcnabb, ; kaiser, ) . chickens are also studied to evaluate reproductive development and retinal disease. embryonated chicken eggs have been used to commercially produce vaccines (such as for human influenza), studied for developmental analysis, and are now being treated with viral vectors like lentivirus to produce transgenic embryos. inbred lines with improved disease resistance are being developed and transgenic technology in the future may allow embryos to be used as bioreactors to produce therapeutic proteins of interest and potentially to generate transgenic chickens which have improved resistance to pathogens (bacon et al., ; scott et al., ) . because chickens develop spontaneous ovarian cancers at an incidence of up to %, they are also a prominent model of ovarian cancer in humans (bahr and wolf, ; hawkridge, ) . quail the zebra finch is a common avian species used in research. from http://www.redorbit.com/news/science/ /male-zebra-finches-fake-song- /. the domesticated chicken commonly used in research. provided by kay stewart. (coturnix coturnix and coturnix japonica) have been studied in many of the same research disciplines as chickens, but offer advantages because of their smaller size and because they are among the shortest-lived bird species (austad, ) . japanese quail (c. japonica) have been selected as a model to evaluate reproductive biology and social behaviors such as mate selection because they readily show sexual behavior in captivity (ball and balthazart, ) . as with the chicken, methods to study transgenic quail are now becoming available and offer a useful tool to study gene function (seidl et al., ) . of the psittaciformes, amazon parrots and budgies (melopsittacus undulates) are among the most commonly studied, with research topics including veterinary medicine, diagnostics, behavioral, cognition, aging, and sensory studies (austad, ; kalmar et al., ) . the african gray parrot has been studied for its cognition and communication abilities (hesse and potter, ; harrington, ) . of the passerines studied in laboratory research, the most commonly evaluated include the zebra finch (taeniopygia guttata), european starling (sturnus vulgaris and sturnus roseus), and house sparrow (passer domesticus) (bateson and feenders, ). zebra finches and other songbirds are commonly studied in regard to aging and neurogenesis in addition to speech, learning, and memory because of their ability to learn and communicate intricate bird songs (harding, ; scott et al., ; austad, ; mello, ) . the most popular songbird species for neurobiological research include the zebra finch, canary, and other types of small finches such as lonchura striata domestica (schmidt, ) . zebra finches are favored in research settings since they are easy to house due to their small size, for their compatibility in groups, and proclivity for breeding. they are also studied for their biologic features such as sexual dimorphism, year-round singing in captivity, age-dependent period of song-learning propensity, and for ease of measurement with respect to their bird song (fee and scharff, ; mello, ) . pigeons (columba livia) have been evaluated in areas such as comparative psychology, neuroanatomy, neuroendocrinology, and atherosclerosis (santerre et al., ; austad, ; shanahan et al., ) . they are studied to understand their navigational skills and memory which allow homing, vision and discrimination ability. barn owls (tyto alba) are an example of a nocturnal avian species and are studied for neuroanatomy, vision, hearing, and for understanding learning mechanisms during auditory space mapping (pena and debello, ; rosania, ) . birds are warm-blooded vertebrates that have feathers for the purpose of flight and plumage. their respiratory system includes avascular air sacs, some of which attach to the lung and bronchi, but do not serve as sites for gas exchange as does the lung (maina, ; ritchie et al., ) . air sacs serve as internal compartments which hold air and facilitate internal air passage to allow birds to have a continuous flow of large volumes of air through the lungs as a way to increase oxygen exchange capacity and efficiency. birds lack a functional diaphragm and use muscles of the thorax to assist with respiration (ritchie et al., ) . care must be taken to ensure that use of physical restraint does not interfere with respiratory movement, cause the bird to struggle, or become stressed. the skeletal system includes pneumatic bones which are lined with air sac epithelium and are considered pneumatized by connection to the respiratory system (frandson et al., ). the specific bones which are pneumatized depend on the species but typically include the humerus, cervical vertebrae, sternum, sternal ribs, and sometimes the femur (ritchie et al., ) . the esophagus in birds leads to the crop, which is an outpocketing where food is held temporarily, and then continues to the proventriculus (also called the true stomach) which produces enzymes to break down food. food travels from the proventriculus to the ventriculus (gizzard) and then on into the small and large intestines. the presence or absence of a gallbladder is species dependent (tully et al., ; kalmar et al., ) . the rectum and urinary tract terminate in the cloaca, resulting in excreta where the fecal portion of waste is mixed with urate (white and/or creamy component). there are many additional unique and complex anatomic and physiologic adaptations of birds. other excellent references are available in the literature (scanes, ). housing requirements of birds held in captivity vary significantly depending on the particular species. basic parameters that apply to all birds include the necessity to provide an enclosure which is safe and permits species-specific behaviors to the greatest extent possible. consideration should be given to ensure that the type of structure is nontoxic, as some birds such as parrots have a powerful beak with the ability to chew through substrates. enclosures may be made of metals or durable plastic, but it is important to note that zinc wire, as well as leaded paint, can be toxic to birds and is best avoided. bar spacing on caging should be appropriate to prevent escape and injury based on the size of the bird. caging size varies and can include large aviaries where full short-distance flight is possible, to individual housing in smaller sized cages where flight may not be feasible. use of environmental enrichment and provision of opportunity for interaction is important to include as part of the cage structure, complexity, and social dynamic. some types of birds are considered social, polygamous, and benefit from group housing, whereas others such as those that pair-bond (such as new world quail) may prefer housing with a single mate (ritchie et al., ) . some species, genders, or individuals show aggression and may not be compatible. for example, sexually active male quail may injure each other and are generally considered incompatible (huss et al., ) . to help reduce aggression, housing densities should be kept low and multiple points of access to resources, such as feed and perches, should be provided. enrichment in the form of manipulanda can take the form of toys and food items. some types of birds may demonstrate foraging behavior in nature and may like to manipulate their feed. parrots, for example, typically grasp their food with their feet and may peel or strip the outer portion of the foodstuff prior to ingesting. toys should be size appropriate for the species, easily sanitized, free from sharp edges, and replaced once wear shows. birds can become easily caught in items that hang from the cage and as toys deteriorate they can become a hazard. for example, rope toys may begin to fray and become a hazard, causing entrapment; and some types of toys contain weights which pose a choking hazard or may be made of toxic materials such as lead. some types of birds spend considerable time perching and require perches, which vary in diameter, for comfort and to prevent pressure sores from developing on their feet. the respiratory system of the bird is very sensitive and caution must be taken by animal care staff to avoid exposure of birds to aerosols from chemicals that may arise from disinfectants used in the laboratory animal facility. scented cleaners, perfumes, hairspray, and emissions from teflon-coated materials are all examples of products which can be especially harmful to birds and may cause death. feeding requirements vary by species and life stage, but commercial pelleted diets designed to meet the nutritional needs can generally be provided. although many birds are seed eaters, a diet of seeds alone is unlikely to provide adequate or balanced nutrition. many birds have a requirement for dietary calcium, especially those that are reproductively active, and should be provided with calcium supplementation in the form of soluble grit such as cuttlebone or crushed oyster shells (sandmeier and coutteel, ; tully et al., ). birds often display neophobic behavior and may require long acclimation periods before fully accepting novel foodstuffs. for this reason, dietary changes should not be made abruptly and daily intake should be closely monitored. for birds in the laboratory setting, clean, fresh water should be provided daily either by use of nonbreakable bowls or sipper tubes. water intake will vary by species and environmental housing conditions. birds can mask disease and are easily stressed. it is best to first observe the bird in its normal home environment whenever possible and only perform restraint for physical exam or collection procedures when indicated. general indications of sickness may include decreased appetite, depressed behavior, loose stools, distended abdomen, ruffled feathers or unkempt appearance, skin lesions, openmouth breathing, abnormal respiratory sounds such as wheezing or sneezing, or signs of dehydration such as reduced skin turgor and sunken eyes. a healthy bird should have well-groomed feathers, appear alert, active and inquisitive, and should show species-typical behaviors. its eyes should be clear and bright. no evidence of discharge should be present from the eyes, nares, mouth, or urogenital area. numerous types of infectious (example, fig. . ) and noninfectious disease presentations are described in birds. additional reference resources should be consulted for in-depth information (ritchie et al., ; tully et al., ; doneley, ) . to provide the reader a broader view of animal use in research, descriptions of some less commonly used small mammal models follow. guinea pigs (cavia porcellus) are rodents, related to porcupines and chinchillas in the suborder hystricomorpha (fig. . ) . they originate from the mountain and grassland regions along the mid-range of the andes mountains in south america. they are small, stocky, nonburrowing, crepuscular herbivores with short legs and little to no tail, ranging from to g, females being smaller than males (harkness et al., ) . guinea pigs have a long-standing historical role in research stretching as far back as the s, when they were first used in anatomical studies (pritt, ) . further, they were used by louis pasteur and robert koch in their example of skin pox on the feet of a dark-eyed junco (junco hyemalis). photo from randalyn shepherd. investigations of infectious disease, and have contributed to the work of several nobel prize worthy studies (pritt, ) . specifically, the guinea pig has been used as a model for infectious diseases such as tuberculosis, legionnaires disease, sexually transmitted diseases such as chlamydia and syphilis, and one of the more common causes of nosocomial infections in people, staphylococcus aureus (padilla-carlin et al., ) . guinea pigs have also been useful tools in researching cholesterol metabolism, asthma, fetus and placental development and aspects of childbirth, as well as alzheimer's disease (bahr and wolf, ) . guinea pigs have many similarities to humans hormonally, immunologically, and physiologically. unlike other rodents, and more like primates (including people), guinea pigs are prone to scurvy if they do not receive adequate vitamin c, typically in their diet (gresham et al., ) . guinea pigs are housed similarly to other rodents, although they require more room than the smaller rodents. hamsters are of the rodentia order, suborder myomorpha along with the mouse and the rat. there are over species of hamsters described in the literature, with the most common hamster used in research being the golden or syrian hamster, mesocricetus auratus (harkness et al., ) (fig. . ) . originating from the northwest region of syria, golden hamsters are thought to be descendants of only three or four littermates collected from syria in (adler, ; smith, ) . as their name implies, the typical wild-type coat is reddish gold along their dorsum, with a gray underside. they are granivores and insectivores, weighing e g, females weighing more than males, with short legs and short tail, and large cheek pouches (harkness et al., ) . specific anatomical and physiological features including their susceptibility to disease and infection make them a useful model for study. initially hamsters were utilized in studies of infectious disease, parasitology and dental disease, transitioning into cancer research in the s (smith, ) . hamsters are still used in many areas of research, including investigations into metabolic diseases like diabetes mellitus (hein et al., ) , cardiovascular disease (russell and proctor, ) , reproductive endocrinology (ancel et al., ) , and oncology (tysome et al., ) . guinea pigs have also been used as models for infectious disease associated with bacteria, parasites, and viruses, such as leptospirosis (harris et al., ), leishmaniasis (gomes-silva et al., , and severe acute respiratory syndrome (sars) and ebola viruses (roberts et al., ; wahl-jensen et al., ) . other species of hamsters used have been used in research. for example, chinese and african hamsters have been used for investigations into diabetes mellitus (kumar et al., ) ; european and turkish hamsters have been useful to evaluate aspects of hibernation (batavia et al., ) ; and siberian and turkish hamsters have been used to study circadian rhythm and pineal gland activity (butler et al., ) (fig. . ) . chinchillas (fig. . ) are in the order rodentia, suborder hystricomorpha, as are the guinea pig and the degu. there are the long-tailed chinchilla, chinchilla lanigera, and the short-tailed chinchilla, chinchilla chinchilla. chinchillas originate from the andes mountains of south america (martin et al., ) . they are e g in size, females weighing more than males, with compact bodies and long, strong hind limbs and dense fur coats (alworth et al., ) . the lushness of the coat is what led them close to extinction in the wild due to excessive hunting siberian hamsters. photo from greg demas. chinchilla. photo from bill shofner jr. in the early to mid- s (jimenez, ) . the chinchilla has a large head, large eyes and ears. the large inner ear anatomy is of specific note as chinchillas are the traditional model for auditory studies (shofner and chaney, ) and otitis media (morton et al., ) . the gerbil is a rodent, suborder myomorpha, used in research. there are over species of gerbil-like rodents documented, but the mongolian gerbil (meriones unguiculatus) is the species most commonly used in the united states (fig. . ). mongolian gerbils originate from a desert terrain in mongolia and northeast china. they are long-tailed, burrowing, herbivorous rodents, e g in size, males being larger than females (harkness et al., ) . due to anatomical variations in the blood supply to the brain in an anatomical region known as the "circle of willis," gerbils have been used most notably as a model for cerebral ischemia or stroke (small and buchan, ) . an interesting animal model to note among the small mammals is the nine-banded armadillo (dasypus novemcinctus), a new world mammal ranging from the southeastern half of north america, extending south through the americas to the northern region of argentina (balamayooran et al., ) . armadillos have a banded carapace, and, importantly, a low core body temperature of e c. the breeding season is in the summer, but embryo implantation is delayed until late fall, at which gerbil. photo used with permission of american association for laboratory animal science. point identical quadruplicates are always formed (balamayooran et al., ) . the armadillo's low body temperature, and susceptibility and physiologic response to the infectious organism, mycobacterium leprae, have made it an ideal model for studying leprosy (balamayooran et al., ) . the consistent polyembryony of the species has also made the animal a model of interest in understanding various aspects of twinning (blickstein and keith, ) . choosing the correct animal model is an essential component to the success of biomedical research. each species used in biomedical research must be provided with adequate housing and care to ensure the well-being of the animals. because good science and good animal care go hand in hand, it is important to understand and address the biological and behavioral needs of the animals being studied. origin of the golden hamster cricetus auratus as a laboratory animal chinchillas: anatomy, physiology and behavior a novel system for individually housing bullfrogs american fancy rat and mouse association the effects of osteoporosis on distraction osteogenesis: an experimental study in an ovariectomised rabbit model birds as models of aging in biomedical research a review of the development of chicken lines to resolve genes determining resistance to diseases comparison of behavioural development in socially isolated and grouped rats the armadillo as an animal model and reservoir host for mycobacterium leprae japanese quail as a model system for studying the neuroendocrine control of reproductive and social behaviors the effects of day length, hibernation, and ambient temperature on incisor dentin in the turkish hamster (mesocricetus brandti) environmental enrichment for laboratory rodents and rabbits: requirements of rodents, rabbits, and research the black death, e : the complete history effects of the environmental estrogenic contaminants bisphenol a and alpha-ethinyl estradiol on sexual development and adult behaviors in aquatic wildlife species morphological and functional events associated to weaning in rabbits on the possible cause of monozygotic twinning: lessons from the -banded armadillo and from assisted reproduction biology of the rabbit facility design and associated services for the study of amphibians tickling induces reward in adolescent rats ultrasonic vocalizations as indicators of welfare for laboratory rats (rattus norvegicus) a melatonin-independent seasonal timer induces neuroendocrine refractoriness to short day lengths repetitive backflipping behaviour in captive roof rats (rattus rattus) and the effects of cage enrichment characterization of a new experimental model of osteoporosis in rabbits . the septum atriorum of the frog and the rabbit antiviral immunity in amphibians a behavioral comparison of new zealand white rabbits (oryctolagus cuniculus) housed individually or in pairs in conventional laboratory cages the social buffering effect of playful handling on responses to repeated intraperitoneal injections in laboratory rats the technic of handling the zebra fish (brachydanio rerio) for the production of eggs which are favorable for embryological research and are available at any specified time throughout the year ovulation induction in rabbit does: current knowledge and perspectives characterization and development of courtship in zebrafish, danio rerio amphibians as laboratory animals a good practice guide to the administration of substances and removal of blood, including routes and volumes avian medicine and surgery in practice a critical period for social isolation in the rat reproductive and developmental toxicity of hydrofluorocarbons used as refrigerants zebrafish in the wild: a review of natural history and new notes from the field the songbird as a model for the generation and learning of complex sequential behaviors detection of zoonotic pathogens and characterization of novel viruses carried by commensal rattus nor rabbit as a reproductive model for human health the rabbit as a model for reproductive and developmental toxicity studies anatomy and physiology of farm animals rabbit medicine and surgery for veterinary nurses biomedical and surgical aspects of captive reptile husbandry a comparison of the histological structure of the placenta in experimental animals social and husbandry factors affecting the prevalence and severity of barbering ('whisker trimming') by laboratory mice evidence for a relationship between cage stereotypies and behavioural disinhibition in laboratory rodents heat or insulation: behavioral titration of mouse preference for warmth or access to a nest impact of nesting material on mouse body temperature and physiology nest building as an indicator of health and welfare in laboratory mice energy reallocation to breeding performance through improved nest building in laboratory mice medicine and surgery of amphibians pheromonal regulation of reproductive success in female zebrafish: female suppression and male enhancement metamorphosis and the regenerative capacity of spinal cord axons in xenopus laevis golden hamster (mesocricetus auratus) as an experimental model for leishmania (viannia) braziliensis infection nonavian reptiles as laboratory animals an introduction to the mexican axolotl (ambystoma mexicanum) guinea pigs: managment, husbandry and colony health inventory of the behaviour of new zealand white rabbits in laboratory cages comparison of mouse and rabbit model for the assessment of strong pgm-containing oil-based adjuvants computerized analysis of audible and ultrasonic vocalizations of rats as a standardized measure of pain-related behavior review of polyclonal antibody production procedures in mammals and poultry learning from bird brains: how the study of songbird brains revolutionized neuroscience rabbit mitochondrial dna diversity from prehistoric to modern times harkness and wagner's biology and medicine of rabbits and rodents speaking of psittacine research in vitro and in vivo activity of first generation cephalosporins against leptospira the chicken model of spontaneous ovarian cancer glp- and glp- as yin and yang of intestinal lipoprotein production: evidence for predominance of glp- -stimulated postprandial lipemia in normal and insulin-resistant states a behavioral look at the training of alex: a review of pepperberg's the alex studies: cognitive and communicative abilities of grey parrots. anal. verbal behav use of a body condition score technique to assess health status in a rat model of polycystic kidney disease extreme cuisine: the weird & wonderful foods that people eat amphibians as models for studying environmental change tickling during adolescence alters fear-related and cognitive behaviors in rats after prolonged isolation utilization of caecal digesta by caecotrophy (soft faeces ingestion) in the rabbit the zebrafish reference genome sequence and its relationship to the human genome environmental enrichment for dendrobatid frogs japanese quail (coturnix japonica) as a laboratory animal model social isolation rearinginduced impairment of the hippocampal neurogenesis is associated with deficits in spatial memory and emotion-related behaviors in juvenile mice teratogenic effects of thalidomide: molecular mechanisms infectious diseases and pathology of reptiles: color atlas and text effect of astragaloside iv on the embryo-fetal development of sprague-dawley rats and new zealand white rabbits the extirpation and current status of wild chinchillas chinchilla lanigera and c-brevicaudata audible and ultrasonic vocalization elicited by single electrical nociceptive stimuli to the tail in the rat the long view: a bright past, a brighter future? forty years of chicken immunology pre-and post-genome guidelines and ethical considerations for housing and management of psittacine birds used in research documented and potential research impacts of subclinical diseases in zebrafish diseases of zebrafish in research facilities combined effects of virus, pesticide, and predator cue on the larval tiger salamander (ambystoma tigrinum) combined effects of atrazine and chlorpyrifos on susceptibility of the tiger salamander to ambystoma tigrinum virus stages of embryonic development of the zebrafish establishing and maintaining a xenopus laevis colony for research laboratories acute and chronic animal models for the evaluation of anti-diabetic agents the biology and use of zebrafish, brachydanio rerio in fisheries research. a literature review development of the antibody repertoire in rabbit: gut-associated lymphoid tissue, microbes, and selection the husbandry of zebrafish (danio rerio): a review generation time of zebrafish (danio rerio) and medakas (oryzias latipes) housed in the same aquaculture facility zebrafish housing systems: a review of basic operating principles and considerations for design and functionality historical perspectives the effects of methotrexate on pregnancy, fertility and lactation the green anole (anolis carolinensis): a reptilian model for laboratory studies of reproductive morphology and behavior the mechanisms underlying sexual differentiation of behavior and physiology in mammals and birds: relative contributions of sex steroids and sex chromosomes development, structure, and function of a novel respiratory organ, the lung-air sac system of birds: to go where no other vertebrate has gone the aerosol rabbit model of tb latency, reactivation and immune reconstitution inflammatory syndrome chinchillas: taxonomy and history notes on the natural diet and habitat of eight danionin fishes, including the zebrafish danio rerio animal models of atherosclerosis progression: current concepts the zebra finch, taeniopygia guttata: an avian model for investigating the neurobiological basis of vocal learning neurological diseases of rabbits and rodents a functional tonb gene is required for both virulence and competitive fitness in a chinchilla model of haemophilus influenzae otitis media a rabbit model of non-typhoidal salmonella bacteremia neuroevolutionary sources of laughter and social joy: modeling primal human laughter in laboratory rats blood sample collection in small laboratory animals positive and negative ultrasonic social signals elicit opposing firing patterns in rat amygdala enriching tortoises: assessing color preference shelter enrichment for rats environmental enrichment of new zealand white rabbits living in laboratory cages recommendations for the care of amphibians and reptiles in academic institutions manual of ornithology: avian structure & function ferrets, rabbits, and rodents: clinical medicine and surgery proteomic analysis of fibroblastema formation in regenerating hind limbs of xenopus laevis froglets and comparison to axolotl inner workings: , the first rabies vaccination in humans avian medicine: principles and application behavioural and neurochemical effects of early social deprivation in the rat immunogenicity and protective efficacy in mice and hamsters of a beta-propiolactone inactivated whole virus sars-cov vaccine ny) , . roy, c.s., . the form of the pulse-wave: as studied in the carotid of the rabbit small animal models of cardiovascular disease: tools for the study of the roles of metabolic syndrome, dyslipidemia, and atherosclerosis normal development of the testes management of canaries, finches, and mynahs spontaneous atherosclerosis in pigeons. a model system for studying metabolic parameters associated with atherogenesis an iacuc perspective on songbirds and their use in neurobiological research housing and husbandry of xenopus for oocyte production applications of avian transgenesis physiological mechanisms of thermoregulation in reptiles: a review transgenic quail as a model for research in the avian nervous system: a comparative study of the auditory brainstem large-scale network organization in the avian forebrain: a connectivity matrix and theoretical analysis antimicrobial peptide defenses in the salamander the watanabe heritable hyperlipidemic (whhl) rabbit, its characteristics and history of development: a tribute to the late dr development of an animal model for spontaneous myocardial infarction (whhlmi rabbit) processing pitch in a nonhuman mammal (chinchilla laniger) abnormal arrangement of the right subclavian artery in a rabbit anatomy, physiology, and behavior mitochondrial dna phylogeography of the norway rat mandibular bone density and fractal dimension in rabbits with induced osteoporosis. oral surg the behaviour and ecology of the zebrafish, danio rerio assessment of anti-scarring therapies in ex vivo organ cultured rabbit corneas vascular access port (vap) usage in large animal species the victorian underworld amphibians, with special reference to xenopus handbook of avian medicine a novel therapeutic regimen to eradicate established solid tumors with an effective induction of tumorspecific immunity effects of husbandry and management systems on physiology and behaviour of farmed and laboratory rabbits herpetology: an introductory biology of amphibians and reptiles use of the syrian hamster as a new model of ebola virus disease and other viral hemorrhagic fevers pseudomonas aeruginosa infectious keratitis in a high oxygen transmissible rigid contact lens rabbit model. invest. ophthalmol amphibian medicine and captive husbandry animal models for the atherosclerosis research: a review construction of corneal epithelium with human amniotic epithelial cells and repair of limbal deficiency in rabbit models rats, lice, and history key: cord- -mat ul c authors: johnson, kristen a title: the future of animal science departments date: - - journal: anim front doi: . /af/vfaa sha: doc_id: cord_uid: mat ul c nan who challenges land grant universities in the united states to return to the founding land grant principles and to recognize the importance of a community-based mission for the st century. his ideas provide an excellent foundation for articles from argentina, china, italy, and south africa that describe the future of animal science research and teaching programs in these countries. workforce training is discussed in the article by dr gustavo jaurena and dr maria boveri (jaurena and boveri, ) from argentina. they highlight the need for the incorporation of multidisciplinary training and thinking in the animal sciences curriculum and describe opportunities to embrace technology in information transfer in outreach and teaching. rulien grobler and her colleagues (grobler et al., ) describe the dynamic animal sciences programs in south africa and identify the need for flexibility and embracing technologies in training future animal scientists. the impact of covid- and the need to embrace technology has reinforced the points made by grobler et al. ( ) . faculty across the world have been forced to learn and incorporate online teaching with only a few days or weeks to prepare. a perspective from some faculty experiencing this rapid, radical change identifies some ideas and challenges for the future is included (radcliffe et al., ) . much has been learned from the covid- pandemic that will be useful and could revolutionize how information is shared: potentially meeting some of the foundational principles gavazzi ( ) discusses. the wef publication ( ) also suggests public-private and cross industry partnerships as change agents and mechanisms by which the talent pool could be increased (schwab and samans, ) . these same themes are found in this issue from contributions across the world. baldi et al. ( ) describes the growing importance of public-private relationships in research and training in italy. animal science research and teaching is also becoming enriched by public-private partnerships in china. dr. jingdong yin and dr. zhengpeng zhu (yin and zhu, ) highlight the need for these relationships to ensure students are well-trained for the developing, high demand careers in china which require critical thinking skills and multidisciplinary approaches to animal production. as new methods for teaching are developed and used to meet the needs and careers of the future, animal and veterinary sciences must insure the effectiveness of the educational program offered. accreditation is one way in which animal science programs can identify and address their strengths and weaknesses and assure a common set of learning goals are obtained. one such program is described by benson et al. ( ) . the principles described in this article provide a process for accreditation of animal science programs. the similarity of challenges and needs for animal and veterinary training programs identified by the authors in this issue is both striking and comforting. sharing ideas, successes, failures and preparations for the future such as those in this issue is critical. the talent, creativity and dedication across the global animal science community makes the future of animal science departments bright. in the middle of preparing this issue of animal frontiers for publication, the sars-cov- virus made many issues for the future take a back seat to other compelling challenges faced by the authors. a primary challenge included moving face-to-face teaching and outreach to on-line platforms immediately, research programs were temporarily curtailed or downsized and people remained in their homes. we are very grateful to the authors who persevered in the submission and review of these articles despite the personal and professional difficulties they faced in their countries and in their personal lives. perspectives for the future in italy: animal science higher education, employment and research animal science program accreditation? this is the future the land-grant mission in the st century: promises made and promises to be kept training the next generation for animal sciences for south africa the future of animal sciences in argentina organization of afro-american unity founding forum at the audubon ballroom perspective: moving online: roadmap and long term forecast the fourth industrial revolution: what it means and how to respond global challenge insight report. the future of jobs: employment, skills and workforce strategy for the fourth industrial revolution perspectives for the future in china. challenges and opportunities facing animal teaching, research and industry key: cord- -ju vuywm authors: rohde, rodney e. title: common myths and legends of rabies date: - - journal: rabies doi: . /b - - - - . - sha: doc_id: cord_uid: ju vuywm humankind has somewhat of a dark, yet almost fascinating, supernatural relationship with rabies. even after pasteur's rabies vaccine discovery, globally people continue to be stricken with it today. history has carried along the myths and legends that surround this diabolical virus. some still believe that rabies treatment requires or more shots to the stomach by some monstrously long needle. however, today's treatment regimen is typically only four vaccinations (five for immunocompromised) in the arm, plus human rabies immune globulin. this chapter explores the misunderstood concepts of rabies prevalence, signs and symptoms, exposures, and treatment protocols. rabies has been a part of human history for as long as it has been recorded in writing and art. from odysseus's story to achilles, the actual word (term) that provoked and transformed hector to a rage and frenzy, "lyssa," is closely linked to the word "lykos" or "wolf" and is used to invoke images and feelings of an animal's anger, madness, and wolfish rage. lyssavirus is a genus of rna viruses in the family rhabdoviridae, order mononegavirales. lyssaviruses are bullet-shaped, single-stranded, negative-sense rna viruses and the causative agents of the ancient zoonosis rabies. africa is the likely home to the ancestors of taxa residing within the genus lyssavirus, family rhabdoviridae. diverse lyssaviruses are envisioned as coevolving with bats, as the ultimate reservoirs, over seemingly millions of years ( fig. . ). through eons of time, a wide range of myths and legends have developed pertaining to rabies. many people still believe today that rabies treatment requires or more shots into the stomach by some monstrously long needle. while in fact, today's treatment regimen is typically only four vaccinations (five for immunocompromised individuals) in the arm, plus a dose of humane rabies immune globulin (hrig). this myth is but one of a long list of questions, untruths, or exaggerations about the disease known as rabies. • is there a way to vaccinate wildlife using a recombinant vaccine dropped from aircraft? [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] • can scientists actually determine the origin of a rabies virus variant by way of genetic testing and methodologies? [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] • are there special government laboratories that conduct specialized rabies testing? [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] • can international public health teams actually halt and remove a moving viral epizootic outbreak in a geographical area? [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] • is there a relationship between rabies and zombies? in this chapter, some of the more common misconceptions (and facts) about rabies will be described. these misconceptions are categorized under concepts of prevalence, signs and symptoms, exposure, treatment, and pop culture influence with respect to rabies. where exactly do most cases of rabies occur in the world? which type of animals are often more "at risk" for contracting rabies virus, otherwise known as highrisk animals? are there places in the world where one might travel without worrying about rabies? do coldblooded animals, insects, or birds have a risk for acquiring this deadly disease? there are many untruths to unpack with respect to the prevalence of rabies. learn more in the following accounts: • many people believe that rabies infections only occur in poor, third-world countries. however, the truth is that rabies or rabies-like viruses are present on nearly every continent, with the exception of antarctica. numerous and diverse variants of lyssaviruses are found in a wide variety of animal species throughout the world, all of which may cause fatal human rabies. rabies virus is by far the most common lyssavirus infection of humans. in the united states, only hawaii is rabies free. interestingly, in hawaii and other rabies-free areas that are often islands, there are very strict laws and restrictions on the transport of animals from rabies-endemic states or countries to rabies-free geographic locations. in the united states, rabies as a disease is most prevalent along the east coast (raccoon and bat rabies virus variants) from florida to maine and in southern arizona (arizona gray fox) along the mexican border. rabies is also prevalent in texas with south central skunk and bat rabies virus variants predominating. common myths and legends of rabies rodney e. rohde, phd, ms, sm(ascp) cm , sv cm , mb cm , facsc as a side note, one should always consider the risk of infectious disease when traveling to other countries by discussing travel plans with an appropriate person such as a travel physician or other health-care professional. there may be a need to plan for preexposure vaccinations (e.g., rabies, yellow fever, etc.) or medications (e.g., malaria, antibiotics) to take on their travels. • a frequent belief is that this disease kills many people in the united states. however, approximately % of human deaths from the disease occur in africa and asia, where access to health-care facilities and treatment protocols is limited. the estimated annual figure of almost , rabies fatalities in humans is probably an underestimate. almost all cases of rabies in humans worldwide result from bites from infected dogs. table ). • all warm-blooded animals can contract rabies, particularly mammals. a viral disease of the central nervous system, rabies transmits between animals, including humans, when saliva containing the virus enters an opening in the skin. usually, the rabies virus enters through the bite of a rabid animal, but transmission can also occur when infected saliva enters through mucous membranes or a break in the skin. viral familial relatives can be found with invertebrates and plants (distant hosts), but warmblooded vertebrates are the hosts for the rabies virus. among warm-blooded vertebrates, birds are susceptible to infection, but rabies predominates naturally among various mammalian populations. only rare accounts occur in nonmammalian hosts, but in the mammalia family, rabies cases have occurred from the armadillo to the zebra. rabies is a significant disease of domestic and wild mammals alike, yet its zoonotic aspect is the cause of major historical infamy. according to the centers for disease control and prevention (cdc), the first clinical signs and symptoms of rabies may be very similar to those of the flu including general weakness or discomfort, fever, or headache. these clinical signs and symptoms may last for days and are directly related to the difficulty of a differential diagnosis of rabies in the absence of an obvious animal bite (or other) exposure. there may be also discomfort or a prickling or itching sensation at the site of bite, progressing within days to signs and symptoms of cerebral dysfunction, anxiety, confusion, and agitation. as the disease progresses, the person may experience delirium, abnormal behavior, hallucinations, and insomnia. the acute period of disease typically ends after - days (see chapter ). once clinical signs of rabies appear, the disease is nearly always fatal, and treatment is typically supportive. disease prevention includes both passive immunity, through an injection of human rabies immune globulin, and active immunity produced via a round of injections with rabies vaccine. if a person has already begun to exhibit signs of the disease, survival is rare. to date, less than documented cases of human survival from clinical rabies have been reported and only two have not had a history of pre-or postexposure prophylaxis. one of the more bizarre, but not uncommon, signs and symptoms of rabies that has been reported is male patients exhibiting hypersexual behavior. the virus sometimes acts on the limbic system of the brain causing men to show this behavior: increased sexual desire, involuntary erections, and in some reports continuous orgasms occurring at a rate of one per hour! in general, most people think of dogs when the topic of animals that can carry or transmit rabies is mentioned. this may be due to the long-standing fascination with the popular media (and ancient writings) that target the canine as the primary vector for rabies. for example, if you have seen the movie old yeller or cujo, then you probably understand how the popular media can influence the public to fear rabies and scrutinize the family dog for any signs of "frothing or foaming" at the mouth. however, the disease affects both domestic and wild animals. early clinical signs of the illness include fever, pain, and/or a tingling sensation around the wound. however, unlike the popular notion of old yeller or cujo, the most typical clinical signs of rabies are unexplained paralysis and a change in behavior (refer to chapter for details). still, there are many reports of other strange clinical behaviors by a rabid animal. . exposure histories are categorized as bite, contact (eg, waking to find bat on exposed skin) but no known bite was acknowledged, or unknown (ie, no known contact with an animal was elicited during case investigation). † variants of the rabies virus associated with terrestrial animals in the united states and puerto rico are identified with the names of the reservoir animal (eg, dog or raccoon), followed by the name of the most definitive geographic entity (usually the country) from which the variant has been identified. variants of the rabies virus associated with bats are identified with the names of the species of bats in which they have been found to be circulating. because information regarding the location of the exposure and the identity of the exposing animal is almost always retrospective and much information is frequently unavailable, the location of the exposure and the identity of the animal responsible for the infection are often limited to deduction. ‡infection was not identified until . when an organ recipient developed rabies. one of the difficult decisions about rabies, if not the most difficult, is "what constitutes an exposure" when trying to discern whether to treat or not to treat an individual. likewise, there are important implications for the animal (domestic). for example, whether to quarantine the animal for observation or to euthanize an animal followed by laboratory testing for confirmation of rabies. the world health organization (who) has recommendations intended as a general guide. it is recognized that, in certain situations, modifications of these procedures are warranted. such situations include exposure of infants or mentally disabled persons and other circumstances where a reliable history cannot be obtained, particularly in areas where rabies is enzootic, even though the animal is considered to be healthy at the time of exposure. there are other myths and legends surrounding the question of a true rabies exposure. the following are a few of the common ones: • on a given warm summer evening in areas of texas just as dark approaches, bats start swooping in their feeding mode of eating insects, which is an ecologic and biologic benefit. this could initiate a warning from some folks about "bats getting tangled in your hair," leading to being exposed to rabies. of course, bats are not attracted to a person's head of hair and do not try to "nest" there, but if a bat did get tangled in someone's hair, they would receive pep if the bat could not be tested as its too close a contact to rule out a bite occurring. however, contributing to this misconception could be media and historical writings with respect to vampire lore surrounding bats. additionally, there were even popular television series like the andy griffith show that had an episode with barney fife discussing bats in caves and their potential for laying "their eggs" in a person's hair (fig. . ) . • can i get rabies if i handle blood, feces, or urine? the simple answer is no in this scenario. rabies is not transmitted through the blood, urine, or feces of an infected animal, nor is it spread airborne through the open environment. saliva provides the primary transmission medium when the animal is in the infectious stage of rabies. for the rabies virus to get to the salivary glands, it has to travel first from the site of entry (usually a bite wound) through the animal's nervous system, then to the brain. this is what causes most rabid animals to exhibit abnormal behaviors, depending on what part of the brain is infected. finally, the virus travels to the salivary glands during the terminal stage of rabies. it is this later stage of rabies when an animal is most infectious because the virus is in the saliva. treatment for rabies, one of the world's most diabolical viruses, has a long history of creative and bizarre origins. rabies is a terrifying disease that dates as far back as the beginnings of humankind. in a sense, we humans fear rabies because it crosses the line between humanity and animal. think about it, the disease is at the intersection of humans and animals. somewhere deep in the psyche, one can become terrified by the thought of a bite from a rabid animal because it symbolizes the very metamorphosis of a human becoming that very rabid animal-a vampire or werewolf if you let your brain take you to that place. it should not be a surprise that humans have a longstanding and deep-seated fear of diseases with animal origins, or as science calls them, zoonotic diseases. a majority of new diseases are zoonotic. for example, swine flu, west nile virus, anthrax, severe acute respiratory syndrome, tuberculosis, influenza, ebola, nipah, powassan, and plague represent diseases from a wide range of eras. it is not a far reach to realize that the collective conscience of the general public has been swayed and biased by diseases with animal origins. so, along with that understanding comes an almost guttural need to find ways to treat or cure those diseases. this is where some of the earliest misconceptions for treating rabies originate in our history. let us examine some of those treatments that seem to perpetuate in time regardless of the modern advances in medicine. • in many of the earliest recordings of the disease, the treatments centered on the wound. this makes sense when you consider that humans from early in history understood that the origin of rabies manifested in humans after a canid bite, often while the animal was drooling copious amounts of saliva (frothing at the mouth). without listing every single early thought on treatment of these wounds, the common thread is to "bleed and cauterize (burn) the wound." for example, in early traditional indian medicine, there is acknowledgment of the fatality of hydrophobia (fear of water in rabies patients) and a prescription of treatment for it-bleeding and cauterization of the wound. the process involved cauterizing the wound with clarified butter, which the patient is then asked to drink. it is accepted even today that after an animal bite, it is wise to let it bleed, followed by rigorous washing and flushing of the wound with soap and water. however, simply closing the wound or cauterizing it will not cure one from rabies. • interestingly, the very pathogenesis of rabies in how it takes the central nervous system hostage to spread its deadly virions is now being studied to treat and possibly cure disease in brain cancer patients. the rabies virus, which kills tens of thousands of people a year, has a rare ability to enter nerve cells and use them as a conduit to infect brain tissue. now, scientists are trying to mimic this strategy to ferry tumorkilling nanoparticles into brain tumors. in laymen's terms, this means using viruses to carry tiny cancer killing agents to the tumor. so far, the approach has been shown to work only in mice. if successful in people, these nanoparticles could one day help doctors send treatment directly to tumors without harming healthy cells. • alarmingly, homeopathic and neuropathic treatments have gained some traction in the world in a variety of applications for mild to deadly diseases. rabies is not immune to these "treatments." while there is truth that some modern day medicines and drugs come from nature (e.g., penicillin is derived from the fungus penicillium), in most instances it does not replace peer-reviewed and sound clinical trial-based medical treatments or procedures. homeopathy principles roughly state that substances that produce similar symptoms of a particular ailment can cure said ailment ("like cures like") and that diluting a substance increases its potency ("law of infinitesimals"), which brings to mind the "hair of the dog" remedy for some hangover sufferers. recently, there was a report in which diluted saliva from a rabid dog was used to "treat" a -year-old boy for aggressive behavior. the first question that comes to mind is "where did the united kingdom homeopathic pharmacy obtain rabid dog saliva?" regardless, the rabid dog treatment, called lyssinum (aka lyssin or hydrophobinum), is one of more than homeopathic products approved by health canada. there is cause for concern, including those at the us food and drug administration, that such homeopathic products can be harmful and/or delay actual medical interventions and treatments. the homeopath who administered the treatment admitted that "there is no common consensus about how the remedies work" and continued to claim that it was effective and safe, plus added that the saliva was diluted to the point that it would not contain any trace of rabies virus. most rabies experts would have concerns about this treatment. • in an earlier section of this chapter, it was mentioned that some individuals (including some in health care or other biological science majors or backgrounds) incorrectly believe that rabies treatment requires or more shots into the stomach by some monstrously long needle. actually, almost all modern treatment regimens for rabies include four key features: • generous cleaning and lavage of the bite wound with soap or topical antiseptic and warm water. • avoidance of wound repair. closed bite wounds from dogs (or other animals) often become bacterially infected with early closure. the recommendation is to use very few sutures early and wait - days to more definitively close the wound. additionally, tetanus immunization boosters are indicated. • human rabies immune globulin (hrig) is essential for bridging immunity until the response to rabies vaccination occurs - days later. the hrig is most effective when infiltrated in the wound and not intramuscularly (im) remote from the bite wound. • rabies vaccine should be a modern cell-based, inactivated vaccine. rabies vaccine is effective when given intradermally or im, although only the latter route is licensed in the united states. the who recommends several schemata for rabies immunization. in the united states, only four doses (from the original five) are now recommended ( , , , and days) because an immune response is invariably present by day . however, a fifth dose is still recommended on day for individuals who are immunocompromised. simply put, rabies usually kills its victims without early intervention. with the recent and increased attention from stories of survivors of rabies, there have been recent reports from india of natural survivors, but most often with poor functional outcomes. , the exciting new milwaukee protocol and its use globally has produced four survivors who have had excellent cognitive recovery and outcomes. however, two had spastic diplegia that has been described in animal survivor models of rabies. it really should not be a surprise that rabies, a disease that goes back to the dawn of human civilization, continues to influence our pop culture (movies, television, art and literature, and tales told over generations). perhaps it is because rabies has always been known to be the very transformation of a disease from animal to man that is easily observed. the cdc estimates that zoonotic diseases are very common, both in america and globally. they estimate that more than out of every known infectious diseases in people are derived from animals and out of every new or emerging infectious diseases in people are transmitted from animals. historically, humans just did not see the connection of infectious disease to animals. scientists and scholars blamed nearly everything but animals. even some of the nastiest culprits of disease like smallpox (albeit this infection is not zoonotic) or black death (bubonic plague), which spreads to humans via respiratory droplets or by way of hitching a ride on a rat or other rodents via a flea vector, were often misunderstood regarding transmission or cause. usually, this meant that things such as demons, bad air (literal meaning of malaria), heavenly bodies/stars, and even human behavior were the root of disease. almost all of them were blamed on nonhuman hosts, except for rabies and anthrax. regardless of how far one goes back in time, one did not need to look further than the consequences of what happened after a human was bitten by a "mad dog" or other animal. in addition, to add insult to injury (literally), it was often the owner's very own best friend-the dog. if one looks to the time of greek myth, we find lycaon, king of arcadia, transforming into a slavering wolf with rabid and foamy jaws. in fifteenth century spain, we read about witch-hunters called saludadores and they were known as healers of rabies. during the interim of the th and th centuries, our european counterparts were building two well-known legendsthe werewolf and vampire-which had the ability to be human and animal and pass on this ability via the bite. even as we approach the th century with viruses becoming more understood by science and the discovery of pasteur's rabies vaccine, people of france were still transfixed and terrified by the fantasy and horror that a rabies infection converted people into maddened animals. ironically, their horror (fantasy) had some foundation. even after pasteur's vaccine, which miraculously could save one from certain death by rabies if given prior to signs and symptoms, humankind's ongoing rabid fantasy about possible monster metamorphoses with rabies continues in the pop culture and is going strong to this day. one needs to look no further than even possibly american's most trusted media icon, walt disney, when he released old yeller or about years later when the novel cujo (and later film) transfixed audiences to fear rabies (figs. . and . ). vampire movie after vampire movie arrived followed by zombie movies (i am legend, world war z, etc.) and the more current television series (the walking dead, fear the walking dead, etc.) (fig. . ). rabies even shows up in our comedy with appearances in shows like king of the hill and beavis and butt-head, as well as in an episode of the office. in the popular seinfeld episode-classic tv-one of the main characters, elaine, gets bitten by a dog, the owner evades her, she has to get pep (the doctor tells her the shot will hurt very much), and she keeps thinking she is showing signs of rabies (spitting back water, frothing at the mouth, etc.). it seems that rabies will always have a place in shaping pop culture, especially in the realm of the horror and science fiction genre. one can assume that because of this ongoing fascination with rabies in the arts and in our passing on of stories, often handing them down from one generation to the next, that we will continue to be fed misconceptions (although often based on science) about this diabolical virus known as rabies. it is incumbent for all of us in the modern era to dispel these myths and legends so that we can move forward in our efforts to assist humankind and downplay the sometimes-misguided fascination with this ongoing threat. realistically, those in the rabies medical, public health, and research community have a long way to go for increased disease survivorship and increased health literacy between health-care professionals and the public. to give just one example about our need to learn and communicate in the world of rabies, one needs to look no further than the variances in virulence and possible better outcomes from rabies virus variant phylogeny differencesthe reasons for the overrepresentation of the silver-haired bat, lasionycteris noctivagans (fig. . ) rabies virus variant in human infections are unclear. the frequency of infection, shedding, and dissemination of rabies virus in l. noctivagans, compared with myotis lucifugus and eptesicus fuscus, suggests the discrepancy of human rabies cases may be due to increased infectivity in heterospecific hosts, human susceptibility, and/or behavioral factors. in the interim, misconceived notions will most likely continue to be generated regarding the prevalence, signs and symptoms, diagnosis, and treatment protocols pertaining to this notorious and ancient killer known as rabies. it is incumbent upon all of us to be better stewards in the art of science communication with respect to decreasing the misunderstanding and sometimes panic surrounding this ancient disease. a silver-haired bat, the type that has been responsible for numerous human rabies cases and deaths. (reprinted with pemission by source, fair use: https://com mons.wikimedia.org/wiki/file:silver-haired_bat.jpg.) a cultural history of the world's most diabolical virus lyssaviruses and rabies: current conundrums, concerns, contradictions and controversies the new rabies plague. college station molecular epidemiology of rabies epizootics in texas typing of rabies virus isolates by dna enzyme immunoassay rabies: methods and guidelines for assessing a clinical rarity bat rabies evaluation of oral rabies vaccination programs for control of rabies epizootics in coyotes and gray foxes bat-associated rabies virus in skunks rabies: an old disease for a new generation controlling rabies at its source: the texas experience -oral rabies vaccination program rabies in skunks in texas epidemiology of rabies in bats in texas molecular diagnosis and epidemiology of rabies invited interview for outbreak news today radio podcast -rabies: history, myths and diagnosis on outbreak news this week centers for diseases control and prevention -explore travel health with the cdc yellow book obscure and little known facts about rabies current status of rabies and prospects for elimination rabies surveillance in the united states during centers for disease control and prevention. what are the signs and symptoms of rabies the many faces of rabies things you may not know about rabies -but should world health organization. rabies -guide for post-exposure prophylaxis misconceptions about rabies how to stop brain cancer-with rabies treated" -yr-old boy's behavior problems with saliva from rabid dog rabies: rare human infection -common questions survival from rabies encephalitis atypical rabies encephalitis in a six-year old boy: clinical, radiological, and laboratory findings rabies: still a uniformly fatal disease? historical occurrence, epidemiological trends, and paradigm shifts overwintering of rabies virus in silver haired bats key: cord- - ldynibm authors: woldehanna, sara; zimicki, susan title: an expanded one health model: integrating social science and one health to inform study of the human-animal interface date: - - journal: soc sci med doi: . /j.socscimed. . . sha: doc_id: cord_uid: ldynibm zoonotic disease emergence is not a purely biological process mediated only by ecologic factors; opportunities for transmission of zoonoses from animals to humans also depend on how people interact with animals. while exposure is conditioned by the type of animal and the location in which interactions occur, these in turn are influenced by human activity. the activities people engage in are determined by social as well as contextual factors including gender, age, socio-economic status, occupation, social norms, settlement patterns and livelihood systems, family and community dynamics, as well as national and global influences. this paper proposes an expanded “one health” conceptual model for human-animal exposure that accounts for social as well as epidemiologic factors. the expanded model informed a new study approach to document the extent of human exposure to animals and explore the interplay of social and environmental factors that influence risk of transmission at the individual and community level. the approach includes a formative phase using qualitative and participatory methods, and a representative, random sample survey to quantify exposure to animals in a variety of settings. the paper discusses the different factors that were considered in developing the approach, including the range of animals asked about and the parameters of exposure that are included, as well as factors to be considered in local adaptation of the generic instruments. illustrative results from research using this approach in lao pdr are presented to demonstrate the effect of social factors on how people interact with animals. we believe that the expanded model can be similarly operationalized to explore the interactions of other social and policy-level determinants that may influence transmission of zoonoses. the current ebola outbreak in west africa (cdc, ) , and to a lesser extent the outbreak of middle east respiratory syndrome (mers), first identified in saudi arabia in (cunha and opal, ) have galvanized the world's attention on the dangers posed by zoonotic infections. however, these are only the latest in a series of outbreaks of novel diseases in humans during the last decade of the th century highlighting the need for focus on the zoonotic (animal) origin of viral infections. for instance, avian influenza h n was identified in hong kong in (mounts et al., ) ; rift valley fever caused an estimated , human infections and reported loss of approximately % of livestock (primarily sheep and goats) in garissa district, kenya, in e with reports of disease in four other provinces in kenya as well as in somalia and tanzania (woods et al., ) ; an outbreak of nipah virus in malaysia in e caused at least human cases of encephalitis and deaths (chua, ) with an estimated . million pigs culled in efforts to stop transmission (fao and aphca, ) ; and outbreaks of west nile virus occurred in europe from to (hubalek and halouzka, ) and the united states beginning in (who, ) . in taylor and colleagues (taylor et al., ) inventoried known human infectious disease pathogens and pointed out that % of all pathogens and % of emerging disease pathogens were zoonotic in origin. about % of human viral pathogens are zoonotic (morse et al., ; taylor et al., ) . the importance of viral zoonoses was emphasized with the emergence of sars coronavirus in e and the reappearance of h n in hong kong in , followed by its spread throughout asia, the middle east, europe and sub-saharan africa by (who, . the pace of new outbreaks led to increasing recognition that emerging infectious diseases originate at the interface of human and animal ecosystems. this recognition underscored the need for an inter-disciplinary approach to dealing with transmission and was one of the main factors leading to creation of the one world one health tm movement. at a conference convened by the wildlife conservation society at the rockefeller university in , the movement gained its trademarked name and issued a call to action, embodied in the "manhattan principles" for preventing emerging diseases in human and animal population and maintaining ecosystem integrity (wcs). by , the un agencies and the world bank had drafted a strategic framework, introduced at the "one world, one health: from ideas to action" conference in winnipeg, canada, in (phac, . the premise of one health is that people, animals and the environment form an interdependent ecosystem that needs to be considered in a coordinated manner (fao et al., ; frank, ) . it rests on a conceptually simple model that focuses on contact e and therefore the potential for transmission of disease e between wild and domestic animals and humans (usually depicted as three overlapping circles) in the context of the environment. this model has worked well as an advocacy tool to present the case for coordination in detecting and responding to outbreaks. it has also fostered discussions on the factors that are contributing to spillover of diseases from animals to humans, the first step in an outbreak. most of the discussions to date about drivers of emergence (daszak et al., ; a. dobson and foufopoulos, ; karesh et al., ; patz et al., ; smolinski et al., ) have focused on anthropogenic land use changes e essentially resource exploitation (logging, mining, establishment of plantations, dam building) and associated road building and pollution. these factors are fundamental drivers of disease emergence in wildlife through their effects on habitat fragmentation, biodiversity and hostpathogen dynamics. a second set of frequently mentioned drivers focuses on movement of hosts and pathogens through travel and the transport and trade of animals and animal products. finally, increased human-animal contact occurs because of increasing human population density and its consequences e encroachment of humans into previously undisturbed areas and the development of larger scale or more intensive animal production systems (slingenbergh et al., ) . while these large-scale changes and interactions may provide the potential for contact between humans and animals, opportunities for transmission e that is, the initial spillover event e also depend on specific human activity at the local level: if, how, where and when people interact with animals (k. a. alexander and mcnutt, ) . for example, chua et al. ( ) suggested that the first documented outbreak of nipah virus in malaysia was precipitated by a combination of ecological and social factors. the ecological factors included el ni no-cycle related drought, land use change (deforestation and reduced habitat for fruit bats due to logging) and fire arising from slash and burn agriculture that displaced bats to orchards in ipoh. in addition, key factors included local practices regarding location of piggeries in and near fruit orchards and pigsties constructed so that water run-off from roofs e and fruit dropped by bats e was directed into the pigsty. the result was that pigs were able to eat bat-saliva-contaminated fruit, became infected and then infected their handlers. this example supports the importance of the land use change drivers, but also suggests the importance of other, more proximate determinants of contact e in this example, the siting of piggeries and pigsty construction. for preventing, or at least slowing, the emergence of new diseases e and for more efficient response to outbreaks e we need to have a better understanding of these proximate determinants. one route to preventing recurrence of nipah virus at pig farms would be to implement policies and regulations addressing the "upstream" land use changes that eventually led to emergence. a second, complementary route is through interventions affecting what chua calls "the pattern of pig and orchard farming" (chua et al., ) ; that is, human activities at the local level. a first step in this approach is discerning those patterns. having a model of proximate determinants facilitates this activity. in this paper we propose an expanded one health model that highlights the social determinants of human-animal exposure, describe a study approach that operationalizes the model to explore factors that influence the risk of transmission at the individual and community level and present some results that illustrate the effect of social factors on how people interact with animals. . an expanded one-health model the expanded one health model we propose gives serious consideration to all the factors, both social and ecological, that can contribute to disease emergence at the local level. in the expanded model ( fig. ) , the probability of zoonotic disease spillover is a function of contact between humans engaging in different activities and infected animals they encounter during those activities. as our primary interest is emerging pandemic threats, in developing the model we considered what we know about emerging viral diseases transmitted from animals to humans by direct or indirect contact; these comprise about a quarter of all emerging zoonotic diseases (calculated from table in taylor et al. ( ) . the model would need further expansion to account for proximate determinants of the emergence of other kinds of diseases of zoonotic-origin: vectorborne diseases, drug resistance, bacterial, fungal or helminthic infections. any specific spillover event involves one or a small number of animals and one or a small group of individuals (e.g., family members or a hunting party). from the animal side, the probability of transmission to humans is primarily affected by the prevalence of infected animals, which could be wild or domestic. there is a lively debate about the proximate determinants of prevalence, which may include animal biology, pathogen ecology, animal density, biodiversity and animal movement, among others (keesing et al., ) . on the human side of the model the probability of spillover transmission from animals is primarily affected by the likelihood of someone encountering an infected animal or its excreta, determined by the frequency with which people come in contact with specific types of animals that might carry infections and the type of contact they have with animals; that is, by the types of activities in which they engage. human activity is influenced by a complex range of factors along the socio-ecological continuum (riekert et al., ) which may act separately or in tandem. key categories include: biological characteristics of individuals; social characteristics of individuals, households and communities, including norms, livelihood systems and settlement patterns; and finally, at the public policy level, local and international governance and politics (see table for examples of key elements in each category). complex social dynamics determine the type and frequency of engagement of any individual, family or community in specific activities involving possible interactions with animals, as well as the intensity of interactions and thus potential exposure to pathogens. at the simplest level, socially-determined roles for individuals of specific gender, age and education affect both the range of possible occupations and division of labor; for instance, women may cook and men slaughter. individuals engaged in occupations related to animals (hunting, butchering and caring for animals, etc.) or working in agricultural areas or forests are obviously at increased risk compared to the general population. household characteristics, including family structures and socio-economic status, can determine if and how families are exposed. these relationships can be complex. for example, while families that hunt for food tend to be poorer and less educated than families that purchase food, poverty is a predictor of only one kind of interaction with specific types of wild animals. wild animal meat purchased in markets is frequently more expensive than domestic animal meat; a market survey carried out by wcs and fhi in laos (unpublished) showed that brush-tailed porcupine meat cost three times as much as domestic pork per kilogram. in addition, within-family dynamics can affect who gets exposed and with what frequency and intensity. as an example, family type e e.g. single parent, nuclear family or extended e will affect whether and how intensely children are socialized to hunting or what food allocation rules are practiced to determine who gets to eat what part of the animal (whitehead, ) . availability of markets, goods and services, the strength and type of social networks, and proximity to natural resources can affect community dynamics leading to greater or less exposure to animals. for instance, a study conducted in several african countries showed that while more wealth was associated with less bush meat consumption in rural communities closer to source of wild life harvest, it was associated with more bush meat consumption in urban communities (brashares et al., ) . various social forces such as conflict or forced or voluntary migration can also change peoples' relationships to the environment and animals (de merode et al., ; fauna and flora, ) . social norms can affect interactions with animals and potential exposure to zoonotic pathogens in many ways (e.g., which if any animals are preferred for eating and what types of preparation are acceptable, or what kinds of animals are considered suitable for pets e or for children to play with). various customs with regards to specific animals may serve different social functions that can shed light on other determinants of exposure. for instance, hunting some animals may confer certain social standing in the community and sharing the products of a hunt maybe used as a way of incurring favors (gurven and von rueden, ) . food taboos related to animals, often applied differentially to subgroups (e.g. children, pregnant women, individuals from different castes), can serve several purposes including protecting health, marking special events, protecting or allocating scarce resources, or creating group cohesion (meyer-rochow, ). settlement patterns affect the variety and number of animals to which people might be exposed. for example, how houses are constructed, which may be associated with ethnicity and/or socioeconomic status, may determine risk of rodent infestation (bonner et al., ) . rodent abundance is also affected by location of housing relative to different types of animal habitat and open water sources and to waste disposal sites (bonner et al., ; masi et al., ; promkerd et al., ) . it is important to note that while increasing urbanization produces habitats unsuitable for many species of animals, urban-adapted wild animals can occur at higher densities in urban and peri-urban areas than in more rural areas (bradley and altizer, ) . finally, governance and politics, from local to international levels, can affect not only the extent and impact of some of the anthropogenic land use changes mentioned above but also social dynamics at all levels to influence who gets exposed and with what frequency and intensity. our expanded model increases the level of detail of the one health framework and strengthens the foundation for understanding the interplay of factors that lead to disease emergence. below we describe a research approach based on this expanded model. the human-animal exposure study was developed to document the extent of human exposure to animals and begin to explore the interplay of social and environmental factors that influence risk of transmission at the individual and community level. the study aims to identify groups who are at particular risk of transmission of infections and the (potentially modifiable) factors contributing to that risk. in southeast asia the human-animal exposure study has been conducted in selected locations in thailand and lao pdr. in thailand the study was implemented in urban and rural locations inhabited by the same ethnic group (the i-san). in lao pdr, the study examined differences in exposure between two culturally different ethnic groups (hmong, lao-tai) living in the same location. both of these studies received formal ethical approval from the fhi protection of human subjects committee (fhi 's irb) and an irb in the country in which they were conducted. informed consent was obtained from all participants; in the case of children, we obtained the child's assent and informed consent from a parent. below we use examples from these settings to illustrate our discussion of the factors important in implementing such an approach. one research objective suggested by the model is to improve understanding of the interaction between culture and ecology in determining potentially risky human-animal exposure. fully addressing this objective will requires in-depth exploration of the multitude of factors mediating risk of exposure that are affected by social dynamics at the international, national, community, familial and individual level. as a first step, we chose to start this endeavor simply, by comparing groups distinguished by different social determinants of human activity involving animals, e.g., gender, age, ethnic norms, livelihood systems, settlement patterns. the study is designed to facilitate such comparisons, for example, of different population groups (e.g., two ethnicities) living in the same location, thus having similar access to animals, or the same group in locations where access to animals is likely to differ (e.g., one ethnic group living in different settings). in addition, the design includes separate samples of men and women to assess the effect of gender on exposure and provides for subsamples of children likely to have different frequencies or types of exposure than adults. the instruments are designed to gather information on other social contrasts including, for example, religion, occupation, and education. the study uses a mixed-methods approach carried out in two phases: qualitative formative research and a quantitative survey. a mixed-methods approach is especially suitable for this research because it accommodates the need to standardize while recognizing the importance of context for the information gathering process. a generic protocol and instruments are adapted for each site. the first phase, formative research, involves collecting qualitative data that can provide an in-depth understanding of the humaneanimal interface, including the "how," "when," "where" and "why" of exposure. this information is also used to guide adaptation of the survey instrument to local conditions. the formative research draws on a variety of qualitative approaches: participatory rural appraisal (pra) methods (chambers, ) are employed to answer research questions where local knowledge is especially critical (e.g., seasonality of activities and animals, organization of physical space). projective techniques (wiehagen et al., ) are tools for uncovering and exploring underlying motivations or feelings that respondents might be unaware they have or might be unwilling to discuss openly (e.g., hunting protected wild animals). they use ambiguous images or descriptions of situations, on which participants may "project" their attitudes, feelings and opinions safely without divulging personal information. structured anthropologic methods (bernard, ) allow for systematic collection of data related to the cultural knowledge (e.g., taxonomies) held by the respondents (e.g., how a community categorizes animals into groups). the second phase of the human-animal exposure study is a random survey of individuals to quantify their exposure to animals as completely and as accurately as possible. it uses a generic protocol and instruments that are adapted for each site. the sample size for the survey should be calculated to allow for estimation of exposure parameters within desired confidence limits for each gender group; we routinely use ± %. the generic survey instrument is structured in modules related to the most common ways (activities and locations) people are exposed to animals. these correspond to three categories of proximate determinants: norms, livelihood and settlement patterns. finalizing locale-specific instruments is based on results of the formative research regarding the types, categorization, roles and uses of different animals in specific communities. (see below for discussion of the key factors considered in adapting the questionnaire for a specific site). if the formative research identifies activities or locations associated with human-animal contact that are not included in generic modules, the survey questionnaire is amended; in some cases entire modules may be developed and added. for instance, during the formative research phase in one setting we learned about use of animals and animal products for medicinal purposes and encounters with animals when gathering wood and non-timber products in the forest; questions about these potential contacts were included in the subsequent survey. currently available modules are listed in table . not all encounters between humans and animals are direct e or memorable. they can easily be overlooked or forgotten. to increase recall of events that may not be considered important, survey questions are supported with extensive, systematic probes. to ensure that survey data are as valid and reliable as possible, we carry out cognitive interviewing (willis, ) using the siteadapted survey instruments. this approach involves pretesting sections of the questionnaire to assess how people understand questions and process information in order to respond to them. cognitive interviewing helps to identify potential misunderstandings and questions that are especially hard for respondents to answer and determine if response categories make policies, regulations and enforcement of trade in animals and animal products, global travel, multinational agriculture and extractive industries, migration sense; this information is used to finalize the questionnaire before deployment. two key decisions in developing a study design were the range of animals and the parameters of exposure that the study should cover. types of animals the survey collects some information about exposure to all kinds of domestic and wild animals and more detailed information to quantify human exposure to wild animals particularly likely to carry zoonotic viruses e bats, rodents and nonhuman primates e as well as to poultry. this decision was based on current information about transmission of viral infections. non-human primates, bats, and rodents (including rats, mice, squirrels and porcupines) are mammals of particular interest. all are reservoirs or suspected reservoirs of infections that have caused important outbreaks of human disease: nonhuman primates for hiv (keele et al., ) ; bats for sars, nipah, hendra and as suspected reservoirs for ebola and marburg viruses (a. p. dobson, ; kuzmin et al., ) ; rodents for lassa fever virus and hantavirus (both confirmed) and as suspected reservoirs or important intermediate hosts for monkeypox (meerburg et al., ). in addition to these reservoir species, it is important to obtain some information about possible intermediate hosts e animals that may be infected and in turn infect humans. a wide range of wild animals can be intermediate hosts; for example, both nonhuman primates and antelopes can be infected with ebola and transmit the infection to hunters who find the carcasses (lahm et al., ) . domestic animals, including pets, and other animals that come in frequent contact with humans also have to be considered, as they have been shown to be intermediate hosts or carry viruses such as avian influenza (van kerkhove et al., ) , rabies (chomel et al., ) or rift valley fever (woods et al., ) . our study therefore accounts for human interactions with a wide variety of mammals, both wild and domestic. it also includes human interactions with domestic poultry and pigs, because of the contribution of influenza viruses from both birds and swine to influenza h n and the importance of h n (and recently h n ) as poultry diseases that can infect humans (d. j. alexander and brown, ) . parameters of exposure the survey assesses frequency and duration of exposure to animals and to a certain extent, proxies of "intensity" of exposure (e.g., contact involving oral fluid from an infected animal on intact human skin is probably less risky than being deeply bitten by an infected animal). it was not designed to directly measure risk because for many diseases too little is currently known for accurate quantification of risk (even, for example, about the amount of viruses present in viscera vs. muscle vs. blood vs. nasal excretions vs. feces vs. oral fluid, or about the persistence of virus in various organs after an animal's death or on different surfaces under different conditions). therefore, for the analysis, information about intensity is translated into weighted scales based on expert opinion. because language and cultural frameworks affect the validity of information gathered through surveys, the formative research is designed to elicit the information needed for local adaptation of the survey instrument. particular attention is paid to local names of animals and how people speak about encounters, time and locations. identification of animals one of the main goals of the formative phase of the research is to generate local animal dictionaries for use during the survey. we have found that there are usually no comprehensive lists of local animal names and that even those lists that are available do not include regional or local variations. ethnic groups vary in the specificity with which they recognize animals. recognition can sometimes serve as a proxy for exposure, as people tend to be more familiar with the animals they encounter most frequently. for instance, in thailand people were able to identify rats (a food source as well as a pest) with a great deal of specificity and were able to name several types of rats. in contrast, they did not use different names for different species of bats. during the formative research a concerted effort is made to identify local names and any areas of possible confusion by using an extensive array of animal photos and discussions about animals found in the community. one exercise uses probes focused around senses (e.g., what about animals you smelled? or animals you heard?) to improve recall. in thailand, when first asked to recall animals in the community (a question unlinked to senses), most people first mention animals they see. on being prompted for animals they "touch," new animals are recalled e mostly those that are cooked or cared for (such as fish, crab, cows, cats and dogs). another prompt, for animals people have observed "evidence of being around," has elicited reports of wild boar and bear (stool, foot print), pangolin and mongoose (holes), squirrels (bitten fruits and food), fox (howling), snake (skin and smell), cockroach (stools), and civet (stool and foot print). in thailand, the formative research generated a dictionary of about animal names just in the rodent, bat and primate animal orders and in lao pdr, dictionaries included names in those animal categories for hmong and for lao-tai. in addition to the dictionaries, naming conventions for animals are ascertained, enabling the identification of additional animals during the survey. for instance, pua is the generic name for bat in hmong; names for different types of bats modify this generic name (e.g., pua-lor and pua-sam-wa). whenever available, this kind of information allows researchers to determine the category of animal if they encounter a new animal during the fielding of the study. the questionnaire is structured such that if respondents do not spontaneously mention specific animals of interest (bats, nonhuman primates, or rodents), interviewers probe for them by local name (spontaneous and probed responses are coded differently). information obtained during the formative research phase is analyzed to determine how specific these probes should be. how people describe actions people vary in the way they talk about encounters. use of the wrong term may lead to miscommunication and collection of incomplete or inaccurate data. different terms may be used in slightly different situations; for example, among the lao-tai, people "hunt" [larr suud] for larger animals but "go looking for" [pai ha kni, pai xook suud] smaller animals. thus "rice field rat" is not a response obtained when "what do you hunt?" is asked. the formative phase of the study explores use of language to describe animal encounters. glossaries of key clusters of words are built, along with notes about the context of their use. for example, one such cluster is "slaughtering," "butchering," "cutting up," "preparing" e terms that can have implications for intensity of exposure since they are associated with how recently an animal has died and thus the amount and viability of pathogens that might be transferred. temporal variation to ensure that information about rare, as well as routine, encounters with animals is obtained and to account for seasonality, the survey documents human-animal contacts over the previous year. the single exception is information about hunting and eating animals. recall about eating is especially problematic because the activity is so routine. thus respondents are first asked about what animals have been hunted or eaten in the past four weeks, and then asked about the previous months. one portion of the formative research focuses on the rhythms of life in the community across the year to identify seasonal patterns of activities and encounters with certain animals. for example, formative research in lao pdr identified two seasons (rainy and dry) significant to the community. for activities identified as seasonal (e.g., application of fertilizer, hunting) probes for these seasons are used during the survey to aid recall of encounters with animals. such information is not only critical for understanding temporal patterns of risk of transmission; it may also be critical in the design of any strategies for the mitigation of such risk. spatial variation location is a key factor that determines the number and variety of animals to which humans are exposed. different groups identify key spaces differently. for example, space just outside a house may be considered "living space," equivalent to space inside the house, or may be considered part of "public space". one section of the formative study explores how people understand and talk about the spaces around them; the cognitive interviews include tests of questions involving specific locations. comparing two ethnic groups living in the same area is a good way to understand how socially-determined gender and age roles and norms affect different kinds of exposure to animals and thus potential risk. this section presents illustrative examples from lao pdr, where lao-tai and hmong ethnic groups living in the same location, with similar access to animals, were interviewed. the hmong are an ethnic minority in lao pdr and tend to be of lower socio-economic status compared to their dominant ethnic counterparts e the lao-tai. the hmong and lao-tai are culturally and linguistically distinct groups; for instance almost all hmong in our study practiced animist religion while a majority of the lao-tai were buddhists. the survey included men, women, boys and girls across both ethnic groups, with roughly half of each ageegender category drawn from each ethnic group. note that all difference between groups discussed in this paper were significant at p < . or below. a comparison of the consumption patterns of lao-tai and hmong highlights the importance of social factors in key exposures. we examined the steps involved, from hunting or purchasing through preparation and eating. practices associated with hunting offer opportunities for transmission: for example, hunters may be bitten or scratched by an infected animal, they may handle the carcasses and viscera of infected animals (whether killed or found dead) and get infected blood into wounds, or may have contact with animal feces. slaughtering and preparing (butchering and cutting up) wild animals, whether done by hunters, their family members or people who buy animals, can place people at risk of transmission through direct exposure to blood and internal organs as well as feces. finally, eating is an important source of potentially risky exposure, as ingestion of meat or blood from infected animals, especially raw or uncooked, or intake of other foods or liquids contaminated with viruses can cause disease. hunting is a common activity among both lao-tai and hmong, but is clearly a domain of men and boys; very few girls and women hunt animals other than rats/mice and other rodents (fig. ) . although the two ethnic groups live in the same area, there is a difference in the types of animals they report hunting e a proxy of their familiarity with these animals (see table ). while respondents from the two groups both report hunting six types of bats, the hmong report more types of nonhuman primates and squirrels while the lao-tai report more types of rats/mice. the animals hunted most often by both ethnic groups are rats/ mice and other rodents (squirrels and porcupines). rats/mice are eaten by nearly three quarters of hmong and lao-tai men, women, and children and more than half of adults and children also participate in preparing them. however, boys, especially hmong boys, are the group most likely to hunt, slaughter, prepare and eat them; more than three quarters of lao-tai and more than % of hmong boys reported hunting rats/mice in the last year. boys are also more likely than any other group to report being scratched and bitten by rats/ mice. about % of hmong and about % of lao-tai boys reported being scratched or bitten by rats/mice in the four weeks. however, it is worth noting that women and girls also hunt rats/mice. among the hmong, about % of girls reported hunting them. women and girls are more involved in slaughtering and preparing rats/mice than in hunting, and their involvement differs by ethnicity. among lao-tai, girls and women are the groups least likely to slaughter and girls are the least likely to prepare rats/mice; however even among girls, nearly % report preparing rats/mice in the last year. on the other hand, hmong girls slaughter animals at rates equal to men (and higher than women) and prepare them at higher rates than men. bats are another animal to which hmong and lao-tai boys are more exposed than others; more than % of boys from both groups reporting hunting bats compared to less than % of all other subgroups. bat-eating by adults is delineated along ethnic lines; both lao-tai men and women were more likely to eat bats in the last year than their hmong counterparts. reasons for this pattern are suggested by results of the formative study: the hmong noted that bats are primarily eaten only when there is less choice of alternative meats, as many people consider them to be dirty and smelly. there is a striking difference between ethnic groups in consumption of nonhuman primates. hmong were more likely to eat nonhuman primates, with more than % of hmong, but almost no lao-tai (< %), eating them in the last year. hmong men were also most likely to report hunting, slaughtering and preparing nonhuman primates. finally, consumption of raw meats varies by gender. both lao-tai and hmong participants in the qualitative research reported that eating raw meat and internal organs, and drinking blood from certain animals, including cows, pigs and squirrels, is considered a male activity. participants also stated that eating raw meat is not appropriate for those who are "weaker," including women and children. the survey confirmed this finding; men (both lao-tai and hmong) were three times as likely as women to consume raw animal products. the survey also showed some ethnic differences in patterns of raw meat consumption: hmong men and boys were more likely than their lao-tai counterparts to consume raw meats. as can be seen from these differentials, social factors clearly influence the possible risk of transmission associated with consumption. both lao-tai and hmong hunt, but different subgroups have different risks as a result of hunting: males in both ethnic groups are at higher risk from hunting any animal (compared to females); boys in both ethnic groups (compared to any other groups assessed) are at higher risk from hunting bats and rats; and hmong men are at higher risk from hunting nonhuman primates. women and girls, on the other hand are exposed to rats by being involved in preparing them. finally, social factors also determine who is at risk from eating different meats: eating rats is an equal source of exposure for adults and children in both ethnic groups; the hmong are more exposed to nonhuman primates and the lao-tai to bats as a result of eating; and men are more intensely exposed as they are more likely to eat raw meat compared to women. raising animals is another area where the influence of social factors on exposure, and therefore the risk of transmission, is evident. domestic animals are raised by nearly everyone in the study area: about % of both hmong and lao-tai reported raising poultry in the last year, and more than half of both hmong and lao-tai reported raising pigs, cows, cats and dogs. risk of transmission from raising animals can come from a number of sources, including sharing living quarters with the animals. more than % of respondents in both ethnic groups reported that poultry come into the house. there is a difference between the two groups in pigs in and around the house: nearly % of lao-tai reported pigs in or around the house, while fewer than % hmong did so. the formative research also shows that the lao-tai build their houses on stilts and keep pigs and other domestic animals under the houses. the study draws attention to a specific risk for children in both communities. among the hmong, more children than adults, especially boys, reported being bitten or scratched by domestic animals in the past year; e.g., nearly one third of hmong boys reported being scratched by poultry compared to < % of hmong men. while this may stem from inexperience when caring for domestic animals, the qualitative portion of the study identified other activities that might place children at particular risk, including playing with chickens and organizing cockfights. children were also reported to keep squirrels and small monkeys as pets and to capture and use bats as playthings. another area with clear differences along ethnic lines is the use of animal feces for agriculture. both groups, but more lao-tai than hmong, use feces from poultry, cows and pigs as fertilizer. in contrast, more hmong than lao-tai use bat guano as fertilizer. the formative study showed a difference in the division of labor among men and women and children in the acquisition and use of guano: men are responsible for collecting bat guano from caves once or twice a year, and women or children are responsible for applying the guano to fields and gardens once or twice a month. while the study highlighted nearly universal exposure to domestic animals in these communities, it also shows a specific risk for hmong boys during care of or playing with poultry (they reported being scratched and bitten more) and a possibly elevated risk for lao-tai households whose pigs may come indoors. finally, exposure to feces used as fertilizer has been shown to vary by ethnic, gender and age groups; this may translate into different kinds of risks for different groups in contact with the same source of virus (feces) but from different animals (poultry and pigs vs. bats) and in different activities/locations (obtaining from around households vs. obtaining in caves vs. applying on fields). insight about the large-scale drivers of zoonotic disease emergence e land use patterns, increasing human population and global movement of people and goods e is useful for informing national and global policies with regards to human development activities. given current realities, a more in-depth understanding of the specific ("micro") aspects of the human-animal interface that can result in spillover events would complement current efforts for planning prevention or mitigation strategies. the expanded one health model asserts that different people living in the same location, affected by the same large-scale drivers, may be at different risk of spillover because of the social factors that influence the types of activities they engage in. who is at risk and how they are at risk of spillover are determined by social factors, such as those affecting communities within societies and families and individuals within communities (e.g., gender, age, family structures, ses, occupation, community resources), norms with regards to different animals (e.g., food taboos or preferences), settlement patterns that can limit or increase contact with certain animals (e.g. how homes are constructed) and livelihood systems that may involve direct or indirect contact with animals (e.g., whether or not people rely on subsistence or commercial hunting). information about these factors can be used to develop targeted interventions to reduce risk. as illustrated by the development of the human-animal exposure study, the expanded model can guide a more in-depth exploration of the human-animal interface. information generated from such studies can be critical for identifying specific groups that are at high risk of spillover and assessing possible routes of transmission. for instance, in lao pdr, where a human-animal exposure study informed by the expanded model was implemented with different groups living in the same general location, the unexpected finding that children were more exposed than adults to some animals suggests that children might warrant a special look if there is a concern about transmission of viruses from bats and rats. on the other hand, if the concern is about viruses from nonhuman primates, a focus on hmong men might be very important. in-depth study of the human-animal interface at the local level not only provides information about specific groups at risk of transmission, but also highlights the activities that put them at risk. for instance, in the communities described in this paper, hunting and ingestion of key animals previously implicated in outbreaks (rodents, bats and nonhuman primates) is common: men and boys hunt, and different sub-groups of the communities eat these animals, albeit at different rates. in addition, raising animals such as chickens and pigs, which are known to be intermediary hosts, is a universal activity. depending on the specific animal, virus or route of transmission, interventions might be able to target specific groups involved in different activities. for example, since men and boys are the groups most involved in hunting, they could be the specific focus of general programs to promote safer hunting practices, but if exposure to rats/mice were a particular concern, the intervention would need to include women and girls as well. general interventions addressing butchering and preparation of animals should involve all age/gender groups. the proposed expanded one health model aims to focus attention on the local level factors that determine probability of disease emergence in conjunction with large-scale drivers. by understanding the complex interactions of these factors, the added value of the expanded model is that community or individual level behavior change interventions can be designed to complement policy-level strategies. the human-animal exposure study described in this paper does not attempt to explore the entirety of the complex social dynamics that can determine the location, time and intensity of exposure. as a first step, we focused on demonstrating that the most basic socially determined factors affect exposure, and did not consider how community and family dynamics or governance and politics at the national and international levels play a role. different types of studies would be needed to address and/or incorporate those factors. we hope that the current study provides a solid start to the conversation about the need to embed social science approaches in explorations of human and animal health and opens the door to further in-depth studies exploring how social dynamics affect risk of spillover. other groups are encouraged to use the model as a basis for developing studies to explore the complex interaction of various social and environmental factors that result in disease emergence. some of the questions to be explored might include: how does urbanization and exposure to "international" norms affect attitudes and patterns of consumption of different types of wildlife meat? what effects do various approaches to communitybased natural resource management in different types of communities have on the frequency and type of interaction with domestic and wild animals? how do national policies regarding universal primary education and their implementation at the local level affect community and family expectations, their socialization of children and different family members' relation with domestic and wild animals? answering these questions will involve different types of methods. a cross-sectional survey of a large group of individuals such as reported here is appropriate for capturing an overall snapshot of the human-animal interface, including all the animal species people could come interact with as well as all the major activities and locations associated with interactions. other approaches would be optimal for more focused studies targeting a specific group of animals (e.g. primates) or specific activities (e.g. hunting). for instance, exposure could be documented by intense observation of individuals over long periods of time or by asking people to keep diaries of specific activities such as hunting or eating. more in-depth qualitative or ethnographic studies can help tease out the social and cultural influences shaping specific types of interactions with animals. it is important, however, to remember that all these methods bring their own set of methodological and ethical challenges that would need to be carefully considered, similar to the process described in this paper for the cross-sectional study. finally, it is important to note that the expanded one health model does not address the factors that determine whether or not a spillover evolves into a full-scale outbreak e a critical topic worthy of immediate attention. recent zoonoses caused by influenza a viruses human behavior influences infectious disease emergence at the humaneanimal interface research methods in anthropology poor housing quality increases risk of rodent infestation and lassa fever in refugee camps of sierra leone urbanization and the ecology of wildlife diseases economic and geographic drivers of wildlife consumption in rural africa the origins and practice of participatory rural appraisal wildlife, exotic pets, and emerging zoonoses anthropogenic deforestation, el nino and the emergence of nipah virus in malaysia middle east respiratory syndrome (mers): a new zoonotic viral pneumonia emerging infectious diseases of wildlifeethreats to biodiversity and human health the impact of armed conflict on protected-area efficacy in central africa virology. what links bats to emerging infectious diseases? emerging infectious pathogens of wildlife manual on the diagnosis of nipah virus infection in animals contributing to one world, one health: strategic framework for reducing risks of infectious diseases at the animalhuman-ecosystems interface batwa cultural values in bwindi impenetrable and mgahinga gorilla national parks one world, one health, one medicine hunting, social status and biological fitness west nile feverea reemerging mosquito-borne viral disease in ecology of zoonoses: natural and unnatural histories chimpanzee reservoirs of pandemic and nonpandemic hiv- impacts of biodiversity on the emergence and transmission of infectious diseases highly pathogenic avian influenza (h n ): pathways of exposure at the animal-human interface, a systematic review commerson's leaf-nosed bat (hipposideros commersoni) is the likely reservoir of shimoni bat virus morbidity and mortality of wild animals in relation to outbreaks of ebola haemorrhagic fever in gabon socioeconomic and environmental risk factors for urban rodent infestation in sao paulo rodent-borne diseases and their risks for public health food taboos: their origins and purposes prediction and prevention of the next pandemic zoonosis casecontrol study of risk factors for avian influenza a (h n ) disease, hong kong unhealthy landscapes: policy recommendations on land use change and infectious disease emergence factors explaining the abundance of rodents in the city of luang prabang, lao pdr, as revealed by field and household surveys the handbook of health behavior change ecological sources of zoonotic diseases microbial threats to health: emergence, detection, and response risk factors for human disease emergence one health™. retrieved from october food rules: hunting, sharing, and tabooing game in papua new guinea h n avian influenza: timeline of major events applying projective techniques to formative research in health communication development cognitive interviewing: a tool for improving questionnaire design an outbreak of rift valley fever in northeastern kenya we thank zo rambeloson and kanokwan suwannarong who implemented the research; they and sidney schuler, lonna shafritz, renata seidel, david sisanath, mario chen and tim mastro provided helpful comments. we also express sincere appreciation the social science and medicine anonymous reviewers for their thoughtful suggestions. support for this study was provided by fhi with funds from usaid cooperative agreement ghn-a- - - - ; this study was made possible by the generous support of the american people through the united states agency for international development (usaid). the contents are the responsibility of the authors and do not necessarily reflect the views of fhi , usaid or the united states government. the funder had no involvement in study design, collection, analysis, interpretation of data, writing or in the decision to submit it for publication. key: cord- -ab hel r authors: li, xiao yan; xue, kang ning; jiang, jin sheng; lu, xuan cheng title: the main biological hazards in animal biosafety level facilities and strategies for control date: - - journal: biomedical and environmental sciences doi: . /bes . sha: doc_id: cord_uid: ab hel r concern about the biological hazards involved in microbiological research, especially research involving laboratory animals, has increased in recent years. working in an animal biosafety level facility (absl- ), commonly used for research on infectious diseases, poses various biological hazards. here, the regulations and standards related to laboratory biosafety in china are introduced, the potential biological hazards present in absl- facilities are analyzed, and a series of strategies to control the hazards are presented. concern about the biological hazards involved in microbiological research, especially research involving laboratory animals, has increased in recent years. working in an animal biosafety level facility (absl- ), commonly used for research on infectious diseases, poses various biological hazards. here, the regulations and standards related to laboratory biosafety in china are introduced, the potential biological hazards present in absl- facilities are analyzed, and a series of strategies to control the hazards are presented. a series of laboratory-acquired infections were reported in recent years, in singapore (september, ) , taipei (december, ) , and beijing (december -january ) [ ] . in addition, teachers and students in the northeast agricultural university of china were infected with brucella spp. in [ ] . in addition, the bacterium that causes anthrax escaped from a laboratory in usa in [ ] . these laboratory-associated infections have increased global concern for laboratory biosafety [ ] , and have prompted many countries, including china, to reexamine and revise the relevant laws and regulations for laboratory biosafety, to facilitate the effort to propose appropriate countermeasures. although biosafety in microbiological and biomedical laboratories (bmbl) (published jointly by the centers for disease control and prevention and the national institutes of health , usa) is the gold standard for laboratory biosafety, the actual biosafety programs applied to control biological hazards in individual facilities depend on numerous factors, including the agents being used, the source of funding, and local codes, among others. here, we summarize the regulations and standards currently in place in china, and propose control strategies. the regulation of pathogenic microorganism laboratory biological safety (issued by the state council of the people's republic of china) [ ] is the primary mandated regulation in china for the management of laboratory biosafety and pathogenic microorganisms. the directory of pathogenic microorganisms transmitted in humans, issued by the ministry of health in china in , specify the grade of laboratory in which specific pathogens and animals should be housed [ ] . the standards include the general laboratory requirements for biosafety [ ] and the architectural and technical code for laboratory biosafety [ ] . these provide national guidelines and standards for the construction, operation, and management of biosafety laboratories in china. based on a combination of our own practical experience and consultation of these references [ ] , here we consider aerosols, zoonoses, and laboratory-associated infections as the main biological hazards in absl- facilities. aerosols are classified as small solid particles or liquid droplets, with a diameter of . to μm, that form relatively stable dispersions in gaseous media [ ] . here, aerosols refer to both bio-aerosols and aerosols originating from laboratory animals. rodent allergens, which can cause anaphylaxis in animal care staff, show a wide range of particle sizes, and both small and large allergen-laden particulates have been shown to migrate throughout facilities [ ] . rat and mouse allergens have been shown to be carried mainly on particles μm or larger [ ] , and another study found that rat allergens could be carried on smaller particles of - μm in size [ ] . therefore, aerosols originating from mice or rats may carry rodent allergens, and thus cause harm to staff through inhalation, skin contact, and eye contact, among others. some studies on rodents have indicated that increased levels of room allergens are correlated with decreased humidity [ ] [ ] , increased animal density [ ] [ ] [ ] , and activities such as cage changing, room cleaning, and animal handling [ ] [ ] [ ] [ ] [ ] . in order to reduce levels of animal aerosols, we should first reduce animal allergen levels, and thus relative humidity and staff activities should be taken into consideration. bio-aerosols are another type of biohazard. bio-aerosols are classified as airborne particles that are living (bacteria, viruses, and fungi) or that originate from living organisms. bio-aerosols are ubiquitous, highly variable, complex, and natural or man-made in origin [ ] . infected animals can release biohazardous materials through respiration or excretion. when staff handle these animals during activities such as feeding, cage changing, blood collection, and anatomical examinations, bio-aerosols may be generated. in order to reduce bio-aerosol loads in indoor environments, certain control measures should be followed [ ] . these include proper identification and elimination of the microbial source in occupational settings, maintenance of equipment, humidity control, use of filters in ventilation, and air cleaning using disinfectants and biocides. the air in operating rooms and other critical areas, such as isolation rooms, can be disinfected by fumigation. in addition, an adequate air change rate and installation of filtration equipment are necessary [ ] . zoonoses are another important source of laboratory-acquired infections. zoonotic infections, such as cases of infection by brucella spp. and the bacteria that cause anthrax, have previously been reported, and here we will use human b. canis as an example. from - , individuals were infected by b. canis; most had close contact with dogs. cases of brucella infection have also been reported in laboratory workers [ ] . these incidents indicate that zoonoses are an important biological hazard during animal experiments that can lead to serious infections in human. pike [ ] reported that only % of laboratory-associated infections could be traced to a known cause, whereas unexplained laboratory-associated infections account for % of all infections. research on unexplained laboratory-associated infections has shown that most unexplained infections are caused by inhalation of aerosols containing infectious pathogens. wedum [ ] reported that more than % of all laboratory-associated infections are caused by aerosols containing microbes. pedrosa [ ] [ ] showed that % of laboratory arbovirus infections are caused by aerosols. zhanbo [ ] and colleagues measured the strength of microbial aerosol sources in different situations, including both normal operation and accidents. they found that the strength of the aerosol source caused by accidental breakage of a bottle is higher than that caused by appropriate operation of a centrifuge [ ] . thus, it is very important to regulate activities and operations in animal laboratories to reduce the risk of biological hazards. according to the directory of pathogenic microorganisms transmitted in humans, most ( of ) species of pathogenic microbes should be contained in absl facilities. thus, most experiments on infected animals are conducted in absl- facilities. from these figures, we can infer that absl- facilities are more likely to generate harmful biohazards than animal facilities of other grades. it is therefore necessary to implement methods to control transmission of harmful factors [ ] . based on our own experience and the relevant regulations and standards in china, we provide the following summary of control strategies. the design and construction of the animal facility should be reasonable and correspond with the national standards. the facility should be divided into several relatively independent functional units according to the rule that different species and different levels of laboratory animals should be housed separately [ ] . the housing area and the experimental area within the containment barrier should be equipped with a controlled access system that only allows authorized staff and visitors to enter. the pressure in the core experimental area must be negative [ ] . in addition, there should be a pressure gradient between the housing room and any 'dirty' corridors [ ] . housing rooms should contain a buffer unit with biosafety cabinets, in which researchers can perform experiments, in order to control aerosols efficiently. the main devices in absl- facilities should include individual ventilated cages (ivc) for rats and mice, negative housing cabinets for rabbits and dogs, and isolators for poultry. the animal experiment equipment should include a negative pressure autopsy table, a disinfection sterilizer, biosafety cabinets, centrifuges, independent ventilation animal transport cages, and independent ventilation recovery cages. other devices may include a cage changing work bench, cleaning equipment, and a hydrogen peroxide generator [ ] . the hvac system is a complex but very important part of an animal facility. a properly designed and functional hvac system is essential to provide adequate pressure, temperature, and humidity. pressurization contributes to controlling airborne contamination by providing directional airflow. areas for quarantine, housing, and use of animals exposed to pathogenic microorganisms, and areas for housing nonhuman primates should be kept at negative pressure, whereas areas for clean equipment storage should be kept at positive pressure. the hvac system should be designed for reliability (and redundancy, if applicable), ease of maintenance, and energy conservation and be able to meet the requirements of all animals housed. the system should also be adjustable and ideally maintain temperatures of ± °c. relative humidity should generally be maintained within a range of %- % throughout the year [ ] . although maintenance of humidification within a limited range over extended periods is extremely difficult, daily fluctuations (recognizing the effects of routine husbandry especially when caring for large animal species) in relative humidity should be minimized. ideally, relative humidity should be maintained within ± % of the set point. however, this may not be achievable under some circumstances. the type and efficiency of supply and exhaust air treatment should be matched to the quantity and type of contaminants and to the risks they pose. overall, the principles for selection of the hvac should be reliability, longevity, and minimal energy consumption. a set of strict assessment criteria for testing or verification has been established to ensure the reliability of the biosafety protective performance of facilities. the focuses of the assessment are verification of the airflow pattern and the capacity of the hepa filter to remove contaminants. it should be noted that routine physical examinations are inadequate for effective evaluation, and that microbial aerosol technology is the most direct and specific technique for biological detection of airflow and hepa filters [ ] [ ] . while the havc system runs, the hepa filter will collect particulates. the capacity of the building supply and the exhaust fan determine the life of a hepa filter. when the havc can no longer maintain a proper airflow balance due to filter loading, the filters need to be replaced. if any filter shows visible signs of damage or leakage, it should be repaired or replaced immediately. before the experiments begin, researchers are required to detail the protocol for each animal experiment. the biosafety officer and the attending veterinarian then judge the feasibility of the experiment. the directory of pathogenic microorganisms transmitted in humans is used to determine the laboratory grade of the animal experiments. during experiments, the status of the animals and the surroundings should be recorded in detail by the staff. for the experiments involving infectious agents, all handlings, such as cage changing, injection, and sampling, must be performed in biosafety cabinets. when sharps, such as needles or glass, must be used, operations should be performed carefully to avoid any injury. researchers must strictly obey the standard operation procedures when working with infectious agents. the protective performance of the biosafety cabinets should be assessed regularly. experiments should be performed on the cleanest laboratory animals firstly, then dirtier animals. at the conclusion of the experiments, the feeding rooms and cages should be sterilized thoroughly. the waste from experiments involving infectious agents should be decontaminated by autoclave sterilization before removal from the facility. the running conditions and the cleanliness of the animal facility should be monitored by the quality control officer. the depositing bacterial concentration is a required inspection item for every laboratory in the facility each term. blood agar plates should be placed in each corner and in the center of the laboratory for min and then cultivated in a °c incubator for hours, at which point the bacterial colonies should be counted. surveillance using sentinel animals is also essential. sentinel animals are externally sourced animals that are introduced into a population, exposed to animals or soiled bedding from the population, and sampled in lieu of the principal animals. most commonly, sentinel animals are indirectly exposed to infectious agents by using dirty bedding. the use of direct contact sentinels is a valuable complement, particularly in certain sensitive situations such as quarantine. bedding sentinel animals are the current standard practice for surveillance of isolated ventilated cages or when animals to be monitored are immunodeficient and there are no immunocompetent resident animals available. if sentinel animals are bred in an absl- facility, they should be monitored at an increased frequency, perhaps monthly, as they are a potential source of infection for the entire facility. sentinel animals should be chosen and placed with a variety of criteria in mind. sentinels should be immunocompetent so that they are suitable subjects for serology, and should have a mature immune system when sampled. sentinels should be introduced to the colony at - weeks of age. sentinels should be female, which will decrease fighting and lessen the chance of genetic contamination of the resident animals. sentinel animals should also be demonstrably free of all infectious agents that would be of concern in the area that they are chosen to monitor. the components of an occupational health and safety program for animal handlers include screening, training, work practices, effective use of engineering controls, selection and use of personal protective equipment, and emergency response protocols [ ] . an effective occupational health program screens those with animal contact in order to identify individuals who may be particularly susceptible to animal allergens or the infectious disease under study, or who may present an elevated risk to the animals. this medical evaluation, which is part of the individual's medical record, must be private and confidential. training should be tailored to the group in question. it is also prudent to provide additional training to cover risks or hazards faced by animal handlers even if it is not mandated by regulation. examples of training programs in this category include ergonomics and slips, trips or falls, effective use of the biosafety cabinet or chemical fume hood, safe handling of sharps, sterilization/disinfection, and an emergency response program. employees must also receive training for the use of any equipment that may involve risk or present a hazard to the employee, such as autoclaves, rack washers, containment equipments, ventilated caging systems, and precision vaporizers used by researchers. an individual who has never worked with infected animals should demonstrate proficiency with the required biosafety work practices and the institution's peer-review standard operating procedures (sop) with noninfectious animals before proceeding to practice sessions with infected animals. selection and use of personal protective equipment (ppe) is essential to avoid infections in an animal laboratory. ppe appropriate for the work environment, including clean institution-issued protective clothing, should be provided as often as necessary. emergency protocols should be established so that employees can protect themselves and the animals. physical injures such as animal bites, skin punctures by needles, as well as other potentially hazardous injuries, should be reported immediately. in this situation, the director of the facility who will take responsibility should be informed as soon as possible. at the same time, the injured person should receive first aid treatment, and emergency procedures should be initiated. finally, regular health examinations help to assure not only the occupational health and welfare of the staff but also the quality of the laboratory animals, and employees should be required to undergo an annual health examination. editorial on laboratory-acquired incidents in taipei, taiwan and singapore following the outbreak of sars coronavirus teachers and students infected serious disease during animal experiments in northeastern university. the china youth daily editorial on laboratory-acquired incidents in taipei, taiwan and singapore following the outbreak of sars coronavirus the state council of the people's republic of china. the regulation of pathogenic microorganism laboratory biological safety the ministry of health of china. the directory of pathogenic microorganisms transmitted in human chinese national standards. general requirements for laboratory biosafety chinese national standards. architectural and technical code for biosafety laboratories biological safety and protection in animal experiments agriculture science and technology press control strategies for aeroallergens in an animal facility elisa method for measurement of airborne levels of major laboratory animal allergens the effectiveness of the duo-flo bioclean unit for controlling airborne antigen levels laboratory animal allergy: the measurement of airborn urinary allergens and effects of different encironmental of airborn urinary allergens and effects of different encironmental conditions the effect of relative humidity on mouse allergen levels in an environmentally-controlled mouse room elisa method for measurement of airborne levels of major laboratory animal allergens reduction of airborne allergenic urinary proteins from laboratory rats immunochemical measurement of airborne mouse allergens in a laboratory animal facility airborne allergens associated with asthma : particle sizes carrying dust mite and rat allergens measured with a cascade impactor task-related variation in airborne concentration of laboratory animal allergens: studies with rat n i bio-aerosols in indoor environment: composition, health effects and analysis indoor air quality: solutions and strategies bio-aerosol concentrations in the quad cities year after the mississippi river floods human brucella canis infection and subsequent laboratory exposures associated with a puppy laboratory-associated infections: incidence, fatalities, causes, and prevention control of laboratory airborne infection past and present hazards of working with infectious agents viral infections in workers in hospital and research laboratory settings: a comparative review of infection modes and respective biosafety aspects contamination of microbiological aerosol generated by pathogenic microbiological labs chinese national standards. environment and facilities for laboratory animals designing and construction of infectious animal testing facilities potential environmental influnce of biosafety level three laboratory and its protective countermeasures incineration of healthcare wastes: management of atmospheric emissions through waste segregation institutional responsibilities in contamination control for reasearch animals and in occupational health and safety for animal handlers key: cord- -smqc vr authors: singla, rubal; mishra, abhishek; joshi, rupa; jha, sonali; sharma, amit raj; upadhyay, sujata; sarma, phulen; prakash, ajay; medhi, bikash title: human animal interface of sars-cov- (covid- ) transmission: a critical appraisal of scientific evidence date: - - journal: vet res commun doi: . /s - - - sha: doc_id: cord_uid: smqc vr coronaviruses are a large family of viruses that are known to infect both humans and animals. however, the evidence of inter-transmission of coronavirus between humans and companion animals is still a debatable issue. there is substantial evidence that the virus outbreak is fueled by zoonotic transmission because this new virus belongs to the same family of viruses as sars-cov associated with civet cats, and mers-cov associated with dromedary camels. while the whole world is investigating the possibility about the transmission of this virus, the transmission among humans is established, but the interface between humans and animals is not much evident. not only are the lives of human beings at risk, but there is an equal potential threat to the animal world. with multiple reports claiming about much possibility of transmission of covid- from humans to animals, there has been a significant increase in the number of pets being abandoned by their owners. additionally, the risk of reverse transmission of covid- virus from companion pets like cats and dogs at home is yet another area of concern. the present article highlights different evidence of human-animal interface and necessitates the precautionary measures required to combat with the consequences of this interface. the centers for disease control and prevention (cdc) and the world health organization (who) have suggested various ways to promote awareness and corroborate practices for helping people as well as animals to stay secure and healthy. the recent outbreak of sars-cov- has caused a serious global threat. the speed with which this virus is spreading all over the world has crossed the rate at which the understanding of the disease and its effects is evolving amongst scientists, public health officials, and other members of the global health community. while the pandemic continues, scientists are looking at tracing the species from which the infection may have originated (guo et al. ) . in the light of the evidence depicting its first circulation in bats before transmitting via intermediate host to humans, a new question on whether this virus has the capability of transfer from human to animal is trending. various new cases have come forward in revealing the transmission of this novel virus from infected humans to animals (ng and hiscox ) . moreover, the risk of reverse transmission from companion animals to humans can also increase the risk of covid- infection. although there is limited evidence of transmission of the virus among pets, yet the likelihood of its transmission to companion humans has also been discussed by various scientists and public health officials. the goal of this article is to review the possible transmission of covid- in animal species in the context of positive animal corona cases worldwide and reach a possible conclusion. moreover, precautionary measures to protect pets and other animals from the virus have also been discussed. this review is built on a narrow range of data available till date and general instructions for preventing and monitoring the consequences of the human animal interface. there is an urgent need to promote awareness and corroborate practices for helping people as well as animals to stay secure and healthy. furthermore, it provides a quick insight into the suspected sars-cov- reservoirs and introduces some safety measures for humans as well as companion animals to control the spread of covid- . coronaviruses (cov) are a group of viruses belonging to the family of coronaviridae. these families of viruses infect both humans and vertebrate animals (ng and hiscox ) . four types of corona viruses, namely, hkui, nl , oc , and e, also known to cause respiratory diseases (saif et al. ) , while sars-cov, mers-cov, and sars-cov- can be responsible for causing severe pulmonary injury and respiratory distress which may cause fatality in humans. the two major events in the past resulted in severe diseases due to the transmission of viruses of beta (β) genera from animals to humans (guarner ) . in in - , the first event occurred in china into guangdong province when a beta coronavirus which originated in bats moved to human via palm civet cats which acted as their intermediate host (martina et al. ) . a decade later, in , another coronavirus named as the middle east respiratory syndrome coronavirus, designated as mers-cov was originated in saudi arabia and its transmission took place from bats with dromedary camels being the intermediate host (qian et al. ) . it was reported to infect about people with fatalities. recently, at the end of the year , wuhan, a major business centre in china, experienced an emergence of a new corona virus, i.e., sars-cov- (covid- ) which is also described to be a subtype of the β subgroup of coronaviruses (xu et al. a ). sars-cov- has drastically spread over more than countries and territories around the world, infecting around million people across the world. not only does this virus attack on various organs of the body including the respiratory system, gastrointestinal system, liver, but as well as invades the central nervous system, and causes illness to both humans and various animals such as bats, cattle, birds, mice etc. (salata et al. ) . the findings of the genetic sequence testing reveal that the new sars-cov- has about % similarity with sars-cov. this data was released by the military medical research institute of nanjing military region. another report by shi et al. described the sars-cov- to share . % similarity with the sars-cov coronavirus that originated from rhinolophus affini (bats) (xu et al. b ). this clearly illustrates that the new emerging virus may have also originated from bats. however, the intermediate host for this virus is elusive, some reports have stated snakes or minks to be the intermediate hosts. recently, a group of researchers from the agricultural university of south china analyzed over one thousand metagenomic samples and observed that about % pangolins out of more than metagenomic samples were found to be positive for the coronavirus . also, there was % similarity of the genetic sequence between the pangolins and the present infectious human strain (zhang and holmes ) . based on these evidence, there is a high probability of the pangolins to be one of the intermediate host of covid- (zhang et al. a; lam et al. ). bats have been identified to be an important reservoir host for an array of coronaviruses. in the past several years, frequent sampling has recognized various bat corona viruses. the data from the genomic comparisons exhibited viruses from bat to have a major correlation with sars-cov- (zhang and holmes ). another study by yadav et al. tested bat samples from seven different states of india and found the rectal swab test of eight samples of rousettus spp. and pteropus spp. to be positive for rdrp gene. additionally, in the same study, the results of the next generation sequencing revealed that the four sequence regions of rousettus spp. had about approximately . % similarity to the previously found coronaviruses in bat (yadav et al. ) . since there is a general ecological separation between bats and humans, it is possible that some mammalian species act as an 'intermediate' or 'amplifying' host. it is likely that the recently emerged coronavirus acquired the much) needed mutations within these hosts for human transmission (ahmad et al. ) . civets and camels acted as the intermediate host for sars and mers, respectively, white camels are thought to be the true reservoir hosts from mers (de wit et al. ) . the recently discovered sars-cov- virus is found to be closely related to the malayan pangolins (manis javanica). these pangolins were imported illegally from the guangdong and guangxi provinces of southern china (lake ). the guangdong pangolin viruses are explicitly related to the sars-cov- in the receptor binding domain and containing all six key mutations that are believed to shape the virus for binding to the angiotensin converting enzyme (ace) receptor and manifesting about % similarity of the amino acid sequence (hasan et al. ). however, the guangdong pangolin viruses showed high divergence in the remaining genome. the various evidence from the past clearly suggest that the evolution of the virus in both reservoir and intermediate animal hosts needs to be explored to better evaluate the emergence of sars-cov- in humans. however, studies revealing the acquirement of key mutations by the virus during abstruse transmission in humans prior to its detection should not be ignored. a group of researchers suggested that pangolins and bats act as a carrier for sars-cov- due to similarity in their genetic makeup. inspite of there being a reasonable correlation of sars-cov- with mers-cov and sars-cov, there are some remarkable differences between these viruses. sars-cov- gains entry into human cells using spike proteins followed by a series of complex processes (shereen et al. ) (fig. ) . once sars-cov enters inside the host body, it targets cells through an endosomal pathway. the initial attachment of the virion to cells occurs via s protein binding to the ace- receptors (ji et al. ). the conformational change in s protein assists the fusion of the viral envelope with the cell membrane, thereby liberating the infectious rna in the host cell. this rna is then translated by viral replicase polyproteins pp a and pp ab, followed by its cleavage by the viral proteinases into smaller products forming a full length negative-strand template for the genomic rna (song et al. ) . during translation, viral protein particles are formed. viral protein and genome rna are assembled in a step-wise manner by virion in the endoplasmic reticulum and golgi. the new virion is then released from the cell through exocytosis (zhang et al. b ). now, the new virions have the ability to infect other body organs. these virions can contaminate other humans via close contact in communities. some evidence also suggested that it can also affect pets like cats and dogs. coronaviruses are generally found in various domestic and wild animal species including cattles, horses, ferrets, bats, dogs, and others (ng and hiscox ) . current evidence suggest that the major transmission route is from human to human. although not common, coronaviruses can also be transmitted from animals to humans. the outbreak of the major coronaviruses in the past in humans suggested that bats act as reservoirs for these viruses that crossed the species barrier and infected humans as well as other domestic and wild mammals (malik et al. ) . there were also studies which reported the fact that palm civet cats (sars) and dromedary camels (mers) acted as the intermediate host (abdullahi et al. ) . the origins of covid- virus are also believed to be from animal source. at present, there are numerous research programs dedicated to finding the potential source as well as species involved in the emergence of this pandemic. the genomic sequence data reveals % similarity between the sars-cov- and the previous human coronavirus (sars-cov), establishing its close relation to the rhinolophus bat (horseshoe bat) population (fig. ) . there is also a possibility of the involvement of an intermediate host for transmission to humans. health officials have found the first sars-cov- infections to be linked to the live animal market in china (liu and saif ) . however, till date, there are not enough scientific documental evidence to explain the original route of transmission from animal to human sources. are other species such as mosquitoes, houseflies responsible for the transmission of virus to humans? till date, there is no evidence that covid- or any other similar corona viruses such as sars or mers are spread through mosquitoes or ticks because the virus needs to replicate inside the mosquito if it has to pass to a person through the mosquito (eslami and jalili ). to check the susceptibility of sars-cov- in ferret a two virus samples, f -e and ctan-h that were previously isolated from environmental samples in the huanan seafood market in wuhan and human patients respectively, were inoculated intranasal in a pair of ferrets ( pfu). the qpcr and virus titration test conducted on the various isolated organs of the ferrets on day post inoculation detected infectious virus in the nasal turbinate, soft palate and tonsils of ferrets indicating the possible replication of the virus in the upper respiratory tract of the ferrets while no infection was found in other organs such as trachea, lung, heart, spleen, kidneys, pancreas, small intestine, brain and liver of the ferrets (kim et al. ) . further, when a group of three animals intranasal inoculated with pfu of f -e and ctan-h placed in three separate cages with uninfected ferrets, the nasal washes of six ferrets were detected with viral rna after day , , and of inoculation. however, the rectal swabs of the ferrets were noted to have lower copy numbers of the infectious virus rna. three ferrets also had a loss of appetite and developed fever on days and (stittelaar et al. ) . to check whether the symptoms were caused due to the virus, the tissues and organs of the two ferrets were euthanized at p.i . low copy number of the virus rna was detected in the turbinate of the ctan-h inoculated ferret. furthermore, the elisa results revealed the presence of antibodies against sars-cov- in all the ferrets (el-duah et al. ). the investigation on the lungs of the ferrets to check the viral replication in these organs was done by intratracheal inoculation of eight ferrets with pfu of ctan-h. the animals were euthanized on p.i days , , , and to check the viral rna in various tissues and organs. the results indicated that the sars-cov- replicates upto days in the upper respiratory tract of the ferrets without causing any severe disease. young-ll kim et al. checked the transmission of sars-cov- in ferrets in an experimental setting (van den brand et al. ). the study was conducted for a period of days. two ferrets were intranasally inoculated with an isolated strain ( . tcid of nmc-ncov ) of a covid- positive south korea patient. after days post inoculation, two naive ferrets per group were either housed together with infected ferrets (direct contact) or were kept in cages having a permeable partition to separate them from the inoculated ferrets (indirect contact). the results of this experiment revealed a rise in the body temperature of all the direct contact ferrets to about °c on average, accompanied by reduced activity. on the contrary, none of the indirect contact ferrets acquired elevated body temperatures. thus, we can infer that the efficient transmission of sars-cov- clinical symptoms requires direct contact with the infected animals ). further, the replication of the virus amongst all groups of ferrets was investigated by collecting saliva, nasal washes, blood, urine and fecal samples consecutively for days. the viral rna copies were calculated using quantitative rt-pcr. these viral rna copies were then compared with the number of standard control copies. the results of the viral rna showed the maximum viral rna in the nasal washes at pi days and . the saliva as well as the fecal matter of the direct contact ferrets was detected with viral rna on p.i day . for the indirect contact group, only two out of six ferrets had viral rna in nasal washes as well as fecal specimens from pi day . in addition, the copy number of the viral rna in the indirect contact ferrets was lower than in the direct contact ferrets. the nasal turbinate and lung tissues had the highest number of viral rna copies, dpi. moreover, there was the presence of a small amount of viral rna in the kidney and intestine of ferrets after day of inoculation. the virus was also tracked down in the lungs, kidney, trachea, nasal turbinate, and intestine at dpi. additionally, the immunohistochemistry and histopathological examination of the various tissues of ferrets revealed that cells in the nasal turbinate, lung, and trachea of the infected ferrets were positive for sars-cov-and acute bronchiolitis by nmc-ncov infection (van den brand et al. ). this study results stipulate ferret to have high susceptibility for the sars-cov- and this infectious virus sheds by multiple routes of body discharge specimens such as urine and faeces of the infected ferrets which serve as a potential source of viral transmission to close contact. to investigate the replication of the sars-cov- virus in cats, an in-vivo study was performed in which five cats were inoculated with pfu (intranasal) of ctan-h. amongst them, two of the infected cats were sacrificed on pi and viral rna was found in the various isolated organs of the cat after euthanizing them on p.i day (mallapaty ). the virus was detected in the nasal turbinates, tonsils, soft palates and trachea. however, no infection was found in the lung samples of the animal. the other three sub adult cats were placed separately with three uninfected cats in different cages (kim et al. ) . the fecal samples of all virus inoculated sub adult cats were detected with viral rna on p.i day while only one exposed cat showed viral rna in the feces. after the cats were euthanized on p.i day , viral rna was also found in the tonsils and soft palate of the infected cat, while in the nasal turbinate, soft palate, tonsils and trachea of the exposed cat. thus, it was clear that the transmission through respiratory droplets had occurred. furthermore, the results of the elisa and neutralization assay revealed that antibodies of sars-cov- were found in all the three pre inoculated cats and one exposed cat (el-duah et al. ) . similar experiments were performed with juvenile cats and the histopathological examination revealed massive lesions in the nasal, mucosal and lungs of the cats. based on these studies, it has been found that sars-cov- can be replicated in both adult and young cats and the mode of transmission between cats was via respiratory droplets. the study examined the transmission and replication of sars-cov- in dogs. five beagles of age months were inoculated intranasal with pfu of ctan-h and were placed along with two naive dogs. the rectal swabs and oropharyngeal swabs were collected until p.i day . the viral rna was detected only in two inoculated dogs on pi and in rectal swabs of one dog on pi . the viral rna positive dog did not show infection in any organs or tissues after it was euthanized from p.i . the results of the elisa were seronegative for all except for two virus inoculated dogs which were found to be seroconverted on p.i day . thus, it is clear from the experiment that dogs are less susceptible to sars-cov- infection ). a similar set of experiments were performed in pigs, chicken and ducks to investigate the virus susceptibility in these animals. the results from the experiment clearly stated that neither the inoculated animals nor the naive contacted animals were detected with viral rna in the swab test. similar results were observed in the elisa experiment where all the animals were seronegative for sars-cov- on post inoculation day . based on the results of this experiment, we can infer that pigs, chickens, ducks are not susceptible to this virus ). as sars-cov- is widely disseminating in the human population, it is likely that animals in close contact with humans can get infected as well (almendros ) . various studies are being carried out to understand the vulnerability of different animal species to the virus and in better assess the infection dynamics in these susceptible animals. till date, some animals such as dog, cats, and tiger are found to be infected with the virus following close contact with infected humans (goumenou et al. ) . the latency period for sars-cov- is almost similar in both humans and animals. on average, it ranges from to days to upto days. surprisingly, the symptoms in animals are not certain, some develop dry cough, etc. (fig. ) . this human animal interface may even remain asymptomatic and can sometimes be contagious and act as silent sources of transmission (ng and hiscox ) . however, there is no current evidence suggesting the possibility of animals infected by humans in spreading the virus. hong kong's agriculture, fisheries, and conservation department (afcd) reported on february , , that the nasal and oral cavity samples of a -year-old pomeranian dog were diagnosed as "weak positive" according to the results of the rt-pcr (real-time reversetranscriptase polymerase chain reaction). the dog was already quarantined as the owner of the dog had been earlier diagnosed with covid- (almendros ). the dog did not show any symptoms of illness. however, the results of the fecal sample and the rectal swab were negative. the reports of rt-pcr testing on the nasal cavity repeated on february , march , , and were again found to be weak positive, indicating the presence of a minute amount of sars-cov- rna in these samples. moreover, the results of the genome data sequencing of the dog and its close human contacts completed on march suggested a high similarity between the viral sequences. the virus neutralizing antibody test completed on march was negative. the rt-pcr conducted on march and consecutively were reported to be negative and thus the pomeranian was released on the following day. however, the dog died three days later and unfortunately the owner of the dog did not allow a postmortem examination (mallapaty ) . the death was possibly due to various co-morbidities and the age of the dog and was unlikely to be associated to covid- . the scientist of hong kong university believed that there was a possibility of the transmission of the virus from the infected owner to the dog considering the persistence of the positive results from the pomeranian's samples. the second possible human-to-dog transmission was reported on march when a two year old german shepherd dog living in pok fu lam was tested positive for covid- . as seen earlier, the years old owner of the dog was also infected with the virus. however, the dog did not show any symptoms of respiratory discomfort during its quarantine period (sit et al. ). the agriculture, fisheries and conservation department reported that the results of rt-pcr on the oral and nasal samples of the dog came out to be positive. until th march, dogs and cats of the owners with confirmed cases or people in contact with confirmed cases were tested and monitored. however, only dogs mentioned earlier were positive for sars-cov- . the federal agency for safety of the food chain (fasfc) was informed on march , by the faculty of veterinary medicine at the university of liege that a cat in belgium was found to be positive for the coronavirus (mallapaty ). the reports of pcr (high throughput sequencing) detected the viral rna of sars-cov- in the faeces and vomit of the cat. the cat was likely to pick up the virus from its owner who after travelling to northern italy fell sick with sars-cov- . the cat started to show symptoms of illness a week later and had signs of respiratory and digestive dysfunction. the cat was quarantined and recovered after nine days. currently, we do not have any evidence regarding the potential other conditions which may lead to respiratory and gastrointestinal signs. once the cat is released from quarantine, the research will be carried out for the sars-cov- antibodies in the blood test, which would provide the true proof of the infection. the virologist named steven van gucht made it clear that this case was about human-to-animal transmission and not vice-versa. however, he also stated that this was a rare case and the possible risk of animal to human transmission is very small. a pet cat in hong kong was tested positive with covid- on march , after it was quarantined on march after its owner, a women of the shek pai wan estate, aberdeen was infected with the corona virus. the samples collected from the nose, mouth, and rectum of the cat were tested positive for the virus. however, the cat did not show any signs of the disease. it is believed to be the second cat in the world to be infected with this virus (pet cat in hong kong ). a female malayan tiger name nadia from the bronx zoo in new york city was found to be having respiratory illness and thus tested positive for covid- on april , . the test results were confirmed by the national veterinary services laboratory at lowa. the samples of the four-year-old female tiger along with six other animals who had developed dry cough and started showing indications on the th of march were taken and are believed to be infected with covid- . however, till date only the results of nadia are confirmed to be positive (gollakner and capua ) . this animal is believed to have acquired the infection from an asymptomatic zoo-keeper after which she started to display a loss of appetite and dry cough. this case was the first case to be documented of covid- infection in us animals as well as tigers all over the world. these findings raise new questions about the transmission of the virus in animals. minks are commonly farmed for their fur in netherlands. however, two of the mink farms (one in the province north brabant, south netherlands observed a increase in the mink mortality in mid april . some these minks displayed signs of respiratory signs (mostly nasal discharge). all the minks in these farms were separately caged and there was no connection of workers, transport between the two farms. the necropsied samples from the dead minks tested positive for sars-cov- . histological findings revealed severe diffuse pneumonia with hyperaemia, alveolar damage as well as air containing alveolar lumina in lungs of the dead minks. the qpcr analysis detected the presence of viral rna in the liver and intestines of some animals. also, the throat swabs of fig. comparison of various aspects of covid- between humans and animals all the necropsied animals showed the presence of viral rna. on further investigation of the farm owners and workers, four members on one of the two farms have had respiratory disease symptoms in the start of april but did not get checked for sars-cov- infection. additionally, one of the people who stayed in the farm was later detected with sars-cov- by the end of april. moreover, one of the workers of farm two was hospitalized on march due to covid- . these results further confirm the possibility of transmission of the virus between humans and animals (here, minks). inhalable dust samples were collected three times between april and may at different locations in the mink house. viral ran was detected in two of the three samples from farm one and one sample in farm two. also, stray cats around the farms were sampled for the sars-cov- and results revealed the presence of antibodies against sars-cov- in seven cats and viral rna in one cat. the mink outbreaks are "spillover" from the human pandemic-a zoonosis in reverse. the most likely explanation for the widespread infection on the mink farms is the possibility of the introduction of the virus by the humans to the minks. the minks were caged separately with non-permeable partition between the cages, precluding the direct contact as a mode of transmission amongst the minks. thus, the indirect transmission may have occurred through the food or the bedding material, infected droplets generated by the infected animals or by the contaminated fecal matter. sequence analysis of mink-derived viruses pointed at humans as the probable source of the initial infection and demonstrated transmission between minks. furthermore, the presence of viral rna in inhalable dust collected from the farms indicated a possible exposure of workers to virus excreted by minks. similar cases were found in around nine other farms in netherlands but there possible connection with the first two cases has not been detected yet. (oreshkova et al. ). it is not yet comprehensive to comment whether animals, including livestock, pets, and wild life pose any risk of covid- infection. however, adequate precautions should be taken while dealing with pets or any other animals such as washing hands properly after any contact with the animals, their food, or animal waste (marconato and finotello ) . also, utmost care should be taken to clean up the pets properly and to follow a good pet hygiene as they may otherwise contain germs which may make humans sick. there should be regular visits to the veterinarians or assistance from them if the need arises about pet health. how to protect pets if you are sick? although there are very few reports on whether sars-cov- can be transmitted from humans to animals, it is recommended that confirmed as well as suspected individuals with covid- to have restricted contact with pets or other animals until more information about the transmission is available. continual exposure to humans hosting the corona virus would sooner or later possibly cause major threats to these animals to extinction. thus, limited exposure and frequent contact would considerably reduce the threat and in turn ensure the safety of both the pet and the owner (decaro et al. ) . it is also suggested that if there is no other member of the family to take care of the pet while the owner is infected, proper hygiene should be maintained around the pet. the owner or the infected person should avoid physical contact such as kissing, licking, snuggling, or sharing food with the pet and wash his hands before and after interaction with them. safe hygiene practice needs to be ensured while handling and caring for the pets and special precautions should be taken when in close proximity with the animal, such as wearing a mask. when handling and caring for the animals, they should maintain good hygiene practices and wear a mask. moreover, the infected person should not have common drinking glasses, utensils, towels, bedding with pets at home ). to keep the pets safe during this pandemic, it is advisable to have an emergency kit prepared in advance containing sufficient food and medications for at least two weeks (aitken ) . since there are not enough evidence that pets can spread the virus to other animals or humans, it is not recommended to remove pets from the homes of people where covid- is confirmed or suspected in any member of the household. in case if the pets are not able to be taken care properly, veterinarians are there to support the good health of the pets. we need to understand that there is a difference between the pets contracting the virus and a pet spreading the virus to humans. the research experts said that the likelihood of humans contracting covid- from their pets is low. also according to the cdc reports, there is no evidence for transmission of the novel corona virus from companion animals to humans. the major route of transmission appears to be from person-to-person by contact with saliva or other bodily secretions through cough or sneeze (covid- : coronavirus and pets faq ) . transmission through contaminated surfaces and objects remains the secondary route of transmission. however, non-porous materials such as door knobs have higher capacity to transmit the virus as compared to nonporous surfaces. since pet furs belongs to the category of non-porous surfaces and are likely to trap and absorb the virus, it is harder to contract the virus onto them through simple touch. on the contrary, there are chances of dissemination of the virus to humans from the body fluids of pet animals. since pet dogs and cats are always in close proximity to their owners, there are chances of transmission through kissing, licking, and playing with the pets (fig. ) . moreover, researchers have found that most of the cats and ferrets that were infected with the virus are asymptomatic, showing no signs of coughing, sneezing, weight loss, or raised body temperature (halfmann et al. ) . thus, it is likely that the owners of the infected pet animals would not have noticed any change in their pets. in view of the fact that sars-cov- virus was also detected in the feces of animals experimentally inoculated with the human virus titre, there are chances that the virus contaminated faecal matter and urine of pet animals can transmit the virus to humans. it has been advised by all healthcare agencies that covid- confirmed positive cases must avoid contact with companion animals (can your pet get covid- ). social distancing (at least ft) should be maintained even with pets or other companion animals outside the household. with the increasing reports of transmission of covid- from infected humans to pets (like cats and dogs) and tigers and lions, there is an urgent need for public health officials to identify and investigate the possibility of virus transmission from pets to humans. at present, people should emphasize on preventing the possible transmission of sars-cov- on to their pets. in accordance with the data from various experimental reports, cats infected with the virus having no appreciable clinical signs can act as a silent transmitter of covid- infection to humans (cdc immediate medical care should be given in case of any type of scratches or bites. furthermore, pets should also be monitored for clinical symptoms or any unusual change in their behavior. are there any precautions to be taken with live animals or animal products? the world health organization (who) has advised people to take general precautions while visiting live animal markets, animal product markets, or wet markets because there is still uncertainty about the exact origin of this virus (zhonghua ). also, it is recommended to wash hands regularly with soap and potable water after any contact with animals or their products as well as avoid touching eyes, nose or mouth after contact with sick animals or spoiled animal products. adequate precautions should be taken to avoid any contact with animal waste or fluids on the soil or any other surfaces. any contact with stray animals such as dogs, cats, birds, rodents living in the streets to be completely restricted. additionally, even though no research based evidence has proven the spread of this virus due to intake of seafood, chicken, or mutton, still the intake of any raw or uncooked milk, meat, or any kind of animal organs should be avoided as per the general good food safety practices ). since the report of the new york zoo tiger tested positive for covid- , the ministry of environment, forest and climate change has advised zoos to remain at high alert and regularly observe animals for any abnormal behaviors or symptoms. the advisory has ordered that the care takers of zoo animals should wear proper personal protective equipment and maintain limited contact with animals while feeding them. in addition, the sick animals are advised to be quarantined and all zoo personnel should strictly follow the safety and disinfection protocol issued by the government (covid- : alipore zoo ). proper screening, testing, and surveillance of the diagnostic samples should be done whenever required. along with this, an advisory has been issued to all the chief wildlife wardens of states and union territories by the ministry of environment which includes the following instructions: -proper regulatory measures should be taken by all the national parks, sanctuaries, and tiger reserves to prevent the spread of sars-cov- from humans to animals. -there should be reduction in the human and wildlife interface. -movement of people to the national parks and sanctuaries should be curtailed. -management of the current situation with the help of task force to be undertaken as soon as possible. -a significant enhancement in the mapping, tracing, monitoring, and surveillance of the system in coordination with various departments. -emergency services for the treatment of animals, if required, should be undertaken. -proper reporting to the nodal officer should be done in any type of noticed cases. -other possible steps to be followed to prohibit the spread of the virus. the comparison of the human version of the coronavirus to previously isolated strains from animals was possible only due to the availability of the complete genetic sequencing of the sars-cov- (kannan et al. ) . various evidence suggested that bats are the primary reservoirs of the novel corona virus and thus the major source of infection to humans. since the sars-cov- genomic sequence in humans and pangolin has shown significant relatedness, these animals are suspected to be the intermediate host for this infection (xu et al. c ). however, the routes of direct or indirect zoonotic transmission are still to be unveiled. thus, comprehensive studies on how an animal virus crossed the species boundaries to infect human species are required to discover the precise routes of transmission. besides, the veterinary community has also been trying to identify the risks to animals and possible zoonotic transmission. various cases of human to animal transmission have also come up indicating the risk of the virus to domestic animals or pets (oreshkova et al. ) . the literature reveals that cats and ferrets are susceptible to covid- infection. these infected animals could efficiently spread the virus to naive animals directly in contact with them. additionally, the ace receptors in a few animal species are found to be similar to the human ace receptors, thus further stipulating the possibility of the human-animal interface. these findings also indicate that domestic cats and ferrets can be an effective animal model for screening candidate vaccines and drugs against sars-cov- (hasan et al. ) . although it is still not certain that humans can transmit covid- to their pets or other animals, it is recommended to follow some preventive measures and basic animal hygiene. moreover, we cannot believe that just if the pets can be infected with sars-cov- , they can also spread it. at this time, there is no literature proving the transmission of the virus from the infected pets to their owners. a lot of research is needed to determine the role of the animals in the spread of the virus. however, social distancing needs to be maintained with animals as with humans. by doing so, we can reduce the human as well as zoonotic transmission of sars-cov- . f u r t h e r m o r e , s o m e e x p e r i m e n t a l s t u d i e s a n d serosurveillance studies in different animal species should be continued to reach an effective conclusion and prevent further zoonotic events. data availability data sharing is not applicable to this article as no new data were created or analyzed in this study. conflict of interest none of the authors declared any potential conflict of interest. ethical statement ethical statement is not applicable as this is a review article. sars-cov- , sars-cov, and mers-cov: a comparative overview covid- : zoonotic aspects. travel medicine and infectious disease ensuring animal welfare during covid- pandemic can companion animals become infected with covid- ? can your pet get covid- ? covid- : alipore zoo takes measures to prevent coronavirus spread covid- : coronavirus & pets faq sars and mers: recent insights into emerging coronaviruses covid- from veterinary medicine and one health perspectives: what animal coronaviruses have taught us development of a whole-virus elisa for serological evaluation of domestic livestock as possible hosts of human coronavirus nl the role of environmental factors to transmission of sars-cov- (covid- ) is covid- the first pandemic that evolves into a panzootic? possibility of transmission through dogs being a contributing factor to the extreme covid- outbreak in north italy three emerging coronaviruses in two decades: the story of sars, mers, and now covid- the origin, transmission and clinical therapies on coronavirus disease (covid- ) outbreak-an update on the status transmission of sars-cov- in domestic cats a review on the cleavage priming of the spike protein on coronavirus by angiotensin-converting enzyme- and furin covid- pandemic: stop panic abandonment of household pets twirls, an automated topic-wise inference method based on massive literature, suggests a possible mechanism via ace for the pathological changes in the human host after coronavirus infection covid- (novel coronavirus ) -recent trends infection and rapid transmission of sars-cov- in ferrets what we know so far: covid- current clinical knowledge and research identifying sars-cov- related coronaviruses in malayan pangolins emerging viruses without borders: the wuhan coronavirus genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding emerging coronavirus disease (covid- ), a pandemic public health emergency with animal linkages: current status update coronavirus can infect cats -dogs, not so much veterinary oncologists adapting to covid- pandemic. veterinary and comparative oncology sars virus infection of cats and ferrets coronaviruses in animals and humans sars-cov- infection in farmed minks, the netherlands hong kong tests positive for covid- after owner becomes infected role of the spike glycoprotein of human middle east respiratory syndrome coronavirus (mers-cov) in virus entry and syncytia formation coronaviruses. diseases of swine coronaviruses: a paradigm of new emerging zoonotic diseases covid- infection: origin, transmission, and characteristics of human coronaviruses susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus . science infection of dogs with sars-cov- from sars to mers, thrusting coronaviruses into the spotlight ferrets as a novel animal model for studying human respiratory syncytial virus infections in immunocompetent and immunocompromised hosts. viruses : van den brand jma structural and functional basis of sars-cov- entry by using human ace systematic comparison of two animal-to-human transmitted human coronaviruses: sars-cov- and sars-cov systematic comparison of two animal-to-human transmitted human coronaviruses: sars-cov- and sars-cov pathological findings of covid- associated with acute respiratory distress syndrome detection of coronaviruses in pteropus & rousettus species of bats from different states of india a genomic perspective on the origin and emergence of sars-cov- probable pangolin origin of sars-cov- associated with the covid- outbreak angiotensin-converting enzyme (ace ) as a sars-cov- receptor: molecular mechanisms and potential therapeutic target zhonghua liu xing bing xue za zh the epidemiological characteristics of an outbreak of novel coronavirus diseases (covid- ) in china. epidemiology working group for ncip epidemic response key: cord- -p pns r authors: malik, yashpal singh; verma, atul; kumar, naveen; deol, pallavi; kumar, deepak; ghosh, souvik; dhama, kuldeep title: biotechnological innovations in farm and pet animal disease diagnosis date: - - journal: genomics and biotechnological advances in veterinary, poultry, and fisheries doi: . /b - - - - . - sha: doc_id: cord_uid: p pns r the application of innovative diagnostic technologies for the detection of animal pathogens at an early stage is essential in restricting the economic loss incurred due to emerging infectious animal diseases. the desirable characteristics of such diagnostic methods are easy to use, cost-effective, highly sensitive, and specific, coupled with the high-throughput detection capabilities. the enzyme-linked immunosorbent assay (elisa) and polymerase chain reaction (pcr) are still the most common assays used for the detection of animal pathogens across the globe. however, utilizing the principles of elisa and pcr, several serological and molecular technologies have been developed to achieve higher sensitivity, rapid, and point-of-care (poc) detection such as lateral flow assays, biosensors, loop-mediated isothermal amplification, recombinase polymerase amplification, and molecular platforms for field-level detection of animal pathogens. furthermore, animal disease diagnostics need to be updated regularly to capture new, emerging and divergent infectious pathogens, and biotechnological innovations are helpful in fulfilling the rising demand for such diagnostics for the welfare of the society. therefore, this chapter primarily describes and discusses in detail the serological, molecular, novel high-throughput, and poc assays to detect pathogens affecting farm and companion animals. livestock, poultry, and aquaculture are among the fastest growing and expanding agriculture sectors to fulfill the need of the growing population of humans. however, the growth in this sector is under the continuous increasing threats of infectious diseases worldwide. this menace is further aggravated by globalization in animal trade for various purposes. the sudden entry of an infectious disease in a new country or geographical location could lead to delayed diagnosis and rapid spread into the susceptible animal population. in response to climate change, vector-borne diseases are also increasing worldwide. to prevent the spread of infectious diseases, one of the basic and critical requirements as prescribed by the world organization of animal health (oie) is the application of rapid, accurate, and highly sensitive identification of infectious agents. though the term "biotechnology" was coined in the year by karl ereky, the tangible biotechnological advancements in improving the human and animal health were started in the late th century. since then, biotechnological applications have been making significant contributions in the development of novel powerful diagnostic assays for the efficient diagnosis and control of animal infectious diseases. importantly, biotechnology has made the availability of pen-side tests for use at the field level to detect the causative infectious agent during a disease outbreak. in this chapter, we primarily describe and discuss the innovative biotechnological advancements made in the animal disease diagnosis in a step-wise manner. the impact of infectious diseases is immense and is felt all across the world. infectious diseases have affected the whole society, economy, and political system. vital sectors are under continuous economic loss and unrelenting development. the infectious diseases have taken a huge physical toll on animals and humans. this has pressed on humanity and has caused substantial economic, social, and mental losses. thus, it is a matter of animal health and economic interest to invest in strategies to give a blow to infectious diseases and put them under control. elimination of the pathogens and/or their vectors from their natural reservoirs would always be a first thought, but the removal is not easy, as they are constantly emerging and it is always very difficult to predict the emergence of infectious agents. evolution of pathogens is putting extra challenges, pressing on humanity to look at the new strategies and forcing the researchers to look for innovative ones. the newly evolved pathogens are always more advanced and deadly from the previous ones and put up a strong resistance. "from the evolutionary perspective, they [viruses and bacteria] are 'the fittest' and the chances are slim that human ingenuity will ever get the better of them." with the increase in the knowledge of infectious diseases and science, the degree of pace in pathogen discovery has increased. to keep pace and for better diagnosis, new tools and techniques need to keep on evolving. this is not only to quickly detect the pathogens, but also to make predictions with probable and possible outbreaks. to understand the scenario and to reach a definite conclusion, knowledge of epidemiology and pathogenic etiology also needs to be studied. this will facilitate in understanding the ancestry of the pathogen, and provide an insight and a mechanism for the epidemic, endemic, and even pandemic outbreaks. this system will also assist in understanding the interface transmission between and the directional flow of the zoonotic infectious diseases. thus, phylogenetic analysis and epidemiology would aim toward strategizing the challenges during pathogen surveillance and discovery. sars, a coronavirus was pandemic in . but epidemiology and microbiology mediated to stem its disastrous results and also the causative agent of sars was identified. bacteria, viruses, and parasites, present in feces, contaminate foodstuffs and cause disease in humans and animals, affecting the social set up and consumer demands. to increase the productivity and for the maintenance of good health of animals, antibiotics are frequently administered resulting in the growth and emergence of antibiotic-resistant bacteria. this further aggravates the condition, and makes the situation more appalling. overseas imported pets were also found to transmit and carry over the diseases to humans (smith et al., ) . even aquaculture is at risk of contaminants with the virus directly affecting the marine lives, as in the case of the new virus discovered in salmon (finstad et al., ) . even honeybees and other pollinators are transmitting pathogens like fungi, bacteria, and viruses through the contaminated food items (cox-foster et al., ) . an array of classical and conventional techniques have been developed and used for the laboratory diagnosis of infectious agents or pathogens. the techniques include serological, cell culture, and electron microscopyÀbased methods, which are either time-consuming or labor-intensive or both. however, with the advancement in the biotechnology field, new and robust diagnostic techniques are continuously evolving and taking over the conventional methods (caliendo et al., ) . presently, molecular detection-based methods such as polymerase chain reaction (pcr) or its variants, and serological methods such as enzyme-linked immunosorbent assay (elisa), are being used worldwide for the accurate diagnosis of many animal diseases. however, point-of-care (poc) and high-throughput novel assays have been developed recently. furthermore, we discuss the pros and cons of frequently used diagnostics assays for animal diseases in accordance with the following sections: serological methods were introduced in the early s for the diagnosis of pathogens. various serological diagnostics have been developed such as complement fixation, counter-immunoelectrophoresis, immunofluorescence in cell culture, elisa, radio immunoassay, immune adherence haemagglutination assay, reverse passive hemagglutination assay, latex agglutination (la), chemiluminescent immunoassay, and immunochromatography test (ict). among these, ict and elisa especially sandwich elisa and competitive elisa are used frequently in the commercial diagnostic kits for animal diseases worldwide. ict assays mostly utilized mammalian igg in commercial diagnostic kits, however, avian igy antibodies, with added advantages over the mammalian igg, have been employed for the detection of norovirus, rotavirus, and astrovirus in the fecal samples with good sensitivity and specificity ranging between % and % (khamrin et al., ) . modifications in elisa format or combinations with other diagnostic methods have proved novel ways to detect pathogens more efficiently and accurately. for example, recently a novel elisa for the detection of group a rotavirus antigen in the fecal samples of multiple host species has been developed . this assay utilizes the potential use of synthetic peptides and is based on the detection of conserved vp protein using anti-recombinant vp antibodies as capture antibodies and anti-multiple antigenic peptide (identified and constructed from highly immune-dominant epitopes within vp protein) antibodies as detector antibodies. another assay, which is simple to perform without the requirement of laboratory facilities, is dot-elisa. a highly sensitive and specific dot-blot assays for rapid detection of staphylococcal enterotoxin-a in food has been reported (singh et al., ) . dot-elisa has been employed in diagnosing various important poultry diseases (alam et al., ; dhama et al., ; he et al., ; majumder et al., ; manoharan et al., ) . immuno-pcr is another powerful assay that has been used for the immunodetection of viral nucleic acids. by combining elisa with pcr, sensitivity of detection can be increased up to times and is especially useful in detecting low quantity viruses in the stool samples (bonot et al., ) . the major advantage of immune-pcr is that several viral nucleic acids can be detected simultaneously. recently, a combination of nanoparticles with the immuno-pcr, also known as nanoparticle amplified immune pcr (npa-ipcr), has been reported which increases the sensitivity -folds compared to elisa and several folds to rt-pcr. antigen detection using an antibody bound to gold nanoparticle cofunctionalized with thiolated dna complementary to a hybridized dna has been developed (perez et al., ) . here, the presence of antigen/virus particles activates the formation of a "sandwich" complex of gold nanoparticle construct, virus, and an antibody functionalized nanoparticles used for extraction. now, this complex is heated to c, thus releasing dna tags followed by the detection through real-time pcr. npa-ipcr offers a viable platform for the development of an early-stage diagnostics requiring an exceptionally low limit of detection. nucleic acid-based detections are used through the amplification methods, hybridization methods, which could be in situ, in vitro, and in vivo. the most common and widely used hybridization-based method is in situ hybridization, which could utilize fluorescent (fish) or chromogenic (cish) molecules. the cish-based assays for the rapid characterization of microorganisms, such as mycobacterium species and the dimorphic fungi in positive culture samples have been described (louro et al., ; scarparo et al., ) . recently, a fish-based assay has been developed for the identification and differentiation of mycobacterium tuberculosis complex from nontuberculous mycobacteria (baliga et al., ) . nucleic acid amplification methods are amongst the best in detecting pathogens with high sensitivity and specificity in the clinical samples. various modifications in nucleic acid amplification methods have provided collectively robust methods to yield better and accurate results. these modifications could be categorized into two amplification methods viz pcr and its variants, and isothermal amplification methods. these are the most common tools used for the pathogen detection worldwide. the three basic variants include ( ) real-time pcr, which is a modified version of conventional pcr, where quantification of dna sequence is possible without any further step of running the amplified product on agarose gel; ( ) multiplex pcr, where multiple sequences can be detected in a single reaction mixture, and ( ) reverse transcriptase pcr (rt-pcr) where rna is transcribed to cdna and this cdna is used in the amplification as template. the real-time pcr can utilize different fluorescence chemistries such as sybr green, taqman, or molecular beacon probes. recently, a taqman real-time rt-pcr assay has been developed for rapid detection and quantification of japanese encephalitis virus in swine blood and mosquito vectors (pantawane et al., ) . in isothermal amplification, a number of target dna copies increase at a constant temperature in just one cycle without the need of a thermocycler. various techniques have been developed using isothermal amplification methods viz nucleic acid sequence-based amplification (nasba), transcription-mediated amplification (tma), signal-mediated amplification of rna technology (smart), strand displacement amplification (sda), rolling circle amplification (rca), loopmediated isothermal amplification (lamp), isothermal multiple displacement amplification (imda), helicase-dependent amplification (hda), circular helicasedependent amplification (chda), single primer isothermal amplification (spia), and strand invasion-based amplification (siba). in all these methods, isothermal temperature amplified products can be visualized on gel through the various structures visible on gel or by the incorporation of dyes in the special structures formed on amplification serving in real-time detection. among all these techniques, lamp is the most widely used isothermal amplification method which is in fact an autocycling strand displacement dna synthesis, which deploys four primers forming a stem-loop dna by self-primed dna synthesis and a dna polymerase with strand displacement activity (malik et al., ; parida et al., ) . recently, an improved strategy using a double-labeled probe to overcome the problem of false positivity of lamp together with target gene real-time quantification is devised for detection of avian orthoreovirus (kumar et al., ) and salmonella spp. (mashooq et al., ) . other potential isothermal amplification techniques are recombinase polymerase amplification (rpa) and nasba. the former has brought a breakthrough in the detection of nucleic acids as it does not require denaturation of the template. rt-rpa is an extension of the above method, in which bacterial rt is used in the amplification of rna. rt-rpa was developed to study the outbreak of footand-mouth disease (fmd) disease in egypt (abd el wahed et al., ) . high degree of fidelity, portability, cost efficiency, simplicity, sensitivity, and tolerance to inhibitors, put this method into the category of resounding techniques, and implementation is quite easy at quarantine stations (moore and jaykus, ); while the latter one requires initial denaturation of the template followed by temperature labile polymerase dependent isothermal amplification and was designed specially to detect rna (compton, ) . a multiplex real-time nucleic acid sequence-based amplification (qnasba) system for the simultaneous detection of rotavirus a, norovirus genogroup ii/astrovirus has recently been developed (mo et al., a) . rt-nasba proved as more efficient than the conventional rt-pcr and taqman rt-pcr assays (mo et al., b) . a microarray is a multiplex lab-on-a-chip test. it is an arrangement of the large amount of biological materials for high-throughput screening on a solid support generally a glass slide, through the detection-based assays. microarray has done wonders in the high-throughput screenings and for the breakthrough causes of the outbreaks. simultaneous detections of coinfections and other more phenomenal changes during the outbreaks are the crucial developments to study the infectious diseases in endemic regions. their easiness has brought the working systems onto the platform on global diagnostics. multiple diagnostics with hybridizing ability put it at more ease to strategize the control management programs. but, this technique comes at high expenditures. data management skills and their interpretations need off to the most important tasks to be worked on. with the advent, new kits for point-of-care detections, bioelectric arrays, and liquid microarrays are in the development process. this would be an easy and an improved hybridization method for individual probe and target combinations with accurate detections. this will reduce the effort from clinical diagnosis to the personal level. these all will help in understanding the proper and common pathogens with scaling down the time. peptide nucleic acids (pnas) are highly versatile synthetic oligonucleotides, in which the native sugar-phosphate backbone of dna is replaced with amino acids. pnas bind to complementary dna strands with higher specificity and strength. furthermore, they are resistant to nucleases and proteases, making them a highly stable diagnostic reagent. the pna-based assay has greater sensitivity than direct sequencing and is significantly more affordable and rapid (ray and nordén, ) . the potential diverse uses of pna have been exhaustively described in a recent review (gambari, ) . a rapid label-free visual pna-based assay for detection and pathotyping of newcastle disease virus has also been reported (joshi et al., ) . similarly, pna-based beacons have also been used in hiv genotyping with high specificity (zhang and apella, ) . aptamers are artificial nucleic acid ligands that are isolated from combinatorial libraries of synthetic nucleic acid by an iterative process of adsorption, recovery, and reamplification. dna aptamers in particular have many advantages over antibodies (brody and larry, ) . aptamers, first reported in , are attracting interest in the areas of diagnostics and offer themselves as ideal candidates for use as biocomponents in biosensors (aptasensors), possessing many advantages over state of the art affinity sensors (o'sullivan, ) . the aptamers have proved to be potential diagnostic assays, especially in the detection of toxins such as brevetoxin- , potent marine neurotoxins (shimaa et al., ) , marine biotoxinpalytoxin (shunxiang et al., ) , β-bungarotoxin (β-butx), and a neurotoxin from the venom of bungarus multicinctus (ye et al., a) . furthermore, aptamers have been used for the serological detection of mycobacterium bovis (fu et al., ) , cryptosporidium parvum (iqbal et al., ) , and prion disease (saijin et al., ) . biosensors are portable, easy to handle, ultrasensitive, quick, and may be quite specific with less probability of a false positive. biosensors work on various principles viz detecting the changes in the ph, the ion concentrations, mass by specific hybridization, enzymatic reaction, loss of functionality, change in the electrical potential, change in color, and temperature. based on these principles, many biosensors have been devised for the detection of animal pathogens; for example, an extended-gate field-effect transistor for the direct potentiometric serological diagnosis of the bhv- (tarasov et al., ) , nanowire-based immunosensor for bovine viral diarrhea virus (bvdv) (montrose et al., ) , luminescence resonance energy transferÀbased biosensors for the ultrasensitive detection of the h strain (ye et al., b) , quartz crystal microbalance (qcm)Àbased immunosensors to detect h n (li et al., ) , and spectrosenstm optical microchip sensors for foot-and-mouth disease virus (fmdv) (bhatta et al., ) . the limited methods for detection of microbial signatures and the advent of new technology for quick and parallel gene expression capacities have eased in the detection of microbial disease. next-generation sequencing (ngs) is now being increasingly applied in understanding the molecular epidemiology, transmission, and characterization of animal pathogens. instead of gene-by-gene analysis, large deposits of genes available in the clinical sample can be detected in a single test. applications of ngs are considered as more resourceful. thus, it is widely accepted as a diagnostic tool and speedily is being replaced with most other molecular diagnostic technologies and has brought revolution in the diagnosis of pathogens. various modifications and improvements have brought a huge change in the sequencing and identification of genomes. it all started with pyrosequencing on roche , with small read lengths and less efficiency. roche was followed by ion-torrent illumina platform. the ngs has made it possible to sequence the complete viral genomes of many viruses cost-effectively such as including an avian influenza virus (croville et al., ) , classical swine fever virus (leifer et al., ) , and bluetongue viruses (rao et al., ) . recently, nanopore technology, with the promising improvement has brought a wonderful efficiency with emerging science and technology (goodwin et al., ) . nanopore systems can sequence both dna and rna viral genome in real time. this technology is based on the principle that when a strand of dna/rna is allowed to pass through a nanopore, the current is changed as the based a, t, c; and c passes through the pore in different combinations. using these systems, sequencing can be performed on the portable minion device, the benchtop gridion and the high-throughput, high-sample number promethion. recently, nanopore sequencing has proved a revolutionary diagnostic tool in detecting the ebola virus (hoenen et al., ) , influenza viruses (keller et al., ; wang et al., ) and porcine viral enteric disease complexes (theuns et al., ) . overall, the biotechnological innovations have equipped us now to have high-resolution sequencing tools that are revolutionizing the ability of veterinary diagnostic laboratories to detect emerging animal pathogens. with the advent of many biotechnological advances in veterinary diagnostics, point-of-care diagnostics (pocd) are now available for economically important animal diseases. the pocd is basically a simple, rapid, and portable diagnostic device that can be applied at the field level in effective monitoring the disease status. most of the commercially available pocds utilize either antigen/antibody or nucleic acid detection technologies. the former is usually available in the format of lateral flow assays or immunochromatographic strip tests. these assays are simple to use, rapid, inexpensive, disposable, and thus make them the ideal assay for pocd for animal pathogens. the commercially available immunochromatographic strip tests for economically important animal diseases are summarized in table . . these assays are equally sensitive as compared to elisa (ferris et al., ) . furthermore, combining these assays with smartphones has made the increased sensitivity and quick reporting of results possible (yeo et al., ) . therefore, these assays offer a novel herd level surveillance tool, and provide immediate results to the farmers. however, these assays have less analytical sensitivity as compared to nucleic acid-based pocd. the real-time pcr (qpcr) is a well-established tool with high sensitivity of pathogens detection and recently, qpcr has been transitioned into pocd platform. these platforms are fully automated combining nucleic acid extraction, thermal cycling, and reporting of results on-site. for example, minilab (enigma diagnostics) is a platform ( À kg) which can be easily carried to field level and it combines silica paramagnetic-bead-based nucleic acid extraction with lyophilized qpcr reagents in a single cartridge. this platform has been validated for aiv, asfv, csfv, and fmdv (goldenberg and edgeworth, ) . however, this platform is still not available commercially. there are other platforms that do not include nucleic acid extraction step (need to be done separately), such as genesig (primerdesign ltd, united kingdom), genedrive (epistem ltd, manchester, united kingdom), cepheid smartcycler (cepheid), t-cor (tetracore), and r.a.p.i.d. (idaho technologies) (takekawa et al., (takekawa et al., , . the genesig is now supplying lyophilized qpcr assay kits for bovine, equine, porcine, avian, canine, and feline different pathogens. however, these kits are not yet licensed for diagnosis of animal pathogens and are for research purposes only. as per the agreement on trade related aspects of intellectual property rights (trips) under paragraph of article , many countries have excluded diagnostic, therapeutic, and surgical methods of humans or animals from the scope of patentable systems. however, the important patented technologies that are being used in the various formats of diagnostic assays are provided in table . . a high-speed reagent system for qpcr, full velocity technology has been developed by the stratagene which saves time in addition to highly reproducible results. this technology has been used for infectious diseases, cancer, and drug sensitivities testing and already granted five us patents, us , us , us , us , and us . besides, a pocd product, dual path platform (dpp) has been developed by the chembio diagnostic systems, on which tests to detect hiv and syphilis have already been developed. this company in collaboration with national institutes of health and the infectious disease research institute, united states is working constantly to use this platform for the detection of infectious diseases of humans and animals. farm animals reared all over the world for major agricultural and production purposes majorly include cattle, buffalo, sheep, and goats. since the last few decades, a number of infectious diseases have been found associated with farm animals, causing colossal loss to the livestock rearing community and few of them being zoonotic in nature, becoming a problem for the public health. highly contagious livestock diseases such as fmd, hemorrhagic septicemia, peste-des-petits ruminants, and surra cause irreparable economic losses. several other infectious diseases of dairy cows such as bvd, johne's disease, tuberculosis, infectious bovine rhinotracheitis, and liver fluke infestations are generally regarded as being widespread and endemic. the best known and arguably most important discovery of farm animal diseases in the last few decades is bovine spongiform encephalopathy (bse), and others include digital dermatitis, neosporosis and bovine abortion, bovine neonatal pancytopenia, arcanobacterium pluranimalium, and schmallenberg virus. among all, the world organization for animal health classifies fmd and bse as diseases of major interest in cattle. these diseases are known to have a significant effect on dairy production either directly due to death or indirectly due to effects on fertility or milk production, and subsequently, culling. the disease conditions are usually identified based on history and clinical profile of the affected population, but for affirmative diagnosis of the pathogens responsible, the identification of the causal agent is done on the samples or clinical specimens for submission to diagnostic labs. the clinical profiles of several diseases overlap, making diagnosis a little tricky and cumbersome, so initially, the isolation of the infectious agent in pure form using cell culture systems or growth on specific and selective medium became a chosen method for the diagnosis of many pathogenic diseases in farm animals. albeit their usefulness as most sensitive method of detection, they are not used routinely due to time-lapse in confirming illness (bursle and robson, ) . these methods may take hours to several weeks to obtain a confirmatory result. therefore, other approaches based on morphology/biochemical properties of pathogens took the lead and were favored for pathogen detection and identification. several infectious viral disease agents viz astrovirus, adenovirus, rotavirus, etc. were identified through electron microscopy (ong and chandran, ) . but, these also have some drawbacks like more time consuming, less sensitivity, and costly instrumentation. apart from these techniques, approaches like detection of pathogen-specific antibodies or detection of antigenic proteins of pathogens were adopted and categorized under serological assays. serological assays measure antigenÀantibody interactions for diagnostic purposes. these assays are continuously being improved with technologies like rapid strip detection, thus becoming the most preferred tools and are broadly referred to as immunoassays. enzyme immunoassays (elisa) have always been the field applicable diagnostic methods in the detection of various farm animal diseases caused by fmdv, clostridium perfringens, m. bovis, and escherichia coli. hitherto reports have shown the problem of false negative results and cross-reactivity in some of the serological methodologies. innovations including the use of synthetic biology by making highly reactive peptides help to increase the sensitivity and avoid the cross-reactivity issues to some extent. with the more recent advances in diagnostics with the availability of sequences, nucleic acid-based methods have complemented the established techniques as more specific and sensitive methods in detection of pathogens with lesser false positive results in comparison to serological-based methods (bursle and robson, ) . pcr and real-time pcr methods are regularly used in the detection of campylobacter, shigella, bovine respiratory syncytial virus, eimeria, salmonella species, and many other pathogens. likewise, seminested and nested pcr have been developed for the detection of babesia bovis and babesia bigemina. these nucleic acid-based techniques are amongst the standard detection methods and are routinely used for testing in diagnostic laboratories. to improve the efficacy and promote the simplicity, modifications in the form of isothermal amplifications, like lamp and polymerase spiral reaction (psr) have been adopted and are better in the application process, as these are easy to perform, portable, specific, sensitive, and most importantly, quick and cost-effective. lamp is established to be an apex, leading diagnostics for the detection of many farm animal-related diseases like fmd, brucellosis, bovine popular stomatitis, sheep pox, and goat pox (dukes et al., ; song et al., ; zhao et al., ) . likewise, psr has been developed for detection of brucella spp. (das et al., ) , bovine herpesvirus- (malla et al., ) , and canine parvovirus (gupta et al., ) . to further increase the sensitivity and specificity, the combination of elisa and pcr-like immune-pcr, proximity ligation assay, pcr-elisa, have been successfully discovered making the detection -fold more sensitive. the pathogen detected with these combinations includes low pathogenic strains of campylobacter (ding et al., ) . nasba, restriction fragment length polymorphism, amplified fragment length polymorphism, and random amplification of polymorphic dna, are various biotechnological tools that have been further advancing the diagnosis of various infectious diseases. in addition, ngs has brought a revolution in the diagnosis of many pathogens. it appears helpful in the identification of many pathogens, especially viruses in the fecal matter and those that could not be isolated in cell culture system. mining of sequences in samples gives varied genome sequences providing the clues of not only pathogens, but also new strains, genotypes, new viruses, and even the zoonotic efficiencies of the viruses. anis and coworkers demonstrated that targeted bovine ngs is a specific and cost-effective tool for diagnosis of major bovine pathogens in clinical samples (anis et al., ) . even pathogens with low pathogenicity such as bovine enteroviruses (bev), adenoviruses in wild captive animals, and hepatitis e viruses have been revealed in the mining of sequences in the sample. one of the approaches to disease diagnosis is the development of biosensors. assays based on biosensors uses the transducers to convert the biological interaction of pathogen with its specific antibodies to measurable signals. biosensors have been quite useful in the diagnosis in poc detection. biosensors with specific biochemical recognition helped in the identification of e. coli in cattle (dharmasiri et al., ) . colibacillosis is also seen in a variety of farm animals like cattle, pigs, and goats. in c. perfringens detection, epsilon-toxin-specific monoclonal antibody was immobilized onto single-walled carbon nanotubes and adjusted to detect relevant concentrations of toxin in nanomolars and were comparable to elisa-based results. many other methods like mass spectrometry, microarrays, and maldi-tof are also under employment for the detection of many farm animals-associated pathogens, like francisella tularensis, staphylococcus aureus, enterococcus faecalis, e. coli (demirev and fenselau, ; lundquist et al., ; van baar, ) . companion animals are the domesticated animals kept for company of human beings or for utilitarian purposes, that is, guarding, herding, military/police activity. they have grown along with the human civilization and evolution and have developed a good bond with humans. although there is a variety of species which are suitable as companion animals (dogs, cats, rabbit, ferrets, caged birds, fishes, and guinea pigs), dogs and cats are the most common companion species. their physical, behavioral, social, and emotional needs can be easily met at home. on the other hand, dogs and cats play a fundamental role in the life of human beings with many physiological and psychological benefits (wood et al., ) . also, living with companion animals makes human surroundings happier and prosperous. as these animals enrich our lives, it becomes our responsibility to take care of the companion animals and to protect them from any kind of harm. there is a spectrum of infectious diseases that occur in companion animals. lyme disease, psittacosis, hookworms, and salmonella are amongst the most common diseases in pet animals. (lembo et al., ; palatnik-de-sousa et al., ). since these animals share a close environment and are in direct contact with humans, hence they have a potential to spread these infections to human beings. chances of introduction of new diseases also arise on the import of animals from foreign lands. thus, maintenance of strict trade rules and regulations and hygienic conditions becomes a necessity. once the disease occurs, it is important to identify the causative agent to improve the effectiveness of treatment and to control the disease. since the clinical observations are not sufficient and can overlap with other diseases leading to misdiagnosis, several validated laboratory assays are used for confirmatory diagnosis. until years ago, these laboratory tests exploited cell culture (for isolation of specific pathogen) and serological assays (for detection of antibodies generated against a specific pathogen or antigenic proteins). few examples composed of vero cells and recombinant vero-slam cells used for culturing rickettsia rickettsia, and toxoplasma gondii, madin-darby canine kidney cells for canine adenovirus and canine herpesvirus. similarly, serological methods include a long list. immunodiffusion testing is most often used to detect antibodies to fungal pathogens in dogs, such as aspergillus fumigatus, coccidioides immitis, and blastomyces dermatitidis. agglutination tests include the microscopic agglutination test for serologic diagnosis of leptospirosis (agglutination of live leptospires) and the cryptococcal antigen la test (agglutination of antibody-coated latex beads). hemagglutination inhibition is used to determine antibody titers to cpv and canine influenza virus, and it evaluates the ability of serum to inhibit erythrocyte agglutination by these viruses. elisa is commonly used for the detection of feline retroviral, heartworm, giardia, leishmania, and tick-borne infections. indirect ifa for serologic testing in dogs and cats include quantitative serology for some tick-borne infectious diseases (e.g., ehrlichia canis, anaplasma spp.). direct immunofluorescence assay in veterinary medicine include diagnosis of giardia oocysts, felv within monocytes in peripheral blood or bone marrow, or canine distemper virus within epithelial cells from a conjunctival scraping. many of these assays involve the use of polyclonal antibodies, or, more commonly, monoclonal antibodies-dependent diagnosis. with the recent boom in the database of sequences for pathogens, new diagnostic tools like pcr, real-time pcr, and multiplex pcr have almost replaced the established techniques and are adopted as routine diagnostics for testing clinical samples. canine respiratory coronavirus, canine adenovirus- , canine herpesvirus, feline herpesvirus- , canine distemper virus, west nile virus, and encephalitis viruses are some of the examples, which are routinely diagnosed using these techniques. other biotechnological tools cover hybridization assays, pna, nanoparticles-based assays, etc. hybridization-based methods have also been found to be compatible with the diagnosis of many diseases and composed of taqman-based probes, molecular beacons, and fret-based probes. although not yet widely used for veterinary applications, pna probes are now increasingly available to detect target dna. fluorescent pna probes, followed by signal amplification were used to differentiate between m. tuberculosis complex and nontuberculous mycobacterium spp. (zerbi et al., ) . in another example, ngs has also been used for the comparison of the oral microbiome of canines with their owners as they are in direct contact with their pets and many diseases might get transmitted to them (oh et al., ) . gold nanoparticle-based immunochromatographic strip test using a combination of mab and pab was developed as an alternative for on-site and cost-effective diagnosis of cpv infection . another use of biotechnology has been observed for rapid and early detection of cpv using a qcm biosensor (kim et al., ) . also, for genotyping of cpv- , conventional methods are time consuming, therefore, a probe-based duplex fluorescence melting curve analysis (fmca) for genotyping six different cpv- variants (original cpv- , cpv- a, cpv- b, cpv- c, and vaccine strains of cpvpf and cpvint) using only two taqman probes has been developed (liu et al., ) . despite the fact that a wide range of diagnostic tools are available, there is a considerable chance for better advancement in diagnostics, in terms of speed and accuracy, to control and eradicate economically important diseases. in the near future, use of new biotechnological tools like biosensors and nanotechnology will pave the way. further, ngs platforms like minion (a portable, real-time ngs sequencer) coupled with nanopipe analysis are promising tools to perform bacterial and viral disease investigation in low throughput laboratories and specifically in the field (beato et al., ; shabardina et al., ) . although, yet not been adopted for animal disease diagnosis, but novel platforms such as smartphonebased diagnosis (which expands nucleic acid-based detection assays toward pocd) like rt-lamp and fluorescent lateral flow immunoassay (already developed for zika virus and dengue virus) provide exciting opportunities for veterinary diagnostics in the near future (rong et al., ) . biotechnological innovations have brought new generation diagnostic methods for rapid and sensitive diagnosis of various diseases of livestock and pet animals. infectious diseases entail remarkable economic loss, weak food production system, food insecurity, and high maintenance cost of the agriculture sectors including farm animals, poultry, and aquaculture. besides, these diseases carry a huge risk of transmission to humans as sporadic and endemic zoonoses. classical and conventional diagnostic methods are labor intensive, time consuming, less sensitive, and difficult to meet the needs of the emerging pathogen diagnostics. thus, new innovations have to be worked on and need to be practiced. over the long term, innovations will be helping in the diagnosis of pathogens with accurate, sensitive and specific detections. ngs, biosensors, and advanced amplification techniques will persist for longer periods in their constant modified forms. innovations will always be bringing the new applications in the diagnostics for the improved versions of techniques. new technique applications come with the cost and unbroken funding will be putting new prospective techniques into the trials. these techniques should be simplified in the innovations for their easy practices at the field itself, without looking for any skilled personnel/highly equipped laboratories. there is no conflict of interest. a portable reverse transcription recombinase polymerase amplification assay for rapid detection of foot-and-mouth disease virus dot elisa for newcastle disease, infectious bursal disease and mycoplasmosis evaluation of targeted next-generation sequencing for detection of bovine pathogens in clinical samples rapid method for detecting and differentiating mycobacterium tuberculosis complex and non-tuberculous mycobacteria in sputum by fluorescence in situ hybridization with dna probes identification and genetic characterization of bovine enterovirus by combination of two next generation sequencing platforms rapid detection of foot-and-mouth disease virus with optical microchip sensors detection of small amounts of human adenoviruses in stools: comparison of a new immuno real-time pcr assay with classical tools aptamers as therapeutic and diagnostic agents non-culture methods for detecting infection better tests, better care: improved diagnostics for infectious diseases a metagenomic survey of microbes in honey bee colony collapse disorder field monitoring of avian influenza viruses: whole-genome sequencing and tracking of neuraminidase evolution using pyrosequencing rapid visual isothermal nucleic acid-based detection assay of brucella species by polymerase spiral reaction mass spectrometry for rapid characterization of microorganisms diagnostic applications of molecular tools and techniques for important viral diseases of poultry enrichment and detection of escherichia coli o : h from water samples using an antibody modified microfluidic chip an overview of control strategy and diagnostic technology for foot-and-mouth disease in china novel reverse transcription loop-mediated isothermal amplification for rapid detection of foot-and-mouth disease virus development and laboratory evaluation of a lateral flow device for the detection of swine vesicular disease virus in clinical samples immunohistochemical detection of piscine reovirus (prv) in hearts of atlantic salmon coincide with the course of heart and skeletal muscle inflammation (hsmi) enzyme linked aptamer assay: based on a competition format for sensitive detection of antibodies to mycoplasma bovis in serum peptide nucleic acids: a review on recent patents and technology transfer the enigma ml fluabÀrsv assay: a fully automated molecular test for the rapid detection of influenza a, b and respiratory syncytial viruses in respiratory specimens polymerase spiral reaction (psr): a novel, visual isothermal amplification method for detection of canine parvovirus genomic dna complementary monoclonal antibody-based dot elisa for universal detection of h avian influenza virus nanopore sequencing as a rapidly deployable ebola outbreak tool detection of cryptosporidium parvum oocysts on fresh produce using dna aptamers rapid label-free visual assay for the detection and quantification of viral rna using peptide nucleic acid (pna) and gold nanoparticles (aunps) direct rna sequencing of the coding complete influenza a virus genome evaluation of a rapid immunochromatography strip test for detection of astrovirus in stool specimens a novel assay for detecting canine parvovirus using a quartz crystal microbalance biosensor peptide-recombinant vp protein based enzyme immunoassay for the detection of group a rotaviruses in multiple host species a double-stranded probe coupled with isothermal amplification for qualitative and quantitative detection of avian reovirus approaches to define the viral genetic basis of classical swine fever virus virulence the feasibility of canine rabies elimination in africa: dispelling doubts with data a nanobeads amplified qcm immunosensor for the detection of avian influenza virus h n application of duplex fluorescence melting curve analysis (fmca) to identify canine parvovirus type variants direct identification of mycobacterium avium complex and mycobacterium gordonae from mb/bact bottles using accu probe discrimination of francisella tularensis subspecies using surface enhanced laser desorption ionization mass spectrometry and multivariate data analysis development of recombinant σb protein based dot-elisa for diagnosis of avian reovirus (arv) rapid detection of human rotavirus using nsp gene specific reverse transcription loop-mediated isothermal amplification assay novel polymerase spiral reaction (psr) for rapid visual detection of bovine herpesvirus genomic dna from aborted bovine fetus and semen rapid serological profiling by an immunocomb-based dot-enzyme-linked immunosorbent test for three major poultry diseases development and evaluation of probe based real time loop mediated isothermal amplification for salmonella: a new tool for dna quantification rapid and simultaneous detection of three major diarrhea-causing viruses by multiplex real-time nucleic acid sequence-based amplification comparative detection of rotavirus rna by conventional rt-pcr, taqman rt-pcr and real-time nucleic acid sequence-based amplification novel single gold nanowire-based electrochemical immunosensor for rapid detection of bovine viral diarrhoea antibodies in serum development of a recombinase polymerase amplification assay for detection of epidemic human noroviruses comparison of the oral microbiomes of canines and their owners using next-generation sequencing identification of gastroenteric viruses by electron microscopy using higher order spectral features aptasensors À the future of biosensing? decrease of the incidence of human and canine visceral leishmaniasis after dog vaccination with leishmune in brazilian endemic areas taqman real-time rt-pcr assay for detecting japanese encephalitis virus in swine blood samples and mosquitoes loop mediated isothermal amplification (lamp): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases detection of respiratory syncytial virus using nanoparticle amplified immuno-polymerase chain reaction deep sequenc-ing as a method of typing bluetongue virus isolates peptide nucleic acid (pna): its medical and biotechnical applications and promise for the future smartphone-based fluorescent lateral flow immunoassay platform for highly sensitive point-of-care detection of zika virus nonstructural protein aptamer-based assay for prion diseases diagnostic direct identification of mycobacteria from mb/bact alert d bottles: comparative evaluation of two commercial probe assays nanopipe-a web server for nanopore minion sequencing data analysis development and evaluation of a gold nanoparticle-based immunochromatographic strip test for the detection of canine parvovirus aptamer-based competitive electrochemical biosensor for brevetoxin- enzyme-linked, aptamer-based, competitive biolayer interferometry biosensor for palytoxin development and evaluation of simple dotÀblot assays for rapid detection of staphylococcal enterotoxin-a in food establishment of loop-mediated isothermal amplification (lamp) for rapid detection of brucella spp. and application to milk and blood samples field detection of avian influenza virus in wild birds: evaluation of a portable rrtÀpcr system and freeze-dried reagents rapid diagnosis of avian influenza virus in wild birds: use of a portable rrtÀpcr and freeze-dried reagents in the field a potentiometric biosensor for rapid on-site disease diagnostics nanopore sequencing as a revolutionary diagnostic tool for porcine viral enteric disease complexes identifies porcine kobuvirus as an important enteric virus characterisation of bacteria by matrix-assisted laser desorption/ionisation and electrospray mass spectrometry minion nanopore sequencing of an influenza genome the pet connection: pets as a conduit for social capital? recognition of bungarus multicinctus venom by a dna aptamer against β-bungarotoxin upconversion luminescence resonance energy transfer (lret)-based biosensor for rapid and ultrasensitive detection of avian influenza virus h subtype smartphone-based fluorescent diagnostic system for highly pathogenic h n viruses amplified in situ hybridization with peptide nucleic acid probes for differentiation of mycobacterium tuberculosis complex and non-tuberculous mycobacterium species on formalin-fixed, paraffin embedded archival biopsy and autopsy samples advantages of peptide nucleic acids as diagnostic platforms for detection of nucleic acids in resource-limited settings development of loopmediated isothermal amplification assay for specific and rapid detection of differential goat pox virus and sheep pox virus all the authors of the chapter thank and acknowledge their respective universities and institutes. key: cord- -vk lt x authors: ruiz, sara i.; zumbrun, elizabeth e.; nalca, aysegul title: animal models of human viral diseases date: - - journal: animal models for the study of human disease doi: . /b - - - - . - sha: doc_id: cord_uid: vk lt x as the threat of exposure to emerging and reemerging viruses within a naïve population increases, it is vital that the basic mechanisms of pathogenesis and immune response be thoroughly investigated. recent outbreaks of middle east respiratory syndrome corona virus, ebola virus, chikungunya virus, and zika virus illustrate the emerging threats that are encountered. by utilizing animal models in this endeavor, the host response to viruses can be studied in a more complex and integrated context to identify novel drug targets, and assess the efficacy and safety of new products rapidly. this is especially true in the advent and implementation of the fda animal rule. although no one animal model is able to recapitulate all aspects of human disease, understanding the current limitations allows for a more targeted experimental design. important facets to consider prior to an animal study are route of viral exposure, species of animal, biomarkers of disease, and a humane endpoint. this chapter covers the current animal models for medically important human viruses, and demonstrates where the gaps in knowledge exist. well-developed animal models are necessary to understand disease progression, pathogenesis, and immunologic responses to viral infections in humans. furthermore, to test vaccines and medical countermeasures, animal models are essential for preclinical studies. ideally, an animal model of human viral infection should mimic the host-pathogen interactions and the disease progression that is seen in the natural disease course. a good animal model of viral infection should allow assay of many parameters of infection, including clinical signs, growth of virus, clinicopathological parameters, cellular and humoral immune responses, and virus-host interactions. furthermore, viral replication should be accompanied by measurable clinical manifestations and pathology should resemble that of human cases such that a better understanding of the disease process in humans is attained. there is often more than one animal model that closely represents human disease for a given pathogen. small animal models are typically used for first-line screening, and for testing the efficacy of vaccines or therapeutics. in contrast, nonhuman primate (nhp) models are often used for pivotal preclinical studies. this approach is also used for basic pathogenesis studies, with most studies in small animal models when possible, and studies in nhps to fill in the remaining gaps in knowledge. the advantages of using mice to develop animal models are low cost, low genetic variability in inbred strains, and abundant molecular biological and immunological reagents. specific pathogen free (spf), transgenic and knockout mice are also available. a major pitfall of mouse models is that the pathogenesis and protection afforded by vaccines and therapeutics cannot always be extrapolated to humans. additionally, blood volumes for sampling are limited in small animals, and viruses often need to be adapted through serial passage in the species to induce a productive infection. the ferret's airways are anatomically and histologically similar to that of humans, and their size enables collection of larger or more frequent blood samples, making them an ideal model for certain respiratory pathogens. ferrets are outbred, with no standardized breeds or strains, thus greater numbers are required in studies to achieve statistical significance and overcome the resulting variable responses. additionally, spf and transgenic ferrets are not available, and molecular biological reagents are lacking. other caveats making ferret models more difficult to work with are their requirement for more space than mice (rabbit-style cages), and the development of aggressive behavior with repeated procedures. nhps are genetically, the closest species to humans, thus disease progression and host-pathogen responses to viral infections are often the most similar to that of humans. however, ethical concerns pertaining to experimentation on nhps along with the high cost and lack of spf nhps raise barriers for such studies. nhp studies should be carefully designed to ensure the fewest number of animals are used, and the studies should address the most critical questions regarding disease pathogenesis, host-pathogen responses, and protective efficacy of vaccines and therapeutics. well-designed experiments should carefully evaluate the choice of animal, including the strain, sex, and age. furthermore, depending on the pathogen, the route of exposure and the dose should mimic the route of exposure and dose of human disease. the endpoint for these studies is also an important criterion. depending on the desired outcome, the model system should emulate the host responses in humans when infected with the same pathogen. in summary, small animal models are helpful for the initial screening of vaccines and therapeutics, and are often beneficial in obtaining a basic understanding of the disease. nhp models should be used for a more detailed characterization of pathogenesis and for pivotal preclinical testing studies. ultimately, an ideal animal model may not be available. in this case, a combination of different well-characterized animal models should be considered to understand the disease progression and to test medical countermeasures against the disease. in this chapter, we will be reviewing the animal models for representative members of numerous virus families causing human diseases. we will focus on viruses for each family that are of the greatest concern for public health worldwide. norovirus, the genus of which norwalk is the prototypic member, is the most common cause of gastroenteritis in the united states (hall et al., ) . there are five distinct genogroups (gi-gv) and numerous strains of norwalk virus, including the particularly significant human pathogens gi. norwalk virus, gii. snow mountain virus, and gii. hawaii virus. in developing countries, norwalk virus, also known as "winter vomiting virus," is responsible for approximately , deaths annually (patel et al., ) . a typical disease course is self-limiting, but there have been incidences of necrotizing enterocolitis and seizures in infants (chen et al., ; lutgehetmann et al., ; turcios-ruiz et al., ) . symptoms of infection include diarrhea, vomiting, nausea, abdominal cramping, dehydration, and fever. incubation is normally - days, with symptoms persisting for - days (koopmans and duizer, ) . viral shedding can range from to days in healthy individuals (atmar et al., ) . however, longer illness duration can be indicative of immunocompromised status, with the elderly and young having a prolonged state of shedding (harris et al., ; rockx et al., ) . interestingly, individuals vary greatly in susceptibility to norovirus infection depending on their fucosyl transferase (fut ) allele functionality and histoblood group antigen status, with type a and o individuals susceptible and types ab and b resistant (hutson et al., ) . transmission occurs predominately through the oralfecal route with contaminated food and water being a major vector (atmar and estes, ; becker et al., ; koopmans and duizer, ) . vomiting results in airborne dissemination of the virus with areas of . m being contaminated and subsequent transmission from oral deposition of airborne particles or contact with contaminated fomites, which can remain contaminated for up to days (makison booth, ; tung-thompson et al., ) . each vomiting event in a classroom setting elevates the risk of norovirus illness among elementary students with proximity correlating with attack rates (evans et al., ; marks et al., ) . viral titers in emesis and fecal suspensions are as high as . × and . × ges (genomic equivalent copies per milliliter), respectively and the % infectious dose is ges (atmar et al., ) . therefore, outbreaks can be extremely difficult to contain. therapeutic intervention consists of rehydration therapy and antiemetic medication (bucardo et al., ; moe et al., ) . no approved vaccine or therapeutic is available, and development has been challenging given that immunity is short-lived after infection, new strains rapidly evolve and the correlates of protection are not completely understood (chen et al., ) . however, one promising strategy utilized a virus-like particle (vlp)-based vaccine that protected or reduced infection by almost % in human volunteers (aliabadi et al., ; atmar et al., ) . given the relatively benign disease in adults, experimental challenge has been carried out on human volunteers (ball et al., ; tacket et al., ) . viral titers are determined by shedding in feces and sera with histopathology changes monitored by biopsies particularly of the duodenum. the ph of emesis samples collected containing virus is consistent with viral replication in the small intestine with reflux to the stomach (kirby et al., ) . additionally, norwalk virus has been shown to bind to duodenal tissue (chan et al., ) . however, this type of research is technically difficult and expensive, and thus other models have been developed. a major hindrance to basic research into this pathogen is the lack of permissive cell culture systems or animal models for norwalk virus. nhps including marmosets, cotton-top tamarins, and rhesus macaques infected with norwalk virus are monitored for the extent of viral shedding; however, no clinical disease is observed in these models. disease progression and severity is measured exclusively by assay of viral shedding (rockx et al., ) . incidentally, more viruses were needed to create an infection when challenging by the oral route than by the intravenous (iv) route (purcell et al., ) . chimpanzees were exposed to a clinical isolate of norwalk virus by the iv route (bok et al., ) . although none of the animals developed disease symptoms, viral shedding within the feces was observed within - days postinfection and lasted anywhere from days to weeks. viremia never occurred and no histopathological changes were detected. the amount and duration of viral shedding was in-line with what is observed upon human infection. as such, chimeric chimpanzee-human antinorovirus neutralizing antibodies have been explored as a possible therapeutic strategy (chen et al., ) . a recently identified calicivirus of rhesus origin, named tulane virus, has been used as a surrogate model of infection. unlike norwalk virus, tulane virus can be cultured in cells. rhesus macaques exposed to tulane virus intragastrically developed diarrhea and fever days postinfection. viral shedding was detected for days. the immune system produced antibodies that dropped in concentration within days postinfection, mirroring the short-lived immunity documented in humans. the intestine developed moderate blunting of the villi as seen in human disease (sestak et al., ) . a murine norovirus has been identified and is closely related to human norwalk virus (karst et al., ) . however, clinically the virus presents a different disease. the murine norovirus model does not include observable gastrointestinal clinical signs, possibly in part because rodents lack a vomiting reflex. additionally, mice infected with norovirus develop a persistent infection in contrast to human disease (hsu et al., (hsu et al., , khan et al., ) . porcine enteric caliciviruses can induce diarrheal disease in young pigs, and an asymptomatic infection in adults (wang et al., . gnotobiotic pigs can successfully be infected with a passaged clinical norovirus isolate by the oral route. diarrheal disease developed in % of the animals and virus was detected in the stool of % of the animals. no major histopathological changes or viral persistence was noted (cheetham et al., ) . calves are naturally infected with bovine noroviruses (scipioni et al., ) . experimental challenge of calves by oral inoculation with a bovine isolate resulted in diarrheal disease - h postinfection. recovery of virus was achieved after . and h postinfection (otto et al., ) . eastern equine encephalitis virus (eeev), western equine encephalitis virus (weev), and venezuelan equine encephalitis virus (veev) present with near synonymous symptoms. the majorities of human cases are asymptomatic, but can present as a flu-like illness progressing to central nervous system (cns) involvement to include seizures and paralysis. mortality rates vary among the virus, with the highest reported for eev at %- % followed by weev and lastly veev at less than % (ayers et al., ; griffin, ; steele and twenhafel, ) . there are currently no licensed vaccines or therapies but a recent phase clinical trial of a veev dna vaccine resulted in veev-neutralizing antibody responses in % of the subjects (hannaman et al., ) . mouse models have been developed for numerous routes of infection including cutaneous, intranasal (in), intracranial (ic), and aerosol. eeev susceptibility in mouse models is correlated with age, with younger mice being more susceptible than adults. importantly, eeev pathogenesis is dependent on route of infection with delayed progression upon subcutaneous (subq) exposure (honnold et al., ) . newborn mice display neuronal damage with rapid disease progression, resulting in death (murphy and whitfield, ) . similarly, eeev produces fatal encephalitis in older mice when administered via the intracerebral route, while inoculation via the subq route causes a pantropic infection eventually resulting in encephalitis (liu et al., ; morgan, ) . a general drawback to the usage of the mouse model is the lack of vascular involvement during the disease course (liu et al., ) . after subq inoculation with weev, suckling mice started to show signs of disease by h and died within h (aguilar, ) . the heart was the only organ in which pathologic changes were observed. conversely, adult mice exhibited signs of lethargy and ruffled fur on day - postinfection. mice were severely ill by day and appeared hunched and dehydrated. death occurred between days and with brain and mesodermal tissues, such as heart, lungs, liver, and kidney involvement (aguilar, ; monath et al., ) . intracerebral and in routes of infection resulted in a fatal disease that was highly dependent on dose while intradermal (id) and subq inoculations caused only % fatality in mice regardless of the amount of virus (liu et al., ) . comparing susceptibility of inbred and outbred strains revealed that cd- , balb/c, a/j, and c bl mice were all highly susceptible to experimental infection via subq inoculation when challenged prior to weeks old with cns involvement and lethality (blakely et al., ) . subq/dermal infection in the mouse model results in encephalitic disease very similar to that seen in horses and humans (macdonald and johnston, ) . virus begins to replicate in the draining lymph nodes at h postinoculation. eventually, virus enters the brain primarily via the olfactory system. furthermore, aerosol exposure of mice to veev can result in massive infection of the olfactory neuroepithelium, olfactory nerves, and olfactory bulbs and viral spread to brain, resulting in necrotizing panencephalitis (charles et al., ; steele et al., ) . aerosol and dermal inoculation routes cause neurological pathology in mice much faster than other routes of exposure. the clinical signs of disease in mice infected by aerosol are ruffled fur, lethargy, and hunching progressing to death (charles et al., ; steele and twenhafel, ; steele et al., ) . in challenge of c h/hen mice with high dose veev caused high morbidity and mortality (julander et al., b) . viral titers in brain peaked on day postchallenge and remained elevated until animals succumbed on day - postchallenge. protein cytokine array performed on brains of infected mice showed elevated il- a, il- b, il- , il- , mcp- , ifnγ, mip- a, and rantes levels. this model was used successfully to test antivirals against veev (julander et al., a) . additionally, a veev vaccine inactivated with , -iodonaphthyl azide v protects against both footpad and aerosol challenge with virulent veev in a mouse model (gupta et al., ) . guinea pigs and hamsters have also been developed as animal models for eeev studies (paessler et al., ; roy et al., ) . guinea pigs developed neurological involvement with decreased activity, tremors, circling behavior, and coma. neuronal necrosis was observed in brain lesions in the experimentally challenged animals (roy et al., ) . subq inoculation of eeev produced lethal biphasic disease in hamsters with severe lesions of nerve cells. the early visceral phase with viremia was followed by neuroinvasion, encephalitis, and death. in addition, parenchyma necrosis were observed in the liver and lymphoid organs (paessler et al., ) . harlan sprague-dawley hamsters develop viremia and progress to respiratory, gastrointestinal, and nervous system involvement when inoculated via subq route. vasculitis and encephalitis were both evident in this model, which mirrors the human disease clinical spectrum (paessler et al., ) . weev is highly infectious to guinea pigs and has been utilized for prophylactic screening (sidwell and smee, ) . studies demonstrated that although the length of the incubation period and the disease duration varied, weev infection resulted in mortality in hamsters by all routes of inoculation. progressive lack of coordination, shivering, rapid and noisy breathing, corneal opacity, and conjunctival discharge resulting in closing of the eyelids were indicative of disease in all cases (zlotnik et al., ) . cns involvement was evident with intracerebral, intraperitoneal (ip), and id inoculations (zlotnik et al., ) . ip inoculation of weev is fatal in guinea pigs regardless of amount of virus inoculum, with the animals exhibiting signs of illness on day - , followed by death on day - (nalca, unpublished results) . id, im, or iv inoculations of eeev in nhps cause disease, but does not reliably result in neurological symptoms (dupuy and reed, ) . intracerebral infection of eeev produces nervous system disease and fatality in monkeys (nathanson et al., ) . the differences in these models indicate that the initial viremia and the secondary nervous system infection do not overlap in nhps when they are inoculated by the peripheral route (wyckoff, ) . in and intralingual inoculations of eeev also cause nervous system symptoms in monkeys, but are less drastic than intracerebral injections (wyckoff, ) . the aerosol route of delivery will result in uniformly lethal disease in cynomolgus macaques (reed et al., ) . in this model, fever was followed by elevated white blood cells and liver enzymes. neurological signs subsequently developed and nhps became moribund and were euthanized between - days postexposure. meningoencephalomyelitis was the main pathology observed in the brains of these animals (steele and twenhafel, ) . similar clinical signs and pathology were observed when common marmosets were infected with eeev by the in route (adams et al., ) . both aerosol and in nhp models had similar disease progression and pathology as seen in human disease. very limited studies have been performed with nhps. reed et al. exposed cynomolgus macaques to low and high doses of aerosolized weev. the animals subsequently developed fever, increased white blood counts, and cns involvement, demonstrating that the cynomolgus macaque model could be useful for testing of vaccines and therapeutics against weev (reed et al., ) . veev infection causes a typical biphasic febrile response in nhps. initial fever was observed at - h after infection and lasted less than h. secondary fever generally began on day and lasted - days (gleiser et al., ) . veev-infected nhps exhibited mild symptoms, such as anorexia, irritability, diarrhea, and tremors. leukopenia was common in animals exhibiting fever (monath et al., ) . supporting the leukopenia, microscopic changes in lymphatic tissues, such as early destruction of lymphocytes in lymph nodes and spleen, a mild lymphocytic infiltrate in the hepatic triads, and focal myocardial necrosis with lymphocytic infiltration have been observed in monkeys infected with veev. surprisingly, characteristic lesions of the cns were observed histopathologically in monkeys in spite of the lack of any clinical signs of infection (gleiser et al., ) . the primary lesions were lymphocytic perivascular cuffing and glial proliferation and generally observed at day postinfection during the secondary febrile episode. similar to these observations, when cynomolgus macaques were exposed to aerosolized veev, fever, viremia, lymphopenia, and clinical signs of encephalitis were observed but the nhps did not succumb to disease (reed et al., ) . a common marmoset model was utilized for comparison studies of south america (sa) and north america (na) strains of eeev (adams et al., ) . previous studies indicated that the sa strain is less virulent than na strain for humans. common marmosets were infected in with either the na or sa strain of eeev. na strain-infected animals showed signs of anorexia and neurological involvement and were euthanized - days after the challenge. although sa strain-infected animals developed viremia, they remained asymptomatic and survived until the end of study. chikungunya virus (chikv) is a member of the genus alphaviruses, specifically the semliki forest complex, and has been responsible for a multitude of epidemics centered within africa and southeast asia (griffin, ) . the virus is transmitted by aedes aegypti and aedes albopictus mosquitoes. given the widespread endemicity of aedes mosquitoes, chikv has the potential to spread to previously unaffected areas. this is typified by the emergence of disease reported for the first time in in the islands of south-west indian ocean, including the french la reunion island, and the appearance in central italy in (charrel et al., ; rezza et al., ) . the incubation period following a mosquito bite is - days, leading to a self-limiting acute phase that lasts - days. symptoms during this period include fever, arthralgia, myalgia, and rash. headache, weakness, nausea, vomiting, and polyarthralgia have all been reported (powers and logue, ) . individuals typically develop a stooped posture due to the pain. for approximately % of infected individuals, joint pain can last months after resolution of primary disease, and has the possibility to relapse. underlying health conditions including diabetes, alcoholism, or renal disease, increase the risk of developing a severe form of disease that includes hepatitis or encephalopathy. children between the ages of and years old have an increased risk of developing neurological manifestations (arpino et al., ) . there is currently no approved vaccine or antiviral. wild-type c bl/ adult mice are not permissive to chikv infection by id inoculation. however, it was demonstrated that neonatal mice were susceptible and severity was dependent upon age at infection. six-dayold mice developed paralysis by day , and all succumbed by day , whereas % of -day-old mice were able to recover from infection. by days, mice were no longer permissive to disease. symptomatic mice developed loss of balance, hind limb dragging, and skin lesions. neonatal mice were also used as a model for neurological complications (couderc et al., ; ziegler et al., ) . an adult mouse model has been developed by injection of the ventral side of the footpad of c bl/ j mice. viremia lasted - days accompanied with foot swelling and noted inflammation of the musculoskeletal tissue morrison et al., ) . adult ifnα/βr knockout mice also developed mild disease with symptoms including muscle weakness and lethargy, symptoms that mirrored human infection. all adult mice died within days. this model was useful in identifying the viral cellular tropism for fibroblasts (couderc et al., ) . icr and cd- mice can also be utilized as a disease model. neonatal mice inoculated subq with a passaged clinical isolate of chikv developed lethargy, loss of balance, and difficulty walking. mortality was low, and % for newborn cd- and icr mice, respectively. the remaining mice fully recovered within weeks after infection (ziegler et al., ) . a drawback of both the ifnα/βr and cd- mice is that the disease is not a result of immunopathogenesis as occurs in human cases, given that the mice are immunocompromised (teo et al., ) . a chronic infection model was developed using recombinant activating gene (rag −/− ) knockout mice. in this study, mice inoculated via the footpad lost weight in comparison to the control group. both footpad and subq injected mice developed viremia - days postinfection, which was detectable up to days postinfection. inflammation was evident in the brain, liver, and lung of the subq inoculated animals at - days postinfection. despite minimal footpad swelling on day postinfection, on day there was severe muscle damage noted at necropsy, which resolved by day (seymour et al., ) . golden hamsters serve as another option for small animal modeling. although hamsters do not appear to develop overt clinical symptoms following subq inoculation, viremia developed in the majority of animals within day postinfection with clearance following from day to . histologically, inflammation was noted at the skeletal muscle, fascia, and tendon sheaths of numerous limbs. this study was limited in the number of animals utilized, and more work is needed to further develop the hamster model (bosco-lauth et al., ) . nhp models of disease include adult, aged, and pregnant rhesus macaques in addition to cynomolgus macaques (broeckel et al., ) . differing routes of infection (subq, iv, and im) have been successfully administered, although there is not a clear understanding of the role that route of transmission plays in subsequent pathogenesis and clinical symptoms. typically, viremia is observed - days postinfection with a correlation between infectious titer and time to viremia observed in cynomolgus but not rhesus (labadie et al., ; messaoudi et al., ) . fever began at - days postinfection and persisted for - days and - days in cynomolgus and rhesus, respectively and coincided with rash (chen et al., ; labadie et al., ; messaoudi et al., ) . overall blood chemistries changed in conjunction with initiation of viremia, and returned to baseline - days postexposure (chen et al., ) . cns involvement has been difficult to reproduce in nhp models, although it was reported that high inoculum in cynomolgus did result in meningoencephalitis (labadie et al., ) . the nhp models have been utilized to conduct efficacy testing on novel vaccines and therapeutics (broeckel et al., ) . dengue virus (denv) is transmitted via the mosquito vectors a. aegypti and a. albopictus (moore and mitchell, ) . given the endemicity of the vectors, it is estimated that half of the world's population is at risk for exposure to denv. this results in approximately million cases of dengue each year, with the burden of disease in the tropical and subtropical regions of latin america, south asia, and southeast asia (gubler, ) . it is estimated that there are , deaths each year due to dengue hemorrhagic fever (dhf) (guzman and kouri, ) . there are four distinct serotypes of denv, numbered - , which are capable of causing a wide clinical spectrum that ranges from asymptomatic to severe with the development of dhf (world health organization, ) . incubation can range from to days, with the average being - days. the virus targets dendritic cells and macrophages following a mosquito bite (balsitis et al., ) . typical infection results in classic dengue fever (df), which is self-limiting and has flu-like symptoms in conjunction with retroorbital pain, headache, skin rash, and bone and muscle pain. dhf can follow, with vascular leak syndrome and low platelet count, resulting in hemorrhage. in the most extreme cases, dengue shock syndrome (dss) develops, characterized by hypotension, shock, and circulatory failure (world health organization, ) . thrombocytopenia is a hallmark clinical sign of infection, and aids in differential diagnosis (gregory et al., ) . severe disease has a higher propensity to occur upon secondary infection with a different denv serotype (thein et al., ) . this is hypothesized to occur due to antibody dependent enhancement (ade). there is no approved vaccine or drug, and hospitalized patients receive supportive care including fluid replacement. in order to further progress toward an effective drug or vaccine, small human cohort studies have taken place. however, to provide statistically relevant results, testing must progress in an animal model. in developing an animal model, it is important to note that mosquitoes typically deposit - pfu, and is considered the optimal range during experimental challenge . denv does not naturally replicate effectively in rodent cells, creating the need for mouse-adapted strains, engineered mouse lines, and a variety of inoculation routes to overcome the initial barrier. several laboratory mouse strains including a/j, balb/c, and c bl/ are permissive to dengue infection. however, the resulting disease has little resemblance to human clinical signs, and death results from paralysis (huang et al., ; paes et al., ; shresta et al., ) . a higher dose of an adapted denv strain induced dhf symptoms in both balb/c and c bl/ souza et al., ) . this model can also yield asymptomatic infections. a mouse-adapted strain of denv introduced into ag mice developed vascular leak syndrome similar to the severe disease seen in humans (shresta et al., ) . passive transfer of monoclonal dengue antibodies within mice leads to ade. during the course of infection, viremia was increased and animals died due to vascular leak syndrome (balsitis et al., ) . another mouse-adapted strain injected into balb/c caused liver damage, hemorrhagic manifestations, and vascular permeability (souza et al., ) . ic injection of suckling mice with denv leads to death by paralysis and encephalitis, which is rare in human infection (lee et al., ; parida et al., ; zhao et al., a) . immunocompromised mice have also been used to gain an understanding of the pathogenesis of denv. the most well-defined model is ag which is deficient in ifnα/β and γ receptors and can recapitulate dhf/dss if a mouse-adapted strain is utilized yauch et al., ) . scid mice engrafted with human tumor cells develop paralysis upon infection, and thus are not useful for pathogenesis studies (blaney et al., ; lin et al., ) . df symptoms developed after infection in nod/scid/il rγko mice engrafted with cd + human progenitor cells (mota and rico-hesse, ) . rag-hu mice developed fever, but no other symptoms upon infection with a passaged clinical isolate and labadapted strain of denv (kuruvilla et al., ) . a passaged clinical isolate of denv was used to create a model in immunocompetent adult mice. ip injection in c bl/ j and balb/c caused lethality by day - postinfection in a dose dependent manner. the first indication of infection was weight loss beginning on day followed by thrombocytopenia. a drop in systolic blood pressure along with noted increases in the liver enzymes, ast and alt, were also observed. viremia was established by day . this model mimicked the characteristic symptoms observed in human dhf/dss cases (costa et al., ) . vascular leakage was also observed when c bl/ were inoculated with denv (st john et al., ) . a murine model was developed that utilized infected mosquitoes as the route of transmission to hu-nsg mice. female mosquitoes were intrathoracically inoculated with a clinical isolate of denv . infected mosquitoes then fed upon the mouse footpad to allow for transmission of the virus via the natural route. the amount of virus detected within the mouse was directly proportional to the number of mosquitoes it was exposed to, with - being optimal. detectable viral rna was in line with historical human infection data. severe thrombocytopenia developed on day . this model is notable in that disease was enhanced with mosquito delivery of the virus in comparison to injection of the virus (cox et al., ) . nhp models have used a subq inoculation in an attempt to induce disease. although the animals are permissive to viral replication, it is to a lower degree than that observed in human infection (marchette et al., ) . the immunosuppressive drug, cyclophosphamide enhances infection in rhesus macaques by allowing the virus to invade monocytes (marchette et al., ) . throughout these preliminary studies, no clinical disease was detected. in order to circumvent this, a higher dose of denv was used in an iv challenge of rhesus macaques. hemorrhagic manifestations appeared by day and resulted in petechiae, hematomas, and coagulopathy; however, no other symptoms developed (onlamoon et al., ) . a robust antibody response was observed in multiple studies (marchette et al., ; onlamoon et al., ) . marmosets also mirror human dengue infection, developing fever, leukopenia, and thrombocytopenia following subq inoculation (omatsu et al., (omatsu et al., , . nhps are able to produce antibodies similar to those observed during the course of human infection, making them advantageous in studying ade. sequential infection led to a cross-reactive antibody response which has been demonstrated in both humans and mice (midgley et al., ) . this phenotype can also be seen upon passive transfer of a monoclonal antibody to dengue and subsequent infection with the virus. rhesus macaques exposed in this manner developed viremia that was -to -fold higher than previously reported, however, no clinical signs were apparent (goncalvez et al., ) . the lack of inducible dhf or dss symptoms hinders further examination of pathogenesis within this model. west nile virus (wnv) was first isolated from the blood of a woman in the west nile district of uganda in uganda in (smithburn et al., . after the initial isolation of wnv, the virus was subsequently isolated from patients, birds, and mosquitoes in egypt in the early s (melnick et al., ; taylor et al., ) and was shown to cause encephalitis in humans and horses. wnv is recognized as the most widespread of the flaviviruses, with a geographical distribution that includes africa, the middle east, western asia, europe, and australia (hayes, ) . the virus first reached the western hemisphere in the summer of , during an outbreak involving humans, horses, and birds in the new york city metropolitan area (centers for disease control and prevention, ; lanciotti et al., ) . since , the range of areas affected by wnv quickly extended. older people and children are most susceptible to wnv disease. wnv generally causes asymptomatic disease or a mild undifferentiated fever (west nile fever), which can last from to days (monath and tsai, ) . the mortality rate following neuroinvasive disease ranges from % to % (asnis et al., ; hayes, ; hubalek and halouzka, ; komar, ) . the most severe complications are commonly seen in the elderly, with reported case fatality rates from % to %. hepatitis, myocarditis, and pancreatitis are unusual, severe, nonneurologic manifestations of wnv infection. inoculation of wnv into nhps intracerebrally resulted in the development of either encephalitis, febrile disease, or an asymptomatic infection, depending on the virus strain and dose. viral persistence is observed in these animals regardless of the outcome of infection (i.e., asymptomatic, fever, encephalitis) (pogodina et al., ) . thus, viral persistence is regarded as a typical result of nhp infection with various wnv strains. after both intracerebral and subq inoculation, the virus localizes predominantly in the brain and may also be found in the kidneys, spleen, and lymph nodes. wnv does not result in clinical disease in nhps although the animals show a low level of viremia (lieberman et al., ; pletnev et al., ) . this is mirrored in new zealand white rabbits in that they only develop fever and low levels of viremia following inoculation via footpad (suen et al., ) . id inoculation of both marmosets and rhesus macaques did not yield any clinical signs of disease including fever. viremia was detected in both nhp species, but marmosets developed a higher titer for a greater duration than rhesus (verstrepen et al., ) . wnv has also been extensively studied in small animals. all classical laboratory mouse strains are susceptible to lethal infections by the intracerebral and ip routes, resulting in encephalitis and % mortality. id route pathogenesis studies indicated that langerhans dendritic cells are the initial viral replication sites in the skin (brown et al., ; johnston et al., ) . the infected langerhans cells then migrate to lymph nodes and the virus enters the blood through lymphatic and thoracic ducts and disseminates to peripheral tissues for secondary viral replication. virus eventually travels to the cns and causes pathology that is similar to human cases (byrne et al., ; cunha et al., ; diamond et al., ; fratkin et al., ) . the swiss mouse strain was inoculated ip in order to screen a variety of viral lineages to assess differences in pathogenesis (bingham et al., ) . tesh et al. developed a model for wn encephalitis using the golden hamster, mesocricetus auratus. hamsters appeared asymptomatic during the first days, became lethargic at approximately day , and developed neurologic symptoms between days and . many of the severely affected animals died - days after infection. viremia was detected in the hamsters within h after infection and persisted for - days. although there were no substantial changes in internal organs, progressive pathologic differences were seen in the brain and spinal cord of infected animals. furthermore, similar to the previously mentioned monkey experiments by pogodina et al. ( ) , persistent wnv infection was found in the brains of hamsters. zika virus recently came to the forefront of public health concerns with the outbreak in brazil at the end of . the clinical disease spectrum is highly variable with reports of a flu-like illness accompanied by rash, guillan-barre syndrome, and microcephaly in newborns (ramos da silva and gao, ) . to date, a correlation between gestational age at which exposure to the virus occurs and severity of microcephaly is not fully understood (brasil et al., ) . however, a recent study of pregnant women in columbia found that infection with zika virus during the third trimester was not associated with any obvious structural abnormalities of the fetus (pacheco et al., ) . transmission of the virus occurs via the bite from an infected a. aegypti or a. albopictus (ramos da silva and gao, ) . other reported routes of exposure include sexual transmission and blood transfusion (cunha et al., ; d'ortenzio et al., ; hills et al., ; mccarthy, ) . the emergence of this virus with no approved vaccine or therapy, and few diagnostic options demonstrates the utility of well-characterized animal model development. it was first demonstrated in that experimentally infected mosquitoes could be used to transmit the virus to mice and nhps (boorman and porterfield, ) . a mice were susceptible to nonadapted zika virus infection following subq inoculation of the limbs. mice began to lose weight days postinfection and met euthanasia criteria by day . microscopic lesions within the brain were noted upon necropsy. in conjunction, viral rna was detected in the blood, brain, ovary, spleen, and liver of the infected mice. wild-type sv/ev mice were also challenged with no observable clinical disease. however, viral rna was detected at day postinfection in the blood, ovary and spleen, and then remained at detectable levels in the ovaries and spleen on day (dowall et al., ) . footpad inoculation of the virus leads to a fatal disease in ag mice by day postinoculation with significant histopathological changes in the brain noted at necropsy (aliota et al., ) . ag mice were also observed to develop neurologic disease by day postexposure (rossi et al., ) . immunocompetent mice are resistant to infection via the subq route (rossi et al., ) . recently, a mouse model was identified to verify vertical transmission of the virus. pregnant c mice were injected either ip or in utero into the lateral ventricle of the fetal brain. ip inoculation induced transient viremia in the pregnant mice on day . viral rna was detected in five out of nine placentas on day postinfection. the virus was able to infect the radial glia cells in the fetal brain and leads to a reduction in the cortical neural progenitors . viral exposure via cerebroventricular space/ lateral ventricle of the fetal brain exhibited small brain size at day postexposure in addition to cortical thinning (cugola et al., ; li et al., a) . ifnar −/− pregnant mice exposed to the virus had nonviable fetuses. in the same study, wild-type mice were given an anti-ifnar antibody prior to and during infection resulting in detectable virus in the fetal head with mild intrauterine growth restriction (miner et al., ) . all of these murine studies will further study of the pathogenesis of vertical transmission and the resulting neurological disorders in conjunction with screening novel countermeasures. nhp studies are currently ongoing for animal model development. numerous viruses from the coronavirus (cov) family exist that infect a wide range of animals. six species have been identified that can infect humans. two of these are alpha coronavirues: hcov- e and hcov-nl . four are beta coronavirueses: hcov-oc , hcov-hku , hcov-sars, and mers-cov. hcov- e and hcov-oc were first detected in the s from the nasal passages of humans with the "common cold" (gaunt et al., ) . hcov-nl , which was first isolated in , causes upper and lower respiratory infections of varying intensity and has been continuously circulating among humans (van der hoek et al., ) . hcov-hku , first isolated in , has been identified more sporadically but also causes respiratory infections (lau et al., ) . a significant portion of common cold infections in humans are caused by coronaviruses. in and , two human coronaviruses, sars-cov and mers-cov, emerged that caused a great deal of alarm since these infections have resulted in nearly and % fatality, respectively (assiri et al., ; peiris et al., ) . the etiologic agent of severe acute respiratory syndrome (sars), sars-cov, emerged in as it spread throughout countries in a period of months, with confirmed infections and deaths (roberts and subbarao, ; world health organization, ) . no additional cases of community acquired sars-cov infection have been reported since . the natural reservoir of sars-cov is the horseshoe bat and the palm civet is an intermediate host (lau et al., ) . the main mechanism of transmission of sars-cov is through droplet spread, but it is also viable in dry form on surfaces for up to days and can be detected in stool, suggesting other modes of transmission are also possible (pearson et al., ; rabenau et al., ; rota et al., ) . sars-cov infection has a % case fatality with the majority of cases in people over the age of (peiris et al., ; wang et al., ) . after an incubation period of - days, clinical signs of sars include general malaise, fever, chills, diarrhea, dyspnea, and cough (drosten et al., ) . in some sars cases, pneumonia may develop and progress to acute respiratory distress syndrome (ards). fever usually dissipates within weeks and coincides with the induction of high levels of neutralizing antibodies (tan et al., ) . in humans, sars-cov replication destroys respiratory epithelium, and a great deal of the pathogenesis is due to the subsequent immune responses (chen and subbarao, ; perlman and dandekar, ) . infiltrates persisting within the lung and diffuse alveolar damage (dad) are common sequelae of sars-cov infection (perlman and dandekar, ) . virus can be isolated from secretions of the upper airways during early, but not later stages of infection as well as from other tissues (cheng et al., ) . sars-cov can replicate in many species, including: dogs, cats, pigs, mice, rats, ferrets, foxes, and monkeys (roper and rehm, ) . no model captures all aspects of human clinical disease (pyrexia and respiratory signs), mortality (∼ %), viral replication, and pathology (roberts et al., ) . in general, the sars-cov disease course in the model species is much milder and of shorter duration than in humans. viral replication in the various animal models may occur without clinical illness and/or histopathologic changes. the best-characterized models utilize mice, hamsters, ferrets, and nhps. mouse models of sars-cov typically are inoculated by the in route under light anesthesia (roberts et al., ) . young, -to -week-old balb/c mice exposed to sars-cov have viral replication detected in the lungs and nasal turbinate, with a peak on day and clearance by day postexposure (mcauliffe et al., ) . there is also viral replication within the small intestines of young balb/c mice. however, young mice have no clinical signs, aside from reduced weight gain, and have little to no inflammation within the lungs (pneumonitis) . in sars-cov infection of c bl/ (b ), also yields reduced weight gain and viral k. viral disease replication in the lungs, with a peak on day and clearance by day (glass et al., ) . in contrast, balb/c mice - months of age show weight loss, hunched posture, dehydration, and ruffled fur on day - postexposure (bisht et al., ) . interstitial pneumonitis, alveolar damage, and death also occur in old mice, resembling the age-dependent virulence observed in humans. s mice and b mice show outcomes to sars-cov infection similar to those observed for balb/c mice but have lower titers and less prolonged disease. while the aged mouse model is more frequently used then young mice, it is more difficult to obtain large numbers of mice older than year (table . ). a number of immunocompromised knockout mouse models of in sars-cov infection have also been developed. svev mice infected with sars-cov by the in route develop bronchiolitis, with peribronchiolar inflammatory infiltrates and interstitial inflammation in adjacent alveolar septae . viral replication and disease in these mice resolves by day postexposure. beige, cd −/− , and rag −/− mice infected with sars-cov have similar outcomes to infected balb/c mice with regard to viral replication, timing of viral clearance, and a lack of clinical signs (glass et al., ) . stat ko mice infected in with sars-cov have severe disease, with weight loss, pneumonitis, interstitial pneumonia, and some deaths . the stat ko mouse model is therefore useful for studies of pathogenicity, pathology, and evaluation of vaccines. angiotensin converting enzyme (ace ) and cd l were identified as cellular receptors for sars-cov, with affinity for the spike (s) protein of the virus (jeffers et al., ) . the variations in the ace sequence across animal species could partially explain the differences in infection severity (li et al., b; sutton and subbarao, ) . since mice in particular have a greater number of sequence differences in ace , transgenic mice were created that express human ace (mccray et al., ; netland et al., ; yang et al., ) . unlike other murine models of sars-cov, mice expressing hace had up to % mortality, with severity correlating to the level of hace expression (tseng et al., ) . with high levels of hace expression, mice developed a severe lung and brain infection. however, cns k. viral disease infection is only rarely observed in humans infected with sars-cov. syrian golden hamsters (strain lvg) are also susceptible to in exposure of sars-cov. after the administration of tcid , along with a period of transient viremia, sars-cov replicates in nasal turbinates and lungs, resulting in pneumonitis (roberts et al., ) . there are no obvious signs of disease, but exercise wheels can be used to monitor decrease in nighttime activity. limited mortality has been observed, but it was not dose dependent and could have more to do with genetic differences between animals because the strain is not inbred (roberts et al., ) . damage is not observed in the liver or spleen despite detection of virus within these tissues. several studies have shown that intratracheal (it) inoculation of sars-cov in anesthetized ferrets (mustela furo) results in lethargy, fever, sneezing, and nasal discharge (skowronski et al., ) . clinical disease has been observed in several studies excluding one, perhaps due to characteristics of the inoculating virus (kobinger et al., ) . sars-cov is detected in pharyngeal swabs, trachea, tracheobronchial lymph nodes, and high titers within the lungs. mortality has been observed around day postexposure as well as mild alveolar damage in %- % of the lungs, occasionally accompanied by severe pathology within the lungs (martina et al., ; ter meulen et al., ) . with fever, overt respiratory signs, lung damage, and some mortality, the ferret intratracheal model of sars-cov infection is perhaps most similar to human sars, albeit with a shorter time course. sars-cov infection of nhps by intransal or it routes generally results in a very mild infection that resolves quickly. sars-cov infection of old world monkeys, such as rhesus macaques, cynomolgus macaques (cynos), and african green monkeys (agms) have been studied with variable results, possibly due to the outbred nature of the groups studied or previous exposure to related pathogens. clinical illness and viral loads have not been consistent; however, replication within the lungs and dad are features of the infections for each of the primate species. some cynos have no illness but others have rash, lethargy, and respiratory signs and pathology martina et al., ; mcauliffe et al., ; rowe et al., ) . rhesus have little to no disease and only have mild findings upon histopathological analysis (rowe et al., ) . agms infected with sars-cov have no overt clinical signs but dad and pneumonitis has been documented (mcauliffe et al., ) . viral replication has been detected for up to days in the lungs of agms; however, the infection resolves, and does not progress to fatal ards. farmed chinese masked palm civets, sold in open markets in china, were involved in the sars-cov outbreak. it and in inoculation of civets with sars-cov results in lethargy, decreased aggressiveness, fever, diarrhea, and conjunctivitis . leucopenia, pneumonitis, and alveolar septal enlargement, with lesions similar to those observed in ferrets and nhps, have also been observed in laboratory-infected civets. squirrel monkeys, mustached tamarinds, and common marmosets have not been susceptible to sars-cov infection (greenough et al., ; roberts et al., ) . vaccines have been developed for related animal covs in chickens, cattle, dogs, cats, and swine, and have included live-attenuated, killed, dna and viral-vectored vaccine strategies (cavanagh, ) . an important issue to highlight from work on these vaccines is that cov vaccines, such as those developed for cats, may induce a more severe disease (perlman and dandekar, ; weiss and scott, ) . as such, immune mice had th type immunopathology upon sars-cov challenge (tseng et al., ) . severe hepatitis in vaccinated ferrets with antibody enhancement in liver has been reported (weingartl et al., ) . additionally, rechallenge of agms showed limited viral replication but significant lung inflammation, including alveolitis and interstitial pneumonia, which persisted for long periods of time after viral clearance (clay et al., ) . mouse and nhp models with increased virulence may be developed by adapting the virus by repeated passage within the species of interest. mouse-adapted sars with uniform lethality was developed from serial passages in the lungs of young balb/c mice (mccray et al., ; roberts et al., ; rockx et al., ) . middle east respiratory syndrome (mers-cov) emerged in saudi arabia and is associated with fever, severe lower respiratory tract infection, and oftentimes renal failure (al-tawfiq et al., ; omrani et al., ) . mers patients can also occasionally manifest with neurological symptoms. mers-cov infection has a high fatality rate. infections in humans can also be asymptomatic. as of october , there were confirmed cases and deaths (li et al., b) . bats serve as the likely natural reservoir since virus with % nucleotide identity to the index case was isolated from egyptian tomb bats (memish et al., ) . spread to humans likely comes from infected dromedary camels (adney et al., ; azhar et al., ) . the host range for mers-cov is dependent on the binding of the viral s protein to the host receptor, which is human dipeptidyl peptidase four (hdpp ), also known as cd (raj et al., ) . the expression and distribution of dpp in the human respiratory tract has recently been well characterized (meyerholz et al., ) . interestingly, dpp expression is preferentially localized to alveolar regions, perhaps explaining why mers predominantly manifests as an infection of the lower respiratory tract. humans with preexisting pulmonary disease have increased dpp expression in alveolar epithelia. small animals typically used for viral disease research, such as mice, hamsters, guinea pigs, and ferrets are naturally nonpermissive to mers-cov infection due to a low binding efficiency of the viral s protein to the host dpp (sutton and subbarao, ). in contrast the rhesus macaque and common marmoset have complete homology to human dpp , allowing productive mers-cov infection to occur falzarano et al., ; munster et al., ; yao et al., ) . new zealand white rabbits can be infected with mers-cov, and virus was isolated from the upper respiratory tract, but there were no clinical symptoms or significant histopathological changes (haagmans et al., ) . due to the lack of strong binding affinity of the mers-cov s protein to the murine dpp receptor, wildtype mice are not susceptible to mers-cov infection. as such, several approaches have been used to create susceptible murine animal models of mers-cov infection by inducing the expression of hdpp . one approach utilized an adenovirus vector expressing hdpp to transduce mice (zhao et al., b) . these mice developed pneumonia but survived mers-cov infection. in mers-cov infection of mice with global expression of hdpp resulted in id and ld values of < and tcid , respectively (tao et al., ) . thus, mers-cov infection of these transgenic mice can be either sublethal or uniformly lethal depending on the dose. inflammatory infiltrates were found in the lungs and brain stems of mice with some focal infiltrates in the liver as well. another strategy uses transgenic mice expressing hdpp under either a surfactant protein c or cytokeratin promoter (li et al., b) . in mers-cov infection in these mice resulted in a uniformly lethal disease characterized by alveolar edema and microvascular thrombosis and mononuclear clear cell infiltration in the lungs. the brain stem was also impacted by the infection. dpp expression with an ubiquitously expressing promoter from cytomegalovirus also had a uniformly lethal infection with predominant lung and brain involvement, but numerous other tissues were also impacted and contained virus (agrawal et al., ) . common marmosets infected with . × tcid (emc- ) mers-cov by the combined in, oral, ocular, and it routes capitulate the severe disease in human infections (falzarano et al., ) . the animals manifested moderate to severe clinical disease, with interstitial infiltration of both lower lung lobes. two of nine animals became moribund between days and . viral rna was detected in nasal and throat swabs, various organs, and in the blood of some animals, indicating a systemic infection. histologically, animals showed evidence of acute bronchial interstitial pneumonia as well as other pathological defects. infection of rhesus macaques with mers-cov results in a mild clinical disease characterized by a transient lung infection with pneumonia. rhesus macaques were inoculated with at least tcid (emc- ) mers-cov either by the it route or a combined in, it, oral, and ocular inoculation . the result was a mild respiratory illness including nasal swelling and a short fever with all animals surviving. viral rna was recovered from nasal swab samples and replicating virus was found in lung tissue . mild pathological lesions were found only in the lungs. radiographic imaging of the lungs revealed interstitial infiltrates, which are signs of pneumonia (yao et al., ) . interestingly, mer-cov infection is more severe in marmosets compared to rhesus macaques (falzarano et al., ) . this is despite the finding that both species have complete homology with humans within the dpp domain that interacts with the viral s protein. other host factors influencing disease severity have not yet been identified. transgenic mouse models expressing hdpp are ideal for initial development and screening of mers-cov countermeasures, and marmosets can be used for final selection and characterization. filoviridae consists of three genera, ebolavirus and marburgvirus, and a newly discovered group, cuevavirus (kuhn, ) . it is thought that various species of bats are the natural host reservoir for these viruses that have lethality rates from % to % in humans. there is evidence that the egyptian rousette bat (rousettus aegyptiacus) is the natural reservoir for marburgviruses but may not be for ebolaviruses (jones et al., ) . marburg virus (marv) first emerged in in germany when laboratory workers contracted the virus from agms (chlorocebus aerthiops) that were shipped from uganda. ebolaviruses sudan and zaire (sudv and ebov) caused nearly simultaneous outbreaks in in what is now the democratic republic of congo (drc). the most recent outbreak of ebov in west africa was by far the largest with over , suspected, probable and confirmed cases and over , deaths. bundibugyo virus (bdbv) first emerged in in bundibugyo, uganda with confirmed cases . two other ebolaviruses are known: taï forest (tafv) (previously named cote d'ivoire) (ciebov) and reston (restv), which have not caused major outbreaks or lethal disease in humans. filovirus disease in humans is a characterized by aberrant innate immunity and a number of clinical symptoms: fever, nausea, vomiting, k. viral disease arthralgia/myalgia, headaches, sore throat, diarrhea, abdominal pain, and anorexia as well as numerous others (mehedi et al., ; wauquier et al., ) . approximately % of patients develop petechia and a greater percentage, depending on the specific strain, may develop bleeding from various sites (gums, puncture sites, stools, etc.) (table . ). natural transmission in an epidemic is through direct contact or needle sticks in hospital settings. however, much of the research interest in filoviruses primarily stems from biodefense needs, particularly from aerosol biothreats. as such, im, ip, and aerosol models have been developed in mice, hamsters, guinea pigs, and nhps for the study of pathogenesis, correlates of immunity, and for testing countermeasures . since filoviruses have such high lethality rates in humans, scientists have looked for models that are uniformly lethal to stringently test efficacy of candidate vaccines and therapeutics. one issue to take note of in animal model development of filovirus infection is the impact of particle to plaque-forming unit (pfu) ratios on lethality, wherein it is possible that increasing the dose could actually decrease infectivity due to an immunogenic effect produced by inactive virions in the stock. additionally, the plaque assay used to measure live virions in a stock may greatly underestimate the true quantity of infectious virions in a preparation (alfson et al., ; smither et al., a) . immunocompetent mice have not been successfully infected with wild-type filoviruses due to the control of the infection by the murine type interferon response (bray, ) . however, wild-type inbred mice are susceptible to filovirus that has been mouse adapted (ma) by serial passage in mice (bray et al., ) . marv angola was particularly resistant to adaptation, but after serial passages in scid mice, infection caused severe disease in balb/c and c bl/ mice when administered in or ip (qiu et al., ) . these mice had pathology with some similarities to infection in humans including lymphopenia, thrombocytopenia, liver damage, and viremia. balb/c mice, which are the strain of choice for ip inoculation of ma-ebov, are not susceptible by the aerosol route (bray et al., ; zumbrun et al., a) . for aerosol infection of immunocompetent mice, a panel of bxd (balb/c x dba) recombinant inbred strains were screened and one strain, bxd , was particularly susceptible to airborne ma-ebov, with % lethality to low or high doses (approximately or pfu) ( zumbrun et al., a) . these mice developed weight loss of greater than % and succumbed to infection between days and postexposure. the aerosol infection model utilizes a whole-body exposure chamber to expose mice aged - weeks to ma-ebov aerosols with a mass median aerodynamic diameter (mmad) of approximately . µm and a geometric standard deviation (gsd) of approximately . for min. another approach uses immunodeficient mouse strains, such as scid, stat ko, ifn receptor ko, or perforin ko with a wild-type ebov inoculum by ip or aerosol routes (bray, ; lever et al., ; zumbrun et al., a) . mice are typically monitored for clinical disease "scores" based on activity and appearance, weight loss, and moribund condition (survival). coagulopathy, a hallmark of filovirus infection in humans, has been observed, with bleeding in a subset of animals and failure of blood samples to coagulate late in infection (bray et al., ) . liver, kidney, spleen, and lung tissue taken from moribund mice have pathology characteristic of filovirus disease in nhps (zumbrun et al., a) . while most mouse studies have used ma-ebov or ebov, an ip mouse-adapted marv model is also available (warfield et al., (warfield et al., , ). ma-marv and ma-ebov models are particularly useful for screening novel antiviral compounds (panchal et al., ) . recently, a model was created using immunodeficient nsg [nonobese diabetic (nod)/scid/il- receptor chain knockout] mice with transplanted human hematopoietic stem cells from umbilical cord blood. these mice were susceptible to lethal wt (nonadapted) ebov by ip and in exposure (ludtke et al., ) . the transplanted mice had all of the cellular components of a fully functional adaptive human immune system and upon ebov (brannan et al., ; lever et al., ) . interestingly, inoculation of infa/br −/− mice with tafv and restv does not result in clinical signs. yet another strategy uses knockout mice lacking possible receptors for filovirus entry, such as niemann-pick c and c (npc and npc ). npc (−/−) mice were fully susceptible to infection with ebov but npc (−/−) mice were completely resistant (herbert et al., ) . hamsters are frequently used to study cardiovascular disease, coagulation disorders, and thus serve as the basis for numerous viral hemorrhagic fever models (gowen and holbrook, ; herbert et al., ) . an ip ma-ebov infection model has been developed in syrian hamsters gowen and holbrook, ; herbert et al., ; tsuda et al., ) . this model, which has been used to test a vesicular stomatitis virus vectored vaccine approach, utilizes male -to -week-old syrian hamsters which are infected with ld of ma-ebov. virus is present in tissues and blood collected on day and all animals succumbed to the disease by day . infected hamsters had severe coagulopathy and uncontrolled host immune responses, similar to what is observed in primates. (ebihara et al., ) guinea pig models of filovirus infection have been developed for ip and aerosol routes using guinea pigadapted ebov (gp-ebov) and marv (gp-marv) (choi et al., ; connolly et al., ; twenhafel et al., ; zumbrun et al., c) . guinea pig models of filovirus infection are quite useful in that they develop fever, which can be monitored at frequent intervals by telemetry. additionally, the animals are large enough for regular blood sampling in which measurable coagulation defects are observed as the infection progresses. a comparison of ip infection of outbred guinea pigs with guinea pig-adapted marv angola and marv ravn revealed similar pathogenesis (cross et al., ) . infection with either strain resulted in features of the disease that are similar to what is seen in human and nhp infection, such as viremia, fever, coagulopathy, lymphopenia, elevated liver enzymes (alt and ast), thrombocytopenia, and splenic, gastrointestinal and hepatic lesions. gp-marv-ravn had a delayed disease progression relative to gp-marv-ang. hartley guinea pigs exposed to aerosolized gp-ebov develop lethal interstitial pneumonia. this is in contrast to subq infection of guinea pigs, aerosol ebov challenge of nhps, and natural human infection (twenhafel et al., ) . both subq and aerosol exposure of guinea pigs to gp-ebov resulted in only mild lesions in the liver and spleen. by aerosol exposure, gp-ebov is uniformly lethal at both high and low doses ( or pfu target doses) but lethality drops with low (less than pfu) presented doses of airborne gp-marv and more protracted disease is seen in some animals (our unpublished observations) (zumbrun et al., c) . weight loss of between % and % is a common finding in guinea pigs exposed to gp-ebov or gp-marv. fever, which becomes apparent by day , occurs more rapidly in gp-ebov exposed guinea pigs than with gp-marv exposure. lymphocytes and neutrophils increase during the earlier part of the disease, and platelet levels steadily drop as the disease progresses. increases in coagulation time can be seen as early as day postexposure. blood chemistries (i.e., alt, ast, alkp, and bun) indicating problems with liver and kidney function are also altered late in the disease course. transmission of ebov has been documented from swine to nhps via the respiratory tract (kobinger et al., ) . as such, guinea pigs have been used to establish transmission models (wong et al., a,b) . nonexposed guinea pigs were placed in the cages with infected guinea pigs day postexposure to gp-ebov. guinea pigs challenged intanasally were more likely to transmit virus to naive cagemates than those that were exposed by the ip route. nhp models of filovirus infection are the preferred models for more advanced disease characterization and testing of countermeasures because they most closely mimic the disease and immune correlates seen in humans (dye et al., ) . old world primates have been primarily used for development of ip, im, and aerosol models of filovirus infection ( twenhafel et al., ) . uniformly lethal filovirus models have been developed for most of the virus strains in cynomolgus macaques, rhesus macaques, and to a lesser degree, agms (alves et al., ; carrion et al., ; davis et al., ; hensley et al., a; reed et al., ; zumbrun et al., b) . low-passage human isolates that have not been passaged in animals have been sought for development of nhp models to satisfy the food and drug administration (fda) animal rule. ebov-makona, the strain responsible for the recent large outbreak in west africa, was compared to the "prototype" ebov strain (marzi et al., ) . the disease in cynos was similar for both viruses, but disease progression was delayed for ebov-makona. this delay as well as the lower fatality rate in the epidemic compared to the outbreak suggest that ebov-makona is less virulent. the large number of cases in the - ebov outbreak brought to light previously underappreciated eye pathology and ocular viral persistence in survivors. while survivors of nhp filovirus infection are infrequent, necrotizing scleritis, conjunctivitis, and other ocular pathology has been observed in ebov-infected animals (alves et al., ) . prominent features of the filovirus infections in nhps are onset of fever by day postexposure, viremia, lymphopenia, tachycardia, azotemia, alteration in liver function enzymes (alt, ast, and alkp), decrease in platelets, and increased coagulation times. petechial rash is a common sign of filovirus disease and may be more frequently observed in cynomolgus macaques than in other nhp species (zumbrun et al., b) . immunological parameters have been evaluated and t, b, and natural killer cells are greatly diminished as the infection progresses (fernando et al., ) . a cytokine storm occurs with rises in ifnγ, tnf, il- , and ccl (fernando et al., ) . however, there is also evidence from transcriptional profiling of circulating immune cells that the early immune response is skewed toward a th response (connor et al., ) . strikingly, animals surviving challenge may have a delay in the production of inflammatory cytokines and chemokines (martins et al., ) . clinical disease parameters may have a slightly delayed onset in aerosol models. dyspnea late in infection is a prominent feature of disease after aerosol exposure (zumbrun et al., b) . aerosol filovirus infection of nhps results in early infection of respiratory lymphoid tissues, dendritic cells, alveolar macrophages, blood monocytes, and fibroblastic reticular cells followed by spread to regional lymph nodes then multiple organs (ewers et al., ; twenhafel et al., ) . a number of pronounced pathology findings include multifocal hepatic necrosis and fibrin accumulation, particularly within the liver and the spleen. for aerosolized marv infection of rhesus, the most significant pathology included destruction of the tracheobronchial and mediastinal lymph nodes (ewers et al., ) . lymphocytolysis and lymphoid depletion are also observed (alves et al., ) . multilead, surgically implanted telemetry devices are useful in continuous collection of temperature, blood pressure, heart rate, and activity levels. as such, blood pressure drops as animals become moribund and heart rate variability (standard deviation of the heart rate) is altered late in infection (zumbrun et al., b) . the most recently developed telemetry devices can also aid in plethysmography to measure respiratory minute volume for accurate delivery of presented doses for aerosol exposure. standardized filovirus-infected nhp euthanasia criteria have also been developed to enhance reproducibility for studies that evaluate therapeutic and vaccine countermeasures (warren et al., ) . filovirus infection of common marmosets (callithrix jacchus) is also a viable model to study the disease course. respiratory infection of marmosets with marv results in a lethal infection with fever, hemorrhaging, transient rash, disseminated viral infection, increases in liver function enzymes, coagulopathy, hepatitis, and histological lesions particularly in the kidney and liver (smither et al., b) . marmosets are similarly susceptible to infection with ebov- kikwit (smither et al., ) . thus, ebov or marv infection of marmosets produces features of the disease that are very similar to that of other nhps and humans. hendra and nipah virus are unusual within the paramyxoviridae family given that they can infect a large range of mammalian hosts. both viruses are grouped under the genus henipavirus. the natural reservoirs of the viruses are the fruit bats from the genus pteropus. hendra and nipah have the ability to cause severe disease in humans with the potential for a high case fatality rate (rockx et al., ) . outbreaks due to nipah virus have been recognized in malaysia, singapore, bangladesh, and india, while hendra virus outbreaks have yet to be reported outside of australia (luby et al., a,b) . hendra was the first member of the genus identified and was initially associated with an acute respiratory disease in horses. all human cases have been linked to transmission through close contact with an infected horse. there have been no confirmed cases of direct transmission from bat to human. nipah has the distinction of transmission among, although the exact route is unknown (homaira et al., ) . the virus is susceptible to ph, temperature, and desiccation, and thus close contact is hypothesized as needed for successful transmission (fogarty et al., ) . both viruses have a tropism for the neurological and respiratory tracts. the incubation period for hendra virus is - days and is marked by a flu-like illness. symptoms at this initial stage include myalgia, headache, lethargy, sore throat, and vomiting (hanna et al., ) . disease progression can continue to pneumonitis or encephalitic manifestations, with the person succumbing to multiorgan failure (playford et al., ) . nipah virus has an incubation period of days to weeks (goh et al., ) . much like hendra, the first signs of disease are nondescript. severe neurological symptoms subsequently develop including encephalitis and seizures that can progress to coma within - h (lo and rota, ). survivors of infection typically make a full recovery; however, % suffer permanent sequelae, including persistent convulsions (tan and chua, ) . at this time, there is no approved vaccine or antiviral, and treatment is purely supportive. animal models are being used to not only test novel vaccines and therapeutics, but also deduce the early events of disease because documentation of human cases is at terminal stages. the best small animal model is the syrian golden hamster due to their high susceptibility to both henipaviruses. clinical signs upon infection recapitulate the disease course in humans including acute encephalitis and respiratory distress. challenged animals died within - days postinfection. the progression of disease and timeline is highly dependent on dose and route of infection. in inoculation leads to imbalance, limb paralysis, lethargy, and breathing difficulties whereas ip resulted in tremors and paralysis within h before death. virus was detected in lung, brain, spleen, kidney, heart, spinal cords, and urine, with the brain having the highest titer. this model is used for vaccination and passive protection studies (guillaume et al., ; rockx et al., ; wong et al., ) . the guinea pig model has not been widely used due to the lack of a respiratory disease upon challenge (torres-velez et al., ; williamson et al., ) . inoculation with hendra virus via the subq route leads to a generalized vascular disease with % mortality. clinical signs were apparent - days postinfection with death occurring within days of cns involvement. higher inoculum has been associated with development of encephalitis and cns lesions. id and in injection does not lead to disease, although the animals are able to seroconvert upon challenge. the inoculum source does not affect clinical progression. nipah virus challenge only causes disease upon ip injection and results in weight loss and transient fever for - days. virus was shed through urine and was present in the brain, spleen, lymph nodes, ovary, uterus, and urinary bladder (hooper et al., ) . ferrets infected with hendra or nipah virus display the same clinical disease as seen in the hamster model and human cases (bossart et al., ; pallister et al., ) . upon inoculation by the oronasal route, ferrets develop severe pulmonary and neurological disease within - days including fever, coughing, and dyspnea. lesions do develop in the ferret's brains, but to a lesser degree than seen in humans. cats have also been utilized as an animal model for henipaviruses. disease symptoms are not dependent upon the route of infection. the incubation period is - days and leads to respiratory and neurological symptoms (mungall et al., ; johnston et al., ; westbury et al., ) . this model has proven useful for vaccine efficacy studies. squirrel and agms are representative of the nhp models. for squirrel monkeys, nipah virus is introduced by either the in or iv route and subsequently leads to clinical signs similar to humans, although in challenge results in milder disease. upon challenge, only % of animals develop disease manifestations including anorexia, dyspnea, and acute respiratory syndrome. neurological involvement is characterized by uncoordinated motor skills, loss of consciousness, and coma. viral rna can be detected in lung, brain, liver, kidney, spleen, and lymph nodes but is only found upon iv challenge (marianneau et al., ) . agms are very consistent model of both viruses. it inoculation of the viruses results in % mortality, and death within . and - days postinfection for hendra and nipah viruses, respectively. the animals develop severe respiratory and neurological disease with generalized vasculitis rockx et al., ) . the reservoir of the viruses, gray-headed fruit bats, has been experimentally challenged. due to their status as the host organism for henipaviruses, the bats do not develop clinical disease. however, hendra virus can be detected in kidneys, heart, spleen, and fetal tissue, and nipah virus can be located in urine . pigs develop a respiratory disease upon infection with both nipah and hendra viruses (berhane et al., ; li et al., ; middleton et al., ) . oral inoculation does not produce a clinical disease, but subq injection represents a successful route of infection. live virus can be isolated from the oropharynx as early as days postinfection. nipah virus can also be transmitted between pigs. nipah virus was able to induce neurological symptoms in % of the pigs, even though virus was present in all neurological tissues regardless of symptoms (weingartl et al., ) . within the pig model, it appeared that nipah virus had a greater tropism for the respiratory tract, while hendra for the neurological system. horses are also able to develop a severe respiratory tract infection accompanied with fever and general weakness upon exposure to nipah and hendra viruses. oronasal inoculation led to systemic disease with viral rna detected in nasal swabs within days (marsh et al., ; williamson et al., ) . animals died within days postexposure and have interstitial pneumonia with necrosis of alveoli (murray et al., a,b) . virus could be detected in all major systems. mice, rats, rabbits, chickens, and dogs have been tested but are nonpermissive to infection (westbury et al., ; wong et al., ) . suckling balb/c mice succumb to infection if the virus is inoculated intracranially (mungall et al., ) . in exposure with nipah does not induce a clinical disease; however, there is evidence of a subclinical infection in the lungs following euthanasia of the mice (dups et al., ) . in addition, a human lung xenograph model in nsg mice demonstrated that the human lung is highly susceptible to nipah viral replication and damage (valbuena et al., ) . embryonated chicken eggs have been inoculated with nipah virus leading to a universally fatal disease within - days postinfection (tanimura et al., ) . annually, respiratory syncytial virus (rsv) is responsible for the lower respiratory tract infections of million children under the age of , which in turn results in million hospitalizations and approximately , deaths (nair et al., ) . within the united states, hospital costs alone amount to over million dollars per year (paramore et al., ) . outbreaks are common in the winter (yusuf et al., ) . the virus is transmitted by large respiratory droplets that replicate initially within the nasopharynx and spreads to the lower respiratory tract. incubation for the virus is - days. rsv is highly virulent leading to very few asymptomatic infections (collins and graham, ) . disease manifestations are highly dependent upon the age of the individual. rsv infections in neonates produce nonspecific symptoms including overall failure to thrive, apnea, and feeding difficulties. infants present with a mild upper respiratory tract disease that could develop into bronchiolitis and bronchopneumonia. contracting rsv at this age results in an increased chance of developing childhood asthma (wu et al., ) . young children develop recurrent wheezing while adults have exacerbation of previously existing respiratory conditions (falsey et al., ) . common clinical symptoms are runny nose, sneezing, and coughing accompanied by fever. mortality rates from rsv in hospitalized children are %- % with the greatest burden of disease seen in - month olds (ruuskanen and ogra, ). hematopoietic stem cell transplant patients, solid organ transplant patients, and copd patients are particularly vulnerable to rsv infection and have mortality rates between . % and . % upon infection (anderson et al., ) . although there are almost rsv vaccine candidates which are in preclinical and clinical phases, there is no licensed vaccine available and ribavirin usage is not recommended for routine treatment (american academy of pediatrics subcommittee on diagnosis and management of bronchiolitis, ; higgins et al., ; kim and chang, ) . animal models of rsv were developed in the hopes of formulating an effective and safe vaccine unlike the formalin-inactivated rsv (fi-rsv) vaccine. this vaccine induced severe respiratory illness in infants whom received the vaccine and were subsequently infected with live virus (kim et al., ) . mice can be used to model rsv infection, although a very high in inoculation is needed to achieve clinical symptoms (jafri et al., ; stark et al., ) . strain choice is crucial to reproducing a physiological relevant response (stokes et al., ). age does not affect primary disease manifestations (graham et al., ) . however, it does play a role in later sequelae showing increased airway hyperreactivity . primary rsv infection produces increased breathing with airway obstruction (jafri et al., ; van schaik et al., ) . virus was detected as early as day and reached maximum titer at day postinfection. clinical illness is defined in the mouse by weight loss and ruffled fur as opposed to runny nose, sneezing, and coughing as seen in humans. a humanized mouse model was recently developed by in inoculation. the challenged mice experienced weight loss and demonstrated a humoral and cellular immune response to the infection (sharma et al., ) . cotton rats are useful given that rsv is able to replicate to high titers within the lungs and can be detected in both the upper and lower airways after in inoculation (boukhvalova et al., ; niewiesk and prince, ) . viral replication is -to -fold greater in the cotton rat model than mouse model (wyde et al., ) . the cotton rats develop mild to moderate bronchiolitis or pneumonia (grieves et al., ; prince et al., ) . although age does not appear to factor into clinical outcome, it has been reported that older cotton rats tend to take longer to achieve viral clearance. viral loads peak by the th day, dropping to below the levels of detection by day . the histopathology of the lungs appears similar to that of humans after infection (piazza et al., ) . this model has limited use in modeling the human immune response to infection as challenge with the virus induces a th response in cotton rats, whereas humans tend to have a response skewed toward th (culley et al., ; dakhama et al., ; ripple et al., ) . fi-rsv disease was recapitulated upon challenge with live virus after being vaccinated twice with fi-rsv. chinchillas have been challenged experimentally with rsv via in inoculation. the virus was permissive within the nasopharynx and eustachian tube. the animals displayed an acute respiratory tract infection. this model is therefore useful in studying mucosal immunity during infection (gitiban et al., ) . ferrets infected by it were found to have detectable rsv in throat swabs up to day postinfection, and positive qpcr up to day . immunocompromised ferrets were observed to have higher viral loads accompanied with detectable viral replication in the upper respiratory tract (stittelaar et al., ) . chimpanzees are permissive to replication and clinical symptoms of rsv including rhinorrhea, sneezing, and coughing. adult squirrel monkeys, newborn rhesus macaques, and infant cebus monkeys were also challenged but did not exhibit any disease symptoms or high levels of viral replication (belshe et al., ) . bonnet monkeys were developed an inflammatory response by day with viral rna detected in both bronchial and alveolar cells (simoes et al., ) . the chimpanzee model has been proven useful for vaccine studies (hancock et al., ; teng et al., ) . sheep have also been challenged experimentally since they develop respiratory disease when exposed to ovine rsv (meyerholz et al., ) . lambs are also susceptible to human respiratory syncytial infection (olivier et al., ; sow et al., ) . when inoculated intratracheally, the lambs developed an upper respiratory tract infection with cough after days. some lambs went on to develop lower respiratory disease including bronchiolitis. the pneumonia resolved itself within days. rsv replication peaked at days, and rapidly declined. studying respiratory disease in sheep is beneficial given the shared structural features with humans (plopper et al., ; scheerlinck et al., ) . the influenza viruses consist of three types: influenza a, b, and c, based on antigenic differences. influenza a is further classified by subtypes; ha and na subtypes are known. seasonal influenza is the most common infection and usually causes a self-limited febrile illness with upper respiratory symptoms and malaise that resolves within days (taubenberger and morens, ) . the rate of infection is estimated at % in the general population and can result in billions of dollars of loss annually from medical costs and reduced work-force productivity. approximately , people in the united states die each year from seasonal influenza (dushoff et al., ) . thus, vaccines and therapeutics play a critical role in controlling infection, and development using animal models is ongoing (braun et al., b) . influenza virus replicates in the upper and lower airways, peaking at approximately -h postexposure. infection can be more severe in infants and children under the age of , people over the age of , or immunocompromised individuals where viral pneumonitis or pneumonia can develop or bacterial superinfection resulting in pneumonia or sepsis (barnard, ; glezen, ) . pneumonia from secondary bacterial infection, such as streptococcus pneumonia, streptococcus pyogenes, and neisseria meningitides, and more rarely, staphylococcus aureus, is more common than viral pneumonia from the influenza virus itself, accounting for ∼ % of all influenza associated fatalities (alonso et al., ; ison and lee, ; speshock et al., ) . death, often due to ards can occur as early as days after onset of symptoms. lung histopathology in severe cases may include dad, alveolar edema and damage, hemorrhage, fibrosis, and inflammation (taubenberger and morens, ) . the h n avian strain of influenza, has lethality rates of around ∼ % (of known cases), likely because the virus preferentially binds to the cells of the lower respiratory tract, and thus the potential for global spread is a major concern (matrosovich et al., ; wang et al., ) . h n is another avian influenza a strain that infected more than people and was implicated in deaths. approximately % of infected people had a known exposure to birds. there is no evidence of sustained spread between humans but these viruses are of great concern for their pandemic potential (zhang et al., ) . the most frequently used animal models of influenza infection include mice, ferrets, and nhps. a very thorough guide to working with mouse, guinea pig, ferret, and cynomolgus models was published by kroeze et al. ( ) . swine are not frequently utilized but are also a potentially useful model for influenza research since they share many similarities to human anatomy, genetics, susceptibility, and pathogenesis (rajao and vincent, ). lethality rates can vary with virus strain used (with or without adaptation), dose, route of inoculation, age, and genetic background of the animal. the various animal models can capture differing diseases caused by influenza: benign, severe, super infection, and sepsis, severe with ards, and neurologic manifestations (barnard, ) . also, models can utilize seasonal or avian strains and have been developed to study transmission, important for understanding the potential for more lethal strains, such as h n for spreading among humans. mouse models of influenza infection are very predictive for antiviral activity and tissue tropism in humans, and are useful in testing and evaluating vaccines (gilbert and mcleay, ; hagenaars et al., ; ortigoza et al., ) . inoculation is by the in route, utilizing approximately µl of inoculum in each nare of anesthetized mice. exposure may also be to small particle aerosols containing influenza with a mmad of < µl. most inbred strains are susceptible, with particularly frequent use of balb/c followed by c bl/ j mice. males and females have equivalent disease but influenza is generally more infectious in younger -to -week-old ( - g) mice. mice are of somewhat limited use in characterizing the immune response to influenza. most inbred laboratory mice lack the mxa gene which is an important part of human innate immune response to influenza infection. the mouse homolog to mxa, mx is defective in most inbred mouse strains (staeheli and haller, ) . mice with the knocked-in mx gene have a -fold higher ld- for an influenza a strain (pr ) than wildtype background c bl/ mice (grimm et al., ) . weight loss or reduced weight gain, decreased activity, huddling, ruffled fur, and increased respiration are the most common clinical signs in influenza infected mice. for more virulent strains, mice may require euthanasia as early as h postexposure, but most mortality occurs from to days postexposure accompanied by decreases in rectal temperature (sidwell and smee, ). pulse oximeter readings and measurement of blood gases for oxygen saturation are also used to determine the impact of influenza infection on respiratory function (sidwell et al., ) . virus can be isolated from bronchial lavage (bal) fluids throughout the infection and from tissues after euthanasia. for influenza strains with mild to moderate pathogenicity, disease is nonlethal and virus replication is detected within the lungs, but usually not other organs. increases in serum alpha- -acidglycoprotein and lung weight also frequently occur. however, mice infected with influenza do not develop fever, dyspnea, nasal exudates, sneezing, or coughing. mice can be experimentally infected with influenza a or b, but the virus generally requires adaptation to produce clinical signs. mice express the receptors for influenza attachment in the respiratory tract; however, the distribution varies and sa , predominates over sa , which is why h , h , and h subtypes usually need to be adapted to mice and h n , h , h , and h viruses do not require adaptation (o'donnell and subbarao, ). to adapt, mice are infected intratracheally or intranasally by virus isolated from the lungs, and reinfected into mice and then the process is repeated a number of times. once adapted, influenza strains can produce severe disease, systemic spread, and neurotropism. h n and the pandemic influenza virus can cause lethal infection in mice without adaptation (gao et al., ; taubenberger, ) . h n infection of mice results in viremia and viral replication in multiple organ systems, severe lung pathology, fulminant diffuse interstitial pneumonia, pulmonary edema, high levels of proinflammatory cytokines, and marked lymphopenia ( dybing et al., ; gubareva et al., ; lu et al., ) . as in humans, the virulence of h n is attributable to damage caused by an overactive host immune response. additionally, mice infected with the h n influenza virus produce severe lung pathology and oxygen saturation levels that decrease with increasing pneumonia (barnard et al., ) . reassortment influenza viruses of the h n virus and a low-pathogenicity avian h n virus can also induce disease in mice without adaptation . in superinfection models, a sublethal dose of influenza is given to mice followed days later by in inoculation of a sublethal dose of a bacterial strain, such as s. pneumoniae or s. pyogenes (chaussee et al., ) . morbidity, characterized by inflammation in the lungs, but not bacteremia, begins a couple of days after superinfection and may continue for up to weeks. at least one transmission model has also been developed in mice. with h n influenza, transmission rates of up to % among cagemates can be achieved after infection by the aerosol route and cocaging after h (schulman, ). rats (f and sd) inoculated with rat-adapted h n developed inflammatory infiltrates and cytokines in bronchoalveolar lavage fluids, but had no lethality and few histopathological changes (daniels et al., ) . additionally, an influenza transmission model has been developed in guinea pigs as an alternative to ferrets (lowen et al., ) . cotton rats (sigmodon hispidus) have been used to test vaccines and therapeutics in a limited number of studies (eichelberger et al., ) . cotton rats have an advantage over mice in that the immune system is similar to humans (including the presence of the mx gene) and influenza viruses do not have to be adapted (eichelberger et al., ; ottolini et al., ) . nasal and pulmonary tissues of cotton rats were infected with unregulated cytokines and lung viral load peaking at h postexposure. virus was cleared from the lung by day and from the nares by day , but animals had bronchial and alveolar damage, and pneumonia for up to weeks. there is also a s. aureus superinfection model in cotton rats (braun et al., a) . coinfection resulted in bacteremia, high bacterial load in lungs, peribronchiolitis, pneumonitis, alveolitis, hypothermia, and higher mortality. domestic ferrets (mustela putorius furo) are frequently the animal species of choice for influenza animal studies because the susceptibility, clinical signs, peak virus shedding, kinetics of transmission, local expression of cytokine mrnas, and pathology resemble that of humans (lambkin et al., ; maines et al., ; mclaren and butchko, ) . like humans, ferrets exclusively express neu ac, which acts as a receptor for influenza a virus, a feature likely contributing to the susceptibility of ferrets to human-adapted influenza a virus strains (ng et al., ) . the glycomic characterization of ferret respiratory tract tissues demonstrated some similarities and some differences to humans in terms of the potential glycan binding sites for the influenza virus (jia et al., ) . ferrets also have airway morphology, respiratory cell types, and a distribution of influenza receptors (sa , and sa , ) within the airways similar to that of humans (van riel et al., ) . influenza was first isolated from ferrets infected in with throat washes from humans harboring the infection and ferret models have since been used to test efficacy of vaccines and therapeutic treatments (huber et al., ; lambkin et al., ; maines et al., ) . when performing influenza studies in ferrets, animals should be serologically negative for circulating influenza viruses. infected animals should be placed in a separate room from uninfected animals. if animals must be placed in the same room, uninfected ferrets should be handled before infected ferrets. anesthetized ferrets are experimentally exposed to influenza by in inoculation of . - . ml containing approximately - egg id dropwise to each nostril. however, a larger inoculum volume of . ml has also been explored as being more appropriate, yielding more severe and consistent respiratory tract pathology, likely because the larger inoculum is more widely distributed in the lower respiratory tract (moore et al., ) . video tracking to assign values to activity levels in ferrets can aid ferret studies, eliminating the need for collection of subjective and arbitrary clinical scores (oh et al., ) . viral replication in the upper respiratory tract is typically measured by nasal washes, but virus can also be measured in bronchoalveolar lavage fluid using a noninvasive technique (lee et al., ) . influenza types a and b naturally infect ferrets, resulting in an acute illness, which usually lasts - days for mild to moderately virulent strains (maher and destefano, ) . ferrets are more susceptible to influenza a than influenza b strains and are also susceptible to avian influenza h n strains without adaptation (zitzow et al., ) . however, the localized immune responses within the respiratory tract of ferrets infected with influenza a and b have been characterized and are similar (carolan et al., ) . virulence and degree of pneumonitis caused by different influenza subtypes and strains vary from mild to severe and generally mirrors that seen in humans (stark et al., ) . nonadapted h n , h n , and h n have mild to moderate virulence in ferrets. the sequencing of the ferret genome has allowed for the characterization of the ferret host response using rnaseq analysis . distinct signatures were obtained depending on the particular influenza strain to inoculate the ferrets. also helpful is the sequencing and characterization of the influenza ferret infectome during different stages of the infection in naïve or immune ferrets (leon et al., ) . since influenza infection is particularly devastating to the elderly population, an aged ferret model of h n influenza infection was developed (paquette et al., ) . features associated with increased clinical disease are weakened hemagglutinin antibody generation and attenuated th responses. pregnant and breastfeeding women and infants are also susceptible to more severe illness from influenza virus. to study this dynamic, a breastfeeding mother-infant ferret influenza infection model was created (paquette et al., ) . notably, the mammary gland itself harbored virus and transcript analysis showed downregulation of milk production genes. in support of the development of therapies, the ferret influenza model for pharmacokinetic/pharmacodynamics studies of antiviral drugs as also been developed (reddy et al., ) . critical to this model is ensuring pronounced clinical signs and robust viral replication upon influenza infection. strains of low virulence have predominant replication in the nasal turbinates of ferrets. clinical signs and other disease indicators in ferrets are similar to that of humans with mild respiratory disease, sneezing, nasal secretions containing virus, fever, weight loss, high viral titers, and inflammatory infiltrate in the airways, bronchitis, and pneumonia (svitek et al., ) . replication in both the upper and lower airways is associated with more severe disease and greater mortality. additionally, increased expression of proinflammatory mediators and reduced expression of antiinflammatory mediators in the lower respiratory tract of ferrets correlates with severe disease and lethal outcome. h n -infected ferrets develop severe lethargy, greater interferon response, transient lymphopenia, and replication in respiratory tract, brain, and other organs (peng et al., ; zitzow et al., ) . immunocompromised humans have influenza illness of greater duration and complications. immunocompromised ferrets infected with influenza similarly had prolonged virus shedding (van der vries et al., ) . interestingly, antiviral resistance emerged in both humans and ferrets with immunocompromised status infected with influenza. alveolar macrophage depleted of ferrets infected with pandemic h n influenza also had a more severe disease with greater viral replication in the lungs and greater induction of inflammatory chemokines (kim et al., ) . a superinfection model resembling that of mice has been developed by in instillation of influenza in -to -week-old ferrets followed by in inoculation of s. pneumonia days later (peltola et al., ) . this typically resulted in otitis media, sinusitis, and pneumonia. transmission models in ferrets have recently met with worldwide media attention and controversy with regard to the study of h n (enserink, ; fouchier et al., ; herfst et al., ; oh et al., ) . in general, some subtypes, such as the h n , can transmit easily through aerosol and respiratory droplets (munster et al., ) . of concern, h n isolated from humans was more pathogenic and readily transmissible between ferrets by larger respiratory droplets and smaller particle aerosols (kreijtz et al., ; richard et al., ; zhang et al., ) . h n became transmissible by adopting just four mutations, spreading between ferrets in separate cages (imai et al., ) . transmission occurs more readily at the height of pyrexia, but for the h n in particular, can occur before fever is detected (roberts et al., ) . ferret-to-ferret transmission of a mouseadapted influenza b virus has also been demonstrated (kim et al., ) . since ferrets can be expensive and cumbersome, influenza infection has been characterized and a transmission model developed in the guinea pig; however, this is a newer model with infrequent utilization thus far (lowen et al., ) . old and new world primates are susceptible to influenza infection and have an advantage over ferret and mouse models which are deficient for h n vaccine studies because there is a lack of correlation with hemagglutination inhibition (murphy et al., ) . of old world primates, cynomolgus macaque (macaca fascicularis) is most frequently utilized for studies of vaccines and antiviral drug therapies (stittelaar et al., ) . h n and h n infection of cynos is very similar to humans (rimmelzwaan et al., ) . cynos develop fever and ards upon in inoculation of h n with necrotizing bronchial interstitial pneumonia . nhps are challenged by multiple routes k. viral disease (ocular, nasal, and tracheal) simultaneously × pfu per site. virus antigen is primarily localized to the tonsils and pulmonary tissues. infection of cynos with h n results in fever, lethargy, nasal discharge, anorexia, weight loss, nasal and tracheal washes, pathologic and histopathologic changes, and alveolar and bronchial inflammation. the h n caused a very high mortality rate due to an aberrant immune response and ards and had more than % lethality (humans only had a %- % lethality) (kobasa et al., ) . ards and mortality also occur with the more pathogenic strains, but nhps show reduced susceptibility to less virulent strains, such as h n (o'donnell and subbarao, ) . influenza-infected rhesus macaques represent a mild disease model for vaccine and therapeutic efficacy studies (baas et al., ) . host microarray and qrt-pcr proved useful for analysis of infected lung tissues. other nhp models include influenza infection of pigtailed macaques as a mild disease model and infection of new world primates, such as squirrel and cebus monkeys (baskin et al., ) . domestic pig models have been developed for vaccine studies for swine flu. pigs are susceptible in nature as natural or intermediate hosts but are not readily susceptible to h n (isoda et al., ; lipatov et al., ) . while pigs infected with influenza may have fever, anorexia, and respiratory signs, such as dyspnea and cough, mortality is rare (van der laan et al., ) . size and space requirements make this animal difficult to work with, although the development of minipig models may provide an easier to use alternative. cat and dog influenza models have primarily been utilized to study their susceptibility to h n with the thought that these animals could act as sentinels or could serve to transmit the virus to humans (giese et al., ; rimmelzwaan et al., ) . these models are not generally used to better understand the disease in humans or for testing vaccines or antivirals. rift valley fever virus (rvfv) causes epizootics and human epidemics in africa. rvfv mainly infects livestock, such as sheep, cattle, goats, etc. after - days incubation period, animals show signs of fever, hepatitis, and abortion, which is a hallmark diagnostic sign known among farmers (balkhy and memish, ) . mosquito vectors, unpasteurized milk, aerosols of infected animal's body fluids, or direct contact with infected animals are the important routes of transmission to humans (abu-elyazeed et al., ; mundel and gear, ) . after -to -day-incubation period, rvfv causes a wide range of signs and symptoms in humans ranging from asymptomatic to severe disease with hepatitis, vision loss, encephalitis, and hemorrhagic fever (ikegami and makino, ; laughlin et al., ; peters and linthicum, ) . depending on the severity of the disease when the symptoms start, %- % of the hospitalized patients might die in - days or - days after the disease onset (ikegami and makino, ) . hepatic failure, renal failure or dic, and encephalitis are demonstrated within patients during postmortem examination. live domestic animals especially sheep and goats were used to develop animal models of rvfv (weingartl et al., ) . this study indicated that goats were more resistant to the disease compared to sheep. the viremia in goats was lower and had a shorter duration with only some animals developing fever. the susceptibility is influenced by route of infection, breed of animals, the rvfv strain, and growth conditions as well as the passage history. therefore, it might be difficult to establish an animal model with domestic ruminants. mice are one of the most susceptible animal species to rvfv infection. several mouse models including balb/c, ifnar −/− , mbt/pas, and c bl/ were exposed to rvfv via parental or aerosol routes of infection (ross et al., ) . subq or ip routes of infection cause acute hepatitis and lethal encephalitis at a late stage of the disease in mice (mims, ; smith et al., ) . mice start to show signs of decreased activity and ruffled fur by day - postexposure. immediately following these signs, they become lethargic and generally die - days postexposure. ocular disease or the hemorrhagic form of the disease has not been observed in mouse models so far (ikegami and makino, ) . increased viremia and tissue tropism were reported in mice with (smith et al., ) increased liver enzymes and lymphopenia observed in sick mice. aerosolized rvfv causes faster and more severe neuropathy in mice compared to the parental route (dodd et al., ; reed et al., ) . the liver is a target organ following aerosol exposure and liver failure results in fatality. rats and gerbils are also susceptible to rvfv infection. the rat's susceptibility is dependent on the rat strain utilized for the challenge model and route of exposure. there is also noted age dependence in the susceptibility of rats. while wistar-furth and brown norway strains, and young rats are highly susceptible to rvfv infection, fisher , buffalo and lewis strains, and old rats demonstrated resistance to infection via subq route of infection (findlay and howard, ; peters and slone, ) . similar pathologic changes, such as liver damage and encephalopathy were observed in both rats and mice. the recent study by bales et al. ( ) showed that aerosolized rvfv caused similar disease outcome in wistar-furth and aci rats while lewis rats developed fatal encephalitis which was much more severe than the subq route of infection. there was no liver involvement in the gerbil model and animals died from severe encephalitis. the mortality rate was dependent on the strain used and the dose given to gerbils (anderson et al., ) . similar to the rat model, the susceptibility of gerbils was also dependent on age. natural history studies with syrian hamsters indicated that the liver was the target organ with highly elevated alt levels and viral titers (scharton et al., ) . lethargy, ruffled fur, and hunched posture were observed in hamsters by day post-subq inoculation and the disease was uniformly lethal by day - postexposure. this model has been successfully used to test antivirals against rvfv (scharton et al., ) . studies thus far showed that rvfv does not cause uniform lethality in a nhp model. ip, in, iv, and aerosol routes have been utilized to develop nhp model. rhesus macaques, cynomolgus macaques, african monkeys, and south american monkeys were some of the nhp species used for this effort . monkeys showed a variety of signs ranging from febrile disease to hemorrhagic disease and mortality. temporal viremia, increased coagulation parameters (pt, aptt), and decreased platelets were some other signs observed in nhps. animals that succumbed to disease showed very similar pathogenesis to humans, such as pathological changes in the liver and hemorrhagic disease. there was no ocular involvement in this model. smith et al. compared iv, in and subq routes of infection in common marmosets and rhesus macaques (peng et al., ) . marmosets were more susceptible to rvfv infection than rhesus macaques with marked viremia, acute hepatitis, and late onset of encephalitis. increased liver enzymes were observed in both species. necropsy results showed enlarged livers in the marmosets exposed by iv or subq routes. although there were no gross lesions in the brains of marmosets, histopathology showed encephalitis in the brains of in challenged marmosets. a recent study by hartman et al. ( ) demonstrated that aerosolized rvfv only caused mild fever in cynomolgus macaques and rhesus macaques, while agms and marmosets had encephalitis and succumbed to disease between days and postexposure. in contrast to other lethal models, the brain was the target organ in agms and marmosets. although no change was observed in ast levels, alp levels were increased in marmosets. little or no change was observed in hepatic enzyme levels in agms. lack of information regarding human disease concerning the aerosol route of exposure makes it difficult to evaluate these animal models. crimean-congo hemorrhagic fever virus (cchfv) generally circulates in nature unnoticed in an enzootic tick-vertebrate-tick cycle and similar to other zoonotic agents, appears to produce little or no disease in its natural hosts, but causes severe disease in humans. cchfv transmits to humans by ixodid ticks, direct contact with sick animals/humans, or body fluids of animals/humans (ergonul and whitehouse, ) . incubation, prehemorrhagic, hemorrhagic, and convalescence are the four phases of the disease seen in humans. the incubation period lasts - days. during the prehemorragic phase, patients show signs of nonspecific flu-like disease for approximately a week. the hemorrhagic period results in circulatory shock and dic in some patients (mardani and keshtkar-jahromi, ; swanepoel et al., ) . over the years, several attempts have been made to establish an animal model for cchf in adult mice, guinea pigs, hamsters, rats, rabbits, sheep, nhps, etc. (fagbami et al., ; nalca and whitehouse, ; shepherd et al., ; smirnova, ) . until recently, the only animal that manifests disease is the newborn mouse. infant mice ip infected with cchfv resulted in fatality around day postinfection (tignor and hanham, ) . pathogenesis studies showed that virus replication was first detected in the liver, with subsequent spread to the blood (serum). virus was detected very late during the disease course in other tissues including the heart (day ) and the brain (day ). the recent studies utilizing knockout adult mice were successful to develop a lethal small animal model for cchfv infection (bente et al., ; bereczky et al., ) . bente et al. infected stat knockout mice by the ip route. in this model, after the signs of fever, leukopenia, thrombocytopenia, viremia, elevated liver enzymes and proinflammatory cytokines, mice were moribund and succumbed to disease in - days postexposure. the second model was developed by using interferon alpha/beta (ifnα/β) receptor knockout mice (ifnar −/− ) (bereczky et al., ) . similar observations were made in this model as in the stat knockout mouse model. animals were moribund and died - days after exposure with high viremia levels in liver and spleen. characterization studies with ifnar −/− mice challenged with different routes (ip, in, im, and subq) showed that cchfv causes acute disease with high viral loads, pathology in liver and lymphoid tissues, increased proinflammatory response, severe thrombocytopenia, coagulopathy, and death, all of which are characteristics of human disease . proinflammatory cytokines and chemokines, such as g-csf, ifnγ, cxc-cl , ccl increased dramatically day postchallenge and gm-csf, il- a, il- b, il- , il- , il- p , il- , il- , cxcl , ccl , ccl , and tnf-α concentrations were extremely elevated at the time of death/euthanasia. this model is also utilized to test therapeutics, such as ribavirin, arbidol, and t- (favipiravir) successfully (oestereich et al., ) . experimental vaccines developed for cchf were evaluated in this model provided protection compare to unvaccinated mice (buttigieg et al., ; canakoglu et al., , p. ) . thus, the ifnar −/− mouse model would be a good choice to test medical countermeasures against cchfv, although they have an impaired ifn and immune response phenotype. other laboratory animals, including nhps, show little or no sign of infection or disease when infected with cchfv (nalca and whitehouse, ) . butenko et al. utilized agms (cercopithecus aethiops) for experimental cchfv infections. except one monkey with a fever on day postinfection, the animals did not show signs of disease. antibodies to the virus were detected in three out of five monkeys, including the one with fever. fagbami et al. ( ) infected two patas monkeys (erythrocebus patas) and one guinea baboon (papio papio) with cchfv. whereas all three animals had low-level viremia between days and after inoculation, only the baboon serum had neutralizing antibody activity on day postinfection. similar results were obtained when horses and donkeys have been used for experimental cchfv infections. donkeys develop a low-level viremia (rabinovich et al., ) and horses developed little or no viremia, but high levels of virus-neutralizing antibodies, which remained stable for at least months. these studies suggest that horses may be useful in the laboratory to obtain serum for diagnostic and possible therapeutic purposes (blagoveshchenskaya et al., (blagoveshchenskaya et al., ). shepherd et al. ( infected species of small african wild mammals and laboratory rabbits, guinea pigs, and syrian hamsters with cchfv. whereas scrub hares (lepus saxatilis), cape ground squirrels (xerus inauris), red veld rats (aethomys chrysophilus), white-tailed rats (mystromys pumilio), and guinea pigs had viremia; south african hedgehogs (atelerix frontalis), highveld gerbils (gerbilliscus brantsii), namaqua gerbils (desmodillus auricularis), two species of multimammate mouse (mastomys natalensis and mastomys coucha), and syrian hamsters were negative for virus. all species regardless of viremia levels developed antibody responses against cchfv. iv and intracranially infected animals showed onset of viremia earlier than those infected by the subq or ip routes. the genus hantavirus is unique among the family bunyaviridae in that it is not transmitted by an arthropod vector, but rather rodents (schmaljohn and nichol, ) . rodents of the family muridae are the primary reservoir for hantaviruses. infected host animals develop a persistent infection that is typically asymptomatic. transmission is achieved by inhalation of infected rodent saliva, feces, and urine (xu et al., ) . human infections can normally be traced to a rural setting with activities, such as farming, land development, hunting, and camping as possible sites of transmission. rodent control is the primary route of prevention (lednicky, ) . the viruses have a tropism for endothelial cells within the microvasculature of the lungs (zaki et al., ) . there are two distinct clinical diseases that infection can yield: hemorrhagic fever with renal syndrome (hfrs) due to infection with old world hantaviruses or hantavirus pulmonary syndrome (hps) caused by new world hantaviruses (nichol, ) . hfrs is mainly seen outside of the americas and is associated with the hantaviruses dobrava-belgrade (also known as dobrava), hantaan, puumala, and seoul (lednicky, ) . incubation lasts - weeks and presents as flu-like in the initial stages that can further develop into hemorrhagic manifestations and ultimately renal failure. thrombocytopenia subsequently develops which can further progress to shock in approximately % patients. overall mortality rate is %. infection with dobrava and hantaan viruses are typically linked to development of severe disease. hps was first diagnosed in within southwestern united states when healthy young adults became suddenly ill, progressing to severe respiratory distress and shock. the etiological agent responsible for this outbreak was identified as sin nombre virus (snv) (centers for disease control and prevention, ) . this virus is still the leading cause within north america of hps. hps due to other hantaviruses has been reported in argentina, bolivia, brazil, canada, chile, french guiana, panama, paraguay, and uruguay (padula et al., ; stephen et al., ) . the first report of hps in maine was recently documented (centers for disease control and prevention, ). andes virus (andv) was first identified in outbreaks in chile and argentina. this hantavirus is distinct in that it can be transmitted between humans (wells et al., ) . the fulminant disease is more lethal than that observed of hfrs with a mortality rate of %. there are four phases of disease including prodromal, pulmonary, cardiac depression, and hematologic manifestation (peters and khan, ) . incubation typically occurs - days following exposure (young et al., ) . unlike hfrs, renal failure is not a major contributing factor to the disease. there is a short prodromal phase that gives way to cardiopulmonary involvement accompanied by cough and gastrointestinal symptoms. it is at this point that individuals are typically admitted to the hospital. pulmonary function is hindered and continues to suffer within h after cardiopulmonary involvement. interstitial edema and air-space disease normally follow. in fatal cases, cardiogenic shock has been noted (hallin et al., ) . syrian golden hamsters are the most widely utilized small animal model for hantavirus infection. hamsters inoculated im with a passaged andes viral strain died within days postinfection. clinical signs did not appear until h prior to death at which point the hamsters were moribund and in respiratory distress. mortality was dose dependent, with high inoculums leading to a shorter incubation before death. during the same study, hamsters were inoculated with a passaged snv isolate. no hamsters developed any symptoms during the course of observation. however, an antibody response to the virus that was not dose dependent was determined via elisa. hamsters infected with andv have significant histopathological changes to their lung, liver, and spleen. all had an interstitial pneumonia with intraalveolar edema. infectious virus could be recovered from these organs. viremia began on day and lasted up to days postinfection. infection of hamsters with andv yielded a similar clinical disease progression as is seen in human hps including rapid progression to death, fluid in the pleural cavity, and significant histopathological changes to the lungs and spleen. a major deviation in the hamster model is the detection of infectious virus within the liver . normally, snv does not cause a disease in hamsters (wahl-jensen et al., ) . but a recent study showed that immunosuppression with dexamethasone and cyclophosphamide in combination causes lethal disease with snv in hamsters (brocato et al., ) . the disease was very similar to the disease caused by andv in hamsters. lethal disease can be induced in newborn mice, but does not recapitulate the clinical symptoms observed in human disease (kim and mckee, ) . the disease outcome is very much dependent on the age of the mice. younger mice are much more susceptible to virus than the adult mice. adult mice exposed to hanta virus leads to a fatal disease dependent upon viral strain and route of infection. the disease progression is marked by neurological or pulmonary manifestations that do not mirror human disease (seto et al., ; wichmann et al., ) . knockout mice lacking ifnα/β are highly susceptible to hanta virus infection (muller et al., ) . in a study of panel of laboratory strains of mice, c bl/ mice were most susceptible to a passaged hanta viral strain injected ip. animals progressed to neurological manifestation including paralyses and convulsions, and succumbed to infection within - h postinfection. clinical disease was markedly different from that observed in human cases (wichmann et al., ) . in a recent study, -weekold icr mice was exposed to htnv strain aa via the subq route (seto et al., ) . mice started to show signs of disease by day postinoculation. piloerection, trembling, hunching, loss of body weight, labored breathing, and severe respiratory disease were observed in mice. studies to develop nhp models were not successful until recently. nhps have been challenged with new world hantaviruses; however, no clinical signs were reported (hooper et al., ; mcelroy et al., ) . cynomolgus monkeys challenged with a clinical isolate of puumala virus developed a mild disease (klingstrom et al., ; sironen et al., ) . challenge with andv to cynomolgus macaques by both iv and aerosol exposure led to no signs of disease. all animals did display a drop in total lymphocytes within days postinfection. four of six aerosol exposed monkeys and of iv injected monkeys developed viremia. infectious virus could not be isolated from any of the animals. in a recent study, rhesus macaques were inoculated by the intramuscular route with snv passaged only in deer mice (safronetz et al., ) . characteristics of hps disease including rapid onset of respiratory distress, severe pulmonary edema, thrombocytopenia, and leukocytosis were observed in this promising model. viremia was observed - days prior to respiratory signs of the disease that were observed on days - postinoculation. with all aspects, this animal model would be very useful to test medical countermeasures against hanta virus. the family arenaviridae is composed of two serogroups: old world arenaviruses including lassa fever virus and lymphocytic choriomeningitis virus and the new world viruses of pichinde virus and junin virus. all of these viruses share common clinical manifestations (mccormick and fisher-hoch, ) . lassa fever virus is endemic in parts of west africa and outbreaks are typically seen in the dry season between january and april (curtis, ) . this virus is responsible for , - , infections per year, leading to approximately deaths (khan et al., ) . outbreaks have been reported in guinea, sierra leone, liberia, nigeria, and central african republic. however, cases have sprung up in germany, netherlands, united kingdom, and the united states due to transmission to travelers on commercial airlines (amorosa et al., ) . transmission of this virus typically occurs via rodents, in particular the multimammate rat, mastomys species complex (curtis, ) . humans become infected by inhaling the aerosolized virus or eating contaminated food. there has also been noted human-to-human transmission by direct contact with infected secretions or needle-stick injuries. the majority of infections are asymptomatic; however, severe disease occurs in % of individuals. the incubation period is from to days and initial onset is characterized by flu-like illness. this is followed by diarrheal disease that can progress to hemorrhagic symptoms including encephalopathy, encephalitis, and meningitis. a third of patients develop deafness in the early phase of disease that is permanent for a third of those affected. the overall fatality is about %; however, of those admitted to the hospital it is between % and %. there is no approved vaccine and besides supportive measures, ribavirin is effective only if started within days (mccormick et al., a,b) . the primary animal model used to study lassa fever is the rhesus macaque (jahrling et al., ) . aerosolized infection of lymphocytic choriomeningitis virus has been a useful model for lassa fever. both rhesus and cynomolgus monkeys exposed to the virus developed disease, but rhesus mirrored more closely the disease course and histopathology observed in human infection (danes et al., ) . iv or intragastric inoculation of the virus led to severe dehydration, erythematous skin, submucosal edema, necrotic foci in the buccal cavity, and respiratory distress. the liver was severely affected by the virus as depicted by measuring the liver enzymes ast and alt (lukashevich et al., ) . disease was dose dependent with iv, intramuscular, and subq inoculation requiring the least amount of virus to induce disease. aerosol infections and eating contaminated food could also be utilized, and mimic a more natural route of infection (peters et al., ) . within this model, the nhp becomes viremic after - days. clinical manifestations were present by day and death typically occurred within - days (lukashevich et al., ; rodas et al., ) . intramuscular injection of lassa virus into cynomolgus monkeys also produced a neurological disease due to lesions within the cns (hensley et al., b) . this pathogenicity is seen in select cases of human lassa fever (cummins et al., ; gunther et al., ) . a marmoset model has recently been defined utilizing a subq injection of lassa fever virus. virus was initially detected by day and viremia achieved by day . liver enzymes were elevated and an enlarged liver was noted upon autopsy. there was a gradual reduction in platelets and interstitial pneumonitis diagnosed in a minority of animals. the physiological signs were the same as seen in fatal human cases (carrion et al., ) . mice develop a fatal neurological disorder upon intracerebral inoculation with lassa, although the outcome of infection is dependent on the mhc background, age of the animal, and inoculation route (salvato et al., ) . stat knockout mice inoculated ip with both lethal and nonlethal lassa virus strains develop hearing loss accompanied by damage to the inner ear hair cells and auditory nerve (yun et al., ) . guinea pig inbred strain was highly susceptible to lassa virus infection. the outbred hartley strain was less susceptible, and thus strain has been the preferred model given its assured lethality. the clinical manifestations mirror those seen in humans and rhesus (jahrling et al., ) . infection with pichinde virus passaged in guinea pigs has also been used. disease signs include fever, weight loss, vascular collapse, and eventual death (lucia et al., ; qian et al., ) . the guinea pig is an excellent model given that it not only results in similar disease pattern, viral distribution, histopathology, and immune response to humans (connolly et al., ; katz and starr, ) . infection of hamsters with a cotton rat isolate of pirital virus is similar to what is characterized in humans, and the nhp and guinea pig models. the virus was injected ip resulting in lethargy and anorexia within - days. virus was first detected at days, and reached maximum titers within days. neurological symptoms began to appear at the same time, and all animals died by day . pneumonitis, pulmonary hemorrhage, and edema were also present (sbrana et al., ) . these results were recapitulated with a nonadapted pichinde virus (buchmeier and rawls, ; gowen et al., ; smee et al., ) . the lentiviruses are a subfamily of retroviridae, which includes human immunodeficiency virus (hiv), a virus that infects . % of the world's population. a greater proportion of infections and deaths occur in subsaharan africa. worldwide, there are approximately . million deaths per year with over , being children. transmission of hiv occurs by exposure to infectious body fluids. there are two species, hiv- and hiv- , with hiv- having lower infectivity and virulence (confined mostly to west africa). the vast majority of cases worldwide are hiv- (de cock et al., ) . hiv targets t-helper cells (cd +), macrophages, dendritic cells (fields et al., ) . acute infection occurs - weeks after exposure, with flu-like symptoms and viremia followed by chronic infection. symptoms in the acute phase may include fever, body aches, nausea, vomiting, headache, lymphadenopathy, pharyngitis, rash, and sores in the mouth or esophagus. cd + t-cells are activated which kill hiv-infected cells, and are responsible for antibody production and seroconversion. acquired immune deficiency syndrome (aids) develops when cd + t-cells decline to less than cells/µl; thus cell-mediated immunity becomes impaired and the person is more susceptible to opportunistic infections as well as certain cancers. hiv has a narrow host range likely because the virus is unable to antagonize and evade effector molecules of the interferon response (thippeshappa et al., ) . humanized mice, created by engrafting human cells and tissues into scid mice, have been critical for the development of mouse models for the study of hiv infection. a number of different humanized mouse models allow for the study of hiv infection in the context of an intact and functional human innate and adaptive immune responses (berges and rowan, ) . the scidhu hiv infection model has proven useful, particularly in screening antivirals and therapeutics (denton et al., ; melkus et al., ) . a number of different humanized mouse models have been developed for the study of hiv, including rag −/− γc −/− , rag −/− γc −/− , nod/scidγc −/− (hnog), nod/scidγc −/− (hnsg), nod/scid blt, and nod/scidγc −/− (hnsg) blt (karpel et al., ; li et al., ; shimizu et al., ) . cd + human stem cells derived from umbilical cord blood or fetal liver are used for humanization (baenziger et al., ; watanabe et al., ) . hiv- infection by ip injection can be successful with as little as % peripheral blood engraftment (berges et al., ) . vaginal and rectal transmission models have been developed in blt scid hu mice in which mice harbor human bone marrow, liver, and thymus tissue. hiv- viremia occurs within approximately days postinoculation . in many of these models, spleen, lymph nodes, and thymus tissues are highly positive for virus, similar to humans (brainard et al., ) . importantly, depletion of human t-cells can be observed in blood and lymphoid tissues of hivinfected humanized mice and at least some mechanisms of pathogenesis that occur in hiv-infected humans, also occur in the hiv-infected humanized mouse models (baenziger et al., ; neff et al., ) . the advantage of these models is that these mice are susceptible to hiv infection and thus the impact of drugs on the intended viral targets can be tested. one caveat is that while mice have a "common mucosal immune system," humans do not, due to differences in the distribution of addressins (holmgren and czerkinsky, ) . thus, murine mucosal immune responses to hiv do not reflect those of humans. another strategy uses a human cd -and human ccr -expressing transgenic luciferase reporter mouse to study hiv- pseudovirus entry (gruell et al., ) . hiv- transgenic (tg) rats are also used to study hiv related pathology, immunopathogenesis, and neuropathology (lentz et al., ; reid et al., ) . the clinical signs include skin lesions, wasting, respiratory difficulty, and neurological signs. brain volume decreases have been documented and the hiv- tg rat is thus used as a model of neuropathology in particular. there are a number of important nhp models for human hiv infection (hessell and haigwood, ) . an adaptation of hiv- was obtained by four passages in pigtailed macaques transiently depleted of cd (+) cells during acute infection (hatziioannou et al., ) . the resulting disease has several similarities to aids in humans, such as depletion of cd (+) t-cells (kimata, ) . simian immunodeficiency virus (siv) infection of macaques has been widely used as a platform for modeling hiv infection of humans (demberg and robert-guroff, ; walker et al., ) . importantly, nhps have similar, pharmacokinetics, metabolism, mucosal tcell homing receptors, and vascular addressins to those of humans. thus, while the correlates of protection against hiv are still not completely known, immune responses to hiv infection and vaccination are likely comparable. these models mimic infection through use of contaminated needles (iv), sexual transmission (vaginal or rectal), and maternal transmission in utero or through breast milk (keele et al., ; miller et al., ; stone et al., ) . there are also macaque models to study the emergence and clinical implications of hiv drug resistance (van rompay et al., ) . these models most routinely utilize rhesus macaques (macaca mulatta), cynomolgus macaques (m. fasicularis), and pigtailed macaques (macaca nemestrina). all ages are used, depending on the needs of the study. for instance, use of newborn macaques may be more practical for evaluating the effect of prolonged drug therapy on disease progression; however, adult nhps are more frequently employed. female pigtailed macaques have been used to investigate the effect of the menstrual cycle on hiv susceptibility (vishwanathan et al., ) . studies are performed in bsl- animal laboratories and nhps must be simian type-d retrovirus free and siv seronegative. siv infection of pigtailed macaques is a useful model for hiv peripheral nervous system pathology, wherein an axotomy is performed and regeneration of axons is studied (ebenezer et al., ) . exposure in model systems is typically through a single high-dose challenge. iv infection of rhesus macaques with tcid of the highly pathogenic siv/ deltab induces aids in most macaques within - months (mean of months) (fuller et al., ) . peak viremia occurs around week . aids in such models is often defined as cd + t-cells that have dropped to less than % of the baseline values. alternatively, repeated low dose challenges are often utilized, depending on the requirements of the model (henning et al., ; moldt et al., ; reynolds et al., ) . since nhps infected with hiv do not develop an infection with a clinical disease course similar to humans, siv or siv/hiv- laboratory-engineered chimeric viruses (shivs) are used as surrogates. nhps infected with pathogenic siv may develop clinical disease which progresses to aids, and are thus useful pathogenesis models. a disadvantage is that siv is not identical to hiv- and is more closely related to hiv- . however, the polymerase region of siv is % homologous to that of hiv- and it is susceptible to many reverse transcriptase (rt) and protease inhibitors. siv is generally not susceptible to nonnucleoside inhibitors, thus hiv- rt is usually put into siv for such studies (uberla et al., ) . sivmac is similar to hiv in the polymerase region and is therefore susceptible to nucleoside, rt, or integrase inhibition (witvrouw et al., ) . nhps infected with sivmac have an asymptomatic period and disease progression resembling aids in humans, characterized by weight loss/wasting, cd + t-cell depletion. additionally, sivmac utilizes the cxcr chemokine receptor as a coreceptor, similar to hiv, which is important for drugs that target entry (veazey et al., ) . nhps infected with shiv strains, may not develop aids, but these models are useful in testing vaccine efficacy . for example, rt-shivs and env-shivs are useful for testing and evaluation of drugs that may target the envelope or rt, respectively (uberla et al., ) . one disadvantage of the highly virulent env-shiv (shiv- . p), is that it uses the cxcr coreceptor. of note, env-shivs that do use the cxcr coreceptor are less virulent; viremia develops then resolves without further disease progression (humbert et al., ) . simian-tropic (st) hiv- contains the vif gene from siv. infection of pigtailed macaques with this virus results in viremia, which can be detected for months, followed by clearance (haigwood, ) . a number of routes are utilized for siv or shiv infection of nhps, with iv inoculation the most common route. mucosal routes include vaginal, rectal, and intracolonic. mucosal routes require a higher one-time dose than the iv route for infection. for the vaginal route, female macaques are treated with depo-provera (estrogen) month before infection to synchronize the menstrual cycle, thin the epithelial lining of the vagina, and increase susceptibility to infection by atraumatic vaginal instillation (burton et al., ) . upon vaginal instillation of tcid of shiv- p , peak viremia was seen around days postexposure with greater than copies/ml and dropping thereafter to a constant level of rna copies/ml at days and beyond. in another example, in an investigation of the effect of vaccine plus vaginal microbicide on preventing infection, rhesus macaques were vaginally infected with a high dose of sivmac (barouch et al., ) . an example of an intrarectal model utilized juvenile ( -year-old) pigtailed macaques, challenged intrarectally with tcid s of siv mne to study the pathogenesis related to the virulence factor, vpx (belshan et al., ) . here, viremia peaked at approximately days with more than copies/ml. viral rna was expressed in the cells of the mesenteric lymph nodes. the male genital tract is seen as a viral sanctuary with persistent high levels of hiv shedding even with antiretroviral therapy. to better understand the effect of haart therapy on virus and t-cells in the male genital tract, adult ( -to -year-old) male cynomolgus macaques were intravenously inoculated with aid s of sivmac and the male genital tract tissues were tested after euthanasia by pcr, ihc, and in situ hybridization (moreau et al., ) . pediatric models have been developed in infant rhesus macaques through the infection of siv, allowing for the study of the impact of developmental and immunological differences on the disease course (abel, ) . importantly, mother-to-infant transmission models have also been developed (jayaraman et al., ) . pregnant female pigtailed macaques were infected during the second trimester with mid shiv-sf p by the iv route. four of nine infants were infected, one in utero and three either intrapartum or immediately postpartum through nursing. this model is useful for the study of factors involved in transmission as well as the underlying immunology. nhps infected with siv or shiv are routinely evaluated for weight loss, activity level, stool consistency, appetite, virus levels in blood, and t-cell populations. cytokine and chemokine levels, antibody responses, and cytotoxic t-lymphocyte responses may also be evaluated. the ultimate goal of an hiv vaccine is sterilizing immunity (preventing infection). however, a more realistic result may be to reduce severity of infection and permanently prevent progression. strategies have included live attenuated, nonreplicating, and subunit vaccines. these have variable efficacy in nhps due to the genetics of the host (mhc and trim alleles), differences between challenge strains, and challenge routes (letvin et al., ) . nhp models have led to the development of antiviral treatments that are effective at reducing viral load and indeed transmission of hiv among humans. one preferred variation on the models for testing the long-term clinical consequences of antiviral treatment is to use newborn macaques and treat from birth onward, in some cases more than a decade (van rompay et al., ) . unfortunately, however, successes in nhp studies do not always translate to success in humans, as seen with the recent step study which used an adenovirus-based vaccine approach (buchbinder et al., ) . vaccinated humans were not protected and may have even been more susceptible to hiv, viremia was not reduced, and the infections were not attenuated as hoped. with regard to challenge route, iv exposure is more difficult to protect than mucosal exposure and is used as a "worst case scenario." however, efficacy at one mucosal route is usually comparable to other mucosal routes. human and animal papillomaviruses cause benign epithelial proliferations (warts) and malignant tumors of the various tissues that they infect (bosch and de sanjose, ) . there are over human papillomaviruses, with different strains causing warts on the skin, oropharynx, nasopharynx, larynx, and anogenital tissues. approximately one third of papillomaviruses are transmitted sexually. of these, virulent subtypes, such as hpv- , hpv- , hpv- , hpv- , and hpv- place individuals at high risk for cervical and other cancers. up to % of head and neck cancers are caused by hpv- , particularly oropharyngeal cancers. major challenges in the study of these viruses are that papillomaviruses generally do not infect any other species outside of the natural hosts and can cause a very large spectrum of severity. thus, no wild-type animal models have been identified that are susceptible to hpv. however, a number of useful surrogate models exist which use animal papillomaviruses in their natural host or a very closely related species (borzacchiello et al., ; brandsma, ; campo, ) . these models have facilitated the recent development of useful and highly effective prophylactic hpv vaccines (rabenau et al., ) . wild-type inbred mice cannot be used to study disease caused by papillomaviruses unless they are engrafted with relevant tissue, orthotopically transplanted or transgenic, but they are often used to look at immunogenicity of vaccines (jagu et al., ; oosterhuis et al., ) . transgenic mice used for hpv animal modeling typically express the viral oncogenes e , e , e , or the entire early region of hpv- from the keratin promoter which is only active in the basal cells of the mouse epithelium (chow, ) . cancers in these models develop upon extended estrogen exposure (maufort et al., ; ocadiz-delgado et al., ; stelzer et al., ; thomas et al., ) . transgenic mice with constitutively active wnt/b-catenin signaling in cervical epithelial cells expressing the hpb oncoprotein e develop invasive cervical squamous carcinomas (bulut and uren, ) . the tumors occur within months approximately % of the time. another model uses c bl/ mice expressing the hpv -e transgene which are then treated topically with , -dimethylbenz(a)anthracene (dmba) (de azambuja et al., ) . these mice developed benign and malignant cutaneous lesions. cervical cancers can also be induced in human cervical cancer xenografts transplanted onto the flanks of athymic mice and serially transplanted thereafter ( hiroshima et al., ; siolas and hannon, ) . a wild-type immunocompetent rodent model uses m. coucha, which is naturally infected with mastomys natalensis papillomavirus (mnpv) (vinzon et al., ) . mnpv induces papillomas, keratoacanthomas, and squamous cell carcinomas and provides a means to study vaccination in an immunocompetent small animal model. wild cottontail rabbits (sylvilagus floridanus) are the natural host for cottontail rabbit papillomavirus (crpv), but this virus also infects domestic rabbits (oryctolagus cuniculus), which is a very closely related species ( breitburd et al., ) . in this model, papillomas can range from cutaneous squamous cell carcinomas on one end of spectrum, and spontaneous regression on the other. lesions resulting from crpv in domestic rabbits do not typically contain infectious virus. canine oral papillomavirus (copv) causes florid warty lesions in mucosa of the oral cavity within - weeks postexposure in experimental settings (johnston et al., ) . the mucosatrophic nature of these viruses and the resulting oropharyngeal papillomas that are morphologically similar to human vaginal papillomas caused by hpv- and hpv- make this a useful model (nicholls et al., ) . these lesions typically spontaneously regress - weeks after appearing; this model is therefore useful in understanding the interplay between the host immune defense and viral pathogenesis. male and female beagles, aged weeks to years, with no history of copv, are typically used for these studies. infection is achieved by application of a µl droplet of virus extract to multiple . cm scarified areas within the mucosa of the upper lip of anesthetized beagles (nicholls et al., ) . some investigators have raised concerns that dogs are not a suitable model for high-risk hpv-induced oral cancer (staff, ) . bovine papillomavirus (bpv) has a wider host range than most papillomaviruses, infecting the fibroblasts cells of numerous ungulates (campo, ) . bpv- infection of cattle feeding on bracken fern, which is carcinogenic, can result in lesions of the oral and esophageal mucosa that lack detectable viral dna. bpv infections in cattle can result in a range of diseases, such as skin warts, cancer of the upper gastrointestinal tract and urinary bladder, and papillomatosis of the penis, teats, and udder. finally, rhesus papillomavirus (rhpv), a sexually transmitted papillomaviruses in rhesus macaques and cynomolgus macaques is very similar to hpv- and is associated with the development of cervical cancer ( ostrow et al., ; wood et al., ) . monkeypox virus (mpxv) causes disease in both animals and humans. human monkeypox, which is clinically almost identical to ordinary smallpox, occurs mostly in the rainforest of central and western africa. the virus is maintained in nature in rodent reservoirs including k. viral disease squirrels (charatan, ; khodakevich et al., ) . mpxv was discovered during the pox-like disease outbreak among laboratory monkeys (mostly cynomolgus and rhesus macaques) in denmark in . no human cases were observed during this outbreak. the first human case was not recognized as a distinct disease until in zaire (the present drc) with continued occurrence of a smallpox-like illness despite eradication efforts of smallpox in this area. during the global eradication campaign, extensive vaccination in central africa decreased the incidence of human monkeypox, but the absence of immunity in the generation born since that time and increased dependence on bush meat have resulted in renewed emergence of the disease. in the summer of , a well-known outbreak in the midwest was the first occurrence of monkeypox disease in the united states and western hemisphere. among reported cases, human cases were laboratory confirmed during an outbreak (nalca et al., ; sejvar et al., ) . it was determined that native prairie dogs (cynomys sp.) housed with rodents imported from ghana in west africa were the primary source of outbreak. the virus is mainly transmitted to humans while handling infected animals or by direct contact with the infected animal's body fluids, or lesions. person-to-person spread occurs by large respiratory droplets or direct contact (jeézek and fenner, ) . most of the clinical features of human monkeypox are very similar to those of ordinary smallpox (breman and arita, ) . after a -to -dayincubation period, the disease begins with fever, malaise, headache, sore throat, and cough. the main sign of the disease that distinguishes monkeypox from smallpox is swollen lymph nodes (lymphadenitis), which is observed in most of the patients before the development of rash (di giulio and eckburg, ; jeézek and fenner, ) . a typical maculopapular rash follows the prodromal period, generally lasting - days. the average size of the skin lesions are . - cm and the progress of lesions follows the order: macules, papules, vesicles, pustules, umblication then scab, and desquamation and lasts typically - weeks. the fatality rate is % among the unvaccinated population and death generally occurs during the nd week of the disease (jeézek and fenner, ; nalca et al., ) . mpxv is highly pathogenic for a variety of laboratory animals and many animal models have been developed by using different species and different routes of exposure (table . ). due to unavailability of variola virus (smallpox) to develop animal models and similar disease manifestations in humans that are similar, mpxv is one of the pox viruses that are utilized very heavily to develop a number of small animal models via different routes of exposure. wild-derived inbred mouse, stat -deficient c bl/ mouse, icr mouse, prairie dogs, african dormice, ground squirrels, and gambian pouched rats are highly susceptible to mpxv by different exposure routes (americo et al., ; falendysz et al., ; hutson et al., ; osorio et al., ; sbrana et al., ; schultz et al., ; sergeev et al., ; stabenow et al., ; tesh et al., ; xiao et al., ) . cast/eij mice, one of the inbred mouse strains tested for susceptibility to mpxv, showed weight loss and dose dependent mortality after in exposure to mpxv. studies with ip route of challenge indicated a fold higher susceptibility to mpxv when compared to in route (americo et al., ) . scid-balb/c mice were also susceptible to the ip challenge route and the disease resulted in mortality on day postinfection (osorio et al., ) . similarly, c bl/ stat −/− mice were infected in with mpxv and the infection resulted in weight loss and mortality days postexposure. recently sergeev et al. ( ) showed that in challenge of icr mice with mpxv resulted in purulent conjunctivitis, blepharitis, and ruffled fur in these mice although there was no death. the mouse models mentioned here are very promising for screening therapeutics against poxviruses but testing in additional models will be required for advanced development. high doses of the mpxv by ip or in routes caused % mortality in days postexposure and days postexposure, respectively, in ground squirrels (tesh et al., ) . the disease progressed very quickly and most of the animals were lethargic and moribund by day postexposure without any pox lesions or respiratory changes. a comparison study of usa mpxv and central african strain of mpxv strains in ground squirrels by the subq route resulted in systemic disease and mortality in - days postexposure. the disease resembles hemorrhagic smallpox with nosebleeds, impaired coagulation parameters, and hemorrhage in the lungs of the animals. another study by sergeev et al. ( ) showed that in challenge with mpxv caused fever, lymphadenitis, and skin rash in ground squirrels - days postexposure. mortality was observed in % of the animals - days postexposure (sergeev et al., ) . since mpxv was transmitted by infected prairie dogs in the us outbreak, this animal model has been more thoroughly studied and utilized to test therapeutics and vaccines compared to other small animal models ( hutson et al., ; keckler et al., ; smith et al., ; xiao et al., ) . studies using in, ip, and id routes of exposure showed that mpxv was highly infectious to prairie dogs, ip infection with the west african mpxv strain caused a more severe disease and % mortality than challenge by the in route. anorexia and lethargy were common signs of the disease for both exposure routes. in contrast to ip route, the in route of exposure caused severe pulmonary edema and necrosis of lungs in prairie dogs, while splenic necrosis and hepatic lesions were observed in ip-infected animals (xiao et al., ) . hutson et al. ( ) african and congo basin strains and showed that both strains and routes caused smallpox-like disease with longer incubation periods and most importantly generalized pox lesions. therefore, this model has the utility for testing therapeutics and vaccines against pox viruses. furthermore, mpxv challenged prairie dogs were used to perform in vivo bioluminescent imaging (bli) studies (falendysz et al., ) . bli studies showed real time spread of virus in prairie dogs as well as potential routes for shedding and transmission. the african dormouse is susceptible to mpxv by a footpad injection or in routes (schultz et al., ) . mice had decreased activity, hunched posture, dehydration, conjunctivitis, and weight loss. viral doses of and pfu provided % mortality with a mean time to death of days. upper gastrointestinal hemorrhage, hepatomegaly, lymphadenopathy, and lung hemorrhage were observed during necropsy. with the hemorrhage in several organs, this model resembles hemorrhagic smallpox. in a recent study, comparison of the disease pathogenesis was performed by using live bioluminescence imaging in the cast/eij mouse and african dormouse challenged with low dose of mpxv (earl et al., ) . following in challenge, mpxv dissemination occurred through the blood or lymphatic system in dormice compared to dissemination that was through the nasal cavity and lungs in cast/eij mice. the disease course was much faster in cast/eij mice (earl et al., ) . considering the limited availability of prairie dogs, ground squirrels and african dormice, lack of reagents specific for these species, and not having commercial sources of these species, these small animal models are as attractive for further characterization and vaccine, and countermeasure testing studies. nhps were exposed to mpxv by several different routes to develop animal model for mpxv (edghill-smith et al., ; johnson et al., ; nalca et al., ; stittelaar et al., ; zaucha et al., ) . during our studies using an aerosol route of exposure, we observed that macaques had mild anorexia, depression, fever, and lymphadenopathy on day postexposure (nalca et al., ) . complete blood count and clinical chemistries showed abnormalities similar to human monkeypox cases with leukocytosis and thrombocytopenia (huhn et al., ) . whole blood and throat swabs had viral loads peak around day , and in survivors, gradually decrease until day postexposure. since doses of × pfu, × pfu, or × pfu resulted in lethality for % of the animals, whereas a dose of × pfu resulted in % lethality, survival was not dose dependent. the main pitfall of this model was the lack of pox lesions. with the high dose, animals succumbed to disease before developing pox lesions. with the low challenge dose, pox lesions were observed but they were few in comparison to the iv model. a recent study also evaluated the cytokine levels in aerosol challenged animals. (tree et al., ) . tree et al. ( ) showed that ifnγ, il- rα, and il- increased dramatically on day postexposure the day that death was most likely to occur, and viral dna was detected in most of the tissues. these results support the idea of a cytokine storm causing mortality in monkeypox disease. mpxv causes dose dependent disease in nhps when given by the iv route (johnson et al., ) . studies showed that a × pfu iv challenge results in systemic disease with fever, lymphadenopathy, macula-papular rash, and mortality. an it infection model skips the upper respiratory system and deposits virus into the trachea, delivering the virus directly to the airways without regard to particle size and the physiological deposition that occurs during the process of inhalation. fibrinonecrotic bronchopneumonia was described in animals that received pfu of mpxv intratracheally (stittelaar et al., ) . although a similar challenge dose of it mpxv infection resulted in a similar viremia in nhps to the aerosol route of infection, the timing of the first peak was delayed by days in intratracheally exposed macaques compared to aerosol infection, and the amount of virus detected by qpcr was approximately -fold lower. this suggests that local replication is more prominent after aerosol delivery compared to the it route. an intrabronchial route of exposure resulted in pneumonia in nhps (johnson et al., ) . delayed onset of clinical signs and viremia were observed during the disease progression. in this model, similar to aerosol and it infection models, the number of pox lesions was much less than in the iv infection model. a major downside of the iv, it, and intrabronchial models is that the initial infection of respiratory tissue, incubation, and prodromal phases are circumvented with the direct inoculation of virus to the blood stream or to the lung. this is an important limitation when the utility of these models is to test possible vaccines and treatments in which the efficacy may depend on protecting the respiratory mucosa and targeting the subsequent early stages of the infection, which are not represented in these challenge models. although the aerosol model is the natural route of transmission for human varv infections and a secondary route for human mpxv infections, the lack of pox lesions is the main drawback of this model. therefore, when this model is used to test medical countermeasures, the endpoints and the biomarkers to initiate treatment should be chosen carefully. hepatitis b virus (hbv) is one of the most common infections worldwide with over million people chronically infected and , cases per year of liver cancer due to infection (lee, ) . the virus can naturally infect both humans and chimpanzees (guha et al., ) . hbv is transmitted parenterally or postnatally from infected mothers. it can also be transmitted by sexual contact, iv drug use, blood transfusion, and acupuncture (lai et al., ) . the age at which one is infected dictates the risk of developing chronic disease (hyams, ) . acute infection during adulthood is self-limiting and results in flu-like symptoms that can progress to hepatocellular involvement as observed with the development of jaundice. the clinical symptoms of hbv infection last for a few weeks before resolving (ganem and prince, ) . after this acute phase, lifetime immunity is achieved (wright and lau, ) . of those infected, less than % will develop the chronic form of the disease. chronicity is the most serious outcome of the disease as it can result in cirrhosis or liver cancer. hepatocellular carcinoma is times more likely to develop in a chronically infected individual than a noncarrier (beasley, ) . the viral determinant for cellular transformation has yet to be determined, although studies involving the woodchuck hepatitis virus suggest that x protein may be responsible (spandau and lee, ). many individuals are asymptomatic until complications emerge related to chronic hbv carriage. chimpanzees have a unique strain that circulates within the population (hu et al., ; . it was found that %- % of all wild-caught animals from africa are positive for hbv antigen ( lander et al., ) . natural and experimental challenge with the virus follows the same course as human disease; however, this is only an acute model of disease (prince, ) . to date, chimpanzees are the only reliable method to ensure that plasma vaccines are free from infectious particles (prince and brotman, ). this animal model has been used to study new therapeutics and vaccines. chimpanzees are especially ideal for these studies given that their immune response to infection directly mirrors humans (nayersina et al., ) . recent regulations by the national institute of health (nih) and restrictions to use great apes as animal models forced researches to find alternate models for hbv infection. other nhps that have been evaluated are gibbons, orangutans, and rhesus monkeys. although these animals can be infected with hbv, none develops hepatic lesions or liver damage as noted by monitoring of liver enzymes (pillot, ). mice are not permissible to infection, and thus numerous transgenic and humanized lines that express hbv proteins have been created to facilitate their usage as an animal model. these include both immunocompetent and immunosuppressed hosts. the caveat to all of these mouse lines is that they reproduce only the acute form of disease (guha et al., ) . recently, the entire genome of hbv was transferred to an immunocompetent mouse line via adenovirus. this provides a model for persistent infection (huang et al., ) . another model that has been developed is hydrodynamic injection of hbv genomes in the liver of mice (liu et al., ; yang et al., ) . although this model is very stressful to mice and has liver toxicity, it is successfully used to evaluate antivirals against hbv (mccaffrey et al., ) . liver chimeric mouse models are an additional set of surrogate models for hbv infection (dandri and lutgehetmann, ) . in these models human hepatocytes are integrated into the murine liver parenchyma (allweiss and dandri, ) . this model might be used to test antivirals as well as to study the molecular biology of hbv infection. hbv can also be studied using surrogate viruses, naturally occurring mammalian hepadna viruses (mason et al., ) . the woodchuck hepatitis virus induces hepatocellular carcinoma (summers et al., ) . within a population, %- % of all neonatal woodchucks are susceptible to chronic infection (cote et al., ) . a major difference between the two hepatitis isolates is the rate at which they induce cancer; almost all chronic carriers developed hepatocellular carcinoma within years of the initial infection in woodchucks, whereas human carcinogenesis takes much longer (gerin et al., ) . the acute infection strongly resembles what occurs during the course of human disease. there is a self-limiting acute phase resulting in a transient viremia that has the potential of chronic carriage (tennant, ) . challenge with virus in neonates leads to a chronic infection while adults only develop the acute phase of disease (buendia, ) . a closely related species to the woodchuck is the marmota himalayan. this animal is also susceptible to the woodchuck hepadna virus upon iv injection. the marmot himalayan develops an acute hepatitis with a productive infection (lucifora et al., ) . hepatitis d virus (hdv) is dependent upon hbv to undergo replication and successful infection in its human host (gerin, ) . there are two modes of infection possible between the viruses: coinfection where a person is simultaneously infected or superinfection in which a chronic carrier of hbv is subsequently infected with hdv (purcell et al., ) . coinfection leads to a similar disease as seen with hbv alone; however, superinfection can result in chronic hdv infection and severe liver damage (guilhot et al., ) . both coinfection and superinfection can be demonstrated within the chimpanzee and woodchuck by inoculation of human hepatitis d (ponzetto et al., ) . a recently published report demonstrated the use of a humanized chimeric upa mouse to study interactions between the two viruses and drug testing (lutgehetmann et al., ) new models ranging from nhps to small animals and representing the disease characteristics in humans are necessary to study viral and host factors that drive disease pathogenesis and evaluate medical countermeasures. the ideal animal model for human viral disease should closely recapitulate the spectrum of clinical symptoms and pathogenesis observed during the course of human infection. whenever feasible, the model should use the same virus and strain that infects humans. it is also preferable that the virus is a low passage clinical isolate thus animal passage or adaptation should be avoided if model species can be identified that are susceptible. ideally, the experimental route of infection would mirror that occurs in natural disease. in order to understand the interplay and contribution of the immune system during infection, an immunocompetent animal should be used. the aforementioned characteristics cannot always be satisfied; however, and often virus must be adapted, knockout mice must be used, and/or the disease is not perfectly mimicked in the animal model. well-characterized animal models are critical for licensure to satisfy fda "animal rule." this rule applies to situations in which vaccine and therapeutic efficacy cannot safely or ethically be tested in humans; thus licensure will come only after preclinical tests are performed in animal models. many fields in virology are moving toward standardized models that can be used across institutions to test vaccines and therapeutics. a current example of such an effort is within the filovirus community, where animal models, euthanasia criteria, assays, and virus strains are in the process of being standardized. the hope is that these efforts will enable results of efficacy tests on medical countermeasures compared across institutions. this chapter has summarized the best models available for each of the viruses described. the rhesus macaque pediatric siv infection model-a valuable tool in understanding infant hiv- pathogenesis and for designing pediatric hiv- prevention strategies prevalence of anti-rift-valley-fever igm antibody in abattoir workers in the nile delta during the outbreak in egypt common marmosets (callithrix jacchus) as a nonhuman primate model to assess the virulence of eastern equine encephalitis virus strains replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels. emerg generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease pathological changes in brain and other target organs of infant and weanling mice after infection with nonneuroadapted western equine encephalitis virus particle-to-pfu ratio of ebola virus influences disease course and survival in cynomolgus macaques progress toward norovirus vaccines: considerations for further development and implementation in potential target populations characterization of lethal zika virus infection in ag mice experimental in vitro and in vivo models for the study of human hepatitis b virus infection a model of meningococcal bacteremia after respiratory superinfection in influenza a virus-infected mice middle east respiratory syndrome coronavirus: current situation and travel-associated concerns aerosol exposure to the angola strain of marburg virus causes lethal viral hemorrhagic fever in cynomolgus macaques necrotizing scleritis, conjunctivitis, and other pathologic findings in the left eye and brain of an ebola virus-infected rhesus macaque (macaca mulatta) with apparent recovery and a delayed time of death american academy of pediatrics subcommittee on diagnosis and management of bronchiolitis identification of wild-derived inbred mouse strains highly susceptible to monkeypox virus infection for use as small animal models the gerbil, meriones unguiculatus, a model for rift valley fever viral encephalitis morbidity and mortality among patients with respiratory syncytial virus infection: a -year retrospective review chikungunya and the nervous system: what we do and do not know the west nile virus outbreak of in new york: the flushing hospital experience hospital outbreak of middle east respiratory syndrome coronavirus diagnosis of noncultivatable gastroenteritis viruses, the human caliciviruses norovirus vaccine against experimental human norwalk virus illness determination of the % human infectious dose for norwalk virus an epizootic attributable to western equine encephalitis virus infection in emus in texas evidence for camel-to-human transmission of mers coronavirus integrated molecular signature of disease: analysis of influenza virus-infected macaques through functional genomics and proteomics disseminated and sustained hiv infection in cd + cord blood cell-transplanted rag −/− gamma c −/− mice choice of inbred rat strain impacts lethality and disease course after respiratory infection with rift valley fever virus rift valley fever: an uninvited zoonosis in the arabian peninsula recombinant norwalk virus-like particles given orally to volunteers: phase i study tropism of dengue virus in mice and humans defined by viral nonstructural protein -specific immunostaining lethal antibody enhancement of dengue disease in mice is prevented by fc modification animal models for the study of influenza pathogenesis and therapy effect of oral gavage treatment with znal and other metallo-ion formulations on influenza a h n and h n virus infections in mice macaque studies of vaccine and microbicide combinations for preventing hiv- sexual transmission early and sustained innate immune response defines pathology and death in nonhuman primates infected by highly pathogenic influenza virus hepatitis b virus. the major etiology of hepatocellular carcinoma transmission of norwalk virus during football game vpx is critical for sivmne infection of pigtail macaques experimental respiratory syncytial virus infection of four species of primates pathogenesis and immune response of crimean-congo hemorrhagic fever virus in a stat- knockout mouse model crimean-congo hemorrhagic fever virus infection is lethal for adult type i interferon receptor-knockout mice the utility of the new generation of humanized mice to study hiv- infection: transmission, prevention, pathogenesis, and treatment hiv- infection and cd t cell depletion in the humanized rag −/− gamma c −/− (rag-hu) mouse model bacterial infections in pigs experimentally infected with nipah virus evaluation of a mouse model for the west nile virus group for the purpose of determining viral pathotypes severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice study of susceptibility to crimean hemorrhagic fever (chf) virus in european and long-eared hedgehogs. tezisy konf manipulation of host factors optimizes the pathogenesis of western equine encephalitis virus infections in mice for antiviral drug development genetic basis of attenuation of dengue virus type small plaque mutants with restricted replication in suckling mice and in scid mice transplanted with human liver cells chimpanzees as an animal model for human norovirus infection and vaccine development a simple technique for infection of mosquitoes with viruses; transmission of zika virus human papillomavirus research: do we still need animal models? human papillomavirus in cervical cancer development of a hamster model for chikungunya virus infection and pathogenesis a neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute nipah virus infection the cotton rat model of respiratory viral infections correlates of immunity to filovirus infection filovirus vaccines induction of robust cellular and humoral virusspecific adaptive immune responses in human immunodeficiency virus-infected humanized blt mice animal models of human-papillomavirus-associated oncogenesis interferon alpha/beta receptor-deficient mice as a model for ebola virus disease zika virus outbreak in rio de janeiro, brazil: clinical characterization, epidemiological and virological aspects co-infection of the cotton rat (sigmodon hispidus) with staphylococcus aureus and influenza a virus results in synergistic disease effectiveness of influenza vaccination the role of the type i interferon response in the resistance of mice to filovirus infection a mouse model for evaluation of prophylaxis and therapy of ebola hemorrhagic fever the rabbit viral skin papillomas and carcinomas: a model for the immunogenetics of hpv-associated carcinogenesis the confirmation and maintenance of smallpox eradication a lethal disease model for hantavirus pulmonary syndrome in immunosuppressed syrian hamsters infected with sin nombre virus nonhuman primate models of chikungunya virus infection and disease tissue tropism and neuroinvasion of west nile virus do not differ for two mouse strains with different survival rates pediatric norovirus diarrhea in nicaragua efficacy assessment of a cell-mediated immunity hiv- vaccine (the step study): a double-blind, randomised, placebo-controlled, test-of-concept trial variation between strains of hamsters in the lethality of pichinde virus infections hepatitis b viruses and hepatocellular carcinoma generation of k -e /n betacat double transgenic mice as a model of cervical cancer limited or no protection by weakly or nonneutralizing antibodies against vaginal shiv challenge of macaques compared with a strongly neutralizing antibody a novel vaccine against crimean-congo haemorrhagic fever protects % of animals against lethal challenge in a mouse model interleukin- beta but not tumor necrosis factor is involved in west nile virusinduced langerhans cell migration from the skin in c bl/ mice animal models of papillomavirus pathogenesis immunization of knock-out alpha/beta interferon receptor mice against high lethal dose of crimean-congo hemorrhagic fever virus with a cell culture based vaccine characterization of the localized immune response in the respiratory tract of ferrets following infection with influenza a and b viruses lassa virus infection in experimentally infected marmosets: liver pathology and immunophenotypic alterations in target tissues a small nonhuman primate model for filovirus-induced disease severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus outbreak of acute illness-southwestern united states outbreak of west nile-like viral encephalitis-new york, . mmwr morb. mortal in vitro whole-virus binding of a norovirus genogroup ii genotype strain to cells of the lamina propria and brunner's glands in the human duodenum animal models for studying dengue pathogenesis and therapy us doctors investigate more than possible cases of monkeypox mechanism of neuroinvasion of venezuelan equine encephalitis virus in the mouse chikungunya outbreaks-the globalization of vectorborne diseases inactivated and live, attenuated influenza vaccines protect mice against influenza: streptococcus pyogenes super-infections pathogenesis of a genogroup ii human norovirus in gnotobiotic pigs the immunobiology of sars* induction of tetravalent protective immunity against four dengue serotypes by the tandem domain iii of the envelope protein norovirus infection as a cause of diarrhea-associated benign infantile seizures comparative pathogenesis of epidemic and enzootic chikungunya viruses in a pregnant rhesus macaque model development of norwalk virus-specific monoclonal antibodies with therapeutic potential for the treatment of norwalk virus gastroenteritis viral shedding patterns of coronavirus in patients with probable severe acute respiratory syndrome a single sublingual dose of an adenovirus-based vaccine protects against lethal ebola challenge in mice and guinea pigs model systems to study the life cycle of human papillomaviruses and hpv-associated cancers primary severe acute respiratory syndrome coronavirus i nfection limits replication but not lung inflammation upon homologous rechallenge viral and host factors in human respiratory syncytial virus pathogenesis pathogenesis of pichinde virus infection in strain guinea pigs: an immunocytochemical, virologic, and clinical chemistry study pathogenesis of experimental ebola virus infection in guinea pigs transcriptional profiling of the immune response to marburg virus infection the use of a neonatal mouse model to study respiratory syncytial virus infections a model of denv- infection that recapitulates severe disease and highlights the importance of ifn-gamma in host resistance to infection effects of age and viral determinants on chronicity as an outcome of experimental woodchuck hepatitis virus infection a mouse model for chikungunya: young age and inefficient type-i interferon signaling are risk factors for severe disease mosquito bite delivery of dengue virus enhances immunogenicity and pathogenesis in humanized mice comparison of the pathogenesis of the angola and ravn strains of marburg virus in the outbred guinea pig model the brazilian zika virus strain causes birth defects in experimental models age at first viral infection determines the pattern of t cell-mediated disease during reinfection in adulthood lassa fever encephalopathy: clinical and laboratory findings profound and prolonged lymphocytopenia with west nile encephalitis first complete genome sequence of zika virus (flaviviridae, flavivirus) from an autochthonous transmission in brazil viral haemorrhagic fevers caused by lassa, ebola, and marburg viruses the enhancement or prevention of airway hyperresponsiveness during reinfection with respiratory syncytial virus is critically dependent on the age at first infection and il- production mouse models of hepatitis b and delta virus infection [experimental inhalation infection of monkeys of the macacus cynomolgus and macacus rhesus species with the virus kinetic profile of influenza virus infection in three rat strains pathology of experimental ebola virus infection in african green monkeys. involvement of fibroblastic reticular cells validation of an hpv -mediated carcinogenesis mouse model middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques selection of unadapted, pathogenic shivs encoding newly transmitted hiv- envelope proteins b-cells and the use of non-human primates for evaluation of hiv vaccine candidates antiretroviral pre-exposure prophylaxis prevents vaginal transmission of hiv- in humanized blt mice innate and adaptive immune responses determine protection against disseminated infection by west nile encephalitis virus rift valley fever virus encephalitis is associated with an ineffective systemic immune response and activated t cell infiltration into the cns in an immunocompetent mouse model evidence of sexual transmission of zika virus a susceptible mouse model for zika virus infection identification of a novel coronavirus in patients with severe acute respiratory syndrome subclinical infection without encephalitis in mice following intranasal exposure to nipah virus-malaysia and nipah virus-bangladesh nonhuman primate models of encephalitic alphavirus infection: historical review and future perspectives mortality due to influenza in the united states-an annualized regression approach using multiple-cause mortality data distinct pathogenesis of hong kong-origin h n viruses in mice compared to that of other highly pathogenic h avian influenza viruses postexposure antibody prophylaxis protects nonhuman primates from filovirus disease comparative live bioluminescence imaging of monkeypox virus dissemination in a wild-derived inbred mouse (mus musculus castaneus) and outbred african dormouse (graphiurus kelleni) siv-induced impairment of neurovascular repair: a potential role for vegf smallpox vaccine does not protect macaques with aids from a lethal monkeypox virus challenge influenza-induced tachypnea is prevented in immune cotton rats, but cannot be treated with an anti-inflammatory steroid or a neuraminidase inhibitor distinct cellular immune responses following primary and secondary influenza virus challenge in cotton rats an outbreak of viral gastroenteritis following environmental contamination at a concert hall natural history of aerosol exposure with marburg virus in rhesus macaques experimental congo virus (ib-an ) infection in primates further assessment of monkeypox virus infection in gambian pouched rats (cricetomys gambianus) using in vivo bioluminescent imaging respiratory syncytial virus infection in elderly and high-risk adults infection with mers-cov causes lethal pneumonia in the common marmoset immune response to marburg virus angola infection in nonhuman primates fields' virology the susceptibility of rats to rift valley fever in relation to age henipavirus susceptibility to environmental variables pause on avian flu transmission research aetiology: koch's postulates fulfilled for sars virus spinal cord neuropathology in human west nile virus infection therapeutic dna vaccine induces broad t cell responses in the gut and sustained protection from viral rebound and aids in siv-infected rhesus macaques hepatitis b virus infection-natural history and clinical consequences biological heterogeneity, including systemic replication in mice, of h n influenza a virus isolates from humans in hong kong chikungunya virus arthritis in adult wild-type mice epidemiology and clinical presentations of the four human coronaviruses e, hku , nl , and oc detected over years using a novel multiplex real-time pcr method development of an acute and highly pathogenic nonhuman primate model of nipah virus infection animal models of hepatitis delta virus infection and disease hepadnavirusinduced liver cancer in woodchucks experimental infection and natural contact exposure of dogs with avian influenza virus (h n ). emerg megaribavirin aerosol for the treatment of influenza a virus infections in mice discovery of novel human and animal cells infected by the severe acute respiratory syndrome coronavirus by replication-specific multiplex reverse transcription-pcr chinchilla and murine models of upper respiratory tract infections with respiratory syncytial virus mechanisms of host defense following severe acute respiratory syndromecoronavirus (sars-cov) pulmonary infection of mice studies on the virus of venezuelan equine encephalomyelitis. i. modification by cortisone of the response of the central nervous system of macaca mulatta serious morbidity and mortality associated with influenza epidemics a novel respiratory model of infection with monkeypox virus in cynomolgus macaques clinical features of nipah virus encephalitis among pig farmers in malaysia monoclonal antibody-mediated enhancement of dengue virus infection in vitro and in vivo and strategies for prevention animal models of highly pathogenic rna viral infections: hemorrhagic fever viruses interferon alfacon- protects hamsters from lethal pichinde virus infection primary respiratory syncytial virus infection in mice pneumonitis and multiorgan system disease in common marmosets (callithrix jacchus) infected with the severe acute respiratory syndrome-associated coronavirus clinical and laboratory features that differentiate dengue from other febrile illnesses in an endemic area-puerto rico acute and chronic airway disease after human respiratory syncytial virus infection in cotton rats (sigmodon hispidus) alphaviruses replication fitness determines high virulence of influenza a virus in mice carrying functional mx resistance gene antibody and antiretroviral preexposure prophylaxis prevent cervicovaginal hiv- infection in a transgenic mouse model characterization of influenza a/hongkong/ / (h n ) virus in a mouse model and protective effect of zanamivir on h n infection in mice epidemic dengue/dengue hemorrhagic fever as a public health, social and economic problem in the st century cell culture and animal models of viral hepatitis. part i: hepatitis b expression of the hepatitis delta virus large and small antigens in transgenic mice acute hendra virus infection: analysis of the pathogenesis and passive antibody protection in the hamster model lassa fever encephalopathy: lassa virus in cerebrospinal fluid but not in serum , -iodonaphthyl azide-inactivated v protects against aerosol challenge with virulent venezuelan equine encephalitis virus dengue: an update pegylated interferonalpha protects type pneumocytes against sars coronavirus infection in macaques asymptomatic middle east respiratory syndrome coronavirus infection in rabbits head-to-head comparison of four nonadjuvanted inactivated cell culture-derived influenza vaccines: effect of composition, spatial organization and immunization route on the immunogenicity in a murine challenge model update on animal models for hiv research norovirus disease in the united states. emerg cardiopulmonary manifestations of hantavirus pulmonary syndrome serum neutralizing antibody titers of seropositive chimpanzees immunized with vaccines coformulated with natural fusion and attachment proteins of respiratory syncytial virus hendra virus infection in a veterinarian a phase clinical trial of a dna vaccine for venezuelan equine encephalitis delivered by intramuscular or intradermal electroporation deaths from norovirus among the elderly, england and wales. emerg aerosolized rift valley fever virus causes fatal encephalitis in african green monkeys and common marmosets hiv- -induced aids in monkeys west nile fever short communication: viremic control is independent of repeated low-dose shivsf p exposures pathogenesis of marburg hemorrhagic fever in cynomolgus macaques pathogenesis of lassa fever in cynomolgus macaques niemann-pick c is essential for ebolavirus replication and pathogenesis in vivo airborne transmission of influenza a/ h n virus between ferrets animal models in hiv- protection and therapy advances in rsv vaccine research and development-a global agenda transmission of zika virus through sexual contact with travelers to areas of ongoing transmissioncontinental united states establishment of a patient-derived orthotopic xenograft (pdox) model of her- -positive cervical cancer expressing the clinical metastatic pattern resolution of primary severe acute respiratory syndromeassociated coronavirus infection requires stat mucosal immunity and vaccines nipah virus outbreak with person-to-person transmission in a district of bangladesh eastern equine encephalitis virus in mice i: clinical course and outcome are dependent on route of exposure the lesions of experimental equine morbillivirus disease in cats and guinea pigs a lethal disease model for hantavirus pulmonary syndrome hantaan/ andes virus dna vaccine elicits a broadly cross-reactive neutralizing antibody response in nonhuman primates persistent infection with and serologic cross-reactivity of three novel murine noroviruses molecular characterization of three novel murine noroviruses identification of hepatitis b virus indigenous to chimpanzees manifestation of thrombocytopenia in dengue- -virusinfected mice transfer of hbv genomes using low doses of adenovirus vectors leads to persistent infection in immune competent mice west nile fever-a reemerging mosquito-borne viral disease in europe. emerg live, attenuated influenza virus (laiv) vehicles are strong inducers of immunity toward influenza b virus clinical characteristics of human monkeypox, and risk factors for severe disease shiv- i and passaged progeny viruses encoding r hiv- clade c env cause aids in rhesus monkeys norwalk virus infection associates with secretor status genotyped from sera a prairie dog animal model of systemic orthopoxvirus disease using west african and congo basin strains of monkeypox virus risks of chronicity following acute hepatitis b virus infection: a review the pathogenesis of rift valley fever experimental adaptation of an influenza h ha confers respiratory droplet transmission to a reassortant h ha/h n virus in ferrets pathogenicity of a highly pathogenic avian influenza virus, a/chicken/yamaguchi/ / (h n ) in different species of birds and mammals respiratory syncytial virus induces pneumonia, cytokine response, airway obstruction, and chronic inflammatory infiltrates associated with long-term airway hyperresponsiveness in mice a multimeric l vaccine for prevention of animal papillomavirus infections lassa virus infection of rhesus monkeys: pathogenesis and treatment with ribavirin pathogenesis of lassa virus infection in guinea pigs perinatal transmission of shiv-sf p in macaca nemestrina human monkeypox and other poxvirus infections of man cd l (l-sign) is a receptor for severe acute respiratory syndrome coronavirus glycomic characterization of respiratory tract tissues of ferrets: implications for its use in influenza virus infection studies comparative analysis of monkeypox virus infection of cynomolgus macaques by the intravenous or intrabronchial inoculation route phenotypic changes in langerhans' cells after infection with arboviruses: a role in the immune response to epidermally acquired viral infection? protection of beagle dogs from mucosal challenge with canine oral papillomavirus by immunization with recombinant adenoviruses expressing codon-optimized early genes detailed analysis of the african green monkey model of nipah virus disease experimental inoculation of egyptian rousette bats (rousettus aegyptiacus) with viruses of the ebolavirus and marburgvirus genera treatment of venezuelan equine encephalitis virus infection with (-)-carbodine c h/hen mouse model for the evaluation of antiviral agents for the treatment of venezuelan equine encephalitis virus infection blt humanized mice as a small animal model of hiv infection stat -dependent innate immunity to a norwalk-like virus pichinde virus infection in strain guinea pigs reduces intestinal protein reflection coefficient with compensation establishment of the black-tailed prairie dog (cynomys ludovicianus) as a novel animal model for comparing smallpox vaccines administered preexposure in both high-and low-dose monkeypox virus challenges low-dose rectal inoculation of rhesus macaques by sivsme or sivmac recapitulates human mucosal infection by hiv- new opportunities for field research on the pathogenesis and treatment of lassa fever gastrointestinal norovirus infection associated with exacerbation of inflammatory bowel disease isolation of monkeypox virus from wild squirrel infected in nature in hot pursuit of the first vaccine against respiratory syncytial virus pathogenesis of hantaan virus infection in suckling mice: clinical, virologic, and serologic observations respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine the severe pathogenicity of alveolar macrophage-depleted ferrets infected with pandemic h n influenza virus mouse adaptation of influenza b virus increases replication in the upper respiratory tract and results in droplet transmissibility in ferrets stepping toward a macaque model of hiv- induced vomiting as a symptom and transmission risk in norovirus illness: evidence from human challenge studies wild-type puumala hantavirus infection induces cytokines, c-reactive protein, creatinine, and nitric oxide in cynomolgus macaques aberrant innate immune response in lethal infection of macaques with the influenza virus adenovirus-based vaccine prevents pneumonia in ferrets challenged with the sars coronavirus and stimulates robust immune responses in macaques replication, pathogenicity, shedding, and transmission of zaire ebolavirus in pigs west nile viral encephalitis foodborne viruses: an emerging problem low pathogenic avian influenza a(h n ) virus causes high mortality in ferrets upon intratracheal challenge: a model to study intervention strategies filoviruses: a compendium of years of epidemiological, clinical, and laboratory studies pathology of human influenza a (h n ) virus infection in cynomolgus macaques (macaca fascicularis) dengue virus infection and immune response in humanized rag (-/-)gamma(c) (-/-) (rag-hu) mice chikungunya disease in nonhuman primates involves long-term viral persistence in macrophages viral hepatitis b strong local and systemic protective immunity induced in the ferret model by an intranasal virosome-formulated influenza subunit vaccine origin of the west nile virus responsible for an outbreak of encephalitis in the northeastern united states antibody to hepatitis-associated antigen. frequency and pattern of response as detected by radioimmunoprecipitation severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats coronavirus hku and other coronavirus infections in hong kong epidemic rift valley fever in egypt: observations of the spectrum of human illness hantaviruses. a short review hepatitis b virus infection quantitative measurement of influenza virus replication using consecutive bronchoalveolar lavage in the lower respiratory tract of a ferret model characterization of the activity of '-c-methylcytidine against dengue virus replication diffusion tensor and volumetric magnetic resonance measures as biomarkers of brain damage in a small animal model of hiv sequencing, annotation, and characterization of the influenza ferret infectome lethality and pathogenesis of airborne infection with filoviruses in a alpha/beta −/− interferon receptor-deficient mice experimental inoculation study indicates swine as a potential host for hendra virus early initiation of antiretroviral therapy can functionally control productive hiv- infection in humanized-blt mice zika virus disrupts neural progenitor development and leads to microcephaly in mice middle east respiratory syndrome coronavirus causes multiple organ damage and lethal disease in mice transgenic for human dipeptidyl peptidase immunogenicity and protective efficacy of a recombinant subunit west nile virus vaccine in rhesus monkeys study of dengue virus infection in scid mice engrafted with human k cells a comparative study of the pathogenesis of western equine and eastern equine encephalomyelitis viral infections in mice by intracerebral and subcutaneous inoculations hydrodynamics-based transfection in animals by systemic administration of plasmid dna the emergence of nipah virus, a highly pathogenic paramyxovirus the guinea pig as a transmission model for human influenza viruses transmission in the guinea pig model a mouse model for the evaluation of pathogenesis and immunity to influenza a (h n ) viruses isolated from humans transmission of human infection with nipah virus recurrent zoonotic transmission of nipah virus into humans the effect of an arenavirus infection on liver morphology and function hepatitis b virus replication in primary macaque hepatocytes: crossing the species barrier toward a new small primate model ebola virus disease in mice with transplanted human hematopoietic stem cells arenavirus-mediated liver pathology: acute lymphocytic choriomeningitis virus infection of rhesus macaques is characterized by high-level interleukin- expression and hepatocyte proliferation humanized chimeric upa mouse model for the study of hepatitis b and d virus interactions and preclinical drug evaluation role of dendritic cell targeting in venezuelan equine encephalitis virus pathogenesis detection of hepatitis b virus infection in wild-born chimpanzees (pan troglodytes verus): phylogenetic relationships with human and other primate genotypes proportion of deaths and clinical features in bundibugyo ebola virus infection the ferret: an animal model to study influenza virus local innate immune responses and influenza virus transmission and virulence in ferrets vomiting larry: a simulated vomiting system for assessing environmental contamination from projectile vomiting related to norovirus infection studies on the pathogenesis of dengue infection in monkeys. . sequential distribution of virus in primary and heterologous infections studies on dengue virus infection in cyclophosphamide-treated rhesus monkeys crimean-congo hemorrhagic fever experimental infection of squirrel monkeys with nipah virus. emerg a school outbreak of norwalk-like virus: evidence for airborne transmission virology: sars virus infection of cats and ferrets characterization of clinical and immunological parameters during ebola virus infection of rhesus macaques delayed disease progression in cynomolgus macaques infected with ebola virus makona strain. emerg asymmetric replication of duck hepatitis b virus dna in liver cells: free minusstrand dna human and avian influenza viruses target different cell types in cultures of human airway epithelium a role for hpv e in cervical carcinogenesis replication of sars coronavirus administered into the respiratory tract of african green, rhesus and cynomolgus monkeys inhibition of hepatitis b virus in mice by rna interference zika virus was transmitted by sexual contact in texas, health officials report lassa fever. effective therapy with ribavirin lassa virus hepatitis: a study of fatal lassa fever in humans lethal infection of k -hace mice infected with severe acute respiratory syndrome coronavirus andes virus infection of cynomolgus macaques regional t-and b-cell responses in influenza-infected ferrets clinical aspects of marburg hemorrhagic fever humanized mice mount specific adaptive and innate immune responses to ebv and tsst- isolation from human sera in egypt of a virus apparently identical to west nile virus middle east respiratory syndrome coronavirus in bats, saudi arabia. emerg chikungunya virus infection results in higher and persistent viral replication in aged rhesus macaques due to defects in anti-viral immunity reduced clearance of respiratory syncytial virus infection in a preterm lamb model dipeptidyl peptidase distribution in the human respiratory tract: implications for the middle east respiratory syndrome experimental nipah virus infection in pigs and cats experimental nipah virus infection in pteropid bats (pteropus poliocephalus) an in-depth analysis of original antigenic sin in dengue virus infection propagation and dissemination of infection after vaginal transmission of simian immunodeficiency virus rift valley fever virus in mice. i. general features of the infection zika virus infection during pregnancy in mice causes placental damage and fetal demise outbreaks of acute gastroenteritis associated with norwalk-like viruses in campus settings a nonfucosylated variant of the anti-hiv- monoclonal antibody b has enhanced fcgammariiiamediated antiviral activity in vitro but does not improve protection against mucosal shiv challenge in macaques flaviviruses experimental studies of rhesus monkeys infected with epizootic and enzootic subtypes of venezuelan equine encephalitis virus necrotizing myocarditis in mice infected with western equine encephalitis virus: clinical, electrocardiographic, and histopathologic correlations aedes albopictus in the united states: ten-year presence and public health implications. emerg severity of clinical disease and pathology in ferrets experimentally infected with influenza viruses is influenced by inoculum volume impact of short-term haart initiated during the chronic stage or shortly post-exposure on siv infection of male genital organs influence of age on susceptibility and on immune response of mice to eastern equine encephalomyelitis virus a mouse model of chikungunya virusinduced musculoskeletal inflammatory disease: evidence of arthritis, tenosynovitis, myositis, and persistence dengue virus tropism in humanized mice recapitulates human dengue fever functional role of type i and type ii interferons in antiviral defense the occurrence of human cases in johannesburg. s feline model of acute nipah virus infection and protection with a soluble glycoprotein-based subunit vaccine vertical transmission and fetal replication of nipah virus in an experimentally infected cat pathogenesis and transmission of swine-origin a(h n ) influenza virus in ferrets pneumonia from human coronavirus in a macaque model eastern equine encephalitis virus infection: electron microscopic studies of mouse central nervous system evaluation of three strains of influenza a virus in humans and in owl, cebus, and squirrel monkeys a novel morbillivirus pneumonia of horses and its transmission to humans. emerg a morbillivirus that caused fatal disease in horses and humans global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis cchf infection among animals reemergence of monkeypox: prevalence, diagnostics, and countermeasures experimental infection of cynomolgus macaques (macaca fascicularis) with aerosolized monkeypox virus eastern equine encephalitis. distribution of central nervous system lesions in man and rhesus monkey hla a restricted cytotoxic t lymphocyte responses to multiple hepatitis b surface antigen epitopes during hepatitis b virus infection an aptamer-sirna chimera suppresses hiv- viral loads and protects from helper cd (+) t cell decline in humanized mice severe acute respiratory syndrome coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ace ferrets exclusively synthesize neu ac and express naturally humanized influenza a virus receptors field's virology naturally occurring, nonregressing canine oral papillomavirus infection: host immunity, virus characterization, and experimental infection regression of canine oral papillomas is associated with infiltration of cd + and cd + lymphocytes diversifying animal models: the use of hispid cotton rats (sigmodon hispidus) in infectious diseases induction of focal epithelial hyperplasia in tongue of young bk -e /e hpv transgenic mice the contribution of animal models to the understanding of the host range and virulence of influenza a viruses evaluation of antiviral efficacy of ribavirin, arbidol, and t- (favipiravir) in a mouse model for crimean-congo hemorrhagic fever evaluation of oseltamivir prophylaxis regimens for reducing influenza virus infection, transmission and disease severity in a ferret model of household contact a novel video tracking method to evaluate the effect of influenza infection and antiviral treatment on ferret activity human respiratory syncytial virus a strain replicates and induces innate immune responses by respiratory epithelia of neonatal lambs common marmoset (callithrix jacchus) as a primate model of dengue virus infection: development of high levels of viraemia and demonstration of protective immunity changes in hematological and serum biochemical parameters in common marmosets (callithrix jacchus) after inoculation with dengue virus middle east respiratory syndrome coronavirus (mers-cov): animal to human interaction dengue virus-induced hemorrhage in a nonhuman primate model preclinical development of highly effective and safe dna vaccines directed against hpv e and e a novel small molecule inhibitor of influenza a viruses that targets polymerase function and indirectly induces interferon comparison of monkeypox viruses pathogenesis in mice by in vivo imaging a rhesus monkey model for sexual transmission of a papillomavirus isolated from a squamous cell carcinoma infection of calves with bovine norovirus giii. strain jena virus: an experimental model to study the pathogenesis of norovirus infection the cotton rat provides a useful small-animal model for the study of influenza virus pathogenesis zika virus disease in colombia-preliminary report genetic diversity, distribution, and serological features of hantavirus infection in five countries in south america liver injury and viremia in mice infected with dengue- virus the hamster as an animal model for eastern equine encephalitis-and its use in studies of virus entrance into the brain a recombinant hendra virus g glycoprotein-based subunit vaccine protects ferrets from lethal hendra virus challenge identification of an antioxidant small-molecule with broad-spectrum antiviral activity impaired heterologous immunity in aged ferrets during sequential influenza a h n infection influenza transmission in the mother-infant dyad leads to severe disease, mammary gland infection, and pathogenesis by regulating host responses economic impact of respiratory syncytial virus-related illness in the us: an analysis of national databases inhibitory potential of neem (azadirachta indica juss) leaves on dengue virus type- replication systematic literature review of role of noroviruses in sporadic gastroenteritis. emerg sars: what have we learned? the severe acute respiratory syndrome severe acute respiratory syndrome bacterial sinusitis and otitis media following influenza virus infection in ferrets neuropathology of h n virus infection in ferrets the draft genome sequence of the ferret (mustela putorius furo) facilitates study of human respiratory disease immunopathogenesis of coronavirus infections: implications for sars hantavirus pulmonary syndrome: the new american hemorrhagic fever rift valley fever inbred rat strains mimic the disparate human response to rift valley fever virus infection experimental studies of arenaviral hemorrhagic fevers experimental rift valley fever in rhesus macaques bovine respiratory syncytial virus protects cotton rats against human respiratory syncytial virus infection human hendra virus encephalitis associated with equine outbreak molecularly engineered live-attenuated chimeric west nile/dengue virus vaccines protect rhesus monkeys from west nile virus structure as revealed by airway dissection. a comparison of mammalian lungs study on west nile virus persistence in monkeys experimental hepatitis delta virus infection in the animal model changing patterns of chikungunya virus: re-emergence of a zoonotic arbovirus grune and stratton. hepatitis and blood transfusion perspectives on hepatitis b studies with chimpanzees pulmonary lesions in primary respiratory syncytial virus infection, reinfection, and vaccine-enhanced disease in the cotton rat (sigmodon hispidus) experimental hepatitis delta virus infection in the chimpanzee relative infectivity of hepatitis a virus by the oral and intravenous routes in species of nonhuman primates cardiovascular and pulmonary responses to pichinde virus infection in strain guinea pigs establishment and characterization of a lethal mouse model for the angola strain of marburg virus stability and inactivation of sars coronavirus possibility of extracting hyperimmune gammaglobulin against chf from doneky blood sera dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc swine as a model for influenza a virus infection and immunity zika virus: an update on epidemiology, pathology, molecular biology, and animal model oseltamivir population pharmacokinetics in the ferret: model application for pharmacokinetic/pharmacodynamic study design aerosol exposure to western equine encephalitis virus causes fever and encephalitis in cynomolgus macaques severe encephalitis in cynomolgus macaques exposed to aerosolized eastern equine encephalitis virus differences in aerosolization of rift valley fever virus resulting from choice of inhalation exposure chamber: implications for animal challenge studies an hiv- transgenic rat that develops hiv-related pathology and immunologic dysfunction a trivalent recombinant ad gag/pol/nef vaccine fails to protect rhesus macaques from infection or control virus replication after a limiting-dose heterologous siv challenge infection with chikungunya virus in italy: an outbreak in a temperate region a single dose of an iscom influenza vaccine induces long-lasting protective immunity against homologous challenge infection but fails to protect cynomolgus macaques against distant drift variants of influenza a (h n ) viruses influenza a virus (h n ) infection in cats causes systemic disease with potential novel routes of virus spread within and between hosts immunomodulation with il- r alpha antisense oligonucleotide prevents respiratory syncytial virus-mediated pulmonary disease animal models for sars aged balb/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans a mouse-adapted sars-coronavirus causes disease and mortality in balb/c mice transmission of a h n pandemic influenza virus occurs before fever is detected, in the ferret model experimental norovirus infections in non-human primates synthetic reconstruction of zoonotic and early human severe acute respiratory syndrome coronavirus isolates that produce fatal disease in aged mice a novel model of lethal hendra virus infection in african green monkeys and the effectiveness of ribavirin treatment clinical outcome of henipavirus infection in hamsters is determined by the route and dose of infection recent progress in henipavirus research: molecular biology, genetic diversity, animal models mucosal arenavirus infection of primates can protect them from lethal hemorrhagic fever sars vaccines: where are we? animal models of rift valley fever virus infection characterization of a novel coronavirus associated with severe acute respiratory syndrome macaque model for severe acute respiratory syndrome pathogenesis of aerosolized eastern equine encephalitis virus infection in guinea pigs pathophysiology of hantavirus pulmonary syndrome in rhesus macaques virulence and pathophysiology of the congo basin and west african strains of monkeypox virus in non-human primates arenaviridae. virus taxonomy, viiith report of the international committee on taxonomy of viruses clinical laboratory, virologic, and pathologic changes in hamsters experimentally infected with pirital virus (arenaviridae): a rodent model of lassa fever comparative pathology of north american and central african strains of monkeypox virus in a ground squirrel model of the disease biomedical applications of sheep models: from asthma to vaccines bunyaviruses the use of an animal model to study transmission of influenza virus infection experimental infection of an african dormouse (graphiurus kelleni) with monkeypox virus animal noroviruses human monkeypox infection: a family cluster in the midwestern united states the possibility of using the icr mouse as an animal model to assess antimonkeypox drug efficacy using the ground squirrel (marmota bobak) as an animal model to assess monkeypox drug efficacy experimental inoculation of juvenile rhesus macaques with primate enteric caliciviruses a rodent model of chikungunya virus infection in rag −/− mice, with features of persistence, for vaccine safety evaluation respiratory syncytial virus (rsv) pulmonary infection in humanized mice induces human anti-rsv immune responses and pathology viremia and antibody response of small african and laboratory animals to crimean-congo hemorrhagic fever virus infection early activation of natural killer and b cells in response to primary dengue virus infection in a/j mice murine model for dengue virus-induced lethal disease with increased vascular permeability in vitro and in vivo assay systems for study of influenza virus inhibitors viruses of the bunya-and togaviridae families: potential as bioterrorism agents and means of control potential role of immunomodulators for treatment of phlebovirus infections of animals patient-derived tumor xenografts: transforming clinical samples into mouse models treatment of lethal pichinde virus infections in weanling lvg/lak hamsters with ribavirin, ribamidine, selenazofurin, and ampligen a comparative study of the crimean hemorrhagic fever-congo group of viruses the pathogenesis of rift valley fever virus in the mouse model effective antiviral treatment of systemic orthopoxvirus disease: st- treatment of prairie dogs infected with monkeypox virus a neurotropic virus isolated from the blood of a native uganda comparison of the plaque assay and % tissue culture infectious dose assay as methods for measuring filovirus infectivity experimental respiratory marburg virus haemorrhagic fever infection in the common marmoset experimental respiratory infection of marmosets (callithrix jacchus) with ebola virus kikwit essential role of platelet-activating factor receptor in the pathogenesis of dengue virus infection respiratory syncytial virus is associated with an inflammatory response in lungs and architectural remodeling of lung-draining lymph nodes of newborn lambs trans-activation of viral enhancers by the hepatitis b virus x protein filamentous influenza a virus infection predisposes mice to fatal septicemia following superinfection with streptococcus pneumoniae serotype contributions of mast cells and vasoactive products, leukotrienes and chymase, to dengue virus-induced vascular leakage. elife , e . stabenow correction: a retrospective investigation on canine papillomavirus (cpv ) in oral oncogenesis reveals dogs are not a suitable animal model for high-risk hpv-induced oral cancer clinical profiles associated with influenza disease in the ferret model comparative neurovirulence and tissue tropism of wild-type and attenuated strains of venezuelan equine encephalitis virus administered by aerosol in c h/hen and balb/c mice a mouse model for human anal cancer first reported cases of hantavirus pulmonary syndrome in canada antiviral treatment is more effective than smallpox vaccination upon lethal monkeypox virus infection evaluation of intravenous zanamivir against experimental influenza a (h n ) virus infection in cynomolgus macaques ferrets as a novel animal model for studying human respiratory syncytial virus infections in immunocompetent and immunocompromised hosts differential pathogenesis of respiratory syncytial virus clinical isolates in balb/c mice limited dissemination of pathogenic siv after vaginal challenge of rhesus monkeys immunized with a live, attenuated lentivirus experimental west nile virus infection in rabbits: an alternative model for studying induction of disease and virus control a virus similar to human hepatitis b virus associated with hepatitis and hepatoma in woodchucks development of animal models against emerging coronaviruses: from sars to mers coronavirus severe seasonal influenza in ferrets correlates with reduced interferon and increased il- induction the clinical pathology of crimean-congo hemorrhagic fever human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes nipah virus encephalitis profiles of antibody responses against severe acute respiratory syndrome coronavirus recombinant proteins and their potential use as diagnostic markers distribution of viral antigens and development of lesions in chicken embryos inoculated with nipah virus characterization and demonstration of the value of a lethal mouse model of middle east respiratory syndrome coronavirus infection and disease the origin and virulence of the "spanish" influenza virus the pathology of influenza virus infections isolation of west nile virus from culex mosquitoes recombinant respiratory syncytial virus that does not express the ns or m - protein is highly attenuated and immunogenic in chimpanzees animal models of hepadnavirus-associated hepatocellular carcinoma mouse models for chikungunya virus: deciphering immune mechanisms responsible for disease and pathology human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets experimental infection of ground squirrels (spermophilus tridecemlineatus) with monkeypox virus. emerg persistent west nile virus infection in the golden hamster: studies on its mechanism and possible implications for other flavivirus infections risk factors in dengue shock syndrome breaking barriers to an aids model with macaque-tropic hiv- derivatives dominant role of hpv e in anal carcinogenesis ribavirin efficacy in an in vivo model of crimean-congo hemorrhagic fever virus (cchf) infection histopathologic and immunohistochemical characterization of nipah virus infection in the guinea pig sequence of pathogenic events in cynomolgus macaques infected with aerosolized monkeypox virus severe acute respiratory syndrome coronavirus infection of mice transgenic for the human angiotensin-converting enzyme virus receptor immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus protective efficacy of a bivalent recombinant vesicular stomatitis virus vaccine in the syrian hamster model of lethal ebola virus infection persistence of human norovirus rt-qpcr signals in simulated gastric fluid outbreak of necrotizing enterocolitis caused by norovirus in a neonatal intensive care unit pathology of experimental aerosol zaire ebolavirus infection in rhesus macaques experimental aerosolized guinea pig-adapted zaire ebolavirus (variant: mayinga) causes lethal pneumonia in guinea pigs animal model for the therapy of acquired immunodeficiency syndrome with reverse transcriptase inhibitors a human lung xenograft mouse model of nipah virus infection human and avian influenza viruses target different cells in the lower respiratory tract of humans and other mammals virulence and reduced fitness of simian immunodeficiency virus with the m v mutation in reverse transcriptase chronic administration of tenofovir to rhesus macaques from infancy through adulthood and pregnancy: summary of pharmacokinetics and biological and virological effects use of a small molecule ccr inhibitor in macaques to treat simian immunodeficiency virus infection or prevent simian-human immunodeficiency virus infection experimental infection of rhesus macaques and common marmosets with a european strain of west nile virus protective vaccination against papillomavirus-induced skin tumors under immunocompetent and immunosuppressive conditions: a preclinical study using a natural outbred animal model cataloguing of potential hiv susceptibility factors during the menstrual cycle of pig-tailed macaques by using a systems biology approach temporal analysis of andes virus and sin nombre virus infections of syrian hamsters anti-alpha integrin antibody blocks monocyte/macrophage traffic to the heart and decreases cardiac pathology in a siv infection model of aids clinical manifestations, laboratory findings, and treatment outcomes of sars patients. emerg prevalence of noroviruses and sapoviruses in swine of various ages determined by reverse transcription-pcr and microwell hybridization assays porcine enteric caliciviruses: genetic and antigenic relatedness to human caliciviruses, diagnosis and epidemiology avian influenza viruses, inflammation, and cd (+) development of a model for marburgvirus based on severe-combined immunodeficiency mice development and characterization of a mouse model for marburg hemorrhagic fever euthanasia assessment in ebola virus infected nonhuman primates hematopoietic stem cell-engrafted nod/scid/ il rgamma null mice develop human lymphoid systems and induce long-lasting hiv- infection with specific humoral immune responses the magnitude of dengue virus ns protein secretion is strain dependent and does not correlate with severe pathologies in the mouse infection model human fatal zaire ebolavirus infection is associated with an aberrant innate immunity and with massive lymphocyte apoptosis immunization with modified vaccinia virus ankarabased recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets invasion of the central nervous system in a porcine host by nipah virus development of a rift valley fever virus viremia challenge model in sheep and goats antibody-mediated enhancement of disease in feline infectious peritonitis: comparisons with dengue hemorrhagic fever an unusual hantavirus outbreak in southern argentina: person-to-person transmission? hantavirus pulmonary syndrome study group for patagonia. emerg equine morbillivirus pneumonia: susceptibility of laboratory animals to the virus susceptibility of cats to equine morbillivirus hantaan virus infection causes an acute neurological disease that is fatal in adult laboratory mice a north american h n influenza virus supports reassortment with pandemic h n and induces disease in mice without prior adaptation transmission studies of hendra virus (equine morbillivirus) in fruit bats, horses and cats a guinea-pig model of hendra virus encephalitis susceptibility of hiv- , siv and shiv to various anti-hiv- compounds: implications for treatment and postexposure prophylaxis a golden hamster model for human acute nipah virus infection ebola virus transmission in guinea pigs intranasal immunization with an adenovirus vaccine protects guinea pigs from ebola virus transmission by infected animals characterization and experimental transmission of an oncogenic papillomavirus in female macaques dengue haemorrhagic fever: diagnosis, treatment, prevention and control, second ed. world health organization, geneva. world health organization clinical aspects of hepatitis b virus infection civets are equally susceptible to experimental infection by two different severe acute respiratory syndrome coronavirus isolates evidence of a causal role of winter virus infection during infancy in early childhood asthma vertical transmission of zika virus targeting the radial glial cells affects cortex development of offspring mice the antiviral activity of sp- , a natural polyphenolic polymer, against respiratory syncytial and parainfluenza type viruses in cotton rats experimental infection of prairie dogs with monkeypox virus. emerg epidemiological studies of hemorrhagic fever with renal syndrome: analysis of risk factors and mode of transmission hydrodynamic injection of viral dna: a mouse model of acute hepatitis b virus infection mice transgenic for human angiotensin-converting enzyme provide a model for sars coronavirus infection an animal model of mers produced by infection of rhesus macaques with mers coronavirus a protective role for dengue virus-specific cd + t cells the incubation period of hantavirus pulmonary syndrome animal model of sensorineural hearing loss associated with lassa virus infection the relationship of meteorological conditions to the epidemic activity of respiratory syncytial virus. epidemiol hantavirus pulmonary syndrome. pathogenesis of an emerging infectious disease the pathology of experimental aerosolized monkeypox virus infection in cynomolgus monkeys (macaca fascicularis) hiv- infection and pathogenesis in a novel humanized mouse model h n influenza viruses are transmissible in ferrets by respiratory droplet induction of neutralizing antibodies against four serotypes of dengue viruses by mixbiediii, a tetravalent dengue vaccine rapid generation of a mouse model for middle east respiratory syndrome an animal model for studying the pathogenesis of chikungunya virus infection pathogenesis of avian influenza a (h n ) viruses in ferrets lethal crimean-congo hemorrhagic fever virus infection in interferon alpha/beta receptor knockout mice is associated with high viral loads, proinflammatory responses, and coagulopathy the pathogenesis of western equine encephalitis virus (w.e.e.) in adult hamsters with special reference to the long and short term effects on the c.n.s. of the attenuated clone variant development of a murine model for aerosolized filovirus infection using a panel of bxd recombinant inbred mice a characterization of aerosolized sudan ebolavirus infection in african green monkeys, cynomolgus macaques, and rhesus macaques characterization of disease and pathogenesis following airborne exposure of guinea pigs to filoviruses manuscripts in preparation opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the us army. key: cord- - udq roo authors: green, jennah; coulthard, emma; norrey, john; megson, david; d’cruze, neil title: risky business: live non-cites wildlife uk imports and the potential for infectious diseases date: - - journal: animals (basel) doi: . /ani sha: doc_id: cord_uid: udq roo simple summary: the uk imports wild animals for commercial purposes from countries all across the world. we analyse a database of wildlife records from the uk’s animal and plant health agency (apha) to summarise the volume and variety of non-cites (convention on international trade in endangered species) listed wild animal imports over a recent -year period ( – ). we found that over million individual animals were imported into the uk from countries across nine global regions from – . in terms of volume (semi-domesticated pigeons and game birds aside), amphibians were the most commonly imported group ( %), followed by reptiles ( %), mammals ( %), and birds ( %). the highest number of import records came from europe and africa, but the largest volume of animals came from north america and asia. we review the potential for infectious diseases emerging from these vast and varied wildlife imports and discuss the potential threats they pose to public health. we also draw attention to an observed current lack of detail in the apha database and suggest that better record keeping and reporting could help prevent and manage the introduction of infectious diseases. abstract: international wildlife trade is recognised as a major transmission pathway for the movement of pathogenic organisms around the world. the uk is an active consumer of non-native live wild animals and is therefore subject to the risks posed by pathogen pollution from imported wildlife. here, we characterise a key yet overlooked portion of the uk wildlife import market. we evaluate the trade in live non-cites (convention on international trade in endangered species) wild terrestrial animals entering the uk over a -year period using data reported by the animal and plant health agency (apha). between and , over million individual animals, across five taxonomic classes and taxonomic orders, were imported into the uk from countries across nine global regions. the largest volumes of wild animals were imported from north america and asia, and most of the import records were from europe and africa. excluding columbiformes (pigeons) and galliformes (‘game birds’), amphibians were the most imported taxonomic class ( %), followed by reptiles ( %), mammals ( %), birds ( %), and arachnids (< %). the records described herein provide insight into the scope and scale of non-cites listed wildlife imported in to the uk. we describe the potential for pathogen pollution from these vast and varied wildlife imports and highlight the potential threats they pose to public health. we also draw attention to the lack of detail in the uk wildlife import records, which limits its ability to help prevent and manage introduced infectious diseases. we recommend that improved record keeping and reporting could prove beneficial in this regard. and trade of wildlife specimens, either living organisms or harvested parts there-of" [ ] . illegal trade can be challenging to audit due to the amount of undetected and under-recorded trade that occurs by way of its illicit nature [ ] . here, we focus on legal trade records, which are readily available and immediately manageable [ ] . we acknowledge that records of legal wildlife trade are not without fault. fraudulent activity, inadequate record keeping, and the misidentification and mislabeling of species have all been attributed to accusations for wild animals traded globally [ ] . the convention on international trade in endangered species (cites) trade database hosts a large number of international wildlife trade records, but it only encompasses species that have been afforded different levels or types of protection from over-exploitation (https://trade.cites.org/). as such, species traded under the guidance of cites represent a small fraction of all species that are traded internationally [ , ] . broader records of wildlife trade are kept at regional and country-specific levels (for example the 'united nations statistics division for comtrade' for un member countries and the 'law enforcement management information system' (lemis) of the usa national government [ ] ). these datasets typically enable researchers to quantify the origin, purpose, amount, and diversity of taxonomic groups in commercial traffic, revealing the extraordinary magnitude of wildlife traded in each region [ ] . the 'trade control and expert system' (traces) is an online management tool for all non-cites listed animals imported in to european union (eu) countries. this web-based service is the system used for recording all trade of live animals, germplasm, and other animal-derived commodities into or through member states territories. when animal-based consignments are exported to-or traded within-the eu, traces manages and records the trade route. authorities post relevant documents online through traces, enabling border control authorities to check the consignments and their accompanying certificates to allow them to travel through the eu. in the united kingdom (uk), traces data can currently be accessed via submission of a freedom of information (foi) request to the animal and plant health agency (apha). apha is an executive agency sponsored by the department for environment, food, and rural affairs of the uk government. the aim of apha is to "safeguard animal and plant health for the benefit of people, the environment and the economy" [ ] . they are responsible for facilitating international trade in animals and products of animal origin into and out of the uk, as well as other activities tangentially related to animal disease surveillance and control, such as infectious disease research and the licensing and registration of wildlife. investigating and summarising wildlife trade data have value for scientists and policy makers because of the trade's impact on global biodiversity and conservation, animal welfare, and infectious disease emergence [ ] . understanding wildlife trade requires both the identification of what species are being traded and where trade routes occur [ ] . such data are freely accessible on the traces database, but to our knowledge, no consolidation or assessment of this information for the uk exists. here, we aim to characterise the nature of the uk live terrestrial wildlife import market that is not currently regulated under cites. we obtained data pertaining to all consignments of live non-domesticated animals (excluding cites listed species and all fish) imported into the uk recorded on the traces database, via a freedom of information request to apha. we evaluate the type and volume of species entering the uk over a -year period, with additional focus on the country of export for all species. our aim was to provide an overview of the import data and to highlight some of the potential pathogens associated with taxa commonly imported in to the uk. data on the volume of non-cites listed live wild vertebrates (excluding fish) imported into the uk between and from all other countries was obtained from the uk apha via a freedom of information act (foi) request, which was received on . . (ref: atic ) . available information regarding the lowest available taxonomic status (e.g., species, genus, family, order, or class), reported country of origin, country of export, source type (e.g., wild-caught, captive bred, ranched), and intended purpose (e.g., commercial, zoological, private collection) for all imports was also specifically requested during this process. data on species considered as "domesticated" (i.e., animals that have been controlled and bred for human benefit over many generations, eventually resulting in changes to their genetic makeup and appearance; e.g., cat (felis catus), dog (canis familiaris) and cattle (bos taurus)) were specifically excluded from this information request. fish were also excluded due to the high number of individuals traded, which would alter the relative proportions of taxonomic groups and distort our data. live fish can also be imported for aquaculture, which is a distinct market that would warrant a separate study of its own. the apha dataset consisted of nine columns of information including "country of origin", "country of destination", "commodity", "species", "species list", "species class list", "species family list", "declaration year", and "total number of animals". a total of records were provided in the original apha dataset. information relating to country of origin, country of destination, commodity, declaration year, and total number of animals were complete with no missing data. any suspected duplicate records were removed from the apha dataset by creating a unique code (using data entered for "country of origin", "commodity", "declaration year", and "total number of animals"). this resulted in the removal of records. taxonomic related information was missing for a proportion of the remaining records (species ( %, n = ), species list ( %, n = ), species class list ( %, n = ) and species family list ( %, n = )) and the level of taxonomic detail was inconsistently applied across these data fields. given the level of taxonomic uncertainty amongst the apha records, only taxonomic class and order-level information (provided directly or added from information in the data column "species class list") was used in our analyses. instances where data on taxonomic status were still missing were classed as "not recorded" ( . %, n = ). trade records that referred to multiple taxa were classed separately as "unknown mixed imports" ( . %, n = ). all data relating to the taxa 'columbiformes' (pigeons) and 'galliformes' ('game birds') in the apha database were separated out and analysed separately due to the high volume of this data (which accounted for % of the individual animals imported). it is likely that these trade records related to semi-domesticated bird species such as gamebirds, which were not a major focus of this study. all analysis was carried out in excel, r, and rstudio (r core team, vienna, austria, ). we described the tabulated categorical data using descriptive statistics, including percentages, bar charts, circle plots, and heat maps. chi-squared goodness of fit was used to investigate the distributions of these data across year, taxonomic group, and regions. a pairwise comparison after any significant chi-squared goodness-of-fit test was performed using the package 'rvaidememoire'. figures were produced using ggplot (new york, ny, usa) [ ] . trade diagrams were created using the package "circlize" (heidelberg, germany) [ ] . the apha records show that a total of individually identified wildlife import records were reported by the uk between january and december . the number of annually declared wildlife import records remained relatively constant during the period examined, with a mean average of . (standard deviation (sd) = . ) wildlife records reported per year (appendix a). these import records included a total of , , individual animals. thus, for the period - , an annual mean average of , , (sd = , , ) non-cites listed live animals were imported into the uk as recorded in the apha database. in total, % (n = , , ) of the total individual live animals traded between january and december comprised bird species belonging to the galliforme (c. %, minimum n = , , ) and columbiforme (< % minimum n = ). these data only accounted for % of the actual number of records (n = ). where data existed, references were made to "semi-domesticated" species (e.g., phasianus spp. and perdix spp.), which were presumed to be "gamebirds". the quantities of these imports remained stable across the time period and were nearly always from france (c. . %, minimum n = , , ; appendix b). of the remaining % of records (n = ) (excluding columbiformes and galliformes), data on taxonomic class was not available on records ( . %). however, a total of five different classes were present in the data (figure ). the most frequently traded was "aves" (birds) ( . %, n = ), followed by "mammalia" (mammals) ( . %, n = ), "reptilia" (reptiles) ( %, n = ), "amphibia" (amphibians) ( . %, n = ), and lastly "arachnida" (arachnids) ( . %, n = ). a chi-squared goodness of fit revealed that number of records was not evenly distributed across the classes of organisms traded (x = . , df = , p < . ). a post-hoc pairwise comparison found a significant difference between all classes (p < . ) except aves and mammalia. the volume of wild animals traded was also unevenly distributed (x = , df = , p < . ). a post-hoc pairwise comparison found a significant difference between all classes (p < . ). the data comprised a range of taxonomic orders. aside from columbiformes and galliformes, a total of orders were recorded in the dataset; however, sauria and serpentes were combined to form squamata, incorporating different taxonomic orders. several records referred to an order grouping of "other birds" in the dataset. despite a lack of clarity, this was left in due to a lack of further detail on these records. the taxonomic order most frequently traded was the 'psittaciformes' (parrots) ( . %; n = ), followed by "other birds" ( . %; n = ), "artiodactyla" (even-toed ungulates) ( . %; n = ), "squamata" (lizards and snakes) ( . %; n = ), and "carnivora" ( . %; n = ) (appendix c). in relation to the taxonomic class and the actual volume of wild animals traded (columbiformes and galliformes aside), the highest number of individual non-cites listed live animals entering the uk during this time period were amphibians ( . %, n = , , ), followed by reptiles ( . %, n = , ), mammals ( . %; n = , ), birds ( . %; n = , ), and arachnids ( . %; n = ). a further . % (n = , ) did not have assigned taxonomic data ( figure ). in reference to specific taxa, anura (frogs) were the most frequently reported taxonomic order ( . %, min n = , , ), followed by squamata (snakes and lizards) ( . %, n = , ), testudinata (turtles) ( . %, n = , ), rodentia (rodents) ( . %, n = , ), and psittaciformes (parrots) ( . %, n = , ). a further , individuals ( . %) were recorded as mixed taxonomic imports, and , individuals ( . %) were missing taxonomic data altogether (appendix c). a chi-squared goodness of fit revealed that the number of wildlife records was not evenly distributed across order of organisms traded (x = . , df = , p < . ). a pairwise comparison test found significant differences across many order pairings (p < . ) with anura, artiodactyla, carnivora, aves, psittaciformes, squamata, and testudinata being found to be higher than expected. the volume of individual wild animals traded was also unevenly distributed (x = , , , df = , p < . ). a pairwise comparison test found significant differences across many order pairings (p < . ) with anura, squamata, and testudinata being found to be higher than expected. in reference to specific taxa, anura (frogs) were the most frequently reported taxonomic order ( . %, min n = , , ), followed by squamata (snakes and lizards) ( . %, n = , ), testudinata (turtles) ( . %, n = , ), rodentia (rodents) ( . %, n = , ), and psittaciformes (parrots) ( . %, n = , ). a further , individuals ( . %) were recorded as mixed taxonomic imports, and , individuals ( . %) were missing taxonomic data altogether (appendix c). a chi-squared goodness of fit revealed that the number of wildlife records was not evenly distributed across order of organisms traded (x = . , df = , p < . ). a pairwise comparison test found significant differences across many order pairings (p < . ) with anura, artiodactyla, carnivora, aves, psittaciformes, squamata, and testudinata being found to be higher than expected. the volume of individual wild animals traded was also unevenly distributed (x = , , , df = , p < . ). a pairwise comparison test found significant differences across many order pairings (p < . ) with anura, squamata, and testudinata being found to be higher than expected. animals , , of according to the apha database, the primary regions exporting non-cites listed live wild animals into the uk were europe and africa based on number of records, and north america and asia for the volume of individual wild animals imported ( figure ). between january and december , non-cites listed live wild animals were imported into the uk from nine different regions and from different countries globally ( figure , appendix d). with regard to individually identified wildlife records for imports to the uk, the highest number reported in the apha database were from the czech republic ( . %, n = ), followed by germany ( . %; n = ), belgium ( . %; n = ), france ( . %; n = ), italy ( . %; n = ), the netherlands ( . %; n = ), slovakia ( . %; n = ), spain ( . %; n = ), "other parts of the uk" ( . %; n = ), and the us ( . %; n = ) (appendix d). according to the apha database, the primary regions exporting non-cites listed live wild animals into the uk were europe and africa based on number of records, and north america and asia for the volume of individual wild animals imported ( figure ). between january and december , non-cites listed live wild animals were imported into the uk from nine different regions and from different countries globally ( figure , appendix d). with regard to individually identified wildlife records for imports to the uk, the highest number reported in the apha database were from the czech republic ( . %, n = ), followed by germany ( . %; n = ), belgium ( . %; n = ), france ( . %; n = ), italy ( . %; n = ), the netherlands ( . %; n = ), slovakia ( . %; n = ), spain ( . %; n = ), "other parts of the uk" ( . %; n = ), and the us ( . %; n = ) (appendix d). between and , amphibians were imported into the uk from six global regions ( figure ) and countries (figure , appendix d). the primary exporters of amphibian imports were the us (n = ), indonesia (n = ), and singapore (n = ) based on number of records, and the us and between and , amphibians were imported into the uk from six global regions ( figure ) and countries (figure , appendix d). the primary exporters of amphibian imports were the us (n = ), indonesia (n = ), and singapore (n = ) based on number of records, and the us and singapore based on the total number of specimens/animals imported. there are seven import records from the usa to the uk that involved , , individual frogs, and six from singapore to the uk that involved , individual frogs. all specimens appeared to be anurans (frogs); however, species-specific data was provided for only one record of frogs, relating to the american bullfrog (lithobates catesbeianus) imported from the usa. reptiles were imported from eight regions ( figure ) and countries (figure , appendix d). the primary exporters of reptile imports were the usa (n = ), ghana (n = ), the czech republic (n = ), and germany (n = ) based on the number of records, and the usa, ghana, uzbekistan, and vietnam for the total number of specimens/animals imported. there are import records from the usa to the uk that involved , individual reptiles and from ghana to the uk that involved , individual reptiles. species-specific data were provided for one record of freshwater red-eared slider turtles (trachemys scripta) from the usa. there are import records from uzbekistan to the uk that involved , individual reptiles. mammals were imported from seven different regions ( figure ) and countries (figure , appendix d). the primary exporters of mammal imports were germany (n = ), the netherlands (n = ), the czech republic (n = ), france (n = ), and spain, the czech republic, and italy based on number of records. there are import records from spain to the uk that involved , individual mammals. there are records from the czech republic to the uk that involved , individual mammals. a total of chiropterans (bats) were imported into the uk from madagascar (n = ), guyana (n = ), and the czech republic (n = ). a total of , individual rodentia (rodents) were imported from different countries within europe. a total of individual carnivora (carnivores) were imported from countries across seven different regions. birds (excluding colombiformes and galliformes) were imported from seven regions ( figure ) and countries (figure , appendix d). the primary exporters of bird imports were the czech republic (n = ), belgium (n = ), and germany (n = ) based on the number of records, and the czech republic, slovakia, and italy for number of specimens/animals imported. there are import records from the czech republic to the uk that involved , individual birds and from slovakia to the uk that involved individual birds. a total of , individual psittaciformes (parrots) were imported from countries. a total of , "other birds" were imported from countries across africa, asia, europe, north america, oceania, and south america. the uk is an active consumer of non-native live wild animals. the records described here provide an important insight into the scope and scale of non-cites listed wildlife being imported into the uk. over million individual animals, across five taxonomic classes and taxonomic orders, were imported into the uk during - , with an annual mean average of , , individual animals. these wild animals were exported from different countries across nine different global regions. the international trade of wild animals in such quantities involves the risk of undesired pathogen pollution (i.e., the introduction of pathogenic viruses, bacteria, fungi and parasites into new environments) [ ] , of which the uk is no exception. a number of zoonotic diseases have been identified in taxa that are commonly traded for the uk exotic pet market (appendix e). brief examples of the potential public health risks associated with the ongoing uk import of wild animals belonging to each of these taxonomic groups are summarised below. mammals-the majority of emerging human diseases are thought to originate from mammals [ ] , and as such, their import represents a particularly prominent concern from a public health perspective [ ] . according to the uk data records, at least , mammals were imported to the uk from countries across seven global regions over the five-year study period. these records include taxonomic groups that are associated with serious emerging zoonotic infections. for example, bats (of which individuals were imported into the uk from madagascar, guyana and the czech republic) have been implicated in the transmission of covid- , ebola, hendra, marburg, sars-coronavirus, nipah, and various rabies-related viruses, all of which can cause currently untreatable diseases in people, often with high fatality rates [ ] [ ] [ ] . the uk import records also include rodents (representing % of all uk mammal imports during the study period), which is a taxonomic group that also has the potential to transmit a number of diseases to humans (both directly (e.g., hantavirus pulmonary syndrome, leptospirosis, plague, and rat-bite fever) and indirectly (e.g., cowpox, babesiosis, lyme disease and murine typhus) [ , ] . a recent confirmed case of bubonic plague in mongolia is thought to have originated from contact with a dead marmot [ ] . the potential public health risks associated with the import of rodents were demonstrated by an outbreak of monkeypox in the usa during where a shipment of rodents originating from ghana resulted in an outbreak affecting people, which prompted domestic and international trade restrictions to control transmission [ ] . birds-birds are also susceptible to many diseases common to humans [ ] , and there are previous examples of zoonotic disease emergence from birds imported to the uk. for example, in , cockatoos (cacatua alba, cacatua sulphurea citrinocristata and cacatua sulphure) entering the uk illegally from singapore were found to be infected with psittacosis, which is a zoonotic respiratory infection that causes severe pneumonia in humans and has a fatality rate of up to - % [ ] . wild caught birds are also natural reservoir hosts avian paramyxovirus (which causes newcastle disease in humans). severe outbreaks of newcastle disease have been recorded in parrots imported into the uk [ ] . however, perhaps most notably from a public health perspective, in , the uk government (along with the eu) posed a permanent ban on imports of wild caught live birds to prevent the spread of avian influenza following the discovery of h n -infected birds at a uk quarantine station [ , ] . yet, this particular trade ban did not extend to birds of captive bred origin [ ] , which is demonstrated by the fact that uk import records report that at least , individual parrots were imported from countries in the period - . as such, additional bird-associated diseases in humans including histoplasmosis, q fever, allergic alveolitis, salmonellosis, campylobacteriosis, and giardiasis [ ] associated with captive sourced bird imports remains an ongoing potential public health concern in the uk. reptiles and amphibians-reptiles imported from tropical countries also have a high possibility of carrying potentially dangerous pathogens [ ] . for example, it is thought that reptiles act as vectors for diseases that affect human health (such as q fever and lyme disease) and are responsible for some of the uk's reported human salmonella cases [ ] . in , public health concerns arose when visceral pentastomiasis (caused by armillifer armillatus larvae) was diagnosed in a worker at a snake farm in the gambia [ ] . similar snake farms (which export wild caught and captive reptiles on to the global exotic pet market) are in operation in ghana [ ] , which exported , individual reptiles into the uk between and . in terms of trade volume, amphibians were the most frequently reported taxonomic group. in total, , , amphibians were shipped from countries across six different regions during the study period. from a public health perspective, amphibians have the potential to act as vectors for zoonotic disease transfer; for example, they can be responsible for human cases of infection with aeromonas spp., mycobacterium marinum, and salmonella spp. [ ] , the latter of which cause severe infections in people, particularly children [ ] . although it is not a threat to human health, chytridiomycosis (a highly contagious fungal disease among amphibians thought to have contributed to the decline or extinction of at least amphibian species across six continents [ ] ) was confirmed in a breeding population of bullfrogs in the uk in [ ] . it is important to acknowledge that pathogenic agents can also be transported across geographical boundaries via the natural movement of wild animals, such as migratory species (e.g., some birds and bats). while there are some zoonotic diseases in humans that appear to have been tied to spillovers from migratory species, the majority have instead more likely resulted from human activities, particularly direct contact with wild animals during harvesting and handling, and increased proximity of humans and livestock to natural habitats [ ] . consequently, there are concerns that the amount of contact between wild animals and people throughout the trade chain could place public health at a greater risk of pathogen transmission than natural migratory processes. excluding the potentially semi-domesticated colombiformes and galliformes exported from europe, the largest volume of wild animals came from north america and asia, with the greatest number of records from europe and africa. in terms of volume, the majority of the wild animals imported into the uk were amphibians ( %), many of which were presumably intended for use as exotic pets. the aquatic nature of such species presents a particular challenge in terms of mitigating the risks of zoonotic disease introduction, as water is an effective transmission medium for pathogenic organisms, and survival outside the host may be for a significant duration [ ] . in addition, considerations on how to responsibly dispose of any water used in shipments must also be taken into account when dealing in aquatic specimens [ ] . many of the wild animals that arrived into the uk were also exported from regions that have been identified as emerging disease hotspots-for example, in tropical lower latitude areas of africa, latin america, and asia [ ] . in particular, the uk import data show that several countries from these regions, such as el salvador, nicaragua, cameroon, madagascar, singapore, and indonesia, exported large volumes of reptiles and amphibians to the uk annually, although some also exported species from other taxonomic groups (see figure and appendix f). additionally, importing wild animals from other regions such as europe is also not without risk. zoonotic disease emergence is closely associated with regions of greater mammal biodiversity [ ] , and europe has been identified as a hotspot for mammal zoonosis [ ] . these are important considerations given that the uk imports large volumes of mammals from countries in mainland europe such as the czech republic, italy, and spain. furthermore, while most current data implicate mammals as the primary zoonotic infection reservoir, a substantial reservoir of human pathogens also exist in non-mammalian animals in trade. the potential significance of disease emergence from non-mammalian reservoirs should not be discounted, and further research should address the under-representation of data for non-mammalian sources. despite its recognised role as a major transmission pathway for pathogenic organisms, the majority of regulatory oversight of the international wildlife trade (e.g., cites) has no focus on preventing zoonotic disease introduction and no authority for biosecurity regulation [ , ] . yet, research has shown that international trade agreements could be an effective way to manage zoonotic disease risk by limiting the amount of contact between humans and animals [ ] . in lieu of establishing an additional international treaty specifically to address pathogen transmission, cites is arguably well placed to adapt and incorporate disease spread via wildlife trade in its international remit [ ] . furthermore, in general, there is a lack of surveillance for key animal diseases in most countries, and minimal health monitoring systems exist surrounding the trade of some wild animals, heightening the potential risk for transboundary disease movement [ ] . to address the problem of emerging infectious diseases arising from wild animal pathogens, the world organisation for animal health (oie) considers wild animal translocation as a particularly high-risk activity and advocates a prevention-led approach as part of a four-stage strategy [ ] . in light of the high volume and diversity of non-native live wild animals being imported into the uk from across the globe, it is perhaps logical that prevention should also be prioritised with regard to pathogenic pollution. currently, biosecurity measures (including risk assessments, border controls, and risk-based post-border surveillance) are in place in the uk to ensure that live animals (and animal products) entering the uk from third (i.e., non-eu) countries are safe and meet the specific animal and public health required conditions for import [ ] . with regard to live animals from third (i.e., non-eu) countries, imports must ( ) come from an approved establishment in an approved country; ( ) be accompanied by agreed animal and public health certification as appropriate; and ( ) enter the eu at an approved border control post where checks are carried out to ensure that the consignment meets import conditions [ ] . these animal consignments can then only enter the uk once they have undergone veterinary checks with satisfactory results and a common veterinary entry document (cved) has been issued clearing the consignment as acceptable for import [ ] . for live animal consignments from eu member states, at the time of writing, the animals must comply with an intra-community trade health certificate and welfare transport assurances, they need to have undergone a veterinary health check prior to moving, and they may be subject to post movement checks at destination in the uk but do not need to enter via a border control post. a lack of detail in the uk import records provided by the apha limits the extent to which these data can be used to describe the scope, scale, and dynamics of this trade activity. for example, with regard to the taxonomic status of wildlife imports, a total of , individuals ( . %) were recorded as 'mixed taxonomic import', and for , individuals ( . %), no information was provided. this meant that it was not possible to determine total import quantities for some taxa, which is something of particular concern when trying to quantify the scale of potential zoonotic transmission threats. furthermore, although taxonomic status was recorded by apha using a 'commodity' number code, these appeared to be limited to order level. most ( %) of data records that did provide more detailed information described taxonomic status to the family level, but only % of records did so to the species level. in addition, the data record entries also do not include other important information such as the country of origin (rather, only country of export was included), the import purpose (e.g., whether the imports were intended for private ownership, commercial use, or zoological purposes) or the source of the animals (e.g., whether specimens were wild caught, ranched, captive born, or captive bred-all of which carry some degree of zoonotic disease risk [ ] ). one further point of note is that columns of the data were in some cases incorrectly titled (e.g., species order was under a column 'species class list'), and some orders also appeared to have been grouped (e.g., 'other birds') for no discernible reason. in addition to reducing the possible applications of the data, these inaccuracies may in turn lead to transcription errors or missing data due to lack of clarity. we intentionally chose to exclude cites listed species from this analysis, as we specifically wanted to focus on non-listed species given concerns that they may be subject to less scrutiny (e.g., [ ] ). by omitting cites listed taxa, there will undoubtedly be species and associated pathogenic organisms that pose a risk to public health, and/or are regularly imported to the uk, that we do no describe or discuss here. for example, our dataset does not include any primates because all primates are listed on cites appendices. yet, there are recent news reports expressing concern for the number of primates entering the uk and their potential to spread disease to humans [ ] . this highlights that while this study provides an important summary of infectious pathogens potentially entering the uk, it is not a complete inventory of pathogen risk. similarly, we did not attempt to quantify any illegal uk imports or any subsequent seizures of wildlife and remained focused on legal trade only. nor did we focus on domesticated animal imports, or imports of fish, which can also be responsible for the transmission of human infectious diseases of zoonotic origin [ ] . this study was further limited to uk import records provided by the apha only and does not include a comparison with records held by exporting countries or the eu's traces online management tool. discrepancies between export and import data in trade databases occur e.g., [ ] and may arise for a number of legitimate reasons [ ] . more generally, it is also recognised that wildlife trade records can be error-prone, incomplete, and characterised by a degree of uncertainty (e.g., [ , ] ). therefore, there is a risk that our analysis may be an under-or overestimate and may have missed some pertinent details that would have been recorded elsewhere. despite these limitations, to our knowledge, this is the most comprehensive report of non-cites listed wildlife trade importation for this time period and of this scale. finally, it was beyond the remit of this study to include a detailed or systematic review of pathogen spillover potential. future studies would benefit from including a meta-analysis or other form of quantitative review of pathogens associated with imported animals, or a specific inventory of pathogens associated with import taxa of concern. although biosecurity protocols can help to lower the risk of zoonotic disease introduction, effective surveillance and control is hindered by the wide variety of species involved and the often-complex natural history of zoonotic agents [ ] . consequently, there are concerns that the current global approach to surveillance is inadequate for some wildlife diseases [ ] , and the large volumes of wildlife imported likely render it challenging and costly to effectively screen all individuals, even if it were technically possible to do so. for example, the parasitic tapeworm echinococcus multilocularis was incidentally spread to the uk via imported european beavers from germany in [ ] . the case was particularly concerning because the infected beaver had spent six months in quarantine [ ] , where biosecurity measures failed to identify this risk to public health. the infection was contained, but mainland europe remains a high-risk region for potential future transmission of this parasite to the uk, which is the causative agent of alveolar echinococcosis (ae) [ ] . it has been suggested that efforts to decrease contact between wild animals and people could prove to be the most practical and cost-effective approach in reducing the global human health threat posed by zoonotic diseases [ ] . consequently, from a policy perspective, trade bans have been proposed as a tool to help achieve these threat reduction goals [ ] . there are concerns that wildlife trade bans could have unintended negative consequences on both wildlife and economically vulnerable communities if they are implemented in a manner that fails to also adequately address aspects such as consumer demand, enforcement, and livelihood dependencies [ ] . however, the impacts of a global pandemic can also have far-reaching negative impacts on livelihoods and economies, which is a point brought into sharp relief by the coronavirus covid- pandemic [ ] [ ] [ ] . the potential merits and inferiorities of trade bans aside, given that the import of non-native wild animals remains an ongoing phenomenon, it is imperative that disease monitoring and surveillance efforts are maximised, with particular attention to species imported from regions identified as zoonotic 'hotspots' [ , ] . our study serves to highlight that efforts to prevent, detect, and eradicate zoonotic diseases associated with the import of live non-native wildlife into the uk could be aided by more detailed and extensive record keeping by the ahpa. as such, we recommend the inclusion of additional data as currently recorded in the cites trade database (e.g., taxonomic status (ideally to species level), country of origin, source, and purpose) and improved record keeping will ensure that data records are as complete and informative as possible. health issues at the human-animal-environment interface cannot be effectively addressed by one sector alone [ ] . collaboration across all sectors and disciplines responsible for health is required to address zoonotic diseases and other shared health threats at the human-animal-environment interface via an approach often referred to as "one health" [ ] . in the uk, the current regulatory environment comprises several different governmental departments and agencies that are responsible for different and overlapping aspects of zoonotic disease prevention and control (e.g., public health england's national infections service (nis), public health wales' communicable disease surveillance centre (cdsc), defra's apha (which also provides services to the welsh and scottish governments), to name but a few). although important and informative data are no doubt held elsewhere (e.g., under the auspices of other relevant government agencies), given the important role of wildlife trade as a transmission mechanism for zoonotic disease, the ahpa data records relating to non-cites listed species represent a valuable source of information that could help efforts to address zoonotic diseases. such improved record keeping and reporting would also aid ongoing efforts to better understand and limit the other unwanted negative impacts associated with this type of trade activity. for example, future areas of study could include associated negative animal welfare impacts (e.g., whether transport conditions meet minimum species-specific animal welfare criteria), conservation concerns (e.g., whether unsustainable numbers are being harvested from species with declining populations), and legal criteria (e.g., whether wildlife imports are fully compliant with relevant international and domestic trade legislation). the authors declare no conflict of interest. animals , , of the other unwanted negative impacts associated with this type of trade activity. for example, future areas of study could include associated negative animal welfare impacts (e.g., whether transport conditions meet minimum species-specific animal welfare criteria), conservation concerns (e.g., whether unsustainable numbers are being harvested from species with declining populations), and legal criteria (e.g., whether wildlife imports are fully compliant with relevant international and domestic trade legislation). campylobacteriosis *; endemic relapsing fever; gastroenteritis mycobacteriosis/tuberculosis; salmonellosis *; streptococcosis *; yersiniosis coccidiomycosis; cryptococcosis; septicaemia reptiles campylobacteriosis *; endemic relapsing fever; gastroenteritis birds campylobacteriosis *; gastroenteritis mycobacteriosis/tuberculosis; salmonellosis septicaemia/general infection; pneumonia psittacosis *; q-fever *; vibriosis; leptospirosis *; western encephalitis; avian influenza *; newcastle disease; cryptococcosis; septicaemia; histoplasmosis mammals campylobacteriosis *; endemic relapsing fever mycobacteriosis/tuberculosis; salmonellosis; yersiniosis; septicaemia/general infection brucellosis leptospirosis *; hepatitis-a; west nile virus; herpesvirus simiae-b; monkeypox molloscum contagiosum; measles; rabies *; haemorrhagic fever cowpox *; coccidiomycosis; streptothricosis *; candidiasis supplying the wildlife trade as a livelihood strategy in a biodiversity hotspot summarizing us wildlife trade with an eye toward assessing the risk of infectious disease introduction illegal wildlife trade: scale, processes, and governance reducing the risks of the wildlife trade united states wildlife and wildlife product imports from wildlife trade and global disease emergence a deadly amphibian disease goes global global trends in emerging infectious diseases covid- infection: origin, transmission, and characteristics of human coronaviruses global wildlife trade permeates the tree of life dealing in deadly pathogens: taking stock of the legal trade in live wildlife and potential risks to human health rough trade: animal welfare in the global wildlife trade reducing the primate pet trade: actions for primatologists exotic pet suitability: understanding some problems and using a labeling system to aid animal welfare, environment, and consumer protection reptiles, amphibians, and human salmonella infection: a population-based, case-control study information could reduce consumer demand for exotic pets global trade in exotic pets when pets become pests: the role of the exotic pet trade in producing invasive vertebrate animals global wildlife trade across the tree of life elegant graphics for data analysis circlize implements and enhances circular visualization in r global patterns of zoonotic disease in mammals host and viral traits predict zoonotic spillover from mammals the many estimates of the covid- case fatality rate a framework for the study of zoonotic disease emergence and its drivers: spillover of bat pathogens as a case study probable pangolin origin of sars-cov- associated with the covid- outbreak list of zoonotic diseases bubonic plague: inner mongolia takes precautions after case bacterial pathogens in wild birds: a review of the frequency and effects of infection summarizing the evidence on the international trade in illegal wildlife wild animals as reservoirs of infectious diseases in the uk polly can make you sick: pet bird-associated diseases ticks imported to europe with exotic reptiles future of keeping pet reptiles and amphibians: towards integrating animal welfare, human health and environmental sustainability transmission of armillifer armillatus ova at snake farm, the gambia snakes and ladders: a review of ball python production in west africa for the global pet market animal movements and the spread of infectious diseases preventing the next pandemic: zoonotic diseases and how to break the chain of transmission; united nations environment programme and international livestock research institute: niarobi ecopathology of ranaviruses infecting amphibians global hotspots and correlates of emerging zoonotic diseases wildlife disease surveillance focus group. rigorous wildlife disease surveillance cites and the zoonotic disease content in international wildlife trade how we can use the cites wildlife trade agreement to help prevent pandemics wildlife hosts for oie-listed diseases: considerations regarding global wildlife trade and host-pathogen relationships training manual on wildlife diseases and surveillance; world organisation for animal health description of the uk system of controls on imports of live animals and products of animal origin and evaluation of its performance to protect public and animal health primates imported to uk for laboratory experiments triple in a year to . the independent domesticated animals and human infectious diseases of zoonotic origins: domestication time matters a guide to using the cites trade database discrepancies in reported levels of international wildlife trade zoonotic disease surveillance-inventory of systems integrating human and animal disease information surveillance for emerging biodiversity diseases of wildlife parasite zoonoses and wildlife: one health, spillover and human activity echinococcus multilocularis in an imported captive european beaver (castor fiber) in great britain simulating control of a focal wildlife outbreak of echinococcus multilocularis the extent of covid- pandemic socio-economic impact on global poverty. a global integrative multidisciplinary review lives and livelihoods: estimates of the global mortality and poverty effects of the covid- pandemic economic effects of coronavirus outbreak (covid- ) on the world economy one health approach: a tripartite guide to addressing zoonotic diseases in countries; world health organization (who) world organisation for animal health (oie): geneve a review of captive exotic animal-linked zoonoses fish-borne parasitic zoonoses: status and issues infectious diseases transmissible from animals to humans microbial zoonoses and sapronoses springer science & business media: berlin/heidenberg companion animal zoonoses table a . example zoonotic diseases associated with taxonomic groups. information obtained from warwick et al. [ ] and references within [ ] [ ] [ ] [ ] [ ] [ ] . terms marked with an asterisk represent * diseases identified as found in the uk by public health england [ ] . key: cord- - sle d j authors: castillo-huitrón, nathalia m.; naranjo, eduardo j.; santos-fita, dídac; estrada-lugo, erin title: the importance of human emotions for wildlife conservation date: - - journal: front psychol doi: . /fpsyg. . sha: doc_id: cord_uid: sle d j animals have always been important for human life due to the ecological, cultural, and economic functions that they represent. this has allowed building several kinds of relationships that have promoted different emotions in human societies. the objective of this review was to identify the main emotions that humans show toward wildlife species and the impact of such emotions on animal population management. we reviewed academic databases to identify previous studies on this topic worldwide. an analysis of the emotions on wildlife and factors causing them is described in this study. we identified a controversy about these emotions. large predators such as wolves, coyotes, bears, big felids, and reptiles, such as snakes and geckos, promote mainly anger, fear, and disgust. this is likely due to the perceptions, beliefs, and experiences that societies have historically built around them. however, in some social groups these animals have promoted emotions such as happiness due to their values for people. likewise, sadness is an emotion expressed for the threatening situations that animals are currently facing. furthermore, we associated the conservation status of wildlife species identified in the study with human emotions to discuss their relevance for emerging conservation strategies, particularly focused on endangered species promoting ambiguous emotions in different social groups. since our origins, wildlife has always had a very important role in human life. the very diverse and continuous human-wildlife interactions can be seen from three main perspectives: ( ) utilitarian, in which wild species provide goods for human well-being, such as food, clothing, transport, tools, raw materials, and companionship, among others; ( ) affective, where human beings feel sympathy, admiration, and respect for animals because of religious, mystical, or philosophical reasons (kellert et al., ) , which has greatly contributed to cultural development worldwide (herzog and galvin, ; alves, ) ; and ( ) conflictive, because of the real or potential damage that wild species may inflict on people and their interests (e.g., attacks on humans, livestock predation, damage on crops, and infrastructure, among others; lescureux and linnell, ) . human-wildlife conflicts have motivated animal killings for centuries, which in many cases continue nowadays (woodroffe, ) . human-wildlife relationships have relied on the uses, values, and meanings that animals represent for people through time and space in different cultures (driscoll, ; . societies have developed a cultural predisposition for emotional reactions toward wild animals (kellert and wilson, ) , causing either positive or negative effects depending on the species (york and longo, ) . fear, anger, and disgust are emotions generating attitudes and behaviors against the presence of some species (fritts et al., ; jacobs, ) . in contrast, emotions, such as happiness, which comes out when cherished species are seen in a given place, or sadness before the vulnerability of others, may generate positive attitudes for their conservation (prinz, ) . this relationship between human emotions and attitudes has an effect on the presence, absence, and recovery of wildlife populations (herzog and burghardt, ) . understanding the transcendence of the emotional factors triggered by animals on human beings would improve our knowledge on the human dimensions of wildlife conservation. in this paper, we offer an overview of the influence that emotions have had on the relationships between wildlife and people through time. a substantial amount of the literature reviewed consists of studies conducted on large carnivores in europe, such as the brown bear (ursus arctos) and the wolf (canis lupus), as well as on snakes around the world. we analyze and discuss relevant aspects that could be considered in further studies on threatened and culturally relevant animal species across latin american countries. darwin ( ) recognized that emotions are manifested by all persons throughout their lifetime, but they vary in an individual between different moments of its life span. frijda and mesquita ( ) mentioned the main points characterizing the emotions in their theoretical perspective: ( ) emotions are considered individual responses to relevant events producing feelings of pleasure or pain; ( ) they help to find solutions to concerns that cannot be treated routinely; ( ) they are always about something, they are used to accept or decline the interaction with a real or imagined object, person, or wild animal in this case; ( ) they tend to control behaviors and thoughts (e.g., angry impulses, behaviors, and thoughts); and ( ) emotions are correlated with psychological, physiological, and social components establishing, changing, or maintaining a particular relationship with a specific object in a concrete situation. there is a wide array of studies analyzing human emotions, their origins, functions, and presence in human life (ekman, ; plutchik, a; nummenmaa et al., , among many others) . six basic emotions have been proposed: happiness, surprise, disgust, anger, fear, and sadness (ekman et al., ) . izard ( ) suggested classifying emotions into two groups: "positive, " representing interest and joy (happiness and surprise), and "negative, " including anger, disgust, fear, and sadness. this classification is an artifact of traditional psychology not informed about an evolutionary approach. here, we have focused on basic emotions to explain human-wildlife relationships because secondary emotions (the combination of basic emotions) are more useful for assessing social relationships among human beings (harelli and parkinson, ) . in this review, we consider two different approaches to explain the origins of basic emotions aiming to understand human-wildlife relationships through time. the first is the evolutionary approach, which suggests that emotions have evolved to solve adaptive problems in different environments (plutchik, b) , such as social communication, reproduction processes, and mechanisms for information processing leading to behavioral responses to specific events or objects (al-shawaf et al., ) . predator presence could have been one of such events contributing to the evolution of human emotions and the development of physiological, psychological, and morphological responses for survival (Öhman and mineka, ; prokop and randler, ) . in particular, fear and disgust are adaptive emotions helping to react toward something representing a risk for human life (ekman and cordaro, ) . fear and disgust, for instance, have been the most studied emotions due to their implications for human survival since the origin of our species . fear probably was a defense mechanism against dangerous animals, particularly large predators (Öhman, ; dalgleish, ) . it is believed that potential alert signals emitted by human groups facing predators, with whom they coexisted and sometimes competed for space, water, prey, and other resources, triggered physiological reactions such as heart rate increase, profuse sweating, and pupil dilation, allowing the generation of alert responses. in that way, human beings have historically developed greater awareness toward potentially perilous animals, such as snakes and spiders Öhman and mineka, ; lobue and deloache, ) . this adaptation mediated by fear has probably been genetically fixed throughout generations, provoking the innate physiological responses mentioned above when dangerous species are or could be present (Öhman, ) . the amygdala is the brain region where fear-generating stimuli are processed into a strong reaction that in some cases may affect human vision (phelps et al., ) . on the other side, disgust can help protect the individual against infections and disease (curtis et al., ) . disgust is saved in memory to avoid future exposure to the subject, in this case with potentially threatening animals (al-shawaf et al., ) . the second approach explaining the origins of basic emotions is the cultural context, where people integrate their physical environment with individual and collective experiences, perceptions, meanings, attitudes, and animal-related traditions to construct emotional diversification (prinz, ; johansson et al., ) . in this view, it can be said that human emotions associated with wildlife have evolved over time and continue to be gradually built and rooted in our societies all over the world. under the cultural context approach, emotions can be understood on two levels: ( ) the individual level, involving meanings, beliefs, attitudes, and behaviors based on personal experiences, knowledge, and perceptions, and ( ) the social level, where emotions are determined by collective factors such as experiences, meanings, beliefs, and myths typical of a certain region or culture, which are transmitted among individuals throughout generations (ekman, ; prinz, ) . physical characteristics of wildlife species and their "personalities" created by humans have generated a variety of emotions (kellert et al., ; kruuk, ; prokop and randler, ) . emotions such as fear and anger may be induced by predators that are bigger and heavier than persons, as in the case of large carnivores (e.g., bears, wolves, and big cats) (røskaft et al., ) or by those species unattractive for most people, like worms, small carnivores, bats, and reptiles, which are often perceived as harmful (knight, ; prokop and tunnicliffe, ; prokop et al., ) . in contrast, beloved animals such as colorful birds or small herbivore mammals (e.g., rabbits) may cause happiness providing they are not noxious for people or their livelihoods (prokop and kubiatko, ) . however, these animals are sometimes perceived in different ways. for some social groups (e.g., farmers), small mammals such as rabbits as rodents may represent a threat due the damage they can inflict on crops, cattle, properties, and human health (morzillo and merting, ; breed and moore, ) . actual or potential damage can promote negative attitudes motivated by emotions of anger, disgust, and fear. animal body shape is another physical feature that has been found to be important for the expression of emotions such as fear and disgust. in the case of class reptilia, two groups could be recognized by people according with their similar morphotype (with legs and legless). reptiles with legs (lizard, turtle, and crocodile) tend to cause fear in many people; crocodiles, specially generate intense fear in many people, in part because of the number of attacks occurring worldwide (crocbite, ) . in contrast, legless reptiles (e.g., amphisbaenia and larutia) that have thin bodies, smooth textures, small eyes, and dull colorations generate disgust rádlová et al., ) . specifically, snakes have long bodies, scales with contrasting patterns, bright coloration, and silent, rolling movements that immediately calls up human attention deloache, , ; rádlová et al., ) . it is likely that both fear and disgust can be simultaneously felt by a person observing a particular species . the ample diversity of snakes around the world makes it difficult to generalize emotions across cultures toward different taxa. species coloration has been an attribute to help identify dangerous animals (prokop and fančovičová, ) , allowing emotional responses in human beings (Öhman, ) . striking color ("aposematic") combinations such as bright red and black in some snakes and spiders intensify fearful reactions (Öhman and mineka, ; lobue and deloache, ; . on the other hand, it has been reported that striking coloration allowed perceiving snakes as beautiful animals in spite they are fearsome . it is noteworthy that aposematic species are simultaneously fearsome and attractive particularly for young persons between and years of age, promoting their interest in those animals (prokop and fančovičová, ) . on the other hand, animals' coloration could be attractive for humans and motivate "positive" feelings. in this sense, lišková et al. ( ) discovered that hues of blue and green in birds of the pittidae family promote human preference. psychologists have found that green is usually associated with happiness, relaxation, and comfort because it is related to nature, while blue elicit happiness, relaxation, and peacefulness, among other feelings (kaya and epps, ) . however, human affection for birds also represents a pressure for wild populations, especially for those charismatic species used as pets, promoting illegal trade (alves et al., ) . feeding habits of species may also influence emotions: large predators are usually regarded as hazardous and fearsome, while their prey provoke sadness (prokop and kubiatko, ) . large herbivores and omnivores in some places are often seen as less fearsome than strict carnivores. this is the case of the mainly vegetarian brown bear (ursus arctos) in some regions of europe (lescureux and linnell, ) . however, in other areas and cultures, large herbivores such as elephants (loxodonta africana) cause intense emotions of anger and fear because of the damage they inflict on crops and rural villages (lamarque et al., ) . although "dangerous" animals promote the attention of people (prokop and randler, ) , it is interesting to note that human emotions may vary depending on the life stage of the animal. for example, jaguar (panthera onca) cubs and lion (panthera leo) cubs are perceived as lovely and safe animals given their physical features, causing minor concern in societies, while adult jaguars and lions are generally considered less attractive and very dangerous, promoting fear (knight, ) . this trend is also reported for amphibians, for which people show more disgust toward the adult stage than for tadpoles (prokop and fančovičová, ) . venom in animal species is one of the most remarkable features triggering fear across cultural groups. as a consequence, snakes constitute an interesting case study in which most species produce fear all over the world, although particular species are in fact perceived as beneficial due to their role as controllers of agricultural pests, producing positive feelings in local farmers (ballouard et al., ) . in this regard, ballouard et al. ( ) observed different intensities of fear toward selected snake groups (cobras, vipers, and boas) depending on the nationality and cultural background of their interviewees. animal activity patterns constitute one more physical factor influencing human emotions toward wildlife. humans are not adapted for living in the darkness; they have a poor vision to act in this kind of environment, hence they may associate nocturnal species such as felines, some snakes, rodents, and bats with danger (buss, ) . in addition, these animals historically have been linked to "evil forces" damaging human beings worldwide (prokop et al., ) . contrastingly, many diurnal species (e.g., most of the birds and ungulates) are usually related to positive values such as peacefulness and wisdom that have inspired leaders and rulers to make better decisions (cano-contreras, ). physical characteristics have been useful to classify animals depending on the emotions they produce on people. in this sense, tarantulas, snakes, sharks, and mosquitoes have been categorized as perilous, generating agonistic emotions. contrastingly, large, charismatic species that have traditionally been regarded as dangerous but intelligent at the same time motivate emotions that may result in actions for their protection, as it has occurred for lions (panthera leo), tigers (panthera tigris), leopards (panthera pardus), and polar bears (ursus maritimus) (driscoll, ; landová et al., ) . these categories have emerged after the anthropomorphization of animals, a process in which cultural groups attribute human features and "personalities" to wildlife species (kruuk, ) . for instance, the panda bear (ailuropoda melanoleuca) inspires tenderness and happiness when it is observed, but those emotions are overcome by sadness after considering its high vulnerability to extinction. in this case, positive attributes facilitate particular species to become flagships for wildlife conservation (root-bernstein et al., ) . in rural communities where people frequently interact with wildlife, knowledge about the behavior of culturally relevant species develops better than in other areas. this facilitates the anthropomorphization of certain animals calling them "shy, " "noxious, " and "monstrous, " among other adjectives, which intensifies fear and rejection toward them (lescureux and linnell, ) . furthermore, if the presence of an animal implies economic losses for residents of a community, their predominant perception will be negative and will produce anger that may end in lethal management (naughton-treves, ). contrastingly, animals inspiring greatness and qualified as "kings" of the wilderness will likely motivate local people to feel happiness and pride because of their presence in the region (lescureux and linnell, ) . these examples help identifying the relevance of animal physical features in emotions, which transform throughout history according to the natural, social, and economic context of each human generation. in some cases, emotions produce attitudes against the conservation of unpopular species (knight, ) . therefore, we propose to highlight the ecological role of dangerous or disgusting species as a potential way to mitigate negative emotions toward them. emotions induced by wildlife differ among individuals according to variables such as their sex, age, cultural and natural environment, and perceived vulnerability to each species (johansson et al., ) . it has been shown that young children (under years of age) of both sexes take more time to detect a snake and react toward it than their parents (lobue and deloache, ). that behavior was explained by deloache and lobue ( ), proposing that fear and alert signals in front of this kind of animals develop later, when individuals start to explore their environment and link adult behaviors with animal species. fear and disgust have been the most studied emotions between genders. in general, women tend to express stronger negative emotions (fear and disgust) toward invertebrates, amphibians, predatory mammals like bears, wolves, lynx (lynx lynx), and wolverine (gulo gulo) and toward snakes compared to men (Öhman and mineka, ; røskaft et al., ; ballouard et al., ; bajwa et al., ; . this difference seems to be related to the female gender role taken since the start of human evolutionary history, where men developed skills for both hunting and escaping from predators (prokop and fančovičová, ) . likewise, men gradually reduced their fear of large animals, while women kept distance from those species in part because of their household activities and their care for children in safer places (røskaft et al., ; prokop et al., ) . however, differences within genders are usually present in different cultural and geographic contexts (kellert and berry, ; bjerke et al., ; de pinho et al., ) . in some societies, women, particularly adolescents, have a greater disposition to spend more time in wildlife related activities as compared to men (e.g., volunteer programs; kidd and kidd, ) . this information could be useful to direct conservation programs in spaces as zoos where experiences with uncharismatic and endanger animals could help to promote positive emotions and attitudes. age is a significant variable determining the presence and intensity of agonistic emotions toward animals, which may be related to personal experiences. childhood is the critical life stage when fear of predators starts and when attitudes and behaviors to avoid encounters with them develop (Öhman, ) . it is likely that fear of predators intensifies with learning from parents, given that as the child gets older, his/her reactions become faster when facing species such as snakes (lobue and deloache, ) . in this regard, fear of animals may either decrease (kaltenborn et al., ) or increase (røskaft et al., ) with age. besides age, the natural and cultural environments in which an individual grows determine the knowledge, perceptions, and emotions related to animals (frynta et al., ) . for a person raised in close contact with nature, an encounter with a wild animal can induce happiness, while the same species may produce fear in an individual that has always lived far away from natural spaces (kellert, ; manfredo, ; almarcha, ) . the presence or absence of different species in human territories has a role in the generation of emotions. residents of rural areas who frequently interact with wildlife are usually less fearful of animals than city dwellers. this is because closeness with native animals promotes knowledge about their ecology and behavior, allowing for building better management strategies and reactions toward them (røskaft et al., ) . likewise, recreational activities involving contact with wildlife such as hiking, bird watching, fishing, and hunting have direct influence in emotions, facilitating the overcoming of fears and phobias by promoting learning through first-hand experiences, although in some cases, these activities decrease with age (bjerke et al., ; røskaft et al., ; prokop et al., ) . in particular, emotions produced by hunting deserve further discussion. subsistence hunting as a traditional practice in many rural areas of the world usually involves local regulations to avoid overexploitation and feelings of respect by the hunters toward their prey (e.g., santos-fita et al., ) . in contrast, sport hunting is more focused on the pleasure of the hunter for finding and killing his target species, which has been a motive social dispute in different contexts, generating anger in broad sectors of society considering this an unacceptable practice (nelson et al., ) . some of these recreational activities involve parents and their children, who get used to those practices at an early age (amiot and bastian, ) . this can be an important inter-generational strategy to avoid negative attitudes toward fearsome and disgusting animals and promote positive emotions (i.e., happiness and surprise), especially in areas where humanwildlife conflicts may arise. significant differences have been found among people with different levels of study with respect to fear of wildlife species: individuals with higher levels of education are generally less fearful of wild animals than those with lower degrees of studies (røskaft et al., ) . it is likely that individuals with higher education had more opportunities to receive information on the environment and wild animals in particular, which may have reduced their negative prejudices and perceptions about non-charismatic species, maximizing their perspectives on the ecological benefits provided by those animals. the geographic space where an event occurs triggers distinct emotions, which have varied according to the lifestyles of societies (mesquita and frijda, ) . this argument could be used to understand emotions historically induced by wildlife, considering the different worldviews of each culture. for example, snakes were regarded as deities in mesoamerican cultures, including quetzalcoatl or kukulkan (the feathered serpent), which was the most important deity for the aztecs and the maya, respectively (díaz, ) . snakes were also given high rankings among the deities of the ancient greek, egyptian, hindu, and roman civilizations, where some of these reptiles were associated with values of wisdom, justice, and power (stanley, ; al-rawi, ) . these reptiles have also starred countless stories and myths around the world (ménez, ) , but for christians, muslims, and jews, snakes have traditionally represented evil and death (gonzález, ; al-rawi, ) . nowadays, myths about the damage caused by snakes are important elements to promote and intensify fear in rural communities (fita et al., ) . the social fear could be learned, inherited, and used by societies across generations, driving particular attitudes toward wild species (Öhman, ) . in this case, the relevant ecological role of snakes as predators and pest controllers has been largely neglected. another interesting example is that of wolves, which have been protagonists of many stories and myths worldwide. these carnivores have traditionally been portrayed as fearsome and dangerous animals, producing social rejection in most areas where they are present, nonetheless, in particular cases such as that of ancient rome (whose founders were suckled by a shewolf) and that of native north american cultures, for whom wolves were spiritual symbols related to power and intelligence (fritts et al., ; prokop et al., ) . beyond mythology, other elements that have facilitated the development of cultures (e.g., art, literature, symbolism, religion) have had their foundations in the relationships between humans and wildlife, involving emotions promoting respect and admiration (fritts et al., ; alves, ; almarcha, ) . these emotions frequently lead to attitudes favorable for animal care and conservation. other events that have always happened, but which have received special attention in recent decades because of the human population growth and expansion, are the attacks of large carnivores on people and livestock, and crop damage by large herbivores (inskip and zimmermann, ). these events make jaguars, tigers, lions, leopards (panthera pardus), hyenas (crocuta crocuta), african wild dogs (lycaon pictus), and african elephants (loxodonta africana), among others, be considered problems in rural communities, giving place to misunderstandings and false beliefs about their behavior (marchini and macdonald, ; dickman et al., ) . this situation has contributed to magnification of the actual damages of those species, stimulating even more fear, disgust, and rejection toward them (lescureux and linnell, ) . in this sense, the individual background and experiences of humans contribute to their emotions and behaviors. for example, the presence of large predators may produce fear and thoughts of escape in most people, while some others may feel encouraged to confront the danger (al-shawaf et al., ) . the context of the encounter with an animal may also be relevant for the emotions manifested. for a given person, the sighting of a carnivore such as a female puma with their offspring while hiking on a forest trail may produce fear and desire to escape. in contrast, the same person may feel surprised and delighted to have the same sighting from the safety of a car (narratives collected by the first author in chiapas, mexico). furthermore, local knowledge and the emotional links between people and wildlife could be useful to identify flagship species to foster interest in nature (bowen-jones and entwistle, ) . flagship species [e.g., giraffe (giraffa camelopardalis), elephants, and lions, among others] are usually charismatic and popular and may be relevant for promoting positive emotions in a public that has been distant from wild animals. differently, more complex sets of emotions (both positive and negative) are usually present where people are in constant interaction with these animal species (bowen-jones and entwistle, ; de pinho et al., ) . zoos represent spaces where emotional confrontations take place. for instance, marseille et al. ( ) observed visitors watching imposing and charismatic polar bears. the authors found that visitors felt happy in front of the bears, but at the same time they felt sad after recognizing the small size of the enclosures and the stereotyped behavior of the captive animals. interestingly, visitors' emotions transformed into fear and even greater sadness when they were told about observing polar bears in their natural habitat, which was associated with concerns about human safety and habitat vulnerability. another element that has an effect in the affection of children for wild animals is the presence of pets (bjerke et al., ) . pets can boost appreciation emotions, such as happiness, while naturalistic, ecological, humanistic, and moralistic attitudes may also be encouraged (prokop and tunnicliffe, ) . although knowledge about animals usually differs between urban and rural communities, the lack of accurate information about the species and their contribution to ecosystem services is persistent in both environments (gomes et al., ) . it promotes the intensification of emotions such as danger and disgust, especially for species that are unattractive to people. disgust has also been identified as one of the emotions inducing human rejection. it may arise when people perceive nasty odors in animals, or when unpleasant feelings emerge while touching (or thinking about) the fur of certain mammals (johansson et al., ) or the skins of amphibians such as frogs (lobue and deloache, ) . in other cases, disgust may be brought after linking animals such as spiders and rats with dirtiness, pollution, disease spreading, and potential crop damage (kellert, ; davey, ; prokop and tunnicliffe, ) . furthermore, animals that cause disgust are often perceived as ugly . contempt of human societies for amphibians and reptiles intensifies misinformation about them and favors negative attitudes toward them (manzano- garcía and martínez, ) . for example, it has been documented that non-venomous snakes are killed just because of their resemblance to poisonous species (breed and moore, ) . moreover, misinformation is an intensifier of disgust, for instance, when considering geckos (hemidactylus turcicus) as venomous animals or vectors of skin diseases (ceríaco et al., ) , or bats as a threat for fruit crops and responsible to infect people with parasites and viruses (musila et al., ) . in this sense, the case of bats and pangolins (pholidota) could be cited, which are considered the main transmitting agents of the novel coronavirus (covid- ; van staden, ). the respiratory illness has become a pandemic infecting million and killing many thousands of people around the world (nature, ). it is likely that the disease has a zoonotic origin as a result to the food and medicinal uses of animals (van staden, ). therefore, in some places there has been motivation to eliminate these animals (zhao, ) . this event might increase the negative perception and emotions of anger, disgust, and fear for this kind of animals and will encourage the eradication of populations without considering their importance in ecosystems. in this regard, it has been found that women and residents living near caves tend to believe in myths about bats more than men and people living far from caves (musila et al., ) . fearsome and disgusting species frequently induce rejection attitudes in social groups (Öhman and mineka, ), a phenomenon known as "biophobia" that is used to express the feeling of panic, fear, and disgust in front of a particular non-human living being. phobia for animals (agrizoophobia) is one of the most frequently reported biophobias in the general population (antony and mccabe, ) , but there are actually around twenty-five documented phobias to particular animal groups, such as that for snakes (ophidiophobia), spiders (arachnophobia), insects (entomophobia or insectophobia), ants (myrmecophobia), bees (apiphobia or melissophobia), and birds (ornithophobia), among others (fredrikson et al., ; antony and mccabe, ; prokop and fančovičová, ) . however, there are no specific phobias for carnivores, probably because the coevolution between humans and these animals has been too short in comparison with other groups such as snakes (prokop and randler, ) . biophobia may promote persecution and extermination attitudes (zhang et al., ) . avoiding contact with animals or killing them are the most frequent reactions without considering their long-term impacts on ecosystems (antony and mccabe, ; al-shawaf et al., ) . orr ( ) mentioned that one of the causes of biophobia is social distancing from nature. in a parallel way, biophilia has a genetic basis and consists of the interest and empathy of humans for other living beings (wilson, ) . as industrialization and urbanization increase around the world, lifestyles change in human societies, sometimes in radical ways (steffen et al., ) . these processes have contributed to the distancing of people from their natural environment even in rural communities (louv, ; lescureux and linnell, ) . however, there are still spaces such as zoos and natural parks facilitating social approach and understanding of wildlife in most of the cities and large towns all over the world. in those spaces, visitors are generally safe in front of animals that otherwise would be considered dangerous or harmful, and they may feel sadness and even culpability after recognizing the impact of the human population on those species. in this sense, vining ( ) suggested that visiting zoos and natural parks may represent opportunities for reconnecting people and wildlife to enhance social cooperation in conserving biodiversity. human emotions transcend over time. a specific emotion is saved by the individual as an experience that may be used in future behavior and decision-making (izard, ) . protection attitudes toward spiders, insects, amphibians, and reptiles are milder than those shown for other groups, such as birds and mammals due the sentiments of danger or disgust that these animal groups provoke in humans (prokop and fančovičová, ; . in addition, emotional experiences may have an effect on wildlife management techniques (larson et al., ) . this has occurred during experiences of invasive species management. one example is that of the house sparrow (passer domesticus), which competes for food and space with native birds and generates anger or disgust when managed through nest and egg removal, repellents, and traps. in contrast, bluebirds (sialia sialis) stimulate happiness in people watching them and listening to their songs, who at the same time feel sadness for these birds due to the negative impact of human activity on their populations. these feelings motivate protection attitudes favoring the persistence of the liking bird species (larson et al., ) . it is important to recognize that fear impacts human attitudes and behaviors toward keystone species, particularly those regarded as dangerous or harmful (e.g., wolves, bears, and big cats). fear may limit the involvement of local communities in managing predator populations because of the high costs implied or because the social acceptance of certain techniques, such as reintroduction, may be difficult (johansson et al., ) . examples of this include reintroducing wolves in mexico and the united states, where emotions have played fundamental roles in the acceptance of new wolf populations (straka et al., ) . mexican wolves (canis lupus baileyi) were eradicated from the mexican territory in the s because of conflicts with farmers and negative perceptions due to livestock predation (leopold, ; moctezuma et al., ) . wolf reintroduction projects have been started recently in northwestern mexico, where it has been clear that social acceptance is the primary limiting factor for their success (araiza et al., ; garcía, ; lara-díaz et al., ) . society's emotions toward wildlife may be key elements for decision-making on conservation issues. anger is one of the primary collective emotions that can lead to positive changes for natural resource management when social pressure is put on government leaders to improve and enforce environmental legislation. however, anger may have other implications and cause social fragmentation (buijs and lawrence, ) . in these cases, participation of wildlife management agencies is crucial given their social confidence. if the capacity of these agencies is not appropriate, collective distrust and fear of dangerous and disgusting animals may stimulate hostile environments for their proper management (johansson et al., ) . community confidence in environmental agencies is especially relevant where threatened species are under recovery, as is the case with wolves in different countries (swenson and andrén, ) , or where people take action by themselves, such as in the case of the killings of andean bears (tremarctos ornatus; figueroa, ) . it seems clear that some wildlife species are far more significant to humans than others (herzog and burghardt, ) , perhaps linked to their evolutionary closeness (e.g., primates, and particularly the great apes; gunnthorsdottir, ; miralles et al., ) or because of their cultural, aesthetic, or affective attributes favoring more interest and attention toward them. interest and attention favor people's attitudes for conserving these species, differently from others without a transcendental meaning for social groups. this idea highlights the relevance of designing conservation strategies fomenting interest for wildlife through generating affective links between humans and animals both in rural and urban areas. beautiful and attractive animals causing "positive" emotions (e.g., happiness and surprise) receive special attention driving in situ and ex situ conservation actions (gunnthorsdottir, ) . this could be a limitation for conservation efforts focused on species considered unattractive particularly in zoos. the preferences of human societies to watch specific animals have promoted that zoos keep attractive species more than those needing protection due to their conservation status (frynta et al., (frynta et al., , . mammals constitute the preferred group among zoo visitors around the world (moss and esson, ) . however, these spaces keep only , individuals belonging to , species (frynta et al., ) , which represent just . % of known living species (burgin et al., ) . this preference is strongly biased toward large, attractive, and active mammals belonging to the families ailuridae, felidae, phascolarctidae, ursidae, giraffidae, elephantidae, equidae, macropodidae, mephitidae, and cervidae, among others (frynta et al., ) . the same correlation between human preference and species kept in zoos was found for large, colorful, and long-tailed parrot species (frynta et al., ) . in contrast, small and unpopular species do not motivate the same appreciation, even if they are endangered. as a consequence, zoos generally keep a few of those local species (frynta et al., ) . in this sense, zoos and other places keeping wildlife need to implement exhibition strategies to promote human interest on less attractive but highly relevant animal species of threatened ecosystems (bitgood and patterson, ; frynta et al., ) . considering this distinction in preference, it is relevant to spread information about the ecological importance of animals in ecosystems, especially regarding native and endangered species (conde et al., ) , messages to promote "positive" emotions in people could be a way to support the appropriation of endangered species by societies and improve their attitudes toward them in the long term. massive media communication may be of utmost importance for these purposes, especially if the appropriate images of and messages about target species are transmitted to the general public (gunnthorsdottir, ) . following breed and moore ( ) , successful conservation projects require focusing on promoting wide social empathy for wildlife species, particularly those that generate fear and disgust (e.g., large predators, venomous species, and many amphibians) motivating their killing or removal (bishop et al., ; prokop and fančovičová, ; . individual and collective idiosyncrasies have promoted a diversity of attitudes toward wildlife species (herzog and burghardt, ) motivated in part by a diversification of emotions built with dynamic biological and cultural elements. identifying and understanding diversified emotions and their local precursors (e.g., in areas where protected areas and human presence are relevant) would allow analyzing wildlife problems and their solutions through multidisciplinary strategies. considering that knowledge is a relevant element for the expression of emotions, we propose that regional strategies to integrate information on the biology, ecology, and management of culturally important animal species (particularly those regarded as fearsome, dangerous, harmful, and disgusting) should be included in national education systems and massive media campaigns throughout the neotropics (espinosa and jacobson, ) . these strategies must be carefully designed by taking into account the impact of mass media (e.g., news, television shows, documentaries, films, and public text books, among others) may have on the public about wildlife conservation (røskaft et al., ; knight, ; ceríaco et al., ; wieczorek, ) . when an animal species is projected as aggressive, a negative emotional experience can be produced in the public. this negative experience may in turn lead the individual to believe the species is a dangerous agent or threat to human life, bringing about attitudes against its conservation (prokop and fančovičová, ) . on the contrary, if wildlife species are positively seen by children through different media outlets, where the real facts about unpopular animals are shown, it is more likely that fear and disgust decrease, while empathy may grow (prokop et al., ) . ensuring the continuity of transmitting traditional ecological knowledge about animal species will be equally important to stimulate positive emotions and a long-term interest of the new generations in wildlife conservation (jacques-coper et al., ) . another strategy that could have a positive impact on emotions toward fearsome and disgusting animals is promoting physical interactions with them (e.g., touching snails, rays, amphibians, mice; randler et al., ; ; the new knowledge about the animals and physical contact with them could reduce the anxiety of danger. recognizing that emotions are culturally influenced, we propose developing outreach strategies by retrieving traditional aspects that formerly favored empathy with animal species, including the noncharismatic or unpopular ones, even if they are threatened. this review aimed to discuss the role of emotions in the conservation of species which a have been transcendent for the human species throughout history and that in many cases are currently threatened by extinction. in particular, we stress that the social component is of utmost importance in wildlife conservation across latin america, especially in megadiverse countries where ethnozoological studies have documented the relevance of human-wildlife relationships (jácome-negrete et al., ; sarukhán and dirzo, ; manzano-garcía and martínez, ) . nc-h wrote and edited the manuscript. en translated and edited the manuscript. ds-f and ee-l edited the manuscript. all authors read and approved the final manuscript. observando al lobo. un estudio antropológico sobre el lobo y el turismo en la sierra de la culebra the religious connotation of the islamic dragon: the roots human emotions: an evolutionary psychological perspective relationships between fauna and people and the role of ethnozoology in animal conservation the live bird trade in brazil and its conservation implications: an overview toward a psychology of human-animal relations overcoming animal and insect phobias: how to conquer fear of dogs, snakes, rodents, bees, spiders and more consensus on criteria for potential areas for wolf reintroduction in mexico prevalence and factors associated with phobias among women schoolchildren and one of the most unpopular animals: are they ready to protect snakes? the amphibian extinction crisis -what will it take to put the action into the amphibian conservation plan? principles of exhibit design animal-related activities and appreciation of animals among children and adolescents identifying appropriate flagship species: the importance of cultural and local contexts conservation and behavior emotional conflicts in rational forestry: towards a research agenda for understanding emotions in environmental conflicts. for how many species of mammals are there? evolutionary psychology: the new science of the mind el papel de la cosmovisión en el conocimiento etnozoológico folklore and traditional ecological knowledge of geckos in southern portugal: implications for conservation and science an emerging role of zoos to conserve biodiversity worldwide crocodilian attack database disgust as an adaptive system for disease avoidance behavior the emotional brain the expression of the emotions in man and animals the "disgusting" spider: the role of disease and illness in the perpetuation of fear of spiders influence of aesthetic appreciation of wildlife species on attitudes towards their conservation in kenyan agropastoralist communities the narrow fellow in the grass: human infants associate snakes and fear los mensajeros de la serpiente emplumada carnivores, culture and 'contagious conflict': multiple factors influence perceived problems with carnivores in tanzania's ruaha landscape attitudes toward animals: species ratings basic emotions what is meant by calling emotions basic pan-cultural elements in facial displays of emotion human-wildlife conflict and environmental education: evaluating a community program to protect the andean bear in ecuador interacciones humano-oso andino tremarctos ornatus en el perú: consumo de cultivos y depredación de ganado offensive" snakes: cultural beliefs and practices related to snakebites in a brazilian rural settlement gender and age differences in the prevalence of specific fear and phobias the analysis of emotions. dimensions of variation wolves and humans being attractive brings advantages: the case of parrot species in captivity are animals in zoos rather conspicuous than endangered? cross-cultural agreement in perception of animal beauty: boid snakes viewed by people from five continents mammalian collection on noah's ark: the effects of beauty, brain and body size la conservación del lobo en américa del norte y desafios de la reintroducción del lobo gris mexicano (canis lupus baileyi) ethnozoology of bats (mammalia, chiroptera) in feira de santana municipality, bahia state, northeastern brazil la simbología de la serpiente en las religiones antiguas: en torno a las posibles causas biológicas que explican su sacralidad e importancia physical attractiveness of animal species a decision factor for its preservation what's social about social emotions? attitudes toward animals: origins and diversity animals, archetypes, and popular culture: tales from the tabloid press human-felid conflict: a review of patterns and priorities worldwide emotion theory and research: highlights, unanswered questions, and emerging issues human emotions toward wildlife etnozoología quichua para la conservación de los mamíferos ungulados en la amazonía central del ecuador provincia pastaza the andean condor as bird, authority, and devil: an empirical assessment of the biocultural keystone species concept in the high andes of chile human attitude toward reptiles: a relationship between fear, disgust, and aesthetic preferences factors governing human fear of brown bear and wolf living with problem animals: self-reported fear of potentially dangerous species in the serengeti region relationship between color and emotion: a study of college students values and perceptions of invertebrates attitudes, knowledge, and behaviors toward wildlife affected by gender human culture and large carnivore conservation in north america the biophilia hypothesis characteristics and motivates of adolescent volunteers in wildlife education bats, snakes and spiders, oh my!" how aesthetic and negativistic attitudes, and other concepts predict support for species protection hunter and hunted: relationships between carnivores and people human-wildlife conflict in africa: causes, consequences and management strategies beauty ranking mammalian species kept in the prague zoo: does beauty of animals increase the respondents willingness to protect them? ¡nacidos libres! en el camino a la recuperación del lobo mexicano emotions as drivers of wildlife stewardship behavior: examining citizen science nest monitors' responses to invasive house sparrows la fauna silvestre de méxico. (méxico: instituto mexicano de recursos naturales renovables knowledge and perceptions of macedonian hunters and herders: the influence of species-specific ecology of bears, wolves, and lynx human preferences for colorful birds: vivid color or pattern? detecting the snake in the grass: attention to fear-relevant stimuli by adults and young children what's so especial about slithering serpents? children and adults rapidly detect snakes based on their simple features last child in the woods: saving our children from nature-deficit disorder who cares about wildlife? social science concepts for exploring human-wildlife relationships and conservation percepción de la fauna silvestre en áreas protegidas de córdoba, argentina: un enfoque etnozoológico predicting ranchers' intention to kill jaguars: case studies in amazonia and pantanal what makes some species of milk snakes more attractive to humans than others? experiencing polar bears in the zoo: feelings and cognitions in relation to a visitor's conservation attitude the subtle beast: snakes, from myth to medicine cultural variations in emotions: a review empathy and compassion toward other species decrease with evolutionary divergence time urban resident attitudes toward rodents, rodent control products, and environmental effects visitor interest in zoo animals and the implications for collection planning and zoo education programmes knowledge and perceptions of, and attitudes to, bats by people living around arabuko-sokoke forest coronavirus latest: pandemic could have killed million without any action farming the forest edge: vulnerable places and people around kibale national park, uganda emotions and the ethics of consequence in conservation decisions: lessons from cecil the lion bodily maps of emotions face the beast and fear the face: animal and social fears as prototypes for evolutionary analyses of emotion emotion drives attention: snakes in the grass fears, phobias, and preparedness: toward an evolved module of fear and fear learning the malicious serpent: snakes as a prototypical stimulus for an evolved module of fear love it or lost it: the coming biophilia revolution emotion facilities perception and potentiates the perceptual benefits of attention a general psychoevolutionary theory of emotion the nature of emotions human emotions have deep evolutionary roots, a fact that may explain their complexity and provide tools for clinical practice scary and nasty beasts: self-reported fear and disgust of common phobic animals which emotions are basic? perceived body condition is associated with fear of a large carnivore predator in humans tolerance of amphibians in slovakian people: a comparasion of pond owners and non-owners does colour matter? the influence of animal warning coloration on human emotions and willingness to protect them the effect of hands-on activities on children's knowledge and disgust for animals animals in dangerous postures enhance learning, but decrease willingness to protect animals vampires are still alive: slovakian student's attitudes toward bats aposematic colouration does not explain fear of snakes in humans bad wolf kills lovable rabbits: children's attitudes toward predator and prey. electron tolerance of frogs among high school students: influences of disgust and culture biological predispositions and individual differences in human attitudes toward animals high school students' attitudes towards spiders: a cross-cultural comparison disgusting" animals: primary school children's attitudes and myths of bats and spiders effects of having pets at home on children's attitudes toward popular and unpopular animals good predators in bad stories: crosscultural comparison of children's attitudes towards wolves snakes represent emotionally salient stimuli that may evoke both fear and disgust practical work at school reduces disgust and fear of unpopular animals anthropomorphized species as tools for conservation: utility beyond prosocial, intelligent and suffering species patterns of self-reported fear towards large carnivores among the norwegian public symbolism and ritual practices related to hunting in maya communities from central quintana roo biodiversity-rich countries snakes: objects of religion, fear, and myth the anthropocene: are humans now overwhelming great forces of nature understanding the acceptability of wolf management actions: roles of cognition and emotion a tale of two countries: large carnivore depredation and compensation schemes covid- and the crisis of national development the connection to other animals and caring for nature exploring the influence of emotion on human decision making in human-wildlife conflict biophilia and the conservation ethic strategies for carnivore conservation: lessons from contemporary extinctions, " in carnivore conservation animals in the world: a materialist approach to sociological animal studies how contact with nature affects children's biophilia, biophobia and conservation attitude in china covid- drives new threat to bats in china mexico's national council of science and technology (conacyt) provided a scholarship to nc-h for her doctoral studies. we thank ph.d. angela may steward for reviewing and editing the manuscript. key: cord- -gvc ij authors: klaunberg, brenda a.; lizak, martin j. title: considerations for setting up a small-animal imaging facility date: journal: lab anim (ny) doi: . /laban - sha: doc_id: cord_uid: gvc ij imaging techniques allow for the conduct of noninvasive, in vivo longitudinal small-animal studies, but also require access to expensive and complex equipment, and personnel who are properly trained in their use. the authors describe their planning and staffing of the nih mouse imaging facility, and highlight important issues to consider when designing a similar facility. state-of-the-art biomedical research often uses rodents and other small animals for disease modeling. a recurring issue for many investigators is the desire to obtain anatomical and physiological information from valuable research animals without sacrificing them. in vivo imaging is a noninvasive way to gain insight into the animal's anatomy and physiology ; however, the unit cost and complexity of many such methods may preclude an investigator's ability to gain access to such devices. location of the imaging equipment in a shared facility can overcome these obstacles. the vision of the nih mouse imaging facility (mif) is to offer various state-of-theart, in vivo small-animal imaging techniques in one facility. the mif is a shared resource for the nih intramural community, and currently has more than active animal protocols. at this time, the mif has three magnetic resonance imaging (mri) scanners, a micro x-ray computed tomography (ct) scanner, two ultrasound scanners, a combined luciferase/gfp imager, and a laser doppler imager. in addition to administrative personnel, the staff consists of a veterinarian, three imaging scientists, an electrical engineer, and three animal technicians. setting up this facility took planning and intellectual contributions from experts in many fields. we provide resources for a wide variety of investigators from many of the various institutes within the nih. this is not a 'how-to' manual, but we will discuss some of the issues that the principal players considered when designing and staffing the mif. a small-animal imaging facility can represent an enormous investment of capital and personnel. to obtain the maximum usefulness and ensure success, a certain amount of planning must take place before an instrument is ordered or facility construction begins. planners should recruit the advice of experts in various imaging fields and involve as many people as necessary to share in the decision-making processes, so that everyone has a voice. knowledge of the needs of the research community is one of the most important priorities in setting up any facility. a consultation with your research community will help to determine which imaging modalities are most needed. it is pointless to include an instrument that no one will use. consultation with experienced operators for each of the imaging modes chosen will elicit useful advice on special needs for each instrument. veterinarians and animal care staff should also have input because they will have a considerable impact on traffic patterns, animal care requirements, and other features. if possible, one should visit other small-animal imaging facilities. what imaging modalities do they have? what problems have they encountered, and how did they solve them? one must consider what types of animals and models could come to the facility for imaging, because these considerations will impact staffing choices, housing availability, and imaging modalities. one should also consider the health status of the animals; a facility that can accommodate immunecompromised animals has more stringent requirements. the biosafety level that the facility will maintain is also a consideration; animals carrying pathogens or treated with radiolabeled agents will require additional restrictions. the mif operates as a clean conventional facility that excludes the following specific pathogens: coronaviruses, pneumovirus of mice (pvm), sendai virus, endoparasites, and ectoparasites. to prevent cross-contamination between rodents, disposable, absorbent material covers all surfaces. all surgical instruments are steam-sterilized for survival procedures, and devices such as nose cones are either placed in cold sterilization or cleaned with a bleach-based disinfectant. planners should decide whether to design and staff the facility so that researchers can be human and animal safety should be the primary consideration when planning an imaging facility design. in addition to excluding specific rodent pathogens, the mif operates at animal biosafety level- (absl- ) [author: edit okay?]. some imaging animals may receive agents (chemotherapeutics, infectious organisms) in higher biosafety levels in other facilities. those animals cannot be imaged until they satisfy absl- requirements. planners should determine the need for a preparation room. it is possible to perform simple procedures such as anesthesia induction and tail vein catheter placement adjacent to the imaging instrument. more complicated preparations that require sterile or aseptic procedures will require a dedicated room. in the mif, there is a small preparation area adjacent to each instrument. in addition, we have a preparation room set aside for rodent procedures more complex than anesthesia induction and tail vein intravenous catheter placement. each preparation area is set up with anesthesia and physiological monitoring equipment, as well as a surgical microscope for microsurgery. we use inhalant anesthesia (isofluorane) as much as possible, and each imaging device preparation area is set up identically. all preparation areas have central gas supplies for oxygen, medical air, and nitrogen, as well as a vacuum system for scavenging anesthetic gases. anesthesia and monitoring equipment must comply with magnet safety. anything that enters the scanner room must be nonmagnetic, and any equipment within the fringe field must operate correctly. once there is an understanding of the goals of the research community, the needs of the investigators can determine the imaging techniques to be made available. some techniques, such as optical imaging, are relatively inexpensive in terms of equipment, personnel, and space. individual laboratories may have the financial capability to purchase these types of devices, but more elaborate techniques, such as mri or ct scanning, require a more substantial investment of resources. individual laboratories rarely have the funds to purchase such equipment or the means to maintain them; therefore, mri and ct are usually the core of an imaging facility. please refer to table for a summary of imaging modalities, their applications, and their estimated costs. mri is one of the most powerful noninvasive imaging methods currently available to research. this technology uses radio waves and powerful magnets to generate radiograph-like images of tissue. a strong magnetic field partially aligns the hydrogen atoms on water molecules in the tissue. a radio wave then disturbs the built-up magnetization, and radio waves are in turn emitted as the magnetization returns to its starting place. these radio waves can be detected and used to construct an image (fig. a) . unlike radiographs, the mri patient is not exposed to x-ray radiation, so repeated imaging is not a risky procedure . mri is excellent for imaging different types of soft tissue with high contrast - . researchers can use it for anatomical and functional studies. some applications include tumor growth and treatment, brain function and stroke, and cardiovascular disease. contrast agents, which are drugs that function like histological stains, increase the usefulness of this imaging method. for resource volume , no. lab trained to run the instruments independently, or whether to offer complete service, in which researchers can deliver animals to the facility and then return for images. this choice will have a profound effect on staffing requirements. the mif is designed to be a resource, not a service, so that investigators are encouraged to participate fully. even if the investigator has no desire to learn to run the equipment, it is our requirement that someone on the protocol be present and responsible for the animal during scanning. an important initial decision is whether or not the animals will be housed in the facility. the number of animals housed and the duration of housing will have an impact on space usage. animal welfare guidelines dictate space requirements that must be carefully considered. moreover, one must also consider space for traffic between housing, preparation rooms, and instruments. at the mif, we provide temporary housing (monday-friday) for mice only. we have one -ft animal room with one ventilated rack that can hold shoebox-sized cages. additionally, we have ft of animal husbandry support area for storage and cage washing. animals involved in long-term studies that must come to the mif periodically for imaging are usually returned to a quarantine facility. because of the surrounding magnetic field, an mri system requires a large area (≥ ft ). the magnet itself requires ∼ ft of surrounding clear area. the fringe field is the distance that the surrounding magnetic field extends before it drops to the level of gauss. the fringe field can occupy an area up to ft . the mri suite must have a design that prevents casual visitors from entering the fringe field. a magnetic field > gauss can adversely affect persons with pacemakers or other metallic implants. the distance to the gauss line varies widely with field strength and magnet type. there are magnets available that have a fringe field limited to a foot or less, but these cost more. mr scanners use extremely heavy magnets; therefore, most facilities locate the mr imager on the ground floor. additionally, because the magnets are cooled by cryogens, good ventilation must be present. rapid boil-off of the cryogens could lead to asphyxiation. the instrument manufacturer can provide a detailed description of space requirements and suggested architectural layouts. a facility will need to plan for additional space for the electronics and console for the scanner as well as a preparation area outside the magnetic field. this can amount to another ft . researchers currently consider a -tesla mri system an optimal field for imaging rodents. at the mif we have several bruker avance mri scanners (bruker-biospin, billerica, ma). some other manufacturers of mri consoles are varian (varian medical systems, inc., palo alto, ca), tecmag (tecmag, houston, tx), and mrrs (mr research systems, guildford, uk). a basic setup for the mri includes the superconducting magnet, the imaging console, and several probes for approximately $ , -$ , , , depending on the options. it is ideal to enclose the magnet in a room shielded from radiofrequency, but this construction would generate additional costs. the magnet itself comes with end caps that adequately shield it; however, passing anesthesia and monitoring lines becomes awkward. because of its complexity, the mri requires a scientist with not only the necessary academic qualifications, but also a technical background for operation. there are many mri measurement methods, each with many control variables, designed for specific purposes. the mr operator must be familiar with the basic physics of the mr measurement and the effect of the controls on the image outcome. if new methods are going to be developed or implemented, a postdoctoral-level scientist will be necessary. new methods require sophisticated knowledge of the hardware and computer programming. this individual will be responsible for running the scanner and maintaining the system. mri maintenance requirements include both hardware and software. hardware maintenance includes the associated magnetic field. the magnet requires liquid nitrogen and liquid helium to maintain the superconducting magnetic field. allowing for a safety margin, most magnets need liquid nitrogen once per week and liquid helium approximately twice per year. if the cryogen level is allowed to become too low, the magnetic field can spontaneously quench (i.e., lose its magnetic force). quality assurance includes regular test images of a standard sample. a small loss in performance can result in unusable images. the operating computer and software also require regular maintenance. system software requires periodic updates to install security patches, repair bugs, and add features. these updates are particularly important if the computers are on a network with outside access. despite hardware and software designed to limit external access, new security holes appear on a regular basis. a malicious attack could destroy valuable work and render the instrument useless until the base software can be reinstalled. archiving and removing old data are important parts of computer maintenance. if the number and size of the stored image files grow too large, system performance can slow or even become blocked. a policy for data management can help prevent major problems, but periodic enforcement is still necessary. this policy can range from a simple principle of moving the oldest data first to a more complicated formula based on size and age. at the mif, we encourage everyone to transfer data from the instrument as soon as possible. when data space becomes an issue, the users with the largest amount of data must export or delete files before they can resume scanning. x-ray ct is an imaging method that uses multiple radiographic views of a subject to construct an image (fig. b) . in our system, an x-ray source and detector rotate around the subject degrees while generating a number of projections or views. the mif has a microcat ii ct scanner built by imtek, inc. (knoxville, tn). other manufacturers of the manufacturer can provide exact measurements of the machine footprint. there are several micro-ct manufacturers, and the price ranges from $ , to $ , . the instrument will require one technician for operation and maintenance. this individual should have a sophisticated education in biology or a related field and be comfortable with computers. the ct scanner relies on several computers on a small network. the ct operator will be responsible for maintaining the computer system software, managing the accumulated data, and quality assurance. a good knowledge of anatomy is vital for data interpretation, and the person should be able to interpret radiographs. reporter gene technology has revolutionized the ability to track cell populations in vivo . optical imaging devices, which allow the user to monitor gene expression [author: edit okay?], are easy to operate and have no special environmental requirements. there are several different types of optical imaging. bioluminescence is an enzymatic biochemical process that produces light. the luciferase gene is first inserted into the targeted cell population. image acquisition requires live cells to process the substrate and produce light. advantages of bioluminescence include high sensitivity, and virtually no background noise; disadvantages include the need for cofactors (i.e., oxygen, magnesium, and atp) and expensive substrates (luciferin). fluorescence imaging also requires genetic manipulation; however, the technology does not require a living system to produce an image. fluorescence imaging differs from bioluminescence in that light must be applied to the tissue to generate a signal. one advantage of fluorescence imaging is the commercial availability of numerous markers (gfp, dsred, icg, and cy . ) with no need for substrate or cofactors; a disadvantage is the low signal/noise ratio due to autofluorescence. basic equipment for bioluminescence or fluorescence imaging consists of a light-tight chamber, a sensitive camera (charge-coupled device), and a computer. it is possible for the ambitious investigator to manufacture his or her own imaging device; however, commercial products are available for about $ , . the mif owns a xenogen ivis (xenogen corp., alameda, ca); other manufacturers include eastman kodak (eastman kodak co., scientific imaging systems, rochester, ny), roper scientific (trenton, nj), and advanced research technologies, inc. (saint-laurent, quebec, canada). our imaging device occupies a space measuring ft ; additional benchtop space is necessary for preparing the animal. image acquisition is simple, and it is not necessary to have a dedicated system operator. investigators can easily learn to perform their own imaging study. our system's operating software first acquires a black-and-white photographic image of the subject, then acquires the luminescent image, and finally overlays the image onto the photograph for anatomical signal mapping (fig. ) . both bioluminescence and fluorescence imaging are useful to detect tumor cells, monitor tumor growth, or track cell movement (e.g., metastasis). optical imaging can be highly specific, but it has low spatial resolution. optical data are typically two-dimensional ( d), but d methods are in development. a high throughput of animals is possible because imaging time is resource volume , no. lab micro-ct equipment include ge medical systems (london, ontario, canada), scanco medical (scanco usa, inc., southeastern, pa), and skyscan (aartselaar, belgium). each projection is the equivalent of a radiographic image. a method called 'filtered back projection' permits these projections to be reconstructed into a three-dimensional ( d) data set. a micro-ct scanner for rodents typically uses less x-ray power than a conventional clinical ct scanner, so the effective radiation exposure per scan is reduced. the highest radiation dose we have measured was rad for skin, but typical scans are much less ( rad). the radiation dose is ultimately dependent on the scan parameters (i.e., number of projections, exposure time, x-ray beam strength). ct is excellent for studying the skeletal system, certain internal organs, and fat distribution [ ] [ ] [ ] [ ] . ct can also be used for tumor studies, depending on the location of the tumor. a typical d image with -µm resolution takes about minutes to acquire; higher resolution images may take up to minutes. image reconstruction can take as little as extra minutes or up to hours depending on hardware and software options. as with mri, a contrast agent can be used to enhance the appearance of some organs. in rodents, the half-life of most clinical ct contrast agents is short, so some experimentation is required to find the optimum dose and administration regimen. a micro-ct scanner requires ∼ ft of short, usually from second to minutes. laser doppler is another type of optical imaging that uses a laser light source and the doppler effect to measure capillary blood flow (fig. ) . the equipment consists of a laser source and computer, and costs less than $ , . the footprint of the laser device is small ( ft plus computer), but its sensitivity to movement warrants placement on an antivibration table. this machine is simple to use and is highly sensitive, but it is limited to small vessels within a -mm depth. the mif owns a laser doppler imager from moor instruments, inc. (wilmington, de). another manufacturer is perimed, inc. (north royalton, oh). ultrasound imaging is a rapid, real-time in vivo technique. an ultrasound transducer broadcasts sound waves beyond the audible range into tissue. as the sound waves encounter the interfaces between various types of tissue, they are reflected. the transducer detects the reflected sound waves and uses them to construct an image (fig. ) . the depth of penetration depends on the frequency of the sound wave. the image-processing software produces an image in real time, and the various tissues within the image display different 'echogenic' properties. ultrasound machines used for rodent imaging are similar to those used clinically; however, the larger field of view offered by a clinical scanner is not optimal for imaging smaller subjects. resolution increases (and field of view decreases) as the ultrasound frequency increases; therefore, high-frequency ultrasound is ideal for rodents. ultrasound is excellent for cardiac studies and evaluation of embryonic development , . it may also be used for various organ evaluations, tumors, and guided injections. advantages of ultrasound include rapid image acquisition and ease of use; however, time is required for the operator to develop proficiency in the acquisition techniques and image interpretation. knowledge of anatomy is useful for positioning the transducers, as well as evaluating the resulting images. a well-trained operator should be able to distinguish normal and abnormal anatomy. scattering of the sound waves in the tissue often cause ultrasound images to appear 'noisy' . also, most ultrasound images are d, although some d-capable instruments are available. most ultrasound machines are built on portable carts for easy relocation. the image display is most clearly visible in low ambient light, so this should be a consideration when determining the location for ultrasound imaging. the mif owns two ultrasound machines: a siemens acuson sequoia (siemens medical solutions, malvern, pa) and a visualsonics vevo (visualsonics, inc., toronto, ontario, canada). there are many other ultrasound manufacturers, including philips (philips medical systems, andover, ma) and general electric (ge medical systems, london, ontario, canada). a commercial ultrasound imager can cost about $ , . although one can perform data analysis on the scanner, this does take time away from available scan time. an additional data-processing workstation could cost as much as $ , . the mif does not have a dedicated ultrasonographer at this time. the investigators that use our acuson ultrasound most frequently have experience in its operation and often collaborate with other investigators in need of their expertise. the mif staff is developing proficiency in ultrasonography. positron emission tomography (pet) is an excellent method to perform functional imaging in vivo [ ] [ ] [ ] . uptake of radioactive compounds can demonstrate the presence of tumor, abnormal cell function, or metabolic changes. this imaging technique can generate d data. pet imaging is highly sensitive but suffers from low spatial specificity and needs to be superimposed on an anatomical image for signal location (fig. ) . the footprint of a micro-pet system may only be ft , but it is ideal to locate the micro-pet adjacent to a micro-ct or mri because an anatomical image will be required for co-registration. this allows convenient transport of an anesthetized animal from one machine to another. micro-pet imaging presents its own set of unique considerations. the use of radioactivity dictates its own specifications. the imaging area should be in a location that allows the environment to be properly controlled and appropriate precautions taken.your institution should be able to provide you with a detailed description of the rules and regulations that govern the use of radionuclides. the imaging animals will remain radioactive for some time after imaging, so housing for these 'hot' animals must be considered. the half-lives of radionuclides in the radioactive compounds are typically short (e.g., hours), so housing vice to new users. at the mif, we train investigators to perform simple data analysis. for more complex data analysis, mif staff collaborates with the investigators. this system works well because, having trained an investigator, the technician is freed up for other duties; the investigator can often then pass that training on to new individuals in his or her group as well as other collaborators. once the user base is established, equipment needs are defined, and the facility floor plan is designed, one must consider the staffing needs. a variety of personnel are needed for a successful imaging facility. diversity of experience will enhance the resources as well as expand the knowledge base of the entire personnel staff. certain base criteria must be met for each unique position, but it is our experience that personnel experienced in imaging laboratory animals are difficult to find. many of the necessary technical procedures may be learned on site. we believe that a strong desire to learn may be more important than experience and formal education. it is important to have at least one person on the staff that is skilled in the routine technical procedures and can provide training for new staff members. as with any animal facility, husbandry staff is necessary. ideally, housing for imaging animals should be on site and accessible. the size of the animal housing facility will determine the number of necessary personnel. routine husbandry duties include daily health checks, cage changing, cage washing, room sanitation, sentinel maintenance, quality control, etc. husbandry staff is the foundation of all successful animal research programs. the qualifications of the animal care staff are dependent on how many people will be required to run the facility and in what capacity the people are expected to function. if you can have dedicated husbandry staff, their qualifications will be less stringent, and a person with aalas alat or lat experience would be sufficient. staff members who will be assisting with imaging experiments are expected to work independently, so it is ideal to have personnel with aalas latg certification or equivalent experience. if the technical staff is required to assist with (or perform) husbandry duties, they should understand that it is part of their job description so that no misunderstandings occur later. laboratory animals must be immobilized for imaging procedures. it is important for the laboratory animal technical staff to maintain a working knowledge of anesthesia and physiological monitoring for a variety of species. additionally, basic procedure skills such as venipuncture are required for the administration of imaging contrast agents and other pharmaceuticals. familiarity with aseptic techniques and rodent colony health management techniques will aid in maintaining the level of health standards for the facility. additional useful procedures include intubation of rodents, placement of rodent femoral, jugular, and carotid catheters. the computers acquiring the images and the networks they are connected to can be complicated. many of the personnel operating the imaging devices have sufficient knowledge of computer operation and software programming to repair problems as they occur; however, given the number of computers needed for imaging and personnel support, it is a good idea to have someone dedicated to their maintenance. it is important that these networks are secure and protected from any outside abuse. the mif has shared information technology personnel that maintain computer software security and upgrades. the imaging instruments require regular hardware maintenance as well as periodic repair. the best solution is to have an engineer on the staff to address these issues. it is possible to maintain a service contract with an outside company, but an internal person can aid in quicker diagnosis and repair. the mif has maintenance contract on all equipment, but the level of each maintenance plan varies from a 'parts only' plan to a full-service plan that includes yearly preventative maintenance. the mif is fortunate to have mri scientists who are knowledgable in mri mainte- resource volume , no. march animals can be a temporary situation. the availability of radioactive compounds will have the greatest impact on the success of a micro-pet program. radiolabeled glucose, oxygen, and ammonia are commercially available; however, many studies may need custom-made radioactive compounds. the ideal arrangement is an imaging suite located adjacent to the radiopharmacy that will produce the radionuclides. production of these reagents can represent the largest expense because of the need for highly specific equipment and personnel. manufacturers of micro-pet imagers include general electric and philips medical systems. micro-pet operation will also require a postdoctorate-level scientist to assemble, operate, and maintain the equipment. it is easy to generate an enormous amount of image data in a relatively short amount of time. a single image data set can range from a few kilobytes to over two gigabytes for highresolution three-dimensional data. the amount of image data generated over time can be a problem. mass storage devices must be available for data storage during analysis. if backup and archiving are required, the amount of equipment needed can be considerable. a room will need to be dedicated to computer storage and workstations for post processing images. a minimal room will have space for a rack containing data servers and network equipment. qualified information technology personnel should be able to provide additional advice. in addition to data storage, extra workstations for data processing are almost a necessity. the alternative is allowing data to be analyzed on the acquisition instruments. this reduces the need for extra computers but takes time away from the instruments. the time required for data analysis can be considerable. some consideration must be given to who will analyze data. often, users of the instrument will not have the experience or skill needed to gain the most information from the images. one solution is for image analysis to be part of the imaging service. another approach is to train the investigators to analyze their own data; this reduces the facility burden, but reduces the usefulness of the ser-nance and repair, as well as an electrical engineer who assists with mri and ct maintenance and repair. the imaging facility also needs personnel to attend to nonscientific tasks. administrators must handle daily paperwork such as budget management and supplies, as well as provide other organizational services. these people are critical, because if the facility is successful, none of the scientific personnel will have time to attend to administrative duties. the mif has shared personnel for these tasks. any facility that conducts laboratory animal research should be accessible by authorized personnel only. we cannot overemphasize this because of magnet safety, radiation safety, and animal health. traffic patterns for animals entering the facility may be necessary if the animal health level varies in different areas. the amount of personal protective equipment (ppe) needed will be defined by the types of animals in the facility, as well as the health standard. the minimum ppe for handling animals in the mif includes a disposable lab coat and gloves. because animal studies are ongoing, and we temporarily house mice, personnel must don a lab coat upon entering our facility, regardless of their animal contact. studies with nonhuman primates require a higher level of ppe that includes mucous membrane protection. standard ppe worn around the mri scanners must be nonmagnetic. an institutional animal care and use committee (iacuc) should approve every study conducted within the facility. new investigators may seek the assistance of facility personnel in writing their protocols. this gives the imaging facility personnel a chance to suggest feasible imaging modalities, preparation methods, and anesthesia regimes. the mif has a committee of experienced researchers that review experiments for feasibility, safety, and time requirement. imaging time may be scheduled on a firstcome first-served basis, or otherwise. instruments with short imaging times allow for a larger number of studies in any given period of time. when demands exceed the available scan time, one solution is to assign blocks of time for specific groups. then individuals within the group can work out their own priority for imaging. in our experience, it is easiest to have the equipment operators schedule the investigators. the operators will be most experienced with the requests of the prospective study and can schedule time accordingly. in the mif, magnet scan time during regular working hours is assigned to institutes in blocks; the remaining scan time is assigned on a first-come first-served basis. the other imaging devices require less time per scan, so it has not been necessary to assign blocks of time yet; each investigator is assigned time on a first-come first-served basis. scan time is charged back to the institute by the hour, and the dollar amount is calculated with a budget-driven formula. noninvasive imaging of rodents and other small animals is a powerful tool for biomedical research. setting up an imaging facility is a complex process that involves many decisions affecting everything from available instruments to staff composition. careful planning should help prevent operational snags. the advice of experienced people will be the most valuable asset. it is our opinion that the facility should be designed around a primary mri scanner because mr offers versatility for many applications. acquisition of other devices will be determined by the needs of the research community. growth of the facility is limited only by usage and executive decisions. staff must include a few experienced personnel, but inexperienced eager personnel can be trained to be experts. we have tried to provide an outline of some of the important considerations that went into the creation of the mif. challenges in small animal noninvasive imaging reproductive and teratologic effects of electromagnetic fields mr microscopy and high resolution small animal mri: applications in neuroscience research imaging transgenic animals in vivo imaging of gene and cell therapies electron paramagnetic resonance for small animal imaging applications molecular imaging in small animals-roles for micro-ct the use of microcomputed tomography to study microvasculature in small rodents a review of high-resolution x-ray computed tomography and other imaging modalities for small animal research high resolution x-ray computed tomography: an emerging tool for small animal cancer research advances in in vivo bioluminescence imaging of gene expression advances in ultrasound biomicroscopy noninvasive cardiovascular phenotyping in mice molecular imaging of small animals with dedicated pet tomographs high resolution spect in small animal research radio-imaging in small animals the authors would like to thank stasia anderson of laboratory of diagnostic radiology research, nih, for providing an mr image, dan schimel of the nih mouse imaging facility for providing a ct image, cecilia lo of the laboratory of developmental biology, nhlbi, nih, for providing an ultrasound image, takashimurakami and sam hwang of the dermatology branch, nci, nih, for providing a luciferase image, michael green and the imaging physics laboratory, nih, for providing a pet image, and afonso silva of the laboratory for functional and molecular imaging, ninds, nih, for providing images for this paper. we would also like to thank alan koretsky of the laboratory for functional and molecular imaging for helpful discussions. key: cord- -l xqi authors: holloway, lewis title: covid- and a shifted perspective on infectious farm animal disease research date: - - journal: agric human values doi: . /s - - - sha: doc_id: cord_uid: l xqi nan the lockdown response to the upsurge of covid- cases in the uk in march brought an immediate end to my current research project's on-farm, in-depth social scientific fieldwork in the north of england. this research, funded by the welcome trust, is itself focused on persistent, endemic infectious diseases-but in our case in cattle populations. as a team of social scientists, historians, and economic and epidemiological modellers, we explore the history of bovine viral diarrhoea (bvd) in the uk, examine how farmers, vets and other professionals attempt to deal with bvd in the present, and attempt to model infection patterns and how farmer behaviours affect the transmission and prevention of this disease. bvd can be transmitted between animals or passed from a cow to her calf in utero. its effects vary in severity but it is linked to reduced productivity, various symptoms of ill-health, and increased susceptibility to other illnesses. as a social scientist on the team, my thinking on bvd is influenced by discussions of biosecurity, or 'making life safe' (bingham et al. (bingham et al. , p. ), a process involving anticipating what threats to life might occur, being prepared to respond to their occurrence, and being ready to make interventions to reduce the effects. discussion of biosecurity (e.g. hinchliffe et al. ) has described three overlapping ways of attempting to make life safe, and although these were originally conceived in relation to protecting of human life, they, and the concept of biosecurity, have more recently tended to be associated with attempts to secure animal life. they are, first, exclusion (preventing the ill moving into a space); second, inclusion (quarantining the ill within a space); and third, normalisation (managing a disease through interventions such as vaccination). as covid- took hold, we have very rapidly seen the application of all of these modes of biosecurity, which i had been thinking about in rather abstract terms and in relation to animals, back onto our own lives in very significant and concrete ways, forcing a recalibration of my perspective on animal and human infectious diseases together. we see the exclusion, inclusion and normalisation practiced by farmers in relation to their animals, being practiced by governments in relation to us through border closures, 'social distancing', quarantine, self-isolation, and in debates and research surrounding treatments, testing regimes, vaccination, 'herdimmunity' etc. our research has been focusing on an animal disease which we have been told (e.g. by vets) should be relatively easy to eradicate through testing and/or vaccination-but bvd hasn't been eradicated, it persists. we ask why does it persist if it's so easy to control? covid- , on a different scale and rapacity as far as humans are concerned, opens up those same questions of why these things are so hard to deal with in practice, because of the complexity of viral infections and their relationships with vulnerable bodies, the logistics of organising medical equipment and care, and the messiness and recalcitrance of human behaviour in relation to 'lockdown' regimes. exemplifying this human-animal parallel, mobility is crucial in thinking about infection. for an agricultural and food system to function people and animals must move, in 'normal' circumstances and even in lockdown, in order for food production to continue, but at the same time movement and the mingling of human and animal bodies facilitates infection. in lockdown, too, addressing viral diseases in animals may be even harder as animal biosecurity and care become more challenging because of attempts to manage human biosecurity, let alone due to human illness affecting farm work. while glib notions of 'one health' (emphasising the interconnectedness of human and animal health) might be criticised for their lack of specificity to particular situations, there is still a sense in which these parallels and interconnections suddenly become more graphic as we see them playing out in our lives in relation to something clearly much more infectious and problematic for us as humans, something shocking our healthcare, social care and politicaleconomic systems. there is a sudden brutal exposure of the entanglement of human and animal lives, not just in terms of the presumed origins of covid- in 'wild' animals and their use as food in certain parts of the world, but in a more mundane way in terms of a sense of shared embodiedness, vulnerability, and subjection to similar biosecurity measures. our food systems and lifestyles together produce these disease risks-in livestock farming systems, and in mobile and interconnected human lives: both provide ideal viral environments. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. biosecurity: spaces, practices, and boundaries pathological lives: disease, space and biopolitics publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.lewis holloway is professor of human geography at the university of hull, hull, uk. his research focuses on changing agricultural practices, particularly those involving farmed animals and technologies. key: cord- - roflnan authors: neethirajan, suresh title: transforming the adaptation physiology of farm animals through sensors date: - - journal: animals (basel) doi: . /ani sha: doc_id: cord_uid: roflnan simple summary: strategy for the protection and welfare of farm animals, and the sustainable animal production is dependent on the thorough understanding of the adaptation physiology. real-time, continuous, and precise measurement of the multi-dimensions and complex intricacies of adaptive capacity of farm animals namely the mental, behavioral, and physiological states are possible only through the sensor-based approaches. this paper critically reviews the latest sensor technologies as assessment tools for the adaptation physiology of farm animals and explores their advantages over traditional measurement methods. digital innovation, diagnostics, genetic testing, biosensors, and wearable animal devices are important tools that enable the development of decision support farming platforms and provides the path for predicting diseases in livestock. sensor fusion data from a multitude of biochemical, emotional, and physiological functions of the farm animals not only helps to identify the most productive animal but also allows farmers to predict which individual animal may have greater resilience to common diseases. insights into the cost of adoption of sensor technologies on farms including computing capacity, human resources in training, and the sensor hardware are being discussed. abstract: despite recent scientific advancements, there is a gap in the use of technology to measure signals, behaviors, and processes of adaptation physiology of farm animals. sensors present exciting opportunities for sustained, real-time, non-intrusive measurement of farm animal behavioral, mental, and physiological parameters with the integration of nanotechnology and instrumentation. this paper critically reviews the sensing technology and sensor data-based models used to explore biological systems such as animal behavior, energy metabolism, epidemiology, immunity, health, and animal reproduction. the use of sensor technology to assess physiological parameters can provide tremendous benefits and tools to overcome and minimize production losses while making positive contributions to animal welfare. of course, sensor technology is not free from challenges; these devices are at times highly sensitive and prone to damage from dirt, dust, sunlight, color, fur, feathers, and environmental forces. rural farmers unfamiliar with the technologies must be convinced and taught to use sensor-based technologies in farming and livestock management. while there is no doubt that demand will grow for non-invasive sensor-based technologies that require minimum contact with animals and can provide remote access to data, their true success lies in the acceptance of these technologies by the livestock industry. adaptation physiology or acclimatization is defined as an individual organism's biological response to environmental stress. adaptation can be broadly classified into genetic (generational or long-term) and non-genetic (phenotypic or short-term) responses to a stressor [ ] . under chronic animals , , of stress experienced over several generations, the animal's acclimatization response becomes genetically "fixed," making the animal adapted to its environment. the physiological and behavioral processes adopted by farm animals in response to environmental changes are not only crucial for their survival, but frequently also affect the profitability and productivity of livestock systems. "farm animals" is a term used to describe a group of animals housed together in a barn or an animal husbandry. these animals are typically raised for the commercial utility of produce such as dairy, leather, meat, eggs, wool, etc. livestock must face the multipronged challenge of physical, chemical, nutritional, and thermal stress [ ] . stressors or the challenges are several and may or may not have a direct influence on the animal performance. factors that act as stressors and thereby influence livestock productivity include age, breed, geographical location, water availability, nutrient availability, photoperiod, environmental conditions, interactions with humans, and management practices [ ] . stressors trigger physiological mechanisms that allow animals to maintain physical equilibrium and homeostasis [ ] . farm animals respond to environmental stressors by altering physiological parameters like rectal temperature and respiration rate, drooling, panting, sweating, heart rate variability, and decreased feed intake [ ] . in order to determine which technological advances can improve both livestock productivity and animal welfare, it is crucial to measure the physiological parameters or adaptation physiology of farm animals. this emphasis on measurement is driving the development of specific sensor-based technologies that can monitor these physiological changes. quantitation of stressors in farm animals often involves invasive techniques that require animal restraint or close contact between animals and humans, which can increase the animals' stress levels [ ] . these techniques are also time-consuming, subjective, and labor-intensive, making them unsuitable for a rational evaluation of stress in farm animals [ ] . smart systems such as sensor technology play an important economic role in this area [ ] , offering considerable long-term financial advantages. the economic welfare modelling framework is a powerful tool for reinforcing policy decision-making in appraising alternate scenarios for animal diseases. sensing platforms and tools are helpful in disease prevention and control strategies, giving them an important role in advancing public health policy [ ] . the economic impact of livestock disease is wide-reaching and multifaceted-direct costs of a disease include production losses in addition to the costs of treatment and preventative care. furthermore, sick animals have a lower economic value. common treatment practices have a critical financial impact on the farmers and can impact long-term herd structure as a consequence of the outbreak. in terms of production parameters, as estimated in a previous study [ ] , milk production in cattle with lumpy skin disease is reduced by up to % during the acute phase and % upon the cattle recover from the disease, revealing the disease's protracted and marked negative impacts. these economic losses can be largely avoided with the implementation and integration of remote sensor technology or automated technologies in agricultural and livestock practices. livestock welfare in farms remains a major concern; in order to protect livestock from poor welfare situations, it is imperative to interpret their behavior as well as their cognitive needs and capacities [ ] . the recent covid- pandemic particularly demands minimal contact with farm animals, and the current situation is only expected to increase the demand for the use of sensor-based technologies and processes that can protect the health of both farm personnel and livestock. driven by an interest in animal welfare, research has gathered speed in the development of new sensing approaches and biosensing methods for non-invasive assessment of physiological stress response in animals [ ] . for instance, remote sensing-based technologies and image processing are being widely researched to aid in the detection of animal health problems and stress levels [ ] . with respect to assessing adaptation physiology in farm animals, sensor technology is considered to be pivotal as it can assist in acquiring time series of behavioral and physiological data. these sensors include biosensors and wearable technologies, which are based on advanced statistical and computer science methods that can be used to predict and assess adaptive responses and resilience in farm animals. automated remote monitoring and detection of animal welfare indicating parameters using real-time analysis of sounds, images, videos, and data tracking for body and body weight conditions may improve biological metrics in livestock [ ] . remote sensor devices such as microphones, cameras, accelerometers, and thermometers can provide credible information when their data is merged with individual animal identification and referenced observations and incorporated in algorithms [ ] . sensors in animal health management can help facilitate timely diagnosis and follow-up treatment for sick farm animals [ ] . the use of sensor-based technologies enables early identification of disease symptoms and subsequent disease management, minimizing the economic losses associated with infection spread in the herd. these are vital for the survival of any business [ ] . moreover, these sensors can be linked to cell phones and other mobile devices so that data can be analyzed and recorded automatically and remotely. the aim of this paper is to critically review sensor technologies as assessment tools for the adaptation physiology of farm animals and explore their advantages over traditional measurement methods. furthermore, the review aims to highlight the challenges involved in the deployment of sensing technologies, especially regarding their applicability in farm settings. the literature cited in this paper were collected using the google scholar and the web of science tools. to showcase the leading-edge research in this field and to ensure the recency and narrow down the search, the author restricted the search to papers published only in the past years. keywords used were, animal sensors; livestock wearables; animal physiology and sensors; farm animal sensing technology; facial recognition and animals; precision livestock farming and sensors; adaptation physiology measurement. both the boolean and individual searches were conducted as part of this study. dairy cattle, pigs/swine, chicken and poultry birds, and sheep were primarily focused as part of the consideration of the common livestock. the number of papers cited in this review is , with that were published before . the papers published before were included as the information on the sensing technologies for farm animals were scant, and to emphasize the content on the need for sensors in the adaptation physiology of animals. precision livestock farming (plf) is a tool for active livestock management with a focus on improving animal welfare and health and enhancing the economic, social, and environmental sustainability of livestock farming. the importance of well-managed animal welfare is not limited to ethical viewpoints; it is also crucial to realize a more efficient process of producing animal products. the metabolic energy balance in a homeothermic living organism includes several different components: basal metabolism, the thermal component, the physical component related to movement or delivering power, the production term (meat, milk, eggs), and the mental component. when applying stress-monitoring techniques originally developed for humans, animal frustration can be monitored in real time. this indicates that real-time animal welfare monitoring based on physiological signals is, in fact, a realistic pathway ( figure ). parameters such as temperature, heart rate, peripheral vasodilation, respiratory rate, sweat production, basal and energetic metabolisms, feed intake, diseased conditions, and sound all respond to stressful conditions [ ] . farmers can more quickly detect livestock health problems by evaluating their animals' physiological responses via measurements of the temperature in body parts, respiration rate, heart rate variability, body weight, feed, emotions, non-invasive biomarkers and water intake, activity, and movement. an animal's ability to maintain homeothermy indicates that the animal is better adapted to the climatic variations of its environment. tolerance of higher temperatures, for instance, is identified by animals' capability to dissipate excess body heat and keep their standard body temperature within the limits of homeothermy [ ] . for this reason, monitoring changes in animals' body temperature can help determine their physiological stress responses to their environment. as an example, it has been shown that broilers in high and medium-density housing have higher body, wing, head, neck, and skin temperatures than those housed in lower densities [ ] . these results can be used to estimate potential stressors for farm animals. the cloacal temperature of a bird is typically measured by inserting digital thermometer into the bird's cloaca [ ] . this invasive procedure requires that the animal be manually restrained and handled [ ] , which would lead to stress-induced hyperthermia. shortcomings of these conventional temperature measurement methods make them not practical for regular heat stress monitoring [ ] . for an accurate, reliable, and continuous measurement of body temperature, farmers can deploy biologgers such as telemetry devices or the radio-telemetry data loggers by integrating with halters or collar/leg sensors for larger animals such as cows, pigs, and horses. body temperature measurement with thermal infrared sensors is considered safer and more efficient than conventional techniques for livestock including broilers and poultry birds [ ] . infrared thermometry is non-obstructive in nature, which makes it suitable for assessing animal stress. furthermore, the use of digital thermography (thermographic camera) makes it possible to detect even minimal temperature variations [ ] . animals , , x of the limits of homeothermy [ ] . for this reason, monitoring changes in animals' body temperature can help determine their physiological stress responses to their environment. as an example, it has been shown that broilers in high and medium-density housing have higher body, wing, head, neck, and skin temperatures than those housed in lower densities [ ] . these results can be used to estimate potential stressors for farm animals. the cloacal temperature of a bird is typically measured by inserting digital thermometer into the bird's cloaca [ ] . this invasive procedure requires that the animal be manually restrained and handled [ ] , which would lead to stress-induced hyperthermia. shortcomings of these conventional temperature measurement methods make them not practical for regular heat stress monitoring [ ] . for an accurate, reliable, and continuous measurement of body temperature, farmers can deploy biologgers such as telemetry devices or the radio-telemetry data loggers by integrating with halters or collar/leg sensors for larger animals such as cows, pigs, and horses. body temperature measurement with thermal infrared sensors is considered safer and more efficient than conventional techniques for livestock including broilers and poultry birds [ ] . infrared thermometry is non-obstructive in nature, which makes it suitable for assessing animal stress. furthermore, the use of digital thermography (thermographic camera) makes it possible to detect even minimal temperature variations [ ] . body temperature can be efficiently and accurately determined with non-invasive infrared technique [ ] . in broilers, for instance, the temperature of the region around the eye is considered to be indicative of the total body temperature because of lack of feather insulation around the eye region [ ] . all objects produce radiant heat in the infrared region of the electromagnetic spectrum. as bodies have temperatures above absolute zero, they emit radiation that forms an electromagnetic spectrum that can be absorbed by other surrounding bodies [ ] . thermal infrared (tir) sensors generate these images (thermograms) by capturing the infrared radiation emitted by objects. the information generated by these images is then determined by algorithms to detect the temperature body temperature can be efficiently and accurately determined with non-invasive infrared technique [ ] . in broilers, for instance, the temperature of the region around the eye is considered to be indicative of the total body temperature because of lack of feather insulation around the eye region [ ] . all objects produce radiant heat in the infrared region of the electromagnetic spectrum. as bodies have temperatures above absolute zero, they emit radiation that forms an electromagnetic spectrum that can be absorbed by other surrounding bodies [ ] . thermal infrared (tir) sensors generate these images (thermograms) by capturing the infrared radiation emitted by objects. the information generated animals , , of by these images is then determined by algorithms to detect the temperature change. thermographic images may point to alterations in blood flow resulting from increased body temperature due to stressful environmental conditions. these changes can be related to blood flow and heat transfer in animals [ ] . the temperature of the eye area measured by tir sensors has been shown to correlate significantly to changes in core body temperature [ ] . temperature measurements from different body parts provide information about animal health and allow for timely decision-making for livestock welfare (e.g., isolating animals with higher body temperature or managing the internal temperatures of animal housing units). respiratory rate (rr) is one of the variables used to assess the physiological adaptability of animals [ ] . in response to alterations in environment, such as transport of cattle by road, animals' respiratory rates increase to maintain homeothermy. evaporation is a key regulatory response for animal body temperature [ ] . rr has been shown to be affected by high milk yield, pregnancy, forced activity, excitement, and pathological conditions [ ] . an increased rr is a significant stress indicator, particularly in thermally stressed animals [ ] . thermal stress results in a reduction in milk production, animal fertility, and general welfare [ ] , so early detection of alterations in rr can help farmers take specific measures to prevent animals from stress, thereby avoiding production losses while addressing animal welfare. visually counting flank movements is the most common technique for assessing respiratory rates in cattle [ ] . however, this is a labor-intensive and time consuming method, and the continuous assessment of rr in long-term studies can be difficult and physically demanding, eventually resulting in miscounts [ ] . misinterpretation of results can also occur from nonspecific flank movements that do not arise from respiration [ ] . moreover, the continuous presence of a person can cause the animals stress and influence their respiratory rates, misleading the measurements. therefore, rr sensors were introduced to assess rr without causing stress to animals. the initial respiratory sensors tested on animals were designed for human application. these devices consisted of a belt attached around the animal's chest (similar to a holter) that measured thoracic and abdominal movements. another system to monitor respiratory rate is a laser distance sensor during milking [ ] . respiratory sensors have been reported to accurately monitor respiratory rates in cattle [ ] . sweating is a physiological mechanism to regulate body surface temperature. sweat is generated by the sweat glands in the rainy and dry seasons. the maximum mean values are reported in the mornings and afternoons during dry season, which aids heat dissipation by evaporation; maximum sweating capacity is achieved under high temperatures and low humidity, when the sweat glands are stimulated by increased blood volume to the epidermis [ ] . the composition of sweat could be helpful in determining the internal physiological changes or disturbances in an animal. sweat glands on pigs are considered non-functional as they are not stimulated by heat stress. imperceptible perspiration in the form of water vapor may have biomarkers such as mirna (micro ribonucleic acid) or micro-concentrations of cortisol or hormones. validated biomarkers from the sweat of dairy cows, pigs, and other farm animals are not yet available and would open up new avenues of non-invasive sensing. immunosensors can target biological fluids like saliva and sweat instead of blood, making these sensors less stressful and invasive for animals. immunosensors provide highly sensitive and specific assessment of hormones like cortisol and lactate in animal biological fluids using label-free electrochemical and chronoamperometric methods. for instance, a mobile, handheld potentiostat integrated with bluetooth communication and power source can be used for point-of-care applications. bioengineers have designed sensors with a sandwich-like structure that can contain the sensing animals , , of mechanisms, secure the biosensor to skin, and use capillary action to draw sweat or other fluids towards the sensing mechanism [ ] . overall, the immunosensor showed remarkable specificity and sensitivity in addition to its non-invasive and point-of-care capabilities, making the diagnostic tool a versatile sweat-sensing platform. skin-deployable microfluidic platform provides a significant capability for on-the-go real-time collection and monitoring of sweat biomarkers. resettable epifluidic sweat patches can be placed on the skin of pigs or the sweat-secreting glands of dairy cows to collect and analyze sweat composition as a visual indicating sensor [ ] . the cardiovascular system is regulated by the autonomic nervous system and controls the physiological systems impacted by stress [ ] . the vagal element of the autonomic nervous system regulates heart rate during stress in farm animals [ ] . heart rate increases when animals are subjected to heat above their tolerance limits [ ] , so changes in heart rate can indicate physiological and psychological stress, disease, and coping strategies in animals [ ] . conventionally, electrocardiogram (ecg) has been used to detect heart rate variability. despite providing good signal quality, its long-term use for continuous heart rate monitoring is inconvenient as the wet electrodes must be in close contact with skin. additional limitations include restriction of movement, allergic reactions, skin irritation, and signal degradation due to continuous contact with wet electrodes [ ] . although dry electrodes and non-contact electrodes are available, they require the use of a chest band to keep the electrodes in place. this can be inconvenient, especially for use in animals. photoplethysmography (ppg) is a non-invasive and cost-effective method for the detection of blood volume alteration. ppg is an optical measurement that uses infrared lights to detect changes in blood volume in the microvascular bed of tissue [ ] . this contrasts with the electrocardiogram (ecg), which measures heart rate by placing electrodes on the chest of the target animal. doppler radar-based sensing technologies include: ultra-wide band radar [ ] and near-field coherent sensing. machine-knitted washable sensor platform based on textile has been developed and validated for precise epidermal arterial pulse waves and respiratory signals simultaneously [ ] . however, this tool has been demonstrated only for biomedical human applications and would need further modifications for adoption in livestock sector. researchers have experimented with non-invasive wearable sensing systems composed of photoplethysmogram and electrocardiogram (ecg) for continuous monitoring of vital signs in companion animals [ ] , but these systems have yet to be fully explored in the livestock sector. body size and the shape of animals, chewing habits of pigs, licking habits of cows, body weight, dust and the harsh barn and farm environments, and discomfort to animals are some of the factors that make the adoption of hr and rr wearable sensing systems challenging for animal farming (table ) . commercially available textile-based wearable sensors, such as hexoskin, biometric shirt, dinbeat uno's multiparameter harness-sensing tools developed for humans and pet animals for measuring heart rate variability, respiratory rate, and maximal oxygen consumption would find new avenues of application in livestock farming. studies on cattle behavior have indicated that illness causes animals to spend less time feeding and more time resting [ ] . feeding and ruminating behavior in cattle provides significant information about their productivity, health, and welfare [ ] . changes in ruminating and feeding timing of cows are indicative of an underlying alteration in their welfare and comfort [ ] . it has also been reported that cows diagnosed with metritis spent less time ruminating than their healthy counterparts during the pre-and post-calving periods [ ] . as alterations in feeding behavior could also be indicative of illness in animals, several sensing methods have been employed to measure feeding behavior. wearable accelerometers wearable accelerometers have been evaluated as a tool for measuring cow behavior [ ] . hobo accelerometers, which are attached to the jaws of cows, can record the time each animal spends grazing and ruminating [ ] . neck-mounted accelerometers, based on either a multi-class support vector machine (svm) or a decision-tree algorithm [ ] , have been tested for the evaluation of bovine ingestive behaviors. neck-mounted triaxial accelerometers have been validated to assess drinking events, along with the algorithm that can distinguish feeding from drinking from a water trough [ ] . a noseband sensor called rumiwatch was developed to monitor eating and ruminating activities in dairy cows [ ] . animals deal with stressors like social changes, environmental changes, satiety, and infection by changing their behavior. monitoring this behavior on a large scale becomes impractical because of the manpower required for continuous monitoring of a large groups of animals. wearable sensor technologies make it possible to simultaneously measure real-time physiological parameters in a herd on a large scale when coordinated with appropriate interpretations of outputs [ ] . thus, wearable sensor technologies have an edge over traditional herd-based approaches as the data from wearable sensors can be analyzed immediately, enabling quick reaction times. water flow sensors can monitor group drinking behavior in pigs and are considered more precise than experienced observers [ ] . the issues and limitations of water sensors includes variable water flow rates, installing sensors in existing plumbing, short drinking bouts, unspecified drinking behavior when a snout is in an outlet, and wastage of water [ ] . the requirements of an rfid system include an rfid transponder or ear tag and an rfid antenna located at the drinking sink or feeding unit. rfid systems (at drinking and feeding areas) have been used to monitor the events and duration of feeding and drinking behavior of individual pigs [ ] . high-frequency (hf), low-frequency (lf), and ultra-high-frequency (uhf) are the frequency ranges typically used for rfid tags. these systems can have significant differences in read ranges as well as varying responses to the influence of materials, especially water and metal [ ] . a combination of stationary rfid, accelerometer, and water flow meter sensors has been used to measure beef cattle drinking behavior and herd water consumption in grazing systems. this approach was determined to be reliable in recording behavioral measures, such as the frequency, duration, and number of visits to the water point per animal as well as the duration of drinking events per animal visit [ ] . garrido-izard and colleagues have monitored a combination of ear skin temperature sensors, body weight measurement, and the amount of feed consumed and the duration per animal in order to identify animal behavior changes based on the integration of their intake patterns and the thermal data. animals with higher-temperature measurements showed less thermal variability, and vice versa. the study also showed that animals with less alteration in feed intake during breeding have higher efficiencies [ ] . posture and lying behaviors can be automatically quantified to increase the productivity and welfare of domesticated animals because changes in posture and activity indicate health and welfare issues [ ] . for instance, changes in behaviors like walking, standing, and lying can indicate sickness in cows [ ] . monitoring postural changes is important in assessing calf or swine wellness or painful conditions. numerous studies have studied postural behavior differences in cows who receive painful stimuli [ ] . accelerometers accelerometers, electromechanical devices that measure acceleration forces, have proven very accurate in monitoring activity and movement in the animals. the use of accelerometers has recently been widely used to quantify or evaluate animal behavior [ ] , grazing behavior in cattle [ ] , lying behavior in sows and lameness in cows [ ] . the accelerometer's smaller size and the versatility of the data generated make these technologies effective for examining animal behavior in farm settings [ ] . accelerometers are considered to be most efficient in assessing activity in pigs. increased activity is indicative of stress in pigs, while reduced activity has been associated with disease [ ] or changes in the environmental conditions of a barn [ ] . analysis of data from two-dimensional accelerometers has revealed that the percentage of time spent standing increases in calves after castration [ ] . in another study, it was shown that five days after castration, calves preferred more time in lying down and less time in walking activity [ ] . the differences between these two studies could possibly stem from the duration of monitoring and a considerable time-dependent change in behavior. the current animal welfare determining factors are mostly conducted at a single point in time (i.e., providing water and food and treating diseases or illnesses as they arise), these assessments are often considered insufficient. pattison and colleagues have designed a methodology for the continuous monitoring of interaction between animals fitted with proximity sensors attached to neck collars. the resulting data and levels of serum cortisol concentrations are expected to provide a visual map of the social structures of each group. this will allow scientists to explore the potential of proximity sensors as a welfare monitoring platform for measuring an animal's freedom to express its normal behavior in natural state [ ] . accelerometer sensors have been used to accurately measure active and not-active behaviors of tiestall-housed dairy cows [ ] and predict the behavior of dairy cows from the signal pre-processing sensor data [ ] . lying behaviors in calves have also been analyzed after administering analgesic drugs. lying behavior decreased in calves following the induction of experimental lameness using an amphotericin b synovitis-arthritis induction model [ ] . accelerometer sensors have also been effectively used in the regular assessment of behavioral changes in response to pain [ ] . the use of two accelerometers can automatically quantify the lying behavior in free-farrowing sows; challenges of automation in the lying behavior have been addressed in both free-farrowing sows as well as sows housed in movement-restricting barn environments [ ] . more recently, data from triaxial accelerometers has been used to define general behavior recognition framework in the form of a hybrid model combined with biomechanical principles and machine learning tools [ ] . in domestic animals, aggression between individuals is a serious stressor and cause of injuries. research is increasingly exploring facial expressions as a non-invasive means of obtaining quantitative data on an animal's emotional state. camerlink and colleagues have demonstrated the use of video imaging in analyzing the emotional state of pigs in an event of aggression or confrontation in dyadic contest. facial metrics can be a powerful way of measuring the aggressive intention of animals. facial metrics after retreat and/or during the episode of aggression in pigs differed significantly from the facial features prior to and during aggression [ ] . mounting behavior, observed in both female and male pigs throughout their lifetime, is mostly prominent during estrus. this behavior is manifested as follows: a pig typically places two front hoofs on the head or the body of another pig in the lying position. mounting behavior can cause epidermal wounds, lameness, and fractures, resulting in economic losses and decreased productivity. li and colleagues have developed a learning algorithm that detects swine mounting behavior based on the visible light images to enable timely detection and intervention [ ] . this method has demonstrated high accuracy, sensitivity, and specificity. however, the sample size of the research included only four mini pigs, so the system still needs to be validated on a larger scale. pressure mats are composed of a series of array of pressure sensing components with the measuring frequency that enables the mats to differentiate the simultaneous impact of different limbs [ ] . systems from various pressure sensor manufacturers have been able to successfully assess locomotion in healthy cows, horses, and sheep [ , , ] . these systems were capable of assessing lameness in cows [ ] . pedometers objectively measure an animal's total number of steps and the total distance travelled via an algorithm that calculates the steps from the raw data [ ] . while pedometers are comparatively easy to deploy and use, there is considerable variation in the number of steps traveled by each calf on different days and environmental conditions. there might be an association between the distance traveled by calves and stressful and painful procedures; one study determined that calves traveled fewer steps for four days following castration [ ] , while another study showed the association between stress experienced by calves and the number of steps traveled following castration. stress influences the distance traveled by calves, though gender differences must be accounted for in such studies; it is observed that steers travel fewer steps per day than bulls [ ] . pedometers have reportedly been useful in intelligently designed experiments to investigate changes in behavior after the animals go through a painful experience. pedometers have been demonstrated in identification of early lameness in dairy cattle, though a % decrease in activity was required in order to accurately detect % of the lame cattle [ ] . pedometers can be valuable tools for detecting and assessing musculoskeletal pain as they rely on the direct quantification of locomotion. tir and rgb (red, green, and blue) image based sensor data has reportedly been useful in evaluating the walking posture and gait analysis of cattle through recorded videos [ ] . changes in posture while walking can indicate skeletal problems in livestock, so tir sensors can be implemented to judge animal well-being. various research investigations in the last decade have demonstrated the advantage of gps telemetry devices for assessing livestock behavior when used in combination with other sensors/devices [ ] . this has been used to distinguish between activities or assess energy expenditure [ ] . gps collars with activity sensors form an efficient technique for simultaneously monitoring the movements of grazing livestock and inferring animal behavior [ ] . rtls have been developed to locate the position of an object anywhere inside a specific area. the design of an rtls consists of a receiver positioned closer to the desired monitoring space, active or passive tags deployed on the target objects, and a hardware and software to receive and interpret positional data. tags used with rtlss are typically smaller in size and have a longer battery life than existing gps systems [ ] . rtls technology has been investigated to assess the association between behaviors like distance travelled and duration of time spent at feed bunk with clinical illness scores. an association was also found between the distance travelled by calves and the level of lung consolidation by rtls monitoring, which indicated that assessing movement can help evaluate the wellness status of livestock animals [ ] . the associations derived from these studies strongly suggest that rtls platform is a legitimate tool for producing quantitative measurements of cattle activity. they can be further helpful in evaluating significant changes in pain status or wellness in response to an intervention [ ] . rtls has the distinctive advantage in monitoring an animal's location anywhere within the farm; therefore, it does not restrict assessment to only drinking and feeding behaviors. vocalization process in animals happens through the active generation of sounds with organs to express distinct physiological status. vocalization may be spontaneous or triggered by an external event [ ] , but their reflection of an animal's inner state makes them an efficient tool for monitoring animal wellbeing, stress response, and interaction among species. heat stress is an important environmental stressor affecting poultry production and welfare, specifically growth, egg production, and impaired poultry health. changes in behavioral and physiological responses due to heat stress might manifest as distinct vocalizations such as gakel, squawk, and alarm calls [ ] . du et al. [ ] have demonstrated a correlation between vocalization in egg-laying hens and the temperature humidity index (thi). the authors specifically concentrated on the quantitative measurement of frustration-related vocalizations (squawk, gakel, and alarm calls) and concluded that specific vocalizations such as poultry squawk and alarm calls are significantly correlated with thi. furthermore, because of thermal inertia in a henhouse, which is a potential early warning detection tool to avoid lags in monitoring the ambient environment [ ] . acoustic monitoring (or vocalization measurement) is a non-invasive and an accurate method of measuring biological responses in livestock; it can be used as an indicator of animal well-being in precision livestock farming, which focusses on addressing animal welfare concerns by facilitating the automated, continuous monitoring of livestock and enabling timely and appropriate interventions [ ] . changes in sound pressure can be transformed into electrical signals, which are then received by audio equipment and processed as digital signals using signal processing techniques through standard computers. the definite evaluation of audio data is typically based on spectral analysis, where acoustic signals automatically separate into bands of appropriate frequencies. this is then followed by subsequent processing [ ] . vocalization in pigs can be used for several different purposes, such as identification of sex, age (figure ), and stress levels through a decision-tree to classify distress condition in pigs with an accuracy of . % using the machine-learning technique [ ] . in turkey farming, cannibalism is identified as a major problem resulting in animal stress and loss of productivity. certain behavioral changes have been identified in animals prior to cannibalistic behavior, including pecking activity. acoustic data analysis in combination with machine learning techniques has been tested as a tool to continuously monitor pecking activity for potential use on turkey farms [ ] . currently, there are no tangible ways to measure reproductive performance, especially with respect to uterine contractility and suitable diagnostic tests for the onset of labor in sows. unsuitable uterine contractility can result in embryonic loss, miscarriages, ectopic pregnancies, and abnormalities of puerperium in sow [ ] . one potential diagnostic tool is the measurement of bioelectric signals from swine uterus using electromyography and magnetomyography. to overcome the drawbacks in the crude uterine contraction information obtained through tocodynamometer or with an intrauterine pressure catheter, a d uterine electrical activation pattern measurement system has been demonstrated using electro-myometrial imaging (emmi) [ ] . emmi surface electrical recording has been shown to be a safe, accurate, non-invasive, and feasible method to measure uterine contractions in sheep. electrophysiological studies using electromyogram sensing systems by domino et al. [ ] demonstrated that myoelectric activity in various regions of sows' reproductive tract can be used as a reliable measurement to assess pregnancy health as the contractility regulation was superiorly observed in the uterine horn tip. a tocodynamometer provides an indirect measure of the intrauterine pressure and is the current standard method to determine labor. a combination of electromyography and magnetomyography sensing platforms is expected to replace tocodynamometers in the near future. in a preliminary study, brassel et al. [ ] have evaluated the performance of automated health monitoring integrated with an accelerometer-based estrus detection system (ods) for dairy cows on pasture. although the ods was able to flag health problems faster than human farmers, the use of ods generated an extremely high rate of false positives [ ] . currently, the activity-based sensors used in the animal farming industry are optical or animal-attached sensors. while sensors attached directly to animals are difficult to manage, the sensitivity of optical sensors decreases in the presence of dirt or changes in the ambient lighting conditions. in cases where spatial distribution of the animal movement or the activity is irrelevant, manteuffel [ ] has demonstrated that simple stationary sensors to measure animal activity can be beneficial due to easy signal interpretation requiring little computational power. soon, doppler-based radar sensors will find new avenues in the dairy cow guided gate system to measure the cow's heart rate and breathing pattern before entering the rotor milking machines. currently, there are no tangible ways to measure reproductive performance, especially with respect to uterine contractility and suitable diagnostic tests for the onset of labor in sows. unsuitable uterine contractility can result in embryonic loss, miscarriages, ectopic pregnancies, and abnormalities of puerperium in sow [ ] . one potential diagnostic tool is the measurement of bioelectric signals from swine uterus using electromyography and magnetomyography. to overcome the drawbacks in the crude uterine contraction information obtained through tocodynamometer or with an intrauterine pressure catheter, a d uterine electrical activation pattern measurement system has been demonstrated using electro-myometrial imaging (emmi) [ ] . emmi surface electrical recording has been shown to be a safe, accurate, non-invasive, and feasible method to measure uterine contractions in sheep. electrophysiological studies using electromyogram sensing systems by domino et al. [ ] demonstrated that myoelectric activity in various regions of sows' reproductive tract can be used as a reliable measurement to assess pregnancy health as the contractility regulation was superiorly observed in the uterine horn tip. a tocodynamometer provides an indirect measure of the intrauterine pressure and is the current standard method to determine labor. a combination of electromyography and magnetomyography sensing platforms is expected to replace tocodynamometers in the near future. in a preliminary study, brassel et al. [ ] have evaluated the performance of automated health monitoring integrated with an accelerometer-based estrus detection system (ods) for dairy cows on pasture. although the ods was able to flag health problems faster than human farmers, the use of ods generated an extremely high rate of false positives [ ] . absence of disease is an essential component of overall animal health and well-being. disease, lameness, and limb disorders pose a significant challenge to the dairy industry today. along with dairy cows, sheep flocks around the farm also face the most persistent and common health challenge of lameness. lameness results in decreased milk production and is an important motivator for early culling of animals. lameness is painful, and animals suffering from pain frequently diverge from their normal behavior by changing activity, gait, appetite, posture, and appearance [ ] . the amplitude and frequency of ruminal contractions in cattle are impacted by metabolic diseases (such as ruminal acidosis and hypocalcemia) and other diseases that cause fever or pain. wireless intraruminal bolus sensors inserted through the esophagus have been developed to monitor the temperature and ph values of the rumen and reticulum [ ] . the assessment process has been simplified by the advent of commercially available boluses for the measurement of reticuloruminal ph, where the boluses regularly transmit ph information wirelessly to a central processing region. wireless and indwelling sensors for rumen facilitates high-resolution ph measurements in determining the kinetic behavior and identification of rumen acidosis. the data from this analysis can assess the physiological status of the rumen and, therefore, the whole animal. while indwelling rumen ph sensors allow for continuous measurement of ph in individual animals, their application is limited; the differences in ph between the ruminal locations cannot be measured, and there is also substantial drift in the baseline sensor data from the non-retrievable sensors [ ] . the biochemical interactions within and outside the cellular environment lead to the end products, which are the metabolites. comprehensive measurement of metabolites, called metabolomics, helps researchers gather accurate and sensitive data in order to better depict the phenotype [ ] . metabolomics is a non-invasive tool to identify phenotypes, phenotypic changes, and assessment of dietary responses [ ] . traditional methods of quantification of phenotypes, such as feed intake and residual feed intake, are time-intensive, costly, and require specialized equipment [ ] . in contrast, phenotype/trait quantification using the metabolomics approach is cost-effective [ ] . there remains a dearth of metabolomic studies on livestock, especially studies that utilize sensor technologies. biosensing technologies have started gaining attention in farmed livestock animal studies as they show promising potential to address the relevant issues of reliable tests, cost of equipment/devices, and early detection of disease/stress [ ] . new and emerging biosensing technologies have the potential to improve livestock animal management and the associated factors [ ] . these technologies monitor animal welfare by evaluating metabolic and stress biomarkers. the non-invasive early detection of stress by assessing physiological responses will help farmers take timely safety measures for animal health and welfare. further advancements in biosensor technology shall generate new approaches for real-time evaluation of metabolic and physiological responses. a non-invasive technique to detect volatile organic compounds or a reliable validated biomarker allows farmers to responsibly assess animal stress and respond quickly. such compounds can be identified in animal breath, skin, urine, feces, blood, and vaginal fluid [ ] . gaseous metabolites present in the breathing air of cattle include hydrogen, carbon dioxide, and volatile organic compounds such as phenol and methane. methane emission in ruminants is an integral part of their energy metabolism and is thus a valuable indicator of their physiological state [ ] . a study conducted to monitor methane emission in cows using a fourier transform infrared (fitr) sensor assessed the breath of the animals and accurately measured the methane-carbon dioxide ratio [ ] . others have assessed methane levels in milk using the mid-infrared (mir) spectra biosensor in cows [ ] . the results have concluded that the extent of methane present in milk is directly dependent on the animal's lactation stage. phenol and p-cresol emitted from nasal secretions clearly indicated the bovine respiratory disease in cattle [ ] . real-time detection of volatile organic compounds using biosensors by integrating with the halter or the neck collar of cattle or pigs would serve as a non-invasive, portable disease-sensing tool. the concentration of metabolites in tears is correlated to their concentration in the blood. thus, tears can be used for the non-invasive continuous evaluation of metabolites. currently, there are no devices or sensors available for measurement in livestock tear fluids as there are no scientifically validated biomarkers. this opens up new avenues of wearable 'eye-based' sensors for livestock. hormone levels related to the reproductive physiology of animals are usually determined to predict the animal's reproductive state. the herd navigation system is a commercially available biosensor that can quantify levels of milk progesterone [ ] . a handheld, smartphone-based, rapid on-farm progesterone immunosensor has been developed to monitor milk progesterone levels in cows [ ] , with a biosensor composed of a monoclonal anti-progesterone antibody (mab) immobilized on an electrode. others have also reported the use of immunosensors for the assessment of hormone levels in saliva, where the method is quick and non-invasive in nature. farm livestock is often challenged by outbreaks of viruses like bovine herpes virus- (bhv- ), foot-and-mouth disease virus (fmdv), and bovine viral diarrhea (bvd) virus, to name a few. various immunosensors have been built to determine the presence of these pathogens in animal sera [ ] , and these biosensors have produced faster results than conventional enzyme-linked immunosorbent assay (elisa) and polymerase chain reaction (pcr) based methods. biosensor technology offers the advantage of being used in combination with other devices and being open to multiplexing, making it suitable for large-scale applications for agriculture and livestock. specific internal or external stimuli trigger short-term, intense states known as emotions, which can lead to behavioral decisions and social interactions (i.e., approaching or avoiding the stimuli) [ ] . emotions have been shown to be of paramount importance in animal welfare, especially in the production chain of farm animals [ ] . studies have suggested that communication by vocal contagion [ ] , monitoring ear posture [ ] , facial expressions [ ] , and body language in livestock animals can provide a better understanding of their emotional adaptation [ ] . however, this requires skillful, experienced workers and may provide erroneous diagnosis [ ] . researchers are evaluating coding systems that could provide objective readings of animal facial expressions instead of guessing the meaning of their expressions. a coding system precisely describes the meaning of different facial expressions such as squinting eyes, posture of ears, eye white region, or pursing lips when an animal feels a particular emotion. such techniques have been developed and tested on domestic animals in some studies. for instance, equifacs has been developed as an anatomy-based objective tool to determine the systematic recording of facial expressions and pain scoring in horses [ ] . however, the scientific community has still not come up with a detailed validation of the outcomes of such a system in farm animals. it is hypothesized that in the future, developers could create a smart phone app that could decipher the emotional status of an animal when the user held the phone in front of the animal's face. although sensor technology has been proven to provide accurate and reliable results in farm animal assessment, it poses a unique set of challenges. for instance, optical sensors such as video cameras, infrared thermography, or depth cameras are commonly used to automatically assess general activity. these stationary optical devices are prone to reduction in sensitivity due to dirt and dust [ ] . depth cameras are susceptible to disturbances from heat sources including sunlight (in the case of infrared-based systems) or the color and structure of the animals' feathers or fur [ , ] . radiation measurements rely on the analysis of the animal's emissivity and conductivity values. furthermore, infrared sensors need specialized ir translucent window cover materials in comparison to plastics or steel based covers, as the sensors are prone to damage by physical forces due to the harsh farming environments [ ] . studies that have attempted to show the correlation between broiler outer body temperature and core body temperature used ir cameras under well-controlled research conditions [ ] . the high-quality ir cameras used in such studies are expensive and remain impractical for commercial applications [ ] . ppg has contact requirements, so its advanced version, photoplethysmographic imaging (ppgi), is being considered for use in livestock. unlike the contact-based requirements in ppg, ppgi is camera-based, low-cost, non-contact, and convenient. it is also preferable over a ppg sensor in areas of hygiene, animal welfare, and practical deployment for housed animals [ ] . however, even ppgi technique is not completely free of loopholes. it requires that subjects face the camera and remain motionless during recording. ppgi signals are also prone to illumination variations and motion-induced artifacts (table ) , especially while dealing with webcams through ambient light [ ] . the era of "big data" is interdependent on the development of novel sensing technologies that allow for rapid and inexpensive collection of observations and data [ ] . the trend towards agricultural applications of big data techniques and methods is not strictly about primary production; farmers also aim to improve the efficiency of the entire supply chain and alleviate concerns related to food security [ ] . the extent of literature on the use of big data in smart farming is limited in peer-reviewed literature, but the commercial viability of internet of things (iot) and new technologies for wireless connectivity is generating huge amounts of data that can be used for end-to-end livestock management [ ] . furthermore, as all this data is available in real time, it can be used to support decision-making capabilities that were incomprehensible before. the scope of the use of big data in adaptive physiology includes automatic phenotype identification of animal breeds using computer vision and machine learning techniques [ ] , genomic prediction, and disease detection for sub-clinical mastitis applying machine learning-based prediction platforms on milking datasets to study the best predictive models of sub-clinical mastitis [ ] . other methodologies exploring the detection of sub-clinical mastitis include the use of cytometric fingerprinting and machine learning [ ] . big data and machine learning have been used for the analysis of animal behavior [ ] . computer vision-based methods have been employed for automatic recognition in commercial farms using spatial and temporal information of the nursing behavior of animals and individual pig recognition, with accuracy rates of . % [ , ] . machine learning tools have been used to evaluate cattle behavior and feed intake using predictive clustering trees (pct) [ ] and by combining accelerometer data with machine learning to analyze cattle behavior including walking, grazing, ruminating while standing, resting while standing, ruminating while lying, and resting while lying [ ] . while this method resulted in excellent behavior prediction accuracy, the machine struggled to differentiate cow postures based on data from a single accelerometer. other methodologies to determine feed intake include the adoption of a machine vision system for feed intake by individual animals based on deep convolutional neural networks (cnns) models, and a low-cost rgb-d (red, green, blue, depth) camera [ ] . big data and machine learning are being evaluated in several other areas as standalone methods or in combination with conventional sensors to evaluate adaptive behavior in livestock. these technologies can determine several other physiological parameters, such as heat stress in dairy cows. this method is considered superior to current approaches of determining stressors as several machine learning algorithms consider nonlinearity in the data, thereby removing the subjectivity [ ] . big data has been used in the analysis of animal behavior, quantitative risk assessment for animal disease transmission, and implementation of practices for risk-reduction [ ] . in near future, big data application would be commonplace in analyzing animal behavior and welfare. big data's true potential in practical terms will undoubtedly require close collaboration in fields such as computer science, engineering, mathematics, statistics, and the livestock industry to enable the development of cutting-edge approaches to analyzing large quantities of heterogeneous data [ ] . predictive machine learning approaches, in combination with sensor-based data, can prove invaluable in addressing the challenges ahead in animal sciences. some of the costs associated with implementation of sensors technology for investigation and measurement of adaptation physiology of farm animals are expenses involving software, hardware sensing electronic components, data storage and information costs, data processing, and learning costs for the producers to develop the management framework. size of the farm greatly influences the adoption and profitability of using sensors technologies as the cost/benefit estimations need a specific minimal farm size to depreciate the investments. the benefits of sensing technologies that enable real time data collection on the animal behaviors and the adaptation physiology are beginning to be validated. benefit-cost ratio, internal rate of return, payback period, net present value, and milk production measures in evaluating the economic value of the investment in precision livestock farming platforms such as the automated estrus detection tools [ ] assured farm profitability and enhancement in production efficiency. cost-benefit analysis of four fictive pig and dairy farms by hammer et al. [ ] showed that the ultra-high frequency and radio frequency identification tags (uhf-rfid) sensor platform in the simultaneous detection and hotspot monitoring of fattening pigs and dairy cows is economically advantageous. however, this study was conducted based on only fictive farms, and did not include additional benefits such as animal welfare indicating parameters for supporting quality marketing, and the advantages associated to traceability programs. the advent of sensor technologies has revolutionized the assessment of livestock behavior and stress responses. sensor-based technologies have contributed immensely to minimizing the stress on animals, improving animal welfare, and consequently preventing economic losses. early detection of physiological responses can help farmers take targeted measures to alleviate the strain on their animals, improve animal welfare, and prevent performance losses by predicting potential disease outbreaks. sensor technologies have an edge over traditional assessment methods as they are timesaving and can automatically take measurements at desired time (table ) . these efficient technologies aid in the evaluation of certain responses that can then be used to precisely estimate physiological states such as stress, welfare, fertility, health, metabolism, and disease. on the technological front, a number of novel sensor-based methodologies are still in the explorative phase of development. some promising sensor candidates include microrna-based sensors for detecting bovine respiratory syncytial virus, sensors for the detection of salivary hormones such as luteinizing hormone for the detection of bovine estrus, and electrochemical sensors to detect antibodies against influenza a and b, to name a few. these rely on highly specific biomarkers for specific physiological conditions. none of the currently available commercial devices and sensing systems provide a combination of size, functionality, and wearability that meets the requirements of the livestock sector or allows for the movement of animals and the simultaneous measurement of physiological parameters such as respiratory and heart rate. this gap calls for research in the design and development of sensing technologies for next generation of precision livestock farming. it should be noted that some of the precision livestock farming sensing platforms do not require internet connectivity and can possibly function by using bluetooth and/or radio frequency spectrum. these sensors may be used in isolation in which encrypted data can be collected from multiple animals and barn systems, compiled, and sent to a local computational platform for data processing [ ] . with the advent of digital twins technologies in livestock farming, real-time data transmission collected by the multitude of sensors from farms is one of the basic design requirements. moreover, the oie-world organization for animal health, strongly advises real-time detection and transmission of animal health and epidemiological data from the sensors and devices [ ] to producers, inspection, and government agencies. hence, internet connection in livestock farms becomes necessary. the limited internet connectivity and the data moving capacity for both rural and urban farms remain a bottleneck for adoption of sensor-based platforms in investigation of adaptation physiology of farm animals. globalization, the post-covid- world, rising per-capita incomes, human population growth, ecological pressures, and global warming are some of the many important parameters that will influence the role of technology in the field of adaptation physiology for farm animals. it is evident that covid- is building individual and societal resilience as it forces entire industries to find new and innovative solutions for farming, livestock, and other industrial sectors. the emphasis on reducing animal experiments for research purposes and limiting physical contact with animals is expected to fuel future research and development as well as create new commercial applications for sensor-based technologies in the agricultural and livestock sectors. sensor-based technology can assess many biochemical, metabolic, physical, and immunological parameters related to adaptation physiology in farm animals. these include measurements of heart rate variability, respiration rate, body temperature, sweating rate, metabolism, health and diseases, vocalization, activity, movement, postural, feed and water intake behavior, and emotional contagion. the presented critical review illustrated the promising outcomes of sensor-based technology in farm animals as an innovative approach for maximizing animal welfare and providing better alternatives for gauging animal health and response. in addition, sensor-based technologies help in the recognition of robust breeds (i.e., animals with better adaptation capabilities and stress response). despite promising outcomes, the use of novel, highly precise sensor-based technologies face several challenges that must be addressed in future research. these key challenges include the validation of large-scale machine learning techniques and issues related to the sensitivity of conventional sensor-based methodologies. another important challenge will be imparting the necessary skillset to farmers in rural areas, so they are equipped and willing to maximize their use of the available technology. educating the end-users of sensor-based technology in the use of information technology-based sensor devices is the only way these innovative new developments can reach their full potential. adaptation strategies: ruminants adaptation of livestock to environmental challenges physiological responses of cattle to heat stress biochemical and physiological changes during thermal stress in bovines: a review computer vision and remote sensing to assess physiological responses of cattle to pre-slaughter stress, and its impact on beef quality: a review models and methods to investigate acute stress responses in cattle smart computing and sensing technologies for animal welfare: a systematic review possible links between crohn's disease and paratuberculosis epidemiological characteristics and economic impact of lumpy skin disease, sheeppox and goatpox among subsistence farmers in northeast nigeria farm animal cognition-linking behavior, welfare and ethics the role of sensors, big data and machine learning in modern animal farming infrared thermography: a non-invasive window into thermal physiology recent advances in wearable sensors for animal health management on-barn pig weight estimation based on body measurements by a kinect v depth camera animal welfare monitoring by real-time physiological signals a sensor-based solution to monitor grazing cattle drinking behaviour and water intake impacts of stocking density on the performance and welfare of broiler chickens comparison of digital rectal and microchip transponder thermometry in cats assessment of the use of temperature-sensitive microchips to determine core body temperature in goats automatic broiler temperature measuring by thermal camera effects of an epinephrine infusion on eye temperature and heart rate variability in bull calves infrared thermography in animal production: an overview smart animal agriculture: application of real-time sensors to improve animal well-being and production avian maternal response to chick distress methodology for data processing and analysis techniques of infrared video thermography used to measure cattle temperature in real time adaptive thermal traits in farm animals. abb-online submiss technical note: development of a noninvasive respiration rate sensor for cattle effects of heat stress on dairy cattle welfare seasonal heat stress: clinical implications and hormone treatments for the fertility of dairy cows technical note: device for measuring respiration rate of cattle under field conditions development of a new respiration rate monitor for cattle a system for contact-free measurement of respiration rate of dairy cows a label-free electrochemical immunosensor for the detection of cardiac marker using graphene quantum dots (gqds) resettable skin interfaced microfluidic sweat collection devices with chemesthetic hydration feedback heart rate monitoring in pigs using photo pethysmography (ppg) technology welfare implication of measuring heart rate and heart rate variability in dairy cattle: literature review and conclusions for future research is continuous heart rate monitoring of livestock a dream or is it realistic? a review photoplethysmography and its application in clinical physiological measurement non-contact vital signs monitoring of dog and cat using a uwb radar animals machine-knitted washable sensor array textile for precise epidermal physiological signal monitoring wearable heart rate sensor systems for wireless canine health monitoring systematic review of bovine respiratory disease diagnosis focused on diagnostic confirmation, early detection, and prediction of unfavorable outcomes in feedlot cattle classification of ingestive-related cow behaviours using rumiwatch halter and neck-mounted accelerometers evaluation of data loggers, sampling intervals, and editing techniques for measuring the lying behavior of dairy cattle behavioral changes before metritis diagnosis in dairy cows triaxial accelerometers for recording grazing and ruminating time in dairy cows: an alternative to visual observations classification of behaviour in housed dairy cows using an accelerometer-based activity monitoring system the non-invasive and automated detection of bovine respiratory disease onset in receiver calves using infrared thermography behavioral classification of data from collars containing motion sensors in grazing cattle use of novel sensors combining local positioning and acceleration to measure feeding behavior differences associated with lameness in dairy cattle inferring behaviour of grazing livestock: opportunities from gps telemetry and activity sensors applied to animal husbandry application of overall dynamic body acceleration as a proxy for estimating the energy expenditure of grazing farm animals: relationship with heart rate system specification and validation of a noseband pressure sensor for measurement of ruminating and eating behavior in stable-fed cows pressure mat analysis of the longitudinal development of pig locomotion in growing pigs after weaning review: quantifying animal feeding behaviour with a focus on pigs relationship between sow conformation, farrowing floor type and posture change characteristics using accelerometer data development of a temperature measurement system for a broiler flock with thermal imaging accuracy of pressure plate kinetic asymmetry indices and their correlation with visual gait assessment scores in lame and nonlame dogs the equine facial action coding system a potentiometric biosensor for rapid on-site disease diagnostics development of quantum dots-based biosensor towards on-farm detection of subclinical ketosis parasitic mites alter chicken behaviour and negatively impact animal welfare automated bioacoustics: methods in ecology and conservation and their potential for animal welfare monitoring drinking behavior in nursery pigs: determining the accuracy between an automatic water meter versus human observers growing pigs' drinking behaviour: number of visits, duration, water intake and diurnal variation comparing three different passive rfid systems for behaviour monitoring in grow-finish pigs continuous monitoring of pigs in fattening using a multi-sensor system: behavior patterns early detection of health and welfare compromises through automated detection of behavioural changes in pigs an intelligent system for livestock disease surveillance remote noninvasive assessment of pain and health status in cattle evaluation and application potential of an accelerometer-based collar device for measuring grazing behavior of dairy cows freedom to lie: how farrowing environment affects sow lying behaviour assessment using inertial sensors past and future: standing on the shoulders of giants sick and grumpy: changes in social behaviour after a controlled immune stimulation in group-housed gilts image-processing technique to measure pig activity in response to climatic variation in a pig barn evaluation of two-dimensional accelerometers to monitor behavior of beef calves after castration evaluation of analgesic protocol effect on calf behavior after concurrent castration and dehorning preparing the digital future of livestock farming validation of an ear-tag accelerometer to identify feeding and activity behaviors of tiestall-housed dairy cattle development of a methodological framework for a robust prediction of the main behaviours of dairy cows using a combination of machine learning algorithms on accelerometer data effect of flunixin meglumine on the amelioration of lameness in dairy steers with amphotericin b-induced transient synovitis-arthritis a novel biomechanical approach for animal behaviour recognition using accelerometers facial expression as a potential measure of both intent and emotion mounting behaviour recognition for pigs based on deep learning the vertical ground reaction force and the pressure distribution on the claws of dairy cows while walking on a flat substrate gait analysis in clinically healthy sheep from three different age groups using a pressure-sensitive walkway development of a real time cow gait tracking and analysing tool to assess lameness using a pressure sensitive walkway: the gaitwise system effects of castration on eating pattern and physical activity of holstein bulls fed high-concentrate rations under commercial conditions a field investigation of the use of the pedometer for the early detection of lameness in cattle the use of infrared thermography for detecting digital dermatitis in dairy cattle: what is the best measure of temperature and foot location to use? clinical, behavioral, and pulmonary changes in calves following inoculation with mycoplasma bovis the feasibility of using vocalization scoring as an indicator of poor welfare during cattle slaughter impact of exposure time to harsh environments on physiology, mortality, and thermal comfort of day-old chickens in a simulated condition of transport assessment of laying hens' thermal comfort using sound technology livestock vocalisation classification in farm soundscapes use of vocalisation to identify sex, age, and distress in pig production pecking activity detection in group-housed turkeys using acoustic data and a deep learning technique parturition detection in sows as test case for measuring activity behaviour in farm animals by means of radar sensors measurement of uterine electrophysiological activity three dimensional noninvasive electromyometrial imaging (emmi) of uterine contractions effects of geometric alterations an application of higher order multivariate cumulants in modelling of myoelectrical activity of porcine uterus during early pregnancy automated detection of health disorders in lactating dairy cattle on pasture: a preliminary study evaluation of ruminal motility in cattle by a bolus-type wireless sensor review: rumen sensors: data and interpretation for key rumen metabolic processes the human urine metabolome the next challenge plasma metabolites associated with residual feed intake and other productivity performance traits in beef cattle modern analytical techniques in metabolomics analysis evaluation of breath biomarkers and serum haptoglobin concentration for diagnosis of bovine respiratory disease in heifers newly arrived at a feedlot methods for measuring and estimating methane emission from ruminants accuracy of noninvasive breath methane measurements using fourier transform infrared methods on individual cows hot topic: innovative lactation-stage-dependent prediction of methane emissions from milk mid-infrared spectra detection of volatile compounds emitted from nasal secretions and serum: towards non-invasive identification of diseased cattle biomarkers heat detection: trends and opportunities gryphsens: a smartphone-based portable diagnostic reader for the rapid detection of progesterone in milk vocal contagion of emotions in non-human animals does farm animals experience emotions and feelings? positive and negative emotions in dairy cows: can ear postures be used as a measure? facial expression of pain in nellore and crossbred beef cattle routine activities and emotion in the life of dairy cows: integrating body language into an affective state framework the nuts and bolts of animal emotion development of automated computer vision systems for investigation of livestock behaviours the automated analysis of clustering behaviour of piglets from thermal images in response to immune challenge by vaccination quantification of the effects of fur, fur color, and velocity on time-of-flight technology in dairy production skin surface temperature of broiler chickens is correlated to body core temperature and is indicative of their thermoregulatory status continuous wavelet filtering on webcam photoplethysmographic signals to remotely assess the instantaneous heart rate a vision for development and utilization of high-throughput phenotyping and big data analytics in livestock big data in smart farming-a review enhancing the security of patients' portals and websites by detecting malicious web crawlers using machine learning techniques comprehensive analysis of machine learning models for prediction of sub-clinical mastitis: deep learning and gradient-boosted trees outperform other models the use of multilayer network analysis in animal behaviour automatic recognition of sow nursing behaviour using deep learning-based segmentation and spatial and temporal features towards on-farm pig face recognition using convolutional neural networks using machine learning to estimate herbage production and nutrient uptake on irish dairy farms computer vision system for measuring individual cow feed intake using rgb-d camera and deep learning algorithms ranking of environmental heat stressors for dairy cows using machine learning algorithms big (pig) data and the internet of the swine things: a new paradigm in the industry big data analytics and precision animal agriculture symposium: machine learning and data mining advance predictive big data analysis in precision animal agriculture economic viability of adoption of automated oestrus detection technologies on dairy farms: a review cost-benefit analysis of an uhf-rfid system for animal identification, simultaneous detection and hotspot monitoring of fattening pigs and dairy cows precision livestock farming in swine welfare: a review for swine practitioners this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license funding: this research received no external funding. the author declares no conflict of interest. key: cord- -mio przi authors: mcaloose, denise; laverack, melissa; wang, leyi; killian, mary lea; caserta, leonardo c.; yuan, fangfeng; mitchell, patrick k.; queen, krista; mauldin, matthew r.; cronk, brittany d.; bartlett, susan l.; sykes, john m.; zec, stephanie; stokol, tracy; ingerman, karen; delaney, martha a.; fredrickson, richard; ivančić, marina; jenkins-moore, melinda; mozingo, katie; franzen, kerrie; bergeson, nichole hines; goodman, laura; wang, haibin; fang, ying; olmstead, colleen; mccann, colleen; thomas, patrick; goodrich, erin; elvinger, françois; smith, david c.; tong, suxiang; slavinski, sally; calle, paul p.; terio, karen; torchetti, mia kim; diel, diego g. title: from people to panthera: natural sars-cov- infection in tigers and lions at the bronx zoo date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: mio przi despite numerous barriers to transmission, zoonoses are the major cause of emerging infectious diseases in humans. among these, severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), and ebolaviruses have killed thousands; the human immunodeficiency virus (hiv) has killed millions. zoonoses and human-to-animal cross-species transmission are driven by human actions and have important management, conservation, and public health implications. the current sars-cov- pandemic, which presumably originated from an animal reservoir, has killed more than half a million people around the world and cases continue to rise. in march , new york city was a global epicenter for sars-cov- infections. during this time, four tigers and three lions at the bronx zoo, ny, developed mild, abnormal respiratory signs. we detected sars-cov- rna in respiratory secretions and/or feces from all seven animals, live virus in three, and colocalized viral rna with cellular damage in one. we produced nine whole sars-cov- genomes from the animals and keepers and identified different sars-cov- genotypes in the tigers and lions. epidemiologic and genomic data indicated human-to-tiger transmission. these were the first confirmed cases of natural sars-cov- animal infections in the united states and the first in nondomestic species in the world. we highlight disease transmission at a nontraditional interface and provide information that contributes to understanding sars-cov- transmission across species. and provide epidemiological and genetic evidence for human-to-animal transmission of the virus. our data show that tigers and lions were infected with different genotypes of sars-cov- , indicating two independent transmission events to the animals. importantly, infected animals shed infectious virus in respiratory secretions and feces. a better understanding of the susceptibility of animal species to sars-cov- may help to elucidate transmission mechanisms and identify potential reservoirs and sources of infection that are important in both animal and human health. keywords one health, panthera leo, panthera tigris, sars-cov- , in situ hybridization, lion, rrt-pcr, tiger, virus isolation, whole-genome sequencing, zoo, zoonotic infection c oronaviruses are a recognized cause of disease in humans and animals ( ) . among them are the viruses that cause colds in humans, multisystemic disease in domestic cats, and gastrointestinal and respiratory diseases in pigs and poultry. coronavirus disease (covid- ) caused by severe acute respiratory syndrome-related coronavirus (sars-cov- ) ( ) was first reported in wuhan, hubei province, china at the end of december ( ) . within weeks the virus spread globally, and by july , over million people were infected and more than , had died (https://www.who.int/ emergencies/diseases/novel-coronavirus- ; accessed july ). genome sequence analysis has shown sars-cov- to be most closely related to a bat coronavirus (ratg - ), and horseshoe bats are currently considered the likely source of the ancestral virus from which the currently circulating sars-cov- virus was derived ( , ) . subsequent genetic adaptation of the currently circulating virus in an intermediate animal host(s) or after human transmission has been proposed ( , ) . the exact details of how long the virus had been circulating in animals prior to transmission to people is unknown. however, a recent study suggests the virus may have been circulating in bats for several decades ( ) , and an early cluster of human covid- cases had an epidemiological link to the huanan seafood wholesale market in wuhan where a variety of live wild animals were sold ( ) . the current sars-cov- pandemic and outbreaks of severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) before it raise awareness and concerns about zoonotic (animal-tohuman) diseases and cross-species transmission of coronaviruses ( ) ( ) ( ) ( ) . given the suspected zoonotic origin of sars-cov- , identifying susceptible animal species, reservoirs, and transmission routes between species is a topic of global scientific and public interest. natural sars-cov- infections in animals have been reported in dogs, cats, and farmed mink in hong kong, europe, china, and the united states ( ) ( ) ( ) . infection in most of these cases has been linked to households or settings in which human owners or caretakers have tested positive for sars-cov- and infection from humans to animals has been presumed. experimental inoculation studies have shown that sars-cov- infects and replicates with high efficiency in domestic cats, ferrets, and fruit bats and poorly in dogs; pigs, chickens, and ducks do not seem to support productive sars-cov- infection ( , ) . importantly, virus shedding and horizontal transmission have been shown in cats and ferrets ( ) ( ) ( ) following experimental inoculation. in this study, we report natural infection of tigers (panthera tigris) and lions (panthera leo) with sars-cov- at the wildlife conservation society's (wcs's) bronx zoo, new york, ny, and provide a detailed genomic characterization of viruses obtained from infected animals and keepers who had close contact with the sars-cov- -positive animals. these were the first confirmed animal infections in the united states and occurred in march , when, due to widespread community transmission ( ) , new york was a global sars-cov- epicenter. audible wheezing despite remaining eupneic. by april, an additional malayan tiger (tiger ) and two amur tigers (panthera tigris altaica) (tigers and ) housed in the same building as tiger but in different enclosures and three african lions (panthera leo krugeri) (lions , , and ) housed in a separate building developed similar respiratory signs. all animals otherwise exhibited normal behavior and activity. clinical respiratory signs resolved in less than days ( to april ) in all animals except tiger , whose clinical signs lasted days (resolution of clinical signs on april ). an additional amur tiger (tiger ) in the same building as tigers to did not develop clinical respiratory disease. detection of sars-cov- in affected animals. a broad diagnostic investigation was performed in tiger on april, days after the onset of clinical signs. this included physical examination, thoracic and abdominal radiography and ultrasonography, and collection of respiratory (nasal swab, oropharyngeal swab, and tracheal wash) and blood samples. thoracic radiography and ultrasonography revealed small, multifocal regions of peribronchial consolidation. cytologic examination of tracheal wash fluid identified necrotic epithelial and inflammatory cells consistent with tracheitis ( fig. a fig tracheal wash cytology (a and b) and in situ hybridization (ish) (c and d). tiger . (a) flocculent material from the trachea consists of stringy mucus with enmeshed degenerate cells characterized by condensed nuclei and loss of distinct cellular features (arrows). (b) few intact cells (short arrow) and degenerate epithelial cells (long arrow) are admixed with abundant round to amorphous cellular debris and granular degenerate mucus (arrowheads). inset: degenerate epithelial cell (upper right) with nuclear fragmentation (karyolysis) and an adjacent intact neutrophil (lower left). modified wright's stain. (c and d) incubation with sars-cov- -specific probe is positive (red puncta) throughout the mucinous material, in the cytoplasm of intact and degenerate epithelial and inflammatory cells, and in cellular debris. red chromogenic assay; hematoxylin counterstain. (note: a software or equipment malfunction produced a faint horizontal line that may be visible in panels a and b.) and b). in situ hybridization (ish) colocalized sars-cov- rna within necrotic epithelial and inflammatory cells in the fluid ( fig. c and d and see also fig. s ). all respiratory samples (nasal, oropharyngeal, and tracheal wash) were negative on virus isolation for feline herpesvirus and feline calicivirus and on targeted pcr testing and metagenomic analysis for common feline pathogens (table s , bioproject accession no. prjna ); all were positive for sars-cov- by real-time reverse transcription-pcr (rrt-pcr) using primers and probes targeting portions of the nucleocapsid (n , n , and n ) and envelope (e) genes (table s ). minion and sanger-based sequencing of the full sars-cov- spike (s) gene, an internal region of the n gene, and the rna-dependent rna polymerase (rdrp) confirmed the virus in the respiratory samples (table s and data set [see "data availability" in materials and methods for definitions and locations of all data sets]). fecal samples collected opportunistically from each animal (symptomatic tigers to , asymptomatic tiger , and symptomatic lions to ) tested positive for sars-cov- by rrt-pcr (table s ). results were confirmed by amplicon sequencing (table s and data set ). infectious sars-cov- detected in respiratory and fecal samples from affected animals. virus isolation was performed on all respiratory and fecal samples. cytopathic effect (cpe) was observed in vero cells inoculated with tracheal wash fluid from tiger ( fig. a and b ) and fecal samples from tiger and lion (table s ). results were confirmed by rrt-pcr (cdc n assay) and/or ish and immunofluorescence assays ( fig. c and d) . additionally, a neutralizing antibody titer of detected in tiger confirmed active sars-cov- infection in this animal (table s ) . epidemiologic and diagnostic investigation of zoo staff. subsequent to confirmation of sars-cov- infection in the animals, an epidemiologic investigation of zoo staff identified zoo keepers and two managers who provided care for and had close (Յ . -m) but not direct contact with the tigers or lions between march (the date on which the zoo was closed to the public due to the pandemic) and march to april (timeline of disease onset in the animals). four staff ( tiger and lion keepers) reported mild respiratory symptoms (including fever, cough, chills, myalgia, and fatigue) between and march . nasopharyngeal samples and blood were collected from these staff members on april , and rrt-pcr and a microsphere immunoassay (mia; to detect igg antibodies) were performed; staff who did not report symptoms were not tested. all tested keepers had evidence of current or prior sars-cov comparative genomics and phylogenetic and haplotype network analysis. nine complete sars-cov- genome sequences (from four tigers, three lions, and two keepers) and eight full-length s gene sequences (from seven symptomatic animals and one asymptomatic animal) were generated directly from respiratory and/or fecal samples (data sets and ). compared to the wuhan-hu- sequence (genbank accession number nc_ ), all tiger and keeper sequences contained six single-nucleotide polymorphisms (snps) with nine additional ambiguous sites ( fig. s and table s ). a total of sites differed between the three lion sequences and wuhan-hu- ( fig. s and table s ). viral sequences in the tigers, lions, and keepers clustered into common sars-cov- clades (fig. a) . those from tigers and tiger keepers clustered with clade g (defined by the d g substitution in the spike protein); the lion sequences clustered with clade v (defined by the g v substitution in orf a) (fig. a ). median-joining haplotype network analysis of the viral sequences corroborated results of phylogenetic analyses ( fig. b and data set ). nucleotide sequence and amino acid analysis of the spike protein of sars-cov- in tigers and lions was performed. compared with the wuhan-hu- strain, the tiger and lion sars-cov- s gene sequences had to nucleotide differences that resulted in several nonsynonymous substitutions (fig. ). of five substitutions in the tiger strains, only one (g d) was found in available human sars-cov- strains. these changes were not observed in the viral sequences from the lions (fig. ). our results document susceptibility and natural sars-cov- infection in tigers and lions. these were the first confirmed animal infections in the united states and the first to be described in a nondomestic species in the world. genomic and epidemiological data support a close evolutionary relationship between the viral strains in the tigers and those in the tiger keepers. notably, the genetic differences and the distant phylogenetic relationship between sequences recovered from the tigers/tiger keepers and lions and the relationship of these strains in the context of global sequences indicate that tigers and lions were infected by two different sars-cov- genotypes. these data suggest that at least two independent sars-cov- introductions occurred, one in tigers and another in lions. importantly, the sars-cov- genome sequence from tiger was identical to the viral sequence from keeper (a tiger keeper) and to other human sars-cov- strains detected in new york (ny-cdc- [mt ] and ny-qdx- [mt . ]). these observations, temporal overlap in animal and human infections, and a lack of new animal introductions to the collection support the conclusion of transmission from an infected keeper(s) to the tigers. whether this was direct or indirect (e.g., fomite, food handling/preparation, or shared enrichment items) and whether subsequent tiger-to-tiger transmission (aerosol, respiratory droplet, etc.) occurred were not determined. a clear association and transmission source were not identified for the lions. the lion sars-cov- sequences were more divergent than those in the tigers and keepers. interestingly, nine of the snps (relative to the wuhan-hu- reference strain) shared by all three lion viruses were also found in a human strain (closest strain, gisaid accession no. epi_isl_ ) detected in connecticut, usa. two lion keepers were serologically positive for sars-cov- , but viral rna was not detected, and sars-cov- could not be confirmed in their respiratory samples. however, given close contact between keepers and animals and the serological evidence indicating infection of two of the lion keepers, it is possible that while asymptomatic, they or other asymptomatic staff may have transmitted virus to the lions. the host range of sars-cov- and other coronaviruses is determined primarily by the interaction of the virus s glycoprotein, specifically the spike subunit (s ), and the cellular receptor, angiotensin-converting enzyme ii (ace ) ( ) . in silico predictions have shown high binding potential between the s receptor binding domain (rbd) and domestic cat ace receptor and conservation of three of five amino acid residues that are critical for interaction with the sars-cov- s glycoprotein in human and domestic cat ace ( ) . these observations are supported by reports describing natural and experimental infection of domestic cats with sars-cov- ( , ) and the data here that show a high degree of conservation between ace in humans and domestic and wild felids. further work is needed to determine if these changes affect sars-cov- receptor binding and pathogenicity in felids and humans. infections in the tigers and lions occurred at a time before sars-cov- testing was widely available in the united states and when there was limited evidence of pre-or asymptomatic viral shedding ( ) . additionally, at that time, keepers caring for the tigers and lions did not generally wear personal protective equipment (ppe) given the (historical) low risk of infectious respiratory disease transmission between humans and domestic or nondomestic felid species. results of this investigation prompted the immediate development of new protocols for ppe use in the enclosures of nondomestic felids and other known or susceptible species including mustelids, viverrids, and chiroptera (ppe was already in place for work with nonhuman primates) at the bronx zoo. they also contributed to the development of similar recommendations by other zoo and wildlife organizations (https://www.aza.org/aza-news-releases/posts/aza-and-aazv-statement-on -covid- -positive-tiger-in-new-york). the role of domestic and wild animal species in the epidemiology of sars-cov- is not completely understood. to date, the reported number of cases of sars-cov- infection in domestic and wild animal species is low, and to our knowledge, no other zoos worldwide have confirmed cases in their animals. this is notable when considered in the context of the large number of human cases and close interactions between people, their pets, and wild animals in their care. however, the fact that companion animals, farmed mink, and zoo animals are susceptible to sars-cov- infection and shed infectious virus in respiratory secretions and/or feces ( - ) makes the humananimal-environment interface an important area for further one health-based studies ( ) . in general, a better understanding of sars-cov- susceptibility across a wide range of animal species will help to elucidate transmission mechanisms and identify potential reservoirs and sources of infection important in both animal and human health. in the last decades, at least three major coronavirus epidemics (sars, mers, and covid- ) have occurred. a feature shared by these and other novel viruses of humans including ebolaviruses and human immunodeficiency virus (hiv) is an origin in a wild sars-cov- in tigers, lions, and keepers at a zoo ® animal host. despite a traditionally held perception of low risk, scientists and conservationists around the world have long recognized and shared concerns related to human activities that increase human-wildlife interactions and zoonotic disease transmission risk ( ) ( ) ( ) . as long as anthropogenic development and population growth bring humans and wildlife into increasing proximity, legal and illegal harvesting persists, and consumption of wildlife and wildlife products exists, there will be continued and significant risk of pandemic viral emergence with devastating global impact on human and animal health, economies, food security, and biodiversity. personal protective equipment (ppe), including n or surgical masks, face shields or goggles, and disposable gloves, were not generally worn when working with the tigers and lions prior to the sars-cov- pandemic but were worn during all animal handling and sample collection subsequent to the development of clinical signs in tiger . diagnostic specimens were submitted to the university of illinois veterinary diagnostic laboratory (uiuc-vdl) and animal health diagnostic center at cornell university (cornell ahdc) (both part of the national animal health laboratory network) for broad diagnostic investigation (respiratory samples-tiger ) and specific sars-cov- testing (fecal samplesall animals). epidemiologic investigation. subsequent to the development of clinical signs and positive test results in the tigers and lions, an epidemiologic investigation into possible human infections in staff working with these animals was conducted by the new york city (nyc) and new york state (nys) public health laboratories in conjunction with the u.s. centers for disease control and prevention (cdc). the investigation focused on the time period between march (date on which the zoo was closed to the public due to the pandemic) and march to april (the timeline of disease onset in the animals). twelve staff members ( keepers and managers) were identified who had responsibilities that offered opportunities for close (Յ . -m) but not direct contact with the animals during this time period. this included moving animals between enclosures and exhibits, feeding, training sessions, enrichment activities, greetings, and social interactions (e.g., chuffing, a form of vocalization that tigers performed that involves air exhalation and which keepers also use to greet tigers). additionally, lion keepers worked at a desk that was located less than . m from metal-mesh-fronted lion enclosures. four keepers reported being mildly symptomatic (including fever, cough, chills, myalgia, and fatigue) with signs beginning in each prior to or concurrently with illness in animals on , , , and march . nasopharyngeal swab and blood samples were collected on april from the symptomatic keepers, and sars-cov- -specific rrt-pcr and a microsphere immunoassay (to detect igg antibodies) were performed; sampling and testing were not performed in the eight additional staff who did not report symptoms. all four of the tested keepers had evidence of sars-cov- infection (one rrt-pcr-positive tiger keeper [keeper ], one rrt-pcr-and serology-positive tiger keeper [tiger ], and two serology-positive lion keepers [keeper and keeper ]). none of the keepers reported being sick at work. all stayed home for at least days from the onset of illness, and none returned to work prior to a minimum of symptom-free days and fever-free hours in compliance with organizational covid- policies and cdc and ny department of health (doh) guidelines. rrt-pcr-positive specimens were forwarded to cdc for whole-genome sequencing (wgs) and haplotype network analysis to characterize the human samples and further compare the human and animal viral genome sequences. interviews with the tiger and lion keepers suggested that up to two additional keepers may have had signs or symptoms suggestive of mild and transient covid- ; however, they did not self-report being sick, may not have recognized their symptoms as being consistent with covid- , and were not tested. non-sars-cov- respiratory pathogen testing. nucleic acid extracted from the respiratory specimens (nasal and oropharyngeal swabs and tracheal wash fluid) from tiger was tested by real-time pcr (rpcr) or rrt-pcr for several common feline respiratory pathogens (cornell ahdc) including bordetella bronchiseptica ( ) , influenza a virus (cdc universal assay) ( ) , mycoplasma cynos ( ) , mycoplasma felis ( ) , pneumovirus ( ) with probe modification ( -carboxyfluorescein [fam]-cttcatcacttttggcctgg cccag-bhq ), and streptococcus equi subsp. zooepidemicus ( ) . additionally, samples were tested for chlamydia psittaci, chlamydia felis, and chlamydia abortus using a conventional pcr assay ( ) , and virus isolation was performed using inoculated feline pulmonary cells to test for feline herpesvirus and feline calicivirus. all assays have been adapted and optimized and are used routinely in feline infectious respiratory disease diagnostic testing. tracheal wash fluid cytology. direct smears of buoyant, flocculent material in the tracheal wash fluid and cytocentrifuge smears of the remaining fluid were prepared and stained with a romanoski stain (modified wright's stain) using an automated stainer (hema-tek ; siemens). the stained slides were examined using standard bright-field microscopy by a board-certified veterinary clinical pathologist (cornell ahdc). in situ hybridization for sars-cov- rna. unstained cytologic smears of tracheal wash fluid from tiger were fixed in ice-cold % methanol for min and stored at Ϫ °c until shipped to uiuc college of veterinary medicine zoological pathology program (zpp) on cold packs. upon arrival, the slides were submerged for an additional min in % neutral buffered formalin at °c, air dried, and then placed in % ethyl alcohol for min at room temperature. an in situ hybridization (ish) chromogenic manual assay was performed using the rnascope . hd detection kit red and a -pair oligonucleotide probe targeting the sars-cov- s gene of the wuhan hu- complete genome (nc_ . ; advanced cell diagnostics catalog no. ) according to the manufacturer's directions (advanced cell diagnostics, inc., newark, ca). positive-control slides consisted of vero cells infected with sars-cov- isolated from tiger . a control probe targeting the dapb gene from the bacillus subtilis strain smy (advanced cell diagnostics catalog no. ) was used as a negative control on all cytology sections in parallel with the sars-cov- target probe (see fig. s ). additional negative controls to rule out cross-reactivity with tiger rna and the felid alphacoronavirus included a cytocentrifuge preparation of cell cultures infected with the alphacoronavirus feline enteric coronavirus (fecov) (kindly provided by gary whittaker, cornell university); formalin-fixed, paraffin-embedded (ffpe) unstained sections of normal malayan tiger trachea, lung, and oropharyngeal tissue; and lung, lymph node, and intestine from a fecov-positive domestic cat (fig. s ) . samples from the control tiger and domestic cat were collected opportunistically in and , respectively, and archived as part of routine necropsy procedures (wildlife conservation society's [wcs's] bronx zoo and uiuc-zpp, respectively). virus isolation. virus isolation on respiratory and fecal samples was performed in vero (atcc ccl- ), vero e , and vero cells under biosafety level conditions at the cornell ahdc and the national veterinary services laboratory (nvsl). cells were cultured in minimum essential medium eagle (mem-e; gibco, gaithersburg, md) supplemented with . to % fetal bovine serum (fbs; gibco), iu/ml penicillin, and g/ml streptomycin (growth medium). cells were seeded in -well culture plates or t flasks and cultured at °c with % co for to h. before inoculation, respiratory swabs and tracheal wash fluid samples were diluted at : , : , and : , in serum-free mem-e containing ui/ml penicillin, g/ml streptomycin, and . g/ml amphotericin b (all from gibco); swabs from fecal samples were placed in ml of sterile pbs supplemented with . % bovine serum albumin (bsa) (sigma-aldrich, st. louis, mo) containing ui/ml penicillin, g/ml streptomycin, and . g/ml amphotericin b (all from gibco). cells were rinsed with mem-e and inoculated with l of each respiratory sample dilution in individual wells of a -well plate or . ml of the diluted fecal sample in a t flask and adsorbed for h at °c with % co for h. mock-inoculated cells were used as negative controls. after adsorption, replacement medium was added, and cells were incubated at °c with % co and monitored daily for cytopathic effect (cpe) for days. cell cultures with no cpe were frozen, thawed, and subjected to three blind passages with inoculation of fresh vero cell cultures with the lysates as described above. sars-cov- infection in cpe-positive cultures was confirmed with sars-cov- specific rrt-pcr using the cdc n primer and probe set (sequences available upon request), an immunofluorescence assay using a mouse monoclonal antibody against the sars-cov n protein ( , ) , and rnascope in situ hybridization as described above. virus neutralization assay. seroconversion of tiger to sars-cov- was assessed by a virus neutralization assay (vn; cornell ahdc). twofold serial dilutions ( : to : , ) of a serum sample collected on april ( days after the onset of clinical signs) were incubated with % tissue culture infective doses (tcid ) of sars-cov- on vero cells for h at °c. following incubation of serum and virus, l of a cell suspension of vero ccl- cells was added to each well of a -well plate and incubated for h at °c with % co . virus cytopathic effect (cpe) was used as an indicator of virus infection/replication. neutralizing antibody titers were expressed as the reciprocal of the highest dilution of serum that completely inhibited cpe. archived frozen sera from another tiger (archived frozen at cornell ahdc) and positive human control sera (deidentified convalescent human sera provided by cayuga medical center, irb protocol ep) were included, and all samples were tested in triplicate with results averaged. a cell culture control was included in the assays, and the virus working dilution was back-titrated. sars-cov- rrt-pcr. nucleic acid was extracted from nasal and oropharyngeal swabs and tracheal wash fluid (tiger ) using the magmax isolation kit (thermo fisher scientific, waltham, ma) and from fecal samples (tigers to and lions to ) using the magmax core nucleic acid purification kit (thermo fisher scientific, waltham, ma) and an automated nucleic acid extractor (king fisher flex purification system; thermo fisher scientific, waltham, ma) according to the manufacturer's instructions. analysis for sars-cov- rna was performed with real-time reverse transcriptase (rrt) pcr and either the -ncov cdc qpcr probe assay targeting three regions of the nucleocapsid (n) gene (n , n , and/or n ; integrated dna technologies [idt], inc., coralville, ia) (uiuc-vdl, cornell ahdc) or the cdc qpcr probe assay, and in-house primers for the envelope (e) gene with the agpath-id one-step rt-pcr kit (applied biosystems, foster city, ca) (uiuc-vdl) (primer and probe sequences are available upon request). thermal cycler conditions consisted of reverse transcription and enzyme activation at to °c for min and °c for min, respectively, followed by to cycles of °c for to s and to °c for s. positive ( -ncov_n_positive control; idt, coralville, ia, and a synthesized plasmid [genscript] e gene control) and negative (distilled or nuclease-free water) controls, plus internal amplification controls (xeno or beta-actin; thermo fisher scientific, waltham, ma), were included as separate reaction mixtures. following initial rrt-pcr testing at both institutions, samples were submitted to the nvsl for confirmatory testing, using the -ncov cdc qpcr probe assay and n and n primers (sequences available upon request). amplicon sequencing. minion and sanger amplicon sequencing was used to confirm rrt-pcr results at cornell ahdc and nvsl, respectively (primer sequences are available upon request). for minion-based sequencing, targets were amplified directly from tracheal wash or fecal samples using the superscript iv one-step rt-pcr system (thermo fisher scientific, waltham, ma). primers targeted the complete spike (s) gene ( , bp) and an internal region of the n gene ( bp). universal oxford nanopore-compatible adapter sequences were added to the = end of each primer sequence to allow pcr-based barcoding. amplicons were purified (ampure xp beads [beckman coulter, brea, ca]; . : volumetric bead-to-dna ratio), and dna quantification was performed on a qubit fluorometer . (double-stranded dna [dsdna] high-sensitivity assay kit; thermo fisher scientific, waltham, ma). samples were subsequently diluted to . nm in a total of l and used as the input for the library preparation following the d pcr barcoding ( ) genomic dna (sqk-lsk ) protocol (oxford nanopore technologies, oxford, uk). final dna libraries were loaded in a flo-min r . flow cell to start the sequencing runs. sanger sequencing was performed using primers targeting partial regions of s, n, and rnadependent rna polymerase (rdrp) genes (primer sequences available upon request). amplicons were generated directly from nasal and oropharyngeal swabs and tracheal wash fluid using the superscript iii one-step rt-pcr system (thermo fisher scientific, waltham, ma). reaction mixtures were purified using the qiagen pcr purification kit (qiagen, germantown, md). dna was amplified for sanger sequencing using the bigdye terminator v . cycle sequencing kit (thermo fisher scientific, waltham, ma) and sequenced on the applied biosystems xl genetic analyzer (thermo fisher scientific, waltham, ma). whole-genome sequencing and phylogenetic analysis. whole-genome sequencing (wgs) was performed on tracheal wash fluid and fecal specimens from all individual tigers and lions as previously described ( ) . individual fecal samples from tigers to and lions to and cell culture viral isolates were subjected to sequencing with either minion-based amplicon sequencing using overlapping primers covering the full viral genome (amplicons with an average size of ϳ , bp; primer sequences are available upon request) or the ion ampliseq kit for chef dl and ion ampliseq sars-cov- research panel (thermo fisher scientific, waltham, ma) (data sets and ). minion libraries were prepared as previously described ( ) using the native barcode kit, exp-nbd , ligation sequencing kit, sqk-sqk (oxford nanopore technologies), and sequenced on an r . flow cell for h. ion targeted libraries were sequenced using an ion chip on the ion s system using the ion -ion -ion kit (thermo fisher scientific, waltham, ma). viral isolates were sequenced with the minion or ion ampliseq approach as described above. whole-genome sequencing on the rrt-pcr-positive specimens from the two sars-cov- -positive keepers was performed as previously described using an amplicon sequencing approach and sanger sequencing ( ) (data set ). all genomes for each animal that were assembled using data generated from different sequencing platforms (illumina, minion, and/or ion torrent) were combined into a single consensus sequence for each animal (data set ). the assemblies for a given animal were aligned with the wuhan-hu- reference sequence (nc_ . ) using mafft v. . ( ) . the reference sequence was then removed from the alignment, and a consensus sequence for the virus sequence recovered from each animal was generated using the consambig program in emboss v. . . . ( ) . when a single assembly shifted alignment due to a single base insertion or repeat nucleotide, the alignment was rerun after removal of the offending nucleotides. to compare the outbreak genomes to others isolated from humans in the same geographic region, all available sars-cov- genomes from new york were downloaded from ncbi on april . these were clustered at . % identity using vsearch v. . . ( ) , and the consensus sequences from each cluster were aligned along with wuhan-hu- reference sequence, using mafft v. . ( ) . a phylogenetic tree was constructed using the consensus sequence from each animal, keepers, sequences from new york, and the wuhan-hu- reference sequence (nc_ . ) using the gtr-gamma model in raxml v. . . ( ) . haplotype network analysis. haplotype network analyses were conducted with two overlapping data sets using popart software ( ) using the median joining algorithm ( ) . data set contained nine genomes generated from the bronx zoo cases: four tigers, three lions, and two tiger keepers. data set contained the nine genomes from data set , as well as additional genomes (including the sars-cov- reference wuhan-hu genome) generated from a total of countries to better understand genetic relatedness of bronx zoo cases in the context of the global pandemic. the top blast results for the lion sequences were included in data set . for both data sets, the entire genome alignment was examined visually for accuracy and evidence of large-scale rearrangements to rule out the likelihood of multiple single-nucleotide polymorphisms (snps) being the result of a single evolutionary event. subsequently, whole-genome alignments were converted into an snp matrix by removing columns containing identical bases, gaps, and ambiguous bases. the lengths of final snp matrices were nucleotides (nt) (data set ) and nt (data set ). data availability. primer and probe sequence information for rrt-pcr and amplicon and wholegenome sequencing is available upon request. all remaining data are available in the main text or the supplemental material or as follows: data set , metagenomics data obtained from respiratory specimens from tiger have been deposited in srr under prjna ; data set , whole-genome consensus sequences obtained from sars-cov- detected in fecal samples from tigers to and lions to have been deposited in genbank under accession numbers mt , mt , mt , mt , mt , and mt , respectively; data set , whole-genome sequences obtained from sars-cov- tiger and lion isolates (tgr /ny/ , tgr /ny/ , and ln /ny/ ) have been deposited in genbank under accession numbers mt , mt , and mt , respectively; data set , whole-genome sequences obtained from sars-cov- strains in keepers and have been deposited in genbank under accession numbers mt and mt , respectively. supplemental material is available online only. the emergence of sars, mers and novel sars- coronaviruses in the st century the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- china novel coronavirus investigating and research team. . a novel coronavirus from patients with pneumonia in china a pneumonia outbreak associated with a new coronavirus of probable bat origin the proximal origin of sars-cov- full-genome evolutionary analysis of the novel corona virus ( -ncov) rejects the hypothesis of emergence as a result of a recent recombination event evolutionary origins of the sars-cov- sarbecovirus lineage responsible for the covid- pandemic pathways to zoonotic spillover host range and emerging and reemerging pathogens risk factors for human disease emergence reverse zoonotic disease transmission (zooanthroponosis): a systematic review of seldomdocumented human biological threats to animals infection of dogs with sars-cov- first reported cases of sars-cov- infection in companion animals sars-cov- infection in farmed minks, the netherlands susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus infection and rapid transmission of sars-cov- in ferrets sars-cov- in fruit bats, ferrets, pigs, and chickens: an experimental transmission study introductions and early spread of sars-cov- in spike protein recognition of mammalian ace predicts the host range and an optimized ace for sars-cov- infection sars-cov- neutralizing serum antibodies in cats: a serological investigation presymptomatic transmission of sars-cov- -singapore one health proof of concept: bringing a transdisciplinary approach to surveillance for zoonotic viruses at the human-wild animal interface sustainable development must account for pandemic risk host and viral traits predict zoonotic spillover from mammals factors associated with upper respiratory tract disease caused by feline herpesvirus, feline calicivirus, chlamydophila felis and bordetella bronchiseptica in cats: experience from european catteries design and performance of the cdc real-time reverse transcriptase pcr swine flu panel for detection of a (h n ) pandemic influenza virus characterization of a novel mycoplasma cynos real-time pcr assay development and evaluation of a real-time polymerase chain reaction method for the detection of mycoplasma felis detection of canine pneumovirus in dogs with canine infectious respiratory disease real-time pcr for detection and differentiation of streptococcus equi subsp. equi and streptococcus equi subsp. zooepidemicus detection of chlamydia psittaci dna in avian clinical samples by polymerase chain reaction intracellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein: absence of nucleolar accumulation during infection and after expression as a recombinant protein in vero cells production and characterization of monoclonal antibodies against the nucleocapsid protein of sars-cov complete genome sequence of sars-cov- in a tiger from a u ncov- sequencing protocol rapid, sensitive, full-genome sequencing of severe acute respiratory syndrome virus coronavirus mafft multiple sequence alignment software version : improvements in performance and usability emboss: the european molecular biology open software suite vsearch: a versatile open source tool for metagenomics raxml version : a tool for phylogenetic analysis and post-analysis of large phylogenies popart: full-feature software for haplotype network construction median-joining networks for inferring intraspecific phylogenies we declare no competing interests. this manuscript represents the opinions of the authors and does not necessarily reflect the position of the u.s. centers for disease control and prevention. key: cord- -z dx ey authors: schaefer, a. l.; cook, n. j.; bench, c.; chabot, j. b.; colyn, j.; liu, t.; okine, e. k.; stewart, m.; webster, j. r. title: the non-invasive and automated detection of bovine respiratory disease onset in receiver calves using infrared thermography date: - - journal: research in veterinary science doi: . /j.rvsc. . . sha: doc_id: cord_uid: z dx ey abstract bovine respiratory disease complex (brd) causes considerable economic loss and biosecurity cost to the beef industry globally and also results in significant degradation to the welfare of affected animals. the successful treatment of this disease depends on the early, timely and cost effective identification of affected animals. the objective of the present study was to investigate the use of an automated, rfid driven, noninvasive infrared thermography technology to determine brd in cattle. sixty-five calves averaging kg were exposed to standard industry practices of transport and auction. the animals were monitored for brd using conventional biometric signs for clinical scores, core temperatures, haematology, serum cortisol and infrared thermal values over weeks. the data collected demonstrated that true positive animals for brd based on a gold standard including core temperature, clinical score, white blood cell number and neutrophil/lymphocyte ratio displayed higher peak infrared thermal values of . ± . °c compared to true negative animals . ± . °c (p < . ). the study also demonstrated that such biometric data can be non-invasively and automatically collected based on a system developed around the animal’s water station. it is concluded that the deployment of such systems in the cattle industry would aid animal managers and practitioners in the identification and management of brd in cattle populations. bovine respiratory disease complex (brd) is common in intensely raised and newly transported calves and refers to the animal displaying an undifferentiated fever in addition to a number of clinical signs, notably respiratory distress (jericho and kozub, ; buckham sporer et al., ) . brd is known to be caused by a number or combination of viruses and microorganisms (jericho and kozub, ; autio et al., ) including mycoplasma bovis (arcangioli et al., ) , coronavirus (decaro et al., ) , bovine para-influenza (pi ) (ellis, ) , bovine respiratory syncytial virus (brsv) (rola and polak, ) , bovine viral diarrhoea virus (bvd) and infectious bovine rhinotracheitis (ibr) (jericho and kozub, ) . brd is a worldwide health problem (griffin, ; horwood and mahony, ) and one of the major and most economically costly diseases affecting the north american beef industry (snowder et al., ; taylor et al., ) . the average incidence of brd in the usa is calculated to be % of receiver calves but can be as high as % (snowder et al., ) . the primary cost is reported to be due to both the cost of treatment as well as a reduction in subsequent animal performance and carcase merit (duff and galyean, ; gay and barnouin, ). in addition to the direct cost of brd, the increasing concern regarding the use of antibiotics and the potential impact on the promotion of resistant microbes is also apparent and is a growing focal point for global health organizations (world health organization (who), ) and the cattle industry generally (jericho and kozub, ; watts and sweeney, ) . this concern has led to the development of antibiotic resistant monitoring systems (wallmann, ) and concern regarding antibiotic resistance has had a significant influence on the national agricultural policy for research and development programs (www.agr.gc.ca). as discussed by cusack et al. ( ) and panousis ( ) the effective management of brd depends on the early recognition and treatment of the onset of the disease. unfortunately, the clinical signs of brd are often not apparent until late in the course of the disease due to the challenge associated with early diagnosis (poulsen and mcguirk, ) . numerous diagnostic approaches have been attempted with varying success including the use of acute phase proteins (humblet et al., ) , pcr techniques (asano et al., ; fulton, ) and elisa (quinting et al., ) , breath analysis (burciaga-robles et al., ), analysis of cough or respiratory sounds (ferrari et al., ) , assessment of feeding and other animal activity (basarab et al., ; hanzlicek et al., ) , analysis of protein profiles (mitchell et al., ) , rumen bolus temperature recording (rose-dye et al., ; timsit et al., ) and various immuno-histochemical techniques (wallmann, ) . the identification of disease in an animal or a population will depend on the information available and on the reliability of that information. currently, the most accurate methods of diagnosis remain analytical techniques such as serum neutralisation, elisa or pcr procedures. however, with cattle at risk of brd the information available is often limited and does not include extensive and expensive serum neutralisation or pcd data. furthermore, if the assessment of an animal's health necessitates the capture and restraint of that animal in order to collect a biological sample then the stress of the process itself will introduce bias. the use of infrared thermography (irt) to diagnose animals with brd has been established (schaefer et al., . these findings are also consistent with those recently reported by hovinen ( ) and polat et al. ( ) for the determination of mastitis in dairy cattle. many of the aforementioned technologies including clinical scores, haematology, acute phase proteins, cytokines, antibody response and core temperature monitoring are useful aids to the diagnosis of brd particularly when its prevalence is high in a population and once the clinical signs of respiratory disease are present. infrared thermography has been shown to demonstrably diagnose brd at an earlier stage of the disease than other conventional technologies . however, the early detection of brd, especially in animal populations where the prevalence of brd and the virulence is low, is more challenging. more difficult still is the development of any technology that can non-invasively and automatically aid in the diagnoses of brd. the objective of the present study was to investigate the use of infrared thermography to non-invasively identify animals with brd in a population with a low prevalence of respiratory disease. the calves used in the present study were exposed to a lower level of stressors for that purpose. furthermore, it was the objective of the current study to examine the feasibility of automating the collection of infrared thermography data. for infrared thermography to be considered as a practical and feasible analytical tool it is necessary that such a system be demonstrated to be compatible with current rfid tags, be automatable and be more user friendly than carrying a hand held camera around cattle pens. sixty-five receiver calves averaging kg were used. the calves were herford x angus from either the agriculture and agri-food canada, lacombe research centre beef herd (n = ) or the animal diseases research centre beef herd (n = ) located at lethbridge, alberta. the calves were weaned and transported approximately h to a commercial auction mart to simulate transport and handling conditions typical of calves received at feedlots in canada (receiver calves). the animals were held overnight without feed or water and then returned to the lacombe research centre beef research unit. this protocol simulated a commercial auction sale and exposure. upon arrival at the lacombe research centre the calves were offloaded, caught in a restraining chute, weighed, blood sampled via a jugular vein venus puncture, sampled for saliva using a cotton swab and placed into outdoor pens measuring approximately  m with one third of the pen having a roof cover. the calves were fed in a conventional bunk feeder a balanced cereal grain silage ration containing % cereal grain which met or exceeded national research council (nrc) recom-mendations (nrc, ) . the calves received straw bedding and free access to fresh water via an automatic system shown in fig. . all management and operating procedures met or exceeded the canadian council of animal care recommendations ( ) and codes of practice for beef cattle guidelines (agriculture canada, ) . in addition, all study protocol was reviewed and approved by the lacombe research centre animal care committee. while contained in their receiving pens the calves were monitored daily by trained personnel for clinical signs of illness using methods described previously (schaefer et al., . briefly, clinical scores were designed to identify bovine respiratory disease (brd) and were based on the appearance of four criteria as follows: respiratory insult: ( - ): = no insult, normal breath sounds (nbs); = very fine crackle (rale) (vfcr) on auscultation and/or a moderate cough; = fine crackle (subcrepitant) (fcr) on auscultation and/or a moderate nasal discharge and moderate cough; = medium crackle (crepitant) (mcr) on auscultation and/or a moderate to severe viscous nasal discharge with cough; = course crackles (ccr), tachypnoea (> % of the norm) and/or a severe discharge with respiratory distress and obtunded lung sounds and = ccr with dyspnoea, tachypnoea, marked respiratory distress and/or lung consolidation. digestive insult: ( - ): = no insult, normal, eating and drinking; = mild or slight diarrhoea with slight dehydration (< %) and reduced eating; = moderate diarrhoea with % dehydration and reduced feed intake (< %); = moderate to severe diarrhoea with % or less of feed intake and more than % dehydration; = severe diarrhoea, and less than % of normal feed intake and = severe diarrhoea and not eating, not drinking and dehydrated. temperature score: core temperature (rectal) ( - ): = < . °c; = . - . °c; = . - . °c; = . - . °c; = . - . °c and = > °c. rectal or core temperatures for the calves were collected at the start and end of the study only since only at these times were the animal restrained. disposition or lethargy score: ( - ): = no lethargy, normal posture; = mild anorexia or listlessness, depressed appearance; = moderate lethargy and depression, slow to rise, anorectic; = recumbent or abnormal posture, largely depressed; = prostrate, recumbent or abnormal posture and = death. with respect to laboratory analysis, salivary and serum cortisol was analysed using an enzymatic assay as described by cook et al. ( ) . haematology analysis and differential counts were conducted on a cell-dyne model haematology analyser (abbott labs, mississauga, ontario). serology assessment was conducted by prairie diagnostic services (saskatoon saskatchewan) and assessment was carried out for bovine viral diarrhoea (bvd) virus types and and infectious bovine rhinotracheitis virus (ibr) via serum neutralisation tests and expressed as a titre or the highest dilution of serum to exert a neutralising effect. additional assessment for coronavirus, bovine para-influenza (pi ), and bovine respiratory syncytial virus (brsv) were conducted by elisa. again, these methods have been referenced previously . the ranking of antibody titre scores was as follows: for bvd and ibr - : = negative, - : = suspicious, - : = low, - : = moderate, > : = high. for brsv, pi and coronavirus < = negative, - = suspicious, - = low, - = moderate, > = high. the determination of an animal true positive or negative for brd was based on the comparison to a set of ''gold standard'' values as per the approach of humblet et al. ( ) and schaefer et al. ( ) . as described by galen and gambino ( ) and thrusfield ( ) this approach is commonly promoted in both veterinary and human medical diagnostic studies. in the current study, the criteria for a true positive animal for brd was defined as an animal displaying three or more of the following signs; a core temperature of > °c, a white blood cell count of less than or greater than  /ll, a clinical score of > or a neutrophil/lymphocyte ratio of < . (leucopaenia) or > . (neutrophilia). a true negative animal was defined as an animal displaying a score of or . these parameters were considered consistent with normal and abnormal ranges suggested by other researchers (kaneko, ; blood et al., ) . for laboratory assessments, all calves were monitored at the beginning of the study and again three weeks later. with respect to infrared thermal measurements, all calves were monitored for radiated temperatures around the orbital area (eye plus one centimetre surrounding the eye) using a flir s broadband camera (flir comp., boston, ma) mounted on a motorised shaft. the scanning camera was interfaced to a control system such that the animal self-collected an infrared image every time it attended the water station. the automatic scanning system used is shown in fig. and schematically in fig. and consisted of the following components: the system was built around a conventional two water bowl float design from ritchie water systems (ritchie cattle fountains. conrad ia, usa). panels were installed along the sides of the water bowls with a partition in the centre to position the calves when they accessed water. extension panels were placed on each side of the water bowls to place the calf's head at the proper focal distance. this system allowed easy access for the calves from two directions. a panel on one side of the water bowls was modified to facilitate a window measuring approximately cm square in order to view the calves while drinking. two inphase loop antennae, specifically designed to read over a defined space where the rfid tags would be located, were mounted on adjacent panels, above and slightly behind each water bowl. each antenna pair was connected to an allflex pnl-oem-modle- control module (allflex eid system. allflex canada inc. st-hyacinthe, p.q.). electromagnetic shielding was placed on the side of the panel exposed to the holding pen to prevent reading tags on calves that were not accessing water. the design of the antenna system provided the identification of the calf at the water station as well as the signal to the control system to rotate the camera if necessary and to initiate capturing images when the calf's head was visible through the viewing window in the panel. the camera/motor assembly, enclosed under a protective cover, was located medially between the two viewing windows at a distance that provided a field of view to cover most head positions of the drinking cattle. rotation of the camera to view a particular window was performed by a geared-head motor, controlled by a set of software commands sent to the motor control circuitry. this design met the good laboratory practice requirements for correct thermography: a fixed focal length and angle with a still image which enabled accurate thermal data collection. software running on a computer located in an instrumentation cabinet read the tag information from the rfid control module, initiated the camera positioning, acquired the infrared image, performed the thermal analysis of the image and stored the acquired information in a database. the cabinet also housed the power supplies and motor control circuitry used in the system. instrument integration, and the hardware and software used in this thermal station was designed and developed at the lacombe research centre. the use of the infrared thermal stations enabled the monitoring of all animals for orbital radiated temperatures every time they attended the water station. the thermal orbital (eye) maximum value for the calves was used in all calculations since this value was found to be most sensitive to stress and disease onset (schaefer et al., . all calves were thus monitored for average daily maximum temperatures and for the mean ratio values (mr). the mean ratio which was calculated as the average of daily radiated maximum temperature for a given animal divided by the average daily maximum value for the group of calves. verified data was entered into microsoft excel (microsoft corporation ). basic statistical calculations for data means, standard deviation and two tailed least squares analysis tests ( tailed t tests) were conducted in microsoft excel. response operant characteristic curves (roc) were used to calculate the relationship between true positive (sensitivity) and true negative ( -specificity) animals for a given biological measurement. these curves were used to calculate the optimal cut off values or values with the greatest efficiency for that parameter. the analysis of data for roc curves, test specificity and sensitivity, optimal cut off value calculations, positive predictive values and negative predictive values were conducted with medcalc ( ) software (medcalc Ò for windows. statistics for biomedical research. version . broekstraat, mariakerke, belgium). infrared thermography data from thermal images were calculated using flir researcher Ò software (version . , boston, ma). the calves used in the present study were exposed to a lower level of stress compared to typical weaned and receiver calves experiencing a multitude of co-mingling, handling and transport stressors. the verified tp incidence in these calves was thus not unexpectedly comparatively low at % ( out of animals show- ( ) side panels, ( ) two bowl water system, ( ) extension panels, ( ) viewing windows, ( ) antennaek ( ) rfid control modules, ( ) electromagnetic, shielding ( ) infrared camera on motor mount within enclosure, ( ) instrumentation cabinet. ing a gold standard value of or / ). of interest, however, was the observation that none of these calves were identified as suspect for brd using subjective clinical signs and none of these animals were deemed to be in need of treatment nor removed for treatment by the animal managers or pen checkers during the study. a total of of the calves displayed tn values (gold standard values of or / ) and a further animals displayed intermediate values (gold score value of / ) for brd incidence. the calves typically would visit the water station between and times per day. the amount of time spent at the water station by any individual varied but again typically twenty or more thermal images were captured per animal per day. the overall average orbital maximum temperature for all calves for the week period was . °c ± . (sd). for this same period, the average for the orbital maximum for the tp calves was . °c ± . and for the tn calves was . °c ± . (p < . ; table ). since daily thermal values for all animals were monitored, it was possible to follow the radiated temperature rise for the tp calves to the point at which a peak temperature occurred (table and fig. ). the peak thermal response for the tp calves as monitored by the thermal station was . °c. the calves displaying intermediate values (a gold standard score of out of ) showed an average orbital irt value of . ± . °c which was not significantly different from the tn calves but was significantly lower than the tp peak values (p < . ). of interest in the present study was the observation that nine of the calves developed brd signs during the study period and for these animals it was possible to automatically and non-invasively follow the thermal radiated response up to the time of peak thermal response and brd onset. these data showed that there was over a °c elevation in temperature for the tp calves over this time. this contrasts to a flat or basically zero average daily change in temperature for the entire group of animals of À . ± . °c/day. the data for the mean ratios (mr. table ) in general paralleled the orbital maximum values. in terms of biological values, compared to the tn animals the tp calves (table ) displayed higher core temperature values, white blood cell counts, clinical scores and serum cortisol values (p < . ) with trends towards a higher value in neutrophil/lymphocyte ratios. these values for the intermediate calves were . ± . °c, . ± .  /ll, . ± . , . ± . and . ± . lmol/l for core temperatures, white blood cell counts, clinical scores, neutrophil/lymphocyte ratio and serum cortisol, respectively. with respect to specific differential counts the tp calves displayed a lower neutrophil count and a higher lymphocyte count, as well as a slight increase in red blood cell numbers (p < . ) ( table ) . salivary cortisol displayed a greater variation among animals and thus there were no statistically significant differences in salivary cortisol values among the groups. the calculation of predictive index values for optimal cut off values, positive predictive values, negative predictive values, test sensitivity and test specificity are shown in table . again, these values were calculated using roc analysis. the collected data may also be used to estimate test efficiency. in the current study, the test efficiency for the irt max values was % which was as high as any test used. in terms of seroprevalence the average and range of titres for ibr and bvd were < : (range : - : ) and : (range < : - : ), respectively. for the pi , brsv and coronavirus these values were (range - ), (range - ) and (range - ). most of the calves displayed a low to moderate titre to one or more viruses. in terms of seroconversion, higher values were seen for the true positive (tp) calves. thirty-three percent of the tp animals seroconverted (> units) to bvd virus, % to ibr, % to brsv, % to pi and % to corona virus. by contrast in the true negative animals a lower extent of seroconversion was seen. twenty-two percent seroconverted to bvd, % to ibr, % to brsv, % to pi and % to coronavirus. the data suggest that the brd viruses measured may have played some significant role in the onset of brd in the tp animals. identifying animals positive for brd is not an easy task, particularly if the cattle have mild signs of brd or the brd prevalence is low. part of the reason for this is that there are few gold standards to identify brd that are absolute (white and renter, ). again, the most accurate determination of a causative agent for brd is still likely to be a serum neutralisation, elisa or pcr technique. however, the gold standards suggested currently in this manuscript have some precedence and support in the literature (humblet et al., ; blood et al., ; kaneko, ; schaefer et al., ) and are reasonable suggested biometric measures for brd. the primary haematological response seen in the tp calves compared to the tn animals was the presence of a leucocytosis characterised by lymphophilia and neutropaenia (tables and ) . considering a lymphocytosis is commonly observed during a fever and a neutropaenia during viraemia (strauss, ) these haematological observations are to be expected in tp animals. in terms of infrared thermography, infrared heat loss is a significant avenue for the dissipation of heat in an animal (kleiber, ) . the technology has been demonstrated to be effective in the non-invasive identification of transport and other environmental stressors that alter heat loss (schaefer et al., ; stewart et al., ) , as well as pain and fear in cattle (stewart et al., ). an earlier comprehensive review of this subject has been published by mccafferty ( ) . using the gold standards criteria in the current study the data suggest that infrared thermography as a biometric measurement also shows utility in identifying the onset of disease in cattle. this is again consistent with previous findings from our own laboratory (schaefer et al., stewart et al., ) and with findings from other laboratories (polat et al., ; hovinen, ; rainwater-lovett et al., ) . indeed, as demonstrated by the roc curve calculations, the efficiency of the irt methods in the current study was equal to or better than any of the other methods ( % efficiency with irt peak values) and in populations with a higher prevalence of the disease has been demonstrated to be superior to other methods in the early identification of disease onset . in the current study the irt maximum values and the mean ratio values appeared to show the greatest utility as single measures. of interest, however, were the observations that the rate of change of an animal's thermal profile as brd onset occurred and even the degree of variation associated with a given measure such as standard deviation did show promise as valuable indicators. further evaluation of such parameters on larger data sets and in data sets with perhaps a greater degree of brd prevalence is merited. in the current study, compared to the industry practice of using clinical signs during pen checking procedures, a lower rate of false negatives was seen. in addition, compared to other procedures using breath analysis (burciaga-robles et al., ) , rumen temperature probes (rose-dye et al., ; timsit et al., ) or any number of biochemical procedures such as pcr, immunohistochemistry, elisa or acute phase proteins (decaro et al., ; fulton, ; quinting et al., ) the non-invasively collected infrared data is likely to be more cost effective, less labour intense and timely as a diagnostic procedure. most calves diagnosed with brd are likely to be treated with one of a few antibiotics regardless of the causative virus. hence, it may make some practical sense to simply provide early diagnostic information to an animal manager earlier. if specific diagnosis is desired then it may still make sense to conduct a first screen with an automated system such as infrared thermography and subsequently test suspected animals with more precise procedures. of interest in the present study was the presentation of a significant number of animals displaying intermediate values for virtually all the biological markers. these animals appeared to be neither tp nor tn. this situation may be a failure of the identification systems to accurately classify animals. or, conversely, these intermediate animals may well be a population of cattle that are either successfully resisting or slowly succumbing to brd. in either case, being able to identify such individuals may still be useful from the position of monitoring potential or suspected or emerging brd cases. . ± . (p < . ). p- equals the day before the peak temperature was evident, p- equals days before, etc. a notable and significant difference of the method used to collect infrared thermal data in the present study to that used in previous studies (schaefer et al., ) is that a multiple animal scanning capability of the system has been developed. mounting the infrared camera on a motor capable of rotating to two different scan windows as signalled from the rfid reader has enabled this system to be placed on two water bowls and hence the capacity to study more animals is significantly increased and can service pens of plus animals. the system was designed to accommodate a second water/thermography station situated parallel to the first station with the camera located centrally between the two stations, hence doubling the animal handling capabilities of the system. also, compared to other prototypes, the present system is automated and non-invasive by using a rfid reader and lap top computer for data storage. these adaptations are significant from an animal behavioural perspective since the calves can attend the water station voluntarily and without restriction or capture. hence, both thermal radiated values from the orbital region, as well as watering frequency, are obtained non-invasively. as a result it can be argued that such data are more representative of the animal's normal or steady state values, compared to values collected during animal capture and restraint. current operation procedures also enable the use of a cell phone communication system to download collected data wirelessly or via the internet. hence, data and animal responses can be monitored remotely to make management and treatment decisions regarding the emergence of brd. furthermore, the use of a non-invasive, automated, remote sensing system such as the infrared scanning station technology, lends itself to easier collection and oversight for early indicators of animal health aberrations. such a system would have clear utility in bio-surveillance and bio-security programs. data collected in the current study demonstrated that the noninvasive, automated collection of infrared thermography data from cattle at risk of brd is effective at identifying true positive and true negative animals. the study further demonstrated that such technology lends itself to the automated, wireless collection of biometric data useful for bio-security and bio-surveillance purposes. recommended code of practice for the care and handling of farm animals. beef cattle, publication /e, communications branch the role of mycoplasma bovis in bovine respiratory disease outbreaks in veal calf feedlots rapid detection of bovine corona virus by a semi-nested, rt-pcr. detecção rápida do coronavírus bovino (bcov) por meio de uma semi-nested rt-pcr etiology of respiratory disease in non-vaccinated, non-medicated calves in rearing herds automatic monitoring of watering behaviour in feedlot steers: potential use in early detection of respiratory disease and in predicting growth performance veterinary medicine transportation of young beef bulls alters circulating physiological parameters that may be effective biomarkers of stress evaluation of breath biomarkers and serum haptoglobin concentration for diagnosis of bovine respiratory disease in heifers newly arrived at a feedlot guide to the care and use of experimental animals dexamethasone reduces transport-induced weight losses in beef calves the medicine and epidemiology of bovine respiratory disease complex in feedlot respiratory disease associated with bovine coronavirus infection in cattle herds in southern italy board-invited review: recent advances in management of highly stressed, newly received feedlot cattle bovine parainfluenza- virus cough sound description in relation to respiratory diseases in dairy calves bovine respiratory disease research a nation-wide epidemiological study of acute bovine respiratory disease in france bovine pasteurellosis and other bacterial infections of the respiratory tract serial evaluation of physiologic, pathological, and behavioral changes related to disease progression of experimentally induced mannheimia haemolytica pneumonia in postweaned calves multiplex real-time rt-pcr detection of three viruses associated with the bovine respiratory disease complex dissertationm, department of production animal medicine acute phase proteins assessment for an early selection of treatments in growing calves suffering from bronchopneumonia under field conditions experimental infectious respiratory disease in groups of calves: lobular distribution, variance, and sample size requirements for vaccine evaluation clinical biochemistry of domestic animals the fire of life the value of infrared thermography for research on mammals: previous applications and future directions statistics for biomedical research. version stress alters the cellular and proteomic compartments of bovine bronchoalveolar lavage fluid dairy calf pneumonia: effective treatment depends on early and accurate diagnosis sensitivity and specificity of infrared thermography in detection of subclinical mastitis in dairy cows respiratory disease of the bovine neonate development of a -step enzyme-linked immunosorbent assay for the rapid diagnosis of bovine respiratory syncytial virus in postmortem specimens detection of foot-and-mouth disease virus infected cattle using infrared thermography aktualne dane na temat zaka _ zeń powodowanych przez wirus syncytialny układu oddechowego bydła rumen temperature change monitored with remote rumen temperature boluses following challenges with bovine viral diarrhea virus and mannheimia haemolytica the effects of fasting and transportation on beef cattle: . acid-base-electrolyte balance and infrared heat loss of beef cattle the early detection of bovine respiratory disease (brd) with infrared (irt) and treatment with nitric oxide infrared detection and nitric oxide treatment of bovine respiratory disease early detection and prediction of infection using infrared thermography the use of infrared thermography as an early indicator of bovine respiratory disease complex in calves early disease detection: implications for industry efficiency, food quality and safety the use of infrared thermography in the non-invasive, automated detection of calves displaying bovine respiratory disease orbital infrared as an early indicator of bovine respiratory disease in sero-negative cattle bovine respiratory disease in feedlot cattle: environmental, genetic, and economic factors infrared thermography as a non-invasive tool to study animal welfare non-invasive measurement of stress in dairy cows using infrared thermography eye temperature and heart rate variability of calves disbudded with or without local anesthetic effects of local anesthetic and a non steroidal anti-inflammatory drup on pain responses of dairy calves to hot iron dehorning effects of an epinephrine infusion on eye temperature and heart rate variability in bull calves white blood cell metabolism and neutropenia and qualitative disorders of neutrophiles the epidemiology of bovine respiratory disease: what is the evidence for predisposing factors? diagnostic testing early detection of bovine respiratory disease in young bulls using reticulo-rumen temperature boluses monitoring of antimicrobial resistance in pathogenic bacteria from livestock animals antimicrobial resistance in bovine respiratory disease pathogens: measures, trends, and impact on efficacy bayesian estimation of the performance of using clinical observations and harvest lung lesions for diagnosing bovine respiratory disease in post-weaned beef calves antibiotic resistance. fact sheet no the authors wish to express their appreciation to agriculture and agri-food canada, growing forward research funding program, the beef cattle research council of the canadian cattleman's association, the national science and engineering research council of canada and the alberta livestock and meat association for funding used in the current study. the authors are also grateful for the valuable technical assistance from p. lepage, l. holt-klemic, d. froehlich and s. marchand. assistance provided by the lacombe beef research unit operational staff is likewise greatly appreciated. key: cord- - ickafd authors: kapil, sanjay; yeary, teresa; johnson, bill title: diagnostic investigation of emerging viruses of companion animals date: - - journal: vet clin north am small anim pract doi: . /j.cvsm. . . sha: doc_id: cord_uid: ickafd in this article, the authors are specifically concerned with the timely and accurate detection of emerging diseases of small animals that are viral in origin. veterinarians are bound to encounter emerging viruses in their practice. the problem is unavoidable, because viruses are highly mutagenic. even the immune response dictates the nature of virus that evolves in a host. if the clinical signs and diagnostic methods fail to correlate, the veterinarian should work with the diagnostic laboratory to solve the diagnostic puzzle. c linicians and laboratorians are usually the first to detect most outbreaks of emerging diseases in animals. much attention is rightfully given to emerging diseases of commercial food animals; however, small animal practitioners also have an obligation to be vigilant to the possibility that new and devastating viral diseases might emerge that infect the companion animals in their charge. canine parvovirus (cpv) type , emerged in and spread worldwide within less than years [ ] . in , a new antigenic type, cpv- c, was reported in italy [ ] , which has since caused outbreaks in western europe, asia, south america, and the united states [ ] because current vaccines offer no protection for this type. in this article, the authors are specifically concerned with the timely and accurate detection of emerging diseases of small animals that are viral in origin. the term emerging virus is defined broadly and includes these categories: variants of a known virus that has gained enhanced virulence or that is able to infect completely vaccinated animals a known virus that has reappeared in the population after a decline in incidence novel or previously unidentified viral agents detected for the first time because of improved diagnostic capabilities ''mystery diseases'' with large numbers of naive animals involved that are caused by previously uncharacterized viruses spread of an emerging virus among small companion animals is multifactorial and includes animal health and sanitation practices; migration of a pathogen from a wild reservoir to domestic animals because of changes in populations, trade, climate, land use, and the introduction of invasive species (eg, plant, animal, insect); and, finally, globalization, as was the case with west nile virus (wnv). emerging viral infections may take a heavy toll on the health of cats or dogs whenever they are brought into situations in which groups of animals are housed together, even temporarily, such as at greyhound racetracks, kennels, catteries, animal shelters, animal obedience training classes, dog parks, pet stores, pet day care facilities. this is especially true when pets are allowed by their owners to roam at will, commingling with ownerless feral dogs and cats and wildlife. for example, the rapid spread of cpv- , which is extremely stable in the environment and highly contagious, was caused not only by the movement of dogs by their owners but by the transfer of fecal material on shoes and clothing of travelers and, unintentionally, through national and international mail [ ] . according to the to national pet owners survey conducted by the american pet products manufacturers association, the us pet cat population is estimated to be . million and the pet dog population is estimated to be . million [ ] . municipalities throughout the united states commonly pass animal control ordinances to protect the public health and safety and general welfare of the citizens and animals residing within the city. typically, animal control codes limit the numbers of companion animals that individuals may own or keep on their private property, require that cats and dogs be licensed annually by owners and vaccinated against rabies, prevent animals from running at large, require proper disposal of animal waste, and prevent the feeding of wild or feral cats or dogs. vaccination of dogs and cats by compliant pet owners for rabies prevention has, since , dramatically reduced the occurrence of this disease; currently, most animal cases reported to the centers for disease control and prevention (cdc) now occur in wildlife [ ] . compliance with other animal control ordinances is variable, particularly among pet owners with respect to leash laws for dogs and cats and among well-intentioned individuals who maintain wild or feral colonies of cats and dogs by providing food, water, and shelter. statistics from the humane society of the united states indicate that to million companion animals are admitted to shelters each year and nearly half are adopted or reclaimed by their owners, whereas the remaining animals are euthanized [ ] . no census of ownerless dogs and cats is available. estimates of the feral cat population in the united states range from million to million animals living primarily in or near urban settings with ample opportunity to interact with pets that are allowed to roam and with wildlife [ ] . thus, ownerless, wild, or feral dog and cat populations may transmit infectious and zoonotic diseases between wildlife and companion animals. from a public health standpoint, this is of particular importance because emerging viral infections from wildlife are often transmitted to human beings by means of a pet that is allowed to stray. it is widely believed by virologists and public health epidemiologists that most viruses emerging from wildlife have an rna or single-strand dna genome [ ] because they have a high propensity for mutation. two significant canine viruses have emerged recently and meet this hypothesis: cpv and canine distemper virus (cdv). canine distemper has re-emerged in the past decade [ , ] because of antigenic and genetic drift in the surface protein (h glycoprotein). in a multicontinent study, variant cdv strains, (but not the vaccine strain of cdv virus) were the cause of illness within weeks after vaccination. in and , large outbreaks of cpv variants (cpv- c and cpv- b*) in kennels occurred in oklahoma and other states [ ] . diagnostic and molecular studies detected mutations in the parvovirus isolates that explained the failures of current commercial cpv vaccines from conferring protection and of approved commercial diagnostic kits from detecting these new viral isolates. another recent example is outbreaks of hemorrhagic symptoms associated with virulent feline calicivirus (fcv) in the united states [ ] ; however, molecular basis of gain of virulence in fcv is not yet understood. in addition to virus evolution, in some cases, the virus can be reintroduced back after the population immunity has declined after a period of disease-free status. thus, diseases that have been eradicated from developed countries but are still circulating in developing countries [ ] may re-emerge by reintroduction from trade or movement of animals. there is a major commitment by the us department of agriculture (usda) in this country and in cooperation with foreign governments and international agencies worldwide to monitor the health of food animals and certain wildlife but not of companion animals [ ] . the primary mission of the cdc is to promote and protect human health. to this end, the cdc performs surveillance for noninfectious and infectious diseases, including zoonoses [ ] ; however, the only chosen reportable viral diseases of animals that are collected by the cdc are rabies and avian influenza (h n ), and those that are reported to the cdc arbonet system are avian, animal, or mosquito wnv infections. largely, surveillance of companion animal diseases, many of which have zoonotic potential, has not been considered to be a priority until recently [ , ] . in , the cdc partnered with the purdue university school of veterinary medicine to establish a pilot surveillance system to monitor clinical syndromes and diseases of small animals [ ] to determine whether animals can serve as sentinels of health hazards to human beings. the national companion animal surveillance program (ncasp) initially drew exclusively on the database of the privately owned organization, banfield, the pet hospital, which provides medical care to approximately . million pet dogs and cats in states, and it now integrates data from antech diagnostics to detect potential emerging and zoonotic infections. a long-term goal of the ncasp is to become a national resource in veterinary public health. in the meantime, the front line of companion animal surveillance for emerging diseases is at the home front, with astute small animal clinicians playing a major role. it can be a challenge for busy and isolated veterinary practices to receive the information on emerging viruses. linking to a health-related network for companion animals might fill the gap. recently, a space-time permutation scan statistic, which was applied in the anthrax terrorist attacks in [ ] , wnv outbreaks [ ] , and enzootic raccoon rabies [ ] , has been applied to veterinary diagnostic data in the unite states and europe [ ] . this analysis provides important information about potential clusters of medical conditions and issues medical alerts about the developing situations based on mortality and confirmed diagnosis of important disease conditions. earlier and more timely notifications should lead to more thorough investigations and reduce losses, especially from emerging viral diseases. it is important to keep in mind that clinical syndromes tend to be multifactorial, and it is essential to review the entire history, including environmental factors, with the specialist in a small animal specialty practice and also with a small animal teaching hospital before arriving at a conclusion about the case. the purpose of this article is to encourage companion animal veterinarians to think outside the routine diagnostic plan when atypical cases of infectious disease are presented at their practices. detecting emerging viral diseases of companion animals requires interaction and discussion among clinicians, pathologists, and virologists, and practicing small animal veterinarians must stay engaged in communication with these specialists through their state diagnostic laboratories or nearby colleges of veterinary medicine. veterinary diagnostic medicine is rapidly progressing, and it is critical for the successful practitioner to stay abreast of new developments in small animal infectious diseases and their diagnosis through continuing education [ ] [ ] [ ] . the development of monoclonal antibody technology in the s and the advent of the polymerase chain reaction (pcr) assay in the s have reshaped veterinary diagnostic strategies, especially in the subspecialty of virology. now, these molecular techniques, which are becoming mainstream applications in routine viral diagnoses, are proving their merit in facilitating the diagnosis of emerging animal viruses. the authors offer practical information on the applications of diagnostic techniques for investigating viral disease outbreaks in companion animals. the authors provide this brief overview of diagnostic techniques in the modern virology laboratory that are used for routine diagnosis and in identifying novel and emerging viruses. every step of diagnostic investigation-history, specimen collection, transportation, and laboratory examination-has to be carefully aligned for optimal outcome. small animal clinicians are familiar with symptoms of common infectious diseases and are often the first to recognize the emergence of new disease problems. in some cases, there may be a history of vaccination compliance, yet some animals develop disease [ , ] . it is important to record the complete history, including the body system involved (eg, respiratory, gastrointestinal, reproductive tract, nervous system), clinical symptoms and their duration, the presence of lesions, and vaccination history. particularly when the case is confounding, the client must be carefully and thoroughly interviewed as to how he or she manages the pet (ie, is the pet free to roam; has the pet traveled recently and where; if this is a new pet, where and how was it obtained; are there other pets in the household). consulting a book on differential diagnoses can be useful to list the potential causes [ , ] . when a history of unusual symptoms is presented, clinicians, recognizing that these cases may be important to individual and universal animal health, should refer these cases to an accredited veterinary diagnostic laboratory. it is convenient to attach copies of all relevant hospital records to the laboratory submission form to aid the diagnostician. correct diagnosis depends on a thorough case history of the affected animal and submission of appropriate specimens that are collected and transported in a manner to preserve the integrity of the viral agent. submitting a comprehensive collection of specimens in a timely manner to the diagnostic laboratory from affected animals when the disease does not fit a familiar clinical picture, as is the case with emerging viral diseases, is of paramount importance. all the system(s) that are potentially involved and all the tissues with gross lesions should be sent to the diagnostic laboratory. it is important to check for concurrent infections. viral diagnosis depends on the quality and type of specimen collected [ ] . the best time for collection of specimens is immediately after symptoms of disease are first noticed. samples from all body systems involved in the acute stage of the disease of affected animals should be submitted to the diagnostic laboratory in a timely manner by overnight delivery. at least to g or ml of each sample should be collected. recovery of virus in cell culture depends on the condition of the specimen received by the diagnostic laboratory. freezing specimens can be detrimental to virus isolation efforts (and also to electron microscopic identification) and should only be done (À c) if it is not possible to deliver the specimen to the laboratory within hours. use wet ice for shipping virology samples, because dry ice (solid carbon dioxide gas) can inactivate many viruses, preventing isolation in cell culture. tissues intended for virus isolation should always be shipped in separate packages from specimens that are immersed in formalin to prevent fumes of formaldehyde from reaching the fresh tissues. it is imperative that tissues and organs from animals that have died be harvested as soon as possible after death. postmortem tissues should be placed in sterile containers with a small amount of transport medium ( - ml), if possible. when the clinician is unsure as to what specific organs and fluids should be retrieved, the entire carcass of the dog or cat may be delivered to the laboratory for examination. to obtain more specific details regarding specimen collection, packaging, and submission, contact the diagnostic laboratory of your choice by telephone or consult its specimen submission and fee schedule guidelines, which are often available on an internet web site. individuals who ship biologic substances for diagnostic testing are required by federal law to be in compliance with all regulations governing packaging and labeling of interstate shipments of causative agents. failure to follow the regulations results in heavy fines (fig. ) . complete instructions on appropriate packaging for laboratory specimens to be mailed or shipped by a common carrier may be accessed in several sections of the code of federal regulations (cfr). health and human service regulations define such terms as diagnostic specimen and etiologic agent and describe requirements for packaging and labeling viruses have a simple structure with a protein coat enclosed with only one type of nucleic acid (dna or rna) rather than both. thus, methods for viral diagnosis target one of the components of the virus structure. for a definitive viral disease diagnosis, four basic approaches are used: direct detection by virus isolation or direct identification, viral serology for detection of a specific antibody, viral antigen detection, and molecular-based detection of genetic material. a brief discussion of the principles of diagnostic assays representative of each approach follows. gross pathologic and histopathologic findings histologic (fig. ) and cytologic examination (fig. ) of tissues and fluids by a board-certified veterinary pathologist contributes valuable information about the pathologic signs, gross and microscopic, that distinguish infections caused by viral or bacterial pathogens and other possible etiologies. tissue tropism, mononuclear infiltrates, development of inclusion bodies (intranuclear, cytoplasmic, or both), and the formation of syncytia are some of the characteristics that differ among viruses and can sometimes distinguish different viral infections. for example, most dna viruses replicate in the nucleus, and thus tend to produce intranuclear inclusions, whereas most rna viruses form cytoplasmic inclusions, although there are exceptions. as part of the pathologist's examination, immunohistochemistry testing (figs. and ), fluorescent antibody testing, and possibly in situ hybridization (ish) studies on tissues may be ordered; these methods are considered elsewhere in this article. a complete histopathology report should include possible differentials for the lesions. the pathologist might note that some findings do not exactly fit the routine lesions he or she has observed in previously. in cases in which there are deviations in lesion type or distribution or when gross lesions and histopathologic findings suggest the involvement of a viral disease but routine virology tests do not detect the expected conventional viral agents, variant or ''emerging'' viruses or even iatrogenic infections may be suspected. in early , blue tongue virus serotype was introduced in canine populations from a commercial modified-live multivalent canine vaccine that was associated with high mortality in dogs [ , ] . in some situations, second or even third opinions from pathologists at other laboratories who have special expertise should be solicited [ ] . with the application of telepathology to veterinary case materials, networks of specialists, including veterinary pathologists, small animal clinicians, infectious disease specialists, and laboratory diagnosticians, are able to exchange patient histories, clinical data, and images (gross and microscopic) through the internet for consultation, diagnosis, and education. this allows timely access to expert opinions at other locations throughout the world [ , ] . the use of telepathology can facilitate rapid intervention through the synergy of computer technology and special pathology expertise (eg, system-and speciesspecific pathologic findings) to understand the lesions in difficult cases better. conventional virus isolation techniques are often the backbone of investigation of novel viral diseases, provided that the virus is cultivable in available cell lines or primary cell cultures. virus isolation may be relatively slow depending on the growth characteristics of the virus; however, roller culturing or centrifugation of samples onto cell monolayer(s) can enhance viral replication and recovery. in many of the recent emerging viruses from wildlife (eg, bats), the virus was first cultivated, allowing further characterization of the virus. it is important to keep in mind that virus isolation, even if the effort is successful, may have a slow turn-around time, approximately to weeks. definitive identification of virus in cell culture can only be accomplished with specific antibody nucleic acid testing, and in the case of an ''emerging'' virus, existing reagents may not be reactive with the ''new'' virus. if culture is successful, however, the viral material may be studied by electron microscopy (em) and by molecular techniques, as described in this article, to characterize the new isolate. virus isolation requires fresh tissues and cannot be done on formalin-fixed tissues. em is often used in veterinary diagnostic laboratories to detect enteric viruses in fecal samples retrieved during the course of viral diarrheal disease. additionally, em is indispensable for identification of emerging and previously unidentified viruses in clinical samples [ ] , and this method has helped in the identification of many new viruses, including, most recently, bat lyssavirus [ ] . viruses can be classified up to the virus family based on size, shape, and distinctive structural features, such as envelopes or protein spikes, particularly for parvovirus, rotavirus (fig. ) , coronavirus, astrovirus, herpesvirus, poxvirus, and picornavirus. em allows detection of multiple viruses simultaneously. application of antibodies to supplement the em diagnosis provides higher sensitivity and further confirmation of the viral diagnosis. sensitivity is the major limitation of em, and at least to virus particles per milliliter must be present in the sample being examined. because the electron microscope is an expensive piece of equipment that requires special technical skills and a high level of expertise, it is not available in many laboratories. viral components can also be determined by several basic biochemistry experiments. acridine orange (ao) staining can determine the nature of the nucleic acid of purified viral particles [ ] . differentiation as to whether the nucleic acid is single-or double-stranded in nature is based on the color developed on ao staining; double-stranded dna or rna nucleic acids stain yellow green, whereas single-stranded dna or rna acids stain flame red. nuclease susceptibility of the purified virions differentiates dna from rna. the presence of envelope on viruses can be determined by susceptibility to the virus to heat, ether, or other lipid solvents [ ] . the titrated virus preparation is treated with ether or chloroform. a decrease in virus titer of greater than log is considered to be significant to indicate the presence of envelope on the virus. the presence of envelope indicates that virus is susceptible to common disinfectants. lack of envelope indicates that the virus is resistant to the use of common disinfectants. classic serology tests indirectly determine the viral etiology of disease by detecting the presence of antibody in serum (red-topped tube) to a specific test viral antigen, and thus provide retrospective evidence of an immune response or exposure to a virus. serologic methods still provide powerful tools in the virology laboratory of today for diagnosing viral diseases that are seen routinely and for discovering and characterizing novel viral diseases. serologic tests are now used to detect antibody or antigen in serum and body fluids. typically, methods used in the virology laboratory are serum neutralization (sn), hemagglutination-inhibition (hai) test, indirect fluorescent antibody test (ifat), and elisa. serologic results require interpretation by an expert diagnostician based on critical clinical observations, confirmation by pathology examination, virus isolation, and mass screening of the populations by serology. if animals in populations that have never been exposed to or vaccinated against a given virus have specific antibodies detected in their serum, it is expected that this is most likely attributable to recent exposure to the emerging virus. paired serum samples are important to demonstrate a fourfold significant increase in antibody titers, which indicates that the diagnosis of recent exposure may be attributable to infection as opposed to previous exposure or vaccination depending on the vaccination history. serology is also useful to study the antigenic distance of the emerging virus and provides clues as to whether the newly emerged agent is or is not likely to be protected by an available vaccine(s), such as heterologous virus in another species of animal. viral hemagglutination (ha) occurs between the viral protein; hemagglutinin (hn), which is present on the viral capsid or envelope of only certain families of viruses; and specific receptors on red blood cells (rbcs) that bind to hn, causing their agglutination and precipitation from solution. this phenomenon is the basis for a powerful and sensitive assay, the hai test. when a hemagglutinating virus is mixed with serum containing antibodies specific to that virus, rbcs that are added to the mixture do not agglutinate and precipitate from solution. feline panleukopenia, cpv, influenza a, and parainfluenza antibodies may be detected by hai testing. the hai method may also be used to identify unknown virus utilizing antibodies of known specificity; however, most often, this test is applied to detect the presence of antibodies in a serum sample against specific hemagglutinating viruses. variants of cpv and feline parvovirus can differ in the hemagglutinating activity of swine erythrocytes [ , ] . sn measures the inhibitory activity of a hyperimmune serum against viral isolates in cell culture. commonly performed in a cell culture microwell format, this is a long-standing method for quantifying virus-specific antibodies, and it is usually performed to test for antibodies to viruses that typically cause cell damage (cytopathic effect [cpe] ) to the host cell culture they infect. when a virus is mixed with hyperimmune serum containing antibodies specific to that virus, the antibodies bind the virus, preventing infection of the cell culture. the sn test can diagnose current infection using acute and convalescent serum samples from individual animals. it may also be used to determine immune status conferred on vaccinated animals. vaccination antibody titers often differ from antibody titers developed in response to natural infection. usually, vaccination titers are lower relative to infection titers, and maximal titers occur approximately to days after vaccination. sn assays are commonly performed to detect antibodies to fcv, herpesvirus, enteric coronavirus, and syncytial viruses and to canine herpesvirus, cdv, coronavirus, parainfluenza virus, and adenovirus. elisa this is useful for screening large numbers of samples for the presence of antibodies against viruses. the elisa format is flexible, and it may be used to detect antibody or antigen in clinical specimens. in either case, the detection system is an antibody conjugated to an enzyme. when the enzyme-linked antibody binds to the analyte being measured, the enzyme reacts with a chromogenic substrate, causing a color change to occur that may be measured spectrophotometrically or evaluated visually. several elisa kits are available to detect antiviral antibodies in companion animals, including cpv and cdv, feline leukemia virus (felv), feline immunodeficiency virus (fiv), and feline coronavirus. the immunoglobulin m (igm) elisa is a method used to distinguish current infection from past infection. during acute disease or immediately after vaccination with modified-live viruses, igm is the first class of immunoglobulin produced in response to infection, appearing to weeks before there are detectable levels of igg in the serum. because it is short-lived, igm levels typically disappear months after infection. a single acute-phase serum test sample is sufficient to diagnose current infection with an igm elisa. testing of igm titers is available for several viral agents, including cdv and cpv among others. elisa is useful for screening naive animal populations for the presence of antibodies against viruses to track the origin and spread of emerging infections. antibodies to wnv have recently been detected in dogs and cats by igm-capture elisa [ ] . a related method known as virus neutralization can be used to identify the serotype of a newly discovered virus. western blot (wb) may be used as a supplementary test to confirm antibody elisa results for fiv testing [ ] . to perform the assay, purified virus is disrupted using detergent; the constituent proteins are then separated on the basis of molecular weight by electrophoresis in a polyacrylamide gel. the proteins are transferred (blotted) from the gel to a nitrocellulose or polytetrafluoroethylene (ptfe) membrane for stabilization. the electrophoretically separated proteins are the antigen substrates for analyzing the test sera for the presence of specific antibodies. as with the elisa format, the western immunoblot uses an enzyme-labeled antispecies antibody that binds to the test serum antibodies that have bound to the separated viral antigens. substrate reacting with the enzyme-labeled antibody in the presence of a colorless soluble benzidine derivative results in conversion to colored insoluble precipitate at the protein bands where test serum antibodies are bound. the molecular weight of the protein detected is characteristic for a particular viral component. immunoblot results of the unknown test antisera are compared with positive control test sera for interpretation. a major advantage of the immunoblot technique is that a full antibody profile of a single serum sample is made simultaneously, identifying each of the individual particulate viral antigens that patient antibodies bind. as an epidemiologic tool, wb analysis may be used to detect currently circulating viral subtypes within a population and to characterize new emerging viral subtypes. immunoblotting is also a valuable research technique for antigen detection that is often used to characterize novel viruses by comparing them with known related viral family members using standard antisera or monoclonal antibodies. immunofluorescence assays on cells from clinical samples can be applied for rapid diagnostic investigations ( - minutes), provided that the fluorescent microscope and expertise are available in a laboratory. with the pooling of primary monoclonal antibodies against potential viral agents, the assay can be used as a screening tool and the sample tested again with individual conjugates to obtain specific virus diagnosis (fig. ) . the elisa is also a means for detecting viral antigens present in clinical specimens, and it offers a relatively quick turn-around time. antigen test elisa kits are available to detect antiviral antigens in companion animals, including cpv, felv, and fiv. additionally, it is a common practice by many veterinary diagnostic laboratories to appropriate the use of some rapid antigen test kits intended for the human diagnostic market, specifically, rotavirus test kits. when monoclonal antibodies are used as capture antibodies in elisa test kits, however, they fail if there is a mutation in the epitope of the viral surface protein present in the specimen that is being tested. lateral flow immunoassay is a special application of the elisa that provides a rapid, economic, portable, sensitive, and specific technique that is convenient for performing testing outside of the laboratory. it is the technique of choice for emerging viral infections [ , ] , and it has gained attention for use in diagnosing foreign animal diseases and zoonotic and emerging viral infections of animals, such as influenza virus and wnv, in the field. the test kits are small in size (size of credit cards), extremely stable at ambient temperature ( c), and take minutes to perform. an advantage of nucleic acid-based testing is that specimens submitted for analysis do not have to have viable viral particles present to be detected by this means. there is a trend toward application of molecular or gene sequence-based techniques to routine virology testing in diagnostic laboratories, which is justified under several circumstances. first, a molecular technique may be the test of choice if conventional methods of diagnosis are technically weak, such as when a viral agent is noncultivable or there are biocontainment concerns with culturing the virus, the virus has amorphous morphology by em, antibodies are unavailable or not specific to the virus, and serologic tests result in a confounding diagnosis. second, molecular techniques may be essential to detect and classify the sequence type or genotype of a virus. third, a viral agent may be characteristically slow to replicate, such as c-herpes virus; thus, a molecular method might provide a better turn-around time for diagnosis. in this instance, a rapid diagnosis might be achieved by pan-herpesvirus pcr. finally, a novel viral isolate that cannot be definitively identified by the routine diagnostic methods described previously may merit investigation and characterization by molecular-based techniques, which are indispensable in the classification of new and emerging viruses. these advanced techniques may confirm a diagnosis of viral etiology when other tests have failed; however, they are, unfortunately, relatively expensive. furthermore, the presence of nucleic acid does not equate to infection, and infections are attributable to subclinical, latency-associated nucleic acids or defective interfering virus particles, such as in paramyxoviruses, produced in nonproductive infections in genetically resistant hosts. clients, who bear the financial burden, should be counseled as to the benefit and shortfalls of this testing before ordering molecular-based tests. an excellent review of molecular-based techniques for diagnostic testing of infectious diseases has appeared in a previous issue in this series [ ] . the most familiar nucleic acid testing technique, pcr, has been used for more than a decade; however, over the past few years, real-time pcr has taken its place, revolutionizing diagnostic virology. in this procedure, the pcr chemistry may be combined with detection using a single-stranded dna probe with a fluorescent label [ ] . moreover, the procedure may be completed within an hour, and it allows for quantitation of results. because the hands-on steps are reduced and the pcr reactions are not opened, it eliminates the chances of cross-contamination in the laboratory. real-time pcr protocols are gaining more acceptance in routine veterinary diagnosis. in situ hybridization ish involves using nucleotide probes with an attached label. non-isotopelabeled probes (digoxigenin or fluochrome) can be applied in veterinary diagnostic laboratories. diagnostic applications of ish involve identification of virus-specific sequences (dna or rna) in the tissues or cells [ ] . although uncommon in veterinary diagnostic laboratories, ish is in routine use in human diagnostic laboratories for detection of the genotype of human papilloma viruses in cervical samples. for ish, smears and tissues (fresh, unfrozen, and fixed tissues) are suitable. in electropherotyping and restriction fragment length polymorphism (rflp), double-stranded dna (rflp) or rna (electropherotypes) is purified and size-separated on agarose or acrylamide gel electrophoresis. because nucleic acids are charged and double-stranded molecules bind more ethidium bromide compared with single-stranded nucleic acids, under the electric field, the nucleic acids migrate and larger sized molecules separate out higher than smaller sized molecules. for dna molecules to be tested, the double-stranded viral dna-or pcr-amplified fragments are digested with restriction enzymes. these techniques allow quick differentiation of viral genomes (dna or rna). both techniques have applications in molecular epidemiology of rotaviruses [ ] . new generation molecular techniques viral genome sequencing technologies viral genome or mrna sequencing is a powerful molecular epidemiologic tool and has been applied for epidemiology of rabies virus [ ] . sequences of novel or emerging viruses may be derived based on known conserved sequences of previously characterized viruses within the same family. although virus sequencing is gaining more routine application in veterinary laboratories, it does add cost, and thus should be used judiciously. when these methods fail to identify a newly discovered virus, which is truly novel, metagenomic analysis, which is largely used in research laboratories, may be applied. pyrosequencing is a recent variation on sequencing short stretches of pcrgenerated dna without the need for labeled primers, labeled nucleotides, and gel electrophoresis [ ] . although this variation on pcr and nucleic acid sequencing is currently used exclusively as a research tool, it is likely to be adapted for clinical diagnostic work in future years because it has been demonstrated to detect many different unrelated viruses simultaneously in a single reaction and to identify viral serotypes and detect viral isolates that could not previously be typed by classic procedures [ , ] . a biochip or microarray is small solid support, such as a nylon membrane, silicon chip, or glass slide, on which nucleic acid fragments, antibodies, or proteins are immobilized in an orderly arrangement. thousands of different molecules, referred to as probes, may be machine-printed as spots on the support, allowing for high throughput of samples using lower volumes of analyte in less time than conventional laboratory techniques take to complete. microarrays are essentially miniaturized laboratories that can perform hundreds or thousands of simultaneous biochemical reactions that are most commonly detected through the use of fluorophores. the fluorescent signal patterns formed by each analyte are then compared by the computer software using complex algorithms to make an identification of its contents. biochips enable researchers to screen large numbers of biologic analytes quickly for a variety of purposes, ranging from disease diagnosis to detection of bioterrorism agents. biochip technology is still relatively new and has not yet entered the mainstream of clinical diagnostics techniques, although it is widely used in research institutions. as an epidemiologic tool, the use of nucleic acid microarrays was instrumental in the rapid identification of the first severe acute respiratory syndrome (sars) coronavirus outbreak in china [ ] . coronavirus protein microarrays have been used to screen canadian sera [ ] for specific antibodies to sars and to other coronaviruses in a comparative study with the traditional elisa. scientists around the world are assessing the feasibility of using microarrays as tools for surveillance and diagnosis of influenza viruses [ , ] . once issues of sensitivity and assay validation have been addressed satisfactorily and the cost of the technology has become more affordable, microarray technology may find a place in clinical diagnosis. pathogenic virus or ''orphan'' virus or ''vaccine-source'' virus molecular methods for detecting and identifying viral pathogens are powerful. it is possible to detect a virus in a specimen, but it may have no association with the clinical condition. these types of viruses are called ''orphan viruses.'' minute virus of canine is a parvovirus, and it causes no clinical disease [ ] . as a result of the advent of sensitive molecular techniques, it is quite common to detect viral sequences of agents that may be present in a sample but not associated with the disease (orphan viral agents). it is possible to study the association of the viral agent with the pathologic findings observed to support the diagnosis. moreover, the pcr protocols targeting structural genes that are expressed only during active infection are useful and avoid the potential false-positive results attributable to latency or persistent viral infections. moreover, the sense and antisense probes offer the opportunity for resident and replication intermediates of viruses. obviously, the history of recent vaccination should be known, and the vaccine virus from the same lot of vaccine should be simultaneously included in the testing run and sequenced over critical regions to ensure that the virus in the sample is the same or different from the vaccine. when fluorescent antibody testing or immunohistochemistry testing is performed, false-negative findings result even when a related virus is present. because of changes in the sequence of the target protein epitopes, antibodybased detection methods may fail to provide the diagnosis; monoclonal antibodies used may fail to react and polyclonal antibodies may cross-react weakly when a variant strain of virus is present. thus, a sudden trend in lack of correlation between tests may signal an emerging variant of the virus. if a new variant of the virus arises, it may be associated with a change in the clinical profile and we may or may not understand the molecular basis of this shift. it is possible that the polyclonal antibodies may react weakly with the new variant of the virus. in many cases, the pcr primers may fail to amplify the new variant if the mutation occurs in the hypervariable region of the target gene amplified. for example, in the recent emergence of cpv variants, many practitioners noted clinical symptoms compatible with cpv but the commercial field tests were not working. if a new variant of virus emerges, a polyclonal antibody antiserum prepared in a heterologous species (rabbit or goat) can be used as a primary antibody against the whole virus, because it is possible that the monoclonal antibody might fail. the molecular techniques are more likely to fail compared with the antibody-based techniques because of the degeneracy of codons. it is important to keep in mind that factors other than emerging viruses can also affect the performance of usda-approved tests. for example, local anesthetic can also affect the outcome of antibody tests. in one study, the use of lidocaine was recommended over oxybuprocaine to avoid false-positive results [ ] . it should be clear to the readers that veterinarians are bound to encounter emerging viruses in their practice. the problem is unavoidable because viruses are ''perfect'' obligate parasites. even the immune response dictates the nature of virus that evolves in a host. thus, vaccines are to be viewed as preventive tools rather than as a cure for emerging viruses. in some situations, the best vaccine is bound to fail. similarly, the diagnostic methods have to be tailorfitted to keep up with the emerging viruses. if the clinical signs and diagnostic methods fail to correlate, the veterinarian should work with diagnostic laboratory to solve the diagnostic puzzle. your state veterinary diagnostic laboratory may be the first place that issues an alert to veterinary professionals and the public at large to possible emerging viral diseases. newsletters from your state diagnostic laboratory can be a good source of information about emerging viral diseases in your area. additional sources that are dedicated to dog and cat health issues and public health are available on the internet [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . an annotated historical account of canine parvovirus evidence for evolution of canine parvovirus type in italy canine parvovirus types c and b circulating in north american dogs in and american pet products manufacturers association. industry statistics and trends: - national pet owners survey rabies surveillance in the united states during feral cat population statistic the origins of new pandemic viruses: the acquisition of new host ranges by canine parvovirus and influenza a viruses experimental infection of dogs with a novel strain of canine coronavirus causing systemic disease and lymphopenia canine distemper viruses circulating in north american dogs an isolated epizootic of hemorrhagic-like fever in cats caused by a novel and highly virulent strain of feline calicivirus future risks from infectious diseases of animals. presented at the world association of veterinary laboratory diagnosticians linking human and animal health surveillance for emerging diseases in the united states-achievements and challenges united states department of health and human services centers for disease control and prevention animals as sentinels of environmental health hazards world health organization. future trends in veterinary public health. who technical report series purdue university-banfield national companion animal surveillance program for emerging and zoonotic diseases early statistical detection of anthrax outbreaks by tracking over-the-counter medication sales dead bird clustering: a potential early warning system for west nile virus activity spatial and temporal patterns of enzootic raccoon rabies adjusted for multiple covariates a space-time cluster investigation of an outbreak of acute respiratory disease in norwegian cattle herds methodology in diagnostic virology a primer on diagnostic virology: specimen selection and serology. a primer of veterinary diagnostic laboratory testing. compendium on continuing education for the practicing veterinarian a primer on diagnostic virology: direct and molecular-based detection of viral pathogens. a primer of veterinary diagnostic laboratory testing. compendium on continuing education for the practicing veterinarian antemortem diagnosis of cdv infection by rt-pcr in distemper dogs with neurological deficits without the typical clinical presentation clinicopathological findings in dogs with distemper encephalomyelitis presented without characteristic signs of the disease differential diagnosis in small animal medicine small animal medical differential diagnosis diagnostic virology bluetongue disease in dogs associated with contaminated vaccine iatrogenic infection of a pregnant dog with bluetongue virus, serotype interobserver variation among histopathologic evaluations of intestinal tissues from dogs and cats telepathology: its role in disease diagnosis in meat hygiene. presented at the world association of veterinary laboratory diagnosticians th international symposium static image telepathology in perspective overview of electron microscopy and its role in infectious disease diagnosis. presented at the world association of veterinary laboratory diagnosticians th international symposium diagnostic electron microscopy: historical review and future. presented at the world association of veterinary laboratory diagnosticians th international symposium acridine orange staining of purified rat virus strain x recovery and characterization of a minute virus of canines characterization of a nonhemagglutinating mutant of canine parvovirus characterization of a canine parvovirus strain isolated from an adult dog serologic survey of cats and dogs during an epidemic of west nile virus infection in humans use of western blot and radioimmunoprecipitation for diagnosis of feline leukemia and feline immunodeficiency virus infections a comparative study of a new rapid and one-step test for the detection of parvovirus in faeces from dogs, cats, and mink simple rapid, on-site detection for diagnosis of animal disease update on molecular techniques for diagnostic testing of infectious disease real-time pcr in clinical microbiology: applications for routine laboratory testing in situ hybridization and its diagnostic applications in pathology the isolation of rotavirus from calves, foals, dogs and cats in new zealand clinical laboratory advances in the detection of rabies virus pyrosequencing sheds light on dna sequencing type-specific multiple sequencing primers: a novel strategy for reliable and rapid genotyping of human papillomaviruses by pyrosequencing technology identification of enterovirus serotypes by pyrosequencing using multiple sequencing primers micro-array-based detection and genotyping of viral pathogens severe acute respiratory syndrome diagnostics using a coronavirus protein microarray comparison of the mchip to viral culture, reverse transcription-pcr, and the quickvue influenza aþb test for rapid diagnosis of influenza detection of respiratory viruses and subtype identification of influenza a viruses by greenechipresp oligonucleotide microarray minute virus of canines (mvc, canine parvovirus type- ): pathogenicity for pups and seroprevalence estimate oxybuprocaine induces a false-positive response in immunochromatographic sas adeno test american association of public health veterinarians cdc: healthy pets healthy people available at national association of state public health veterinarians united states animal health association code of federal regulations (search engine) key: cord- - d dym g authors: bowlin, melissa s.; bisson, isabelle-anne; shamoun-baranes, judy; reichard, jonathan d.; sapir, nir; marra, peter p.; kunz, thomas h.; wilcove, david s.; hedenström, anders; guglielmo, christopher g.; Åkesson, susanne; ramenofsky, marilyn; wikelski, martin title: grand challenges in migration biology date: - - journal: integr comp biol doi: . /icb/icq sha: doc_id: cord_uid: d dym g billions of animals migrate each year. to successfully reach their destination, migrants must have evolved an appropriate genetic program and suitable developmental, morphological, physiological, biomechanical, behavioral, and life-history traits. moreover, they must interact successfully with biotic and abiotic factors in their environment. migration therefore provides an excellent model system in which to address several of the “grand challenges” in organismal biology. previous research on migration, however, has often focused on a single aspect of the phenomenon, largely due to methodological, geographical, or financial constraints. integrative migration biology asks ‘big questions’ such as how, when, where, and why animals migrate, which can be answered by examining the process from multiple ecological and evolutionary perspectives, incorporating multifaceted knowledge from various other scientific disciplines, and using new technologies and modeling approaches, all within the context of an annual cycle. adopting an integrative research strategy will provide a better understanding of the interactions between biological levels of organization, of what role migrants play in disease transmission, and of how to conserve migrants and the habitats upon which they depend. migration is a common phenomenon in eukaryotes. it is the consequence of complex interactions between intrinsic factors (genetics, physiology, and behavior) and extrinsic factors (weather, habitat conditions, food availability, predation, topography; Å kesson and . it has evolved repeatedly and independently, in groups as diverse as slime molds, crustaceans, fish, amphibians, insects, reptiles, birds, and mammals (bonner et al. ; dingle ; alerstam et al. ) , and it can the relationship between the first challenge and migration is straightforward: in order to migrate successfully, animals must respond appropriately to environmental cues and interact appropriately with both abiotic and biotic factors in their environment. as to the second, there is a great deal of functional diversity in migratory behavior; animals range from completely sedentary to altitudinal or partial migrants to short-distance, medium-distance, longdistance, and extremely long-distance migrants. migration biologists also need to integrate living and physical systems in order to understand migration, because weather conditions and oceanic currents can influence migrants (kunz et al. ) and because appreciating migratory locomotion necessitates a thorough understanding of biomechanics. as to the fourth challenge, little is known about the genetics underlying the behavior and physiology of migration, but that is gradually changing (bensch et al. (bensch et al. , berthold ) ; we encourage further research in this area. finally, what we know about the evolution of migration suggests that it is an excellent system in which to study the interplay between stability and change. thus, researchers who study migration have the opportunity to elevate organismal biology as a discipline and to help reach all of the goals outlined by schwenk et al. ( ) -but, we would argue, only if they adopt an integrative research strategy. employing such a strategy will also allow us to address some of the grand challenges in migration biology. the primary goal of researchers who study migration is to understand when, where, why, and how animals migrate wilcove ) . we intend to explore all the factors involved, ranging from the underlying genetics to the environmental conditions that drive animal movements (nathan et al. ) . we also wish to understand how migration affects individual survival and reproductive success, and ultimately how these factors affect, or are affected by, population demographics and the evolution of different life-history strategies. understanding the effects of migrants on their breeding, wintering, and stopover areas is an equally poignant goal in migration research because these effects can be substantial (holland et al. a) ; spawning salmon, for example, transfer many tons of biomass each year from ocean ecosystems to terrestrial ones and historically transferred many more (gresh et al. ) . furthermore, migratory movements may have farreaching ramifications for other animals, including humans, because migratory animals have the potential to spread emerging diseases to new geographic areas (liu et al. ; li et al. ; olsen et al. ) . the answers to these questions are important from a scientific standpoint, but they will also help us determine how best to conserve migratory populations. many populations of migrants are declining, others are becoming more sedentary, and still others may not be able to adapt to rapid climatic change and alterations in habitat (newton ; wilcove and wikelski ; wilcove ) . therefore, we are also interested in how migrants are affected by their environments-especially because humans are currently fragmenting or destroying migrants' habitats, exploiting some migratory populations, depleting resources, altering the aerosphere (kunz et al. ) , and changing the climate. as denny and helmuth ( ) argued, one of the major obstacles to our ability to predict the effects of climatic change on organisms is an understanding of the ecomechanical and physiological consequences of such change; this is no less true for migrants. much of the research on animal migration to date has focused on birds, but even within this clade, many questions remain to be answered. to answer these questionsand meet the grand challenges in organismal and migration biology-we must adopt an integrative research strategy. by ''integrative research,'' we mean conducting activities that involve multiple taxa, the complete annual cycle of organisms, multiple scientific disciplines, and conservation efforts. this type of research also incorporates traditional and novel technologies and modeling techniques, and it combines laboratory-based studies and field-based studies to take full advantage of what each approach has to offer, similar to the strategy outlined by pennycuick ( ) for research into bird flight. of course, it is impossible for a single project to fully integrate all aspects of migration biology, but new insights can emerge if we incorporate even one additional methodology or scientific discipline into a given research project. below, we highlight some examples of integrated research on migration and demonstrate how this strategy has already begun to answer some important and pervasive questions. integrating across species and clades schwenk et al. ( ) argued that we should take advantage of biodiversity and investigate the different ways natural selection has solved various problems. migration is one of several traits that animals have evolved to compensate for seasonality and/or temporally or spatially ephemeral resources. indeed, environmental factors may influence migratory behavior more than phylogenetic relationships do, although characteristics of certain clades may have predisposed these animals to evolve migration (fleming and eby ) . for example, swimming and flying have lower costs of transport per unit distance than terrestrial locomotion has (schmidt-nielsen ); taxa that swim or fly consequently are particularly prone to evolving migratory behavior . research on many different migratory species has led to a better understanding of the underlying patterns of migration and has provided general hypotheses about the ecology and physiology of migrating animals (dingle and drake ) . these kingdom-wide comparisons are particularly important as we explore the ecology and physiology of migrating animals using modern methods and technologies that make it possible to follow animals both directly and indirectly throughout an increasing fraction of their lives (fuller et al. ; hobson ; kunz et al. a; klaasen et al. ; ). inter-taxon approaches are also important when exploring the consequences of environmental change, because such changes are likely to affect multiple migratory clades and have similar effects on them (kunz et al. a (kunz et al. , b wilcove and wikelski ) . finally, migration often involves a diversity of species; rarely does a single species move by itself. as a result, transfer of information both intraspecifically and interspecifically can occur during migration (muhkin et al. ; couzin et al. ) . for all these reasons, we need to include multiple taxa in our research programs. the challenges associated with long-distance movements are common to most migrants, and as a result many of them have evolved similar physiological and morphological adaptations, usually convergently. energy balance during migration is critical, particularly during locomotion; for example, regional heterothermy of the wings of bats may help to reduce the costs of maintaining euthermic body temperature during prolonged nighttime flights (reichard et al. ) . bats in the family molossidae have a highly vascularized hairless region (a thermal window) under their wings that facilitates thermoregulation during flight (reichard et al. ) . similarly, some birds are able to position their uninsulated legs appropriately for heat dissipation or conservation depending on their physical exertion and the air temperature (torre-bueno ; bryant ) . such adaptations for flexibile, context-dependent thermoregulation presumably optimize the use of limited reserves of water and energy during migration. understanding the intricacies of migration can be especially challenging for small and long-ranging species. thus, developing models and methods for these species relies on synthesizing findings and methodologies from studies on other taxa. protocols for investigating migration using stable isotopes, for example, were developed and initially tested in avian (marra et al. ) and marine species (killingley ) , but are now routinely employed in mammals, fish, amphibians and reptiles (reviewed in hobson ; hobson and wassenaar ; cryan and diehl ) . a critical component of using stable isotopes to track animal migrations is understanding how they fractionate across trophic levels. thus, as research uncovers stable isotope relationships with the environment in one taxon, those who study the predators or prey of that animal can gain critical information about the sources of input of stable isotopes (fleming et al. ; fleming ; sullivan et al. ; hobson and wassenaar ) . similarly, small data loggers that had been previously used on ocean-going migrants (gonzalez-solis et al. ) are now being applied to small terrestrial migrants (stutchbury et al. ). applying new (and old) methodologies to an increasing variety of migrants will help us understand, among other things, how a life-history strategy like migration can evolve in so many different taxa and what its costs and benefits are. selecting a model species to study migration can also prove highly fruitful, especially when expertise can be built up by a research group (or groups), with new researchers adding more knowledge about specific aspects of the migratory system or species being studied. such long-term knowledge and expertise will, over time, naturally lead to integrative studies, combining knowledge about different parts of the system to improve our understanding not only of the entire migration process, but, for example, how it connects to other life-history traits and its potential relevance for conservation. unfortunately, we do not have the complete genome sequence of any migratory species, although efforts are underway to obtain sequences for the little brown bat (myotis lucifugus) (http:// www.broadinstitute.org/science/projects/mammalsmodels/mammalian-genome-project). we therefore suggest that some of the next genetic model organisms (discussed by satterlie et al. ) include migratory model organisms. a prominent example of one such species is the red knot (calidris canutus). this species has been studied for decades and, as a result, a large body of information has accumulated on its stopover ecology, refueling rates, flight energetics, physiology, importance of wind for use of stopover sites, and much more (dick et al. ; wiersma and piersma ; kvist et al. ; baker et al. ; piersma et al. ; van gils et al. ; leyrer et al. ; shamoun-baranes et al. b) . over the years, these studies have incorporated observations and measurements of red knots in the field, samples of food resources, laboratory experiments, and modeling. this rich body of information provides valuable insight into this particular migratory system, collectively enabling researchers to work towards understanding seasonal interactions and linking individual responses to population-level patterns. unfortunately, this knowledge was not enough to prevent a major population crash of the red knot in north america (see conservation section, below; niles et al. ), but it can perhaps help us determine how best to help this population recover. integrating the annual cycle of organisms a third strategy that researchers can use to address the grand challenges in migration biology is, perhaps paradoxically, to focus on other life-history stages. the mere fact that many migratory species spend substantial amounts of time and energy each year in two or more widely separated geographic areas and shorter periods at multiple stopover sites while en route has obvious, but poorly-studied, consequences for all aspects of their biology. the conditions and selective pressures at winter locations are likely to affect individual condition and performance during spring migration and during the breeding season and vice versa (mcnamara et al. ; mcnamara and houston ) . this fact has important implications for the ecology, evolution, and conservation of seasonal migrants. periods of the annual cycle are inextricably linked, such that ecological circumstances within one season may influence events in subsequent seasons, a phenomenon referred to as a seasonal interaction wingfield ; hahn and macdougall-shackleton ; marra and studds ) . seasonal interactions can operate at the individual or population level (fleming and eby ; newton ; webster and marra ; runge and marra ; norris ; marra et al. ) . at the individual level, effects such as poor physical condition or late arrival carry-over from one season to the next and can thus generate ecologically relevant variation in a life-history attribute in a later season. empirical evidence for such effects in migratory animals has been strongest in non-passerine birds, including waterfowl (heitmeyer and fredrickson ; mihelsons et al. ; kaminski and gluesing ; bêty et al. ) and shorebirds (goss-custard et al. a , b gill et al. ), but evidence is also mounting for the importance of seasonal interactions in passerine migrants (marra et al. ; sillett et al. ; bearhop et al. ; saino et al. ; smith and moore ) . these studies have focused on understanding how the annual cycle influences condition and population abundance, but more recent studies have also examined how seasonal interactions can influence phenomena such as natal dispersal (studds et al. ) and sexual selection (reudink et al. ). seasonal interactions can also involve mortality within a season but act at the population level to influence density-dependent effects across seasons. this has been demonstrated most clearly in the context of hunting mortality in winter and whether it is additive or compensatory with other sources of mortality in waterfowl (gauthier et al. ; frederiksen et al. ; lebreton ) . the evidence supporting the importance of population-level seasonal interactions in passerine migrants is scarce (sillett et al. ; stokke et al. ) , but this is likely a demonstration of the difficulty of obtaining such data rather than a testimony against such interactions. modeling the annual cycle and comparing model predictions to measurements provides an opportunity to assess the interactions and carry-over effects between different aspects of migration (mcnamara et al. ; pettifor et al. ; klaassen et al. ; barta et al. ; bauer et al. ) . doing so also allows us to examine the fitness consequences of different behavioral decisions, particularly under varying environmental conditions. however, we need better estimates of various sources of mortality throughout the annual cycle in migrants, whether such mortality results from density-dependent, age-related, or geographically-dependent effects (strandberg et al. ). another obstacle to understanding seasonal interactions is a lack of knowledge about migratory connectivity, or the geographic linkage of individuals or populations between different stages of the annual cycle (webster et al. ) . currently, migratory connectivity is poorly understood in most species, yet this variable is essential for interpreting fundamental ecological and evolutionary phenomena (lawton and may ) and for developing effective conservation strategies. recent advances in tracking migratory birds using satellite transmitters, gps loggers, small light level loggers (geolocators), dna markers, and stable isotopes all represent potential approaches for tracking migratory animals throughout the annual cycle (kunz et al. b; robinson et al. ) . the continued development of such approaches is essential to our ability to understand the biology of migratory animals in the context of the annual cycle, which will aid in our ability to understand organism-environment linkages (schwenk et al. ). another strategy we can use to develop a more complete understanding of migration is to combine field-based and laboratory-based studies (gwinner ) . many important advances in migration biology have their origins in measurements of variables (such as heart rate or heat loss) under field conditions that have only previously been measured in the laboratory (butler et al. ; bowlin and wikelski ; reichard et al., ) , or studying animals under controlled laboratory conditions (lindström et al. ; hasselquist et al. ). measurements in the field make it possible to observe the behavior and physiology of animals in the environment in which natural selection acts upon them, whereas measurements in the laboratory make it possible to manipulate variables that cannot be controlled in the field. by combining these two approaches, we can achieve a more complete understanding of the particular aspect of migration under investigation. avian orientation provides an excellent example of how a single field study helped to clarify the conflicting results of many laboratory-based studies. early integrative migration biologists had demonstrated that migratory birds have the ability to use many different cues to orient, such as polarized light (able ) , geomagnetism (reviewed in wiltschko and wiltschko ) , and the stars (sauer ; emlen ) . to determine which of these was the primary cue that birds use to orient, migration biologists conducted multiple 'cue-conflict' experiments with primarily captive animals (Å kesson ). in these experiments, birds placed in orientation cages were exposed to cues that 'pointed' in different directions and researchers observed their orientation. unfortunately, the results of these experiments were ambiguous (see Å kesson ; muheim et al. a for reviews); sometimes it appeared that birds recalibrated a celestial compass based on magnetic cues, and other times it appeared that they recalibrated their magnetic compass based on celestial cues. cochran et al. ( ) conducted a field study designed to determine which cues free-flying birds use during migration. thrushes were fitted with radio-transmitters and exposed to an altered magnetic field at sunset; they were then released and followed as they migrated naturally. results were consistent with the hypothesis that these birds had recalibrated their magnetic compass based on the position of the sun at sunset or on associated patterns of polarized light. prior studies on cue-conflicts yielded confusing results because the animals were exposed to the sunset prior to tests in some experiments, particularly those conducted during the pre-migratory period, but not in others (muheim et al. a) . subsequent experiments on captive birds confirmed that several additional species also recalibrated their magnetic compasses when afforded a view of the horizon at sunrise and sunset, but were unable to do so when they did not have a view of the horizon (muheim et al. b (muheim et al. , (muheim et al. , . recent evidence suggests that bats might also use a sunset-calibrated magnetic compass (holland et al. b, in press) . field-based studies have also revealed the importance of physiological state (e.g. sandberg for review), topography (Å kesson , Å kesson et al. a; zehnder et al. ) , timing of migration (Å kesson et al. b) , and weather (Å kesson and hedenström ; Å kesson et al. ; wikelski et al. ; chapman et al. ) on the orientation and migratory decisions of birds and insects. such studies clearly show the need to be aware of the physiological state of the animal as well as the external conditions it experienced prior to capture when interpreting results from experiments with caged migrants. notwithstanding, laboratory-based studies should not be abandoned: cochran et al.'s ( ) field study may have provided the original impetus for a paradigm shift in avian orientation, but some variables are difficult, if not impossible, to manipulate in the field. for example, no method can rotate the stars in new directions while a bird is migrating naturally, nor can changes in magnetic fields or polarized light patterns be made during natural migratory flight without specialized, potentially cumbersome equipment. thus, further studies on captive birds will be needed before we can understand, among other things, the exact mechanism(s) behind the recalibration of the magnetic compass revealed by cochran et al. ( ) . integrating theoretical models pennycuick ( ) argued that, in addition to field observations and laboratory-based studies, we need theoretical models to completely understand bird flight; the same is true for animal migration. indeed, the study of migration ecology rests on a solid theoretical foundation based on biomechanics and concepts of optimality . these theoretical concepts mainly concern the process of migration by an individual animal, considering such variables as duration of stopover, timing of departure from stopover sites, optimal fuel loads at departure, migration step length, migration routes, and the overall speed of travel (hedenström ) . in a broader sense, models of the annual routines of migrants, which use multiple variables for the state of individuals and dynamic programming (barta et al. ), and seek the best possible timing of major events (breeding, molt, and migration) in a seasonal world, also fall within the domain of migration theory. all of these models represent attempts to integrate living and physical systems, as prescribed by schwenk et al. ( ) . a fundamental component of simple optimality models is the ''range equation'' , which gives the potential flight range based on a specified amount of metabolizable energy (fuel). the equation will vary depending on the type of migration (flying, swimming, or running/ walking), but the overall characteristic of the model is a diminishing return function, meaning that the increase in range from a certain amount of fuel decreases as fuel load increases. thus, migrants must trade off the benefits of additional fuel, such as increased range and higher safety margins, with the energetic costs of carrying the fuel. working out predictions about optimal tactics requires assumptions about some relevant surrogate currency, which can be energy, time or survival rate. it is assumed that by optimizing such an immediate currency, or a combination of two or more simple currencies, the animal also maximizes its fitness. in most cases, however, it is not possible to measure fitness directly in migratory animals, but by assuming a particular currency and an appropriate decision rule, it is possible to predict an optimal behavior, for example the duration of stopover and the associated fuel load at departure as a function of the rate of fuel deposition (lindström and alerstam ) . in doing so, relevant constraints must be considered, such as the physiological capacity for digesting food. to experimentally test whether migrants respond to variation in fuelling rates, one could provide additional food at a stopover site and monitor individual mass (fuel) gain and timing of departure (lindström and alerstam ) . a number of such experiments have been conducted and have confirmed the predicted positive relationship between fuelling rate and the fuel load at departure (reviewed in hedenström ) . these studies support the hypothesis that minimization of time is an important strategy in avian migration. by considering the rate of energy intake and the power required for locomotion, the overall speed of migration (including both fueling and active migratory locomotion) can be derived and how it scales with body size can be determined hedenström ) . theory predicts that the overall rate of migration should decline with increasing body size, which is consistent with observational data. however, some recent studies in which migration speeds were measured in passerines (stuchbury et al. ) and the arctic tern sterna paradisaea (egevang et al. ) seem to surpass the expected rates. it is unclear at this stage if this discrepancy is due to the use of beneficial winds, if physiologists have misjudged the capacity of migrants for fuelling, or if other aspects of the theory need to be refined. for migratory birds, bats, and insects, mechanical flight theory also predicts different optimal flight (air) speeds depending on ecological context (norberg and rayner ; norberg ; hedenström and alerstam ) . the cruising speed of flight during migration should be either the maximum range speed (energy minimization), or a speed slightly faster than this (time minimization), both of which are significantly faster than the speed of minimum power. the exact calculation of these alternative speeds is difficult; hence a powerful test to see if animals do adjust their flight speeds according to theory is to compare speeds of the same species in contrasting situations, such as migration versus display flight (hedenström and alerstam ) , or commuting versus food searching (grodzinkski et al. ). what emerges from such comparisons is that birds and bats do adjust speed adaptively according to context. it is important to keep in mind that migrating animals are not always able to behave optimally. cochran and wikelski ( ) showed that nocturnal avian migrants can sometimes be pushed backward against their normal migratory direction by headwinds but will still fly for hours in such winds. these birds presumably behave in this sub-optimal way because they use simple decision rules to guide their take-off decisions and flight times (cochran and wikelski ) . on average, such rules are presumably beneficial (wikelski et al. ) , and are therefore maintained by natural selection, but these rules can have negative consequences for some individuals. as pennycuick ( ) pointed out, we need empirical observations to complement theoretical predictions. a great deal of migration research has focused on behavior and ecology, but a complete understanding of migration requires data from other biological disciplines. below we discuss two of many possible examples of integrating additional biological disciplines. physiology brings a distinct mechanistic and constraint-oriented perspective to the study of migration. because migratory movements have such high physiological demands, much of the behavior and ecology of migrants, as well as the evolution of their migration strategies, can be understood by studying the physiological and biochemical mechanisms that influence migration performance. in other words, physiologists emphasize the need to answer proximate ''how'' questions to fully answer the ultimate ''why'' questions. two of the greatest physiological challenges to migrants are the optimal accumulation and budgeting of energy and nutrients needed to fuel movement. for example, several years at sea are required for semelparous salmon (onchorynchus sp.) to accumulate nutrient stores for their all-or-none migration and spawning. similarly, monarch butterflies (danaus plexippus) must carefully budget their fuel stores to be able to migrate in the autumn, over-winter, and migrate again the following spring, and many temperate bat species must deposit sufficient energy reserves (i.e. fat) in the autumn to sustain physiological processes through hibernation and then migrate to summer ranges before insect prey become available (kunz et al. ). as discussed above, the rate of utilization of fuel during flight, the rate of fuel deposition at stopover, and the delay between arrival and net refueling (search/settling time) are key parameters in optimal migration models engel et. al, this volume) , all of which have a sound physiological basis. the rate of utilization of fuel during locomotion is influenced by a number of factors including morphology, speed of movement, and the conditions in, and attributes of, the media traversed (e.g. wind speed and turbulence for airborne animals; bowlin and wikelski ; pennycuick ) . notwithstanding, the physiological mechanisms governing the mobilization, transport, oxidation, and ultimately conversion to mechanical power of fuel have an overriding influence on the duration of flight by affecting metabolic efficiency and the rate of use of different stores (e.g. fat and protein). laboratory and field studies of how these mechanisms function, how they are regulated, and how intrinsic (e.g. age and sex) and extrinsic (e.g. diet, photoperiod, altitude, temperature, and humidity) factors influence them are critical to understanding flight and, ultimately, migration performance. studies of the physiological aspects of search/settling time and refueling rate also exemplify how laboratory and field studies can be integrated to shed light on the ecology and evolution of migration and inform conservation efforts. it has often been observed that birds do not gain weight for one or more days following arrival at stopover sites, but it was unclear whether this search/settling behavior reflected time required to find the best feeding conditions, or a period of physiological recovery (klaassen and biebach ; alerstam and hedenström ; guglielmo et al. ). in recent years, data from the field and laboratory have revealed that migrants catabolize significant amounts of lean tissue during flight, particularly from the digestive system, and thus must rebuild this digestive capacity before they can increase their refueling rates (Å kesson et al. ; klaassen and biebach ; piersma et al. ; karasov and pinshow ) . settling time could also be affected by other physiological requirements, such as the repair of damaged flight muscles (guglielmo et al. ) or recovery from sleep deprivation (schwilch et al. ) . in optimality models, search/settling time is generally treated as constant for a given simulation, yet the physiological information suggests that it may be positively related to the duration of the previous flight; long flight may cause greater catabolism and fatigue or injury of muscles. this potential interplay between duration of flight and subsequent search/ settling time should be explored further. even after accounting for post-flight recovery and major ecological factors (e.g. availability of food, predation risk, and competition), refueling rate will be strongly influenced by physiological processes because food must be digested, absorbed and reassembled into functional tissues or energy stores. moreover, the macronutrient and micronutrient composition of the diet can influence flight performance and fuel mixture (gannes ; pierce et al. ; price and guglielmo ; weber ). thus, to fully understand what nutrients migrants seek at stopover sites, and to develop conservation strategies to provide these resources, we need to identify the mechanisms of digestion and post-absorptive processing of nutrients underlying the deposition of fuel and which set limits on refueling rate (lindström ; mcwilliams and karasov ) . denny and helmuth ( ) provided several excellent examples of how biomechanical and physiological studies can aid conservation efforts. for migratory animals, conservation efforts are being facilitated by the development of new physiological approaches to assess rates of refueling in birds ) and bats (mcguire et al. ). feeding/fasting states are reflected in the concentrations of key metabolites in plasma, and numerous laboratory and field studies show that rate of change in mass of individuals sampled only once can be estimated from metabolite profiles (jenni-eiermann and jenni ; guglielmo et al. ; cerasale and guglielmo ) . this is an advance over other methods of assessing refueling rate, such as analysis of recapture data or relationships between capture mass and time of day, because every captured individual provides useful data . by combining this simple approach with other standard methods (e.g. morphometrics, population censuses, mark-recapture analyses, telemetry, behavioral observations, surveys to assess food availability, and stable isotope analyses), it is now possible to study how factors such as age, weather, and habitat structure affect individual deposition rates of fuel. we can potentially improve conditions for migrants by providing high-quality stopover sites that allow migrants to rapidly replace depleted energy and nutrient reserves. physiology can therefore play an important role in conservation by providing the means of identifying sites with suitable refueling conditions in addition to the role it may play in improving our ability to predict the ecological effects of climatic change (denny and helmuth ). many captive migrants express a suite of migratory behaviors, allowing researchers to study specific aspects of their physiology. for example, nocturnal avian migrants undergo normal daily fueling and activity cycles in the laboratory. throughout the daylight hours, these birds feed, amassing fuel stores composed of primarily lipid and protein. at the close of the day, prior to nocturnal ''departure,'' all activity ceases. this has been termed the quiescent phase, and is considered to be a transitional period when digestion is completed and various mechanisms are activated in preparation for departure. with the onset of darkness, captive birds exhibit flight-like activity (e.g. beating their wings, extending the beak skywards and attempting to take off). such activity is termed ''migratory restlessness'' and continues until dawn, after which birds return to their daytime activities, including feeding and resting. researchers can take advantage of these behavioral changes to study the underlying hormonal changes and how they are affected by the environment, thus addressing the challenge put forth by denver et al. ( ) . studies investigating the hormonal basis of captive migrants' diurnal rhythms find that plasma levels of the metabolic and behavioral glucocorticoid hormone, corticosterone, are elevated when birds are expressing migratory restlessness, suggesting that this hormone may play an important role during periods of high energy demand (landys et al. ; ramenofsky et al. ) . during migratory restlessness, both proteins and lipids are mobilized, elevating uric acid and the ketone b-hydroxybutyrate. these findings corroborate results obtained from free-ranging migrants (jenni-eiermann and jenni ; guglielmo et al. ) . however, the studies on captive migrants illustrate how rapidly the behavioral transitions occur in conjunction with endocrine and metabolic fluctuations, thus providing new insight into the behavioral and physiological capabilities of migrants. although the exact mechanisms involved have yet to be discerned, the fact that captive migrants express a full range of migratory behavior means that researchers have a valuable model for investigating the physiological parameters of migratory behavior. results from these investigations have offered new clues that researchers might have missed had only free-living migrants been studied (reviewed by landys et al. ) . thus, employing this type of research strategy provides an exceptional opportunity for understanding the physiological mechanisms underlying the behavioral expression of migration. while much attention has focused on the ecology, evolution and physiology of migrating animals, relatively little attention has been dedicated to understanding the diverse microorganisms that live on these migratory animals and how migration affects the dynamics of these microbial communities. yet, microorganisms account for more than half of the total biomass on the planet (nee ) and are key contributors to the health of most ecosystems (madigan et al. ; olff et al. ). given the ubiquity (waldenström et al. ) and importance of bacterial assemblages, the need to understand how they are acquired, transported, and dispersed across ecosystems is critical to the global understanding of the biosphere. we now know that animal migration can act as both an efficient mode of transport for microorganisms (waldenström et al. ; liu et al. ) as well as a catalyst for infection by endoparasites and viral pathogens (gylfe et al. ) . moreover, with the emergence of zoonotic diseases such as avian influenza (liu et al. ) , west nile virus (marra et al. ) , and severe acute respiratory syndrome (sars; li et al. ; lau et al. ) , animal migration has become a topic of multidisciplinary interest (reed et al. ; hubalék ; calisher et al. ). microbial ecology is therefore an appropriate component of integrative migration biology. avian plumage, for example, provides a particularly interesting microbial ecosystem. feathers harbor a diverse microbiota (burtt and ichida ; bisson et al. ) , which includes bacteria and fungi. microorganisms in the plumage of migratory birds are exposed to many different environments within a single annual cycle because the bird migrates between breeding and non-breeding sites and uses numerous stopover sites en route. recent studies have indicated that local landscapes play a significant role in microbial acquisition by migratory birds. for example, the greater abundance of common soil bacteria (lucas et al. ) in ground-foraging birds (burtt and ichida ) suggests exchanges between the microbiotas of soil and plumage. indeed, bisson et al. ( ) found that the composition of the bacterial assemblage in avian plumage varied between breeding and wintering sites, indicating that it may be possible to use microorganisms as tags in population connectivity studies if the microbial ''signature'' on the plumage remains informative throughout migration. the microbial community of avian plumage also varies in relation to migration strategy and stage of the annual cycle: resident birds differed significantly from migrants in the composition of the microbial communities of their plumage and nearctic residents had higher microbial diversity than did nearctic migrants (bisson et al. b ). moreover, the microbial composition of plumage differed significantly between fall pre-migratory stages and either breeding or non-breeding stages. migration and season may thus play an important role in the dynamics of the microbial community in avian plumage, and may also affect the dispersal of pathogens. migratory animals can be highly mobile reservoirs for both pathogenic and non-pathogenic microorganisms. a fundamental understanding of how animals acquire and disperse microbes and the interaction between microbes and the local environment with which their hosts interact during migration will provide insights into the movement of microbes across the globe. perhaps more importantly, studying the relationship between microbial ecology and migration may reveal how shifting migration patterns resulting from climatic change will affect when, where, and how migrants transport and disperse microorganisms, including invasive species such as the fungus associated with white-nose syndrome in hibernating bats (gargas et al. ). incorporating biological disciplines such as physiology and disease ecology into research on migration can greatly expand our understanding of the phenomenon. incorporating other scientific disciplines may be equally useful; schwenk et al. ( ) identified integrating living and physical systems as one of their grand challenges in organismal biology. research on migration can help us address this challenge. environmental conditions can influence migrants in numerous ways, resulting in instantaneous costs (or benefits) and more cumulative and even evolutionary consequences (nathan et al. ; shamoun-baranes et al. a) . specifically, because aerial, marine, and freshwater migrants move long distances through highly dynamic fluids, the properties of the medium may have multifaceted, direct consequences on their movement as well as indirect consequences on fitness. an example of a direct consequence is the effect of wind and water on the speed of progression during the journey (chapman et al. ; shamoun-baranes et al. a) . to thoroughly investigate these consequences, as well as their dynamics at multiple scales, interdisciplinary research is essential. the emerging discipline of aeroecology, for example, promotes the integration of atmospheric science, earth science, geography, ecology, computer science, and engineering into a conceptual framework that focuses on where, when, how, and why organisms use the aerosphere (kunz et al. ) . although most studies on the effects of environmental conditions on migrants do not explicitly integrate expertise in all fields simultaneously, they do require some basic knowledge from most of these fields; many of the best-known migrations, for example, occur in the aerosphere (drake and farrow ) . from an ecological and evolutionary perspective, however, the aerosphere is one of the least understood substrata of the troposphere with respect to how organisms interact with, and are influenced by, this highly variable, fluid environment (taylor ; kunz et al. ) . one exception to this rule is that the evidence for strong effects of wind and other weather conditions on migrating birds (richardson (richardson , liechti ; shamoun-baranes et al. a) , bats, and insects (williams ; srygley et al. ; kunz et al. ) is diverse and widespread. for example, birds and insects typically depart under beneficial horizontal wind conditions (Å kesson and hedenström ; dänhardt and lindström ; Å kesson et al. ; chapman et al. ) . due to synoptic-scale correlation in weather conditions, extending over hundreds and possibly thousands of kilometers spatially, and over several days temporally, this coordination presumably benefits these animals throughout a major portion of their migration (gill et al. (gill et al. , shamoun-baranes et al. ). in addition to horizontal winds, the spatial and temporal distribution of vertical air currents can substantially affect the soaring of migratory birds (e.g. shamoun- baranes et al. a ) and insects ). updrafts may limit the movement of soaring animals to locations and times when intense air currents exist, because these currents must be capable of lifting the animals to higher altitudes (pennycuick ; leshem and yom-tov ; shannon a shannon , b . recent findings from european bee-eaters (merops apiaster) that migrated over southern israel (sapir ), as well as earlier studies on several north american butterfly species (gibo and pallett ; gibo ) , suggest that the development of updrafts always precede the onset of movement. intensity of updrafts and the height of the boundary layer influence the altitude at which several migrants soar (shannon a (shannon , b shamoun-baranes et al. a , b , and can positively affect the speed and progression of vultures during migration (mandel et al. ) . although soaring flight is thought to be energetically inexpensive, moving through such turbulent air currents may induce elevated metabolic costs for flapping birds cochran et al. ) , presumably due to instabilities of flight. these studies demonstrate how important atmospheric dynamics are in space and time for aerial migrants. evidence for hydrodynamic effects on marine migrants is scarcer, but from the several studies that considered such effects, they seem to be at least as influential (luschi et al. ; polovina et al. ; gaspar et al. ) . to study environmental effects on long-distance migrants, one must consider the conditions encountered by the migrants throughout their extended migratory routes. because atmospheric and oceanic measurements generally cannot be carried out at the same spatial and temporal scales at which they influence migration, the most practical way to explore how the dynamics of air and water affect the movement of migrating animals is by modeling the relevant processes (see shamoun-baranes, a ). many studies have used robust meteorological models, such as the ncep-ncar re-analysis archives (kalnay et al. ) , which have a spatial extent on the order of an entire continent or even the entire globe, and a temporal extent of several tens of years, to investigate different properties of the movement of birds and insects (thorup et al. ; shamoun-baranes c; erni et al. ; stefanescu et al. ; bowlin and wikelski ; mandel et al. ) . the principal limitation of these models is their relatively poor spatial (several tens to several hundreds of kilometers) and temporal (several hours) resolution. thus, the ability of these models to explain details of specific, highly variable properties of animal movements, such as flight strategy selection (e.g. flapping or soaring) and altitude, is relatively poor. shannon et al. ( a shannon et al. ( , b and shamoun-baranes et al. ( a , b pioneered the application of atmospheric simulations to the study of bird flight at local and regional scales, but these models are limited in the processes they modeled and in their spatial extents. when exploring fine-scale responses to meteorological conditions, we advocate the use of atmospheric and oceanic models such as the regional atmospheric modeling system (rams; pielke et al. ; walko et al. ; cotton et al. ) and the ocean-land-atmosphere model (olam; walko and avissar. ). these models have been used extensively in the atmospheric sciences to study events that occur anywhere from a tiny (cm) scale to a global scale . the capacity of these models to simulate variable meteorological processes, ranging from synoptic (e.g. the dynamics of large pressure systems), to meso-(e.g. sea breeze circulation), to local (e.g. valley-mountain orographic flows) scales, permits researchers to investigate the movement of aerial migrants in fine detail over extensive areas. recently, for example, simulations of this kind were conducted to help explain flight mode, flight speed, soaring height, and the extent of wind drift of migratory birds passing over southern israel (horvitz ; sapir ). understanding how animals respond to, and are influenced by, environmental dynamics at various scales is an essential piece in both the migration puzzle (kunz et al. ; shamoun-baranes et al., a) and to general organismal biology (schwenk et al. ). in terms of migration biology, this knowledge will help inform researchers about why animals migrate (e.g. climatic influence on availability of resources), how animals migrate (e.g. conserving energy through atmospheric assistance from wind, currents, or thermal convection), how migration affects fitness (e.g. the survival consequences of animals' responses to environmental conditions), and how migration evolves (e.g. potential role of regional winds or leading lines in shaping migratory flyways). declines in a host of migratory species, from songbirds to salmon and whales to wildebeest, have led some conservation biologists to conclude that the world's great animal migrations are endangered phenomena (fleming and eby ; wilcove ) . while many of the species undertaking migrations may not be in immediate danger of extinction, severe population declines of migratory animals could lead to changes in the structure, health, and functioning of entire ecosystems (kunz et al. ) . conserving these migrants poses major scientific and political challenges. given how profoundly the act of migration shapes the life-history of animals, and given how little is known about so many aspects of migration, it is not too much of an exaggeration to suggest that almost any integrative research on migration could yield important insights for conservation. there are four areas, however, where additional research is almost certain to produce urgently needed information for the conservation of migratory animals. it is impossible to protect a migratory animal without knowing where it goes, yet our knowledge of the pathways and destinations of most migrants is fragmentary at best and almost non-existent at worse. fortunately, recent advances in telemetry and tracking have revolutionized this aspect of research. for example, croxall et al. ( ) used satellite transmitters to study the movements of gray-headed albatrosses across the southern oceans; they discovered that individuals within a breeding population employed one of three distinct strategies. birds using a given strategy followed consistent migratory routes and used the same staging areas. such basic information is vital for reducing mortality related to fisheries operations, which is now the primary threat to albatrosses and many other seabirds. options for tracking smaller animals are limited (wikelski et al. ), but $ g light-sensitive loggers were recently used to track $ g migrants (stutchbury et al. ) , and stable isotopes have been used for many years to estimate where migratory animals molt or shed (reviewed in hobson and wassenaar ) . from a conservation perspective, understanding seasonal interactions allows us to predict how events at any one stage of the life cycle, or the combined events of all stages, will influence the population dynamics of migrants. for example, illegal logging in the central mexican forests where almost all of the monarch butterflies of eastern north america winter is thought to reduce the thermal protection provided by the trees and to expose the diapausing insects to greater mortality due to cold, inclement weather. winter storms in winter storms in - , winter storms in - , winter storms in - , and resulted in the deaths of millions of monarchs, yet butterfly-watchers did not report any long-term population declines in the number of monarchs on the breeding grounds (with the important caveat that the monitoring of butterfly populations is fragmentary in north america). this begs the question of how much winter mortality (and loss of winter habitat) the monarchs can sustain before the population declines sharply. there has been a puzzling drop in the proportion of female butterflies on the wintering grounds over the past years (davis and rendon-salinas ) , but here too we do not know how this shift in the sex ratio may affect populations over the long term because we do not understand seasonal interactions in this species. synchronicity, phenology, and climatic change many animal migrations are tied to phenological events. for example, red knots, semipalmated sandpipers (calidris pusilla), ruddy turnstones (arenaria interpres), and other shorebirds time their spring stopover in delaware bay to overlap the inshore migration of horseshoe crabs (limulus polyphemus). horseshoe crabs spawn in shallow water, and their eggs are an important food for the birds. in fact, a % decline in the population of red knots migrating through the area has been linked, at least in part, to the overharvest of horseshoe crabs in the 's (niles et al. ). the migration of wildebeest and zebras in the serengeti is tied to the seasonal rains that generate new forage. how these, and other, migrations will fare in the wake of global climatic change is of growing concern to conservationists. in a landmark study, both et al. ( ) attributed declines in populations of pied flycatchers (ficedula hypoleuca) in the netherlands to a disruption of the synchronicity between the flycatchers' migration and the emergence of caterpillars caused by climatic change. these flycatchers require an abundant supply of caterpillars to feed their offspring; the caterpillars appear after the emergence of leaves. in apparent response to rising temperatures in the netherlands, leaves and therefore caterpillars are emerging earlier in the spring. the flycatchers, however, have not adjusted the timing of their arrival to the netherlands, perhaps because they use a stable cue, such as photoperiod, on their african wintering grounds to trigger their northward migration. thus, the peak of the caterpillar emergence no longer corresponds to the time when the birds are feeding their young. a similar, climate-driven disruption of the timing of caterpillar emergence and bird migration may threaten migratory songbirds in north america (strode ) . organisms that use the aerosphere are also influenced by an increasing number of anthropogenic factors, such as communication towers and wind turbines, that now dot the earth's landscape (desholm et al. ; kunz et al. a kunz et al. , b nrc ; arnett et al. ; wwea ). human alteration of landscapes is rapidly and irreversibly transforming the quantity and quality of available habitats that airborne organisms rely upon for navigational cues, for sources of food, water, and for use as nesting and roosting habitats-conditions that in turn are influencing the structure and function of terrestrial and aquatic ecosystems and the assemblages of organisms therein. climatic change and its expected increase in global temperatures, altered circulation of air masses, and its effects on local, regional, and weather patterns have had, and will continue to exert, profound influences on the dispersal, foraging and migratory behavior of arthropods, birds, and bats kunz et al. ) . understanding how climatic change will affect animal migrations will therefore require integrated research across a range of disciplines. the degree to which changes to the migratory landscape will affect animals depends in large part on the ability of the animals in question to alter the timing, direction, and destinations of their journeys. migration in birds has both endogenous and exogenous control mechanisms. as a result, birds have demonstrated the ability to alter migratory behaviors over remarkably short periods of time. house finches (carpodacus mexicanus) from an allegedly nonmigratory population in southern california were released in long island, new york in the early s and quickly established an expanding population in the northeastern us. within several decades, - % of the house finches in the northeast were migrating back and forth in southerly and northerly direction in excess of km (able and belthoff ) . a sizeable fraction of the blackcaps (sylvia atricapilla) in central europe, which used to migrate almost exclusively to mediterranean and african wintering grounds, have altered their route over the past years and now winter in the british isles, where a combination of warmer temperatures and bird feeders boosts survival (berthold ; bearhop et al. ) . migratory populations may also adjust to long-term anthropogenic changes by altering morphological traits, as has recently been shown in a group of neotropical migrants (desrochers ) . that animals can alter aspects of their migrations is clear; what is less clear is which species are able to do so and to what extent and how quickly. understanding these limits, which will be critical for the effective conservation of some species, will require a detailed understanding of the mechanisms underlying the development of migratory behaviors, particularly the physiological and ecomechanical ones (denny and helmuth ). in the long term, integrated research that is itself incorporated into policy making will be crucial for preserving many of the world's animal migrations. the phenomenon of migration presents a unique opportunity to address some of the grand challenges in organismal biology, but integrative research on migration has also greatly advanced our understanding of migration and will continue to do so. within the next decade, we anticipate that technological and methodological advances such as light loggers (stutchbury et al. ), global satellite tracking systems for small animals (wikelski et al. ) , and atmospheric simulations (pielke et al. ; walko et al. ; cotton et al. ) will make it possible to directly address some important questions. for example, technologies that allow us to track migrants year-round (wikelski et al. ; robinson et al. ) , coupled with research on individual reproductive success and survival, can help us to understand not only how populations are regulated (runge and marra ; wilcove and wikelski ) and the importance of currencies such as energy and time during migration, but also how migration itself evolves (robinson et al. ) . the other challenges in animal migration will follow, but only if we continue to implement interdisciplinary research. for example, we need geneticists to explore the genes that are expressed in rapidly refueling migratory birds, hydrologists to help understand the effects of ocean currents on migrating whales, and evolutionary biologists to examine the historical factors that have led caribou to migrate, before we can say how, where, when and why animals migrate. studying the effects of migrants on their environments and vice-versa also requires an interdisciplinary approach; aeroecology and migratory disease ecology, for example, cannot advance without atmospheric scientists and microbial biologists. integrative research is needed because migration is an extremely complex phenomenon. luckily, such research is becoming more common as collaboration becomes the rule rather than the exception and an increasing number of graduate students and post-doctoral researchers receive training in multiple disciplines. if migration researchers continue to adopt and facilitate integrative research programs, we may soon have to brainstorm a new set of grand challenges for migration biology. skylight polarization patterns at dusk influence migratory orientation in birds rapid 'evolution' of migratory behaviour in the introduced house finch of eastern north america flight initiation of nocturnal passerine migrants in relation to celestial orientation conditions at twilight selective flight departure in passerine nocturnal migrants how migrants get there: migratory performance and orientation body contents and migration strategies: a comparison between robins erithacus rubecula from two stop-over sites in sweden bimodal orientation and the occurrence of temporary reverse bird migration during autumn in south scandinavia nocturnal migratory flight initiation in reed warblers acrocephalus scirpaceus: effect of wind on orientation and timing of migration coastal migration and wind compensation in nocturnal passerine migrants comparative orientation experiments with different species of long-distance migrants: effect of magnetic field manipulation do passerines captured at an inland ringing site perform reverse migration in autumn longdistance migration: evolution and determinants the development of bird migration theory evidence for carbon dioxide and moisture interactions from the leaf cell up to global scales: perspective on human-caused climate change patterns of bat fatalities at wind energy facilities in north america rapid population decline in red knots: fitness consequences of decreased refuelling rates and late arrival in delaware bay optimal moult strategies in migratory birds bird migration: a general survey genetic basis and evolutionary aspects of bird migration individual variation in timing of migration: causes and reproductive consequences in greater snow geese (anser caerulescens atlanticus) a molecular comparison of plumage and soil bacteria across biogeographic, ecological, and taxonomic scales evidence for repeated independent evolution of migration in the largest family of bats variation in plumage microbiota depends on season and migration size in relation to the rate of migration in the slime mold dictyostelium discoideum climate change and population declines in a long-distance migratory bird pointed wings, low wingloading and calm air reduce migratory flight costs in songbirds heat stress in tropical birds: behavioural thermoregulation during flight occurrence of feather-degrading bacilli in the plumage of birds behaviour and physiology of svalbard barnacle geese, branta leucopsis, during their autumn migration bats: important reservoir hosts of emerging viruses dietary effects on prediction of body mass changes in birds by plasma metabolites flight orientation behaviors promote optimal migration trajectories in highflying insects migrating songbirds recalibrate their magnetic compass daily from twilight cues birds of two worlds: the ecology and evolution of temperate-tropical migration systems wingbeat frequency and flap-pause ratio during natural flight in thrushes rams : current status and future directions effective leadership and decision-making in animal groups on the move global circumnavigations: tracking yearround ranges of nonbreeding albatrosses ecological and behavioral methods for the study of bats optimal departure decisions of songbirds from an experimental stopover site and the significance of weather are female monarch butterflies declining in eastern north america? evidence of a -year change in sex ratios at mexican overwintering sites confronting the physiological bottleneck: a challenge from ecomechanics comparative endocrinology in the st century remote techniques for counting and estimating the number of bird-wind turbine collisions at sea: a review morphological response of songbirds to years of landscape change in north america spring migration of the siberian knots calidris canutus canutus: results of a co-operative wader study group project red knots give up flight capacity and defend food processing capacity during winter starvation intraspecific variation in avian pectoral muscle mass: constraints on maintaining manoeuvrability with increasing body mass migration: the biology of life on the move what is migration? the influence of atmospheric structure and motions on insect migration tracking of arctic terns sterna paradisaea reveals longest animal migration the stellar orientation system of a migratory bird the role of wind in passerine autumn migration between europe and africa the use of stable isotopes to study the diets of plant-visiting bats ecology of bat migration seasonal changes in the diets of migrant and non-migrant nectarivorous bats as revealed by carbon stable isotope analysis the interplay between culling and density-dependence in the great cormorant: a modeling approach research and management techniques for wildlife and habitats comparative fuel use of migrating passerines: effects of fat stores, migration distance, and diet geomyces destructans sp. nov. associated with bat white-nose syndrome marine animal behaviour: neglecting ocean currents can lead us up the wrong track seasonal survival of greater snow geese and effect of hunting under dependence in sighting probability soaring flight of monarch butterflies, danaus plexippus (lepidoptera, danaidae), during the late summer migration in southern ontario some observations on soaring flight in the mourning cloak butterfly (nymphalis antiopa l.) in southern ontario depletion models can predict shorebird distribution at different spatial scales crossing the ultimate ecological barrier: evidence for an -km-long nonstop flight from alaska to new zealand and eastern australia by bar-tailed godwits extreme endurance flights by landbirds crossing the pacific ocean: ecological corridor rather than barrier? migration strategies in relation to the population of origin: the case of cory's shearwaters tracked by gls population consequences of winter habitat loss in a migratory shorebird. i. estimating model parameters population consequences of winter habitat loss in a migratory shorebird. ii. model predictions an estimation of historic and current levels of salmon production in the northeast pacific ecosystem: evidence of a nutrient deficit in the freshwater systems of the pacific northwest context-dependent flight speed: evidence for energetically optimal flight speed in the bat pipistrellus kuhlii a field validation of plasma metabolite profiling to assess refueling performance of migratory birds a sport physiological perspective on bird migration: evidence for flightinduced muscle damage reactivation of borrelia infection in birds circannual rhythms in birds: their interaction with circadian rhythms and environmental photoperiod adaptive specialization, conditional plasticity and phylogenetic history in the reproductive cue response system of birds long flights do not influence immune responses of a long-distance migrant bird: a wind-tunnel experiment scaling migration speed in animals that run, swim and fly adaptations to migration in birds: behavioural strategies, morphology and scaling effects skylark optimal flight speeds for flying nowhere and somewhere optimal flight speed of birds how fast can birds migrate? do wetland conditions in the mississippi delta hardwoods influence mallard recruitment? t n isotopic tracking of migratory wildlife tracking animal migration with stable isotopes how and why do insects migrate? bat orientation using earth's magnetic field studying the migratory behavior of individual bats: current techniques and future directions the secret life of oilbirds: new insights into the movement ecology of a unique avian frugivore slow and safe or fast and risky: a comparative analysis of soaring-gliding flight performance an annotated checklist of pathogenic microorganisms associated with migratory birds plasma metabolite levels predict individual body-mass changes in a small longdistance migrant, the garden warbler the ncep/ncar -year reanalysis project density-and habitat related recruitment in mallards test for physiological limitation to nutrient assimilation in a long-distance passerine migrant at a springtime stopover site migrations of california gray whales by oxygen- variations in their epizoic barnacles flexibility in daily travel routines causes regional variation in bird migration speed modelling behavioural and fitness consequences of disturbance for geese along their spring flyway energetics of fattening and starvation in the long distance migratory garden warbler, sylvia borin, during the migratory phase assessing impacts of wind energy development on nocturnally active birds and bats: a guidance document ecological impacts of wind energy development on bats: questions, hypotheses, and research needs aeroecology: probing and modeling the aerosphere changes in body mass and fat reserves in pre-hibernating little brown bats carrying large fuel loads during sustained bird flight is cheaper than expected actions of glucocorticoids at a seasonal baseline as compared to stress-related levels in the regulation of periodic life processes severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats the birds of selbourne dynamical and statistical models for exploited populations the use of thermals by soaring migrants northward migration of afro-siberian knots calidris canutus canutus: high variability in red knots numbers visiting stopover sites on french atlantic coast bats are natural reservoirs of sars-like coronaviruses birds: blowin' by the wind? optimal fat loads in migrating birds: a test of the time-minimization hypothesis maximum fat deposition rates in migrating birds avian pectoral muscle size rapidly tracks body mass changes during flight, fasting and fuelling highly pathogenic h n influenza virus infection in migratory birds high diversity among feather-degrading bacteria from a dry meadow soil a review of long-distance movements by marine turtles, and the possible role of ocean currents brock biology of microorganisms movement ecology of migration in turkey vultures linking winter and summer events in a migratory bird by using stable-carbon isotopes west nile virus and wildlife migratory connectivity. in: crooks k, muttulingam s, editors. maintaining connections for nature migratory connectivity determining feeding state and rate of mass change in insectivorous bats using plasma metabolite analysis optimal annual routines: behaviour in the context of physiology and ecology the timing of migration within the context of an annual routine phenotypic flexibility in digestive system structure and function in migratory birds and its ecological significance regulatory mechanisms of numbers in breeding populations of migratory ducks calibration of magnetic and celestial compass cues in migratory birdsa review of cue-conflict experiments polarized light cues underlie compass calibration in migratory songbirds magnetic compass of migratory savannah sparrows is calibrated by skylight polarization at sunrise and sunset whitethroated sparrows calibrate their magnetic compass by polarized light cues during both autumn and spring migration nocturnal life of young songbirds well before migration long-distance biological transport processes through the air: can nature's complexity be unfolded in-silico? a movement ecology paradigm for unifying organismal movement research more than meets the eye population limitation in migrants the migration ecology of birds effects of horseshoe crab harvest in delaware bay on red knots: are harvest restrictions working? vertebrate flight ecological morphology and flight in bats (mammalia: chiroptera): wing adaptations, flight performance, foraging strategy, echolocation carry-over effects and habitat quality in migratory populations environmental impacts of wind-energy projects parallel ecological networks in ecosystems global patterns of influenza a virus in wild birds soaring behavior and performance of some east african birds observed from a motorglider towards an optimal strategy for bird flight research modeling the flying bird spatially explicit, individual-based, behavioural models of the annual cycle of two migratory goose populations a comprehensive meteorological modeling system -rams effect of dietary fatty acid composition on depot fat and exercise performance in a migrating songbird, the red-eyed vireo rapid changes in the size of different functional organ and muscle groups during refuelling in a long-distance migrant shorebird fuel storage rates before northward flights in red knots worldwide: facing the severest ecological constraint in tropical intertidal environments? forage and migration habitat of loggerhead (caretta caretta) and olive ridley (lepidochelys olivacea) sea turtles in the central north pacific ocean the effect of muscle phospholipid fatty acid composition on exercise performance: a direct test in the migratory white-throated sparrow (zonotrichia albicollis) behavioral and physiological conflicts in migrants: the transition between migration and breeding corticosterone and migratory behaviour of captive white-crowned sparrows. international proceedings of ica-cpb, pressures of life: molecules to migration birds, migration and emerging zoonoses: west nile virus, lyme disease, influenza a and enteropathogens thermal windows on brazilian free-tailed bats facilitate thermoregulation during prolonged flight non-breeding season events influence sexual selection in a long-distance migratory bird does a 'turbophoretic' effect account for layer concentrations of insects migrating in the stable night-time atmosphere? aphid aerial density profiles are consistent with turbulent advection amplifying flight behaviours: abandoning the epithet 'passive a nocturnal mammal, the greater mouse-eared bat, calibrates a magnetic compass by the sun timing and amount of bird migration in relation to weather: a review timing and amount of bird migration in relation to weather: updated review integrating concepts and technologies to advance the study of bird migration birds of two worlds: the ecology and evolution of temperate-tropical migration systems ecological conditions during winter affect sexual selection and breeding in a migratory bird stored fat and migratory orientation of birds the effects of weather on bee-eater (merops apiaster) migration the black box, the creature from the black lagoon, august krogh, and the dominant animal die sternenorientierung nächtlich ziehender grasmücken (sylvia atricapilla, borin und currura) locomotion -energy cost of swimming, flying and running grand challenges in organismal biology differential use of thermal convection by soaring birds over central israel using a convection model to predict altitudes of white stork migration over central israel the effect of wind, season and latitude on the migration speed of white storks ciconia ciconia, along the eastern migration route is there a connection between weather at departure sites, onset of migration and timing of soaring-bird autumn migration in israel? integrating meteorological conditions into migration research stochastic atmospheric assistance and the use of emergency staging sites by migrants american white pelican soaring flight times and altitudes relative to changes in thermal depth and intensity measurements of thermal updraft intensity over complex terrain using american white pelicans and a simple boundary-layer forecast model impacts of a global climate cycle on the population dynamics of a migratory songbird arrival timing and seasonal reproductive performance in a long-distance migratory landbird wind drift compensation, flyways, and conservation of diurnal, migrant neotropical lepidoptera migration of the painted lady butterfly, vanessa cardui, to north-eastern spain is aided by african wind currents weather in the breeding area and during migration affects the demography of a small long-distance passerine migrant how hazardous is the sahara desert crossing for migratory birds? indications from satellite tracking of raptors implications of climate change for north american wood warblers (parulidae) natal dispersal driven by environmental conditions interacting across the annual cycle of a migratory songbird tracking long-distance songbird migration by using geolocators models developed from c and n of skin tissue indicate non-specific habitat use by the big brown bat (eptesicus fuscus) synoptic dynamics, migration and the rothamsted insect survey -presidential address to the british ecological society bird orientation: compensation for wind drift in migrating raptors is age dependent temperature regulation and heat dissipation during flight in birds modelling phenotypic flexibility: an optimality analysis of gizzard size in red knots calidris canutus prevalence of campylobacter jejuni, campylobacter lari, and campylobacter coli in different ecological guilds and taxa of migrant birds the ocean-land-atmosphere model (olam). part i: shallow-water tests coupled atmosphere-biophysicshydrology models for environmental modeling the physiology of long-distance migration: extending the limits of endurance metabolism birds of two worlds: the ecology and evolution of temperate-tropical migration systems links between worlds: unraveling migratory connectivity effects of microhabitat, flocking, climate and migratory goal on energy-expenditure in the annual cycle of red knots simple rules guide dragonfly migration going wild -what a global small-animal tracking system could do for experimental biologists going, going, gone: is animal migration disappearing? no way home: the decline of the world's great animal migrations insect migration magnetic orientation in animals organization of vertebrate annual cycles: implications for control mechanisms world wind energy report observations of free-flying nocturnal migrants at falsterbo: occurrence of reverse flight directions in autumn this article is based on a talk given by m.s.b. and i.-a.b. in the integrative migration biology symposium at the society for integrative and comparative biology meeting in seattle, washington. the idea to hold the symposium originated in a migrate workshop. key: cord- -mbg e j authors: hardstaff, jo l; häsler, barbara; rushton, jonathan r title: livestock trade networks for guiding animal health surveillance date: - - journal: bmc vet res doi: . /s - - - sha: doc_id: cord_uid: mbg e j background: trade in live animals can contribute to the introduction of exotic diseases, the maintenance and spread endemic diseases. annually millions of animals are moved across europe for the purposes of breeding, fattening and slaughter. data on the number of animals moved were obtained from the directorate general sanco (dg sanco) for . these were converted to livestock units to enable direct comparison across species and their movements were mapped, used to calculate the indegrees and outdegrees of european countries and the density and transitivity of movements within europe. this provided the opportunity to discuss surveillance of european livestock movement taking into account stopping points en-route. results: high density and transitivity of movement for registered equines, breeding and fattening cattle, breeding poultry and pigs for breeding, fattening and slaughter indicates that hazards have the potential to spread quickly within these populations. this is of concern to highly connected countries particularly those where imported animals constitute a large proportion of their national livestock populations, and have a high indegree. the transport of poultry (older than hours) and unweaned animals would require more rest breaks than the movement of weaned animals, which may provide more opportunities for disease transmission. transitivity is greatest for animals transported for breeding purposes with cattle, pigs and poultry having values of over %. conclusions: this paper demonstrated that some species (pigs and poultry) are traded much more frequently and at a larger scale than species such as goats. some countries are more vulnerable than others due to importing animals from many countries, having imported animals requiring rest-breaks and importing large proportions of their national herd or flock. such knowledge about the vulnerability of different livestock systems related to trade movements can be used to inform the design of animal health surveillance systems to facilitate the trade in animals between european member states. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. animal trade is an effective way of introducing, maintaining and spreading animal diseases, as observed with the spread of different strains of foot and mouth disease (fmd) in africa, the middle-east and asia [ ] and the spread of bovine spongiform encephalopathy (bse), for example into oman and canada through the importation of infected cattle [ , ] . within a year, millions of live animals of many different species are transported between countries within europe for breeding, fattening, sports, companionship, conservation and slaughter. this creates opportunities for communicable diseases to be spread across the european union (eu), which is the focus of this study, even though animals must be in a fit state to be transported i.e. healthy animals without clinical signs of illness [ ] . however, animals with sub-clinical infections may go unnoticed, providing an opportunity to transport disease to different regions. live animal trade complicates tracing the origin of any disease outbreak that may occur due to an infected animal being displaced. for this reason, the eu has established a trade control and expert system (traces) to monitor imports, exports and trade in animals and animal products across the eu and to ensure traceability within the food chain [ ] , in addition to livestock movements recorded by the food and agricultural organisation of the united nations (fao). traces records the number of animals and consignments entering and leaving eu countries. despite the availability of this comprehensive database, animal health surveillance systems are rarely based on international live animal movements. to understand better livestock trade within europe with a view to inform disease surveillance we analysed trade networks across the eu for all major livestock species and purposes of movements. animal health surveillance includes the systematic, continuous or repeated, measurement, collection, collation, analysis, interpretation and timely dissemination of animal health and welfare related data from defined populations, essential for describing health hazard occurrence and to contribute to the planning, implementation and evaluation of risk mitigation measures [ ] . recent outbreaks and spread of exotic or emerging diseases such as avian influenza (ai), schmallenberg virus (sbv) and bluetongue virus (btv) in previously unaffected territories of the eu have emphasised the need for well-developed and adequately resourced health systems, including surveillance, to ensure early detection and rapid containment, the complexities of which are highlighted by braks et al. ( ) [ ] . at the same time investment is being constrained due to significant financial budget reductions in many european countries. livestock disease is important economically with regards to a loss of productivity, its potential impact on human and animal health, and the mitigation activities implemented when disease occurs (for example trade or movement bans, testing and culling). for example, the economic cost of bse in the uk accrued from the value loss in infected carcasses, disposal costs, and, most importantly, the sharp drop in domestic beef demand due to consumer scares (sales of beef products declined by % once the possible link between bse and new variant creutzfeldt-jakob disease (cjd) was announced, but the costs were partly offset by an increase in consumption of substitute meat), and a complete loss in export markets [ ] . further costs accrued from operating various public schemes, establishment and enforcement of new legislation and the adjustment of the industry to the new structure and markets [ ] . livestock disease can be spread directly for example the introduction of fmd from irish calves imported to the netherlands that were also held responsible for the infection of a farm near to the port of introduction to mainland europe [ ] . it can be spread by infected equipment, crates or transporter vehicles which can be contaminated by microbes. for example escherichia coli (e. coli) bacteria were detected on the sides and floors of lorries [ ] and contaminated transporters were found to be responsible for spreading classical swine fever to different farms in lithuania [ ] . by moving animals with latent or asymptomatic infections this enables disease to spread to wherever the animal travels or where the necessary vectors may be present. particularly in the case of epidemic diseases where the reduction of time from introduction of a hazard to its detection can enable early response and thereby lead to a reduction in intervention costs to contain an outbreak [ ] , effective surveillance is critical. few surveillance systems however, are designed based on international livestock movement data, even though such data can provide information on the quantity and seasonality of livestock movements, the types of movement (for example flows from production of point of lay birds to laying units), the route the animals take and associated stopover or resting points. surveillance for many livestock species occurs at the farm where it is the responsibility of the farmer (and veterinarian) to report notifiable diseases or at the abattoir where it is the role of the official veterinarian to inspect livestock according to council regulation (ec) / [ ] and report notifiable diseases to the national authorities, which in the uk is the department of the environment, food and rural affairs (defra), which in turn must inform the european food safety authority (efsa) as stated in council regulation (ec) / [ ] . network analyses are useful ways of visualising the countries that are importing animals from a great number of other countries (high level of indegree) and countries that are exporting to a high number of countries (outdegree), these are values that can change temporally. they have been used to find out movement between farms of different species, for example, fish movement between farms in scotland [ ] and a study of pig and cattle movement between farms in sweden [ ] . countries with a high indegree, which for the purposes of this study has a maximum number of (the number of countries, i.e. (nodes, within this study and the eu as of ) that could be used to rank countries, can be more vulnerable to introducing disease due to importing animals from a greater number of countries than those with a low indegree whilst countries with a high outdegree may have a great ability to be able to transmit a disease to many countries; this highlights the importance of understanding levels of disease within trading countries. information about the indegree and outdegree of farms was used by frössling et al. ( ) [ ] to investigate whether it could be used to target the surveillance of two cattle diseases in sweden, based on a threshold of in-and out-degrees. they found a positive association between a positive test result and the purchase of animals and proposed approaches to design risk-based surveillance based on cattle movement data. networks can also be used to quantify the proportion of international partners trading with each other (dyadic contacts) compared with the maximum number of national trading partners available for trade within an area allowing a comparison to be made between species and production systems [ ] . the higher the density the more connected countries are with respect to the animal being traded and the more countries that may be at risk from contracting a disease from buying in infected livestock. a measure of mixing within a network is to look at its transitivity which indicates whether countries that a country is trading animals to are also trading animals with each other (a triad) [ ] . the greater the level of transitivity the faster a disease can spread between countries and potentially infect many countries within the european area [ ] . transitivity and density for different communities of wild and domestic ungulates were investigated for the propensity to transmit e. coli by vanderwaal et al. ( ) [ ] . however, the network may only consider the point of origin and destination and not necessarily consider the route itself that may involve briefly stopping in other countries where a disease transmission event may occur, for example fmd in france [ ] . we hypothesise that the description of trade networks can inform the design of more efficient animal health surveillance systems that may enable a more rapid investigation or response to be implemented. different species being transported for different purposes will have networks of different densities and different countries with the greatest indegree or outdegree. the aim of this project was to map live animal trade networks in eu countries and assess potential differences between species and purposes of transport. this was done by illustrating the number of live animal imports and exports between eu countries including the number of country contacts and numbers of livestock units (lsu, a unit that takes into account the age, sex, purpose of animals with dairy cows having a reference number of ) moved determining the density of networks and similarities of networks between species. table illustrates the median livestock intra-community movements (expressed in livestock units) and the densities of the transport networks. by far the most heavily moved animal species within europe in were poultry for slaughter and breeding, followed by poultry for 'other' purposes, pigs for fattening, pigs for slaughter and cattle for fattening; goats were the least traded species. generally more lsus were transported for fattening than for slaughter. the density of movement (table ) shows that there was greater connectivity for cattle than for the heavily traded poultry. breeding networks were found to be denser than those for other purposes. this may be due to the number of consignments needed to move the relative units of animals. the geographical trade flows are shown in figures , , , , and . the transitivity indicates that disease would spread more slowly for 'other' purposes of animal movement than for breeding, fattening or slaughter with the exception of poultry and equines. figures , , , , and show the in-and outdegrees of livestock unit movements in the eu on the left and the geographical trade flows in the right, which are separated by species and by purpose of trade. the axes of the graphs of the in-and outdegrees reflect the numbers of trading partners. the countries in the top right received and exported animals with the greatest number of countries, whilst the bottom left indicates those that have little or no export or import trade with other countries. some countries are found in the top right corner with regards to many different animal movements e.g. germany, whilst others rarely buy or sell to the other countries considered in this study e.g. cyprus, finland and sweden, whilst other countries import from many countries and export to few e.g. italy. very few shipments of weaned cattle, sheep and goats require a rest period of hours (additional file ), whereas many unweaned animals would require a hour break in their journey from their point of origin to their figure the outdegree is shown against the indegree for the trade of cattle for different purposes on the left column of the table and the geographical movement across europe is shown on the right column of the table. the arrows between the countries indicate trade between the countries. the numbers in the figures refer to the corresponding countries: [ ] austria, [ ] belgium, [ ] bulgaria, [ ] cyprus, [ ] czech republic, [ ] denmark, [ ] estonia, [ ] finland, [ ] france, [ ] germany, [ ] greece, [ ] hungary, [ ] ireland, [ ] italy, [ ] lithuania, [ ] latvia, [ ] luxembourg, [ ] malta, [ ] netherlands, [ ] poland, [ ] portugal, [ ] romania, [ ] slovakia, [ ] slovenia, [ ] spain, [ ] sweden and [ ] uk. and exporting high proportions of their national population, the officially recorded number of animals of that species in the particular country. the poultry and pig sectors had the greatest number of lsu movements, which are being used to indicate breeding fattening slaughter other figure the outdegree is shown against the indegree for the trade of pigs for different purposes on the left column of the table and the geographical movement across europe is shown on the right column of the table. the arrows between the countries indicate trade between the countries. the numbers in the figures refer to the corresponding countries: [ ] austria, [ ] belgium, [ ] bulgaria, [ ] cyprus, [ ] czech republic, [ ] denmark, [ ] estonia, [ ] finland, [ ] france, [ ] germany, [ ] greece, [ ] hungary, [ ] ireland, [ ] italy, [ ] lithuania, [ ] latvia, [ ] luxembourg, [ ] malta, [ ] netherlands, [ ] poland, [ ] portugal, [ ] romania, [ ] slovakia, [ ] slovenia, [ ] spain, [ ] sweden and [ ] uk. the potential opportunities of pathogen introduction and spread, implying that they require more attention in terms of disease prevention and management, while the equine and goat sectors had the greatest and lowest densities of movements respectively. in addition to lsu movements larger proportions of national pig populations are imported breeding fattening slaughter other figure the outdegree is shown against the indegree for the trade of sheep for different purposes on the left column of the table and the geographical movement across europe is shown on the right column of the table. the arrows between the countries indicate trade between the countries. the numbers in the figures refer to the corresponding countries: [ ] austria, [ ] belgium, [ ] bulgaria, [ ] cyprus, [ ] czech republic, [ ] denmark, [ ] estonia, [ ] finland, [ ] france, [ ] germany, [ ] greece, [ ] hungary, [ ] ireland, [ ] italy, [ ] lithuania, [ ] latvia, [ ] luxembourg, [ ] malta, [ ] netherlands, [ ] poland, [ ] portugal, [ ] romania, [ ] slovakia, [ ] slovenia, [ ] spain, [ ] sweden and [ ] uk. compared with species such as goats increasing the possibility for the introduction of infected animals to an existing population. for poultry, the highest numbers of lsus moved were for slaughter, which may present less of a risk of introducing disease to an existing population, as the animals are likely to be transported from the production site directly to the slaughter point. however, many poultry journeys would require a break in transit emphasising the breeding fattening slaughter other figure the outdegree is shown against the indegree for the trade of goats for different purposes on the left column of the table and the geographical movement across europe is shown on the right column of the table. the arrows between the countries indicate trade between the countries. the numbers in the figures refer to the corresponding countries: [ ] austria, [ ] belgium, [ ] bulgaria, [ ] cyprus, [ ] czech republic, [ ] denmark, [ ] estonia, [ ] finland, [ ] france, [ ] germany, [ ] greece, [ ] hungary, [ ] ireland, [ ] italy, [ ] lithuania, [ ] latvia, [ ] luxembourg, [ ] malta, [ ] netherlands, [ ] poland, [ ] portugal, [ ] romania, [ ] slovakia, [ ] slovenia, [ ] spain, [ ] sweden and [ ] uk. vulnerability of the chain and need for adequate surveillance. poultry for breeding had the second highest lsu movements overall, which likely reflects the current structure of commercial poultry production. pure line grandparent and parent stock for breeding are produced by only a limited number of breeding organisations worldwide. for example, the two companies aviagen and cobb, have a market share of more than % of the commercial broilers produced in the eu and use their global network of distributors to serve almost all european countries [ ] . the breeder farms supplied with young breeding stock have links to hatcheries that produce day old chicks, broiler or layer farms, and slaughterhouses. this system leads to transport of young breeders, hatching eggs and day old chicks. in pigs, heavy movements were recorded for fattening, which reflects ongoing changes in production centres in the eu. in fact, more than two thirds of breeding pigs are produced in denmark, germany, spain, france, the netherlands and poland with half of the breeding pigs at regional level being concentrated in eleven regions in these six countries [ ] . germany is the main importer of fattening pigs, with an indegree of and denmark is the main exporter with an outdegree of . moreover, pigs for breeding and fattening as well as poultry for breeding were shown to have among the highest transitivities, indicating that disease spread in these networks would be fast if uncontained. hence, solely taking into breeding slaughter other figure the outdegree is shown against the indegree for the trade of poultry for different purposes on the left column of the table and the geographical movement across europe is shown on the right column of the table. the arrows between the countries indicate trade between the countries. the numbers in the figures refer to the corresponding countries: [ ] austria, [ ] belgium, [ ] bulgaria, [ ] cyprus, [ ] czech republic, [ ] denmark, [ ] estonia, [ ] finland, [ ] france, [ ] germany, [ ] greece, [ ] hungary, [ ] ireland, [ ] italy, [ ] lithuania, [ ] latvia, [ ] luxembourg, [ ] malta, [ ] netherlands, [ ] poland, [ ] portugal, [ ] romania, [ ] slovakia, [ ] slovenia, [ ] spain, [ ] sweden and [ ] uk. account trade data, surveillance efforts would need to focus on poultry for breeding and pigs for breeding and fattening. however, a mapping of surveillance in seven european countries showed that the highest proportion of surveillance components in place were for cattle [ ] . similarly, a recent literature review on animal health issues (including zoonoses) researched in the eu showed that cattle and buffalo were the species most breeding slaughter other registered figure the outdegree is shown against the indegree for the trade of equines for different purposes on the left column of the table and the geographical movement across europe is shown on the right column of the table. the arrows between the countries indicate trade between the countries. the numbers in the figures refer to the corresponding countries: [ ] austria, [ ] belgium, [ ] bulgaria, [ ] cyprus, [ ] czech republic, [ ] denmark, [ ] estonia, [ ] finland, [ ] france, [ ] germany, [ ] greece, [ ] hungary, [ ] ireland, [ ] italy, [ ] lithuania, [ ] latvia, [ ] luxembourg, [ ] malta, [ ] netherlands, [ ] poland, [ ] portugal, [ ] romania, [ ] slovakia, [ ] slovenia, [ ] spain, [ ] sweden and [ ] uk. frequently studied in the eu [ ] ; this may reflect differences in resource allocation for surveillance and disease mitigation. the reasons for this may be that cattle harbour or are perceived to harbour more pathogens than other species, that outbreaks in cattle systems have higher impact, that cattle receives more attention than other species for cultural or historical reasons, or that disease prevention and management in cattle systems are of lower quality. currently, there are no multipathogen, multi-species systematic risk assessments available at eu level that would allow a comparison of these factors. breeding networks were found to be more highly connected with more trade between countries indicating disease may spread more easily through them. this is of concern as these animals are not intended to be slaughtered on arrival and will produce new animals, therefore stringent precautions are needed to protect these populations, particularly if they are diseases not covered by eu legislation, for example the diseases listed in council regulation (ec) / [ ] . the density of international agri-trade calculated by ercsey-ravasz et al. ( ) [ ] was . which was comparable with density of many networks in this study. however, in national networks the densities and transitivities are smaller, which are due to the greater number of farms involved in national animal production compared with the number of countries involved in this study. the cattle trade network in france had a very low annual level of transitivity indicating that disease spread would be slower than that between european countries [ ] . the pig and cattle networks in sweden had lower transitivities than international networks of these species [ ] as did the transitivity of pig movements in denmark [ ] and the uk [ ] . the location of countries in figures , , , , and gave an indication of where surveillance could be targeted with countries in the upper right quadrant both importing and exporting high numbers of lsu, which means that they need to monitor both production to export healthy animals and import processes to avoid introduction of disease. countries in the lower right quadrant may need to consider strengthening surveillance related to import processes. many national studies have found that the majority of animal movements are between premises with lower indegrees and outdegrees as shown in a study by smith et al. [ ] , this reduces the likelihood of disease transmission to many different areas, reducing the level of surveillance needed. many countries trading cattle were found to have an in or out degree equal or greater than five. this was the threshold that was calculated to require enhanced surveillance for bovine coronavirus in a study on trade and cattle in sweden by frössling et al. [ ] . consequently, there seems to be ample opportunity to take advantage of trade network data to enhance surveillance. the evolution of trade networks over time at the eu level could be monitored using indegrees, outdegrees, and transitivity. such monitoring would provide information at the systems level and allow observations of changes in networks over time and where consequent surveillance efforts should be focused. higher-level surveillance capturing trends or changes in trade patterns could complement existing surveillance systems that are commonly disease centered. the differences across countries in terms of indegrees and outdegrees also bring up the question of who has the responsibility for disease control, including surveillance the buyer, the seller or relevant food business operator depending on the stage of livestock production [ ] . while the draft new eu animal health law [ ] refers to listed diseases and pre-dominantly supports disease centered surveillance, it also creates a framework for the better use of the synergies between surveillance undertaken by the different actors in the field to ensure the most effective and cost efficient use of surveillance resources as well as promotion of data availability and facilitation of data exchange. transportation itself is stressful for animals as indicated in many studies in many species for example cortisol in pigs [ ] ; heart rate and cortisol in cattle [ ] ; cortisol in lambs [ ] ; cortisol in horses [ ] ; increasing susceptibility to disease and may enhance the likelihood of shedding pathogenic agents in transit or in the receiving country, which may lead to infection in other animals. it is common to refer to malaise post-transportation as shipping illness [ ] . however, pathogens may be introduced or spread from transporters and not just from the animals that they transport. studies have demonstrated that transporters need to be thoroughly cleaned to prevent them from acting as a source of pathogens to subsequently carried animals, for example to prevent transmission of porcine reproductive and respiratory syndrome virus, that can survive in transporters, being transferred to pigs [ ] . rest stops are infrequent for some species, however, if animals from more than one origin are rested in the same place it may allow for disease spread. this is most likely to impact animals traded for breeding and fattening purposes that have more lsus and are more highly connected than animals already at slaughter weight. these are animals that will live in the receiving country for a period of time that may enable pathogen transfer. many of the highly connected countries (with high in and out degrees in the top right of figures , , , , and ) for example germany are geographically located in an area (central europe) that minimises the distances and therefore time that animals have to travel reducing the need for rest breaks and the consequent potential for pathogen transfer. many of the long distances are from countries that rarely trade with mainland europe for example cyprus. many animals undergo long journeys between countries. the time in transit is a concern with regards of the potential for disease to spread along trade routes [ ] . this has implications for policy around the planning of livestock production and slaughter. ideally, large production facilities would not be placed adjacent to well-known and used trade routes and or resting points. however, such information is only of use to policy makers if it is captured in a systematic and continuous way allowing to monitor trends, change and modify policies accordingly if deemed necessary. the analyses have only considered the spatial aspect of trade and not taken into account temporal variations that may occur altering the relationships between the countries (nodes) and the respective network, and affect the likelihood of an animal being infectious with a disease. animal populations fluctuate within a year and the population recorded in december was used to calculate the proportion of animals being imported or exported into a country, therefore it may have under or overestimated the actual population at the time of movement. for example the majority of lambs are born between january and april increasing the sheep population until they reach slaughter weight and are culled, which occurs before december. networks are highly dynamic and these changes in movements between countries will need to be considered by surveillance programs using this approach. one method that may address this is to use exponential random graph models that can incorporate a range of different distributions of connectivity between the nodes to create many different networks, which can be compared with the data to find a model that best fits the current trade pattern [ ] . the distances that animals are transported between countries may be shorter or longer than the distances between centroids. in addition, there are many different routes across europe that may be used and this may be worth investigating in future analyses with regards to distance, time and mixing between countries. this means that our calculations for whether particular species need a rest break for movement between particular countries are generalised so that there may be fewer or greater numbers of animals being rested en-route to their destination country altering the potential for pathogen exposure. the analyses did not take into account the numbers of convoys or animals and the mixing of animals: from different farms per convoy, at resting places, at borders, when received by individuals and at markets in the country of destination. these factors will have an impact on contact between potentially naïve and infectious animals, pathogen exposure and susceptibility. the analyses could not take into animals being bought and sold on to more than one country i.e. the chain of infection [ ] and assumed that an animal moved once between countries in its lifetime. creating networks has enabled us to visualise the countries that have a higher level of involvement in animal trade. using network analysis we were able to determine the extent to which a disease may spread, the production systems where disease spread may be more rapid, for example registered horses and breeding cattle, pigs and poultry, and facilitates comparisons with networks in other areas. similarities between countries, species and production purposes has the potential to inform international surveillance policies that take into account trade patterns. the study has highlighted the vulnerability of the pig network to disease, which is of increasing concern due to the proximity of african swine fever to the eu and the potential for wildlife to introduce the disease [ ] . this information could complement the national movement recording systems that are mandatory for cattle throughout the eu [ ] that will soon be implemented in sheep and goats now that their form of identification tags have been decided upon [ ] , and being planned for porcines [ ] to produce a more robust surveillance plan. data on numbers of live cattle, goats, horses, pigs, poultry and sheep movements in eu countries were obtained from directorate general sanco animal health dg sanco unit g activity report for the year obtained from http://ec.europa.eu/food/animal/resources/publications_en. htm. the data obtained related to the production purpose of the animals, which fell into five categories: breeding, fattening, slaughter, registered and other (e.g. pets, show animals). these categories were analysed separately and combined for each species. the numbers of animals were converted into livestock units to enable comparison between species using the following conversion factors derived from the eurostat glossary on statistics ( ) [ ] : pigs . (breeding), pigs . (other), goats . , sheep . , horses . and poultry . . all data were obtained at a national level from publically accessible databases and no animal experimentation occurred nor consultation with animal owners therefore ethical approval was not needed. all the analyses and associated network figures were created and carried out using r . . . [ ] . networks were created from adjacency matrices and their densities were calculated using network function found in r package network [ ] . the in and out degrees were calculated and respective graphs were produced using the degree and network.layout.degree functions in r package network [ ] . the transitivity of each network was calculated using the gtrans function in the sna package [ ] . trade maps in the figures , , , , and were produced by merging shapefiles of all the countries of europe downloaded from maplibrary.org (www.gadm.org/, , gadm version ) into one polygon (europe) using arcgis . [ ] . the map of europe was then read into r using the function readshapepoly found in the maptools package [ ] . centroids (the co-ordinates for the centre of a country) were calculated for each country and linked with respective importing and exporting countries were calculated using the calccentroid function in r package pbsmapping [ ] . curved lines and arrows were drawn between the centroids for each movement using the gcintermediate function found in the geosphere package [ ] . to be able to relate the numbers of animals being traded with the animal populations of the countries, the numbers of animals of each species were obtained for from the eurostat database. the data used was for december as this was the only calendar month available for all species. a movement:standing population ratio was calculated for both animal imports and exports through adding the total number of breeding, fattening, slaughter, registered and other animals being moved and dividing by the total population of animals of that species in the exporting or importing country. to illustrate the number of animal journeys that require hour rest periods during transit, distances that animals would have to travel were approximated by estimating arc distances from one capital city to the other using www.timeanddate.com. the time in transit before animals are required to have a hour rest period were obtained from council regulation ec / [ ] . the regulation states that unweaned cattle, goats, sheep, pigs and horses require a hour rest period after hours of travel. weaned cattle, goats and sheep can be in transit for hours without a rest, whereas weaned pigs and domestic horses need to be rested after hours of transportation. any animal being transported by boat should be rested for hours at the port after being unloaded. the law for poultry and rabbits states that they can travel for up to hours without food or water and whereas chicks within hours of hatching can travel for up to hours without food or water. to gauge whether a journey between two rest points would need a break the following equation was used given the assumption that a vehicle would be travelling at an average kilometres an hour. hour rest period ¼ distance between cities duration of travel before hours rest period à km=h combining livestock trade patterns with phylogenetics to help understand the spread of foot and mouth disease in sub-saharan africa, the middle east and southeast asia bovine spongiform encephalopathy identified in a cow imported to canada from the united kingdom-a case report european union council regulation (ec) / . the protection of animals during transport and related operations and amending directives / / eec and / /ec and regulation (ec) no / l european union council regulation (ec) / . commision decision of august concerning the development of an integrated computerised veterinary system known as traces proposed terms and concepts for describing and evaluating animal-health surveillance systems towards an integrated approach in surveillance of vector-borne diseases in europe the economic impact of bse on the uk beef industry the foot-and-mouth disease epidemic in the netherlands in the effects of transport and lairage on counts of escherichia coli o in the feces and on the hides of individual cattle international disease monitoring economic principles for resource allocation decisions at national level to mitigate the effects of disease in farm animal populations european union council regulation (ec) / . the laying down specific rules for the organisation of official controls on products of animal origin intended for human consumption council regulation (ec) / . laying down the general principles and requirements of food law, establishing the european food safety authority and laying down procedures in matters of food safety small-and large-scale network structure of live fish movements in scotland network analysis of cattle and pig movements in sweden: measures relevant for disease control and risk based surveillance application of network analysis parameters in risk-based surveillance -examples based on cattle trade data and bovine infections in sweden collective dynamics of "small-world" networks disease evolution on networks: the role of contact structure quantifying microbe transmission networks for wild and domestic ungulates in kenya chapter production and consumption of poultry meat and eggs in the european union pig farming in the eu, a changing sector mapping of surveillance and livestock systems, infrastructure, trade flows and decision-making processes to explore the potential of surveillance at a systems level review of the emerging animal health and food security issues council regulation (ec) / approving annumal and multiannual programmes and the financial contribution from the union for the eradication, control and monitoring of certain animal diseases and zoonoses presented by the member states for and the following years complexity of the international agro-food trade network and its impact on food safety vulnerability of animal trade networks to the spread of infectious diseases: a methodological approach applied to evaluation and emergency control strategies in cattle relationship of trade patterns of the danish swine industry animal movements network to potential disease spread descriptive and social network analysis of pig transport data recorded by quality assured pig farms in the uk european union council regulation (ec) / . laying down of specific hygiene rules on the hygiene of foodstuffs council regulation (ec) / . approving annual and multiannual programmes and the financial contribution from the union for the eradication, control and monitoring of certain animal diseases and zoonoses presented by the member states for and the following years shipping stress and social status effects on pig performance, plasma cortisol, natural killer cell activity, and leukocyte numbers a comparison of the welfare and meat quality of veal calves slaughtered on the farm with those subjected to transportation and lairage effects of weaning and h transport by road and ferry on some blood indicators of welfare in lambs effects of transport, lairage and stunning on the concentrations of some blood constituents in horses destined for slaughter isolation of respiratory bovine coronavirus, other cytocidal viruses, and pasteurella spp of shipping fever an evaluation of disinfectants for the sanitation of porcine reproductive and respiratory syndrome virus-contaminated transport vehicles at cold temperatures an introduction to exponential random graph (p*) models for social networks council regulation (ec) / : implementing regulation (ec) no / of the european parliament and of the council as regards eartags, passports and holding registers report from the commission to the council on the implementation of electronic identification in sheep and goats council regulation (ec) / : the identification and registration of pigs eurostat glossary: livestock unit (lsu) -statistics explained r core team: r. a language environment for statistical programming package network package sna desktop: release . environmental systems research institute maptools: tools for reading and handling spatial objects pbsmapping: mapping fisheries data and spatial analysis tools submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution acknowledgements bh acknowledges financial support from the leverhulme centre for integrative research on agriculture and health (lcirah). additional file : journeys that would require rest breaks due to being over hours long or over hours long. these data are displayed in tables.additional file : journeys that would require rest breaks for unweaned animals. the data are displayed in a table.additional file : journeys that would require rest breaks for poultry other than chicks < hours old. the data are displayed in a table.additional file : the proportions of national animal imports and exports compared with the national population. these data are displayed in separate tables for each species. the authors declare that they have no competing interests.authors' contributions jh obtained the data and undertook the analyses. jh, bh and jr interpreted the results and had an equal contribution to the manuscript. all authors have read and approved the final manuscript. key: cord- - usqpy j authors: hassan, atef a.; mansour, mogda k.; el hamaky, ahmed m.; sayed el ahl, rasha m.; oraby, noha h. title: nanomaterials and nanocomposite applications in veterinary medicine date: - - journal: multifunctional hybrid nanomaterials for sustainable agri-food and ecosystems doi: . /b - - - - . - sha: doc_id: cord_uid: usqpy j nowadays, nanotechnology has made huge, significant advancements in biotechnology and biomedicine related to human and animal science, including increasing health safety, production, and the elevation of national income. there are various fields of nanomaterial applications in veterinary medicine such as efficient diagnostic and therapeutic tools, drug delivery, animal nutrition, breeding and reproduction, and valuable additives. additional benefits include the detection of pathogens, protein, biological molecules, antimicrobial agents, feeding additives, nutrient delivery, and reproductive aids. there are many nanomaterials and nanocomposites that can be used in nanomedicine such as metal nanoparticles, liposomes, carbon nanotubes, and quantum dots. in the near future, nanotechnology research will have the ability to produce novel tools for improving animal health and production. therefore, this chapter was undertaken to spotlight novel methods created by nanotechnology for application in the improvement of animal health and production. in addition, the toxicity of nanomaterials is fully discussed to avoid the suspected health hazards of toxicity for animal health safety. significant economic importance for the security of farmers (thornton, ) whereas the products of these animals such as milk, meat, hides, and leather are essential for human populations (patel et al., ) . nowadays, microbial infections cause several health hazards to humans and animals. despite progressive advances in diagnosis and therapeutic technologies, microbial spoilage still has a major economic impact on the world's food supplies and is considered the main source of outbreaks of human and animal diseases (taylor and rodwell, ) . in the ongoing race between the emergence of drug resistance and the development of novel antimicrobial agents, microbes appear to be the targets of several studies (hassan et al., a) . in addition, nanotechnology helps in the development of new diagnostic tools and treatments that improve the quality of life of veterinary animals. some studies employed nanomaterials in disease treatment such as african animal trypanosomiasis (kroubi et al., ) as well as foot and mouth disease in cattle (greenwood et al., ) and sheep (mohanty et al., ) . recently, several studies evaluated the use of nanobiosensors in the detection of estrus, hormone levels, and metabolite profiles (monerris et al., ; sagadevan and periasamy, ) . also, nanomaterial has the ability to preserve gonadal tissues, sperm, oocytes, and embryos (saragusty and arav, ) . meanwhile, quantum dots (qds) improve the understanding of mammalian spermatozoon and oocyte movements as well as their interactions in a different physiological setting of fertility, as detected by hill and li ( ) . furthermore, the nanocomposites can make products and applications for obtaining efficient results in treatments and diagnosis (gordon and sagman, ) . also, the composites of drug molecules with nanomaterials elevated the efficacy of drug solubility and passed through the blood supply to the targeted affected tissues . moreover, several nanocomposites can be used in various applications such as nanoshells to destroy cancer cells, alumino-silicate nanoparticles to reduce bleeding, carbon nanoparticles as sensors and for drug delivery, gold nanoparticles (au nps) for diagnosis, silver nanoparticles (ag nps) as antimicrobial agents, and iron oxide nanoparticles to improve mri imaging (chakravarthi and balaji, ; hassan et al., b) . additionally, other tools such as microfluidics, nanomaterial, and bioanalytical nanosensors have the potential to solve many challenges in the diagnosis and treatment of diseases (meena et al., ) . therefore, this chapter was undertaken to spotlight the recent advances in nanotechnology and their application in the field of veterinary medicine. also, this review includes a brief discussion of the use of nanomaterials and nanocomposites in human and animal science. moreover, practical applications and ways to overcome the suspected toxicity of nanomaterials in veterinary medicine are fully discussed. there have been rapid advances in the fields of nanomaterials and nanocomposite applications in biomedical science related to human and animal health. a brief illustration of the beneficial fields of nanomaterials and nanocomposite application in animal science is shown in fig. . . the metal nanomaterials constitute effective antimicrobial agents against common pathogenic microorganisms (gong et al., ; hassan et al., a, b; nabawy, ) . in addition, metal nanoparticles have greater useful advantages than largesized metal particles (stankic et al., ) . recently, abd-elsalam et al. ( ) reported that metal nanoparticles such as ag, silver oxide (ag o), titanium dioxide (tio ), silicon (si), copper oxide (cuo), zinc oxide (zno), au, calcium oxide (cao), and magnesium oxide (mgo) have potential antifungal activity, whereas most metals provide novel antimicrobial potential (taylor et al., ) . another study reported that the minimum inhibitory concentration (mic) of zinc nanoparticles (zn nps) was ( . - μg/ml) (hosseini et al., ) . however, hassan et al. ( a) showed the antimicrobial potential of zn nps against the dermatological infection of buffaloes through species such as trichophyton verucosum, dermatophilus congolensis, and staphyloccocus aureus. similarly, the authors detected the antimicrobial potential of zn nps against esherichia coli, s. aureas, candida albicans, and aspergillus flavus, which caused mastitis in cattle. regarding ag nps applications, they have several potential uses such as their antimicrobial, antiinflammatory, and anticancer properties (ge et al., ; hassan et al., a hassan et al., , . currently, ag nps showed antimicrobial effects against fungal causes of skin diseases in bovines (hassan et al., b) ; fungal and bacterial diarrhea and mastitis infection in buffaloes (hassan et al., a) ; and mastitis in goats (gurunathan et al., ; yuan et al., ) . however, refai et al. ( ) detected the antimicrobial effect of ag nps against s. aureus and c. albicans. meanwhile, the ag nps significantly reduced the viability of coliform bacteria in ilea contents of piglets (fondevilaa et al., ) . but in another study, no effects of enteric bacteria were observed in piglets administered ag nps (sawosz et al., ) . furthermore, hassan et al. ( b) detected the antifungal potential of the ag nps against c. albicans and trichophyton mentagrophytes. ag nps also showed the ability to inhibit the growth of fungal cells, particularly c. albicans and dermatophytes (lara et al., ) . kim et al. ( ) showed that the mic of ag nps against pathogenic candida spp. was mg/ml of ag nps had higher potential than crud silver. the antifungal potential of nanosilver against t. mentagrophytes and the candida species was detected by kim et al. ( ) . moreover, the application of nanosilver in the biostabilization of footwear materials ( % solution) inhibited the growth of some mold (falkiewicz-dulik and macura, ) . nanoparticles of iron oxide (fe o nps) exhibited strong antimicrobial activity sawai, ) . currently, fe o nps are known to have antifungal potential against the growth of mycotoxigenic a. flavus while also altering their ability to produce aflatoxin (ahmad et al., ; lopes et al., ; nabawy et al., ) . meanwhile, hassan et al. ( c) detected the antifungal effect of fe o nps against c. neoformance that was recovered from respiratory diseases in cattle. similarly, nabawy et al. ( ) and mouhamed et al. ( ) detected the antifungal potential of fe o nps against the mycotoxigenic aspergillus species that was isolated from feeds. in another study, hassan et al. ( b) yielded the efficient antimicrobial potential of fe o nps against trichophyton verrucosum, t. mentagrophytes, and the bacteria of dermatophilus sp., which is isolated from bovine skin infections. abd el-tawab et al. ( ) discovered that fe o nps have an more of an antifungal effect than fe o nps. nabawy et al. ( ) and mouhamed et al. ( ) detected that aflatoxin b (afb ) and ochratoxin production by respective fungal isolates was significantly diminished until complete inhibition by increasing the dose treatment with fe o nps. moreover, the antimicrobial action of metal nanoparticles was suggested as being due to disrupting and penetration of the cell membrane of microorganism, damage and rupture of the cell wall and leakage of cytoplasm contents (gajbhiye et al., ; hassan et al., hassan et al., , a . in another study, khandelwal et al. ( ) determined that ag nps were able to prevent the penetration of a ruminant virus into animal cells by the destructive action of nanomaterials on viral cells. in another study, the antibacterial effects of zn nps against gram-positive and gram-negative bacteria occurred due to the penetration of nanoparticles into the cell membrane of bacteria and led to cell death (arabi et al., ; auffan et al., ; rosi and mirkin, ) . furthermore, the antibacterial activity of zno-nps due to their interaction with bacterial cells caused microbial cell injury and could enter the cells ( jin et al., ; stoimenov et al., ; zhang et al., ) . currently, we evaluate the synthesis and characterization of some metal nanoparticles such as zn nps, fe o nps, ag nps, and selenium nps and their antimicrobial potential against the viability of microbial causes of cow mastitis, abortion, and diarrhea ( fig. . a-c). the viability and growth of bacterial cells (e. coli and s. aureas) and fungal spores (a. flavus and c. albicans) significantly decreased as the used concentration on metal nanoparticles increased. when these isolates were observed by scanning electron microscopy (sem), they showed complete destruction and death of the treated microbial cells (fig. . d) . recently, bai et al. ( ) detected that au nps decreased the cell viability of pathogenic bacteria in chicken. meanwhile, the antibacterial potential of au nps against the bacillus species and e. coli was detected by zhou et al. ( ) . recently, mohamed et al. ( ) detected the antibacterial activity of au nps against corynebacterium pseudotuberculosis, which caused chronic caseous lymphadenitis and resulted in a major economic loss in goats and sheep. on the other hand, regarding ( ) after treatment; c. albicans ( ) before and ( ) after treatment; e. coli o :h ( ) before and ( ) after treatment, and of s. aureus ( ) before and ( ) after treatment with metal nanoparticles. the single-walled carbon nanotubes (swcnts), it was detected that as the length decreased, the antibacterial activity increased . meanwhile, suspensions such as phosphate-buffered saline decreased swcnt toxicity and their interactions with bacterial cells, hence reducing the aggregation of cells ). however, graphene oxides (gos) exhibited stronger antimicrobial potential against food-borne bacteria . boor ( ) detected the antimicrobial potential of cnts against the activity of bacteria and fungi. in another study, the antimicrobial potential of swcnt was investigated against bacteria, which caused the failure of bacterial film formation and caused cell death (kang et al., ) . swcnts are also toxic to bacterial cells that have the ability to interrupt bacterial membranes . carbon nanotubes (cnts) showed selective antibacterial agents (fernandez-lopez et al., ) and different forms of graphene materials against listeria monocytogenes and salmonella species (ye et al., ) . however, swcnts have potential antiviral activity against reovirus (gurunathan et al., ) . similarly, ye et al. ( ) detected the antiviral activity of go against the pseudorabies virus dna and the porcine epidemic diarrhea virus. furthermore, other cnts (fullerenes) and their derivatives showed antiviral activity against hiv (friedman et al., ; sijbesma et al., ) and the vesicular stomatitis virus (kaesermann and kempf, ) . the antifungal potential of chitosan and cs nps against fusarium graminearum colonies was evaluated, may be used as a novel nano-biopesticides (kheiri et al., ) and a growth inhibitor for rhizopus sp. and aspergillus niger (chookhongkha et al., ) . cs nps also have antimicrobial activity against staphylococcus saprophyticus and e. coli (soutter, ) . in addition, nanosensors have antibacterial and antiviral potential, even if used in a very small amount, which improves the feedstock (kuswandi et al., ; vyas et al., ) . furthermore, nanoscience produces a novel nontoxic antimicrobial agent against various pathogens, including brucella, mycobacterium bovis, s. aureus, and rhodococcus equi (muktar et al., ) as well as fungi and bacteria that cause mastitis and aflatoxins in feeds (hassan et al., a in cattle. over the past decade, various mycotoxins were detected as the main contaminant for foods and feed products, infested with mycotoxigenic fungi which have the potential ability to produce one or more types of mycotoxins. mycotoxins are carcinogenic and make other adverse effects on human and animal health. hence, the degradation of mycotoxins can improve animal health and production. recently, it was reported that zno nps have antifungal potential against the growth of mycotoxigenic a. flavus while altering its ability for aflatoxin production (ahmad et al., ; hassan et al., a; lopes et al., ; nabawy, ) . meanwhile, hassan et al. ( a) demonstrated the ability of zinc nps to inhibit the growth of mycotoxigenic molds and respective mycotoxin production (aflatoxins, ochratoxins, fumonisin b ) at the concentrations of , , and μg/ml of zno nps, respectively. aflatoxigenic a. flavus strains that require higher concentration of metals nanoparticles ( μg/ ml) than nonaflatoxigenic strains ( μg/ml) to inhibit their growth in animal feeds and aflatoxins production (nabawy et al., ) . similarly, mouhamed et al. ( ) reported that zn nps inhibit the growth of ochratoxigenic mold in feeds. moreover, the inhibition of microbial growth by metal nanoparticles varies according to their particle size and dose levels of treatment (shawky et al., ; violeta et al., ) . the first step in the degradation of mycotoxins by metal nanomaterials, hassan et al. ( a hassan et al. ( , b, successfully eliminated the carcinogenic effects of aflatoxins on vital organs such as the liver and kidney of rabbits by the addition of zn nps in the feed. similarly, abd el-fatah et al. ( ) detected the ability of zn nps to eliminate the aflatoxicosis carcinogenic effects in rats. however, zno nps can cause toxicity to vital internal organs in sheep (allen et al., ) . similarly, the oral administration of zn nps in mice resulted in toxicity to their vital organs (wang et al., a) . nowadays, researchers detected that the treatment of trichothecenes-producing fusarium poae by zn nps and ag nps resulted in the inhibition of their growth viability as well as trichothecenes mycotoxin production while decreasing the density and quantity of biosynthetic genes as detected by rt-pcr. we also reported that the nanoemulsion of cinnamon and olive oils caused the complete inhibition of mycotoxigenic fusarium growth and mycotoxin production at concentrations of %- %. zia-jahromi ( , ) reported the ability of ag nps to degrade aflatoxin in broiler chickens feed, ag nps are significantly elevated body weight gain and feed consumption. the significant application of magnetic nanoparticles (mnps) in toxin degradation through dehydroxylation of the toxin (mishima et al., ) . moreover, gibson et al. ( ) modified nanodiamond substrates in carboxylation, hydrogenation, and hydroxylation for immobilization of the carcinogenic effects of mycotoxins, particularly afb and ochratoxin a (ota). currently, the antimicrobial potential of cnts against aflatoxigenic a. flavus and the prevention of aflatoxin production in feeds were investigated (hassan et al., ) . the obtained results showed that the viability of microbial cells was inhibited and complete prevention of aflatoxin production occurred at a concentration level of μg/ml of cnts. veterinary applications of metal nanoparticles have been used in various fields related to animal disease diagnosis and treatment as well as biological sensing. several metal nanomaterials such as zn nps have been significantly used in diagnostic and therapeutic activities in animal lymphomas, cutaneous cancers, transmissible venereal tumors, and equine sarcomas (borzacchiello and corteggio, ; carr et al., ; raguvaran et al., ; scott and miller, ) . recently, in dairy cattle, gurunathan et al. ( ) showed the antibacterial potency of ag nps against causes of endometritis (prevotella melaninogenica and arcanobacterum pyogenes). moreover, asgary et al. ( ) used ag nps as adjuvants of the rabies virus in mice and dogs and observed that no toxicity occurred. several studies applied au nps in the diagnosis and detection of some viral infections in chickens (nurulfiza et al., ) ; the foot and mouth disease virus (ding et al., ) ; and the bluetongue virus in cows (yin et al., ) and pigs (wang et al., ) . au nps were used in the detection and diagnosis of some bacterial diseases in chickens (moongkarndi et al., ) , mycoplasma suis in pigs (meng et al., ) , and bacterial toxins (zhu et al., ) . also, au nps were applied in serological diagnosis of cystic echinococcosis ( jahani et al., ) and e. coli o :h in feed and water in mastitis of cow (hassan et al., c) and canine parasitic infestation ( jiang et al., ) . other studies have detected that carbon-based nanomaterials (cbns) such as cnts, graphene (g), carbon fibers, and carbon nanoparticles have huge potential in biomedicine, nanoelectronics, and mechanical engineering (de-volder et al., ; le croy et al., ) . they can be used in disease diagnosis, gene therapy, and physiological treatments wang et al., ) . nowadays, the varieties of nanomaterials and nanosensors have a significant value in all aspects of animal science such as the diagnosis of tuberculosis in bovines and the resulting therapy (kuswandi et al., ; sekhon, ) . it is interesting to report here that zn nps can kill cancer cells without affecting on normal immune cells and hence can be used as anticancer agents and for cancer diagnostic devices in humans and veterinary animals ). on the other hand, a carbon nanoparticle suspension injection (cnsi) can act as a drug carrier, where it has the ability to adsorb drugs such as epirubicin and doxorubicin drugs (xie et al., ) , which are used in lymphatic chemotherapy (yang et al., ) . currently, cnsi has been applied for clinical treatments as part of an injection during oncological surgery (xie et al., a) . cnsi could be applied for lymphatic mapping as well as in parathyroid gland and lymph node tumor imaging, such as in gastric cancer zhu et al., ) and breast cancer (wu et al., ) . gu et al. ( ) and zhu et al. ( ) successfully investigated relevancy of cnsi in lymph node dissection in the diagnosis of thyroid carcinoma. furthermore, the cnsi might include in photothermal therapy, gene delivery, and serve as an immune adjuvant (liu et al., ; xie et al., a, b) . meanwhile, the qds polyethylene glycol (peg)-functionalized carbon nanoparticles were nontoxic to mice after intravenous exposure . moreover, cbns can be utilized in the imaging of organs such as lymph nodes as well as physiological treatments wang et al., ) . wang et al. ( b) evaluated silica nanoparticles (snps) in the imaging of tumor cells, where the primary or secondary antibodies immobilized onto the snps and attached them to cancer cells. similarly, he et al. ( ) detected that the in vitro adoption of snps in methylene blue dyes initiated fluorescent imaging of hela cells and can be injected directly into the tumors in mouse causing its necrosis after laser radiation treatment. snps can also be used for the photodynamic therapy of cancer by producing singlet oxygen through fluorescence energy (kim et al., ) and mnp employed in medical imaging (mishima et al., ) . recently in sheep, fe o nps were used for the diagnosis of tendon disease by fluorescent imaging (scharf et al., ) . the advance tools in nanotechnology developed new delivery systems and methods at the nanoscale level to produce chemical and biological reactions related to targeted sites and cells (tomanek and enbody, ) . in the early stage, the use of nanotechnology in the treatment of salmonella typhi infection in mice conjugated with ampicillin resulted in a decrease in the amount of ampicillin used. therefore, their residues in animal tissues were reduced, which reflected the safety of produced food (fattal et al., ) . significant applications of mnps include pathogen detection, protein purification, and drug delivery (mishima et al., ) . narducci ( ) injected the ag np-loaded rabies vaccine into dogs, checked them daily for days, the obtained data noted that no abnormal signs. recently, cs nps have been widely studied for the delivery of antibiotic drugs (cover et al., ; zaki and hafez, ) and anticancer drugs such as -fluorouracil, paclitaxel doxorubicin, letrozole, and saponin (rejinold et al., ; saboktakin et al., ) . several reports examined the application of cbns in animal disease diagnosis and drug delivery wang et al., ) , and carbon-based drug delivery systems for cancer treatment (reina et al., ) . furthermore, the use of nanotechnology in drug delivery reduces the amount of antibiotics administered for the treatment of diseased animals and hence reduces their residue in animal tissues. the novel tools of diagnosis and treatment of animal diseases by recent nanotechnology have potentiated progressive advances in animal production and reproduction. the feed supplements at the nanoscale of nanocopper and nanochromium are more bioavailable to animals and poultry, allowing more interaction to occur in the gut and better absorbance (gonzales-eguia et al., ; huang et al., ; sirirat et al., ; wang and xu, ; zha et al., ) . recently, several studies detected that the supplementation of znonps into poultry feeds to improve their growth performance, immune status, reproduction activity, and preventing microbial infections (partha et al., ) and piglets and poultry (lina et al., ; yang and sun, ) . however, the bulk of the zinc was added as a feed additive to pigs to treat diarrhea caused by enterotoxigenic e. coli (broom et al., ; case and carlson, ) . the dietary supplementation of zinc-methionine in feeds of mastitis cows caused an elevation in milk production (salama et al., ) . meantime, the addition of zn nps in cows that suffered from subclinical mastitis caused a reduction in the level of the somatic cell and improved milk production (rajendran et al., ) . however, the mastitis disease caused huge economic losses by reducing the milk yield. the most recovered organisms are s. aureus, e. coli, c. albicans, a. flavus and a. niger, and they can be successfully treated by zno nps (bajpa et al., ; hassan et al., ; jalal et al., ) . currently, bai et al. ( ) screened the antibacterial potential of zn nps against staphylococcus epidermidis, streptococcus agalactiae, klebsiella pneumoniae, and e. coli recovered from mastitis. on the other hand, in poultry, the addition of zno-nps to feeds of broiler chicks significantly improved the growth, production, and dress performance (lina et al., ; mishra et al., ) . within another function, it has the ability to potentiate economic importance in piglets via increasing their growth performance and feeding utility (yang and sun, ) . ag nps in broiler chickens caused antimicrobial and growth-promoting effects, but the level of plasma immunoglobulin (igg) was decreased (pineda et al., ) . furthermore, nanographene is monitored in the measurement of blood parameters and the milk of large and small ruminant animals mccomb et al., ; rahimi, ) . in addition, the oral administration of milk containing casein nanoparticles conjugated with vitamin d to humans or animals reached the stomach in which nanocasein hydrolyzed separated from vitamin d, resulting in the elevation of their bioavailability (haham et al., ) . several reports evaluated employed various nanoparticles to improve meat and egg quality. wang and xu ( ) added chromium nanoparticles ( μg/kg) in pig feed, which caused an increase in muscle mass and improved pork quality. similar results were detected in birds when chromium nanoparticles added to feed ( μg/kg) caused the decreased cholesterol content of muscles and raised the feed efficiency (zha et al., ) . huang et al. ( ) gave a nanocalcium compound to mice that resulted in its bioavailability and increased the density of bone minerals, which is higher than the use of calcium compounds on a large scale. the administration of cs nps loaded with chromium pigs feed resulted in an improvement in pork quality and the low-fat content of meat by decreasing the synthesis of fatty acids . regarding animal reproduction, nanotechnology develops nanobiosensors for detection of the reproductive and fertility status of animals for use in fertility control applications, the cryopreservation of gametes and embryos, and the detection of fertility hormones (saragusty and arav, ) . they added that nanobiosensor devices can help in the detection of diseases, pathogens, estrus, hormone levels, and metabolites as tools for reproductive management. similarly, the level of estrogen hormones in the blood of animals could be measured by implantation of nanotubes under the skin and hence the detection of estrus. this is due to the nanotubes binding with estradiol antibodies by fluorescence-producing signals that help in the control system of breeding (o'connell et al., ) . the state of animal breeding such as fertilization and the viability of sperm and eggs can be measured by nanofluid. in addition, metal nanoparticles are used for the cryopreservation of gonadal tissues, sperm, oocytes, and embryos to cause ultrafast cooling rates and homogeneous rewarming of the biological materials (tomanek and enbody, ). applications of nanomaterials are currently used for the meat and food industry, including the use of nanomaterials as carriers of food ingredients/additives that are placed directly into food or as a part of food packaging. in addition, this can improve the dispersing ability of fat-soluble additives in food products, enhance taste, and reduce the use of fat, salt, sugar and preservatives, preventing hypertension and cardiovascular disease in humans and animals. nowadays, several applications of metal nanomaterials in veterinary medicine used zn nps as a food preservative and feed additive. however, they are toxic to microorganisms and used as antimicrobial agents (hosseini et al., ) and their addition in cow feeds resulted in a significant increase in milk production (rajendran et al., ) . in addition, they can be used in catalysis, sensors, environmental remediation, and personal care products of humans and animals (raguvaran et al., ) . the nanocarriers enable nutritive substances to be resistant to protease enzymes and other desaturating compounds. also, it may be increasing its ability to transfer across the intestinal membrane into blood. in addition, nanocarriers controlled release and better dispersion of nutrients in aqueous systems to water-insoluble food ingredients (lee, ) . nanomaterials can produce novel tools for the detection of food-borne pathogens such as nanomaterials loaded with anti-s. aureus antibodies; gold and iron oxide nanoparticles yielded the rapid detection of s. aureus in milk (sung et al., ) . similarly, nanotechnology could be applied in the science of biomedicine, food systems, food system security, and disease treatment delivery (scott, ) . nowadays, the varieties of nanomaterials and nanosensors are of significant value in all aspects of animal science, and are used as additives to animal products and food safety (muktar et al., ) . in the near future, nanotechnology will potentiate in the production of "interactive" poultry meat that changes color, flavor, or nutrients (meena et al., ) . until now, the conjugated and functionalized nanoparticles have resulted in the formation of hybrid nanomaterials (nanocomposites) that have significant benefits to animal health and production. they can be used in drug delivery, mri imaging, the delivery of therapeutic agents such as anticancer drugs, and disease diagnosis, particularly tumor imaging. nowadays, several studies have employed nanomaterials to avoid the toxic doses of metal nanoparticles for animal health. hassan et al. ( a hassan et al. ( , reported that synergistic and combination therapy has the ability to overcome microbial resistance to traditional antibiotics, resulting in more efficient antimicrobial and antitoxin activity of metal nanoparticles for the treatment of human and animal diseases. however, mody et al. ( ) detected the availability of metal nanomaterial conjugation with drugs and other biomedical components of health importance. hassan et al. ( a) detected that the combination between ag nps and traditional antibiotics resulted in the requirement of lower concentrations from both to obtain the strong antimicrobial effects against c. albicans, a. flavus, salmonella, and s. aureas. in addition, the combined and synergistic therapy of low dose levels of antibiotics and ag nps enhanced their antibacterial and antifungal effects (gurunathan et al., (gurunathan et al., , . the conjugation of nanosilver together with fluconazole (antifungal) and florfenico (antibacterial) have more potential to inhibit the growth of microbial causes of disease in buffaloes rather than their single forms (hassan et al., a) . this combination resulted in a decrease in ag nps dosage to avoid toxicity in the animal. similarly, smekalova et al. ( ) screened the antibacterial activity in the combined therapy of ag nps with penicillin g against actinobacillus pleuropneumoniae. these activities are attributed to the potential of ag nps in killing and their destructive effects on microbial cells . in addition, the functionalized swcnts had an antiviral effect against reovirus (gurunathan et al., ) , hiv (friedman et al., ) , and vesicular stomatitis virus (kaesermann and kempf, ) . therefore, it is suggested that swcnts and multiwalled carbon nanotubes (mwcnts) could be functionalized and combined with bioactive molecules to be a benefit in diverse biological applications (endo et al., ) . the carbohydrate-functionalized cnts have antibacterial and antifungal potential against e. coli, c. albicans, a. flavus (elkin et al., ) , and bacillus anthracis (wang et al., c) . meanwhile, the functionalized cnts can influence the viability of cells by injection cnts with complex cells (kam et al., ) . also, cnts were employed as cancer biomarkers (thakare et al., ) and viruses therapy agent (patolsky et al., ) . cnts have the ability to detect particular dna sequences in cells (tu et al., ) . zhu et al. ( ) and determined the significant antibacterial effects of various carbon nanoparticles and nanocomposites against bacteria causing mastitis in cows. the well-functionalized cnts have more beneficial effects on biological cells than the nonfunctionalized cnts (khalid et al., ) . hence, the benefit activity of cnts is associated with the property of conjugation with other biological components (dumortier et al., ) . therefore, there is significant importance in functionalized swnts for clinical applications in human and animal health (fig. . ) . several studies illustrated that the antimicrobial potential of liposomal formulations against several pathogenic bacterial species (swenson et al., ) . the liposome-encapsulated gentamicin has antibacterial potential against causes of mastitis in bovines such as s. aureus (macleod and prescott, ) . however, liposomeencapsulating tobramycin has broad-spectrum antibacterial effects against different kinds of bacteria (sachetelli et al., ) . in a study by singla et al. ( ) , liposomeencapsulating phage showed strong antibacterial potential against multidrugresistant bacterial infections. moreover, the liposomal amb is an effective and less toxic antifungal than conventional agents in the treatment of mycotic respiratory infection in animals (lambros et al., ; leenders and de marie, ) as well as yeast infections in mice (khan et al., ) and dogs (krawiec et al., ) . in a recent study, al-qushawi et al. ( ) investigated the antimicrobial potentials of tilmicosin in poultry in its binding with the micelle's nanomaterials. similarly, the antiviral activity of liposomal-encapsulated ribavirin was used in animals, resulting in increasing the drug safety efficacy and preventing replication of the virus (kende et al., ; makabi-panzu et al., ) . also, it can be used as a carrier against different types of microorganisms, such as the influenza virus (boraschi and italiani, ) , hav, hiv (qiao et al., ) , and infectious plasmodium vivax (powles et al., ) . moreover, the conjugation of the hem-agglutinin-derived synthetic peptide with liposome induces antiviral protection against the lethal influenza virus (rhee et al., ) . recently, the antimicrobial potential of polymeric nanoparticles composed of chitosan was evaluated against various causes of diseases such as e. coli o : h (doavi et al., ) , pseudomonas aeruginosa (cui et al., ) , the influenza virus (oberoi et al., ) , hbv (lebre et al., ) , filariasis (malathi et al., ) , and dengue (hunsawong et al., ) . furthermore, the attached hybrid dendrimer-based nanomaterials with the biological molecules of the cell wall as proteins initiated the rapid destruction of bacterial cells (xiao et al., ) . the antimicrobial potential of dendrimer-based nanocomposites caused inhibition of the growth of several pathogenic bacterial species via their ability to penetrate microbial cell membranes and cytoplasm contents with the final death of cells (bai et al., ) . recently, meena et al. ( ) reported that lipophilic substances and protection from degradation are the significant advantages of nanoemulsions. essential oils obtained from mentha piperita and encapsulated in chitosan nanogels with cinnamon acid showed antifungal potential against a. flavus (beyki et al., ) . hassan et al. ( a, b) used forskolin emulsions as antifungals against mycotoxigenic fungi (hassan et al., a, b) . hassan and mansour ( ) and hassan et al. ( ) determined that nanoemulsions of rhamnus cathartica oil, molasses, and garlic extracts successfully eliminated the fungal contamination in tested feeds. moreover, the nanoemulsions of natural oils are characterized by their physical activity and avoid chemical action. in addition, the contact of oil nanodrops with the membranes of bacteria, fungi, or enveloped viruses caused the leakage of cell contents and the death of organisms (meena et al., ) . the administration of conjugated penicillin or methicillin (β-lactam drug) to polymeric polyacrylate nanoparticles may increase the antibacterial activities of drugs. this due to the nanoparticles improves the destroying action of β-lactamase in the animal body and used against penicillin and methicillin-resistant s. aureas (turos et al., ) . similarly, ghosh et al. ( ) evaluated the antibacterial potential of tetracycline loaded into polymeric cs nps against e. coli strains and the mic was μg/ml. the authors of this chapter evaluated the antifungal and antimycotoxin potentials of conjugated ag nps with olive oil against the mycotoxin-producing fusarium sp. they caused the inhibition of fungus growth and the prevention of trichothecene and zearalenone production. in addition to antiflatoxins and antishiglla toxins, the antimicrobial potentials for nanoemulsions of cinnamon oil and zn nps were evaluated against isolated a. flavus and e. coli from feeds and diarrheic buffaloes. the results showed the complete inhibition of microbial growth and that toxin production requires lower levels of the combined form ( μg/ml of zn nps and % of cinnamon oil) than the single use of zn nps ( μg/ml) and cinnamon oil ( %) in treatment. in other studies, the role of mnps in toxin decorporation was reported by leroux ( ) while the reduction and removal of toxins from contaminated materials was detected by wang et al. ( ) . similarly, mycotoxins such as aflatoxins and ota were completely detoxified by the addition of fe o nps to mycotoxicosis poultry feeds (mouhamed et al., ; nabawy et al., ) . it is interesting to report that the synergistic and combination therapy of the lower doses of zn nps and ozone fumigation were able to cause the complete detoxification of afb in poultry feed at lower nontoxic doses ( ppm of ozone + μg of zn nps/kg of yellow corn) . the authors have evaluated olive oil and cinnamon oil emulsions against the mycotoxigenic fusarium species and the production of trichothecene and zearalenone mycotoxins. these emulsions have antifungal and antimycotoxin potentials at concentrations of %- %. in recent years, there have been progressive advances in the diagnosis and therapy of animal diseases, particularly using nanocomposites to improve diagnostic tools. the main types of mnps are composed of iron oxide and have several applications in biomedicine through coating mnps by biofunctional molecules to increase their sensitivity for many biological applications. furthermore, biocompatibility, a large surface-to-volume ratio, and chemically reactive biomolecules provide a lot of chemically active sites for biomolecule conjugation, responsible for their functionalization and the encapsulation of other biological materials (gupta and gupta, ; shokrollahi, ; tartaj et al., ) . the mnp structures and coating schemes with many other beneficial materials are of significant health importance to humans and animals; they are illustrated in fig. . . in this direction, gu et al. ( ) used vancomycin-conjugated fept nanoparticles to detect vancomycin-resistant enterococci and other gram-positive bacteria at low concentrations by increasing the ability of vancomycin to penetrate the microbial cells after conjugation. moreover, kaittanis et al. ( ) detected the high potential of immunomagnetic nanosensors (superparamagnetic iron oxide nps) in the detection of mycobacterium avium paratuberculosis. in another study, e. coli o :h could be detected by dextran-coated iron oxide nanosensors or silica-coated iron oxide nanosensors (kaittanis et al., ) . furthermore, mnps will be useful for various biological applications in veterinary medicine and produce useful tools for disease diagnosis, mri imaging, drug delivery, and cancer therapy (michalet et al., ) . the recent exposure of murine models to fe o nps resulted in a significant decrease in inflammatory reactions such as footpad swelling and increased phagocytosis activity in the spleen (shen et al., ) . in addition, mnps offer a simple and versatile platform for separating six histidines ( xhis)-tagged protein and increasing the protein binding capacity (xu et al., a, b) . the production of heterodimers of two distinct nanospheres is a way to fabricate fe o -au heterodimers in a homogeneous organic solvent (yu et al., ) . in recent years, au nps have been characterized by their biocompatibility with human cells and the ability to conjugate with dna, rna, antibodies, and peptides (shah et al., ) , which has resulted in the initiation of novel nanodiagnostic tools (syed and bokhari, ) . some studies used au nps as biosensors in the synthesis of the immune-chromatographic strip for efficient diagnostic assays (halfpenny and wright, ; syed and bokhari, ) to detect antibodies against microbial antigens (cui et al., ) and bacterial and viral diseases (peng et al., ) . furthermore, the functionalized ag nps with some oils were used for treatment of dermatophytes diseases in animals (bansod et al., ) . the authors have determined that the conjugation of ag nps with olive oil resulted in significant and obvious antimicrobial effects against bacterial and fungal causes of mastitis in cows at concentrations of - μg/ml. however, parvongnukul and lumb ( ) detected the useful application of functionalized graphite in the treatment of bone disorders in ovine. in addition, functionalized nanographene has several advantages that help in clinical applications in the therapy of animal diseases (novoselov, ; sanchez et al., ) and can also be used in various biomedical applications (sun et al., a, b) . in another study, the polymers of nanomaterials can be prepared by adhesion of the natural biological compound as carbohydrates and protein molecules with a polymer that increases the efficacy of delivery of these compounds (meena et al., ) . in addition, several studies used natural polymeric nanoparticles in disease diagnosis such as inulin in anthrax (feinen et al., ) synthetic polymer nanoparticles were used in the treatment of several diseases (cho et al., ) . in addition, the synthetic polymeric nanoparticle drugs were used to detect several causes of animal diseases such as s. aureus (colonna et al., ; liu et al., ) , tuberculosis (lawlor et al., ; parlane et al., ) , brucella abortus, b. anthracis (zhao et al., a, b) , influenza (oberoi et al., ) , and plasmodium malariae (powles et al., ) infections. also, qds could be used to screen blood samples for certain proteins that may indicate certain diseases (mohanty et al., ) . qds were applied in microbial detection such as pathogenic mycobacteria with cdse qds conjugated with streptavidin (liandris et al., ) . hahn et al. ( ) used the brighter signal of streptavidin-conjugated cdse/zns (core/shell) qds for detection of the e. coli o :h strain. modified cs nps have the ability to decrease doses of used drugs in disease treatment, which can lower the costs associated with drug treatments (aruna et al., ; fang et al., ; wang et al., a) and also in antitumor treatments (ghadi et al., ) . moreover, chitosan nanocomposites could be used for tumor therapy (aruna et al., ; cao and zhou, ; fang et al., ) . other studies by lu et al. ( ) illustrated that the injection of a chitosan solution in articular cartilage increases the chondrocyte density and their genesis. the chitosan-based material can support chondrogenesis, activate produced new cartilage, and improve the long-term outcomes of cartilage repair in clinical settings (suh and matthew, ) . in addition, it can be used in gene therapy and have an adjuvant effect in vaccines in vivo (peniche and peniche, ; shi et al., ) . silica nanoparticles (snps) have been characterized by well-defined structures that enable them to be easily combined with other materials (burns et al., ) . in addition, their optical transparency allows excitation and emission light to pass through targeted cells (shirahata, ) . snps can also be conjugated with drug molecules and enter targeted cells in diseased animals, resulting from the effective accumulation of drugs inside cells and hence treatment (taylor-pashow et al., ; wang et al., b) . peled et al. ( ) diagnosed a respiratory mycobacterial infection in cattle using nanocomposite tools. nanomaterials have the ability to conjugate with biological elements such as viral and phage cells and are used for bacterial detection (billington et al., ) . recently, bai et al. ( ) reported that nanosensors can provide a diagnosis of subclinical bovine ketosis by using β-hydroxybutyrate (bhba). in addition, nanosensor tools are used in the measurement of different physical and chemical blood parameters (cui and mumper, ; hasuwa et al., ; rolfe, ) and the detection of the constituents of animal excretions such as sweat, breath, and temperature (neethirajan et al., ) . currently, various types of nanosensors can be utilized in the detection of cations, anions, organic compounds, and other toxic and microbial content in feed, food, and intelligent packaging (lu and bowles, ; neethirajan et al., ) . generally, it is suggested that the action potentials of nanosensors are due to two detection principles: catalytic sensing (using enzymes, cells, tissues/ organelles, and microorganisms) and affinity sensing (antibodies, nucleic acid, phages, bonding proteins, polymers, and synthetic proteins) as a diagnosis tools (akkoyun et al., ) . the first step in this direction, wang et al. ( b) used the nanocomposites of polyclonal antibodies and au nps for immunochromatographic strip detection of toxin contaminants in milk such as carcinogenic aflatoxin m . nanoshells are composed of a dielectric core material such as silica conjugated with a metallic material layer can be injected into animals with targeted agents that search and attach to cancer cell receptors (hirsch et al., ) . while qds have many advantages over organic fluorescent dyes, they are brighter and easier to visualize than organic dyes. hence, they can observe cell pathways and events inside the animal body as well as drug delivery to target tissues (chakravarthi and balaji, ) . qds also may be injected into the bloodstream of animals, and they may detect cells that are malfunctioning by illuminating the body with light and stimulating the qd to produce sufficient heat to kill the cancer cell (freitas, ) . the nanocomposites and nanomaterials have the advantages to enter into the cancer cells and agglomerated to form large clusters inside tumor cells (yigit et al., ) . this helps in drug delivery, imaging, and cancer therapy (fig. . ). schematics of different nanotechnology-based tools used for cancer therapy and imaging. liposomes are made up of lipid structures that can be made stealthy by pegylation and encapsulating different therapeutic agents; these are used as a potential nanocarrier for cancer therapy. nanocantilevers are array-like structures in which engineered tiny bars anchored at one end help in the detection of altered proteins present in certain types of cancers. quantum dots are fluorescent nanocrystals that can be conjugated to a ligand by coating a polymeric layer onto it; therapeutic agents are encapsulated and used for cancer therapy. new synthetic methods have been developed to design multifunctional nanoparticles, in which we can encapsulate both therapeutic and imaging agents in a single nanocarrier system that will conjugate with more than one ligand on the surface. thus, it will act as a novel multifunctional nanocarrier system with the capacity of targeted tumor imaging and the delivery of therapeutic agents. the most recent drug delivery in targeted tissues was achieved by nanocarrier composites with biological molecules such as fluorescent nanostructured glucoseand sucrose-derived nanoparticles, which help successfully deliver therapeutic agents to lung carcinoma (ajmal et al., ) . similarly, when injected, superparamagnetic nanoparticles made essentially from iron oxide target cancer cell receptors, enhance the location of cancer cells, and deliver the attached drug to kill the cancer cells. the encapsulation of qds with snps and coating with pegylated phospholipids and a paramagnetic lipid coating have been applied in animal science ( van-schooneveld et al., ) . moreover, qds also may be injected into the bloodstream of animals. platinum-containing nanoparticles (fept) without any surface coating may act as potential anticancer drugs yin et al., ) . magnetic nps encapsulated with silica could be used as carriers for anticancer drugs and fluorescence molecules (xu and sun, ) . in addition, the combination of fept@fe o with yolk-shell nanoparticles has high cytotoxicity and strong mr contrast enhancement (gao et al., b; peng and sun, ) . they added that it can encapsulate anticancer drugs. the functionalization of yolk-shell mnps can target a specific tissue for delivering therapeutic agents and monitoring the transformation of the tumor by mri. the mnps can combine with qds to exhibit magnetic and fluorescent properties (gao et al., a (gao et al., , a gu et al., ) and sequentially grow metallic nanocomponents (gu et al., b) . they added that exotic nanostructures such as yolk-shell nanoparticles are very stable because the biocompatible, compact fe o shells prevent the oxidative species from reaching the fept (gao et al., b (gao et al., , b . ag nps and fept nanocomposites remediate tumors and radiation therapy in cats (woods et al., ) . liposomes have the potential to provide several valuable tools in the diagnosis and therapy of tumors (hofheinz et al., ; johnston et al., ; sadozai and saeidi, ) . they also have the ability to functionalize and conjugate with biologically valuable materials for use in animals as anticancer drugs (alexis et al., ) . it is interesting to report that the intravenous administration of conjugated au nps with gum arabic (ga) to swine helped in the detection of tumor tissues by au np constructs (fent et al., ) . similarly, when conjugated, radioactive au nps with ga were injected intralesionally in canine animals suffering from prostate cancer. a successful entrance of au nps with drugs to the prostate tissues occurred, resulting in successful cancer therapy (axiak-bechtel et al., ) . au nps can be used for cancer detection and imaging in dogs and mice (chanda et al., ) . the variations in the thickness of the nanoshells help with their use in diagnostic assays and tumor detection (avaritt et al., ) . cnts without any additive were toxic to mice lungs (lam et al., ; shvedova et al., ) . the functionalization of cnts was nontoxic to animals in conjunction with other beneficial materials (schipper et al., ; wu et al., ) . hence, the nonfunctionalized, long mwcnts may be carcinogenic to mice (ding et al., ) . the drug is delivered for cancer therapy by liposomes characterized by the successful reach of drugs to the site of cancer cells that overcome the resistance of cancer for therapy (malam et al., ) . zamboni et al. ( ) also detected the efficacy of docetaxel encapsulated in the liposome bilayer for treatments of cancers in the mice prostate and pancreas as well as nonsmall-cell lung cancer. in a similar study, torchilin ( ) and sadozai and saeidi ( ) detected the ability of liposome nanocomposites to treat canine cancers in the spleen. meanwhile, kleiter et al. ( ) showed that liposome-encapsulated muramyl tripeptide showed antitumor potential and prolonged disease-free survival in canines. dendrimers are nanocomposites characterized by low cost, high potentiality in complexion or encapsulation with other beneficial biomaterials, and active destruction of the target cell membrane in cancer cells (baker et al., ; koda et al., ; sekowski et al., ) . in addition, these characteristics enable dendrimers to attach to drugs through a sphere on their surface and enter from the site of administration to targeted tissues via the blood vascular system, resulting in effective cancer therapy (al-jamal et al., ) . the polymers also adhered to albumin molecules, which potentiated their delivery and could be used in cancer detection and therapy (gradishar et al., ) . recently, meena et al. ( ) detected major benefits of cnts for animal health and production. they can be used as sensors, antimicrobial agents, anticancer agents, and for drug delivery. fernandez-lopez et al. ( ) and dilbaghi et al. ( b) illustrated that dendrimers can be used in the diagnosis and therapy of tumors in animals as well as the detection of their nature by their ability to penetrate tumor cells and change the biochemical content of cytoplasm, leading to cell death. various studies have shown that mesoporous silica nanoparticles (msnps) have potential applications (hom et al., ; slowing et al., slowing et al., , vivero-escoto et al., c) . msnps have a high surface area and volume, a stable mesostructure, a tunable pore diameter ( - nm), and a modifiable morphology (vivero-escoto et al., a, b) . recently, msnps were effectively used to protect conjugated molecules and agents ( juan et al., ) . they can be functionalized with nanostructures and employed for drug/gene delivery and sensing applications (igor et al., ) . the msnps were readily internalized by eukaryotic cells without detectable toxic effects (radu et al., ) . in addition, the surface functionalization of msnps was manipulated by the uptake efficiency of hela cancer cells (slowing et al., ) . in the meantime, the msnps were functionalized with folic acid to form folate receptors, which facilitated drug aggregation in target cells and hence gave a promising result in the regulation of human cancers in mice (lu et al., ) . today, there are different progressive advances in nanotechnology applications in the diagnosis and therapy of cancer that successfully target cancer cells without affecting normal, healthy cells (fig. . ) . . . . bioimaging (x-ray, fluorescent, magnetic resonance imaging (mri)) today, the right diagnosis of human and animal diseases depends on visions of the activities of body cells and organs by imaging technology, which has progressively advanced. the essential nanomaterial is the mnps that are used for various biological applications in veterinary medicine and produce useful tools for mri imaging (michalet et al., ) . the mnps can penetrate cell membranes and image the targeted tissues using magnetic resonance imaging (mri) (soenen et al., ) and produce fluorescence through light signal activation (ajmal et al., ; croissant et al., ) . cheon and lee ( ) reported that the conjugation of mnps and dyes will lead to efficient mri and optical imaging. furthermore, gu et al. ( a) and neuberger et al. ( ) revealed that porphyrin-modified fe o nanoparticles can act as a multifunctional nanomedicine that combines anticancer treatment and noninvasive mri imaging. similarly, gao et al. ( ) detected that the conjugation of fluorescent vancomycin with fluorescein-amine stains or fept@van causes the quick, sensitive, and low-cost detection of bacteria by using fluorescence microscopy; it also offers the ability to enhance contrast in mri. kolecka et al. ( ) incubated canine stem cells with superparamagnetic iron oxide nanoparticles for h. different approaches of nanotechnology such as gene therapy, photodynamic therapy, radio therapy, radiofrequency therapy, and cancer theragnostics are being applied for the treatment of cancer. these advanced technologies help target cancer cells only, without affecting normal cells. ultimately, this leads to the death of the cancer cells while the normal healthy cells survive. the nano-sized materials entered the cells by endocytosis and acted as contrasts for the detection of the activity of canine stem cells. long et al. ( ) also reported that the ultrasmall superparamagnetic nanoparticles could be used for mri imaging for temporal lobe epilepsy and the detection of any abnormalities. in addition, the fe o -cd-se heterodimer nanoparticles produced fluorescent mnps that allow their intracellular movements to be controlled using magnetic force and a fluorescent microscope (gao et al., a) . another study detected the reduced coated graphene oxide used in the treatment and diagnosis of diseases in humans and animals. it may employ as an opt-acoustic transmitter for producing high-pressure and highfrequency ultrasound (lee et al., ) . it is interesting to report that the hypernanocomposites of dendrimers can be used as alternative tools to the traditional dyes used as mri contrasts as well as in genes and drug delivery for therapy (kim and zimmerman, ; margerum et al., ; samad et al., ). nowadays, real-time sensitive imaging and sensing applications have been adopted with the development of qds, which are crystalline materials with a facet and lattice structure that are analogous to bulk semiconductor materials. several useful biomedical applications of qds in biology have highlighted its potential in nanobiotechnology (michalet et al., ) . they can be used as sensors and detecting tools for biomarkers, pathogens, and immune-labeling of cells and tissues (geszke-moritz and moritz, ; kaittanis et al., ) . most qds used for analytical applications are synthesized as core/shell structures (geszke-moritz and moritz, ). they have greater stability and resistance to photo-bleaching. they exhibit both fluorescence and magnetism, such as co@cd-se core-shell nanocomposites (kim et al., ) and fept-zn s nanosponges (gu et al., b) . similarly, the mnps can combine with qds to exhibit magnetic and fluorescent properties for imaging target cells and organs (gao et al., a (gao et al., , b, a . the modification of the qd surface gives rise to a variety of conjugation strategies with biomolecules such as vitamins, proteins, peptides, and antibodies, and initiates their entrance to targets in animal cells. similarly, maša ( ) illustrated that qds produced a signal of fluorescence (fluorophores) to detect different biological events and helped in enhancing sensitivity in analyzing different biological processes. he added that different fluorophores transfer energy from biological events into specific lights that can be detected. the most effective target detection is achieved by coupling biomolecules (such as enzymes, antibodies, small molecules, and oligonucleotides) into fluorescents (kaittanis et al., ) . furthermore, the combination of nano-sized particles and fluorescence has become a good alternative to detect biological events (h€ otzer et al., ; ruedas-rama et al., ) . giri et al. ( ) added that qd barcodes were conjugated with single-stranded oligonucleotides that can hybridize to a target sequence and conjugate to a specific dye, resulting in a genetic biomarker that is measured by its optical emissions by a fluorescence signal. moreover, qds have a unique property such as highly bright and extremely photo-stable with longer excited-state lifetimes than classical fluorescent dyes and chemical degradation (petryayeva et al., ) . fluorescent bioconjugated silica nps were also used in the encapsulation of fluorescent dye molecules, producing a highly amplified and reproducible signal (zhao et al., ) . moreover, the rapid detection of e. coli o :h was performed with a fluorescence microscope using snps encapsulated with dye (tuitemwong et al., ) . this produced effective detection rather than using dyes alone (he et al., ) . the use of doped snps with fluorescent dyes, qds, and metal nps did not cause any changes in encapsulated agents (tallury et al., ) . hence, snps have the advantage of robust materials while also being mechanically stable and transparent, enabling the stabilization and protection of encapsulated fluorophores. moreover, juan et al. ( ) quantified the biodistribution of snps doped in fluorescent dye and found that the main organs for accumulation were the liver and spleen, then passing to the stomach via fecal excretion. currently, mri is a recent imaging technology that is considered a noninvasive diagnostic tool with a high payload of molecules and molecular contrast agents (na and hyeon, ; villaraza et al., ) . in another study, msnps were used as an alternative to mri contrast agents and the produced signal loss in the liver was detected (taylor et al., ) . the liver, kidney, and spleen are the main organs of carryover of nanomaterial after intravenous administration (souris et al., ) . furthermore, msnps may applicable in biomedical imaging and develop new tools for clinical diagnosis and disease therapy (lai et al., ; lu et al., ; radu et al., ) . another study reported that the biodistributions and detection of disease pathogenesis by biomarkers for therapeutic treatments (cheon and lee, ) . the advances in nanotechnology enable us to develop delivery systems and tools at the nanoscale and to produce chemical and biological reactions related to targeted sites and cells (tomanek and enbody, ) . magnetic nanocomposites are useful for various biological applications in veterinary medicine and produce new tools for drug delivery and cancer therapy (michalet et al., ) . it is suggested that an efficient delivery system should have the capability to transport the desired guest molecules without any loss before reaching the targeted location (radin et al., ) . however, rejman et al. ( ) showed that msnps can be efficiently employed as carriers for intracellular drug delivery as well as cell tracers and cytoplasmic biosensors. in addition, liposomes have been successfully used for targeted drugs, imaging agents, vaccines, and gene delivery (bakker-woudenberg et al., ; hiszczy nska-sawicka et al., ) . mesosilica nanoparticles (msnps) can be encapsulated in drugs to increase the delivery to target organs (vallet-regi et al., ) such as cadmium sulfide (lai et al., ) , gold (liu et al., ; torney et al., ) , and iron oxide (giri et al., ; vivero-escoto et al., ) . dendrimers (radu et al., ) proteins (zhao et al., ) , and polymers have been developed radu et al., ) . several studies have detected the availability of fluorescent snps in gene therapy such as dna probes and carriers (lee et al., b; mintzer and simanek, ; wang et al., b; zhao et al., a, b) . they can also help in transferring genes in mice lungs using silica nanoparticles (ravi et al., ) . . nanomaterials and nanocomposite applications micelle nanoparticles are characterized by a hydrophobic core stabilized by a hydrophilic shell and used in transdermal therapeutics (lee et al., a) . the micelle nanoparticles are highly water soluble due to their hydrophilic shell; hence, they have low toxicity and are used for the drug delivery of therapeutic agents (koo et al., ) . scott-moncrieff et al. ( ) determined that insulin mixed with micelles was efficiently absorbed in dogs, resulting in successful insulin delivery and therapy. vail et al. ( ) currently uses the water-soluble micelle paclitaxel to efficiently treat tumors in dogs. the activity and safety of micelle paclitaxel are superior to lomustine by a high ability to enter the targeted cancer cells. several studies illustrated that polymer compositions, structures, and properties potentiated its use in a biomedical application such as drug delivery (moreno-vega et al., ; yang, ) . the advantages of using polymer-drug conjugates include their ability to overcome drug resistance and to elicit immune-stimulatory effects (ríhová et al., ; sirova et al., ) . moreover, some studies detected that skin administration is the better route for the administration of polymer-based nanoparticles such as hydrogels containing dexamethasone for the treatment of psoriasis (degim, ) . meanwhile, drug delivery can use polymers to coat mnps and to encapsulate drugs to form nanocapsules or micelles (gupta and gupta, ) , which may require complicated processes and result in modest efficiency. klajnert and bryszewska ( ) and stecko et al. ( ) formed a dendrimer-based nanocomposite with vaccine preparation, increased the efficacy of vaccine delivery, and potentiated its immunogenicity. furthermore, nanoemulsions of natural oils were successfully used in veterinary medicine as a drug delivery agent (kang et al., ; vandamme et al., ). . . . animal production, reproduction, nutrition, and breeding today, there is progressive exploration and refinement in nanotechnology, which plays a significant role in animal production and reproduction. some nanoparticles have been demonstrated to enhance fertility and protect spermatozoa through the functional groups they carry. recently, qds have been used to improve the detection of spermatozoon and oocyte movement and their interactions in a physiological setting. they potentiated the production of greater signal intensity than the old traditional method used in imaging gametes (druart et al., ; feugang et al., ; long et al., ) . feugang et al. ( ) and hasuwa et al. ( ) detected that the consumption of composed bioluminescence resonance energy transferconjugated quantum dot nanoparticles can be used in male pig gametes for the observation and imaging of fertilization events in deep gonadal tissues. the purification of semen is essential for successful artificial insemination in animals. recently, coated mnps with antibodies or lectins were used for the separation of damaged sperm from undamaged healthy sperm, while the conjugated antibodies with mnps directed directed to ubiquitin characteristic material for defective sperm (odhiambo et al., ; petruska et al., ) . in addition, the application of nanotechnology in sperm cryopreservation fixes the stability of sperm quality and prevents microbial pollution during storage (bryla and trzcinska, ) . antibiotic treatment may reduce the motility and activity of sperm; this problem will be solved by the use of nanoparticles that replace extender antibiotics (hargreaves et al., ) . also, mesoporous silica nanoparticles were loaded with dna and transferred the sperm in vitro without changes in its quality (barkalina et al., ) . pawar and kaul ( ) detected that the incubation of buffalo sperm with low levels ( μg/ml) of titanium led to successful fertilization. the administration of nanofunctionalized α-tocopherol dose for horses may increases the absorption and plasma concentration due to the oxidative status of racehorses become under intense training (rey et al., ) . the supplementation of ag nps singly or in combination with amino acids in chicken feed can improve their immune status (bhanja et al., ) . similarly, rey et al. ( ) demonstrated the potential of the administration of micellar nanoparticles conjugated with vitamin e to pigs in improving the health status of the animals. several studies have used nanobiosensors for animal reproduction and measurement of their fertility (monerris et al., ; sagadevan and periasamy, ) and detection of the viability of reproductive organ functions and fetus (saragusty and arav, ) . furthermore, engineered nanoparticles with fluorescent probes visualize the events during ovulation and pregnancy in reproductive tissues (feugang et al., ; hasuwa et al., ) . in addition, qds have the ability to detect the mammalian spermatozoon and oocyte movement, which significantly helps in animal production (hill and li, ) . moreover, the small size of the biosensors and the high surface-to-volume ratio enable them to act as signal reporters in biosensors and reduce the time of target cells to be detectable by spectrophotometer, fluorescence microscope, and luminometer (koedrith et al., ) . greenwood et al. ( ) suggested that the formation of reactive oxygen species (ros) and free radical production by nanomaterials causes the destruction of mitochondria, the denaturation of proteins, and the damage of dna. several studies have detected that ros production can be found in c fullerenes, swnts, and qds (dilbaghi et al., a) . the mechanism of action of nanoparticles is briefly illustrated in fig. . . the nanomaterial potentials such as anticancer, antimicrobial, and other activities against target cells resulted in the penetration and disruption of the cell membrane and cell death. particularly, when the microbial cells that were treated by metal nanoparticles were examined by a scanning electron microscope, cell membrane damage and adhered nanoparticles to the respiratory sequence of cytoplasm were observed, followed by the death of target cells (gajbhiye et al., ; hassan et al., b hassan et al., , hassan et al., , a nabawy, ) . another suggestion is the interaction between the constituents of target cells with the oxygen atom and metal ions of metal nanoparticles, which can cause damaged cell components and death (brayner et al., ; matei et al., ; moraru et al., ; violeta et al., ) . in spite of the progressive and valuable tools of biosensor applications in animal science, their toxicity risk to animals and the environment is the essential reason for limiting their use in human and animal science (donaldson et al., ; oberd€ orster and kuhlbusch, ) . in addition to nanotechnology applications in several aspects of biomedicine, there are potential toxicity hazards to the environment and users as well (baltic et al., ) . oxidative stress and inflammation are caused by affected tissue fibers or secondary mutations (aschberger et al., ) . in general, nanomaterial toxicity varies according to nano-sized particles, the health states of the humans and animals, the administered doses, and the time of exposure (aschberger et al., ; maynard, ; rim et al., ) . also, the efficient evaluation of nanoparticle toxicity risk must include their particle size and shape, crystalline form, functionalization, and purity (aschberger et al., ) . some nanoparticles are toxic to sperm; this is adversely reflected as it decreases its viability in vitro due to overdoses and long exposure to zinc oxide and titanium oxide nanoparticles (pawar and kaul, ) . the incubation of the sperm with the concentrations of - μg/ml of zn nps resulted in the death of sperm within a few minutes (barkhordari et al., ) . pawar and kaul ( ) detected that the incubation of buffalo sperm with μg/ml of titanium oxide nanoparticles reduced its viability. several studies have reported that metal nanoparticles may induce toxicity by their ability to easily access the skin, lungs, and brain, causing adverse effects in biological functions. the toxicity of zn np nanoparticles to animals and the environment must be briefly studied before they are used as a feed additive. therefore, the measurement of effective nontoxic doses of metal nanoparticles in laboratory animal models must be undertaken to study the suitability of their field application (abd el-fatah et al., ; asharani et al., ; hassan et al., ; shaw et al., ) . until now, there has been a lack of toxicological effects of nanoparticles on health (savolainen et al., ) . so, the toxicological aspects of nanomaterials require further continuous studies to be completely understood (fig. . ). regarding the route of exposure to nanomaterials, ingestion is the main exposure route for humans and animals (aschberger et al., ) . upon ingestion, the nanomaterials enter the gastrointestinal tract to the intestines and are eliminated rapidly through the liver and spleen (oberd€ orster et al., ) . the ingestion mainly results from the presence of nanoparticles in food due to direct contact of nanopackaging to food (baltic et al., ; bouwmeester et al., ) . the nanoparticles are distributed in the liver and spleen in circulation (baltic et al., ; silvestre et al., ) . inhalation and the skin are other routes of nanoparticle toxicity, which are detected mainly in the laboratory and in industry workers in nanomaterial production factories (aschberger et al., ) . another study described that inhalation and skin exposure to magnesium oxide nanoparticles allows them to enter the nerve cells and central nervous system (elder et al., ) . nurkiewicz et al. ( ) demonstrated that the inhalation of nano-sized titanium dioxide reached systemic circulation in rats. tinkle et al. ( ) and tsuji et al. ( ) detected the ability of nanomaterials to enter healthy human skin during constant flexing, causing many controversial effects (monteiro-riviere et al., ) . in other studies, the zn nps detected the availability of penetrated skin tissue (baltic et al., ; sharma et al., ) and have carcinogenic effects such as asbestos-caused fibrosis in the lungs (muller et al., ) . the inhalation of high doses of nano-tio also has the potential for lung carcinogenesis (aschberger et al., ) . furthermore, the entrance of nanoparticles via the bloodstream may affect blood vessel function and cause blood clot formation and other adverse effects on the cardiovascular system, as in the case of inhalation of ambient ultrafine particles (pekkanen et al., ) . similar microvascular dysfunction was observed in rats after inhalation exposure to low concentrations of nano-sized titanium dioxide (nurkiewicz et al., ) and platelet aggregation and vascular thrombosis after inhalation exposure to swcnts and mwcnts (radomski et al., ) . some studies reported that nanomaterials can penetrate the blood and are distributed in body organ cell tissues, and sometimes transmitted to the fetus via the blood supply (baltic et al., ; silvestre et al., ) . in the near future, more progressive advancement in nanobiomedical science and availability to improve animal health is needed. they will have the potential to solve many problems related to animal disease diagnosis, animal production, reproduction, and good hygienic practices during rearing and maintaining of food animals. the possible applications of the technology are almost incredible in relation to livestock. although much research is needed before nanotechnology applications are used in veterinary and animal sciences, in particular the toxicity risk. schematic mechanism of cytotoxic activity of nanoparticles (nps). until now, nanotechnology has offered significant advances in the development of novel technology in human and veterinary medicine as a synthesis of new materials and tools that help increase animal health and production. several applications of nanomaterials are used to produce strategies for disease diagnosis, drug delivery, animal nutrition, breeding and reproduction, additives to animal products, and finally food safety for human and animal health. in addition, they are used for tumor detection, production of tumor vaccines, tissue engineering, mri images, sensor development, and the detection of pathogens, proteins, and biological molecules. these applications resulted in the development of novel nanodrugs such as metal nanoparticles (particularly, zn nps, ag nps, au nps, and mnps), liposomes, polymeric nanoparticles, dendrimers, cs nps, qds, etc. the use of metallic nanoparticles in nanomedicine has the ability to improve imaging and therapeutic drug delivery for the treatment of cancer in humans and veterinary medicine. the nanomaterials and nanocomposites bind with biological fluids to help control drug release and hence are used in the synthesis of biological markers, imaging, drug delivery systems, and disease detection. in addition, the potential of nanoparticles in disease treatment and diagnosis, antimicrobial, anticancer and other activities of metals nanoparticles against target cells resulted from disruption of cell membrane, damage, rupture of cell wall and leakage in inter cellular components and finally cell death. the toxicity of nanomaterials is mainly due to oxidative stress. the exact mechanism of the formation and generation of ros requires more study to be completely understood. oxidative stress resulting from the use of nanomaterials can cause inflammation, fibrosis, genotoxicity, and cancers. in the future, there will be progressive advances in nanobiomedical science and use in improving animal health. it will have the potential to solve many problems related to animal health, animal production, reproduction, and good hygienic practices during rearing and maintaining of food animals. the possible applications of the technology are almost incredible in relation to livestock. however, special attention is required for the known toxicological aspects of nanomaterials prior to application in veterinary and animal sciences. because there is no brief knowledge about toxicology studies available, hence much research is urgently needed before nanotechnology applications can be used, in particular their toxicity risk. comparative study between the use of bulk and nanoparticles of zinc oxide in amelioration the toxic effects of aflatoxins in rats a comparative study on antifungal activity of fe o , and fe o nanoparticles nanobiotechnological strategies for molud and mycotoxin control extracellular biosynthesis of silver nanoparticles using the fungus fusarium oxysporum synthesis, characterization and in vitro evaluation of methotrexate conjugated fluorescent carbon nanoparticles as drug delivery system for human lung cancer targeting detection of sulphamethazine with an optical biosensor and anti-idiotypic antibodies nanoparticle technologies for cancer therapy tumor targeting of functionalized quantum dot-liposome hybrids by intravenous administration zinc toxicity in ruminants preparation and characterization of three tilmicosinloaded lipid nanoparticles: physicochemical properties and in-vitro antibacterial activities antimicrobial therapeutic determinants of outcomes from septic shock role of chitosan nanoparticles in cancer therpy analysis of currently available data for characterising the risk of engineered nanomaterials to the environment and human health; lessons learned from four case studies green synthesis and evaluation of silver nanoparticles as adjuvant in rabies veterinary vaccine toxicity of silver nanoparticles in zebrafish models towards a definition of inorganic nanoparticles from an environmental, health and safety perspective plasmon resonance shifts of au-coated au nanoshells: insight into multicomponent nanoparticle growth gum arabic-coated radioactive gold nanoparticles cause no short-term local or systemic toxicity in the clinically relevant canine model of prostate cancer theranostics aspects of various nanoparticles in veterinary medicine moderate intensity static magnetic field has bactericidal effect on e. coli and s. epidermidis on sintered hydroxyapatite the synthesis and testing of anti-cancer therapeutic nanodevices longcirculating sterically stabilized liposomes in the treatment of infections nanotechnology and its potential applications in meat industry in vitro effect of biogenic silver nanoparticles on sterilisation of tobacco leaf explants and for higher yield of protoplasts effects of mesoporous silica nanoparticles upon the function of mammalian sperm in vitro effect of zinc oxide nanoparticles on viability of human spermatozoa encapsulation of mentha piperita essential oils in chitosan-cinnamic acid nanogel with enhanced antimicrobial growth against aspergillus flvus in ovo administration of silver nanoparticles and/or amino acids influence metabolism and immune gene expression in chicken embryos prevention of bacterial foodborne disease using nano biotechnology comparison of two methods for blood lead analysis in cattle: graphite-furnace atomic absorption spectrometry and leadcare(r) ii system bacterial stress responses: what does not kill them can make them stronger from antigen delivery system to adjuvanticy: the board application of nanoparticles in vaccinology equine sarcoid: state of the art review of health safety aspects of nanotechnologies in food production toxicological impact studies based on escherichia coli bacteria in ultrafine zno nanoparticles colloidal medium effects of zinc oxide and enterococcus faecium sf dietary supplementation on the performance, intestinal microbiota and immune status of weaned piglets quality and fertilizing capacity of boar spermatozoa during liquid storage in extender supplemented with different antibiotics fluorescent core-shell silica nanoparticles: towards "lab on particle" architectures for nanobiotechnology progress in antitumor studies of chitosan. chin bovine papillomavirus dna in neoplastic and nonneoplastic tissues obtained from horses with and without sarcoids in the western united states effect of feeding organic and inorganic sources of additional zinc on growth performance and zinc balance in nursery pigs applications of nanotechnology in veterinary medicine gold nanoparticle based x-ray contrast agent for tumor imaging in mice and dog: a potential nano-platform for computer tomography theranostics nanotechnology in nutraceuticals and functional foods synergistically integrated nanoparticles as multimodal probes for nanobiotechnology therapeutic nanoparticles for drug delivery in cancer effect of chitosan and chitosan nanoparticles on fungal growth and chilli seed quality sub-unit vaccine against s. aureus-mediated infections: set-up of nano-sized polymeric adjuvant synergetic effects of doxycyclineloaded chitosan nanoparticles for improving drug delivery and efficacy two-photontriggered drug delivery via fluorescent nanovalves chitosan-based nanoparticles for topical genetic immunization a simple and rapid immunochromatographic strip test for detecting antibody to porcine reproductive and respiratory syndrome virus d-mannose-modified chitosan microspheres enhance oprf-opri-mediated protection of mice against pseudomonas aeruginosa infection via induction of mucosal immunity combinatorial prospects of nano-targeted chemoimmunotherapy nanoparticle therapeutics: an emerging treatment modality for cancer new tools and approaches for predicting skin permeability carbon nanotubes: present and future commercial applications evaluation of tropicamide-loaded tamarind seed xyloglucan nano-aggregates for ophthalmic delivery nanoscale device for veterinary technology: trends and future prospective molecular characterization of the cytotoxic mechanism of multiwall carbon nanotubes and nano-onions on human skin fibroblast a highly sensitive detection for foot-and-mouth disease virus by gold nanopariticle improved immuno-pcr chitosan-based intranasal vaccine against escherichia coli o :h in vivo imaging of in situ motility of fresh and liquid stored ram spermatozoa in the ewe gential tract functionalized carbon nanotubes are non-cytotoxic and preserve the functionality of primary immune cells nanomedicine(s) under the microscope human health risks of engineered nanomaterials: critical knowledge gaps in nanomaterials risk assessment immune-carbon nanotubes and recognition of pathogens potential applications of carbon nanotubes nanosilver as substance biostabilising footwear materials in the foot mycosis prophylaxis comparison of antitumor effects of chitosan nanoparticles from different sources in vitro impact of nanotechnology on drug delivery treatment of experimental salmonellosis in mice with ampiciline-bound nanoparticles advax-adjuvanted recombinant protective antigen provides protection against inhalational anthrax that is further enhanced by addition of murabutide adjuvant bio distribution of maltose and gum arabic hybrid gold nanoparticles after intravenous injection in juvenile swine antibacterial agents based on the cyclic d, l-alpha-peptide architecture application of quantum dot nanoparticles for potential non-invasive bio-imaging of mammalian spermatozoa selfilluminating quantum dots for non-invasive bioluminescence imaging of mammalian gametes silver nanoparticles as a potential antimicrobial additive for weaned pigs microbivores: artifcial mechanical phagocytes using digest and discharge protocol inhibition of the hiv- protease by fullerene derivatives: model building studies and experimental verification fungus-mediated synthesis of silver nanoparticles and their activity against pathogenic fungi in combination with fluconazole antimicrobial activities of commercial nanoparticles against an environmental soil microbe, pseudomonas putida kt combining fluorescent probes and biofunctional magnetic nanoparticles for rapid detection of bacteria in human blood fluorescent magnetic nanocrystals by sequential addition of reagents in a one-pot reaction: a simple preparation for multifunctional nanostructures fept@cos yolkshell nanocrystals as a potent agent to kill hela cells multifunctional yolk-shell nanoparticles: a potential mri contrast and anticancer agent intracellular spatial control of fluorescent magnetic nanoparticles nanosilver particles in medical applications: synthesis, performance, and toxicity quantum dots as versatile probes in medical sciences: synthesis, modification and properties synthesis and optimization of chitosan nanoparticles: potential applications in nanomedicine and biomedical engineering nanosilver effects on growth parameters in experimental aflatoxicosis in broiler chickens effect of nanosilver on blood parameters in chickens having aflatoxicosis amelioration studies on optimization of low molecular weight chitosan nanoparticle preparation, characterization with potassium per sulfate and silver nitrate combined action with aid of drug delivery to tetracycline resistant bacteria immobilization of mycotoxins on modified nanodiamond substrates stimuli-responsive controlledrelease delivery system based on mesoporous silica nanorods capped with magnetic nanoparticles preparation and antibacterial activity of fe o @ag nanoparticles effects of nanocopper on copper availability and nutrients digestibility, growth performance and serum traits of piglets nanomedicine taxonomy: briefing paper. canadian nanobusiness alliance phase iii trial of nanoparticle albumin-bound paclitaxel compared with polyethylated castor oil-based paclitaxel in women with breast cancer vaccination against foot-and-mouth disease virus using peptides conjugated to nano-beads using biofunctional magnetic nanoparticles to capture gram-negative bacteria at an ultra-low concentration facile one-pot synthesis of bifunctional heterodimers of nanoparticles: a conjugate of quantum dot and magnetic nanoparticles synthesis and cellular uptake of porphyrin decorated iron oxide nanoparticles-a potential candidate for bimodal anticancer therapy direct synthesis of a bimodal nanosponge based on potential role for carbon nanoparticles identification and preservation in situ of parathyroid glands during total thyroidectomy and central compartment node dissection synthesis and surface engineering of iron oxide nanoparticles for biomedical applications biosynthesis, purification and characterization of silver nanoparticles using escherichia coli green chemistry approach for the synthesis of biocompatible graphene enhanced antibacterial and antibiofilm activities of silver nanoparticles against gram-negative and gram-positive bacteria reduced graphene oxide-silver nanoparticle nanocomposite: a potential anticancer nanotherapy antibacterial efficacy of silver nanoparticles on endometritis caused by prevotella melaninogenica and arcanobacterum pyogenes in dairy cattle stability and bioavailability of vitamin d nanoencapsulated in casein micelles detection of single bacterial pathogens with semiconductor quantum dots nanoparticle detection of respiratory infection effects of co-trimoxazole, erythromycin, amoxycillin, tetracycline and chloroquine on sperm function in vitro new trials of use of molasses and garlic extracts for competing mycotoxicosis study the availability of using buckthorn (rhamnus cathartica) plant extract in laboratory control of some bacterial and fungal diseases efficacy of use of forskolin plant extract in control of toxic effects of aflatoxicosis in food prevalence of yeast infections in small ruminants with particular references to their treatment by some natural herbal extracts effect of zinc oxide nanoparticles on the growth of mycotoxigenic mould biosynthesis of silver nanoparticles (ag-nps) (a model of metals) by candida albicans and its antifungal activity on some fungal pathogens (trichophyton mentagrophytes and candida albicans) using of molecular biology techniques for detection of c. neoformance in respiratory disorders in cow with references to its control by nanoparticles of iron oxide the possibility of using zinc oxide nanoparticles in controlling some fungal and bacterial strains isolated from buffaloes herbal biosynthesis of zinc nanoparticles and evaluation of their antifungal and antibacterial effect for buffaloes skin affections antimicrobial potential of iron oxide nanoparticles in control of some causes of microbial skin affection in cattle highly sensitive and rapid determination of escherichia coli o :h in minced beef and water using electrocatalytic gold nanoparticle tags the efficiency of using silver nanoparticles singly and in combination with traditional antimicrobial agents in control of some fungal and bacterial affection of buffaloes efficacy of zinc oxide nanoparticles and curcumin in amelioration the toxic effects in aflatoxicated rabbits evaluation of the efficacy of ozone fumigation and zinc oxide nanoparticles in control of aflatoxins contamination in cattle feeds chapter : toxic and benefit effects of carbon nanomaterials on human and animal health. in: carbon nanomaterials for agri-food and environmental applications transgenic mouse sperm that have green acrosome and red mitochondria allow visualization of sperm and their acrosome reaction in vivo ultra stable, highly fluorescent, and water-dispersed silicon-based nanospheres as cellular probes current and future prospects for nanotechnology in animal production nanoshell-mediated near-infrared thermal therapy of tumors under magnetic resonance guidance induction of immune responses in sheep by vaccination with liposome-entrapped dna complexes encoding toxoplasma gondii mic gene liposomal encapsulated anti-cancer drugs silica nanoparticles as a delivery system for nucleic acid based reagents advax™, a polysaccharide adjuvant derived from delta inulin, provides improved influenza vaccine protection through broad-based enhancement of adaptive immune responses severe acute respiratory syndrome-associated coronavirus vaccines formulated with delta inulin adjuvants provide enhanced protection while ameliorating lung eosinophilic immunopathology antifungal effect of sodium dodecil sulfate and nano particle zno on growth inhibition of standard strain of candida albicans fluorescence in nanobiotechnology: sophisticated fluorophores for novel applications endostar-loaded peg-plga nanoparticles: in vitro and in vivo evaluation effects of nano calcium carbonate and nano calcium citrate on toxicity in icr mice and on bone mineral density in an ovariectomized mice model immunogenic properties of a bcg adjuvant chitosan nanoparticle-based dengue vaccine in human dendritic cells mesoporous silica nanoparticles for drug delivery and bios improved serodiagnosis of hydrated cyst disease using gold nanoparticle labeled antigen b in naturally infected sheep escherichia coli signal peptidase recognizes and cleaves the signal sequence of xylanase from a newly isolated bacillus subtilis strain r a novel dynamic flow immunochromatographic test (dfict) using gold nanoparticles for the serological detection of toxoplasma gondii infection in dogs and cats antimicrobial efficacy of zinc oxide quantum dots against listeria monocytogenes, salmonella enteritidis, and escherichia coli o :h characterization of the drug retention and pharmacokinetic properties of liposomal nanoparticles containing dihydrosphinomyelin inorganic-organic hybrid nanomaterials for therapeutic and diagnostic imaging applications photodynamic inactivation of enveloped viruses by buckminsterfullerene one-step, nanoparticle-mediated bacterial detection with magnetic relaxation rapid nanoparticle-mediated monitoring of bacterial metabolic activity and assessment of antimicrobial susceptibility in blood with magnetic relaxation emerging nanotechnology-based strategies for the identification of microbial pathogenesis carbon nanotubes as multifunctional biological transporters and near-infrared agents for selective cancer cell destruction controlled release of paclitaxel from microemulsion containing plga and evaluation of anti-tumor activity in vitro and in vivo single-walled carbon nanotubes exhibit strong antimicrobial activity microbial cytotoxicity of carbon based nanomaterials: implications for river water and wastewater effluent magnesium and iron nanoparticles production using microorganisms and various salts enhanced efficacy of liposome-encapsulated ribavirin against rift valley fever virus infection in mice. antimicrob. agents chemother toxicology of carbon nanotubes-a review incorporation of amphotericin b in tuftsin-bearing liposomes showed enhanced efficacy against systemic cryptococcosis in leucopenic mice silver nanoparticles impair peste des petits ruminant's virus replication application of chitosan and chitosan nanoparticles for the control of fusarium head blight of wheat fusarium graminearum in vitro and greenhouse applications of dendrimers in bio-organic chemistry synthesis and characterization of co/cdse core/shell nanocomposites: bifunctional magnetic-optical nanocrystals organically modified silica nanoparticles co-encapsulating photosensitizing drug and aggregationenhanced two-photon absorbing fluorescent dye aggregates for two photon photodynamic therapy antifungal effect of silver nanoparticles on dermatophytes multimodal drug delivery using gold nanoparticles dendrimers: properties and applications concomitant liposomal doxorubicin and daily palliative radiotherapy in advanced feline soft tissue sarcomas gene transfection into adherent cells using electroporation on a dendrimer-modified gold electrode recent trends in rapid environmental monitoring of pathogens and toxicants: potential of nanoparticle-based biosensor and applications behavior of adipose-derived canine mesenchymal stem cells after superparamagnetic iron oxide nanoparticles labeling for magnetic resonance imaging role of nanotechnology in targeted drug delivery and imaging: a concise review use of an amphotericin b lipid complex for treatment of blastomycosis in dogs development of a nanoparticulate formulation of diminazene to treat african trypanosomiasis biodistribution of a high dose of diamond, graphite, and graphene oxide nanoparticles after multiple intraperitoneal injections in rats chapter -nanosensors for the detection of food contaminants a mesoporous silica nanosphere-based carrier system with chemically removable cds nanoparticle caps for stimuli-responsive controlled release of neurotransmitters and drug molecules pulmonary toxicity of single wall carbon nanotubes in mice and days after intratracheal instillation disposition of aerosolized liposomal amphotericin b bacteriicidal effect of silver nanoparticles against multidrug-resistant treatment of mycobacterium tuberculosis-infected macrophages with poly (lactic-co-glycolic acid) microparticles drives nfκb and autophagy dependent bacillary killing functionalized carbon nanoparticles: syntheses and applications in optical bioimaging and energy conversion intranasal administration of novel chitosan nanoparticle/dna complexes induces antibody response to hepatitis b surface antigen in mice quality and safety aspects of meat products as affected by various physical manipulations of packaging materials synergistic anticancer effects achieved by co-delivery of trail and paclitaxel using cationic polymeric micelles synthesis of stable silicadye hybrid nanomaterial as dna carrier dual-color photoacoustic lymph node imaging using nanoformulated naphthalocyanines the use of lipid formulations of amphotericin b for systemic fungal infections injectable nanocarriers for biodetoxification nanomaterials in the application of tumor vaccines: advantages and disadvantages clinical study of harvesting lymph nodes with carbon nanoparticles in advanced gastric cancer: a prospective randomized trial porphyrin-based carbon dots for photodynamic therapy of hepatoma detection of mycobacteria based on functionalized quantum dots coupled with immunomagnetic separation effect of nano-zinc oxide on the production and dressing performance of broiler sharper and faster nano darts kill more bacteria: a study of antibacterial activity of individually dispersed pristine single-walled carbon nanotube ph-responsive nanogated ensemble based on gold-capped mesoporous silica through an acid-labile acetal linker understanding the toxicity of carbon nanotubes construction of poly (vinyl alcohol)/poly (lactide-glycolide acid)/vancomycin nanoparticles on titanium for enhancing the surface self-antibacterial activity and cytocompatibility genetic and spectrally distinct in vivo imaging: embryonic stem cells and mice with widespread expression of a monomeric red fluorescent protein mri tracking of bone marrow mesenchymal stem cells labeled with ultra-small superparamagnetic iron oxide nanoparticles in a rat model of temporal lobe epilepsy sensory trial to assess the acceptability of zinc fortificants added to iron-fortified wheat products how will nanotechnology affect agricultural supply chains effects of chitosan on rat knee cartilages biocompatibility, biodistribution, and drug-delivery efficiency of mesoporous silica nanoparticles for cancer therapy in animals the use of liposomally-entrapped gentamicin in the treatment of bovine staphylococcus aureus mastitis comparison of cellular accumulation, tissue distribution, and anti-hiv activity of free and liposomal t, t-dideoxycytidine liposomes and nanoparticles: nanosized vehicles for drug delivery in cancer immunopotentiating nano-chitosan as potent vaccine carter for efficacious prophylaxis of filarial antigens gadolinium(iii) do a macrocycles and polyethylene glycol coupled to dendrimers effect of molecular weight on physical and biological properties of macromolecular magnetic resonance imaging contrast agents nanotechnology in food safety and quality assessment: potentiality of nanoparticles in diagnosis of foodborne pathogens cobalt doped zno prepared by electrochemistry: chemistry, morphology, and magnetism nanotechnology: assessing the risks an investigation of blood selenium concentrations of goats in new york state applications of nanotechnology in veterinary therapeutics development of colloidal gold-based immunochromatographic assay for rapid detection of mycoplasma suis in porcine plasma quantum dots for live cells, in vivo imaging, and diagnostics nonviral vectors for gene delivery development of magnetic field control for magnetically targeted drug delivery system using a superconducting magnet colloidal nanocarriers: a review on formulation technology, types and applications toward targeted drug delivery growth performance and serum biochemical parameters as affected by nano zinc supplementation in layer chicks introduction to metallic nanoparticles nanomedicine: prospective diagnostic and therapeutic potential polyvinylpyrrolidone-coated gold nanoparticles inhibit endothelial cell viability, proliferation, and erk / phosphorylation and reduce the magnitude of endothelial-independent dilator responses in isolated aortic vessels an overview of nanomedicine in veterinary science integrated electrochemical immunosensor with gold nanoparticles for the determination of progesterone nanotoxicology-characterization, dosing and health effects evaluation of an immunochromatographic assay for rapid detection of salmonella enterica serovars typhimurium and enteritidis nanotechnology: a new frontier in food science polymeric and ceramic nanoparticles in biomedical applications effect of metal nanoparticles on the growth of ochratoxigenic moulds and ochratoxin a production isolated from food and feed application of nanotechnology for animal health and production improvement: a review clastogenic and aneugenic effects of multi-wall carbon nanotubes in epithelial cells nanostructured t mri contrast agents effect of metal element nanoparticles in the growth of aflatoxogenic a. flavus and aflatoxin production in feed effect of metal nanoparticles in comparison with commercial antifungal feed additives on the growth of a. flavus and aflatoxin b production an introduction to nanotechnologies: what's in it for us? references national nanotechnology initiative recent advancement in biosensors technology for animal and livestock health management superparamagnetic nanoparticles for biomedical applications: possibilities and limitations of a new drug delivery system development and applications of mesoscopic hall microprobes nanoparticle inhalation augments particle-dependent systemic microvascular dysfunction immunochromatographic gold-based test strip for rapid detection of infectious bursal disease virus antibodies band gap fluorescence from individual singlewalled carbon nanotubes in vivo effects: methodologies and biokinetics of inhaled nanomaterials translocation of inhaled ultrafine particles to the brain peg modified liposomes containing crx- adjuvant in combination with methylglycol chitosan enhance the murine sublingual immune response to influenza vaccination increased conception rates in beef cattle inseminated with nanopurified bull semen phosphatidylinositol di-mannoside and derivates modulate the immune response to and efficacy of a tuberculosis protein vaccine against mycobacterium bovis infection preparation and effects of nano mineral particle feeding in livestock: a review evaluation of polytetrafluoroethylene-graphite-coated total hip prostheses in goats superovulatory response to fsh and embryo recovery rate in pandharpuri buffaloes (bubalus bubalis) electrical detection of single viruses toxicity of titanium oxide nanoparticles causes functionality and dna damage in buffalo (bubalus bubalis) sperm in vitro nanocarriers as an emerging platform for cancer therapy particulate air pollution and risk of st-segment depression during repeated submaximal exercise tests among subjects with coronary heart disease: the exposure and risk assessment for fine and ultrafine particles in ambient air (ultra) study detection of volatile organic compounds in cattle naturally infected with mycobacterium bovis synthesis and characterization of monodisperse hollow fe o nanoparticles development of an immunochromatographic strip for rapid detection of h subtype avian influenza viruses chitosan nanoparticles: a contribution to nanomedicine advax™, a novel microcrystalline polysaccharide particle engineered from delta inulin, provides robust adjuvant potency together with tolerability and safety antioxidant supplementation and purification of semen for improved artificial insemination in livestock species quantum dots in bioanalysis: a review of applications across various platforms for fluorescence spectroscopy and imaging effect of silver nanoparticles on growth performance, metabolism and microbial profile of broiler chickens the use of synthetic carriers in malaria vaccine design enhanced noninflammasome mediated immune responses by mannosylated zwitterionic-based cationic liposomes for hiv dna vaccines silica sol-gel for the controlled release of antibiotics. i. synthesis, characterization, and in vitro release nanoparticle-induced platelet aggregation and vascular thrombosis a polyamidoamine dendrimer-capped mesoporous silica nanosphere-based gene transfection reagent zinc oxide nanoparticles: opportunities and challenges in veterinary sciences lead and cadmium concentrations in goat, cow, sheep, and buffalo milks from different regions of iran enhancing the milk production and immunity in holstein friesian crossbred cow by supplementing novel nano zinc oxide cationic silica nanoparticles as gene carriers: synthesis, characterization and transfection efficiency in vitro and in vivo antimicrobial effect of biologically prepared silver nanoparticles (agnps) on two different obturator's soft linings in maxillectomy patients promises, facts and challenges for graphene in biomedical applications saponin-loaded chitosan nanoparticles and their cytotoxicity to cancer cell lines in vitro size-dependent internalization of particles via the pathways of clathrin-and caveolae-mediated endocytosis short-and long-term effect of oral administration of micellized natural vitamin e (d-α-tocopherol) on oxidative status in racehorses under intense training lower oral doses of micellized α-tocopherol compared to α-tocopheryl acetate in feed modify fatty acid profiles and improve oxidative status in pigs immunization with a hemagglutinin-derived synthetic peptide formulated with a cpg-dna-liposome complex induced protection against lethal influenza virus infection in mice cytostatic and immunomobilizing activities of polymer-bound drugs: experimental and first clinical data oxidative dna damage from nanoparticle exposure and its application to workers' health: a literature review a gold glyco-nanoparticle carrying a listeriolysin o peptide and formulated with advax™ delta inulin adjuvant induces robust t-cell protection against listeria infection micro-and nanosensors for medical and biological measurement nanostructures in biodiagnostics fluorescent nanoparticles for intracellular sensing: a review a novel hepatitis b vaccine containing advax™, a polysaccharide adjuvant derived from delta inulin, induces robust humoral and cellular immunity with minimal reactogenicity in preclinical testing synthesis and characterization of biodegradable thiolated chitosan nanoparticles as targeted drug delivery system demonstration of a ion mechanism between a fluid bactericidal liposomal formulation and bacterial cells recent developments in liposome-based veterinary therapeutics recent trends in nanobiosensors and their applicationsa review nanotechnology: the future medicine effects of dietary supplements of zinc-methionine on milk production, udder health and zinc metabolism in dairy goats dendrimers: a class of polymers in the nanotechnology for the delivery of active pharmaceuticals biological interactions of graphenefamily nanomaterial: an interdisciplinary review current progress in oocyte and embryo cryopreservation by slow freezing and vitrification risk assessment of engineered nanomaterials and nanotechnologies-a review quantitative evaluation of antibacterial activities of metallic oxide powders (zno, mgo and cao) by conduct metric assay influence of hydrocolloidal silver nanoparticles on gastrointestinal microflora and morphology of enterocytes of quails superparamagnetic iron oxide nanoparticles as a means to track mesenchymal stem cells in a large animal model of tendon injury a pilot toxicology study of single-walled carbon nanotubes in a small sample of mice nanotechnology and animal health treatment with individualized homeopathic remedies unsuccessful enhancement of intestinal insulin absorption by bile salt-fatty acid mixed micelles in dogs drug-drug co-crystals dendrimers in biomedical sciences and nanotechnology gold nanoparticles: various methods of synthesis and antibacterial applications dna damaging potential of zinc oxide nanoparticles in human epidermal cells perturbational profiling of nanomaterial biologic activity evaluation of the antimicrobial effect of zinc oxide nanoparticles on listeria monocytogenes and candida albicans isolated from infected egyptian buffalo suffering from abortion iron oxide nanoparticles suppressed t helper cell-mediated immunity in a murine model of delayed-type hypersensitivity biocompatibility of chitosan-coated iron oxide nanoparticles with osteoblast cells silica nanocrystals: a controlled organic-inorganic interface and its implications of color-tuning and chemical design toward sophisticated architectures structure, synthetic methods, magnetic properties and biomedical applications of ferrofluids unusual inflammatory and fibrogenic pulmonary responses to single-walled carbon nanotubes in mice synthesis of a fullerene derivative for the inhibition of hiv enzymes food packaging based on polymer nanomaterials encapsulation of bacteriophage in liposome accentuates its entry in to macrophage and shields it from neutralizing antibodies effect of different levels of nanoparticles chromium picolinate supplementation on performance, egg quality, mineral retention, and tissues minerals accumulation in layer chickens treatment with hpma copolymer-based doxorubicin conjugate containing human immunoglobulin induces long-lasting systemic anti-tumour immunity in mice effect of surface functionalization of mcm- -type mesoporous silica nanoparticles on the endocytosis by human cancer cells mesoporous silica nanoparticles as controlled release drug delivery and gene transfection carriers mesoporous silica nanoparticles: structural design and applications enhanced antibacterial effect of antibiotics in combination with silver nanoparticles against animal pathogens intracellular nanoparticle coating stability determines nanoparticle diagnostics efficacy and cell functionality surface charge-mediated rapid hepatobiliary excretion of mesoporous silica nanoparticles chitosan nanoparticles properties and applications pure and multi metal oxide nanoparticles: synthesis, antibacterial and cytotoxic properties synthesis of pyrrolizidine alkaloids via , -dipolar cycloaddition involving cyclic nitrones and unsaturated lactones metal oxide nanoparticles as bactericidal agents application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review magnetic nanoparticles in mr imaging and drug delivery nanographene oxide for cellular imaging and drug delivery novel antibody/gold nanoparticle/magnetic nanoparticle nanocomposites for immunomagnetic separation and rapid colorimetric detection of staphylococcus aureus in milk in vitro and in vivo antifungal activity of amphotericin b lipid complex: are phospholipases important? gold nanoparticle based microbial detection and identification nanobioimaging and sensing of infectious diseases the preparation of magnetic nanoparticles for applications in biomedicine outbreak of foetal infection with bovine pestivirus in a central queensland beef herd mesoporous silica nanospheres as highly efficient mri contrast agents reducing infections through nanotechnology and nanoparticles hybrid nanomaterials for biomedical applications carbon nanotubes in cancer theragnosis livestock production: recent trends, future prospects skin as a route of exposure and sensitisation in chronic beryllium disease science and application of nanotubes lipid-core micelles for targeted drug delivery mesoporous silica nanoparticles deliver dna and chemicals into plants research strategies for safety evaluation of nanomaterials, part iv: risk assessment of nanoparticles dna sequence motifs for structure specific recognition and separation of carbon nanotubes facile and sensitive epifluorescent silica nanoparticles for the rapid screening of ehec penicillin-bound polyacrylate nanoparticles: restoring the activity of β-lactam antibiotics against mrsa a randomized trial investigating the efficacy and safety of water-soluble micellar paclitaxel (paccal vet) for treatment of nonresectable grade or mast cell tumors in dogs a new property of mcm- : drug delivery system adjuvant effect of gantrez ® an nanoparticles during oral vaccination of piglets against f + enterotoxigenic escherichia coli improved biocompatibility and pharmacokinetics of silica nanoparticles by means of a lipid coating: a multimodality investigation macromolecules, dendrimers, and nanomaterials in magnetic resonance imaging: the interplay between size, function, and pharmacokinetics nanoparticles applications for improving the food safety and food processing photoinduced intracellular controlled release drug delivery in human cells by gold-capped mesoporous silica nanosphere tuning the cellular uptake and cytotoxicity properties of oligonucleotide intercalator-functionalized mesoporous silica nanoparticles with human cervical cancer cells hela mesoporous silica nanoparticles for intracellular controlled drug delivery mesoporous silica nanoparticles: synthesis and applications development of immunochromatographic strip test using fluorescent, micellar silica nanosensors for rapid detection of b. abortus antibodies in milk samples effect of chromium nanoparticle on growth performance, carcass characteristics, pork quality and tissue chromium in finishing pigs a biocompatible method of decorporation: bisphosphonate-modified magnetite nanoparticles to remove uranyl ions from blood acute toxicological impact of nano-and submicro-scaled zinc oxide powder on healthy adult mice bioconjugated silica nanoparticles: development and applications unique aggregation of anthrax (bacillus anthracis) spores by sugar coated single walled carbon nanotubes fluorescent dye encapsulated zno particles with cell-specific toxicity for potential use in biomedical applications recent advances of chitosan nanoparticles as drug carriers sensitive competitive direct enzyme-linked immunosorbent assay and gold nanoparticle immunochromatographic strip for detecting aflatoxin m in milk effects of chromiumloaded chitosan nanoparticles on growth, blood metabolites, immune traits and tissue chromium in finishing pigs development of a gold nanoparticle-based oligonucleotide microarray for simultaneous detection of seven swine viruses effects of chromiumloaded chitosan nanoparticles on growth, carcass characteristics, pork quality, and lipid metabolism in finishing pigs host-guest carbon dots as high-performance fluorescence probes anti-microbial porcelain enamels nanocrystalline silver dressing and subatmospheric pressure therapy following neoadjuvant radiation therapy and surgical excision of a feline injection site sarcoma biocompatible carbon nanotubes generated by functionalization with glycodendrimers sentinel lymph node detection using carbon nanoparticles in patients with early breast cancer bioactive cell-like hybrids coassembled from (glyco) dendrimersomes with bacterial membranes drug-loaded carbon nanoparticle suspension injection, drug selection, releasing behavior, in vitro cytotoxicity and in vivo lymph node targeting bioaccumulation and toxicity of carbon nanoparticles suspension injection in intravenously exposed mice skeleton labeled c-carbon nanoparticles for the imaging and quantification in tumor drainage lymph nodes new forms of superparamagnetic nanoparticles for biomedical applications dopamine as a robust anchor to immobilize functional molecules on the iron oxide shell of magnetic nanoparticles nitrilotriacetic acid-modified magnetic nanoparticles as a general agent to bind histidine-tagged proteins physiochemical aspects of drug delivery and release from polymer-based colloids effects of nanometre zno on growth performance of early weaned piglets carbon dots as nontoxic and high performance fluorescence imaging agents antimicrobial activity of single walled carbon nanotubes length effect effects of gestational age and surface modification on materno-fetal transfer of nanoparticles in murine pregnancy antiviral activity of graphene oxide: how sharp edged structure and charge matter magnetic nanoparticles for cancer diagnosis and therapy formation of hollow nanocrystals through the nanoscale kirkendall effect nanoparticle-based bio-barcode assay for the detection of bluetongue virus dumbbell-like bifunctional au-fe o nanoparticles effects of silver nanoparticles on multiple drug-resistant strains of staphylococcus aureus and pseudomonas aeruginosa from mastitis-infected goats: an alternative approach for antimicrobial therapy enhanced antibacterial effect of ceftriaxone sodium-loaded chitosan nanoparticles against intracellular salmonella typhimurium tumor, tissue, and plasma pharmacokinetic studies and antitumor response studies of docetaxel in combination with -nitrocamptothecin in mice bearing skov- human ovarian xenografts efficacy of trivalent chromium on growth performance, carcass characteristics and tissue chromium in heat-stressed broiler chicks biofabrication of a novel biomolecule-assisted reduced graphene oxide: an excellent biocompatible nanomaterial investigation into the antibacterial behavior of suspensions of zno nanoparticles (zno nanofluids) degradable disulfide core-crosslinked micelles as a drug delivery system prepared from vinyl functionalized nucleosides via the raft process silver nanoparticles: synthesis, characterization, properties, applications, and therapeutic approaches bioconjugated silica nanoparticles for bioanalysis mesoporous silica nanoparticlebased double drug delivery system for glucose-responsive controlled release of insulin and cyclic amp advances in rapid detection methods for foodborne pathogens pegylated mesoporous silica as a redox-responsive drug delivery system for loading thiolcontaining drugs antibacterial activities of gold and silver nanoparticles against escherichia coli and bacillus calmette-gu erin recent developments in antibody-based assays for the detection of bacterial toxins carbon nanotube-based nanocarrier loaded with ribavirin against grass carp reovirus carbon nanoparticle-guided central lymph node dissection in clinically nodenegative patients with papillary thyroid carcinoma liposomes as immunological adjuvants cancer nanotechnology: application of nanotechnology in cancer therapy chapter : synthesis, characterization, and applications of carbon nanotubes functionalized with magnetic nanoparticles fungal nanotechnology: a new approach toward efficient biotechnology application ultrasensitive dna detection using highly fluorescent bioconjugated nanoparticles the authors of this chapter gratefully acknowledge dr. m.k. refai, professor of microbiology, cairo university, for his continuous assistance and advice in initiating the achievement of this work. acknowledgments key: cord- -s gw k authors: capps, benjamin; lederman, zohar title: one health, vaccines and ebola: the opportunities for shared benefits date: - - journal: j agric environ ethics doi: . /s - - - sha: doc_id: cord_uid: s gw k the ebola virus outbreak in west africa, as of writing, is declining in reported human cases and mortalities. the resulting devastation caused highlights how health systems, in particular in west africa, and in terms of global pandemic planning, are ill prepared to react to zoonotic pathogens. in this paper we propose one health as a strategy to prevent zoonotic outbreaks as a shared goal: that human and great ape vaccine trials could benefit both species. only recently have two phase / ebola human vaccine trials been started in west africa. this paper argues for a conceptual change in pandemic preparedness. we first discuss the ethics of one health. next, we focus on the current ebola outbreak and defines its victims. third, we present the notion of a ‘shared benefit’ approach, grounded in one health, and argue for the vaccination of wild apes in order to protect both apes and humans. we believe that a creation of such inter-species immunity is an exemplar of one health, and that it is worth pursuing as a coextensive public health approach. ebola virus has devastated parts of west africa, and has caused alarm worldwide. it is one of a number of notable zoonotic emergent infectious diseases (zeid), also including influenza, coronaviruses like middle east respiratory syndrome (mers), and the now pandemic human immunodeficiency virus (hiv). the majority of all eids are caused by zoonoses ; and most of these are pathogens of wildlife origin that become endemic in localised non-human animal and human populations (jones ) . these pathogens are emerging at an alarming rate, reflecting changes in local topologies and the global climate, triggered by human and animal causative and adaptive activities (epstein ) . ebola is endemic to central africa, and is normally dormant in still unknown reservoirs. periodically however, it infects local human populations, causing extensive mortalities but then fading out before widespread contagion (hayden ; marzi and feldmann ; macneil and rollin ) . the ongoing outbreak in west africa surpasses all previous occasions, although at time of this writing, the endemic appears to be receded (who ebola response team ) . many have been dismayed by the global efforts to curtail the epidemic, questioning international resolve to respond timely and effectively (mitman ; spencer ) . in particular, many have been critical of the systematic neglect of public health infrastructure, and have identified strengthening health systems as the long term solution to the disease (dawson ; farmer ; gates ; rid and emanuel ) . the measures used during this outbreak are focused on human communities, and includes clinical case management (that to date lacks any curative treatment), quarantine and isolation, surveillance and contact tracing, a rapid and reliable laboratory service, safe and dignified burials, and social education (dawson ; macneil and rollin ; marzi and feldmann ) . critics have much to say about the importance of infrastructure and basic supplies needed, but less has been said about the limitations of ebola containment measures. although these previously worked well within geographically isolated communities where ebola periodically emerged, they were less likely to do so in a sustained and widespread outbreak. in light of the current catastrophe, it now compels us to consider also the limitations of traditional public health measures during an epidemic of this magnitude, which although they may bring an acute situation under control eventually, are challenging to enforce, strain medical and social networks, and provide limited prevention and no cure. indeed, although these measures have brought the emergency to its current abating state, it took a great deal of time and vast efforts, many still died, and infection resurgence is a possibility. the importance of biomedical countermeasures, such as vaccines, therefore cannot be understated. in this respect, it has been resolved that failures in advanced drug development and production must be tackled (who ) , especially the political and economic barriers that hamper development and deployment in places such as west africa, and which further emphasise the neglect of certain transmissible diseases in that region (marzi and feldmann ) . the current perspectives to zoonotic risks and pandemic planning have changed little despite the warnings from the 'swine flu' pandemic of that the opportunities for expedient vaccine production and sustainable clinical access still seem someway off (gates ) . our particular concern, however, is that while the ethical debate is being dominated by global human threats, other considerations about endemic zoonoses are being overlooked. using ebola as a case study, we apply one health (oh) as an ethical framework to make the case for strategic changes. in particular, the debate about vaccines plausibly could be extended to the concurrent need in primate populations. this paper therefore proposes the possibility of shared immunity between species that are equally affected by ebola. our proposal for a novel approach to vaccination that protects both human communities and the fauna they interact with and often depend upon is speculative, as technical issues are far from resolved. however, we have two further intentions: firstly, to highlight the oh in general, prevention and then containment of highly pathogenic eids is about slowing and limiting the contagion, while often treating patients to the degree possible and who are likely to die, thus allowing the existing infrastructure to operationalize and then keep up. traditional public health methods of infectious disease control are known to work up to a point, depending on various factors such as the pathogen, victim and context. in particular, these methods rely to a large degree on the trust of the populations effected to follow non-pharmaceutical precautions under conditions of immensurable suffering and burdens, and the dedication, training and supplies made available to health care and other workers who sustain the infrastructure (such as, in the ebola case, the highly risky and stressful job of digging and filling graves). confidence in these may have become complacent (putting aside the question of political negligence), as it was only a matter of time before ebola would befall upon a highly populated city for the first time. ''ebola emerged nearly four decades ago. why are clinicians still empty-handed, with no vaccines and no cure? because ebola has historically been confined to poor african nations. the r&d incentive is virtually non-existent. a profit-driven industry does not invest in products for markets that cannot pay'' (chan ) . these failings have become, for some, symbolic of the abject failures of a global system which does not allow new possibilities for pandemic planning, such as more effective and urgent vaccine production (capps and lysaght ) . that ebola is a neglected tropical disease cannot be disputed, meaning that it has failed to attract significant interest for deployment of pharmaceutical interventions (until its full pandemic potential came to light in the current outbreak) (macneil and rollin ). so far, local responses fall back on traditional public health measures; these measures do little to benefit non-human interests, as victims or by finding mutual solutions. we propose that a different approach to pandemic prevention should invest in such technologies as vaccines, but do so using a broad ecological scope. we use primate (clade haplorhini) to identify the non-human apes (hominidae) that are susceptible to the ebola virus; our analysis will proceed to discuss the great apes (genus gorilla and pan), as more is known about the effects of the virus on them as highly sentient and endangered species. initiative as a source of alternatives to pandemic planning, so that, secondly, in the spirit of oh collaboration, we can encourage further and broaden the ethical debate. the paper unpacks in the following way: first, we explain the ethics of oh as an approach that recognises an ecological perspective. second, we define and expand upon the victims of the ebola epidemic so as to consider a new oh-grounded agenda. third, we articulate a possible preventive measure to prevent ebola in both human and animal populations. we argue that, along with efforts to test ebola vaccines in humans, existing vaccines that have been proven safe and efficacious in primates should already be deployed in order to protect both species. our proposal supports the conjecture that focusing on broadly ecological factors, and understanding and reacting to the natural ecology of zoonoses, is central to future zeid planning . one health (oh) has come to signify the interdisciplinary effort to optimize the health of humans, non-human animals, and their ecosystems. as an approach to biomedical enquiry, it has been adopted as a broad heuristic for evidence-based policy involving the usual suspects from public health, as well as veterinarians, animal and plant biologists, ecologists, and environmental scientists (scoones ; leach and scoones ) ; and thereby, it has become a stimulus for collaborative research. thus, its trans-disciplinarily-across multiple disciplines, encouraging de-siloing of sectors, and engagement with partisan stakeholders-creates change by identifying and solving real-world ecological problems. it is thus an extensive ecological perspective to that of public health. however, there are those who have been critical of the oh agenda because, like some existing study or practice lenses, it excludes the humanities and social sciences (lapinski et al. ) , and that, in part, obstructs the development of an inclusive bioethics framework (thompson and list ) . while the first is largely an empirical point, and we can point to anthropologists, among others, expressing solutions, but perhaps being less heard, in respect to ebola (aaa ); the latter observation indicates oh's lack of a philosophical grounding. in fact, oh has no origins in any particular ethical theory. one explanation for this is that normative enquiries are outside of the purview of oh. the collaborative model, therefore, is not about a distinctive oh ethics per se, but an attempt to integrate ecological perspectives on the same terms as public health activism; to probe conventional wisdom to find innovative solutions. this is perhaps a practical consideration because oh otherwise would likely lose political traction under anything more concretely conceptual. the oh goal is to assemble a comprehensive set of data across a broad spectrum of expertise, and to thereby provide solutions that are of benefit to human wellbeing within ecological settings. most recently, this idea is being framed as effectiveness gains through dynamic cooperation in environmental contexts, and has the effect of raising environmental concerns on par with concurrent efforts in public health such as in disease surveillance and animal management. this might be enough to create a vision of oh ethics: van rensselaer potter, in his earliest definition of bioethics, talked about a system of human survival that included environmental, or ecological ethics (potter ). this could easily capture the idea of oh as broadening public health into diverse fields. potter, a pioneer in challenging parochial and non-secular ideas shaping the human condition, noted a schism between the medical-science domains and humanistic ethics, and that both were distanced from environmental ethics. the ethics of oh, therefore, may just be signalling the resurgence of bioethics as a unified endeavour (thompson and list ) , allowing for reflective and critical engagement with current pandemic measures, which up to now gave little credence to solutions outside the scope of public health ethics. a deeper appreciation of secular bioethics, however, also points to the intrinsic interests beyond those of human beings. in the developing oh literature, it is more commonly acknowledged that human beings are part of and dependent upon the biosphere. one way oh has developed is in a perspective that a 'healthy' environment entails healthy animals along with healthy people. it is not 'us versus them', then, but a problem of shared risk that is something concrete to act on, thus providing opportunities to maintain healthy or rescue unhealthy ecosystems (rabinowitz et al. ) . however, in practice, reactions to these risks, and solutions to pathogens, still prioritise human interests, because there is no fundamental sense in which non-human animals, or the environment, matter morally. sure, while oh in this sense creates the grounds for humans to express compassion towards animals and ecosystems and to engage in novel approaches to health problems, overall it often achieves the same goals of prevention and response so far already installed in public health; so oh, in this sense, adds nothing to the ethical debate except by broadening the factors considered in any human cost-benefit analysis. the difference oh makes is in engaging with alternatives: it questions public health ideas entrenched as the only way to solve such problems, and indicates the dangers of the unreflective or blinkered view (leach and scoones ) . its effectiveness in ethical discourse, much like the collaborative idea, is that it asks questions about ecological benefits without overstepping public health priorities. finally, there is the sense in which oh has an enabling effect in respect to grounding an ethical theory in environmental issues. what that theory is, however, is contested. in this paper, therefore, we will sketch the idea that oh ethics ought to contain two elements: ( ) a focus on the inclusive and shared determinants of health; and ( ) a unifying theory. by spelling out these elements better, one is able to assess those projects that profess to be oh; and this will be essential in judging our shared immunity proposal. health is often understood as being normative: implying something good or desirable. this might be applicable from an internal view (being healthy), or an external one, such as the view from public health that concerns community (that is, conditions for being healthy). an 'unhealthy' state can be explained by a pathogen or other kind of destabilising event that impacts or creeps into a biological system, resulting in an altered, often unwanted and endured state. this might be the presence of a virus in an individual, or even the conditions (opportunities and barriers) of healthy living. public health often takes a similar focus, aiming to create healthy circumstances and conditions for people by focussing on the determinants of health. in this respect, oh uses health as an inclusive determinant, such that it includes actions that are broad in orientation and scope, so that health activism ought not be limited to human agents. oh is therefore an investigation of the scientific, social, economic and ecological determinants of human, non-human and ecosystem health, but also a 'shared benefit' approach. our use of 'shared' points to ethical consistency; that actions that affect a broad spectrum of agents should be fairly applied. just as racism is paradoxical in human societies, some exclusionary actions between human beings and non-human animals might be similarly judged as speciesistic. this echoes ideas of equality, and the interests of minorities or the vulnerable being protected against parochial or vested interests. it also befits an examination of incongruity, need and fairness, and justice-these components of comprehensive doctrines are only knowable through ethical study, and in this respect, we are less confident in setting the oh agenda, for such a task requires far greater elucidation than is possible here. we can, however, offer a basic account of 'benefits' that will begin the conversation in earnest about oh as a unifying theory. human beings act in ways that affect non-human animals and the environment, and this raises the question as to how much we should either change such actions, or, indeed, make efforts to assist in the wellbeing of other species. the basic assumption in public health has been that we should interfere only to the extent that their collective welfare is at stake, because animals' interests are outweighed by human interests . thus, public heath applies welfare conditions for the health of animals, which only occasionally includes ethical considerations, such as the humane culling of disease vectors and hosts. however, without engaging in a lengthy debate about non-human moral status, there is also a condition of interspecies connectedness. in the case of preventing zoonotic pathogens, oh on this reading implores us to study the causes and roots of transmission, counting each being as an equal unit in this biological process. the wider study of biospheres, ecosystems, and social networks achieves this. what is ethically important is that this study is concerned with the health of the ecosystem in its entirety, not solely that of humans. oh, therefore, becomes a study of 'natural' environments, enriching public health with animal and ecological studies, and creates a whole new frame of evidence to better design effective responses. in turn, the emphasis turns to discovering and developing creative ways to recover and maintain healthy ecosystems. these hint at plausible strategies that draw on the humanities and social sciences, which can better comprehend the emergent contingencies beyond statistical confines (neyland ) . but what are the objects, goods, or benefits(and harms) that enable states of heath? one way we might extend ethical concerns to non-human interests is by securing universal goods (capps and lederman ) . these are the kinds of goods that reach beyond the needs of human communities, describing benefits as inclusive across species, and feature broadly in ecosystems and the environment. for example, ecosystems are necessary for life by providing the basic requirements (and even complex determinants of heath, in terms of social and cultural goods), and can therefore create 'unhealthy' lives by becoming unproductive and even toxic. these effects can be observed in stressed and challenged environments when they are misused, exploited and degraded. the ecosystem is, therefore, a foundation of universal goods-goods necessary for the health of multiple species, and these goods are likely shared through interspecies connectedness. primarily, then, universal goods extend terms of reference beyond the restrictions of public health purposes. one set of solutions would emanate from comparative medicine originating in human beings (this is the opposite of current comparative medicine studies where animal models are utilised for human health). human trials and treatments may well be useable in animal populations, benefiting them directly, and in some cases, where a pathogen is eliminated, it might reduce risks for human populations. a second possibility would be adapting biobanks, which, because of the terms of reference in providing public goods, are restricted to furthering human interests only (capps ). this does not make good scientific sense, because there is a welter of data being lost or overlooked simply because of intentional institutional design that arbitrarily excludes other contributions. for example, animal samples may well show up zoonotic risks sooner, or enable the natural history of a pathogen to be understood. a recent proposal to create an ebola biobank would do well to consider extending its remit to include the animals that are the essential links in zeids (hayden ) . these are intriguing possibilities because they also allow real environment information gathering and sharing, and not just the artificial data, for example, from de novo animal experiments (capps and lederman ) . there are, however, going to be more or less hard cases where conflict between public health goals and securing universal goods is more or less likely; and solutions are going to be less amicable between human interests and an ecological perspective. at this level of disagreement, a debate about animal or environmental interests or rights is to be had. but in our paper, we develop this idea of universal goods to give weight to the broadly inclusive and shared determinants that are affecting both humans and animals as victims of ebola. according to oh, it behooves us to consider the opportunities to improve the health of those directly affected by the virus, in the sense that operationalizing public health should be extended to other primates such as chimpanzees and gorillas; related not only in their level of evolutionary sentience, but also as victims of ebola. the current ebola outbreak, which started in a single index case in december , but was not reported as an outbreak until march , is the largest known in history (rio et al. ; yakubu et al. ) . both humans and great apes have been affected. at time of writing, there have been , reported confirmed, probable, and suspected cases in human infections, mainly in guinea, nigeria, liberia, and sierra leone. , confirmed patients have died. in great apes, the effect of ebola is likewise devastating. gorillas and chimpanzees are susceptible to the virus (bermejo et al. ; kaiser ) . ebola has killed roughly one third of the western lowland gorilla population in the past years, which, along with habitat loss and poaching, led the world conservation union to declare it a critically endangered species (walsh et al. ) . three interrelated enquiries interest us as advocates of oh. first, a significant question is 'why now?' (bausch and schwarz ; farmer ) . why only now has the ebola virus, which has previously emerged in isolated regions, become a regional endemic (olival and hayman ; olson et al. )? this question has been asked in the context of other zoonotic diseases, most prominently hiv and its analogous emergence from primates in africa. answers will likely become evident as our understanding of zoonoses encompasses the exponential amount of accumulated knowledge from across disciplines, including the study of the reservoir, host and effected animals, the ecologies they inhabit, and their natural responses to the virus. it is therefore not only a question of what humans might have done differently this time to create the tragedy, but also their ongoing interactions with the environment whence the virus came from. these insights will be significant in developing strategies for potential future ebola outbreaks, and paradigms for other zeids, including possible preventative measures. second, it has been debated as to whether medical interventions for eid should be deployed in animal species. according to one view, we should not interfere with natural systems at all; apes have lived with ebola for years without need for human intervention. yet the state of wild populations today is such that no environment is free from human effects, and therefore such groups must adapt to the 'anthropocene' (hockings et al. ) . in fact, the landscape has changed so significantly that human intervention is perhaps necessary for them to survive at all. although dissent has been voiced against interfering in 'natural systems' and the effectiveness of medical interventions relative to other conservation strategies (ryan and walsh ) , the magnitude and significance of the current ebola outbreak should at least question the premise of non-intervention. intervention, therefore, can be justified because the alternative is decimation across the biosphere, affecting human beings who rely on it, and the animals that live within it. if this can be considered as a universal good, then, we can start to envision medical strategies to protect both human and animal populations. the plausibility of vaccinating other species during significant endemics has been voiced before, often from the conservation angle (marzi and feldmann ; ryan and walsh ) , but never received any serious consideration, as far as we are aware. two reasons for this might be postulated: limited resources are to be used to address human needs, especially at times when endemics or potential pandemics are occurring; and vaccine safety in administering to potentially critically endangered species. recently, a vaccine trial for ebola was carried out on captive chimpanzees to inform future conservation (warfield et al. ) . third, what (at least partially) grounds the need to respond to the queries posed above in respect to the shared risk of zoonoses, is the fact that human beings and primates are equally affected by the virus. therefore, if an ethics of shared benefits is persuasive, then one can start to see how conceptual change is necessary in zeid planning. for example, standard public health policies prioritise human interests, and often, these interests are perceived to collide with and outweigh the conservation of the biosphere. examples would include devastating and often ineffective culling (johansen and penrith ; jenkins et al. ) , or ravaging biodiversity on the basis of 'at-risk-to-human' calculations. oh, however, starts to give rise to different opportunities: for example, developing data storage from veterinary and conservation studies that can benefit humans, and vica versa (capps and lederman ) ; or strategizing to create healthy ecologies that will concurrently present fewer risks to human beings. concomitantly, humans, who often receive better medical care, may serve as 'concurrent research participants' and adaptive public/veterinary health models. the scientific literature to support oh as an approach to coordinate pandemics of zoonotic origin is rapidly accumulating (rabinowitz et al. ), such that it should be gaining traction in pandemic planning. it has, however, yet to feature in the solutions to ebola. oh ought to have some quite significant implications for pandemic planning (against ebola and generally) in various chronological phases. . the natural ecology of the ebola virus. the animal origin of the current epidemic is perplexing. the virus tends to only occasionally emerge in isolated villages, rarely appearing in hospitals and other health facilities (garrett ) . in this regard, the current outbreak is unique. beyond human interference, the ecology of the virus itself undoubtedly plays a key part. there are a number of species that could be implicated as the host, such as bats, other large mammals, or primates; even insects and plant viruses have been implicated in its transmission to human beings (hayden ; monath ) . it is imperative to conduct studies to locate the reservoirs and the plausible transmission routes to human beings and primates (in terms of group-to-group interspecies and cross-species transmission) and other known and unknown species contagions, to explain risks and spillover events. wildlife conservation workers have been tracking ebola in gorilla and chimpanzee populations for some time; but these data rarely reach the attention of public health planners (walsh et al. ). . manage habitat disruption. there is a vast and largely uncharacterized pool of possible zoonotic pathogens, and increasing opportunities for infection caused by disruptive human activities and ecological encounters (morse ). the understanding ecologies of vector-borne pathogens reveals some intriguing events, such as how biodiversity and diverse species networks can buffer, dilute and 'soak up' pathogens (harris and dunn ; keesing et al. ). that is, comparative studies and the reverse data use of human trials to benefit animal populations, such as in veterinary application (yeates ) . an early report from the current outbreak hypothesized that the host was a bat colony living in a local hollowed out tree (saéz et al. ). development of industry, such as mining, can bring people into regular contact with zoonotic reservoirs and hosts (kangbai and koroma ). these industries employ local and international workers who then travel to and from wild territories (allouche ) . anthropocentric activity also disrupts normal animal behaviour, for example, changing fruit bat roosting and foraging ranges so that they move to proximate sites to human dwellings (looi and chua ) . further, evidence suggests that biodiversity is a key element in emergent zoonotic diseases, where, in some cases, there is a reversed correlation: less biodiversity, or even deprived ecologies, create more risks for human zeids spillover events (cardinale et al. ; jones ) . . prevention of zoonotic infections. non-pharmaceutical measures can work well in eid outbreaks, but are only practical considerations once the spillover event has occurred in humans (in other contexts, personal protection equipment might be used as biosafety measures). reactive pharmaceutical measures, such as vaccines, take time to develop to specific pathogens, and then are often hampered by politics and investment, biological limitations, errors, and logistics. prevention, as is central to public health, might therefore be considered key. currently, several types of ebola vaccines have been proven effective and safe in primates, but none has been approved in humans yet (see below). human trials however are ongoing; and several captive chimpanzee trials have been conducted warfield et al. ) . once an ebola vaccine is approved for use in humans, several strategies to increase coverage may be used, such as ensuring that eco-tourists are appropriately vaccinated before visiting at-risk primate populations, and introducing health programmes in mining and refining communities often located in remote areas and near to potential ebola hotspots, such as bat roosts. . monitoring of disease in animals. studies have identified stereotypic behaviours in animals when burdened with zoonotic disease. for example, gorillas faced with endemic ebola reacted in ways that point to a decrease in social cohesion and lower reproductive potential: females were significantly more likely to transfer from breeding groups to non-breeding groups and males were more likely to transfer from groups to solitary-living. in general, there was a decrease in formation of breeding groups. interestingly, during the post-epidemic period, immigration of breeders between groups returned to normal while immigration of non-breeders remained low. observable social dynamics, then, may be used as indicators to detect ebola outbreaks (genton et al. ). this is an example of how animals can act as sentinels for imminent human risk. . animal-to-human transmission. several routes of animal-human transmission of ebola exist, including ingestion of raw infected meat (bats, primates and other animals) , and exposure to hosts and reservoirs through daily life, professions and tourism (köndgen et al. ). these various routes are potentially causing more zeid spillover events. for example, 'bushmeat' is consumed in higher amounts due to population growth in some areas (wolfe et al. ) ; mining is a growing industry in many regions (see below); and local economies rely on the growth of ecotourism. presenting these as local and global issues is challenging: for example, the local population is unlikely to support the prohibition of eating specific species as part of infection control. moreover, to have ethical credence, it would be consistent to address concurrent risks in developed countries, such as reducing intensive farming that also drives zeids. as regional industrial growth is essential for creating sustainable development for all countries, a call to reduce anthropocentric activity in rich wildlife areas in order to meet expedient conservation efforts would likely be rejected because of the local economic losses (and the international desire to visit such areas). nonetheless, efforts should be aimed at education of locals and visitors about the modes of transmission of ebola (muyembe-tamfum et al. ). learning about the local ecology-animal behaviour, biology, anthropology-would point to innovative ways to adapt in order to reduce the risk of transmission. the accumulated knowledge, therefore, raises some intriguing possibilities for the study and feasibility of potential zoonotic control measures; and in particular, using and adapting the 'shared' biosphere as part of the solutions to endemic ebola. however, despite the obvious ecological links to human zeid outbreaks, the interest in devising such possibilities has, until now, had little traction in public health and extant pandemic planning. most pandemic plans mention little about the ecosystem beyond its risk potential and stipulate requirements to devastate the animal populations (culling and the like) as a means to limit future human-to-human transmission. the one health solution to endemic ebola: inter-species immunity vaccination is by far one of the most significant responses to eid in human beings, and in the context of ebola its importance is beyond doubt; since the outbreak was first detected, public health, clinical staff and allied workers struggled against quite immense odds to bring it to the current state. the case for human vaccination speaks for itself. however it should not be understated quite how important it is since the alternative is to fall back on the objective: ''not to dramatically increase the person's chance of survival, it's to contain the spread'' (fjeldsaeter ) . one can only imagine what advantages the early deployment of an effective vaccine would have been. putting aside questions of the economic inequality that provides little incentive for vaccines until worst case scenarios prevail (capps and lysaght ; dawson ; farmer ) , the current upscaling in research to find a vaccine for ebola illustrates the standard phased approach to innovation: invention, animal experiments, trials in humans before large scale production and delivery. as with human beings, the obvious advantage to animals affected by the disease, such as great apes, is immunity from the disease (and relationally, although not always the case, such as with endangered species, is exclusion from culling measures), and prevention of cross infection (walsh et al. ). the obvious benefit is also in terms of conservation. specifically the case of highly endangered gorillas (and other susceptible animals on wwf lists) is extremely significant (ryan and walsh ) . no ebola vaccine has yet to be approved for therapeutic use in human beings. however, ebola vaccine development has been an active field of research for several laboratories worldwide, and candidate vaccines were found some time ago to be safe and efficacious in mice (blaney et al. ) . several human-targeted vaccines have been proven safe and efficacious in trials in primates, including adenovirus type and , human parainfluenza virus type , and vesicular stomatitis virus geisbert and feldmann ; marzi and feldmann ; stanley et al. ). these prospective vaccines, however, raise different concerns, such as safety, price, effectiveness, delivery and side effects. in an attempt to aver safety in the use of replicating viruses (see below), warfield et al. ( ) tested the protective effects of a virus-like particle in captive chimpanzees using adenovirus as a vector. first, they demonstrated that the vaccine was safe for chimpanzees. second, they documented the development of a robust immune response in chimpanzees, evidenced by a detection of virus-specific glycoprotein and vp antibodies - weeks post-vaccination. third, they demonstrated that total igg fractions taken from the chimpanzees that were vaccinated had a protective effect in mice challenged with murine ebola: - % of mice in the study groups survived compared to none of mice in the control groups. similarly, blaney et al. ( ) developed a live-attenuated and inactivated rabies virus vaccine that expresses the ebola glycoprotein. the vaccine had no adverse effects in primate models, it induced humoral response to both rabies and ebola, and was shown to be protective against both viruses. while this is obviously an early-phase study, it has great potential in terms of resources and feasibility in conferring immunity in mammals against two lethal pathogens. so far, two vaccines have passed phase clinical testing: chimpanzee adenovirus -vectored vaccine encoding ebola surface glycoprotein (chad ) (rampling et al. ) , and vesicular stomatitis virus (vsv)-vectored vaccine also encoding for the outer protein of the zaire ebola strain (agnandji et al. ) . the prevail study is an ongoing phase / trial taking place in liberia that examines the safety and effectiveness of these two vaccines. concomitantly, the strive trial is taking place in sierra leone, where healthy volunteers will be given the vsv vaccine in order to test its safety and effectiveness. agricultural policy tends to follow vaccinating all of the exposed animals so that those not already infected will develop sufficient immunity. however, when time and resources permit, it is normal for all exposed animals to be slaughtered (kahn et al. ) . vp is a matrix protein that together with glycoprotein constitute the virus-like particle vaccine. occasionally, nucleoprotein is also present. vp is essential for cell expression of viral antigens to which the body responds by creating antibodies (escudero-perez et al. ; marzi and feldmann ) . see: http://www.niaid.nih.gov/news/qa/pages/ebolavaxresultsqa.aspx; accessed june . http://www.cdc.gov/media/releases/ /p -ebola-vaccine.html; accessed / ; accessed june . the vsv phase trial in geneva was halted due to safety concerns when several healthy participants developed different adverse effects such as arthritis. the trial was continued a month later, to understand contagion networks and possibilities for control, first we need to see the connections between vectors and victims, and by understanding these within shared ecologies we might be able to better safeguard communities-both animal and human. as these authors postulated: ''in addition to the protection of threatened nhps [nonhuman primates], vaccination of nhp populations in endemic areas might also offer an additional, critical benefit to humans. the interaction of humans and infected nhps has been associated with transmission of ebov to humans and initiation of subsequent outbreaks, so prevention of disease in nhps may also serve to limit ebov transmission into the human population'' (blaney et al. ) . concurrently with the race to develop and test vaccines on human beings, we argue that already now, we can and should (upon assuring the degree of safety) deploy vaccines in captive and wild primates with the aim of benefiting both primates and humans. the current strategies, we submit, are driven by a too narrow vision: we propose that oh espouses a 'shared benefit' approach that is complementary to the 'shared risk' approach (rabinowitz et al. ) . a 'shared benefit' approach seeks to actively maximize health in one species while in turn benefiting another species as well. specifically, we refer here to research and interventions in humans that benefit animals and vice versa. our proposal is to implement the notion of inter-species immunity. one of the identified risks for ebola, while not knowing for sure the reservoirs of the virus, is close proximity between human and primate populations (towner et al. ). our proposal is for direct action to administer vaccinations to humans through public health and research paradigms, and additionally to animals to stave off future outbreaks in both populations. such an approach, aimed at vaccinating animals in the first instance, would be preventative rather than reactive to an outbreak in human populations, by protecting across species and thereby creating a potential barrier to future occurrences of ebola in the fauna. our proposal is to co-develop vaccines for human and primate use in ebola endemic and at-risk sites in africa; and simultaneously, to deploy such vaccines to these sites in animal and (in due course) human populations. the delay in getting vaccines to the people in africa is in part due to the need to conduct proper clinical trials first and the troubling consequences of creating randomization (donovan ; shaw ) . however, captive primate populations could be enrolled in trials as benefiting vaccine development at a lower safety level (in comparison to the footnote continued upon approval by the review committee. the ongoing phase / vsv trials were modified according to the results in that study (agnandji et al. ) . one health, vaccines and ebola: the opportunities for… standard profiles for first in human trials, and additionally avoiding the later phased stages of human clinical trials). primates might be research subjects who can contribute to a longer-term ecologycentred strategy to vaccinate wild animal populations urgently. simply put, researchers are already injecting captive primate populations, and, if proven safe and efficacious in these trials (i.e. would not to our knowledge wipe out remaining primate populations), this approach provides a fast track to wild primate populations. an oh approach would potentially justify animal research on captive primates within parameters of participation of 'vulnerable' populations (i.e. the agents likely to be the first cases or most at risk in future outbreaks because of their situation and circumstances). the next stage would be vaccinating the same species in the wild for the protection both of the same species (primates) and other at-risk species (human beings). this is, firstly, an ethical enquiry involving the status of primates as sentient beings who possess moral value (fenton ) ; and secondly, a conceptualization of animals as vulnerable populations such that risky clinical trials, with conditions, can be ethical. in answering the first enquiry, we note that ebola and the recent chimpanzee trials happen at a time when the national institutes of health is planning to reduce significantly the use of chimpanzees in invasive research, and therefore raises the case of whether minimally invasive research on still captive or retired chimpanzees is ethical at all. we might see experimentation, however, as a parallel development to research and treatment in vulnerable human beings, such as children and other people who cannot consent (wendler ) . the idea here is that trials might benefit wild populations and therefore it might be possible to justify within human research ethics paradigms. in human clinical research, the acceptability of such study is a function of acceptable risk, and, when vulnerability is in question, so are the chances of direct benefit (the 'best interests' test) and the possibility of appreciating benefits for others of one's own kind (children suffering from the same condition, for example). in this sense, developing protocols with primates in captivity might be justified, including using those that have 'retired', to meet the conditions of expediency; but concurrently we must anticipate that there is a direct benefit-or a best interest in play. while potential 'secondary ecological risks' exist, such as accidental extinction of the animal species, there would be some important caveats scholars concerned with animal ethics will blame us here for putting the animals at increased risk compared to humans. however, given a vaccine that has been proven safe in the lab, and the significant risk ebola poses for apes, we believe that the risks posed by the vaccine are proportional to the benefit that might be accrued to the animals themselves. http://www.nih.gov/news/health/jun /od- .htm; news release; wednesday, june , ; accessed june . this perspective is different from the predominant study of exogenous factors such as habitat disturbance and climate change as drivers of ebola emergence, and links directly to the contribution of transmission between gorilla or chimpanzee social groups in the wild. if this equivalency were to remain within an oh approach: that the vaccine is safe enough to use in human phase trials concurrently (shared risk) and that wild apes would receive the treatment as part of the same strategy (shared benefit). if this shared benefit paradigm of securing universal goods is legitimate, then it goes some way in justifying our strategy as mutually benefiting from a single intervention. this raises feasibility problems, but some intriguing ecological repercussions warrant serious consideration of an oh vaccine approach. . one would have to possess extensive knowledge about the reservoirs, vectors and hosts, and hierarchical zoonotic bridges between species, to understand the impacts of vaccines in terms of safety, stability, and effectiveness. this will involve knowledge of human, human-animal, and animal-animal interactions (i.e. comprehensive studies of fauna and flora), and their linked activities within the biosphere. at present, pandemic planning is focussed on public health, and to a degree, anthropocentric studies of how we contract and spread the pathogens amongst our own kind. this focus, for instance, locates some major challenges of vaccine use, specifically high levels of distrust and ambivalence towards medical interventions in some african populations that would impede wide human community vaccination programmes (mark ; macneil and rollin ; mitman ) . one might therefore face resistance in deploying an effective vaccination programme. oh, therefore, helps planners look to other solutions that may complement communitybased interventions. firstly, vaccinating domestic animal populations, both companion animals and those in husbandry, could avoid collateral loss to families and livelihoods. these losses are substantial and as targets for public health intervention might gain widespread support. secondly, and which we focus on here, is developing a novel approach to research and deployment in the field as a protective measure that demands immediate attention. thus, following the approach we outlined above, the vaccine will increase the welfare of humans and wild apes, both as protection (eventually) and in conducting knowledge based trials. to address the expediency argument, we again note that both the chad vaccine and the vsv vaccine have proven to be safe and efficacious in non-human primates (stanley et al. ; geisbert and feldmann ) . the human trials will take time to conclude. clearly, with the primate trials already concluded, there is an while the context of animal vaccinations has been debated considerably in respect to farming practices (and risks to humans as pathogenic risks, food safety and economics), there has been little coverage of the benefits to the animals. the debate is now further sparked by the quarantine and killing of companion animals exposed to potential contagions by their owners, such as the spanish dog killed for the fear of ebola transmission (associated press ). we note that the ongoing debate is deliberate in its assessment to get vaccines into the field as soon as possible to protect health care workers and needed staffing (i.e. burial teams and cleaners). this is a separate, urgent debate which does not entirely equate to the stage wise proposal we make here. however, the design to get vaccines first to primates as a joint shared immunity strategy could expedite human benefits and use, with a focus on employing biologists, veterinarians and the like to target the animal populations. opportunity to deploy these vaccines right away to wild apes. in the short term, we might be seeing every primate that lives as a benefit of vaccine deployment. the long-term benefits are immunity, possibly extending across species and thus limiting the future scope for spillover events. furthermore, it will provide in situ data to be gathered from the wild populations. . achieving broad coverage to widely dispersed animals would be costly and logistically challenging but has been achieved in other settings using low interventional methods such as baiting in the case of rabies (morters et al. ) . one challenge is the difficulty in reaching entire ape populations. the dense tropical forests and the animals' nomadic tendencies would make effective immunization difficult. however, by use of local and interdisciplinary knowledge and expertise, and various vaccination methods such as hypodermic darts and synthetic baits, this obstacle may be overcome (ryan and walsh ) . one long-term strategy may be to create buffer zones around villages by vaccinating domestic and wild animals, that might be enough to minimise risks of future outbreaks, following the alreadyexisting use of designated zones in farmed animal populations elsewhere (kahn et al. ) . the approach would require an increased evidence base, of course, but effectiveness could be achieved by focusing on 'hot spots', localized risk maps (jones ) , and using targeted empirical data, such as weather patterns that are known to influence zoonotic spillover events (bausch and schwarz ) . ring vaccination is another strategy, where vaccines are delivered to animals found in the ryan and walsh ( ) counted different pathogens that are harmful for apes, of which have licensed vaccines. they claim that the major obstacle in dispensing these vaccines is the delivery to the animals (ryan and walsh ) . the authors point out that ''the high seroprevalence among children indicates the same source(s) of exposure [to eobla] as in adults, either inside or near villages''. moreover, because great ape infection is often lethal, and direct contact with humans is rare, some other animal, perhaps bat roosts near settlements, represent the most likely common animal source of exposure: ''these animals, previously identified as a potential reservoir, are abundant in the forest ecosystem and consume fruits on trees located in or around villages'' (nkoghe et al. ) . 'hotspot' maps highlight regions: ( ) where the risk of disease transfer between wild primates and from wild primates to humans is greatest; ( ) where there are cross-species transmission events between wild primates due to a high diversity of closely related primate species; and ( ) where it is most likely that human beings will come into frequent contact with their wild primate relatives. ''these areas also are likely to sustain a novel epidemic due to their rapidly growing human populations, close proximity to apes, and population centers with high density and contact rates among individuals'' (pedersen and davies ). this would have to be an ideal, managed area, additionally creating the rural populations in control of the solutions. there are two elements to achieving this: ( ) engagement, cf. ''far greater community engagement is the cornerstone of a more effective response. where communities take charge, especially in rural areas, and put in place their own solutions and protective measures, ebola transmission has slowed considerably'' (who ); and knowledgeable land use, cf. protecting ''threatened habitats by reminding nearby communities of all the benefits they derive from keeping these habitats intact. forests, meadows and marshes prevent floods, supply clean water, provide habitat for species that pollinate crops, put oxygen into the atmosphere and take carbon out, and otherwise make themselves useful. in some cases, conservation groups or other interested parties actually put down cash for these ecosystem services-paying countries, for instance, to maintain forests as a form of carbon sequestration'' (conniff ). proximity of a known outbreak. further, vaccination campaigns in animals are likely to be cheaper and possibly more temporally feasible than in humans (ryan and walsh ; macneil and rollin ) . . technical issues, including the use of live attenuated viruses as vectors. for example, live attenuated vaccines are more effective than killed vaccines in conferring long-term immunity, thus necessitating fewer vaccine shots and lower rates of compliance and coverage. moreover, using viruses that are replication-competent as vaccine vectors will increase the chances for herdimmunity and therefore the potential for inter-species immunity. however, one of the risks in using a live attenuated, replication-competent vaccine in wildlife is the activation of the attenuated virus and spread to other species, including humans. beyond using killed viruses or viral particles, one solution may be using as vector a species-specific virus. for example, a recombinant murine cytomegalovirus (cmv) that was genetically engineered to express ebola particles was found to be protective in mice. since cmv is highly species-specific, a cmv-based ebola vaccine will potentially spread rapidly in a wildlife population, such as gorillas, without any cross infection to other species (marzi and feldmann ) . the use of replication-competent vectors raises another problem: pre-existing immunity to the virus that is used as the vector will hinder spread of the ebola particles, thereby preventing immunity to be acquired. this challenge could be addressed by the development of vectors to which there is no pre-existing immunity among the specific population. for example, newcastle disease virus, to which there is no detectable pre-existing immunity in humans, was developed as a potential vector of ebola particles with some (limited) positive results. vsv was also used as a vector with little if any pre-existing immunity in humans, with even greater success (marzi and feldmann ; stanley et al. ). the existing challenge with vaccine development was captured by the ghana academy of arts and sciences technical committee. they enumerated that development of vaccine is notoriously tricky given pathogen diseases' drift and other factors that impact on their individual effectiveness with different strains; the possibilities of emergent side-effects and other unforeseen incidents; the distrust of trials originating from certain foreign organisations; and how all of this will affect uptake (both in terms of willingness and immunisation) in the target population. within a 'shared benefit' approach, however, one originating in a one health perspective, we coin the term inter-species immunity to conceptually re-think the notion of immunity within a community; specifically, to extend the goods of health, such as immunity strived for in human populations, to other species and vice versa. we suggest that ebola incidence may be prevented or reduced in one species population by inducing immunity against that pathogen in another species population. so far, the best example of the success of such an approach can be seen in the response to the hendra virus, where vaccination of horses prevented disease in both horses and humans (middleton et al. ). the key to success of inter-species immunity might be with other measures that look to adapt and benefit other ecologies, such as preparing protected ecological zones (removing food and perching areas for bats) based on planned and managed farming areas, and identifying timely and imminent risks to initiate human and animal vaccination in and around these zones. one could adopt already-used surveillance programmes in at-risks regions (these, as we noted earlier, should already be modified to include 'indicative' behaviour in animals of possible zoonoses infection): ''previous serosurveys, together with the geographic pattern of outbreaks, have highlighted the potential role of the ecosystem, and an increased risk among forest populations has previously been described. our study confirms that the forest, particularly the deep forest, is the environment most at risk. this is the area harboring animals susceptible to the virus, such as great apes and bats, the latter representing a viral reservoir'' (nkoghe et al. ). we could not say whether the remoteness and distances between villages could create conditions for regional immunity by lowering the chances that an affected host might infiltrate the buffered populations from long distances away. however, as mentioned, oh is about interdisciplinary collaboration, and solutions to extreme situations such as the current ebola outbreak require such an approach more than ever (middleton et al. ) . understanding the needs of the various stakeholders such as villagers and hunters, and the ecology of all the organisms involved, is without a doubt essential for the success of any viable long-term solution. at the moment, inter-species immunity is likely to involve a programme of trials in captive primate populations, early role out to wild populations (assuming they are safe), and then a concurrent programme to vaccinate human communities in at risk regions (this human challenge is already featured in the literature with respect to other pathogens). however, with the impending imh prohibition on some primate research in the united states, and paralleled restrictions in other countries, this is a window that is potentially closing. invasive great ape research rarely has scientific justification and primate research in general is falling out of favour, although it remains possible in many jurisdictions under strict conditions. invasive research on great apes-using chimpanzees in particular-is likely to be prohibited; but we suspect that monkey research will continue for some time. this might provide the necessary level to proceed to trials in human and great ape populations. so, one could also look at it through a shared vision-if human beings are willing to volunteer for phase one trials, which was highly evident in recent calls, then possibly retired chimpanzees could be coopted as well. at this stage, it will be envisioned that the vaccine is safe for human beings, so this might be an acceptable concurrent risk for primate populations. we surmise that this vaccination strategy, inspired by the ongoing ebola outbreak, might be replicated in future ones. we make the case for the vaccination of wild primates even prior to the completion of the ongoing human trials, with the conditions of optimizing their safety and welfare, and assuring mutual benefits to them and their future offspring, as well as to humans. phase trials of rvsv ebola vaccine in africa and europe-preliminary report ebola and extractive industry strengthening west african health care systems to stop ebola: anthropologists offer insights excalibur, spanish ebola patient's dog, is euthanised despite global outcry. the guardian outbreak of ebola virus disease in guinea: where ecology meets economy ebola outbreak killed gorillas inactivated or live-attenuated bivalent vaccines that confer protection against rabies and ebola viruses defining variables of access to uk biobank: the public interest and the public good introducing one health to the ethical debate about zoonotic diseases in south east asia one health and paradigms of public biobanking challenging the production of vaccines for a future influenza pandemic biodiversity loss and its impact on humanity who director-general addresses the regional committee for africa; director-general of the world health organization; address to the regional committee for africa, sixty-fourth session useless creatures ebola: what it tells us about medical ethics ebola, epidemics, and ethics-what we have learned climate change and emerging infectious diseases one health, vaccines and ebola: the opportunities for… shed gp of ebola virus triggers immune activation and increased vascular permeability the early spread and epidemic ignition of hiv- in human populations diary. london review of books ebola haemorrhagic fever. the lancet can a chimp say ''no''? reenvisioning chimpanzee dissent in harmful research ebola in sierra leone: battling sadness, fear and disgust on the frontline the coming plague: newly emerging diseases in a world out of balance the next epidemic-lessons from ebola recombinant vesicular stomatitis virus-based vaccines against ebola and marburg virus infections how ebola impacts social dynamics in gorillas: a multistate modeling approach species loss on spatial patterns and composition of zoonotic parasites the ebola question biobank planned for ebola samples apes in the anthopocene: flexibility and survival the duration of the effects of repeated widespread badger culling on cattle tuberculosis following the cessation of culling has culling been properly assessed as a valid and justified control intervention measure for zoonotic diseases? global trends in emerging infectious diseases confronting zoonosis through closer collaboration between medicine and veterinary medicine (as 'one medicine') vaccination against foot-and-mouth disease: the implications for canada changing epidemiology of west africa ebola outbreaks - impacts of biodiversity on the emergence and transmission of infectious diseases pandemic human viruses cause decline of endangered great apes recommendations for the role of social science research in one health the social and political lives of zoonotic disease models: narratives, science and policy lessons from the nipah virus outbreak ebola and marburg hemorrhagic fevers: neglected tropical diseases? fear and ignorance as ebola 'out of control' in parts of west africa. the guardian ebola virus vaccines: an overview of current approaches hendra virus vaccine, a one health approach to protecting horse, human, and environmental health. emerging infectious disease ebola in a stew of fear ecology of marburg and ebola viruses: speculations and directions for future research emerging infections: condemned to repeat? in microbial evolution and co-adaptation: a tribute to the life and scientific legacies of joshua lederberg: workshop summary. institute of medicine (us) forum on microbial threats evidence-based control of canine rabies: a critical review of population density reduction ebola virus outbreaks in africa: past and present an ethnography of numbers risk factors for zaire ebolavirus-specific igg in rural gabonese populations filoviruses in bats: current knowledge and future directions dead or alive: animal sampling during ebola hemorrhagic fever outbreaks in humans cross-species pathogen transmission and disease emergence in primates global bioethics the chimp and the river: how aids emerged from an african forest toward proof of concept of a one health approach to disease prediction and control from ''us vs. them'' to ''shared risk'': can animals help link environmental factors to human health? a monovalent chimpanzee adenovirus ebola vaccine-preliminary report ethical considerations of experimental interventions in the ebola outbreak ebola hemorrhagic fever in : the tale of an evolving epidemic consequences of non-intervention for infectious disease in african great apes investigating the zoonotic origin of the west african ebola epidemic towards a one world, one health approach randomisation is essential in ebola drug trials. the lancet having and fighting ebola-public health lessons from a clinician turned patient chimpanzee adenovirus vaccine generates acute and durable protective immunity against ebolavirus challenge ebola needs one bioethics isolation of genetically diverse marburg viruses from egyptian fruit bats catastrophic ape decline in western equatorial africa potential for ebola transmission between gorilla and chimpanzee social groups vaccinating captive chimpanzees to save wild chimpanzees should protections for research with humans who cannot consent apply to research with nonhuman primates? ebola situation in liberia: non-conventional interventions needed second who high-level meeting on ebola vaccines access and financing. summary report west african ebola epidemic after one year-slowing but not yet under control bushmeat hunting, deforestation, and prediction of zoonotic disease the ebola outbreak in western africa: ethical obligations for care animal welfare in veterinary practice acknowledgments capps conceived of the idea for this article and led its drafting. lederman was integral to developing the idea and contributed equally to the research for the paper. the authors would like to thank the following people for helpful comments: wang linfa, paul anantharajah tambyah, and sharon amit. key: cord- -srq bo v authors: fèvre, eric m.; bronsvoort, barend m. de c.; hamilton, katie a.; cleaveland, sarah title: animal movements and the spread of infectious diseases date: - - journal: trends microbiol doi: . /j.tim. . . sha: doc_id: cord_uid: srq bo v domestic and wild animal population movements are important in the spread of disease. there are many recent examples of disease spread that have occurred as a result of intentional movements of livestock or wildlife. understanding the volume of these movements and the risks associated with them is fundamental in elucidating the epidemiology of these diseases, some of which might entail zoonotic risks. the importance of the worldwide animal trade is reviewed and the role of the unregulated trade in animals is highlighted. a range of key examples are discussed in which animal movements have resulted in the introduction of pathogens to previously disease-free areas. measures based on heightened surveillance are proposed that mitigate the risks of new pathogen introductions. domestic and wild animal population movements are important in the spread of disease. there are many recent examples of disease spread that have occurred as a result of intentional movements of livestock or wildlife. understanding the volume of these movements and the risks associated with them is fundamental in elucidating the epidemiology of these diseases, some of which might entail zoonotic risks. the importance of the worldwide animal trade is reviewed and the role of the unregulated trade in animals is highlighted. a range of key examples are discussed in which animal movements have resulted in the introduction of pathogens to previously disease-free areas. measures based on heightened surveillance are proposed that mitigate the risks of new pathogen introductions. animal movements and disease transmission infectious diseases are transmitted between hosts by a variety of mechanisms, including direct, airborne and vector-borne transmission. control of animal-to-animal transmission of disease agents is a key concept in infectious disease epidemiology; however, a more sensible approach might be to prevent the types of contact that lead to transmission in the first place. in humans, it is often difficult to prevent contacts, particularly with the ease of long-distance travel [ ] . however, in livestock and animals, movements can be the subject of legislation or strict controls and there is a real opportunity to reduce disease transmission. the importance of animal movements is, of course, well understood and international regulations [e.g. from the world organisation for animal health (oie; box )] exist to mitigate the risks involved [ ] . in spite of these regulations, outbreaks occur regularly as a result of both legal and illegal animal movements. in this review, we examine several issues that relate to the movement of domestic and wild animals and discuss the risks that these movements entail -at local, regional and global scales -with regard to the spread of disease. first, we present data on livestock movements at a global level, and highlight the scale of wildlife movements as a result of translocation by humans. second, we provide key case studies of animal and animal-to-human (zoonotic) disease introductions in different parts of the world that have resulted in subsequent disease transmission and new outbreaks, with an emphasis on how introduction of the disease agents could have been prevented either through intervention or regulation. the trade in livestock, wildlife and animal products is enormous and complex, and occurs on many different scales. there are no overriding rules to control these movements and much of the trade is still based on bilateral agreements between countries. however, countries that are members of the world trade organization are bound by the sanitary and phytosanitary agreement (http://www.wto.org/english/tratop_e/sps_e/ spsagr_e.htm), which concerns food safety and animal and plant health regulations. countries are encouraged to base their sanitary and phytosanitary measures on existing international standards. the international standards relating to animal health and zoonoses were developed by the oie and are stated in the aquatic and terrestrial animal health codes (http://www.oie.int/eng/ normes/fcode/en_index.htm and http://www.oie.int/eng/ normes/mcode/a_summry.htm, respectively). the aim of the codes is to assure the sanitary safety of the international trade in terrestrial animals and their products by detailing the health measures that should be used by the veterinary authorities. the important role of a good veterinary infrastructure to minimize the risks of disease spread has been emphasized [ ] . the relevant legislation for international wildlife trade [ ] relates to three main areas: animal health, animal welfare and the international movement of endangered species. the animal health regulations relevant for livestock trade (see earlier) also apply to non-domesticated animals. however, additional regulations exist for wildlife to protect endangered species from overexploitation from trade, in the form of the convention on international trade in endangered species of wild fauna and flora (cites) and the convention on biological diversity (cbd). detailed guidelines have also been developed by the world conservation union (iucn) to minimize disease risks associated with the intentional movement of wildlife for conservation or game management purposes: for example, the supplementation of populations, reintroduction of endangered species, removal of problem animals and the release of confiscated animals [ ] ( table ) . the international trade in livestock is big business; for example, in , china exported us$ million, us$ million and us$ million worth of cattle, swine and poultry, respectively [ ] unregulated (illegal or informal) trade is, by its nature, difficult to quantify, although available data for some regions of the world show that unregulated trade is substantial. although there are no official records, it has been documented that, for example, head of cattle move from somalia to kenya annually, and that up to goats move from somalia to the middle east (which accounts for o % of all goat imports from the eastern africa region) [ ] . indeed, in the case of cattle trading with kenya, political instability resulted in a large increase in the value of unofficial trade. this coincided with a collapse in the animal health infrastructure of somalia and a resulting lack of animal export controls. diseases known to have been circulating in somalia include both zoonotic and non-zoonotic infectious diseases: anthrax, babesiosis, brucellosis, contagious bovine pleuropneumonia, contagious caprine pleuropneumonia, foot and mouth disease (fmd), heartwater, peste des petits ruminants, rabies, rift valley fever, rinderpest and trypanosomiasis, among others [ ] . unofficially traded animals are a much greater risk factor for disease spread because they are not necessarily subject to veterinary controls. it has been reported that much of the human brucellosis problem in saudi arabia, which has an incidence of cases per people nationally, is the result of unscreened and unregulated imports that come mainly from africa [ ] . the picture that emerges from a consideration of these various data sources is one of a highly interconnected world in which animals move locally, regionally and across large international distances. the global wildlife trade is also huge, with an annual turnover estimated at billions of dollars and involving hundreds of millions of individual plants and animals (http://www.traffic.org/ /wild .htm). precise estimates of its scale are difficult because much is conducted through informal or illegal networks but recent figures suggest that w live primates, four million live birds, live reptiles and million live tropical fish are traded globally each year [ ] . in asia, the substantial regional trade in wild animals is estimated to result in several billion direct and indirect contacts among wildlife, humans and domestic animals each year [ ] . despite the widespread recognition of the risks of disease transmission associated with wildlife translocation, and the legislation and regulation in place to minimize disease risks [ , [ ] [ ] [ ] , new diseases continue to emerge as a result of wildlife trade. here, we present key examples from the past five years that illustrate the continuing threat to human, livestock and wildlife health. severe acute respiratory syndrome (sars) and highly pathogenic avian influenza (e.g. h n ) are emerging infections that have the potential for pandemic spread with massive public health and economic consequences. both diseases are maintained in wildlife reservoir hosts: h n in wild fowl and sars in horseshoe bats (rhinolophus species) in china [ , ] . for sars, the trade in bats is likely to have brought infected animals into contact with susceptible amplifying hosts such as the masked palm civet (parguma larvata) at some point in the wildlife supply chain, establishing a market cycle in which susceptible people and animals could subsequently become infected [ , ] . for avian influenza, these 'wet markets' could also function as network hubs for potential cross-species transmission. however, the international trade in birds also poses considerable risks for longdistance transmission of h n , as highlighted by the detection of infected hawk eagles imported illegally from thailand to belgium [ ] . the disease risks of the wild bird trade have been brought into sharp relief by the importation of h n -infected birds destined for the uk pet market [ ] and, at the time of submission of this review, a temporary ban on the importation of wild birds had been implemented by the uk government. the recent detection of pseudamphistomum truncatum (an opisthorchid fluke parasite) in the english otter population has been linked with the introduction of two freshwater fishes: the sunbleak (leucaspius delineatus) and the topmouth gudgeon (pseudorasbora parva), which can function as intermediate hosts for the fluke. these species were newly introduced into the uk by an ornamental fish supplier in hampshire in the mid- s and their escape led to the colonization of many river systems in southern england [ ] . the translocation of amphibians is implicated in the emergence of amphibian diseases such as chytridiomycosis and ranavirus infections, which might be a major contributing factor in the widespread decline and extinction of amphibian species worldwide [ ] . chytridiomycosis, which has been associated with amphibian mortalities and population declines in central america and australia, has also been linked to the introduction of cane toads (bufo marinus) into australia [ ] . also, chytridiomycosis has appeared recently in the uk, with confirmed cases in an established breeding population of north american bullfrogs (rana catesbeiana), which is an introduced species [ ] . recent movements of r. catesbeiana and b. marinus might also have disseminated ranaviral diseases such as tadpole oedema virus [ ] . in australia, where b. marinus was introduced to queensland in , ranaviral antibodies can be identified in this species throughout its geographic range [ ] . in addition to the well-recognized threat that animal translocations and invasions into new geographic areas pose for species extinctions and biodiversity, the large wildlife trade clearly poses great dangers for the emergence of human and animal pathogens. it is doubtful whether the disease risks associated with the international wildlife trade, let alone additional welfare and conservation-related problems, can ever be justified simply to supply a demand for pets or recreational hunting. the dilemma is perhaps more acute in areas where the wildlife trade is associated with food and medicines, particularly in asia, where the trade could be an important element in rural livelihoods and food security. in addition, translocation of wildlife can be an important tool for both conservation and animal welfare. case studies of disease threats movement of pets in february , the uk adopted the pet travel scheme (pets) to enable pets with the appropriate documentation to move between the uk and certain countries. this represented the first major change in uk quarantine regulations since the importation of dogs act. the scheme is primarily designed to prevent the importation of rabies, with secondary measures to prevent the introduction of the cestode parasite echinococcus multilocularis, which is currently endemic in continental europe and can be transmitted from canids to humans to cause potentially fatal alveolar echinococcosis. eighty-one countries now qualify under the scheme but quarantine remains the only option for non-listed countries. the pets is a model for the use of legislation to minimize risks: the risks of importing rabies under the scheme are small. however, other zoonotic diseases continue to pose a substantial risk and there are few formal checks for these under the system. figure shows the volume of dog and cat movements into the uk following the introduction of the pets. leishmania infantum is the predominant cause of both visceral and cutaneous leishmaniasis throughout the mediterranean region, including southern france, portugal, spain, italy, greece, turkey and north africa [ ] ; the main reservoir host is the domestic dog. seropositivity rates of canine leishmaniasis can be o % [ ] in the mediterranean, with patterns of infection that reflect the distribution of the sand fly vector (phelobotomus species) [ , ] . in brazil, where canine visceral leishmaniasis is an important emerging disease, canine infection in endemic areas can be as high as % [ ] . to control the disease, a mass culling of seropositive dogs has been adopted in several areas but has not been effective [ ] . a feasible alternative to culling dogs is to fit collars treated with the insecticide deltamethrin; preliminary results have shown a large drop in the number of bites by sand flies and infection rates in dogs [ ] [ ] [ ] . this might be the only way to control the increasing spread of canine visceral leishmaniasis and could be recommended as a control measure for dogs leaving and re-entering the uk under pets. quarantine is not effective in preventing the introduction of leishmaniasis due to the long incubation period of the disease. outbreaks of leishmaniasis have occurred in dogs in the usa as a result of mediterraneanbased military personnel who have returned home with their pets. in addition, there has been an apparent increase in the incidence of leishmaniasis in us hunting dogs [ ] -although the cause remains unclear, it could be a result of either animal movements or spread of the vector. the chances of leishmaniasis becoming established in domestic dogs in the uk are small because, currently, phelobotomus species are not known to be present in the uk. however, if climate change does affect air temperatures substantially, the sand fly vector could become established in the uk and, potentially, could maintain endemic leishmaniasis and other 'exotic' parasitic diseases [ ] , the introduction of which would probably result from the movement of domestic pets. in addition to leishmaniasis, diseases such as heartworm (dirofilaria immitis), babesiosis (babesia canis), ehrlichiosis (ehrlichia canis) and echinococcosis (echinococcus granulosus and e. multilocularis) are all likely to be moved with domestic pets into the uk and elsewhere [ , ] . the responsibility falls on domestic pet owners in such a situation who should ensure the use of anthelminthics -agents that destroy or expel parasitic intestinal worms -and, possibly, insecticide-treated collars. owners should also be advised about preventative measures before pet travel. e. multilocularis is common in red foxes in hokkaido, northern japan, where the prevalence of infection in foxes is as high as % and high worm burdens are recorded in some individuals [ ] . dna sequencing of parasite isolates from this area show that it was probably introduced in the s from a neighbouring island [ ] through the movement of infected foxes. this disease now presents an important public health problem, with a human incidence of . per people [ ] , or w cases annually. domestic dogs have become part of the transmission cycle and close contact between humans and their pets is a major risk factor. a recent risk analysis [ ] showed that the movement of pet animals between hokkaido and the rest of japan is likely to result in review further geographical spread of the parasite, particularly because there are few movement controls or programmes for screening and treatment. rabies is a prime example of an infectious disease in which transmission can be enhanced by animal movements. flores island in indonesia was free of rabies until [ ] ; in that year, three dogs were imported from a rabiesendemic area and this was sufficient to result in human deaths (mainly children) and the culling of almost % of the dog population in some areas as part of an unsuccessful control campaign [ ] . flores island is now endemic for rabies, which has become a major public health issue, and dealing with its introduction has incurred a high cost. translocation of raccoons from florida to virginia for hunting purposes [ ] also led to the emergence of raccoon rabies in the mid-atlantic states of the usa, with thousands of animal cases that resulted in enormous expenditure on rabies post exposure prophylaxis and oral vaccination programmes [ ] . france now has rabies-free status because terrestrial rabies has been eliminated through a long-term concerted effort to vaccinate foxes [ ] . however, the risk is ever present and, in , contacts with an illegally imported rabid domestic dog resulted in people requiring post-exposure prophylaxis [ ] . since the s, the uk has conducted regular bovine tuberculosis (btb) testing of cattle herds [ ] . herd breakdowns -the occurrence of btb in a previously btb-free herd -have become more common since and the causes of these breakdowns are the subject of intense debate. molecular evidence (analysis of spoligotype data) from btb isolates in the uk [ ] indicates an important role for cattle movements in the long-distance spread of disease and the establishment of novel strains in new geographic areas. while also highlighting environmental, wildlife and other factors in the short-range spread of btb in the uk, gilbert et al. [ ] quantified the strong association between the proportion of inward movements from infected areas and the breakdown of herds, which highlighted the strong predictive power of cattle movements for later disease distributions. the importance of contact networks in the spread of infectious diseases of livestock in the uk has been clearly highlighted [ ] ; in addition, a small proportion ( %) of farm holdings contributes to the majority ( %) of movements. if one of these high-contact farms was infected with btb, its impact on long-range spread to many other farms would be substantial. a thorough understanding of livestock contact networks at different scales is essential in predicting such spread. trypanosoma evansi (the tabanid-transmitted pathogen responsible for surra in livestock) has spread in south east asia, particularly in the philippines where there is high mortality in those areas in which the parasite has been detected. this spread has been blamed on the movements of livestock as part of herd-improvement programmes [ ] . similarly, another trypanosome species (trypanosoma brucei rhodesiense) caused an outbreak of human sleeping sickness in a previously unaffected area of uganda. this was caused by movement of the cattle reservoir of the trypanosome parasite through markets without proper screening or disease control. veterinary and public health services are now struggling to control a rapidly spreading sleeping sickness epidemic in humans [ ] . these introductions would have been entirely preventable if local authorities had been alerted to the risks and if resources were made available to administer prophylaxis or treatment to the animals. the integrated management of disease threats (e.g. the treatment of animals as a means of preventing disease spread to humans) would be a cost-effective and efficient use of resources, particularly where zoonotic diseases are concerned. in late january or early february , fmd was introduced to a pig farm in the north of england. disease spread through this pig herd probably occurred with three waves of infection and amplification. however, fmd was not notified to the authorities until it was identified during a routine meat inspection at an abattoir in the south east of the country (several hundred miles away) on february; fmd was confirmed on february [ ] . at the time of disease identification in the abattoir, the virus had already spread to a neighbouring farm where sheep and cattle were infected, probably by airborne spread [ ] . sheep from this farm were sold to a local market on february and sold again two days later at a larger market [ ] . by the time the first case was confirmed, an estimated - farms had been infected across the uk [ , ] . rapid tracing identified the pig farm but a movement ban for the whole of the uk was delayed for three days (although all international movements were stopped immediately). this three-day delay might have caused a further farms to become infected, mostly from contacts with markets [ ] . the virus spread to france through the movement of infected sheep before the ban. in addition, calves bound for the netherlands were in a vehicle next to these sheep at a stopping point and it is thought that the virus was transmitted between these two consignments -fmd was confirmed in the netherlands on march [ ] . this outbreak clearly demonstrates the risks associated with large-scale movements of animals over long distances across international borders. given modern abattoirs and chilling transport units, the question arises as to why this sort of live animal movement is necessary at all. in africa, livestock are one of the few tradable commodities available to millions of poor households, particularly those living in the more arid regions of sub-saharan africa. there are well-established trade routes across the continent that supply the large coastal populations of west and central africa and the arabian peninsula. these animal movements are of major importance for the dissemination of new strains of fmd and review other diseases such as rift valley fever [ ] . modern dna sequencing techniques make it possible to compare isolates and identify probable sources of infection. for example, these techniques enabled the tracing of outbreaks caused by the sat fmd serotype as it spread up the east african coast into saudi arabia through cattle movements from somalia and eritrea [ ] and its subsequent spread west into cameroon [ ] . in these regions, herdsmen are nomadic or semi-nomadic and move with their animals on a seasonal transhumance in search of grazing, which brings different herds into contact with one other and increases the risk of spread of fmd [ ] . the movements of domestic and wild animals are complex and profitable but are extremely risky from a disease perspective. movements can result in the introduction of exotic animal diseases or human pathogens, which might themselves have important economic and/or public health impacts. one example occurred in when humans in the usa became infected with monkey pox [ ] , which originated from the importation of wild west african rodents. minimizing such risks should be a high priority and, in some cases, this might involve preventing the animal trade altogether: from the perspective of disease introductions and animal welfare, it is difficult to justify the movement of exotic birds or mammals simply for the pet trade. attempting to ban such movement, however, is likely to drive it underground, which makes riskmitigation far more difficult. with livestock, there is a strong case for trading only in livestock products -a commodity-based approach -rather than live animals themselves [ ] but this, of course, is dependent on the appropriate investment and infrastructure being available to process animal products and to provide adequate quality assurance. for diseases linked to livestock, such as fmd, bovine tuberculosis and sleeping sickness, markets have an important role in the dissemination of infectious organisms. markets serve as contact nodes between infected herds and the ease of transportation can result in the widespread dissemination of animals that have been in contact in a market. contact nodes such as quarantine facilities, markets and ports of entry can also result in the transmission of agents between individuals and species, with rapid subsequent dissemination. better communication between scientists, livestock traders, livestock keepers and decision makers is required to avoid this. the key issue that requires most attention is to acquire a better understanding of the risks of global movements. simple risk assessments that focus on the individual country trying to protect itself from disease introductions are no longer sufficient and passive detection of disease at ports of entry is an increasingly dangerous strategy as the volume of movements increases. a holistic understanding of risk at the global level is required to understand disease risks by species and by country, supplemented by an efficient global surveillance network in which different animal species are regularly screened, particularly before moving from their source areas. control of disease threats should be dealt with locally rather than when movement has already occurred, which requires knowledge of species, volumes moved and fine-scale information on point of origin. knowledge of movement routes is the key to predicting the pattern of spread of infectious diseases of humans [ ] , and similar data could be crucial to understand animal disease risks. databases already exist [ ] [ ] [ ] that list pathogens, hosts and probable risks of emergence; additional layers of information on the movements of host species and their impacts on the risks of emergence should be added, and the information should be made publicly available. such global cooperation and international level disease control will lead to better risk management and mitigation. travel and the emergence of infectious diseases the application of risk analysis to international trade in animal products emerging diseases and implications for global trade international regulation of wildlife trade: relevant legislation and organisations economic reform and the changing pattern of china's agricultural trade (working paper no. - ). department of agricultural and resource economics community-based animal health and participatory epidemiology unit, pan african programme for the control of epizootics and organization of african unity/interafrican bureau for animal resources unofficial trade when states are weak: the case of cross-border commerce in the horn of africa somalia: towards a livestock sector strategy -final report brucellosis control in saudi arabia: prospects and challenges wildlife trade and global disease emergence international disease implications for wildlife translocation disease risks of wildlife translocations emerging infectious diseases of wildlifethreats to biodiversity and human health bats are natural reservoirs of sars-like coronaviruses severe acute respiratory syndrome coronaviruslike virus in chinese horseshoe bats highly pathogenic h n influenza virus in smuggled thai eagles quarantine premises in essex, department for the environment, food and rural affairs sunbleak and topmouth gudgeon -two new additions to britain's freshwater fishes emerging infectious diseases and amphibian population declines emergence of amphibian chytridiomycosis in britain giant toads bufo marinus in australia and venezuela have antibodies against 'ranaviruses' systematic review of the distribution of the major vector-borne parasitic infections in dogs and cats in europe canine leishmaniasis imported diseases in small animals host preferences of phlebotomine sand flies at a hypoendemic focus of canine leishmaniasis in central italy assessment of an optimized dog-culling program in the dynamics of canine leishmania transmission infectiousness in a cohort of brazilian dogs: why culling fails to control visceral leishmaniasis in areas of high transmission further evidence that deltamethrin-impregnated collars protect domestic dogs from sandfly bites protection of dogs from bites of phlebotomine sandflies by deltamethrin collars for control of canine leishmaniasis are insecticide-impregnated dog collars a feasible alternative to dog culling as a strategy for controlling canine visceral leishmaniasis in brazil? infectious diseases -has leishmaniasis become endemic in the us? sensitivity of malaria, schistosomiasis and dengue to global warming is heartworm disease really spreading in europe? prevention of alveolar echinococcosisecosystem and risk management perspectives in japan short report: echinococcus multilocularis confirmed on kunashiri island, kilometers from the eastern part of hokkaido the rabies epidemic on flores island rabies on flores island, indonesia: is eradication possible in the near future descriptive epidemiology from an epizootic of raccoon rabies in the middle atlantic states, - the ascension of wildlife rabies -a cause for public health concern or intervention elimination of terrestrial rabies in western european countries risque de rage en france et importation illégale d'animaux en provenance de zones d'endémie rabique investigations of cattle herd breakdowns with bovine tuberculosis in four counties of england and wales using vetnet data the population structure of mycobacterium bovis in great britain: clonal expansion cattle movements and bovine tuberculosis in great britain epidemiological implications of the contact network structure for cattle farms and the - rule trypanosoma evansi control and containment in australasia a burgeoning epidemic of sleeping sickness in uganda descriptive epidemiology of the footand-mouth disease epidemic in great britain: the first months airborne transmission of foot-and-mouth disease virus from burnside farm early dissemination of foot-and-mouth disease virus through sheep marketing in temporal and geographical distribution of cases of foot-and-mouth disease during the early weeks of the epidemic in great britain the construction and analysis of epidemic trees with reference to the uk foot-and-mouth outbreak lessons from the foot and mouth disease outbreak in the netherlands in epidemiological processes involved in the emergence of vector-borne diseases: west nile fever, rift valley fever, japanese encephalitis and crimean-congo haemorrhagic fever the implications of virus diversity within the sat serotype for control of foot-and-mouth disease in sub-saharan africa the molecular epidemiology of foot-andmouth disease viruses in the adamawa province of cameroon risk factors for herdsman-reported footand-mouth disease in the adamawa province of cameroon the detection of monkeypox in humans in the western hemisphere international trade in livestock and livestock products: the need for a commodity-based approach forecast and control of epidemics in a globalized world risk factors for human disease emergence diseases of humans and their domestic mammals: pathogen characteristics, host range and the risk of emergence the microbial rosetta stone database: a compilation of global and emerging infectious microorganisms and bioterrorist threat agents acknowledgements e.m.f. and s.c. are funded by the uk department for international development (dfid), although the views expressed are not necessary those of dfid. we thank philippe ankers of vétérinaires sans frontières (vsf)-suisse for supplying some key reference material. key: cord- -ys n authors: whary, mark t.; baumgarth, nicole; fox, james g.; barthold, stephen w. title: biology and diseases of mice date: - - journal: laboratory animal medicine doi: . /b - - - - . - sha: doc_id: cord_uid: ys n today’s laboratory mouse, mus musculus, has its origins as the ‘house mouse’ of north america and europe. beginning with mice bred by mouse fanciers, laboratory stocks (outbred) derived from m. musculus musculus from eastern europe and m. m. domesticus from western europe were developed into inbred strains. since the mid- s, additional strains have been developed from asian mice (m. m. castaneus from thailand and m. m. molossinus from japan) and from m. spretus which originated from the western mediterranean region. laboratory animal medicine development of the 'modern' laboratory mouse. research use of mice has grown exponentially during the past and current century with the recognition of the power of the mouse for gene and comparative mapping and have made the laboratory mouse, in genetic terms, the most thoroughly characterized mammal on earth (silver, ; lyon et al., ; morse, a) . the current ability to create highly sophisticated, genetically engineered mice by inserting transgenes or targeted mutations into endogenous genes has also made the laboratory mouse the most widely and heavily used experimental animal. historical reviews have documented the origins of the laboratory mouse, which extend thousands of years into antiquity (keeler, ; morse, ; silver, ) . the laboratory mouse belongs within the genus mus, subfamily murinae, family muridae, superfamily muroidea, order rodentia, and within the m. musculus clade collectively called the 'house mouse' (lundrigan et al., ) . anatomic features of molar teeth and cranial bones were traditionally used by zoologists to identify over different species within the genus, and to differentiate them from other murids. because of considerable phenotypic variation within a single mus species, this approach has proven to be inaccurate, and given way to contemporary genetic analysis. the native range of the genus mus is eurasia and north africa. members of this genus are generally classified as aboriginal, consisting of species that live independent of humans, or commensal, which includes taxa that have coevolved and geographically radiated with human civilization since the dawn of agriculture , years before present (bp). this close association with human agrarian society gave rise to the genus name, derived from sanskrit, mush: to steal. the commensal group is known as the 'house mouse' clade, consisting of several subspecies of mus musculus, including m. m. domesticus, m. m. musculus, m. m. castaneus, m. m. bactrianus , and a lesser known lineage, m. m. gentilulus (prager et al., ) . the japanese house mouse, m. m. molossinus, is a natural hybrid of m. m. musculus and m. m. castaneus. the progenitor of the m. musculus clade arose in the northern indian subcontinent and diverged into genetically isolated and distinct species or subspecies due to geographic barriers (mountain ranges). there is debate whether these taxa are species or subspecies, and some have referred to them as 'incipient species,' but their genetic divergence is now blurring as they colonize the world and hybridize. the native ranges of these taxa are important for understanding the origins of various laboratory mice, whose genomes are mosaics derived from m.m. domesticus (~ %), m.m. musculus (~ %), and m.m. castaneus (~ %) (wade and daly, ; wade et al., ) . it is now apparent that the m.m. musculus and m.m. castaneus contributions to the laboratory mouse genome were primarily derived from m.m. molossinus japanese fancy mice (takada et al., ) . mus m. domesticus is indigenous to western europe and southwest asia, m.m. musculus to eastern europe and northern asia, m.m. castaneus to southeast asia, and m.m. molossinus to japan and the korean peninsula. the cohabitation of humans with commensal mice gave rise to captive breeding for coat color and behavioral variants in china over years bp. by the s, mouse 'fanciers' in asia had created many varieties of fancy mice, as did european fanciers, who subsequently acquired asian stocks, particularly japanese fancy mice (m.m. molossinus) , to mix with european (m.m. domesticus) fancy mouse varieties. this genetic mixing for fancy variants was also occurring in the united states, and these mouse lines contributed to many of the major laboratory mice used today. meanwhile, the european colonial expansion era contributed to the worldwide dissemination of m.m. domesticus, which now occupies every continent of the world. it is well documented that wild-caught m.m. domesticus also contributed to the genetic composition of fancy and laboratory mice on multiple occasions. despite their diverse genetic origins and phenotypic differences, most laboratory mouse strains are closely related, since many were derived from a genetically mixed but small number of fancy mice from a single mouse breeder (abbie lathrop's granby mouse farm, massachusetts) at the beginning of the th century. most inbred laboratory mice share a common maternal mitochondrial genome derived from m.m. domesticus (ferris et al., ; yu et al., ) , and a common y chromosome contributed by m.m. musculus (bishop et al., ) through its contribution to the genome of m.m. molossinus (nagamine et al., ) . thus, the most inclusive name that can be assigned to the genetically mosaic laboratory mouse is m. musculus, the over-arching name for the entire commensal clade. there are exceptions, however. c bl/ mice contain minor genetic elements derived from m. spretus (hardies et al., ) , and a number of wild aboriginal species that are not members of the m. musculus clade, including m. spretus, m. caroli, and others, have been established as inbred lines of mice. genetic mapping in mice began in the early s with a focus on inheritance of coat color. the first autosomal genes, albino and pink-eyed dilution, were linked in (haldane et al., ) . extensive linkage maps and an impressive array of inbred strains are now available to expedite genetic research (table . ) (lyon et al., ) . mice have pairs of telocentric chromosomes that are differentiated by their size and patterns of transverse bands. the chromosomes are designated by arabic numbers in order of decreasing size. during the s, chromosome rearrangements were used to assign known genetic linkage groups -identified by roman numerals -to specific chromosomes and for determining locus order with respect to the centromere. genes can be located physically on chromosomes by fluorescent in situ hybridization (fish). development of quantitative trait loci (qtl) methodology for mapping genes and the similarity between mouse and human genomes have made the mouse invaluable for identifying genes and underlying complex traits that are inherent to the most common human genetic diseases (moore and nagle, ) . for more information on comparative genomics, see chapter animal models in biomedical research, subsection c. one of the most thoroughly studied genetic systems of the mouse is the histocompatibility complex. histocompatibility (h) loci control expression of cell surface molecules that modulate critical immune responses, such as the recognition of foreign tissue. for example, the time, onset, and speed of skin graft rejection are controlled by two groups of h loci. the major group is located in the major histocompatibility complex (mhc, h ) on chromosome . the h complex contains several loci, including k, d, l, i-a, and i-e. inbred strains of mice, being homozygous, each have unique sets of h alleles, termed h haplotypes. for example, the balb h haplotype is h d and the c bl h haplotype is h b . the international immunogenetics (imgt) information system provides details on h haplotypes for various inbred mice (www.imgt.org/imgtrepertoiremhc/polymorphism/ haplotypes/mouse/mhc/mu_haplotypes.html). minor h loci groups are scattered throughout the genome and are responsible for delayed graft rejection. genes associated with the h complex also control other immunological functions, such as cell-cell interactions in primary immune responses and the level of response to a given antigen. immune-mediated responses to infectious agents such as viruses and complement activity are influenced directly or indirectly by the h complex (stuart, ) . non-mhc or minor histocompatibility systems also are under active study (roopenian et al., ) . mouse genomics have accelerated tremendously in the last two decades, heralded by the development of a robust physical map and high-quality genome sequence of the c bl/ j mouse in by the international mouse genome sequencing consortium (waterston et al., ) . the mouse genomes project/wellcome trust sanger institute is extending this effort to include the genomic sequences of key mouse strains. completed and evolving sequence data are available through the european nucleotide archive (www.ebi.ac.uk/ena/home). the burgeoning numbers of inbred mouse strains, natural mutants, induced mutants, transgenic lines, and targeted mutant lines of mice are cataloged in the mouse genome informatics (mgi) database: http://www.informatics.jax.org/mgihome). the growing number of mutant mice has fostered the development of a number of mouse repositories, from which specific mice can be located and acquired. in the united states, there are four regional national institutes of health (nih)-supported mutant mouse regional resource centers (http://www.mmrrc.org), which link to international repositories in europe, japan, china, australia, and canada, as well as additional resource programs in the united states through the international mouse strain resource (imsr; http://www.informatics.jax.org/ imsr/index.jsp) for depositing, archiving, and distributing mutant mouse and embryonic stem cell lines to the scientific community. in addition to numerous mutant mice produced independently by scientists in various academic institutions, three major targeted gene knockout programs, all utilizing c bl/ n embryonic stem cells, are under way internationally, and funded by the nih, the european community, and genome canada (collins et al., ; skarnes et al., ) . these include the knock out mouse project (komp; http://www.knockoutmouse.org), the european conditional mouse mutagenesis program (eucomm; http://www.eucomm.org), and the north american conditional mouse mutagenesis project (norcomm; http://norcomm.phenogenomics.ca/index. htm). these mouse lines will be available through laboratory animal medicine three distribution centers: the german resource center for genome research (rzpd; http://www.rzpd.de), the komp repository (https://komp.org), and the canadian mouse consortium (cmc; http://www.mousecanada. ca/index.htm). the repositories are all linked to the imsr, and provide access to mice, germplasm, genomic detail, and phenotypic data. genetic, genomic, and biological data are also available through the international mouse phenotyping consortium (impc; www.mousephenotype. org) and the mouse genome database (mgd; http://www. informatics.jax.org) (eppig et al., ) . inbreeding is a fundamental genetic tool applied to the laboratory mouse and detailed information is available on the web (table . ). the first inbred strain (dba) was developed by c.c. little in , with the subsequent creation of over inbred strains and stocks of mice (festing, ) . genetic origins, basic characteristics, references, and breeding performance of inbred strains of mice are available through michael festing's online version of inbred strain characteristics (http:// www.informatics.jax.org/external/festing/mouse/ strains.shtml). overviews of genetic manipulation for the creation of different types of mice are available (lyon et al., ; silver, ) . inbred mouse lines are termed strains, and are achieved by or more brother × sister (filial; f) generations (table . ). mice within an inbred strain, for practical purposes, are genetically identical (syngeneic or isogenic) to other mice of the same strain and sex. because of residual heterozygosity, a strain is not fully inbred until after f generations. most commonly used inbred mouse strains represent or more f generations, providing a high degree of experimental reproducibility. the mouse genome is not static, so when branches of an inbred strain are separated, spontaneous mutations, residual heterozygosity, and retroelement integrations result in genetic differences. therefore, if branches of an inbred strain are separated before f , if branches have separated for generations, or if genetic differences arise, the different branches become substrains. the same holds true if branches of a substrain diverge, resulting in substrains of the inbred substrain. when two inbred mouse strains are crossed, the f hybrids are genetically identical to one another (isogenic), but maximally heterozygous (with chromosomes of each chromosomal pair separately contributed by each parental strain), whereas f hybrids are maximally genetically diverse from one another (with chromosomes of both chromosomal pairs containing a mixture of contributions from each parental strain). with each subsequent f generation, mice once again approach inbred status. this technique is used for creating recombinant inbred (ri) strains. ri strains are sets of inbred strains of mice derived from crossing two inbred strains, and developed by singlepair random matings of sibling mice from the f generation, thereby creating separate breeding lines. each line created is maintained separately, and then propagated by brother-sister matings for generations, with each line becoming a separate inbred strain, but belonging to a set of ri strains. ri mice are useful for mapping phenotypic or quantitative traits that differ between the progenitor strains (bailey, ) . ri sets are generally limited to two parental strains. an ongoing international effort has been undertaken to increase allelic diversity among ri strains by creating the collaborative cross (cc) in which a panel of ri strains are being generated mixing the genomes from eight disparately related inbred (octo-parental) mouse strains, including a/j, c bl/ j, s /svimj, nonobese diabetic (nod)/shiltj, nzo/ hlltj, cast/eij, pwk/phj, and wsb/eij. these eight strains capture nearly % of the known genetic variation present among laboratory mice. future applications of the cc will utilize ri intercrosses of pairs of ri cc lines (threadgill and churchill, ; welsh et al., ) . recombinant congenic strains are sets of inbred strains derived in a manner similar to that for ri sets, except that one or more backcrosses to one parental strain (designated the background strain) are made after the f generation, before inbreeding is begun. the other parental strain is designated as the donor strain. the proportion of background and donor genomes is determined by the number of backcrosses preceding inbreeding (demant and hart, ) . advanced intercross lines (ails) are another type of ri lines. they are made by producing an f generation between two inbred strains and then, in each subsequent generation, intercrossing mice but avoiding sibling matings. the purpose is to increase the possibility of recombination between tightly linked genes. when a mutation arises spontaneously or is induced within an inbred strain, that mutant mouse becomes co-isogenic with the parental inbred strain, being virtually identical except for the single mutant allele. frequently, a mutation that arose in one inbred strain may be desired within the genetic background of another inbred strain. this can be accomplished by backcrossing, in which an f hybrid is created by mating the donor mutant strain to the desired background strain, with subsequent matings to the background strain while retaining the mutant locus. after backcross generations (n generations), the mutant mouse line is now congenic to the background inbred strain. backcrossing to create congenic strains of mice has been used extensively when targeted mutations have been induced in embryonic stem cells, with backcrossing onto c bl/ inbred mice. congenic mice are never co-isogenic, as the preserved locus in a congenic mouse is invariably surrounded by flanking dna, which may significantly influence phenotype (linder, ) . in contrast to inbred mice, outbred mice are genetically heterogeneous and are maintained by breeding systems that intentionally minimize inbreeding. outbred mice are called stocks, which are defined as a closed population (for at least four generations) of genetically variable mice that are bred to maintain maximal heterozygosity. outbred mice may be used when high genetic heterogeneity is desired or for experiments requiring large numbers of mice. outbreeding can be achieved only in a large breeding population using a systematic breeding scheme, or randomized selection of breeders from the population. a small breeding population or passage through the genetic 'bottleneck' of rederivation to improve health status will reduce genetic heterogeneity and lead eventually to some degree of inbreeding. in a population of breeding pairs, e.g., heterozygosity will decrease at % per generation with standard randomization techniques. random breeding involves the statistically random selection of breeders by using a random numbers table or computer program. an outbreeding program that is easy to manage is the circular pair mating system, in which each pair is mated only once. conceptually, cages are visualized in a circle, and each cage contains one breeding pair in the nth generation. another 'circular' set of cages serves as the breeding nucleus for the n + generation. each mated pair in the nth generation contributes one female and one male to the n + generation. outbreeding is accomplished by assigning the female and male derived from each nth generation cage to different cages in the n + generation. most outbred mouse stocks are of 'swiss' origin, derived from nine mice imported to the united states in , and are therefore quite homogeneous genetically (chia et al., ) . various lines of these mice have been maintained at different institutions, giving rise to numerous closely related stocks. although considered outbred, they have a high degree of homozygosity, exemplified by the fact that many swiss mouse stocks are blind due to the homozygous recessive rd allele (serfilippi et al., b) . it is preferable to ensure genetic heterogeneity by intercrossing multiple inbred strains to achieve heterogeneity with known genetic input. in that regard, the diversity outbred mouse has been developed, which is a heterogeneous stock derived from the same eight founder inbred strains of the cc . additional types of inbred mice are utilized in research, including consomic and conplastic strains. consomic strains, also known as chromosome substitution strains, are inbred mice that are congenic for entire chromosomes, and are useful for studying polygenic traits (singer et al., ) . conplastic mice are inbred mice that are congenic for different mitochondrial genomes (mtdna) contributed by other inbred strains, other subspecies, or other species of mus (yu et al., ). in addition to spontaneously occurring mutations that are maintained as co-isogenic strains (such as the c bl/ beige mouse), mutant lines of mice have been created by radiation mutagenesis, chemical mutagenesis, or transgenesis. radiation was one of the earlier methods for in vivo mutagenesis (silver, ) , but in vitro radiation of embryonic stem (es) cells is also performed (thomas et al., ) . chemical mutagenesis involves in vivo treatment of male mice or in vitro treatment of es cells with mutagenic chemicals such as ethylmethanesulphonate (ems) or n-ethyl-n-nitrosourea (enu) , which induce point mutations in dna (o'brien and frankel, ; justice et al., justice et al., , . technically, a transgenic mouse is any mouse in which foreign dna has been integrated into its genome, regardless of method. however, the term transgenic commonly refers to mice that are genetically altered by additive transgenesis through microinjection of foreign dna into the pronucleus of a fertilized egg. each ensuing embryo results in a genetically different founder mouse, since the transgene is integrated in random sites of the genome of each founder mouse. since the injected dna is not homologous to the mouse genome and is not an allele, transgenic founders are hemizygous (rather than heterozygous) for the transgene until the mice carrying the transgene are bred into homozygosity for the transgene. transgenes typically integrate as tandem repeats, copy numbers affect phenotype of each founder, and may be lost in subsequent generations, thereby changing the phenotype of the mouse line (tinkle and jay, ) . transgenes are often constructed with an upstream promoter, which confers widespread (ubiquitous) or tissuespecific expression of the cdna, so that the transgene expression pattern reflects the expression pattern of the promoter. transcriptional regulation of the transgene can be inducible by drug-dependent regulatory control, such as the widely used tetracycline (tet) regulatory system, in which treatment of mice with tetracycline or doxycycline induces up-or down-regulation of the transgene (jaisser, ) . es cells are used for the less efficient integration of genetic material by homologous dna recombination, but allow large-scale screening of es cell clones for transformation. integration can be achieved in a random fashion by gene trapping, or by targeted mutation. both methods involve homologous dna recombination. gene trapping is a high-throughput approach that randomly introduces insertional mutations within the genome. vectors contain a gene trapping cassette with a promoter-less reporter gene and/or selectable genetic marker flanked by an upstream ′ splice site and a downstream termination sequence. when inserted into an laboratory animal medicine intron of an expressed gene, the gene trap is transcribed from the endogenous promoter of that gene. gene traps simultaneously inactivate and report the expression of the trapped gene at the insertion site, and provide a dna tag for the rapid identification of the disrupted gene (skarnes et al., ) . targeted gene mutations are achieved by homologous recombination of specific sites within the genome of es cells. homologous sequences flank the upstream and downstream regions of the targeted gene, and the construct between the flanking sequences may inactivate (knock out) or replace (knock in) a gene, and typically contains a reporter gene to track the integration. a variation on this approach is site-specific recombinase (ssr) technology. two of the most common recombinases are cre from the coliphage p and flp from saccharomyces cerevisiae. cre and flp mediate recombination between target sites, termed loxp and frt, respectively. for example, cre loxp target sites are engineered to flank the gene target, which can be used in different ways to achieve different outcomes (conditional mutations), depending upon the orientation and location of the flanking loxp sites. if the loxp sites are oriented in opposite directions, cre recombinase mediates inversion of the floxed segment. if the loxp sites are on different chromosomes (trans), cre recombinase mediates a chromosomal translocation. if the loxp sites are oriented in the same direction on the same chromosome (cis), cre recombinase mediates deletion of the floxed segment. once the floxed mutation is created in es cells, the transformed es cells are developed into a mouse with the conditional mutation. the conditional mutant mouse is then genetically crossed with a cre transgenic mouse, in which cre recombinase is under the control of a ubiquitous or tissue-specific promoter. wherever and whenever cre is expressed, cre recombinase will recognize and recombine the loxp sites. this approach can include insertion of reporter genes and selectable markers, and can be under the control of inducible gene expression systems (http:// www.eucomm.org/docs/protocols/mouse_protocol_ _ sanger) (nagy, ) . es cells are pluripotent with the full genetic capacity to develop into mice when implanted into the blastocyst of a developing embryo. interest in 'embryonal carcinomas' (teratomas) that arose in relatively high frequency in the testes of mice and early gene transfer experiments in the late s and early s led to the development of es cell lines derived from several different strains. this early emphasis on teratomas prompted creation of 'better' mouse lines that were more prone to development of testicular teratomas, resulting in genetic corruption of the mouse (simpson et al., ; threadgill et al., ) . this realization gave rise to the need to revise mouse nomenclature (festing et al., ) . this was necessary because genetic variation significantly impacts homologous recombination in order to match genome sequence of the es cell line with the mouse from which it was derived. es cells can be created from any mouse strain or hybrid, but es cell lines have been commonly used. recent international knockout mouse program efforts use c bl/ n es cells. transformed es cells are microinjected into the inner cell mass of recipient blastocysts, which are then implanted into the uteri of pseudopregnant surrogate mothers. the pups that are born are composed of a mixture of cells derived from recipient blastocysts and the transformed es cells (chimeras). the goal is for male chimeric progeny to produce spermatozoa of es cell origin (containing the mutation), in order to create f progeny by mating the chimera with the desired background strain (http://www.eucomm.org/docs/protocols/mouse_protocol_ _sanger). for this reason, most es cell lines are xy, which favors male chimerism. if the es cells are of (or other) strain origin, the chimeras are often bred to a desired background mouse strain (commonly c bl/ ) and backcrossed for n generations, thereby creating congenic inbred mouse lines. recent international knockout mouse efforts utilize c bl/ n es cells, so that chimeric males are bred directly with c bl/ mice, thereby creating co-isogenic lines. the latter approach saves time and money, and creates a more genetically refined mutant mouse. an alternate approach is to allow es cells to aggregate with a developing embryo to form blastocysts in culture (aggregation chimera), then implant the chimeric blastocysts (tanaka et al., ) . rna interference (rnai), which functions through short double-stranded rna (dsrna), has also been utilized to produce transgenic mice, known as gene knockdown mice (gao and zhang, ; peng et al., ) . the dsrna is enzymatically processed into small molecules, termed small interfering rna (sirna), which find homologous target mrnas, resulting in interference. this phenomenon is believed to be a self-defense mechanism against viral infection. in order to adapt this approach to generation of transgenic mice, small hairpin rna (shrna) can be expressed in the same way as other transgenes in mice, resulting in processing of the shrna into sirna with gene-silencing effects. constructs are introduced into mouse es cells by electroporation or lentiviral infection. this method can be embellished conditionally, as with other transgenes. although rnai knockdown mice are genetically stable, rnai-mediated transgenesis is never complete, has variable tissue expression, and cannot induce point mutations (peng et al., ) . recent advances in engineered endonuclease (ee) technology, including zinc finger nucleases (zfns), laboratory animal medicine transcription activator-like effector nucleases (talens), and rna-guided endonucleases (rgens), have revolutionized the field of transgenics (sung et al., ; wijshake et al., ; gaj et al., ) . zfns and talens consist of engineered proteins that target dna fused to the nonspecific endonuclease, fok (cathomen and joung, ; joung and sander, ) . zfns are comprised of three to six tandem zinc finger proteins, each of which targets a specific bp nucleotide sequence. paired zfns are generated, with each half of the pair targeting opposite dna strands, allowing dimerization of fok which is required for introduction of double-stranded breaks (dsbs) in the dna of interest (cathomen and joung, ) . talens function similarly, but are composed of tandem repeats of - amino acids, each with nucleotide specificity occurring in two hypervariable amino acids, the 'repeat variable di-residue (rvd)', at positions and (joung and sander, ) . in contrast to zfns and talens, clustered regularly interspaced short palindromic repeats (crisprs) paired with crispr-associated (crispr/cas) systems are rgen systems that target specific dna sequences. cas proteins, rather than fok , produce dsb (hsu et al., ) . dsb generated by ee are repaired by host cells by either nonhomologous end joining (nhej) or, less commonly, by homologous recombination (hr). nhej is an error-prone repair system and results in insertions or deletions (indels) with a relatively high frequency, which can result in gene disruption. hr is a less common repair pathway, but certain manipulations of the engineered nucleases can increase hr efficiency. for example, nucleases can be engineered to generate a break in a single strand of dna rather than inducing dsb, and the resulting nickases increase the incidence of hr with high fidelity (gaj et al., ; wijshake et al., ) . hr allows for the introduction of donor dna to generate knock-ins, specific point mutations, or for the generation of larger modifications such as insertions of loxp sites (brown et al., ; wijshake et al., ) . vectors encoding the ee can be injected into mouse embryos by pronuclear injection of dna, intracytoplasmic injection of rna, or transfection of mouse es cells (sung et al., ; wijshake et al., ) . one advantage of ee technologies over more traditional transgenic methods is the ability to target dna and induce mutations in any background strain of mouse negating the need to backcross onto the desired strain. multiple genes can be targeted with crisprs simultaneously, thus avoiding the need to cross single knockout animals (zhou et al., ) . in addition, it is possible to obtain bi-allelic mutations in some cases, allowing for the generation of functional gene knockout animals in a single generation (zhou et al., ; wijshake et al., ) . vectors for generating ee are available through plasmid repositories; websites are available to assist in identifying appropriate dna sequences to target; and multiple websites post protocols for generating the various types of engineered endonucleases (xie et al., ; sander et al., ; bae et al., ; reyon et al., ; herscovitch et al., ; wolfson, ) . crisprs tend to be particularly cost effective and easy to design, with minimal restrictions for targeting specific dna sequences. there are currently more than separate outbred stocks and traditional inbred strains, often with multiple substrains (table . ). in addition, there are thousands of induced mutant strains. therefore, it is critical that strain or stock designations be complete and accurate to avoid semantic and genetic confusion, and to ensure reproducibility of research results. as an example of substrain variation that makes precise nomenclature important, cba/j mice are homozygous for the retinal degeneration allele (rd ), whereas cba/caj mice do not carry this allele. the international committee on standardized genetic nomenclature for mice and rats, established in the early s, is responsible for genetic nomenclature rules. the rules are available online at the mgi website (http:// www.informatics.jax.org/mgihome/nomen). inbred mouse strains are designated by a series of capital letters and/or numbers, which often provide a shorthand description of the origin and history of the strain. the c bl/ j mouse serves as an example. the inbred strain c bl originated from abbie lathrop's female (and male ) at the cold spring harbor laboratory (c), and was the black (bl) line from this female. early in their history, inbred c bl mice split into major substrains, e.g., c bl/ and c bl/ . substrains are identified by appending a forward slash (/) after the inbred strain name. since , uniform international nomenclature has been built upon these historical names, so that substrains of an inbred strain are now designated using lab codes that are registered in the international laboratory code registry maintained at the institute for laboratory animal research (ilar) of the national academies (dels. nas.edu/global/ilar/lab-codes). laboratory codes are composed of one to five letters that identify an institute, laboratory, or investigator. each lab code starts with an uppercase letter, followed by lowercase letters if more than one letter is used (such as n, j, jci, crl, and tac). the j in c bl/ j means it is a substrain maintained at the jackson laboratory (j). another common substrain of c bl/ mice is c bl/ n, which is maintained at nih (n). substrains can be cumulative, reflecting the genetic history of the mouse strain. for example, there are a number of c bl/ j substrains (such as c bl/ jjci and c bl/ jjmsslc), and a number of c bl/ n substrains (such as c bl/ njci, c bl/ ncrlcrlj, dba/ j inbred strain named for its characteristic coat color genes (using their original gene symbols), dilute (d), brown (b), and nonagouti (a); it is the second of two sublines separated before generations of brother × sister breeding and is the subline maintained at the jackson laboratory (j) c h/hesn-ash/+ co-isogenic segregating inbred mutant strain carrying the ashen (ash) mutation, which arose on c h/hesn c bl/ j-tyrc- j /+ co-isogenic segregating inbred mutant strain carrying the albino j mutant allele of the cloned tyrosinase gene (tyr) aej/gnj-a e /a w-j inbred strain segregating for two alleles at the agouti gene congenic inbred strain in which the b haplotype at the h complex was transferred from c bl/ j (b ) to the akr background b .cba-d mit -d mit congenic strain in which the chromosomal segment between d mit and d mit was transferred from cba to b b .cg m lepr db /++ congenic inbred strain in which the linked mutant genes misty (m) and diabetes (lepr db ) were transferred from multiple, mixed, or unknown genetic backgrounds to b and are carried in coupling, i.e., on the same chromosome b .cg-m +/+ lepr db congenic inbred strain in which the m and lepr db mutations are carried in repulsion bxd- /ty recombinant inbred (ri) strain number in a set of ri strains derived from a c bl/ j (b) female mated to a dba/ j (d) male and made by taylor (ty) recombinant congenic (rc) strain number in a set made by crossing the balb/c (c) and sts (s) strains, backcrossing one or two times to balb/c and then inbreeding as with ri strains and c bl/ ntac). significant differences may exist among these substrains (mekada et al., ). thus, a string of substrain designations indicate the genetic progression of the substrain, which can be identified when reading the entire strain name. this nomenclature is highly nuanced, as c bl/ ncrlcrlj mice, whose last letter is a lowercase j, are not a substrain maintained at the jackson laboratory (j), but rather at charles river japan (crlj), underscoring the importance of upper-and lowercase lettering in rodent nomenclature. balb/c mice are another popular inbred strain with numerous substrains. like the ' ' in c bl/ , the 'c' that follows laboratory animal medicine of the mutational event as a superscript. for example, cftr tm unc is a targeted mutation (tm), first line ( ) congenic mice are often derived from es cells, backcrossed onto a background strain, such as c bl/ . under such circumstances, when the backcross generation is at n , the '.' symbol is used between the background inbred strain and the donor strain (e.g., c bl/ n. p /olahsd-abc tm zzz , abbreviated as b . -abc tm zzz . when backcrossing is incomplete but at the n generation, the mouse is an incipient congenic, designated with a ';' in lieu of a '.': b ; -abc tm zzz . if the background strain is mixed genetic origin, it is designated stock. -abc tm zzz . if the donor strain is mixed origin, it is designated 'cg'. for example, b .cg-abc tm zzz outbred stock that meets specific criteria is designated by placing the lab code before the stock symbol, separated by a full colon (':'). for example, hsd:icr designates an icr (swiss) outbred stock maintained by harlan sprague dawley (hsd). the above overview covers the nomenclature of commonly encountered types of mice. there are numerous additional specifications for nomenclature of mice. details are available at the mgi website (http://www. informatics.jax.org/mgihome/nomen). optimum housing conditions and husbandry practices for research mice should be guided by program requirements to ensure biosecurity, occupational health, efficient use of equipment, labor and financial resources, behavioral needs of mice, and investigator needs for consistent colony maintenance, including standardized husbandry practices and nutrition. the emerging interest in the mouse microbiome in combination with the immune competency of diverse genetically engineered mouse strains demands high standards of mouse care. mouse colonies are optimally maintained as specificpathogen-free (spf) which obligates veterinary and facility management to exclude specific organisms. housing options for spf immunocompetent mice typically include static or individually ventilated microisolator cages, which differ significantly in cost and labor required to maintain. severely immunodeficient strains such as nod.cg-prkdcscid il rgtm wjl/szj (nsg) mice require staff training, caging systems and husbandry practices that minimize risk for opportunistic infections the '/' in balb/c is a lowercase letter because of historical precedent. subsequent substrains follow accepted nomenclature, e.g., balb/cbyj and balb/cann. hybrids of two inbred strains are often used in research, and are particularly common with engineered mutations that are created in -derived es cells, followed by intercrossing the chimeric mice with c bl/ or other background strains of mouse. when an f hybrid is created, the female partner is listed first, e.g., a c bl/ j × s /svpas hybrid would be designated: c bl/ j s /svpasf . ri strain sets that are derived from two parental inbred strains are identified by an x between the two parental strains followed by a hyphen designating the specific ri line, e.g., c bl/ jxdba/ j- , c bl/ jxdba/ j- , etc. cc ri strains do not use the x between the parental strains because they are derived from eight parental strains, so they are designated cc- , cc- , etc. in order to simplify the complexity of this nomenclature, abbreviations are used for common inbred strains and substrains of mice (table . ), but it is important to include the full genetic nomenclature in publications. using the abbreviated nomenclature, c bl/ j s /svpasf mice would be b f and c bl/ jxdba/ j- ri mice would be bxd- . parental order is an important consideration in nomenclature, as a b mouse is genetically different from a b mouse due to mitochondrial dna (from the female) and y chromosome (from the male) differences. mutant genes are designated by a brief abbreviation for the mutation (e.g., bg for beige which arose at the jackson laboratory, j). the symbol for the parent gene is noted in italics, starting with an uppercase letter (e.g., lyst) and the mutant allele is designated in superscript (e.g., lyst bgj ). thus, the beige mutation arose in c bl/ j mice, so that c bl/ j beige mice, which are co-isogenic with c bl/ j mice, are designated c bl/ j-lyst bgj . a transgenic strain is designated by the strain and substrain name, followed by a symbol for the transgene. transgene symbols take the form tg(yyy)#zzz, where 'tg' indicates transgenic, yyy defines the transgene as a brief description of the inserted dna (such as a gene symbol), '#' is the assigned number in the series of events generated using a given construct, and 'zzz' is the lab code. for example, fvb/n-tg(mmtv-erb ) led mice are inbred fvb/n mice in which the rat erb gene was introduced under control of the mouse mammary tumor virus (mmtv) ltr promoter (mmtv-erb ), the first line ( ) created in the laboratory of phil leder (lab code led). when a transgene causes an insertional mutation in an identified endogenous gene, the mutant allele of the gene is designated by using the gene symbol and an abbreviation for the transgene as a superscript (-abc tg zzz ). a targeted mutation, or knockout, is designated by the mutated gene with the identification laboratory animal medicine (foreman et al., ) . barrier practices and microisolator techniques may include autoclaved or irradiated feed and bedding, autoclaved or acidified water, cage-tocage transfer of mice using disinfected forceps, positive displacement change hoods, and verified sanitation of caging and equipment through tunnel or rack washers to prevent fomite transmission of infectious agents (compton et al., ) . in addition to husbandry staff, it is critical to maintenance of colony health status that investigators who handle cages are also trained in these techniques. the microenvironment for mice is the cage which will vary in design, size, and composition. vendors often successfully house production colonies in open-top cages to expedite detection of pathogen transmission should a break occur. end-users usually prefer filter-top microisolator cages which prevent (at least) gross contamination between cages by fecal contamination and aerosolized debris. the objective is to keep mice in an uncrowded, socially compatible, low-odor, dry and clean environment. ambient temperature should minimize any confounding impact on the animal model and energy expenditure for the mice, while also being suitable for staff and investigators. shoebox static cages made of polycarbonate, polypropylene, or polystyrene plastic (in order of decreasing cost and durability) with filtered microisolator tops continue to be used for housing and breeding mice. older cage designs are being rapidly supplanted by individually ventilated caging systems that promote the advantages of increasing housing capacity, decreasing labor costs, and mitigating exposure of mice to noxious gases such as ammonia and exposure of humans to allergens. as more advanced caging systems are developed, the level of biosecurity may be increased but at the cost of increased health surveillance efforts to detect the source of an infectious outbreak (shek, ) . disposable, recyclable polyethylene caging is a recent innovation, particularly for facilities not equipped with a cage wash facility. animal care programs should carefully consider the necessity for housing mice on wire-mesh flooring because of injury risk to limbs and thermoregulation issues in neonates and hairless mice which are more difficult to maintain without nesting material. solidbottom cages should contain sanitary bedding, such as hardwood chips, paper products, or ground corn cob. criteria for selecting bedding vary with experimental and husbandry needs. it may be preferable to irradiate or autoclave bedding, but if this is not done, the bedding should be used only after its origin and microbial content have been evaluated (table . ). germfree and gnotobiotic mice require positive pressure isolators, most usually flexible film, with additional protection provided by sterile air through high-efficiency particulate air (hepa) filters. this equipment can be negatively pressurized when the objective is to contain known or unknown pathogens. animal care programs should establish enrichment policies which for mice should include social housing when mice are compatible and experiments do not require single housing. species-specific behaviors are encouraged by nesting material and hiding places such as tubes or shacks. nutrient requirements for the mouse are influenced by genetic background, disease status, growth rate, pregnancy, lactation, and environmental factors such as ambient temperature. the best current estimate of nutritional requirements is shown in table . . nutritional requirements for laboratory mice are also published periodically by the national research council and have been reviewed by knapka and coworkers (knapka et al., ; knapka, ) . feed intake and weight gain data are used to estimate the nutritional needs of a particular stock or strain. mice consume about - g of feed per day after weaning, and maintain this intake throughout life. outbred mice tend to gain weight faster than inbred mice and are heavier at maturity (figs. . and . ). diet is often neglected as a variable in animal-related research. diet can influence responses to drugs, chemicals, or other factors and lead to biased research results. therefore, diet must provide a balance of essential kraft ( ) . knapka ( ) . b linoleic acid: . % is adequate. c john and bell ( ) . d theuer ( ) . e knapka et al. ( ). f nutrition ( . g hurley and bell ( ) . h pleasants et al. ( ) . nutrients, and contaminants must be kept to a minimum (see also chapter ). natural-product commercial diets for mice are usually satisfactory for breeding and maintenance. animal care programs should avoid using fresh produce, grains, fish meal, or other supplements to minimize exposure of colonies to pathogens or harmful chemicals such as pesticide residues or phytoestrogens (guerrero-bosagna et al., ) . mouse diets can be purchased as open-formula, fixedformula, constant nutrition, and closed-formula which laboratory animal medicine are designed to reduce variation in experimental data attributable to diet (reviewed in barnard et al. ( ) ). diets are supplied in standard, irradiated, or autoclavable formulations. irradiated diets will be virtually free of live microorganisms but have the risk of residual, radio-resistant bacteria. autoclavable diets are higher in heat-labile nutrient content. many programs use sterilized mouse chow exclusively to minimize risk of opportunistic infections. because commercial diets vary in nutrient content, diets should be selected for optimal maintenance of adult mice or for growth and reproduction in breeding colonies. mice should have continuous access to potable water even if a high-moisture diet is fed. water is needed for lubrication of dry food and for hydration. adult mice drink - ml of water per day. decreased water intake will decrease food consumption. water imbalance may occur immediately post weaning and weanlings on automatic watering systems need extra attention. water intake will decrease in sick mice. therefore, dosing mice with medicated water requires careful assessment of hydration and clinical or experimental efficacy of the compound administered. the main reference used to update this section of the rd edition is volume iii; normative biology, husbandry and models in the mouse in biomedical research, nd edition, aclam series published by academic press. normative data on the mouse are presented in table . , and clinical chemistry reference ranges are summarized in table . . mice have a relatively large surface area per gram of body weight. this results in dramatic physiologic changes in response to fluctuations in the ambient temperature (t a ). the mouse responds to cold exposure, e.g., by nonshivering thermogenesis. a resting mouse acclimated to cold can generate heat equivalent to triple the basal metabolic rate, a change that is greater than for any other animal. a mouse must generate about kcal/m per h to maintain body temperature for each °c drop in t a below the thermoneutral zone. mice cannot tolerate nocturnal cooling as well as larger animals that have a greater heat sink. therefore, it is not advisable to conserve energy in animal quarters at night by lowering t a . because of the ratio of evaporative surface to body mass, the mouse has a greater sensitivity than most mammals to water loss. its biological half-time for turnover of water ( . days) is more rapid than for larger mammals. water conservation is enhanced by cooling of expired air in the nasal passages and by highly efficient concentration of urine. the conservation of water can preempt thermal stability. if the mouse had to depend on the evaporation of body water to prevent elevations of body temperature, it would go into shock from dehydration. the mouse has no sweat glands, it cannot pant, and its ability to salivate is severely limited. mice can partially compensate for changes in t a increases from °c to °c. it adapts to moderate but persistent increases in environmental temperature by a persistent increase in body temperature, a persistent decrease in metabolic rate, and increased blood flow to the ears to increase heat loss. its primary means of cooling in the wild is behavioral -retreat into a burrow. in the confinement of a cage, truck, or plane, mice do not survive well in heat and begin to die at an ambient temperature of °c or higher. thus, the mouse is not a true endotherm. in fact, the neonatal mouse is ectothermic and does not have well-developed temperature control before days of age. the thermoneutral zone for mice varies with strain and with conditioning but is about . - . °c, narrower than that of any other mammal measured thus far. thermoneutrality should not be equated with comfort or physiological economy. recent data have suggested that mice housed under routine vivarium conditions are chronically cold-stressed. mice maintained at °c were shown to expend more energy compared with mice housed at intermediate ( °c) and a higher temperature ( °c) with an increase in glucose utilization and activation of brown adipose tissue (david et al., ) . in contrast, other studies report that mice in a t a range of - °c grow faster, have larger litters, and have more viable pups than those maintained in the thermoneutral zone. the respiratory tract has three main portions: the anterior respiratory tract consists of nostrils, nasal cavities, and nasopharnyx; the intermediate section consists of larynx, trachea, and bronchi, all of which have cartilaginous support; and the posterior portion of the respiratory tract consists of the lungs. the left lung is a single lobe. the right lung is divided into four lobes: superior, middle, inferior, and postcaval (cook, ) (fig. . loeb and quimby ( ) . a mouse at rest uses about . ml o /g/h, which is about times more o /g/h than is used by an elephant. to accommodate for this high metabolic rate, the mouse has a high alveolar p o ; a rapid respiratory rate; a short air passage; a moderately high erythrocyte (rbc) concentration; high rbc hemoglobin and carbonic anhydrase concentrations; a high blood o capacity; a slight shift in the o -dissociation curve, enabling o to be unloaded in the tissue capillaries at a high p o ; a more pronounced bohr effect, i.e., the hemoglobin affinity for o with changes in ph is more pronounced; a high capillary density; and a high blood sugar concentration. the kidneys, ureters, urinary bladder, and urethra form the urinary system. the paired kidneys lie against the dorsal body wall of the abdomen on either side of the midline. the right kidney is normally located anterior to the left kidney. kidneys from males of many inbred strains are consistently heavier than kidneys from females. the glomeruli of mice are small, about μm in diameter, or about half the size of glomeruli in rats. there are, however, . times as many glomeruli in the mouse, and the filtering surface per gram of tissue is twice that of the rat. mice excrete only a drop or two of urine at a time, and it is highly concentrated (table . ). the high concentration is made possible by long loops of henle and by the organization of giant vascular bundles (vasa recta) associated with the loops of henle in the medulla. the mouse can concentrate urine to mosm/l, whereas humans can concentrate to a maximum of mosm/l. mice normally excrete large amounts of protein in the urine. taurine is always present in mouse urine, whereas tryptophan is always absent. creatinine is also excreted in mouse urine, a trait in which mice differ from other mammals. the creatinine/creatine ratio for fasting mice is about : . . mice excrete much more allantoin than uric acid. the submaxillary salivary gland, a mixed gland in most animals, secretes only one type of saliva (seromucoid) in the mouse. the tubular portion of the gastrointestinal (gi) tract consists of esophagus, stomach, small intestine, cecum, and colon. the esophagus of the mouse is lined by a thick cornified squamous epithelium, making gavage a relatively simple procedure. the proximal portion of the stomach is also keratinized, whereas the distal part of the stomach is glandular. gastric secretion continues whether or not food is present. the gastrointestinal flora consists of (at least) species of bacteria that begin to colonize the alimentary canal selectively shortly after birth. the ceca of normal mice contain up to bacteria/g of feces. the bacteria throughout the gastrointestinal tract form a complex ecosystem that provides beneficial effects, such as an increase in resistance to certain intestinal pathogens, production of essential vitamins, and homeostasis of important physiological functions. gnotobiotic animals colonized with known microbiota have been used to great advantage as models for biomedical research (see chapter ). for certain studies, it is desirable to colonize germfree mice with a defined microbiota. in the mid- s, schaedler was the first to colonize germfree mice with selected bacteria isolated from normal mice (schaedler and orcutt, ) . he subsequently supplied animal breeders with this group of microorganisms. these defined bacteria included aerobic bacteria and some less oxygen-sensitive anaerobic organisms. the so-called extremely oxygen-sensitive (eos) fusiform bacteria, which make up the majority of the normal microbiota of rodents, were not included, because of technical difficulties in isolation and cultivation. of the defined microbiotas later used for gnotobiotic studies, the one known as the 'schaedler flora' was the most popular. in , the national cancer institute (nci) decided to revise the schaedler flora, or 'cocktail' consisting of eight bacteria, in order to standardize the microbiota used to colonize germfree rodents. the new defined microbiota, now known as the 'altered schaedler flora' (asf), consisted of four members of the original schaedler flora (two lactobacilli, bacteroides distasonis, and the eos fusiform bacterium), a spiral-shaped bacterium, and three new fusiform eos bacteria. studies have quantified the regional colonization of the asf strains along the gastrointestinal tract (sarma-rupavtarm et al., ) (fig. . ) . individual strain abundance was dependent on oxygen sensitivity, with microaerotolerant lactobacillus murinus asf present at - cells/g of tissue in the upper gastrointestinal tract and obligate anaerobic asf strains being predominant in the cecal and colonic flora at - cells/g of tissue. it is difficult to monitor a gnotobiotic mouse colony with a defined microbiota. it is necessary to demonstrate that microorganisms of the specified microbiota are present and that adventitious microorganisms are absent. in the past, monitoring relied on bacterial morphology, limited evaluation of biochemical traits, and growth characteristics. with the advent of polymerase chain reaction (pcr) technology, the eight asf strains were identified taxonomically by s rrna sequence analysis . three strains were previously identified as lactobacillus acidophilus (strain asf ), l. salivarius (strain asf ), and bacteroides distasonis (strain asf ), based on phenotypic criteria. s rrna analysis and genome sequencing indicated that each of the strains differed from its presumptive identity (wannemuehler et al., ) . the s rrna sequence of strain asf is essentially identical to the s rrna sequences of the type strains of l. murinus and l. animalis (both isolated laboratory animal medicine from mice), and all of these strains probably belong to a single species. strain asf is a novel lactobacillus that clusters with l. acidophilus and l. lactis. strain asf is a parabacteroides sp. the spiral-shaped strain, strain asf , is in the flexistipes phylum, exhibits sequence identity with rodent isolates of robertson, and has been formally named, mucispirillum schaedleri (robertson et al., ) . the remaining four asf strains, which are eos fusiform bacteria, group phylogenetically with the low-g + c content gram-positive bacteria (firmicutes, bacillus-clostridium group) (asf -eubacterium plexicaudatium; asf -firmicutes bacterium; asf and asf -clostridium sp.) (fig. . ) . the s rrna sequence information was determined by dewhirst et al. ( ) and draft genome sequences for each member of asf were recently published (wannemuehler et al., ) . this genetic data will permit detailed analysis of the interactions of asf organisms during development of intestinal disease in mice that are coinfected with a variety of pathogenic microorganisms. the lymphatic system consists of lymph vessels, thymus, lymph nodes, spleen, solitary peripheral nodes ( fig. . ) , and intestinal peyer's patches. mouse lymph nodes are numerous but typically are small, reaching only a few millimeters. the typical lymph node is beanshaped and consists of a cortex and a medulla. the cortex is divided into b lymphocyte domains, called primary follicles, and t lymphocyte domains, known s i i i i i i c c l l l e s s i i i i i i c c l l l e s s i i i i i i c c l l l e s s i i i i i i c c l l section of the gi tract bacteroides sp. asf the sections are taken from the esophagus (esop.) (section e ), stomach (sections s and s ), small intestine (sections i to i ), ileocecal junction and apical cecum (sections c and c , respectively), and colon (sections l to l ). from sarma-rupavtarm et al. ( ). as the diffuse cortex. the mouse does not have palatine or pharyngeal tonsils. the spleen lies adjacent to the greater curvature of the stomach. different strains of mice have varying degrees of accessory splenic tissue. age, strain, sex, and health status can affect the size, shape, and appearance of the spleen. male spleens, e.g., may be % larger than those of females. most lymphocytes enter and leave the spleen in the bloodstream. the so-called white pulp of the spleen is organized along the central arteriole and is subdivided into t-and b-cell zones. the periarteriolar sheath is composed mainly of cd + and cd + t cells, and lymph follicles, which often contain germinal centers, are located at the periphery. the red pulp consists of sinusoids and hemoreticular tissue. cellular and humoral components of immunity are distributed to the bloodstream and tissues by efferent lymphatic vessels and lymphatic ducts, which empty into the venous system. the thymus is a bilobed lymphoid organ lying in the anterior mediastinum. it reaches maximum size around the time of sexual maturity and involutes between and days of age. the thymus plays a major role in maturation and differentiation of t lymphocytes. this function is not complete in newborn mice. thymectomy is routinely performed in immunological research for experimental manipulation of the immune system. thymectomy of newborn mice causes a decrease in circulating lymphocytes and marked impairment of certain immune responses, particularly cellular immune responses. thymectomy in adult mice produces no immediate effect, but several months later mice may develop a progressive decline of circulating lymphocytes and impaired cellular immune responses. the mutant athymic nude mouse is a powerful experimental tool in the study of the thymus in immune regulation (fogh, ) . the mucosa-associated lymph tissue (malt) contains more lymphoid cells and produces greater amounts of immunoglobulin than both the spleen and the lymph nodes. the term malt designates all peripheral lymphoid tissues connecting to cavities communicating with the external milieu. they include the peyer's patches, the cecal lymphoid tissue, and the lymphoid tissue in upper and lower respiratory tract, as well as the respiratory and genitourinary system. lymphatics drain these lymphoid-rich areas, thus providing a direct link with lymph nodes and the bloodstream. bone marrow and splenic red pulp produce erythrocytic, granulocytic, and megakaryocytic precursors over the life of the mouse. bone marrow is located in the protected matrix of cancellous bone and is sustained by reticular tissue rich in blood vessels and adipose cells (pastoret et al., ) . normal hematologic values are listed in table . . bone marrow-derived mononuclear phagocytes remove particulate antigens and act as antigen-presenting cells for lymphocytes. tissue macrophages, which often function in a similar way, are found in many tissues, including peripheral lymphoid tissues, lung, liver, intestine, and skin. the cardiovascular system of mice is reviewed extensively by hoyt et al. in the nd edition of volume iii; normative biology, husbandry and models in the aclam series the mouse in biomedical research (hoyt, ) . the heart consists of four chambers, the thin-walled atria and the thick-walled ventricles ( fig. . ) . mice conditioned to a recording apparatus have mean systolic blood pressures ranging from to mmhg. an increase in body temperature does not lead to an increase in blood pressure. heart rate, cardiac output, and the width of cardiac myofibers are related to the size of the animal. heart rates from to /min have been recorded for mice, and there are wide variations in rates and blood pressure among strains. the skeleton is composed of two parts: the axial skeleton, which consists of the skull, vertebrae, ribs, and sternum, and the appendicular skeleton, which consists of the pectoral and pelvic girdles and the paired limbs. the normal vertebral formula for the mouse is c t l s c , with some variations among strains, especially in the thoracic and lumbar regions. normal mouse dentition consists of an incisor and three molars in each quadrant. these develop and erupt in sequence from front to rear. the third molar is the smallest tooth in both jaws; the upper and lower third molar may be missing in wild mice and in some inbred strains. the incisors grow continuously and are worn down during mastication. the mouse brain has a typical mammalian structure as documented by a detailed study of the neuroanatomy of the c bl/ j mouse (sidman et al., ) . more recently, gene expression patterns have been used to study the functional anatomy of the mouse brain (bohland et al., ) . use of wild-type and genetically modified mice in behavior, learning, and memory paradigms has exponentially increased over the last decade. the male reproductive organs consist of paired testes, urethra, penis, prostate and associated ducts and glands ( fig. . ) . the female reproductive organs consist of paired ovaries and oviducts, uterus, cervix, vagina, clitoris, and paired clitoral glands ( fig. . ). the clitoral glands are homologous to the male preputial glands and secrete a sebaceous substance through ducts entering the lateral wall of the clitoral fossa. the female mouse normally has five pairs of mammary glands, three in the cervicothoracic region and two in the inguinoabdominal region ( fig. . ). the mammary glands are often not appreciated for how far they extend over the cervical, axillary, and inguinoabdominal flank regions which become the following section summarizes normal reproduction in the mouse. the reader is referred to a more comprehensive text in the aclam series (pritchett and taft, ) and online resources such as the jackson laboratories publication of the biology of the laboratory mouse (http://jaxmice.jax.org/jaxnotes/ / j.html). external influences, such as noise, vibration, diet, light cycle, and cage density, and intrinsic factors, such as health status, genetics, and parity impact reproductive success by directly or indirectly influencing the hypothalamic-pituitary axis for hormonal control of ovarian and testicular function. genotype also dramatically affects the reproductive performance of the mouse. coincident with the explosion in the number of mouse strains, each with unique induced or spontaneous mutations, a sound breeding program must include training of care staff to recognize anticipated and unanticipated breeding performance and strain or stock characteristics. in the new age of genomics, older methods of confirming genetic purity of mouse lines are being replaced with formal genetic monitoring by comparing strain-specific panels of single-nucleotide polymorphisms (snps). follicle-stimulating hormone promotes gametogenesis in both sexes. luteinizing hormone promotes the secretion of estrogen and progesterone in the female and androgen in the male. prolactin promotes lactation and development of the ovary during pregnancy. these gonadal hormones also ensure proper maintenance of the reproductive tract and modulate behavior to promote successful mating. the hypophysis is usually responsive to hormonal influence by day in the male and day in the female. ovarian follicle development begins at weeks of age and matures by days. rising levels of gonadotropins evoke signs of sexual maturity at about the same age. in the female, estrogen-dependent changes such as cornification of vaginal epithelium at the vaginal opening can occur as early as - days. puberty is slightly later in the male (up to weeks). sexual maturation varies among strains and stocks of mice and is subject to seasonal and environmental influences. mating behavior and the ability to conceive and carry fetuses to parturition are under complex hormonal control mediated by the anterior pituitary. the mouse is polyestrous and cycles every - days. in the first two phases (proestrus and estrus), active epithelial growth in the genital tract culminates in ovulation. degenerative epithelial changes occur during the third phase, followed by diestrus, a period of quiescence or slow cell growth. the cycle can be followed by changes in the vaginal epithelium that are often used to determine optimum receptivity of the female for mating and fertilization (table . ). patency of the vaginal orifice and swelling of the vulva are useful signs of proestrus and estrus ( fig. . ) . irregularities of the estrous cycle occur during aging. seasonal and dietary factors, such as estrogenic substances found in a variety of feeds, and genetic backgrounds also influence estrous cycles. estrus is routinely observed in mice at about - h after parturition (postpartum estrus). however, cornification of the vagina is not complete, and fertile matings are not as frequent compared with normal estrus. mice are spontaneous ovulators. ovulation does not accompany every estrus, and estrus may not coincide with every ovulation, because estrus is dependent on gonadal hormones, whereas ovulation is responsive to gonadotropin. the cyclicity of estrus and ovulation is controlled by the diurnal rhythm of the photoperiod. mating, estrus, and ovulation most often occur during the dark phase of the photoperiod. reversing the timing cook ( ) . laboratory animal medicine of the light-dark cycle reverses the time of estrus, ovulation, and mating. pheromones (table . ) and social environment also affect the estrous cycle. for example, estrus may be suppressed in group-housed female mice and reentry into estrus can be synchronized by exposure to pheromones in male mouse urine ('whitten effect'). once exposed to male urine, most female mice will be in estrus within days with a second estrus in about days. hence, estrus can be synchronized by group-housing females prior to pairing with males. in contrast, pheromones from a strange male mouse, particularly of a different strain, may prevent implantation or pseudopregnancy in recently bred females and is known as the 'bruce effect'. see section ii.c on behavior for more detail on the effect of pheromones on mouse reproductive behavior. mating is normally detected by formation of a vaginal plug (a mixture of the secretions of the vesicular and coagulating glands of the male) whose prevalence is highly strain dependent. the plug usually fills the vagina from cervix to vulva (fig. . ). plug detection is often coupled with vaginal cytology to evaluate fertility and conception. when the cervix and vagina are stimulated physically during estrus, prolactin is released from the anterior pituitary to enable the corpus luteum to secrete progesterone. secretion continues for about days. if fertilization has occurred, the placenta takes over progesterone production. if fertilization does not occur, a pseudopregnant period ensues, during which estrus and ovulation do not occur. fertilization usually takes place ( ) testis, ( ) head of epididymitis, ( ) caudal epididymitis, ( ) vas deferens, ( ) testicular vein, ( ) ampullary gland, ( ) seminal vesicle, ( ) anterior prostate, ( ) ureter, ( ) bladder, ( ) ventral prostate, ( ') dorsal prostate, ( ) urethra, ( ) bulbourethral muscle, ( ) ischiocavernosus, ( ) bulbourethral gland, ( ) diverticulum of bulbourethral gland, ( ) penis, ( ) preputial gland, ( ) glans penis, ( ) prepuce, ( ) testicular artery, and ( ) vas deferens artery. adapted from (komarek, ) . fallopian tube, ( ) uterine horn, ( ) endometrium, ( ) cervix, ( ) vagina, ( ) vaginal vestibulum, ( ) clitoris, ( ) clitoral gland, ( ) urethra, ( ) bladder, ( ) medial ligament of bladder, ( ) lateral ligament of bladder, ( ) left ureter, ( ') right ureter, ( ) mesovarium, ( ) mesometrium, ( ) ovarian artery, ( ) uterine horn artery, and ( ) ovarian artery and vein. adapted from (komarek, ) . adapted from komarek ( ) . in the ampulla or the upper portion of the oviduct. ova can be fertilized to produce normal embryos for - h after ovulation. gestation is usually - days. because of postpartum estrus, lactation and gestation can occur simultaneously. lactation can delay gestation because of delayed implantation. this may cause prolongation of gestation for up to - days in certain inbred strains. the effective reproductive life of some inbred strains approaches years where optimum environmental conditions are maintained, but litter size usually decreases as the female ages. therefore, females are usually retired by months of age. average litter size is strain dependent and commonly ranges from to pups. maternal care can account for about % of the variation in body weight of neonatal mice. nursing females usually lactate for weeks. milk production increases up to days postpartum and then declines until weaning at days. interestingly, oxytocin is required for nursing but is not essential for parturition or reproductive behavior (nishimori et al., ) . some transmission of humoral immunity from dam to progeny occurs in utero, but the majority of antibody is transferred through colostrum. transmission of passive immunity by colostral antibodies has been demonstrated to a wide variety of antigens, including viruses, bacteria, and parasites. antibodies continue to be secreted in the milk throughout lactation. decay of maternally acquired immunity occurs within several months after weaning. loss of maternal immunity increases susceptibility to infection and warrants continued care of weaned mice under barrier conditions. mice are socially gregarious animals with strong family bonds who communicate through complex olfactory, auditory, tactile, and visual signals. wild mice aggregate into groups called demes with low exchange of individuals between different groups. each deme consists of kinrelated members with a high degree of natural inbreeding, higher mutation rates compared to other mammals, and a wide range of developmental flexibility based on early life experience, which all contribute to their remarkably successful environmental adaptability. the deme is composed of a dominant breeding male, a hierarchy of females, subordinate males, and juveniles. wild mice occupy territories measuring just a few square meters when food is abundant to several square kilometers. mice are crepuscular (active during the twilight hours of dawn and dusk), strongly territorial, and omnivorous. coprophagy contributes to approximately one-third of their ingesta as an essential nutritional activity. aside from territoriality, social interactions, breeding, burrowing (when conducive substrates are available), and nest building are major activities. in managing laboratory mice, it is important to understand the complex behavioral biology of their free-living counterparts (latham and mason, ) . chemo-olfactory communication is mediated through extremely diverse chemical factors that trigger innate (non-learned) social responses among conspecifics, known as pheromones (table . ). pheromones have been traditionally divided into two broad categories: releaser pheromones, which elicit an immediate behavioral response, and primer pheromones, which mediate a slowly developing and longer-lasting endocrine response. this original definition of pheromone categories has been expanded to another category, termed signaler pheromones, which convey individual or group identity, as well as mediating parent-offspring recognition and mate choice. the biology and genetics of pheromone signaling is being extensively studied in the mouse as a model of mammalian pheromone communication (brennan and zufall, ; rodriguez and boehm, ) . mouse pheromones are excreted in the urine, as well as plantar, salivary, lacrimal, preputial, and mammary glands. in the urine, major urinary proteins (mups), small peptides, mhc class i peptides, volatile chemicals, and sex hormones all contribute to chemosignals that communicate dominance, kinship, diversity, and gender. wild mice possess a great deal of individual variations of roberts et al. ( ) . c ferrero et al. ( ) . laboratory animal medicine these elements, providing a 'bar code' that distinguishes individuals. inbreeding of laboratory mice has reduced individual variation, but each inbred strain possesses a characteristic array of signals, and to a certain extent, unique signals exist among individuals within a strain (sharrow et al., ; sturm et al., ) . pheromones are detected by sensory neurons in the vomeronasal organ, the olfactory epithelium, and the lesser known septal organ of masera within the olfactory epithelium, and the gruenberg ganglion, which is located at the anterior end of the nasal cavity (breer et al., ; chamero et al., ; liberles and buck, ; restrepo et al., ) . neuronal signals are transmitted to the ganglion layer of the olfactory bulb, and thence to the brain. mups are important components of chemosensory communication in mice, and also an important occupational hazard to human handlers. chromosome contains a cluster of mup genes, plus a number of pseudogenes. mups are small soluble proteins known as lipocalins, which bind small organic chemicals (pheromones) with high affinity, and function as pheromone transporters and stabilizers (thereby contributing to slow release), but also act as protein pheromones themselves. they are synthesized in the liver and excreted in the urine, as well as nasal mucosa, lacrimal glands, and salivary glands. their endogenous role on metabolic activity is not yet understood. male mice excrete significantly more mups in the urine than females. one wellcharacterized mup is 'darcin', named after fitzwilliam darcy, the romantic hero in pride and prejudice. as its name implies, it is a female attractant. mups also act as kairomones, which function as chemical signals between species. for example, cat and rat mups invoke fear in mice. mups are important in the laboratory animal management context, as they are excreted in copious amounts ( - mg/ml in urine) and are potent allergens for humans, particularly mus m (ag or ma ), which is encoded by the mup gene (sharrow et al., ) . chemosensory communication has numerous behavioral effects that influence mouse social interactions. one of the most studied behavioral effects is the bruce effect, or pregnancy block, which is a complex physiologic response in which recently conceived females resorb fetuses during early pregnancy in the presence of an unrelated male, particularly a dominant male. the continued presence of the original mate protects the female from this effect (bruce, ) . the vandenbergh effect results in acceleration of puberty of juvenile females in response to male urine (vandenbergh, ) . the lee-boot effect occurs among group-housed females that are isolated from males, in which there is suppression of estrus cyclicity (van der lee and boot, ) . the whitten effect results in synchronization of estrus among a group of females in response to a male (whitten et al., ) . the lee-boot and whitten effects are utilized in the laboratory to assist in induction of synchronized timed pregnancy, but the bruce effect can have deleterious consequences on breeding colonies when foreign males are introduced to a breeding colony, as pheromone communication can occur in the absence of direct contact. the above effects are well-defined pheromone-driven behavioral responses, but chemosensory communication has a myriad of other effects. estrus, pregnant, or lactating females also accelerate puberty among juvenile females. females use odor cues to avoid parasite-laden males, males prefer odors of estrus females, and estrus females prefer odors of dominant males. mice have strong mating and social preferences based upon mhc proteins, which indicate genetic relatedness. maternal recognition of young is also mhc-related, and pups prefer nest odors of maternal and sibling pups based upon mhc relatedness. male aggression against unrelated males is also a strong mhc-related phenomenon. mhc haplotypes determine not only mhc proteins in the urine, and mhc-specific olfactory receptors, but also the composition of volatile chemicals in the urine (kelliher and wersinger, ) . the complexity of social communication extends to auditory stimuli as well. male mice utilize ultrasonic 'birdsong' to vocally communicate and attract females. mouse vocalization patterns are largely genetically innate and unique to each strain of mouse, but they can also be modified, or learned, to a limited extent (arriaga et al., ) . the behavioral biology of the mouse is highly complex, and depends upon genetic, physiologic, social, and environmental variables, which all impact on how laboratory mice can best be managed in captivity. it is clear that this rich complexity cannot be fully addressed under laboratory conditions, but that does not mean that basic needs, such as nest building, burrowing, foraging, and olfactory environments, cannot be provided. for example, intermale aggression, which is particularly apparent in some strains of mice such as balb/c and swiss-origin stocks and strains, can be minimized by maintaining males from infancy as sibling groups, since adult siblings tend not be aggressive to one another. this sibling bond, however, can be easily broken by short-term separation. environmental enrichment often features provision of plastic houses, which may make vivarium managers feel good, but maximal enrichment can be provided by provision of nesting material, which includes structural scaffolding, such as crinkled cardboard, which facilitates construction of three-dimensional nests. mouse nests are replete with 'appeasement' pheromones, thereby contributing to harmony within the cage, whereas introduction of dirty bedding has the opposite effect. frequent cage changing, including removal of established nests, is highly stressful and disruptive to social harmony within a cage. provision of appropriate and adequate amounts of bedding material that is conducive to burrowing is desirable. it is important to remember that mice are socially gregarious, and that mouse welfare is optimally enriched by other mice within a socially harmonious deme (latham and mason, ; van loo et al., ) . a laboratory mouse ethogram, defined as an operationalized list of mouse behaviors, arranged by their adaptive meaning to the animal, is available on the web: www. mousebehavior.org. behavioral phenotyping, particularly of transgenic mice, is used extensively in genomic research. a wide variety of standardized test batteries and approaches are used, depending upon the focus of research (reviewed in crawley ) . initial behavioral evaluations include general health, body weight, body temperature, appearance of the fur and whiskers, and neurological reflexes assessment. specific tests include observations of home cage behaviors, righting reflex, acoustic startle, eye blink, pupil constriction, vibrissae reflex, pinna reflex, digiscan open field locomotion, rotarod motor coordination, hanging wire, footprint pathway, visual cliff, auditory threshold, pain threshold, and olfactory acuity. novel and complex environmental enrichment in animal housing conditions facilitates enhanced sensory and cognitive stimulation as well as physical activity. environmental enrichment and exercise have beneficial effects such as cognitive enhancement, delayed disease onset, enhanced cellular plasticity, and associated molecular processes in animal models of brain disorders (pang and hannan, ) . the immune system of the mouse is very similar to that of humans. the availability of inbred mouse strains, in which each individual animal expresses identical mhc alleles so that tissues and cells can be transplanted without tissue rejection, greatly simplifies and indeed enables functional analyses of immune system components not possible with any other outbred mammalian species. in addition, the ability to genetically manipulate the mouse genome, adding to, altering, and deleting existing genes, enables unprecedented in vivo analysis of immune cell functions. it is for these reasons that the mouse is the primary animal model for immunology research. the immune system is an unusual organ system in that it consists of both solid tissues and various migrating cell populations. the bone marrow and thymus are considered primary lymphoid organs, as sites of hematopoiesis and b-and t-lymphocyte development, respectively. lymph nodes, spleen, and intestinal peyer's patches are considered secondary lymphoid tissues, as sites of immune response initiation. lymph nodes and spleen are analyzed frequently for studies of immune responses and as organs for immune cell isolation. tertiary lymphoid tissue sites are those that form in other solid organs in response to an insult or microbial exposure. among them are the lymphoid cell aggregates of the gastrointestinal and respiratory tract, also called 'gut-associated lymphoid tissue' (galt) and bronchusassociated lymphoid tissues (balt). leukocytes are classified as belonging to the innate or adaptive immune system. the innate immune system responds rapidly to an antigen insult via recognition of pathogen-associated molecular patterns (pamps), such as lipopolysaccharide, bacterial flagellin, single (s)-and double-stranded (ds) rna, and non-methylated dna, via extracellular or intracellular pattern recognition receptors (prrs). receptors include the toll-like receptors (tlrs), such as tlr (recognizing lps), tlr / (ss and dsrna) and tlr (dna), nod-like receptors (nod / ), and rig-like receptors (rig-i, mda- ) among others (takeuchi and akira, ) . cells of the innate immune system are monocytes/macrophages, granulocytes and dendritic cells as well as innate-like lymphocyte populations (ilc) , and , which include natural killer (nk) cells (spits et al., ) . cells of the adaptive immune system (t and b lymphocytes) express a highly antigen-specific receptor that has arisen through gene rearrangement (t-cell and b-cell receptors, respectively). b cells of the b- lineage and γδ t cells are regarded as innate-like cells, as they express a rearranged antigen receptor but seem to respond in an innate-like manner. leukocytes are identified and classified by sets of monoclonal antibodies (mab) against uniquely expressed surface receptors, typically measured by flow cytometry. identification of a unique receptor by one or more mab of the same specificity leads to the assignment of a receptor name, as a 'cluster of differentiation (cd)'. for example, t cells are differentiated into two subsets based on their expression of either cd or cd . cd + t cells (t helper cells) recognize peptides presented in mhc class ii and promote b-lymphocyte activation and activate and regulate cellular immune responses via secretion of differing cytokines (see below). cd + t cells recognize antigenic peptides presented in mhc class i and serve as cytotoxic cells during the cell-mediated immune response where they can destroy infected cells (e.g., against cells containing infectious agents). the major function of b cells is to respond to an encounter with an antigen/pathogen with the production of highly antigen-specific immunoglobulins (ig; antibodies), which can bind to and inactivate pathogens and toxins. activation of b cells can lead to their differentiation to plasma cells, which produce large amounts of ig. five laboratory animal medicine classes or ig 'isotypes' can be distinguished, which differ in effector function: igm, igg, iga, ige, and igd. the latter is expressed only on the surface of b cells in mice. the igg class, the most abundant antibody class in the serum, is further divided into subtypes: igg , igg a/c , igg b , and igg . polymorphisms exist on the ig locus such that some strains of mice produce the igg a subtype (e.g., balb/c), whereas others produce igg c (e.g., c bl/ ) (zhang et al., ) . additional allelic polymorphisms of the locus also exist. for example, balb/c and sv mice express the igh-a allotype, whereas c bl/ mice express the igh-b allotype. recombinant inbred strains of mice exist for both balb/c and c bl/ , which harbor the reciprocal igh locus (i.e., igh-b for balb/c and igh-a for c bl/ mice). these mice are useful tools for tracking b cells following adaptive cell transfer via allotype-specific mab (see below). immunoglobulin isotype production varies according to the type of immunogen used to evoke the response. igm is secreted short term after initial exposure to an antigen, followed by the other ig isotypes. in viral and intracellular bacterial infections, igg a/c is dominant, whereas in extracellular bacterial infections igg dominates the response. igg b and igg are usually induced to carbohydrate or lipid antigens. ige is linked to parasitic infections and to allergy. serum antibodies specific for an immunogen can often be measured for the life of the animal. while serum iga levels are low, iga is the highest produced ig in mice. iga production, however, occurs in plasma cells lodged in the lamina propria of mucosal tissues, from where the iga is actively transported in dimeric form onto the luminal surface of mucosal tissues as 'secretory' iga (brandtzaeg, ). cytokines are secreted signaling molecules involved in cell-cell communication in a complex biological system (table . ). these include the large family of interleukins (ils, currently il- to il- ), tumor necrosis factors (tnfs), interferons (type i, ii, and iii) and growth factors such as granulocyte-macrophage colonystimulating factor (gm-csf) and stem cell factor (scf). cytokine secretion often occurs in response to recognition of antigen via prr or tcr. because of their importance in modulating immunity to antigenic stimuli, mice with specific deletions or overexpression of individual cytokines have been made and have contributed to a detailed understanding of many of their often pleotropic functions (akdis et al., ) . chemokines are a similarly large group of small, secreted molecules that regulate cell trafficking to sites of antigen encounter but also facilitate cell-cell contact by acting as chemoattractants. chemokines are grouped according to the number of cysteines and disulfide bonds in the molecule into c-x-c-, c-c, c, and cx cl chemokine ligands (l) and receptors (r) and designated accordingly as cxcr - /cxcl - and ccr - /ccl - (allen et al., ) . immune responses must be coordinated to provide the most appropriate effector functions for the type of pathogen/antigen encountered. immune effector responses differ depending on the life cycle (facultative or obligate intracellular, extracellular, localized, systemic, etc.) and antigen types displayed by the encountered antigen/ pathogen, because this affects the type of prr engaged and activated. prr engagement leads to cytokine and chemokine responses by the first responders, i.e., epithelial cells, local macrophage populations and other innate cells. the type of cytokines and chemokines produced then dictates the types of cells recruited to the site of infection and their subsequent differentiation and functions. the prr engagement also leads to antigen uptake, activation and migration of dendritic cells (dcs) from the site of insult to the regional lymph nodes, where dcs present antigen peptides on mhc molecules to t cells. in addition, the dcs secrete cytokines induced by the initial prr activation, which cause the differentiation of cd t cells towards a particular effector response. for example, secretion of il- in response to activation of tlr or will result in the induction of interferongamma (ifn-γ) production by cd t cells, whereas il- and tgf-β production by dc will induce cd t cells to secrete il- (kara et al., ) . because the dc translates signals from prr at the site of infection into differentiation signals for t cells in the lymph tissues, these cells are regarded as a 'bridge' between the innate and adaptive immune systems. the specific ig isotype secreted in response to a pathogen depends to a large degree on the type of cytokine produced by cd t cells that provide 't-cell help' for b cells. t cells that interact with b cells are identified as a discrete subset termed 't follicular helper cells (t fh )' and it is their cytokine profile that directs b cells to secrete a particular ig isotype (kara et al., ) . the classic t h /t h dichotomy outlined above was in part shaped by the observation that ifn-γ production will lead to switching of b cells to secrete igg a/c, whereas production of il- leads to the secretion of igg . interestingly, it appears that the cytokine profile induced by the effector t-cell population is mirrored by the innate immune response. innate-like lymphocytes also have effector phenotypes that correspond to those of cd t cells and are induced by the same signals and transcriptional regulators (spits et al., ) and the same appears to be true also for macrophages and other innate immune cells (sica and mantovani, ) . while initial studies identified two particular antagonistic effector response types (termed t h and t h and classified by t-cell production of ifn-γ and il- , respectively), more recent studies now demonstrate a much wider array of effector responses in which innate and adaptive immunity acts together to reinforce an immune response phenotype as well as modulate its size by induction of t regulatory cells (t regs ) that generate inhibitory cytokines (kara et al., ; sica and mantovani, ; spits et al., ) . the use of cytokine-deficient and reporter mice that enabled the identification of cytokine-producing cells via expression of a fluorescent reporter was particularly valuable for the development of this more nuanced view of the quality of immune responses. spontaneous mouse models of immune deficiencies have been used extensively in research. their use, plus the expanding number of knockout, transgenic, and dominant negative mouse mutants, has advanced understanding of human immune deficiency diseases as well as basic understanding of the immune system (table . ). interbreeding of multiple immune-deficient mice has allowed the development of 'humanized' mice in which immune cells of the mouse are replaced with those of humans. while many challenges remain to fully replenish mice with components of the human immune system, the use of immune-deficient nod/severe combined immunodeficient (scid)/il- rγ -/recipients for transfer of human peripheral blood lymphocytes, cordblood or bone marrow-derived cd + stem cells with human liver and thymus (blt-mice) is yielding promising results (akkina, ) . investigators using genetically engineered mice are constantly reminded that phenotypic analysis of these animals must be done cautiously because the immune system may be profoundly affected and in ways that are not always anticipated. this may make it difficult to determine whether a given gene product is directly involved or may be secondary to a more global dysregulation of the immune system. as with other biological systems, compensation mechanisms also may mask the phenotype. experimental approaches are being increasingly used to refine the knockout technology by restricting a specific genetic deficiency to a particular tissue of interest using the cre-lox system, in which tissue-specific or temporal restricted expression of the cre recombinase induces the deletion of a 'floxed' gene (mak et al., ) . transgenic mice are available that restrict cre expression to various hematopoietic cells or tissue or drive cre recombination following injection of tamoxifen. other approaches are the generation of 'bone marrow irradiation' chimeras. here, inbred wild-type mice or mice deficient in certain immune cells (table . ) are lethally irradiated by exposure to a gamma-irradiation source to deplete the hematopoietic stem cells. these are then replaced by transfer of bone marrow cells to the irradiated mice. reconstitution of the hematopoietic system is usually achieved within about weeks, during which time mice are provided with antibiotic-containing drinking water to avoid infections of these temporarily immune-compromised animals. transfer of bone marrow from a congenic knockout restricts the genetic defect to the hematopoietic system. a mix of bone marrow from two sources is also often used to generate tissue-specific knockouts. for example, mixing a bone marrow from t-cell-deficient mice ( %) with that of a gene knockout ( %) generates 'mixed bone marrow chimeras' in which all t cells only develop from the knockout, thus lack the gene of interest, whereas most of the other cells t from the wild-type source, effectively constraining the genetic defect to the t-cell population. sets of congenic mice with defined allotypic differences are often used to confirm the source of individual cells. such markers include the gene locus cd . /cdc . or cd . /cd . (thy . /thy . ). alternatively, cells may express a fluorescent transgene, such as green-fluorescent protein (gfp). identification is usually performed by flow cytometry, or less commonly by immunofluorescence or immunohistochemistry. generation of bone marrow chimeras circumvents the time-consuming breeding of cre recombinase-expressing flx/flx mice. however, numerous controls are needed to exclude off-target effects due to irradiation damage. repeat injection of antibodies targeting specific cell populations is another rapid approach that avoids the potential for irradiation damage and allows short-term depletion of individual cell subsets. its main disadvantage is the need to identify mab that bind to surface receptors uniquely expressed by a cell subset of interest and the verification of the efficacy of the depletion. frequently used is antibody treatment for the short-term depletion of t-cell subsets using mab against cd or cd as well as individual cytokines. contemporary knowledge about diseases of laboratory mice has developed primarily from examining the effects of disease on traditional strains and stocks. the widespread use of genetically engineered mice is likely to modify current concepts because of novel or unpredictable interactions among genetic alterations, the genetic backgrounds on which they are expressed, and exogenous factors, such as infectious agents. because the number of combinations is extraordinarily high, clinical and laboratory diagnosticians should be alert to the potential for altered disease expression in genetically engineered mice and not be misled by unexpected signs, lesions, and epizootiology. many microbial agents have the potential to cause disease in mice or interfere with mouse-based research. housing and husbandry in microbiologically sheltered environments are designed to reduce the risks of disruptive infection, especially among immunologically dysfunctional mice, but must be accompanied by effective microbiological surveillance. surveillance should encompass resident mice and mouse products (serum, cell lines, transplantable tumors) procured from external sources. because surveillance strategies will vary with research needs and operating conditions, it is prudent to consult a number of sources, such as the federation of european laboratory animal science associations (felasa) (nicklas et al., ) and commercial laboratories, for guidance. detailed discussion of microbial quality control is provided in chapter . there are also recommendations regarding specific agents in following sections. diagnostic methods involve gross and microscopic pathology, parasitology, microbial isolation and culture, serology, and pcr. serology is particularly important for viral surveillance, and now relies principally on enzyme-linked immunosorbent assay (elisa), multiplex fluorescent immunoassay (mfi) for simultaneous detection of antibodies to multiple agents (hsu et al., ) , indirect fluorescent antibody (ifa) assay, or hemagglutination inhibition (hai), with the latter two methods generally used for confirmation (livingston and riley, ; pritchett-corning et al., ) . mouse antibody production (map) testing has been historically used for testing biological materials for contamination by infectious agents. pcr panels for murine infectious agents are now commercially available and have cost and time-saving advantages as well as improved assay sensitivity and specificity. beyond the classic bacterial and viral murine infections, pcr assays are now available for endo-and ectoparasites (see chapter ). etiology mousepox is caused by ectromelia virus (ectv), an orthopoxvirus that is antigenically and genetically closely related to a number of other poxviruses, including vaccinia, variola, and cowpox viruses. the original isolate of ectv, known as the hampstead strain, was discovered by j. marchal in (marchal, as the cause of epizootic disease among laboratory mice in england. the disease featured amputation of extremities, which marchal termed ectromelia (from the greek, ectro, amputation and melia, limb). other strains of the virus include moscow, nih- , washington university, st. louis , beijing , ishibahsi i-iii, and naval (nav) strains, which vary in virulence, but are essentially indistinguishable genetically and serologically, suggesting a common origin. virus can be isolated from infected tissues by inoculation of cell cultures (bs-c- , hela, l cells) or embryonated eggs. the natural host (and original source of infection of laboratory mice) of ectv remains unknown. clinical signs the expression of clinical signs reflects an interplay among virus-related factors, including virus strain, dose and portal of entry, and host-related factors, including age, genotype, immunological competence, and gender (brownstein et al., a) . during natural epizootics, it was observed that a, bc, dba/ , dba/ , and cba strains developed acute fatal infections, whereas c bl/ mice were resistant to severe disease (briody, ) . experimental studies have shown that all strains of mice are susceptible to infection, but balb/c, a, dba/ , and c h/he mice were highly susceptible, akr and sjl mice were moderately susceptible, and c bl/ mice were highly resistant to lethal infection (bhatt and jacoby, c; wallace and buller, ) . the mechanisms of genetic resistance are not fully understood but appear to reflect multiple genes, some of which appear to be expressed through lymphoreticular cells, including nk cells (brownstein et al., a; jacoby et al., ) . the nuances of cytokine and cellular immune responses to ectv infection have received recent attention (reviewed in buller and fenner ( ) and esteban and buller ( ) ). outbreaks among susceptible mice are often volatile, with variable morbidity and high mortality in susceptible strains of mice. clinical signs such as ruffled fur or prostration may occur for only a few hours before death. mice that survive acute infection may develop chronic disease characterized by a focal or generalized rash anywhere on the body ( fig. . ). conjunctivitis also may occur. skin lesions usually recede within several weeks, but hairless scars may remain. additionally, severe viral infection of the feet and tail during the rash syndrome can lead to necrosis and amputation. epizootiology mousepox is not a common disease. outbreaks occur sporadically and recent outbreaks have been traced to the importation of contaminated mice or mouse products. for example, contaminated mouse serum was responsible for recent outbreaks in the united states (dick et al., ; . natural exposure is thought to occur through direct contact and skin abrasions. cage-to-cage transmission is low and can be virtually nil if filter-topped cages are used (bhatt and jacoby, b) . ectromelia virus is highly stable at room temperature, especially under dry conditions, leading to the potential for prolonged environmental contamination in infected colonies (bhatt and jacoby, d) . aerogenic exposure is not a major factor in natural outbreaks, and arthropod-borne transmission does not appear to occur. virus-free progeny can be obtained laboratory animal medicine from immune dams (bhatt and jacoby, b) . however, intrauterine infection and fetal deaths, albeit rare, have been reported. natural transmission is facilitated by intermediately resistant mice, which survive long enough to develop skin lesions that can shed virus for relatively long periods of time. the risks for transmission are further increased by persistence of infectious virus in excreta and exfoliated scabs. although virus excretion typically lasts for about weeks, virus has been found in scabs and/or feces for up to weeks. resistant mouse strains also are dangerous because they can shed virus during subclinical infections. however, infections in resistant mice tend to be short-lived. highly susceptible mice are a relatively small hazard for dissemination of infection, if properly discarded, because they die before virus shedding becomes prominent. thus, juxtaposition of resistant or intermediately resistant infected mice with highly susceptible mice can provoke explosive outbreaks. infant and aged mice are usually more susceptible to lethal infection than young adult mice. maternal immunity among enzootically infected breeding mice may perpetuate infection by protecting young mice from death, but not from infection. such mice may subsequently transmit infection by contact exposure. pathology the classic descriptions of ectv pathogenesis by fenner remain timely, including the frequently cited and reproduced figure summarizing the pathogenesis of infection ( fig. . ) (fenner, b) . interest in smallpox has renewed the interest in ectv as a model of host response to infection (esteban and buller, ) . ectv multiplies in the cell cytoplasm and produces two types of inclusion bodies. the a type (marchal body) is well demarcated and acidophilic in histological sections. it is found primarily in epithelial cells of skin ( fig. . ) or mucous membranes and can also be found in intestinal mucosa. the b type of inclusion is basophilic and can be found in all ectromelia-infected cells. however, it is difficult to visualize unless cells are stained intensely with hematoxylin. ectv antigen can be readily visualized by immunohistochemistry on formalin-fixed, paraffin-embedded tissue sections (esteban and buller, ; jacoby and bhatt, ) . following skin invasion, viral multiplication occurs in the draining lymph node and a primary viremia ensues. splenic and hepatic involvement begin within - days, whereupon larger quantities of virus are disseminated in blood to the skin. this sequence takes approximately week and, unless mice die of acute hepatosplenic infection, ends with the development of a primary skin lesion at the original site of viral entry. the primary lesion is due to the development of antiviral cellular immunity. severe hepatocellular necrosis occurs in susceptible mice during acute stages of mousepox. white spots indicative of necrosis develop throughout the liver ( fig. . ). in nonfatal cases, regeneration begins at the margins of necrotic areas, but inflammation is variable. splenic necrosis in acute disease commonly precedes hepatic necrosis but is equally or more severe. necrosis and scarring of red and white pulp can produce a macroscopic 'mosaic' pattern of white and red-brown the primary skin lesion, which occurs - days after exposure, is a localized swelling that enlarges from inflammatory edema. necrosis of dermal epithelium provokes a surface scab and heals as a deep, hairless scar. secondary skin lesions (rash) develop - days later as the result of viremia. they are often multiple and widespread and can be associated with conjunctivitis, with blepharitis, and, in severe cases, with buccal and lingual ulcers. the skin lesions also can ulcerate and scab before scarring. diagnosis mousepox can be diagnosed from clinical signs, lesions, serological tests, and demonstration of virus or viral antigen in tissues. observation of characteristic intracytoplasmic eosinophilic inclusions aids detection of infection. several serological tests are available to detect mousepox. historically, the standard test was hai, using vaccinia antigen as a source of hemagglutinin. elisa is more sensitive and specific and has replaced hai for serological monitoring among nonvaccinated mice (buller et al., ) . ectv infection also can be detected by ifa (buller et al., ) and pcr. serological differentiation of mousepox from vaccinia infection in vaccinated mice is based on the lack of hemagglutinin in the vaccine strain of virus. thus, serum from vaccinated mice may react by elisa but should not react by hai. differential diagnosis mousepox must be differentiated from other infectious diseases associated with high morbidity and high mortality. these include sendai pneumonia, mouse hepatitis, and tyzzer's disease. the latter two can be expressed by acute necrosis in parenchymal organs, but they can be differentiated by morphological, serological, and virological criteria. the skin lesions of chronic mousepox must be differentiated from other skin diseases caused by opportunistic or pathogenic bacteria, ascariasis, and bite wounds. prevention and control mousepox is a dangerous disease because of its virulence for susceptible mice. therefore, infected colonies should be quarantined immediately. depopulation has been used as a primary means for control, but confirmation of infection should be obtained before exposed mice are destroyed. tissues, supplies, instruments, or other items that have had potential contact with infected mice should be disinfected by heat or chemicals such as formalin, sodium hypochlorite, or chlorine dioxide. materials should be autoclaved or, preferably, incinerated. disinfected rooms should be challenged with susceptible sentinel animals that are observed for clinical signs and tested for seroconversion after several weeks. depopulation and disinfection must be carried out vigorously. because modern housing and husbandry methods based on the use of microbarrier caging are effective for containing infection, testing and culling properly isolated mice is a potential alternative, especially for irreplaceable breeding mice. such mice can be quarantined along with cessation of breeding to permit resolution of infection (bhatt and jacoby, b) . sequential testing with contact-exposed sentinels should be employed with this option. additionally, maternal immunity from fully recovered dams can protect mice from infection, thereby enhancing opportunities to derive virus-free mice from previously infected dams, with the caveat that progeny will be transiently seropositive with maternally derived antibody. vaccination can control or prevent clinically apparent mousepox. the hemagglutinin-deficient strain of vaccinia virus (ihd-t) is used to scarify skin on the dorsum of the tail. 'takes' should occur in previously uninfected mice by - days, but not in infected mice (bhatt and jacoby, a) (fig. . ). infected mice should be quarantined separately or eliminated. vaccination may not prevent infection, although infection in vaccinated mice is often transient. furthermore, vaccinia virus can be shed from scarification sites for at least several days. therefore, other preventive measures, such as strict controls on the entry of mice or mouse products, combined with periodic serological monitoring, should not be relaxed until diagnostic testing has confirmed the elimination of vaccinia and ectromelia virus. additionally, seroconversion evoked by vaccination must be taken into account in serological monitoring of vaccinated colonies. finally, vaccinia virus is a human pathogen, so vaccination procedures should include personnel protective measures to prevent exposure. research complications the primary threat from mousepox is mortality in susceptible mice. the loss of time, animals, and financial resources can be substantial. (shellam, , ) mice are naturally susceptible to two herpesviruses from the subfamily betaherpesvirinae and in the genus muromegalovirus, the two species murid herpesvirus (of which one of the members is mouse cytomegalovirus (mcmv)) and murid herpesvirus (of which one of the members is mouse thymic virus (mtv)). they are species-specific viruses and distinct from each other and from other rodent herpesviruses. mctv has received considerable attention as a model of human cmv infection. etiology mouse cytomegalovirus (mcmv) is a mouse-specific betaherpesvirus. it can, however, replicate in cell cultures from several species, including mouse (fibroblasts and t cells), hamster, rabbit, sheep, and nonhuman primate. cocultivation may be required to rescue latent virus. clinical signs mcmv causes subclinical infection in adult immunocompetent mice, but experimental inoculation of neonates can cause lethal disease due to multisystemic necrosis and inflammation. epizootiology the prevalence of mcmv in laboratory mice is probably uncommon but undefined, since infection is clinically silent and serological surveillance is not widely practiced. wild mice are commonly infected and serve as a natural reservoir for infection, which implies that the entry of virus into a modern vivarium is most likely to occur from contaminated animal products. persistence is a central feature of nonlethal infection. persistently infected mice excrete virus in saliva, urine, and tears for many months, resulting in horizontal transmission through mouse-to-mouse contact. virus also can infect prostate, testicle, and pancreas, implicating other modes of excretion. vertical transmission does not appear to be a common factor in natural infection. further, maternal immunity protects sucklings from infection. pathology mouse cytomegalovirus can replicate in many tissues, and viremia commonly occurs. lesions are not remarkable during natural infection and may be limited to occasional enlarged cells (megalocytosis) containing eosinophilic intranuclear and/or cytoplasmic inclusions associated with lymphoplasmacytic interstitial inflammation, especially in the cervical salivary glands. susceptibility to experimental infection varies with age, dose, route, virus strain, and host genotype. infection can occur in young and adult mice. however, the pathogenicity of mcmv for mice decreases with age. neonates are highly susceptible to lethal infection, but resistance to disease develops by the time mice are weaned. immunodeficient mice, however, remain susceptible to pathogenic infection as adults. persistent infection often affects the salivary glands and pancreas. the persistence of salivary gland infection appears to be dose dependent. there is experimental evidence that mcmv can produce latent infection of b cells, probably t cells as well as aforementioned tissues. persistent infection may lead to immune complex glomerulonephritis. latent persistent infection can be reactivated by lymphoproliferative stimuli and by immunosuppression. diagnosis mcmv antigens appear to be weak stimuli for humoral antibody production, which is consistent with the fact that cellular immunity is critical for protection against infection. neutralizing antibody titers are low during acute infection and difficult to find during chronic infection. serology and pcr-based diagnosis are available, but neither is widely used because of assumptions that infection has a very low prevalence. detection of enlarged cells with intranuclear inclusions, especially in salivary glands, is diagnostic, if they are present. in situ hybridization can be used as an adjunct to routine histopathology. differential diagnosis mcmv infection must be differentiated from infection with mtv. the latter virus can produce necrosis of thymic and peripheral lymphoid tissue when infant mice are experimentally inoculated. lytic lesions of lymphoid tissues are not a hallmark of mcmv. the viruses can also be distinguished from each other serologically. sialoadenitis with inclusions can occur during infection with mouse polyoma virus. like mcmv, mtv infects the salivary gland as its primary target organ. prevention and control control measures for mcmv have not been established, because it has not been considered an important infection of laboratory mice. cage-to-cage transmission has not been demonstrated, but horizontal infection from contaminated saliva must be considered. the exclusion of wild mice is essential. research complications mcmv can suppress immune responses. apart from the potential for interfering with immunology research, it can exacerbate the pathogenicity of opportunistic organisms such as pseudomonas aeruginosa. etiology mouse thymic virus (mtv) is a herpesvirus (murid herpesvirus ) that is antigenically distinct from mcmv. no suitable in vitro method for cultivation has been developed; therefore, viral propagation depends on mouse inoculation. clinical signs natural infections are subclinical. epizootiology the prevalence of mtv is thought to be low. mice can be infected at any age, although lesions develop only in mice infected perinatally. mice infected as infants or adults can develop persistent infection of the salivary glands lasting several months or more. excretion of virus in saliva is considered the primary factor in transmission. seroconversion occurs in adults but does not eliminate infection. infection in neonates may not elicit seroconversion, rendering such mice serologically negative carriers. the mode of infection is obscure, but virus is excreted in saliva, suggesting that transmission from infected dams to neonatal mice occurs by ingestion. mtv also has been isolated from the mammary tissue of a lactating mouse, suggesting the potential for transmission during nursing. prenatal transmission has not been found. pathology mtv causes severe, diffuse necrosis of the thymus and lymphoid tissue with tropism for cd + t cells in mice inoculated within approximately week after birth. the severity of thymic and lymph node necrosis can be mouse strain-dependent. grossly, the thymus is smaller than normal. infected thymocytes display mtv-positive intranuclear inclusions. necrosis is followed by granulomatous inflammation and syncytium formation. reconstitution of lymphoid organs takes - weeks. diagnosis thymic necrosis associated with intranuclear viral inclusions is the hallmark lesion. viral antigen can be detected by immunohistochemistry. serologic detection is effective, but generally not utilized, and is potentially negative in neonatally exposed mice. suspicion of infection in seronegative mice can be tested by inoculation of virus-free neonatal mice with homogenates of salivary gland or with saliva. inoculated mice should be examined for thymic necrosis - days later. pcr or the mouse antibody production (map) test can also be used to detect infection. differential diagnosis reduction of thymus mass can occur in severe mouse coronavirus infection, during epizootic diarrhea of infant mice, or following stress. prevention and control because mtv induces persistent salivary infection, rederivation or restocking should be considered if infection cannot be tolerated as a research variable. research complications mtv transiently suppresses cellular and humoral immune responses because of its destructive effects on neonatal t lymphocytes. parvoviruses are among the most common viral infections in contemporary laboratory mouse populations (livingston et al., ) (pritchett-corning et al., ) , and pose major challenges to both detection and control. the mouse parvoviruses are composed of two antigenically and genetically distinct but related groups, including minute virus of mice (mvm) and mouse parvovirus (mpv), with each group containing a number of strains. the international committee on taxonomy of viruses classifies mpv, mvm, and several other rodent parvoviruses into one genus, protoparvovirus, and species, rodent protoparvovirus , but these viruses will be treated separately herein. etiology minute virus of mice (mvm) is a small ( -kb) single-stranded dna virus. the prototypic strain is designated mvmp. an allotropic variant with immunosuppressive properties in vitro is named mvmi, and additional laboratory animal medicine named strains include mvmc and mvmm. the genome encodes two nonstructural proteins, ns- and ns- , which are highly conserved among the rodent parvoviruses and account for prominent cross-reactivity in serological assays that utilize whole virus antigen. the viral capsid proteins, vp- and vp- , are virus-specific and form the basis for serological differentiation of mvm from mpv. mvm has a broad in vitro host range. it replicates in monolayer cultures of mouse fibroblasts (a cells), c rat glial cells, sv (simian virus )-transformed human newborn kidney ( k cells), t-cell lymphomas (el ), and rat or mouse embryo cells, producing cytopathic effects that can include the development of intranuclear inclusions. clinical signs natural mvm infections are subclinical. neonatal mice of some inbred strains are experimentally susceptible to lethal renal and/or intestinal hemorrhage during mvmi infection, but this syndrome has not been reported in natural outbreaks. experimental inoculation of adult c.b- -prkdc scid (scid) mice with mvmi results in lethal infection (lamana et al., ; segovia et al., ) , and similar severe illness has been noted in naturally infected b-cell-deficient nod. cg-h h -igh null mice (naugler et al., ) . epizootiology mvm is a common virus that naturally infects laboratory mice, but appears to be less common than mpv (besselsen et al., ; livingston et al., ) . mvm is moderately contagious for mice, its only known natural host. virus can infect the gastrointestinal tract and is excreted in feces and urine. the resistance of rodent parvoviruses to environmental inactivation increases the risks of transmission after virus is excreted. therefore, contamination of caging, bedding, food, and clothing must be considered a risk for the spread of infection. transmission occurs by oronasal exposure, but viral contamination of biologicals used for experimental inoculation, such as transplantable tumors, also can be a source of infection. continuous contact exposure to infected animals or soiled bedding usually induces a humoral immune response within weeks, but limited exposure may delay seroconversion. young mice in enzootically infected colonies are protected by maternal antibody, but actively acquired immunity develops from infection sustained after the decay of maternal immunity. mvm, in contrast to mpv, is not thought to cause persistent infection; infection in immunocompetent adult mice usually lasts less than weeks (smith, ; smith and paturzo, ). infection appears to last less than month, even in oronasally inoculated neonatal mice, but immunodeficient mice may be persistently infected. there is no evidence that mvm is transmitted in utero. pathology natural infections or experimental inoculation of adult mice appears to be nonpathogenic. contact-exposed neonates have been reported to develop cerebellar lesions, but these are very rare. experimental infection of neonatal balb/c, swr, sjl, cba, and c h mice with mvmi can cause renal hemorrhage and infarction (brownstein et al., b) . dba/ mice also developed intestinal hemorrhages and accelerated involution of hepatic hematopoiesis. c bl/ neonates are resistant to vascular disease. this lesion has been attributed to viral infection of endothelium. infection of immunodeficient mice, including scid and b-cell-deficient mice, results in lethal damage to granulomacrophagic, megakaryocytic, and erythrocytic hematopoietic tissue with severe leukopenia (lamana et al., ; naugler et al., ; segovia et al., ) . intranuclear viral inclusions and viral antigen have been observed in splenic mononuclear cells of b-cell deficient mice (naugler et al., ) . diagnosis serology is the primary method of detecting infection, which utilizes recombinant mvm and mpv major capsid viral proteins (vp ) as antigens, which discriminate between the two groups of mouse parvoviruses. in contrast, the conserved nonstructural protein, ns can be used to detect antibody to both groups, but is less sensitive than vp assays (livingston et al., ) . mvm infection also can be detected by pcr, in situ hybridization, and immunohistochemistry. pcr assays can be used to detect mvm-or mpv-specific vp or all rodent parvovirus group specific ns exons (besselsen, ; besselsen et al., ) . mvm can be isolated from the spleen, kidney, intestine, and other tissues by inoculation of the c rat glial cell line. it also can be detected by the mouse antibody production test. prevention and control because mvm does not persist in immunocompetent mice, control and elimination should exploit quarantine combined with thorough disinfection of the environment, because parvoviruses are resistant to environmental inactivation. mpv has been shown to be successfully eliminated by a cage-bycage test (serology and fecal pcr) and cull approach, although there are no published reports confirming the success of this strategy for eliminating mvm (macy et al., ) . cesarean rederivation or embryo transfer may also be used to rederive virus-free progeny. prevention of mvm infection depends on strict barrier husbandry and regular surveillance of mice and mouse products destined for use in vivo. research complications mvm contamination of transplantable neoplasms can occur; therefore, infection can be introduced to a colony through inoculation of contaminated cell lines. failure to establish long-term cell cultures from infected mice or a low incidence of tumor 'takes' should alert researchers to the possibility of mvm contamination. mvmi has the potential to inhibit the generation of cytotoxic t cells in mixed lymphocyte cultures. etiology mouse parvovirus (mpv) is among the more common viruses detected within contemporary laboratory animal medicine mouse colonies, and is more common than mvm (livingston et al., ; livingston and riley, ; pritchett-corning et al., ) . mpv was initially isolated following its detection as a lymphocytotropic contaminant in in vitro assays for cellular immunity. the virus grew lytically in a cd + t-cell clone designated l and inhibited the proliferation of cloned t cells stimulated with antigen or interleukin (il- ) (mckisic et al., ) . molecular analysis of mpv indicates that regions encoding the ns proteins are similar to those of mvm (and other rodent parvoviruses). however, they differ significantly in regions encoding the capsid proteins, accounting for their antigenic specificity. the prototype isolate was first called an 'orphan' parvovirus of mice because its biology and significance were obscure, but it has subsequently been named mouse parvovirus (mpv). immortalized t cells (l ) are the only cells found thus far to support replication of mpv. there are three genetically distinct variants of mpv, including mpv- , mpv- , and mpv- . mpv- includes a number of closely related variants, including mpv- a, mpv- b, and mpv- c. in addition, a hamster parvovirus isolate is closely related to mpv- , which is infectious to mice and likely to be of mouse origin (besselsen et al., ; christie et al., ) . clinical signs mpv infection is clinically silent in infant mice and adult immunocompetent or immunodeficient mice (besselsen et al., ) . immunologic perturbations are the most likely signs of infection (mckisic et al., ) . epizootiology mpv causes persistent infection in infant and adult mice, a property that differentiates it from mvm. in situ hybridization has identified the small intestine as a site of viral entry and early replication, but respiratory infection cannot be excluded. experimental studies following inoculation of neonatal balb/c and c.b- -prkdc scid (scid) mice revealed that balb/c mice shed high levels of virus for weeks, with transmission to sentinels exposed during the first weeks of infection. thereafter, balb/c mice shed extremely low virus intermittently. in contrast, scid mice shed high levels of virus until weaning, but lower levels at weeks of age, yet they effectively transmitted infection to sentinels at all stages of infection (besselsen et al., ) . others have shown that transmission of mpv by sencar mice inoculated as infants was intermittent up to weeks, whereas transmission by mice inoculated as weanlings occurred during the first weeks of infection . transmission to balb/c progeny from infected dams was shown to occur, but embryo transfer rederivation was found to be successful in experimentally infected scid mice (besselsen et al., ) . humoral (e.g., passively or maternally acquired) immunity can protect against mpv infection. however, immunity to mvm may not confer cross-immunity to mpv (hansen et al., ) . pathology mpv appears to enter through the intestinal mucosa, which is a site of early virus replication ( fig. . ). acute infection is widespread but mild, involving the lung, kidney, liver, and lymphoid organs. histological lesions are not discernible. lymphocytotropism is a characteristic of acute and persistent mpv infection in infant and adult mice. during acute infection, virus is dispersed within lymph nodes, but during persistent infection virus localizes in germinal centers (fig. . ) . diagnosis because infected mice do not manifest signs or lesions and the virus is very difficult to propagate in cell culture, detection and diagnosis rely on serology and molecular methods. serology that utilizes mpv vp as antigen is a sensitive and specific assay that differentiates mpv from mvm (livingston et al., ) . the map test also can be used to detect parvovirus infections but is relatively time-consuming and expensive. as noted for mvm, pcr for murine parvoviruses, using nucleoprotein gene sequences that are conserved among murine parvoviruses, can be used as a screening test. pcr also can be used to detect mpv-specific sequences in the vp gene. although diagnostic pcr is sensitive and specific, it is effective only in actively infected animals. it can be used on feces to detect virus shedding, or applied to tissues, such as mesenteric lymph nodes, obtained at necropsy. differential diagnosis mpv infection must be differentiated from mvm infection. because both viruses are enterotropic and lymphocytotropic, serology and pcr must be used to distinguish between them. prevention and control the persistence of mpv in individual mice, its potential for provoking immune dysfunction, and the resistance of murine parvoviruses to environmental inactivation favor active control and prevention of mpv infection. quarantine of infected rooms is appropriate. elimination (depopulation) of infected mice should be considered if they are an immediate threat to experimental or breeding colonies and can be replaced, but a cage-by-cage test and cull approach has been shown to be successful under natural conditions (macy et al., ) . for mice that are not easily replaced, virus persistence in the absence of transplacental transmission favors cesarean rederivation or embryo transfer as relatively rapid options to eliminate infection. control of infection also should include environmental decontamination. chemical disinfection of suspect animal rooms and heat sterilization of caging and other housing equipment are prudent steps. prevention is based on sound serological monitoring of mice and surveillance of biologicals destined for inoculation of mice. with the increasing use of mouse germplasm, it is important to note that mouse sperm, oocytes, ovarian tissue, and preimplantation embryos from enzootically mpvinfected mouse colonies may have a high prevalence of mpv contamination, based upon pcr (agca et al., ) . research complications murine parvoviruses can distort biological responses that depend on cell proliferation. for mpv, such effects are seen on immune function and include augmentation or suppression of humoral and cellular immune responses. etiology adenoviruses are nonenveloped dna viruses that produce intranuclear inclusions in vitro and in vivo. two adenovirus species in the genus mastadenovirus have been associated with mice: murine mastadenovirus a (with the representative strain being mav- or fl) and murine mastadenovirus b (with the representative strain being mav- or k ). both strains replicate in mouse kidney tissue culture but are antigenically distinct. clinical signs mav- can cause severe clinical disease after experimental inoculation of infant mice. signs include scruffiness, lethargy, stunted growth, and often death within days. mav- virus is enterotropic and is responsible for virtually all naturally occurring infections in contemporary mouse populations. infection is usually subclinical in immunocompetent mice, with the possible exception of transient runting among infant mice. wasting disease can occur in athymic mice infected with mav- . epizootiology the prevalence of adenovirus infection in mouse colonies is low, particularly mav- riley, , pritchett-corning et al., ) . transmission occurs by ingestion. adult mice experimentally infected with mav- may remain persistently infected and excrete virus in the urine for prolonged periods. adult mice experimentally infected with mav- excrete virus in feces for at least weeks but eventually recover. athymic mice can shed mav- for at least weeks and episodically for at least months. pathology infection with mav- causes multisystemic disease characterized by necrosis. infant mice are especially susceptible to rapidly fatal infection characterized by necrosis of brown fat, myocardium, adrenal cortex, salivary gland, and kidney, with the development of intranuclear inclusions. more mature mice usually develop subclinical infection leading to seroconversion; however, athymic and scid mice can develop intestinal hemorrhage and wasting, with fatal disseminated infection (lenaerts et al., ) . infection with mav- produces amphophilic, intranuclear inclusions in intestinal epithelium, especially in the distal small intestine (fig. . ) . inclusions are easier to detect in infant mice than in adults. infection of c.b- -prkdc scid mice with mav- results in enteric infection, but also hepatic lesions resembling reye's syndrome (pirofski et al., ) . diagnosis although mav strains can be isolated in tissue culture, routine diagnosis depends on detection of infection by serological assay and/or demonstration of adenoviral inclusions, most commonly in the intestinal mucosa. cross-neutralization tests have revealed that antiserum to mav- neutralizes both strains, but antiserum to mav- neutralizes mav- weakly at best. therefore, mav- antigen should be used for the serological detection of adenovirus infection irrespective of the assay employed. mav also can be detected by pcr. differential diagnosis intranuclear adenoviral inclusions in intestinal epithelium are pathognomonic laboratory animal medicine and differentiate mav- infection from other known viral infections of mice. infection may resemble rotavirus infection, with runting and abdominal bloating in infant mice. prevention and control prevention requires serological monitoring of mice and examination for contamination of animal products such as transplantable tumors. because mav- infection appears to be transient in individual mice, segregation of infected colonies may be effective for control. however, rederivation coupled with subsequent barrier housing is a more conservative approach. research complications mav infection is unlikely to affect research using immunocompetent mice. however, it has the potential for pathogenicity in immunodeficient mice. mice can incur natural infection with two polyomaviruses: polyoma virus (pyv) and k virus. these viruses belong to the family polyomaviridae. k virus belongs to the genus polyomavirus and the species murine pneumotropic virus, while the classical polyoma virus belongs to the species murine polyomavirus. etiology polyoma virus (pyv) is a small dna virus that derives its name 'polyoma' (many tumors) from its ability to experimentally induce multiple types of tumors in mice experimentally infected as neonates. its primary importance stems from use in murine models of experimental oncogenesis, with natural infection being rare. the transformative activity is mediated by 't' (tumor) antigens, encoded by large t, middle t, and small t genes, with middle t (mt) being considered the major viral oncogene, and as a result has been used extensively in transgenic constructs. clinical signs natural infections in immunocompetent mice are usually subclinical. however, tumor induction, neurological disease, and wasting can occur in naturally exposed immunodeficient mice (mccance et al., ; sebesteny et al., ) . epizootiology modern husbandry and health care have essentially eliminated natural exposure in laboratory mice. pyv is used for experimental studies and thus can inadvertently be introduced to mouse colonies. inoculation of mice with contaminated biologicals or cell cultures is a potential source of entry and spread. natural transmission occurs via the respiratory route. exposure of neonatal mice results in persistent infection and shedding of the virus in urine, feces, and saliva, thereby contaminating the environment for spread to other mice. infection of adult mice is transient, with minimal virus shedding, although pcr has revealed infection lasting up to months in cba mice inoculated with virus as adults (berke and dalianis, ) . maternal antibody is highly effective at preventing infection of newborn mice, but as maternal antibody wanes, mice are partially susceptible, with transient virus shedding. thus, the natural cycle of transmission in enzootically infected populations requires contamination of bedding and nesting material in order to infect and be inefficiently transmitted, which is readily precluded by modern husbandry. intrauterine infection also can occur, and persistent renal infection, contracted neonatally, can be reactivated during pregnancy. as in immunologically immature neonatal mice, pyv infection can persist in adult immunodeficient mice. pathology pyv-induced tumors are essentially a laboratory phenomenon, optimized by virus strain and mouse strain, with akr, c h, c , cba, swr, and others being most susceptible, and c bl/ being among the most resistant to pyv oncogenesis. intranasal inoculation of neonatal mice results in initial replication in pulmonary respiratory epithelium (gottlieb and villarreal, ) followed by viremic dissemination and acute, lethal disease. tumors appear - months after inoculation of surviving mice. tumors of both epithelial and mesenchymal origin arise in multiple organs, particularly mammary carcinomas, basal cell tumors of the skin, carcinomas of salivary glands, thymomas, and various types of sarcomas. athymic mice can develop cytolytic and inflammatory lesions, followed by multisystemic tumor formation. intranuclear inclusions may be present in cytolytic lesions. demyelinating disease and skeletal tumors have been reported in experimentally . laboratory animal medicine inoculated and naturally exposed athymic mice, and myeloproliferative disease has been reported in experimentally inoculated c bl/ -scid mice (szomolanyi-tsuda et al., ) . diagnosis pyv can be isolated in mouse fibroblast cell lines, but infection is ordinarily detected serologically. additionally, pcr and immunohistochemistry can be used. differential diagnosis wasting in athymic mice can be caused by other infectious agents, including coronaviruses, sendai virus (sv), and pneumocystis. intranuclear inclusions can occur in infections caused by mouse adenovirus, mouse cytomegalovirus, and k virus. prevention and control control depends on elimination of infected mice and material, together with prevention of airborne spread. biological material destined for mouse inoculation should be tested for pyv by the map test or molecular diagnostics. research complications pyv infection can affect experiments by inadvertent contamination of cell lines or transplantable tumors, leading to infection of inoculated mice and the potential for epizootic spread. k virus infection k virus has historical importance, and is apparently absent from contemporary mouse populations (livingston and riley, ; pritchett-corning et al., ) , but it continues to be tested for, adding to the expense of infectious disease surveillance. oral inoculation of neonatal mice results in initial infection of capillary endothelium in the intestine, followed by viremic spread. vascular endothelium is the primary target in affected tissues, which often include the lung, liver, spleen, and adrenal glands. dyspnea occurs from pulmonary infection because of edema and hemorrhage. infection of immunocompetent adult mice is subclinical and results in a vigorous immune response. however, both adults and infant mice develop persistent infection. the primary organ for persistence is the kidney, with shedding of virus from tubular epithelium, and shedding can be reactivated by immunosuppression (greenlee et al., ) . additionally, infection of athymic mice can lead to clinical signs and lesions akin to those described for neonatally inoculated mice. gross lesions are limited to pulmonary hemorrhage and edema. histologically, intranuclear inclusions, which are visualized more easily using immunohistochemistry, are present in vascular endothelium of infected tissues. mild hepatitis with hepatocyte degeneration also may develop. infection can be detected by serology or pcr. prevention and control measures, if ever to be found within a mouse population, are similar to those described for pyv. etiology lactate dehydrogenase-elevating virus (ldv) is a mouse-specific small enveloped rna virus belonging to the family arteriviridae. infected mice are persistently viremic, resulting in increased concentration of several serum enzymes, most notably lactate dehydrogenase (ldh). infection is common among wild mice, but is now rare in contemporary laboratory mouse populations. however, surveys of biologic material indicate that ldv may be a common contaminant of biologic materials (nicklas et al., ) . clinical signs infection is subclinical. however, poliomyelitis has occurred in immunosuppressed c and akr mice inoculated with ldv, and has recently been observed in icr-scid mice following inoculation with contaminated biologic material (carlson-scholz et al., ) . epizootiology the primary mode of mouse-tomouse transmission is mechanical transfer from aggressive behavior (e.g., bite wounds). inoculation of mice with contaminated animal products such as cell lines, transplantable tumors, or serum is probably the most common source of induced infection. it is important to note, with respect to mechanical transmission, that infection induces lifelong viremia. natural transmission between cagemates or between mother and young is rare even though infected mice may excrete virus in feces, urine, milk, and probably saliva. pathology viremia peaks within day after inoculation, then persists at a diminished level. the elevation of enzyme levels in blood is thought to result primarily from viral interference with clearance functions of the reticuloendothelial system. ldv selectively targets mature f /f -positive macrophages, which are continually produced by uninfected progenitor cell populations, thereby maintaining persistent infection. virus also escapes immune clearance by evolution of neutralizing antibody-resistant quasi-species. no lesions are seen in naturally infected mice. the only significant lesion that can arise from experimental infection is poliomyelitis. this syndrome requires a combination of immunosuppression (due to age, genetics or induced means), mouse strain (c , akr, c h/fg, and pl), neurotropic ldv strains, and endogenous ecotropic murine leukemia virus. the mouse strain-dependent element is homozygosity for the fv- n allele, which permits replication of endogenous n-tropic ectotropic murine leukemia virus. mice develop spongiosis, neuronal necrosis, and astrocytosis of the ventral spinal cord and brain stem, with axonal degeneration of ventral roots. lesions contain both ldv and retrovirus. although this syndrome is largely experimentally induced, a natural outbreak of poliomyelitis has been reported in fv- homozygous icr-scid mice following inoculation with contaminated biologic material (carlson-scholz et al., ) . diagnosis plasma ldh levels are elevated, a response that is used to detect and titrate ldv infectivity. of the five isoenzymes of ldh in mouse plasma, only laboratory animal medicine ldh-v is elevated. sjl/j mice in particular show spectacular increases in ldh levels ( - times normal), a response controlled by a recessive somatic gene. ldv is detected by measuring ldh levels in mouse plasma before and days after inoculation of specific pathogenfree (spf) mice with suspect material. it is important to use nonhemolyzed samples because hemolysis will produce falsely elevated readings. plasma enzyme levels are measured in conventional units/ml, conventional unit being equivalent to . international units (iu). normal plasma levels are - iu, whereas in ldv infection, levels as high as iu can occur. ldv also interferes with the clearance of other serum enzymes and results in their elevation in serum. in a recent survey, serum samples were tested by serum ldh enzyme assays, among which % were deemed potentially positive. however, pcr revealed that all were false-positives (pritchett-corning et al., ) , emphasizing the inaccuracy of traditional enzyme assays. infection provokes a modest humoral antibody response, but it is difficult to detect because of formation of virus-antibody immune complexes. molecular diagnostics also can be used to diagnose infection in mouse tissues and serum and biologic materials. however, inhibitory factors in cells and serum may cause falsenegative results in pcr testing, so appropriate quality control measures are essential if this method is used (lipman and henderson, ) . prevention and control transplantable tumors have been a common source of ldv historically. therefore, tumors or cell lines destined for mouse inoculation should be monitored for ldv contamination. although ldv can contaminate tumor cell lines, it does not replicate in the tumor cells. therefore, one can attempt to free tumors of virus by passaging them through athymic nude rats, which are not nonpermissive to ldv but are permissive to xenografts. research complications ldv has numerous potential effects on immunological function. it may reduce autoantibody production, cause transient thymic necrosis and lymphopenia, suppress cell-mediated immune responses, and enhance or suppress tumor growth. etiology the house mouse is the natural host for lymphocytic choriomeningitis virus (lcmv), an old world member of the arenaviridae family that has spread worldwide along with m. musculus. lcmv virions are pleomorphic, containing single-stranded rna, and bud from the cell membrane. disease associated with infection is due to host immune response to the otherwise non-cytolytic virus. its name is derived from the immune-mediated inflammation resulting from the intracerebral inoculation of virus into immunologically competent mice. lcmv is a zoonotic virus that may cause a variety of clinical manifestations in humans, including meningitis. it has been extensively studied as an experimental model of virus-induced immune injury, using a number of closely related strains, including ones that have been selected for their relative neurotropism or viscerotropism. lcmv can be propagated in a variety of mammalian, avian, and even tick cell lines, with minimal cytopathic effect. these characteristics favor its propensity to persistently and silently contaminate biologic products, such as tumor cell lines. clinical signs natural infection in immunocompetent adult mice is usually self-limiting and subclinical. during enzootic infection of a mouse population, lcmv is transmitted in utero from persistently infected dams to their fetuses or to neonates, which are persistently infected and immunologically tolerant to lcmv. since lcmv is non-cytolytic in and of itself is minimally pathogenic, congenitally infected mice grow into adulthood, reproduce, and therefore transmit infection to the next generation. however, with age, immune tolerance breaks down, and mice develop a syndrome known as 'late disease' in which mice will progressively lose weight and die. in utero infection results in a low level of fetal mortality and maternal cannibalism of infected pups. the immune tolerance to lcmv is virus-specific, with the mice capable of eliciting effective immune responses against other agents. clinical signs following experimental inoculation of lcmv vary with age and strain of mouse, route of inoculation, and strain of virus. when virus is inoculated intracerebrally into immunocompetent adult mice, mice develop immune-mediated lymphocytic choriomeningitis, characterized by illness beginning - days after inoculation. sudden death may result or subacute illness associated with one or more of the following signs may develop: ruffled fur, hunched posture, motionlessness, and neurological deficits. mice suspended by the tail display coarse tremors of the head and extremities, culminating in clonic convulsions and tonic extension of the rear legs. spontaneous convulsions also can occur. animals usually die or recover in several days. a visceral form of infection can occur in adult mice inoculated by peripheral routes with 'viscerotropic' strains. it can be subclinical or lead to clinical signs, including ruffled fur, conjunctivitis, ascites, somnolescence, and death. if mice survive, recovery may take several weeks. surviving mice may have immune exhaustion due to consumption of lymphoid tissue, in contrast to immune tolerance that occurs when mice are infected in utero or as neonates. runting and death from lcmv infection may occur in neonatally infected mice and can lead to transient illness or to death. clinical signs are nonspecific, recovery is slow, and survivors may remain runted. this early form of disease is attributed to endocrine dysfunction caused laboratory animal medicine by lcmv infection. late-onset disease can occur in previously subclinical carrier mice that develop immune complex glomerulonephritis. it is usually the result of prenatal or neonatal infection and occurs in persistently infected mice when they are - months old. clinical signs are nonspecific and include ruffled fur, hunched posture, weight loss, proteinuria, and ascites. epizootiology lcmv is distributed widely in wild m. musculus throughout the world. among common laboratory species, mice, hamsters, guinea pigs, and nonhuman primates are susceptible to infection, but only the mouse and the hamster are known to transmit virus. lcmv infection is rare in laboratory mice produced and maintained in modern quarters (livingston and riley, ; pritchett-corning et al., ) . infection is usually introduced through inoculation of virusinfected biologicals, such as transplantable tumors, or by feral mice. wild mice are a natural reservoir of infection and a potential threat to research colonies if they gain entry inadvertently. naturally infected carrier mice can have persistently high concentrations of virus in many organs, thereby facilitating virus excretion in saliva, nasal secretions, and urine. persistently infected neonates usually reach breeding age and can perpetuate infection in a breeding colony. thus introduction of a single lcmv carrier mouse to a breeding colony can eventually result in a high prevalence of persistently infected mice. infection in adult mice, in contrast, is often acute because of the onset of effective immunity, and the spread of virus is halted. horizontal spread of infection is enhanced by close contact, but rapid horizontal spread is not characteristic. mice can transmit lcmv to hamsters, which can remain viremic and viruric for many months, even if they contract infection as adults. infected hamsters can transmit virus to other hamsters and mice and are the primary source of human lcmv infection. persistent infection in immunodeficient mice may carry greater risks for viral excretion and zoonotic transmission. pathology lcmv disease is a prototype for virusinduced, t-lymphocyte-mediated immune injury, noncytolytic endocrine dysfunction, and immune complex disease. however, lesions comparable to experimentally induced disease are rare during natural infection. intracerebral inoculation of virus into immunocompetent adult mice induces nonsuppurative leptomeningitis, choroiditis, and focal perivascular lymphocytic infiltrates. host tissues are damaged during the course of the cellular immune response to the virus. the character of visceral lesions depends on virus strain and mouse strain; the ratio of cytolytic to proliferative responses in lymphoid organs is mouse strain-dependent. in severe infection, nonsuppurative inflammation can occur in many tissues. the severity of accompanying cytolytic lesions seems to parallel the intensity of cellular immunity. liver lesions can include hepatocyte necrosis accompanied by nodular infiltrates of lymphoid cells and kupffer cells, activated sinusoidal endothelium, an occasional granulocyte or megakaryocyte, and fatty metamorphosis. cytolysis, cell proliferation, and fibrinoid necrosis can develop in lymphoid organs. necrosis of cortical thymocytes can lead to thymic involution. lesions of late-onset disease are characterized by formation of immune complexes and associated inflammation. renal glomeruli and the choroid plexus are most severely affected, but complexes may also be trapped in synovial membranes, blood vessel walls, and skin. lymphoid nodules can form in various organs. lesions associated with early deaths in neonatally infected mice have not been thoroughly described but include hepatic necrosis. the lesions of acute and persistent lcmv infection reflect separate immunopathologic processes. in adult mice with acute lcmv infection, virus multiplies in dcs, b cells, and macrophages, whereas t cells are resistant. internal viral epitopes induce humoral immune responses, but surface epitopes elicit cell-mediated immunity and neutralizing antibodies. thus, elimination of virus and virus-associated immunological injury are both t-cell-mediated. this apparent paradox has been explained by the view that prompt cellular immunity limits viral replication and leads to host survival, whereas slower cellular immune responses permit viral spread and increase the number of virus-infected target cells subject to attack once immunity is fully developed. antibody can be detected by week after infection but does not play a significant role in eliciting acute disease. lesions of lcmv infection appear to develop from direct t-cell-mediated damage to virus-infected cells and may involve humoral factors released from immune effector t cells. lcmv also can suppress humoral and cellular immunity in acutely infected mice. persistent infection commonly evolves from exposure early in pregnancy, and virus has been demonstrated in the ovaries of carrier mice. prenatal or neonatal infection induces immunological tolerance to lcmv, which can then replicate to high titer in many tissues. nevertheless, persistently infected mice develop humoral antibody to lcmv. antibody can complex with persistent virus to elicit complement-dependent inflammation in small vessels. immune complex glomerulonephritis exemplifies this process, as noted above. diagnosis lcmv infection can be diagnosed serologically. whereas immunocompetent adult mice will normally seroconvert after exposure, carrier mice may develop poor humoral immune responses. therefore, testing must avoid false-negative results. employment of adult contact sentinel mice is a useful strategy for detecting lcmv infection by seroconversion. tissues, including biologic products and cell lines, can be tested laboratory animal medicine by pcr. a traditional method for detection involved collection of small blood samples from persistently infected live suspects, which are often viremic, and using them to inoculate cultured cells or adult and neonatal mice. intracerebral inoculation of lcmv-positive tissues should elicit neurological signs in adult mice within days, whereas infant mice should remain subclinical. histological examination of brains from affected adults may reveal nonsuppurative inflammation, but lesions may be minimal in mice infected with viscerotropic isolates. immunohistochemistry can be used to detect viral antigen in brains of suckling and adult mice. intraperitoneal inoculation of adult mice may yield short-lived infection with seroconversion, i.e., the map test. virus can be grown and quantified in several continuous cell lines, including mouse neuroblastoma (n- ) cells, bhk- cells, and l cells. application of immunofluorescence staining to detect lcmv antigen in inoculated cultured cells yields results more quickly than animal inoculation. of course, all diagnostic procedures involving potential contact with live virus should be carried out under strict containment conditions to avoid infection of laboratory personnel (see chapter ). the use of in vitro detection has the added advantage, in this regard, of reducing biohazardous exposure and the use of live animals for testing. differential diagnosis neurological signs must be differentiated from those due to mouse hepatitis virus, mouse encephalomyelitis virus, and meningoencephalitis from bacterial infection. trauma, neoplasia, and toxicities also must be ruled out in neurological disease with low prevalence. late-onset disease is associated with characteristic renal lesions, including deposition of viral antigen in tissues. early-onset disease must be differentiated from other causes of early mortality, such as mouse hepatitis virus, ectromelia virus, reovirus infection, tyzzer's disease, or husbandry-related insults. prevention and control adequate safeguards for procurement and testing of animals and animal products are essential to prevent entry. because mouse-to-mouse spread is slow, selective testing and culling for seropositive or carrier mice is possible. if mice are easily replaced, however, depopulation is a safer and more reliable option. valuable stock can be rederived, but progeny must be tested to preclude in utero transmission. because infected hamsters can excrete large quantities of virus, exposed hamsters should be destroyed and hamsters should not be housed with mice. infection of immunodeficient mice poses similar risk. lcmv can be transmitted to human beings, who can contract flu-like illness or severe cns disease. more frequently, human infection is subclinical. the zoonotic potential of lcmv infection makes it especially important to detect and eliminate carrier animals and other potentially contaminated sources, such as cell cultures, transplantable neoplasms, and vaccines to prevent human exposure. serum banking and periodic serological testing of highrisk human populations, such as those working with lcmv experimentally, are recommended. research complications lcmv may stimulate or suppress immunological responses in vivo and in vitro, and it can replicate in cells used as targets or effectors for immunological studies. introduction of immune cells to a carrier animal may elicit an immunopathological response. immune complex disease can complicate longterm experiments and morphological interpretations. illness and death in mice and zoonotic risk to humans are obvious research-related hazards. etiology sv is a paramyxovirus that is antigenically related to human parainfluenza virus . viral particles are pleomorphic, contain single-stranded rna, and have a lipid solvent-sensitive envelope that contains glycoproteins with hemagglutinating, neuraminidase, and cell fusion properties. sv grows well on embryonated hens' eggs and in several mammalian cell lines (e.g., monkey kidney, baby hamster kidney , and mouse fibroblast [l]). virus replicates in the cytoplasm and by budding through cell outer membranes. once common in laboratory rodent populations, sv is now rare or absent (livingston and riley, ; pritchett-corning et al., ) . clinical signs clinically affected adult mice often assume a hunched position and have an erect hair coat. rapid weight loss and dyspnea occur, and there may be chattering sounds and crusting of the eyes. although highly susceptible adults may die, lethal infection is more common in suckling mice. sex differences in susceptibility have not been found. genetically resistant mice usually have subclinical infection. athymic mice and immunodeficient mice are at high risk for development of a wasting syndrome. they develop illness later than their immunocompetent counterparts, since clinical signs in immunocompetent mice are related to immune-mediated destruction of respiratory epithelium. opportunistic infections can complicate the clinical presentation. for example, secondary bacterial infections of the ear can cause vestibular signs. epizootiology sv is transmitted by aerosol and is highly contagious. morbidity in infected colonies is commonly %, and mortality can vary from % to %, partly because strains of mice vary greatly in their susceptibility to lethal sv infection. for example, c bl/ mice are highly resistant to clinically apparent infection, whereas dba/ mice are highly susceptible. aerogenic infection is promoted by high relative humidity and by low air turnover. prenatal infection does not occur. enzootic infection is commonly detected in postweaned mice ( - weeks old) and is associated with seroconversion within - days and the termination of infection. therefore, entrenched infection is perpetuated by the introduction of susceptible animals. there is no evidence for persistent infection in immunocompetent mice, but prolonged infection is common in immunodeficient mice. maternally acquired immunity protects young mice from infection, and actively acquired immunity is thought to be long-lived. rats, hamsters, and guinea pigs also are susceptible to sv infection. therefore, bidirectional cross-infection is a risk during outbreaks. pathology viral replication is nominally restricted to the respiratory tract and peaks by the first week after infection. gross lesions feature partial to complete consolidation of the lungs (fig. . ) . individual lobes are meaty and plum-colored, and the cut surface may exude a frothy serosanguinous fluid. pleural adhesions or lung abscesses caused by secondary bacterial infection are seen occasionally, and fluid may accumulate in the pleural and pericardial cavities. sv targets airway epithelium and type ii pneumocytes. type i pneumocytes are less severely affected. histologically, the pattern of pneumonia is influenced by mouse genotype. susceptible mice usually have significant bronchopneumonia and interstitial pneumonia, whereas the interstitial component may be less prominent in resistant mice. typical changes begin with inflammatory edema of bronchiolar lamina propria, which may extend to alveolar ducts, alveoli, and perivascular spaces. necrosis and exfoliation of bronchiolar epithelium ensue, frequently in a segmental pattern (fig. . ). alveolar epithelium also may desquamate, especially in severe disease, and necrotic cell debris and inflammatory cells can accumulate in airways and alveolar spaces. alveolar septae are usually infiltrated by leukocytes to produce interstitial pneumonia (fig. . ) . lymphoid cells also invade peribronchiolar and perivascular spaces. the lymphocytic response to sv infection reflects the fact that cellular immunity contributes both to lesions and to recovery. local immunoglobulin synthesis by infiltrating cells also occurs. the extent of inflammatory cell infiltration corresponds to the level of genetic resistance expressed by the infected host, with clinically susceptible hosts mounting a more florid immune response than resistant hosts. additionally, strain-related differences in the severity of infection may reflect differences in airway mucociliary transport. multinucleated syncytia are occasionally seen in affected sucklings and scid mice, and inclusion bodies have been reported in infected athymic mice. regeneration and repair begin shortly after the lytic phase and are characterized by hyperplasia and squamous metaplasia of bronchial epithelium, which may extend into alveolar septae. proliferation of cuboidal laboratory animal medicine epithelium may give terminal bronchioles an adenomatoid appearance. repair of damaged lungs is relatively complete in surviving mice, but lymphocytic infiltrates, foci of atypical epithelium, and mild scarring can persist. acute phase lesions are prolonged in immunodeficient mice, which can lead to wasting and death. aged mice also have a prolonged recovery phase accompanied by focal pulmonary fibrosis (jacoby et al., ) . diagnosis sv is notable for its ability to cause epizootics of acute respiratory distress in adult genetically susceptible strains. serology is an effective means to detect infection in all strains of immunocompetent mice. antibody can be detected by days postinfection and coincides with development of clinical signs related to the immune-mediated necrotizing bronchiolitis and alveolitis. repeated serologic sampling over several weeks can help stage infection within a population. alternatively, sentinel animals can be added to seropositive colonies to detect active infection. irrespective of serologic results, histopathology, immunohistochemistry (which can be performed on formalin-fixed, paraffin-embedded sections), and, where possible, virus isolation should be used to confirm infection. virus can be isolated from the respiratory tract for up to weeks, with peak titers occurring at about days postinfection. nasopharyngeal washings or lung tissue homogenates are most reliable and should be inoculated into embryonated hens' eggs or bhk- cell monolayer cultures. sv infection of cultured cells is non-cytolytic, so erythrocyte agglutination or antigen detection methods must be used. rt-pcr also can be used to detect virus in infected lungs. differential diagnosis respiratory infection caused by pneumonia virus of mice (pvm) is generally milder or subclinical. histologically, necrosis of airway epithelium is less severe. bacterial pneumonias of mice, including murine respiratory mycoplasmosis, are sporadic and can be differentiated morphologically and by isolation of causative organisms. because sv pneumonia may predispose the lung to opportunistic bacterial infections, the presence of bacteria should not deter evaluation for a primary viral insult. control and prevention sv infection is self-limiting in surviving immunocompetent mice. suckling mice from immune dams are protected from infection by maternal antibody until after weaning. control and eradication measures must eliminate exposure of susceptible animals, so that infection can 'burn out.' this is most easily accomplished by a quarantine period of - weeks wherein no new animals are introduced either as adults or through breeding. control also is aided by the fact that sv is highly labile. barrier housing is preferred for prevention and for control of transmission. vaccination with formalin-killed virus can provide short-term protection of valuable mice but is not commonly used for prevention. research complications sv can cause immunosuppression and can inhibit growth of transplantable tumors. this effect has been attributed to virus-induced modification of tumor cell surface membranes. pulmonary changes during sv pneumonia can compromise interpretation of experimentally induced lesions and may lead to opportunistic infections by other bacteria. they also have been associated with breeding difficulties in mice. this sign is thought to be an indirect effect due to stress, fever, or related changes during acute infection. clinical signs natural pvm infection in mice is subclinical. therefore, its name is clinically misleading, being derived from pneumonic illness that occurred after serial passage of the agent in mice. however, dyspnea, listlessness, and wasting may develop in immunodeficient mice infected with pvm (weir et al., ) . pvm is used experimentally as a model to study acute respiratory infection, using highly pathogenic strains of the virus (dyer et al., ) . epizootiology pvm causes natural infections of mice, rats, hamsters, and probably other rodents and may be infectious for rabbits. serological data indicate that pvm was once common, but is now relatively uncommon (livingston and riley, ; pritchett-corning et al., ). pvm appears to spread less rapidly than sv. intimate contact between mice is probably required for effective transmission. this characteristic may reflect the fact that environmental inactivation of virus occurs rapidly. infection is acute and self-limiting in immunocompetent mice but may persist in immunodeficient mice. pathology pvm replicates exclusively in the respiratory tract and reaches peak titers in the lung - days after infection. although pulmonary consolidation can occur in experimentally infected mice, gross lesions are rare during natural infection. histological lesions can occur in the upper and lower respiratory tract. they consist of mild necrotizing rhinitis, necrotizing bronchiolitis, and interstitial pneumonia, which usually occur within weeks after exposure to virus and are largely resolved by weeks. the predominant inflammatory infiltrate is comprised of mononuclear cells, but some neutrophils laboratory animal medicine are usually present. immunohistochemistry on paraffinembedded tissues can be used to detect viral antigen in bronchiolar epithelium, alveolar macrophages, and alveolar epithelium during acute infection. residual lesions include nonsuppurative perivasculitis, which can persist for several weeks after acute infection has ceased. severe progressive pneumonia, with wasting, can occur in immunodeficient mice. it is characterized by generalized pulmonary consolidation that reflects severe interstitial pneumonia with desquamated alveolar pneumocytes and leukocytes filling alveolar spaces (fig. . ) . diagnosis diagnosis is based primarily on serological detection that can be supplemented by histopathology, immunohistochemistry, in situ hybridization, and virus isolation. virus replication in bhk- cells is detected by immunofluorescence or other antigen detection methods. virus also can be detected in tissues by rt-pcr. differential diagnosis because pvm is antigenically distinct from other murine viruses, serology is the most useful method to separate pvm infection from other respiratory infections of mice. however, in immunodeficient mice, where clinical signs and lesions are typical, it must be differentiated from other pneumonias, especially those due to sv and pneumocystis. additionally, pvm can coexist with and exacerbate pneumocystis infection in immunodeficient mice (bray et al., ) . prevention and control pvm infection is acute and self-limiting in immunocompetent mice, but persistent in immunodeficient mice. seropositive mice should be viewed as either immune or in the final stages of acute infection. therefore, control and prevention follows guidelines applicable to sv infection. research complications pvm can exacerbate pneumocystosis, as noted above. (ward et al., ) two members of the family reoviridae infect laboratory mice: reovirus per se (species: mammalian orthoreovirus) and murine rotavirus (species: rotavirus a), also known as epizootic diarrhea of infant mice (edim) virus. etiology reoviruses of mammals, although taxonomically considered one type species, have been divided into three cross-reacting prototypic serotypes: reovirus , and , which can be differentiated by cross-serum neutralization. mice can be infected with any serotype, but reovirus is emphasized because it has been associated with naturally occurring disease. natural infections in mice are usually not caused by pure serotypes, because reoviruses actively recombine. a number of wild-type and laboratory strains have been characterized, and related viruses have been recovered from virtually every mammal tested, as well as birds, reptiles, and insects. the virion contains segmented, double-stranded rna and is relatively heat stable. reoviruses replicate well in bhk- cells and other continuous cell lines, as well as in primary monolayer cultures from several mammals. clinical signs clinical disease is rare and age dependent. acute disease affects sucklings at about weeks of age, whereas adults have subclinical infection. signs in sucklings include emaciation, abdominal distension, and oily, matted hair due to steatorrhea. icterus may develop and is most easily discerned as discoloration in the feet, tail, and nose. incoordination, tremors, and paralysis occur just before death. convalescent mice are often partially alopecic and are typically runted. alopecia, runting, and icterus may persist for several weeks, even though infectious virus can no longer be recovered. infants born to immune dams are protected from disease by maternal immunity. epizootiology the prevalence of reovirus infection in contemporary mouse colonies is rare (livingston and riley, ; pritchett-corning et al., ). reoviruses are highly contagious among infant mice and can be transmitted by the oral-fecal or aerosol routes, but mechanical transmission by arthropods has also been documented. additionally, virus may be carried by transplantable neoplasms and transmitted inadvertently by injection. transmission is inefficient among adult mice. there is no evidence that vertical transmission is important or that genetic resistance or gender influence expression of disease. infection in immunocompetent mice appears to be self-limiting, lasting up to several weeks but terminating with the development of host immunity. the course of infection in immunodeficient mice should be considered prolonged, but the duration has not been determined. pathology reovirus can cause severe pantropic infection in infant mice. after parenteral inoculation, virus can be recovered from the liver, brain, heart, pancreas, spleen, lymph nodes, and blood vessels. following ingestion, reoviruses gain entry by infecting intestinal epithelial cells (m cells) that cover peyer's patches. virus can be carried to the liver in leukocytes, where it is taken up by kupffer cells prior to infecting hepatocytes. in acute disease, livers may be large and dark, with yellow foci of necrosis. the intestine may be red and distended, and, in infants, intestinal contents may be bright yellow. myocardial necrosis and pulmonary hemorrhages have been reported. myocardial edema and necrosis are especially prominent in papillary muscles of the left ventricle. the brain may be swollen and congested. central nervous system lesions have a vascular distribution, and are most prevalent in the brain stem and cerebral hemispheres. neuronal degeneration and necrosis are followed quickly by meningoencephalitis and satellitosis. severe encephalitis may evoke focal hemorrhage. in the chronic phase, wasting, alopecia, icterus, and hepatosplenomegaly may persist. orally infected suckling mice can develop multifocal hepatocyte necrosis, which may include the accumulation of dense eosinophilic structures resembling councilman bodies. hepatocytomegaly, kupffer cell hyperplasia, and intrasinusoidal infiltrates of mononuclear cells and neutrophilic leukocytes also can develop. in experimentally inoculated mice, necrotic foci can persist in the liver for at least weeks. chronic active hepatitis may develop after acute infection and result in biliary obstruction. acinar cells of the pancreas and salivary glands can undergo degeneration and necrosis. because pancreatic duct epithelium is susceptible to infection, parenchymal lesions in the pancreas may be caused by obstruction rather than by viral invasion of parenchyma. pulmonary hemorrhage and degeneration of skeletal muscles also have been observed. both humoral and cellular immunity seem to participate in host defenses, but it is unclear how host immunity may influence the course of chronic infection. oronasal inoculation of infant mice with reovirus results in a similar distribution but significantly milder lesions compared to reovirus . in contrast, reovirus is highly enterotropic, inducing mild enteritis without lesions in other tissues, similar to epizootic diarrhea of infant mice (edim) . diagnosis serology uses reovirus as antigen, which detects seroconversion to all serotypes, and viral rna can be detected by rt-pcr. a presumptive diagnosis of reovirus infection is aided clinically by detection of the oily hair effect, accompanied by jaundice and wasting. the presence histologically of multisystemic necrosis is consistent with severe reovirus infection but should be confirmed by immunohistochemistry or virus isolation. differential diagnosis reovirus infection must be differentiated from other diarrheal diseases of infant mice, including those caused by mouse coronaviruses, edim virus, salmonella spp., or clostridium piliforme. prevention and control although surviving mice appear to recover completely from infection, the potential for a carrier state is unresolved. therefore, it may be necessary, after adequate testing for the continued presence of virus by the use of sentinels, map testing, or other appropriate means, to rederive or replace infected stock. prevention depends on adequate barrier husbandry coupled with adequate serological monitoring. research complications reovirus infection can interfere with research in several ways. infections in breeding colonies can result in high mortality among sucklings from nonimmune dams. virus has been commonly recovered from transplantable neoplasms and is suspected of being oncolytic. the potential exists for interference with hepatic, pancreatic, cardiovascular, or neurological research. etiology rotaviruses are double-stranded, segmented rna viruses that have a wheel-like ultrastructural appearance. edim virus is a group a rotavirus that replicates in differentiated epithelial cells of the small intestine by budding into cisternae of endoplasmic reticulum. currently, only a single antigenic strain is recognized, but antigenically distinct variants may exist. edim virus shares an inner capsid antigen with rotaviruses of rabbits, fowl, nonhuman primates, human beings, and domestic and companion animals. these agents tend to be species-specific under natural conditions and can be differentiated by serum neutralization tests. cultivation of edim virus requires the presence of proteolytic enzymes to cleave an outer capsid polypeptide. clinical signs clinical signs occur in infant mice less than weeks old. this age-related susceptibility also applies to infection in immunodeficient mice. furthermore, clinical signs occur only in offspring of nonimmune dams, because maternal immunity protects infants until they have outgrown susceptibility to clinical disease. the cardinal signs are bloated abdomens with fecal soiling of the perineum, which may extend to the entire pelage in severe cases. despite high morbidity, mortality is low because affected mice continue to nurse. transient weight loss does occur, and there may be a delay in reaching adult weight. recovery from infection usually occurs in about weeks and, once weight is regained, is clinically complete. epizootiology edim virus appears to be infectious only for mice and occurs episodically in mouse colonies, and infection is probably widespread geographically (livingston and riley, ; pritchett-corning laboratory animal medicine et al., ) . all ages and both sexes can be infected, but genetic resistance and susceptibility have not been determined. the virus is highly contagious and is transmitted by the oral-fecal route. subclinically infected adult mice can shed virus in feces for at least days, an interval that may be extended in immunodeficient mice. after oral inoculation, virus is essentially restricted to the gastrointestinal tract, although small amounts of virus may be present in the liver, spleen, kidney, and blood. nursing dams can contract infection from their litters. transplacental transmission has not been demonstrated. pathology gross lesions occur primarily in the gastrointestinal tract, but thymic involution can result from infection-related stress. the intestine is often distended, flaccid, and filled with gray-green gaseous liquid or mucoid fecal material that soils the pelage. the stomach contains curdled milk, except in terminal cases with anal impaction due to caking of dried feces. virus preferentially infects terminally differentiated enterocytes in the small and large intestines, which accounts for the agerelated susceptibility to disease; the number of such cells decreases as the intestinal tract matures. characteristic histological lesions are often very subtle, but are most easily discerned in the small intestine in mice less than weeks old. they consist of vacuolation of villar epithelial cells with cytoplasmic swelling, which give villi a clubbed appearance (fig. . ) . the vacuoles must be differentiated from normal absorption vacuoles in nursing mice. the lamina propria may be edematous, but necrosis and inflammation are not prevalent. diagnosis edim virus infection is readily detected serologically. clinical disease is diagnosed from signs and typical histological lesions in the intestine, which can be confirmed by immunohistochemical or ultrastructural demonstration of virus in the intestine or in intestinal filtrates or smears. rotavirus antigen can be detected in feces by elisa, but certain dietary ingredients can cause false-positive reactions. infection can also be diagnosed by rt-pcr. differential diagnosis edim virus infection must be differentiated from other diarrheal diseases of suckling mice such as intestinal coronavirus (mouse hepatitis virus) infection, reovirus infection, tyzzer's disease, and salmonellosis. the presence of milk in the stomach can be helpful in differentiating edim virus infection from more severe enteric infections, such as those caused by pathogenic coronaviruses, during which cessation of nursing often occurs. the possibility of dual infections must also be considered. thymic necrosis in edim virus-infected mice, although nonspecific, must be differentiated from that due to mouse thymic virus (mtv) infection or other stressors. prevention and control the spread of edim can be controlled effectively by the use of microbarrier cages and good sanitation. because infection appears to be acute and self-limiting, cessation of breeding for - weeks to allow immunity to build in adults while preventing access to susceptible neonates also is recommended. alternatively, litters with diarrhea can be culled, in combination with the use of microbarrier cages. the duration of infection in immunodeficient mice has not been determined, but it is reasonable to assume that chronic infection occurs. therefore, such animals should be eliminated. litters from immune dams are more resistant to infection. if edim virus is allowed to become enzootic within a colony, clinical signs will disappear within the population, which may be an appropriate management approach in conventional colonies. prevention of edim virus infection depends on maintenance of sanitary barrier housing with adequate serological surveillance. research complications the research complications of edim infection pertain to clinical illness with diarrhea and retarded growth. transient thymic necrosis may perturb immunological responses. infection (barthold, a,b) etiology coronaviruses are large, pleomorphic, enveloped rna viruses with radially arranged peplomers (spikes). in mice, early clinical and laboratory investigations emphasized their potential to induce hepatitis, so their original designation, which is still used actively, is mouse hepatitis virus (mhv). during that time, enteritis in infant mice was recognized as a separate entity caused by an uncharacterized virus, known as lethal intestinal virus of infant mice (livim). subsequent studies revealed that hepatitis-causing mhv and enteritis-causing livim were closely related coronaviruses, now collectively termed mhv. mhv isolates differ in biologic behavior according to their organ tropism into two biotypes: enterotropic strains, which infect primarily the intestinal tract, and polytropic strains, which initially infect the respiratory tract but may progress to multisystemic dissemination, including the liver and brain. these differences are often reflected in their cell tropism in vitro. however, natural isolates may contain features of both biotypes. several prototype polytropic strains have been extensively studied as experimental models of hepatitis and encephalitis. they include jhm (mhv ), mhv- , mhv- , mhv-s, and mhv-a . numerous additional strains have been identified that differ in virulence, tissue tropism, and antigenicity. differentiation by strain, particularly under natural conditions, is irrelevant, since mutation is common among coronaviruses, and even named prototype strains differ significantly depending upon passage history. although mhv isolates and strains share internal antigens (m and n), they can be distinguished by neutralization tests that detect strainspecific spike (s) antigens. mhv shares antigens with the coronaviruses of rats, a finding that has been exploited to develop heterologous antigens for serological tests. mhv also is related to human coronavirus oc . a number of established cell lines may be used for propagating polytropic mhv strains in vitro. however, field isolates are difficult to maintain in vitro. nctc mouse liver cells are useful for growing many polytropic strains. mhv can also be grown in mouse macrophages, cells that have been used for genetic studies of resistance and susceptibility to infection. enterotropic strains, because of their tendency to be strictly enterotropic, have been grown in cmt- cells derived from a rectal carcinoma in a c bl mouse, but are generally difficult to propagate in cell culture. irrespective of cellular substrate used for isolation or propagation, syncytium formation is emblematic of mhv infection (fig. . ) . clinical signs clinical signs depend primarily on the age, strain, and immunological status of infected mouse and strain and tropism of virus. as with many murine viruses, infection is often clinically silent among immunologically competent mature mice. clinical morbidity is most often associated with suckling mice less than weeks old or with immunodeficient mice. suckling mice infected with enterotropic mhv develop inappetence, diarrhea, and dehydration, often terminating in death (fig. . ) . epizootics of enterotropic mhv have been known to result in % mortality among neonatal mice in a breeding colony. older mice ( - weeks of age) may have ruffled pelage and runting. neurotropic strains such as mhv-jhm may induce flaccid paralysis of the hindlimb, but this sign is rarely encountered alone during natural infection. conjunctivitis, convulsions, and circling may be seen occasionally. enterotropic strains may not cause acute disease in athymic mice when exposed as adults, whereas mildly pathogenic polytropic strains can cause a progressive wasting syndrome that may be accompanied by progressive paralysis. epizootiology mhv infection, despite constant surveillance and preventive programs, continues to be a common threat to laboratory mouse populations (livingston and riley, ; pritchett-corning et al., ). there are no reports of natural transmission from mice to other species, but suckling rats have been found to develop necrotizing rhinitis after intranasal inoculation with mhv-s. mhv is highly contagious, with natural transmission occurring by respiratory or oral routes. mouse appears normal and has a milk-filled stomach. lower mouse is runted and dehydrated and has an empty stomach. from barthold et al. ( ) . enterotropic biotypes predominate in natural infections in contemporary laboratory animal facilities, since they tend to be the most contagious due to copious excretion of virus in feces, whereas polytropic strains generally spread by direct respiratory contact. natural vertical transmission has not been demonstrated. introduction of mhv through injection of contaminated biologicals can be an important factor in epizootics, especially because some isolates infect b lymphocytes and, by implication, hybridomas nonlytically. infection in immunocompetent mice is self-limiting. immune-mediated clearance of virus associated with seroconversion usually begins about a week after infection, and mice recover fully within - weeks. humoral and cellular immunity participate in host defenses to infection, and t-cell-dependent immunity is an absolute requirement. thus, age-related resistance to mhv correlates with maturation of lymphoreticular tissues, but intestinal proliferative kinetics are critical determinants of disease susceptibility with enterotropic mhv. enzootic infection had been construed to include persistent infection in individual mice. current evidence suggests, however, that enzootic infection results either from the fresh and continuous introduction of immunologically naive or deficient mice or from the recurrent infection of immune mice with mhv variants that arise by natural mutation. mutation is favored by immune pressure in enzootically infected colonies as well as missteps during natural replication, which include copying errors and recombination. thus, mice that have developed immunity to one strain of mhv can remain susceptible to one or more genetically and antigenically divergent strains, resulting in reinfection. this caveat has practical importance for breeding colonies. maternal immunity protects suckling mice against homologous mhv strains but not against antigenically variant strains. however, maternal immunity, even to homologous strains, depends on the presence of maternally acquired antibody in the lumen of the intestine. therefore, the susceptibility of young mice to infection increases significantly at weaning. strain differences in resistance and susceptibility to polytropic mhv can be inherited as an autosomal dominant trait. for example, dba/ mice are highly susceptible to mhv- and die acutely even as adults, whereas a/j mice develop resistance to lethal infection shortly after weaning. however, genetic resistance is also virus strain-dependent. therefore, mice resistant to one strain of mhv may be susceptible to another strain. it also is worth noting that the expanded use of genetically altered mice with novel or unanticipated deficits in antiviral responses may alter the outcome of virus-host interactions unpredictably. this pertains to mhv as well as other agents. for example, mhv infection has presented as granulomatous peritonitis and pleuritis in interferongamma (ifn-γ) knockout mice (france et al., ) . pathology polytropic strains replicate initially in the nasal mucosa, where necrotizing rhinitis may occur. viremic dissemination can follow if virus gains access to regional blood vessels and lymphatics. thus, viremia leads to secondary infection of vascular endothelium and parenchymal tissues in multiple organs including liver, brain, lymphoid organs, and other sites. mice also may develop central nervous system disease by direct extension of infection from the olfactory mucosa along olfactory tracts. at necropsy, yellow-white foci indicative of necrosis can occur in multiple tissues, with the involvement of the liver as the classical lesion. liver involvement may be accompanied by icterus and peritonitis. histologically, necrosis can be focal or confluent and may be infiltrated by inflammatory cells (fig. . ) . syncytia commonly form at the margin of necrotic areas and, in mild infections, may develop in the absence of frank necrosis. syncytia formation is a hallmark of infection in many tissues, including the intestine (fig. . ) , lung, liver, lymph nodes, spleen, thymus, brain, and bone marrow and in vascular endothelium in general. although syncytia are transient in immunocompetent mice, they are a persistent feature in chronically infected, immunodeficient mice ( fig. . ) . neurotropic variants cause acute necrotizing encephalitis or meningoencephalitis in suckling mice, with demyelination in the brain stem and in peri-ependymal areas secondary to viral invasion of oligodendroglia. convalescent mice may have residual mononuclear cell infiltrates around vessels or as focal lesions in the liver. immunodeficient mice can develop progressive necrotic lesions in the liver and elsewhere. compensatory splenomegaly may occur because of expansion of hematopoietic tissue. enterotropic strains infect primarily the intestine and associated lymphoid tissues, although some may also figure . necrosis, inflammation, and syncytium in the liver of a mouse infected with mhv. courtesy of s.w. barthold. cause systemic lesions, especially in the liver and brain. the most common sites are terminal ileum, cecum, and proximal colon. the severity of disease is age-related, and dependent upon intestinal proliferative kinetics, similar to edim, with young infants being at highest risk for lethal infection. pathogenic strains can cause lesions ranging from villus attenuation to fulminant necrotizing enterotyphlocolitis, which can kill suckling mice within a few days (fig. . ) . the stomach is often empty, and the intestine is filled with watery to mucoid yellowish, sometimes gaseous contents. syncytia are a consistent feature in viable mucosa (fig. . ) and not only are formed in intestine but also may be present in mesenteric lymph nodes and endothelium of mesenteric vessels. enterocytes may contain intracytoplasmic inclusions, but they are not diagnostic. surviving mice develop compensatory mucosal hyperplasia, which eventually recedes, but may contribute to clinical signs due to osmotic, secretory, and malabsorptive diarrhea. older mice are equally susceptible to infection, but are resistant to severe disease due to their mature (more rapid) intestinal proliferative kinetics. pathology may be subtle, consisting of transient syncytia without necrotic lesions. in adult mice, syncytia can be found most often in the surface mucosal epithelium of the ascending colon. the exception occurs in immunodeficient mice, such as athymic and scid mice, which can develop chronic proliferative bowel disease of varying severity with mhv antigen in mucosal epithelium (figs. . and . ). this may not always be present, as athymic nude mice exposed as adults may only manifest a few enterocytic syncytia without hyperplasia. diagnosis because mhv infection is often subclinical, serological testing is the most reliable diagnostic tool. many animal resources rely on sentinel mouse protocols for continuous serological surveillance. serology is well established, sensitive, and reliable. neutralization tests are used to differentiate individual virus strains in the research laboratory but are inappropriate for routine use, because of cost, technical complexity, and serologic identification per se does not predict biological behavior, including virulence or tissue tropism. serology also can be used in the context of map testing in which adult mice are inoculated with suspect tissues to elicit seroconversion. rt-pcr protocols to detect virus in tissues or excreta are available. the detection of syncytia augmented, when possible, by immunohistochemistry to laboratory animal medicine detect mhv antigen is a useful and practical means to confirm infection. this strategy should attempt to select mice that are in early stages of infection, because necrosis in infant mice or seroconversion in older mice may reduce the chances of detecting syncytia or viral antigens. the option of using immunodeficient mice as sentinels can be considered, because they sustain prolonged infection. however, they should be securely confined because they also amplify virus loads. if properly controlled, amplification in immunodeficient mice can, however, facilitate subsequent virus isolation in tissue culture. differential diagnosis mhv infection must be differentiated from other infectious diseases that cause diarrheal illness, runting, or death in suckling mice and wasting disease in immunodeficient mice. these include edim, mousepox, reovirus infection, tyzzer's disease, and salmonellosis. neurological signs or demyelinating lesions must be differentiated from mouse encephalomyelitis virus infection or noninfectious cns lesions, such as neoplasms, including polyoma virus-induced tumors in athymic mice. prevention and control control and prevention of mhv infection can be difficult because of the numerous variables that influence its expression. perhaps the most important factor is the duration of infection in individual mice and in mouse colonies. there is evidence that infection in an individual immunocompetent mouse is acute and self-limiting. such mice can be expected to develop immunity and eliminate virus within days. therefore, selective quarantine at the cage (not room) level with the temporary cessation of breeding can be used effectively to eliminate infection. quarantine at the room basis is likely to fail, since mutations arise and continually reinfect the mouse population. additionally, maternally derived immunity can protect infant mice from infection until they are weaned and moved to uncontaminated quarters. careful testing with sentinel mice should be used to assess the effectiveness of quarantine or 'natural rederivation,' as just described. immunodeficient mice, in contrast, are susceptible to chronic infection and viral excretion. mice with unrecognized or unanticipated immune dysfunction or with selective immune dysfunction may impact on mhv infection and its control. such colonies, which may contain highly valuable or irreplaceable mice, may be rescued by cesarean rederivation or embryo transfer if vertical transmission of mhv infection is subsequently ruled out. although rodent coronaviruses are not viable for extended periods in the environment, excreted virus may remain infectious for up to several days, so proper sanitation and disinfection of caging and animal quarters as well as stringent personal sanitation are essential to eliminate infection. the prevention of mhv requires procurement of animals from virus-free sources and maintenance under effective barrier conditions monitored by a well-designed quality assurance program. control of feral mouse populations, proper husbandry and sanitation, and strict monitoring of biological materials that may harbor virus (e.g., transplantable neoplasms, cell lines) are also important strategies to prevent adventitious infection. research complications numerous research complications have been attributed to mhv, and the unpredictable outcome of infection in genetically altered mice is likely to lengthen the list. for example, apart from its clinical impact, mhv may stimulate or suppress immune responses, contaminate transplantable neoplasms, and be reactivated by treatment of subclinically infected animals with several classes of drugs, including immunosuppressive agents, and by intercurrent infections. it also can alter tissue enzyme levels. additionally, the ubiquitous threat of mhv infection and uncertainty about its potential effects on a given research project provoke concerns that may exceed its true impact. for example, transient infection with a mild enterotropic strain is unlikely to disrupt systemic immune responses, whereas infection with a polytropic strain may be highly disruptive. this is not to say that subclinical or strictly enterotropic infection should be taken lightly but simply to caution against overreaction in assessing the impact of an outbreak. etiology mice are susceptible to infection by two members of the cardiovirus genus within the theilovirus. emcv has a less selective host range and can infect wild mice, but is not known to infect laboratory mice. tmev is a small, nonenveloped, rna virus that was discovered by max theiler during experimental studies of yellow fever virus in mice. established prototype strains include to (theiler's original), fa, da, and gd vii, the last of which is named after george martine (george's disease), an assistant in theiler's laboratory. tmev is rapidly destroyed by temperatures over °c and by alcohol but not by ether. it can be cultivated in vitro in several continuous cell lines, but bhk- cells are routinely used for isolation and propagation. tmev is antigenically related to emcv. as with other nonenveloped viruses, tmev is resistant to environmental inactivation, a factor that must be considered in control and prevention of infection. clinical signs the development of clinical disease depends on virus strain, mouse strain, and route of exposure, but natural disease is exceedingly rare (estimated at . - . % of infected mice). when clinical signs occur, they are expressed as neurological disease. the characteristic sign is flaccid posterior paralysis, which may be preceded by weakness in the forelimbs or hindlimbs, but in mice that are otherwise alert (fig. . ) . some mice may recover, but death frequently ensues, often because of failure to obtain food or water. furthermore, mice that recover from the paralytic syndrome are disposed to a chronic demyelinating phase, which is expressed as a gait disturbance. epizootiology infection occurs primarily in laboratory mice with the exception of the mgh strain, which has been isolated from laboratory rats and is pathogenic in mice and rats after experimental inoculation. the prevalence of tmev in mouse colonies is low, a reflection of the slow rate at which virus is transmitted from mouse to mouse, but it continues to be among the more common viral contaminants of mouse colonies (livingston and riley, ; pritchett-corning et al., ) . tmev infection is acquired by ingestion and replicates primarily in the intestinal mucosa. enteric infection can persist after the development of host immunity and can result in chronic or intermittent excretion of virus in feces over several months . mice often become infected shortly after weaning, but virus is seldom recovered in mice over months of age. however, neurologic infection can persist in the brain and spinal cord for at least year. immunity to one strain of tmev provides cross-protection to other strains. there are no reports of differences in mice with respect to susceptibility to infection under natural conditions. prenatal transmission has not been found. pathology intestinal tmev infection does not cause lesions, but virus can be detected in enterocytes by immunohistochemistry or in situ hybridization. poliomyelitis-like disease, the syndrome that may be encountered during natural infections, is characterized by acute necrosis of ganglion cells and neurons, neuronophagia, and perivascular inflammation, which occur particularly in the ventral horn of the spinal cord gray matter but also can involve higher centers such as the hippocampus, thalamus, and brain stem. during the subsequent demyelinating phase, mononuclear cell inflammation develops in the leptomeninges and white matter of the spinal cord, accompanied by patchy demyelination. the white-matter lesions are due to immune injury. spontaneous demyelinating myelopathy, affecting the thoracic spinal cord and associated with mev infection, has also been reported in aged mice. virulent strains may cause acute encephalitis after experimental inoculation, whereas less virulent isolates produce acute poliomyelitis followed by chronic demyelinating disease. diagnosis infection is usually detected serologically or by pcr of feces, but virus shedding from infected mice may be intermittent. clinical signs are striking, if they occur, but are too rare to rely on for routine diagnosis. histological lesions in the cns and especially the spinal cord are characteristic when present. differential diagnosis neurotropic variants of mhv may, on occasion, cause similar neurological signs. injury or neoplasia affecting the spinal cord can also produce posterior paralysis. polyoma virus infection in athymic mice can induce tumors or demyelination in the cns, which may result in clinical signs resembling those of tmev infection. prevention and control disease-free stocks were originally developed by foster-nursing infant mice. this technique, cesarean rederivation, or embryo transfer can be used successfully to eliminate infection. in either case, foster mothers should be surveyed in advance to ensure their mev-free status. selective culling can be considered as an option to eliminate infection, because laboratory animal medicine infection spreads slowly. however, the virus is hardy in the environment and resists chemical inactivation, so it may be prudent to depopulate and disinfect rooms if the presence of infection is unacceptable. research complications the principal hazard from tmev for research relates to its potential effects on the cns. noroviruses are nonenveloped rna viruses that belong to the family caliciviridae. they are notoriously resistant to environmental inactivation, and cause significant gastrointestinal morbidity in humans. noroviruses are species-specific, including mnv, which exclusively infects mice. until the discovery of mnv, replication of noroviruses in vitro has not been possible. for this reason, mnv has emerged as an important small animal model of norovirus pathogenesis. mnv was relatively recently discovered in , and subsequent surveillance has revealed that it is the most common adventitious virus infection in laboratory mice (hsu et al., ; pritchett-corning et al., ) . over mnv isolates have been found in mouse research colonies around the world, which display nearly % genetic identity, comprising a single genetic cluster. although genetically homogeneous, significant biological differences exist among mnv strains (thackray et al., ) . mnv effectively replicates in macrophages and dendritic cells, including the mouse macrophage-like raw . cell line, as well as a microglial cell line (wobus et al., ) . clinical signs clinical signs of infection in immunocompetent mice are usually absent, but infection leads to systemic disease with high mortality in interferon αβγ receptor and stat null mice. affected mice have loss of body weight, ruffled fur, and hunched posture (ward et al., ) . experimental infection of and c h mice with mnv- caused mild diarrhea (kahan et al., ) . epizootiology mnv is transmitted by the fecaloral route, and contaminates the environment as an environmentally resistant virus. for this reason, it can efficiently infect sentinel mice with soiled bedding (manuel et al., ) . duration of infection varies with mnv strain, mouse immunocompetence and mouse genotype. experimental studies have revealed that several mnv strains persist in various tissues of c bl/ j, hsd:icr, and jcl:icr and c.b- -prkdc scid mice, with fecal shedding for at least - days (goto et al., ; hsu et al., ; thackray et al., ) . although not clinically ill, rag null mice are unable to clear infection . comparative studies with mnv- and mnv- have shown differences in virus replication and shedding (kahan et al., ) . mnv has a tropism for macrophages and dendritic cells, and virus can be detected in the intestine, intestinal lymphoid tissue, liver, and spleen (hsu et al., ; kahan et al., ; wobus et al., ) . pathology naturally and experimentally infected stat or ifnγr null mice may develop splenomegaly and multifocal pale spots on the liver. microscopic findings include varying degrees of hepatitis, focal interstitial pneumonia, vasculitis, peritonitis, and pleuritis (karst et al., ; ward et al., ) . encephalitis, cerebral vasculitis, pneumonia, and hepatitis have also been described in intracerebrally infected stat null mice (karst et al., ) . infection of immunocompetent mice may be associated with mild inflammation of the intestine, splenic hypertrophy, and lymphoid hyperplasia of spleen and lymph nodes (mumphrey et al., ) . diagnosis mnv infection can be detected by serology or rt-pcr. sentinel mouse surveillance, using soiled bedding, is an effective strategy for detecting mnv (manuel et al., ) differential diagnosis the mild change in fecal consistency associated with mnv in adult mice may mimic rotavirus, coronavirus, helicobacter spp., citrobacter rodentium, or other enteric diseases. disseminated lesions in stat or ifnγr null mice must be differentiated from other polytropic viral diseases in immunodeficient mice, including mhv. prevention and control depopulation and decontamination has been shown to be effective at eliminating mnv from an enzootically infected colony, whereas testand-removal of positive mice was found to be ineffective (kastenmayer et al., ) . embryo transfer and cesarean rederivation are also effective (goto et al., ; perdue et al., ) . neonatal mice are resistant to infection, so that cross-fostering neonates onto uninfected dams is another effective means of rederivation mnv-free mice (artwohl et al., ; compton, ) . research complications the tropism of mnv for macrophages and dendritic cells is likely to modify immune responses, and mnv infection may interfere with studies involving enteric disease. hantaviruses are rna viruses belonging to the very large bunyaviridae family. they differ from other members of this family by not being arthropod-borne. each hantavirus is antigenically distinct and maintained within single or at most a few rodent or insectivore hosts, but are infectious for other hosts. infection is lifelong, and virus is transmitted by shedding of virus in urine, feces, and saliva. several hantaviruses are zoonotic and may cause severe disease in humans. although there is overlap, hantaviruses in asia and europe cause hemorrhagic fever with renal syndrome (hfrs) in humans, a multisystem disease with significant renal involvement, and hantaviruses that are endemic in the americas cause laboratory animal medicine hantavirus pulmonary syndrome (hps) in humans, which is a multisystem disease with pulmonary involvement. among the better-known old world hfrs hantaviruses are hantaan, seoul, puumala, and dobrava-belgrade viruses. sin nombre virus is the best known new world phs hantavirus, among many others. most notably from the perspective of laboratory animal medicine, the norway rat, rattus norvegicus, serves as a reservoir host for hantavirus in the wild, but infection has also been associated with laboratory rats. in addition to being endemic in wild rats in asia, it has been found to be endemic in wild rats in the eastern united states and associated with human cases of hfrs (childs et al., ; leduc et al., ; tsai et al., ) . over cases of hantavirus infection have been transmitted to humans from laboratory rats in japan, belgium, and the united kingdom (desmyter et al., ; kawamata et al., ; lloyd et al., ; umenai et al., ) . m. musculus is not considered to be a primary reservoir host, but hantavirus infection has been documented serologically in conventional and barrier-maintained laboratory mice and rats in korea (won et al., ) , infection of wild m. musculus has been documented in the united states (baek et al., ) , and infection of wild mice in europe has been associated with human exposure (diglisic et al., ) . hantaviruses are difficult to culture in vitro. infection in rodents is subclinical and is detected by serology or rt-pcr. the main research complication from natural infection is the zoonotic risk and potentially subclinical effects on the immune response associated with viral defenses such as cd + t cell (taruishi et al., ) and nk function as demonstrated in human studies (braun et al., ) . the mouse is host to a number of enveloped rna viruses of the family retroviridae, subfamily orthovirinae, including the two type species (and their variants) mouse mammary tumor virus (mmtv) and murine leukemia virus (mlv). these viruses belong to a diverse assemblage of related mobile dna elements that are integrated into the host genome, and collectively termed 'retroelements', which include retrovirus-related elements and nonviral elements. during cell division, retroelements are transcribed into rna, and subsequently reverse-transcribed into dna copies that become integrated into a new location within the genome. this process utilizes reverse transcriptase, which is encoded by the retroelement. over millennia, retroelements have been repeatedly integrated within the genome in large numbers, comprising approximately % of the mammalian genome. various families of mouse retroelements share sequence similarity, despite their random distribution throughout the mouse genome, and the majority of them are truncated, mutated, and methylated to become incapable of infectivity. nevertheless, many of them continue to be mobile within the genome. noninfectious retrovirus-related retroelements include iap, vl , musd, and etn elements. replication-competent retroviruses represent the pinnacle of the retroelement constellation and are best considered as the most evolutionarily recent members. these include mmtv and mlv. mtv and mlv share similar genetic structure, except that the long terminal repeat (ltr) region of the mmtv genome encodes an additional superantigen (sag). both mmtv and mlv include exogenous viruses, which are horizontally transmitted, replication-competent viruses, and endogenous viruses, which are closely related to exogenous viruses, encoded within the mouse genome, and transmitted by mendelian inheritance. exogenous mmtv and mlv exist in wild mouse populations, but have been eliminated from contemporary laboratory mice. however, they may continue to be used experimentally, including bittner mmtv, and gross, friend, moloney, and rauscher mlvs. in particular, mouse colonies may be purposely infected with mmtv for mammary cancer research and are termed 'mmtv-positive', reflecting their exogenous virus status, even though the mice may also carry endogenous mmtv. the genomes of all inbred strains of mice encode one or more (over in some mouse strains) endogenous mmtv loci, the distribution of which is unique to each inbred strain of mouse. most mmtv genomic loci do not encode infectious virus or are transcriptionally inactive, except for mouse strains (dba, c h, grs) that carry mtv or mtv loci. these loci encode infectious virus, which can be visualized as b-type particles by electron microscopy. likewise, all mouse strains carry endogenous mlv loci within their genome, but not all mice carry replication-competent mlv sequences. some endogenous mlvs encode infectious virus, which can be visualized as c-type particles by electron microscopy. mice have often evolved mechanisms to counter the deleterious effects of retroviruses by preventing reentry or replication of virus into other cells. if an endogenous retrovirus is still infectious to other mouse cell targets, it is termed ecotropic, whereas if it is no longer infectious for mouse cells, but can infect cells of other species, it is termed xenotropic. viruses capable of infecting cells of mice as well as other species are termed polytropic. the combinations of endogenous replication-competent mlvs and cell tropism factors are a reflection of selective breeding of mouse strains for susceptibility to various types of cancer. clinical signs mice were originally inbred for specific phenotypes, including mammary tumors and lymphomas. thus, some strains of mice were genetically selected for unique combinations of endogenous mmtv and mlv in concert with susceptibility factors laboratory animal medicine that favored their expression and disease manifestations. in addition, noninfectious retroelements continue to reintegrate randomly within the genome during cell division as retro transposons. these ongoing integrations contribute to genetic drift, spontaneous mutations, and well-recognized mouse strain phenotypes, including the athymic nude allele, the hairless allele, and the rodless retina allele, among others. epizootiology exogenous mmtv and mlv are horizontally transmissible, primarily through the milk of lactating females. endogenous retroviruses and retroelements are inherited through the genome. replicationcompetent endogenous mmtvs and mlvs are also transmissible like their exogenous counterparts, but differ by being integrated within the genome of the mouse. pathology replication-competent mmtv and mlv, regardless of their exogenous or endogenous origin, are usually clinically silent. their ability to cause neoplasia is a reflection of genetic selection for susceptibility factors that are genetically encoded within individual mouse strain genomes. mmtv derives its name from its association with induction of mammary carcinomas in mammary cancer-susceptible strains of mice. mlv is associated with lymphomas, the pattern of which is mouse strain specific. for example, akr mice develop % prevalence of thymic lymphoma between and months of age, whereas aging balb/c mice commonly develop multicentric lymphoma. in these strains of mice, multiple endogenous mlvs are coexpressed in tissues and undergo recombination events that allow them to target and transform cells into neoplasia. despite its name, mmtv can induce lymphomas in some strains of mice, such as sjl mice which develop lymphomas arising from enteric lymphoid tissue and mesenteric lymph nodes. diagnosis exogenous retroviruses have been eliminated from contemporary mouse populations, unless purposely introduced for experimental purposes. because endogenous retroviruses and retroelements are encoded within the genome, and reflect the unique genetic composition of each strain of mouse, they are not targets of diagnostic pursuit. differential diagnosis patterns of some types of neoplasia within individual inbred strains of mice are a reflection of their endogenous retroviral integration. prevention and control exogenous retroviruses have been eliminated from laboratory mice by cesarean rederivation and foster nursing. mmtv-s, the 'bittner agent', continues to be purposely maintained in some mouse breeding populations, but can be eliminated by foster-nursing or other means. caution is advised when re-deriving such mouse colonies for other purposes, as elimination of exogenous mmtv will be an unintended consequence. research complications endogenous retroviruses and retroelements influence the life span of individual strains of mice, and random integrations during cell division can give rise to spontaneous mutations and genetic drift. it is estimated that significant mutations may arise due to mobile retroelement integrations every generations. astroviruses are small, nonenveloped, singlestranded rna viruses that have been associated with human gastroenteritis and detected in association with other enteric pathogens. the viral family astroviridae is split into two genuses: avastrovirus for those astroviruses infecting avians and mamastrovirus for those infecting mammals. astrovirus infection has been detected in research mice (muastv) using metagenomic analyses and appears to have a wide geographical, institutional, and host strain distribution. clinical signs none reported. epizootiology pcr screening has found muastv infection in up to % of a variety of mouse strains housed in vendor and academic facilities in the united states and japan. the virus has been detected most commonly in immunocompromised mice (nsg, nod-scid, nsg- gs, c bl -timp- −/− , and upa-nog), but also in immuncompetent strains (b j, icr, bash , and balb/c). both immunodeficient and immunocompetent mice are susceptible to muastv, but adaptive immunity is required to clear the virus. based on human epidemiology indicating children are at highest risk for infection, the virus may preferentially infect young mice. pathology immunodeficient mice showed no sign of pathology based on histopathology. diagnosis pcr data has indicated that muastv causes a systemic, chronic infection in immunocompromised mice, indicating samples from most tissues will be pcr positive. yokoyama et al. ( ) detected high viral load (up to genome copies) per fecal pellet from immunocompetent mice. differential diagnosis none, in the absence of lesions and clinical disease. prevention and control because immunocompetent mice clear the infection, quarantine may be successful but lack of routine screening for muastv in laboratory mice will allow for uncontrolled spread of the infection. research complications based on limited surveys, muastv may have a high prevalence in laboratory mice. the impact of infection on both innate and adaptive immune responses warrants further investigation to assess the potential for confounding research data. this section briefly describes the etiology, clinical signs, epizootiology, pathology, diagnosis, differential diagnoses, prevention and control, and research complications of the most common bacterial diseases encountered in research colonies of mice. as sequencing technology becomes more available, the number and genus/species classification of bacteria potentially responsible for infections, in particular, opportunistic infections, will grow (benga et al., ) . potential candidates include members of pasteurellacaeae, bordetella hinzii, streptococcus danielae, acinetobacter spp., and others, for which little is currently known about their pathogenic potential. etiology lawsonia intracellularis, an obligate intracellular bacterium and the causative agent of proliferative enteropathy, is not a pathogen encountered in research colonies of mice but has been reported to infect wild mice and rats in close contact with infected livestock (collins et al., ) . clinical signs none reported but should consider lawsonia as a differential in necropsy cases with gross or histologic evidence of proliferative lower bowel lesions. epizootiology although mice are experimentally susceptible to infection and develop classic lesions of hyperplastic ileitis and typhlocolitis (murakata et al., ) , susceptibility varied with mouse strain and source of inoculum from rabbits or swine, suggesting important differences in l. intracellularis strains. pathology lawsonia infection may result in hyperplastic ileitis, typhlitis and/or colitis, and hemorrhagic intestines may be noted (percy and barthold, ) . diagnosis lawsonia spp. has been diagnosed using a variety of techniques, including pcr, immunohistochemistry, in situ hybridization, and warthin-starry silver stains. differential diagnosis bacterial infections associated with hyperplastic intestinal epithelium, including c. rodentium and enterohepatic helicobacter species in susceptible (typically immunodeficient) mouse strains. prevention and control species separation from hosts more commonly associated with natural infection (hamsters, ferrets, pigs). research complications none reported. the following section describes infection due to mycoplasma pulmonis and summarizes infections associated with other murine mycoplasmas including m. arthriditis, m. neurolyticum, m. collis, and m. muris. antigenic cross-reactivity among these species, and especially between m. pulmonis and m. arthriditis, mandates that reliable diagnostic strategies incremental to serology (elisa, ifa, mfia) such as culture (often false negative) and pcr be employed to distinguish potentially pathogenic infections. when screening cell lines for opportunistic pathogens, pcr is the most efficient method to discriminate between m. pulmonis and mycplasma contaminants associated with cell culture. etiology m. pulmonis is a pleomorphic, gram-negative bacterium that lacks a cell wall and has a single outer limiting membrane. it causes murine respiratory mycoplasmosis (mrm). clinical signs mice are relatively resistant to florid mrm; thus, subclinical infection is more common. when clinical signs occur, they reflect suppurative rhinitis, otitis media, and chronic pneumonia. affected mice may display inactivity, weight loss, and ruffled hair coat, but the most prominent signs are 'chattering' and dyspnea, due to rhinitis and purulent exudate in nasal passages. otitis media may cause a head tilt, whereas suppurative inflammation in the brain and spinal cord, although rare, can cause flaccid paralysis. experimental infection of the genital tract can cause oophoritis, salpingitis, and metritis, which may lead to infertility or fetal deaths. experimental inoculation of scid mice has caused systemic infection accompanied by severe arthritis (evengard et al., ) . epizootiology mrm historically was a common infectious disease of mice, but improved housing, husbandry, and health surveillance have reduced its prevalence dramatically. serologic data from a large diagnostic laboratory indicated m. pulmonis infection affects about . % of conventionally housed mouse colonies in the united states and . % in europe (pritchett-corning et al., ) . m. pulmonis infection is contracted by inhalation and can occur in suckling and adult mice. therefore, infection should be considered highly contagious. mice injected with cells harvested from m. pulmonis contaminated cell cultures may develop disease. m. pulmonis can also be transmitted venerally; in utero infection has been demonstrated in rats but not in mice. because transplacental infection occurs in rats, the same route may be possible in mice, particularly immunocompromised strains. concomitant viral pneumonia (sv, mouse coronavirus) or elevated environmental ammonia concentrations may increase susceptibility to mrm. m. pulmonis also infects rats, hamsters, guinea pigs, and rabbits. among these species, only rats are significant reservoirs of infection for mice. pathology m. pulmonis is an extracellular organism that colonizes the apical cell membranes of respiratory epithelium. attachment occurs anywhere from the anterior nasal passages to the alveoli and may be mediated by surface glycoproteins. the organism may injure host cells through competition for metabolites such as carbohydrates and nucleic acids or by release of toxic substances such as peroxides. ciliostasis, reduction in the number of cilia, and ultrastructural changes leading to laboratory animal medicine cell death have also been described. detrimental effects on ciliated epithelium can lead to disrupted mucociliary transport, which exacerbates pulmonary disease. experimental infection of mrm is dose dependent. doses of colony-forming units (cfus) or less cause mild, transient disease involving the upper respiratory tract and middle ears, whereas higher doses often lead to acute, lethal pneumonia. additionally, m. pulmonis strains can differ in virulence. survivors of severe infection may develop chronic bronchopneumonia with bronchiectasis and spread infection to other mice. intravenous inoculation of m. pulmonis can cause arthritis in mice, but arthritis is not a significant feature of natural infection. host genotype also is a major factor in the outcome of infection, with resistance being expressed phenotypically through the bactericidal efficiency of alveolar macrophages. strains derived from a c bl background appear to be resistant to pathogenic infection, whereas balb/c, c h, dba/ , swr, akr, cba, sjl, and other strains have varying degrees of increased susceptibility (cartner et al., ; lai et al., ) . the initial lesion of mrm is suppurative rhinitis, which may involve the trachea and major airways. early inflammatory lesions, if not quickly resolved, progress to prominent squamous metaplasia. transient hyperplasia of submucosal glands may occur, and lymphoid infiltration of the submucosa can persist for weeks. syncytia can sometimes be found in nasal passages, in association with purulent exudate (fig. . ) . affected mice also develop suppurative otitis media and chronic laryngotracheitis with mucosal hyperplasia and lymphoid cell infiltrates. pulmonary lesions are typified by bronchopneumonia, which spreads from the hilus. lymphoid cells and plasma cells accumulate around bronchi which often contain neutrophils in their lumen. chronic lung disease features suppurative bronchitis, bronchiolitis, and alveolitis (fig. . ) . chronicity also increases the prevalence of bronchiectasis and abcessation. diagnosis accurate diagnosis should exploit the complementary use of clinical, serological, microbiological, molecular, and morphological methods. clinical signs are variable but can be characteristic when they occur. serology is sensitive but although antibodies do not clear the infection, seroconversion may be weak or take months and may not accurately differentiate between m. pulmonis infection and m. arthriditis infection (cassell et al., ) . therefore samples for culture and pcr of the upper respiratory tract should be obtained to confirm diagnosis. buffered saline or mycoplasma broth should be used to lavage the trachea, larynx, pharynx, and nasal passages. specimens for culture from the genital tract are warranted if this site is suspected. mycoplasma spp. may be difficult to grow, so it is prudent to confirm that the relevant expertise and quality control exist in the diagnostic laboratory. speciation can be accomplished by immunofluorescence or immunoperoxidase staining or by growth inhibition. immunohistochemistry should be considered to supplement basic histopathologic examination. immunofluorescence and immunoperoxidase techniques are available to identify mycoplasma antigens in tissue sections or in cytological preparations of tracheobronchial or genital tract lavages (brunnert et al., ) . pcr assays for m. pulmonis at veterinary diagnostic laboratories and pcr kits to screen cell culures for mycoplasma are readily available. differential diagnosis mrm must be differentiated from bronchopneumonia associated with ciliaassociated respiratory (car) bacillus. silver stains may reveal car bacilli adherent to the respiratory epithelium. sv also can cause bronchopneumonia in mice but can be detected by serology and immunohistochemistry. other causes of respiratory infection include pvm, corynebacteriosis and, in immunodeficient mice, pneumocystis murina infection. combined infections with known pathogens or secondary opportunists also must be considered. prevention and control mice mount an effective immune response to m. pulmonis, as measured by their recovery from mild infection and their resistance to infection after active or passive immunization (cartner et al., ) . antibodies of various classes are produced locally and systemically, but clearance of the infection has been attributed to innate immune responses (love et al., ; sun et al., ) . there is some evidence that antibody may facilitate phagocytosis of m. pulmonis. t-cell responses, however, appear to exacerbate m. pulmonis in mice, because immunity cannot be transferred with immune cells. in addition, athymic and neonatally thymectomized mice are not more susceptible than immunocompetent mice to m. pulmonis pneumonia. nude and scid mice develop less severe respiratory disease than immunocompetent mice but infection becomes systemic and they may develop suppurative disease in multiple organs and joints (arthritis). host immunity aside, effective control and prevention of mrm depend primarily on maintenance of mycoplasma-free colonies under barrier conditions supported by careful surveillance for infection by serology, microbiology, pcr, and histopathology. cesarean or embryo rederivation may eliminate infection, although vertical transmission may occur in immunocompromised mice. treatment with tetracycline suppresses clinical disease but does not eliminate infection. earlier interest in developing dna-based vaccines against m. pulmonis has not achieved clinical application (lai et al., ) . research complications m. pulmonis can interfere with research by causing clinical disease or death. experiments involving the respiratory tract, such as inhalation toxicology, can be compromised by chronic progressive infection. additionally, affected mice are at greater risk during general anesthesia. m. pulmonis may alter immunological responsiveness. for example, it is mitogenic for t and b lymphocytes and can increase nk cell activity. perhaps one of the most important complications of mycoplasma infection is contamination of cell lines and transplantable tumors. other murine mycoplasmas cell lines are often contaminated with mycoplasma species such as m. arginini, m. hyorhinis, m. orale, or m. fermentans that can distort the results of in vitro assays (garner et al., ) . initial evidence of a contamination is often by pcr evidence of mycoplasma at the genus level when cell lines are pcr screened for opportunistic murine pathogens prior to use in mice. other than m. pulmonis, these mycoplasmas are not normally considered mouse pathogens in immunocompetent mice. in contrast, injection of mycoplasma contaminated cells into immunodeficient mice (e.g., xenografts) may result in clinical disease or confounding effects on immune responses (peterson, ) . mycoplasma contamination of murine embryonic stem cells has adversely affected germline transmission and postnatal health of chimeric progeny (markoullis et al., ) . mycoplasma arthritidis is antigenically related to m. pulmonis. therefore, serological evidence of mycoplasma infection must be supplemented by other diagnostic tests, as outlined above, to differentiate between these agents. differentiation is important because m. arthritidis, though arthritogenic in mice after intravenous inoculation, is nonpathogenic during natural infection. mycoplasma collis has been isolated from the genital tract of mice but does not appear to cause natural disease. mycoplasma neurolyticum is the etiological agent of rolling disease, a rare syndrome which occurs within hours after intravenous inoculation of m. neurolyticum exotoxin. characteristic clinical signs include spasmodic hyperextension of the head and the raising of one foreleg followed by intermittent rolling on the long axis of the body. the rolling becomes more constant, but mice occasionally leap or move rapidly. after - h of rolling, animals become comatose and usually die within h. all published reports of rolling disease are associated with experimental inoculation of m. neurolyticum or exotoxin. large numbers of organisms are needed to produce disease, and there is no indication that, under natural conditions, organisms replicate in the brain to concentrations required for the induction of these signs. because animals are frequently inoculated with biological materials by parenteral routes, contamination with m. neurolyticum may induce rolling disease inadvertently. diagnosis can be made from the appearance of typical clinical signs, astrocytic swelling, and isolation of the causative organism. clinical signs must be differentiated from rolling associated with pseudomonas-and p. pneumotropica-caused otitis. m. pulmonis has been recovered from the brain of mice but does not seem to cause overt neurological disease. hemotropic mycoplasmas ribosomal rna sequencing has reclassified hemobartonella muris and eperythrozoon coccoides as mycoplasma hemomuris and mycoplasma coccoides, respectively (neimark et al., ; percy and barthold, ) . distinct from the mycoplasmas just discussed, these agents are trophic for red blood cells and cause anemia and hemolytic disease. these laboratory animal medicine infections could be encountered in wild mice but are rarely found in research mice. diagnosis is by morphologic assessment of blood smears and pcr. clinical signs mice infected with m. coccoides may remain clinically normal or develop febrile, hemolytic anemia and splenomegaly, which can be fatal. hepatocellular degeneration and multifocal necrosis have been recorded in acute infections. hemotropic mycoplasma infections are long-lived and are expressed clinically in one of two ways: acute febrile anemia and latent or subclinical infection that can be reactivated by splenectomy. the carrier state may be lifelong. epizootiology the primary natural vector of m. coccoides, historically, is the mouse louse, polyplax serrata. infection was associated with primitive housing and husbandry conditions that no longer occur in modern vivaria. although the risks for infection have been reduced substantially by modern animal care procedures, m. coccoides can be transmitted to mice from contaminated biological products such as transplantable tumors or blood plasma. diagnosis splenectomy or inoculation of test material into splenectomized mice is the most sensitive means of detecting m. coccoides infection. these procedures provoke mycoplasmemia, usually within - days. because mycoplasmemia may be transient, blood smears stained by the romanowsky or indirect immunofluorescence procedures of the blood should be prepared every h, beginning at h after splenectomy of index animals or inoculation of test specimens into splenectomized animals to ensure that mycoplasmemia is not missed. prevention and control treatment of m. coccoides infection is not practical. control is based on culling or rederivation of infected stock. if replacement animals are readily available, euthanasia is a more prudent course. suspect biological materials destined for animal inoculation should be screened for mycoplasma contamination by inoculation of splenectomized mice. research complications subclinical infection can be reactivated by irradiation, immunosuppressive therapy, or intercurrent disease. conversely, m. coccoides may potentiate coincident viral infections in mice. this effect has been clearly demonstrated for mouse coronavirus and has been suspected for lymphocytic choriomeningitis virus and ldv. active infection also may suppress interferon production. etiology car bacillus is a slender, gram-negative, non-spore-forming bacillus, which, in rats, produces clinical disease and lesions that closely resemble those of mrm (see chapter ). clinical signs chronic respiratory disease has been produced in mice by experimental inoculation, but natural clinical disease is rare (griffith et al., ; pritchett-corning et al., ) . furthermore, putative natural cases were reported in mice that were seropositive for sv and pneumonia virus of mice. therefore, car bacillus may exacerbate respiratory disease as an opportunist rather than as a primary pathogen. on balance, it is assumed that mice contract natural infection, but attributing severe chronic respiratory disease in mice solely to car bacillus should be supported by screening for other respiratory pathogens. epizootiology car bacillus is transmitted by direct contact; dirty bedding transfer to sentinel mice may not reflect colony infection status. pathology lung lesions are typically mild in mice and are similar to respiratory mycoplasmosis. uncomplicated car bacillus infection results in peribronchiole cuffing with lymphocytes and plasma cells. severe bronchiolitis and pneumonia are possible (fig. . ) . fatal bronchopneumonia was reported in ob/ob mice (griffith et al., ) . diagnosis an elisa for serological screening is routinely used; pcr and histology are used for definitive diagnosis. in active infection, histologic assessment using warthin-starry or similar stains will reveal argyrophilic bacilli adherent to the apical membranes of bronchial respiratory epithelium along with the presence of peribronchial lymphocytes (fig. . ) . alternatively, immunohistochemistry assays have also been used successfully to detect infection. recovery of car bacillus requires cell culture or culture in embryonated eggs. differential diagnosis respiratory mycoplasmosis, bordetella (avium, hinzii). prevention and control given car bacillus does not form spores, disinfection of the environment should be effective. treatment using sulfamerazine ( mg/l) in drinking water may eradicate infection (matsushita and suzuki, ) but culling or embryo rederivation is recommended. research complications infection is most often subclinical, but like other infectious agents for mice, may confound studies particularly when mice are immunocompromised (griffith et al., ) . etiology the causative agent of transmissible murine colonic hyperplasia, c. rodentium (formerly citrobacter freundii strain ), is a nonmotile, gramnegative rod that ferments lactose but does not utilize citrate or does so marginally (barthold, ; schauer et al., ) . clinical signs c. rodentium infection can be a selflimiting colitis with sterilizing immunity or lead to severe colitis with life-threatening dehydration. clinically apparent infection is characterized by retarded growth, ruffled fur, soft feces or diarrhea, rectal prolapse, and moderate mortality in older suckling or recently weaned mice (barthold et al., ) . epizootiology c. rodentium is not detected in the gastrointestinal flora of normal mice, and therefore, there is not a carrier state. it is thought to be introduced by contaminated mice, food, or bedding, from which it spreads by contact or additional fecal contamination. c. rodentium shares several pathogenic mechanisms, such as attaching and effacing lesions mediated by the intimin receptor, with select escherichia coli (reviewed in collins et al. ( ) ). c. rodentium is used experimentally to model colitis caused by enteropathogenic (epec) and enterohemorrhagic e. coli (ehec) in humans (mallick et al., ; collins et al., ) . host genotype can influence the course and severity of disease (barthold et al., ) . for example, dba, nih swiss, and c bl mice are relatively resistant to mortality, whereas c h/hej mice are relatively susceptible both as sucklings and as adults. interestingly, c bl mice obtained from different commercial sources have varying susceptibility to c. rodentium (ostensibly due to the presence or absence of segmented filamentous bacteria). diet also can modulate infection, but specific dietary factors responsible for this effect have not been identified. pathology c. rodentium attaches to the mucosa of the descending colon and displaces the normal flora. attachment is accompanied by effacement of the microvillus border and formation of pedestal-like structures (attaching and effacing lesions) (schauer and falkow, ; newman et al., ) . colonization results in prominent mucosal hyperplasia, by unknown mechanisms. the characteristic gross finding is severe thickening of the descending colon, which may extend to the transverse colon and lasts for - weeks in surviving animals ( fig. . ) . affected colon segments are rigid and either are empty or contain semiformed feces. histologically, accelerated mitotic activity results in a markedly hyperplastic mucosa, which may be associated with secondary inflammation and ulceration (fig. . ). lesions subside after several weeks. intestinal repair is rapid and complete in adults but slower in sucklings. diagnosis diagnosis depends on clinical signs, characteristic gross and histological lesions, and isolation of c. rodentium from the gastrointestinal tract or feces. the organism can be cultured on macconkey's agar during early phases of infection, whereas the intestine may be free of c. rodentium during later stages of the disease. c. rodentium also can be detected by molecular hybridization (schauer et al., ) . barthold et al. ( ) . diagnosis transmissible murine colonic hyperplasia must be differentiated from other diarrheal diseases of mice, including infections caused by coronavirus, rotavirus, adenovirus, reovirus, salmonella, c. piliforme, and helicobacter spp. prevention and control some success in curtailing epizootics has been achieved by adding antimicrobials to the drinking water (barthold, ; silverman et al., ) . because c. rodentium may contaminate food, bedding, or water, proper disinfection of such materials is prudent before they are used for susceptible animals. additionally, the employment of microbarrier caging can reduce transmission. surveillance for c. rodentium should be incorporated into quality-assurance programs, and the organism screened for during quarantine of incoming mice from atypical sources. research complications the potential effects on research of colonic hyperplasia as a clinically severe disease are obvious. colonic hyperplasia has been shown to increase the sensitivity of colonic mucosa to chemical carcinogens and to decrease the latent period between administration of carcinogen and the appearance of focal atypical cell growth (barthold and beck, ) . c. rodentium infection has been incriminated in immune dysfunction, poor reproductive performance, and failure to thrive in t-cell receptor transgenic mice (maggio-price et al., ) . immunocompromised mice infected with c. rodentium will die from sepsis. etiology pseudomonas aeruginosa is a motile, gramnegative rod. clinical signs p. aeruginosa infections are almost always silent, but immunologically compromised animals are prone to septicemia (brownstein, ) . p. aeruginosa can, e.g., cause severe or lethal infections in athymic and scid mice. sick mice may have equilibrium disturbances, conjunctivitis, serosanguinous nasal discharge, edema of the head, weight loss, and skin infections. immunosuppressed mice may also develop gastrointestinal ulcers. generalized infection is associated with severe leukopenia, especially neutropenia. neurologic signs are rare, but there are reports of central nervous system infection. chronic proliferative inflammation in the cochlea and vestibular apparatus with dissolution of surrounding bone may cause torticollis. epizootiology p. aeruginosa is not considered a component of the normal flora. however, it is an opportunist that inhabits moist, warm environments such as water and skin. once established in a host, it may be found chronically in the nasopharynx, oropharynx, and gastrointestinal tract, all sites from which additional environmental contamination or direct transmission to susceptible mice can occur. pathology pathogenic infection is most common in immunodeficient mice. organisms enter at the squamocolumnar junction of the upper respiratory tract and, in some cases, the periodontal gingiva. bacteremia is followed by necrosis or abscess formation in the liver, spleen, or other tissues. if otitis media occurs, the tympanic bullae may contain green suppurative exudate. the bowel may be distended with fluid, and gastrointestinal ulceration has been reported. diagnosis infection is diagnosed on the basis of history (e.g., immune dysfunction or recent immunosuppression), clinical signs, lesions, and isolation of p. aeruginosa from affected mice. carrier mice can be detected either by nasal culture or by placing bottles of sterile, nonacidified, nonchlorinated water on cages for - h and then culturing the sipper tubes. p. aeruginosa can also be cultured from feces. differential diagnosis pseudomoniasis must be differentiated from other bacterial septicemias that may occur in immunodeficient mice. these include, but are not limited to, corynebacteriosis, salmonellosis, colibacillosis, staphylococcosis, and tyzzer's disease. prevention and control infection can be prevented by acidification or hyperchlorination of the drinking water (homberger et al., ) . these procedures will not, however, eliminate established infections. entry of infected animals can be prevented by surveillance of commercially procured colonies. maintenance of pseudomonas-free animals usually requires barrierquality housing and husbandry. p. aeruginosa has a long history in the literature of antibiotic resistance and resistance to quaternary amine disinfectants. research complications p. aeruginosa infection is not a substantial threat to immunocompetent mice but can complicate experimental studies by causing fatal septicemia in immunodeficient mice. viral infections that alter host defense mechanisms, such as mcmv may enhance susceptibility to pseudomoniasis. (lindsey et al., a; percy and barthold, ) etiology pasteurella pneumotropica is a short, gramnegative rod. clinical signs many early observations concerning the pathogenicity of p. pneumotropica are questionable because they were made on colonies of mice with varying levels of bacterial and viral contamination. infection is usually subclinical. therefore, p. pneumotropica is most properly viewed as an opportunistic pathogen. studies of experimental p. pneumotropica suggest that it may complicate pneumonias due to mycoplasma pulmonis or sv. it has also been associated with suppurative or exudative lesions of the eye, conjunctiva, skin, mammary glands, and other tissues, especially in immunodeficient mice or in mice with a predisposing primary infection. epizootiology p. pneumotropica is a ubiquitous inhabitant of the skin, upper respiratory tract, and gastrointestinal tract of mice. litters from infected dams can become infected during the first week after birth. pathology infections can cause suppurative inflammation, which may include abcessation. dermatitis, conjunctivitis, dacryoadenitis, panophthalmitis, mastitis, and infections of the bulbourethral glands have been attributed to p. pneumotropica. preputial and orbital abscesses also occur, especially in athymic mice (fig. . ). its role in metritis is unclear, but it has been cultured from the uterus, and there is some evidence that it may cause abortion or infertility. cutaneous lesions can occur without systemic disease. they include suppurative lesions of the skin and subcutaneous tissues of the shoulders and trunk. diagnosis diagnosis requires isolation of the organism on standard bacteriological media. although infection can be detected serologically by elisa (wullenweber-schmidt et al., ; boot et al., a, b) , subclinical carriers often do not seroconvert. pcr assays also are available (dole et al., ) and have shown that p. pneumotropica did not transmit from infected mice to contact or dirty bedding sentinels (ouellet et al., ; dole et al., ) . differential diagnosis suppurative lesions in mice may be caused by other bacteria, including staphylococcus, streptococcus, corynebacterium, klebsiella, and mycoplasma. treatment antibiotic sensitivity testing in vitro indicated p. pneumotropica was significantly more sensitive than p. aeruginosa to enrofloxacin (sasaki et al., ) . enrofloxacin in the drinking water at mg/kg daily for days eliminated clinical signs and infection in a closed breeding colonic of transgenic mice and after days of treatment there were no detectable carriers when the colony was screened weeks later (matsumiya and lavoie, ) . prevention and control because p. pneumotropica is an opportunistic organism, it should be excluded from colonies containing immunodeficient mice and from breeding colonies. achieving this goal will normally require barrier housing supported by sound microbiological monitoring. rederivation should be considered to eliminate infection in circumstances where infection presents a potential threat to animal health or experimentation. research complications clinically severe infection in immunodeficient mice is the major complication. although clinically silent, experimental evidence has shown that p. pneumotropica infection in immunocompetent mice (c bl/ ) stimulated transcription of multiple proinflammatory cytokines for at least days with residual elevation detectable days later (patten et al., ) . pioneering studies conducted in the s first linked a novel microaerobic bacterium, helicobacter hepaticus, with chronic active hepatitis and hepatic tumors in a/jcr mice (fox et al., , ward et al., ) . the organism could be visualized by electron microscopy in the bile canaliculi of the liver in susceptible mouse strains (fig. . ). subsequently, it was associated with inflammatory bowel disease in several murine models (table . ) which were further developed to examine the role of immune cell subsets, such as t regulatory cells, in the pathogenesis of inflammatory bowel disease (ibd) and colon cancer (fig. . ). helicobacteriosis is laboratory animal medicine now appreciated to be a common infection of laboratory mice. it is caused by a growing list of helicobacter spp. that vary in clinical, pathologic, and epidemiologic significance (whary and fox, ; fox et al., ) . because recognition and investigation of helicobacteriosis continues to evolve, many important questions about the impact of this infection on mice remain unresolved. h. hepaticus infection is emphasized here, because it is among the most prevalent causes of helicobacteriosis and has been studied more extensively than other murine enterohepatic helicobacter spp. (ehs) (fox et al., , ward et al., ; suerbaum et al., ) . however, current information about other murine helicobacters is summarized in the concluding section. etiology helicobacter spp. are gram-negative, microaerophilic, curved to spiral-shaped organisms that have been isolated from the gastrointestinal mucosa of many mammals, including humans and mice whary and fox, ) . to date, the genus includes formally named helicobacter spp. assigned on the basis of s rrna analysis, complemented by biochemical, molecular, and morphological characteristics. the organisms can be grown on freshly prepared antibiotic impregnated blood agar or in broth supplemented with fetal bovine serum in a microaerobic atmosphere ( % co , % n , % h ). there are currently formally named helicobacter species have been isolated from laboratory mice, as well as several other novel helicobacter spp. awaiting formal naming. species isolated from mice include h. hepaticus, h. bilis (which also infects rats), h. muridarum, h. rappini, and h. rodentium, h. ganmani, h. mastomyrinus, h. magdeburgensis, and h. typhlonius , each of which cahill et al. ( ) tcrα, β mutants defective t-receptors typhlocolitis chin et al. ( ) scid icr-defined flora b t-and b-cell deficient typhlocolitis shomer et al. ( ), shomer et al. ( c bl/il- −/−c lacks il- typhlocolitis burich et al. ( ) , kullberg et al. ( ) , kullberg et al. ( ) , kullberg et al. ( ) c blrag mice infected with h. bilis also developed ibd (shomer et al., ) . c ibd also developed in c bl/il- −/− mice experimentally infected with a novel urease-negative helicobacter spp. now named h. typhlonius (franklin et al., ) ; also ibd produced with h. trogontum (whary et al., ) and h. cinaedi (shen et al., ) . d h. bilis produces ibd (maggio-price et al., and colon cancer (maggio-price et al., ) . have been formally named (except for h. rappini) (fox and lee, ; franklin et al., ; whary and fox, ) . most recently, helicobacter pullorum, a human pathogen, has been isolated from commercial, barriermaintained mice (boutin et al., ) . these ehs are most commonly urease-, catalase-, and oxidase-positive. however, h. rodentium, h. typhlonicus, and another novel helicobacter sp. are urease-negative. clinical signs helicobacteriosis in adult immunocompetent mice is usually asymptomatic. liver enzymes are elevated in h. hepaticus-infected a/jcr mice (fox et al., a) . infection of immune-dysregulated mice with h. hepaticus can cause inflammatory bowel disease, which may present as rectal prolapse and/or diarrhea (miller et al., ) . epizootiology recent surveys and anecdotal evidence suggest that helicobacteriosis is widespread among conventional and barrier-maintained mouse colonies (shames et al., ; fox et al., b; taylor et al., ; lofgren et al., ) . furthermore, h. hepaticus (and probably other helicobacters) can persist in the gastrointestinal tract, particularly the cecum and colon, and is readily detected in feces. these results indicate that transmission occurs primarily by the fecal-oral route and imply that carrier mice can spread infection chronically in enzootically infected colonies. pathology helicobacter spp. colonize the crypts of the lower bowel, where, depending on host genotype, the organisms can be pathogenic or nonpathogenic. h. hepaticus and h. bilis, e.g., can cause inflammation in the gastrointestinal tract, which is expressed as ibd and colon cancer in immunodeficient mice or typhlitis in a/jcr mice infected with h. hepaticus (ward et al., ; knutson et al., ; shomer et al., ; erdman et al., b; nguyen et al., ) . thickening of the cecum and large bowel develops because of proliferative typhlitis, colitis, proctitis, and lower bowel carcinoma. these lesions can occur without coincident hepatitis. indeed, helicobacter spp. induced ibd and colon carcinoma are increasingly popular models to study pathogenesis of the disease in humans (table . ). helicobacter spp. also can cause liver disease. bacterial translocation is thought to occur and results in colonization of the liver and progressive hepatitis. it is characterized by angiocentric nonsuppurative hepatitis and hepatic necrosis (fig. . ) . inflammation originates in portal triads and spreads to adjacent hepatic parenchyma. hepatic necrosis also may occur adjacent to intralobular venules, which can contain microthrombi. additionally, phlebitis may affect central veins. this lesion has been linked to the presence of organisms in bile canaliculi by silver stains and electron microscopy. age-related hepatocytic proliferation can develop in infected livers, a response that is more pronounced in male mice than in female mice (fox et al., a) . this lesion may laboratory animal medicine increase susceptibility to hepatomas and hepatocellular carcinomas among aged male a/jcr and b c f mice from infected colonies. an increased incidence of hepatic hemangiosarcoma also has been noted in h. hepaticusinfected male b c f mice. in this context, a/jcr, c h/ hencr, and sjl/ncr mice are susceptible to hepatitis, whereas c bl/ mice are resistant (ward et al., ) . the finding of severe liver disease and tumor induction in b c f mice infected with h. hepaticus infers that genetic susceptibility to h. hepaticus-induced neoplasia has a dominant pattern of inheritance. studies with h. hepaticus in recombinant inbred mice also indicate that disease susceptibility has multigenetic properties (hailey et al., ; fox and lee, ; ihrig et al., ; franklin, ; hillhouse et al., ) . diagnosis rapid generic diagnosis can be accomplished by pcr detection of the highly conserved s rrna region of the helicobacter genome in feces or tissues, using suitable oligonucleotide primers (fox et al., a; shames et al., ; beckwith et al., ) . however, genus-specific pcr does not differentiate among different helicobacter spp. molecular speciation can be accomplished by s rrna sequencing, restriction fragment length polymorphism analysis of the pcr product or use of species-specific pcr assays. this procedure requires suitable skill and experience to avoid technological pitfalls and should be performed by qualified laboratories. an igg elisa using the outer membrane protein as the antigen has been proposed for serological diagnosis, but shared antigens among ehs create lack of specificity for the assay. as noted above, helicobacters can be isolated on antibiotic-impregnated blood agar under microaerobic conditions and can then be speciated biochemically, and by helicobacter species-specific pcr. isolation of h. hepaticus and from other helicobacter spp. with spiral to curved morphology from feces should be preceded by passing slurried samples through a . -μm filter before plating. if infection with larger fusiform helicobacters (h. bilis, h. rappini) is suspected, filtration at . μm is preferred. helicobacters grow slowly and require prolonged incubation of cultures (up to weeks) before they can be deemed negative. signs (rectal prolapse) and lesions (hepatitis, typhlocolitis), depending on host genotype, can be suggestive of infection. histopathological examination should include silver stains, especially of liver, to attempt to visualize spiral or curved organisms (whary and fox, ) . differential diagnosis clinically apparent helicobacteriosis must be differentiated from other gastrointestinal or hepatic infections of mice. coronavirus infection, clostridium piliforme, and salmonella spp. can cause enterocolitis and/or hepatitis. c. rodentium also causes colonic hyperplasia, which can present as rectal prolapse. infections caused by other helicobacters of mice h. bilis has been isolated from the livers and intestines of aged mice and experimentally induces ibd in scid mice as does h. hepaticus. h. bilis also experimentally produces lower bowel cancer in immunocompromised mice (nguyen et al., ) . helicobacter muridarum colonizes the ileum, cecum, and colon. it appears to be nonpathogenic, although it can colonize the stomach of mice and induce gastritis under certain circumstances. h. 'rappini' has been isolated from the feces of mice without clinical signs. h. rodentium also colonizes the intestine and may be a component of normal flora. a dual infection of h. bilis and h. rodentium was noted in a natural outbreak of ibd in immunocompromised mice (shomer et al., ) . a novel urease negative helicobacter, which has been named h. typhlonius, causes ibd in il- −/− and scid mice (franklin et al., (franklin et al., , fox et al., ) . decreased reproductive efficiency has been reported in il knockout mice infected with h. rodentium and/or h. typhlonius (sharp et al., ) . prevention and control eradication of infection from small numbers of mice, such as quarantine groups, can be achieved by standard rederivation or intensive antibiotic therapy. the best results have been obtained by triple therapy with amoxicillin, metronidazole, and bismuth given for weeks (del carmen martino-cardona et al., ) . this strategy requires repeated daily gavage rather than administration in drinking water, but it has successfully eliminated h. hepaticus from naturally infected mice. antibiotic impregnated wafers have been used to eradicate helicobacter spp. in mouse colonies (kerton and warden, ) . wide-scale, eradication of enzootic helicobacteriosis can be expensive and time-consuming, without guarantee of success. careful husbandry procedures can limit infection within a colony (whary et al., ) . therefore, strategies have to be weighed carefully against risks of enzootic infection for the health and use of mice. in contrast, infection should be avoided in immunodeficient mice, including genetically engineered mice with targeted or serendipitous immune dysfunction. lastly, the outcome of opportunistic helicobacteriosis has not been thoroughly examined. this condition could occur during simultaneous infection with two or more helicobacter species or during combined infection with an intestinal virus (e.g., coronavirus) and helicobacter spp. if highly valuable animals are exposed, antibiotic therapy or rederivation may be warranted. research complications chronic inflammation of the liver and or gastrointestinal tract may be injurious to health. additionally, it may impede the development and assessment of noninfectious disease models, such as ibd models in mice with targeted deletions in t-lymphocyte receptors (fox et al., ) . h. hepaticus infections provoke a strong th proinflammatory response, which may perturb other immunological responses. h. hepaticus infection also has been incriminated as a cofactor or promoter in the development of hepatic neoplasia in a/ jcr, b c f , ab f , b af , and carko mice (hailey et al., ; fox et al., a; garcia et al., garcia et al., , h. salmonellosis (ganaway, ; lindsey et al., etiology the genus salmonella contains two species, s. bongori which infects mainly poikilotherms and rarely, humans, and s. enterica which includes approximately serovars and are a major cause of food-borne illness in humans (fookes et al., ) . the salmonella of historical importance in mice that are now rare include s. enterica subsp. enterica serovar typhimurium (aka s. typhimurium) and serovar enteritidis (s. enteritidis). s. enteritidis is a motile, gram-negative rod that rarely ferments lactose. the genomes of many strains have been sequenced. virulence factors carried on pathogenicity islands and plasmids include antimicrobial resistance genes, type iii secretion systems, vi antigen, lipopolysaccharide and other surface polysaccharides, flagella, and factors essential for a intracellular life cycle in macrophages (de jong et al., ) . pathogenassociated molecular patterns (pamps) unique to salmonella interact with tlrs and nod-like receptors (nlrs) which recruit neutrophils and macrophages leading to inflammasome formation and release of pro-inflammatory il- , il- β, tnf-α, and ifn-γ. clinical signs acute infection is especially severe in young mice (casebolt and schoeb, ) . it is characterized by anorexia, weight loss, lethargy, dull coat, humped posture, and occasionally conjunctivitis. gastroenteritis is a common sign, but feces may remain formed. subacute infection can produce distended abdomens from hepatomegaly and splenomegaly. chronic disease is expressed as anorexia and weight loss. enzootic salmonellosis in a breeding colony can produce episodic disease with alternating periods of quiescence and high mortality. the latter can be associated with diarrhea, anorexia, weight loss, roughened hair coat, and reduced production. epizootiology s. typhimurium is commonly used experimentally and cross-contamination in a mouse facility is a risk. modern production and husbandry methods have reduced the importance of salmonellosis as a natural infection of mice. however, the organisms are widespread in nature. therefore, cross-infection from other species or from feral mice remains a potential hazard. salmonellas are primarily intestinal microorganisms that can contaminate food and water supplies. infection occurs primarily by ingestion. salmonella have a broad host range and vermin, birds, feral rodents, and human carriers are potential sources of infection. other common laboratory species such as nonhuman primates, dogs, and cats also can serve as carriers. conversely, murine salmonellosis presents a zoonotic hazard to humans. the induction and course of infection are influenced by the virulence and dose of the organism, route of infection, host sex and genetic factors, nutrition, and intercurrent disease. suckling and weanling mice are more susceptible to disease than mature mice. immune deficiency, exposure to heavy metals, and environmental factors such as abnormal ambient temperatures can increase the severity of disease. nutritional iron deficiency has an attenuating effect on salmonella infection in mice, whereas iron overload appears to promote bacterial growth and enhance virulence. resistance to natural infection is increased by the presence of normal gastrointestinal microflora. resistance to infection also can be an inherited trait among inbred strains. among the most important considerations is that mice that recover from acute infection can become subclinical carriers and a chronic source of contamination from fecal shedding. pathology the virulence of s. enteritidis depends on its ability to penetrate intestinal walls, enter lymphatic tissue, multiply, and disseminate. organisms reach peyer's patches within h after inoculation and spread quickly to the mesenteric lymph nodes. bacteremia results in spread to other lymph nodes, spleen, and liver within several days. in chronic infections, organisms persist in the spleen and lymph nodes as well as in the liver and gallbladder and from the latter are discharged into the intestinal contents. bacteria reaching the intestine can reinvade the mucosa and can be shed intermittently in the feces for months. s. enteritidis infection also has been associated with chronic arthritis. acute deaths may occur without gross lesions, but visceral hyperemia, pale livers, and catarrhal enteritis are more common. if mice survive for up to several laboratory animal medicine weeks, the intestine may be distended and reddened, whereas the liver and spleen are enlarged and contain yellow-gray foci of necrosis. affected lymph nodes are also enlarged, red, and focally necrotic. focal inflammation can develop in many organs, including the myocardium (percy and barthold, ) . histologic lesions reflect the course of disease and the number of bacteria in affected tissues. during acute infection, necrotic foci are found in the intestine, mesenteric lymph nodes, liver, and spleen. neutrophilic leukocytes and histiocytes accumulate in lymphoid tissues. thrombosis from septic venous embolism may occur, especially in the liver. granulomatous lesions are particularly characteristic of chronic salmonellosis, especially in the liver. diagnosis diagnosis is based on isolation of salmonellas together with documentation of compatible clinical signs and lesions. in mice with systemic disease, bacteria may persist in the liver and spleen for weeks. during acute stages, bacteria can also be isolated from the blood. subclinically infected animals can be detected by fecal culture using selective enrichment media (selenite f broth plus cystine followed by streaking on brilliant green agar). culture of the mesenteric lymph nodes may be more reliable, because fecal shedding can be intermittent. isolates can be speciated with commercial serotyping reagents. alternatively, isolates can be sent to a reference laboratory for confirmation. antibodies to salmonellas can be detected in the serum of infected mice by an agglutination test. however, this method is not entirely reliable, because serological crossreactivity is common even among bacteria of different genera. pcr-based assays are also available. differential diagnosis salmonellosis must be differentiated from other bacterial diseases, including tyzzer's disease, helicobacter spp., pseudomoniasis, corynebacteriosis, c. rodentium, and pasteurellosis. viral infections that cause enteritis or hepatitis must also be considered, especially infections caused by coronavirus, ectromelia virus, and reoviruses. among noninfectious conditions, mesenteric lymphadenopathy is an agingassociated lesion in mice and is not indicative of chronic salmonellosis. prevention and control salmonellosis can be prevented by proper husbandry and sanitation. contact between mice and potential carriers, such as nonhuman primates, dogs, and cats, should be prevented. diets should be cultured periodically to check for inadvertent contamination. contaminated colonies should be replaced to eliminate infection and its zoonotic potential. research complications apart from the clinical manifestations, the zoonotic potential for salmonellosis is a major concern. this includes transmission among laboratory species, but especially between mice and the personnel working with them. i. streptobacillosis (lindsey et al., e; percy and barthold, ) etiology streptobacillus moniliformis is a nonmotile, gram-negative, pleomorphic rod that can exist as a nonpathogenic l-phase variant in vivo. however, it can revert to the virulent bacillus form. clinical signs streptobacillosis generally has an acute phase with high mortality, followed by a subacute phase and finally a chronic phase that may persist for months. signs of acute disease include a dull, damp hair coat and keratoconjunctivitis. variable signs include anemia, diarrhea, hemoglobinuria, cyanosis, and emaciation. cutaneous ulceration, arthritis, and gangrenous amputation may occur during chronic infection. the arthritis can leave joints deformed and ankylosed. hindlimb paralysis with urinary bladder distention, incontinence, kyphosis, and priapism may occur if vertebral lesions impinge on motor nerves. breeding mice may have stillbirths or abortions. epizootiology streptobacillosis has historical importance as a disease of rats and mice, but modern husbandry, production, and health surveillance strategies have reduced its impact dramatically (wullenweber, ) . subclinical, persistently infected rats are the most likely source of dissemination to mice, but mouse-tomouse transmission then ensues. transmission may occur from aerogenic exposure, bite wounds, or contaminated equipment, feed, or bedding. s. moniliformis is also pathogenic for humans, causing rat bite fever (haverhill fever). pathology during acute disease, necrotic lesions develop in thoracic and abdominal viscera, especially in the liver, spleen, and lymph nodes. histological lesions include necrosis, septic thrombosis of small vessels, acute inflammation, fibrin deposition, and abscesses. chronically infected mice may develop purulent polyarthritis because of the organism's affinity for joints. diagnosis diagnosis depends on clinical and pathological evidence of septicemia and isolation of the organism on blood agar. the organism has been recovered from joint fluid as long as months after infection. isolation from chronic lesions requires serumenriched medium. s. moniliformis as a cause of septic joints in humans has been diagnosed using pcr and electrospray-ionization followed by mass spectrometry (mackey et al., ) . differential diagnosis clinical signs must be differentiated from septicemic conditions, including mousepox, tyzzer's disease, corynebacteriosis, salmonellosis, mycoplasmosis, pseudomoniasis, and traumatic lesions. prevention and control control is based on exclusion of wild rodents or carrier animals such as latently infected laboratory rats. bacterins and antibiotic therapy are not adequately effective. the potential laboratory animal medicine for cross-infection is a reason not to house rats and mice in the same room. research complications infection can be disabling or lethal in mice and has zoonotic potential for humans. j. corynebacteriosis (lindsey et al., ; weisbroth, ; percy and barthold, ) etiology corynebacteria are short gram-positive rods. corynebacterium kutscheri is the cause of pseudotuberculosis in mice and rats. corynebacterium bovis has been associated with hyperkeratosis, especially in immunodeficient mice (clifford et al., ; scanziani et al., ; dole et al., ) . clinical signs c. kutscheri infection is often subclinical in otherwise healthy mice. active disease is precipitated by immunosuppression or environmental stresses and is expressed as an acute illness with high mortality or a chronic syndrome with low mortality. clinical signs include inappetence, emaciation, rough hair coat, hunched posture, hyperpnea, nasal and ocular discharge, cutaneous ulceration, and arthritis. c. bovis infection causes hyperkeratotic dermatitis characterized by scaly skin, which is accompanied by alopecia in haired mice. severe infection may cause death. corynebacterial keratoconjunctivitis has been reported in aged c bl/ mice (mcwilliams et al., ) . epizootiology subclinically infected animals harbor c. kutscheri in the upper alimentary tract, colon, respiratory tract, regional lymph nodes, middle ear, and preputial gland. c. bovis colonizes skin and is shed in feces. therefore, transmission is by direct contact, fecaloral contact, and aerosol. resistance to infection appears to be under genetic control in some mouse strains. rats are susceptible to c. kutscheri, so cross-infection to mice may occur. pathology lesions caused by c. kutscheri develop from hematogenous spread to various internal organs and appear as gray-white nodules in the kidney, liver, lung, and other sites. cervical lymphadenopathy and arthritis of the carpometacarpal and tarsometatarsal joints also may occur. septic, necrotic lesions often contain caseous material or liquefied exudate. histologic lesions are characterized by coagulative or caseous necrosis bordered by intense neutrophilic infiltration. colonies of gram-positive organisms with 'chinese letter' configurations can usually be demonstrated using tissue gram stains of caseous lesions. mucopurulent arthritis of carpal, metacarpal, tarsal, and metatarsal joints are related to bacterial colonization of synovium accompanied by necrosis, cartilage erosion, ulceration, and eventually ankylosing pan arthritis. c. kutscheri is not a primary skin pathogen, but skin ulcers or fistulas follow bacterial embolization and infarction of dermal vessels. subcutaneous abscesses have also been reported. hyperkeratotic dermatitis caused by c. bovis is characterized grossly by skin scaliness and alopecia. microscopically, skin lesions consist of prominent acanthosis and moderate hyperkeratosis accompanied by mild nonsuppurative inflammation (fig. . ) . hyperkeratosis is typically more severe in glabrous athymic mice than in haired mice. organisms can be demonstrated in hyperkeratotic layers by gram stain. diagnosis c. kutscheri is usually diagnosed by culture and tissue gram stains on lesions from clinically apparent cases. agglutination serology is available, and immunofluorescence, immunodiffusion, and elisa tests have been reported (boot et al., a) . pcr of skin swabs or feces is a sensitive and specific method for the detection of c. bovis infection in mice (dole et al., ) . differential diagnosis the caseous nature of c. kutscheri-induced lesions helps separate them from necrotic changes or abscesses caused by other infectious agents of mice. thus, they can be differentiated from streptococcosis, mycoplasmosis, and other septicemic bacterial infections in which caseous necrosis does not occur. because mice can sustain natural infections with mycobacterium avium, histochemical techniques for acidfast bacilli and appropriate culture methods for mycobacteria should be considered if nodular inflammatory lesions of the lung are detected. diffuse scaling dermatitis in athymic nude mice is classic for c. bovis infection; however, in one case report staphylococcus xylosus was instead isolated in high numbers from the skin lesions (russo et al., ) . hyperkeratotic dermatitis caused by laboratory animal medicine c. bovis must be differentiated from scaly skin caused by low humidity in glabrous mice. prevention and control c. kutscheri infection occurs sporadically and infected colonies should be culled or rederived into an spf facility as treatment is not curative and control is difficult. c. bovis can be endemic in athymic nude mouse colonies. prevention and control are difficult because both immunocompetent and athymic mice as well as humans can carry c. bovis on the skin and in the upper respiratory system, respectively. c. bovis readily contaminates the environment as aerosolization within a class ii biosafety cabinet was shown to spread the bacterium during cage-change procedures (burr et al., ) . antibiotic treatment has been unrewarding (burr et al., ) research complications corynebacteriosis can cause morbidity and mortality, especially among immunodeficient mice. dermatologic disease in suckling mice can be fatal but is less severe and transient in weanling mice. etiology staphylococci are gram-positive organisms that commonly infect skin and mucous membranes of mice and other animals. the two most frequently encountered species are staphylococcus aureus, which can be highly pathogenic, and s. epidermidis, which is generally nonpathogenic. species subtypes are identified by phage typing and biochemistry profiles. pathogenic staphylococci are typically coagulase-positive, although s. xylosus has caused serious infections and is coagulasenegative (gozalo et al., ) . clinical signs staphylococcosis causes suppurative conjunctivitis, periorbital and retroorbital abscesses, preputial adenitis, and pyoderma in mice, particularly in immunocompromised strains such as nude mice. some evidence suggests that staphylococci can produce primary cutaneous infections, but they are more likely opportunistic organisms that induce lesions after contamination of skin wounds. eczematous dermatitis develops primarily on the face, ears, neck, shoulders, and forelegs and can progress to ulcerative dermatitis, abscessation (including botryomycotic granulomas), and cellulitis. because lesions are often pruritic, scratching causes additional trauma and autoinoculation. staphylococcal infection in the genital mucosa of males may produce preputial gland abscesses. these occur as firm, raised nodules in the inguinal region or at the base of the penis and may rupture to spread infection to surrounding tissues. male mice also may develop septic balanoposthitis secondary to penile self-mutilation. retrobulbar abscesses caused by s. aureus are frequently noted in athymic mice. sjl mice, which are nk cell deficient, are prone to necrotic dermatitis on the tail secondary to s. xylosus infection. epizootiology staphylococci are ubiquitous and can be carried on the skin and in the nasopharnyx and gastrointestinal tract. they also can be cultured from cages, room surfaces, and personnel. the prevalence of staphylococcal dermatitis appears to be influenced by host genotype, the overall health of the animal, and the degree of environmental contamination with staphylococcus spp. c bl/ , c h, dba, and balb/c mice are among the most susceptible strains. age may also influence susceptibility, with young mice being more susceptible than adults. immunodeficient mice (e.g., athymic mice) contaminated with staphylococci often develop abscesses or furunculosis (fig. . ). as noted above, behavioral dysfunction resulting in selfmutilation, including scratching and trichotillomania, is a likely predisposing factor. once virulent staphylococci contaminate the environment, colonization of the gastrointestinal tract can occur and produce a carrier state. phage typing can help to determine the source of infection. human phage types of staphylococci can infect mice, but the zoonotic importance of this connection is not clear. pathology gross lesions are typified by suppurative, ulcerative and necrotic dermatitis involving the head and neck but may extend to the shoulders and forelegs (percy and barthold, ) . superficial or deep abscesses may occur in conjunction with dermatitis or separately, as, e.g., in the external male genitalia. histologically, acute skin infections result in ulceration with neutrophils in the dermis and subcutis. chronic lesions contain lymphocytes, macrophages, and fibroblasts. deep infections appear as coalescing botryomycotic pyogranulomas with necrotic centers containing bacterial colonies. infected athymic mice may develop laboratory animal medicine furunculosis of the muzzle and face accompanied by regional lymphadenitis. diagnosis diagnosis is made by documenting gross and histological lesions, including gram staining of suspect tissues, complemented by isolation of grampositive, coagulase-positive (s. aureus), or coagulasenegative staphylococcus species. differential diagnosis staphylococcosis must be differentiated from other suppurative infections of mice, including pasteurellosis, streptococcosis, corynebacteriosis, and pseudomoniasis. ectoparasitism, fight wounds, and self-mutilation per se should also be considered. prevention, control, and treatment removal of affected animals, sterilization of food and bedding, and frequent changing of bedding may limit or reduce transmission. in affected animals, nail trimming can reduce self-inflicted trauma. conditions that facilitate aggressive or self-mutilating behavior should be avoided. research complications staphylococcosis can cause illness and disfigurement in mice. immunodeficient mice are at increased risk. etiology streptococci are ubiquitous commensal gram-positive organisms and in some cases, primary pathogens. pathogenic streptococcal infections in laboratory mice are caused by β-hemolytic organisms in lancefield's group c, but epizootics caused by group a streptococci have occurred, and group g organisms have been isolated occasionally. group d has been reclassified as an enterococcus. alpha-hemolytic streptococci can cause systemic disease in scid mice, and group b streptococcus sp. infection has been reported to cause meningoencephalitis in athymic mice (schenkman et al., ) . additionally, streptococcus dysgalactiae subsp. equisimilis has lancefield group g or c antigens and was isolated from visceral abscesses of immunocompetent mice (greenstein et al., ) . clinical signs cutaneous infections can cause ulcerative dermatitis over the trunk, which may appear gangrenous, whereas systemic infections may be expressed as conjunctivitis, rough hair coat, hyperpnea, somnolescence, and emaciation. epizootiology mice can carry streptococci subclinically in their upper respiratory tracts. lethal epizootics can occur, but factors leading to clinical disease are unknown, although some infections may be secondary to wound contamination. pathology systemic lesions reflect hematogenous dissemination and include abscessation, endocarditis, splenomegaly, and lymphadenopathy (percy and barthold, ) . streptococcal cervical lymphadenitis can lead to fistulous drainage to the neck complicated by ulcerative dermatitis. infection with α-hemolytic streptococci can cause inflammatory lesions affecting kidney and heart. diagnosis diagnosis and differential diagnosis depend on isolation of organisms from infected tissues, combined with histopathologic confirmation. differential diagnosis streptococcosis must be differentiated from other suppurative infections of mice, including staphylococcosis, pasteurellosis, corynebacteriosis, and pseudomoniasis. prevention and control removal of affected animals, sterilization of food and bedding, and frequent changing of bedding may limit or reduce transmission. research complications immunodeficient mice are at increased risk for streptococcosis. etiology e. coli is a small gram-negative rod that is a normal inhabitant of the mouse intestine. epizootiology infection is considered nonpathogenic in immunocompetent mice. however, hyperplastic typhlocolitis resembling transmissible murine colonic hyperplasia has been reported in scid mice infected with a non-lactose-fermenting e. coli (waggie et al., ; arthur et al., ) . clinical signs affected mice develop lethargy and fecal staining. pathology gross lesions consist of segmental thickening of the colon or cecum, which may contain blood-tinged feces. microscopically, affected mucosa is hyperplastic and may be inflamed and eroded. diagnosis diagnosis depends on demonstrating lesions and isolating non-lactose-fermenting e. coli. differential diagnosis this condition must be differentiated from proliferative and inflammatory intestinal disease caused by lawsonia intracellularis, c. rodentium, or enterotropic mouse hepatitis virus, especially in immunodeficient mice. colibacillosis provides an example of the morbidity associated with a nominally innocuous organism when it affects an immunocompromised host. prevention and control removal of affected animals and disinfection of caging and equipment will limit or reduce transmission. research complications clinical illness may develop in immunodeficient mice. historically, klebsiella pneumoniae is a ubiquitous gram-negative organism that is a natural inhabitant of the mouse alimentary tract. most commercial vendors have excluded it from their barriers. it can be pathogenic for the respiratory and urinary tract of mice after experimental inoculation but is not a significant cause of naturally occurring disease. etiology klebsiella oxytoca is an opportunistic pathogen implicated in various clinical diseases in animals and humans. epizootiology k. oxytoca also is purported to be an etiological agent of antibiotic-associated hemorrhagic colitis (aahc) in adult humans and adolescents. in animals, k. oxytoca has been isolated from apparently healthy sentinel rodents being monitored for pathogens in health surveillance programs and from utero-ovarian infections including suppurative endometritis, salpingitis, perioophoritis, and peritonitis in aged b c f mice (davis et al., ; rao et al., ) . a model of aahc has been developed in rats by administering amoxicillinclavulanate followed by orally infecting rats with a strain of k. oxytoca cultured from a patient with aahc. studies in humans suggest that k. oxytoca exerts its pathogenicity in part through a cytotoxin. recently, authors have showed that several animal isolates of k. oxytoca, including clinical isolates, produced secreted products in bacterial culture supernatant that display cytotoxicity on hep- and hela cells, indicating the ability to produce cytotoxin. using mass spectroscopy techniques, they also confirmed tilivalline as the cytotoxin present in animal k. oxytoca strains. tilivalline may serve as a biomarker for k. oxytoca-induced cytotoxicity (darby et al., ) . clinical signs k. oxytoca has been cultured from cases of suppurative otitis media, urogenital tract infections, and pneumonia in c h/hej and nmri-foxn (nu) mice (bleich et al., ) . additionally, k. oxytoca was recently cultured from three breeding colonies of nod. cg-prkdc scid il rg tm wjl /szj (nsg) mice with chronic renal inflammation and ascending urinary tract infections (foreman et al., ) . differential diagnosis other bacterial infections capable of causing suppurative lesions, including staphylococci, streptococci, pasteurella sp., and e. coli, among others are considered a differential diagnosis. research complications morbidity and mortality from spontaneous infections can affect ongoing research. etiology clostridium difficile was identified as the etiology of antimicrobial-associated pseudomembranous colitis in humans and currently a considerable cause of morbidity in hospitalized patients who acquire nosocomial infections. in the early s, an increased interest in c. difficile infection (cdi) resulted from the emergence of a hyper-virulent strain (nap /bi/ ) associated with frequent recurrences and more severe clinical disease (abou chakra et al., ; mcfarland, ; kuijper et al., ) . c. difficile has also been implicated in antibioticassociated colitis in syrian hamsters (bartlett et al., ) , guinea pigs (lowe et al., ) , rabbits (thilsted et al., ; ryden et al., ) , prairie dogs (muller et al., ) , ostriches (frazier et al., ) , and horses (diab et al., ) . c. difficile is a rod-shaped strict anaerobe. cycloserinecefoxitin-fructose agar (ccfa) is a commonly used selective medium for c. difficile. cultures are incubated under anaerobic conditions at - °c. when grown on blood agar, c. difficile colonies are nonhemolytic and gray, and have a slightly raised umbonate profile with filamentous edges and a ground-glass appearance. colonies grown on blood agar have fluorescence under ultraviolet light. c. difficile forms acid from glucose and fructose, but is negative on lactose, maltose, and sucrose. two closely related exotoxins, toxin a and toxin b, are produced by c. difficile. recent taxonomic classification support placement of c. difficile and its close relatives within the family peptostreptococcaceae. the authors suggested renaming it peptoclostridium difficile (yutin and galperin, ) . epizootiology it is estimated that c. difficile spores germinate and establish infection less than h after ingestion. spores rapidly transit through the upper gastrointestinal tract and colonize the colon and cecum. spore shedding begins less than h postingestion. when c bl mice were challenged with cfu of c. difficile spores, severe cdi signs developed and all mice were clinically affected by h postchallenge (chen et al., ) . specific methods to control and prevent c. difficile infections in mice have not been described. given the method of transmission of c. difficile and c. perfringens are via ingestion or spores, these clostridia can probably be excluded from mouse colonies by maintaining strict husbandry practices, robust sanitation, and use of autoclaved feed, bedding, cages, and cage accessories. sudden dietary changes should be avoided and antibiotics should be used judiciously to minimize disruption of the normal gut microbiota of mice. diagnosis of c. difficile-associated disease is generally based on detection of cytotoxin using a tissue culture cytotoxicity assay. pcr assays for detection of both c. difficile and its cytotoxins have been developed (eastwood et al., ) . there are no published regimens specifically for the treatment of natural c. difficile infections in mice. oral doses given twice daily of mg vancomycin for days to experimentally infected gnotobiotic mice caused a -to -log decrease in vegetative bacterial cell count and no detectable cytotoxin. bacterial counts and cytotoxin levels returned to previous levels after treatment was discontinued. clinical signs untreated mice are relatively resistant to infection with c. difficile and do not develop fatal infections, although these mice can become asymptomatic carriers that persistently shed low numbers of spores (lawley et al., ) . susceptibility of mice to infection must be induced by disrupting the microbiota through antibiotic treatment. brief exposure to environmental laboratory animal medicine spore contamination is sufficient for transmission of c. difficile to naïve but susceptible mice. the cdi transmission model has been used to demonstrate that clindamycin treatment of asymptomatic carriers of c. difficile can inadvertently trigger the excretion of high levels of spores (lawley et al., ) . a c bl mouse model of recurrence/relapse cdi has been reported (sun et al., ) . the primary bout of cdi induced little or no protective antibody response against c. difficile toxins and mice continued shedding c. difficile spores. antibiotic treatment of surviving mice induced a second episode of diarrhea. a simultaneous reexposure of mice to c. difficile bacteria or spores elicited a full clinical spectrum of cdi similar to that of the primary infection. immunosuppressive agents resulted in more severe and fulminant recurrent disease. vancomycin treatment only delayed disease recurrence; however, neutralizing polysera against both tcda and tcdb completely protected mice against cdi relapse (sun et al., ) . a recent study in c bl mice demonstrated that antibiotic-mediated alteration of the gut microbiome favors a global metabolic profile, and therefore increases susceptibility to c. difficile clinical diseases (theriot et al., ) . c. difficile is not tissue invasive and only toxigenic strains are associated with disease. experimental c. difficile infections include diarrhea, cecitis, polymorphonuclear cell infiltration of the lamina propria, inflammation, pseudomembrane formation, and death. differential diagnosis c. difficile-induced diarrhea is most often associated with antibiotic treatment. other clostridial diseases in mice must be ruled out as well as other enteric pathogens in mice causing diarrhea and mortality. salmonella spp. and c. rodentium should be considered in the differential diagnosis. etiology clostridium perfringens is associated with a number of diseases in domestic animals and humans. c. perfringens is a nonmotile, rod-shaped, encapsulated, anaerobic bacterium measuring - µm in length and . - . µm in diameter (murray et al., ) . c. perfringens grow rapidly on blood agar, and colonies are smooth, round, and grayish in color, and are surrounded by a double zone of hemolysis. c. perfringens is grouped into five types based on the production and secretion of four major toxins. c. perfringens produces a number of other virulence-enhancing toxins and hydrolytic enzymes. the most significant of these is probably enterotoxin, released with the bacterial spore after cell lysis. epizootiology c. perfringens is most likely acquired by the ingestion of spores that originated in the soil or in the intestinal tract of a carrier animal. the organism can be a member of the normal microbiota in human and domestic animals. factors that have been associated with the proliferation of the organism of these species include poor husbandry and sudden dietary changes (quinn et al., ) . methods to control and prevent c. perfringens infections have not been evaluated in mice. because the bacterium is most likely acquired by the ingestion of spores, it can probably be excluded from mouse colonies by maintaining good sanitation and sterilizing feed, bedding, cages, and cage accessories. sudden dietary changes have also been associated with proliferation of the organism and should be avoided if possible (quinn et al., ) . clinical signs only a few reports in the literature exist describing clinical disease associated with c. perfringens infection in mice (matsushita and matsumoto, ; rozengurt and sanchez) . disease has been observed in mice of both sexes, from to days old, and in female mice of breeding age. clinical signs have included hunched posture, ruffled hair coat, enlarged painful abdomen, soft or impacted feces, hindquarter paralysis, and dyspnea. sudden death without premonitory signs has also been reported. the toxin types of c. perfringens isolated from these cases were reported to be non-type a (matsushita and matsumoto, ) , type b (rozengurt and sanchez, ) , and type d (clapp and graham, ) . mucosal necrosis in both the large and small intestine is a consistent finding on microscopic examination of tissues from mice with clinically apparent c. perfringens infections. differential diagnosis c. perfringens produces a number of major and minor toxins. different types of the bacterium produce different toxins which account for different disease outcomes. c. perfringens type a is a constituent of the normal microbiota of the intestine of humans and other animal species. bacterial culture should be obtained from live or recently dead animals, and placed in anaerobic transfer medium for transport to a microbiology laboratory and should be cultured soon after their arrival. a presumptive diagnosis for c. perfringens can be based on the presence of large grampositive rods in fecal smears or in histologic sections of intestines (quinn et al., ) . definitive diagnosis is based on toxin identification. mice treated with chlortetracycline hydrochloride in drinking water at a level of mg/l for weeks have eliminated c. perfringens-associated disease (matsushita and matsumoto, ) . penicillin g in the diet or changing the diet has also been reported to be effective in disease remission. c. perfringens treatments in domestic species include ampicillin, amoxicillin-clavulanate, tylosin, clindamycin, metronidazole, and bacitracin (marks, ; mcgorum et al., ) . commercially available bacterins for use in mice were not effective in controlling the disease (clapp and graham, ) . research complications clostridia are large, rodshaped, gram-positive anaerobic bacteria. naturally occurring clostridial infection in mice is rare. epizootics of c. perfringens type d infection with high mortality laboratory animal medicine have been reported in a barrier colony where heavy mortality occurred in -to -week-old suckling mice. clinical signs included scruffy hair coats, paralysis of the hindquarters, and diarrhea or fecal impaction. however, attempts to reproduce the disease experimentally with clostridia isolated from naturally infected animals were unsuccessful. c. perfringens also has been isolated from sporadic cases of necrotizing enteritis in recently weaned mice. clostridium piliforme -tyzzer's disease (fujiwara and ganaway, ; ganaway, ; ganaway et al., ; percy and barthold, ) etiology tyzzer's disease is named for ernest tyzzer, who first described it in a colony of japanese waltzing mice. the causative organism, c. piliforme (formerly bacillus piliformis), is a long, thin, gram-negative spore-forming bacterium that appears to require living cells for in vitro growth. it has not been grown successfully on cell-free media, but it can be propagated by inoculation of susceptible vertebrates, in select cell lines, the yolk sac of embryonated eggs, or hepatocyte cell cultures obtained from mice (ganaway et al., ; kawamura et al., ) . clinical signs clinical disease occurs as unexpected deaths that may be preceded by diarrhea and inactivity. although outbreaks can be explosive and mortality is usually high, morbidity varies. additionally, subclinical infections can occur, accompanied by the development of antibodies to c. piliforme. stresses, such as overcrowding, high temperature and humidity, moist food, and immunosuppression, and young age, may predispose mice to tyzzer's disease. susceptibility and resistance also are influenced by host genotype. it has been shown, e.g., that c bl/ mice are more resistant than dba/ mice to tyzzer's disease (waggie et al., ) . resistance to severe infection appears to be due, in part, to b-lymphocyte function. the role of t cells in resistance is not clear, because susceptibility among athymic mice appears to vary (livingston et al., ) . however, the involvement of t cells can be inferred by the fact that several interleukins modulate resistance and susceptibility. depletion of neutrophils or nk cells also increases susceptibility to infection. epizootiology current prevalence rates, reservoirs of infection, carrier states, and the mechanism of spread remain speculative. tyzzer's disease occurs in many species of laboratory animals and in domestic and free-living species. some strains appear capable of cross-infecting mice, rats, and hamsters, whereas others have a more restricted host range (franklin et al., ) . therefore, the risks for cross-infection depend on the strain causing a given outbreak. although the vegetative form of c. piliforme is unstable, spores can retain infectivity at room temperature for at least year and should be viewed as the primary means of spread. natural infection is probably due to ingestion of organisms, which are subsequently shed in feces. feces-contaminated food and soiled bedding are the most likely sources of environmental contamination. prenatal infection can be induced by intravenous inoculation of pregnant mice, but its importance in the natural transmission of infection has not been determined. pathology infection begins in the gastrointestinal tract, followed by bacteremic spread to the liver and, to a smaller extent, the heart. the lesions are characterized by necrosis in these tissues and in the mesenteric lymph nodes. grossly, segments of the ileum, cecum, and colon may be red and dilated, with watery, fetid contents, whereas the liver, mesenteric lymph nodes, and heart often contain gray-white foci. histologically, intestinal lesions include necrosis of mucosal epithelium, which may be accompanied by acute inflammation and hemorrhage. in the liver, foci of coagulation necrosis are generally distributed along branches of the portal vein, a finding compatible with embolic infection from the intestine. peracute lesions are largely free of inflammation, but neutrophils and lymphocytes may infiltrate less fulminant lesions. myocardial necrosis is sporadic in natural infection. diagnosis tyzzer's disease is diagnosed most directly by the demonstration of characteristic intracellular organisms in tissue sections of liver and intestine. bundles of long, slender rods occur in the cytoplasm of viable cells bordering necrotic foci, especially in the liver (fig. . ) and intestine. they are found more easily during early stages of infection. organisms in tissue sections do not stain well with hematoxylin-eosin stain. silver stains, giemsa stains, or periodic acid-schiff stains are usually required for visualization of the organism. pcr and serologic assays are readily available at diagnostic laboratories. older supplemental procedures included inoculation of cortisonized mice or embryonated eggs laboratory animal medicine with suspect material, followed by histological or immunocytochemical demonstration of organisms in tissues. differential diagnosis the histological detection of organisms is essential for differentiating tyzzer's disease from other infections that can produce similar signs and lesions, especially mousepox, coronaviral hepatitis, reoviral hepatitis, helicobacteriosis, and salmonellosis. it also is important not to misconstrue extracellular rods as c. piliforme. prevention and control barrier housing and husbandry that incorporate sanitation measures to avoid the introduction or buildup of spores in the environment are the bases for control or prevention of tyzzer's disease. if infection occurs, spore formation will make control or elimination by antibiotic therapy problematic. therefore, strict quarantine, followed by replacement of affected or exposed stock, must be considered. rederivation by embryo transfer or cesarean section should take the potential for prenatal transmission of infection into account in housing and testing offspring. thorough decontamination of the environment with an oxidizing disinfectant must be included in any control program. additionally, procurement of food and bedding from suppliers with thorough quality assurance and vermin control programs is essential for both prevention and control. husbandry supplies should be stored in vermin-proof quarters, and the option of heat sterilization of food and bedding should be considered. research complications research complications stem from clinical morbidity and mortality. mice with immune dysfunction are at increased risk. there is recent evidence that infection causes elevations in selected cytokines (van andel et al., ) . etiology two mycobacteria are known to be pathogenic for laboratory mice: mycobacterium avium-intracellulare and m. lepraemurium. both are acid-fast, obligate intracellular bacteria. epizootiology mycobacteria are widespread in water and soil. their presence in laboratory mice would indicate a significant break in husbandry practices. infection with m. avium-intracellulare should be considered extremely rare, with the only published report describing an episode in a breeding colony of c bl/ mice . the source of the outbreak was presumed to be drinking water. mycobacterium lepraemurium has been isolated from healthy laboratory mice and can persist as a latent infection, but its significance is primarily historical, as a model for human leprosy. it is highly unlikely to encounter this infection in a modern, well-managed mouse colony. clinical signs m. avium-intracellulare infection is typically subclinical but mice have developed granulomatous pneumonia . pathology lesions are classically a chronic granulomatous disease with granulomas, langhans giant cells, and concurrent presence of acid-fast bacteria in various organs including the lungs, liver, spleen and lymph nodes. m. lepraemurium may cause alopecia, thickening of skin, subcutaneous swellings, and ulceration of the skin. disease can lead to death or clinical recovery. gross lesions are characterized by nodules in subcutaneous tissues and in reticuloendothelial tissues and organs (lung, spleen, bone marrow, thymus, and lymph nodes). lesions can also occur in the lung, skeletal muscle, myocardium, kidneys, nerves, and adrenal glands. the histologic hallmark is perivascular granulomatosis with accumulation of large, foamy epitheloid macrophages (lepra cells) packed with acid-fast bacilli. diagnosis acid-fast bacilli in lesions are the hallmark of presumptive diagnosis of mycobacteriosis. definitive diagnosis results from positive culture which takes days to weeks to rule out or positive pcr assays which are more time-efficient but require associated expertise. differential diagnosis other bacterial species that cause granulomatous lesions in mice. research complications natural infection is very rare. etiology proteus mirabilis is a ubiquitous gram-negative organism that can remain latent in the respiratory and intestinal tracts of normal mice (percy and barthold, ) . epizootiology proteus mirabilis colonizes the intestinal tract of most humans and is commonly found in research mice unless specifically excluded. clinical signs clinical disease can occur following stress or induced immunosuppression. immunodeficient mice have a heightened susceptibility to pathogenic infection. pathology proteus has been associated with ulcerative lesions in the gastrointestinal tract of immunodeficient mice. infected animals lose weight, develop diarrhea, and die within several weeks. if septicemia develops, suppurative or necrotic lesions, including septic thrombi, may be found in many organs, but the kidney is commonly affected. proteus pyelonephritis is characterized by abscessation and scarring. ascending lesions may occur following urinary stasis, but hematogenous spread cannot be ruled out. proteus mirabilis and pseudomonas aeruginosa have been isolated concomitantly from cases of suppurative nephritis or pyelonephritis. infection in immunodeficient mice is typified by splenomegaly and focal necrotizing hepatitis. pulmonary lesions include edema and macrophage activation. septic thrombi can occur, however, in many tissues. diagnosis culture recovery of proteus mirabilis as a predominant or single isolate confirms an opportunistic local or systemic infection. differential diagnosis gram-negative bacterial infections. research complications natural infections are typically isolated cases. etiology leptospirosis remains one of the most common zoonoses transmissible from rodents (desvars et al., ) but is exceedingly rare in laboratory mice. infection with leptospira interrogans serovar ballum has been reported on several occasions (see chapter ). epizootiology leptospira are gram-negative organisms that, after a septicemic phase, establish persistent infection in the renal tubules and are periodically excreted in the urine. clinical signs natural infection is subclinical and causes no significant lesions. experimental infections can result in severe vascular, hepatic and renal lesions dependent on serovar, mouse strain and immunocompetency. diagnosis diagnosis requires isolation of organisms in kidney culture. serological testing should be used with caution because neonatal exposure can lead to persistent infection without seroconversion. histologic examination of kidney using silver stains can also be attempted. pcr assays are reliable for preliminary diagnosis. differential diagnosis not applicable in research colonies. research complications persistent murine infections associated with active shedding present a zoonotic hazard for humans; therefore, infected mice should be culled. elimination of infection from highly valuable mice requires rederivation. (percy and barthold, ) etiology chlamydia trachomatis is an intracellular organism that produces glycogen-positive intracytoplasmic inclusions (elementary bodies). c. trachomatis causes ocular and urogenital disease in humans. however, at least one strain historically referred to as the 'nigg agent' after clara nigg, is most recently classified as chlamydia muridarum and is used experimentally to model human chlamydia infection. epizootiology mice are susceptible to natural infection and experimental infection with c. trachomatis and chlamydophila psittaci, especially immunodeficient mouse strains. clinical signs natural infections are typically subclinical but persistent. pathology c. muridarum is also known as the 'mouse pneumonitis agent' due to severe acute infection which is characterized by ruffled fur, hunched posture, and labored respiration due to interstitial pneumonitis and death in h. mice with more chronic infections may develop progressive emaciation and cyanosis of the ears and tail. experimental infections to model human venereal chlamydia infections will develop hydrosalpinx, cervical, and vaginal infections in female mice and urethritis in male mice. diagnosis chlamydia can be diagnosed by impression smears stained with giemsa or macchiavello stains, cell culture, or inoculation of embryonated eggs. pcr and sequencing can be used to speciate the type of chlamydia. differential diagnosis c. muridarum, c. trachomatis, and c. psittaci are included in the differential diagnosis. research complications chlamydia is a rare spontaneous infection in research mice; its potential significance is low. etiology pneumocystis murina (pm) is a common opportunistic organism of laboratory mice and other mammals. when first described by chagas in , p. carinii was misidentified as trypanosoma cruzi and was considered a protozoan (chagas, ) . it was renamed as a new species, p. carinii, when observed in a rat in (delanoë, p. and delanoë, m. ) . p. carinii, however, has now been grouped taxonomically with the fungi based on dna analysis and the homology of p. murina housekeeping genes with those found in fungi (edman et al., ; stringer et al., ; wakefield et al., ) . these dna studies and apparent differences of host susceptibility prompted a new name, p. jiroveci, for pneumocystis isolated from humans (stringer et al., ; frenkel, ) . p. carinii is now used to name the organism in rats and p. murina, the organism in mice. clinical signs pm infection is subclinical in immunocompetent mice. however, it can be clinically severe in immunodeficient mice, because an adequate complement of functional t lymphocytes is required to suppress infection (roths et al., ; shultz and sidman, ; walzer et al., ; weir et al., ) . b cells have also been shown to be critical to clearance of infection and the mechanism appears only partially related to igg and has a more important role in promoting activation and expansion of t cells (lund et al., ) . b cells may also protect early hematopoietic progenitor activity during systemic responses to pneumocystis infection (hoyt et al., ) . infection proceeds slowly, but relentlessly in immunodeficient mice leading to clinical signs of pneumonia, usually within several months. primary signs include dyspnea and hunched posture, which may laboratory animal medicine be accompanied by wasting and scaly skin. severe cases, such as those that occur in advanced disease in scid mice, may be fatal. epizootiology pm is known to infect a number of mammalian hosts, including ferrets, rats, mice, and humans. pm is a ubiquitous organism that is often present as a latent infection. although firm prevalence data are not available, because detection methods are not simple to apply, infection is assumed to be present in mouse colonies unless ruled out by extensive surveillance. although these organisms appear morphologically similar, there are antigenic and genetic differences among p. murina isolated from different hosts (weinberg and durant, ; cushion, ) . furthermore, studies indicate that p. carinii isolated from one host species is unable to survive and replicate after inoculation into a different immunodeficient host species (gigliotti et al., b) . pm infection also occurs in human beings, but transmission between rodents and human beings has not been documented. pm is transmitted by aerosol and establishes persistent, quiescent infection in the lungs of immunocompetent mice. prenatal infection has not been demonstrated. pathology pm is normally not pathogenic but can be activated by intercurrent immunosuppression. activation fills the lung with trophic and cystic forms. gross lesions occur in the lungs, which are often rubbery and fail to deflate (fig. . ). histopathological changes are characterized by interstitial alveolitis with thickening of alveolar septa from proteinaceous exudate and infiltration with mononuclear cells (fig. . ) (roths et al., ) . alveolar spaces may contain vacuolated eosinophilic material and macrophages. special stains are required to visualize pm. silver-based stains reveal round or partially flattened -to -mm cysts in affected parenchyma (fig. . ) . in florid cases, alveolar spaces may be filled with cysts, but cysts may be sparse in mild cases. disease can be especially severe when subclinically infected immunodeficient mice are reconstituted with competent immune cells that subsequently promote pneumonitis. diagnosis respiratory distress in immunodeficient mice should elicit consideration of pneumocystosis. pathologic examination of the lung, including silver laboratory animal medicine methenamine staining, is essential to confirm a presumptive clinical diagnosis. past infections of immunocompetent mice also can be detected by elisa (furuta et al., ) . pcr can be used to detect active infection (gigliotti et al., a; reddy et al., ) and is particularly useful for screening immunodeficient mice. differential diagnosis pneumocystosis must be differentiated from viral pneumonias of mice. it is worth noting, in this regard, that pneumonia virus of mice has been shown to accelerate the development of pneumocystosis in scid mice (bray et al., ; roths et al., ) . prevention and control pm infection is a significant disease threat to immunodeficient mice. its widespread distribution strongly suggests that susceptible mice should be protected by microbarrier combined, where possible, with macrobarrier housing. husbandry procedures should include proper sterilization of food, water, and housing equipment and the use of hepa-filtered change stations. infected colonies can be rederived by embryo transfer or cesarean methods, because infection does not appear to be transmitted in utero. research complications pneumonia in immunodeficient mice is the major complication of pm infection. trichophyton mentagrophytes is the most common fungal agent of mice. however, infection rarely causes clinical disease. clinical signs include sparse hair coats or well-demarcated crusty lesions, with a chalky surface on the head, tail, and legs (favus or ringworm). skin lesions are composed of exfoliated debris, exudate, mycelia, and arthrospores with underlying dermatitis. invasion of hair shafts is not characteristic. diagnosis depends on effective specimen collection. hairs should be selected from the periphery of the lesion, and hairless skin should be scraped deeply to obtain diagnostic specimens. t. mentagrophytes rarely fluoresces under ultraviolet light, and hyphae must be differentiated from bedding fibers, food particles, and epidermal debris. histological sections should be stained with a silver stain or schiff's reagent to reveal organisms. trichophyton also can be cultured on sabouraud agar. plates are incubated at room temperature ( - °c), and growth is observed at - days. ringworm is not easily eradicated from laboratory mice. the use of antifungal agents to treat individual mice is time-consuming, expensive, and variably effective. rederivation is a more prudent course. cages and equipment should be sterilized before reuse. concurrent infection with ectoparasites also must be considered during eradication steps. candida albicans and other systemic mycoses are not important causes of disease in mice, but they can be opportunistic pathogens in immunodeficient mice. etiology giardia muris is a pear-shaped, flagellated organism with an anterior sucking disk. it inhabits the duodenum of young and adult mice, rats, and hamsters. clinical signs infection is often subclinical, unless organisms proliferate extensively, and can cause weight loss, a rough hair coat, sluggish movement, and abdominal distension, usually without diarrhea. additionally, immunodeficient mice may die during heavy infestation. epizootiology the contemporary prevalence of affected mouse colonies is not well documented, but surveys during the s found the rates exceeding %. transmission occurs by the fecal-oral route. crossinfection between mice and hamsters after experimental inoculation of organisms has been demonstrated, whereas rats were resistant to isolates from mice and hamsters (kunstyr et al., ) . c h/he mice are particularly susceptible to giardiasis, whereas balb/c and c bl/ mice are more resistant. additionally, female mice appear to be more resistant to infection than male mice (daniels and belosevic, ) . c bl/ females, e.g., have lower trophozoite burdens and for a shorter interval than male mice. females also shed cysts later than male mice. these differences may be related to a more potent humoral immune response to giardia in female mice. pathology gross lesions are limited to the small intestine, which may contain yellow or white watery fluid. histopathology reveals organisms in the lumen that often adhere to microvilli of enterocytes or reside in mucosal crevices or mucus. the crypt/villus ratio may be reduced, and the lamina propria may have elevated numbers of inflammatory cells. diagnosis diagnosis is based on detection of trophozoites in the small intestine or in wet mounts of fecal material. organisms can be recognized in wet preparations by their characteristic rolling and tumbling movements. ellipsoidal cysts with four nuclei also may be detected in feces. infection also can be detected by serology (daniels and belosevic, ) and by pcr (mahbubani et al., ) . treatment, prevention, and control murine giardiasis can be treated by the addition of . % dimetridazole to drinking water for days. prevention and control depend on proper sanitation and management, including adequate disinfection of contaminated rooms. research complications accelerated cryptal cell turnover and suppression of the immune response to sheep erythrocytes have been observed in infected mice. the potential for severe or lethal infection in immunodeficient mice was noted previously. etiology spironucleus muris is an elongated, pearshaped, bilaterally symmetrical flagellated protozoan that commonly inhabits the duodenum, usually in the crypts of lieberkühn. it is smaller than giardia muris and lacks an anterior sucking disk. clinical signs s. muris infection is usually subclinical in normal adult mice. it is more pathogenic, however, for young, stressed, or immunocompromised mice (kunstyr et al., ) . additionally, clinical morbidity may indicate an underlying primary infection with an unrelated organism. clinically affected mice can have a poor hair coat, sluggish behavior, and weight loss. mice at - weeks of age are at notably higher risk for clinically evident infection. they can develop dehydration, hunched posture, abdominal distension, and diarrhea. severe infections can be lethal. epizootiology transmission occurs by the fecaloral route and can occur between hamsters and mice as well as between mice. it does not appear to be transmitted between mice and rats (schagemann et al., ) . the most recent surveys, which are somewhat dated, indicated that prevalence rates exceeded % among domestic mouse colonies in the mid- s. there is some evidence that inbred strains vary in their susceptibility to infection and their rate of recovery (baker et al., ; brett and cox, ) . pathology gross findings associated with infection include watery, red-brown, gaseous intestinal contents. however, it is essential to rule out primary or coinfection by other organisms before attributing these lesions to spironucleosis. microscopically, acute disease is associated with distension of crypts and intervillous spaces by pear-shaped trophozoites and inflammatory edema of the lamina propria. organisms can be visualized more easily with periodic acid-schiff staining, which may reveal invasion of organisms between enterocytes and in the lamina propria. chronic infection is associated with lymphoplasmacytic infiltration of the lamina propria and occasional intracryptal inflammatory exudate. diagnosis diagnosis is based on identification of trophozoites in the intestinal tract. they can be distinguished from giardia muris and tritrichomonas muris by their small size, horizontal or zigzag movements, and the absence of a sucking disk or undulating membrane. pcr-based detection also is available (rozario et al., ) . it is not clear whether duodenitis is a primary pathogenic effect of s. muris or represents opportunism secondary to a primary bacterial or viral enteritis. therefore, it is prudent to search for underlying or predisposing infections. treatment, prevention, and control treatment consists of adding . % dimetridazole to drinking water for days, as described for giardiasis. prevention and control require good husbandry and sanitation. research complications as with giardiasis, infection can accelerate enterocytic turnover in the small intestine. there is some evidence that infected mice may have activated macrophages that kill tumor cells nonspecifically and that infection can diminish responses to soluble and particulate antigens. additionally, infected mice also have increased sensitivity to irradiation. such effects should, however, be interpreted cautiously in order to rule out intercurrent viral infections. tritrichomoniasis t. muris is a nonpathogenic protozoan that occurs in the cecum, colon, and small intestine of mice, rats, and hamsters. no cysts are formed, and transmission is by ingestion of trophozoites passed in the feces. it can be detected by microscopy or by pcr (viscogliosi et al., ) . coccidiosis eimeria falciformis is a pathogenic coccidian that occurs in epithelial cells of the large intestines of mice. it was common in european mice historically but is seldom observed in the united states. heavy infection may cause diarrhea and catarrhal enteritis. klosiella muris causes renal coccidioisis in wild mice but is rare in laboratory mice. mice are infected by ingestion of sporulated sporocysts. sporozoites released from the sporocysts enter the bloodstream and infect endothelial cells lining renal arterioles and glomerular capillaries, where schizogony occurs. mature schizonts rupture into bowman's capsule to release merozoites into the lumen of renal tubules. merozoites can enter epithelial cells lining convoluted tubules, where the sexual phase of the life cycle is completed. sporocysts form in renal tubular epithelium and eventually rupture host cells and are excreted in the urine, but oocysts are not formed. infection is usually nonpathogenic and subclinical. gray spots may occur in heavily affected kidneys and are the result of necrosis, granulomatous inflammation, and focal hyperplasia. destruction of tubular epithelium may impair renal physiology. diagnosis is based on detection of organisms in tissues. prevention and control require proper sanitation and management techniques. there is no effective treatment. cryptosporidiosis cryptosporidium muris is a sporozoan that adheres to the gastric mucosa. it is uncommon in laboratory mice and is only slightly pathogenic. cryptosporidium parvum inhabits the small intestine and is usually nonpathogenic in immunocompetent and athymic mice (ozkul and aydin, ; taylor et al., ) . athymic mice may develop cholangitis and hepatitis, however, if organisms gain access to the biliary tract. entamoebiasis entamoeba muris is found in the cecum and colon of mice, rats, and hamsters throughout the world. organisms live in the lumen, where they feed on particles of food and bacteria. they are considered nonpathogenic. encephalitozoonosis encephalitozoon cuniculi is a gram-positive microsporidian that infects rabbits, mice, rats, guinea pigs, dogs, nonhuman primates, humans, and other mammals. infection is extremely rare among laboratory mice. the life cycle of the organism is direct, and animals are infected by ingesting spores or by cannibalism. spore cells are disseminated in the blood to the brain and other sites. infection can last more than year, and spores shed in the urine serve as a source of infection. vertical transmission has not been confirmed in mice. e. cuniculi is an obligate intracellular parasite, but infection usually elicits no clinical signs of disease. organisms proliferate in peritoneal macrophages by asexual binary fission. they have a capsule that accepts giemsa and goodpasture stains but is poorly stained by hematoxylin. fulminating infection can cause lymphocytic meningoencephalitis and focal granulomatous hepatitis. in contrast to encephalitozoonosis in rabbits, affected mice do not develop interstitial nephritis. infection is diagnosed by cytological examination of ascitic fluid smears, histopathologic examination of brain tissues stained with goodpasture stain, and elisa serology. no effective treatment has been reported. prevention and control require rigid testing and elimination of infected colonies and cell lines. pcr-based assays may also be useful. toxoplasmosis toxoplasma gondii is a ubiquitous gram-negative coccidian parasite for which the mouse serves as a principal intermediate host. however, the prevalence of natural infection is negligible because laboratory mice no longer have access to sporulated cysts shed by infected cats, which were historically the major source for cross-infection. toxoplasmosis can cause necrosis and granulomatous inflammation in the intestine, mesenteric lymph nodes, eyes, heart, adrenals, spleen, brain, lung, liver, placenta, and muscles. diagnosis is based on elisa serology, histopathology, and pcr. control and prevention depend largely on precluding access of mice to cat feces or to materials contaminated with cat feces. oocytes are very resistant to adverse temperatures, drying, and chemical disinfectants; therefore, thorough cleaning of infected environments is required. b. cestodiasis baker, ) etiology hymenolepis (rodentolepis) nana, the dwarf tapeworm, infects mice, rats, and humans although the zoonotic risk has been questioned (macnish et al., ) . adults are extremely small ( - mm) and have eggs with prominent polar filaments and rostellar hooks (fig. . ) . clinical signs young adult mice are most frequently infected. signs and lesions include weight loss and focal enteritis, but clinical disease is rare unless infestation is severe. epizootiology the life cycle may be direct or indirect (r. nana is the only cestode known that does not require an intermediate host). the indirect cycle utilizes arthropods as intermediate hosts. liberated oncospheres penetrate intestinal villi and develop into a cercocystis stage before reemerging into the intestinal lumen - days later. the scolex attaches to the intestinal mucosa, where the worm grows to adult size in weeks. the cycle from ingestion to patency takes - days. pathology cysticerci are found in the lamina propria of the small intestine and sporadically in the mesenteric lymph nodes, whereas adults, which have a serrated profile, are found in the lumen. inflammation is not a feature of infection. diagnosis infection can be diagnosed by demonstrating eggs in fecal flotation preparations or by opening the intestine in petri dishes containing warm tap water to facilitate detection of adults. r. nana can be differentiated from another species of rodent tapeworm, h. diminuta, by the fact that r. nana has rostellar hooks and eggs with polar filaments. however, h. diminuta requires an intermediate arthropod host, so it is rarely found in contemporary mouse colonies. treatment, prevention, and control drugs recommended for treatment and elimination include praziquantel ( . % in the diet for days), albendazole, mebendazole, and thiabendazole. although the benzimidazoles have an excellent activity against cestodes and nematodes in rats, they have not been tested extensively in mice. the potential for successful treatment is high, however, because eggs do not survive well outside the host and because the prevalence of infestation is low in caged mice kept in properly sanitized facilities. because r. nana can directly infect humans, proper precautions should be taken to avoid oral contamination during handling of rodents (see chapter ). hymenolepis microstoma is found in the bile ducts of rodents and could be confused with r. nana in the mouse. however, the location of the adult as well as the large size of h. microstoma eggs compared with those of r. nana make differential diagnosis relatively simple. the mouse and the rat are intermediate hosts of the cestode taenia taeniaformis. the definitive host is the cat. this parasite should not be found in laboratory mice housed separately from cats. c. nematodiasis (wescott, ) syphacia obvelata (mouse pinworm) infestation etiology syphacia obvelata, the common mouse pinworm, is a ubiquitous parasite of wild and laboratory mice. the rat, gerbil, and hamster are also occasionally infected. female worms range from . to . mm in length, and male worms are smaller ( . - . mm). eggs are flattened on one side and have pointed ends (fig. . ). the nucleus fills the shell and is frequently at a larval stage when eggs are laid. clinical signs infestation is usually subclinical, although heavily infested mice can occasionally sustain intestinal lesions, including rectal prolapse, intussusception, enteritis, and fecal impaction. epizootiology pinworm infestation is one of the most commonly encountered problems in laboratory mice. a national survey revealed that more than % of barrier colonies and about % of conventional colonies were affected (jacoby and lindsey, ; carty, ) . syphacia obvelata infestation can occur unexpectedly in commercial barrier murine colonies, resulting in widespread dissemination of the parasite into academic mouse colonies. the epizootiological impact of pinworm infestation is increased by the airborne dissemination of eggs, which can remain infectious even after drying. the life-cycle is direct and completed in - days. females deposit their eggs on the skin and hairs of the perianal region. ingested eggs liberate larvae in the small intestine and they migrate to the cecum within h. worms remain in the cecum for - days, where they mature and mate. the females then migrate to the large intestine to deposit their eggs as they leave the host. there is unconfirmed speculation that larvae may reenter the rectum. infestation usually begins in young mice and can recur, but adult mice tend to be more resistant. syphacia infestation often occurs in combination with aspiculuris tetraptera. because the life cycle of syphacia is much shorter than that of aspiculuris, the number of mice that are apt to be infected with s. obvelata is correspondingly greater. there is evidence that resistance to infestation may be mouse strain-specific (derothe et al., ) . pathology gross lesions are not prevalent, aside from the presence of adults in the lumen of the intestine. diagnosis infestation is diagnosed by demonstrating reniform-shaped eggs in the perianal area or adult worms in the cecum or large intestine. four-to -weekold mice should be examined because the prevalence is higher in this age group than in older mice. because most eggs are deposited outside the gastrointestinal tract, fecal examination is not reliable. eggs are usually detected by pressing cellophane tape to the perineal area and then to a glass slide that is examined by microscopy. aspiculuris tetraptera eggs are not ordinarily found in tape preparations and are easily differentiated from eggs of s. obvelata (see below). adult worms can be found in cecal or colonic contents diluted in a petri dish of warm tap water. they are readily observed with the naked eye or with a dissecting microscope. an elisa also is available to detect serum antibodies to s. obvelata somatic antigens (sato et al., ) . pcr assays are increasingly being used to augment traditional diagnostic methods and to discriminate between pinworm species (dole et al., ) . pcr panels for pinworm detection using fecal pellets are available from commercial diagnostic laboratories. treatment, prevention, and control pinworm infestation can be treated effectively by a number of regimens, which include the use of anthelmintics such as piperazine, ivermectin, and benzimidazole compounds alone or in combination (klement et al., ; le blanc et al., ; lipman et al., ; flynn et al., ; wescott, ; zenner, ) . because some of the recommended therapies have the potential for toxicity, it is prudent to keep mice under close clinical observation during treatment (davis et al., ; skopets et al., ; toth et al., ) . fenbendazole diets can be fed with week on/ week off rotation with normal chow although the potential impact on experimental data must be considered (duan et al., ; gadad et al., ; landin et al., ) . prevention of reinfestation requires strict isolation because syphacia eggs become infective as soon as h after they are laid, and they survive for weeks, even in dry conditions. strict sanitation, sterilization of feed and bedding, and periodic anthelmintic treatment are required to control infestation. the use of microbarrier cages can reduce the spread of infective eggs. syphacia muris is the common rat pinworm. it can potentially infest mice but is not found in well-managed colonies. it can be differentiated from s. obvelata because s. muris eggs are smaller. treatment is the same as for pinworms of mice. etiology aspiculuris tetraptera is the other major oxyurid of the mouse and may coinfect mice carrying s. obvelata. females are . - . mm long, and males are slightly smaller. the eggs are ellipsoidal (fig. . ) . clinical signs ingested eggs hatch, and larvae reach the middle colon, where they enter crypts and remain for - days. they move to the proximal colon about weeks after infection of the host. because the life cycle is - days longer than in s. obvelata, infestations appear in somewhat older mice; heaviest infestation is expected in - weeks after initial exposure. infection is usually subclinical, but heavy loads can produce signs similar to those discussed for s. obvelata. light to moderate loads do not produce clinical disease. epizootiology as noted under s. obvelata, pinworm infestation is highly prevalent and contagious in laboratory mice. the life cycle is direct and takes approximately - days. mature females inhabit the large intestine, where they survive from to days and lay their eggs. the eggs are deposited at night and are excreted in a mucous layer, covering fecal pellets. they require - days at °c to become infective and can survive for weeks outside the host. pathology see s. obvelata (section iii, a, ,c). diagnosis aspiculuris tetraptera eggs can be detected in the feces, and adult worms are found in the large intestine. eggs are not deposited in the perianal area; therefore, cellophane tape techniques are not useful. measures for treatment, prevention, and control are similar to those described for s. obvelata. because a. tetraptera takes longer to mature and because eggs are deposited in feces rather than on the host, adult parasites are more amenable to treatment by frequent cage rotations. immune expulsion of parasites and resistance to reinfection are hallmarks of a. tetraptera infection. research complications see s. obvelata (section iii, a, ,c). several species of mites infest laboratory mice. they include myobia musculi, radfordia affinis, myocoptes musculinus, and, less commonly, psorergates simplex. the common murine mites are described below, while less frequently encountered species are listed in table . . these include the mouse mite trichoecius romboutsi, which resembles myocoptes and ornithonyssus bacoti, the tropical rat mite, which can infect laboratory mice. characteristics of specific infestations are described after a general introductory section. clinical signs mites generally favor the dorsal anterior regions of the body, particularly the top of (jacoby and lindsey, ; carty, ) reported mite infestations in % of colonies. acarids spend their entire lives on the host. populations are limited by factors such as self-grooming, mutual grooming, the presence of hair, and immunological responses, which tend to produce hypersensitivity dermatitis. inherited resistance and susceptibility also affect clinical expression of acariasis. mite populations, e.g., vary widely among different stocks and strains of mice housed under similar conditions. pathology gross lesions include scaly skin, regional hair loss, abrasions, and ulcerations. histologically, hyperkeratosis, acanthosis, and chronic dermatitis may occur. long-standing infestation provokes chronic inflammation, fibrosis, and proliferation of granulation tissue. ulcerative dermatitis associated with acariasis may have an allergic pathogenesis but often results in secondary bacterial infections. lesions resemble allergic acariasis in other species and are associated with mast cell accumulations in the dermis. diagnosis classic methods of detection include direct observation of the hair and skin of dead or anesthetized mice. hairs are parted with pins or sticks and examined with a dissecting microscope. examination of young mice, prior to the onset of immune-mediated equilibrium, is likely to be more productive. alternatively, recently euthanized mice can be placed on a black paper, and double-sided cellophane tape can be used to line the perimeter to contain the parasites. as the carcass cools, parasites will vacate the pelage and crawl onto the paper. sealed petri dishes can also be used. cellophane tape also can be pressed against areas of the pelt of freshly euthanatized mice and examined microscopically. skin scrapings made with a scalpel blade can be macerated in % koh/glycerin or immersion oil and examined microscopically. this method has the disadvantage of missing highly motile species and low-level populations of slower moving immature forms. it is important to remember that mite infestations may be mixed, so the identification of one species does not rule out the presence of others. detecting mites in sentinels exposed to dirty bedding from colony animals has been reported to be unreliable (lindstrom et al., ) . thus, pcr assays offered by commercial diagnostic laboratories are increasingly being used to augment traditional diagnostic methods and to test individual animals or equipment using a swabbing technique; samples can be pooled to decrease cost (jensen et al., ) . gross anatomical features facilitate differentiation of intact mites. myocoptes has an oval profile with heavily chitinized body, pigmented third and fourth legs, and tarsal suckers (fig. . ) . myobia and radfordia have a similar elongated profile, with bulges between the legs. myobia has a single tarsal claw on the second pair of legs (fig. . ) , whereas radfordia has two claws of unequal size on the terminal tarsal structure of its second pair of legs (fig. . ) . histopathological examination of skin is helpful for diagnosing unique forms of acariasis, such as the keratotic cysts associated with psorergates simplex infestation. treatment, prevention, and control ivermectin can be used topically, in drinking water or as a medicated feed and often is the first-choice approach for attempting eradication although cost and potential toxicity are concerns. because of potential differences in laboratory animal medicine blood-brain barrier permeability to ivermectin, pilot treatments should be evaluated. for large facilities, ivermectin medicated feed may be an attractive option (ricart arbona et al., ) . for valuable lines of mice, rederivation may be cost-and time-effective. control and prevention programs should be carried out on a colony-wide basis, which includes thorough sanitation of housing space and equipment to remove residual eggs. research complications hypersensitivity dermatitis has the potential to confound immunological studies (jungmann et al., ) , especially those involving skin, and has been shown to elevate serum ige (morita et al., ) . heavy mite infestations can cause severe skin lesions and have been associated with weight loss, infertility, and premature deaths. chronic acariasis also may provoke secondary amyloidosis due to long-standing dermatitis. myocoptes musculinus this is the most common ectoparasite of the laboratory mouse but frequently occurs in conjunction with myobia musculi. the life cycle includes egg, larva, protonymph, tridonymph, and adult stages. eggs hatch in days and are usually attached to the middle third of the hair shaft. the life cycle may range from to days. transmission requires direct contact, for mice separated by wire screens do not contract infestations from infested hosts. bedding does not seem to serve as a vector. neonates may become infested within - days of birth, and parasites may live for - days on dead hosts. myocoptes appears to inhabit larger areas of the body than myobia and tends to displace myobia during heavy infestations. it has some predilection for the skin of the inguinal region, abdominal skin, and back, but it will also infest the head and neck. it is a surface dweller that feeds on superficial epidermis. infestation can cause patchy thinning of the hair, alopecia, or erythema. lesions can be pruritic, but ulceration has not been reported. chronic infestations induce epidermal hyperplasia and nonsuppurative dermatitis. myobia musculi this is a common mite of laboratory mice. the life cycle of myobia can be completed in days and includes an egg stage, first and second larval stages, protonymph, deutonymph, and adult. eggs attach at the base of hair shafts and hatch in - days. larval forms last about days, followed by nymphal forms on day . adults appear by day and lay eggs within h. myobia are thought to feed on skin secretions and interstitial fluid but not on blood. they are transmitted primarily by contact. mite populations increase during new infestations, followed by a decrease to equilibrium in - weeks. the equilibrated population can be carried in colonies for long periods (up to years). population fluctuations may represent waves of egg hatchings. because mites are thermotactic, they crawl to the end of hair shafts on dead hosts, where they may live for up to days. infestation may result in hypersensitivity dermatitis, to which c bl mice are highly susceptible. clinical signs vary from ruffled fur and alopecia to pruritic ulcerative dermatitis. therefore, lesions can be exacerbated by self-inflicted trauma. radfordia affinis radfordia is thought to be common in laboratory mice, but it closely resembles myobia and may occur as a mixed infestation. therefore, its true prevalence is conjectural. additionally, its life cycle has not been described. it does not appear to cause clinical morbidity. psorergates simplex this species has not been reported as a naturally occurring infection in well-managed colonies for several decades, but it is unique in that it inhabits hair follicles. its life cycle is unknown, but developmental stages from egg to adult may be found in a single dermal nodule. transmission is by direct contact. invasion of hair follicles leads to development of cyst-like nodules, which appear as small white nodules in the subcutis. histologically, they are invaginated sacs of squamous epithelium, excretory products, and keratinaceous debris. there is usually no inflammatory reaction, but healing may be accompanied by granulomatous inflammation. diagnosis is made by examining the subcuticular surface of the pelt grossly or by histological examination. sac contents also can be expressed by pressure with a scalpel blade or scraped and mounted for microscopic exam. mesostigmoid mites rarely, blood-sucking ornithonyssus bacoti and laelaps echidnina, normally limited to wild rodents, can also infect laboratory rodent colonies (watson, ; fox, ) . these mites may also transiently bite humans and can transmit zoonotic infections (see chapter ). unlike the more common rodent fur mites, mesostigmoid mites live off the host and can travel a long distance in search of a blood meal. they access research colonies via contaminated supplies or wild rats and mice gaining access to the facility. polyplax serrata, the mouse louse, is encountered in wild mice but no longer is a significant issue in research colonies. eggs are deposited at the base of hair shafts and nymph stages and adults can be found principally on the dorsum. p. serrata causes pruritus with associated dermatitis, anemia and debilitation and historically is the vector for mycoplasma coccoides. amyloidosis is caused by the deposition of insoluble (polymerized), mis-folded amyloid protein fibrils in organs and/or tissues. primary amyloidosis is a naturally occurring disease in mice, associated with the deposition of amyloid proteins consisting primarily of immunoglobulin light chains. secondary amyloidosis is associated with antecedent and often chronic inflammation. it results from a complex cascade of reactions involving release of multiple cytokines that stimulate amyloid synthesis in the liver (falk and skinner, ) . primary amyloidosis is common among aging mice (lipman et al., ) but also may occur in young mice of highly susceptible strains such as a and sjl or somewhat older c bl mice. other strains, such as balb/c and c h are highly resistant to amyloidosis (percy and barthold, ) . secondary amyloidosis is usually associated with chronic inflammatory lesions, including dermatitis resulting from prolonged acariasis. it can be induced experimentally, however, by injection of casein and may occur locally in association with neoplasia or in ovarian corpora lutea in the absence of other disease. in reactive amyloid a (aa) amyloidosis, serum aa (saa) protein forms deposits in mice, domestic and wild animals, and humans that experience chronic inflammation. aa amyloid fibrils are abnormal β-sheet-rich forms of the serum precursor saa, with conformational changes that promote fibril formation. similar to prion diseases, recent findings suggest that aa amyloidosis could be transmissible in mice and other species (murakami et al., ) . amyloid fibrils induce a seeding-nucleation process that may lead to development of aa amyloidosis. amyloidosis can shorten the life span of mice and can be accelerated by stress from intercurrent disease. amyloid appears histologically as interstitial deposition of a lightly eosinophilic, acellular material in tissues stained with hematoxylin and eosin. however, it is birefringent after staining with congo red when viewed with polarized light. deposition patterns vary with mouse strain and amyloid type. although virtually any tissue may be affected, the following sites are common: hepatic portal triads, periarteriolar lymphoid sheaths in spleen, renal glomeruli and interstitium (which can lead to papillary necrosis), intestinal lamina propria, myocardium (and in association with atrial thrombosis), nasal submucosa, pulmonary alveolar septa, gonads, endocrine tissues, and great vessels (fig. . ) . naturally occurring mineralization of the myocardium and epicardium and other soft tissues is a common finding at necropsy in some inbred strains of mice. although this condition is usually an incidental finding at necropsy, interference with organ function such as the heart cannot be ruled out if lesions are severe. it occurs in balb/c, c h, and especially dba mice (eaton et al., ; brownstein, ; brunnert et al., ) . it is found in the myocardium of the left ventricle ( fig. . ) , in the intraventricular systems, and in skeletal muscle, kidneys, arteries, and lung and may be accompanied by fibrosis and mononuclear inflammatory infiltrates. dba mice also can develop mineralization in the tongue and cornea. dietary, environmental, disease-related, and endocrine-related factors are thought to influence the prevalence of this lesion. ectopic mineralization is associated clinically with skin and vascular connective tissue conditions in humans and mouse models have been developed to study metastatic and dystrophic tissue mineralization (li and uitto, ) . pseudoxanthoma elasticum (pxe), a heritable ectopic mineralization disorder in humans, is caused by mutations in the abcc gene. knockout abcc −/− mice model the histopathologic and ultrastructural features of pxe, notably with mineralization of the vibrissae dermal sheath, serving as a biomarker of tissue mineralization (benga et al., ) . other inbred mouse strains, including kk/hlj and s /svimj, also develop vibrissae dermal mineralization and have an snp (rs ) in the abcc gene associated with low levels of abcc protein expression in the liver. dba/ j and c h/hej mice have the same polymorphism and low abcc protein levels; however, these mice only develop tissue mineralization when fed an experimental diet enriched in phosphate and low in magnesium. a reye's-like syndrome has been reported in balb/ cbyj mice (brownstein et al., ) . the etiology is unknown; however, antecedent viral infection may be involved. affected mice rapidly become lethargic and then comatose. they also tend to hyperventilate. high mortality ensues within - h, but some mice may recover. lesions are characterized grossly by swollen, pale liver and kidneys. the major histopathological findings include swollen hepatocytes with fatty change and nuclear swelling among astrocytes in the brain. hepatic lesions resembling changes in reye's syndrome have been reported in scid mice infected with madv- (pirofski et al., ) . deficiencies (tobin et al., ) vitamin deficiencies in mice have not been thoroughly described. unfortunately, much of the information that does exist reflects work carried out - years ago; thus, the reliability and specificity of some of these syndromes is questionable. vitamin a deficiency may produce tremors, diarrhea, rough hair coat, keratitis, poor growth, abscesses, hemorrhages, and sterility or abortion. vitamin a is recognized for its importance in development of the immune system (ross, ) and knockout mouse models have been used to demonstrate genetic polymorphisms in humans that negatively regulate intestinal β-carotene absorption and conversion to retinoids in response to vitamin a requirements for growth and reproduction (von lintig, ) . vitamin e deficiency can cause convulsions and heart failure, as well as muscular dystrophy and hyaline degeneration of muscles. two knockout mouse models of severe vitamin e deficiency were independently developed and lack α-tocopherol transfer protein (α-ttp), a gene that controls plasma and tissue α-tocopherol concentrations by exporting α-tocopherol from the liver. ttpa −/− mice have very low to undetectable levels of α-tocopherol and are infertile. the phenotype includes neuronal degeneration associated with progressive ataxia and age-related behavioral defects (yu and schellhorn, ) . deficiency of b complex vitamins produces nonspecific signs such as alopecia, decreased feed consumption, poor growth, poor reproduction and lactation, as well as a variety of neurological abnormalities. choline deficiency produces fatty livers and nodular hepatic hyperplasia, as well as myocardial lesions, decreased conception, and decreased viability of litters. folic acid-deficient diets cause marked decreases in red and white cell blood counts and the disappearance of megakaryocytes and nucleated cells from the spleen. pantothenic acid deficiency is characterized by nonspecific signs, such as weight loss, alopecia, achromotrichia, and posterior paralysis, as well as other neurological abnormalities. thiamin deficiency is associated with neurological signs, such as violent convulsions, cartwheel movements, and decreased food consumption. dietary requirements for ascorbic acid have not been shown in mice, and mouse diets are generally not fortified with ascorbic acid. the gulonolactone oxidase knockout mouse (gulo −/− ) on the c bl/ background requires vitamin c supplementation although the plasma ascorbate concentration of gulo −/− mice fed a vitamin c-deficient diet is maintained at % of wild-type concentrations, suggesting an uncharacterized pathway to generate a small amount of ascorbate (yu and schellhorn, ) . the gulo −/− mouse has become the model of choice in studying the role of vitamin c in complex diseases. vitamin c production has been successfully restored in gulo −/− mice using adenovirus vectors, making it possible to robustly manipulate physiological ascorbate concentrations in an inbred mouse. mineral deficiencies have been described only for several elements, and the consequences of the deficiencies are similar to those observed for other species. for example, iodine-deficient diets produce thyroid goiters; magnesium-deficient diets may cause fatal convulsions; manganese deficiency may cause congenital ataxia from abnormal development of the inner ear; and zinc deficiency may cause hair loss on the shoulders and neck, emaciation, decreased liver and kidney catalase activity, and immunosuppression. chronic essential fatty acid deficiency may cause hair loss, dermatitis with scaling and crusting of the skin, and occasional diarrhea. infertility has also been associated with this syndrome. mice have an absolute requirement for a dietary source of linoleic and/or arachidonic acid. (sundberg, ; ward, ) the significant syndrome of ulcerative dermatitis (ud) is a common idiopathic skin lesion that causes morbidity and early euthanasia losses in c bl/ and related lines of mice. significant pruritus leads to skin trauma associated with opportunistic bacterial infection and deep dermal ulcerations. initial signs include alopecia and papular dermatitis, which usually occur over the dorsal trunk (fig. . ) . progressive inflammation can be halted, sometimes reversed, by nail trimming and therapy with a wide spectrum of topical or systemic antibiotics, steroids, and other drugs such as vitamin e and aloe, all of which speak to the frustrating search for a primary etiology. treatment should be based on microbiological culture and sensitivity and screening for ectoparasites as hypersensitivity to acariasis has been proposed. seasonal fluctuation in the incidence of disease suggests that environmental factors may play a role. the incidence appears to increase during periods of significant seasonal changes in temperature and humidity, i.e., the onset of winter and early spring. there is some evidence that incidence is related to dietary fat with mice on high fat or ad libitum diets being more susceptible than those on restricted diets (neuhaus et al., ) . ileus associated with high mortality has been reported to occur in primiparous female mice during the second week of lactation (kunstyr, ) . this disorder has been described as acute intestinal pseudo-obstruction (ipo) in c bl/ mice free of known pathogens (feinstein et al., ) . lactating mice are either found dead or becoming moribund. segments of the small intestine become distended with fluid contents and histologically there is apoptosis of the villus epithelium of the small intestine and superficial epithelial cells of the large intestine. the enteric nervous system appears morphologically normal but necrotic enterocytes, mucosal erosions, and acute mucosal inflammation are commonly observed. there is no strong evidence for metabolic issues such as hypocalcemia or low blood glucose. the direct cause is unknown but death probably results from sepsis secondary to loss of barrier function reflected in apoptosis of the gut epithelium during peak lactation. environmental variables can affect responses of mice in experimental situations. changes in respiratory epithelial physiology and function from elevated levels of ammonia, effects of temperature and humidity on metabolism, effects of light on eye lesions and retinal function, and effects of noise on neurophysiology are examples of complications that can vary with the form of insult and the strain of mouse employed. mice do not easily acclimatize to sudden and dramatic changes in temperature. therefore, they are susceptible to both hypothermia and hyperthermia. mice also are susceptible to dehydration. poorly functioning automatic watering system valves or water bottles, resulting in spills (hypothermia) or obstructed sipper tubes (dehydration), are a significant cause of husbandry-related morbidity. shipping mice between facilities, irrespective of distance, warrants institutional guidelines to minimize exposure to temperature extremes. reheat coils should be designed to fail in the closed position to avoid overheating holding rooms. ringtail is a condition associated with low relative humidity. clinical signs include annular constriction of the tail and occasionally of the feet or digits, resulting in localized edema that can progress to dry gangrene ( fig. . ). it should be differentiated from dryness and gangrene that may occur in hairless mice exposed to low temperatures and perhaps other environmental or nutritional imbalances. necrosis of legs, feet, or digits also can occur in suckling mice because of disruption of circulation by wraps of stringy nesting material such as cotton wool. corneal opacities can result from acute or chronic keratitis, injury (unilateral) and developmental defects; the latter may occur in combination with inherited microphthalmia in c black mice (koch and gowen, ) . there is some evidence that the buildup of ammonia in mouse cages may contribute to inflammatory keratitis, because it can be controlled by increasing the frequency of cage cleaning. corneal opacities and anterior polar cataracts are a developmental defect in inbred c black mice (pierro and spiggle, ) . corneal opacity may be associated with keratolenticular adhesions involving a persistent epithelial stalk of the lens vesicle, which normally disappears around day of gestation (koch and gowen, ) . typically noted in runted or cachectic mice soon after weaning, malocclusion of the open-rooted, continually growing incisor teeth is an inherited trait expressed as poorly aligned incisors, especially of the lower incisors causing osteomyelitis, soft tissue abscesses, or necrosis in the lips or oral cavity. the incidence of inherited malocclusion varies with mouse strain (petznek et al., ) . malocclusion in older mice may be the result of trauma or oral neoplasia. overgrown molar teeth have been associated with trauma to developing tooth buds. skin lesions can be caused by fighting, tail biting, and overgrooming such as whisker chewing. barbering of facial hair and whiskers in subordinate mice by a dominant cagemate is common and may be solved by removing the dominant, normally haired mouse. hair or whisker chewing (barbering) has long been interpreted to be a manifestation of social dominance. apparent dominant animals retain whiskers, whereas cagemates have 'shaved faces' (fig. . ). chronic hair chewing can produce histological abnormalities such as poorly formed or pigmented club hairs. once chewing has ceased, many mice regrow previously lost hair in several weeks. both sexes may engage in this activity, and sometimes females may be dominant. barbering of whiskers and fur-plucking behavior in mice has been suggested to model human trichotillomania (compulsive hair plucking) because of similarities including elevated serotonin levels (dufour et al., ) , 'barbers' predominately pluck hair from the scalp and around the eyes and the genitals; the behavior is female biased, and begins during puberty and is impacted by genetic background (garner et al., ) . fighting is more common in male mice and more aggressive in some strains (sjl, fvb, balb/c) with bite wounds typically located on the head, neck, shoulders, perineal area, and tail. often one mouse in the cage is free of lesions and is the likely aggressor. removal of the unaffected male may end the fighting or simply reorder the dominance order. removing males for breeding and then regrouping them often results in fighting. for programs that produce sentinel mice in-house, castration is an option to reduce aggression in group-housed male sentinels (lofgren et al., ) . regional alopecia, especially around the muzzle, may result from abrasion against cage surfaces. improperly diluted disinfectants may also cause regional hair loss. ear tags used for identification may cause pruritis and self-induced trauma. hair removal products or clipping prior to imaging or application of experimental compounds to the skin may cause pruritus and can augment lesions that interfere with test results. dermatophytosis, ectoparasitism, or idiopathic hair loss must be considered in the differential diagnoses for muzzle or body alopecia. (burek et al., ; percy and barthold, ) common idiopathic lesions in aging mice include cardiomyopathy (with or without mineralization or arteritis), chronic nephropathy (frequently with mineralization), myelofibrosis (fibrotic change in the bone marrow) especially in female mice, melanosis in the meninges, ovarian atrophy (with or without hyaline material), pigment (ceroid-lipofuscin), tubular or stromal hyperplasia, cystic endometrial hyperplasia, testicular tubular degeneration or mineralization, prostate atypical epithelial hyperplasia, gastric glandular epithelial hyperplasia, pancreatic islet cell hyperplasia, dental dysplasia of incisor teeth, pituitary hyperplasia of pars intermedia and pars distalis, cataracts, increased extramedullary hematopoiesis in spleen, and lymphocytic infiltrates or other inflammatory changes in various tissues, including harderian gland, salivary gland, kidney, liver, gall bladder, nasal, trachea, thyroid, periovarian fat, epididymis, and urinary bladder. lymphoma is also very common . spontaneous atrial thrombosis is rare in mice (< % in -year-old mice) and appears to be strain-related, with a high prevalence in rfm mice. it also is more common in aged mice affected by kidney disease and amyloidosis. organizing thrombi will be found usually in an enlarged, hyperemic left atrium and auricle and may be accompanied by amyloidosis. affected mice may display signs of heart failure, particularly severe dyspnea. induction of atrial thrombosis in b c f mice has been used to assess cardiovascular risk of chemical exposures (yoshizawa et al., ) . myocardial and epicardial mineralization is described above (section iii,b, ). periarteritis, also known as arteritis, polyarteritis, or systemic arteritis, impacts older mice and lesions may be observed in multiple tissues, including the spleen, heart, tongue, uterus, testes, kidney, and urinary bladder. the media of the affected vessels is homogenous and intensely eosinophilic with hematoxylin and eosin stain. fibrosis and mononuclear cells infiltrate the vessel wall. experimental coronary arteritis with cardiac hypertrophy has been model in dba/ and other strains by intraperitoneal administration of mannoprotein-beta-glucan complex isolated from c. albicans (nagi-miura et al., ) . hyperplasia of alveolar or bronchial epithelium occurs in old mice and must be differentiated from pulmonary tumors. pulmonary histiocytosis, acidophilic macrophage pneumonia, and acidophilic crystalline pneumonia are synonymous morphologic descriptions of an idiopathic lung lesion that can be incidental or the cause of significant morbidity. incidence varies with mouse strain or stock, with c bl, s /svjae and swiss mice and older mice in general particularly susceptible. histologically, alveoli and bronchioles are filled with varying quantities of macrophages containing eosinophilic crystalline material . the crystalline material consists of ym and/or ym chitanases and can be found in other tissues including the upper respiratory tract, stomach, gall bladder, and bone marrow where it is described as hyalinosis (nio et al., ) . gastric lesions include crypt dilatation, submucosal fibrosis, adenomatous gastric hyperplasia, mineralization, and erosion or ulceration. gastric ulcers have been induced by cold stress, food restriction (rehm et al., ) , chemical injury (yadav et al., ) , and gastritis and gastric tumors by helicobacter infection (fox et al., ) . germfree mice have reduced muscle tone in the intestinal tract. cecal volvulus is a common cause of death in germfree mice and is caused by rotation of the large, thin-walled cecum. age-associated lesions are common in the livers of mice. cellular and nuclear pleomorphism, including binucleated and multinucleated cells, are detectable by months. mild focal necrosis occurs with or without inflammation, but an association of mild focal hepatitis with a specific infectious disease is often hard to confirm. other geriatric hepatic lesions include biliary hyperplasia with varying degrees of portal hepatitis, hepatocellular vacuolization, amyloid deposition (especially in periportal areas), strangulated or herniated lobes, hemosiderosis, lipofuscinosis, and fibrosis. extramedullary hematopoiesis occurs in young mice and in response to anemia. exocrine pancreatic insufficiency has been reported in cba/j mice. acinar cell atrophy is common but is strain-and sex-dependent. blood-filled mesenteric lymph nodes may occur in aged mice, especially c h mice. this condition is an incidental finding and should not be confused with infectious lymphadenopathy such as that associated with salmonellosis. aggregates, or nodules of mononuclear cells, are found in many tissues of aged mice, including the salivary gland, thymus, ovary, uterus, mesentery and mediastinum, urinary bladder, and gastrointestinal tract. these nodules should not be mistaken for lymphosarcomas. grossly observable black pigmentation in the spleen of c bl/ is normal and is melanosis caused by melanin deposition (weissman, ) . the spleen is subject to amyloidosis and hemosiderin deposition. lipofuscin deposition is common, especially in older mice. the thymus undergoes age-associated atrophy. a variety of genetic immunodeficiencies have been described in mice, many of which increase susceptibility to infectious diseases. perhaps the most widely known of these is the athymic nude mouse that lacks a significant hair coat and, more importantly, fails to develop a thymus and thus has a severe deficit of t-cellmediated immune function. additionally, scid mice, which lack both t and b lymphocytes, are used widely and are highly susceptible to opportunistic agents such as pneumocystis murina. specific immune deficits have become excellent models for studying the ontogeny and mechanisms of immune responsiveness (table . ). age-associated osteoporosis or senile osteodystrophy can occur in some mice. it is not associated with severe renal disease or parathyroid hyperplasia. nearly all strains of mice develop some form of osteoarthrosis. it is generally noninflammatory, affects articulating surfaces, and results in secondary bone degeneration. glomerulonephritis is a common kidney lesion of mice. it is more often associated with persistent viral infections or immune disorders rather than with bacterial infections. its prevalence in some strains approaches %. nzb and nzb × nzw f hybrid mice, e.g., develop immune complex glomerulonephritis as an autoimmune disease resembling human lupus erythematosus, whereas glomerular disease is relatively mild in nzb mice (nzb mice have a high incidence of autoimmune hemolytic anemia). renal changes occur as early as months of age, but clinical signs and severe disease are not present until - months. the disease is associated with wasting and proteinuria, and lesions progress until death intervenes. histologically, glomeruli have proteinaceous deposits in the capillaries and mesangium. later, tubular atrophy and proteinaceous casts occur throughout the kidney. immunofluorescence studies show deposits of immunoglobulin and the third component of complement, which lodge as immune complexes with nuclear antigens and antigens of murine leukemia virus in glomerular capillary loops. mice infected with lcmv or with retroviruses can also develop immune complex glomerulonephritis. mice also can develop chronic glomerulopathy characterized by progressive thickening of glomerular basement membrane by pas-positive material that does not stain for amyloid. this lesion can be accompanied by proliferation of mesangial cells; local, regional, or diffuse mononuclear cell infiltration; and fibrosis. advanced cases may lead to renal insufficiency or failure. interstitial nephritis can be caused by bacterial or viral infections but may also be idiopathic. typical lesions include focal, regional, or diffuse interstitial infiltration of tubular parenchyma by mononuclear cells, but glomerular regions also may be involved. severe lesions can be accompanied by fibrosis, distortion of renal parenchyma, and intratubular casts, but not by mineralization. if renal insufficiency or failure ensues, it can lead to ascites. some strains of mice, such as balb/c, can develop polycystic kidney disease, which, if severe, can compromise normal renal function. urinary tract obstruction occurs as an acute or chronic condition in male mice. clinical signs usually include wetting of the perineum from incontinence. in severe or chronic cases, wetting predisposes to cellulitis and ulceration. at necropsy, the bladder is distended, and proteinaceous plugs are often found in the neck of the bladder and proximal urethra. in chronic cases the urine may be cloudy, and calculi may develop in the bladder. additionally, cystitis, urethritis, prostatitis, laboratory animal medicine balanoposthitis, and hydronephrosis may develop. this condition must be differentiated from infectious cystitis or pyelonephritis and from the agonal release of secretions from accessory sex glands, which is not associated with an inflammatory response. hydronephrosis also may occur without urinary tract obstruction. ascending pyelitis occurs in mice secondary to urinary tract infection. parvovarian cysts are observed frequently and may be related to the fact that mouse ovaries are enclosed in membranous pouches. amyloidosis is also common in the ovaries of old mice. cystic endometrial hyperplasia may develop unilaterally or bilaterally and may be segmental. in some strains, the prevalence in mice older than months is %. endometrial hyperplasia is often associated with ovarian atrophy. mucometra is relatively common in adult female mice. the primary clinical sign is abdominal distension resembling pregnancy among mice that do not whelp. testicular atrophy, sperm granulomas, and tubular mineralization occur with varying incidence. preputial glands, especially of immunodeficient mice, can become infected with opportunistic or pathogenic bacteria. spontaneous lesions in prostate, coagulating gland (anterior prostatic lobe), seminal vesicles, and ampullary glands were described in control b c f mice from national toxicology program -year carcinogenicity and toxicity studies conducted in one of four different laboratories (suwa et al., ) . lymphocytic infiltration, inflammation, edema, epithelial hyperplasia, mucinous cyst, mucinous metaplasia, adenoma, adenocarcinoma, granular cell tumor, and glandular atrophy were variously observed in accessory sex glands. accessory adrenal cortical nodules are found in periadrenal and perirenal fat, especially in females. these nodules have little functional significance other than their potential effect on failures of surgical adrenalectomy. lipofuscinosis, subcapsular spindle cell hyperplasia, and cystic dilatation of cortical sinusoids are found in the adrenal cortices of aged mice. some inbred strains have deficiencies of thyrotropic hormone, resulting in thyroid atrophy. thyroid cysts lined by stratified squamous epithelium and generally of ultimo-branchial origin may be seen in old mice. amyloid can be deposited in the thyroid and parathyroid glands as well as in the adrenal glands. spontaneous diabetes mellitus occurs in outbred swiss mice and genetic variants of several strains such as nod mice (lemke et al., ) . high levels of estrogen in pregnancy may influence postpartum hair shedding. various endocrine effects on hair growth have also been described. abdominal and thoracic alopecia have been reported in b c f mice. symmetrical mineral deposits commonly occur in the thalamus of aged mice. they may also be found in the midbrain, cerebellum, and cerebrum and are particularly common in a/j mice. lipofuscin accumulates in the neurons of old mice. age-associated peripheral neuropathy with demyelination can be found in the nerves of the hindlimbs in c bl/ mice. deposits of melanin pigment occur in heavily pigmented strains, especially in the frontal lobe. a number of neurologically mutant mice have been described. they commonly have correlative anatomical malformations or inborn errors of metabolism. a seizure syndrome in fvb mice has been described (goelz et al., ) and can be spontaneous or associated with tail tattooing, fur clipping, and fire alarms. mice are most often female with a mean age of . months (range, - months) and can exhibit facial grimace, chewing automatism, ptyalism with matting of the fur of the ventral aspect of the neck and/or forelimbs, and clonic convulsions that may progress to tonic convulsions and death. ischemic neuronal necrosis was consistently observed in these mice and is consistent with status epilepticus in humans. unilateral and bilateral microphthalmia and anophthalmia are frequent (as high as %) developmental defects in inbred and congenic strains of c bl mice, especially impacting the right eye and female mice. these conditions may first be recognized due to ocular infections, secondary to inadequate tear drainage. other common findings include central corneal opacities, iridocorneal and corneal-lenticular adhesions, abnormal formation of the iris and ciliary body, cataracts, extrusion of lens cortical material with dispersion throughout the eye, failure of vitreous development, and retinal folding. these syndromes can be reproduced by exposure to alcohol at critical stages of embryogenesis when the optic cup and lens vesicle are developing and impacting normal development of other ocular structures, including the iris, ciliary body, vitreous, and retina . retinal degeneration can occur as either an environmental or a genetic disorder (chang et al., ) in mice. nonpigmented mice, both inbred and outbred, can develop retinal degeneration from exposure to light, with the progression of blindness being related to light intensity and duration of exposure. mouse genetics have laboratory animal medicine been shown to be more important than potential light associated tissue injury (serfilippi et al., a) . other strains such as c h, cba, and fvb are genetically predisposed to retinal degeneration because they carry the rd gene, which leads to retinal degeneration within the first few weeks of life and has been used extensively as a model for retinitis pigmentosa (farber and danciger, ) . presence of the rd gene in some mouse strains highlights that impaired vision must be a consideration when selecting strains for behavioral assays that rely on visual clues (garcia et al., ) . blindness does not interfere with health or reproduction and blind mice cannot be distinguished from non-blind mice housed in standard caging. cataracts can occur in old mice and have a higher prevalence in certain mutant strains. vestibular syndrome associated with head tilt, circling, or imbalance can result from infectious otitis or from necrotizing vasculitis of unknown etiology affecting small and medium-sized arteries in the vicinity of the middle and inner ears. (jones et al., (jones et al., - maronpot et al., ; percy and barthold, ) neoplasms of lymphoid and hematopoietic tissues are estimated to have a spontaneous prevalence of - %. there are, however, some strains of mice that have been specifically inbred and selected for susceptibility to spontaneous tumors. leukemogenesis in mice may involve viruses and chemical or physical agents. viruses associated with lymphopoietic and hematopoietic neoplasia belong to the family retroviridae (type c oncornaviruses) and contain rna-dependent dna polymerase (reverse transcriptase). these viruses are generally noncytopathogenic for infected cells, and mice appear to harbor them as normal components of their genome. although they may be involved in spontaneous leukemia, they are not consistently expressed in this disease. recombinant viruses have recently been discovered that can infect mouse cells and heterologous cells and are associated with spontaneous leukemia development in high leukemia strains such as akr mice. their phenotypic expression is controlled by mouse genotype. endogenous retroviruses are transmitted vertically through the germ line. horizontal transmission is inefficient but can occur by intrauterine infection or through saliva, sputum, urine, feces, or milk. the leukemia induced by a given endogenous virus is usually of a single histopathological type. loss of function in nucleic acid-recognizing, tlr , tlr , and tlr can result in spontaneous retroviral viremia and acute t-cell lymphoblastic leukemia (yu et al., ) . chemical carcinogens, such as polycyclic hydrocarbons, nitrosoureas, and nitrosamines, and physical agents such as x-irradiation can also induce hematological malignancies in mice. the most common hematopoietic malignancy in the mouse is lymphocytic leukemia that originates in the thymus. disease begins with unilateral atrophy and then enlargement of one lobe of thymus as tumor cells proliferate. cells can spread to the other lobe and then to other hematopoietic organs, such as the spleen, bone marrow, liver, and peripheral lymph nodes. clinical signs include dyspnea and ocular protrusion. the latter sign is due to compression of venous blood returning from the head. tumor cells spill into the circulation late in disease. most of these tumors originate from t lymphocytes or lymphoblasts, but there are leukemias of b-lymphocyte or null cell lineage. in the last two syndromes, the lymph nodes and spleen are often involved, but the thymus is generally normal. reticulum cell sarcomas are common in older mice, especially in inbred strains such as c bl/ and sjl. primary tumor cell types have been divided into several categories based on morphological and immunohistochemical features. histiocytic sarcomas correspond to the older dunn classification as type a sarcomas and are composed primarily of reticulum cells. the tumor typically causes splenomegaly and nodular lesions in other organs, including the liver, lung, kidney, and the female reproductive tract. follicular center cell lymphomas correspond to dunn type b sarcomas. they originate from b-cell regions (germinal centers) of peripheral lymphoid tissues, including the spleen, lymph nodes, and peyer's patches. typical tumor cells have large vesiculated, folded, or cleaved nuclei and ill-defined cytoplasmic borders. tumors also often contain small lymphocytes. type c reticulum cell tumors often involve one or several lymph nodes rather than assuming a wide distribution. they consist of reticulum cells with a prominent component of well-differentiated lymphocytes. myelogenous leukemia is uncommon in mice and is associated with retrovirus infection. disease begins in the spleen, resulting in marked splenomegaly, but leukemic spread results in involvement of many tissues including the liver, lung, and bone marrow. leukemic cells in various stages of differentiation can be found in peripheral blood. in older animals, affected organs may appear green because of myeloperoxidase activity, giving rise to the term chloroleukemia. the green hue fades on contact with air. affected mice are often clinically anemic and dyspneic. erythroleukemia is rare in mice. the major lesion is massive splenomegaly, which is accompanied by anemia and polycythemia. hepatomegaly can follow, but there is little change in the thymus or lymph nodes. erythroleukemia can be experimentally induced in mice by friend spleen focus-forming virus (sffv) which initially activates the erythropoietin (epo) receptor and the receptor tyrosine kinase sf-stk in erythroid cells, resulting in proliferation, differentiation, and survival. in a second stage, sffv activates the myeloid transcription factor pu. , blocking erythroid cell differentiation, and in conjunction with the loss of p tumor suppressor activity, results in the outgrowth of malignant cells (cmarik and ruscetti, ). mast cell tumors are also very rare in mice. they are found almost exclusively in old mice and grow slowly. they should not be confused with mast cell hyperplasia observed in the skin following painting with carcinogens or x-irradiation. natural plasma cell tumors are infrequent in the mouse. they can, however, be induced by intraperitoneal inoculation of granulomatogenic agents such as plastic filters, plastic shavings, or a variety of oils, particularly in balb/c mice. mineral oil-induced plasmacytomas in balb/c mice produce large amounts of endogenous retroelements such as ecotropic and polytropic murine leukemia virus and intracisternal a particles. associated inflammation may promote retroelement insertion into cancer genes, thereby promoting tumors (knittel et al., ) . similar to other spontaneous cancers, plasmacytoma development in mice is inhibited by innate immune responses of nk cells which when activated by viruses will release γinf (thirion et al., ) . mammary tumors can be induced or modulated by a variety of factors, including viruses, chemical carcinogens, radiation, hormones, genetic background, diet, and immune status. certain inbred strains of mice, such as c h, a, and dba/ , have a high natural prevalence of mammary tumors. other strains, such as balb/c, c bl, and akr, have a low prevalence. among the most important factors contributing to the development of mammary tumors are mammary tumor viruses. several major variants are known. the primary tumor virus mmtv-s (bittner virus) is highly oncogenic and is transmitted through the milk of nursing females. infected mice typically develop a precursor lesion, the hyperplastic alveolar nodule, which can be serially transplanted. spontaneous mammary tumors metastasize with high frequency, but this property is somewhat mouse strain dependent. metastases go primarily to the lung. some mammary tumors are hormone dependent, some are ovary dependent, and others are pregnancy dependent. ovary-dependent tumors contain estrogen and progesterone receptors, whereas pregnancy-dependent tumors have prolactin receptors. ovariectomy will dramatically reduce the incidence of mammary tumors in c h mice. if surgery is done in adult mice - months of age, mammary tumors will develop, but at a later age than normal. grossly, mammary tumors may occur anywhere in the mammary chain. they present as one or more firm, welldelineated masses, which are often lobular and maybe cystic (fig. . ) . histologically, mammary tumors have been categorized into three major groups: carcinomas, carcinomas with squamous cell differentiation, and carcinosarcomas. the carcinomas are divided into adenocarcinoma types a, b, c, y, l, and p. most tumors are type a or b. type a consists of adenomas, tubular carcinomas, and alveolar carcinomas. type b tumors have a variable pattern with both well-differentiated and poorly differentiated regions. they may consist of regular cords or sheets of cells or papillomatous areas. these two types are locally invasive and may metastasize to the lungs. type c tumors are rare and are characterized by multiple cysts lined by low cuboidal to squamous epithelial cells, and they have abundant stroma. type y tumors, which are also rare, are characterized by tubular branching of cuboidal epithelium and abundant stroma. adenocarcinomas with a lacelike morphology (types l and p) are hormone dependent and have a branching tubular structure. the control or prevention of mammary neoplasms depends on the fact that some strains of mammary tumor virus are transmitted horizontally, whereas others are transmitted vertically. although horizontally transmitted virus such as mmtv-s can be determined by cesarean rederivation or by foster nursing, endogenous strains of tumor virus may remain. fortunately, these latter tumor viruses have generally low oncogenicity relative to the bittner virus. mammary tumors are increased in frequency in c bl apc +/− female mice infected with h. hepaticus (rao et al., ) . mice develop an assortment of liver changes as they age, including proliferative lesions which can progress from hyperplastic foci to hepatomas to hepatocellular carcinomas. almost all strains of mice have a significant prevalence of hepatic tumors, some of which appear to result from dietary contamination or deficiency and h. hepaticus infections in susceptible strains of mice such as the a/jcr male mouse ward et al., ) . the prevalence of spontaneous liver tumors in b c f hybrids is increased by feeding choline-deficient diets or when infected with h. hepaticus (hailey et al., ) . tumors also can develop in mice exposed to environmental chemicals, many of which are carcinogenic or potentially carcinogenic (hoenerhoff et al., ) . spontaneous liver tumors in mice occur grossly as gray to tan nodules or large, poorly demarcated darkred masses. they are usually derived from hepatocytes, whereas cholangiocellular tumors are rare. hepatomas are well circumscribed and well differentiated, but they compress adjacent liver tissue as they develop. hepatocellular carcinomas are usually invasive and display histopathological patterns ranging from medullary to trabecular. large carcinomas also may contain hemorrhage and necrosis. carcinomas also may metastasize to the lungs. primary respiratory tumors of mice occur in relatively high frequency. it has been estimated that more than % of these tumors are pulmonary adenomas that arise either from type pneumocytes or from clara cells lining terminal bronchioles. pulmonary adenomas usually appear as distinct whitish nodules that are easily detected by examination of the lung surface. malignant alveologenic tumors are infrequent and consist of adenocarcinomas and squamous cell carcinomas. they invade pulmonary parenchyma and are prone to metastasize. the prevalence of spontaneous respiratory tumors is mouse straindependent. for example, the prevalence is high in aging a strain mice but low in aging c bl mice. the number of tumors per lung is also higher in susceptible mice. pulmonary tumors often occur as well-defined gray nodules. microscopically, adenomas of alveolar origin consist of dense ribbons of cuboidal to columnar cells with sparse stroma. adenomas of clara cell origin are usually associated with bronchioles. they have a tubular to papillary architecture consisting of columnar cells with basal nuclei. pulmonary adenocarcinomas, though comparatively rare, are locally invasive. they often form papillary structures and have considerable cellular pleomorphism. given the rapid development of mouse strains genetically predisposed to neoplasia, the mouse tumor biology database maintained by jackson laboratory is a valuable centralized resource for the most current tumor descriptions. the database contains information on more than strains and substrains, tissues and organs, over , tumor frequency records, and nearly histopathological images and descriptions. risk factors for recurrence, complications and mortality in clostridium difficile infection: a systematic review detection of mouse parvovirus in mus musculus gametes, embryos, and ovarian tissues by polymerase chain reaction interleukins, from to , and interferon-gamma: receptors, functions, and roles in diseases new generation humanized mice for virus research: comparative aspects and future prospects chemokine: receptor structure, interactions, and antagonism of mice, birds, and men: the mouse ultrasonic song system has some features similar to humans and song-learning birds intestinal inflammation targets cancerinducing activity of the microbiota the use of cross-foster rederivation to eliminate murine norovirus cas-offinder: a fast and versatile algorithm that searches for potential off-target sites of cas rnaguided endonucleases leaky virus: a new hantavirus isolated from mus musculus in the united states recombinant-inbred strains: an aid to finding identity, linkage, and function of histocompatibility and other genes experimental infection of inbred mouse strains with spironucleus muris open-and closed-formula laboratory animal diets and their importance to research the microbiology of transmissible murine colonic hyperplasia mouse hepatitis virus infection, intestine, mouse mouse hepatitis virus infection, liver, mouse murine rotavirus infection, intestine, mouse modification of early dimethylhydrazine carcinogenesis by colonic mucosal hyperplasia lymphocytic choriomeningitis virus dietary, bacterial, and host genetic interactions in the pathogenesis of transmissible murine colonic hyperplasia transmissible murine colonic hyperplasia epizootic coronaviral typhlocolitis in suckling mice infectivity, disease patterns, and serologic profiles of reovirus serotypes , , and in infant and weanling mice clindamycin-associated colitis due to a toxin-producing species of clostridium in hamsters fecal pcr assay for diagnosis of helicobacter infection in laboratory rodents s ribosomal dna sequence-based identification of bacteria in laboratory rodents: a practical approach in laboratory animal bacteriology diagnostics polyoma viruses persistence of polyomavirus in mice infected as adults differs from that observed in mice infected as newborns aerobic gram-positive organisms detection of rodent parvoviruses by pcr identification of novel murine parvovirus strains by epidemiological analysis of naturally infected mice temporal transmission studies of mouse parvovirus in balb/c and c.b- /icr-prkdc(scid) mice embryo transfer rederivation of c.b- /icr-prkdc(scid) mice experimentally infected with mouse parvovirus effect of vaccination on the clinical response, pathogenesis, and transmission of mousepox mousepox in inbred mice innately resistant or susceptible to lethal infeciton with ectromelia virus. iii. experimental transmission of infection and derivation of virus-free progeny from previously infected dams mousepox in inbred mice innately resistant or susceptible to lethal infection with ectromelia virus. i. clinical responses stability of ectromelia virus strain nih- under various laboratory conditions most classical mus musculus domesticus laboratory mice carry a mus musculus musculus y chromosome klebsiella oxytoca: opportunistic infections in laboratory rodents clustering of spatial gene expression patterns in the mouse brain and comparison with classical neuroanatomy serological studies of corynebacterium kutscheri and coryneform bacteria using an enzyme-linked immunosorbent assay (elisa) an enzymelinked immunosorbent assay (elisa) for monitoring rodent colonies for pasteurella pneumotropica antibodies helicobacter pullorum outbreak in c bl/ ntac and c h/hentac barrier-maintained mice mucosal immunity: induction, dissemination, and effector functions nk cell activation in human hantavirus infection explained by virus-induced il- /il ralpha expression exacerbation of pneumocystis carinii pneumonia in immunodefi-� cient (scid) mice by concurrent infection with a pneumovirus the sense of smell: multiple olfactory subsystems pheromonal communication in vertebrates immunological aspects of giardia muris and spironucleus muris infections in inbred and outbred strains of laboratory mice: a comparative study the natural history of mousepox reproduction whole-rat conditional gene knockout via genome editing pathogenesis of bacteremia due to pseudomonas aeruginosa in cyclophosphamide-treated mice and potentiation of virulence of endogenous streptococci genetics of dystrophic epicardial mineralization in dba/ mice sendai virus and pneumonia virus of mice (pvm) spontaneous reye'slike syndrome in balb/cbyj mice duration and patterns of transmission of theiler's mouse encephalomyelitis virus infection chromosomal locations and gonadal dependence of genes that mediate resistance to ectromelia (mousepox) virus-induced mortality pathogenesis of infection with a virulent allotropic variant of minute virus of mice and regulation by host genotype an exteroceptive block to pregnancy in the mouse comparison of polymerase chain reaction and immunohistochemistry for the detection of mycoplasma pulmonis in paraffin-embedded tissue chromosomal localization of the loci responsible for dystrophic cardiac calcinosis in dba/ mice evaluation of an enzymelinked immunosorbent assay for the detection of ectromelia (mousepox) antibody mousepox selected non-neoplastic diseases helicobacter-induced inflammatory bowel disease in il- -and t cell-deficient mice strategies to prevent, treat, and provoke corynebacteriumassociated hyperkeratosis in athymic nude mice corynebacterium bovis: epizootiologic features and environmental contamination in an enzootically infected rodent room inflammatory bowel disease: an immunity-mediated condition triggered by bacterial infection with helicobacter hepaticus poliomyelitis in mulv-infected icr-scid mice after injection of basement membrane matrix contaminated with lactate dehydrogenase-elevating virus resistance to mycoplasmal lung disease in mice is a complex genetic trait roles of innate and adaptive immunity in respiratory mycoplasmosis opportunistic infections of mice and rats: jacoby and lindsey revisited an outbreak in mice of salmonellosis caused by salmonella enteritidis serotype enteritidis detection of natural mycoplasma pulmonis infection in rats and mice by an enzyme linked immunosorbent assay (elisa) zinc-finger nucleases: the next generation emerges uber eine neue trypoanosomiasis des menschen from genes to social communication: molecular sensing by the vomeronasal organ two mouse retinal degenerations caused by missense mutations in the beta-subunit of rod cgmp phosphodiesterase gene a mouse model of clostridium difficileassociated disease the origins and uses of mouse outbred stocks evidence of human infection with a rat-associated hantavirus in helicobacter hepaticus infection triggers inflammatory bowel disease in t cell receptor alphabeta mutant mice experimental infection of mice with hamster parvovirus: evidence for interspecies transmission of mouse parvovirus the diversity outbred mouse population an experience with clostridium perfringens in cesarean derived barrier sustained mice hyperkeratosis in athymic nude mice caused by a coryneform bacterium: microbiology, transmission, clinical signs, and pathology friend spleen focus-forming virus activates the tyrosine kinase sf-stk and the transcription factor pu. to cause a multi-stage erythroleukemia in mice colonisation and shedding of lawsonia intracellularis in experimentally inoculated rodents and in wild rodents on pig farms a mouse for all reasons citrobacter rodentium: infection, inflammation and the microbiota prevention of murine norovirus infection in neonatal mice by fostering transmission of mouse parvovirus by fomites anatomy lactate dehydrogenase-elevating virus behavioral phenotyping strategies for mutant mice genetic heterogeneity of rat-derived pneumocystis serum antibody responses by male and female c bl/ mice infected with giardia muris comparison of the course of infection with giardia muris in male and female mice cytotoxic and pathogenic properties of klebsiella oxytoca isolated from laboratory animals the hidden cost of housing practices: using noninvasive imaging to quantify the metabolic demands of chronic cold stress of laboratory mice the role of klebsiella oxytoca in utero-ovarian infection of b c f mice behavioral effects of ivermectin in mice host-pathogen interaction in invasive salmonellosis sur les rapports des kystes de carini du poumon des rats avec le trypanosoma lewisi eradication of helicobacter spp. by using medicated diet in mice deficient in functional natural killer cells and complement factor d recombinat congenic strains -a new tool for analyzing genetic traits by more than one gene comparison between patterns of pinworm infection (aspiculuris tetraptera) in wild and laboratory strains of mice, mus musculus laboratory rat associated outbreak of haemorrhagic fever with renal syndrome due to hantaan-like virus in belgium animal leptospirosis in small tropical areas phylogeny of the defined murine microbiota: altered schaedler flora clostridium difficile infection in horses: a review mousepox outbreak in a laboratory mouse colony isolation of a puumala-like virus from mus musculus captured in yugoslavia and its association with severe hemorrhagic fever with renal syndrome assessment of rpob and s rrna genes as targets for pcrbased identification of pasteurella pneumotropica comparison of traditional and pcr methods during screening for and confirmation of aspiculuris tetraptera in a mouse facility pathogenicity and genetic variation of strains of corynebacterium bovis in immunodeficient mice use of fenbendazole-containing therapeutic diets for mice in experimental cancer therapy studies nutritional up-regulation of serotonin paradoxically induces compulsive behavior the pneumonia virus of mice (mpnv) model of acute respiratory infection comparison of nine commercially available clostridium difficile toxin detection assays, a real-time pcr assay for c. difficile tcdb, and a glutamate dehydrogenase detection assay to cytotoxin testing and cytotoxigenic culture methods dystrophic cardiac calcinosis in mice: genetic, hormonal, and dietary influences isolation and expression of the pneumocystis carinii dihydrofolate reductase gene the mouse genome database (mgd): comprehensive resource for genetics and genomics of the laboratory mouse typhlocolitis in nf-kappa b-deficient mice cd + cd + regulatory t lymphocytes inhibit microbially induced colon cancer in rag -deficient mice cd (+)cd (+) regulatory lymphocytes require interleukin to interrupt colon carcinogenesis in mice nitric oxide and tnf-alpha trigger colonic inflammation and carcinogenesis in helicobacter hepaticusinfected, rag -deficient mice ectromelia virus: the causative agent of mousepox intranasal inoculation of mycoplasma pulmonis in mice with severe combined immunodeficiency (scid) causes a wasting disease with grave arthritis the systemic amyloidoses: an overview inherited retinal degenerations in the mouse molecular detection of novel picornaviruses in chickens and turkeys fatal acute intestinal pseudoobstruction in mice the epizootic behaviour of mouse-pox (infectious ectromelia) the pathogenesis of acute exanthema. an interpretation based on experimental investigations with mouse-pox (infectious ectromelia of mice) studies in mousepox, infectious ectromelia of mice; a comparison of the virulence and infectivity of three strains of ectomelia virus studies in mousepox, infectious ectromelia of mice; the effect of age of the host upon the response to infection a juvenile mouse pheromone inhibits sexual behaviour through the vomeronasal system evidence from mtdna sequences that common laboratory strains of inbred mice are descended from a single female origins and characteristics of inbred strains of mice revised nomenclature for strain mice congenic strains treatment of syphacia obvelata in mice using ivermectin the nude mouse in experimental and clinical research opportunistic bacterial infections in breeding colonies of the nsg mouse strain outbreak of tropical rat mite dermatitis in laboratory personnel the role of helicobacter species in newly recognized gastrointestinal tract diseases of animals helicobacter hepaticus sp. nov., a microaerophilic bacterium isolated from livers and intestinal mucosal scrapings from mice chronic proliferative hepatitis in a/jcr mice associated with persistent helicobacter hepaticus infection: a model of helicobacterinduced carcinogenesis persistent hepatitis and enterocolitis in germfree mice infected with helicobacter hepaticus hepatic helicobacter species identified in bile and gallbladder tissue from chileans with chronic cholecystitis comparison of methods of identifying helicobacter hepaticus in b c f mice used in a carcinogenesis bioassay a novel urease-negative helicobacter species associated with colitis and typhlitis in il- -deficient mice inflammatory bowel disease in mouse models: role of gastrointestinal microbiota as proinflammatory modulators host and microbial constituents influence helicobacter pylori-induced cancer in a murine model of hypergastrinemia helicobacter hepaticus infection in mice: models for understanding lower bowel inflammation and cancer granulomatous peritonitis in interferon-gamma gene knockout mice naturally infected with mouse hepatitis virus microbial considerations in genetically engineered mouse research tyzzer's infection: host specificity of clostridium piliforme isolates enteric lesions in scid mice infected with 'helicobacter typhlonicus,' a novel urease-negative helicobacter species helicobacter typhlonius sp. nov., a novel murine urease-negative helicobacter species diagnosis of enteritis and enterotoxemia due to clostridium difficile in captive ostriches (struthio camelus) pneumocystis pneumonia, an immunodeficiencydependent disease (idd): a critical historical overview manual of microbiological monitoring of laboratory animals detection of antibodies to pneumocystis carinii by enzyme-linked immunosorbent assay in experimentally infected mice effect of fenbendazole on three behavioral tests in male c bl/ n mice zfn, talen, and crispr/cas-based methods for genome engineering animal transgenesis: an overview bacterial and mycotic diseases of the digestive system tyzzer's disease propagation of the etiologic agent of tyzzer's disease (bacillus piliformis) in cell culture. contribution of laboratory animal science to the welfare of man and animals: past, present, and future transgenic rna interference in mice genetic susceptibility to chronic hepatitis is inherited codominantly in helicobacter hepaticus-infected ab f and b af hybrid male mice, and progression to hepatocellular carcinoma is linked to hepatic expression of lipogenic genes and immune function-associated networks helicobacter hepaticus-induced liver tumor promotion is associated with increased serum bile acid and a persistent microbial-induced immune response the retinal degeneration (rd) gene seriously impairs spatial cognitive performance in normal and alzheimer's transgenic mice mycoplasma detection in cell cultures: a comparison of four methods barbering (fur and whisker trimming) by laboratory mice as a model of human trichotillomania and obsessive-compulsive spectrum disorders further evidence of host species-specific variation in antigens of pneumocystis carinii using the polymerase chain reaction pneumocystis carinii is not universally transmissable between mammalian species fungal diseases in laboratory mice neuropathologic findings associated with seizures in fvb mice molecular detection of murine norovirus from experimentally and spontaneously infected mice the distribution and kinetics of polyomavirus in lungs of intranasally infected neonatal mice spontaneous staphylococcus xylosus infection in mice deficient in nadph oxidase and comparison with other laboratory mouse strains genetics and probability in animal breeding experiments the major site of murine k papovavirus persistence and reactivation is the renal tubular epithelium isolation of streptococcus equisimilis from abscesses detected in specific pathogen-free mice ciliaassociated respiratory (car) bacillus infection of obese mice epigenetic and phenotypic changes result from a continuous pre and post natal dietary exposure to phytoestrogens in an experimental population of mice impact of helicobacter hepaticus infection in b c f mice from twelve national toxicology program two-year carcinogenesis studies pathology of aging b ; mice reduplication in mice humoral immunity and protection of mice challenged with homotypic or heterotypic parvovirus line- (l ) lineages in the mouse addgene provides an open forum for plasmid sharing quantitative trait loci in a bacterially induced model of inflammatory bowel disease a review of the molecular mechanisms of chemically induced neoplasia in rat and mouse models in national toxicology program bioassays and their relevance to human cancer control of pseudomonas aeruginosa infection in mice by chlorine treatment of drinking water mouse phyisology b cells modulate systemic responses to pneumocystis lung infection and protect on-demand hematopoiesis via t cell-independent, innate mechanism when type-i-ifn-signaling is absent development of a microsphere-based serologic multiplexed fluorescent immunoassay and a reverse transcriptase pcr assay to detect murine norovirus infection in mice persistent infection with and serologic cross-reactivity of three novel murine noroviruses multiplex fluorescent immunoassay for the simultaneous detection of serum antibodies to multiple rodent pathogens development and applications of crispr-cas for genome engineering genetic influence on response to dietary manganese deficiency in mice differential susceptibility to hepatic inflammation and proliferation in axb recombinant inbred mice chronically infected with helicobacter hepaticus parvoviruses mousepox in inbred mice innately resistant or susceptible to lethal infection with ectromelia virus. pathogenesis of vaccinia (ihd-t) virus infection in balb/cann mice evidence that nk cells and interferon are required for genetic resistance to infection with ectromelia virus sendai virus pneumonia in aged balb/c mice health care for research animals is essential and affordable inducible gene expression and gene modification in transgenic mice pcr testing of a ventilated caging system to detect murine fur mites amino acid requirements of the growing mouse monographs on pathology of laboratory animals talens: a widely applicable technology for targeted genome editing murine acariasis. ii. immunological dysfunction and evidence for chronic activation of th- lymphocytes mouse enu mutagenesis effects of enu dosage on mouse strains comparative murine norovirus studies reveal a lack of correlation between intestinal virus titers and enteric pathology tailored immune responses: novel effector helper t cell subsets in protective immunity stat -dependent innate immunity to a norwalk-like virus eradication of murine norovirus from a mouse barrier facility control of laboratory acquired hemorrhagic fever with renal syndrome (hfrs) in japan growth of tyzzer's organism in primary monolayer cultures of adult mouse hepatocytes the laboratory mouse. its origin, heredity, and culture olfactory regulation of the sexual behavior and reproductive physiology of the laboratory mouse: effects and neural mechanisms review of successful treatment for helicobacter species in laboratory mice on the maximum avoidance of inbreeding an oral ivermectin regimen that eradicates pinworms (syphacia spp.) in laboratory rats and mice nutrition effect of open and closed formula rations on the performance of three strains of laboratory mice insertional hypermutation in mineral oil-induced plasmacytomas chemical and cytokine features of innate immunity characterize serum and tissue profiles in inflammatory bowel disease gross anatomy the manufacture, shipping and receiving and quality control of rodent bedding materials emergence of clostridium difficile-associated disease in north america and europe helicobacter hepaticus-induced colitis in interleukin- -deficient mice: cytokine requirements for the induction and maintenance of intestinal inflammation bacteria-triggered cd (+) t regulatory cells suppress helicobacter hepaticus-induced colitis il- plays a key role in helicobacter hepaticus-induced t cell-dependent colitis paresis of peristalsis and ileus lead to death in lactating mice experimental spironucleosis (hexamitiasis) in the nude mouse as a model for immunologic and pharmacologic studies host specificity of giardia muris isolates from mouse and golden hamster genetic control of resistance to mycoplasma pulmonis infection in mice therapeutic effect of dna immunization of genetically susceptible mice infected with virulent mycoplasma pulmonis in vitro and in vivo susceptibility of mouse megakaryocytic progenitors to strain i of parvovirus minute virus of mice effects of fenbendazole on the murine humoral immune system from house mouse to mouse house: the behavioral biology of free-living mus musculus and its implications in the laboratory antibiotic treatment of clostridium difficile carrier mice triggers a supershedder state, spore-mediated transmission, and severe disease in immunocompromised hosts use of topical ivermectin treatment for syphacia obvelata in mice hantaan-like viruses from domestic rats captured in the united states obesity and non-insulin-dependent diabetes mellitus in swiss-webster mice associated with late-onset hepatocellular carcinoma mouse adenovirus type infection in scid mice: an experimental model for antiviral therapy of systemic adenovirus infections mineralization/anti-mineralization networks in the skin and vascular connective tissues a second class of olfactory chemosensory receptors in the olfactory epithelium genetic variables that influence phenotype mycoplasma and other bacterial diseases of the respiratory system pasteurella pneumotropica pseudomonas aeruginosa salmonella enteritidis staphylococcus aureus streptobacillus moniliformis streptococcus pneumoniae soiled bedding sentinels for the detection of fur mites in mice false negative results using rt-pcr for detection of lactate dehydrogenase-elevating virus in a tumor cell line eradication of pinworms (syphacia obvelata) from a large mouse breeding colony by combination oral anthelmintic therapy mousepox resulting from use of ectromelia virus-contaminated, imported mouse serum husbandry factors and the prevalence of age-related amyloidosis in mice cardioviruses: encephalomyocarditis virus and theiler's murine encephalitis virus diagnostic testing of mouse and rat colonies for infectious agents a novel presentation of clostridium piliforme infection (tyzzer's disease) in nude mice serodiagnosis of mice minute virus and mouse parvovirus infections in mice by enzyme-linked immunosorbent assay with baculovirus-expressed recombinant vp proteins hfrs outbreak associated with laboratory rats in uk the clinical chemistry of laboratory animals lack of commensal flora in helicobacter pylori-infected ins-gas mice reduces gastritis and delays intraepithelial neoplasia castration eliminates conspecific aggression in group-housed cd male surveillance mice toll-like receptor (tlr ) plays a major role in innate resistance in the lung against murine mycoplasma clostridium difficile-associated cecitis in guinea pigs exposed to penicillin clearance of pneumocystis carinii in mice is dependent on b cells but not on p-carinii-specific antibody phylogenetic relationships in the genus mus, based on paternally, maternally, and biparentally inherited characteristics genetic variants and strains of the laboratory mouse direct detection of indirect transmission of streptobacillus moniliformis rat bite fever infection fenner's veterinary virology failure to infect laboratory rodent hosts with human isolates of rodentolepis (=hymenolepis) nana detection and control of mouse parvovirus diminished reproduction, failure to thrive, and altered immunologic function in a colony of t-cell receptor transgenic mice: possible role of citrobacter rodentium helicobacter bilis infection accelerates and h. hepaticus infection delays the development of colitis in multiple drug resistance-deficient (mdr a −/−) mice helicobacter infection is required for inflammation and colon cancer in smad -deficient mice detection of giardia cysts by using the polymerase chain reaction and distinguishing live from dead cysts knockout mice: a paradigm shift in modern immunology a novel murine infection model for shiga toxin-producing escherichia coli infection-induced colitis in mice causes dynamic and tissue-specific changes in stress response and dna damage leading to colon cancer soiledbedding sentinel detection of murine norovirus infectious ectromelia. a hitherto undescribed virus disease of mice mycoplasma contamination of murine embryonic stem cells affects cell parameters, germline transmission and chimeric progeny clostridium perfringens and clostridium difficile-associated diarrhea pathology of the mouse from sexual attraction to maternal aggression: when pheromones change their behavioural significance an outbreak of pasteurella pneumotropica in genetically modified mice: treatment and elimination spontaneous necrotic enteritis in young rfm/ms mice prevention and treatment of ciliaassociated respiratory bacillus in mice by use of antibiotics a paralytic disease in nude mice associated with polyomavirus infection renewed interest in a difficult disease: clostridium difficile infections -epidemiology and current treatment strategies use of metronidazole in equine acute idiopathic toxaemic colitis identification and propogation of a putative immunosuppressive orphan parvovirus in cloned t cells corynebacterium species-associated keratoconjunctivitis in aged male c bl/ j mice genetic differences among c bl/ substrains isolation of helicobacter spp. from mice with rectal prolapses complex trait analysis in the mouse: the strengths, the limitations, and the promise yet to come fur mites induce dermatitis associated with ige hyperproduction in an inbred strain of mice, nc/kuj origins of inbred mice: proceedings of a workshop building a better mouse: one hundred years of genetics and biology retroelements in the mouse a mammalian herpesvirus cytolytic for cd + (l t +) t lymphocytes prairie dog model for antimicrobial agent-induced clostridium difficile diarrhea murine norovirus infection is associated with histopathological changes in immunocompetent hosts, but clinical disease is prevented by stat -dependent interferon responses transmission of systemic aa amyloidosis in animals infection of different strains of mice with lawsonia intracellularis derived from rabbit or porcine proliferative enteropathy the musculus-type y chromosome of the laboratory mouse is of asian origin lethal and severe coronary arteritis in dba/ mice induced by fungal pathogen, caws, candida albicans water-soluble fraction cre recombinase: the universal reagent for genome tailoring manual of microbiological monitoring of laboratory animals reduced fecundity and death associated with parvovirus infection in b-lymphocyte deficient mice phylogenetic analysis and description of eperythrozoon coccoides, proposal to transfer to the genus mycoplasma as mycoplasma coccoides comb. nov. and request for an opinion experimental analysis of risk factors for ulcerative dermatitis in mice citrobacter rodentium espb is necessary for signal transduction and for infection of laboratory mice colitis and colon cancer in waspdeficient mice require helicobacter species contamination of transplantable tumors, cell lines, and monoclonal antibodies with rodent viruses recommendations for the health monitoring of rodent and rabbit colonies in breeding and experimental units cellular expression of murine ym and ym , chitinase family proteins, as revealed by in situ hybridization and immunohistochemistry oxytocin is required for nursing but is not essential for parturition or reproductive behavior report of the american institute of nutrition ad hoc committee on standards for nutritional studies moving forward with chemical mutagenesis in the mouse implementation of a pcr assay of pasteurella pneumotropica to accurately screen for contaminated laboratory mice natural cryptosporidium muris infection of the stomach in laboratory mice enhancement of cognitive function in models of brain disease through environmental enrichment and physical activity handbook of vertebrate immunology perturbations in cytokine gene expression after inoculation of c bl/ mice with pasteurella pneumotropica a transgenic approach for rna interference-based genetic screening in mice mouse pathology of laboratory rodents and rabbits naturally occurring murine norovirus infection in a large research institution from bench to cageside: risk assessment for rodent pathogen contamination of cells and biologics reduced body growth and excessive incisor length in insertional mutants mapping to mouse chromosome congenital eye defects in the mouse. i. corneal opacity in c black mice murine adenovirus infection of scid mice induces hepatic lesions that resemble human reye syndrome improved lactation in germfree mice following changes in the amino acid and fat components of a chemically defined diet a systematic method of breeder rotation for noninbred laboratory animal colonies genetic variation and phylogeography of central asian and other house mouse mice, including a major new mitochondrial lineage in yemen reproductive biology of the laboratory mouse helminth parasites of laboratory mice contemporary prevalence of infectious agents in laboratory mice and rats clostridium species. veterinary microbiology and microbial disease uteroovarian infection in aged b c f mice breast cancer: should gastrointestinal bacteria be on our radar screen? detection of pneumocystis carinii in a rat model of infection by polymerase chain reaction spontaneous nonneoplastic gastric lesions in female han:nmri mice, and influence of food restriction throughout life emerging views on the distinct but related roles of the main and accessory olfactory systems in responsiveness to chemosensory signals in mice zfngenome: a comprehensive resource for locating zinc finger nuclease target sites in model organisms treatment and eradication of murine fur mites: iii. treatment of a large mouse colony with ivermectin-compounded feed pheromonal induction of spatial learning in mice mucispirillum schaedleri gen. nov., sp. nov., a spiral-shaped bacterium colonizing the mucus layer of the gastrointestinal tract of laboratory rodents pheromone sensing in mice minor histocompatibility antigens: from the laboratory to the clinic vitamin a and retinoic acid in t cell-related immunity spontaneous pneumocystis carinii pneumonia in immunodeficient mutant scid mice. natural history and pathobiology lethal exacerbation of pneumocystis carinii pneumonia in severe combined immunodeficiency mice after infection by pneumonia virus of mice primary structure and phylogenetic relationships of glyceraldehyde- -phosphate dehydrogenase genes of free-living and parasitic diplomonad flagellates duodenal adenomas in balb/-c mice monoinfected with clostridium perfringens diffuse scaling dermatitis in an athymic nude mouse clostridium difficile typhlitis associated with cecal mucosal hyperplasia in syrian hamsters zifit (zinc finger targeter): an updated zinc finger engineering tool spatial distribution and stability of the eight microbial species of the altered schaedler flora in the mouse gastrointestinal tract comparison of the in vitro susceptibility of rodent isolates of pseudomonas aeruginosa and pasteurella pneumotropica to enrofloxacin antibody production in syphacia obvelata infected mice hyperkeratosis-associated coryneform infection in severe combined immunodeficient mice gastrointestinal microflora host specificity of cloned spironucleus muris in laboratory rodents the eae gene of citrobacter freundii biotype is necessary for colonization in transmissible murine colonic hyperplasia genetic and biochemical characterization of citrobacter rodentium sp. nov outbreak of group b streptococcal meningoencephalitis in athymic mice demylination and wasting associated wigh polyomavirus infection in nude (nu/nu) mice severe leukopenia and dysregulated erythropoiesis in scid mice persistently infected with the parvovirus minute virus of mice assessment of retinal degeneration in outbred albino mice assessment of retinal degeneration in outbred albino mice identification of widespread helicobacter hepaticus infection in feces in commercial mouse colonies by culture and pcr assay helicobacter infection decreases reproductive performance of il -deficient mice pheromone binding by polymorphic mouse major urinary proteins role of housing modalities on management and surveillance strategies for adventitious agents of rodents murine cytomegalovirus and other herpesviruses cytolethal distending toxin promotes helicobacter cinaediassociated typhlocolitis in interleukin- -deficient mice manual of microbiological monitoring of laboratory animals helicobacter bilis-induced inflammatory bowel disease in scid mice with defined flora helicobacter bilis/helicobacter rodentium co-infection associated with diarrhea in a colony of scid mice genetically determined murine models of immunodeficiency macrophage plasticity and polarization: in vivo veritas atlas of the mouse brain and spinal cord mouse genetics: concepts and applications a natural outbreak of transmissible murine colonic hyperplasia in a/j mice genetic variation among mouse substrains and its importance for targeted mutagenesis in mice genetic dissection of complex traits with chromosome substitution strains of mice a conditional knock out resource for the genome-wide study of mouse gene function ivermectin toxicity in young mice response of weanling random-bred mice to inoculation with minute virus of mice explant cultures for detection of minute virus of mice in infected mouse tissue in vivo studies with an 'orphan' parvovirus microphthalmia and associated abnormalities in inbred black mice mouse adenoviruses innate lymphoid cells -a proposal for uniform nomenclature a new name (pneumocystis jiroveci) for pneumocystis from humans major histocompatibility complex (mhc): mouse. els mouse urinary peptides provide a molecular basis for genotype discrimination by nasal sensory neurons the complete genome sequence of the carcinogenic bacterium helicobacter hepaticus mouse relapse model of clostridium difficile infection dendritic cells are the major antigen presenting cells in inflammatory lesions of murine mycoplasma respiratory disease chronic ulcerative dermatitis in black mice generation of knockout mice using engineered nucleases spontaneous lesions in control b c f mice and recommended sectioning of male accessory sex organs acute, lethal, natural killer cell-resistant myeloproliferative disease induced by polyomavirus in severe combined immunodeficient mice the ancestor of extant japanese fancy mice contributed to the mosaic genomes of classical inbred strains pattern recognition receptors and inflammation aggregation chimeras: combining es cells, diploid and tetraploid embryos analysis of the immune response of hantaan virus nucleocapsid protein-specific cd + t cells in mice the pathogenesis of experimental infections of cryptosporidium muris (strain rn ) in outbred nude mice enterohepatic helicobacter species are prevalent in mice from commercial and academic institutions in asia, europe, and north america murine noroviruses comprising a single genogroup exhibit biological diversity despite limited sequence divergence antibiotic-induced shifts in the mouse gut microbiome and metabolome increase susceptibility to clostridium difficile infection effect of essential amino acid restriction on the growth of female c bl mice and their implanted bw adenocarcinomas fatal diarrhea in rabbits resulting from the feeding of antibiotic-contaminated feed modulation of the host microenvironment by a common non-oncolytic mouse virus leads to inhibition of plasmacytoma development through nk cell activation x-ray-induced mutations in mouse embryonic stem cells ten years of the collaborative cross geneology of the inbred strains: /svj is a contaminated inbred strain molecular biology, analysis, and enabling technologies: analysis of transgene integration nutrition toxicity evaluation of prophylactic treatments for mites and pinworms in mice serological and virological evidence of hantaan virus-related enzootic in the united states korean hemorrhagic fever in staff in an animal laboratory prolonged perturbations of tumour necrosis factoralpha and interferon-gamma in mice inoculated with clostridium piliforme acceleration and inhibition of puberty in female mice by pheromones spontaneous pseudopregnancy in mice male management: coping with aggression problems in male laboratory mice phylogeny of trichomonads based on partial sequences of large subunit rrna and on cladistic analysis of morphological data il- deficiency prevents development of a non-t cell non-b cell-mediated colitis provitamin a metabolism and functions in mammalian biology genetic variation in laboratory mice the mosaic structure of variation in the laboratory mouse genome a study of mouse strains susceptibility to bacillus piliformis (tyzzer's disease): the association of b-cell function and resistance a naturally occurring outbreak of mycobacterium avium-intracellulare infections in c bl/ n mice cecocolitis in immunodeficient mice associated with an enteroinvasive lactose negative e. coli pneumocystis carinii shows dna homology with the ustomycetous red yeast fungi kinetics of ectromelia virus (mousepox) transmission and clinical response in c bl/ j, balb,cbyj and akr inbred mice outbreaks of pneumocystis carinii pneumonia in colonies of immunodeficient mice draft genome sequences of the altered schaedler flora, a defined bacterial community from gnotobiotic mice pathology of genetically engineered mice chronic active hepatitis and associated liver tumors in mice caused by a persistent bacterial infection with a novel helicobacter species inflammatory large bowel disease in immunodeficient mice naturally infected with helicobacter hepaticus hyalinosis and ym /ym gene expression in the stomach and respiratory tract of s /svjae and wild-type and cyp a -null b , mice pathology of immunodeficient mice with naturally occurring murine norovirus infection reoviridae protozoa initial sequencing and comparative analysis of the mouse genome new building, old parasite: mesostigmatid mites -an ever-present threat to barrier facilities genetic diversity of pneumocystis carinii derived from infected rats, mice, ferrets, and cell cultures spontaneous wasting disease in nude mice associated with pneumocystis carinii infection respiratory disease and wasting in athymic mice infected with pneumonia virus of mice arthropods manual of microbiological monitoring of laboratory animals genetic and histochemical studies on mouse spleen black spots status and acces to the collaborative cross population helminths natural and experimental helicobacter infections monitoring sentinel mice for helicobacter hepaticus, h rodentium, and h bilis infection by use of polymerase chain reaction analysis and serologic testing rapid onset of ulcerative typhlocolitis in b . p -il tm cgn (il- −/−) mice infected with helicobacter trogontum is associated with decreased colonization by altered schaedler's flora estrus-inducing pheromone of male mice. transport by movement of air endonucleases: new tools to edit the mouse genome replication of a norovirus in cell culture reveals a tropism for dendritic cells and macrophages murine norovirus: a model system to study norovirus biology and pathogenesis addgene: the bank that gives points for (plasmid) deposits microbiological contamination of laboratory mice and rats in korea from streptobacillus moniliformis -a zoonotic pathogen. taxonomic considerations, host species, diagnosis, therapy, geographical distribution an enzyme-linked immunosorbent assay (elisa) for the detection of antibodies to pasteurella pneumotropica in murine colonies sgrnacas : a software package for designing crispr sgrna and evaluating potential off-target cleavage sites inhibition of tnf-alpha, and nf-kappab and jnk pathways accounts for the prophylactic action of the natural phenolic, allylpyrocatechol against indomethacin gastropathy adaptive immunity restricts replication of novel murine astroviruses chemical-induced atrial thrombosis in ntp rodent studies nucleic acid-sensing toll-like receptors are essential for the control of endogenous retrovirus viremia and erv-induced tumors recent applications of engineered animal antioxidant deficiency models in human nutrition and chronic disease dissecting the effects of mtdna variations on complex traits using mouse conplastic strains a genomic update on clostridial phylogeny: gram-negative spore formers and other misplaced clostridia effective eradication of pinworms (syphacia muris, syphacia obvelata and aspiculuris tetraptera) from a rodent breeding colony by oral anthelmintic therapy possible allelic structure of igg a and igg c in mice one-step generation of different immunodeficient mice with multiple gene modifications by crispr/cas mediated genome engineering key: cord- -g oes authors: nemzek, jean a.; lester, patrick a.; wolfe, a. marissa; dysko, robert c.; myers, daniel d. title: biology and diseases of dogs date: - - journal: laboratory animal medicine doi: . /b - - - - . - sha: doc_id: cord_uid: g oes historically, the dog played an important role as a laboratory animal in biomedical research. although numbers are declining, the use of dogs continues to be common in pharmacokinetics and cardiovascular studies. the normal biology of the dog as both a laboratory and a companion animal has been well studied and reference values are presented here as a clinical and experimental resource. this provides the necessary background to discuss the spontaneous diseases, including infectious and neoplastic conditions, prevalent in purpose bred as well as random source dogs used in biomedical research. in addition, diseases and conditions that arise secondary to the housing and experimental manipulation of dogs is discussed with emphasis on treatment and prevention. laboratory animal medicine mellitus. a comprehensive but concise review of the use of the dog as a research subject is available in gay ( ) . the breed of dog most commonly bred for use in biomedical research is the beagle. some commercial facilities also breed foxhounds or other larger dog breeds for use in surgical research studies. some specific breeds with congenital or spontaneous disorders have also been maintained by research institutions (see examples below). random-source dogs used in research are most frequently mongrels or larger dog breeds (e.g., german shepherd, doberman pinscher, labrador and golden retrievers) that are used for surgical research and/or training. according to a computerized literature search for "beagle" for the years - , a significant portion of the biomedical scientific publications identified were in the fields of pharmacology or toxicology. especially common were studies focusing on pharmacokinetics, alternative drug delivery systems, and cardiovascular pharmacology. other common areas of research using beagles were dental and periodontal disease and surgery, orthopedic surgery, skeletal physiology, and imaging studies. other research areas that utilized beagles included canine infectious disease, prostatic urology, and ophthalmology. most large-sized dogs (either purpose-bred or randomsource) are used in biomedical research because of their suitability for surgical procedures. anesthetic protocols and systems for dogs are well established and the organs of larger dog breeds are often an appropriate size for trials of potential pediatric surgical procedures. surgical canine models have been used extensively in cardiovascular, orthopedic, and transplantation research. there are also some unique spontaneous conditions for which dogs have proven to be valuable animal models. a colony of gray collies had been maintained at the university of washington (seattle) for the study of cyclic hematopoiesis. this condition is manifested by periodic fluctuations of the cellular components of the blood, most notably the neutrophil population. these dogs can be used to study the basic regulatory mechanisms involved with hematopoiesis, as well as possible treatments for both the human and the canine conditions (brabb et al., ) . golden retrievers affected with muscular dystrophy have been used as models of duchenne muscular dystrophy in human children. duchenne muscular dystrophy is caused by an absence of the muscle protein dystrophin, inherited in an x-linked recessive manner. the dystrophy in golden retrievers is caused by the absence of the same protein and is inherited in the same way. the clinical signs (such as debilitating limb contracture) are also similar between the canine and human conditions (kornegay et al., ) . other genetic disorders studied in dog colonies include hereditary canine spinal muscle atrophy (cork, ) and narcoplepsy in doberman pinschers (ripley et al., ) . bedlington terriers have been used to study copper storage diseases (such as wilson's disease) and the development of spontaneous diabetes mellitus and hypothyroidism has been studied in several breeds of dogs for comparisons with the human conditions. although historically the dog has been a common laboratory animal, their use in research has waned over the past years. according to the u.s. department of agriculture (usda), animal and plant health inspection service ( , ) , the number of dogs used in research has declined from , in to , in (prior to the previous edition of this text) and , in . this decrease was caused by a variety of factors, including (but not limited to) decreased availability, local restrictive regulations, conversion to other animal models (such as livestock or rodents), increased cost, and shift in scientific interest from pathophysiology to molecular biology and genetics. dogs used for research are generally segregated into two classes: purpose-bred and random-source. purposebred dogs are those produced specifically for use in biomedical research; they are intended for use in long-term research projects and/or pharmacologic studies in which illness or medication would require removal from the study. usually these dogs are either beagles or mongrel foxhounds, although other breeds may be available. purpose-bred dogs typically receive veterinary care throughout their stay at the breeding facility. they are usually vaccinated against rabies virus, canine distemper virus, parvovirus, adenovirus type , parainfluenza virus, leptospira serovars canicola, icterohaemorrhagiae, grippotyphosa, and pomona, and bordetella bronchiseptica (jasmin, personal communication). purpose-bred dogs are also usually treated prophylactically for intestinal helminths and ectoparasites, and possibly given a heartworm preventative. random-source dogs are not bred specifically for use in research. they may be dogs bred for another purpose (e.g., hunting and racing) or stray dogs collected at pounds or shelters. the health status of these dogs can be the same quality as purpose-bred dogs, or it can be an unknown entity. random-source dogs that have been treated and vaccinated in preparation for use in research are termed conditioned dogs. these dogs are then suitable for long-term studies or terminal preparations that require unperturbed physiologic parameters. conditioned dogs are often tested for heartworm antigen because of the implications that infestations can have on cardiovascular status and surgical risk. nonconditioned random-source dogs are useful only in a limited number of research studies, such as nonsurvival surgical training preparations and tissue/organ harvest. options for procurement of dogs for biomedical research typically include purchase from a usdadesignated class a or class b licensed dealer or directly from a municipal pound. the requirements for usda licensure are detailed in code of federal regulations (cfr), title , chapter ( - - edition), subchapter a, animal welfare, . definitions, and . requirements and application (office of the federal register, ) . briefly, class a licensees are breeders who raise all animals on their premises from a closed colony. class b licensees purchase the dogs from other individuals (including unadopted animals from municipal pounds) and resell them to research facilities. there are additional regulations that apply to class b dealers (such as holding periods and record-keeping documentation) because of the public concern that stolen pets could enter biomedical research facilities in this manner. in december , the national institutes of health (nih) issued notice not-od- - entitled notice regarding nih plan to transition from use of usda class b dogs to other legal sources (national institutes of health, ) . this nih policy begins in the fiscal year and prohibits the procurement of dogs from class b dealers using nih grant funds. from that point forward, dogs on nihfunded studies will have to be obtained from class a vendors, privately owned colonies (such as institutional breeding colonies), or client-owned animals (e.g., animals participating in veterinary clinical trials). the best resource for identification of possible vendors are online 'buyer's guide' sites or 'buyer's guide' issues of trade periodicals. online sites include the buyer's guide of the american association of laboratory animal science (http://laboratoryanimalbuyersguide.com), and the trade journals lab animal (http://guide.labanimal. com) and animal lab news (http://www.alnmag.com/ content/buyers-guide). a 'buyer's guide' typically lists sources for both purpose-bred and random-source dogs, and denotes such features as pathogen-free status, health status, and availability of specific breeds and timed pregnant females. some suppliers also have separate advertisements within issues of the journals. federal regulations promulgated by the animal and plant health inspection service, usda, in response to the animal welfare act ( cfr . , . , and . [g] ) are described in cfr chapter ( - - edition), subchapter a, animal welfare (office of the federal register, ) . regulations pertaining specifically to the care of dogs used in research are found in subpart a, specifications for the humane handling, care, treatment, and transportation of dogs and cats of part (standards) of subchapter a. particular attention should be paid to section . c (primary enclosures-additional requirements for dogs) because the space required for housing dogs is calculated using body length rather than weight (a parameter used for other species and also for dogs in the national research council (nrc) guidelines). section . (exercise for dogs) describes the requirements that dealers, exhibitors, and facilities must follow in order to provide dogs with sufficient exercise. the institute for laboratory animal research (ilar) has written the guide for the care and use of laboratory animals (national research council, ) . the 'guide' is the primary document used by institutional animal research units to develop their programs and by animal care evaluation groups, such as the association for assessment and accreditation of laboratory animal care international (aaalac international), to facilitate site visits and inspections. the primary difference between the th and th editions of the 'guide' (national research council, regarding the care of dogs is the notation that "enclosures that allow greater freedom of movement and unrestricted height (i.e., pens, runs, or kennels) are preferable." the ilar committee on dogs authored dogs: laboratory animal management (national research council, ) . this publication describes "features of housing, management, and care that are related to the expanded use of dogs as models of human diseases" and includes "an interpretive summary of the animal welfare regulations and the requirements of the public health service policy on humane care and use of laboratory animals." the reader is encouraged to use these publications to obtain further information on care and husbandry of dogs in the biomedical research setting. the information presented in the tables represents a range of normal values that can vary depending on the analytical method, as well as the age, breed, and sex of the animal. cohen, covance laboratories, inc., cumberland, va ( ) . physiological data for a mixed population of dogs of both sexes. fig. . demonstrates the normal weights and corresponding ages for both male and female beagle and hound dogs. tables . and . feature hematology data from beagles of both sexes from two commercial facilities. tables . and . list serum chemical data for beagles of both sexes from two commercial facilities. representative blood gas, coagulation data, and normal urinalysis parameters can be found in tables . - . , respectively. finally, the reviews in arterial and venous blood gas anaylses (rieser, ) and the manual of canine and feline cardiology (tilley et al., ) are excellent resources. good nutrition and a balanced diet are essential to the health, performance, and well-being of the animal. the nrc of the united states national academy of sciences is the leading provider of nutrient recommendations for dogs and provides average requirements needed to maintain growth and prevent deficiencies (subcommittee on dog and cat nutrition, ) . the nrc publications form the basis for the association of american feed control officials (aafco) nutrient profiles, which are updated periodically (baldwin et al., ) . the aafco is an advisory body comprising state representatives from across the united states. it provides a mechanism for developing and implementing uniform and equitable laws, regulations, standards, and enforcement policies, and establishes nutrient profiles for cat and dog foods (dzanis, ; thatcher et al., ) . additional resources should be consulted for details on the nutritional requirements for dogs of all ages (dzanis, ; subcommittee on dog and cat nutrition, ; baldwin et al., ; thatcher et al., ; hand et al., ) . recommendations for feeding the appropriate amount of diet are determined by the dog's metabolic requirements. the maintenance energy requirement (mer) is the amount of energy used by a moderately active adult animal in a thermoneutral environment. the mer for most breeds may be calculated using the following equation: mer (metabolizable kcal/day) = bw × . × kj de, where bw = body weight (kg), kj = kilojoules, de = digestable energy (kienzle and rainbird, ) . in-depth overviews of diets used in biomedical research are available in diet-specific literature. open-formula diets have defined concentrations of all ingredients and the information is publicly available. this allows researchers to control for this important environmental variable and enables retrospective analysis of possible diet composition effects on research results (barnard et al., ) . open-formula diets occasionally may require changes in formulation to maintain nutrient composition or meet changing nutrient requirements. these changes in quantitative ingredient formulation are made public when open-formula diets are modified. in contrast, closed-formula diets are commercially available, balanced diets that meet and label the minimum requirements for protein and fat and the maximum values for ash and fiber; however, the exact composition of ingredients may vary from batch to batch. ingredient composition varies as the manufacturer applies a leastcost strategy, referring to formulating diets to maximize profit by using the least-expensive ingredients. although the ingredients are listed, the quantitative ingredient formulation is not publicly available and can vary without public disclosure, due to proprietary nature of commercial diets produced and marketed under vendor trade names. closed-formula diets have also been referred to as as 'fixed formula' or 'constant nutrition' (labdiets, pmi nutrition international, st. louis, missouri) by manufacturers (barnard et al., ) . in fixed-formula diets, the quantitative ingredient formulation does not change; however, this information is proprietary and therefore laboratory animal medicine not disclosed publically (barnard et al., ) . semipurified and purified diets provide the strictest control of ingredients and are formulated from purified components: amino acids, lipids, carbohydrates, vitamins, and minerals. although purified and semipurified diets do differ in the types of ingredients used, the terms are generally used to mean the same thing. purified-ingredient diets are generally 'open' formulas, meaning that they are published and available to the scientific community. the animal care provider should be aware of the manufacture date of the diet, which should be clearly visible on the bag. as a general rule, diets are safe for consumption up to months following the manufacture date when stored at room temperature. refrigeration may prolong the shelf-life, but the best strategy is to feed only fresh diets and use each lot based on the date of manufacture. specifications for feeding and watering of dogs are provided in the regulations of the animal welfare act. management of a breeding colony requires broad knowledge of the dog's anatomy, reproductive physiology, and behavioral needs during breeding, gestation, and parturition. although a comprehensive discussion of the biology of canine reproduction is beyond the scope of this chapter, essential features of the broad topics noted above are presented. from dr. asheley wathen, covance laboratories, inc., madison, wi, and dr. kimberley cohen, covance laboratories, inc., cumberland, va ( ) . overall health, body condition, nutrition, and age greatly influence reproductive efficiency (gavrilovic et al., ; johnson, ) . therefore, only normal, healthy animals in excellent body condition should be used in breeding programs. beagles between and . years of age have the best conception rates and litter size with the lowest neonatal mortality. after years of age, conception rates and litter size decline and neonatal mortality increases (johnson, ) . the vagina is a long, musculomembranous canal that extends from the uterus to the vulva. during physical examination, the gloved finger or examination instrument should be introduced through the dorsal commissure of the vulva, avoiding the deep ventral clitoral fossa. examination should proceed at an angle of approximately ° until the instrument or fingertip has passed over the ischial arch, after which it can be directed further craniad toward the cervix. the uterus consists of the cervix, uterine body, and uterine horns. the cervix is an abdominal organ, located approximately halfway between the ovaries and the vulva. when the bitch is in proestrus and estrus, the cervix can be distinguished during abdominal palpation as an enlarged, turgid, walnut-shaped structure. female dogs are monoestrous, typically nonseasonal, spontaneous ovulators that have a spontaneous luteal phase approximately days longer than the ± days of pregnancy followed by obligate anestrus. puberty (beginning of the first estrus) occurs between and months in most breeds. the time of onset positively correlates with the body size (concannon, ) . the canine cycle is divided into four phases: proestrus, estrus, diestrus, and anestrus. the duration of laboratory animal medicine proestrus is - days with an average of days and reflects the follicular phase rise in estrogen. during this stage, the vulva is enlarged and turgid, and a serosanguinous vaginal discharge is present (concannon, ) . estrus may be from to days in duration but generally lasts days. the endocrine feature of estrus is the first abrupt increase in progesterone (> ng/ml), which occurs concomitantly with the luteinizing hormone (lh) surge % of the time, followed by ovulation within - h. the vulva is softer and smaller than in proestrus. the vaginal discharge persists and may remain serosanquinous or become straw colored. diestrus begins approximately days after the onset of standing heat. the end of this stage is days later, which would be coincident with whelping if the bitch had become pregnant. defined behaviorally as starting when estrous behavior ceases (concannon, ) , diestrus represents the peak of serum progesterone. anestrous may last from to days and is the stage of reproductive quiescence. it is characterized by an absence of ovarian activity and serum progesterone levels of less than ng/ml. the onset of puberty in the male ranges from to months of age and is affected by breed, season, nutrition, and disease status. this process is initiated by the secretion of lh from the anterior pituitary, which stimulates the production of testosterone by the interstitial or leydig's cells. at this time, the testicular growth is rapid, the seminiferous tubules begin to differentiate, and sertoli cells form the blood-testis barrier. secretion of follicle-stimulating hormone (fsh) by the anterior pituitary stimulates the production of other key hormones by the sertoli cells, including, inhibin, androgen binding protein, and estrogen. fsh stimulates spermatogenesis in the presence of testosterone, whereas inhibin and estrogen provide negative feedback to the pituitary gland to decrease fsh production. spermatogenesis in the dog is completed in days, with subsequent maturation of sperm occurring in the epididymis for approximately days. thus, the entire process from the initiation of spermatogonial mitosis to the delivery of mature sperm to the ejaculate is days. a breeding soundness exam should be conducted to assess the probability of a male dog's successful production of offspring. factors affecting male fertility include libido, ability to copulate, testicular size, and quality semen production. supression of sexual behavior and problems with libido may occur in dogs due to early weaning, isolation, or inherited abnormalities. animals with poor hind limb conformation or trauma to the back may be unable to properly mount the female. there is a positive correlation between scrotal circumference and the number of sperm produced. finally, the quality of sperm is assessed by motility, morphology, volume, and concentration. an ejaculate ( ml) that contains approximately million progressively motile sperm without significant morphological abnormalities is a good indicator of normal male fertility. complete anatomy of the bitch and dog can be found in miller's anatomy of the dog (evans and de lahunta, ) . cells of the vaginal epithelium mature to keratinized squamous epithelium under the influence of estrogen. because of the rise in estrogen throughout proestrus, with peak levels occurring just prior to the onset of standing heat, the vaginal smear can be used as an indicator of the bitch's readiness for breeding. the smear will not confirm the presence of ovulation nor is it of prognostic value in normal bitches during anestrus. the percentage of vaginal epithelial cell cornification is an index of estrogen secretion by the ovarian follicles. cornification occurs approximately days prior to the estrogen peak and days prior to standing heat. as cornification of vaginal epithelial cells proceeds, the cells become larger, with more angular borders. the nuclear/ cytoplasmic ratio decreases until the nuclei reach a point where they no longer take up stain (coincident with the onset of estrus). the cells appear 'anuclear' and are classified as 'cornified' or 'anuclear squames.' the vaginal cytology smear of the bitch changes from predominantly cornified to noncornified days after ovulation. the day of this change is the first day of diestrus. other epithelial cell types noted on vaginal cytology include superficial cells (large, angular cells with small nuclei); intermediate cells (round or oval cells with abundant cytoplasm and large, vesicular nuclei); and parabasal cells (small round or elongated cells with large, well-stained nuclei, and a high nuclear/cytoplasmic ratio). based on vaginal cytology, the estrous cycle is classified as follows: although vaginal cytology is a useful tool, observation of behavioral estrus is the best criterion to use in breeding management. during proestrus, the male is attracted to the bitch and will investigate her hindquarters, but she laboratory animal medicine will not accept breeding. estrus is characterized by proactive receptivity to mounting by males and increased male-seeking behavior (concannon, ) . during this stage, the bitch will exhibit 'flagging,' or elevation of her tail with muscular elevation of the vulva to facilitate penetration by the male. in order to maximize the conception rate and litter size, it is recommended to breed the bitch on days , , and of the standing heat. due to the long life span of canine sperm, fertilization occurs in the oviduct up to days after coitus. the ovulated oocyte is a primary oocyte that must undergo two meiotic divisions before fertilization can occur. this overall maturation process takes approximately days. after maturation, the oocyte remains viable for - days. optimal conception rates tend to occur when the bitch is bred from days before to days after ovulation; best litter size is achieved when the bitch is bred days after ovulation. implantation is evident by areas of local endometrial edema - days after breeding. there is no correlation between the number of corpora lutea and the number of fetuses in the corresponding uterine horn, suggesting transuterine migration of embryos. the dog has endotheliochorial placentation. the endothelium of uterine vessels lies adjacent to the fetal chorion, mesenchymal, and endothelial tissues, so that maternal and fetal blood are separated by four layers. the canine placenta is also classified as zonary, indicating the placental villi are arranged in a belt, and deciduate, reflecting that maternal decidual cells are shed with fetal placentas at parturition. the length of gestation is - days. luteal progesterone is responsible for maintaining pregnancy and canine corpora lutea retain their structural development throughout gestation. serum progesterone rises from less than ng/ml in late proestrus to a peak of - ng/ml during gestation, and then declines to - ng/ml just prior to parturition. progesterone is essential for endometrial gland growth, secretion of uterine milk, attachment of the placentas, and inhibition of uterine motility (johnson, ; verstegen-onclin and verstegen, ) . pregnancy detection can be performed by several methods. abdominal palpation of the uterus may be most informative at approximately days after breeding. the embryos and chorioallantoic vesicles form a series of ovoid swellings and are approximately inches in length at - days. by day , the uterus begins to enlarge diffusely and the vesicles become difficult to identify by palpation. radiology can be used to confirm pregnancy and facilitate determination of gestational age, beginning days after the lh surge (lopate, ) . bitches in which a difficult whelping is anticipated should be radiographed in late pregnancy to determine the litter size and to evaluate the size of the fetal skulls in relation to the bony maternal birth canal. ultrasonography can be used to confirm pregnancy beginning on days - , at which point the gestational sacs will be approximately cm in diameter, and until parturition (shille and gontarek, ; lopate, ) . ultrasonography can assess fetal viability by visualizing fetal heartbeats and fetal movement beginning on gestational days - and , respectively (lopate, ) . it can also predict gestational age using the inner diameter of the chorionic cavity in early pregnancy and the biparietal diameter in late pregnancy luvoni and beccaglia, ) . however, ultrasonography for determination of gestational age is most accurate at day of pregnancy when using correction factors for small (< kg) and large (> kg) body weight dogs (kutzler et al., ) . thermal support should be provided prior to parturition. dogs housed on grated flooring should be provided with mats and those on solid floors would benefit from blankets placed in a corner of the primary enclosure. shavings are discouraged because they may adhere to the umbilical cord and predispose to ascending infections. heat lamps may be placed h prior to parturition and remain until all neonates demonstrate vigorous suckling behavior. however, the use of heat lamps necessitates strict supervision in order to prevent thermal burns. if possible, whelping bitches should be housed in a quiet corridor in order to decrease periparturient stress, especially in primiparous or young mothers. monitoring of parturition is important, but human intervention should be minimal in order to prevent stressinduced cannibalism. an abrupt drop in body temperature to less than °f indicates impending parturition within - h. the process of parturition has been divided into three stages. stage of labor lasts - h and is characterized by uterine contractions and cervical dilation. during this stage, the bitch may appear restless, nervous, and anorexic. other common clinical signs include panting and increased pulse rate (johnson, ) . fetal expulsion occurs during stage , which lasts approximately - h. as the fetus engages the cervix, there is release of oxytocin, referred to as the ferguson reflex, which strengthens the uterine contractions and may elicit abdominal contractions as well. the bitch is able to inhibit this stage of labor if disturbed. the chorioallantois ruptures either during passage of each neonate through the birth canal or by the bitch's teeth at birth. interestingly, posterior presentation is common in dogs but does not predispose to dystocia. the time interval between deliveries of each pup is irregular, but the average is less than h between pups. veterinary assistance laboratory animal medicine is necessary if the bitch remains in stage for more than h without delivering the first pup, or for more than h before delivering subsequent pups. during stage of labor, the placentas are expelled either immediately or within min of delivery of each pup. if two pups are delivered from alternate uterine horns, then the birth of both puppies may precede expulsion of the respective placentas. the bitch will lick the newborn vigorously to remove the membranes from its head and to promote respiration. she will also sever the umbilical cord. the bitch may ingest the placentas, although they confer no known nutritional benefit and may induce a transient diarrhea. the peripartum use of oxytocin is required only in the event of uterine inertia, stillbirths, or agalactia. oxytocin should not be used in the event of systemic illness or abnormalities precluding vaginal delivery. indications for its use include lack of delivery h after onset of stage labor, greater than h of unproductive stage labor, inadequate contractions, or abnormal vaginal discharge. in these cases, radiographs are recommended to assess fetal size in relation to the birth canal and any possible obstructions, followed by . - . iu of oxytocin intramuscularly or subcutaneously. the oxytocin can be repeated - min after the first dose for a total of two doses (plunkett, ) . in some cases, treatment with . - . ml/kg of % calcium gluconate, delivered slowly iv while monitoring closely for bradycardia, and % dextrose iv may be indicated. uterine involution occurs during anestrus within - weeks of parturition. during this time, a greenish to red-brown vaginal discharge, or lochia, is considered normal. the presence of an odiferous, purulent discharge, accompanied by systemic signs of illness, indicates metritis or pyometra. desquamation of the endometrium begins by the th postpartum week, with complete repair by months. newborn puppies are easily sexed by examination of the anogenital distance. in female puppies, the vulva is evident a short distance from the anus, whereas the prepuce of male puppies is nearly adjacent to the umbilicus. eyes are open at approximately days, and ears are patent at approximately - days. solid food can be introduced between . and weeks of age, and puppies can be weaned at - weeks. artificial insemination (ai) is indicated when the male is physically incapable of mounting or penetrating the bitch, when there are vaginal abnormalities such as a vaginal-vestibular stricture, narrow vagina, vaginal septum, and vaginal hyperplasia, or if there is a behavioral incompatibility between the male and female dogs (kutzler, ) . semen is collected using a plastic centrifuge tube and rubber latex artificial vagina. the male is introduced to the scent of an estrous bitch and manually stimulated. the first two fractions are collected followed by a sufficient amount of the third fraction (predominantly of prostatic fluid) to bring the total semen volume to - ml. the semen can be introduced into the cranial vagina or directly into the uterus either through trans-cervical catheterization with a norwegian ai catheter or utilizing fiberoptic endoscopy. use of the norwegian ai catheter for intrauterine insemination of frozen-thawed, fresh, and chilled-extended semen results in significantly higher whelping rates than intravaginal insemination (linde-forsberg et al., ; thomassen and farstad, ) . for trans-cervical insemination, the bitch is either standing on all four legs or standing with hindquarters raised. the ai catheter and guiding tube are inserted into the vestibulum as far as the pseudocervix. firm abdominal palpation is then used to locate and fix the cervix in the other hand, at which point the catheter is further inserted along the dorsal vaginal fold until the cervical opening is located and semen is deposited into the uterus lumen (thomassen and farstad, ) . surgical and laparoscopic ai has been used successfully for intrauterine and intratubal insemination; however, these techniques are invasive and require anesthesia. therefore, the nonsurgical techniques mentioned above are recommended, as these approaches are less invasive and can be completed without anesthesia in nonsedated or sedated dogs depending on the experience of the personnel and personality of the dogs. ai with freshly collected sperm can be done on days , , and of standing heat or on days of maximal vaginal cornification. the viability of frozen-thawed sperm is significantly reduced compared to fresh or chilled sperm that may live up to or days in the reproductive tract of the bitch; frozen-thawed sperm live only a few hours. therefore, the ova must be mature and insemination with frozen-thawed semen must be done - days after ovulation in the bitch as determined by serum progesterone concentrations (thomassen et al., ) . false pregnancy (pseudocyesis), a stage of mammary gland development and lactation associated with nesting or mothering behavior, is common in the bitch. the condition occurs after the decline in serum progesterone toward the end of diestrus. there is no age or breed predisposition. pseudopregnancy does not predispose the bitch to reproductive disease or infertility. a comprehensive review of canine pseudocyesis exploring its cause, clinical features, and treatments is covered by c. gobello (gobello et al., ) . reproductive performance in the bitch is optimal prior to years of age. cycling does not completely cease; however, after - years of age, bitches demonstrate significant decreases in conception rate and the number of live pups whelped. by - years of age, pathologic conditions of the uterus, such as cysts, hyperplasia, atrophy, and neoplasia, are extremely common. dogs prefer living in a social environment. dogs have well developed olfactory glands, vision, and auditory and tactile senses that allow them to gain environmental cues and information from other dogs and humans (field and jackson, ; joint working group on refinement, ) . much of their instinctive behavior is dependent on learning to interact with other members of their species. beagles have been a popular animal model because of their docile nature. they are easily handled and, for the most part, respond favorably to repetitive manipulations such as body weight measurements, physical examination, electrocardiograms (ecgs), oral gavage, and venipuncture. although sexually mature by - months of age, dogs are not socially mature until - months of age. the socialization process should begin early during development, when puppies are receptive to conspecific and human contact. for example, from to weeks of age, puppies are most capable of learning about how to interact with other dogs. between weeks and , puppies are most capable of learning how to interact with people. by - weeks of age, dogs voluntarily wander and explore new environments. thus, early handling and mild stress (such as vaccination) appear to be extremely beneficial components of a dog's social exposure. canid social systems use signals and displays that minimize the probability of outright aggression. these behavior patterns are most likely elicited during distressful situations, such as strange environments, being handled by strange people, or encountering new animals. an excellent, illustrated discussion of normal canine behavior patterns can be found in the canine behavior section of the manual of clinical behavioral medicine for dogs and cats (overall, ) . by virtue of the dog's status as a companion animal, there are many veterinary publications and reference texts on the diagnosis, medical management, pathology, and epidemiology of its disorders. the authors of this chapter have chosen to emphasize those diseases that are more frequently encountered in the research setting, especially infectious diseases associated with the use of random-source dogs and conditions seen frequently in the beagle. for more thorough and detailed discussion of these diseases, as well as those not discussed in this chapter, the reader should consult standard veterinary textbooks. etiology canine infectious respiratory disease (cird) is a highly contagious illness and several organisms have been incriminated including bordetella bronchiseptica; streptococcus equi subsp. zooepidemicus; canine parainfluenza virus (cpiv); canine influenza virus (civ); canine respiratory coronavirus; canine adenovirus type (cav- ); canine herpesvirus; canine reovirus types , , and ; and mycoplasma and ureaplasma. naturally occuring infection can result in coinfection by two or more organisms (garnett et al., ; ford, ) . clinical signs cird can be subdivided into mild or severe forms. the mild form is more common and is characterized by an acute onset of a loud, dry, hacking cough. increased formation of mucus sometimes results in a productive cough, followed by gagging or retching motions. cough may be elicited by tracheal palpation and may be more frequent with excitement or exercise. otherwise, dogs are typically asymptomatic. mild tracheobronchitis usually lasts - days, even when untreated. the severe form results from poor general health, immunosuppression, or lack of vaccination. secondary bronchopneumonia can occur and can be the determinant of severity (sherding, ) . animals are clinically ill and may be febrile, anorexic, and depressed. productive cough and mucopurulent naso-ocular discharge are more common than in the mild form. epizootiology and transmission the natural reservoir for b. bronchiseptica is the respiratory tract (bemis, ) , and it is very easily spread by aerosol and direct contact. transmission is heightened by confined housing of multiple animals. bordetella bronchiseptica is highly infectious with an incubation period of - days. pathogenesis the most common clinical isolates are cpiv and b. bronchiseptica (mochizuki et al., ) . however, b. bronchiseptica is often recovered from clinically healthy animals (chalker et al., ) . during clinical infection, b. bronchiseptica attaches to the cilia of the upper airway epithelium, causing suppurative tracheobronchitis and bronchiolitis. infections with cpiv laboratory animal medicine or cav- alone are usually subclinical but can cause necrotizing tracheobronchiolitis (dungworth, ) . diagnosis and differential diagnosis diagnosis is often based on clinical signs and known history; however, cough elicited by tracheal palpation may be inconsistent and should not be used for definitive diagnosis. presumptive diagnosis can be made by isolation of b. bronchiseptica or mycoplasma by nasal swabs. viral isolation or paired serology is often impractical and expensive. if cough persists for more than days, other disease conditions should be considered. differential diagnoses include civ, canine distemper virus, pneumonia, heartworm disease, tracheal collapse, mycotic infections, and diseases resulting in tracheal compression (johnson, ) . prevention prevention is best achieved by avoiding exposure to infected animals. dogs should be vaccinated prior to or upon admission to the animal facility. intranasal vaccines protect against infection and disease and can be given to dogs as young as weeks of age (greene and levy, ) . combination vaccines for b. bronchiseptica, cav- , and cpiv are preferred. vaccinations should be boostered every months when multiple animals are housed in a confined area. control staff must practice proper hygiene to prevent transmission by fomites. sanitation, proper ventilation, and proper humidity are critical for control. symptomatic animals should be isolated and kennels should be disinfected with agents such as bleach, chlorhexidine, or quaternary ammonium chloride. treatment bordetella bronchiseptica is sensitive to potentiated sulfas, chloramphenicol, quinolones, tetracyclines, gentamicin, and kanamycin. use of antibiotics is indicated when severe or persistent clinical signs occur and should be continued for days. for severe or unresponsive infection, treatment should be based on bacterial culture/sensitivity patterns. nebulized gentamicin or kanamycin may be helpful in severe cases. antitussives should be avoided if the cough is productive; however, their use is indicated if coughing is causing discomfort or interfering with sleep. bronchodilators such as aminophylline, theophylline, or terbutaline can be helpful in reducing reflex bronchoconstriction. research complications due to the altered respiratory tract physiology, infected animals should not be used for pulmonary studies. etiology β-hemolytic lancefield's group c streptococcus (s. equi ssp. zooepidemicus) is a gram-positive, non-spore-forming coccus that causes pneumonia and sepsis in dogs. clinical signs clinical signs vary based on the organ system affected. pneumonic disease is typically associated with sudden onset of clinical signs including coughing, weakness, fever, dyspnea, and hematemesis. the rapid progression of disease is similar to that seen in humans with toxic shock syndrome (tss) caused by streptococcus pyogenes. peracute death has been reported in research and shelter dogs (bergdall et al., ; pesavento et al., ) . epizootiology and transmission streptococcus equi ssp. zooepidemicus is not considered a commensal of healthy dogs as most of the β-hemolytic commensal organisms belong to group g, specifically streptococcus canis. asymptomatic carriers are suspected to be the route by which infection enters populations. streptococcus equi ssp. zooepidemicus is considered an opportunistic pathogen and stressful factors such as transport can predispose to disease (priestnall et al., ) . pathologic findings in peracute cases, hemorrhage from the mouth and nose and within the pleural cavity can be the most striking lesion. ecchymotic and petechial hemorrhages can be noted on several organs ( fig. . ). 'bull's-eye' lesions may be observed on the pleural surface of affected lung lobes. histologic lesions can include fibrino-suppurative, necrotizing, and hemorrhagic pneumonia. gram-positive cocci can be found in intracellular clusters throughout the lung ( fig. . ), tonsils, and spleen of affected animals (bergdall et al., ; priestnall and erles, ) . pathogenesis predisposing factors such as transport stress and viral coinfection have been shown to contribute to the virulence of s. zooepidemicus (priestnall and erles, ) . due to the similarities with the clinical signs seen in human cases of tss, superantigens are thought to contribute to the virulence of s. zooepidemicus in cases of acute hemorrhagic pneumonia. these superantigens work by bypassing the conventional mechanisms of antigen presentation and binding to major histocompatibilitity complex class ii receptors. as a result, there is a hyperactive proinflammatory response and an 'avalanche' of cytokines including interleukin β (il- β), interleukin (il- ), and tumor necrosis factor alpha (tnf-α). three novel superantigen-encoding genes have been identified from a case of acute fatal hemorrhagic pneumonia, szef, szen, and szep. however, it is currently unclear what effect these superantigens have in vivo priestnall et al., ) . while superantigens have been detected in some isolates, there is not enough data to determine if superantigens play a role in the pathogenesis (byun et al., ; kim et al., ) . diagnosis and differential diagnosis definitive diagnosis is based on bacterial culture of nasal swabs or transtracheal lavage. polymerase chain reaction (pcr) can be done on post-mortem lung tissue. bacterial pneumonias or bacteremias can be caused by other pathogenic streptococcus spp., staphylococcus spp., escherichia coli, pasteurella multocida, pseudomonas spp., klebsiella pneumoniae, and b. bronchiseptica. nonbacterial causes of respiratory disease include rodenticide intoxication, coagulopathies, heartworm disease, pulmonary thromboembolism, ruptured aneurysm, and left-sided congestive heart failure. prevention and control there is no vaccine for prevention of s. zooepidemicus. the organism has been isolated from the environment during active outbreaks (pesavento et al., ) , so dogs diagnosed with s. zooepidemicus should be quarantined and any potential fomites (e.g. food bowls, enrichment) should be properly disinfected. treatment antibiotic therapy should be based on culture and sensitivity. resistance to doxycycline and tetracycline has been demonstrated (garnett et al., ; pesavento et al., ) . research complications dogs with severe hemorrhagic pneumonia or systemic disease are not appropriate for research study. the association between epizootics of this disease and transportation supports operational policies that require adequate acclimation periods for animals upon arrival. etiology serovars canicola, bratislava, and grippotyphosa result in renal or hepatic disease, whereas serovars icterohaemorrhagiae and pomona predominantly result in hepatic disease . clinical signs canine leptospirosis can present as subclinical, acute, or chronic disease. clinical signs in acute infection can be nonspecific and include lethargy, depression, abdominal discomfort, stiffness, anorexia, vomiting, muscle tenderness, and pyrexia. clinical signs can be related to renal failure including polyuria and polydipsia, with or without azotemia, oliguria, or anuria. leptospirosis can also lead to hepatic failure with signs such as icterus or bleeding abnormalities. peracute leptospirosis is characterized by shock, vascular collapse, and rapid death. uveitis, abortions, stillbirths, and pulmonary hemorrhage have also been associated with leptospirosis (klopfleisch et al., ; van de maele et al., ) . bivalent vaccines against the most common canine serovars, icterohaemorrhagiae and canicola, have resulted in the increased prevalence of other serovars including grippotyphosa, pomona, bratislava, and autumnalis. increased movement of wild animal reservoirs (rats, raccoons, skunks, opossums) into urban/suburban areas have also contributed to the greater prevalence of previously uncommon serovars (sykes et al., ) . transmission occurs primarily through environmental contact, although direct transmisson between hosts may also occur. leptospires passing from urine into water is the most common route of contamination (goldstein, ) . leptospirosis is a zoonotic disease. pathologic findings the kidneys consistently have gross and microscopic lesions. in the acute phase, the kidneys are swollen with subcapsular and cortical ecchymotic hemorrhages. petechial or ecchymotic hemorrhages and swelling of the lungs may also be noted. hepatic lesions during the acute phase consist of diffuse hemorrhage and necrotic foci (searcy, ) . in chronic stages of leptospirosis, the kidneys become small and fibrotic. endothelial cell degeneration and focal to diffuse lymphocytic-plasmacytic interstitial nephritis are the characteristic histopathological findings. pathogenesis the severity and course of leptospirosis depend on the causative serovar as well as the age and immune status of the dog. infection occurs after the leptospires penetrate a mucous membrane or abraded skin. the organisms then invade the vascular space and multiply rapidly, reaching the renal tubular epithelium several days postinfection. acute or progressive renal failure leading to oliguria or anuria may occur. nephritis may or may not be accompanied by hepatitis, uveitis, pulmonary hemorrhage, and meningitis. disseminated intravascular coagulation is often a secondary complication. diagnosis and differential diagnosis paired serology for the microscopic agglutination test is the most reliable means of definitive diagnosis, and successive serum sampling should be done - days after the first sample. pcr can be used to identify active infection early in the disease when serologic testing is negative or in previously vaccinated animals (sykes et al., ) . differential diagnoses include other causes of acute renal failure and hepatitis. prevention and control according to the american animal hospital association's vaccination guidelines, vaccination for leptospirosis is recommended based on geographic location and exposure risk (welborn et al., ) . both quadrivalent and bivalent inactivated bacterins are available. quadrivalent bacterins protect against canicola, icterohaemorrhagiae, grippotyphosa, and pomona serovars, whereas bivalent bacterins cover only canicola and icterohaemorrhagiae. immunization does not prevent the development of the carrier state or protect against other serovars. control requires preventing contact with wildlife reservoirs as well as identification of carrier animals. treatment doxycycline is the drug of choice as it can eliminate renal colonization. if vomiting or allergic reactions prohibit treatment with doxycycline, ampicillin or other penicillins should be utilized. aggressive fluid therapy and supportive care may also be needed. research complications due to the zoonotic potential, dogs with clinical leptospirosis should not be used in research studies. etiology campylobacter spp. are thin, curved or spiral, microaerophilic, thermophilic motile gram-negative rods. many species of campylobacter have been isolated from normal and diarrheic animals; however, the most common pathogenic species include campylobacter jejuni ssp. jejuni and c. coli (marks et al., ) . clinical signs most adult animals infected with c. jejuni are asymptomatic carriers; clinical signs are most commonly noted in dogs that are less than months of age (greene, ; burnens et al., ) . in cases of clinical illness, mild and intermittent mucoid or watery diarrhea, with or without frank blood, is most commonly noted. signs typically last - days but can persist for several months. tenesmus, inappetance, vomiting, and a mild fever may accompany the diarrhea (marks et al., ) . bacteremia and cholecystitis secondary to c. jejuni have also been documented in dogs (fox, ) . epizootiology and transmission the role of campylobacter spp. as a primary pathogen has been questioned; it may require a coenteropathy to produce disease (sherding and johnson, ) . stress or immunosuppression may make animals more susceptible. transmission is via the fecal-oral route, mostly through contaminated food or water. campylobacter jejuni can be zoonotic with immunocompromised individuals at greatest risk. pathologic findings lesions depend on the mechanism of the enteropathy (van kruiningen, ) . enterotoxin production results in dilated, fluidfilled bowel loops, with little or no histopathologic alteration. cytotoxin-mediated disease results in a friable, hemorrhagic mucosal surface. histologically, the mucosa is ulcerated with lymphoplasmacytic infiltration. translocation can result in edema and congestion of the lamina propria with focal accumulation of granulocytes. epithelial hyperplasia and decreased goblet cell numbers are also noted. campylobacter jejuni may be visualized between enterocytes with warthin-starry silver-stained sections. pathogenesis clinical disease may be produced by several different mechanisms as campylobacter spp. have a variety of virulence factors including enterotoxins, cytotoxins, and adherence or invasion properties. campylobacter jejuni can cause an erosive enterocolitis by invasion of epithelium and production of the cytolethal distending toxin (cdt) (fox, ; van kruiningen, ) . in addition, c. jejuni can produce illness via translocation to regional lymph nodes causing a mesenteric lymphadenitis. diagnosis and differential diagnosis fresh feces (per rectum) can be used for presumptive diagnosis by demonstration of highly motile, curved or spiral organisms with dark-field or phase-contrast microscopy. gram-stained c. jejuni appear as gull-winged rods. definitive diagnosis requires isolation of the organism (sherding and johnson, ) . culture requires selective isolation media, and growth is favored by reduced oxygen tension and a temperature of °c. a pcr multiplex assay for differentiation of c. jejuni, c. coli, c. lari, c. upsaliensis, and c. fetus ssp. fetus has been developed (wang et al., ) . any disorder that can cause diarrhea in dogs should be considered as a differential diagnosis. prevention and control proper environmental sanitation, waste disposal, and food storage can prevent campylobacteriosis. in enzootic situations, group housing should be avoided. outbreaks are controlled by isolation and treatment of affected individuals. treatment antibacterial treatment should be considered in severely ill dogs. erythromycin, neomycin, enrofloxacin, clindamycin, and doxycycline are all effective. resistance to quinolones and ciprofloxacin has been documented (acke et al., ) . treatment should be a minimum of - days with bacterial cultures repeating and weeks after treatment. research complications dogs with clinical campylobacteriosis have temporary derangements to digestive and absorptive functions. etiology helicobacters are gram-negative, microaerophilic, spiral bacteria that infect the gastrointestinal tract. helicobacter spp. can be separated into gastric and enterohepatic groups. the gastric helicobacters commonly identified in dogs are referred to as non- (haesebrouck et al., ; joosten et al., ) . the most common enterohepatic species found in dogs include h. bilis, h. canis, and h. cinaedi (castiglioni et al., ; dewhirst et al., ; fox, (haesebrouck et al., ; fox, ) . clinical signs most infections are subclinical in the dog. gastric infections may present with vomiting, diarrhea, and fever, accompanied by anorexia, pica, or polyphagia. enterohepatic helicobacters have been linked with inflammatory bowel disease in experimental animal models. heavy infections in dogs have been associated with inflammatory lesions of the large intestine (castiglioni et al., ; nguyen et al., ) . epizootiology and transmission the epizootiology and transmission of helicobacter spp. in the dog remain unknown. both oral-oral and fecal-oral routes for transmission have been suggested in humans, but transmission via canine saliva is a less reliable source of infection (craven et al., ) . enterohepatic infections of pet dogs are as high as % (castiglioni et al., ) . prevalence of gastric helicobacter infections in colony or shelter dogs can be as high as - % (fox, ; hermanns et al., ) . pathologic findings gastritis is usually mild and characterized by reduced mucus content of the surface epithelium with vacuolation, swelling, karyolysis, and karyorrhexis of parietal cells. multifocal infiltrates of plasma cells and neutrophils occur around blood vessels and between gastric pits (hermanns et al., ) . intestinal lesions include mild to moderate lymphoplasmacytic infiltration as well as crypt dilation and crypt hyperplasia (castiglioni et al., ) . pathogenesis gastric helicobacters are urease positive, which assists with survival in the acidic environment of the stomach (kusters et al., ; uberti et al., ) . enterohepatic helicobacters are urease negative and typically reside in the lower intestine. the mechanism by which enterohepatic helicobacters colonize the liver is thought to be through portal circulation after uptake by enterocytes or through retrograde movement from the intestine into the bile duct (fox, ) . diagnosis and differential diagnosis organisms may be demonstrated with histopathology on endoscopic or surgical biopsy tissue samples. warthin-starry silver stain may increase the sensitivity for histopathologic diagnosis. culture may be difficult depending on the helicobacter spp. for species that produce urease, a positive urease test on a gastric biopsy specimen may give a presumptive diagnosis. the urea breath test has been successfully used to diagnose helicobacter spp. in laboratory beagles with a sensitivity and specificity of % (kubota et al., ) . western blot has been used to detect serum antibodies to enterohepatic species and pcr can be used to detect helicobacter spp. in fecal samples (oyama et al., ; wadström et al., ) . any causes of acute or chronic vomiting and diarrhea in the dog are differential diagnoses. prevention and control until more is known about the epizootiology and transmission of helicobacter spp., specific recommendations cannot be made for prevention and control. treatment for gastric species, combination therapy of amoxicillin ( mg/kg q h), metronidazole ( mg/kg q h), and sucralfate ( . - . mg/kg q h) has proven to be most effective (hall and simpson, ) . replacing sucralfate with famotidine, omeprazole, or bismuth subsalicylate may also be effective (marks, ; jenkins and bassett, ; denovo and magne, ) . recurrence rates within days of treatment can be as high as % (anacleto et al., ) . treatment of enterohepatic helicobacters may depend on species susceptibility. aminoglycosides have been successful in treating h. cinaedi, but resistance to fluoroquinolones has been documented (tomida et al., ) . combination therapy of amoxicillin, clarithromycin, metronidazole, and omeprazole in medicated chow has been successful in eliminating various enterohepatic helicobacters from mice (del carmen martino-cardona et al., ) . long-term antibiotic treatment at a minimum of days is suggested for enterohepatic and gastric helicobacters. research complication dogs used in gastrointestinal physiology or oral pharmacology studies should be free from helicobacteriosis. etiology parvoviral enteritis in dogs is caused by canine parvovirus strain (cpv- ) of the family parvoviridae, genus protoparvovirus, species carnivore protoparvovirus . currently, there are three antigenic variants, a, b, and c. parvoviruses are nonenveloped, single-stranded dna viruses. clinical signs while parvoviral infection can affect the gastrointestinal tract, bone marrow, myocardium, and nervous tissues, the most common manifestation of disease is acute enteritis. clinical signs usually appear days after fecal-oral inoculation and include anorexia, fever, depression, vomiting, and profuse intractable diarrhea which may become hemorrhagic. excessive fluid and protein losses through the gastrointestinal tract result in rapid and severe dehydration. dogs can develop severe leukopenia with a total leukocyte count of cells/μl or less. repeated hemograms may provide prognostic value, as rebounds in leukocyte counts are indicative of impending recovery. terminally ill dogs may develop hypothermia, icterus, or disseminated intravascular coagulation due to endotoxemia. epizootiology and transmission parvovirus can infect dogs of any age, but puppies between and weeks of age are particularly susceptible. puppies less than weeks of age are protected by passive maternal antibody. strain cpv- c has been associated with severe disease in adult vaccinated dogs (calderon et al., ) . pathogenesis canine parvovirus has an affinity for rapidly dividing cells of the intestine and causes acute enteritis with intestinal crypt necrosis and villus atrophy. the virus also has tropism for the bone marrow and lymphoid tissues; thus, leukopenia and lymphoid depletion accompany the intestinal destruction. diagnosis and differential diagnosis parvovirus can be detected with a commercially available fecal enzyme-linked immunosorbent assay (elisa). due to intermittent and brief shedding of the virus, fecal elisas can have false-negative results. pcr can be used to confirm an elisa result and to differentiate the viral strain. at necropsy, diagnosis is based on gross and histopathologic evidence of necrosis and dilatation of intestinal crypt cells with secondary villous collapse. prevention and control parvoviral-positive animals should be quarantined for at least days as the infectious virus is shed for several days after onset of clinical signs. although pcr has been used to detect viral dna in feces for up to weeks (decaro et al., ) , it is currently unknown if the material being shed at this time is still infectious. disinfection of exposed areas with dilute bleach ( : ) or a commercial disinfectant is essential for elimination of the virus. six-week-old puppies should be vaccinated every - weeks with a modified live vaccine until at least weeks of age. treatment treatment is largely supportive and aimed at restoring fluid and electrolyte balance. antimicrobial therapy is recommended due to intestinal compromise and risk of sepsis. early nutritional support continued throughout the disease has been shown to decrease recovery times (mohr et al., ) . research complications infection with parvovirus precludes the use of a particular dog in an experimental protocol. due to the significant discomfort of the animal, as well as the intensive therapy required, humane euthanasia is usually chosen in a research setting. etiology rabies virus is a lyssavirus belonging to the family rhabdoviridae. clinical signs clinical progression of neurologic disease occurs in three stages. the first, prodromal, stage is characterized by a change in species-typical behavior. the loss of the instinctive fear of humans by a wild animal is a classic sign of impending rabies. in the second, furious, stage, animals are easily excited or hyperreactive to external stimuli and will readily bite at inanimate objects. the third, paralytic, stage is characterized by incoordination and ascending ataxia of the hindlimbs due to viral-induced damage of motor neurons. death due to respiratory failure usually occurs after onset of the third stage. wild animals such as raccoons, skunks, and bats are common reservoirs of infection for domestic animals, which in turn are the principal source of infection for humans. transmission occurs primarily by contact with infected saliva, usually via bite wounds. pathogenesis the incubation period for rabies is - weeks to the onset of clinical signs but can range from week to year. bites to the head and neck result in shorter incubation periods due to the close proximity to the brain. following infection, the virus migrates centripetally via peripheral nerve fibers to neurons within the brain, resulting in neurologic dysfunction. on reaching the brain, the virus migrates centrifugally to the salivary glands, thus enabling shedding and subsequent transmission. diagnosis and differential diagnosis definitive diagnosis is based on immunofluorescence of the virus in negri bodies of hippocampal cells. submission of the whole, unfixed brain, including the cerebellum and proximal brain stem, should be done within h of collection. the tissue should be kept refrigerated as freezing can cause delays in testing. differential diagnoses laboratory animal medicine include pseudorabies, canine distemper, bacterial meningitis, and toxicants that affect neurologic function. prevention and treatment puppies should be vaccinated by weeks of age, again at year, and then annually or triennially, depending on state and local laws. research complications immuno-prophylaxis is recommended for animal care and research personnel who may have work-related risks of exposure. due to risk of human exposure, animals with suspected infection should be humanely euthanized and brain tissue should be submitted for confirmation. giardiasis giardia lamblia, also known as g. duodenalis and g. intestinalis, is a binucleate flagellate protozoan that usually causes subclinical infestation of the small intestine. clinical disease is usually seen in young dogs and the characteristic sign is voluminous, light-colored, foul-smelling, soft to watery diarrhea, which is the result of malabsorption and hypersecretion. giardia has a direct life cycle with infection resulting after consuming cyst-contaminated food or water. the change in ph between the stomach and duodenum activates excystation and trophozoites then attach to the enterocytes. for diagnosis, direct fecal smears are considered best for observing trophozoites and zinc sulfate centrifugation is preferred for detection of cysts. a commercial elisa kit is licensed for use in dogs, but the positive predictive value is poor and zinc sulfate centrifugation techniques should be used in conjunction with elisa (rishniw et al., ) . pcr assays are also available for diagnosing giardiasis. differential diagnoses for giardiasis include bacterial and protozoal enteritis, coccidiosis, and whipworm infestation. metronidazole at - mg/kg po q h for - days is effective at treating giardiasis as well as other enteric protozoans, which may be potential differential diagnoses or coinfections. albendazole, fenbendazole, pyrantel, and praziquantel are also effective. coccidiosis intestinal coccidia associated with enteropathy in dogs include isospora canis, i. ohioensis, i. neorivolta, i. burrowsi, and hammondia heydorni (dubey and greene, ) . coccidian oocysts can be found in feces of clinically healthy dogs, as well as animals with diarrhea. clinically affected animals are young or immunosuppressed and develop diarrhea, which can vary from soft to watery and may contain blood or mucus. vomiting, dehydration, lethargy, and weight loss can also be seen. coccidia oocysts are typically spread by fecal-oral transmission, but dogs can ingest monozoic cysts in intermediate host tissues. the coccidian life cycle is both sexual and asexual, and results in the release of unsporulated eggs, which sporulate under appropriate environmental conditions. other causes for diarrhea should be excluded before a coccidial etiology is implicated. treatment may not be necessary, as infections are typically self-limiting and clinically insignificant. treatment may help to limit the number of oocysts shed in a kennel-housing situation and may be necessary in cases of protracted clinical illness. possible choices for treatment include daily administration of sulfadimethoxine ( - mg/kg po q h for - days) or trimethoprim sulfa ( mg/kg po q h for days). ascarids roundworms of dogs are most often toxocara canis; however, toxascaris leonina can also affect dogs. clinical illness is usually only seen in young animals with large worm burdens. diarrhea, vomiting, dehydration, and abdominal discomfort with vocalization can be seen. puppies may have a classical 'potbellied' appearance. heavy infestations can cause intussusception and/or intestinal obstruction. puppies that experience lung migrations of larval worms can develop fatal pneumonia. toxascaris canis can infect dogs by transplacental migration, transmammary migration, or ingestion of infective eggs. the infective stage of t. canis is the third-stage larva (l ). in transplacental infections, puppies may be born with l larvae in their lungs (sherding, ) . for diagnosis, large ( - μm in diameter) and relatively round ascarid eggs can be seen by standard fecal flotation methods. monthly administration of milbemycin or ivermectin plus pyrantel pamoate is recommended for prevention (hall and simpson, ) . most anthelmintics are effective for treatment. puppies should be treated early and often (every other week until weeks of age) because of the possibility of prenatal or neonatal infection. pregnant bitches can be treated with extended fenbendazole therapy ( mg/kg po once a day from day of gestation through day of lactation). hookworms the most common and most pathogenic hookworm of dogs is ancylostoma caninum. ancylostoma braziliense can also be found in dogs, but only a. caninum infestation typically results in clinical illness. puppies with hookworm infections can present as anemic with bloody diarrhea or melena. other clinical signs include lethargy, anorexia, dehydration, vomiting, and poor weight gain. these signs are a direct result of the worms' consumption of blood and body fluids. infective larvae (l ) are ingested from the environment and develop directly in the intestinal tract. infestation can also be transmammary, from ingestion of a paratenic host, and, less often, by transplacental migration. on histological sections, embedded worms with mouthparts may be identified. diagnosis is made by identification of eggs or larvae by either fecal flotation or direct smear. a differential diagnosis of parvovirus should be considered for puppies with bloody diarrhea, and autoimmune hemolytic anemia should be considered in young dogs with anemia. pyrantel pamoate is the anthelmintic of choice because it is safest in young ill animals. monthly administration of milbemycin or ivermectin plus pyrantel pamoate is recommended for prevention and control (hall and simpson, ) . due to transplacental or milkborne infection, puppies should be treated q weeks from to weeks of age. whipworms trichuris vulpis, the canine whipworm, can cause acute or chronic large intestinal diarrhea. the adult worm resides in the cecum or ascending colon. most infections are subclinical, but in symptomatic cases, the typical clinical sign is diarrhea with blood and/or mucus. abdominal pain, anorexia, and weight loss may also be seen. dogs may have eosinophilia, anemia, and/or hypoproteinemia on clinical hematology. trichuris vulpis has a direct life cycle with eggs passed in the feces. the penetration of the adult worm into the enteric mucosa, and the associated inflammation, can lead to diarrhea. factors that influence development of clinical symptoms are the number and location of adult whipworms; the severity of inflammation, anemia, or hypoproteinemia in the host; and the overall condition of the host. whipworm infestation is diagnosed by the presence of barrel-shaped, thickwalled eggs with bipolar plugs on fecal flotation. adult worms intermittently release eggs; therefore, negative results do not exclude infection. differential diagnoses for whipworm infestation include giardiasis, coccidiosis, and bacterial enteritis. fenbendazole, oxibendazole, and milbemycin have all been recommended for treatment of whipworms. treatment for whipworm infestation should be at monthly intervals for months (jergens and willard, ) . several species of cestodes parasitize the small intestine of dogs. the most common is dipylidium caninum. other species include taenia pisiformis and, more rarely, echinococcus granulosus, multiceps spp., mesocestoides spp., and spirometra spp. most cestode infestations are subclinical, but severe infestations with dipylidium can cause diarrhea, weight loss, and poor growth. the cestode requires an intermediate host, which for d. caninum are fleas and lice. ingestion of these arthropods results in transmission of the tapeworm. definitive diagnosis is usually made by the identification of egg capsules or proglottids (tapeworm segments) on the surface of the feces or around the anus. the most significant means to limit cestode infestation is to control flea and/or louse exposure. praziquantel at - . mg/kg orally or subcutaneously is the standard treatment for cestodiasis, especially taenia or echinococcus species. fenbendazole, mebendazole, or oxfendazole may also be effective against d. caninum (hall and simpson, ) . demodicosis canine demodicosis is caused by demodex canis, a commensal mite that lives in the hair follicles and is passed from dams to nursing pups. localized demodicosis is typically asymptomatic, but disease can present with variable and nonspecific clinical signs, such as alopecia, erythema, pruritus, crusts, and hyperpigmentation. it can occur anywhere on the body but is often seen on the feet and face, and around the ears (demanuelle, a). generalized demodicosis can develop in juvenile or adult populations and is indicative of an underlying immunosuppressive disorder. demodex has a characteristic 'cigar shape' and can be identified from deep skin scrapings mounted on mineral oil (campbell, ; noli, ) . differential diagnoses include dermatophytosis, allergic contact dermatitis, and seborrheic dermatitis. the primary differential diagnosis for generalized demodicosis is primary bacterial pyoderma, which is also a common secondary complication of generalized demodicosis. ivermectin at - μg/kg and oral milbemycin at - mg/kg/day are effective treatments. treatment duration can be extensive and must be accompanied by repeated skin scrapings. sarcoptic mange canine sarcoptic mange is caused by sarcoptes scabiei var. canis, which is zoonotic. the most common clinical sign is an intense pruritus, usually beginning at sparsely furred areas of the ear pinnae, elbows, ventral thorax, and abdomen. lesions are characterized by alopecia and yellowish dry crusts with a macular papular eruption. these lesions may be exacerbated by excoriation due to the pruritic nature of the condition. adult mites, mite eggs, or mite feces may be observed on superficial skin scrapings, but diagnosis may be difficult because multiple skin scrapings may yield negative results. even if scrapings are negative, a therapeutic trial should be initiated if the clinical signs and history suggest a sarcoptes etiology. demonstration of anti-mite ige either in the serum or via an intradermal antigen test can be used as a diagnostic aid (campbell, ) . histologic examination is nondiagnostic; however, suggestive lesions include small foci of edema, exocytosis, degeneration, and necrosis . an important differential diagnosis is flea allergy dermatitis (fad). unless antiparasitic therapy would interfere with research objectives, all dogs with sarcoptic mange should be treated. in addition, their kennel mates should also be treated due to the contagious nature of the disease and its zoonotic potential. the usual means of treatment is either ivermectin at - μg/kg q days or milbemycin at mg/kg q days for three oral doses . ticks ticks are obligate arachnid parasites that require vertebrate blood as their sole food source. genera that more commonly infest dogs in the united states include species of rhipicephalus, dermacentor, amblyomma, and ixodes. the primary significance of tick infestation is vector-borne infectious diseases, including rocky mountain spotted fever (rickettsia rickettsii), lyme disease (borrelia burgdorferi sensu stricto), thrombocytic anaplasmosis (anaplasma platys), and canine monocytic ehrlichiosis (ehrlichia canis). ticks alone cause minimal signs unless the dog develops a hypersensitivity reaction leading to a more granulomatous response at the bite location (merchant and taboada, ) . some species (primarily dermacentor andersoni and d. variabilis) produce a salivary neurotoxin that causes an ascending flaccid paralysis (malik and farrow, ) . uncomplicated tick bites and tick-bite paralysis are diagnosed by identification of the tick and clinical signs of paralysis. dogs with tick-bite paralysis usually show improvement within h of tick removal, with complete recovery within h (malik and farrow, ) . formamidines (amitraz), pyrethroids, and phenylpyrazoles (fipronil) are available as spot-ons, collars, sprays, and foggers to treat tick infestations in both the animal and the environment (halos et al., ; beugnet and franc, ) . differential diagnoses for tick-bite paralysis include botulism, snakebite, polyradiculoneuritis, and idiopathic polyneuropathy (malik and farrow, ) . fleas the most common flea to infest dogs is ctenocephalides felis felis, the cat flea (sousa, ) . flea infestations usually cause foci of alopecia and pruritus. dogs that are hypersensitive to antigenic proteins in flea saliva develop severe fad, which features papules, crusting, and excoriations over the lumbosacral region, flanks, thighs and abdomen. these animals may require oral corticosteroids to relieve clinical signs (muller et al., ) . secondary bacterial and fungal infections can also develop. fleas can also transmit other parasitic diseases, such as dipylidium tapeworms. flea infestations and fad are definitively diagnosed by observing the fleas on the host's skin; however, the presence of flea excrement can support a presumptive diagnosis (demanuelle, b) . treatment of flea infestations should use an integrated pest management (ipm) approach that targets adult fleas, immature stages, and environmental contamination in order to limit the risk of chemoresistance. combining ovicidal treatments, such as lufenuron and selamectin, with adulticidal treatments, such as fipronil, spinosad, selamectin, and imidacloprid, is recommended (halos et al., ; beugnet and franc, ; dryden et al., ) . certain chemicals (i.e., imidacloprid and selamectin) have both adulticidal and larvacidal abilities, but the principles of ipm preclude the use of one product solely for both adulticidal and larvacidal properties (schwassman and logas, ). differential diagnoses include mite and louse infestations, bacterial folliculitis, and allergic or atopic conditions that present with skin lesions in dogs. canine dermatophytoses are commonly caused by microsporum spp., trichophyton spp., and epidermophyton spp. (moriello and deboer, ) . uncomplicated infections are characterized by circular areas of alopecia and crusting with or without follicular papules, usually around the face, neck, and forelimbs. dermatophytes infect the hair shaft and follicle, as well as the surrounding skin. infected hairs become brittle and broken shafts remain infective in the environment for months. dermatophytoses are zoonotic and easily transmitted to other animals through the environment or by direct contact. definitive diagnosis is made using dermatophyte test medium for culture. hair and crust material from infected sites can be plucked and placed on culture; however, the 'toothbrush' method is more effective for sampling multiple sites. the brush is used to comb hairs and scales from several infected sites and then pressed into the culture media. media plates should be visually inspected daily for days. positive cultures will become red at the same time as growth of a fluffy white colony. microscopic examination of hairs and scales to visualize fungal elements can be done using skin scrapings in % koh or mineral oil; however, this method is not very sensitive. topical and systemic therapy should be initiated together after all suspected areas are clipped to reduce spreading of contaminated fragile hairs. wholebody topical therapies with antifungal shampoos, rinses, and creams are recommended rather than spot treatment. systemic therapy can be achieved with griseofulvin, ketoconazole, itraconazole, or fluconazole. due to the highly infective nature of this disease, animals should be isolated and the environment thoroughly disinfected. chlorhexidine and virkon ® s are ineffective at clearing environmental spores, but lime sulfur ( : ), enilconazole ( . %), and bleach ( : ) are effective across many strains of microsporum canis (moriello and deboer, ) . although the incidence of hypothyroidism in the canine population is not high (kemppainen and clark, ) , deficiency in thyroid hormone can significantly laboratory animal medicine affect basal metabolism and immune function. because these factors are important in many biomedical research studies, it is imperative that laboratory animal veterinarians be able to recognize, diagnose, and treat this problem. etiology primary hypothyroidism affects the thyroid gland directly, whereas secondary hypothyroidism has indirect effects through dysfunction of the pituitary gland (seguin and brownlee ) . both of these causes result in a gradual loss of functional thyroid tissue (avgeris et al., ; kemppainen and clark, ) . the majority of cases of canine hypothyroidism are due to lymphocytic thyroiditis, an autoimmune disorder, or idiopathic atrophy of the thyroid gland. lymphocytic thyroiditis is the major cause of hypothyroidism in laboratory beagles and appears to be familial in that breed (tucker, ; beierwaltes and nishiyama, ; manning ) . rarely, congenital defects or nonfunctional tumors may cause hypothyroidism (peterson and ferguson, ; kemppainen and clark, ) . clinical signs because it affects metabolism in general, hypothyroidism can produce a large number of clinical signs referable to many organ systems. an individual dog with hypothyroidism may have one or any combination of clinical signs. hypothyroidism reduces the dog's metabolic rate, which then produces such signs as obesity, lethargy, cold intolerance, and constipation. additionally, hypothyroidism can produce several dermatologic abnormalities, including nonpruritic, bilaterally symmetrical alopecia, hyperpigmentation, seborrhea, and pyoderma (avgeris et al., ; peterson and ferguson, ; panciera, ) . several clinicopathologic abnormalities have also been reported in a large percentage of hypothyroid dogs. these aberrations include increased serum cholesterol and triglycerides due to a decrease in lipolysis and decreased numbers of low-density lipopolysaccharide receptors (peterson and ferguson, ; panciera, ) . normocytic, normochromic, nonregenerative anemia may be seen in approximately one-half of the cases (avgeris et al., ) . increased serum alkaline phosphatase and creatine kinase have also been reported in a significant number of hypothyroid dogs (peterson and ferguson, ; panciera ) . neurologic signs of hypothyroidism, which include lameness, foot dragging, and paresis, may be caused by several mechanisms such as segmental nerve demyelination or nerve entrapment secondary to myxedema (peterson and ferguson, ) . mental impairment and dullness have also been reported in hypothyroid dogs, secondary to atherosclerosis and cerebral myxedema (peterson and ferguson, ) . hypothyroidism has been implicated in other neurological abnormalities such as horner's syndrome, facial nerve paralysis, megaesophagus, and laryngeal paralysis; however, these conditions do not always resolve with treatment (bichsel et al., ; panciera, ) , and a true causal relationship with hypothyroidism has not been completely defined (panciera, ) . myopathies associated with hypothyroidism are caused by metabolic dysfunction and atrophy of type ii muscle fibers and can present with signs similar to neurological disease (peterson and ferguson, ) . hypothyroidism can also cause abnormalities of the cardiovascular system including bradycardia, hypocontractility, increased vascular volume, and atherosclerosis (seguin and brownlee ) . abnormalities that may be detected by ecg include a decrease in p-and r-wave amplitude (peterson and ferguson, ) and inverted t waves (panciera, ) . these ecg abnormalities are caused by lowered activity of atpases and calcium channel function. an association between hypothyroidism and von willebrand disease has been suggested. however, the relationship is probably one of shared breed predilection and not a true correlation. contradictory studies have shown either deficient (avgeris et al., ) or normal (panciera and johnson , ; avgeris et al., ) von willebrand factor antigen and bleeding times in hypothyroid dogs. most importantly, hypothyroidism does not appear to cause overt, clinical von willebrand disease. however, it may exacerbate existing subclinical von willebrand disease (seguin and brownlee, ) . epizootiology the prevalence of hypothyroidism in the general canine population is reportedly less than % (panciera, ) . the disorder occurs most often in middle-aged, larger breed dogs (avgeris et al., ) , and reports suggest a higher incidence of hypothyroidism in spayed, female dogs (panciera, ; peterson and ferguson, ) . doberman pinschers and golden retrievers appear to have a higher incidence of hypothyroidism compared with other breeds (panciera, ; peterson and ferguson, ; scarlett, ) . there have been several reports about hypothyroidism in laboratory colonies of beagles (manning, ; tucker, ; beierwaltes and nishiyama, ) . diagnosis and differential diagnosis because of the large number of clinical manifestations in dogs, the recognition of hypothyroidism is not always straightforward. likewise, the diagnosis of hypothyroidism can be difficult because of the lack of definitive diagnostic tests available for the dog. a complete understanding of the diagnosis of hypothyroidism requires a familiarity with thyroid hormone metabolism and function that is beyond the scope of this writing. for additional information, the reader is referred to one of several manuscripts available (peterson and ferguson, ; ferguson, ) . currently, the ability to diagnose hypothyroidism relies heavily on the measurement of serum total t (thyroxine) and free t (peterson and ferguson, ; ferguson, ) . t serves primarily as a precursor for t and is heavily protein bound. free t represents the laboratory animal medicine unbound fraction that is available to the tissues (peterson and ferguson, ) . the measurement of total t carries a sensitivity of around % and can be used as a good screening tool. with the measurement of both serum total t and free t , hypothyroidism can usually be ruled out if the values are within the normal range or higher. if both hormone concentrations are low, it is highly likely that the patient has hypothyroidism, and a therapeutic trial may be in order (peterson and ferguson, ) . however, nonthyroidal illnesses and some drugs (e.g., glucocorticoids, anticonvulsants, phenylbutazone, salicylates) can falsely lower these values (peterson and ferguson, ; ferguson, ) . therefore, low values do not always indicate that hypothyroidism is present and animals should not be treated solely on the basis of serum hormone levels if clinical signs are not present. if the clinical signs are equivocal or only total t or free t is decreased, further diagnostic testing is warranted (peterson and ferguson, ) . although t is the most biologically active form of thyroid hormone, the measurement of serum t levels is an unreliable indicator of hypothyroidism (peterson and ferguson, ; ferguson, ) . serum t can be falsely lowered by many nonthyroidal illnesses and many drugs (see above). in addition, t may be preferentially released and conversion of t to t may be enhanced by the failing thyroid (peterson and ferguson, ; ferguson, ) , particularly early in the disease. in one study, t was within normal limits in % of the hypothyroid dogs (panciera, ) . autoantibodies can be responsible for false elevations in the concentrations of t and t found in these respective assays. it has been recommended that free t , measured by equilibrium dialysis, be assayed in dogs that are suspected of hypothyroidism and have autoantibodies with normal or high t and t . autoantibodies have been found in less than % of the samples submitted to one laboratory (kemppainen and behrend, ) . other means of diagnosing hypothyroidism have been described. in humans, endogenous thyroid-stimulating hormone (tsh) levels provide reliable information on thyroid status, and an assay is available for dogs. however, endogenous tsh levels can be normal in some dogs with hypothyroidism and high tsh levels have been noted in normal dogs and sick animals that are actually euthyroid. it is therefore recommended that tsh levels be considered along with other information (clinical signs, t ) prior to diagnosis and treatment (kemppainen and behrend, ) . tsh stimulation testing using exogenous bovine tsh provides a good and reliable method for establishing a diagnosis. unfortunately, the availability and expense of tsh limit the use of this diagnostic tool (peterson and ferguson, ; ferguson, ) . another drawback of tsh testing is that the test must be postponed for weeks if thyroid supplementation has been given (peterson and ferguson, ) . when tsh is available for testing, there are several recommendations for dosage, routes of administration, and sampling times. one recommendation is . u of tsh per pound of body weight (up to a maximum of u) to be administered iv. for this protocol, blood samples are taken prior to administration of tsh and h after. a normal response to the administration of tsh should create an increase of t levels at least μg/dl above the baseline levels or an absolute level that exceeds μg/dl (peterson and ferguson, ; wheeler et al., ) . treatment the treatment of choice for hypothyroidism in the dog is l-thyroxine (sodium levothyroxine). a recommended dosing regimen is . - . mg/kg once a day (avgeris et al. ). if drugs that decrease thyroxine levels are being administered concurrently, it may be necessary to divide the thyroxine dose for twice daily administration. after the supplementation has begun, the thyroid hormone level should be rechecked in - weeks, and blood samples should be drawn - h after the morning pill. a clinical response is usually seen in - weeks and would include weight loss, hair regrowth, and resolution of other signs (panciera, ) . ecg abnormalities also return to normal (peterson and ferguson, ) . for dogs with neurologic signs, the prognosis is guarded, because the signs do not always resolve with supplementation (panciera, ) . weight gain and eventual obesity are frequent findings in dogs in the research environment. because obesity can adversely affect several body systems as well as general metabolism, the laboratory animal veterinarian must address obesity and its potential effects on animal welfare and research results. etiology obesity is defined as a body weight - % over the ideal. in general, obesity occurs when the intake of calories exceeds the expenditure of energy, the result of overeating or eating an unbalanced diet. overeating is a common cause of obesity in pet dogs and may be triggered by boredom, nervousness, or conditioning (macewen, ) . in addition, pet animals are often subjected to unbalanced diets supplemented with high-fat treats. in the laboratory animal setting, overeating is less likely than in a household because access to food is more restricted, and diets are usually a commercially prepared balanced ration. however, obesity can still be a problem if specific guidelines for energy requirements are not followed. in addition, the necessary caging of dogs in the research environment and limitation to exercise reduces energy expenditure. it is also important to realize that other factors may predispose dogs to obesity, even when guidelines for caloric intake and energy laboratory animal medicine expenditure are followed (butterwick and hawthorne, ) . as in humans, genetics plays an important role in the development of obesity in dogs, and certain breeds are more predisposed toward obesity. in a study of dogs visiting veterinary clinics in the united kingdom, labrador retrievers were most likely to be obese. other breeds affected included cairn terriers, dachshunds, basset hounds, golden retrievers, and cocker spaniels. the beagle was also listed as a breed predisposed to obesity in the household environment (edney and smith, ) . several metabolic or hormonal changes are also associated with obesity. it has been well established that neutering promotes weight gain. in one study, spayed female dogs were twice as likely to be obese compared with intact females (macewen, ) . the authors proposed that the absence of estrogen promotes an increase in food consumption. a similar trend toward obesity was found in castrated male dogs (edney and smith, ). in addition, hypothyroidism and hyperadrenocorticism may present with obesity as one of the clinical signs (macewen, ) . differential diagnosis the diagnosis of obesity is somewhat subjective and relies on an estimate of ideal body weight. the ideal body condition for dogs is considered to be achieved when the ribs are barely visible but easily palpated beneath the skin surface. when the ribs are not easily palpated and/or the dog's normal function is impaired by its weight, the animal is considered obese. there are few objective, quantifiable methods for establishing this diagnosis. ultrasound has been evaluated for measurement of subcutaneous fat in dogs, and measurements taken from the lumbar area can be used to reliably predict the total body fat (wilkinson and mcewan, ) . after a diagnosis of obesity has been made, additional diagnostic tests should be performed to determine if there is an underlying cause for the problem. a complete physical exam should be performed to look for signs of concurrent disease and to establish if obesity has adversely affected the individual. serum thyroid hormones should be evaluated (see above), and serum chemistry may reveal an increased alkaline phosphatase associated with hyperadrenocorticism. treatment restricting food intake readily treats obesity, and this is easily done in the research setting. it has been suggested that a good weight loss program involves restriction of intake to % of the calculated energy requirement to maintain ideal body weight. it has been shown that restriction of calories down to % produces no adverse health effects. however, t levels will decrease in direct proportion with caloric intake. ideally, weight loss will occur at a rate of - % of body weight per week (laflamme et al., ) . with more severe calorie restriction and more rapid weight loss, the individual is more likely to have rebound weight gain after restrictions are relaxed. there has been a great deal of attention in humans as to the correct diet to encourage weight loss. likewise, the type of diet fed to dogs has been examined. as mentioned above, the restriction of calories is most important, and feeding less of an existing diet can do this. alternatively, several diet dog foods are available, and there is some evidence that these diets are superior to simple volume restriction (macewen, ) . there has been much concern about the addition of fiber to the diet as a method for reducing caloric intake while maintaining the volume fed. studies in dogs have examined the addition of both soluble and insoluble fibers to calorie-restricted diets. these studies have shown that the addition of fiber does not have an effect on satiety in dogs and therefore does not have a beneficial effect in weight loss protocols (butterwick et al., ; butterwick and markwell, ) . research complications it is important to control weight gain in research animals because of the association of obesity with metabolism. although an association between obesity and reproductive, dermatologic, and neoplastic problems has been reported (macewen, ) , this relationship is not consistently apparent (edney and smith, ) . joint problems including osteoarthritis and hip dysplasia have also been related to obesity (macewen, ; kealy et al., ) . in addition, diabetes mellitus has been linked to obesity and obesity-induced hyperinsulinism in several experimental models (macewen, ) . a recent study demonstrated metabolic disease, typified by hyperinsulinemia and hypoadiponectinemia, in approximately % of obese dogs (tvarijonaviciute et al., ) . research that requires anesthesia may be complicated by a greater risk of cardiovascular diseases (edney and smith, ) including hypertension and compromise to the respiratory tract. etiology in the laboratory setting, the majority of traumatic wounds will be small in size and quickly observed. occasionally, dogs may sustain minor trauma during transport or have a small, previously undetected, chronic wound upon arrival at the facility. when dogs are group housed, they may sustain bite wounds during early socialization periods. under these conditions, proper initial treatment will lead to uncomplicated wound healing. complications such as infection and delayed healing arise when wounds are not noticed immediately or when the basic principles of wound management are not followed. clinical signs the signs and appearance of a traumatic wound will vary with the cause and the duration of time since wounding. abrasions, sustained by shear forces, are partial thickness skin wounds characterized laboratory animal medicine by minimal bleeding or tissue disruption. puncture wounds have a small surface opening but penetrate into deep tissues with the potential for contamination. lacerations are wounds caused by sharp separation of skin that may extend to deeper tissues. acute wounds are characterized by bleeding tissue, sharp edges and no obvious devitalization. they have variable degrees of contamination. chronic wounds generally do not exhibit active bleeding and will have curled or rounded edges. these wounds often have necrotic tissue and are considered contaminated. treatment to aid decision making about wound therapy, several classification systems have been developed for traumatic injuries. at one time, decisions about wound therapy were largely based upon the length of time since wounding, or the concept of a 'golden period.' it is now recognized that several factors must be considered prior to initiating wound care, including (but not limited to) the type and size of the wound, the degree of wound contamination, and the competence of the host's defense systems (swaim, ; waldron and trevor, ) . one of the most widely used classification systems is based upon wound contamination and categorizes wounds as clean, clean-contaminated, contaminated, or dirty (see table . ). the vast majority of the wounds seen in the laboratory setting will fall into the clean and clean-contaminated categories. these wounds may be treated with the basic wound care described below and primary closure of the wound. contaminated and dirty wounds require more aggressive therapy. postsurgical infections or complications of initial therapy would be considered dirty wounds. when in doubt as to the classification of a wound, the worst category should be presumed in order to provide optimal therapy and reduce the chance for complications. the initial treatment of a wound is the same regardless of its classification. when first recognized, the wound should be covered with a sterile dressing until definitive treatment can be rendered. bleeding should be controlled with direct pressure; tourniquets are discouraged because of the complications that may arise with inappropriate placement (swaim, ) . it is best to avoid using topical disinfectants in the wound until further wound treatment (culture, debridement, lavage) has been performed (swaim, ) . anesthesia or analgesia may be necessary and the choice of agent will depend on the size and location of the wound as well as the preference of the clinician. if the wound is contaminated or dirty, bacterial cultures, both aerobic and anaerobic, should be performed. then a water-soluble lubricant gel may be applied directly to the wound to prevent it from further contamination during the hair removal process. a wide margin of hair should be clipped and a surgical scrub performed around the edges of the wound. povidone-iodine alternating with alcohol or chlorhexidine gluconate scrub alternating with water is most often recommended for surgical preparation of the skin surface (osuna et al., a, b) . simple abrasions that involve only a partial thickness of the skin do not generally require further treatment. full-thickness wounds require further attention, including irrigation with large quantities of a solution delivered under pressure. several irrigation solutions have been recommended (lozier et al., ; waldron and trevor, ; sanchez et al., ) , but type may not be as important as the volume and pressure of delivery. it has been suggested that psi is required to obtain adequate tissue irrigation, and this may be achieved by using a -ml syringe with an -or -gauge needle (waldron and trevor, ) . for wounds that are contaminated or dirty, debridement is an important part of initial therapy. debridement usually proceeds from superficial to deeper layers. skin that is obviously necrotic should be removed. although it is often recommended to remove skin back to the point at which it bleeds, this may not be feasible with large wounds on the limbs. in addition, other factors such as edema or hypovolemia may reduce bleeding in otherwise viable skin (waldron and trevor, ) . if one is unsure about tissue viability in areas that are devoid of waldron and trevor ( ) . extra skin, the tissue may be left (swaim, ; waldron and trevor, ) , and nonviable areas will demarcate within - days (waldron and trevor, ) . necrotic fat should be resected liberally, because it does not have a large blood supply and will provide an environment for infection. often, resection of subcutaneous fat is necessary to remove debris and hair that could not be removed during wound irrigation. damaged muscle should also be liberally resected (swaim, ) . the wound should be irrigated several times during debridement and again after completion. after initial wound treatment, the options concerning wound closure must be weighed. the principles of basic surgery are discussed in several good texts, and readers are encouraged to pursue additional information. primary wound closure is defined as closure at the time of initial wound therapy and is the treatment of choice for clean and clean-contaminated wounds. closure is performed in two or more layers, carefully apposing tissues and obliterating dead space. if dead space will remain in the wound, a drain should be placed. subcutaneous closure should be performed with absorbable suture such as polydioxanone, polyglactin , or polyglycolic acid. it is best to use interrupted sutures and avoid leaving excess suture material in the wound. it may be necessary to choose tension-relieving suture patterns, such as horizontal mattress. skin closure is generally performed with nylon ( - or - ). in situations where gross contamination cannot be completely removed, closure of the wound should be delayed or avoided. after debridement and irrigation, the wound should be bandaged. the wound may be covered by a nonadherent dressing such as vaseline-impregnated gauze (swaim, ) . the contact layer is covered by cotton padding, and the entire bandage is covered by a supportive and protective layer. the bandages should be changed once or twice daily, depending upon the amount of discharge coming from the wound. wound closure within - days of wounding (prior to the formation of granulation tissue) is considered delayed primary closure. when the wound is closed after days, this is considered secondary closure (waldron and trevor, ) . second-intention healing involves allowing the wound to heal without surgical intervention. this type of healing is often used on limbs when there is an insufficient amount of skin to allow complete closure (swaim, ) . it is important to note that second-intention healing will take longer than with surgical repair, and, in the case of large wounds, it will be more expensive because of the cost of bandaging materials. several factors must be weighed when considering the use of antibiotics in traumatic wound care, including the classification and site of the wound, host defenses, and concurrent research use of the animal. when wounds are clean or clean-contaminated, antibiotics are seldom necessary unless the individual is at high risk for infection. when wounds have been severely contaminated or are dirty, antibiotics are indicated and the type of antibiotic will ultimately depend on culture and sensitivity results. until such results are available, the choice of antibiotic is based on the most likely organism to be encountered. topical application of bacitracin, neomycin sulfate, and polymixin b combinations may be used in wounds with minor contamination. in skin wounds with more extensive contamination, staphylococcus spp. are generally of concern, whereas pasteurella multocida should be considered in bite wounds. when systemic antibiotics are necessary, cephalosporins, amoxicillinclavulanate, and trimethoprim sulfas are often recommended for initial antibiotic therapy (waldron and trevor, ) . prevention in facilities with good husbandry practices and a diligent staff, potentially injurious equipment or surfaces are identified quickly. appropriate attention to surgical technique and to initial wound care will generally reduce the occurrence of postprocedure wound infection. etiology pressure sores (decubital ulcers) can be a problem in long-term studies and housing situations that require chronic skin contact with hard surfaces. decubital ulcers often develop over a bony prominence such as the elbow, tuber ischii, tarsus, or carpus. the compression of soft tissues between hard surfaces results in vascular occlusion, ischemia, and ultimately tissue death (swaim and angarano, ) . several factors that increase pressure at the site and/or affect the integrity of the skin will predispose an individual to develop pressure sores, including poor hygiene, self-trauma, low-protein diet, preexisting tissue damage, muscle wasting, inadequate bedding, and ill-fitting coaptation devices (swaim and angarano, ) . clinical signs initially, the skin will appear red and irritated. over time, constant trauma can result in full-thickness skin defects and can progress to necrosis of underlying tissues. the severity of the sores may be graded from i to iv according to the depth of the wound and the tissues involved, from superficial skin irritation to involvement of underlying bone (waldron and trevor, ) . epizootiology the problem usually occurs in large dog breeds, but any type of dog can be affected. prevention and control minimizing or eliminating predisposing factors is important to both the prevention and treatment of this condition. if a dog will experience long periods of recumbency, adequate bedding or padding must be provided. recumbent animals should be moved frequently, ideally every h, to prevent continuous compression on a specific laboratory animal medicine area (waldron and trevor, ) . skin hygiene is of the utmost importance when trying to prevent or treat pressure sores. the skin should be kept clean and dry at all times. if urine scalding is a problem, the affected area should be clipped, bathed, and dried thoroughly at least once or twice daily. finally, an appropriate diet to maintain body weight will minimize compressive forces experienced over areas susceptible to ulceration (swaim and angarano, ) . treatment the treatment of pressure sores must involve care of the wound and attention to the factors causing the wound. the extent of initial wound management will largely depend on the depth of the wound. for simple abrasions and small wounds involving the skin only, simple wound cleansing and openwound management provide adequate treatment. when wounds involve deeper tissues, including fat, fascia, or bone, more aggressive diagnostics and therapy must be performed. the affected area should be radiographed to assess bone involvement and the wound should be cultured. all of the damaged tissue should be debrided and basic wound management guidelines should be followed (see above). when a healthy granulation bed has formed over the entire wound, a delayed closure over a drain may be performed (swaim and angarano, ) . with extensive lesions, reconstruction with skin flaps may be necessary (waldron and trevor ) . bandaging should be performed on all full-thickness wounds; however, it is important to remember that illfitting or inadequately padded bandages or casts may worsen the problem. the area over the wound itself should not be heavily padded. the wounded area should be lightly covered and then a doughnut, created from rolled gauze or towel, should be fitted around the wound, in order to displace pressure over a larger area and onto healthier tissue. the doughnut is then incorporated into a padded bandage. if a cast has been applied to the area for treatment or research purposes, a hole can be cut over the wound to reduce pressure in that area and allow treatment of the wound (swaim and angarano, ) . bandages should be removed at least once or twice a day to allow wound care. etiology an acral lick granuloma is a skin lesion caused by self-trauma. in a few cases, the self-trauma is due to initial irritation caused by an identifiable neurologic or orthopedic condition (tarvin and prata, ) . allergy may also be a source of irritation that leads to self-trauma. however, the majority of cases begin because of repetitive licking by dogs that are confined and lack external stimuli (swaim and angarano, ) . it has been theorized that the self-trauma promotes the release of endogenous endorphins, which act as a reward for the abnormal behavior (dodman et al., ) . the laboratory environment could promote the abnormal behavior and lead to acral lick granuloma. epizootiology the lesions associated with acral lick granuloma are seen most often in large dog breeds, particularly dobermans. however, any type of dog can be affected (walton, ) . clinical signs early lesions appear as irritated, hairless areas usually found on the distal extremities (swaim and angarano, ) . the predilection for the limbs may be due to accessibility or possibly a lower threshold for pruritus in these areas. as the lesions progress, the skin becomes ulcerated and the wound develops a hyperpigmented edge. the wounds may partially heal and then be aggravated again when licking resumes. diagnosis and differential diagnosis acral lick granulomas must be differentiated from several other conditions, including bacterial or fungal infection, foreign bodies, and pressure sores. in addition, mast cell tumors and other forms of neoplasia can mimic the appearance of acral lick granuloma. many of the aforementioned problems can be ruled out by the history of the animal. however, a complete history may be unavailable in the laboratory setting. fungal cultures and allergy testing may aid in diagnosis. biopsy of the affected area would rule out neoplasia. an uncomplicated acral lick granuloma would feature hyperplasia, ulceration, and fibrosis without evidence of infection or neoplasia (walton, ) . prevention and control behavior modification and relief of boredom are important aspects of preventing (and treating) acral lick granuloma. environmental enrichment including exercise, co-housing and various toys is already a basic requirement and may be increased to combat self injurious behaviors. treatment several treatments have been reported for acral lick granuloma and the selection of a treatment should be based on the underlying cause. one of the most important aspects of treatment is to break the cycle of self-trauma. mechanical restraint with an elizabethan collar is one of the easiest methods to accomplish this goal. several direct treatments have been examined, including intralesional and topical steroids, perilesional cobra venom, acupuncture, radiation, and surgery (swaim and angarano, ; walton, ) . opioid antagonists have been applied as treatments for acral lick granulomas and self-injurious behaviors with the theory that this will block the effects of endogenous opioids. naltrexone and nalmefene have been used successfully to reduce excessive licking behaviors and resolve associated lesions. however, lesions did recur after the drugs were discontinued (dodman et al., ; white, ) . the topical administration of a mixture of flunixin meglumine, steroid, and dimethyl sulfoxide has also been shown to be effective (walton, ) . in addition, psychoactive drugs have been suggested to relief of laboratory animal medicine boredom or anxiety. these have included phenobarbital, megestrol acetate, and progestins. however, side effects have been reported (swaim and angarano, ) . other behavior-modifying medications such as clomipramine may be effective in the treatment of compulsive anxiety disorder. the potential for effects that could interfere with experimental results must be determined prior to initiation of treatment. it is important to note that none of the above-mentioned treatments have been successful in all cases. the overall prognosis for acral lick granuloma should be considered guarded since the lesions often recur when treatment is discontinued. etiology hygromas are fluid-filled sacs that develop as a result of repeated trauma or pressure over a bony prominence. the area over the olecranon is most frequently affected, but hygromas have been reported in association with the tuber calcis, greater trochanter, and stifle (newton et al., ) . epizootiology elbow hygromas are most frequently reported in large and giant breeds of dogs, less than years of age (johnston, ; white, ; cannap et al., ) . elbow hygromas are seen infrequently in the laboratory animal setting because the commonly affected breeds are seldom used in research. however, the housing environment of research dogs, especially cage bottoms and cement runs, may predispose them to hygromas. for this reason, laboratory animal veterinary and husbandry staff should be familiar with this condition. clinical signs the clinical presentation will depend upon the chronicity of the problem. a dog with an elbow hygroma usually presents with a painless, fluctuant swelling over the point of the elbow without signs of lameness. the condition may be unilateral or bilateral. over a long period of time, elbow hygromas may become inflamed and ulcerated. if the hygroma becomes secondarily infected, the animal may exhibit pain and fever (johnston, ; white, ) . pathology the fluid-filled cavity in the hygroma is lined by granulation and fibrous tissue. hygromas lack an epithelial lining and therefore are not true cysts. the fluid within the cavity is a yellow or red serous transudate. this fluid is less viscous than joint fluid and elbow hygromas do not communicate with the joint (johnston, ) . treatment the treatment of elbow hygromas should be conservative whenever possible. conservative management of the elbow hygroma is aimed at relieving the source of pressure at the point of the elbow. in early and mild cases, simply providing padding to cover hard surfaces will result in resolution of the hygroma. a soft padded bandage or doughnut bandage around the affected site may also be of benefit. neoprene/polyester sleeves that cover the elbows and fit over the shoulders are also available as an option for either prevention or treatment of hygromas (cannap et al., ) . more aggressive therapies, including needle drainage and injection of corticosteroids into the hygroma, have been described but are not recommended due to the risk of infection (johnston, ) . surgical options should be reserved for complicated or refractory cases. even simple excision can be associated with complications such as wound dehiscence and ulceration (johnston, ) due to the location of the bony prominence at the surgical site. this issue may be avoided by using a skin advancement flap (white, ) that allows intact, healthy skin to cover the boney prominence. a muscle advancement flap has also been described (green et al., ) . regardless of the method used to treat an elbow hygroma, recurrence of the problem is likely unless the predisposing factors are identified and relieved. etiology in the research environment, corneal ulcers are most often associated with direct trauma, contact with irritating chemicals, or exposure to the drying effects of air during long periods of anesthesia. chronic or recurrent corneal ulcers may also be associated with infection or hereditary causes in some breeds of dogs; however, these would be rare in the laboratory setting. clinical signs the signs of corneal ulceration are blepharospasm, epiphora, and photophobia. the eye may appear irritated and inflamed. in minor cases, the cornea may appear normal however, in cases of deeper ulceration, the cornea may appear roughened or have an obvious defect. in addition, the periocular tissues may be swollen and inflamed because of self-inflicted trauma from rubbing at the eye. diagnosis a tentative diagnosis of corneal ulcer or abrasion may be based on the clinical signs. a definitive diagnosis of corneal ulcers is made by the green appearance of the cornea when stained with fluorescein dye. when a corneal ulcer has been diagnosed, the eye should be inspected for underlying causes such as foreign bodies, abnormal eyelids, or abberant cilia. treatment the treatment of corneal ulcers will depend on the depth and size of the affected area, as well as the underlying cause. superficial abrasions are generally treated with topical application of antibiotics. a triple antibiotic ointment that does not contain steroids given three times a day for - days usually provides adequate treatment. simple corneal ulcers are restained with fluorescein after days and should show complete healing at that time. if the ulcer is not healed, this may indicate that the ulcer has an undermined edge impeding proper healing. topical anesthetic should be applied to the eye, and a cotton-tipped applicator can be rolled over the surface of the ulcer toward its edge. this will remove the unattached edge of the cornea and healing should progress normally after debridement. deep ulcers may require further debridement and primary repair. in such cases, a third eyelid or conjunctival flap may be applied to the eye until experienced help can be obtained. in all cases, an elizabethan collar or other restraint may be necessary to prevent additional trauma to the eye. ulcers caused by entropion, ectropion, or dystichiasis will not resolve until the condition is repaired, and descriptions for this can be found elsewhere. prevention the proper application of lubricant eye ointment at the time of anesthesia will prevent drying due to exposure and may also protect the eye from scrub solutions applied near the eye. early treatment of superficial ulcers should prevent self-trauma and progression of the wound. etiology research protocols often require the placement of chronic implants. implants such as cardiac or other biomedical devices may be the primary focus of the research study. implants may also be used as chronic monitoring devices, for delivery of compounds, or to collect serial samples. infection may occur at the time of implant. alternatively, the implant may serve as a nidus after hematogenous spread from other sources. one of the most common sources of infection is from colonization of the device from an external component, which is a frequent complication with indwelling catheters. the actual incidence of complications associated with indwelling vascular catheters in dogs is unknown. one study (hysell and abrams, ) examined the lesions found at necropsy in animals with chronic indwelling catheters, which included traumatic cardiac lesions, visceral infarcts, and fatal hemorrhages. these lesions were primarily associated with catheter-induced trauma or secondary to embolization of fibrin. in a veterinary clinical setting, infections in peripheral catheters were more likely when the catheters were used for blood collection immediately after placement and when a 't' connector rather than a 'y' connector was used. . intestinal access ports have been used to study the pharmacokinetics of drugs at various levels in the intestinal tract. these catheters are usually vascular access ports with several modifications to allow secure placement in bowel (meunier et al., ) . the most frequently reported complication associated with these catheters is infection around the port site (meunier et al., ; kwei et al., ) . clinical signs dogs with implant infections may not exhibit signs initially . localized swelling around the implant may occur. in the case of indwelling catheters, signs may include redness and swelling of the skin around the external port or discharge from the skin wound. vascular access ports may develop fluctuant subcutaneous abscesses. in more severe cases, systemic signs may be noted (bach et al., ; hysell and abrams, ) . the systemic signs of infection are covered elsewhere in this chapter. treatment the treatment of catheter infections almost invariably requires removal of the catheter, as demonstrated in both dogs and monkeys (ringler and peter, ; darif and rush, ). superficial wound irritation or infection may be treated locally with antibiotic ointment, sterile dressing changes, and efforts to minimize catheter movement; however, more extensive problems require aggressive therapy. localized abscesses or sinus tracts may be managed by establishing drainage and copious flushing. aerobic and anaerobic cultures of blood and locally infected sites should be performed prior to initial treatment (ringler and peter, ) . systemic antibiotic therapy should be initiated for a -day period. the choice of drug will ultimately be based on previous experience and culture results. if retention of a catheter is important, the catheter lumen may be safely disinfected with chlorine dioxide solution (dennis et al., ) . the solution is removed after min and replaced with heparinized saline. all of the extension lines and fluids used with an infected catheter should be discarded. the blood cultures should be repeated days after the antibiotic therapy has ceased. if bacteria are still cultured, the catheter must be removed. prevention it is highly desirable to prevent complications that may result in loss of an implanted device. catheters and other implants should be made of nonthrombogenic material and be as simple as possible. a catheter with extra ports or multiple lumens requires additional management and supplies more routes for infection. the initial placement of an indwelling catheter must be done under aseptic conditions by individuals who are familiar with the procedure. intravenous catheters that are used for delivery of drugs or blood sampling should be positioned in the vena cava and not in the right atrium, thereby minimizing trauma to the tricuspid valve. ideally, catheters are secured to reduce movement and irritation of the skin, which may predispose to infection around external ports. the use of vascular access ports that lie entirely under the skin eliminates many problems with infection. it has also been found that long extension tubing connected to the port may actually reduce the potential for infection of the catheter (ringler and peter, ) . for intestinal access ports, catheter security may be improved with a synthetic cuff added to the end of the catheter allowing better attachment to the intestine (meunier et al., ) . after any catheter placement, animals should be observed daily for signs of either local or systemic infection. the catheter entry site should be disinfected, coated with antibiotic ointment, and rebandaged every other day. once a month, the catheter line may be disinfected with chlorine dioxide. in addition, a solution of the antibiotic ceftazidime used on alternate days with the heparin locking solution has been shown to effectively reduce infections in indwelling vascular catheters (bach et al., ) . throughout the life of the catheter, injections into and withdrawals from the catheter should be done in a sterile manner, and the number of breaks in the line should be kept to a minimum. periodically, the placement of an indwelling catheter may be verified by radiography. when placed and managed correctly, catheters and ports of any kind may remain in place for months without complications. etiology sepsis is defined as the systemic response to infection caused by bacteria (gram negative and/or gram positive), fungi, or viruses. in laboratory animals, sepsis is most often seen as a complication of surgical procedures or associated with chronic implants. sepsis may also be seen as a complication of infectious diseases such as parvovirus. clinical signs the signs of sepsis can vary, depending on the source of the infection and the stage of the disease. early in the course, dogs may present with signs of a hyperdynamic sepsis, including increased heart rate, increased respiratory rate, red mucous membranes, and a normal-to-increased capillary refill time. systemic blood pressure and cardiac output will be increased or within the normal range. the animals will often be febrile. later in the course of the syndrome, the animals may show classic signs of septic shock including decreased temperature, pale mucous membranes, and a prolonged capillary refill time. cardiac output and blood pressure are decreased as shock progresses. peripheral edema and mental confusion have also been reported (hauptman and chaudry, ) . pathogenesis the pathophysiology of sepsis is complex and is mediated by immune responses involving mediators such as cytokines, eicosinoids, complement, superoxide radicals, and nitric oxide. the body responds to overwhelming infection with an attempt to optimize metabolic processes and maximize oxygen delivery to tissues. however, if inflammation is left unchecked, the system may be unable to compensate, and the result is cardiovascular collapse. diagnosis in general, a presumptive diagnosis of sepsis is made based on the occurrence of several in a group of signs, including altered body temperature, increased respiratory and/or heart rate, increased or decreased white blood cell (wbc) count, increased number of immature neutrophils, decreased platelet count, decreased blood pressure, hypoxemia, and altered cardiac output. however, extreme inflammation without infection (e.g., pancreatitis, trauma) may create similar signs. one study examined the diagnosis of sepsis in canine patients at a veterinary hospital based on easily obtainable physical and laboratory findings. that study found that septic individuals had higher temperatures, wbc counts, and percentage of band neutrophils than nonseptic individuals, whereas platelet counts were lower in the septic dogs. there were no differences in respiratory rate or glucose levels between the groups. using these criteria, the results had a high sensitivity and a tendency to overdiagnose sepsis (hauptman et al., ) . ultimately, the presence of a septic focus simplifies diagnosis greatly; however, the focus may not be obvious. if the signs of sepsis are evident but the focus is not, several areas should be evaluated for infection, including the urinary, reproductive, respiratory, alimentary, and cardiovascular systems, as well as the abdominal cavity (kirby, ) . treatment the treatment of sepsis has three aims. the first aim is to support the cardiovascular system. all septic animals should be treated with fluids to replace deficits and to maximize cardiac output. crystalloids are most frequently used to maintain vascular volume, primarily because of their low cost. colloids offer the advantage of maintaining volume without fluid overload and may have other positive effects on the cardiovascular system. acid-base and electrolyte imbalances should also be addressed. after the animal has stabilized, the treatment of sepsis should be aimed at removing the septic focus. obvious sources of infection should be drained or surgically removed. if an implant is infected, it should be removed. antibiotic therapy should also be instituted. the choice of antibiotic will ultimately depend upon the results of culture; however, the initial choice of antibiotics is based on previous experience, source of infection, and gram stains. the organisms associated with sepsis are often gram-negative bacteria of gastrointestinal origin or are previously encountered nosocomial infections. ideally, the antibiotic chosen for initial therapy should be a broad-spectrum, bactericidal drug that can be administered intravenously. second-or thirdgeneration cephalosporins provide good coverage, as does combination therapy with enrofloxacin plus metronidazole or penicillin. finally, the treatment of sepsis is aimed at blocking the mediators of the systemic response. this category of sepsis treatment is the focus of much research. several studies have examined the effects of steroids, nonsteroidal anti-inflammatory drugs, and antibodies directed against endotoxin, cytokines, or other mediators of the inflammatory response; however, none of these treatments have proven greatly effective in clinical trials. consequently, there is no 'magic bullet' for the treatment of sepsis at this time. successful therapy remains dependent on aggressive supportive care coupled with identification and elimination of the inciting infection. etiology in research animals, aspiration may occur accidentally during the oral administration of various substances or by the misplacement of gastric tubes. aspiration of gastric contents may also occur as a complication of anesthesia. in pet animals, aspiration is often seen as a result of metabolic and anatomical abnormalities; however, such occurrence would be rare in the research setting. pathogenesis aspirated compounds can produce direct injury to lung tissue, but more importantly, the aspiration provokes an inflammatory response, probably mediated by cytokines. the result is a rapid influx of neutrophils into the lung parenchyma and alveolar spaces. the inflammation leads to increased vascular permeability with leakage of fluid into the alveolar spaces and can eventually lead to alveolar collapse. if the condition is severe, it may result in adult respiratory distress syndrome and respiratory failure. it should be noted that infection is not present in the early stages of this condition but may complicate the problem after - h. clinical signs the severity and clinical manifestation of aspiration lung injury are dependent upon the ph, osmolality, and volume of the aspirate. the signs of aspiration lung injury may include cough, increased respiratory rate, pronounced respiratory effort, and fever. when respiration is severely affected, the oxygen saturation of blood will be decreased. the diagnosis of this problem is based on witness of aspiration, history consistent with aspiration, and/or the physical findings. classically, radiographs of the thorax demonstrate a bronchoalveolar pattern in the cranioventral lung fields. however, these lesions may not appear for several hours after the incident of aspiration. in addition, the location of the lesions may be variable, depending on the orientation of the animal at the time of aspiration. treatment the treatment of aspiration lung injury is largely supportive and depends upon the severity of the inflammation and the clinical signs. if the aspiration is witnessed, the mouth and, idealy, the upper airway should be cleared of residual material. when small amounts of a relatively innocuous substance (e.g., barium) have been aspirated, treatment may not be necessary. when severe inflammation is present, systemic as well as localized therapy may be necessary. oxygen therapy may be instituted; however, the concentration and time frame are controversial, because lung injury may be exacerbated by long-term administration of oxygen at high concentrations (nader-djahal et al., ) . fluid therapy may also be necessary in severe cases; however, cardiovascular support should be performed judiciously as fluid overload could lead to an increase in pulmonary edema. the use of colloids is also controversial because of the increase in vascular permeability that occurs in the lungs. several studies have addressed the use of anti-inflammatory agents to reduce lung injury associated with aspiration; however, none are used clinically in human or veterinary medicine at this time. corticosteroids are contraindicated (raghavendran et al., ) . in humans, antibiotics are reserved for cases with confirmed infection, in order to prevent the development of antibiotic-resistant pneumonia. it has been suggested that dogs should be immediately treated with antibiotics when the aspirated material is not acidic or has potentially been contaminated by oral bacteria associated with severe dental disease. amoxicillin-clavulanate has been recommended as a first line of defense, reserving enrofloxacin for resistant cases (hawkins, ) . the presence of pneumonia should be verified by tracheal wash and cultures. prevention aspiration of drugs and other compounds may be avoided through careful administration of oral medications by experienced individuals. likewise, gavage or orogastric administration of liquids should be performed by experienced individuals, and the procedure should be aborted if coughing or other respiratory signs occur. the aspiration of stomach contents can largely be avoided by appropriate fasting prior to anesthesia for at least h for food and h for water. if appropriate fasting times are not observed, anesthesia should be postponed whenever possible, particularly if intended procedures require manipulation of the viscera or head-down positioning of the dog. if anesthesia cannot be avoided, it should be rapidly induced and the dog should be intubated. during recovery from anesthesia, the endotracheal tube should be removed with the cuff partially inflated and with the dog in a head-up position (haskins, ) . based on the source of energy, burn wounds may be categorized into four groups: thermal, chemical, radiation, and electrical. in laboratory animals, accidental burns are usually the result of thermal injury (heating pads, water bottles), chemicals (strong alkalis, acids, disinfectants, drugs), or experimental irradiation protocols. etiology inappropriate use of external heating devices is the most common cause of burns in laboratory animal medicine. the insult to the skin results in desiccation of the tissue and coagulation of proteins. in addition, the severely injured area is surrounded by a zone of vascular stasis, which promotes additional tissue damage. even small burns can result in significant inflammation that could affect the outcome of some research investigations and cause considerable discomfort to the animal. the proper and immediate treatment of burn wounds can reduce the effects of the injury on both the individual and the research. clinical signs the clinical signs vary with the depth, location, and surface area of burn injury. classification systems for thermal burns are generally based on the depth of the injury, varying from superficial involvement of only epidermis to complete destruction of skin and subcutaneous tissues (bohling, ) . superficial burns appear erythematous and inflamed. in some cases, matting of the overlaying hair with exudate may be the first sign of a previously undetected skin lesion. progressive hair and skin loss may be evident over the first few days after injury (johnston, ) . although blistering is a characteristic of partial thickness burns in humans, this is rarely seen in dogs (bohling, ) . uncomplicated, superficial burn wounds heal by reepithelialization within - days. deeper burn wounds are characterized by a central area of nonviable tissue surrounded by edematous, inflamed tissues. a thick eschar, composed of the coagulated proteins and desiccated tissue fluid, develops over deep burn wounds. these wounds heal by granulation under the eschar, which will eventually slough. the amount of pain associated with burns depends upon several factors including the depth and area of the wound, procedural manipulations, and movement at the affected site (bohling, ) . pain associated with superficial burn wounds usually subsides in - days. theoretically, deep burns destroy nerve endings and result in less pain than superficial burns. however, inflammatory pain may still be present due to the tissue reaction around the necrotic site. in addition, sharp procedural pain and breakthrough pain have been described in humans during the healing phases of burn injuries and should be considered as potential complications in dogs as well (bohling, ) . severe and widespread accidental burn injury can result in clinical signs associated with multiple organs including the pulmonary, gastrointestinal, hematopoietic, and immune systems. in addition, extensive burn injury can predispose to infection and even sepsis. this type of injury with the associated complications would be extremely rare in the laboratory setting. treatment appropriate and timely treatment of a burn wound will reduce the extent of tissue damage and associated pain. thermal injuries should be immediately exposed to cool water ( °c) to reduce edema and pain. exposure to very cold water and ice does not improve outcomes (bohling, ) . topical wound dressings are recommended in the early stages of treatment for both partial-and full-thickness burns that are of small size. systemic antibiotics are unable to penetrate eschar and are not adequately distributed through the abnormal blood supply of burned tissues. therefore, a thin film of a water-soluble, broad-spectrum antibiotic ointment should be applied to the wound surface. silver sulfadiazine has a broad spectrum, penetrates eschar, and is often the preparation of choice for burn wound therapy. povidone-iodine ointment will also penetrate thin eschar and provides a broad spectrum. mafenide has a good spectrum that covers gram-negative organisms well and is often used to treat infected wounds, although it has been associated with pain upon application (demling and lalonde, ) . once a topical antibiotic has been applied, a nonadherent dressing should be placed on the wound. burn wounds covered in such a manner tend to epithelialize more rapidly and are less painful than uncovered wounds (demling and lalonde, ; bohling, ) . after the initial treatment, burn wounds should be gently cleansed two to three times a day, followed by reapplication of the topical antibiotic and rebandaging (demling and lalonde, ) . systemic antibiotics are indicated in cases where local or systemic infection is present and their ultimate selection should be based on culture results. burn wounds can be extremely painful, and analgesia should be instituted immediately and adjusted accordingly throughout the treatment period. surgical intervention may be necessary in some cases. with small or moderately sized wounds, the eschar over the burn wound may actually impede wound contraction and reepithelialization. in such cases, once the eschar has become fully defined, a complete resection may improve wound healing. with large and severe burn wounds, repeated debridement by surgery or other means might be necessary. in the laboratory setting, a decision to pursue extensive surgical intervention would be dependent upon full consideration of the effects on animal welfare and research results. prevention thermal burns can be prevented in the research setting. electric heating pads and heat lamps should be avoided if possible. only heated water blankets or circulating warm air devices should be used to provide warmth to the animals. in rare instances, heated water blankets have also caused burns; therefore, these devices should be carefully monitored. as a precaution, a thin towel may be placed between the animal and the water blanket. basic fire prevention precautions should be taken particularly around oxygen sources and flammable agents etiology chemical injury may be due to skin contact with concentrated solutions such as disinfectants or inadvertent exposure to laboratory chemicals. in addition, perivascular injection of certain drugs (pentobarbital, thiamylal, thiopental, thiacetarsemide, vincristine, vinblastine, and doxorubicin) have been associated with extensive tissue damage (swaim and angarano, ; waldron and trevor, ) . the mechanism of action will vary depending upon the ph, osmolality, and chemical composition of the agent and may include oxidation, reduction, disruption of lipid membranes, or other reactions (bohling, ; swaim, ; waldron and trevor, ) . clinical signs surface contact with chemicals may result in mild irritation and redness of superficial layers of the skin. however, many agents may cause progressive injury until the chemical reaction has been neutralized. this may result in tissue necrosis and secondary infection. the immediate signs of perivascular injection are withdrawal of the limb or other signs of discomfort and swelling at the injection site. the area may appear red, swollen, and painful as inflammation progresses. there may eventually be necrosis of the skin around the injection site. in cases of doxorubicin extravasation, signs may develop up to a week after the injection, and the affected area may progressively enlarge over a -to -month period. this is because the drug is released over time from the dying cells (swaim and angarano, ) . treatment in cases of skin contact with chemical agents, the affected area should be thoroughly and repeatedly lavaged with warm water to dilute or remove the substance. the material safety data sheet for the substance should be consulted for any possible neutralization protocol. additional treatment will depend upon the severity of the tissue damage and will follow the same guidelines as for the thermal injury described earlier. for the treatment of perivascular injections, dilution of the drug with subcutaneous injections of saline is recommended. in addition, steroids may be infiltrated locally to reduce inflammation. topical application of dimethyl sulfoxide (dmso) may also be helpful in reducing the immediate inflammation and avoiding the development of chronic lesions. the addition of lidocaine to subcutaneous injections of saline has been used in cases of thiacetarsemide injection (hoskins, ) . the local infiltration of hyaluronidase accompanied by warm compresses has been suggested for perivascular vinblastine (waldron and trevor, ) and for doxirubicin. the use of dmso or another free radical scavenger, dexrazoxane, infused at the site has also been suggested for doxorubicin toxicities. despite these treatments, necrosis of skin may be observed and would require serial debridement of tissues with secondary wound closure or skin grafting. in cases of doxorubicin extravasation, early excision of affected tissues is advocated to prevent the progressive sloughing caused by sustained release of the drug from dying tissues (swaim and angarano, ) . in all cases, the condition can be painful and analgesia should be addressed. prevention prior to the use of any substance, the investigator should be aware of its chemical composition and the potential for problems. the material safety data sheets should be available for all compounds and storage recommendations followed closely. for intravenous administration of toxic compounds, insertion of an indwelling catheter is extremely important. prior to the injection, the catheter should be checked repeatedly for patency by withdrawal of blood and injection of saline. any swelling at the catheter site or discomfort by the subject indicates that the catheter should not be used. access to a central vessel such as the cranial or caudal vena cava is preferred over the use of peripheral vessels. when peripheral catheters are used, the injection should be followed by a vigorous amount of flushing with saline or other physiological solution and removal of the catheter. additional injections are best given through newly placed catheters in previously unused vessels. the repeated use of an indwelling peripheral catheter should be approached cautiously and done only out of necessity. etiology radiation burns are generally a complication of therapeutic administration and are a result of free oxygen radical formation (waldron, ) . the severity of radiation burns and their treatment will depend upon the dose, frequency, total surface area, and location of the radiation. damage to epithelial layers of the skin can lead to desquamation. direct injury to fibroblasts results in decreased collagen production and poor wound healing. in addition, there may be fibrosis of blood vessels (pavletic, ) and subsequent hypoxia causing necrosis of deeper tissues. clinical signs the tissues most often affected are the skin and mucus membranes. with superficial injury, affected skin may exhibit hair loss and erythema, and produce a clear exudate. the intensity of the inflammation may increase for - weeks after the completion of radiation treatment. deeper and more serious injury manifests with subcutaneous fibrosis and can lead to disfigurement . the skin and underlying deep structures including the bone may become necrotic over several weeks (pavletic, ) . these deeper injuries are prone to infection due to their lack of blood supply. systemic signs such as vomiting are rare in dogs unless there has been direct radiation treatment to organs . treatment with superficial skin burns, the wound should be kept clean and should be covered if possible. in cases of oral mucous membrane damage, there may be special feeding requirements. when wounds are ulcerated, avascular tissues should be excised. treatments with silver sulfadiazine, mafenide acetate, or other topical agents are recommended to control infection. in addition, infection is avoided by closure of the wound as soon as possible. the goal of surgery is to cover the wound with healthy tissue to promote vascularization of the area. in some cases, this may require muscle and/ or skin grafts. prevention radiation burns can be limited by selection of appropriate, fractionated therapy and application of shielding to reduce exposure. prompt treatment of the injuries can reduce the occurrence of infection. since radiation is associated with poor wound healing, complications may arise when additional procedures are required. it is recommended to wait at least week (pavletic, ) or even longer (laing, ) prior to administering radiation to a surgical site. after radiation, routine surgeries should be avoided for - months (pavletic, ). the prevalence of cancer in the general canine population has increased over the years (dorn, ) . this can be attributed to the longer life spans resulting from improvements in nutrition, disease control, and therapeutic medicine. because of these changes, cancer has become a major cause of death in dogs (bronson, ) . in a lifetime cancer mortality study of intact beagles of both sexes, albert et al. ( ) found death rates similar to the death rate of the at-large dog population (bronson, ) . approximately % of the male beagles died of cancer. the majority of the tumors were lymphomas ( %) and sarcomas ( %), including hemangiosarcomas of the skin and fibrosarcomas. of the female beagles dying of cancer ( % of the population studied), three-quarters had mammary cancer ( %), lymphomas ( %), or sarcomas ( %). of the sarcomas in females, one-third were mast cell tumors. in addition to these tumors that cause mortality, the beagle is also at risk for thyroid neoplasia (hayes and fraumeni, ; benjamin et al., ) . because of the popularity of the beagle as a laboratory animal, discussion of specific neoplasms will focus on the tumors for which this breed is at risk, as well as tumors that are common in the general canine population. a complete review of clinical oncology in the dog is beyond the scope of this chapter but can be found elsewhere (withrow et al., ) . fine-needle aspirates are generally the first diagnostic option for palpable masses, because they can easily be performed in awake, cooperative patients. this technique allows for rapid differentiation of benign and neoplastic processes. in cases where cytologic results from fine-needle aspirates are not definitive, more invasive techniques must be used. needle-punch or core biopsies can also be performed in awake patients with local anesthesia. an instrument such as a tru-cut ® needle (travenol laboratories, inc., deerfield, illinois) is used to obtain a -mm × - . -cm biopsy of a solid mass. a definitive diagnosis may be limited by the size of the sample acquired using this technique. incisional and excisional biopsies are utilized when less invasive techniques fail to yield diagnostic results. excisional biopsies aid in histopathological examination and are the treatment of choice when surgery is necessary, because the entire mass is removed. surgical margins should extend at least cm around the tumor and cm if mast cell tumors are suspected (morrison et al., ) . incisional biopsies are performed when large softtissue tumors are encountered and/or when complete excision would be surgically difficult or life-threatening. when performing an incisional biopsy, always select tissue from the margin of the lesion and include normal tissue in the submission. etiology lymphomas are a diverse group of neoplasms that originate from lymphoreticular cells. canine lymphoma represents - % of canine tumors and a majority ( %) of canine hematopoetic disease (ettinger, ; vail and young, ) . whereas retroviral etiologies have been demonstrated in a number of species (e.g., cat, mouse, chicken), conclusive evidence of a viral etiology has not been established in the dog. in humans, data implicate the herbicide , -dichlorophenoxyacetic acid as a cause of non-hodgkin's lymphoma, but studies in dogs with similar conclusions have come under scrutiny (macewen and young, ) . in addition, tobacco smoke, environmental chemicals, and waste emissions are considered possible risk factors (marconato et al., ; gavazza et al., ) clinical signs multicentric high-grade lymphoma (mhgl) accounts for the majority of reported cases of canine lymphoma. depending upon grade, immunophenotype, and location involved, dogs with mhgl usually present with painless, enlarged lymph nodes and nonspecific signs such as anorexia, weight loss, polyuria, polydypsia, fever, and lethargy. when the liver and spleen are involved, generalized organomegaly may be felt on abdominal palpation. less commonly, dogs develop alimentary, mediastinal, cutaneous, and extranodal lymphomas. alimentary lymphoma is associated with vomiting and diarrhea, in addition to clinical signs associated with mhgl. dogs with mediastinal lymphoma often present with respiratory signs (dyspnea and exercise intolerance) secondary to pleural effusion or cranial vena caval syndrome. hypercalcemia is most frequently associated with this form of lymphoma and may result in polyuria, polydypsia, and weakness. cutaneous lymphoma is an uncommon epitheliotrophic form of lymphoma. it is often referred to as mycosis fungoides and is typically of a cd + t-cell immunophenotype. it varies in presentation from solitary to generalized and may mimic any of a number of other inflammatory skin disorders including oral mucosal lesions. the lesions may occur as erythema, plaques, erosions, scales, nodules, crusts, hypopigmentation, and alopecia (fontaine et al., ) . approximately half of the cases are pruritic. a number of extranodal forms of lymphoma have been reported, including tumors affecting the eyes, central nervous system, kidneys, or nasal cavity. clinical presentation varies, depending on the site of involvement (e.g., nervous system: seizures, paresis, paralysis). epizootiology the incidence of lymphoma is highest in dogs - years old, accounting for % of cases. although the neoplasm generally affects dogs older than year, cases in puppies as young as months have been reported (dorn et al., ) . no gender predilection has been reported. diagnosis and pathologic findings a fine-needle aspirate is initially performed on accessible lymph nodes. thoracic radiographs and abdominal ultrasound ± fine needle aspiration of the liver or spleen can be used if mediastinal or abdominal involvement is suspected. additional staging can be determined through complete blood counts, serum biochemistry, flow cytometry for immunotyping, bone marrow aspiration, or surgical lymphadenectomy and histology. enlarged neoplastic lymph nodes vary in diameter from to cm and are moderately firm. some may have areas of central necrosis and are soft to partially liquefied. the demarcation between cortex and medulla is generally lost, and on cut section, the surface is homogenous. the spleen may have multiple small nodular masses or diffuse involvement with generalized enlargement. the enlarged liver may have disseminated pale foci or multiple large, pale nodules. in the gastrointestinal tract, both nodular and diffuse growths are observed. these masses may invade through the stomach and intestinal walls. flow cytometry and lymphoblastic markers (cd ) can aid in diagnosis and subtyping of tumors. in addition, positron emission tomography is being explored for detection of extranodal and metastatic lymphoma (leblanc et al., ; marconato, ; elstrom et al., ) . classification of lymphoma types is based upon cytological, morphological, and immunological characteristics using the kiel classification criteria (vail and young, ) . histologically, the most common lymphomas are classified as intermediate to high grade and of large-cell (histiocytic) origin. the neoplastic lymphocytes typically obliterate the normal architecture of the lymph nodes and may involve the capsule and perinodal areas. lymphoma subtypes can be further characterized based upon genetic, molecular, and immunological criteria (ponce et al., ) . pathogenesis all lymphomas regardless of location should be considered malignant. a system for staging lymphoma has been established by the world health organization. the average survival time for dogs without treatment is - weeks. survival of animals undergoing chemotherapy is dependent on the treatment regimen as well as the form and stage of lymphoma (macewen and young, ) . median survival time with aggressive therapy is generally less than months. hypercalcemia is a paraneoplastic syndrome frequently associated with lymphoma. the pathogenesis of this phenomenon is not fully understood but may be a result of a parathormone-like substance produced by the neoplastic lymphocytes. differential diagnosis differential diagnoses for multicentric lymphoma include systemic mycosis; salmon-poisoning and other rickettsial infections; lymph node hyperplasia from viral, bacterial, and/or immunologic causes; and dermatopathic lymphadenopathy. alimentary lymphoma must be distinguished from other gastrointestinal tumors, foreign bodies, and lymphocytic-plasmacytic enteritis. in order to make a definitive diagnosis, whole lymph node biopsies and full-thickness intestinal sections for histopathologic examination may be needed. treatment therapy for lymphoma primarily consists of one or a combination of several chemotherapeutic agents. in addition, radiation therapy and bone marrow transplantation have been utilized. the treatment regimen is based on the staging of the disease, the presence of paraneoplastic syndromes, and the overall condition of the patient. although treatment may induce clinical remission and prolong short-term survival, most treatment is palliative and aimed at improving quality of life. a thorough discussion of therapeutic options for the treatment of lymphomas in the dog can be found elsewhere (chun, ; marconato, ) . future directions include development of molecular and cellular targeted therapies to enhance traditional chemotherapy treatment, prolong remission, and treat immunologic subtypes of lymphoma (e.g., t-cell lymphoma). research complications given the grave prognosis for lymphoma with or without treatment, euthanasia should be considered for research animals with significant clinical illness. etiology mast cells are derived from cd + bone marrow progenitor cells. neoplastic proliferations of mast cells are the most commonly observed skin tumor of the dog and may account for up to % of canine skin laboratory animal medicine tumors (bostock, ; welle et al., ) . mast cells are normally found in the connective tissue beneath serous surfaces and mucous membranes, and within the skin, lung, liver, and gastrointestinal tract. current research has linked mast cell tumor development to multifactorial causes including breed predisposition and a genetic component, chronic inflammation, and mutations in the surface growth factor, c-kit (ma et al., ; reguera et al., ; webster et al., ) . clinical signs well-differentiated mast cell tumors are typically solitary, well-circumscribed, slow-growing, -to -cm nodules in the dermis and subcutaneous tissue. alopecia may be observed, but ulceration is not usual. poorly differentiated tumors grow rapidly, may ulcerate, and may cause irritation, inflammation, and edema to surrounding tissues. mast cell tumors can be found on any portion of the dog's skin but frequently affect the trunk and hind limb extremeties along with perineal and preputial areas. the tumors usually appear to be discrete masses, but they frequently extend deep into surrounding tissues. abdominal organs are rarely involved but may be associated with anorexia, vomiting, melena, abdominal pain, and gastrointestinal ulceration. mast cell tumors have also been reported in extracutaneous areas such as the salivary glands, larynx, nasopharynx (london and thamm, ) and conjunctiva (fife et al., ) . mast cell tumors within the perineal, preputial, or inguinal areas are associated with a greater predilection for recurrence or metastasis (misdorp, ) . epizootiology these tumors tend to affect middleaged dogs but have been observed in dogs ranging from months to years (pulley and stannard, ) . pathologic findings because of the substantial variation in histologic appearance of mast cell tumors, a classification and grading system described by patnaik et al. ( ) has become widely accepted. in this system, grade i has the best prognosis and are well differentiated, with round to ovoid, uniform cells with distinct cell borders. the nuclei are round and regular, the cytoplasm is packed with large granules that stain deeply, and mitotic figures are rare to absent. grade ii (intermediately differentiated) mast cell tumors have indistinct cytoplasmic boundaries with higher nuclear-cytoplasmic ratios, fewer granules, and occasional mitotic figures. grade iii (anaplastic or undifferentiated) mast cell tumors have the worst prognosis. the cells contain large, irregular nuclei with multiple prominent nucleoli and few cytoplasmic granules. mitotic figures are much more frequent. cells are pleomorphic with indistinct borders. in addition to associated skin lesions (e.g., ulceration, collagenolysis, necrosis, and infection), mast cell tumors have been associated with gastric ulcers in the fundus, pylorus, and/or proximal duodenum, most likely secondary to tumor production of histamine. histamine stimulates the h receptors of the gastric parietal cells, causing increased acid secretion. gastric ulcers have been observed in large numbers (> %) of dogs with mast cell tumors (howard et al., ) . pathogenesis although all mast cell tumors should be considered potentially malignant, the outcome in individual cases can be correlated with the histologic grading of the tumor. grade iii tumors are most likely to disseminate internally. this spread is usually to regional lymph nodes, spleen, and liver, and less frequently to the kidneys, lungs, and heart. diagnosis and differential diagnosis using fineneedle aspiration, mast cell tumors can be distinguished cytologically from other round cell tumors (such as histiocytomas and cutaneous lymphomas) by using toluidine blue to metachromatically stain the cytoplasmic granules red or purple. mast cell granules can also be stained with wright's, giemsa, and romanowsky stains. in addition, mast cells may contain tryptase, chymase, or both (fernandez et al., ) . histological evaluation is generally required for grading. examination of regional lymph nodes may be warranted if metastatic or systemic disease is suspected. in addition, radiographs and ultrasound with guided aspirates of the liver, spleen, or sublumbar lymph nodes can be used to determine metastatic disease. treatment depending upon the grade, initial treatment for mast cell tumors is generally wide surgical excision ( -cm margins), which may be followed by radiation, chemotherapy, or glucocorticoid therapy. aspiration or surgical removal of regional lymph nodes is recommended if lymphatic tumor drainage is suspected. if the tumor is not completely resectable or is grade ii or iii (moderately to undifferentiated), then debulking surgery and adjunct therapy may be used. treatment algorithms are outlined elsewhere (withrow et al., ) research complications because of the potential for systemic release of substances such as histamine, vasoactive substances, heparin, eosiniphilic chemotactic factor, and proteolytic enzymes, along with the possibility of delayed wound healing and tumor recurrence, dogs with mast cell tumors are not good candidates for research studies. grade i mast cell tumors may be excised, allowing dogs to continue on study; however, monitoring for local recurrence should be performed monthly. grade ii tumors are variable; animals that undergo treatment should be monitored for recurrence monthly, and evaluation of the buffy coat should be performed every - months for detection of systemic mastocytosis. because of the poor prognosis for grade iii tumors, treatment is unwarranted in the research setting. etiology also known as infectious or venereal granuloma, sticker tumor, transmissible sarcoma, and contagious venereal tumor, the canine transmissible venereal tumor (ctvt) is transmitted horizontally to the genitals by coitus (nielsen and kennedy, ) . ctvt is a 'parasitic-like' tumor that appears to have originated from dogs or wolves thousands of years ago and despite immense mutation, ctvt adapted, survived, and spread across multiple continents making it the oldest known continuously passaged somatic cell line (rebbeck et al., ; murchison et al., ; murgia et al., ) . it has been described as a round cell tumor of histiocytic origin. although this tumor has been reported in most parts of the world, it is most prevalent in tropical or temperate climates (macewen, ) . clinical signs the tumors are usually cauliflowerlike masses on the external genitalia, but they can also be pedunculated, nodular, papillary, or multilobulated. these friable masses vary in size up to cm, and hemorrhage is frequently observed. in male dogs, the lesions are found on the caudal part of the penis from the crura to the bulbus glandis or on the glans penis. less frequently, the tumor is found on the prepuce. females typically have lesions in the posterior vagina at the junction of the vestibule and vagina. when located around the urethral orifice, the mass may protrude from the vulva. these tumors have also been reported in the oral cavity, skin, and eyes. epizootiology and transmission ctvts are most commonly observed in young, sexually active dogs. transmission takes place during coitus when injury to the genitalia allows for exfoliation and transplantation of the tumor. genital to oral to genital transmission has also been documented (nielsen and kennedy, ) . extragenital lesions may be the result of oral contact with previously traumatized areas. pathologic findings histologically, cells are arranged in compact masses or sheets. the cells are round, ovoid, or polyhedral, and have large, round nuclei with coarse chromatin. the cytoplasm is eosinophilic with small vacuoles arranged in a 'string of pearls' pattern. pathogenesis tumor growth occurs within - months after mating or implantation, and then growth generally slows. metastasis is rare (< - % of cases) but may involve the superficial inguinal and external iliac lymph nodes as well as distant sites. spontaneous regression may occur within - months of tumor development. diagnosis and differential diagnosis transmissible venereal tumors have been confused with lymphomas, histiocytomas, mast cell tumors, and amelanotic melanomas. however, cytological examination of impression smears, swabs, and fine-needle aspirates generally provide a definitive diagnosis. although not usually required, histopathology of a biopsy from the mass can aid in diagnosis. prevention thorough physical examinations prior to bringing new animals into a breeding program should prevent introduction of this tumor into a colony. control removing affected individuals from a breeding program should stop further spread through the colony. treatment surgery and radiation can be used for treatment, but chemotherapy is the most effective. vincristine ( . - . mg/m ) iv once weekly for four to six treatments will induce remission and cure in greater than % of the cases (macewen, ) . research complications experimental implantation of ctvts has been shown to elicit formation of tumor-specific igg (cohen, ) . this response may occur in natural infections and could possibly interfere with immunologic studies. etiology dogs are susceptible to a wide variety of mammary gland neoplasms, most of which are influenced by circulating reproductive steroidal hormones. clinical signs single nodules are found in approximately % of the cases of canine mammary tumors. the nodules can be found in the glandular tissue or associated with the nipple. masses in the two most caudal glands (fourth and fifth) account for a majority of the tumors. benign tumors tend to be small, well circumscribed, and firm, whereas malignant tumors are larger, invasive, and coalescent with adjacent tissues. inflammatory mammary carcinomas may mimic mastitis or severe dermatitis and must be ruled out to prevent misdiagnosis. epizootiology mammary tumors are uncommon in dogs under years of age with the incidence rising sharply after that. the median age at diagnosis is - years. a longitudinal study of a large beagle colony showed that significant risk for development of mammary tumors begins at approximately years of age (taylor et al., ) . mammary tumors occur almost exclusively in female dogs, with most reports in male dogs being associated with endocrine abnormalities, such as estrogen-secreting sertoli cell tumors. pathologic findings the t (tumor size), n (lymph node involvement), and m (metastasis) system is commonly used to stage mammary tumors. based on histologic classification of mammary gland tumors, approximately half of the reported tumors are benign (fibroadenomas, simple adenomas, and benign mesenchymal tumors), and half are malignant (solid carcinomas, tubular adenocarcinomas, papillary adenocarcinomas, anaplastic carcinomas, sarcomas, and carcinosarcomas) (bostock, ) . histopathologic grades are scored based upon tubule formation, nuclear pleomorphism, and mitosis (elston and ellis, ) . extensive discussions of classification, staging, and histopathologic correlations can be found elsewhere (moulton, ; sorenmo et al., ) . pathogenesis mammary tumors of the dog develop under the influence of hormones. receptors for both estrogen and progesterone can be found in - % of tumors. malignant mammary tumors typically spread through the lymphatic vessels. metastasis from the first, second, and third mammary glands is to the ipsilateral axillary or anterior sternal lymph nodes. the fourth and fifth mammary glands drain to the superficial inguinal lymph nodes where metastasis can be found. many mammary carcinomas will eventually metastasize to the lungs and extraskeleton. diagnosis and differential diagnosis both benign and malignant mammary tumors must be distinguished from mammary hyperplasia, mastitis, and severe dermatitis. cytological evaluation from fine-needle aspirates correlates well with histological examination of benign and malignant tumors (simon et al., ) . radiographs and possibly ultrasound should be performed to rule out metastatic disease prior to surgery. prevention the lifetime risk of developing mammary tumors can effectively be reduced to . % by spaying bitches prior to the first estrus (schneider et al., ) . this is commonly done in the general pet population at months of age. the protective effects of early spay rapidly decrease after several estrus cycles. dogs spayed prior to the first estrus had a risk of . %, whereas dogs spayed after the first and second estrus had risks of % and %, respectively. treatment surgery is the treatment of choice for mammary tumors, because chemotherapy and radiation therapy have not been reported to be effective. the extent of the surgery is dependent on the area involved. single mammary tumors should be surgically removed with -cm lateral margins or margins wide enough for complete resection. deep margins may include removing sections of abdominal fascia or musculature en bloc with mammary tumor. multiple mammary tumors should be removed via regional or unilateral chain mastectomies. bilateral, staged mastectomies are reserved for more aggressive tumors. there is insufficient evidence at this time to recommend routine complete unilateral or bilateral chain mastectomies. at the time of surgery, axillary lymph nodes are removed only if enlarged or positive on cytology for metastasis. sorenmo et al. ( ) provide a thorough review of canine mammary gland neoplasia. research complications treatment of early-stage or low-grade mammary tumors may be rewarding, allowing dogs to continue on study. if removed early enough, malignant masses could yield the same results. all dogs should be monitored regularly for recurrence and new mammary tumors. beagles are subject to many of the inherited and/ or congenital disorders that affect dogs in general. in a reference table on the congenital defects of dogs (hoskins, ) , disorders for which beagles are specifically mentioned include brachyury (short tail), spina bifida, pulmonic stenosis, cleft palate-cleft lip complex, deafness, cataracts, glaucoma, microphthalmos, optic nerve hypoplasia, retinal dysplasia, tapetal hypoplasia, factor vii deficiency, pyruvate kinase deficiency, pancreatic hypoplasia, epilepsy, gm gangliosidosis, globoid cell leukodystrophy, xx sex reversal, and cutaneous asthenia (ehlers-danlos syndrome). other defects observed include cryptorchidism, monorchidism, limb deformity, inguinal hernia, diaphragmatic hernia, hydrocephaly, and fetal anasarca. each of these other congenital defects occurred at less than . % incidence. etiology benign prostatic hyperplasia (bph) is an age-related condition in intact male dogs. the hyperplasia of prostatic glandular tissue is a response to the presence of both testosterone and estrogen. clinical signs bph is often subclinical. straining to defecate (tenesmus) may be seen because the enlarged gland impinges on the rectum. urethral discharge (yellow to red) and hematuria can also be presenting clinical signs. epizootiology and transmission bph typically affects older dogs (> years), although glandular hyperplasia begins as early as years of age. approximately % of inact male dogs will develop bph by years of age (smith, ) . pathologic findings in the early stages of bph, there is hyperplasia of the prostatic glandular tissue. this is in contrast to human bph, which is primarily stromal in origin. eventually, the hyperplasia tends to be cystic, with the cysts containing a clear to yellow fluid. the prostate becomes more vascular with a honeycomb appearance (resulting in hematuria or hemorrhagic urethral discharge), and bph may be accompanied by mild chronic inflammation. pathogenesis bph occurs in older intact male dogs because increased production of estrogens (estrone and estradiol), combined with decreased secretion of androgens, sensitizes prostatic androgen receptors to dihydrotestosterone. the presence of estrogens may also increase the number of androgen receptors, and hyperplastic prostate glands also have an increased ability to metabolize testosterone to α-dihydrotestosterone (kustritz and klausner, ) mediating bph. diagnosis and differential diagnosis bph is diagnosed in cases of nonpainful symmetrical swelling of the prostate gland in intact male dogs, with normal hematologic profiles and urinalysis that may be characterized by hemorrhage. a prostatic biopsy can be performed to confirm diagnosis. differential diagnoses include squamous metaplasia of the prostate, para-prostatic cysts, bacterial prostatitis, prostatic abscessation, and prostatic neoplasia (primarily adenocarcinoma). these differential diagnoses also increase in frequency with age and, except for squamous metaplasia, can also occur in castrated dogs. as such, these conditions do not necessarily abate or resolve with castration. prevention castration is the primary means for prevention of benign prostatic hyperplasia. treatment the first and foremost treatment for bph is castration. in pure cases of bph, castration results in involution of the prostate gland detectable by rectal palpation within - days. for most dogs in research studies, this is a viable option to rapidly improve the animal's condition. the alternative to castration is hormonal therapy, primarily with estrogens. this may be applicable in cases where semen collection is necessary from a valuable breeding male (e.g., genetic diseases). if the research study concerns steroidal hormone functions, then neither the condition nor the treatment is compatible. finasteride, a synthetic α-reductase inhibitor, has been used in dogs to limit the metabolism of testosterone to α-dihydrotestosterone. treatment at daily doses of . - . mg/kg orally for weeks was shown to reduce prostatic diameter and volume without affecting testicular spermatogenesis (sirinarumitr et al., ) . upon discontinuation of finasteride, the prostate generally returns to its pretreatment size within several months (smith, ) . gonadotropin-releasing hormone analogs such as desorelin inhibit production of testosterone and estrogen via negative feedback on the hypothalamus-pituitary axis. this is available in a sustained release subcutaneous implant, which has demonstrated efficacy in reducing prostatic size in dogs (junaidi et al., ) . however, medical therapy has not shown to be as advantageous as castration. research complications bph can cause complications to steroidal hormone studies, in that the condition may be indicative of abnormal steroidal hormone metabolism, and neither castration nor estrogen therapy is compatible with study continuation. the development of tenesmus as a clinical sign may also affect studies of colorectal or anal function. research model older dogs with benign protastic hypertrophy are used in research to evaluate the use of ultrasonic histotripsy as a precise nonsurgical urethralsparing alternative to prostate surgery (lake et al., ; hall et al., ; schade et al., ) . etiology juvenile polyarteritis syndrome (jps), also known as steroid-responsive meningitis-arteritis, is a painful disorder seen in young beagles (occasionally reported in other breeds) caused by a systemic necrotizing vasculitis. the cause of the vasculitis has not been established but appears to have an autoimmune-mediated component and may have a hereditary predisposition. clinical signs clinical signs of jps include fever, anorexia, lethargy, and reluctance to move the head and neck. the dogs tend to have a hunched posture and/ or an extended head and neck. most dogs seem to be in pain when touched, especially in the neck region. neurological examination may reveal proprioceptive deficits, paresis, or paralysis. the syndrome typically has a course of remissions and relapses characterized by - days of illness and - weeks of remission (scott-moncrieff et al., ) . there may be a component of this condition that is subclinical, given that vasculitis has been diagnosed postmortem in beagles that had no presenting signs. epizootiology and transmission jps typically affects young beagles ( - months), with no sex predilection. jps has been reported in other breeds including sibling welsh springer spaniels (caswell and nykamp, ) . pathologic findings on gross necropsy, foci of hemorrhage can be seen in the coronary grooves of the heart, cranial mediastinum, and cervical spinal cord meninges (snyder et al., ) . local lymph nodes may be enlarged and hemorrhagic. histologically, necrotizing vasculitis and perivasculitis of small to mediumsized arteries are seen. these lesions are most noticeable where gross lesions are observed, but they may be seen in other visceral locations. arterial fibrinoid necrosis leading to thyroid gland hemorrhage and inflammation was also reported (peace et al., ) . the perivasculitis often results in nodules of inflammatory cells that eccentrically surround the arteries. the cellular composition of these nodules is predominantly neutrophils, but it can also consist of lymphocytes, plasma cells, or macrophages (snyder et al., ) . fibrinous thrombosis of the affected arteries is also seen. a subclinical vasculitis has also been diagnosed in beagles post mortem; it is not known whether this subclinical condition is a different disorder or part of a jps continuum. this subclinical vasculitis often affects the coronary arteries (with or without other sites). pathogenesis the initiating factors for jps are unknown. it was once presumed to be a reaction to test compounds by laboratory beagles, but this may have been coincident to the fact that the beagle is the breed most often affected with jps. immune mediation of jps is strongly suspected, because the clinical signs have a cyclic nature and respond to treatment with corticosteroids, and the affected dogs have elevated α -globulin fractions and abnormal immunologic responses. there may be hereditary predisposition, given that pedigree analysis has indicated that the offspring of certain sires are more likely to be affected, and breeding of two affected dogs resulted in one in seven affected pups (scott-moncrieff et al., ) . diagnosis and differential diagnosis differential diagnoses include encephalitis, meningitis, injury or degeneration of the cervical vertebrae or disks, and arthritis. in the research facility, the disorder may be readily confused with complications secondary to the experimental procedure, or with postsurgical pain. beagles with jps that were in an orthopedic research study were evaluated for postsurgical complications and skeletal abnormalities prior to the postmortem diagnosis of systemic vasculitis (authors' personal experience). prevention and control no prevention and control measures are known at this time. treatment clinical signs can be abated by administration of corticosteroids. prednisone administered orally at . mg/kg, q h, was associated with rapid relief of clinical symptoms. maintenance of treatment at an alternate-day regimen of . - . mg/kg was shown to relieve symptoms for several months. however, withdrawal of corticosteroid therapy led to the return of clinical illness within weeks. research complications because of the potentially severe clinical signs and the need for immunosuppressive treatment, jps is often incompatible with use of the animal as a research subject. it is unknown whether subclinical necrotizing vasculitis causes sufficient aberrations to measurably alter immunologic responses. etiology interdigital cysts are chronic inflammatory lesions (not true cysts) that develop in the webbing between the toes. the cause for most interdigital cysts is usually not identified unless a foreign body is present. bacteria may be isolated from the site, but the lesions may also be sterile (hence the synonym 'sterile pyogranuloma complex'). clinical signs dogs with interdigital cysts are usually lame on the affected foot, with licking and chewing at the interdigital space. exudation may be noticed at the site of the lesion. the lesion appears as a cutaneous ulcer, usually beneath matted hair, with possible development of sinus tracts and purulent exudate. epizootiology and transmission interdigital cysts are common in a variety of canine breeds, including german shepherds. beagles have been affected in the research setting. interdigital cysts usually occur in the third and fourth interdigital spaces (bellah, ) . a retrospective study of beagles housed in a research industry setting, linked development of interdigital cysts to body codition score, age, location of cyst, and type of caging, and may result from chronic interdigital dermatitis (kovacs et al., ) . pathologic findings histopathologically, interdigital cysts are sites of chronic inflammation, typically described as pyogranulomatous. pathogenesis initial development of the cysts is unknown, except for those cases in which a foreign body can be identified. diagnosis and differential diagnosis bacterial culture swabs and radiographs should be taken of the cysts to rule out bacterial infection and radiopaque foreign bodies or bony lesions, respectively. a biopsy should be taken if neoplasia is suspected. treatment if a foreign body is associated with the lesion, then removal is the first order of treatment. if biopsy of the site provides a diagnosis of sterile pyogranuloma complex, then systemic corticosteroid therapy (e.g., prednisolone at mg/kg q h) can be initiated and then tapered once the lesion heals. interdigital cysts that are refractory to medical therapy require surgical removal. excision includes the removal of the lesion and the interdigital web, and a two-layer closure of the adjacent skin and soft tissues is recommended (bellah, ) . the foot should be put in a padded bandage and a tape hobble placed around the toes to reduce tension when the foot is weight-bearing. the prognosis for idiopathic interdigital cysts is guarded, because the cysts tend to recur (bellah, ) . research complications research complications from the cysts are minimal, unless the dogs need to be weight-bearing for biomechanic or orthopedic studies. treatment with systemic steroids could be contraindicated with some experimental designs. etiology 'cherry eye' is a commonly used slang term for hyperplasia and/or prolapse of the gland of the nictitating membrane (third eyelid). this is not considered a congenital anomaly, but there is breed disposition for this condition, including beagles. a specific etiology is not known. clinical signs the glandular tissue of the nictitating membrane protrudes beyond the membrane's edge and appears as a reddish mass in the ventromedial aspect of the orbit. excessive tearing to mucoid discharge can result, and severe cases can be associated with corneal erosion. pathologic findings typically, the glandular tissue is hyperplastic, possibly with inflammation. rarely is the tissue neoplastic. pathogenesis prolapse of the gland may be a result of a congenital weakness of the connective tissue band between the gland and the cartilage of the third eyelid (helper, ) . prevention hyperplasia of the third eyelid cannot be prevented, but dogs that develop this condition unilaterally should have the other eye evaluated for potential glandular prolapse. preventative surgical measures might be warranted. treatment corticosteroid treatment (topical or systemic) can be used to try to reduce the glandular swelling. however, surgical reduction or excision of the affected gland is typically required to resolve the condition. in the reduction procedure, the prolapsed gland is sutured to fibrous tissue deep to the fornix of the conjunctiva (helper, ). if reduction is not possible (as with deformed nictitating cartilage) or is unsuccessful, removal of the gland can be performed. such excision is fairly straightforward and can be done without removal of the nictitating membrane itself. the gland of the third eyelid is important in tear production; although the rest of the lacrimal glands should be sufficient for adequate tear production, keratoconjunctivitis sicca is a possible consequence after removal of the gland of the nictitating membrane. research complications in most cases, research complications would be minimal, especially if treated adequately. either the presence of the hyperplastic gland or its removal might compromise ophthalmologic studies. antimicrobial resistance profiles and mechanisms of resistance in campylobacter jejuni isolates from pets life span and cancer mortality in the beagle dog and human studies of distribution and recurrence of helicobacter spp. gastric mucosa of dogs after triple therapy plasma von willebrand factor concentration and thyroid function in dogs catheter-related infections in long-term catheterized dogs. observations on pathogenesis, diagnostic methods, and antibiotic lock technique aaha nutritional assessment guidelines for dogs and cats open-and closed-formula laboratory animal diets and their importance to research comparison of the accuracy of two ultrasonographic measurements in predicting the parturition date in the bitch dog thyroiditis: occurrence and similarity to hashimoto's struma surgical management of specific skin disorders bordetella and mycoplasma respiratory infections in dogs and cats associations between lymphocytic thyroiditis, hypothyroidism, and thyroid neoplasia in beagles diagnostic exercise: peracute death in a research dog insecticide and acaricide molecules and/ or combinations to prevent pet infestation by ectoparasites neurologic manifestations associated with hypothyroidism in four dogs veterinary surgery: small animal neoplasms of the skin and subcutaneous tissues in dogs and cats neoplasia of the skin and mammary glands of dogs and cats pancreatic adenocarcinoma in two grey collie dogs with cyclic hematopoiesis variation in age at death of dogs of different sexes and breeds comparison of campylobacter carriage rates in diarrheic and healthy pet animals advances in dietary management of obesity in dogs and cats effect of amount and type of dietary fiber on food intake in energy-restricted dogs effect of level and source of dietary fiber on food intake in the dog an outbreak of fatal hemorrhagic pneumonia caused by streptococcus equi subsp. zooepidemicus in shelter dogs molecular characterization of canine parvovirus strains in argentina: detection of the pathogenic variant cpv c in vaccinated dogs external parasites: identification and control orthopedic coaptation devices and small-animal prosthetics enterohepatic helicobacter spp. in colonic biopsies of dogs: molecular, histopathological and immunohistochemical investigations intradural vasculitis and hemorrhage in full sibling welsh springer spaniels respiratory disease in kennelled dogs: serological responses to bordetella bronchiseptica lipopolysaccharide laboratory animal medicine do not correlate with bacterial isolation or clinical respiratory symptoms lymphoma: which chemotherapy protocol and why? detection of humoral antibody to the transmissible venereal tumor of the dog laboratory animal management: dogs reproductive cycles of the domestic bitch hereditary canine spinal muscular atrophy: an animal model of motor neuron disease. can evaluation of the helicobacteraceae in the oral cavity of dogs management of septicemia in rhesus monkeys with chronic indwelling catheters clinical and virological findings in pups naturally infected by canine parvovirus type glu- mutant eradication of helicobacter spp. by using medicated diet in mice deficient in functional natural killer cells and complement factor d client information series: canine demodicosis client information series: fleas and flea allergy dermatitis management of the burn wound burn trauma chlorine dioxide sterilization of implanted right atrial catheters in rabbits current concepts in the management of helicobacter associated gastritis discordant s and s rrna gene phylogenies for the genus helicobacter: implications for phylogenetic inference and systematics use of narcotic antagonists to modify stereotypic selflicking, self-chewing, and scratching behavior in dogs epidemiology of canine and feline tumors epizootiologic characteristics of canine and feline leukemia and lymphoma evaluation of the ovicidal cctivity of lufenuron and spinosad on fleas' eggs from treated dogs enteric coccidiosis the respiratory system the association of american feed control officials dog and cat food nutrient profiles: substantiation of nutritional adequacy of complete and balanced pet foods in the united states study of obesity in dogs visiting veterinary practices in the united kingdom pathological prognostic factors in breast cancer. i. the value of histological grade in breast cancer: experience from a large study with long-term follow-up utility of fdg-pet scanning in lymphoma by who classification principles of treatment for canine lymphoma miller's anatomy of the dog update on diagnosis of canine hypothyroidism immunohistochemical and histochemical stains for differentiating canine cutaneous round cell tumors the laboratory canine canine conjunctival mast cell tumors: a retrospective study canine cutaneous epitheliotropic t-cell lymphoma: a review canine infectious respiratory disease helicobacter-associated gastric disease in ferrets, dogs, and cats enteric bacterial infections hemorrhagic streptococcal pneumonia in newly procured research dogs association between canine malignant lymphoma, living in industrial areas, and use of chemicals by dog owners reproductive patterns in the domestic dog-a retrospective study of the drever breed the dog as a research subject a review of canine pseudocyesis leptospirosis surgical treatment of an elbow hygroma utilizing microvascular free muscle transfer in a newfoundland bacterial diseases immunoprophylaxis leptospirosis gastric helicobacters in domestic animals and nonhuman primates and their significance for human health non-helicobacter pylori helicobacter species in the human gastric mucosa: a proposal to introduce the terms h. heilmannii sensu lato and sensu stricto diseases of the small intestine histotripsy of the prostate: dose effects in a chronic canine model flea control failure? myths and realities small animal clinical nutrition operating room emergencies shock evaluation of the sensitivity and specificity of diagnostic criteria for sepsis in dogs pulmonary parenchymal disease canine thyroid neoplasms: epidemiologic features magrane's canine ophthalmology helicobacter-like organisms: histopathological examination of gastric biopsies from dogs and cats current veterinary therapy x: small animal practice congenital defects of the dog gastric and duodenal ulcers in dogs with mastocytoma complications in the use of indwelling vascular catheters in laboratory animals helicobacter infection diseases of the large intestine pregnancy management in the bitch tracheal collapse. diagnosis and medical and surgical treatment hygroma of the elbow in dogs thermal injuries joint working group on refinement factors contributing to the contamination of peripheral intravenous catheters in dogs and cats diversity in bacterium-host interactions within the species helicobacter heilmannii sensu stricto morphological study of the effects of the gnrh superagonist deslorelin on the canine testis and prostate gland five-year longitudinal study on limited food consumption and development of osteoarthritis in coxofemoral joints of dogs cvt update: interpretation of endocrine diagnostic test results for adrenal and thyroid disease etiopathogenesis of canine hypothyroidism maintenance energy requirement of dogs: what is the correct value for the calculation of metabolic body weight in dogs? outbreak and control of haemorrhagic pneumonia due to streptococcus equi subspecies zooepidemicus in dogs kirk's current veterinary therapy xii: small animal practice an emerging pulmonary haemorrhagic syndrome in dogs: similar to the human leptospiral pulmonary haemorrhagic syndrome? tarsal joint contracture in dogs with golden retriever muscular dystrophy an epidemiological study of interdigital cysts in a research beagle colony value of the ( )c-urea breath test for detection of gastric helicobacter spp. infection in dogs undergoing endoscopic examination pathogenesis of helicobacter pylori infection prostatic diseases semen collection in the dog accuracy of canine parturition date prediction using fetal measurements obtained by ultrasonography chronic catheterization of the intestines and portal vein for absorption experimentation in beagle dogs evaluation of weight loss protocols for dogs problems in wound healing associated with chemotherapy and radiation therapy histotripsy: minimally invasive technology for prostatic tissue ablation in an in vivo canine model fdg-pet imaging in canine lymphoma and cutaneous mast cell tumor comparison of fertility data from vaginal vs intrauterine insemination of frozenthawed dog semen: a retrospective study withrow & macewen's small animal clinical oncology estimation of gestational age and assessment of canine fetal maturation using radiology and ultrasonography: a review effects of four preparations of . % chlorhexidine diacetate on wound healing in dogs the prediction of parturition date in canine pregnancy clustering of activating mutations in c-kit's juxtamembrane coding region in canine mast cell neoplasms transmissible venereal tumors kirk's current veterinary therapy : small animal practice canine lymphoma and lymphoid leukemias the staging and treatment of multicentric highgrade lymphoma in dogs: a review of recent developments and future prospects association between waste management and cancer in companion animals tick paralysis in north america and australia thyroid gland and arterial lesions of beagles with familial hypothyroidism and hyperlipoproteinemia bacterial gastroenteritis in dogs and cats: more common than you think enteropathogenic bacteria in dogs and cats: diagnosis, epidemiology, treatment, and control dermatologic aspects of tick bites and tick-transmitted diseases a chronic access port model for direct delivery of drugs into the intestine of conscious dogs mast cells and canine mast cell tumours: a review etiologic study of upper respiratory infections of household dogs effect of early enteral nutrition on intestinal permeability, intestinal protein loss, and outcome in dogs with severe parvoviral enteritis determination of strain variability of microsporum canis to disinfectants cutaneous fungal infections diagnosis of neoplasia tumors of the mammary gland transmissible dog cancer genome reveals the origin and history of an ancient cell lineage clonal origin and evolution of a transmissible cancer notice regarding nih plan to transition from use of usda class b dogs to other legal sources (not-od- - ) guide for the care and use of laboratory animals surgical closure of elbow hygroma in the dog colitis and colon cancer in waspdeficient mice require helicobacter species tumors of the genital system practical laboratory methods for the diagnosis of dermatologic diseases animals and animal products, subchapter a, parts , , and comparison of three skin preparation techniques in the dog comparison of three skin preparation techniques in the dog. part : clinical trial in dogs manual of clinical behavioral medicine for dogs and cats. mosby-year book identification of and screening for human helicobacter cinaedi infections and carriers via nested pcr identification of three novel superantigen-encoding genes in streptococcus equi subsp. zooepidemicus, szef, szen, and szep hypothyroidism in dogs: cases ( - ) plasma von willebrand factor antigen concentration in dogs with hypothyroidism plasma von willebrand factor antigen concentration and buccal bleeding time in dogs with experimental hypothyroidism canine cutaneous mast cell tumor: morphologic grading and survival time in dogs atlas of small animal wound management and reconstructive surgery what's your diagnosis? fever and leukocytosis in a young beagle. canine juvenile polyarteritis syndrome (beagle pain syndrome) a clonal outbreak of acute fatal hemorrhagic pneumonia in intensively housed (shelter) dogs caused by streptococcus equi subsp. zooepidemicus thyroid diseases urogenital emergencies emergency procedures for the small animal veterinarian prognostic significance of morphological subtypes in canine malignant lymphomas during chemotherapy streptococcus zooepidemicus: an emerging canine pathogen characterization of pneumonia due to streptococcus equi subsp. zooepidemicus in dogs tumors of the skin and soft tissue aspiration-induced lung injury origins and evolution of a transmissible cancer canine mast cell tumors express stem cell factor receptor arterial and venous blood gas analyses dogs and cats as laboratory animals hypocretin levels in sporadic and familial cases of canine narcolepsy comparison of giardia diagnostic tests in diagnosis of naturally acquired canine chronic subclinical giardiasis effects of chlorhexidine diacetate and povidone-iodine on wound healing in dogs genetic evidence for an east asian origin of domestic dogs urethral-sparing histotripsy of the prostate in a canine model factors influencing canine mammary cancer development and postsurgical survival how to treat common parasites safely muller and kirk's small animal dermatology systemic necrotizing vasculitis in nine young beagles thomson's special veterinary pathology, second ed. mosby-year book thyroid and parathyroid glands diseases of the small bowel canine infectious tracheobronchitis (kennel cough complex) diseases of the intestines the use of ultrasonography for pregnancy diagnosis in the bitch cytologic examination of fine-needle aspirates from mammary gland tumors in the dog: diagnostic accuracy with comparison to histopathology and association with postoperative outcome effects of finasteride on size of the prostate gland and semen quality in dogs with benign prostatic hypertrophy canine prostatic disease: a review of anatomy, pathology, diagnosis, and treatment pathologic features of naturally occurring juvenile polyarteritis in beagle dogs development, anatomy, histology, lymphatic drainage, clinical features, and cell differentiation markers of canine mammary gland neoplasms withrow & macewen's small animal clinical oncology nutrient requirements of dogs and cats (nutrient requirements of domestic animals) trauma to the skin and subcutaneous tissues of dogs and cats chronic problem wounds of dog limbs acvim small animal consensus statement on leptospirosis: diagnosis, epidemiology, treatment, and prevention lumbosacral stenosis in dogs mammary neoplasia in a closed beagle colony complete mitochondrial genomes of ancient canids suggest a european origin of deomestic dogs artificial insemination in canids: a useful tool in breeding and conservation artificial insemination with frozen semen in dogs: a retrospective study of years using a non-surgical approach manual of canine and feline cardiology comparative evaluation of agar dilution and broth microdilution methods for antibiotic susceptibility testing of helicobacter cinaedi thyroiditis in a group of laboratory dogs: a study of beagles obesity-related metabolic dysfunction in dogs: a comparison with human metabolic syndrome pro-inflammatory properties and neutrophil activation by helicobacter pylori urease of agriculture, animal and plant health inspection service annual report animal usage by fiscal year. available from: <www leptospirosis in dogs: a review with emphasis on clinical aspects thomson's special veterinary pathology, second ed. mosby-year book endocrinology of pregnancy in the dog: a review immunoblot analysis as an alternative method to diagnose enterohepatic helicobacter infections management of superficial skin wounds psychodermatosis colony multiplex pcr assay for identification and differentiation of campylobacter jejuni, c. coli, c. lari, c. upsaliensis, and c. fetus subsp. fetus the role of c-kit in tumorigenesis: evaluation in canine cutaneous mast cell tumors aaha canine vaccination guidelines canine mast cell tumours: a review of the pathogenesis, clinical features, pathology and treatment serum concentrations of thyroxine and , , ′-triiodothyronine before and after intravenous or intramuscular thyrotropin administration in dogs surgical treatment of specific skin disorders naltrexone for treatment of acral lick dermatitits in dogs use of ultrasound in the measurement of subcutaneous fat and prediction of total body fat in dogs mammal species of the world: a taxonomic and geographic reference withrow & macewen's small animal clinical oncology the authors thank dr. bambi jasmin of marshall farms for contributions on preventive health practices for class a dogs. key: cord- -dxbvfwqu authors: mcfarland, richard; verthelyi, daniela; casey, warren; arciniega, juan; isbrucker, richard; schmitt, michael; finn, theresa; descamps, johan; horiuchi, yoshinobu; sesardic, dorothea; stickings, paul; johnson, nelson w; lipscomb, elizabeth; allen, david title: non-animal replacement methods for human vaccine potency testing: state of the science and future directions date: - - journal: procedia in vaccinology doi: . /j.provac. . . sha: doc_id: cord_uid: dxbvfwqu abstract niceatm and iccvam convened an international workshop to review the state of the science of human and veterinary vaccine potency and safety testing methods, and to identify opportunities to advance new and improved methods that can further reduce, refine, and replace animal use. this report addresses methods and strategies identified by workshop participants for replacement of animals used for potency testing of human vaccines. vaccines considered to have the highest priority for future efforts were ( ) vaccines for which antigen quantification methods are already developed but not validated, ( ) vaccines/components that require the largest number of animals, ( ) vaccines that require an in vivo challenge test, and ( ) vaccines with in vivo tests that are highly variable and cause a significant number of invalid tests. vaccine potency tests identified as the highest priorities for replacement were those for diphtheria and tetanus, pertussis (whole cell and acellular), rabies, anthrax, polio vaccine (inactivated) and complex combination vaccines based on dt or dtwp/ap. research into understanding the precise mechanism of protection afforded by vaccines and the identification of clinically relevant immunological markers are needed to facilitate the successful implementation of in vitro testing alternatives. this report also identifies several priority human vaccines and associated research objectives that are necessary to successfully implement in vitro vaccine potency testing alternatives. © published by elsevier ltd. selection and/or peer-review under responsibility of the national toxicology program interagency center for the evaluation of alternative toxicological methods (niceatm). vaccines contribute to improved animal and human health and welfare by preventing and controlling infectious diseases. however, testing necessary to ensure vaccine effectiveness and safety can involve large numbers of animals and significant pain and distress. accordingly, the u.s. interagency coordinating committee on the validation of alternative methods (iccvam) and the u.s. national toxicology program interagency center for the evaluation of alternative toxicological methods (niceatm) recently identified vaccine potency and safety testing as one of their four highest priorities [ ] . iccvam is an interagency committee of federal agencies that is charged by law with evaluating new, revised, and alternative test methods with regulatory applicability. iccvam members represent u.s. federal regulatory and research agencies that require, use, generate, or disseminate safety testing data. these include the department of agriculture (usda), which regulates veterinary vaccines, and the food and drug administration (fda), which regulates human vaccines. iccvam is a permanent interagency committee of the national institute of environmental health sciences (niehs) under niceatm. niceatm administers iccvam, provides scientific and operational support for iccvam-related activities, and conducts international validation studies on promising new safety testing methods. niceatm and iccvam serve a critical public health role in translating research advances from the bench into standardized safety testing methods that can be used in regulatory practice to prevent disease and injury. to promote and advance the development and use of scientifically valid alternative methods for human and veterinary vaccine testing, niceatm and iccvam organized the international workshop on alternative methods to reduce, refine, and replace the use of animals in vaccine potency and safety testing: state of the science and future directions. the workshop was held at the national institutes of health in bethesda, maryland, on september [ ] [ ] [ ] . it was organized in conjunction with the european centre for the validation of alternative methods (ecvam), the japanese center for the validation of alternative methods (jacvam), and health canada. the workshop addressed the state of the science of human and veterinary vaccine potency and safety testing. participants developed recommendations for future progress in three major areas: ( ) in vitro replacement methods for potency testing; ( ) reduction and refinement methods for potency testing; and ( ) reduction, refinement, and replacement methods for vaccine safety testing [ ] . reports were prepared for each of the three topics for human vaccines and for each of the three topics for veterinary vaccines [ , , , , , ] . this report addresses methods and strategies for the replacement of animal use for potency testing of human vaccines. the goals of the international workshop were to ( ) identify and promote the implementation of currently available and accepted alternative methods that can reduce, refine, and replace the use of animals in human and veterinary vaccine potency and safety testing; ( ) review the state of the science of alternative methods and identify knowledge and data gaps that need to be addressed; and ( ) identify and prioritize research, development, and validation efforts needed to address these gaps in order to advance alternative methods that will also ensure continued protection of human and animal health. the workshop was organized with four plenary sessions and three breakout group sessions. in the breakout sessions, workshop participants: identified criteria to prioritize vaccine potency and safety tests for future alternative test method development and identified high priorities using these criteria reviewed the current state of the science of alternative methods and discussed ways to promote the implementation of available methods identified knowledge and data gaps that need to be addressed identified and prioritized research, development, and validation efforts needed to address these gaps in order to advance alternative methods while ensuring continued protection of human and animal health the workshop opened with a plenary session in which expert scientists and regulatory authorities from the united states, europe, japan, and canada outlined the importance of vaccines to human and animal health [ , ] and described national and international regulatory testing requirements for human and veterinary vaccines [ , , , , , , ] . authorities emphasized that, following the regulatory approval of a vaccine, testing is required to ensure that each subsequent production lot is pure, safe, and sufficiently potent to generate a protective immune response in people or animals [ , ] . the second plenary session addressed methods that have been accepted and methods that are in development that do not require the use of animals for assessing the potency of vaccines [ , , , ] . this was followed by breakout sessions to discuss the state of the science and recommendations for future progress for in vitro potency tests for human and veterinary vaccines. workshop recommendations to advance the use and development of alternative methods that can replace animals for the potency testing of human vaccines are provided in this paper, while the report on replacement of animals for veterinary vaccines is available elsewhere in these proceedings [ ] . the third plenary session addressed ( ) potency testing methods that refine procedures to avoid or lessen pain and distress by incorporating earlier humane endpoints or by using antibody quantification tests instead of challenge tests and ( ) methods and approaches that reduce the number of animals required for each test [ , , , , , , ] . breakout groups then discussed the state of the science and developed recommendations for future progress. workshop recommendations to advance the use and development of alternative methods that can reduce and refine animal use for potency testing of human vaccines [ ] and veterinary vaccines [ ] are available in the respective publications in these proceedings. the final plenary session addressed methods and approaches for reducing, refining, and replacing animal use to assess the safety of serial production lots of human and veterinary vaccines [ , , , ] . breakout groups then discussed the state of the science and developed recommendations for advancing alternative methods for vaccine safety testing. workshop recommendations to advance the use and development of alternative methods for safety testing of human vaccines [ ] and veterinary vaccines [ ] are available in the respective publications in these proceedings. human vaccines save lives, prevent disease and morbidity, and represent a critical tool for successful and costsaving health interventions [ ] . for example, for each u.s. birth cohort that receives a series of seven vaccines that protect against infectious diseases including diphtheria, tetanus, pertussis, measles, mumps, rubella, and polio, an estimated million disease episodes and , premature deaths are prevented. these vaccinations are estimated to save billion dollars in medical and societal costs [ , ] . because of the number of animals used annually for the release of human vaccines, many regulatory agencies actively encourage the evaluation, development, and implementation of novel approaches that reduce, refine, and replace ( rs) the use of animals in vaccine safety and potency product release testing [ , ] . the u.s. public health service act [ ] and certain sections of the u.s. food, drug and cosmetic act [ ] give the fda the authority to regulate human vaccines. section of the public health service act states that the approval of a biologics license is based on the demonstration of product safety, purity, and potency and assurance that the facility for manufacture, processing, and packaging meets the standards to ensure that any product released for distribution is safe, pure, and potent. the regulatory definitions of safety, purity, and potency are detailed in title of the u.s. code of federal regulations, and testing methodology and validation must be included in the biologics license application (bla) [ ] . safety and potency testing may be performed on the final bulk sample or final container sample and may consist of either in vivo or in vitro tests or both. post-licensing changes to potency or safety tests require a supplement to the license with adequate rationale and data to support the alternative method (potency or safety) or to support the demonstration of lack of need for a particular test (safety). current human vaccines consist of: live viruses modified live (attenuated) viruses and bacteria inactivated (killed) viruses and bacteria toxoids (chemically inactivated toxins) polysaccharides the general types of potency tests employed by vaccine manufacturers include ( ) titration of live organisms (in vitro, but occasionally in vivo); ( ) in vitro assays such as enzyme-linked immunosorbent assays (elisas) or other antigen quantification methods; ( ) in vivo methods involving immunization of small laboratory animals (e.g., guinea pigs, rats, mice) followed by challenge with toxin/virus/bacteria or titration of immune sera to measure the antibody response [ ] . in vitro potency testing is used for live, attenuated vaccines but is not commonly used for inactivated vaccines [ ] . the center for biologics evaluation and research (cber) has an active research program that evaluates, develops, and integrates novel scientific technologies for use in product regulation. the cber program includes the development and analysis of approaches that reduce, refine, and replace the use of animals. cber encourages the development of these alternative methods for vaccine safety and potency testing with appropriate relevance, supporting data, and test method validation. reduction and refinement approaches can be applied to all serological or vaccination-challenge methods that use animals to determine the potency of a human vaccine. the current potency testing methods for several human vaccines still utilize animals ( table ) , and the development of alternative assays for these products would significantly reduce the number of animals used for testing and benefit animal welfare. to identify human vaccines that should have priority for further development and validation of in vitro replacement tests, criteria for prioritization were established at this workshop. workshop participants agreed on the following vaccine prioritization criteria for development of in vitro potency tests: vaccines for which antigen quantification methods are already developed but not validated for routine use vaccines/components that require the largest number of animals vaccines that require an in vivo challenge test vaccines with in vivo tests that are highly variable and cause a significant number of invalid tests vaccines for which the relationship between antigen quantity and potency are well understood combined vaccines (recognizing that their complexity may make successful replacement more difficult than single-component vaccines) vaccines that have a well-defined and understood mode of action or known target vaccines for which the manufacturing process is well understood and consistent based upon the key criteria described above, workshop participants identified the following vaccines as the highest priorities for additional research, development, and validation efforts: diphtheria and tetanus toxoid vaccines whole cell and acellular pertussis vaccines rabies vaccines anthrax vaccines complex combination vaccines inactivated polio vaccines diphtheria and tetanus toxoids were considered the highest priorities because the current potency tests require large numbers of animals. rabies and anthrax vaccines were identified as priorities because the tests also require large numbers of animals and may involve significant pain and distress. in addition, the vaccine-challenge test for these vaccines requires the use of live virus, toxin, or bacteria that pose significant safety risks for laboratory technicians. the current in vivo potency test for inactivated rabies vaccines is also known to be highly variable [ ] and produces a significant number of invalid tests. finally, the use of antigen quantification methods for those vaccines for which the protective antigen is already known was considered to be a high priority for validation and further development. as shown in table , serological methods are currently in development or have been approved for use by specific regulatory authorities for many of these high-priority vaccines. the availability of these refinement methods indicates that they should therefore aid in the development of replacement methods based on in vitro antigen quantification [ ] . currently, some human vaccines do not require the use of animals because in vitro methods have been developed that quantify the presence of the protective antigen. these so-called antigen quantification methods are based on binding of key protective antigens in the vaccine batch to specific antibodies using in vitro immunoassays. at the present time, these methods represent the most promising in vitro approaches for the replacement of animals used in vaccine potency testing. it is important to note, however, that in vitro antigen quantification methods only measure antigen quantity and do not necessarily reflect biological activity. another limitation of these methods is the requirement for adjuvant removal from the product before testing. therefore, it is necessary to also demonstrate that the process of adjuvant removal does not affect recovery and/or integrity of the antigen, which could interfere with its detection in the assay [ ] . to date, in vitro tests have been successfully implemented (regulatory acceptance) for only a few products. these include hepatitis a/b vaccines, inactivated polio vaccine, polysaccharide and polysaccharide conjugate vaccines, and human papillomavirus vaccine ( table ) . for the recombinant hepatitis b vaccine, two types of in vitro immunoassays have been developed as alternatives to the mouse immunogenicity test. the first is based on the direct determination of the hepatitis b surface antigen (hbsag), while the other is based on the neutralization of hbsag and subsequent detection of remaining anti-hbsag antibodies in an elisa [ , , ] . perhaps the most critical need for a valid alternative for potency testing is in the area of rabies vaccines. the traditional method for inactivated rabies vaccine potency testing is a multiple-dilution vaccination challenge test in mice. this test, commonly referred to as the nih test, because of the origin of its development (the national institutes of health), is based on the relative proportion of animals protected from rabies in groups receiving the test vaccine compared to those immunized with a reference vaccine. the need for an alternative test is critical because the traditional potency test requires large numbers of animals that experience unrelieved pain and distress. approximately half of the test animals die or develop acute signs of rabies (e.g., convulsions, paresis, paralysis). the inherent variability of this assay contributes to a high number of invalid assays, which leads to further use of animals for repeat testing [ ] . the potential exposure of laboratory technicians to biohazardous materials during testing is also an important consideration. in recent years, national and international regulatory agencies have introduced one or more rs initiatives for rabies vaccine potency testing that can be seen as significant improvements to the welfare of test animals. these include ( ) a single-dilution assay, which reduces the number of dilutions (and therefore animals) for the test (reduction); ( ) routine use of humane endpoints to allow earlier study termination (refinement); ( ) a serological assay that obviates the need for in vivo challenge with rabies virus (refinement). there are antigen quantification methods available that may one day eliminate the need for an in vivo potency test (replacement), leading to a significant reduction in the use of animals for testing. several types of in vitro elisa procedures for rabies vaccines have been developed during the past few years, taking advantage of advances in defining the neutralizing epitopes [ , , ] . while these assays would confer distinct advantages over in vivo test methods (e.g., no animals required, faster, less costly), limitations that have impeded their development and implementation include the following: ( ) they are not universally applicable to all vaccine strains, ( ) they cannot be used in combination with adjuvants, and ( ) they do not correlate well with the in vivo challenge test [ , ] . clear correlations have yet to be demonstrated among the amount of antigen required to induce a protective immune response in animals, the amount of antigen measured using alternative in vitro assays, and the immune response in human vaccines [ ] . therefore, it is difficult to correlate potency defined by protection in animals versus potency defined by alternative methods [ , ] . however, further studies targeted toward the potential usefulness of elisa procedures for rabies vaccine potency testing are ongoing [ ] . these initiatives represent critical first steps toward developing a scientifically valid alternative replacement method for potency testing of rabies vaccines. it is of note that the european pharmacopoeia has now issued a draft proposal for comments for the use of antibody quantification for lot release of rabies vaccines for veterinary use, subject to product-specific validation [ ] . with the identification of the highest-priority vaccines for replacement potency assay development, the knowledge and data gaps that have previously prevented use of non-animal potency assays must be addressed. a thorough understanding of the pathogenesis of an infectious disease and associated protective mechanisms is necessary to accurately identify the specific antigen or key epitope responsible for eliciting a protective immune response. once a clinically relevant antigen/epitope is identified, further studies are required to fully understand its role in protection and how the antigen in the vaccine may be affected by the vaccine manufacturing process (e.g., epitope modification due to detoxification and absorption to adjuvant) and how this process may alter how the vaccine is presented to the immune system and how it is processed in the target species. studies can then be conducted to determine the structure and stability of antigen so that immunological markers can ultimately be identified to measure the immune response in the target species. for some vaccine antigens, the mechanism of protection is well understood, and the antigens are well characterized. this is the case for diphtheria and tetanus antigens. despite the complexity of the detoxification and production processes involved in manufacturing these toxoid vaccines, methods are available for in vitro quantification of the antigen in the final product [ , ] . however, the amount of antigen detected using an in vitro method and the biological activity (in vivo potency) may depend on additional factors: for example, in certain combination vaccines containing tetanus toxoid as an active ingredient and hib polysaccharide conjugated to tetanus toxoid (e.g. dtwp + hib-tt [ ] and an experimental dtap-ipv + hib-vaccine [ ] ) tetanus potency measured using the in vivo method was increased relative to the potency of a vaccine which did not contain the hib-tt component due to the presence of the carrier protein. in such cases the amount of tetanus toxoid detected by an in vitro binding assay may not be different because the in vitro assay cannot detect the conjugated tetanus toxoid [ ] . in such circumstances this may be an additional challenge to address during replacement of an in vivo test with an in vitro detection method. the availability of selected animal disease models can facilitate identification of immunological markers, which will then aid in developing in vitro tests. for example, vaccines containing recombinant papillomavirus virus-like particles (vlps) have been used to control disease in cows, dogs, and rabbits. these successes provided the basis for subsequent licensure of a bivalent and quadravalent human papilloma virus (hpv) vaccine to control cervical cancer in humans [ ] . ready access to relevant information plays a key role in the development, validation, and subsequent implementation of in vitro vaccine potency test methods. ideally, manufacturers and regulatory authorities should frequently interact and share information in order to avoid duplicating efforts in defining procedures used to validate alternative in vitro tests. however, proprietary issues may limit the dissemination of information to outside stakeholders. increased engagement by academic researchers could provide a means through which information is publicized through various vehicles such as journal publications, workshops, and scientific/professional society meetings. any strategy for the development of alternative replacement methods must be based on a step-by-step approach. ideally, the definitive potency assay (in vivo or in vitro) should be developed before licensing to eliminate the need for parallel in vivo testing for licensing or complex comparability protocols. finally, there is a clear need to differentiate between tests limited to measure the amount of antigen and additional tests for demonstrating manufacturing consistency, as a single test only assessing antigen quantity is unlikely to provide all the required information. changes in the structural properties of the product that affect its efficacy may not be detected by some tests. therefore, an additional priority would be to determine the ability of in vitro antigen quantification test in predicting product molecular stability and/or biological activity. significant time and resources are required for each of the above priority vaccines because of ( ) the complexities associated with moving from an in vivo test method to one that does not use animals and ( ) the significant costs associated with the research, development, and validation of in vitro vaccine potency test methods [ , ] . therefore, workshop participants strongly encourage early and frequent interactions with regulators throughout this process to maximize the likelihood of a final design that will be accepted by regulatory authorities and to avoid any unnecessary delays. workshop participants agreed that broader access to all relevant information describing successfully implemented alternative potency testing methods would have an obvious impact on expanding their use and acceptance. for example, open online access to the united states pharmacopeia (usp) and european pharmacopoeia (ph. eur.) monographs would greatly facilitate the dissemination of methods and procedures that have been implemented for use by regulators in various regions. manufacturers are also encouraged to frequently consult with the relevant regulatory authorities regarding their planned study design and how best to incorporate alternative methods into vaccine potency testing (i.e., product-specific validation). all workshop participants recognized the need for harmonization during the vaccine development phase and noted that any guidelines that would standardize an approach, or a roadmap on how to transition from in vivo to in vitro testing, would be helpful. collaborative interlaboratory studies involving both national control authorities and supranational organizations, such as the european directorate for the quality of medicines (edqm; ph. eur.) and who, could accomplish this. workshop participants recognized that the significant costs and the lack of available funding associated with additional research, development, and validation efforts of in vitro vaccine potency test methods are the primary barriers to progress. more specifically, they agreed that concerns associated with rabies vaccine antigen quantification methods need to be addressed. this includes increasing the availability of reference standards and antisera for the various rabies strains, as well as maintaining a consistent source of antisera (or, preferably, a set of nonproprietary monoclonal antibodies). workshop participants identified the following issues that would aid in facilitating the broader acceptance of alternative methods: establishment of readily available and/or nonproprietary reference standards sharing of resources, including key reagents and assay procedures educational outreach in bioethics and training in the conduct of rs assays international harmonization of requirements for acceptable test methods significant differences exist between human and veterinary vaccine production and formulation, most notably the presence of complex adjuvants or adjuvant combinations in veterinary vaccines. extrapolating results from in vitro methods developed for a veterinary vaccine to the similar human vaccine is often not possible. for example, human influenza vaccines are often not adjuvanted, while veterinary influenza vaccines (e.g., avian, swine) may contain mineral oil or other more complex adjuvants. use of adjuvants may also result in significantly lower antigen content in veterinary vaccines compared to that present in nonadjuvanted human vaccines. veterinary vaccines are often not as well characterized as the corresponding human vaccines and are typically composed of complex mixtures that include antigens derived from the whole cell, cell lysates, or cell culture supernatants. each of these variables could influence both safety and potency of the vaccine product. table provides a list of veterinary vaccines for which antigen quantification methods have been accepted for use by some regulatory authorities. taking into account these significant differences between human and veterinary vaccines, workshop participants discussed ways to extrapolate alternative potency testing results from veterinary to human vaccines. increased interaction and communication between global regulatory agencies, research institutions, and vaccine manufacturers was recognized as the critical first step in this process. one suggestion was to develop an interagency task force to deal with specific issues relevant to both human and veterinary medicine. for example, the increased complexity of adjuvants in certain veterinary vaccines has resulted in a better understanding of adjuvant/antigen interactions, which may be useful to human vaccine manufacturers as additional adjuvants are considered for approval in their products. conversely, antigen quantification methods that are currently available for human rabies vaccines for inprocess control (who technical guidelines) may be useful to veterinary rabies vaccine manufacturers. workshop participants agreed on the need for global harmonization, which could be accomplished by convening meetings and workshops with the various regulatory agencies and international research organizations to discuss alternative in vitro vaccine potency testing strategies. the development and implementation of an online, worldwide database of available alternative methods was viewed as a potential significant advancement. workshop participants also recognized the importance of standardizing lot-release criteria and encouraging mutual recognition of test results among regulatory authorities. this would avoid duplicative testing that results in more animals being used and extends the time before products can be released to the public. participants also recognized the need for stepwise advancement in which specific vaccines are prioritized and administrative procedures, such as a reduction in the frequency of official control testing, may precede technical strategies based on refinement and replacement alternatives. incentives by regulatory authorities to encourage the implementation of new validated rs approaches for potency testing of human vaccines were also viewed as important considerations. finally, it was considered critical for regulators to communicate what is required of a quality control production system to allow development of new products without the need for in vivo testing. this was the first international workshop held in the united states that focused on the reduction, refinement, and replacement of animal use for safety and potency release testing of both human and veterinary vaccines. a key accomplishment of the workshop was bringing together experts from industry, academia, and government in the areas of potency and safety testing for both human and animal vaccines. several other recent symposia have sought to further the science of the rs as they apply to the use of animals for quality control of biologicals. these symposia addressed various aspects of promoting and facilitating the use of non-animal methods for the quality control of either human or veterinary biologicals, as well as increasing the understanding and implementation of the consistency approach to the manufacturing of human and veterinary biologics. several workshops have been convened over the past several years that were all dedicated to finding new ways to advance the development and implementation of alternative methods for the quality control of human or veterinary biologicals [ , , ] . this workshop highlighted the need for basic scientific research, as well as product-specific validation efforts, to advance the development, validation, and implementation of alternative methods to replace animals used in potency testing of human vaccines. these specific recommendations were considered necessary to address scientific knowledge gaps that exist in our understanding of disease processes and/or product-specific information as they relate to vaccine function. there was strong agreement among the workshop participants that a thorough in-depth understanding of the precise mechanism of protection afforded by a vaccine in the target population is a key first step in moving from in vivo to in vitro potency testing. recurring themes that surfaced during the discussions included ( ) the need for more open access to methods and information such as pharmacopoeial monographs; ( ) increased sharing of information among regulators, manufacturers, and the scientific community; ( ) international harmonization of requirements on test methods and specifications (e.g., ich q r development and validation of potency assays); ( ) recognition that the regulatory authorities should encourage the use of rs approaches by various means; and ( ) the need for widely available validated reference standards, antisera, and reagents. although specific detailed recommendations from each workshop/symposium differed, these same general themes of the need for specific scientific research, the need for international harmonization of regulations and specifications, the need for sharing of information, the responsibility that regulators have for encouraging the use of alternative methods, and the need for validated reagents were evident in the output from each workshop/symposium. by building on and reaffirming these important conclusions and recommendations for rs advancement the outcome of this workshop will advance alternative methods for vaccine potency testing that will reduce, refine, and replace the use of animals in testing, while maintaining and promoting scientific quality and the protection of human health, animal health, and the environment. ): a plan to advance alternative test methods of high scientific quality to protect and advance the health of people, animals, and the environment. national institute of environmental health sciences introduction and summary of the international workshop on alternative methods to reduce, refine, and replace the use of animals in vaccine potency and safety testing: state of the science and future directions non-animal replacement methods for human vaccine potency testing: state-of-the-science and future directions non-animal replacement methods for veterinary vaccine potency testing: state-of-the-science and future directions improving animal welfare and reducing and refining animal use for human vaccine potency testing: state-of-the-science and future directions improving animal welfare and reducing animal use for veterinary vaccine potency testing: state-of-the-science and future directions alternative methods and strategies to reduce, refine, and replace animal use for human vaccine post-licensing safety testing: state of the science and future directions alternative methods and strategies to reduce, refine, and replace animal use for veterinary vaccine post-licensing safety testing: state of the science and future directions veterinary vaccines and their importance to animal health and public health human vaccines and their importance to public health fda requirements for human vaccine safety and potency testing usda requirements for veterinary vaccine safety and potency testing health canada's human vaccine lot release program: impact on the rs european regulatory requirements for veterinary vaccine safety and potency testing and recent progress toward reducing animal use strategic approaches for developing alternative tests for safety and potency of vaccines international regulatory requirements for vaccine safety and potency testing: a who perspective overview of currently approved veterinary vaccine potency testing methods and methods in development that do not require animal use case study of development, validation, and acceptance of a non-animal method for assessing veterinary vaccine potency presented at: international workshop on alternative methods to reduce, refine, and replace the use of animals in vaccine potency and safety testing: state of the science and future directions. niceam. bethesda, md a case study of development, validation, and acceptance of a non-animal method for assessing human vaccine potency overview of currently approved serological methods with a focus on diphtheria and tetanus toxoid potency testing presented at: international workshop on alternative methods to reduce, refine, and replace the use of animals in vaccine potency and safety testing: state of the science and future directions. niceam. bethesda, md animal refinement and reduction: alternative approaches for potency testing of diphtheria and tetanus vaccines development and validation of serological methods for human vaccine potency testing: case study of an anthrax vaccine humane endpoints in vaccine potency testing approaches to reducing animal numbers in vaccine potency testing application of the consistency approach in the united states to reduce animal use in vaccine potency testing veterinary vaccine post-licensing safety testing: overview of current regulatory requirements and accepted alternatives target alternative vaccine safety testing strategies for pertussis toxin toward replacement of the monkey neurovirulence test in vaccine safety testing economic evaluation of the -vaccine routine childhood immunization schedule in the united states (as amended). u.s.c. a federal food, drug, and cosmetic act. (as amended). u.s.c. et seq code of federal regulations, title , food and drugs replacement, reduction and refinement alternatives to animal use in vaccine potency measurement development and use of a novel in vitro assay for testing of diphtheria toxoid in combination vaccines european department for the quality of medicines within the council of europe united states minimum requirements: tetanus toxoid recommendations for diphtheria, tetanus, pertussis and combined vaccines european department for the quality of medicines within the council of europe united states minimum requirements: diphtheria european department for the quality of medicines within the council of europe european department for the quality of medicines within the council of europe japanese minimum requirements for biological products. national institute of infectious diseases annex . guidelines for the production and control of the acelluar pertussis compenent of monovalent or combined vaccines european department for the quality of medicines within the council of europe annex . recommendations for inactivated rabies vaccine for human use produced in cell substrates and embryonated eggs the nih test for potency alternatives to animals in the development and control of biological products for human and veterinary use. meeting of the international association of biological standardization validation of an in vitro potency test for the cuban hepatitis b vaccine european department for the quality of medicines within the council of europe european department for the quality of medicines within the council of europe world health organization. requirements for hepatitis a vaccine inactivated european department for the quality of medicines within the council of europe biological assays: assay of hepatitis b vaccine (rdna) world health organization. requirements for hepatitis b vaccine inactivated european department for the quality of medicines within the council of europe world health organization. requirements for oral poliomyelitis vaccine three rs approaches in the quality control of inactivated rabies vaccines. the report and recommendations of ecvam workshop use of elisa for in vitro potency test of rabies vaccine for animals use rabies vaccine potency control: comparison of elisa systems for antigenicity testing monograph xxxx: -rabies vaccine (inactivated) for veterinary use development of an in vitro potency test for tetanus vaccines: an immunoassay based on hc fragment determination combination of dtp and haemophilus influenzae type b conjugate vaccines can affect laboratory evaluation of potency and immunogenicity mutual interactions between dtap-ipv and haemophilus influenzae type b (hib)-conjugated vaccines in laboratory animal models use of animals models in the development of human vaccines animals and animal products. part -standard requirements. section . . sam : supplemental assay method for purity, potency and dissociation of brucella abortus vaccine, strain l animals and animal products. part -standard requirements. section . . sam : supplemental assay method for bacterial plate count of erysipelothrix rhusiopathiae vaccines supplemental assay method for test for potency of live avirulent pasteurella haemolytica vaccine animals and animal products. part -standard requirements. section . . sam : supplemental assay method for titration of chlamydophila felis (formerly feline chlamydia psittaci) in embryonated chicken eggs animals and animal products. part -standard requirements. section . . sam : supplemental assay method for the titration of feline calicivirus in cell culture animals and animal products. part -standard requirements. section . . sam : supplemental assay method for the titration of feline rhinotracheitis virus in cell culture animals and animal products. part -standard requirements. section . . sam : supplemental assay method for titration of monovalent european department for the quality of medicines within the council of europe animals and animal products. part -standard requirements. sam : supplemental assay method for titration of porcine transmissible gastroenteritis virus animals and animal products. part -standard requirements. sam : supplemental assay method for titration of porcine rotovirus in modified-live vaccines animals and animal products. part -standard requirements. section . . sam : supplemental assay method for titration of infectious canine hepatitis virus in primary canine kidney cell culture animals and animal products. part -standard requirements. section . . sam : supplemental assay method for titration of canine distemper virus in vero cell culture animals and animal products. part -standard requirements. section . . sam : supplemental assay method for titrating tissue culture adapted vaccine strains of infectious bursal disease virus european department for the quality of medicines within the council of europe standard requirements for vaccines against infectious bursal disease animals and animal products. part -standard requirements. section . . sam : supplemental assay method for titration of feline panleukopenia virus in cell culture animals and animal products. part -standard requirements. section . . sam . supplemental assay method for the titration of canine parvovirus in cell culture animals and animal products. part -standard requirements. section . . sam : supplemental assay method for the titration of distemper virus in embryonated chicken eggs animals and animal products. part -standard requirements. section . . sam : supplemental assay method for in vitro potency testing of erysipelothrix rhusiopathiae bacterins animals and animal products. part -standard requirements. section . . sam : supplemental assay method for potency testing of erysipelas bacterins in mice animals and animal products. part -standard requirements. sam - : supplemental assay method for potency testing enterotoxigenic (k , k , p, f pilus) escherichia coli bacterins animals and animal products. part -standard requirements. section . . sam : supplemental assay method for in vitro potency testing of leptospira interrogans serovar pomona bacterins animals and animal products. part -standard requirements. section . . sam : supplemental assay method for potency assay of leptospira interrogans serovar pomona bacterins animals and animal products. part -standard requirements. section . . sam : supplemental assay method for in vitro potency testing of leptospira interrogans serovar canicola bacterins european department for the quality of medicines within the council of europe animals and animal products. part -standard requirements. section . . sam : supplemental assay method for potency assay of leptospira interrogans serovar canicola bacterins animals and animal products. part -standard requirements. section . . sam : supplemental assay method for in vitro potency testing of leptospira interrogans serovar grippotyphosa bacterins animals and animal products. part -standard requirements. section . . sam : supplemental assay method for potency assay of animals and animal products. part -standard requirements. section . . sam : supplemental assay method for in vitro potency testing of leptospira interrogans serovar icterohaemorrhagiae bacterins animals and animal products. part -standard requirements. section . . sam : supplemental assay method for potency assay of leptospira interrogans serovar icterohaemorrhagiae bacterins animals and animal products. part -standard requirements. section . . leptospira hardjo bacterin european department for the quality of medicines within the council of europe replacement, reduction and refinement alternatives to animal use in vaccine potency measurement veterinary services memorandum no. . . in vitro serial release potency test for completed product containing clostridium chauvoei animals and animal products european department for the quality of medicines within the council of europe animals and animal products. part -standard requirements. sam : supplemental assay method for the in vitro potency assay of bovine respiratory viruses in killed vaccines animals and animal products. part -standard requirements. sam : supplemental assay method for quantitating the gp antigen of feline leukemia virus veterinary vaccines united states department of agriculture licensing requirements for feline leukemia virus vaccines animals and animal products. part -standard requirements. sam : supplemental assay method for determination of the specific viral antigen content in inactivated canine coronavirus vaccines european department for the quality of medicines within the council of europe three rs achievements in vaccinology validation study to evaluate the reproducibility of a candidate in vitro potency assay of newcastle disease vaccines and to establish the suitability of a candidate biological reference preparation edqm international symposium on alternatives to animal testing new approaches in the development and control of biologicals practical alternatives to reduce animal testing in quality control of veterinary biologicals in the americas the consistency approach for quality control of vaccines -a strategy to improve quality control and implement rs the authors extend their sincere appreciation to all participants in the international workshop for their enthusiastic contributions leading to the workshop recommendations and conclusions. the members of the iccvam interagency biologics working group and niceatm staff are acknowledged for their contributions to the planning of the workshop, and the many invited experts are acknowledged for their contributions to breakout group discussions and workshop proceedings. finally, the authors thank brett jones and vivian doelling for their assistance in the preparation of this manuscript. key: cord- -f i lq w authors: bachofen, claudia; braun, ueli; hilbe, monika; ehrensperger, felix; stalder, hanspeter; peterhans, ernst title: clinical appearance and pathology of cattle persistently infected with bovine viral diarrhoea virus of different genetic subgroups date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: f i lq w bovine viral diarrhoea (bvd) is an economically important cattle disease with a world-wide distribution that is caused by bvd virus, a pestivirus of the flaviviridae family. bvd viruses are genetically highly variable. they are classified into two genetic species (bvdv- and - ) that are further divided into numerous subgroups, particularly for bvdv- . the complexity of these viruses is also reflected in their interaction with the host animals. infections are either transient or persistent and can cause a wide spectrum of clinical signs, from no or very mild disease to severe forms, reminiscent of viral haemorrhagic fevers. in this work, we have analysed the clinical signs and the pathology of bvd viral infections in a cattle population where different subgroups of bvdv- genotype viruses are endemic. in addition, we have examined potential virulence properties of bvdv- subgroups during persistent infection by comparing the viral subgroups present in clinical cases with those detected in persistently infected (pi) animals sampled for epidemiological criteria, irrespective of their health condition. furthermore, the clinical and postmortem findings were compared with respect to genetic characteristics of the viruses isolated from these animals. our results indicate that the bvdv positive animals fall roughly into two categories, depending on the primary organ affected and the age, with lung-centred pathology occurring mainly in young animals and mucosal pathology predominantly in older animals. furthermore, we found a markedly higher proportion of representatives of the bvdv- e subgroup in stillborn calves and aborted foetuses originating from epidemically unrelated cattle herds, suggesting that bvdv- e may play a special role in prenatal and perinatal losses. bovine viral diarrhoea (bvd) is an economically important cattle disease with a worldwide distribution that is caused by bvd virus, a pestivirus of the flaviviridae family. bvd viruses are genetically highly variable. they are classified into two genetic species (bvdv- and - ) that are further divided into numerous subgroups, particularly for bvdv- . the complexity of these viruses is also reflected in their interaction with the host animals. infections are either transient or persistent and can cause a wide spectrum of clinical signs, from no or very mild disease to severe forms, reminiscent of viral haemorrhagic fevers. in this work, we have analysed the clinical signs and the pathology of bvd viral infections in a cattle population where different subgroups of bvdv- genotype viruses are endemic. in addition, we have examined potential virulence properties of bvdv- subgroups during persistent infection by comparing the viral subgroups present in clinical cases with those detected in persistently infected (pi) animals sampled for epidemiological criteria, irrespective of their health condition. furthermore, the clinical and postmortem findings were compared with respect to genetic characteristics of the viruses isolated from these animals. our results indicate that the bvdv positive animals fall roughly into two categories, depending on the primary organ affected and the age, with lung-centred pathology occurring mainly in young animals and mucosal pathology predominantly in older animals. furthermore, we found a markedly higher proportion of representatives of the bvdv- e subgroup in stillborn calves and aborted foetuses originating from epidemically unrelated cattle herds, suggesting that bvdv- e may play a special role in prenatal and perinatal losses. ß elsevier b.v. all rights reserved. growth in cultured cells. the two biotypes also differ with respect to their effects in infected animals. only the ncp biotype can establish a persistent infection of the foetus if the dam is transiently infected between approximately days and of its development. even though such foetuses may develop normally, they remain infected for life and shed large quantities of virus, thereby assuring the persistence of bvdv in the cattle population even in the absence of animals susceptible to transient infection. different from other persistent viral infections in man and animals, bvdv persistently infected (pi) cattle are immunotolerant to the infecting viral strain (reviewed in peterhans et al., ) . the clinical consequences of infection with bvd virus are as diverse as the genetic and antigenic properties of these viruses. disease can result from different pathogenetical mechanisms, depending on the different types of infection. most transient infections may take an inapparent or mild course, associated with low-grade fever, diarrhoea and coughing. rarely, however, acutely infected animals may suffer from high grade fever and bleeding in internal organs. this often fatal form of acute infection was reported in outbreaks in the late s and early s, caused by highly virulent bvd virus- strains (ridpath et al., (ridpath et al., , . although generally considered to be of low virulence, bvdv- , too, may cause similar severe disease signs in acutely infected animals, albeit only rarely (liebler-tenorio et al., ; ridpath et al., ) . in the case of persistent infections, clinical signs in pi animals can be differentiated pathogenetically into mucosal disease (md) and non-md cases. the onset of md is associated with the appearance of the cytopathic biotype of bvdv that arises as a result of mutations and/or recombination events from the persisting ncp virus (kü mmerer et al., ; tautz et al., ; becher et al., ) . this lethal disease is characterised by mucosal lesions, destruction of the lymphoid tissue in the gastrointestinal tract (liebler et al., ) and untreatable diarrhoea. the onset of the clinical signs is often acute but a more chronic form, called late onset md, is also described (liebler-tenorio et al., ; fritzemeier et al., ) . the clinical signs in pi animals that have not (yet) developed md can encompass a wide spectrum of symptoms, ranging from normal health to subclinical disorders, ill-thrift and growth retardation. chronic or recurrent intestinal and/or pulmonary symptoms are frequently observed but occasionally dermatological, neurological or haematological disorders are the only signs of a persistent infection (braun et al., ; taylor et al., ) . in many cases the clinical and necropsy findings do not allow a clear differentiation between md and non-md cases. the isolation of both biotypes, ncp and cp, proves a pi animal to suffer from md. possible reasons for the variable clinical signs seen in bvdv infections have been analysed repeatedly and in some cases an impact of the infecting virus strain was proven (baule et al., (baule et al., , fulton et al., ; jones et al., ; ridpath et al., ) . however, these studies focused on transient infections. moreover, the analyses were restricted to single outbreaks of identical or very closely related viral strains that affected several animals within one herd or in different herds simultaneously, or to experimental infections. hence, the observations may be representative for a given viral strain rather than for an entire subgroup of bvdv. in contrast to transient infections, disease associations between individual bvdv strains or subgroups have not been investigated in persistent infection. bvd is endemic in switzerland, with roughly % of cattle seropositive and . % persistently infected, with seemingly no change in prevalence over a time period of some years (bü rki et al., ; homberger et al., ; rü fenacht et al., ; stalder et al., ) . in this work, we have analysed pi animals referred by private veterinary practitioners to the clinical and pathology departments of the veterinary hospital of the university of zü rich. due to the endemic nature of bvd, these animals may represent the most severe clinical forms of persistent infection, comparable to the tip of the iceberg. we have recorded and applied a scoring protocol for the clinical and postmortem findings of the diseased animals and analysed possible correlations between different organ manifestations and the subgroups of persisting bvdv. to obtain an insight into the possibility of virulence of bvdv in persistent infection, we have analysed the viral subgroups present in diseased pi animals as compared to subgroups present in animals diagnosed bvdv pi independently of the clinical status. at the department of farm animals of the university of zurich, all calves less than months of age and chronically diseased animals are routinely tested for the presence of bvdv. we have retrospectively analysed all clinical reports of pi animals shown at the clinic for ruminants from first of january to st of december (group a, table ). in addition, the prospective cases from the years and were included (group b, table ) , yielding clinical case reports. all animals had undergone the same thorough clinical examination following a standardised protocol as described previously (braun et al., ) . haematological, biochemical, bacteriological and parasitological analyses were performed as described by braun et al. ( ) . the bvdv status of animals of groups a and b was determined intra vitam by immunohistochemistry (ihc) of skin biopsies ( cases) and by antigen-capture elisa of blood leukocytes ( cases) (strasser et al., ) or of skin biopsies ( cases) (idexx herdcheck bvdv ag/serum plus, idexx europe b.v., ne schiphol-rijk, the netherlands). some animals were repeatedly tested by multiple methods. ten animals of groups a and b were only diagnosed after being euthanised. the bvdv status of all animals was confirmed postmortem by ihc of organ samples. all samples revealed the staining pattern typical for persistent infection, as described previously by hilbe et al. ( ) . as a result of the unfavourable prognosis, all animals were euthanised within - days after the initial clinical investigation and sent to the institute of veterinary pathology for necropsy. a necropsy report was available of of the cases from groups a and b. in addition, we included in our study necropsy reports of animals that had been euthanised or had perished and been submitted by private veterinarians for necropsy to the institute of veterinary pathology where they were tested bvd viruspositive by ihc of organ samples (group c, table ). the reasons for the submission of these carcasses for postmortem analysis were variable and encompassed examination of herd problems, clarification of ambiguous clinical disease signs or suspicion of inherited disease such as spinal muscle atrophy of swiss brown calves. no information on intra vitam examinations and their results were available of the animals of group c. all in all, we analysed necropsy reports (groups a, b, c), of which contained complete data sets (groups a, b, c. ). in cases, postmortem reports were only available for single organs or body parts (group c. , table ). the cases analysed were extended by cases of bvdv infected neonates (stillbirths and calves younger than days) and aborted foetuses, tested positive by ihc (staining pattern typical for persistent infection (group d, table )). descriptive information from the standardised clinical and necropsy reports was transformed into ordinal data (e.g. general health condition, degree of lung alteration) or nominal data (e.g. gender) with the help of a specially created evaluation protocol. the evaluation protocol applied was created in collaboration with clinicians and pathologists and is based on clinical scoring protocols used previously (braun et al., (braun et al., , . for the majority of findings we used a simple -level grading: nothing abnormal detected = , mild = , moderate = , severe alteration = . the evaluation of all reports was performed by the same person and without any information on the bvdv subgroup isolated from the corresponding pi animal. the scoring was based on the graduation of the clinical and postmortem findings made by the various clinicians and pathologists who had initially recorded the cases. grading the findings is an essential part of the standardised protocols used for initial clinical examination and necropsy. in those cases where the organ was only partially affected (e.g. an abscess in a single lung lobe) we considered that the severity and spread of the lesion would serve to assess the general impairment of the organ. to compare the mucosal alterations between different pi animals, we used a standardised mucosal (mc) index calculated for each animal. this index considers the number of affected sections of the alimentary tract (from nose to large intestines = max. organs; affected = any pathological change from the normal tissue integrity) and the severity of the alterations ( = no alteration, = mild, = moderate, = severe alteration). with respect to the multiple dispersed lesions typically seen in md cases (liebler-tenorio et al., ) the calculation places more weight on the number of affected sections than on the severity of the alterations. the mc index may range from to . swiss bvdv strains, isolated between and were sequenced and phylogenetically analysed by stalder et al. ( ) . these viruses originated from animals detected to be pi as a result of a cross-sectional epidemiological study to determine the prevalence of bvdv in switzerland (rü fenacht et al., ) . in addition, viruses were derived from herd analyses performed in order to assess diagnostic tests or to apply molecular epidemiology. the bvdv isolates of the clinical cases described here were isolated, sequenced and phylogenetically analysed for a study published previously (bachofen et al., ) . for all statistical calculations the ncss/pass software (kaysville, ut, usa) was used. as the data were not normally distributed, we used the non-parametric oneway anova test (kruskal-wallis) to compare medians of three or more groups of patients. if this test revealed a significant over-all difference, pair-wise comparisons were performed using the wilcoxon rank-sum test. to compare proportions we applied the chi-square or fisher's exact test. the probability level was set to %. the displayed pvalues represent the results of the two-tailed hypothesis testing. we tested serum of pi animals from the prospective cases for the existence of cytopathic bvdv virus as a hallmark for the onset of md. the sera were diluted seven times in tenfold steps in cell culture medium (earle's minimal essential medium (mem)) enriched with % foetal bovine serum (fbs). mem was purchased from seromed (biochrom, munich, germany) and fbs from sigma or oxoid gmbh (wesel, germany). fbs was free of bvdv and antibody to bvdv as tested by virus isolation and serum neutralization test, respectively. each serum dilution step was distributed to six wells ( ml) of a -well microtiter plate, seeded with primary bovine turbinate cells. after days of incubation at c and % co , ml of supernatant was transferred to a fresh -well microtiter plate, pre-seeded with bovine turbinate cells. after addition of ml of fresh mem, the microtiter plate was incubated as before and the passaging procedure repeated once more. after each passage, the cells were fixed and stained for viral protein as described by adler et al. ( ) . the cells were then microscopically controlled for the presence of a cytopathic effect. in order to investigate differences between bvdv subgroups regarding virulence during persistent infection, clinical recordings and necropsy reports of pi animals infected with viruses of different bvdv- subgroups were analysed. we included patients from the clinic for ruminants of the department of farm animals (university zü rich) and additional necropsy reports of animals referred by private veterinarians directly to the institute of veterinary pathology (university zü rich) for postmortem analysis. as shown in table , the cases could be separated into four groups: retrospective cases (group a, n = ) and prospective cases (group b, n = ) with both a clinical and a necropsy report, cases directly referred for necropsy by private practitioners without clinical report (group c, n = ) and postmortem analyses of abortions and neonates (animals days of age) without clinical report (group d, n = ). of the cases of groups a, b and c (abortions and neonates not included since the gender is often not stated) % were females, while for the animals with a clinical report (groups a and b, n = ) the proportion of females was %. animals of five different breeds and diverse crossbreeds were analysed. the majority of cases (n = ) were of the swiss brown ( %), holstein-frisian ( %) and swiss fleckvieh ( %) breeds. these numbers reflect the over-all breed proportions of the patient population of the clinic. single cases of rhä tisches grauvieh, dexter, limousin and beef crossbreeds were also represented. the median age of the animals (n = ) was months, with a range from days to years. animals referred to the clinic (groups a and b, n = ) were significantly older (median = months) than those submitted directly for necropsy (group c, n = , median age = months). of the animals with a clinical record (n = ), % had an age of months or over and four animals were older than years ( , , and months). the cases originated from of the swiss cantons. most of the cases (n = ) had a history of recurrent or untreatable diarrhoea ( %), pneumonia ( %) or both conditions together ( %). neurological symptoms ( %) were often anamnestically described in necropsy reports of young calves, directly referred for postmortem examination (group c). other anamnestic signs were abortion, lameness due to claw problems, anaemia and ill-thrift. in % of the cases (n = ) the anamnestic report indicated concomitant health problems in the herd. growth retardation was indicated in % of the cases. the most frequently observed clinical and postmortem manifestations are shown in fig. . nearly all animals showed unspecific signs like loss of appetite ( %), reduction of the general condition ( %) and ruminal hypoperistalsis ( %), followed by intestinal disorders like diarrhoea ( %) and the resulting dehydration ( %). respiratory sounds of different degree were present in % of the cases. mucosal erosions as a more specific manifestation were found in the oral cavity and on the muzzle of % and % of the animals, respectively (fig. a) . for the determination of the most frequent postmortem findings, only cases with a complete necropsy report were included, whereas partial necropsies (e.g. single organs or body parts) were excluded (group c. ). group d was excluded, because the abortions were often in a condition that would no longer allow clear postmortem diagnosis or consisted only of foetal body parts. the most frequent necropsy findings differed between animals with clinical reports (groups a and b) and those referred directly for necropsy (group c. ) (fig. b) . in the latter, these were pathological changes in the lung, while alterations in one or more mucosal organs (from muzzle to large intestines) were the primary findings in the clinical cases. generally, except for the lung, a higher proportion of cases referred to the clinic (groups a and b) showed macroscopic lesions than animals directly referred for a postmortem examination (group c. ). haematological data were available from of the cases with a clinical report (groups a and b). the most frequently observed changes were elevated bilirubin levels ( %) and monocytosis ( %), neutrophilia (in %) and leukocytosis (in % of the animals). decreased leukocyte counts were found in % of the cases. alterations in erythrocyte parameters were frequent and were displayed as polycythemia ( % of the animals), decreased cellular haemoglobin content ( %) and a decreased erythrocyte volume ( %). for groups a and b, intra vitam parasitological and bacteriological analyses were not performed routinely but rather upon clinical suspicion. however, with the exception of gastrointestinal nematodes that were present in % of the cases analysed, parasites and bacteria such as liver fluke, lung worm, salmonella, campylobacter and enterotoxic escherichia coli were only occasionally confirmed ( - % of analysed cases). furthermore, all animals analysed for the presence of ovine herpesvirus- , the causative agent of malignant catarrhal fever in cattle, were negative. four and five animals were analysed for the presence of rota-and coronavirus, respectively. for each virus, one animal tested positive. bovine herpesvirus- that can cause symptoms similar to bvd/md is eradicated in switzerland and can be excluded as contributing factor to the symptoms observed. for all groups, postmortem table ). (b) the most frequently observed necropsy findings from pi animals submitted for postmortem examination from the clinic (black bars) or directly from private practitioners (grey bars). only necropsies of entire carcasses were included. the animal groups indicated refer to table . a one or multiple pathological alteration(s) of the mucosa of the whole alimentary tract, including nose and muzzle. b one or multiple pathological alteration(s) of the mucosa of the gastrointestinal tract. c macroscopic alteration of the mediastinal or mesenterial lymph nodes. parasitological and bacteriological analyses were performed sporadically, depending on demand and suspicion and were not included to the study. in many cases, mucosal lesions were observed at multiple locations of the gastrointestinal tract. to be able to compare the general degree of mucosal lesions between groups of animals, it was necessary to condense the multiple mucosal alterations to one parameter. we used the number of affected organs and the severity of the single alterations to calculate an index for each animal, referred to as mucosal (mc) index (see section for details). the highest value that is theoretically possible (= all analysed organs are severely affected) is . the maximal and minimal mc index values observed from the cases analysed (complete necropsy reports only: groups a, b and c. ) was and , respectively, with an arithmetic mean of . . because intestinal and pulmonary symptoms were the most frequent findings observed in the bvdv infected animals, we investigated whether these findings were correlated. interestingly, these organ manifestations tended to exclude each other. as shown in fig. a , animals with a high mucosal index had significantly less severe lung alterations than those with a low mucosal index. moreover, animals with severe pulmonary manifestations were significantly younger than those with less severe pathological changes in the lungs (fig. b ). by contrast, the mucosal index was significantly higher in older animals than in young ones (fig. c) . linear regression analysis (spearman rank) demonstrated a weak (r = . ) but statistically significant (p = . ) correlation between the mc index and the age of the animals. the dichotomy between lung and mucosal lesions pointed to roughly two groups of bvdv infected patients. in order to investigate if this correlated with the presence or absence of mucosal disease, we tested serum samples of pi animals of group b for the presence of cytopathic (cp) virus by passaging different dilutions of the sera three times on cultured bovine turbinate cells. this analysis was restricted to animals, since blood samples were only available from the prospective cases (group b) and young calves with maternal antibodies had to be excluded due to inhibition of virus isolation. the results are shown in table . with one exception ( - ), the cp biotype was only present in sera of animals with an mc index of ! . , suggesting that these patients had suffered from md. no cp biotype was observed in animals with an index of . . therefore, we decided to set the threshold between mdand non-md cases to . . extrapolating this finding to the (table ) . mucosal-indices of all cases, % of the animals ( / ) had suffered from md. of the cases analysed in this work (groups a, b, c and d), the partial genomic sequence of the persisting virus was known from cases (bachofen et al., ) . for the cases of group b, viral rna was isolated from blood, from the other cases from frozen tissue samples. phylogenetic subgroup assessment was performed based of the untranslated region and in some cases additionally on the n pro coding region. the subgroups present are shown in fig. b . the majority of the animals harboured viruses of the subgroups bvdv- e ( %) and h ( %), followed by k ( %) and b ( %). from one single animal we isolated the bvd- x virus as described previously (bachofen et al., ) . these are (with exception of x) the same subgroups as described by stalder et al. ( ) (fig. a) . the subgroup e was most prominent in the latter ( %) while h was the most frequent subgroup in clinical cases ( %) (fig. ) . however, statistical analysis revealed no significant differences of the proportions of bvdv subgroups present in the diseased pi's and animals detected persistently infected independent of their health condition. cases with a complete necropsy report and a known bvdv sequence could be included in this part of the study. this number is composed of animals of group a, of group b and the cases of group c. . the single animal harbouring bvdv x was excluded from the analysis, resulting in pi animals. of these, % had harboured viruses belonging to subgroup b, % to e and h each, and % to k (fig. ) . we observed no statistically significant differences in the associations of viruses of a given subgroup with mucosal or pulmonary alterations or suspected md cases (fig. ) , nor did we see differences with regard to different age groups, breeds or other clinical, postmortem or haematological findings (data not shown). however, there was a tendency for h viruses to be more frequently isolated from animals with mucosal problems, presumable md cases (mucosal index ! ), and thus older animals, while e was the predominant subgroup of bvd virus in patients with pulmonary alterations and thus younger animals (fig. ) . to broaden the age range of the patients, we additionally analysed the proportions of the different bvdv- subgroups in the cases of group d (abortions, stillbirths and neonates). interestingly, we observed a clear predominance of representatives of subgroup e bvd viruses in these animals (fig. ) . the subgroup proportions in group d were significantly different from those observed in animals with mucosal lesions and suspected md cases (mc index ! ). the spectrum and severity of clinical signs caused by bvdv infection are highly variable. factors contributing to this complexity include two types of infection with entirely different involvement of the immune system (immune response in transient versus immunotolerance in persistent infection), two different biotypes of virus (noncytopathic versus cytopathic) and the diverse genetic these animals were chosen due to availability of serum and absence of maternal antibodies. in persistent infection, in contrast, analysis of a potential impact of the viral strain on the clinical picture is, for a number of reasons, more difficult. among them is the existence of disease signs associated with persistent infection as such, and the occurrence of md as a form of infection associated with the emergence of a cytopathic biotype of bvdv in these immunotolerant animals. furthermore, some of the signs of persistent infection unrelated to md may be of chronic nature, such as growth retardation and general ill-thrift. however, as shown by pluriparous pi cows and pi bulls detected among the candidates for artificial insemination stations, clinical signs of persistent infection can be absent in some animals. experimental generation of a sufficient number of pi animals with different strains of bvdv would be laborious and require long-term follow-up. in addition, many different factors such as time point of maternal infection, genetic background of the animals and environmental factors, e.g. the presence of various other cattle pathogens, may modulate the outcome. moreover, the epidemiological situation -endemic versus non-endemic -may influence the clinical appearance of infections. it is not surprising, therefore, that questions such as the impact of bvdv genetic properties on the health status of a pi animal, or possible associations between disease signs and a given subgroup of bvdv during this type of infection, have to date not been addressed. we have investigated these questions in an endemic situation that was virtually undisturbed by vaccination or other control measures, both of which might influence the outcome of infection. in order to obtain information on the pathogenic potential of bvdv in persistent infection, we analysed clinical and necropsy findings of diseased pi animals and compared the bvdv subgroups present in these animals those isolated from animals diagnosed as pi independently of the health status. since we included prospective as well as retrospective cases examined over a period of years, the long time range and the fact that different clinicians and pathologists examined the animals might influence the comparability of the raw data. however, the protocol applied to both the clinical and pathological investigations did not change over this time. in addition, the fact that the initial examinations were performed by many different clinicians and pathologists decreases the risk of a systematic bias. moreover, the transformation of nominal into numerical data was done by one person, using a standardised protocol. to obtain a larger number of cases, we analysed different groups of patients. while the retro-and prospective cases from the ruminants' clinic (groups a and b) showed similar demographic parameters and postmortem findings (data not shown), we observed significant differences to the animals of group c. animals of groups a and b were significantly older and generally more severely diseased than the cases directly referred for postmortem analysis (group c) (fig. b) . this might be due to the fact that, generally, the more valuable and thus primarily older animals with complex or unsuccessfully pre-treated health problems are referred for a stay at the clinic. the high proportion of females in groups a and b simply reflects the fact that most of these animals originate from dairy farms. interestingly, also the main postmortem findings differed between groups a and b versus group c, with mucosal alterations playing a prominent role in groups a and b and pulmonary lesions in group c (fig. b) . keeping in mind the age difference between the groups, we tested the statistical correlation between lung and mucosal manifestations and the age of the animals. mucosal lesions were more frequent in older and lung lesions in younger animals (fig. ) ; that held also true when the two animal groups (a + b and c) were tested separately (data not shown). the reason for the correlation between age and the postmortem finding may be related to the higher risk of md onset with increasing age. md is correlated to the appearance of the cp virus that arises as a result of mutations or recombination during viral rna replication from the persisting ncp virus (deregt and loewen, ; neill and ridpath, ; lackner et al., ) . statistically, a longer time period of viral replication may increase the risk of generating mutations leading to a cp biotype. due to the retrospective nature of the study, we were not in a position to fulfill the virological criterion for diagnosing md by showing the presence of cytopathic and non- fig. . proportions of the four main swiss bvdv subgroups isolated from different groups of pi animals. a only complete necropsy reports were included: cases of group a, of group b and cases of group c (table ) = . however, one single animal harboured a virus that did not belong to any of the four subgroups analysed and was therefore not included (n = ). b cases of group d (table ). cytopathic bvdv in all cases. to overcome this problem we combined a virological determination of md of selected cases (namely the isolation of both biotypes) with a standardised pathological investigation ( table ) . the latter was achieved by creating a ''mucosal (mc) index'', comparable to other clinical or pathological indices, e.g. the psoriasis area severity index (pasi) in human dermatology. typically, md leads to mucosal lesions at multiple locations of the digestive tract. therefore, the mc index places more weight on the number of affected organs than on the severity of single lesions. this approach also minimizes individual differences in rating the severity of lesions and favours completeness of the necropsy reports. the results of the comparison of the biotype analysis with the mc index (table ) support the validity of the concept. taking all bvd viral sequences of groups a, b, c and d together, we did not observe statistically significant differences to the bvdv subgroup distribution previously described by stalder et al., (fig. ) . with the exception of the single ''orphan'' bvd x strain, we found the same four bvd- subgroups in the diseased animals and in the pi animals sampled in the course of an epidemiological survey. although sampled in the same time period, the two large groups of pi animals are not matched with respect to breed, age or gender. on the one hand, using unmatched hosts, only major differences in tropism and virulence would show up. the results of our study argue against such major differences between entire subgroups. on the other hand, this observation does not exclude that individual strains may differ in virulence and tropism, as shown for transient infections. a different aspect concerns possible bvdv subgroup affiliations with the two main clinical and pathological patterns observed, i.e. lung-versus gastrointestinal tractcentred. in this context, the possibility that these patterns might be influenced by the presence (or absence) of other pathogens needs to be considered. such pathogens could include other viruses, bacteria and parasites. viruses known to induce similar clinical signs and pathology, i.e. bovine herpesvirus- and ovine herpesvirus- , can be excluded. the former is eradicated in switzerland, and all pi animals suspected of suffering from malignant catarrhal fever tested negative for this lethal form of infection caused by ovine herpes virus- (ackermann, ) . where data were available, we recorded additional bacteriological and parasitological examinations. however, in most cases the suspected infections were not confirmed. we did not find a statistically significant association between a given bvd viral subgroup and the two major disease manifestations (fig. ) , nor to any other clinical or mortem finding (data not shown). however, we observed a tendency for viruses of subgroup h to be more frequently isolated from animals with mucosal lesions, while e was the predominant subgroup in animals with lung alterations (fig. ) . regarding the age dependency of the mucosal and pulmonary manifestations (fig. ) , the observed subgroup tendencies may well correlate to different age groups of pi animals rather than reflect a selective organ tropism. indeed, % of the animals younger than months ( months = median age of all animals analysed) harboured bvdv- e, while this proportion was only % in older animals (! months). since all pi animals are infected as foetuses, animals infected with bvdv- e may die younger than animals infected with other subgroups and are therefore less frequently observed among older animals. this is supported by the observation of a surprisingly high proportion of bvdv- e strains in abortions, stillbirths and neonates (fig. ) , which clearly encourages additional investigations. we can exclude that a e live vaccine may be responsible, since, as indicated above, vaccines are used only very infrequently, and a e vaccine is not licensed in switzerland. moreover, we can also exclude that a particular strain of e may cause these abortions because the sequence data indicate that genetically different strains were present in these animals (bachofen et al., ) . in summary, our study showed that, in an endemic situation, clinical cases of bvdv pi animals fall roughly in two distinct categories, with lung-centred pathology occurring mainly in young animals and mucosal pathology mainly in older animals. moreover, even though we did not find evidence for one bvdv subgroup being generally more virulent during persistent infection, the epidemiologically unrelated concentration of e subgroup bvd viruses in stillborn calves and aborted foetuses provides preliminary evidence that viruses of this subgroup may play a special role in prenatal and perinatal losses due to bvd virus. virus in sheep's skin macrophages infected with cytopathic bovine viral diarrhea virus release a factor(s) capable of priming uninfected macrophages for activationinduced apoptosis co-existence of genetically and antigenically diverse bovine viral diarrhoea viruses in an endemic situation genetic heterogeneity of bovine viral diarrhoea viruses isolated in southern africa pathogenesis of primary respiratory disease induced by isolates from a new genetic cluster of bovine viral diarrhea virus type i phylogenetic analysis of pestiviruses from domestic and wild ruminants rna recombination between persisting pestivirus and a vaccine strain: generation of cytopathogenic virus and induction of lethal disease genetic and antigenic characterization of novel pestivirus genotypes: implications for classification bovine virus diarrhea mucosal disease: clinical findings in calves and heifers clinical findings and treatment of cattle with botulism clinical findings in cattle with traumatic pericarditis kasuistischer beitrag zur mucosal disease bovine viral diarrhea virus: biotypes and disease genetic heterogeneity of bovine viral diarrhoea virus in italy the development of early vs. late onset mucosal disease is a consequence of two different pathogenic mechanisms bovine viral diarrhea virus (bvdv) b: predominant bvdv subtype in calves with respiratory disease immunohistochemical diagnosis of persistent infection with bovine viral diarrhea virus (bvdv) on skin biopsies zur epizootologischen bedeutung der sö mmerung bei der mucosal disease/virusdiarrhö e des rindes the extended genetic diversity of bvdv- : typing of bvdv isolates from france phylogenetic analysis of bovine pestiviruses: testing the evolution of clinical symptoms bovine viral diarrhea virus strain oregon: a novel mechanism for processing of ns - based on point mutations temporal modulation of an autoprotease is crucial for replication and pathogenicity of an rna virus experimental mucosal disease in cattle: changes of lymphocyte subpopulations in peyer's patches and in lymphoid nodules of large intestine comparative investigation of tissue alterations and distribution of bvd-viral antigen in cattle with early onset versus late onset mucosal disease recombination with a cellular mrna encoding a novel dnaj protein results in biotype conversion in genotype bovine viral diarrhea viruses identification of a new group of bovine viral diarrhea virus strains associated with severe outbreaks and high mortalities how the bovine viral diarrhea virus outwits the immune system phylogenetic, antigenic and clinical characterization of type bvdv from north america impact of variation in acute virulence of bvdv strains on design of better vaccine efficacy challenge models multiple outbreaks of severe acute bvdv in north america occurring between and linked to the same bvdv strain prevalence of cattle infected with bovine viral diarrhoea virus in switzerland genetic heterogeneity of pestiviruses of ruminants in switzerland detection of bovine virus diarrhea virus (bvdv) in peripheral blood, cell cultures and tissue using a monoclonal antigen-capture elisa pathogenesis of mucosal disease, a deadly disease of cattle caused by a pestivirus performance, survival, necropsy, and virological findings from calves persistently infected with the bovine viral diarrhea virus originating from a single saskatchewan beef herd genetic diversity of international bovine viral diarrhoea virus (bvdv) isolates: identification of a new bvdv- genetic group bovine viral diarrhoea virus genotype can be separated into at least eleven genetic groups sequencing and comparative analysis of a pig bovine viral diarrhea virus genome this work was supported by the swiss national science foundation and a grant from vetsuisse. we thank matthias schweizer and reto zanoni for helpful discussions, the team of the diagnostic unit for technical help, marcus doherr for statistical support and ruth parham for linguistic assistance. key: cord- - ad fw z authors: monath, thomas p. title: vaccines against diseases transmitted from animals to humans: a one health paradigm date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: ad fw z abstract this review focuses on the immunization of animals as a means of preventing human diseases (zoonoses). three frameworks for the use of vaccines in this context are described, and examples are provided of successes and failures. framework i vaccines are used for protection of humans and economically valuable animals, where neither plays a role in the transmission cycle. the benefit of collaborations between animal health and human health industries and regulators in developing such products is discussed, and one example (west nile vaccine) of a single product developed for use in animals and humans is described. framework ii vaccines are indicated for domesticated animals as a means of preventing disease in both animals and humans. the agents of concern are transmitted directly or indirectly (e.g. via arthropod vectors) from animals to humans. a number of examples of the use of framework ii vaccines are provided, e.g. against brucellosis, escherischia coli o , rabies, rift valley fever, venezuelan equine encephalitis, and hendra virus. framework iii vaccines are used to immunize wild animals as a means of preventing transmission of disease agents to humans and domesticated animals. examples are reservoir-targeted, oral bait rabies, mycobacterium bovis and lyme disease vaccines. given the speed and lost cost of veterinary vaccine development, some interventions based on the immunization of animals could lead to rapid and relatively inexpensive advances in public health. opportunities for vaccine-based approaches to preventing zoonotic and emerging diseases that integrate veterinary and human medicine (the one health paradigm) are emphasized. zoonoses (diseases transmissible from animals to humans) account for approximately % of all infectious pathogens of human beings and % of all emerging infectious diseases [ , ] . the origins of these diseases, and underlying factors (including man-made factors) in their emergence have been the subject of considerable interest [ , ] . certain zoonotic diseases have the potential for pandemic spread by human contagion, such as avian influenza, sars and the middle east respiratory syndrome coronavirus, and others for regional cross-border epizootics, such as yellow fever, venezuelan equine encephalitis and rift valley fever. the cost of a short list of zoonotic disease emergences in the interval between and , including bovine spongiform encephalopathy, sars, highly pathogenic avian influenza, west nile, and pneumonic plague (india), was estimated to exceed $ billion [ ] . of major emergencies that concerned public health (including hurricanes, earthquakes, and terrorist attacks) occurring between and , more than % were zoonotic disease outbreaks [ ] . however, the toll on public health is much greater than that caused by such dramatic outbreaks. it is estimated that different zoonotic diseases are responsible annually for . billion cases of human disease with . million deaths and substantial reductions in livestock production [ ] . animals, including livestock and companion animals, also suffer illness and death following infection with many zoonotic infections, and livestock and poultry are subject to large-scale intentional destruction as a means of preventing human infections, resulting in huge economic losses. wild animals, including endangered species, may also be mortally affected, examples being west nile disease in birds, yellow fever in neotropical monkeys, plague in black-footed ferrets, and ebola in the great apes. vaccines are an important means of prevention and control of zoonotic infectious diseases in humans and domesticated animals. however, the target for vaccination is, in almost all cases, the directly affected species, and there are few practically implemented illustrations of the potential for indirectly preventing human disease by immunizing the domesticated or companion animal sources of infection. the concept of immunizing wild animal reservoirs for the prevention of disease in humans or domestic animals is even more challenging and has received limited attention. moreover, human and animal health divisions of the biopharmaceutical industry are generally separate and segregated, and there is no organized approach to the development of new vaccines indicated for the prevention of spread of diseases from domestic or wild animals to humans. in addition, the major funding sources for research in human and animal diseases tend to be stove-piped into different government agencies, stifling cross-cutting approaches. the purpose of this review is to stimulate the science and policy communities to seek innovative ways to interdict zoonotic diseases by integrating human and veterinary medicine and vaccine development, and by creating new streams of funding aimed at the intersection of human and animal health. these aims are consistent with the one health initiative, which seeks to establish "collaborative efforts of multiple disciplines working locally, nationally and globally to attain optimal health for people, animals and our environment" [ ] . a framework for considering one health vaccine interventions is provided, as well as a brief review of past and current efforts, including a few successes. it will be obvious to the reader that this is a wide-open field with many opportunities that deserve more attention than they have received. the complexity, timeline, and cost of development of animal vaccines and the regulatory hurdles for product approval are far less than for human vaccines. thus some interventions based on the immunization of animals could lead to rapid and relatively inexpensive advances in public health. it is obvious that the benefit to human health deriving from vaccination of animals is far easier to justify when there is also a benefit to animal health, the latter often being closely linked to economic value. causative agents of zoonotic diseases, including viruses, parasites, bacteria, fungi, and prions, have extraordinarily varied life cycles and modes of transmission. some may persist between periods of active transmission in soil or invertebrate species. many have silent transmission cycles involving wild animals that have coevolved with the infectious agent and exhibit no signs of disease. some zoonotic diseases occur when a causative agent harbored by a wild animal reservoir jumps species to domesticated animals and thence to humans. others are primarily diseases of domesticated animal species. humans may be infected by direct contact with wild or domesticated animals, or indirectly by ingestion of contaminated milk or meat, inhalation of aerosolized secretions or excreta, fomites, or hematophagous insect or tick vectors. despite this complexity of epidemiological patterns, the opportunities for intervention often boil down to a few simple bottlenecks in the transmission process. for example, milk-borne diseases can be prevented by pasteurization, certain meat-borne diseases by inspection and animal husbandry improvements (e.g., trichinella, bovine tuberculosis), and other diseases avoided by limiting contact with known high risk species (e.g., tularemia, turtle-borne salmonellosis, exposure to bats carrying henipaviruses). where the risk of infection is high, or the resulting disease severe, vaccines may be the most efficient and cost-effective means of prevention and control. alternative methods for control of zoonotic diseases have generally employed trapping, poisoning, or other means of destroying the offending animal reservoir/vector; these methods have a mixed (often negative) record of success and are in any case becoming socially unacceptable. this review focuses on the use of vaccines for animals as an acceptable means of interdicting zoonotic disease in both animals and humans. three major epidemiological frameworks are identified for the control of zoonotic disease by means of vaccination of animals ( table ). the scope of this review encompasses only those diseases for which a strategy targeting vaccination of animals is actually used or is under development. there are many diseases of table frameworks for vaccine development and utilization as a means of preventing zoonotic diseases, and status of approval of existing vaccines for humans and animals. the first major framework includes zoonotic infectious diseases that affect both humans and economically important animals, where wild animals are the source of infection. livestock and humans are dead-end hosts, and neither contributes to the transmission cycle. in this case, vaccines are required to prevent disease in both humans and economically important animals, but do not interrupt transmission of the disease in nature. for framework i diseases, there is an important opportunity to accelerate the development of new vaccines by concurrent veterinary and human product research ( table ) . moreover the affected animal species represents a natural disease model of infection, pathogenesis and immunity that may be useful in testing efficacy and immunological correlates of protection of a new vaccine intended for human use, and thus provide data supporting regulatory approval under fda's animal rule [ ] . a list of framework i diseases and vaccines is shown in table , and an example is given below. west nile virus is a mosquito-borne, single strand, positivesense, enveloped rna virus (genus flavivirus, family flaviviridae), closely related to japanese encephalitis virus. west nile virus is a recognized cause of human disease ranging from mild feverrash-headache syndromes to lethal encephalomyelitis. horses, domesticated geese, farmed alligators (as well as a number of wild birds and mammals) are also susceptible to severe and fatal disease. although recognized as a cause of disease as early as the s, west nile became an increasing problem in the s, with outbreaks affecting humans and/or horses in northern africa, western europe, the eastern mediterranean, the black sea region and the volgograd oblast of russia [ ] . the most dramatic development was the introduction of west nile virus into north america in [ ] [ ] [ ] , the occurrence of large outbreaks in and , rapid spread across the us, and subsequent introduction to the caribbean and south america. horses and a number of wild bird species, notably crows and jays, were affected in addition to humans. between and , a total of , cases of west nile fever and , cases of neuroinvasive west nile disease were reported in the us, with the highest incidence in the middle of the country from texas north to the dakotas [ ] . the incidence of neuroinvasive disease in the us has varied between . and . per , in this interval [ ] . over , horses have been affected since , and in these animals the disease is more severe ( % case-fatality, % of survivors with neurological sequelae) than in humans ( - % case fatality, % of encephalitis survivors with sequelae) [ ] . moreover, the incidence of west nile in horses (∼ per , ) is substantially higher than in humans [ ] . the animal health industry rapidly responded to this veterinary emergency, and multiple west nile vaccines were rapidly developed, including a live vaccine (discussed below), whole virion inactivated, dna, and poxvirus vectored vaccines. the first vaccine for horses was marketed in , only years after introduction of the virus into the us. multiple human vaccine development programs were also initiated, but many of these efforts were discontinued or decelerated due to the high market risk associated with low incidence, a slackening of public concern with the disease, and the uncertain regulatory pathway for vaccine approval. although efficacy of a vaccine for equids is established [ ] , field trials to prove vaccine efficacy in humans would be large, expensive, and difficult due to the unpredictable occurrence of west nile outbreaks. the application of the animal rule to licensing a west nile vaccine, while plausible, has not been adjudicated by the fda with a sponsor. although approaches to developing veterinary and human vaccines against west nile were technologically similar, in only one case was a concurrent development program undertaken by a single company. this fact illustrates the current status of stove-piped and separate animal and human health biopharmaceutical industries. the exceptional case is of interest as a model of how vaccine development for zoonotic diseases could be improved. in , within months of the identification of west nile as the etiological agent of the initial outbreak affecting humans and horses in new york, acambis, a publically traded human-vaccine biotechnology company, applied its platform technology for yellow fever dvectored vaccines to the development of a west nile vaccine, with the intent to develop both vaccines for both horses and humans. this technology involved replacing the gene encoding the yellow fever d vaccine virus' envelope (e) protein with the corresponding gene of west nile virus. this chimeric vector vaccine platform had been previously used by the company to construct a chimeric vaccine against the closely-related japanese encephalitis virus [ ] ; at the time, that vaccine had proven to be highly effective, protecting non-human primates against intracerebral challenge with virulent japanese encephalitis virus [ ] . two new vectors were engineered, one with the wild-type west nile ny strain e protein gene and one with an e gene containing three attenuating mutations [ ] [ ] [ ] . the former was neurovirulent in mice, while the latter was more attenuated and thus deemed more suitable as a human vaccine. the principal question was whether the yellow fever d vector would infect and immunize horses, since yellow fever is a host-restricted primate virus. to find out, studies were sponsored by acambis in early at the colorado state university school of veterinary medicine. horses were vaccinated with the west nile/yellow fever chimeric virus or with yellow fever d vaccine, and viremia and antibody responses were determined [ ] . in addition, vaccinated and control horses were challenged by the intrathecal injection of a high dose of virulent west nile virus, following the same model referred to above for protection studies of the chimeric japanese encephalitis vaccine wherein monkeys were challenged by the intracerebral route. these studies showed that horses inoculated with chimeric vaccine developed neutralizing antibodies against west nile and were protected against a severe intrathecal challenge. a similar development plan was successfully followed for the mutated human vaccine version, using non-human primates as the test host. the veterinary and human vaccine candidates were developed side by side, and the data obtained in both programs were designed to support transition to advanced clinical trials in horses and humans. in , acambis licensed the veterinary vaccine technology to a major animal health company (intervet), and in the same year clinical trial materials were made for human trials. intervet completed development of the veterinary vaccine (prevenile ® ) [ ] , which was approved by usda in , and acambis brought the human vaccine (chimerivax-wn ) into clinical trials in the same timeframe [ ] , subsequently outlicensing the technology to sanofi pasteur. as expected, development of the veterinary vaccine and its progression through the regulatory pathway substantially outpaced the human vaccine; importantly, the veterinary application significantly informed the human vaccine table strengths and limitations of vaccination of animals as a means to control of zoonotic diseases. framework (see table development program in providing useful information on safety, durability of immunity and immune correlates or protection. co-development of the veterinary and human west nile vaccines is as a potential model for other vaccines. there are a few other examples where vaccines were co-developed for animals and humans, including vaccines against venezuelan equine encephalitis and rift valley fever (described below). certain issues arise, however, that may need to be considered in the context of such integrated efforts. first, a safety problem arising during use of a veterinary vaccine could provoke regulatory concerns or a liability problem for the human analog. although a safety issue did arise briefly due to anaphylaxis type reactions in horses, resulting in temporary withdrawal of prevenile ® from the market [ ], there were no repercussions for the human analog vaccine (chimerivax-wn ). this raises the interesting question whether the human and veterinary regulatory agencies are integrating information that might be of value, either during development of similar vaccines for different species or after marketing approval. this is an obvious overlooked area for application of one health principles. second, the immune response to veterinary vaccines generally should differentiate naturally infected from vaccinated animals (diva) on the basis of a laboratory test, which allows compliance with trade restrictions. this is, of course not a concern for regulatory approval of human vaccines, although it can be useful in seroepidemiological and vaccine coverage studies. an example of problems associated with diva is the off-label use of commercial horse vaccine to protect emus against eastern equine encephalitis (eee), since some combination vaccines against eee contain equine influenza antigen and such vaccines elicit cross-reactive antibodies to avian influenza resulting in quarantine. the chimeric west nile vaccine described above potentially allows for diva testing (using responses to the yellow fever nonstructural proteins expressed by the vector) [ ] . a number of important diseases are transmitted between domesticated animals and thence to humans. vaccination of domesticated animals has the potential to protect humans against these zoonoses, either indirectly by interrupting transmission where domesticated animals are amplifying hosts in the transmission cycle or directly by preventing spread from infected animals to humans. a list of framework ii diseases and vaccines is shown in table . selected examples are used to illustrate the role of vaccination of domesticated animals in preventing human disease. brucella spp. are facultative, intracellular gram-negative bacteria, pathogenic for domestic animals and humans. brucellosis, caused mainly by brucella melitensis (which infects sheep and goats), brucella abortus (cattle), and brucella suis (swine), occurs worldwide, with the highest prevalence in the middle east, asia, africa, tropical america, and the mediterranean region [ , ] . the annual incidence of human infections is estimated at , cases but the disease is widely acknowledged to be underreported [ ] . brucella canis, an infection of dogs, occurs worldwide with highest prevalence in tropical america. b. canis disease in humans has been reported, especially in persons handling breeding dogs and in immunosuppressed individuals. human brucellosis is acquired by contact or aerosol spread from infected animals, fomites, or ingestion of unpasteurized milk or undercooked meat. not surprisingly, brucellae survive for long periods in dust, animal excreta, soil, meat and dairy products. wild animals are also affected and can be the source of infection of livestock and humans. in animals, brucellosis causes epididymitis in males and abortion, placentitis, infertility and reduced milk production in female animals. the human disease is protean, manifested by chronic fatigue, relapsing fever, endocarditis, spondylitis, osteomyelitis, arthritis, and meningitis [ ] . prevention of human disease by control of brucellosis in livestock has long been a public health priority [ ] . control of brucellosis relies principally on surveillance, testing, removal of infected animals, import/export animal and animal product control provisions, protection from exposure to wild reservoirs (such as elk, deer, and bison), and vaccination. antibiotic treatment of animals is regulated and discouraged due to the large doses and long treatment required and concern about resistance. old, empirically developed live attenuated vaccines, b. melitensis rev vaccine for goats and sheep; b. abortus s and rb vaccines for cattle; and the oral b. suis s vaccine used widely in china for multiple species, elicit cellular immunity against the intracellular pathogen and are more effective than other types of vaccine [ ] . the rb vaccine (a spontaneous rifampin-resistant rough mutant) is approved in the us [ ] . however, the live vaccines have a number of drawbacks. the latter include lack of the ability to differentiate s and rev vaccine immunity from natural immunity (diva) and interference with surveillance and export control procedures; pathogenicity (especially abortion when animals are vaccinated during pregnancy); antibiotic resistance of the vaccine strains; and modest efficacy. live vaccines used in livestock can also cause illness in humans. vaccination as a stand-alone strategy has rarely been carefully evaluated, in large part due to concerns over the quality of the existing vaccines. however, vaccination is largely credited with elimination of brucellosis in the us [ , ] (the us was declared free of brucellosis in cattle in ) and for control of brucellosis in china [ ] . a recent study in greece showed that a mass vaccination program with the b. melitensis rev vaccine resulted in a decrease in human infections [ ] . a number of new vaccine approaches, including diva vaccines, designed to induce th oriented cellular immunity are under investigation, including safer rationally designed, mutated live vaccines [ ] ; recombinant, invasive escherischia coli [ ] ; recombinant subunit microencapsulated vaccines; and dna vaccines [ ] . experimental b. canis vaccines have been investigated in mice. where brucellosis-free status has been achieved, as in the us, wild animal reservoirs (especially bison and elk) threaten to reintroduce the disease. vaccination with existing vaccines is feasible, but delivery is challenging [ ] . this vero cytotoxin secreting gram-negative bacteria is an important cause of sporadic and epidemic food-borne illnesses of humans, including gastroenteritis and hemorrhagic colitis, with potentially lethal complications (hemolytic-uremic syndrome). cattle and sheep are the principal reservoirs of infection and transmission to humans occurs via food (meat, seeds and vegetables) contaminated with animal feces. undercooked ground beef is a source of infection in approximately one-third of human cases and recalls are a significant economic threat to the meat packing and distribution industry. animals concentrated at feed lots and slaughter that shed bacteria can produce lots of meat with high rates of o [ , ] , but there is considerable variability in the occurrence of contaminations [ ] . in developed countries, various sanitary measures and testing have been instituted to reduce the risk to consumers, but these remain imperfect. vaccination of feed lot cattle has been proposed as a measure to reduce the prevalence and duration of shedding and the risk to consumers. o -specific bacterial extract vaccines containing protective outer membrane proteins have been conditionally approved by usda (manufactured by epitopix, willmar, mn) and fully approved by the canadian food inspection agency (bioniche life sciences, belleville, ont.). feedlot cattle receiving or doses of the bioniche vaccine - weeks apart had - % reduction in colorectal colonization or fecal shedding and significant reduction in magnitude and duration of shedding [ ] [ ] [ ] . hurd and malladi [ ] modeled the impact of vaccinating cattle on human health outcomes. assuming % efficacy of the vaccine and % adoption rate, the model indicated a % reduction in the incidence of e. coli o -related human illness. the model also predicted significant reductions in the number of lots of contaminated ground beef and detection by usda, which would have substantial economic benefit to packers and distributors. vaccine effectiveness under conditions of field use will be highly dependent on adoption rate (vaccine coverage), and in part by whether cattle complete the -dose vaccination series on the proscribed schedule [ ] . an interesting question is: who will pay for vaccination of feedlot cattle? is the economic benefit there for the meat industry, and will vaccination reduce the cost of other preventive measures and testing or will vaccine costs simply be on top of other costs? will government agencies concerned with human health subsidize the cost, without empirical demonstration of a human health benefit? since ground beef only contributes about one-third of human infections, how could vaccines be used as a means of reducing other sources of food contamination? another issue for use of the current vaccines is the role of other enterohemorrhagic e. coli (e.g. o , o , and o ) in human disease. this disease is caused primarily by the gram-negative bacterium, bartonella henselae, transmitted between domestic cats by the agency of cat fleas, ctenocephalides felis. human infection occurs by contact spread from cats, including scratches by claws contaminated with blood or flea feces, or possibly by flea bite [ , ] . the prevalence of infection in household cats in the us is approximately %, but in stray animals it is % and high prevalence rates have been found in developing countries [ , ] . disease in humans is manifest by fever, a papule followed by a pustule at the site of infection and lymphadenopathy. rare complications include meningitis, encephalitis, endocarditis, glomerulonephritis, osteomyelitis, neuropsychiatric abnormalities, and relapsing fever and splenomegaly. human infections in immune-compromised individuals are particularly severe. approximately , - , cases and hospitalizations caused by cat scratch disease are estimated to occur annually in the us. in the late s, heska corporation, an animal heath company in colorado, initiated a program to develop a vaccine for household cats, with the goal of limiting the potential for transmission of the bacteria from cats to humans. unfortunately, the program was not completed and no vaccine is available at present. given the fact that cats rarely become ill with b. henselae, this would have been an unusual product providing protection to pet owners, with marginal if any real benefit to the target species. moreover, it would likely have been extremely challenging to demonstrate a health benefit to humans. rabies is a fatal infection of the central nervous system caused by rabies virus, a member of the lyssavirus genus, family rhabdoviridae. it is estimated that up to , - , cases of human rabies occur annually, and dog bite is the cause of over % [ ] . successful vaccination of dogs and humans against rabies was first demonstrated in by louis pasteur, using crude nerve tissue preparations. however, until the first decades of the th century in developed countries, and continuing in many developing countries today, the ancient practice of dog population reduction campaigns have been the main approach to rabies control, a method that has repeatedly proven to be ineffective. dog licensing and vaccination requirements were introduced in the united kingdom in , and gradually at the local and then state levels in the us beginning in the s, with national requirements attained by [ ] . many successful dog vaccination campaigns have been reported in latin american countries and asia [ ] . some countries have declared eradication of canine rabies, notably the united kingdom in , japan in , the us in , as well as malaysia, singapore, taiwan, hong kong. in , the first world rabies day event, the ultimate vision was promulgated of canine rabies elimination through systematic vaccination. however, rabies remains a significant public health problem, due to absent or incomplete dog vaccination in many areas of the world. more than . million post-exposure treatments with rabies vaccines are given annually, at a cost of over $ billion worldwide [ , ] . in china, due to the low prevalence of canine vaccination, the sales of rabies vaccines for human post-exposure prophylaxis outstrips any other human vaccine, accounting for % of all annual vaccine sales, i.e. million doses costing $ million [ ] . the requirement for human post-exposure vaccination at this scale represents an obvious failure of public health, since it plays no role in containing the spread of rabies in the canine vector. a very high rate of vaccine coverage in the dog population (exceeding %) is required for interruption of the rabies transmission cycle [ ] . the development of oral rabies vaccines has allowed vaccination of free-roaming dogs that could not be restrained and vaccinated by injection, as well as the vaccination of wild animal species that are the source of infection in dogs [ ] . oral bait vaccine for dogs has been successfully deployed in trials in many countries, including turkey, thailand, sri lanka, south africa, and the philippines [ ] ; the world health organization has supported this approach as a supplemental program where there are substantial populations of free-ranging or feral dogs [ ] . significant increases in canine vaccination coverage have been achieved when oral rabies vaccine was added to a program of standard parenteral vaccination. oral vaccination of dogs has been accomplished using commercial baits containing live modified rabies vaccines, such as the street-alabama-dufferin (sad) strain, variant b [ ] and the attenuated copenhagen strain of vaccinia expressing the rabies glycoprotein gene (g protein) [ ] . a more detailed review of oral rabies vaccines is provided below (framework iii). hendra virus disease, a severe and fatal infection of horses and humans in australia, has been noted as an example illustrating one health principles in disease prevention and control [ ] , and is especially relevant now that a new vaccine for horses has been introduced. hendra is a member of the henipavirus genus, family paramyxoviridae. the reservoir hosts are fruit bats (pteropus spp.), and the virus is spread from bats to horses by contact (including respiratory droplets), by food or fomites contaminated with bat urine, or by contact with sick horses. all reported human infections have resulted from contact with infected horses [ ] [ ] [ ] . hendra virus disease was first described in . human and equine cases have occurred in coastal queensland and new south wales and positive bats have been detected in the northern territory. a total of deaths in equids have been reported in outbreaks, with a very high case-fatality rate ( %), and cases ( fatal) have occurred in humans, including horse trainers and veterinarians, all of whom had contact with sick horses [ ] . the disease is manifested by severe systemic illness, respiratory symptoms or acute and relapsing encephalitis [ ] . swine appear to be susceptible to experimental infection. nipah virus, a closely related bat-borne agent in se asia has caused outbreaks of severe and fatal disease in swine and humans [ ] [ ] [ ] . in november , pfizer animal health launched equivac ® hev, an adjuvanted subunit protein vaccine for the prevention of hendra virus disease of horses in australia [ ] . since horses are a major source of contact spread of hendra virus to humans, the vaccine promises to make an important contribution to human health as well. fear of acquiring the disease has also constrained equine veterinary practice in australia [ ] , and the vaccine should mitigate this problem. development of equivac ® hev was a collaborative effort between pfizer and csiro's australian animal health laboratory. however, support for the development program was also provided by human medical researchers in the us, at the uniformed services university of the health sciences supported by the henry jackson foundation for the advancement of military medicine. a provisional approval for limited use of the vaccine was obtained in early , with full approval in november. the vaccine is a soluble, recombinant glycoprotein (g) of hendra virus, the ligand for cell attachment and antibodies to the protein neutralize cell receptor binding of the virus [ , ] . the vaccine protects horses and ferrets against experimental infection [ ] , and appears to cross-protect against nipah virus [ ] . the availability of equivac ® hev should lead to rapid uptake by horse owners in australia. the equine and horse racing industry in australia is large, contributing billions of dollars, and over % of total gross domestic product [ ] . hendra virus in horses is a notifiable disease in all australian jurisdictions; the property where the horse cases are located is quarantined and animals that are infected are euthanized. the occurrence of at least one hendra virus outbreak annually since , and the high lethality of the disease have raised considerable awareness in australia. since all human cases of this zoonosis have resulted from contact with infected horses, vaccination of horses against hendra virus promises to be a highly effective strategy for preventing human cases. rift valley fever is an enveloped, single-strand, segmented rna virus belonging to the phlebovirus genus, family bunyaviridae, occurring in africa, with intermittent extensions to the arabian peninsula. rift valley fever virus causes explosive and economically damaging outbreaks of disease in cattle and sheep, with stillbirth, abortion, and very high mortality of young animals; in adult animals, it causes % mortality in sheep, - % in cattle, and - % in goats [ ] . humans typically develop self-limited nonspecific febrile illness, but - % have a complicated course with hemorrhagic fever syndrome, encephalitis, hepatitis, renal failure, or retinitis, and case-fatality rates in severely ill and hospitalized patients is as high as % [ , ] . the virus is transmitted between livestock and from livestock to humans by the agency of mosquito vectors. in addition, humans commonly acquire infection by contact and aerosol routes when handling, treating, or butchering infected livestock, and the virus can persist in meat for weeks. the ecology of rift valley fever and the reasons behind its periodic emergences have been the subject of intensive study. in brief, the reservoir of infection is aedes mosquitoes, especially ae. linneatopennis, which maintain the virus by transovarial transmission [ ] . the dessication-resistant ova containing virus remain in depressions in the earth ('dambos') that are flooded during the rainy season, with the subsequent emergence of infected adult aedes mosquitoes. infected, viremic, livestock in turn served as source for amplified virus transmission by a variety of mosquito vectors [ , ] . there are no approved vaccines for prevention of rift valley fever in humans, although a number of candidates are in development by academic and government laboratories. vaccination of cattle and sheep represents a strategy for preventing disease in these species, and thereby for interrupting virus transmission to humans. rift valley fever epizootics are to some extent predictable based on rainfall patterns and surveillance of disease in livestock [ ] . surveillance provides opportunities for rapid intervention, particularly with a single-dose vaccine (most likely a live, attenuated vaccine) that would protect against viremia in cattle and sheep and prevent mosquito infection. in addition, routine vaccination of livestock in inter-epizootic periods with a product capable of inducing durable protective immunity is a long range goal supported conceptually by modeling [ ] . however, there are many obstacles to livestock immunization in africa, including access, policy, regulatory approval, cold chain, and commercial viability. additionally, diva requirements are driven by regulations prohibiting export of livestock or meat by countries experiencing rift valley fever [ ] . some of the obstacles to vaccination implementation could be overcome by development of improved vaccines. two old veterinary vaccines, the live smithburn vaccine, developed using techniques of serial passage in mouse brain similar to that applied to the early development of the french neurotropic vaccine against yellow fever [ , ] , and a formalin inactivated vaccine [ ] are commercially available from the onderstepoort institute in south africa. however, the smithburn vaccine, now produced in bhk- cells, is reported to cause teratogenicity and abortion when used in pregnant animals [ ] and is used only in countries endemic for rift valley fever due to concerns about reversion. the inactivated vaccine, also grown in bhk- cell culture, requires multiple doses to be effective and probably has relatively short durability [ ] , making it less desirable for the interventions proposed above. nonetheless, the inactivated vaccine was successfully used to interrupt a rift valley fever outbreak in south african sheep [ ] . indeed, following a large epidemic of rift valley fever in egypt in - , the veterinary serum and vaccine research institute (cairo, egypt) produced a formalin-inactivated vaccine in bhk- cells using the epidemic strain (zh ), which matched locally circulating strains compared to the south african strain [ ] . another inactivated vaccine (tsi-gsd ) developed by the us army for human immunization and grown in diploid fetal rhesus lung cells was tested clinically and shown to be well tolerated and immunogenic. ninety % of the subjects developed a neutralizing titer > , which was shown to be higher than the protective level in a passive immunization-challenge study in hamsters [ , ] . in addition to the requirement for multiple doses for primary and booster immunization, inactivated rift valley fever vaccines have the disadvantage of requiring high biocontainment facilities for manufacturing. in recent years, there has been substantial progress in development of newer vaccines, and some of these vaccine development projects have been collaborations between human and veterinary research groups. additionally, there has been a moderate level of support from the us government because rift valley fever is a credible threat of natural or intentional (bioterrorist) introduction. nevertheless, despite very promising technical results, there has been insufficient support from industry and government to propel any of these new vaccine candidates into use. because of the obvious advantages of rapid onset and durable immunity associated with live vaccines, development of an improved live vaccine has been the focus of research. us army investigators attempted to induce attenuating mutations in two rift valley fever virus strains isolated during the egyptian epidemic [ ] . mp- is a live vaccine that was developed from the virulent zh- strain by passages in mrc- cells in the presence of the mutagen, -fluorouracil, resulting in a temperature sensitive virus with amino acid mutations. attenuation was demonstrated in multiple animal models, and reassortment studies showed that attenuating mutations were redundant and resided in all three gene segments [ , ] . development of mp- was undertaken by the us army medical research institute of infectious diseases with the intention to produce a vaccine for both human and animal immunization. the vaccine was clinically tested in human volunteers and shown to be well-tolerated and highly immunogenic [ ] . army investigators, in collaboration with usda, conducted a number of studies of mp- vaccine in sheep and cows, including neonatal, pregnant and lactating animals. these studies showed that mp- caused a low viremia, but with no attendant clinical signs; there was no virus secretion in milk, and no abortions or teratogenicity when vaccine was given in in mid-to late term pregnancy. mp- was highly immunogenic and protected livestock against virulent challenge [ , ] . however, ewes vaccinated with mp- early in pregnancy showed a low incidence of abortion and teratogenicity, indicating some residual virulence of the vaccine [ ] . to improve genetic stability and safety of mp- , reverse genetic techniques were used to introduce deletions in the s and m rna segments in genes encoding, respectively, nss and nsm proteins [ , ] . two deletion mutants were evaluated for safety and immunogenicity in pregnant ewes [ ] and in calves (morrill jc personal communication, ) with positive results for the nsm deletant, making it an attractive candidate as a veterinary and human vaccine. there were no clear safety signals when ewes were inoculated in early-mid pregnancy. further safety studies are required to rule out the low incidence of abortion/teratogenicity seen with parental mp- in early-term ewes. unfortunately, human trials have not yet been performed with the rationally designed mp- derivative. a third live vaccine designated clone , is a plaque-derived clone of a central african strain of rift valley fever isolated from a human subject, and was found to be naturally attenuated for mice and to have an in-frame deletion of most of the nss gene [ ] . this observation was the basis for modifying the mp- vaccine by ns gene deletion, as described above. once again, a collaboration between the human and veterinary researchers led to a study in ewes, showing that clone was highly immunogenic but did not cause abortions [ , ] . another attenuated vaccine designated r , has been developed by reassorting clone and mp- so that it contains the s segment of clone and the l and m segments of mp- . this strain has attenuation domains from both parental vaccine candidates. a number of live, replicating, and non-replicating heterologous viral vectors expressing rift valley fever g and g glycoproteins and nonstructural proteins have been have been investigated in mice, elicited immune responses and protected against challenge. the vectors included lumpy skin disease (capripoxvirus) [ ] , alphavirus (vee and sindbis) replicons [ ] , newcastle disease virus [ ] , adenovirus, and modified vaccinia ankara. several of these constructs were used to immunize sheep and/or cattle (lumpy skin disease virus, newcastle disease virus, and sindbis replicons) with somewhat variable success. for a more comprehensive review see indran and ikegami [ ] and boshra et al. [ ] . live vectors are a promising approach for new rift valley fever vaccines, particularly veterinary vaccines, but may have problems for homologous boosting in light of anti-vector immunity. various prime-boost strategies have been proposed, as for plasmid dna vaccines (see below), but these would be exceptionally difficult to implement for immunization of livestock in the field, and are thus impractical. subunit protein produced in insect cells, virus-like particles [ ] , and dna vaccines [ ] against rift valley fever are also in early stage development. these approaches have potential advantages of safety and thermostability during storage and distribution, but may require multiple dosing and provide less durable immunity than live vaccines, and thus are less desirable products for framework ii implementation. overall, it remains to be seen which of the many rift valley fever vaccines in development progress to regulatory approval and whether an integrated veterinary and human health policy based on the immunization of livestock in africa together with predictive surveillance, can abort impending outbreaks, and lead to long range control of this important disease. vee is a mosquito-borne single strand, positive-sense, enveloped rna virus belonging to the alphavirus genus, family togaviridae. other medically important members of the alphavirus genus include eastern and western equine encephalitis viruses. there are vee virus subtypes identified by antigenic and genomic analyses, and a number of additional varieties. subtype iab and ic cause epizootic disease in equids and associated zoonotic infections of humans [ ] . during epizootics, horses and donkeys infected with these strains develop high viremias, serve as the primary hosts for infection of mosquito vectors and therefore are the indirect source of human infections acquired by mosquito bite. in contrast, the enzootic subtypes ii-vi, are maintained in nature in cycles involving rodent species and mosquitoes, are not amplified by equid viremic hosts, and cause sporadic illness in humans and equid dead-end hosts. epizootics of ic virus are the result of mutation and selection of virulent equine-competent viruses from enzootic strains, particularly the id variant [ ] . in the - s vee iab viruses caused large epizootics in south america, with associated human epidemics of encephalitis. between and , a series of major subtype iab and ic epizootics occurred in northern south america, and between and the virus spread north to central america, mexico, and texas [ ] . the cumulative economic and medical impact of vee outbreaks between and was devastating, with over , equid and , human cases. some of the vee iab epizootics are believed to have been spawned by the injection of horses with inactivated veterinary vee vaccines containing residual live virus [ ] . this likely occurred in trinidad in and again in nicaragua in , but probably was a widespread problem in the past. between and , vee ic re-emerged in venezuela and colombia, with an estimated equid deaths and over , human cases of which had encephalitis [ , ] . since vee causes an acute incapacitating illness in humans and the virus efficiently infects via the aerosol route, it was developed by both the us and soviet union as an offensive biological weapon [ ] . as part of these programs, vaccines for the protection of military personnel were also developed. in the us, a live, attenuated virus (tc- ) was developed by the us army medical research & development command (usamrdc) by empirical passages of the prototype trinidad donkey (subtype iab) virus in fetal guinea pig heart cell culture [ ] . the development of the live vaccine followed poor experiences with chemically inactivated vaccines; in animal models, only the live vaccine protected against aerosol challenge. however, tc- vaccine has a number of drawbacks as a human vaccine, including failure to immunize about % of vaccinees, and moderate-to-severe reactogenicity in about % of subjects. in humans, it remains an investigational product, used solely for the protection of laboratory workers [ ] , with approximately persons vaccinated since . the tc- virus acquired mutations during the empirical passage series in guinea pig heart cells, but attenuation appears linked to only of these, in the -noncoding region and the e envelope glycoprotein, and these substitutions appear to be subject to reversion in the vaccinated host [ ] . in addition, tc- has been isolated from mosquito vectors during field use, illustrating the potential for secondary spread and mutation and recombination events. an investigational formalin-inactivated tc- vaccine (designated c- ) was also developed by usamrdc and used following tc- priming to seroconvert tc- non-responders. since horses and related species are severely affected during epizootics and are the source of mosquito vectors infecting humans, there is an obvious need for a single dose veterinary vaccine that evokes rapid immunity. us army investigators explored the use of tc- live, attenuated human vaccine for immunization of equids beginning in [ ] , and there was limited field use of the vaccine in colombia in . however, when epizootic vee appeared for the first time in central america (guatemala) in may , and then spread southwards to costa rica and northwards to the us, there was considerable urgency to utilize a vaccine strategy for control of the disease in horses, donkeys and mules. in , the us military responded rapidly to requests for tc- vaccine from guatemala and el salvador. the vaccine had been produced and stockpiled at the merrell national laboratories, swiftwater pa under contract to the usamrdc for the purposes of biological defense. by , over million doses of tc- had been given to equidae in the us, mexico and central america [ ] . collaborative studies were also undertaken by agencies concerned with human and animal health (usamrdc, nih and usda) to fully explore the biology of the vaccine in horses [ , ] , ultimately leading to licensure and commercialized by the animal health industry, both as a live vaccine and then an inactivated vaccine combo with eastern and western equine encephalitis vaccines. tc- vaccine was credited with a rapid curtailment of the - outbreak. the history of vee exemplifies many one health principles, including the prevention of human cases through domesticated animal vaccination, use of a single vaccine product for animals and humans, and an agency (the us army) concerned with human health engaged in both veterinary and human vaccine development, and providing a solution for curtailing an emerging zoonosis. after the large epizootic in the s, tc- vaccine was again deployed during the epidemic in in colombia to create an immune barrier to spread of the virus. recent efforts have focused on development of improved vee vaccines for humans that are less reactogenic and more immunogenic than tc- , can be manufactured in a more acceptable substrate, and have a lower risk of reversion to virulence and of mosquito transmission [ ] . in addition, vaccines that crossprotect against the enzootic vee subtypes are needed. vee id is endemic in colombia, peru, venezuela, and ecuador, and the ie subtype circulates in southern mexico. aguilar et al. [ ] postulated that disease caused by vee is confused clinically with dengue, and that, in endemic areas, up to % of dengue cases may actually be due to vee enzootic subtype viruses. subtype id poses the ever-present risk of mutational change to produce high viremia and epizootic transmission in equids, as happened in the s. v vaccine is a rationally designed vaccine from the epizootic subtype iab genome, with insertion of a pe cleavage-signal mutation combined with an e gene resuscitating mutation. v had a good safety profile and was immunogenic and protective in laboratory animals, including nonhuman primates [ ] . while retaining a degree of neurovirulence for suckling mice, v is not virulent when inoculated intracranially in juvenile monkeys [ ] . v has also been evaluated in horses [ ] . the vaccine was safe and highly immunogenic, with subcutaneous doses as low as plaque-forming units shown to protect horses against challenge with virulent subtype iab virus. unfortunately, v proved to be too reactogenic for humans in a phase trial [ ] , and thus development for both human and veterinary use has stopped. the v virus was subsequently formalin inactivated and has been investigated with adjuvants replacement for the c- vaccine. other live and live vector approaches to improved vee vaccines have been investigated only in laboratory animals, including a chimeric virus constructed from nonstructural genes of sindbis and the structural genes from vee [ ] , vee replicon vaccines, and vaccinia recombinants. none of these approaches have reached advanced development. it is only a matter of time before another vee outbreak emerges in tropical america, and there is a substantial risk of cross-border spread. the prospects for vaccine interventions have diminished with dwindling support for new vaccines and increased concerns for vaccine safety. most zoonotic diseases are maintained in transmission cycles involving wild mammals or birds. however, because of the difficulties in vaccinating specific host species, wildlife immunization as a means of preventing spread to domestic animals and humans has been applied in only a few diseases. some of the barriers to implementing wild animal vaccination include (i) involvement of multiple species in natural transmission cycles; (ii) safety concerns for non-target species; (iii) high reproductive rates and population turn-over; (iv) fastidious feeding behaviors and difficulty in designing effective baits; (v) difficult delivery due to very high or, conversely, very low population densities of the target species; (vi) environmental concerns, and release of genetically modified organisms; (vii) difficulty in designing an effective formulation for oral immunization; (viii) instability of a vaccine or vector under prevailing environmental conditions; and (ix) requirement for low unit cost and government funding for vaccine purchase and delivery. nevertheless, targeted immunization of wild animal reservoirs is a subject of considerable interest for future research, not only for control of infectious agents affecting domestic animals and humans but also for control of wildlife diseases. one example of the latter was the effort to develop a means of immunizing great apes affected by ebola virus in central africa with vaccines previously developed for human use. aside from rabies vaccines delivered in oral baits, which is well-established, wildlife vaccination has had limited success. two promising examples of early-stage vaccine applications are described below (lyme disease and mycobacterium bovis). in addition experimental immunization and protection of prairie dogs (cynomys ludovicianus) using a raccoon poxvirus recombinant oral bait vaccine [ ] , and ballistic vaccination of bison against b. abortus [ ] have been described. plague, a global but localized zoonotic disease with rodent wildlife reservoirs, would appear to be a target of particular interest for future research [ ] . there are many other possible targets for new framework iii vaccines, and future research in this field is encouraged. in the united states, lyme disease is the most common vectorborne disease and the th most common infectious disease overall. it is also a major and increasing public health problem in europe. approximately , cases are reported in the us annually, and the number has doubled in the last years [ ] . however, at a meeting in august, , the centers for disease control and prevention (cdc) reported that the annual incidence of infection is believed to be -fold higher, i.e. , cases. although lyme disease occurs across the country, the incidence is highest in the northeast and north central states. in the us, lyme disease is caused by the spirochete borrelia burgdorferi, which is amplified each spring and summer in a cycle principally involving ixodes scapularis ticks and field mice. mice are persistently infected and represent the reservoir of infection in nature [ ] . b. burgdorferi is passed transtadially to nymphal and adult ticks which infect humans and dogs; these species develop clinical disease but are dead-end hosts. the human disease is manifested by a protean syndrome, starting with a localized skin infection (erythema migrans), and progressing to a multisystem disease variably with lassitude, arthritis, carditis, meningitis and other neurological manifestations [ ] . because of the increasing incidence and geographic expansion of lyme disease, the high incidence of tick exposure, and the difficulty in recognizing and removing attached ticks due to their small size, difficult differential diagnosis, troublesome and potentially severe clinical manifestations and medical controversies over treatment and chronicity of the disease, lyme disease has emerged as a high priority for public health interventions [ ] . vaccination of humans would appear to be a logical and cost-effective means to prevent the disease [ ] , and veterinary vaccines for dogs are widely used and have proven to be modestly effective [ ] . however, whereas safe and highly effective vaccines for humans have been developed, none is available for distribution today. glaxo smithkline's lymerix ® vaccine was approved in , but withdrawn in by the company, principally for commercial reasons, a decision that is lamentable given the increasing incidence of the disease [ , , ] . a new lyme disease vaccine for humans active against both b. burgdorferi and species causing lyme disease in europe developed by baxter bioscience is now in phase ii development, but it is uncertain whether it will reach the market. nevertheless, these vaccines established critical immunological principles; the human vaccines are composed of recombinant ospa protein, the dog vaccines of both ospa and ospc, and work via antibody-mediated mechanisms. ospa is expressed by the borrelia spirochete in the midgut of infected ticks. since the tick vector only begins to transmit borrelia - h after initiating blood feeding, ospa specific antibodies imbibed in the blood meal of a vaccinated host kill the bacteria and block transmission [ , ] . if a similar ospa antibody response could be evoked in the natural reservoir hosts of b. burgdoferi (peromyscus spp. field mice), it may be possible to interrupt the transmission cycle and reduce the prevalence of infected nymphal and adult ticks responsible for human and canine infections. proof of concept was obtained in a field study where peromyscus leucopus mice were trapped and vaccinated by subcutaneous injection of ospa; a reduction in the prevalence of b. burgdorferi in nymphal ticks was seen in the following year [ ] . however, practical vaccine delivery and effective immunization of mice in the wild, requires a thermostable oral bait vaccine matched to the high population density and rapid population turnover of the reservoir hosts, the effects of which are not diluted by non-targeted species that play a role in b. burgdorferi transmission [ ] . two promising live oral vaccine approaches have been investigated in the laboratory: a bacterial (e. coli) vector [ , ] and a viral vector (vaccinia) [ , ] expressing ospa. the e. coli vector contained in an oral bait formulation and ingested multiple times elicited anti-ospa antibodies and protected laboratory and wild p. leucopus mice against needle and tick challenge. a -year field study of the oral bait vaccine, sponsored by cdc, has been performed and results are anticipated with interest. a company, us biologics inc., is engaged in bringing this vaccine to market. the vaccinia technology, which rests on the shoulders of the successful oral bait vaccine against wildlife rabies (see below), has been tested in the laboratory. laboratory mice immunized by gavage with vaccinia expressing ospa were successfully immunized after a single dose and were protected against tick challenge. peromyscus consuming oral bait vaccine were also significantly protected against challenge with infected ticks. although the vaccinia vector looks promising, no commercial endeavor has yet emerged to support development. both the e. coli and vaccinia oral vaccines require specialized formulations in baits that incorporate the vaccine in the bait itself, rather than in a liquid sachet embedded in the bait used for delivery of rabies vaccines. many questions surround the application of an oral bait vaccine targeting the reservoir host, including efficacy of this approach in the field, the high density of baits required, cost and sustainability of local and state funded programs aimed at distributing baits, and the role of species not targeted by the vaccine in lyme disease maintenance cycles. if only partially effective, the risk of acquiring lyme disease may be reduced, but the public would still need to take precautions against tick bite. nevertheless, given the lack of a vaccine for humans, the high level of public concern about lyme disease, the high risk to children, the localized nature of b. burgdorferi transmission allowing geospatially focused control efforts, and the possibility that homeowners may be motivated to play an active role in distributing baits, the idea has appeal. m. bovis is the cause of tuberculosis in a wide array of domesticated and wild animals, and it remains a major veterinary health problem worldwide, causing severe economic losses from livestock disease, death and export restrictions. humans become infected by ingesting raw milk or undercooked meat, or by the aerosol route from infected animals or humans. in developed countries where pasteurization and test-and-slaughter programs have controlled the disease, zoonotic infections are relatively rare, accounting for . - . % of tuberculosis cases [ ] [ ] [ ] ; in developing countries which do not practice these measures, it remains more common, although few data on prevalence exist. wild animals are a major source of infection of domestic livestock [ ] . control measures aimed at control of m. bovis by culling wildlife reservoirs is problematic, with inconsistent results and ethical concerns. vaccination of wildlife is an attractive alternative control measure, especially since the traditional tuberculosis vaccine (bacille calmette-guerin, bcg) derived from m. bovis is effective when orally administered [ ] . examples of wildlife that serve as maintenance hosts of m. bovis and sources of infection in livestock, include white-tail deer in the us [ ] ; wild boar, red and fallow deer in europe [ ] ; badgers in the united kingdom [ ] ; african buffalo (syncerus caffer) in south africa [ ] ; and brushtail possums (trichosurus vulpecula) in new zealand [ ] . brushtail possums have been experimentally vaccinated using oral bcg and shown to be resistant to challenge with m. bovis [ ] . proof of concept has been provided by a field study in an endemic area of new zealand. possums were trapped, manually vaccinated using orally delivered bcg in a lipid matrix formulation, and vaccinated and control animals were recaptured at intervals [ ] . vaccinated animals received - vaccinations during the -year study. at the end of study, the ha study area was depopulated, and all animals assessed for clinical and subclinical m. bovis infections. vaccine efficacy against naturally acquired tuberculosis was - %. the authors concluded that oral vaccination of possums could be a practical strategy contributing to elimination of m. bovis in livestock. although the field study did not demonstrate control via freely consumed bait vaccine, captive possums have been shown to consume vaccine in flavored baits [ ] . rabies is transmitted between specific wild carnivore reservoir hosts, which serve as a source of spill-over infections of other wild carnivores, and infection of domesticated animals and humans. oral rabies vaccine was initially deployed in europe for control of rabies in the red fox (vulpes vulpes), using modified live virus vaccine [ , ] . the concept began in the s with the work of george baer at the cdc, which showed that foxes could be orally immunized with modified live virus [ ] . the live, attenuated evelyn-rokitnicki-abelseth (era) vaccine or street-alabama-dufferin (sad) viruses were employed in experimental and field studies. numerous studies in the s confirmed that captive and wild foxes could be orally immunized with a variety of baits containing vaccine [ ] . in , steck and colleagues initiated a wild fox rabies control program in the swiss alps using oral bait vaccine consisting of chicken heads with vaccine and tetracycline biomarker in a container made of polyvinyl chloride and aluminum foil inserted under the scalp [ ] . the trial demonstrated that % of foxes had ingested bait. over the next years, successful fox rabies control programs were carried out in many european countries, after the late s using baits distributed by fixed wing aircraft and helicopters rather than by ground [ ] , and resulting in elimination of terrestrial rabies in several countries [ , ] . for large scale distribution, the laborious chicken head method bait gave way to commercially manufactured molded or extruded baits of various kinds, consisting of fish meal or bone meal, fat, and a pouch or blister containing liquid vaccine virus [ ] . the vaccines currently used in europe are ( ) sag (e.g., rabigen ® , virbac laboratories, france), a modified live attenuated rabies virus derived from the original sad vaccine and having an additional mutation in the codon for amino acid of the rabies g protein, which increases genetic stability of the virus [ ] ; and ( ) recombinant vaccinia virus (copenhagen strain) expressing the era ® strain rabies g protein (raboral ® , merial corp.) [ ] . the rabies g protein gene has been inserted into the thymidine kinase gene of vaccinia, which results in further attenuation compared to the parental virus [ , ] . duration of oral rabies immunity, at least months in adult red foxes, is sufficient to provide herd immunity and reduce the reproductive rate (r ) to less than . the modified live virus vaccines are more thermolabile than vaccinia, require − • c storage, retain some pathogenicity for non-target species, and pose safety risks to humans exposed inadvertently. consequently, recombinant vaccinia is the only oral rabies vaccine approved for wildlife immunization in the us. this vaccine consists of fishmeal and fish oil bound by a polymer (ethylene vinyl acetate) and containing the vaccine in a plastic pouch held in place by a wax mixture. immunization with vaccinia or modified live virus occurs in the buccal mucosa and tonsils, and vaccines are poorly effective after ingestion [ ] . in one study, consumption by red foxes of a single bait containing vaccinia resulted in protection against virulent rabies in only half of the animals [ ] . this observation suggests that high bait densities and repeated vaccinations are important to effective control in the wild. bait densities distributed in europe generally range between and baits/km , resulting in - % of animals potentially immunized (positive for the tetracycline biomarker) [ ] . in addition to distribution density, feeding habits may also be important, since animals have been observed to pick apart baits and consume only the bait portion. while control of terrestrial wildlife rabies has been successful in parts of europe, it has been more challenging in other areas due to the diversity of carnivores involved in transmission of different rabies virus variants. in the arctic regions, a specific rabies variant is maintained in the arctic fox, with spill-over infections of red foxes, skunks, and raccoon dogs. where vaccination is not practiced in domesticated sledge dogs, these animals may be severely impacted by contacts with rabid foxes, and human dog owners placed at considerable risk. while experimental oral vaccination of arctic foxes has been successful, there is limited experience in the field [ ] . in ontario, canada control of arctic rabies variant in red foxes using oral bait vaccine has been successful, but the virus still occurs as a result of spill-over transmission to skunks, which are not efficiently immunized with recombinant vaccinia vaccine [ , ] . oral bait recombinant vaccinia vaccine has been primarily used to control raccoon rabies, which expanded beginning in the mid- s from enzootic areas in florida northwards and westwards to involve many states in the eastern us, as well as new brunswick and quebec, canada [ , ] . the control program relies on distribution of vaccine baits specific zones of rabies activity, particularly along the appalachian ridge, enhanced surveillance and ring vaccination with evidence of spread of the disease. judged from the absence of spread beyond the zones of vaccine distribution, the program has worked well, despite relatively low prevalence of rabies antibody (approximately %) in sampled raccoons [ ] . it is possible that pre-existing immunity to raccoonpox virus may interfere with immunization with vaccinia [ ] . the economics of large-scale oral vaccination programs in the us have been modeled and are generally favorable [ ] . in addition to control of raccoon rabies, successful use of the vaccine has been made in the control of gray fox (urocyon cinereoargenteus) variant rabies in west texas [ ] . in contrast to raccoons, a higher prevalence of rabies antibody ( %) attributed to vaccination is found in gray foxes. rabies in coyotes (canis latrans) was responsible for epizootic canine rabies in the s and s in parts of the us, and was controlled by an oral bait vaccination campaign, contributing to the elimination of canine rabies by [ ] . skunks remain a problematic species for vaccine control of rabies. skunks are an important spill-over host for the arctic fox, raccoon rabies, and big brown bat rabies virus variants [ ] . although the vaccinia vector vaccine is not sufficiently effective in skunks [ ] , promising results were obtained with a replicationcompetent adenovirus type vector expressing the rabies g protein [ ] . aerial distribution of this vaccine (onrab ® , artemis technologies, guelph) showed high rates of immunization of raccoons, and arctic foxes and modest seroconversion ( - % in different plots) in skunks, probably due to lower rates of bait acceptance [ , ] . onrab ® is approved by the canadian regulatory authorities for control of rabies in skunks, raccoons, and foxes, and is under investigation in the us. there are some notable failures of animal vaccination as a means to preventing zoonotic diseases of humans, and these illustrate some of the limitations of the approach shown in table . japanese encephalitis, a mosquito-borne flavivirus closely related to west nile virus, is endemic in asia, with nearly billion people at risk of infection [ ] . horses and humans are dead-end hosts, and framework i immunization of both is widely practiced in many parts of asia, with a long record of success. pigs are an important domesticated animal amplifying host for infection of rice paddy-breeding culex spp. vectors, resulting in spill-over infections of humans. moreover, infection of pregnant sows can lead to abortion and stillbirth, and infected boars may have reduced spermatogenesis and infertility [ ] . framework ii immunization of swine was previously a major initiative in japan, using live, attenuated vaccines. however, it was exceedingly difficult to vaccinate piglets born in spring and early summer during the narrow window between loss of interfering maternal antibody and contribution to virus amplification. the practice of vaccination was abandoned in favor of re-locating piggeries from areas of culex breeding and biting activity. elsewhere in asia, other obstacles precluded consideration of framework ii vaccination against japanese encephalitis, including the prevalence of small piggeries located near vector breeding sites and of feral swine, and the importance of wild ardeid birds and waterfowl in virus transmission. moreover in many areas of asia, less developed than japan, and undergoing rapid urbanization, the locations of pig holdings are not controlled and are often located near rice paddies and urban centers [ ] . consequently, the focus has long been on human vaccination as the primary means of prevention. this case study exemplifies some of the factors that can limit application of framework ii vaccination: ( ) the need to customize vaccination to the breeding and husbandry practices and timing of domesticated animal targets; ( ) the role of wild animals and feral domesticated animals as additional amplifying hosts in the transmission cycle; and ( ) difficult access given the very large scale and geographic complexity of domesticated animal populations. q fever is caused by the intracellular gram-negative bacterium, coxiellla burnetii and an important worldwide infection of ruminants, which serve as the source of infection of humans, especially where large numbers of animals are concentrated [ ] . q fever is a major occupational hazard of abattoir and farm workers, veterinarians, and persons involved in the handling and distribution of animals or animal products. a dramatic outbreak recently occurred in high-intensity goat farms in the netherlands ( ), with human cases and deaths [ ] . transmission of c. burnetii occurs between direct spread between domesticated animals, and from animals to humans. infected animals shed bacteria in urine, feces, vaginal secretions and products of conception, and in milk. ticks are also a reservoir of bacteria in nature and a source of infection of livestock. ruminants, particularly goats and sheep develop pneumonia, abortion, stillbirth, premature delivery, and delivery of weak offspring, and herds can be affected for prolonged periods, causing significant economic losses [ ] . there are two developmental stages of c. burnetii, a small-cell variant (scv, the extracellular form) and the metabolically active intracellular large-cell variant. the scv is highly resistant to degradation and can persist in the environment for long periods of time. infection of humans is acquired principally by the aerosol route via dust containing spore-like scv forms, with oral routes of infection (ingestion of unpasteurized milk) being secondary. the human disease is characterized by an influenza-like illness, and may be complicated by pneumonia, endocarditis, and (pregnant women) abortion and fetal death. person-to-person transmission occurs, but is rare. approximately % of infected persons may develop chronic infections, with various manifestations. prophylactic vaccines have played a role in the control of q fever in livestock, but the practice is not a reliable means of preventing human infections. in russia, a live, attenuated m- vaccine was used for many years in animals and humans, but causes a persistent infection and has not been considered safe for use elsewhere. in europe, coxevac ® , a formalin inactivated strain rsa /nine-mile phase bacterial form (smooth forms, with complete surface lipopolysaccharide) has been used in goats and cattle and reduced the incidence of shedding, but is not effective in pregnant or chronically infected animals [ , ] . the live and phase vaccines are also not diva, which presents substantial issues for export controls. in australia, an inactivated phase vaccine prepared from henzereling strain is marketed for humans by csl ltd (qvax ® ) [ ] . however, severe reactions occur in persons who have previously been naturally infected with c. burnetii, and skin testing to ensure absence of exposure is required [ ] . clearly, improved vaccines are needed for both livestock and humans, but various attempts at recombinant vaccines have been disappointing. the problem for framework ii vaccination against q fever is due to multiple factors, including imperfect vaccines, limited efficacy of vaccines in parous animals, the difficulty in recognizing the disease and intervening expeditiously, and the rapid and widespread contamination of the environment with scv forms. the last problem is the major reason that livestock vaccination is not a reliable means of protecting humans against exposure. a year study of sheep vaccinated with the phase vaccine showed that the proportion of animals shedding bacteria in feces was markedly reduced after year and then eliminated after years, but c. burnetii was still present in environmental samples [ ] . finally, live veterinary vaccines may pose a risk of inadvertent infection of humans handling the vaccine or vaccinated animals. examples include live brucella and orf (contagious ecthyma) vaccines. while this topic is beyond the scope of this review, it is important in several contexts, and indeed little is known about the risk of human pathogens to animals. immunization of swine and poultry workers against influenza as a means of preventing introduction of human influenza viruses to these animals has been emphasized as a means of preventing emergence of reassortant strains [ ] . immunization of humans to prevent spread of viruses to captive nonhuman primates is often practiced, including vaccines for influenza, hepatitis a and b, and measles. prevention of animal diseases and human diseases by use of vaccines is a well-established principle, and there are potential synergies that can be achieved in concurrent delivery of human and animal vaccines in developing country settings [ ] . for some diseases affecting both livestock and humans there is a clear commercial incentive to develop vaccine products (framework i vaccines) ( table ) . however, such development efforts are generally segregated in the animal and human health divisions in industry and academia, and have separate regulatory pathways. this results in a potential waste of resources and duplicated scientific endeavors. interestingly, when one company (akso nobel), an animal health company, decided in on a strategic move into human vaccine development, it drew on its veterinary scientists to staff the program. as pointed out in this review, there have been isolated successful examples, e.g. a west nile vaccine, of co-development of a vaccine for both veterinary and human indications, an obviously efficient strategy that broadened both the commercial and public health opportunity. future efforts along similar lines should be considered on a case by case basis, depending on medical need, but in general there is value in closer connections between human and veterinary vaccines and regulatory science, and in the application of domesticated animals as models for development of infectious disease and cancer vaccines. several issues related to diva requirements and liability concerns have been mentioned. prevention of zoonotic diseases of humans by means of vaccination of domesticated (framework ii) or wild (framework iii) animals is an attractive but under-exploited concept. an obstacle is that there may be limited commercial incentives (table ) . where a market exists, governments may be the principal customers, as is the case for the approved oral bait rabies vaccines and the reservoirtargeted lyme disease vaccine in development. thus, the public health gains for such an intervention need to be compelling and must offset the cost of development and implementation. the goal is far easier to justify when vaccination also prevents disease in an economically valuable animal species, there is a profit incentive for animal vaccination or a clear social gain from improved animal health, and when the public health spin-off is an added benefit. examples of the latter may include vaccines against hendra and nipah virus diseases, brucellosis, chlamydophila felis, rift valley fever, and venezuelan equine encephalitis. the potential for elimination of a disease through vaccination (employed together with other strategies), as has been demonstrated for brucellosis in the us and terrestrial rabies in some european countries is a compelling economic concept, though applicable in only selected cases. the cost of preventative programs is almost always lower than emergency response programs, as illustrated by the significantly lower cost of oral wildlife rabies programs over contingency actions to control epizootic spread [ ] . the recent announcement of a fold higher incidence of lyme disease than previously believed will lead to a reassessment of the economics of preventive strategies for this disease, including wildlife vaccines. economics represent the key determinant for development and utilization of framework ii and iii vaccine strategies. a low unit cost of such vaccines will always be a requirement. the economic barriers are particularly relevant when considering vaccines for developing countries. on the positive side, the cost of developing a new animal vaccine through licensure is a small fraction, approximately %, of the cost of a typical human vaccine (the latter being $ - million by one estimate [ ] , but often far higher) [ ] . the relatively lower cost and shorter timelines for developing animal vaccines reflects the simpler path to regulatory approval, and is driven by the significantly lower market potential for these vaccines. despite the lower cost of developing new veterinary vaccines, high and middle income countries still pay higher prices until the fixed costs of development are paid off, there is an over-supply of vaccine, or competing products enter the market. to redress the pricing barriers in the case of human vaccines, there has been strong advocacy for new approaches to secure vaccine supply and access for developing countries where the burden of infectious diseases is greatest [ ] . as part of this strategy, emerging market manufacturers provide access to low unit cost vaccines, and such manufacturers of veterinary vaccines could play a substantial role in a public health strategy for animal immunization. indeed all of the principles being applied to human vaccines could be extended to vaccines for animals, particularly if there is both a clear rationale for public health and the "pull" of a potentially expanded market or of guaranteed purchase agreements. up to now public-private financing for developing and distributing veterinary vaccines has represented a tiny fraction of support available for human developing-world vaccines, and has focused on vaccines for livestock as a means of improving animal production and protein supply rather than preventing zoonotic diseases [ ] . given the public health impact of zoonotic diseases described in the introduction to this review and the potentially lower cost of interventions targeting animals vs. humans, there should be a new emphasis on the public health improvements that could result from animal immunization. zoonotic diseases that merit consideration because they occur at high incidence or are poorly controlled, include rabies, zoonotic leishmaniasis, brucellosis, rift valley fever, m. bovis, lyme disease, and several enteric bacterial infections. as mentioned above, the regulatory pathway for animal vaccines is considerably simpler than for human vaccines [ ] . this is due to multiple factors, including less onerous requirements for manufacturing and control of veterinary products, the simpler and far less expensive clinical trial requirements for marketing approval, the ability to challenge animals to demonstrate vaccine efficacy, as well as an established regulatory mechanism for conditional approval allowing commercial sales while still gathering more definitive data. there is no requirement for large, statistically powered efficacy field trials to obtain marketing approval, as is the case for human vaccines. the development of veterinary vaccines is consequently far faster than for human vaccines. for example, west nile vaccine for horses was commercialized years after introduction of the virus into the u.s., whereas the first (phase ) human trial of a west nile vaccine was completed years after introduction. the lower costs and accelerated timeframe for development of animal vaccines represent an important rationale for novel investments in public health, particularly in developing countries. to justify investments in a framework ii or iii vaccine where an economic incentive for animal immunization is marginal and a public health benefit is a goal, it is important to plan for vaccine effectiveness studies showing that vaccination of animals actually reduces human disease prevalence, as was postulated in greece following vaccination against b. melitensis [ ] . such demonstrations would really drive the field forward. thus, while the deployment of tc- venezuelan equine encephalitis vaccine was credited with the curtailment of the human epidemic in - , no controlled study to demonstrate an effect on human disease incidence was actually performed. indeed, studies to confirm the attractive hypothesis that immunization of domesticated ruminants against rift valley fever in africa would prevent intermittent outbreaks of the disease in animals and humans, while leaving the mosquito reservoir of infection intact, would be difficult to design and carry out. because of its discrete epidemiology, hendra virus (albeit a low-incidence disease) presents a unique opportunity to demonstrate the effectiveness of animal immunization on the occurrence of a disease in humans. the increasing problem of emerging infections, the majority of which are the result of spill-over from animals to humans, is a compelling reason to consider novel vaccine interventions, and the collaborations between veterinary and human health institutions in the development of the hendra, west nile, vee and rift valley fever vaccines described in this review serve as examples of the power of this approach. other potential targeted vaccine interventions focused on animal reservoirs or intermediate hosts in order to control disease emergences include avian influenza, nipah virus disease, and, possibly, middle east respiratory syndrome. funding agencies and industry should be encouraged to seek integrated approaches to prevention of zoonotic diseases. the ultimate success of examples provided in this review, such as e. coli o vaccines for cattle, reservoir targeted lyme disease vaccines for field mice, and rift valley fever vaccines for livestock will require sustained efforts utilizing the one health paradigm. human-animal medicine. clinical approaches to zoonoses, toxicants, and other shared risks host range and emerging and reemerging pathogens the human/animal interface: emergence and resurgence of zoonotic infectious diseases monath tp, editors. one health-one medicine: linking human, animal, and environmental health the economics of one health research as a part of public health emergency response the multiple burdens of zoonotic disease and an ecohealth approach to their assessment health: a new professional imperative: avma one health initiative task force report fda experience with medical countermeasures under the animal rule epidemiology of west nile in europe and in the mediterranean basin west nile virus and its emergence in the united states of america west nile virus neuroinvasive disease incidence in the united states west nile virus: epidemiology and clinical features of an emerging epidemic in the united states long-term prognosis for clinical west nile virus infection a case-control study of factors associated with development of clinical disease due to west nile virus immunogenicity, genetic stability and protective efficacy of a recombinant, chimeric yellow fever-japanese encephalitis virus (chimerivax tm -je) as a live, attenuated vaccine candidate against japanese encephalitis chimaeric live, attenuated vaccine (chimerivax tm ) incorporating the envelope genes of japanese encephalitis (sa - - ) virus and the capsid and nonstructural genes of yellow fever ( d) virus is safe, immunogenic and protective in non-human primates yellow fever vector live-virus vaccines: west nile vaccine development chimerivax tm -west nile live-attenuated vaccine: preclinical evaluation of safety, immunogenicity and efficacy west nile vaccine efficacy of a live attenuated chimeric west nile virus vaccine in horses against clinical disease following challenge with virulent west nile virus, abstr. . in: suppl. proc. rd annu. meet comparative efficacies of three commercially available vaccines against west nile (wnv) in a short-duration challenge trial involving an equine wnv encephalitis model a live, attenuated recombinant vaccine against west nile virus differentiation of west nile virus-infected animals from vaccinated animals by competitive elisa using monoclonal antibodies against non-structural protein brucellosis: an overview the new global map of human brucellosis towards a brucella vaccine for humans clinical manifestations of human brucellosis: a systematic review and meta-analysis human health benefits from livestock vaccination for brucellosis: case study brucellosis: the case for live, attenuated vaccines rough vaccines in animal brucellosis: structural and genetic basis and present status estimating herd prevalence of bovine brucellosis in us states using slaughter surveillance vaccination strategies for managing brucellosis in yellowstone basin progress in brucella vaccine development incidence of human brucellosis in a rural area in western greece after the implementation of a vaccination programme against animal brucellosis invasive escherichia coli vaccines expressing brucella melitensis outer membrane proteins or or periplasmic protein bp confer protection in mice challenged with b. melitensis a combined dna vaccine provides protective immunity against mycobacterium bovis and brucella abortus in cattle efficacy of calfhood vaccination with brucella abortus strain rb in protecting bison against brucellosis risk factors for sporadic shiga toxin-producing escherichia coli o infections in foodnet sites escherichia coli o prevalence and enumeration of aerobic bacteria, enterobacteriaceae, and escherichia coli o at various steps in commercial beef processing plants animal-and truckload-level associations between escherichia coli o :h in feces and on hides at harvest and contamination of preevisceration beef carcasses bioniche food safety inc. econiche ® escherichia coli o bacterial extract vaccine use of a siderophore receptor and porin proteins-based vaccine to control the burden of escherichia coli o :h in feedlot cattle effects of a siderophore receptor and porin proteins-based vaccination on fecal shedding of esherichia coli o :h in experimentally inoculated cattle an outcomes model to evaluate risks and benefits of escherichia coli vaccination in beef cattle bartonella spp. in pets and effect on human health one health: the importance of companion animal vector-borne diseases arthropod-borne infectious diseases of the dog and cat zoonoses control in dogs how to eradicate canine rabies: a perspective of historical efforts mass vaccination campaign against rabies: are dogs correctly protected? the peruvian experience human rabies: a disease of complex neuropathogenetic mechanisms and diagnostic challenges re-evaluating the burden of rabies in africa and asia field evaluation of a dog owner, participation-based, bait delivery system for the oral immunization of dogs against rabies in tunesia world health organization. field application of oral rabies vaccines for dogs. geneva: world health organization field trial with oral vaccination of dogs against rabies in the philippines oral vaccination of foxes against rabies with sad b in europe, - : a review oral vaccination of dogs with recombinant rabies virus vaccines one health and hendra virus: a collaborative approach in action hendra and nipah viruses: different and dangerous pteropid bats are confirmed as the reservoir hosts of henipaviruses: a comprehensive experimental study of virus transmission henipaviruses: emerging paramyxoviruses associated with fruit bats henipavirus: a review of laboratory animal pathology hendra vaccine success announced unexpected result of hendra virus outbreaks for veterinarians receptor binding, fusion inhibition, and induction of cross-reactive neutralizing antibodies by a soluble g glycoprotein of hendra virus a recombinant hendra virus g glycoprotein-based subunit vaccine protects ferrets from lethal hendra virus challenge a hendra virus g glycoprotein subunit vaccine protects african green monkeys from nipah virus challenge rift valley fever severe rift valley fever may present with a characteristic clinical syndrome rift valley fever epidemic in saudi arabia: epidemiological, clinical, and laboratory characteristics rift valley fever virus (bunyaviridae: phlebovirus): an update on pathogenesis, molecular epidemiology, vectors, diagnostics and prevention rift valley fever virus (family bunyaviridae, genus phlebovirus). isolations from diptera collected during an inter-epizootic period in kenya prediction of a rift valley fever outbreak breaking the chain: rift valley fever virus control via livestock vaccination rift valley fever: the neurotropic adaptation of the virus and the experimental use of this modified virus as a vaccine adverse response of nonindigenous cattle of european breeds to live attenuated smithburn rift valley fever vaccine an inactivated rift valley fever vaccine world health organization. the use of veterinary vaccines for prevention and control of rift valley fever: memorandum from a who/fao meeting zh -vsvri: is it still the best choice for vaccination against rift valley fever in egypt? immunogenicity of an inactivated rift valley fever vaccine in humans: a -year experience active and passive immunization against rift valley fever virus infection in syrian hamsters mutagen-directed attenuation of rift valley fever virus as a method for vaccine development use of reassortant viruses to map attenuating and temperature-sensitive mutations of the rift valley fever virus mp- vaccine mapping of the mutations present in the genome of the rift valley fever virus attenuated mp strain and their putative role in attenuation factors in the emergence of arbovirus diseases safety and efficacy of a mutagen-attenuated rift valley fever virus vaccine in cattle pathogenicity and immunogenicity of a mutagen-attenuated rift valley fever virus immunogen in pregnant ewes teratogenicity of a mutagenised rift valley fever virus (mvp ) in sheep rescue of infectious rift valley fever virus entirely from cdna, analysis of virus lacking nss gene, and expression of a foreign gene rift valley fever virus lacking the nss and nsm genes is highly attenuated, confers protective immunity from virulent virus challenge, and allows for differential identification of infected and vaccinated animals safety and immunogenicity of recombinant rift valley fever mp- vaccine candidates in sheep characterization of clone , a naturally attenuated avirulent isolate of rift valley fever virus, which is altered in the small segment rift valley fever evaluation of the efficacy and safety of the rift valley fever clone vaccine in sheep protective immune responses induced by different recombinant vaccine regimes to rift valley fever an alphavirus replicon-derived candidate vaccine against rift valley fever virus rift valley fever virus immunity pro-vided by a paramyxovirus vaccine vector novel approaches to develop rift valley fever vaccines rift valley fever: recent insights into pathogenesis and prevention vaccination with virus like particles protects mice from lethal infection of rift valley fever virus priming with dna plasmids encoding the nucleocapsid protein and glycoprotein precursors from rift valley fever virus accelerates the immune responses induced by an attenuated vaccine in sheep endemic venezuelan equine encephalitis in the americas: hidden under the dengue umbrella venezuelan encephalitis emergence mediated by a phylogenetically predicted viral mutation the health and economic impact of venezuelaqn equine encephalitis (vee) inactivated and live vee vaccines-a review epidemic venezuelan equine encephalitis in la guajira, colombia re-emergence of epidemic venezuelan equine encephalomyelitis in south america. vee study group medical aspects of chemical and biological warfare. textbook of military medicine, part i attenuation of venezuelan equine encephalomyelitis virus by in vitro cultivation in guinea pig heart cells long-term duration of detectable neutralizing antibodies after administration of live-attenuated vee vaccine and following booster vaccination with inactivated vee vaccine attenuation of venezuelan equine encephalitis virus strain tc- is encoded by the -noncoding region and the e envelope glycoprotein immunization of burros with living venezuelan equine encephalitis virus safety and efficacy of an attenuated vee vaccine for use in equidae field studies of an attenuated venezuelan equine encephalomyelitis vaccine (strain tc- ) vaccines for venezuelan equine encephalitis genetically engineered, live attenuated vaccines for venezuelan equine encephalitis: testing in animal models neurovirulence evaluation of venezuelan equine encephalitis (vee) vaccine candidate v in nonhuman primates venezuelan equine encephalitis virus vaccine candidate (v ) safety, immunogenicity and efficacy in horses evaluation of formalin inactivated v virus with adjuvant as a next generation vaccine candidate for venezuelan equine encephalitis virus recombinant sindbis/venezuelan equine encephalitis virus is highly attenuated and immunogenic protection of black-tailed prairie dogs (cynomys ludovicianus) against plague after voluntary consumption of baits containing recombinant raccoon poxvirus vaccine immune responses of bison to ballistic or hand vaccination with brucella abortus strain rb a baiting system for delivery of an oral plague vaccine to black-tailed prairie dogs vaccination against lyme disease of ticks, mice and men: understanding the dual-host lifestyle of lyme disease spirochaetes the emergence of lyme disease vaccines against lyme disease: what happened and what lessons can we learn? the cost effectiveness of vaccinating against lyme disease safety, efficacy, and immunogenicity of a recombinant osp subunit canine lyme disease vaccine prevention of lyme disease: a review of the evidence correcting a public health fiasco: the need for a new vaccine against lyme disease elimination of borrelia burgdorferi from vector ticks feeding on ospa immunized mice borrelia burgdorferi ospa is an arthropod-specific transmission-blocking lyme disease vaccine an ecological approach to preventing human infection: vaccinating wild mouse reservoirs intervenes in the lyme disease cycle oral vaccine that breaks the transmission cycle of the lyme disease spirochete can be delivered via bait reservoir targeted vaccine for lyme borreliosis induces a yearlong, neutralizing antibody response to ospa in white-footed mice protective efficacy of an oral vaccine to reduce carriage of borrelia burgdorferi (strain n ) in mouse and tick reservoirs development of a baited oral vaccine for use in reservoir-targeted strategies against lyme disease human mycobacterium bovis infection in the united kingdom: incidence, risks, control measures and review of the zoonotic aspects of bovine tuberculosis epidemiology of mycobacterium bovis disease in humans, the netherlands zoonotic tuberculosis due to mycobacterium bovis in developing countries classification of worldwide bovine tuberculosis risk factors in cattle: a stratified approach bcg moreau rio de janeiro-an oral vaccine against tuberculosis-review descriptive epidemiology of bovine tuberculosis in michigan ( - ): lessons learned risk factors associated with the prevalence of tuberculosis-like lesions in fenced wild boar and red deer in south central spain comparing badger (meles meles) management strategies for reducing tuberculosis incidence in cattle the epidemiology of tuberculosis in freeranging african buffalo (syncerus caffer) in the kruger national park, south africa directions and issues in bovine tuberculosis epidemiology and control in new zealand a new attenuated mycobacterium bovis vaccine protects brushtail possums (trichosurus vulpecula) against experimental tuberculosis infection oral vaccination reduces the incidence of tuberculosis in free-living brushtail possums lipid-formulated bcg as an oralbait vaccine for tuberculosis: vaccine stability, efficacy and palatability to new zealand possums (trichosurus vulpecula) epidemiology of fox rabies the use of commercially available vaccines for the oral vaccination of foxes against rabies oral immunization of foxes against rabies a review of the development of the oral vaccination technique for immunizing wildlife against rabies oral immunization of foxes against rabies. laboratory and field studies assessing anti-rabies baiting-what happens on the ground? efficacy of oral vaccination in the final stage of fox rabies elimination in switzerland a review of baits and bait delivery systems for free-ranging carnivores and ungulates vaccination against rabies: construction and characterization of sag , a double avirulent derivative of sadbern vaccination of wildlife against rabies: successful use of a vectored vaccine obtained by recombinant technology protection from rabies by a vaccinia virus recombinant containing the rabies virus glycoprotein gene decreased virulence of recombinant vaccinia virus expression vectors is associated with a thymidine kinase-negative phenotype the oral rabies immunization of foxes and dogs with sausage baits efficacy of three oral rabies vaccine baits in the red fox: a comparison arctic rabies -a review rabies challenge of captive striped skunks (mephitis mephitis) following oral administration of a live vaccinia-vectored rabies vaccine oral rabies vaccination in north america: opportunities, complexities, and challenges rabies surveillance in the united states during raccoon rabies: the re-emergence of an epizootic in a densely populated area potential effect of prior raccoonpox virus infection in raccoons on vaccinia-based rabies immunization tactics and economics of wildlife oral rabies vaccination, canada and the united states economic analysis of a large scale oral vaccination program to control raccoon rabies molecular inferences suggest multiple host shifts of rabies viruses from bats to mesocarnivores in arizona during human adenovirus type vectors expressing rabies glycoprotein aerial distribution of onrab baits as a tactic to control rabies in raccoons and striped skunks in ontario, canada high-density baiting with onrab ® rabies vaccine baits to control arctic-variant rabies in striped skunks in ontario effect of irrigated rice agriculture on japanese encephalitis, including challenges and opportunities for integrated vector management disorder of spermatogenesis and viral discharge into semen in boars infected with japanese encephalitis virus occurrence of japanese encephalitis virus mosquito vectors in relation to urban pig holdings q fever: current state of knowledge and perspectives of research of a neglected zoonosis the q fever epidemic in the netherlands: history, onset, response and reflection prévention de l'excrétion de coxiella burnetii à l'aide d'un vaccin phase i (coxevac en troupeaux bovines laitiers infectés) vaccine prophylaxis of q fever-a follow-up study of the efficacy of q-vax (csl) - prevention of coxiella bumetii infection: vaccines and guidelines for those at risk four-year evaluation of the effect of vaccination against coxiella burnetii on reduction of animal infection and environmental contamination in a naturally infected dairy sheep flock the importance of including swine and poultry workers in influenza vaccination programs human and animal vaccination delivery to remote nomadic families emergency response to raccoon rabies introduction into ontario how the research-based industry approaches vaccine development and establishes priorities current status of veterinary vaccines assembling a global vaccine development pipeline for infectious diseases in the developing world key: cord- -kng z kf authors: quesenberry, katherine e.; de matos, ricardo title: basic approach to veterinary care of ferrets date: - - journal: ferrets, rabbits, and rodents doi: . /b - - - - . - sha: doc_id: cord_uid: kng z kf the approach to preventive medicine and basic veterinary care in ferrets is very similar to that used in dogs and cats. special equipment needs are minimal, and pet ferrets can be easily incorporated into a general small animal practice. this chapter describes the unique aspects of handling, restraint, and clinical and treatment techniques used in ferrets. the approach to preventative medicine and basic veterinary care in ferrets is very similar to that used in dogs and cats. special equipment needs are minimal, and pet ferrets can be easily incorporated into a general small animal practice. however, there are unique aspects of handling, restraint, and clinical and treatment techniques used in ferrets. for procedures not discussed here, modify techniques used in other small animals by using instrumentation appropriate for the ferret's small size and choosing appropriate sedation or anesthesia to facilitate the procedure while minimizing stress or discomfort in the ferret. most ferrets are docile and can be easily examined without assistance. however, an assistant is usually needed when taking the rectal temperature, when administering injections or oral medications, or if an animal tends to bite. young ferrets often nip, and nursing females and ferrets that are handled infrequently may bite. ferrets often bite without warning. therefore always ask the owner if the ferret bites before handling it and take precautions accordingly. obtain the rabies vaccination history before physical examination. be aware that rabies protocols for animal bites from vaccinated and unvaccinated ferrets differ by locale. ferrets that are prone to biting and are not currently vaccinated for rabies may need to be sedated for procedures requiring restraint. depending on the ferret's disposition, several basic manual restraint methods can be used for examination. for tractable animals, lightly restrain the ferret on the examination table. examine the mucous membranes, oral cavity, head, and skin. then pick the ferret up and support its body with one hand while using the other hand to auscultate the thorax and palpate the abdomen. for an active animal or one that bites, scruff the ferret and suspend it with all four legs off the table (fig. . ). most ferrets become relaxed with this hold, and the veterinarian can examine the oral cavity, head, and body; palpate the abdomen; vaccinate; and clean the ears. however, even scruffing may not work for fractious animals. to restrain a ferret for a procedure, hold it firmly by the scruff of its neck and around the hips without pulling the legs back. most ferrets struggle if their legs are extended by pulling on the feet. some animals can be distracted during a procedure by feeding a meat-based canned food or a small amount of a supplement such as ferretone ( -in- pet products, islandia, ny) by syringe. for very fractious or anxious animals or for procedures requiring lengthy restraint, light sedation or anesthesia may be indicated (see chapter ) . flexible digital thermometer that is well lubricated. the normal rectal temperature of a ferret is . °f to . °f ( . °c to . °c); however, a mean of °f ( . °c), with a wider range of °f to °f ( . °c to . °c), is also reported. physical examination of a ferret can be performed quickly and efficiently if a few simple guidelines are followed. observe the attitude of the animal. ferrets may sleep in the carrier in the veterinary office; however, once awakened for the examination, a ferret should be alert and active. assess hydration by observing the skin turgor of the eyelids, tenting of the skin at the back of the neck, and moistness of the oral mucous membranes. note that skin turgor can be difficult to evaluate in a cachectic animal. estimate the capillary refill time by digitally pressing on the gingiva. examine the eyes, nose, ears, and facial symmetry. cataracts can develop in both juvenile and adult animals. retinal degeneration occurs in ferrets and may be indicated by abnormal pupil dilation. inspect for nasal discharge, and ask the owner about any history of sneezing or coughing. the ears may have a brown waxy discharge, but excessive brown exudate may indicate infestation with ear mites. observe the facial symmetry. although uncommon, salivary mucoceles occur in ferrets and present as a unilateral swelling on the side of the face, usually in the cheek or temporal area (see chapter ) . the teeth should be clean and the gingiva pink. dental tartar is common in pet ferrets, possibly related to feeding kibble instead of natural prey. plaque buildup may be exacerbated by feeding a diet with a high mineral content. remove excess dental tartar by prophylactic techniques used in dogs and cats, and recommend measures to prevent tartar buildup. a pet toothpaste can be used to decrease the rate of calculus formation. , , gingivitis is a common sequela of excessive dental tartar. ferrets often break off the tip of one or both canine teeth, and the broken tooth may appear dark. however, ferrets rarely exhibit sensitivity associated with a fractured canine. if the ferret exhibits sensitivity when the tip of the canine is probed, recommend a root canal or extraction, depending on the degree of tooth damage (see chapter ) . bruxism often indicates gastrointestinal discomfort. palpate the submandibular, axillary, popliteal, and inguinal lymph nodes. nodes should be soft and may sometimes feel enlarged in overweight animals because of surrounding fat. any firmness or asymmetry warrants fine-needle aspiration or biopsy. if two or more nodes are enlarged and firm, a diagnostic workup is indicated. auscultate the heart and lungs in a quiet room. ferrets have a rapid heart rate ( to beats/min) and often a pronounced sinus arrhythmia. if a ferret is excited and has a very rapid heart rate, subtle murmurs may be missed. valvular disease, cardiomyopathy, and congestive heart failure are seen in ferrets, and any murmur or abnormal heart rhythm should be investigated further (see chapter ) . the ferret's normal respiratory rate is to breaths/min (see chapter ) . palpate the abdomen while either scruffing the ferret or supporting it around the thorax with one hand. this allows the abdominal organs to displace downward, facilitating palpation. if the history is consistent with an intestinal foreign body or urinary blockage, palpate gently to avoid causing iatrogenic injury, such as a ruptured bladder. palpate the cranial abdomen, paying attention to the presence of gas or any firm, irregularly shaped material in the stomach area, especially in ferrets with a history of vomiting, melena, or chronic weight loss. the spleen is often enlarged, which may or may not be significant, depending on other clinical findings (see chapter ) . a very enlarged spleen may indicate systemic disease or, very rarely, idiopathic hypersplenism, and further diagnostic workup is warranted. examine the genital area, observing the size of the vulva in females. vulvar enlargement in a spayed female is consistent with either adrenal disease or an ovarian remnant; the latter is rare. examine the preputial area and size of the testicles of male ferrets; preputial and testicular tumors are sometimes seen. check the fur for evidence of alopecia. tail tip alopecia is common and may be an early sign of adrenal disease. symmetric, bilateral alopecia or thinning of the fur that begins at the tail base and progresses cranially is a common finding in ferrets with adrenal disease. examine the skin on the back and neck for evidence of scratching. pruritus is common with adrenal disease and also may indicate ectoparasites (e.g., fleas or sarcoptes scabiei). palpate and visually examine the skin thoroughly for masses. mast cell tumors are common and are variable in size. often, the fur around a mast cell tumor is matted with dried blood from the animal's scratching. other types of skin tumors, such as sebaceous adenomas and basal cell tumors, are also common (see chapter ) . perform an excisional biopsy of any lump found on the skin. young, recently purchased ferrets need serial canine distemper vaccinations until they are weeks of age. rabies vaccines should be given annually beginning at months of age. ferrets should be examined annually until they are to years of age; then, older animals may need examinations twice yearly because of the high incidence of metabolic disease and neoplasia. annual blood tests are recommended for older animals. measure the blood glucose concentration twice yearly in healthy middle-aged and older ferrets; more-frequent monitoring is needed in ferrets with insulinoma. abdominal ultrasound scanning or an endocrine panel is indicated in ferrets with thinning fur on the tail or other clinical signs suggestive of adrenal disease (see chapter ) . testing for infectious diseases may be warranted, especially in new or young ferrets that will be introduced into a multi-ferret household or those that are taken to ferret shows. ferrets can be tested for aleutian disease virus and ferret enteric coronavirus by polymerase chain reaction testing (michigan state university, diagnostic center for population and animal health, www.animalhealth.msu.edu; veterinary molecular diagnostics, www.vmdlabs.com; zoologix, www.zoologix.com). serologic tests for aleutian disease by enzyme-linked immunosorbent assay (elisa) and counterimmunoelectrophoresis are also available (see chapter ). ferrets must be vaccinated against canine distemper virus (cdv). currently, one vaccine is approved by the u.s. department of agriculture for use in ferrets: purevax ferret (boehringer ingelheim animal health, duluth, ga). purevax is a canarypox-vectored recombinant vaccine that does not contain complete cdv or adjuvants; thus, post-vaccination risks are reduced. this product has a wide safety margin and has proved effective in protecting ferrets against cdv. although supply from the manufacturer has been intermittently problematic in the united states, the vaccine is available. canine distemper vaccines that were previously used in ferrets but are now discontinued include fervac-d (united vaccines, inc, fitchberg, wi), a modified-live virus vaccine propagated in avian cell lines, and galaxy d (schering-plough animal health/merck), a modified-live virus vaccine derived from the onderstepoort canine distemper strain and attenuated in a primate cell line. galaxy vaccines are now marketed under the nobivac (merck animal health, madison, nj) trade name. in a safety and efficacy study, galaxy d proved effective in preventing canine distemper in young ferrets challenged after serial vaccination. other canine distemper vaccines have been used off-label in ferrets in countries other than the united states or when purevax has been unavailable. recombitek cdv (boehringer ingelheim animal health) is also a recombinant canarypox vaccine approved for use in dogs that has been used in ferrets. this cdv vaccine is marketed in several multivalent combinations including cdv with parvovirus: a monovalent product is not available in the united states. nobivac puppy-dpv (merck animal health) is a modified live virus canine distemper vaccine combined with parvovirus vaccine, a virus that does not affect ferrets. although these vaccines have been used clinically in ferrets, their safety and efficacy in ferrets have not been studied. a cdv vaccine approved for use in mink (distemink; united vaccines inc, fitchberg, wi) is available in -dose vials only. because of the possibility of vaccine-induced disease, especially in immunosuppressed or sick ferrets, avoid using multivalent canine vaccines and do not use modified live cdv vaccines of ferret-cell or low-passage canine-cell origin. standard vaccination protocols for canine distemper in ferrets have been based on serial vaccinations of young ferrets at , , and weeks, with annual boosters. however, recent data suggest a modified vaccine schedule consisting of two initial vaccines with less-frequent boosters is effective. in an efficacy study of ferrets, % of ferrets that were initially vaccinated at weeks and given a booster vaccine between and weeks of age with one of three commercial vaccines (purevax ferret, fervac-d, or galaxy d) maintained protective antibody titers of > : for at least years. the three commercial vaccines did not differ in efficacy of eliciting protective titers. therefore an initial vaccination protocol of two vaccinations, starting at to weeks of age and separated by weeks, followed by a booster every years, should suffice in most cases (d. perpiñan, personal communication, may ). for ferrets at high risk of contracting canine distemper or when highly pathogenic strains of cdv are circulating, consider more-intensive vaccination protocols. if the ferret is first vaccinated after to months of age, a series of two vaccinations separated by weeks is sufficiently protective (d. perpiñan, personal communication, may ). all ferrets should be vaccinated against rabies. two inactivated (killed) rabies vaccines are approved for use in ferrets in the united states: imrab- or imrab- tf (boehringer ingelheim animal health) and defensor or defensor (zoetis, parsippany, nj). inactivated vaccines are effective at producing immunity for at least year. current recommendations are to vaccinate healthy ferrets at months of age at a dose of ml administered subcutaneously (sc). give booster vaccinations annually. titers develop within days of rabies vaccination. clinical signs of rabies in ferrets can vary. in studies of experimentally induced rabies in ferrets, clinical signs range from restlessness, apathy, and paresis to ascending paralysis, ataxia, cachexia, bladder atony, fever, hyperactivity, tremors, and paresthesia. , mean incubation period in experimental studies varies from to days. , virus is present in the brain tissue and salivary glands of inoculated ferrets, and virus is shed in the saliva to days after onset of illness. , ferrets are at least times less susceptible than skunks to rabies infection when fed mice carcasses infected with rabies virus. survival and clearance of rabies virus infection was reported in one ferret experimentally infected with rabies virus of skunk origin. the ferret initially exhibited hindlimb paralysis that resolved to paraparesis. no virus antigen was found at necropsy months after inoculation. ferrets are considered currently immunized days after the initial rabies vaccination and immediately after a booster vaccination. if a healthy pet ferret bites a person, current recommendations of the compendium of animal rabies prevention and control are to confine and observe the animal for days, during which the ferret should not be vaccinated. any illness that develops during observation should be reported immediately to the local health department. if signs suggest rabies, the ferret must be euthanized, and protocols for rabies evaluation followed. for a ferret with a current vaccine status exposed to a possible rabid animal, recommendations are to revaccinate the ferret within hours of exposure and then keep the ferret under the owner's observation and care for days. exposed ferrets that are overdue for a booster rabies vaccination should be evaluated on a case-by-case basis by the health department. an unvaccinated animal that is exposed to a rabid animal or a stray ferret that bites a person should be euthanized immediately and submitted for rabies testing. see the website of the centers for disease control and prevention (https://www.cdc.gov/ rabie s/specific_groups/veterinarians/) or the national association of public health veterinarians (http://www.nasphv.org/ documentscompendia.html) for specific guidelines. in ferrets, adverse events associated with vaccination are primarily type i hypersensitivity reactions or anaphylaxis. ferrets with mild reactions may exhibit pruritus and skin erythema. more severe reactions are typified by vomiting, diarrhea, piloerection, hyperthermia, cardiovascular collapse, or death. vaccine reactions are most common after canine distemper vaccination but may also occur after rabies vaccination. in a study of vaccine-associated adverse events in ferrets in the united states, the incidence of adverse events associated with rabies vaccine alone, canine distemper vaccine alone, and rabies and canine distemper vaccines together were . %, . %, and . %, respectively, with no significant difference among groups. however, occurrence of a vaccine-associated adverse event was significantly associated with the cumulative number of canine distemper vaccinations, with an % increase in risk of an adverse event with each additional distemper vaccine. the canine distemper vaccines used in this cohort of ferrets were purevax ferret and fervac-d; the two vaccines were grouped collectively in the analysis, and the incidence of adverse events associated with individual vaccines was not reported. all reactions occurred immediately after vaccination and most commonly consisted of vomiting and diarrhea. in another study of ferrets, the incidence of adverse events after administering either canine distemper ( . %) (fervac d), rabies ( . %) (imrab- ), or both vaccines ( . %) did not differ significantly between groups. in a report of vaccine reactions in ferrets reported to the u.s. pharmacopeia veterinary practitioners' reporting program, % involved administration of fervac d, % involved concomitant administration of fervac d and imrab , and % involved administration of imrab alone (purevax was not approved for use at the time these data were collected). according to merial's (now boehringer ingelheim) product information, the incidence of vaccine reactions with purevax is . %. no adverse events were reported in an efficacy study of vaccination of ferrets with either purevax ferret, fervac-d, or galaxy d. surveillance of vaccine-associated adverse events relies on voluntary reporting by practitioners. vaccine-associated adverse events can be reported to the center for biologics, u.s. department of agriculture (https:// www.aphis.usda.gov/aphis/ourfocus/animalhealth/veterinarybiologics/adverse-event-reporting). always follow the manufacturer's instructions for vaccine administration, and inform the owner of the possibility of an adverse reaction before vaccinating. because most reactions occur almost immediately after vaccination, have the owner monitor the ferret in the waiting area for minutes or more after vaccination with any product. if a ferret has an adverse reaction, administer an antihistamine (e.g., diphenhydramine hydrochloride, . to . mg/kg intravenously [iv] or intramuscularly [im]), epinephrine ( . mg/kg iv, im or intratracheally), or a short-acting corticosteroid (e.g., dexamethasone sodium phosphate, to mg/kg iv or im), and give supportive care. for any biologic product, veterinarians must assess risk versus benefit of vaccination. the treatment options for ferrets that have had a vaccine reaction are to administer diphenhydramine ( mg/kg orally [po] or sc) at least minutes before vaccination or not to vaccinate if exposure risk is minimal. vaccine injection-site sarcomas have been described in ferrets. , in one report, of fibrosarcomas in ferrets were from locations used for vaccination. fibrosarcomas had similar histologic, immunohistochemical, and ultrastructural features as those reported for feline vaccine-associated sarcomas. in cats, adjuvanted vaccines are most likely to be involved in tumor development; however, no definitive association was made between the fibrosarcoma and the type of vaccine in ferrets. ferrets appear less prone than cats to development of vaccine-induced tumors. gastrointestinal parasitism is most common in young or recently purchased pet ferrets and is relatively uncommon in mature ferrets in the united states. in a survey of ferrets that were either privately owned or in pet shops in italy, of ( %) were positive for ancylostomids (hookworms) and one ( %) was positive for sarcocystis. ferrets can be intermediate hosts or vectors of parasites from other natural hosts. protozoan parasites are occasionally seen. therefore perform routine fecal flotations and direct fecal smears for all young or recently acquired ferrets. coccidiosis (isospora species) usually is seen in young ferrets, which shed oocysts between and weeks of age. infection is often subclinical, although ferrets occasionally may have loose stool or bloody diarrhea. coccidiostats, such as sulfadimethoxine and amprolium, are effective and safe, and treatment should be continued for at least weeks. coccidia in ferrets may cross-infect dogs and cats; therefore check other pets in the household for coccidia and treat as needed. giardiasis is occasionally seen in ferrets. results of studies on molecular characterization and host specificity of giardia duodenalis isolates from pet ferrets vary. in one study, genetic sequences of giardia isolates from ferrets were similar to those of giardia associated with human infections. results of another study showed genetic sequences of giardia differed in ferrets and people and other mammals, suggesting that giardia isolates from ferrets may be host specific. giardia can be detected by identifying cysts or trophozoites in a fresh fecal smear or zinc sulfate flotation, or by fecal elisa. treat ferrets with giardiasis with metronidazole ( mg/kg po every hours for to days) or fenbendazole ( mg/kg po every hours for to days). cryptosporidiosis is described primarily in young ferrets. infection is associated with the ferret genotype of cryptosporidium parvum and therefore is an unlikely source of human infection. infection is usually subclinical and, although most ferrets recover within to weeks, can persist for months in immunosuppressed animals. oocysts of cryptosporidium are small ( - μm) and difficult to detect but can be found in fresh fecal samples examined immediately after acid-fast staining. , various drugs, including azithromycin, tylosin, and nitazoxanide, are used for treatment in dogs and cats, but efficacy in ferrets is not known. heartworms (dirofilaria immitis) can cause disease in ferrets. ferrets that are housed outdoors in heartworm-endemic areas are most susceptible to infection; however, all ferrets in endemic areas should be treated with heartworm preventive (see chapter and appendix). ear mites (otodectes cynotis) are common in ferrets, but affected animals may not exhibit pruritus or irritation. this mite species also infects dogs and cats, and animals in households with multiple pets can transmit mites to other animals. a red-brown, thick, waxy discharge in the ear canal and pinna characterizes infection. a direct smear of the exudate reveals adult mites or eggs. because ferrets normally have brown ear wax, the color or appearance of debris in the ear canal is not pathognomonic for mites. at the initial examination, check for ear mites and do follow-up checks at the annual examination in ferrets kept in multiple-pet households. several products, including selamectin, are effective in treatment (see chapter ) . flea infestation (ctenocephalides species) is most common in ferrets kept in households with dogs or cats. ferrets with chronic infestations can become severely anemic. check ferrets during the physical examination for signs of fleas or flea dirt. treat infested animals with products safe for use in cats, and institute flea control measures (see chapter ) . ticks are rarely seen on domestic ferrets, and lyme disease in ferrets has not been reported. ferrets can be hospitalized in standard hospital cages with some adaptations. ferrets are agile escape artists and often can squeeze through vertical cage bar openings on standard hospital cages. for housing ferrets, use only commercial cages with small spacing between bars or use cages with attached plexiglass fronts at least half the height of the cage door or higher to prevent escape. small animal intensive care cages or incubators also can be used to house ferrets and are especially useful for animals that need supplemental heat or oxygen. closely monitor the temperature in these cages when using supplemental heat to prevent hyperthermia or hypothermia. the cage should be large enough to accommodate a sleeping area and an area for defecation and urination. ferrets typically do not soil their sleeping area, even when very sick. all ferrets like to burrow and should be given opportunity to do so while hospitalized. clean towels or a mound of shredded paper make excellent burrowing material. take care with plastic-backed underpads, which ferrets may chew and ingest. small padded pet beds and fleece pet "pockets" work well as sleeping areas. provide water in either water bottles or small weighted bowls. before hospitalization, ask the owner which type of watering system the ferret is accustomed to. ferrets can be finicky eaters and should be fed their regular diet while hospitalized, if possible. otherwise, feed a very palatable ferret food or a premium high-protein cat/kitten kibble. for animals that are anorectic, force-feed a high-calorie semisolid food or supplement until the animal is eating on its own (see later discussion). most veterinary laboratories offer small mammal hematologic and biochemical panels that require . ml or less of blood. in-clinic, point-of-care analyzers require very small sample sizes (usually μl). the blood volume of healthy ferrets is approximately ml in average-sized females weighing g and ml in males weighing kg. up to % of the blood volume can be safely withdrawn at one time in a normal ferret; however, collect only the minimum amount needed for analysis. repeated blood drawing can contribute to anemia in sick animals hospitalized for long periods. obtaining a blood sample from a ferret is relatively easy. venipuncture usually can be done with manual restraint, but some veterinarians prefer sedating or anesthetizing especially active animals. several venipuncture sites are readily accessible; the technique and site chosen depend on how much blood is needed and the availability of assistants for restraint. anesthesia or sedation can be used if assistants are unavailable. if needed, ferrets often can be distracted during restraint for venipuncture by offering semisolid food or a product such as ferretone ( -in- pet products) by syringe. avoid using supplements with corn syrup or other sugars, because this will affect blood glucose levels, and collect blood for glucose determination or other fasting samples before offering food. the blood collection technique can affect hematologic test results. isoflurane anesthesia can cause decreases in all hematologic values beginning at induction of anesthesia and reaching maximal effects at minutes after induction. both isoflurane and sevoflurane can cause a decrease in packed cell volume. therefore hematologic results of blood samples collected while a ferret is anesthetized must be interpreted carefully (see chapter ) . two sites are commonly accessed to obtain large blood volumes in ferrets. jugular venipuncture can be accomplished by extending the ferret's forelegs over the edge of a table and the neck up, or by restraining the ferret in lateral recumbency (fig. . ) . have a second assistant restrain the ferret's hind end on the table to prevent twisting, or wrap the lower body in a towel. use a -gauge needle bent slightly to a -degree angle with a -to -ml syringe for venipuncture in most ferrets; a -gauge needle can be used in large males. shave the neck at the venipuncture site to enhance visibility of the jugular vein. the vein is relatively superficial and is located more laterally in the neck than it is in dogs or cats. once the needle is inserted, the blood should flow easily into the syringe; if the neck is overextended and the head is arched back, the blood may not flow readily from the vein. relax the hold on the head or gently "pump" the vein by moving the head slowly up and down to enhance blood flow into the syringe. with ferrets that resist limb extension, a towel-wrap technique can be used. scruff the ferret with its front legs extended caudally against the ventral thorax and wrap the animal's body firmly with a towel from the base of the neck down. have an assistant restrain the toweled ferret in dorsal recumbency while scruffing the neck or holding the head. however, with very fractious animals, even this technique may be difficult. the second venipuncture site from which to obtain large blood samples is the cranial vena cava. the site of venipuncture is the anterior vena cava or the right or left brachiocephalic trunk, depending on the point of entry and the depth of needle penetration (fig. . , a) . this technique is relatively safe in ferrets because of the long anterior vena cava and the caudal location of the heart in the thoracic cavity, which is approximately cm from the thoracic inlet. however, rare instances of hemorrhage into the anterior thoracic cavity can occur. for this technique, restrain the ferret on its back with the forelegs pulled caudally and the head and neck extended ( fig. . , b) . with manual restraint, two assistants are usually needed, one to restrain the forelegs and head and the other to restrain the rear just cranial to the pelvis. use a -gauge needle with an attached -ml or -ml syringe; insert it into the thoracic cavity between the first rib and the manubrium at an angle to degrees to the body. direct the needle toward the opposite rear leg or most caudal rib and insert it almost to the hub. pull back on the plunger as the needle is slowly withdrawn until blood begins to fill the syringe. if the ferret struggles, quickly withdraw the needle and wait until the ferret is quiet before making a second attempt. in very fractious or active ferrets, jugular venipuncture or use of tranquilization are safer choices to avoid lacerating the vessels. the lateral saphenous or cephalic vein can be used if only a small amount of blood is needed to measure a packed cell volume or blood glucose level. to prevent collapse of the vein during venipuncture, use an insulin syringe with an attached -or -gauge needle. the saphenous vein lies just proximal to the tarsus on the lateral surface of the leg; the cephalic vein is in the same anatomic location as in a dog. before venipuncture, shave the fur from the area to enhance visibility of the vein. venipuncture of the tail artery is possible but rarely used in pet ferrets. venipuncture at this site is painful and requires anesthesia. insert a syringe with a -gauge needle into the ventral midline of the tail directed toward the body. once the artery is entered to mm deep into the skin, slowly withdraw the plunger until blood fills the syringe. apply pressure to the venipuncture site for to minutes after withdrawing the needle. most commercial veterinary diagnostic laboratories provide laboratory-specific reference intervals for ferret hematologic and biochemical values (see chapter ) . published sources of reference intervals for both laboratory and pet ferrets are available. contains blood coagulation values determined in normal ferrets that can be used as guidelines in the absence of reference intervals specific to a laboratory or to coagulation analyzer equipment. indwelling intravenous catheters can be placed in the lateral saphenous or cephalic vein (fig. . ) in ferrets that are collapsed with poor blood pressure or in young or very small ferrets, attempts to place an intravenous catheter may be unsuccessful. an intraosseous catheter can be placed in these animals. the proximal tibia is the most common site for intraosseous catheter placement in small mammals, but the proximal femur can also be used and allows for a greater range of movement. unless the ferret is very depressed, anesthesia is required to place the catheter. sterilely prepare the insertion site and infiltrate the area with % to % lidocaine (maximum dose, mg/kg). insert a -or -gauge, . -inch spinal needle into the marrow cavity. alternatively, use a -or -gauge hypodermic needle with a surgical steel wire inserted into the lumen to prevent a bone plug from occluding the needle during insertion. the intraosseous catheter should occupy approximately % to % of the marrow cavity at the narrowest portion. administer drugs through intraosseous catheters in small volumes with minimal pressure to prevent leakage from the insertion site. if possible, change to an intravenous catheter as soon as the animal is rehydrated or blood pressure improves. intraosseous catheters should not be left in place for more than hours. vascular access ports can be placed when repeated vascular access is required for any reason, such as for chemotherapy. the technique used to place the catheter and port has been described and illustrated. hospitalized ferrets usually require fluid therapy to maintain hydration and correct dehydration. daily fluid requirements of ferrets have not been determined; however, calculating fluid requirements at a rate of to ml/kg per day appears to be adequate for maintenance. one source estimates daily water consumption of adult ferrets as to ml/day. provide additional fluids to compensate for ongoing fluid loss and to correct dehydration calculated as a percentage of the body weight. intravenous fluids are preferred in ill animals. if possible, administer intravenous fluids by continuous-rate infusion. alternatively, divide the calculated daily fluid volume into two or three doses and administer by a buretrol (baxter healthcare, glendale, ca) or syringe pump. depending on the clinical condition of the ferret, supplements and drugs can be added to fluids by using the same criteria and calculations as for dogs and cats. although subcutaneous fluids can be given, ferrets react painfully to subcutaneous administration, and adequate restraint is needed. administer subcutaneous fluids in the loose skin along the back and dorsal cervical area, dividing the calculated daily fluid volume into doses given two or three times daily. colloids are effective in improving intravascular fluid volume and oncotic pressure in ferrets that are hypoproteinemic or in shock. dosage and administration are like those used in other small animals (see chapter ). antibiotics and other therapeutic agents are usually given at dosages like those in cats per kg of body weight (see appendix). in hospitalized animals with an indwelling catheter, give medications intravenously if possible. however, monitor the leg where the catheter is placed carefully for phlebitis and cellulitis, particularly when caustic medications are administered. antibiotics can be administered im; however, if treatment continues over several days, subcutaneous administration is preferred in cachectic animals because of the limited muscle mass. pills are very difficult to administer to ferrets; therefore oral medications are most easily given compounded into liquid form. ferrets generally accept chicken and beef flavors of compounded medications but do not like the taste of fish flavors. pain management is important in the postoperative period, with diseases that create significant discomfort (such as gastrointestinal ulceration), and for traumatic injuries (see chapter ) . ferrets in pain may exhibit tachypnea, a stiff gait, a strained facial expression, teeth grinding, shivering, half-closed eyelids, aggression, focal muscle fasciculation, hiding, general malaise, bristling of tail fur, and appear "tucked" in the abdomen. , many analgesic agents are used effectively in ferrets, including opioids, α- agonists, nonsteroidal antiinflammatory drugs (nsaids), and local anesthetics. clinically, buprenorphine, butorphanol, or meloxicam or combinations thereof are most commonly used for hospitalized animals and outpatients (see chapter and appendix). although no studies have evaluated efficacy of meloxicam for analgesia in ferrets, a meloxicam dose of . mg/kg in ferrets achieves plasma concentrations considered effective in other companion animals. ferrets are sensitive to acetaminophen toxicity. the activity of uridine ′-diphospho (udp)-glucuronosyltransferase in their livers compares with that of cats. acetaminophen glucuronidation is slower in ferrets than in other nonfelid species. unlike cats, however, no genetic mutations are associated with this slow metabolism, and the exact cause is unknown. ibuprofen can be toxic in ferrets at high doses, and accidental ibuprofen ingestion of mg/kg can cause death. clinical signs of toxicosis are depression, coma, ataxia, recumbency, tremors, weakness, and death. therefore avoid acetaminophen in ferrets and use nsaids with caution. do not use nsaids in ferrets being treated with a corticosteroid for insulinoma or other disease. epidural administration of analgesics before surgery appears to be effective in controlling postoperative pain in ferrets undergoing procedures involving the abdomen, spine, pelvis, hind legs, tail, and perineum (see chapter ) . , the technique has been well described in ferrets and is similar to that used in other small animals. , epidural injection is contraindicated in cases of coagulopathy, sepsis, hypovolemia, skin infection, and local fractures. be careful in the immediate postoperative period when opioids, such as butorphanol or buprenorphine, are used. in cats, as well as in other species, opioids including hydromorphone, butorphanol, and buprenorphine are associated with an increase in body temperature. opioid analgesics also can cause pronounced sedation, and ferrets can remain very lethargic and immobile for long periods. because of these two factors, immobile ferrets can overheat quickly if a heating lamp or other focused heat source is used during anesthetic recovery. therefore closely monitor the body temperature of any immobile or lethargic ferret given opioids or sedatives to prevent overheating when using heat lamps or forced-air warming devices. many sick ferrets are either cachectic or hypoglycemic or have minimal body fat and require nutritional support. products formulated as recovery diets for carnivores are available and readily accepted by most ferrets (carnivore care, oxbow animal health, murdock, ne; emeraid carnivore, emeraid, cornell, il). ferrets can be syringe-fed meat-based soft foods marketed for hospitalized dogs and cats (maximum-calorie plus, the iams company, mason, oh; canine/feline a/d, hill's pet nutrition, topeka, ks). do not offer supplement gels that contain corn syrup because of the high sugar content. force-feed anorectic ferrets as much as they will accept comfortably, usually to ml syringe fed three or four times daily. ferrets that develop a taste for the food may eat it directly from a bowl. ferret owners often prepare homemade diets of "duck soup" or "chicken gravy" to nurse their pets at home. many different recipe variations are available, and most are based on whole chicken with additives ranging from beef fat, dog kibble, nutritional supplement gels, or brewer's yeast to echinacea capsules. the "duck soup" variations all provide a soft, porridge-consistency food that is usually readily eaten by sick and convalescing ferrets. some recipes are very high in fat and carbohydrates. unless the homemade diets are based on or used with a commercial ferret diet, they should not be used long term because of possible nutritional imbalances and deficiencies. although seldom used clinically, esophagostomy feeding tubes can be placed in ferrets to manage debilitated animals over the long term. the technique is like that used in cats. gastric feeding tubes have been placed in ferrets both experimentally and clinically. , in a study of ferrets, gastrostomy tubes were placed percutaneously by a nonendoscopic technique. a gastrostomy tube was placed and maintained successfully in a ferret after surgical repair of an esophageal perforation caused by an esophageal foreign body. total nutrient admixtures have been formulated to provide partial parenteral nutrition to ferrets. , depending on the osmolarity of the solution, parenteral nutrition solutions can be delivered via a central, peripheral, intraosseous, or intraperitoneal catheter; however, the relatively high protein requirements of ferrets usually result in a solution of to mosm/l, which should be administered into a large (central) vein. urine samples can be collected by cystocentesis or by free catch after natural voiding or gentle manual expression of the bladder. the techniques for manually expressing the bladder and cystocentesis are the same as those used in dogs and cats. anesthetize fractious ferrets to avoid trauma to the thin bladder wall. use a -gauge needle for cystocentesis. reference values for urinalysis are listed in table . . in one study, the reference range for urine ph in ferrets was reported as . to . ; however, urine ph can vary according to the diet. the normal urine ph in ferrets fed a high-quality, meat-based diet is approximately . . urinary catheterization is commonly indicated in male ferrets, but the procedure can be challenging. although techniques have been described for both sexes, in male ferrets, the urethral opening is very small and is located on the ventral surface of the penis, proximal to the j-hook in the end of the os penis. use a . -or . -fr polytetrafluoroethylene, silicone, or polyurethane urinary catheter (slippery sam tomcat urethral catheter, surgivet, smiths medical, dublin, oh; tomcat urethral catheter, surgivet; tomcat/small animal urinary catheter, mila international inc., florence, ky). alternatively, use a . -fr rubber feeding catheter; estimate the length of the catheter that must be inserted to reach the bladder before placing (see also chapter ). if using a long rubber catheter, use a stylet or flexible wire to stiffen the catheter if needed; the stylet can be retracted slightly while passing the catheter, but be very careful when rounding the pelvic flexure to avoid perforating the urethra. another option is to use a -gauge or -gauge, -inch jugular catheter with the stylet removed. to pass the urinary catheter, aseptically prepare the preputial area, locate the urethral opening, and introduce the catheter tip (fig . , a) . if the urethral opening is difficult to see, dilate the opening by passing a -gauge intravenous catheter just inside the tip of the urethra and flushing gently with saline solution (fig . , b) . slip the tip of the lubricated urinary catheter gently into the dilated urethral opening alongside the intravenous catheter and, while gently flushing with saline solution, pass the catheter into the bladder. often resistance is met at the pelvic flexure; if this occurs, try repeated gentle flushing and relubricating the catheter until it passes. once in place, if using a red rubber catheter, place butterfly tape strips around the catheter just as it enters the urethra and at another point to cm distal, and suture these to the skin (fig . , c) . if using a urethral catheter, suture the catheter hub to the prepuce (fig . , d) . attach a sterile urinary collection device and tape the tubing to the tail to further prevent tension. if needed, bandage the ferret's abdomen to minimize rotation of - - . ± . ph . ± . . - . . - . . ± . . - . the catheter and to restrict the ferret from traumatizing it. soft elizabethan collars are needed in some ferrets to prevent chewing at the catheter. maintain sterility of the collection system, and drain the bag by needle and syringe rather than opening the system (see also chapter ) . temporary tube cystostomy has been used successfully to manage male ferrets with urinary obstruction caused by adrenal disease. in four ferrets treated surgically, a -or -fr foley catheter was placed in the bladder at the time of adrenalectomy and left in place for to days. in these ferrets, immediate treatment of urinary blockage was by cystocentesis. a cystostomy catheter also can be placed by using interventional radiographic techniques. this is an option in obstructed ferrets in which a urethral catheter cannot be passed and surgery is considered high risk. a cystostomy catheter allows azotemic ferrets to be managed with diuresis before surgery; alternatively, in cases in which surgery is not an option, the catheter can remain in place pending response to medical management with leuprolide acetate. indirect blood pressure monitoring techniques, using both doppler ultrasonography and oscillometric methods, have been described in ferrets. , , , however, both methods poorly approximate direct arterial blood pressure measurements. indirect blood pressure readings may be inaccurate because the neonatal blood pressure cuff does not fit securely on the short legs of ferrets (doppler method), and pressure changes of the tail artery may not be sufficiently high to be detected by the oscillometric system. in one study, the indirect systolic blood pressure values measured by doppler on the tail of ferrets measured mmhg less than the direct arterial systolic blood pressure values. in a study using male ferrets that compared indirect blood pressure measurements on the tail, forelimb, and hindlimb using an oscillometric sphygmomanometer and a high-definition oscillometric monitor with direct arterial blood pressure measurements, measurements using the tail were considered most reliable. however, the oscillometric sphygmomanometer consistently overestimated the systolic arterial pressure. indirect measurements of systolic, mean, and diastolic arterial pressures were consistently higher during hypotensive states but substantially lower in hypertensive states than corresponding direct blood pressure measurements. in a study of healthy ferrets, sedation with butorphanol ( . mg/kg im) and midazolam ( . mg/kg im) was found optimal to allow indirect blood pressure measurement. reference values for indirect blood pressure measured with the cuff placed at the base of the tail were to mmhg systolic, to mmhg mean, and to mmhg diastolic arterial pressure. be aware of the above-described inconsistencies of indirect compared with direct blood pressure measurements and interpret clinical results carefully. in clinical situations, indirect blood pressure is most useful for evaluating changes in blood pressure from a known baseline or for surgical monitoring of general trends rather than for exact blood pressure measurement. the doppler method of indirect blood pressure measurement is most commonly used in ferrets. shave the hair on the ventral tail and place a no. ( to cm) cuff on the most proximal part of the tail and attach it to a sphygmomanometer. place the doppler transducer probe crystal with ultrasound gel on the shaved skin approximately cm distal to the cuff and tape or hold in place. alternatively, place the cuff just above the carpus or tarsus (overlying the radial artery on the front leg or the digital branch of the tibial artery on the rear leg, alternatively) or on the distal humerus. evaluating a bone marrow sample is a valuable diagnostic tool for many disease conditions, including anemia, thrombocytopenia, pancytopenia, proliferative abnormalities, and suspected hematopoietic malignancies. although the proximal femur is usually the most readily accessible site (fig. . ) , the iliac crest, tibial crest, and humerus can also be used to collect bone marrow samples. anesthesia is necessary to aspirate the bone marrow or perform a core biopsy. with the ferret in lateral recumbency, shave and aseptically prepare the area around the collection site. for the proximal femur, make a small incision over the greater trochanter. stabilize the femur with one hand while inserting a -gauge, . -inch spinal needle into the bone, medial to the greater trochanter. use steady pressure and an alternating rotating motion to advance the needle into the marrow cavity. withdraw the stylet and attach a -to -ml syringe to the needle to aspirate the marrow, stopping suction as soon as the sample is visible (to prevent blood contamination). to collect a core biopsy sample, use the same technique, but use a . -inch, -gauge needle in place of the spinal needle (see also chapter ) . collect samples from alternate sites by using the same basic technique. blood transfusions may be needed in ferrets that are anemic from chronic disease, blood loss, or estrogen toxicosis, or in ferrets that are thrombocytopenic. as in other species, evaluate the need for a transfusion based on the packed cell volume or platelet count and clinical status of the ferret. consider a transfusion if the packed cell volume is % or less in a ferret that exhibits clinical signs of anemia or requires surgery, or if a ferret is thrombocytopenic and exhibits ecchymosis, petechiation, or bleeding. ferrets lack detectable blood groups, and risk of transfusion reaction is minimal, even without cross-matching. because of a greater blood volume, large male ferrets are preferred over females as blood donors. depending on the size of the donor ferret, to ml of blood can be safely collected for transfusion. collect blood into an anticoagulant such as acid-citrate-dextrose or citrate-phosphate-dextrose-adenine (cpda) at a ratio of ml of anticoagulant to to ml of donor blood. ferret blood collected into cpda at a : ratio has a shelf life of days when stored at ° c; after days, deterioration of red blood cells (rbcs) causes a decrease in blood ph, glucose, and sodium and an increase in lactate and potassium. whenever possible, collect blood from donor ferrets shortly before transfusion. always use a filter when transfusing whole blood, and use at least a -gauge catheter to prevent cell lysis during the transfusion. intraosseous blood transfusions can be given to ferrets if an intravenous catheter cannot be placed. splenic aspiration is a common diagnostic technique in ferrets with enlarged spleens (see chapter ) . the technique is simple and can be done in unanesthetized ferrets; however, if a ferret is fractious, use an injectable sedative. an ultrasound-guided aspirate is preferred, especially if a splenic mass is present. alternatively, palpate the abdomen and immobilize the enlarged spleen next to the body wall and aspirate directly. a positive aspirate appears bloody. the most common findings on cytologic examination of a splenic aspirate are extramedullary hematopoiesis and lymphoma. analysis of cerebrospinal fluid (csf) as a diagnostic tool is occasionally indicated in ferrets that present with neurologic signs. the volume of csf fluid that can be removed safely is . ml/kg. for a csf tap, place the anesthetized ferret in lateral recumbency. using a -gauge, . -cm-long hypodermic needle, puncture the cerebromedullary cistern and allow free flow of the csf fluid. in clinically normal ferrets, reference values for csf tap are mean white blood cells (wbcs), . cells/μl (range - cells/μl); mean rbcs, cells/μl (range - , cells/μl; and mean protein concentration, . mg/dl (range - mg/dl). in a study of ferrets in which a csf tap was performed, rbcs of < cells/μl were obtained in % of cases, and the number of wbcs was significantly affected by blood contamination, but protein concentration was not. identification of genotypes of cryptosporidium parvum isolates from ferrets in japan molecular characterization of giardia duodenalis isolates from domestic ferrets antibody titers in domestic ferret jills and their kits to canine distemper virus vaccine parasites of domesticated pet ferrets susceptibility of carnivore to rabies virus administered orally percutaneous placement of a gastric feeding tube in the ferret coagulation values in normal ferrets (mustela putorius furo) using selected methods and reagents experimental rabies in the ferret (mustela [putorius] furo): susceptibility-symptoms-excretion of the virus simple technique for bleeding ferrets (mustela putorius furo) medical and surgical management of esophageal foreign body in a ferret acute ibuprofen toxicosis in a ferret meloxicam pharmacokinetics using nonlinear mixed-effects modeling in ferrets after single subcutaneous administration impact of diet on the dentition of the domesticated ferret compendium of animal rabies prevention and control. national association of state public health veterinarians acetaminophen udp-glucuronosyltransferase in ferrets: species and gender differences, and sequence analysis of ferret ugt a rabbit and ferret hemostasis first survey of endoparasites in pet ferrets in italy epidural anesthesia and analgesia in ferrets urine specific gravity values in clinically healthy young pet ferrets (mustelo furo) estimation of glomerular filtration rate and evaluation of renal function in ferrets (mustela putorius furo) esophagotomy feeding tube placement in the ferret normal clinical and biologic parameters incidence of adverse events in ferrets vaccinated with distemper or rabies vaccine: cases recovery from and clearance of rabies virus in a domestic ferret epidural analgesia in ferrets measurement of dietary and dentrifice effects upon calculus accumulation rates in the domestic ferret reference ranges for laboratory parameters in ferrets diagnosis and treatment of dental disease in ferrets viral diseases of ferrets anesthesia and analgesia in ferrets principles of transfusion medicine in small animals comparison of sevoflurane and isoflurane in domestic ferrets (mustela putorius furo) haematological and serum chemistry profiles of ferrets (mustela putorius furo) intraosseous catheterization of exotic animals a new type of urinary catheter for catheterization of the male ferret critical care monitoring comparative hemostasis in vertebrates reduction of calculus accumulation in domestic ferrets with two dentifrices containing pyrophosphate lack of detectable blood groups in domestic ferrets: implications for transfusion a technique for catheterization of the urinary bladder in the ferret effect of isoflurane on hematologic variables in ferrets use of behavior analysis to recognize pain in small mammals vaccine-associated adverse events laboratory management of the ferret for biomedical research incidence of and risk factors for adverse events associated with distemper and rabies vaccine administration in ferrets histology and immunochemistry of seven ferret vaccination-site fibrosarcomas vaccine injection-site sarcoma in a ferret pathogenesis of experimentally induced rabies in domestic ferrets viral excretion in domestic ferrets (mustela putorius furo) inoculated with a raccoon rabies isolate temporary tube cystostomy as a treatment for urinary obstruction secondary to adrenal disease in four ferrets evaluation of noninvasive monitoring techniques in domestic ferrets (mustela putorius furo) emergency and critical care of ferrets use of vascular access ports in exotic animals a technique for femoral bone marrow collection in the ferret occurrence and molecular typing of giardia isolates in pet rabbits, chinchillas, guinea pigs and ferrets collected in europe during assessment of a blood preservation protocol for use in ferrets before transfusion composition of cerebrospinal fluid in clinically normal adult ferrets effects of opioids and anesthetic drugs on body temperature in cats use of a vascular access system for administration of chemotherapeutic agents to a ferret with lymphoma cryptosporidiosis in ferrets parenteral nutrition support in rabbits and ferrets ibuprofen ingestion in ferrets: cases evaluation of an inactivated rabies vaccine in domestic ferrets update on the diagnosis and management of cryptosporidium spp infections in dogs and cats intra-arterial blood pressure in ferrets compared to peripheral blood pressure evaluation of epidural morphine for postoperative analgesia in ferrets (mustela putorius furo) hematology of the domestic ferret (mustela putorius furo) comparison of the blood coagulation profiles of ferrets and rats minimum protective dose (mpd) and efficacy determination of a recombinant canine distemper virus vaccine for ferrets the ferret, mustela putorius furo, as a new species in toxicology pain management in ferrets serum-neutralizing antibody responses to canine distemper virus vaccines in domestic ferrets (mustela putorius furo) non-invasive blood pressure measurement in sedated ferrets (mustela putorius furo): a study to find the optimal dosing regimen and reference ranges. faculty of veterinary medicine theses serologic evaluation, efficacy, and safety of a commercial modified-live canine distemper vaccine in domestic ferrets key: cord- - wg wodr authors: dolzhikova, i. v.; grousova, d. m.; zubkova, o. v.; tukhvatulin, a. i.; kovyrshina, a. v.; lubenets, n. l.; ozharovskaia, t. a.; popova, o.; esmagambetov, i. b.; shcheblyakov, d. v.; evgrafova, i. m.; nedorubov, a. a.; gordeichuk, i. v.; gulyaev, s. a.; botikov, a. g.; panina, l. v.; mishin, d. v.; loginova, s. y.; borisevich, s. v.; deryabin, p. g.; naroditsky, b. s.; logunov, d. y.; gintsburg, a. l. title: preclinical studies of immunogenity, protectivity, and safety of the combined vector vaccine for prevention of the middle east respiratory syndrome date: journal: acta naturae doi: . /actanaturae. sha: doc_id: cord_uid: wg wodr the middle east respiratory syndrome (mers) is an acute inflammatory disease of the respiratory system caused by the mers-cov coronavirus. the mortality rate for mers is about . %. due to its high mortality rate, the lack of therapeutic and prophylactic agents, and the continuing threat of the spread of mers beyond its current confines, developing a vaccine is a pressing task, because vaccination would help limit the spread of mers and reduce its death toll. we have developed a combined vector vaccine for the prevention of mers based on recombinant human adenovirus serotypes and . studies of its immunogenicity have shown that vaccination of animals (mice and primates) induces a robust humoral immune response that lasts for at least six months. studies of the cellular immune response in mice after vaccination showed the emergence of a specific cd (+) and cd (+) t cell response. a study of the vaccine protectivity conducted in a model of transgenic mice carrying the human dpp receptor gene showed that our vaccination protected % of the animals from the lethal infection caused by the mers-cov virus (mers-cov emc/ , ld( ) per mouse). studies of the safety and tolerability of the developed vaccine in rodents, rabbits, and primates showed a good safety profile and tolerance in animals; they revealed no contraindications for clinical testing. the middle east respiratory syndrome (mers) is an acute inflammatory disease of the respiratory system that was first diagnosed in june in saudi arabia [ , ] . the disease is caused by the mers-cov coronavirus, a member of the genus betacoronavirus of the family coronaviridae. one-humped camels are the natural reservoir of the virus; human infection occurs through contact with camels and consumption of unpasteurized camel milk; an aerosol transmission of infection is also possible [ , ] . according to the who, a total of , laboratory-confirmed cases of mers had been registered by september , , of which resulted in a fatal outcome (a . % mortality rate) [ ] . most mers cases were registered in saudi arabia [ ] . however, the disease was also detected in other countries (the united arab emirates, south korea, yemen, etc.); cases of imported infection were reported in europe, north africa, and north america [ ] . because of the lack of effective preventive and therapeutic drugs for mers, the high mortality rate of the disease, and the widespread character of the infection reservoir, who experts classify mers-cov as a virus with the potential to cause a pandemic. there have been no cases of mers in russia. however, due to the high mortality of mers and the continuing threat that it could spread outside the endemic areas [ ] , development of a vaccine is an urgency. vaccination can limit the spread of mers and reduce its mortality [ ] . to date, several candidate vaccine preparations based on a protective antigen, mers-cov s glycoprotein and its derivatives (s subunit, receptor-binding domain), are known: vector vaccines (based on recombinant adenoviruses and vaccinia virus), a dna vaccine based on plasmid dna, as well as vaccines based on recombinant proteins and virus-like particles [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . since the formation of a humoral and cellular immune response is important to protect against mers-cov, the use of recombinant viral vectors for antigen delivery seems promising for the development of anti-mers vaccines. these vectors provide long-term expression of the antigen in the cells of the immunized organism, which results in a protective immune response as early as after the first or second immunization. repeated vaccination is effective in inducing the most pronounced and lasting immune response, while heterologous vaccination involving the use of different viral vectors for primary and secondary immunization is the most optimal regimen. this regimen was successfully implemented in the development of a vaccine against the disease caused by the ebola virus; the vaccine has been registered in the russian federation for medical use and already undergone post-registration clinical trials in the african republic of guinea [ ] . we have developed a combined vector vaccine for the prevention of mers based on recombinant human adenovirus serotypes and expressing mers-cov glycoprotein (mers-cov emc/ isolate). here, we present the results of a study of the post-vaccination humoral and cellular immune responses in mice and primates, as well as the results of preclinical studies of the safety of the developed vaccine against mers. both components are lyophilisates for the preparation of solutions for intramuscular administration. the drug was obtained in compliance with the conditions of biotechnological production at the medgamal branch of the gamaleya national research center for epidemiology and microbiology of the ministry of health of the russian federation. all experiments on animals were carried out in strict accordance with the recommendations of the national standard of the russian federation (gost r - , "principles of good laboratory practice"). sixweek-old female c bl/ mice ( - g) were purchased from the pushchino breeding facility (russia). transgenic f hybrid mice were obtained by crossing transgenic homozygous +/+ males carrying the human dpp receptor gene (hdpp ) (medical university of texas, usa) and non-transgenic c bl/ females (pushchino, russia). expression of the transgene in f hybrid mice was confirmed by immunoblotting. all mice had free access to water and food and were housed in an isocage animal housing system (tecniplast, italy). common marmosets (callithrix jacchus) were born and kept in a specialized animal facility at the chumakov federal scientific center for research and development of immune-and-biological products ras (moscow, russia). the animals were kept at the laboratory for modeling immunobiological processes with the experimental clinic of callitrichidae (chumakov federal scientific center for research and development of immune-and-biological products ras) in accordance with the requirements for housing laboratory primates. all experimental procedures with marmosets were carried out by a specialist who had received certification from the federation of european laboratory animal science associations (felasa) and completed a course on working with primates ("laboratory animal science for researchers: non-human primates," karolinska institute, stockholm, sweden). all animals were identified by a radio chip implanted subcutaneously and having a unique -digit code (globalvet, moscow). the mice were immunized intramuscularly using the widest possible dose range, × to v.p. per mouse. immunization was carried out twice successively with component and then component with a -day interval. mouse serum samples were collected at the following time points: and days, three and six months after immunization. the marmosets were immunized intramuscularly at a dose of v.p. per animal. immunization was conducted twice successively with component and then component with a -day interval. plasma samples were collected at the following time points: before immunization, seven and days, as well as three and six months, after immunization. the titer of glycoprotein-specific antibodies in serum/ plasma was determined by enzyme immunoassay. the following recombinant proteins were used: s glycoprotein ( -v b; sino biological, china) and rbd ( -v b ; sino biological). a pbs solution in . % tween- (pbs-t) containing % non-fat dry milk (a ; applichem, spain) was used for blocking. serum/plasma was titrated in two steps in a pbs-t solution containing % non-fat dry milk. anti-mouse igg horseradish peroxidase-linked secondary antibodies (nxa ; ge healthcare, usa) were used to detect mouse igg. serum of a rabbit immunized with marmoset igg and anti-rabbit igg horseradish peroxidase-linked secondary antibodies (na v; ge healthcare, usa) were used to detect marmoset igg. a tetramethylbenzidine solution (niiopik, russia) was used as a chromogenic agent. the reaction was stopped by adding m h so ; optical density was measured at nm (od ) using a multiskan fc microplate reader (thermo fisher scientific, usa). the igg titer was defined as the maximum serum dilution at which the od value of the serum sample from the immunized animal exceeded that of the control serum/plasma (serum/plasma of the control animal or animal before immunization) more than twofold. the titer of virus-neutralizing antibodies (vnas) in the plasma of immunized animals was determined in a neutralization reaction (nr) by suppressing the cytopathic effect caused by the mers-cov virus (mers-cov emc/ ) in the monolayer of vero b cells. the neutralization reaction was carried out in the "constant viral dose/serum dilution" mode. monkey plasma was incubated at °c for min to remove non-specific inhibitors. all serum samples were diluted in a dmem medium supplemented with % inactivated fetal bovine serum, starting from the : ratio, then with two-fold dilution to : , . dilutions of the mers-cov virus suspension were prepared in a dmem medium supplemented with % inactivated fetal bovine serum. the concentration of the mers-cov virus in the prepared dilution was , tcid /ml. a mixture of equal volumes of plasma and the virus suspension was incubated at °c for min. vero b cells were plated in -well plates at × cells per well at a volume of µl and then supplemented with µl of the mixture of plasma and the virus suspension. the cytopathic effect was assessed after four days. the vna titer of the studied plasma was defined as its highest dilution at which the cytopathic effect was suppressed in two out of three wells (compared to the control serum samples). the mice were euthanized on day eight after immunization. the spleens were collected and homogenized through a -µm sieve in sterile pbs. splenocytes were isolated by ficoll ( . g/ml; paneco, russia) density gradient centrifugation ( g, min). for t cell proliferation assay, the splenocytes were stained with carboxyfluorescein using the carboxyfluorescein succinimidyl ester (cfse) tracer kit (invitrogen, usa) as previously described [ ] . cells were seeded in -well plates at × cells per well in a rpmi medium and re-stimulated with the recombinant mers-cov s protein ( -v b; sino biological) at µg per well. after h, the media were collected for a ifn-gamma analysis and the cells were harvested, washed with pbs, stained with antibodies specific to cd , cd , and cd : allophycocyanin (apc)-labelled anti-cd , apc-cy -labelled anti-cd , and phycoerythrin-labelled anti-cd (bd biosciences, usa), and then fixed in % paraformaldehyde. proliferating cd + and cd + t lymphocytes were evaluated in the cell mixture using a bd facs aria iii flow cytometer (bd biosciences). the resulting percentage of proliferating cells (x) was determined using the following formula: x = %st -%, where %st is the percentage of proliferating cells after splenocyte re-stimulation with recombinant mers-cov s glycoprotein, and % is the percentage of proliferating cells in the absence of splenocyte re-stimulation (intact cells). the concentration of ifn-gamma in the medium was measured by elisa using a commercial kit (mouse ifn-γ elisa kit; invitrogen) according to the manufacturer's protocol. the increase in the concentration of ifn-gamma was determined using the following formula: x = cst/cint, where x is the fold increase in the concentration of ifn-gamma, cst is the concentration of ifn-gamma in the medium of stimulated cells (pg/ ml), and cint is the concentration of ifn-gamma in the medium of unstimulated (intact) cells (pg/ml). the protective efficacy of the vaccine was studied in a model of lethal infection in transgenic mice carrying the human dpp receptor gene and obtained by crossing homozygous transgenic hdpp +/+ males and non-transgenic c bl/ females. the animals were immunized intramuscularly twice successively with component and then component with a -day interval. seven days after the injection of component , the mice were infected intranasally with the mers-cov virus (mers-cov emc/ ) at a dose of ld per animal, and then the survival rate was analyzed for a period of days. preclinical studies of the safety of the combined vector vaccine against mers were conducted in collaboration with the autonomous non-commercial organization "institute of biomedical research and technology" and fsaei he i.m. sechenov first moscow state medical university, in compliance with the guidelines for preclinical trials of medicinal products [ ] and guidelines for experimental (preclinical) study of new pharmacological substances [ ] . the safety study included the analysis of the toxicity of a single and repeated administration, as well as sn assessment of the reproductive and ontogenetic toxicity, immunogenicity, and allergenicity. a total of mice, rats, rabbits, guinea pigs, and six common marmosets were used in the preclinical safety study. tolerability of the vaccine in primates was analyzed daily by assessing the physical condition of the animals and based on the presence of general symptoms of in-toxication, which included an assessment of behavior, appearance, and physiological functions. vaccine tolerance in marmosets was studied in the laboratory by monitoring the body weight, rectal temperature, and blood biochemical parameters: total bilirubin, direct bilirubin, aspartate aminotransferase (ast), alanine aminotransferase (alt), creatinine, total protein, and alkaline phosphatase (alp). the studies were carried out on fully automatic analyzers ca- and b- (furuno, japan) using diasys reagent kits (germany). statistical analysis of the data was performed using the graphpad . software. either the student's t-test for independent samples or the mann-whitney u-test was used for the analysis of the data of unpaired samples depending on the data distribution normality [ ] . either the student's t-test for paired samples or wilcoxon's test was used for the analysis of the data of related samples depending on the data distribution normality [ ] . distribution normality was determined using the generalized d'agostino-pearson test [ ] . in order to select an effective dose, mice were immunized intramuscularly with the vaccine at doses of - , × v.p. per mouse; serum samples were collected, and the titers of glycoprotein-specific antibodies were analyzed two and four weeks after immunization. next, the intensity of the post-vaccination humoral immune response was assessed based on the titer of glycoprotein-specific igg (fig. ) . analysis of the obtained results demonstrates a dose-dependent increase in the serum titer of glycoprotein-specific igg. the minimum dose of the combined vector vaccine required to induce a robust humoral immune response was v.p. per mouse for all vaccinated animals. analysis of the duration of post-vaccination humoral immunity showed that glycoprotein-specific antibodies were detected at a high titer in the mouse serum six months after immunization (the geometric mean titer was : , , fig. ) . next, we studied the level of humoral immunity in primates vaccinated with the developed vaccine. in order to determine the level of humoral immunity in common marmosets (c. jacchus), they were immunized with the combined vaccine according to the regimen intended for clinical use, i.e. successively with component (at a dose of v.p. per animal) and then com-ponent (at a dose of v.p. per animal) with a -day interval. further, plasma samples were collected from animals for the analysis of the titer of glycoproteinspecific igg seven and days, as well as three and six months, after the boosting of immunization (fig. a) . immunization of primates was shown to induce robust humoral immunity, which persists for at least six months. for instance, the titers of glycoprotein-specific igg in primates six months after immunization did not differ from the titers after three months, which is an indication of the induction of long-term immunity. analysis of the titer of neutralizing antibodies to the mers-cov virus in the plasma of immunized monkeys showed that vnas were detected in the animals as early as seven days after booster immunization, while virus-neutralizing antibody titers are shown on the ordinate axis; time after immunization is represented on the abscissa axis. individual titers for each studied animal and the geometric mean titer (n = ) are indicated the maximum vna titer was reached three and six months after immunization (fig. b) . no vnas were detected in the plasma of the control animals and the animals before immunization. thus, our analysis of the level of post-vaccination immunity showed that immunization of mice and primates induces a robust humoral immune response, which persists for at least six months after immunization. in order to assess the level of post-vaccination cellular immunity, the mice were immunized with the candidate vaccine against mers once at a dose of v.p. per mouse. spleens were collected from the animals days after immunization; splenocytes were isolated, and the number of proliferating cd + and cd + t lymphocytes was determined in the splenocyte culture in vitro after cell re-stimulation with the recombinant mers-cov s protein (fig. ) . the obtained data demonstrate that introduction of the combined vector vaccine induces the formation of s-specific cd + and cd + t cells. activation of cellular immunity was also analyzed by measuring the expression of ifn-gamma. the results of the study of an increase in the ifn-gamma concentration in an in vitro culture of mouse splenocytes after repeated stimulation of the cells with the recombinant mers-cov s protein are presented in fig. . administration of the vaccine increased the concentration of ifn-gamma in the medium upon stimulation of the splenocytes of immunized mice with the mers-cov s glycoprotein. the concentration of ifn-gamma in the medium increased by an average of times. summarizing the data of our analysis of the antigenspecific lymphoproliferative activity of cd + and cd + t cells and the level of ifn-gamma expression by restimulated splenocytes, we can conclude that immunization of animals with the combined vaccine against mers results in the formation of glycoprotein-specific cellular immunity. the study was carried out in a model of lethal infection caused by mers-cov in transgenic mice carrying the human dpp receptor gene. mice were immunized successively with component and then component with a -day interval. one week after administration of component of the vaccine, animals were infected intranasally with the mers-cov virus (mers-cov emc/ ) at a dose of ld per animal, and the survival rate was assessed during days. immunization of the animals with the combined vector vaccine was shown to protect % of animals from lethal infection caused by the mers-cov virus. all control (unvaccinated) animals died (fig. ) . irritation effect, immunotoxicity, allergenic properties, and reproductive toxicity) were evaluated in rodents (mice, rats, and guinea pigs) and large animals (rabbits). the combined vector vaccine against mers did not cause any toxic effects, did not have an allergenic or immunotoxic effect, did not affect the generative function, did not have a local irritation effect, and can be recommended for clinical studies. vaccine tolerability was also studied in primates. no abnormalities in the analyzed parameters of physical condition (behavioral reactions, appearance, and physiological functions) were found in the animals immunized with the combined vector vaccine against mers and the control animals during the observation period. rectal temperature, changes in body weight, and biochemical parameters were within the normal range for the species in all animals during the experiment (fig. ) . summarizing the obtained data, we can conclude that the combined vector vaccine against mers has shown good tolerability in the common marmoset model. currently, there are no specific prophylactic and therapeutic agents against the middle east respiratory syndrome in the world. intensive studies on the development of vaccines for this disease are currently underway in the united states, germany, korea, china, great britain, and other countries. among the prophy-lactic drugs with the highest efficiency demonstrated in preclinical studies, the following candidate vaccines can be mentioned: vaccines based on adenoviral vectors (ad , ad , chadox ) [ , ] , modified vaccinia virus ankara (mva) [ ] encoding mers-cov protective antigen s, as well as preparations of recombinant mers-cov protective antigen s [ , ] . two drugs are currently undergoing clinical trials: two vaccines based on recombinant viral vectors mers (based on chimpanzee adenovirus, phase ) [ ] and mva-mers-s (based on vaccinia virus, phase ) [ ] . clinical studies of the first phase of a vaccine based on plasmid dna (gls- ), as well as a vaccine based on a vaccinia virus (mva-mers-s), have been completed [ , ] . all vaccines that have reached clinical trials are based on mers-cov s glycoprotein. this glycoprotein performs one of the most important roles in the viral life cycle: it enables virus internalization via interaction with the dpp receptor on the cell surface. neutralization of this interaction limits penetration of the virus into the cell, thus decreasing its replication. since the formation of not only a humoral, but also cellular immune response is important for protection against mers, the development of vaccines based on recombinant viral vectors seems promising. such vectors effectively deliver antigen-encoding genetic material to the cells, which results in the cellular expression of the antigen and induction of a robust cellular and humoral immunity. an important property of recombinant viral vectors is that they induce protective immunity as early as after the first or second immunization, which is extremely important when developing a vaccine for the prevention of dangerous and extremely dangerous infections and is intended for use during an epidemic or in the case of an infection that spreads beyond non-endemic areas. we have conducted a study of the immunogenicity of various forms of mers-cov s glycoprotein: fulllength glycoprotein (s), full-length glycoprotein with the transmembrane domain of the g protein (s-g) of the vesicular stomatitis virus, a secreted glycoprotein receptor-binding domain (rbd), a secreted glycoprotein rbd fused to the fc fragment of human igg (rbd-fc), and the membrane form of the glycoprotein rbd (rbd-g) [ ] . the obtained data demonstrated that the membrane form of the rbd is the most effective in inducing a robust cellular immune response, while full-length glycoprotein is most efficient in inducing a robust cellular immunity. when choosing an immunization regimen, one should take into account the fact that repeated heterologous vaccination, which involves the use of two different recombinant viral vectors for primary and secondary immunization, is advisable for inducing long-term immunity. for this studies of the immunogenicity of the combined vector vaccine revealed the induction of long-term humoral immunity in mice, while the mean titer of glycoprotein-specific antibodies equaled : , two weeks after vaccination at a dose of v.p. per mouse. a similar antibody titer was observed by alharbi et al. in mice days after immunization with a vaccine against mers based on chimpanzee adenovirus chadox mers [ ] ; however, the authors used a dose of v.p. per mouse for immunization. in another study by munster et al. [ ] , immunization of transgenic mice carrying the human dpp receptor gene with a chadox mers vaccine at a dose v.p. per mouse was shown to protect % of the animals from a lethal infection with mers-cov. hashem et al. developed a rad -based drug carrying the mers-cov s sequence and demonstrated that repeated immunization of mice with the drug at a dose of v.p. per mouse induced a humoral immune response [ ] . the titer of glycoprotein-specific igg was : , three weeks after the second immunization (one and a half months from the beginning of immunization); the drug also provided % protection to animals from mers-cov infection [ ] . glycoprotein-specific antibodies were found at a titer range of : , to : , in the plasma of the animals as early as a week after the boosting of immunization. it is important to note that muthumani et al. [ ] detected glycoprotein-specific antibodies at a titer of : , for a period of six weeks in primates after long-term thrice immunization with a dna vaccine; the authors also showed that immunization of primates with the dna vaccine protects them from a mers-cov infection. the study of post-vaccination humoral immunity in mice and primates demonstrated that an intense humoral immune response persists in the animals for at least six months after vaccination. analysis of the cellular component of the immunity in the mice showed that administration of the developed vaccine induces a robust cellular response. it is important to note that not only a cd + but also cd + t cell response is observed, which can play an important role in protection against mers-cov [ , ] having completed studies of the immunogenicity of the combined vector vaccine in a model of lethal infection in transgenic mice carrying the human dpp receptor gene, we studied the protective effect of the vaccine. the vaccine was shown to provide % protection to animals from lethal infections of mers-cov. our series of preclinical studies of vaccine safety revealed no contraindications for the clinical testing of the developed vaccine. in this work, we have studied the immunogenicity and safety of a combined vector vaccine for the prevention of the middle east respiratory syndrome. the following conclusions were obtained: vaccination of animals with the vaccine induces a robust humoral immune response to the mers-cov s glycoprotein persisting for at least six months. vaccination of animals induces a robust cellular immune response to the mers-cov s glycoprotein. vaccination of animals induces a protective immune response, which protects % of animals from a lethal infection of mers-cov. preclinical studies of the vaccine safety did not reveal any contraindications to clinical testing. middle east respiratory syndrome coronavirus (mers-cov) annual review of diseases prioritized under the research and development blueprint) international multicenter study of the immunogenicity of medicinal product gamevac-combi. clinicaltrials.gov identifier: nct guidelines for preclinical trials of medicinal products. // moscow: grif i k guidelines for experimental (preclinical) study of new pharmacological substances // moscow: medicine // ekologiya cheloveka with the english safety and immunogenicity of a candidate mers-cov vaccine (mers ) phase ib study to assess the safety and immunogenicity of mva-mers-s_df- . clinicaltrials.gov identifier: nct url phase i, open label dose ranging safety study of gls- in healthy volunteers clinicaltrials.gov identifier: nct url proc. natl. acad. sci. usa. . v. . № key: cord- -eyvaazfj authors: rao, ghanta n.; huff, james title: refinement of long-term toxicity and carcinogenesis studies() date: - - journal: fundam appl toxicol doi: . / - ( ) -l sha: doc_id: cord_uid: eyvaazfj the chance that alternatives will completely replace animals for toxicology research in the foreseeable future is nil. continual refinement of animal toxicity and carcinogenesis studies, however, can be an effective means of reducing the numbers of animals used and conserving time and resources without compromising scientific quality. we must continue to strive to find species and strains that can metabolize chemicals similar to humans, are small enough to be housed in large numbers, and have low prevalence of spontaneous lesions with sufficient life span to express the toxic and carcinogenic potential of chemicals. adequate care of animals with control of variables such as light, temperature, diet, bedding, diseases, and genetic characters of laboratory animals will decrease the variability. humane considerations and euthanasia of animals with large masses and other conditions interfering with eating and drinking, major injuries and ulcers related to husbandry and treatment, and diseases indicating pain and suffering will help not only to alleviate further pain and distress but also to facilitate collection of tissues without secondary complications for detection of chemical treatment-related lesions. limiting the duration of studies to decrease the variability due to ageassociated changes will also refine long-term studies. other considerations for refinement of carcinogenesis studies include selection of the most sensitive sex of one or more species for evaluation of selected chemicals in a class where toxic and carcinogenic potential of other representative chemicals are known. genetically engineered animal models with known oncogenes may reduce the duration and increase the sensitivity of carcinogenesis studies with a reduction in the use of animals. of long-term toxicity and carcinogenesis studies. rao, g. n., and huff, j. ( ) . fundam. appl. toxicol. , - . the chance that alternatives will completely replace animals for toxicology research in the foreseeable future is nil. continual refinement of animal toxicity and carcinogenesis studies, however, can be an effective means of reducing the numbers of animals used and conserving time and resources without compromising scientific quality. we must continue to strive to find species and strains that can metabolize chemicals similar to humans, are small enough to be housed in large numbers, and have low prevalence of spontaneous lesions with sufficient life span to express the toxic and carcinogenic potential of chemicals. adequate care of animals with control of variables such as light. temperature. diet, bedding, diseases, and genetic characters of laboratory animals will decrease the variability. humane considerations and euthanasia of animals with large masses and other conditions interfering with eating and drinking, major injuries and ulcers related to husbandry and treatment, and diseases indicating pain and suffering will help not only to alleviate further pain and distress but also to facilitate collection of tissues without secondary complications for detection of chemical treatment-related lesions. limiting the duration of studies to decrease the variability due to ageassociated changes will also refine long-term studies. other considerations for refinement of carcinogenesis studies include selection of the most sensitive sex of one or more species for evaluation of selected chemicals in a class where toxic and carcinogenic potential of other representative chemicals are known. genetically engineered animal models with known oncogenes may reduce the duration and increase the sensitivity of carcinogenesis studies with a reduction in the use of animals. society oftoxicology. the search for alternatives to the use of animals in research and testing remains a valid goal of researchers. but the chance that alternatives will completely replace animals in the foreseeable future is nil. (national research council, ) . we must use alternatives to whole animals such as bacterial systems and cell culture methods only when they are reliable predictors of toxicity and carcinogencity. however, we must evaluate potential alternative tests objectively, free of subjective attachment. in the field of toxicology alone there are hundreds (goldberg, ; tennant et al., ; mehlman, ) of in vitro assays and other alternative methods for evaluation of toxic and carcinogenic potential of drugs and chemicals. many of these alternative tests with cell systems and subcellular components will aid in understanding effects and mechanisms at cellular and subcellular levels. but many of these tests do not predict the re-studies on these chemicals were considered to sponse of the whole animal (tennant et al., be incomplete or inadequate. continual re- ) . there are a number of reasons for this finement and optimization of animal toxicity difference. in a living animal during the ex-and carcinogenesis studies can be an effective pression of physiologic, pharmacologic, and means of reducing the numbers of animals toxic responses to an agent, in addition to used and of conserving time and resources subcellular component interactions, there are without compromising scientific quality. esother interactions. they include (a) cell to cell sential components for refinement of longcommunication between cells of the same or term studies include selection of proper spedifferent types in organs such as liver, and (b) cies and strains, proper care of the animals, organ to organ communication, interaction, humane considerations during the course of and compensation between organs of a living a study, and adequate duration of the studies. animal such as liver and kidney, thymus and each of these essential components is dislymphoid tissue, pituitary and other endo-cussed below. crine organs, which we cannot predict by alternative assays. in addition the living animal as a whole by its complex feedback mecha-selection of proper species, nisms between organs and tissues adjusts to strains, and number varying chemical and environmental insults, of animals compensates, and survives or overreacts and dies, which we cannot predict by any single we should continue to strive to find species or battery of in vitro and alternative tests. and strains of experimental animals (a) that notwithstanding these drawbacks, we must metabolize or dispose a chemical in a manner continue to strive for reliable in vitro or alter-similar to humans; (b) with (median) life native methods to the use of whole animals. it spans long enough to adequately assess the is the most humane, ethical, and economical carcinogenic potential of chemicals (mouse direction for us to follow. but diseases like strains such as a and akr with relatively cancer and acquired immunodeficiency syn-short life spans may be useful for assessing the drome (aids) humble us with regard to limi-carcinogenic potential of potent carcinogens tations in our understanding of complex bio-but will not provide adequate information on logic systems such as mammals. alternatives weaker carcinogens); (c) with low spontaneare useful for mechanistic research and a lim-ous lesions and tumors, especially in the tarited number of alternative tests currently get organs of the chemical, to enhance the have some utility for assessing the toxic and specificity and sensitivity and to facilitate incarcinogenic potential of selected classes of terpretation; and (d) small enough that they chemicals (e.g., polycyclic aromatic hydro-can be housed conveniently in large numcarbons). however, the probability that alter-bers, thus allowing the use of a greater numnatives will completely replace animals for ber of animals to increase the sensitivity (statoxicology research in the foreseeable future tistical power) for detecting toxic responses, is nil. especially carcinogenic effects. the number if a study must be done using animals, then of animals per group should be adequate to we are obligated to do it right thejirst time. in assess the toxic and carcinogenic potential. the long run this will save money and time, however, increasing the number of animals will decrease the number of animals used, beyond to per group does not result in and is one of the best refinements we can do. a proportional increase in sensitivity. as in recent years a number of chemicals were shown in table , for animals having a backselected by the national toxicology program ground tumor rate of % if the number per (ntp) for reevaluation, because the previous group is increased by animals from to , there will be more than a doubling in sensitivity (after compensating for background rate); whereas the same increase of animals from to loo/group will result in an increase of sensitivity by only a third. approximately animals per group (portier and hoel, : ntp, ) achieves a reasonable balance between the cost, animal use, and study sensitivity. much of the variation between animals and between studies is a product of genetic and environmental factors. contribution of genetic variability may be substantial when outbred stock (e.g., sprague-dawley rats, icr-swiss mice) are used. however, with genetically defined inbred and hybrid strains, most of the variability is due to environmental factors. the environmental variables could be physical or biological factors and some of these are listed in table . the effects of these factors were discussed in detail elsewhere (rao, : haseman et al., , and the references cited therein). some recent findings on the effects of light, diet, and viral infections are discussed below. light. the duration, intensity, and quality of light will influence many physiologic responses and reactions. light may cause eye lesions and influence tumor incidences (greenman et al., ; wiskemann et al., ) . high light intensity may cause eye lesions in albino rodents, (bellhorn, ; greenman et al., ) . in some ntp studies, for example, fischer rats housed in the top row and side columns of racks where they were exposed to more light than the rats in other cages have been observed to develop opacity of the eyes and this appears to be due to inflammation of various structures of the eye. this lesion later progressed to generalized inflammation of the eye with retinal degeneration and cataract formation. prevalence of eye lesions by cage position and light intensity in male f rats is illustrated by an example shown in table . if the chemical under investigation has the potential to cause eye lesions, then the light intensity related eye lesions may complicate the interpretation of chemical effects. as shown in table (ntp, ) high doses of the chemical appear to have increased the incidence of eye lesions in male f rats. however, the contribution of light intensity for development of eye lesions in the treated groups is not known. the prevalence of light intensity-associated eye lesions in rodents could be markedly decreased by (a) reducing the light intensity in the cages to < foot-candles or the animal room light intensity to ~ foot-candles at ft from the rao, . floor and (b) rotating the cages of each column of a rack from top to bottom when cages or racks are changed (ntp, unpublished data). diet. a casein-based purified diet (newberne et al., ) considered to be adequate for a multigeneration reproduction study in rats was found to be deficient in manganese, warranting invalidation of a -year in utero exposure chemical carcinogenesis study (rao, a) . optimal diets for rodents in long-term toxicity and carcinogenesis studies should be nutritionally adequate for growth and maintenance without excesses of high energy and growth enhancing nutrients. there should be established formulation with standards for ingredients, nutrient concentra-tions, and contaminant limits. contaminant concentrations should be as low as practical. each batch/lot of diet should be analyzed for macronutrients and selected micronutrients with complete micronutrient analyses on selected batches/lots (rao, a) . viral infections. sendai virus (sv), pneumonia virus of mice (pvm). and mouse hepatitis virus (mhv) are the most common viral infections of mice and sv, pvm, and rat corona virus/sialodacryoadenitis virus (rcv/sdav) infections are most common in rats (boorman et al., ) . viral infections may complicate toxicology research (collins, ) . in a systematic comparison of control and chemically treated groups of b c fl mice, viral infections did not cause consistent adverse effects on survival and tumor prevalences. however, sv infection in mice was associated with significantly (r, < . ) higher survival and survival-associated increase in liver tumors (rao et al.. a ). viral infections did not cause consistent adverse effects on survival or tumor prevalences in control groups of f rats (rao et al., b) . however, viral infections were associated with nonneoplastic lesions in lungs, nasal cavity, liver, and other organs of rats and mice and may complicate the identification and interpretation of toxic effects of chemicals (ntp, unpublished data). animals used in toxicology or carcinogenesis studies should be monitored closely by experienced professional scientists, especially if the chemical is toxic or if there are increases in tumor-bearing animals. there should be specific criteria supplemented with professional judgment for euthanasia of moribund animals during the course of these long-term studies. the objective of these criteria is not only to relieve excessive pain and distress to animals but also to allow collection of tissues for pathologic assessment that are free of secondary complications. major reasons for eu- thanasia of animals during the course of a large mammary tumor may be injured during study include: (a) large masses and other conthe normal movement in the cage leading to ditions interfering with eating and drinking, ulceration and infection resulting in second-(b) major injuries and ulcers related to husary lesions in the tumor and in the animal. bandry, fighting, or chemical exposure, and major injuries such as accidental fracture of (c) diseases and conditions indicating pain a limb or wounds resulting from fighting of and suffering as judged by an experienced group-caged animals may lead to unrelieved laboratory animal specialist. for example, a pain and nonhealing lesions. for humane reasons and to prevent complications of chemical-induced lesions by secondary changes, it is prudent to euthanatize and necropsy the animal. in studies such as dermal toxicity and carcinogenesis where treatment causes persistent or enlarging ulcers and fast growing tumors which are injured by the animal or components of the cage, it is appropriate to euthanatize and necropsy the animal not only to relieve pain and distress but also to prevent infections. infections and inflammation may lead to secondary lesions which can complicate the interpretation of toxic effects and may decrease the sensitivity of a toxicology study. other considerations for euthanasia of rodents in chronic studies are listed in table ; most of these conditions will change the normal physiology and may affect morbidity and mortality. any one of these conditions may not lead to mortality in a short period, but a combination of these conditions will compromise the physiology and health of the animals. if these animals are allowed to continue in a study, secondary affects may mask or exacerbate the chemical effects, complicate the interpretation of toxic effects, and decrease the sensitivity and quality of a long-term study. duration of toxicology studies with rodents may range from to months depending on the toxicity of the chemical (frederick, ). in general a -to -month toxicity study may be long enough for many chemicals with considerable acute toxic effects and limited cumulative toxicity. however, with a high proportion of chemicals, due to the cumulative nature of their toxicity, to months duration may not be long enough and studies of to months duration may be necessary. chemicals that can cause delayed toxicity, such as neurotoxicity, may have to be evaluated in studies of to months duration. however, when con- ducting studies longer than months, one should be aware of and consider the influence of age-associated changes and complications in interpretation of the toxic responses. duration of chemical carcinogenesis studies in rodents usually covers a major portion of the life span. length of generally accepted chemical carcinogenesis studies in rats and mice include: (a) to months, (b) months or % survival, whichever occurs first, and (c) until most or all animals in the chemically exposed groups die or are euthanatized due to moribund condition. for potent or "early" carcinogens, studies longer than months may not be necessary (e.g., , -butadiene, huff et al., ) . for weaker carcinogens with delayed expression of carcinogenic potential, studies of longer than months may be necessary. the median life span ( % survival) of rodent species and strains used for chemical carcinogenesis studies is to months and, therefore, studies lasting to months will cover - % of the life span, assuming % mortality is close to the life span. in these long-term studies, especially in studies longer than to months, there are several age-associated changes that may compromise the health of the animals and complicate the expression and interpretation of carcinogenic response. tables and and figs. to illustrate some of the age-associated changes that may complicate long-term carcinogenesis studies. male f rats attain a maximum body weight at . * . (mean ? sd) weeks of age (haseman et al., ) or some time during the th to th month of a chronic study and may lose as much as % of their body weight by the th month. the decline in body weight is accompanied by an increase in water consumption or vice versa (fig. i) . the increase in water consumption (polydipsia) is due to nephrosis leading to polyuria with substantial changes in physiologic processes such as distribution, metabolism, and excretion of chemicals (grice, ) . these changes could complicate the interpretation of a toxicology study. even in a carcinogenesis study where the animals are steadily losing body weight, it may not be scientihcally prudent to continue the study after a substantial decline in body weight. female b c fl mice reach maximum body weight at . t . weeks of age (haseman et al., ) or during the th to rd month of a chronic study. a steep in-crease in mortality and persistent loss of body weight (fig. ) starts after to months of study. even though the life span ( % mortality) of female b c fl mice is about months, median survival is reached at about the th month of a chronic study and coincides with a to % loss of body weight. furthermore, the prevalence of lymphoma in control mice shows a marked increase between the st and th months (fig. ) , indicating that it may not be appropriate to con- tinue carcinogenesis studies in b c fl mice beyond months, especially when the lymphoreticular organs are the target tissues. table shows the prevalence of amyloidosis in icr-swiss (or cd-l) outbred mice (rao et al., b) . high prevalence of amyloidosis in major organs such as kidney, liver, and heart at - months of age (or - months of a chronic study) may impair the ability of these organs to metabolize and dispose the chemicals and may complicate the interpretation of neoplastic lesions. body weight and water consumption patterns of a strain of syrian hamsters (homburger et al., ) are shown in fig. marked increase in water consumption begins at - weeks of age together with a start of persistent decrease in body weight, indicating changes in physiologic processes. furthermore, there is a high prevalence of amyloidosis (newcomer et al., ) in major organs such as liver, kidney, and spleen in -to %month-old syrian hamsters as shown in table . the body weight and water consumption patterns (fig. ) and the prevalence of amyloidosis (table ) indicate that the physiology and health of these animals may have been compromised by about months of age and so carcinogenesis study of longer than months may not be appropriate with syrian hamsters. tables and and figs. to identified a few of many age-associated changes observable in the whole animal. these changes are due to several pertubations at the subcellular, cellular, and organ level. some of these changes relevant to chemical toxicology and carcinogenesis may include chemical absorption, distribution, metabolism, and excretion; protein, lipid, and carbohydrate metabolism; hepatic drug metabolism, renal excretion, and morphologic changes in liver. kidney, and other organs (grice, ) . a variety of diseases, tumors, and age-associated changes present in laboratory rodents most often increase in severity and incidence with increasing age, resulting in marked alter-ations in physiologic processes. these diseases and tumors are associated with alterations in function and morphology of many organs, making it difficult to differentiate between age-associated changes and chemically induced toxicity and lesions. changes in organ function are associated with changes in metabolism and toxic responses to chemicals. with few exceptions, the aging animal metabolizes chemicals less effectively and is more susceptible to the toxic effects of chemicals (grice, ) . studies beyond months in hamsters and months in mice and rats may result in greater variability and nonneoplastic disease-associated complications in interpretation of results. increasing the duration beyond these general limits may not be a refinement of long-term toxicity and carcinogenesis studies. however, for weak carcinogens with delayed expression of carcinogenic potential, studies of longer than months in strains with long life spans and low prevalence of spontaneous lesions and tumors may be necessary. most of the national cancer institute-national toxicology program long-term chemical carcinogenesis studies included male and female f inbred rats and male and female b c fl hybrid mice. haseman and huff ( ) reported species correlation in long-term toxicology and carcinogenesis studies. retrospective analyses of sex-species combinations for identifying potential chemical carcinogens are shown in table . combinations of male rats and male mice or male rats and female mice detected ( %) of the identified carcinogens. the male rats and female mice combination "missed" ( %) chemicals shown to be positive in one of the other sex-species, of which were positive in male mice only, in female rats only, and in female rats and male mice. the male rats and male mice combination also "missed" ( %) carcinogens, of which were positive in female mice only, in female rats only, and in female rats and female mice. prospective predictions based on these retrospective evaluations may not be appropriate for unknown chemicals. when a class of chemicals is evaluated (e.g., benzidine dyes, nitrotoluenes, glycol ethers), both sexes of rats and mice may have to be included to assess full carcinogenic potential and mechanism of carcinogenesis of a few representative chemicals of the class. however, for evaluation of other chemicals in that class, the most sensitive sex of one or more species may be adequate. this refinement may decrease the number of animals for selected chemicals and reduce costs of some long-term studies. chemical carcinogenesis is a multistep process and may involve the activation of one or more protooncogenes. genetically engineered animal models such as transgenic mice with ras, myc. net{ or other oncogenes may be useful in refinement of chemical carcinogenesis studies. the advantages of transgenie animal models could be (a) carcinogenic potential of chemicals may be detected with shorter studies, e.g., to months instead of to months: (b) sensitivity and possibly specificity may be increased with proper animal models for different types or classes of potentially carcinogenic chemicals: and (c) the number of animals per chemical may be reduced with a reduction in cost. the ntp has started studies with transgenic strains tg.sh with rus, tg.m with rrz~~, and tg.nk with neu oncogenes using known carcinogens and noncarcinogens. the purpose of these studies is to assess the usefulness and advantages of transgenic mouse models for detecting chemical carcinogens. in addition, the ntp (niehs) is developing an extensive program for development and evaluation of transgenic models to refine and optimize not only carcinogenesis studies but also genetic toxicology, possibly immunotoxicity, and reproductive and developmental toxicology studies. in summary, alternative in vitro assays or other methods will not replace live animals ( ) a haseman and huff ( ) . ' i studies were negative in male and female rats and mice. for toxicology research. if a study must be done with animals, then we are obligated to do it right the first time. this is the best refinement one can do to decrease the use of animals. essential components of refining toxicity and carcinogenesis studies include: (a) selection of animal models that can metabolize a chemical in a similar manner to humans, that are small enough to house in large numbers in a laboratory setting, that have a low prevalence of spontaneous lesions, and that have a long life span; (b) control of variables such as environmental conditions, diseases, and genetic characters of laboratory animals; (c) euthanasia of animals during the course of a study to alleviate pain and suffering, thus preventing development of secondary lesions and facilitating detection of chemical effects; and (d) limiting the duration of studies in rodents; which may decrease variability and complications due to nonneoplastic lesions and spontaneous tumors. all of these procedures will help to decrease the variability and increase the sensitivity--thus refining toxicity and carcinogenesis studies. further refinements include the use of genetically engineered animal models such as transgenie mouse strains with known oncogenes to decrease the duration and increase the sensitivity of carcinogenesis studies with a decrease in the use of animals. lighting in the animal environment. spontaneous and induced neoplasms in mice age associated (geriatric) pathology: its impact on long-term toxicity studies sources ofvariability in rodent carcinogenicity studies neoplasms observed in untreated and corn oil gavage control groups of f /n rats and b c fl mice species correlation in long-term carcinogenicity studies a new firstgeneration hybrid syrian hamster, bio fld alexander for in vivo carcinogenesis bioassay, as a third species or to replace the mouse multiple organ carcinogenicity of . -butadiene in b c fi mice after weeks of inhalation exposure report cf the ntp ad hoc punel on chemical carcinogenesis testing and evaluation. pp. l-l . u.s. department of health and human services, public health service. national toxicology program (i ). toxicology and curcinogenesis studies qf nalidisic safety evaluation of fish protein concentrate over five generations of rats optimal design of chronic animal bioassay significance ofenvironmental factors on the test system rodent diets for carcinogenesis studies mouse strains for chemical carcinogenicity studies: overview of a workshop influence of viral infections on body weight, survival, and tumor prevalences of b c fl (c bl/ n x c h/hen) mice in carcinogenicity studies. fundam. . pp/. taxicd influence of viral infections on body weight. survival, and tumor prevalence in fischer rats of long-term studies prediction of chemical carcinogenicity in rodents from in vitro genetic toxicity assays fluorescent lighting enhances chemically induced papilloma formation and increases susceptibility to tumor challenge in mice the authors thank drs. joseph haseman and lawrence boone for scientific advice and drs. herbert amyx and michael jokinen for reviewing the manuscript. key: cord- - arpo gn authors: manskikh, v. n.; averina, o. a.; nikiforova, a. i. title: spontaneous and experimentally induced pathologies in the naked mole rat (heterocephalus glaber) date: - - journal: biochemistry (mosc) doi: . /s sha: doc_id: cord_uid: arpo gn the naked mole rat (heterocephalus glaber, rüppell, ) is a unique eusocial rodent with unusually long lifespan. therefore, the study of spontaneous and experimentally induced pathologies in these animals is one of the most important tasks of gerontology. various infections, noninfectious pathologies (including age-dependent changes), and tumors have been described in the naked mole rat. the most frequent pathologies are traumas (bite wounds), purulent and septic complications of traumatic injuries, renal tubular calcinosis, chronic progressive nephropathy, hepatic hemosiderosis, testicular interstitial cell hyperplasia, calcinosis cutis, cardiomyopathy, and dysbiosis-related infectious lesions of the digestive system. however, the summarized data on pathology (including tumor incidence) and on the causes of mortality are insufficient. there are only few publications about the results of experiments where pathologies were induced in the naked mole rat. all these problems could be subjects for promising future studies without which adequate studies on mechanisms providing the long lifespan of the naked mole rat are impossible, as well as the elucidation of causes of tumor resistance of this species. infectious diseases of the naked mole rat are known the least. it is supposed that this animal is more resistant to pathogens usual for laboratory rodents [ ] , but up to now no accurate experimental data about this have been published. in this connection, it seems interesting to mention here that infecting (by scarification of the extremity skin) with recombinant herpes simplex first type virus apathogenic for mice caused % death of naked mole rats [ ] . they are also very sensitive to exper imental infection caused by leishmania donovani [ ] . biochemistry hill et al. [ ] found six cases of bilateral pneumonia in spontaneously deceased animals in a colony of naked mole rats in their laboratory. the etiology of this pneumonia remained unknown. in the same colony, five animals died with a pathomorphological picture of sep ticemia accompanied by typhlitis and typhlocolitis (sometimes of hemorrhagic type); moreover, from the lungs and blood pneumococcus cultures were isolated, although pneumococcus is not a usual causative agent of spontaneous respiratory infections in laboratory rodents [ ] . moreover, in the animals with clinical signs of the disease (which ones is not specified in the work), bacteria from the groups haemophilus and spirilla were found in the blood. nonspecific differently located purulent lesions are not rare in naked mole rats [ ] . many of them seem to be associated with infecting traumas caused by other ani mals (bite wounds) that lead to development of ulcerative dermatitis. sometimes a severe purulent inflammation was caused by infecting of the wound on implanting a chip for labeling the animal. especially severe are puru lent lesions of the muzzle leading to its total necrosis, mutilation, and impossibility of food consumption by the animal. the further spreading of the purulent inflamma tion often leads to development of phlegmon, myositis, arthritis, osteomyelitis; in some cases -to sepsis, peri tonitis, and even (on lesion of the muzzle) to purulent encephalitis and encephalomyelitis (our observations). bacteria causing these lesions have been identified as pasteurella, pseudomonas, staphylococcus [ ] . hill et al. [ ] also described a disease of the gas trointestinal tract morphologically manifesting as ulcera tive colitis that they called "dysentery". they connected the etiology of these lesions with balantidium and trichomonas found in the large intestine. however, today these protozoa are thought to be components of the intes tinal contents of healthy animals [ ] ; therefore, the etio logical role of these organisms needs to be proved (some times we observed balantidium in the intestinal wall of naked mole rats, and this was always associated with signs of pronounced tissue autolysis, but inflammatory reaction was absent). similar inflammatory epizootic like lesions were also described by other authors [ , ] . in these cases, diarrhea was observed, as well as overfilling of all parts of the intestine with gas and liquid contents, hemor rhages, and areas with mucosal ulcers (with mixed leuko cytic infiltration, rare basophilic intranuclear inclusions in enterocytes, and hyperplasia of crypts), signs of intox ication, exicosis, and death of animals. however, the eti ology of these lesions was explained differently than in the work by hill et al. [ ] : based on all the cytological, his tological, and microbiological data (including tests on rotavirus, clostridial enterotoxin, and mycotoxin), it was hypothesized that these changes could be caused by intes tinal dysbiosis, but not by a single specific causative agent [ , ] . however, the role of an agent with unknown bio logical (cultural) properties cannot be excluded [ ] . a high lethality ( . %) epizooty outburst was described of a disease accompanied by diarrhea, exicosis, and intestin al hemorrhages caused by coronavirus [ ] . males and the younger animals were more sensitive to this infection than females and old animals; the inbreeding degree also decreased the resistance to the coronavirus. hill et al. [ ] mentioned a case of stomach ulcer but did not discuss its etiology. later ulcerative lesions of the stomach were described among the other changes during dysbiosis of the gastrointestinal tract [ ] . up to now, helicobacter genus bacteria affecting many laboratory rodents have not been detected in the naked mole rats [ ] . it is difficult to determine the summarized incidence of spontaneous infectious lesions in deceased naked mole rats based on the literature data, because, even on exclud ing infections obviously associated with wounds, this incidence will vary over very broad limits: from . % [ ] to % [ ] . the problem of tumor development in the naked mole rat is widely known and most intriguing. the initial reports published by zoologists stated that tumors were absent in and even in dead bodies of the animals [ , ] . these reports led to a steady conviction about the extraordinary resistance of the naked more rat to carcino genesis and even to attempts to find the reason for such resistance [ , ] . this conviction was partially con firmed by results of the first pathological study of a signif icant series ( animals) of naked mole rats [ ] . however, it should be noted that animals that died of nat ural causes were only a part of this series ( animals), whereas animals were killed by euthanasia. the authors did not ascribe to tumors two cases of prolifera tive lesions with atypical cells but denoted them as pre neoplasias. one case presented a focal expansion of atyp ical cell structures in the kidney (a tubular papillary hyperplasia with atypia), and the other case presented limited alterations in the spleen characteristic for follicu lar lymphoma. the summarized incidence of such changes was . %. however, the limitation of this work (besides the investigation was mainly of euthanized ani mals) is the absence of precise data on the age structure of the material studied. since the risk of tumor development is clearly age dependent [ ] , this is important for com paring with data on other animal species and on colonies of naked mole rats, as well as for answering the question about the incidence of spontaneous neoplasms in this species. recently, two works were published that clearly indi cated the possibility of development of malignant tumors in naked mole rats and even presented an approximate idea about their incidence [ , ] . by now, separate cases biochemistry (moscow) vol. no. have been described of the following neoplasms: adeno carcinoma of axillary region (mammary or salivary gland), neuroendocrine carcinoma of stomach [ ] , skin hemangioma, nephroblastoma (wilms' tumor), adeno carcinoma of esophagus, hepatocellular carcinoma with lung metastases and disseminated lymphoma (lym phosarcoma) [ ] . histopathology and clinical behavior of these tumors (invasiveness, metastasizing) were similar to those of the corresponding neoplasms in the other lab oratory rodents [ ] . unfortunately, only one work [ ] reported the age characteristics of the animals stud ied, although very generalized (all animals were of age above one year) and the size of the sample ( animals) was given. according to this publication, the incidence of tumor arising in naked mole rats is . % ( / ). comparison of these data with the results of studies on tumor incidence in other animals gives the following picture. routine comparison of naked mole rats with inbred strains (balb/c or c bl/ ) and hybrid labora tory mice (among which tumors were found at necropsy in % of the animals that died of natural causes [ ] ) results in the conclusion about incomparably lower tumor incidence in the naked mole rats [ ] . the same result is obtained if we compare the data on necropsy in naked mole rats with modern data on the risk of tumors in humans during their entire lives, which is ∼ % [ ] . however, it should be taken into consideration that com parison of the modern data on the incidence of clinically detected neoplasms in the human population with the results of necropsies in animals is somewhat incorrect, because in humans most tumors are revealed during their lifetimes, and many patients die without subsequent autopsy or are cured. moreover, the latter circumstance makes possible appearance of another tumor, which sig nificantly influences the evaluation of the total oncologi cal risk [ , ] . unfortunately, modern data from autop sy are virtually absent from the literature, because patients who died from tumors are relatively seldom sub jected to autopsy. therefore, it seems reasonable to com pare the incidence of neoplasms in the naked mole rat with the data obtained on wild animals and with the results of autopsy of patients obtained when there were no approaches for the efficient treatment and vital diagnosis of malignant tumors. then the autopsy and histological examination were an obligatory procedure very similar to the algorithm used now for studies on tumor incidence in laboratory animals. according to results of such studies, the total incidence of tumor damaged animals in the adult population of wild mice (mus musculus) having lived to natural death was . % [ ] , in the population of wild rats (rattus norvegicus) it was % [ , ] , and in northern mole voles (ellobius talpinus) it was % [ ] . autopsy results of cats and dogs give similar values [ ] . as to humans, autopsy results fitting the above presented requirements give tumor incidence of % (moscow, data of all autopsies made in hospitals during ) [ ] , or % of the total mortality in different countries dur ing the period from to [ ] . thus, it appears that the total incidence of tumors arising during the whole life is quite comparable (about %) in all the mentioned mammals. it is likely that in this case the situ ation is the same as with guinea pig (cavia porcellus), which for a long time was thought resistant to carcino genesis [ , , ] , although analysis of the literature revealed the absence of valid studies favoring this view point [ ] . according to the literature, the comparable inci dence of tumors in naked mole rats and in mice does not mean that the naked mole rat has no relative resistance to carcinogenesis. however, this means that its resistance is manifested by the longer latent period of tumor appear ance or by the prolonged life period free of tumors that is indirectly confirmed by the different lifespans of these animals. a comparative study on this problem can be per formed only in models of tumors induced by chemical or physical factors, which has not yet been done on the naked mole rat. meanwhile, it is a priori unlikely to expect that the antitumor resistance of cells and tissues of the naked mole rat will be higher than such resistance of human cells and tissues, since the human lifespan (and hence the duration of the tumor free life period) is more than twofold longer and the body weight and total cellu larity (stochastically determining the risk of the mutant clone appearance [ , ] ) are three orders higher than that of the naked mole rat. attempts to experimentally induce tumors in naked mole rat are extremely important. unfortunately, the results published by now were obtained in very strange and unusual models, whereas classical methods of inducing tumors by chemical carcinogens or by ionizing radiation have not been reproduced on naked mole rats. nevertheless, in some works the results obtained in such ersatz models are taken as reliable data. this may be exemplified by a publication [ ] where data are discussed of a work [ ] about the tumorigenic potential of induced pluripotent stem cells of the naked mole rat and mecha nisms of their resistance to tumorigenesis. however, in the original work [ ] , the authors studied the ability of the naked mole rat's stem cells transplanted to nude mice to form teratomas. having in mind that teratomas are benign lesions with a very specific position in oncology [ , ] , it is obvious that these results have only an indirect con nection with studies on carcinogenesis in the naked mole rat. another work declared as being the first study on the resistance of the naked mole rat's cells to experimental carcinogenesis [ ] really contained only data that the naked mole rat's fibroblasts transfected with three genesvirus sv tag antigen, oncogene ras, and the gene of one of htert telomerase subunits -could grow after subcutaneous transplantation into immunodeficient mice. the key event needed by the naked mole rat's cells to acquire this ability was the transfection with the telom erase catalytic subunit gene that was not necessary for the cells of rats and mice. from the viewpoint of experimen tal oncology, this work is only a variant of the experiments by seluanov et al. [ ] on induction of the naked mole rat's immortalized cells, different only by the cultivation of these cells in immunodeficient mice instead of nutrient medium. the need to introduce the gene of the human catalytic telomerase into the naked mole rat's cells seems paradoxic, because in both mice and naked mole rats (as discriminated from humans) this enzyme retains high activity in somatic cells [ , ] . in general, we think that none these works allow us even to speak about attempts of experimental reproduction in naked mole rats of real malignant tumors as has been for long the routine practice for many other laboratory rodents [ ] . even in the absence of reliable data on spontaneous neoplasms and of attempts to reproduce real experimen tal malignant tumors, many investigations have been done, and some hypotheses have been proposed about possible mechanisms of the naked mole rat's resistance to carcinogenesis. these works were summarized recently in a review [ ] . it should be noted that any specific feature found in the naked mole rat in comparison with mouse, if it could have any (even a hypothetical) relation to tumori genesis, was automatically ascribed to mechanisms responsible for this animal's resistance to tumor growth. such mechanisms are suspected to exist in genome fea tures, level of general metabolism, redox and protein homeostasis, cell resistance to stress, telomerase activity, and early contact inhibition. it is necessary to note that data about the unusual expression of contact inhibition [ ] and the role in it of high molecular weight hyaluro nan [ ] seem doubtful and in any case not universal. such mechanism is unreal for so frequent tumors of rodents as lymphomas and leukemias, because their cells, unlike fibroblasts and epitheliocytes, can proliferate in the suspension state, and in this case contact inhibition cannot act as a regulatory mechanism preventing car cinogenesis. the list of nontumorous lesions in the naked mole rats is rather large. according to the work of delaney et al. [ ] , in the naked mole rats studied ( male, females; deceased spontaneously, euthanized) the following lesions were found: renal tubular calcinosis ( % in males, % in females), wounds inflicted on ani mals by each other ( % in males, % in females), wounds inflicted upon moving animals ( % in males, % in females), cpn ( % in males, % in females), liver hemosiderosis ( % in males, % in females), megalo cytosis of hepatocytes ( % in males, % in females), extramedullary hemopoiesis in the spleen ( % in males, % in females), hyperplasia of the testicular interstitial cells ( % in males), calcification of the skin ( % in males and % in females), adrenal cortical hyperplasia ( % in males, % in females), generalized calcification ( % in females, % in males), myocardial megalocytosis ( % in males, % in females), pulmonary edema ( % in males, % in females), myocarditis ( % in males, % in females), myocardial fibrosis ( % in males, % in females), generalized lipofuscinosis ( % in males, % in females), dystrophic changes in intervertebral disks ( % in males, % in females), pancreatic fibrosis ( % in males, % in females), arteriosclerosis ( % in males, % in females), and atypical hyperplasia of the thymus and lympholysis ( % in males, % in females). virtually all the above listed pathological changes were described earlier in other laboratory animals and, being different in the incidence, they are similar in macro and microscopic morphology [ , ] . the only exception was calcinosis cutis caused in naked mole rats by vitamin d excess and leading to formation of very large tumor like deposits of calcium salts [ ] . the most frequent type of histological changes in naked mole rats is calcium phosphate deposits in renal tubules of animals of both sexes. similar deposits with the same high frequency are found in intact rat wistar females of different age, but not in the males [ ] . the development of these changes in the naked mole rats is thought to be contributed to by both metastatic (i.e., associated with decrease in calcium level in blood and urine) and dystrophic mechanisms (i.e., caused by bio chemical changes in areas of massive death of cells) [ ] . in the second place, there are various wounds and their complications. this is difficult to compare with other animals because, although traumas are significant mortality causes in laboratory rodents, such animals are usually rejected and are not considered in the summa rized statistics of pathologic alterations and the corre sponding data are not published. the complications caused by these traumas, such as ulcerative dermatitis, are usually described in other rubrics. severity of the traumas in naked mole rats varies very strongly -from almost invisible scratches on the skin to giant subcutaneous hematomas and fractures of bone structures of the chest. special attention is given to chronic progressive nephropathy (cpn), the frequency of which is higher than in the other laboratory animals [ ]. this pathol ogy with still enigmatic origin [ ] is considered in a sep arate study [ ] . in this work, the morphology is described in detail and the incidence of individual cpn manifestations (different variants of lesions of glomeruli, interstitium, and tubular component of kidneys) is char acterized. however, the authors also ascribed to these manifestations kidney infarction and post infarction changes that are not usual components of cpn in labora tory rodents [ , ] . it is interesting that in that work no cases were described of amyloidosis or nonamyloid glomerulopathy (although changes similar to membra biochemistry (moscow) vol. no. nous glomerulonephritis were observed and described also as cpn manifestations), which are not rare in labo ratory mice [ ]. the primary role of the tubulo inter stitial component in the development of cpn in naked mole rats occurred to be much more pronounced than in rats and even in mice [ ] . this can be significant because the primary role of the tubulo interstitial component is a necessary condition for realizing the scenario of the clon al-mutational pathogenesis of this disease [ ] . it is also interesting that in the naked mole rat there are foci of pronounced renal hemopoiesis described earlier in an ecologically similar species, the northern mole vole [ ] . it is also worth attention that in some of the naked mole rats there were kidneys with the classic picture of terminal renal disease [ ] usually leading to death [ , ] . hepatic hemosiderosis, or more precisely even hemochromatosis (because many deposits of iron con taining pigment are found in the hepatocytes and not only in the kupffer cells) occurs frequently in the naked mole rat, but not in the other laboratory rodents. the hemo siderin deposits in the naked mole rat most frequently are detected in the periportal zones and are associated with megalocytosis of the hepatocytes [ ] . the cause of the disease must be elucidated, and this requires complex studies of the iron metabolism, of the system of protein iron carriers, and of the activity of erythrocyte destruc tion and utilization (hemolysis). as to megalocytosis, it is supposed to arise, in particular, because of the entrance into the naked mole rat's organism of xenobiotics with the plant food [ ] . diffuse hyperplasia of testicular interstitial cells (leydig cells) with atrophy of the spermatogenic epitheli um occurs in naked mole rats with extremely high fre quency and causes sterility of the males [ ] . it is thought to be associated with the low blood levels of gonadotropins and testosterone and decreased reproduc tive activity [ ] . myocardial fibrosis, productive myocarditis, and megalocytosis of cardiomyocytes together are fully corre sponding to the histological picture of a disease known as spontaneous cardiomyopathy [ ]. the frequency of these lesions in naked mole rats is the same as in labora tory mice [ ] . the cause of development of this disease is not established [ ] , but its incidence decreases on application of mitochondria targeted antioxidants [ ] . this is associated with a little diastolic dysfunction arising with age in females but not in males [ ] . in some ani mals, pulmonary edema is observed (which is often asso ciated with pulmonary congestion), but it is not a self inflicted disease but a complication of traumas, severe pathologies of the heart and kidneys, or a manifestation of agony at euthanasia [ , , ] . it is important to note that the naked mole rat lung morphology (neotenic) is not characteristic for rodents, and the large respiratory pathways sometimes induced an erroneous diagnosis of bronchiectasis [ ] . cortical hyperplasia of adrenals in the naked mole rat has a typical morphology and can be focal or diffuse. the focal hyperplasia is an age dependent pathology, whereas the diffuse hyperplasia can be associated with both age related changes and stress [ ] . both variants of this hyperplasia are observed in the naked mole rat. other alterations described in the naked mole rat can be considered minor and therefore do not require special commentaries, which can be found in guidebooks on pathology of laboratory animals [ ]. we have also observed two leukemia like cases with the peripheral blood of the animals characterized by the presence of many immature myeloid elements, including blasts, but without leukemic infiltrates in organs (unpublished data). we ascribed these observations to leukemoid reactions like those that were earlier described in the northern mole vole [ ] . hill et al. [ ] mentioned "avitaminosis" and other lesions of the tail skin caused by placing the animals into quite dry sand. it is more likely that these lesions are similar to lesions arising in laboratory mice maintained under conditions of insufficient humidity (tail dry gangrene, ringtail) [ ] and which have been described in the naked mole rat by other authors [ ] . hill et al. [ ] also presented two cases of death, one of which they thought to be caused by cardiac arrest caused by the expanded stomach, and the other to be caused by "renal-cardiac failure" (with subcutaneous edemas and ascites) because of the "acute nephritis". the absence of histological data and the insufficient documentation of the investigation results of the deceased animals prevents us from determining the correctness of these diagnoses. experimental reproduction in the naked mole rat of diseases not belonging to infections or tumors are virtual ly absent. we can mention only works that revealed the unusual resistance of these animals to hypoxia [ ] and the resistance of their neurons to the toxic action of beta amyloid related with development of alzheimer's disease [ ] . although the naked mole rat is an animal with unusually long for rodents lifespan, even the first profes sionally executed pathological studies revealed in it dis eases and tissue alterations characteristic for senescent short living rodents, including lipofuscinosis, sarcopenia, cortical hyperplasia of adrenals, megalocytosis of car diomyocytes and hepatocytes, dystrophic changes of intervertebral disks, heart changes of age related type, and also renal changes represented by cpn [ , , ] . it is interesting to note that, as differentiated from short liv ing mice and rats, in the naked mole rat thymus there are true hassall's bodies (resembling such bodies in humans), whereas the spleen of animals at the age of several years has signs specific for stress induced or the age related atrophy (our observation). age related changes also include preneoplasias and true neoplasms, which are inevitably present in the naked mole rat [ , , ] . the common age related pathology and the unusual for rodents long lifespan do not contradict each other, but such animals cannot be considered ageless (in any case, if we understand aging as stepwise accumulation of changes in tissues and associated decrease in physiological func tions). nevertheless, to elucidate the cause of such anom alous lifespan remains a very real problem. thus, the question comes to the fore of spontaneous diseases limit ing the lifespan, or otherwise, about causes of the natural mortality of the naked mole rat. the causes of mortality of naked mole rats is the first question that the scientific community expects to be answered by pathologists. however, unfortunately, the approach adopted in pathology of laboratory animals can answer this question only in probabilistic form. this problem has already been posed in a work [ ] and then discussed in a series of publications [ ] , which allows us to not present it in this article. note only that difficul ties of studying the causes of death of laboratory animals are amplified if the individual maintenance and monitor ing of physiological and biochemical parameters of ani mals are impossible, as in the case of naked mole rat colonies [ ] . unfortunately, it must be said that up to now there are no works with correct data on causes of the naked mole rats' natural mortality. in a work by delaney et al. [ ] , only about one third of the animals died of natural causes, and these data are combined with data obtained on euthanized animals. the conclusion of the authors of this work that the euthanasia was the most frequent cause of the death of the traumatized animals cannot be adopt ed as a sufficient answer to the general question about the mortality causes of naked mole rats. however, based on the available data, let us try to outline the range of dis eases and pathological conditions that can significantly contribute to the natural mortality of these animals. there is no doubt that fatal injuries of animals caused by each other must be mentioned as first. such injuries directly cause the death of at least half of all naked mole rats (our own observations). some other ani mals die as a result of fatal purulent and septic complica tions, but its incidence cannot be determined based avail able data. the second place is for that of cpn type renal dam age, which occur in more than half of the animals. although the incidence of such damage in naked mole rats is known, it remains unclear how often this disease leads to death, because it is impossible to perform regularly individual analyses of blood and urine of the animals in the colony [ ] . terminal renal disease is the cpn stage that, in the absence of other pronounced lesions, can be established as the cause of death, but it was recorded only in . % of the animals investigated in the work of delaney et al. [ ] . however, this investigation was conducted on the same animals as described earlier in the work [ ] ; therefore, the presented value characterizes the totality of spontaneously deceased and euthanized animals, and the deceased animals represent only one third of the total material. consequently, this work does not allow us to conclude about the contribution of kidney lesions to the total spontaneous mortality of naked mole rats. just the same notes can be referred to such compar atively frequent pathologies of naked mole rats as hepatic hemosiderosis and those damages of the myocardium that can be combined as cardiomyopathies. in the last case, the role of this disease as the cause of death can be estab lished not only by regular monitoring of clinical and bio chemical parameters, but also by investigation of the functional state of the animals' hearts. the real role of tumors as a mortality cause of the naked mole rats needs to be elucidated; however, as judged by the available data, their contribution cannot be more than % of the general mortality of the animals. data concerning infections are rather contradictory. it seems that under laboratory conditions, excluding the cases of epizootics and complications of traumas, infec tions also can cause the death of no more than % of the animals. first, this refers to a strange form of the gas trointestinal tract infectious damage whose etiology is thought to be associated with dysbiosis. such states as dermal or generalized calcinosis, dys trophic changes in intervertebral disks (if they lead to neurological damages), and possibly generalized lipofus cinosis can have minor significance as causes of death of individual animals of the total population. based on gen eral pathology concepts [ , ] , other damages described in the naked mole rats cannot themselves cause the death of the animals. thus, although the available data allow us to outline the spectrum and even rank the significance of possible mortality causes of naked mole rats, the contribution of different causes to the general mortality of these animals cannot be accurately determined until a sufficient num ber (some hundreds) of spontaneously deceased animals have been studied. for such studies a rather complex algorithm should be developed and used for individual clinical, physiological, and biochemical monitoring of their health state [ ] . as one can see from the data presented here, the naked mole rat suffers from many diseases described ear lier in laboratory rodents, and overall the spectrum of its diseases, despite some peculiarities, does not seem abnor mal on the background of the interspecies differences of the other rodents [ ] . for the naked mole rat, high fre biochemistry (moscow) vol. no. quencies of injuries caused by the animals to each other, cpn, liver hemosiderosis, calcinosis cutis, and diffuse interstitial hyperplasia of the testicle are relatively specif ic [ ] . the presence of age dependent pathological changes of tissues and organs, including malignant tumors, can be considered firmly established. however, the number of animals studied, especially of those who died from natural causes, is still insufficient. expansion of pathological studies, especially the search for sponta neous tumors with careful characteristics of the material studied, is a very urgent task, the solution of which will allow us to establish the true frequency of malignant tumors in this species. another important task is experimental reproduction on the naked mole rats of pathological processes and gen eral pathological reactions, because such data are very scarce. this especially refers to induction of tumor growth with chemical carcinogens or by ionizing radia tion, without which it is impossible to study the features and mechanisms of carcinogenesis and antitumor resist ance. however, we think that one should not have great expectations on discovery of new approaches for the pre vention and treatment of human tumors, because human cells and tissues are probably more resistant to carcino genesis than the cells and tissues of the naked mole rat. the real state of the question about the possible mecha nisms of the naked mole rat resistance to carcinogenesis appears very vague. a great variety of hypotheses and the absence of precise data about the contribution to this resistance of each of the mechanisms under discussion do not allow us to adopt the viewpoint [ ] that this resist ance is provided by the whole complex of the known fac tors. in general, the study on the antitumor resistance mechanism before obtaining reliable data on the sponta neous and experimental carcinogenesis (tumor frequen cies, their spectrum, latent period, etc.) does not seem to us the right approach. the assumption that the naked mole rat, like humans, is an example of neoteny and that the seemingly abnormal lifespan of this animal is connected just with it [ ] , in general does not contradict the available data on the age related pathology of this rodent: there is no rea son to think that neoteny completely excludes the devel opment of age related changes. thus, various sponta neous malignant tumors have been described in animals with classical neoteny − axolotls [ , ] that, as men tioned above, are considered a typical manifestation of age related pathology [ ] . another thing is that now it is unknown how exactly neoteny can influence the spec trum and development of spontaneous and experimental pathological processes, and here the naked mole rat, like the axolotl, can prove itself as a remarkable model object. it is quite possible that during aging neoteny can prevent only the "fast" component of phenoptosis and only mod ify the "slow" component [ ] − at least this conclusion does not contradict the data discussed in this report. perhaps in the case of neoteny, when the phenoptosis pro gram is canceled or its execution is strongly modified and slowed, aging occurs mainly by accumulation of errors. finally, it is extremely urgent to develop an algo rithm for a complex individual clinical, biochemical, and physiological study and its application for monitoring the health status of a sufficient laboratory population of naked mole rats with the aim to accurately determine the mortality causes of these animals. the naked mole rat: a new long liv ing model for human aging research negligible senescence in the longest living rodent, the naked mole rat: insights from a success fully aging species successful aging and sustained good health in the naked mole rat: a long lived mammalian model for biogerontology and biomedical research the naked mole rat moscow news: two more represen tatives of sodium motive force generators (na + cbb oxi dase and na + bacteriorhodopsin); natural delay of the aging program (neoteny) in mammals, namely in the naked mole rat and protracted brain development in a rodent model of extreme longevity the naked mole rats and humans: highly social creatures prolonging youth by delay of ontoge nesis (neoteny neoteny, prolongation of youth: from the naked mole rats to extreme susceptibility of african the naked mole rats (heterocephalus glaber) to experimen tal infection with herpes simplex virus type experimental infection of the naked mole rat, heterocephalus glaber, with leishmania donovani field and laboratory studies on the naked mole rat, heterocephalus glaber aging and respiratory infections in laboratory animals spontaneous histologic lesions of the adult the naked mole rat (heterocephalus glaber): a retrospective sur vey of lesions in a zoo population viral epizootic reveals inbreeding depression in a habitual ly inbreeding mammal four cases of spontaneous neoplasia in the naked mole rat (heterocephalus glaber), a putative cancer resistant species learning of nature: the curious case of the naked mole rat comparative genetics of longevity and cancer: insights from long lived rodents initial case reports of cancer in naked mole rats (heterocephalus glaber) diseases of the wistar rat pathology of the mouse pathology of laboratory rodents and rabbits pathobiology of aging mouse lifespan and lesions in genetically heterogenous (four way cross) mice: a new model for aging research genetic loci that influence cause of death in a het erogeneous mouse stock effect of the mitochondria target ed antioxidant skq on development of spontaneous tumors in balb/c mice cancer statistics multiple neoplasms, single primaries, and patient survival second primary cancer risk -the impact of applying differ ent definitions of multiple primaries: results from a retro spective population based cancer registry study spontaneous tumors of aging wild house mice. incidence, pathology, and c type virus expression essays on evolutionary oncology methods for determination and study of blastomogenicity of chemical substances spectrum of spontaneous pathological changes in the northern mole vole and influence of the mitochondria targeted antioxidant skq on it analysis of the sectional material of pathoanatomical departments of hospitals, institutes and maternity hospitals for general pathology of human resistance of guinea pigs to tumor growth: the reality of the phenomenon and its possible causes telomerase activity coevolves with body mass not lifespan coevolution of telomerase activity and body mass in mammals: from mice to beavers tumour resistance in induced pluripotent stem cells derived from naked mole rats robbins and cotran pathologic basis of disease resistance to experimental tumorigenesis in cells of a long lived mammal, the naked mole rat hypersensitivity to contact inhibition provides a clue to cancer resistance of naked mole rat high molecular mass hyaluro nan mediates the cancer resistance of the naked mole rat practical pathology of aging mice chronic progressive nephropathy in rodents as a disease caused by an expanding somatic mutant clone renal pathology in a nontraditional aging model: the naked mole rat (heterocephalus glaber) comparative observations on intertubular lymphatics and the organization of the interstitial tissue of the mammalian testis age associat ed murine cardiac lesions are attenuated by the mitochon dria targeted antioxidant skq vascular aging in the longest living rodent, the naked mole rat fructose driven gly colysis supports anoxia resistance in the naked mole rat amyloid beta and the longest lived rodent: the naked mole rat as a model for natural protection from alzheimer's dis ease the problem of determination of cause of laboratory animal's death: a critical review of def initions of "fatal" and "incidental" lesions cause of death analysis in rodent aging studies why animals die: an introduction to the pathology of aging the vital role of pathology in improving reproducibility and translational rel evance of aging studies in rodents neoplastic and possibly related skin lesions in neotenic tiger salamanders from a sewage lagoon multiple melanomas in the axolotl ambystoma mexicanum cutaneous mastocytomas in the neotenic caudate amphibians ambystoma mexicanum (axolotl) and ambystoma tigrinum (tiger salamander) programmed aging of mammals: proof of concept and prospects of bio chemical approaches for anti aging therapy this work was supported by the russian science foundation (project no. ). key: cord- -cpc yefj authors: van doremalen, neeltje; haddock, elaine; feldmann, friederike; meade-white, kimberly; bushmaker, trenton; fischer, robert j.; okumura, atsushi; hanley, patrick w.; saturday, greg; edwards, nick j.; clark, madeleine h. a.; lambe, teresa; gilbert, sarah c.; munster, vincent j. title: a single dose of chadox mers provides protective immunity in rhesus macaques date: - - journal: sci adv doi: . /sciadv.aba sha: doc_id: cord_uid: cpc yefj developing a vaccine to protect against the lethal effects of the many strains of coronavirus is critical given the current global pandemic. for middle east respiratory syndrome coronavirus (mers-cov), we show that rhesus macaques seroconverted rapidly after a single intramuscular vaccination with chadox mers. the vaccine protected against respiratory injury and pneumonia and reduced viral load in lung tissue by several orders of magnitude. mers-cov replication in type i and ii pneumocytes of chadox mers–vaccinated animals was absent. a prime-boost regimen of chadox mers boosted antibody titers, and viral replication was completely absent from the respiratory tract tissue of these rhesus macaques. we also found that antibodies elicited by chadox mers in rhesus macaques neutralized six different mers-cov strains. transgenic human dipeptidyl peptidase mice vaccinated with chadox mers were completely protected against disease and lethality for all different mers-cov strains. the data support further clinical development of chadox mers. coronaviruses (cov) pose a continuous emerging virus threat, as demonstrated by the emergence of three previously unidentified corona viruses in the past years. severe acute respiratory syndrome coronavirus (sars-cov) was first detected in and went on to infect > people, resulting in fatalities ( ) . in , middle east respiratory syndrome coronavirus (mers-cov) was first detected. mers-cov continues to infect humans from the dromedary camel host and has thus far infected > people, resulting in fatalities ( ). an outbreak of pneumonia with an unknown cause in wuhan, china was first reported in december . although information is still limited, we now know that this outbreak is caused by a third emerging cov. currently, > , cases are associated with this outbreak ( ) . the clinical spectrum of mers-cov infection in humans varies from asymptomatic to severe respiratory disease and death. patients present with influenza-like symptoms such as a fever and shortness of breath. thereafter, they may develop pneumonia, which can require mechanical ventilation and support in an intensive care unit ( ). human-to-human transmission of mers-cov is relatively limited and occurs mainly in nosocomial settings but has been reported in local communities as well ( ) . in , a traveler from the middle east to south korea caused an outbreak involving people and fatalities ( ). the nosocomial outbreak lasted from may to july, and , people were isolated with mers-cov-like symptoms. at least three superspreaders were identified in the outbreak, who infected , , and patients, respectively ( ) . the introduction and spread of mers-cov in south korea underscore the potential of this virus to cause epidemics outside of the arabian peninsula. mers-cov has continued to cause disease in humans, and in , cases have been reported in the kingdom of saudi arabia (ksa), of which % were fatal ( ). the ongoing circulation of mers-cov and subsequent outbreaks in the human population highlight the need for an efficient mers-cov vaccine. mers-cov is mainly prevalent in the arabian peninsula, with the majority of cases occurring in ksa ( %) ( ) . however, phylogenetically diverse mers-cov strains have been isolated from africa and the middle east ( ) ( ) ( ) , and antigenic differences have been reported between spike (s) proteins, the main antigen used in mers-cov vaccines, from the middle east and africa ( ) . thus, it is important that a mers-cov vaccine is protective against a variety of diverse mers-cov strains. thus far, only four vaccines have been tested in rhesus macaques. these are a dna vaccine ( ); a vaccine based on the receptor-binding domain of the mers-cov s adjuvanted with alum ( ); a combination of s plasmid dna and s protein ( ) ; and virus-like particles based on mers-cov s, matrix, and envelope proteins ( ) . of the four vaccine studies, only three studies included challenge studies, all of which used mers-cov isolates from . to date, there have been no reports of efficacy of a single-dose mers-cov vaccine in nonhuman primates (nhps) . we recently demonstrated that vaccination of mice with a replication-deficient simian adenovirus vaccine vector (chadox ) encoding full-length mers-cov s protein (chadox mers) elicited high-titer mers-cov-neutralizing antibodies and a robust cd + t cell response against the s protein ( ) . in addition, chadox mers vaccination resulted in full protection of human dipeptidyl peptidase (hdpp ) transgenic mice against a lethal challenge with mers-cov ( ) . chadox mers vaccination of dromedary camels was immunogenic and reduced mers-cov shedding after challenge in a highly stringent natural transmission model with multiday exposure to infectious mers-cov ( ) . chad-vectored vaccines against malaria ( ) , hiv ( ) , influenza ( ) , hepatitis c ( ), tuberculosis ( ) , ebola ( ) , and others show an excellent immunogenicity and safety profile in humans. in the current manuscript, we show that a single dose of chadox mers vaccine protects rhesus macaque model against a mucosal challenge with hcov-emc/ . serum obtained from vaccinated rhesus macaques was able to neutralize six diverse mers-cov strains. furthermore, a single dose of chadox mers vaccine protects hdpp transgenic mice against all evaluated mers-cov strains. three groups of six animals each were vaccinated with chadox mers via a prime-boost regimen [− and − dpi (days post infection)] or prime-only regimen (− dpi) or with chadox green fluorescent protein (gfp) (− and − dpi). animals were then challenged with × tcid (median tissue culture infectious dose) of hcov-emc/ on dpi via combined intratracheal, intranasal, oral, and ocular route ( ) . blood samples were taken at − , − , − , − , and dpi (fig. a) . the humoral immune response to vaccination was examined by enzyme-linked immunosorbent assay (elisa) using s protein and by virus neutralization assay. s proteinspecific immunoglobulin g (igg) antibodies were detected as early as days after vaccination in both chadox mers-vaccinated groups. all animals in these groups had s protein-specific antibodies at dpi, whereas no antibodies against s protein were found before vaccination or at any time in the chadox gfp-vaccinated group. a significant difference in elisa titers was observed between primeboost and prime-only animals at dpi (fig. b) . neutralizing antibodies were detected in of chadox mers-vaccinated animals at the time of challenge. one animal in the prime-only vaccination group did not have detectable neutralizing antibodies at this time and had the lowest antibody titer as measured by elisa ( ). a significant difference in vn (virus-neutralizing) titers was observed between prime-boost and prime-only animals at dpi (fig. c) . a second chadox mers vaccination at − dpi resulted in a statistically significant increase in s protein-specific elisa titer (geometric mean titer − dpi = ; dpi = , ; p < . ) and neutralizing antibody titer (geometric mean titer − dpi = ; dpi = ; p < . ) as determined via two-tailed t test, although neutralizing antibodies against the chadox vector could be detected at the time of the second vaccination ( fig. s a ). this suggests that the presence of neutralizing antibodies against chadox does not prevent the vaccine vector from boosting the immune response. vaccination with chadox mers reduces disease severity animals vaccinated with chadox gfp and challenged with mers-cov showed similar clinical signs as previously reported ( ), such as a decreased appetite and increased respiratory rate. clinical signs were scored using a standard nhp scoring sheet, focusing on areas such as general appearance, nasal discharge, and food intake. on average, chadox mers-vaccinated animals had a lower clinical score than chadox gfp-vaccinated animals ( fig. a) . all animals underwent exams on , , , , and dpi. no significant changes in weight or body temperature were observed for the duration of the study. peripheral capillary oxygen saturation (spo ) measurements supported a decrease in oxygen saturation from the baseline in chadox gfp-vaccinated animals, but not in chadox mersvaccinated animals (fig. b ). ventrodorsal and lateral thoracic radiographs were collected on all exam days. all animals vaccinated with chadox gfp showed moderate to severe pulmonary interstitial infiltrations, whereas animals vaccinated with chadox mers showed only mild signs of respiratory disease. a severe collapsed lung was observed on dpi in two animals in the prime-boost group, likely caused by the bronchoalveolar lavage (bal) performed on dpi. full details of all observed signs can be found in table s ( fig. a and table s ). all lung lobes (right cranial, right middle, right caudal, left cranial, left middle, and left caudal) of each individual animal were scored for severity of disease signs for each day radiographs were taken, and average scores were compared. scores obtained from animals vaccinated with chadox gfp were significantly higher from animals vaccinated with a prime-boost regimen of chadox mers on , , and dpi (fig. b) . upon necropsy on dpi, gross lung lesions were more prevalent in animals vaccinated with chadox gfp than in animals vaccinated with chadox mers (fig. , c and d). animals vaccinated with chadox gfp showed focally extensive areas of consolidation in all lung lobes and lungs generally failed to collapse. mediastinal lymph nodes were often edematous and enlarged. animals that received a vaccination with chadox mers, either via a prime-boost or primeonly regimen, had either no lesions or limited small multifocal areas of consolidation and congestion. an increased lung:body weight ratio is an indicator of pulmonary edema. animals in the control group had significantly higher lung:body weight ratios compared to chadox mers-vaccinated animals, and there was minimal difference between prime-boost and prime-only chadox mers-vaccinated animals (fig. f) . . statistical significance between − and − dpi in the prime-boost group was determined via one-tailed paired student's t test. statistical significance between prime-boost and prime-only groups on dpi was determined via two-tailed unpaired student's t test. **p < . ; ***p < . . lung tissue sections were stained with hematoxylin and eosin or with mers-cov-specific antibodies. all slides were evaluated by a board-certified veterinary pathologist blinded to study group allocations. in animals that received a vaccination with chadox mers, either prime-boost or prime-only, a minimal to mild bronchointerstitial pneumonia was present, characterized by mild thickening of the alveolar septae by lymphocytes and macrophages. pulmonary vessels were bound by moderate numbers of lymphocytes. in stark contrast, in lung tissue obtained from animals vaccinated with chadox gfp, moderate to marked bronchointerstitial pneumonia was present throughout the lung lobes characterized by thickening of the alveolar septae by lymphocytes and macrophages and edema and fibrin. alveoli contained abundant edema and fibrin and moderate to abundant numbers of alveolar macrophages, neutrophils, and necrotic debris. inflammation often surrounded bronchioles and pulmonary vasculature, and type ii pneumocyte hyperplasia was prominent. in addition, the presence of mers-cov antigen by immunohistochemistry was found only in lungs of animals vaccinated with chadox gfp within type i and ii pneumocytes and was not found in lung tissue of chadox mers-vaccinated animals. severity of bronchointerstitial pneumonia, type ii pneumocyte hyperplasia and hemorrhages, edema, and fibrin deposits was scored. statistically significant differences between animals vaccinated with chadox mers or chadox gfp were found for all three categories ( fig. , e, f, and h). thus, vaccination with chadox mers, either via a prime-boost or prime-only regimen, significantly decreased the severity of pulmonary pathology and protected rhesus macaques against bronchointerstitial pneumonia. bal was performed on all animals on , , , and dpi, and the amount of viral rna, mrna, and infectious virus was determined. in the prime-boost group, infectious virus was only detected at dpi (n = ) after inoculation, and in the prime-only group at dpi (n = ) and dpi (n = ) but not thereafter. in contrast, infectious virus was detected on all days in bal fluid from the control group (fig. a ). viral mrna in bal fluid of animals in the prime-boost group was only detected at dpi (n = ) and dpi (n = ). in contrast, viral mrna in bal fluid from animals that received a single vaccination with chadox mers mrna was detected on all days, but the amount was reduced compared to animals vaccinated with chadox gfp (fig. s b ). viral rna as measured by upe quantitative reverse transcription polymerase chain reaction (qrt-pcr) assay could be detected in all groups up to dpi. however, the number of genome copies per milliliter detected was lower in animals vaccinated with chadox mers compared to animals vaccinated with chadox gfp (fig. a) . a significant association was found between higher elisa titer or vn titer and lower levels of viral rna, mrna, or infectious virus in bal fluid for all days, except dpi for infectious virus (spearman's rank correlation coefficient; table s ). all animals were euthanized on dpi, and tissues were analyzed for the presence of viral rna, mrna, or infectious virus. infectious virus titers were only found in nasal turbinate tissue (n = ) and lung lobe tissue (n = ) from control animals. no viral mrna was found in all tissues obtained from animals that received a prime-boost regimen. in tissue from animals that received a prime-only regimen, limited viral mrna could be found in upper and lower lung lobe tissues (n = ). in contrast, mrna could be found in respiratory tract tissues of all control animals and in conjunctiva (fig. b ). viral rna was detected in tissues from all groups but was mainly found in lung lobes and bronchi. viral load was higher for lower respiratory tract tissue obtained from animals vaccinated with chadox gfp (n = ) than from animals receiving a prime-only (n = ) or a prime-boost regimen of chadox mers (n = ) (fig. b ). the presence of cytokines was evaluated in lung tissue. several cytokines were up-regulated in animals vaccinated with chadox gfp compared to animals vaccinated with chadox mers, including interleukin- (il- ), il- , il- , il- , monocyte chemotactic protein- (mcp- ), macrophage inflammatory protein- a (mip- ), and transforming growth factor- (tgf-), although only il- and mcp- were significantly different in tissue obtained from control animals compared to vaccinated animals. a significant difference was observed in il- ra levels in lung tissue obtained from the prime-only and prime-boost groups. it is not clear what the clinical significance of this difference is ( fig. s ). overall, these results show local increased immune activity in animals vaccinated with chadox gfp, but not in animals vaccinated with chadox mers, days after challenge with mers-cov. the likely explanation for this is that vaccinated animals have controlled the infection rapidly, whereas in the control animals, more viral replication has taken place followed animal vaccinated with chadox gfp shows focally extensive area of increased pulmonary opacity and deviation of the cardiac silhouette, highlighted in the circle located in the middle and caudal lung lobes. (b) thoracic radiographs of each animal were scored per lung lobe, resulting in a maximum score of . values were averaged per group per day (d), and mean with sd is shown (see table s for more details). (c) gross pathology of lungs shows no pathologic changes in chadox mers-vaccinated animals and focally extensive areas of consolidation in left cranial, middle, and caudal lung lobes in control animals (asterisks). (d) gross lung lesions were scored for each lung lobe, ventral and dorsal. values were averaged per group, and mean with sd is shown. (e) lung tissue sections were stained with hematoxylin and eosin. moderate numbers of lymphocyte accumulation around pulmonary arterioles (asterisks) and mild thickening of alveolar septae by lymphocytes and macrophages (arrows) in lung tissue of animals vaccinated with chadox mers. marked bronchointerstitial pneumonia with abundant pulmonary edema and fibrin (asterisks), type ii pneumocyte hyperplasia (arrows), and increased numbers of alveolar macrophages (arrowheads) in lung tissue of control animals. magnification, × . (f) lung-to-body weight (bw) ratio was determined for all animals at necropsy. mean with sd is shown. (g) lung tissue sections were stained with antibody against mers-cov antigen, which is visible as a red-brown staining. no immunoreactivity was found in chadox mers-vaccinated animals, whereas multifocal immunoreactivity of type i and ii pneumocytes could be found in lung tissue of chadox gfp-vaccinated animals. (h) lung tissue sections were scored on severity of lesions ( , no lesions; , to %; , to %; , to %; , to %; and , to %) and averaged per group. mean with sd is shown. a, bronchointerstitial pneumonia; b, type ii pneumocyte hyperplasia; c, hemorrhages, edema, and fibrin deposits. statistical significance between groups was determined via two-tailed unpaired student's t test. *p < . ; **p < . ; ***p < . ; ****p < . . photo credit: neeltje van doremalen, niaid/nih. chadox mers is based on the s protein from camel/qatar/ / , and we currently do not have access to an isolate. we thus already show cross-protection of chadox mers against hcov-emc/ ( . % s protein amino acid identity). here, we extend that analysis to five other strains. we selected six different strains of mers-cov ( fig. s ). s protein identity for all strains to the vaccine s protein was > . %. amino acid identity was lowest for camel/burkina faso/ cirad-hku / ( . %) and highest for hu/korea/seoul/ snu - / ( . %). we tested neutralizing capability of serum obtained at dpi. strains were selected on the basis of geographical location (ksa, south korea, and burkina faso), host (dromedary camel or human), and time of isolation ( to ). all six strains were neutralized by antibodies elicited by chadox mers vaccination. although we were not able to detect neutralizing ability of serum obtained from animal against hcov-emc/ , antibodies in the serum were able to neutralize four of six tested mers-cov strains (table ) . to investigate whether vaccination with chadox mers vaccination provides protection against a variety of different mers-cov strains, we vaccinated balb/c mice transgenic for hdpp with chadox mers or chadox gfp days before challenge with tcid of one of six diverse mers-cov strains ( fig. s ) via the intranasal route. all mice vaccinated with chadox mers survived challenge with mers-cov, independent of the challenge virus used, whereas most control mice were euthanized because of > % weight loss or poor body condition (fig. a) . four animals per group were euthanized at dpi, and infectious mers-cov titers in lung tissue were evaluated. whereas infectious virus could be found in lung tissue of control animals, we were unable to find infectious virus in lung tissue of chadox mers-vaccinated animals (fig. b) . thus, chadox mers protects against a variety of different mers-cov strains in hdpp transgenic mice. ( ) . mers is associated with a high case-fatality rate ( . %), and human-tohuman transmission is a major contributor to patient infections ( ). currently, no mers-cov vaccine is available. ideally, such a vaccine would only require a single administration and would protect against a wide variety of different mers-cov strains. several studies have evaluated different types of mers-cov vaccines in animal models, but few have taken these vaccines into nhps. in the current study, we show the efficacy of chadox mers in rhesus macaques. unlike other nhp vaccine studies ( - ), we investigate vaccine efficacy after a single dose. animals that received a single dose of chadox mers showed an induction of a neutralizing antibody response associated with mostly normal clinical parameters, showing no breathing irregularities or reduced lung function by spo values, limited evidence of infiltration by radiograph analysis after challenge, and no signs of gross pathological lesions. vaccination reduced viral rna load in tissues collected at dpi compared to chadox gfp-vaccinated animals by several logs. in contrast to the abundant presence of viral mrna and infectious virus in control animals, we found no evidence of infectious virus, and only a limited presence of viral mrna, in the lungs of prime-only vaccinated macaques. viral mrna and infectious virus was completely absent from animals vaccinated with a prime-boost regimen of chadox mers. although both vaccine regimens protected rhesus macaques from the clinical symptoms of mers-cov inoculation, we detected lower virus replication in animals that received a prime-boost regimen compared to animals receiving a prime-only regimen, suggesting that the prime-boost regimen is a superior therapeutic approach. it should be noted that in the current model, animals are inoculated with a high dose of virus ( × tcid per animal). this is likely a higher inoculum than most humans the study was not designed to determine correlates of protection, which must be determined separately for each vaccine candidate, but it is of interest that here, one animal was protected despite not having detectable neutralizing antibodies against hcov-emc/ , and in general, the neutralizing antibody titer was not high. in clinical trials, chadox -vectored vaccines prime strong t cell responses against the vaccine antigen ( ) ( ) ( ) ( ) ( ) ( ) . in a clinical study of patients that recovered from mers infection, some had strong cd + t cell responses without detectable antibodies ( ) . further studies addressing correlates of protection for chadox mers should assess cd + t cell responses. following the successful induction of protective immunity after vaccination as demonstrated here, the duration of immunity and ability to induce the development of memory b and t cells should be assessed. the phenotype of memory cells induced by vaccination may not necessarily mimic that induced by infection with the pathogen. in the first clinical trial of chadox mers (clinicaltrials.gov identifier: nct ) both humoral-and t cell-mediated responses were assessed at multiple times and persisted up to year after vaccination. further work will be required to assess memory b and t cell phenotypes. we are currently planning studies to look at long-term protection by chadox mers vaccination in our hdpp mouse model. a variety of different mers-cov strains have been isolated from dromedary camels and humans over the past years of mers-cov emergence ( ) . dromedary camels are distributed throughout africa, the middle east, asia, and australia ( ) . although mers-cov has not been detected in dromedary camels in australia ( ) , strains have been isolated from africa ( ) and the middle east ( ), and seropositive dromedary camels have been found in asia ( , ) . phylogenetic analyses show a clustering of mers-cov by geographical location ( , ) , and analysis of complete mers-cov genomes, which only included one african strain, showed the presence of two clades, with human isolates in both clades ( ) . notably, antigenic differences have been reported between s proteins from the middle east and africa ( ), potentially affecting the efficacy of a vaccine based in conclusion, we show that a single vaccination with chadox mers results in protection against disease progression and virus replication associated with mers-cov challenge in the rhesus macaque, and a prime-boost regimen reduced viral replication further. furthermore, chadox mers vaccination protected against a diverse panel of contemporary mers-cov strains in hdpp mice. this is the first time that broad protection after a single vaccination has been shown for any mers-cov vaccine. last, chadox vaccines can be produced rapidly, have been shown to be safe in human patients, and are protective against mers-cov in rhesus macaques and hdpp mice. we conclude that the chadox platform is ideal for the development of vaccines against novel emerging coronaviruses, such as hcov- /sars-cov- . animal experiment approval was provided by the institutional animal care and use committee at rocky mountain laboratories. all animal experiments were executed in an association for assessment and accreditation of laboratory animal care-approved facility by certified staff, following the guidelines and basic principles in the national institutes of health (nih) guide for the care and use of laboratory animals, the animal welfare act, the u.s. department of agriculture, and the u.s. public health service policy on humane care and use of laboratory animals. rhesus macaques were housed in individual primate cages allowing social interactions, in a climatecontrolled room with a fixed light-dark cycle ( / hours). rhesus macaques were monitored a minimum of twice daily throughout the experiment. commercial monkey chow, treats, and fruit were provided twice daily by a trained personnel. water was available ad libitum. environmental enrichment consisted of a variety of human interaction, commercial toys, videos, and music. the institutional biosafety committee (ibc) approved work with infectious mers-cov virus strains under bsl conditions. all sample inactivation was performed according to ibc-approved standard operating procedures for the removal of specimens from high containment. the s protein gene from mers-cov strain camel/qatar/ / (genbank accession no. kj . ) was codon optimized for humans and synthesized by geneart (thermo fisher scientific). the synthesized s gene was cloned into a transgene expression plasmid comprising a modified human cytomegalovirus immediate early promoter (cmv promoter) with tetracycline operator sites and the polyadenylation signal from bovine growth hormone. the resulting expression cassette was inserted into the e locus of a genomic clone of chadox using site-specific recombination ( ) . the virus was rescued and propagated in t-rex- cells (invitrogen). purification was by cscl gradient ultracentrifugation, and the virus was titered, as previously described ( ) . doses for vaccination were based on infectious units (ius) ( ) . eighteen adult rhesus macaques ( males and female) were purchased from morgan island and randomly divided into three groups of six animals each. the group was vaccinated with chadox mers at − and − dpi, the group was vaccinated with chadox mers at − dpi, and the group was vaccinated with chadox gfp at − and − dpi. all vaccinations were done with . × iu per animal per vaccination. blood samples were obtained before vaccination and days thereafter. animals were challenged with mers-cov strain hcov-emc/ on dpi with administrations of ml intratracheally, ml intranasally, ml orally, and ml ocularly of tcid /ml virus solution. clinical exams were performed on − , − , − , − , , , , and and dpi; animals were euthanized at dpi. all exams existed of the following: weight and temperature measurements, radiographs, spo measurements using pulse oximetry, and blood sampling. bal was performed on , , , and dpi by insertion of an endotracheal tube and bronchoscope into the trachea, then past the third bifurcation, and subsequent installation of ml of sterile saline. manual suction was applied to retrieve the bal sample. necropsy was performed on dpi. radiographs were evaluated and scored by a board-certified veterinarian who was blinded to the group assignment of the animals according to the following criteria: , normal examination; , mild interstitial pulmonary infiltrates; , moderate interstitial infiltrates, perhaps with partial cardiac border effacement and small areas of pulmonary consolidation (alveolar patterns and air bronchograms); and , pulmonary consolidation as the primary lung pathology, seen as a progression from grade lung pathology ( ) . veroe cells were maintained in dmem supplemented with % fetal calf serum, mm l-glutamine, penicillin ( u/ml), and streptomycin ( g/ml). virus titrations were performed by endpoint titration in veroe cells, which were inoculated with -fold serial dilutions of virus. after -hour incubation at °c and % co , tissue homogenate dilutions were removed, and cells were washed twice with phosphate-buffered saline (pbs) and incubated in l of % dmem. cytopathic effect was scored at dpi, and the tcid was calculated from four replicates by the spearman-karber method ( , ) . sera were heat inactivated ( min, °c), and twofold serial dilutions were prepared in % dmem. hereafter, tcid of mers-cov was added. after -min incubation at °c, virus:serum mixture was added to veroe cells and incubated at °c and % co . at dpi, the cytopathic effect was scored. the virus neutralization titer was expressed as the reciprocal value of the highest dilution of the serum, which still inhibited virus replication ( ) . chimpanzee adenovirus chadox -specific neutralizing antibody titers were assessed using a secreted placental alkaline phosphatase (seap) quantitation assay. briefly, griptite msr cells (invitrogen, catalog no. r - ) were cultured as per manufacturer's instructions and were seeded at × cells per well in a -well plate the day before starting the assay ( ± hours). cells were infected with the test sera dilutions (fourfold dilution series) at : , : , : , : , and : in phenol red-free % fbs dmem (life technologies, catalog no. ) and the chadox -seap reporter virus in a : mixture (pre-incubated for hour to allow any neutralization to occur) for hour before replacing with phenol redfree % fbs dmem for a further hours (± hours). sample dilutions were tested in duplicate lanes. for each sample, seap concentration was assessed in l aliquots of culture supernatant, with cpsd as an indicator substrate (tropix phospha-light chemiluminescent assay kit, life technologies, catalog no. t ), using a minor variant of the manufacturer's instructions; luminescence intensity was measured using a varioskan flash luminometer (thermo fisher scientific). serum dilution neutralization titers were measured by linear interpolation of adjacent values (to % inhibition) to determine the serum dilution required to reduce seap concentration by % compared to wells with virus alone. tissues ( mg) were homogenized in rlt buffer, and rna was extracted using the rneasy kit (qiagen) according to the manufacturer's instructions. rna was extracted from bal fluid using the qiaamp viral rna kit (qiagen) on the qiaxtractor. the upe mers-cov ( ) or mrna ( ) detection assay was used for the detection of mers-cov viral rna. five microliters of rna was tested with the rotor-gene probe kit (qiagen) according to the instructions of the manufacturer. dilutions of mers-cov virus stock with known genome copies were run in parallel. genome copies were determined using droplet digital pcr (bio-rad) and the corresponding qrt-pcr. a soluble, trimeric recombinant s protein of mers-cov (isolate ca/jeddah/d / ) incorporating amino acids to and a c-terminal trimerization domain was produced in chinese hamster ovary cells (expicho; thermo fisher scientific) and purified by immunoaffinity chromatography. maxisorp plates (nunc) were coated overnight at room temperature with g of s protein per plate in pbs. plates were blocked with l of casein (thermo fisher scientific) for min at room temperature. serum ( × serial dilution in casein starting at × dilution) was incubated at room temperature for hours. antibodies were detected using affinity-purified polyclonal antibody peroxidase-labeled goat anti-monkey igg (seracare, - - ) in casein and , ′, , ′-tetramethylbenzidine (tmb) two-component peroxidase substrate (seracare) and read at nm. all wells were washed three times with pbst (pbs-tween . %) in between steps. threshold for positivity was set at × optical density (od) value of negative control (serum obtained from nhps before the start of the experiment). samples for analysis of cytokine/chemokine levels were inactivated with -radiation ( mrad) according to the standard operating procedures. concentrations of granulocyte colony-stimulating factor (g-csf), granulocyte-macrophage csf, interferon-, il- , il- receptor antagonist, il- , il- , il- , il- , il- , il- , il- / (p ), il- , il- , il- , il- mcp- , and mip- , mip- , soluble cd ligand (scd l), tgf-, tumor necrosis factor-, and vascular endothelial growth factor were measured on a bio-plex instrument (bio-rad) using the non-human primate cytokine milliplex map -plex kit (millipore) according to the manufacturer's instructions. necropsies and tissue sampling were performed according to ibcapproved protocols. lungs were perfused with % formalin and processed for histologic review. harvested tissues were fixed for a minimum of days in % neutral-buffered formalin and then embedded in paraffin. tissues were processed using a vip- tissue-tek (sakura finetek, usa) tissue processor and embedded in ultraffin paraffin polymer (cancer diagnostics, durham, nc). samples were sectioned at m, and the resulting slides were stained with hematoxylin and eosin. specific anti-cov immunoreactivity was detected using mers-cov nucleocapsid protein rabbit antibody (sino biological inc.) at a : . the tissues were processed for immunohistochemistry using the discovery ultra automated ihc/ish staining instrument (ventana medical systems) with a discovery red (ventana medical systems) kit. all tissue slides were evaluated by a board-certified veterinary anatomic pathologist blinded to study group allocations. tukey's multiple comparison test or a two-tailed unpaired student's t test was conducted to compare differences between vaccine groups and the control group. a bonferroni correction was used to control the type i error rate for the two comparisons (group versus control and group versus control), and thus, statistical significance was reached at p < . . spearman's rank correlation coefficient test was used to interfere correlation. supplementary material for this article is available at http://advances.sciencemag.org/cgi/ content/full/ / /eaba /dc sars and mers: recent insights into emerging coronaviruses environmental contamination and viral shedding in mers patients during mers-cov outbreak in south korea middle east respiratory syndrome coronavirus (mers-cov) outbreak in south korea, : epidemiology, characteristics and public health implications evidence for camel-to-human transmission of mers coronavirus middle east respiratory syndrome coronavirus in dromedaries in ethiopia is antigenically different from the middle east isolate mers coronaviruses from camels in africa exhibit regiondependent genetic diversity a synthetic consensus anti-spike protein dna vaccine induces protective immunity against middle east respiratory syndrome coronavirus in nonhuman primates recombinant receptor binding domain protein induces partial protective immunity in rhesus macaques against middle east respiratory syndrome coronavirus challenge evaluation of candidate vaccine approaches for mers-cov mers-cov virus-like particles produced in insect cells induce specific humoural and cellular imminity in rhesus macaques chadox and mva based vaccine candidates against mers-cov elicit neutralising antibodies and cellular immune responses in mice protective efficacy of a novel simian adenovirus vaccine against lethal mers-cov challenge in a transgenic human dpp mouse model humoral immunogenicity and efficacy of a single dose of chadox mers vaccine candidate in dromedary camels phase ia clinical evaluation of the plasmodium falciparum blood-stage antigen msp in chad and mva vaccine vectors safety and tolerability of conserved region vaccines vectored by plasmid dna, simian adenovirus and modified vaccinia virus ankara administered to human immunodeficiency virus type -uninfected adults in a randomized, single-blind phase i trial heterologous two-dose vaccination with simian adenovirus and poxvirus vectors elicits long-lasting cellular immunity to influenza virus a in healthy adults chronic hepatitis c viral infection subverts vaccine-induced t-cell immunity in humans a phase i trial evaluating the safety and immunogenicity of a candidate tuberculosis vaccination regimen, chadox a prime -mva a boost in healthy uk adults safety and immunogenicity of a heterologous prime-boost ebola virus vaccine regimen in healthy adults in the united kingdom and senegal middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques mers-cov spillover at the camel-human interface recovery from the middle east respiratory syndrome is associated with antibody and t-cell responses bactrian camels shed large quantities of middle east respiratory syndrome coronavirus (mers-cov) after experimental infection absence of mers-cov antibodies in feral camels in australia: implications for the pathogen's origin and spread middle east respiratory syndrome coronavirus antibodies in dromedary camels serologic evidence for mers-cov infection in dromedary camels molecular evolution of mers coronavirus: dromedaries as a recent intermediate host or long-time animal reservoir? a novel chimpanzee adenovirus vector with low human seroprevalence: improved systems for vector derivation and comparative immunogenicity preventing spontaneous genetic rearrangements in the transgene cassettes of adenovirus vectors a single-dose chadox -vectored vaccine provides complete protection against nipah bangladesh and malaysia in syrian golden hamsters thoracic radiography as a refinement methodology for the study of h n influenza in cynomologus macaques (macaca fascicularis) beitrag zur kollektiven behandlung pharmakologischer reihenversuche detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction growth and quantification of mers-cov infection diversity of middle east respiratory syndrome coronaviruses in dromedary camels based on full-genome sequencing competing interests: s.c.g. is a board member of vaccitech and named as an inventor on a patent covering use of chadox -vectored vaccines. the other authors declare that they have no competing interests. data and materials availability: all data needed to evaluate the conclusions in the paper are present in the paper and/or the supplementary materials. chadox mers can be provided by the jenner institute, university of oxford pending scientific review and a completed material transfer agreement a single dose of chadox mers provides protective immunity in rhesus macaques key: cord- -uy f f o authors: nara, peter l.; nara, deanna; chaudhuri, ray; lin, george; tobin, greg title: perspectives on advancing preventative medicine through vaccinology at the comparative veterinary, human and conservation medicine interface: not missing the opportunities date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: uy f f o abstract vaccination has historically and remains one of the most cost-effective and safest forms of medicine today. along with basic understanding of germ theory and sanitation, vaccination, over the past years, has transformed lives and economies in both rich and poor countries by its direct impact on human and animal life—resulting in the eradication of small pox, huge reductions in the burden of previously common human and animal diseases such as polio, typhoid, measles in human medicine and contagious bovine pleuropneumonia, foot-and-mouth disease, screwworm and hog cholera and the verge of eradicating brucellosis, tuberculosis, and pseudorabies in veterinary medicine. in addition vaccination along with other animal production changes has provided the ability to produce otherwise unaffordable animal protein and animal health worldwide. the landscape however on which vaccinology was discovered and applied over the past years, even in the past years has and is undergoing continuous change. for vaccination as a public health tool to have its greatest impacts in human and veterinary medicine, these great medical sciences will have to come together, policy-relevant science for sustainable conservation in developing and developed countries needs to become the norm and address poverty (including lack of basic health care) in communities affected by conservation, and to consider costs and benefits (perceived or not) affecting the well-being of all stakeholders, from the local to the multinational. the need to return to and/or develop new education-based models for turning the tide from the heavily return-on-investment therapeutic era of the last century into one where the investment into the preventative sciences and medicine lead to sustainable cultural and cost-effective public health and economic changes of the future is never more evident than today. the new complex problems of the new millennium will require new educational models that train para- and professional people for thinking and solving complex inter-related biological, ecological, public-, political/economic problems. the single profession that is best positioned to impact vaccinology is veterinary medicine. it’s melding with human medicine and their role in future comparative and conservation-based programs will be critical to the successful application of vaccines into the st century. vaccination has historically and remains one of the most cost-effective and safest forms of medicine today. along with basic understanding of germ theory and sanitation, vaccination, over the past years, has transformed lives and economies in both rich and poor countries by its direct impact on human and animal life-resulting in the eradication of small pox, huge reductions in the burden of previously common human and animal diseases such as polio, typhoid, measles in human medicine and contagious bovine pleuropneumonia, foot-and-mouth disease, screwworm and hog cholera and the verge of eradicating brucellosis, tuberculosis, and pseudorabies in veterinary medicine. in addition vaccination along with other animal production changes has provided the ability to produce otherwise unaffordable animal protein and animal health worldwide. the landscape however on which vaccinology was discovered and applied over the past years, even in the past years has and is undergoing continuous change. for vaccination as a public health tool to have its greatest impacts in human and veterinary medicine, these great medical sciences will have to come together, policy-relevant science for sustainable conservation in developing and developed countries needs to become the norm and address poverty (including lack of basic health care) in communities affected by conservation, and to consider costs and benefits (perceived or not) affecting the well-being of all stakeholders, from the local to the multinational. the need to return to and/or develop new education-based models for turning the tide from the heavily return-on-investment therapeutic era of the last century into one where the investment into the preventative sciences and medicine lead to sustainable cultural and cost-effective public health and economic changes of the future is never more evident than today. the new complex problems of the new millennium will require new educational models that train para-and professional people for thinking and solving complex inter-related biological, ecological, public-, political/economic problems. the single profession that is best positioned to impact vaccinology is veterinary medicine. it's melding with human medicine and their role in future comparative and conservation-based programs will be critical to the successful application of vaccines into the st century. published by elsevier ltd. the new millennium did not bring the anticipated global internet technology shutdown however, it has brought with and heralded a time of significant change, opportunity and challenges. i and my co-authors goal in this overview are to celebrate, provocate, instigate innovate and activate those in society who are in interested to contributing to the betterment of human and animal health through vaccination. for vaccination to have its greatest chance of working policy-relevant science for sustainable conservation in developing countries needs to address poverty (including lack of basic health care) in communities affected by conservation, and to consider costs and benefits (perceived or not) affecting the wellbeing of all stakeholders, from the local to the multinational. the need to return to and/or develop new education-based models for turning the tide from the heavily return-on-investment therapeutic era of the last century into one where the investment into the pre-ventative sciences and medicine lead to sustainable cultural public health and economic changes of the future is never more evident than today. if the article gets the attention of researchers, educators/teachers, funders, policy makers, economists and the general public in both developed and developing countries to become involved in finding collaborative solutions to the conservation crisis than we will consider it a success. vaccination has and remains one of the most cost-effective and safest forms of medicine toward improving health today. along with basic understanding of germ theory and sanitation, vaccination, over the past years, has transformed lives in both rich and poor countries-resulting in the eradication of smallpox and huge reductions in the burden of previously common diseases such as polio, typhoid and measles. immunization is particularly well suited to all countries including those with weak health systems, because it requires relatively less training and equipment and does not depend on skilled diagnosis, long-term drug regimens or extensive medical care. immunization and sanitation remain as the most important public health modality responsible for improving the gnp of developing countries through additional gains in healthier children who are better educated and grow up to impact on their productivity. like schoolchildren, healthier workers have better attendance rates and are more energetic and mentally robust. workers in healthy communities, moreover, need to take less time off to care for sick relatives. body size, which is greatly influenced by one's health during childhood, has been found to have large impacts on long-term productivity. recent economists [ ] have calculated that a -year increase in life expectancy improves labor productivity by %. despite the weakness of health systems in many developing countries, three-quarters of the world's children now receive a standard package of childhood vaccines through the who/unicef expanded program on immunization to protect them against diphtheria, tetanus, pertussis, polio, measles and neonatal tuberculosis [ ] . these vaccines currently save an estimated million lives a year -almost , lives a day -and protect millions more from illness and permanent disability, thus providing as mentioned above a healthier cohort of people to contribute to the economic development of the nation. the full package of basic vaccines (diphtheria, tetanus, pertussis, polio, measles and neonatal tuberculosis) costs less than $ per year of life saved in poor countries. "life-years" and "year of life" consistently refer to disability-adjusted life-years (dalys). interventions are generally considered extremely costeffective if the cost per year of life is less than $ . by comparison, antiretroviral treatment for hiv/aids-an intervention that donors widely support in the developing world costs up to five times as much at $ to $ per life-year saved; by way of comparison, in the us and the uk medical interventions are considered cost effective at $ , to $ , per life-year saved [ , [ ] [ ] [ ] . the week during the conference in amsterdam it was reported that "vaccine-preventable deaths reach new low in u.s." as reported in a federal report released tuesday, november , people readily associate the role of veterinarians with private veterinary practice focused on pets and farm animals, but the true dimensions and contributions of veterinary medicine are much broader and reflect expanding societal needs and contemporary challenges to animal and human health and to the environment [ ] . veterinary medicine has responsibilities in biomedical research; ecosystem management; public health; food and agricultural systems; and care of companion animals, wildlife, exotic animals, and food animals. the expanding role of veterinarians at cdc reflects an appreciation for this variety of contributions. veterinarians' educational background in basic biomedical and clinical sciences compare with that of physicians. however, unlike their counterparts in human medicine, veterinarians must be familiar with multiple species, and their training emphasizes comparative medicine. veterinarians are competent in preventive medicine, population health, parsitology, zoonoses, and epidemiology, which serve them well for careers in public health. the history and tradition of the profession always have focused on protecting and improving both animal health and human health [ ] . since , a total of diseases have been eliminated from equine, poultry, and livestock populations in the united states [ ] . the elimination of these livestock diseases, along with outstanding research in animal health, is key to the remarkable gains in the efficiency of u.s. animal production [ ] . partly as a consequence, u.s. residents spend only approximately % of their disposable income on food, whereas residents in other countries pay three or four times more [ ] . although this achievement is recognized to have added billions of dollars to other parts of the u.s. economy, its success in allowing the u.s. public access to a nutritious, affordable, and sustainable food supply -also important for the public's health and well-being -is far less appreciated. the success of the national brucellosis and tuberculosis elimination campaigns has benefited not only the u.s. livestock industries but also human health by substantially reducing these zoonotic threats in animals. additional public health contributions can be attributed to the food safety and inspection service of the u.s. department of agriculture (usda), which has substantially reduced the burden of food-borne illnesses, improved food safety, and eliminated other zoonotic threats. over the years, cdc has worked closely with usda and the food and drug administration to improve the safety of u.s. foods and reduce antimicrobial resistance in pathogens that infect both humans and animals. veterinary scientist and veterinarians within the health and human services serve in many critical capacities. veterinary officers in the commissioned corps work throughout the u.s. department of health and human services and in other federal agencies. most veterinary officers are assigned to the cdc, nih, fda, usda, epa, ogha, ndms and state department. other veterinarians and veterinary scientists function as medical research scientists post-doctoral nih/nci fellows, principal investigators, some specializing in lab animal medicine and providing critical lab animal health infrastructure and support, design of animal models for human disease in most of the hhs institutes. some are part of the hhs national disaster medical services and were deployed as the veterinary medical assistant teams (v-mats) and supported the search and rescue in the world trade disaster. additionally, since , the avma and the college of american pathologists have been working together to create a standard nomenclature that would allow veterinarians, physicians, and other medical professionals to create electronic medical records that use a common language. the systematized nomenclature of medicine, snomed, was initially created by the college for human medicine but has sincethrough the partnership with avma -expanded to include veterinary terms. on july , , the department of health and human services announced it would make snomed available nationally at no charge, a step toward instituting a standardized electronic medical records system. the hhs signed a -year, $ . million contract with the college to license snomed and make it available nationally. the national library of medicine is administering the program. prior to the hhs's agreement with the avma, practitioners in areas of practice nationally on the front line of surveillance would have had to pay a $ to $ annual registration fee to access snomed. the cdc has expanded the role, scope, and influence of veterinarians and veterinary scientists and epidemiologists in public health since its inception in [ ] . early in the history of cdc, veterinarians in the u.s. public health service and the cdc veterinary public health division helped reduce zoonotic diseases, especially rabies and food-borne illnesses [ ] . today, veterinarians serve throughout cdc in positions that address not only infectious diseases but also the entire spectrum of public health challenges: environmental health, chronic diseases, human immunodeficiency virus infection and acquired immunodeficiency syndrome, injuries, immunizations, laboratory animal medicine, global health, migration and quarantine, health education, and bioterrorism. veterinarians contribute as epidemiologists, laboratory scientists, policymakers, researchers, and surveillance experts and in environmental and disease prevention and control programs both domestically and globally. at cdc, veterinarians have participated in the epidemic intelligence service since [ ] . forty-one states now have state veterinary public health officials. in , almost students and faculty attended the first veterinary student day at cdc; in april , cdc will co-host an inaugural conference with the association of schools of public health and association of american veterinary medical colleges. in addition, cdc has been recognized as a world association for animal health collaborating center for emerging and re-emerging zoonoses. the cdc publication, emerging infectious diseases, has highlighted zoonotic diseases in nearly every issue to zoonotic diseases and has devoted an annual issue in each of the previous years. the cdc has provided an important scientific forum for zoonotic disease research and programs both domestically and globally and should serve as a template for the nih, as will be discussed later in this paper (section . ), for moving these highly trained and broad-based medical skill set professionals from a more decentralized setting into a central institute at the nih. benjamin franklin's famous quote, "an ounce of prevention is worth a pound of cure" was actually fire-fighting advice-he founded the first fire fighting organization in philadelphia, its obvious application to medicine, although obvious, has not been a mainstay of heavily invested research and development in human health practices. many of the leading causes of death and disability in the united states can be prevented [ ] . primary prevention can prevent or arrest the disease process in its earliest stages by promoting healthier lifestyles or immunizing against infectious disease [ ] . secondary prevention, by detecting and treating asymptomatic risk factors or early asymptomatic disease, can substantially reduce subsequent morbidity or mortality. the human and veterinary clinician plays a pivotal role in both primary and secondary prevention. health professionals deliver vaccinations, screen for modifiable risk factors such as high blood pressure and high cholesterol, counsel patients about smoking and other behavioral risk factors, provide screening tests for early detection of cancer and other chronic conditions, and advise patients about the benefits and risks of preventive therapies such as postmenopausal hormone replacement therapy. the preventative health care landscape has changed in some regards in the years since the u.s. preventive services task force (uspstf/task force) was first established in to provide advice about prevention for health professionals. prevention became more of an integral component of primary health care [ ] . delivery of clinical preventive services such as immunizations, mammograms, and cholesterol screening has risen steadily over the past two decades (national center for health statistics [ ]). roughly % of employers now include well-child visits, childhood immunizations, screening tests, and adult physical examinations among covered health benefits, compared to less than half that did so in [ ] . interest in prevention grew significantly among the public, clinicians, educators, employers, and policymakers [ ] and health plans and individual clinicians were increasingly being held more accountable for the quality of the preventive care they provide to their patients [ ] . at the close of the th century, health care costs in the united states continued to rise steadily, accounting for . % of the gross domestic product in [ ] , and debate on health care funding for the aging american population intensified. no doubt fueled by the incredibly imbalanced historic spending in preventative healthcare of cents for every cents spent for curative treatment [ ] . numbers are harder to come by for recent years, but given the spiraling costs of treatment since it is likely that this ratio has gone down considerably since then-possibly grossly estimated to be closer to : today. in this environment, preventive services often compete with one another and with diagnostic-and treatment-oriented care for increasingly constrained resources [ ] . while preventive services are often believed to save costs, delivery of most preventive services, with few exceptions (e.g. some immunizations), incurs net costs [ ] . evidence that us society clearly favors the cure (or treat) approach to disease over prevention can be shown in the following ways. first, though there is a shortage of preventive medicine specialists (public health, general preventive medicine, occupational medicine, and aerospace medicine physicians), in the us the number of residents in training in was less than . % of all residents, not sufficient for replacement or to fill the expanding demand for the specialty's skills and talents [ , ] . second, preventive medicine residencies and subspecialties in human and veterinary medicine are generally found in only graduate medical education programs not financed by cms or mainstream academic training programs. third, we believe that our preventive acts are only statistical, whereas our curative acts are certain. this mistaken belief perhaps derives from our sense that we have more control over cure outcomes than prevention outcomes-we think that we do cure, whereas we only facilitate prevention. this notion of doing vs. facilitating is an important one, because if we believe that our curative actions are more effective than our preventive ones then we will more likely act toward the more effective. the editor of the british medical journal, fiona godlee, expressed this well when she states, "because it is acted on healthy people, preventive medicine needs even stronger supporting evidence on benefits and harms than therapeutic interventions" [ ] . thus substantial gaps in the delivery of effective preventive care in the united states remained, however, because clinicians continued to face many of the same barriers that originally spurred the formation of the first uspstf. identifying effective interventions were and are difficult in prevention, where prospective controlled trials are often difficult to conduct. these studies come from the field of epidemiology which has changed remarkably during its growth in the past quarter century. one of those changes has been a mixed blessing of ever-increasing specialization among its practitioners at the cost of the generalist. this phenomenon has shaped the field and a partial explanation for this trend is found in the decline in the availability of training funds not focused on specific and general disease areas. without returning to the training of general conservation-medicine based epidemiologists, the needed trend associations and study designs that are needed to show the economic and public health returns related to preventative practices field will not be realized and in addition lose some of its ability to quickly respond to new and expected emerging public health challenges. conflicting recommendations from different organizations, further exacerbated by the advocacy positions of some groups, leave many clinicians uncertain about what to do. clinicians facing increasing time pressures in practice may question the value of some routine preventive interventions, as may employers and other payers struggling with accelerating health care costs. although more prevention information is reaching the public, the messages conveyed are often inconsistent and increasingly colored by commercial self-interest. clinicians may feel compelled to provide unproven or ineffective services because patients demand them or they fear being sued, but patients may find that insurance coverage for individual preventive services, especially new technologies, is inconsistent. the importance of clarifying what we know and do not know about the effectiveness of specific preventive services is as important in as it was in . although the uspstf was disbanded in with the release of the guide, the need to keep pace with the rapid growth in scientific evidence led to convening a second panel in . the second uspstf was smaller, with only members, eight of whom were primary care physicians. it refined the previous group's methods for reviewing evidence and making recommendations, and expanded the scope of topics. it adopted policies for disclosure of conflicts arising from financial interests, funding sources, or other affiliations. the work of the second uspstf was marked by strengthened ties with both federal and nongovernmental partners, including primary care subspecialty societies. the work of the second uspstf culminated in the publication of the second edition of the guide in , which covered over interventions in areas. by the time the second edition of the guide appeared, the environment for preventive medicine and evidence-based medicine had changed dramatically. managed care organizations, which had emerged as a dominant paradigm for delivering and paying for health care, included some preventive care among basic covered services more commonly than had traditional fee-for-service insurance. at the same time, the heightened competition spurred by managed care brought increased attention to costs and value of treatments with less attention given to prevention. the guide was frequently cited by health plans and systems of care in defending their health maintenance programs and benefits packages, and its recommendations informed many of the health plan employer data and information set (hedis) quality measures developed by the national committee on quality assurance for evaluating health plan performance but not integrated into cost saving practices by the insurance companies. the rapid progress towards universal vaccination coverage in the s and s has slowed in recent years. unicef funding for vaccination fell from $ million to $ . million between and [ , ] . global coverage of the diphtheria, tetanus, and pertussis (dtp ) vaccine has been at around % since [ ] . fifty-seven developing countries have yet to eliminate neonatal tetanus, and , babies died of the disease in . ten developing countries reported cases of polio in june , despite the massive (and largely successful) global effort to eradicate the virus [ , , ] . sixty-two percent of countries, meanwhile, had still not achieved full routine immunization coverage in , with gavi estimating that at least . million additional infants need to be reached to achieve full coverage. there are several factors behind this loss of momentum. although dramatic progress has been made in increasing worldwide vaccination coverage from below % to above %, the task has inevitably become harder now that the easiest-to-reach populations have been vaccinated. because these communities are more elusive, the average cost per vaccination has increased, and it may be that other apparently cheaper health interventions have become more attractive. there are many practical problems impeding vaccine delivery. delivering vaccines to patients requires functioning freezers and reliable transport to move the vaccines from port to clinic; clinics refrigerators (which in turn require a constant supply of energy); good roads and with access to people who need to be immunized; parents who know the value of vaccination; trained medical staff to deliver the dose; and sterile syringes. only % of vaccineimporting countries could guarantee vaccine safety and quality [ ] , while a further study of developing countries found that at least half of injections were unsafe [ , [ ] [ ] [ ] [ ] . the third factor behind the lack of progress in recent years is political. political disruptions have affected coverage in some areas. in somalia and congo, for example, where vaccination rates have fallen rapidly in the past decade, war and social breakdown have impeded public health campaigns, despite "vaccination days" in congo that temporarily halted fighting. gauri et al. have found that the quality of institutions and governance are positively correlated with vaccination coverage [ , ] . politics in the developed world have also played a part. according to a report by the us institute of medicine, in the us vaccine industry was forced to stop offering low-price vaccines to develop-ing countries following congressional hearings that "savaged" the industry for "allegedly subsidizing vaccines for the poor children of the world by charging high costs to us families and taxpayers". as the institute of medicine points out, this move was based on a flawed premise, as the us vaccines would have been developed anyway to protect american children and travelers. public perceptions of vaccination change-as coverage spreads through a community and it reaches a point at which those who are unvaccinated are highly unlikely to catch a disease because herd immunity has set in. at this juncture, it may be more rational for an individual to refuse vaccination in order to avoid any risk of side effects. with the oral polio vaccine, for example, there is a one in a million chance of paralysis, and in societies where mass vaccination has eliminated the disease, the risk of paralysis is greater than that of catching polio itself. what had once been a public and private good is now a public good but a private risk. as more and more people choose to avoid this risk, of course, overall coverage rates decline, and the community is once again exposed to the threat of the disease. public perceptions have been influenced by vaccine scares. controversy and the attendant bad publicity about the safety of vaccines have been abetted by incidents such as the withdrawal of half the us supply of flu vaccines in due to contamination at the manufacturer [ ] . in addition, alarms over the safety of vaccines such as that for measles, mumps and rubella (mmr), which some believe to cause autism, have further fanned the anti-vaccine movement's flames [ ] . in the us, disputes continue to rage about the scientific basis of such claims, but the preponderance of the evidence, according to the us centers for disease control (cdc), says that the mmr vaccine is safe [ ] . in response to these types of controversies in the us, the institute of medicine has called for independent oversight of vaccine safety studies to ensure the fairness and openness of the vaccine safety datalink program, which is overseen by the cdc. as one can see there are many complex factors that have to be considered when bringing vaccination programs into existence. the impact of vaccination of animal diseases on agriculture is typically assessed in quantitative terms-lost revenues; costs of eradication, decontamination, and restocking; and the numbers of affected farms, animals and humans. this approach can be applied universally to all outbreaks in all countries because it normally reflects the hard data supplied by large commercial operations and the estimates by relevant governmental agencies of small farmer impact [ ] . when used exclusively, however, it fails as a barometer, because it does not and cannot factor in the multi-dimensional character of major disease events-and the accompanying societal effects that often get lost when it comes to assessing the damage in developing countries. the quantitative approach must also be interpreted, and cannot be used "as is" for comparing impacts in developed and developing countries. further, while export trade losses in a developing country may be small in terms of the dollar amount, the impact upon its pre-epidemic market share is inevitably greater and more persistent. other impacts such as effects on human health and community stability tend to be more visible and last longer in developing countries, particularly at the village level where animal are husbanded primarily for the benefit of the immediate family, and often in impoverished circumstances [ ] . the consequences of animal diseases in domesticated birds and livestock can be complex and generally go well beyond the immediate effects on affected producers. these diseases have numerous impacts, including: • productivity losses for the livestock sector (e.g. production losses, cost of treatment, market disturbances). • loss of income from activities using animal resources (in such sectors as agriculture; energy; transportation; tourism). • loss of well-being of human beings (morbidity and even mortality rates; food safety and quality). • prevention or control costs (production costs; public expenditure). • suboptimal use of production potential (animal species, genetics, livestock practices). the most direct economic impact of animal diseases is loss of production and/or productivity, and ensuing income losses for farmers [ ] . however, if the economy depends on one or some of the vulnerable products, the impacts can be serious, and local food security can be threatened. the economic impact also depends on response strategies adopted by farmers and possible market adjustments. if the farm economy is diversified or if there are other opportunities to generate income, the impacts can be mitigated. the economic impact also depends on response strategies adopted by farmers and possible market adjustments. the loss of the farmer's "well-being" will generally be lower than the value of the lost product, except where the farmer has few alternatives or is wholly dependent on the affected product, which is quite often the case in developing countries. direct losses are the result of the disease itself (they may be very high when mortality rates are between and %), or from animal health measures (stampingout policies) [ , ] . in vietnam, one of the countries most seriously affected by the avian flu, almost million birds - % of the country's poultry population -had to be destroyed at an estimated cost of us $ million ( . % of gnp) [ ] . the smaller scale producers lost the least in absolute terms, but the most in relative terms, as the outbreak resulted in losses equivalent to upwards of times their daily income (from us $ a day or less). in africa, abortions caused by the rift valley fever virus not only affect birth rates, but also push human consumption of milk downward in the year following an outbreak [ ] . in the dairy farming sector in kenya, it is estimated that losses in milk production accounted for % of all losses caused by an outbreak of foot-and-mouth disease in the s. direct costs are generally well below the indirect costs of animal diseases and are directly linked to the rapid containment of outbreaks: case studies have shown that early detection and the implementation of appropriate measures in the event of an outbreak are essential to help minimize direct losses as much as possible. conversely, inappropriate control and eradication measures are at the root of such endemic situations, which are much more difficult, and infinitely more costly, to keep under control or eradicate. the livestock sector plays a significant role in the economic development of many countries and vaccination can serve as one of the most important means of assuring its health. as such the cost of not developing new and important or properly applied vaccines can have tremendous economic consequences. the production of meat and other animal-based food items generates income, jobs, and foreign exchange for all stakeholders in the animal industries. consequently, an epizootic which could have been otherwise mitigated by vaccination can affect the industry's upstream (inputs, genetic resources) and downstream activities (slaughterhouses, butchering operations, processing, marketing) in terms of jobs, income for the stakeholders in the industry, or market access. a survey by the food and agriculture organization of the united nations (fa ) on avian flu revealed that in the most seriously affected regions of indonesia, % of permanent workers at industrial or commercial farms lost their jobs [ ] . similarly, an outbreak of contagious bovine pleuropneumonia in botswana led to the destruction of more than , animals in the most seriously affected province, and the immediate closure of the export slaughterhouse, which employed persons. owing to the catalyst role of livestock raising in the rural economy as a whole, the costs of the indirect effects of these measures were later estimated to be seven times higher than the costs caused by direct losses [ ] . in vietnam, % of the poorest segment of the population, for which poultry farming accounts for - % of household income, is particularly vulnerable to income losses caused by avian flu. the fao and world organization for animal health (oie) estimate that between one-third and one-half of the populations living in the most seriously affected areas of southeast asia depend on poultry farming for at least a part of their income [ , ] . in france, the leading european poultry producer, it is estimated that farmers affected by the crisis lost % of their income in months (between january and march ). the effects of the production losses are also linked to price variations, which are caused by supply and demand (im)balances. depending on the market, prices can rise sharply (consumer product on the domestic market) or plummet (product banned for export but cleared for consumption on the domestic market, product deemed too dangerous for human consumption or perceived as such). in brazil, where % of products are exported, the price of a day-old chick, an early indicator of a possible change in production, reportedly fell by %. and even in cases where the country is not infected, market uncertainties and the fall in prices prompted the largest producers to cut back production by % this year. loss of access to, or the opportunity to access, regional and international markets generally have more significant economic implications than just production losses. in / , the rift valley fever outbreaks in east africa seriously affected pastoral economies in somalia, with a decline of more than % in exports (which generate more than % of foreign exchange in "somali land"), following an embargo declared by saudi arabia on all animal products from the horn of africa [ , ] . conversely, the world bank has reported that eradication of certain major diseases to facilitate access to "high value" export markets can provide considerable benefits. loss of access to, or the opportunity to access, regional and international markets generally have more significant economic implications than just production losses. uruguay is a good example of a country that gained access to a lucrative market after eradicating foot-and-mouth disease. beef exports increased in volume by more than % and in value by % after the oie declared uruguay to be officially foot-and-mouth disease-free without vaccination in . access to the u.s. market (where prices are double those of the domestic market) provides uruguay with additional revenue to the tune of us $ million each year. a medium-term analysis showed that access to "pacific rim" markets would generate additional revenue of us $ million each year, and yet, before the disease was eradicated, uruguay had been spending (only) us$ million to us $ million each year on vaccines to combat foot-and-mouth disease. in this case, control costs would account for less than % of the revenue generated by exports alone [ ] . animal diseases that could be controlled by vaccination can have major effects on food availability and quality for poor communities. it is well known that agriculture plays an important role in the generation of income and jobs in other sectors but the closeness of this interdependence became particularly obvious during recent epizootics. for pastoral societies, animal husbandry contributes directly and indirectly to food security and to nutrition as a source of quality proteins, vitamins and trace elements, traction, and com-mercially tradable products [ , ] . certain diseases could have significant repercussions on food supply and the nutrition of poor communities that do not have readily available substitute products, which could therefore lead to famine (rinderpest for example). poultry meat is the primary animal protein in africa (which has little to begin with) and the indispensable source of discretionary income for the survival of millions of small farmers. the high mortality rates as a result of avian flu, which is extremely pathogenic, and the sanitary slaughter of poultry would therefore have a negative impact on the food available to the entire population, as well as on rural revenue. furthermore, developing or transition countries which generally have poor public health systems are particularly at risk from zoonoses making vaccination against these diseases particularly important to target. in / a major rift valley fever epidemic in egypt resulted in , human cases and fatalities [ , ] . twenty years later, a new epidemic affected over , persons in east africa, and persons succumbed to the hemorrhagic form of the disease. but zoonoses also affected industrialized countries with high health standards as was the case with the bovine spongiform encephalopathy crisis in europe [ ] . food-borne diseases (over have been classified) are a major source of acute gastroenteritis (which costs the netherlands us $ million per year) and the cause of major morbidity with fatalities among children in the third world [ ] . in the specific case of a pandemic, most of the economic loss is caused by the increase in morbidities and fatalities in the human population and its repercussions on the world economy. the most recent estimates suggest that the "spanish" influenza in caused the death of million persons, that is, . % of the population at the time. the most obvious economic losses were the reduction in quantity and productivity of the workforce, and according to the experts, in the case of a pandemic could represent times more than all the other losses combined [ ] . another category of economic impact is linked to individual strategies to avoid contamination-or to survive possible contamination. the example of the severe acute respiratory syndrome (sars) clearly shows the sharp drop in demand in the services sector (tourism, public transport, retail trade, hospitality and food services) resulting from the combined efforts of individuals to avoid any close contact [ ] . based on the experience with severe acute respiratory syndrome in south-east asia, the world bank thinks that an avian flu pandemic could result in a % loss of the world's gross domestic product and cost the world economy us $ billion in the space of year. the losses are difficult to calculate and would undoubtedly be much more significant in light of the extremely high mortality rates in developing countries which do not have good health care systems. the impact of animal diseases on the tourism and leisure sectors could also be quite significant. the negative effect of foot-and-mouth disease in the united kingdom on these two sectors amounted to us $ billion because of restrictions on access to rural areas and represented more than half of the total cost of the disease [ ] . the federation of american scientists' animal health/emerging animal diseases (ahead) project proposed a major program in sub-saharan africa to detect and document the extent of infectious diseases shared by farm and wild animals, and to supply treatment, prevention and control services to remote communities that have previously been neglected by other programs, both national and international. this program, international lookout for infectious animal disease (iliad), was implemented in south africa [ ] . at the core of iliad is the need for a permanent and sustainable regional program of in situ surveillance designed to detect, monitor, treat, prevent and control infectious diseases with the goals of increasing livestock production in remote farming communities, protecting the health of wild species, building indigenous physical and professional resources, and introducing communications and epidemiology information technologies. transmission of infectious diseases is rampant in remote communities in the sub-saharan region, just as they once were in the united states and as they always are wherever poverty and farming co-exist. diseases shared by wild, farmed and captive/bred animals, and by animals and humans, suppress food production, frustrate species preservation efforts and greatly affect public health. detection, prevention and control of these diseases are an essential element in expanding trade, improving nutrition, exploiting ecotourism and ensuring food security. iliad is structured in the investor mode-an international consortium of donor groups providing short-term developmental assistance with program direction and oversight provided by veterinary diagnostic, public policy and epidemiology experts representing the sub-saharan africa partnership members-the renowned onderstepoort veterinary and exotic disease institutes (ovi) and tuskegee university (tu), and fas-ahead. given positive assessments of the benefits of the program after years, national or provincial institutions will integrate some or all of the activities into their official veterinary and agricultural activities. it is difficult to calculate the cost of the public's loss of confidence in animal industries in their countries, or of an importer country towards the veterinary services of the exporter country. animal diseases can have major effects on food availability and quality for poor communities. consumers' obsessive fear of bovine spongiform encephalopathy (mad cow disease), fed by the media and which a good communication strategy could have prevented, would have tremendous social repercussions on a europe still reeling from long term economic repercussions. in italy, the baseless perception of a food risk related to avian flu coupled with low confidence in public health services eventually resulted in a % reduction in the consumption of poultry and eggs. the loss of confidence by an importer country can trigger a lasting embargo and major economic and social repercussions (arabian peninsula embargo on the horn of africa, affected by the rift valley fever virus). loss of access to, or the opportunity to access, regional and international markets generally have more significant economic implications than just production losses. animal diseases might also have indirect long-term impacts, affecting deferred productivity. this is the case for example of the reduction in the fertility rate of long-cycle species, the effects of which span periods of - years [ ] . in short, the long-term costs of a slow response are rarely taken into account. economic analyses focus primarily on the effects of the outbreaks and rarely take into account the long-term effects of an endemic situation (characterized by less virulent outbreaks which recur for several years). this is the case of classic swine fever in haiti where recurrent outbreaks reduced the usage rate by %, which for pig farmers meant a loss of revenue of us $ . million per year [ ] [ ] [ ] . with major crisis, long-term impacts would make themselves felt, since the additional costs of financing prevention and control measures would lead to an equivalent reduction in savings and investments. for example, the analysis of the global impact of the avian flu crisis in europe was complicated by outbreaks of foot-and-mouth disease in brazil, the largest global exporter of beef and poultry. it is therefore easy to imagine what the combination of these two events would mean in terms of the upward push of prices of all meats, similar to what occurred in with north american beef and bovine spongiform encephalopathy. the european union, a net importer of beef, especially from brazil, would see an increase in the price of beef in its internal markets stemming from the embargo imposed on brazilian beef because of the foot-and-mouth disease. it must be pointed out that the crises could have a cumulative impact, particularly since they are amplified by the effects of globalization the following example therefore illustrates the ripple, spillover and remote effects: in the united states, where % of oleaginous and cereal production is geared towards animal production. an epizootic which reduces animal production by % would have the immediate consequence of the loss of , jobs, a surplus of . ton in cereals and oleaginous products, a % reduction in world trade and, crises in other producing countries. in , nearly americans out of every , died each year of infectious disease. laurie garrett, author of "the coming plague: newly emerging diseases in a world out of balance", writes that in the postwar environment, powerful medical weaponry (antibiotics, vaccines, water treatment, anti-malaria drugs) gave scientists confidence that they could significantly control and/or eradicate infectious disease from viral, bacterial or parasitical sources. in the late s, the surgeon general of the usa, william h. stewart, said that ". . .it was time to close the book on infectious diseases and pay more attention to chronic ailments such as cancer and heart disease." a measure of that success came towards the end of the s, when the world realized that smallpox had become the first disease to be eradicated from the human species. such halcyon days from the s to the early s are but a memory. by , the numbers were down to per , . the "health for all" accord, signed in , set a goal of the year for eliminating many international scourges. but amid all this optimism, the numbers started rising. in , people per , died and we know the rest of the story, . . . or do we? the grandiose optimism rested on two false assumptions; that microbes were biologically stationary targets, that for the most part human and other animal diseases were for the most part limited to those species and geographically sequestered. scientists have witnessed an alarming mechanism of microbial adaptation and change, anything but stationary, microbes and the insects, rodents and other animals that transmit them are in a constant state of biological, ecological flux and evolution [ ] . according to the u.k. centre for tropical veterinary medicine, - % of all the known species of infectious organisms that affect human health (causing a quarter of the world's deaths) can be transmitted by animals. approximately of these infectious organisms are linked to diseases that have only recently emerged, or have increased in severity (and geographic distribution) in recent years. who averages outbreak investigations every year, and around will require an international response. more than new and highly infectious diseases have been identified in the last years. furthermore, known strains of diseases such as tuberculosis, many species of gram positive and negative bacteria as well as many parasites, e.g. malaria, food animal coccidia have developed resistance to various classes of antibiotics, while old diseases have reappeared, such as cholera (in angola, with deaths), yellow fever (new cases recently reported in guinea, sudan, mali, and senegal), plague, dengue fever, meningitis, hemorrhagic fever, measles, mumps, rubella and diphtheria. there are emerging diseases just among marine life, reports the book conservation medicine, and these include tuberculosis in fur seals and chlamydiosis in sea turtles. more staggering than these cited numbers in living animals including humans are those coming out of the microbial genetic sequencing and diversity studies involving the oceans. recent data in this field suggest that the oceans of the world contains approximately ( ) phage particles or virions (cf. million metric tons), much of it turning over once per day and most likely be a regular source for current and future zoonotic and human infections. this vast mutation engine, even if one assumes a minimal mutation rate, generates the equivalent of hundreds of new complete human genomes per day. a -l sample of surface seawater was concentrated; ∼ × ( ) viral particles, the dna once randomly sheared and cloned yielded , fragments for sequencing. data analysis showed that most of the sequences were from previously unknown viruses. approximately . % of the total sequence samples overlapped, suggesting that the marine viral community was highly diverse. a unique mathematical analysis further suggested that approximately ( ) different new viral types may be present. it is obvious from this most recent description that complex and confounding zoonotic interactions can be expected to occur as ecosystems become concentrated and/or diluted during the upcoming environmental and ecological change. this will require a new breed of medical scientists that have foregone the days of super-and sub-specialization and are now grounded in both depth and broad general eco-and bio-medical systems training. few people recognize the broad range of clinical and basic veterinary research and its many important contributions to society in the realms of public health and food safety, vaccination, fertility, drug and vaccine development, surgical techniques and biodegradable materials, space medicine, animal health and welfare, and comparative medicine [ ] . opportunities in veterinary research include comparative studies with animals that shed light on human health problems; the development of tools to better detect, prevent, and control zoonotic diseases (that spread from animals to humans); the establishment of scientifically based policies for the humane treatment of animals; and the development of measures to secure and protect the nation's food supply and farm-animal economy from a potential act of bioterrorism [ , ] . the new complex problems of the new millennium will require new educational models that train para-and professional people for thinking and solving complex inter-related biological, ecological, public-economic problems. the single profession that is most centered on the new paradigm is veterinary medicine. the three major disciplines within veterinary medicine and research -public health, comparative clinical and basic medicine, and animal health -are closely intertwined [ , ] . for example, research in comparative medicine contributes to animal health through the development of preventive medicine and treatment. the study of wildlife diseases contributes not only to wildlife health and conservation, but also to public health because many animal diseases can spread to humans. therefore, collaborative and interdisciplinary research is crucial in translating scientific advances from one traditional discipline to another. however, interdisciplinary research is in many cases hampered by administrative, funding, and cultural barriers between institutions. furthermore, agencies that support veterinary research have specific missions. funding to support proposed interdisciplinary research can be difficult to obtain when it is partially related to the mission of several agencies but does not perfectly fit the mission of any one agency. the future requires the veterinary research community to encourage research funders to develop a long-term interagency strategy for veterinary research [ , , ] . in , rudolph virchow, the father of comparative medicine, stated, "between animal and human medicine there are no dividing lines-nor should there be. the object is different but the experience obtained constitutes the basis of all medicine" [ ] . sir william osler ( - ), considered the best-known physician in the english-speaking world at the turn of the century, called the "most influential physician in history" is quoted as saying "veterinary medicine and human medicine complement each other and should be considered as one medicine." dr. calvin schwabe, a retired uc davis professor of veterinary medicine who pioneered the use of human disease tracking techniques in the study of animal illnesses, a global authority on animal diseases that are communicable to human beings and was an early visionary in a field that today is marked by the emergence of pathogens such as avian influenza, mad cow disease and sars. in , dr. schwabe established the department of epidemiology and preventive medicine at the uc davis school of veterinary medicine-the first of its kind in the world at a vet school. the author of more than publications, he promoted the concept of "one medicine," which attempts to bring the fields of human and animal health care together. to this goal in june of the american veterinary medical association (avma) announced that in partnership with the american medical association (ama) there was an adopted resolution calling for collaboration on a one health initiative. the two national, medical organizations will work collaboratively on areas of mutual medical interest, such as pandemic influenza, bioterrorism risks, biomedical research and will be charged with developing strategies to promote collaboration among the various health science associations, colleges, government agencies and industries. a quote from a ama board member, duane m. cady, md "new infections continue to emerge and with threats of cross-species disease transmission and pandemic in our global health environment, the time has come for the human and veterinary medical professions to work closer together for the greater protection of the public health in the st century," the avma policies supporting the concept of "one health" include: furthermore to strengthen this initiative and give it a renewable funding foundation, it would be recommended to move toward, the creation of: ( ) a specific focus at the national institutes of health (nih) on integrated veterinary research via the roadmap initiativemore specifically to create or combine existing potions of institutes into an institute of comparative medicine (icm); ( ) a joint interagency collaborative programs could also be established to enhance interdisciplinary collaborative research and have either re-routed an/or new congressionally supported intra-mural and extra-mural funding program; and ( ) while working out and introducing the legislative changes for the icm the nih should create a veterinary liaison with all the current institutes like the veterinary-medicine and public-health liaison at the centers for disease control and prevention (cdc) in which a veterinarian, dr. lonnie j. king, was selected to head up the agency's national center for zoonotic, vector-borne, and enteric diseases-a relatively recent creation, the center is dedicated to understanding infectious disease ecology and will help to ensure integration of veterinary and human medical research. the american veterinary medical association, with information provided by many other organizations and institutions, conservatively estimates a current deficit of public health veterinarians (e.g. usda food safety and animal disease control, homeland security, research on domestic and foreign animal diseases, wildlife disease control, laboratory animal care and research) and is expected to increase possibly to , by as the human population increases without intervention. comparably the health resources and services administration (hrsa) in the u.s. department of health and human services (dhhs) released a report in , projecting a shortfall of approximately , physicians in [ ] . if current trends continue, the full time equivalent (fte) physician supply is projected to grow to , by , while demand for physicians will increase to , due to the growth and aging of the u.s. population (physician workforce policy guidelines, ). the report projects shortages will be in greatest in non-primary care specialties. it has been over years since the federal government has allocated funds to increase the number of veterinarians and physicians that graduate each year. to begin to alleviate this problem the veterinary workforce expansion act (s. /h.r. ), which is currently being considered by the united states house of representatives and senate would provide an average of $ million per year for the next years in the form of competitive grants to help increase the number of veterinarians entering public practice. if passed and funded, this bill would allow the nation's veterinary schools and other institutions training public health veterinarians to apply for competitive grants to increase capacity in the form of classrooms, teaching laboratories, research facilities, and administrative space. this bill would be vital to the nation's ability to protect human and animal health, as veterinarians are often the first line of defense for both. the text of the bill can be found at: http://thomas.loc.gov by searching by the bill numbers listed above. a list of co-sponsors to the bill can also be found via that site by selecting the link entitled "bill summary & status". over the past century, humanity has had a devastating impact on the earth's wildlife and ecosystems. we are in fact living through the largest mass extinction since the end of the dinosaurs million years ago. unless effective solutions are found, this new century will see the demise of countless more species and pristine ecosystems, particularly in the tropics. the global society, and what surrounds and influences it, are in profound change. these changes will have very significant impacts on future veterinary medicine and veterinary medical education. there are major demographic, political, environmental, disease, technological, and economic influences, all forcing changes onto society. a few examples illustrate the point [ ] . • with immigration into north america accelerating, combined with a declining birth rate, the ethnic diversity in society will continue to increase, with the associated impact on values. • in , for the first time in history, urban people will outnumber rural people. • political destabilization, inflamed by bio-terrorism and religious fanaticism, is expected to increase. • changes in the atmosphere are causing powerful shifts in the environment (melting of the ice caps, rising sea levels) and in the climate (hurricanes, flooding). • global water shortages, especially in heavily populated areas, will soon approach critical levels. to information and service (http://www.jvmeonline.org/cgi/ content/full/ / / #b #b ). • consumer spending power in emerging economies will go from $ trillion to $ trillion by , but the gap between rich and poor is increasing. how will these changes alter the needs of society? how must academic veterinary medicine adapt to prepare veterinarians to respond to these new needs? clearly, humanity has yet to find a way to live on planet earth in a potentially sustaining manner where by stabilizing flora and fauna remain intact to promote healthy ecosystems diversity for all species including our very ownhumans. for example, it is estimated that the equivalent of six earths would be needed to sustain the current world's population if people everywhere consumed natural resources at the rate we do in the united states. it is interesting to think that vaccination directly or indirectly impacts six of the seven united nations millennium goals. understanding and coping effectively with an emerging crisis may sometimes require the birth of action-oriented "crisis disciplines." conservation medicine: ecological health in practice brings together an impressive group of experts from diverse specialties medicine, veterinary science, conservation biology, epidemiology, parasitology, public health, and others) to examine the links among human health, wildlife health, and ecosystem health and begin to address questions like: • how factors such as climate change, endocrine disruptors, and toxic microalgae affect wildlife and human health; • the importance of biodiversity for human health (as medical models, sources of medicines, factors in the ecology of infectious diseases, and indicators of environmental quality), with a review of biodiversity-related biomedical research projects funded by the national institutes of health from to ; • how the health of rainforest-dwelling peoples depends on such diverse factors as forest integrity, floods, seasonality, community organization, education, gender dynamics, national budgets, and global markets; • how wildlife health relates to environmental security; • the health hazards of ecotourism; • the causes and impacts of emerging infectious diseases of humans and wildlife; • how the health of terrestrial and marine animals and ecosystems are monitored, and descriptions of innovations using stool dna and retrovirus evolution as markers of animal population dynamics, stool hormones to indicate species stress, and animal behaviors as proxies for the health of ecosystems; • how habitat fragmentation and reduced biodiversity can increase the risk of lyme disease infection; • how land use changes such as deforestation and water projects influence the ecology of malaria and other vector-borne infections; • how ecological health and wildlife disease are managed in national parks; • the role of zoos in the recovery and conservation of endangered species; • how reducing the burden of infectious disease among park workers in africa could prevent a devastating epidemic among the world's remaining mountain gorillas; • how efforts to control livestock diseases are affecting wildlife health and ecosystems in botswana; • teaching ecosystem health in an undergraduate medical curriculum. more than one billion poor people in asia and africa are closely linked with animals for their livelihoods and they pay a really high tribute to different animal diseases-one can easily demonstrate that improving animal-health mechanisms at the national and local level, and decreasing this weight of animal diseases, will lead very quickly to the alleviation of poverty for this class of people. the world organization of animal health (oie) brings together chief veterinary officers from countries in an effort to create global standards of animal health and animal welfare robust enough to withstand the daunting challenges of worldwide commerce. with an annual budget of around $ million, it is a significantly smaller organization that its human-health counterpart, the world health organization, which has some $ billion a year expenditure on its programs. last year, over billion food animals were produced worldwide to help feed a population of billion people, resulting in trillions of pounds of animal products distributed worldwide and projections for show that demand for animal products will increase by %, especially in developing countries. this poses unprecedented risks for human safety and will require a closer linking of the two agencies. along with those core concerns, oie also addresses farming practices, analyzes import risks, and mediates bilateral trade disputes among its member countries. among its most important achievements in recent years, has been the eradication of rhinderpest, a centuries-old intestinal disease also known as "cattle plague in some countires." in the early s, rhinderpest wiped out upwards of $ million worth of african livestock, contributing to widespread human famine. in general professional students of veterinary and human medicine are exposed more likely to a more scientifically exposed than a politically exposed culture. animal and human health policies must be science-based the rallying cry that is heard from animal and human health professionals around the world. the second verse of this mantra is often 'science, not politics', as if science is unquestionably 'good' and politics 'evil'. antipathy toward politics is worn as a badge of honor. the crowning moment frequently comes with the proclamation, 'i am a scientist, i want nothing to do with politics. ' the phrase 'science-based' infers that the underlying justification of animal/human health policy is derived from knowledge gathered through the systematic observation of, or experimentation with, phenomena. scientific knowledge implies the compilation and analysis of data by individuals with advanced education in specific disciplines. scientists seek facts, the fundamental truths which explain the world around us. at face value, the 'science, not politics' paradigm has a great deal of appeal. however, the very notion of fundamental truth is illusory, as scientific knowledge changes frequently with new observations and experiments. furthermore, conjecture and refutation characterize the scientific method, with disagreement and debate the recognized features of scholarly pursuit. scientists often reach conflicting interpretations of observational and experimental data. consequently, individual scientists may champion different, even diametrically opposed, sets of ideas and principles, so that any number of alternatives may be justified as 'science-based'. finally, animal health professionals typically consider only the biological and physical sciences as 'true sciences', dismissing the social sciences. politics reflect the human need for organization of authority, whether in public or private life. politics exist whenever two or more people come together. the terms 'office politics' and 'family politics' are recognized as clearly as the collective activities surrounding local or national governance. politics exist even in science, affecting scientific organizations, refereed publications and academe. indeed, politics are inescapable. all public courses of animal/human health action adopted by governments emerge from the interplay of science and politics. the policy-making process is governed by rules and regulations, affected by the organizational culture of the government agencies involved, and constrained by legal authorities, political correctness and resource availability. the animal/human health policy-making process involves consideration of current biological and physical scientific knowledge. policy decisions also consider social science factors including ideologies, economics and public opinion. hence the etiology of animal/human health policy is multifactorial. the current older traditional schools of veterinary and human medicine and future schools of "one medicine" will need to include leadership, economics and local, state, national and international governances courses and better training in mechanisms of public policies and rule making. epidemiologists accept the concept of multifactorial etiology as a basic tenet. we discuss disease in terms of agent, host and environment interactions. the practice of applied epidemiology demands a breadth of knowledge and the ability to work in interdisciplinary teams. the more complex the problem, the greater the demand for additional knowledge and insights. veterinary and human epidemiologists study risk factors for disease in animal/human populations and develop strategies for health promotion and disease control [ ] . unfortunately these are done frequently in a vacuum when in fact the two are inter-related and contributing to the observed and at the time undiagnosed disease spectrum however, animal/human health problems cannot be resolved by consideration of biological and physical factors alone. the veterinary and human (someday to become one and the same) epidemiologist working with field problems soon recognizes the critical role played by people in animal/human health issues. applying epidemiological principles to animal/human disease prevention requires consideration of social issues such as attitudes toward animals, cultural and religious mores, and individuals' willingness and capability to implement the prevention strategies. implementing prevention on a national scale brings additional factors into the equation such as availability of resources, adequacy of veterinary services, and animal health infrastructure, among others. therefore, animal health policy development and imple-mentation require attention to macroepidemiology, the study of all of the economic, social and political inputs which affect the distribution and impact of animal or human disease at the national level [ , ] . the low hanging fruit of yesterdays fields of microbiology, zoonotic and emerging infectious diseases, immunology, antibiotic sensitivity, vaccine development, oncology, anti-viral drugs, public health, ecology, environment, human and animal health, to name a few have been picked. today comparative and interdisciplinary research is critical to translating scientific advances from one discipline or species to another and providing new insights into human health problems. scientific fields such as laboratory animal medicine, pathology, immunology, biophysics, mathematics, bioinformatics, genetics, molecular biology and toxicology, when combined with veterinary medicine, have proven especially relevant to success in biomedical research. veterinarians also have contributed to public health through the care of companion animals. fifty-seven percent of all u.s. households own a dog, cat, or both. in addition, millions of exotic animals, birds, and reptiles are kept as pets [ ] . although pets enrich the lives of humans, they also potentially can threaten public health. veterinarians help educate the public about prevention of zoonoses; vaccinate large numbers of pets for zoonotic diseases, such as rabies and leptospirosis; and reduce the level of ecoparasites that can transmit human diseases and intestinal worms, such as roundworms and hookworms, which can cause serious health problems in humans. the , private-practice veterinarians in the united states form a valuable front line for detecting adverse health events, reducing zoonotic diseases, and delivering public health education. because veterinarians work at the interface of human, animal, and environmental health, they are uniquely positioned to view this dynamic through the lens of public health impact. significant changes in land use, expansion of large and intensified animal-production units, and microbial and chemical pollution of land and water sources have created new threats to the health of both animals and humans [ ] . because animals share human environment, food, and water, they are effective sentinels for environmental, human, and public health problems, including bioterrorism. concerns are increasing about antimicrobial resistance of pathogens, waste and nutrient management, and potential runoffs into streams, rivers, and oceans. food animal and wildlife populations are inextricably linked to some environmental problems. together these have led to creation of a new scientific discipline called conservation medicine and ecosystem health, and veterinarians are assuming a leadership role in the field [ ] . several decades ago, special factors came together to create a new epidemiologic era characterized by increases in emerging and reemerging zoonoses [ ] . humans, animals, and animal products now move rapidly around the world, and pathogens are adapting, finding new niches, and jumping across species into new hosts. in , approximately billion food animals were produced to help feed a world population of . billion persons; the united nations' food and agriculture organization estimates that demand for animal protein will increase by % by , especially in developing countries [ ] . the lessons learned from severe acute respiratory syndrome, west nile virus, monkeypox, and avian influenza are reminders of the need to view diseases globally; integrate animal and public health surveillance, epidemiology, and laboratory systems; and create new strategic partnerships among animal, human, and public health professions [ , ] . veterinarians are essential to the detection and diagnosis of and response to these threats and are integral to first-line defense and surveillance for bioterrorism agents. the convergence of human and animal health drove creation of the newly proposed national center for zoonotic, vector-borne, and enteric diseases. plans are being completed to establish several multidisciplinary state-level zoonosis research and development centers. the veterinary profession has recently gained a important foothold on the health and human services government research institutes at the cdc and evolved in prominence as a member of the health professions and has established its importance and usefulness to human and public health. it is hoped and expected that with the developing visions and challenges outlined in this overview we will see the continued melding of veterinary and human medicine to create new educational programs and tools for developing future leaders for solving globally some of the most challenging public and animal health, ecosystem, and conservation problems of the st century. the value of vaccination world health organization. measles deaths drop dramatically as vaccine reaches world's poorest children the value of vaccination: a global perspective intervention cost-effectiveness: overview of main messages the costs and benefits of front-loading and predictability of immunization. cgd working paper veterinarians in population health and public practice: meeting critical national needs cattle, priests and progress in medicine veterinary medicine: an illustrated history committee on assessing the nation's framework for addressing animal diseases. national research council. animal health at the crossroads: preventing, detecting, and diagnosing animal diseases veterinary medicine and public health at cdc the th anniversary of the veterinary medical corps officers of the u.s. public health service veterinarians and public health: the epidemic intelligence service of the centers for disease control and prevention actual causes of death in the united states primary care: balancing health needs, services, and technology mercer survey of employer-sponsored health plans recommendations to the congressional prevention coalition: nine high-impact actions congress can take to protect and promote the nation's health hedis : the health plan employer data and information set and the health accounts team health spending in : signals of change effectiveness in disease and injury prevention estimated national spending on prevention-united states disease prevention research at nih: an agenda for all. workshop j: prevention strategies, economic realities, and identification of prevention research needs dissecting cost-effectiveness analysis for preventive interventions: a guide for decision makers us graduate medical education editor's choice: preventive medicine makes us miserable immunization in developing countries: its political and organizational determinants progress towards immunization goals : summary presentation of key indicators global market-global vaccines. immunization focus global market-global vaccines. immunization focus national academy of sciences. america's vital interest in global health vaccine safety: real and perceived issues the status and role of vaccines in the u.s. food animal industry: implications for biological terrorism economic and social consequences of animal diseases issue paper: global risks of infectious animal diseases tending animals in the global village: a guide to international veterinary medicine appendix a. global status of livestock and poultry species risk factors for human disease emergence summary of probable sars cases with onset of illness from lessening the impact of animal disease on developing country agriculture: a proposed program using developed country technologies sustainable agriculture solutions-action report veterinary medicine and human health envisioning the future of veterinary medical education: the association of american veterinary medical colleges foresight project, final report science, politics and animal health policy: epidemiology in action* the current and future market for veterinarians and veterinary medical services in the united states potential of cooperation between human and animal health to strengthen health systems emerging zoonoses and pathogens of public health concern microbial threats to health: emergence, detection, and response livestock to : the next food revolution. food, agriculture, and the environment discussion paper learning from sars: preparing for the next disease outbreak. workshop summary confronting zoonoses, linking human and veterinary medicine conservation medicine: ecological health in practice the burden of vaccine-preventable diseases making markets for vaccines: from ideas to action diseases of humans and their domestic mammals: pathogen characteristics, host range and the risk of emergence committee on the national needs for research in veterinary science. national research council. critical needs for research in veterinary science committee on increasing veterinary involvement in biomedical research. national research council. national need and priorities for veterinarians in biomedical research animal agriculture and the global food supply. task force report . ames, iowa: council for agricultural science and technology council on graduate medical education. physician workforce policy guidelines for the u.s. for towards a better map: science, the public, and the media. economic and social research council the general epidemiologist: is there a place in today's epidemiology? misleading media reporting? the mmr story vaccine delays hit chiron's recovery. la times ten great veterinary public health/preventive medicine achievements in the united states advance market commitments: a policy to stimulate investment in vaccines for neglected diseases historical comparisons of morbidity and mortality for vaccine-preventable diseases in the united states priorities in developing countries surveillance and monitoring systems for animal health programs and disease surveys committee on assessing the nation's framework for addressing animal diseases, national research council. animal health at the crossroads: preventing, detecting, and diagnosing animal diseases. washington sustainable agriculture solutions (sas)-action report the annual report the annual report understanding and improving health and objectives for improving health. vols an agenda for action: veterinary medicine's crucial role in public health and biodefense and the obligation of academic veterinary medicine to respond investing in health immunization at a glance immunization at a glance world development indicators polio eradication website, global case count measuring the national economic benefits of reducing livestock mortality the primary author wishes to acknowledge and dedicate this paper to the late dr.'s jonas salk and maurice hilleman whose lifetime experiences and contributions are unprecedented in medicine and vaccinology and whom personally encouraged and instilled in me during numerous public and private discussions and meetings the importance of working in and helping to advance the field of vaccinology for the betterment of global human and animal health. key: cord- - fh pe i authors: reyes, leticia; reinhard, mary; brown, mary b title: different inflammatory responses are associated with ureaplasma parvum-induced uti and urolith formation date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: fh pe i background: epidemiologic studies show a strong association between ureaplasmas and urogenital tract disease in humans. since healthy humans can be colonized with ureaplasmas, its role as a pathogen remains controversial. in order to begin to define the role of the host in disease, we developed a rodent model of urinary tract infection (uti) using fischer (f ) rats. animals were inoculated with sterile broth, ( ), ( ), ( ), ( ), or ( )log cfu of a rat-adapted strain of ureaplasma parvum. results: infected animals exhibited two distinct profiles, asymptomatic uti and uti complicated with struvite urolithiasis. inoculum dose of u. parvum affected the incidence of uti, and % to % of animals inoculated with ≥ ( )cfu of u. parvum remained infected (p < . ). however, inoculum dose did not influence immune response to u. parvum. asymptomatic uti was characterized by a minimal immune response that was predominantly monocytic and lymphocytic, with limited lesions, and elevated urinary levels of ifn-γ, il- and mcp- (p ≤ . ). uti complicated with struvite formation was characterized by an exaggerated immune response that was mostly neutrophilic (p ≤ . ), with lesions that showed extensive uroepithelial hyperplasia (p ≤ . ), and a predominance of il- α, il- β, and gro/kc in the urine (p ≤ . ). animals with asymptomatic uti also had a significantly high rate of kidney infection (p ≤ . ). conclusion: complications associated with u. parvum infection are primarily dependent upon host-specific factors rather than ureaplasma microbial load. the immune response in f rats is similar to that which occurs in humans with ureaplasmal associated disease. therefore, this model of infection is a useful tool for elucidating u. parvum-host interactions that confer uti and disease. vated pro-inflammatory cytokines, most notably il- α, il- β, il- , il- , mcp- and tnf-α, accompanied by infiltration of neutrophils and macrophages at sites of infection [ , [ ] [ ] [ ] . however, little work has been done to characterize the immune response during uncomplicated infections. therefore, the complex interactions between ureaplasma and the host that lead to simple colonization versus inflammation and disease are largely unknown. in a recent study, we showed that the inbred rat strain fischer (f ) is susceptible to uti induced by a rat adapted strain of ureaplasma parvum isolated from the urine of a patient with recurrent uti [ ] . as part of that study, we found that % of infected f rats developed struvite uroliths, which were associated with an exaggerated inflammatory response that is similar to what has been reported in other disease states caused by ureaplasma infection [ , , , , ] . interestingly, the other % of f rats developed uncomplicated uti that was characterized by low concentrations of pro-inflammatory cytokines in urine as well as mild to moderate lesions in the lower urinary tract. since f rats are an inbred strain, this particular infection model would be useful for identifying the host/ureaplasma interactions that confer disease or asymptomatic infection without confounding variables that would be introduced by genetic variability. in the study reported here, we examined the innate immune response to uti induced with varying microbial concentrations of u. parvum in the f rat. by applying an integrated approach that combines histopathology with cytokine profiling, we were able to identify innate immune response profiles that were significantly different between an uncomplicated uti and a uti accompanied by struvite formation. our findings provide insights into innate immune responses that are likely involved in the development of complicated disease with ureaplasma. ureaplasma preparation and culture a host-adapted strain of u. parvum, designated strain - was used for the entire study [ ] . fifty mls of u. parvum in logarithmic growth phase was aliquoted into ml volumes and stored at - °c. this stock was used for all experiments. for infection studies, one ml of the working stock was grown in ml of b broth for to hours at °c. the ureaplasma culture was pelleted by centrifugation at , × g, at °c, for minutes. due to the delicate nature of ureaplasma, the pellet was resuspended in to ml of fresh b broth instead of saline, to give a final concentration of cfu per ml then serially diluted to produce various inocula that contained , , , and cfu per ml. the cfu of all inocula (including all serial dilutions) were confirmed by culture on a agar. for each infection experiment, at least two animals were included in each u. parvum dose group and experiments were replicated a minimum of times. inocula and animal tissues were serially diluted -fold in b broth to - and - , respectively. for cfu determination, μl from each sample and its corresponding dilutions were plated on a agar. agar plates were incubated at °c in % co ; broth cultures were incubated at °c in ambient air. agar cultures were incubated for at least days before colonies were counted to determine cfu. specific pathogen free f virgin female rats were purchased from a commercial vendor (charles river, indianapolis, in). all animals ranged in weight from - grams. animal colonies were monitored and found free of the following pathogens: sendai virus, h- virus, rat corona virus, sialodacroadenitis virus, reovirus type , kilham rat virus, hantaan virus, m. pulmonis, respiratory and enteric bacterial pathogens, endoparasites and ectoparasites. all animals were handled in accordance with procedures approved by the university of florida institutional animal care and use committee. all animals were handled within a biosafety laminar flow hood. rats were housed in microisolator ® (lab products, inc., maywood, nj) cages in the same room under the same temperature and light conditions. control animals were always handled before infected and housed in separate microisolator cages in order to prevent contamination with ureaplasma. all food, water, bedding, and caging were autoclaved before use. rats were anesthetized and inoculated with sterile broth or u. parvum inoculum into the bladder as previously described [ ] . for each infection experiment, a minimum of two rats per inoculum dose were infected, so that each dose was represented in each experiment. rats were necropsied at two weeks post-infection as previously described [ ] . prior to euthanasia, free catch urine was collected for cytokine analysis. the bladder was processed for histopathologic evaluation. each kidney was transected sagittally so that a portion of the renal pelvis was present in each section. one half of each kidney was processed for histopathologic evaluation. the remaining halves of the right and left kidneys for an individual animal were combined, minced in sterile b broth, and the medium was aseptically removed and cultured for u. parvum. bladder calculi were submitted to a commercial laboratory (louis c. herring and co., orlando, fl) and analyzed by integrated crystallography. bladder and kidney tissues were fixed in a paraformaldehyde-lysine-periodate [ ] solution for hours, then washed times in sterile saline and transferred to % ethanol prior to processing. tissues were processed routinely and stained with hematoxylin and eosin (h&e). bladder lesions were scored by a system developed in a previous study [ ] . epithelial changes in bladder tissues were scored as: for none, for minimal hyperplasia, ulceration or effacement of epithelium by inflammation; for mild hyperplasia and rare dense inflammatory infiltrates, and for the same changes noted in a score of but accompanied with marked erosion and/or ulceration of the epithelial surface. scoring for cell types that comprised the inflammatory infiltrate was: for primarily mononuclear cells (lymphocytes, plasma cells and macrophages), for mononuclear cells and neutrophils, and for mononuclear cells, neutrophils and fibrous infiltrates. kidney tissues were scored on the basis of total area affected, which was: for less than %, for to %, and for greater than %. urine from control and infected rats was analyzed for the presence of cytokines with a multiplex antibody-immobilized bead immunoassay (lincoplex kit, linco research, inc., st. charles, mo). the manufacturer's protocol was followed for the simultaneous detection of the following cytokines and chemokines: gm-csf, il- α, il- β, il- , il- , il- p , ifn-γ, il- , gro/kc (growth related oncogene/keratinocyte chemoattractant-the rat analog for human il- ), and tnf-α within the same aliquot of urine. briefly, a standard cocktail was serially diluted in order to develop a standard curve for each analyte that ranged from . to pg/ml. urine samples were diluted in assay buffer to obtain a total volume of μl per well, and run in duplicate as previously described [ ] . data from multiple experiments were grouped together in order to make statistical analysis possible. wherever possible, data were analyzed by one-way anova when more than two groups were included in the analysis. fisher's plsd test was used when the anova indicated a significant difference among group means. an unpaired student's t test was used for comparisons between two groups. contingency table analysis was used for comparisons of groups involving nominal data (positive vs. negative). cytokine pattern recognition analysis was performed using jmp genomics . (sas institute, cary, nc). datasets were initially evaluated by distribution analysis and normalized prior to analysis by one-way anova using row by row modeling and fischer c correction for multiple comparisons. for all analyses a probability of p < . was considered significant. ureaplasmas were not isolated from any site from any control rat (data not shown). neither uroliths nor crystals were detected at any site in animals inoculated with sterile b broth. there was no statistical difference in the log cfu of u. parvum isolated from culture positive animals among the various inoculum groups. the log cfu isolated from the bladder tissue of culture positive animals was . ± . (mean ± sd), and . ± . from kidney tissue. however, there was a statistical difference in the frequency of animals that remained infected weeks postinoculation (table ) . animals inoculated with or higher cfu had the greatest frequency of bladder infections at time of necropsy (p ≤ . ). animals inoculated with u. parvum were divided into three groups (negative, uti, or struvite) stratified on the basis of culture status and urolith status ( table ). the negative group consisted of animals found to be culture negative and urolith negative at time of necropsy. as expected, the frequency of animals that were culture negative after inoculation with u. parvum decreased as the log cfu of the inoculum increased. the uti group represents ( ) / ( ) / ( ) / ( ) / ( ) ns a) data was collected at weeks post-inoculation, and is a combination of separate experiments. b) probability values were derived by g statistic, p values greater than . were considered not significant (ns). c) both sites refer to bladder and kidney. animals that were culture positive in the bladder and/or kidney at time of necropsy but were found to be negative for uroliths. eleven of ( . %) animals within the uti group had kidney infections at time of necropsy, which was significantly greater (p ≤ . ) than the number of renal infections within the urolith group [ of ( %) animals]. all animals within the struvite group had bladder stones, which were composed of to % magnesium ammonium phosphate (struvite), to % calcium phosphate (carbonate apatite) and to % protein and blood. no animal had macroscopic evidence of kidney stones at time of necropsy. although not statistically significant (p ≤ . ), animals inoculated with or greater cfu tended to have the highest frequency of struvite uroliths. the inoculum dose of u. parvum did not impact the distribution of animals that developed either uti or struvite uroliths (table ) . further, there was no statistical difference between the log cfu cultured from bladder tissue among these two clinical groups. the log cfu (mean ± sd) of u. parvum isolated from the bladder of animals within the uti group was . ± and . ± from the bladder of animals within the struvite group. the extent and severity of bladder lesions as well as the types of inflammatory cells present differed among groups. there were no detectable lesions in bladder tissue from control rats (see figure , panel a). in animals inoculated with u. parvum, bladder lesions associated with inflammation were highly variable. when present, lesions consisted of infiltrates of lympho-plasma cells, macrophages, and neutrophils that were primarily located within the epithelial layer ( figure , panel b and d). mast cells could be found within the submucosa regardless of clinical profile (data not shown). uroepithelial changes ranged from spongiosis of epithelial cells with some necrosis, exfoliation of uroepithelium, or hyperplasia (see figure , panels b, c, and d). animals with struvite uroliths had the most extensive lesions and the highest lesion scores ( figure ). in these animals, inflammation extended into the submucosa and muscularis layers, and was occasionally accompanied by venous congestion and edema with a fibrinous reaction ( figure , panel b). neutrophilic infiltrates were present in all of the animals within the struvite group. in contrast, infiltrates in animals within the negative and uti groups were predominantly lymphocytes, plasma cells and macrophages ( figure , panel b) . animals with uroliths also had the most extensive uroepithelial hyperplasia as shown in figure , panel c. animals within the negative and uti groups had the mildest inflammatory changes ( figure ). however, somewhat surprisingly, more animals within the negative group had a higher degree of both inflammation and epithelial change than did animals within the uti group. most of the animals in the uti group exhibited exfoliation of uroepithelium with some spongiosis. both the inflammatory cell type score and inoculating dose of u. parvum influenced the lesion scores within the negative group. there was no difference in the lesions scores pertaining to degree of inflammation and degree of epithelial change among the inoculating dose groups (data not shown). however, there was a significant difference (p ≤ . ) in the inflammatory cell type score and inoculating dose of u. parvum (figure , panel d) . a significant number of animals that received log cfu had a mixed inflammatory cell infiltrate that included neutrophils as well as mononuclear cells (lymphocytes, plasma cells, and macrophages). kidney tissue from f rats were also evaluated for the presence of inflammation. there were no significant lesions present in the collecting ducts and renal pelvis of control rats (fig , panel a) . histopathologic findings in kidney tissue from rats inoculated with u. parvum ranged from minimal changes to varying degrees of inflammatory infiltration that consisted of lymphocytes, plasma cells, macrophages and neutrophils. in some animals, lesions were characterized by scant multifocal areas of predominantly mononuclear cells that were present in the subepithelial region of the renal pelvis ( figure , panel b). in ( ) / ( ) / ( ) these animals, the uroepithelial lining the pelvic space was hyperplastic. animals with the most severe kidney lesions had extensive erosion of the uroepithelium, as shown in figure , panel d. in these animals, erosion of the uroepithelial barrier was accompanied by hemorrhage and infiltration with inflammatory cells (predominantly neutrophilic) that spanned the pelvic luminal space, through the epithelial layer and into the sub-epithelial region of the pelvis (figure , panel d). other animals, had inflammatory infiltration of the renal interstitium as shown in figure , panel c scoring of kidney tissues on the basis of total area affected did not reveal any patterns that could be correlated to local/active infection or clinical profile. specifically, there was no correlation between the total area affected score and u. parvum culture status (data not shown). there also was no correlation between the total area affected score summary of the bladder lesions found in f rats experimentally infected with u. parvum figure summary of the bladder lesions found in f rats experimentally infected with u. parvum. panel a is a × magnification of bladder tissue from a control rat. this sample represents a lesion score of for degree of inflammation, degree of epithelial change and inflammatory cell type. panels b, c, and d are tissue sections from animals inoculated with u. parvum that had a lesion score of for degree of inflammation, degree of epithelial change and inflammatory cell type. panel b is a × magnification of bladder tissue from an animal within the struvite group. the asterisk demarcates the extensive edema and fibrinous exudate infiltrating the submucosa. the black arrow points to uroepithelial effacement. panel c is a × magnification of extensive uroepithelial hyperplasia. panel d is a magnified inset of panel b that highlights the array of white blood cells that comprised in the inflammatory cellular infiltrate in the tissues of infected animals. the blue arrow is pointing to a neutrophil. the green arrow is pointing to a tissue macrophage. the yellow arrow is pointing to a plasma cell. the red arrow is pointing to a lymphocyte. and clinical profile (negative, uti, or struvite), additional file . the relationships between specific cytokines and bladder lesion scores among animals inoculated with sterile b broth or u. parvum were examined by spearman correlation analysis. there were no correlations between urine cytokine levels and degree of inflammation score among animals inoculated with sterile b broth (data not shown). however, there was a significant correlation (summarized in table ) between the degree of inflammation score and urine concentrations of gro/kc, il- α, il- β, il- and tnf-α. there was also a significant correlation between degree of epithelial change and urine concentrations of gro/kc and il- β (table ). mcp- levels negatively correlated with degree of epithelial change ( table ). the cytokine profile in urine differed among groups. absolute concentrations of each individual cytokine in urine were compared among clinical profiles (control, negative, uti and struvite). control animals had significantly higher levels of mcp- than animals in either the negative or uti groups (figure ) . animals in the struvite group had the highest levels of gro/kc (figure ) , il- α, il- β, il- , il- and tnf-α ( figure ) than did animals in all other groups. there was no statistical difference in the absolute amounts of urine gm-csf, ifn-γ, il- , il- , il- , and il- among groups (data not shown). in order to identify distinctive chemokine/cytokine patterns between clinical profiles associated with active infection, samples from animals within the negative group were excluded from this analysis. each urine cytokine multiplex from each animal was normalized prior to analysis by one-way anova using row by row modeling and fischer c correction for multiple comparisons. figure is a clustered heat map illustrating two cytokine profile clusters that significantly differed between control, uti and struvite groups (p ≤ . ). both control and uti groups showed a significant emphasis in il- and mcp- , whereas the struvite group showed a significant emphasis in il- α, il- β, and gro/kc. only the uti group showed a significant emphasis in ifn-γ. urine cytokine data from control and culture negative animals was normalized prior to statistical analysis and analyzed as described above. there was a significant difference in the overall pattern of il- , il- , il- , tnfα, ifn-γ, and gro/kc in the urine of culture negative animals that received different inoculating doses of u. parvum (p ≤ . ). figure is a clustered heat map illustrating lesion scores of bladder tissue from f rats inoculated with u. parvum two distinct cytokine profile clusters (green and red cluster trees) among these animals. with the exception of log and log inoculating groups, there was an inverse relationship in the pattern of expression between the two cytokine clusters. ureaplasmas are an underappreciated pathogen of the urogenital tract. despite strong epidemiological evidence and even experimental infections in humans that fulfilled koch's postulates [ ] , the etiologic role of ureaplasmas is confounded by the isolation of the microbe from the lower urogenital tract of normal, asymptomatic individuals. in addition, the severity of disease for most mycoplasmal infections depends on the host immune response. therefore, experimental infections in genetically defined animal models will be critical to unraveling the key interactions in the host/parasite relationship that contribute to disease severity. by using a combined approach involving histopathology and cytokine profiling, we were able to further characterize the immune response associated with asymptomatic uti and uti complicated with struvite formation. summary of the kidney lesions found in f rats experimentally infected with u. parvum figure summary of the kidney lesions found in f rats experimentally infected with u. parvum. panel a is a × magnification of kidney tissue from a control rat and demonstrates the lack of inflammatory lesions that are characteristic in animals inoculated with u. parvum. panels b, c, and d are tissue sections from animals inoculated with u. parvum that had a lesion score of for total area affected. panel b is a × magnification demonstrating the inflammatory infiltrate extending from the renal pelvic space into the interstitium with uroepithelium largely intact. the black arrow is pointing to uroepithelial hyperplasia. panel c is a × magnification of renal tubules. the black arrow is pointing to the extensive inflammatory infiltrate throughout the renal tubular interstitium. the yellow arrow is pointing to a glomerulus. panel d is a × magnification of renal uroepithelium at the edge of the pelvic space. the black arrow is pointing to extensive hemorrhage and disruption of the uroepithelial barrier by a fibrinous inflammatory infiltrate. the f rat strain is highly susceptible to development of complicated uti following experimental inoculation with u. parvum [ ] . in this study, we showed that varying the inoculum dose of u. parvum significantly affected the frequency of animals that remained colonized two weeks after inoculation. therefore, the initial microbial load is important in establishing infection. however, once infected, the proportion of animals that developed complicated uti in response to varying inocula of u. parvum did not show a definitive dose response effect. more importantly, the immune response to infection in this group of animals with complicated uti was consistent, regardless of initial inoculating dose. for example, the cytokine profile and urinary tract pathology of a struvite positive animal that was inoculated with cfu was indistinguishable from a struvite positive animal that was inoculated with cfu. this suggests that the initial microbial load of u. parvum is not a critical factor in the development of complicated uti in this rat strain. further, it supports the concept that, once infection is established, the host inflammatory response is a key determinant of lesion severity in the urinary tract. as previously reported [ ] , animals with asymptomatic uti had significantly less pro-inflammatory urine cytokines and tissue damage when compared to rats with struvites. by profiling the entire cytokine milieu, we were able to identify a significant predominance of cytokines such as ifn-γ, il- , and mcp- in the uti group that work synergistically to regulate monocyte/macrophage activation [ ] [ ] [ ] . an intriguing finding was the significant emphasis of ifn-γ in the urine of animals with asymptomatic uti, since this cytokine is a potent priming agent for macrophages [ ] . this cytokine profile also coincides with the cellular immune response in these animals that consisted of macrophages, lymphocytes, and plasma cells, which resembles a profile that may be seen during the healing or resolution phase of infection. we cannot rule out that these animals could be displaying a pre-resolution phase to infection, but there are indicators suggesting that these animals have compromised immune defense. for example, the immune profile of these animals was obtained while they were actively colonized with u. parvum, and % exhibited an ascending infection into the kidneys. further, the microbial load of u. parvum in animals with asymptomatic uti was equivalent to animals in the struvite group. another intriguing feature in animals with asymptomatic uti was the overall lack of uroepithelial proliferation that was present in varying degrees in the negative group as well as the struvite group. a primary defense mechanism of uroepithelium exposed to bacteria involves desquamation, necrosis or apoptosis followed by proliferation [ ] . therefore, the overall lack of this response in animals with asymptomatic uti also implies that uroepithelial defense mechanisms may be perturbed by u. parvum. f rats with struvite uroliths had a similar clinical profile to what we have previously described [ ] . specifically, these animals had the greatest concentration of proinflammatory cytokines in their urine (gro/kc -the rat analog of human il- , il- α, il- β, il- and tnf-α) and the most extensive inflammatory changes in bladder tissue. since il- β is a known inducer of il- and gro chemokines in human and murine epithelial cells [ ] [ ] [ ] , it is not an unexpected finding that these cytokines are closely linked in their expression. cytokine pattern analysis showed this cytokine cluster is unique to animals with struvites. moreover, there is a significant positive correlation between il- β, gro/kc and the degree of histopathologic change, which suggests that il- β and gro/ kc are critical elements in a pro-inflammatory loop that leads to chronic active inflammation, epithelial hyperplasia and struvite formation as seen in struvite positive f rats. most of the animals within the struvite group had uroepithelial hyperplasia or erosion with hemorrhage and inflammation within the kidneys, yet none of these animals had uroliths in the renal pelvis that could account for these lesions. therefore, although mechanical irritation by the urolith itself may partially contribute to epithelial erosion or hyperplasia in the bladder, it cannot entirely account for the lesions that were present in the urinary tract of these animals. the immune response of animals within the negative group was highly variable and most likely comprises a mixed population of rats, including animals that never became colonized as well as animals that cleared the infection at various time points post-inoculation. therefore, interpretation of data from this group of animals is difficult and is done with caution. in spite of this limitation, profiling urine cytokine data and bladder lesion scores by inoculum dose was informative. the threshold dose for successful colonization appears to be between log and log cfu, since % and % of rats respectively were culture negative weeks post-inoculation. the animals within the log and log cfu inoculation groups also had the greatest flux in both pro-inflammatory (tnfα, ifn-γ and gro/kc) and anti-inflammatory (il- , and il- ) cytokines. interestingly, pattern analysis of urine cytokine data showed two distinct clusters. the first cluster identified in the negative group included il- , il- , il- , and tnf-α; these cytokines were notable as they were not part of the cytokine cluster groupings of u. parvum infected animals. therefore, these cytokines may be critical in directing a more efficient immune response that leads to bacterial clearance with minimal tissue damage. the second cytokine cluster in the negative group included ifn-γ and gro/kc, which are significant cytokines in the uti and struvite groups, respectively. except for animals that were inoculated with log cfu of u. parvum, the expression pattern of ifn-γ and gro/kc was not inversely related as they were in culture positive animals. this may be reflecting a more balanced immune response than what is seen in u. parvum positive animals, and we suggest that this balanced response may be critical to resolution of infection and prevention of severe disease. the variable clinical outcome to experimental inoculation with u. parvum in the f rat is an interesting phenomenon since this is an inbred strain. in this study, both genetic and environmental influences on disease were minimized to the extent possible. all of the animals in this study originated from the same colony. further, rats were housed under the same barrier maintained conditions in order to minimize environmental variability. despite our efforts, it was common to find that a rat that developed asymptomatic uti had co-habited the same cage with a rat that developed struvites or was culture negative at time of necropsy. therefore, external environment could not account for the varying clinical outcome in our study. however, our experimental inoculation procedure may be a critical source of variability. although attempts were made to reduce mechanical trauma caused by catherization, it is possible that the trauma may have been sufficient to shift the immune response towards a proinflammatory profile in a subset of animals. once this occurred, the pro-inflammatory cycle progressed until infection was resolved (negative group) or the study was terminated (struvite group mcp- pg/ml complex interactions between most mucosal pathogens and the host that lead to uncomplicated colonization versus inflammation and disease are largely unknown. therefore, this model may be particularly useful for identifying the molecular events that confer asymptomatic infection, complicated infection as well as resolution of infection with an opportunistic pathogen of the urogenital tract. the complex interactions between ureaplasma and the host that lead to uncomplicated colonization versus inflammation and disease are largely unknown. we characterized the f rat immune response in the urinary tract to varying inoculum concentrations of u. parvum. establishment of uti was influenced by microbial load, but the host immune response was independent of microbial load. two distinct innate immune profiles were identified with two different clinical outcomes: asymptomatic uti and complicated uti with struvite formation. asymptomatic uti was characterized by a minimal immune response that was predominantly monocytic and lymphocytic and was accompanied by a significantly high rate of kidney infection. uti complicated with struvite formation was characterized by an exaggerated immune response that was predominantly neutrophilic and was accompanied by uroepithelial hyperplasia and extensive tissue damage. the authors declare that they have no competing interests. urine cytokines detected in f rats inoculated with sterile b or u. parvum figure urine cytokines detected in f rats inoculated with sterile b or u. parvum. data represent the mean ± sd of a combination of separate experiments. urine cytokine concentrations were grouped according to clinical profile, control (n = ), negative (n = ), uti (n = ), and struvite (n = ). p values within each graph were obtained by one-way anova. fisher's plsd test revealed that il- α, il- β, il- , and tnf-α concentrations in the struvite group were significantly greater than control, negative, and uti groups. fisher's plsd test revealed il- concentrations in the struvite group were significantly greater than negative and uti groups. global profiling of urine cytokines detected in control and u. parvum infected f rats figure global profiling of urine cytokines detected in control and u. parvum infected f rats. the clustered heat map represents the standardized ls means for each cytokine that had a significantly different pattern of expression among clinical groups (p ≤ . ). values were obtained by one-way anova using a row by row modeling with fischer c correction for multiple comparisons. two main cytokine cluster patterns were identified in the analysis and are demarcated by the green and red cluster tree. the number of biological replicates were n = for control, n = for uti group, n = for the struvite group. ureaplasma infection and neonatal lung disease ureaplasma urealyticumharmless commensal or underestimated enemy of human reproduction? a review comparative randomized pilot study of azithromycin and doxycycline efficacy and tolerability in the treatment of prostate infection caused by ureaplasma urealyticum uropathogens and urinary tract concretion formation and catheter encrustations antenatal ureaplasma urealyticum respiratory tract infection stimulates proinflammatory, profibrotic responses in the preterm baboon lung characterization of a murine model of ureaplasma urealyticum pneumonia biochemical and histologic findings in experimental pyelonephritis due to ureaplasma urealyticum concrement formation in the urinary bladder in rats inoculated with ureaplasma urealyticum morphological lesions of the rat urinary tract induced by inoculation of mycoplasmas and other urinary tract pathogens epidemiology and causes of preterm birth intrauterine infection and prematurity. mental retardation and developmental disabilities research reviews disordered pulmonary myofibroblast distribution and elastin expression in preterm infants with ureaplasma urealyticum pneumonitis rat strains differ in susceptibility to ureaplasma parvum-induced urinary tract infection and struvite stone formation immunohistochemical detection of cytokines in paraffinembedded mouse tissues human intra-urethral inoculation of ureaplasmas interleukin- and host defense against infection. the journal of infectious diseases the role of gamma interferon in antimicrobial immunity il- induces monocyte chemotactic protein- production in macrophages through the phosphatidylinositol -kinase/akt and mek/erk / pathways regulation of macrophage activation bad bugs and beleaguered bladders: interplay between uropathogenic escherichia coli . and innate host defenses the effect of interleukin- on cytokine gene expression by human corneal epithelial cells keratinocyte chemoattractant (kc)/human growth-regulated oncogene (gro) chemokines and pro-inflammatory chemokine networks in mouse and human ovarian epithelial cancer cells. cancer biology & therapy interferon gamma induces differential upregulation of alpha and beta chemokine secretion in colonic epithelial cell lines this work was supported by the national institutes of health grants r ai and k dk . we also wish to acknowledge ms. janet stevens and lj mcdonnell for their assistance with microbial cultures and cytokine immunoassays. lr designed and executed animal infection studies, data analysis and manuscript preparation. mr performed histopathologic evaluation of tissues and developed the lesion scoring system implemented in this study. mbb participated in the design and coordination of the study, and helped draft the manuscript. all authors read and approved the final manuscript. profiling the inflammatory response to different doses of u. parvum in culture negative f rats figure profiling the inflammatory response to different doses of u. parvum in culture negative f rats. panel a is a clustered heat map representing the standardized ls means for each cytokine with a significantly different pattern of expression among infection dose groups (p . ). values were obtained by one-way anova using a row by row modeling with fischer c correction for multiple comparisons. two main cytokine cluster patterns were identified in the analysis and are demarcated by the green and red cluster tree. the number of biological replicates were n = for control, n = for log cfu, n = for log cfu, n = for log cfu, n = for log cfu, and n = for log cfu. the red arrow is highlighting the pattern of cytokines present in the urine of culture negative rats that were inoculated with log cfu. animals within this dose group were the only animals to exhibit an obvious inflammatory cell infiltrate comprising a mixture of mononuclear cells with neutrophils (p . , figure the pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/ - / / /prepub key: cord- -bhl up authors: rantsios, a.t. title: zoonoses date: - - journal: encyclopedia of food and health doi: . /b - - - - . - sha: doc_id: cord_uid: bhl up zoonoses are “those diseases and infections which are naturally transmitted between vertebrate animals and man.” relevant lists of zoonotic diseases are presented. factors, including environmental aspects that may influence the manifestation of zoonoses and their significance for public health, are discussed. foodborne, nonfoodborne, and vector-borne diseases are presented, followed by an article on preventative actions and control measures. specific reference is made to the ‘one health’ concept, since, for zoonoses containment, an integrated approach and cooperation between all responsible health professionals, at all levels, is a sine qua non precondition. according to the joint who/fao expert committee on zoonoses, second report, in the year , zoonoses (the expression zoonotic diseases is also used) are "those diseases and infections which are naturally transmitted between vertebrate animals and man." the transmission may take place directly or indirectly by means of vectors. the severity of zoonotic diseases in humans varies from mild symptoms to life-threatening conditions. zoonotic diseases are usually transmitted at the human-animal interface through exposure to animals (e.g., rabies), animal products (e.g., brucellosis and salmonellosis), or their contaminated environment (e.g., echinococcosis/ hydatidosis). transmission may occur by a diseased animal or by a clinically normal animal that, nevertheless, is able to transmit pathogens to humans (e.g., campylobacter and verotoxin producing escherichia coli (vtec)). a distinction is made between foodborne and nonfoodborne diseases. a concise list of zoonoses appears in table . emerging infectious diseases (eids) are diseases appearing in a geographic area or population for the first time. the term reemerging diseases is also used for ancient diseases that are 'forgotten' and thought to be controlled or extinct from a particular area or population and manifest a new appearance. the emergence of eids is thought to be driven by socioeconomic, environmental, and ecological factors. they are, mostly, related to free-living wild animals. the term neglected tropical diseases is used for a certain list of diseases recognized as such by the who, including a number of zoonotic diseases appearing in table . neglected tropical zoonotic diseases are ancient endemic zoonoses in europe and north america. they are largely controlled and tended to be overlooked, while the attention is shifted to newly emerged zoonoses. they can be, potentially, turned into a pandemic. although, at the time of the aforementioned who report, the number of zoonoses was at the level of , they are doubled by then and still counting. zoonoses may be bacterial, viral, parasitic, or due to unconventional agents (e.g., prions). it is estimated that out of the about human nosogenic factors, due to biological agents, % have multiple hosts and move between species. in addition, % of the eids, in the last $ - years, are zoonotic; again, % of which originate from wild animals. further to the significance of zoonoses for human health, one must not underestimate the impact they are having in preventing the efficiency in animal food production and enhancing problems in living animals and animal products international trade. as an indication of the significance of zoonotic diseases for human health, you can see, in table , the most important zoonoses in terms of human health impact, livestock impact, amenability to agricultural interventions, severity of diseases, and emergence. zoonoses present a major threat to human and animal health. these diseases, as already indicated, are multifactorial manifestations and therefore a reflection of the complexities of ecosystems in which animals and humans coexist. they are influenced by multiple interrelated global factors, including ecological evolution, human demographics, and behavior. emerging and reemerging diseases represent failures in understanding the socioecological systems we live in and respond to new conditions. what we learn from these failures will largely determine how successful we are in developing sustainable and healthy human communities. as can be seen in figure , humans, livestock, and wildlife can expose each other and spread, among them, potential pathogenic agents. any one of these players, therefore, can spill over pathogens to the others. more specifically, changes in human behavior, expressed as population increase and urbanization, and the demand for improved living conditions, requiring in turn proportional increase in food production and related economic activity, in particular agricultural development, are a decisive contributing factor. for example, water management, associated with still water collections, one way or another, increases mosquito breeding opportunities, which in turn may result in enhancing disease occurrence, like rift valley fever. such conditions are observed after the construction of dams and irrigation networks. similarly, intensive animal farming promotes disease transmission through untreated or poorly treated waste material spread in the environment or through ventilation system diffusion of contaminated material carrying pathogens. due to the problems related to high animal population density, maintained in intensive livestock production (e.g., pigs and poultry), which facilitated disease transmission, efforts were made to combat them with the use of antibiotics, either therapeutically or preventatively. however, these schemes resulted in widespread antibiotic-resistant pathogens, including those of zoonotic significance. these conditions, more specifically, can contaminate animal food products and potentially provide for the production of unsafe foods. it is important in this respect to mention that, according to eu food food production premises are considered as food business operations. consequently, they are obliged to certain responsibilities, related to putting together and effectively implementing and functioning a food safety management system (fsms), tailor-made for each particular activity. in table appears a list of zoonoses emergence linked to agricultural intensification and environmental changes. evidently, ecosystems are complicated, and the impact they are having on fauna and thereof on conditions potentially harboring zoonotic agents does not provide for a general interdisciplinary multipurpose approach, with patterns applicable in all cases. on the contrary, principal factors, among others, biological, ecological, economic, and social characteristics, and climatic conditions must be assessed, ad hoc, locally within each ecosystem. the implementation, therefore, of the concept of one medicine and one health, which, as an integrated approach, is very important for facing zoonotic diseases per se because not, only it contributes to animal and human health but also it significantly supports, apart from aspects of economic development, food safety and security. foodborne zoonotic pathogens are transmitted through consumption of contaminated food and drinking water. infectious agents present in foodstuffs include bacteria (e.g., salmonella and campylobacter), viruses (e.g., norovirus and hepatitis a virus), parasites (e.g., trichinella), and prions (infectious agents of bovine spongiform encephalopathy). challenges to food safety continue to increase in unpredictable ways, largely due to changes in food production, processing, distribution, and the environment, which may contaminate food; to emerging germs and toxins of food safety significance; and to new conditions, created by new food technology applications, with an impact on food safety (e.g., minimally processed foods and maintenance of cold chain). in table , a list of the most important biological hazards responsible for foodborne illnesses appears, along with the relevant most important foods involved in each case. nonfoodborne zoonotic diseases are transmitted through the following: • direct contact or close proximity with infected animals or through the environment. examples are the following: ○ avian influenza, a viral disease occurring mainly in poultry and other birds but transmissible to other animals or humans. ○ q fever, caused by the coxiella burnetii, affecting animals and humans. human infection mainly results from the inhalation of contaminated dust from the placenta and birth fluids or feces from infected animals. ○ salmonella infections, which can originate from contact with infected reptiles and amphibians such as pet snakes, iguanas, and frogs or their environment. ○ vtec, which can be acquired through contact with infected farm animals. for zoonoses prevention, the focus is on surveillance, rapid detection, and quick response. zoonotic infections in animals may produce a distinctive recognizable disease, such as rabies, or they may manifest themselves as a mild illness or the animal may be entirely asymptomatic. however, in this last case, if the pathogen is transmitted to humans, it may result in illness if they lack the specific immunity required. anyone, who has contact with animals, can get a zoonotic disease, but some people may be more at risk than others. risk table the most important zoonoses in terms of human health impact, livestock impact, amenability to agricultural interventions, severity of disease, and emergence data from the who and authoritative literature: when there are several authoritative estimates, the midpoint is given. note: high human mortality gets a double weight as the most important criterion for many stakeholders. total score ¼ (human death  ) þ (humans affected) þ (high livestock impacts) þ (farm intervention possible) þ (other concerns: severe or emerging disease). the maximum possible score is therefore and the minimum . factors for susceptibility to zoonoses, among others, are certain population groups, including professional and occupational groups working in close contact with animals (like livestock attendants, slaughterhouse workers, and veterinarians); homeless and poor people; and in general, people with a weakened immune system, children aged less than five, the elderly, and pregnant women. the factors promoting zoonotic disease outbreaks in humans, apart from frequent contact with animals, include intensive livestock production, poor animal and personal hygiene, and overlap with wildlife habitat. a common way for vector-borne diseases to spread is through the bite of a mosquito or tick. people can get diseases in most places, where they might have contact with infected animals and insects, including animal displays, farms, petting zoos, county or state fairs, pet stores, child care facilities or schools, nature parks, and wooded and bushy areas. in order to reduce the risks of transmission of zoonoses from pet animals to humans and also to production animals, the concept of responsible pet ownership (rpo) is advocated. it is recognized that education and awareness promotion of pet owners, for rpo, to prevent zoonoses related to companion animals, is of a paramount importance for eliminating these diseases. the who and efsa promote activities, of the general public's concern, for preventing zoonoses. they include the following: -promotion of awareness to understand the potential risk for human infection from zoonotic diseases, after contact with animals. -specific risk communication, as it may be required, through appropriate public actions. biosphere wildlife livestock humans peri-domestic wildlife figure pathogen flow at the wildlife-livestock-human interface. arrows indicate direct, indirect, or vector-borne candidate pathogen flow. in each host species, there are a vast array of constantly evolving microorganisms, some of which are pathogenic in the host. these are a source of new organisms for other host species, some of which may be pathogenic in the new host or may evolve in the new host to become pathogenic. if the pathogen is also transmissible in the new host species, then a new transmission cycle may be established. the rate and direction of candidate pathogen flow will depend on the nature and intensity of interaction between wildlife, livestock, and human compartments and the characteristics of the compartments ( table ) . adapted from proceedings of the national academy of science, may , , vol. , no , p. . -improvement in the level of personal hygiene by ○ acquiring the habit to wash hands thoroughly and frequently, after contact with animals. ○ closely supervising children to ensure they wash their hands properly and avoid hand-to-mouth activities (thumb sucking, eating, and use of pacifiers) after animal contact. -the use of registered insect repellents and products that contain repellents for use on clothing. accordingly, treat clothing and gear, such as boots, pants, socks, and tents. -inspection for and removal of ticks from the human body, with specific care for children. -limitation in the number of places around residential areas for mosquitoes to breed by eliminating places holding water. for health professionals, recommendations may include the following: -responsible services to systematically search for potential sources of human infection from animal sources and the environment -joint efforts and coordination among public health authorities and related professionals, both public and private -risk communication and information sharing among responsible health services and close coordination to manage risks related to the movement and trade of livestock -concerted actions for ○ good practices in the efficient implementation of biosecurity measures in farms and at border or territory crossings; ○ continuously reminding and training people, who work with livestock and in slaughterhouses, for the significant importance of personal hygiene practices; ○ the implementation of the one health concept zoonotic diseases are strongly influenced by social and economic practices. many of them are thus not only diseases related to climate and environment but also diseases of poverty. in epidemiological terms, conditions of poverty increase the probability of enhanced contact and hence increase the likelihood of epidemics. combating diseases like tuberculosis and malaria has much more to do with housing, nutrition, and water management than with any advances in biomedical science. animal migrations facilitate the global spread of pathogens and increase cross-species transmission. understanding, predicting, and controlling diseases at the human-animal interface are a huge challenge, for health professionals, in today's world, where international trade and travel globalized diseases. however, even when risks are identified, adequate underlying infrastructure and resources are required to take the measures needed, if outbreaks and emergencies are to be prevented or controlled. special consideration needs to be given to vector-borne diseases. vectors may move to long distances and therefore may introduce disease, in new geographic areas, by means of human traveling, international trade, animal movement (more specifically livestock and migratory birds), the wind, and changes in agricultural practices. health is a state of complete physical, mental, and social wellbeing and not merely the absence of disease or infirmity. it is the outcome of a complex of several interdependent medical, economic, sociocultural, environmental, and ecological factors. people's health and well-being and equally animal health and welfare are strongly interlinked. both also influence and are impacted by the health of the environment. health is a precondition for well-being and respectively welfare. wellbeing and welfare reinforce health. the advancement of the health and well-being of people and animals depends on effective and sustained cooperation between varied professions and disciplines, in both the public and private sectors (world veterinary association (wva) position). therefore, multisectoral horizontal links at all levels between human and animal health professionals, public and private, are needed to face zoonotic diseases. reducing relevant risks is impossible to be achieved alone by a particular sector, regardless of its importance. therefore, there is an increasing convergence to a one health approach, which incorporates, in an integrated manner, cross-sectoral, multidisciplinary cooperation. one cannot distinguish human, animal, and environmental health. the health of each one of them is the precondition for the health of the others (see again figure ). building sustainable national mechanisms for more effective cross-sectoral cooperation has greatly facilitated risk assessment and management of specific diseases, such as h n influenza and rift valley fever. at the international level, strong cooperation among the who, oie, and fao is improving the efficiency of surveillance, including data collection, risk assessment, and risk management options, allowing for consistent, sciencebased risk communication on global health threats at the human-animal-ecosystem interface. a strategic agreement outlines the sharing of responsibilities and enhanced coordination of complementary roles and activities between the fao, oie, and who at national, regional, and global levels. internally, cooperation takes place across departments, clusters, and regions of the aforementioned organizations. externally, further cooperation materializes with links and contacts to additional international partners, such as international agencies and networks, ngos, and academia, and to national agencies, such as institutions and administrative governmental units. according to statements made by eu officials in support of it, one health is linked to livelihood and equity and fits with eu objectives to promote global security, social justice, international cooperation, and multilateralism and fight poverty. further, there should be no resignation vis-à-vis the existence of different health standards across nations. table biological hazards responsible for most important foodborne illnesses and related foods campylobacter (poultry) e. coli o :h (ground beef, leafy greens, and raw milk) listeria (deli meats, unpasteurized soft cheeses, and produce) salmonella (eggs, poultry, meat, and produce) vibrio (raw oysters) norovirus in many foods (e.g., sandwiches and salads) toxoplasma (meats) for the eu, the one health movement has in many senses grown out of the response to influenza-related crises. the definition of one health chosen by the european commission in its external relations and actions reads as follows: the one health approach consists of (i) improving health and wellbeing through the prevention of risks and the mitigation of the effects of crises that originate at the interface between humans, animals and their various environments; (ii) for that purpose: (a) promoting a multi (cross) sectoral and collaborative approach; and (b) promoting a 'whole of society' approach to health hazards, as a systemic change of perspective in the management of risk. in may , a meeting was organized by the centers for disease control and prevention (cdc), atlanta, georgia, the united states, titled 'operationalizing "one health": a policy perspective.' 'critical enabling initiatives' such as 'training,' 'one health global network,' 'information clearing house,' 'needs assessment,' 'capacity building,' 'proof of concept,' and 'business plan' were identified as fundamental to moving forward one health. further, it seems reasonable to think that an improved coordination that includes intersectoral cooperation in surveillance, communications, outbreak response, and sample sharing community-based interventions for the prevention and control of zoonotic diseases is needed. there is further and more specific need for systematic cooperation between strong and autonomous public health services and strong and autonomous veterinary services, in the respect of their specific expertise. however, a culture of cross-sectoral cooperation does not yet exist all along the chain. fostering such a culture, stretching from the field level to that of international organizations, is the big challenge for successfully controlling zoonotic diseases in general and more particularly either emerging or neglected ones. there is a deficit in current university medical training, due to the fact that the whole training concept is geared to treat, rather than prevent, diseases and preserve and promote health. a cultural change putting emphasis on the prevention and appreciation of the importance of the connection, in terms of health and well-being, between humans, animals, and ecosystems, is required. combating zoonotic diseases is not how to clean up the disease mess after the fact, but on how to prevent the mess from occurring in the first place. it would almost appear that the ideological lenses through which diseases in general are being studied preclude acting on the evidence. this, if nothing else, should raise a warning flag that those who study disease are not necessarily well equipped to promote health. see also: escherichia coli and other enterobacteriaceae: food poisoning and health effects; milk: processing of milk. the european union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in emerging infectious diseases of wildlife -threats to biodiversity and human health mapping of poverty and likely zoonotic hotspots joint who/fao committee on zoonoses, second report global trends in emerging infectious diseases zoonosis emergence linked to agricultural intensification and environmental change one health agriculture-associated diseases: adapting agriculture to improve human health public health impact of zoonoses and international approaches for their detection and containment oxford textbook of zoonoses. biology, clinical practice and public health control (oxford textbooks in public health) paho/who zoonoses and communicable diseases common to man and animals: vol. i. bacterioses and mycoses chlamydioses, rickettsioses and viroses brucellosis: recent developments towards ''one health coordinating surveillance policies in animal health and food safety: "from farm to fork sharing responsibilities and coordinating global activities to address health risks at the animal -human-ecosystems interface. a tripartite concept note foodborne disease outbreaks: guidelines for investigation and control steps in a foodborne outbreak investigation html -who initiative to estimate the global burden of foodborne diseases key: cord- -w fnkq authors: batchelder, margaret; keller, lynn s.; sauer, mary ball; l. west, wanda title: gerbils date: - - journal: the laboratory rabbit, guinea pig, hamster, and other rodents doi: . /b - - - - . - sha: doc_id: cord_uid: w fnkq the gerbil is usually nonaggressive and is one of the easiest rodents to maintain and handle. its disposition, curious nature, relative freedom from naturally occurring infectious diseases, and adaptability to its environment have contributed to its popularity as a laboratory animal. gerbils are found in deserts and semiarid geographical regions of the world. the mongolian gerbils that are available today originated from pairs of captured animals that were maintained in in a closed, random-bred colony at the kitasato institute in japan. gerbils have several unique anatomical and physiological features. mature gerbils are smaller than rats, but larger than mice. mongolian gerbils are attracted to saliva and use salivary cues to discriminate between siblings and nonsiblings, and females use oral cues in the selection of sociosexual partners. gerbils have been used as experimental models in a number of areas of biomedical research. gerbils are excellent subjects for laboratory animal research as they are susceptible to bacterial, viral, and parasitic pathogens that affect humans and other species. gerbils may have spontaneous seizures secondary to stress such as handling, cage change, abrupt noises, or changes in the environment. cystic ovaries are seen commonly in female gerbils over year of age. gerbils have unique characteristics, which make them appropriate for a number of animal models. classically, gerbils have been used in research involving stroke, parasitology, infectious diseases, epilepsy, brain development and behavior, and hearing. the introduction and development of the mongolian gerbil, meriones unguiculatus, as a laboratory animal is recent, compared to other rodents. the gerbil is usually non-aggressive and is one of the easiest rodents to maintain and handle. its disposition, curious nature, relative freedom from naturally occurring infectious diseases, and adaptability to its environment have contributed to its popularity as a laboratory animal (wagner and farrar, ) . it has several interesting anatomical and physiological characteristics that make it a useful model in biomedical research. several other species of gerbils (e.g. meriones libycus, meriones crassus) have been used as experimental animals (belhocine et al., ; khokhlova et al., ). however, the mongolian gerbil (meriones unguiculatus), on which this chapter will focus, is the most common species of gerbil used in biomedical research (schwentker, ) . it should be noted that mammalian taxonomy is a rapidly changing field. the following description outlines a classification of the mongolian gerbil based on morphology as well as more recent molecular techniques. gerbils are in the class mammalia and order rodentia. rodents are divided into five major suborders (musser and carleton, ) . gerbils are part of the suborder myomorpha, originally so named because the deep and lateral masseter muscles attach to the front of the muzzle, giving it a forward thrust. myomorphs also lack premolar teeth (hurst, ) . gerbils belong to the superfamily muroidea, family muridae, and subfamily gerbillinae based on morphology and on evaluation of nuclear protein coding sequences of the lecithin cholesterol acyl transferase gene and the von willebrand factor gene (michaux et al., ; robinson, ) . the genus meriones was first described by illiger in , and meriones unguiculatus was first identified in by milne-edwards (robinson, ; thiessen and yahr, ) . chaworth-musters and ellerman ( ) provided a comprehensive description of the genus meriones which was updated by ellerman and morrison-scott ( ) , corbet ( ) , and pavlinov et al. ( ) . the genus name, meriones, was derived from a greek warrior who wore a battle helmet decorated with boar tusks (robinson, ) . the species name, unguiculatus, is latin for clawed or fingernail leading to one of the common names, clawed jird. the name gerbil is from the arabic word, yarbu, which refers to saltatorial, desertinhabiting rodents. yarbu was translated into latin as gerbo and into english as gerbil (robinson, ) . approximately genera and species of gerbils are known (agren, ) . gerbils are found in deserts and semi-arid geographical regions of the world (field and sibold, ; thiessen and yahr, ) . they are native to northern africa, india, mongolia, southwestern and central asia, northeastern china, and regions of eastern europe (robinson, ) . because wild gerbils live in arid habitats they dig burrows that extend between cm and . m below the surface so the temperature is relatively constant throughout the day and night. the burrows may be small and simple with one entrance or complex with eight to entrances (agren, ; brain, ) . the domestication of the mongolian gerbil is a relatively recent occurrence. they were found by a french missionary, father armand david, who traveled extensively in mongolia and china in the s. some wild-caught stock were sent to japan, where they were bred freely (alderton, ) . the mongolian gerbils that are available today originated from pairs of captured animals that were maintained in by dr. c. kasuga in a closed, random-bred colony at the kitasato institute in japan. in a sub-colony of gerbils was established at the central laboratories for experimental animals in tokyo by m. nomura. in dr. v. schwentker imported pairs from japan. five females and four males from this group were there is a low amount of genetic variability present in laboratory gerbils compared to wild gerbils as a result of their origination from a few founder animals (razzoli et al., ) . recently, neumann et al. ( ) identified the first polymorphic dinucleotide repeat loci in mongolian gerbils by a microsatellite technique. these studies demonstrated that there has been minimal genetic variability identified in the mongolian gerbil used in research, even though there are several different lines or strains. razzoli et al. ( ) confirmed these results using an amplified fragment length polymorphism technique to characterize and detect strainspecific polymorphisms in gerbils. prior to these studies, relatively few genetic studies had been performed. the common agouti or mixed brown mongolian gerbil stock is used in research and sold as pets. the agouti has given rise to black (cramlet et al., ; waring and poole, ) , albino (hedges, ) , piebald, dove, cinnamon, and a "hairless" mutant (alderton, ; matsuzaki et al., b ). an inbred strain (mon/tum) is also available (brain, ) . there are also seizure-sensitive and seizure-resistant lines of gerbils (loskota et al., ) . at the time of publication, mongolian gerbils are commercially available in the united states from charles river laboratories, inc., wilmington, ma and harlan, indianapolis, in. gerbils have several unique anatomical and physiological features. mature gerbils are smaller than rats, but larger than mice (figure . ). adults of both sexes vary between . - . cm in body length with males weighing an average of grams and females weighing an average of . grams (kramer, ) . body weights vary among colonies and this variation may be a genetic phenomenon as well as a dietary factor (arrington et al., ) . in adults, the sexes are easily differentiated by the male's longer anogenital distance, prominent testicles, and pigmented scrotum (figure . ). females have four pair of mammae, two inguinal and two thoracic, and the urethra is located outside the vagina (wagner and farrar, ) . external anatomical features are depicted in figure . . cranial characteristics are consistent with saltatorial rodents. they have broad, short heads with prominent ears and large, black, slightly bulging eyes. most mice and rats have longer, extended faces. the large eyes are an adaptation found in nocturnal animals, and their vision is very well developed (alderton, ) . gerbils show active periods during day and night, and use both rod-and cone-based vision. gerbils generally have reddish brown fur with black outer tips on their backs, but coat color can vary from tan to gray, with a gray or creamy white undercoat. the tail is covered with fur that is short at the base and progressively becomes longer and bushy toward the tip of the tail. the tail length ranges from . - . cm (norris and adams, c) . the hindlimbs are elongated, and the forefeet are relatively small. the hindlimbs are very muscular, and aid in the gerbil's ability to jump considerable distances in proportion to their size. gerbils use jumping or hopping as a form of ambulation (brain, ) . gerbils' hind paws are not equipped with friction pads or opposable toes, and the soles are covered with fur; therefore, they cannot climb like mice (roger and polioudakis, ) . the gerbil's sense of smell is very keen and well developed. gerbils have been shown to use a variety of odors as discriminative social cues from urine, ventral sebaceous glandular secretions, and harderian gland secretions (halpin, ; thiessen et al., ; thiessen and yahr, ) . mongolian gerbils are attracted to saliva and use salivary cues to discriminate between siblings and non-siblings, and females use oral cues in the selection of sociosexual partners (smith and block, ) . the harderian gland functions as a potential site for immune response, serves as a part of the retinal-pineal axis, acts to cushion the eyeball, secretes lubrication for the eye, and is a source for pheromones and thermoregulatory lipids (johnston et al., ; sakai, ) . harderian gland secretions, comprised of lipids, proteins, and protoporphyrin, are carried by an excretory duct to the medial aspect of the nictitating membrane. these secretions bathe the eye and conjunctival space and are transported down the nasolacrimal duct, exiting at the external nares. the harderian secretions are mixed with saliva and spread over the pelage during grooming (thiessen, ) . compared with mice, gerbils possess a much higher proportion of cones to rods. since the gerbil retina is not exclusively rod-dominated, it is a valuable model for in vitro studies of retinogenesis (bytyqi and layer, ) . the tympanic bullae are prominent, giving the gerbil remarkable hearing with a high frequency peak of khz (johnson and marcotti, ) . the dental formula is (incisors / , canines / , premolars / , molars / )  . the incisors are hypsodont (long crown) and elodont (continuously growing and erupting teeth that do not develop anatomical roots). the molars in gerbils are brachydont (short crown), rooted, anelodont (limited growth period) and cease to grow in mature animals. they are prone to dental caries, periodontal disease, and can develop incisor malocclusion (field and sibold, ) . the mongolian gerbil's liver enzymes which are involved in cholesterol metabolism and hepatic cholesterol ester storage differ from those found in other rodents and make the gerbil an excellent model for hypercholesterolemia research (norris, ; temmerman et al., ) . the gerbil's serum cholesterol level is very sensitive to increased dietary cholesterol. gerbils are resistant to atheromatous changes on highcholesterol diets, but they can develop hepatic lipidosis and cholesterol gallstones when fed these diets (vincent et al., ) . approximately % of mongolian gerbils have an incomplete circle of willis that allows for a reliable development of focal cerebral ischemia which is used to study the pathophysiology and treatment of ischemic stroke (başkaya et al., ) . when one carotid artery is ligated, a cerebral infarct on the side ipsilateral to the ligation is formed (vincent et al., ) . gerbils have less collateral blood supply to the brain compared to mice and rats (vincent et al., ) . the respiratory system is very similar to other rodents in that gerbils do not appear to have respiratory bronchioles in the lung (bal and ghoshal, ) . the right lung is composed of four lobes, while the left lung has three lobes (williams, ) . since they are desert animals, gerbils have several characteristics that have allowed them to adapt to dry environments. gerbils have an excellent ability for thermoregulation, and they have a high level of heat tolerance. they have a unique water metabolism in that they require very little water to function (winkelmann and getz, ) . gerbils can obtain sufficient water from their diet and their kidneys have a highly efficient urineconcentrating capacity to ensure adequate hydration (goyal et al., ) . the ratio of long-loop nephrons to short-loop nephrons in gerbils is high. ninety-six percent of their nephrons are long loop which allows them to efficiently concentrate their urine (ichii et al., ) . the digestive system is also very efficient at absorbing and retaining water, and water can be stored in fat cell layers. gerbils produce and excrete a small amount of concentrated urine and dry feces per day (alderton, ) ; therefore they require less frequent cage changing than other laboratory rodents. the internal anatomy of mongolian gerbils was investigated and described in detail by williams ( ) . he demonstrated that the anatomy of the gerbil and the albino laboratory rat is similar except for some minor variations. these include a lack of a preputial gland, the presence of a gall bladder, and pairs of ribs in gerbils (williams, ) . gerbils have an unusually large thymus and adrenal glands compared to other rodents of similar size (figures . and . ) . the thymus is persistent in adults (wagner and farrar, ) . the adrenal gland weight when compared to the body weight is approximately three times the size of the adrenal gland in rats (cullen et al., ; holmes, ) . the significance of these anatomically enlarged structures is not known (schwentker, ) , although the enlarged adrenal glands are thought to contribute to their unique water conservation ability (wagner and farrar, ) . the adrenal cortex produces equal amounts of corticosterone and -hydroxycorticosterone (drummond et al., ) . unlike a rat, an adrenalectomized gerbil cannot be maintained by providing supplemental sodium (cullen et al., ) . both sexes have distinct mid-ventral abdominal sebaceous glands, which are used for territorial marking. the male sebaceous gland is twice the size of the female gland ( figure . ). at puberty in males, the gland becomes orange and produces an oily, musky scented secretion (clark, ) . selective normal mature gerbil physiological parameter and hematological and serum chemistry values are listed in tables . , . and . , respectively. the data reported are compiled from literature using average values with sexes combined. there is a limited amount of hematological and clinical chemistry data available in gerbils. ruhren ( ) , mays ( ) , dillon and glomski ( ) , and gattermann ( ) conducted studies and reported original data in gerbils. the blood volume of a mongolian gerbil is approximately . ml/ g. the red blood cell half life is approximately . days, which is short in comparison with that of other rodents (womack, ) . young gerbils can have up to a % increase in circulating, stippled red blood cells and reticulocytes compared to adults (ruhren, ; smith et al., ) . gerbils have lower hematocrit values than mice, and have higher blood lipid values than rats (mays, ) . sexual dimorphism in hematocrit, hemoglobin, total leukocyte count, and lymphocytes has been reported (dillon and glomski, ; mays, ) . mays ( ) demonstrated that there were significant differences between males and females in phosphorus and uric acid levels. serum cholesterol levels are higher than other rodents, even when gerbils are fed diets containing normal levels of fat (roscoe and fahrenbach, ) . basic biological values are presented in tables . , . , and . (adapted from clark, ; field and sibold, ; harkness and wagner, ; wagner and farrar, ) . similar and additional data are presented in the sections on normative values; and clinical biochemistry and hematology. gerbils are quiet animals that are generally docile, easy to handle, and seldom bite. they are quite (oldham and morlock, ) . in nature, gerbils live in family groups in complex burrows. their compulsive burrowing behavior extends into the laboratory setting as well, and in many cases they will scratch at the sides and bottoms of cages in their attempts to burrow (brain, ; field and sibold, ) . other normal activities include gnawing, making nests, and nibbling on food continually to support their bursts of energy (bradley and pence, ) . while primarily nocturnal, they are active during daylight hours, too, alternating periods of intense activity with sleep or rest (brain, ; thiessen and yahr, ) . they do not hibernate, but may exhibit estivation, depending on the species (bradley and pence, ) . gerbils are territorial, and will mark almost any object in their environment by depositing a pheromone with their ventral sebaceous gland gray, , ) (figure . ). this enlarged, specialized gland is under the control of gonadal hormones (glenn and gray, ) . animals of both sexes which have higher androgen levels are more frequent markers and have an advantage in hostile encounters (swanson et al., ; thiessen et al., a) . in males, the gonadal androgens act on the medial preoptic area of the brain (commins and yahr, ; thiessen and yahr, ) . in the female there is contradictory information regarding the influence of estrogen and progesterone on marking behavior (thiessen and lindzey, ; wallace et al., ; whitsett and thiessen, ) . male gerbils scent mark more frequently than females (lindzey and carbonell, ) and gerbils with black coat color tend to mark more often than those with brown coat color (turner and carbonell, ) . marking behavior in males is used for exploration, establishing social dominance, and identifying territory (thiessen and yahr, ) . in females, scent marking is used for exploration and is also important for identification and for the attraction between mother and young (dagg and windsor, ; wallace et al., ) . males also urinate and defecate to mark their territory. only groups of males that are raised together are compatible as adults and will defend their territory as a group. once a family unit (male and female, or parents with a litter) is established, the gerbils will viciously attack intruders. pair bonding between male and female gerbils occurs; however, gerbils may not be completely monogamous (brain, ; robinson, ) . mating usually occurs at night, and the male will mate with the female several times. both males and females will build a nest and care for the young (elwood, ) . gerbils make a drumming sound by thumping their hindlegs rapidly on the cage floor to signal a warning or excitement, or during courtship by the male (brain, ) . food hoarding is also a natural behavior in gerbils with females serving as the primary food hoarders. castration of male gerbils will increase hoarding behavior indicating that hoarding is inversely related to androgen levels. in one experimental setting where a pair of gerbils was provided with a ten foot square room with a closet, the female gerbil gathered and stored approximately pounds of food in the closet (thiessen and yahr, ) . adult total blood volume (ml) . - . (adapted from clark, ; field and sibold, ; harkness and wagner, ). maintaining a well-managed gerbil colony requires attention to many aspects of animal care. animal wellbeing is dependent on appropriate housing conditions, from the micro-environment (cage) to the macro-environment (holding room and facility). animals must receive appropriate nutrition, and veterinary oversight must be provided to ensure an effective preventive medicine and health-monitoring program. finally, documentation is essential to ensure that procedures are being followed as designed and to comply with national regulations. caging designed for other rodents (e.g. hamsters and rats) is generally adequate for gerbils, and solid-bottom, bedded caging is preferred (brain, ; field and sibold, ; harkness and wagner, ; moore, ; olfert and cross, ) . recommended cage dimensions vary and may be subject to national regulations. for example, the canadian council on animal care (olfert and cross, ) proposes a floor area per animal of cm and cm cage height, while the committee for the purpose of control and supervision on experiments on animals (cpcsea) in india recommends a floor area of . cm (up to g) to . cm (over g) and a height of cm. although the gerbil is a united states department of agriculture (usda)-covered species, neither the animal welfare act regulations ( cfr part nor the guide for the care and use of laboratory animals (guide)) (national research council, ) provide standards for cage sizes for gerbils in the united states. others have recommended that adult gerbils have in / cm of floor space per animal and in/ cm of internal cage height and that breeding pairs with litters should have in / cm of floor space (harkness and wagner, ) . bedding types used for gerbils are the same as those used for other rodent species. these include chopped corncob, hardwood chips, cellulose fiber, and other commercially available beddings. in addition to absorbing wastes, bedding may be used as a burrowing substrate, or a separate substrate such as sand may be provided. pine bedding is not recommended as it causes matting and greasiness of the fur (brain, ; field and sibold, ) as well as the induction of hepatic drug-metabolizing enzymes (vesell, ) . gerbils do best when housed in groups established prior to puberty. introducing new cagemates into an established group, or re-introducing former cagemates who have been separated for more than days, may precipitate fighting (harkness and wagner, ; moore, ; olfert and cross, ) . one method of pairing unfamiliar adults is to allow them to recover from anesthesia in a neutral cage (harkness and wagner, ) . male gerbils raised in isolation exhibit increases in social sniffing, aggression, and anxiety compared with groupreared males (shimozuru and kikusui, ) . recommendations for environmental conditions for gerbils are similar to those for rats and mice. temperature recommendations range from - °f/ - °c (typically  °f/  °c). a -h light: -h dark cycle is appropriate for general housing, but a longer -h light: -h dark cycle is desirable for breeding colonies (field and sibold, ; moore, ) . cold temperatures and/or short light cycles (e.g. °c, -h light: -h dark) may elicit physiological changes related to winter survival mechanisms such as increased basal metabolic rate and non-shivering thermogenesis (li and wang, ) . relative humidity should be maintained at - % rh; however, rough hair coats have been reported to occur at humidity levels greater than % rh (field and sibold, ; harkness and wagner, ; moore, ) . housing areas should be kept quiet, because sudden noises may elicit epileptic seizures. the guide (national research council, ) recommends - air changes per hour as a general standard for animal holding rooms. cage enrichment devices for gerbils that are not made of a sturdy material will be quickly destroyed by the gerbils' natural chewing activities. stainless steel and pvc materials have been recommended in the form of "toys", cups, tubes, and other shelters (field and sibold, ) . however, it has been suggested that providing shelters may result in differences in behavior, maturation rate, and adrenal weights (moore, ) . nesting material is used readily but may be chewed and incorporated into the cage bedding. heavier material such as thick cardboard tubing can provide shelter and a chewing substrate which will last longer than other nesting materials (batchelder, personal communication). naturalistic environmental enrichment such as sand and stones has been recommended. sand or deep bedding allow expression of the animals' burrowing behaviors (field and sibold, ) . sand bathing is an important part of the gerbil behavioral repertoire (tortora et al., ) , but providing sand may not always be practical in the laboratory setting. gerbils in solid-bottom caging generally have the bedding replaced once or twice a week, depending on factors such as cage density or the presence of newborn pups. since gerbils are a desert species, they produce little urine and dry feces, which may allow a decreased frequency of bedding changes relative to other species (moore, ) . typically, bedding is changed and solid-bottom cages are sanitized on the same schedule, while animals in wire-bottom cages may have the cage sanitized less often. the guide recommends that primary enclosures and accessories (including lids and feeders) be sanitized at least every weeks (national research council, ) . sanitization may be performed by hand or in automated equipment. detergent and hot water ( - °f/ - . °c) are used to remove soil and disinfect the equipment. care must be taken to rinse all chemical residue from the caging before use (committee for the purpose of control and supervision on experiments on animals, ; national research council, ) . cages, accessories, and bedding may be autoclaved if a sterile environment is desired (field and sibold, ) . gerbils are adapted to live in arid conditions in the wild. their diet is primarily seeds, and a large portion of their water intake is derived from their food. although they can live for extended periods without supplemental water, provision of fresh water is generally recommended for animals in captivity. reproduction may be decreased if water is restricted (yahr and kessler, ) . supplementation with natural feeds such as vegetables and grains is not necessary, but they may be provided as a treat. zeman ( ) proposed a semi-purified diet as a basis for research into the nutritional requirements of gerbils. however, very little work has been published since then, and the specific nutritional requirements of gerbils remain largely undetermined. they are generally considered to be similar to those of the rat, and gerbils are usually maintained by ad libitum feeding of commercial diets formulated for rats or mice. diets should contain at least % protein. harkness and wagner ( ) recommend % protein and - % dietary fat. gerbils appear to have a higher need for magnesium than rats, as levels below g/kg diet may result in alopecia and increased susceptibility to seizures; . g/ kg diet is recommended. gerbils are also susceptible to developing elevated blood and liver cholesterol when fed an excess of fat or cholesterol in the diet (brain, ; subcommittee on laboratory animal nutrition, ). gerbils are not coprophagic when fed a nutritionally complete diet (field and sibold, ; otken and scott, ) . weanling gerbils may have difficulty reaching food provided in a cage-lid feeder. if necessary, food may be placed in a dish on the cage floor. it is relatively easy to determine the sex of adult gerbils. the male is distinguished by its longer anogenital distance, prominent genital papilla, and pigmented scrotum (figure . ). gerbils become sexually mature at - weeks of age. females have a - -day estrous cycle and are spontaneous ovulators. they are sexually receptive for - hours, and mating often occurs several times. males thump their feet as a courtship display. a vaginal plug is present after ejaculation, but usually disappears by the following morning (brain, ) . gestation is - days, and litter size averages five pups (range - ). larger litters may necessitate fostering some of the pups to another lactating female gerbil or even to a lactating rat (brain, ; moore, ) . both parents participate in building a nest and caring for the young, although during post-partum estrus the male may be more interested in mating than in childrearing (prates and guerra, ) . arrington et al. ( ) reported that % of females become pregnant at this time. as in mice, gestation during lactation may be prolonged due to delayed implantation (brain, ; norris and adams, ) . pups are about . g at birth, are altricial, begin eating solid food at about days of age, and are weaned when they reach - days of age. handling and cage changing should be minimized during the breeding and peri-partum periods to reduce the chance of females abandoning their pups. gerbils may be bred as pairs or as harems with - females per male. however, they may form monogamous pairs, mating for life. after the loss of one mate, it may be difficult to introduce a new one to the survivor (norris and adams, a) . pairs are usually established at about - weeks of age (moore, ) , although brain ( ) suggests animals reproduce sooner if paired at - months of age. animals may fight or fail to mate if they were not previously housed together. females which mature earlier (vaginal opening before days of age) have been reported to be more productive breeders than later-maturing females (brain, ) . female gerbils housed as a single-sex group show decreased incidence of estrus whereas pairing with a male will result in estrus by day in % of females (meckley and ginther, ) . pseudopregnancy has been known to occur, lasting a shorter period ( - days) than pregnancy (brain, ) . gerbils have a relatively high incidence of cystic ovaries which may affect breeding production (norris and adams, b) . wu ( ) has described artificial insemination in the gerbil, and mochida and wakayama ( ) describe gerbil embryo cryopreservation with subsequent implantation and live births. maintaining a healthy gerbil colony is a matter of good husbandry and sanitation, screening of any new animals brought into the colony, and appropriate veterinary medical care and oversight. routine health monitoring is recommended to detect any unwanted agents that may arise in the colony. animals being brought into an established colony should be obtained from a reliable source and have documentation that they do not carry any infectious agents excluded by the institution. shipping containers should be disinfected before bringing into the facility, in case the outside surfaces were contaminated during transport. animals should be checked for general health upon receipt, and in some cases -especially if obtained from a non-commercial or untested source -may be subjected to a quarantine period to ensure absence of unwanted infectious agents before introduction into the general population. similarly, any tissues or biologicals that will be introduced into the colony should be screened for infectious agents prior to use (rehbinder et al., ) . this can be done by mouse antibody production testing or by screening with a panel of polymerase chain reaction (pcr) tests. gerbils have not been reported to be susceptible to spontaneous viral diseases. nevertheless, they can be screened for viruses that may infect other species, including zoonotic viruses. these include lymphocytic choriomeningitis virus (lcmv), pneumonia virus of mice (pvm), minute virus of mice (mvm), parainfluenza virus, hantavirus, coronavirus, reovirus, sendai virus, and simian virus (charles river laboratories, ; rehbinder et al., ) . health monitoring for agents likely to cause disease in gerbils is primarily directed toward bacterial and parasitic agents. it is recommended that gerbil colonies be screened for clostridium piliforme (tyzzer's disease) and for salmonella spp. they may also be screened for car bacillus, bordetella bronchiseptica, pasteurella spp., beta streptococcus spp., streptococcus pneumoniae, pseudomonas spp., klebsiella spp., helicobacter spp., citrobacter rodentium, and corynebacterium kutscheri, which may less commonly cause disease in gerbils, or be transmitted to other species (de la puente-redondo et al., ; glage et al., ) . screening for staphylococcus aureus may generate positive results without disease being present, but the organism can be associated with some conditions such as nasal dermatitis (charles river laboratories, ; rehbinder et al., ; solomon et al., ) . screening should also be performed for the more common parasites including demodex merioni mites and pinworms such as syphacia spp. or dentostomella translucida (wightman et al., ) . other possible parasitic agents include hymenolepis diminuta and hymenolepis nana, giardia spp., entamoeba muris, eimeria spp., encephalitozoon cuniculi, toxoplasma gondii, spironucleus spp., and intestinal flagellates. screening may be performed using colony animals and/or using dedicated sentinel animals, most often animals exposed to soiled bedding and/or caging from colony animals. mice have been used as sentinels in gerbil colonies as they are susceptible to lymphocytic choriomeningitis virus, coronavirus, sendai virus, pneumonia virus of mice, and reovirus , as well as to syphacia and bacterial pathogens (batchelder, personal communication). several types of records are important in the maintenance of a gerbil colony, including animal records, facility records, breeding records, and regulatory records. identification records and health records are two types of animal records. each animal should be able to be identified as to source, birth date, receipt date, sex, animal use protocol, responsible investigator name, and any identifying information such as an ear tag or microchip number. these records help ensure that the appropriate animal, and the correct animal, is used in an experimental procedure. health records can include examination records at the time of receipt, surgical and experimental procedure records, and records of any individual health issues and treatments the animal may have received. facility records consist of documentation that appropriate husbandry and sanitization procedures have been followed. these are commonly kept as a check-off sheet that lists daily, weekly, and other scheduled tasks which are marked off as they are completed. other facility records include cage wash and autoclave validation, feed receipt and expiration dates, pest control documentation, environmental parameters such as light, temperature, and humidity, etc. breeding records are essential to the maintenance of any breeding colony. an animal's genealogy should be able to be traced, and records should be kept on the breeding success of both males and females. daily records, such as birth and weaning dates, and long-term records such as planned breeder replacement are necessary to keep breeding at top efficiency. unless inbreeding is desired, careful records will help avoid mating close relatives. additional records may be required by regulatory agencies in the country where the animals are being kept. such records might include animal receipt and transfer records, disposition records, and documentation concerning the research for which the animals were used. animals which are sick or which die unexpectedly may be expected to have additional documentation such as individual medical or necropsy records. records may be needed to document research oversight activities such as approval of protocols involving gerbils, facility inspections, and animal care program reviews. regulations may also stipulate how long such records need to be vi. other rodents retained. those involved with research using gerbils should be familiar with the regulations for their particular location. gerbils have been used as experimental models in a number of areas of biomedical research. they are relatively easy to maintain and to handle. experimental methods used for gerbils are similar to those used in other rodents such as mice and rats. gerbils are generally non-aggressive and can easily be handled with little risk of being bitten. they may be moved using cupped hands, or may be lifted gently by the base of the tail. care must be taken to grasp the tail base only (figure . ) , since the skin on more distal parts of the tail may pull off easily (donnelly, ) . if more secure restraint is needed, gerbils may be grasped by the loose skin on the back of the neck (figure . ). with this grasp, they may be oriented in any position; however gerbils dislike being held on their backs and may struggle in this position (moore, ) . mechanical restraint devices designed for mice or small rats may be used for gerbils. again, care must be taken to grasp the tail only at the base when placing the animal in the device to prevent a degloving injury. a number of chemical agents used in other species are also used to sedate gerbils for handling. these include diazepam ( mg/kg im or ip), ketamine ( - mg/ kg im), medetomidine ( - µg/kg ip or sc), midazolam ( mg/kg im or ip), or xylazine ( mg/kg im) (flecknell, ; hawk et al., ) . acepromazine has been reported to induce convulsions in gerbils (harkness and wagner, ) and is not recommended in this species. a short-acting inhalant anesthetic such as isoflurane or sevoflurane may also be used to effect for brief chemical restraint. atropine ( . - . mg/kg sc, im, or iv) or glycopyrrolate ( . - . mg/kg im or sc) may be used to decrease salivary and bronchial secretions (hawk et al., ) . gerbils may be identified by cage cards, ear tags or notches, indelible markers, or microchip implantation. tattooing is another option, but is less useful in gerbils than in mice and rats due to the presence of hair on the paws and tail. several sites may be used for blood collection in the gerbil. use of the lateral tail vein may be facilitated by warming the tail to promote blood flow and by placing pressure on the vein at the base of the tail. methods for bleeding from the lateral saphenous vein and from the submandibular vein have also been reported (golde and gollobin, ; hem and smith, ) . for a small amount of blood, a toenail may be clipped short, or the tip of the tail ( - mm) may be transected. care should be taken to control hemorrhage, the tail tip should only be used once or twice to avoid damage to the coccygeal vertebrae, and anesthesia is recommended. larger volumes of blood may be collected by retro-orbital puncture (figure . ), jugular venipuncture (palm and hollander, ) , or cardiac puncture; all of these procedures should be performed under anesthesia, and cardiac puncture (figure . ) should be terminal because of the risk of pericardial hemorrhage and cardiac tamponade. the maximum volume for blood withdrawal which has the least scientific impact on the gerbil's physiologic response is . ml/ g body weight (diehl et al., ; moore, ) . urine collection is difficult due to the very small volume of urine produced by the gerbil, variously reported as a few drops to - ml/day (brain, ; moore, ) . if urine cannot be collected by expressing the bladder or by free catch, a metabolism cage may be used. care should be taken that the small amount of urine does not evaporate from the collection chamber. oral administration of compounds is most commonly accomplished with the use a ball-tipped gavage needle, as used in other small rodents. the length of the needle should be checked against the side of the animal to the level of the last rib to ensure it is not so long as to bypass the stomach (figure . ). the needle should slide easily into the stomach (figure . ); any resistance may indicate the needle is located in the trachea and it should be withdrawn and redirected. animals may also be dosed orally by the use of medicated feed or water. if this method is used, the animals' food and water consumption should be monitored to ensure that the drug is not causing the feed or water to be unpalatable. decreased consumption may lead to underdosing. the most common site for intravenous (iv) injection in the gerbil is the lateral tail vein (figure . ). injection may be facilitated by warming the tail to promote blood flow and by placing pressure on the vein at the base of the tail to dilate the vessel. a -gauge or smaller needle should be used (field and sibold, ) . cutdown procedures have been described for intravenous dosing using the jugular vein (palm and hollander, ) and the femoral vein (pérez-garcía et al., ) . vi. other rodents intramuscular (im) injections are given in the muscles of the thigh (figure . ), with care being taken to avoid the sciatic nerve. the volume of the im injection should be small enough to avoid muscle damage and pain. volumes over . ml should be split between two sites. most subcutaneous (sc) injections are given under the loose skin over the neck and shoulders of the gerbil (figure . ). the skin is tented and the needle is inserted parallel to the body wall to avoid injecting into deeper tissues. gerbils may be dosed intraperitoneally (ip) in the caudal left or right abdomen, similar to other small rodent species. however, because gerbils may struggle if held on their backs, they should be restrained in a vertical position while dosing (figure . ) . extra care should be taken to draw back on the syringe to ensure the needle has not entered the bowel or bladder prior to injecting. other routes of dosing include topical, intradermal, intranasal, and cerebroventricular. any substance used for parenteral dosing should be sterile and pyrogen-free (moore, ) . if dosing is performed repeatedly, the animal should be monitored closely for any complications. depression, dyspnea, hunched posture, abdominal splinting, or signs of pain or irritation at an injection site should be brought to the attention of the veterinary staff. an alternative to repeated parenteral injections is the implantation of an osmotic pump. these devices are designed to be implanted subcutaneously or intraperitoneally, and will subsequently provide a steady-state dose of compound for up to several weeks. methods for euthanasia should be selected based on the recommendations of the avma (american veterinary medical association, ), the experimental parameters, and operator comfort with the procedure. common methods include carbon dioxide inhalation, overdose of an anesthetic agent (e.g. pentobarbital - mg/kg ip), use of a commercial injectable euthanasia agent, or physical methods such as decapitation or cervical dislocation. the latter two methods should be performed under anesthesia unless scientifically justified and approved by the institutional animal care and use committee. less commonly used methods include microwave irradiation and exsanguination/perfusion under anesthesia. all methods require training and proper equipment in order to be performed humanely. gerbils are excellent subjects for laboratory animal research as they are susceptible to bacterial, viral, and parasitic pathogens that affect humans and other species, yet they have very few of their own diseases. some of the most common conditions seen in laboratory gerbils are related to handling, husbandry, or keeping the gerbils until old age. in general, veterinary practices used in providing health care for other laboratory rodents are also appropriate for laboratory gerbils. meeting gerbils' specific housing and husbandry needs will help keep them healthy. gerbils' hind legs are longer than their front legs and they like to stand erect on the hind legs. to accommodate this behavior, the floor to lid height of gerbil cages needs to be sufficient for the gerbil to stand. gerbils should be housed in solid-bottom caging with bedding at least - cm deep to allow for burrowing behavior. gerbils also exhibit gnawing behavior and need to have non-toxic, hard materials on which to gnaw. gerbils must be picked up by the base of the tail to avoid the skin on the tail degloving. they should have their incisors and claws checked periodically to make sure that both are being worn down. claws can be clipped if they are too long. a rough-surfaced object such as a stone or a bathroom tile can be placed in the cage for the gerbils to wear down their claws as well as to use for normal marking behaviors. gerbils exhibit shredding behavior and should be provided with autoclaved cardboard, hay, straw, paper, or tissue. gerbils are burrowing animals and sometimes will resort to stereotypic digging in the corner of the cage. a man-made burrow may be used in a laboratory setting. one example utilizes clear and opaque sections with an access tunnel (waiblinger, ) . gerbils need to have a substrate to remove the lipids that they place on their hair coat during self-grooming. in the wild, gerbils use sand baths, and they will use them in the laboratory if provided (tortora et al., ) . if gerbils exhibit a dirty, ungroomed hair coat, consider changing the type and amount of bedding, make sure the relative humidity is less than % and consider lowering the temperature of the room. epilepsy gerbils may have spontaneous seizures secondary to stress such as handling, cage change, abrupt noises, or changes in the environment. generally, gerbils will recover from these seizures after - seconds without treatment, and appear to have no long-lasting effects. gerbils that are picked up by the tip or middle of the tail are very susceptible to skin degloving (donnelly, ) . gerbils should always be picked up by the base of the tail or by cupping the hands. tails that have been degloved can be surgically amputated. this is a common skin condition of gerbils which likely has a multifactorial etiology. clinical signs start as erythema at the nares and develop into facial alopecia, dermatitis, and scab formation. some cases can develop into a moist dermatitis involving the head, forelimbs, and chest. this condition has been reported to be most common in young weanling gerbils (field and sibold, ) . gerbils release a complex mixture of pigments and lipids from the harderian gland during self-grooming. the material is excreted at the nares and is mixed with saliva and spread over the gerbil's hair coat. harderian exudates are normally removed from the gerbil's pelage by sand bathing (harriman and thiessen, ; thiessen, ) . two studies have shown that harderian secretions which act as primary skin irritants are a factor in the development of nasal dermatitis. removal of harderian glands or housing animals on sand either prevented lesions or caused improvement in existing lesions, while animals with intact harderian glands and reduced opportunity to groom or sand bathe developed or maintained existing lesions (farrar et al., ; thiessen and pendergrass, ) . staphylococcus spp. have been implicated in the development of this condition. one paper reports s. aureus as the bacteria most commonly cultured from the nasal area of affected animals, although s. aureus was also cultured from non-affected gerbils (bresnahan et al., ) . another study reported that s. xylosus was the predominant species isolated from all gerbils, most commonly from the nasal area, and the only species isolated from the nasal area of gerbils with clinical signs of nasal dermatitis (solomon et al., ) . cystic ovaries are seen commonly in female gerbils over year of age (norris and adams, b) . clinical signs include symmetrical alopecia, abdominal swelling, lethargy, anorexia, dyspnea, and reduced fertility. in , norris and adams showed that female gerbils older than days had a % rate of cystic ovaries in gerbils with two ovaries and a % rate of cystic ovaries in gerbils with one ovary. they also showed that removing one ovary in a gerbil does little to affect overall reproductive performance (norris and adams, ) . although there have been reports of bacterial diseases in laboratory gerbils in the past, animals can be purchased today that are free from antibodies to clostridium piliforme and are culture-negative for salmonella spp., bordetella bronchiseptica, and pasteurella pneumotropica. the most common bacterial threat to a high-quality research colony is helicobacter. there have been reports of laboratory and pet gerbils infected with clostridium piliforme, the etiologic agent for tyzzer's disease (motzel and gibson, ; port et al., ; vincent et al., ) . gerbils usually exhibited diarrhea or sudden death. necropsy findings included multi-focal hepatic necrosis. in , waggie et al. experimentally induced tyzzer's disease in gerbils and showed that they were highly susceptible to the disease. strittmatter ( ) eliminated the disease in gerbils by cross-fostering gerbil offspring to unaffected mice. in , bergin et al. reported a case of two laboratory gerbils with fatal clostridium difficile enteritis. the gerbils were being treated for helicobacter infection with antibiotic wafers containing amoxicillin, metronidazole, and bismuth subsalicylate. there is also one report of a fatal epidemic of citrobacter rodentium affecting nine gerbils (de la puente-redondo et al., ) . in , tappe et al. reported a new species of the genus streptococcus which was cultured from the oropharynx of mongolian gerbils. the new name proposed for these gram-positive, catalase-negative, chain-forming cocci is streptococcus merionis sp. nov. there were no clinical signs reported in the gerbils. gerbils are natural carriers of multiple species of helicobacter (bergin et al., ) . glage et al. ( ) reported rederivation of helicobacter hepaticus-infected gerbils by caesarean section and cross-fostering on non-infected mice. no naturally occurring viral diseases of gerbils have been published. parasites rarely cause clinical problems in the laboratory gerbil, and are excluded from major commercial gerbil suppliers. wightman et al. ( ) reported finding syphacia obveleta and dentostomella translucida in gerbils. they showed that s. obveleta can be transmitted from gerbil to mouse, mouse to gerbil, and gerbil to gerbil, while ross et al. ( ) showed that syphacia muris is similarly easily transmitted between gerbils, rats, hamsters, and mice. wilkerson et al. ( ) reported that using fenbendazole-medicated feed was the only practical and reliable way to eradicate pinworms in gerbil colonies. in , lussier and loew reported a naturally occurring case of hymenolepis nana in mongolian gerbils, while in , vincent et al. reported the recovery of hymenolepis diminuta from gerbils. demodex merioni mites have been reported in gerbils with alopecia, dry skin, and ulcerations. animals who are already debilitated are at most risk for developing mite infestations (rollin and kesel, ) . gerbils are very sensitive to dihydrostreptomycin. wightman et al. ( ) reported that an injection of mg caused mortality in - % of gerbils weighing - grams. two gerbils given a commercially available treatment to eradicate helicobacter developed a necrohemorrhagic enteritis. it was determined that the amoxicillin component caused an overgrowth of clostridium difficile (bergin et al., ) . there are a number of surveys about neoplasia in laboratory colonies of mongolian gerbils (meckley and zwicker, ; ringler et al., ; vincent and ash, ; vincent et al., ) . neoplasia has been most often reported in gerbils over years of age (matsuoka and suzuki, ) . commonly reported tumors are squamous cell carcinoma of the ventral marking gland in males and ovarian granulosa cell tumors in females. in surveys, adrenocortical tumors, cutaneous squamous cell carcinoma, malignant melanoma, and renal and splenic hemangiomas are the next most commonly reported tumors. other tumors that have been reported include astrocytoma, craniopharyngioma, and systemic mastocytosis (guzman-silva, ; guzman-silva et al., ; kroh et al., ; rembert and johnson, ) . in , campos et al. reported a variety of epithelial-type neoplasias in the ventral prostrate of -month-old gerbils. tumors included prostatic intraepithelial neoplasias, microinvasive carcinomas, and adenocarcinomas. this condition is considered an old-age change. gerbils will exhibit clinical signs of polyuria, polydipsia, and weight loss. on gross exam the kidneys will be shrunken and pitted (bingel, ) . older gerbils can spontaneously develop cholesteatomas in the ear canal which resemble the human condition. clinical signs include scratching, head tilt, and circling. the incidence appears to be age-related (fulghum and cole, ; schiffer et al., ) . gerbils have unique characteristics which make them appropriate for a number of animal models. classically, gerbils have been used in research involving stroke, parasitology, infectious diseases, epilepsy, brain development and behavior, and hearing. in mammals the circle of willis is comprised of a communication of arteries at the bottom of the brain consisting of the internal carotid arteries, anterior cerebral arteries, anterior communicating arteries, posterior communicating arteries, posterior cerebral arteries, and basilar arteries. this structure provides for alternate blood flow to the brain in case one artery becomes occluded. levine and payan ( ) first identified gerbils as having an anatomical anomaly of the circle of willis with no communication between the posterior cerebral arteries. this has been termed an incomplete circle of willis. researchers found that ligating the left carotid artery of the gerbil caused acute cerebral ischemia much more consistently than when performing the same ligation in the rat (wexler, ) . in , levy and brierley performed a study on gerbils to assess the cerebrovascular anatomy and communications in the gerbil brain by injecting colored dye into the aorta. based on evaluating ten male gerbils from an unspecified source, these authors concluded that each of the gerbil's carotid arteries is divided into an anterior, middle, and posterior cerebral artery. they found that the basilar artery communicated with the carotid artery in all gerbils, that the posterior cerebral artery was a branch of the carotid, and that there was no single posterior communicating artery. as gerbils were more frequently used for stroke research, more information about their cerebrovascular anatomy became available. it was determined that not all gerbils have an incomplete circle of willis and that "stroke-prone" and "stroke-resistant" gerbils could be identified (delbarre et al., ; kitagawa et al., ; pelliccioli et al., ) . considerable variability was found both in the extent of damage after bilateral artery occlusion and in the percentage of stroke-prone gerbils in a population. the percentage varied between the sexes (males . % stroke-prone, females . %) (hall et al., ) and by the source of the animals (breuer and mayevsky, ; laidley et al., ; seal et al., ) . methods to define stroke-prone gerbils include examination of retinal blood flow after ligation of the carotid artery (delbarre et al., ) , measurement of the diameter of the common carotid artery before and after temporary ligation (kitagawa et al., ) , survival after unilateral carotid artery occlusion (pelliccioli et al., ) , and determination of the presence and diameter of the posterior communicating artery (seal et al., ) . despite the fact that the anatomy of the gerbils' circles of willis cannot be considered uniformly lacking in posterior communicating arteries, gerbils continue to be used as a common animal model for stroke, with the two-vessel occlusion being considered the simplest (small and buchan, ) . laboratory animal veterinarians that work with gerbils as stroke models need to be aware that this difference in cerebral vascular anatomy could lead to variable or conflicting results. gerbils seem to be very well suited to host parasitic infections from other species and are therefore a popular animal model for a wide variety of parasites. many parasitic genera have been described in experimental studies of infection, susceptibility, pathology, immunology, and chemotherapy. gerbils are alternative definitive hosts for echinococcus granulosis (conchedda et al., ) . gerbils have also been used as an animal model for echinococcus multilocularis (kamiya and sato, ) as well as echinococcus vogeli (matsuo et al., ) . although the mongolian gerbil is not a natural host for rodentolepsis nana, it can be experimentally transmitted to gerbils that are given dexamethasone daily (vianna and demelo, ) . gerbils have also been experimental hosts for hymenolepis diminuta, taeniasolium, taenia saginata, taenia asiatica, and taenia crassiceps (avila et al., ; chang et al., ; johnson and condor, ; kamiya and sato, ; sato et al., ) . gerbils can also serve as experimental hosts for a number of nematode parasites. these include haemonchus contortus, toxocara canis, strongyloides stercoralis, strongyloides papillosus, aspiculuris tetraptera, syphacia obvelata, dentostomella translucida, and baylisascaris procyonis (akao et al., ; de jesús-gabino et al., ; lok, ; pinto et al., a pinto et al., , b zanandréa et al., ) . one of the most important uses of gerbils in biomedical research is for the study of filarid nematodes, which include brugia malayi, brugia pahangi, loa loa, and litomosoides sigmodontis (lim et al., ; mand et al., ; shigeno et al., ; wanji et al., ) . gerbils have been experimentally infected with these filarid nematodes to study the immunology, antigenicity, and life cycles of the parasites. serologic tests and treatment regimens have been developed by using these models (hübner et al., ; lim et al., ; shigeno et al., ) . gerbils have been used to study all aspects of the trematode life cycle including infectivity and immunology as well as to study potential treatments. these trematodes include schistosoma mansoni, schistosoma japonicum, schistosoma haemotobium, schistosoma magreboweie, echinostoma caproni, and opisthorcis viverinni (adam et al., ; boonmars et al., ; chisty et al., ; mahler et al., ; ogbe, ; sato and kamiya, ) . gerbils are also a good host for the avian schistosome, austrobilharzia variglandis (bacha et al., ) . gerbils are good animal models for intestinal cryptosporidium parvum infection and for gastric infection with cryptosporidium andersoni and muris (kvác et al., ) . immunosuppressed gerbils have been reported to be infected with cryptosporidium hominis (baishanbo et al., ) . gerbils have been identified as a model for infection with giardia lamblia (belosevic et al., ; faubert et al., ) . gerbils were used to develop the fecal antigen test for giardiasis (moss et al., ) . gerbils can also be experimentally infected with giardia duodenalis (araújo et al., ) . this is caused by a negative-strand rna neurotropic virus which causes acute or subacute encephalitis in horses and sheep. this virus has been linked with schizophrenia and other affective disorders in humans (rott et al., ) . borna disease is considered a model for studying neuronal plasticity, as the virus can persist in the cns and cause changes in brain cell function (gonzalez-dunia et al., ) . the gerbil is the definitive animal model for borna disease. newborn gerbils are very susceptible to intracranial infection with borna virus resulting in inflammatory reactions in the brain as well as evidence of viral persistence in the brain (nakamura et al., ) . in , gerbils were identified as the first animal model for swine hepatitis virus. gerbils inoculated intraperitoneally with hepatitis e virus became infected, with viremia and virus shedding in the feces for weeks. additionally, the virus could be found in the liver of the infected gerbils (li et al., ) . this is an arbovirus that is one of the most common causes of pediatric arboviral infection in the us (haddow and odoi, ). in , researchers at the university of wisconsin discovered that mongolian gerbils were an ideal animal model for la crosse virus studies. gerbils were susceptible to infection, survived, and developed viremia and neutralizing antibody titers following exposure by intramuscular injection and by the bite of infected mosquitoes. moreover, they are attractive to mosquito vectors (osorio et al., ) . gerbils are able to be infected with encephalomyocarditis virus, with viral replication evident in the heart and pancreas (matsuzaki et al., a) . the young gerbil has also been identified as the first animal model of rift valley fever encephalitis that was uniformly fatal without causing lesions outside of the cns (anderson et al., ) . during the summer of , three gerbils were shown to be infected with monkey pox virus after being exposed to the virus from an infected gambian giant rat (kulesh et al., ) . newborn gerbils have been shown to be a better model for puumula virus transmission than rats or mice (lokugamage et al., ) . newborn gerbils can also be experimentally infected with reovirus with histopathologic lesions noted in the brain and pancreas (yukawa et al., ) . the gerbil is favored by many researchers as an animal model for helicobacter pylori research because, when infected, the gerbil develops severe gastritis and gastric ulcers. these ulcers do not heal spontaneously but do respond to therapeutic treatment. this is similar to human infection with helicobacter pylori (peek, ) . gerbils also can develop gastric adenocarcinoma secondary to chronic helicobacter infection (franco et al., ; tsukamoto et al., ; watanabe et al., ) . the mongolian gerbil is an animal model for listeriosis, the disease resulting from infection with listeria monocytogenes (blanot et al., ) . the gerbil has two cell receptors which are similar to the human, inla-ecadherin and inlb-met. these cell receptors work with l. monocytogenes surface proteins to facilitate entry into the cells (disson et al., ) . the gerbil has been identified as a good animal model to study pulmonary hemorrhage as a consequence of severe leptospirosis infection. more importantly, the gerbil mimics the human in its response to the infection by producing increased amounts of platelet-activating factor acetylhydrolase (yang et al., ). gerbils have been used in research on vaccines for borrelia burgdorferi, the etiologic agent for lyme disease (preac-mursic et al., ) . gerbils are considered a good model for inherited epilepsy. gerbils will display spontaneous, recurrent generalized seizures beginning at about weeks of age (buckmaster and wong, ; loskota and lomax, ) . mongolian gerbils were first identified as a new animal model of inherited seizures in (thiessen et al., b) . immediately thereafter a number of papers were published regarding gerbils and seizures (kaplan and miezejeskic, ; loskota et al., loskota et al., , . in the s loskota started a selective breeding program at ucla to develop seizure-sensitive and seizure-resistant gerbils. selective breeding of three pairs of seizure-sensitive gerbils, originating from tumblebrook farm in massachusetts, for generations resulted in the creation of the seizure-sensitive gerbil. the seizure-sensitive gerbils were identified as wjl/uc and seizure-resistant gerbils as str/uc (loskota et al., ) . mongolian gerbils continue to be used as a model of inherited epilepsy. it is now known that the seizuresensitive gerbils have abnormalities that involve the gabaergic synaptic transmission in the brain (hwang et al., ; kang et al., ; kwak et al., ) . the entire mechanism of how and why seizures occur continues to be under investigation. since the s, the gerbil has been widely used in studies of brain growth and development, neural plasticity, behavior, and normal and pathological aspects of aging (cheal, ) . examples include studies of changes in the muscarinic receptors in the gerbil thalamus from age - months (pilar-cuéllar et al., ) , effects of periadolescent sensory stimulation on neural development (lehmann et al., ) , and ontogeny of the dopamine innervations in the nucleus accumbens (lesting et al., ) . behavioral studies include reports of neonatal separation that results in behavioral and biochemical differences in adult gerbils (jaworska et al., ) and studies of the effects of the father in gerbil developmental behavior (piovanotti and vieira, ) . lee et al. ( lee et al. ( , a lee et al. ( , b have studied neurochemical changes in the gerbil brain, especially the hippocampus, as it relates gerbils have been used extensively in neuroscience research. it has been found that gerbils are more homologous to humans in the tachykinin nk- , , , receptor activity and affinity as compared to rats and mice (griffante et al., ; leffler et al., ). a new model, gerbil foot tapping, has been developed as a fear-related response. gerbils injected with nk- agonists in the brain develop a fear-related foot-tapping behavior (bristow and young, ; sundqvist et al., ) . gerbils have also been used as animal models of anxiety in the elevated plus maze and black/white box tests (bridges and starkey, ; heldt et al., ) , and to evaluate the anxiolytic properties of rose odor inhalation (bradley et al., ) . gerbils have been used in the forced swim test which has predictive validity for the evaluation of novel anti-depressants (wallace-boone et al., ) . in , gaese et al. published a report using mongolian gerbils for acoustic startle and prepulse inhibition studies. mongolian gerbils are a common animal model in auditory research as gerbils have human-like lowfrequency hearing (engel, ; wetzel et al., ) . gerbils have been used as an animal model of auditory neuropathy and it has been shown that there is the potential for use of stem cells to cure some hearing loss (matsuoka et al., ) . the gerbil is the first animal model found for the human disease hemochromatosis. in the experimental gerbil model histopathologic lesions in the heart and liver are very similar to the human disease. the model is created by once-or twice-daily injections with iron dextran (carthew et al., ) . the gerbil iron-overload model continues to be used to further elucidate the mechanisms of the disease as well as to explore potential therapies (al-rousan et al., ; kaiser et al., ; otto-duessel et al., ) . just as gerbils have been used widely in acoustic and auditory basic research, they have also been found to be an animal model for cholesteatoma, a benign tumor of the middle ear. the animal model is most commonly created by surgical ligation of the gerbil's auditory duct (choufani et al., ; kim and chole, ) . gerbils are also commonly used for studies of infectious otitis media. in the induced model the gerbils are usually infected with streptococcus pneumoniae (soriano et al., ) , although haemophilus influenza (ponte et al., ) and moraxella catarrhallis (fulghum and marrow, ) have also been used. gerbils have been used as an animal model for the study of androgen receptors in the prostate gland (campos et al., ; cordeiro et al., ) . gerbils are used as experimental models to correlate the periodontium's biological response to various mechanical stresses, as the periodontal ligament was shown to be highly sensitive to occlusal alterations (iyomasa et al., ) . gerbils have also been used as animal models for dental caries (fitzgerald and fitzgerald, ) . the development of specific parts of the brain that relate to hearing, such as agerelated changes in the medial nucleus of the trapezoid body and in the lateral superior olive (gleich and strutz references cfr part ultrastructural hepatic alterations in hamsters and jirds after experimental infection with the liver fluke opisthorchis viverrini gerbils cerebellar ataxia due to toxocara infection in mongolian gerbils, meriones unguiculatus a petkeeper's guide to hamsters and gerbils deferasirox removes cardiac iron and attenuates oxidative stress in the iron-overloaded gerbil the gerbil, meriones unguiculatus, a model for rift valley fever viral encephalitis giardia duodenalis: pathological alterations in gerbils, meriones unguiculatus, infected with different dosages of trophozoites growth, longevity, and reproductive life of the mongolian gerbil avma guidelines on euthanasia laboratory animal models for human taenia solium infection of the gerbil by the avian schistosome austrobilharzia variglandis (miller and northrup , penner ) infectivity of cryptosporidium hominis and cryptosporidium parvum genotype isolates in immuno-suppressed mongolian gerbils morphology of the terminal bronchiolar region of common laboratory mammals application of endovascular suture occlusion of middle cerebral artery in gerbils to obtain consistent infarction observations on natural and experimental infections with giardia isolated from cats immunoexpression of gelatinase (mmp- and mmp- ) in the seminal vesicles and ventral prostate of libyan jird (meriones libycus) during the seasonal cycle of reproduction eradication of enteric helicobacters in mongolian gerbils is complicated by the occurrence of clostridium difficile enterotoxemia pathologic lesions in an aging mongolian gerbil. (meriones unguiculatus) colony a gerbil model for rhombencephalitis due to listeria monocytogenes animal models for opisthorchis viverrini infection the effects of prolonged rose odor inhalation in two animal models of anxiety a step-by-step book about gerbils the laboratory gerbil nasal dermatitis in the mongolian gerbil brain vasculature and mitochondrial responses to ischemia in gerbils. ii. strain differences and statistical evaluation sex differences in mongolian gerbils in four tests of anxiety chromodacryorrhea and repetitive hind paw tapping: models of peripheral and central tachykinin nk receptor activation in gerbils evoked responses of the dentate gyrus during seizures in developing gerbils with inherited epilepsy lamina formation in the mongolian gerbil retina meriones unguiculatus age-related histopathological lesions in the mongolian gerbil ventral prostate as a good model for studies of spontaneous hormonal related disorders a unique rodent model for both the cardiotoxic and hepatotoxic effects of prolonged iron overload development of taenia asiatica cysticerci to infective stage and adult stage in mongolian gerbils the gerbil: a unique model for research on aging health monitoring protocols. retrieved on october a revision of the genus meriones schistosoma mansoni: kinetics of glomerulonephritis in mongolian gerbils and its correlation with intensity and duration of infection animal model for cholesteatoma induced in the gerbil: will the profiles of differentiation/growth-regulatory markers be similar to the clinical situation biology and diseases of other rodents adult testosterone levels influence the morphology of a sexually dimorphic area in the mongolian gerbil brain cpcsea guidelines for laboratory animal facility development and sexual maturation of echinococcus granulosus adult worms in the alternative definitive host the mammals of the palaearctic region: a taxonomic review androgen receptor in the mongolian gerbil ventral prostate: evaluation during different phases of postnatal development and following androgen blockage malignant melanoma in a black gerbil (meriones unguiculatus) adrenal weight ratios in the mongolian gerbil (meriones unguiculatus) olfactory discrimination limits in gerbils anthelmintic effects of prosopis laevigata n-hexanic extract against haemonchus contortus in artificially infected gerbils (meriones unguiculatus) epidemic infection caused by citrobacter rodentium in a gerbil colony a suitable method to select gerbils with incomplete circle of willis a good practice guide to the administration of substances and removal of blood, including routes and volumes the mongolian gerbil: qualitative and quantitative aspects of the cellular blood picture modeling human listeriosis in natural and genetically engineered animals disease problems of small rodents gerbils' adrenal beta-and -hydroxylating activities respond similarly to inhibitory or stimulatory agents: two activities of a single enzyme checklist of palaearctic and indian mammals to paternal and maternal behavior in the mongolian gerbil experimental nasal dermatitis in the mongolian gerbil: effect of bilateral harderian gland adenectomy on development of facial lesions comparative studies on the pattern of infection with giardia spp. in mongolian gerbils the laboratory hamster and gerbil induction of dental caries in gerbils laboratory animal anaesthesia regulation of gastric carcinogenesis by helicobacter pylori virulence factors bacterial flora in spontaneously occurring aural cholesteatomas in mongolian gerbils experimental otitis media with moraxella (branhamella) catarrhalis acoustic startle and prepulse inhibition in the mongolian gerbil hematologic and clinical chemical normal ranges of the mongolian gerbil rederivation of helicobacter hepaticus-infected mongolian gerbils by caesarean section and cross-fostering to rats and mice age dependent changes in the medial nucleus of the trapezoid body in gerbils age-dependent changes in the lateral superior olive of the gerbil (meriones unguiculatus) effects of various hormones on the growth and histology of the gerbil (meriones unguiculatus) abdominal sebaceous gland pad a rapid, simple, and humane method for submandibular bleeding of mice using a lancet borna disease virus interference with neuronal plasticity body water relations in two species of gerbil (tatera indica indica and meriones hurrianae) of the indian desert h]gr displays similar nk receptor binding profile in gerbil and human brain systemic mast cell disease in the mongolian gerbil, meriones unguiculatus craniopharyngioma in the mongolian gerbil (meriones unguiculatus) the incidence risk, clustering, and clinical presentation of la crosse virus infections in the eastern united states sex differences in postischemic neuronal necrosis in gerbils individual differences in the biological odors of the mongolian gerbil (meriones unguiculatus) the biology and medicine of rabbits and rodents removal of harderian exudates by sandbathing contributes to osmotic balance in mongolian gerbils formulary for laboratory animals the albino gerbil anxiolytic-like effects of the neurokinin receptor antagonist gr- in the elevated plus maze and contextual fear-potentiated startle model of anxiety in gerbils saphenous vein puncture for blood sampling of the mouse, rat, hamster, gerbil, guinea pig, ferret and mink the mongolian gerbil in biomedical research litomosoides sigmodontis: a simple method to infect mice with l larvae obtained from the pleural space of recently infected jirds (meriones unguiculatus) introduction to rodents gaba a, not gaba b, receptor shows subunit-and spatialspecific alterations in the hippocampus of seizure prone gerbils species specific differences in the ratio of short to long loop nephrons in the kidneys of laboratory rodents histological and histomorphometrical alterations of the periodontal ligament in gerbils submitted to teeth extraction repeated neonatal separation results in different neurochemical and behavioral changes in adult male and female mongolian gerbils biophysical properties of cav . calcium channels in gerbil inner hair cells infectivity of hymenolepis diminuta for the jird (meriones unguiculatus), and utility of this model for anthelmintic studies the harderian gland, its secretory duct and porphyrin content in the mongolian gerbil (meriones unguiculatus) does the gerbil model mimic human iron overload? complete life cycle of the canid tapeworm, echinococcus multilocularis, in laboratory rodents the alteration of gamma-aminobutyric acid-transaminase expression in the gerbil hippocampus induced by seizure development of seizures in the mongolian gerbil (meriones unguiculatus) is the feeding and reproductive performance of the flea, xenopsylla ramesis, affected by the gender of its rodent host, meriones crassus experimental models of aural cholesteatomas in mongolian gerbils prediction of stroke-prone gerbils and their cerebral circulation body and organ weights and linear measurements of the adult mongolian gerbil spontaneous astrocytoma in the mongolian gerbil (meriones unguiculatus) monkeypox virus detection in rodents using real-time '-minor groove binder taqman assays on the roche lightcycler infectivity of gastric and intestinal cryptosporidium species in immunocompetent mongolian gerbils (meriones unguiculatus) effects of gabaergic transmissions on the immunoreactivities of calcium binding proteins in the gerbil hippocampus increased behavioral and histological variability arising from changes in cerebrovascular anatomy of the mongolian gerbil age-dependent changes in calretinin immunoreactivity and its protein level in the gerbil hippocampus calbindin d- k immunoreactivity and its protein level in hippocampal subregions during normal aging in gerbils cyclooxygenase- immunoreactivity and protein level in the gerbil hippocampus during normal aging characterization of species-related differences in the pharmacology of tachykinin nk receptors , and developmental effects on dopamine projections and hippocampal cell proliferation in the rodent model of postweaning social and physical deprivation can be triggered by brief changes of environmental contex ontogeny of the dopamine innervation in the nucleus accumbens of gerbils effects of ischemia and other procedures on the brain and retina of the gerbil (meriones unguiculatus) communications between vertebrobasilar and carotid arterial circulations in the gerbil experimental infection of mongolian gerbils by a genotype strain of swine hepatitis e virus seasonal adjustments in body mass and thermogenesis in mongolian gerbils (meriones unguiculatus): the roles of short photoperiod and cold brugia malayi infection in meriones unguiculatus: antibody response to recombinant bmr development and hormonal control of territorial marking in the male mongolian gerbil (meriones unguiculatus) strongyloides stercoralis: a model for translational research on parasitic nematode biology development of an efficient method for recovery of puumala and puumala-related viruses by inoculation of mongolian gerbils the mongolian gerbil (meriones unguiculatus) as a model for the study of the epilepsies: eeg records of seizures the gerbil as a model for the study of the epilepsies. seizure patterns and ontogenesis case report. natural hymenolepis nana infection in mongolian gerbils (meriones unguiculatus) studies on the reproductive capacity of echinostoma caproni (trematoda) in hamsters and jirds ultrasonography in filaria-infected rodents: detection of adult litomosoides sigmodontis and brugia malayi filariae development and sexual maturation of echinococcus vogeli in an alternative definitive host, mongolian gerbil (meriones unguiculatus) enhanced survival of bone-marrow-derived pluripotent stem cells in an animal model of auditory neuropathy spontaneous tumors in the mongolian gerbil (meriones unguiculatus) experimental encephalomyocarditis virus infection in mongolian gerbils (meriones unguiculatus) the genetics of coat colors in the mongolian gerbil (meriones unguiculatus) baseline hematological and blood biochemical parameters of the mongolian gerbil (meriones unguiculatus) occurence of oestrus in the mongolian gerbil after pairing with a male naturally-occurring neoplasms in the mongolian gerbil, meriones unguiculatus evolutionary history of the most speciose mammals: molecular phylogeny of muroid rodents birth of offspring after transfer of mongolian gerbil (meriones unguiculatus) embryos cryopreserved by vitrification hamsters and gerbils antigenic variation of giardia lamblia in the feces of mongolian gerbils tyzzer's disease in hamsters and gerbils from a pet store supplier rodentia: myomorpha: muroidea: muridae: gerbillinae high susceptibility of mongolian gerbil (meriones unguiculatus) to borna disease virus low microsatellite variation in laboratory gerbils the ufaw handbook on the care and management of laboratory animals aggressive behaviour and reproduction in the mongolian gerbil, meriones unguiculatus, relative to age and sexual experience at pairing incidence of cystic ovaries and reproductive performance in the mongolian gerbil, meriones unguiculatus the growth of the mongolian gerbil, meriones unguiculatus, from birth to maturity mating post partum and length of gestation in the mongolian gerbil (meriones unguiculatus) lifetime reproductive performance of mongolian gerbils (meriones unguiculatus) in vivo and in vitro development of schistosoma margrebowiei the effects of openfield size on activity in the mongolian gerbil guide to the care and use of experimental animals effects of la crosse virus infection on pregnant domestic rabbits and mongolian gerbils feeding characteristics of mongolian gerbils (meriones unguiculatus) safety and efficacy of combined chelation therapy with deferasirox and deferoxamine in a gerbil model of iron overload a procedure for intravenous injection using external jugular vein in mongolian gerbil (meriones unguiculatus) helicobacter pylori infection and disease: from humans to animal models variable response of the mongolian gerbil to unilateral carotid occlusion: magnetic resonance imaging and neuropathological characterization a simple procedure to perform intravenous injections in the mongolian gerbil (meriones unguiculatus) muscarinic receptor changes in the gerbil thalamus during aging first natural helminth infection in the mongolian gerbil (meriones unguiculatus), parasitized with dentostomella translucida in the neotropical region evaluation of co-infection with pinworms (aspiculuris tetraptera, dentostomella translucida, and syphacia obvelata) in gerbils and mice presence of the father and parental experience have differentiated effects on pup development in mongolian gerbils (meriones unguiculatus) antimicrobial treatment of an experimental otitis media caused by a beta-lactamase positive isolate of haemophilus influenzae an ultrastructural study of tyzzer's disease in the mongolian gerbil (meriones unguiculatus) parental care and sexual interactions in mongolian gerbils (meriones unguiculatus) during the postpartum estrus active immunization with pc protein of borrelia burgdorferi protects gerbils against b. burgdorferi infection aflp to assess genetic variation in laboratory gerbils (meriones unguiculatus) report of the federation of european laboratory animal science associations (felasa) working group on animal health accepted by the felasa board of management what's your diagnosis? pigmented mass in an experimental gerbil. spontaneous malignant melanoma spontaneous neoplasms in aging gerbillinae th anniversary of tumblebrook gerbils: some historical notes gerbil classification and nomenclature gerbil care and maintenance ecology of the mongolian gerbil reproduction in the mongolian gerbil tumblebrook gerbils: th anniversary the behavior of mongolian gerbils in a semi-natural environment, with special references to ventral marking, dominance and sociability ii: care, husbandry and wellbeing. an overview by species cholesterol metabolism in the gerbil experimental transmission of syphacia muris among rats, mice, hamsters and gerbils detection of serum antibodies to borna disease virus in patients with psychiatric disorders normal values for hemoglobin concentration and cellular elements in the blood of mongolian gerbils the mammalian harderian gland: morphology, biochemistry function and phylogeny defect of protective immunity to schistosoma mansoni infection in mongolian gerbils involves limited recruitment of dendritic cells in the vaccinated skin survival of destrobilated adults of taenia crassiceps in t-cell-depleted mongolian gerbils diagnostic exercise: head tilt in a gerbil the gerbil, a new laboratory animal new variability in cerebrovascular anatomy determines severity of hippocampal injury following forebrain ischemia in the mongolian gerbil temporary shift of microfilariae of brugia pahangi from the lungs to muscles in mongolian jirds, meriones unguiculatus, after a single injection of diethylcarbamazine effects of isolation-rearing on the development of social behaviors in male mongolian gerbils (meriones unguiculatus) animal models male saliva cues and female social choice in mongolian gerbils erythrocyte basophilic stippling in the mongolian gerbil a survey of staphylococci isolated from the laboratory gerbil role of streptococcus pneumoniae and haemophilus influenzae in the development of acute otitis media and otitis media with effusion in a gerbil model elimination of tyzzer's disease in the mongolian gerbil (meriones unguiculatus) by fostering to mice nutrient requirements of the gerbil senktide-induced gerbil foot tapping behaviour is blocked by selective tachykinin nk and nk receptor antagonists population growth and social structure of confined colonies of mongolian gerbils: scent gland size and marking behaviour as indices of social status streptococcus merionis sp. nov., isolated from mongolian jirds (meriones unguiculatus) effects of dietary cholesterol in the mongolian gerbil and the rat: a comparative study thermoenergetics and the evolution of pheromone communications body temperature and grooming in the mongolian gerbil territorial marking in the female mongolian gerbil: short term reaction to hormones harderian gland involvement in facial lesions in the mongolian gerbil central control of territorial marking in the mongolian gerbil the gerbil in behavioral investigations harderian gland pheromone in the mongolian gerbil (meriones unguiculatus) androgen control of territorial marking in the mongolian gerbil spontaneous seizures in the mongolian gerbil mechanisms of territorial marking in the male and female mongolian gerbil (meriones unguiculatus) identification of a ventral scent marking pheromone in the male mongolian gerbil (meriones unguiculatus) the effect of sand deprivation on sandbathing and marking in mongolian gerbils (meriones unguiculatus) animal models of stomach carcinogenesis a relationship between frequency of display of territorial marking behavior and coat color in the mongolian gerbil induction of drug-metabolizing enzymes in liver microsomes of mice and rats by softwood bedding experimental infection and adaptation of rodentolepis nana to the mongolian jird (meriones unguiculatus) further observations on spontaneous neoplasms in the mongolian gerbil, meriones unguiculatus spontaneous lesions and parasites of the mongolian gerbil (meriones unguiculatus) the pathology of the mongolian gerbil (meriones unguiculatus): a review experimentally induced tyzzer's disease in mongolian gerbils (meriones unguiculatus) husbandry and medicine of small rodents comfortable quarters for gerbils in research institutions the control and function of maternal scent marking in the mongolian gerbil behavioral and pharmacological validation of the gerbil forced-swim test: effects of neurokinin- receptor antagonists the migration and localization of loa loa infective and fourth-stage larvae in normal and immuno-suppressed rodents genetic analysis of the black pigment mutation in the mongolian gerbil helicobacter pylori infection induces gastric cancer in mongolian gerbils global versus local processing of frequency-modulated tones in gerbils: an animal model of lateralized auditory cortex functions pathophysiological responses to acute cerebral ischemia in the gerbil sex differences in the control of scent marking behavior in the mongolian gerbil (meriones unguiculatus) dihydrostreptomycin toxicity in the mongolian gerbil, meriones unguiculatus dentostomella translucida in the mongolian gerbil (meriones unguiculatus) comparison of practical treatment methods to eradicate pinworm (dentostomella translucida) infections from mongolian gerbils (meriones unguiculatus) the anatomy of the mongolian gerbil (meriones ungiculatus) water balance in the mongolian gerbil red cell survival in the gerbil (meriones unguiculatus) artificial insemination and induction of pregnancy in the mongolian gerbil (meriones unguiculatus) serum activity of platelet-activating factor acetylhydrolase is a potential clinical marker for leptospirosis pulmonary hemorrhage suppression of reproduction in waterdeprived mongolian gerbils (meriones unguiculatus) infection of reovirus type in mongolian gerbils (meriones unguiculatus) -lesions in pancreas and brain ocular lesions in gerbils (meriones unguiculatus) infected with low larval burden of toxocara canis: observations using indirect binocular ophthalmoscopy a semipurified diet for the mongolian gerbil (meriones unguiculatus) key: cord- -u slrjmk authors: jiménez, ma Ángeles; sánchez, belén; pérez alenza, ma dolores; garcía, pilar; lópez, jose vicente; rodriguez, alejandro; muñoz, Álvaro; martínez, fernando; vargas, astrid; peña, laura title: membranous glomerulonephritis in the iberian lynx (lynx pardinus) date: - - journal: vet immunol immunopathol doi: . /j.vetimm. . . sha: doc_id: cord_uid: u slrjmk the iberian lynx is the most endangered felid species in the world, confined nowadays to two isolated metapopulations in the southwest of spain, where less than individuals survive. little is known about the diseases that affect these animals in the wild or in captivity. kidney samples from necropsies of iberian lynxes, wild and captive, were examined by histopathology, immunohistochemistry (igg, igm, iga, laminin, type iv collagen, and fibronectin), electron microscopy (n = ) and immunogold labelling for igm, igg and iga in one case, in order to characterize the glomerulopathy prevalent in this species. urinalyses from records were available for of the necropsied animals and blood and urine samples from free ranging and captive iberian lynxes were prospectively obtained in order to evaluate the renal function of the living population. a focal, diffuse membranous glomerulonephritis (mgn) that progressed with age was diagnosed in all but one of the animals in different stages not associated to concurrently known infectious diseases. positive immunoexpression of igm and igg was observed in the glomerular capillary basement membranes and intramembranous electron-dense deposits, compatible with immune complexes (ics) were seen with electron microscopy. the immunogold labelling was also positive for igm and igg in the electron-dense areas. the serum biochemistry and urinalyses also revealed signs of mild chronic kidney disease in of the animals evaluated. in conclusion, the membranous glomerulopathy affecting the iberian lynx is a progressive disease of immune origin. we postulate a possible genetic predisposition towards the disease, enhanced by inbreeding and a possible connection to an immune-mediated systemic disease. the iberian lynx is considered to be the most endangered felid species in the world (nowell and jackson, ) . its situation is critical due to habitat loss and fragmentation, being confined nowadays to two isolated metapopulations in the southwest of spain (gaona et al., ; ferreras, ; rodriguez and www.elsevier.com/locate/vetimm veterinary immunology and immunopathology ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] abbreviations: bun, blood urea nitrogen; ckd, chronic kidney disease; fcv, feline calicivirus; fcov, feline coronavirus; felv, feline leukaemia virus; fiv, feline immunodeficiency virus; ics, immune complexes; iris, international renal interest society; malt, mucosa-associated lymphoid tissue; mgn, membranous glomerulonephritis; pas, periodic acid schiff reagent; pbs, phosphate buffer solution; rt, room temperature; tbs, tris buffer solution; usg, urine specific gravity. delibes, ) . today, less than animals are estimated to be left (palomares et al., ; guzmán et al., ) . the remaining population presents signs of inbreeding and allele dropout (johnson et al., ; godoy, ) . very little is known about the diseases that affect these animals in the wild or in captivity. bovine tuberculosis has been reported to be the cause of death of some iberian lynxes during the years and that probably acquired the disease by eating on infected fallow deer (dama dama) or wild boars (sus scrofa) of the area (briones et al., ; aranaz et al., ; peña et al., ) . a recent histopathological study of our group, evaluating the status of peripheral lymph nodes, spleen and malt (mucosa-associated lymphoid tissue) in iberian lynxes necropsied between the years and , has reported a generalized immune depletion in these animals, apparently not related to infectious agents or malnutrition (peña et al., ) . routine histological examination of kidneys obtained from the necropsy of animals between the years and , including the in the abovecited study, revealed a high prevalence of glomerulonephritis (peña, unpublished data) . this disease is common in the domestic cat (felis cati), especially the membranous type, and is usually of immune origin (grant maxie, ; glick et al., ) . glomerulonephritis can be associated to viral infections caused by feline coronavirus (fcov), feline leukaemia virus (felv) and the feline immunodeficiency virus (fiv) (glick et al., ; slauson and lewis, ; grant maxie, ; newman et al., ) . these pathogens are known to affect a wide variety of wild felids such as the cheetah (acinonyx jubatus), the african lion (pantera leo), the puma (puma concolor), the canada lynx (lynx canadensis), etc. (roelke et al., ; kennedy et al., ; biek et al., ) . also, many non-specific parasitic infections that can affect domestic animals are associated with immunemediated glomerulonephritis (newman et al., ) . a case of glomerulonephritis associated to heartworm infection has been described in the black-footed cat (felis nigripes) (deem et al., ) . furthermore, a non-immunogenic glomerulosclerosis has been reported in captive cheetahs (acinonyx jubatus) (munson, ; bolton and munson, ; munson et al., ) . rare diseases of uncertain origin are frequently described in endangered animals, usually related to poor genetic diversity or to captive conditions. the study of these diseases is important because of their relevance in endangered species conservation programs. a good example of this is the cheetah, considered a paradigm for disease vulnerability (munson et al., ) . this is the first report of glomerulonephritis in the iberian lynx. the main objectives of this study were to characterize by means of histology, immunohistochemistry, and electron microscopy, the type of glomerulopathy detected in the iberian lynx, to determine its pathogenesis, and to evaluate serum biochemical and urinary parameters related to renal function in wild and captive iberian lynx surviving in the southwest of spain. the membranous glomerulopathy affecting the iberian lynx is a progressive disease of immune origin. we postulate that the iberian lynx has a likely genetic predisposition towards the disease, enhanced by inbreeding, and is potentially connected to a possible immune-mediated systemic disease. twenty-seven necropsies were performed on iberian lynxes between the years and . twenty-one animals came from the wild and six from captivity with ages that ranged from days to years. there were females and males. the main cause of death were car accidents (n = ), followed by tuberculosis (n = ), traumatisms (other than car accidents, n = ), fights (n = ), squamous cell carcinomas (n = ), and other diseases (n = ). serology and pcr results for various infectious agents that included those related to glomerulonephritis (fcov, feline calicivirus (fcv), fiv, and felv,) were provided. with the exception of one animal that was positive for felv in serology, and two that were pcr positive for fcov (without histological signs of feline infectious peritonitis), the remaining animals were negative for these agents. right and left kidney samples from necropsies were fixed in formalin, paraffin embedded and sectioned mm wide for hematoxilin-eosin, pas (periodic acid schiff reagent), masson's trichromic, congo red and methenamine silver stains. in order to quantify the lesions in the kidneys, each glomerulus was classified according to the severity of lesions in three categories: ( ) healthy or mild, ( ) moderate and ( ) severe glomerulonephritis or glomerulosclerosis. the percentages of each category of glomeruli were established in all samples. kidneys were then classified as stage or apparently healthy when over % of the glomeruli belonged to category (unaltered or presenting slight changes). the remaining kidneys were classified considering the most prevalent category of glomerular lesion (stage , moderate kidney affection; stage , severe kidney affection). the streptavidin-biotin peroxidase method was used on dewaxed kidney sections in order to detect the expression of fibronectin, laminin, and type iv collagen, components of the glomerular basement membrane. this technique was also used for iga, igm, and igg, markers of the immunoglobulin fraction of immune complexes. antigens were unmasked with trypsin (sigma . %, c, min) for the detection of laminin, fibronectin, iga, igm, and igg while a buffer citrate (tri-sodium citrate panreac , . %; ph ) heated in a microwave oven for min was used to unmask type iv collagen antigen. endogenous peroxidase was deactivated in all cases (h o . % in methanol) at room temperature (rt), for min. unspecific staining of laminin and fibronectin was blocked incubating the slides with normal swine serum (dako , x , dilution / , min, rt) and with normal rabbit serum (dako , x , / ) for iga, igm, and igg). the primary antibodies used (all incubated at c overnight) were polyclonal rabbit anti-laminin (dako , z , / ), polyclonal rabbit anti-fibronectin (dako , a , / ), monoclonal mouse anti-type iv collagen (dako , clone civ , / ), polyclonal goat anti-feline iga (serotec , aa , / ), polyclonal goat anti-feline igm (serotec , aa , / ) and polyclonal goat anti-feline igg (serotec , aa , / ). biotinylated secondary antibodies swine anti-rabbit (for laminin and fibronectin, dako e , / ,), rabbit anti-goat (for iga, igg, and igm, pierce , , / ) and rabbit anti-mouse (for type iv collagen, dako e , / ) were used (incubated min at rt) after washing. slides were then washed and incubated with a streptavidin peroxidase conjugate (zymed - , / , min rt). a chromogen solution containing - diaminobenzidine tetrachloride (sigma chemical co. d ) and h o in distilled water was used for developing, and finally, slides were counterstained with hematoxylin (sigma gh - - ). all washes were done with tbs (tris buffer solution ph . ). the glomerular capillary basement membranes served as internal controls for type iv collagen, laminin and fibronectin, while lymphoid tissues with plasmatic cells from iberian lynx were used as positive controls for igm, igg and iga. negative controls were used substituting the primary antibodies with tbs. selected kidney samples fixed in formalin (n = ) and in . % glutaraldehyde (n = ) from animals without concurrent diseases or detected viruses were processed for electron microscopy. after fixation, the samples were washed in a phosphate buffer (pbs, ph . ) and included in % osmium (diluted in pbs) for h at room temperature. they were then washed in distilled water and dehydrated in acetones of increasing percentage ( , , , , and %) . the samples were gradually infiltrated in a müllenhauer mixture resin and solidified at c for h. the resin blocks were then processed and studied in the electron microscopy center of the complutense university of madrid, spain. one additional kidney sample fixed in formalin was selected for immunogold labelling of igm, igg and iga. the sample was thoroughly washed in pbs at c until clear of formalin. it was then dehydrated in alcohols of increasing percentage ( , , , , and %) while the temperature was lowered gradually (from c to À c for alcohols at and %, respectively) until reaching À c. resin blocks were obtained by gradually infiltrating the sample with lowicryl resin at À c. tissue samples were obtained from the resin blocks and placed in grids for immunogold labelling. polyclonal goat anti-feline igm, iga and igg (serotec , aa , aa , aa ; / ) were used as primary antibodies. the grids were incubated at c overnight and later washed with . m glycine in tbs. then they were incubated for h with nm colloidal gold-affinipure donkey anti-goat igg (jackson immunoresearch , - - , / ). the grids were washed again with the glycine solution. they were stained with % uranyl acetate and reynolds lead citrate, washing with distilled water after both and finally dried and observed by electron microscopy. urinalyses from records were available for nine of the necropsied animals. these were retrieved and evaluated. a prospective study of free ranging and captive iberian lynxes was carried out (table ). blood samples (n = ) and urine samples (n = ) were collected from anaesthetized iberian lynxes (table ) . serum biochemistry parameters related to renal function (total proteins, albumin, blood urea nitrogen (bun), creatinine, calcium, and phosphorus) were determined. the analytical results have been evaluated based on published values for the canada lynx (felis lynx canadensis) (weaber and johnson, ) , the european lynx (lynx lynx) (ryser-degiorgis, personal communication), and the domestic cat (dibartola and rutgers, ) . the urinalysis included urine specific gravity (usg), albumin, glucose, leukocytes, blood, and examination of the urine sediment. the protein-to creatinine ratio was also measured in order to quantify proteinuria. chronic kidney disease (ckd) was categorized in four stages using serum chemistry concentrations and considering the presence of markers of kidney damage according to the recommendations of the international renal interest society (iris) (polzin, ) . the markers of kidney damage used for the iberian lynx were: elevated bun, hypoalbuminemia, elevated serum creatinine concentration, hyperphosphatemia, hypocalcemia, impaired urine concentrating ability (usg was considered normal when ! ), proteinuria, and renal hematuria. these must be confirmed to be of kidney origin and at least one must be present to identify the existence of renal disease. stage (non-azotemic) was considered when markers of kidney damage were present and serum creatinine concentration was < . , stage (mild renal azotemia) presented markers and creatinine values between . and . mg/dl. in stage (moderate renal azotemia) creatinine values must be . - . mg/dl, and in stage (severe renal azotemia), creatinine values exceed mg/dl. the stages were further subcategorized using the protein-to-creatinine ratio as proteinuric (ratio > . ), non-proteinuric (ratio < . ) or borderline proteinuric (ratio . - . ). the associations of epidemiological, pathological and immunohistochemical data were studied using computer software spss. categorical variables were analyzed by pearson and yates chi-square tests. pearson correlation coefficients were used to study continuous variables. levene f-tests were used to analyze the homogeneity of variances. if variances were equal, f-tests or pooled t-tests were chosen to evaluate the variables. if variances were not equal, then welch tests or separate variance t-tests were selected. values p < . were considered significant. non-parametric statistical tests (kruskal-wallis and median test) have been performed when necessary. epidemiological variables included: age as a numerical variable in months and as a categorical variable: (young, - months; young adult, - months; and adult/senile, > months), gender, origin (sierra morena or doñana), and free or captive. in order to evaluate the kidney lesion, four histopathological variables were used: (a) percentage of glomeruli in category , (b) percentage of glomeruli in category , (c) percentage glomeruli in category and (d) stage of kidney affection (stage , none or minimal lesion; stages and , moderate and severe) (categorical). other variables were considered as present or absent such as interstitial nephritis, peritubular fibrosis, and hyaline cylinders. immunohistochemical variables concerning normal or increased expression of laminin, iv collagen, and fibronectin and the presence or absence of igg, igm, and iga were also included. a focal, diffuse membranous glomerulonephritis (mgn) was diagnosed in all of the animals, regardless of their age, sex, and whether they came from captivity or from the wild, except a -day-old cub that did not present any changes in the glomeruli. sclerotic glomeruli were focally scattered in the cortex, and stained blue in the mesangium with masson's trichromic (fig. ) . bowman's capsules appeared focally thickened. the kidneys were classified according to the percentage of altered glomeruli. ten animals were found to have unaltered or slight changes in over % of the glomeruli (stage ), presented moderate mgn (stage ) and only one animal had a severe mgn (stage ). interstitial fibrosis was seen in animals and chronic (lympho-plasmacytic) interstitial nephritis in (not including the granulomas found in the kidneys of the three animals with tuberculosis). other renal lesions such as calcifications and hyaline cylinders in tubules were seen in seven animals. the congo red stain was negative in all of the animals. severity of mgn was found to increase significantly with age: high percentages of normal and minimally altered glomeruli were significantly associated to young animals (mean . % ae s.d. . , median . %) while lower percentages were associated to young adults (mean . % ae sd . , median . % of unaltered glomeruli) and adult/senile animals (mean . % ae sd . , . %) ( p = . anova test). the non-parametric kruskal-wallis ( p = . ) and the median tests ( p = . ) were also significant for this association. there was also a positive correlation between the age (in months) and the percentage of severely affected glomeruli ( p = . ). the presence of interstitial lympho-plasmacytic infiltrates (chronic interstitial nephritis) was significantly associated to the advanced grade of mgn ( p = . ). interstitial fibrosis was evident in advanced mgn ( p = . ). a slight increase of the three components of the glomerular basement membrane, laminin, fibronectin and type iv collagen was observed in glomeruli at beginning or middle stages of glomerulonephritis, while in glomeruli presenting glomerulosclerosis, laminin and fibronectin would always appear intensely expressed, in a focal or diffuse matter, and type iv collagen could either be intensely expressed (fig. ) or negative. laminin expression was significantly associated with the advanced grade of mgn ( p = . ). a positive immunoexpression of igm and igg was observed in the thickened glomerular capillary basement membranes in early stages of glomerulonephritis (figs. and ) , while in glomerulosclerosis, the expression of these two immunoglobulins was negative. iga expression was negative in all cases. electron microscopy revealed irregularly thickened glomerular capillary basement membranes (n = / ) and effacement of foot processes (n = / ) (fig. ) . electron-dense deposits, compatible with immune complexes (ics), were located intramembranously (n = / ) (fig. ) . some electron-lucent areas also were seen in the basement membranes, compatible with areas where immune complexes were being reabsorbed (n = / ). in glomerulosclerosis, the capillary basement membranes were thickened and wrinkled; foot processes were effaced and sometimes lost. electron-dense deposits were not found in this stage. the immunogold labelling for igm and igg stained positive the electron-dense structures that were compatible with (ics) (fig. ) , while iga was not detected. the urinalyses available from nine of the necropsied animals presented low urine specific gravity (usg) in five cases (usg < ), three of which presented proteinuria qualitatively measured (albumin ! mg/ dl). two other animals presented proteinuria (albumin ! mg/dl) with normal specific gravity. haematuria was seen in six animals and glycosuria was present in one. serum biochemistry parameters of the live iberian lynxes were: total proteins (range . median . ), creatinine (range . - . mg/dl; mean . ae s.d. . ; median . ), calcium (range . - . mg/dl; mean . ae s.d. ; median . ), and phosphorus (range . - . mg/dl; mean . ae s.d. . ; median . ). twelve animals had hyperphosphatemia (phosphorus > . mg/dl), presented hypocalcemia (calcium < . mg/dl), animals had uremia (bun > mg/dl), presented elevated creatinine (creatinine > . mg/dl). nine lynxes presented decreased usg (< ) and proteinuria was detected qualitatively in animals. according to the iris ckd classification system, five animals did not present any signs of kidney damage, two presented only slight hyperphosphatemia and uremia that could not be attributed to the kidney so were considered healthy also, six animals were classified in stage (non-azotemic), seven in stage (mild-azotemia), and three animals in stage (moderate renal azotemia). one animal in stage was subclassified as proteinuric and three animals were subclassified as borderline proteinuric in stages , and (table ) . membranous glomerulonephritis (mgn) in different stages was the main lesion described in the kidneys of the iberian lynxes. this disease is characterized by an irregular increase of the glomerular capillary basement membranes due to the deposit of circulating immune complexes, or specific antibody attachment to components of the glomerulus (franklin et al., ; valaitis, ; newman et al., ) . the immunohistochemistry revealed basement membranes to be thickened by specific components such as laminin, fibronectin and type iv collagen. the detection of immunoglobulins g and m in the capillary basement with immunohistochemistry along with the intramembranous electron-dense deposits seen with the electron microscopy, confirm the presence of ics in our samples and an immune origin of the gn. the immunogold labelling results matched the immunohistochemistry since both igm and igg were detected over the electron-dense deposits and iga was absent. signs of complete and partial reabsortion of ics were also detected with electron microscopy, but not when there were histological features of sclerosis. immune complexes are usually present during initial stages of the disease and are progressively reabsorbed by the basement membrane, which thickens after the process. repeated injury on the basement membranes can impair their capacity to reabsorb immune complexes and regenerate, and therefore sclerosis of the membranes occurs as it loses its structural detail and capacity to eliminate ics (valaitis, ) . the most severe lesions, including the glomerulosclerosis, were significantly related to the age of the animals, which corresponds with a progressive degenerative disease. the other lesions found in many of the kidneys such as chronic interstitial nephritis and tubular fibrosis were less frequent and related to the severity of the mgn, and therefore were considered secondary to the disease. the presence of hyaline cylinders indicates some degree of filtration impairment of the tubuli, as consequence of the glomerular injury (newman et al., ) . the study of the urinalysis available from some of the necropsied animals was important to determine if there were analytical signs of renal dysfunction that corresponded with the histological findings. unfortunately, only nine urinalyses of the necropsied animals were available. nevertheless, the presence of proteinuria and low urine specific gravity in five animals, suggests that filtration might have been impaired to some degree. glycosuria and hematuria were also present in some cases. these are unspecific signs of renal damage and there was no information available about existing extrarenal infections at the time of sampling, therefore their interpretation should be taken with caution. blood and urine sampling of the living population was important to evaluate if there was a correspondence between the prevalent histopathological findings and the existence of chronic kidney disease (ckd) in the live animals. there is one study published on serum biochemical characteristics of the iberian lynx done in wild iberian lynxes (beltrán and delibes, ) , and recently another study is being done with a larger number of animals by members of this team, describing the parameters observed in the population regardless of the presence of infection or other diseases. due to this situation the iris staging system is useful since it permits the diagnoses of ckd even when reference ranges are not very accurate (polzin, ) . according to the results obtained with the iris classification system, out of animals presented some signs of chronic kidney disease, though only three presented moderate azotemia and none of them were found in the most severe stage. the presence of renal disease seems to exist among the living population, both captive and wild, and this would correlate with the histological changes detected in the necropsied animals. another common feature of renal failure is hyperphosphatemia and therefore was included as a marker of kidney disease. it appears when the glomerular filtration rate declines around % below normal (grauer and dibartola, ) and it was detected in of the animals. in two of them it was not considered since the urinary analyses were not available and it could have been due to recent ingestion. hypocalcemia was present in four of the animals with hyperphosphatemia. the main consequence of hyperphosphatemia is the development of secondary renal hyperparathyroidism (nagode and chew, ; kates et al., ; barber and elliot, ) . it is usually associated to hypocalcemia, though hypercalcemia or normal serum calcium concentrations accompanying hyperphosphatemia can also be found due to the compensation of the parathyroid hormone (pth) (barber and elliot, ) . the consequences of a secondary hyperparathyroidism are completely unknown in the iberian lynx, but it may be interesting to explore animals during sampling or in future necropsies for signs of pain or osteodistrophia seen in other species (barber and elliot, ) . membranous glomerulonephritis is a common glomerular affection in cats that can be associated to viral infections (felv, fiv, fcov, etc.) , neoplasms, intoxications (methyl mercury), familial diseases, autoimmune pathologies, or in many cases it is considered idiopathic (glick et al., ; enestrom and hultman, ; newman et al., ) . membranous glomerulonephritis can also be secondary to other systemic immunological diseases such as systemic lupus erythematosus or immune complex polyarteritis in dogs and cats (newman et al., ) . the only iberian lynx without signs of membranous glomerulonephritis was the -day-old female cub that had died in a sibling quarrel. with the exception of the few animals with neoplasias or tuberculosis, or the three lynxes that were pcr positive for viral detection (seven animals altogether), features that could explain the presence of mgn were not found in the rest of the animals. other vascular and metabolic diseases like hypertension and diabetes mellitus are non-immunogenic causes of glomerular disease (alpers, ) and the glomerular capillary basement membranes can also appear thickened due to hypertension or sclerosis (buckalew, ; newman et al., ) . hypertension, probably associated to stress, genetic and dietary factors (a high protein diet), was postulated to be the cause of the glomerulosclerosis described in a high percentage of captive cheetahs . the pathogenesis of the glomerular injury caused by these diseases lacks an immune component and therefore is different to the one seen in the iberian lynx. however, the iberian lynx is also a carnivore, and high protein ingestion can cause hypertension and accelerate progressive renal failure, which leads to glomerulosclerosis (buckalew, ) . therefore, this could be contributing to the progression of the disease, as occurs in the cheetah, even though the main cause of the glomerulopathy is immunogenic. inherited glomerulopathies have been described in many breeds of dogs and there are reports in cats, these are primary glomerular diseases (grauer and dibartola, ; casal et al., ) . it may be interesting to take these into consideration since the iberian lynx displays very low genetic diversity for a felid, with two remnant populations that have undergone a recent founder effect or population bottleneck (godoy, ) . recent studies have found that it possesses a deficiency of heterozygosity possibly due to inbreeding and some degree of allele loss (johnson et al., ) . this could encourage the inheritance of a certain glomerular disease among the population. animals as young as or months presented glomerulonephritis suggesting there may be a predisposition for the disease. also, the cause of the generalized immune depletion described in of the animals (peña et al., ) included in this study remains undetermined and a possible relationship with the membranous glomerulonephritis should be taken into consideration. as with many species in danger of extinction, with the handicap of low genetic diversity that seem to lead to uncommon diseases, the importance of a detailed study of the diseases that may affect the iberian lynx is evident. in conclusion, the membranous glomerulopathy that affects the iberian lynx is a progressive disease of immune origin. we postulate that the iberian lynx may be genetically prone to the disease, which could be enhanced by inbreeding and also may have a connection with an immune-mediated systemic disease. future studies are necessary. nevertheless, none of the animals are known to have died from chronic renal failure yet, though the results indicate that it may be widely extended, considering that the remaining population is of less than individuals. because of the critical state of this endangered species, signs of progression of this disease should be watched for, especially in the wild population. robbins and cotran pathologic basis of disease bovine tuberculosis (mycobacterium bovis) in wildlife in spain feline chronic renal failure: calcium homeostasis in cases diagnosed between and hematological and serum chemical characteristics of the iberian lynx (lynx pardina) in southwestern spain serologic survey for viral and bacterial infections in western populations of canada lynx (lynx canadensis) glomerulosclerosis in captive cheetahs (acinonyx jubatus) bovine tuberculosis and the endangered iberian lynx pathophysiology of progressive renal failure familial glomerulopathy in the bullmastiff heartworm (dirofilaria immitis) disease and glomerulonephritis in a black-footed cat (felis nigripes) the cat, diseases and clinical management immunemediated glomerulonephritis induced by mercury chloride in mice landscape structure and asymmetrical inter-patch connectivity in a metapopulation of the endangered iberian lynx membranous glomerulonephritis: long-term serial observations on clinical course and morphology dynamics and viability of a metapopulation of the endangered iberian lynx characterization of feline glomerulonephritis associated with viral-induced hematopoietic neoplasms iberian lynx population genetics: doñana, sierra morena and ex-situ program the urinary system textbook of internal medicine, diseases of the dog and cat iberian lynx (lynx pardinus) distribution and current conservation status in spain phylogenetic and phylogeographic analysis of iberian lynx populations evidence that serum phosphate is independently associated with serum pth in patients with chronic renal failure detection of feline coronavirus infection in southern african nondomestic felids diseases of captive cheetahs (acinonyx jubatus): results of the cheetah research council pathology survey - diseases of captive cheetahs (acinonyx jubatus) in south africa: a -year retrospective survey extrinsic factors significantly affect patterns of disease in free ranging and captive cheetah (acinonyx jubatus) populations nephrocalcinosis caused by hyperparathyroidism in progression of renal failure: treatment with calcitriol. semin urinary system wild cats-status survey and conservation action plan iberian lynx in a fragmented landscape: predispersal, dispersal, and postdispersal habitats histopathological and immunohistochemical findings in lymphoid tissues of the endangered iberian lynx (lynx pardinus) evaluating patients with chronic kidney disease (vet- ). western veterinary conference internal structure and patterns of contraction in the geographic range of the iberian lynx t-lymphocyte profiles in fiv-infected wild lions and pumas reveal cd depletion comparative pathology of glomerulonephritis in animals renal glomerular disease hematologic and serum chemistry values of captive canadian lynx this research was supported by the spanish ministry of environment, research project / in agreement with the environmental council of the ''junta de andalucía'', ''dirección general de investigación'' project cgl - /bos and ''red nacional de parques nacionales'' project / . we thank all the veterinarians and biologists involved with the field work, management and sampling of the iberian lynxes at doñana national park and sierra morena. we also thank dr. marie-pierre ryser-degiorgis for the serum biochemical reference values of the european lynx. we are grateful to ana vicente, agustín fernández and dr. maría luisa garcía for their assistance processing the electron microscopy samples and to pedro aranda for his histotechnology assistance, and to pedro cuesta for his assistance in the statistical analysis. key: cord- -zpi uis authors: roberts, anjeanette; wood, john; subbarao, kanta; ferguson, morag; wood, david; cherian, thomas title: animal models and antibody assays for evaluating candidate sars vaccines: summary of a technical meeting – august , london, uk date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: zpi uis abstract severe acute respiratory syndrome (sars) emerged in the guangdong province of china in late and spread to countries. by the end of the outbreak in july , the cdc and who reported cases with a . % case fatality rate. the disease was caused by a previously unrecognized coronavirus, sars-cov. drawing on experience with animal coronavirus vaccines, several vaccine candidates have been developed and evaluated in pre-clinical trials. available data suggest that vaccines should be based on the the kda viral spike protein, s, the only significant neutralization antigen capable of inducing protective immune responses in animals. in the absence of clinical cases of sars, candidate vaccines should be evaluated for efficacy in animal models, and although it is uncertain whether the united states food and drug administration's “animal rule” would apply to licensure of a sars vaccine, it is important to develop standardized animal models and immunological assays in preparation for this eventuality. this report summarizes the recommendations from a who technical meeting on animal models and antibody assays for evaluating candidate sars vaccines held on – august in south mimms, uk, provides guidance on the use of animal models, and outlines the steps to develop standard reagents and assays for immunological evaluation of candidate sars vaccines. severe acute respiratory syndrome (sars) is a severe respiratory illness caused by the sars coronavirus (sars-cov) [ ] . the disease emerged in the guangdong province of china in late [ ] and spread to countries mostly within asia, although europe and north america were also affected, notably toronto, canada. the epidemic was finally controlled by july through strict implementation of prevalence for sars-cov antibody, although they had no history of sars-like disease. sars-cov-like viruses that were isolated from civets and raccoon dogs had more than % homology with human sars-cov, with major differences found in orf , whose deletion has been suggested to represent a sign of adaptation to humans [ ] . only four amino acid residues in the receptor glycoprotein ace binding domain of the viral spike protein differ between the human epidemic sars-cov strains and civet strains, but they cause more than a -fold difference in binding affinity to the ace molecule [ , ] . although a high prevalence of sars-like coronaviruses were found in chinese horseshoe bats [ , ] , their great genetic diversity makes it difficult to identify which one might be the ancestor of sars-cov and to decide with certainty whether bats indeed are the animal reservoir of the virus. sars-cov infection exhibits a wide clinical course characterized mostly by fever, dyspnea, lymphopenia and lower respiratory infection, often with concurrent gastrointestinal symptoms including diarrhea [ , ] . pathology in sars patients has been associated with diffuse alveolar damage, epithelial cell proliferation and multinucleated giant cell infiltrates of epithelial or macrophage origin, suggestive of syncytium-like formation in the lung. the virus can be recovered from peripheral blood mononuclear cells, respiratory secretions, stools, urine and even sweat (for a review, see [ ] ). sars vaccine development efforts were initiated very rapidly after the identification of the etiologic agent, even though the immune correlates of protection were not known. research efforts to identify protective antigens and to develop animal models were undertaken in parallel with efforts to develop candidate vaccines [ ] , drawing on experience with animal coronavirus vaccines and using several vaccine strategies, including inactivated virus vaccines, purified subunit vaccines, plasmid dna and viral vector-based vaccines as well as virus-like particles. much effort has been made to identify appropriate animal models for sars-cov replication and pathogenesis. several research groups have shown that mice [ , ] , ferrets [ ] , hamsters [ ] and nonhuman primates [ ] [ ] [ ] [ ] [ ] [ ] support replication of sars-cov with varying degrees of associated disease. these animal models were used for the evaluation of candidate vaccines, and the common conclusion that has emerged from the evaluation of several vaccines is that the kda viral spike protein, s, is the only significant neutralization antigen [ ] [ ] [ ] [ ] and the only one to elicit protective immunity in animal models [ , [ ] [ ] [ ] [ ] [ ] [ ] . the s protein can be divided into two domains by analogy with other coronavirus spike proteins: an n-terminal s domain, which contains the receptor-binding site and neutralization epitopes and a cterminal s domain which forms the membrane-anchored stalk region and contains a putative fusion peptide followed by two heptad repeats predicted to form a six-helix coiled-coil bundle [ ] . in the absence of clinical cases of sars, candidate vaccines will have to be evaluated for efficacy in animal models. the united states fda "animal rule" states that, when efficacy studies in humans are not feasible, vaccines may be approved based on animal data alone, provided the pathophysiological mechanism of the disease is reasonably wellunderstood as is its prevention or reduction by the vaccine. moreover, the protective effect of the vaccine should be demonstrated in more than one animal species expected to react with a response predictive for humans. the endpoint of animal studies should be clearly related to the desired benefit in humans (i.e. enhancement of survival or reduction in major morbidity), and the data generated should allow selection of an effective dose in humans. at the present time it is uncertain whether the "animal rule" would apply to licensure of a sars vaccine. however, it is important to develop standardized animal models and immunological assays in preparation for this eventuality. scientists at the who technical meeting on animal models and antibody assays for evaluating candidate sars vaccines held on - august in south mimms, uk, discussed many aspects of research pertaining to the use of animal models in vaccine development including available animal models, suitability of the various models, correlates of protection, critical components of potential vaccines, and the potential for disease enhancement in vaccinated animals following exposure to sars-cov. in addition, standardization of antibody assays and the establishment of a who international standard for sars-cov antibody were also discussed. this report endeavors to summarize the recommendations from this meeting, based on consensus agreement. recommendations for use of each animal model are given in section below. correlates of protection, an overview of vaccine development, and observations pertaining to potential disease enhancement are summarized in the following sections - . in selecting animal models for vaccine evaluation, it is important to remember the principle underlying the so called "animal rule", where data from more than one animal species is often required: each animal species should contribute something different to our understanding of disease and protection. at this time, no single model offers a direct reproduction of what was seen in humans with sars. pathology (including pneumonitis, alveolar edema, and diffuse alveolar damage (dad)) in humans is probably the most difficult element to reproduce in an animal model. attention also should be given to the reduction of viral shedding because this would likely correlate with decreased risk of further spread of the disease among humans. in using animal models to study aspects of sars-cov infection, it must be emphasized that the kinetics of viral replication and appearance and resolu-tion of pathological findings are much more rapid in animal models than in humans. whichever animal model is employed, special consideration should be given to the presence of co-existing pathogens, the age of the animals and the route(s) of infection. a sufficient number of time points and large enough number of animals should be used to allow statistical evaluation. the strain of sars-cov used also could be of importance. it should be emphasized that different species may prove useful for studying different aspects of sars-cov. whereas vaccines or antivirals may be addressed in many models, pathogenesis is best evaluated in those animal models for which immunological tools and reagents are available for detailed analysis of the immune response to the vaccine. this includes inbred mice and rhesus and cynomolgus macaques. it may actually be worthwhile to enhance the virulence of a sars-cov isolate by serial passages in an animal model to produce a challenge virus stock for vaccine studies that would elicit more reproducible disease in the animals. if a highly virulent host-adapted virus were to become available, such as a mouse-adapted or a monkey-adapted sars-cov strain, demonstration of the capacity of vaccines to protect against challenge with these more virulent strains would provide an almost ideal animal model. different models may also need to be employed to evaluate pathogenesis versus immunogenicity. for pathogenesis studies in animal models, mortality is not required as a readout. it would be ideal to develop animal models with comparable levels of mortality to that seen in humans (∼ % overall), including the increased mortality at increased age (∼ % > age ). the optimal result would be to demonstrate efficacy of vaccines or antivirals in sars-cov animal models that mimic human morbidity and mortality and that show protection without vaccine-associated immunopathology. inbred mouse strains (balb/c, c bl/ , svev, stat −/−) support sars-cov replication and can develop pneumonitis ( s), but pneumonia and clinical symptoms are only observed in older balb/c mice [ ] . the mouse model is suitable for immunogenicity and efficacy studies of vaccines. prolonged viral replication, dissemination of virus to liver and spleen and accompanying pathology are seen in stat −/− mice; these mice, therefore, also are suitable for studies of pathogenesis and evaluation of antiviral drugs. specific pathogen-free (spf) animals must be used. their age can be either - weeks or over months and should be specified. the number of animals included must be sufficient for statistical analysis, and should include mock-infected controls. a variety of sars-cov strains has been tested in mice, including urbani, frankfurt, hku- , tor- , and the mouse-adapted sars-cov strain ma- . these were inoculated by the intranasal (in) route under light anesthesia [ , ] , using a dose of tcid in l/mouse. critical time points for specimen collections are days (peak titer) and post-infection (p.i.) for quantitative virology, days and p.i. for the study of interstitial pneumonitis and dad in aged mice (inflate lungs with % neutral-buffered formalin), and day for resolution. golden syrian hamsters are an excellent animal model because they demonstrate high levels of sars-cov replication and develop pneumonitis. hamsters are suitable for vaccine efficacy, immunoprophylaxis and treatment studies [ ] . in contrast with balb/c mice, in which the virus is detected only in the respiratory tract and is cleared by day p.i., hamsters demonstrate a longer duration of viral shedding from the upper respiratory tract, some transient viremia, spread of the virus to liver and spleen and, most significantly, inflammation of respiratory tissues [ , ] . spf animals older than weeks of age should be used in sufficient number for statistical analysis and study design should include mock-infected animals. the animals can be housed in pairs or by three if the experiment is to last less than - weeks. reserve space should be available to separate the animals in case of fights. males and littermates tend to fight less. virus strains that have been tested in hamsters are urbani, frankfurt and hku- . virus should be administered by the in route under light anesthesia, using a dose of tcid in l/hamster. as an outcome of efficacy studies, quantitative virology should be preferred over quantitative pcr. for pathology studies, one can grade pathology as none, mild, moderate or severe as per roberts et al. [ ] . critical time points for specimen collection are days or p.i. (peak viral titer) and (clearance) for quantitative virology studies; and days p.i. (consolidation and pneumonitis) and or (resolution) for pathology studies (lung). there is evidence from one study that ferrets support sars-cov replication and develop pulmonary lesions [ ] but according to another study, the animals remain asymptomatic, in the presence of sars-cov replication [ ] . in view of these conflicting data, the ferret model needs to be further studied to determine its optimal utility for vaccine efficacy and immune prophylaxis studies. additional studies are needed to define the extent of biological variability of the model and the possible role of co-pathogens that may contribute to the variability observed between different laboratories. animals aged months or older should be used. although not well documented, more consistent viral replication, pathology and clinical symptoms seem to be observed in older animals. the animals should be screened for viral co-pathogens: aleutian mink disease, respiratory viruses, hepatitis viruses, and others. the route of inoculation may be in or it, but not iv. the dose of virus (strains tor- , hku- ) sufficient to ensure reproducibility of infection in all animals is likely to be pfu or more/ferret. again, quantitative virology is preferred over qrt-pcr. for pathology studies, the same recommendations apply as for nonhuman primate studies (see below): slides should be shared between pathologists to develop a scoring system. regarding specimen collection of respiratory tissues, further studies are needed to establish how much variation occurs in samples from different lobes of the lungs. critical time points are day p.i. for quantitative virology and days - p.i. for pathology studies (pneumonitis). nhps support sars-cov replication and develop pneumonitis with a variable degree of clinical symptoms depending upon the species employed. no single nhp species is preferred at this time. the number of animals in a given study needs to be large enough to account for animal-toanimal variability: a sample of or animals is not sufficient. in view of the cost of the experiments, challenge studies should be limited to those vaccine candidates that are most promising, using larger sample sizes ( - animals/group) and avoiding animals with free-range periods in life if possible. immunological responses are best studied in species for which microarrays and reagents for identifying immune components are available, such as rhesus or cynomolgus macaques. however, the limited viral replication observed in cynomolgus macaques might be a disadvantage in selecting this species for studies. other recommended nhp species are the common marmoset and african green monkeys (agms, chlorocebus aethiops sabeus). the country of origin may play an important role and should be specified, e.g. philippines (cynomolgus macaques, macaca fascicularis); china (rhesus, macaca mulatta); brazil (marmosets, callithrix jacchus), etc. prior to the experiment, the animals should be housed indoor to limit exposure to potential co-pathogens. they should be screened for parasites (strongyloides, pneumonyssus simicola (lung mites)) and for possible viral co-pathogens (retroviruses, respiratory viruses, adenoviruses). the sars-cov strains tested in nhp models are hku- (cynos), pumc (rhesus) and urbani (marmosets and agms). these were inoculated by the respiratory route (in, it) at a dose of pfu or more/nhp. here again, quantitative virology is preferred over qrt-pcr. for pathology studies, it would be an obvious advantage that laboratories share pathology slides for review by different pathologists in order to develop a scoring system. specimens of respiratory tissues should be collected, but further studies are needed to establish how much variation occurs in samples from different lobes of the lungs, as was done and reported for african green monkeys (agms) [ ] . critical time points are days - p.i. for quantitative virology in cynomolgus macaques and agms, and later than day p.i. for rhesus macaques of chinese origin. for collection of tissues for histopathological analyses, days - p.i. are optimal for cynomolgus macaques and agms, and later than day p.i. for rhesus macaques. due to limitations of immunological reagents (including microarray assays now available), research may be limited to rhesus and cynomolgus macaques. data on other animal models are insufficient for consideration for use in sars-cov vaccine and antiviral evaluations [ ] [ ] [ ] . any additional model other than the four listed above (section . . to section . . ) would require thorough characterization including viral replication data and histopathological analysis of sars-cov-infected and mockinfected animals of the same age and gender. viral replication and histopathological data in any new animal model should be reminiscent of at least some aspect of sars in humans. although all the correlates of protection from sars associated disease have not been identified in human infections, neutralizing antibodies are present in convalescent human serum. antibodies to sars-cov spike (s) protein have been shown to prevent virus entry and neutralize virus infectivity in vitro [ , ] . prophylactically administered monoclonal antibodies and passively transferred sars-cov hyper-immune sera have been shown to prevent sars-cov infection and associated disease following sars-cov challenge of naive mice and hamsters [ , , [ ] [ ] [ ] . monoclonal antibodies administered therapeutically (i.e. post-infection) also have been shown to limit viral replication and reduce associated disease in hamsters [ ] . although cell mediated immunity may have a protective role in viral clearance or resolution of disease, work in animal models shows that antibody alone is effective for prevention and treatment of sars. thus, mice immunized with live-recombinant vaccines expressing the sars-cov spike protein, using rabies virus [ ] , vesicular stomatitis virus (vsv) [ ] , adenovirus (ad ) [ , ] or attenuated vaccinia virus mva [ , ] as a vector, as well as mice immunized with dna vaccines expressing the s gene [ , ] , developed neutralizing antibodies to sars-cov and were protected against sars-cov challenge. similar findings were reported after mucosal immunization of hamsters and agms using a bovine parainfluenza virus type (bpiv ) vector expressing the sars cov s gene [ , ] . several whole inactivated virus and recombinant protein candidate vaccines also have been developed and shown to elicit a neutralizing antibody response that provided protection against infectious challenge [ ] [ ] [ ] [ ] [ ] [ ] . in addition, passively administered sera from vaccinated animals prevented sars-cov infection upon subsequent challenge of naïve mice, demonstrating that antibodies induced by these vaccines did confer protection [ , ] . the neutralizing antibody titer that is necessary to achieve protection in humans exposed to sars-cov is, however, still not known. it was recommended that when evaluating vaccine efficacy in future animal experiments, the challenge virus should be administered at two different time-points, once when postimmunization neutralizing antibody titers are high, and later when neutralizing antibody titers have waned or are low. it also was suggested that viral titers and pathology should be evaluated at two different time points. specific times points for sample collection are given for each animal model in section above. previous observations of disease enhancement have been reported for human viral pathogens and shown to be due to antibody-mediated enhancement of virus entry (for reviews see [ , ] ). enhanced disease and mortality have been observed in kittens immunized against or infected with a type-i coronavirus, feline infectious peritonitis virus (fipv), when subsequently exposed to fipv infection [ ] [ ] [ ] . aggravated fip is apparently a result of enhanced viral entry into macrophages mediated by sub-neutralizing antibody levels [ ] . children vaccinated with inactivated respiratory syncytial virus (rsv) vaccines developed serious disease on subsequent exposure to rsv [ ] [ ] [ ] . individuals exposed to one of the four serotypes of dengue virus developed severe disease when subsequently infected with a second, different serotype [ , ] . enhanced disease following rsv vaccine or dengue infection occur by different mechanisms than fipv. in view of such examples of enhanced disease following infection in a vaccinated host, there has been heightened concern that a similar phenomenon could occur with sars-cov vaccines. it was highly recommended, therefore, that known mechanisms of disease enhancement observed with other viruses and especially with other coronaviruses should be examined in sars-cov infections, especially in vaccinated animals. although none of the studies to date have shown enhanced respiratory disease following sars-cov challenge in previously immunized animals, further studies in this area are warranted in view of some of the available in vitro data. antibodies against human sars-cov isolates were shown to enhance the entry of pseudo-typed viruses expressing the civet sars-like cov-spike protein into a human renal adenocarcinoma cell line ( -o) . enhancement was only demonstrated at the level of entry, but not of replication [ ] . this phenomenon was seen with pseudo-typed lentiviruses expressing sars-cov spike protein of civet sequence specificity, but not with pseudo-typed viruses expressing spike proteins of human sars-cov isolates. it also was not observed with human isolates of sars-cov. the role of enhanced viral entry, as observed in these in vitro studies, has not been related to any known component of human disease or infection in vivo. however, given that sars-cov may replicate, albeit poorly, in human pbmcs [ , ] , in vitro experiments looking for antibody-enhancement of sars-cov replication in human cells (e.g. macrophages and b-cells) should be performed. several groups have studied sars-cov infection in animals in the presence of neutralizing and sub-neutralizing levels of sars-cov anti-sera or anti sars-cov s-protein monoclonal antibodies, but no evidence of enhanced respiratory disease has been observed. however, foci of hepatic necrosis were noted following sars-cov challenge in mva-sars-s immunized ferrets [ ] . although these findings are worrisome, several questions were raised regarding the significance of the observation. the mva-sars-s vaccine used in these experiments was poorly immunogenic in the ferrets. the question of whether there could have been any co-pathogen in the animals was raised. it also would be important to know if the observed phenomenon depends on the mva vector or on the animal model. it was strongly urged, therefore, that the experiment be repeated in ferrets. additional experiments, in nonhuman primates and hamsters, looking for evidence of enhanced respiratory and hepatic diseases upon vaccination and challenge were also encouraged. several candidate sars vaccines that are at various stages of pre-clinical and clinical development are being developed worldwide. in china alone, three companies have been given regulatory approval for the clinical evaluation of a candidate sars vaccine. it is important, therefore, to be able to compare data from each of the candidate vaccines, which, in turn, requires international standardization of the immunological assays used for the evaluation of these vaccines. the accepted method of international standardization is to employ a who international standard (is), which allows comparison of results from different laboratories [ ] . this is essential for establishing international requirements for vaccines, diagnostics or therapeutics. an is is prepared from material bearing a close resemblance to the samples being assayed; the material is distributed in glass ampoules with high precision and reproducibility and then freeze dried. it is important that a sufficient number of ampoules ( - ) be prepared so as to provide for about years of use, and that the activity of the contents remain stable over this period. the process of establishing a who is involves an international collaborative study, in which the candidate is is compared with other samples. if the results of the tests are suitable, the candidate is assigned a provisional arbitrary unitage, a report is distributed for approval by study participants and for eventual approval by the who expert committee on biological standardization (ecbs). the preparation, storage and distribution of over % of is have been undertaken by the national institute for biological standards and control (nibsc) at south mimms (uk), which is a who laboratory for biological standards. nibsc has developed in the past several who iss for calibration of the antibody response against virus vaccines, including iss for antibodies against dengue, hepatitis a virus (hav), [ ] hepatitis b virus (hbv), measles, [ ] polio, rabies, [ ] rubella, and smallpox. a candidate standard against human papilloma virus- (hpv- ) is under evaluation. the corresponding is's were used for a variety of antibody assays including virus neutralisation (vn), haemagglutination-inhibition, single radial diffusion, enzyme-linked immunoassay and radio-immunoassay. antibody iss are most useful in epidemiological studies and in clinical trials. their use allows correlates of immunity and potency requirements of prophylactic and therapeutic products to be expressed in international units (ius). data from several collaborative studies demonstrate that use of an is generally reduces the level of variability between assay results. however, there may be problems in using iss due to the complex array of antibody populations in each serum and the different sensitivity of different assay systems. examples of potential problems can be found in hbv and parvovirus b studies [ ] , which showed that different assay kits gave different results even when the is was included in the assays. another issue is the degree of antigenic homology between the viral antigen used for the preparation of the is and the virus used in the assays. in a jev collaborative study, a candidate antibody is, which had been prepared from the sera of vaccinees immunized with an inactivated vaccine that was antigenically different from some of the viruses used in vn assays, demonstrated that the response to at least some inactivated vaccine is strain specific and the candidate is was consequently not established by the who ecbs. whether a panel of monoclonal antibodies to sars-cov could be used to prepare an is is an attractive alternative which should be explored. significant progress with standardisation of sars-cov antibody assays had been made in china with the development of a national antibody standard. in order to develop the national standard, sera were collected from convalescent sars patients who were found to have sars-cov vn antibody titers ranging from undetectable to : . one serum sample was selected for further evaluation based on crossreactivity with four sars-cov strains and on western blot analysis. this serum was freeze dried in . ml aliquots and was then assessed for stability by vn assays. the chinese standard was assigned a vn titer of . with % confidence limits of . - . . a further important development in china was the preparation of human immunoglobulin for treatment of sars patients. national guidelines have been prepared for collec-tion of plasma, quality control testing and standardisation of assays. three chinese manufacturers have been licensed for preparation of sars immunoglobulin. the source material was plasma from convalescent patients at more than days after infection. all were in good health and their plasma tested negative for blood-borne agents. plasma samples were processed by a combination of cold ethanol fractionation and ultrafiltration. in september , three lots of igg were produced and assayed by nationally-agreed procedures. the stock of immunoglobulin currently available is sufficient to treat patients. monoclonal antibodies have not yet been considered for treatment purposes in china. the importance of assessing immunogenicity of candidate sars-cov vaccines using vn assays is well acknowledged, but the variety of vn tests in use is a significant problem since there is at this time no consensus on the most sensitive, specific, and reproducible assay system. it is therefore desirable to establish an antibody is to serve as a basis of comparison in all vn assays. the most important activity at this time is to obtain a suitable source of antibody. a number of options can be considered, such as convalescent human sera, post-immunization human sera, monoclonal antibodies or hyperimmune animal sera. as an example, the availability of a suitable source of serum from convalescent patients in hong kong needs to be explored, although antibody levels in these individuals are probably quite low by now. it also would be important that other assays than vn be included in the collaborative study, and that the impact of sars-cov strain variation be examined by using different sars-cov strains and/or sera with different specificities. the centralized facility for aids reagents (cfar), which is based at nibsc, could be a suitable model for a sars-cov repository [ ] . the cfar was established in to support aids vaccine research and it is now eu-funded [ ] . there are currently reagents available including peptides, recombinant proteins, human sera/plasma, monoclonal and polyclonal antibodies, expression systems, cdna clones and viruses. a comparison can be drawn between sars-cov and hiv vn assays. currently, there are several different hiv neutralization assays formats under consideration and a lack of agreement on the most suitable assay. the cfar is supporting a joint who/eu project (neunet) to evaluate and standardize hiv vn assays in an international collaborative study. in the usa, a sars-cov repository has been established on behalf of the american type culture collection (atcc) in order to meet the needs of biodefence and the threat of emerging infections [ ] . the type of reagents stored includes viruses, peptide arrays, monoclonal antibodies and proteins. it is hoped that an active collaboration can be established between niaid and nibsc in order to meet the expanding needs of the sars research community. based on the discussion at the meeting, the following recommendations were made with respect to standardization of the immunological assays for sars vaccine evaluation: . a who repository for sars-cov reagents ought to be developed. collaboration between niaid and nibsc is recommended to achieve this goal. . consensus must be reached for the reagents to be given priority in the repository. needed. . the most suitable source of antibody for the is is convalescent human sera, but post-vaccination human sera could also be used. . a protocol for an international collaborative study aimed at validating the is should be developed and distributed to prospective participants. . collaborative study participants should be asked about their assay capabilities, e.g. number of sera, virus strains handled, etc. . . . the proposed is collaborative study should include a core set of antibody preparations to be distributed and assayed in each laboratory (e.g. monoclonal antibodies, animal sera, other human sera). . tests should be conducted using the same strain of sars-cov in each laboratory, but the different genetic lineages of sars-cov should also be represented in the study. biosafety issues associated with sars-cov vaccine development stem from the reports of laboratory-acquired infections in china. sanofi pasteur has adopted bsl practices and bsl equipment (e.g. class or microbiological safety cabinets with respiratory protective equipment) for the preparation of sars-cov vaccines. of note is the fact that sars-cov appears to be quite resistant to normal methods of virus decontamination (jf saluzzo, personal communication). who has developed guidance, both general [ ] , and specific for handling sars specimens [ ] . the rapid success in the development of immunogenic and protective vaccines against sars using a variety of platforms is encouraging, but should be tempered with concerns about the possibility of enhanced disease following exposure in vaccinated individuals [ ] . concerns mainly stem from reports of enhanced disease in fipv-immunized or -infected kittens [ , ] , from observations that antibodies elicited against certain coronaviruses mediate antibody-dependent enhancement of viral entry [ ] , and from the observation of inflammatory foci in liver tissue following sars-cov challenge in mva-sars-s vaccinated ferrets [ ] . candidate sars vaccines will need to be evaluated in more than one animal model. they also will need to be thoroughly evaluated for the duration of the antibody response they induce, as well as for the breadth of their protective effi-cacy against different strains of sars-cov. the implications of the sequence heterogeneity among sars-cov strains are difficult to test at this time because the most divergent strains (civet sars-like viruses) have not been recovered in culture. validation and international standardization of immunological assays for the evaluation of candidate sars vaccines are essential to compare data across different trials. this requires the establishment of international standards for sars-cov antibody and a repository for sars-cov reagents, with an international collaborative study to validate the iss. the establishment of the repository by who in collaboration with nibsc and niaid was recommended. severe acute respiratory syndrome who investigates china's fall in sars cases who says sars outbreak is over, but fight should go on summary of probable sars cases with onset of illness from identification of a novel coronavirus in patients with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group lineage the genome sequence of the sars-associated coronavirus sars coronavirus: a new challenge for prevention and therapy isolation and characterization of viruses related to the sars coronavirus from animals in southern china the aetiology, origins, and diagnosis of severe acute respiratory syndrome structure of sars coronavirus spike receptor-binding domain complexed with receptor adaptation of sars coronavirus to humans bats are natural reservoirs of sars-like coronaviruses severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study clinical features and short-term outcomes of patients with sars in the greater toronto area coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus sars immunity and vaccination prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice aged balb/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans virology: sars virus infection of cats and ferrets severe acute respiratory syndrome coronavirus infection of golden syrian hamsters koch's postulates fulfilled for sars virus newly discovered coronavirus as the primary cause of severe acute respiratory syndrome effects of a sars-associated coronavirus vaccine in monkeys replication of sars coronavirus administered into the respiratory tract of african green, rhesus and cynomolgus monkeys macaque model for severe acute respiratory syndrome an animal model of sars produced by infection of macaca mulatta with sars coronavirus an exposed domain in the severe acute respiratory syndrome coronavirus spike protein induces neutralizing antibodies identification of an antigenic determinant on the s domain of the severe acute respiratory syndrome coronavirus spike glycoprotein capable of inducing neutralizing antibodies contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity evaluation of human monoclonal antibody r for immunoprophylaxis of severe acute respiratory syndrome by an animal study, epitope mapping, and analysis of spike variants development and characterisation of neutralising monoclonal antibody to the sars-coronavirus severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice a dna vaccine induces sars coronavirus neutralization and protective immunity in mice immunization with modified vaccinia virus ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets mucosal immunisation of african green monkeys (cercopithecus aethiops) with an attenuated parainfluenza virus expressing the sars coronavirus spike protein for the prevention of sars adenoviral expression of a truncated s subunit of sars-cov spike protein results in specific humoral immune responses against sars-cov in rats identification of the membrane-active regions of the severe acute respiratory syndrome coronavirus spike membrane glycoprotein using a / -mer peptide scan: implications for the viral fusion mechanism mechanisms of host defense following severe acute respiratory syndrome-coronavirus (sars-cov) pulmonary infection of mice symptoms of infection caused by sars coronavirus in laboratory mice and guinea pigs civets are equally susceptible to experimental infection by two different severe acute respiratory syndrome coronavirus isolates infection of sars-cov on juvenile and adult brandt's vole microtus brandtii potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s protein that blocks receptor association development and characterization of a severe acute respiratory syndrome-associated coronavirus-neutralizing human monoclonal antibody that provides effective immunoprophylaxis in mice human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus therapy with a severe acute respiratory syndrome-associated coronavirus-neutralizing human monoclonal antibody reduces disease severity and viral burden in golden syrian hamsters a single immunization with a rhabdovirus-based vector expressing severe acute respiratory syndrome coronavirus (sars-cov) s protein results in the production of high levels of sars-cov-neutralizing antibodies long-term protection from sars coronavirus infection conferred by a single immunization with an attenuated vsv-based vaccine severe acute respiratory syndrome coronavirus nucleocapsid protein expressed by an adenovirus vector is phosphorylated and immunogenic in mice characterization of humoral responses in mice immunized with plasmid dnas encoding sars-cov spike gene fragments a subcutaneously injected uv-inactivated sars coronavirus vaccine elicits systemic humoral immunity in mice inactivated sars-cov vaccine prepared from whole virus induces a high level of neutralizing antibodies in balb/c mice immunogenicity of sars inactivated vaccine in balb/c mice augmentation of immune responses to sars coronavirus by a combination of dna and whole killed virus vaccines immunogenicity, safety, and protective efficacy of an inactivated sars-associated coronavirus vaccine in rhesus monkeys a double-inactivated whole virus candidate sars coronavirus vaccine stimulates neutralising and protective antibody responses antibody-dependent enhancement of virus infection and disease antibody-dependent enhancement of viral infection: molecular mechanisms and in vivo implications intracellular transport of recombinant coronavirus spike proteins: implications for virus assembly localization of antigenic sites of the s glycoprotein of feline infectious peritonitis virus involved in neutralization and antibody-dependent enhancement monoclonal antibody analysis of neutralization and antibody-dependent enhancement of feline infectious peritonitis virus a review of feline infectious peritonitis virus: molecular biology, immunopathogenesis, clinical aspects, and vaccination enhanced pulmonary histopathology is observed in cotton rats immunized with formalin-inactivated respiratory syncytial virus (rsv) or purified f glycoprotein and challenged with rsv - months after immunization cotton rats previously immunized with a chimeric rsv fg glycoprotein develop enhanced pulmonary pathology when infected with rsv, a phenomenon not encountered following immunization with vaccinia-rsv recombinants or rsv a human respiratory syncytial virus (rsv) primate model of enhanced pulmonary pathology induced with a formalin-inactivated rsv vaccine but not a recombinant fg subunit vaccine antibody-mediated enhancement of viral disease neutralization and antibody-dependent enhancement of dengue viruses evasion of antibody neutralization in emerging severe acute respiratory syndrome coronaviruses sars-coronavirus replicates in mononuclear cells of peripheral blood (pbmcs) from sars patients sars-coronavirus replication in human peripheral monocytes/macrophages recommendations for the preparation, characterization and establishment of international and other biological reference standards (revised ) a immunoglobulin: an international collaborative study to establish the second international standard the st international standard for anti-measles serum calibration of a replacement preparation for the international standard for rabies immunoglobulin report of a collaborative study to calibrate the second international standard for parvovirus b antibody the importance of standardisation of laboratory evaluations in hiv vaccine trials biodefense and emerging infections research resources repository (bei resources world health organization. laboratory biosafety manual who biosafety guidelines for handling of sars specimens caution urged on sars vaccines the authors thank dr. marc p. girard and dr. marie-paule kieny for their invaluable assistance in preparing the manuscript. the contributions of anjeanette roberts and kanta subbarao were supported in part by the intramural research program of nih/niaid. key: cord- - v r jij authors: recht, judith; schuenemann, verena j.; sánchez-villagra, marcelo r. title: host diversity and origin of zoonoses: the ancient and the new date: - - journal: animals (basel) doi: . /ani sha: doc_id: cord_uid: v r jij simple summary: there is a wide variety of diseases caused by bacteria, viruses, and parasites that are transmitted to humans by different routes from other animals. these diseases, known as zoonoses, represent % of new or reemerging infectious diseases. there is a considerable impact of these diseases on the economy and health at local and global levels, including zoonotic diseases caused by the ingestion of food and products derived from animals. the wide range of animal species that host these disease-causing organisms include all groups of mammals. birds are the second significant animal group to act as hosts for zoonoses. much progress has been made in understanding disease evolution and animal origin, with important contributions from fields such as paleopathology and analysis of dna, applied to ancient human bone remains. the study of ancient diseases such as brucellosis and tuberculosis benefits from these approaches. more research is needed as new diseases emerge causing pandemics and some previously eradicated reemerge in some regions. global efforts are focused, based on evidence generated by research, on the prevention of new pandemics. abstract: bacterial, viral, and parasitic zoonotic diseases are transmitted to humans from a wide variety of animal species that act as reservoir hosts for the causative organisms. zoonoses contribute an estimated % of new or reemerging infectious diseases in humans. all groups of mammals have been shown to act as hosts for transmission of different organisms that cause zoonoses, followed in importance by birds; with both wild and domestic species identified as hosts in specific cases. there has been considerable research progress leading to a better understanding of the host range, animal origin, evolution, and transmission of important zoonoses, including those caused by the ingestion of food and products derived from animals. paleopathology studies of ancient human bone lesions, in combination with ancient dna analysis of the causative pathogen, have contributed to our understanding of the origin of zoonotic diseases, including brucellosis and mycobacterial zoonoses. however, there are still knowledge gaps and new confirmed and potential hosts are reported locally with some frequency. both the economic cost and burden of disease of zoonoses are substantial at local and global levels, as reflected by recent coronavirus pandemics that spread rapidly around the world. evidence-based prevention strategies are currently a global priority increasingly recognized, especially in zoonoses-affected regions. zoonoses, or diseases transmitted to humans from vertebrate animals in both rural and urban settings where humans live, are widespread, with an estimated % of all human infectious diseases antimicrobial-resistant isolates. as the leading cause of zoonotic disease in both animals and humans, salmonella was placed in an antimicrobial resistance (amr) "serious threats" category of the usa cdc in [ ] . some strains of salmonella causing infections have developed resistance to antibiotics such as ciprofloxacin, azithromycin, and ceftriaxone, often used to treat patients with severe infections [ ] . in the us, campylobacter causes an estimated . million campylobacteriosis infections annually, of which % have decreased susceptibility to antibiotics used to treat these infections, including ciprofloxacin and azithromycin, limiting treatment options especially in low-and middle-income countries [ ] . in , a systematic review and meta-analysis of studies on interventions to reduce antibiotic use in food-producing animals compared the presence of antibiotic-resistant bacteria in animals and humans [ ] . based on this review, the world health organization (who) launched new guidelines on the use of antimicrobials in these animals, recommending to stop using antibiotics routinely to promote growth and prevent disease [ ] . here we present a review on zoonoses host diversity and animal origin of selected zoonoses, with an emphasis on brucellosis and mycobacterial zoonoses. we discuss examples of paleopathology and ancient dna studies contributing to our knowledge on disease origin and evolution, followed by viral zoonoses including recent pandemics as examples of recent and emerging zoonoses. the range of animal hosts of pathogens linked to zoonotic diseases is in most cases impressively diverse. some zoonotic disease outbreak studies have revealed new (sometimes unexpected) animal sources of human infection. hosts may include both wild and domesticated animals, some leading to foodborne zoonoses. monitoring animals for the presence of important pathogens is the focus of public health strategies. aside from pathogens shared by humans with invertebrates (vectors or intermediate hosts for disease transmission), the majority (about %) of the reservoirs known for zoonotic diseases are mammalian, followed by avian hosts [ ] . among other mammalian major taxa ("orders"), humans share the most pathogens with artiodactyls (frequently in proximity), followed by rodents, carnivorans, and primates. taxonomic identification of source groups for the emergence of zoonotic diseases could help to improve targeting of surveillance and interventions leading to prompt containment and even prevent zoonotic pandemics. however, given the taxonomic breadth reported for the many diseases, pinpointing specific groups and concentrating on them may not be justified. the information on hosts collected in tables s -s reflects how imprecisely and often very generally this is reported, and how vague our knowledge is in most cases. for example, often reports are of "rodents"-this group alone represents more than one-third of recognized mammalian species, with over of them [ ] . bats are the natural reservoirs of a significant number of important viral zoonoses, including probably the recent covid- that caused a high impact global pandemic this year ( ), still ongoing and discussed below. it has been hypothesized that bats are unique hosts in this regard, in particular as compared with rodents, the other is a particularly speciose major group (order) of placental mammals besides bats. however, the total number of zoonotic viruses identified in bats ( ) was lower than in rodents ( ) . there is a higher number of rodents than bat species ( -fold) [ , ] . a recent report on the largest dataset of the zoonotic virus-reservoir relationships built with avian and mammalian reservoir hosts of viruses aimed at determining whether some animal species are special reservoirs of zoonotic pathogens [ ] . the results did not support this view but a host-neutral variation instead; the analysis showed that the proportion of viruses infecting humans showed minimal variability across reservoir taxonomic orders and were in agreement with the number of animal species within each group (including for bats and rodents), with rare reservoir host effects restricted to one or two viral families. the camel has been highlighted in a recent review that showed zoonoses reported in this animal in iran (where raising camels is common with close contact between farmers and camels well as meat and milk consumption) including plague, q fever, campylobacteriosis, tuberculosis, salmonellosis, rabies, mers, and toxoplasmosis ( [ ] , section ). a plague outbreak in southern afghanistan in manifested as cases of acute gastroenteritis with deaths; the outbreak was linked to the consumption or handling of camel meat [ ] . birds are the main reservoir of the influenza a virus (table s ) . pigs, susceptible to infection with both avian and mammalian influenza viruses, may be acting in interspecies viral transmission as "mixing vessels", in which avian and mammalian influenza viruses recombine through a process known as "reassortment" to produce novel strains that can then infect humans [ ] . a role for wild migratory birds in the transmission of zoonoses has been acknowledged either as a reservoir host or by dispersing infected arthropod vectors. for example, west nile virus was likely introduced in the usa via infected birds, expanding in - along the atlantic seaboard, a common bird migration route, to reach southern florida to establish an enzootic cycle with mosquitoes and then from to , expanded westward possibly as a result of elliptical avian migration routes (reviewed in [ ] ). migrating birds can also carry one or more ectoparasites such as mites, ticks, fleas, and lice, all arthropods that themselves can carry pathogens; a potential role for migrating birds in dispersing ticks and associated pathogens that cause zoonotic disease has been recently highlighted [ ] . gut microbes and obligate ectoparasites such as ticks, fleas, and lice, depending on their animal hosts for transport. in this context, it is remarkable that recent geological events may have had a large impact on the dispersal capacity of both ectoparasites and gut pathogens. the megafauna (animals over . kg ( lb) body weight) decline in the late pleistocene/early holocene, which led to a decrease in seeds and nutrients dispersal, may have also caused a reduction in the movement of ectoparasites and generalist fecal microbes, including escherichia coli, to~ % of pre-extinction levels based on reductions after extinction estimated using the average home range for modern and extinct species [ ] . in this study, the distance that gut pathogens can travel between consumption and defecation, which is size-dependent, showed the largest declines in the americas and eurasia, however, whether pathogens disappeared with the previous hosts or adapted to new hosts is not known. this subject could be explored by metagenomic analyses of pathogens present in extinct mammal dung [ ] . chagas disease is an example of a successful parasitic zoonosis with a wide range (hundreds) of mammal species hosts in south america, transmitted by dozens of triatomine bug species (table ) . known as american trypanosomiasis, it also owes its name to carlos chagas, a brazilian researcher who first described it in brazil in as a disease due to the parasite trypanosoma cruzi (named after oswaldo cruz, another brazilian scientist), later shown to parasitize species of mammals from seven "orders" and triatomines from genera [ ] . a comprehensive study of reservoirs and wild hosts of t. cruzi. in brazil showed that species of several placental "orders" (artiodactyla, chiroptera, primates, carnivora, rodentia, cingulata, pilosa) and one marsupial (didelphimorphia) were involved in transmission, with four of them (primates, didelphimorphia, chiroptera, and the carnivora species nasua nasua) considered as key reservoir taxa exhibiting higher rates of parasitemia [ ] . in latin america, about million people are estimated to be infected with t. cruzi through feces or night bites of the "kissing bug" (triatomines), or through blood transfusion, maternally, or orally through contaminated food that can lead to an acute phase usually without symptoms or very mild [ ] . after - years following the acute phase, however, symptomatic chronic disease can occur, leading to irreversible damage to several organs such as the heart, esophagus, and colon [ ] . cattle, bison, african buffalo, cervids, brushtail possums, badgers, kudu can be reservoirs ingestion (unpasteurized dairy products, undercooked meat including bushmeat), inhalation, contamination of breaks in the skin * information sources: [ , , [ ] [ ] [ ] [ ] ; additional tables with information on many bacterial, viral, and parasitic zoonoses are in the supplementary material (tables s -s , respectively). trypanosoma cruzi has been identified in hosts such as armadillos and monkeys (in and , respectively, by chagas in brazil) [ ] , as well as cats and dogs. a recent report of a study lasting two decades ( - ) of t. cruzi infection in wild mammals in brazil showed that % of mammals were seropositive and % with high parasitemia, indicating infectivity potential [ ] . a recent study reported a potential effect on chagas disease transmission of oil palm plantations in colombia, where the main vector in the region has been captured and reported to have t. cruzi natural infection, based on vertebrate host analyses of blood meals from nymphs that revealed vertebrate species including pigs, house mouse and opossum [ ] . hunting dogs in indigenous mayangna and miskitu populations from nicaragua's remote bosawás biosphere reserve were shown to have t. cruzi antibodies and therefore previous exposure ( / sera screened, or %), suggesting hunting dogs as the potential zoonotic risk for chagas disease in these communities [ ] . the origin of chagas disease is unclear. bats may have been the original reservoirs hosts of t. cruzi, as postulated in the bat-seeding hypothesis, followed by a host switch to a non-volant mammal and then several switches to humans resulting in the diversity of lineages circulating in human populations. two genetic lineages of trypanosomes associated with bats were recently detected in rural areas of southern ecuador [ ] , one of which, named tcbat, is t. cruzi-related and was detected in a -year-old female in a forest area in northwestern colombia as a mixed infection of t. cruzi i and tcbat genotypes [ ] . a recent systematic review on zoonotic diseases that manifest with human febrile illness reported in malaria-endemic countries showed a wide distribution of these diseases causing febrile illness; half of them were bacterial diseases [ ] . for a list of selected bacterial zoonoses reflecting the impressive diversity of this group of diseases, see table s . next, we discuss two well-known bacteria genera-brucella and myocabacteria-as they represent ancient human diseases for which origins are still highly debated. two major diseases caused by these bacteria are brucellosis and tuberculosis. they affect the skeleton, causing bone pathologies in about - % of individuals who have the disease [ ] and can result in similar lesions in affected individuals despite being caused by very different bacterial species. paleopathology studies along with ancient dna analysis of bone lesion remains have been critical in determining the origin for each of these two diseases. the overlapping appearance of affected bones in the spine in both infections have led researchers to initially suspect a tuberculosis lesion, with molecular analyses demonstrating in some cases that the cause was brucellosis. a study that evaluated calcified nodules from a th-century skeleton found at an abandoned medieval village in northwest sardinia, italy, initially hypothesized the nodules were due to tuberculosis, but shotgun metagenomics revealed medieval brucella melitensis genome sequences instead [ ] . even today, with advanced health technology, there is overlap in the understanding of symptoms and laboratory test results for these two diseases [ ] , making differential diagnosis difficult in countries such as india, where they are both endemic [ ] . caused by bacteria of the genus brucella (mostly brucella melitensis), brucellosis is a common zoonotic infection resulting in febrile illness in humans, mainly through the ingestion of unpasteurized dairy products and direct contact with infected animals (table ). travelers to endemic areas may get infected by consumption of unpasteurized milk or other dairy products and may be the source of imported cases into their own countries (mostly from infected cheeses consumed by their families), as is the case for most of the acute brucellosis cases in north america and northern europe [ ] . re-emergence of zoonotic diseases in countries where they have been previously eradicated has been reported, calling for a need for disease-free countries to remain aware and implement regional monitoring systems. brucellosis has reemerged in bulgaria after years, probably due to the illegal import of infected animals from endemic border countries, as has occurred with bovine brucellosis in france, with possible cross-border brucellosis transmission into europe from middle-eastern countries with the highest incidences of brucellosis worldwide such as turkey and syria (reviewed in [ ] ). the exact prevalence of both animal and human disease due to brucellosis is not well known. the economic burden of brucellosis is considerably high in low-income countries in tropical asia and africa [ ] . in , out of . million estimated cases of brucellosis in kenya, , people died, % of whom were livestock keepers [ ] . brucellosis can have skeletal manifestations, with bones and joints affected being the most frequent complications occurring in up to % of cases [ ] . analysis of vertebral lesions and pathological changes described in a . - . million-year-old male skeleton of australopithecus africanus from south africa were interpreted as resulting from initial phases of brucellosis [ ] . this disease is an example of human and domestic animal paleopathology studies suggesting brucellosis in ancient bone remains, with most cases involving adult male skeletal individuals showing lumbar vertebrae and sacroiliac joints involved [ ] , evidence which combined with ancient dna analysis by pcr have confirmed the presence of brucella dna (reviewed in [ ] ). as discussed above, dna detection of brucella dna has been critical in confirming brucellosis in ancient human remains when paleopathology initially suggested tuberculosis. however, brucella bacteria do not preserve as well as mycobacteria, which have a thicker, hydrophobic and more resistant cell wall that has allowed more data to be collected for ancient infection for the latter type of disease-causing bacteria (discussed next). mycobacteria vary in epidemiology, reservoirs, and their ability to cause disease, with groups such as the mycobacterium tuberculosis complex including m. tuberculosis and m. bovis (see bovine tuberculosis below) that infect a large range of mammals including humans via inhalation of droplets containing bacteria that reach the lungs; m. leprae (see leprosy section below); "nontuberculous mycobacteria" [ ] . human tuberculosis caused by m. tuberculosis is a well-known disease of unclear origin, with a considerable impact on global health. it affects an estimated million people annually. those living with hiv are more likely to be infected with and die of tuberculosis [ ] . the origin of tuberculosis is under debate regarding whether it started as a zoonosis from cattle or not. one hypothesis states that it originated with bovid milk consumption known to occur in the early neolithic in europe [ ] . another hypothesis based on biomolecular studies proposes a new evolutionary scenario with human tuberculosis having a human origin, present in early african human populations at least , years ago and expanding with human migration, especially in the neolithic [ ] . in both scenarios, human-animal proximity starting in the neolithic was a critical factor for emergence/expansion, due to close interaction between early domesticates and humans and increased human density. human density in some areas peaked during the industrial revolution when crowded dairy cow populations in densely populated urban settings were the source of milk [ ] . although rare, tuberculosis can result in osteological lesions which can help identify, along with molecular analysis, tuberculosis infection in ancient bone remains. these approaches have been applied to materials from both humans and animals. however, molecular genetics applications in these cases have limitations, including cost. human remains from a medieval churchyard in england in which both m. tuberculosis and m. bovis were predicted due to milk and beef consumption at the time, showed only m. tuberculosis dna in human skeletons that displayed morphological symptoms of tuberculosis (reviewed in [ ] ). a particular question on ancient dna in this context relates to the origins of tuberculosis in the americas. while today european lineages of m. tuberculosis are found, morphological evidence exists that may support a pre-columbian prevalence of the disease. a recent report on sequencing and analysis of three mycobacterial genomes from peruvian human skeletons dated to - revealed the presence of tuberculosis before european contact in pre-columbian south america [ ] , a region with considerable morphological evidence of pre-columbian tuberculosis previously reported. in this study, the three sequenced mycobacterial genomes were most closely related to bacteria belonging to the m. tuberculosis complex that have adapted to seals and sea lions (m. pinnipedii) than those adapted to humans today in europe, asia, or africa. these data suggest that sea mammals may have played a role in tuberculosis transmission to humans across the ocean, an intriguing possibility for a zoonotic origin of new world tuberculosis to be further investigated. bovine tuberculosis resulting from m. bovis infection affects cattle and other mammals and, when transmitted to humans, becomes zoonotic tuberculosis (table ). its overall incidence has decreased due to cattle control and routine pasteurization of milk from cows, as infection happens mostly by consuming unpasteurized milk and dairy products or by wound contact occurring during slaughter or hunting [ ] ; occupational exposure of livestock workers may occur via inhalation of aerosol or cough from infected cattle. this form of widespread zoonotic tuberculosis can be fatal, present mainly in africa (causing approximately % of all pulmonary tuberculosis cases) and southeast asia. in , out of cases of bovine tuberculosis in kenya, people died (about %); % of the fatalities were livestock keepers [ ] . often bovine tuberculosis caused by m. bovis in humans is indistinguishable clinically from human tuberculosis resulting from m. tuberculosis infection; bovine tuberculosis is estimated to account for up to % of human tuberculosis cases in some countries [ ] . however, accurate information is lacking on the incidence of tuberculosis due to human m. bovis infection from countries with high tuberculosis and hiv prevalence and where populations have direct contact with cattle such as sub-saharan africa [ , ] . in these low resource settings, it is often assumed (and not tested) that tuberculosis is caused by m. tuberculosis, leading to underestimating the real incidence of zoonotic tuberculosis and underscoring a need to accurately diagnose and treat human tuberculosis caused by m. bovis [ ] . m. bovis has a broad host range including domestic and wild animals, with feral maintenance hosts such as badgers (meles meles) in the uk and ireland, the brushtail possum (trichosurus vulpecula) in new zealand, and the white-tailed deer (odocoileus virginianus) in michigan, usa posing a threat for livestock infection as well as disease eradication from cattle (reviewed in [ ] ). cases of bovine tuberculosis in humans linked to deer hunting and handling in an endemic white-tailed deer area have been reported in michigan [ ] . there is also epidemiological evidence in new zealand that feral ferrets (mustela furo) may be tuberculosis vectors for cattle, and can be used as sentinels for this disease as an alternative to possums (reviewed in [ ] ). mycobacterium avium, a causative bacterium of tuberculosis in birds, can also be transmitted to mammals, including livestock. the livestock industry suffers economic losses and trade restrictions due to its incidence. widespread paratuberculosis in animals (also known as jones disease) including bovine paratuberculosis, is an endemic disease especially in developing countries affecting livestock production, zoo, and wildlife animals (reviewed in [ ] ). m. avium subspecies can cause paratuberculosis in humans, manifested as inflammatory bowel disease and autoimmune diseases including asthma, insulin-dependent diabetes mellitus, sarcoidosis, rheumatoid arthritis, multiple sclerosis, celiac disease and may also be a contributing factor to crohn's disease [ ] . mycobacterium leprae, the main causative agent of leprosy in humans, a chronic disease with skin lesions and peripheral nerve damage mostly spread via a human-to-human transmission with zoonotic transmission from natural reservoirs, which are not clearly understood yet. the first animal reservoir to be discovered was the nine-banded armadillo (dasypus novemcinctus) in the southern united states [ ] [ ] [ ] . this armadillo, which can be naturally infected with m. leprae and shows similar disease presentation as humans systemically, have been used as animal models for leprosy studies by laboratory infection; they present typical plantar ulceration with foot ulcers increasing as the infection progresses (reviewed in [ ] ). this species has been recently shown to be a potential reservoir in brazil, where in some areas people hunt and eat them as a dietary source of protein [ ] . recently, m. leprae was also isolated from red squirrels on brownsea island in the southern uk [ ] . for both nine-banded armadillos and red squirrels reservoirs, related m. leprae strains were found via ancient dna studies in human skeletons from england and denmark dating to medieval times, suggesting a potential european origin of the strains present today in the reservoirs [ , ] . non-human primates in several regions have been identified as additional m. leprae reservoirs [ ] . m. leprae genomes were obtained from a chimpanzee (pan troglodytes) from sierra leone, a cynomolgus macaque (macaca fascicularis) from the philippines, and a sooty mangabey (cercocebus atys) from west africa [ ] . marmosets (callithrix jacchus) in brazil, also examined as possible hosts for m. leprae, were not positive by dna analysis although mycobacterial dna (rpob locus) was detected [ ] . in contrast to the previously discussed zoonoses, which are linked to well-known diseases, viral zoonoses are the cause of the majority of recent human pandemics, suggesting that viruses may evolve more rapidly than other pathogens to adapt to the human host, sometimes leading to the person-to-person transmission without the need for another reservoir, and with transmission enhanced in dense populations and by human travel [ ] . many of them are distributed worldwide (table s ). here we present examples of recent viral zoonoses, some resulting in widespread pandemics, to showcase the clear involvement of wild animals, which are often debated in the previously introduced cases likely due to the old nature of those diseases. in many areas of central africa in particular, wild meat (known as bush meat) is in high demand. human contact with wild animals during hunting and preparation or consumption of undercooked meat has led to important zoonoses developing. an example may be hiv/aids, which was linked to the butchering of hunted chimpanzees in africa, afterward adapting to human-to-human transmission [ ] . the human ebola epidemic in west africa in - caused by the ebola virus was linked to the hunting or handling of infected gorillas and other wild animal carcasses ( [ ] , table s ). the congo's ebola hemorrhagic fever affects gorillas and chimpanzees; about gorillas were killed in gabon and the republic of the congo in - by the ebola virus [ ] . asia is another example of wild animals trafficking for consumption, especially in china, myanmar, vietnam, and thailand, where "ye wei" ("wild taste" for wild and exotic animals) was associated with social status including in the dynastic eras as well as currently being widely sold in wet markets and restaurants, sourced legally or illegally from the wild or wildlife farms (reviewed in [ ] ). important zoonoses such as some caused by coronaviruses (table ) have also been associated with the consumption of wild meat in markets, two of them in china. the severe acute respiratory syndrome coronavirus (sars-cov) in and the middle east respiratory syndrome coronavirus (mers-cov) in each caused a large pandemic, but these were small compared to the one caused by sars-cov- , a second sars virus recently identified as the cause of covid- . the sars pandemic caused cases and deaths and mers resulted in cases and deaths [ ] , while covid- has caused, as of july and only six months after who declared it a pandemic on january, over million cases and slightly over half a million deaths [ ] . the animal sources for sars and mers were identified as the civet and dromedary camel, respectively (table ) . however, these are considered intermediate hosts, as these zoonotic diseases are thought to have originated in bats [ , ] , subsequently spilling over to intermediate hosts, and eventually jumping to humans [ ] . a very recent phylogenetic dating study based on bioinformatic approaches strongly suggests that sars-cov- emerged directly, without an intermediate host, from the same horseshoe bat subgenus of sars-like coronaviruses, with both sars-cov and sars-cov- diverging at the same time ( - years ago) from currently known extant bat virus [ ] . nonhuman primates were shown to be an effective yellow fever sentinel system in brazil. yellow fever (table s ) , a reemerging viral zoonotic disease endemic in africa and south america transmitted from vector mosquitoes, often causes outbreaks in both humans and nonhuman primates in brazil. this country has an established and successful yellow fever national surveillance program that includes postmortem nonhuman primate studies for early circulating virus detection and prompt implementation of vaccination and vector control [ ] . after a epizootic occurred in brazil's espirito santo state, deceased nonhuman primates (two howler monkeys (alouatta spp.) and not further identified) were examined with of them showing typical yellow fever features; yellow fever was diagnosed the same year for of animals tested from southern states of brazil ( % occurrence) [ ] . emerging zoonotic diseases causing epidemics have prompted scientists to focus on efforts towards identifying factors involved in disease emergence as well as possible prediction tools, including modeling approaches. recent such modeling used for spatial mapping of hotspots showed that the global distribution of the risk of emerging zoonotic diseases is higher in tropical regions with wildlife biodiversity (especially mammals) experiencing land-use changes [ ] . this study, along with previous ones [ ] , predicted a higher risk in tropical, developing countries. the authors stated that even though emerging infectious disease events have been predominantly reported in developed countries, this is probably an artifact due to stronger surveillance and reporting systems in these areas. it would be hard to test this idea. the one health multisector approach response to zoonoses threat is an appropriate one, which may need input from additional sectors, such as travel and tourism, social and political scientists, anthropologists, and economists to better plan and implement strategies related to surveillance, capacity building, and risk reduction when addressing, for example, the possible closure of wet markets in communities that rely heavily on them for income and food [ ] . there is a wide range of species reported as hosts of zoonoses, making predictions on new and potential reservoirs based on phylogenetic considerations challenging. basic information on which species serve as hosts is lacking for many regions and diseases. although considerable progress has been made in our understanding of these diseases' reservoirs, animal origin, and evolution, with contributions made by paleopathology and ancient dna detection for ancient diseases such as brucellosis and zoonotic tuberculosis. further research is needed to gain insights into mechanisms of disease emergence and transmission. in view of the recent pandemics, research on zoonoses should be prioritized towards developing evidence-based prevention strategies. these should include approaches based on sustainable living and food consumption that address inequality, as commentators across the globe currently discuss prompted by the covid- pandemic. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s . table s : selected bacterial zoonoses, table s : selected viral zoonoses, table s : selected parasitic and other non-bacterial non-viral zoonoses. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection or interpretation of data and the writing of the manuscript or in the decision to publish the results. prediction and prevention of the next pandemic zoonosis prioritizing zoonoses for global health capacity building-themes from one health zoonotic disease workshops in countries operational framework for strengthening human, animal and environmental public health systems at their interface; the world bank the economic impact. . available online recreational infections (chapter ) ecology of zoonoses: natural and unnatural histories biggest threats and data drug-resistant nontyphoidal restricting the use of antibiotics in food-producing animals and its associations with antibiotic resistance in food-producing animals and human beings: a systematic review and meta-analysis. lancet planet guidelines on use of medically important antimicrobials in food-producing animals a comparison of bats and rodents as reservoirs of zoonotic viruses: are bats special? viral zoonotic risk is homogenous among taxonomic orders of mammalian and avian reservoir hosts zoonotic implications of camel diseases in iran outbreak of gastroenteritis caused by yersinia pestis in afghanistan unique feature of swine anp a provides susceptibility to avian influenza virus infection in pigs birds, migration and emerging zoonoses: west nile virus, lyme disease, influenza a and enteropathogens arthropods and associated arthropod-borne diseases transmitted by migrating birds. the case of ticks and tick-borne pathogens megafauna decline have reduced pathogen dispersal which may have increased emergent infectious diseases chagas' disease: an emergent urban zoonosis. the caracas valley (venezuela) as an epidemiological model. front trypanosoma cruzi transmission in the wild and its most important reservoir hosts in brazil chagas disease: what is known and what is needed-a background article trypanosoma cruzi: entry into mammalian host cells and parasitophorous vacuole formation zoonotic diseases. in merck veterinary manual list of zoonotic diseases zoonotic aspects of mycobacterium bovis and mycobacterium avium-intracellulare complex (mac) generalist host species drive trypanosoma cruzi vector infection in oil palm plantations in the orinoco region, colombia giardia infection and trypanosoma cruzi exposure in dogs in the bosawás biosphere reserve trypanosomes, and triatomines in ecuador: new insights into the diversity, transmission, and origins of trypanosoma cruzi and chagas disease first report of human trypanosoma cruzi infection attributed to tcbat genotype zoonotic causes of febrile illness in malaria endemic countries: a systematic review human skeletal paleopathology recovery of a medieval brucella melitensis genome using shotgun metagenomics diagnosis and management of spinal tuberculosis combined with brucellosis: a case report and literature review clinical profile of brucellosis from a tertiary care center in southern india. asian pac zoonoses under our noses. microbes infect economics of brucellosis impact and control in low-income countries the monetary impact of zoonotic diseases on society evidence from three zoonoses in kenya possible brucellosis in an early hominin skeleton from sterkfontein, south africa origin, evolution and paleoepidemiology of brucellosis approaching ancient disease from a one health perspective: interdisciplinary review for the investigation of zoonotic brucellosis practice guidelines for clinical microbiology laboratories: mycobacteria shuffling nags lame ducks: the archaeology of animal disease the paleopathological evidence on the origins of human tuberculosis: a review pre-columbian mycobacterial genomes reveal seals as a source of new world human tuberculosis mycobacterium bovis (bovine tuberculosis) in humans tuberculosis (world organisation for animal health) why has zoonotic tuberculosis not received much attention? the importance of mycobacterium bovis as a zoonosis zoonotic tuberculosis in human beings caused by mycobacterium bovis-a call for action human mycobacterium bovis infection and bovine tuberculosis outbreak feral ferrets (mustela furo) as hosts and sentinels of tuberculosis in new zealand mycobacterium avium paratuberculosis and mycobacterium avium complex and related subspecies as causative agents of zoonotic and occupational diseases leprosy in wild armadillos evaluation of the origin of mycobacterium leprae infections in the wild armadillo, dasypus novemcinctus probable zoonotic leprosy in the southern united states armadillos and leprosy: from infection to biological model evidence of zoonotic leprosy in para, brazilian amazon, and risks associated with human contact or consumption of armadillos red squirrels in the british isles are infected with leprosy bacilli ancient genomes reveal a high diversity of mycobacterium leprae in medieval europe genome-wide comparison of medieval and modern mycobacterium leprae mycobacterium leprae genomes from naturally infected nonhuman primates validation of qpcr methods for the detection of mycobacterium in new world animal reservoirs aids as a zoonosis: scientific and public health implications wild animal mortality monitoring and human ebola outbreaks, gabon and republic of congo ebola outbreak killed gorillas baby pangolins on my plate: possible lessons to learn from the covid- pandemic covid- has killed more people than sars and mers combined, despite lower case fatality rate origin and evolution of pathogenic coronaviruses a pneumonia outbreak associated with a new coronavirus of probable bat origin evolutionary origins of the sars-cov- sarbecovirus lineage responsible for the covid- pandemic surveillance for yellow fever virus in non-human primates in southern brazil outbreak of yellow fever among nonhuman primates global hotspots and correlates of emerging zoonotic diseases global trends in emerging infectious diseases this article is an open access article distributed under the terms and conditions of the creative commons attribution key: cord- -xe lljz authors: overgaauw, paul a.m.; vinke, claudia m.; van hagen, marjan a.e.; lipman, len j.a. title: a one health perspective on the human–companion animal relationship with emphasis on zoonotic aspects date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: xe lljz over time the human–animal bond has been changed. for instance, the role of pets has changed from work animals (protecting houses, catching mice) to animals with a social function, giving companionship. pets can be important for the physical and mental health of their owners but may also transmit zoonotic infections. the one health initiative is a worldwide strategy for expanding collaborations in all aspects of health care for humans, animals, and the environment. however, in one health communications the role of particularly dogs and cats is often underestimated. objective: evaluation of positive and negative one health issues of the human–companion animal relationship with a focus on zoonotic aspects of cats and dogs in industrialized countries. method: literature review. results: pets undoubtedly have a positive effect on human health, while owners are increasing aware of pet’s health and welfare. the changing attitude of humans with regard to pets and their environment can also lead to negative effects such as changes in feeding practices, extreme breeding, and behavioral problems, and anthropozoonoses. for the human, there may be a higher risk of the transmission of zoonotic infections due to trends such as sleeping with pets, allowing pets to lick the face or wounds, bite accidents, keeping exotic animals, the importation of rescue dogs, and soil contact. conclusions: one health issues need frequently re-evaluated as the close human–animal relationship with pet animals can totally differ compared to decennia ago. because of the changed human–companion animal bond, recommendations regarding responsible pet-ownership, including normal hygienic practices, responsible breeding, feeding, housing, and mental and physical challenges conforming the biology of the animal are required. education can be performed by vets and physicians as part of the one health concept. the one health initiative or concept is a worldwide strategy that recognizes that public health is connected with animal health and the environment. it concerns multidisciplinary collaboration between physicians, veterinarians, environmental scientists, public health professionals, wildlife experts, and many others [ , ] . with a multisectoral and transdisciplinary approach, public health threats can be better monitored and controlled. the resulting synergism enhances the knowledge of how diseases, known as zoonotic diseases, can be shared between animals and people with the goal of this article is based on an existing post-graduate course for veterinarians, vet technicians, family doctors, midwifes, and specialists such as pediatricians which explores healthy human-animal relationships. the existing evidence-based knowledge contained in this course has been actualized by performing a literature search to add new relevant publications. a literature search was conducted through march , using the national library of medicine's pubmed for the terms "one health" and "companion animals"; "pet ownership"; "households" and "pets"; "dogs" or "cats" or "pets" and "mental" or "physical health" or "children"; "animal assisted therapy"; "dogs" or "cats" and "nutritional problems" or "overweight" or "obesity" or "homemade" or "raw meat diets"; "dogs" or "cats" and "behavior problems" or "aggression" or "fear" or "anxiety" or "abnormal repetitive behavior"; "dogs" or "cats" and "breeding" or "genetic problems"; "dogs" or "cats" and "zooanthroponoses"; "pets" and "anthropomorphism"; "dogs" or "cats" or "exotic animals" or "rescue dogs" or "soil" and zoonoses. for some topics the internet was accessed and used as reference if additional information was not available as scientific publication. the authors selected articles that described pivotal and novel insights in the different topics. all searches were carried out without filters. the titles of all found articles were screened for relevance to the topic, and appropriate titles were assessed and selected based on their abstracts. if a selected article was a review, it was read and relevant citations were used to find primary literature on the subject. additional studies were found using the bibliographies of selected articles. occasionally, reviews were directly used as sources, mostly to convey background information that is not in the core focus of this article. original articles in english and different national languages (dutch, german, french, spanish, if available) were included. specific searches were made for citations dated after the year to ensure more recent literature on the topic had not been missed. a pet or companion animal is an animal that lives in or around the house and is fed and cared for by humans. until the s, pets were mainly kept as utility animals, for example as draft dogs or watch dogs or for pest control when it comes to cats. due to major changes that have taken place in society since the second world war, such as increased leisure time and prosperity, but also individualization of humans, animals are nowadays kept as pets and are regarded by many owners as valued family members, e.g., over % in the united kingdom [ ] . pet ownership is still increasing in many industrialized countries and these animals are more often considered a member of the family [ ] . even in china, a country where pets were banned in urban areas until , pet ownership has grown quite rapidly in the major cities. the rate of pet ownership of all usa households increased from . % to % in . dogs continue to dominate in popularity among american households. approximately % of households nationwide owned a dog, bringing the population of pet dogs to nearly million, while % of households owned cats, with a total population of million [ ] . in , an estimated million european households owned at least one pet animal; % of households owned dogs and % owned cats [ ] . there are million pet dogs and million pet cats in europe which is a % increase of dogs within years ( million dogs in ) and a % increase of cats ( million in ) [ ] . pet cats in europe thereby are more popular than dogs. an explanation of the higher popularity of cats may be the number of single-person households in the eu that rose on average by . % per annum between and to . %, and the growth of two-family households grew by % to % in this period [ ] . also, dual-earner families are widespread as a result of quite a steep growth in female employment over the past two decades [ ] . when animals live with humans, they too benefit from human interaction. over the past decades, animal welfare has evolved to recognize that animals are sentient beings capable of experiencing positive and negative emotions. the social and ethical dimensions of animal welfare, which are concerned with how human society morally regards and treats non-human animals, are also increasingly being recognized [ ] . in dutch law, the intrinsic value of kept animals is expressly incorporated and used as a guiding ethical principle that forms the basis of any further legislation. the intrinsic value is thereby defined that animals are sentient beings that can feel pain and discomfort. therefore, they should be kept free of stress, pain, disease, hunger, thirst, and should be able to show natural behavior known as the five freedoms of r. brambell [ ] . in industrialized countries animal keepers, their owners, are legally required to provide such circumstances and can be prosecuted if they infringe the law. of course, in the field there is animal abuse, negative animal welfare conditions, and animal diseases. however, in general, caretaking for companion animals is nowadays performed at a high level. new insights into animal behavior has had its influence on the general public. for example, owners are aware or are told by vets and pet shops that rabbits should not be kept alone but at least in pairs due to their need for social contact [ ] . there are various reasons to keep pets, such as love, warmth, and companionship. companion animals have an important emotional value, and promote the socialization of the lonely elderly because they facilitate additional contact with people. pets form a goal in life, reduce stress, and ensure that the owner keeps physically active. around % of dog owners and % of cat owners expressed that owning their pet makes them happy and % of owners selected this as one of the reasons they got their pet in the first place [ ] . however, the function of companion animals consists of more than just providing a socializing being. studies show other benefits of having a pet, such as the positive effect on individuals' mental and physiological health status. most research addressing the health benefits of pet ownership show reductions in distress and anxiety, decreases in loneliness and depression, and increases in physical condition [ ] . the positive benefits to human health from interacting with animals, focusing on the companion animal, have also be described with the term "zooeyia" [ ] . in fact, % of owners agreed that having a pet makes them physically healthier, with dog owners more likely to agree, most probably because dog owners exercise more ( % agreeing, compared to % of cat owners). besides, % of owners agreed that having a pet makes them mentally healthier. expressed reasons are the non-judgmental nature of their pets, their playfulness, or physical contact [ ] . another demonstrated positive influence is the blood pressure and heart rate lowering effects that occurs when stroking a friendly-looking dog or even being in the presence of a friendly animal, while it is not necessary to own a pet to obtain these stress-moderating benefits [ ] . many studies have demonstrated the association between pet ownership and cardiovascular health and dog owners appear to have a significantly greater chance of survival after a heart attack compared to people without pets [ ] [ ] [ ] . pets can therefore play an important role in reducing absenteeism and visits to family doctors or the hospital [ ] . it has been estimated that pet ownership saved australia $ billion in [ ] , while it may reduce the use of the national health service (nhs) in the uk to the value of £ . billion per year [ ] . dogs also play an increasing role as co-therapist or as supporter for people with psychological or physical disabilities. the benefits of these animal-assisted activities are improved mood, decreased physiological distress, depression, dementia, and loneliness [ , ] . examples include resident or visiting dogs in prisons, nursing homes [ ] , mental institutions, and hospitals where they can reduce patient anxiety in a hospital emergency department [ ] , reduce pain perceptions in children after surgery [ ] , or calm young patients at a pediatric dental clinic [ ] . since dogs have extremely sensitive noses, they are used for several purposes such as tracking, bomb detection, and search and rescue. in recent years, canine olfaction has also been more recognized as a diagnostic tool for identifying pre-clinical disease status, such as diabetes (ketones), different forms of cancer, and infections from biological media samples [ ] . animal-assisted therapies can act as co-therapies to facilitate psychotherapy or to provide specific types of therapeutic interventions such as improving motor skills or behavior [ ] . such interventions were effective in improving the state of children or adults with or at risk of developing mental disorders such as attention deficit hyperactivity disorder (adhd), post-traumatic stress disorder (ptsd), or autism spectrum disorder (asd) [ ] [ ] [ ] , and for the treatment of ptsd in military veterans [ , ] . assistance or service animals are trained to perform tasks for the benefit of individuals who have disabilities such as hearing loss, physical disabilities, emotional disabilities, seizure disorders, or diabetes [ ] . finally, a wide range of emotional health benefits from childhood pet ownership has been identified, particularly for those suffering from low self-esteem and loneliness. there is evidence of an association between pet ownership and educational and cognitive benefits, increased social competence, social networks, social interaction, and social play behavior [ , ] . significantly less absenteeism from school through sickness among children who live with pets has also been reported [ ] . having a dog or cat in the house during the first year of life may protect against childhood asthma and allergy [ , ] . it can therefore be concluded that companion animals contribute significantly toward the public health, but also increasingly, the health of individually challenged persons through animal-assisted interventions [ ] . providing companion animals with feed by humans, has been considered an advantage for companion animals in their relationship with humans. however, the feeding practices can also have a negative impact on companion animals [ , ] . obesity in cats and dogs is a disease which is rapidly increasing with significant and lifelong implications for animal welfare. although no universally accepted definition of canine and feline obesity exists, the american veterinary medical association defined obesity being more than % above the ideal weight of an animal. overweight is defined as %- % above the ideal weight. using body condition scores, it has been estimated that in the united states, % of dogs and % of cats are obese or overweight. a study in the united kingdom reported % of adult dogs and % of juvenile dogs as being obese or overweight [ , ] . overweight dogs are more likely to be diagnosed with, e.g., urinary tract diseases. obese and overweight dogs are at risk developing orthopedic disorders and hypothyroidism [ , ] . obese cats are at higher risk for developing urinary tract disease, diabetes mellitus, and neoplasia [ , ] . other diet-related problems in companion animals can be caused by the changed feeding behavior of humans, e.g., by providing companion animals with bone and raw feed (barf) or vegan diets. risks for companion animals associated with barf or vegan diets are the presence of microbial hazards, insufficient nutrition, and in raw meat diets the presence of risk materials like thyroid tissue. through contact with their animals, it is possible that risks could even develop for owners. there could be an increased risk of human salmonellosis because of the presence of salmonella spp. in the diet which can spread to humans through diet leftovers or by contact with animal feces. recently a review was published on the risks of barf feeding [ ] . the authors concluded that the data for the nutritional, medical, and public health risks of raw feeding are fragmentary, but they are increasingly forming a compelling body of formal scientific evidence. publications were found reporting the presence of escherichia coli o , salmonella typhimurium, campylobacter spp., and antibiotic resistant bacteria in the feed. nutritional problems, such as calcium/phosphorous imbalances and specific vitamin deficiencies [ ] are also reported. moreover, homemade diets are inherently susceptible to nutritional imbalances and deficiencies [ ] . awareness about climate change, public health and animal welfare has incited a major change in dietary choices among many individuals. the number of vegans in the world keeps growing, even quadrupling from , to , individuals between and in affluent countries such as the uk [ ] . the popularity of veganism goes beyond the scope of the human diet, as more people are interested in the possibility of feeding their companion animal a vegan diet than ever before. to create animal-free complete cat food requires replacing nutrients in animal-based materials with plant-based materials. different sources are used such as corn, rice, peas, soy, potato, and different oils and seeds. any further nutrients that are missing from plant-based materials, such as taurine and carnitine, are replaced with synthetically produced versions [ , ] . feeding trials using vegan animal food are either not performed due to testing costs or kept private due to the highly competitive vegan pet food market [ ] . additionally, they reported testing vegetarian diets for cats and dogs and found that one was lacking protein and six did not meet all amino acid concentration requirements. vegan animal food may not contain meat, but it does contain grains, soy, and corn. plant-based products, such as grains, can be a source of health problems because of the presence of mycotoxins, for example [ ] . warm, humid storage conditions can lead to the formation of mycotoxins such as aflatoxins, produced by the fungi aspergillus flavus and aspergillus parasiticus. many regular animal feeds also contain plant-based products, therefore the negative impact of feeding vegan diets to companion animals, especially obligate carnivores such as the cat or the ferret, seems therefore more related to diet insufficiency than to microbial health risks. additionally, addressing behavioral problems, the "free" provision of food might fulfil the consumptive part of feeding behavior of our companion animals but does not fulfil the appetitive phase. especially this phase of feeding patterns can have consequences for the companion animals' mental health and may result in behavioral problems if appetitive physical and mental challenges remain chronically absent in the human-animal relationship. in the human-companion animal bond, pets may develop abnormal behavior, including excessive aggression, fear and anxiety, or even abnormal repetitive behavior. abnormal repetitive behavior (arbs) were first noticed in zoo-, shelter-, and laboratory animals: all animals housed under stimulus-poor conditions and with limited space. however, companion animals can develop arbs as well, if the individual's adaptive capacity is exceeded due to, e.g., a lack of social contact, physical exercise, mental challenges, and in uncontrollable and unpredictable environments (e.g., separation, mistreatment, or inadequate application of cages). arbs can either be classified in stereotypies or compulsive disorders [ ] with stereotypies generally defined as unvarying repetitive behavior patterns with no obvious goal or function [ ] . the terminology of compulsive disorders is preferably chosen for repetitive behavior patterns that are goal-directed and show variability in the repetitive (motor) patterns [ , ] . under chronic conditions without possibilities to adapt (cope), companion animals may develop stereotypies or compulsive disorders like, e.g., tail chasing, polyphagia, compulsively self-directed licking and/or biting the coat [ ] , or feather pecking in parrots [ ] . self-directed patterns can result in serious degrees of alopecia, lick granuloma, or even self-inflicted injuries (auto-mutilation) with a risk of infection. two main reasons underlie the development of arbs in our companion animals. first of all, a lot of companion animals are social species eager for social contact. in the human-companion animal bond, the need for social contact with either conspecifics and/or humans [ , ] can remain unfulfilled if owners work from nine to five, five days a week with the pet staying alone at home on a daily basis. on the other hand, a cat which is originally a solitary hunter with a complex dynamic social structure may start overgrooming or house soiling in the presence of another cat in the territory. such situations might occur in multi-cat households, in the presence of neighboring cats, and in in-stable grouped housing conditions in shelters [ , ] . secondly, most companion animals are species that are eager for mental and physical challenges on a daily basis. the lack of foraging opportunities, the appetitive phase of feeding behavior [ ] might be another reason for the possible development of arbs in companion animals. foraging is often regarded as a high priority behavior [ , ] , i.e., an internally motivated behavioral pattern that should be performed, or otherwise may induce a state of chronic stress, which may result in behavioral pathology like arbs as described in many other animal species [ ] [ ] [ ] . foraging patterns may include walking, running, jumping, nose pushing, digging, and overseeing the area, all active patterns that imply the daily need for physical exercise and mental challenges in most of our companion animals. nonetheless, our pets mostly, if not always, get their food for free with minimal foraging challenges, except for going out - times a day. for some individuals (and especially some dog breeds, e.g., malinois, border collies, and pit bull terriers) [ , ] , situations and contexts with limited challenges can make them more vulnerable to the development of arbs. as well as arbs, other problematic behavior may develop in our companion animals, and the prevalence of some of this behavior is even higher than that of arbs, for example excessive interspecific and/or intraspecific aggression, fear, and anxiety. at what moment, and which type of problem behavior may develop, depends on the intermingled factors of, e.g., genetics, early life experiences (maternal-child bonding, weaning, socialization [ ] ), daily environment, and multiple factors in and around the human-companion animal bond. the history of breeding animals goes back to a time when humans and animals shared each other's habitat. dogs originally have been selectively bred to support human needs, such as hunting, herding, obedience, guarding, rescuing, and for companionship. this artificial selection has generated a large number of dog breeds, displaying a large variation of behavior, size, head shape, coat color, and coat texture [ , ] . unfortunately, in the last years, intensive selection for extreme looks and a narrow gene pool of many breeds has interfered in the genetic make-up of dogs, leading to unfavorable anatomy (extreme large, or extreme "teacup" small), and genetic predisposition to numerous health, welfare, and behavioral problems [ ] . over inherited disorders and traits have already been described in the domestic dog [ ] . one type of dog with a distinct dysmorphology is the brachycephalic dog. brachycephalic dogs are characterized by a large head and round face due to a shortened muzzle, a high and protruding forehead, and widely spaced large eyes. these facial features fit the concept of baby schema ("kindchenschema") proposed by konrad lorenz [ ] . infantile (cute) faces are biologically relevant stimuli for rapidly and unconsciously capturing attention and eliciting positive or affectionate behavior, including the willingness to care [ ] . the appeal of brachycephalic animals has led to specimens that are the so-called "over-typed" dogs and cats with a too short nose, excessively protruding eyes, too straight angulations, etc. breeding animals with this type of severe skull and muzzle abnormalities leads to physical and physiological hardship and limits their natural behavior [ , ] . this violates their integrity and is a big risk for their welfare. selectively breeding animals in order to express specific traits does not only alter existing animals, but also creates new ones, turning animals into an instrument for human use [ ] . the bambino sphynx cat is an example of so called "mutant breeding", where breeders deliberately stack in two steps the recessive inheriting mutations, which leads to hairlessness in sphynx cats, on the dominant inheriting (lethal) mutation responsible for the shortened legs of the munchkin cat. the lack of hair in combination with short legs interferes with the normal physiology of the cat with regard to the manner of movement, thermoregulation, and skin health. one may argue that artificial selection in exchange for money, status, or aesthetic reasons violates the animal's dignity and integrity [ ] . conclusively, artificial selection for excessive traits can have direct consequences for individual health and welfare, may obstruct and prevent a pet from fulfilling its behavioral needs, and conflicts with the current moral way of thinking on animal dignity and integrity. pet animals are not only perceived in %- % as family members or partners but are almost treated like humans. in one study, up to % of owners agreed with the statement "my dog is more important to me than any human being" [ ] . this kind of behavior is the result of the attribution of human cognitive processes and emotional states to animals, such as feelings of happiness, love, or guilt. people believe that animals have awareness, thoughts, and feelings. this behavior is called anthropomorphism, personification, or humanization and can also be applied to plants, gods, or objects. anthropomorphism appears to be caused by the perceived similarity between humans and animals and the extent to which people have developed an affectionate bond with their dogs and cats [ ] . human empathy provides the basis for the attribution of empathy to other animals, as well as attributions of the communicative ability of other animals [ ] . anthropomorphistic behavior can be harmless, such as talking to pets, which many owners do and one of the reasons for this may be the unique ability of humans to recognize facial expressions. talking to pets is also found to be linked to social intelligence [ ] . however, it can lead to animal welfare problems when the feelings of owners no longer match the needs and the intrinsic values of their animal. examples of this are designer dog clothes, animal perfumes, and jewelry, thought the use of protective coats in colder climates for small, short-haired breeds, e.g., chihuahuas, is considered useful. the large number of obese pets can also be partly attributed to anthropomorphism. studies from north america, europe, and australia to determine what proportion of animals, mainly dogs, are overweight or obese reported prevalences of between %- % [ ] . in , an estimated % of cats and % of dogs in the usa were overweight or obese [ ] . when the owner takes a treat with coffee, it is believed that the dog should also get it. even chocolate treats are given, when these are potentially fatal for dogs and cats. this also applies to a good meal that is shared with the pet. dog owners who did not consider obesity to be a disease, maybe because the facial features of their pets fit the concept of baby schema [ ] , were more likely to have obese dogs [ ] . an often-unrecognized risk for pets is reverse zoonotic disease transmission, the so-called zooanthroponosis. a review on this subject reported articles dealing with human to animal disease transmission [ ] . most of the articles dealt with bacterial pathogens but also viral, parasitical, and fungal pathogens were studied in these publications. animals reported to have been infected or inoculated with human diseases included wildlife, livestock, companion animals, and other animals or animals not explicitly mentioned. the majority of the studies focused on human to wildlife transmission with an emphasis on mycobacterium spp. for companion animals, mrsa-infection was especially reported but m. tuberculosis, influenza a, and candida albicans were also discussed. for all groups of animals, microsporum spp. and trichophyton spp. were identified as infectious agents originating from humans [ ] . recent publications report a different kind of zooanthroponosis: the transmission of high-risk, multidrug-resistant pathogens from humans to animals [ ] . a major issue mentioned is the transmission of high-risk clones of extended-spectrum beta-lactamase (esbl) producing bacteria including escherichia coli, enterobacter cloacae, and klebsiella pneumonia [ , ] . the transmission of carbapenem-resistant ndm- producing e. coli from previously hospitalized humans to dogs has also been suggested [ ] . transmission of hospital acquired antibiotic resistant bacteria from human patients to their pets has been confirmed, such as the vim- producing pseudomonas aeruginosa st strain in brazil [ ] . this increased transmission of high-risk multidrug-resistant pathogens from humans to animals was related to the closer relationships between humans and companion animals. some authors doubt the generalized pet-effect on human mental and physical health because of conflicting results that are prevalent in this area of science and the lack of publication of negative results [ , [ ] [ ] [ ] . the majority of research evidence was also considered inconclusive due to methodological limitations such as reliance on self-reports, small sample sizes that may not be representative of the general population, homogeneous populations, varying research designs, narrow range of outcome variables that were examined, and the use of cross-sectional designs that do not consider long-term health outcomes [ ] [ ] [ ] . other studies found for example that pet ownership was associated with a higher incidence of heart attacks and readmissions in heart attack patients instead of a lower incidence [ ] or that pet owners had higher diastolic blood pressure than those without pets [ ] . müllersdorf ( ) showed that pet owners had better general health but suffered more from mental problems such as anxiety, insomnia, and depression, than those who did not own pets [ ] . other studies failed to support earlier findings that pet ownership is associated with a reduced use of general practitioner services [ ] or psychological or physical benefits on health for community dwelling older people [ ] . negative effects of pet ownership include dog and cat bites or scratches, the spreading of disease (zoonoses), and fall injuries, caused by falling or tripping over dogs and cats [ ] . allergic reactions may be a consequence of animal contact and affect %- % of individuals (often genetically) predisposed [ ] . allergies relating to more uncommon pets such as fish, birds, and amphibians seem to be increasing in prevalence [ ] . other studies prove that pet ownership in early life did not appear to either increase or reduce the risk of asthma or allergic rhinitis symptoms in children aged - years. therefore, advice to avoid or to specifically acquire pets for primary prevention of asthma or allergic rhinitis in children should not be given [ ] . there are also less-positive effects that pets can have on health. more excessive forms of anthropomorphism became clear in our study for the presence of zoonotic parasites in healthy dogs and cats. fifty percent of owners allow pets to lick their faces. sixty percent of the pets visit the bedroom; - % (dogs-cats) are allowed on the bed, and - % (dogs-cats) sleep with their owner in bed. six percent of pets always sleep in the bedroom. of the cats, % are allowed to jump onto the kitchen sink [ ] . this means that in addition to the detected zoonotic parasites (the hazard), there was a significant potential exposure to these pathogens. in addition to parasites, other pathogens such as bacteria, viruses, and fungi can also be transmitted by animals by direct contact through biting, licking, scratching, sneezing or coughing, handling pets or their body fluids or secretions and by indirect contact through contaminated bedding, food, water, or bites from an arthropod vector [ ] . not every individual will develop symptoms after being infected with a zoonosis. this is the result of various factors such as the causative pet species, housing, the degree of contact and contamination, the ability of a micro-organism to cause disease in humans and animals, but especially due to the degree of immunity of the recipient. in order to assess the risk of disease transmission from pets it is important that the nature and frequency of contacts between pets and their owners or other people are evaluated [ ] . we traditionally know that young children (age < years), the elderly (age ≥ years), patients with an impaired immunity, and pregnant women that carry a fragile fetus are at more than average risk of becoming ill after an infection. moreover, they may have more severe disease, have symptoms for a longer duration, or develop more severe complications compared to other patients. young children (notably those aged - years) and some people with developmental disabilities often have suboptimal hygiene practices or higher risk contact with animals which further increases risk [ ] . in children, hand-to-mouth behavior is part of their natural development and they mouth their fingers and other objects. in a meta-analysis, the average indoor hand-to-mouth frequency ranged from . to . contacts/hour and the average outdoor frequency ranged from . to . contacts/hour. the lowest value was attributed to the -to -year-olds and the highest to the -to < -month-olds [ ] . fifteen percent of dog owners and % of cat owners always wash their hands after contact with the animals [ ] . in addition, due to improved healthcare in recent decades, the group of immunocompromised patients has increased sharply. this includes, for example, patients with diabetes, post-splenectomy, after placement of implants and patients being treated with chemotherapy or immunosuppressants. the risk groups are also referred to as yopis (young, old, pregnant, and immune suppressed). patient surveys and epidemiological studies suggest that the occurrence of pet-associated zoonotic disease is low overall [ ] . many of these pathogens are not reportable and presumably underdiagnosed or not recognized by family doctors due to the general, mostly flu-like, symptoms. therefore, any reported frequency of such infections is likely underestimated. to get a better picture of zoonotic risks, a risk analysis is required where a risk score can be calculated using exposure, contagiousness of the infection, and its consequences in the human. in addition, the disease burden of an infection for the population can be calculated and expressed in disability adjusted life years (dalys). this quantifies the health loss based on two components: the life years lost due to premature death and secondly the proportional loss of quality of life as a result of the disease. there is a trend towards closer physical contact between owners and their pets or their environment which poses an increased risk of transmission of zoonotic pathogens. these trends (a general direction in which something is developing or changing) will be explained. our publication [ ] showed that a high percentage of pets were allowed in the bedroom and in bed, with % always sleeping in bed with their owner. is that a problem? already from a hygienic point of view, it is not advisable to sleep with animals or take them into bed. they do not pay attention to where they are walking outside and do not wipe their feet after arriving home. dogs like to roll in carcasses and both dogs and cats regularly lick the anus and thereafter the fur. in a pilot study with healthy dogs and healthy cats that slept with their owners, we tested % of the dogs ( ) and % of the cats ( ) positive for enterobacteriaceae on the fur or footpads. fleas and flea larvae were found on % of pets [ ] . as a result of our publication, chomel investigated in whether transmission of infections by sleeping with pets and licking the face could be found in literature. he reported that also in the usa, france and the uk, a relatively large number of pets slept in bed with their owner ( %- % of dogs and %- % of cats) [ ] . similar results of sleeping with pets were also reported from canada ( % of pets slept with children), the czech republic ( %), and qatar ( . %) [ ] [ ] [ ] . chomel found bacterial infections such as yersinia pestis (plague), bartonella henselae (cat scratch disease), methicillin-resistant staphylococcus aureus, and sometimes fatal bite wound infections such as capnocytophaga canimorsus and pasteurella multocida. furthermore, parasite infections such as cheyletiella spp. were reported. feline cowpox is a rare viral infection, but it can be transferred to the human after direct contact. both animals and humans reveal local exanthema on arms and legs or on the face. in most cases the disease is self-limiting, but immunosuppressed patients can develop a lethal systemic disease resembling smallpox [ ] . it can be concluded that, although uncommon with healthy pets, the risk of transmission of zoonotic agents by close contact between pets and their owners through bed sharing is real and has even been documented for life threatening infections such as plague [ , ] . although pets do not transmit arthropod-borne diseases to people (e.g., lyme borreliosis, ehrlichiosis, anaplasmosis), they do bring zoonotic disease vectors such as ticks and fleas, in close proximity to people, e.g., when they are sleeping with their animals [ ] . while fleas are considered a vector of bartonella henselae (the causative agent of cat scratch disease) tickborne diseases are reported as increasing as ticks expand their ranges [ , ] . with an estimated , cases a year, lyme borreliosis is responsible for the largest disease burden of any vector-borne disease in the european union [ ] . another increased risk associated with close contact with fur is when it is contaminated with zoonotic parasite eggs. especially with echinococcus multilocularis (fox tapeworm) or e. granulosus (hydatid worm, or dog tapeworm). these eggs are immediately infective and may cause serious health problems in the human, many years post infection [ , ] . despite a low prevalence of infectious (embryonated) eggs of toxocara spp. on dog's fur, the potential zoonotic risk should not be disregarded [ ] . the same risk is applicable for the persistence of sporulated toxoplasma gondii oocysts in dogs' fur [ ] . in relation to this, it is noteworthy that many publications report striking increases of ringworm, a common zoonotic fungal skin infection in mainly children caused by microsporum spp., trichophyton spp. or arthroderma spp., where the presence of pets is always mentioned [ ] . however, nowhere has it been suggested that close contact with infected pets in bed increases the infection risk [ , ] . rodents or rabbits are mainly infected with t. mentagrophytes, while m. canis is primarily found in dogs and cats. infection occurs by direct or indirect contact with infected hair, scales, or materials. infected animals may be asymptomatic carriers without clinical signs. examples are % m. canis carriage in a study of european cats [ ] , % in suspected brazilian cats [ ] , and the isolation of t. mentagrophytes dermatophytes from % of clinically healthy rabbits and % of guinea pigs in dutch pet shops [ ] . licking the face of humans by mainly dogs is an expression of their naturally submissive, positive social behavior. the owner is recognized by the dog as the dominant superior in the ranking. in a pack of dogs, submissive dogs lick their dominant counterparts at the corners of the mouth from a typical submissive attitude [ , ] . owners apparently allow this as a token of affection from their pet. such behavior is more common in young animals and has been considered as attention-seeking or care-soliciting gestures. it indicates to the owner the strength of the social bond between dogs and people [ ] . licking or nudging of veterans by service dogs may help take their mind off any negative thoughts, emotions, or memories that they might be experiencing [ ] . on the internet, many images can be found of mainly dogs, but also cats and even rats licking their owner's face. various studies show that around %- % of owners allow this [ , ] . the question is whether this is harmful due to the potential transmission of infections. the review by chomel ( ) shows in the literature that infections, especially pasteurella spp. and capnocytophaga canimorsus, were reported to have transmitted to humans by dogs, cats, kittens, and rabbits [ ] . pasteurella multocida meningitis has been reported in infants where % had been exposed directly or indirectly to the oropharyngeal secretions of household dogs or cats through licking or sniffing [ ] . zoonotic transmission via this route is also assumed for various other pathogens such as gastric helicobacter spp. [ ] , periodontal pathogens [ ] , and bartonella henselae the etiological agent in cat scratch disease. the b. henselae bacteria may cause ocular complications, including parinaud oculoglandular syndrome, a severe eye infection. the route of infection is unknown, although direct conjunctival inoculation, most likely with infected flea feces, seems to be most plausible [ ] . knowing, however, that b. henselae is present in up to % of cat saliva [ ] , it is more plausible that salivary fluid could be rubbed directly into the eye from the skin after been licked by a cat. there are several anecdotal reports of infections in mainly young children that were transmitted by being licked. one recent example is an -month-old baby that presented with fever and preseptal cellulitis with purulent discharge. the causative agent was surprisingly corynebacterium bovis, a bacterium that is normally found in bovine mastitis. it became clear that the dog was frequently allowed to lick the baby's face and was fed on raw meat [ ] . there is an ineradicable belief among a large part of the public that the licking of human wounds by dogs can disinfect them and that the saliva thereby has healing properties [ ] . in addition, it is regularly reported that a dog's tongue is believed to be sterile. this is of course not the case and the oral flora of a dog comprises hundreds of species (including pathogenic) bacteria, fungi, and viruses [ , ] . various wound healing saliva components have indeed been demonstrated in human and animal studies [ , ] . the bactericidal effects of male and female dog saliva facilitate the hygienic function of maternal licking of the mammary and anogenital areas by protecting newborns from fatal coliform enteritis caused by e. coli and neonatal septicemia caused by streptococcus canis. however, the saliva is only slightly, and non-significantly, bactericidal against wound bacteria such as coagulase positive staphylococci and pseudomonas aeruginosa [ ] . capnocytophaga canimorsus and pasteurella multocida are common commensals in the oral cavity of dogs, cats, and other species [ , ] . transmission has been reported after the licking of mucous membranes or open wounds [ ] [ ] [ ] . in patients at high risk, severe wound infections, sepsis, disseminated intravascular coagulation, or death can occur. patients with immunodeficiency, splenectomy, or alcohol dependence are at a particularly increased risk of infection with c. canimorsus [ ] . even immunocompetent persons who have been licked by a dog can develop fatal sepsis [ ] . a further negative effect of companion animal ownership is, of course, accidents inflicted on humans by these animals. these accidents can involve tripping over a cat or being dragged by an enthusiastic dog, but in literature, most evidence points towards biting and scratching incidents. dog biting and cat scratching incidents can cause physical health problems both at the time of infliction but also afterwards by triggering trauma-related secondary infections. dog biting incident reports are numerous, and numbers vary from country to country. in the uk, . dog bites per population per year were reported [ ] , while a commission in the netherlands reported in , , bite accidents per year in a population of million ( events per inhabitants) [ ] . children are especially vulnerable to dog bites. the majority of dog bites occurred in children years of age or younger ( . %) and almost all ( . %) of the dogs were known to the children [ ] . recently, a systematic review has been published that analyzed more than , bites from the literature of the past years about the risk of bites relative to specific breeds of dogs, combining bite incidence with bite severity [ ] . the analysis by breed revealed that pit bulls were responsible for the highest percentage of reported bites across all studies ( . %), followed by mixed breed ( . %), and german shepherds ( . %). dog bite incidents can result in medical treatment, hospitalization and even death. in the netherlands it was calculated that from the , dog bite victims per year, around , seek medical attention and are hospitalized [ ] . between % and % of dog bites become infected and complications become more severe when infection occurs. more than species of bacteria have been isolated from bacterial infections of dog bites, suggesting that most oral flora of dogs have the potential to be pathogenic [ ] . the top pathogens found are pasteurella, staphylococcus, and streptococcus. in literature, specific attention is given to wound infections caused by capnocytophaga canimorsus, because this bacterium is seen as the relatively deadliest pathogen. it was suggested that only % of dog bite wounds contained capnocytophaga spp. [ ] while others reported infection percentages of . % [ ] . wound infection with this bacterium can lead to severe complications like septicaemia, meningitis, osteomyelitis, peritonitis, endocarditis, pneumonia, purulent arthritis, and disseminated intravascular coagulation. c. canimorsus septicaemia has been associated with % mortality. the true number of c. canimorsus infections is probably largely underestimated due to the fastidious growth of the organism. however, infected dog bites in predisposed persons should be taken seriously especially after splenectomy [ ] . cat bite incidents occur less frequently. in only %- % of reported bite incidents in australia, cats are to blame. the long incisor teeth inflict less severe superficial wounds but because of the penetrating effect, joint and tendon infections more easily occur. in a review it is reported that %- % of cat bites become infected mostly by pasteurella multocida [ ] . the bacterium bartonella henselae can also be transmitted by cats through biting incidents but the transmission of this bacterium is much more related to a cat scratch accident. even less frequently reported animal biting incidents are those inflicted by rodents ( % of cases in australia). these bites have an infection rate of approximately %. this could result in rat bite fever in humans, an infection with streptobacillus monoliformis or spirillum minus, characterized by the triad of fever, rash, and arthritis [ ] . cat scratch disease (csd) was first described in a french boy in and is a common, often self-limited, disease that usually presents as tender lymphadenopathy caused by bartonella henselae [ ] . the cat is considered the primary reservoir for this bacterium, with infected fleas and ticks serving as vectors and humans and dogs as accidental hosts. vector transmission of this bacterium occurs via two primary routes: inoculation of bartonella contaminated arthropod feces via animal scratches, most often cat scratches, or by self-inflicted contamination of wounds induced by the host scratching arthropod bites [ ] . immunocompromised human hosts (kidney transplant patients or patients with hiv) are especially susceptible to infection. in these individuals, the disease may be present as a more disseminated form with hepatosplenomegaly, meningoencephalitis, or angiomatosis [ ] . an increasing number of pocket pets and exotic pets are kept by humans. numbers of ornamental birds in europe are estimated as million, fish tanks . million ( million ornamental fish), small mammals million, and reptiles million [ ] . these animal species can also be a source of many zoonotic diseases, especially in young children and immunocompromised individuals. most cases of these conditions are not serious, and deaths are very rare but some of these diseases can be life threatening, such as rabies, rat bite fever infections, and plague [ ] . however, there is also a trend to keep more unusual exotic animals, legally or illegally. these are "wild" animals that are kept in the home such as bats, foxes, skunks, raccoons, meerkats, prairie dogs, kinkajous, sloths, monkeys, apes, prosimians (mammals), parrots, mynah birds, finches (birds), crocodiles, turtles, tortoises, lizards, snakes (reptiles), frogs, toads, newts, salamanders (amphibians), fish, eels, rays (fish), crabs, crayfish, snails, insects, spiders, and millipedes (invertebrates) [ ] . even fruit bats are kept as pets [ ] and it is known that bats harbor a higher proportion of zoonoses than all other mammalian orders, including rabies-like viruses that are highly pathogenic for people. [ ] . another example of a zoonosis is monkeypox following the importation of prairie dogs in the usa [ ] . most reptiles and amphibians such as turtles, lizards, and frogs carry salmonella bacteria in their gut, in most cases without visible signs of infection. the infection may cause symptoms of sickness, diarrhea and fever in humans [ ] . zoonotic transmission of salmonella infections causes an estimated % of salmonellosis annually in the united states. in cases involving pet turtles, almost half ( %) of infections occurred in children younger than years [ ] . salmonella infections are often transferred by feeder rodents [ ] and outbreaks highlight the importance of improving public awareness and education in countries who receive imported reptiles [ ] . it is advised to exclude reptiles, amphibians, rodents, exotic species, baby poultry, and raw animal-based pet food items from the households of patients at high risk. in lower risk households, an understanding of the risk of salmonellosis and other pet-associated zoonoses and preventive hygiene measures is needed. the pet trade in general, with its high turnover and diversity of available species, creates a reservoir for pathogens originating from all over the globe. reptiles account for approximately % of live animal shipments imported to the united states [ ] . importation of live reptiles and amphibians for commercial purposes is for a great part unregulated at eu level except cites and customs regulations [ ] . in a risk assessment study, the five pathogens with the highest public health risk caused by the import of exotic animals were salmonella spp., crimean-congo hemorrhagic fever virus, west nile virus, yersinia pestis, and arenaviruses. the risk via legally imported animals was considered low, but substantial for illegally imported animals due to the unknown health status of the animals [ ] . avoiding exposure to exotic pet pathogens in the home is difficult and best achieved by not keeping them in the first place. otherwise, it is advised to always wash the hands immediately and thoroughly after contact with exotic pets and after handling raw (including frozen or defrosted) mice, rats and chicks. children should be supervised so that they do not put their mouths close to or kiss exotic animals. reptiles and other animals should be kept out of rooms in which food is prepared and eaten. the number of abandoned and homeless dogs and cats in europe is estimated to be over million. countries with more than million abandoned animals are italy, romania, russia, and ukraine [ ] . there is an increasing trend to rescue and import dogs from countries with stray animal problems, in europe often from southern or eastern europe and in the us from puerto rico, the dominican republic, mexico, the middle east, turkey, china, and korea [ ] . many charities and independent groups are involved to rescue dogs and seek adoption in more animal-friendly countries [ ] . new owners primarily choose to adopt from abroad based on a desire for a particular dog they had seen advertised and on concern for its situation, while some were motivated by previously having been refused dogs from local rescues [ ] . in the usa % of all household dogs were neutered and today there are no longer enough dogs being born in the usa annually to replace the approximately million dogs that die each year. developing countries have hundreds of millions of street dogs available for export, for example egypt has an estimated million, india, million, and afghanistan, million [ ] . in , more than a decade ago, the centers for disease control (cdc) estimated annual dog imports at around , . today the number of imported dogs is estimated to be more than a million a year [ ] . imported dogs are reintroducing diseases and parasites that were previously eliminated in the usa [ ] . in the usa new and lethal strains of distemper and canine influenza as a result of imported rescue dogs were reported as well as canine brucellosis, rabies, and vector-borne diseases like ehrlichiosis, heartworm, babesiosis, and leishmaniasis [ , ] . in many southern european countries, there is also a risk of exposure to diseases not encountered in the northern, importing, countries. animals could be infected with anaplasma phagocytophilum, babesia canis, brucella canis, borrelia burgdorferi, dirofilaria immitis (heartworm), dirofilaria repens (subcutaneous worm), echinococcus multilocularis (fox tapeworm), echinococcus granulosus (hydatid or dog tapeworm), ehrlichia canis, hepatozoon canis, leishmania infantum, linguatula serrata (tongue worm), onchocerca lupi, rabies, rickettsia conorii, strongyloides stercoralis, and thelazia callipeda. except b. canis, e. canis, and h. canis, the infections are zoonotic and regularly reported [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . most of these are transmitted by ticks, sand flies, or mosquitoes that are non-endemic in the receiving countries, but a reservoir of infections has been created and the risk is that vectors will become present as result of climate change. [ ] . animals that are infected with rabies or echinococcus spp. may infect people directly. the importation of dogs from endemic, predominantly mediterranean, regions to northern europe, as well as travelling with dogs to these regions carries a significant risk of acquiring an infection. pet owners are therefore advised not to travel with dogs and to seek the advice of their veterinarian prior to importing a dog from an endemic area or travelling to such areas [ ] . for this reason, esccap developed maps on their website featuring european countries and regions with advice on endemic parasites, diseases and recommended treatments when travelling with dogs [ ] . a three-year european union funded project entitled callisto (companion animal multisectorial interprofessional and interdisciplinary strategic think tank on zoonoses), has investigated zoonotic infectious diseases transmitted between companion animals and humans and food producing animals [ ] . the committee advised that special attention should be given to stray cats and dogs. stray dogs, in particular, may pose serious health and welfare problems for humans and animals [ ] , including the transmission of zoonotic diseases such as rabies. consideration should be given to controlling companion animal movement between areas of the eu endemic for particular zoonoses and areas that are not currently endemic for that disease. reliable figures are not available, probably because of the fact that many voluntary organizations are responsible for saving and transporting these rescue animals. such data are essential in order to be able to quantify the actual risks of zoonotic diseases attributable to companion animals and to develop sustainable interventions to prevent transmission to humans and livestock [ ] . as long as there are no official guidelines, to prevent the spread of (zoonotic) diseases to new countries, the time, expense, disease risk, and the implications of adopting a dog from abroad should all be carefully considered before importation. as an alternative, the conditions for native dogs could be improved by supporting local charities to organize neutering campaigns and rehoming programs, to build local animal shelters and to improve attitudes towards dogs and their living conditions [ ] . cats and dogs harbor the enteric nematodes toxocara canis and toxocara cati, and cats are the final host for the protozoal parasite toxoplasma gondii. these parasites can be transmitted to humans because they have an oral-fecal transmission cycle. humans can be infected by ingestion of infective toxocara spp. eggs or toxoplasma oocysts from contaminated soil (gardens, sandpits, and playgrounds) [ , ] . a recent meta-analysis of data from published records indicates that public places are often heavily contaminated with a pooled global prevalence of toxocara eggs of % [ ] . both parasites are considered by cdc as part of five neglected parasitic infections. these infections are considered neglected because relatively little attention has been devoted to their surveillance, prevention, and/or treatment. the diseases that they cause have been targeted as priorities for public health action based on the number of people infected, the severity of the illnesses and the ability to prevent and treat them [ ] . tens of millions of people worldwide are estimated to be exposed to, or infected with, toxocara spp. and recent findings suggest that the effect of toxocarosis on human health is increasing in some countries. almost one fifth ( %; . billion individuals) of the world's human population is seropositive to toxocara. the highest seroprevalence rates were found in africa (mean: . %) and the lowest in the eastern mediterranean region (mean: . %) [ ] . toxocara larvae migrate into the body of the human to several organ systems with a preference for the central nervous system (brain, eyes). human toxocarosis can manifest itself as syndromes known as visceral larva migrans, ocular larva migrans, neurotoxocarosis, and covert or common toxocarosis [ ] . asthma is one of the most common chronic respiratory diseases worldwide, with a negative impact on the quality of life and socio-economic status of patients. two decades ago, the first evidence was published that suggested that toxocara infection is a neglected risk factor for childhood asthma [ ] . the finding that children infected with toxocara spp. are more likely to have asthma compared to non-infected children was recently confirmed in a systematic review and meta-analysis [ ] . cognitive or developmental delays in children or young adults who become infected is of particular concern. toxocarosis appears to be associated with decreased cognitive function [ , ] . the annual toxoplasma oocyst burden measured in community surveys has been reported as up to oocysts per square foot ( per square meter) and is greater in areas with loose soil, that cats like to use to cover their feces in gardens, children's play areas, and especially sandboxes, also called sandpits and sand piles [ ] . because a single oocyst can possibly cause infection, this oocyst burden represents a major potential public health problem. an estimated one third of the world's population harbor anti-toxoplasma antibodies. due to keeping pigs indoors, more education and awareness, the prevalence of the disease in the usa and europe declined by % over the last decades [ ] . during an acute invasion of toxoplasma parasites there is mild to major tissue damage without clinical symptoms (latent toxoplasmosis). the most important form is congenital toxoplasmosis when a woman receives her first exposure to toxoplasma during pregnancy. in early pregnancy, this can lead to abortion or to malformations that are not compatible with life shortly after birth. congenital infections may also be characterized by mental retardation and ocular defects. acquired infection after birth may result in clinical symptoms such as lymphadenitis, fever, and malaise and probably leads to a clinically symptomatic disease state more frequently than the congenital condition, with an estimated incidence of % of all ocular toxoplasmosis cases [ ] . playing in a sandbox is also found to be a predominant risk factor for s. typhimurium salmonellosis in children aged - years [ ] . this can be the result of fecal contamination of the sand by dogs and cats that have been fed raw meat (see section . . .). young children are especially at risk as they put their hands or other objects in their mouths every - min [ ] . it has also been reported that children ingest a median of mg of soil per day and that one child consumed - g of soil per day on average [ ] . it is therefore advisable not to let children play in public places or on playgrounds with loose sand, but only in sandboxes that can be covered. furthermore, washing hands after playing outside is important and fingernails must be trimmed to prevent sand being left behind. in this context, a strange trend can be observed as young children play in mud baths on june "as a way to connect and celebrate the natural joys of playing in the mud". this international mud day originated in and was initiated by an australian pedagogue who had observed this phenomenon during a visit to nepal [ ] . if the origin of the soil needed for producing the mud is unknown, there is of course an infection risk for the above discussed parasites. to prevent the transmission of zoonotic diseases from pets, risk analysis is of great value. this starts with an assessment of the potential zoonoses in an area, depending on the endemicity (the hazard, h). hazard characterization also includes prevalence in animals (the reservoir), virulence for man, transmission routes, and survival of the agent in the environment. the second step is exposure assessment (e). who is exposed to the potential hazard and for how long or how often? how much of the potential pathogen is needed to become a health risk? this inevitably is directly related to human behavior in relation to their pets. the third step is to assess the impact of getting infected (i). how serious is the disease, what is the chance of complications, and what economic consequences may be expected (e.g., labor hours lost)? each of the parameters can be ranked in classes from negligible to the most serious possibility. ranking is based on literature data, own observations (measuring), or experts' opinions. the final risk assessment can be achieved by multiplying the outcome of hazard characterization, exposure assessment and impact (h × e × i = a number). the outcome can be compared with other zoonotic agents and a ranking of significance can be made [ ] . there is one important parameter to reduce the risk of contracting a zoonotic infection that can directly be influenced, which is exposure. recommendations are particularly targeted to households with very young children, the elderly, pregnant women, or immunocompromised patients. they are based on reducing exposure to hazards and involve four categories of advice (table ) . table . recommendations to prevent the transmission of zoonotic pathogens from pets [ , ] . • washing the hands thoroughly -after animal contact, at least before eating and drinking and before preparing food or drinks -after handling raw pet food -after handling pet habitats or equipment -after cleaning up feces -after removing soiled clothes or shoes reflecting on the changed human-companion animal bond, it can be concluded that pets undoubtedly have a positive effect on human health. conversely, the human-pet bond seen nowadays is facing many challenges, putting pet welfare under more pressure due to issues such as anthropomorphism, which mainly results in obesity, breeding on extreme appearance rather than health, behavioral problems connected to unfulfilled species specific mental and physical needs, and the provision of inadequate food because owners mistakenly think they feed more naturally. with regard to the negative effects of pets this article attempts to give an impression of increasing trends in the human-companion relationship that can be observed in society, which appears to increase the risk of transmission of infection between pets and humans. it is mainly a consequence of the increasing contacts between humans and pets and with pathogens secreted by animals in the shared environment. more than out of every known infectious diseases in people can be spread from animals, and out of every new or emerging infectious diseases in people come from animals [ ] . the recent pandemic of the covid coronavirus (sars-cov- ), that may be originated from bats, is a good example of a recent emerging zoonotic infectious disease. a few cats and dogs have tested positive but are not considered as a source of infection for people. the proportion of zoonotic human disease that is attributable to pets is largely unknown. reports about the frequency of such infections are likely underestimated [ ] ; however, the risk of infection is relatively small for many zoonoses and the severity of the disease is often limited. a person's age and health status may affect their immune system, and thereby increase his or her chances of getting a disease from animals. pregnant women should avoid contact with pet rodents, reptiles, cat feces, and raw meat to prevent infection of the unborn child, abortion, or birth defects. if symptoms occur in immunocompetent, non-pregnant persons between and years of age, these are mainly of a general nature such as diarrhea or flu-like symptoms. physicians do not regularly ask about the presence of pets or pet contact, nor do they discuss the risks of zoonotic diseases with patients, regardless of the patient's immune status, which means that many cases of zoonotic diseases go undiagnosed. the general public and people at high risk of pet-associated disease are not aware of the risks associated with high risk pet practices or recommendations to reduce them. since unfamiliarity with hazards reinforces fear, communication plays an important role in this. veterinarians play a key role in education regarding risk reduction by giving advice about responsible pet ownership and the required preventive hygiene. healthcare providers such as family doctors, school doctors, and pediatricians can also provide information about safe pet ownership. physicians should ask as part of the medical history about eventual contact with pets, particularly with patients at high risk [ ] . the "one health" initiative aims to reduce this professional gap between vets and physicians [ ] . when giving recommendations to prevent zoonotic transmission, one of the often-made remarks is that people nowadays are already too hygienic. it is assumed that there is a protective influence of postnatal infection and that it might be lost in the presence of modern hygiene. this belief is based on the hygiene hypothesis that was formulated in [ ] . it was observed that hay fever in young adults was inversely related to the number of siblings in their family. this hypothesis focused exclusively on allergic conditions as a result of the modern way of life and the assumption that modern hygiene was reducing contact with bacteria. another view is that some chronic inflammatory disorders have increased over the last decades as a result of decreased frequency of infections due to pathogenic organisms [ ] . another related theory is the "old friends" mechanism that is based on the positive influence of gut parasites, non-pathogenic environmental bacteria (saprophytes, pseudocommensals), and gut commensals or microbiota. however, decreased exposure to these microorganisms is not the only reason for the increasing frequency of allergies and chronic inflammatory disorders in industrialized countries. nowadays there is more information about the immunological roles of skin, oral mucosa, and gut microbiota as well as helminths and the influence these have on the immune system [ ] . gut flora may be modified due to diet, obesity, hygiene, antibiotics, but also to psychological stress, vitamin d deficiency, and pollution [ ] [ ] [ ] . pollution also has a significant effect on the development of several respiratory problems and diseases. not only due to outdoor pollution such as fine dust, harmful solids, liquids, or gases [ ] , but also due to indoor molds as result of insufficient ventilation in energy efficient homes [ ] . finally, there is a clear increase in the allergen production of house dust mites and pollen leading to more exposure and sensitization in susceptible individuals [ ] . all together it must be clear that there is much more known about other causes of increasing allergies worldwide than simply excessive cleanliness as suggested in the hygiene hypothesis. regarding field infections with helminths such as trichuris trichiura in early life, these are associated with a reduced prevalence of allergies later in life and infants of helminth-infected mothers have a reduced prevalence of eczema. hookworm infections in developing countries are associated with a reduced prevalence of asthma [ ] . the rate of eczema in such countries was found to be about five times higher in infants whose mothers had never had helminths compared with persistent helminth-infected mothers [ , ] . helminths are nowadays even used under controlled conditions to stimulate immunity. examples are trichuris suis therapy for crohn's disease and necator americanus larvae to treat crohn's disease and other autoimmune disorders [ ] . there are no clinically apparent childhood infections found to be associated with protection from allergic disorders [ ] . it can even result in an opposite effect in the case of toxocara infection by children after soil contact. a recent meta-analysis showed that children infected with toxocara spp. are more likely to have asthma compared to non-infected children [ ] . this parasite and asthma both have elevated immunoglobulin e (ige) levels and eosinophilia in common. that means that precautions should be taken in children to prevent soil contact not only to prevent toxocara infection, but also to prevent acquired ocular toxoplasmosis. there is no need, therefore, to stimulate the contraction of pathogenic bacteria or helminths to achieve a healthy gut microbiota and to reduce allergic conditions. recommendations based on the hygiene hypothesis should preferably be based on results from controlled studies to prevent unintentional negative effects. it is both humans and companion animals who experience negative effects of a changed human-companion animal bond. the education given by vets to their clients should therefore also focus on preventing these negative health and behavioral effects. for instance, by giving science-based advice on feeding practices. in general, regulating authorities should encourage the development of enforcement criteria for breeding dogs and cats to reduce health and welfare risks. pets undoubtedly have a positive effect on human health and well-being, while owners are increasingly aware of pet health, welfare, and well-being. anthropomorphism, also resulting in behavioral problems and breeding on appearance rather than health, and trends such as keeping exotic animals and importing rescue dogs may result in an increased risk of contracting zoonotic infections. recommendations regarding responsible pet ownership, including normal hygienic practices, responsible breeding, feeding, housing, and mental and physical challenges conform the biology of the animal, are key in preventing such negative aspects of the human-animal bond. there is no need to stimulate unhygienic practices following the hygiene hypothesis. education can be performed by vets and physicians as part of the one health concept. the brewing storm one health initiative. available online: www.onehealthinitiative.com available online: www one health: the small animal dimension one medicine, one health, one world one health in history. in one health: the theory and practice of integrated health approaches cab international the global burden of bacterial and viral zoonotic infections the one health european joint programme (ohejp) raw pet food as a risk factor for shedding of extended-spectrum beta-lactamase-producing enterobacteriaceae in household cats companion animals as sentinels for public health wsava. one health initiative pet ownership and human health: a brief review of evidence and issues available online: www. avma.org/news/press-release/avma-releases-latest-stats-pet-ownership-and-veterinary-care report the european pet food industry report the european pet food industry people in the eu-statistics on household and family structures changing families in the european union: trends and policy implications applying ethological and health indicators to practical animal welfare assessment report on priorities for animal welfare research and development onderzoek naar de gezondheids-en welzijnsstatus van konijnen in de nederlandse gezelschapsdierensector. faculty of veterinary medicine animal wellbeing (paw) report. yougov and the people's dispensary for sick animals (pdsa) the human-companion animal bond: how humans benefit an essential component of "one health animal companions and one-year survival of patients after discharge from a coronary care unit pet ownership, social support, and one-year survival after acute myocardial infarction in the cardiac arrhythmia suppression trial (cast) stressful life events and use of physician services among the elderly: the moderating role of pet ownership effects of proximity between companion dogs and their caregivers on heart rate variability measures in older adults: a pilot study health benefits and health cost savings due to pets: preliminary estimates from an australian national survey indirect costs: extending the scope of economic value. in companion animal economics: the economic impact of companion animals in the uk presence of human friends and pet dogs as moderators of autonomic responses to stress in women effect of animal-assisted activity on balance and quality of life in home-dwelling persons with dementia effect of animal-assisted interventions on depression, agitation and quality of life in nursing home residents suffering from cognitive impairment or dementia: a cluster randomized controlled trial controlled clinical trial of canine therapy versus usual care to reduce patient anxiety in the emergency department canine visitation (pet) therapy: pilot data on decreases in child pain perception animal wellness magazine canine olfaction as an alternative to analytical instruments for disease diagnosis: understanding 'dog personality' to achieve reproducible results animal-assisted therapy and occupational therapy animal-assisted therapies for youth with or at risk for mental health problems: a systematic review process evaluation of animal-assisted therapy: feasibility and relevance of a dog-assisted therapy program in adults with military veterans and canine assistance for post-traumatic stress disorder: a narrative review of the literature the study of service dogs for veterans with post-traumatic stress disorder: a scoping literature review the influence of animals on the development of children companion animals and child/adolescent development: a systematic review of the evidence early exposure to dogs and farm animals and the risk of childhood asthma early exposure to cats, dogs and farm animals and the risk of childhood asthma and allergy non-communicable diseases: how can companion animals help in connection with coronary heart disease, obesity, diabetes and depression? in one health: the theory and practice of integrated health approaches cab international similarities between obesity in pets and children: the addiction model the financial costs, behaviour and psychology of obesity: a one health analysis association for pet obesity prevention use of starting condition score to estimate changes in body weight and composition during weight loss in obese dogs prevalence and risk factors for obesity in adult dogs from private us veterinary practices. intern a review of osteoarthritis and obesity: current understanding of the relationship and benefit of obesity treatment and prevention in the dog prevalence and risk factors for obesity in adult cats from private us veterinary practices. intern overweight in adult cats: a cross-sectional study raw diets for dogs and cats: a review, with particular reference to microbiological hazards evaluation of raw food diets for dogs alternative & raw food diets: what do we know? the vegan society. available online: www.vegansociety.com available online: www.wysong.net/cat-food amì vegetable pet food company assessment of protein and amino acid concentrations and labeling adequacy of commercial vegetarian diets formulated for dogs and cats available online: petfoodindustry.com:www.petfoodindustry.com/articles/ -the-importance-of-mycotoxinmanagement-in-dog-food?v=preview veterinary and pharmacological approaches to abnormal repetitive behaviour forms of stereotypic behaviour cab international stereotypic route-tracing in experimentally caged songbirds correlates with general behavioural disinhibition perseveration and stereotypy-systems-level insights from clinical psychology behavior problems of the dog and cat feather damaging behaviour in parrots: a review with consideration of comparative aspects neurophysiological correlates of affiliative behaviour between humans and dogs human interaction and cortisol: can human contact reduce stress for shelter dogs? stress and adaptation in cats (felis sylvestris catus) housed singly, in pairs and in groups in boarding catteries effects of density and cage size on stress in domestic cats (felis sylvestris catus) housed in animal shelters and boarding catteries stereotypies in carnivores: does pacing stem from hunting, ranging or frustrated escape? a concept of welfare based on reward evaluating mechanisms in the brain: anticipatory behaviour as an indicator for the state of reward systems to swim or not to swim: an interpretation of farmed mink's motivation for a water bath do the stereotypies of pigs, chickens and mink reflect adaptive species differences in the control of foraging? captivity effects on wide-ranging carnivores the effects of food-related cage enrichments on time budgets and abnormal behaviours in laboratory dogs (canis familiaris) behavioural differences among breeds of domestic dogs (canis lupus familiaris): current status of the breed differences in everyday behaviour of dogs the importance of early life experiences for the development of behavioural disorders in domestic dogs genome sequence, comparative analysis and haplotype structure of the domestic dog the genetics of canine skull shape variation online mendelian inheritance in animals, omia die angeborenen formen möglicher erfahrung face: mechanisms underlying human-animal relationships conformational risk factors of brachycephalic obstructive airway syndrome (boas) in pugs, french bulldogs, and bulldogs impact of facial conformation on canine health: corneal ulceratio expert statement. breedings designer cats like bambino sphynx cat reverse zoonotic disease transmission (zooanthroponosis): a systematic review of seldom-documented human biological threats to animals dermatophytes that infect animals and humans zooanthroponotic transmission of high-risk multidrug-resistant pathogens: a neglected public health issue highly tigecycline-resistant klebsiella pneumoniae sequence type (st ) and st isolates from companion animals sharing more than friendship-transmission of ndm- st and ctx-m- st escherichia coli between dogs and humans in a family zooanthroponotic transmission of drug-resistant pseudomonas aeruginosa is your dog empathic? developing a dog emotional reactivity survey on seeing human: a three-factor theory of anthropomorphism anthropomorphism, empathy, and perceived communicative ability vary with phylogenetic relatedness to humans canine and feline obesity: a one health perspective european dog owner perceptions of obesity and factors associated with human and canine obesity the impact of pets on human health and psychological well-being fact, fiction, or hypothesis? the state of research on human-animal relations: implications for human health exploring the differences between pet and non-pet owners: implications for human-animal interaction research and policy gaps in the evidence about companion animals and human health: some suggestions for progress the relationship between dog ownership, psychopathological symptoms and health-benefitting factors in occupations at risk for traumatization survival following an acute coronary syndrome: a pet theory put to the test pet ownership and risk factors for cardiovascular disease: another look aspects of health, physical/leisure activities, work and socio-demographics associated with pet ownership in sweden the impact of pet ownership on health and health service use: results from a community sample of australians aged to years pet ownership and health in older adults: findings from a survey of community-based australians aged - dogs and cats as environmental fall hazards management of allergies to animals allergy to uncommon pets: new allergies but the same allergens does pet ownership in infancy lead to asthma or allergy at school age? pooled analysis of individual participant data from european birth cohorts zoonotic parasites in fecal samples and fur from dogs and cats in the netherlands dog-human and dog-dog interactions of dog-owning households in a community in cheshire reducing the risk of pet-associated zoonotic infections a meta-analysis of children's hand-to-mouth frequency data for estimating nondietary ingestion exposure the zoonotic risks of sleeping with pets zoonoses in the bedroom pet husbandry and infection control practices related to zoonotic disease risks in ontario awareness of zoonotic diseases and parasite control practices: a survey of dog and cat owners in qatar. parasites vectors sharing bacterial microbiota between owners and their pets (dogs, cats) emerging and re-emerging zoonoses of dogs and cats. animals (basel) dog-associated risk factors for human plague two cases of human plague tickborne disease surveillance data summary impact of climate change in the epidemiology of vector-borne diseases in domestic carnivores vector-borne diseases and climate change: a european perspective hair coat contamination with zoonotic helminth eggs of farm and pet dogs and foxes human cystic echinococcosis: epidemiologic, zoonotic, clinical, diagnostic and therapeutic aspects. asian pac the presence of toxocara eggs on dog's fur as potential zoonotic risk in animal-assisted interventions: a systematic review dogs as possible mechanical carriers of toxoplasma, and their fur as a source of infection of young children kerion caused by the zoophilic dermatophyte trichophyton species of arthroderma benhamiae in a child. a new emerging pathogen of dermatomycoses in germany tinea capitis: main mycosis child. epidemiological study on years prevalence and characterization of microsporum canis carriage in cats epidemiological investigation and molecular typing of dermatophytosis caused by microsporum canis in dogs and cats prevalence and zoonotic risks of trichophyton mentagrophytes and cheyletiella spp. in guinea pigs and rabbits in dutch pet shops vries de, h. dominance in domestic dogs: a quantitative analysis of its measures dominance in domestic dogs revisited: useful habit and useful construct? an observational study of service dogs for veterans with posttraumatic stress disorder pasteurella multocida meningitis in newborns after incidental animal exposure gastric helicobacters in domestic animals and nonhuman primates and their significance for human health comparison of periodontal pathogens between cats and their owners ocular bartonellosis oral shedding of bartonella in cats: correlation with bacteremia and seropositivity an infant with a red and swollen eyelid dog licks man bacteriologic analysis of infected dog and cat bites defining the healthy "core microbiome" of oral microbial communities antimicrobial peptides with therapeutic potential discovery of the wound-healing capacity of salivary histatins antibacterial properties of saliva: role in maternal periparturient grooming and in licking wounds the presence of capnocytophaga canimorsus and capnocytophaga cynodegmi in companion animals in the netherlands meningitis caused by capnocytophaga canimorsus: when to expect the unexpected beware of dogs licking ears a bizarre postoperative wound infection pasteurella multocida meningitis in infancy (a lick may be as bad as a bite) being licked by a dog can be fatal: capnocytophaga canimorsus sepsis with purpura fulminans in an immunocompetent man how many people have been bitten by dogs? a cross sectional survey of prevalence, incidence and factors associated with dog bites in a uk community dog bites in perspective analysis of pediatric facial dog bites dog bite injuries to the face: is there risk with breed ownership? a systematic review with meta-analysis scoping decades of dog evidence: a scoping review of dog bite-related sequelae review article: animal bites: an update for management with a focus on infections cat scratch disease and other bartonella infections bartonella infections in cats and dogs including zoonotic aspects. parasites vectors cat-scratch disease diseases transmitted by less common house pets a review of captive exotic animal-linked zoonoses callisto (companion animal multisectorial interprofessional and interdisciplinary strategic think tank on zoonoses) final report host and viral traits predict zoonotic spillover from mammals multistate outbreak of monkeypox tiny turtles purchased at pet stores are a potential high risk for salmonella human infection in the valencian region, eastern spain. vector borne zoonotic dis flea market finds and global exports: four multistate outbreaks of human salmonella infections linked to small turtles, united states- outbreak of salmonella typhimurium associated with feeder rodents salmonella isolates from wild-caught tokay geckos (gekko gecko) imported to the u.s. from indonesia. vector borne zoonotic dis live exotic animals legally and illegally imported via the main dutch airport and considerations for public health esdaw (european society of dog and animal welfare) foreign rescue dogs breed health concerns, undermine adoptions points to consider when importing a rescue dog from abroad. factsheet importing rescue dogs into the uk: reasons, methods and welfare considerations it's now or never: stop dog trafficking now! national animal interest alliance naia federal register document compendium of animal rabies prevention and control available online: www.cdc.gov/importation/bringing-an-animal-into-theunited-states/egypt-dogs-temp-suspension.html imported non-endemic, arthropod-borne and parasitic infectious diseases in austrian dogs imported and travelling dogs as carriers of canine vector-borne pathogens in germany. parasites vectors rabies in europe: what are the risks? brucellosis outbreak in a swedish kennel in : determination of genetic markers for source tracing the first report of autochthonous non-vector-borne transmission of canine leishmaniosis in the nordic countries tongue worm (linguatula species) in stray dogs imported into the uk animal welfare foundation. taking your pets abroad, your guide to diseases encountered abroad three cases of imported eyeworm infection in dogs: a new threat for the united kingdom international dog travelling and risk for zoonotic onchocerca lupi strongyloides stercoralis infection in imported and local dogs in switzerland: from clinics to molecular genetics recent advances on dirofilaria repens in dogs and humans in europe. parasites vectors potential risk posed by the importation of ticks into the uk on animals: records from the tick surveillance scheme available online: www.esccap.org/travelling-petsadvice points to consider when importing a rescue dog from abroad veterinary and public health aspects of toxocara spp toxoplasma oocysts as a public health problem toxocara eggs in public places worldwide-a systematic review and meta-analysis neglected parasitic infections human toxocariasis-a look at a neglected disease through an epidemiological 'prism' human toxocariasis relationship between allergic manifestations and toxocara seropositivity: a cross-sectional study among elementary school children toxocara spp. infection and risk of childhood asthma: a systematic review and meta-analysis reduced cognitive function in children with toxocariasis in a nationally representative sample of the united states association between toxocariasis and cognitive function in young to middle-aged adults ocular toxoplasmosis: an update risk factors for salmonella enteritidis and typhimurium (dt and non-dt ) infections in the netherlands: predominant roles for raw eggs in enteritidis and sandboxes in typhimurium infections how much soil do young children ingest: an epidemiologic study celebrating international mud day dogs and transmission of infection to man, respected member of the family? healthy pets. available online: www zoonotic diseases. available online: www hay fever, hygiene, and household size microbes, immunoregulation, and the gut th dahlem conference on infection, inflammation and chronic inflammatory disorders: darwinian medicine and the 'hygiene' or 'old friends' hypothesis metabolic and metagenomic outcomes from early-life pulsed antibiotic treatment current understanding of the human microbiome environmental and health impacts of air pollution: a review. front. public health , , abridged version of the awmf guideline for the medical clinical diagnostics of indoor mould exposure house dust mite allergy under changing environments interactions between helminth parasites and allergy the impact of helminths on the response to immunization and on the incidence of infection and disease in childhood in uganda: design of a randomized, double-blind, placebo-controlled, factorial trial of deworming interventions delivered in pregnancy and early childhood immunologic profiles of persons recruited for a randomized, placebo-controlled clinical trial of hookworm infection an update on the use of helminths to treat crohn's and other autoimmune diseases infections presenting for clinical care in early life and later risk of hay fever in two uk birth cohorts this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest. key: cord- -dyju w authors: fielding, c.l.; higgins, j.k.; higgins, j.c.; mcintosh, s.; scott, e.; giannitti, f.; mete, a.; pusterla, n. title: disease associated with equine coronavirus infection and high case fatality rate date: - - journal: j vet intern med doi: . /jvim. sha: doc_id: cord_uid: dyju w background: equine coronavirus (ecov) is associated with clinical disease in adult horses. outbreaks are associated with a low case fatality rate and a small number of animals with signs of encephalopathic disease are described. objectives: the aim of this study is to describe the epidemiological and clinical features of two outbreaks of ecov infection that were associated with an high case fatality rate. animals: miniature horses and miniature donkey testing fecal positive for ecov from two related disease outbreaks. methods: retrospective study describing the epidemiological findings, clinicopathological findings, and fecal viral load from affected horses. results: in ecov positive horses, % ( / ) of the animals died or were euthanized. severe hyperammonemia ( μmol/l, reference range ≤ μmol/l) was identified in one animal with signs of encephalopathic disease that subsequently died. fecal viral load (ecov genome equivalents per gram of feces) was significantly higher in the nonsurvivors compared to animals that survived (p = . ). conclusions and clinical importance: equine coronavirus had a higher case fatality rate in this group of miniature horses than previously reported in other outbreaks of varying breeds. hyperammonemia could contribute to signs of encephalopathic disease, and the fecal viral load might be of prognostic value in affected horses. e quine coronavirus (ecov) infection was initially reported in neonatal foals (≤ weeks of age) with and without clinical signs suggestive of enteritis. , outbreaks of ecov are described in adult horses at racing facilities and boarding stables. [ ] [ ] [ ] descriptions of outbreaks of ecov infection involving breeding farms and young horses (< year of age) are not reported. case fatality rates in the outbreaks have ranged between and %. [ ] [ ] [ ] in a case series of four boarding stables, two of the horses that died had acute onset of neurologic disease. a definitive cause for the neurologic signs is not determined, but authors speculated that hyperammonemia had contributed to the death of these horses. fecal shedding of ecov after infection occurs in a median of days (range of - days). however, more information is needed to confirm this finding as longer periods of shedding could lead to unanticipated disease transmission if quarantine periods are ended prematurely. in addition, a high viral load has been associ-ated with adverse outcome in people with respiratory coronavirus infection, but this has not been demonstrated in horses. the purpose of this study was to describe the epidemiological and clinicopathological features of two outbreaks of ecov infection that likely originated at a large equine competition and subsequently spread to two separate locations in idaho and california. the high case fatality rate, documentation of hyperammonemic encephalopathy, and association between viral load and death has not been previously reported. both reported outbreaks from california (ca) and idaho (id) initially involved horses that had recently returned from the american miniature horse association world show in texas in october, . clinical examination findings were recorded at presentation for veterinary care. in each outbreak, initial cases of ecov infection were confirmed by real-time pcr on feces. the pcr assay was based on the detection of a specific base-pair product of the n gene of ecov. viral load was reported as genome equivalents per gram of feces. a plasmid was constructed with the bp amplicon and -fold dilutions were used to obtain the slope and y-intercept for load calculations. the equation is the absolute value of the cp-y intercept/slope. furthermore, a real-time pcr assay targeting a universal sequence of the bacterial s rrna gene was used as quality control (ie, efficiency of dna purifications and amplification) and as an indicator of fecal inhibition. after identification of ecov in affected horses, subsequent screening of the remaining horses in the herd was completed. repeated testing was performed daily on six horses from the ca outbreak until at least three consecutive negative samples were obtained. other common enteric pathogens of horses, including salmonella enterica, clostridium difficile, clostridium perfringens, lawsonia intracellularis, and neorickettsia risticii were also investigated in feces of four of the sick horses by specific pcr. complete blood cell count and biochemical panels were performed on horses. blood ammonium concentration was measured in ecov-infected horses with clinical signs. for a complete list of variables that were evaluated refer to table . data are reported as median (range). a mann-whitney test was used to compare viral load between horses testing positive for ecov that died versus those horses that survived. significance level was set at p < . . available clinical examination and clinicopathological data for animals from both outbreaks (ca and id) are combined in table . the total number of horses in each farm was (ca) and (id). ecov testing was performed with all horses for a total of animals from both farms combined ( from ca and from id). fifteen of the animals ( %) tested positive. of the animals ( %) testing positive ( from ca and from id) manifested clinical disease including but not limited to colic, fevers, lethargy, and inappetance, while ( %) of positive animals remained asymptomatic. four of the horses positive for ecov ( %) tested negative for s. enterica, c. difficile, c. perfringens, l. intracellularis, and n. risticii. in the ca outbreak, the first horse displayed clinical signs including fevers ( . °c), lethargy, and inappetance approximately days after leaving the competition. over a -day period, additional horses that had not attended the show displayed similar clinical signs (fevers up to . °c) and were quarantined as a group for approximately days. at the end of the quarantine period, the horses were returned to the herd and approximately days later, another individual with severe clinical signs died. despite isolation and enhanced biosecurity protocols at the farm, other animals tested positive for ecov over the next days. these animals showed a range of clinical signs from no significant changes to fevers, lethargy, and mild colic. in addition to the initial animal that died in transport to the hospital, an -year-old miniature horse mare died during hospitalization. she initially was presented for veterinary care with mild signs of colic, but had a normal rectal temperature ( . °c), increased heart rate ( bpm), and increased respiratory rate ( bpm). initial blood work showed leukopenia ( cells/ll) because of lymphopenia ( cells/ll) and neutropenia ( cells/ll), and hyperlactatemia ( . mmol/l). eight hours after examination, the mare exhibited circling, head pressing, nystagmus and decreased pupillary light reflex. there was hyperlactatemia ( . mmol/l) and severe hyperammonemia ( lmol/l, reference range ≤ lmol/l). over a -hour period, clinical signs progressed and the mare became recumbent, developed respiratory and cardiovascular arrest, and died. both horses that died in the ca outbreak were submitted to the california animal health and food safety laboratory in davis, ca for complete postmortem examination (data not shown). in both animals, small intestinal content tested positive for ecov by pcr. in the first animal that died, there was severe, diffuse necrotizing enteritis. in the second animal with hyperammonemia, there was alzheimer type ii astrocytosis in the cerebral cortex. serial testing for ecov was completed for horses that tested positive in the ca outbreak. the mean shedding time was . days, however one horse continued shedding for a total of days (range - days). the daily fecal sample collection began on the first sick day (first day that clinical signs were observed), but healthy individuals were not repeatedly tested. in the ca outbreak, animals had clinical signs compatible with ecov infection approximately weeks before the remaining animals became symptomatic. pcr testing was not performed at that time and these horses tested pcr negative for ecov at the time the resident horses developed disease. in the id outbreak, resident farm horses displayed clinical signs of disease including decreased appetite, lethargy, and fevers ≤ . °c approximately - days after show horses returned from competition (american miniature horse association world championship show). the horses that returned from the show did not exhibit clinical abnormalities. a total of horses tested fecal positive for ecov on real-time pcr. of these horses, ( %) had clinical signs of disease whereas ( %) animals did not have clinical abnormalities. the first horse had signs of lethargy and anorexia hours after exposure to the show horses. this horse was severely ill by hours post exposure. clinical signs in affected horses included fevers up to . °c (three horses), tachycardia (one horse), encephalopathic signs (circling, head-pressing, and/or nystagmus in two animals), and lethargy (all horses). of the horses with abnormal clinical signs, ( %) died during the course of disease. a miniature donkey (one of the animals that died) displayed signs of rapidly progressive nystagmus, decreased mentation, and recumbency. the donkey presented for veterinary care hours after the onset of clinical signs, but because of rapid progression over the next hours, the donkey was euthanized. the other animal that died, a miniature horse, developed an acute decrease in packed cell volume from % to % with ulcerations of the oral mucosa and tongue. maximum viral loads measured in the feces for each horse (n = ) had a median of . ( . to . ) ecov genome equivalents per gram of feces with a corresponding cp value of . . horses with ecov that died had a significantly higher viral load as compared to horses that survived (p = . ). the median viral load was . ( . to . ) ecov genome equivalents per gram of feces in survivors with a corresponding cp value of . . this compared to horses that died . ( . to . ) with a corresponding cp value of . . the specific mortality rate for the outbreaks combined was % ( / ). this was calculated as the number of animals that died divided by the number of all animals. the number of animals testing positive for ecov that died was % ( / ). in addition to the severe hyperammonemia documented in the animal that died, blood ammonium was measured in other animals that tested positive for ecov and no other animals had values outside of the reported reference range ( table ) and none of the other tested animals displayed any neurologic abnormalities. the proportion of positive animals that died ( %) in this report is higher than the case fatality rate reported in previous outbreaks ( %). the deaths did not occur in stressed show horses, but in resident horses. the increased rate could reflect the virulence of the ecov strain, host or environmental factors. amount of viral particles being shed in manure was evaluated in horses. while the animals that died had variable viral loads, as a group they were statistically significantly higher than surviving animals. the miniature horse that died in the id outbreak initially had a low viral load despite significant clinical disease, but had a much higher viral load on the second fecal sample submitted. in human coronavirus infections, clinical outcome and death are highly correlated with viral load. results in this group of horses suggest that viral load in ecov infection is related to case fatality as well. two of the deaths described in a previous outbreak were associated with the onset of signs of encephalopathic disease. hyperammonemia may have been responsible for the neurological signs and contributed to the death of the horses. the current study documents a fatal case of hyperammonemia associated with ecov infection. it is possible that hyperammonemia could be common when encephalopathic signs are present in horses with ecov and that severe cases are associated with an increased risk of death. severe hyperammonemia is likely because of increased ammonia production within or absorption from the gastrointestinal tract. reports of equine hyperammonemia have speculated that overgrowth of urease producing bacteria and increased absorption might contribute to the increased levels of ammonia. , early treatment for presumptive hyperammonemia might be prudent, if there is a high suspicion for an ecov outbreak with associated encephalopathic signs in cases where testing of ammonium levels is not readily available. idiopathic equine hyperammonemia has been reported in case reports and retrospective case series. , the majority of these reported cases had clinical signs consistent with ecov infection (inappetance, depression, fever, colic, and diarrhea). some of these cases have occurred as outbreak situations. some of these cases of previously reported idiopathic hyperammonemia could have been associated with ecov infection. further study would be needed to determine if younger animals or certain breeds are more severely affected. the recent studies have described outbreaks in larger boarding stables or competition barns in full sized horses. [ ] [ ] [ ] four of the horses in the present study were tested for other common enteric pathogens and were negative, but the findings of the paper would have been stronger if all other horses had been tested for other pathogens. ecov was commonly identified as a coinfection with other pathogens in foals with diarrhea. to the authors' knowledge, this has not been described in adult horses. fecal shedding in this study was as long as days in one animal, compared to days in a previous study. the presumed initially infected animals were quarantined for days after the cessation of clinical signs in ca. after return to the herd, other animals became sick within a few days. this suggests that fecal shedding could have occurred for longer than days. if confirmed, this would indicate that longer quarantine periods might be needed for animals returning from competition or being newly introduced into the herd. one horse in the id outbreak that was initially negative on pcr for ecov despite showing clinical signs. this could have been because of the ileus and delayed passage of manure exhibited by this horse. that the horse was positive on retesting shows the importance of repeat testing in horses that are negative initially, particularly if they are not passing normal amounts of feces. in conclusion, these outbreaks of ecov were associated with younger animals, a higher case fatality rate, and a longer fecal shedding period. severe hyperammonemia was recognized in one animal with acute encephalopathic signs and death. if acute neurologic signs develop, hyperammonemia should be suspected and treated aggressively. in addition, viral load might be a factor to consider when determining prognosis. infectious agents associated with diarrhoea in neonatal foals in central kentucky: a comprehensive molecular study neonatal enterocolitis associated with coronavirus infection in a foal: a case report emerging outbreaks associated with equine coronavirus in adult horses epidemic of equine coronavirus at obihiro racecourse, hokkaido, japan in isolation of an equine coronavirus from adult horses with pyrogenic and enteric disease and its antigenic and genomic characterization in comparison with the nc strain initial viral load and the outcomes of sars comparison of five real-time pcr assays for detecting virulence genes in isolates of escherichia coli from septicaemic neonatal foals viral loads in clinical specimens and sars manifestations putative intestinal hyperammonaemia in horses: cases hyperammonaemia associated with encephalopathy and abdominal pain without evidence of liver disease in four mature horses equine history, physical examination, records, and recognizing abuse or neglect in patients appendix viii blood analyte reference values in large animals clinical biochemical and hematologic values of the american miniature horse: reference values veterinary laboratory medicine source of funding: no external funding was provided. off-label antimicrobial declaration: the authors declare no off-label use of antimicrobials. key: cord- - ndt a g authors: gazda, lawrence s.; collins, james; lovatt, archie; holdcraft, robert w.; morin, merribeth j.; galbraith, daniel; graham, melanie; laramore, melissa a.; maclean, christine; black, john; milne, euan w.; marthaler, douglas g.; vinerean, horatiu v.; michalak, michelle m.; hoffer, deborah; richter, steven; hall, richard d.; smith, barry h. title: a comprehensive microbiological safety approach for agarose encapsulated porcine islets intended for clinical trials date: - - journal: xenotransplantation doi: . /xen. sha: doc_id: cord_uid: ndt a g background: the use of porcine islets to replace insulin‐producing islet β‐cells, destroyed during the diabetogenic disease process, presents distinct challenges if this option is to become a therapeutic reality for the treatment of type diabetes. these challenges include a thorough evaluation of the microbiological safety of the islets. in this study, we describe a robust porcine islet‐screening program that provides a high level of confidence in the microbiological safety of porcine islets suitable for clinical trials. methods: a four‐checkpoint program systematically screens the donor herd (large white – yorkshire × landrace f hybrid animals), individual sentinel and pancreas donor animals and, critically, the islet macrobeads themselves. molecular assays screen for more than known viruses, while electron microscopy and in vitro studies are employed to screen for potential new or divergent (emergent) viruses. results: of monthly samples taken from random animals over a ‐year period, only a single positive result for transmissible gastroenteritis virus was observed, demonstrating the high level of biosecurity maintained in the source herd. given the lack of clinical signs, positive antibody titers for porcine reproductive and respiratory syndrome virus, porcine parvovirus, and influenza a confirm the efficacy of the herd vaccination program. porcine respiratory coronavirus was found to be present in the herd, as expected for domestic swine. tissue homogenate samples from six sentinel and donor animals, over the same ‐year period, were negative for the presence of viruses when co‐cultured with six different cell lines from four species. the absence of adventitious viruses in separate islet macrobead preparations produced from individual pancreas donor animals was confirmed using validated molecular (n = viruses), in vitro culture (cells from four species), and transmission electron microscopy assays ( cell profiles per donor animal) over the same ‐year period. there has been no evidence of viral transmission following the implantation of these same encapsulated and functional porcine islets into non‐immunosuppressed diabetic cynomolgus macaques for up to years. isolated peripheral blood mononuclear cells from all time points were negative for pcv (type ), plhv, prrsv, pcmv, and perv‐a, perv‐b, and perv‐c by pcr analysis in all six recipient animals. conclusion: the four‐checkpoint program is a robust and reliable method for characterization of the microbiological safety of encapsulated porcine islets intended for clinical trials. the ultimate goal for the treatment of type diabetes is the restoration of physiological blood glucose regulation without the requirement for lifelong therapy such as immunosuppression or immunoregulation. there are numerous challenges for the attainment of this goal, and diverse approaches ranging from insulin pumps to gene therapy to stem cell strategies are all being pursued. [ ] [ ] [ ] [ ] [ ] in another approach, the replacement of lost islet cells through human islet allotransplantation has established the clinical utility of islet grafting. , although only % of human islet recipients remain insulin-independent at years, the majority of patients ( %) retain some graft function as evidenced by persistent c-peptide secretion. importantly, islet transplantation largely eliminates hypoglycemic unawareness post-grafting. further improvement in long-term insulin independence is likely; data from a few select methods: a four-checkpoint program systematically screens the donor herd (large white -yorkshire × landrace f hybrid animals), individual sentinel and pancreas donor animals and, critically, the islet macrobeads themselves. molecular assays screen for more than known viruses, while electron microscopy and in vitro studies are employed to screen for potential new or divergent (emergent) viruses. organization institutions indicate that an increasing number of islet recipients (approaching %) are able to maintain external insulin independence for years. nonetheless, the availability of suitable human pancreases for either whole organ or isolated islet transplantation is entirely inadequate as evidenced by the recovery of only donor pancreases in the united states in . the use of xenogeneic islets could provide a solution to the limited supply of human islets for transplantation. porcine islets are particularly suitable as a source of insulin-producing cells because porcine insulin differs from human insulin by only a single amino acid and porcine insulin has been shown to provide reliable and safe glycemic control in human diabetic patients. furthermore, only the transplantation of whole pancreas or intact islets of langerhans can be expected to replicate normal and precise physiological glucose control, which is dependent on a variety of cell types and proteins from pancreatic islets. this enormously complex regulatory machinery is absent in all other approaches to the re-establishment of normoglycemia. the use of animal-derived islet tissue, however, will necessitate either suppression of the patient's immune system or the physical isolation of the islets to prevent their rejection. we have previously described the ability of agarose-agarose encapsulated porcine islets to function in diabetic animal models. [ ] [ ] [ ] [ ] [ ] these encapsulated islet macrobeads are at least partially immune isolated as an outer layer of dense agarose prohibits direct cell access to the xenogeneic islets. although small immune mediators, such as cytokines, can still enter the macrobead, this encapsulation approach has been sufficiently robust to allow spontaneously diabetic bb rats to survive for more than months without immunosuppression and without exogenous insulin administration. ongoing studies in non-immunosuppressed diabetic cynomolgus macaques also demonstrate porcine islet macrobead function for more than months in of animals (unpublished data, see checkpoint below). the clinical use of islets from animals also requires a well-thoughtout strategy to assure the microbiological safety of the animal tissue. a strategy to provide an aseptic product and to reduce the probability of inadvertent transmission of adventitious viruses is critical to the successful implementation of this exciting therapy. our approach takes advantage of the unique aspects of the islet macrobead, namely its long-term viability in culture and lack of patient immunosuppression, to limit the microbiological safety risk of porcine islets for transplantation. essential to our approach is the establishment of four checkpoints that span the macrobead production and transplantation processes. the checkpoint approach is built upon an increasing level of microbiological screening from the donor herd up to the islet product, using multiple assay formats with the ability to screen for known and unknown pathogens. using the checkpoint approach, we provide evidence to support the use of donor animals housed in high hygienic but not hepa-filtered environments, not only for islet xenotransplantation but also other xeno-cell therapies. in this study, we present and document the effectiveness of our microbiological safety strategy to minimize the microbiological risks of agarose encapsulated porcine islets for the treatment of type diabetes. donor pigs were large white -yorkshire × landrace f hybrid animals obtained from well-characterized, non-genetically modified choice genetics (formerly newsham) source animals. the source animal facility is an independently owned herd located in rural southwest ohio. the breeding herd numbers approximately sows and utilizes a two-tiered quarantine testing system through a modern multisite production strategy. three segregated facilities (nursery, gilt isolation, and farrowing and gestation) maintain data records and follow standard operating procedures (sops) for animal husbandry, vaccination, housing, cleaning and waste management, and biosecurity measures. controlled transportation of animals, supplies and semen, feeding and watering, pest and rodent control, gravel barrier around facility perimeter, and caretaker qualifications including routine health assessments are all components of the comprehensive biosecurity program. routine quality assurance site audits are used to verify compliance. corn-based pig feed was produced exclusively with corn grown and ground onsite at the source animal facility. gilts, gestation sows, and lactating sows were given the same base feed, consisting of corn with various supplements (eg, amino acids, vitamins, minerals, protein), with slight variations based on the breeding status of the animal. all ingredients used in the production of complete feed were certified to be free of any restricted use protein products as de- the facility holds pork quality assurance plus (pqa plus ® ) certification, which is a producer-driven program to ensure u.s. pork products are of the highest quality and safe. the pqa plus program mandates good production practices with an emphasis on biosecurity and practices to reduce the risk of introducing and spreading of infectious agents. all retired breeding sows not used as pancreas donors are sent to a local abattoir for pork products. source animals are routinely vaccinated for various agents ( quarterly sentinel following electrical stun and exsanguination at a usda-regulated abattoir in compliance with all federal regulations concerning animal handling and welfare, numerous tissues including brain, heart, lung, liver, tonsil, lymph node, spleen, ileum, kidney, pancreas, bone marrow, pbmcs, feces, and serum were collected and archived as fixed and frozen aliquots at the time of pancreas procurement. aliquots of isolated islets were also frozen and archived. donor animal tissue samples were sent to the university of minnesota, veterinary diagnostic laboratory (mvdl) to screen for the bacterial and viral pathogens listed in table . the mvdl is one of the three major swine diagnostic laboratories donor pancreases were procured from sows that were over years of age with a history of multiple parities. following electrical stunning and exsanguination of the donor, abdominal viscera were retrieved by a sterile-gowned technician and transferred to a sterile container for transport. the viscera were then placed in a custom built, hepafiltered, laminar flow work station for tissue retrieval. surface monitoring was performed on the work surface prior to retrieval, and settle plates were placed inside the workstation during dissection to monitor for microbiological contamination. intact pancreas was dissected from the viscera by a sterile-gowned technician and immediately to provide absolute viral sequestration although the encapsulated islets are protected from direct immune cell contact. agarase, the enzyme necessary to break down agarose, is abundant in ocean-dwelling bacteria but has not been found in mammals. the macrobeads, in the absence of trauma, remain intact indefinitely following implantation in the abdominal cavity. macrobeads were then cultured at °c in a humidified atmosphere of % co until collection for microbiological screening or implanta- , a colon, ileum, liver, spleen, lung, and mesenteric lymph node, b coronavirus (n = ), caudovirales (n = ), c small round viral particle, d dilution factor (df): , e kidney, lung, mesenteric lymph node, tonsil, and spleen, f testing performed at national veterinary service laboratory, g bacteriophage, h df: (n = ), (n = ), i df: (n = ), (n = ), (n = ), ≥ (n = ), j df: (n = ), (n = ), (n = ), k df: (n = ), (n = ), l df: (n = ), (n = ), m df: (n = ), (n = ), (n = ), n heart, intestine, liver, pancreas, kidney, lung, mesenteric lymph node, tonsil, and spleen. ny, usa; pre-screened for endotoxin < . eu/ml and for sterility) containing mmol/l glucose, . % heat-inactivated porcine serum (biologos, montgomery, il, usa; pre-screened for sterility, and the absence of mycoplasma and adventitious viruses per cfrs and endotoxin < eu/ml) and % antibiotic/anti-mycotic (life technologies) was changed weekly and -hour post-change media samples were taken for porcine insulin elisa assays (mercodia, uppsala, sweden). islet isolation and encapsulation procedures were performed in class ii biosafety cabinets within an iso class (at rest) and iso class (active processing) laboratory by sterile-gowned technicians. routine environmental monitoring included particle counts, viable particle counts, settling plates, and surface monitoring and was performed for every islet isolation procedure. macrobeads from a given donor were assigned a unique islet isolation number and cultured separately from islet macrobeads produced from other donor animals. prior to islet macrobead implantation, representative samples of macrobeads were sent for sterility testing per usp < > six non-immunosuppressed streptozotocin-induced diabetic male cynomolgus macaques of mauritian or asian origin with a median age of multiple checkpoints during the process of porcine islet isolation, encapsulation, culture, and transplantation were implemented to assess the microbiological safety of islet macrobeads (figure ). the step: each test sample is spiked with an internal extraction control (iec; plant rna or dna) at the detection limit, mimicking the target nucleic acid. this process allows the level of nucleic acid recovery in the presence of the test sample to be assessed, which ensures that low levels of rna or dna can be recovered. given evidence of satisfactory insulin production by cultured islet macrobeads (≥ mu/macrobead/ -hour), the macrobeads were sent for viral screening to sgs vitrology prior to use in pre-clinical animal studies. all islet macrobeads were negative for viruses screened by pcr (n = viruses) and by co-culture assays using four different susceptible cell lines (n = pancreas donor animals; table ). transmission scanning electron microscopy was incorporated as an (table and figure ). were negative for bacterial growth and for the presence of endotoxin and mycoplasma (table ). although not a source material or product release screen, recipient monitoring post-macrobead implantation provides valuable data that could be used to not only treat individual patients but also as a check (table ) . moreover, there has been no evidence of viral infection as observed and documented in the health status of the animals. the four-checkpoint microbiological safety program outlined utilizes four separate assessments for the microbiological safety of agarose [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the focus of all these guidelines, including the ixa statements, is on the safety of xenotransplantation. , an invited review of us xenogeneic regulations was published by fda staff in and provides additional details on fda guidelines. schuurman has more recently published a thorough overview of worldwide xenogeneic regulations with an emphasis on the european requirements. f i g u r e transmission electron microscopy of islet cell profiles, typical of those present in the sample of cells examined. the islet cells were generally adherent with intercellular connections, cell junctions, and desmosomes observed. the cell surface appearances varied, smooth surfaces, blebs, and cell processes were observed. various cell types were present, including α and β cells. no mitotic cells were observed by light or electron microscopy. no significant numbers of dead or dying cells were present in the population of cells examined. nuclear profiles varied in size. in some cells, more than one nuclear profile was visible. they were often indented and invaginated or contained lacunae of cytoplasmic material. nucleoli were prominent and sometimes more than one was observed. heterochromatin was abundant, clumped, and found on the inner nuclear membrane and sporadically throughout the nucleus. the cells had a normal range of organelles. the mitochondria were numerous and varied in size and shape. the presence of cristae was noted although no matrix granules were seen. the golgi body, when observed, was prominent and exhibited stacked cisternae. the rough-surfaced endoplasmic reticulum was extensive and occurred in varied lengths. lipid bodies, peroxisomes, and multivesicular bodies were present. free ribosomes, polysomes, and fibrils were found throughout the cytoplasm. vacuoles, with electron dense and flocculent material, were observed. coated, un-coated, and secretory vesicles, of varying types, were seen. centrioles and microtubules were present. the cell structure is consistent with the morphology expected of secretory cells. no viruses, virus-like particles or extraneous agents, including mycoplasmas, yeasts, fungi, or bacteria, were found. abbreviations: f, fibrils; mitochondria (m); desmosome (arrow); centriole (c); nucleus (nu); golgi bodies (g), lipid bodies (l), vacuoles (v), multivesicular body (mvb), and rough-surfaced endoplasmic reticulum (rer) our microbiological safety program is in-line with the issues for consideration in the fda xenotransplantation guideline, as well as the ixa statements. in regard to donor animals, it is important to note that the guidelines do not state that source animals must be raised in hepa-filtered environments. this is significant because, in addition to the difficulty of obtaining and maintaining pathogen-free swine, [ ] [ ] [ ] there is no guarantee that the pancreas from individual animals will be suitable for islet isolation. in fact, it is becoming increasing clear that only about % of porcine pancreases yield a sufficient number of islets for clinical use. , [ ] [ ] [ ] given these limitations, our approach is to raise source animals in high hygienic environments, biopsy individual pancreases for islet isolation suitability and perform extensive microbiological screening of all selected donor animal tissues and the islet macrobeads themselves. our methodology, however, employs an alternative approach to the fda-recommended -day quarantine period for individual donor animals that deserves some discussion. a minimum -day quarantine period for animals used in the processing of biological products is also stated in cfr part . the rationale for the quarantine period is to provide time for an acute infection to become clinically apparent. but not as reliably in peripheral blood cells. thus, it is the islet macrobeads themselves, that is, the tissue to be implanted, that undergoes quarantine and it is the ability to thoroughly screen the islets using a wide variety of specific and non-specific assays that obviates the need to raise or quarantine donor animals in hepa-filtered environments. as discussed by time points evaluated were: month (± days) and months (± days) post-transplant. additionally, samples from year (± days), years (± days), years (± days) and years (± days) posttransplant were evaluated in a subset of recipients who reached these time points during extended follow-up. t a b l e pcr screening of implanted non-human primates treatment options in the case of suspected pathogen transmission but also manufacturing decisions as well as feedback to checkpoints - that may result in the modification of one or more checkpoints. as with any medical procedure, there will always be risk with the transplantation of porcine islets. the challenge we face is to understand those risks such that they can be minimized to an acceptable level in light of the expected clinical benefits. the potential risk of viral transfer from the transplanted cells to the recipient has been the major safety concern. to address this issue, a testing rationale was developed to screen sentinel and donor animals for viruses that are present in the herd, are of special interest such as known zoonotic agents, or are known to infect swine in the usa. an initial characterization of the perv infectivity status of the source herd was not performed given the unlikely ability, at least at that time, to eliminate these endogenous retroviruses and the growing conclusion that the risk of perv transmission to human xenotransplantation trial participants is low. additionally, co-culture and electron microscopy assays are employed to look for unknown viruses, which would then prompt efforts to identify the virus as either a known or unknown agent (eg, prrsv inducing cpe in ma cells as one component of the islet macrobead screen). also, as part of their release criteria, the islet macrobeads are confirmed to be sterile and free from mycoplasma and any significant level of en- culture. this sequence was not found in purified islets isolated in the absence of porcine serum. because we also detected the sequence in domestic and international porcine serum sources, phov screening of islet macrobeads cultured in these sera was postponed until a suitable phov-free source could be obtained. with regard to perv, the identification of this virus and its ability to cross the species barrier has been the principal concern with porcine xenotransplantation. it is worth mentioning that the nhp is not an ideal model to assess the risk of perv transmission. cynomolgus monkeys lack the perv-a receptor (par- ) that is the major receptor for perv-a and perv-a/c entry. the actual risk of perv infection in the clinic remains unclear, but so far there has been no transmission of perv or other porcine microorganisms in humans that have been exposed to pig islet cells. , [ ] [ ] [ ] we consider these findings, as well as the negative viral findings from diabetic dogs exposed to porcine islet macrobeads for . years, to be evidence that viral transmission following macrobead implantation is very low risk. studies with d canine cells have shown these cells to have the perv-a receptor, but apparently not to the same degree as human cells. other, non-microbiological risks to the patient also exist and must be considered as part of the comprehensive safety evaluation. these include procedural risks that accompany pre-and post-implantation screening, as well as the general anesthesia and laparoscopic surgery required to implant the macrobeads. the procedural risks are well known and, although real, are modest. we have been implanting cancer macrobeads (encapsulated mouse renal adenocarcinoma cells) to treat patients with various malignancies for years without adverse events attributable to the macrobeads either as an intraperitoneal irritant or as a microbiological hazard. see also clinicaltrials.gov identifiers nct , nct , and nct . a final important consideration is the immune status of the recipients. recent progress in clinical islet allotransplantation has confirmed the potential of islet grafting to reduce exogenous insulin administration and the occurrence of hypoglycemic unawareness. , while these results are encouraging, numerous adverse events including intrahepatic bleeding, neutropenia, mouth ulcers, anemia, diarrhea, edema, hypercholesterolemia, and pharyngitis have been reported. most of the reported adverse events are primarily associated with immunosuppressive therapy. encapsulating porcine islets using the agaroseagarose method eliminates the need for immunosuppressive therapy, and a fully competent immune system is expected to significantly reduce the risk of xenozoonotic infection. during the last years, an understanding of the risks associated with porcine xenotransplantation, and methodologies to manage those risks, including theoretical risks and the potential presence of unknown pathogens, have significantly progressed. the recent ixa consensus update notes that "theoretical risk" still exists in relation to the transmission of infectious agents when employing appropriate safeguards, but that such events will likely be rare should they occur. although the actual risks of islet xenotransplantation cannot be not known in the absence of clinical trials, as stated by fishman, "in clinical xenotransplantation, a level of safety has been developed beyond that available for human organs…". insulin pump risks and benefits: a clinical appraisal of pump safety standards, adverse event reporting and research needs. a joint statement of the european association for the study of diabetes and the american diabetes association diabetes technology working group performance and acceptability of a combined device for insulin infusion and glucose sensing in the home setting insulin gene therapy for type diabetes mellitus gene therapy with neurogenin , betacellulin and socs reverses diabetes in nod mice toward beta cell replacement for diabetes achievement of insulin independence in three consecutive type- diabetic patients via pancreatic islet transplantation using islets isolated at a remote islet isolation center islet transplantation in seven patients with type diabetes mellitus using a glucocorticoid-free immunosuppressive regimen the clinical impact of islet transplantation update on islet transplantation. cold spring harb perspect med glucose control and long-term survival in biobreeding/worcester rats after intraperitoneal implantation of hydrophilic macrobeads containing porcine islets without immunosuppression retrievable, replaceable, macroencapsulated pancreatic islet xenografts. long-term engraftment without immunosuppression encapsulation of porcine islets permits extended culture time and insulin independence in spontaneously diabetic bb rats pravastatin improves glucose regulation and biocompatibility of agarose encapsulated porcine islets following transplantation into pancreatectomized dogs no evidence of viral transmission following long-term implantation of agarose encapsulated porcine islets in diabetic dogs pork quality assurance plus certification evidence of pig-to-pig transmission of a modified-live prrs virus vaccine the use of pancreas biopsy scoring provides reliable porcine islet yields while encapsulation permits the determination of microbiological safety agarase: review of major sources, categories, purification method, enzyme characteristics and applications nucleic acid amplification techniques. section . . nucleic acid-based techniques-amplification diseases of swine application of current statutory authorities to human somatic cell therapy products and gene therapy products. fda: federal register source animal, product, preclinical, and clinical issues concerning the use of xenotransplantation products in humans of the european parliament and of the council on advanced therapy medicinal products and amending directive / /ec and regulation (ec) no / . official j of the eur union guideline on human cell-based medicinal products. european medicines agency: committee for medicinal products for human use european medicines agency: committee for medicinal products for human use first who global consultation on regulatory requirements for xenotransplantation clinical trials. world health organization second who global consultation on regulatory requirements for xenotransplantation clinical trials. world health organization test procedures and acceptance criteria for biotechnological/biological products (q b) quality of biotechnological biological products: derivation and characterization of cell substrates used for production of biotechnological/biological products (q d). ich harmonised tripartite guideline safety preclinical evaluation of biotechnology: derived pharmaceuticals for biotechnological product (s ) viral safety evaluation of biotechnology products derived from cell lines of human or animal origin (q a) first update of the international xenotransplantation association consensus statement on conditions for undertaking clinical trials of porcine islet products in type diabetes-executive summary first update of the international xenotransplantation association consensus statement on conditions for undertaking clinical trials of porcine islet products in type diabetes -chapter : update on national regulatory frameworks pertinent to clinical islet xenotransplantation first update of the international xenotransplantation association consensus statement on conditions for undertaking clinical trials of porcine islet products in type diabetes -chapter a: source pigs-preventing xenozoonoses first update of the international xenotransplantation association consensus statement on conditions for undertaking clinical trials of porcine islet products in type diabetes first update of the international xenotransplantation association consensus statement on conditions for undertaking clinical trials of porcine islet products in type diabetes. chapter : porcine islet product manufacturing and release testing criteria first update of the international xenotransplantation association consensus statement on conditions for undertaking clinical trials of porcine islet products in type diabetes. chapter : pre-clinical efficacy and complication data required to justify a clinical trial first update of the international xenotransplantation association consensus statement on conditions for undertaking clinical trials of porcine islet products in type diabetes. chapter : recipient monitoring and response plan for preventing disease transmission first update of the international xenotransplantation association consensus statement on conditions for undertaking clinical trials of porcine islet products in type diabetes. chapter : patient selection for pilot clinical trials of islet xenotransplantation regulation of xenogeneic porcine pancreatic islets regulatory aspects of clinical xenotransplantation serological evidence for hepatitis e virus infection in laboratory monkeys and pigs in animal facilities in japan microbiological safety of porcine islets: comparison with source pig extended microbiological characterization of gottingen minipigs in the context of xenotransplantation: detection and vertical transmission of hepatitis e virus optimizing porcine islet isolation to markedly reduce enzyme consumption without sacrificing islet yield or function the morphology of islets within the porcine donor pancreas determines the isolation result: successful isolation of pancreatic islets can now be achieved from young market pigs assessment of some porcine strains as donors of islets of langerhans food and drug administration phs guideline on infectious disease issues in xenotransplantation. u.s. food and drug administration a novel porcine gammaherpesvirus the international xenotransplantation association consensus statement on conditions for undertaking clinical trials of porcine islet products in type diabetes-chapter : source pigs xenotransplantation: infectious risk revisited infection barriers to successful xenotransplantation focusing on porcine endogenous retroviruses xenotransplantation-associated infectious risk: a who consultation microbiological safety of the first clinical pig islet xenotransplantation trial in new zealand identification of novel porcine and bovine parvoviruses closely related to human parvovirus phylogenetic characterization of the first ungulate tetraparvovirus detected in pigs in brazil suboptimal porcine endogenous retrovirus infection in non-human primate cells: implication for preclinical xenotransplantation search for cross-species transmission of porcine endogenous retrovirus in patients treated with living pig tissue. the xen study group no evidence of infection with porcine endogenous retrovirus in recipients of encapsulated porcine islet xenografts no evidence of porcine endogenous retrovirus in patients with type diabetes after long-term porcine islet xenotransplantation host range and interference studies of three classes of pig endogenous retrovirus extended analysis of the in vitro tropism of porcine endogenous retrovirus antigenic relationships among homologous structural polypeptides of porcine, feline, and canine coronaviruses recombinant canine coronaviruses related to transmissible gastroenteritis virus of swine are circulating in dogs pseudorabies virus (aujeszky's disease). virus infections of porcines replication and plaque assay of influenza virus in an established line of canine kidney cells susceptibility of different cell lines to avian and swine influenza viruses emergence of human-like h n influenza viruses in pet dogs in guangxi first-in-human phase trial of agarose beads containing murine renca cells in advanced solid tumors benefits and risks of solitary islet transplantation for type diabetes using steroid-sparing immunosuppression: the national institutes of health experience infection and xenotransplantation. developing strategies to minimize risk implications of the advent of homozygous alpha l, -galactosyltransferase gene-deficient pigs on transmission of infectious agents reduced sensitivity to human serum inactivation of enveloped viruses produced by pig cells transgenic for human cd or deficient for the galactosyl-alpha( - ) galactosyl epitope porcine endogenous retrovirus transmission characteristics of galactose alpha - galactose-deficient pig cells assessment of infectious risk in clinical xenotransplantation: the lessons for clinical allotransplantation a comprehensive microbiological safety approach for agarose encapsulated porcine islets intended for clinical trials key: cord- -nkb nzyt authors: wiebers, david o.; feigin, valery l. title: what the covid- crisis is telling humanity date: - - journal: neuroepidemiology doi: . / sha: doc_id: cord_uid: nkb nzyt nan the world is enveloped in a global health emergency that is exacting enormous medical and economic tolls upon humanity. the sars-cov- that has caused the current covid- pandemic is thought to have originated in bats and, via an intermediary such as the pangolin, to have found its way from a "wet market" where live wildlife species were being sold for human consumption in wuhan, china, to one or more humans at that location [ ] . within months, this highly infectious virus spread throughout china and around the world, currently involving at least countries and territories, with a trail of incredible damage in its wake [ ] . the medical community finds itself on the front lines throughout the world dealing with the immediate human health consequences of this rapidly evolving crisis and trying to develop therapies and vaccines, as countries and their leaders attempt to mitigate the overwhelming societal and economic devastations that are unfolding. from a neuropsychiatric perspective, there are obvious signs of global psychological distress related to social isolation and fears of illness, death, and countless uncertainties about the future [ ] . less attention has been given to neurological manifestations including headache, anosmia, ageusia, ataxia, paresthesia, ischemic stroke, seizures, and various encephalopathies, collectively occurring in up to nearly % of hospitalized covid- patients in early series. encephalopathies have been more likely to occur in severely ill patients and have included at least one case of documented viral encephalitis with sars-cov- in the cerebrospinal fluid [ ] . past respiratory viral pandemics have been associated with other delayed neurological sequelae, including acute inflammatory polyradiculopathy, peripheral neuropathy, myopathy, brainstem encephalitis, and parkinsonism, the latter of which was prominently associated with the so-called spanish influenza pandemic of [ ] . given the immense global burden of covid- , it is likely that the long-term neuropsychiatric complications will be substantial and it will be important for the medical community to prospectively monitor patients to establish the nature and extent of such morbidities while also dealing with the acute manifestations of this illness that are unfolding each day in emergency rooms throughout the world [ , ] . yet, in the midst of all of the pandemonium and destruction, and as we begin to find our way through this crisis, it is imperative for us as a society and species to focus and reflect deeply upon what this and other related human health crises are telling us about our role in these increasingly frequent events and about what we can do to avoid them in the future. failure to do so may result in the unwitting extermination of all or a good part of our species from this planet. although it is tempting for us to lay the blame for pandemics such as covid- on bats, pangolins, or other wild species, it is human behavior that is responsible for the vast majority of zoonotic diseases that jump the species barrier from animals to humans. the us centers for disease control and prevention (cdc) observes that "… in every new or emerging infectious diseases in people come from animals." these infections are caused not only by viruses but also by bacteria, fungi, and parasites from a variety of animal sources [ ] . the alarming increase in frequency of these lethal zoonotic diseases relates in large part to our human-dominated ecosystem with increasingly unnatural human-animal close contact, grossly aberrant crowding of animals for human purposes, destruction of animal habitats, and vast numbers of highly mobile humans to swiftly carry these diseases throughout the world [ ] . as is likely with covid- , the outbreak of sars (an earlier severe acute respiratory syndrome) in was the result of a coronavirus that originated in bats with subsequent infection of wild animals sold in live-animal street markets in china. such markets provide breeding grounds for these and countless other zoonotic pathogens with sick, highly stressed, and overcrowded animals in highly unsanitary conditions. the opportunity for human transfer of these pathogens is increased immensely by allowing direct access to large crowds of humans [ ] . the human immunodeficiency virus (hiv), which has infected million people and killed million since the onset of acquired immunodeficiency syndrome (aids) in the s, originated in a type of chimpanzee in central africa. scientists have generally concluded that the chimpanzee version of the virus (called simian immunodeficiency virus) was transmitted to humans and mutated into hiv when humans hunted these chimpanzees for meat and came into contact with their infected blood [ ] . the hunting, capturing, and selling of wild animals for human consumption, particularly in connection with live-animal markets, clearly constitute major public health risks. continuing these practices will assure further human health crises with potentially even greater destructive power in the future. another well-recognized source for increasingly lethal human zoonoses is the massive overcrowding of animals for human consumption in industrial "factory farm" environments -also known as concentrated animal feeding operations. over the last years, as the factory farm model has become a global phenomenon, a host of avian influenza (bird flu) viruses, including h n , have emerged in countries with large-scale industrial poultry operations [ , ] . intensive confinement of unprecedented num-bers of chickens in these facilities to lower cost has provided a fertile ground for the development of an everincreasing supply of new pathogens. and while bird flu was once a very rare disease among chickens, today we see outbreaks occurring every year [ ] . transmission of these diseases from chickens to humans was almost nonexistent years ago; now serious outbreaks are occurring regularly -more in the past years than in the entire th century [ ] . initially detected in humans in , the h n pandemic (the so-called swine flu because of its origin in pigs) constituted the st worldwide flu pandemic in years. the cdc estimates that over the past years, this disease has killed between , and , people worldwide. pigs had long been considered a possible mixing vessel for influenza viruses that originated within pigs, birds, and humans, and the cdc has concluded that this virus resulted from reassortment, a process through which two or more influenza viruses can swap genetic information by infecting a single human or animal host [ ] . larger numbers of pigs in closer proximity provide greater opportunities for this to occur, and the mixing of live pigs from north america and eurasia through international trade or other means could have also contributed to the circumstances necessary for this reassortment to occur [ ] . the large-scale confinement of animals for human consumption has also played a major direct role in another ongoing health crisis in the usa and around the world -antibiotic resistance. nearly % of the antibiotics sold in the usa are now used for livestock feed to prevent disease and promote growth [ ] . intensive confinement operations require more antibiotics than family farms, and if the bacteria that naturally exist in these animals acquire antibiotic resistance genes, treatment becomes ineffective. this circumstance constitutes a threat not only to animal health but also to human health [ ] . humans may be exposed to antibiotic-resistant organisms by handling or eating raw or undercooked meat, coming into contact with farm animals or their feces, and/ or eating produce or consuming water (including recreational water) that has come into contact with animal feces [ ] . robust scientific data confirm that proximity to livestock operations and manure application to crop fields are each associated with the likelihood of acquiring antibiotic-resistant infections in humans [ ] . researchers at the johns hopkins bloomberg school of public health drove cars, windows down, behind trucks that were transporting broiler chickens from farms to slaughterhouses in virginia and maryland; they documented neuroepidemiology doi: . / antibiotic-resistant bacteria in the air inside the cars, as well as on the top of soda cans in the cars' cupholders [ ] . antibiotic-resistant infections constitute a major and growing global health threat and now kill an estimated , people in the usa and , people worldwide per year [ ] . one further fundamental source for the increasing threat of pandemics and other human health crises is habitat destruction. vast numbers of wildlife species are threatened with extinction from cutting down forests and expanding urban areas and industrial activities. the survivors are forced into closer proximity to themselves and humans, increasing the likelihood of transforming what would otherwise be benign animal microbes into deadly human pathogens. to compound the issue, much of the land that is being lost to deforestation is being converted to land for raising more animals for human consumption [ ] . our species has come to the edge of the cliff on these issues, and the covid- pandemic is forcing us to make a choice between either changing our thinking and practices in these realms, or facing increasing destruction and perhaps self-annihilation. medical experts are painfully aware of the undeniable hazards of continuing along our current trajectory. dr. amesh adalja of the johns hopkins center for health security writes openly of the relatively new avian influenza, h n , that has been considered one of the most concerning of all pandemic threats over the past few years. although an epidemic of h n began in southern china in , there has to date been no sustained human-to-human transmission. nevertheless, more than , human cases have occurred with a case fatality rate of %, and adalja warns that "if h n achieves sustained human-to-human transmission, it would arguably be the worst health and national security threat faced by the world in literally a century. an h n pandemic could well be worse -perhaps much worsethan the great pandemic of " [ ] that killed an estimated million people [ ] . rather than simply attempting to react to crises like covid- after death and destruction are already upon us, we need to have the vision, wisdom, and compassion to address fundamental underlying causes and act now to mitigate and prevent the numerous disasters that are literally waiting to happen. the chinese government should be applauded for taking the much-needed step of banning the trade and consumption of wild animals in china on february , . although shutting down this usd billion wildlife farming industry has been decried by some as economically harmful, such harm pales over-whelmingly in comparison to the vast health and economic threats to china and the world involved in continuing to allow business as usual. other nations throughout the world should also prohibit such practices, particularly in association with live-animal markets. intensive confinement of animals in factory farm operations should be discontinued worldwide for the sake of animals, humans, and the environment, and we should rapidly evolve to eating other forms of protein that are safer for humans, including plant-based meat alternatives and cultured meat (produced by culturing animal cells). additional investment in plant-based agriculture to grow crops to feed humans rather than livestock for human consumption would feed more people while utilizing far less land and water, allowing for the preservation of vital ecosystems for innumerable species. the time has come for us to rethink our relationship with all life on this planet -other humans, nonhumans, and the earth, a life form in itself. what is good for nonhumans and the earth is virtually always in the best interests of humans, given the profound interconnectedness of all life. all that we do depends upon abundant plant and animal life as well as clean air and water. each of us can have a positive impact upon these fundamentals by demonstrating and inspiring an enhanced mindfulness, beginning most basically with what we eat and how all of our daily choices and actions may be affecting animals and natural habitats. ultimately, the survival, not only of other life forms on this planet, but also of our own, will depend upon humanity's ability to recognize the oneness of all that exists and the importance and deeper significance of compassion for all life. the authors have no conflicts of interest to disclose. escaping pandora's box: another novel coronavirus johns hopkins coronavirus resource center are we facing a crashing wave of neuropsychiatric sequelae of covid- ? neuropsychiatric symptoms and potential immunologic mechanisms first case of novel coronavirus disease with encephalitis nervous system involvement after infection with covid- and other coronaviruses global hotspots and correlates of emerging zoonotic diseases origin and evolution of pathogenic coronaviruses simian immunodeficiency virus infection of chimpanzees the next influenza pandemic: lessons from hong kong bird flu: a virus of our own hatching avian influenza: recent developments origins of the h n influenza pandemic in swine in mexico antibiotics overuse in animal agriculture: a call to action for health care providers industrial food animal production, antimicrobial resistance, and human health high-density livestock operations, crop field application of manure, and risk of community-associated methicillin-resistant staphylococcus aureus infection in pennsylvania food animal transport: a potential source of community exposures to health hazards from industrial farming (cafos) tackling drug-resistant infections globally: final report and recommendations johns hopkins school of public health center for health security clinicians biosecurity report the challenge of emerging and re-emerging infectious diseases key: cord- -yjxbm tn authors: correa, m.t.; grace, d. title: slum livestock agriculture date: - - journal: encyclopedia of agriculture and food systems doi: . /b - - - - . - sha: doc_id: cord_uid: yjxbm tn slums are unplanned squatter human settlements in peri-urban and urban areas where more than million people live. these densely populated areas lack basic public services. livestock raised in these conditions compete with humans for space and water, and pose a risk to human and animal health. notwithstanding the risk of disease transmission, slum livestock agriculture plays an essential role in the livelihoods of people and deserves consideration in urban planning and policy making. slums are human settlements developing in peri-and urban areas. often not recognized as part of planned urban environments, slums continue to grow in number and size around the world. there are now million to a billion people living in slums, a large percent of the seven billion worldwide. slums are difficult to define based on a single characteristic because they are not homogeneous within cities in a country or among countries; their unique milieu depends on the geographical location of the settlement, the socioanthropological background of the dwellers, and the history of its development. notwithstanding the sociocultural differences, slums have some common characteristics: poor housing, often illegitimately built on private or public land with poor drainage and unfit for agriculture; overcrowded conditions; limited access to potable water; poor sanitation and lack of sewage or waste removal; high numbers of domestic pets; and clandestine keeping of livestock. slums originate from the need of people for a place to live after displacement due to drought, famine, or wars, and migration from the countryside into the cities by the push of rural unemployment and the pull of urban opportunities. this migration from rural to urban areas populates informal settlements in the periphery of cities and towns in many countries (united nations, ) . demographic information from slums is only as good as the reports governments make. less than countries report data on slum human population, and from those, indicate they have more than % of the population living in slums. one country reported that % of the population lives in slum conditions (united nations, ) . in the census in india, million people were identified as living in slums (government of india, ) , which is probably an underestimate. in lesser developed countries, the increase in the urban population dating from the s is parallel to the decrease of the rural population. demographers expect that % of the world's population will live in urban areas by the year and billion people will be living in slums (united nations, ) . in africa, the population in urban areas increased sevenfold in years. population density in some slums has reached people per square kilometer, as in bangladesh (huque, ) . with one in every seven people living in a slum, ensuring sufficient food becomes a concern; slum livestock agriculture could be part of the solution to fulfill the nutritional needs of that many people (united nations human settlements programme, ) . production of food in slums is clearly different from rural, peri-urban, or other types of urban agriculture developing in many industrial countries. urban agriculture had been characterized as a decentralized supply system composed of small and scattered production units (mougeot, ) . there are other classifications of urban agriculture, only some of which include slum agriculture (egbuna, ) . slum livestock keeping is especially difficult to classify. the lack of basic resources to maintain and exercise good agricultural practices coupled with unsanitary slaughter is likely to render slum livestock agricultural products unfit for human consumption. however, despite all the negatives, slum livestock agriculture is a means of securing food, earning income, and supporting livelihoods of slum dwellers. slum agriculture benefits from a large market close at hand, low transportation costs, availability of production inputs, the ability to make use of waste foods and water, and low entry costs to setting up production (food and agriculture organization, ) . informal markets for real estate and goods develop in slums, including markets for processed and unprocessed animal products (potsiou and ioannidis, ) . peri-urban and urban livestock agriculture is a complex and multifaceted activity developing in an environment often unfit for human habitation. roaming chickens, pigs, and goats are a common sight in slums, and even cows and animals used for transportation or hauling such as donkeys and horses can be seen. in different regions of the world, different animal species are popular: goats and sheep are more common in africa and southeast asia, and pigs are more common in latin american slums. food animals in slums are a public health concern due to their potential for transmitting zoonotic diseases, unsafe food products, the risk of physical injuries and traffic accidents, and environmental contamination. animal products processed locally enter the general population food chain through peri-urban and urban food markets. owing to the lack of a proper waste disposal, animal and human waste mixes in the streets with leftover water from household activities. stagnant water and waste, garbage, and other contaminants develop a foul smell and attract vermin and insects. untreated wastewater flows into local waterways and is drunk by roaming animals. this gray water is often used for urban vegetable or crop farming. there are few estimates of the volume of animal or vegetable products originating from slums used to supplement the family diet or entering the food chain through peri-urban or urban markets. in ghana, % of the lettuce consumed in accra is produced in urban and peri-urban areas (amoah et al., ) . in the chimbote slum in lima, peru, where people live, it has been estimated that there are swine production units (chimbotenlinea.com, ) . that is approximately % of the households in the slum assuming there are four members per household. the food and agriculture organization of the united nations has conducted technical consultations and extensive studies on urban and peri-urban agriculture. thirty percent of the meat and % of eggs produced in the world is associated with urban production (food and agriculture organization, ) . it is not clear if slum agriculture production is included in these estimates. slum livestock keeping is typically smaller scale than the peri-urban and nonslum urban agriculture. however, slum agriculture has rarely been the focus of systematic evaluations and studies. the complexity of this type of slum livestock agriculture and its implications in the social and political arena is, however, getting recognition (international livestock research institute, ; lemma and rao, ) , and some evidence is emerging on scale and importance. in maputo ( . million inhabitants), mozambique, more than one in three households in urban areas raises livestock (international livestock research institute, ) . in ibadan in southern nigeria, one in three urban households kept livestock, whereas in kaduna in northern nigeria nearly half the households kept livestock (international livestock research institute, ) . livestock production in slums is typically small to medium scale and low technology. chickens and pigs seem to be ubiquitous in latin american slums, whereas goats and rabbits are favored in africa and southeast asia. cows are noticeable in india and nepal where, for religious reasons, they cannot be slaughtered. some animals in slums are allowed to roam and scavenge whereas others are confined in unsuitable small spaces shared with humans. the scale of this agricultural activity varies in type and size and it can range from subsistence to small-to medium-size commercial enterprises with hundreds of small animals. in more densely populated slums, fewer animals are kept and enterprises are likely to be small scale; where more land is available, livestock keeping is more common and on a larger scale (box ). chickens are animals most commonly raised in slums. one or two hens and one rooster roaming free with a few chicks is a common sight. it is not uncommon to find medium ( - box livestock keeping in dagoretti district, nairobi, kenya dagoretti district lies to the west of nairobi city, km from nairobi city center. it is one of urban districts in nairobi and has around onetenth of the total population. most inhabitants live in tin-roofed timber houses with inadequate access to piped water, sanitation, and electricity. unemployment (approximately %), human immunodeficiency virus-acquired immune deficiency syndrome, crime, and homelessness are persistent problems. dagoretti was originally outside the city of nairobi, but when the boundaries were extended in , it became part of the city. dagoretti district has both government and ancestral land. some ancestral land has been sold to migrants, and government land has also been allocated to individuals. in some areas, squatters live on land temporarily leased out by landlords, a loose agreement, which can be revoked at any time. the population in the last available census was up from in , a vivid illustration of the rapid growth of cities. of the population, % are below years of age, an age distribution that is also typical of developing countries. a recent survey characterized livestock keeping in dagoretti. more than half the households kept animals: in order of declining popularity: dogs ( %); poultry ( %); cats ( %); sheep and goats ( %); pigs ( %); and cattle ( %). a few households kept other animals including, rabbits, donkeys, doves, turkeys, ducks, and geese. dogs were kept mainly for security, cats for vermin control, donkeys for work, geese for security and food production, and the other species were kept mainly for food production. in addition, cattle manure is a valuable by-product used for urban crop and vegetable production, and livestock are an easily liquidated asset that can be sold to meet urgent household needs or act as pledge enabling access to credit. moreover, farmers report deriving social and psychological benefits from livestock keeping. the study focused on dairy cattle kept by in households in dagoretti. farmers kept an average of three cattle. nearly all households kept productive breeds, used artificial insemination, and zero grazing. farms produced approximately l of milk a day, and % of this was sold with the rest being consumed in the household. most of the milk produced is sold in the community through informal markets. all sales of milk earn farmers in dagoretti approximately us per year. farmers reported that both demand and production are increasing. chickens) to larger layer flocks (up to chickens) in confined spaces. chickens are the most versatile of poultry, given the value added of egg production and the worth of the animal, live or processed. in slums with limited electricity or where the cold chain is not available, chickens and eggs are a quick and easy source of protein and income generation. chickens have an additional value because they are used in religious ceremonies and offerings (alves et al., ) . the breeds of chickens vary from region to region and some commercial breeds are seen in slums. scavenging chickens are fed in the morning with leftover food and possibly with added corn or poultry meal. during the day, chickens eat insects, garbage, and organic matter found in empty lots or by the side of the road. housing is rudimentary and may consist of hay or rice husks arranged in cardboard or wooden boxes and placed off the ground for protection from dogs and predators mostly during the night. raising chickens is considered an easy activity requiring minimal labor and is usually performed by women and children (food and agriculture organization, ). backyard flocks requiring additional attention compete for space with humans and other animals and are more laborintensive requiring feeding, watering, and pen cleaning. noise levels and manure disposal become a problem with larger flocks and are a deterrent for keeping them in small and crowded spaces. larger flocks are more common in peri-urban areas than slums, but broiler chickens keepers may have more than birds, as in the case in the mombasa slum in bangladesh where a bird keeper is reported to have between and birds (sabuni, ) . live chickens are transported in cages or burlap bags to nearby food markets. these markets are located at walking distance from the dwelling where the animals are raised, although sometimes birds are transported in cages on top of buses or trucks to other urban markets. chickens are sold live or slaughtered on site at the buyer´s request. eggs have a longer shelf life (lasting - weeks without refrigeration depending on environmental conditions) than poultry meat. eggs are wrapped in newspaper and kept in the shade to prolong their shelf life. they may be sold to neighbors in small numbers, as needed, for income generation or in larger numbers at markets. other poultry kept in slums include turkeys, geese, doves, pigeons, quail, and guinea fowl. rabbits are gaining popularity in slums in africa. they can be kept in small spaces in crates, either individually or in small groups. rabbit crates are easy to build and can be kept stacked up against each other or leaned against a wall to occupy less space. these animals require more attention than scavenging or backyard flocks of chickens for feeding, watering, and cage cleaning. however, it is a profitable and uncomplicated business as evidenced by the number of rabbits a person can keep. one resident in the kahawa soweto slum in nairobi has more than rabbits in a shed (kelto, ) . rabbits are fed greens collected locally and supplemented with vegetable leftover from markets or pelleted food. this is a small species that can reach to kg of weight and can easily be processed locally as chickens for sale or family consumption. in a space of  feet, slum residents can raise more than rabbits of different breeds. more unusual animals are also kept: a survey in nigeria reported snails, grass cutters, and antelope among a total of species kept. pigs adapt easily to urban conditions. one sow can produce - piglets twice or three times a year if their reproductive cycle is timed properly (pregnancy lasts months, weeks, and days). selling the piglets before birth is not uncommon at certain times of the year for events or festivities where pork is commonly consumed. pigs are considered a form of saving account because piglets can be sold for cash or given in exchange for the boar´s service (the piggy bank). boars can grow tusks and become very large and aggressive, requiring confinement to protect people and other animals. this is why fewer people keep boars compared to sows. rearing of pigs in slums in small numbers (one to five) is often done by women and larger herds by men (food and agriculture organization, ) . piglets are easy to manage because they can roam during the day searching for food and come back to their home space at night. they can also be tethered and kept close to the dwelling, or housed in makeshift pens behind or in the confines of the human dwelling. nowadays pigs are ubiquitous to slums in latin america and present even in countries with muslim and hindu population such as india and nepal (rajshekhar, ) . it is difficult to establish the genetics of pigs commonly reared in slums. these are smaller and more adaptable to the local conditions than large commercial breeds. pigs revert to wild type very quickly and grow hair and tusks adapting to the environment. however, pigs kept in confined spaces in and small numbers are usually larger commercial breeds requiring more attention and feeding. the creole pig from haiti was an example of a small hairy dark pig well adapted to the environment, eating garbage and fallen rotten fruit. they had sociocultural value and were considered to have an important role in recycling waste. issues associated with swine fever in the late s and early s prompted the united states department of agriculture and other international organizations to develop and conduct an eradication program. the sociocultural value of these animals was not clearly understood. people were given large commercial breeds as substitutes for the creole pig and asked to pen the pigs. the larger breeds had to be fed and cared for and were not allowed to roam, which resulted in detrimental conditions of the environment in which people lived (ebert, ) . goats are adaptable to urban environments. they are known for eating almost anything, including bark from trees and shrubbery, garbage from streets and land fields, or clothes hanging to dry. these animals require attention, given their inquisitive nature and destructive eating habits. usually people keep a couple of female goats for their milk with the offspring. in slums close to peri-urban areas people keep the goats in pens or house patios during the night and walk them to pasture at different locations during the day. in the urban environment, and in particular in slums, goats roam the streets in search of food, and it is common to see them searching for food with pigs (diogo et al., ) . goats can decimate green areas very quickly, causing damage to vegetables growing by the side of the roads or public open spaces. they require more attention than chickens or pigs and they need shelter from the rain. goats' milk is nutritious and can be easily transformed into yogurt and cheese to extend the shelf life of an otherwise perishable product. in some large slums such as in delhi, india, slum goats' sightings have become a tourist attraction and are offered as part of city tours (news.com.au, ) . mixed bos taurus taurus and bos taurus indicus cattle are let free in search of food or water or walked to pasture in the mornings and brought back at night. in countries with a predominantly hindu population cows are sacred and only the milk is consumed. oxens are mainly kept for draught power; they are used for plowing and also transport. owing to religious reasons female cattle cannot be disposed of after their productive lives have ended. older unproductive animals may be abandoned and may die of diseases, hunger, intoxication from garbage, or get involved in traffic accidents. there are also many goshalas in india where abandoned cattle are looked after. milk is a valued product that contributes to food security in slums. but it is perishable and has to be consumed quickly or transformed into a variety of cheeses, yogurt, or sour milk. naturally fermented sour milk is popular in east and west africa; fermenting also removes lactose, which makes it easier to digest for populations that do not have lactase enzymes persisting in adulthood. tending larger animals is time consuming and may involve walking long distances to find a place to feed away from the city or to cut grass that can be brought back to the animal. this is easier to do when the slum is located in the periphery of a city compared to an urban-area slum. often cattle are confined to a small space or their mobility may be limited by tethering them close to the dwelling. studies of peri-urban dairying in east africa found that nearly all cattle were 'zero grazed,' that is, kept inside all the time (figure ) . cow dung in india is also a valuable fuel: it is collected, reshaped into cake-like forms, and dried. in india and elsewhere cattle dung is also used as a building material mixed with mud and other substances. livestock value is more than monetary; it is a symbol of social status and social identity both individually and collective (comaroff and comaroff, ) . the value of livestock and the production parameters used elsewhere (such as in the industrial countries) need to be revaluated because a cow or goat whose production is low may still be providing useful services (dutta, ) . in slums, an intricate relationship exists between poverty, food security, livestock keeping, and the environment. enforcing livestock regulations becomes a difficult task when animal ownership is clandestine. in many countries regulations cover animal health and welfare issues, the disposal of dead animals, slaughter procedures, and environmental contamination, but in practice they are not enforced in slums. in other cases, regulations are used for 'rent-seeking' by authorities: that is police or other officials will confiscate animals and not give them back unless a bribe is paid. keeping animals in cities undoubtedly creates environmental problems. for example, in kisumu, kenya, keeping animals is illegal. the city has six slums and enforcing animal farming law is a challenge. the dung of the animals becomes a problem when the rain washes it and contaminates the city water supply. the city lacks the infrastructure to recycle the dung and % of it is not utilized (new agriculturist, ). yet the draconian response of banning livestock from cities may not be the most appropriate. in kampala, uganda, a process lasting several years has recently led to new city ordinances that seek to support urban agriculture as an important economic activity, while regulating against the potential adverse effects. animals of the same or different species in slums are in proximity with each other and with humans. contagious diseases can spread rapidly under these conditions. additionally, slum-raised animals suffer from malnutrition coupled with an adverse environment that makes them more susceptible to disease or injury (food and agriculture organizations, ). some problems are associated with animals living in areas with poor waste disposal. pigs and goats eat plastic bags and these are common findings at slaughter or necropsy. the bags fill the stomachs impeding food digestion and nutrient absorption. rwanda banned nonbiodegradable plastic bags in , an excellent example for other african countries where plastic waste is ubiquitous. the health of animals in slums requires attention, but estimating disease and its impact is difficult. both zoonotic (diseases transmitted from animals to people) and nonzoonotic animal diseases need to be considered. reportable, zoonotic, and production diseases are discussed elsewhere in this encyclopedia. some of these diseases are of national public health interest or are of international trade importance requiring immediate reporting through specific official public health channels and to the world organisation for animal health, one of the three sisters of the world trade organization. reportable diseases of animals are those with the potential to cause epidemics or pandemics that cause serious disease in people, as is the case of the severe acute respiratory syndrome known as sars, the h n (pathogenic avian), and h n influenza virus (known as swine influenza) (gauthier-clerc et al., ; girad et al., ) . public health resources and market inspections are deployed for some of these diseases, but control is often ineffective in poor countries. determining the public health penetration of these programs in slums has been highlighted as a need (unger and riley, ) . risk communication in a crisis situation involving the livestock population in a slum may require additional resources and targeted programming. the lack of information of the size and location of the avian and swine population may impede the progress of disease control measures. public awareness programs or the application of rigid standards for disease control without consideration of the sociocultural and economic value of the animals for people may also decrease compliance (box ). references to the flu pandemic are always brought to light in the media as the possible serious consequences of disease spread and death. however, public health crisis response is different today and includes emergency and disaster preparedness, first responders training programs, and damage mitigation and relief. nonetheless robust economic estimates by the world bank suggest that the cost of an epidemic originating in animals could be a trillion dollars (world bank, ). there are approximately zoonoses or diseases transmitted from animals to humans. disease transmission may be direct through contact with infected animals or its fluids, or through contaminated animal products, water, or objects contaminated with infectious material (center for disease control and prevention, cdc / , ). especially important for poor countries are the so-called neglected zoonoses often associated with poverty, poor hygiene, and poor understanding of disease transmission. the rest of the article discusses some neglected zoonoses that are known or likely to be a problem in slums. brucella melitensis, the cause of brucellosis, is very pathogenic to humans (center for disease control and prevention, ). it is transmitted from goats and sheep to humans through direct or indirect contact. unpasteurized milk or contact with body fluids of infected animals are considered the most common transmission routes. there are other brucellae affecting swine, cattle, and dogs, but melitensis is associated with the more severe forms of the disease. flaying and skinning sheep by blowing air through a cut in the skin exposes people to body fluids and inhalation of bacteria from infected animal, and hence is not recommended. cysticercosis is a disease caused by the larval stage of the tapeworm taenia solium. the disease is sometimes wrongly known as the 'the pig tapeworm,' but humans are the definitive host for the adult form of the parasite, not the pig. the adult parasite releases segments daily containing thousands of eggs into the gut. these eggs pass into the environment with the feces. humans and pigs become infected and develop cysticercosis by ingesting the parasite eggs. pigs are coprophagic and human defecation in open spaces is one of the main forms of transmission of the disease from humans to pigs. when eaten by pigs, the eggs develop into cysts within the pig (figure ). if people eat meat containing viable cysts these can develop to tapeworms in the human host, thus completing its cycle. human to human transmission occurs when people harboring the parasite contaminate food due to poor hand hygiene (garcia et al., ; carrique-mas et al., ) . autoinfection can also occur following improper hand washing after a bowel movement. after humans consume eggs, cysts start to develop in any organ of the body, including the brain. when cysts develop in the brain, the disease is known as neurocysticercosis. it is considered as one of the most common infestations of the brain in humans. the main manifestation of the disease is epilepsy (garcia et al., ) . studies from a slum in india revealed that . % of people in the studied sample who had active epilepsy had antibodies to t. solium (singh et al., ) . cysticercosis in pigs may reach % prevalence or more in some countries where pigs are allowed to roam and human defecation is done in the open (carrique-mas et al., ; fleury et al., ; flisser et al., ) . even with low human taeniasis in the order of - %, the prevalence of cysticercosis in pigs can be high. this is mainly due to the fact that only adult parasite can live in the intestine of the host for years and release thousands of eggs into the environment. hepatitis e virus is an enteric disease transmitted from different species of animals including pigs to humans; it can be transmitted between humans through contaminated water and food. the seroprevalence in the human population unexposed to swine is % whereas the seroprevalence in swine workers or other swine-exposed populations is % (whithers et al., ) . data from a study of a pediatric population in karachi with access to municipal piped water and nonflush toilets in a slum in india showed a high seroprevalence for hepatitis e virus (jafri et al., ) . the exposure to hepatitis e virus seems lower than hepatitis a (mohanavalli et al., ) . hepatitis e is a disease that can cause severe gastrointestinal symptoms and has been associated with high death rates of pregnant women in lesser developed countries. in some peri-urban farms pigs are raised close to ponds where fish are kept. the pig feces are washed down to the ponds for the fish to eat. the virus seems to be ubiquitous in swine populations and undistinguishable even in samples from different countries. this was the case in hepatitis e study in two farming communities (not slum conditions), in north carolina and costa rica (whithers et al., ; kase et al., ) . hepatitis e is a zoonosis, but the importance of animals in its maintenance and transmission has not been fully established. a pandemic of h n influenza was declared in . this disease originated in pigs but subsequently was maintained entirely by human to human transmission. in response to the pandemic, the government of egypt ordered all of the country's pigs to be slaughtered. because humans can only get the new flu from other humans this was not effective as a control response. it also had far-reaching and unintended consequences on the cities' waste management. cairo's garbage collectors used to feed the city's organic waste to pigs and their livelihood became endangered while the streets of the capital filled up with trash. source: adapted from the new york times article, mona el-naggar contributed reporting, september . available at: http://www. nytimes.com/ / / /world/africa/ cairo.html_r= &scp= &sq= michael% slackman% cairo% pigs&st=cse& (accessed . . ). cryptosporidium is associated with cattle and causes diarrheal disease in humans and cattle. although extensively studied in developed countries, it has not been diagnosed until recently in slums. surveyed households in the nairobi´s dagoretti district, kenya, determined the extent of cattle keeping and the prevalence of cryptosporidiosis. with a prevalence of % in urban cattle, this parasite could be a major contaminant of water sources and disease that needs further consideration (international livestock research institute, ) (figure ) . antibiotics and other veterinary drugs administered to animals without proper veterinary supervision can have direct and indirect consequences for human health. after administration of an antibiotic there is a period of time when the animal product should not be consumed. this period known as the withdrawal period is to allow the drug in question to clear the animal's system because it may have harmful health effects if consumed by people who are allergic or sensitive to the drug. for example, clenbuterol is commonly used as a growth promoter (often illegally), and several outbreaks of illness have occurred when people consume livestock products from animals treated with clenbueterol. of potentially greater importance than sickness as the result of consumption of residues is the risk of bacteria developing resistance to antibiotics because of their use in agriculture. as a result of these concerns, many countries require that antibiotics and some other animal health drugs should only be prescribed by veterinarians. however, this is not practicable in most slums. veterinary services may be out of reach for many people in slums either because of distance or cost. in some cases, livestock owners may visit a veterinary office and explain the symptoms of the disease to the veterinarian, but without proper examination of the animal and the information received, the medicines may not be appropriately prescribed. the livestock owner may not fully understand how to administer the medicines and use a single dose of a medicament keeping the rest in case the animal does not improve. getting advice from another family member or neighbors, purchasing a single dose of an antibiotic from a street vendor, or using leftover medicines from a previous case is not uncommon. veterinary services are often expensive and out of reach of the poor (ahuja et al., ) . in addition, farmers may use drugs for nonhealth purposes. in khartoum, the practice of adding antibiotic to milk to preserve it was reported, and this could have health consequences such as allergy in the people ingesting the product (hassabo et al., ) . in china, melamine was added to milk so that it would appear to have higher protein levels. this lead to one of the largest food safety events of recent years resulting in the death of six infants; the children died from kidney stones and thousands were hospitalized as a result of drinking the contaminated milk (wei and liu, ) . slum agriculture and associated markets are part of the informal sector of the economy. it is a business model based on necessity, mostly home-based, and often clandestine. when there is no proper access to water and refrigeration, meat and eggs must be consumed rapidly, processed, and sold close to the place of origin. markets in slums are public places where vendors congregate. products may also be sold by hawkers moving from door-to-door or as requested. in many cities in developing countries milk is sold in this way. it is not uncommon to sell products from homes through modified window sills, from makeshift sale racks in corridors between houses, or on top of blankets or plastic sheets in the floor in open spaces. slum dwellers acquire knowledge of who produces and sell different items. prices vary based on perceived notions of quality, hygiene, or availability. the exchange of money is not always the norm for payment and people engage in bartering or exchanging products for services. agricultural markets in slums differ from municipal markets where there is an operational legal framework and the state is responsible for the enforcement of public health and food safety regulations. however, the great majority of food sold in municipal markets lack any structured sanitary inspection. this does not mean that the markets in slums lack internal form of self-regulation, they have a sui generis operational business model. although these markets are outside the purview of state regulation and disease control and preventative measures, the prevailing food-safety actions are based on accepted cultural norms. these norms are developed by the people who live, sell, and purchase products under these conditions or based on religious beliefs or restrictions (rheinländer, ) . the number, identification of species, and location and ownership of animals in a slum is not usually performed. the lack of census data and owner identification poses a challenge for the implementation of national disease control or eradication programs. required reporting of diseases and animal or product traceability to origin may be impossible in case of a food-related outbreak. this is contrary to the application of the sanitary and phtyo-sanitary measures of the world trade organization. most countries agree to adhere to these measures when they join the world trade organization. conducting an animal census in a slum is challenging but without knowing how many animals there are, vaccine campaigns or eradication programs cannot be conducted. however, the legally ambiguous position of livestock in slums mean owners are reluctant to incur official notice. the inspection of animal facilities and product transformation industries destined for human consumption is highly regulated. although in many developing countries animals must be processed in municipal slaughterhouses, at home and clandestine slaughter are common. in bolivia, some of the municipal slaughterhouses are rudimentary and managed by the local communities. the official veterinarian is allowed to inspect the animals preslaughter and outside the facility. the inspection concerns mostly reportable diseases of national importance. inside the slaughterhouse, skilled butchers castrate male pigs, and these and other animals bleed on the floor as part of the exsanguination process. once the internal organs are removed the carcass is hung on a wall hook and employees rub the pigs´canal with a rag. the same rag and water stored in a bucket is used to wash several carcasses. an inquiry into the practice revealed the employees neither understood why they were doing the cleaning nor how it was supposed to be done (correa et al., ) . in ibadan, nigeria facilities are even less developed. approximately - cattle are slaughtered daily; more are slaughtered on weekends, and fewer during muslim holidays. cattle are kept in pens, then moved to the slaughter slab. they are tied down at the slab and killed by cutting the throat. the dead cattle are then dragged on the ground to the abattoir area. this is simply a shed with a concrete floor and open sides. removal of the intestines and quartering is done on the floor. portions of the carcass are then carried to the adjacent butchers' stalls where they are sold. the abattoir is under municipal management and officers collect tax and tariffs on each cow amounting to us$ per animal. the role of environmental sanitary officers is to inspect slaughter slabs and the general environment and ensure the area is clean. however, the filthy conditions of the market witness the challenges they face in carrying out their work. the veterinary department is supposed to check animals before slaughtering and inspect meat after slaughter, but many animals escape inspection, and even when problems are found veterinarians find it difficult to ensure condemned meat is discarded. most butchers kill only one animal a day, and if this is condemned by veterinarians as unfit for human consumption they lose their entire day's earnings. hence, they strongly resist attempts to condemn meat (grace et al., ) . slaughter practices are probably clearly specified in pertinent regulations, but without oversight or evaluation by competent authorities, wrongly learned and applied practices may never be corrected. new employees learn from other employees and the practice is passed down, transformed, and perpetuated. the 'trichina inspectors' in livestock markets in bolivia are lay people trained by other people to inspect the tongue of pigs in order to detect cysts associated with cysticercosis. trichina is a different parasite and the tongue inspection is not associated with this parasite, but t. solium. the wrong parasite reference has prevailed and the practice continues unchecked for efficiency in preslaughter diagnosis of cysticercosis in pigs. in ecuador´s countryside, a traveling butcher works at the community slaughterhouses at scheduled days and times of the week and processes the carcasses of mainly large species at the municipal facility. this is a trained person who can condemn carcasses from sick animals and has an important food safety role. however, the most predominant form of slaughter in slums is the one done at home, in the streets, between or behind buildings. usually for smaller species the head is removed using a sharp knife or axe against a hard surface like a piece of wood or the stump of a tree. the internal organs are removed swiftly; sometimes the esophagus and the rectum are tied to avoid intestinal content spillage and contamination. animals processed in this manner are not properly exsanguinated. raw offal may attract dogs and vermin. the meat, without refrigeration must be consumed immediately or it is left to dry on racks. meat maybe carried to markets in small carts, buckets, or burlap bags and sold in small pieces or cut by the client´s request. the meat is not inspected when sold in the slums to neighbors or at smaller slum markets. in nepal, animal parts are sold with other identifiable body parts like a hoof or horns in order to guarantee that the species being sold is the actual species. in many cases the meat is covered in turmeric, reported to be done to decrease contamination (oral tradition reported to authors by street vendors). interestingly, homeopathy studies suggest that turmeric can help combat bacterial infections (krup et al., ; vasavda et al., ) . slum livestock agriculture interweaves its existence with the geographic location and culture of the population. people take advantage of their new environment and recreate a way of living based on their upbringing, including raising and processing livestock. the main motivations for this activity are family subsistence, income generation, and social status. slum livestock agriculture activities range from subsistence to semicommercial production. it plays a role in the livelihood of millions of people providing animal protein and generating income. however, it lacks technical sophistication and typically operates outside regulatory purview, and its practice overlooked and tolerated by local authorities. animals pose a risk to human health and other animals, are a public nuisance, and contribute to the waste accumulating on streets and the run off contaminating the environment and water sources. livestock products, raw or processed are sold in street markets and may enter the urban food chain. animal ownership is difficult to establish in slums and livestock population difficult to determine. under these circumstances, veterinary services and disease-control programs may be hard to implement. good hygiene, good manufacturing practices, and animal slaughter inspection turn into an impossible task to implement and enforce. disaster and white-coat diplomacy practices combined with information and communication technologies should be considered in the planning and implementation of public awareness and outreach programs (lemery, ; lin and heffernan, ) . the elimination of the slums is not feasible. the magnitude of the animal population in slums is unknown, but its food security importance is getting recognition from public and private organizations. there is a need for a community-led approach in collaboration with private and public institutions, academic, international, and nongovernmental organizations to find sustainable solutions. the delivery of veterinary services to poorer communities: the case of rural orissa. revue scientifique et technique de l commercialization of animal-derived remedies as complementary medicine in the semi-arid region of northeastern brazil irrigated urban vegetable production in ghana: microbiological contamination in farms and markets and associated consumer risk groups an epidemiological study of taenia solium in rural population in the bolivian chaco center for disease control and prevention, cdc / , . when humans and animals intersect criaderos clandestinos de cerdos se incrementan en pueblos jóvenes goodly beasts, beastly good: cattle and commodities in a south african context proceedings of the th international symposium on veterinary epidemiology and economics. available at: www.sciquest.org.nz resource use efficiency in urban and peri-urban sheep, goat and cattle enterprises the problem of huge unproductive livestock population in india north american 'swine aid' and economic disaster for haitian peasants urban agriculture: a strategy for poverty reduction in nigeria spotlight issues in urban agriculture studies suggest that up to two-thirds of city and peri-urban households are involved in farming livestock keeping in urban areas. fao animal production and health papers challenges of animal health information systems and surveillance for animal diseases and zoonoses neurocysticercosis is still prevalent in mexico neurocysticersosis: regional status, epidemiology, impact and control measures in the americas teniasis/ cisticercosis por taenia solium, un serio problema de salud pública en el perú recent expansion of highly pathogenic avian influenza h n : a critical review the a (h n ) influenza virus pandemic: a review the influence of gender and group membership on food safety: the case of meat sellers in bodija market usage of antibiotic as milk preservative in slums of khartoum state urban slum mapping in bangladesh. columbia conference presentation urban agriculture and zoonoses in nairobi living with livestock, and livestock livings, in the city seroprevalence of hepatitis e and helicobacter pylori in a low socioeconomic area of a metropolitan city in a developing country isolation, detection and characterization of swine hepatitis e virus from herds in costa rica livestock in the slum: a visit to an urban farm in kenya. farming livestock in african slums pharmacological activities of turmeric (curcuma longa linn): a review of a case for white coat diplomacy environmental and social sustainability of urban and periurban agriculture (upa) in selected towns of ethiopia creating the livestock guru: icts to enhance livestockrelated knowledge among poor households in orissa prevalence of antibodies to hepatitis a and hepatitis e virus in urban school children in chennai urban agriculture: definition, presence, potentials and risks available at cleaning up its act: recycling livestock waste slum tour in dharavi, mumbai with reality tours and travel informal settlements, real estate market need for good land administration and planning epidemiology of taenia solium taeniasis/cysticercosis in india and nepal street food quality. a matter of neatness and trust broiler chicken market in slums association between epilepsy and cysticercosis and toxocariasis: a population-based case-control study in a slum in india slum health: from understanding to action world urbanization prospects. economic and social affairs united nations human settlements programme pharmacological activities of turmeric (curcuma longa linn): a review review of melamine scandal: still a long way ahead antibody levels to hepatitis e virus in north carolina swine workers, non-swine workers, and murids growing greener cities in africa proper slaughter and flaying of sheep and goat. ethiopia sheep and goat productivity improvement program key: cord- -u ibep authors: kulpa-eddy, jodie; srinivas, geetha; halder, marlies; hill, richard; brown, karen; roth, james; draayer, hans; galvin, jeffrey; claassen, ivo; gifford, glen; woodland, ralph; doelling, vivian; jones, brett; stokes, william s title: non-animal replacement methods for veterinary vaccine potency testing: state of the science and future directions date: - - journal: procedia in vaccinology doi: . /j.provac. . . sha: doc_id: cord_uid: u ibep abstract niceatm and iccvam convened an international workshop to review the state of the science of human and veterinary vaccine potency and safety testing methods and to identify opportunities to advance new and improved methods that can further reduce, refine, and replace animal use. six topics were addressed in detail by speakers and workshop participants and are reported in a series of six reports. this workshop report, the second in the series, provides recommendations for current and future use of non-animal methods and strategies for veterinary vaccine potency testing. workshop participants recommended that future efforts to replace animal use give priority to vaccines ( ) that use large numbers of animals per test and for which many serials are produced annually, ( ) that involve significant animal pain and distress during procedures, ( ) for which the functional protective antigen has been identified, ( ) that involve foreign animal/zoonotic organisms that are dangerous to humans, and ( ) that involve pathogens that can be easily spread to wildlife populations. vaccines identified as the highest priorities were those for rabies, leptospira spp., clostridium spp., erysipelas, foreign animal diseases (fad), poultry diseases, and fish diseases. further research on the identification, purification, and characterization of vaccine protective antigens in veterinary vaccines was also identified as a priority. workshop participants recommended priority research, development, and validation activities to address critical knowledge and data gaps, including opportunities to apply new science and technology. recommendations included ( ) investigations into the relative impact of various adjuvants on antigen quantification assays, ( ) investigations into extraction methods that could be used for vaccines containing adjuvants that can interfere with antigen assays, and ( ) review of the current status of rabies and tetanus human vaccine in vitro potency methods for their potential application to the corresponding veterinary vaccines. workshop participants recommended enhanced international harmonization and cooperation and closer collaborations between human and veterinary researchers to expedite progress. implementation of the workshop recommendations is expected to advance alternative in vitro methods for veterinary vaccine potency testing that will benefit animal welfare and replace animal use while ensuring continued protection of human and animal health. veterinary vaccines contribute to improved human and animal health and welfare by preventing and controlling infectious agents that can cause disease and death. however, the testing necessary to ensure vaccine effectiveness and safety can involve large numbers of animals and significant pain and distress. in the united states, the interagency coordinating committee on the validation of alternative methods (iccvam) and the national toxicology program interagency center for the evaluation of alternative toxicological methods (niceatm) promote the scientific validation and regulatory acceptance of test methods that accurately assess the safety of chemicals and products while reducing, refining (less pain and distress), and replacing animal use. accordingly, niceatm and iccvam recently identified vaccine potency and safety testing as one of their four highest priorities [ ] . iccvam is an interagency committee of federal agencies that is charged by law with evaluating new, revised, and alternative test methods with regulatory applicability. iccvam members represent u.s. federal regulatory and research agencies that require, use, generate, or disseminate safety testing data. these include the department of agriculture (usda), which regulates veterinary vaccines, and the food and drug administration (fda), which regulates human vaccines. iccvam is a permanent interagency committee of the national institute of environmental health sciences (niehs) under niceatm. niceatm administers iccvam, provides scientific and operational support for iccvam-related activities, and conducts international validation studies on promising new safety testing methods. niceatm and iccvam serve a critical public health role in translating research advances from the bench into standardized safety testing methods that can be used in regulatory practice to prevent disease and injury. to promote and advance the development and use of scientifically valid alternative methods for human and veterinary vaccine testing, niceatm and iccvam organized the international workshop on alternative methods to reduce, refine, and replace the use of animals in vaccine potency and safety testing: state of the science and future directions. the workshop was held at the national institutes of health in bethesda, maryland, on september [ ] [ ] [ ] . it was organized in conjunction with the european centre for the validation of alternative methods (ecvam), the japanese center for the validation of alternative methods (jacvam), and health canada. the workshop addressed the state of the science of human and veterinary vaccine potency and safety testing. participants developed recommendations for future progress in three major areas: ( ) in vitro replacement methods for potency testing; ( ) reduction and refinement methods for potency testing; and ( ) reduction, refinement, and replacement methods for vaccine safety testing [ ] . reports were prepared for each of the three topics for human vaccines and for each of the three topics for veterinary vaccines [ , , , , , ] . this report addresses methods and strategies for the replacement of animal use for potency testing of veterinary vaccines. the goals of the international workshop were to ( ) identify and promote the implementation of currently available and accepted alternative methods that can reduce, refine, and replace the use of animals in human and veterinary vaccine potency and safety testing; ( ) review the state of the science of alternative methods and identify knowledge and data gaps that need to be addressed; and ( ) identify and prioritize research, development, and validation efforts needed to address these gaps in order to advance alternative methods that will also ensure continued protection of human and animal health. the workshop was organized with four plenary sessions and three breakout group sessions. in the breakout sessions, workshop participants: identified criteria to prioritize vaccine potency and safety tests for future alternative test method development and identified high priorities using these criteria reviewed the current state of the science of alternative methods and discussed ways to promote the implementation of available methods identified knowledge and data gaps that need to be addressed identified and prioritized research, development, and validation efforts needed to address these gaps in order to advance alternative methods while ensuring continued protection of human and animal health the workshop opened with a plenary session in which expert scientists and regulatory authorities from the united states, europe, japan, and canada outlined the importance of vaccines to human and animal health [ , ] and described national and international regulatory testing requirements for human and veterinary vaccines [ , , , , , , ] . authorities emphasized that, following the regulatory approval of a vaccine, testing is required to ensure that each subsequent production lot is pure, safe, and sufficiently potent to generate a protective immune response in people or animals [ , ] . the second plenary session addressed methods that have been accepted and methods that are in development that do not require the use of animals for assessing the potency of vaccines [ , , , ] . this was followed by breakout sessions to discuss the state of the science and recommendations for future progress for in vitro potency tests for human and veterinary vaccines. this paper provides workshop recommendations to advance the use and development of alternative methods that can replace animals for the potency testing of veterinary vaccines. recommendations for human vaccines are available elsewhere in these proceedings [ ] . the third plenary session addressed ( ) potency testing methods that refine procedures to avoid or lessen pain and distress by incorporating earlier humane endpoints or by using antibody quantification tests instead of challenge tests and ( ) methods and approaches that reduce the number of animals required for each test [ , , , , , , ] . breakout groups then discussed the state of the science and developed recommendations for future progress. workshop recommendations to advance the use and development of alternative methods that can reduce and refine animal use for potency testing of human vaccines [ ] and veterinary vaccines [ ] are available in the respective papers in these proceedings. the final plenary session addressed methods and approaches for reducing, refining, and replacing animal use to assess the safety of serial production lots of human and veterinary vaccines [ , , , ] . breakout groups then discussed the state of the science and developed recommendations for advancing alternative methods for vaccine safety testing. workshop recommendations to advance the use and development of alternative methods for safety testing of human vaccines [ ] and veterinary vaccines [ ] are available in these proceedings. strict regulations and guidelines are designed to ensure that every veterinary vaccine distributed in or from the united states is pure, safe, potent, and effective [ ] . an estimated , serials (batches) of veterinary vaccines are released annually in the united states for approximately different products that protect animals from different animal diseases [ ] .given that many inactivated vaccines still require animals for potency testing, significant numbers of animals are necessary. veterinary vaccines contribute to the health and well being of people and animals. in addition to controlling and preventing diseases of companion and domestic animals, vaccines help ensure a safe and efficient global food supply. they reduce the transmission of zoonotic and foodborne infections from animals to people. vaccines also reduce the need for low-level antibiotics to control some diseases in food animals. due to the number of animals used annually for the release of veterinary vaccines, global regulatory agencies actively encourage the evaluation, development, and implementation of novel approaches that reduce, refine, and replace ( rs) the use of animals in vaccine safety and potency product release testing [ , , ] . potency testing procedures for many veterinary vaccines still require the use of animals; therefore, the development and validation of additional replacement tests could significantly benefit animal health and welfare. workshop participants prioritized the veterinary vaccines that should be targeted for further development and validation of in vitro replacement tests. the criteria for prioritization included: vaccines that use large numbers of animals per test and for which many serials are produced annually vaccines that involve significant animal pain and distress during testing procedures vaccines for which the functional protective antigen has been identified and characterized vaccines that involve foreign animal/zoonotic organisms vaccines that involve pathogens that can be easily spread to wildlife populations based on these criteria, the following vaccines were given highest priority for further development of alternative replacement methods: rabies vaccines leptospira spp. vaccines clostridium spp. vaccines erysipelas vaccines vaccines for foreign animal diseases (fads) especially those posing viral biohazards that require enhanced security and biosafety measures (e.g., foot and mouth disease [fmd] and bluetongue disease) poultry vaccines fish vaccines new vaccines that are currently undergoing prelicensing development and evaluation rabies, leptospira spp., and clostridium spp. vaccines were identified as the highest priorities because their required potency tests use large numbers of animals and involve significant pain and distress. for example, analysis of serials released in the uk between and indicated that potency tests involving live challenge testing for leptospira spp. and rabies vaccines accounted for a high proportion (> %) of animals used in batch potency testing [ ] . vaccine challenge tests that require live viruses and bacteria that are hazardous to laboratory workers, livestock, companion animals, and wildlife were also considered high priorities (e.g., rabies and fmd vaccines). in addition, prioritization of vaccines for which the functional protective antigen has previously been identified would greatly facilitate the successful development of antigen quantification methods. finally, new vaccines were included as high priorities in order to encourage the development of replacement alternatives early in the development cycle. as shown in table , several of the vaccines identified as high priorities, those that currently use animals in vaccination-challenge or toxin-neutralization testing, have alternative serology methods either in development or accepted for use by specific regulatory authorities. therefore, validated refinement methods already exist and represent critical first steps toward the ultimate goal of identifying in vitro replacement methods for these highpriority vaccines. for many veterinary vaccines, regional differences affect the availability and implementation of in vitro replacement assays. for example, the usda published an in vitro elisa potency test for inactivated swine erysipelas vaccine (erysipelothrix rhusiopathiae), while the european directorate for the quality of medicines & healthcare (edqm) published a mouse-based serology test in the european pharmacopoiea (ph. eur.) ( table ) . the edqm has developed, validated, and approved an in vitro test for inactivated newcastle disease vaccine that is not a standard requirement in the united states ( table ) . clearly, improved international communication and harmonization may expand the number of veterinary vaccines for which replacement methods are available and/or accepted for use. however, regional differences in disease status, product composition, number of manufacturers, and funding may all affect priorities established in those specific regions. current veterinary vaccines consist of ( ) modified live (attenuated) virus and bacteria, ( ) inactivated (killed) viruses and bacteria, ( ) toxoid or bacterin toxoids, ( ) peptide and subunit vaccines, and ( ) genetically engineered products. the general types of potency tests employed by vaccine manufacturers include the following: titration of live organisms (in vitro but occasionally in vivo) in vitro assays such as elisas or other quantitative methods serology methods (in vivo to in vitro) vaccination-challenge in vivo methods using either the host animal (fish, poultry) or laboratory animals (e.g., hamsters, mice) [ ] for a typical u.s. veterinary vaccine manufacturer, % of tests use in vitro titration assays, % use in vitro elisas, % use some other in vitro method, % use in vivo serology test, and % use in vivo vaccinationchallenge methods [ ] . these data exclude poultry and fish vaccine potency testing but do suggest that in vitro methods are being applied for most potency testing conducted on veterinary vaccines. animal welfare concerns, increased scientific accuracy, and the financial benefits associated with in vitro assays provide significant incentives to veterinary vaccine manufacturers for the replacement of animals for potency testing procedures, especially if a vaccine product can be released without the potential concern for repeat in vivo testing [ , ] . in vitro potency testing procedures are currently used in the release of many modified live (attenuated) and genetically modified vaccines ( table ) but are not widely used for the potency release of inactivated vaccines. in the united states, the usda's center for veterinary biologics (cvb) publishes many supplemental assay methods (sams) that provide detailed, validated protocols for the safe and effective potency testing of specific veterinary vaccines. to further facilitate the use of alternative in vitro methods, the cvb and other regulatory authorities provide many of the critical reagents and reference standards necessary to conduct these potency assays. in vitro potency methods for the quantification of several modified live bacterial vaccines are currently outlined in publicly available usda sams. for example, enumeration methods that quantify the colony-forming units (cfus) of specified live organisms are described for brucella abortus [ ] , erysipelothrix rhusiopathiae [ ] , and avirulent pasteurella haemolytica (new name mannheimia haemolytica) [ ] vaccines. in addition, the cvb has published an in vitro potency assay that uses indirect fluorescent antibody staining of inoculated cell culture to quantify bacterial titers for chlamydophila felis (formerly feline chlamydia psittaci) [ ] . as the majority of bacterial vaccines for veterinary use are inactivated, toxoid-or bacterin-toxoid-based, there are relatively few modified live bacterial vaccines available for veterinary use. for live or genetically engineered viruses, virus titration is performed in cell cultures using endpoints such as plaque formation; cytopathology; and, indirectly, virus neutralization by virus-specific serological reagents. for example, in vitro titration assays utilizing the enumeration of plaque-forming units (pfus) are available for feline calicivirus [ ] , feline rhinotracheitis virus [ ] , and marek's disease vaccines [ ] . for other live viral vaccines, the virus is quantified by determining its cytopathic effect in primary cell culture. these include vaccines for the following: porcine transmissible gastroenteritis [ ] porcine rotavirus [ ] infectious canine hepatitis [ ] canine adenovirus [ ] canine distemper [ ] infectious bursal disease [ ] finally, some modified live viral vaccines, such as those for feline panleukopenia [ ] and canine parvovirus [ ] , quantify virus titers using direct or indirect fluorescent antibody staining of virus-inoculated cell cultures. although these assay methods are approved by the usda, it is often difficult to estimate which procedures are routinely used to release vaccine products because product-specific validation is required. however, it is estimated that approximately % of all u.s. veterinary vaccine serials are now released based on in vitro potency testing [ ] . examples of modified live veterinary vaccine potency assays that do not require the use of animals are provided in table . other live vaccines, such as mink distemper virus vaccines [ ] , use an alternative in vitro system to quantify viral content by counting viral plaques that grow on the chorioallantoic membrane of inoculated chicken embryos. for live chicken embryo-adapted chlamydophilia felis vaccine [ ] , embryonated chicken eggs are used as the indicator host system to determine vaccine titer ( table ). in addition, a procedure is available for titrating newcastle disease virus (ndv), infectious bronchitis virus (ibv), and combination ndv-ibv vaccines through the inoculation of embryonated chicken eggs in order to calculate the % egg infective dose (eid ) [ ] . the majority of modified live vaccines use in vitro methods for potency release, however, some live attenuated vaccines, such as ovine ecthyma vaccine for sheep [ ] , still require a target animal vaccination-challenge potency test. for many inactivated veterinary vaccines, especially bacterial vaccines, a key hurdle to the successful development of in vitro antigen quantification assays is the lack of protective antigen identity and the inclusion of complex adjuvants in vaccine formulations [ ] . therefore, many inactivated veterinary vaccines still require in vivo methods (i.e., serology or vaccination-challenge methods) for determining relative potency. however, there are specific examples in which the protective antigen for an inactivated bacterial vaccine has been identified and used to develop a specific elisa quantification assay based on comparison to a reference standard of antigen. these include reference standards available from cvb or product-specific standards developed by the manufacturer. examples include erysipelothrix rhusiopathiae bacterins for kd protein [ ] and escherichia coli bacterins testing for k pilus [ ] , k pilus [ ] , p pilus [ ] and p pilus [ ] . the development of the swine erysipelas potency test also included extensive work to develop humane endpoints [ ] and an elisa serology test [ ] . for the potency determination of various leptospira interrogans serovars, an in vitro elisa assay is used to measure the relative potency of specific bacterins compared to a suitably qualified reference standard, such as the one available from the cvb. the leptospira interrogans serovars tested in this way include pomona [ ] , canicola [ ] , grippotyphosa [ ] , and icterohaemorrhagiae [ ] . published in vitro assays are also available for selected inactivated virus vaccines. for example, the potency of an inactivated respiratory cattle vaccine containing several bovine respiratory viruses (bovine diarrhea [bvd], bovine respiratory syncytial virus [brsv], bovine rhinotracheitis [brv], bovine parainfluenza [pi ]] is determined using an elisa assay relative to a reference standard [ ] . additional in vitro methods have been published for feline leukemia virus gp antigen quantification [ ] and inactivated canine coronavirus vaccines [ ] . an in vitro elisa antigen quantification assay for inactivated ndv vaccine has been developed and validated by the edqm [ , , , ] . the successful transition from an in vivo assay to an in vitro elisa was aided by the fact that there was a strong correlation between antigen content and antibody response. the antigen-specific antibodies correlated with protection, and the european ndv vaccines were a homologous group with a single serotype and comparable oil-based adjuvants. even with these distinct advantages, the replacement test took almost years ( - ) to be incorporated in the eu monograph for inactivated newcastle vaccines [ , , , ] . although publication in the eu monograph is encouraging, the in vitro assay is only optional because it is one of several approved assays for inactivated newcastle vaccines currently included in the monograph. accordingly, it is difficult to estimate how widely this or any other replacement assay is used by vaccine manufacturers to release vaccine products. table provides specific examples of potency tests for inactivated veterinary vaccines that do not require the use of animals. this is not an exhaustive list, and in some cases general methods are available, often without detailed methodologies. adding to the complexity, these references do not clearly indicate what assays are being used to release a product. nor do they indicate that multiple methods may be available and approved for a specific vaccine by a specific regulatory agency. the proceedings of the edqm international symposium on alternatives to animal testing included a report provided by vaccine manufacturer intervet international on the development of alternative veterinary vaccine potency tests [ ] . according to this report, alternative in vitro potency tests for inactivated veterinary vaccines are described in only a few individual monographs. for example, of the inactivated mammalian veterinary vaccines released from the intervet boxmeer facility, separate potency tests are conducted of which three utilize vaccination/challenge tests, use serology, and two use in vitro techniques. the eu monographs provide detailed descriptions of only of the tests. both of the in vitro tests used by intervet are described. for inactivated poultry vaccines, intervet conducts potency tests: three use vaccination-challenge methods, use serology, and one has a serology or challenge option. twelve of these potency tests are currently described in eu monographs with one in vitro alternative also described (currently not in use by intervet) [ ] . for fish vaccines, intervet uses potency tests, all of them vaccination-challenge tests. five of the are described in eu monographs. as yet, no in vitro alternatives are provided. although this represents only one vaccine manufacturer's potency release of inactivated veterinary products, for which fewer in vitro methods currently exist, it does provide some indication of the potency tests utilized and the need for improved availability of both general and detailed in vitro methods. the development of in vitro potency assays for the highest-priority vaccines that still use animals requires an understanding of the knowledge and data gaps that have delayed the introduction of such non-animal assays. understanding the protective antigen was identified as the primary technical issue. however, for many veterinary vaccines, especially bacterial vaccine products, the protective antigen is unknown or is a complex combination of antigens [ ] . therefore, development of antigen quantification tests is technically difficult because demonstrating a dose response between an antigen and protection in the target species may not be possible. future efforts could focus upon cloning the genes for the protective antigens or obtaining the rights to those genes that have already been cloned during the development of reference standards. purification methods could then be developed for the protective antigens, these antigens characterized, and appropriate assays developed and validated. purified antigens may be made available to industry as reference standards. the availability of reference standards would enable vaccine manufacturers to develop their own standards for inhouse evaluations. regulatory agencies such as the animal and plant health inspection service (aphis), the cvb, and the biological standardisation programme (bsp) under the edqm develop, produce, characterize, and distribute reference standards and other critical reagents. these references are provided to manufacturers to use in developing assays; comparing direct or indirect potency; or independently testing efficacy, identity, and purity. the challenges caused by the adjuvants that are present in many veterinary vaccines present the second key technical issue identified by workshop participants. these challenges must be addressed during the development of in vitro replacement alternatives. typically, in vivo potency tests evaluate the protective or immune response to the complete vaccine, including antigenic material (e.g., adjuvants, excipients). however, many typically use adjuvants such as mineral oil and aluminum salts, which may interfere with in vitro quantification methods. therefore, these adjuvants would need to be separated from the antigen component of the vaccine before in vitro potency testing. because the adjuvant is a critical component for developing the appropriate protective response for inactivated vaccines, additional in vitro tests may be required to ensure their quality. regardless, when antigen quantification methods are being developed, the effect of an adjuvant on the immunogenicity of the protective antigen will also need to be investigated [ , , ] . in addition, the effect of the inactivant on in vitro potency methods must be investigated. a recent study showed that the method of inactivation (in this case, formaldehyde) on an oil-based adjuvanted inactivated newcastle vaccine lowered the in vitro elisa potency result but did not affect the in vivo potency result compared to the use of the inactivant b-propiolactone [ ] . this study indicated that the in vitro potency results for commercial newcastle vaccines inactivated with formaldehyde cannot be directly compared to those inactivated using b-propiolactone [ ] . validation of an in vitro potency assay begins when the assay is initially developed and involves establishing its relationship to efficacy in the target species. the protective antigen (protein) must be identified, purified, and shown to elicit protection in vaccinated animals. antibodies to that protein should neutralize infectivity of the pathogen. extensive validation continues through the assessment of the assay's precision, accuracy, and ruggedness, toward the transition to implementation and use over time [ ] . workshop participants recommended the following highpriority research, development, and validation activities. the current in vivo potency test for inactivated veterinary rabies vaccine comprises a multidilution vaccinationchallenge test in mice, traditionally termed the national institute of health (nih) test. it is known to be highly variable with a high frequency of invalid results [ , , ] . recently implemented reduction and refinement alternatives to this test include ( ) the use of a single-dilution vaccination (reduction) [ ] that results in a significant reduction in animal usage to approximately mice per test and ( ) the incorporation of earlier humane endpoints of paresis, paralysis, and convulsions (refinement) [ ] . in addition, several alternative serological methods have also been developed in which the rabies virus neutralizing antibodies are quantified from the serum of immunized animals. two such serological methods include the rapid fluorescent focus inhibition test (rffit) [ , ] and the fluorescent antibody virus neutralization test (favn) [ ] . according to the european pharmacopoeia, the rffit may be used after a correlation has been established with the mouse vaccination-challenge in vivo test. a recent study demonstrated good correlation between results from the rffit and the traditional in vivo challenge assay [ ] . the rffit is also reproducible within and between laboratories, providing a potential alternative to the mouse vaccination-challenge assay [ , ] . in fact, the european pharmacopoeia recently published a revised draft monograph incorporating the rffit potency assay for inactivated rabies vaccines for veterinary use [ ] . considering these recent developments, workshop participants recommended a focused international workshop to discuss the barriers to international implementation of the rffit. several types of antigen quantification tests are currently in development for inactivated rabies veterinary vaccines, including single radial diffusion tests, antibody-binding tests, and elisa methods [ , , ] . although the elisa assays are reproducible, inexpensive, and quantitative, they are currently product specific, and reagents are not universally available [ ] . in addition, it has yet to be demonstrated that the antigen concentration in the vaccine can be correlated with an ability to stimulate a protective immune response [ ] . furthermore, guidance and/or recommendations from global regulatory agencies are necessary to resolve how any new alternative assay (i.e., serological or antigen quantification) can be validated against the current, highly variable in vivo assay [ , ] . there was broad recognition and general consensus among workshop participants that interaction between the human/veterinary regulatory agencies and vaccine manufacturers should be expanded. such interaction would significantly increase, where appropriate, information exchange to keep all parties current on possible approaches that can be used to further the development and implementation of replacement alternatives for vaccine potency testing. the potency release test used for human rabies vaccines is similar to that used for veterinary products. all u.s.licensed rabies vaccines for human use define potency as the geometric mean of two valid nih potency tests with humane endpoints defined [ ] . in the eu, a similar vaccination-challenge procedure with humane endpoints is also described for human rabies vaccines [ ] . the fda has approved the replacement of several animal-based immunogenicity assays with elisa-based potency assays for some vaccine products, but this does not include human rabies vaccines [ ] . at issue is the fact that, although the neutralizing antigens are well defined, a clear correlation has not been demonstrated among the amount of antigen required to induce immune response in animals, the amount of antigen measured using alternative in vitro assays, and the immune response in human vaccines [ ] . consequently, serological assays may be required to serve as an intermediate step toward the successful development of an in vitro antigen quantification test. although the development of a single potency test (i.e., serological, antigen quantification) for both human and veterinary rabies vaccines is the desired goal, it may be necessary to adapt the test for both product-specific and strain-specific vaccines [ ] . because of the clear synergies between human and veterinary rabies vaccines, workshop participants recommended as a priority that manufacturers and regulatory agencies worldwide collaborate on the development and validation of a refinement or replacement assay for all rabies vaccine products. briefly, the current in vivo leptospira potency test consists of a vaccination-challenge procedure in hamsters, followed days later with a lethal endpoint. the in vivo test is time consuming (more than five weeks) and exposes laboratory personnel to live, viable leptospira, a zoonotic pathogen. the usda recently developed a sandwich elisa as an alternative in vitro test using rabbit polyclonal capture and a specific mouse monoclonal detecting antibody to measure the relative potency of specific bacterins compared to a qualified reference standard for several leptospira interrogans serovars including pomona [ ] , canicola [ ] , grippotyphosa [ ] , and icterohaemorrhagiae [ ] . studies still to be completed include the testing of adjuvants and other vaccine components on assay interference [ ] . the in vivo and in vitro assay methods are currently published by the usda in sams and european monographs (e.g., canine leptospiral antigen quantification method, ph. eur. monograph ; leptospira hardjo antigen quantification method, ph. eur. monograph ) [ , , , ] . in summary, the development and validation of an in vitro potency assay is product-and manufacturer-specific, and manufacturers must perform the necessary studies using specific regulatory memorandums as guidance throughout this process. as a secondary priority, workshop participants recommended the continued development and implementation of elisa antigen quantification methods, including research into the effects of adjuvants and other vaccine excipients, and the harmonization of these tests among global regulatory authorities. the typical potency test for veterinary clostridium spp. vaccines is an in vivo rabbit/mouse toxin-neutralization test currently used, for example, for clostridium novyi [ , , ] and clostridium perfringens [ , ,] (table ) . however, alternative methods for clostridial toxoid potency testing have also been developed and published [ , , ] . for example, european regulatory authorities have a serological potency test for clostridium perfringens [ ] and clostridium septicum [ ] vaccines that has been accepted by european regulatory authorities, although product-specific validation is still required by each vaccine manufacturer [ ] . for clostridium chauvoei, an alternative approach using a validated elisa method [ , ] and an in vitro replacement test for clostridium hemolyticum utilizing toxin-neutralizing antibodies with the characterized protective antigen, is described [ ] . potentially, all the clostridium protective antigens could be evaluated by antigen quantification methods, such as quantitative elisas, after the protective antigen has been identified by gene cloning or after rights to the protective gene have been obtained from sources that have cloned the genes for the purpose of developing reference standards. based upon the published literature and available regulatory methods, replacement of the toxin-neutralization test for specific clostridium spp. vaccines is a realistic goal but will require the global recognition of reference vaccines and the identification of the target antigens for these vaccines. in addition to rabies vaccines, workshop participants agreed that a synergy among experts in human and veterinary tetanus vaccines could facilitate and expedite the development of a replacement potency test for both of these vaccine products. currently, in the united states and the eu, the potency tests for human and veterinary vaccines consist of vaccination of guinea pigs and serological evaluation of antitetanus toxoid antibodies by an indirect elisa [ ] or a toxin-binding inhibition (tobi) test [ , ] . efforts to develop a replacement test for either human or veterinary tetanus vaccines are impeded by the facts that toxoid vaccines are not well characterized, and potential analytical tests, including physiochemical and immunochemical tests, require much greater data generation, characterization, and validation for in-process and final product characterization [ ] . a proposed blueprint for the development of an in vitro replacement potency test for clostridium tetani included ( ) the validation of currently available physiochemical and immunochemical tests, ( ) parallel testing of vaccines by in vitro and serological methods, and ( ) regulatory acceptance and implementation [ , ] . a focused, coordinated effort by human and veterinary tetanus vaccine experts to develop a replacement implementation plan was given a high priority by all workshop participants. vaccines for foreign animal diseases were identified as high priorities due to the biohazard imposed upon laboratory workers and the threat to livestock and wildlife. foot and mouth disease is the most economically important viral livestock disease worldwide, infecting both domestic and wild cloven-footed animals including cattle, swine, sheep, goats, and deer [ , , ] . control of fmd has proven difficult because of the rapid replication of the virus, persistence of the virus in both infected and vaccinated animals, existence of multiple serotypes, and the lack of a globally available and effective vaccine supply [ , , ] . inactivated vaccines are commonly used but limited by the vaccines' short shelf life, the short duration of immunity, the need to include many antigens to obtain broad immunity, and biosafety concerns with production of live virus [ , , ] . improved vaccines currently in development include ( ) recombinant protein and peptide vaccines, ( ) dna vaccines, ( ) empty capsid vaccines, and ( ) adenoviral or fowlpox-vectored vaccines [ , , , ] . there is also growing need for a marker fmd vaccine that would differentiate infected from vaccinated animals (diva). the development of such a vaccine would be significant because vaccination can interfere with disease surveillance using serological testing, and may result in a country's loss of fmd-free status and substantial economic loss [ ] . as superior, functionally characterized vaccines are developed, greater opportunities to reduce, refine, or replace animal use in potency testing will undoubtedly arise. to date, the most successful vaccine strategy has been the development of a recombinant, replication-defective human adenovirus type that expresses the fmd capsid sequence. solid efficacy has been demonstrated in cattle and swine [ ] . however, it is uncertain whether a single vaccine approach can successfully overcome all the shortcomings of the current inactivated vaccines. a combination of different vaccine strategies is likely to be required for effective disease control [ , ] . currently, the vast majority of fmd infections occur in asia, africa, and south america. fmd-free regions include north america, europe, and australia [ , ] . because of significant safety concerns associated with the production of large amounts of fmd virus, the united states prohibits live virus vaccine production on its mainland [ ] . to achieve global disease control, vaccines with improved themostability and a longer duration of immunity are required, especially in those regions of the world without advanced infrastructures [ ] . for the complete control and eradication of fmd, vaccination, surveillance, and an effective monitoring program are necessities [ ] . workshop participants recommended poultry vaccines as priorities for future research and development of in vitro assays because of the large number of target animals currently used in vaccination-challenge and vaccinationserology testing procedures. in vitro potency testing of live viruses is typically performed in primary cell cultures using endpoints such as plaque formation and cytopathology. examples of live virus poultry vaccines that use in vitro potency assessment include those for marek's disease [ ] and infectious bursal disease [ ] . other examples of non-animal potency testing for poultry vaccines include a procedure for titrating newcastle disease virus (ndv) vaccine, infectious bronchitis virus (ibv) vaccine, and a combination ndv-ibv vaccine that uses embryonated chicken eggs to determine the eid [ ] . as described earlier, an in vitro elisa antigen quantification for inactivated ndv is validated and accepted for use in the eu [ , , . ] . additional antigen quantification assays have been developed for infectious bursal disease virus and ibv vaccines; however inadequate funding has prevented further validation [ , , ] . although the technology is now available, sufficient resources and efforts must still be adequately applied to validate these replacement potency assays and gain regulatory approval. finally, as new and better characterized poultry vaccines are developed through the use of viral-vectored systems, purified recombinant proteins, or dna vaccines, alternative in vitro approaches to potency testing should become available [ ] . fish vaccine potency tests were highlighted at the workshop because of the large number of animals used, including unvaccinated controls, in vaccination-challenge procedures [ ] . the majority of fish vaccine potency release tests consist of host animal vaccination-challenge methods. little progress has been achieved in reducing, refining and replacing the use of animals (fish) for this process [ ] . fish inactivated bacterial vaccines have been successfully used in aquaculture, but only recently has the industry developed effective viral vaccines. the number of available fish vaccines increased significantly in the s [ ] . increasingly, adjuvants and immunostimulants are being used to enhance vaccine potency in fish, thereby further complicating the ability to develop refinement or replacement potency testing procedures [ ] . for many fish vaccines, the correlation of serological response and protection is not well established either, impeding the development of serological potency tests [ ] . however, some protective antigens have been identified for inactivated bacterial vaccines, such as those protecting from vibrio salmonicida and vibrio anguillarum diseases. this suggests that serology or antigen quantification methods could be developed for selected vaccine products [ ] . finally, research and development efforts are expected to expand as additional fish vaccines enter the market and more animal health companies develop vaccines for aquaculture use. each of the priority vaccines described above requires a significant investment of time and resources because of ( ) the complexities associated with moving from an in vivo test method to one that does not require animals and ( ) the costs associated with the significant research, development, and validation of in vitro vaccine potency test methods [ , ] . therefore, early and frequent interactions with regulators are strongly encouraged throughout this process to maximize the likelihood of a final product that will be accepted by regulatory authorities and to avoid any unnecessary delays. workshop participants agreed that the primary impediment to broader acceptance and use of available nonanimal replacement methods is the associated cost and time required for each vaccine manufacturer to conduct a product-specific validation of the in vitro potency assay for each specific vaccine. in addition, the lack of international harmonization on alternative potency methods often means that the veterinary vaccine manufacturer must perform multiple potency release tests for the same vaccine depending on its point of manufacture and use. as a starting point, workshop participants recommended that regulatory agencies harmonize the general principles for the validation of alternative potency tests. in the united states, the cvb has issued general guidelines on the validation of in vitro potency assays [ ] and relative potency assays and reference preparations based on elisa antigen quantification [ ] . international organizations also play an important role in this harmonization process. the international cooperation on harmonisation of technical requirements for registration of veterinary medicinal products (vich) is a trilateral program of collaboration among the regulatory authorities and animal health industries of the european union, japan, and the united states. the vich aims to harmonize technical requirements for the registration of veterinary medicinal products by establishing and implementing specific guidelines after extensive input and review from national regulatory authorities. the vich was established under the auspices of the world organization of animal health (oie), which participates as an associate member in the vich process by supporting and disseminating the outcomes at a worldwide level (http://www.vichsec.org). as vich guidelines are developed and reviewed by members of the international animal health community, there is increased acceptance of the regulatory principles that should facilitate faster and more uniform implementation. examples of vich guidelines that have been adopted by aphis include vich gl : examination of live veterinary vaccines in target animals for absence of reversion to virulence (vich (adopted by the u.s. in ) and gl : target animal safety for veterinary live and inactivated vaccines ( ) (adopted in the u.s. in ). in addition, a draft guideline is in development by vich to consider a waiver for the target animal batch safety test [ ] . in addition to harmonizing general principles, there is a need to harmonize the testing procedures for individual vaccine antigens, including development of the necessary reagents. for example, reference standards such as specific antibodies, viruses, bacteria, and antigens can be accessed from the cvb by u.s. entities to aid in the development of in vitro potency test assay development. broad international availability of reference standards, supported by the national and regional regulatory authorities, would greatly help to convert animal-based tests to non-animal assays. additionally, universal reference standards could be monitored and maintained by organizations such as the oie, usda, world health organization (who), or edqm. the availability of reference standards is a key factor in the ability of vaccine manufacturers to switch to an in vitro replacement assay. for example, in an elisa, the reference must be analyzed in conjunction with the sample so that a direct comparison of test vaccine to a known reference can be used to determine a relative potency. relative potency is defined by the cvb as the potency of a product as determined by comparison with an approved reference [ ] . for in vitro antigen potency assays, the unknown is typically compared with a working reference that was generated from the master reference. the master reference potency must have been previously correlated, directly or indirectly, to host animal immunogenicity. as the master reference is correlated to host immunogenicity, its relative stability must be monitored over time to ensure that the reference remains stable during storage. currently, in the united states a frozen master reference is allowed a maximum dating of five years or, if stored under refrigeration, a maximum dating of two years [ ] . after the dating period, each reference must be requalified in the host animal immunogenicity test. to avoid the use of additional animals for requalification, workshop participants recommended that requalification be conducted in any currently acceptable potency test. development of new requalification tests is the responsibility of the vaccine manufacturer. this requires significant resources, especially in the development stage. vaccine manufacturers cannot afford to dedicate these resources to products that are older and less profitable. therefore, prioritization of veterinary vaccines for replacement testing and the potential availability of reference standards can significantly accelerate the animal test replacement process. workshop participants recommended that stability monitoring for both products and reference standards be considered early in the development process. they recommended that regulatory authorities work with industry stakeholders to set expectations for the stability monitoring program [ ] . the stability monitoring of references typically requires that multiple previously validated tests be conducted on a -, -, or -month schedule (brown , personal communication). as test methods change so might the stability monitoring methods and even the reference standard itself. consequently, regulatory agencies may require flexibility to work with the vaccine manufacturers in bridging reference standards and methodologies as industry moves toward in vitro replacement assays. regulatory guidance will also be required on the development and application of new technologies to the development of veterinary vaccines such as genetically engineered (rdna) products, including inactivated/subunit, live (or inactivated) gene-deleted, or live vector (gene insertion) products [ ] . clearly, vaccine manufacturers must decide which products to prioritize for specific non-animal replacement potency testing. typically, considering the output of industry resources, this decision is based upon product revenue and profitability. to aid in this process and to expedite replacement testing, sufficient resources are essential to develop and maintain reference standards specifically for industry use. in addition, broad accessibility of general procedural guidelines (as well as specific testing procedures) for individual antigens would further facilitate the international harmonization of replacement assay development and use. a key issue that should be addressed is the available funding for research and development of alternative methods. this research and development should be funded not just by industry stakeholders but also by government granting agencies, industry associations, and animal welfare advocacy groups. for example, the u.s. national institutes of health may offer funding opportunities for veterinary vaccines for those animal diseases associated with human health, such as rabies. furthermore, academic research into test method alternatives should be promoted, and manufacturers should be encouraged, where appropriate, to present and/or publish their research findings regarding their alternative test methods. workshop participants also encouraged the increased availability of regulatory guidance documents in the public domain. as indicated in section , the inclusion of adjuvants in veterinary vaccines complicates the development of alternative methods because of their reported interference with antigen quantification assays. consequently, priority should be given to developing replacement potency tests for vaccines that do not contain adjuvants. where adjuvants are required, priority should be given to those adjuvants for which methods already exist to separate the adjuvant from the antigen. newly developed adjuvants improve the immune response but may also be more difficult to separate from the antigen. in such instances, regulatory agencies may consider allowing manufacturers to measure potency on the bulk material, before the addition of adjuvant, or allowing antigen testing on the bulk material with an additional characterization/quantitative test on the final product. there is a clear need for further research on simpler adjuvants (and/or the methods to extract then) that may exert an effect on the animal's immune system but that do not directly interact with the antigen. detailed protocols for available replacement alternatives that have been reviewed and endorsed by scientific groups should be readily available in the public domain to facilitate scientific exchange and consideration. for example, detailed protocols and supporting data for validated methods, such as those that appear or are referenced in the european pharmacopoeia monographs, should be freely available to manufacturers and the scientific community to facilitate the implementation of alternative methods. further incentives for industry stakeholders to develop, validate, and implement alternative methods need to be clearly conveyed and implemented by regulatory agencies. workshop participants identified several examples of incentives that may be considered attractive to relevant vaccine manufacturers, including an expedited regulatory review time, waiving the variation fee (if applicable), and the opportunity to utilize intermittent in vivo/in vitro parallel data to expedite validation of new in vitro methods. this was the first international workshop in the united states that focused on the reduction, refinement, and replacement of animal use for safety and potency release testing of both human and veterinary vaccines. a key accomplishment of the workshop was bringing together experts from industry, academia, and government in the areas of safety and potency testing for both human and animal vaccines. there was broad recognition among the vaccine manufacturers and regulatory authorities and a general consensus among the participants that international workshops vastly improve information exchange not only between global regions but also between regulatory authorities (e.g., the usda and the fda) in the same country. this interaction may accelerate development of alternative methods once priorities are firmly established. the presentations and subsequent breakout group sessions allowed participants to clarify the current status of in vitro replacement testing procedures and establish the key criteria to identify those vaccines for prioritization. a focus on inactivated vaccines for rabies, leptospira spp., and clostridium spp. diseases was generated from this debate. an important outcome of this workshop was the recommendation for a similar international workshop to specifically discuss the development, validation, and implementation of alternative reduction, refinement, and replacement potency testing assays for rabies vaccines for both human and veterinary use. this workshop is currently scheduled for october [ ] [ ] [ ] in ames, iowa. the workshop reflected a growing awareness of the need for alternative tests for both poultry and fish vaccines, in which the vaccines are typically tested in large numbers of target animals. because the number of fish vaccines has grown significantly in the last years, much more research and greater focus is needed to identify protective antigens for replacement testing. finally, workshop participants recognized the uniqueness of veterinary vaccines and the need to focus on more-modern, stronger revenue-generating vaccines that can support the cost of new test method development. this workshop also brought attention to ( ) the development and use of more-complex adjuvants and ( ) the use of multiple adjuvants to generate solid and sustained immunity with poorer immunogens (vaccines) and to lower vaccine antigen levels. the use of more-complex or multiple adjuvants further complicates potency replacement efforts and therefore highlights the need for much more extensive research into simpler adjuvants and/or methods to extract them from the protective antigen. workshop participants were encouraged by the significant number (estimated to be between % and %) of veterinary vaccines, especially the modified live viral and bacterial vaccines that now use in vitro potency tests. clearly, better estimates of the number of veterinary vaccine serials released using replacement methods would be beneficial and would also focus the discussion on those vaccines for which replacement potency testing is not yet available or in use. accessing the information on the current state of the art of veterinary vaccine potency tests is challenging because some procedures or general guidelines are not universally available. this results in an unnecessary hindrance to the implementation of the rs for vaccine product release. the growing role of international organizations such as the vich and the oie is apparent. workshop participants agreed that the harmonization of guidelines and reference standards for broad use by the vaccine community would likely increase the interaction between those organizations and the national regulatory groups. in addition, workshop participants clearly expressed the need for additional funding for these regulatory groups to allow greater availability of some of these key reagents (e.g., reference standards) to vaccine manufacturers. although the vaccine companies must develop and validate product-specific assays, the reference standards would provide the basis for this further development and validation. this workshop set the stage for a series of specific workshops on the identified priority vaccines. based upon the general scientific literature and the presentations at the workshop, there is broad international consensus to reduce, refine, and replace the use of animals for both human and veterinary vaccine potency testing. implementation of the workshop recommendations discussed in this report is expected to advance alternative methods for veterinary vaccine potency testing that will benefit animal welfare while ensuring continued protection of human and animal health. this veterinary vaccine session summarized the current status of in vitro potency testing for veterinary vaccines and identified the critical issues to further advance and implement in vitro replacement assays for currently used in vivo challenge or toxin-neutralization testing. to focus these efforts, criteria were established for vaccines that should have the highest priority for development of replacement testing methods. based upon these criteria, the highest-priority vaccines were identified as those for rabies, leptospira spp., clostridium spp., erysipelas, foreign animal diseases (e.g., fmd), poultry diseases, and fish diseases. workshop participants also prioritized the research, development, and validation activities necessary to expedite veterinary vaccine potency testing with fewer animals. workshop participants recognized that there are special considerations with veterinary vaccines due to the complexity of antigenic material and the inclusion of complex adjuvants. they acknowledged that, in many cases, reduction/refinement testing may precede the introduction of in vitro replacement assays. this, combined with the number of veterinary vaccines and their value to the veterinary industry, suggests that the priorities identified are correct and have the highest chance of successful implementation. there was consensus among workshop participants on the need for more universally available reagents and harmonized approaches. the successful implementation of these activities will require additional resources at both national and international levels. finally, workshop participants agreed that the continued interaction of the global vaccine community (i.e., manufacturers, regulatory agencies, animal health organizations), both human and veterinary, could expedite the unified goal of the replacement of animals for veterinary vaccine potency testing. ): a plan to advance alternative test methods of high scientific quality to protect and advance the health of people, animals, and the environment. national institute of environmental health sciences introduction and summary of the international workshop on alternative methods to reduce, refine, and replace the use of animals in vaccine potency and safety testing: state of the science and future directions non-animal replacement methods for human vaccine potency testing: state-of-the-science and future directions non-animal replacement methods for veterinary vaccine potency testing: state-of-the-science and future directions improving animal welfare and reducing and refining animal use for human vaccine potency testing: state-of-the-science and future directions improving animal welfare and reducing animal use for veterinary vaccine potency testing: state-of-the-science and future directions alternative methods and strategies to reduce, refine, and replace animal use for human vaccine post-licensing safety testing: state of the science and future directions alternative methods and strategies to reduce, refine, and replace animal use for veterinary vaccine post-licensing safety testing: state of the science and future directions veterinary vaccines and their importance to animal health and public health human vaccines and their importance to public health fda requirements for human vaccine safety and potency testing usda requirements for veterinary vaccine safety and potency testing health canada's human vaccine lot release program: impact on the rs european regulatory requirements for veterinary vaccine safety and potency testing and recent progress toward reducing animal use strategic approaches for developing alternative tests for safety and potency of vaccines international regulatory requirements for vaccine safety and potency testing: a who perspective overview of currently approved veterinary vaccine potency testing methods and methods in development that do not require animal use case study of development, validation, and acceptance of a non-animal method for assessing veterinary vaccine potency presented at: international workshop on alternative methods to reduce, refine, and replace the use of animals in vaccine potency and safety testing: state of the science and future directions. niceam. bethesda, md a case study of development, validation, and acceptance of a non-animal method for assessing human vaccine potency overview of currently approved serological methods with a focus on diphtheria and tetanus toxoid potency testing presented at: international workshop on alternative methods to reduce, refine, and replace the use of animals in vaccine potency and safety testing: state of the science and future directions. niceam. bethesda, md animal refinement and reduction: alternative approaches for potency testing of diphtheria and tetanus vaccines development and validation of serological methods for human vaccine potency testing: case study of an anthrax vaccine humane endpoints in vaccine potency testing approaches to reducing animal numbers in vaccine potency testing application of the consistency approach in the united states to reduce animal use in vaccine potency testing veterinary vaccine post-licensing safety testing: overview of current regulatory requirements and accepted alternatives target alternative vaccine safety testing strategies for pertussis toxin toward replacement of the monkey neurovirulence test in vaccine safety testing united states public law of and public law - chapter § § et. seq animals and animal products. part -standard requirements. section . . sam : supplemental assay method for potency testing clostridium novyi type b alpha antigen monograph / : . clostridium novyi (type b) vaccine for veterinary use alternative potency testing and other possible related quality issues for veterinary clostridial vaccines. pharmeuropa, special issue validation of alternative methods for the potency testing of vaccines: the report and recommendations of ecvam workshop european department for the quality of medicines within the council of europe animals and animal products. part -standard requirements. section . . sam : supplemental assay method for potency testing of clostridium perfringens type c beta antigen animals and animal products. part -standard requirements. section . . sam : supplemental assay method for potency testing of clostridium perfringens type d epsilon antigen european department for the quality of medicines within the council of europe quality control of inactivated erysipelas vaccines: results of an international collaborative study to establish a new regulatory test animals and animal products. part -standard requirements. section . . sam : supplemental assay method for potency testing of clostridium sordellii antigen animals and animal products. part -standard requirements. section . . sam : supplemental assay method for potency testing products containing clostridium haemolyticum antigen supplemental assay method for potency testing tetanus antitoxins supplemental assay method for potency testing tetanus toxoid by elisa european department for the quality of medicines within the council of europe council of europe. . . assay of tetanus vaccine (adsorbed) interlaboratory validation of the in vitro serological assay systems to assess the potency of tetanus toxoid in vaccines for veterinary use animals and animal products. part -standard requirements. section . . sam : supplemental assay method for in vitro potency testing of leptospira interrogans serovar canicola bacterins european department for the quality of medicines within the council of europe animals and animal products, part -standard requirements european department for the quality of medicines within the council of europe animals and animal products. part -standard requirements. section . . sam : supplemental assay method for potency testing of inactivated rabies vaccines in mice using the national institutes of health test monograph xxxx: -rabies vaccine (inactivated) for veterinary use development of a florescent antibody virus neutralization test (favn test) for the quantitation of rabies-neutralising antibody the rapid fluorescent focus inhibition test is a suitable method for batch potency testing of inactivated rabies vaccines collaborative study for validation of a serological potency assay for rabies vaccine (inactivated) for veterinary use a simple immuno-capture elisa to estimate rabies viral glycoprotein antigen in vaccine manufacture vaccination against foot and mouth disease virus confers complete clinical protection in days and partial protection in days: use in emergency outbreak response foot and mouth disease virus vaccines replacement, reduction and refinement alternatives to animal use in vaccine potency measurement animals and animal products. part -standard requirements. section . . sam : supplemental assay method for bacterial plate count of erysipelothrix rhusiopathiae vaccines animals and animal products. part -standard requirements. section . . sam : supplemental assay method for test for potency of live avirulent pasteurella haemolytica vaccine animals and animal products. part -standard requirements. section . . sam : supplemental assay method for titration of chlamydophila felis (formerly feline chlamydia psittaci) in embryonated chicken eggs animals and animal products. part -standard requirements. section . . sam : supplemental assay method for the titration of feline calicivirus in cell culture animals and animal products. part -standard requirements. section . . sam : supplemental assay method for the titration of feline rhinotracheitis virus in cell culture animals and animal products. part -standard requirements. section . . sam : supplemental assay method for titration of monovalent animals and animal products. part -standard requirements. sam : supplemental assay method for titration of porcine transmissible gastroenteritis virus animals and animal products. part -standard requirements. sam : supplemental assay method for titration of porcine rotovirus in modified-live vaccines animals and animal products. part -standard requirements. section . . sam : supplemental assay method for titration of infectious canine hepatitis virus in primary canine kidney cell culture animals and animal products. part -standard requirements. sam : supplemental assay method for titration of canine adenovirus in canine kidney cell culture animals and animal products. part -standard requirements. section . . sam : supplemental assay method for titrating tissue culture adapted vaccine strains of infectious bursal disease virus animals and animal products. part -standard requirements. section . . sam : supplemental assay method for titration of feline panleukopenia virus in cell culture animals and animal products. part -standard requirements. section . . sam : supplemental assay method for titration of canine parvovirus in cell culture animals and animal products. part -standard requirements. section . . sam : supplemental assay method for the titration of distemper virus in embryonated chicken eggs animals and animal products. part -standard requirements. sam : supplemental assay method for titration of chlamydophilia felis (formerly feline chlamydia psittaci) in embryonated chicken eggs animals and animal products. part -standard requirements. sam : supplemental assay method for the titration of newcastle disease-infectious bronchitis vaccine in chicken embryos animals and animal products. part -standard requirements. section . . ovine ecthyma vaccine animals and animal products. part -standard requirements. section . . sam : supplemental assay method for in vitro potency testing of jodie kulpa animals and animal products. part -standard requirements. sam - : supplemental assay method for potency testing enterotoxigenic (k , k , p, f pilus) escherichia coli bacterins humane endpoints in the efficacy testing of swine erysipelas vaccines animals and animal products. part -standard requirements. section . . sam : supplemental assay method for in vitro potency testing of leptospira interrogans serovar pomona bacterins animals and animal products. part -standard requirements. section . . sam : supplemental assay method for in vitro potency testing of leptospira interrogans serovar grippotyphosa bacterins animals and animal products. part -standard requirements. section . . sam : supplemental assay method for in vitro potency testing of leptospira interrogans serovar icterohaemorrhagiae bacterins animals and animal products. part -standard requirements. sam : supplemental assay method for the in vitro potency assay of bovine respiratory viruses in killed vaccines animals and animal products. part -standard requirements. sam : supplemental assay method for quantitating the gp antigen of feline leukemia virus in veterinary vaccines animals and animal products. part -standard requirements. sam : supplemental assay method for determination of the specific viral antigen content in inactivated canine coronavirus vaccines antigen quantification as in vitro alternative for potency testing of inactivation viral poultry vaccines validation study to evaluate the reproducibility of a candidate in vitro potency assay of newcastle disease vaccines and to establish the suitability of a candidate biological reference preparation determination of minimum hemagglutinin units in an inactivated newcastle disease virus vaccine for clinical protection of chickens from exotic newcastle disease virus challenge correlation of haemagglutinin-neuraminidase and fusion protein content with protective antibody response after immunisation with inactivated newcastle disease vaccines feasibility study to evaluate the correlation between results of a candidate in vitro assay and established in vivo assays for potency determination of newcastle disease vaccines perspectives from industry: development of alternative test for quality control testing of veterinary vaccines animals and animal products. part -standard requirements. section . . sam : supplemental assay method for purity, potency and dissociation of brucella abortus vaccine, strain l european department for the quality of medicines within the council of europe european department for the quality of medicines within the council of europe standard requirements for vaccines against infectious bursal disease animals and animal products. part -standard requirements. section . . sam : supplemental assay method for potency testing of erysipelas bacterins in mice animals and animal products. part -standard requirements. section . . sam : supplemental assay method for potency assay of leptospira interrogans serovar pomona bacterins animals and animal products. part -standard requirements. section . . sam : supplemental assay method for potency assay of leptospira interrogans serovar canicola bacterins animals and animal products. part -standard requirements. section . . sam : supplemental assay method for potency assay of leptospira interrogans serovar grippotyphosa bacterins animals and animal products. part -standard requirements. section . . sam : supplemental assay method for potency assay of leptospira interrogans serovar icterohaemorrhagiae bacterins veterinary services memorandum . . in vitro serial release potency test for completed product containing clostridium chauvoei animals and animal products european department for the quality of medicines within the council of europe animals and animal products. part -standard requirements. section . . bovine rhinotracheitis animals and animal products. part -standard requirements. section . . staphylococcus aureus bacterin-toxoid united states department of agriculture licensing requirements for feline leukemia virus vaccines european department for the quality of medicines within the council of europe three rs achievements in vaccinology the influence of the inactivating agent on the antigen content of inactivated newcastle disease vaccines assessed by the in vitro potency test veterinary services memorandum . . guidelines for validation of in vitro potency assays nih test, a problematic method for testing potency of inactivated rabies vaccine effects of vaccine route and dosage on protection from rabies after intracerebral challenge in mice the rabies peripheral challenge test: more accurate determination of vaccine potency a rapid tissue culture test for determining rabies neutralising antibody three rs approaches in the quality control of inactivated rabies vaccines. the report and recommendations of ecvam workshop application of the three rs to challenge assays used in vaccine testing: tenth report of the bvaawf/frame/rspca/ufaw joint working group on refinement rabies vaccine standardization: international collaborative study for the characterization of the fifth international standard for rabies vaccine monograph / : . rabies vaccine for human use prepared in cell culture the potential of physiochemical and immunochemical assays to replace animal tests in the quality control of toxoid vaccines. the report and recommendations of ecvam workshop an in vitro immune response model to determine tetanus toxoid antigen (vaccine) specific immunogenicity: selection of sensitive assay criteria a review of emergency foot-and-mouth disease (fmd) vaccines development of novel strategies to control foot-and-mouth disease: marker vaccines and antivirals foot and mouth disease virus vaccines options for the control of foot-and-mouth disease: knowledge, capability and policy the current state of vaccines used in the field for foot and mouth disease virus in china vaccination against foot and mouth disease virus confers complete clinical protection in days and partial protection in days: use in emergency outbreak response current status of veterinary vaccines animals and animal products. part -standard requirements. sam . supplemental assay method for titration of monovalent, cell associated marek's disease vaccines of serotypes , , or animals and animal products. part -standard requirements. sam . supplemental assay method for titrating tissue culture adapted vaccine strains of infectious bursal disease virus quantification of infectious bursal disease viral proteins and in inactivated vaccines as an indicator of serological responses and as a measure of potency in vitro methods for vaccine evaluation aquaculture pharmaceuticals and biologicals: current perspectives and future possibilities adjuvants and immunostimulants for enhancing vaccine potency in fish veterinary services memorandum . . guidelines for veterinary biological relative potency assays and reference preparations based on elisa antigen quantification the authors extend their sincere appreciation to all participants in the international workshop for their enthusiastic contributions leading to the workshop recommendations and conclusions. the members of the iccvam interagency biologics working group and niceatm staff are acknowledged for their contributions to the planning of the workshop, and the many invited experts are acknowledged for their contributions to breakout group discussions and workshop proceedings. finally, the authors thank david allen and nelson w. johnson for their assistance in the preparation of this manuscript. key: cord- -a fynm authors: riggs, shannon m. title: chapter guinea pigs date: - - journal: manual of exotic pet practice doi: . /b - - . - sha: doc_id: cord_uid: a fynm publisher summary this chapter deals with the health and medical care issues of guinea pigs. guinea pigs have wide bodies with short limbs. they have a short, flat nose, laterally placed eyes, and hairless external pinnae. the dentition of the guinea pig is described as aradicular hypsodont. guinea pigs are best housed in well-ventilated, wire-sided cages with solid bottoms. if housed indoors, guinea pig enclosures do not require a cover, as these animals do not typically jump or climb. heavy food containers are recommended to make dumping of the receptacle more difficult. all food containers should be easy to disinfect and should be cleaned regularly, because guinea pigs have a habit of soiling their food bowls. these animals, native to the andes mountains, are very susceptible to hyperthermia and should never be housed in temperatures greater than °f. high humidity can also exacerbate a guinea pig's sensitivity to elevated temperatures by increasing the heat index. guinea pigs often do not exhibit clinical signs early in a disease process. therefore, a thorough physical examination can be extremely useful in determining the overall health status of the animal. c h a p t e r guinea pigs shannon m. riggs guinea pigs, cavia porcellus, are members of the caviidae family of the order rodentia. guinea pigs are native to mountainous regions of south america where they were domesticated as long as years ago. wild species of guinea pigs, or cavies, still inhabit columbia, peru, venezuela, argentina, brazil, and paraguay. domesticated cavies in these countries are used for food. in the wild, guinea pigs live in small groups and, therefore, are often more comfortable in the presence of other guinea pigs when maintained as companion animals. extensive breeding has resulted in numerous varieties of coat color and characteristics. the most common breeds are the american (or english) which has a short, smooth coat and the multicolored teddy (figure - ); abyssinian ( figure - ), which has a medium length coat in a whorled pattern; and the peruvian, which has a very long, smooth coat. guinea pigs have wide bodies with short limbs. a distinctive anatomic characteristic of species in the family caviidae is the number of digits on the front and rear feet ( digits front feet and digits rear). tails are usually very short or absent. the guinea pig has a short, fl at nose, laterally placed eyes, and hairless external pinnae. adult guinea pigs usually weigh between and g, with the males being slightly larger than females. the average life span of the companion guinea pig is approximately to years. the dentition of the guinea pig is described as aradicular hypsodont (e.g., all teeth have a relatively long crown and are "open rooted"). the maxilla is slightly wider than the mandi-ble, and the occlusal angle of the premolars and molars is marked compared to other rodent species. the dental formula of the guinea pig is (i / , c / , pm / , m / ) = . the maxillary incisors are much shorter than those set in the mandible. the molars and premolars are not easily visualized without special instrumentation because of the small size of the oral cavity and tendency for the involution of the buccal surface. females are sexually mature at weeks of age, whereas males on average reach puberty approximately weeks later. gestation is long, when compared to other rodents, at days. as a result of this long gestation period, young are precocial when born. juvenile pigs usually eat solid foods by or days of age. litter sizes range from to , with an average of to young. a female guinea pig should deliver her fi rst young before she is months of age. if birth has not occurred before months of age, the pubic symphysis becomes mineralized, with future pregnancies resulting in an inability of the sow to naturally deliver the babies. female guinea pigs that become pregnant after months of age invariably require cesarean section deliveries. guinea pigs are best housed in well-ventilated, wire-sided cages with solid bottoms. wire-bottom cages may also be used; however, care must be taken to ensure that the mesh is small enough that a limb cannot become entrapped. an area of solid fl ooring should be provided, as uninterrupted time on wire mesh may predispose the guinea pig to pododermatitis. adequate space is needed in the enclosure for the guinea pig to move about unencumbered with enough space for a hide box. hide boxes or a secluded space is required for prey species (e.g., rodents) to reduce stress that may lead to disease problems. substrate products that contain aromatic oils (e.g., cedar and pine shavings) should not be used, as they can act as contact and respiratory irritants. appropriate bedding materials include recycled newspaper products, shredded paper, and aspen shavings. the enclosure should be cleaned thoroughly on a regular basis (e.g., times per week) because unsanitary conditions predispose the guinea pig to pododermatitis, respiratory, and other health problems. if housed indoors, guinea pig enclosures do not require a cover, as these animals do not typically jump or climb. however, the sides of the enclosure should be high enough to prevent escape (approximately cm). heavy food containers are recommended to make dumping of the receptacle more diffi cult. all food containers should be easy to disinfect and cleaned regularly, as guinea pigs have a habit of soiling their food bowls. most guinea pigs readily accept drinking water from a sipper bottle, which will decrease spillage and will keep feces, urine, and bedding from contaminating the water. these animals, native to the andes mountains, are very susceptible to hyperthermia and should never be housed in temperatures greater than ° f. high humidity can also exacerbate a guinea pig's sensitivity to elevated temperatures by increasing the heat index. all animals are very sensitive to environmental and/or nutritional changes. therefore, if changes have to be made, gradual exposure of the animal to the changes is recommended. an appropriate guinea pig diet includes a formulated, pelleted diet for that species, high-quality hay (e.g., timothy, orchard grass, oat) ad libitum, and ample fresh vegetables. as the animal's food intake is more dependent on volume consumed rather than calories consumed, a pet fed a predominantly pelleted diet (higher nutritional concentration) has a tendency to become obese. fruits and grains, if they are offered at all, should comprise a very small portion (< %) of the total diet and offered only as treats. because guinea pigs lack the enzyme l-gulonolactone oxidase, they are unable to synthesize ascorbic acid from glucose. therefore, guinea pigs require supplemental vitamin c in their diets. although commercial guinea pig pellets are manufactured with vitamin c, the supplement often degrades rapidly, especially if the pellets are subjected to high heat and humidity. vitamin c placed in drinking water also degrades rapidly and should be changed daily. to ensure that a guinea pig is receiving a proper amount of vitamin c, it is necessary to supplement a diet of pellets and hay with plenty of fresh foods or often a specifi cally manufactured vitamin c supplement tablet (oxbow, inc., murdock, ne). many green, leafy vegetables, such as kale, mustard greens, dandelion greens, parsley, and many others, are excellent sources of ascorbic acid (box - ). the vitamin c requirement of an adult, nonbreeding guinea pig is mg/kg/day. , in the united states, guinea pigs are not routinely vaccinated for infectious diseases. however, owners should be encouraged to have annual examinations that include an oral examination and a complete blood count. being prey species in the wild, guinea pigs are adept at hiding illness, and routine evaluations by a qualifi ed veterinarian may help in detecting abnormalities early. many health problems of guinea pigs are related to improper husbandry. during a routine veterinary visit, the owners should be asked to provide a detailed description of the animal's housing environment, including substrate, frequency of cleaning, ambient temperature, and exercise time. the diet history is also important. owners should be asked not only what the guinea pig is offered, but also in what proportions and of what foods the animal actually eats. most guinea pigs are quite docile and do not require aggressive restraint. often a hand on the animal's dorsum is adequate to restrain a guinea pig patient on the examination table ( figure - ). when transporting a guinea pig, support the body with one hand under the thorax and abdomen while placing the other hand on the back to prevent the patient from falling or jumping ( figure - ) . if chemical restraint is required, gas anesthesia with isofl urane or sevofl urane is generally well tolerated. anesthetic gases can be delivered via mask or induction chamber. mild sedation can be achieved with an intramuscular injection of a combination of midazolam ( . - . mg/kg) and butorphanol ( . - . mg/kg) intramuscularly. guinea pigs often do not exhibit clinical signs early in a disease process. therefore, a thorough physical examination can be extremely useful in determining the overall health status of the animal. before beginning a physical examination, it is important to observe the animal before it has been stressed by restraint. a healthy guinea pig should be alert and aware of its surroundings. as guinea pigs are often shy animals, they may attempt to hide or escape. the examiner should use a thorough, systematic approach to focus on the respiratory character and rate, posture, and attitude of the animal. it is also important to have any instruments (e.g., transilluminator, stethoscope, thermometer, blood-collecting supplies) that may be necessary to decrease the amount of handling time for the patient. obtaining an accurate body temperature, heart rate, and respiratory rate is best accomplished at the beginning of the examination, as these parameters will invariably change with handling. an accurate weight should be obtained using an electronic gram scale. a "hands-on" physical examination should begin with the head; the veterinarian should assess the eyes for symmetry or discharge and check that the external pinnae of the guinea pig are hairless, as normal. the external ear canals often contain a small to moderate amount of dark, ceruminous debris. the nasal planum should be dry and fl at, whereas palpation of the ventral mandible may reveal deformities secondary to overgrowth of molar and premolar apices. the guinea pig coat varies somewhat with breed but, in general, should be smooth and shiny. guinea pigs often have a mild to moderate amount of dark sebaceous debris on the skin of the dorsum. older male guinea pigs may develop a focal accumulation of this debris at the base of the vertebral column, which may be referred to as the "grease gland." thoracic auscultation and abdominal palpation can be performed as in other patients. heart and respiratory rates will vary depending on the degree of stress a patient experiences. as pulse and respiratory rates can be very rapid, careful auscultation is necessary to detect subtle abnormalities (e.g., murmurs, crackles, wheezes). auscultation of gut sounds is also an important part of the guinea pig physical exam. a healthy guinea pig should have to borborygmi per minute. the practitioner should keep in mind that stress will decrease gastrointestinal (gi) motility; therefore, a stressed animal will often have a decrease in borborygmi. structures normally found on abdominal palpation include kidneys, urinary bladder, cecum, and intestines. fecal pellets are often palpable in the colon. careful examination may reveal the presence of abnormalities such as gi distention, masses, or, in females, ovarian cysts. limbs and joints should be carefully evaluated because thickened or painful joints may be indicative of a vitamin c defi ciency. a complete oral examination is an important part of the guinea pig exam. because of the potential stress associated with the oral exam, this evaluation should be reserved for the end. the oral cavity of the guinea pig is very narrow with a small opening, making visualization diffi cult ( figure - ) . instruments such as an otoscope with cone or a human nasal speculum will increase visualization of the caudal oral cavity ( figure - ). however, many dental lesions may be overlooked using these methods, and anesthesia is often required to adequately determine oral health. obtaining blood samples from guinea pigs for routine diagnostics is a challenging procedure. peripheral venipuncture sites available for most mammals (e.g., lateral saphenous vein and cephalic vein) are diffi cult to visualize in guinea pigs and only allow for the collection of small sample volumes. the jugular vein may be used; however, the short neck of the guinea pig and poor tolerance of the aggressive restraint by the patient restrict the availability of this site for blood collection. often to obtain an adequate blood sample, it is necessary to anesthetize the patient. once anesthetized, the large central vessels may be accessed with less stress on the patient and phlebotomist. acceptable venipuncture sites while the patient is anesthetized include the cranial vena cava ( figure baseline blood work is an important tool in the routine monitoring of health as well as in the diagnosis of disease. a complete blood count is essential for assessing red blood cell and white blood cell parameters (box - ). guinea pigs have heterophils rather than neutrophils as the predominant circulating granulocyte. heterophils lack myeloperoxidase, the enzyme that causes purulent, liquid exudates. therefore, the debris contained in guinea pig abscesses is often found to be very thick and caseous, a fact that must be understood when treating these conditions. a normal guinea pig white blood cell differential will consist of primarily heterophils and lymphocytes, usually with a greater proportion of lymphocytes. the remaining leukocyte types (e.g., monocytes, eosinophils, basophils) are normally present in very low numbers. early stages of a guinea pig's infl ammatory response are often characterized by a shift in the differential white cell ratio (e.g., increased heterophils, decreased lymphocytes) rather than an increase in total leukocyte count. this ratio shift makes evaluating the entire leukogram essential for health evaluation (box - ). the platelet count is also an important marker of infl ammation in guinea pigs and other small mammal species. large increases in the platelet count (> , , /μl) can be seen without an increase in the total white blood cell count. a cell type that is unique to guinea pigs is the kurloff cell. these large lymphocytes contain a cytoplasmic inclusion (e.g., a kurloff body). kurloff cells are noted most often in the peripheral blood of reproductive age females, although they are also identifi ed in male guinea pigs. the number of circulating kurloff cells will increase in response to exogenous estradiol and testosterone administration, although the effects were more dramatic with estradiol administration. , other studies have shown the disappearance of kurloff cells in spayed female and castrated male guinea pigs. the function of the kurloff cell is not completely understood, and these cells appear to lack lysosomes. at this time, there is no evidence that the kurloff cell has phagocytic activity. the activity of kurloff cells appears to most closely correlate with that of natural killer cells found in other species. plasma biochemical analysis is necessary for evaluation of organ function as well as plasma glucose and proteins. it is important to interpret the results of a chemistry panel in conjunction with history, physical exam fi ndings, and the results of other diagnostic tests. reference intervals for biochemical parameters are shown in box - . urinalysis is a useful diagnostic test in guinea pigs with signs of upper or lower urinary tract disease. urine samples may be obtained by free catch, fl oor catch, or cystocentesis. if urine is to be cultured, it is ideal to use the cystocentesis method of collection. ultrasound guidance and/or mild sedation of the patient will aid the veterinarian and/or veterinary technician in obtaining the urine sample. the urine of guinea pigs is typically yellow to amber in color, but it may be darker and more orange depending on the patient's diet. pigments in the urine can sometimes be mistaken for hematuria, so differentiation is important. because guinea pigs are herbivores, the ph of guinea pig urine is alkaline, usually . to . . crystalluria may be seen but is not a normal fi nding. if crystals are found in a urine sample, the guinea pig patient should be examined for urinary calculi. to minimize rotation, care should be taken to extend the limbs symmetrically when positioning the patient. because guinea pigs have stocky builds with short limbs, and because they resent aggressive restraint, sedation or anesthesia is helpful in obtaining diagnostic radiographs as well as in reducing the patient's stress ( figure - ). table - is a guideline for radiographic techniques used in common small mammal radiographic studies. dental malocclusion is a common disease problem in guinea pigs. radiographs of the skull are helpful in assessing the degree of malocclusion as well as potential bone involvement. in addition to lateral and dorsoventral views of the skull, right and lateral oblique views can help to localize lesions. magnifi ed views of the skull can be obtained by placing the patient on an elevated platform under the x-ray beam without changing the distance between the x-ray cassette and beam. ultrasound is another imaging modality that is very useful in the diagnosis of common guinea pig disease processes, such as ovarian cysts (figure - ) and urinary tract calculi. as with radiographs, sedation or anesthesia can assist in reducing patient stress and improve the quality of images. guinea pigs often have a large amount of gas accumulation in the gi tract, which obscures the ultrasound image, sometimes making this imaging technique of limited value. advanced imaging techniques, such as computed tomography (ct) and magnetic resonance imaging (mri), are useful tools in the diagnosis of disease in guinea pig patients. limitations of these modalities include limited availability, expense, and decreased image quality compared with that of larger patients. mri has the added limitation of requiring a significant amount of time (often > min) under anesthesia with limited monitoring ability. regardless of the present limitations, veterinarians should be aware that these techniques are available and that reference material exists that depicts normal anatomy. as guinea pig owners continue to demand high-quality care for their pets, these imaging techniques will likely become more commonplace in small mammal practice for these patients. microbiologic samples can be obtained for diagnosis of various guinea pig infections. exudates from nasal or ocular secretions can be examined for abnormal fl ora. some bacterial organisms (e.g., bordetella bronchiseptica) are diffi cult to culture. laboratories need to be advised as to the organisms in question so they can perform more specifi c diagnostic tests to obtain organism identifi cation. abscesses are another disease condition that may require culture for proper treatment. because guinea pigs form caseous abscesses, the purulent debris itself is typically not useful for bacterial isolation. for the best chance of identifying organisms within an abscess, a portion of the abscess capsule should be submitted to the laboratory. both aerobic and anaerobic bacterial cultures should be requested, especially if it is suspected that the abscess may have originated from a dental problem. roundworms and coccidia are seen in guinea pigs and can be identifi ed by fecal fl otation or fecal direct smear, as in other species. cryptosporidiosis has also been reported. identifi cation of cryptosporidium organisms usually requires acid-fast staining or immunofl uorescent antibody testing in addition to fecal fl otation. ectoparasites (e.g., mites, lice, fl eas) commonly infest guinea pigs. visualization of the parasites and/or their waste, as well as skin scrapings, microscopic examination of hair follicles, and tape preparations can be useful in the identifi cation of these parasites. the term enterotoxemia refers to the overgrowth of toxinproducing bacteria in the gi tract, particularly clostridium diffi cile. this can occur with stress, an abrupt change in diet, gi stasis, or inappropriate antibiotic administration. the gi fl ora of guinea pigs is predominantly gram positive, and administration of antibiotics with a primarily gram-positive spectrum (e.g., beta-lactam antibiotics, macrolides, lincosamides) can lead to the depletion of normal gut fl ora, allowing colonization by opportunistic bacteria (e.g., gram-negative organisms, clostridium spp.). the dentition of the guinea pig is described as aradicular hypsodont, meaning all teeth grow throughout the animal's life and are open-rooted. the dental formula of the guinea pig is i / , c / , p / , m / . the mandible of these animals is wider than the maxilla. the occlusal angle of the molars and premolars in the guinea pig is quite severe when compared with that of rabbits and other rodents (figure - ). guinea pigs lack the layer of yellow enamel present on the rostral surface of the incisors in most other rodents. dental malocclusion is a common disease process in guinea pigs. the development of malocclusion can be the result of multiple etiologies or a combination of factors (e.g., improper diet, genetics, trauma). incisor malocclusion alone is rare in guinea pigs, so a thorough oral examination is necessary to determine the presence of cheek teeth malocclusion. when the molars and premolars do not occlude properly, overgrowth of the crowns and reserve crowns takes place. with improper wear, sharp points can form on the buccal aspects of the maxillary cheek teeth and the lingual aspects of the mandibular cheek teeth. the mandibular cheek teeth can overgrow to such an extent that they entrap the tongue, making eating and drinking diffi cult (figure - ) . clinical signs can include inappetence or dysphagia, decreased fecal output, ptyalism, poor coat quality, and leth-argy. abscesses commonly occur at the apical aspects of overgrown molars and premolars. because the premolars and molars are elodont (open-rooted), it is possible that these teeth can overgrow and impinge on the nasolacrimal duct, causing ocular and nasal discharge. the reserve crowns can also overgrow into the nasal cavity, where oral bacteria may be seeded, causing rhinitis and sinusitis. diagnosis of dental disease is based on physical and oral examination fi ndings. careful palpation of the ventral mandible and maxilla may reveal bony protuberances corresponding to overgrowth of the apical surfaces of the cheek teeth. proper instrumentation is very important for adequate visualization of the oral cavity. dental speculums and pouch dilators are invaluable aids in obtaining a good view of the molars and premolars (figure - ) . however, many abnormalities can be overlooked in a conscious animal, so anesthesia is often required to obtain a thorough oral examination. visualization of the oral cavity and occlusal surfaces can be facilitated by the use of an endoscope (figure - ) . most endoscopes available in veterinary practice have a degree of angulation of the fi eld of view, allowing greater focal area. abnormal oral examination fi ndings may include an uneven occlusal surface or angle of the cheek teeth, formation of sharp points with or without associated ulceration of the oral mucosa, food impaction, and abnormal spaces (diastema) between teeth. imaging, including routine radiographs, magnifi ed skull radiographs, and computed tomography can be incorporated to better evaluate the extent and seriousness of the process (figure - ) . radiographic studies should include dorsoventral, lateral, and right and left oblique views. treatment is centered on restoring a normal occlusal plane to the teeth, a procedure that should be performed while the animal is under general anesthesia. unstable animals should be provided with supportive care before the anesthetic event. a high-speed surgical dental handpiece can be used to restore a normal occlusal plane and to reduce any sharp points. handheld trimmers are not acceptable, as they tend to crush teeth and can cause fractures and pulp exposure. dremel tools are also not considered acceptable because the small size of the guinea pig oral cavity will not allow for appropriate trimming. with any dental instruments, care should be observed to minimize oral soft tissue trauma. when a guinea pig is diagnosed with dental malocclusion, it is important to convey to the owners that this will be a lifelong problem for their pets. with many cases, routine occlusal adjustments will be necessary for the remainder of the animal's life. tyzzer's disease is the common term for bacterial enteritis caused by clostridium piliforme. infection with this organism is more commonly described in small rodents such as mice and hamsters, although it has also been described in guinea pigs as well as other mammalian groups. tyzzer's disease often presents acutely, with signs such as diarrhea, depression, and poor coat quality, often progressing rapidly to death. transmission is via the fecal-oral route. guinea pigs can be infected with c. piliforme without showing clinical signs. animals that are carrying the organism but showing no signs of illness have still been found to shed organisms. animals that carry the organism and then become immunocompromised (e.g., stress, immunosuppressive drug therapy) often develop clinical disease. clostridium piliforme is an intracellular bacterium that will not grow on routine culture media, so antemortem diagnosis is diffi cult. tyzzer's disease begins as an intestinal infection, but later spreads to the liver hematogenously, causing areas of necrosis. , positive identifi cation of the organism on necropsy requires examination of hematoxylin and eosin or silver staining of affected tissues. treatment should consist of fl uid and nutritional support as well as appropriate antibiotic therapy. unfortunately, the progression of the disease is rapid, making treatment unrewarding. prevention is key, focusing on owners' reducing housing stress, providing a proper diet, and maintaining a clean environment. cryptosporidiosis has been described as causing disease in guinea pigs in a laboratory setting. diagnosis was made histologically from affected animals that lost weight, had diarrhea, and suffered an acute death. cryptosporidium organisms were found in the brush border of the intestinal tract from the duodenum to cecum, with associated infl ammation. suspected coronavirus infection has been described in young guinea pigs. clinical signs in affected animals included anorexia, weight loss, and diarrhea. approximately half of the affected animals in the reported outbreak recovered. necropsies performed on animals that died or were euthanized showed lesions consistent with coronavirus infection resulting from an acute to subacute necrotizing enteritis involving primarily the distal ileum. coronavirus-like virions were identifi ed on transmission electron microscopy of feces collected from the lower gi tract at necropsy. outbreaks of salmonellosis have occurred in guinea pig colonies. some of the organisms that have been isolated include s. enteritidis, s. dublin, s. fl orida, s. poona, and s. bredeney. , salmonellosis has been described to affect guinea pigs in acute and chronic disease processes. with acute salmonellosis, guinea pigs often die after a brief illness characterized by nonspecifi c signs of illness and diarrhea. usually only a few pathogenic abnormalities are found at necropsy, and confi rmation of a diagnosis is dependent on culture of affected tissues. chronic salmonellosis is a wasting disease, lasting several weeks. splenomegaly, hepatomegaly, and mesenteric lymphadenopathy are common postmortem fi ndings in affected animals. guinea pigs that recover can remain chronic, intermittent shedders of salmonella organisms. yersinia pseudotuberculosis causes several disease syndromes in guinea pigs. , the most common presentation affects the mesenteric and colonic lymph nodes, infi ltrating these lymph nodes with caseous nodules. clinically, affected guinea pigs will lose weight, have diarrhea, and develop a generalized lymphadenopathy. transmission of the disease can be either vertical or horizontal. as stated previously, guinea pigs require dietary supplementation of ascorbic acid because they lack the enzyme l-gulonolactone oxidase necessary for synthesis of this compound. although diets formulated for guinea pigs are supplemented with ascorbic acid, it is important to remember that it breaks down very rapidly, usually within the fi rst days after production. if a guinea pig is not receiving vitamin c supplementation in its diet, the veterinarian can assume that the diet is defi cient in this important nutrient. ascorbic acid is a necessary component of collagen, and defi ciencies are often noted as manifestations of abnormal collagen synthesis. clinical signs of hypovitaminosis c can include lameness, hemorrhage, lethargy, anorexia, poor coat quality, and bruxism. diagnosis of hypovitaminosis c is based largely on clinical signs and history. radiographs will reveal changes to the costochondral junctions and widening of the epiphyses of long bones. recommended treatment includes fl uids and nutritional support, pain control, and parenteral vitamin c supplementation. attention should also be paid to improvement of the guinea pig's diet at home. in addition to causing skeletal and cartilage abnormalities, vitamin c defi ciency has been known to reduce immune function. in vitro, vitamin c defi ciency was demonstrated to cause a reduction in migration of macrophages in guinea pigs. urogenital dystocia dystocia most frequently occurs in primiparous sows that are bred after approximately months of age. at this time, the symphysis between the pubic bones becomes fused and will not expand to allow the passage of fetuses. cesarean section must be performed in these cases to save the sow and the young. factors other than age that can predispose a sow to dystocia include large fetuses in relation to sow size, uterine inertia, and obesity. urolithiasis occurs commonly in pet guinea pigs, and the common clinical signs associated with the disease include stranguria and pollakiuria, vocalizing when urinating, and hematuria. the underlying cause(s) of this condition is not completely understood but is likely associated with a genetic predisposition and/or the presence of a high-calcium diet. other less common underlying etiologies associated with urinary calculi formation include ureteral neoplasms (e.g., papilloma). calculi are primarily composed of calcium carbonate, although magnesium ammonium phosphate hexahydrate and calcium phosphate calculi will also occur. radiographic or ultrasonic imaging can be used to confi rm the location of the urinary calculi, which may be present in the renal pelvis, ureters, urinary bladder, or urethra (figure - ) . urinary tract calculi often require surgical removal. ideally, once removed, the calculus and/or a portion of the bladder wall should be cultured. it may also be helpful to analyze the composition of the stone to help determine ways of preventing recurrence. ovarian cysts are a common fi nding in middle-aged to older female guinea pigs. this disorder has a reported prevalence of % in female guinea pigs aged . to years. , multiple cysts can be present on one or both ovaries. clinical signs related to cystic ovaries can include abdominal distention, lethargy, and anorexia related to the space-occupying nature of the cysts. ovarian cysts that are actively producing hormones can produce bilaterally symmetrical alopecia of the fl anks. ovarian cysts can be quite large (up to several centimeters) and can often be identifi ed on physical examination. abdominal ultrasound is the most useful diagnostic tool for a defi nitive diagnosis. the most defi nitive treatment for cystic ovaries is ovariectomy or ovariohysterectomy, curing the current problem and preventing recurrence. in animals where surgery is not a viable option, ultrasound-guided percutaneous aspiration of ovarian cysts can be performed as a palliative treatment. however, without removal of the ovaries, the cysts will recur, requiring repeated procedures. pregnancy toxemia typically occurs in pregnant sows during the last weeks of gestation. as with other species, pregnancy toxemia is the result of a negative energy balance and the metabolism of fat. sows that experience pregnancy toxemia are typically overweight and become anorexic. clinical signs include lethargy, dyspnea, and anorexia, usually progressing to death within a few days. therapy of affected sows should center on providing nutritional support, correcting electrolyte imbalances, and preventing opportunistic infections. prognosis is generally considered poor, as many sows fail to respond to treatment. neoplasia of the reproductive tract is not commonly reported in guinea pigs, but several tumor types have been described. of the described reproductive neoplasms, the vast majority occur in female guinea pigs. uterine leiomyoma (often associated with ovarian cysts) and leiomyosarcoma, ovarian teratoma, and granulosa cell tumor are reported neoplasms of the reproductive tract. diagnosis, as in any other patient, should be based on the results of cytologic or histopathologic sampling. benign neoplasms may be resolved with ovariohysterectomy, but further diagnostics to determine the extent of local invasion should be performed before surgery takes place. a thorough diagnostic work-up, including imaging techniques (e.g., abdominal ultrasound, thoracic radiographs), should be performed to look for metastases of malignant tumor types. mycoplasma caviae has been isolated from the reproductive tracts of guinea pigs. these guinea pigs are often unaffected by the organism, but metritis has been suspected to be associated with infection. as with other species, mycoplasma spp. is suspected to cause reproductive problems (e.g., abortion and decreased fertility). respiratory disease is a common presenting complaint in guinea pig patients. clinical signs can vary from sneezing and upper respiratory signs to severe dyspnea and death. bordetella bronchiseptica is one of the most common respiratory bacterial agents associated with pneumonia in guinea pigs. many guinea pigs are carriers of the organism, which will cause clinical disease if the animal is stressed. a thorough history, obtained from the patient's owner, often reveals interactions with other species that are also subclinical carriers of b. bronchiseptica (e.g., rabbits, dogs). clinical signs noted in guinea pigs infected by the bordetella organism include nasal discharge, dehydration, tachypnea, and lethargy. diagnosis should be based on clinical and radiographic signs and history. confi rmation of the diagnosis can be determined with enzyme-linked immunosorbent assay (elisa) and indirect immunofl uorescence diagnostic tests, or culture of exudates. treatment/preventive options for b. bronchiseptica infections in guinea pigs include an autogenous bacterin vaccine as well as three commercially available vaccines for bordetella spp. (porcine b. bronchiseptica and human b. pertussis), which were found to offer some protection against the development of bronchopneumonia in experimentally infected guinea pigs. streptococcus pneumoniae can cause pleuropneumonia, pleuritis, and peritonitis in guinea pigs. serologic detection of s. pneumoniae antibodies using elisa have been described in previously published papers. on postmortem examination, lesions found to be associated with s. pneumoniae infection included pleuritis, pleural effusion, lung abscessation, otitis media, pericarditis, and others. streptococcus pneumoniae was also identifi ed from septic arthritis lesions in a group of guinea pigs. five guinea pigs in a laboratory colony demonstrated multiple enlarged joints from which pure cultures of s. pneumoniae were isolated. several other individuals in this colony had previously died with typical s. pneumoniae lesions (e.g., pleuritis, pericarditis) and lesions consistent with hypovitaminosis c. the group with septic arthritis also had scorbutic changes on necropsy. streptobacillus moniliformis, the causative agent of rat-bite fever in humans, was isolated from a laboratory guinea pig with pneumonia. this organism is of particular importance because of its zoonotic potential. adenovirus has been shown to cause necrotizing bronchopneumonia in guinea pig populations, although it can also produce a transient, subclinical infection. , clinical signs include depression and dyspnea, but guinea pigs often die acutely without clinical signs. identifi cation of adenovirus dna from diseased lung tissue was achieved using a polymerase chain reaction technique in one study. development of this pcr assay for identifi cation of the virus has determined that the guinea pig adenovirus is distinct. histopathologic examination of affected tissues of animals infected with adenovirus will reveal the presence of characteristic intranuclear inclusion bodies. the most commonly reported neoplasm of the respiratory tract in guinea pigs is bronchogenic papillary adenoma. the prevalence of the tumor is as high as % in guinea pigs over the age of years. given the relatively more common occurrence of pneumonia in guinea pigs, bronchogenic papillary adenoma can often be misdiagnosed. for this reason, thoracic radiographs of guinea pigs with respiratory disease are highly recommended. yersinia pseudotuberculosis can cause septicemic pneumonia in guinea pigs. death usually occurs rapidly, after the development of coughing and dyspnea. at necropsy, severe congestion of the lungs is found grossly and through histologic evaluation of the tissues. trixacarus caviae are sarcoptoid mites that commonly affect guinea pigs (figure - ) . affected guinea pigs are intensely pruritic, sometimes to the extent of seizure development. as with other mite infestations, diagnosis is based on the identifi cation of the parasites on skin scrapings. once defi nitive diagnosis has been made, treatment with ivermectin and selamectin ( mg/kg q - wk) is usually effective. chirodiscoides caviae are fur mites diagnosed in guinea pigs. because it rarely causes clinical disease, treatment is usually unnecessary. , gyropus ovalis and gliricola porcelli are species of lice that are commonly identifi ed in guinea pigs (figure - ) . infested guinea pigs may be pruritic, but are usually unaffected. guinea pigs severely infested with these mites may demonstrate poor coat quality and alopecia. patchy hair loss without associated pruritus may be attributed to dermatophytosis, most commonly trichophyton mentagrophytes (figure - ) . lesions are circular and scaled and usually occur on the face and head. the diagnosis of dermatophytosis is made by a positive fungal culture. because of the zoonotic potential of these fungal organisms, care should be streptococcus zooepidemicus lancefi eld's group c is the causative agent of cervical lymphadenitis. this disease will cause severe swellings of the lymph nodes in the cervical region in guinea pigs. affected guinea pigs will frequently exhibit no other clinical signs but may become septicemic, with lesions affecting the heart, lungs, kidney, and skin. the most effective treatment for cervical lymphadenitis is complete surgical excision of the affected lymph nodes, followed by appropriate antibiotic therapy based on culture and sensitivity testing. lancing and draining the abscesses is often not curative, as the abscesses form thick capsules that harbor organisms, leading to recurrence. yersinia pseudotuberculosis has also been shown to cause cervical lymphadenitis in guinea pigs. although the affected guinea pigs are usually not ill, the concern is that rupture of the abscesses will release large amounts of this potentially zoonotic organism into the environment. another potential causal agent of cervical lymphadenitis is streptobacillus moniliformis. one report exists of a cervical mass, initially believed to be cervical lymphadenitis, which was histologically determined to be a thyroid papillary adenoma. the mass was apparently nonfunctional and the guinea pig appeared otherwise healthy, but surgical resection was curative. guinea pigs housed in cages with wire fl ooring are predisposed to developing ulcerated lesions on the plantar surfaces of their feet. mild lesions may appear as hyperemic, swollen areas of the weight-bearing surfaces. these lesions can progress to ulcerations with secondary infections (figure - ). vitamin c defi ciency has also been considered a predisposing factor for the development of pododermatitis, as affected animals may be in pain and reluctant to move, resulting in the development of pressure sores. ulcerated, infected lesions should be managed with appropriate antibiotics and antiinfl ammatory drugs, but the focus of treatment should be improving husbandry (e.g., providing appropriate fl ooring/bedding, vitamin c supplementation). trichofolliculomas are the most common cutaneous tumor seen in guinea pigs. these benign tumors often occur on the dorsum and are typically round and hairless (figure - ). another cutaneous abnormality that has been described in guinea pigs is cutaneous vascular malformation. the lesion described was a raised, ulcerated plaque on the animal's fl ank, which bled intermittently. ultimately, the cutaneous vascular malformation resulted in fatal hemorrhage. histologically, the lesion was described as an expansile mass extending into the skeletal muscle and consisting of multiple vascular spaces of varying sizes. these vascular spaces were lined with endothelial cells. compared to the incidence of neoplasia in other mammalian species, the incidence of neoplasia in guinea pigs appears low or is underreported. however, there have been several reported cases of neoplasia in guinea pigs. as more guinea pig owners seek quality veterinary care for their pets, reports of neoplasia will increase. lymphoma is the most commonly reported neoplasia in guinea pigs. clinical signs associated with guinea pig neoplasia include lymphadenopathy, splenomegaly, and hepatomegaly. leukemic and aleukemic forms of guinea pig lymphoma have been identifi ed. thyroid carcinoma has been reported in an adult guinea pig that demonstrated multiple masses in the ventral cervical region. as there was no evidence of neoplastic disease elsewhere on postmortem examination, this was considered a primary tumor. mesothelioma has been reported in the abdomen of an adult guinea pig. the guinea pig had died of complications associated with pneumonia, and the mesothelioma was diagnosed through a necropsy examination. the mass consisted of diffuse nodules on the serosal surfaces of numerous organs in the abdominal cavity. guinea pigs are native to cooler regions of south america and are therefore relatively intolerant of temperatures above ° f, lower if the environment is also humid. guinea pigs should be housed in well-ventilated enclosures at temperatures between ° and ° f to prevent heat stress. clinical signs of heat stress include rapid, shallow respirations, lethargy, poor peripheral perfusion, and ptyalism. treatment includes reducing the animal's core body temperature with cool water baths or applying alcohol to the feet and ears; in addition, fl uid therapy (either intravenous or subcutaneous) is recommended to improve perfusion. the prognosis for this condition is guarded. chlamydophila psittaci has been identifi ed as a disease-causing agent in guinea pigs; it usually causes a mild, self-limiting conjunctivitis. this intracellular bacterial disease usually occurs in young guinea pigs to weeks of age but has been reported in adults as well. in one outbreak, the typical conjunctival abnormalities were present with other, more signifi cant, clinical signs, such as rhinitis, abortion, and pneumonia. a defi nitive diagnosis can be achieved through giemsa staining, immunofl uorescent antibody testing of conjunctival scrapings, and serologic testing. guinea pigs have been reported to develop pathologic changes related to ingestion of forssk fern. , in these studies, guinea pigs fed fresh fern developed hematuria and hemorrhage of the bladder wall. one guinea pig fed dried fern developed proliferative urocystica and adenoma of the bladder, a fi nding sometimes considered precancerous in human patients. this guinea pig did not show clinical signs associated with the bladder abnormalities. rabies virus infection is uncommon in rodent species, but it has been described and should be considered a differential diagnosis for an ill guinea pig with suspect contact to wildlife, especially raccoons. a recent report of a rabies in a privately owned guinea pig described abnormal behavior (biting the owner) days after the guinea pig had possible interactions with a raccoon. in this guinea pig, rabies virus antigen was detected by immunofl uorescent antibody testing in the sublingual salivary gland, tongue, and buccal tissues, implying that the guinea pig could have transmitted the virus via a bite wound. sick guinea pigs are often anorexic and therefore dehydrated. restoration of normal hydration status is crucial for the successful treatment of many disease processes. replacement of fl uid defi cit and maintenance of normal hydration can be achieved by administering crystalloids substances through subcutaneous, intraperitoneal, intravenous, or intraosseous routes. because of the diffi culty in placing and maintaining catheters in peripheral veins and bones, the most common route for fl uid administration is subcutaneous. subcutaneous fl uid administration is generally well tolerated. fluids are administered under the skin of the cranial, dorsal thorax (figure - ) . butterfl y catheter needles are useful because they allow the patient to move around without pulling out the injection needle. maintenance fl uid rates for guinea pigs are - ml/kg/day. another important aspect of management for the sick guinea pig is nutritional support. anorexic guinea pigs can experience a change in their normal gi fl ora in as little as to hours. this change of gi fl ora can lead to ileus, colic, overgrowth of pathogenic bacteria, and enterotoxemia. commercial products are available that are palatable and high in fi ber. these products help to maintain gut motility (oxbow critical care for herbivores, oxbow hay company, murdock, ne). patients will often eat directly from a dish or -ml catheter tip syringe. for patients that are more resistant to eating, a technique that is useful, in my experience, is to remove the plungers from -ml or -ml syringes and fi ll them individually using a catheter tip syringe. although this method may seem tedious, it allows the delivery of small boluses of food to be incrementally dispensed. the gi tract of guinea pigs can be very sensitive to the effects of certain classes of antibiotics. the gi fl ora of guinea pigs is primarily gram positive, and administration of antibiotics with a primarily gram-positive spectrum can result in overgrowth of gram-negative and anaerobic organisms. enteral administration of penicillins (e.g., amoxicillin, ampicillin), macrolides (e.g., erythromycin, lincomycin), and fi rst-generation cephalosporins can result in overgrowth of opportunistic pathogens. ampicillin administered subcutaneously at doses of and mg/kg three times a day resulted in mortality rates of % and %, respectively, in a group of guinea pigs. at necropsy, clostridium diffi cile was cultured from the ceca of all fatalities. twenty-fi ve percent of guinea pigs administered mg/kg of cefazolin, a fi rst-generation cephalosporin, intramuscularly died of enterocolitis after several injections. all antibiotics should be used with caution in guinea pigs because of the possibility of disruption of the gi fl ora (table - ). guinea pigs can be induced using isofl urane or sevofl urane administered by mask or induction chamber (figure - ) . for lengthy or potentially painful procedures, preanesthetic medications may be used before induction of the inhalant anesthetic agent. as a preanesthetic, an injectable combination, midazolam ( . - . mg/kg) and butorphanol ( . - . mg/kg), has been used with success. the administration of premedications will also decrease the stress of induction. isofl urane and sevofl urane are both acceptable inhalant anesthetics for guinea pigs. the advantage of sevofl urane is that it does not appear to have as noxious a scent as isofl urane, thereby reducing breath holding during induction and producing a smoother anesthetic episode. after induction, the inhalant anesthetic may be switched to isofl urane if cost is an issue. guinea pigs should not be fasted more than to hours before anesthesia. because these animals are hindgut fermenters, withholding food for longer periods of time may disrupt gi fl ora. careful monitoring during anesthesia is essential. the number of respirations must be visually monitored for depth and character. heart rate and rhythm should also be monitored continuously using a pediatric stethoscope or doppler unit, which can be placed on a peripheral artery. changes in heart rate or respiratory rate can occur rapidly, so it is advantageous to have precalculated doses of emergency drugs (e.g., glycopyrrolate, epinephrine, atropine, dopram) drawn and available for use before anesthetic induction. intubation of guinea pigs is generally considered to be very diffi cult. the long, narrow oral cavity makes visualization of the glottis diffi cult. the soft tissues of the tongue and soft palate are continuous in the caudal oropharynx, leaving only a small aperture, the palatal ostium. intubation must be performed through this opening in the mucosal tissues. several techniques have been described for visualizing the glottis to assist with intubation. these usually involve alterations of laryngoscope blades so that they may be introduced into the oral cavity without traumatizing the delicate buccal mucosa. , another possible intubation technique utilizes a stethoscope altered so that an appropriately sized endotracheal tube can be affi xed to the end. the endotracheal tube is then inserted into the caudal oropharynx. the anesthetist will then listen through the ear pieces of the stethoscope for expiration. the tube is then gently introduced into the trachea. this technique requires practice to perfect but, once the procedure has been performed several times, is a reliable way to intubate guinea pigs. application of a small amount of lidocaine to the rima glottis can ease intubation. rigid endoscopes and otoscopes, when available, are also very helpful in visualizing the glottis for intubation. indications for an ovariohysterectomy procedure in guinea pigs include routine sterilization, dystocia, and reproductive tract disease (e.g., neoplasia, muco/hydro/pyometra, ovarian cysts). in general, the surgical approach and technique are similar to those used with domestic species. the patient is placed in dorsal recumbency, and a ventral midline incision is made through the linea alba. care must be taken upon entering the abdomen to avoid incising the large cecum, which is often situated along the ventral body wall. the body of the uterus can be located dorsal to the bladder. the ovaries lie caudally and laterally to the kidneys and are to mm in length. the abdomen of the guinea pig is deep, and the ovaries can be diffi cult to exteriorize. care must be taken to avoid tearing the short ovarian vessels. once the ovaries are removed, the uterus is ligated and transected in a manner similar to that used with other mammals. another method of sterilization that is reported in guinea pigs is ovariectomy. the incidence of uterine disease compared with ovarian disease in guinea pigs is quite low. the approach for removal of the ovaries is through small incisions of the dorsal-lateral body wall caudal to the last rib. the advantage of this approach is that the sensitive gi tract is not manipulated to reach the structures to be excised. orchiectomy is typically performed to prevent reproduction, to decrease undesirable sexual behavior, and to treat reproductive tract disease. the orchiectomy procedure in guinea pigs is performed by making an incision through the scrotum over each testicle. reactions to suture material are relatively common in guinea pigs after surgical procedures. these reactions can vary in severity from mild local irritation to abscessation. suture materials that cause a large amount of infl ammation, such as chromic gut, should never be used in guinea pigs. in general, monofi lament suture materials that are degraded by hydrolysis are preferred. advances in diagnosis and treatment of small exotic mammal dental disease practitioner's guide to domestic rodents biology, husbandry, and clinical techniques hematologic and serum biochemical values of rodents mammalian hematology: laboratory animals and miscellaneous species natural cytotoxicity in the guinea-pig: the natural killer (nk) cell activity of the kurloff cell the kurloff cell radiology equipment and positioning techniques cryptosporidiosis in guinea pigs: a retrospective study tyzzer's disease subclinical infection and transmission of tyzzer's disease in rats disease problems of guinea pigs coronavirus-like virions associated with a wasting syndrome in guinea pigs natural infections of guinea pigs salmonellosis in laboratory animals the guinea pig or cavy, the ufaw handbook on the care and management of laboratory animals macrophage function in vitamin c-defi cient guinea pigs ureterolithiasis and papilloma formation in the ureter of a guinea pig guinea pigs reproductive medicine of rabbits and rodents spontaneous tumors of small mammals airborne-induced experimental bordetella bronchiseptica pneumonia in strain guinea pigs interlaboratory comparison of enzyme-linked immunosorbent assay (elisa) and indirect immunofl uorescence (iif) for detection of bordetella bronchoseptica antibodies in guinea pigs effi cacy of commercial vaccines for protecting guinea pigs against bordetella bronchiseptica pneumonia serodiagnosis of streptococcus pneumoniae infection in guinea pigs by an enzyme-linked immunosorbent assay isolation of streptobacillus moniliformis from a guinea pig with granulomatous pneumonia polymerase chain reaction for detection of guinea pig adenovirus pathogenesis of guinea pig adenovirus infection adenoviral bronchopneumonia of guinea pigs anorexia and chirodiscoides caviae infection in a guinea pig (cavia porcellus) ilar: a guide to infectious diseases in guinea pigs, gerbils, hamsters, and rabbits, part ii, diseases outlines thyroid papillary adenoma in a guinea pig with signs of cervical lymphadenitis cutaneous vascular malformation in a guinea pig (cavia porcellus) thyroid carcinoma of a guinea pig: a case report abdominal mesothelioma in an aged guinea pig guinea pig inclusion conjunctivitis (gpic) in a commercial colony verlauf einer chlamydienbedingten "meerschweinchen-einschluß körperchen-könjunktivitis" in einer versuchstierhaltung, zeitschrift für versuchsteirkunde proliferative urocystica and adenoma in a guinea pig preliminary studies on christella dentate (forssk) fern toxicity in guinea pigs rabies virus infection in a pet guinea pig and seven pet rabbits an evaluation of ampicillin pharmacokinetics and toxicity in guinea pigs pharmacokinetics of cefazolin in guinea pigs exotic animal formulary ferrets, rabbits, and rodents: clinical medicine and surgery plumb dc: veterinary drug handbook endotracheal intubation in guinea pigs by direct laryngoscopy an improved method for direct laryngeal intubation in the guinea pig biology of the guinea pig key: cord- -x tvq a authors: de wit, emmie; feldmann, friederike; cronin, jacqueline; jordan, robert; okumura, atsushi; thomas, tina; scott, dana; cihlar, tomas; feldmann, heinz title: prophylactic and therapeutic remdesivir (gs- ) treatment in the rhesus macaque model of mers-cov infection date: - - journal: proc natl acad sci u s a doi: . /pnas. sha: doc_id: cord_uid: x tvq a the continued emergence of middle east respiratory syndrome (mers) cases with a high case fatality rate stresses the need for the availability of effective antiviral treatments. remdesivir (gs- ) effectively inhibited mers coronavirus (mers-cov) replication in vitro, and showed efficacy against severe acute respiratory syndrome (sars)-cov in a mouse model. here, we tested the efficacy of prophylactic and therapeutic remdesivir treatment in a nonhuman primate model of mers-cov infection, the rhesus macaque. prophylactic remdesivir treatment initiated h prior to inoculation completely prevented mers-cov−induced clinical disease, strongly inhibited mers-cov replication in respiratory tissues, and prevented the formation of lung lesions. therapeutic remdesivir treatment initiated h postinoculation also provided a clear clinical benefit, with a reduction in clinical signs, reduced virus replication in the lungs, and decreased presence and severity of lung lesions. the data presented here support testing of the efficacy of remdesivir treatment in the context of a mers clinical trial. it may also be considered for a wider range of coronaviruses, including the currently emerging novel coronavirus -ncov. the continued emergence of middle east respiratory syndrome (mers) cases with a high case fatality rate stresses the need for the availability of effective antiviral treatments. remdesivir (gs- ) effectively inhibited mers coronavirus (mers-cov) replication in vitro, and showed efficacy against severe acute respiratory syndrome (sars)-cov in a mouse model. here, we tested the efficacy of prophylactic and therapeutic remdesivir treatment in a nonhuman primate model of mers-cov infection, the rhesus macaque. prophylactic remdesivir treatment initiated h prior to inoculation completely prevented mers-cov−induced clinical disease, strongly inhibited mers-cov replication in respiratory tissues, and prevented the formation of lung lesions. therapeutic remdesivir treatment initiated h postinoculation also provided a clear clinical benefit, with a reduction in clinical signs, reduced virus replication in the lungs, and decreased presence and severity of lung lesions. the data presented here support testing of the efficacy of remdesivir treatment in the context of a mers clinical trial. it may also be considered for a wider range of coronaviruses, including the currently emerging novel coronavirus -ncov. mers-cov | antiviral | animal model | remdesivir | therapy s ince its discovery in , cases of middle east respiratory syndrome coronavirus (mers-cov) have continued to emerge, with the current case count close to , cases, and a case fatality rate ∼ % ( ). this continuous emergence of mers-cov infections in saudi arabia and its ability to spread through humanto-human transmission has prompted the world health organization to include mers on their list of emerging diseases likely to cause major epidemics and for which countermeasures are urgently needed ( ) . through the coalition for epidemic preparedness innovations, mers-cov vaccines are going to advance through preclinical and clinical trials ( ), but, despite the urgent need, a similar initiative does not exist for the development and clinical testing of antivirals effective against mers-cov. remdesivir (gs- ) is a nucleotide prodrug that has broad antiviral activity against viruses from different families in vitro ( ) , and therapeutic efficacy in nonhuman primate models of lethal ebola virus and nipah virus infection ( , ) . studies in human airway epithelial cells showed that remdesivir also inhibits replication of a wide range of coronaviruses, including mers-cov ( ) . efficacy studies in mice showed that remdesivir had therapeutic efficacy against severe acute respiratory syndrome (sars)-cov and mers-cov in ces c −/− mice, deficient in a secreted carboxylesterase responsible for poor pharmacokinetics profile of remdesivir in mice, when administered before the peak of virus replication ( , ) . in vitro studies with mouse hepatitis virus showed that remdesivir inhibits coronavirus replication through interference with the viral polymerase, despite the presence of a viral proofreading exoribonuclease ( ) . importantly, coronaviruses partially resistant to inhibition by remdesivir, obtained in vitro following > passages in the presence of gs- , a parent nucleoside that is metabolized into the same active triphosphate metabolite, were still sensitive to higher concentrations of remdesivir, and fitness was impaired in the resistant viruses as compared to wild-type mers-cov ( ) . with these promising data in mind, we tested the prophylactic and therapeutic efficacy of remdesivir treatment in a nonhuman primate model of mers-cov infection, the rhesus macaque ( ) . and therapeutic treatment. to assess the efficacy of remdesivir to alleviate clinical signs of mers-cov infection, rhesus macaques were randomly assigned to three groups of six animals. three animals in the control group were treated with ml/kg vehicle solution h before mers-cov inoculation, and three animals were treated at h post mers-cov inoculation. another group of six rhesus macaques was treated prophylactically h before mers-cov inoculation with mg/kg remdesivir, and one group of six animals was treated therapeutically at h postinoculation with mers-cov with mg/kg remdesivir. treatment middle east respiratory syndrome, caused by the mers coronavirus (mers-cov), continues to cause severe respiratory disease with a high case fatality rate. to date, potential antiviral treatments for mers-cov have shown limited efficacy in animal studies. here, we tested the efficacy of the broad-acting antiviral remdesivir in the rhesus macaque model of mers-cov infection. remdesivir reduced the severity of disease, virus replication, and damage to the lungs when administered either before or after animals were infected with mers-cov. our data show that remdesivir is a promising antiviral treatment against mers that could be considered for implementation in clinical trials. it may also have utility for related coronaviruses such as the novel coronavirus -ncov emerging from wuhan, china. data deposition: all data discussed here will be made available to readers upon request. to whom correspondence may be addressed. email was continued once daily until d postinoculation (dpi), when animals were euthanized and necropsied (fig. ). after inoculation with mers-cov on day , all animals were closely observed for signs of disease, and clinical scores were assigned according to a previously determined scoring sheet. all vehicle-treated animals displayed signs of disease, starting as early as dpi, such as decreased appetite and ruffled fur; all vehicle-treated animals had respiratory signs such as increased respiration for (n = ) or (n = ) d after inoculation. the animals treated prophylactically with remdesivir did not show any respiratory signs of disease, but decreased appetite, possibly due to daily anesthesia, was noted in five of six animals. the animals treated therapeutically with remdesivir all displayed reduced appetites, and five out of six animals had increased respiration rates at (n = ), (n = ), or (n = ) d after inoculation. these observations are reflected in the clinical scores of the animals, with clinical scores in the prophylactically treated animals being statistically significantly lower than in vehicletreated control animals at to dpi, and in the therapeutically treated animals at to dpi ( fig. a) . on days , , , , and , clinical examinations were performed on the animals, and respiration rates were determined on anesthetized animals. there was a clear increase in respiration rates in the vehicle-treated animals ( fig. b ), while respiration rates in prophylactically treated animals remained normal throughout the study. although respiration rate was increased in therapeutically treated animals at dpi, respiration was statistically significantly lower than in vehicle-treated controls at and dpi (fig. b ). on examination days, radiographs were collected from all animals and analyzed for the presence of infiltrates; from dpi onward, lung infiltrates became visible on x-ray (si appendix, fig. s ). at dpi, there was statistically significantly less infiltration in the lungs of animals treated both prophylactically and therapeutically with remdesivir as compared to vehicle-treated control animals (fig. c ). at dpi, all animals were euthanized, and respiratory tissues were collected for quantitative analysis of the levels of viral rna by qrt-pcr. compared to vehicle-treated control animals, prophylactic remdesivir treatment resulted in significantly lower levels of mers-cov replication in the lungs, with lung viral loads . to logs lower in each lung lobe (fig. a) . although lung viral loads were, on average, lower in individual lung lobes after therapeutic treatment, this was statistically significant in only a few lung lobes, due to larger variation between animals in the therapeutically treated group (fig. a) . however, when all lung lobes were combined, the lung viral load in therapeutically treated animals was clearly lower than in vehicle-treated animals (fig. b ). additionally, viral loads were significantly lower in trachea, bronchi, tonsils, and mediastinal lymph nodes of animals treated prophylactically and therapeutically with remdesivir than in vehicle-treated control animals ( fig. c and si appendix, fig. s ); viral rna was not detected in kidney tissue samples (si appendix, fig. s ). treatment. upon necropsy, the area of each lung lobe affected by gross lesions was estimated by a board-certified veterinary pathologist. gross lung lesions were present in several lung lobes of all of the vehicle-treated control animals (fig. a ). in contrast, gross lung lesions were completely absent in the lungs of animals that received prophylactic remdesivir treatment. in animals treated therapeutically with remdesivir, there were obvious gross lesions present in five out of six animals; however, the total area of lungs affected by gross lesions was statistically significantly smaller than in vehicle-treated control animals (fig. a ). in addition, the severity of histologic lung lesions was assessed by assigning a score for each lung lobe. the resulting cumulative lung histology score was compared between treatment groups to assess differences in the severity of histologic lesions. cumulative lung histology scores were significantly lower in animals treated prophylactically with remdesivir (fig. b) . the large variation between animals in the therapeutically treated group meant that the lower average histology score did not reach statistical significance (fig. b) . histologically, all of the vehicle-treated control animals developed some degree of pulmonary pathology when inoculated with mers-cov. lesions were multifocal, frequently centered on terminal bronchioles, and consisted of minimal to marked, interstitial pneumonia, characterized by thickening of alveolar septae by edema fluid and fibrin and small to moderate numbers of macrophages and fewer neutrophils. alveoli contained moderate numbers of pulmonary macrophages and neutrophils. in areas with moderate to marked changes, there was abundant alveolar edema and fibrin with multifocal formation of hyaline membranes, as well as abundant type ii pneumocyte hyperplasia. perivascular infiltrates of inflammatory cells multifocally within and adjacent to affected areas of the lung were also observed (fig. c ). in contrast, all animals treated prophylactically with remdesivir had essentially normal pulmonary tissue with no evidence of coronavirus infection (fig. c ). animals treated with remdesivir therapeutically demonstrated various levels of severity of coronaviral pneumonia. in two out of six animals, no histologic evidence of pneumonia was detected. in three animals, multifocal, minimal to moderate interstitial pneumonia was observed like that described for the control animals; however, the lesions were less severe than in the controls and not as widely distributed throughout the lung lobes. only one out of six animals had moderate interstitial pneumonia that was indistinguishable from the vehicle-treated control animals in severity and distribution. immunohistochemical analysis for the presence of mers-cov antigen showed small numbers of antigen-positive type i pneumocytes in all vehicle-treated control animals and in five out of six animals treated therapeutically with remdesivir; there was no difference in number or distribution of antigen-positive cells in animals where antigen was detected. mers-cov antigen could not be detected in any of the animals treated prophylactically with remdesivir (fig. d ). prophylactic remdesivir treatment prevented mers-cov−induced clinical disease and lung lesions in rhesus macaques inoculated with mers-cov, and strongly inhibited mers-cov replication in respiratory tissues. since nosocomial transmission accounts for approximately one-third of mers-cov cases ( ) , prophylactic remdesivir treatment of patients, contacts of patients, and healthcare personnel with high-risk exposure to a diagnosed mers patient and at high risk of developing severe mers due to underlying conditions ( ) could be considered. therapeutic remdesivir treatment also provided a clear clinical benefit, with a reduction in clinical signs and virus replication, and the absence of lung lesions in two out of six remdesivir-treated animals and a reduction in lesion severity in three additional animals. absence of histologic lung lesions, as seen in two out of the six animals with therapeutic remdesivir treatment, has so far rarely been observed in studies testing the efficacy of mers-cov antivirals in nonhuman primate models ( ) ( ) ( ) ( ) ; it has only been shown once before in one out of three common marmosets treated with hyperimmune plasma at h after inoculation ( ) . thus, although it is hard to compare different studies due to the fact that different species were used and treatment was initiated at different time points after inoculation, remdesivir appears to be one of the most promising antiviral treatments tested in a nonhuman primate model to date. therapeutic remdesivir treatment was administered at h after inoculation with mers-cov, and, although this may seem relatively early after inoculation, it is close to the peak of mers-cov replication in the rhesus macaque model ( ) . a drug that inhibits virus replication may be of little use once virus replication has reached its peak, as was shown in vitro ( ) . however, in a considerable number of severe cases of mers, viral rna and infectious virus can still be detected in respiratory tract samples several weeks after the onset of symptoms ( , ) , with this prolonged virus replication most likely due to the presence of underlying conditions such as diabetes mellitus ( ) . likewise, an increase in virus replication over a longer period of time was observed in immunocompromised rhesus macaques ( ) . thus, remdesivir treatment could not only be of benefit to patients diagnosed with mers early after symptom onset but may also improve recovery in those patients with severe cases of mers where prolonged virus replication occurs. human safety data are available for remdesivir. it has been used on a compassionate basis in several unique cases of ebola virus disease ( , ) , as well as on a large scale in the ongoing ebola virus outbreak in the democratic republic of congo ( ), with around treated patients. in addition, its efficacy is currently being tested in a clinical trial in ebola virus disease survivors with prolonged virus shedding ( , ) . although the efficacy of remdesivir was lower in the ebola virus trial than that of the different antibody treatments tested, survival was increased as compared to overall survival rate in this outbreak. taken together, the data presented here on the efficacy of remdesivir in prophylactic and therapeutic treatment regimens, the difficulty of coronaviruses to acquire resistance to remdesivir ( ) , and the availability of human safety data warrant testing of the efficacy of remdesivir treatment in the context of a mers clinical trial. our results, together with replication inhibition by fig. . clinical findings in rhesus macaques inoculated with mers-cov and treated with remdesivir. three groups of six rhesus macaques were inoculated with mers-cov strain hcov-emc/ ; one group was i.v.-administered ml/kg vehicle solution (vehicle control; gray circles), one group was administered mg/kg remdesivir starting at h before inoculation (prophylactic remdesivir; black squares), and one group was administered mg/kg remdesivir starting at h after inoculation (therapeutic remdesivir; red triangles). after inoculation, the animals were observed twice daily for clinical signs of disease and scored using a predetermined clinical scoring system (a). on , , , and dpi, clinical examinations were performed during which respiration rate was determined (b), and radiographs were taken. radiographs were used to score individual lung lobes for severity of pulmonary infiltrates by a clinical veterinarian according to a standard scoring system ( : normal; : mild interstitial pulmonary infiltrates; : moderate pulmonary infiltrates perhaps with partial cardiac border effacement and small areas of pulmonary consolidation; : serious interstitial infiltrates, alveolar patterns and air bronchograms); the cumulative x-ray score is the sum of the scores of the six individual lung lobes per animal; scores shown are from dpi (c). asterisks indicate statistically significant difference in a two-way (a and b) or one-way (c) anova with dunnett's multiple comparisons; black asterisks indicate statistical significance between the vehicle control and prophylactic remdesivir groups, and red asterisks indicate statistical significance between the vehicle control and therapeutic remdesivir groups. *p < . ; **p < . ; ***p < . ; ****p < . . ethics and biosafety statement. all animal experiments were approved by the institutional animal care and use committee of rocky mountain laboratories, nih and carried out by certified staff in an association for assessment and accreditation of laboratory animal care international accredited facility, according to the institution's guidelines for animal use, and followed the guidelines and basic principles in the united states public health service policy on humane care and use of laboratory animals, and the guide for the care and use of laboratory animals. rhesus macaques were housed in adjacent individual primate cages allowing social interactions, in a climate-controlled room with a fixed light−dark cycle ( -h light/ -h dark). animals were monitored at least twice daily throughout the experiment. commercial monkey chow, treats, and fruit were provided twice daily by trained personnel. water was available ad libitum. environmental enrichment consisted of a variety of human interaction, commercial toys, videos, and music. the institutional biosafety committee (ibc) approved work with infectious mers-cov strains under bsl conditions. sample inactivation was performed according to ibc-approved standard operating procedures for removal of specimens from high containment. study design. to evaluate the effect of remdesivir treatment on mers-cov disease outcome, we used the rhesus macaque model of mers-cov infection that results in transient lower respiratory tract disease ( ) . rhesus macaques were chosen because of the requirement of daily anesthesia and intravenous (i.v.) injections that were perceived to be problematic in the alternative nonhuman primate model of mers-cov infection, the common marmoset ( ), due to their small size. all animals were randomly assigned to groups and inoculated as described previously with a total dose of × tcid of mers-cov strain hcov-emc/ via intranasal, oral, ocular ( × tcid each), and intratracheal ( × tcid ) routes ( ) . in the first experiment, the efficacy of prophylactic remdesivir treatment was tested in one group of six rhesus macaques (all males; female rhesus macaques were not available from the supplier at the time of this study) treated with mg/kg remdesivir in vehicle solution ( mg/ml % sulfobutylether-β-cyclodextrin in water and hydrochloric acid, ph . ) and three control rhesus macaques (all males) who received the same volume ( ml/kg) of vehicle solution. this mg/kg dosing in rhesus macaques is roughly equivalent to the -mg daily dosing used in humans in the ebola virus clinical trials. treatment was initiated at h before virus inoculation and continued once daily until dpi. after observing good efficacy of remdesivir upon prophylactic treatment, a second experiment was performed to assess its therapeutic efficacy. one group of six rhesus macaques (all males) was treated with mg/kg remdesivir, and three control rhesus macaques (all males) received the same volume of vehicle solution. due to the acute nature of the mers-cov model in rhesus macaques, therapeutic treatment was initiated at h after inoculation with mers-cov and continued once daily until dpi. treatment was delivered as a slow i.v. bolus injection (total dose delivered over ∼ min) administered alternatingly in the left or right cephalic and saphenous veins. the animals were observed twice daily for clinical signs of disease, using a standardized scoring sheet as described previously ( ); the same person, who was blinded to the group assignment of the animals, assessed the animals throughout the study. the predetermined endpoint for this experiment was dpi. clinical examinations were performed at , , , , and dpi on anesthetized animals. on examination days, clinical parameters such as body weight and respiration rate were collected, as well as dorsal−ventral and lateral chest radiographs. chest radiographs were analyzed by a board-certified clinical veterinarian blinded to the group assignment of the animals. after euthanasia at dpi, necropsies were performed. the percentage of gross lung lesions were scored by a board-certified veterinary pathologist blinded to the group assignment of the animals, and samples of the following tissues were collected: conjunctiva, nasal mucosa, mandibular lymph node, tonsil, pharynx, trachea, all six lung lobes, mediastinal lymph node, liver, spleen, kidney, and bladder. histopathological analysis of tissue slides was performed by a board-certified veterinary pathologist blinded to the group assignment of the animals. -administered ml/kg vehicle solution (vehicle control; gray circles), one group was administered mg/kg remdesivir starting at h before inoculation (prophylactic remdesivir; black squares), and one group was administered mg/kg remdesivir starting at h after inoculation (therapeutic remdesivir; red triangles). treatment was continued once daily until dpi, when all animals were euthanized and necropsies were performed. at necropsy, tissue samples were collected from all six lung lobes, rna was extracted, and viral load was determined as tcid equivalents per gram tissue. individual animals and lung lobes are indicated (a), and averages and sds per group (b). similarly, viral loads were determined in additional tissues from the respiratory tract of each animal (c). r: right; l: left. asterisks indicate statistically significant differences in a two-way anova with dunnett's multiple comparisons. *p < . ; **p < . ; ***p < . ; ****p < . . qpcr. tissues ( mg) were homogenized in rlt buffer, and rna was extracted using the rneasy kit (qiagen) according to the manufacturer's instructions. for detection of viral rna, μl of rna was used in a one-step real-time rt-pcr upe assay ( ) using the rotor-gene probe kit (qiagen) according to instructions of the manufacturer. in each run, standard dilutions of a titered virus stock were run in parallel, to calculate tcid equivalents in the samples. histopathology and immunohistochemistry. histopathology and immunohistochemistry were performed on rhesus macaque tissues. after fixation for d in % neutral-buffered formalin and embedding in paraffin, tissue sections were stained with hematoxylin and eosin (h&e). to detect hcov-emc/ antigen, immunohistochemistry was performed using an in-house rabbit polyclonal antiserum against hcov-emc/ ( : , ) as a primary antibody. stained slides were analyzed by a board-certified veterinary pathologist blinded to the group assignment of the animals. statistical analysis. statistical analyses were performed using graphpad prism software version . . for analysis, the three vehicle control animals from the first and second experiment were combined to form one group of six animals. data availability statement. all data discussed here will be made available to readers upon request. fig. . pathological findings in the lungs of rhesus macaques inoculated with mers-cov and treated with remdesivir. three groups of six rhesus macaques were inoculated with mers-cov strain hcov-emc/ ; one group was i.v.-administered ml/kg vehicle solution (vehicle control; gray circles), one group was administered mg/kg remdesivir starting at h before inoculation (prophylactic remdesivir; black squares), and one group was administered mg/kg remdesivir starting at h after inoculation (therapeutic remdesivir; red triangles). treatment was continued once daily until dpi, when all animals were euthanized and necropsies were performed. at necropsy, the percentage of each lung lobe affected by gross lesions was estimated by a board-certified veterinary pathologist (a). lung samples were collected and stained with h&e and analyzed for the presence of lesions by a board-certified veterinary pathologist. each lung was given a score from to based on the abundance of lesions; the cumulative histology score is the sum of the scores of the six individual lung lobes per animal (b). one representative h&e image was chosen for each group (magnification: ×) (c). lung samples were also stained with a polyclonal α-mers-cov antibody; one representative image was chosen for each group (magnification: ×) (d). images in c and d were chosen as representative images of lung lesions and antigen expression, respectively, rather than being images from consecutive tissue slides. asterisks indicate statistically significant differences in a two-way anova with dunnett's multiple comparisons. **p < . ; ****p < . . world health organization, coronavirus infections a roadmap for mers-cov research and product development: report from a world health organization consultation cepi-a new global r&d organisation for epidemic preparedness and response gs- and its parent nucleoside analog inhibit filo-, pneumo-, and paramyxoviruses therapeutic efficacy of the small molecule gs- against ebola virus in rhesus monkeys remdesivir (gs- ) protects african green monkeys from nipah virus challenge broad-spectrum antiviral gs- inhibits both epidemic and zoonotic coronaviruses comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov coronavirus susceptibility to the antiviral remdesivir (gs- ) is mediated by the viral polymerase and the proofreading exoribonuclease middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study treatment with lopinavir/ritonavir or interferon-β b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset human neutralizing monoclonal antibody inhibition of middle east respiratory syndrome coronavirus replication in the common marmoset prophylactic and therapeutic efficacy of mab treatment against mers-cov in common marmosets treatment with interferon-α b and ribavirin improves outcome in mers-cov-infected rhesus macaques efficacy of antibody-based therapies against middle east respiratory syndrome coronavirus (mers-cov) in common marmosets middle east respiratory syndrome coronavirus infection dynamics and antibody responses among clinically diverse patients, saudi arabia environmental contamination and viral shedding in mers patients during mers-cov outbreak in south korea pathogenicity and viral shedding of mers-cov in immunocompromised rhesus macaques first newborn baby to receive experimental therapies survives ebola virus disease late ebola virus relapse causing meningoencephalitis: a case report palm writing group; palm consortium study team, a randomized, controlled trial of ebola virus disease therapeutics gs- to assess the antiviral activity, long-term clearance of ebola virus and safety in male ebola survivors with evidence of ebola virus persistence in semen investigational therapeutics for the treatment of people with ebola virus disease infection with mers-cov causes lethal pneumonia in the common marmoset thoracic radiography as a refinement methodology for the study of h n influenza in cynomologus macaques (macaca fascicularis) detection of a novel human coronavirus by real-time reversetranscription polymerase chain reaction key: cord- -naz vct authors: rostal, melinda k.; olival, kevin j.; loh, elizabeth h.; karesh, william b. title: wildlife: the need to better understand the linkages date: - - journal: one health: the human-animal-environment interfaces in emerging infectious diseases doi: . / _ _ sha: doc_id: cord_uid: naz vct wildlife are frequently a neglected component of one health; however, the linkages between the health of wildlife and human, domestic animal, and environmental health are clear. the majority of emerging zoonotic diseases are linked to wildlife, primarily driven by anthropogenic land changes. despite this risk, wildlife have important links to people as environmental indicators, food security and safety, and through human livelihoods. this chapter will describe these linkages and demonstrate the need to understand these linkages through targeted surveillance and understanding the ecology of wildlife diseases. while the management of wildlife diseases presents a significant challenge, such practices will greatly improve the health of people, domestic animals, wildlife and the environment. one health is the all-encompassing concept that recognizes the inextricable links between the health of people, animals (wild and domestic), and the environment. while the link between people and domestic animals is well recognized, as they have been used for food, work, and products for millennia, the link between the health of people and wildlife is often neglected. this may be due to a perceived distance between wildlife and people and a lack of understanding of the important linkages that unite human health with the health of all animals. in an ever more urbanized and globalized world, the distance between people and wildlife is shrinking and these linkages are becoming ever more evident. in this chapter, we will discuss the linkages between human health and wildlife from multiple perspectives. this includes direct linkages with human health, such as emerging and nonemerging zoonotic diseases, as well as linkages between wildlife health and the environment, food security and the health of domestic animals, and sustainable human livelihoods. wildlife are an important component of one health, but is often neglected due to difficulty in conducting health studies and limited data and funding. we will further discuss the importance of understanding these linkages with wildlife through targeted surveillance, understanding the ecology of wildlife diseases, and the management of wildlife diseases. when wildlife are discussed in the context of one health, it is frequently in terms of their role as hosts to emerging infectious diseases (eids). with the number of eids significantly increasing during the past years, recent work has demonstrated that the majority are zoonotic ( %) and approximately % of those are of wildlife origin (jones et al. ). an eid is frequently defined as a disease that has recently been jumped into a new host, has evolved a new pathogenicity, is increasing in incidence, or has expanded into a new geographic range (lederberg et al. ; jones et al. ) . while outbreaks of eids may seem like rare events, as a group they cause hundreds of thousands of deaths annually (bogich et al. ) and a single event may cost us$ - billion to the global economy (newcomb ) . the process of disease emergence is complex and often multifactorial, but it can be better understood using broadscale, ecological approaches in identifying these factors, or drivers. in the first attempt to classify the underlying drivers of disease emergence, the institute of medicine (iom) identified six factors including: human demographics and behavior; technology and industry; economic development and land use; international travel and commerce; microbial adaptation and change; and breakdown of public health measures (lederberg et al. ) . in , seven additional drivers were added to the iom report including: human susceptibility to infection; climate and weather; changing ecosystems; poverty and social inequity; war and famine; lack of political will; and intent to harm (smolinski et al. ). iom's classification of the ''factors in emergence'' largely paved the way for current research investigating the underlying drivers of infectious disease emergence. it is important to note that these drivers are not mutually exclusive, and that factors may act in concert and will vary at different stages of the emergence process. for example, anthropogenic land use change has resulted in cross-species transmission of disease or initial emergence, from animal hosts to humans both directly by increasing human contact with animal populations, and in other cases indirectly by changing vector populations (daszak et al. ; patz et al. ) . climate and weather can augment these affects and modulate outbreak size; particularly with vector or waterborne diseases, and global trade and travel has facilitated the spread of those diseases (hufnagel et al. ). these eid drivers function on a different scales, vary geographically (keesing et al. ) , and can be attributed to a combination of environmental, ecological, political, and social forces. despite ongoing research investigating the role of eid drivers, additional studies and modeling approaches are needed to more fully understand the complex mechanisms of emergence (bogich et al. ). one important driver of zoonotic disease emergence that has often been overlooked by the one health community is the wildlife trade. trade in wildlife, both legal and illegal, can lead to the introduction of zoonoses and/or foreign animal diseases that may impact domestic animals or native wildlife species (karesh et al. ) . the illegal wildlife trade is estimated to be approximately us$ billion, the second largest black market after narcotics (karesh et al. ) . it is likely that this estimate is overly conservative as a recent study found that within one tropical country, venezuela, - million animals are traded annually at an estimated cost of us$ million (asmüssen et al. ). an estimated . billion live wild animals were legally imported into united states between and . nearly, % of those animals were destined for the pet trade (smith et al. ). with the magnitude of stressed, and possibly immune compromised, wild animals being transported on a global scale, it is not surprising that this has resulted in the spread and transmission of diseases that affect native wildlife, domestic animals, and people. previous outbreaks of important zoonotic diseases have already been attributed to the wildlife trade; notable, among these in the twenty-first century are severe acute respiratory syndrome (sars) and monkeypox. sars is a novel coronavirus that shifted from bats into civets and humans causing severe morbidity and mortality (case fatality rate of . - . %) (donnelly et al. ) . globalization and airline networks expidited the spread of sars from its point of origin in southern china to infect people in countries, making it the first pandemic of the twenty-first century (zhou and yan ) . the ''wet market'' where sars emerged in guangzhou, china had stalls where wild mammals, domestic animals, reptiles, and birds were sold in conditions of poor hygiene and in close proximity. after the sars outbreak was traced back to these markets, the chinese government reportedly confiscated , wild animals (karesh et al. ) . the most likely scenario of initial spillover and emergence was that rhinolophid bats harboring a sars or sars-like coronavirus were kept in cages in close proximity to civets that contracted and amplified the virus in the markets (li et al. ) . however, the evidence demonstrating bats as the original natural reservoir of sars-like coronaviruses was not discovered until years after the initial outbreak (li et al. ; field ). ongoing research has now revealed large numbers of novel coronaviruses from bats, and the hypothesis of sars originating from these hosts has been further validated (woo et al. ; yuan et al. ) . monkeypox emerged under similar conditions through the legal pet trade in the us in . it is hypothesized that prairie dogs (cynomys spp.) were in contact with a shipment containing gambian rats (cricetomys spp.) and african dormice (graphiurus spp.) at a wholesale pet store. the prairie dogs became ill, as did people in contact with them (guarner et al. ) . despite the serious threat of eids from wildlife in the legal and illegal trade, few international programs exist to screen imported wildlife for pathogens of concern. a recent study analyzed wildlife products confiscated by the us customs and border protection at john f. kennedy airport in queens, ny as well as seizures from airports in philadelphia, washington, dc, houston, and atlanta . smith et al. ( ) detected simian foamy virus (sfv) and several herpesviruses from bushmeat samples, including nonhuman primates, e.g., chimpanzees (pan troglodytes), mangabeys (cercocebus spp.), and guenons (cercopithecus spp.), that were imported from guinea, liberia, and nigeria. while sfv has not yet been shown to be pathogenic in people, approximately % of bushmeat hunters in close contact with dead primates were found to be infected with sfvs (wolfe et al. ) . wolfe et al. ( ) demonstrated sporadic transmission of sfvs to humans, and offered a better understanding of how human immunodeficiency virus/acquired immunodeficiency syndrome (hiv/aids) may have emerged from primates. we know that simian immunodeficiency virus (siv) jumped several times into people hunting and consuming nonhuman primates before mutating into the pandemic hiv strains that are now circulating (heeney et al. ). this recent work by smith et al. ( ) demonstrates that zoonotic pathogens can be transported in bushmeat and potentially could cause an outbreak in a location far from its endemic region, underscoring the need for better port surveillance and regulation of this trade. left unregulated, legal and illegal wildlife trade can potentially have a large impact on human health, as well as direct impacts on wildlife and domestic animal health (discussed below). the health of wildlife is closely linked to the health of the environment and can be extremely sensitive to anthropogenic changes. this includes direct physiological and behavioral responses to chemicals and pollution as well as competition and other effects from the introduction of nonnative wildlife and/or new pathogens. animals have long been used as indicators of a toxic environment. the proverbial ''canary in a coal mine'', as later memorialized by sting in a song, stems from the use of canaries to detect trace amounts of methane and carbon monoxide in mines since the early s. their death indicated to miners that they needed to evacuate the mine to prevent asphyxiation. dichlorodiphenyltrichloroethane (ddt) was one of the first global acknowledgments of chemicals affecting nontarget animals. ddt was found to reduce the eggshell thickness of multiple bird species (porter and wiemeyer ) . it was later discovered that bioaccumulation of the chemical through the trophic levels had devastating effects on the populations of certain top predators and insectivorous birds (e.g., raptors such as peregrine falcons (falco peregrinus) and bald eagles (haliaeetus leucocephalus) (grier ) . these effects became widely distributed by rachel carson's ''silent spring'' (carson ) and wildlife and plants were increasingly recognized as important indicators of man-made environmental health threats. during the s, the us environmental protection agency (epa) began using ecological risk assessment (in addition to human risk assessment) to evaluate the risk of agrochemicals or other manufactured chemicals, superfund sites, as well as air and water pollution (epa ) . ecological risk assessment depends on scientific assessment of the risk a chemical poses to a wide variety of plants and animals, including invertebrates, fish, birds, and small mammals (epa ). while the system continues to be improved, risk assessments have been important in identifying the detrimental effect of acid rain (beamish ), perfluorinated chemicals ( van de vijver et al. ) , and most recently endocrine disruptors (kloas and lutz ) . these ecological risk assessments have contributed to several us national environmental laws that were created to protect the health of people. in addition to top predators, small mammals are frequently used in ecological risk assessments and have been proposed as sentinels for heavy metal contamination. talmage ( ) suggests that small mammals make good indicators of environmental pollution because of their abundance, widespread distribution, short dispersal distance, generalized food habits, short life span, high reproductive rate, and relative ease of capture. in particular, they can be used to assess the environmental contamination of landfills and mine areas (torres et al. ) . insectivorous mammals appear to be the best indicators as they are exposed more directly through invertebrates that may consume soil (e.g., earthworms) (hamers et al. ) . in particular, small mammals have been used successfully to assess cadmium, fluoride, lead, and mercury exposures (talmage ) . one relatively recent study found that both rodents and children living around a mining site in mexico had nearly twice the levels of lead and arsenic as the respective controls from the reference site (jasso-pineda et al. ) . despite the many studies that have successfully demonstrated that various rodent species can be used as environmental indicators for heavy metals and chronic pollution and the frequent use of rodents in risk assessments for new chemicals on the market, rodents are rarely used for regulatory purposes (e.g., for long-term monitoring of mining sites) (handy et al. ) . when used appropriately, environmental indicators could be a very valuable tool in long-term monitoring of the risk of pollution and contamination of both terrestrial and aquatic habitats (lam and gray ; jasso-pineda et al. ) and should be utilized more frequently. as with zoonotic eids, emerging diseases of wildlife have increased during recent decades (daszak et al. ) and have frequently been linked to anthropogenic ecological changes. specifically, it is likely that trade, travel, invasive species, and poor biosecurity measures are driving many of these diseases. we highlight this with two emerging fungal pathogens that have caused devastating impact on two vertebrate groups, chytrid fungus in amphibians and white-nose syndrome (wns) in bats, with indirect one health consequences for human health and food security. during the late twentieth century, amphibian populations began to decline on a global scale (heyer et al. ; young et al. ) . chytridiomycosis (batrachochytrium dendrobatidis; chytrid fungus) was determined to be the causative agent in many of the declines (daszak et al. ) . this fungus caused multiple species declines (even local extirpation of multiple amphibian populations in some areas) in pristine habitats in the americas and australia (daszak et al. ) . it is believed that the chytrid fungus was spread through the trade of african clawed frogs (xenopus laevis), the original host, and the north american bullfrog (rana catesbeiana) (weldon et al. ; schloegel et al. ). r. catesbeiana is frequently raised and traded for food, with greater than , kg of this species sold within brazil every year (schloegel et al. ). it has been suggested that out of more than species of amphibians noted to be rapidly declining due to enigmatic causes (stuart et al. ), a significant portion of them may have been due to chytrid (skerratt et al. ). this fungus demonstrates how seemingly unrelated anthropogenic actions (wildlife trade) can have far-reaching effects on the environment and wildlife. additionally, the loss of amphibians may disrupt ecological processes, such as a reduction in predation on mosquito larvae, resulting in consequent indirect impacts on human or animal health. wns is an emerging disease of hibernating bats caused by the fungus geomyces destructans (blehert et al. ; lorch et al. ) . it was first documented in the us in , and was most likely introduced accidentally by people traveling to and from europe, where it is ubiquitous and causes no bat mortality (puechmaille et al. ). this cold-loving fungus thrives in the winter environment of bat hibernacula and disrupts the hibernation and physiology of over-wintering bats. bat mortality is frequently attributed to starvation and dehydration as the infection causes arousal during the winter leading to the depletion of the fat reserves of the hibernating animals (cryan et al. ; reeder et al. ). the fungus grows on the muzzle and wing membranes of susceptible bats. mortality rates at many hibernacula are extremely high, commonly in the range of - % (turner et al. ). wns has rapidly spread south and west across the us, being confirmed in us states and four canadian provinces; and by early , the us fish and wildlife service estimated that over . million bats have died from the disease. wns is predicted to cause the local extirpation or possibly the extinction of the little brown bat (myotis lucifugus), which was once the most populous bat in the us (frick et al. ) . once a wildlife disease such as wns is established, control becomes very difficult. researchers are currently working to understand the environmental and life-history variables that allow the fungus to persist and spread, with hopes that areas or microclimates can be set aside for management intervention ahead of the epidemic wave (boyles and willis ; wilder et al. ; langwig et al. ) . other unproven solutions being developed include antifungal treatment, maintaining rescue captive breeding colonies, and artificially heating caves. wns, like chytrid, is an introduced fungal pathogen and arguably they are two of the most significant wildlife diseases-threatening a wide range of species with possible ecological and global extinction (fisher et al. ) . the value of bats in control of agricultural pests in north america alone has been estimated to us$ . billion annually, with a loss of bats translating into increased production costs (pesticides and other pest control methods) and smaller crop yields (boyles et al. ). as introduced above, food safety and security is an important component of one health, especially as the fao estimates that . billion people are undernourished worldwide (fao ) . wildlife is linked with food security as wildlife can contaminate foodstuff with zoonotic diseases, bushmeat is a major protein source for people living in many tropical countries, and wildlife and domestic animals can share significant pathogens. foodborne illnesses pose a serious threat to public health with growing economic and international trade ramifications. standard epidemiological public health methods are frequently used to investigate foodborne outbreaks among people. however, foodborne diseases are good examples of the intricate link between human and animal populations, and the surrounding environment. in , a virulent strain of e. coli o :h was linked to spinach and affected approximately people in states (cdc ) . a typical epidemiological investigation of this outbreak would have extended only to human morbidity, mortality, assessments of risk and probable source, laboratory diagnosis, and clinical treatment. however, when domestic and wild animal health and ecology were considered, warnert ( ) found the same strain of e. coli o :h isolates that caused the human outbreak in wild pig feces, the feces of several cows, and in a stream on one of the four spinach farms in the area. thus, a one health perspective integrating our knowledge of epidemiology, clinical diagnostics, the environment and ecology, was required to fully investigate and understand this outbreak and has great utility in understanding the foodborne illness outbreaks. foodborne pathogens from wildlife span the taxonomic spectrum from helminthes to viruses. emerging foodborne diseases represent the majority of foodborne illnesses in the us and an even larger percentage of the foodborne illnesses are likely due to yet undescribed pathogens (tauxe ) . many of the known foodborne pathogens and up to % of foodborne eid events are zoonotic and many may be linked to wildlife. commonly, both wild and domestic animals are implicated as sources of food contamination (beuchat and ryu ; doyle and erickson ; newell et al. ; gorski et al. ; cima ) . despite this, definitive identification of a specific source animal or species is rare, particularly as epidemiological investigations often occur long after the index case, as it can take time for an outbreak across various states or counties to be detected. however, in some cases, detailed ecological studies can determine the exact route of food contamination from wildlife. for example, nipah virus, a bat-borne emerging encephalitic paramyxovirus, is primarily transmitted through contaminated date palm sap in bangladesh. with annual outbreaks in people and very high fatality rates ([ %), this is a pathogen of special concern (luby et al. (luby et al. , . recent serological and pathogen discovery studies have shown that pteropus giganteus, a large fruit bat, is likely the primary reservoir for this virus (epstein et al. ) . further, using techniques of wildlife surveillance and infrared camera traps, the exact mechanism of transmission was determined. bats were observed feeding from date palm sap collecting pots at night and currently specific interventions that do not entail bat eradication are being developed to prevent this transmission (nahar et al. ; khan et al. ). wild animals provide a substantial portion of our food globally, with nearly half of all seafood coming from wild sources. in some regions of the world, wild meat from terrestrial animals represents a primary source of protein on which people are dependent. the volume of wild meat (''bushmeat'') harvested from central africa alone totals more than billion kg per year (wilkie and carpenter ) . this volume of meat, almost all of which is processed and distributed to consumers with few if any modern hygiene practices, provides a constant opportunity for human exposure to both rare and common foodborne pathogens (karesh et al. ; smith et al. ). modeling has been used to indicate direct linkages between health and bushmeat consumption. golden et al. ( ) used generalized linear mixed-model regression to suggest that if bushmeat were removed from the diet of children in madagascar, hemoglobin concentration would decrease by . g/dl leading to a likely increase in anemic children of nearly %. the overconsumption of wildlife resources may soon lead to the loss of bushmeat protein to diets. fa et al. ( ) predict that the percentage of dietary protein consumed through bushmeat is unsustainable and will decrease from an estimated % in to % by in the congo basin. this prediction is based on increasing wildlife extraction to production ratios that will ultimately lead to a decline in wildlife abundance. without the availability of bushmeat or the redistribution of global food sources, food insecurity in this region is likely to increase. this link between bushmeat hunting and food insecurity has been supported by the results of a study, which found the alleviation of food security concerns of villagers in zambia by improving domestic animal production over years led hunters to turn in , snares and firearms, saving an estimated , wild animals (lewis and jackson ). wildlife can be reservoirs of important diseases of domestic animals, some of which were originally diseases of livestock and are now maintained in wildlife populations despite eradication of the disease in domestic animals. diseaseinduced morbidity and mortality in domestic animals as well as economic sanctions associated with the presence of certain diseases can adversely affect the supply of food animals. certain wildlife diseases can cause morbidity and mortality in domestic animals that are accidental or dead-end hosts. one such example is malignant catarrhal fever, caused by alcelaphine herpesvirus of wildebeest (connochaetes taurinus), which can cause acute mortality in cattle (russell et al. ). while its significance is limited to regions endemic for wildebeest (or zoos housing them), outbreaks of the virus can cause significant losses and hardships for local herders in africa. some diseases have larger economic ramifications, such as foot-and-mouth disease (fmd). though the virus affects all species with cloven hooves (order: artiodactyla), there are specific strains that are more likely to circulate in different geographic locations and possibly primary reservoir species (e.g., cattle or african buffalo, syncerus caffer) (klein ). while the virus is not highly fatal to the animals, trade restrictions to prevent the introduction of the virus in fmd-free regions has led to it becoming an economically important virus. it is estimated that during the fmd outbreak in the uk, losses to agricultural industry and the food supply chain amounted to £ . billion (thompson et al. ) . outbreak response led to the destruction of million animals (thompson et al. ) . it is likely that the virus was imported into the country from cattle in virus endemic regions (samuel and knowles ) . some diseases of concern have their origins in domestic animals, but have now been established into wildlife populations where they can then be retransmitted back to domestic animals (daszak et al. ). in the us, brucella abortus is generally believed to be introduced to the continent by cattle (meagher and meyer ) . after a successful eradication campaign, all states were declared free of brucellosis in domestic cattle herds in ; however, the maintenance of the bacteria in herds of elk and bison in the greater yellowstone area has allowed the pathogen to persist and continue to cause outbreaks. the debate over how to eradicate brucellosis from the country is ongoing. management methods that have been discussed include: test and cull, depopulation, and prohibiting winter feeding sites for elk as well as vaccination (olsen ) . a similar story follows the introduction of bovine tuberculosis (mycobacterium bovis) into wildlife hosts (see box ). one health includes considering pathogens originating from domestic animals that can severely affect wildlife. rinderpest virus was introduced into africa during the early s. the virus swept across the continent killing susceptible cattle and wild artiodactyls en masse. it has been reported that over . million cattle died in southern africa and up to % of the african buffalo population was decimated (plowright ) . fortunately, rinderpest was not able to be maintained in large wildlife populations without the presence of cattle (plowright ) . it is believed that the massive loss of multiple species of grazers (buffalo, wildebeest etc.) actually led to a change in the ecosystem of the region that is still in effect today, although as these species are recovering, the ecosystem is likely reverting to that which was believed to have been prior to the outbreak (holdo et al. ). rinderpest is now the second virus that has been eradicated through the use of vaccinations, after smallpox virus (yamanouchi ) . box . bovine tuberculosis: a persistent linkage between wildlife and domestic animals wildlife reservoirs of domestic animal diseases can make it exceedingly difficult or even impossible to eradicate economically important pathogens. often, diseases that originated in domestic animals persist in wildlife at such low prevalences that they are not detected in the wildlife until the disease is controlled or eliminated in domestic animals. in the us, a program to eliminate bovine tuberculosis (mycobacterium bovis; btb) was initiated in . by , every state had been declared to be free of btb (knust et al. ) , when a case of btb was reported in a white-tailed deer (odocoileus virginianus; wtd) in michigan. the outbreak was confirmed after of hunter-killed deer tested positive for btb (schmitt et al. ) . genetic analyses have confirmed that this strain of btb has been circulating in the wtd population at a low prevalence (o'brien et al. ) . efforts by the michigan department of agriculture and michigan department of natural resources (mdnr) initially focused on depopulation of wtd in the -county affected area and a ban on feeding or baiting deer during winter. while they have been successful in decreasing the prevalence by %, low levels of btb ( . % prevalence) continue to circulate in the wtd and occasionally spillover into cattle, leading to depopulation of the cattle herd (o'brien et al. ) . the circulation of btb in wildlife has a high economic cost to agriculture in the region as michigan now has a splitstate tuberculosis accreditation (the upper peninsula of michigan is still considered tuberculosis free, while the rest of the state is divided between accredited free, modified accredited advanced, and modified accredited, depending on the distance from the nidus of btb. it is estimated that btb and the accreditation change cost michigan's agriculture industry us$ million from to and us$ million from to (thiel ) . eradication of btb in the wtd population has been prevented partially due to the lack of public support for continued lethal population control. hunters contribute us$ million to michigan's economy and with other sympathetic electorate, who desire to view deer in their yards, have significant political clout (o'brien et al. ) . political pressure to decrease the level of lethal control has lead the mdnr to look to developing new vaccine-related technologies. while no such vaccine has yet been developed, pressure continues to mount as wtd in minnesota were found to have btb (most likely a cattle strain) following outbreaks in cattle in (knust et al. ) . several other wildlife species are known to be reservoirs of bovine tb in countries beyond the us, making it very difficult to eradicate globally. (o'brien et al. ) . bovine tuberculosis demonstrates the difficulty of sustainable control of a zoonotic and economically costly disease. the challenges of either eradication or control in wildlife also highlight the cost-effectiveness of prevention, i.e., investment in efforts to prevent domestic animal diseases from becoming established in wildlife populations would be more cost-effective and more less effort than trying to remove a pathogen from wildlife populations. many of the most biologically diverse regions coincide with human populations living at the highest poverty levels. as people raise themselves out of poverty, they can provide their families with better medical care, leading to better health. one proposed method of community development for improving livelihoods and health is the sustainable use and conservation of wildlife. an important component of ensuring sustainable use of wildlife is engaging all of the relevant stakeholders, especially the people living around the conservation areas whose livelihoods can be directly impacted by wildlife. people living in areas with free-ranging wildlife are frequently in conflict with wildlife, e.g., crop raiding. the sustainable use of wildlife can lead the community to accept the risks of coexisting with wildlife to support conservation and the health of the natural ecosystem. ecotourism has been defined by many different groups, here we will use the same definition as stronza and pêgas ( ) -nature tourism that intentionally seeks to deliver net positive contributions to environmental conservation and sustainable development for local communities. this definition links conservation directly with the health and development of the local communities. this concept aims to harness resources from the ever-growing tourism industry (walpole and thouless ) . for example, in kenya wildlife tourism grossed nearly us$ . billion in tourism-related industries in , with . million visitors to parks and game reserves (knbs ) . that amount of earnings contributes significantly to the national economy. however, despite the significant earnings from wildlife-based tourism and safari hunting, the equitable distribution of the funds is important in achieving the sustainability of wildlife resources. the success of ecotourism can be evaluated by measuring local economic benefits and participation as well as conservation indicators. there are many factors that may contribute to the success or failure of ecotourism, including: the presence of a flagship species, the biodiversity index and ease of viewing wildlife, the popularity of a particular location, the attitudes and current livelihood of the local communities, the perceived risk of wildlife to the community (e.g., crop raiding, disease, safety, or competition with, or consumption of domestic animals), and the perceived cost of living near the protected area (e.g., loss of access to cultivable or grazing land, watering holes, and inability to hunt) (walpole and thouless ) . a recent analysis of perceived cost from villages around kibale national park, uganda suggested a distinct geographical variation in households with perceived loss compared to those with perceived benefit. households within . km of the park boundary perceived the highest losses, while benefits were perceived up to km from the boundary (mackenzie ) . salafsky et al. ( ) worked with local communities to establish and support business operations and evaluate them with financial, social, and conservation indicators. they found that community participation in an operation was significantly linked to conservation success, even if the focus of the operation itself was not involved in conservation, such as the example from zambia discussed earlier where snares were turned in. interestingly, few operations were able to cover their costs after years and those that did required strong management systems to remain financially viable (salafsky et al. ) . in particular, creating successful community-based ecotourism programs can be very difficult given the competition and the high cost associated with start-ups in resource poor areas. it can take several years before such operations are able to cover their costs and it is not always clear that benefits for human health will trickle down from these operations (kiss ; walpole and thouless ) . few projects have successfully linked wildlife conservation directly to health care, although there are a few examples of large-scale initiatives seeking to do so. in the qomolangma national nature preserve in tibet, a collaboration among the villages, government, and various ngos led to the training of local villagers to protect the nature preserve with the benefits being improved access to basic health care. several individuals from local villages were educated in: preventative health care, distribution of medicines, environmental protection, ecotourism, poverty reduction, and income generation. these trainees provide services and education to the villagers. the success of the program was measured by a doubling of the estimated wildlife populations in the preserve, a decrease in logging by two-thirds, a decrease in the incidence of diarrheal diseases, and a reduction of infant mortality by % (melnyk ; taylor-ide and taylor ) . rarely mentioned in one health publications, discussions, or meetings is the topic of anthropozoonoses-diseases transmitted from humans to animals. much of the original literature on this subject area comes from studies with nonhuman primates, especially the great apes. butynski ( ) provides an extensive review of anthropozoonotic risks to great apes, including measles, herpesviruses, poliovirus, mycobacterium tuberculosis, sarcoptes scabeii, and a number of intestinal parasites. one survey found that % of tourists visiting sepilok orangutan rehabilitation centre in sabah, malaysia reported having one or more symptoms of an infectious disease while they were visiting the center (muehlenbein et al. ) . it has long been recognized that human tuberculosis (m. tuberculosis) can infect nonhuman primates. standard practices for captive nonhuman primates include routine testing of both the nonhuman primates and people who have contact with them (e.g., zoo keepers). interestingly, there is genetic evidence that suggests m. tuberculosis is significantly older than strains found in domestic livestock (m. bovis). it has been hypothesized that m. bovis evolved from an existing human pathogen or a common ancestor (brosch et al. ) . this is consistent with evidence of human tuberculosis preceding the to , -year-old domestication of animals (gutierrez et al. ) . excluding anthropozoonotic diseases from one health discussions alters the discourse to a narrow anthropocentric view of the world. the prevention of transmission of human diseases and improved human health can provide simple and cost-effective methods to protect wildlife from anthropozoonotic diseases. the linkages discussed above clearly indicate that wildlife health is intricately tied to the health of people, domestic animals, and the environment. despite these linkages and their key role as reservoirs of human eids, global efforts for wildlife health surveillance are lacking and underfunded. wildlife health surveillance can be used to better understand the pool of pathogens that may spillover into people or domestic animals; it can also be used to track the spread of wildlife diseases through populations. this surveillance can be used to investigate the ecology of the pathogen and hosts, which in turn can facilitate the prevention and control of important diseases. frequently, responses to emerging disease outbreaks are reactive and costly (childs and gordon ) . surveillance based on disease-specific control programs has successfully mobilized financial resources and delivered short-term results against disease-specific objectives (oliveira-cruz et al. ) . however, some have criticized this approach for focusing exclusively on a single disease and failing to reduce the risk of most pandemics (oliveira-cruz et al. ; travis et al. ). further, pathogen-specific surveillance often lacks sustainability and cannot be scaled-up, as benefits and outcomes are generally limited to the target area and funding cycle (oliveira-cruz et al. ) . to better target wildlife surveillance, there are several factors that need to be considered including geographic risk of emergence, the host species of the greatest concern for zoonotic spillover, and transmission pathways. the initial work to identify geographic ''eid hotspots'' found that countries with high biodiversity and human density are at the greatest risk for outbreaks (jones et al. ). most of these hotspots are located within developing countries, which often lack the infrastructure to conduct wildlife surveillance, either active or passive, and the ability to conduct diagnostic assays for rare or new diseases. one example of a multinational effort is the usaid predict project (see box ), which is based on initiating wildlife surveillance and investigating viral diversity in wildlife in these geographic hotspots. as zoonotic disease surveillance in wildlife clearly represents a great challenge (i.e., there are , + mammal species globally), predictive modeling and known patterns in host range can be used to focus the effort on the species and pathogens that pose the greatest risk of zoonotic emergence. new tools may make it possible to predict general patterns of host range in unsampled hosts, given known patterns from the past years of the published literature, knowledge of a species ecological and life-history traits, and some measure of surveillance or sampling bias from both a host and disease perspective. initial reviews of the literature were largely descriptive and grouped host species at higher taxonomic levels (e.g., ungulates, carnivores, rodents, and nonmammals) (cleaveland et al. ; woolhouse and gowtage-sequeria ; woolhouse and gaunt ) . more recent studies have tested patterns of pathogen-host range in a more mechanistic way, by explicitly including information on phylogenetic relatedness, although these studies are usually limited to a single host group or pathogen, e.g., bats and rabies (streicker et al. ) ; primates (davies and pedersen ) ; and fish ectoparasites (krasnov et al. ; poulin ) . similar approaches are currently being used to look at patterns of zoonotic disease emergence for all known mammal viruses and to test mechanistic drivers of cross-species viral emergence (bogich et al. ) . another useful way to focus surveillance efforts, speed up early detection, and reduce the risk of cross-species transmission is to target transmission pathways at specific human-animal interfaces. a key advantage of this approach is that surveillance efforts and control measures for one route of transmission should also mitigate a number of infectious diseases sharing the same transmission pathway. a multipathogen approach targeting disease transmission routes would be a useful way to target pathogen surveillance and control. it may also effectively focus prevention efforts, achieve early detection, and reduce additional risk of transmission. box . the usaid predict project: establishing a global wildlife surveillance network the predict project is part of the us agency for international development's emerging pandemic threats program. this project is developing a global wildlife virus surveillance system in countries that are in geographic hotspots (jones et al. ) in the amazon basin, mexico, southeast asia, and china, the gangetic plain, and the congo basin. this active surveillance system is aimed at understanding the importance of various human-wildlife interfaces. based on phylogenetic modeling, rodents, bats, and nonhuman primates were selected as target taxa due to their higher likelihood of harboring zoonotic pathogens (olival et al., unpublished data) . specifically, the project is targeting bushmeat sold in markets, wildlife that is collected by hunters, and wildlife living in proximity and/or conflict with people. predict is also investigating how changes in land use (using a landscape development index) can affect biodiversity and viral diversity of wildlife across a gradient of urban areas, rural areas with forest fragmentation, and in areas of pristine forest. this project works within each country's infrastructure to build surveillance and diagnostic capacity. viral discovery is conducted using degenerative primers to target viral families of zoonotic importance, which is then confirmed through genetic sequencing. in addition, deep sequencing methods are being used to discover new viruses. this diagnostic method maximizes the likelihood of discovering viruses, instead of targeting specific pathogens that may not be present. the predict project is standardizing surveillance methods across the globe to target potentially zoonotic viruses before they spillover. it is an example of combining high-level modeling with on-the-ground field data to target a surveillance system to efficiently detect potential pandemic viral threats. understanding wildlife diseases necessitates a multidisciplinary team, including epidemiologists, ecologists, and medical professionals. the ecology of the reservoir hosts as well as other competent species can be used to target disease management and mitigation (see box ). the importance of this is clear when considering pathogens such as ebola virus, where we have only recently discovered the probable reservoir (leroy et al. ), yet often cannot trace the transmission events from bats to nonhuman primates and/or people. further, new surveillance is finding evidence for ebola-like viruses in natural mammal reservoirs (e.g., bats and primates) well outside of their previously known range in africa (nidom et al. ; olival et al. unpublished data) . these recent findings point to a more urgent need to implement general, not pathogen-host specific, strategies to prevent zoonotic disease spillover from wildlife, i.e., by targeting transmission pathways or specific groups of hosts as were mentioned above. highly pathogenic avian influenza a/h n is a good example of the importance of understanding the ecology and epidemiology of a zoonotic disease in wildlife. this strain of avian influenza was first diagnosed in people in and in subsequent outbreaks has had an observed case fatality rate up to % (kandun et al. ) , although seroprevalence data indicate that it may be as low as - % (li et al. ) . when a/h n spread into europe and africa in there was an immediate reaction and assumption that it was transported by migrating birds, despite the lack of data at that time. the role of wild birds in the transmission and maintenance of a/h n remains controversial. outbreaks of a/h n in wild bird populations have occurred in isolation of poultry outbreaks and caused severe morbidity and mortality in some species. more than , birds at qinghai lake in china died during an outbreak, % of which were bar-headed geese (ansar indicus) (chen et al. ) . similar outbreaks occurred in mongolia and europe, indicating that there is occasional long-distance transmission by migratory birds (alexander ) . the effectiveness of longdistance transmission varies by species, as some species are severely affected by a/h n and others may be nonclinical shedders (based on studies in domestic mallards) (sturm-ramirez et al. ) . modeling by kilpatrick et al. ( ) suggests that the spread of a/h n may be a combination of the trade of poultry, the commercial trade in wild birds and transmission through migratory birds. their model suggests that the spread throughout asia was primarily due to poultry trade, and the spread in africa was partly due to poultry trade and partly due to migrating birds and the spread in europe was most likely through migrating birds. however, transmission by migratory birds appears to be rare, as proposed by gilbert et al. ( ) , who suggest that wild birds stopping over in areas at high risk for poultry strains of a/h n may occasionally transmit the virus via migration. a lack of sufficient information on the frequency or likelihood of spread through migratory birds indicates that further surveillance of a/h n should be conducted to better understand the transmission dynamics. without further ecological and epidemiological studies on the dynamics of a/h n , wild birds may have continued to be blamed for the maintenance of a/h n . it is known that waterfowl can be a mixing vessel for various subtypes of avian influenza (hatchette et al. ) , and it is rational to assume that they are the reservoir for a/h n . however, the seroprevalence of the h subtype is low in apparently healthy wild water bird populations (kang et al. ) . analyses of epidemics of a/h n in thailand found a strong association with the presence of free-grazing domestic ducks (gilbert et al. ) . additional analyses by gilbert et al. ( ) suggest that the presence of domestic ducks is the main factor associated with risk of a/h n in south asia, while human population and chicken density were also associated. recent modeling suggests that moderately sized flocks of poultry could maintain transmission of a/h n , whereas isolated small flocks or large commercial flocks are unlikely to maintain the virus (hosseini et al., unpublished data). thus, ecological studies of a/h n in both wild and domestic birds will continue to be important in understanding the maintenance of this virus. box . vulpine rabies: the importance of understanding ecology for control of rabies in europe vulpine rabies was first introduced to poland in and has since radiated out through eastern and western europe (anderson et al. ) . passive surveillance of wildlife throughout europe found that red foxes (vulpes vulpes) represented % of all wildlife diagnosed with rabies (who ) . attempts to control the fox population through culling did not succeed at preventing the spread of rabies, which expanded at an annual rate of - km. several papers in the early s illustrate the need for approaches and models that combined research on the viral pathogenesis, fox ecology, mapping of the development of epidemics, and importantly, the contact rate between foxes (anderson et al. ; macdonald and bacon ) . the social structure and density of foxes in some urban areas (five foxes per kilometer) would require a culling rate of - % to have a high probability of eliminating rabies (smith and wilkinson ) . culling adult foxes leaves empty territories that are quickly filled by dispersing yearlings and other young foxes. additionally, cub production is density dependent; thus, if culling were to decrease the population, more susceptibles would be introduced into the system as the reproductive rate of foxes would increase (macdonald and bacon ) . vaccination has been demonstrated to be effective in parts of europe and has two major advantages over culling: ( ) immunity decreases the likelihood of contact between two susceptible foxes and ( ) the reproductive rate remains stable, preventing the surge of susceptibles that follows a decrease in fox density (macdonald and bacon ) . for point-source infections, such as what would happen if rabies was introduced into britain, culling is more likely to be successful than in areas where rabies is endemic, especially as control efficacy may vary according to season (smith and wilkinson ) . the current control method for britain is to cull foxes within km of the point source followed by a ring of vaccine bait to prevent escape (smith ) . the ecological and epidemiological theory behind the development of control strategies for rabies in foxes can be transferred to other similar systems. in developing countries, the domestic dog remains the primary rabies reservoir. research in india has shown that trap, vaccinate (with or without sterilization), and release programs have led to a decreased number of rabies cases in humans, and may have led to a decrease in the stray dog population as well (reece and chawla ) . understanding the ecology of rabies in foxes and other carnivores has lead to a significant decrease in human rabies cases. wildlife are elusive, and have different ownership and custodian status among states and countries, and are often perceived in a variety of ways (emotional, religious, cultural, or utilitarian, etc.) by the general public; all of which necessitates new methods of disease control that consider the whole ecosystem including human interactions. artois et al. ( ) recently wrote a review of methods for controlling disease in wildlife and the risks associated with these methods. the primary goals of control are to limit the number of susceptibles or to treat/eliminate infected individuals to limit infectious period. lethal control and vaccination are the primary methods available to limit the number of susceptibles. lethal control, or culling, has frequently been shown to be very difficult to maintain in large populations of wildlife with high reproductive or immigration rates and is increasingly considered socially unacceptable (caughley and sinclair ) . further, culling is generally not a viable option when dealing with outbreaks in rare or endangered species, and the act of culling itself actually increases the human-wildlife contact interface and potential transmission of zoonoses. vaccination is increasingly being considered as a control option and is predicted to be the most efficient method to control hosts with relatively low reproductive rates. however, the production of a vaccine that is efficacious, stable in the environment, and easily deliverable (frequently orally) makes vaccination a difficult control method (artois et al. ). in addition, most effective wildlife vaccines (e.g. for rabies) are modified live vaccines that have the potential to harm nontarget species. another possible control method is fencing or other physical barriers to prevent direct contact. this is being recommended to farmers in michigan and minnesota as a method to prevent btb transmission from wtd sharing feed or entering cattle lots (palmer et al. ). low-tech bamboo skirts are also being used to keep bats out of date palm sap harvest areas to prevent the transmission of nipah virus (nahar et al. ). however, fences and barriers can sometimes interrupt nontarget species as well as the local ecosystem, e.g., in southern africa where fences prevent the migration of large herbivores, such as elephants (loarie et al. ). innovative methods of wildlife disease management will continue to be needed, especially as diseases emerge in difficult to manage species such as wns in bats. one health links the health of humans with that of domestic and wild animals and the environment. in this chapter, we have explored various linkages that connect wildlife health to human and domestic animal health. wildlife are linked to people through the risk of pathogen spillover, food security and safety, changes in the environment, and a human dependence on wildlife for livelihoods. each of these linkages can have positive and negative effects; e.g., human and wildlife transportation can increase the risk of spreading an emerging disease in either population; zoonoses may be balanced by the effects of anthrpozoonoses (though little research has been done to determine this); wildlife are a vital protein or income source for some human populations and can be responsible for food contamination or income loss for others. a better understanding of the role of wildlife and a more robust surveillance system to investigate this will be critical to the one health field in the future. targeted wildlife surveillance, by coupling active and passive surveillance with predictive models, can be an important tool in understanding and preventing eids in people and in animals. this surveillance data and data from other ecological studies can be used to inform management of diseases in both wildlife and domestic animals. the linkages between people and wildlife are clearly an important part of one health and there is a growing need to understand them. summary of avian influenza activity in europe population-dynamics of fox rabies in europe sustainable control of zoonotic pathogens in wildlife: how to be fair to wild animals? loss of fish populations from unexploited remote lakes in ontario, canada as a consequence of atmospheric fallout of acid produce handling and processing practices bat white-nose syndrome: an emerging fungal pathogen? using mathematical models in a unified approach to predicting the next emerging infectious disease economic importance of bats in agriculture could localized warm areas inside cold caves reduce mortality of hibernating bats affected by white-nose syndrome? a new evolutionary scenario for the mycobacterium tuberculosis complex africa's great apes silent spring wildlife ecology and management update on multi-state outbreak of e. coli o :h infections from fresh spinach h n virus outbreak in migratory waterfowl surveillance and control of zoonotic agents prior to disease detection in humans wildlife, trade, susceptibility amplify food risks: food-borne illness risks cross borders, production types diseases of humans and their domestic mammals: pathogen characteristics, host range and the risk of emergence wing pathology of white-nose syndrome in bats suggests life-threatening disruption of physiology emerging infectious diseases and amphibian population declines emerging infectious diseases of wildlife-threats to biodiversity and human health anthropogenic environmental change and the emergence of infectious diseases in wildlife phylogeny and geography predict pathogen community similarity in wild primates and humans epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong summer meeting -the problems with fresh produce: an overview epa ( ) guidelines for ecological risk assessment. environmental protection agency henipavirus infection in fruit bats (pteropus giganteus) bushmeat and food security in the congo basin: linkages between wildlife and people's future the state of food insecurity in the world: economic crises-impacts and lessons learned emerging fungal threats to animal, plant and ecosystem health an emerging disease causes regional population collapse of a common north american bat species free-grazing ducks and highly pathogenic avian influenza flying over an infected landscape: distribution of highly pathogenic avian influenza h n risk in south asia and satellite tracking of wild waterfowl benefits of wildlife consumption to child nutrition in a biodiversity hotspot prevalence, distribution, and diversity of salmonella enterica in a major produce region of california ban of ddt and subsequent recovery of reproduction in bald eagles monkeypox transmission and pathogenesis in prairie dogs ancient origin and gene mosaicism of the progenitor of mycobacterium tuberculosis lack of a distinct gradient in biomarker responses in small mammals collected at different distances from a highway a proposal for the use of biomarkers for the assessment of chronic pollution and in regulatory toxicology influenza a viruses in feral canadian ducks: extensive reassortment in nature origins of hiv and the evolution of resistance to aids decimations, extinctions, and colonizations of frog populations in southeast brazil and their evolutionary implications a disease-mediated trophic cascade in the serengeti and its implications for ecosystem c forecast and control of epidemics in a globalized world an integrated health risk assessment approach to the study of mining sites contaminated with arsenic and lead global trends in emerging infectious diseases factors associated with case fatality of human h n virus infections in indonesia: a case series surveillance of avian influenza virus in wild bird fecal samples from south korea wildlife trade and global disease emergence the unregulated and informal trade in wildlife: implications for biodiversity and health impacts of biodiversity on the emergence and transmission of infectious diseases use of infrared camera to understand bats' access to date palm sap: implications for preventing nipah virus transmission predicting the global spread of h n avian influenza is community-based ecotourism a good use of biodiversity conservation funds? understanding the molecular epidemiology of foot-and-mouth-disease virus amphibians as model to study endocrine disrupters characterization of the risk of deer-cattle interactions in minnesota by use of an on-farm environmental assessment tool deconstructing spatial patterns in species composition of ectoparasite communities: the relative contribution of host composition, environmental variables and geography the use of biomarkers in environmental monitoring programmes sociality, density-dependence and microclimates determine the persistence of populations suffering from a novel fungal disease, white-nose syndrome emerging infections: microbial threats to health in the united states fruit bats as reservoirs of ebola virus safari hunting and conservation on communal land in southern africa evidence-based public health policy and practice: finding the real case-fatality rate of h n avian influenza bats are natural reservoirs of sars-like coronaviruses fences and artificial water affect african savannah elephant movement patterns experimental infection of bats with geomyces destructans causes white-nose syndrome recurrent zoonotic transmission of nipah virus into humans foodborne transmission of nipah virus fox society, contact rate and rabies epizootiology accruing benefit or loss from a protected area: location matters on the origin of brucellosis in bison of yellowstone national park: a review community health and conservation: a review of projects unhealthy travelers present challenges to sustainable primate ecotourism date palm sap collection: exploring opportunities to prevent nipah transmission sars and the new economics of biosecurity food-borne diseases-the challenges of years ago still persist while new ones continue to emerge serological evidence of ebola virus infection in indonesian orangutans management of bovine tuberculosis in michigan wildlife: current status and near term prospects delivery of priority health services: searching for synergies within the vertical versus horizontal debate brucellosis in the united states: role and significance of wildlife reservoirs investigation of the transmission of mycobacterium bovis from deer to cattle through indirect contact unhealthy landscapes: policy recommendations on land use change and infectious disease emergence the effects of rinderpest and rinderpest control on wildlife in africa decay of similarity with host phylogenetic distance in parasite faunas pan-european distribution of white-nose syndrome fungus (geomyces destructans) not associated with mass mortality control of rabies in jaipur, india, by the sterilisation and vaccination of neighbourhood dogs frequent arousal from hibernation linked to severity of infection and mortality in bats with white-nose syndrome malignant catarrhal fever: a review a systematic test of an enterprise strategy for communitybased biodiversity conservation foot-and-mouth disease virus: cause of the recent crisis for the uk livestock industry the north american bullfrog as a reservoir for the spread of batrachochytrium dendrobatidis in brazil magnitude of the us trade in amphibians and presence of batrachochytrium dendrobatidis and ranavirus infection in imported north american bullfrogs (rana catesbeiana) bovine tuberculosis in free-ranging white-tailed deer from michigan spread of chytridiomycosis has caused the rapid global decline and extinction of frogs modeling control of rabies outbreaks in red fox populations to evaluate culling, vaccination, and vaccination combined with fertility control reducing the risks of the wildlife trade zoonotic viruses associated with illegally imported wildlife products veterinary clinical epidemiology: a problem-oriented approach microbial threats to health: emergence, detection, and response. institute of medicine host phylogeny constrains cross-species emergence and establishment of rabies virus in bats ecotourism and conservation: two cases from brazil and peru status and trends of amphibian declines and extinctions worldwide are ducks contributing to the endemicity of highly pathogenic h n influenza virus in asia? small mammals as monitors of environmental contaminants emerging foodborne pathogens just and lasting change: when communities own their futures a summary of the resources and roles dedicated to the eradication of bovine tuberculosis in michigan economic costs of the foot-and-mouth disease outbreak in the united kingdom in cadmium and lead concentrations in moniliformis moniliformis (acanthocephala) and rodentolepis microstoma (cestoda), and in their definitive hosts, rattus rattus and mus domesticus in el hierro (canary archipelago, spain) overcoming health-systems constraints to achieve the millennium development goals a five-year assessment of mortality and geographic spread of white-nose syndrome in north american bats and a look to the future perfluorinated chemicals infiltrate ocean waters: link between exposure levels and stable isotope ratios in marine mammals increasing the value of wildlife through non-consumptive use? deconstructing the myths of ecotourism and community-based tourism in the tropics expanded research to target e. coli outbreaks origin of the amphibian chytrid fungus who ( ) rabies bulletin europe. who collaborating centre for rabies surveillance and research risk factors associated with mortality from white-nose syndrome among hibernating bat colonies bushmeat hunting in the congo basin: an assessment of impacts and options for mitigation naturally acquired simian retrovirus infections in central african hunters coronavirus diversity, phylogeny and interspecies jumping host range and emerging and reemerging pathogens ecological origins of novel human pathogens scientific background to the global eradication of rinderpest population declines and priorities for amphibian conservation in latin america intraspecies diversity of sars-like coronaviruses in rhinolophus sinicus and its implications for the origin of sars coronaviruses in humans severe acute respiratory syndrome epidemic in asia acknowledgments we would like to acknowledge the generous support from our funders for this work. including the us agency for international development emerging pandemic threats predict project, a niaid nonbiodefense emerging infectious disease research opportunities award r ai - , an nih/nsf ''ecology of infectious diseases'' award from the john e. fogarty international center ( r -tw ). key: cord- -xsxkn my authors: vojtkovská, veronika; voslářová, eva; večerek, vladimír title: methods of assessment of the welfare of shelter cats: a review date: - - journal: animals (basel) doi: . /ani sha: doc_id: cord_uid: xsxkn my simple summary: the welfare of animals in shelters draws the attention of both the scientific and general public. it is possible to assess the well-being of cats in shelters using tools that are based on indicators used to reveal problematic aspects of welfare. this review aims to provide an insight into available methods of assessment of the welfare of cats in shelters with an emphasis on behavioural, physiological and health indicators. abstract: at any moment, there are millions of cats housed in foster care facilities for abandoned and stray animals for various reasons worldwide. care, management and regulation among these facilities differ. moreover, shelters can never substitute the full comfort of a good home for the animal, and the welfare of cats in shelters is a subject of discussion in many respects. cats are animals sensitive to changes; for most of them, placement in a shelter is a stressful experience because of changes in routine, environment and the presence of other animals. stress is reflected in changes in behaviour, causes fluctuations in physiological values and disrupts the immune system, which is a predisposition to the development or reactivation of disease. evaluation of the presence and intensity of negative impacts is possible through the use of evaluation tools based on indicators that help set the environment and management of keeping so as to disrupt the quality of life as little as possible. although a comprehensive and valid welfare tool that would evaluate animal-based and at the same time resource-based (or management-based) indicators of cats in shelters is not currently available, it is possible to use partial evaluation of individual welfare indicators to assess welfare. this review aims to provide the readers with an insight into current options of assessment of the welfare of cats in shelters with an emphasis on behavioural, physiological and health indicators with an application in both practical and scientific contexts. in recent years, cats have become the most popular pet animals in western europe and the united states. their number in the european union reached over million [ ] and in the united states over million [ ] . the result is an increased focus on issues dealing with their well-being and behaviour [ , ] . at any moment, there are millions of cats housed in shelters around the world [ ] . for example, approximately . million cats enter shelters in the united states every year [ ] . in canadian shelters, , cats were housed in [ ] . in , , cats entered shelters in the uk [ ] , , cats were housed in shelters in spain in [ ] and about cats are housed in shelters in sweden annually [ ] . the traditional view that animals with unrestricted movement pose a risk to public health and safety, and that the solution is euthanizing them, has already been partially overcome. in most western countries, the approach to solving the problem has changed over the last to years; non-governmental organisations and in some countries, public authorities have invested in programs to increase the number of successful adoptions to minimise the need for animal euthanasia [ ] . in order to eliminate euthanasia of animals in shelters, some countries (e.g., czech republic, italy, sao paulo, austria, india, taiwan, germany, costa rica) [ ] have adopted the so-called no-kill policy. in these countries, shelter cats can only be euthanised for medical reasons. in other countries, on the other hand, the shelters themselves determine the functioning policy; for instance, some no-kill shelters refuse to accept animals that are sick, elderly or have unacceptable behaviour because they are bad candidates for adoption. however, this type of restrictive policy can also lead to worsening of welfare in an unwanted group of animals and overcrowding in shelters that have open admission policies [ ] . all facilities providing temporary care should aim to reduce the animal's stay there to a minimum. a problem can arise if the no-kill policy orders facilities to accept all animals and keep them in a shelter until they are adopted by a new owner, returned to the original owner, die of natural causes or are put down for health-related or behavioural reasons. an animal that no one is interested in (often an older animal or an animal with a disability) can therefore legally stay in a shelter for a very long time. however, shelters are generally not designed as facilities that would be able to replace a new home in the long run; the animal generally has limited living space and access to resources, which it often shares with other animals with various medical histories. since a multitude of facilities does not have a sufficient amount of staff at their disposal, only a minimum of time is reserved for the care of each animal; ammons [ ] reports an average of min per day per animal. the common goal of most facilities is to temporarily provide the animals with a suitable space that takes into account nutritional, housing, health care and human contact requirements. the quality of care is a critical aspect of cat welfare in many facilities [ ] . various factors of shelter environment can be stressful for cats (different care routine, lack of a familiar/bonded caretaker, veterinary treatments, increased infectious pressure, the presence of other animals, inadequacy in terms of space or poor environment and overall lack of control over the environment) [ ] [ ] [ ] [ ] [ ] . the intensity and number of negative factors may reflect the condition of the shelter; in many countries, the system for dealing with unwanted animals is complex, lacks comprehensiveness and the cooperation between the state (or state facilities) and private facilities is not functional; likewise, the level of supervision in various types of facilities is different, sometimes even completely absent. the standard of maintenance and care of animals can vary significantly across facilities, as can their funding. insufficient funding may be a factor causing a number of other potential animal welfare problems [ ] . animal welfare can generally be assessed from three different perspectives [ ] . the first defines well-being through a biological point of view-the welfare of the animal is preserved if there is no deterioration in health and reproductive ability. the second view assumes that well-being depends on the ability to engage in natural behaviour. although this approach is traditional, today there is a tendency to attribute feelings and emotions to animals; trying to understand mental processes creates another perspective on welfare. the third view of the welfare concept, applied in the manuscript, involves the assumption that animals are sentient beings capable of experiencing positive and negative emotions [ ] . because the subjective state of well-being is the result of the mental processes which are intrinsic to the animal in which they take place and invisible to the eye, its assessment is difficult. the fact that animal welfare is based on the subjective states perceived by the animal prevents us from its accurate assessment in spite of utilising any available methods. while current evaluation methods do not possess the capability (or this capability is limited) to provide direct insight into the mental state of the animal, at least the indirect methods are available [ ] . the development of the first protocols for the practical assessment of welfare began in farm animals. efforts to evaluate welfare first led to the creation of some indicators of well-being; these focused on key aspects-health, behaviour, housing and management of keeping [ ] . it is now customary to divide these indicators into two groups-a group of indicators, which directly relates to the evaluated animals (indicators of behaviours, health and physiological markers of stress [ ] ) and a group concerning the resources of the environment in which the animal lives. it is appropriate to assess the well-being of the animal in terms of comparing the provided living conditions with the optimal environment, and in terms of the strategies that the animal must apply to cope with the difficulties and successfully adapt to the conditions [ ] . the welfare quality project [ ] , focusing on cattle, pigs and poultry, proposed a scientifically valid welfare assessment system and its four principles (good feeding, good housing, good health, appropriate behaviour) became a key basis in designing tools and evaluating the welfare of two other categories of animals-laboratory animals and companion animals. while in the past the general interest was concentrated on livestock (with the intensification of agriculture linked to the emergence of large-scale livestock farming, it was natural for questions pointing to the deteriorating living conditions of farmed animals to arise), attention has now also shifted to species that seemed previously least affected. unlike in cats, tools that assess the well-being of dogs have been the subject of several studies [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in addition, evaluation tools have been developed to assess the behaviour of dogs in terms of its prediction in the home environment of the new owner. the tests are mainly aimed at detecting aggression [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the tools have been criticised because some authors [ , ] consider them to be scientifically insufficiently validated to decide on the future of the animal (often, based on testing, the shelter decides to move the animal to another facility or euthanise it because of unacceptable behaviour or low adoption potential). shelters in none of the european union countries use an officially validated tool to assess the behaviour of dogs in shelters [ ] . behavioural tests have also been developed in shelter cats [ ] ; their main aim, once admitted to a shelter, is to discriminate socialised animals from unsocialised ones in order to preserve their welfare. temperament tests form another group of tools used in cat shelters; the goal is to assess the cat's temperament and find a compatible new owner [ ] . however, the situation in evaluating the welfare of cats in shelters is different compared to dogs-at present, there is no comprehensive well-being evaluation tool usable in cat shelters. efforts to improve animal welfare are noticeable, however, e.g., within the european union, a number of member states have not laid down a legal obligation regarding any specific procedures to be performed after the animal is admitted to a shelter or during its stay in a shelter [ ] . since there is no comprehensive tool for evaluating the welfare of cats in shelters, it is desirable to consider currently available tools that enable to assess individual aspects of welfare, i.e., to use the indicators that have been developed to evaluate the welfare of cats under specific conditions. the aim of the review was to provide the readers with an insight into welfare assessment tools, whose criteria may form the basis for the development of a complex, more comprehensive and valid tool evaluating the welfare of cats in shelters and summarise additional options of assessment of welfare of cats in shelters with an emphasis on behavioural, physiological and health indicators. welfare or quality of life (qol) are interrelated concepts and are often interchanged; according to some authors [ ] [ ] [ ] [ ] their definitions are identical. while the welfare concept has been presented only for the last few decades (one of the first definitions of welfare was introduced by broom in [ ] ), the origin of the term 'quality of life' dates back to the days of plato and aristotle, who used the term to examine the conditions necessary to live a 'good' life [ ] . the principle of the original concept, which became widely accepted, was to assess the quality of human life in terms of health aspects [ ] . the term qol was later introduced into veterinary medicine; the concept began to apply when deciding between treatment and euthanasia of an animal in veterinary clinics [ , ] . although there are several approaches to understanding the focus of the concept of qol, taylor and mills [ ] have provided a general definition of qol applicable in various contexts. according to the authors, qol can be defined as an individual state that an animal perceives at a given moment in its existence; the condition should include a balance between the positive and negative attributes of life. cummins [ ] adds that well-being is the result of many factors; it is a multidimensional construct. while tools for assessing farm and laboratory animal welfare are based largely on five freedoms [ ] (freedom from hunger, thirst, pain, injury, illness, discomfort, fear and anxiety and freedom to express normal behaviour), the evaluation of companion animals is mostly focused on the use of health and behavioural parameters that can provide insight into the animal's internal state and its ability to cope with adverse conditions [ ] . stress (especially distress) has an impact on the quality of life of animals [ ] . in addition to the evaluation of behaviour and health, it is possible to evaluate the worsened welfare or the ongoing stress response by using physiological markers of stress [ ] . it is challenging to select indicators that, in turn, represent positive emotions and good living conditions, i.e., indicators or conditions that reflect a good quality of life. the proposed indicators of positive emotions reflect the positive cognitive attitude of the individual; these are behaviours such as play, exploration, vocalisation, social behaviour and grooming [ , ] . however, some of these indicators have not been fully validated in order to be usable in practice. in developing welfare assessment tools, reliability and validity are key to assessing whether the resulting concept actually measures what it was designed to [ ] . reliability is expressed as a measure of repeatability and consistency [ ] and can be measured within a single observer (intra-observer) and between multiple observers (inter-observer), within a test (test-retest) and between components which are the subjects of the test (internal-consistency) [ ] . it does not mean that a tool that was previously validated in the original language is automatically valid when its language is changed or when it is used in other cultural contexts [ , ] . before assessing reliability and validity, the purpose and standardisation of evaluation need to be addressed [ ] , taking into account practicality and executability [ ] . there is a risk that measurements that take a long time or are too complex will not be performed correctly [ ] or will not be used at all. some of the practical problems in assessing welfare or qol are similar to those that arise in assessing people with communication and cognitive impairments. like them, animals cannot provide their own self-assessment [ ] [ ] [ ] ; therefore, questionnaire methods such as those common in human medicine cannot be used to assess their quality of life [ ] . questionnaires can be used as comprehensive tools containing objective records [ ] on nutrition, level of activity, environment, social interactions and behaviour when filled out by a person closely related to the animal [ ] , i.e., the caregiver of the sheltered cat. however, the disadvantage of most animal-based measurements is their subjectivity [ ] . the caregiver is required to interpret the animal's expressions correctly, which is a problem if shelter staff are not sufficiently educated and trained-testing can yield anthropomorphised, biased findings. results can also be influenced by the principles and feelings of the individual who evaluates the animal [ ] . traditionally, quality of life assessment can focus not only on the assessment of the animal itself but also on the assessment of resources [ ] and management (thus a combination of several indicators is used for evaluation), as these remain constant and can be measured objectively (e.g., assessment of appropriate housing, nutrition and the human factor). an isolated assessment of resources is insufficient because it does not provide a real picture of the level of welfare, but only indicates the risk of its deterioration; although optimal management may be set up in the shelter facility and the animal may have all the necessary resources at its disposal, this does not automatically mean that it is healthy and provided with a high standard of welfare. a comprehensive welfare assessment tool should, therefore, be based primarily on the parameters of the animal, supplemented by other parameters. in some studies, the approach of assessing quality of life only from the point of view of health is maintained [ ] . if welfare assessment is set up properly, it takes into account feedback on changes that affect the animal's welfare and allows for appropriate course of action to be taken thereon [ ] . the majority of tools assessing the quality of life that are available today tend to focus on evaluating the lives of cats as patients with certain types of health problems, such as diabetes mellitus [ ] , cancer (see [ ] for the review of current methods of qol assessment in cats receiving chemotherapy), degenerative joint disease [ , ] , heart disease [ , ] , skin problems [ ] or chronic disease in general [ ] . these tools cannot be used for the evaluation of healthy cats, as they consist of disease-specific parameters. in addition to specifically targeted tools, efforts have been made to create more general tools that can be used in a variety of situations, facilities and contexts. their purpose is to define and differentiate normal behaviour and health and their deviations that an individual exhibits. some of these comprehensive tools (modified karnofsky score, chew [cat health and wellbeing] tool, owner completed measure of feline qol, catqol tool, the awag software, shelter quality and the shelter dog qol evaluation tool) could be considered for shelter cats' welfare assessment after some adjustments (these tools were developed for the use in different context, so they are not able to cover the full range of evaluation criteria requirements emerging from the shelter environment) or their principles could be used when creating a new assessment protocol. a qol tool that can be used to assess aspects of cat health and well-being without specific health problems is the so-called karnofsky rating scale, modified by hartmann and kuffer in [ ] . it was originally designed to determine disease progression, treatment effects and the ability to perform normal daily activities in human cancer and hiv patients [ ] . the karnofsky score (ks) modified for feline patients consists of two parts: the first part is the score given to the cat by the veterinarian (clinician's score) after assessing its state of health and body condition; the second part consists of a questionnaire containing questions about behaviours, which are answered by the owner [ ] . a high degree of correlation was found between the results of individual observers when assessing behaviours [ ] . lower inter-observer reliability for clinician's score was found, requiring additional modifications; a standardised, detailed approach with appropriate criteria combined with more specific training on how to use the scale may improve inter-observer consensus and consistency. the score obtained from questionnaire seems reliable, so it may serve as a reliable tool in the assessment of qol [ ] . although the ks assessment method is not primarily designed for cats in shelters, it is one of the few tools that can be used in this regard due to its relative non-specificity. it should be mentioned that the protocol could only be used for animals that have been in a shelter for a long time; the caregiver completing the questionnaire must be able to correctly estimate the level of reported activities of the cat. the need to find a comprehensive but especially reliable tool for assessing mainly the clinical aspects of healthy cats also resulted in a study conducted by freeman et al. [ ] , a group of american scientists. they first defined the attributes that cat owners consider relevant in health assessment; then they tried to create an evaluation tool (chew [cat health and wellbeing]) based on these attributes. parameters that are part of the assessment include easily determinable, animal-oriented items such as play, mood, energy, appetite, body condition and coat condition. the experiment resulted in the establishment of eight domains containing assessment items. the authors mention that the final tool showed good validity, was able to discriminate overall health status, demonstrated good internal and test-retest reliability and is usable across various age categories of cats [ ] , which is an important aspect for its use in shelters, as they usually house cats in various age categories. on the other hand, its use in shelter environment has not been tested. however, because the tool is based on two types of animal-based indicators (it involves behavioural and health indicators), its use as a comprehensive tool for assessing quality of life of shelter cats could be considered. a similar procedure (where its authors let the owners of cats determine, what behaviour they considered normal in the animals) was employed in tool creation by tatlock et al. [ ] as well- evaluation items were developed in areas-general health, interaction with humans, vocalisation, pain, sleeping behaviour, appearance, interaction with surroundings, toilet habits, gastrointestinal symptoms, hydration, appetite, weight loss, grooming, general happiness and physical activity level. authors reported strong internal consistency and test-retest reliability for all domains, indicating good reliability. in qol tools, it is customary for each rating item to have the same weight; however, it should be noted that the importance and impact of the items included on quality of life may vary, which should be taken into account when assigning weight and calculating the overall qol score. in contrast to the aforementioned studies, in addition to animal-based indicators (general state of health, food intake, behaviour), the qol tool (catqol) proposed by bijsmans et al. [ ] takes into account management-based indicators and also the principle of evaluating each item included in the tool according to the frequency or severity with which it affected the cat's life. the catqol questionnaire was validated by taking all aspects of the psychometric analysis into account and assessing each question individually. the disadvantage of the questionnaire is that it contains questions on cat's experience during the previous week. thus, a cat could not be evaluated sooner than after days spent in a shelter. in addition, the authors mention that the result obtained reflects the nature of the relationship between the owner and the cat to a certain extent. adamelli et al. [ ] , who analysed the relationship between a person and a cat with an emphasis on quality of life in animals, concluded that the qol of a cat may be more influenced by the characteristics of the owner than the characteristics of the cat. qol was rated low, moderate or high in this study using four questionnaires that analysed care, behaviour and characteristics of the cat and the owner, a simple physical examination and lexington's attachment to pets scale (laps) test. the laps test, developed by johnson, garrity and stalones in [ ] , is probably the most widely used questionnaire for assessing the emotional bond between humans and animals [ ] . although the described tools have been tested for future use by cat owners (in the home environment of cats), they represent at least a sketch or insight into the criteria that could be evaluated in the case of shelter cats. also, other tools that have been developed to be used in various animal species and contexts could be considered for cats in shelters. one such tool is the animal welfare grid (awag) software, which was originally designed to monitor animal welfare in research institutions (originally used in primates) [ ] . the awag draws attention to the temporal component of any suffering, reflecting the cumulative lifetime experience of an individual. the system measures criteria covering four different parameters that impact animal welfare-health, psychological well-being, environmental quality and clinical and management procedural events. within each parameter, a number of indicators are evaluated on a scale of to ; a score of one indicates the lowest impact on welfare, whereas a score of represents the highest impact on welfare. for each parameter, the software calculates the mean score, which is used to create polygon; the total area of the polygon presents the cumulative welfare score of an animal at a particular moment [ ] . the awag tool can be used either predictively or retrospectively, including monitoring specific events or procedures affecting an animal or group of animals. the advantage is that software is freely available on the project website (https://github.com/publichealthengland/animal-welfare-assessment-grid/wiki) and that it is transferrable and can be used to manage the welfare of a wide range of species in a variety of settings [ ] . some studies have been conducted using the awag, however no study has described its use in a shelter environment, therefore, no information on possible problems in this context is available. a potential problem with applicability in a shelter could be the evaluation of indicators itself. the scale contains evaluation points, i.e., it requires ability of the evaluator to distinguish subtle changes and deviations between scores. this could be an issue in behavioural assessment of shelter cats; shelter staff usually do not have information on temperament of admitted cats, so distinguishing subtle changes in their behaviour could be challenging. the basic four principles (good feeding, good housing, good health and appropriate behaviour) used in the protocol evaluating the welfare of dogs in shelters [ ] have general application in the assessment of welfare and are therefore applicable to almost any species of animal; they could therefore also be applied when assessing the welfare of cats in shelters. however, the individual evaluation criteria, of course, have to be adapted to the conditions in the cat shelters and to the cats themselves-the biological and ethological aspects of the cats should be taken into account in the evaluation criteria. the shelter quality protocol for evaluation of welfare of dogs that are older than months and have been housed in a shelter longer than months, developed in by the italian instituto zooprofilattico sperimentale dell'abruzzo e del molise, follows the criteria of reliability, validity and feasibility [ ] . it is a tool that uses direct observation of animal responses while identifying important aspects of the environment and management to detect welfare risks [ ] . observations are made at the level of the shelter, the cage and the animal. in developing the evaluation criteria, the authors were inspired by the approach of the welfare quality consortium [ ] , which developed protocols for the evaluation of livestock welfare [ ] . other principles have been incorporated by kiddie and collins [ ] into the shelter dog qol evaluation tool-unlike the animal-based, resource-based and management-based approaches applied in shelter quality, the qol evaluation tool focuses only on animal assessment; the tool does not take into account environmental assessment and does not look at the management of the facility. the authors first compiled a list of behaviours that were associated with stress and behaviours that were associated with positive emotions according to literature and the experience of the shelter staff. the evaluation itself consists of parts- parts require observation of the dog's behaviour from a distance, in which positive and negative behaviours are recorded, the th part evaluates the dog's reaction to a person during an interaction. in the last part, three parameters are evaluated by the observer-body condition-according to a -point scale, the presence of scurf and eye discharge. each evaluation item can receive a score of or , depending on whether or not the occurrence of the observed trait or behaviour is recorded. kiddie and northrop [ ] launched a project called 'assessing the quality of life of kennelled cats', which aims to create a similar rating system for cats in shelters. the final tool has not yet been published. in this section, we discuss the options of assessing three categories of animal-based indicators-behavioural (section . ), physiological (section . ) and health (section . ), which can be used to evaluate the welfare of shelter cats in a practical and scientific context. for welfare assessment, it is important to select behavioural variables relevant to the studied species as indicators and to take into account its evolutionary history. the domestic cat evolved into a carnivore with a solitary lifestyle, so the development of exaggerated behaviour was not necessary. assessing their well-being based on behaviour may seem all the more challenging [ , ] . in cats, some degree of variability in behavioural responses to stressful stimuli was found, which is probably influenced by temperament and personality; even its inheritance is presumed [ ] . in this section, we discuss the behavioural indicators that indicate impaired welfare of cats in shelters and methods that can be used in practical as well as scientific evaluation of behavioural responses of cats to stress (cat-stress-score assessment) and methods that are used to discriminate socialised and unsocialised cats (approach tests). in terms of the applicability of approach tests, we discuss tests usable in scientific (cat-approach-test, human-approach-test) and practical (feline spectrum assessment) contexts. in general, behavioural assessments can be perceived as subjective (namely considering the possibly subjective interpretation of observed behaviours by the rater) and are not always accepted as a valid indication of stress unless they are accompanied by the results of physiological measurements. the combination of physiological and behavioural data may provide credible evidence of the presence of stress [ ] , but there are cases, when this statement is not valid (e.g., cat-stress-score does not correlate with the level of cortisol). however, shelter staff usually do not have sufficient resources to analyse physiological data, so it is useful to have an easy-to-use tool for assessing behaviour [ ] . cats could find particular aspects of the shelter environment stressful, such as confinement, the proximity of other unfamiliar cats, handling by caregivers and changes in routine [ , , , ] . on the other hand, a recent study by vitale and udell [ ] found that shelter cats seek out human interaction (more than pet cats) so lack of human proximity may also be a stressor. cats respond to the presence of humans and prefer contacting with them with toys [ ] . behavioural changes form the body's primary response to stress, serve as an adaptation mechanism and reflect the animal's internal state [ ] . stress is a common aspect of life and is experienced by all living beings [ ] . a problem arises if the amount of stress that an animal is experiencing crosses a certain line and becomes undesirable, and the coping strategies to handle stress have been exhausted [ ] . although it has been found that the level of stress in sheltered cats decreases as they spend more time in the shelter [ ] and that most cats adjust to the shelter environment within to weeks [ , ] , some cats are unable to get used to it at all and thus remain in a state of chronic stress for a long time [ ] . gouveia et al. [ ] studied the differences in the behaviour of cats kept in shelters in groups with various densities and sex ratios for various lengths of time. cats that stayed in the shelter for a long time were less active and more involved in conflict situations. the presence of stress in cats is more often manifested by inactivity with inhibition of natural behaviour (reduced food intake, insufficient coat care, reduced frequency of playful behaviour [ , , ] , persistent vigilance [ ] , agonistic behaviour [ ] and the need to hide [ ] ) rather than any active manifestation of abnormal behaviour [ , ] . the aforementioned indicators of good and impaired welfare, along with some others, are summarised in table . general activity exploration of the surroundings normal occurrence reduced occurrence or absence of activity (rarely increased occurrence) [ , ] behaviour associated with metabolic processes feeding normal occurrence reduced occurrence or absence of activity [ , [ ] [ ] [ ] drinking normal occurrence reduced occurrence or absence of activity [ , ] urination normal occurrence reduced occurrence or absence of activity; urination outside of the litter box and instead in other locations of the cage [ , ] defecation normal occurrence reduced occurrence or absence of activity [ ] comfort behaviour resting normal occurrence excessive vigilance [ , ] sleeping normal occurrence reduced occurrence or absence of activity; feigned sleep; somnolence [ , , ] grooming normal occurrence over-grooming, self-mutilation or reduced occurrence of grooming [ , ] playing occurrence of playful behaviour (individual play, play with other cats, objects or people) reduced occurrence or absence of activity [ , [ ] [ ] [ ] social interactions interactions with people positive interactions with people (seeking human presence, direct contact, staying in proximity); positive responses to human-initiated interactions absence of or negative response to a human-initiated interaction, particularly redirected aggression and some forms of affective aggression [ , , ] interactions with conspecifics present; positive activities (rubbing, allogrooming, not avoiding contact) absent or negative activities: hostility, aggression, contact avoidance [ , , , , , ] communication scratching normal occurrence reduced occurrence or absence of activity [ ] facial marking normal occurrence reduced occurrence or absence of activity [ ] urine spraying normal occurrence increased occurrence [ , , ] other types of reported activities compulsive behaviour absence of compulsive behaviour presence of compulsive behaviour [ ] [ ] [ ] hiding hiding as a normal reaction to fearful stimuli or as a part of playful behaviour effort to hide [ , , , ] vocalisation normal occurrence excessive occurrence [ , ] the level of stress is affected by the quality of housing; the inability to show the natural range of activities for a longer period of time can lead to stress, which applies especially to individually housed cats in cage housing [ ] , often poor in enrichment, which is often used in shelters e.g., in the united states [ , ] or in general within the quarantine. the level of stress can be partially regulated by providing an undisturbed, dark place where the cat can hide [ , ] , which, of course, does not address the overall lack of stimuli and space to engage in natural active movement. on the other hand, no difference was found in the stress responses of cats when comparing the amount of space provided per cat ( m , m , m ) when the resources were the same [ ] . this may indicate that it is not the size of the cage but its capacity to be utilised that has an impact. group housing, chosen by a number of shelters because of lack of space, on the other hand, is not entirely satisfactory from the point of view of infectivity (as there is a constant change in the composition of groups) and of social structure-in nature, cats form groups only under certain conditions; one of them is the sufficient availability of resources. in addition, cat colonies are usually comprised of females and their offspring [ ] . grouping in shelters is therefore generally problematic when mutually unfamiliar and unrelated cats are housed together [ ] . some individuals in a group may prevent others from accessing resources due to their territoriality. in addition, cats have been found to be able to form a linear rank order when living in group; higher-ranking cats tended to gain weight, whereas lower-ranking cats tended to lose weight [ ] . escaping or avoiding another cat may be a problem, as it may not be possible in a confined environment. on the other hand, the advantage of group housing is an environment that usually provides cats with more enrichment. cats have more space available for movement and more control over the use of their environment [ ] . uetake et al. [ ] reported lower levels of activity in individual cage housing as opposed to group housing. this finding is in contrast to the findings of guveia et al. [ ] -cats that were housed in groups with a high number of individuals were found to generally show lower levels of activity [ ] . there may be several reasons why the findings are inconsistent. cats in any housing system are exposed to stress, but it comes from a variety of sources, and cats also have different options for coping with stress. as density is often related to stress, key information such as the total number of cats and the spatial characteristics of the room are often missing. another complication when comparing cats from group and individual housing is the fact that the groups are not equal to each other and the studies do not take into account the characteristics of individual cats and the treatment of each group at the shelter [ ] . suchak and lamica [ ] attempted to address this issue by creating a retrospective matched cohort of cats in two housing systems. cats housed in groups were matched with cats housed individually with the same characteristics (age, breed, sex, size and coat colour). authors found no difference in cats' experience while at the shelter. however, in this study authors sought to compare individually and group housed cats based on measures related to their management in the shelter and outcome; behavioural measures were not assessed. the discussion about which type of housing is less stressful is still ongoing and with ambiguous results [ , ] . the first scale developed to assess behavioural responses to stress was the global assessment score, designed by sandra mccune in [ ] . it contained a description of levels of stress in cats [ ] , the number of which was later reduced to by kessler and turner in [ ] ; they used it in their study to compare cats housed in a shelter individually, in pairs and in groups. the scale was named 'cat-stress-score' (css) and was well received by a wide range of professionals. it is currently a relatively simple and at the same time the most commonly used method for assessing behaviours of stress in cats [ , ] . the scoring scale consists of grades, from the state of 'total relaxation' (score ) to the state of 'extreme stress' (score ). the resulting grade is obtained from a brief observation of the cat's posture and behaviour [ ] described in the ethogram developed by the british cat behaviour working group [ ] . the authors suggest that a score below is still acceptable because it represents only the basic level of stress present in any living animal. an increase above this level means a response to an acute stressor and does not present a problem as long as the score does not remain the same over time [ ] . they also point out that the scale can be used in all housing systems except those in which the temperature does not reach at least • c. at lower temperatures, animals naturally do not show a relaxed attitude, which could lead to skewed results [ ] . the use appears to be reliable-mccune [ ] mentions % repeatability among observers, kessler and turner [ ] reported a % match among observers who received training in the use of the scale and a % match among untrained shelter staff. however, more research should be performed on how the css scale could be reconstructed for increasing its validity and reliability. the disadvantage of the method is that the evaluation is based on a very short assessment period, is subjective [ ] and the results may be influenced to some extent by the presence of some factors (e.g., age, sex, neuter status). another significant disadvantage of the method is that the score does not correlate with the level of cortisol [ , ] . no correlation was found in the combination of evaluating the behaviour using the css scale and determining the ratio of cortisol to creatinine in cat urine; nor was it found when comparing the css test result with cortisol metabolites in the faeces [ , ] . the authors explained the absence of correlation by the fact that the ratio of cortisol to creatinine in urine was cumulative, while the behavioural score was dependent on environmental conditions at the time of assessment. it is not clear whether the intervals between scores are equivalent (i.e., whether, for example, a change from score to score presents the same level of stress reduction as, for example, a change from score to score ) and also whether the use of intermediate stages in assessment is possible (when the cat exhibits behavioural elements of two separate grades at the same time) [ , ] . decreased cat activity and suppressed natural behaviours that may persist for long periods of time and are a sign of stress may be misinterpreted by an untrained evaluator as calm and contented [ ] . in order to improve the sensitivity to detect subtle variations in stress levels, bradshaw et al. [ ] added half steps between scores - . on the contrary, cats that have impulsive personality achieve higher scores in certain aspects of the scale, which does not automatically mean that they are extremely stressed. if possible, because of the individuality of each cat, it is more appropriate to carry out the assessment at different times during the day or over the course of several days [ ] . observers may also misjudge manifestations of oestrus resulting in increased scores; an example is an increased rate of vocalisation [ ] . the css scale has been used to assess cat stress in shelters in a number of studies [ , , , ] . it was found the score was highest in cats in the morning, and that the tool was not suitable for detecting pretended sleep and overly excessive stress levels [ ] . in a study by broadley et al. [ ] , the scale was used to compare the behaviours of cats (originating from multi-cat households and households with only one cat) after they were placed in separate cages in a shelter. cats that came from households without additional cats could experience higher levels of stress in the first days in the shelter than cats that had previously been accustomed to the presence of other individuals, in spite of limited direct visual contact with other cats. kry and casey [ ] used a scale to examine the effect of enrichment of individual housing in shelter using a bc spca hide & perch tm box to achieve a lower score for cats for which this enrichment was available. other tests to assess behavioural responses are approach tests. these are used to evaluate responses of cats to unknown stimuli (unfamiliar humans or cats) approaching the cat to determine whether a cat is socialised or not. the individuality of a cat and socialisation are the most important factors influencing a cat's behaviour toward humans and new situations in general [ , , [ ] [ ] [ ] . the socialisation period begins in cats at the age of weeks and ends around weeks of age. the experience of kittens in this period has a long-term impact on their development and behaviour. kittens handled during this period are more friendly to humans than kittens that did not come into contact with humans [ ] . in general, many facilities have developed their own rating systems, but only a few use a specific procedure or guide [ ] . these tests have certain limitations; any cat that enters an unfamiliar environment is prone to fear. even well-socialised cats can show fearful aggression or motivation to escape. as a result, it can be very difficult to initially determine which cats are unsocialised and which have the potential to be adopted or returned to the owner [ ] . it was assumed that the stress manifestations of the new environment in socialised cats would present itself differently than the stress manifestations of the new environment as well as people in non-socialised cats. however, it is not possible to separate these two fears [ ] . frightened cats may first begin showing their characteristic behaviour after a few days or weeks when stress levels begin to decline or when they are re-evaluated in a calmer, less stressful environment [ , ] . slater et al. [ ] found that a number of facilities euthanises a cat as soon as it is marked as unsocialised, often on the day of admission to the shelter, without providing time for acclimatisation. in creating an assessment tool for distinguishing between socialised and non-socialised cats, slater et al. [ ] therefore selected a three-day observation period because previous research found that about half of the shelters were able to keep the cat in the facility for at least three days [ ] and that some stress reduction can be expected during this period [ ] . in shelters where cats are not provided with acclimatisation time, there is an increased probability that socialised animals will be accidentally euthanised and also the likelihood that the original owner will no longer be able to reunite with their animal. the opposite extreme is the situation where unsocialised cat is kept in a shelter for longer than is necessary for the assumption that it will 'settle' and prove to be socialised. keeping non-socialised cats in a shelter for longer than necessary has significant consequences for them in terms of deteriorating living conditions and is not recommended [ , ] . in their approach study, kessler and turner [ ] described the test they used to assess the degree of socialisation of cats towards other cats. in this test, authors assessed the cat's reactions to another cat (cat-approach-test). the test was based on a procedure in which a calm, neutered male that was socialised in relation to other cats was placed in a portable box at a distance of one meter from the test subject. both subjects were allowed visual contact with each other for min. after this time has passed, the behaviour of the tested cat was rated on a scale of (the cat reacts extremely friendly to another individual) to (the cat reacts extremely hostile to other individuals). cats were considered socialised when they reached an average grade below . after eight consecutive trials. on the other hand, those individuals who achieved an average mark higher than . in a series of eight tests were considered unsocialised. in research studies, rather than exposing a cat to another cat, human approach tests are used. it is generally possible to assess the stress manifestations of cats caused by humans in two main ways; either the test subject is allowed to approach or not approach the experimenter [ ] or the experimenter approaches the location of the cat [ , , ] . a less commonly used method is to hold a cat in one's arms for a predetermined period of time [ ] . the human-approach-test, which was modified by kessler and turner [ ] on the basis of the stranger-approach test [ ] , the experimenter approaches the cat's cage from the front, addresses the animal and remains in front of the cage for min while touching the door with their hands. after the time has elapsed, the experimenter opens the door of the housing unit and closes it again. the cat's response during the test is rated on a -point scale ( = the cat reacts extremely friendly to humans, = the cat reacts extremely hostile to humans). as with the cat-approach-test, the authors suggested how to interpret the results; the cat that received an average grade greater than . in consecutive trials was considered unsocialised to humans. the cat that received an average grade of . or less (friendly response to humans) in eight tests was considered socialised. when comparing the results obtained using the human-approach-test and the css rating scale, no correlation was found, which raises the question of reliability [ ] and validity for both tools. a critical aspect of the tests is the fact that some cats can distinguish between 'a known and an unknown' experimenter and adjust their behaviour accordingly, although horsfield [ ] suggests that the difference between cats' responses to known and unknown individuals is minimal in the tests. the individuality of a cat and socialisation are reported to be the most important factors influencing a cat's behaviour towards humans [ , [ ] [ ] [ ] . the basic idea of the human-approach-test has been retained in many modifications used across various studies [ , , , , , ] . one of them is, for example, the human-approach test proposed by arhant and troxler [ ] , who applied the test to cats housed in groups (the original human-approach-test by kessler and turner [ ] was used only for cats housed individually in cages). the experimenter approached the selected cat in the group in a slow, smooth motion and presented his hand; he stopped if the cat withdrew or when the experimenter's hand was already cm far from the cat's head. direct eye contact with the cat was not maintained during the test. the cat was rated as an 'animal with which contact is possible' if the cat did not withdraw and observed the experimenter. commonly observed reactions included scenting in the direction of the experimenter, sniffing the hand and rubbing the hand. cats that withdrew during the test, exhibited any form of aggressive behaviour, or were hiding in hiding spots were identified as 'cats with which contact is not possible'. in groups of cats, all cats in the group were assessed, in larger groups, a sample of cats was randomly selected. if there were cats of similar appearance in the group, a smaller sample of clearly identifiable cats was selected because individuals were not marked. in contrast to the human-approach test by kessler and turner, which was used mainly in research studies, the feline spectrum assessment by american society for the prevention of cruelty to animals (aspca) was created for practical use in shelters. the tool consists of evaluation items (greet, hand on cage and cracking the cage door, interactive toy, touch with wand). the animals are tested for three days; the test result is a numerical score that indicates whether the animal is likely to be socialised or not [ ] . in addition, to maximise the chance for the cats to show their true natures, the assessment tool should be used four times per cat across three days. it is important that cats are not given a place to hide so they are visible during the assessment items. the disadvantage of the tool is that cats younger than months old and group-housed cats cannot be evaluated. on the other hand, the advantage is that assessment generally takes no more than five minutes per cat to complete, requires a very basic understanding of feline behaviours and its guidelines are available for free on the internet [ ] . for teaching purposes of the evaluators, instructional videos were also created. the principles of approach tests have been applied to other group of tests, which aim to reveal cats' personalities. studies show that many aspects of a cat's individuality are stable over time [ ] ; this fact has led and continues to lead to new studies on the temperament of cats [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] and on development of tools for assessing the temperament of cats in shelters (feline temperament profile (ftp) [ ] , aspca ® 's meet your match ® feline-ality™ [ ] and its modifications [ , ] and other alternatives of testing [ ] ). testing of cats' personalities contributes to the improvement of welfare as it increases the chance of compatibility between the lifestyle of the new family and temperament of cats, which leads to a reduction in the numbers of cats that are returned to the shelter [ ] . another category of indicators that can provide insight into welfare of an animal includes physiological indicators, which are mostly used to detect a stress response. a stress response can be assessed either by determining laboratory parameters or by direct assessment of vital signs-most often by measuring blood pressure, heart rate, respiratory rate and rectal temperature. although these indicators can reliably determine the presence of stress, it seems their applicability in shelters is limited due to several reasons. the analysis of laboratory parameters is often too expensive for the shelter to afford and measurement of changes in vital functions requires specific equipment. in addition, at least minimal handling with an animal is necessary to collect data. this is a significant disadvantage; since measurements are performed to detect stress but handling itself can be stressful to an animal, the results may be skewed and interpreted incorrectly. in order to reduce the handling stress, the animals need to be trained to the measuring procedure; this, however, cannot be applied in shelters. methods discussed in the following subsections are typically used in research for validating tools, that are easier to use, cost less and are more applicable to shelter settings. activation of the sympathetic nervous system causes an increase in heart rate, respiratory rate, systolic blood pressure and a decrease in heart rate variability to facilitate the 'fight or flight' response [ ] [ ] [ ] . as was already mentioned in previous sections, cats are extremely sensitive to handling, environmental changes and changes in routine; the result may be a change in physiological values [ ] . changes in physiological values are an established indicator of distress in various welfare studies [ , [ ] [ ] [ ] [ ] including cats, [ , , [ ] [ ] [ ] [ ] ; for example, pratsch et al. [ ] , used an ear thermometer to monitor body temperature. an increase in body temperature is an accepted indicator of stress [ ] . the temperature was measured to detect stress during transport of the cat to the veterinarian; an increase in temperature of . - • c was observed, as well as a difference in temperature between the right and left ear, with the temperature of the right ear being higher. the right ear was warmer also in cats with elevated cortisol levels after a visit to the veterinarian, indicating the presence of stress [ ] . although changes in heart rate, respiratory rate and temperature may be sufficient stress indicators, their applicability in the shelters is low as the measures usually require special equipment and skills. measuring procedures (e.g., wearing a heart rate monitor or taking temperature measurements manually by inserting the thermometer into the rectum or the ear canal) can be a stressful experience for animals, so the handling procedure itself can cause a change in values. another disadvantage of direct measurements lies in the monitoring of elevations or decreases of values, which, however, is not specific only to stress (but also, for example, to illness or increased activity). therefore, the assessment of changes in vital signs is possible under conditions where the animal is healthy and does not have enough space to develop intensive locomotive activity, which could cause false elevations of values [ ] . common methods of assessing the ongoing stress response include assessing hypothalamic-pituitaryadrenal (hpa) axis activity, most commonly by determining cortisol, adrenocorticotropic hormone (acth) and glucose [ , , , ] . activation of the hpa axis by a stress stimulus induces the release of acth, which increases the synthesis and secretion of cortisol from the adrenal glands [ ] . the assessment of physiological indices has an advantage over other evaluation methods in its quantification. the disadvantage is the existence of great individual variability in physiological responses. the levels can also vary over the course of the day-comparing and interpreting the results can, therefore, be difficult [ ] . a stress response is complex; a single measurement of a parameter may not reflect subtle changes that occur in the body permanently, which may result in drawing incorrect conclusions about the extent of stress. factors such as age, animal history, physical individuality and the circumstances under which the samples were obtained must be taken into account when interpreting the levels of physiological indices [ ] . another disadvantage is that the analyses are usually too expensive for shelters, so their use is almost exclusively limited to the scientific context. assessment of glucocorticoid levels is a common part of welfare assessment in many animal species especially in a scientific context [ ] [ ] [ ] . different types of samples can be used to determine cortisol levels; their aspects in terms of practicality are summarised in a table . for acute stress measurement, it is appropriate to examine blood plasma [ ] , serum [ ] and saliva [ ] . chronic stress, on the other hand, can be detected using non-invasively obtained samples-urine [ ] , faeces (determination of glucocorticoid metabolites) [ ] and fur [ ] . [ , , ] serum acute; peak concentrations: - min after stressor exposure reflects the actual level of cortisol in the blood invasive (restraint or sedation of cat is necessary); sampling requires skills sampling procedure as well as diurnal patterns, temperature, activity levels may confound results [ , , ] saliva acute; concentration: - % of the total amount of cortisol in the blood less invasive prior training of cats on sampling procedure is necessary; a relatively big amount of sample is needed for analysis, which can be a problem to obtain (saliva production is reduced during the stress exposure) sample is easily contaminated by the intake of food and water or by blood from the oral cavity [ , , , [ ] [ ] [ ] urine chronic; concentration: % to % of the total amount of cortisol in the blood, peak concentrations: ± h after stressor exposure non-invasive; detection of long-term stress is possible; easy sample collection in single housing; sample collection does not requires skills in group housing, the sample is difficult to associate with an individual as cats share toilets diurnal patterns, temperature, activity levels may confound results; sample is often contaminated by blood [ , , , , , ] faeces chronic; concentration: % of the total amount of cortisol in the blood, peak concentrations: ± h after stressor exposure non-invasive; detection of long-term stress is possible; easy sample collection in single housing; sample collection does not requires skills in group housing, the sample is difficult to associate with an individual as cats share toilets diurnal patterns, temperature, activity levels may confound results [ , , [ ] [ ] [ ] [ ] fur chronic non-invasive; easy sample collection (does not requires skills); stability of the sample over time; does not require special transport and storage conditions; ability to determine the time period in which stress occurred in the animal inability of detecting changes in cortisol levels during short periods of time (hours/days) cortisol levels may vary depending on some factors-still in research [ , [ ] [ ] [ ] in addition to stressors, changes in cortisol concentrations depend on other factors (e.g., sampling and animal handling procedures) [ ] , which can be a problem in assessing acute stress. the animals should be accustomed to the collection of the necessary sample, otherwise, it is a stressful stimulus that causes a physiological change in the body [ ] . the marginal methods used today are identifying the level of cortisol from plasma, serum and saliva. in saliva, cortisol is only found in its free form [ ] . siegford et al. [ ] described a collection procedure as a procedure in which the experimenter provided the cat with a cotton swab to chew until it was entirely soaked. for each 'soaking', the cat was rewarded with a treat. pratsch et al. [ ] conducted a pilot study aimed at detecting cortisol in saliva-the goal was to investigate the method of saliva collection by cotton swabs and the effects of various food and feeding time on cortisol levels in saliva. all cats were familiarised with sample collection (although the authors do not mention how). only of the samples contained sufficient saliva ( µl) for further analysis, so the authors concluded that the method could not be used in the planned study. not only saliva collection, but also collection of urine can be problematic as stress in cats can be manifested by urinary retention for the first - h after admission to the shelter [ , ] . in addition, % of all cats admitted to the shelter have blood in their urine [ ] , which can interfere with the samples meant to be used for analysis. the disadvantage of detecting glucocorticoid metabolites in faeces is the fact that they do not detect short-term stressful experiences. short-lasting or small fluctuations in the level of circulating glucocorticoids are mostly masked by metabolites accumulating in faeces and bile. faecal samples containing a detectable amount of metabolites reflecting one-time stress experiences may also be overlooked in the case of irregular sampling [ ] . however, unlike some other samples the collection procedure of urine and faeces is relatively simple when using a two-layer cat litter box or a litter box with non-absorbent litter [ ] (although with a large number of housed animals sharing toilets, collection from specific individuals may be a bit problematic without constant animal supervision). the analysis of urine and faeces to determine the level of stress has been conducted in various studies focused on cats in shelters. for example, the cortisol to creatinine ratio in urine was found to decrease during the cat's stay in the shelter [ ] and to be significantly lower in cats in enriched shelters than in traditional shelters [ ] . an increase in the ratio was found in individually housed cats compared to cats housed in groups [ ] . identifying urinary cortisol levels was also used in a study by lichtsteiner and turner [ ] , who found no difference in the amount of cortisol in cats housed individually and in groups. in similar studies, the same results were obtained by ramos et al. [ , ] , who used faeces as a matrix to analyse the level of cortisol. the level of glucocorticoid metabolites in faeces in cats decreased with increasing time spent in the shelter [ ] . determining the level of cortisol from fur is an innovative method of detection of long-term elevated cortisol in the body [ ] , first used in in wild hyraxes (procavia capensis) [ ] . although the process of cortisol incorporation into the hair is not entirely clear [ ] , in general, it can be said that the free, unbound fraction of cortisol is incorporated into the hair pulp during its growth [ ] from blood vessels through passive diffusion [ ] . the cortisol-containing sebum secreted by the skin glands is thought to play a role [ ] . to determine the presence of stress, the fur must be harvested from previously shaved areas [ ] close to the skin and not by plucking to avoid blood contamination [ ] . the area and size of the fur collection area vary across studies (in a study by finkler and terkel [ ] , cat fur was collected from a × cm sacral area with small surgical scissors). the advantage of using fur as a matrix is especially the ability to determine the time period in which stress occurred in the animal [ ] and the possibility of rapid sampling from a larger number of animals, which is an advantage for animals in group housing. another advantage is the fact that the stress caused by handling the animals during collection does not affect the concentration of cortisol in the collected fur [ ] . although there was a doubt that the level of cortisol in fur does not represent its actual level in the body but only a kind of 'local level' present in hair follicles [ ] , stalder and kirschbaum [ ] found indications that hair cortisol reflects the systemic cortisol levels well and seems only slightly affected by the local follicle cortisol production. there was found a connection between the concentration of cortisol in faeces and fur [ ] and between its concentration in saliva and fur [ ] . however, there is evidence that some factors affect cortisol levels in fur -e.g., colour, which was confirmed in dogs [ ] . debatable factors may be, for example, sex, age, state of health and various areas of the body [ ] . accorsi et al. [ ] found a significant correlation between agonistic behaviour and the level of cortisol in the fur, suggesting that a greater amount is found in the fur of aggressive cats. neutered cats also had lower levels of cortisol in their fur than intact individuals. interesting insights can be gained by combining the assessment of behavioural and physiological indicators of cat stress. for example, hiding was negatively correlated with cortisol levels, leading the authors to conclude that this may be important compensatory behaviour in an unpredictable environment [ ] . behavioural assessment is probably a less sensitive indicator of stress than the cortisol-creatinine ratio. the behaviour must, therefore, be extreme for stress to be detectable. cats with higher cortisol levels have also been found to vocalise more and move less than cats with lower cortisol levels [ ] . the spectrum of coping strategies is wide in cats [ ] -while some show elements of stress behaviour but have physiologically adapted to new conditions, others have high levels of stress hormones, but altered behaviour is not noticeable. the welfare assessment protocol should also include parameters indicating deteriorating animal health [ ] , including impairment in body condition score [ ] . indicators should be selected to reflect the most frequently observable signs of health problems occurring in cats in shelters. the problems discussed in this section include signs of upper respiratory tract disease, sickness behaviours, gastrointestinal and skin problems, pain in general and changes in body condition. we discuss the methods of evaluating body condition in more details in section . . ., because only for this indicator more than one validated tool that is well applicable in shelters was created. efforts to develop valid tools for assessing pain in cats are also intensive, however usability of most of them is low in shelters due to their specificity to the certain context of use or time-consuming procedure. in addition, many of these tools have not been validated yet. in the case of other indicators described in this section, we discuss the reasons why they need to be included in the health evaluation and what results have been obtained by studies that included or studied these indicators. admission of the cat to the shelter is a stressful experience that can suppress the activity of the immune system and reactivate the subclinical course of the disease (e.g., feline herpesvirus) [ , [ ] [ ] [ ] . stress can inhibit the production of mucosal antibodies, particularly immunoglobulin a (s-iga) [ ] , resulting in increased susceptibility to pathogens causing upper respiratory tract disease (urtd) [ ] [ ] [ ] . after lack of space, urtds are the second most common reason for cat euthanasia in shelters in the united states [ , ] . emotional state and health are interconnected; a decrease in immunoglobulin a (s-iga) secretion was found in stressed shelter cats [ ] . it has, however, been proven that 'gentling' (gentle stroking of the head and neck area of the cat together with gentle vocalisation) at regular intervals can help improve the cat's mental health, increase s-iga immunoglobulin production and thus reduce susceptibility to upper respiratory tract diseases [ ] . immunoglobin s-iga is essential for protection against pathogens that may be inhaled or ingested [ ] and are highly concentrated in shelters [ ] . higher concentrations of cats are associated with an increase in the prevalence of urtds [ , ] . cats that have achieved a high degree of stress according to css are almost five times more likely to develop upper respiratory tract infections than cats with low levels of stress [ ] . risk factors in the development of a urtd are the cat's age (the risk is greater in younger individuals [ , ] , the number of days spent in the shelter and specific conditions in the shelter [ ] . the development time of a urtd reflects the incubation time of urtd-causing viruses [ , , ] . although shelters differ in the prevalence of pathogens, many cats show signs of disease after as little as week spent in the shelter [ , ] , according to dinnage et al. [ ] up to a third of the shelter cats suffer from a urtd. therefore, it is appropriate to include an assessment of the absence or occurrence of eye and nose discharge among health indicators [ ] . to evaluate upper respiratory tract infections, bannasch and foley [ ] developed a three-point scale, the degrees of which express the severity of symptoms ( . absence of upper respiratory tract infection, . mild upper respiratory tract infection, . moderate to severe upper respiratory tract infection). in addition to urtds, as a result of events perceived as aversive by cats, the so-called 'sickness behaviours' (sb) occur [ , ] . it is a term denoting a set of non-specific clinical but also behavioural symptoms, which include e.g., vomiting, diarrhoea, refusal of food, lethargy, fever, drowsiness and a general decrease in activity [ ] . stella et al. [ ] observed how unpredictable events (presence of dogs, change of caregiver, loud sounds, grouping of new individuals, irregularities in periods of light and dark, temperature fluctuations, transfer of cats) induced by experimenters influenced the occurrence of sb in cats. a negative impact of the events on the animals resulting in a demonstrably increased incidence of sb was confirmed by the authors [ ] ; the inclusion of sb as an indicator of deteriorated welfare is therefore appropriate. similarly, for lower urinary tract diseases and idiopathic cystitis in cats, the relationship between the stressor (especially cohabitation with other cats, limited access to the external environment, sharing of feeding bowls and generally frequent changes) and the occurrence of the disease has been demonstrated [ ] [ ] [ ] . gastrointestinal problems, vomiting and diarrhoea are often associated with stress [ , ] . it seems that stress can affect the integrity of the intestinal barrier, causing an increase in its permeability and local inflammatory response [ ] . a large number of shelter cats suffers from diarrhoea caused by dietary issues, stress or illness (gastrointestinal infection, inflammation or neoplasia [ , ] . in a study by german et al. [ ] . % of all cats in the shelter had diarrhoea, in a study by andersen et al. [ ] it was up to half of the cats. most cats had at least one feline or zoonotic pathogen in their faeces, with the most common pathogens in cats with diarrhoea being feline coronavirus and tritrichomonas foetus [ ] . diarrhoea was most common in kittens and cats housed in a group [ ] , and a higher prevalence of pathogens was also found in the faeces of group-housed cats [ ] . in order to determine the severity of diarrhoea, faecal consistency can be assessed, which was the approach chosen in a study by german et al. [ ] based on a -point scale ( = severe diarrhoea, = constipation). monitoring faecal consistency may be a useful health indicator of welfare, but in group housing, where multiple cats share several toilets, identification of an individual with diarrhoea may be challenging [ ] . however, there are some procedures that could be used to mark faeces; in the study by griffin [ ] , commercially available concentrated food colourings (known as bakers' pasters) were used as faecal markers in group-housed cats. cats consumed food colourings in canned cat food. colourings served as faecal markers by imparting a distinct colour to each cat's faeces, allowing identification in the litter box. in another study, to facilitate identification of faecal samples from females housed in pairs, each cat was fed a teaspoon of canned cat food daily with one animal receiving ml green commercial food dye mixed into the food [ ] . in addition to dyes, glitter and plastic beads were used to mark faeces in various felids in zoos [ ] . the state and condition of the coat can serve as additional health indicators of welfare [ ] , as they reflect changes in normal grooming behaviour, food quality, the presence of social conflicts and chronic diseases [ , ] . there is a clear link between the skin and the nervous system [ ] . dermatological diseases such as atopic dermatitis can be caused by stress, which is likely to cause itching [ ] . disorders associated with excessive coat care (e.g., psychogenic alopecia) manifest in the form of alopecic areas on the caudal part of the body [ ] . it was found that the worsened coat condition of shelter cats correlated with the average length of stay of a cat in a shelter (the longer the cat was in the shelter, the greater the risk of deterioration of coat quality), fewer toilets per cat and unpleasant odour [ ] . similarly, the number of food bowls and their location were associated with a worsened condition of the coat-the monitored shelters, where the study was performed, an average of two bowls was assigned to three cats. the condition of the coat may indirectly indicate the occurrence of conflicts between cats, in which there are fights and, as a result, injuries. the presence of injuries, similar to the condition of the coat, can be included among the indicators of welfare [ ] . in a study by arhant et al. [ ] , a combination of assessment of health indicators (coat condition, body condition, eye and nose discharge) and behaviours was used to assess the welfare of cats in shelters housed in groups. the authors found that of all the monitored indicators, only two of them were stable over time (body condition and coat quality) as the composition of cats in the shelter was constantly changing. these two indicators correlated with repeated visits to the shelters. inter-rater reliability was found to be good for coat quality but only moderately good for body condition. to ensure better agreement between observers, the authors recommend improving the evaluation guidelines by providing pictures and more detailed instructions. authors suggested that body condition and coat quality are feasible and valid for assessing impaired welfare in cat shelters. to assess the welfare of stray cats and abandoned cats, a scale with five components related to cat health was used in a new zealand study [ ] -body condition score (bcs), coat condition, presence of nasal discharge, presence of injuries and the ear crusting. the authors found no difference in the welfare of free roaming companion cats and managed stray cats. they also suggested further validation of the scale using a more detailed physical examination and a determination of biochemical parameters that would complement the overall picture of welfare. a problem in evaluating health indicators is that cats generally tend to hide the clinical signs of the disease [ ] . some cats show no signs of pain at all, if they are supervised by humans, other animals, or in stressful situations [ ] . the absence of indicators does not necessarily mean that the animal is healthy. with a large number of cats in a shelter, it can also be problematic to notice subtle deviations indicating deterioration in health, especially if the shelter caretaker has only a minimum amount of time to provide care to one animal. in shelters, there are animals with an unknown history; therefore, the caretakers have no way of determining 'normal behaviour', that is, the temperament of the given cat, and deviations in behaviour specific to each individual exhibited in case of deterioration. furthermore, in shelters where cats are housed individually in cage housing with minimal freedom of movement, the caretaker may not notice a reduction in activity or signs of pain, or he may not be able to distinguish an inhibition of activity due to stress from symptoms of an emerging disease [ ] . group housing, on the other hand, is characterised by the placement of several cats in a single room containing equipment providing the cat with a place to hide [ ] , which may prevent direct visual inspection of shy or frightened individuals. distinguishing between hiding as a manifestation of pain and hiding as a manifestation of stress can be problematic. tools based on animal observation are used in veterinary medicine to assess pain; observation is a non-invasive and effective method that can be used by different people and in different contexts [ ] . several tools for detecting cat pain have been developed [ ] , but only few are valid and reliable. an example of such a tool is the unesp-botucatu scale [ , ] ; however, it is a tool applicable only if the cat has undergone an ovariohysterectomy. although cats are routinely neutered in most shelters, the unesp-botucatu scale, with its specificity, focuses only on the detection of pain after this procedure; the limited context of use thus excludes it from the detection of pain in general. however, the glasgow composite measure pain scale-feline (cmps-f) is available, which is a validated tool that can be used for assessment in a broader context, specifically in the presence of acute pain in cats [ ] . in order to improve its discriminatory ability, it has been updated and its final version includes two additional pain-detecting markers [ ] . in addition to assessing behavioural changes, detecting changes in facial expressions also has the potential to indicate the emotional experiences of animals and to provide valuable information about their internal condition. assessment of facial expressions using the so-called grimace scales can be a useful, valid and reliable tool for determining pain [ ] . manifestations are objectively measurable using the 'facial action coding system' (facs) assessing the animal's facial movements (so-called action units-au). the system assigns codes to the activity of an individual muscle or group of muscles. in the case of cats, a specific coding scheme (catfacs) has been developed [ ] . grimace scales are a simplified method of evaluating facial expressions that can be specifically applied to assess pain. in the case of cats, the feline grimace scale (fgs) was recently developed, which, according to the authors [ ] , is a valid assessment tool for detecting the presence of pain in cats; to confirm the applicability of the tool in practice, evangelista et al. [ ] applied the scale in assessing pain in cats after an ovariohysterectomy and found good applicability. the methodology was based on the evaluation of facial expressions using images and real-time scoring. the standard methodology for assessing facial expressions of pain using grimace scales is usually based on image scoring [ ] ; screenshots are obtained from videos, but their analysis is time-consuming. in practice, pain should be assessed immediately and easily [ ] . a change in weight can be another indicator of the welfare of cats in shelters. assessing body condition is a traditional and relatively easily applicable approach to welfare assessment. previous studies have shown a link between body condition, health, behaviour and welfare [ , ] . there are several methods to describe the appearance of the body and the distribution of body fat, their practical aspects are summarised in a table . [ , , ] the most commonly used is the semi-quantitative body condition scoring (bcs), which uses visual and tactile assessment of the body. the body condition of the animal is sorted into three categories as underweight, optimal condition and overweight [ ] . areas assessed in cats include the chest, hips, abdomen and waist [ ] . a general problem in assessment can occur in long-haired cats when the silhouette of the body is not clearly visible. a -point scale has been well-received-individual levels correlate with the amount of body fat detected by dual-energy x-ray absorptiometry (dxa) [ ] . to facilitate the assessment, the assessor may be provided with a series of illustrations of cat silhouettes-these reflect the characteristics of each point of the scale. the method has been validated and is considered reliable; scores determined by independent observers correlate [ ] . the disadvantage of the scale is its inability to detect small deviations; therefore, it should not be the only method chosen to monitor changes in body weight [ ] . at present, in addition to the -point scale, a simplified -point model of the bcs system is also used. its practicality seems to be better-the deviations between the stages are more noticeable, which in practice seems to be an advantage. each point on the scale represents a % increase in body fat. grade - . represents optimal weight with to % body fat [ ] . -point scale has not been validated by dxa. however, good reproducibility was found when comparing results of experienced and inexperienced evaluators [ ] . relatively new approaches to fitness assessment include the s.h.a.p.e. system (size, health and physical evaluation), which was developed to minimise variability in assessment across observers. it is an algorithm-based system, using the principles of existing scoring systems with a difference in grading-each category of body condition is assigned a letter from a (underweight) to g (obesity). the use of the rating scale does not require previous training of the observer [ ] , so it could potentially be used by shelter caretakers. as with the -point scale, a strong correlation was found between densitometry and the s.h.a.p.e. system [ ] . the disadvantage of s.h.a.p.e. and bcs is their relatively small informative value-they do not assess the state of muscle, only the storage of body fat [ ] . musculature can be assessed using the muscle condition scoring (mcs) system; it contains four levels of evaluation-normal musculature, mild, moderate and severe muscle loss [ ] . when verifying the applicability of mcs, good repeatability but only moderate reproducibility was found [ ] . the animal's spine, scapulae, skull and wings of ilea are palpated during evaluation [ ] . although the animal may appear to be overweight, it may also suffer from significant muscle loss; in contrast, animals with low a bcs score may show minimal loss of muscle [ ] . overall, the system is mainly used in underweight animals that undergo muscle mass catabolism due to disease or injury [ ] , and an example of the system's use is a study monitoring the changes of body condition in cats with hyperthyroidism [ ] . the use of direct body weight measurement is an additional option that should be considered in body condition assessment. however, direct measurement, similarly to bcs, does not depict the ratio between individual body components (fat and muscle) [ ] , so its use is not appropriate when monitoring these components is necessary. in addition, weighing procedure requires the use of equipment. on the other hand, the advantage is that the measurement is easy and quick. impaired body condition may predispose a cat to euthanasia [ ] or to death in general [ , ] . body condition assessment can provide useful information related to the health aspect of welfare, as stress is often associated with reduced food intake in cats [ , , ] . some cats refuse to eat completely during the first days in the shelter [ ] . weight loss due to anorexia has a serious effect on health because it increases the risk of hepatic steatosis [ ] . in a study of cats in a shelter, . % of cats were found to be underweight (which is equivalent to point on the scale) [ ] . in shelter cats, weight loss was observed during the first two weeks spent in the shelter [ ] despite providing adequate food; authors of the study suggested the weight loss was probably caused by low food intake associated with stress (food intake and css were correlated). however, weight loss may also indicate a serious health problem [ ] . in a study by tanaka et al. [ ] , % of all admitted cats lost their original body weight in the first week in the shelter alone. an association between impaired condition and the development of upper respiratory tract diseases has also been found [ ] , as well as an association between the level of body condition and the quality of housing [ ] . the increased number of underweight cats correlated with housing with less than one resting place per cat, and housing with a lower number of hiding spots. cats that were given the opportunity to hide were able to adapt faster to the new conditions in the shelter, and their weight loss was not as pronounced as that of cats that did not have this option [ ] . by being placed in a shelter, cats encounter a number of stressful stimuli, which can disrupt the state of their well-being. although the efforts of facilities to improve the quality of life of cats in shelters have increased significantly in recent years, as the overall interest in welfare issues has increased, it is necessary for experts to come up with new knowledge that would be applicable in practice. efforts to date have succeeded in enforcing certain measures based on a deeper understanding of the biology and behaviour of cats-for instance, practical improvements such as sound insulation of housing units from dog sounds and other stressful sounds, access to windows and natural light sources, sufficient number of hiding places and elevated locations or placement of housing units above floor level, giving cats a greater sense of security [ , , ] . the current research also focuses on procedures and methods of management and enrichment of the environment (e.g., playing specific music to cats to relieve stress [ ] , various forms of olfactory [ , ] , cognitive [ , ] , visual [ ] , food [ ] , pheromone enrichment [ ] or social enrichment in the form of human interaction [ ] ). another trend in the design of new facilities is group housing of cats, but opinions on it differ in the scientific community. in certain respects, this type of housing may be a form of social enrichment; it has been found that cats from multi-cat households had a higher level of stress after entering a shelter when placed alone than cats from single-cat households [ ] . on the other hand, groups of cats may compete for the resources provided; in a study by dantas-divers et al. [ ] , conflicts over days were recorded, of which involved competition over resources. given the experiences that cats perceive as aversive in a shelter environment, the need to study appropriate welfare indicators and the development of new tools is immense. the aim of using welfare assessment tools is to reveal critical aspects of living conditions and at the same time establish care management so that disruption occurs as little as possible. as it is not entirely possible to satisfy each individual due to cats' unique temperament, personality, history and coping strategies and at the same time to combine the personnel, financial and value structure of the shelter, it is necessary to seek compromises. the disadvantage of evaluating individual indicators separately is the fact that the results provided by the indicators may not match [ ] . while behavioural measurements are valuable and commonly used indicators because they can be used immediately, are non-invasive, and require a relatively short time to train the observer [ , ] , physiological indicators are accurate, quantifying stress levels [ ] and can demonstrate the presence of stress even if there is no external behavioural change. as deterioration of health is associated with welfare impairment, health indicators should also be included in the assessment [ ] . although the assessment of environmental indicators has recently faded into the background and the focus has shifted to animals, they can also help to complete the overall picture of welfare. although shelters tend to develop their own welfare assessment procedures, there is no consensus among the indicators that should be part of the tools, let alone the validity and reliability of these procedures [ ] . another fact is that shelters for cats are characterised by many significant differences across countries (e.g., in the number of animals-cats housed in large shelters versus small ones or home refuges; housing-individual versus group housing; composition of animals-mixed shelters with cats and dogs at the same time versus cat-only shelters, value structure-kill versus no-kill policy, but also in the length of stay, adoption programs, level of veterinary care provided, capture programs, etc.); given the various factors influencing welfare, finding a tool that is comprehensive, reliable and at the same time applicable (or that at least some of its parts are applicable) is, therefore, a great challenge. although such a tool is not currently available, efforts to create it are noticeable [ , ] . the study in which such a tool will be developed will have the potential to significantly help to improve many lives of shelter cats globally. number of cats in the united states from feral cats: their role in the population dynamics of felis catus a review of the housing requirements of domestic cats (felis silvestris catus) kept in the home associations among weight loss, stress, and upper respiratory tract infection in shelter cats they'd never do it to you": a study by fundación affinity into the abandonment and adoption of pets in spain in : interpretation of results a survey of cat shelters in sweden shelters reflect but cannot solve underlying problems with relinquished and stray animals-a retrospective study of dogs and cats entering and leaving shelters in denmark from is there a relationship between attitudes of shelter staff to cats and the cats' approach behaviour? appl animal shelters and animal welfare: raising the bar assessing local performance and establishing community standards behavioral and physiological correlates of stress in laboratory cats effect of gentle stroking and vocalization on behaviour, mucosal immunity and upper respiratory disease in anxious shelter cats stress in owned cats: behavioural changes and welfare implications environmental factors that affect the behavior and welfare of domestic cats (felis silvestris catus) housed in cages recommendations for the housing of cats in the home, in catteries and animal shelters, in laboratories and in veterinary surgeries attachment bonds between domestic cats and humans understanding animal welfare applying ethological and health indicators to practical animal welfare assessment anthropomorphism and anthropocentrism as influences in the quality of life of companion animals the welfare quality ® project and beyond: safeguarding farm animal well-being no better than flipping a coin: reconsidering canine behavior evaluations in animal shelters behavioral evaluations of shelter dogs: literature review, perspectives, and follow-up within the european member states's legislation with emphasis on the belgian situation a survey of the methods used in shelter and rescue programs to identify feral and frightened pet cats validation of a temperament test for domestic cats from an animal's point of view: motivation, fitness, and animal welfare animal welfare defined in terms of feelings a concept of welfare based on reward evaluating mechanisms in the brain: anticipatory behaviour as an indicator for the state of reward systems animal well-being i. general considerations indicators of poor welfare family quality of life: moving from measurement to application is quality of life a useful concept for companion animals? reasons for the euthanasia of dogs and cats quality-of-life assessment in pet dogs assessing quality of life farm animal welfare. fawc updates the five freedoms combined behavioural and physiological measurements as a basis of the assessment of animal welfare animal emotions, behaviour and the promotion of positive welfare state assessment of positive emotions in animals to improve their welfare measuring behaviour: an introductory guide reliability and validity assessment the development and assessment of temperament tests for adult companion dogs cross-cultural adaptation of health-related quality of life measures: literature review and proposed guidelines translation, adaptation and validation of instruments or scales for use in cross-cultural health care research: a clear and user-friendly guideline objective, categoric methods for assessing pain and analgesia evaluation of a quality-of-life tool for cats with diabetes mellitus pain assessment in animals can we really measure animal quality of life? methodologies for measuring quality of life in people and other animals development of a discriminative questionnaire to assess nonphysical aspects of quality of life of dogs enhancing quality of life for dogs and cats in confined situations stress and adaptation of cats (felis silvestris catus) housed singly, in pairs and in groups in boarding catteries animal-based measures for the assessment of welfare state of dairy cattle, pigs and laying hens: consensus of expert opinion too cute to kill? the need for objective measurements of quality of life quality of life assessment in dogs and cats receiving chemotherapy-a review of current methods owner-assessed indices of quality of life in cats and the relationship to the presence of degenerative joint disease item generation and design testing of a questionnaire to assess degenerative joint disease-associated pain in cats assessment of the responsiveness of the cats' assessment tool for cardiac health (catch) questionnaire development and evaluation of a questionnaire for assessment of health-related quality of life in cats with cardiac disease development and validation of a questionnaire to evaluate the quality of life of cats with skin disease and their owners, and its use in cats with skin disease development, initial validation and reliability testing of a web-based, generic feline health-related quality-of-life instrument karnofsky's score modified for cats the use of the nitrogen mustards in the palliative treatment of carcinoma. with particular reference to bronchogenic carcinoma evaluation of a modified karnofsky score to assess physical and psychological wellbeing of cats in a hospital setting development and initial validation of the cat health and wellbeing (chew) questionnaire: a generic health-related quality of life instrument for cats development and preliminary psychometric evaluation of an owner-completed measure of feline quality of life psychometric validation of a general health quality of life tool for cats used to compare healthy cats and cats with chronic kidney disease owner and cat features influence the quality of life of the cat psychometric evaluation of the lexington attachment to pets scale (laps) benefits to pets from the human-animal bond: a study of pet owner behaviors and their relation to attachment refinement of welfare through development of a quantitative system for assessment of life time experience application of a welfare assessment tool (shelter quality protocol) in italian long-term dogs' shelters: welfare hazard analysis temperament and the welfare of caged cats the impact of paternity and early socialisation on the development of cats' behaviour to people and novel objects the scientific assessment of animal welfare assessment of stress levels among cats in four animal shelters the behaviour of the domestic cat the quality of being sociable: the influence of human attentional state, population, and human familiarity on domestic cat sociability environmental enrichment in a large animal facility caged cats: avoiding problems and providing solutions evolution and animal welfare biological response to stress: implications for animal welfare the effect of a hiding box on stress levels and body weight in dutch shelter cats; a randomized controlled trial effects of density and cage size on stress in domestic cats (felis silvestris catus) housed in animal shelters and boarding catteries welfare of cats in a quarantine cattery the behaviour of domestic cats in a shelter: residence time, density and sex ratio a study of owner observed behavioural and lifestyle changes in cats with musculoskeletal disease before and after analgesic therapy effects of stressors on the behavior and physiology of domestic cats feline welfare issues sickness behaviors in response to unusual external events in healthy cats and cats with feline interstitial cystitis coping styles in the domestic cat (felis silvestris catus) and implications for cat welfare behavioural aspects of the welfare of rescued cats pleasures', 'pains' and animal welfare: toward a natural history of affect effect of novel stimulation on cats reared in a restricted environment animal play and animal welfare the assessment of welfare. in the welfare of cats stress, security, and scent: the influence of chemical signals on the social lives of domestic cats and implications for applied settings a case-controlled comparison of behavioural arousal levels in urine spraying and latrining cats vertical or horizontal? diagnosing and treating cats who urinate outside the box stereotypic and compulsive disorders feline compulsive disorders diagnosis and management of compulsive disorders in dogs and cats housing conditions and behavioural problems of indoor cats as assessed by their owners stable individual differences in vocalisation and motor activity during acute stress in the domestic cat companion animal welfare and possible implications on the human-pet relationship shelter housing for cats: principles of design for health, welfare and rehoming behavior and welfare of domestic cats housed in cages larger than us norm the effect of hiding enrichment on stress levels and behaviour of domestic cats (felis sylvestris catus) in a shelter setting and the implications for adoption potential will a hiding box provide stress reduction for shelter cats? the effect of space on behaviour in large groups of domestic cats kept indoors social behavior and aggressive problems of cats cat housing in rescue shelters: a welfare comparison between communal and discrete-unit housing social behaviour of domestic cats (felis lybica f. catus l.): a study of dominance in a group of female laboratory cats effects of single caging and cage size on behavior and stress level of domestic neutered cats housed in an animal shelter comparison of cats (felis silvestris catus) housed in groups and single cages at a shelter: a retrospective matched cohort study a critically appraised topic (cat) to compare the effects of single and multi-cat housing on physiological and behavioural measures of stress in domestic cats in confined environments the effects of social interaction and environmental enrichment on the space use, behaviour and stress of owned housecats facing a novel environment an ethogram for behavioural studies of the domestic cat (felis silvestris catus l.); universities federation for animal welfare effect of single-cat versus multi-cat home history on perceived behavioral stress in domestic cats (felis silvestris catus) in an animal shelter behavioral differences between owner surrender and stray domestic cats after entering an animal shelter development and validation of a behavioral acclimation protocol for cats to respiration chambers used for indirect calorimetry studies ovarian and testicular function in the domestic cat: clinical management of spontaneous reproductive disease use of single-dose oral gabapentin to attenuate fear responses in cage-trap confined community cats: a double-blind, placebo-controlled field trial experimental analysis of human-cat interactions during first encounters the human-cat relationship friendliness to humans and defensive aggression in cats: the influence of handling and paternity ontogeny of individuality in the domestic cat in the home environment physical and behavioral measures that predict cats' socialization in an animal shelter environment during a three day period an evaluation of feral cat management options using a decision analysis network reasons for liking and choosing a cat as a pet responses of pet cats to being held by an unfamiliar person, from weaning to three years of age behavioural aspects of the population genetics of the domestic cat clicker training increases exploratory behaviour and time spent at the front of the enclosure in shelter cats; implications for welfare and adoption rates admissions of cats to animal welfare shelters in melbourne the aspca's feline spectrum assessment pet personality: a review human-cat interactions: relationships with, and breed differences between, non-pedigree, persian and siamese cats factors influencing the temporal patterns of dyadic behaviours and interactions between domestic cats and their owners are cats (felis catus) from multi-cat households more stressed? evidence from assessment of fecal glucocorticoid metabolite analysis a proposal for assessing individual differences in behaviour during early development in the domestic cat personality structure in the domestic cat (felis silvestris catus), scottish wildcat (felis silvestris grampia), clouded leopard (neofelis nebulosa), snow leopard (panthera uncia), and african lion (panthera leo): a comparative study assessment of domestic cat personality, as perceived by owners, suggests six dimensions meet your match®feline-ality™ manual and training guide modification of the feline-ality™ assessment and the ability to predict adopted cats' behaviors in their new homes modified meet your match ® feline-ality tm validity assessment: an exploratory factor analysis of a sample of domestic cats in a brazilian shelter evidence for individual differences in behaviour and for behavioural syndromes in adult shelter cats heart rate variability as a measure of autonomic regulation of cardiac activity for assessing stress and welfare in farm animals-a review evaluation of the effects of hospital visit stress on physiologic parameters in the cat heart rate and heart rate variability of healthy cats in home and hospital environments physiological stress coping and anxiety in greyhounds displaying inter-dog aggression heart rate variability predicts the emotional state in dogs use of thermography techniques in equines: principles and applications nasal temperatures in dairy cows are influenced by positive emotional state evaluation of the white-coat effect in cats the right ear but not the left ear temperature is related to stress-induced cortisolaemia in the domestic cat (felis catus) evaluation of environment and a feline facial pheromone analogue on physiologic and behavioral measures in cats carrier training cats reduces stress on transport to a veterinary practice stress and animal welfare responses of the porcine pituitary-adrenal axis to chronic intermittent stressor comparison of stress exhibited by cats examined in a clinic versus a home setting tissue-specific cushing's syndrome, b-hydroxysteroid dehydrogenases and the redefinition of corticosteroid hormone action problems associated with the interpretation of physiological data in the assessment of animal welfare the use of open field tests in the assessment of welfare of cattle analysis of endogenous cortisol concentrations in the hair of rhesus macaques relationships between scores of the feline temperament profile and behavioural and adrenocortical responses to a mild stressor in cats the use of hair cortisol for the assessment of stress in animals plasma hormones in neotropical and domestic cats undergoing routine manipulations assessment of adrenal function in cats: response to intravenous synthetic acth cortisol levels and aggression in neutered and intact free-roaming female cats living in urban social groups noninvasive monitoring of adrenocortical activity in carnivores by fecal glucocorticoid analyses human interaction and cortisol: can human contact reduce stress for shelter dogs? feline stress encyclopedia of stress cortisol metabolism in the domestic cat and implications for non-invasive monitoring of adrenocortical function in endangered felids measurement of faecal cortisol metabolites in cats and dogs: a non-invasive method for evaluating adrenocortical function factors affecting faecal glucocorticoid levels in domestic cats (felis catus): a pilot study with single and large multi-cat households behavioural and faecal glucocorticoid metabolite responses of single caging in six cats over days a novel method using hair for determining hormonal levels in wildlife analysis of cortisol in dog hair-a potential biomarker of chronic stress: a review validation and use of hair cortisol as a measure of chronic stress in eastern chipmunks (tamias striatus) exploration of the hypothalamic-pituitary-adrenal function as a tool to evaluate animal welfare why is there no simple way of measuring animal welfare? influence of indoor-cat group size and dominance rank on urinary cortisol levels relationship between hair cortisol concentrations and depressive symptoms in patients with coronary artery disease hair cortisol: a novel biomarker of hypothalamic-pituitary-adrenocortical activity mechanisms of drug incorporation into hair hair analysis of anabolic steroids in connection with doping control-results from horse samples measuring cortisol in hair and saliva from dogs: coat color and pigment differences human hair follicles display a functional equivalent of the hypothalamic-pituitary-adrenal (hpa) axis and synthesize cortisol analysis of cortisol in hair-state of the art and future directions cortisol determination in hair and faeces from domestic cats and dogs definition of criteria for overall assessment of animal welfare assessment of behavior and physical condition of shelter cats as animal-based indicators of welfare stress and the immune system feline herpesvirus when cats' ways of life interact with their viruses: a study in natural populations of owned and unowned cats (felis silvestris catus) associations of behaviour with secretory immunoglobulin a and cortisol in domestic cats during their first week in an animal shelter experimental induction of feline viral rhinotracheitis virus re-excretion in fvr-recovered cats epidemiologic evaluation of multiple respiratory pathogens in cats in animal shelters mucosal immunology: overview and potential in the veterinary species shelter medicine for veterinarians and staff descriptive epidemiology of feline upper respiratory tract disease in an animal shelter mucosal defence along the gastrointestinal tract of cats and dogs common virus infections in cats, before and after being placed in shelters, with emphasis on feline enteric coronavirus seroprevalence and isolation rate of bordetella bronchiseptica in cats in the uk mechanisms for persistence of acute and chronic feline calicivirus infections in the face of vaccination feline respiratory disease update on feline calicivirus: new trends from inflammation to sickness and depression: when the immune system subjugates the brain a study of environmental and behavioural factors that may be associated with feline idiopathic cystitis risk factors and clinical presentation of cats with feline idiopathic cystitis risk factors for idiopathic cystitis in norwegian cats: a matched case-control study stress and gastrointestinal motility in animals: a review of the literature stress-induced gastrointestinal barrier dysfunction and its inflammatory effects enteropathogen co-infection in uk cats with diarrhoea the fecal microbiome in cats with diarrhea faecal consistency and risk factors for diarrhoea and constipation in cats in uk rehoming shelters prevalence of enteropathogens in cats with and without diarrhea in four different management models for unowned cats in the southeast united states the use of fecal markers to facilitate sample collection in group-housed cats chorionic gonadotropin administration in domestic cats causes an abnormal endocrine environment that disrupts oviductal embryo transport the effectiveness of indigestible markers for identifying individual animal feces and their prevalence of use in north american zoos body condition of feral cats and the effect of neutering effects of group stability on aggression, stress and injuries in breeding rabbits the cat psychophysiology of stress in dermatology: the psychobiologic pattern of psychosomatics a preliminary description of companion cat, managed stray cat, and unmanaged stray cat welfare in auckland, new zealand using a -component assessment scale guidlines for recognition, assessment and treatment of pain managing pain in feline patients systematic review of the behavioural assessment of pain in cats refinement and initial validation of a multidimensional composite scale for use in assessing acute postoperative pain in cats validation of the english version of the unesp-botucatu multidimensional composite pain scale for assessing postoperative pain in cats development of a behaviour-based measurement tool with defined intervention level for assessing acute pain in cats definitive glasgow acute pain scale for cats: validation and intervention level geometric morphometricsfor the study of facial expressions in non-human animals, using the domestic cat as an exemplar development and application of catfacs: are human cat adopters influenced by cat facial expressions? facial expressions of pain in cats: the development and validation of a clinical applicability of the feline grimace scale: real-time versus image scoring and the influence of sedation and surgery development of the horse grimace scale (hgs) as a pain assessment tool in horses undergoing routine castration associations of body condition score with health conditions related to overweight and obesity in cats comparison of various methods for estimating body fat in dogs aaha nutritional assessment guidelines for dogs and cats evaluation of a nine-point body condition scoring system in physically inactive pet cats trained vs untrained evaluator assessment of body condition score as a predictor of percent body fat in adult cats a simple, reliable tool for owners to assess the body condition of their dog or cat usefulness of muscle condition score and ultrasonographic measurements for assessment of muscle mass in cats with cachexia and sarcopenia how often do veterinarians assess the bodyweight and body condition of dogs? use of body condition scores in clinical assessment of the provision of optimal nutrition development and validation of a body condition score system for cats: a clinical tool feline obesity: epidemiology, pathophysiology and management correlation of a feline muscle mass score with body composition determined by dual-energy x-ray absorptiometry muscle condition score evaluation of body weight, body condition, and muscle condition in cats with hyperthyroidism cachexia and sarcopenia: emerging syndromes of importance in dogs and cats strong associations of nine-point body condition scoring with survival and lifespan in cats aggregation of measures to produce an overall assessment of animal welfare. part : analysis of constraints an investigation into the epidemiology of feline obesity in great britain: results of a cross-sectional study of companion animal practises acute feline leukemia virus infection causes altered energy balance and growth inhibition in weanling cats environmental enrichment: practical strategies for improving feline welfare environmental enrichment choices of shelter cats effects of music on behavior and physiological stress response of domestic cats in a veterinary clinic the influence of olfactory stimulation on the behaviour of cats housed in a rescue shelter influence of olfactory enrichment on the exploratory behaviour of captive-housed domestic cats effect of cognitive enrichment on behavior, mucosal immunity and upper respiratory disease of shelter cats rated as frustrated on arrival assessment of clicker training for shelter cats the influence of visual stimulation on the behaviour of cats housed in a rescue shelter food puzzles for cats: feeding for physical and emotional wellbeing effect of a synthetic feline facial pheromone product on stress scores and incidence of upper respiratory tract infection in shelter cats agonistic behavior and environmental enrichment of cats communally housed in a shelter vocalization of farm animals as a measure of welfare how-to" guide for designing judgment bias studies to assess captive animal welfare assessing the welfare of kennelled dogs-a review of animal-based measures key: cord- -j ribasg authors: gjerde, eli-anne b; eide, dag marcus; brønstad, aurora; reed, rolf k title: problems in physiological experimental animal models investigated with factorial design date: - - journal: journal of experimental animal science doi: . /j.jeas. . . sha: doc_id: cord_uid: j ribasg abstract in the present study we investigated four variables using factorial design to decide if any of these could explain the variations in the control measurements of interstitial fluid pressure (p if) in rat trachea that were experienced. this approach requires only a fraction of the animals normally needed when studying each factor separately. p if in tracheal tissue was measured with the servocontrolled counterpressure system using sharpened micropipettes. the measurements were performed over a period of min and are presented as mean for every min period. the factors investigated in the study were: three strains of female rats (strain) two brands of diets (food); two breeder companies (source); and finally two batches of the same set of animals to repeat the experiment twice (week), using a total of animals. there was a highly significant effect within strain the first week (p= . ), but this response was not observed the second week. the interaction between strain×week was significant (p= . ) while the main effects strain or week alone were not significant. the response pattern for strain and food was inconsistent for the two experimental weeks studied. these experiments made it possible for us to simultaneously test several factors and exclude these factors as the reason for the observed changes in our experiments since the experiments did not allow the conclusion that one or several of these factors could explain the variation in p if. the standardization of animal experiments in physiological research is important to increase reproducibility at different times and locations with similar results. in animal experiments the rules of the r's: reduction, refinement and replacement (russell and burch, ) , should be implemented in planning and performing experiments (flecknell, ) . much focus has been put on standardizing environmental factors known to cause unintended variations of the experiments. some of the parameters that are known to affect results are temperature, air changes, air humidity, light-dark cycles, microbiological conditions (poole et al., ) . the current investigation, demonstrate how such a study can be performed, when it becomes necessary to perform experiments involving several variables and groups. the experiments were performed since interstitial fluid pressure (p if ) in rat trachea in control animals was observed to be lowered, a response otherwise seen in acute inflammatory reactions such as following mast cell degranulation and neurogenic inflammation (koller and reed, ; koller et al., ; woie et al., ; woie and reed, ) . these findings occurred irregularly, but some observations could suggest that the differences could be due to variability in animal stocks or to conditions in the animal facility. the transcapillary fluid filtration is created by the imbalance between the osmotic and the hydrostatic pressures over the microcirculatory wall, giving a net filtration pressure of . - mmhg in peripheral tissues as skin and skeletal muscle (aukland and reed, ; wiig et al., ) . the lowering of p if will increase the transmural driving pressure during the initial phase of an inflammatory challenge. the lowering of p if measured in tracheal tissue of rats following an inflammatory challenge ranges from À to À mmhg (gjerde et al., (gjerde et al., , koller and reed, ; wei et al., ; woie et al., ; woie and reed, ; woie and reed, ; woie and reed, a; . under normal (non-inflammatory) conditions p if is slightly subatmospheric and the mean average and standard deviation measured over or min registration period for trachea is À . . mmhg. this average is taken from a total of studies and series of experiments containing all together control animals giving a average of - animals per series (gjerde et al., (gjerde et al., , (gjerde et al., , (gjerde et al., , koller and reed, ; koller et al., ; wei et al., ; woie et al., woie and reed, . figures from previous studies gjerde et al., ) illustrate that the levels of p if in vehicle treated animals are constant throughout the min recording period. the slightly negative p if is one of the forces controlling the filtration over the microcirculatory wall into the interstitium and also acts as a filling pressure for the initial lymphatics (aukland and reed, ; levick, ) . for all previous studies on changes in p if in rat trachea, female wistar rats were used, mainly provided from m&b as (p.o box , dk- , ry, denmark) and with a very few exceptions delivered by harlan uk ltd (shaw's farm, blackthorn, bicester, oxon, ox tp, england). these animals were, over the years, first fed with sds (special diets services, uk), rm maintenance diet and later, due to changes in the diet routine in the animal facility, the animals were fed with b&k (b&k universal uk) rodent low protein diet. over a period of several months we began to observe variable values and quality in the control measurements of p if with values considerably more negative, i.e. with values similar to those observed in inflammation. environmental factors were suspected to cause the problems since even minor changes in the airway histology are common findings in laboratory animals and often seen in rats without any serological evidence of viral infection (greaves and faccini, ) , although they are usually not considered to be of pathological importance (greaves and faccini, ) . several environmental factors were improved such as reducing stress by taking special care in the animal handling before and during the experiments. we also improved the conditions at the animal facility by isolating the animals in separate rooms or by placing them in a ventilated cupboard as well as changing the time of the acclimatization period after transport. the conditions with normal p if ; i.e. the control measurements of p if were only partially restored, but with little consistency and with no clear preferences towards any environmental factor. mcdonald and collaborators (mcdonald, (mcdonald, , have demonstrated that animals infected with mycoplasma pulmonis had increased response to neurogenic inflammation. our animals were delivered by a supplier with regular health monitoring programs and tested negative for mycoplasma as did the sentinels from our animal facility during the actual time period. also animals were kept in isolators to avoid infection after arrival to the animal facility. the aim of this study was to investigate if genetic or environmental factors were able to explain the observed variability in p if : also, this report demonstrates the use of factorial design to proceed systematically in the study of multiple factors by avoiding the use of large amount of animals. the conclusion of the study is, however, that none of the factors investigated alone or together explained the observed changes in p if : female rats from two inbred strains, fisher f (f /nmol and f /nhsd) and brown norway (bn/mol and bn/ssnolahsd) as well as one outbred stock wistar (mol:wist han and hsdola:wi) were supplied in equal numbers from both m&b as (p.o box , dk- , ry, denmark) and harlan uk ltd (shaw's farm, blackthorn, bicester, oxon, ox tp, england). the animals from both week groups were acclimatized for weeks at the following conditions before the experiment: relative humidity (rh) - %, temperature range of - c, and h light cycle and were fed either sds (special diets services, uk), rm maintenance diet or b&k (b&k universal uk) rodent low protein diet and tap water ad libitum. both diets have similar protein source i.e. extract of soya bean meal and whey powder. the respective unit percentage of the nutrients in the two different diets, for crude protein was . % and . %, respectively, and for digestible crude protein . % vs . %, respectively. a total of rats were used and divided into two experimental weeks each with rats. the two groups of animals forming the week-groups, differed in arrival time by approximately weeks and had an acclimatizing time of weeks before the experiments took place. three weeks of acclimatization has previously been observed as more than enough time to induce changes in p if : the animals were of approximately the same age (around weeks, but varied in weight at the time of the experiments from to g. this variation is due to the fact that inbred strains are often smaller in size than outbred at the same age. the animals were kept isolated from other experimental animals, in separate rooms provided with four racks to separate the two food groups. each rack contained rats from the three different strains and each cage contained only two animals. an overview of the experimental design is presented in table . although, we were not able to measure p if in two of the rats (i.e. one mol:wist han; b&k; week and one f /nhsd; b&k; week ), the statistical analysis was not impaired by a reduction to animals. the procedures were carried out with the approval of and in accordance with the regulations of the national animal research authority. the rats were anaesthetized with sodium pentobarbital (initial dose of mg/kg i.p. and supplementary doses when needed). a branch of the femoral vein was cannulated for injection of potassium chloride ( . - ml) to induce cardiac arrest. the subsequent measurements of interstitial fluid pressure in the tracheal tissue lasted for a total of min, with measurements reported for four time periods of min each. interstitial fluid pressure (p if ) after induction of circulatory arrest an extra-thoracic portion of trachea was exposed and the muscle overlying the trachea was split longitudinally. the tracheal surface was covered with mineral oil to avoid desiccation. the time required for the experimental preparation after cardiac arrest was - min. measurements of p if were initiated soon thereafter on the abluminal portion of trachea and between the cartilage rings using sharpened glass capillaries ( - mm) connected to a servocontrolled counterpressure system (wiederhielm et al., ; wiig et al., ) and continued until min after circulatory arrest. the glass pipettes were filled with . m nacl colored with evans blue. micropuncture was performed under visual guidance with a microscope (wild m c, heerbrugg, switzerland) and care was taken to minimize stretch or compression at the site of puncture (wiig et al., ) . the measurements were accepted when the following criteria were met: ( ) no change in recorded pressure when increasing feedback gain; ( ) suction applied by the servocontrolled pump should increase electrical resistance in the pipette, verifying open communication to interstitial fluid because of the lower tonicity of the fluid entering the pipette; ( ) the baseline measurements before and after p if registration were unchanged. the latter measurement was performed in a plastic cup filled with saline placed at the level of the site of puncture. all surgical manipulations on or near the trachea were performed after circulatory arrest to avoid local inflammation that may increase interstitial fluid volume and confound accurate estimates of p if . a factorial design was used, with all cross-classifications present. the statistical model was the following repeated measures anova, using jmp ( ) from sas institute (sas institute, ) : where y À y were the consecutive recordings of p if at min intervals within an hour, food were of two brands (i ¼ ), source were two commercial breeders (j ¼ ), strain represented the two inbred strains and one single outbred (k ¼ ), week are the two consecutive weeks of repeated experiments (l ¼ ) and e ijkl is the random error. the normality test was performed and passed prior to performing the main analysis. the analysis started with a full factorial resolution, then we only kept the significant interaction terms plus the main effects in the final model. the number of animals needed for the experiment was estimated according to mead's resource equation (mead, ) . we included sufficient animals to provide information on all two-way interactions, i.e. n ¼ : results are presented as the least-squares means from ( ), ls mean sem the effect of rat strain was highly significant in the first week of the experiment (table ) and the different rat strains had different response profiles on p if (fig. , upper left) . however, this effect did not appear on other rats the following week (fig. , upper right) . there was a significant effect of the interaction term strain  week, while none of the main effects, strain or week were significant although strain was of borderline significance (table ). this implies that the response pattern for each rat strain was different in the two experimental periods. there was a significant effect of time on the repeated measurements, i.e. the curves were not horizontal. the p if was falling throughout the h recording procedure a pattern recognized in an inflammatory response . there was a significant interaction term for time  strain in the experiment done in the first week, indicating that the reaction curves for the different strains had different shapes and/or slopes. however, the following week's experiment produced response curves that were not different between strains. the strains contribute differently to this effect. wistar and f are the main contributors to the difference between the weeks in the three last time periods, as can be exemplified in a strain  week leverage plot for the - min time period (fig. ) . there was a significant interaction term for strain  food (table ). this effect could be seen in fig. , where the mean response curves for the feeding groups appear in the reverse order in the consecutive weeks. only of the rats behaved like 'normal controls' (i.e. with horizontal curves throughout the h recording and no mean total p if less than À mmhg, . a log-linear analysis could not identify an increased likelihood of 'normal controls' in any experimental group (data not shown). when the statistical analysis was performed with n ¼ ; i.e. in effect by stating that only one measurement can be obtained from all animals in the same cage, the statistical differences above disappeared (see below). the purpose for using the factorial design was to increase the amount of information and the generality of the results of an experiment (festing et al., ) . it is a powerful alternative for doing several smaller experiments for each factor and also allows testing for interactions between these factors (festing et al., ) . factorial designs are used to investigate whether a response to one treatment is the same across the levels of all the studied variables (festing et al., ) . the observation that when the measurements from each cage are averaged, the statistical difference disappears, may have two explanations and implications. first, it does in fact strengthen our conclusion that we cannot in this study pinpoint a single causative factor for the observations that were the starting point for this study. also, since the factors reaching significance in weeks and were different, this would seem to be an argument in the same direction, i.e. that the statistical significances were borderline and could not be verified, either when studied in the subsequent week, or when reducing the power of the statistical analysis. however, if the experiments were to be performed according to the procedure outlined above by averaging all corresponding measurements from one cage, and perform the subsequent statistical analysis on such average data, rather than on data from one single experimental animal, this is in fact testing a different hypothesis. however, depending on the hypotheses and questions asked, both approaches seem to be valid. in the experimental situation leading to the present study, there was seemingly a random appearance of animals that behaved like ''experimental'' animals while they were in fact controls. in the measurements performed in this study, these animals appeared randomly throughout the cages. the effect of averaging the data from one cage is therefore to minimize an effect that should be present, simply due to the averaging. therefore, it is not surprising that after averaging the statistical significances disappear. furthermore, the averaging procedure has another principal discussion attached to it, namely whether the number of experiments actually performed should be the total number of registrations in an animal, the average of the measurements within one animal or the average between different animals in the same cage. in accordance with accepted practice for physiological experiments, we have averaged the measurements within one animal and used this for the subsequent statistical analysis. our argument in favor of this choice of procedure is that in all previous similar studies, among them the ones leading to the present one, the averaging and analysis have been outlined as described above. we feel that if the study was to be performed based on averaging the animals within one cage, the proper design would actually be to have one animal per cage. however, this will have such implication on animal care and cost, that it would seriously limit the number of experiments based on cost. the rationale for testing the two types of diet chosen here, was based on the fact that there had been performed changes in the diet routine in the animal facility and due to other research groups necessity to develop old specimens of rats. a diet from another supplier, with lower protein, starches and sugar content, was put to use to avoid excessive growth in these animals. it was then decided that all animals in this facility should be fed with the same low protein diet. animals used in our experiments were relatively young and were only housed in the animal facility for a short acclimatization period before the experiment. another argument for testing the response of p if on diet is the fact that the source of fatty acid can modulate the immune response. likewise, arachidonic acid metabolites, derived from fatty acids, are important in the inflammation cascade and may modulate the inflammatory response (demarne et al., ; hoverstad and midtvedt, ) . also a change of gut microbiological flora can affect the metabolism of fatty acid (bruckner and gannoe-hale, ) . the significant effect of the interaction between food and week could suggest a variation in the food to be of importance. the inconsistency between the two weeks could indicate that one of the food brands did not have the same composition within the same batch, which is occasionally observed (baumans et al., ) . it is well known that stress like shipping, can provoke latent disease caused by opportunistic microbes as with shipping fever caused by pasturella spp. in cattle or glasser's disease caused by hemophilus spp. in pigs (blood and radostits, ) . immunosuppression caused by increased endogenous cortisol due to stress is the usual explained mechanism behind this (blood and radostits, ) . the animals were supplied by breeders with a regular health monitoring program, and did not carry pathogens according to the felasa guidelines. there is still a possibility that the animals were infected after they arrived to our facility and this was tested by performing experiments with animals housed in isolators. however, this gave a distribution of p if similar to what was experienced in the ''problem period'' (unpublished data). the use of isolators was therefore not included in the factorial study. even if the animals are tested and specific pathogen free (spf) for defined pathogens, this gives limited information and only tells which microbes that the animals do not have. the animal's normal bacterial flora is seldom characterized or reported. complete control of the bacterial and microbiological flora is obtained in germ free animals delivered by cesarean section, be colonized with a defined microbiological flora, and housed isolated for the rest of their life (heidt et al., ) . such animals were not used in this study. mcdonald (mcdonald, (mcdonald, , demonstrated that m. pulmonis, sendai virus and coronavirus cause an increased susceptibility to neurogenic inflammation in the rat trachea, which is an inflammatory reaction that also induces lowering of p if . the current inability to achieve 'normal control' measurements of p if could therefore result from an infection from some of the agents above. however, neither mycoplasma, sendai virus nor coronavirus have ever been detected in sentinel animals during this period. an inbred strain is obtained by having brother and sister mating from the same branch for at least generations. they are homozygous at more than % of the loci (festing, ; poole et al., ) . the genetic status of an inbred strain will stay constant over time, like an immortal clone of identical individuals and changes only occur by mutations. an outbred stock like the wistar rat, is obtained by breeding a closed colony of genetically undefined animals for at least generations. the genetics of an outbred stock might change over time due to genetic drift as a result of random mating within the population. the use of inbred strains gives a larger statistic precision by use of fewer animals. however, they are more sensitive to environmental influences than outbred stocks. the overall choice was to use several different inbred strains. this will increase the generality of the experiment and still have the same genetic material over time. the rationale for using the outbred wistar is that it has been used for a long time in similar experiments. the different strains behaved differently in the two experimental weeks. although inbred animals are more sensitive than outbred to changes in the environment (festing, ) the combined effect of strain in the total experiment is not significant since the two repeated experiments gave opposite ranking of the strain mean values. the strain  week interaction is actually a study of a genotype-environment interaction. the tendency in these experiment is that bn rats show the highest degree of consistency in the experiments between week, while the outbred wistar rats give slightly less consistent results than f , i.e. bnbf >wistar. this may suggest that the variation in the response in wistar rats was due to the genetic heterogeneity of this stock, since two batches of wistar rats are genetically different. the differences between the strains could be due to variation at a receptor expression level. pauwels and collaborators joos et al., ; pauwels et al., ) report different susceptibility to plasma protein extravasation following neurogenic inflammation in trachea of different rat strains. they identified several genetic differences between the f -rat and bde-rats, among others in the mechanisms involved in the plasma protein extravasation in the airways following substance p or capsaicin, since these agents released hydroxytryptamine from the mast cells in f -rats but no bde-rats . this study has shown that even though we find differences in p if measurements with regard to several of the parameter investigated, none of them were able to restore 'normal control' p if ; i.e. not less than À mmhg. although the experiments were inconclusive with regard to which factor that by itself or in combination with others caused the variability in p if the study demonstrates how several factors can be investigated using a minimum of experimental animals. interstitial-lymphatic mechanisms in the control of extracellular fluid volume principles of laboratory animal science: a contribution to the humane use and care of animals and to the quality of experimental results veterinary medicine: a textbook of the diseases of cattle, sheep, pigs, goats and horses. bailli" ere tindall fatty acid compositional changes in germfree and conventional young and old rats comparative study of endogenous fecal fatty acids in germ-free and conventional rats inbred strains in biomedical research replacement, reduction and refinement characterization of neurogenic inflammation in the airways of two highly inbred rat strains cgrp, but not substance p, induces an increased negativity of the interstitial fluid pressure in rat trachea corticotropin-releasing hormone inhibits lowering of interstitial pressure in rat trachea after neurogenic inflammation lowering of interstitial fluid pressure after neurogenic inflammation is inhibited by mystixin- peptide the neurotensin fragment acnt( - ) inhibits lowering of interstitial fluid pressure in rat trachea lowering of interstitial fluid pressure in rat trachea after substance p alone and in combination with calcitonin gene related peptide rat histopathology: a glossary for use in toxicity and carcinogenicity studies the use of a rat derived microflora for providing colonization resistance in spf rats short-chain fatty acids in germfree mice and rats tachykinins contract trachea from fischer rats by interaction with a tachykinin nk receptor increased negativity of interstitial fluid pressure in rat trachea in dextran anaphylaxis increased negativity of interstitial fluid pressure in rat trachea after mast cell degranulation capillary filtration-absorption balance reconsidered in light of dynamic extravascular factors respiratory tract infections increase susceptibility to neurogenic inflammation in the rat trachea infections intensify neurogenic plasma extravasation in the airway mucosa the design of experiments: statistical principles for practical applications genetic control of indirect airway responsiveness in the rat the ufaw handbook on the care and management of laboratory animals the principles of humane experimental technique sas institute, . jmp in user's guide: statistical visualization software for the macintosh dynorphin a( - ) analogs suppress thermal edema pulsatile pressures in the microcirculation of frog's mesentery micropuncture measurement of interstitial fluid pressure in rat subcutis and skeletal muscle: comparison to wick-in-needle technique neurogenic inflammation and lowering of interstitial fluid pressure in rat trachea is inhibited by alpha-trinositol the relationship between interstitial fluid pressure and volume in rat trachea alloxan diabetes abolishes the increased negativity of interstitial fluid pressure in rat trachea induced by vagal nerve stimulation neurogenic inflammation in rat trachea is accompanied by increased negativity of interstitial fluid pressure lowering of interstitial fluid pressure will enhance edema in trachea of albumin-sensitized rats the technical assistance of gerd signe salvesen, tore-jacob raa and linda vabî s appreciated. financial support from the norwegian heart association and the research council of norway is appreciated. the authors greatly acknowledge m&b as for providing the animals for this study. key: cord- - rdlf l authors: tully, thomas n. title: chapter mice and rats date: - - journal: manual of exotic pet practice doi: . /b - - . - sha: doc_id: cord_uid: rdlf l publisher summary this chapter focuses on mice and rats, and provides detailed information that may be useful for veterinarians treating these animals. mice are continuous, polyestrous rodents that should be bred in polygamous or monogamous setups because of the males' aggressive territoriality behavior. when breeding mice that have been housed in a polygamous ratio, there may be one male with two to six females. females are removed from a polygamous cage before parturition, whereas the monogamous pair is maintained together with the young until weaning. mice are maintained in environments that are similar to other small rodents but require a thorough cleaning of their cage more often because of their malodorous urine. ventilation is essential for small rodent housing to prevent irritation of the respiratory tract from ammonia vapors generated by urine. quarantining is important when a new animal is being introduced into a setting in which there is an established group. as with other animals, a -day quarantine period is recommended, along with a physical examination and fecal parasite check. to maintain oversight of breeding animals' health and reduce the exposure of young animals to infectious disease and parasites, routine screening of representative animals within the colony is recommended. in very large colonies, special caging, food, and water may be necessary to prevent exposure to disease organisms. c h a p t e r thomas n. tully, jr. this chapter, on mice and rats, contains information on the companion species that is useful for veterinarians treating the animals or providing information to the owners. although similarities exist for mice and rats-in care, husbandry, diagnostic testing, and treatment-emphasis will be on the differences between the species. mice may be maintained as pets because of their size and playful nature. although playful, mice can be aggressive to cagemates and owners. the aggressive nature often manifests as barbering and/or fi ghting with cagemates and biting their owners. any potential owner should be educated on the aggressive nature of these small dynamos. domestic mice (mus musculus) and the african pygmy mouse (baiomys spp.) are commonly sold for pets and are available in several varieties. the mice varieties sold in pet stores include white, black, or tan colored, satin hair coat (shiny), pied or spotted, and longhaired , (figure - ) . the size and ability of mice to escape quickly from the grasp of a human handler make these animals, as pets, better suited for older individuals. the timid nature of mice predispose these animals to biting if handled roughly, which commonly occurs with a young owner. mice are nocturnal animals and may be a disturbance at night if maintained in a bedroom. female mice produce less odor than males and therefore may be more desirable as pets. as mentioned earlier, male mice are territorial, and if placed with other male mice, may fi ght. advantages of having mice as pets include their size, ability to be a good companion animal for the educated (regarding mouse behavior) owner, and adorable appearance. mice also rarely become infected with bacterial diseases, and their life span is approximately years (box - ). the integument of mice is commonly associated with a number of disease presentations. a hair coat that has not been adequately maintained is often the fi rst clinical sign associated with disease. mice are fastidious with their grooming and, when healthy, maintain a very tidy hair coat. common problems associated with a hair coat that is not maintained include general illness, parasitism (internal and external), aggression by cagemates as a result of psychological trauma, and infectious dermatitis. neoplasia is a disease condition that affects mice. there are some mouse strains in which mouse mammary tumor viruses have been identifi ed that have a neoplasia incidence as high as %. tumor viruses, as well as a very short life span (up to years), predispose these animals to cancer. mammary adenocarcinomas and fi brosarcomas are the most common tumors that affect mouse mammary tissue. unfortunately, by the time many of these tumors are diagnosed, the malignant masses are large and ulcerated. fibrosarcomas may be hormonally induced, and the incidence of this disease process can be reduced through an ovariectomy at an early age. the small size of the female mouse often makes the ovariectomy surgery challenging for the veterinary surgeon. although relatively uncommon, urethral obstruction can occur in male mice as a result of preputial and/or bulbourethral gland infections. male mice suffering from an urethral obstruction may have a mutilated penis, a sign often associated with the disease process. mice are continuous, polyestrous rodents that should be bred in polygamous or monogamous setups because of the males' aggressive territoriality behavior. when breeding mice that have been housed in a polygamous ratio, there may be one male with two to six females. females are removed from a polygamous cage before parturition, whereas the monogamous pair is maintained together with the young until weaning. mice can chew out of enclosures; therefore, it is important that the housing be "mouse proof." if an animal escapes, the best way to capture the pet is to place food in the center of the room. once it has been determined in which room the animal is staying, then it should be sealed and measures taken to look for the pet. because mice are nocturnal animals, capture at night in a dark room with a fl ashlight may work best. this technique will work for capture of other small rodents and pocket pets too. mice are maintained in environments that are similar to other small rodents but require a thorough cleaning of their cage more often because of their malodorous urine. ventilation is essential for small rodent housing to prevent irritation of the respiratory tract from ammonia vapors generated by urine. the recommended housing unit for mice is - ″ × - ″ × ″ (length × width × height) for each adult mouse; females and young require to times the space listed. the major considerations for selecting an enclosure for mice is that it be resistant to their escape and easy to clean. an open screen top of the enclosure is recommended for proper ventilation. available rodent cages that fi t this criterion are wire or metal mesh, plastic, and plexiglas. if the plastic tube housing systems are used, there should be routine cleaning of the sections using hot water and a mild detergent. owners should be informed of the small distance required for a mouse to escape and their ability to chew through plastic. an owner must always monitor the cage for possible escape avenues, especially if the animal has a predilection for modifying the cage opening through chewing. it is very important to provide cage toys and exercise opportunities for mice. the enclosure should be large enough to house not only food and water containers but also cage toys and an exercise wheel (figure - ). although not a cage toy, a hide box, in some form, should also be included for the psychological well-being of the animal. this hide box can be manufactured or a small cardboard container that has one end cut out. mice like to hide and also sleep during the day. this piece of cage furniture will enable the mouse to sleep undisturbed during the daylight hours. mice, especially males, have odiferous urine. to prevent the buildup of urine and fecal material in the cage, commercially available paper rodent bedding or hardwood shavings should be used as a cage substrate. although softwood (pine) and cedar shavings are available, the volatile oils that radiate from these substrates are irritating to small animals. these irritating compounds can cause dermal and respiratory infl ammation, often leading to secondary bacterial infections. cardboard tissue tubes can be placed in a mouse enclosure; mice like to chew and run through these items. because many fabrics have strong thread and elastic, it is not recommended to put clothes items in enclosures with animals that chew. often mice will shred the clothing items, exposing thread and elastic that can become entangled around an extremity; this can lead to necrosis distal to the stricture. it is recommended to change the substrate within the enclosure at least twice a week-more often if there is a problem with odor or excessive excreta. temperature, humidity, and lighting within the enclosure should follow what is commonly maintained with the ambient conditions within the house. if the animals are housed outside or in an outbuilding, the temperature range should be ° f to ° f and humidity % to %, although the recommended humidity is on the higher end of the range provided. mice, especially males, are extremely territorial animals. if mice are housed alone, it is better to keep them separate, as introduction of new animals will often lead to aggression and fi ghting. to prevent aggression, fi ghting, and psychologically induced adverse behavior (e.g., barbering), a single mouse is recommended as a companion animal. for those who intend to breed the animals, enclosures with a single pair is necessary for reproductive success. because mice are commonly used as laboratory animals in investigations of human disease processes, much research literature is available on the recommended nutritional requirements of these animals. the benefi t of these dietary studies is the availability of commercially produced diets that provide the recommended daily nutritional requirements of the mouse. the problem that most owners face is the multitude of dietary products available for mice and the lack of knowledge regarding which food to buy. most new mouse owners think that rodents eat seed-based diets. although mice will happily eat seed, seed-based diets are lacking in a number of the nutritional requirements needed to maintain long-term health. commercial rodent biscuits or pellets with more than % protein are the recommended diets for mice. the commercial rodent biscuits or pellets are the only food mice need to obtain their required nutrients (figure - ) . again, seed-based diets are not recommended, nor is a signifi cant supplementation of fruits, nuts, vegetables, cheese, or other human foods (e.g., peanut butter). if a treat is to be given, yogurt or dried fruit treats manufactured specifi cally for rodents and/or mice should be provided to times a week. for younger mice, less than weeks old, softer pellets are needed because babies start eating pellets and drinking water at to weeks of age. a sipper bottle, placed on the outside of the cage, is easy to maintain and does not take any space within the enclosure. if the cage is plastic or plexiglas, modifi cations will be needed to attach the sipper bottle on the outside of the cage. most sipper bottles come with attachment hardware to attach to wire cages ( figure - ). fresh bottled water is recommended for mice, although chlorinated tap water is acceptable. the water should be checked on a daily basis and clean water supplied at least every days, if not every day. quarantining an animal is important when a new animal is being introduced into a setting in which there is an established group. as with other animals, a -day quarantine period is recommended, along with a physical examination and fecal parasite check. unfortunately, there is no time period that will screen % against potential infectious agents, and with mice, time is critical because their life span and reproductive time frame are so short. the animals should be quarantined in conditions similar to those in which they will be permanently maintained. providing adequate food and water will make for a smooth transition. it is imperative that the owner screen the new animal for diseases and watch for any signs of illness. the most common sign of clinical illness is a rough hair coat. the lack of grooming is most often related to the animal feeling depressed or sick, thereby not having the energy to perform routine behaviors. if a number of animals are being quarantined, sacrifi cing an apparently diseased animal is the most effi cient way to determine a rapid defi nitive diagnosis. if it is a single or an expensive animal, routine diagnostic testing will be required. to maintain oversight of breeding animals' health and reduce the exposure of young animals to infectious disease and parasites, routine screening of representative animals within the colony is recommended. in very large colonies, special caging (e.g., fi lter), food, and water may be necessary to prevent exposure to disease organisms. before a veterinarian examines a mouse, the examination table should be disinfected with either a dilute sodium hypochlorite or chlorhexidine solution. the examiner should always wash his or her hands and, if necessary, wear examination gloves to capture and restrain the patient. the diffi culty with using latex gloves when examining a mouse is the small size of the patient and its ability to twist and turn. the only way to restrain some mouse patients is without gloves. if gloves are not worn, the examiner's hands must be washed thoroughly after the examination is completed. to catch a mouse, the tail should be grabbed with the thumb and forefi nger, allowing the mouse to hold on to an object with their front feet (figure - ). when the mouse securely attaches itself to an object, the opposite hand then grabs the dorsal skin in the cervical region while keeping the tail in a fi rm grasp. if one is unable to adequately restrain a mouse while the animal is conscious, inhalant anesthesia may be used for sedation purposes. the animal should be placed in a small induction chamber and isofl urane gas permeated into the closed space. once the animal has stopped moving, the enclosure top is removed and a nose cone placed on the anesthesia tube and placed on the patient's face. a syringe case can be modifi ed as a nose cone for small rodents, including mice. once the physical examination is complete, the nose cone is removed, allowing the patient to breath oxygen from the anesthesia unit. obtaining a detailed history of the mouse patient is very important, as it is with other animal patients that are brought to a veterinarian's offi ce. it is important to get as much information from the owner as possible, even if it is a routine health examination. typical background information required includes how long the mouse been owned, where it was acquired, how often it is handled, and what the character of the feces and urine is. husbandry questions should focus on the animal's housing and whether it is allowed to roam unobserved; cage location; type, size, and material of the cage; cage substrate, furniture, and toys; and the frequency with which the cage is cleaned and what disinfectant is used. when investigating the diet, the veterinarian should ask not only if pellets are fed and in what quantity, but also what the animal is eating and what the primary diet is. supplemental offerings and frequency of feeding are important data for the case work-up. the veterinarian should fi nd out about the water supply, how often the water is changed, and how much the animal drinks on a daily basis. because there are transmissible diseases among animals, the fi nal questions should center on the other pets in the household, if new animals have been added to the family, and if the animals are housed together. a description of any previous problems and a complete chronological description of the presenting problem are needed to complete the history form. before the animal is restrained, an observation should be made on the attitude, activity, and posture of the animal. the next step is to weigh the mouse in a basket on a digital gram scale. if possible, temperature, respiration, and pulse should be measured and any abnormalities in rate and/or character noted. the veterinarian should start the physical examination at the head, looking for any abnormalities. eyes, ears, and nares are observed, looking for discharge or infl ammation. the oral cavity is diffi cult to examine in mice because of the small opening and tendency for the buccal mucosa to encroach toward the middle of the mouth. a small speculum (e.g., modifi ed paper clip) or an otoscope may be used to examine the oral cavity and cheek teeth. mucous membranes help determine hydration status using capillary refi ll time and moisture. body condition and abdominal palpation are important information that should be obtained. lymph nodes and limbs are palpated before checking the nails and plantar surface of each foot. the patient should have a normal posture, be aware of its surroundings, and move properly. any problems should be noted in the record. finally, a dermatologic exam considers hair coat quality, alopecia, external parasites, and any skin abnormalities. all abnormal fi ndings are written in the record for case review, differential diagnoses determination, diagnostic testing, and treatment considerations. most of the common problems noted during the physical examination of mice involve the skin and hair coat. as mentioned previously, an unkempt hair coat may point toward generalized illness or external parasitism. abrasive lesions or pustules on the skin can be signs of external parasites or infectious dermatitis. if external parasites are a problem, the mouse may be uncontrollably scratching. hair loss may be associated with barbering, either cagemate or self-induced, or with infection (e.g., ringworm). although not common, trichophyton mentagrophytes will cause hair loss in mice, from the face, head, and neck. mice will commonly present with tumors and abscesses. the entire body of the mouse should be palpated for any evidence of masses. if a mass is identifi ed, a fi ne needle aspirate is recommended for basic diagnostic evaluation. with most fi ne needle aspirate samples, the clinical pathology results are not very rewarding. if a diagnosis cannot be obtained through cytologic examination, a biopsy of the lesion is required, and if possible, an excisional biopsy is the procedure of choice. as with many rodents, respiratory disease is commonly identifi ed in mice. mice may have presenting symptoms of dyspnea, sneezing, coughing, chattering, and sniffl ing if they have a respiratory infection. in cases where respiratory signs are observed, anesthetizing the animal to perform the physical examination is not recommended, and handling should be kept to a minimum. the two most common respiratory disease conditions in mice are sendai virus and mycoplasma pulmonis. these two organisms are very diffi cult to identify through routine diagnostic measures, and obtaining samples significantly stresses the patient. if the animal is being examined for placement into a colony or breeding environment, sacrifi ce to identify the disease condition is recommended. if the animal is an individual companion animal, doxycycline treatment should be initiated. although treatment with doxycycline will not clear the organism from the animal, it will reduce clinical disease and improve the quality of life. the treated animal should always be considered a carrier of these respiratory diseases if a defi nitive diagnosis cannot be obtained. male mice that are licking and/or mutilating their penis may be suffering from a urethral obstruction. infection of accessory sex glands, young males with aggressive breeding activity, urolithiasis, and trauma may initiate this self-induced trauma to the penis. pasteurella pneumotropica is often isolated from accessory sex gland infections as well as subdermal abscesses. treatment for this organism may aid in the recovery of a mouse affected by urethral obstruction. diarrhea in mice is rare, but endoparasite examinations should be performed if the animal has diarrhea. although pinworms are considered nonpathogenic in mice, syphacia obvelata and aspiculuris tetraptera may cause rectal prolapse due to straining in immunosuppressed individuals. as with other animals, a tape test is the best way to identify these parasites. a tape test follows a procedure of pressing the sticky side of the tape against the rectum and perianal area and then examining the tape under a microscope for eggs and parasites. as with other pocket pets, blood collection from mice can be quite diffi cult. approximately . to . ml/ g body weight can be safely removed from a nonanemic healthy mouse. the mouse patient should always be anesthetized for blood collection procedures. usually inducing the animals in a closed chamber and maintaining the patients in a mask will allow the technician plenty of time to collect the blood sample. a small, -, -, or -gauge needle, usually placed on a -ml syringe, is recommended for collecting the blood. recommended blood collection sites in a mouse are a warmed ventral tail vein or the tip of the tail. box - describes the technique used for retroorbital bleeding in mice. the retroorbital bleeding technique is commonly used in laboratory animal settings and is easily performed by an experienced veterinarian or veterinary technician. on smaller rodents, the saphenous vein or lateral vein of the tarsus may be used for multiple blood collections without the use of anesthesia. the patient must be properly immobilized in a restraint tube ( -ml syringe) with the leg extended and the skin held tight on the medial aspect of the thigh using the thumb and forefi nger. the taut skin on the lateral aspect of the thigh allows exposure of the saphenous vein. a -gauge needle is used to puncture the vein, and blood is collected in a microhematocrit tube as it fl ows from the vessel. blood samples obtained from nail and ear clips are not considered appropriate diagnostic samples. cardiac puncture is recommended only in terminal cases and when the animal is maintained under general anesthesia because of possible complications involving the lungs and heart vessels. reference ranges for complete blood counts and serum biochemistry panels for mice are listed in boxes - and - . , marrow samples may be obtained from the ilium, tibia, sternum, femur, or the bones of the proximal one third of the tail. preparation of the samples is consistent with that of other mammalian patients. standard rodent cages can be used without substrate to collect urine and feces when the patient is hospitalized. after placing a rodent in a zip-lock bag, urination frequently occurs and the sample can be collected. in a similar manner, urine can be collected over a disinfected stainless steel examination table. the urine is voided while the animal is restrained and can be collected in a capillary tube for evaluation on urine reagent strips. urinalysis reference values for mice are listed in box - . under most practice settings, the size of a mouse patient limits imaging capabilities to radiographs. restraint is essential for quality diagnostic images and is even more important when patients weigh as little as g. to obtain adequate restraint and prevent movement of the mouse patient during radiographic evaluation, sedation is required. as with all diagnostic box - retroorbital blood collection for mice from procedures involving avian and exotic animals, an evaluation of the patient is required to determine its ability to withstand the stress associated with the assessment. if it is determined that the patient cannot withstand sedation, then it should be stabilized until it is deemed healthy enough for diagnostic imaging evaluation. sedating the mouse using an induction chamber and isofl urane anesthetic, as described for restraint, is necessary to obtain diagnostic fi lms. high-speed, -ma machines with fi ne or detail-intensifying screens should be adequate for most small exotic mammal images. with mice, dental x-ray units that can focus at short distances may be advantageous for both isolation of focal anatomy and full body radiographs in extremely small patients. for extreme detail in small exotic mammal medicine, using a traditional radiography unit, the min r mammography system (eastman kodak) with min r single-intensifying screen cassettes, in conjunction with min r single-emulsion fi lm, has been recommended. digital radiography is bringing a new dimension to radiograph evaluations and detail. the secret to success in either traditional or digital radiographic imaging is to restrict patient movement during exposure. two views, ventrodorsal and lateral, are recommend for mice patients. again, because of a mouse patient's size, whole body radiographs are commonly obtained during the radiographic evaluation, even using dental radiographic equipment. for the mouse patient in which microbiological testing is recommended, standard collection techniques used for other animals is appropriate. the diffi culty when culturing mice is acquiring a representative sample of the suspect area that will provide diagnostic information. sick mice often are extremely stressed when being examined or when diagnostic samples are being collected. it is also important to know what the common disease conditions are and the common etiologies associated with those clinical signs. with the most common respiratory conditions in mice, one is sendai virus and the other a mycoplasma sp. both sendai virus and mycoplasma spp. will not be isolated using standard aerobic or anaerobic culture techniques. if in doubt on collecting, preserving, and/or shipping a microbiological sample, the diagnostic laboratory must be contacted for full instructions as they relate to mouse submissions. routine fecal parasite evaluations should be performed on small rodents when they are brought to the veterinary clinic for a health examination or an abnormal stool. common protozoal organisms can be detected using a direct fecal examination, and mouse pinworms are commonly diagnosed using the sticky side of clear cellophane tape to make an impression of the perianal area. the anal tape test will aid in diagnosing syphacia spp. the sticky surface will pick up any of the banana-shaped pinworm eggs that can be observed under a microscope. diagnosis of ectoparasites in small rodents is similar to that in other species listed in this text. the pelage tape test is performed by pressing clear cellophane tape against the pelt of a mouse, dorsally and ventrally, from the nose to the base of the tail. myobia spp. and myocoptes spp. can be diagnosed using the pelage tape test. a skin scraping of the affected area may also help in identifying ectoparasites. diagnosis of intestinal parasites can be made by fi nding individual eggs during fecal examination or whole worms within the lumen of the small intestine during necropsy. even though most of the diagnostic testing is performed through pathologic examination for mice, there are serologic tests available that can be run on a minimum of μl of undiluted serum. sound diagnostics, inc. (woodinville, washington) provides serologic testing on a number of common mouse diseases, including ectromelia virus, mouse hepatitis virus, mouse parvovirus, mouse minute virus, rotavirus, theiler's murine encephalomyelitis virus, pneumonia virus of mice, reovirus , sendai virus, mycoplasma pulmonis, lymphocytic choriomeningitis virus, and mouse adenovirus. the serologic tests are done using enzyme-linked immunosorbent assay (elisa) and, when indicated, are confi rmed by indirect fl uorescent antibody testing. many of the diseases for which there are available serologic tests are benefi cial in maintaining disease-free animals in laboratory settings or large breeding facilities. in cases of unknown disease conditions for the individual pet mouse, there are panels available that test for multiple diseases from a single serum sample. it is very important for the veterinary clinician to understand both the signifi cance of any test that is being promoted for mice and how the results are interpreted. bacterial infections associated with dermatitis lesions and subcutaneous abscesses in mice are commonly caused by staphylococcus aureus, pasteurella pneumotropica, and streptococcus pyogens. acute and chronic respiratory infections may be caused by sendai virus or mycoplasma pulmonis. acute respiratory infections usually are associated with sendai virus; adults live while neonates often die. chronic respiratory infection, with clinical signs being pneumonia, suppurative rhinitis, and occasionally otitis media, may be the result of a mycoplasma pulmonis infection. both infections should be treated using supportive care, whereas mycoplasmosis can be treated with enrofl oxacin in combination with doxycycline hyclate for days. p. pneumotropica has been associated with urethral obstruction caused by infl ammation and swelling related to infection of accessory sex glands. although uncommon, trichophyton mentagrophytes (ringworm) can cause alopecia of the face, head, and neck of mice. in addition to the disease organisms mentioned in this chapter (which are the most common disease organisms identifi ed in pet mice), there are other bacterial, fungal, and viral organisms that can cause disease in mice. proper diagnostic protocol should always be followed to identify the causal agent. once the cause is identifi ed, the proper treatment can be initiated and control measures implemented to prevent other animals' exposure to the disease. numerous parasites have been identifi ed in mice. hymenolepis nana (dwarf tapeworm) and cysticercus fasciolaris (the larval stage of the cat tapeworm) can be isolated from these small rodents. the dwarf tapeworm is often found in young mice that are dead or have severe gastroenteritis resulting in diarrhea. diagnosis of the dwarf tapeworm can be made following either a fecal fl otation exam or necropsy, when the adults may be found in the small intestine. treatment for the dwarf tapeworm is with praziquantel. the mouse pinworm (syphacia obvelata) is a commensal oxyurid nematode that feeds on bacteria that inhabit the intestinal tract of mice. although these nematodes are nonpathogenic in most cases, an overwhelming number of them can cause severe irritation of the terminal gastrointestinal tract. these nematode parasites can be diagnosed using transparent tape and applying it to the rectal area. after removing the tape from the affected rectal area, it is placed on a slide and the ova may be viewed under a microscope. ivermectin and fenbendazole have been used effectively in treating this parasite in mice. giardia muris is a common protozoal parasite affecting mice. this organism can be seen using a direct fecal examination of an affected patient's fecal material. metronidazole is the treatment of choice for giardia spp. infections in small rodents. myobia musculi, myocoptes musculinis, radfordia affi nis, and psorergates simplex are fur mites that can cause severe selfmutilation and hair loss in mice. polyplax serrata (house mouse louse) can cause anemia, pruritus, dermatitis, and death in severely infested mice. diagnosis of ectoparasites in mice is similar to that in other companion animal species (e.g., skin scraping, examining hair samples for nits). treatment of ectoparasites can be accomplished with ivermectin and a topical miticide. as with other rodent species, there has been an increasing ectoparasite resistance to ivermectin treatment. because most rodent diets are manufactured in the form of pellets or small biscuits that provide all recommended nutrients, there are few nutritional disease problems diagnosed in pet mice. if mice are fed an all-seed diet, there could be nutritionally related consequences. a lack of vitamin a could result in roughened hair coat or skin lesions. a breakdown of the protective epithelial lining of the gastrointestinal and/or respiratory tract could predispose the mouse to secondary bacterial infections affecting those body systems. nutritional defi ciencies may also result in poor reproductive activity. it is important to provide an adequate supply of fresh water and appropriate rodent food on a daily basis. following these basic nutritional instructions should provide a nutritional basis for mice to live healthy lives. the average life span of a mouse is years old. this short life span and aging process increase the prevalence of tumors in these small rodents. if an animal is over months of age, neoplastic disease should be considered as a differential diagnosis, especially if an asymmetrical mass is present. the most common tumors diagnosed in mice are mammary adenocarcinomas and fi broadenomas. other tumor types are identifi ed in mice, and if possible, the suspect tissue should be completely excised. representative tissue sections should be submitted for histopathological evaluation. although there are treatment options available for mice that have been diagnosed with neoplasia (e.g., radiation, chemotherapy), its small size does not allow it to withstand the stress and negative side effects of these healing modalities. in mice, the recommended treatment for neoplasia is removal of the affected tissue, if possible, to increase its quality of life. the most common disease in mice colonies is barbering. barbering is defi ned as the dominant mouse nibbling off the whiskers and hair around the face of the subservient cagemates. the hair clipping is very close to the skin without causing skin lesions. this behavior is noted often among female mice, whereas male mice are more likely to fi ght. mice may injure themselves on cage furniture or poorly designed cages and sustain abrasions on the face or nose. individual animals may show stereotypic behavior that results in abrasions and/or alopecia. enrichment furniture and toys may help alleviate this initial cause of dermatologic abnormality. the size of mice contributes to the diffi culty in properly administering therapeutic medications. the scruff of the neck or caudal fl ank are the subcutaneous sites that may be utilized to administer medication, fl uids, or both. the semitendinosus, triceps, and epaxial muscles are commonly used for intramuscular injections, whereas intraperitoneal injections are used in extremely small animal species, including mice. intraosseous catheters are much easier to place in mice than are intravenous catheters. the tibial plateau and the greater trochanter are the sites of choice for intraosseous catheter placement in mice. although there are many methods that may be used to administer medication in small mammals, oral treatment using a dropper or tuberculin syringe is the easiest to administer and the least stressful for the patient. medication can be added to the food or water, but often the patient is anorexic or the medicated food or water is not palatable, in which case, the patient does not receive the treatment. although a relatively diffi cult procedure in mice, gastric gavage can be performed using a bulbed stainless steel feeding needle or fl exible red rubber feeding tube. the length of the tube is premeasured, externally from the nares to the last rib, and marked. a water-based lubricant is applied to the feeding tube before it is inserted through the intradermal space toward the back of the oral cavity. the tube should advance without diffi culty until the premeasured mark is reached. restraint of mice for this procedure is accomplished by pinching the dorsal neck skin fold and holding the body with the remaining free fi ngers. both oral medication and nutritional supplementation can be administered through an oral gastric feeding tube. medication dosages for mice can be found in a number of formularies. before administration of any therapeutic agent, the dosage should be checked twice and calculations thoroughly evaluated to make sure they are correct. because mice are very small patients, any overdose of medication can have adverse consequences on the patient's recovery. inducing and maintaining mouse patients under anesthesia can be very challenging. with the widespread use of isofl urane anesthesia in small animal practices, the previous problems associated with methoxyfl urane and halothane anesthetic agents has been overcome. there are still concerns and differences in anesthetic protocol because of these animals' small size, but in general, isofl urane is a safe agent when used on a relatively healthy surgical candidate. although there are doses of injectable agents for sedation, the small size of the mouse makes it diffi cult to respond to anesthetic crises that may appear during a diagnostic or surgical procedure. therefore, inhalant anesthetic agents (e.g., isofl urane, sevofl urane) are recommended for anesthetizing mouse patients. an induction chamber is used to induce the patients, and then they are maintained under gas anesthesia using a face mask. it may be possible to intubate a larger rodent, but it is very diffi cult in mice; under most conditions, a modifi ed syringe case face mask is used. to guard against aspiration of stomach contents in a patient that is not intubated, the veterinary surgeon should place the head and neck in a position slightly higher than the body. box - provides basic guidelines for proper techniques used in surgical preparation and surgical assistance in small mammals, including mice. premedication and sedation doses for mice and rats are listed in surgical procedures that are performed on mouse patients require similar techniques to those used with larger animals. veterinarians should follow the same protocol but remember that the surgical fi eld is smaller and patient manipulation more delicate with patients that weigh less than g. zoonotic diseases associated with mice maintained as pets are rare. possibly the most commonly reported zoonotic condition associated with mice is an allergic reaction to their dander and urine. as with all animals, it is important that a handler wash his or her hands after interacting with a mouse. salmonella spp. may colonize the intestinal tract of small mammals, including mice. contact with the feces or anus of a mouse may expose the handler to this enteric pathogen. washing hands after handling a mouse will help prevent exposure to possible pathogenic organisms a mouse may carry. one can monitor the individual mouse and mice colonies for subclinical carriers of zoonotic organisms by culturing the terminal intestinal tract. mice are carriers of two potentially deadly viruses: the hantaan disease virus and an arenavirus that causes lymphocytic choriomeningitis (lcm) in humans. mice that are propagated for sale in the pet trade are rarely if ever exposed to the hantaan disease virus. lymphocytic choriomeningitisinfected mice generally show no clinical signs, although weight loss, photophobia, tremors, and convulsions may occur. humans are exposed to lcm through contaminated feces, urine, or a bite wound. disease signs in humans infected with lcm are fl u-like (e.g., malaise, headaches, fever, myalgia, arthritis). fatal aseptic meningitis or meningoencephalitis in humans infected with lcm is rare. hymenolepis spp. are cestodes found in mice that can infect humans if contaminated feces are ingested. rarely identifi ed in pet mice, giardia spp. are protozoan parasites that can cause severe gastroenteritis in humans. because feces have to be ingested for someone to be exposed, it is more common for children to become infected with zoonotic intestinal parasites than for adults. using proper sanitary practices after handling mice helps reduce exposure to zoonotic intestinal parasites and other potentially transmissible diseases. although there are diseases listed in this zoonotic section that can be transmitted from mice to humans, the occurrence of this happening is extremely rare when commercially bred mice are purchased at reputable pet stores. rats, like mice, are common laboratory animals and are propagated for commercial sale in the pet trade and for use as reptile food. the life expectancy of a rat is to years. unlike mice, rats have excellent pet characteristics that include a charming personable disposition and extreme intelligence (figure - ). the common rat species maintained as a companion animal is rattus norvegicus, with the white rat and hooded rat being the most common variations. although rats have an excellent temperament for companionship, they can infl ict a serious bite if provoked. also, as with other animal species, humans can be allergic to their hair, skin dander, and urine and salivary proteins. rats are not as territorial as other rodent species and are very social. the integument of rats is commonly associated with a number of presenting symptoms of disease. as with mice, a rat's hair coat that has not been adequately maintained is often the fi rst clinical sign associated with disease. rats are fastidious with their grooming and, when healthy, maintain a very tidy hair coat. common problems associated with a hair coat that is not maintained include general illness, parasitism (internal and external), aggression by cagemates as a result of psychological trauma, and infectious dermatitis. staphylococcus aureus infections initiated by self-trauma due to a fur mite infestation will manifest as ulcerative dermatitis. the most common tumor in rats is a mammary-associated fi broadenoma. fibroadenomas rarely metastasize but are locally invasive and can grow to a large size. a coronavirus is the etiologic agent behind sialodacryoadenitis which results in infl ammation and swelling of the cervical salivary glands. the most common disease presentations in rats involve the respiratory system. the three major pathogens that infect rat respiratory systems are mycoplasma pulmonis, streptococcus pneumoniae, and corynebacterium kutcheri. respiratory disease in rats often produces "red tears" and a red nasal discharge. this red staining around the eyes and nose is not blood but porphyrins produced by an irritated harderian gland located behind each eye. the red staining at the nasal opening is due to the drainage of the porphyrins via the nasolacrimal duct. renal disease may present in older males in the form of chronic progressive nephrosis. on necropsy, kidneys in affected animals are enlarged, appear pale, and have a pitted mottled surface. rats are continuous, polyestrous rodents that should be bred in polygamous or monogamous systems. when breeding rats that are housed in a polygamous ratio, it is recommended to have one male with two to six females. females are removed from a polygamous cage on day of gestation, whereas the monogamous pair is maintained together with the young until weaning. baseline rat physical information is listed in box - . rats, like mice, can chew out of enclosures; therefore, it is important that the housing be "rat proof" regarding the animal's ability to chew through the substance and escape into the house. rats are maintained in environments that are similar to those of other small rodents, but rats do not produce the quantities of odiferous urine associated with other rodents. for rats, large wire cages with easy-to-remove plastic bottoms are the optimal enclosure (figure - ) . the recommended housing unit size for rats is - ″ × - ″ × - ″ (length × width × height) for each adult rat, with females and young requiring to times the space listed. rats like to climb ramps and rope for exercise, so a cage that is taller than the listed requirements would be better suited for most singly housed animals. the major considerations for selecting an enclosure for rats, as with mice, are that it is resistant to their escape and easy to clean. an open screen top on the enclosure is recommended for proper ventilation. available rodent cages that fi t this criterion are wire or metal mesh, plastic, and plexiglas. if the plastic tube housing systems are used, there should be routine cleaning of the sections using hot water and a mild detergent. an owner must always monitor the cage for possible escape avenues, especially if the animal adult male - g adult female - g birth weight - g rectal body temperature °- ° f normal heart rate - beats/min normal respiratory rate - breaths/min daily food consumption g/ g body weight daily water consumption - ml/ g body weight has a predilection for modifying the cage opening through chewing. it is very important to provide cage toys and exercise opportunities for rats; therefore, the enclosure should be large enough to house not only food and water containers but also cage toys and an exercise wheel. although not a cage toy, a hide box, in some form, should also be included, for the psychological well-being of the animal. this hide box can be a manufactured one or a small cardboard container that has one end cut out. rats like to hide and also sleep during the day. by providing this piece of cage furniture or deep enough substrate for the rat to burrow, it will be able to sleep undisturbed during the daylight hours. to prevent the buildup of urine and fecal material in the cage, commercially available paper rodent bedding or hardwood shavings should be used as a cage substrate (figure - ) . although softwood (pine) and cedar shavings are available, the volatile oils that radiate from these substrates are irritating to small animals, especially because they are in very close contact with the material. these irritating compounds can cause dermal and respiratory infl ammation, often leading to secondary bacterial infections. cardboard tissue tubes can be placed in a rat enclosure for chewing and play. clothes items are not recommended in cages with animals that chew. rats will shred the clothing items, exposing thread and elastic material that can become entangled around a rat's extremity, leading to necrosis distal to the stricture. it is recommended to change the substrate within the enclosure at least twice a week and more often if there is a problem with odor or excessive excreta. temperature, humidity, and lighting within the enclosure should follow what is commonly maintained with the ambient conditions within the house. for animals housed outside or in an outbuilding, the temperature range should be ° f to ° f. the preferred light cycle for rats is hours of light alternating with hours of darkness. there have been a number of studies investigating the recommended nutritional requirements of rats. these studies have taken place because of the fact that rats are one of the most commonly used laboratory animals when investigating human disease processes. the benefi t of these dietary studies is the availability of commercially produced diets that provide the recommended daily nutritional requirements of the rat. the problem that most owners face is the multitude of dietary products available for rats, and owners often buy seed-based diets. like mice, rats will happily eat seed although seed-based diets are lacking in a number of the nutritional requirements needed to maintain long-term health. commercial rodent biscuits or pellets with % to % protein are the recommended diets for rats. the commercial rodent biscuits or pellets are the only food rats need to obtain their required nutrients. again, seed-based diets are not recommended, nor is a signifi cant supplementation of fruits, nuts, vegetables, cheese, or other human foods (e.g., peanut butter). if a treat is to be given, yogurt or dried fruit treats manufactured specifi cally for rodents and/or rats should be provided to times a week (figure - ) . for younger rats, less than weeks old, softer pellets are needed because babies start eating pellets and drinking water at to weeks of age. a sipper bottle, placed on the outside of the cage is easy to maintain and does not take up space within the enclosure. if the cage is plastic or plexiglas, modifi cations will be needed to attach the sipper bottle on the outside of the cage. most sipper bottles come with attachment hardware to attach to wire cages. fresh bottled water is recommended for rats, although chlorinated tap water can also be used. the water should be checked on a daily basis and clean water supplied at least every days, if not every day. quarantining an animal is important for the pet owner who is introducing a new animal into a setting in which there is an established group. as with other animals a -day quarantine period is recommended, along with a physical examination and fecal parasite check. unfortunately, there is no time period that will screen % against potential infectious agents, and with rats, time is critical because their life span and repro ductive time frame is so short. quarantining the introduced animals in conditions similar to those in which they will be maintained permanently, reduction of handling, and providing adequate food and water will make for a smooth transition. it is imperative that the owner screen the new animal for diseases and watch for any signs of illness. all rats that are captive, either as pets or in a breeding setup, should have no access to insects, wild rodents, or other animals. the most common sign of clinical illness is an unkempt hair coat. the lack of grooming is most often related to the animal feeling depressed or sick and thereby not having the energy to perform routine behaviors. if a number of animals are being quarantined, sacrifi cing an apparently diseased animal is the most effi cient way to determine a rapid defi nitive diagnosis. if it is a single or an expensive animal, routine diagnostic testing will be required. to maintain oversight of a breeding animal's health and reduce the exposure of young animals to infectious disease and parasites, routine screening of representative animals within the colony is recommended. in very large colonies, special caging (e.g., fi lter), food, and water may be necessary to maintain disease control. before examining a rat, the examination table should be disinfected with either a dilute sodium hypochlorite or chlorhexidine solution. the examiner should always wash his or her hands and, if necessary, put on gloves before the capture and restraint of the patient. the diffi culty with using latex gloves when examining a rat is the small size of the patient and its ability to twist and turn. some rat patients can be restrained only if the examiner is not wearing gloves. if gloves are not worn, the examiner must thoroughly wash his or her hands, as with all cases, after the examination is completed. to restrain a rat, the animal should be picked up with one hand being placed over the back and rib cage, restraining the head with the thumb and forefi nger directly behind the jaws. the other hand is grasping the tail, stabilizing the animal (figure - ) . the skin on the dorsal cervical region may also be used, as with other rodents, to pick up the rat. a plastic sandwich bag can be modifi ed as a restraint device for rats and small rodents. the rat's head is placed toward a bottom corner and the sides folded over the animal's back to limit movement. if one is unable to adequately restrain a rat while the animal is conscious, an inhalant anesthetic may be used for sedation purposes. the animal should be placed in a small induction chamber and isofl urane gas permeated into the closed space (figure - ) . once the animal has stopped moving, the enclosure top is removed and a nose cone placed on the anesthesia tube and placed on the patient's face. a syringe case can be modifi ed as a nose cone for small rodents. after the physical examination is completed, the nose cone is removed and the patient is allowed to recover, breathing oxygen from the anesthesia unit. injectable agents are another form of sedation used with rats for examination and diagnostic sample collection purposes (see table - ). a thorough assessment of the patient is necessary before using injectable drugs for sedation and anesthesia because of the inability of veterinarians or technicians to rapidly manipulate the effects of these treatments. obtaining a detailed history for the rat patient is very important. it is imperative to get as much information from the owner as possible as it relates to the patient's presenting problem, even if it is routine health examination. typical background information required for the rat patient includes how long the owner has had the animal, where it was acquired, how often it is handled, and the appearance of the feces and urine. husbandry questions should focus on where the animal is housed, whether it is allowed to roam unobserved, where its cage is located; type, size, and material of the cage; cage substrate, furniture, and toys; how often the cage is cleaned and what disinfectant is used. when investigating the diet it is important to ask not only if pellets are fed and in what quantity, but also what the animal is eating and what its primary diet is. supplemental offerings and frequency of feeding are very important data for the case work-up. the technician should fi nd out about the water supply, how often the water is changed, and how much the animal drinks on a daily basis. because there are transmissible diseases among animals, the fi nal questions should center on other pets in the household, if new animals have been added to the family, or if the animals are housed together. a description of any previous problems and a complete chronological description of the presenting problem are needed to complete the history form. before restraining the animal, an observation should be made on its attitude, activity, and posture. the next step is to weigh the rat in a basket on a digital gram scale. if possible, temperature, respiration, and pulse should be measured and any abnormalities in rate and/or character noted. the physical examination starts at the head where the veterinarian should look for any abnormalities. eyes, ears, and nares are observed for signs of discharge or infl ammation. the oral cavity is diffi cult to examine in rats because of the small opening and tendency for the buccal mucosa to encroach toward the middle of the mouth. rat incisors have a tendency to overgrow, so examination of the front teeth for normal occlusion and length is essential. a small speculum (e.g., modifi ed paper clip) or an otoscope may be used to examine the oral cavity and cheek teeth. mucous membranes help determine hydration status using capillary refi ll time and moisture. body condition should be examined and abdominal palpation performed. lymph nodes and limbs are palpated before checking the nails and plantar surface of each foot. the patient should have a normal posture, be aware of its surroundings, and move properly. any problems should be noted in the record. finally, a dermatologic exam considers hair coat quality, alopecia, external parasites, or any skin abnormalities. all abnormal fi ndings are written in the record for case review, differential diagnoses determination, diagnostic testing, and treatment considerations. respiratory disease is often the primary complaint offered by owners when presenting a sick rat to the veterinary hospital. bacterial and viral pathogens can infect rats and are easily spread within breeding colonies because most of the organisms are transmitted by subclinical carriers. ocular and nasal discharge is often observed in combination with dark red pigment. this pigment may be mistaken for blood by owners but actually is porphyrins produced by the harderian glands located behind the eyes. the irritation of the harderian glands causes the excessive production of porphyrins, which are secreted around the eye and down the nasolacrimal duct to the nose. rats have incisors that grow continuously. the continuous growth is not a problem for animals with normal occlusion. if the incisors do not line up, then there is a condition that leads to overgrowth of these teeth. the upper incisors will curve around, and if they are not cut, they will lodge in the dorsal aspect of the oral cavity. the ventral incisors will fl are out from the oral cavity. a common tumor found in older intact female rats is the mammary tumor. mammary tumors can be large and extensive. rat mammary tissue extends, on their ventrum, from the inguinal area to the thoracic inlet and up to their dorsal lateral body wall. the most common subcutaneous mammary tumor in the rat is a fi broadenoma (figure - ) . removing the ovaries at an early age reduces the incidence of the hormonally infl uenced tumor later in a female rat's life. swelling of the cervical salivary gland is caused by a virus. the etiologic agent of this condition is a coronavirus, sialodacryoadenitis virus, resulting in upper respiratory infl ammation and the previously mentioned swelling of the cervical salivary and lacrimal gland. there is no treatment for this highly contagious viral disease. external and internal parasites may affect rats in the form of a rough hair coat or dermal lesions. rats that are pruritic may cause abrasions or scratches that become infected with staphylococcus aureus bacteria, leading to secondary ulcerative dermatitis. routine parasite examinations are necessary so that the veterinarian can identify parasitic infestations and administer treatment. rats can also present with neurologic, gastrointestinal, and urinary disease signs. a detailed history and examination will help direct the clinician toward a differential diagnosis list in which the appropriate diagnostic tests can be submitted to obtain a defi nitive diagnosis. as with other pocket pets, blood collection from rats can be quite diffi cult. approximately . to . ml/ g body weight can be safely removed from a nonanemic healthy rat. a rat patient should always be anesthetized for blood collection procedures, to reduce patient stress and movement. usually inducing the animals in a closed chamber and maintaining the patients in a mask will allow the technician plenty of time to collect the blood sample. using a warmed ventral tail vein, . ml can be collected through a -gauge butterfl y needle. to collect from the tail vein, warm the distal two-thirds of the ventral aspect of the tail in a container of warm water for approximately minutes. the tail vein can be observed more clearly in the distal aspect of the tail. a -to -gauge butterfl y needle is used, and free fl owing blood can be collected directly into the microtainer tubes. a tape bandage should be placed on the injection site to aid in hemostasis. the easiest way to collect blood from many small mammals, including rats, is from the anterior vena cava. with the rat under general anesthesia, a -gauge, -inch needle attached to a -ml syringe is directed toward the umbilicus from the right side of the manubrium. box - describes the technique used for retroorbital bleeding in rats and is not recommended for companion animals. the retroorbital bleeding technique is commonly used in laboratory animal settings and is easily performed by an experienced veterinarian or veterinary technician. as with smaller rodents, the saphenous vein or lateral vein of the tarsus may be used for multiple blood collections without the use of an anesthetic. the patient must be properly immobilized in a restraint tube ( -ml syringe) with the leg extended and the skin on the medial aspect of the thigh held tightly between the handler's thumb and forefi nger. the taut skin on the lateral aspect of the thigh allows exposure of the saphenous vein. a -gauge needle is used to puncture the vein, and blood is collected in a microhematocrit tube as it fl ows from the vessel. blood samples obtained from nail and ear clips are not considered appropriate diagnostic samples. cardiac puncture is recommended only in terminal cases and when the animal is maintained under general anesthesia because of possible complications involving the lungs and heart vessels. the collection of samples for diagnostic testing in rats is similar to that in other rodents. the reference ranges for complete blood counts and serum biochemistry panels are listed in boxes - and - . marrow samples may be obtained from the ilium, tibia, sternum, femur, or the bones of the proximal one third of the tail. preparation of the samples is consistent with that of other mammalian patients. standard rodent cages can be used without substrate to collect urine and feces when the patient is hospitalized. by placing a rodent in a ziplock bag, urination frequently occurs and the sample can be collected. in a similar manner the urine in a rat can be collected over a disinfected stainless steel examination table. the urine is voided while the animal is restrained and can be collected in a capillary tube for evaluation on urine reagent strips. urinalysis reference values for rats are listed in box - . under most practice settings, the size of a rat patient limits imaging capabilities to radiographs. restraint is essential for quality diagnostic images. to obtain adequate restraint and prevent movement of the patient during radiographic evaluation, sedation is required. as with all diagnostic procedures involving avian and exotic animals, an evaluation of the patient is required to determine its ability to withstand the stress associated with the assessment. if it is determined that the patient cannot withstand sedation, then it should be stabilized until it is deemed healthy enough for diagnostic imaging evaluation. a rat can be sedated in an induction chamber following a technique similar to that described for mice. because rats are larger than mice, they are easier to intubate. using a special otoscope adapter, exposure is enhanced for endotracheal tube placement. high-speed, -ma machines with fi ne or detail-intensifying screens should be adequate for most small exotic mammal images. dental x-ray units that can focus at short distances may be advantageous for both isolation of focal anatomy and full body radiographs in extremely small patients, including rats. for extreme detail in small exotic mammal medicine, using a traditional radiography unit, the min r mammography system (eastman kodak) with min r singleintensifying screen cassettes in conjunction with min r singleemulsion fi lm has been recommended. digital radiography is bringing a new dimension to radiograph evaluations and detail. the secret to success with either traditional or digital radiographic imaging is to restrict patient movement during exposure. two views, ventrodorsal and lateral, are recommend for rat patients. if there are rotational problems with the rat patient, a dorsoventral view may provide better imaging quality. the patient should be taped to the cassette or positioning board with white cloth tape or masking tape. taping the rat to the cassette or positioning board will aid in image consistency with that particular patient and among similar animals. in the lateral views, the dependent legs should be positioned cranial to the contralateral limbs. again, as with mice, whole body radiographs are commonly obtained for rat patients during the radiographic evaluation. normal radiographic appearance of the rat patient is described in box - . for the rat patient in which microbiological testing is recommended, standard collection techniques used for other animals are appropriate. with the most common respiratory conditions in rats being viral and bacterial organisms, it is important to try and aid the diagnostic laboratory in organism isolation through listing the potential pathogens. as always, if in doubt on collecting, preserving, and/or shipping a microbiological sample, the diagnostic laboratory should be contacted for full instructions regarding rat submissions. parasitology routine fecal parasite evaluations should be performed on small rodents when they are brought to the veterinary clinic for a health examination or an abnormal stool. common protozoal organisms can be detected using a direct fecal examination. an anal tape test is useful for identifying syphacia spp. in rats. diagnosis of ectoparasites in small rodents is similar to that in other species listed in this text. a skin scraping of the affected area or a pelage tape test (to identify radfordia spp.) will usually yield the parasite. diagnosis of intestinal parasites can be made by fi nding individual eggs in a fecal fl otation of a macerated fecal pellet and in the lumen of the small intestine during necropsy. staphylococcus aureus is the most common cause of ulcerative dermatitis in rats. this organism is ubiquitous in most rat colonies and survives on the animals' skin. unless there is a break in the skin, there is no infection or disease caused by the organism. in cases where there is a fur mite (radfordia ensifera) infestation, rats become pruritic, developing lesions that seed the s. aureus organism. the ulcerative dermatitis can be treated with an appropriate antibiotic through both systemic and topical application, cleaning the affected areas, and clipping the toe nails of the rear feet. mycoplasma pulmonis, streptococcus pneumoniae, corynebacterium kutscheri, sendai virus, and cilia-associated respiratory bacillus have been isolated and identifi ed as infectious agents causing rat respiratory disease. with the respiratory pathogens, clinical signs can vary from mild dyspnea to severe pneumonia and death. rats with mycoplasmosis are treated in much the same way as infected mice. sialodacryoadenitis virus is a highly contagious disease that causes rhinitis and infl ammation of the cervical salivary and lacrimal glands. there is no treatment for this viral disease, and supportive care is recommended for the affected patient. there are other infectious diseases that can infect rats, as evidenced by the available serologic tests (e.g., kilham's rat virus, pneumonia virus of mice, rat parvovirus, rat coronavirus, toolan's h- virus, theiler's murine encephalomyelitis virus, mouse adenovirus, reovirus ). these infectious diseases are diagnosed less commonly than those previously identifi ed. even so, veterinarians should be aware of the infectious disease organisms to which rat patients may be exposed and aware that tests are available for antemortem diagnosis. specifi c intestinal parasites identifi ed in rats include the dwarf tapeworm (hymenolepis nana), pinworms (syphacia muris), nematodes (specifi cally trichosomoides crassicauda), and giardia muris. diagnosis of intestinal parasites is important for the implementation of the appropriate treatment plan. pinworms are diagnosed through the anal tape test, protozoan parasites (e.g., giardia muris) through a direct fecal exam, and cestodes and nematodes from fecal fl otation on macerated fecal pellets. once the parasite has been identifi ed, proper treatment can be initiated and environmental recommendations applied to prevent exposure of other animals and reexposure of the patient being treated. rats do not appear as susceptible to ectoparasites as mice, but the following have been identifi ed: ornithonyssus bacoti (tropical rat mite), radfordia ensifera (rat fur mite), demodex nanus (demodex), polyplax spinulosa (the spined rat louse), and fl eas. diagnosis and treatments are similar to those recommended for mice although the dosages and duration differ. treatment of ectoparasites can be accomplished with ivermectin and a topical miticide. as with other rodent species, there has been an increasing ectoparasite resistance to ivermectin treatment. because most rodent diets are manufactured in the form of pellets or small biscuits that provide all recommended nutrients, there are few nutritional disease problems diagnosed in pet rats. it is also important to provide an adequate supply of fresh water and appropriate rodent food on a daily basis. if rats are fed an all-seed diet, there could be nutrition-related consequences. a lack of vitamin a could result in roughened hair coat or skin lesions. a breakdown of the protective epithelial lining of the gastrointestinal and/or respiratory tract could predispose the rat to secondary bacterial infections affecting those body systems. nutritional defi ciencies may also result in poor reproductive activity. vitamins b complex, c, and e plus selenium are recommended for treatment of female rats diagnosed with pregnancy toxemia. rats, if fed a high quantity of human food, will develop dental plaque on the cheek teeth. proper diet, primarily rodent biscuits or pellets, will reduce the formation and buildup of dental plaque. if left unattended, the excess plaque may lead to gum disease and dental caries. if caries develop within cheek teeth, the recommended treatment is removal of the affected teeth. rats are susceptible to tumors, most likely because of their short life span and physiologic predisposition. the most common subcutaneous tumor in rats is the fi broadenoma of the mammary tissue. the mammary tumors can reach very large sizes and affect both males and females. to reduce the incidence of fi broadenomas, ovariohysterectomies are advocated at an early age in female animals. whereas mammary tumors in mice are almost always malignant, rat mammary tumors are usually localized and respond to surgical resection. rats have continuously growing incisors. under normal anatomic conditions, the incisors maintain their length by grinding on their occlusive surface. if the position of the incisors is compromised, malocclusion will occur, causing overgrowth of their front teeth. the teeth, if left untrimmed, can cause trauma to the dorsal surface of the oral cavity (upper incisors) and the lower teeth extending outside of the mouth. in severe cases the teeth may adversely affect the animal's ability to eat. the teeth can be trimmed with a high-speed motor tool (e.g., dremel ® ) with a cutting disk attachment. a common disease in older male rats is chronic progressive nephrosis; the disease can also affect females. the protein level in the urine commonly exceeds mg/day and progressively increases as the animal ages. the pathophysiology of chronic progressive nephrosis involves glomerulosclerosis with tubulointerstitial disease primarily affecting the convoluted proximal tubule. the progression of the disease process may be slowed through a low-protein diet and anabolic steroid therapy. therapeutics fluid therapy can be given to rats through intramuscular, subcutaneous, intraperitoneal, and intravenous routes. using a -gauge needle, a volume of up to . ml of fl uid can be injected into the quadriceps muscle. up to ml of fl uid can be injected under the loose skin covering the neck or lateral body wall. intraperitoneal fl uids should be administered while the rat is properly restrained to restrict movement. with one of the rat's hindlimbs extended, a -gauge needle is inserted into the center of the caudal quadrant of the abdomen, following the line of the extended leg. up to ml of fl uids can be given via the intraperitoneal route. the most diffi cult method of providing fl uids is intravenously to the lateral tail vein. to access the tail vein, warming the tail to dilate the vessel in warm water for approximately minutes is required to aid in visualization. the older the animal is, the more diffi cult it is to see the vessel through the tail skin. although there are many methods that may be used to administer medication in small mammals, oral treatment with a dropper or tuberculin syringe is the easiest for the veterinarian and least stressful to the patient. medication can be added to the food or water, but often the patient is anorexic or the medicated food/water is not palatable, in which case, the patient does not receive treatment. both oral medication and nutritional supplementation can be administered through an oral gastric feeding tube to rat patients. the techniques described earlier for mice can also be applied to rats. medication dosages for rats can be found in a number of formularies. before administration of any therapeutic agent, the dosage should be checked twice and calculations thoroughly evaluated to make sure they are correct. the same surgical and anesthetic techniques described for mice can be used for rats (see box - ). dosages for rat injectable anesthetic and sedative agents and analgesic treatment can be found in tables - and - . rats are larger than mice and can be intubated using a modifi ed otoscope head (figure - ) . when using inhalant anesthesia, an intubated animal can be ventilated and there is less risk of aspiration from fl uid that enters the oral cavity. surgical procedures that are performed on rat patients use similar techniques utilized on larger animals. veterinary surgeons should follow the same protocols but remember that the surgical fi eld is small and patient manipulation is delicate. the most common surgical procedures performed on rats are castration, ovariohysterectomy, and tumor (especially mammary tumor) removal. occurrences of humans becoming infected with zoonotic diseases associated with pet rats are uncommon. as with mice, an basic anatomy, physiology, husbandry, and clinical techniques small rodents: exotic companion medicine handbook for veterinarians box - normal radiographic appearance of the rat from johnson-delaney ca, harrison lr: small rodents: exotic companion medicine handbook for veterinarians basic anatomy, physiology, husbandry, and clinical techniques small rodents: exotic companion medicine handbook for veterinarians tumours of the mammary gland disease problems of small rodents a technician's guide to exotic animal care parasites of ferrets, rabbits, and rodents zoonotic diseases small mammal dentistry allergic reaction to rat dander and urine may be the most commonly reported zoonotic condition. as with all animals it is important that a handler always wash his or her hands after interacting with a rat. salmonella spp. may colonize the intestinal tract of rats. there has been a human case of chorioamnionitis in which corynebacterium kutscheri has been isolated. this grampositive bacillus is routinely isolated from the nasopharynx of rats and therefore can be considered a zoonotic agent.streptobacillus moniliformis and spirillum minus are organisms that initiate the disease called rat-bite fever. a rat bite is required to seed the bacteria in a human, and after a -to day incubation cycle, the human begins to show clinical signs associated with the disease (e.g., relapsing fever, chills, vomiting, myalgia, regional lymphadenopathy). rats appear to be one of the primary rodent carriers of streptococcus pneumoniae. the rodent develops respiratory disease through which the organism is shed and humans are exposed. infected humans have respiratory and meningeal disease signs associated with s. pneumoniae illness. it should be mentioned that the rodent fl ea (xenopsylla cheopis) transmits the plague organism yersinia pestis. it is wild rodents in which the plague organism is of most concern, but the disease is found worldwide, especially in the american southwest. this is a very good reason why rats under the care of humans should have no access to wild animals.leptospira interrogans is transmitted from the infected urine of rats. again, restricting contact with wild rodents will reduce the risk of pet exposure. humans infected with l. interrogans have chills and fever and often develop septicemic conditions that can lead to organ dysfunction. hymenolepis sp. is a cestode found in rats, which can infect humans if contaminated feces are ingested. giardia sp. is a protozoan parasite, rarely identifi ed in pet rats, that can cause severe gastroenteritis in humans. because feces have to be ingested for exposure, it is more common for children to become infected with zoonotic intestinal parasites than for adults. proper sanitary practices after handling rats will help reduce humans' exposure to zoonotic intestinal parasites and other potentially transmissible diseases. all potential rat owners and current rat owners should know that the occurrence of zoonotic disease transmission is extremely rare when commercially bred rats are purchased from reputable pet stores. key: cord- - jgusov authors: dignard, caroline; leibler, jessica h. title: recent research on occupational animal exposures and health risks: a narrative review date: - - journal: curr environ health rep doi: . /s - - - sha: doc_id: cord_uid: jgusov purpose of review: in the last year, an increasing number of studies have reported on methicillin-resistant staphylococcus aureus (mrsa) transmission in africa and asia and in migrant workers. we reviewed original research on occupational health and safety of animal workers published from january , , through june , , with a targeted focus on infectious disease studies published in these populations. recent findings: studies focused on occupational exposures to infectious agents, dust and allergens, pesticides, and occupational injury. research on zoonotic mrsa used whole genome–sequencing technologies to evaluate transmission in africa and asia. swine worker exposure to porcine coronavirus and emerging influenza a viruses was documented in china. s rna amplicon sequencing identified distinct microbiota compositions in households with active animal farmers. multiple bioaerosol exposures were assessed for industrial dairy workers. occupational injury studies highlighted the struggles of latino animal workers in the usa. summary: these studies highlighted the global expansion of zoonotic antibiotic resistance and identified novel occupational zoonoses of concern. the integration of microbiome assessment and compound mixtures into the evaluation of dust and endotoxin exposures for animal workers marks a new direction for this work. occupational exposure to animals is associated with a myriad of health and safety risks, including zoonotic infections, occupational injury, respiratory disease, and cancer [ ] [ ] [ ] [ ] . in the usa, food animal workers have elevated workplace mortality and injury rates compared with workers in other industries, highlighting the occupational risks involved in the profession [ ] . in the last years, research on zoonotic infection risk has dominated the occupational health literature on the animal workforce, highlighting in particular exposure risk to drugresistant bacteria and influenza viruses and subsequent transmission from workers to the general public [ ] [ ] [ ] . in the last years, industrialization and corporate consolidation have characterized the food animal production industry, first in the usa and europe and then globally [ , ] . these trends have fundamentally altered occupational exposures for the food animal workforce, by increasing and intensifying specific occupational exposures that impart health risks [ ] . for workers, the intensity of animal exposures has increased, as industrial farms can hold tens of thousands of animals on site. the dramatic increase in the number of animals housed together in confinement contributes to intensified worker exposure to animals and animal products, including allergens and fecal materials [ , ] . notably, the introduction of antibiotics into animal production-in an effort to facilitate the higher carrying capacity of industrial farms-has resulted in worker exposures to antibiotic-resistant bacterial infections [ ] . since , research on zoonotic methicillin-resistant staphylococcus aureus (mrsa), particularly the livestock-associated mrsa strains st and cc , have identified important public health concerns stemming from the misuse and overuse of these antibiotics in agriculture [ ] [ ] [ ] . likewise, the h n epidemic in poultry in asia in the mid- s and the h n swine flu human pandemic, highlight the role of industrial systems in the ecology of pandemic influenza [ ] [ ] [ ] . the emergence and re-emergence of zoonotic pathogens with potential to infect humans remains a critical public health issue, and animal workers are at the front lines [ ] [ ] [ ] . the demographics of this workforce have also changed significantly in recent decades, with latino and immigrant workforce currently dominating the worker population in the usa. this change has resulted in additional challenges for the workforce, including language barriers, immigration status concerns, stagnant and falling wages, and other socioeconomic and political stressors [ ] . the relationship between these stressors and occupational injury and mental health has been documented in recent years [ ] [ ] [ ] [ ] . many animal workers experience occupationally induced respiratory disease, including allergies, asthma, and rhinitis [ , ] . high levels of inhalable dust and endotoxins are considered the primary exposures of concern in regard to respiratory disease; however evaluating these often complex mixtures-including animal products, dust, pathogens, and chemicals-is typically limited to single-compound analyses. as a result, much remains unknown about the etiology of occupational respiratory disease among animal workers. across agricultural industries, the use of pesticides is associated with a variety of health risks, including reproductive, dermatological, and neurological problems as well as cancer [ ] [ ] [ ] [ ] . pesticide use is common among animal facilities, particularly those that engage in both crop and livestock production, yet pesticide exposures have received limited scrutiny to date in research on animal workers. on both industrial and small-scale animal farms, chemical disinfectants are used to prevent transmission of infectious agents and may result in health concerns for workers [ , ] . while the biosecurity literature has promoted the use of disinfectants to prevent disease transmission, health risks associated with worker exposure to these compounds are largely unstudied. in this manuscript, we review occupational health studies published in the last months in the peer-reviewed literature focused on the health and safety of animal workers. our intention was to highlight important findings and new directions for this research area. we searched pubmed, web of science, and google scholar for terms and keywords relating to occupational health and animal exposure, including combinations of the following worker and health-specific terms: "food animal worker", "animal worker", "industrial animal worker", "animal farmer", "occupational injury", "occupational health", "health and safety", and "occupational safety". a date range of january , , through june , , was included so as to maximize identification and in-depth discussion of recent research. a total of distinct manuscripts were identified upon initial search. following review by two researchers (c.d., j.h.l.), these papers were reduced to manuscripts of relevance to the current topic. these papers included three review manuscripts and original research studies. we included the reviews in our analysis because they provide important insight and expert consensus as to the direction of important fields (biosecurity for live bird market workers; respiratory exposures and disease among food animal workers; and effectiveness of health and safety trainings and interventions for latino animal workers). the manuscripts were organized in an excel spreadsheet and read by two researchers. a narrative synthesis approach was used to extract central themes, findings, and conclusions. based on our a priori knowledge of the field and an assessment of other recent manuscripts in the literature, we identified manuscripts we believed to be of elevated significance to readers engaged in animal worker health and safety work and research, and we discuss those studies in greater detail. the manuscripts published during this -month period were predominantly in the following topic areas: infectious disease and pathogen exposures; respiratory disease and irritants; pesticide and chemical exposures, including neurological toxicants and carcinogens; and occupational injury. below, we summarize the key findings from manuscripts published in each of these topic areas, highlighting the papers that in our opinion are of greatest importance for the field. the majority of manuscripts identified in our review ( / ; %) were focused on animal worker exposure to infectious agents, zoonotic pathogen carriage or infection within this workforce, or pathogen contamination of the work environment. the infectious disease papers are summarized in table . the reviewed manuscripts documented the identification of livestock-associated mrsa in animals and humans in regions around the world and in animal-exposed professions in which mrsa had not previously been assessed. a study in nigeria identified low prevalence of la-mrsa among abattoir workers ( . %) and distinguished a diversity of s. aureus spa types in the work environment, including a novel spa type (t ) [ ] . the first published study of la-mrsa among workers and livestock in trinidad identified a low prevalence among animals (< %) and no worker carriage, indicating limited transmission in this country [ ] . a case study of mrsa among swine and workers on an australian swine farm where workers were affected by skin lesions identified high odds of mrsa nasal carriage among the workers (or . ) and a dose-response relationship of mrsa nasal carriage in association with duration of time spent working with pigs [ ] . a study in italy reinforced the elevated prevalence of st among industrial swine (approx. %) and swine workers ( %) in that country and highlighted a component of the production cycle (fattening) in which workers had higher risk of exposure [ ] . cuny and colleagues assessed mrsa nasal colonization among butchers and food preparers in germany to evaluate whether these persons with contact with raw meat were colonized with livestock-associated mrsa, and found limited evidence of colonization (< %) [ ] . these studies continue to expand our knowledge of the distribution of livestock-associated mrsa, both by industry and by geographic region. whole genome-sequencing (wgs) technology was used to elucidate transmission pathways in two studies conducted in africa. amoako and colleagues took a comprehensive approach and used wgs to evaluate mrsa along the "farm to fork" continuum in the intensive poultry industry in south africa [ ] . the authors evaluated samples collected from the farms, transport vehicles, slaughterhouses, and retail outlets, as well as fecal and nasal specimen from workers along the production process. the authors document the widespread distribution of mrsa clone st -cc _t -sccmec_type_ivd ( b) throughout the production cycle. they hypothesize that the multidrug resistance of this clone is mediated by mobile genetic elements, due to the similarity of resistance patterns between the human and animal specimen. the identified prevalent clone is considered both nosocomial and community-associated, highlighting the public health risks associated with the poultry industry in south africa. this work and the study in cameroon, detailed below, are of relevance due to the rapid intensification and expansion of industrial food animal production into africa and the limited research to date on the public health consequences of this industrial growth. a second study in africa used wgs to identify the genetic lineage of mrsa isolates from swine slaughterhouses in south africa and cameroon [ ] . these authors found approximately % prevalence in pigs in south africa but a low prevalence in cameroon (< %), with no workers colonized in either country. all isolates were st , a distinction from the amoako study. these findings highlight potential differences in mrsa carriage by species and/or region and also suggest that production or environmental containment practices may differ among countries and corporations in relevant ways for public health. chen et al. used wgs to identify whether cc , the predominant livestock-associated mrsa strain in asia, was associated with pathogenicity in humans [ ] . the authors screened mrsa isolates from a national database in taiwan and found cc had a low prevalence ( . %); however, these isolates were associated with invasive disease, including bacteremia leading to death and osteomyelitis in four of the eight identified cases. the remaining four cases were associated with mild disease or colonization without disease. of note, only two of the eight cases had documented exposure to pigs, considered the main cc reservoir in the region. this important paper highlights two core concepts: ( ) while rare in humans, cc may be associated with significant pathogenicity in humans, including death and ( ) nosocomial or community transmission for this pathogen should be considered. like the african studies, this paper elucidates the public health risks from animal work and highlights the potential role of animal workers at the front lines of exposure to zoonotic pathogens of broader health concern. other antibiotic-resistant bacterial infections escherichia coli (e. coli) recovered from swine workers and pigs in northern vietnam, a region characterized by rapid growth in industrial swine production and heavy agricultural antibiotic usage [ ] . esbls are of particular concern because these genes are encoded by plasmids that are easily transferred across bacterial species, potentially resulting in widespread antibiotic resistance. the authors observed high prevalence of ctx-resistant e. coli among both workers and pigs ( % of pig workers and % of pigs) on farms studied. esblproducing e. coli was detected from more than % of both pigs and farms. this paper highlights significant concern regarding potential spillover of drug-resistant bacteria from swine to humans in this region, as well as the likelihood of dissemination of the esbl mges. research during this period focused on zoonotic influenza of multiple subtypes, including the emerging influenza d virus. ma et al. published findings from a longitudinal study of swine workers, swine, and environmental sampling in china [ ••] . notably, in this study, workers were monitored for influenza-like illness along with surveillance sampling, so as to identify active symptoms associated with infection. approximately % of workers with ili were positive for influenza a virus, with more than % of those infected with a putative swine lineage virus. additionally, high concordance was noted between a(h n )pdm -like h n viruses isolated from workers with ili and iav circulating among swine, indicating species crossover. a second study, led by borkenhagen et al. identified influenza b and influenza d viruses in swine worker nasal passages during a surveillance study in malaysia [ ] . the authors also recovered porcine circovirus in worker nasal specimen as well as in pig specimen, indicating zoonotic concern associated with this viral pathogen of growing concern in asia. we would also direct readers interested in zoonotic influenza emergence to two valuable review papers published in the last year this topic, by zhou et al. and bailey et al [ , ] . animal worker exposure to hepatitis e virus (hev) was explored in two notable papers, both of which extended the prior paradigm of hev research to include new populations or production specifics. a study in hubei, china, identified elevated seroprevalence among rabbit slaughterhouse workers compared with community controls and observed a doseresponse relationship between increasing seroprevalence associated with duration of employment [ ] . khounvisith and colleagues evaluated hev seroprevalence among commercial pig workers in laos, a region with hev endemicity among swine [ ] . the authors observed % of workers were hev seroprevalent, compared with % of controls, and workers exposed to piglets during the growth process were at elevated risk. other authors highlighted additional emerging zoonotic viral pathogens in the food animal workforce, including a report of brucellosis among sheep farmers in egypt and knowledge and biosecurity practices among indian animal farmers about rabies [ , ] . msimang and colleagues reported on rift valley fever seroprevalence among animal farmers and veterinarians in south africa, concluding that infection with this re-emerging pathogen is likely notably higher than previously recognized and under-diagnosed in the region [ ] . we identified eight original research papers and one review study focused on topics related to respiratory disease, exposure to allergens and dust, and airborne bacteria among animal workers. these papers expanded the literature in two core ways: ( ) a focus on combined and interacting respiratory exposures, rather than single-exposure assessments and ( ) the use of s rna amplicon sequencing technology to evaluate house microbiota in farmer's homes and correlating these data to endotoxin levels. key papers are discussed below. davidson et al. conducted personal exposure monitoring of bioaerosol exposures, including inhalable dust, endotoxin, -hydroxy fatty acids, muramic acid, ergosterol, and ammonia among workers at large dairies in the western usa [ •] . this paper marks one of the early studies to consider multiple, and interacting, respiratory exposures in this population. the authors conclude that a majority of these workers were exposed to endotoxin concentrations that exceed recommended guidelines ( %). workers were also exposed to inhalable dust and ammonia at levels above guidelines. the authors also evaluated the correlation between pairs of these exposures by different dairy tasks, another novelty of this work. lee et al. used s rna amplicon sequencing to evaluate bacterial composition of dust samples recovered from households of active and former farmers recruited in the agricultural lung health study, a nested study of the agricultural health study in north carolina and iowa [ ••] . current farming was a significant predictor of the composition and diversity of house dust microbiota. animal farming was uniquely associated with firmicutes and proteobacteria phyla, with bacillaceae, bacteroidaceae, xanthomonadaceae, streptococcaceae, and lactobacillacae also identified in dust specimen from homes with animal farmers. the authors identified taxa associated with endotoxin concentration. asthma status was not associated with bacterial diversity or composition. this paper is notable for its integration of traditional exposure assessment approaches to endotoxin and s rna amplicon sequencing technology for evaluating microbiota, and for contributing detail to our understanding of household-level exposures experienced by animal workers and their families. other notable manuscripts this year included: a study of bacterial and fungal exposures among portuguese veterinarians, exposures to ammonia, vocs, and fungus among swine workers during the summer and winter seasons in poland, and a study from australia of worker exposure to asthmagens derived from animals or fish/shellfish (el zaemey et al.) [ ] [ ] [ ] . the latter study was notable for its large sample (n = ) and its comparison of farmers and animal workers to community controls in a national agricultural study. additionally, an excellent consensus paper published by the european academy of allergy and clinical immunology highlights the state of the literature on respiratory disease and animal workers, specifically food processing workers, in europe, focusing on all elements of the food production chain [ ] . studies of pesticide and chemical exposures among animal workers highlighted pesticides usage in livestock production may increase the risk of parkinson's disease (pd) among farmers. while this relationship has been previously assessed in crop farmers, the identification of animal farmers as a population at risk due to shared exposures is a notable contribution of work from this year. pouchieu and colleagues evaluated the risk of pd among both livestock and crop farmers in france exposed to pesticides in the agrican cohort [ ] . the crop matrix pestimat was used to evaluate exposure to active ingredients and duration of lifelong use, and the implementation of this matrix again reinforces the interest in evaluating complex and realistic mixture scenarios for worker exposures. in this study, cattle workers in particular had an elevated risk of pd, with dithiocarbamate fungicides, rotenone and the herbicides diquat and paraquat identified as compounds of concern for this occupational group. additionally, further studies elucidate carcinogenic compounds beyond pesticides that animal workers may be exposed to, highlighting cancer research as an underexplored area for consideration in this population. darcey and colleagues conducted a cross-sectional study to evaluate exposure to solar radiation, diesel engine exhaust, and solvents among australian farmers [ ] . exposure to these carcinogens was highest for farmers with mixed livestock and crop production, again highlighting unique risk profiles for workers who engage in multiple agricultural activities. hoffman et al. evaluated serum immune markers in a subset of ahs participants who were swine farmers to consider an immunological explanation for the inverse relationship between swine farming and lung cancer, which is hypothesized due to endotoxin exposure [ ] . the authors observed that macrophage-derived chemokine (ccl ), which is believed to contribute to lung carcinogenesis, was lower in swine farmers compared to cattle farmers with a % reduction in levels among farmers at the largest farms (> head), suggesting a dose-response relationship. these manuscripts highlight the complex health effects associated with occupational animal exposure and indicate how emerging technologies and personal monitoring can inform the biological basis of epidemiologic observations. reviewed manuscripts largely focused on the experiences of latino immigrant and migrant farmworker populations in the usa, who comprise a majority of the us food animal workforce. we note a limited number of peer-reviewed original research publications on occupational injury during the short period of our review. we identified four published epidemiological studies of occupational injury in the animal workforce as well as three studies evaluating effectiveness of injury prevention training. a small study conducted in missouri examined self-reported injury and health status among latino immigrant workers. their results indicated a high prevalence of workers rating their health as fair or poor, along with high prevalence of occupational injury [ ] . clouser and colleagues found that occupational injury was more likely for latino farmworkers in the usa if they self-reported work stress, supervisor unfairness, or supervisor inability to speak spanish [ ] . these findings reinforce that immigrant latino and migrant workers in the animal industry need additional resources and supports to successfully mitigate injury risk. bush and colleagues evaluated the causes of missed work among a sample of latino horse workers in the usa in an attempt to evaluate the causes of occupational illnesses [ ] . the authors found that having at least one child, poor selfrated health, and elevated stress were associated with missed work, highlighting the intersecting role of personal and workrelated factors for these workers. an assessment of osha's dairy-focused local emphasis programs (leps) in wisconsin and new york by liebman et al. found that the osha's recent initiative to reduce injury and hazard in the dairy industry improved farmers' ability to recognize occupational hazards [ ] . the authors found that the leps motivated participating dairy producers in these two states to address hazards, such as correct signage, repairs and fit for ppe, and manure management and also encouraged workers to advocate for health care needs. rodriguez et al. evaluated the effectiveness of delivering health and safety training using mobile platforms to us dairy workers with limited english proficiency [ ] . this method was successful, with workers enrolled (n = ) demonstrating a % mean increase from pre-to post-test knowledge of workplace safety practices (p < . ). this paper is of particular note given the proliferation of smartphones and the increasing proportion of the food animal workforce with limited english proficiency. rodriguez and colleagues noted that more than / of the participants in their study spoke a central american indigenous language and were able to receive training through smartphone applications and translation, highlighting the power of this technology to reach many workers with necessary education. caffaro and colleagues conducted a literature review on occupational safety and health training programs addressing migrant farmworkers, including animal workers, to determine the effectiveness of the standard programs in place [ ] . the majority of the reviewed studies found the training programs to be ineffective, with no or little difference in injury outcomes with or without the standard training programs. the authors recommended an increase in participatory approaches and multilingual offerings so as to improve the effectiveness of these programs for migrant workers. continued efforts to evaluate the effective means of developing and delivering injury prevention and health promotion training to the changing and diverse food animal workforce is an important theme of study. research in and early on occupational health and safety topics involving animal workers highlighted the risks and interventions associated with infectious disease, respiratory disease, chemical exposure, and occupational injury. in the realm of infectious disease, these studies identified an expanding, and concerning, geographical distribution of mrsa as well as novel transmission pathways. the expansion of mrsa into africa is of particular note, as the continent has witnessed rapid intensification of food animal production and demand for industrial meat products in recent years. given the known consequences of unregulated antibiotic usage in animal production for public health and the emergence of la-mrsa, surveillance, and regulation of la-mrsa in this region is a critically important direction for future research. in the absence of antibiotic stewardship, interventions to protect workers from zoonotic antibiotic resistant infections-building on the experiences in europe and the usa-would improve african worker health. likewise, the discovery of mrsa strain cc among human patients in taiwan, and the association of this strain with severe illness and death, signifies an important direction for future research. while highly prevalent in livestock populations in asia, cc has not been considered a human pathogen of significance. chen et al.'s paper should reignite interest in this strain as an important, if rare, contributor to severe illness in humans, with surveillance targeting food animal workers at the front lines of exposure. research on zoonotic influenza viruses identified species spillover from swine farming into the food animal workforce, highlighting the importance of this pathway, and this industry for surveillance and pandemic influenza prevention. bailey and colleagues nicely highlight the recent expansion of zoonotic influenza research, notably the discovery of zoonotic influenza d virus, in their review on this topic [ ] . continued research on influenza transmission at the humananimal interface in food animal production remains a critically important area for continued work. likewise, research on behaviors and practices that affect worker exposure, as well as intervention evaluation studies, are central. studies of respiratory irritants and disease integrated new technologies into multi-exposure assessments, including s rna amplicon sequencing technology. the incorporation of metagenomics approaches will likely mark exposure assessment studies in the future, given the relevance of these techniques in other research areas and the opportunities to shed new light on existing occupational health problems. in particular, microbiome analyses have the potential to highlight the relationship between occupational exposures and chronic conditions, such as cancers and respiratory diseases, whose etiology has remained elusive. gene expression studies could elucidate pathways of respiratory irritation among highly exposed workers, with relevance for both the food animal workforce and also the general population. as whole genome sequencing techniques have clarified the role of zoonotic pathogens in the emergence of novel pathogens, such as livestockassociated mrsa and zoonotic influenza viruses, genetic, and genomic techniques hold significant power to clarify pathways of occupational disease for the food animal workforce. this is an important area for future research. consideration of pesticide exposures in animal workers, and multiple exposures between crop and animal farmers, also reflects the emerging interest in complex mixtures analyses in occupational exposure assessment. while studies reviewed here did not formally engage mixtures analytic approaches (such as weighted quantile sum regression or lagged kernel machine regression) davidson and colleagues illustrated the value of combined metrics in exposure assessment studies for food animal workers. given the complex mixtures of pathogens, allergens, toxicants, and other compounds that food animal workers are exposed to on the job, the application of mixtures methods to occupational health studies of food animal workers is an important next step for the field. these techniques may hold specific relevance for cancer endpoints of relevance to this workforce, whose etiologies are potentially multifactorial and have remained rather elusive to date. the predominance of research on latino and migrant worker injury and safety reflect changing demographics in the animal industry over the last years. food animal production in the usa, including both live animal production as well as processing, remains in a period of demographic transition, with the industry increasingly facing a reckoning between the needs and demands of the immigrant and us-born workforce. as segments of the workforce become increasingly immigrant-based, the specific training needs, and injury experiences of these workers become central. studies on the effectiveness of safety training and intervention that target the specific needs and experiences of this segment of the workforce are critically important to reducing morbidity in this industry. likewise, future research that highlights the health experiences and needs of us-born food animal workers, who currently experience wage stagnation and significant social stressors in many regions of the usa, should also be at the forefront. the occupational injury implications of the industry's interests in increased line speeds and also automation also remain an important area for future work, so as to inform regulations and protect workers. food animal work remains a complex and often dangerous occupation. research in and beyond would best suit the needs of this workforce by continuing to highlight pathogens of concern, identify regulatory and intervention opportunities to reduced occupational pathogen exposure, integrate emerging microbiome and genomic technologies to more fully elucidate occupational disease pathways, and evaluate injuryprevention techniques specific to the demands and realities of the industry. funding information support for this work was provided by cdc/ niosh k oh (jhl) and williams college center for environmental studies (cd). antimicrobial-resistant bacteria: an unrecognized work-related risk in food animal production particulate matter, endotoxin, and worker respiratory health on large californian dairies environmental exposure and health effects from concentrated animal feeding operations lung cancer risk in workers in the meat and poultry industries-a review incidence rates of nonfatal occupational injuries and illnesses by industry and case types the animal-human interface and infectious disease in industrial food animal production: rethinking biosecurity and biocontainment. public health rep wash dc swine workers and swine influenza virus infections tackling antibiotic resistance: the environmental framework chickenizing farms and food: how industrial meat production endangers workers, animals, and consumers industrial livestock production and global health risks. food agric organ u n -poor livest policy initiat res rep industrial food animal production and global health risks: exploring the ecosystems and economics of avian influenza microbial ecology, bacterial pathogens, and antibiotic resistant genes in swine manure wastewater as influenced by three swine management systems public health implications related to spread of pathogens in manure from livestock and poultry operations animal antibiotic use has an early but important impact on the emergence of antibiotic resistance in human commensal bacteria an overview of livestock-associated mrsa in agriculture food-animal production and the spread of antibiotic resistance: the role of ecology staphylococcus aureus cc : host adaptation and emergence of methicillin resistance in livestock avian influenza virus (h n ): a threat to human health persistence of highly pathogenic avian influenza h n virus defined by agro-ecological niche diversity of influenza viruses in swine and the emergence of a novel human pandemic influenza a (h n ). influenza other respir viruses pandemic influenza planning: shouldn't swine and poultry workers be included? vaccine methicillin-resistant staphylococcus aureus (mrsa) strain st is present in midwestern u.s. swine and swine workers multidrug-resistant and methicillin-resistant staphylococcus aureus (mrsa) in hog slaughter and processing plant workers and their community in north carolina (usa) sweat and fear: workers' rights in us meat and poultry plants measuring job characteristics and mental health among latino farmworkers: results from cognitive testing migrant farmworker stress: mental health implications. j rural health off j am rural health assoc natl rural health care assoc systematic review of respiratory health among dairy workers stress, depression, and occupational injury among migrant farmworkers in nebraska lifetime allergic rhinitis prevalence among us primary farm operators: findings from the farm and ranch safety survey pesticides and their metabolites in the homes and urine of farmworker children living in the salinas valley. ca noncancer health effects of pesticides: systematic review and implications for family doctors rotenone, paraquat, and parkinson's disease dietary fat intake, pesticide use, and parkinson's disease the health and safety of workers in a confined animal system respiratory illness in agricultural workers staphylococcus aureus in two municipal abattoirs in nigeria: risk perception, spread and public health implications genomic analysis of methicillin-resistant staphylococcus aureus isolated from poultry and occupational farm workers in umgungundlovu district clinical and molecular features of mdr livestockassociated mrsa st with staphylococcal cassette chromosome mecxii in humans genome analysis of methicillin-resistant staphylococcus aureus isolated from pigs: detection of the clonal lineage st in cameroon and south africa mrsa in swine, farmers and abattoir workers in southern italy emergence of highly prevalent ca-mrsa st as an occupational risk in people working on a pig farm in australia prevalence of methicillin-resistant staphylococcus aureus (mrsa) in broilers and workers at "pluck shops" in trinidad cephalosporin-resistant escherichia coli isolated from farm workers and pigs in northern vietnam surveillance for respiratory and diarrheal pathogens at the human-pig interface in sarawak. malaysia the authors identified cross-species transmission of human-adapted h n viruses and swine-adapted h n viruses in both swine and swine workers in china, highlighting continued pandemic influenza risk from swine production in this region. the authors also noted symptomatic influenza-like illness among workers using a longitudinal design sero-diagnosis of brucellosis in sheep and humans in assiut and el-minya governorates. egypt challenges to human rabies elimination highlighted following a rabies outbreak in bovines and a human in high seroprevalence of hepatitis e virus in rabbit slaughterhouse workers high circulation of hepatitis e virus in pigs and professionals exposed to pigs in laos rift valley fever virus exposure amongst farmers, farm workers, and veterinary professionals in central south africa methicillin susceptible staphylococcus aureus (mssa) of clonal complex cc , t from infections in humans are still rare in germany effectiveness of market-level biosecurity at reducing exposure of poultry and humans to avian influenza: a systematic review and meta-analysis the continual threat of influenza virus infections at the human-animal interface: what is new from a one health perspective the authors conducted personal exposure monitoring for multiple airborne irritants and toxins among dairy workers in the western us, and considered exposureby-exposure mixtures, setting the stage for future work the authors evaluated microbial composition of household dust using s technology and compared findings to endotoxin levels. this study marks an early foray into microbiome research for understanding health effects associat organic dust exposure in veterinary clinics: a case study of a small-animal practice in portugal occupational exposure level of pig facility workers to chemical and biological pollutants prevalence of occupational exposure to asthmagens derived from animals, fish and/or shellfish among australian workers food processing and occupational respiratory allergy-a eaaci position paper pesticide use in agriculture and parkinson's disease in the agrican cohort study prevalence of exposure to occupational carcinogens among farmers industrial hog farming is associated with altered circulating immunological markers self-reported occupational injuries and perceived occupational health problems among latino immigrant swine confinement workers in missouri associations of work stress, supervisor unfairness, and supervisor inability to speak spanish with occupational injury among latino farmworkers missed work due to occupational illness among hispanic horse workers an overview and impact assessment of osha large dairy local emphasis programs in new york and wisconsin using mobile technology to increase safety awareness among dairy workers in the united states effectiveness of occupational safety and health training for migrant farmworkers: a scoping review nasal colonization of humans with occupational exposure to raw meat and to raw meat products with methicillin-susceptible and methicillin-resistant staphylococcus aureus publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations human and animal rights this article does not contain any studies with human or animal subjects performed by the authors. the authors declare that they have no conflict of interests. papers of particular interest, published recently, have been highlighted as: key: cord- - gq e f authors: staroverov, sergey a.; volkov, alexei a.; mezhenny, pavel v.; domnitsky, ivan yu.; fomin, alexander s.; kozlov, sergey v.; dykman, lev a.; guliy, olga i. title: prospects for the use of spherical gold nanoparticles in immunization date: - - journal: appl microbiol biotechnol doi: . /s - - - sha: doc_id: cord_uid: gq e f recent years have seen extremely fast development of new viral nanovaccines and diagnostic agents using nanostructures prepared by biological and chemical synthesis. we used spherical gold nanoparticles (average diameter, nm) as a platform for the antigen for swine transmissible gastroenteritis virus (tgev). the literature data demonstrate that immunization of animals with the tgev antigen coupled to gold nanoparticles (gnps) not only activates antigen-presenting cells but also increases the proliferative activity of splenic lymphoid (antibody-forming) cells. the contents of γ-ifn, il- β, and il- in animals immunized with gnp-antigen conjugates were found to be higher than those in intact animals or in animals given the antigen alone. the increased concentration of il- β in the immunized animals directly correlated with the activity of macrophages and stimulated b cells, which produce this cytokine when activated. the increased concentration of il- indicates that the injected preparations are stimulatory to cellular immunity. immunization with the tgev antigen conjugated to gnps as a carrier activates the respiratory activity of lymphoid cells and peritoneal macrophages, which is directly related to their transforming activity and to the activation of antibody generation. furthermore, the use of this conjugate allows marked improvement of the structure of the animals’ immune organs and restores the morphological–functional state of these organs. the microanatomical changes (increased number of follicles) indicate the activation of the b-dependent zone of the spleen and, consequently, the development of a humoral-type immunological reaction. the degradative processes observed in the animals immunized with tgev antigen alone are evidence of weak resistance to pathogen attack. these results can be used to develop vaccines against this infection by employing tgev antigen coupled to gold nanoparticles as a carrier. swine transmissible gastroenteritis is an acute, rapidly spreading, highly contagious enteric disease of pigs of all ages. it is characterized by vomiting, watery and yellow diarrhea, weight loss, and rapid dehydration, leading to a high mortality rate, especially in piglets (mortality decreases with age). swine transmissible gastroenteritis is caused by transmissible gastroenteritis virus (tgev) of the genus coronavirus in the family coronaviridae. the major route of virus transmission is fecal-oral. infected animals cannot digest food properly and die from dehydration. the disease causes great economic losses in pig farms all over the world, and the primary means of animal protection against it is vaccination (cheng and niu ; straw et al. ) . therefore, the development of new vaccines and the implementation of large-scale vaccination programs are important in pig breeding (moxley and olson ( ) . in several countries, vaccination against swine transmissible gastroenteritis is conducted effectively but only with inactivated monovalent and combined vaccines not recognized internationally. traditional inactivated vaccines have high contents of contaminant substances (up to %), causing side effects, and/or may contain, as inactivating compounds, toxic agents such as phenol and formaldehyde, normally used to inactivate microbial cells. moreover, traditional vaccines are usually low immunogenic, because killed microorganisms cannot induce fully functioning cellular immunity. therefore, there is a need to develop new, more effective, and less costly vaccines against swine transmissible gastroenteritis (mout et al. ) . recent years have seen extremely fast development of new viral nanovaccines and diagnostic agents using nanostructures prepared by biological and chemical synthesis. in particular, this can be attributed to the development of biocompatible inorganic nanomaterials-gold nanoparticles. gold nanoparticles are stable metallic nanoparticles which have excellent surface functional chemistry, high biocompatibility, and nil toxicity. in addition, gold nanoparticles can be easily synthesized, giving them shapes such as spheres, rods, cubes, stars, and pyramids. several reports have described the development and use of nanostructures for the delivery of biologically active agents to target cells (mout et al. ; bhattacharya and mukherjee ; staroverov et al. ). to reach their target cells in vivo, nanoparticles must bypass many barriers that protect animals against antigens (dreaden et al. ) . gold nanoparticles (gnps) have been used widely as carriers of antigens and therapeutic agents in human medicine, veterinary practice, and biology . of particular interest is the ability of gnps to induce humoral immune response to weakly immunogenic antigens and haptens (dykman et al. a; dykman et al. b ). m o r e o v er, g n p s c on j u ga t ed t o t g e v h a v e a n immunostimulatory effect (dykman et al. b) . we studied the immunological variable and morphological changes in the immune organs of guinea pigs immunized with tgev antigen conjugated to gnps as a nanocarrier. antigen tgev strain vn- was taken from the museum of strains of the laboratory of virology (scientific research veterinary institute, russian academy of sciences, saratov) and deposited in the all-russian state collection of microbial strains used in veterinary and animal husbandry by the number bvn- ^(patent ru a ). tgev strain vn- with a titer of . - . lg tcd ml − was used. test cultures were inoculated with the same strain at . lg tcd ml − after h of cultivation. tgev was purified in a sucrose gradient according to horzinek et al. ( ) . the resultant virus suspension was used for protein analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) at a constant voltage of v for min and then at v for h (maniatis et al. ) . for molecular m a s s d e t e r m i n a t i o n s , p r e c i s i o n p l u s p r o t e i n ™ kaleidoscope™ prestained protein standards no. were used. the results of protein separation were processed with the gel-imager and gel explorer, version . , programs (dna-technology, moscow). genome identity was checked by polymerase chain reaction (pcr) using a specific primera conservative part of tgev's s genome, containing base pairs (bp) (herrington and mcgee ) . rna was isolated from tgev by using a nucleos+tm suspension silicate sorbent (biokom, moscow) according to the manufacturer's instructions. pcr results were recorded on a transilluminator with a dna-analyzer video system. pcr products obtained from a preparation of the tgev strain vn- were analyzed by electrophoresis in agarose gel ( . %) using dna ladder as a molecular size marker and dna of bacteriophage λ as a negative control. as viral nucleic acid is responsible for virulence, it was inactivated, after being isolated from tgev and purified, with ribonuclease (rnaase), a hydrolase enzyme that catalyzes rna hydrolysis. the inactivation was done by the procedure described by fedorova et al. ( ) . the antigen resulting from nucleic acid inactivation was used for animal immunization. spherical gold nanospheres (average diameter, nm) were made by the citrate method of frens ( ) . for reduction, . ml of . % aqueous tetrachloroauric acid (haucl ; sigma-aldrich) was heated under reflux to °c on a magnetic stirrer in an erlenmeyer flask. this was followed by the addition of . ml of % aqueous sodium citrate to the flask. the particle diameter was found as described earlier (khlebtsov and dykman ) , by using a libra transmission electron microscope (carl zeiss, germany), a specord s spectrophotometer (analytik jena, germany), and a zetasizer nano-zs particle size and zeta potential analyzer (malvern). to prepare antigen-gold nanoparticle conjugates, we found the bgold number^(minimal amount of antigen that protects the sol against salt aggregation) for the antigen solution. to this end, μl of an antigen solution in pbs (initial concentration, mg ml − ) was titrated twofold on a -well microtiter plate. each well received μl of gnps and μl of . m nacl. the minimal stabilizing concentration for the antigen was . μg ml − . conjugation was done by simple mixing (without the use of coupling agents) (dykman et al. ). thirty six male guinea pigs, weighing - g at the time the experiment started, were used. the animals were cared for and handled in compliance with the guide for the care and use of laboratory animals, the european convention for the protection of vertebrate animals used for experimental and other scientific purposes, and the legislation of the russian federation. for immunobiological studies, the guinea pigs were divided into four groups of nine each. the animals were immunized subcutaneously along the spinal column by two injections with an interval of days between. the guinea pigs in group were injected with . ml of tgev antigen-gnps (antigen concentration, . μg per animal). group received ml of tgev antigen solution ( . μg per animal). group served as the control, which received ml of physiological saline. group served as the control gnps, which received ml of gnps solution. gnp-antigen group was in comparison with group which received only physiological saline (control group). the animals were killed days after the last injection, and blood serum samples were taken for immunological studies. immediately after removal, all pieces of tissues and organs were placed in a fixing solution ( % aqueous neutral formalin), and then -μm-thick histological sections were prepared on a freezing microtome, model (reichert wien, austria). all histological samples were stained with hematoxylin-eosin. for isolating peritoneal macrophages, the animals were killed and then fixed on their backs. an incision was made along the midline of the anterior abdominal wall, and the skin flap was carefully separated, with care taken to keep the peritoneum intact. after a puncture had been made with a needle connected to a syringe, ml of pbs, ph . , was injected into the peritoneal cavity. the anterior abdominal wall was then gently massaged, and after - min, peritoneal fluid was collected with a pasteur pipet through a cut made in the peritoneum and was filtered into a test tube through a nylon filter. the cells were washed three times by centrifugation in pbs at g, after which they were resuspended in ml of pbs and counted in a goryaev chamber. peritoneal macrophages were cultured by standard procedures (leiter ) . for isolating splenic lymphocytes, an incision was made along the white line of the peritoneum after peritoneal macrophages had been isolated, and the spleen was removed. the spleen was minced with scissors, and the tissue pieces were mashed through a fine sieve into a petri plate containing sterile pbs. the resulting suspension was subjected to ficoll-urografin density-gradient centrifugation. the lymphocyte ring was collected in a new test tube. the lymphocytes were washed three times by centrifugation in pbs, ph . , at g for min, and the cell sediment was resuspended in ml of pbs. the lymphocytic cells were counted with a haemascreenvet hematology analyzer (hospitex diagnostics, italy). the titer of antibodies in the sera was estimated by enzyme-linked immunosorbent assay (elisa) with horseradish peroxidase-labeled antibodies against guinea pig igg (jackson immunoresearch, uk). the synthetic peptide was used as the immobilized antigen. the reaction results were recorded on a plate screen microplate spectrophotometer. the interleukin concentration in the sera was measured with a plate screen analyzer (hospitex diagnostics, italy) and using reagents of il- β, il- , and inf-γ (vector-best, russia). respiratory activity was measured conventionally (bernas and dobrucki ) by the ability of cells to reduce nitrotetrazolium blue [ -( , -dimethylthiazol- -yl)- , diphenyl tetrazolium bromide, mtt (sigma-aldrich)] to formazan. briefly, suspensions of known concentrations of isolated animal cells (macrophages and lymphocytes) were centrifuged at g for min, and the sediment was resuspended in ml of . % mtt and incubated at °c for h. after incubation, the cells were centrifuged at g for min and the sediment was resuspended in . ml of dimethyl sulfoxide (fluka, switzerland). the amount of reduced formazan was measured with a genesys s uv-vis spectrophotometer at nm. to construct the calibration curve, we used commercial formazan (sigma-aldrich) at . , . , . , and mg ml − . for studying possible morphological changes in the spleen as an important part of the macrophage system, the animals were immunized with tgev antigen-gnps. the control group comprised of nonimmunized animals. we used the spleens of guinea pigs of the same physiological age. longitudinal and transverse spleen sections ( -μm-thick) were prepared on a freezing microtome, model (reichert wien, austria), by following standard procedures. histological sections were differentially stained with hematoxylin-eosin. the results were statistically processed by the standard procedures using student's t test to evaluate the reliability of the differences between samplings in the experimental and control studies. after the arithmetic mean and the standard deviation for a given data sample were found, we determined the standard error of the arithmetic mean and its confidence limits by student's t coefficient (n, p) at a significance level of % (p = . ). the obtained results were statistically processed by the standard procedures integrated in excel software (microsoft corp., usa). after the arithmetic mean and the standard deviation for a given data sample had been found, we determined the standard error of the arithmetic mean and its confidence limits with account of student's t coefficient (n, p) [number of measurements n = , significance level = % (p = . )]. these results are presented as histograms. the significance of differences between individual samples was evaluated by a two-sample unpaired student's t test with unequal variances. differences were considered significant when the experimentally found p exp value was ≤ . . the reliability of the changes recorded in the results of each of the experiments described above for the entire set of antigen formulations examined was assessed by one-way analysis of variance (anova) by using fisher's ratio test. the dependences found were considered significant at f > f crit , where the critical value f crit at n = and m = - (number of data sets) corresponded to p = . (with the number of degrees of freedom (df) lying between and ) and the effective value p eff was < . . swine transmissible gastroenteritis is caused by an rna virus belonging to group а of the genus alphacoronavirus in the family coronaviridae. the virion has a spherical shape with a diameter of - nm (martins et al. ) . the viral nucleocapsid is a flexible spiral containing single-stranded rna and a large number of nucleocapsid protein molecules (delmas et al. ; sturman et al. ) . the virus replicates in the cytoplasm of mature epithelial cells located at the ends of the intestine villi. a general scheme of the experiment is depicted in fig. . after the antigen was isolated and its protein composition was analyzed by sds-page, we found that the molecular masses of the separated proteins were identical to those of the tgev proteins. the antigen contained the following proteins: p protein (large surface glycoprotein s; molecular mass, - kda), n protein (basic nucleocapsid protein, - kda), and m protein (matrix membrane protein, - kda) (fig. ) . a protein fraction of - kda was also present. according to wesley et al. ( ) , kda is the size of an unglycated structural protein of tgev. thus, all known tgev proteins were separated. the tgev nucleic acid was identified by pcr using a specific primer-a conservative part of tgev's s genome, containing bp. analysis of the pcr products from a purified preparation of the tgev strain vn- , which was done by agarose gel electrophoresis with dna ladder , demonstrated sufficiently high separation of cdna (fig. ) . the presence of only a characteristic cdna fraction (size, bp) indicated the presence of the tgev genome in the sample. after the virus's nucleic acid was inactivated with ribonuclease, the resultant antigen (a mixture of viral capsid proteins) was used for conjugation with gnps and for subsequent animal immunization. the gnp diameter was measured by tem, the average particle size was . nm. analysis of the antibody titer data showed that the titer produced by immunization with gnp-antigen conjugates ( : . ; p = . ) was higher than that produced by immunization with unconjugated tgev antigen ( : . ). in immunology, the mononuclear phagocyte system (mps) (also known previously as the reticuloendothelial or macrophage system) is part of the immune system that consists of phagocytic cells located in reticular connective tissue. the cells are primarily monocytes and macrophages, which accumulate in lymph nodes and in the spleen. the effectiveness of interaction of the gnp-antigen conjugate with cells of the reticuloendothelial system was evaluated using the mtt test. on day after immunization, an increase in the respiratory activity of macrophages was noted (fig. ) . after immunization with the gnp-antigen conjugate, the activity increased by % (p = . ) as compared with the control and by % (р = . ) as compared with the immunization with tgev antigen and % (р = . ) as compared with gnp. a study of the respiratory activity of splenic lymphoid cells (fig. ) showed that after immunization with the conjugate, the activity increased . -fold, as compared to the control, whereas after immunization with tgev antigen alone, it did not change much. the data demonstrate that immunization of animals with tgev antigen-gnps not only activates antigen-presenting cells but also increases the proliferative activity of splenic lymphoid (antibody-forming) cells. this indicates that gnps can present antigens to the organs of the endothelial system. the effect of the conjugate on humoral immunity was assessed by comparing changes in the cytokine concentration between the control and experimental animals. there was a statistically significant increase in the content of the γ-ifn in animals immunized with gnp-antigen conjugates (fig. ) . these animals had the highest content of γ-ifn ( . ± . pgml - ; p = . ) this group was comparison with group which received only physiological saline (control group). we determined the p value error probability when deviating from the null hypothesis (errors of the first kind). we determined that for our research, p was ≤ . .when the animals were given the native antigen and gnps, no appreciable changes in γ-ifn content were found. the content of il- β in animals immunized with gnpantigen conjugate was . ± . pgml - (p = . ), which is higher than it was in intact animals ( . ± . pgml - ) or in animals given the antigen alone ( . ± pgml - ; p = . ) and gnps ( ± . pgml - ; p = . ) (fig. ) (p value for gnp-antigen group was comparison with group which received only physiological saline (control group). gnp-antigen group was comparison with group which received only physiological saline (control group). the increased concentration of il- β in the immunized animals directly correlated with the activity of macrophages and stimulated b cells, which produce this cytokine when activated. this point is so important, since it is desirable to avoid an inflammatory process during immunization, which results in the appearance of granulomas. similar data were obtained with il- ( fig. ) . this fact indicates that the injected preparations are stimulatory to cellular immunity. it is known that interferons, which possess protective properties, are produced in the body only when the virus enters or tumor diseases. that is why the analysis of the effect of immunization with the help of gold nanoparticles on the change in the level of interferon was made. it was found that in the immunized group, the concentration of interferon increased by % (compared to the control) against % for the group immunized with the antigen. when immunizing animals with native antigen, no significant changes in the concentration of interferon in blood serum of the immunized animals were observed. thus, an increase in the concentration of interferon indicates the stimulation of the introduced drugs of the cell link of immunity. the results obtained correlate with research in this area by other scientists. for example, in work (zhang et al. ) showed that tgev n protein induced er stress and activated nf-κb, which was responsible for the upregulation of il- markers protein markers (kda) protein p protein and bcl- expression. tao et al. ( ) also revealed an increase in the concentration of cytokines including interferon gamma and interleukin in response to immunization aunp-m e + scpg. but he mainly considered the reaction of splenocytes to a given complex (their reaction, which was expressed in the production of cytokines, on the effect of aunp) was studied. in addition, aunp-m e was administered together with scpg which is a strong immunomodulator. it was interesting for us to look at the production of these cytokines in the body as a whole and to reveal the general clinical changes that can develop on the introduction of antigen and aunp. one can speculate that gnps facilitate antigen expression with the major histocompability complex on the surface of antigen-presenting cells. this subsequence ensures that the viral peptides are effectively presented to cytotoxic t lymphocytes and natural killers. our data for the increase in γ-ifn activity are in agreement with those for the increase in the respiratory activity of splenocytes on day after the last injection, another indication that colloidal gold can stimulate the release of γ-ifn by t cells, thereby enhancing the activity of lymphoid immunity. in guinea pigs, the spleen forms leucocytes and removes old red blood cells (erythrocytes). the spleen framework is formed by trabeculae, which consist of reticular connective tissue, smooth muscle cells, and blood vessels (at the same time, the spleen contains only efferent lymphatic vessels). the reticular connective tissue includes both red and white pulp. the white pulp is a lymphoid tissue that produces immune and blood cells. the red pulp functions to filter blood for antigens, microorganisms, and defective red blood cells; therefore, it contains various blood corpuscles, including large and small lymphocytes, immature leucocytes, leucocytes at the final stage of graininess, and disintegrating erythrocytes. the spleen is also important for the development of humoral immunity and is a b-system organ. in the animals immunized with gnp-antigen conjugates, the lymphoid follicles had distinct boundaries and contained large number of densely spaced lymphoid cells (fig. c . the lymphoid tissue was well structured and was located in a compact manner. there was a clear boundary between the red and white pulp regions, and there were full of cellular elements. for comparison, we analyzed morphological changes in the spleens of the animals immunized with tgev antigen alone. the observed changes were characterized by the existence of perivascular edema (fig. d) , sometimes accompanied by proliferation and desquamation of the vessels' endothelium. moreover, the lymphoid tissue of the follicles became sparse, and the number and density of lymphoid cells decreased. in spleen tissue sections from the animals immunized with tgev antigen alone, the boundary between red and white pulp was less clear, the size and total number of follicles in the microscopic field decreased, and no germinative centers could be detected. these results point to degradative processes in the immune organs of the animals. in the control group of guinea pigs and gnps, injected with physiological saline, no morphological changes in spleen tissue were observed (fig. a, b) . gold nanoparticles are stable metallic nanoparticles which have excellent surface functional chemistry, high biocompatibility, and nil toxicity (pissuwan et al. ( ) . in addition, gold nanoparticles can be easily synthesized, giving them shapes such as spheres, rods, cubes, stars, and pyramids. this quality allows the use of gold nanoparticles for a variety of purposes, including vaccine delivery (versiani et al. ). there are a series of works devoted to the use of gold nanoparticles as carriers of antigens for pathogens such as the virus grippe (tao et al. ) , virus foot-and-mouth (chen et al. ) , tetanus toxoid (barhate et al. ) , and yersinia pestis (gregory et al. ) . in our work, we used spherical gold nanoparticles (average diameter, nm) as a platform for the antigen for tgev. the data demonstrate that immunization of animals with tgev antigen-gnps not only activates antigen-presenting cells but also increases the proliferative activity of splenic lymphoid a b c d fig. a spleen tissue section from the animals immunized gnps. hematoxylin-eosin staining; magnification, × . b spleen tissue section from the animals control group. hematoxylineosin staining; magnification, × . c spleen tissue section from the animals immunized with tgev antigen-gnps. hematoxylin-eosin staining; magnification, × . lymphoid follicles have distinct boundaries. d spleen tissue section from the animals immunized with tgev antigen alone. hematoxylineosin staining; magnification, × . perivascular edema and sparse lymphoid tissue in the follicles can be observed fig. concentration of il- in animals immunized with gnpantigen conjugates (tgev antigen р = . ; gnp-antigen conjugate р = . ; gnps р = . ) (antibody-forming) cells. the contents of γ-ifn, il- β, and il- in animals immunized with gnp-antigen conjugates were higher than those in the intact animals or in animals given the antigen alone. the increased concentration of il- β in the immunized animals directly correlated with the activity of macrophages and stimulated b cells, which produce this cytokine when activated. the increased concentration of il- indicates that the injected preparations are stimulatory to cellular immunity. several reports have described the development and use of nanostructures for stimulating cellular immunity. for example, xu et al. ( ) showed that gold nanorods associated with the dna vaccine may cause increased formation of γ-ifn in hiv-infected mice. assis et al. ( ) showed that gold nanorods, functionalized with cysteamine and associated with the protein rsm , are stimulatory to il- . in a work, tao et al. ( ) showed that the use of nanostructured vaccine led to an increase in the concentration of igg a, iga, and il- a, il- , il- , tnf-и rantes. our research correlates with these studies but in the present work we used gold nanoparticles for the first time as a platform for antigen for tgev. also, we did nonspecific adsorption of the antigen and did not perform a chemical surface functionalization. this made it possible to simplify the conjugation process, as well as to remove additional procedures related to the purification of the drug from toxic components. additionally we analyzed the morphological changes in the spleens of the animals immunized with gnp-antigen conjugates and tgev antigen. the microanatomical changes (increased number of follicles) indicate the activation of the b-dependent zone of the spleen and, consequently, the development of a humoral-type immunological reaction (galaktionov, ) . the degradative processes observed in the animals immunized with tgev antigen alone are evidence of weak resistance to pathogen attack. in conclusion, immunization with tgev antigen conjugated to gnps as a carrier activates the respiratory activity of lymphoid cells and peritoneal macrophages, which is directly related to their transforming activity and to activation of antibody generation. furthermore, the use of this conjugate allows marked improvement of the structure of the animals' immune organs and restores the morphological-functional state of these organs. the results of this study can be used in the further development of nanovaccines against swine transmissible gastroenteritis. the microanatomical changes (increased number of follicles) indicate the activation of the b-dependent zone of the spleen and, consequently, the development of a humoral-type immunological reaction (galaktionov, ) . the degradative processes observed in the animals immunized with tgev antigen alone are evidence of weak resistance to pathogen attack. funding information this study was funded by the russian science foundation (project no. - - ). conflict of interest the authors declare that they have no conflict of interest. ethical statement all applicable international, national, and/or institutional guidelines for the care and use of animals were followed. the use of gold nanorods as a new vaccine platform against schistosomiasis quillajasaponaria extract as mucosal adjuvant with chitosan functionalized gold nanoparticles for mucosal vaccine delivery: stability and immunoefficiency studies the role of plasma membrane in bioreduction of two tetrazolium salts, mtt, and ctc biological properties of bnakedm etal nanoparticles assessment of gold nanoparticles as a size-dependent vaccine carrier for enhancing the antibody response against synthetic foot-and-mouth disease virus peptide investigation on the porcine epidemic diarrhea prevalent on qinhai antigenic structure of transmissible gastroenteritis virus. ii. domains in the peplomer glycoprotein the golden age: gold nanoparticles for biomedicine gold nanoparticles in biomedical applications: recent advances and perspectives adjuvant properties of gold nanoparticles gold nanoparticles as an antigen carrier and an adjuvant. nova science publishers gold nanoparticles: synthesis, properties, biomedical applications inactivation of a non-enveloped rna virus by artificial ribonucleases: honey bees controlled nucleation for the regulation of the particle size in monodisperse gold suspensions conjugation of y. pestis f -antigen to gold nanoparticles improves immunogenicity diagnostic molecular pathology: a practical approach antigenic relationships among homologous structural polypeptides of porcine, feline, and canine coronaviruses optical properties and biomedical applications of plasmonic nanoparticles the nod mouse: a model for insulin dependent diabetes mellitus //current protocols in immunology diagnosis to detect porcine transmissible gastroenteritis virus (tgev) by optical and transmission electron microscopy techniques surface functionalization of nanoparticles for nanomedicine clinical evaluation of transmissible gastroenteritis virus vaccines and vaccination procedures for inducing lactogenic immunity in sows prospects for gold nanorodparticles in diagnostic and therapeutic applications usage of phage mini-antibodies as means of detecting ferritin concentration in animal blood disease of swine proteolytic cleavage of the e glyco protein of murine coronavirus by trypsin and separation of two different k cleavage fragments gold nanoparticle-m e conjugate coformulated with cpg induces protective immunity against influenza a virus consensus m e peptide conjugated to gold nanoparticles confers protection against h n , h n and h n influenza a viruses gold nanoparticles and their applications in biomedicine genetic analysis of porcine respiratory coronavirus, an attenuated variant of transmissible gastroenteritis virus surface-engineered gold nanorods: promising dna vaccine adjuvant for hiv- treatment transmissible gastroenteritis virus n protein causes endoplasmic reticulum stress, upregulates interleukin- expression and its subcellular localization in the porcine intestinal epithelial cell key: cord- -uih alib authors: khoury, bassam title: the root causes of covid- screech for compassion date: - - journal: mindfulness (n y) doi: . /s - - - sha: doc_id: cord_uid: uih alib nan sars, bird flu, and now covid- , caused by a novel coronavirus, are on the rise (smith et al. ) . pathogens are crossing from animals to humans, and many can spread quickly to new places. the mechanisms of transmission of viruses such as sars-cov- from bats to humans are not yet fully understood but can originate in a direct encounter between bats and humans; for example, during hunting as bats are consumed as food in some corners of the world. another possible route of transmission involves inhalation of infectious particles by humans. these infective aerosols could arise from the secretions (e.g., saliva) of the bats (wong et al. ) . a third route for transmission is via a secondary intermediate vertebrate host serving as an amplifying host. this last scenario seems to fit other recent outbreaks of coronavirus-caused disease in humans, such as sars, which arose from cat-like civets, and mers, which arose from camels (andersen et al. ) . despite the obvious risk that bat viruses pose to human health, it must be acknowledged that most outbreaks of batborne zoonotic diseases are a consequence of human activities (wynne and wang ) . anthropogenic activities such as habitat loss, human encroachment, and the destruction of natural feeding and roosting habitats caused by urban sprawl and agricultural expansion are increasing the interactions between bats, humans, and livestock, thereby heightening the zoonotic potential conferred by those characteristics. researchers have found that disrupting bat habitats and hunting them appears to stress the bats, causing them to shed even more virus in their saliva, urine, and feces, which can then infect other animals (university of california, berkeley, ). preliminary links have been established between loss of bat habitat and the spillover of bat viruses into other animals and humans (jones et al. ) . it is clear that human actions, such as the hunting of wild animals and mindless destruction of biodiversity, create the underlying favorable conditions for new viruses and diseases such as covid- , which threaten the existence of humanity itself, so how come humans still engage in destructive behaviors that threaten their own existence? obvious reasons relate to accessing more food sources, exploiting additional land, extending agriculture, and furthering economic growth in order to provide resources for a growing human population. however, such motives, although important, do not explain why humans are failing to balance their needs with the well-being of the planet and animals. to answer this question, it is necessary to turn to buddhist philosophy. in buddhism, the human condition is divided into two states: wholesome (kusala) and unwholesome (akusala) (le duc ). unwholesome states lead to suffering and their origins are rooted in greed (rāga), hatred (dosa), and delusion (moha) (kornfield ) . greed refers to any form of over-attachment to a sensory object. in this case, greed can be manifested through exploiting the planet, destruction of natural habitats, and taking advantage of animals, without ever attaining satisfaction. hate is the opposite of greed, defined as the drive to avoid, resist, or even destroy things someone does not want. a subtle form of hatred is apathy. apathy can be manifested by a lack of concern to the suffering of others and by justifying it at times as necessary for economic growth. apathy plays a central role in the persistence of the ecological crisis (le duc ), the current surge of infectious diseases, and the transmission of viruses, such as the novel coronavirus, from animals to humans. delusion is the inability to perceive reality and phenomena as they really are due to being trapped between the poles of greed and hatred/apathy (khoury ) . another form of delusion is ignorance, such as denying empirical scientific facts in order to continue indulging in selfish desires while discounting the well-being of others, including animals and nature. delusion can result from ideological notions supporting, for example, the stance that human beings can exercise absolute domination over nature and animals according to some sort of divine ordination, while denying the impacts of such actions on nature, animals, and therefore humans (le duc ). rectifying unwholesome states of mind requires engaging in daily practices of mindfulness and compassion. mindfulness can be defined as an embodied non-judgmental attention and lucid awareness of oneself, others, and the environment (khoury ; khoury et al. khoury et al. , khoury et al. , . compassion can be defined as an embodied, integrated state with cognitive, affective, and behavioral dimensions that is characterized by an altruistic attitude, an emphatic concern, and a desire to alleviate suffering in oneself and in others (khoury ) . the dalai lama ( a) included wisdom as an integral part of compassion, writing "genuine compassion must have both wisdom and loving kindness. that is to say, one must understand the nature of the suffering from which we wish to free others (this is wisdom), and one must experience deep intimacy and empathy with other sentient beings (this is loving kindness)" (p. ). thus, compassion implies non-cruelty and harmlessness towards all sentient beings including animals (finnigan ) . empirical studies have shown positive associations between mindfulness and nature connectedness (howell et al. ) , ecological sustainability (amel et al. ; jacob et al. ), and ecological behaviors (geiger et al. ) . similar studies have shown positive associations between compassion to other human beings and pro-environmental tendencies, including values, intentions, and actions (pfattheicher et al. ) , as well as compassion towards animals in lab settings and the well-being of research personnel (lafollette et al. ) . according to buddhism, mindfulness and compassion should be taught as part of several complementary perspectives or ethics (shonin et al. ) . such perspectives include the concepts of "non-self," "non-attachment," "impermanence," and "inter-connectedness." the term "non-self" refers to the realization that the self has no intrinsic existence (dalai lama b) . "non-attachment" can be defined as the liberation from excessive craving or clinging that is favored by a vision of all objects and living beings as void of any lasting self and by the impermanent nature of all phenomena. "impermanence" refers to the notion that all phenomena are transient occurrences and are subject to decay and dissolution (rinpoche ). finally, the term "interconnectedness" is used in buddhism to refer to the interconnected nature of all phenomena (called also interbeing) (nhat hanh ) . practices of mindfulness and compassion such as concentrative meditation (samatha), insight meditation (vipassana), and loving-kindness meditation (metta), along with nonformal mindfulness practices during daily activities, facilitate the recognition of the interconnectedness between humans, animals, and nature. this interconnectedness implies that the well-being and safety of humans depend on the well-being and safety of animals and of nature. in line with that, planetary health is emerging as a new discipline that focuses on the increasingly visible connections between the well-being of humans, other living things, and entire ecosystems (vidal ) . mindfulness and compassion practices rooted in buddhist ethics allow for overcoming greed by cultivating non-attachment, generosity, and humbleness; hatred through generating loving-kindness to all sentient beings and to nature; and delusion by recognizing the realities of impermanence, non-self, and interconnectedness. mindfulness and compassion practices do not preach apathy and non-doing, but rather the opposite. they cultivate a radical commitment to act in order to reduce the suffering of all sentient beings and to approach nature with awareness and kindness. a commitment to act should include the cultivation of social, racial, ethnic, and gender equalities based on a just, mindful, and non-competitive sharing of the natural resources and beauties of the planet. while social and political activism should be a core element of mindfulness and compassion practices, it is paramount that activism is conducted in a totally peaceful, non-violent, and wholesome manner. any use of unwholesome or violent means would defeat the underlying purpose of such a movement. in summary, biological, pharmacological, and medical interventions are highly needed to overcome the current covid- pandemic. however, it is essential to understand its root causes in order to undertake a real, sustainable, and profound transformation of our state of mind, moment to moment, and therefore, in our actions towards all sentient beings and the surrounding environment. mindfulness and compassion, when used skillfully and ethically, are among the most influential practices to facilitate change and transformation on an individual, inter-individual, social, environmental, and planetary scale. perhaps, if we start listening to the screeches of bats with awareness and warm heartedness, and responding to them with compassion, we can start to reverse the destruction of the earth and save its sentient habitants, humans, and animals alike. not contain any studies with human participants performed by the author. no official funding was provided. the author has no competing interests. mindfulness and sustainable behavior: pondering attention and awareness as means for increasing green behavior the proximal origin of sars-cov- buddhism and animal ethics mindfully green and healthy: an indirect path from mindfulness to ecological behavior nature connectedness: associations with well-being and mindfulness personal and planetary well-being: mindfulness meditation, pro-environmental behavior and personal quality of life in a survey from the social justice and ecological sustainability movement global trends in emerging infectious diseases zoonosis emergence linked to agricultural intensification and environmental change mindfulness: embodied and embedded compassion: embodied and embedded embodied mindfulness. mindfulness la pleine conscience incarnée : un concept unificateur entre les traditions orientales et occidentales de la pleine conscience la dimension interpersonnelle de la pleine conscience bringing home the dharma: awakening right where you are laboratory animal welfare meets human welfare: a cross-sectional study of professional quality of life, including compassion fatigue in laboratory animal personnel essence of the heart sutra: the dalai lama's heart of wisdom teachings the many ways to nirvana buddhist communication of the true roots of the ecological crisis a comparison of bats and rodents as reservoirs of zoonotic viruses: are bats special? the sun my heart feelings for the suffering of others and the environment: compassion fosters proenvironmental tendencies the emerging role of buddhism in clinical psychology: toward effective integration coronavirus outbreak raises question: why are bat viruses so deadly? bats' fierce immune systems drive viruses to higher virulence, making them deadlier in humans tip of the iceberg': is our destruction of nature responsible for covid- ? the guardian bats as a continuing source of emerging infections in humans rolling updates on coronavirus disease (covid- ) bats and viruses: friend or foe? publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors declare that they have no conflict of interest. key: cord- -r g e y authors: wang, l. -f.; eaton, b. t. title: bats, civets and the emergence of sars date: journal: wildlife and emerging zoonotic diseases: the biology, circumstances and consequences of cross-species transmission doi: . / - - - - _ sha: doc_id: cord_uid: r g e y severe acute respiratory syndrome (sars) was the first pandemic transmissible disease of previously unknown aetiology in the twenty-first century. early epidemiologic investigations suggested an animal origin for sars-cov. virological and serological studies indicated that masked palm civets ( paguma larvata ), together with two other wildlife animals, sampled from a live animal market were infected with sars-cov or a closely related virus. recently, horseshoe bats in the genus rhinolophus have been identified as natural reservoir of sars-like coronaviruses. here, we review studies by different groups demonstrating that sars-cov succeeded in spillover from a wildlife reservoir (probably bats) to human population via an intermediate host(s) and that rapid virus evolution played a key role in the adaptation of sars-covs in at least two nonreservoir species within a short period. severe acute respiratory syndrome (sars) first appeared in mid-november in guangdong province in southern china, and continued to spread to more than countries on five continents with , reported cases and deaths by the end of july , placing it with hiv/aids as one of the severe and readily transmissible new diseases to emerge in the twenty-first century (who ) . the high case fatality rate and global spread led to an urgent response by an international network co-ordinated by the world health organization (who) of the united nations, which resulted in the rapid identification of the aetiological agent fouchier et al. ; ksiazek et al. ; kuiken et al. ; peiris et al. ) . the outbreak was caused by a newly emerged and previously unrecognised coronavirus, now known as the sars coronavirus (sars-cov). the complete genome sequence of sars-cov has been determined and the virus is classified within the order nidovirales , family coronaviridae , genus coronavirus (marra et al. ; rota et al. ) . from december , to january , , four sars cases were detected in the city of guanghzou, the capital city of guangdong province of china (liang et al. ; wang et al. ) . none of these cases was fatal or resulted in documented secondary transmission, suggesting the possibility that these sporadic outbreaks were caused by less virulent strains of sars-cov. coronaviruses are known to infect a variety of avian and mammalian species (holmes and lai ; see the chapter by holmes and drummond, this volume) . before the discovery of sars-cov, two human coronaviruses ( e and oc ) were known to cause upper respiratory tract infections that varied in frequency and severity in different disease outbreaks, but were usually mild and self-limited (holmes and lai ) . since the discovery of sars-cov, two new coronaviruses, nl (van der hoek et al. ) and hku , have been isolated from human patients with nonfatal infections. to date, sars-cov is the only known coronavirus capable of causing lethal infection in humans. recently, two groups independently demonstrated that bats in the genus rhinolophus are natural reservoirs of sars-like viruses , providing strong evidence that sars-cov is indeed a new zoonotic virus with a wildlife origin. epidemiological studies of index sars cases in guangdong province provided initial evidence that the agent responsible for the outbreak was zoonotic in origin. between november and february , the first cases or clusters of sars appeared in several independent geographic locations in the pearl river delta region in southern guangdong, and suggested multiple introductions of a virus or similar viruses from a common source. several of the early cases were reportedly associated with occupations that involved contact with wildlife, including handling, killing and selling wild animals as well as preparing and serving wildlife animal meat in restaurants (xu et al. ). moreover, a study of early sars cases (i.e. those with disease onset prior to january ) compared to those identified later in the outbreak found that % of early-onset cases were food handlers, whereas only %- % of cases between february and april were associated with this occupation. also, early-onset cases were more likely to live within walking distance of animal markets than lateonset cases (xu et al. ) . to confirm the initial epidemiologic association of early-onset patients with animal handling, several groups conducted retrospective serologic surveillance in different human populations in guangdong province during the outbreak period. in one study by xu et al. ( ) , a total of , clinically confirmed human cases were analysed covering the period from november to april , . several observations supported the hypothesis of a wild animal origin for sars. it was observed that early cases of sars occurred independently in at least five different well-separated municipalities in guangdong province. the study also found that early patients were more likely than later patients to report living near a produce market, but not near a farm, and nine of (or %) early patients were food handlers with probable animal contact. several studies revealed a higher than normal seroprevalence of sars-cov antibodies among wild animal traders. guan et al. ( ) found that eight of ( %) wild animal traders sampled from a market in shenzhen, guandong, in had anti-sars-cov antibodies in comparison to from ( %) vegetable traders from the same market. yu et al. ( ) analysed serum samples taken on may , from animal traders in three different live animal markets in guangzhou. out of animal traders surveyed, % had antibodies to sars-cov; % of traders of masked palm civets ( paguma larvata ) were seropositive. interestingly, none of the animal traders had sars or atypical pneumonia diagnosed during the sars outbreak in guangdong, suggesting asymptomatic infection by sars-cov or a closely related sars-like coronavirus. the presence of subclinical infections was corroborated in a separate study conducted by a hong kong group (zheng et al. ) , who found that of (or . %) adults recruited in had antibodies to sars-cov detected by immunofluorescence and virus neutralisation assays. these findings suggest that a small proportion of healthy individuals in hong kong had been exposed to sars-cov-related viruses at least years before the sars outbreak reached hong kong in mid-february . in may , in the middle of the sars outbreak, a joint team from hong kong and shenzhen sampled a total of animals from seven wild and one domestic animal species from a live animal market in shenzhen. it was claimed that these animals were sourced from southern china, and that they had been kept in separate storehouses before delivery to the market. the animals remained in the market for a variable period of time and each stall holder had only a few animals of a given species. animals from different stalls within the market were sampled. nasal and faecal swabs were collected for pcr and virus isolation and, where possible, blood samples were taken for serology. among the six masked palm civets sampled, three were pcr-positive, and a sars-cov-like virus was isolated from four nasal swabs and one faecal swab . in addition, a very closely related virus was isolated from the faecal swab of the only raccoon dog ( nyctereutes procyonoides ) sampled in the study. two chinese ferret badgers ( melogale moschata ) were sampled, and although neither was pcr-positive, one displayed a neutralising antibody titre of : against sars-cov. sequencing of pcr products and virus isolates from palm civets and the raccoon dog revealed several important observations. first, the animal sars-covs were almost identical in sequence to sars-covs isolated from human patients, showing a . % sequence identity. second, the animal sars-covs contained a -nt sequence, located in the c-terminal region of the genome immediately upstream from the n gene; this -nt sequence was absent from most of the human sars-cov isolates. later it was discovered that human sars-covs isolated during the early phase of the outbreaks contained the -nt sequence, suggesting that the deletion event occurred during adaptation of the animal-derived sars-cov to its new human host (the chinese sars molecular epidemiology consortium ). these data indicated that at least three different wildlife animal species in the shenzhen market were infected by a coronavirus that is closely related to sars-cov. this important discovery provided the first direct evidence that sars-cov existed in animals, and that the virus responsible for the sars outbreak most likely originated from animals. determining the route and direction of transmission is important for the understanding of zoonotic disease emergence and for the control of future outbreaks. for sars-cov, there is evidence to suggest that four possible routes of transmission, animal-to-human, animal-to-animal, human-to-human and human-to-animal, occurred during the outbreaks of sars in - and - . when sars-cov was identified as the causative agent of the sars outbreaks, the first question asked was whether this new virus arose from a pre-existing human virus by an evolutionary process which enhanced its virulence or whether it was an animal virus newly introduced into the human population. retrospective serologic studies indicated that there were no antibodies to sars-cov in the human population prior to the sars outbreak, suggesting that sars-cov was not an existing human coronavirus . epidemiologic studies, as discussed above, revealed that animal handlers and people working in the food industry had a higher representation than other groups among early sars patients. molecular epidemiologic studies confirmed that the earliest genotypes of human sars-cov from the - outbreaks were most closely related to those of animal sars-cov isolates ; the chinese sars molecular epidemiology consortium ). during the sporadic outbreaks of - , a total of four patients were independently infected with the sars-cov (liang et al. ). there was no direct link between any of the four cases and none of the patients had direct or indirect contact history with previously documented sars cases; all of them had a history of contact with animals. furthermore, genome sequences of sars-covs from human patients in - were almost identical to those isolated from civets in the market at the same time period, but more divergent from the human sars-covs obtained during the - outbreaks. taken together, these results demonstrated that animal-to-human transmission was responsible for the introduction of sars-cov into the human population. in the market study conducted by guan et al. ( ) , it was shown, by virus isolation, rt-pcr or serum neutralisation assay, that all of the six masked palm civets were exposed to sars-cov. considering that these animals were sampled at the same time in the same market, but originated from different regions of southern china, it is most likely that some, if not all, of them got infected in the market through animal-to-animal transmission. in the same study, it was shown that the raccoon dog isolate (sz ) had an s-gene sequence which was identical to that of one of the civet isolates (sz ) but differed from the other two civet isolates (sz and sz ) which displayed s-gene sequence variation. this observation strongly indicated the occurrence of inter-species transmission among the animals in the market. animal-to-animal transmission has also been demonstrated in experimental situations. martina et al. ( ) showed that ferrets ( mustela furo ) and domestic cats ( felis domesticus ) are susceptible to infection by sars-cov and that they can efficiently transmit the virus to previously uninfected animals that are housed with them. numerous epidemiologic studies documented the rapid human-to-human transmission of sars-cov, which spread the virus to more than countries in less than months (who ). one important example was the spread of sars-cov from mainland china to hong kong by a chinese doctor attending a conference there. through the individuals he infected at a hong kong hotel, this single human source was mainly responsible for the subsequent spread of sars to the rest of the world zhong et al. ) . the major routes of sars cov transmission are believed to be droplets, aerosols and fomites (peiris et al. ) . in general, the average number of secondary cases of infection generated by one infected individual ( r ) was low (see the chapter by real and biek, this volume), approximately . - . (anderson et al. ), a figure much lower than the r of influenza, which ranges from to . however, for countries with a moderate to large number of cases, superspreading events (sses) played a pivotal role in large-scale transmission of the virus. in such circumstances, a few infected individuals caused a much higher number of secondary infections. in addition to the sse in the hong kong hotel, other sses occurred in a hospital setting in hong kong, an air flight from hong kong to beijing and in healthcare settings in beijing, singapore and toronto (anderson et al. ). in the sse in a beijing hospital, one patient infected out of persons that had close contact with the patient. these secondary cases resulted in a further cases before this chain of transmission subsided (shen et al. ). the exact cause for the rapid transmission of sars-cov among the more than residents at the amoy gardens apartment block in hong kong remains a mystery. although there have been reports suggesting environmental spread through u-traps contaminated with sars-cov in bathrooms, other studies also indicated a possible role played by domestic animals such as rats and cats (lu and qu ; martina et al. ; ng ) . domestic cats living in the apartment complex were found to be infected with sars-cov (martina et al. ) , suggesting possible human-to-animal transmission. this notion was supported by the subsequent experimental infection of domestic cats with a human sars-cov isolated from a hong kong patient (martina et al. ) . experimentally infected cats were asymptomatic, but were able to infect other co-housed cats. in another potential example of human-to-animal transmission, sars-cov was isolated from a pig during a surveillance study in farming villages outside of tianjin, where a sars outbreak occurred in the spring of . the genome sequence of the pig isolate (designated tjf) revealed it to be closely related to the human isolate bj obtained from a patient in beijing, km from tianjin, but only distantly related to sars-covs isolated from animals. more importantly, the tjf genome contained the -nt deletion, the genetic feature characterising sars-cov which circulated among human patients during the later phases of the - outbreaks, but never observed in any of the animal sars-cov isolates. the authors concluded that direct human-to-pig transmission was the most likely explanation for these results. during investigations of new zoonotic diseases, it is important to differentiate the roles that different animals may play in distinct stages of disease emergence (see the chapters by childs et al. and childs, this volume) . it is especially important to distinguish between the reservoir host, which may or may not be responsible for direct pathogen transmission to humans, and the intermediate or amplifying host which introduced the pathogen into the human population. due to the sudden emergence of sars, it was extremely difficult to obtain reliable epidemiologic data to pinpoint the source of the outbreak. the vast number of live animals being traded in animal markets in southern china further complicated the investigation process. experimental animal infection studies therefore became an important component of the sars-cov investigation. they provided the proof that sars-cov was the causative agent of sars, helped define the range of animals susceptible to this new virus, elucidated the mechanisms of virus transmission, and established useful animal model(s) for pathogenesis studies and the testing of vaccines and antivirals. since the first experimental infection of cynomolgus macaques by fouchier et al. ( ) , rhesus macaques, african green monkeys, cats, ferrets, mice, pigs, hamsters, guinea pigs and civets have also been shown to be susceptible to experimental infection by sars-cov martina et al. ; roberts et al. ; subbarao et al. ; weingartl et al. ; wentworth et al. ; wu et al. ) . together with the three naturally infected animal species identified in the market study , more than ten different mammalian species have so far been shown to be susceptible to sars-cov. it can be expected that many more susceptible species will be identified in the future. rats have also been identified as another potentially susceptible host and may have played a role in the transmission and spread of sars-cov in the wellpublicised outbreaks of sars in the amoy gardens apartment block in hong kong (ng ) . also, the first confirmed sars case in in guangdong was reported not to have had any contact with animals with the exception of rats (liang et al. ) . in our inoculation studies, we have obtained serologic evidence to indicate that sars-cov was able to replicate asymptomatically in rats (b.t. eaton, l.-f. wang et al., unpublished results) . it is clear that further studies are required to clarify the role played by rats in the transmission of sars-cov. in contrast, two independent studies conducted in canada (weingartl et al. ) and the usa (swayne et al. ) indicated that none of the avian species tested, which included chicken, turkey, goose, duck and quail, was susceptible to sars-cov infection under laboratory conditions. these findings suggest that domestic poultry were unlikely to be the reservoir or associated with dissemination of sars-cov in the animal markets of southern china. in the study by guan et al. ( ) , sars-cov-like viruses were isolated from palm civets and a raccoon dog in a live animal market in southern china and serologic evidence indicted that a third species, the chinese ferret-badger, was also infected by a similar virus. in spite of the diversity of animals susceptible to sars-cov-like viruses, subsequent attention focussed on palm civets, probably because of the larger number of these animals being traded in the market. however, despite the initial speculation that palm civets might be the source of sars-cov, several studies demonstrated that there was no widespread infection in wild or farmed civets and that infection in this and other species in animal markets was more likely a reflection of an "artificial" market cycle in naïve species than an indication of the natural reservoir of sars-cov. the first clue came from serological surveillance conducted by tu et al. ( ) . in this study, a total of civet serum samples were taken from a number of civet farms and a market in different regions of china. no sars-cov antibody was detected in any of the sera taken in june from two different farms in hunan and henan provinces. the same was true for serum samples obtained in january from three different farms in guangdong province. in contrast, out of the serum samples taken from an animal market in guangdong during the same period in january , (or %) had significant level of neutralising antibodies to sars-cov, indicating widespread infection by a virus that is closely related to sars-cov. molecular analysis was used to investigate the distribution and evolution of sars-cov in palm civets and to compare the prevalence of the virus in palm civets in markets and on farms. following the detection of sars-cov in market palm civets at the end of , palm civets were culled in guangdong province in an attempt to prevent the potential reemergence of sars. this provided a unique opportunity for kan et al. ( ) to sample a relatively large number of animals for molecular epidemiological studies. a total of palm civets and raccoon dogs were sampled in the xinyuan animal market in guangzhou in january . the animals were selected from vendors with booths located in four blocks dedicated to the sale of civets and raccoon dogs. pcr analysis indicated that all of the animals sampled were positive and that most animals yielded positive rectal and throat swabs. in the same study, a total of , palm civets were sampled from farms in provinces from january to september , but none of them was positive when analysed by the same pcr tests. these farms were selected on the basis that they used to sell animals to one of the booths at the xingyuan animal market or that they claimed to have previously provided more than % of their animals to markets in guangdong province. in an animal surveillance study conducted in hong kong between the summer of and , poon et al. ( ) sampled wild trapped palm civets in addition to other mammalian, avian and reptile species. serological and pcr analyses indicated that none of the animals surveyed was positive for sars-cov. moreover, when palm civets were experimentally infected with two different strains of human sars-cov, one with a -nt deletion isolated in beijing (qin et al. ) and another containing the -nt sequence isolated in the early phase of the outbreak from guangzhou (gz ), all of the animals developed clinical symptoms including fever, lethargy and loss of aggressiveness . these results indicated that palm civets were equally susceptible to infection by sars-cov with or without the -nt sequence. taken together, the lack of widespread infection in wild or farmed palm civets and the display of overt clinical symptoms following experimental infection suggest that palm civets are unlikely to be the natural reservoir of sars-cov. instead, the animal's high susceptibility to sars-cov and its wide distribution in markets and restaurants made it an ideal amplifying host that is believed to have played an important role in both the major - and sporadic - outbreaks. bats are reservoir hosts of several zoonotic viruses (calisher et al. ) , including the hendra and nipah viruses, which have recently emerged in australia and east asia, respectively (chua et al. ; murray et al. ; wang and eaton ; see the chapter by field et al., this volume). they are susceptible and respond asymptomatically to infection with many viruses (sulkin and allen ; calisher et al. ) . these characteristics and the increasing presence of bats and bat products in food and traditional medicine markets in southern china and other asian countries (mickleburgh et al. ) suggest that bats could be a potential natural reservoir host of sars-cov. recently, two groups have independently reported the presence of sars-like viruses in different species of horseshoe bats within the genus rhinolophus . in one study conducted from march to december of , a total of bats representing nine species, six genera and three families from four locations in china (guangdong, guangxi, hubei and tianjin) were sampled by trapping in their native habitat ). blood, faecal and throat swabs were collected for antibody and pcr analyses. three communal cave-dwelling species from the genus rhinolophus in the family rhinolophidae had a high sars-cov antibody prevalence: of ( %) in r. pearsoni from guangxi; two of six ( %) in r. pussilus from guangxi; and five of seven ( %) in r. macrotis from hubei. the high seroprevalence and wide distribution of seropositive bats is consistent with the pattern of serology expected from a pathogen's wildlife reservoir host (hudson et al. ) . the serological findings were corroborated by pcr analyses using primer pairs derived from the nucleocapsid (n) and polymerase (p) genes of sars-cov. a total of five positive faecal samples were detected, three in r. pearsoni from guangxi and one each in r. macrotis and r. ferrumequinum , respectively, from hubei. genome sequence analysis indicated that sars-like coronaviruses (sl-covs) present in bats have an almost identical genome organisation to those of sars-covs isolated from humans or civets, sharing an overall sequence identity of %. the most variable regions were located in the ′ end of the s gene, which codes for the surface spike protein involved in receptor binding, and in the orf -coding region immediately upstream from the n gene, which contains the coding region for putative nonstructural proteins of unknown function (marra et al. ; rota et al. ) and is known to be prone to mutation and deletions of various sizes song et al. ; the chinese sars molecular epidemiology consortium ). when these regions were excluded from the comparison, the sequence identity increased to % between sl-covs and sars-covs . it was interesting to note that the orf -coding region of bat sl-covs contained the -nt sequence present in civet sars-cov isolates and human sars-cov isolates from the early phase of the outbreak, but absent from human isolates obtained in the later phases of the outbreak (the chinese sars molecular epidemiology consortium ). this finding suggests that sl-covs and sars-covs may have a common ancestor. in another study reported by lau et al. ( ) , it was found that ( %) of anal swabs of wild chinese horseshoe bats ( rhinolopus sinicus ) contained genetic material closely related to sars-cov when analysed by pcr. it was also found that up to % of the horseshoe bats examined contained antibodies to a recombinant n protein of sars-cov. this study was conducted using wild animals from unpopulated areas of the hong kong special administration region of china. analysis of three full-length genome sequences derived from pcr products revealed similar findings to those reported by in that the bat viruses shared an overall % nucleotide and % sequence identity to ten human and civet sars-covs isolated from different locations and at different times during the sars outbreaks, and the major differences were located in the s gene and orf -coding region. the bat viruses from hong kong also contained the -nt sequence in the orf region. the genetic diversity observed among bat-derived sl-covs together with the high prevalence and wide distribution of seropositive bats, as revealed by two independent groups, are consistent with bats being the wildlife reservoir host of sl-covs. as shown by , comparison of partial sequences from sl-covs isolated from three different horseshoe bat species revealed a much higher genetic diversity than those observed among all the reported sequences of civet and human sars-covs. furthermore, sequence analysis also indicated that human and civet sars-cov nestle phylogenetically within the spectrum of sl-covs, suggesting that the viruses responsible for the sars outbreaks were members of this diverse coronavirus group, tentatively named the group b coronaviruses or g b-covs (wang et al. ) . this notion was strengthened by the comparison of genetic relatedness among the different bat viruses detected in hong kong and mainland china. as mentioned above, the overall genome sequence identity between the human or civet sars-cov and the bat viruses rp (isolated from r. pearsoni ) and b (isolated from r. sinicus ) was % and %, respectively. the sequence identity between rp and b is %, suggesting that rp has a closer evolutionary relationship to the civet/human isolates than to the b isolate of a different bat species. further surveillance studies in the region are required to investigate the distribution and diversity of the g b-covs in different bat species, and to find the location and reservoir species of the sars-covs responsible for the sars outbreaks in - and - . emergence of zoonotic viruses maintained by wildlife reservoir hosts is a complex and poorly understood sequence of events. childs ( ) and childs et al., this volume, recognised four transitions in the process by which zoonotic viruses are transmitted and infect other species. two of these transitions, interspecies contact and cross-species virus transmission (i.e. spillover) are essential and sufficient to cause epidemic emergence. two other transitions, sustained transmission and virus adaptation within the spillover species, are not required for emergence, but will determine the magnitude and scope of subsequent disease outbreaks. these transition events are discussed below in relation to the potential mechanism of sars emergence. there are a number of possibilities for contact between horseshoe bats, the putative reservoir host (h r ), and one or more secondary hosts (h s ). this could happen in the bat's natural habitat and in a variety of other situations. horseshoe bats are cave-dwelling animals which feed mainly on moths and beetles and may have the opportunity to come into close proximity with other animals which live in or explore caves. it is interesting to note that in the study by , serological findings indicated that rousettus leschenauti , a much larger cavedwelling fruit bat, may also be infected by a closely related g b-cov, although at much lower frequency. contact between bats and other animal species may also arise because bats are used as a source of medicinal components and live bats are among a large number of different animal species that are traded in animal markets. from the studies by and lau et al. ( ) , we know that the main route of excretion of g b-cov from naturally infected bats is via faecal shedding. the opportunity of virus transmission between h r and h s is therefore further enhanced since direct contact of bats and other animals may not be absolutely required for the virus to pass to a h s . live animal trading in china and asia thus provides the most likely circumstances for inter-species contact. as revealed in an epidemiology study conducted during the peak of the sars outbreaks in china, most animal traders handle more than one animal species, thus providing numerous opportunities for animal-to-animal contact. this could happen during transportation, where animal cages are often piled on top of each other, or in the market where more than different animal species can be housed under a single roof simultaneously. wholesale animal markets or warehouses also offer the possibility of sustained opportunity for inter-species contact because animals may be kept together for an extended time before being sold individually. the notion of inter-species transmission in wholesale or retail markets is supported by the finding in two different studies that g b-covs were detected in civets and raccoon dogs in the market, but not in the farms which claimed to have supplied the animals to the particular markets surveyed (tu et al. ; kan et al. ) . the second transition (i.e. cross-species transmission or spillover) requires not only inter-species contact, but also the susceptibility of h s animals to the virus. for sars-cov, this does not seem to be a major constraint. as discussed above, a large number of mammalian species have been demonstrated to be susceptible to sars-cov infection, either under experimental conditions or by natural infection in markets. spillover is defined as introduction, replication and release of virus from the h s (childs ) . for sars-cov, there was ample evidence to suggest that this has happened in more than one h s species, including civets, raccoon dogs, ferret badgers and humans. three separate surveillance studies indicate that, at least for the civet populations in markets, intra-h s transmission of sars-cov occurred readily tu et al. ; kan et al. ) . sars-cov reactive antibody was found in % of civets in january in one study and % of civets tested in another study contained sars-cov genomic rna. civet trading was banned in may after the first sars outbreaks, but was resumed in august . considering that there was no evidence of widespread infection of sars-cov among civet populations on farms and in the wild (tu et al. ; kan et al. ) , it can be concluded that re-appearance of the virus in the civet population in markets in late to early was a result of separate spillover event(s) which occurred after the resumption of civet trading in august . this would suggest sustainable intra-h s transmission among civets after spillover events. similarly, intra-h s transmission among different human populations was documented in many different cities, especially in guanghzhou, hong kong, beijing, singapore and toronto (anderson et al. ) . it is conceivable that such intra-h s transmission would have been sustained for a much longer period if draconian quarantine measures had not been implemented. virus adaptation is the fourth transition considered to be important in determining the scope and magnitude of a disease outbreak after a spillover event (childs ) . several studies demonstrated rapid evolution of the sars-cov sequence, especially in the receptor-binding domain (rbd) of the spike protein gene, a location believed to be important for virus adaptation to the different h s species, i.e. civet and human. in the first detailed molecular epidemiology study (the chinese sars molecular epidemiology consortium ), sars-covs derived from early, middle and late phases of the sars outbreaks in were analysed by genomic sequencing. it was discovered that genotypes characteristic of each phase could be identified, and that the earliest genotypes were the most similar to those of sars-covs isolated from animals. moreover, it was shown that while the neutral mutation rate of the viral genome was constant during the different phases of the outbreak, the amino acid substitution rate of the coding region slowed during the course of the outbreak, indicating rapid adaptation to the human host. as expected, the spike proteincoding gene showed the strongest initial responses to host selection pressures. in a separate study focusing on sars-covs isolated from humans and civets during the - outbreaks, song et al. ( ) discovered that the ratio of nonsynonymous/synonymous nucleotide substitution in viruses isolated from civets collected year apart and from different geographic locations, was very high. this suggested a rapid process of virus evolution in civets, much like the adaptation process revealed for human sars-cov isolates in the first study (the chinese sars molecular epidemiology consortium ). these results also indicated that civets were not likely to be an h r , and highlighted their potential role as an h s involved in transmitting the virus from bats to humans. the authors concluded that major genetic variations in critical genes, particularly the s gene, are essential for the progression from animal-to-human transmission to sustained human-to-human transmission, which eventually led to the first sars outbreaks in - . the rapid evolution of sars-covs in palm civets in markets in guangdong was also confirmed by kan et al. ( ) who analysed a total of animal-derived sequences isolated in january and compared them to those from animals and humans isolated in . their study revealed that viruses in palm civets in the live animal markets had undergone a process of evolution that generated viruses with the potential to infect humans. within the animal-derived sequences, there were three which did not contain any of the novel signature variation residues (snv) that characterised previously isolated pathogenic viruses. the authors postulated that such viruses were the evolutionary starting point of a process which introduced seven snvs and caused the substitution of six amino acid residues in the spike protein. the resulting virus jumped to humans and was the cause of the low pathogenic infection of humans in - . a further snvs caused amino acid residue changes and resulted in the high-pathogenicity viruses which were responsible for human infection during early phase of the - outbreaks. finally, six snvs with four amino acid changes produced the group of viruses that were responsible for the global epidemic in the middle to late phases of the sars outbreaks. the metallopeptidase, angiotensin-converting enzyme (ace ), has been identified as the functional receptor for sars-cov infection . in a comparative study of binding affinity of different s proteins to human and civet ace , it was shown that s proteins of sars-covs isolated from civet and the mild human cases in bind to human ace much less efficiently than the s proteins of sars-cov isolated from human patients during - outbreaks . similar findings were obtained in a separate study by yang et al. ( ) . it was found that the s protein from viruses isolated from a patient in late and from two civets depended less on the human ace receptor and were markedly resistant to antibody inhibition. these data demonstrated that sars-covs were successful in both maintaining intra-h s transmission among at least two different h s species and in adapting to the new hosts via rapid virus evolution. these attributes made possible the rapid global spread of sars-cov to cause the most severe infectious disease outbreak of the twenty-first century. less than years after the first emergence of sars, rapid progress has been made in the identification and genetic analysis of the aetiological agent and its molecular epidemiology, the identification of the host receptor and molecular characterisation of the virus-host interaction, and the rapid development of diagnostic assays and vaccine and therapeutic candidates. the recent identification of horseshoe bats as the natural reservoir of this new group of g b-covs will undoubtedly play an important role in facilitating our understanding of sars emergence and in the prevention of future outbreaks. bats have been identified or implicated as the natural reservoir host for an increasing number of new and often deadly zoonotic viruses. in addition to the emergence of g b-covs from insectivorous rhinolophus species, hendra virus, nipah virus and, most recently, ebola virus have been shown to have fruit bat reservoir hosts (chua et al. ; halpin et al. ; leroy et al. ; see the chapters by field et al., and gonzalez et al., this volume) . bats typically respond asymptomatically to virus infection and display a capacity to permit persistent virus infections (sulkin and allen ) . their wide distribution and abundant status (one mammalian species in five is a bat) makes them prime candidates for reservoirs of viruses which may, like g b-covs, jump the species barrier and infect humans and other animals. information on the ecology of bats and the nature of their response to virus infection may not only be scientifically interesting, but may also provide fundamental information on how best to cope with further outbreaks of disease caused by bat-borne viruses (calisher et al. ) . bats: important reservoir hosts of emerging viruses sars-associated coronavirus transmitted from human to pig zoonotic viruses of wildlife: hither from yon molecular evolution of the sars coronavirus during the course of the sars epidemic in china nipah virus: a recently emergent deadly paramyxovirus isolation of nipah virus from malaysian island flying-foxes identification of a novel coronavirus in patients with sever acute respiratory syndrome aetiology: koch's postulates fulfilled for sars virus isolation and characterization of viruses related to the sars coronavirus from animals in southern china isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus coronaviridae: the viruses and their replication the ecology of wildlife diseases molecular evolution analysis and geographic investigation of severe acute respiratory syndrome coronavirus-like virus in palm civets at an animal market and on farms a novel coronavirus associated with severe acute respiratory syndrome newly discovered coronavirus as the primary cause of severe acute respiratory syndrome severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats fruit bats as reservoirs of ebola virus angiotensin-converting enzyme is a functional receptor for the sars coronavirus bats are natural reservoirs of sars-like coronaviruses laboratory diagnosis of four recent sporadic cases of community-acquired sars pathology of guinea pigs experimentally infected with a novel reovirus and coronavirus isolated from sars patients animal-to-human sars-associated coronavirus transmission? sars virus infection of cats and ferrets a review of the global conservation status of bats a morbillivirus that caused fatal disease in horses and humans possible role of an animal vector in the sars outbreak in amoy gardens severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome identification of a novel coronavirus in bats aged balb/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans characterization of a novel coronavirus associated with severe acute respiratory syndrome superspreading sars events beijing cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice domestic poultry and sars coronavirus, southern china a cluster of cases of severe acute respiratory syndrome in hong kong identification of a new human coronavirus emerging paramyxoviruses susceptibility of pigs and chickens to sars coronavirus mice susceptible to sars coronavirus summary of probable sars cases with onset of illness from characterization and complete genome sequence of a novel coronavirus, coronavirus hku , from patients with pneumonia civets are equally susceptible to experimental infection by two different severe acute respiratory syndrome coronavirus isolates epidemiologic clues to sars origin in china evasion of antibody neutralization in emerging severe acute respiratory syndrome coronaviruses prevalence of igg antibody to sars-associated coronavirus in animal traders-guangdong province china sarsrelated virus predating sars outbreak hong kong epidemiology and cause of acute respiratory syndrome (sars) in guangdong people's republic of china acknowledgements work conducted in the authors' group on the identification of the natural reservoir host of sars-cov is supported by the australian biosecurity cooperative research centre for emerging infectious diseases (project . r) in collaboration with research activities supported by an nih/nsf "ecology of infectious diseases'' award (no. r -tw ) from the john e. fogarty international center and the v. kann rasmussen foundation. key: cord- -ikomc t authors: van doremalen, neeltje; lambe, teresa; sebastian, sarah; bushmaker, trenton; fischer, robert; feldmann, friederike; haddock, elaine; letko, michael; avanzato, victoria a.; rissanen, ilona; lacasse, rachel; scott, dana; bowden, thomas a.; gilbert, sarah; munster, vincent title: a single-dose chadox -vectored vaccine provides complete protection against nipah bangladesh and malaysia in syrian golden hamsters date: - - journal: plos negl trop dis doi: . /journal.pntd. sha: doc_id: cord_uid: ikomc t nipah virus (niv) is a highly pathogenic re-emerging virus that causes outbreaks in south east asia. currently, no approved and licensed vaccine or antivirals exist. here, we investigated the efficacy of chadox niv(b), a simian adenovirus-based vaccine encoding niv glycoprotein (g) bangladesh, in syrian hamsters. prime-only as well as prime-boost vaccination resulted in uniform protection against a lethal challenge with niv bangladesh: all animals survived challenge and we were unable to find infectious virus either in oral swabs, lung or brain tissue. furthermore, no pathological lung damage was observed. a single-dose of chadox niv(b) also prevented disease and lethality from heterologous challenge with niv malaysia. while we were unable to detect infectious virus in swabs or tissue of animals challenged with the heterologous strain, a very limited amount of viral rna could be found in lung tissue by in situ hybridization. a single dose of chadox niv(b) also provided partial protection against hendra virus and passive transfer of antibodies elicited by chadox niv(b) vaccination partially protected syrian hamsters against niv bangladesh. from these data, we conclude that chadox niv(b) is a suitable candidate for further niv vaccine pre-clinical development. introduction chadox -vectored vaccines fulfil all these requirements, making this a promising platform. the chadox vector is a replication-deficient simian adenovirus vector which has been used to produce several vaccines which are now in clinical development. a common feature of these vaccines is their low reactogenicity, strong immunogenicity, and the absence of vector replication after immunization, an important safety feature. in pre-clinical studies a single dose of chadox vectored vaccines has been shown to be protective against infection with rift valley fever virus, middle east respiratory syndrome coronavirus, mycobacterium tuberculosis and zika virus [ ] [ ] [ ] [ ] . large scale manufacturing has been performed for replicationdeficient adenoviral vectored vaccines for ebola, with one vaccine now licensed and another in advanced clinical development [ , ] . further, a simple thermostabilization process allows for vaccine storage at ambient temperatures [ ] , removing the need for a cold chain for storage and shipping. we now report on pre-clinical immunogenicity and efficacy testing of chad-ox niv b . animal experiment approval was received from the institutional animal care and use committee (iacuc) at rocky mountain laboratories. experiments were performed in an association for assessment and accreditation of laboratory animal care-approved facility by certified staff, following the guidelines and basic principles in the nih guide for the care and use of laboratory animals, the animal welfare act, united states department of agriculture and the united states public health service policy on humane care and use of laboratory animals (protocol # - e and - e). the institutional biosafety committee (ibc) approved work with infectious niv and hendra virus (hev) strains under bsl conditions and sample inactivation was performed according to ibc-approved standard operating procedures for removal of specimens from high containment. henipavirus isolates were obtained from the special pathogens branch of the centers for disease control and prevention, atlanta, ga or public health agency, winnipeg, canada. niv bangladesh (genbank no. ay ), niv malaysia (genbank no. af ), and hev (genbank no. af ) have been passaged three, four, and three times in veroe cells respectively. all virus propagation in this manuscript was performed in veroe cells in dulbecco's modified eagle's medium (dmem, sigma) supplemented with % fetal bovine serum (gibco), mm l-glutamine (gibco), u/ml penicillin (gibco), and μg/ml streptomycin (gibco) ( % dmem). veroe cells were maintained in dmem supplemented with % fetal bovine serum, mm l glutamine, u/ml penicillin and μg/ml streptomycin. the glycoprotein (g) gene from nipah virus (bangladesh outbreak - , genbank accession number: jn . ) was codon optimized for humans and synthesized by geneart (thermo fisher scientific). the synthesized g gene was cloned into a transgene expression plasmid comprising a modified human cytomegalovirus immediate early promoter (cmv promoter) with tetracycline operator (teto) sites and the polyadenylation signal from bovine growth hormone (bgh). the resulting expression cassette was inserted into the e locus of a genomic clone of chadox using site-specific recombination [ ] . the virus was rescued and propagated in t-rex- cells (invitrogen). purification was by cscl gradient ultracentrifugation, and the virus was titered as previously described [ ] . doses for vaccination were based on infectious units (iu). female golden syrian hamsters ( - weeks old) were purchased from envigo. animals were vaccinated i.m. with μl of iu of vaccine or injected i.m. with μl of saline, in each thigh ( μl total volume). for the homologous challenge vaccine experiment, animals were vaccinated at d- and/or d- . for the heterologous challenge experiment, animals were vaccinated at d- . three days prior to vaccination and virus challenge animals were bled via orbital sinus puncture. all animals were challenged with ld of virus in μl dmem via i.p. inoculation: niv bangladesh = . x tcid ; niv malaysia = . x tcid ; hev = . x tcid . we chose the i.p. route as a uniformly lethal challenge route and to be able to compare with previously conducted vaccine experiments [ ] . for each study group, hamsters were utilized. of these, four animals were euthanized (hev) or (niv) days post inoculation and the remaining six animals were followed for days post challenge. weight was recorded daily up to days post infection, and oropharyngeal swabs were taken daily up to days post inoculation in ml of dmem. animals were euthanized when > % of weight loss was recorded, or severe disease signs (e.g. difficulty breathing or paralysis) were observed. upon euthanasia, blood and tissues were collected and subsequently analyzed for virology and histology as approved by iacuc. female golden syrian hamsters ( - weeks old) were purchased from envigo. fifteen animals were vaccinated with either chadox niv b or chadox gfp as described above at and days before serum collection. serum was collected via cardiac puncture, pooled per vaccine group and iggs were purified using the mabtrap kit (sigma) according to manufacturer's instructions from ml of serum. purified iggs were filtered through an . μm filter and diluted to . ml in sterile pbs. ten hamsters were immunized via i.p. injection using μl per hamster. all animals were challenged as described above one day post treatment. for each study group, hamsters were utilized. of these, four animals were euthanized days post challenge and the remaining six animals were followed for days post challenge. weight was recorded daily up to days post challenge, and oropharyngeal swabs were taken daily up to days post inoculation in ml of dmem. animals were euthanized when > % of weight loss was recorded, or severe disease signs (e.g. difficulty breathing or paralysis) were observed. upon euthanasia, blood and tissues were collected and subsequently analyzed for virology and histology as approved by iacuc. virus titrations were performed by end-point titration in veroe cells, which were inoculated with tenfold serial dilutions of virus swab media or tissue homogenates. after hr incubation at ˚c and % co , tissue homogenate dilutions were removed, washed twice with pbs and replaced with μl % dmem. cytopathic effect was scored at dpi and the tcid was calculated from replicates by the spearman-karber method [ ] . added to veroe cells and incubated at ˚c and % co . at dpi, cytopathic effect was scored. the virus neutralization titer was expressed as the reciprocal value of the highest dilution of the serum which still inhibited virus replication. niv-g malaysia (residues e -t , gene accession number nc_ ) was cloned into the phlsec mammalian expression vector [ ] and niv-f malaysia (residues g -d , gene accession number ay . ) was cloned into the phlsec vector containing a c-terminal gcnt trimerization motif [ ] . the constructs were transiently expressed in human embryonic kidney (hek) t cells in roller bottles, as described previously [ ] . supernatant was harvested hours after transfection and diafiltrated using the akta flux system (ge healthcare) against either pbs, ph . (niv-g) or buffer containing mm tris and mm nacl, ph . (niv-f). the proteins were further purified by ni-nta immobilized metal-affinity chromatography using his-trap hp columns (ge healthcare) followed by size exclusion chromatography. niv-g was purified using a superdex / increase gl column (ge healthcare) equilibrated in pbs ph . and niv-f was purified using a superose increase / gl column (ge healthcare) equilibrated in mm tris and mm nacl ph . . maxisorp plates (nunc) were coated overnight at ˚c with μg of g or f protein per plate in carb/bicarb binding buffer ( . g khco and . g na co in l distilled water). after blocking with % milk in pbs with . % tween (pbst), serum ( x serial diluted starting at x dilution) in % milk in pbst was incubated at rt for hr. antibodies were detected using affinity-purified antibody peroxidase-labeled goat-anti-hamster igg (fisher, - - ) in % milk in pbst and tmb -component peroxidase substrate (seracare) and read at nm. all wells were washed x with pbst in between steps. prior to using f and g proteins based on niv malaysia, we established that cross-reactivity with niv bangladesh antibodies was sufficient for usage in elisa by testing sera known to be positive for niv bangladesh antibodies. necropsies and tissue sampling were performed according to ibc-approved protocols. harvested tissues were fixed for a minimum of days in % neutral-buffered formalin and subsequently embedded in paraffin. hematoxylin and eosin (h&e) staining and in situ hybridization (ish) were performed on tissue sections and cell blocks. detection of niv and hev viral rna was performed using the rnascope ffpe assay (advanced cell diagnostics inc., newark, usa) as previously described [ ] and in accordance with the manufacturer's instructions. briefly, tissue sections were deparaffinized and pretreated with heat and protease before hybridization with target-specific probes for niv or hev. ubiquitin c and the bacterial gene, dapb, were used as positive and negative controls, respectively. whole-tissue sections for selected cases were stained for niv and hev viral rna, ubc and dapb by the rnascope vs ffpe assay (rnascopevs, newark, usa) using the ventana discovery xt slide autostaining system (ventana medical systems inc., tucson, usa). a board-certified veterinary anatomic pathologist evaluated all tissue slides. statistical analysis was performed by the log-rank (mantel-cox) test to compare survival curves, and by welch-corrected one-tailed unpaired student's t-test to compare infectious virus titers in tissue. sem was calculated for all samples. p-values < . were significant. to determine efficacy of the chadox niv b vaccine, we vaccinated groups of hamsters with either a single dose at d- or a prime-boost regime at d- and d- . as control groups, we either injected hamsters with chadox gfp at d- and d- or saline at d- ( fig a) . virus neutralizing antibodies could be detected after a single dose of chadox niv b and increased upon a secondary dose (average vn titer ± sem = . ± . after single dose, ± after boost). in contrast, no neutralizing antibodies could be detected in serum obtained from the control groups ( fig b) . all hamsters were challenged with a lethal dose of niv bangladesh ( ld ) via intraperitoneal inoculation on d ( fig a) . all vaccinated animals survived challenge and did not show signs of disease, such as weight loss, at any stage throughout the experiment. this was in contrast to the control groups in which all animals succumbed to disease between d and d and exhibited weight loss (fig c and d) , as well as respiratory and/or neurological signs, including labored breathing and paralyzed hind legs. statistical analysis demonstrated that survival in the vaccinated groups was significant compared to both control groups (p < . ). oropharyngeal swabs were taken daily and assessed for infectious virus by limiting dilution titrations. none of the vaccinated animals shed virus at any timepoint. in contrast, control animals from both groups were found to shed virus at d and d (fig e) . four animals of each group were euthanized at d and lung and brain tissue were harvested. infectious virus could only be detected in lung tissue of animals from both control groups (average titer ± sem = . x ± . x tcid /g of tissue) and was not detected in any tissue of the vaccinated animals ( fig f) . we did not observe any differences between the two control groups. lung and brain tissue harvested at d were then evaluated for pathological changes. none of the vaccinated animals displayed pulmonary pathology and no viral rna was detected in lung tissue by ish. control animals developed pulmonary lesions that were indistinguishable between the two groups. these hamsters developed bronchointerstitial pneumonia that was characterized by multifocal inflammatory nodules that were centered on terminal bronchioles and extend into adjacent alveoli. the nodules were composed of large numbers of foamy macrophages and fewer neutrophils and lymphocytes admixed with small amounts of necrotic debris. in most cases hemorrhage, fibrin and edema admixed with inflammatory cells was observed. edema and fibrin often were extended into surrounding alveoli. alveoli that were adjacent to areas of inflammation were thickened by fibrin, edema and small numbers of macrophages and neutrophils as previously observed in niv infected hamsters [ ] . there was abundant viral rna demonstrated by ish in areas of inflammation (brown staining). the viral rna was predominantly found in type i pneumocytes but was also multifocally present in vascular and bronchiolar smooth muscle and endothelial cells (fig ) . to determine efficacy of chadox niv b against niv malaysia and hev, groups of hamsters were vaccinated with a single dose of chadox niv b or a single dose of chadox gfp at d- ( fig a) . as before, virus neutralizing antibodies could be detected after vaccination with chadox niv b but not upon injection with chadox gfp (average vn titer ± sem = . ± . ) ( fig b) . subsequently, hamsters were challenged with either niv malaysia or hev ( ld ) via intraperitoneal inoculation on d (fig a) . all vaccinated animals challenged with niv malaysia survived with no signs of disease such as weight loss at any stage throughout the experiment. in contrast, animals challenged with niv malaysia that received chadox fgp all succumbed to infection between d and d . these animals experienced weight loss and respiratory and neurological signs (fig c and d) . statistical analysis demonstrated that survival in the vaccinated group was significantly different from the control group (p = . ). oropharyngeal swabs were taken daily and assessed for infectious virus. none of the vaccinated animals challenged with niv malaysia shed virus at any timepoint. in contrast, control animals challenged with niv malaysia were found to shed virus at d and d (fig e) . four animals from both groups were euthanized at d and lung and brain tissue were harvested. infectious virus could only be detected in lung and brain tissue of animals from the control group (average virus titer lung ± sem = . x ± . x tcid /g, brain ± sem = . x ± . x tcid /g) and was not detected in any tissue of the vaccinated animals ( fig f) . four out of six vaccinated animals challenged with hev succumbed to disease between d and d . the two survivors showed minimal weight loss (< %) and no signs of disease. animals that received chadox fgp all succumbed to hev infection between d and d . these animals showed weight loss as well as respiratory and neurological signs (fig c and d) . logrank (mantel-cox) test demonstrated that survival in the vaccinated group was significant (p = . ) compared to the control group. oropharyngeal swabs were taken daily and assessed for infectious virus. none of the vaccinated animals challenged with hev shed virus at any timepoint. in contrast, control animals challenged with hev were found to shed virus at d , d and d (fig e) . four animals from both groups were euthanized at d and lung and brain tissue were harvested. infectious virus was detected in three out of four lungs of the vaccinated animals and all lungs of the control animals (average virus titer ± sem = . x ± . x and . x ± . x tcid /g tissue for vaccinated and control animals, respectively). no statistical difference in infectious virus titer was found between the two groups using an unpaired onetailed student's t-test (p = . ). infectious virus was only detected in brain tissue of animals from the control group (average titer ± sem = . x ± . x tcid /g) and not in vaccinated animals (fig f) . harvested lung tissue was then evaluated for pathological changes. all four groups of hamsters developed pulmonary lesions. all animals challenged with hev and control animals challenged with niv malaysia developed bronchointerstitial pneumonia which was indistinguishable from the lesions described for the control animals in the homologous challenge study. vaccinated hamsters challenged with niv malaysia developed mild to moderate bronchointerstitial pneumonia and did not display any evidence of pulmonary edema, fibrin or hemorrhage. ish demonstrated viral rna predominantly in type i pneumocytes and rarely in vascular and bronchiolar smooth muscle and endothelial cells in animals challenged with hev and control animals challenged with niv malaysia. in vaccinated animals challenged with niv malaysia, however; there was very little rna present and only in type i pneumocytes in areas of inflammation (fig ) . finally, we wanted to assess the protective effect of antibodies elicited after chadox niv b vaccination. two groups of hamsters were either vaccinated with chadox niv b or injected with chadox fgp at d- and d- . all animals were bled at d and we collected and ml respectively. igg was purified from ml pooled serum. ten animals per group were then injected peritoneally with purified igg. animals were challenged with a lethal dose of niv bangladesh ( ld ) one day post passive transfer ( fig a) . we were unable to detect neutralizing antibodies in serum obtained at d from four hamsters from each group. however, serum from animals treated with niv antibodies was positive by elisa against niv g protein, albeit with a lower reciprocal titer than antibodies in serum obtained from single-dose vaccinated animals (fig b) . one out of six animals treated with niv antibodies succumbed to disease on d . no weight loss was observed, however the animal showed severe neurological signs. none of the other niv antibody-treated animals experienced weight loss or signs of disease. four out of six animals treated with gfp antibodies succumbed to disease between d and d . these animals showed weight loss and respiratory or neurological signs. the two surviving animals did not show any signs of disease throughout the experiment. one of these animals did not seroconvert as measured by elisa against niv f and g protein, and it was suspected this animal was not infected. therefore, this animal was excluded from the survival curve. the log-rank (mantel-cox) test demonstrated that survival in the treated group was significant (p = . ) compared to the control group (fig c and d) . oropharyngeal swabs were taken daily and assessed for infectious virus. shedding was minimal and found in one animal treated with niv antibodies on d , and five animals treated with gfp antibodies between d and d (fig e) . four animals from both groups were euthanized at d and lung and brain tissue were harvested. infectious virus could only be detected in lung tissue of animals treated with gfp antibodies and was not detected in any tissue of the animals treated with niv antibodies (fig f) . lung tissue harvested at d was then evaluated for pathological changes. both groups of hamsters developed pulmonary lesions similar to those described in the homologous challenge study, however; the niv antibody-treated hamsters developed mild to moderate pulmonary lesions whereas the control animals developed severe lesions. additionally, none of the niv antibody-treated hamsters displayed any pulmonary fibrin, edema or hemorrhage. ish demonstrated viral rna in type i pneumocytes in areas of inflammation. abundance of viral rna was notably less in animals treated with niv antibodies (fig ) . niv is a re-emerging infectious disease which causes outbreaks with a high case-fatality rate. no licensed vaccine against niv currently exists, and it is therefore key that a safe and effective vaccine be developed. several vaccine candidates have been explored in different animal models. these can be categorized as subunit vaccines or live-vectored vaccines that target the niv outer membrane proteins g and/or f. protection against disease and lethality has been shown in hamsters [ , ] , pigs [ , ] , african green monkeys [ ] [ ] [ ] , cats [ ] , and ferrets [ , ] . efficacy is thought to be mediated by neutralizing antibodies, as passive transfer of chadox nivb efficacy in the syrian golden hamster antibodies in naive animals also results in protection against disease [ , ] . these approaches are promising, but no vaccine candidates have so far been moved into clinical trials. in the studies presented here, we tested the efficacy of a vaccine based on niv bangladesh g protein in a replication-deficient simian adenovirus vector in syrian hamsters. a primeonly as well as a prime-boost regime protected syrian hamsters against challenge with a lethal dose of niv bangladesh and niv malaysia, and partially protected against hev challenge. furthermore, antibodies elicited by vaccination alone provided partial protection against a niv bangladesh challenge. two genetic lineages of niv have been described; niv malaysia and niv bangladesh [ ] [ ] [ ] . although niv malaysia has not caused an outbreak in humans since , the virus was isolated from pteropus vampyrus, pteropus hypomelanus and pteropus lylei in malaysia and cambodia [ ] [ ] [ ] and another spillover event could occur. having one vaccine that protects against both lineages of niv virus would be the easiest and cheapest countermeasure. a singledose vaccination with chadox niv b , which is based on niv bangladesh, fully protected syrian hamsters against lethal disease caused by niv malaysia. the g proteins of the niv strains used in this study are . % pairwise identical on the amino acid level, with amino acid differences scattered throughout the protein. although we did not see sterile protection against niv malaysia, none of the vaccinated animals showed signs of disease and all were protected against lethal disease. these results suggest that chadox niv b could protect against both lineages of niv. like niv, hev is a species in the henipavirus genus and thus we investigated cross-protection of chadox niv b against a lethal challenge with hev in syrian hamsters. the g protein of the hev strain used in this study was . % identical to the chadox niv b g protein; amino acids differ between the two proteins. chadox niv b only protected partially against hev challenge; four out of six animals did not survive challenge. we observed a non-significant decrease in infectious hev titer in lung and brain tissue of vaccinated animals compared to control animals. it is possible that disease progression in vaccinated animals is delayed compared to control animals. this is supported by the delay in time to death; whereas the average time to death is days in control animals, it is days in vaccinated animals. cross-protection of niv or hev vaccines has been studied by other groups as well. an adeno-associated virus vaccine expressing niv g protein offered % protection against a lethal challenge with hev in hamsters [ ] . in contrast, vaccines based on hev provide full protection against niv in the ferret and nhp model [ , , ] . likewise, high levels of crossprotective antibodies were found in sera from hev-infected individuals, whereas cross-protective antibodies were limited in niv-infected individuals [ ] . this might be caused by induction of a more robust and cross-reactive immune response by native hev protein compared to niv protein, as suggested by bossart et al. [ ] . human cases of hev are associated with direct contact with infected horses, the intermediate animal host of hev, and direct contact with bats or their products has not yet been associated with hev infection in humans [ ] . it is therefore likely that prevention of hev in horses will completely prevent human cases. currently, a hev vaccine (equivac) is available for horses and fully protects against hev [ ] . furthermore, the total number of human cases that contracted hev is relatively low at [ ] . thus, the requirement of a human vaccine for hev is therefore less urgent than that of a niv vaccine. previous work has shown that the humoral immune response to niv vaccination is sufficient to protect syrian hamsters against a lethal challenge with niv [ , ] . likewise, administration of a human neutralizing monoclonal antibody (m . ) provided full protection against both hev and niv in multiple animal models [ , ] . administration of purified igg obtained from chadox niv b vaccinated hamsters provided partial protection against niv challenge. furthermore, infectious virus could only be detected in the lungs of control animals and not in the lungs of vaccinated animals, and thus as in previous studies, chadox niv belicited antibodies are able to provide protection against a lethal challenge with niv. although we were able to detect niv g protein-specific antibodies in serum obtained from niv antibody-treated animals, the reciprocal titer was much lower than that detected in serum from syrian hamsters after a single dose of chadox niv b . it is possible that administering a higher dose of igg would have led to uniform protection. two animals treated with igg purified from animals which received injections with chad-ox fgp survived a lethal challenge with niv bangladesh. occasional survival has been observed in the syrian hamster model [ ] . the increased survival rate might however also reflect a non-specific effect of treatment with igg, which has been reported previously [ ] . as the survival rate was significantly different between the niv igg-treated group and the control igg-treated group, the passive transfer experiment shows that antibodies elicited by chad-ox -niv b are sufficient for protection against a lethal challenge with niv. animals in the passive transfer experiment were observed for days, to ensure that the two animals that survived would not succumb to disease after days. the syrian hamster is a suitable initial small animal model to investigate the efficacy of niv vaccines, followed by the african green monkey [ ] . the immune system of african green monkeys is more like humans than that of hamsters and is therefore seen as a more relevant animal model to test niv vaccines. based on the results presented in the current manuscript, future studies are planned to test chadox niv b in african green monkeys, supported by the coalition for epidemic preparedness innovations (cepi). we show that chadox niv b provides complete protection against lethal disease in syrian hamsters challenged with niv bangladesh. furthermore, chadox niv b vaccination results in complete survival but with limited evidence of viral replication after niv malaysia challenge, and partial protection against hev. passive transfer of antibodies elicited by chadox niv b vaccination provide partial protection against lethal challenge with niv bangladesh. nipah virus: a recently emergent deadly paramyxovirus pteropid bats are confirmed as the reservoir hosts of henipaviruses: a comprehensive experimental study of virus transmission pathogenic differences between nipah virus bangladesh and malaysia strains in primates: implications for antibody therapy nipah virus outbreaks in the who south-east asia region person-to-person transmission of nipah virus in a bangladeshi community nipah virus transmission from bats to humans associated with drinking traditional liquor made from date palm sap, bangladesh date palm sap linked to nipah virus outbreak in bangladesh convergence of humans, bats, trees, and culture in nipah virus transmission foodborne transmission of nipah virus genetic characterization of nipah virus outbreak investigation of nipah virus disease in kerala, india isolation and full-genome characterization of nipah viruses from bats hendra virus ecology and transmission late-onset nipah virus encephalitis years after the initial outbreak: a case report functional studies of host-specific ephrin-b ligands as henipavirus receptors blueprint for r&d preparedness and response to public health emergencies due to highly infectious pathogens heterologous two-dose vaccination with simian adenovirus and poxvirus vectors elicits long-lasting cellular immunity to influenza virus a in healthy adults chimpanzee adenovirus vaccine provides multispecies protection against rift valley fever protective efficacy of a novel simian adenovirus vaccine against lethal mers-cov challenge in a transgenic human dpp mouse model identification and evaluation of novel protective antigens for the development of a candidate tuberculosis subunit vaccine rational zika vaccine design via the modulation of antigen membrane anchors in chimpanzee adenoviral vectors safety and immunogenicity of a recombinant adenovirus type- vector-based ebola vaccine in healthy adults in sierra leone: a single-centre, randomised, double-blind, placebo-controlled, phase trial safety and immunogenicity of novel adenovirus type -and modified vaccinia ankara-vectored ebola vaccines: a randomized clinical trial potency of a thermostabilised chimpanzee adenovirus rift valley fever vaccine in cattle a novel chimpanzee adenovirus vector with low human seroprevalence: improved systems for vector derivation and comparative immunogenicity preventing spontaneous genetic rearrangements in the transgene cassettes of adenovirus vectors single-dose live-attenuated nipah virus vaccines confer complete protection by eliciting antibodies directed against surface glycoproteins. vaccine beitrag zur kollektiven behandlung pharmakologischer reihenversuche a time-and cost-efficient system for high-level protein production in mammalian cells biochemical, conformational, and immunogenic analysis of soluble trimeric forms of henipavirus fusion glycoproteins rnascope: a novel in situ rna analysis platform for formalin-fixed, paraffin-embedded tissues comparison of the pathogenicity of nipah virus isolates from bangladesh and malaysia in the syrian hamster a vlp-based vaccine provides complete protection against nipah virus challenge following multiple-dose or single-dose vaccination schedules in a hamster model recombinant nipah virus vaccines protect pigs against challenge chadox nivb efficacy in the syrian golden hamster plos neglected tropical diseases protection against henipaviruses in swine requires both, cell-mediated and humoral immune response a hendra virus g glycoprotein subunit vaccine protects african green monkeys from nipah virus challenge single-dose live-attenuated vesicular stomatitis virus-based vaccine protects african green monkeys from nipah virus disease recombinant measles virus vaccine expressing the nipah virus glycoprotein protects against lethal nipah virus challenge a recombinant subunit vaccine formulation protects against lethal nipah virus challenge in cats single injection recombinant vesicular stomatitis virus vaccines protect ferrets against lethal nipah virus disease vaccination of ferrets with a recombinant g glycoprotein subunit vaccine provides protection against nipah virus disease for over months nipah virus: vaccination and passive protection studies in a hamster model characterization of nipah virus from naturally infected pteropus vampyrus bats isolation of nipah virus from malaysian island flying-foxes nipah virus in lyle's flying foxes, cambodia protection against henipavirus infection by use of recombinant adeno-associated virus-vector vaccines feline model of acute nipah virus infection and protection with a soluble glycoprotein-based subunit vaccine neutralization assays for differential henipavirus serology using bio-plex protein array systems changing resource landscapes and spillover of henipaviruses hendra virus vaccine, a one health approach to protecting horse, human, and environmental health a neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute nipah virus infection a neutralizing human monoclonal antibody protects african green monkeys from hendra virus challenge a protective monoclonal antibody targets a site of vulnerability on the surface of rift valley fever virus development of an acute and highly pathogenic nonhuman primate model of nipah virus infection we would like to thank the animal care takers for their excellent care of the animals, and anita mora for assistance with figures. benjamin carrasco provided outstanding assistance with the preparation for animal experiments. greg saturday, kimberly meade-white and kathleen cordova were instrumental in assistance during the animal studies. we thank benhur lee for kindly providing the c-dna for niv-f. key: cord- -ktajrf d authors: monagin, corina; paccha, blanca; liang, ning; trufan, sally; zhou, huiqiong; wu, de; schneider, bradley s.; chmura, aleksei; epstein, jonathan; daszak, peter; ke, changwen; rabinowitz, peter m. title: serologic and behavioral risk survey of workers with wildlife contact in china date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: ktajrf d we report on a study conducted in guangdong province, china, to characterize behaviors and perceptions associated with transmission of pathogens with pandemic potential in highly exposed human populations at the animal-human interface. a risk factor/exposure survey was administered to individuals with high levels of exposure to wildlife. serological testing was performed to evaluate prior infection with several wildlife viral pathogens. follow up serology was performed on a subset of the cohort as well as close contacts of individuals. , individuals were enrolled in the study. contact with a wide range of wildlife species was reported in both occupational and occasional contexts. the overall proportion of individuals seropositive to any of the tested wildlife pathogens was approximately . %. however, persons employed as butchers demonstrated a seropositivity of . % to at least one pathogen of interest. by contrast, individuals working as hunters had lower rates of seropositivity. among the study population, a number of other behaviors showed correlation with seropositivity, including contact with particular wildlife species such as field rats. these results demonstrate the need to further explore zoonotic risks of particular activities regarding wildlife contact, and to better understand risks of persons working as butchers with wildlife species. the majority of human infectious diseases have an animal origin, therefore understanding the human-animal interface as it relates to disease emergence and risk is of upmost importance [ ] . the increasing frequency and variety of human-wildlife interactions in china provide opportunities for the transmission of zoonotic pathogens from animals to humans [ ] . a a a a a handling, transporting, and butchering of hunted or farmed wildlife poses a risk of pathogen spillover into humans [ ] . in southern china provinces, including guangdong, a significant percentage of the population obtains fresh meat for consumption from wet markets, community markets that specialize in selling and butchering live animals, including animals that are rare and endangered. research has demonstrated that human-animal interfaces, such as within these wet markets, provide an ideal environment for infectious disease emergence, transmission, and amplification [ ] [ ] [ ] [ ] . wet markets and restaurants that butcher and sell wild animals are common in china and south east asia, creating a high-risk interface where humans come into regular contact with the blood and bodily fluids of wild and domesticated animals. intermingling of wildlife in wet markets can lead to inter-species transmission of pathogens, amplifying and maintaining these pathogens, and may result in spillover into a species that can more efficiently transmit the pathogen to humans [ ] . furthermore, increasing numbers of individuals are traveling to urban-rural interface areas to eat at "wildlife" restaurants. hunted or farmed wildlife are transported and kept alive at these restaurants until consumption, increasing the risk of pathogen spillover. the wildlife trade has played a role in the emergence of severe acute respiratory syndrome (sars), avian influenza, ebola, and other diseases of wildlife origin [ , ] . guangdong province in china was the site of the first cases of sars in . now known to have originated in bats [ ] , sars emerged in humans and other mammals in wet markets, such as himalayan palm civets (paguma larvata) and raccoon dogs (nyctereutes procyonoides) [ ] . the disease spread to countries and infected over , people; killing nearly [ ] . sars shut down trade of domestic animals and resulted in nearly $ billion in losses to the global economy [ ] . h n outbreaks during - and h n in have demonstrated the realities of the risk of disease transmission and spread in china and the detriments to animal and human health, as well as human livelihoods [ , ] . these epidemics shed new light on how quickly and how far newly emerged zoonotic diseases can spread in today's world. these outbreaks continue to challenge scientists, policymakers, and public health systems, exemplifying the need for a better understanding of the factors leading to emergence, and clarity regarding emerging infectious disease policy, particularly as it concerns high-risk human-animal interfaces. although mounting evidence implicates illegal trade and behavioral and cultural factors as influencing disease emergence, it is clear that more research is needed to identify these drivers [ , , , , ] . a key aspect of understanding human-wildlife interfaces is the characterization of risk behaviors and perceptions of individuals who have wildlife contact. specifically, there is a need to evaluate whether particular behaviors are associated with increased risk of transmission. as well, serological studies may be helpful to evaluate exposure, either current or prior, to zoonotic agents. for example, when guan et al. ( ) surveyed animals in markets for evidence of sars-like coronavirus [ ] , they also performed serological analysis on market employees. seroprevalence in their study ranged widely between traders (wild-animal to vegetable), butchers and community controls. the present study focuses on the potential for zoonotic viral transfer through contact with wildlife in guangdong prefectures in china, and seeks to augment our understanding and identification of risky populations, occupations, and behaviors, as well as the perceptions of risk at these interfaces. we performed a serological survey and concurrent behavioral questionnaire of individuals with wildlife contact in guangdong province, china, in order to better characterize occupations and community-level behavioral risks that contribute to zoonotic transmission of various wildlife pathogens with pandemic potential. this study took place in guangdong province ( ˚ 'n, ˚ 'e), china from - . guangdong is located on the south china sea coast, bordering guangxi province, hunan province, jiangxi province and fujian province. it neighbors hong kong and macau, and has over million residents living in cities: guangzhou, shenzhen, dongguan, foshan, zhongshan, zhuhai, jiangmen, huizhou and zhaoqing. enormous consumption of animal and animal products in this region is believed to serve an economical incentive for butchers, cooks and hunters in less developed guangdong prefectures, e.g. qingyuan, yunfu and meizhou. guangdong province has consistently reported the largest population in china since , with the highest gdp rankings for the last years. its economy is highly dependent on the pearl river delta region, which contributes around . % total provincial gdp while supporting . % of the provincial population. the study was conducted in prefectures within the guangdong province: dabu, jiaoling, pingyuan, lianping, heping, lianshan, lianzhou, yunfu, yunan, xinyi, deqing and fengkai (fig ) . we chose field sites within the identified prefecture areas that have wet-markets, wildanimal restaurants, and other environments with a higher risk of animal-to-human pathogen transmission. these environments, particularly the markets, were deliberately chosen due to their size and character. similar species of wildlife, both live and butchered, were present, with similar butchering methods being utilized. populations in these environments with high-risk animal-to-human pathogen transmission activities, such as hunting, and butchering in wetmarkets and wild-animal restaurants were selected. we targeted high-risk individuals, defined as individuals with high levels of exposure to wildlife (wild animal blood or bodily fluids)-primarily hunters, persons working in wet markets and restaurants that butcher wild game, who could be followed over a period of time. inclusion criteria included participant age between - , and individuals that reported previously hunting, selling, butchering, or eating wild animals. individuals who did not provide informed consent and/or did not agree to biological sample collection were excluded. individuals were residents of the site area so that they were available for follow up visits. more than potential field sites were visited to gauge level of risk at the site (number of high-risk animal-human interfaces). local prefecture-level chinese center for disease control (cdc) teams were asked to pre-enroll individuals who fit the inclusion criteria and would be available for follow up visits for up to three years. based on pre-enrollment numbers and human-animal interfaces identified at each site, field sites that were identified in the previously named prefectures: dabu, jiaoling, pingyuan, lianping, heping, lianshan, lianzhou, yunfu, yunan, xinyi, deqing and fengkai were chosen to participate in the study (fig ) . study participation was voluntary and respondents were given the equivalent of usd (approximately cny) as compensation for their time. informed consent was obtained from all enrolled participants prior to enrollment and specimen collection. in total, , participants were enrolled in the first round of the study. the prefecture-level cdc research teams were trained in all enrollment procedures (biological and behavioral questionnaire). ten ( ) ml of peripheral venous blood was collected using pre-labeled vacuum edta blood collection tubes. all blood samples were chilled immediately at ˚c, and transferred to participating cdc laboratories at surveillance sites within hours after sampling. upon arrival of blood samples into the laboratories, sera were isolated by , g centrifugation for min. serum was aliquoted, flash frozen in liquid nitrogen, and then stored at - ˚c. all study participants responded to a detailed questionnaire including questions about demographics, occupation, rural vs. urban habitation, zoonotic disease risk perceptions, and contact with particular types of wildlife through hunting, butchering, or eating. the questionnaire was based on a previous study in cameroon and is available in the s file [ , ] . it was adapted in english for the study environment in china and then translated into simplified chinese. during the training of the local cdc research teams, pretesting of the questionnaire was done to ensure the intelligibility of the questions and proper translation. pretesting was also done with the study population during the pre-enrollment phase for the study sites. during enrollment, a trained member of the local cdc team who was fluent in the local language of the study site administered the questionnaire. in guangdong province, both mandarin and cantonese are spoken in the study sites. the questionnaire was administered in either mandarin or cantonese depending on the respondent. all follow up study respondents were contacted by a member of the trained research team regarding their test results. seropositive individuals or those with indeterminate results were referred to a local clinic/hospital where they could follow up on their results. this study did not provide additional financial or medical support for respondents who tested positive. all results were kept confidential and only reported to the respondent themselves. individuals who tested seropositive from the first enrollment were asked to participate in an additional follow up sampling visit. they were also asked to invite up to three of their close contacts to enroll in the follow up study. we aimed to continue behavioral information and biological sample collection towards gauging virus transmissibility. prior to sampling, we re-consented seropositive individuals and consented their close contacts. due to geographic reallocation of some respondents, we were unable to locate individuals with indeterminate or seropositive results for inclusion in our follow up study. we successfully re-enrolled seropositive respondents and of their close contacts in our follow-up study (fig ) . the serological investigation focused on detection of hantavirus, sars cov, and severe fever with thrombocytopenia syndrome (sfts) bunyavirus. this study aimed to demonstrate viral transfer associated with exposure to wildlife and the virus targets for this study were selected based on prevalence and infection associated with animal/wildlife contact as well as an interest by the gdcdc [ ] [ ] [ ] . to determine hantavirus specific antibodies in study respondents, stored human sera were tested using hantavirus hantaan igg/igm elisa in a single well for each sample (ibl, hamburg, germany) [ ] . microtiter plates were pre-coated with recombinant nucleo-capsidprotein of hantaan virus. according to the manufacturer's instructions, control groups and experiment group sera were diluted at : using ×pbs, ph . , and incubated at ˚c. coated wells were washed, and then incubated with μl diluted peroxidase conjugated anti-igg enzyme at ˚c. color development reaction was initiated by adding a chromogenic substrate, tetramethylbenzidine, followed by a minute incubation at room temperature. reaction was quenched by adding μl . m h so , and then measured at nm to obtain its optical density. human sera were also tested for sars cov using the sars coronavirus igg diagnostic kit (bgi, shenzhen, china) [ ] [ ] [ ] . microtiter plates were pre-coated with vero e cell lysate. cells were previously infected by sars coronavirus that was isolated during the sars outbreak in in china. sera preparation and indirect elisa were conducted as described above. the average optical density (od) of negative control sera plus . was used as the cut off value as suggested by the manufacturer. we adopted recombinant coating antigen in the diagnosis of sfts bunyavirus using indirect elisa. gene encoding ×his tagged sfts bunyavirus hb nucleocapsid (n) protein was expressed in e. coli, and purified using nickel affinity chromatography. recombinant anti-sftsv igg was immobilized in -well plate as coating antigen. sera were diluted at : using ×pbs, ph . , and incubated at ˚c. we used horseradish peroxidase (hrp)-conjugated anti-human igg antibody (sigma, saint louis, mo) as the secondary antibody for color development. od was measured at nm, with a reference wavelength of nm. cut-off value was determined by adding × sd to the mean of od values resulting from analyses of negative control sera. moreover, a microneutralization assay was performed to detect neutralizing antibodies against sfts bunyavirus [ ] . this was done to ensure the highest level of certainty given the newness of the sfts virus assays. fifty ( ) μl serially diluted human sera were mixed with an equal volume of tcid / . ml of sfts bunyavirus hb , and then incubated at ˚c for . h. incubated mixture was added to cultured vero cells in a -well plate in quadruplicate. subsequently, the plates were incubated at ˚c with . % co for d. viral infection was assessed via an immunofluorescence assay using mouse polyclonal anti-sfts bunyavirus antibody. end-point titer was then expressed as the reciprocal of the highest dilution of serum with viral infection prevention capability. laboratory results and behavioral data from the questionnaire were manually entered twice (double entry system) and compared for entry error. hand checks were also done on . % of the database to further reduce chances of human entry error. questionnaire responses and serological test results were then analyzed and compared using sas (version . ; sas institute inc., cary, nc, usa). we assigned primary occupation to the categories of hunter, agriculture worker, butcher, market worker, and restaurant worker based on questionnaire responses. we also aggregated reported behaviors to assign an "ever" status to each individual in terms of eating, butchering, or hunting wildlife. basic descriptive univariate statistics were carried out on the data set. we then examined bivariate associations between serological status for the three viruses of interest and independent demographic and risk behavior variables. variables showing bivariate association with seropositivity at a p value of . or less were included in multivariate logistic models for seropositivity to individual viruses or any of the three viruses using firth's penalized likelihood adjustment to address small sample size and possible collinearity [ ] . since butchers were the only occupational group showing increased risk of seropositivity, we also performed a subanalysis of specific behaviors performed by butchers, again including behaviors with bivariate association of p = . or less in a multivariate logistic model. a total of , individuals provided a baseline blood sample for serological testing and completed a behavioral risk factor survey. the demographics of these individuals are shown in table . the study population was predominantly younger than age , with a mean age of . ± . years. junior high school was most frequently reported as the highest level of education attained. most of the respondents were married or living with their partner and had between - children, with a total of - people living in the house. traveling to a forest at least once a month, which may provide additional exposure to wild animals, was reported by . % of respondents (data not shown). some of the most common reasons for traveling to the forest were fieldwork ( . %), gathering of fruit and vegetables ( . %), collecting firewood ( . %), and hunting ( . %). more respondents reported ever living in a city than not, with the most common urban occupations being restaurant work followed by "other" and factory jobs. table describes type and location of occupational activities. restaurant-related occupations such as cook or kitchen worker were the most commonly reported primary occupation, followed by agriculture, butcher, and jobs working in a market. table summarizes reported exposures to wild animals. almost all respondents indicated that they had ever touched any live or dead wild animal. having butchered and eaten wild animals were the most common types of animal interactions, and injury was experienced by less than a quarter of the respondents. table shows the beliefs and reported protective behavior regarding contact with animals and zoonotic disease transmission. less than one third of respondents reported believing that they could get infected from contact with blood or other types of contact with wild animals. a smaller proportion reported taking at least one type of protective measure to prevent infection in such circumstances. among those who reported taking protective measures, use of gloves, and avoiding touching the animal were the most commonly reported protective practices. risk factors for seropositivity. table summarizes the results of baseline testing of blood for evidence of antibodies against zoonotic wildlife viruses. the overall rate of seropositivity to any of the other three tested pathogens (hantavirus, sars cov, or sfts bunyavirus) was low among the study population (approximately %). seropositivity in respondents was only found in the first phase of enrollment. among contacts of these seropositive respondents (n = ), all individuals were seronegative for the pathogens of interest. the most common seropositivity was for hantavirus ( . % of the study population). however, among the persons reporting that their occupation was a butcher, the rate of seropositivity to at least one pathogen was higher ( . %), and this elevated risk was statistically significant (fisher's exact test, p = . ). butchers had elevated seropositivity rates to all three of the viruses compared to the overall study population. no other occupational group showed an increased risk of seropositivity. in a multivariate model of the butchers, those who reported butchering porcupines showed an elevated risk of seropositivity (data not shown). there was no increased risk among the butchers associated with not wearing gloves or taking fewer protective measures. by contrast, the persons reporting hunting as a profession showed a decreased risk for seropositivity as did those reporting their profession as housework. no other occupational groups showed significantly increased or decreased risk. persons who reported ever hunting also had a lower risk of seropositivity compared to those who never hunted. age and gender did not show a significant association with seropositivity, nor did reporting living in a city or going frequently into the forest. while persons reported butchering as a profession, a larger number ( ) reported ever butchering a wild animal; this was associated with an elevated risk of seropositivity, however when the professional butchers were removed from this group, the risk was no longer significant. as shown in the table s table, reported contact with certain wildlife species such as civets (for hanta and bunyavirus) and field rats (for sars) showed elevated risk for certain viruses in bivariate analyses. in multivariate models of specific wildlife contact, (table ) , the principal association remaining significant was eating civets (associated with hantavirus seropositivity). the risk estimate for bunyavirus was elevated but did not reach statistical significance. this study, examining the association between reported wildlife contact and seropositivity for wildlife zoonotic viruses, found detectable levels of antibodies for several pathogens in the population surveyed. overall, the population had contact with multiple species of animals. among reported primary occupations, working as a butcher was associated with an increased risk of infection with wildlife pathogens, while professional hunters were at decreased risk, and no increased risk was seen for market or restaurant workers. contact with certain animals, such as civets and field rats, showed evidence of increased risk of seropositivity. few people in the study reported using protective measures such as gloves when working with wild animals, and less than a third of individuals reported believing that they could become infected through wildlife contact. when seropositive individuals were followed over time, no evidence of infection of close contacts was seen, although the number of contacts enrolled was small and there are limited conclusions that can be draw about alternative transmissions risks. further investigation is warranted to gauge transmissibility of the viruses studied. these findings have a number of implications for prevention of zoonotic disease transmission and outbreaks. those individuals at the animal-human interface (such as persons butchering wild animals) that are highly exposed to wild animals are influential in disease amplification and dissemination and reduction of risk factors can play a large role in mediating potential outbreaks. while we identified butchers as an occupational group at increased risk, better understanding of the specific exposures driving this increased risk would require more in-depth study. among butchers, we did find an elevated risk of virus seropositivity associated with butchering porcupines, but due to small numbers, such a finding must be considered preliminary. this study also identified an increased risk of seropositivity with contact with civets and field rats which could be due to multiple factors. civets and field rats are among the most popular types of wild animals found in markets and restaurants and prolific contact may result in higher risk. there is also a bias related to the viruses that the study targeted with two of the three (sars cov and hantavirus) associated with both civets and rats. this study had a number of limitations, including selection bias. we deliberately targeted high risk individuals, making our sample less representative of the general population. conversely, it is possible that there could be differential participation rates between high risk and lower risk individuals, further limiting any extrapolation to community levels. we focused on wildlife contact, and therefore were unable to assess the risk impact of contact with other animal species including livestock and companion animals. further research should aim to include larger sample sizes from more geographically diverse regions, and assess seroprevalence rates in random population samples. underreporting of risk activities on the behavioral survey may have occurred, since many activities related to wild animals are illegal in china. in addition, since this was a hypothesis generating study, the analysis involved multiple comparisons. therefore, any reported association should be viewed with caution. while this study involved serological testing for more than one potential wildlife pathogen, there are numerous other wildlife pathogens, including bacterial and viral agents, which the study did not investigate. it is therefore possible that this study failed to detect other significant transmission events of human health relevance. future studies should consider such possibilities. the finding that less than one third of the surveyed population, most of whom reported wild animal contact, believed that they could become infected through such contact, indicates the need for targeted educational programs for prevention, especially among those involved in butchering wildlife for consumption. targeted education programs should aim to increase knowledge of disease risk, perception of risk, and risky behaviors in the identified high-risk sub groups. previous studies indicate the need to target the high-risk sub group of hunters [ , ] . in this study, however, hunters and persons reporting ever hunting or hunting as a profession exhibited a lower rate of seropositivity compared with the overall population. it is possible that hunting activities may be underreported, but the results of this study indicate some of the difficulties of relying on self report to identify individuals at increased risk. due to the huge demand by the local residents and immigrants in the larger pearl river delta area, which includes guangdong, hong kong and macau, increasing number of individuals engaged in animal production and value chain systems are believed to continue to come into contact with wild animals and engage in risky behaviors increasing the risk of a possible infectious disease outbreak that could have a potentially severe impact. wild animals are an important part of southern chinese economy and culture, and pragmatic policy changes should be implemented to control the risks of possible disease outbreaks. these policy changes should aim to increase risk perception and uptake of precautionary behaviors without risking the cultural and financial security of the community. results of the analyses of these research findings can be used to inform public health strategies such as communication and educational campaigns, targeting those behaviors that place individuals at higher risk of disease. our study provides evidence towards recommendations that include: an increase in the level of collaboration between animal and human health programs targeting human-animal interfaces to increase efforts to control and prevent outbreaks; development of new educational campaigns to increase knowledge and awareness of infectious diseases that target specific sub-groups, such as butchers, that are at increased risk; and increase monitoring of highrisk groups to detect continued transmission of zoonotic diseases in a timely manner. supporting information s file. study questionnaire for persons hunting, butchering, eating and/or keeping wild animals as pets. (pdf) s table. prevalence of seropositivity for tested viruses, by reported wildlife exposure. (pdf) global trends in emerging infectious diseases wildlife trade and global disease emergence. emerging infectious disease bushmeat hunting, deforestation, and prediction of zoonotic disease wet markets-a continuing source of severe acute respiratory syndrome and influenza? the lancet animal movements and the spread of infectious diseases disease and destiny-mystery and mastery infectious diseases emerging from chinese wet-markets: zoonotic origins of severe respiratory viral infections the role of wildlife in emerging and re-emerging zoonoses emergence and predominance of an h n influenza variant in china bats are natural reservoirs of sars-like coronaviruses isolation and characterization of viruses related to the sars coronavirus from animals in southern china sars-beginning to understand a new virus learning from sars: preparing for the next disease outbreak: workshop summary. the national academies collection: reports funded by national institutes of health human infection with a novel avian-origin influenza a (h n ) virus emerging angiostrongyliasis in mainland china emergence of a novel and highly divergent htlv- in a primate hunter in cameroon new directions in conservation medicine: applied cases of ecological health hantavirus infections in humans and animals distribution of tick-borne diseases in china a national assessment of the epidemiology of severe fever with thrombocytopenia syndrome a global perspective on hantavirus ecology, epidemiology, and disease the life cycle of sars coronavirus in vero e cells establishment of vero e cell clones persistently infected with severe acute respiratory syndrome coronavirus diagnosis of severe acute respiratory syndrome (sars) by detection of sars coronavirus nucleocapsid antibodies in an antigen-capturing enzyme-linked immunosorbent assay fever with thrombocytopenia associated with a novel bunyavirus in china. the new england journal of medicine the application of firth's procedure to cox and logistic regression university of vienna, section of clinical biometrics domcs we thank prefecture cdcs at dabu, jiaoling, pingyuan, lianping, heping, lianshan, lianzhou, yunfu, yunan, xinyi, deqing and fengkai for their efforts in participant enrollment and specimen collection. there are no competing interests in this scientific work. key: cord- - bflqj c authors: csiszar, anna; jakab, ferenc; valencak, teresa g.; lanszki, zsófia; tóth, gábor endre; kemenesi, gábor; tarantini, stefano; fazekas-pongor, vince; ungvari, zoltan title: companion animals likely do not spread covid- but may get infected themselves date: - - journal: geroscience doi: . /s - - - sha: doc_id: cord_uid: bflqj c coronavirus disease (covid- ) is a highly contagious infectious disease caused by the novel severe acute respiratory syndrome coronavirus (sars-cov- ). from the epidemiological data, the picture emerges that the more severe etiopathologies among covid- patients are found in elderly people. the risk of death due to covid- increases exponentially with age. eight out of covid- related deaths occur in people older than years of age. older patients with comorbid conditions such as hypertension, heart failure, diabetes mellitus, asthma, chronic obstructive pulmonary disease, and cancer have a much higher case fatality rate. governments and public health authorities all over the world have realized that protections of vulnerable older adults should be a priority during the covid- pandemic. covid- is a zoonotic disease. the sars-cov- virus was originally transmitted likely from a bat or a pangolin to humans. recent evidence suggests that sars-cov- , similar to other coronaviruses, can infect several species of animals, including companion animals such as dogs, cats, and ferrets although their viral loads remain low. while the main source of infection transmission therefore is human to human, there are a few rare cases of pets contracting the infection from a sars-cov- -infected human. although there is no evidence that pets actively transmit sars-cov- via animal-to-human transmission, senior pet ownership potentially may pose a small risk to older adults by ( ) potentially enabling animal-to-human transmission of sars-cov- in the most vulnerable population and ( ) by increasing the exposition risk for the elderly due to the necessity to care for the pet and, in the case of dogs, to take them outside the house several times per day. in this overview, the available evidence on sars-cov- infection in pets is considered and the potential for spread of covid- from companion animals to older individuals and the importance of prevention are discussed. coronavirus disease (covid- ) is a highly contagious infectious disease caused by the novel severe acute respiratory syndrome coronavirus (sars-cov- ) [ , ] . as of june , during the / global pandemic, approximately . million people have contracted the virus globally resulting in over , deaths [ ] . from the beginning of the outbreak of the pandemic, the centers for disease control and prevention (cdc) have stated that sars-cov- is a respiratory virus, and, as such, its main route of transmission is via respiratory droplets when an infected individual coughs, sneezes, or just talks in close proximity of others [ ] . furthermore, the virus may be also spread via contaminated surfaces, when a person touches a contaminated object (e.g., doorknob), then touches their nose, mouth, or eyes [ ] [ ] [ ] . the mean incubation period of the disease is . days with . % of patients producing symptoms by day . [ ] . the most common symptoms include fever, cough, fatigue, and shortness of breath. most common cause of death is respiratory failure [ , ] . there is also increasing evidence suggesting that covid- may cause fatal myocardial injury, arrythmia/ cardiac arrest, neurological damage (including stroke), and kidney failure (acute kidney injury occurs in one third of patients) [ ] . currently reported case fatality rates vary from % to more than %, due to country-to-country differences in screening in the whole population. from the epidemiological data, the picture emerges that severe covid- is primarily a disease of older people [ ] . the risk of death due to covid- increases exponentially with age in every country studied [ ] . a report of , cases from the chinese center for disease control and prevention [ ] shows that case fatality rates by age are as follows: . % ( - years of age); . % ( to years of age); . % ( to years of age); . % ( to years of age); % ( - years of age); . % (≥ years of age). a report of , cases produced by the istituto superiore di sanità (iss) showed that the case fatality rates for males by age in italy are as follows: % ( - years of age); % ( to years of age); . % ( to years of age); . % ( to years of age); . % ( to years of age); . % ( to years of age); . % ( - years of age); . % ( to years of age); . % (≥ years of age) [ ] . older patients with comorbid conditions such as hypertension, heart failure, diabetes mellitus, asthma, chronic obstructive pulmonary disease, cancer, and chemotherapeutic treatment have a much higher case fatality rate. critical cases have a case fatality rate of %, whereas mortality is very low in patients with mild symptoms. in the usa, deaths involving covid- by age group are as follows: % (≤ year of age), % ( - years of age), % ( - years of age), % ( - years of age), % ( - years of age), % ( - years of age), % (≥ years of age) [ ] . in total, % of all covid- related deaths occurred in people older than years of age. the causes of these age-related differences in mortality rates are not yet fully understood. possible causes likely include the less efficient functioning and coordination of both cellular and molecular elements of the immune system, the higher number of comorbidities, and the overall frailty and impaired organismal and cellular resilience of elderly patients [ , , ] . governments and public health authorities all over the world have realized that protections of vulnerable older adults should be a priority during the covid- pandemic. the most effective step to prevent further sars-cov- infections in older adults is to limit exposure by reducing social activity, avoiding gatherings with many people and public transportation. accordingly, governments and public health authorities ordered nursing homes and assisted living facilities to limit visits, even by family members. formal and informal group social and recreational activities and church services were canceled. although these steps are effective, additional measures are needed to limit spread of covid- . staying at home all day however is impossible if seniors actually possess a dog as the animal will have to be taken out regularly. yet, pet owners usually take their dogs out alone and social distancing may be well doable. potential role for zoonotic spread of covid- in older adults? as coronaviruses generally are zoonotic (i.e., they are transmitted between animals and people), covid- as well is a zoonotic disease just like sars. whether the novel coronavirus causing covid- came from a bat or a pangolin is still uncertain. the initial event where the virus may have jumped to humans seem to have happened in a so-called "wet" market [ , ] , where fresh meat as well as living wild animals are commonly sold. because of the observed accumulation of infected people by january who were in some relation to the "hua nan" market in wuhan, this town of million inhabitants located at the yangtze river in central china is commonly considered the "ground zero" for the global pandemic [ , ] . it should be noted that our understanding of the origin of the pandemic may evolve. a recent phyloepidemiologic analysis of sequenced genomic data of close to sars-cov- samples by chinese researchers (published in a non-peer reviewed pre-print form [ ] ) suggested that while the crowded market may have boosted spread of the novel virus to the whole city, it may have not originated there. the possibility that the virus may have been introduced from elsewhere clearly warrants more research in the upcoming months. genetic sequence data reveals that the sars-cov- virus is closely related to coronaviruses found in rhinolophus bat (horseshoe bat) populations [ ] . although bats may be the primary reservoir, the original route of transmission to humans is currently unknown and may have involved an intermediate host, probably a pangolin [ ] . sars-cov- , similar to other coronaviruses, can infect several species of animals. initial data suggested that the sars-cov- virus can bind to receptors and infect cells of horseshoe bats and civets, whereas mice are not susceptible. in vivo studies suggest that several species, including cats, can be infected with sars-cov- virus, whereas chickens, pigs, and ducks are not susceptible [ ] . it is estimated that there are currently to million pet dogs and cats in the usa (according to the us pet ownership & demographics sourcebook by the american veterinary medical association (avma) and the biennial appa national pet owners survey by the american pet products association, respectively). accordingly, - % of us households own at least one dog or cat. studies, including the national poll on healthy aging (https://deepblue.lib.umich. edu/bitstream/handle/ . / /npha_pets-report_final- .pdf?sequence= &isallowed= y), demonstrate the multifaceted health benefits of senior pet ownership (including increased physical activity such as walking, higher emotional well-being, and significant stress reduction). despite these advantages, pet ownership potentially may pose a minor risk to older adults by enabling animal-tohuman transmission of sars-cov- in the most vulnerable population. here, we summarize the available evidence about sars-cov- infection in pets. in late march , the federal agency for the safety of the food chain (fasfc) in belgium reported that a pet cat was diagnosed to be infected with sars-cov- [ , ] , showing that felines living in the household of people with covid- are at risk of contracting the disease and may potentially spread the virus. the cat became ill week after its owner's return home from italy [ ] . susceptibility of cats to sars-cov- infection is supported by a recent experimental observation [ ] . specifically, it was demonstrated that cats exposed to sars-cov- under laboratory conditions can be infected and are able to transmit the disease to other felines. on april , it was reported that two pet cats in new york state have tested positive for the sars-cov- , which are the first confirmed covid- cases in companion animals in the usa [ ] . one of these two cats became sick approximately a week after a person in its household developed respiratory symptoms. the other cat's owner tested positive for sars-cov- before the cat fell ill. both cats developed symptoms of upper respiratory disease, including coughing and nasal discharge. in june , a french study reported that screening of cats and dogs from owners previously infected or suspected of being infected by sars-cov- identified a per cat infected by sars-cov- [ ] . for each animal, rectal, nasopharyngeal swabs, and serum were taken. the infected cat, which exhibited mild respiratory and digestive symptoms, tested positive by rt-qpcr on the rectal swab, whereas the nasopharyngeal swab was tested negative. serological analysis confirmed the presence of antibodies against sars-cov- . additionally, on april , , it was reported that a -year-old malayan tiger at the bronx zoo in new york city was tested positive for the sars-cov- virus [ , ] . in addition, six other big cats (another malayan tiger, two amur tigers, and three african lions) were reported to exhibit symptoms, including dry coughs, which are indicative of sars-cov- infection. only one tiger was tested for the virus, as collection of the samples in big cats requires anesthesia [ ] . it was reported that the felines likely have contracted the virus from a caretaker, who was asymptomatic at the time of contact with the animals [ ] . these data support the view that different species of felines are susceptible for sars-cov- infection. there are also emerging data that dogs may be also infected by the covid- virus [ , ] . in february in hong kong, a companion dog was discovered to be positive for sars-cov- by pcr testing [ ] . this animal is thought to have contracted the sars-cov- from its owner who was diagnosed with covid- [ ] . serological testing on blood samples derived from the dog was performed by a who reference laboratory and yielded a positive result [ ] . as of march , hong kong's agriculture, fisheries, and conservation department had tested dogs and cats, which lived in households with confirmed covid- human cases and, so far, dogs (including the one described above) had tested positive for sars-cov- . neither of the dogs showed any sign of respiratory disease. one of them, a -year-old pomeranian dog died shortly after the diagnosis was made [ ] . it should be noted, that in human covid- disease, the cause of death often is cardiac arrest. the emerging view is that cardiac damage is present in every fifth covid- patients, leading to heart failure and death even among those who do not exhibit respiratory distress syndrome. in addition to hypoxic damage, the heart muscle likely can be infected by the sars-cov- virus. sars-cov- utilizes angiotensin converting enzyme- (ace- ) as a cellular entry receptor and cardiac myocytes as well as coronary arterial endothelial cells express ace- abundantly. of note, heart failure is a leading cause of death among older pomeranian dogs [ ] . genetically impaired cardiac resilience in this breed may represent increased risk for death associated with covid- . we propose that veterinarians should be aware of sars-cov- -induced cardiac pathologies in dogs and look more closely for cardiac symptoms of sars-cov- infection in patients, even in the absence of respiratory symptoms. to definitely identify sars-cov- as cause of heart failure in dogs from households affected by covid- , elaborate pathological testing should be performed postmortem in the dogs, most likely without the owners having an interest to cover the costs for these tests. besides cats and dogs, ferrets are also common pet animals in several countries. notably, laboratory animals of the mustelidae family are frequently used models for respiratory diseases [ ] , including sars coronavirus. in addition, recent experimental studies verified the possibility of airborne transmission of sars-cov- between ferrets (published in a non-peer review, pre-print form [ ] ). another study reported strong evidence for the possibility of virus transmission from and between these animals. viral shedding in detectable amount was observed in nasal washes, saliva, urine, and feces of the animals and all of them presented acute bronchitis [ ] . minks are close relative of ferrets and are kept for their fur on large mink farms. it has been recently reported that in the netherlands, thousands of minks have been gassed on mink farms to prevent infected mink from becoming a viral reservoir that could cause new outbreaks in humans [ ] . it is suspected that rapid spread of the infection occurred via infectious droplets, on feed or bedding, or in dust containing fecal matter [ ] . infection in minks looks like covid- in humans, from asymptomatic infection to severe pneumonia [ ] . mortality was reported to be negligible at one farm and close to % at another [ ] . alarmingly, feral cats roaming the dutch mink farms and stealing the mink's food were also found to be infected as well [ ] . minipigs have also become popular pets in recent years [ ] . although recent studies revealed relatively low expression levels of angiotensin converting enzyme (ace ) protein (the docking protein for the viral spike protein) in the swine respiratory tract [ ] , the susceptibility of pigs to sars-cov- transmitted from other pets or humans is still controversial [ , ] . concluding, there is a small but existing possibility for an animal-human transmission although the main source of infection transmission remains human-human [ ] . in theory, a zoonotic disease such as covid- could be directly transmitted from companion animals to humans through media such as air or through saliva and bites. contact with pet dogs and cats, including petting, snuggling, being kissed or licked, sleeping in the same location, and sharing food with companion animals is clearly increased during times when the owner is staying at home sick with covid- , quarantined, or due to governmentally decreed lockdown measures. this common behavior, which, from a medical point of view, may be unfortunate for both the owner's and the pet's health, as it is creating ample opportunities for transmission of the virus in either direction. additionally, as the virus seems to be present in the saliva of infected animals, which lick their paws and fur, every surface in contact with the animal in an affected household should be considered potentially contaminated with sars-cov- [ ] . it is likely, in the case of the very few positively tested companion animals, that the pets contracted the virus from their owners in an infected household (and not vice versa). actually, there is no evidence for a single case of pet to human transmission to date. cats very often stay within the house of their owners so their risk for contracting the infectious virus from outside is much lower than in dogs, which are taken out multiple times per day while coming into contact with other dogs and humans, sniffing on and touching the ground in parks, open space, or even public transport where previously another human or animal might have infected the area with sars-cov- . nothing is known yet about the zoonotic potential of sars-cov- in seniors or elderly patients who own companion animals. in none of the positively tested cats and dogs was a reference made to the age of the owners or the age of the human commonly caring for the pet. prior to the news of the bronx zoo big cat cases, there have not been any reports of pets or other animals in the usa contracting covid- , according to the websites of the us department of agriculture and the centers for disease control and prevention (cdc) [ , ] . since the discovery of the cats infected with sars-cov- in new york, the website of the american veterinary association carefully documents each new case [ ] . the guidelines of the cdc currently indicate that there is no evidence available that companion animals, including pets, can spread covid- [ ] . yet, it has to be emphasized that animals have not been routinely tested in the usa and according to the website of the cdc and the american veterinary medical association (avma) [ ] as of april , routine testing of animals is not recommended by the avma, cdc, us department of agriculture (usda), american association of veterinary laboratory diagnosticians (aavld), national association of state public health veterinarians (nasphv), or the national assembly of state animal health officials [ ] . the cdc currently advises that public and animal health officials may decide to test animals that are showing signs of covid- and that are known to have been exposed to the virus [ ] . veterinarians considering a possible covid- infection in the presented patients and performing a test may thus contribute very usefully to the understanding of the zoonotic potential of sars-cov- . it is now clear that many people infected with sars-cov- remain asymptomatic while spreading the disease. if the same holds true also for animals, it cannot be ruled out that asymptomatic household pets and domesticated animals also can shed the virus and infect people without exhibiting actual symptoms of the disease. however, according to shi and coworkers, their viral loads are much lower compared with humans [ ] . the reported hong kong case of a sars-cov- infected dog also did not have any clinical signs of the disease. furthermore, the sars coronavirus (sars-cov; which is also an animal virus identified in that causes a zoonotic disease in humans) infects mice, macaques, and marmosets, but does not cause a respiratory disease in them similar to the one it causes in people. middle east respiratory syndrome (mers) similarly is a respiratory illness caused by the coronavirus mers-cov first reported in saudi arabia in . mers-cov can infect primates and hoofed animals, including camels and alpacas. importantly, infected alpacas appear to be asymptomatic, yet they spread the virus. of note is that several coronavirus-mediated diseases exist both in companion animals (including canine respiratory coronavirus, enteric coronaviruses, feline infectious peritonitis) and in livestock (infectious bronchitis virus, alpha coronaviruses causing mild gastrointestinal or respiratory disease, swine enteric coronaviruses, bovine coronaviruses), although most are species-specific and do not affect humans. in the current sars-cov- pandemic, the situation is rapidly evolving and in the light of the recent evidence, we should be aware of the possibility that humans can be potentially infected with covid- by animals, including by pet cats, dogs, or other domesticated species. the cdc recently issued an interim guidance for public health professionals managing the home care and isolation of people with covid- who have pets or other animals (including service or working animals) in the same home [ ] . according to these cdc guidelines, state public health veterinarians should be contacted by public health professionals, animal health professionals, or veterinarians that have discovered a household animal with a new, concerning illness and that reside with a person with covid- [ ] . due to concerns of potential human-to-animal transmission of sars-cov- , the cdc recommends that people with covid- and in-home isolation should limit their interaction with household animals, but rather increase hygiene measures and hand washing after close contact with the pet [ ] . it is recommended that while a person infected with covid- is symptomatic, they should maintain separation from household animals as they would with other household members, practicing social distancing [ ] . dogs are frequently used in veterinary and cardiovascular research as large animal models of heart failure and other chronic diseases [ ] . many researchers who are working with dogs on a daily basis in universities and the pharmaceutical industry belong to vulnerable age groups. in the laboratory setting, there is an increased probability for contact with bodily fluids of potential virus carrier animals, especially during procedures that involve invasive surgery [ ] . research teams should get adequate safety training to prevent transmission of sars-cov- and closely follow laboratory guidelines for handling biological specimens, waste, and hazardous materials. pet health companies are working on developing laboratory tests for companion animals that may be affected by sars-cov- . as the pandemic is progressing and testing of companion animals by veterinarians will be more often performed, our understanding of sars-cov- and its animal-toanimal and animal-to-human transmission will rapidly evolve. as of april , two commercial laboratories in the usa reported they had tested using rt-pcr thousands of specimens from dogs and cats for sars-cov- and had obtained no positive results [ ] . these specimens were submitted from the usa, south korea, canada, and europe for pcr analysis of common pathogens causing respiratory illness in dogs and cats [ ] . there is no or only limited information available as to whether these animals had close contact with human covid- patients before the testing. the world organization for animal health (oie) considers sars-cov- an emerging disease, and therefore the us department of agriculture regularly reports confirmed us animal infections to the oie. for up-to-date information, we advise the reader to consult the online reference sources provided at the end of this paper, including the websites of the avma [ ] , cdc [ ] , usda [ ] , and oie [ ] . further and broader veterinary studies are needed to determine which other companion animal species and pets can be infected by sars-cov- and elucidate, whether they demonstrate the clinical signs (e.g., upper respiratory infection, lung injury), and whether they develop an immune response. it is crucial to determine where the virus is shed in each species, whether it's in urine, tears, saliva, blood, or feces. it will be also essential to identify the species potentially serving as a reservoir for the virus. future research opportunities include widescale serosurveys of pet animals in contact with confirmed covid- patients. it may reveal the extension of this transmission route between various pet animals and humans. taken together, animal-to-human transmission is likely only a minor route of transmission for sars-cov- . available research suggests that companion animals may theoretically play a role by either establishing a reservoir for sars-cov- , or even potentially being able to spread covid- to other people in the household or people being in close contact with the animals. people should thus be advised to always follow standard handwashing practices before and after interacting with their pets. in view of the large number of fatalities in the older generation, seniors should be advised to adopt an even improved hygiene practice with their pets similar to the "social distancing" recommendations between humans. as per cdc recommendations, elderly people in particular are instructed not to let their pets interact with other people or other pets from outside the household. domestic cats should be kept indoors to prevent them from interacting with other animals or people. dogs should be walked on a leash, maintaining at least m from other people and animals. dog parks or public places as well as public transport, where a large number of people and dogs usually gather, should be avoided. the epidemiology and pathogenesis of coronavirus disease (covid- ) outbreak sars-cov- and covid- in older adults: what we may expect regarding pathogenesis, immune responses, and outcomes aerosol and surface stability of sars-cov- as compared with sars-cov- -ncov transmission through the ocular surface must not be ignored persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents the incubation period of coronavirus disease (covid- ) from publicly reported confirmed cases: estimation and application understanding pathways to death in patients with covid- case-fatality rate and characteristics of patients dying in relation to covid- in italy a geroscience perspective on covid- mortality comorbidity and its impact on patients with covid- in china: a nationwide analysis task force covid- del dipartimento malattie infettive e servizio di informatica isds. epidemia covid- geroprotective and senoremediative strategies to reduce the comorbidity, infection rates, severity, and lethality in gerophilic and gerolavic infections susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus a pneumonia outbreak associated with a new coronavirus of probable bat origin a new coronavirus associated with human respiratory disease in china decoding evolution and transmissions of novel pneumonia coronavirus using the whole genomic data genomic analysis of a -ncov strain in the first covid- patient found in hangzhou identifying sars-cov- related coronaviruses in malayan pangolins coronavirus can infect cats -dogs sars-cov- in animals first detection and genome sequencing of sars-cov- in an infected cat in france sars-cov- and animals, including pets and other domestic animals usda statement on the confirmation of covid- in a t i g e r i n n e w y o rk dogs caught coronavirus from their owners, genetic analysis suggests infection of dogs with sars-cov- vertebral heart scores in eight dog breeds ferret models of viral pathogenesis sars-cov- is transmitted via contact and via the air between ferrets infection and rapid transmission of sars-cov- in ferrets coronavirus rips through dutch mink farms, triggering culls minipigs and potbellied pigs as pets in the veterinary practice-a retrospective study comparison of severe acute respiratory syndrome coronavirus spike protein binding to ace receptors from human, pets, farm animals, and putative intermediate hosts coronavirus disease (covid- ) outbreak: could pigs be vectors for human infections? interim guidance for public health professionals managing people with covid- in home care and isolation who have pets or other animals testing animals for sars-cov- confirmation of covid- in two pet cats in new york animal models of dilated cardiomyopathy for translational research intracoronary gene delivery of the cytoprotective factor vascular endothelial growth factor-b in canine patients with dilated cardiomyopathy: a short-term feasibility study united states department of agriculture world organization for animal health publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments this work was supported by grants from the american heart association, the oklahoma center for the advancement of science and technology, and the presbyterian health foundation. the authors acknowledge the support from the nia-funded geroscience training program in oklahoma (t ag ) and the cellular and molecular geroscience cobre ( p gm ). the funding sources had no role in the writing of the report and in the decision to submit the article for publication. key: cord- -yp ofhi authors: ruiz, sara i.; zumbrun, elizabeth e.; nalca, aysegul title: chapter animal models of human viral diseases date: - - journal: animal models for the study of human disease doi: . /b - - - - . - sha: doc_id: cord_uid: yp ofhi abstract as the threat of exposure to emerging and reemerging viruses within a naive population increases, it is vital that the basic mechanisms of pathogenesis and immune response be thoroughly investigated. by using animal models in this endeavor, the response to viruses can be studied in a more natural context to identify novel drug targets, and assess the efficacy and safety of new products. this is especially true in the advent of the food and drug administration's animal rule. although no one animal model is able to recapitulate all the aspects of human disease, understanding the current limitations allows for a more targeted experimental design. important facets to be considered before an animal study are the route of challenge, species of animals, biomarkers of disease, and a humane endpoint. this chapter covers the current animal models for medically important human viruses, and demonstrates where the gaps in knowledge exist. well-developed animal models are necessary to understand the disease progression, pathogenesis, and immunologic responses in humans. furthermore, to test vaccines and medical countermeasures, well-developed animal models are essential for preclinical studies. ideally, an animal model of human viral infection should mimic the host-pathogen interactions and the disease progression that is seen in the natural disease. a good animal model of viral infection should allow many parameters of infection to be assayed, including clinical signs, growth of virus, clinicopathological parameters, cellular and humoral immune responses, and virus-host interactions. furthermore, viral replication should be accompanied by measurable clinical manifestations, and pathology should resemble that of human cases such that a better understanding of the disease process in humans is attained. there is often more than one animal model that closely represents human disease for a given pathogen. small animal models are typically used for first-line screening, and for initially testing the efficacy of vaccines or therapeutics. in contrast, nonhuman primate (nhp) models are often used for the pivotal preclinical studies. this approach is also used for basic pathogenesis studies, with most experiments performed in small animal models when possible, and nhps only used to fill in remaining gaps in knowledge. the advantages of using mice to develop animal models are low cost, low genetic variability in inbred strains, and abundant molecular biological and immunological reagents. specific pathogen-free (spf), transgenic, and knockout mice are also available. a major pitfall of mouse models is that the pathogenesis and protection afforded by vaccines and therapeutics cannot always be extrapolated. additionally, blood volumes for sampling are limited in small animals, and viruses often need to be adapted through serial passage in the species to induce a productive infection. the ferret's airways are anatomically and histologically similar to that of humans, and their size enables larger or more frequent blood samples to be collected, making them an ideal model for certain respiratory pathogens. ferrets are outbred, with no standardized breeds or strains; thus, greater numbers are required in studies to achieve statistical significance and overcome the resulting variable responses. additionally, spf and transgenic animals are not available, and molecular biological reagents are lacking. other caveats making ferret models more difficult to work with are their requirement for more space than mice (rabbit-style cages), and the development of aggressive behavior with repeated procedures. nhps are genetically the closest species to humans; thus, disease progression and host-pathogen responses to viral infections are often the most similar to that of humans. however, ethical concerns of experimentation on nhps, along with the high cost and lack of spf nhps raise barriers for such studies. nhp studies should be carefully designed to ensure that the least amount of animals are used, and the studies should address the most critical questions regarding disease pathogenesis, host-pathogen responses, and protective efficacy of vaccines and therapeutics. well-designed experiments should carefully evaluate the choice of animal, including the strain, sex, and age. furthermore, route of exposure and the dose should be as close as possible to the route of exposure and dose of human disease. the endpoint for these studies is also an important criterion. depending on the desired outcome, the model system should emulate the host responses in humans when infected with the same pathogen. in summary, small animal models are helpful for the initial screening of vaccines and therapeutics, and are also often beneficial in obtaining a basic understanding of the disease. nhp models should be used for a more detailed characterization of pathogenesis and for pivotal preclinical testing studies. ultimately, an ideal animal model may not be available. in this case, a combination of different well-characterized animal models should be considered to understand the disease progression and to test medical countermeasures against the disease. in this chapter, we will be reviewing the animal models for representative members of numerous virus families causing human diseases. we will focus on the viruses for each family that are the greatest concern for public health worldwide. poliovirus (pv) is an enterovirus in the picornavirus family and causes poliomyelitis. humans are the only natural host for the virus, but a number of nhp species are also susceptible. all three serotypes of pv cause paralytic disease, but it is relatively rare with only - % of infected individuals ultimately developing paralysis. humans typically acquire and transmit the virus by the oral-fecal route, although transmission by aerosol droplets may also be possible. the virus replicates in the oropharyngeal and intestinal mucosa, made possible by the resistance of pv to stomach acids. cd expression in peyer's patches and m cells suggest that these cell types may be important during initiation of infection. replication at extraneural sites . in vivo models of viral diseases affecting humans precedes invasion into the central nervous systems (cnss), when it occurs. two effective vaccines, the salk killed polio vaccine delivered by the intramuscular route and the sabin live attenuated polio vaccine delivered orally, have been used very successfully to eliminate the disease from most parts of the world. the world health organization has led a long and hard-fought global polio eradication campaign, with much success, but full eradication has not yet been achieved. since , between and cases of pv infection are reported worldwide each year. thus, animal models are also needed to test new vaccine approaches that could be used toward eradication of polio in the areas where it still persists. additionally, the recent focus of work with pv animal models has been fraught with urgency, as to gain understanding of pv pathogenesis before the eradication effort is complete and work with this virus ceases. animal models for the study of pv consist of nhp models and mouse models. mice are susceptible to certain adapted pv strains: p /lansing, p /lsb, and a variant of p /leon. mice infected intracerebrally with p /lansing develop disease with some clinical and histopathological features resembling that of humans. wild-type mice are not susceptible to wildtype pv; however, the discovery of the pv receptor (cd ) in led to the use of hcd transgenic mice as a model of pv infection. these mice are not susceptible to pv by the oral route and must be exposed intranasally or by intramuscular infection to induce paralytic disease. interestingly, hcd mice that have a disruption in the interferon (ifn)-a/b receptor gene are susceptible to oral infection. this finding has given rise to speculation that an intact ifn-a/b response may be responsible for limiting infection in the majority of individuals exposed to pv. thus, mouse models have proven to be very useful in gaining a better understanding of pv disease and pathogenesis. rhesus macaques are not susceptible to pv by the oral route, but they have been used extensively to study vaccine formulations for safety and immunogenicity, for monitoring neurovirulence of the live attenuated sabin vaccine, and in the past for typing pv strains. bonnet monkeys are also susceptible to oral inoculation of pv, which results in the gastrointestinal shedding of virus for several weeks, with paralysis occurring in only a small proportion of animals. consistent paralytic disease can be induced in bonnet monkeys (macaca radiata) through exposure to pv by infection into the right ulnar nerve (at the elbow), resulting in limb paralysis that resembles human paralytic poliomyelitis both clinically and pathologically. as such, bonnet monkeys can be used to study pv distribution and pathology and the induction of paralytic poliomyelitis or provocation paralysis. hepatitis a virus causes jaundice, which is a public health problem worldwide. the incubation period lasts from to days with an average of days. transmission between humans occurs by the oral-fecal route, person-to-person contact, or ingestion of contaminated food and water. hepatitis a virus causes an acute and self-limited infection of the liver with a spectrum of signs and symptoms ranging from subclinical disease, to jaundice, fulminant hepatitis, and in some cases death. , the disease can be divided into four clinical phases: ( ) incubation period, during which the patient is asymptomatic but virus replicates and possibly transmits to others. ( ) prodromal period, which might last from a few days to a week with patients generally experiencing anorexia, fever (< f), fatigue, malaise, myalgia, nausea, and vomiting. ( ) icteric phase, in which increased bilirubin causes characteristic dark brownish colored urine. this sign is followed by pale stool and yellowish discoloration of the mucous membranes, conjunctiva, sclera, and skin. most patients develop an enlarged liver, and approximately - % of the patients have splenomegaly. ( ) convalescent period, with resolution of the disease and recovery of the patient. rarely, during the icteric phase, extensive necrosis of the liver occurs. these patients show a sudden increase in body temperature, marked abdominal pain, vomiting, jaundice, and the development of hepatic encephalopathy associated with coma and seizures, all signs of fulminant hepatitis. death occurs in - % of patients with fulminant hepatitis. experiments showed that hepatitis a causes disease only in humans, chimpanzees, several species of south american marmosets, stump-tailed monkeys, and owl monkeys via the oral or intravenous (iv) routes. [ ] [ ] [ ] [ ] it is known that cynomolgus macaques are infected with hepatitis a virus in the wild. amado et al. used cynomolgus macaques (macaca fascicularis) for experimental hepatitis a infections. the animals did not exhibit clinical signs of disease, but viral shedding was observed in saliva and stool as early as h postinoculation (pi) and days pi, respectively. although mildto-moderate hepatic pathology was observed in all macaques, seroconversion and mildly increased alanine aminotransferase (alt), an enzyme associated with liver function, were observed in some of them. because this study had a very small group of animals (four macaques), the data should not be considered as conclusive, and more studies are needed to better define the cynomolgus macaque model. although hepatitis a virus is transmitted by the oralfecal route, studies in chimpanzees and tamarins showed that the iv route was much more infectious than oral route was. there was no correlation between dose and development of clinical disease for either species or experimental routes, and similar to cynomolgus macaques, none of these species showed clinical signs of disease. inoculation of common marmosets (callithrix jacchus) with hepatitis a virus did not produce clinical signs of disease as seen in other nhp models. , liver enzyme levels increased on day pi, and monkeys had measurable antihepatitis a antibodies by day pi. an experimental study with cell culture-adapted hepatitis avirus in guinea pigs challenged by oral or intraperitoneal routes did not result in clinical disease, increase in liver enzymes, or seroconversion. viral load was detected in stool and serum between days and and and days, respectively. liver pathology showed mild hepatitis. furthermore, histopathology indicated that virus replicated in extrahepatic tissues such as spleen, regional lymph nodes, and intestinal tract. in summary, none of the animal models for hepatitis a infection is suitable for studying pathogenesis of the virus because all clinical and most of the laboratory parameters remain within normal range or only slightly increased after the infection. one possibility is to test the safety of vaccines against hepatitis a virus in those models with demonstrable viral shedding. noroviruses, of which norwalk is the prototypic member, are responsible for up to % of reported food-borne gastroenteritis cases. in developing countries, this virus is responsible for approximately , deaths annually. a typical disease course is self-limiting, but there have been incidences of necrotizing enterocolitis and seizures in infants. , symptoms of infection include diarrhea, vomiting, nausea, abdominal cramping, dehydration, and fever. incubation normally is for - days, with symptoms enduring for - days. viral shedding is indicative of immunocompromised status within an individual with the elderly and young having a prolonged state of shedding. transmission occurs predominately through the oral-fecal route with contaminated food and water being the major vector. a major hindrance to basic research into this pathogen is the lack of a cell culture system. therefore, animal models are used not only to determine the efficacy of novel drugs and vaccines but also for understanding the pathogenesis of the virus. therapeutic intervention consists of rehydration therapy and antiemetic medication. no vaccine is available, and development of one is expected to be challenging given that immunity is short lived after infection. nhps including marmosets, cotton-top tamarins, and rhesus macaques infected with norwalk virus can be monitored for the extent of viral shedding; however, no clinical disease is observed in these models. disease progression and severity are measured exclusively by assay of viral shedding. it was determined that more virus was needed to create an infection when challenging by the oral route than when challenging by the iv route. chimpanzees were exposed to a clinical isolate of norwalk virus by the iv route. although none of the animals developed disease symptoms, viral shedding within the feces was observed within - days postinfection and lasted anywhere from days to weeks. viremia never occurred, and no histopathological changes were detected. the amount and duration of viral shedding were in line with what is observed upon human infection. a recently identified calicivirus of rhesus origin, named tulane virus, was used as a surrogate model of infection. rhesus macaques exposed to tulane virus intragastrically developed diarrhea and fever days postinfection. viral shedding was achieved for days. the immune system produced antibodies that dropped in concentration within days postinfection, mirroring the short-lived immunity documented in humans. the intestine developed moderate blunting of the villi as seen in human disease. a murine norovirus has been identified and is closely related to human norwalk virus. however, clinically, the viruses present a different disease. the murine norovirus does not induce diarrhea nor vomiting and can develop a persistent infection in contrast to human disease. [ ] [ ] [ ] porcine enteric caliciviruses can induce diarrheal disease in young pigs and an asymptomatic infection in adults. gnotobiotic pigs can successfully be infected with a passaged clinical noroviruses isolate orally. diarrheal disease developed in % of the animals, and % were able to shed virus in their stool. no major histopathological changes or viral persistence was noted. calves are naturally infected with bovine noroviruses. experimentally challenging calves with an oral inoculation of a bovine isolate resulted in diarrheal disease [ ] [ ] [ ] the link between equine cases and the human disease was confirmed in by observing cases of fatal encephalitis in children living in the same area as the equine cases. during this outbreak, eeev was isolated from the cnss of these children as well as from pigeons and pheasants. eeev primarily affects areas near salty marshes and can cause localized outbreaks of disease in the summer. the enzootic cycles are maintained in moist environments such as coastal areas, shaded marshy salt swamps in north america (na), and moist forests in central america and south america (sa). birds are the primary reservoir, and the virus is transmitted via mosquitoes. furthermore, forest-dwelling rodents, bats, and marsupials frequently become infected and may provide an additional reservoir in central america and sa. despite known natural hosts, the transmission cycles in these animals are not well characterized. reptiles and amphibians have also been reported to become infected by eeev. eeev pathogenesis and disease have been studied in several laboratory animals. as a natural host, birds do not generally develop encephalitis except pheasants or emus, in which eeev causes encephalitis with - % mortality. young chickens show signs of extensive myocarditis in early experimental infection and heart failure rather than encephalitis is the cause of death. besides the heart, other organs such as pancreas and kidney show multifocal necrosis. additionally, lymphocytopenia has been observed in the thymus and spleen in birds. eeev causes neuronal damage in newborn mice, and the disease progresses rapidly, resulting in death. similarly, eeev produces fatal encephalitis in older mice when administered via the intracerebral route, whereas inoculation via the subcutaneous route causes a pantropic infection eventually resulting in encephalitis. , guinea pigs and hamsters have also been used as animal models for eeev studies. , guinea pigs developed neurological involvement with decreased activity, tremors, circling behavior, and coma. neuronal necrosis was observed and resulted in brain lesions in these animals. subcutaneous inoculation of eeev produced lethal biphasic disease in hamsters with severe lesions of nerve cells. the early visceral phase with viremia was followed by neuroinvasion, encephalitis and death. in addition, parenchyma necroses were observed in the liver and lymphoid organs. intradermal, intramuscular, or iv inoculations of eeev in nhps cause disease but does not always result in symptoms of the nervous system. intracerebral infection of eeev results in nervous system disease and fatality in monkeys. the differences in these models indicate that the initial viremia and the secondary nervous system infection do not overlap in monkeys when they are infected by the peripheral route. intranasal and intralingual inoculations of eeev also cause nervous system symptoms in monkeys, but less drastic than those caused by intracerebral injections. the aerosol route of infection also progresses to uniformly lethal disease in cynomolgus macaques. in this model, fever was followed by elevated white blood cells and liver enzymes. neurological signs subsequently developed, and nhps became moribund and were euthanized between days and days postexposure. meningoencephalomyelitis was the main pathology observed in the brains of these animals. similar clinical signs and pathology were observed when common marmosets were infected with eeev by the intranasal route. both aerosol and intranasal nhp models had similar disease progression and pathology as those seen in human disease. a common marmoset model was used for comparison studies of sa and na strains of eeev. previous studies indicated that the sa strain is less virulent than na strain for humans. common marmosets were infected intranasally with either the na or sa strain of eeev. na strain-infected animals showed signs of anorexia and neurological involvement and were euthanized - days after the challenge. although sa strain-infected animals developed viremia, they remained healthy and survived the challenge. epizootics of viral encephalitis in horses were previously described in argentina. more than , horses died from western equine encephalitis virus (weev) in the central plains of the united states in . weev was first isolated from the brains of horses during the outbreak in the san joaquin valley of california in . although it was suspected, the first diagnosis of weev as a cause of human encephalitis occurred in , when the virus was recovered from the brain of a child with fatal encephalitis. in horses, the signs of disease are fever, loss of coordination, drowsiness, and anorexia, leading to prostration, coma, and death in about % of affected animals. weev also infects other species of birds and often causes fatal disease in sparrows. weev infection occurs throughout western na and sporadically in sa as it circulates between its mosquito vector and wild birds. chickens and other domestic birds, pheasants, rodents, rabbits, ungulates, tortoises, and snakes are natural reservoirs of weev. , weev has caused epidemics of encephalitis in humans, horses, and emus, but the fatality rate is lower than that for eeev. predominately young children and those older than years demonstrate the clinical symptoms of the disease. severe disease, seizures, fatal encephalitis, and significant sequelae are more likely to occur in infants and young children. , typically, the disease progresses asymptomatically with seroprevalence in humans being fairly common in endemic areas. species used to develop animal models for weev are mice, hamsters, guinea pigs, and ponies. studies with ponies resulted in viremia in % of the animals - days pi. fever was observed in of animals, and six exhibited signs of encephalitis. after subcutaneous inoculation with weev, suckling mice started to show signs of disease by h and died within h. in suckling mice, the heart was the only organ in which pathologic changes were observed. conversely, adult mice exhibited signs of lethargy and ruffled fur on days - postinfection. mice were severely ill by day and appeared hunched and dehydrated. death occurred between days and , and both brain and mesodermal tissues such as heart, lungs, liver, and kidney were involved. , intracerebral and intranasal routes of infection resulted in a fatal disease that was highly dependent on dose, while intradermal and subcutaneous inoculations caused only % fatality in mice regardless of the amount of virus. studies demonstrated that although the length of the incubation period and the disease duration varied, weev infection resulted in mortality in hamsters by all routes of inoculation. progressive lack of coordination, shivering, rapid and noisy breathing, corneal opacity, and conjunctival discharge resulting in closing of the eyelids were indicative of disease in all cases. cns involvement was evident with intracerebral, intraperitoneal, and intradermal inoculations. weev is highly infectious to guinea pigs. intraperitoneal inoculation of weev is fatal in guinea pigs regardless of virus inoculum, with the animals exhibiting signs of illness on days - , followed by death on days - (nalca, unpublished results). very limited studies have been performed with nhps. the intranasal route of infection causes severe, lethal encephalitis in rhesus macaques. reed et al. exposed cynomolgus macaques to low and high doses of aerosolized weev. the animals subsequently developed fever, increased white blood counts, and cns involvement, demonstrating that the cynomolgus macaque model would be useful for testing of vaccines and therapeutics against weev. venezuelan equine encephalitis virus (veev) is maintained in nature in a cycle between small rodents and mosquitoes. the spread of epizootic strains of the virus to equines leads to high viremia followed by a lethal encephalitis, and tangential spread to humans. veev can easily be spread by the aerosol route making it a considerable danger for laboratory exposure. in humans, veev infection causes a sudden onset of malaise, fever, chills, headache, and sore throat. , , symptoms persist for - days, followed by a -to -week period of generalized weakness. encephalitis occurs in a small percentage of adults ( . %); however, the rate in children may be as high as %. neurologic symptoms range from nuchal rigidity, ataxia, and convulsions to the more severe cases exhibiting coma and paralysis. the overall mortality rate in humans is < %. laboratory animals such as mice, guinea pigs, and nhps exhibit different pathologic responses when infected with veev. the lymphatic system is a general target in all animals infected with cns involvement variable between different animal species. the disease caused by veev progresses very rapidly without showing signs of cns disease in guinea pigs and hamsters. mortality is typically observed within - days after infection and fatality is not dose dependent. veev infection lasts longer in mice, which develop signs of nervous system disease in - days and death - days later. lethal dose in mice changes depending on the age of mice and the route of exposure. in contrast to guinea pigs and hamsters, the time of the death in mice is dose dependent. mortality is observed generally within - days after infection and fatality is not dose dependent. subcutaneous/dermal infection in the mouse model results in encephalitic disease very similar to that seen in horses and humans. virus begins to replicate in the draining lymph nodes at h pi. eventually, virus enters the brain primarily via the olfactory system. furthermore, aerosol exposure of mice to veev can result in massive infection of the olfactory neuroepithelium, olfactory nerves, and olfactory bulbs and viral spread to brain, resulting in necrotizing panencephalitis. , aerosol and dermal inoculation routes cause neurological pathology in mice much faster than other routes of exposure do. the clinical signs of disease in mice infected by aerosol are ruffled fur, lethargy, and hunching progressing to death. , , intranasal challenge of c h/hen mice with high dose veev caused high morbidity and mortality. viral titers in brain peaked on day postchallenge and stayed high until animals died on day - postchallenge. protein cytokine array done on brains of infected mice showed elevated interleukin (il)- a, il- b, il- , il- , monocyte chemoattractant protein- (mcp- ), ifng, mip- a, and regulated and normal t-cell expressed and secreted levels. this model was used successfully to test antivirals against veev. xi. viral disease veev infection causes a typical biphasic febrile response in nhps. initial fever was observed at - h after infection and lasted < h. secondary fever generally began on day and lasted - days. veev-infected nhps exhibited mild symptoms such as anorexia, irritability, diarrhea, and tremors. leucopenia was common in animals exhibiting fever. supporting the leucopenia, observed microscopic changes in lymphatic tissues such as early destruction of lymphocytes in lymph nodes and spleen, a mild lymphocytic infiltrate in the hepatic triads, focal myocardial necrosis with lymphocytic infiltration have been observed in monkeys infected with veev. surprisingly, characteristic lesions of the cns were observed histopathologically in monkeys in spite of the lack of any clinical signs of infection. the primary lesions were lymphocytic perivascular cuffing and glial proliferation and generally observed at day postinfection during the secondary febrile episode. cynomolgus macaques develop similar clinical signs including fever, viremia, lymphopenia, and encephalitis upon aerosol exposure to veev. chikungunya virus is a member of the genus alphaviruses, specifically the semliki forest complex, and has been responsible for a multitude of epidemics mainly within africa and southeast asia. the virus is transmitted by aedes mosquitoes. given the widespread endemicity of aedes mosquitoes, chikungunya virus has the potential to spread to previously unaffected areas. this is typified by the emergence of disease for the first time in in the islands of the southwest indian ocean, including the french la reunion island, and the appearance in central italy in . , the incubation period after a mosquito bite is - days followed by a self-limiting acute phase that lasts - days. symptoms during this period include fever, arthralgia, myalgia, and rash. headache, weakness, nausea, vomiting, and polyarthralgia have all been reported. individuals typically develop a stooped posture due to the pain. for approximately % of infected individuals, joint pain can last months after resolution of primary disease, and has the possibility to relapse. underlying health conditions, including diabetes, alcoholism, or renal disease, increase the risk of developing a severe form of disease that includes hepatitis or encephalopathy. children between the ages of and years have an increased risk of developing neurological manifestations. there is no effective vaccine or antiviral. wild-type c bl/ adult mice are not permissive to chikungunya virus infection by intradermal inoculation. however, it was demonstrated that neonatal mice were susceptible, and severity was dependent upon age at infection. six-day-old mice developed paralysis by day , and all died by day , whereas % of nine-day-old mice were able to recover from infection. by days, mice were no longer permissive to disease. infected mice developed loss of balance, hind limb dragging, and skin lesions. neonatal mice were also used as a model for neurological complications. , an adult mouse model has been developed by injection of the ventral side of the footpad of c bl/ j mice. viremia lasted - days accompanied by foot swelling and noted inflammation of the musculoskeletal tissue. , adult ifna/br knockout mice also developed mild disease with symptoms including muscle weakness and lethargy, symptoms that mirrored human infection. all adult mice died within days. this model was useful in identifying the viral cellular tropism for fibroblasts. imprinting control region (icr) cd mice can also be used as a disease model. neonatal mice subcutaneously inoculated with a passaged clinical isolate of chikungunya virus developed lethargy, loss of balance, and difficulty in walking. mortality was low, % and % for newborn cd and icr mice, respectively. the remaining mice fully recovered within weeks after infection. a drawback of both the ifna/ br and cd mice is that the disease is not a result of immunopathogenesis as occurs in human cases, given that the mice are immunocompromised. long-tailed macaques challenged with a clinical isolate of the virus developed a similar clinical disease to humans. initially, the monkeys developed high viremia with fever and rash. after this period, viremia resolved and virus could be detected in lymphoid, liver, meninges, joint, and muscle tissue. the last stage mimicked the chronic phase in which virus could be detected up to two months after infection, although no arthralgia was noted. dengue virus is transmitted via the mosquito vectors aedes aegypti and aedes albopictus. given the endemicity of the vectors, it is estimated that half of the world's population is at risk for exposure to dengue virus. this results in approximately million cases of dengue each year, with the burden of disease in the tropical and subtropical regions of latin america, south asia, and southeast asia. it is estimated that there are , deaths each year caused by dengue hemorrhagic fever (dhf). there are four serotypes of dengue virus, numbered - , which are capable of causing a wide spectrum of disease that ranges from asymptomatic to severe with the development of dhf. incubation can range from to days, with the average being - days. the virus targets dendritic cells and macrophages after a mosquito bite. typical infection results in classic dengue fever (df), which is self-limiting and has flu-like symptoms in conjunction with retroorbital pain, headache, skin rash, and bone and muscle pain. dhf can follow, with vascular leak syndrome and low platelet count, resulting in hemorrhage. in the most extreme cases, dengue shock syndrome (dss) develops, characterized by hypotension, shock, and circulatory failure. thrombocytopenia is a hallmark clinical sign of infection, and aids in differential diagnosis. severe disease has a higher propensity to occur upon secondary infection with a different dengue virus serotype. this is hypothesized to occur due to antibodydependent enhancement (ade). there is no approved vaccine or drug, and hospitalized patients receive supportive care including fluid replacement. in developing an animal model, it is important to note that mosquitoes typically deposit - pfu, and is therefore the optimal range to be used during challenge. a comprehensive review of the literature regarding animal models of dengue infection was recently published by zompi et al. several laboratory mouse strains including a/j, balb/c, and c bl/ are permissive to dengue infection. however, the resulting disease has little resemblance to human clinical signs, and death results from paralysis. [ ] [ ] [ ] a higher dose of an adapted dengue virus strain induced dhf symptoms in both balb/c and c bl/ . , this model can also yield asymptomatic infections. a mouse-adapted (ma) strain of dengue virus introduced into ag mice developed vascular leak syndrome similar to the severe disease seen in humans. passive transfer of monoclonal dengue antibodies within mice leads to ade. during the course of infection, viremia was increased, and animals died due to vascular leak syndrome. another ma strain injected into balb/c caused liver damage, hemorrhagic manifestations, and vascular permeability. intracranial injection of suckling mice with dengue virus leads to death and has been used to test the efficacy of therapeutics. scid mice engrafted with human tumor cells develop paralysis upon infection, and are thus not useful for pathogenesis studies. , df symptoms developed after infection in nod/scid/il rgko mice engrafted with cd þ human progenitor cells. rag-hu mice developed fever, but no other symptoms upon infection with a passaged clinical isolate and laboratory-adapted strain of dengue virus . a passaged clinical isolate of dengue virus type was recently used to create a model in immunocompetent adult mice. interperitoneal injection in c bl/ j and balb/c caused lethality by day - postinfection in a dose-dependent manner. the first indication of infection was weight loss beginning on day followed by thrombocytopenia. a drop in systolic blood pressure along with noted increases in the liver enzymes, aspartate aminotransferase (ast) and alt, were also observed. viremia was established by day . this model mimicked the characteristic symptoms observed in human dhf/dss cases. a novel model was developed that used infected mosquitoes as the route of transmission to hu-nsg mice. female mosquitoes were intrathoracically inoculated with a clinical isolate of dengue virus type . infected mosquitoes then fed upon the mouse footpad to allow for the transmission of the virus via the natural route. the amount of virus detected within the mouse was directly proportional to the amount of mosquitoes it was exposed to, with four to five being optimal. detectable viral rna was in line with what is observed during human infection. severe thrombocytopenia developed on day . this model is intriguing given that disease was enhanced with mosquito delivery of the virus in comparison to injection of the virus. nhp models have used a subcutaneous inoculation in an attempt to induce disease. although the animals are permissive to viral replication, it is to a lower degree than that observed in human infection. the immunosuppressive drug, cyclophosphamide enhances infection in rhesus macaques by allowing the virus to invade monocytes. throughout these preliminary studies, no clinical disease was detected. to circumvent this, a higher dose of dengue virus was used in an iv challenge of rhesus macaques. hemorrhagic manifestations appeared by day and resulted in petechiae, hematomas, and coagulopathy; however, no other symptoms developed. further development would allow this model to be used for testing of novel therapeutics and vaccines. although primates do not develop disease upon infection with dengue, their immune system does produce antibodies similar to those observed during the course of human infection. this has been advantageous in studying ade. sequential infection led to a crossreactive antibody response, which has been demonstrated in both humans and mice. this phenotype can also be seen upon passive transfer of a monoclonal antibody to dengue and subsequent infection with the virus. rhesus macaques exposed in this manner developed viremia that was -to -fold higher than was previously reported; however, no clinical signs were apparent. the lack of inducible dhf or dss symptoms hinders further examination of pathogenesis within this model. japanese encephalitis virus ( jev) is a leading cause of childhood viral encephalitis in southern and eastern asia and is a problem among military personnel and travelers to these regions. it was first isolated from the brain of a patient who died from encephalitis in japan in . culex mosquitoes, which breed in rice fields, transmit the virus from birds or mammals (mostly domestic pigs) to humans. the disease symptoms range from a mild febrile illness to acute meningomyeloencephalitis. after an asymptomatic incubation period of - weeks, patients show signs of fever, headache, stupor, and generalized motor seizures, especially in children. the virus causes encephalitis by invading and destroying the cortical neurons. the fatality rate ranges from % to %, and most survivors have neurological and psychiatric sequelae. , jev virus causes fatality in infant mice by all routes of inoculation. differences in pathogenesis and outcome are seen when the virus is given by intraperitoneal inoculation. these differences depend on the amount of virus and the specific viral strains used. the biphasic viral multiplication after peripheral inoculation is observed in mice tissues. primary virus replication occurs in the peripheral tissues and the secondary replication phase in the brain. hamsters are another small animal species that are used as an animal model for jev. fatality was observed in hamsters inoculated intracerebrally or intranasally, while peripheral inoculation caused asymptomatic viremia. studies with rabbits and guinea pigs showed that all routes of inoculation of jev produce asymptomatic infection. serial sampling studies with -day-old wistar rats inoculated intracerebrally with jev indicated that jev causes the overproduction of free radicals by neurons and apoptosis of neuronal cells. following a study in by the same group, showed that although cytokines tumor necrosis factor (tnf)-a, ifn-g, il- , il- , il- , and chemokine mcp- increased gradually and peaked on days pi with jevin rats, the levels eventually declined, and there was no correlation with the levels of cytokines and chemokines and neuronal damage. intracerebral inoculation of jev causes severe histopathological changes in brain hemispheres of rhesus monkeys. symptoms such as weakness, tremors, and convulsions began to appear on days - , with indicative signs of encephalomyelitis occurring on days - postinfection for most of the animals followed by death occurring on days - postinfection for most of the animals followed by death. although intranasal inoculation of jev results in fatality in both rhesus and cynomolgus monkeys, peripheral inoculation causes asymptomatic viremia in these species. , west nile virus west nile virus (wnv) was first isolated from the blood of a woman in the west nile district of uganda in . after the initial isolation of wnv, the virus was subsequently isolated from patients, birds, and mosquitoes in egypt in the early s , and was shown to cause encephalitis in humans and horses. wnv is recognized as the most widespread of the flaviviruses, with a geographical distribution that includes africa, the middle east, western asia, europe, and australia. the virus first reached the western hemisphere in the summer of , during an outbreak involving humans, horses, and birds in the new york city metropolitan area. , since , the range of areas affected by wnv quickly extended. older people and children are most susceptible to wnv disease. wnv generally causes asymptomatic disease or a mild undifferentiated fever (west nile fever), which can last from to days. the mortality rate after neuroinvasive disease ranges from % to %. , [ ] [ ] [ ] the most severe complications are commonly seen in the elderly, with reported case fatality rates from % to %. hepatitis, myocarditis, and pancreatitis are unusual, severe, nonneurologic manifestations of wnv infection. although many early laboratory studies of wn encephalitis were performed in nhps, mice, rat, hamster, horse, pig, dog, and cat models were used to study the disease. [ ] [ ] [ ] [ ] [ ] [ ] [ ] inoculation of wnv into nhps intracerebrally resulted in the development of either encephalitis, febrile disease, or an asymptomatic infection, depending on the virus strain and dose. viral persistence is observed in these animals regardless of the outcome of infection (i.e. asymptomatic, fever, encephalitis). thus, viral persistence is regarded as a typical result of nhp infection with various wnv strains. after both intracerebral and subcutaneous inoculation, the virus localizes predominantly in the brain and may also be found in the kidneys, spleen, and lymph nodes. wnv does not result in clinical disease in nhps although the animals show a low level of viremia. , wnv has also been extensively studied in small animals. all classical laboratory mouse strains are susceptible to lethal infections by the intracerebral and intraperitoneal routes, resulting in encephalitis and % mortality. intradermal route pathogenesis studies indicated that langerhans dentritic cells are the initial viral replication sites in the skin. , the infected langerhans cells then migrate to lymph nodes, and the virus enters the blood through lymphatic and thoracic ducts and disseminates to peripheral tissues for secondary viral replication. virus eventually travels to the cns and causes pathology that is similar to human cases. [ ] [ ] [ ] [ ] tesh et al. developed a model for wn encephalitis using the golden hamster, mesocricetus auratus. hamsters appeared normal during the first days, became lethargic at approximately day , and developed neurologic symptoms by days - . many of the severely affected animals died - days after infection. viremia was detected in the hamsters within h after infection and persisted for - days. although there were no substantial changes in internal organs, progressive pathologic differences were seen in the brain and spinal cord of infected animals. furthermore, similar to the above-mentioned monkey experiments by pogodina et al., persistent wnv infection was found in the brains of hamsters. the etiologic agent of severe acute respiratory syndrome (sars), sars-coronavirus (cov), emerged in as it spread throughout countries in a period of months, infecting > people and causing nearly deaths. , the main mechanism of transmission of sars-cov is through droplet spread, but it is also viable in dry form on surfaces for up to days and can be detected in stool, suggesting other modes of transmission are also possible. although other members of the family usually cause mild illness, sars-cov infection has a % case fatality with the majority of cases in people over the age of years. , after an incubation period of - days, clinical signs of sars include general malaise, fever, chills, diarrhea, dyspnea, and cough. in some sars, cases, pneumonia may develop and progress to acute respiratory distress syndrome (ards). fever usually dissipates within weeks and coincides with the induction of high levels of neutralizing antibodies. in humans, sars-cov replication destroys respiratory epithelium, and a great deal of the pathogenesis is due to the subsequent immune responses. infiltrates persisting within the lung and diffuse alveolar damage (dad) are common sequelae of sars-cov infection. virus can be isolated from secretions of the upper airways during early, but not later stages of infection as well as from other tissues. sars-cov can replicate in many species, including dogs, cats, pigs, mice, rats, ferrets, foxes, and nhps. chinese palm civets, raccoon dogs, and bats are possible natural hosts. no model captures all aspects of human clinical disease (pyrexia and respiratory signs), mortality (w %), viral replication, and pathology. in general, the sars-cov disease course in the model species is much milder and of shorter duration than in humans. viral replication in the various animal models may occur without clinical illness and/or histopathologic changes. the best-characterized models use mice, hamsters, ferrets, and nhps (table . ). mouse models of sars-cov typically are inoculated by the intranasal route under light anesthesia. young, -to -week old balb/c mice exposed to sars-cov have viral replication detected in the lungs and nasal turbinates, with a peak on day and clearance by day postexposure. there is also viral replication within the small intestines of young balb/c mice. however, young mice have no clinical signs, aside from reduced weight gain, and have little to no inflammation within the lungs (pneumonitis). intranasal sars-cov infection of c bl/ (b ) also yield reduced weight gain and viral replication in the lungs, with a peak on day and clearance by day . in contrast, balb/c mice - interstitial pneumonitis, alveolar damage, and death also occur in old mice, resembling the age-dependent virulence observed in humans. s mice and b mice show outcomes to sars-covinfection similar to those observed for balb/c mice but have lower titers and less prolonged disease. one problem is that it is more difficult to obtain large numbers of mice older than year. a number of immunocompromised knockout mouse models of intranasal sars-cov infection have also been developed. svev mice infected with sars-cov by the intranasal route develop bronchiolitis, with peribronchiolar inflammatory infiltrates, and interstitial inflammation in adjacent alveolar septae. viral replication and disease in these mice resolve by day postexposure beige, cd À/À, and rag À/À mice infected with sars-cov have similar outcomes to infected balb/c mice with regard to viral replication, timing of viral clearance, and a lack of clinical signs. signal transducer and activator of transcription- (stat ) ko mice infected intranasally with sars-cov have severe disease, with weight loss, pneumonitis, interstitial pneumonia, and some deaths. the stat ko mouse model is therefore useful for studies of pathogenicity, pathology, and evaluation of vaccines. syrian golden hamsters (strain lvg) are also susceptible to intranasal exposure of sars-cov. after the administration of tcid (tissue culture infective dose), along with a period of transient viremia, sars-cov replicates in nasal turbinates and lungs, resulting in pneumonitis. there are no obvious signs of disease, but exercise wheels can be used to monitor decrease in nighttime activity. some mortality has been observed, but it was not dose dependent and could have more to do with genetic differences between animals because the strain is not inbred. damage is not observed in the liver or spleen despite detection of virus within these tissues. several studies have shown that intratracheal inoculation of sars-cov in anesthetized ferrets (mustela furo) results in lethargy, fever, sneezing, and nasal discharge. clinical disease has been observed in several studies. sars-cov is detected in pharyngeal swabs, trachea, tracheobronchial lymph nodes, and high titers within the lungs. mortality has been observed around day postexposure as well as mild alveolar damage in - % of the lungs, occasionally accompanied by severe pathology within the lungs. with fever, overt respiratory signs, lung damage, and some mortality, the ferret intratracheal model of sars-cov infection is perhaps most similar to human sars, albeit with a shorter time course. sars-cov infection of nhps by intransal or intratracheal routes generally results in a very mild infection, which resolves quickly. sars-cov infection of old world monkeys, such as rhesus macaques, cynomolgus macaques (cynos), and african green monkeys (agms) have been studied with variable results, possibly due to the outbred nature of the groups studied or previous exposure to related pathogens. clinical illness and viral loads have not been consistent; however, replication within the lungs and dad are features of the infections for each of the primate species. some cynos have no illness, but others have rash, lethargy, and respiratory signs and pathology. rhesus have little to no disease and only have mild findings upon histopathological analysis. agms infected with sars-cov have no overt clinical signs, but dad and pneumonitis have been documented. viral replication has been detected for up to days in the lungs of agms; however, the infection resolves and does not progress to fatal ards. farmed chinese masked palm civets, sold in open markets in china, were thought to be involved in the sars-covoutbreak. intratracheal and intranasal inoculation of civets with sars-covresults in lethargy, decreased aggressiveness fever, diarrhea, and conjunctivitis. leucopenia, pneumonitis, and alveolar septal enlargement, with lesions similar to those observed in ferrets and nhps, have also been observed in laboratory-infected civets. common marmosets have also been shown to be susceptible to sars-cov infection. vaccines have been developed for related animal covs in chickens, cattle, dogs, cats, and swine have used live-attenuated, killed, dna and viral-vectored vaccine strategies. an important issue to highlight from work on these vaccines is that cov vaccines, such as those developed for cats, may induce a more severe disease. as such, immune mice had th -type immunopathology upon sars-cov challenge. severe hepatitis in vaccinated ferrets with antibody enhancement in liver has been reported. additionally, rechallenge of agms showed limited viral replication but significant lung inflammation, including alveolitis and interstitial pneumonia, which persisted for long periods of time after viral clearance. mouse and nhp models with increased virulence may be developed by adapting the virus by repeated passage within the species of interest. ma sars and human ace transgenic mice are available. all mammals experimentally or naturally exposed to rabies virus have been found to be susceptible. this highly neurotropic virus is a member of the lyssavirus genus and is transmitted from the bite of an infected animal to humans. the virus is able to replicate within the muscle cells at the site of the bite, and then travel to the cns. once reaching the cns by retrograde axonal transport, the virus replicates within neurons creating inflammation and necrosis. the virus subsequently spreads throughout the body via peripheral nerves. a typical incubation period is - days, and is highly dependent upon the location of the bite. proximity to the brain is a major factor for the onset of symptoms. the prodromal stage lasts from to days and is when the virus initially invades the cns. flu-like symptoms are the norm in conjunction with pain and inflammation at the site of the bite. subsequently, there are two forms of disease that can develop. in % of cases, an individual develops the encephalitic or furious form. this form is marked by hyperexcitability, autonomic dysfunction, and hydrophobia. the paralytic, or dumb form, is characterized by ascending paralysis. ultimately, both forms result in death days after the onset of symptoms. once the symptoms develop, there is no proven effective therapy. in the developing world, death is caused by the lack of access to medical care including postexposure prophylaxis. in na, fatal cases result because of late diagnosis. syrian hamsters have been challenged with rabies virus intracerebrally, intraperitoneally, intradermally, and intranasally. all animals died as a result of the exposure, although intracerebral and intranasal inoculation led to only the furious form depicted by extreme irritability, spasms, excessive salivation, and cries. the virus used had been isolated from an infected dog brain and passaged in swiss albino mice. animals inoculated by intracerebral injection develop disease within - days, whereas all other routes of entry develop disease within - days. this model has been used to study and test novel vaccine candidates. mice have been extensively studied as an animal model for rabies. it was shown that swiss albino mice intracerebrally injected with a virus isolated from a dog developed only the paralytic form of disease days after the initial challenge. balb/c mice are universally susceptible to intracerebral injection of rabies virus within days. disease symptoms include paralysis, cachexia, and bristling appearing - days before death. a more natural route of infection via peripheral injection into masseter muscles was tested on icr mice. these mice developed neurological signs including limb paralysis, and all died within - days. icr mice have been instrumental in analyzing novel vaccines and correlates of protection. this line of mice was also used to assess the value of ketamine treatment to induce coma during rabies infection. another mouse line used is the p neurotrophin receptor-deficient mouse. this mouse developed a fatal encephalitis when inoculated intracerebrally with the challenge virus standard. bax-deficient mice have also been used to determine the role of apoptotic cell death in the brain during the course of infection. a viral isolate from silver-haired bats can also be used in the mouse model. this strain is advantageous given that it is responsible for the majority of deaths in north na. early death phenomenon is typified by a decrease to time of death in a subset of individuals and animals that have been vaccinated and subsequently exposed to rabies. this trend has been demonstrated experimentally in swiss outbred mice and primates. , cynomolgus and rhesus were both infected with passaged rabies virus to create an nhp model. a high titer of virus was needed to induce disease, but exposure was found to not be universally fatal. the animals that survived beyond weeks within the experiment did not develop clinical disease nor succumb to infection. primates that did develop disease refused food and had progressively less activity until death. this lasted from h up to days, with all animals with symptoms dying within weeks. bats have been experimentally challenged with rabies. vampire bats, desmodus rotundus, intramuscularly injected with a bat viral isolate displayed clinical signs including paralysis in half of the population of the study animals. of those who did develop disease, the duration was days, and incubation period ranged from to days. regardless of disease manifestation, % of challenged animals died. skunks can be challenged intramuscularly or intranasally with either challenge virus strain or a skunk viral isolate. interestingly, the challenge virus strain more readily produced the paralytic form, whereas the street form of rabies developed into the furious form. however, the challenge strain virus resulted in a shorter incubation period of - days in comparison to - days seen with the street virus. filoviridae consists of two well-established genera, ebola virus and marburg virus (marv) and a newly discovered group, cuevavirus (table two other ebola viruses are known; taï forest (tafv; previously named cote (circumflex over the 'o') d'ivoire) (ciebov) and reston (restv), which have not caused major outbreaks or lethal disease in humans. the disease in humans is characterized by aberrant innate immunity and a number of clinical symptoms such as fever, nausea, vomiting, arthralgia/myalgia, headaches, sore throat, diarrhea, abdominal pain, anorexia, and numerous others. approximately % of patients develop petechia and a greater percentage, depending on the specific strain, may develop bleeding from various sites (gums, puncture sites, stools, etc.). natural transmission in an epidemic is thought to be through direct contact or needle sticks in hospital settings. however, much of the research interest in filoviruses primarily stems from biodefense needs, particularly from aerosol biothreats. as such, intramuscular, intraperitoneal, and aerosol models have been developed in mice, hamsters, guinea pigs, and nhps for the study of pathogenesis, correlates of immunity, and for testing countermeasures. because filoviruses have such high lethality rates in humans, scientists have looked for models that are uniformly lethal to stringently test efficacy of candidate vaccines and therapeutics. immunocompetent mice have not been successfully infected with wild-type filoviruses due to the control of the infection by the murine type ifn response. however, wild-type inbred mice are susceptible to filovirus that has been ma by serial passage. balb/c mice, which are the strain of choice for intraperitoneal inoculation of ma-ebov, are not susceptible by the aerosol route. for aerosol infection of immunocompetent mice, a panel of bxd (balb/ c  dba) recombinant inbred strains were screened, and one strain, bxd , was shown to be particularly susceptible to airborne ma-ebov, with % lethality to low or high doses (w or pfu). these mice developed weight loss of > % and succumbed to infection between days and postexposure. the aerosol infection model uses a whole-body exposure chamber to expose mice aged - weeks to ma-ebov aerosols with a mass median aerodynamic diameter (mmad) of approximately . mm and a geometric standard deviation of approximately . for min. another approach uses immunodeficient mouse strains such as scid, stat ko, ifn receptor ko, or perforin ko with a wild-type ebov inoculum by intraperitoneal or aerosol routes. mice are typically monitored for clinical disease "scores" based on activity and appearance, weight loss, and moribund condition (survival). coagulopathy, a hallmark of filovirus infection in humans, has been observed, with bleeding in a subset of animals and failure of blood samples to coagulate late in infection. liver, kidney, spleen, and lung tissue taken from moribund mice have pathology characteristic of filovirus disease in nhps. although most mouse studies have used ma-ebov or ebov, an intraperitoneal ma marv model is also available. ma-marv and ma-ebov models are particularly useful for screening novel antiviral compounds. hamsters are frequently used to study cardiovascular disease, coagulation disorders, and thus serve as the basis for numerous viral hemorrhagic fever models. an intraperitoneal ma-ebov infection model has been developed in syrian hamsters. this model, which has been used to test a vesicular stomatitis virus vectored vaccine approach, uses male -to -week-old syrian hamsters that are infected with ld of ma-ebov. virus is present in tissues and blood collected on day , and all animals succumbed to the disease by day . detailed accounts of this model have been presented at international scientific meetings by ebihara and feldmann et al. but have not been reported in a scientific journal at the time of writing this chapter. guinea pig models of filovirus infection have been developed for intraperitoneal and aerosol routes using guinea pig-adapted ebov (gp-ebov) and marv (gp-marv). , guinea pigs models of filovirus infection are quite useful in that they develop fever, which can be monitored at frequent (hourly) intervals by telemetry. additionally, the animals are large enough for regular blood sampling in which measurable coagulation defects are observed as the infection progresses. hartley guinea pigs exposed to aerosolized gp-marv or gp-ebov become moribund at times comparable to that of nhps, generally succumbing to the infection between and days postexposure. by aerosol exposure, gp-ebov is uniformly lethal at both high and low doses ( or pfu target doses) but lethality drops with low (< pfu) presented doses of airborne gp-marv, and more protracted disease is seen in some animals. weight loss of between % and % is a common finding in guinea pigs exposed to gp-ebov or gp-marv. fever, which becomes apparent by day , occurs more rapidly in gp-ebov exposed guinea pigs than with gp-marv exposure. lymphocytes and neutrophils increase during the earlier part of the disease, and platelet levels steadily drop as the disease progresses. increases in coagulation time can be seen as early as day postexposure. blood chemistries (i.e. alt, ast, alkaline phosphatase (alkp), and blood urea nitrogen) indicating problems with liver and kidney function are also altered late in the disease course. nhp models of filovirus infection are the preferred models for more advanced disease characterization and testing of countermeasures because they most closely mimic the disease and immune correlates seen in humans. old world primates have been primarily used for the development of intraperitoneal, intramuscular, and aerosol models of filovirus infection. uniformly lethal filovirus models have been developed for most of the virus strains in cynomolgus macaques, rhesus macaques, and to a lesser degree, in agms and marmosets. [ ] [ ] [ ] [ ] [ ] low-passage human isolates that have not been passaged in animals have been sought for development of nhp models to satisfy the food and drug administration (fda) animal rule. prominent features of the infections are onset of fever by day postexposure, alteration in liver function enzymes (alt, ast, and alkp), decrease in platelets, and increased coagulation times. clinical disease parameters may have a slightly delayed onset in aerosol models. petichial rash is a common sign of filovirus disease and may be more frequently observed in cynomolgus macaques than in other nhp species. dyspnea late in infection is a prominent feature of disease after aerosol exposure. a number of pronounced pathology findings include multifocal necrosis and fibrin lesions, particularly within the liver and the spleen. lymphocytolysis and lymphoid depletion are also observed. multilead, surgically implanted telemetry devices are useful in the continuous collection of temperature, blood pressure, heart rate, and activity levels. as such, blood pressure drops as animals become moribund and heart rate variability (standard deviation of the heart rate) is altered late in infection. the most recently developed telemetry devices can aid in plethysomography to measure respiratory minute volume for accurate delivery of presented doses for aerosol exposure. hendra and nipah virus are unusual within the paramyxoviridae family given that they can infect a large range of mammalian hosts. both viruses are grouped under the genus henipavirus. the natural reservoirs of the viruses are the fruit bats from the genus pteropus. hendra and nipah have the ability to cause severe disease in humans with the potential for a high case fatality rate. outbreaks caused by nipah virus have been recognized in malaysia, singapore, bangladesh, and india, while hendra outbreaks have yet to be reported outside of australia. , hendra was the first member of the genus to be identified and was initially associated with an acute respiratory disease in horses. all human cases have been linked to transmission through close contact with an infected horse. there have been no confirmed cases of direct transmission from human to human or bat to human. nipah has the distinction of being able to be transmitted by humans, although the exact route is unknown. the virus is susceptible to ph, temperature, and desiccation, and thus close contact is hypothesized to be needed for successful transmission. both viruses have a tropism for the neurological and respiratory tract. hendra virus incubation period is - days and is marked by a flu-like illness. symptoms at this initial stage include myalgia, headache, lethargy, sore throat, and vomiting. disease progression can continue to pneumonitis or encephalitic manifestations, with the person succumbing to multiorgan failure. nipah virus has an incubation period of days to weeks. much like hendra, the first signs of disease are nondescript. severe neurological symptoms subsequently develop including encephalitis and seizures that can progress to coma within - h. survivors of infection typically make a full recovery; however, % suffer permanent sequelae, including persistent convulsions. at this time, there is no approved vaccine or antiviral, and treatment is purely supportive. animal models are being used to not only test novel vaccines and therapeutics, but also deduce the early events of disease because observed human cases are all at terminal stages. the best small animal representative is the syrian golden hamster due to their high susceptibility to both henipaviruses. clinical signs upon infection recapitulate the disease course in humans including acute encephalitis and respiratory distress. challenged animals died xi. viral disease . in vivo models of viral diseases affecting humans within - days postinfection. the progression of disease and timeline are highly dependent on dose and route of infection. intranasal inoculation leads to imbalance, limb paralysis, lethargy, and breathing difficulties whereas intraperitoneal resulted in tremors and paralysis within h before death. virus was detected in lung, brain, spleen, kidney, heart, spinal cords, and urine, while the brain was the most affected organ. this model has been used for vaccination and passive protection studies. [ ] [ ] [ ] the guinea pig model has not been widely used due to the lack of a respiratory disease upon challenge. , inoculation with hendra virus via the subcutaneous route leads to a generalized vascular disease with % mortality. clinical signs were apparent - days postinfection with death occurring within days of cns involvement. higher inocula have been associated with the development of encephalitis lesions. intradermal and intranasal injections do not lead to disease, although the animals are able to seroconvert upon challenge. inoculum source does not affect clinical progression. nipah virus challenge only develops disease upon intraperitoneal injection and results in weight loss and transient fever for - days. virus was shed through urine and found to be present in the brain, spleen, lymph nodes, ovary, uterus, and urinary bladder. ferrets display the same clinical disease as seen in the hamster model and human cases. , upon inoculation by the oronasal route, ferrets develop severe pulmonary and neurological disease within - days including fever, coughing, and dyspnea. lesions do develop in the ferrets' brains, but to a lesser degree than seen in humans. cats have also been used as an animal model for henipaviruses. disease symptoms are not dependent upon the route of infection. the incubation period is - days and leads to respiratory and neurological symptoms. , this model has proven to be useful in a vaccine challenge model. squirrel and agms are representative of the nhp models. within the squirrel monkeys, nipah virus is introduced by either the intranasal or iv route and subsequently leads to clinical signs similar to that in humans, although intranasal challenge results in milder disease. upon challenge, only % animals develop disease manifestations including anorexia, dyspnea, and acute respiratory syndrome. neurological involvement is characterized by uncoordinated motor skills, loss of consciousness, and coma. viral rna can be detected in the lung, brain, liver, kidney, spleen, and lymph nodes but is only found upon iv challenge. agms have been found to be a very consistent model of both viruses. intratracheal inoculation of the viruses results in % mortality, and death within . and - days postinfection for hendra and nipah, respectively. the animals develop severe respiratory and neurological disease with generalized vasculitis. , the reservoir of the viruses, gray-headed fruit bats, has been experimentally challenged. due to their status as the host organism for henipaviruses, the bats do not develop clinical disease. however, hendra virus can be detected in kidneys, heart, spleen, and fetal tissue and nipah virus can be located in urine. pigs have been investigated as a model as they develop a respiratory disease upon infection with both nipah and hendra. [ ] [ ] [ ] oral inoculation does not produce a clinical disease, but subcutaneous injection represents a successful route of infection. live virus can be isolated from the oropharynx as early as days postinfection. nipah can also be transmitted between pigs. nipah was able to induce neurological symptoms in % of the pigs, even though virus was present in all neurological tissues regardless of symptoms. within the pig model, it seemed that nipah had a greater tropism for the respiratory tract, while hendra for the neurological system. horses also are able to develop a severe respiratory tract infection accompanied with fever and general weakness upon exposure to nipah and hendra. oronasal inoculation led to systemic disease with viral rna detected in nasal swabs within days. , animals died within days postexposure and were found to have interstitial pneumonia with necrosis of alveoli. , virus could be detected in all major systems. mice, rats, rabbits, chickens, and dogs have been tested but found to be nonpermissive to infection. , suckling balb/c mice succumb to infection if the virus is inoculated intracranially. embryonated chicken eggs have been inoculated with nipah virus leading to a universally fatal disease within - days postinfection. respiratory syncytial virus is responsible for lower respiratory tract infections of million children under the age of years, which in turn results in three million hospitalizations and approximately , deaths. within the united states, hospital costs alone amount to > million dollars. outbreaks are common in the winter. the virus is transmitted by large respiratory droplets that replicate initially within the nasopharynx and further spreads to the lower respiratory tract. incubation for the virus is - days. respiratory syncytial virus is highly virulent leading to very few asymptomatic infections. disease manifestations are highly dependent upon the age of the individual. primary infections in neonates produce nonspecific symptoms including the overall failure to thrive, apnea, and feeding difficulties. infants present with a mild upper respiratory tract disease that could develop into bronchiolitis and bronchopneumonia. contracting the virus at this age results in an increased chance of developing childhood asthma. young children develop recurrent wheezing, whereas adults exacerbate previous respiratory conditions. common clinical symptoms are runny nose, sneezing, and coughing accompanied with fever. mortality rates in hospitalized children are - % with the greatest burden of disease seen in - -month-olds. there is no vaccine available, and ribavirin usage is not recommended for routine treatment. animal models were developed in the hopes of formulating an effective and safe vaccine unlike the formalin-inactivated respiratory syncytial virus vaccine (fi-rsv). this vaccineinduced severe respiratory illness in infants who received the vaccine and were subsequently infected with live virus. mice can be used to model disease, although a very high intranasal inoculation is needed to achieve clinical symptoms. , strain choice is crucial to reproducing a physiological relevant response. age does not affect primary disease manifestations. however, it does play a role in later sequelae showing increased airway hyperreactivity. primary infection produces increased breathing with airway obstruction. , virus was detected as early as day and reached maximum titer at day postinfection. clinical illness is defined in the mouse by weight loss and ruffled fur as opposed to runny nose, sneezing, and coughing as seen in humans. cotton rats are useful given that it is a small animal disease model. the virus is able to replicate to high titers within the lungs and can be detected in both the upper and lower airways after intranasal inoculation. , it has been reported that viral replication is -to -fold greater in the rat model than in the mouse model. the rats develop mild-to-moderate bronchiolitis or pneumonia. although age does not seem to factor in clinical outcome, it has been reported that older rats tend to take longer to achieve viral clearance. viral loads peak by the fifth day, dropping to below the levels of detection by . the histopathology of the lungs seems to be similar to that in humans after infection. this model has limited usage in modeling the human immune response to infection as challenge with the virus creates a th response, whereas humans tend to skew toward th . [ ] [ ] [ ] fi-rsv disease was recapitulated upon challenge with live virus after being vaccinated twice with fi-rsv. chinchillas have been challenged experimentally via intranasal inoculation. the virus was permissive within the nasopharynx and eustachian tube. the animals displayed an acute respiratory tract infection. this model is thought to be useful in studying mucosal immunity during infection. chimpanzees are permissive to replication and clinical symptoms of respiratory syncytial virus including rhinorrhea, sneezing, and coughing. adult squirrel monkeys, newborn rhesus macaques, and infant cebus monkeys were also challenged but did not exhibit any disease symptoms nor high levels of viral replication. bonnet monkeys were also tested and found to develop an inflammatory response by day with viral rna detected in both bronchial and alveolar cells. the chimpanzee model has proven to be useful for vaccine studies. , sheep have also been challenged experimentally since they develop respiratory disease when exposed to ovine respiratory syncytial virus. lambs were also found to be susceptible to human respiratory syncytial infection. , when inoculated intratracheally, the lambs developed an upper respiratory tract infection with cough after days. some lambs went on to develop lower respiratory disease including bronchiolitis. the pneumonia resolved itself within days. during the course of disease, viral replication peaked at days, and rapidly declined. studying respiratory disease in sheep is beneficial given the shared structural features between them and humans. , the influenza viruses consist of three types: influenza a, b, and c, based on antigenic differences. influenza a is further classified by subtypes; ha and na subtypes are known. seasonal influenza is the most common infection and usually causes a self-limited febrile illness with upper respiratory symptoms and malaise that resolves within days. the rate of infection is estimated at % in the general population and can result in billions of dollars of loss annually from medical costs and reduced work-force productivity. approximately , people in the united states die each year from seasonal influenza. thus, vaccines and therapeutics play a critical role in controlling infection, and development using animal models is ongoing. influenza virus replicates in the upper and lower airways, peaking at approximately h postexposure. infection can be more severe in infants and in children under the age of years, people over the age of years, or immunocompromised individuals in whom viral pneumonitis or pneumonia can develop or bacterial superinfection resulting in pneumonia or sepsis. pneumonia from secondary bacterial infection, such as streptococcus pneumonia, streptococcus pyrogenes, and neisseria meningitides, and more rarely, staphylococcus aureus, is more common than viral pneumonia from the influenza itself, accounting for approximately % of all influenza-associated fatalities. death, often due to ards can occur as early as days after the onset of symptoms. lung histopathology in severe cases may include dad, alveolar edema and damage, hemorrhage, fibrosis, and inflammation. the h n avian strain of influenza has lethality rates of approximately % (of known cases), likely because the virus preferentially binds to the cells of the lower respiratory tract, and thus, the potential for global spread is a major concern. the most frequently used animal models of influenza infection include mice, ferrets, and nhps. a very thorough guide to working with mouse, guinea pig, ferret, and cynomolgus models was published by kroeze et al. lethality rate can vary with the virus strain used (with or without adaptation), dose, route of inoculation, age, and genetic background of the animal. the various animal models can capture differing diseases caused by influenza: benign, severe, superinfection and sepsis, severe with ards, and neurologic manifestations. also, models can use seasonal or avian strains and models have been developed to study transmission, important for understanding the potential for more lethal strains such as h n for spreading among humans. mouse models of influenza infection are very predictive for antiviral activity and tissue tropism in humans, and are useful in testing and evaluating vaccines. inoculation is by the intranasal route, using approximately ml of inoculum in each nare of anesthetized mice. exposure may also be to small particle aerosols containing influenza with an mmad of < mm. most inbred strains are susceptible, with particularly frequent use of balb/c followed by c bl/ j mice. males and females have equivalent disease, but influenza is generally more infectious in younger -to -week-old ( - g) mice. mice are of somewhat limited use in characterizing the immune response to influenza. mice lack the mxa gene, which is an important part of the human innate immune response to influenza infection. the mouse homolog to mxa, mx , is defective in most inbred mouse strains. weight loss or reduced weight gain, decreased activity, huddling, ruffled fur, and increased respiration are the most common clinical signs. for more virulent strains, mice may require euthanasia as early as h postexposure, but most mortality occurs from to days postexposure accompanied by decreases in rectal temperature. pulse oximeter readings and measurement of blood gases of oxygen saturation are also used to determine the impact of influenza infection on respiratory function. virus can be isolated from bronchial lavage fluids throughout the infection and from tissues after euthanasia. for influenza strains with mild-tomoderate pathogenicity, disease is nonlethal and virus replication is detected within the lungs, but usually not other organs. increases in serum alpha- -acidglycoprotein and lung weight are also frequently present. however, mice infected with influenza do not develop fever, dyspnea, nasal exudates, sneezing, or coughing. mice can be experimentally infected with influenza a or b, but the virus generally requires adaptation to produce clinical signs. mice express the receptors for influenza attachment in the respiratory tract; however, the distribution varies and sa , predominates over sa , which is why h , h , and h subtypes usually need to be adapted to mice and h n , h , h , and h viruses do not require adaptation. to adapt, mice are infected intratracheally or intranasally by virus isolated from the lungs, and reinfected into mice and then the process is repeated a number of times. once adapted, influenza strains can produce severe disease, systemic spread, and neurotropism. however, h n and the pandemic influenza virus can cause lethal infection without adaptation. h n infection of mice results in viremia and viral replication in multiple organ systems, severe lung pathology, fulminant diffuse interstitial pneumonia, pulmonary edema, high levels of proinflammatory cytokines, and marked lymphopenia. as in humans, the virulence of h n is attributable to damage caused by an overactive host immune response. additionally, mice infected with the h n influenza produces severe lung pathology and oxygen saturation levels that decrease with increasing pneumonia. in superinfection models, a sublethal dose of influenza is given to mice followed days later by intranasal inoculation of a sublethal dose of a bacterial strain such as s. pneumoniae or s. pyrogenes. morbidity, characterized by inflammation in the lungs, but not bacteremia, begins a couple of days after superinfection and may continue for up to weeks. at least one transmission model has also been developed in mice. with h n influenza, transmission rates of up to % among cage mates can be achieved after infection by the aerosol route and cocaging after h. domestic ferrets (mustela putorius furo) are frequently the animal species of choice for influenza animal studies because the susceptibility, clinical signs, peak virus shedding, kinetics of transmission, local expression of cytokine mrnas, and pathology resemble that of humans. [ ] [ ] [ ] ferrets also have airway morphology, respiratory cell types, and a distribution of influenza receptors (sa , and sa , ) within the airways similar to that of humans. influenza was first isolated from ferrets infected intranasally with throat washes from humans harboring the infection and ferret models have since been used to test efficacy of vaccines and therapeutic treatments. when performing influenza studies in ferrets, animals should be serologically negative for circulating influenza viruses. infected animals should be placed in a separate room from uninfected animals. if animals must be placed in the same room, uninfected ferrets should be handled before infected ferrets. anesthetized ferrets are experimentally exposed to influenza by intranasal. inoculation of . - . ml containing approximately - egg id dropwise to each nostril. influenza types a and b naturally infect ferrets, resulting in an acute illness, which usually lasts - days for mildly to moderately virulent strains. ferrets are more susceptible to influenza a than to influenza b strains and are also susceptible to avian influenza h n strains without adaptation. virulence and degree of pneumonitis caused by different influenza subtypes and strains vary from mild to severe and generally mirror that seen in humans. nonadapted h n , h n , and h n have mild-to-moderate virulence in ferrets. strains of low virulence have predominant replication in the nasal turbinates. clinical signs and other disease indicators are similar to that of humans with a mild respiratory disease, sneezing, nasal secretions containing virus, fever, weight loss, high viral titers, and inflammatory infiltrate in the airways, bronchitis, and pneumonia. replication in both the upper and lower airways is associated with more severe disease and greater mortality. additionally, increased expression of proinflammatory mediators and reduced expression of antiinflammatory mediators in the lower respiratory tract ferrets correlates with severe disease and lethal outcome. h n -infected ferrets develop severe lethargy, greater ifn response, transient lymphopenia, and replication in respiratory tract, brain, and other organs. old and new world primates are susceptible to influenza infection and have an advantage over ferret and mouse models, which are deficient for h n vaccine studies because there is a lack of correlation with hemagglutination inhibition. of old world primates, cynomolgus macaque (m. fascicularis) are most frequently used for studies of vaccines and antiviral drug therapies. , h n and h n infections of cynos are very similar to those in humans. cynos develop fever and ards upon intranasal inoculation of h n with necrotizing bronchial interstitial pneumonia nhps are challenged by multiple routes (ocular, nasal, and tracheal) simultaneously  pfu per site. virus antigen is primarily localized to the tonsils and pulmonary tissues. infection of cynos with h n results in fever, lethargy, nasal discharge, anorexia, weight loss, nasal and tracheal washes, pathologic and histopathologic changes, and alveolar and bronchial inflammation. the h n caused a very high mortality rate due to an aberrant immune response and ards and had > % lethality (humans only had a - % lethality). ards and mortality also occur with the more pathogenic strains, but nhps show reduced susceptibility to less virulent strains such as h n . influenzainfected rhesus macaques represent a mild disease model pathogenesis for vaccine and therapeutic efficacy studies. other nhp models include influenza infection of pigtailed macaques as a mild disease model and infection of new world primates such as squirrel and cebus monkeys. rats (f and sd) inoculated with rat-adapted h n developed inflammatory infiltrates and cytokines in bronchoalveolar lavage fluids, but had no lethality and few histopathological changes. additionally, an influenza transmission model has been developed in guinea pigs as an alternative to ferrets. cotton rats (sigmodon hispidus) have been used to test vaccines and therapeutics in a limited number of studies. , cotton rats have an advantage over mice in that the immune system is similar to humans (including the presence of the mx gene) and influenza viruses do not have to be adapted. , nasal and pulmonary tissues of cotton rats were infected with unregulated cytokines and lung viral load peaking at h postexposure. virus was cleared from the lung by day and from the nares by day , but animals had bronchial and alveolar damage, and pneumonia for up to weeks. there is also a s. aureus superinfection model in cotton rats. coinfection resulted in bacteremia, high bacterial load in lungs, peribronchiolitis, pneumonitis, alveolitis, hypothermia, and higher mortality. domestic pig influenza models have been developed for vaccine studies for swine flu. pigs are susceptible in nature as natural or intermediate hosts but are not readily susceptible to h n . , although pigs infected with influenza may have fever, anorexia, and respiratory signs such as dyspnea and cough, mortality is rare. size and space requirements make this animal difficult to work with, although the development of minipig (ellegaard gottingen) models may provide an easier-to-use alternative. incubation period, animals exhibit signs of fever, hepatitis, and abortion, which is a hallmark diagnostic sign known among farmers. mosquito vectors, unpasteurized milk, aerosols of infected animal's body fluids, or direct contact with infected animals are the important routes of transmission to humans. , after - days of incubation period, rvfv causes a wide range of signs and symptoms in humans ranging from asymptomatic to severe disease with hepatitis, vision loss, encephalitis, and hemorrhagic fever. [ ] [ ] [ ] depending on the severity of the disease when the symptoms start, - % of the hospitalized patients might die in - days or - days after the disease onset. hepatic failure, renal failure or disseminated intravascular coagulation (dic), and encephalitis are demonstrated within patients during postmortem examination. mice are one of the most susceptible animal species to rvfv infection. subcutaneous or intraperitoneal routes of infection cause acute hepatitis and lethal encephalitis at a late stage of the disease in mice. , mice start to exhibit signs of decreased activity and ruffled fur by day - postexposure. immediately after these signs are observed, they become lethargic and generally die - days postexposure. ocular diseases or hemorrhagic form of the disease has not been observed in mice models so far. increased viremia and tissue tropism were reported in mice with increased liver enzymes and lymphopenia observed in sick mice. rats and gerbils are also susceptible to rvfv infection. rats' susceptibility is dependent on the rat strain used for the challenge model. there was also noted an age dependence in susceptibility of rats. although wistar-furth and brown norway strains and young rats are highly susceptible to rvfv infection, fisher , buffalo and lewis strains, and old rats demonstrated resistance to infection. , similar pathologic changes such as liver damage and encephalopathy were observed in both rats and mice. there was no liver involvement in the gerbil model and animals died from severe encephalitis. the mortality rate was dependent on the strain used and the dose given to gerbils. similar to the rat model, the susceptibility of gerbils was also dependent on age. so far, studies showed that rvfv does not cause uniform lethality in an nhp model. intraperitoneal, intranasal, iv, and aerosol routes have been used to develop the nhp model. rhesus macaques, cynomolgus macaques, african monkeys, and south american monkeys were some of the nhp species used for this effort. monkeys showed a variety of signs ranging from febrile disease to hemorrhagic disease and mortality. temporal viremia, increased coagulation parameters (pt, aptt), and decreased platelets were some other signs observed in nhps. animals that succumbed to disease showed very similar pathogenesis to those seen in humans such as pathological changes in liver and hemorrhagic disease. there was no ocular involvement in this model. recently, smith et al. compared iv, intranasal, and subcutaneous routes of infection in common marmosets and rhesus macaques. marmosets were more susceptible to rvfv infection than were rhesus macaques with marked viremia, acute hepatitis, and late onset of encephalitis. increased liver enzymes were observed in both species. necropsy results showed enlarged livers in the marmosets exposed by iv or subcutaneous routes. although there were no gross lesions in the brains of marmosets, histopathology showed encephalitis in the brains of intranasally challenged marmosets. crimean-congo hemorrhagic fever virus (cchfv) generally circulates in nature unnoticed in an enzootic tick-vertebrate-tick cycle and similar to other zoonotic agents, seems to produce little or no disease in its natural hosts, but causes severe disease in humans. cchfv transmits to humans by ixodid ticks, direct contact with sick animals/humans, or body fluids of animals/humans. incubation, prehemorrhagic, hemorrhagic, and convalescence are the four phases of the disease seen in humans. the incubation period lasts - days. during the prehemorrhagic phase, patients show signs of nonspecific flu-like disease for approximately a week. the hemorrhagic period results in circulatory shock and dic in some patients. , over the years, several attempts have been made to establish an animal model for cchf in adult mice, guinea pigs, hamsters, rats, rabbits, sheep, nhps, etc. [ ] [ ] [ ] [ ] until recently, the only animal that manifests disease is the newborn mouse. infant mice infected with cchfv intraperitoneally caused fatality around day postinfection. pathogenesis studies showed that virus replication was first detected in the liver, with subsequent spread to the blood (serum). virus was detected very late during the disease course in other tissues including the heart (day ) and the brain (day ). the recent studies using knockout adult mice were successful to develop a lethal small animal model for cchfv infection. , bente et al. infected stat knockout mice by the intraperitoneal route. in this model, after the signs of fever, leucopenia, thrombocytopenia, viremia, elevated liver enzymes, and proinflammatory cytokines, mice were moribund and succumbed to disease in - days of postexposure. the second model was developed by using ifn-alpha/beta (ifna/b) receptor knockout mice. similar observations were made in this model as in the stat knockout mouse model. the animals were moribund and died - days after exposure with high viremia levels in the liver and spleen. other laboratory animals, including nhps, show little or no signs of infection or disease when infected with cchfv. butenko et al. used agms (cercopithecus aethiops) for experimental cchfv infections. except one monkey with a fever on day postinfection, the animals did not exhibit signs of disease. antibodies to the virus were detected in three out of five monkeys, including the one with fever. in , fagbami et al. infected two patas monkeys (cercopithecus erythrocebuserythrocebus) and one guinea baboon (papio papio) with cchfv. although all three animals had low level viremia between days and after inoculation, only the baboon serum had neutralizing antibody activity on day postinfection. similar results were obtained when horses and donkeys have been used for experimental cchfv infections. donkeys develop a low-level viremia, and horses developed little or no viremia, but high levels of virus-neutralizing antibodies, which remained stable for at least months. these studies suggest that horses may be useful in the laboratory to obtain serum for diagnostic and possible therapeutic purposes. shepherd et al. infected species of small african wild mammals and laboratory rabbits, guinea pigs, and syrian hamsters with cchfv. although scrub hares (lepus saxatilis), cape ground squirrels (xerus inauris), red veld rats (aethomys chrysophilus), white-tailed rats (mystromys pumilio), and guinea pigs had viremia; south african hedgehogs (atelerix frontalis), highveld gerbils (tatera brantsii), namaqua gerbils (desmodillus auricularis), two species of multimammate mouse (mastomys natalensis and m. coucha), and syrian hamsters were negative. all species regardless of viremia levels developed antibody responses against cchfv. iv and intracranially infected animals showed the onset of viremia earlier than those infected by the subcutaneous or intraperitoneal routes. the genus hantavirus is unique among the family bunyaviridae in that it is not transmitted by an arthropod vector, but rather by rodents. rodents of the family muridae are the primary reservoir for hantaviruses. infected host animals develop a persistent infection that is typically asymptomatic. transmission is achieved by the inhalation of infected rodent saliva, feces, and urine. human infections can normally be traced to a rural setting with activities such as farming, land development, hunting, and camping as possible sites of transmission. rodent control is the primary route of prevention. the viruses have a tropism for endothelial cells within the microvasculature of the lungs. there are two distinct clinical diseases that infection can yield; hemorrhagic fever with renal syndrome (hfrs) due to infection with old world hantaviruses or hantavirus pulmonary syndrome (hps) caused by new world hantaviruses. hfrs is mainly seen outside of the americas and is associated with the hantaviruses dobrava-belgrade (also known as dobrava), hantaan, puumala, and seoul. incubation lasts two to three weeks and presents as flulike in the initial stages that can further develop into hemorrhagic manifestations and ultimately renal failure. thrombocytopenia subsequently develops, which can further progress to shock in approximately % patients. the overall mortality rate is %. infection with dobrava and hantaan viruses are typically linked to the development of severe disease. hps was first diagnosed in within the southwestern united states when healthy young adults became suddenly ill, progressing to severe respiratory distress and shock. the etiological agent responsible for this outbreak was identified as sin nombre virus. this virus is still the leading cause within na of hps. hps due to other hantaviruses has been reported in argentina, bolivia, brazil, canada, chile, french guiana, panama, paraguay, and uruguay. , the first report of hps in maine was recently documented. andes virus was first identified in outbreaks in chile and argentina. this hantavirus is distinct in that it can be transmitted between humans. the fulminant disease is more lethal than that observed for hfrs with a mortality rate of %. there are four phases of disease including prodromal, pulmonary, cardiac depression, and hematologic manifestation. incubation typically occurs - days after exposure. unlike hfrs, renal failure is not a major contributing factor to the disease. there is a short prodromal phase that gives way to cardiopulmonary involvement accompanied by cough and gastrointestinal symptoms. it is at this point that individuals are typically admitted to the hospital. pulmonary function is hindered and continues to suffer within h after cardiopulmonary involvement. interstitial edema and air-space disease normally follow. in fatal cases, cardiogenic shock has been noted. vaccine development has been hampered by the vast diversity of hantaviruses and the limited number of outbreaks. syrian golden hamsters are the most widely used small animal models for hantavirus infection. hamsters inoculated intramuscularly with a passaged andes viral strain died within days postinfection. clinical signs did not appear until h before death at which point the hamsters were moribund and in respiratory distress. mortality was dose dependent, with high inoculums leading to a shorter incubation before death. during the same study, hamsters were inoculated with a passaged sin nombre isolate. no hamsters developed any symptoms during the course of observation. although an antibody response to the virus that was not dose dependent was determined via an enzymelinked immunosorbent assay. hamsters infected with andes virus were found to have significant histopathological changes to their lung, liver, and spleen. all had an interstitial pneumonia with intraalveolar edema. infectious virus could be recovered from these organs. viremia began on day and lasted up to days postinfection. infection of hamsters with andes virus yielded a similar clinical disease progression as is seen in human hps including rapid progression to death, fluid in the pleural cavity, and significant histopathological changes to the lungs and spleen. a major deviation in the hamster model is the detection of infectious virus within the liver. lethal disease can be induced in newborn mice but does not recapitulate the clinical symptoms observed in human disease. adult mice exposed to hantaan virus leads to a fatal disease dependent upon viral strain and route of infection. the disease progression is marked by neurological or pulmonary manifestations that do not mirror human disease. , knockout mice lacing ifn-a/b were found to be highly susceptible to hantaan virus infection. in a study looking at a panel of laboratory strains of mice, c bl/ mice were found to be most susceptible to a passaged hantaan viral strain injected intraperitoneally. animals progressed to neurological manifestation including paralyses and convulsions and succumbed to infection within - h postinfection. clinical disease was markedly different than that observed in human cases. nhps have been challenged with new world hantaviruses; however, no clinical signs were reported. , cynomolgus monkeys challenged with a clinical isolate of puumala virus developed a mild disease. , challenge with andes virus to cynomolgus macaques by both iv and aerosol exposure led to no signs of disease. all animals did display a drop in total lymphocytes within days postinfection. aerosol exposure led to of monkeys and of iv injected monkeys developed viremia. infectious virus could not be isolated from any of the animals. the family arenaviridae is composed of two serogroups: old world arenaviruses including lassa fever virus and lymphocytic choriomeningitis virus and the new world viruses of pichinde virus and junin virus. all these viruses share common clinical manifestations. lassa fever virus is endemic in parts of west africa and outbreaks are typically seen in the dry season between january and april. this virus is responsible for , - , infections per year, leading to approximately , deaths. outbreaks have been reported in guinea, sierra leone, liberia, nigeria, and central african republic. however, cases sprung up in germany, the netherlands, the united kingdom, and the united states due to transmission to travelers on commercial airlines. transmission of this virus typically occurs via rodents, in particular the multimammate rat, mastomys species complex. humans become infected by inhaling the aerosolized virus or eating contaminated food. there has also been noted human-to-human transmission by direct contact with infected secretions or needle-stick injuries. the majority of infections are asymptomatic; however, severe disease can occur in % of individuals. the incubation period is from to days, and the initial onset is characterized by flulike illness. this is followed by diarrheal disease that can progress to hemorrhagic symptoms including encephalopathy, encephalitis, and meningitis. a third of patients develop deafness in the early phase of disease, which is permanent for a third of those affected. the overall fatality is about %; however, of those admitted to the hospital, it is between % and %. there is no approved vaccine, and besides supportive measures, ribavirin is effective only if started within days. , the primary animal model used to study lassa fever is the rhesus macaque. aerosolized infection of lymphocytic choriomeningitis virus has been a useful model for lassa fever. both rhesus and cynomolgus monkeys exposed to the virus developed disease, but rhesus more closely mirrored the disease course and histopathology observed in human infection. iv or intragastric inoculation of the virus led to severe dehydration, erythematous skin, submucosal edema, necrotic foci in the buccal cavity, and respiratory distress. the liver was severely affected by the virus as depicted by measuring the liver enzymes ast and alt. disease was dose-dependent with iv, intramuscular, and subcutaneous inoculation requiring the least amount of virus to induce disease. aerosol infections and eating contaminated food could also be used, and mimic a more natural route of infection. within this model, the nhp becomes viremic after - days. clinical manifestations were present by day , and death typically occurred within - days. , intramuscular injection of lassa virus into cynomolgus monkeys also produced a neurological disease due to lesions within the cns. this pathogenicity is seen in select cases of human lassa fever. , a marmoset model has recently been defined using a subcutaneous injection of lassa fever virus. virus was initially detected by day and viremia achieved by day . liver enzymes were elevated, and an enlarged liver was noted upon autopsy. there was a gradual reduction in platelets and interstitial pneumonitis diagnosed in a minority of animals. the physiological signs were the same as seen in fatal human cases. mice develop a fatal neurological disorder upon intracerebral inoculation with lassa, although the outcome of infection is completely dependent upon the major histocompatibility complex (mhc) background and age of animal along with the route of inoculation. guinea pig inbred strain was found to be highly susceptible to lassa virus infection. the outbreed hartley strain was less susceptible, and thus, strain has been the preferred model given its assured lethality. the clinical manifestations mirror those seen in humans and rhesus. infection with pichinde virus that has been passaged in guinea pigs has also been used. disease signs include fever, weight loss, vascular collapse, and eventual death. , the guinea pig is an excellent model given that it not only results in similar disease pattern as humans but also the viral distribution is similar along with the histopathology and immune response. , infection of hamsters with a cotton rat isolate of pirital virus is similar to what is characterized in humans, and the nhp and guinea pig model. the virus was injected intraperitoneally resulting in the animals becoming lethargic and anorexic within - days. virus was first detected at days and reached maximum titers within days. neurological symptoms began to appear at the same time, and all the animals died by day . pneumonitis, pulmonary hemorrhage, and edema were also present. these results were recapitulated with a nonadapted pichinde virus. [ ] [ ] [ ] globally, diarrheal disease is the leading cause of death with rotavirus being one of the main etiological agents responsible. according to the world health organization, rotavirus alone is responsible for a third of all hospitalization related to diarrhea and , - , deaths per year. the virus is very stable due to its three-layer capsid, which allows it to be transmitted via the oral-fecal route, depositing itself in the small intestine. rotavirus is highly contagious, and only viruses are needed to cause symptomatic disease. the host determinant with the greatest influence on clinical outcome is age. neonates typically are asymptomatic, which is suggested to be due to the existence of maternal antibodies. hence, the most susceptible age group is months to years, coinciding with a drop in these protective antibodies. within this age range, children will develop noninflammatory diarrhea. virus replicates in the intestinal villus enterocytes resulting in their destruction and malabsorption of needed electrolytes and nutrients. symptoms of disease include watery, nonbloody diarrhea with vomiting, fever, and potentially dehydration that lasts up to a week. there is a short episode of viremia during the course of infection. mice can be used as both an infection and disease model depending upon age at challenge. mice < days old develop disease, whereas older mice are able to clear the infection before the onset of symptoms. this halts the study of active vaccination against disease in the infection model. in the adult mouse model, the course of the infection is monitored via viral shedding within the stool. infant mice, specifically balb/c, receiving an oral inoculation of a clinical strain of virus developed diarrhea within h postinfection, and % of those exposed developed symptoms within h postinfection. symptoms lasted from to days with no mortality. viral shedding was at its peak at h and lasted up to days. there were noted histopathological changes within the small intestine localized to the villi that was reversible. within the adult mouse model, oral inoculation of a mouse rotavirus strain showed viral shedding by days lasting up until days postinfection. these mouse models have been used to study correlates of protection and therapeutic efficacy including gastro-gard Ò . , , rats can also be used as disease models depending upon the strain of rat. , suckling fischer rats were exposed to a simian strain of rotavirus orally. the rats were susceptible to diarrheal disease till they were days old with age determining the length of viral shedding. rats have mainly been used to study the correct formulation for oral rehydration. these rodents are large enough to perform in situ intestinal perfusions. within these studies, -day-old rats were infected with a rat strain of rotavirus by orogastric intubation. within h postinfection, the rats developed diarrhea, at which point the small intestine was perfused to compare differing solutions of oral rehydration. gnotobiotic pigs are also used given that they can be infected with both porcine and human strains. they are susceptible to developing clinical disease from human strains up to weeks of age. they allow for the analysis of the primary immune response to the virus given that they do not receive transplacental maternal antibodies and are immune competent at birth. another advantage of this model is that the gastrointestinal physiology and mucosal immune system closely resemble that of humans. this model has been useful in studying correlates of protection. gnotobiotic and colostrum-deprived calves have also been used as an experimental model of rotavirus infection. they are able to develop diarrhea and shed live virus. gnotobiotic lambs can also develop clinical disease upon oral inoculation with clinical strains. infant baboons, agms, and rhesus macaques have all proven to be infection models with severity measure by viral shedding. , retroviridae human immunodeficiency virus type the lentiviruses are a subfamily of retroviridae, which includes human immunodeficiency virus (hiv), a virus that infects . % of the world's population. a greater proportion of infections and deaths occur in sub-saharan africa. worldwide, there are approximately . million deaths per year with > , being children. transmission of hiv occurs by exposure to infectious body fluids. there are two species, hiv- and hiv- , with hiv- having lower infectivity and virulence (confined mostly to west africa). the vast majority of cases worldwide are hiv- . hiv targets t-helper cells (cd þ), macrophages, and dendritic cells. acute infection occurs - weeks after exposure, with flu-like symptoms and viremia followed by chronic infection. symptoms in the acute phase may include fever, body aches, nausea, vomiting, headache, lymphadenopathy, pharyngitis, rash, and sores in the mouth or esophagus. cd þ t-cells are activated which kill hiv-infected cells, and are responsible for antibody production and seroconversion. acquired immune deficiency syndrome (aids) develops when cd þ t-cells decline to < cells per microliter; thus, cell-mediated immunity becomes impaired, and the person is more susceptible to opportunistic infections and certain cancers. humanized mice, created by engrafting human cells and tissues into scid mice, have been critical for the development of mouse models for the study of hiv infection. a number of different humanized mouse models allow for the study of hiv infection in the context of intact and functional human innate and adaptive immune responses. the scidhu hiv infection model has proven to be useful, particularly in screening antivirals and therapeutics. a number of different humanized mouse models have been developed for the study of hiv, including rag À/ÀgcÀ/À, rag À/ ÀgcÀ/À, nod/scidgcÀ/À (hnog), nod/ scidgcÀ/À (hnsg), nod/scid blt, and nod/ scidgcÀ/À (hnsg) blt. cd þ human stem cells derived from the umbilical cord blood or fetal liver are used for humanization. hiv- infection by intraperitoneal injection can be successful with as little as % peripheral blood engraftment. vaginal and rectal transmission models have been developed in blt scidhu mice in which mice harbor human bone marrow, liver, and thymus tissue. hiv- viremia occurs within approximately days pi. in many of these models, spleen, lymph nodes, and thymus tissues are highly positive for virus, similar to humans. importantly, depletion of human t-cells can be observed in blood and lymphoid tissues of hiv-infected humanized mice, and at least some mechanisms of pathogenesis that occur in hiv-infected humans also occur in the hiv-infected humanized mouse models. the advantage of these models is that these mice are susceptible to hiv infection, and thus, the impact of drugs on the intended viral targets can be tested. one caveat is that although mice have a "common mucosal immune system," humans do not, due to the differences in the distribution of addressins. thus, murine mucosal immune responses to hiv do not reflect those of humans. there are a number of important nhp models for human hiv infection. simian immunodeficiency virus (siv) infection of macaques is widely considered to be the best platform for modeling hiv infection of humans. importantly, nhps have similar, pharmacokinetics, metabolism, mucosal t-cell homing receptors, and vascular addressins to those of humans. thus, although the correlates of protection against hiv are still not completely known, immune responses to hiv infection and vaccination are likely comparable. these models mimic infection through the use of contaminated needles (iv), sexual transmission (vaginal or rectal), and maternal transmission in utero, or through breast milk. [ ] [ ] [ ] there are also macaque models to study the emergence and clinical implications of hiv drug resistance. these models most routinely use rhesus macaques (macaca mulatta), cynomolgus macaques (macaca fasicularis), and pigtailed macaque (macaca nemestrina). animals of all ages are used, depending on the needs of the study. for instance, the use of newborn macaques may be more practical for evaluating the effect of prolonged drug therapy on disease progression; however, adult nhps are more frequently used. studies are performed in bsl- animal laboratories, and nhps must be of simian type-d retrovirus free and siv seronegative. siv infection of pigtailed macaques is a useful model for hiv peripheral nervous system pathology, wherein an axotomy is performed and regeneration of axons is studied. challenges may be through a single high dose. iv infection of rhesus macaques with tcid of the highly pathogenic siv/deltab induces aids in most macaques within - months (mean of months). peak viremia occurs around week . aids in such models is often defined as cd þ t-cells that have dropped to < % of the baseline values. alternatively, repeated low-dose challenges are often used, depending on the requirements of the model. , because nhps infected with hiv do not develop an infection with a clinical disease course similar to that in humans, siv or siv/hiv- laboratory-engineered chimeric viruses (simian-human immunodeficiency virus or shiv) are used as surrogates. nhps infected with pathogenic siv may develop clinical disease, which progresses to aids and are thus useful pathogenesis models. a disadvantage is that siv is not identical to hiv- and is more closely related to hiv- . however, the polymerase region of siv is % homologous to that of hiv- , and it is susceptible to many reverse transcriptase (rt) and protease inhibitors. siv is generally not susceptible to nonnucleoside inhibitors; thus, hiv- rt is usually put into siv for such studies. sivmac is similar to hiv in the polymerase region and is therefore susceptible to nucleoside, rt or integrase inhibition. nhps infected with sivmac have an asymptomatic period and disease progression resembling aids in humans, characterized by weight loss/wasting, cd þ t-cell depletion. additionally, sivmac uses the cxcr chemokine receptor as a coreceptor, similar to hiv, which is important for drugs that target entry. nhps infected with shiv strains may not develop aids, but these models are useful in testing vaccine efficacy. for example, rt-shivs and env-shivs are useful for the testing and evaluation of drugs that may target the envelope or rt, respectively. one disadvantage of the highly virulent env-shiv (shiv- . p) is that it uses the cxcr coreceptor. of note, env-shivs that do use the cxcr coreceptor are less virulent; viremia develops and then resolves without further disease progression. simian-tropic (st) hiv- contains the vif gene from siv. infection of pigtailed macaques with this virus results in viremia, which can be detected for three months, followed by clearance. a number of routes are used for siv or shiv infection of nhps, with iv inoculation being the most common route. mucosal routes include vaginal, rectal, and intracolonic. mucosal routes require a higher one-time dose than does the iv route for infection. for the vaginal route, female macaques are treated with depo-provera (estrogen) one month before infection to synchronize the menstrual cycle, thin the epithelial lining of the vagina, and increase the susceptibility to infection by atraumatic vaginal instillation. upon vaginal instillation of tcid of shiv- p , peak viremia was seen around days postexposure with > copies per milliliter and dropping thereafter to a constant level of rna copies per milliliter at days and beyond. in another example, in an investigation of the effect of vaccine plus vaginal microbicide on preventing infection, rhesus macaques were vaginally infected with a high dose of sivmac . an example of an intrarectal model used juvenile ( - year-old) pigtailed macaques, challenged intrarectally with tcid s of siv mne to study the pathogenesis related to the virulence factor, vpx. here, viremia peaked at approximately days with > copies per milliliter. viral rna was expressed in the cells of the mesenteric lymph nodes. the male genital tract is seen as a viral sanctuary with persistently high levels of hiv shedding even with antiretroviral therapy. to better understand the effect of highly active antiretroviral therapy on virus and t-cells in the male genital tract, adult ( -to -year old) male cynomolgus macaques were intravenously inoculated with aid s of sivmac , and the male genital tract tissues were tested after euthanasia by pcr, ihc, and in situ hybridization. pediatric models have been developed in infant rhesus macaques through the infection of siv, allowing for the study of the impact of developmental and immunological differences on the disease course. importantly, mother-to-infant transmission models have also been developed. pregnant female pigtailed macaques were infected during the second trimester with mid shiv-sf p by the iv route. four of nine infants were infected; one in utero and three either intrapartum or immediately postpartum through nursing. this model is useful for the study of factors involved in transmission and the underlying immunology. nhps infected with siv or shiv are routinely evaluated for weight loss, activity level, stool consistency, appetite, virus levels in blood, and t-cell populations. cytokine and chemokine levels, antibody responses, and cytotoxic t-lymphocyte responses may also be evaluated. the ultimate goal of an hiv vaccine is sterilizing immunity (preventing infection). however, a more realistic result may be to reduce severity of infection and permanently prevent progression. strategies have included live attenuated, nonreplicating and subunit vaccines. these have variable efficacy in nhps due to the genetics of the host (mhc and terminal-repeat retrotransposon in miniature (trim) alleles), differences between challenge strains, and challenge routes. nhp models have led to the development of antiviral treatments that are effective at reducing viral load and indeed transmission of hiv among humans. one preferred variation on the models for testing the longterm clinical consequences of antiviral treatment is to use newborn macaques and treat from birth onward, in some cases more than a decade. unfortunately, however, successes in nhp studies do not always translate to success in humans, as seen with the recent step study that used an adenovirus-based vaccine approach. vaccinated humans were not protected and may have even been more susceptible to hiv, viremia was not reduced, and the infections were not attenuated as hoped. with regard to challenge route, iv is more difficult to protect than mucosal and is used as a "worst-case scenario." however, efficacy at one mucosal route is usually comparable to that at other mucosal routes. human and animal papillomaviruses cause benign epithelial proliferations (warts) and malignant tumors of the various tissues that they infect. there are > human papillomaviruses (hpvs), with different strains causing warts on the skin, oropharynx, nasopharynx, larynx, and anogenital tissues. approximately a third of these are transmitted sexually. of these, virulent subtypes such as hpv- , hpv- , hpv- , hpv- , and hpv- place individuals at high risk for cervical and other cancers. major challenges in the study of these viruses are that papillomaviruses generally do not infect any other species outside of the natural hosts and can cause a very large spectrum of severity. thus, no animal models have been identified that are susceptible to hpv. however, a number of useful surrogate models exist that use animal papillomaviruses in their natural host, or a very closely related species. , these models have facilitated the recent development of useful and highly effective prophylactic hpv vaccines. wild cottontail rabbits (sylvilagus floridanus) are the natural host for cottontail rabbit papillomavirus (crpv), but this virus also infects domestic rabbits (oryctolagus cuniculus), which are a very closely related species. in this model, papillomas can range from cutaneous squamous cell carcinomas on the one end of the spectrum, and spontaneous regression on the other. lesions resulting from crpv in domestic rabbits do not typically contain infectious virus. canine oral papillomavirus (copv) cause florid warty lesions in the mucosa of the oral cavity within - weeks postexposure in experimental settings. the mucosatrophic nature of these viruses and the resulting oropharyngeal papillomas that are morphologically similar to human vaginal papillomas caused by hpv- and hpv- make this a useful model. these lesions typically spontaneously regress - weeks after appearing; this model is therefore useful in understanding the interplay between the host immune defense and viral pathogenesis. male and female beagles, aged weeks to years, with no history of copv, are typically used for these studies. infection is achieved by the application of a ml droplet of virus extract to multiple . cm scarified areas within the mucosa of the upper lip of anesthetized beagles. bovine papillomavirus (bpv) has a wider host range than do most papillomaviruses, infecting the fibroblasts cells of numerous ungulates. bpv- infection of cattle feeding on bracken fern, which is carcinogenic, can result in lesions of the oral and esophageal mucosa that lack detectable viral dna. bpv infections in cattle can result in a range of diseases such as skin warts, cancer of the upper gastrointestinal tract and urinary bladder, and papillomatosis of the penis, teats, and udder. finally, sexually transmitted papillomaviruses in rhesus macaques and cynomolgus macaques, rhesus papillomavirus, is very similar to hpv- and is associated with the development of cervical cancer. mice cannot be used to study disease caused by papillomaviruses unless they are engrafted with relevant tissue, but they are often used to look at immunogenicity of vaccines. , herpesviridae please see chapter . monkeypox virus (mpxv) causes disease in both animals and humans. human monkeypox, which is clinically almost identical to ordinary smallpox, occurs mostly in the rainforest of central and western africa. the virus is maintained in nature in rodent reservoirs including squirrels. , mpxv was discovered during the pox-like disease outbreak among laboratory java macaques in denmark in . no human cases were observed during this outbreak. the first human case was not recognized as a distinct disease until in zaire (the present drc) with the continued occurrence of a smallpox-like illness despite eradication efforts of smallpox in this area. during the global eradication campaign, extensive vaccination in central africa decreased the incidence of human monkeypox, but the absence of immunity in the generation born since that time and increased dependence on bush meat have resulted in renewed emergence of the disease. in the summer of , a well-known outbreak in the midwest was the first occurrence of monkeypox disease in the united states and the western hemisphere. among reported cases, human cases were laboratory confirmed during an outbreak. , it was determined that native prairie dogs (cynomys sp.) housed with rodents imported from ghana in west africa were the primary source of outbreak. the virus is mainly transmitted to humans while handling infected animals or by direct contact with the infected animal's body fluids, or lesions. person-toperson spread occurs by large respiratory droplets or direct contact. most of the clinical features of human monkeypox are very similar to those of ordinary smallpox. after a -to -day incubation period, the disease begins with fever, malaise, headache, sore throat, and cough. the main sign of the disease that distinguishes monkeypox from smallpox is swollen lymph nodes (lymphadenitis), which is observed in most of the patients before the development of rash. , typical maculopapular rash follows the prodromal period generally lasting - days. the average size of the skin lesions is . - cm, and the progress of lesions follows the order macules through papules, vesicles, pustules, umblication then scab and desquamation and lasts typically - weeks. fatality rate is % among the unvaccinated population and death generally occurs during the second week of the disease. , mpxv is highly pathogenic for a variety of laboratory animals, and so far, many animal models have been developed by using different species and different routes of exposure (table . ). because of the unavailability of variola virus to develop animal models and resulting disease manifestations in humans that are similar, mpxv is one of the pox viruses that are used very heavily to develop a number of small animal models via different routes of exposure. wild-derived inbred mouse, stat -deficient c bl/ mouse, prairie dogs, african dormice, ground squirrels are highly susceptible to mpxv by different exposure routes. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] cast/eij mice, one of the inbred mouse strains tested for susceptibility to mpxv, showed weight loss and dose-dependent mortality after intranasal exposure to mpxv. studies with the intraperitoneal route of challenge indicated a higher susceptibility to mpxv with an almost -fold less ld when compared to the intranasal route. scid-balb/c mice were also susceptible to the intraperitoneal challenge route, and the disease resulted in mortality day postinfection. similarly c bl/ stat À/À mice were infected intranasally with mpxv, and the infection resulted in weight loss and mortality days postexposure. mice models mentioned here are very promising for screening of therapeutics against pox viruses, but testing in additional models will be required for advanced development. high doses of mpxv by intraperitoneal or intranasal route caused % mortality in days postexposure and days postexposure, respectively, in ground squirrels. the disease progressed very quickly, and most of the animals were lethargic and moribund by day postexposure without any pox lesions or respiratory changes. a comparison study of usa mpxv and central african strain of mpxv in ground squirrels by subcutaneous route resulted in systemic disease and the mortality in - days postexposure. the disease resembles hemorrhagic smallpox with nose bleeds, impaired coagulation parameters, and hemorrhage in the lungs of the animals. because in the us outbreak the virus was transmitted by infected prairie dogs, this animal model has recently been studied much further and used to test therapeutics and vaccines compared to other small animal models. , , , studies using intranasal, intraperitoneal, and intradermal routes of exposure showed that mpxv was highly infectious to prairie dogs. by using the west african mpxv strain, the intraperitoneal route caused a more severe disease and % mortality than challenge by the intranasal route. anorexia and lethargy were common signs of the disease for both exposure routes. in contrast to the intraperitoneal route, the intranasal route of exposure caused severe pulmonary edema and necrosis of lungs in prairie dogs, while splenic necrosis and hepatic lesions were observed in intraperitoneally infected animals. recent studies by hutson et al. used intranasal and intradermal infections with west african and congo basin strains and showed that both strains and routes caused smallpox-like disease with longer incubation periods and generalized pox lesions. therefore, this model can be used for testing therapeutics and vaccines against pox viruses. the african dormouse is susceptible to mpxv by the foodpad injection route or intranasal route. mice exhibited decreased activity, hunched posture, dehydration, conjunctivitis, and weight loss. viral doses of and pfu provided % mortality with a mean time to death of days. upper gastrointestinal hemorrhage, hepatomegaly, lymphadenopathy, and hemorrhage in lungs were observed during necropsy. with the hemorrhage in several organs, this model resembles hemorrhagic smallpox. considering the limited availability of ground squirrels and african dormice, lack of reagents to these species, and resemblance to hemorrhagic smallpox disease, these models are not very attractive for further characterization and vaccine and countermeasure testing studies. nhps were exposed to mpxv by several different routes to develop animal models for mpxv. , , , , during our studies by using an aerosol route of exposure, we observed that macaques had mild anorexia, depression, fever, and lymphadenopathy on day postexposure. complete blood count and clinical chemistries showed abnormalities similar to those of human monkeypox cases with leukocytosis and thrombocytopenia. whole-blood and throat swabs had viral loads peak around day , and in survivors, gradually decrease until day postexposure. because doses of  ,  , or  pfu resulted in lethality for % of the animals, whereas a dose of  pfu resulted in % lethality, survival was not dose dependent. the main pitfall of this model was the lack of pox lesions. with the high dose, before animals can develop pox lesions, they succumbed to disease. with the low challenge dose, pox lesions were observed, but they were few in comparison to the iv model. mpxv causes dose-dependent disease in nhps when given by the iv route. studies showed that with  pfu iv, challenge results in systemic disease with fever, lymphadenopathy, macula-papular rash, and mortality. an intratracheal infection model deposits virus into the trachea, delivering directly to the airways without regard to particle size and the physiological deposition that occurs during the process of inhalation by skipping the upper respiratory system. fibrinonecrotic bronchopneumonia was described in animals that received pfu of mpxv intratracheally. although a similar challenge dose of intratracheal mpxv infection resulted in a similar viremia in nhps than with the aerosol route of infection, the timing of the first peak was delayed by days in intratracheally exposed macaques compared to aerosol infection, and the amount of virus detected by qpcr was approximately -fold lower. this suggests that local replication is more prominent after aerosol delivery compared to that after intratracheal delivery. an intrabronchial route of exposure resulted in pneumonia in nhps. delayed onset of clinical signs and viremia were observed during the disease progression. in this model, similar to aerosol and the intratracheal route of infection models, the number of pox lesions was much less than in the iv route of the infection model. a major downside of the iv, intratracheal and intrabronchial models is that the initial infection of respiratory tissue, incubation, and prodromal phases are circumvented with the direct inoculation of virus into the blood stream or into the lung. this is an important limitation when the utility of these models is to test possible vaccines and treatments in which the efficacy may depend on protecting the respiratory mucosa and targeting subsequent early stages of the infection, which are not represented in these challenge models. although the aerosol model is the natural route of transmission for human variola virus (varv) infections and a secondary route for human mpxv infections, the lack of pox lesions is the main drawback of this model. therefore, when this model is decided to be used to test medical countermeasures, the endpoints and the biomarkers to initiate treatment should be chosen carefully. hepatitis b is one of the most common infections worldwide with > million people chronically infected and , cases per year of liver cancer due to infection. the virus can naturally infect both humans and chimpanzees. hepatitis b is transmitted parenterally or postnatally from infected mothers. it can also be transmitted by sexual contact, iv drug use, blood transfusion, and acupuncture. the age at which one is infected dictates the risk of developing chronic disease. acute infection during adulthood is self-limiting and results in flu-like symptoms that can progress to hepatocellular involvement as observed with the development of jaundice. the clinical symptoms last for a few weeks before resolving. after this acute phase, life time immunity is achieved. of those infected, < % will develop the chronic form of disease. chronicity is the most serious outcome of disease as it can result in cirrhosis or liver cancer. hepatocellular carcinoma is times more likely to develop in a chronically infected individual than in a noncarrier. the viral determinant for cellular transformation has yet to be determined, although studies involving the woodchuck hepadna virus suggest that x protein may be responsible. many individuals are asymptomatic until complications emerge related to chronic carriage. chimpanzees have a unique strain that circulates within the population. , it was found that - % of all wild-caught animals from africa are positive for hepatitis b antigen. natural and experimental challenge with the virus follows the same course as human disease; however, this is only an acute model of disease. to date, the use of chimpanzees provides the only reliable method to ensure that plasma vaccines are free from infectious particles. this animal model has been used to study new therapeutics and vaccines. chimpanzees are especially attuned to these studies given that their immune response to infection directly mirrors humans. other nhps that have been evaluated are gibbons, orangutans, and rhesus monkeys. although these animals can be infected with hepatitis b, none develop hepatic lesions or liver damage as noted by monitoring of liver enzymes. mice are not permissible to infection, and thus, numerous transgenic and humanized lines that express hepatitis b proteins have been created to facilitate their usage as an animal model. these include both immunocompetent and immunosuppressed hosts. the caveat to all of these mouse lines is that they reproduce only the acute form of disease. recently, the entire genome of hepatitis b was transferred to an immunocompetent mouse line via adenovirus. this provides a model for persistent infection. hepatitis b can also be studied using surrogate viruses, naturally occurring mammalian hepadna viruses. the woodchuck hepatitis virus was found to induce hepatocellular carcinoma. within a population, - % of all neonatal woodchucks are susceptible to chronic infection. a major difference between the two hepatitis isolates is the rate at which they induce cancer; almost all chronic carriers developed hepatocellular carcinoma within years of the initial infection in woodchucks, whereas human infection takes much longer. the acute infection strongly resembles what occurs during the course of disease in humans. there is a self-limiting acute phase resulting in a transient viremia that has the potential of chronic carriage. challenge with virus in neonates leads to a chronic infection, while adults only develop the acute phase of disease. a closely related species to the woodchuck is the marmota himalayan. this animal is also susceptible to the woodchuck hepadna virus upon iv injection. it was found to develop an acute hepatitis with a productive infection. hepatitis d is dependent upon hepatitis b to undergo replication and successful infection in its human host. there are two modes of infection possible between the viruses: coinfection in which a person is simultaneously infected or superinfection in which a chronic carrier of hepatitis b is subsequently infected with hepatitis d. coinfection leads to a similar disease as seen with hepatitis b alone; however, superinfection can result in chronic hepatitis d infection and severe liver damage. both coinfection and superinfection can be demonstrated within the chimpanzee and woodchuck by inoculation of human hepatitis d. a recently published report demonstrated the use of a humanized chimeric mouse to study the interactions between the two viruses and drug testing. the ideal animal model for human viral disease should closely recapitulate the spectrum of clinical symptoms and pathogenesis observed during the course of human infection. whenever feasible, the model should use the same virus and strain that infects humans. it is also preferable that the virus be a low passage clinical isolate; thus, animal passage or adaptation should be avoided if model species can be identified that are susceptible. ideally, the experimental route of infection would mirror that which occurs in natural disease. to understand the interplay and contribution of the immune system during infection, an immunocompetent animal should be used. the above characteristics cannot always be satisfied, however, and often virus must be adapted, knockout mice must be used, and/or the disease is not perfectly mimicked in the animal model. well-characterized animal models are critical for licensure to satisfy the food and fda's "animal rule." this rule applies to situations in which vaccines and therapeutics cannot safely or ethically be tested in humans; thus, licensure will come only after preclinical tests are performed in animal models. many fields in virology are moving toward standardized models that can be used across institutions to test vaccines and therapeutics. a current example of such an effort is within the filovirus community, where animal models, euthanasia criteria, assays, and virus strains are in the process of being standardized. the hope is that these efforts will enable results of efficacy tests on medical countermeasures to be compared across institutions. this chapter has summarized the best models available for each of the viruses described. fields' virology pathogenesis of poliomyelitis: reappraisal in the light of new data one hundred years of poliovirus pathogenesis expression of the poliovirus receptor in intestinal epithelial cells is not sufficient to permit poliovirus replication in the mouse gut present status of attenuated live-virus poliomyelitis vaccine world health organization. polio global eradication initiative annual report pathogenesis of human poliovirus infection in mice. ii. age-dependency of paralysis the poliovirus receptor protein is produced both as membranebound and secreted forms poliovirus pathogenesis in a new poliovirus receptor transgenic mouse model: agedependent paralysis and a mucosal route of infection establishment of a poliovirus oral infection system in human poliovirus receptor-expressing transgenic mice that are deficient in alpha/beta interferon receptor macaque models of human infectious disease ulnar nerve inoculation of poliovirus in bonnet monkey: a new primate model to investigate neurovirulence experimental poliomyelitis in bonnet monkey. clinical features, virology and pathology hepatitis a in day-care centers. a community-wide assessment persistence of hepatitis a virus in fulminant hepatitis and after liver transplantation acute liver failure: redefining the syndromes experimental hepatitis a virus (hav) infection in cynomolgus monkeys (macaca fascicularis): evidence of active extrahepatic site of hav replication experimental hepatitis a virus (hav) infection in callithrix jacchus: early detection of hav antigen and viral fate animal models of hepatitis a and e relative infectivity of hepatitis a virus by the oral and intravenous routes in species of nonhuman primates wild malaysian cynomolgus monkeys are exposed to hepatitis a virus histopathological and immunohistochemical studies of hepatitis a virus infection in marmoset callithrix jacchus intragastric infection induced in marmosets (callithrix jacchus) by a brazilian hepatitis a virus (haf- ) experimental hepatitis a virus infection in guinea pigs systematic literature review of role of noroviruses in sporadic gastroenteritis norovirus infection as a cause of diarrhea-associated benign infantile seizures outbreak of necrotizing enterocolitis caused by norovirus in a neonatal intensive care unit progress in understanding norovirus epidemiology natural history of human calicivirus infection: a prospective cohort study foodborne viruses: an emerging problem pediatric norovirus diarrhea in nicaragua clinical immunity in acute gastroenteritis caused by norwalk agent experimental norovirus infections in nonhuman primates chimpanzees as an animal model for human norovirus infection and vaccine development experimental inoculation of juvenile rhesus macaques with primate enteric caliciviruses molecular characterization of three novel murine noroviruses persistent infection with and serologic cross-reactivity of three novel murine noroviruses [comparative study research support, n.i.h., extramural research support gastrointestinal norovirus infection associated with exacerbation of inflammatory bowel disease porcine enteric caliciviruses: genetic and antigenic relatedness to human caliciviruses, diagnosis and epidemiology pathogenesis of a genogroup ii human norovirus in gnotobiotic pigs infection of calves with bovine norovirus giii. strain jena virus: an experimental model to study the pathogenesis of norovirus infection an epizootic of equine encephalomyelitis that occurred in massachusetts in eastern equine encephalomyelitis virus: epidemiology and evolution of mosquito transmission vaccines and animal models for arboviral encephalitides fields virology studies on avian encephalomyelitis. ii. flock survey for embryo susceptibility to the virus the occurrence in nature of "equine encephalomyelitis" in the ring-necked pheasant eastern equine encephalitis virus infection: electron microscopic studies of mouse central nervous system a comparative study of the pathogenesis of western equine and eastern equine encephalomyelitis viral infections in mice by intracerebral and subcutaneous inoculations influence of age on susceptibility and on immune response of mice to eastern equine encephalomyelitis virus the hamster as an animal model for eastern equine encephalitiisdand its use in studies of virus entrance into the brain pathogenesis of aerosolized eastern equine encephalitis virus infection in guinea pigs eastern equine encephalitis. distribution of central nervous system lesions in man and rhesus monkey encephalomyelitis in monkeys severe encephalitis in cynomolgus macaques exposed to aerosolized eastern equine encephalitis virus pathology of animal models of alphavirus encephalitis common marmosets (callithrix jacchus) as a nonhuman primate model to assess the virulence of eastern equine encephalitis virus strains arbovirus investigations in argentina historical aspects and description of study sites western encephalitis in illinois horses and ponies medically important arboviruses of the united states and canada the ecology of western equine encephalomyelitis virus in the central valley of california an epizootic attributable to western equine encephalitis virus infection in emus in texas epidemiologic observations on acute infectious encephalitis in california, with special reference to the outbreak neurologic, intellectual, and psychologic sequelae following western encephalitis. a follow-up study of cases louis encephalitis; preliminary report of a clinical follow-up study in california pathological changes in brain and other target organs of infant and weanling mice after infection with nonneuroadapted western equine encephalitis virus necrotizing myocarditis in mice infected with western equine encephalitis virus: clinical, electrocardiographic, and histopathologic correlations the pathogenesis of western equine encephalitis virus (w.e.e.) in adult hamsters with special reference to the long and short term effects on the c.n.s. of the attenuated clone variant viruses of the bunya-and togaviridae families: potential as bioterrorism agents and means of control aerosol exposure to western equine encephalitis virus causes fever and encephalitis in cynomolgus macaques venezuelan equine encephalitis recovery of venezuelan equine encephalomyelitis virus in panama. a fatal case in man role of dendritic cell targeting in venezuelan equine encephalitis virus pathogenesis mechanism of neuroinvasion of venezuelan equine encephalitis virus in the mouse comparative neurovirulence and tissue tropism of wild-type and attenuated strains of venezuelan equine encephalitis virus administered by aerosol in c h/hen and balb/c mice c h/hen mouse model for the evaluation of antiviral agents for the treatment of venezuelan equine encephalitis virus infection treatment of venezuelan equine encephalitis virus infection with (-)-carbodine studies on the virus of venezuelan equine encephalomyelitis. i modification by cortisone of the response of the central nervous system of macaca mulatta experimental studies of rhesus monkeys infected with epizootic and enzootic subtypes of venezuelan equine encephalitis virus aerosol infection of cynomolgus macaques with enzootic strains of venezuelan equine encephalitis viruses chikungunya outbreaksdthe globalization of vectorborne diseases infection with chikungunya virus in italy: an outbreak in a temperate region changing patterns of chikungunya virus: re-emergence of a zoonotic arbovirus chikungunya and the nervous system: what we do and do not know [review] a mouse model for chikungunya: young age and inefficient type-i interferon signaling are risk factors for severe disease an animal model for studying the pathogenesis of chikungunya virus infection chikungunya virus arthritis in adult wild-type mice a mouse model of chikungunya virus-induced musculoskeletal inflammatory disease: evidence of arthritis, tenosynovitis, myositis, and persistence mouse models for chikungunya virus: deciphering immune mechanisms responsible for disease and pathology chikungunya disease in nonhuman primates involves long-term viral persistence in macrophages aedes albopictus in the united states: tenyear presence and public health implications epidemic dengue/dengue hemorrhagic fever as a public health, social and economic problem in the st century dengue: an update dengue haemorrhagic fever: diagnosis, treatment, prevention, and control tropism of dengue virus in mice and humans defined by viral nonstructural protein -specific immunostaining clinical and laboratory features that differentiate dengue from other febrile illnesses in an endemic areadpuerto rico risk factors in dengue shock syndrome animal models of dengue virus infection manifestation of thrombocytopenia in dengue- -virus-infected mice liver injury and viremia in mice infected with dengue- virus early activation of natural killer and b cells in response to primary dengue virus infection in a/j mice induction of tetravalent protective immunity against four dengue serotypes by the tandem domain iii of the envelope protein essential role of platelet-activating factor receptor in the pathogenesis of dengue virus infection murine model for dengue virus-induced lethal disease with increased vascular permeability lethal antibody enhancement of dengue disease in mice is prevented by fc modification inhibitory potential of neem (azadirachta indica juss) leaves on dengue virus type- replication genetic basis of attenuation of dengue virus type small plaque mutants with restricted replication in suckling mice and in scid mice transplanted with human liver cells study of dengue virus infection in scid mice engrafted with human k cells dengue virus tropism in humanized mice recapitulates human dengue fever dengue virus infection and immune response in humanized rag (À/À) gamma(c)(À/À) (rag-hu) mice a model of denv- infection that recapitulates severe disease and highlights the importance of ifn-gamma in host resistance to infection mosquito bite delivery of dengue virus enhances immunogenicity and pathogenesis in humanized mice studies on the pathogenesis of dengue infection in monkeys. . sequential distribution of virus in primary and heterologous infections studies on dengue virus infection in cyclophosphamide-treated rhesus monkeys dengue virus-induced hemorrhage in a nonhuman primate model an in-depth analysis of original antigenic sin in dengue virus infection monoclonal antibody-mediated enhancement of dengue virus infection in vitro and in vivo and strategies for prevention the arboviruses: epidemiology and ecology screening of protective antigens of japanese encephalitis virus by dna immunization: a comparative study with conventional viral vaccines immunogenicity, genetic stability, and protective efficacy of a recombinant, chimeric yellow fever-japanese encephalitis virus (chimerivax-je) as a live, attenuated vaccine candidate against japanese encephalitis studies on host factors in inapparent infection with japanese b encephalitis: influence of age, nutrition and luminal induced sleep on the course of infection in mice relation of the peripheral multiplication of japanese b encephalitis virus to the pathogenesis of the infection in mice field's virology free radical generation by neurons in a rat model of japanese encephalitis sequential changes in serum cytokines and chemokines in a rat model of japanese encephalitis experimental infections of monkeys with langat virus. i. comparison of viremia following peripheral inoculation of langat and japanese encephalitis viruses intranasal infection of monkeys with japanese encephalitis virus: clinical response and treatment with a nuclease-resistant derivative of poly (i).poly (c) a neurotropic virus isolated from the blood of a native uganda isolation from human sera in egypt of a virus apparently identical to west nile virus isolation of west nile virus from culex mosquitoes the arboviruses: epidemiology and ecology centers for disease control & prevention. outbreak of west nile-like viral encephalitisdnew york origin of the west nile virus responsible for an outbreak of encephalitis in the northeastern united states clinical virology the west nile virus outbreak of in new york: the flushing hospital experience nile feverda reemerging mosquito-borne viral disease in europe west nile viral encephalitis experimental infection of cats and dogs with west nile virus experimental infection of horses with west nile virus pathogenesis of west nile virus encephalitis in mice and rats. . influence of age and species on mortality and infection study on west nile virus persistence in monkeys experimental infection of pigs with west nile virus persistent west nile virus infection in the golden hamster: studies on its mechanism and possible implications for other flavivirus infections experimental encephalitis following peripheral inoculation of west nile virus in mice of different ages immunogenicity and protective efficacy of a recombinant subunit west nile virus vaccine in rhesus monkeys molecularly engineered live-attenuated chimeric west nile/dengue virus vaccines protect rhesus monkeys from west nile virus tissue tropism and neuroinvasion of west nile virus do not differ for two mouse strains with different survival rates phenotypic changes in langerhans' cells after infection with arboviruses: a role in the immune response to epidermally acquired viral infection? interleukin- beta but not tumor necrosis factor is involved in west nile virusinduced langerhans cell migration from the skin in c bl/ mice profound and prolonged lymphocytopenia with west nile encephalitis innate and adaptive immune responses determine protection against disseminated infection by west nile encephalitis virus spinal cord neuropathology in human west nile virus infection animal models for sars world health organization. the world health report d shaping the future stability and inactivation of sars coronavirus the severe acute respiratory syndrome clinical manifestations, laboratory findings, and treatment outcomes of sars patients identification of a novel coronavirus in patients with severe acute respiratory syndrome profiles of antibody responses against severe acute respiratory syndrome coronavirus recombinant proteins and their potential use as diagnostic markers the immunobiology of sars* viral shedding patterns of coronavirus in patients with probable severe acute respiratory syndrome sars vaccines: where are we? animal models and vaccines for sars-cov infection replication of sars coronavirus administered into the respiratory tract of african green, rhesus and cynomolgus monkeys aged balb/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice severe acute respiratory syndrome (sars): a year in review koch's postulates fulfilled for sars virus pegylated interferon-alpha protects type pneumocytes against sars coronavirus infection in macaques macaque model for severe acute respiratory syndrome mechanisms of host defense following severe acute respiratory syndromecoronavirus (sars-cov) pulmonary infection of mice resolution of primary severe acute respiratory syndromeassociated coronavirus infection requires stat virology: sars virus infection of cats and ferrets civets are equally susceptible to experimental infection by two different severe acute respiratory syndrome coronavirus isolates pneumonitis and multi-organ system disease in common marmosets (callithrix jacchus) infected with the severe acute respiratory syndrome-associated coronavirus severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus immunopathogenesis of coronavirus infections: implications for sars immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus immunization with modified vaccinia virus ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets primary severe acute respiratory syndrome coronavirus infection limits replication but not lung inflammation upon homologous rechallenge a mouse-adapted sars-coronavirus causes disease and mortality in balb/c mice rabies re-examined rabies and other lyssavirus diseases overview, prevention, and treatment of rabies studies of rabies street virus in the syrian hamster live attenuated rabies virus co-infected with street rabies virus protects animals against rabies spread and pathogenic characteristics of a g-deficient rabies virus recombinant: an in vitro and in vivo study biological basis of rabies virus neurovirulence in mice: comparative pathogenesis study using the immunoperoxidase technique intracerebral vaccination suppresses the spread of rabies virus in the mouse brain human rabies therapy: lessons learned from experimental studies in mouse models experimental rabies virus infection of p. neurotrophin receptor-deficient mice apoptosis in experimental rabies in bax-deficient mice emerging pattern of rabies deaths and increased viral infectivity effective protection of monkeys against death from street virus by post-exposure administration of tissue-culture rabies vaccine a model in mice for the pathogenesis and treatment of rabies a model in mice for the study of the early death phenomenon after vaccination and challenge with rabies virus experimental rabies infection and oral vaccination in vampire bats (desmodus rotundus the distribution of challenge virus standard rabies virus versus skunk street rabies virus in the brains of experimentally infected rabid skunks a compendium of years of epidemiological, clinical, and laboratory studies proportion of deaths and clinical features in bundibugyo ebola virus infection clinical aspects of marburg hemorrhagic fever correlates of immunity to filovirus infection the role of the type i interferon response in the resistance of mice to filovirus infection a mouse model for evaluation of prophylaxis and therapy of ebola hemorrhagic fever development of a murine model for aerosolized filovirus infection using a panel of bxd recombinant inbred mice. viruses lethality and pathogenesis of airborne infection with filoviruses in a alpha/betaÀ/À interferon receptordeficient mice development and characterization of a mouse model for marburg hemorrhagic fever identification of an antioxidant small-molecule with broad-spectrum antiviral activity animal models of highly pathogenic rna viral infections: hemorrhagic fever viruses protective efficacy of a bivalent recombinant vesicular stomatitis virus vaccine in the syrian hamster model of lethal ebola virus infection pathogenesis of filoviruses in small animal models program abstr th int symp filoviruses pathogenesis of experimental ebola virus infection in guinea pigs characterization of disease and pathogenesis following airborne exposure of guinea pigs to filoviruses manuscripts in preparation postexposure antibody prophylaxis protects nonhuman primates from filovirus disease aerosol exposure to the angola strain of marburg virus causes lethal viral hemorrhagic fever in cynomolgus macaques a small nonhuman primate model for filovirus-induced disease pathology of experimental ebola virus infection in african green monkeys. involvement of fibroblastic reticular cells pathogenesis of marburg hemorrhagic fever in cynomolgus macaques a characterization of aerosolized sudan ebolavirus infection in african green monkeys, cynomolgus macaques, and rhesus macaques recent progress in henipavirus research: molecular biology, genetic diversity, animal models transmission of human infection with nipah virus recurrent zoonotic transmission of nipah virus into humans nipah virus outbreak with person-to-person transmission in a district of bangladesh henipavirus susceptibility to environmental variables hendra virus infection in a veterinarian human hendra virus encephalitis associated with equine outbreak clinical features of nipah virus encephalitis among pig farmers in malaysia the emergence of nipah virus, a highly pathogenic paramyxovirus nipah virus encephalitis acute hendra virus infection: analysis of the pathogenesis and passive antibody protection in the hamster model clinical outcome of henipavirus infection in hamsters is determined by the route and dose of infection a golden hamster model for human acute nipah virus infection histopathologic and immunohistochemical characterization of nipah virus infection in the guinea pig a guinea-pig model of hendra virus encephalitis the lesions of experimental equine morbillivirus disease in cats and guinea pigs a neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute nipah virus infection a recombinant hendra virus g glycoproteinbased subunit vaccine protects ferrets from lethal hendra virus challenge vertical transmission and fetal replication of nipah virus in an experimentally infected cat susceptibility of cats to equine morbillivirus experimental infection of squirrel monkeys with nipah virus development of an acute and highly pathogenic nonhuman primate model of nipah virus infection a novel model of lethal hendra virus infection in african green monkeys and the effectiveness of ribavirin treatment experimental nipah virus infection in pteropid bats (pteropus poliocephalus) bacterial infections in pigs experimentally infected with nipah virus experimental inoculation study indicates swine as a potential host for hendra virus experimental nipah virus infection in pigs and cats invasion of the central nervous system in a porcine host by nipah virus experimental infection of horses with hendra virus/australia/horse/ /redlands transmission studies of hendra virus (equine morbillivirus) in fruit bats, horses and cats a novel morbillivirus pneumonia of horses and its transmission to humans a morbillivirus that caused fatal disease in horses and humans equine morbillivirus pneumonia: susceptibility of laboratory animals to the virus feline model of acute nipah virus infection and protection with a soluble glycoprotein-based subunit vaccine distribution of viral antigens and development of lesions in chicken embryos inoculated with nipah virus global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis [meta-analysis research support economic impact of respiratory syncytial virus-related illness in the us: an analysis of national databases the relationship of meteorological conditions to the epidemic activity of respiratory syncytial virus viral and host factors in human respiratory syncytial virus pathogenesis evidence of a causal role of winter virus infection during infancy in early childhood asthma respiratory syncytial virus infection in elderly and high-risk adults respiratory syncytial virus american academy of pediatrics subcommittee on diagnosis & management of bronchiolitis. diagnosis and management of bronchiolitis respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine respiratory syncytial virus induces pneumonia, cytokine response, airway obstruction, and chronic inflammatory infiltrates associated with long-term airway hyperresponsiveness in mice genetic susceptibility to respiratory syncytial virus infection in inbred mice differential pathogenesis of respiratory syncytial virus clinical isolates in balb/c mice primary respiratory syncytial virus infection in mice the use of a neonatal mouse model to study respiratory syncytial virus infections respiratory syncytial virus affects pulmonary function in balb/c mice the cotton rat model of respiratory viral infections diversifying animal models: the use of hispid cotton rats (sigmodon hispidus) in infectious diseases the antiviral activity of sp- , a natural polyphenolic polymer, against respiratory syncytial and parainfluenza type viruses in cotton rats pulmonary lesions in primary respiratory syncytial virus infection, reinfection, and vaccine-enhanced disease in the cotton rat bovine respiratory syncytial virus protects cotton rats against human respiratory syncytial virus infection age at first viral infection determines the pattern of t cell-mediated disease during reinfection in adulthood the enhancement or prevention of airway hyperresponsiveness during reinfection with respiratory syncytial virus is critically dependent on the age at first infection and il- production [comparative study research support immunomodulation with il- r alpha antisense oligonucleotide prevents respiratory syncytial virusmediated pulmonary disease chinchilla and murine models of upper respiratory tract infections with respiratory syncytial virus experimental respiratory syncytial virus infection of four species of primates respiratory syncytial virus infects the bonnet monkey serum neutralizing antibody titers of seropositive chimpanzees immunized with vaccines coformulated with natural fusion and attachment proteins of respiratory syncytial virus reduced clearance of respiratory syncytial virus infection in a preterm lamb model human respiratory syncytial virus a strain replicates and induces innate immune responses by respiratory epithelia of neonatal lambs respiratory syncytial virus is associated with an inflammatory response in lungs and architectural remodeling of lung-draining lymph nodes of newborn lambs structure as revealed by airway dissection. a comparison of mammalian lungs biomedical applications of sheep models: from asthma to vaccines the pathology of influenza virus infections mortality due to influenza in the united statesdan annualized regression approach using multiple-cause mortality data animal models for the study of influenza pathogenesis and therapy serious morbidity and mortality associated with influenza epidemics centers for disease control & prevention. bacterial coinfections in lung tissue specimens from fatal cases of pandemic influenza a (h n )dunited states human and avian influenza viruses target different cell types in cultures of human airway epithelium animal models head-to-head comparison of four nonadjuvanted inactivated cell culture-derived influenza vaccines: effect of composition, spatial organization and immunization route on the immunogenicity in a murine challenge model [comparative study interferon-induced mx protein: a mediator of cellular resistance to influenza virus in vitro and in vivo assay systems for study of influenza virus inhibitors utilization of pulse oximetry for the study of the inhibitory effects of antiviral agents on influenza virus in mice the contribution of animal models to the understanding of the host range and virulence of influenza a viruses biological heterogeneity, including systemic replication in mice, of h n influenza a virus isolates from humans in hong kong distinct pathogenesis of hong kong-origin h n viruses in mice compared to that of other highly pathogenic h avian influenza viruses effect of oral gavage treatment with znal and other metallo-ion formulations on influenza a h n and h n virus infections in mice inactivated and live, attenuated influenza vaccines protect mice against influenza: streptococcus pyogenes super-infections [comparative study research support the use of an animal model to study transmission of influenza virus infection strong local and systemic protective immunity induced in the ferret model by an intranasal virosome-formulated influenza subunit vaccine local innate immune responses and influenza virus transmission and virulence in ferrets regional t-and b-cell responses in influenza-infected ferrets human and avian influenza viruses target different cells in the lower respiratory tract of humans and other mammals live, attenuated influenza virus (laiv) vehicles are strong inducers of immunity toward influenza b virus [comparative study the ferret: an animal model to study influenza virus pathogenesis of avian influenza a (h n ) viruses in ferrets severe seasonal influenza in ferrets correlates with reduced interferon and increased il- induction neuropathology of h n virus infection in ferrets evaluation of three strains of influenza a virus in humans and in owl, cebus, and squirrel monkeys a computationally optimized hemagglutinin virus-like particle vaccine elicits broadly reactive antibodies that protect nonhuman primates from h n infection evaluation of intravenous zanamivir against experimental influenza a (h n ) virus infection in cynomolgus macaques aberrant innate immune response in lethal infection of macaques with the influenza virus pathology of human influenza a (h n ) virus infection in cynomolgus macaques (macaca fascicularis integrated molecular signature of disease: analysis of influenza virus-infected macaques through functional genomics and proteomics integration of clinical data, pathology, and cdna microarrays in influenza virus-infected pigtailed macaques (macaca nemestrina kinetic profile of influenza virus infection in three rat strains the guinea pig as a transmission model for human influenza viruses influenza-induced tachypnea is prevented in immune cotton rats, but cannot be treated with an anti-inflammatory steroid or a neuraminidase inhibitor the antiviral potential of interferon-induced cotton rat mx proteins against orthomyxovirus (influenza), rhabdovirus, and bunyavirus the cotton rat as a model to study influenza pathogenesis and immunity the cotton rat provides a useful small-animal model for the study of influenza virus pathogenesis co-infection of the cotton rat (sigmodon hispidus) with staphylococcus aureus and influenza a virus results in synergistic disease pathogenicity of a highly pathogenic avian influenza virus, a/chicken/yamaguchi/ / (h n ) in different species of birds and mammals domestic pigs have low susceptibility to h n highly pathogenic avian influenza viruses [comparative study research support animal models in influenza vaccine testing rift valley fever: an uninvited zoonosis in the arabian peninsula prevalence of anti-rift-valley-fever igm antibody in abattoir workers in the nile delta during the outbreak in egypt rift valley fever the occurrence of human cases in johannesburg the pathogenesis of rift valley fever epidemic rift valley fever in egypt: observations of the spectrum of human illness crc handbook series in zoonoses rift valley fever virus in mice. i. general features of the infection the pathogenesis of rift valley fever virus in the mouse model the susceptibility of rats to rift valley fever in relation to age inbred rat strains mimic the disparate human response to rift valley fever virus infection the gerbil, meriones unguiculatus, a model for rift valley fever viral encephalitis experimental rift valley fever in rhesus macaques development of a novel nonhuman primate model for rift valley fever crimean-congo hemorrhagic fever: a global perspective crimean-congo hemorrhagic fever the clinical pathology of crimean-congo hemorrhagic fever experimental congo virus (ib-an ) infection in primates crimean congo hemorrhagic fever: a global perspective viremia and antibody response of small african and laboratory animals to crimean-congo hemorrhagic fever virus infection a comparative study of the crimean hemorrhagic faverdcongo group of viruses [comparative study ribavirin efficacy in an in vivo model of crimean-congo hemorrhagic fever virus (cchf) infection pathogenesis and immune response of crimean-congo hemorrhagic fever virus in a stat- knockout mouse model crimean-congo hemorrhagic fever virus infection is lethal for adult type i interferon receptorknockout mice possibility of extracting hyperimmune gammaglobulin against chf from donkey blood sera study of susceptibility to crimean hemorrhagic fever (chf) virus in european and long-eared hedgehogs. tezisy konf vop med virus field's virology epidemiological studies of hemorrhagic fever with renal syndrome: analysis of risk factors and mode of transmission a short review hantavirus pulmonary syndrome. pathogenesis of an emerging infectious disease field's virology centers for disease control & prevention. outbreak of acute illness-southwestern united states genetic diversity, distribution, and serological features of hantavirus infection in five countries in south america first reported cases of hantavirus pulmonary syndrome in canada [case reports an unusual hantavirus outbreak in southern argentina: person-to-person transmission? hantavirus pulmonary syndrome study group for patagonia hantavirus pulmonary syndrome: the new american hemorrhagic fever the incubation period of hantavirus pulmonary syndrome cardiopulmonary manifestations of hantavirus pulmonary syndrome hantavirus pulmonary syndrome a lethal disease model for hantavirus pulmonary syndrome pathogenesis of hantaan virus infection in suckling mice: clinical, virologic, and serologic observations infection of hantaan virus strain aa leading to pulmonary disease in laboratory mice hantaan virus infection causes an acute neurological disease that is fatal in adult laboratory mice functional role of type i and type ii interferons in antiviral defense andes virus dna vaccine elicits a broadly cross-reactive neutralizing antibody response in nonhuman primates andes virus infection of cynomolgus macaques wild-type puumala hantavirus infection induces cytokines, c-reactive protein, creatinine, and nitric oxide in cynomolgus macaques pathology of puumala hantavirus infection in macaques viral haemorrhagic fevers caused by lassa, ebola, and marburg viruses new opportunities for field research on the pathogenesis and treatment of lassa fever imported lassa fever lassa fever. effective therapy with ribavirin lassa virus hepatitis: a study of fatal lassa fever in humans lassa virus infection of rhesus monkeys: pathogenesis and treatment with ribavirin experimental inhalation infection of monkeys of the macacus cynomolgus and macacus rhesus species with the virus of lymphocytic choriomeningitis (we) arenavirus-mediated liver pathology: acute lymphocytic choriomeningitis virus infection of rhesus macaques is characterized by high-level interleukin- expression and hepatocyte proliferation experimental studies of arenaviral hemorrhagic fevers lcmv-mediated hepatitis in rhesus macaques: we but not arm strain activates hepatocytes and induces liver regeneration mucosal arenavirus infection of primates can protect them from lethal hemorrhagic fever pathogenesis of lassa fever in cynomolgus macaques lassa fever encephalopathy: clinical and laboratory findings lassa fever encephalopathy: lassa virus in cerebrospinal fluid but not in serum lassa virus infection in experimentally infected marmosets: liver pathology and immunophenotypic alterations in target tissues virus taxonomy, viiith report of the international committee on taxonomy of viruses pathogenesis of lassa virus infection in guinea pigs the effect of an arenavirus infection on liver morphology and function cardiovascular and pulmonary responses to pichinde virus infection in strain guinea pigs pathogenesis of pichinde virus infection in strain guinea pigs: an immunocytochemical, virologic, and clinical chemistry study pichinde virus infection in strain guinea pigs reduces intestinal protein reflection coefficient with compensation clinical laboratory, virologic, and pathologic changes in hamsters experimentally infected with pirital virus (arenaviridae): a rodent model of lassa fever variation between strains of hamsters in the lethality of pichinde virus infections interferon alfacon- protects hamsters from lethal pichinde virus infection treatment of lethal pichinde virus infections in weanling lvg/lak hamsters with ribavirin, ribamidine, selenazofurin, and ampligen [comparative study research support global mortality associated with rotavirus disease among children in minimal infective dose of rotavirus rotavirus immunoglobulin levels among indian mothers of two socio-economic groups and occurrence of rotavirus infections among their infants up to six months analyses of clinical, pathological and virological features of human rotavirus strain, yo induced gastroenteritis in infant balb/c mice epizootic diarrhea of infant mice: identification of the etiologic agent immunity to rotavirus infection in mice development of an adult mouse model for studies on protection against rotavirus a gastrointestinal rotavirus infection mouse model for immune modulation studies protection of the villus epithelial cells of the small intestine from rotavirus infection does not require immunoglobulin a rotavirus viremia and extraintestinal viral infection in the neonatal rat model [comparative study research support characterization of clinical and immune response in a rotavirus diarrhea model in suckling lewis rats development of a heterologous model in germfree suckling rats for studies of rotavirus diarrhea studies of oral rehydration solutions in animal models induction of mucosal immune responses and protection against enteric viruses: rotavirus infection of gnotobiotic pigs as a model developmental immunity in the piglet swine in biomedical research neonatal calf diarrhea induced by rotavirus characterisation of the primary local and systemic immune response in gnotobiotic lambs against rotavirus infection experimental infection of non-human primates with a human rotavirus isolate development of a rotavirus-shedding model in rhesus macaques, using a homologous wild-type rotavirus of a new p genotype reflections on years of aids hivs and their replication the utility of the new generation of humanized mice to study hiv- infection: transmission, prevention, pathogenesis, and treatment antiretroviral pre-exposure prophylaxis prevents vaginal transmission of hiv- in humanized blt mice hematopoietic stem cell-engrafted nod/ scid/il rgamma null mice develop human lymphoid systems and induce long-lasting hiv- infection with specific humoral immune responses hiv- infection and cd t cell depletion in the humanized rag À/À gamma cÀ/À (rag-hu) mouse model hiv- infection and pathogenesis in a novel humanized mouse model induction of robust cellular and humoral virusspecific adaptive immune responses in human immunodeficiency virus-infected humanized blt mice an aptamer-sirna chimera suppresses hiv- viral loads and protects from helper cd (þ) t cell decline in humanized mice mucosal immunity and vaccines low-dose rectal inoculation of rhesus macaques by sivsme or sivmac recapitulates human mucosal infection by hiv- propagation and dissemination of infection after vaginal transmission of simian immunodeficiency virus limited dissemination of pathogenic siv after vaginal challenge of rhesus monkeys immunized with a live virulence and reduced fitness of simian immunodeficiency virus with the m v mutation in reverse transcriptase siv-induced impairment of neurovascular repair: a potential role for vegf therapeutic dna vaccine induces broad t cell responses in the gut and sustained protection from viral rebound and aids in siv-infected rhesus macaques a nonfucosylated variant of the anti-hiv- monoclonal antibody b has enhanced fcgammariiiamediated antiviral activity in vitro but does not improve protection against mucosal shiv challenge in macaques a trivalent recombinant ad gag/pol/nef vaccine fails to protect rhesus macaques from infection or control virus replication after a limiting-dose heterologous siv challenge animal model for the therapy of acquired immunodeficiency syndrome with reverse transcriptase inhibitors susceptibility of hiv- , siv and shiv to various anti-hiv- compounds: implications for treatment and postexposure prophylaxis use of a small molecule ccr inhibitor in macaques to treat simian immunodeficiency virus infection or prevent simian-human immunodeficiency virus infection shiv- i and passaged progeny viruses encoding r hiv- clade c env cause aids in rhesus monkeys update on animal models for hiv research limited or no protection by weakly or nonneutralizing antibodies against vaginal shiv challenge of macaques compared with a strongly neutralizing antibody macaque studies of vaccine and microbicide combinations for preventing hiv- sexual transmission vpx is critical for sivmne infection of pigtail macaques impact of short-term haart initiated during the chronic stage or shortly post-exposure on siv infection of male genital organs the rhesus macaque pediatric siv infection modeld a valuable tool in understanding infant hiv- pathogenesis and for designing pediatric hiv- prevention strategies perinatal transmission of shiv-sf p in macaca nemestrina immune and genetic correlates of vaccine protection against mucosal infection by siv in monkeys chronic administration of tenofovir to rhesus macaques from infancy through adulthood and pregnancy: summary of pharmacokinetics and biological and virological effects efficacy assessment of a cell-mediated immunity hiv- vaccine (the step study): a double-blind, randomised, placebo-controlled, test-of-concept trial human papillomavirus in cervical cancer human papillomavirus research: do we still need animal models? animal models of papillomavirus pathogenesis evidence of human papillomavirus vaccine effectiveness in reducing genital warts: an analysis of california public family planning administrative claims data the rabbit viral skin papillomas and carcinomas: a model for the immunogenetics of hpv-associated carcinogenesis protection of beagle dogs from mucosal challenge with canine oral papillomavirus by immunization with recombinant adenoviruses expressing codon-optimized early genes naturally occurring, nonregressing canine oral papillomavirus infection: host immunity, virus characterization, and experimental infection regression of canine oral papillomas is associated with infiltration of cd þ and cd þ lymphocytes characterization and experimental transmission of an oncogenic papillomavirus in female macaques a multimeric l vaccine for prevention of animal papillomavirus infections preclinical development of highly effective and safe dna vaccines directed against hpv e and e us doctors investigate more than possible cases of monkeypox isolation of monkeypox virus from wild squirrel infected in nature reemergence of monkeypox: prevalence, diagnostics, and countermeasures human monkeypox infection: a family cluster in the midwestern united states human monkeypox and other poxvirus infections of man the confirmation and maintenance of smallpox eradication human monkeypox identification of wild-derived inbred mouse strains highly susceptible to monkeypox virus infection for use as small animal models a prairie dog animal model of systemic orthopoxvirus disease using west african and congo basin strains of monkeypox virus comparison of monkeypox viruses pathogenesis in mice by in vivo imaging comparative pathology of north american and central african strains of monkeypox virus in a ground squirrel model of the disease experimental infection of an african dormouse (graphiurus kelleni) with monkeypox virus a mouse model of lethal infection for evaluating prophylactics and therapeutics against monkeypox virus experimental infection of ground squirrels (spermophilus tridecemlineatus) with monkeypox virus experimental infection of prairie dogs with monkeypox virus experimental infection of cynomolgus macaques (macaca fascicularis) with aerosolized monkeypox virus the pathology of experimental aerosolized monkeypox virus infection in cynomolgus monkeys (macaca fascicularis) immunogenicity of a highly attenuated mva smallpox vaccine and protection against monkeypox smallpox vaccine does not protect macaques with aids from a lethal monkeypox virus challenge smallpox vaccine-induced antibodies are necessary and sufficient for protection against monkeypox virus virulence and pathophysiology of the congo basin and west african strains of monkeypox virus in non-human primates a novel respiratory model of infection with monkeypox virus in cynomolgus macaques antiviral treatment is more effective than smallpox vaccination upon lethal monkeypox virus infection comparative analysis of monkeypox virus infection of cynomolgus macaques by the intravenous or intrabronchial inoculation route establishment of the black-tailed prairie dog (cynomys ludovicianus) as a novel animal model for comparing smallpox vaccines administered preexposure in both high-and low-dose monkeypox virus challenges effective antiviral treatment of systemic orthopoxvirus disease: st- treatment of prairie dogs infected with monkeypox virus clinical characteristics of human monkeypox, and risk factors for severe disease hepatitis b virus infection cell culture and animal models of viral hepatitis. part i: hepatitis b risks of chronicity following acute hepatitis b virus infection: a review hepatitis b virus infectiondnatural history and clinical consequences clinical aspects of hepatitis b virus infection hepatitis b virus. the major etiology of hepatocellular carcinoma trans-activation of viral enhancers by the hepatitis b virus x protein identification of hepatitis b virus indigenous to chimpanzees detection of hepatitis b virus infection in wild-born chimpanzees (pan troglodytes verus): phylogenetic relationships with human and other primate genotypes antibody to hepatitis-associated antigen. frequency and pattern of response as detected by radioimmunoprecipitation hepatitis and blood transfusion perspectives on hepatitis b studies with chimpanzees hla a restricted cytotoxic t lymphocyte responses to multiple hepatitis b surface antigen epitopes during hepatitis b virus infection primates in the study of hepatitis viruses transfer of hbv genomes using low doses of adenovirus vectors leads to persistent infection in immune competent mice asymmetric replication of duck hepatitis b virus dna in liver cells: free minus-strand dna a virus similar to human hepatitis b virus associated with hepatitis and hepatoma in woodchucks effects of age and viral determinants on chronicity as an outcome of experimental woodchuck hepatitis virus infection hepadnavirusinduced liver cancer in woodchucks animal models of hepadnavirus-associated hepatocellular carcinoma hepatitis b viruses and hepatocellular carcinoma hepatitis b virus replication in primary macaque hepatocytes: crossing the species barrier toward a new small primate model animal models of hepatitis delta virus infection and disease experimental hepatitis delta virus infection in the chimpanzee expression of the hepatitis delta virus large and small antigens in transgenic mice experimental hepatitis delta virus infection in the animal model humanized chimeric upa mouse model for the study of hepatitis b and d virus interactions and preclinical drug evaluation key: cord- - otz w v authors: walsh, michael g.; sawleshwarkar, shailendra; hossain, shah; mor, siobhan m. title: whence the next pandemic? the intersecting global geography of the animal-human interface, poor health systems and air transit centrality reveals conduits for high-impact spillover date: - - journal: one health doi: . /j.onehlt. . sha: doc_id: cord_uid: otz w v the health and economic impacts of infectious disease pandemics are catastrophic as most recently manifested by coronavirus disease (covid- ). the emerging infections that lead to substantive epidemics or pandemics are typically zoonoses that cross species boundaries at vulnerable points of animal-human interface. the sharing of space between wildlife and humans, and their domesticated animals, has dramatically increased in recent decades and is a key driver of pathogen spillover. increasing animal-human interface has also occurred in concert with both increasing globalisation and failing health systems, resulting in a trifecta with dire implications for human and animal health. nevertheless, to date we lack a geographical description of this trifecta that can be applied strategically to pandemic prevention. this investigation provides the first geographical quantification of the intersection of animal-human interfaces, poor human health system performance and global connectivity via the network of air travel. in so doing, this work provides a systematic, data-driven approach to classifying spillover hazard based on the distribution of animal-human interfaces while simultaneously identifying globally connected cities that are adjacent to these interfaces and which may facilitate global pathogen dissemination. we present this geography of high-impact spillover as a tool for developing targeted surveillance systems and improved health infrastructure in vulnerable areas that may present conduits for future pandemics. the global spread of severe acute respiratory syndrome coronavirus- (sars-cov- ) in has shown how rapidly emerging infectious diseases can devastate human health and national economies. such infections typically have zoonotic origins, with other notable examples being sars-cov in [ ] , influenza a h n virus in [ ] , and the west african ebola virus disease epidemic of - [ ] . emerging infectious diseases are increasing in incidence and expanding in geographic range due in large part to direct and diffuse anthropogenic pressure across ecosystems [ ] . this is particularly true in areas with high wildlife biodiversity that are experiencing land-use changes such as deforestation [ , , , ] , although the process of disease emergence is complex and cannot be attributed to any one driver. morse et al. have developed a useful framework for conceptualising a staged process of pathogen emergence in humans [ ] . in the first stage, potential pathogens circulate exclusively in reservoir hosts. while this stage precedes spillover, the stage is still highly influenced by human activities that place stress on animal populations, such as habitat destruction, population displacement, and nutritional insecurity, thereby altering pathogen circulation in affected non-human host populations [ ] . various researchers have hypothesized that certain animal taxa, such as bats [ , ] , harbor a significantly higher proportion of zoonotic viruses. however, differential impact of specific taxa on spillover has been recently challenged by research showing that overall species richness within order is the primary driver of pathogen richness among mammalian and bird reservoir hosts, rather than shared biological study did show differences in viral richness between mammals and birds so hosts may still cluster within some taxonomic levels [ ] . in the framework's second stage, activities that increase animal-human contact (such as forest management practices or raising domestic animals) create opportunities for spillover from reservoir hosts into human populations. the risk of spillover appears greatest both from generalist species that are highly abundant and adapted to human-dominated landscapes, and -conversely -from those specialist species that are threatened specifically due to habitat loss and human exploitation [ ] ; both scenarios confirm the central influence of humans as a driving force behind spillover. hassell et al. argued that wildlife-livestock-human interfaces emerging under urbanisation represent particularly critical points for cross-species transmission, noting an urgent need for better characterisation of periurban wildlife interfaces using transdisciplinary approaches [ ] . despite the critical influence of anthropogenic pressure, spillover is a complex process requiring that pathogens overcome many landscape and immunological barriers [ ] , and even when successful, not all spillovers lead to sustained transmission. most often this is due to a lack of efficient transmission in the new host populations. occasionally, spillover into naïve human or domesticated animal populations does result in efficient transmission. the framework's final stage is marked by sustained onward transmission and widespread regional or global dissemination. at this stage, the capacity of local health systems to rapidly detect cases of a novel disease and control ongoing chains of transmission is key to preventing broader dissemination from the original focus [ ] . in this work we distinguish wildlife-origin zoonoses with the potential to transmit efficiently between humans as impactful spillovers, and we further designate impactful spillovers with the potential to disseminate rapidly to regions beyond their origin focus as high-impact spillovers. highimpact spillovers are therefore a function of the capacity of local health systems and the proximity of the initial spillover to conduits of broader global dissemination, particularly transportation hubs such as airports [ ] . consequently, in order to block emerging zoonoses with pandemic potential (high-impact spillovers), biosurveillance systems must simultaneously consider critical animal-human interfaces, the performance and reach of the health systems, and the biosecurity of proximate transportation hubs that can serve as conduits for rapid global dissemination. despite the documented importance of animal-human interfaces for zoonotic transmission of pathogens, and the framework described above for understanding the staged transition from spillover through to human pandemic, we still lack a practical, data-driven and synthetic description of the geography of the critical intersection of animal-human interface and vulnerable points with low disease detection and rapid widespread dissemination potential. the aims of the current work were therefore to ( ) describe and quantify the global geography of the interfaces between mammalian and bird wildlife and humans and their domestic livestock; and ( ) to synthesize the geography of the wildlifelivestock/poultry-human interface, poor health system performance , and the global network of air travel to identify cities whose global connectedness and proximity to animal-human interfaces indicate significant potential to serve as conduits for high-impact spillover. raster data for mammalian and bird species richness, livestock and poultry densities, and human population density were acquired to describe the intersection of their geographic distributions as landscapes of potential animal-human interface. mammalian and bird species richness were used as a representation of total mammalian and bird diversity (ɣ-diversity), respectively, rather than distinguishing between taxonomic groups since it has recently been shown that overall species richness is the primary driver of pathogen richness among these vertebrate hosts [ ] . a mammalian richness raster was acquired from the socioeconomic data and applications center (sedac) repository [ ] . species richness was quantified using the geographic extents of the international union for conservation of nature (iucn) assessment of mammalian species ( , species in families). a total bird richness raster was obtained from the biodiversity mapping project [ ] and was constructed from the handbook of birds of the world [ ] , birdlife international[ ], and iucn. the gridded livestock of the world (glw) provided livestock densities for cattle, pigs, sheep, goats, and buffaloes, and poultry densities for chicken and ducks [ ] . the glw was updated in to more appropriately account for spatial heterogeneity within and between countries. aggregate livestock and poultry rasters were created by taking the sum of the number of cattle, pigs, sheep, goats, and buffaloes per unit area, and chicken and ducks per unit area, respectively, since the current aim was to describe the geography of the wild mammal-livestock-human interface, and wild bird-poultry-human interfaces. human population density was also obtained from the sedac repository and is derived from the global rural-urban mapping project version estimates for the population [ ] . finally, defining high-impact spillover potential requires a measure of the distribution of health system performance as an indication of the local capacity to detect and control the occurrence of cases of a novel zoonosis. the infant mortality ratio (imr) was chosen as a proxy for health system performance because this has been recognised as a j o u r n a l p r e -p r o o f journal pre-proof robust and widely used indicator of health infrastructure and health system performance and used extensively to compare health services between countries [ , ] . the imr has been shown to correlate very well with disability adjusted life expectancy (dale) as well as the human development index (hdi) and the inequality-adjusted human development index (ihdi) and strongly relates to structural issues that affect entire populations, such as economic development, general living conditions, social wellbeing, and the quality of the environment [ , ] . the raster of the imr was obtained from sedac [ ] . mammalian and bird richness, human population, and imr rasters were aggregated to the spatial resolution of the livestock and poultry rasters such that all features were analysed with a granularity of arc minutes, which is approximately km. finally, an adjacency matrix for the global airport network was acquired from worldpop to compute the network centrality of individual airports within the global network [ ] . this data product was constructed by worldpop to model annual air passenger flows and comprises airports (network nodes) with , flight connections between them (network edges) [ ] . the global distribution of each landscape feature is presented in the left column of s figure , with the corresponding top quartile of each distribution ( th percentile) presented in the right column. the objective was to demarcate geographic zones based on the degree of intersection of each landscape feature, thus constructing proxy interfaces between humans, livestock, poultry, and mammalian and bird wildlife, and finally identifying where these interfaces intersect with poor health system performance. wild bird-poultry and wild mammal-livestock interfaces were considered separately as there is evidence that interfaces arise largely from interaction between phylogenetically related and/or sympatric species [ ] , and further evidence suggesting that zoonotic viral richness is higher overall among mammalian species than avian species [ ] . intersection was classified as the co-occurrence of the top quartile ( th percentile) of each specified feature. the classification scheme was also reconstructed using the co-occurrence of the th percentile for each landscape feature to examine how different distribution classifications affect the demarcation of interface and associated hazard. three alert levels (yellow, orange, and red) were identified, as conceptualised in figure . all levels represent landscapes with both high anthropogenic pressure and high biodiversity, and thus have potential for impactful spillover. alert-level yellow depicts two-way interfaces between mammalian/bird richness and human population or livestock/poultry densities. alert-level orange depicts three-way interfaces between mammalian/bird richness, human population density, and livestock/poultry j o u r n a l p r e -p r o o f journal pre-proof densities. alert-level red depicts the same animal-human interfaces as alert-level orange, but extends the intersection of these interfaces to include the top quartile of infant mortality. therefore, the hierarchy of this classification system designates two-way interfaces with wildlife (yellow) as the minimum source requirement for wildlife-derived high-impact spillovers, whereas alert-level orange represents landscapes with maximal pressure on, and contact with, wildlife populations. finally, alertlevel red depicts where the geography of maximum human-animal interface intersects with the geography of poor health system performance. the latter represents those areas that might be expected to miss detection of early cases of spillover to humans and thus presents the greatest risk for high-impact spillover. this scheme thus builds on the work by morse et al. by ) quantifying and mapping the degree of shared space between animals and humans, which they posit as driving the first stages of their framework, and ) quantifying and mapping the degree of health system capacity to intercept global dissemination of zoonotic pathogens that demonstrate onward human-to-human transmission, which defines the third stage of their framework. the validity of these animal-human interface metrics was tested using two case studies. the value of these metrics lies in how well they represent contact between wildlife, livestock/poultry, and humans, and the extent to which they intersect with poor health system performance. therefore, the two diseases selected as case studies were chosen based on their representation of infection ecology at the relevant animal-human interfaces and not based on their potential to cause pandemics in humans. as such anthrax was selected because it is a model disease for the wild mammal-livestock-human interface [ , ] , while highly pathogenic avian influenza a h n (hpai h n ) was selected as a similarly good representation of the wild bird-poultry-human interface [ ] . the food and agriculture organisation's global animal disease information system (empres-i) was used to capture reported occurrences of anthrax and hpai h n between january, , and may [ ] . the empres-i system has a particular focus on transboundary animal diseases and emergent zoonoses. only those occurrences for which the exact location, or the location of the centroid of the subdistrict of the occurrence, was known were included in the case studies to minimise spatial uncertainty. for the two cases studies, respectively, the anthrax (n = ) and hpai h n (n = ) cases were considered as cities were within km of the alert-level red zone for the wild mammal-livestock-human interface and wild bird-poultry-human interface ( . % and . %, respectively; figure ). the interface zones of highest potential spillover impact, and their adjacent cities, were predominantly in sub-saharan africa and south and southeast asia (figure ). all descriptive analyses were repeated at the next quartile ( th percentile) for each interface and city centrality, at which % of cities in the top th percentile of network centrality were within km of alert-level red zone for each interface, while ≥ % of cities were within km of alert-level yellow and orange for each distinct interface (supplemental material: s figure , s figure , and s figure ). this work has defined a hierarchical geography of potential high-impact spillover based on variable animal-human interfaces, human health system capacity and proximate cities of high global connectivity. this work was a descriptive geographical exercise intended to quantify and map a systematic representation of the global animal-human interface, and subsequently it showed that many of the world's most connected cities are adjacent to or within areas where wildlife share space with humans and their domesticated animals. indeed, more than % of these cities were within or adjacent to landscapes of extensive animal-human interface, while approximately %- % were located in landscapes of both extensive interface and poor health system performance, thus demonstrating the precarious positioning of many global transportation hubs. as a means toward preventing future highimpact spillovers of pandemic potential, we have highlighted those areas of the world that could most benefit from simultaneous investment in ) conservation efforts to limit wildlife encounters with humans and their domesticated animals, ) improved surveillance of animals, and ) improved human health infrastructure to detect spillovers when they occur and prevent onward spread. finally, defining a global geography of potential high-impact spillover offers unique value by locating those areas and cities that could most benefit from the development of composite, or at least coordinated, landscape and airport biosurveillance systems at local and national levels. in this study we identify areas where the sharing of space between wildlife and humans is high, health system performance is low, and critical interfaces are located adjacent to cities with high global connectivity. we thus provide practitioners in human and animal health with a geographical framework that applies a synthesis of epidemiological, ecological and health system research outputs to aid j o u r n a l p r e -p r o o f journal pre-proof evidence-based public health decision-making. we emphasize that this work is not presented as a prediction of global hotspots of spillover. several well-conducted studies have been undertaken that provide useful results in this domain [ , , ] . moreover, previous efforts have clearly demonstrated the importance of increasing human and domesticated animal encounters with wildlife as a key driver of spillover [ ] , as well as the potential modulation of wildlife-human encounters and subsequent spillover by climate change [ ] . while these previous modelling studies have identified the importance of human pressure on wildlife for spillover, none have described the global distribution of animal-human interface as a data-driven construct of the degree of shared space between wildlife, domesticated animals, and humans. nor have they considered the location of proximate transportation hubs, which are positioned to amplify impactful spillovers through rapid regional or global dissemination and thus transitioning impactful spillovers to high-impact spillovers with regional epidemic or global pandemic consequence. in other words, conduits of high-impact spillover have not been previously systematically mapped, nor have they been described using a practical hierarchy designating the degree of animal-human interface. the practical approach presented here will allow for a more targeted development of one health surveillance and prevention programs, and provides considerable scope as a strategic tool for preventing future pandemics. for example, a preventive strategy can be designed to create barriers between the high-hazard landscapes and their adjacent points of global dissemination. moreover, the closer an intervention is to the landscape generating the hazard (i.e. spillover) the broader would be its expected downstream prevention effect, whereas an intervention implemented farthest from initial spillover (e.g. at transportation hubs) is more permeable and would expectedly provide narrower effect. however, when integrated with upstream surveillance, enhanced monitoring at transportation hubs can contribute to robust, multi-tiered systems by providing a data-informed, critical last line of defense ( figure ). some limitations with this work regarding proxy measures require further discussion. first, the animalhuman interfaces described here are based on the distributions of the species involved (wild mammals and birds, domesticated animals, and humans) rather than on direct observations of the species' interactions in the landscape. therefore the metrics used to represent these interfaces are proxies for interspecific interaction. nevertheless, the granularity of the species' distributions was relatively fine scale ( km x km) and, given the high percentile ( th ) of species' distributions used to construct the primary risk zones, it is reasonable to assume that humans and animals do indeed share the spaces, either in whole or in part or directly or indirectly, within the interfaces we have demarcated. second, the infant mortality rate was used as a proxy for health system performance. we recognise that health j o u r n a l p r e -p r o o f systems are complex coalescences of economic and social infrastructure, medical capacity and training, public health capacity and training, governmental organisation, and the general socioeconomic status of human populations. moreover, it would not be possible to measure these individual components with the granularity required to synthesise a geographically meaningful new construct of health systems. however, the infant mortality rate has long been recognised as both a reliably measured outcome of health systems and a robust metric for comparing the performance of health systems between countries [ ] [ ] [ ] . finally, the validation case studies should not be over-interpreted with respect to anthrax or hpai h n or any other zoonosis. as described above, the current work is not intended as a predictive modelling study. the models used here were not trained with one set of outbreaks to identify some optimal suite of predictors, and then, once identified, tested and evaluated against an independent set of outbreaks. rather, under the current framework specific hazard metrics for animalhuman interfaces were created by quantifying the extent to which humans share space with wildlife. the point process models were then used to determine how these metrics, data-driven and standradised but conceptualised a priori, actually fit against real-world outbreaks of important known zoonoses. it is also important to recognise that outbreak reporting as captured by the empres-i system may not be geographically homogeneous, which is in fact reflected in the finding that red zone interfaces were a poorer fit to the case study outbreaks indicating that these areas may be more prone to missing cases. this investigation has shown that there are substantial animal-human interfaces in areas of poor health system performance, highlighting those areas of potential impactful spillover where health infrastructure may be insufficient to identify spillover cases early and block onward human-to-human transmission should this emerge. this work further showed that there are many cities with a high degree of global connectivity that are proximate to the areas at risk of impactful spillover, and thus has bats, civets and the emergence of sars emergence and pandemic potential of swine-origin h n influenza virus the ebola outbreak global hotspots and correlates of emerging zoonotic diseases global trends in emerging infectious diseases the elephant-livestock interface modulates anthrax suitability in india forest loss shapes the landscape suitability of kyasanur forest disease in the biodiversity hotspots of the western ghats, india prediction and prevention of the next pandemic zoonosis changing resource landscapes and spillover of henipaviruses host and viral traits predict zoonotic spillover from mammals viral zoonotic risk is homogenous among taxonomic orders of mammalian and avian reservoir hosts urbanization and disease emergence: dynamics at the wildlife-livestock-human interface pathways to zoonotic spillover global mammal richness grids mapping the world's bird diversity global distribution data for cattle, buffaloes, horses, sheep, goats, pigs, chickens and ducks in infant mortality rate as an indicator of population health health indicators related to disease, death, and reproduction inequality as a powerful predictor of infant and maternal mortality around the world global subnational infant mortality rates global flight data an open-access modeled passenger flow matrix for the global air network in global trends in infectious diseases at the wildlife-livestock interface anthrax outbreaks in the humans -livestock and wildlife interface areas of northern tanzania: a retrospective record review wildlife/domestic livestock interactions human-animal interface: the case for influenza interspecies transmission empres-i -global animal disease information system spatial point patterns: methodology and applications with r spatstat: an r package for analyzing spatial point patterns the architecture of complex weighted networks some unique properties of eigenvector centrality the igraph software package for complex network research. interjournal complex systems r: a language and environment for statistical computing, r foundation for statistical computing global patterns of zoonotic disease in mammals global shifts in mammalian population trends reveal key predictors of virus spillover risk climate change will drive novel cross-species viral transmission anthrax ppm models . * model : wild mammal-livestock model : wild mammal-human model : wild mammal-livestock-human model : wild mammal-livestock-human-low health avian influenza a h n ppm models model : wild bird-poultry-human model : wild bird-poultry-human-low health baseline aic from spatial-only specified model of zoonosis outcome key: cord- -hnf vayd authors: ford, richard b.; mazzaferro, elisa m. title: emergency care date: - - journal: kirk and bistner's handbook of veterinary procedures and emergency treatment doi: . /b - - - / - sha: doc_id: cord_uid: hnf vayd nan in the event that you suspect peritonitis and have a negative tap with abdominal paracentesis, a diagnostic peritoneal lavage can be performed. to perform abdominal paracentesis, follow this procedure: . place the patient in left lateral recumbency and clip a -to -inch square with the umbilicus in the center. . aseptically scrub the clipped area with antimicrobial scrub solution. . wearing gloves, insert a -or -gauge needle or over-the-needle catheter in four quadrants: cranial and to the right, cranial and to the left, caudal and to the right, and caudal and to the left of the umbilicus. as you insert the needle or catheter, gently twist the needle to push any abdominal organs away from the tip of the needle. local anesthesia typically is not required for this procedure, although a light sedative or analgesic may be necessary if severe abdominal pain is present. in some cases, fluid will flow freely from one or more of the needles. if not, gently aspirate with a -to -ml syringe or aspirate with the patient in a standing position. avoid changing positions with needles in place because iatrogenic puncture of intraabdominal organs may occur. . save any fluid collected in sterile red-and lavender-topped tubes for cytologic and biochemical analyses and bacterial culture. monitor hemorrhagic fluid carefully for the presence of clots. normally, hemorrhagic effusions rapidly become defibrinated and do not clot. clot formation can occur in the presence of ongoing active hemorrhage or may be due to the iatrogenic puncture of organs such as the spleen or liver. if abdominal paracentesis is negative, a diagnostic peritoneal lavage can be performed. peritoneal dialysis kits are commercially available but are fairly expensive and often impractical. to perform a diagnostic peritoneal lavage, follow this procedure: . clip and aseptically scrub the ventral abdomen as described previously. . wearing sterile gloves, cut multiple side ports in a -or -gauge over-the needle catheter. use care to not cut more than % of the circumference of the catheter, or else the catheter will become weakened and potentially can break off in the patient's abdomen. . insert the catheter into the peritoneal cavity caudal and to the right of the umbilicus, directing the catheter dorsally and caudally. . infuse to ml of sterile lactated ringer's solution or . % saline solution that has been warmed to the patient's body temperature. during the instillation of fluid into the peritoneal cavity, watch closely for signs of respiratory distress because an increase in intraabdominal pressure can impair diaphragmatic excursions and respiratory function. . remove the catheter. . in ambulatory patients, walk the patient around while massaging the abdomen to distribute the fluid throughout the abdominal cavity. in nonambulatory patients, gently roll the patient from side to side. . next, aseptically scrub the patient's ventral abdomen again, and perform an abdominal paracentesis as described previously. save collected fluid for culture and cytologic analyses; however, biochemical analyses may be artifactually decreased because of dilution. remember that you likely will retrieve only a small portion of the fluid that you instilled. during the early stage of repair, granulation tissue, some exudate, and minor epithelialization is observed. place a nonadherent bandage with some antibacterial properties (petroleum or nitrofurazone-impregnated gauze) or absorbent material (foam sponge, hydrogel, or hydrocolloid dressing) in direct contact with the wound to minimize disruption of the granulation tissue bed. next, place an absorbent intermediate layer, followed by a porous outer layer, as previously described. granulation tissue can grow through gauze mesh or adhere to foam sponges and can be ripped away at the time of bandage removal. hemorrhage and disruption of the granulation tissue bed can occur. later in the repair process, granulation tissue can exude sanguineous drainage and have some epithelialization. a late nonadherent bandage is required. the contact layer should be some form of nonadherent dressing, foam sponge, hydrogel, or hydrocolloid substance. the intermediate layer and outer layers should be absorbent material and porous tape, respectively. with nonadherent dressings, wounds with viscous exudates may not be absorbed well. this may be advantageous and enhance epithelialization, provided that complications do not occur. infection, exuberant granulation tissue, or adherence of absorbent materials to the wound may occur and delay the healing process. moist healing is a newer concept of wound management in which wound exudates are allowed to stay in contact with the wound. in the absence of infection a moist wound heals faster and has enzymatic activity as a result of macrophage and polymorphonuclear cell breakdown. enzymatic degradation or "autolytic debridement" of the wound occurs. moist wounds tend to promote neutrophil and macrophage chemotaxis and bacterial phagocytosis better than use of wet-to-dry bandages. a potential complication and disadvantage of moist healing, however, is the development of bacterial colonization, folliculitis, and trauma to wound edges that can occur because of the continuously moist environment. use surfactant-type solutions (constant clens; kendall, mansfield, massachusetts) for initial wound cleansing and debridement. use occlusive dressings for rapid enzymatic debridement with bactericidal properties to aid in wound healing. bandage wet necrotic wounds with a dressing premoistened with hypertonic saline (curasalt [kendall] , % saline) to clean and debride the wounds. hypertonic saline functions to desiccate necrotic tissue and bacteria to debride the infected wound. remove and replace the hypertonic saline bandage every to hours. next, place gauze impregnated with antibacterial agents (kerlix amd [kendall] ) over the wound in the bandage layer to act as a barrier to bacterial colonization. if the wound is initially dry or has minimal exudate and is not obviously contaminated or infected, place amorphous gels of water, glycerin, and a polymer (curafil [kendall] ) over the wound to promote moisture and proteolytic healing. discontinue moisture gels such as curafil once the dry wound has become moist. finally, the final stage of moist healing helps to promote the development of a healthy granulation tissue bed. use calcium alginate dressings (curasorb or curasorb zn with zinc [kendall] ) in noninfected wounds with a moderate amount of drainage. alginate gels promote rapid development of a granulation tissue bed and epithelialization. foam dressings also can be applied to exudative wounds after a healthy granulation bed has formed. change foam dressings at least once every to days. for closed wounds without any drainage, such as a laceration that has been repaired surgically, a simple bandage with a nonadherent contact layer (telfa pad [kendall] , for example), intermediate layer of absorbent material, and an outer porous layer (elastikon, vetrap) can be placed to prevent wound contamination during healing. the nonadherent pad will not stick to the wound and cause patient discomfort. because there usually is minimal drainage from the wound, the function of the intermediate layer is more protective than absorptive. any small amount will be absorbed into the intermediate layer of the bandage. it is important in any bandage to place the tape strips or "stirrups" on the patient's limb and then overlap in the bandage, to prevent the bandage from slipping. place the intermediate and tertiary layers loosely around the limb, starting distally and working proximally, with some overlap with each consecutive layer. this method prevents excessive pressure and potential to impair venous drainage. leave the toenails of the third and fourth digits exposed, whenever possible, to allow daily examination of the bandage to determine whether the bandage is impairing venous drainage. if the bandage is too tight and constricting or impeding vascular flow, the toes will become swollen and spread apart. when placed and maintained properly (e.g., the bandage does not get wet), there usually are relatively few complications observed with this type of bandage. in some cases, it is necessary to cover a wound in which a penrose drain has been placed to allow drainage. in many cases, there is a considerable amount of drainage from the drain and underlying soft tissues. the function of the bandage is to help obliterate dead space created by the wound itself, absorb the fluid that drains from the wound and that will contaminate the environment, and prevent external wicking of material from the external environment into the wound. when the bandage is removed, the clinician can examine the amount and type of material that has drained from the wound in order to determine when the drain should be removed. when placing a bandage over a draining wound, the contact layer should be a commercially available nonadherent dressing and several layers of absorbent wide-mesh gauze placed directly over the drain at the distal end of the incision. overlay the layers of gauze with a thick layer of absorbent intermediate dressing to absorb fluid that drains from the wound. if the gauze and intermediate layers are not thick or absorbent enough, there is a potential for the drainage fluid to reach the outer layer of the bandage and provide a source of wicking of bacteria from the external environment into the wound, leading to infection. some wounds such as lacerations have minor bleeding or hemorrhage that require an immediate bandage until definitive care can be provided. to create a pressure bandage, place a nonadherent dressing immediately in contact with the wound, followed by a thick layer of absorbent material, topped by a layer of elastic bandage material such as elastikon or vetrap. unlike the bandage for a closed wound, the top tertiary outer layer should be wrapped with some tension and even pressure around the limb, starting from the distal extremity (toes) and working proximally. the pressure bandage serves to control hemorrhage but should not be left on for long periods. pressure bandages that have been left on for too long can impair nerve function and lead to tissue necrosis and slough. therefore, pressure bandages should be used in the hospital only, so that the patient can be observed closely. if hemorrhage through the bandage occurs, place another bandage over the first until the wound can be repaired definitively. removal of the first bandage will only disrupt any clot that has formed and cause additional hemorrhage to occur. fractures require immediate immobilization to prevent additional patient discomfort and further trauma to the soft tissues of the affected limb. as with all bandages, a contact layer, intermediate layer, and outer layer should be used. place the contact layer in accordance with any type of wound present. the intermediate layer should be thick absorbent material, followed by a top layer of elastic bandage material. an example is to place a telfa pad over a wound in an open distal radius-ulna fracture, followed by a thick layer of cotton gauze cast padding, followed by an elastic layer of kling (johnson & johnson medical, arlington, texas) , pulling each layer tightly over the previous layer with some overlap until the resultant bandage can be "thumped" with the clinician's thumb and forefinger and sound like a ripe watermelon. the bandage should be smooth with consecutive layers of even pressure on the limb, starting distally and working proximally. leave the toenails of the third and fourth digits exposed to monitor for impaired venous drainage that would suggest that the bandage is too tight and needs to be replaced. finally, place a top layer of vetrap or elastikon over the intermediary layer to protect it from becoming contaminated. if the bandage is used with a compound or open fracture, drainage may be impaired and actually lead to enhanced risk of wound infection. bandages placed for initial fracture immobilization are temporary until definitive fracture repair can be performed once the patient's cardiovascular and respiratory status are stable. wounds with exuberant granulation tissue must be handled carefully so as to not disrupt the healing process but to keep an overabundance of tissue from forming that will impair epithelialization. to bandage a wound with exuberant granulation tissue, place a corticosteroid-containing ointment on the wound, followed by a nonadherent contact layer. the corticosteroid will help control the exuberant growth of granulation tissue. next, carefully wrap an absorbent material over the contact layer, followed by careful placement of and overlay of elastic bandage material to place some pressure on the wound. leave the toenails of the third and fourth digits exposed so that circulation can be monitored several times daily. bandages that are too tight must be removed immediately to prevent damage to neuronal tissue and impaired vascularization, tissue necrosis, and slough. because wound drainage may be impaired, there is a risk of infection. gaping wounds or those that have undermined in between layers of subcutaneous tissue and fascia should be bandaged with a pressure bandage to help obliterate dead space and prevent seroma formation. an example of a wound that may require this type of bandage is removal of an infiltrative lipoma on the lateral or ventral thorax. use caution when placing pressure bandages around the thorax or cervical region because bandages placed too tightly may impair adequate ventilation. to place a pressure bandage and obliterate dead space, place a nonadherent contact layer over the wound. usually, a drain is placed in the wound, so place a large amount of wide-mesh gauze at the distal end of the drain to absorb any wound exudate or drainage. place several layers of absorbent material over the site to further absorb any drainage. place a layer of elastic cotton such as kling carefully but firmly over the dead space to cause enough pressure to control drainage. place at least two fingers in between the animal's thorax and the bandage to ensure that the bandage is not too tight. in many cases, the bandage should be placed once the animal has recovered from surgery and is able to stand. if the bandage is placed while the animal is still anesthetized and recumbent, there is a tendency for the bandage to be too tight. finally, the tertiary layer should be an elastic material such as elastikon or vetrap. many wounds require a pressure relief bandage to prevent contact with the external environment. wounds that may require pressure relief for healing include decubitus ulcers, pressure bandage or cast ulcers, impending ulcer areas (such as the ileum or ischium of recumbent or cachexic patients), and surgical repair sites of ulcerated areas. pressure relief bandages can be of two basic varieties: modified doughnut bandage and doughnut-shaped bandage. to create a cup or clamshell splint, follow this procedure (figures - to - ): . place a nonadherent contact layer directly over the wound. . place stirrups of tape in contact with the skin of the dog, to be placed over the intermediate layer and prevent the bandage from slipping. . place a fairly thick layer of absorbent intermediate bandage material over the contact layer such that the bandage is well-padded. pull the tape stirrups and secure them to the intermediate layer. . place a length of cast material that has been rolled to the appropriate length, such that the cast material is cupped around the patient's paw, and lies adjacent to the caudal aspect of the limb to the level of the carpus or tarsus. in the case of a clamshell splint, place a layer of cast material on the cranial and caudal aspect of the paw and conform it in place. . take the length of cast padding and soak it in warm water after it has been rolled to the appropriate length. wring out the pad, and secure/conform it to the caudal (or cranial and caudal, in the case of a clamshell splint) aspect of the distal limb and paw. . secure the cast material in place with a layer of elastic cotton gauze (kling). . secure the bandage in place with a snug layer of elastikon or vetrap. short or long splints made of cast material can be incorporated into a soft padded bandage to provide extra support of a limb above and below a fracture site. for a caudal or lateral splint to be effective, it must be incorporated for at least one joint above any fracture site to prevent a fulcrum effect and further disruption or damage to underlying soft tissue structures. a short lateral or caudal splint is used for fractures and luxations of the distal metacarpus, metatarsus, carpus, and tarsus. to place a short lateral or caudal splint, follow this procedure: . secure a contact layer as determined by the presence or absence of any wound in the area. . place tape stirrups on the distal extremity to be secured later to the intermediate bandage layer and to prevent slipping of the bandage distally. . place layers of roll cotton from the toes to the level of the mid tibia/fibula or mid radius/ulna. place the layers with even tension, with some overlap of each consecutive layer, moving distally to proximally on the limb. . secure the short caudal or lateral splint and conform it to the distal extremity to the level of the toes and proximally to the level of the mid tibia/fibula or mid radius/ulna. . secure the lateral or caudal splint to the limb with another outer layer of elastic cotton (kling). . cover the entire bandage and splint with an outer tertiary layer of vetrap or elastikon. make sure that the toenails of the third and fourth digits remain visible to allow daily evaluation of circulation. long lateral or caudal splints are used to immobilize fractures of the tibia/fibula and radius/ulna. the splints are fashioned as directed for short splints but extend proximally to the level of the axilla and inguinal regions to immobilize above the fracture site. • packed cell volume drops rapidly to less than % in the dog and less than % to % in the cat • acute loss of more than % of blood volume ( ml/kg in dog, ml/kg in cat) • clinical signs of lethargy, collapse, hypotension, tachycardia, tachypnea (acute or chronic blood loss) • ongoing hemorrhage is present • poor response to crystalloid and colloid infusion • life-threatening hemorrhage caused by thrombocytopenia or thrombocytopathia • surgical intervention is necessary in a patient with severe thrombocytopenia or thrombocytopathia plasma support • life-threatening hemorrhage with decreased coagulation factor activity • severe inflammation (pancreatitis, systemic inflammatory response syndrome) • replenish antithrombin (disseminated intravascular coagulation, protein-losing enteropathy or nephropathy) • surgery is necessary in a patient with decreased coagulation factor activity • severe hypoproteinemia is present; to partially replenish albumin, globulin, and clotting factors type a cats typically possess weak anti-b antibodies of igg and igm subtypes. transfusion of type b blood into a type a cat will result in milder clinical signs of reaction and a markedly decreased survival half-life of the infused rbcs to just days. because type ab cats possess both moieties on their cell surface, they lack naturally occurring alloantibodies; transfusion of type a blood into a type ab cat can be performed safely if a type ab donor is not available. the life span of an rbc from a type-specific transfusion into a cat is approximately days. . indications for fresh whole blood transfusion include disorders of hemostasis and coagulopathies including disseminated intravascular coagulation, von willebrand's disease, and hemophilia. fresh whole blood and platelet-rich plasma also can be administered in cases of severe thrombocytopenia and thrombocytopathia. stored whole blood and packed rbcs can be administered in patients with anemia. if pcv drops to below % or if rapid hemorrhage causes the pcv to drop below % in the dog or less than % to *indicates that this must be done for each donor being tested. minor crossmatch* . obtain a crossmatch segment from blood bank refrigerator for each donor to be crossmatched, or use an edta tube of donor's blood. make sure tubes are labeled prop-erly. . collect ml of blood from recipient and place in an edta tube. centrifuge blood for minutes. . extract blood from donor tubing. centrifuge blood for minutes. use a separate pipette for each transfer because cross-contamination can occur. . pipette plasma off of donor and recipient cells and place in tubes labeled dp and rp, respectively. . place µl of donor and recipient cells in tubes labeled dr and rr, respectively. . add . ml . % sodium chloride solution from wash bottle to each red blood cell (rbc) tube, using some force to cause cells to mix. . centrifuge rbc suspension for minutes. . discard supernatant and resuspend rbcs with . % sodium chloride from wash bottle. . repeat steps and for a total of three washes. . place drops of donor rbc suspension and drops of recipient plasma in tube labeled ma (this is the major crossmatch). . place drops of donor plasma and drops recipient rbc suspension in tube labeled mi (this is the minor crossmatch). . prepare control tubes by placing drops donor plasma with drops donor rbc suspension (this is the donor control); and place drops recipient plasma with drops recipient rbc suspension (this is the recipient control). . incubate major and minor crossmatches and control tubes at room temperature for minutes. . centrifuge all tubes for minute. . read tubes using an agglutination viewer. . check for agglutination and/or hemolysis. . score agglutination with the following scoring scale: + one solid clump of cells + several large clumps of cells + medium-sized clumps of cells with a clear background + hemolysis, no clumping of cells neg = negative for hemolysis; negative for clumping of red blood cells fresh whole blood coagulopathy with active hemorrhage (disseminated intravascular coagulation, thrombocytopenia; massive acute hemorrhage; no stored blood available) stored whole blood massive acute or ongoing hemorrhage; hypovolemic shock caused by hemorrhage that is unresponsive to conventional crystalloid and colloid fluid therapy; unavailability of equipment required to prepare blood components packed red blood cells nonregenerative anemia, immune-mediated hemolytic anemia, correction of anemia before surgery, acute or chronic blood loss fresh frozen plasma factor depletion associated with active hemorrhage (congenital: von willebrand's factor, hemophilia a, hemophilia b; acquired: vitamin k antagonist, rodenticide intoxication, dic); acute or chronic hypoproteinemia (burns, wound exudates, body cavity effusion; hepatic, renal, or gastrointestinal loss); colostrum replacement in neonates frozen plasma acute plasma or protein loss; chronic hypoproteinemia; (contains stable colostrum replacement in neonates; hemophilia b and clotting factors) selected clotting factor deficiencies platelet-rich plasma* thrombocytopenia with active hemorrhage (immune-mediated thrombocytopenia, dic); platelet function abnormality (congenital: thrombasthenia in bassett hounds; acquired: nsaids, other drugs) cryoprecipitate congenital factor deficiencies (routine or before surgery): (concentration of factor hemophilia a, hemophilia b, von willebrand's disease, viii, von willebrand's hypofibrinogenemia; acquired factor deficiencies factor, and fibrinogen) *must be purchased because logistically one cannot obtain enough blood simultaneously to provide a significant amount of platelets; platelets infused have a very short (< hours) half-life. dic, disseminated intravascular coagulation; nsaids, nonsteroidal antiinflammatory drugs. universal donor (e.g., should be administered whenever possible. because there is no universal donor in the cat and because cats possess naturally occurring alloantibodies, all cat blood should be typed and crossmatched before any transfusion. if fresh whole blood is not available, a hemoglobin-based oxygen carrier (oxyglobin, to ml/kg iv) can be administered until blood products become available. table - indicates blood component dose and administration rates. blood products should be warmed slowly to °c before administering them to the patient. blood warmer units are available for use in veterinary medicine to facilitate rapid transfusion without decreasing patient body temperature (thermal angel; enstill medical technologies, inc., dallas, texas). red blood cell and plasma products should be administered in a blood administration set containing a -µm in-line filter. smaller in-line filters ( µm) also can be used in cases in which extremely small volumes are to be administered. blood products should be administered over a period of hours, whenever possible, according to guidelines set by the american association of blood banks. the volume of blood components required to achieve a specific increment in the patient's pcv depends largely on whether whole blood or packed rbcs are transfused and whole blood ml/kg will increase max rate: ml/kg/ max: ml/kg/ volume by % hours hour packed red ml/kg will increase critically ill blood cells volume by % patients (e.g., cardiac failure or renal failure): - ml/kg/hour fresh frozen ml/kg body mass (repeat - ml/minute or use rates as for plasma in - days or in - days whole blood (infuse within - hours) or until bleeding stops); monitor act, aptt, and pt before and hour after transfusion cryoprecipitate general: unit/ kg/ hours - ml/minute or use rates as for whole or until bleeding stops blood (infuse within - hours) hemophilia a: - units factor viii/kg; unit of cryoprecipitate contains approximately units of factor viii platelet-rich unit/ kg ( unit of ml/minute plasma platelet-rich plasma will check platelet count before and hour increase platelet count after transfusion hour after transfusion by , /µl) whether there is ongoing hemorrhage or rbc destruction. because the pcv of packed rbcs is unusually high ( % for greyhound blood), a smaller total volume is required than whole blood to achieve a comparable increase in the patient's pcv. in general, ml/kg of packed rbcs or ml/kg whole blood will raise the recipient's pcv by %. the "rule of ones" states that ml per lb of whole blood will raise the pcv by %. if the patient's pcv does not raise by the amount anticipated by the foregoing calculation(s), causes of ongoing hemorrhage or destruction should be considered. the goal of red blood component therapy is to raise the pcv to % to % in dogs and % to % in cats. if an animal is hypovolemic and whole blood is administered, the fluid is redistributed into the extravascular compartment within hours of transfusion. this will result in a secondary rise in the pcv hours after the transfusion in addition to the initial rise to hours after the rbc transfusion is complete. the volume of plasma transfused depends largely on the patient's need. in general, plasma transfusion should not exceed more than ml/kg during a -hour period for normovolemic animals. thaw plasma at room temperature, or place it in a ziplock freezer bag and run under cool (not warm) water until thawed. then administer the plasma through a blood administration set that contains an in-line blood filter or through a standard driptype administration set with a detachable in-line blood administration filter. the average rate of plasma infusion in a normovolemic patient should not exceed ml/kg/hour. in acute need situations, plasma can be delivered at rates up to to ml/kg/minute. for patients with cardiac insufficiency or other circulatory problems, plasma infusion rates should not exceed ml/kg/hour. plasma or other blood products should not be mixed with or used in the same infusion line as calcium-containing fluids, including lactated ringer's solution, calcium chloride, or calcium gluconate. the safest fluid to mix with any blood product is . % sodium chloride. administer fresh frozen plasma, frozen plasma, and cryoprecipitate at a volume of ml/kg until bleeding is controlled or source of ongoing albumin loss ceases. the goal of plasma transfusion therapy is to raise the albumin to a minimum of . g/dl or until bleeding stops as in the case of coagulopathies. monitor the patient to ensure that bleeding has stopped, coagulation profiles (act, aptt, and pt) have normalized, hypovolemia has stabilized, and/or total protein is normalizing, which are indications for discontinuing ongoing transfusion therapy. plasma cryoprecipitate can be purchased or manufactured through the partial thawing and then centrifugation of fresh frozen plasma. cryoprecipitate contains concentrated quantities of vwf, factor viii, and fibrinogen and is indicated in severe forms of von willebrand's disease and hemophilia a (factor viii deficiency). platelet-rich plasma must be purchased from a commercial source. one unit of fresh whole blood contains to platelets. the viability of the platelets contained in the fresh whole blood is short-lived, just to hours after transfusion into the recipient. because platelet-rich plasma is difficult to obtain, animals with severe thrombocytopenia or thrombocytopathia should be treated with immunomodulating therapies and the administration of fresh frozen plasma. in dogs, blood and plasma transfusions can be administered intravenously or intraosseously. the cephalic, lateral saphenous, medial saphenous, and jugular veins are used most commonly. fill the recipient set so that the blood in the drip chamber covers the filter (normal -µm filter). with small amounts of blood ( ml) or critically ill patients, use a -µm filter. avoid latex filters for plasma and cryoprecipitate administration. blood can emergency care be administered at variable rates, but the routine figure of to ml/minute often is used. normovolemic animals can receive blood at ml/kg/day. dogs in heart failure should receive infusions at no more than ml/kg/hour. volume is given as needed. to calculate the approximate volume of blood needed to raise hematocrit levels, use the following formula for the dog: anticoagulated blood volume (ml) = body mass (kg) × × pcv desired − pcv of recipient pcv of donor in anticoagulant an alternative formula is the following: . × recipient body mass (kg) × (dog) × pcv desired − pcv of recipient pcv of donor in anticoagulant surgical emergencies and shock may require several times this volume within a short period. if greater than % of the patient's blood volume is lost, supplementation with colloids, crystalloids, and blood products is indicated for fluid replacement. one volume of whole blood achieves the same increase in plasma as two to three volumes of plasma. if the patient's blood type is unknown and type a-negative whole blood is not available, any dog blood can be administered to a dog in acute need if the dog has never had a transfusion before. if mismatched blood is given, the patient will become sensitized, and after days, destruction of the donor rbcs will begin. in addition, any subsequent mismatched transfusions may cause an immediate reaction (usually mild) and rapid destruction of the transfused rbcs. the clinical signs of a transfusion reaction typically only are seen when type a blood is administered to a type a-negative recipient that has been sensitized previously. incompatible blood transfusions to breeding females can result in isoimmunization and in hemolytic disease in the puppies. the a-negative bitch that receives a transfusion with a-positive and that produces a litter from an a-positive stud can have puppies with neonatal isoerythrolysis. cats with severe anemia in need of a blood transfusion are typically extremely depressed, lethargic, and anorexic. the stress of restraint and handling can push these critically ill patients over the edge and cause them to die. extreme gentleness and care are mandatory in restraint and handling. the critically ill cat should be cradled in a towel or blanket. supplemental flow-by or mask oxygen should be administered, whenever possible, although it may not be clinically helpful until oxygen-carrying capacity is replenished with infusion of rbcs or hemoglobin. blood can be administered by way of cephalic, medial saphenous, or the jugular vein. intramedullary infusion is also possible, if vascular access cannot be accomplished. the average -to -kg cat can accept to ml of whole blood injected intravenously over a period of to minutes. administer filtered blood at a rate of to ml/kg/hour. the following formula can be used to estimate the volume of blood required for transfusion in a cat: anticoagulated blood volume (ml) = body mass (kg) × × pcv desired − pcv of recipient pcv of donor in anticoagulant the exact overall incidence and clinical significance of transfusion reactions in veterinary medicine are unknown. several studies have been performed that document the incidence of transfusion reactions in dogs and cats. overall, the incidence of transfusion reactions in dogs and cats is . % and %, respectively. transfusion reactions can be immune-mediated and non-immune-mediated and can happen immediately or can be delayed until after a transfusion. acute reactions usually occur within minutes to hours of the onset of transfusion but may occur up to hours after the transfusion has been stopped. acute immunologic reactions include hemolysis and acute hypersensitivity including rbcs, platelets, and leukocytes. signs of a delayed immunologic reaction include hemolysis, purpura, immunosuppression, and neonatal isoerythrolysis. acute nonimmunologic reactions include donor cell hemolysis before onset of transfusion, circulatory volume overload, bacterial contamination, citrate toxicity with clinical signs of hypocalcemia, coagulopathies, hyperammonemia, hypothermia, air embolism, acidosis, and pulmonary microembolism. delayed nonimmunologic reactions include the transmission and development of infectious diseases and hemosiderosis. clinical signs of a transfusion reaction typically depend on the amount of blood transfused, the type and amount of antibody involved in the reaction, and whether the recipient has had previous sensitization. monitoring the patient carefully during the transfusion period is essential in recognizing early signs of a transfusion reaction, including those that may become life threatening. a general guideline for patient monitoring is first to start the transfusion slowly during the first minutes. monitor temperature, pulse, and respiration every minutes for the first hour, hour after the end of the transfusion, and every hours minimally thereafter. also obtain a pcv immediately before the transfusion, hour after the transfusion has been stopped, and every hours thereafter. monitor coagulation parameters such as an act and platelet count at least daily in patients requiring transfusion therapy. the most common documented clinical signs of a transfusion reaction include pyrexia, urticaria, salivation/ptyalism, nausea, chills, and vomiting. other clinical signs of a transfusion reaction may include tachycardia, tremors, collapse, dyspnea, weakness, hypotension, collapse, and seizures. severe intravascular hemolytic reactions may occur within minutes of the start of the transfusion, causing hemoglobinemia, hemoglobinuria, disseminated intravascular coagulation, and clinical signs of shock. extravascular hemolytic reactions typically occur later and will result in hyperbilirubinemia and bilirubinuria. pretreatment of patients to help decrease the risk of a transfusion reaction remains controversial, and in most cases, pretreatment with glucocorticoids and antihistamines is ineffective at preventing intravascular hemolysis and other reactions should they occur. the most important component of preventing a transfusion reaction is to screen each recipient carefully and process the donor component therapy carefully before the administration of any blood products. treatment of a transfusion reaction depends on its severity. in all cases, stop the transfusion immediately when clinical signs of a reaction occur. in most cases, discontinuation of the transfusion and administration of drugs to stop the hypersensitivity reaction will be sufficient. once the medications have taken effect, restart the transfusion slowly and monitor the patient carefully for further signs of reaction. in more severe cases in which a patient's cardiovascular or respiratory system become compromised and hypotension, tachycardia, or tachypnea occurs, immediately discontinue the transfusion and administer diphenhydramine ( mg/kg im), dexamethasone-sodium phosphate ( . to . mg/kg iv), and epinephrine to the patient. the patient should have a urinary catheter and central venous catheter placed for measurement of urine output and central venous pressures. aggressive fluid therapy may be necessary to avoid renal insufficiency or renal damage associated with severe intravascular hemolysis. overhydration with subsequent pulmonary edema generally can be managed with supplemental oxygen administration and intravenous or intramuscular administration of furosemide ( to mg/kg). plasma products with or without heparin can be administered for disseminated intravascular coagulation. the hbocs can be stored at room temperature and have a relatively long shelf life compared with red blood component products. the hbocs function to carry oxygen through the blood and can diffuse oxygen past areas of poor tissue perfusion. an additional characteristic of hbocs is as a potent colloid, serving to maintain fluid within the vascular space. for this reason, hbocs must be used with caution in euvolemic patients and patients with cardiovascular insufficiency. central venous pressure (cvp) measures the hydrostatic pressure in the anterior vena cava and is influenced by vascular fluid volume, vascular tone, function of the right side of the heart, and changes in intrathoracic pressure during the respiratory cycle. the cvp is not a true measure of blood volume but is used to gauge fluid therapy as a method of determining how effectively the heart can pump the fluid that is being delivered to it. thus the cvp reflects the interaction of the vascular fluid volume, vascular tone, and cardiac function. measure cvp in any patient with acute circulatory failure, large volume fluid diuresis (i.e., toxin or oliguric or anuric renal failure), fluid in-and-out monitoring, and cardiac dysfunction. the placement of central venous catheters and thus cvp measurements is contraindicated in patients with known coagulopathies including hypercoagulable states. to perform cvp monitoring, place a central venous catheter in the right or left jugular vein. in cats and small dogs, however, a long catheter placed in the lateral or medial saphenous vein can be used for trends in cvp monitoring. first, assemble the equipment necessary for jugular catheter (see vascular access techniques for how to place a jugular or saphenous long catheter) and cvp monitoring (box - ). after placing the jugular catheter, take a lateral thoracic radiograph to ensure that the tip of the catheter sits just outside of the right atrium for proper cvp measurements (see to establish an intravenous catheter for cvp, follow this procedure: . assemble the cvp setup such that the male end of a length of sterile intravenous catheter extension tubing is inserted into the t port of the jugular or medial/lateral saphenous catheter. make sure to flush the length of tubing with sterile saline before connecting it to the patient to avoid iatrogenic air embolism. . next, insert the male end of a three-way stopcock into the female end of the extension tubing. . attach a -ml syringe filled with heparinized sterile . % saline to one of the female ports of the three-way stopcock and either a manometer or a second length of intravenous extension tubing attached to a metric ruler. . lay the patient in lateral or sternal recumbancy. . turn the stopcock off to the manometer/ruler and on to the patient. infuse a small amount of heparinized saline through the catheter to flush the catheter. . next, turn the stopcock off to the patient and on to the manometer. gently flush the manometer or length of extension tubing with heparinized saline from the syringe. use care not to agitate the fluid and create air bubbles within the line or manometer that will artifactually change the cvp measured. . next, lower the cm point on the manometer or ruler to the level of the patient's manubrium (if the patient is in lateral recumbancy) or the point of the elbow (if the patient is in sternal recumbancy). . turn the stopcock off to the syringe, and allow the fluid column to equilibrate with the patient's intravascular volume. once the fluid column stops falling and the level rises and falls with the patient's heartbeat, measure the number adjacent to the bottom of the meniscus of the fluid column. this is the cvp in centimeters of water (see figure - ). . repeat the measurement several times with the patient in the same position to make sure that none of the values has been increased or decreased artifactually in error. alternately, attach the central catheter to a pressure transducer and perform electronic monitoring of cvp. there is no absolute value for normal cvp. the normal cvp for small animal patients is to cm h o. values less than zero are associated with absolute or relative hypovolemia. values of to cm h o are borderline hypervolemia, and values greater than cm h o suggest intravascular volume overload. values greater than cm h o may be correlated with congestive heart failure and the development of pulmonary edema. in individual patients, the trend in change in cvp is more important than absolute values. as a rule of thumb, when using cvp measurements to gauge fluid therapy and avoid vascular and pulmonary overload, the cvp should not increase by more than cm h o in any -hour period. if an abrupt increase in cvp is found, repeat the measurement to make sure that the elevated value was not obtained in error. if the value truly has increased dramatically, temporarily discontinue fluid therapy and consider administration of a diuretic. delaforcade am, rozanski ea: central venous pressure and arterial blood pressure measurements, vet clin north am small anim pract ( ) the diagnosis of intracellular fluid deficit is difficult and is based more on the presence of hypernatremia or hyperosmolality than on clinical signs. an intracellular fluid deficit is expected when free water loss by insensible losses and vomiting, diarrhea, or urine is not matched by free water intake. consideration of the location of the patient's fluid deficit, history of vomiting and diarrhea, no visible clinical signs of deficit % dry mucous membranes, mild skin tenting % increased skin tenting, dry mucous membranes, mild tachycardia, normal pulse* % increased skin tenting, dry mucous membranes, tachycardia, weak pulse pressure % increased skin tenting, dry corneas, dry mucous membranes, % elevated or decreased heart rate, poor pulse quality, altered level of consciousness* the respiratory system further contributes to acid-base status by changes in the elimination of carbon dioxide. hyperventilation decreases the blood pco and causes a respiratory alkalosis. hypoventilation increases the blood pco and causes a respiratory acidosis. depending on the altitude, the pco in dogs can range from to mm hg. in cats, normal is to mm hg. venous pco values are to mm hg in dogs and to mm hg in cats. use a systematic approach whenever attempting to interpret a patient's acid-base status. ideally, obtain an arterial blood sample so that you can monitor the patient's oxygenation and ventilation. once an arterial blood sample has been obtained, follow these steps: . determine whether the blood sample is arterial or venous by looking at the oxygen saturation (sao ). the sao should be greater than % if the sample is truly arterial, although it can be as low as % if a patient has severe hypoxemia. . consider the patient's ph. if the ph is outside of the normal range, an acid-base disturbance is present. if the ph is within the normal range, an acid-base disturbance may or may not be present. if the ph is low, the patient is acidotic. if the ph is high, the patient is alkalotic. . next, look at the base excess or deficit. if the base excess is increased, the patient has higher than normal bicarbonate. if there is a base deficit, the patient may have a low bicarbonate or increase in unmeasured anions (e.g., lactic acid or ketoacids). . next, look at the bicarbonate. if the ph is low and the bicarbonate is low, the patient has a metabolic acidosis. if the ph is high and the bicarbonate is elevated, the patient has a metabolic alkalosis. . next, look at the paco . if the patient's ph is low and the paco is elevated, the patient has a respiratory acidosis. if the patient's ph is high and the paco is low, the patient has a respiratory alkalosis. . finally, if you are interested in the patient's oxygenation, look at the pao . normal pao is greater than mm hg. the metabolic acidosis early in renal failure may be hyperchloremic and later may convert to typical increased anion gap acidosis. . next, you must determine whether the disorders present are primary disorders or an expected compensation for disorders in the opposing system. for example, is the patient retaining bicarbonate (metabolic alkalosis) because of carbon dioxide retention (respiratory acidosis)? use the chart in table - to evaluate whether the appropriate degree of compensation is occurring. if the adaptive response falls within the expected range, a simple acid-base disorder is present. if the response falls outside of the expected range, a mixed acid-base disorder is likely present. . finally, you must determine whether the patient's acid-base disturbance is compatible with the history and physical examination findings. if the acid-base disturbance does not fit with the patient's history and physical examination abnormalities, question the results of the blood gas analyses and possibly repeat them. the most desirable method of assessing the acid-base status of an animal is with a blood gas analyzer. arterial samples are preferred over venous samples, with heparin used as an anticoagulant (table - ) . potassium primarily is located in the intracellular fluid compartment. serum potassium is regulated by the actions of the sodium-potassium-adenosinetriphosphatase pump on cellular membranes, including those of the renal tubular epithelium. inorganic metabolic acidosis artifactually can raise serum potassium levels because of redistribution of extracellular potassium in exchange for intracellular hydrogen ion movement in an attempt to correct serum ph. metabolic acidosis potassium is one of the major players in the maintenance of resting membrane potentials of excitable tissue, including neurons and cardiac myocytes. changes in serum potassium can affect cardiac conduction adversely. hyperkalemia lowers the resting membrane potential and makes cardiac cells, particularly those of the atria, more susceptible to depolarization. characteristic signs of severe hyperkalemia that can be observed on an ecg rhythm strip include an absence of p waves, widened qrs complexes, and tall tented or spiked t waves. further increases in serum potassium can be associated with bradycardia, ventricular fibrillation, and cardiac asystole (death). treatment of hyperkalemia consists of administration of insulin ( . to . units/kg, iv regular insulin) and dextrose ( g dextrose per unit of insulin administered, followed by . % dextrose iv cri to prevent hypoglycemia), calcium ( to ml of % calcium gluconate administered iv slowly to effect), or sodium bicarbonate ( meq/kg, iv slowly). insulin plus dextrose and bicarbonate therapy help drive the potassium intracellularly, whereas calcium antagonizes the effect of hyperkalemia on the myocardial cells. all of the treatments work within minutes, although the effects are relatively short-lived ( minutes to hour) unless the cause of the hyperkalemia is identified and treated appropriately (box - ). dilution of serum potassium also results from restoring intravascular fluid volume and correcting metabolic acidosis, in most cases. treatment with a fluid that does not contain potassium (preferably . % sodium chloride) is recommended. hypokalemia elevates the resting membrane potential and results in cellular hyperpolarization. hypokalemia may be associated with ventricular dysrhythmias, but the ecg changes are not as characteristic as those observed with hyperkalemia. causes of hypokalemia include renal losses, anorexia, gastrointestinal loss (vomiting, diarrhea), intravenous fluid diuresis, loop diuretics, and postobstructive diuresis (box - ). if the serum potassium concentration is known, potassium supplementation in the form of potassium chloride or potassium phosphate can be added to the patient's intravenous fluids. correct serum potassium levels less than . meq/l or greater than . meq/l. potassium rates should not exceed . meq/kg/hour (table - ) . metabolic acidosis from bicarbonate depletion often corrects itself with volume restoration in most small animal patients. patients with moderate to severe metabolic acidosis may benefit from bicarbonate supplementation therapy. the metabolic contribution to acid-base balance is identified by measuring the total carbon dioxide concentration or calculating the bicarbonate concentration. if these measurements are not available, the degree of expected metabolic acidosis can be estimated subjectively by the severity of underlying disease that often contributes to metabolic acidosis: hypovolemic or traumatic shock, septic shock, diabetic ketoacidosis, or oliguric/anuric renal failure. if the metabolic acidosis is estimated to be mild, moderate, or severe, add sodium bicarbonate at , , and meq/kg body mass, respectively. patients with diabetic ketoacidosis may not require bicarbonate administration once volume replacement and perfusion is restored, and the ketoacids are metabolized to bicarbonate. if the bicarbonate measurement of base deficit is known, the following formula can be used as a gauge for bicarbonate supplementation: base deficit × . = body mass (kg) = meq bicarbonate to administer osmolality osmolality is measured by freezing point depression or a vapor pressure osmometer, or it may be calculated by the following formula: mosm/kg = [(na + ) + (k + )] + bun/ . + glucose/ where sodium and potassium are measured in milliequivalents, and bun and glucose are measured in milligrams per deciliter. osmolalities less than mosm/kg or greater emergency care than mosm/kg are serious enough to warrant therapy. the difference between the measured osmolality and the calculated osmolality (the osmolal gap) should be less than mosm/kg. if the osmolal gap is greater than mosm/kg, consider the presence of unmeasured anions such as ethylene glycol metabolites. the volume of extracellular fluid is determined by the total body sodium content, whereas the osmolality and sodium concentration are determined by water balance. serum sodium concentration is an indication of the amount of sodium relative to water in the extracellular fluid and provides no direct information about the total body sodium content. unlikely to cause hyperkalemia in presence of normal renal function unless iatrogenic (e.g., continuous infusion of potassium-containing fluids at an excessively rapid rate) acute mineral acidosis (e.g., hydrochloric acid or ammonium chloride) insulin deficiency (e.g., diabetic ketoacidosis) acute tumor lysis syndrome reperfusion of extremities after aortic thromboembolism in cats with cardiomyopathy hyperkalemic periodic paralysis (one case report in a pit bull) mild hyperkalemia after exercise in dogs with induced hypothyroidism infusion of lysine or arginine in total parenteral nutrition solutions nonspecific β-blockers (e.g., propranolol)* cardiac glycosides (e.g., digoxin)* urethral obstruction ruptured bladder anuric or oliguric renal failure hypoadrenocorticism selected gastrointestinal disease (e.g., trichuriasis, salmonellosis, or perforated duodenal ulcer) late pregnancy in greyhound dogs (mechanism unknown but affected dogs had gastrointestinal fluid loss) chylothorax with repeated pleural fluid drainage hyporeninemic hypoaldosteronism † angiotensin-converting enzyme inhibitors (e.g., enalapril)* angiotensin receptor blockers (e.g., losartan)* cyclosporine and tacrolimus* potassium-sparing diuretics (e.g., spironolactone, amiloride, and triamterene)* nonsteroidal antiinflammatory drugs* heparin* trimethoprim* from dibartola sp: fluid, electrolyte and acid-base disorders in small animal practice, st louis, , saunders. *likely to cause hyperkalemia only in conjunction with other contributing factors (e.g., other drugs, decreased renal function, or concurrent administration of potassium supplements). † not well documented in veterinary medicine. if refractory hypokalemia is present, supplement magnesium at . meq/kg/day for hours. alone unlikely to cause hypokalemia unless diet is aberrant administration of potassium-free (e.g., . % sodium chloride or % dextrose in water) or potassium-deficient fluids (e.g., lactated ringer's solution over several days) bentonite clay ingestion (e.g., cat litter) alkalemia insulin/glucose-containing fluids catecholamines hypothermia hypokalemic periodic paralysis (burmese cats) albuterol overdosage patients with hyponatremia or hypernatremia may have decreased, normal, or increased total body sodium content (boxes - and [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ). an increased serum sodium concentration implies hyperosmolality, whereas a decrease in serum sodium concentration usually, but not always, implies hypoosmolality. the severity of clinical signs of hypernatremia and hyponatremia is related primarily to the rapidity of the onset of the change rather than to the magnitude of the associated plasma hyperosmolality or hypoosmolality. clinical signs of neurologic disturbances include disorientation, ataxia, and seizures, and coma may occur at serum sodium concentrations less than meq/l or greater than meq/l in dogs. therapy of hypernatremia or hyponatremia with fluid containing low or higher concentrations of sodium should proceed with caution, for rapid changes (decreases or increases) of serum sodium and osmolality can cause rapid changes in the intracellular and extracellular fluid flux, leading to intracellular dehydration or edema, even though the serum sodium has not been returned to normal. a rule of thumb is to not raise or lower the serum sodium by more than meq/l during any one -hour period. restoration of the serum sodium concentration over a period of to hours is better. in almost all circumstances, an animal will correct its sodium balance with simple fluid restoration. if severe hypernatremia exists that suggests a free water deficit, however, the free water deficit should be calculated from the following formula: hypernatremia can be corrected slowly with . % sodium chloride plus . % dextrose, % dextrose in water, or lactated ringer's solution (sodium content: meq/l). correct hyponatremia initially with . % sodium chloride. sodium is balanced predominantly by chloride and bicarbonate. the difference between these concentrations, (na , has been called the anion gap. the normal anion gap is between and meq/l. when the anion gap exceeds , consider the possibility of an accumulation of unmeasured anions (e.g., lactate, ketoacids, phosphate, sulfate, ethylene glycol metabolites, and salicylate). abnormalities in the anion gap may be helpful in determining the cause of metabolic acidosis (boxes - and - ). the colloid oncotic pressure of blood is associated primarily with large-molecular-weight colloidal substances in circulation. the major player in maintaining intravascular and interstitial oncotic pressure, the water-retaining property of each fluid compartment, is albumin. albumin contributes roughly % to the colloidal oncotic pressure of blood. the majority of albumin is located within the interstitial space. hypoalbuminemia can result from increased loss in the form of protein-losing enteropathy or nephropathy and wound exudates, or it may be due to lack of hepatic albumin synthesis. serum albumin pools are in a constant flux with interstitial albumin. once interstitial albumin pools become depleted from replenishing serum albumin, serum albumin levels can continue to decrease, which can lead to a decrease in colloidal oncotic pressure. serum albumin less than . g/dl has been associated with inadequate intravascular fluid retention and the development of peripheral edema and third spacing of fluid. oncotic pressure can be restored with the use of artificial or synthetic colloids or natural colloids (see colloids). maintenance fluid requirements have been extrapolated from the formulas used to calculate a patient's daily metabolic energy requirements because it takes ml of water to metabolize kcal of energy (table - ) . the patient's daily metabolic water (fluid) requirements can be calculated by the following formula: administration of an isotonic crystalloid fluid for maintenance requirements often can produce iatrogenic hypokalemia. in most cases, supplemental potassium must be added to prevent hypokalemia resulting from inappetance, kalliuresis, and supplementation with isotonic crystalloid fluids. the most reliable method of determining the degree of fluid deficit is by weighing the animal and calculating acute weight loss. acute weight loss in a patient with volume loss in the form of vomiting, feces, wound exudates, and urine is due to fluid loss and not loss of muscle or fat. lean body mass normally is not gained or lost rapidly enough to cause major changes in body weight. one milliliter of water weighs approximately g. this fact allows calculation of the patient's fluid deficit, if ongoing losses can be measured. when a patient first presents, however, the body weight before a fluid deficit has occurred rarely is known. instead, one must rely on subjective measures of dehydration to estimate the patient's percent dehydration and to calculate the volume of fluid required to rehydrate the patient over the next hours. to calculate the volume deficit, use the following formula: body mass (kg) × (% dehydration) × = fluid deficit (ml) the patient's fluid deficit must be added to the daily maintenance fluid requirements and administered over a -hour period. ongoing losses can be determined by measuring urine output, weighing the patient at least to times a day, and measuring the volume or weight of vomitus or diarrhea. a crystalloid fluid contains crystals of salts with a composition similar to that of the extracellular fluid space and can be used to maintain daily fluid requirements and replace fluid deficits or ongoing fluid losses (table - ) . metabolic, acid-base, and electrolyte imbalances also can be treated with isotonic fluids with or without supplemental electrolytes and buffers. depending on the patient's clinical condition, choose the specific isotonic crystalloid fluid to replace and maintain the patient's acid-base and electrolyte status ( table - ) . crystalloid fluids are readily available, are relatively inexpensive, and can be administered safely in large volumes to patients with no preexisting cardiac or renal disease or cerebral edema. following infusion, approximately % of the volume of a crystalloid fluid infused will redistribute to the interstitial fluid compartment. as such, crystalloid fluids alone are ineffective for ongoing intravascular volume depletion when given as a bolus. the crystalloid fluid bolus must be followed by a constant rate infusion, taking into consideration the patient's daily maintenance fluid requirements and ongoing fluid losses. administration of a large volume of crystalloid fluids can cause dilutional anemia and coagulopathies. * × bw kg + = kcal/day = ml/day. note: this formula will slightly underestimate the requirements for patients that are less than kg and will slightly overestimate the requirements for patients greater than kg. retain fluid in the vascular space, the volume of crystalloid fluid infused (maintenance + deficit + ongoing losses) should be decreased by % to % to avoid vascular volume overload. two major classes of colloids exist: natural and synthetic. natural colloids (whole blood, packed rbcs, plasma) are discussed elsewhere in this text. concentrated human albumin is a natural purified colloid that recently has become more popular in the treatment of advanced hypoalbuminemia and hypoproteinemia and will be discussed here. synthetic colloids are starch polymers and include dextrans and hetastarch. concentrated human albumin is available as a % or % solution. the % solution has an osmolality similar to that of serum ( mosm/l), whereas the % solution is hyperoncotic ( mosm/l). a % albumin solution draws fluid from the interstitial space into the intravascular space. concentrated albumin solutions often are used to restore circulating volume when synthetic colloids are not available. albumin not only is important at maintaining the colloidal oncotic pressure of blood but also serves as a valuable free-radical scavenger and carrier of drugs and hormones necessary for normal tissue function and healing. albumin levels less than . g/dl have been associated with increased morbidity and mortality. concentrated human albumin solutions can be administered as an effective method of restoring interstitial and serum albumin concentrations in situations of acute and chronic hypoalbuminemia. albumin ( %) is available in -and -ml vials and is more cost-efficient as an albumin replacement than procurement and administration of fresh frozen plasma. recommended albumin infusion rates are to ml/kg over hours, after pretreatment with diphenhydramine. although concentrated human albumin is structurally similar to canine albumin, closely monitor the patient for signs of allergic reaction during and after the infusion. dextran- is a synthetic high-molecular-weight polysaccharide (sucrose polymer) with a molecular weight of , d. particles less than , d, are cleared rapidly by the kidneys, whereas larger particles are cleared more slowly by the hepatic reticuloendothelial system. dextran- can coat platelets and inhibit platelet function and so must be used with caution in patients with known coagulopathies. the total daily dosage should not exceed ml/kg/day. hetastarch (hydroxyethyl starch) is a large-molecular-weight amylopectin polymer, has molecules with a molecular weight that exceeds , d, and has an average half-life of to hours in circulation. hetastarch can bind with vwf and cause prolongation of the act and aptt; however, it does not cause a coagulopathy. recommended rates of hetastarch infusion are -to -ml incremental boluses for the treatment of hypotension and to ml/kg/day as a constant rate infusion for maintenance of colloidal oncotic pressure. many are the acceptable ways to administer the fluids prescribed for each patient based on the degree of dehydration, estimation of ongoing losses, ability to tolerate oral fluid, and metabolic, acid-base, and electrolyte derangements. administer the fluids in a manner that is best for the patient and most appropriate for the practice. to determine the rate of intravenous fluid infusion, take the total volume of fluids that have been prescribed and divide the total volume by the total number of hours in a day that intravenous fluids can be delivered safely and monitored. the safest and most accurate way to deliver intravenous fluids, particularly in extremely small animals or those with congestive heart failure, is through an intravenous fluid pump. fluid should not be administered intravenously if the patient cannot be monitored to make sure that the fluids are being delivered at a safe rate and that the fluid line has not become disconnected. supplement fluids over as many hours as possible to allow the patient as much time as possible to redistribute and fully utilize the fluids administered. fluids administered too quickly can cause a diuresis to occur, such that the majority of the fluids administered will be excreted in the urine. if time is limited or if extra time is needed for safe administration of fluids, consider using a combination of intravenously and subcutaneously emergency care administered fluids. intravenous is the preferred route of administration of fluids in any patient with dehydration and hypovolemia. as intravascular volume depletion occurs, reflex peripheral vasoconstriction occurs to restore core perfusion. the subcutaneous tissue are not perfused well and therefore fluids administered subcutaneously will not be absorbed well into the interstitial and intravascular spaces. subcutaneously administered fluids can be absorbed slowly and delivered effectively in the management of mild interstitial dehydration and in the treatment of renal insufficiency. subcutaneously administered fluids should never take the place of intravenously administered fluids in a hypovolemic patient or one with severe interstitial dehydration. intramedullary (intraosseous) infusion works well in small patients in which vascular access cannot be established. shock doses of fluids and other substances, including blood products, can be administered under pressure through an intraosseous cannula. because of the inherent discomfort and risk of osteomyelitis with intraosseous infusion, establish vascular access as soon as possible. the safest and most efficient method of intravenous fluid infusion is through a fluid pump. in cases in which a fluid pump is unavailable, infusion by gravity feed is the next option. infusion sets from various manufacturers have calibrated drip chambers such that a specific number of drops will equal ml of fluid. fluid rates can be calculated based on the number of drops that fall into the drip chamber per minute: fluid volume to be infused (ml) = ml/hour number of hours available many pediatric drip sets deliver drops/ml, such that milliliters/hour equals drops/ minute. carefully record fluid orders so that the volume to be administered is recorded as milliliters/hour, milliliters/day, and drops/minute. this will allow personnel to detect major discrepancies and calculation errors more readily. the volume actually delivered should be recorded in the record by nursing personnel. all additives should be listed clearly on the bottle on a piece of adhesive tape or a special label manufactured for this purpose. a strip of adhesive tape also can be attached to the bottle and marked appropriately to provide a quick visualization of the estimate of volume delivered. includes a large-bore flexible orogastric lavage tube, permanent marker or white tape, lubricating jelly, warm water, two large buckets, a roll of -inch white tape, and a manual lavage pump. to perform the orogastric lavage, follow this procedure: . place all animals under general anesthesia with a cuffed endotracheal tube in place to protect the airway and prevent aspiration of gastric contents into the lungs. . place a roll of -inch white tape into the animal's mouth, and secure the tape around the muzzle. you will insert the tube through the hole in the center of the roll of tape. . next, place the distal end of the tube at the level of the last rib, directly adjacent to the animal's thorax and abdomen. measure the length of the tube from the most distal end to the point where it comes out of the mouth, and label this location on the tube with a permanent marker or piece of white tape. . lubricate the distal portion of the tube, and gently insert it through the roll of tape in the animal's mouth. . gently push the tube down the esophagus. palpate the tube within the esophagus. two tubes should be palpable, the orogastric tube, and the patient's trachea. push the tube down into the stomach. you can verify location by blowing into the proximal end of the tube and simultaneously auscultating the stomach for borborygmi. . insert the manual pump to the proximal end of the tube, and instill the warm water. alternate instilling water with removal of fluid and gastric debris by gravity. repeat the process until the efflux fluid is clear of any debris. . save fluid from the gastric efflux fluid for toxicologic analyses. hackett tb: emergency approach to intoxications, clin tech small anim pract ( ): - , . hypoxia, or inadequate tissue oxygenation, is the primary reason for supplemental oxygen therapy. major causes of hypoxia include hypoventilation, ventilation-perfusion mismatch, physiologic or right-to-left cardiac shunt, diffusion impairment, and decreased fraction of inspired oxygen (table - ) . inadequate tissue perfusion caused by low cardiac output or vascular obstruction also can result in circulatory hypoxia. finally, histiocytic hypoxia results from inability of cells to use oxygen that is delivered to them. this form of hypoxia can be observed with various toxin ingestions (bromethalin, cyanide) and in septic shock. a patient's oxygenation status can be monitored invasively by drawing of arterial blood gas samples or noninvasively through pulse oximetry, in most cases (see acid-base physiology and pulse oximetry). inspired air at sea level has a po of mm hg. as the air travels through the upper respiratory system to the level of the alveolus, the po drops to mm hg. tissue oxygen saturation in a normal healthy animal is mm hg. after oxygen has been delivered to the tissues, the oxygen left in the venous system (pvo ) is approximately mm hg. normally, oxygen diffuses across the alveolar capillary membrane and binds reversibly with hemoglobin in rbcs. a small amount of oxygen is carried in an unbound diffusible form in the plasma. when an animal has an adequate amount of hemoglobin and hemoglobin becomes fully saturated while breathing room air, supplemental oxygen administration will only increase the sao a small amount. the unbound form of oxygen dissolved in plasma will increase. if, however, inadequate hemoglobin saturation is obtained by breathing room air, as in a case of pneumonia or pulmonary edema, for example, breathing a higher fraction of inspired oxygen (fio ) will improve bound and unbound hemoglobin levels. the formula for calculating oxygen content of arterial blood is as follows: where cao is the arterial oxygen content, . is the amount of oxygen that can be carried by hemoglobin (hb), sao is the hemoglobin saturation, and . × pao is the amount of oxygen dissolved (unbound) in plasma. dissolved oxygen actually contributes little to the total amount of oxygen carried in the arterial blood, and the majority depends on the amount or availability of hemoglobin and the ability of the body (ph and respiratory status) to saturate the hemoglobin at the level of the alveoli. oxygen therapy is indicated whenever hypoxia is present. the underlying cause of the hypoxia also must be identified and treated, for chronic, lifelong oxygen therapy is rarely feasible in veterinary patients. if hemoglobin levels are low due to anemia, oxygen supplementation must occur along with rbc transfusions to increase hemoglobin mass. whenever possible, use arterial blood gas analyses or pulse oximetry to gauge a patient's response to oxygen therapy and to determine when an animal can be weaned from supplemental oxygen. the goal of oxygen therapy is to increase the amount of oxygen bound to hemoglobin in arterial blood. oxygen supplementation can be by hood, oxygen cage or tent, nasal or nasopharyngeal catheter, or tracheal tube. in rare cases, administration of oxygen with mechanical ventilation may be indicated. administration of supplemental oxygen to patients with chronic hypoxia is sometimes necessary but also dangerous. with chronic hypoxia the patient develops a chronic respiratory acidosis (elevated paco ) and depends almost entirely on the hypoxic ventilatory drive to breathe. administration of supplemental oxygen increases pao and may inhibit the central respiratory drive, leading to hypoventilation and possibly respiratory arrest. therefore, closely monitor animals with chronic hypoxia that are treated with supplemental oxygen. oxygen hoods can be purchased from commercial sources or can be manufactured in the hospital using a rigid elizabethan collar, tape, and plastic wrap. to make an oxygen hood, place several lengths of plastic wrap over the front of the elizabethan collar and tape them in place. leave the ventral third of the collar open to allow moisture and heat to dissipate and carbon dioxide to be eliminated. place a length of flexible oxygen tubing under the patient's collar into the front of the hood, and run humidified oxygen at a rate of to ml/kg/minute. animals may become overheated with an oxygen hood in place. carefully monitor the patient's temperature so that iatrogenic hyperthermia does not occur. commercially available plexiglass oxygen cages can be purchased from a variety of manufacturers. the best units include a mechanical thermostatically controlled compressor cooling unit, a circulatory fan, nebulizers or humidifiers to moisten the air, and a carbon dioxide absorber. alternately, a pediatric (infant) incubator can be purchased from hospital supply sources, and humidified oxygen can be run into the cage at to l/minute (depending on the size of the cage). high flow rates may be required to eliminate nitrogen and carbon dioxide from the cage. in most cases, the fio inside the cage reaches % to % using this technique. disadvantages of using an oxygen cage are high consumption/ use of oxygen, rapid decrease in the fio within the cage whenever the cage must be opened for patient treatments, lack of immediate access to the patient, and potential for iatrogenic hyperthermia. one of the most common methods for oxygen supplementation in dogs is nasal or nasopharyngeal oxygen catheters: . to place a nasal or nasopharyngeal catheter, obtain a red rubber catheter ( f to f, depending on the size of the patient). a. for nasal oxygen supplementation, measure the distal tip of the catheter from the medial canthus of the eye to the tip of the nose. b. for nasopharyngeal oxygen supplementation, measure the catheter from the ramus of the mandible to the tip of the nose. . mark the tube length at the tip of the nose with a permanent marker. . instill topical anesthetic such as proparacaine ( . %) or lidocaine ( %) into the nostril before placement. . place a stay suture adjacent to (lateral aspect) the nostril while the topical anesthetic is taking effect. . lubricate the tip of the tube with sterile lubricant. . gently insert the tube into the ventral medial aspect of the nostril to the level made with the permanent marker. if you are inserting the tube into the nasopharynx, push the nasal meatus dorsally while simultaneously pushing the lateral aspect of the nostril medially to direct the tube into the ventral nasal meatus and avoid the cribriform plate. . once the tube has been inserted to the appropriate length, hold the tube in place with your fingers adjacent to the nostril, and suture the tube to the stay suture. if the tube is removed, you can cut the suture around the tube and leave the stay suture in place for later use, if necessary. . suture or staple the rest of the tube dorsally over the nose and in between the eyes to the top of the head, or laterally along the zygomatic arch. . attach the tube to a length of flexible oxygen tubing, and provide humidified oxygen at to ml/kg/minute. . secure an elizabethan collar around the patient's head to prevent the patient from scratching at the tube and removing it. the rule of s states that if a patient's pao is less than mm hg, or if the paco is mm hg, mechanical ventilation should be considered. for mechanical ventilation, anesthetize the patient and intubate the patient with an endotracheal tube. alternately, a temporary tracheostomy can be performed and the patient can be maintained on a plane of light to heavy sedation and ventilated through the tracheostomy site. this method, a noninvasive means of determining oxygenation is through the use of pulse oximetry. a pulse oximeter uses different wavelengths of light to distinguish characteristic differences in the properties of the different molecules in a fluid or gas mixture, in this case, oxygenated (oxyhemoglobin) and deoxygenated hemoglobin (deoxyhemoglobin) in pulsatile blood. the process is termed pulse oximetry. oxyhemoglobin and deoxyhemoglobin are different molecules that absorb and reflect different wavelengths of light. oxyhemoglobin absorbs light in the infrared spectrum, allowing wavelengths of light in the red spectrum to transmit through it. conversely, deoxyhemoglobin absorbs wavelengths of the red spectrum and allows wavelengths in the infrared spectrum to transmit through the molecule. the spectrophotometer in the pulse oximeter transmits light in the red ( nanometers) and infrared ( nanometers) spectra. the different wavelengths of light are transmitted across a pulsatile vascular bed and are detected by a photodetector on the other side. the photodetector processes the amount of light of varying wavelengths that reaches it, then transmits an electrical current to a processor that calculates the difference in the amount of light originally transmitted and the amount of light of similar wavelength that actually reaches the photodetector. the difference in each reflects the amount of light absorbed in the pulsatile blood and can be used to calculate the amount or ratio of oxyhemoglobin to deoxyhemoglobin in circulation, or the functional hemoglobin saturation by the formula: where hbo is oxygenated hemoglobin, and hb is deoxygenated hemoglobin. four molecules of oxygen reversibly bind to hemoglobin for transport to the tissues. carbon monoxide similarly binds to hemoglobin and forms carboxyhemoglobin, a molecule that is detected similarly as oxygenated hemoglobin. thus sao as detected by a pulse oximeter is not reliable if carboxyhemoglobin is present. in most cases, pulse oximetry or sao corresponds reliably to the oxyhemoglobin dissociation curve. oxygen saturation greater than % corresponds to a pao greater than mm hg. above this value, large changes in pao are reflected in relatively small changes in sao , making pulse oximetry a relatively insensitive method of determining oxygenation status when pao is normal. because pulse oximetry measures oxygenated versus nonoxygenated hemoglobin in pulsatile blood flow, it is fairly unreliable when severe vasoconstriction, hypothermia, shivering or trembling, or excessive patient movement are present. additionally, increased ambient lighting and the presence of methemoglobin or carboxyhemoglobin also can cause artifactual changes in the sao , and thus the measurement is not reliable or accurate. most pulse oximeters also display a waveform and the patient's heart rate. if the photodetector does not detect a good quality signal, the waveform will not be normal, and the heart rate displayed on the monitor will not correlate with the patient's actual heart rate. the efficiency of ventilation is evaluated using the paco value on an arterial blood gas sample. alternatively, a noninvasive method to determine end-tidal carbon dioxide is through use of a capnograph. the science of capnometry uses a spectrophotometer to measure carbon dioxide levels in exhaled gas. the capnometer is placed in the expiratory limb of an anesthetic circuit. a sample of exhaled gas is aliquoted from the breath, and an infrared light source is passed across the sample. a photodetector on the other side of the sample flow measures the amount or concentration of carbon dioxide in the sample of expired gas. the calculated value is displayed as end-tidal carbon dioxide. this value also can be displayed as a waveform. when placed in graphic form, a waveform known as a capnograph is displayed throughout the ventilatory cycle. normally, at the onset of exhalation, the gas exhaled into the expiratory limb of the tubing comes from the upper airway or physiologic dead space and contains relatively little carbon dioxide. as exhalation continues, a steep uphill slope occurs as more carbon dioxide is exhaled from the bronchial tree. near the end of exhalation, the capnogram reaches a plateau, which most accurately reflects the carbon dioxide level at the level of the alveolus. because carbon dioxide diffuses across the alveolar basement membrane so rapidly, this reflects arterial carbon dioxide levels. if a plateau is not reached and notching of the waveform occurs, check the system for leaks. if the baseline waveform does not reach zero, the patient may be rebreathing carbon dioxide or may be tachypneic, causing physiologic positive end-expiratory pressure. the soda-sorb in the system should be replaced if it has expired. conversely, low end-tidal carbon dioxide may be associated with a decrease in perfusion or blood flow. decreased perfusion can be associated with low end-tidal carbon dioxide values, particularly during cardiopulmonary cerebral resuscitation. end-tidal carbon dioxide levels are one of the most accurate predictors of the efficacy of cardiopulmonary cerebral resuscitation and patient outcome. additionally, the difference between arterial carbon dioxide levels (paco ) and end-tidal carbon dioxide can be used to calculate dead-space ventilation. increases in the difference also occur with poor lung perfusion and pulmonary diffusion impairment. thoracocentesis refers to the aspiration of fluid or air from within the pleural space. thoracocentesis may be diagnostic to determine whether air or fluid is present and to characterize the nature of the fluid obtained. thoracocentesis also can be therapeutic when removing large volumes of air or fluid to allow pulmonary reexpansion and correction of hypoxemia and orthopnea. to perform thoracocentesis, follow this procedure: . first, assemble the equipment necessary (box - ). . next, clip a -cm square in the center of the patient's thorax on both sides. . aseptically scrub the clipped area. . ideally, thoracocentesis should be performed within the seventh to ninth intercostal space. rather than count rib spaces in an emergent situation, visualize the thoracic cage as a box, and the clipped area as a box within the box. you will insert your needle or catheter in the center of the box and then direct the bevel of the needle dorsally or ventrally to penetrate pockets of fluid or air present. . attach the needle or catheter hub to the length of intravenous extension tubing. attach the female port of the intravenous extension tubing to the male port of the three-way stopcock. attach the male port of the -ml syringe to one of the female ports of the three-way stopcock. the apparatus is now assembled for use. . insert the needle through the intercostal space such that the bevel of the needle initially is directed downward. . next, push down on the hub of the needle such that the needle becomes parallel with the thoracic wall. by moving the hub of the needle in a clockwise or counterclockwise manner, the bevel of the needle will move within the thoracic cavity to penetrate pockets of air or fluid. in general, air is located dorsally and fluid is located more ventrally, although this does not always occur. . aspirate air or fluid. save any fluid obtained for cytologic and biochemical analyses and bacterial culture and susceptibility testing. in cases of pneumothorax, if the thoracocentesis needs to be repeated more than times, consider using a thoracostomy tube. place a thoracostomy tube in cases of pneumothorax whenever negative suction cannot be obtained or repeated accumulation of air requires multiple thoracocentesis procedures. thoracostomy tubes also can be placed to drain rapidly accumulating pleural effusion and for the medical management of pyothorax. before attempting thoracostomy tube placement, make sure that all necessary supplies are assembled (box - ; table - ) . to place a thoracostomy tube, follow this procedure: . lay the patient in lateral recumbency. . clip the patient's entire lateral thorax. . aseptically scrub the lateral thorax. . palpate the tenth intercostal space. . have an assistant pull the patient's skin cranially and ventrally toward the point of the elbow. this will facilitate creating a subcutaneous tunnel around the thoracostomy tube. . draw up mg/kg % lidocaine ( mg/kg for cats) along with a small amount of sodium bicarbonate to take away some of the sting. . insert the needle at the dorsal aspect of the tenth intercostal space and to the seventh intercostal space. inject the lidocaine into the seventh intercostal space at the point where the trocarized thoracic drainage catheter will penetrate into the thoracic cavity. slowly infuse the lidocaine as you withdraw the needle to create an anesthetized tunnel through which to insert the catheter. . while the local anesthetic is taking effect, remove the trocar from the catheter and cut the proximal end of the catheter with a mayo scissors to facilitate adaptation with the christmas tree adapter. . attach the christmas tree adapter to the three-way stopcock and the three-way stopcock to a length of intravenous extension tubing and the -ml syringe so that the apparatus can be attached immediately to the thoracostomy tube after placement. . aseptically scrub the lateral thorax a second time and then drape it with sterile huck towels secured with towel clamps. . wearing sterile gloves, make a small stab incision at the dorsal aspect of the tenth intercostal space. . insert the trocar back into the thoracostomy drainage tube. insert the trocar and tube into the incision. tunnel the tube cranially for approximately intercostal spaces while an assistant simultaneously pulls the skin cranially and ventrally toward the point of the elbow. . at the seventh intercostal space, direct the trocar and catheter perpendicular to the thorax. grasp the catheter apparatus at the base adjacent to the thorax to prevent the trocar from going too far into the thorax. . place the palm of your dominant hand over the end of the trocar, and push the trocar and catheter into the thoracic cavity, throwing your weight into the placement in a swift motion, not by banging the butt of your hand on the end of the stylette. for small individuals, standing on a stool, or kneeling over the patient on the triage table can create leverage and make this process easier. the tube will enter the thorax with a pop. . gently push the catheter off of the stylette, and remove the stylette. . immediately attach the christmas tree adapter and have an assistant start to withdraw air or fluid while you secure the tube in place. . first, place a horizontal mattress suture around the tube to cinch the skin securely to the tube. use care to not penetrate the tube with your needle and suture. . next, place a purse-string suture around the tube at the tube entrance site. leave the ends of the suture long, so that you can create a finger-trap suture to the tube, holding the tube in place. . place a large square of antimicrobial-impregnated adhesive tape over the tube for further security and sterility. . if antimicrobial adhesive is not available, place a gauze pad × inches square over the tube, and then wrap the tube to the thorax with cotton roll gauze and elastikon adhesive tape. . draw the location of the tube on the bandage to prevent cutting it with subsequent bandage changes. an alternate technique to use if a trocar thoracic drainage catheter is not available is the following: . prepare the lateral thorax and infuse local lidocaine anesthetic as listed before. . make a small stab incision with a no. scalpel blade, as listed before. . obtain the appropriately sized red rubber catheter and cut multiple side ports in the distal end of the catheter, taking care to not cut more than % of the circumference of the diameter of the tube. . insert a rigid, long urinary catheter into the red rubber catheter to make the catheter more rigid during insertion into the pleural space. . grasp the distal end of the catheter(s) in the teeth of a large carmalt. tunnel a metzenbaum scissors under the skin to the seventh intercostal space and make a puncture through the intercostal space. . remove the metzenbaum scissors, and then tunnel the carmalt and red rubber tube under the skin to the hole created in the seventh intercostal space with the metzenbaum scissors. . insert the tips of the carmalt and the red rubber catheter through the hole, and then open the teeth of the carmalt. . push the red rubber catheter cranially into the pleural cavity. . remove the carmalt and the rigid urinary catheter, and immediately attach the suction apparatus. secure the red rubber catheter in place as listed before. placement of a temporary tracheostomy can be lifesaving to relieve upper respiratory tract obstruction, to facilitate removal of airway secretions, to decrease dead space ventilation, to provide a route of inhalant anesthesia during maxillofacial surgery, and to facilitate mechanical ventilation. in an emergent situation in which asphyxiation is imminent and endotracheal intubation is not possible, any cutting instrument placed into the trachea distal to the point of obstruction can be used. to perform a slash tracheostomy, quickly clip the fur and scrub the skin over the third tracheal ring. make a small cut in the trachea with a no. scalpel blade, and insert a firm tube, such as a syringe casing. alternately, insertion of a -gauge needle attached to intravenous extension tubing and adapted with a -ml syringe case to attach to a humidified oxygen source also temporarily can relieve obstruction until a temporary tracheostomy can be performed. in less emergent situations, place the patient under general anesthesia and intubate the patient. assemble all the equipment necessary before starting the temporary tracheostomy procedure (box - ). to perform a tracheostomy, follow this procedure: . place the patient in dorsal recumbency. . clip the ventral cervical region from the level of the ramus of the mandible caudally to the thoracic inlet and dorsally to midline. . aseptically scrub the clipped area, and then drape with sterile huck towels secured with towel clamps. . make a -cm ventral midline skin incision over the third to sixth tracheal rings, perpendicular to the trachea. . bluntly dissect through the sternohyoid muscles to the level of the trachea. . carefully pick up the fascia overlying the trachea and cut it away with a metzenbaum scissors. . place two stay sutures through/around adjacent tracheal rings. . incise in between trachea rings with a no. scalpel blade. take care to not cut more than % of the circumference of the trachea. . using the stay sutures, pull the edges of the tracheal incision apart, and insert the tracheostomy tube. the shiley tube contains an internal obturator to facilitate placement into the tracheal lumen. remove the obturator, and then insert the inner cannula, which can be removed for cleaning as needed. . once the tube is in place, secure the tube around the neck with a length of sterile umbilical tape. postoperative care of the tracheostomy tube is as important as the procedure itself. because the tracheostomy tube essentially bypasses the protective effects of the upper respiratory system, one of the most important aspects of tracheostomy tube care and maintenance is to maintain sterility at all times. any oxygen source should be humidified with sterile water or saline to prevent drying of the respiratory mucosa. if supplemental oxygen is not required, instill to ml of sterile saline every to hours to moisten the mucosa. wearing sterile gloves, remove the internal tube and place it in a sterile bowl filled with sterile hydrogen peroxide and to be cleaned every hours (or more frequently as necessary). if a shiley tube is not available, apply suction to the internal lumen of the tracheostomy tube every to hours (or more frequently as needed) with a sterile f red rubber catheter attached to a vacuum pump to remove any mucus or other debris that potentially could plug the tube. unless the patient demonstrates clinical signs of fever or infection, the prophylactic use of antibiotics is discouraged because of the risk of causing a resistant infection. after the temporary tracheostomy is no longer necessary, remove the tube and sutures, and leave the wound to heal by second intention. primary closure of the wounds could predispose the patient to subcutaneous emphysema and infection. baker gd: trans-tracheal oxygen therapy in dogs with severe respiratory compromise due to tick (i. holocyclus) toxicity, aust vet pract ( ) urohydropulsion is a therapeutic procedure for removal of uroliths from the urethra of the male dog. the technique works best if the animal is heavily sedated or is placed under general anesthesia (figure - ) . to perform urohydropulsion, follow this procedure: . place the animal in lateral recumbency. . clip the fur from the distal portion of the prepuce. . aseptically scrub the prepuce and flush the prepuce with to ml of antimicrobial flush solution. . have an assistant who is wearing gloves retract the penis from the prepuce. . while wearing sterile gloves, lubricate the tip of a rigid urinary catheter as for urethral catheterization. . gently insert the tip of the catheter into the urethra until you meet the resistance of the obstruction. . pinch the tip of the penis around the catheter. . have an assistant insert a gloved lubricated finger into the patient's rectum and press ventrally on the floor of the rectum to obstruct the pelvic urethra. . attach a -ml syringe filled with sterile saline into proximal tip of the catheter. . quickly inject fluid into the catheter and alternate compression and relaxation on the pelvic urethra such that the urethra dilates and suddenly releases the pressure, causing dislodgement of the stone. small stones may be ejected from the tip of the urethra, whereas larger stones may be retropulsed back into the urinary bladder to be removed surgically at a later time. the type of catheter that you choose for vascular access depends largely on the size and species of the patient, the fragility of the vessels to be catheterized, the proposed length of time that the catheter will be in place, the type and viscosity of the fluid or drug to be administered, the rate of fluid flow desired, and whether multiple repeated blood samples will be required (table - ) . a variety of over-the-needle, through-the-needle, and over-the-wire catheters are available for placement in a variety of vessels, including the jugular, cephalic, accessory cephalic, medial saphenous, lateral saphenous, dorsal pedal artery, and femoral artery. one of the most important aspects of proper catheter placement and maintenance is to maintain cleanliness at all times. the patient's urine, feces, saliva, and vomit are common sources of contamination of the catheter site. before placing a peripheral or central catheter in any patient, consider the patient's physical status including whether vomiting, diarrhea, excessive urination, or seizures. in a patient with an oral mass that is drooling excessively or a patient that is vomiting, peripheral cephalic catheterization may not be the most appropriate, to prevent contamination. conversely, in a patient with excessive urination or diarrhea, a lateral or medial saphenous catheter is likely to become contaminated quickly. whenever one places or handles a catheter or intravenous infusion line, the person should wash the hands carefully and wear gloves to prevent contamination of the intravenous catheter and fluid lines. one of the most common sources of catheter contamination in veterinary hospitals is through caretakers' hands. in emergent situations, placement of a catheter may be necessary under less than ideal circumstances. remove those catheters as soon as the patient is more stable, and place a second catheter using aseptic techniques. in general, once the location of the catheter has been decided, set up all equipment necessary for catheter placement before starting to handle and restrain the patient. lists the equipment needed for most types of catheter placement. after setting up all of the supplies needed, clip the fur over the site of catheter placement. make sure to clip all excess fur and long feathers away from the catheter site, to prevent contamination. for catheter placement in limbs, clip the fur circumferentially around the site of catheter placement to facilitate adherence of the tape to the limb and to facilitate catheter removal with minimal discomfort at a later date. next, aseptically scrub the catheter site with an antimicrobial scrub solution such as hibiclens. the site is now ready for catheter insertion. consider using a central venous catheter whenever multiple repeated blood samples will need to be collected from a patient during the hospital stay. central venous catheters also can be used for cvp measurement, administration of hyperoncotic solutions such as parenteral nutrition, and administration of crystalloid and colloid fluids, anesthesia, and other injectable drugs (figures - and - ) . to place a jugular central venous catheter, place the patient in lateral recumbancy and extend the head and neck such that the jugular furrow is straight. clip the fur from the ramus of the mandible caudally to the thoracic inlet and dorsally and ventrally to midline. wipe the clipped area with gauze × -inch squares to remove any loose fur and other debris. aseptically scrub the clipped area with an antimicrobial cleanser. venocaths (abbott laboratories) are a through-the-needle catheter that is contained within a sterile sleeve for placement. alternately, other over-the-wire central venous catheters can be placed by the seldinger technique. sterility must be maintained at all times, regardless of the type of catheter placed. wearing sterile gloves, drape the site of catheter placement with sterile drapes, and occlude the jugular vein at the level of the thoracic inlet. pull the clear ring and wings of emergency diagnostic and therapeutic procedures figure - : lateral thoracic radiograph of a central venous catheter. note that the tip of the catheter is inserted in its proper location, just outside of the right atrium. the catheter cover down toward the catheter itself to expose the needle. remove the guard off of the needle. lift the skin over the proposed site of catheter insertion and insert the needle under the skin, with the bevel of the needle facing up. next, reocclude the vessel and pull the skin tight over the vessel to prevent movement of the vessel as you attempt to insert the needle. in some cases, it may be difficult actually to see the vessel in obese patients. if you cannot visualize or palpate the needle, gently bounce the needle over the vessel with the bevel up. the vessel will bounce in place slightly, allowing a brief moment of visualization to facilitate catheter placement. once the vessel has been isolated and visualized, insert the needle into the vessel at a -to -degree angle. watch closely for a flash of blood in the catheter. when blood is observed, insert the needle a small distance farther, and then push the catheter and stylette into the vessel for the entire length, until the catheter and stylette can be secured in the catheter hub. if the catheter cannot be inserted fully into the vessel for its entire length, the tip of the needle may not be within the entire lumen, the catheter may be directed perivascularly, and the catheter may be caught at the thoracic flexure and may be moving into one of the tributaries that feeds the forelimb. extend the patient's head and neck, and lift the forelimb up to help facilitate placement. do not force the catheter in because the catheter potentially can form a knot and will need to be removed surgically. remove the needle from the vessel, and have an assistant place several × -inch gauze squares over the site of catheter placement with some pressure to control hemorrhage. secure the catheter hub into the needle guard, and remove the stylette from the catheter. immediately insert a -to -ml syringe of heparinized saline and flush the catheter and draw back. if you are in the correct place, you will be able to draw blood from the catheter. to secure the catheter in place, tear a length of -inch white tape that will wrap around the patient's neck. pull a small length of the catheter out of the jugular vein to make a semicircle. the semicircle should be approximately / inch in diameter. let the length of catheter lie on the skin, and then place × -inch gauze squares impregnated with antimicrobial ointment over the site of catheter insertion. secure the proximal end of white tape around the white and blue pieces of the catheter, and wrap the tape around the patient's neck so that the tape adheres to the skin and fur. repeat the process by securing the gauze to the skin with two additional lengths of white tape, starting to secure the gauze in place by first wrapping the tape dorsally over the patient's neck, rather than under the patient's neck. in between each piece of tape and bandage layer, make sure that the catheter flushes and draws back freely, or else occlusion can occur. gently wrap layers of cotton roll gauze, kling, and elastikon or vetrap over the catheter. secure a male adapter or t port that has been flushed with heparinized saline, and then label the catheter with the size and length of catheter, date of catheter placement, and initials of the person who placed the catheter. the catheter is ready for use. monitor the catheter site daily for erythema, drainage, vessel thickening, or pain upon infusion. if any of these signs occur, or if the patient develops a fever of unknown origin, remove the catheter, culture the catheter tip aseptically, and replace the catheter in a different location. as long as the catheter is functional without complications, the catheter can remain in place. central catheters also can be placed via the seldinger or over-the-wire technique. a number of companies manufacture kits that contain the supplies necessary for over-the-wire catheter placement. each kit minimally should contain an over-the-needle catheter to place into the vessel, a long wire to insert through the original catheter placed, a vascular dilator to dilate the hole in the vessel created by the first catheter, and a long catheter to place into the vessel over the wire. additional accessories can include a paper drape, sterile gauze, a scalpel blade, local anesthetic, -gauge needles, and -or -ml syringes. restrain the patient and prepare the jugular furrow aseptically as for the percutaneous through-the-needle catheter placement. the person placing the catheter should wear sterile gloves throughout the process to maintain sterility. pick up the skin over the site of catheter placement, and insert a small bleb of local anesthetic through the skin. the local anesthetic should not be injected into the underlying vessel (figure - ) . make a small nick into the skin through the local anesthetic with a no. or no. scalpel blade. use care to avoid lacerating the underlying vessel. next, occlude the jugular vein as previously described, and insert the over-the-needle catheter into the vessel. watch for a flash of blood in the catheter hub. remove the stylette from the catheter. next, insert the long wire into the catheter and into the vessel (figures - and - ) . never let go of the wire. remove the catheter, and place the vascular dilator over the wire and into the vessel (figure - ) . gently twist to place the dilator into the vessel a short distance, creating a larger hole in the vessel. the vessel will bleed more after creating a larger hole. remove the vascular dilator, and leave the wire in place within the vessel. insert the long catheter over the wire into the vessel (figure - ) . push the catheter into the vessel to the catheter hub (figure - ) . slowly thread the wire through a proximal port in the catheter. once the catheter is in place, remove the wire, and suture the catheter in place to the skin with nonabsorbable suture. cover the catheter site with sterile gauze and antimicrobial ointment, cotton roll bandaging material, gauze, and kling or vetrap. flush the catheter with heparinized saline solution, and then use the catheter for infusion of parenteral nutrition, blood products, crystalloid and colloid fluids, medications, and frequent blood sample collection. examine the catheter site daily for evidence of infection or thrombophlebitis. the catheter can remain in place as long as it functions and no complications occur. place the patient in sternal recumbency as for cephalic venipuncture. clip the antebrachium circumferentially, and wipe the area clean of any loose fur and debris (figure - ) . aseptically scrub the clipped area, and have an assistant occlude the cephalic vein at the crook of the elbow. the person placing the catheter should grasp the distal carpus with the nondominant hand and insert the over-the-needle catheter into the vessel at a -to -degree angle ( figure - ) . watch for a flash of blood in the catheter hub, and then gently push the catheter off of the stylette (figure - ) . have the assistant occlude the vessel over the catheter to prevent backflow. flush the catheter with heparinized saline solution. make sure that the skin and catheter hub are clean and dry to ensure that the tape adheres to the catheter hub and skin. secure a length of / -inch white tape tightly around the catheter and then around the limb. make sure that the catheter hub does not "spin" in the tape, or else the catheter will fall out. next, secure a second length of -inch adhesive tape under the catheter and around the limb and catheter hub (figure - ). this piece of tape helps to stabilize the catheter in place. finally, place a flushed t port or male adapter in the catheter hub and secure to the limb with white tape. make sure that the tape is adhered to the skin securely, but not so tightly as to impede venous outflow (figure - ) . the catheter site can be covered with a cotton ball impregnated with antimicrobial ointment and layers of bandage material. label all catheters with the date of placement, the type and gauge of catheter inserted, and the initials of the person who placed the catheter. the femoral artery can be catheterized for placement of an indwelling arterial catheter. indwelling arterial catheters can be used for continuous invasive arterial blood pressure monitoring and for procurement of arterial blood samples. place the patient in lateral recumbancy, and tape the down leg in an extended position. clip the fur over the femoral artery and aseptically scrub the clipped area. palpate the femoral artery as it courses distally on the medial surface of the femur and anterior to the pectineus muscle. make a small nick incision over the proposed site of catheter placement using the bevel of an -gauge needle. place a long over-the-needle catheter through the nick in the skin and direct it toward the palpable pulse. place the tip of the catheter so that the needle tip rests in the subcutaneous tissue between the artery and the palpating index finger. advance the needle steeply at a -degree angle to secure the superficial wall of the vessel and then the deep wall of the vessel. the spontaneous flow of blood in the catheter hub ensures that the catheter is figure - : catheter is taped in place with a t-port. situated in the lumen of the artery. feed the catheter off of the stylette, and cover the hub with a catheter cap. flush the catheter with sterile heparinized saline solution, and then secure it in place. some persons simply tape the catheter in place with pieces of / -and -inch adhesive tape. others use a "butterfly" piece of tape around the catheter hub and suture or glue the tape to the adjacent skin for added security. the dorsal pedal artery commonly is used for catheter placement. to place a dorsal pedal arterial catheter, place the patient in lateral recumbency. clip the fur over the dorsal pedal artery, and then aseptically scrub the clipped area. tape the distal limb so that the leg is twisted slightly medially for better exposure of the vessel, or the person placing the arterial catheter can manipulate the limb into the appropriate position. palpate the dorsal pedal pulse as it courses dorsally over the tarsus. place an over-the-needle catheter percutaneously at a -to -degree angle, threading the tip of the needle carefully toward the pulse. advance the needle in short, blunt movements, and watch the catheter hub closely for a flash of pulsating blood that signifies penetration into the lumen of the artery. then thread the catheter off of the stylette, and cover the catheter hub with a catheter cap. secure the catheter in place with lengths of / -and -inch adhesive tape as with any other intravenous catheter, and then flush it with heparinized saline solution every to hours. any vessel that can be catheterized percutaneously also can be catheterized with surgical cutdown. restrain the patient and clip and aseptically scrub the limb or jugular vein as for a percutaneous catheterization procedure. block the area for catheter placement with a local anesthetic before cutting the skin over the vessel with a no. scalpel blade. while wearing sterile gloves, pick up the skin and incise the skin over the vessel. direct the sharp edge of the blade upward to avoid lacerating the underlying vessel. using blunt dissection, push the underlying subcutaneous fat and perivascular fascia away from the vessel with a mosquito hemostat. make sure that all tissue is removed from the vessel. using the mosquito hemostat, place two stay sutures of absorbable suture under the vessel. elevate the vessel until it is parallel with the incision, and gently insert the catheter and stylette into the vessel. secure the stay sutures loosely around the catheter. suture the skin over the catheter site with nonabsorbable suture, and then tape and bandage the catheter in place as for percutaneous placement. remove catheters placed surgically as soon as possible and exchange them for a percutaneously placed catheter to avoid infection and thrombophlebitis. the most important aspect of catheter maintenance is to maintain cleanliness and sterility at all times. an indwelling catheter can remain in place for as long as it is functional and no complications occur. change the bandage whenever it becomes wet or soiled to prevent wicking of bacteria and debris from the environment into the vessel. check the bandages and catheter sites at least once a day for signs of thrombophlebitis: erythema, vessel hardening or ropiness, pain upon injection or infusion, and discharge. also closely examine the tissue around and proximal and distal to the catheter. swelling of the paw can signify that the catheter tape and bandage are too tight and are occluding venous outflow. swelling above the catheter site is characteristic of perivascular leakage of fluid and may signify that the catheter is no longer within the lumen of the vessel. remove the catheter if it is no longer functional, if there is pain or resistance upon infusion, if there is unexplained fever or leukocytosis, or if there is evidence of cellulitis, thrombophlebitis, or catheter-related bacteremia or septicemia. aseptically culture the tip of the indwelling catheter for bacteria. animals should wear elizabethan collars or other forms of restraint if they lick or chew at the catheter or bandage. catheter patency may be maintained with constant fluid infusion or by intermittent flushing with heparinized saline ( units of unfractionated heparin per to ml of saline) every hours. flush arterial catheters more frequently (every hours). disconnect intravenous connections only when absolutely necessary. wear gloves whenever handling the catheter or connections. label all fluid lines and elevate them off of the floor to prevent contamination. date each fluid line and replace it once every to hours. if an intravenous catheter cannot be placed because of small patient size, hypovolemia, hypothermia, or severe hypotension, needles can be placed into the marrow cavity of the femur, humerus, and tibia for intraosseous infusion of fluids, drugs, and blood products. this technique is particularly useful in small kittens and puppies and in exotic species. contraindications to intraosseous infusion is in avian species (which have air in their bones), fractures, and sepsis, because osteomyelitis can develop. an intraosseous catheter is relatively easy to place and maintain but can cause patient discomfort and so should be changed to an intravenous catheter as soon as vascular access becomes possible. to place an intraosseous catheter, clip and aseptically scrub the fur over the proposed site of catheter placement. the easiest place for intraosseous placement is in the intertrochanteric fossa of the femur. inject a small amount of a local anesthetic through the skin and into the periosteum where the trocar or needle will be inserted. place the patient in lateral recumbency, and grasp the leg in between your fingers, with the stifle braced against the palm of your hand. push the stifle toward the abdomen (medially) to abduct the proximal femur away from the body. this will shift the sciatic nerve out of the way of catheter placement. insert the tip of the needle through the skin and into the intertrochanteric fossa. gently push with a simultaneous twisting motion, pushing the needle parallel with the shaft of the femur, toward your palm. you may feel a pop or decreased resistance as the needle enters the marrow cavity. gently flush the needle with heparinized saline. if the needle is plugged with bone debris, remove the needle and replace it with a fresh needle of the same type and size in the hole that you have created. a spinal needle with an internal stylette also can be placed. the stylette will prevent the needle from becoming clogged with bone debris during insertion. secure the hub of the needle with a butterfly length of white adhesive tape and then suture it to the skin to keep the catheter in place. the catheter is now ready for use. the patient should wear an elizabethan collar to prevent disruption or removal of the catheter. the intraosseous catheter can be maintained as any peripheral catheter, with frequent flushing and daily evaluation of the catheter site. the definition of pain has been debated philosophically over the ages and has changed as knowledge has increased. pain is defined as an unpleasant sensory or emotional experience associated with actual or perceived tissue damage. until recognition of a noxious stimulus occurs in the cerebral cortex, no response or adaptation results. rational management of pain requires an understanding of the underlying mechanisms involved in pain and an appreciation of how analgesic agents interact to disrupt pain mechanisms. multiple factors and causes produce pain in human beings and domestic animal species. the causes of pain, psychological and physical, may derive from many different mechanisms within emergency medicine, among them trauma, infectious disease, neglect, environmental stress, surgery, and acute decompensation of chronic medical conditions. the two major classes of pain are acute and chronic pain. box - gives specific categories and causes of pain. the pain sensing and response system can be divided into the following categories: nociceptors, which detect and filter the intensity of the noxious stimuli; primary afferent nerves, which transmit impulses to the central nervous system (cns); ascending tracts, which are part of the dorsal horn and the spinal cord that conveys stimuli to higher centers in the brain; higher centers, which are involved in pain discrimination, some memory, and motor control; and modulating or descending systems, which are a means of processing, memorizing, and modifying incoming impulses. current analgesic therapies may inhibit afferent nociceptive transmission within the brain and spinal cord; directly interrupt neural impulse conduction through the dorsal horn, primary afferent nerves, or dorsal root ganglion; or prevent the nociceptor sensitization that accompanies initial pain and inflammation. the physiologic aspects of pain are believed to be produced by the transmission, transduction, and integration of initial nerve endings, peripheral neuronal input, and ascending afferent nerves via the thalamus to the cerebral cortex. ascending afferent nerves to the limbic system are believed to be responsible for the emotional aspects of pain. there are several classification schemes for different types of pain. acute pain, such as that which results from trauma, surgery, or infectious agents, is abrupt in onset, relatively short in duration, and may be alleviated easily by analgesics. in contrast, chronic pain is a long-standing physical disorder or emotional distress that is slow in onset and difficult to treat. both types of pain can be classified further based on site of origin. somatic pain arises from superficial skin, subcutaneous tissue, body wall, or appendages. visceral pain arises from abdominal or thoracic viscera and primarily is associated with serosal irritation. analgesia, then, is the loss of pain without the loss of consciousness. this is in contrast to anesthesia, which is the loss of sensation in the whole body or a part of the body with the loss of consciousness or at least depression of the cns. untreated pain causes immediate changes in the neurohormonal axis, which in turn causes restlessness, agitation, increased heart and respiratory rates, fever, and blood pressure fluctuations, all of which are detrimental to the healing of the animal. a catabolic state is created as a result of increased secretion of catabolic hormones and decreased secretion of anabolic hormones. the net effect the majority of neurohormonal changes produce is an increase in the secretion of catabolic hormones. hyperglycemia is produced and may persist because of production of glucagon and relative lack of insulin. lipolytic activity is stimulated by cortisol, catecholamines, and growth hormone. cardiorespiratory effects of pain include increased cardiac output, vasoconstriction, hypoxemia, and hyperventilation. protein catabolism is a common occurrence and major concern regarding healing. pain associated with inflammation causes increase in tissue and blood levels of prostaglandins and cytokines, both of which promote protein catabolism indirectly by increasing the energy expenditure of the body. powerful evidence indicates that local anesthetic, sympathetic agonist, and opioid neural blockade may produce a modification of the responses to these physiologic changes. variable reduction in plasma cortisol, growth hormone, antidiuretic hormone, β-endorphin, aldosterone, epinephrine, norepinephrine, and renin is based on the anesthetic technique and the drugs selected. prophylactic administration of analgesics blunts the response before it occurs; analgesics administered following perception or pain are not as effective, and higher doses are generally necessary to achieve an equivalent level of analgesia. effective pain control can be achieved only when the signs of pain can be assessed effectively, reliably, and regularly. the experience of pain is unique to each individual, which makes pain assessment difficult, especially in traumatized and critical patients. most attempts to assess clinical pain use behavioral observations and interactive variables in addition to assessment of physiologic responses such as heart rate and respiratory rate, blood pressure, and temperature. but many factors can influence the processing and outward projection of pain, including altered environments, species differences, withinspecies variations (age, breed, sex), and the type, severity, and chronicity of pain. within-species differences (age, breed, and sex) further complicate the pain assessment. most notable is that different breeds of dogs act differently when confronted with pain or fear. labrador retrievers tend to be stoic, whereas greyhounds and teacup breeds tend to react with a heightened state of arousal around even the simplest of procedures (e.g., subcutaneous injections and nail trims). the individual character and temperament of the animal further influences its response. pediatric and neonatal animals seem to have a lower threshold for pain and anxiety than older animals. in any species, the duration and type of pain make it more (acute) or less (chronic) likely to be expressed or exhibited outwardly. unfamiliarity with normal behaviors typical of a particular species or breed makes recognition of their painful behaviors and responses impossible. the definition and recognition of pain in an individual animal is challenging. because of all the differences discussed, there is no straight line from insult, albeit actual or perceived, to degree of pain experienced. nor is there a formula for treating "x" type of pain with "y" type of analgesic. a goal of analgesia is to treat all animals with analgesic drugs and modalities as preemptively as possible and using a multimodal approach. use analgesic treatment as a tool for diagnosis of pain in the event that recognition of these phenomena is difficult for the patient. in other words, with countless drugs and treatment modalities available, analgesic administration should never be withheld in an animal, even if pain is questionable. it is important to remember that no behavior or physiologic variable in and of itself is pathognomonic for pain. interactive and unprovoked (noninteractive) behavior assessments and trending of physiologic data are useful to determine the pain in an individual animal. this is known as pain scoring. baseline observations, especially those observations from someone who has known the animal well, can be helpful to serial behavior and pain assessments. pain scoring systems have been developed and are reviewed elsewhere; the purposes of these systems are to evaluate and to help guide diagnostic and analgesic treatments (table - ) . regardless of the scale or method used to assess pain, the caregiver must recognize the limitations of the scale. if in doubt of whether pain is present or not, analgesic therapy should be used as a diagnostic tool. classic behaviors associated with pain in dogs and cats include abnormal postures, gaits, movements, and behaviors (boxes - and . stoicism is the apparent apathy and pain: assessment, prevention, and management indifference in the presence of pain and is perhaps the no. sign of ineffective pain relief or persistent pain in many animals, because so many display apathy and classically normal physiologic parameters even in the face of severe distress, overt suffering, or blatant trauma and illness. the absence of normal behaviors is also a clinical sign of pain, even when abnormal behaviors are not observed. acute pain results in many of the aforementioned behavioral and physiologic signs, but chronic pain in small animals is an entirely different and distinct entity. chronic pain is often present in the absence of obvious tissue pathology and changes in physical demeanor. again, the severity of the pain may not correlate with the severity of any pathologic condition that may or may not be present. chronic pain, especially if insidious in onset (cancer, dental, or degenerative pain), may well go unnoticed in dogs and cats, even by family members or intermittent caregivers. inappetance, lack of activity, panting in a species classically designed to be nose breathers, decreased interest in surroundings, different activity patterns, and abnormal postures are just a few signs of chronic pain in cats and dogs. cats are a species that in particular are exemplary in their abilities to hide chronic pain. they will exhibit marked familial withdrawal, finding secluded areas where they may remain for days to weeks when they experience acute and chronic pain. when deciding on a pain management protocol for a patient, always perform a thorough physical examination and include a pain score assessment before injury and pain has occurred, whenever possible. form a problem list to guide your choice of anesthesia and analgesia. for example, using a nonsteroidal antiinflammatory drug (nsaid) in an animal with renal failure would not be wise. remember to account for current medications that the patient may be taking that may augment or interfere with the analgesic or anesthetic drugs. use multimodal techniques and regional therapy and drugs to target pain at different sites before it occurs. once a strategy is decided upon, frequently reassess the patient and tailor the protocol to meet each patient's response and needs. drug therapy (in particular, opioids with or without α -agonists) is a cornerstone for acute pain treatment and surgical preemptive pain prevention. however, local anesthetics delivered epidurally, via perineural or plexus injection, intraarticular or trigger point injection, are also effective analgesics for acute and chronic forms of pain and inflammation. the nsaids that classically have been reserved for treatment of more chronic or persistent pain states now are being used regularly for treatment of acute and perioperative pain once blood pressure, coagulation, and gastrointestinal parameters have been normalized. an opioid is any natural or synthetic drug that is derived from the poppy, which interacts with opiate receptors identified on cell membranes. the drugs from this class constitute the most effective means of controlling acute, perioperative, and chronic pain in human and veterinary medicine (table - ) . their physiologic effects result from the interaction with one or more of at least five endogenous opioid receptors (µ, σ, δ, ε, and κ). µ-receptor agonists are noted for their ability to produce profound analgesia with mild sedation. these drugs diminish "wind-up," the hyperexcitable state resulting from an afferent volley of nociceptive impulses. they elevate the pain threshold and are used preemptively to prevent acute pain. as a class, opioids cause cns depression with their intense analgesia. dose-related respiratory depression reflects diminished response to carbon dioxide levels. cardiac depression is secondary only to bradycardia and is more likely with certain opioids such as morphine and oxymorphone. narcotics produce few if any clinically significant cardiovascular effects in dogs and cats; they are considered cardiac soothing or sparing. because opioids increase intracranial and intraocular pressure, use them more cautiously in patients with severe cranial trauma and or ocular lesions. opioids directly stimulate the chemoreceptor trigger zone and may cause nausea and vomiting. most opioids depress the cough reflex via a central mechanism; this may be helpful in patients recovering from endotracheal intubation irritation. a key characteristic of opioids that makes them desirable for use in emergency and critical care situations is their reversibility. antagonists block or reverse the effect of agonists by combining with receptors and producing minimal or no effects. administer all reversal agents, such as naloxone and naltrexone, slowly if given intravenously and to effect. α α -agonists as a class of drugs, α -agonists warrant special attention because most members of the group possess potent analgesic power at doses that are capable of causing sedation, cns depression, cardiovascular depression, and even general anesthetic states. originally developed for antihypertensive use, α -agonists quickly have attained sedative analgesic status in veterinary medicine (table - ) . like the opioids, α -agonists produce their effects by aggravating α-adrenergic receptors in the cns and periphery. emergency care among them cyclooxygenase- (cox- ), the major constitutive enzyme primarily involved in normal physiologic functions, and cox- , the enzyme responsible for most of the hyperalgesia and pain responses experienced after tissue injury or trauma. some nsaids inhibit cyclooxygenase and lipoxygenase activity. most of the currently available oral and parenteral nsaids for small animal medicine and surgery target the cyclooxygenase pathways predominantly, although one (tepoxalin) is thought to inhibit both pathways. inhibition of cox- and cox- can inhibit the protective effects and impair platelet aggregation and lead to gastrointestinal ulceration. there are definite contraindications and relative contraindications for the use of nsaids. nonsteroidal antiinflammatory drugs should not be administered to patients with renal or hepatic insufficiency, dehydration, hypotension or conditions that are associated with low circulating volume (congestive heart failure, unregulated anesthesia, shock), or evidence of ulcerative gastrointestinal disease. trauma patients should be stabilized completely regarding vascular volume, tone, and pressure before the use of nsaids. patients receiving concurrent administration of other nsaids or corticosteroids, or those considered to be cushingoid, should be evaluated carefully for an adequate "washout" period (time of clearance of drug from the system) before use of an nsaid or before switching nsaids. patients with coagulopathies, particularly those that are caused by platelet number or function defects or those caused by factor deficiencies, and patients with severe, uncontrolled asthma or other bronchial disease are probably not the patients in which to use nsaids. other advice is that nsaids not be administered to pregnant patients or to females attempting to become pregnant because cox- induction is necessary for ovulation and subsequent implantation of the embryo. the administration of nsaids should be considered only in the well-hydrated, normotensive dog or cat with normal renal or hepatic function, with no hemostatic abnormalities, and no concurrent steroid administration. nonsteroidal antiinflammatory drugs can be used in many settings of acute and chronic pain and inflammation. among these are the use in well-stabilized musculoskeletal trauma and surgical pain, osteoarthritis management, meningitis, mastitis, animal bite and other wound healing, mammary or transitional cell carcinoma, epithelial (dental, oral, urethral) inflammation, ophthalmologic procedures, and dermatologic or otic disease. whereas opioids seem to have an immediate analgesic effect when administered, most nsaids will take up to minutes for their effect to be recognized. as such, most perioperative or acute nsaids use is part of a balanced pain management scheme, one that uses narcotics and local anesthetic techniques. nonsteroidal antiinflammatory drugs are devoid of many of the side effects of narcotic administration; namely, decreased gastrointestinal motility, altered sensorium, nausea/vomition, and sedation. nonsteroidal antiinflammatory drugs are also devoid of many of the side effects of steroid administration; namely, suppression of the pituitary adrenal axis. the toxic effects of salicylates in cats are well documented. cats are susceptible because of slow clearance and dose-dependent elimination because of deficient glucuronidation in this species. because of this, the dose and the dosing interval of most commonly used nsaids need to be altered in order for these drugs to be used. cats that have been given canine doses of nsaids (twice daily or even once daily repetitively) may show hyperthermia, hemorrhagic or ulcerative gastritis, kidney and liver injury, hyperthermia, respiratory alkalosis, and metabolic acidosis. acute and chronic toxicities of nsaids have been reported in cats, especially after repeat once daily dosing. ketoprofen, flunixin, aspirin, carprofen, and meloxicam have been administered safely to cats, although like most antibiotics and other medications, they are not approved and licensed for use in cats. an important note, though, is that dosing intervals ranging from to hours have been used, and antithrombotic effects often can be achieved at much lower doses than those required to treat fevers and inflammation. i recommend the use of no loading doses, minimum -hour dosing intervals, and assurance of adequate circulating blood volume, blood pressure, and renal function. because many of the nsaids are used off-label in cats, it is imperative that the clinician carefully calculate the dose, modify the dosing interval, and communicate this information to the client before dispensing the drug. even drugs that come in liquid form (meloxicam), if administered to cats via box-labeled directions used for dogs, will be given in near toxic doses. to worsen the misunderstanding about dosages for cats, drops from manufacturer's bottles often are calibrated drops; when these same liquids are transferred into pharmacy syringes for drop administration, the calibration of course is lost, and the animal potentially is overdosed. a more accurate method of dispensing and administering oral nsaids in cats is to calculate the dose in milligrams and determine the exact number of milliliters to administer, rather than use the drop method. ketamine classically was considered a dissociative anesthetic, but it also has potent activity as an n-methyl-d-aspartate (nmda) receptor antagonist. this receptor located in the cns mediates windup and central sensitization (a pathway from acute to chronic pain). blockade of this receptor with microdoses of ketamine results in the ability to provide body surface, somatic, and skin analgesia with potentially lower doses of opioids and α-agonists. loading doses of . to mg/kg are used intravenously with continuous rate infusions of to µg/kg/minute. in and of itself, this drug possesses little to no analgesic ability and indeed in high doses alone often can aggravate, sensitize, or excite the animal in subacute or acute pain. amantadine is another nmda blocker that has been used for its antiviral and parkinson's stabilizing effects. amantadine has been used for neuropathic pain in human beings but is only available in an oral form. suggested starting doses for cats and dogs range from to mg/kg po daily. when the drug is given orally and intravenously, patients are unlikely to develop behavioral or cardiorespiratory effects with ketamine or amantadine. tramadol is an analgesic that possesses weak opioid µ-agonist activity and norepinephrine and serotonin reuptake inhibition. tramadol is useful for mild to moderate pain in small animals. although the parent compound has very weak opioid activity, the metabolites have excellent binding affinity for the µ-receptor. tramadol has been used for perisurgical pain control when given orally in cats and dogs at a dose of to mg/kg po sid to bid. cats appear to require only once daily dosing. regardless of its affinity for the opioid receptors, the true mechanism of action of tramadol in companion animals remains largely unknown. gabapentin is a synthetic analog of γ-aminobutyric acid (gaba). originally introduced as an antiepileptic drug, the mechanism of action of gabapentin remains somewhat unclear in veterinary medicine. the drug is among a number of commonly used antiepileptic medications used to treat central pain in human beings. the rationale for use is the ability of the drugs to suppress discharge in pathologically altered neurons. gabapentin does this through calcium channel modulation without binding to glutamate receptors. chronic, burning, neuropathic, and lancinating pain in small animals responds well to to mg/kg po daily. local anesthetic agents are the major class used as a peripheral-acting analgesic ( table - ) . local anesthetics block the transmission of pain impulses at the peripheral nerve nociceptor regions. local anesthetics may be used to block peripheral nerves or inhibit nerve "zones" using regional techniques. although all local anesthetics are capable of providing pain relief, agents with a longer duration of action are preferred for pain management purposes. bupivacaine is an example of a long-acting local anesthetic drug that is used along with lidocaine for long-acting pain relief. a single dose of bupivacaine injected at a local site will provide local anesthesia and analgesia for to hours. when lidocaine is administered as an intravenous constant rate infusion ( to µg/kg/minute in dogs, to µg/kg/minute in cats) is effective in the treatment of chronic neuropathic pain and periosteal and peritoneal pain (e.g., pancreatitis). mexiletine, an oral sodium channel blocker, can be used as an alternative to injectable lidocaine for provision of background analgesia. many drugs (table - ) are used in combination with opioids, α -agonists, and ketamine to provide anxiolysis and sedation. injection of local anesthetic solution into the connective tissue surrounding a particular nerve produces loss of sensation (sensory blockade) and/or paralysis (motor nerve blockade) in the region supplied by the nerve. local anesthetics also may be administered epidurally, intrathoracically, intraperitoneally, and intraarticularly. lidocaine and bupivacaine are the most commonly administered local anesthetics. lidocaine provides for quick, short-acting sensory and motor impairment. bupivacaine provides for later-onset, longerlasting desensitization without motor impairment. combinations of the two agents diluted with saline are used frequently to provide for quick-onset analgesia that lasts between and hours in most patients. adding narcotic and/or α agent often maximizes the analgesia and increases the pain-free interval to to hours. epinephrine and preservative-free solutions are recommended. precision placement of anesthetic close to nerves, roots, or plexuses is improved with the use of a stimulating nerve locator. cats seem to be more sensitive to the effects of local anesthetics; as such the lower ends of most dosing ranges are used for blockades in this species. unlike most instances of general anesthesia, during which the animal is rendered unconscious and nerve transmission is decreased by virtue of cns depression, local and regional techniques block the initiation of noxious signals, thereby effectively preventing pain from entering the cns. this is an effective means of not only preventing initial pain but also reducing the changes that take place in the dorsal horn of the spinal cord, spinothalamic tracts, limbic and reticular activating centers, and cortex. frequently, the neurohormonal response that is stimulated in pain and stress is blunted as well. overall, the patient has fewer local and systemic adverse effects of pain, disease processes are minimized, chronic pain states are unlikely, and outcome is improved. regional techniques are best used as part of an analgesic regimen that consists of their continuous administration, narcotics, α-agonists, anxiolytics, and good nursing. lidocaine can be added to sterile lubricant in a one-to-one concentration to provide decreased sensation for urinary catheterization, nasal catheter insertion, minor road burn analgesia, and pyotraumatic dermatitis analgesia. proparacaine is a topical anesthetic useful for corneal or scleral injuries. local anesthetics can be used to infiltrate areas of damage or surgery by using long-term continuous drainage catheters and small, portable infusion pumps. this is an effective means of providing days of analgesia for massive surgical or traumatic soft tissue injury. even without the catheter, incisional or regional soft tissue blocking using a combination of to mg/kg lidocaine and . to mg/kg bupivacaine diluted with equal volume of saline and : with sodium bicarbonate is effective for infiltrating large areas of injury. administration of local anesthetic drugs around the infraorbital, maxillary, ophthalmic mental, and alveolar nerves can provide excellent analgesia for dental, orofacial, and ophthalmic trauma and surgical procedures. each nerve may be desensitized by injecting . to . ml of a % lidocaine hydrochloride solution and . to . ml of . % bupivacaine solution using a . -to . -cm, -to -gauge needle. precise placement perineurally versus intraneurally (neuroma formation common) is enhanced by using catheters in the foramen versus needle administration. always perform aspiration before administration to rule out intravascular injection of agents. this block is used to provide analgesia for thoracic, lower cervical, cranial abdominal, and diaphragmatic pain. following aseptic preparation, place a small through-the-needle ( -to -gauge) catheter in the thoracic cavity between the seventh and ninth intercostal space on the midlateral aspect of the thorax. aseptically mix a . to mg/kg lidocaine and a . to . mg/kg bupivacaine dose with volume of saline equal to the volume of bupivacaine, and slowly inject it over a period of to minutes following aspiration to ensure that no intravascular injection occurs. depending on where the lesion is, position the patient to allow the intrapleural infusion to "coat" the area. most effective is positioning the patient in dorsal recumbency for several minutes following the block to make sure local anesthetic occupies the paravertebral gutters and hence the spinal nerve roots. the block should be repeated every hours in dogs and every to hours in cats. secure the catheter to the skin surface for repetitive administration. administration of local anesthetic around the brachial plexus provides excellent analgesia for forelimb surgery, particularly that distal to the shoulder, and amputations. nerve locator-guided techniques are much more accurate and successful than blind placement of local anesthetic; however, even the latter is useful. to administer a brachial plexus blockade, follow this procedure: . aseptically prepare a small area of skin over the point of the shoulder. . insert a -gauge, / -to -inch spinal needle medial to the shoulder joint, axial to the lesser tubercle, and advance it caudally, medial to the body of the scapula, and toward the costochondral junction of the first rib. aspirate first before injection to make sure that intravenous injection does not occur. . inject one third of the volume of local anesthetic mix, and then slowly withdraw the needle and fan dorsally and ventrally while infusing the remaining fluid. . local anesthetic doses are similar to those for intrapleural blockade. epidural analgesia refers to the injection of an opioid, a phencyclidine, an α-agonist, or an nsaid into the epidural space. epidural anesthesia refers to the injection of a local anesthetic. in most patients a combination of the two is used. epidural analgesia and anesthesia are used for a variety of acute and chronic surgical pain or traumatically induced pain in the pelvis, tail, perineum, hind limbs, abdomen, and thorax (table - ) . procedures in which epidural analgesia and anesthesia are useful include forelimb and hind limb amputation, tail or perineal procedures, cesarean sections, diaphragmatic hernia repair, pancreatitis, peritonitis, and intervertebral disk disease. epidural blocks performed using opioids or bupivacaine will not result in hind limb paresis or decreased urinary or anal tone (incontinence), unlike lidocaine or mepivicaine epidural blocks. morphine is one of the most useful opioids for administration in the epidural space because of its slow systemic absorption. epidural catheters used for the instillation of drugs through constant rate infusion or intermittent injection can be placed in dogs and cats. routinely placed at the lumbosacral junction, these catheters are used with cocktails including preservative-free morphine, bupivacaine, medetomidine, and ketamine. extremely effective for preventing windup pain in the peritoneal cavity or caudal half of the body, the catheters may be maintained if placed aseptically for to days. to provide epidural analgesia or anesthesia, follow this procedure: . position the animal in lateral or sternal recumbency. . clip and aseptically scrub over the lumbosacral site. . palpate the craniodorsal-most extent of the wings of the ileum bilaterally and draw an imaginary line through them to envision the spine of l located immediately behind the imaginary line. . advance a -to -gauge, / -to -inch spinal or epidural needle through the skin just caudal to the spine of l . . the needle will lose resistance as it is introduced into the epidural space. drop saline into the hub of the needle, and the saline will be pulled into the epidural space as the needle enters. discrete intercostal nerve blocks can provide effective analgesia for traumatic or postsurgical pain. identify the area of the injury, and infiltrate three segments on either side of the injury with analgesic. to perform an intercostal nerve block, follow this procedure: . clip and aseptically scrub the dorsal and ventral third of the chest wall. . palpate the intercostal space as far dorsally as possible. . use a -gauge, . -inch needle at the caudolateral aspect of the affected rib segments and those cranial and caudal. . direct the tip of the needle caudally such that the tip of the needle "drops" off of the caudal rib. (this places the needle tip in proximity to the neuromuscular bundle that contains the intercostal nerve that runs in a groove on the caudomedial surface of the rib.) . aspirate to confirm that the drug will not go intravenously. . inject while slowly withdrawing the needle. inject . to . ml at each site, depending on the size of the animal. gaynor js, an acute condition in the abdomen is defined as the sudden onset of abdominal discomfort or pain caused by a variety of conditions involving intraabdominal organs. many animals have the primary complaint of lethargy, anorexia, ptyalism, vomiting, retching, diarrhea, hematochezia, crying out, moaning, or abnormal postures. abnormal postures can include generalized rigidity, walking tenderly or as if "on eggshells," or a prayer position in which the front limbs are lowered to the ground while the hind end remains standing. in some cases, it may be difficult initially to distinguish between true abdominal pain or referred pain from intervertebral disk disease. rapid progression and decompensation of the patient's cardiovascular status can lead to stupor, coma, and death in the most extreme cases, making rapid assessment, treatment, and definitive care extremely challenging. often the patient's signalment and history can increase the index of suspicion for a particular disease process. a thorough history often is overlooked or postponed in the initial stages of resuscitation of the patient with acute abdominal pain. often, asking the same question in a variety of methods can elicit an answer from the client that may lead to the source of the problem and the reason for acute abdominal pain. important questions to ask the client include the following: • what is your chief complaint or reason that you brought your animal in on emergency? • when did the signs first start, or when was your animal last normal? • do you think that the signs have been the same, better, or getting worse? • does your animal have any ongoing or past medical problems? • have similar signs occurred in the past? • does your animal have access to any known toxins, or does he or she run loose unattended? as with any other emergency, the clinician must follow the abcs of therapy, treating the most life-threatening problems first. first, perform a perfunctory physical examination. examination of the abdomen ideally should be performed last, in case inciting a painful stimulus precludes you from evaluating other organ systems more thoroughly. briefly observe the patient from a distance. are there any abnormal postures? is there respiratory distress? is the animal ambulatory, and if so, do you observe any gait abnormalities? do you observe any ptyalism or attempts to vomit? auscultate the patient's thorax for crackles that may signify aspiration pneumonia resulting from vomiting. examine the patient's mucous membrane color and capillary refill time, heart rate, heart rhythm, and pulse quality. many patients in pain have tachycardia that may or may not be accompanied by dysrhythmias. if a patient's heart rate is inappropriately bradycardic, consider hypoadrenocorticism, whipworm infestation, or urinary obstruction or trauma as a cause of hyperkalemia. assess the patient's hydration status by evaluating skin turgor, mucous membrane dryness, and whether the eyes appear sunken in their orbits. a brief neurologic examination should consist of whether the patient is actively having a seizure, or whether mental dullness, stupor, coma, or nystagmus are present. posture and spinal reflexes can assist in making a diagnosis of intervertebral disk disease versus abdominal pain. perform a rectal examination to evaluate for the presence of hematochezia or melena. finally, examination of the abdomen should proceed first with superficial and then deeper palpation. visually inspect the abdomen for the presence of external masses, bruising, or penetrating injuries. reddish discoloration of the periumbilical area often is associated with the presence of intraabdominal hemorrhage. it may be necessary to shave the fur to inspect the skin and underlying structures visually for bruising and ecchymoses. auscultate the abdomen for the presence or absence of borborygmi to characterize gut sounds. next, perform percussion and ballottement to evaluate for the presence of a gas-distended viscus or peritoneal effusion. finally, perform first superficial and then deep palpation of all quadrants of the abdomen, noting abnormal enlargement, masses, or whether focal pain is elicited in any one area. once the physical examination has been performed, implement initial therapy in the form of analgesia, fluid resuscitation, and antibiotics. treatment for any patient with an acute condition in the abdomen and shock is to treat the underlying cause, maintain tissue oxygen delivery, and prevent end-organ damage and failure. a more complete description of shock and oxygen delivery is given in the section on shock. emergency care the administration of analgesic agents to any patient with acute abdominal pain is one of the most important therapies in the initial stages of case management. many patients with acute abdominal pain are clinically dehydrated or are in hypovolemic shock because of hemorrhage. careful titration of intravenous crystalloid and colloid fluids including blood products is necessary based on the patient's perfusion parameters including heart rate, capillary refill time, blood pressure, urine output, and pcv. fluid therapy also should be based on the most likely differential diagnoses, with specific fluid types administered according to the primary disease process. in dogs, a shock volume of fluids is calculated based on the total blood volume of ml/kg/hour. in cats, shock fluid rate is based on plasma volume of ml/kg/hour. in most cases, any crystalloid fluid can be administered at an initial volume of one fourth of a calculated shock dose and then titrated according to whether the patient's cardiovascular status responds favorably or not. in cases of an acute condition in the abdomen from known or suspected hypoadrenocorticism, severe whipworm infestation, or urinary tract obstruction or rupture, . % sodium chloride fluid without added potassium is the fluid of choice. when hemorrhage is present, the administration of whole blood or packed rbcs may be indicated if the patient has clinical signs of anemia and shows clinical signs of lethargy, tachypnea, and weakness. fresh frozen plasma is indicated in cases of hemorrhage resulting from vitamin k antagonist rodenticide intoxication or hepatic failure or in cases of suspected disseminated intravascular coagulation (dic). a more thorough description of fluid therapy is given under the sections on shock and fluid therapy. the empiric use of broad-spectrum antibiotics is warranted in cases of suspected sepsis or peritonitis as a cause of acute abdominal pain. ampicillin sulbactam ( mg/kg iv q - h) and enrofloxacin ( mg/kg once daily) are the combination treatment of choice to cover gram-negative, gram-positive, aerobic, and anaerobic infections. alternative therapies include a second-generation cephalosporin such as cefotetan ( mg/kg iv tid) or cefoxitin ( mg/kg iv tid) or added anaerobic coverage with metronidazole ( to mg/kg iv tid). tissue oxygen delivery depends on a number of factors, including arterial oxygen content and cardiac output. if an animal has had vomiting and subsequent aspiration pneumonitis, treatment of hypoxemia with supplemental oxygen in the form of nasal, nasopharyngeal, hood, or transtracheal oxygen administration is important (see oxygen supplementation under emergency diagnostic and therapeutic procedures). perform a complete blood count in all cases of acute abdominal pain to determine if lifethreatening infection or coagulopathy including dic is present. in cases of sepsis, infection, or severe nonseptic inflammation, the white blood cell count may be normal, elevated, or low. examine a peripheral blood smear for the presence of toxic neutrophils, eosinophils, atypical lymphocytes, nucleated rbcs, platelet estimate, anisocytosis, and blood parasites. a falling pcv in the face of rbc transfusion suggests ongoing hemorrhage. perform a biochemistry panel to evaluate organ system function. azotemia with elevated bun and creatinine may be associated with prerenal dehydration, impaired renal function, or postrenal obstruction or leakage. the bun also can be elevated when gastrointestinal hemorrhage is present. serum amylase may be elevated with decreased renal function or in cases of pancreatitis. a normal serum amylase, however, does not rule out pancreatitis as a source of abdominal pain. serum lipase may be elevated with gastrointestinal inflammation or pancreatitis. like amylase, a normal serum lipase does not rule out pancreatitis. total bilirubin, alkaline phosphatase, and alanine transaminase may be elevated with primary cholestatic or hepatocellular diseases or may be due to extrahepatic causes including sepsis. obtain a urinalysis via cystocentesis whenever possible, except in cases of suspected pyometra or transitional cell carcinoma. azotemia in the presence of a nonconcentrated (isosthenuric or hyposthenuric) urine suggests primary renal disease. secondary causes of apparent renal azotemia and lack of concentrating ability also occur in cases of hypoadrenocorticism and gram-negative sepsis. renal tubular casts may be present in cases of acute renal ischemia or toxic insult to the kidneys. bacteriuria and pyuria may be present with infection and inflammation. when a urinalysis is obtained via free catch or urethral catheterization, the presence of bacteriuria or pyuria also may be associated with pyometra, vaginitis, or prostatitis/prostatic abscess. serum lactate is a biochemical indicator of decreased organ perfusion, decreased oxygen delivery or extraction, and end-organ anaerobic glycolysis. elevated serum lactate greater than mmol/l has been associated with increased morbidity and need for gastric resection in cases of gdv and increased patient morbidity and mortality in other disease processes. rising serum lactate in the face of adequate fluid resuscitation is a negative prognostic sign. obtain abdominal radiographs as one of the first diagnostic tests when deciding whether to pursue medical or surgical management. the presence of gdv, linear foreign body, pneumoperitoneum, pyometra, or splenic torsion warrants immediate surgical intervention. if a loss of abdominal detail occurs because of peritoneal effusion, perform additional diagnostic tests including abdominal paracentesis (abdominocentesis) and abdominal ultrasound to determine the cause of the peritoneal effusion. abdominal ultrasonography is often useful in place of or in addition to abdominal radiographs. the sensitivity of abdominal ultrasonography is largely operator dependent. indications for immediate surgical intervention include loss of blood flow to an organ, linear bunching or placation of the intestinal tract, intussusception, pancreatic phlegmon or abscess, a fluid-filled uterus suggestive of pyometra, gastrointestinal obstruction, intraluminal gastrointestinal foreign body, dilated bile duct, or gallbladder mucocele, or gas within the wall of the stomach or gallbladder (emphysematous cholecystitis). the presence of peritoneal fluid alone does not warrant immediate surgical intervention without cytologic and biochemical evaluation of the fluid present. see also abdominal paracentesis and diagnostic peritoneal lavage. abdominal paracentesis (abdominocentesis) often is the deciding factor in whether to perform immediate surgery. abdominocentesis is a sensitive technique for detecting peritoneal effusion when more than ml/kg of fluid is present within the abdominal cavity. abdominal effusion collected should be saved for bacterial culture and evaluated biochemically and cytologically based on your index of suspicion of the primary disease process. if creatinine, urea nitrogen (bun) or potassium is elevated compared with that of serum, uroabdomen is present. elevated abdominal fluid lipase or amylase compared with serum supports a diagnosis of pancreatitis. elevated lactate compared with serum lactate or an abdominal fluid glucose less than mg/dl is highly sensitive and specific for bacterial/ septic peritonitis. the presence of bile pigment or bacteria is supportive of bile and septic peritonitis, respectively. free fibers in abdominal fluid along with clinical signs of abdominal pain strongly support gastrointestinal perforation, and immediate surgical exploration is required. text continued on p. the following are clinical conditions, patient signalment, common history, physical examination, and characteristic findings of various diagnostic tests. a blank column next to a condition indicates no specific signalment, history, physical examination, or diagnostic test characteristic for a particular disease process. lack of contiguity of body wall surgical ( medical unless perforation present present c-shaped abnormal gas pattern with plication on radiographs surgical (immediate) dilation of bowel cranial to foreign object, radiopaque object in surgical (immediate) stomach or intestines, hypochloremic metabolic acidosis on bloodwork if pyloric outflow obstruction is present elevated or decreased wbc; foreign material, wbcs and medical unless perforation bacteria on abdominal fluid, elevated lactate and decreased present glucose on abdominal fluid target shaped soft tissue density on abdominal u/s, soft tissue surgical (immediate): density with gas dilation cranially on abdominal radiographs medical management of primary cause colonic distension with hard feces on radiographs medical increased or decreased wbc, septic abdominal effusion surgical (immediate) elevated t bili, alt, alk phos, and wbc hypoechoic hepatic medical after biopsy parenchyma on ultasound hepatomegaly elevated t bili, alt, alk phos, and wbc hyperechoic foci in surgical (immediate) gallbladder or sludge on u/s, free gas in wall of gall bladder abdominal effusion, bile pigment in effusion surgical (immediate) elevated t bili, alk phos, alt surgical (immediate) elevated or decreased wbc, elevated t bili, alk phos and surgical (immediate) alt, free gas in hepatic parenchyma on rads, hypoechoic mass with hyperechoic material in hepatic parenchyma on u/s heteroechoic liver with hyperechoic center on ultrasound surgical (immediate) mixed echogenic mass on ultrasound, soft tissue mass surgical (immediate or density on radiographs, elevated alk phos, alt, delayed) t bili, hypoglycemia pain-cont'd elevated t bili, alk phos, alt, amylase and/or lipase, elevated medical in most cases or decreased wbc, hypocalcemia, focal loss of detail in right unless abscess or cranial quadrant on radiographs hypo-to hyperechoic phlegmon is present pancreas with hyperechoic peri-pancreatic fat on ultrasound, abdominal and/or pleural effusion on radiographs and ultrasound pancreatic soft tissue mass effect on radiographs and surgical if mass identified, ultrasound, elevated amylase and lipase, hypoglycemia, otherwise medical elevated serum insulin management of hypoglycemia splenomegaly on radiographs, hyperechoic spleen with no surgical (immediate) blood flow on ultrasound soft tissue mass effect and loss of abdominal detail on surgical (immediate) radiographs, cavitated mass with abdominal effusion on u/s hyperechoic spleen with no blood flow on abdominal u/s, surgical (immediate) abdominal effusion, thrombocytopenia loss of abdominal detail on radiographs, peritoneal effusion medical unless refractory on u/s, hemoabdomen on abdominocentesis hypotension diagnosis based primarily on clinical signs medical fracture of the os penis on radiographs largely medical unless urethral tear diagnosis based primarily on clinical signs medical, although prepuce may need to be incised to allow replacement of penis into sheath prostatomegaly on radiographs and ultrasound hypoechoic medical prostate on u/s, pyuria and bacteriuria and u/a prostatomegaly on radiographs and ultrasound hypo-to surgical (delayed) hyperechoic prostate on u/s, bacteriuria and pyuria on u/a prostatomegaly on radiographs and ultrasound, prostatic medical/surgical mineralization on radiographs and ultrasound hypoechoic kidneys on u/s, pyuria on u/a, elevated wbc, medical azotemia pyuria, bacteriuria on u/a medical pyelectasia in abdominal u/s, azotemia surgical (immediate) renomegaly on radiographs, azotemia renal mass on u/s, renomegaly on radiographs surgical (immediate) renal mass on u/s, azotemia, lack of renal blood flow surgical (delayed) on u/s calculi in renal pelvis on radiographs and ultrasound, azotemia medical unless both kidneys affected ureteral calculi on radiographs and ultrasound, hydronephrosis, medical unless both azotemia kidneys affected ureteral calculi on radiographs and ultrasound, hydronephrosis, surgical (delayed until fluid or soft tissue density on u/s, azotemia electrolyte stabilization) diagnosis largely based on physical examination medical unless cannot pass findings urethral catheter azotemia, no peritoneal effusion, lack of urine output or surgical (delayed until outflow with ureteral catheterization, double contrast electrolyte stabilization) cystourethrogram indicated transitional cellular casts on u/a, hematuria, mass effect or surgical and medical thickened irregular urethra on ultrasound or management cystourethrogram hypoechoic swollen testicle on testicular ultrasound surgical (immediate) fluid or gas-filled tubular structure on abdominal ultrasound or surgical (immediate) abdominal radiographs soft tissue tubular structure on radiographs, fluid-filled uterus surgical ( in the event of a negative abdominocentesis, but peritoneal effusion or bile or gastrointestinal perforation are suspected, perform a diagnostic peritoneal lavage. peritoneal dialysis kits are commercially available but are often expensive and impractical (see p. ). animals that have acute abdominal pain can be divided into three broad categories, depending on the primary cause of pain and the initial definitive treatment (table - ) . some diseases warrant a nonsurgical, medical approach to case management. other conditions require immediate surgery following rapid stabilization. other conditions initially can be managed medically until the patient is hemodynamically more stable and then may or may not require surgical intervention at a later time. specific management of each disease entity is listed under its own subheading. box - lists specific indications for exploratory laparotomy. the best means to explore the abdominal cavity accurately and thoroughly is to open the abdomen on midline from the level of the xyphoid process caudally to the pubis for full exposure and then to evaluate all organs in every quadrant in a systematic manner. address specific problems such as gastric or splenic torsion, enteroplication, and foreign body removal, and then copiously lavage the abdomen with warmed sterile saline solution. suction the saline solution thoroughly from the peritoneal cavity so as to not impair macrophage function. in cases of septic peritonitis, the abdomen may be left open, or a drain may be placed for further suction and lavage. the routine use of antibiotics in irrigation solutions is contraindicated because the antibiotics can irritate the peritoneum and delay healing. when the abdominal cavity is left open, secure sterile laparotomy towels and water-impermeable dressings over the abdominal wound with umbilical tape, and then change these daily or as strike-through occurs. open abdomen cases are often effusive and require meticulous evaluation and management of electrolyte imbalances and hypoalbuminemia. the abdomen can be closed and/or the abdominal drain removed when the volume of the effusion decreases, when bacteria are no longer present, and when the neutrophils become more healthy in appearance. bischoff mg: radiographic techniques and interpretation of the acute abdomen, clin tech small anim pract ( ) anaphylactic shock occurs as an immediate hypersensitivity reaction to a variety of inciting stimuli (box - ). in animals, the most naturally occurring anaphylactic reaction results from wasp or bee stings. most other reactions occur as a result of an abnormal sensitivity to items used in making medical diagnoses or treatment. during an anaphylactic reaction, activation of c a and the complement system results in vascular smooth muscle dilation and the release of a cascade of inflammatory mediators, including histamine, slow-reacting substance of anaphylaxis, serotonin, heparin, acetylcholine, and bradykinin. clinical signs associated with anaphylaxis differ between dogs and cats. in dogs, clinical signs may include restlessness, vomiting, diarrhea, hematochezia, circulatory collapse, coma, and death. in cats, clinical signs often are associated with respiratory system abnormalities. clinical signs may include ptyalism, pruritus, vomiting, incoordination, bronchoconstriction, pulmonary edema and hemorrhage, laryngeal edema, collapse, and death. the most important steps to remember in any emergency is to follow the abcs of airway, breathing, and circulation. first, establish an airway through endotracheal intubation or emergency tracheostomy, if necessary. concurrently, an assistant should establish vascular or intraosseous access to administer drugs and fluids (box - ). the patient should be hospitalized until complete resolution of clinical signs. after initial stabilization and treatment, it is important to maintain vascular access and continue intravenous fluid therapy until the patient is no longer hypotensive, and vomiting and diarrhea have resolved. in cases of fulminant pulmonary hemorrhage and edema, administer supplemental oxygen until the patient is no longer hypoxemic or orthopneic on room air. normalize and maintain blood pressure using positive inotropes (dobutamine, - µg/kg/ minute cri) or pressors (dopamine, to µg/kg/minute iv cri; see shock). if bloodtinged vomitus or diarrhea has been observed, administer antibiotics to decrease the risk of bacterial translocation and sepsis (cefoxitin, mg/kg iv tid; metronidazole, mg/kg iv tid). also consider using gastroprotectant drugs (famotidine, . to . mg/kg iv; ranitidine, . to . mg/kg po, iv, im bid; sucralfate, . to . g po tid; omeprazole, . to . mg/kg po sid). a second and less serious form of allergic reaction is manifested as angioneurotic edema and urticaria. in most cases, clinical signs develop within minutes of an inciting allergen. although this type of reaction causes patient discomfort, it rarely poses a life-threatening problem. most animals have mild to severe swelling of the maxilla and periorbital regions. the facial edema also may be accompanied by mild to severe generalized urticaria. some animals may paw at their face, rub at their eyes, or have vomiting or diarrhea. the treatment for angioneurotic edema involves suppressing the immune response by administration of short-acting glucocorticoid drugs and blocking the actions of histamine by the synergistic use of histamine and histamine receptor blockers (box - ). in some cases, the inciting cause is a known recent vaccination or insect sting. many times, however, the inciting cause is not known and is likely an exposure to a stinging insect or arachnid. differential diagnoses for acute facial swelling and/or urticaria include acetaminophen toxicity (cats), anterior caval syndrome, lymphadenitis, vasculitis, hypoalbuminemia, and contact dermatitis. observe animals that have presented for angioneurotic edema for a minimum of to minutes after injection of the short-acting glucocorticoids and antihistamines. monitor blood pressure to make sure that the patient does not have concurrent anaphylaxis and hypotension. after partial or complete resolution of clinical signs, the animal can be discharged to its owner for observation. in dogs, mild vomiting or diarrhea may occur within to days after this type of reaction. wherever possible, exposure to the inciting allergen should be avoided. • administer short-acting glucocorticoid: complications observed while a patient is under anesthesia can be divided into two broad categories: ( ) those related to equipment malfunction or human error and ( ) the patient's physiologic response to the cardiorespiratory effects of the anesthetic drugs. careful observation of the patient and familiarity with anesthetic equipment, drug protocols, and monitoring equipment is necessary for the safest anesthesia to occur. despite this, however, anesthetic-related complications are frequent and need to be recognized and treated appropriately. many anesthetic drugs have a dose-dependent depressive effect on the respiratory system and cause a decrease in respiratory rate and tidal volume, leading to hypoventilation. respiratory rate alone is not a reliable indicator of the patient's oxygenation and ventilatory status. the respiratory tidal volume can be measured with a wright's respirometer. perform pulse oximetry and capnography as noninvasive measures of the patient's oxygenation and ventilation. ventilation can be impaired as a result of anesthetic drugs, patient position, pneumothorax, pleural effusion (chylothorax, hemothorax, pyothorax), equipment malfunction, rebreathing of carbon dioxide, thoracic wall injury, or alveolar fluid (pulmonary edema, hemorrhage, or pneumonia). problems such as a diaphragmatic hernia, gdv, or gravid uterus can impede diaphragmatic excursions once the patient is placed on its back and can lead to impaired ventilation. the work of breathing also may be increased because of increased resistance of the anesthesia circuit and increased dead space ventilation. this is particularly important in small toy breeds. clinical signs of inadequate ventilation and respiratory complications include abnormal respiratory pattern, sudden changes in heart rate, cardiac dysrhythmias, cyanosis, and cardiopulmonary arrest. end-tidal carbon dioxide, or capnography, gives a graphic display of adequacy of ventilation. rapid decreases in end-tidal carbon dioxide can be caused by disconnection or obstruction of the patient's endotracheal tube or poor perfusion, namely, cardiopulmonary arrest (see capnometry [end-tidal carbon dioxide monitoring]). postoperatively, hypoventilation can occur because of the residual effects of the anesthetic drugs, hypothermia, overventilation during intraoperative support, surgical techniques that compromise ventilation (thoracotomy, cervical disk surgery, atlantooccipital stabilization), postoperative bandaging of the abdomen or thorax, ventilatory muscle fatigue, or injury to the cns. cardiac output is a function of heart rate and stroke volume. factors that influence stroke volume include vascular and cardiac preload, cardiac afterload, and cardiac contractility. the patient's cardiac output can be affected adversely by the negative inotropic and chronotropic and vasodilatory effects of anesthetic drugs, all leading to hypotension. emergency care bradycardia, tachycardia, cardiac dysrhythmias, and vascular dilation can lead to hypotension and inadequate organ perfusion. table - lists the normal heart rate and blood pressure in dogs and cats. bradycardia is defined as a heart rate below normal values. many anesthetic drugs can cause bradycardia. causes of bradycardia include the use of narcotics or α -agonist drugs, deep plane of anesthesia, increased vagal tone, hypothermia, and hypoxia. table - lists the causes of bradycardia and the necessary immediate action or treatment. tachycardia is defined as a heart rate above normal values. common causes of tachycardia include vasodilation, drugs, inadequate anesthetic depth and perceived pain, hypercapnia, hypoxemia, hypotension, shock, or hyperthermia. table - lists the causes and immediate action or treatment for tachycardia. hypotension is defined as physiologically low blood pressure (mean arterial pressure less than mm hg). a mean arterial blood pressure less than mm hg can result in inadequate tissue perfusion and oxygen delivery. the coronary arteries are perfused during diastole. inadequate diastolic blood pressure, less than mm hg, can cause decreased coronary artery perfusion and myocardial hypoxemia that can predispose the heart to dysrhythmias. causes of perianesthetic hypotension include peripheral vasodilation by anesthetic drugs, bradycardia or tachyarrhythmias, hypothermia, inadequate cardiac preload from vasodilation or hemorrhage, decreased venous return from patient position or surgical manipulation of viscera, and decreased cardiac contractility. electrocardiogram monitoring is useful for the early detection of cardiac dysrhythmias during the perianesthetic period. clinical signs of cardiac dysrhythmias include irregular pulse rate or pressure, abnormal or irregular heart sounds, pallor, cyanosis, hypotension, and an abnormal ecg tracing. remember that the single best method of detecting cardiac emergency care vagolytic drugs atropine allow time for the drug to wear off. glycopyrrolate allow time for the drug to wear off. sympathomimetic drugs epinephrine allow time for the drug to wear off; administer a β-blocker; turn off infusion. isoproterenol administer a β-blocker. turn off infusion; administer a β-blocker. allow time for drug to wear off. inadequate anesthetic depth increase anesthetic depth. hypercapnia increase ventilation (assisted ventilation). hypoxemia increase gas flow and oxygenation. hypotension decrease anesthetic depth; administer an intravenous crystalloid or colloid bolus, positive inotrope drug, positive chronotrope drug, or pressor. hyperthermia apply ambient or active cooling measures; administer dantrolene sodium if malignant hyperthermia is suspected. hypothermia provide ambient rewarming. hypocalcemia * administer calcium chloride ( mg/kg iv) or calcium gluconate ( mg/kg). decrease vaporizer setting/anesthetic depth. reverse with opioids or a -agonists. vasodilation administer an intravenous crystalloid bolus ( ml/kg). administer an intravenous colloid bolus ( ml/kg). administer a pressor (epinephrine, phenylephrine dysrhythmias is with your fingertips (palpate a pulse or apex heartbeat) and ears (auscultate the heart). confirm the dysrhythmia by auscultating the heart rate and rhythm, identify the p waves and the qrs complexes, and evaluate the relationship between the p waves and qrs complexes. is there a p wave for every qrs, and a qrs for every p wave? during anesthesia, fluid, acid-base, and electrolyte imbalances can predispose the patient to dysrhythmias. sympathetic and parasympathetic stimulation, including the time of intubation, can predispose the patient to dysrhythmias. if the patient's plane of anesthesia is too light, perception of pain can cause catecholamine release, sensitizing the myocardium to ectopic beats. atrioventricular blockade can be induced with the administration of α -agonist medications, including xylazine and medetomidine. thiobarbiturates (thiopental) can induce ventricular ectopy and bigeminy. although these dysrhythmias may not be harmful in the awake patient, anesthetized patients are at a particular risk of dysrhythmia-induced hypotension. carefully monitor and treat all dysrhythmias (see cardiac dysrhythmias). box - lists steps to take to prevent perianesthetic dysrhythmias. awakening during anesthesia can occur and can be caused by equipment failure and simply, although no one likes to admit it, human error. table - lists causes of arousal during anesthesia and appropriate immediate actions. awaken patient, and administer dantrolene arousal (e.g., malignant hyperthermia) sodium. • stabilize acid-base and electrolyte balance before anesthetic induction, whenever possible. • rehydrate patient before anesthetic induction. • select anesthetic agents appropriate for the particular patient. • be aware of the effects of the drugs on the myocardium. • ensure adequate anesthetic depth and oxygenation before anesthetic induction. • ensure ventilatory support during anesthesia. • monitor heart rate, rhythm, blood pressure, pulse oximetry, and capnometry during anesthesia. • ensure adequate anesthetic depth before surgical stimulation. • avoid surgical manipulation to the heart or great vessels, whenever possible. • avoid changes in perianesthetic depth. • avoid hypothermia. delayed recovery can be caused by a number of factors, including excessive anesthetic depth, hypothermia, residual action of narcotics or tranquilizers, delayed metabolism of anesthetic drugs, hypoglycemia, hypocalcemia, hemorrhage, and breed or animal predisposition. careful monitoring of the patient's blood pressure, acid-base and electrolyte status, anesthetic depth, pcv, and vascular volume intraoperatively and taking care with supportive measures to prevent abnormalities can hasten anesthetic recovery and avoid postoperative complications. gaynor the presentation of a patient with a bleeding disorder often is a diagnostic challenge for the veterinary practitioner (boxes - and - ). in general, abnormal bleeding can be caused by five major categories: ( ) vascular trauma, ( ) circulating inhibitors of coagulation heparin fibrin degradation products development of spontaneous deep hematomas, unusually prolonged bleeding after traumatic injury, bleeding at multiple sites throughout the body involving multiple organ systems, delayed onset of severe hemorrhage after bleeding, and an inability on the practitioner's part to find an organic cause of bleeding. the signalment, history, clinical signs, and results of coagulation often can aid in making a rapid diagnosis of the primary cause of the disorder and in the selection of appropriate case management. when taking a history, ask the following important questions: • what is the nature of the bleeding? • what sites are affected? • how long has the bleeding been going on? • has your animal had any previous or similar episodes? • is there any possibility of any toxin exposure? • if so, when and how much did your animal consume? • is there any possibility of trauma? • does your animal run loose outdoors unattended? • have you ever traveled, and if so, where? • has your animal been on any medications recently or currently? • has your animal been vaccinated recently? • have any known relatives of your animal had any bleeding disorders? • are there any other abnormal signs that you have seen? abnormalities found on physical examination may aid in determining whether the hemorrhage is localized or generalized (i.e., bleeding from a venipuncture site versus bleeding diathesis). note whether the clinical signs are associated with a platelet problem and superficial hemorrhage or whether deep bleeding can be associated with abnormalities of the coagulation cascade. also, make an attempt to identify any concurrent illness that can predispose the patient to a bleeding disorder (i.e., pancreatitis, snakebite, sepsis, immunemediated hemolytic anemia, or severe trauma and crush or burn injury). abnormalities associated with coagulopathies include petechiae and ecchymoses, epistaxis, gingival bleeding, hematuria, hemarthrosis, melena, and hemorrhagic cavity (pleural and peritoneal or retroperitoneal) effusions. disseminated intravascular coagulation is a complex syndrome that results from the inappropriate activation of the clotting cascade, leading to disruption of the normal balance between thrombosis and fibrinolysis. the formation of diffuse microthrombi with concurrent consumption of platelets and activated clotting factors leads to end-organ thrombosis with various degrees of clinical hemorrhage. in animals, dic always results from some other pathologic process, including various forms of neoplasia, crush and heat-induced injury, sepsis, inflammation, and immune-mediated disorders (box - ). the pathophysiologic mechanisms involved in dic include vascular endothelial damage, activation and consumption of platelets, release of tissue procoagulants, and consumption of endogenous anticoagulants. because dic always results from some other disease process, diagnosis of dic is based on a number of criteria when evaluating various coagulation tests, peripheral blood smears, platelet count, and end products of thrombosis and fibrinolysis. there is no one definitive criterion for the diagnosis of dic (box - ). thrombocytopenia occurs as platelets are consumed during thrombosis. it is important to remember that trends in decline in platelet numbers are just as important as thrombocytopenia when making the diagnosis. in some cases the platelet count still may be within the normal reference range but has significantly decreased in the last hours. early in dic the procoagulant cascade dominates, with hypercoagulability. activated clotting time, aptt, and pt may be rapid and shorter than normal. in most cases, we do not recognize the hypercoagulable state in our critically ill patients. later in dic, as platelets and activated clotting factors become consumed, the act, aptt, and pt become prolonged. antithrombin, a natural anticoagulant, also becomes consumed, and antithrombin levels decline. antithrombin levels can be measured at commercial laboratories and in some large veterinary institutions. the end products of thrombosis and subsequent fibrinolysis also can be measured. fibrinogen levels may decline, although this test is not sensitive or specific for dic. fibrin degradation (split) products also become elevated. fibrin degradation products are normally cleared by the liver, and these also become elevated in cases of hepatic failure because of lack of clearance. more recently, cageside d-dimer tests have become available to measure the breakdown product of cross-linked fibrin as a more sensitive and specific monitor of dic. management of dic first involves treating the primary underlying cause. by the time dic becomes evident, rapid and aggressive treatment is necessary. if you are suspicious of dic in any patient with a disease known to incite dic, then ideally, you should begin treatment before the hemostatic abnormalities start to occur for the best possible prognosis. treatment involves replacement of clotting factors and antithrombin and prevention of further clot formation. to replenish clotting factors and antithrombin, administer fresh whole blood or fresh frozen plasma. heparin requires antithrombin as a cofactor to inactivate thrombin and other activated coagulation factors. administer heparin ( to units/kg sq q - h of unfractionated heparin; or fractionated enoxaparin [lovenox], mg/kg sq bid). aspirin ( mg/kg po bid in dogs; every third day in cats) also can be administered to prevent platelet adhesion. management of dic also involves the rule of twenty monitoring and case management to maintain end-organ perfusion and oxygen delivery (see the rule of ). hemophilia a is a sex-liked recessive trait that is carried by females and manifested in males. female hemophiliacs can occur when a hemophiliac male is bred with a carrier female. hemophilia a has been reported in cats and a number of dog breeds, including miniature schnauzer, saint bernard, miniature poodle, shetland sheepdog, english and irish setters, labrador retriever, german shepherd, collie, weimaraner, greyhound, chihuahua, english bulldog, samoyed, and vizsla. mild to moderate internal or external bleeding can occur. clinical signs of umbilical cord bleeding can become apparent in some animals shortly after weaning. gingival hemorrhage, hemarthrosis, gastrointestinal hemorrhage, and hematomas may occur. clotting profiles in animals with factor viii deficiency include prolonged aptt and act. the pt and buccal mucosa bleeding time are normal. affected animals have low factor viii activity but normal to high levels of factor viii-related antigen. carrier females can be detected by low ( % to % of normal) factor viii activity and normal to elevated levels of factor vii-related antigen. von willebrand's disease is a deficiency or defect in von willebrand's protein. a number of variants of the disease have been described: von willebrand's disease type i is associated with a defect in factor viir/protein concentration, and von willebrand's disease type ii is associated with a defect in viiir:vwf. type i von willebrand's disease is most common in veterinary medicine. von willebrand's disease has been identified in more than breeds of dogs, with an incidence that varies from % to % depending on the breed of origin. affected breeds include doberman pinchers, german shepherd dogs, scottish terriers and standard manchester terriers, golden retrievers, chesapeake bay retrievers, miniature schnauzers, and pembroke welsh corgis. two forms of genetic expression occur: ( ) autosomal recessive disease in which homozygous von willebrand's disease individuals have a bleeding disorder, whereas heterozygous individuals carry the trait but are clinically normal. the second variant of genetic expression involves an autosomal dominant disease with incomplete expression such that heterozygous individuals are affected carriers and homozygous individuals are severely affected. von willebrand's disease has high morbidity, but fortunately a low mortality. dogs with % or less than normal vwf tend to hemorrhage. platelet counts are normal, but bleeding times can be prolonged. the aptt can be slightly prolonged when factor viii is less than % of normal. routine screening tests are nondiagnostic for this disease, although in a predisposed breed with a normal platelet count, a prolonged buccal mucosa bleeding time strongly supports a diagnosis of von willebrand's disease. documentation of clinical bleeding with low or undetectable levels of factor viii antigen or platelet-related activities of vwf support a diagnosis of von willebrand's disease. recessive animals have zero vwf:antigen (a subunit of factor iii); heterozygotes have % to % of normal. in the incompletely dominant form, levels of vwf antigen are reduced (less than % to %). clinical signs in affected animals include epistaxis, hematuria, diarrhea with melena, penile bleeding, lameness, hemarthrosis, hematoma formation, and excessive bleeding with routine procedures such as nail trimming, ear cropping, tail docking, surgical procedures (spay, neuter), and lacerations. estrous and postpartum bleeding may be prolonged. a dna test to detect carriers of the vwf gene is available through vetgen (ann arbor, michigan) and michigan state university. patients with von willebrand's disease should avoid drugs known to affect platelet function adversely (sulfonamide, ampicillin, chloramphenicol, antihistamines, theophylline, phenothiazine tranquilizers, heparin, and estrogen). hemophilia b is an x-linked recessive trait that occurs with less frequency that hemophilia a. the disease has been reported in scottish terriers, shetland and old english sheepdogs, saint bernards, cocker spaniels, alaskan malamutes, labrador retrievers, bichon frises, airdale terriers, and british shorthair cats. carrier females have low ( % to % of normal) factor ix activity. clinical signs are more severe than for hemophilia a. congenital deficiencies of factor vii have been reported as an autosomal, incompletely dominant characteristic in beagles. heterozygotes have % factor vii deficiency. bleeding tends to be mild. the pt is prolonged in affected individuals. factor x deficiency has been documented in cocker spaniels and resembles fading-puppy syndrome in newborn dogs. internal or umbilical bleeding can occur, and affected dogs typically die. bleeding may be mild in adult dogs. in severe cases, factor x levels are reduced to % of normal; in mild cases, factor x levels are % to % of normal. factor xii deficiency has been documented as an inherited autosomal recessive trait in domestic cats. heterozygotes can be detected because they have a partial deficiency ( % of normal) of factor xii. homozygote cats have less than % factor xii activity. deficiency of hageman factor usually does not result in bleeding or other disorders. factor xi deficiency is an autosomal disease that has been documented in kerry blue terriers, great pyrenees, and english springer spaniels. in affected individuals, protracted bleeding may be observed. homozygotes have low factor xi activity (< % of normal), and heterozygotes have % to % of normal. the management of congenital defects of hemostasis typically involves replenishing the clotting factor that is present. usually, this can be accomplished in the form of fresh frozen plasma transfusion ( ml/kg). if anemia is present because of severe hemorrhage, fresh whole blood or packed rbcs also can be administered. recent research has investigated the use of recombinant gene therapy in the treatment of specific factor deficiencies in dogs; however, the therapy is not yet available for use in clinical practice. in cases of von willebrand's disease, administration of fresh frozen plasma ( to ml/kg) or cryoprecipitate ( unit/ kg body mass) provides vwf, factor viii, and fibrinogen. doses can be repeated until hemorrhage ceases. -desamino- -d-arginine vasopressin (ddavp) also can be administered ( µg/kg sc or iv diluted in . % saline given over to minutes) to the donor and patient to increase the release of stored vwf from endothelial cells. a fresh whole blood transfusion can be obtained from the donor and immediately administered to the patient, or spun down and the fresh plasma administered if rbcs are not needed. administer a dose of ddavp to any affected dog before initiating any elective surgical procedures. a supply of fresh frozen plasma and rbcs should be on hand, should uncontrolled hemorrhage occur. platelets are essential to normal blood coagulation. after a vessel is damaged, release of vasoactive amines causes vasoconstriction and sluggish flow of blood in an attempt to squelch hemorrhage. platelets become activated by platelet activating factor, and attach to the damaged vascular endothelium. normal platelet adhesion depends on mediators such as calcium, fibrinogen, vwf:antigen, and a portion of factor viii. after adhesion, the platelets undergo primary aggregation and release a variety of chemical mediators including adenosine diphosphate, prostaglandins, serotonin, epinephrine, thromboplastin, and thromboxane a that promote secondary aggregation and contraction. platelet abnormalities can include decreased platelet production (thrombocytopenia), decreased platelet function (thrombocytopathia), increased platelet destruction, increased platelet consumption, and platelet sequestration. thrombocytopathia refers to platelet function abnormalities. alterations in platelet function can affect platelet adhesion, aggregation, or release of vasoactive substances that help form a stable clot (box - ). in von willebrand's disease there is a deficiency in vwf:antigen that results in altered platelet adhesion. vascular purpuras are reported and have been seen in collagen abnormalities such as ehlers-danlos syndrome, which can be inherited as an autosomal dominant trait with complete penetrance and has been recognized in german shepherd dogs, dachshunds, saint bernards, and labrador retrievers. thrombasthenic thrombopathia is a hereditary autosomal dominant abnormality that has been described in otterhounds, foxhounds and scottish terriers. in this condition, platelets do not aggregate normally in response to adenosine diphosphate and thrombin stimulation. evaluation of platelet function is based on a total platelet count, buccal mucosa bleeding time, and thromboelastography. platelet function defects (thrombocytopenia and thrombocytopathia) can affect both sexes. clinical signs can resemble von willebrand's disease. in most cases, buccal mucosa bleeding time will be prolonged, but platelet count and clotting tests will be normal. platelet count can be decreased because of problems with production, increased consumption, sequestration, or destruction. causes of accelerated platelet destruction are typically immune-mediated autoantibodies, drug antibodies, infection, and isoimmune destruction. consumption and sequestration usually are caused by dic, vasculitis, microangiopathic hemolytic anemia, severe vascular injury, hemolytic uremic syndrome, and gram-negative septicemia. primary thrombocytopenia with no known cause has been called idiopathic thrombocytic purpura. in approximately % of the cases, thrombocytopenia is associated with immune-mediated destruction caused by immune-mediated hemolytic anemia, systemic lupus erythematosus, rheumatoid arthritis, dic, and diseases that affect the bone marrow. in systemic lupus erythematosus, % to % of the affected dogs have concurrent idiopathic thrombocytic purpura. when immune-mediated hemolytic anemia and idiopathic thrombocytic purpura are present in the same patient, the disease is called evans syndrome. pf- is a non-complement-fixing antibody that is produced in the spleen and affects peripheral and bone marrow platelets and megakaryocytes. antibodies directed against platelets are usually of the igg subtype in animals. antiplatelet antibodies can be measured by a pf- release test. platelet counts with immune-mediated destruction typically are less than , platelets/µl. infectious causes of thrombocytopenia include ehrlichia canis, anaplasma phagocytophilum (formerly, ehrlichia equi), and rickettsia rickettsii (rocky mountain spotted fever). primary immune-mediated thrombocytopenia has an unknown cause and most frequently is seen in middle-to older-aged female dogs. breed predispositions include cocker spaniels, german shepherd dogs, poodles (toy, miniature, standard), and old english sheepdogs. thrombocytopenia usually is manifested as petechiae, ecchymoses of skin and mucous membranes, hyphema, gingival and conjunctival bleeding, hematuria, melena, and epistaxis. to make a diagnosis of idiopathic thrombocytic purpura, measure the severity of thrombocytopenia (< , platelets/µl), analyze the peripheral blood smear for evidence of platelet fragmentation or microthrombocytosis, normal to increased numbers of megakaryocytes in the bone marrow, detection of antiplatelet antibody, increased platelet counts after starting glucocorticoid therapy, and elimination of other causes of thrombocytopenia. if tick-borne illnesses are suspected, antibody titers for e. canis, a. phagocytophilum (formerly e. equi), and r. rickettsii should be performed. treatment of immune-mediated thrombocytopenia involves suppression of the immune system to stop the immune-mediated destruction and to stimulate platelet release from the bone marrow. traditionally, the gold standard to suppress the immune system is to use glucocorticoids (prednisone or prednisolone, to mg/kg po bid divided, or dexamethasone, . to . mg/kg iv or po q h). more recently human serum immunoglobulin (igg) also has been used ( . to . g/kg iv in saline over hours; pretreat with mg/kg diphenhydramine minutes before starting infusion). vincristine ( . mg/m iv once) can stimulate the release of platelets from the bone marrow if megakaryocytic precursors are present; however, the platelets released may be immature and potentially nonfunctional. treatment with fresh whole blood or packed rbcs is appropriate if anemia is present; however, unless specific platelet-rich plasma has been purchased from a blood bank, fresh whole blood contains relatively few platelets, which are shortlived ( hours) and will not effectively raise the platelet count at all. finally, long-term therapy is usually in the form of azathioprine ( mg/kg po once daily, tapered to mg/kg daily to every other day after week) and cyclosporine ( to mg/kg po divided). if a tickborne illness is suspected, administer doxycycline ( to mg/kg po bid) for weeks or if titers come back negative. thrombocytopenia also can occur in the cat. causes for thrombocytopenia in cats include infections ( %), neoplasia ( %), cardiac disease ( %), primary immune-mediated disease ( %), and unknown causes ( %). in one study of cats with feline leukemia and myeloproliferative disease, % of cases had thrombocytopenia. warfarin and coumarin derivatives are the major class of rodenticides used in the united states. vitamin k antagonist rodenticides inhibit the epoxidase reaction and deplete active vitamin k, causing a depletion of vitamin k-dependent coagulation factors (ii, vii, ix, x) within hours to week of ingestion, depending on the ingested dose. affected animals can spontaneously hemorrhage anywhere in the body. clinical signs can include hemoptysis, respiratory difficulty, cough, gingival bleeding, epistaxis, hematuria, hyphema, conjunctival bleeding, petechiae and ecchymoses, cavity hemorrhage (pleural, peritoneal, retroperitoneal) with acute weakness, lethargy or collapse, hemarthrosis with lameness, deep muscle bleeds, and intracranial or spinal cord hemorrhage. diagnosis of vitamin k antagonism includes prolonged pt. a pivka (protein induced by vitamin k absence or antagonism) test also can be performed, if possible. treatment of vitamin k antagonist rodenticide intoxication and other causes of vitamin k deficiency involves supplementation with vitamin k (phytonadione, mg/kg sq once with -gauge needle in multiple sites, and then . mg/kg po bid to tid for days). never administer injections of vitamin k intramuscularly, because of the risk of causing deep muscle hematomas, or intravenously, because of the risk of anaphylaxis. the pt should be rechecked days after the last vitamin k capsule is administered, for some of the secondgeneration warfarin derivates are fat-soluble, and treatment may be required for an additional weeks. act, activated clotting time; aptt, activated partial thromboplastin time; bmbt, buccal mucosa bleeding time; fdp, fibrin degradation products; n, normal; pt, prothrombin time. thermal burns are fortunately a relatively infrequent occurrence in veterinary patients. box - lists various causes of malicious and accidental burns. the location of the burn is also important in assessing its severity and potential to lose function. burns on the perineum, feet, face, and ears are considered to be the most severe because of loss of function and severe pain. often the severity of thermal injury is difficult to assess in animals because hair coat potentially can mask clinical signs and because the thermal injury can continue after the animal has been removed from the heat source. the skin cools slowly and warms slowly, considerations that become important when initiating therapy for burns. the severity of thermal injury is associated with the temperature to which the animal is exposed, the duration of contact, and the ability of the tissue to dissipate heat. the tissue closest to the heat source undergoes necrosis and has decreased blood flow. the severity of thermal burn injury is associated directly with the temperature to which the animal is exposed, the percentage of total body surface area affected, the thickness of injured tissue, and whether underlying complications with other body systems occur. prognosis largely depends on the total body surface area affected (table - ) . superficial partial thickness, or first-degree, burns offer the most favorable prognosis. the affected epidermis initially appears erythematous and then quickly desquamates within to days. in most cases, fur grows back without leaving a scar. deep partial thickness, or second-degree, burns involve the epidermis and dermis and are associated with subcutaneous edema, inflammation, and pain. deep partial thickness burns heal from deeper adnexal tissues and from the wound edges and are associated with an increased chance of scarring and depigmentation. the most severe type is known as full thickness, or third-degree, burns, in which thermal injury destroys the entire thickness of the skin and forms an eschar. thrombosis of superficial and deeper skin vasculature and gangrene occurs. treatment involves sequential wound debridement. healing occurs by second intention and reepithelialization or by wound reconstruction. in most cases, scarring is extensive in affected areas. burns greater than % of total body surface area will have systemic effects, including impaired cardiovascular function, pulmonary dysfunction, and impaired immune function. burned tissue, with capillary damage, has increased permeability. the release of inflammatory cytokines, oxygen-derived free radical species, prostaglandins, leukotrienes, emergency care histamine, serotonin, and kinins results in increased vascular permeability and leakage of plasma proteins into the interstitium and extravascular space. at the time of presentation, first examine the patient and ascertain whether airway obstruction, impaired ventilatory function, circulatory shock, or pain are present. if necessary, establish an airway with endotracheal intubation or emergency tracheostomy. next, cool the burned area(s) with topical cool water. use care to avoid overcooling and iatrogenic hypothermia. the best approach is to cool only one portion of the patient's body at a time, then dry, and repeat the process for all affected areas to avoid overcooling and iatrogenic hypothermia. establish vascular access and administer appropriate and judicious analgesic drugs and intravenous fluid therapy. whenever possible, avoid placing a catheter through an area of burned or damaged skin. in the early stages of burn injury, shock doses of intravenous crystalloid fluids usually are not required. later, however, as severe tissue exudation occurs, protein and fluid losses can become extensive, requiring aggressive crystalloid and colloid support to treat hypovolemia and hypoproteinemia. flush the eyes with sterile saline and examine behind the third eyelids for any particulate matter. stain the corneas to make sure that superficial corneal burns are not present. treat superficial corneal burns with triple antibiotic ophthalmic ointment. next, assess the total body surface area affected, as this will gauge prognosis. depending on the extent of the damage, decide whether the burn is superficial and local therapy is indicated or whether more severe injuries exist that may involve systemic therapy or possibly euthanasia. in most cases the diagnoses of thermal burns are based on a clinical history of being in a house fire, clothes dryer, or under a heating lamp. too frequently, however, thermal burns become apparent days after an elective surgical procedure in which the patient was placed on a faulty heating pad rather than a circulating warm water or warm air blanket. superficial burns appear as singed fur with desquamating, easily epilated hair. this condition also can resemble a superficial or deeper dermatophytosis if history is unknown. other differential diagnoses include immune-mediated vasculitis or erythema multiforme. unless the superficial dermis is blistered, it may be difficult to distinguish between a thermal burn, chemical burn, or electrical burn if the trauma went unnoticed. management of burn injury largely depends on the depth of injury and the total body surface area affected. partial thickness burns and those affecting less than % of the total body surface area will require support in the form of antibiotic ointment and systemic analgesic drugs. burns affecting greater than % of total body surface area or deep thickness burns require more aggressive therapy. central venous catheters can be placed to administer crystalloid and colloid fluids, parenteral nutrition if necessary, antibiotics, and analgesic drugs. monitor perfusion parameters closely, including heart rate, blood pressure, capillary refill time, and urine output. respiratory function can be impaired because of concurrent smoke inhalation, thermal damage to the upper airways and alveoli, and carboxyhemoglobin or methemoglobin intoxication. respiratory function also can be impaired because of burn injury to the skin around the thoracic cage. thoracic radiographs may reveal patchy interstitial to alveolar infiltrates associated with pulmonary edema, pneumonia, and atelectasis. bronchoscopy often reveals edema, inflammation, particulate matter, and ulceration of the tracheobronchial tree. in some cases, upper airway inflammation is so severe that an emergency tracheostomy must be performed to treat airway obstruction. administer supplemental humidified oxygen at to ml/kg/minute via endotracheal tube, tracheostomy, nasal or intratracheal tube, or hood oxygen if respiratory function and hypoxemia are present. perform blood work including a hematocrit, albumin, bun, creatinine, and glucose at the time of presentation. monitor serum electrolytes, albumin, and colloid oncotic pressure closely because derangements can be severe as burns become exudative. the goal of fluid therapy in the burn patient is to establish and maintain intravascular and interstitial fluid volume, normalize electrolyte and acid-base status, and maintain serum albumin and oncotic pressure. in the first hours following burn injury, direct fluid therapy to maintaining the patient's metabolic fluid requirements. crystalloid fluids in the form of normosol-r, plasmalyte-m, or lactated ringer's solution can be administered according to the patient's electrolyte and acid-base status (see fluid therapy). monitor urine output, and keep it at to ml/kg/hour. avoid overhydration in the early stages of burn injury. in affected burn patients, calculate the amount of fluid that should be administered over a -hour period from the formula − ml/kg × percent total body surface area. administer half of this calculated dose over the first hours and then the remaining half over the next hours. in cats, administer only % to % of this calculated volume. to administer this volume and also avoid fluid overload is often difficult in critically ill patients with pulmonary involvement associated with smoke inhalation injury. avoid colloids in the first hours after burn injury. monitor the patient closely for serous nasal discharge, chemosis, and rales that may signify pulmonary edema. as burns become exudative, weigh the patient at least twice daily. infused fluid should equal fluid output in the form of urine and wound exudates. acute weight loss signifies acute fluid loss and that crystalloid fluid infusion should be more aggressive. ideally, keep the patient's serum albumin equal to or greater than . g/dl and total protein between . and . g/dl using a combination of fresh frozen plasma or concentrated human albumin. adjunct colloidal support can be provided with synthetic colloids including hetastarch or hbocs. keep serum potassium within . to . meq/l using potassium chloride or potassium phosphate supplementation. if potassium supplementation exceeds to meq/l and the patient continues to have severe refractory hypokalemia, administer magnesium chloride ( . meq/kg/day) to enhance potassium retention. if anemia occurs, administer packed rbcs or whole blood (see blood component therapy). lavage wounds daily with lactated ringer's solution or . % sodium chloride solution. place wet-to-dry bandages or bandages soaked in silver sulfadiazine or nitrofurazone ointment over the wounds. depending on the thickness of the burn, epilation and eschar formation and separation may take to days. at each bandage change, debride devitalized tissue to normal tissue. perform staged partial or total escharectomy, and leave the wound to heal by second intention or by reconstruction using skin advancement flaps or grafts. maintain meticulous sterility at all times, given that burn patients are at high risk for infection. administer broad-spectrum antibiotics including cefazolin and enrofloxacin. perform wound culture if a resistant bacterial infection is suspected. the most common cause of electrical injury is associated with an animal chewing on low-voltage alternating current electrical cords in the household. damage is caused by the current flowing through the path of least resistance, causing heat and thrombosis of vessels and neurons. in some cases, the owner witnesses the event. in other cases, the owner presents the patient because of vague nonspecific signs, and characteristic abnormalities on physical examination support a diagnosis of electrocution. burns on the face, paws, commissures of the mouth, tongue, and soft palate may be present. electrocution causes a massive release of catecholamines and can predispose the patient to noncardiogenic pulmonary edema within hours of the incident. clinical signs may be isolated to the pulmonary system, including orthopnea, pulmonary crackles, and cyanosis. assess the patient's lips, tongue, soft palate, gingivae, and commissures of the mouth. early after electrocution, the wound may appear small and white, black, or yellow. later, the wound may become larger as tissue sloughs because of damaged vascular supply. assess the patient's respiratory status. auscultate the lungs to determine whether pulmonary crackles emergency care are present. if the patient is stable, thoracic radiographs may demonstrate an interstitial to alveolar lung pattern in the dorsocaudal lung fields. measure the patient's heart rate, blood pressure, oxygenation as determined by pulse oximetry or arterial blood gas and urine output. immediate treatment consists of judicious use of analgesics for the burn injury, antibiotics (cefazolin, mg/kg q h; cephalexin, mg/kg q h), and humidified supplemental oxygen ( to ml/kg/minute). direct fluid therapy at providing the patient's metabolic fluid requirements. because of the risk of development of noncardiogenic pulmonary edema, avoid overzealous administration of crystalloid fluids. differential diagnoses for the patient with electrical burn injury and electrocution include chemical or thermal burn, immune-mediated glossitis, cardiogenic pulmonary edema, and pneumonia. management of the patient with electrical burn injury and electrocution primarily involves the administration of analgesic agents, supplemental humidified oxygen, and topical treatment of electrical burns. the noncardiogenic pulmonary edema is typically unresponsive to diuretics (i.e., furosemide), bronchodilators (i.e., aminophylline), and splanchnic vascular dilators (i.e., low-dose morphine). the use of glucocorticoids has no proven benefit and may impair respiratory immune function and is therefore contraindicated. oral burns may require debridement and advancement flaps if large defects or oronasal fistulas develop. if oral injury is severe, place an esophagostomy or percutaneous gastrostomy tube to ensure adequate nutrition during the healing process. if an animal survives the initial electrocution, prognosis is generally favorable with aggressive supportive care. chemical burns are associated with a number of inciting causes, including oxidizing agents, reducing agents, corrosive chemicals, protoplasmic poisons, desiccants, and vesicants. the treatment for chemical burns differs slightly from that for thermal burns, so it remains important to investigate the cause of the burn when providing initial treatment, whenever possible. at the scene, advise the owner to wrap the patient in a clean towel for transport. chilling can be avoided by then wrapping the patient in a second or third blanket. placement of ointments by well-doers should be avoided. encourage immediate transport to the nearest triage facility. the first and foremost consideration when treating a patient with chemical burn is to remove the animal from the inciting cause or offending agent. make no attempt to neutralize alkaline or acid substances because the procedure potentially could cause an exothermic reaction, leading to thermal injury in addition to the chemical injury. remove collars or leashes that may act as tourniquets or constricting devices. flush affected areas with copious amounts of cool water for several minutes, not cooling more than % to % of the body at any one time to prevent iatrogenic hypothermia. support breathing by extending the patient's head and neck. carefully clip the fur over affected areas for further evaluation of the extent of the injury. lavage exposed eyes with sterile saline, and stain the cornea to evaluate for any corneal burns. debride any wounds carefully, knowing that the full extent of the wound may not manifest itself for several days. then cover the wounds with antibiotic burn ointment such as silver sulfadiazine and an occlusive dressing. without a history of exposure, the differential diagnosis for any chemical burn includes thermal burn, necrotizing vasculitis, erythema multiforme, or superficial or deep pyoderma. contact local or national animal poison control regarding whether to attempt neutralization. perform daily bandage changes with staged debridement as the full extent of the wound manifests itself. place antimicrobial ointment and silver sulfadiazine ointment over the wound to prevent infection. the routine use of antibiotics may promote the development of a resistant bacterial infection. first-generation cephalosporin can be administered. if a more serious infection develops, perform culture and susceptibility testing to direct appropriate antibiotic therapy. the wound can heal by second intention or may require reconstructive repair for definitive closure. the primary cause of radiation injury in small animal patients is radiation therapy for neoplastic conditions. the goal of radiation therapy is to kill neoplastic cells. an unfortunate side effect is damage to adjacent normal tissue that results in necrosis, fibrosis, and impaired circulation to the affected area. radiation burns result in dermatitis, mucositis, impaired surgical wound healing, and chronic nonhealing wounds. in many cases, the degree of secondary radiation injury to normal tissue can be prevented or decreased with careful radiation planning and mapping of the radiation field, such that radiation exposure to normal tissue is limited to the smallest extent possible. with the advent of three-dimensional imaging modalities such as computed tomography (ct) and magnetic resonance imaging (mri), this has become more routine in veterinary oncology to date. radiation injury can be early and appear at the later stage of the course of radiation therapy. late effects can be delayed and occur months to years after treatment. the degree of radiation injury is categorized based on the depth of tissue affected. first-degree changes cause cutaneous erythema. second-degree changes cause superficial desquamation. thirddegree changes cause deeper moist desquamation, and fourth-degree changes are associated with complete dermal destruction and ulceration. during the early stages of radiation injury, affected tissues may appear erythematous and edematous. wound exudates may be moist, or the skin may appear dry and scaly with desquamation or ulceration. later, the area may scar and depigment or may have induration, atrophy, telangiectasia, keratosis, and decreased adnexal structures. treatment for radiation dermatitis is to irrigate the area with warmed saline and to protect the area from self-mutilation. no-bite, or elizabethan, collars or loose clothing can be used to protect the area for patient-induced injury. mucositis can be treated with topical green tea baths and the administration of an oral solution of l-glutamine powder ( g/m ). local irrigation of xylocaine or lidocaine viscous jelly can be used in dogs but should be avoided in cats because of the risk of inducing hemolytic anemia and neurotoxicity. topical and systemic antibiotics (cephalexin, mg/kg po tid) also can be administered. avoid antibiotics that can be sensitized by radiation (i.e., metronidazole). because most radiation burns are associated with a known exposure to radiation therapy, the cause of the patient's injury usually is known. if an animal presents to you with a scar, however, differential diagnoses may include nasal planum solar dermatitis, pemphigus foliaceus, discoid lupus, superficial necrolytic dermatitis, superficial or deep pyoderma, chemical burn, or thermal burn. treatment of radiation injury involves making the patient as comfortable as possible with analgesic drugs, prevention of self-mutilation, and staged debridement techniques. wounds can heal by second intention or may require reconstructive surgery. distress syndrome (ards), and anesthetic agents. the acute onset of bradycardia, change in mucous membrane color and capillary refill time, change in respiratory pattern, and change in mentation are signs of possible deterioration and impending cardiopulmonary arrest. the diagnosis of cardiopulmonary arrest is based on the absence of effective ventilation, severe cyanosis, absence of a palpable pulse or apex heartbeat, absence of heart sounds, and ecg evidence of asystole or other nonperfusing rhythm such as electricalmechanical dissociation (aka pulseless electrical activity) or ventricular fibrillation. the goals of cpcr are to obtain airway access, provide artificial ventilation and supplemental oxygen, implement cardiac compressions and cardiovascular support, recognize and treat dysrhythmias and arrhythmias, and provide stabilization and treatment for cardiovascular, pulmonary, and cerebral function in the event of a successful resuscitation. even with aggressive treatment and management, the overall success of cpcr is less than % in critically ill or traumatized patients and % to % in anesthetized patients. basic life support involves rapid intubation to gain airway access, artificial ventilation, and cardiac compressions to promote blood flow and delivery of oxygen to the brain and other important tissues (figure - ). perform the abcs or cabs of cpcr, where a is airway, b is breathing, and c is compression and circulation. recently, the paradigm has shifted to cabs. while a team member is grabbing an endotracheal tube, clearing the airway of foreign debris, and establishing airway access through endotracheal intubation, a second person starts external cardiac compressions to deliver oxygen that is in the bloodstream to the vital organs. the patient should be positioned in dorsal (> kg) or lateral (< kg) recumbency for external cardiac compressions. approximately to external compressions should be performed over the patient's sternum. a team member should palpate for a peripheral pulse to determine whether cardiac compressions are actually effective. if a peripheral pulse cannot be palpated for every chest compression, change the patient's position and have a larger individual perform compressions, or initiate open-chest cardiac resuscitation. once the patient is intubated, tie in the endotracheal tube and attach it to an oxygen source (anesthetic machine or mechanical ventilator or ambu bag) for artificial ventilation. the oxygen flow rate should be ml/kg/minute. give two long breaths, and then to breaths per minute. simultaneous ventilation with thoracic compression increases the pressure difference in the thorax and allows more forward flow of oxygenated blood through the great vessels into the periphery. if possible, a third team member can initiate interposed abdominal compressions, compressing the abdomen when the thoracic cage is relaxed, to improve forward flow. if only one person is available to perform the thoracic compressions and ventilation, give two breaths for every compressions (i.e., thoracic compressions followed by two long breaths, and then start thoracic compressions again). the jen chung maneuver can be performed by placing a -to -gauge hypodermic needle through the skin of the nasal philtrum and twisting the needle into the periosteum to stimulate respirations. this maneuver appears to work better in cats than dogs at return to spontaneous respiration. advanced life support during cpcr involves ecg, pulse oximetry and capnometry monitoring, administration of drugs, and the administration of intravenous fluids (in select cases). most of the drugs used during cpcr can be administered directly into the lungs from the endotracheal tube (intratracheal tube). therefore, only in select instances is it necessary to establish vascular or intraosseous access during cpcr (figure - ) . if an animal experiences cardiopulmonary arrest because of extreme hemorrhage or hypovolemia, inappropriate vasodilation caused by sepsis or systemic inflammation, or vasodilation resulting from anesthesia, the administration of shock volumes ( ml/kg/hour in dogs and ml/kg/hour in cats) is appropriate. if a patient is euvolemic and experiences cardiopulmonary arrest, however, an increase in circulating fluid volume actually can impair coronary artery perfusion by increasing diastolic arterial blood pressure and is asystole is one of the most common rhythm disturbances that causes cardiac arrest in small animal patients. one of the most important things to do when the ecg looks like asystole is to make sure that the ecg monitor is working properly and that all ecg leads are attached properly to the patient. if asystole is truly present, reverse any opiate, α -agonist, or benzodiazepine drugs with their appropriate reversal agents. lowdose epinephrine ( . to . mg/kg diluted with ml sterile saline) can be administered directly into the endotracheal tube via a rigid or red rubber catheter. if vascular access is available, epinephrine ( . to . mg/kg) can be administered intravenously. no drug should ever be administered directly into the heart by intracardiac injection. unless the heart is in the veterinarian's hand during open-chest cpcr, intracardiac injection is risky and potentially could lacerate a coronary artery or cause the myocardium to become more irritable and refractory to other therapies, if a drug is delivered into the myocardium and not into the ventricle. for these reasons, intracardiac injections are contraindicated. administer atropine ( . mg/kg iv, io, or . mg/kg it) immediately after the epinephrine. atropine, a vagolytic drug, serves to decrease tonic vagal inhibition of the sinoatrial and atrioventricular node and increase heart rate. administer atropine and epinephrine every to minutes during asystole while cardiac compressions, interposed abdominal compressions, and artificial ventilation are continued. although discontinuation of thoracic compressions can decrease the chance of success during cpcr, you must intermittently evaluate the ecg monitor for any rhythm change that may require different drug therapies. if the cardiac arrest was not witnessed or more than to minutes have passed without successful return to a perfusing rhythm, perform open-chest cpcr, if the client wishes. administer sodium bicarbonate ( to meq/kg iv) every to minutes during cpcr. sodium bicarbonate is the only drug used in cpcr that should not be administered intratracheally because of inactivation of pulmonary surfactant. electrical-mechanical dissociation also is known as pulseless electrical activity and is an electrical rhythm that may look wide and bizarre and irregular with no associated mechanical contraction of the ventricles. the rhythm can appear different from patient to patient. electrical-mechanical dissociation is one of the more common nonperfusing rhythms observed during cardiopulmonary arrest in small animal patients (figure - ) . when electrical-mechanical dissociation is identified, first confirm the rhythm and proceed with cpcr as previously described. electrical-mechanical dissociation is thought to be associated with high doses of endogenous endorphins and high vagal tone. the treatment of choice for electrical-mechanical dissociation is high-dose atropine ( mg/kg iv, it [ times the normal dose]) and naloxone hydrochloride ( . mg/kg iv, io, it). administer epinephrine ( . to . mg/kg diluted in ml sterile . % saline it). if the rhythm does not change within minutes, consider open-chest cardiac massage. ventricular fibrillation can be coarse (figure - ) . patients with coarse ventricular fibrillation are easier to defibrillate than those with fine defibrillation. if ventricular fibrillation is identified, initiate cpcr as described previously (figure - ) . if an electrical defibrillator is available, administer j/kg of direct current externally. when a patient in cardiopulmonary arrest is attached to ecg leads, it is important to use contact electrode paste, water-soluble gel such as ky jelly, or water, rather than any form of alcohol. electrical defibrillation of a patient who has alcohol on the ecg leads can lead to fire and thermal burns. reverse any opioid, α -agonist, and phenothiazine drugs that have been administered to the patient. if fine ventricular fibrillation is identified, administer epinephrine figure - : electrical-mechanical dissociation (emd), also known as pulseless electrical activity (pea). the complexes often appear wide and bizarre without a palpable apex beat or functional contraction of the heart. this is just one example of emd, as many shapes and complexes may be observed. organized according to whether an electrical defibrillator is available. after each intervention step, the ecg should be reevaluated and the next step initiated if v-fib is still seen. if a new arrhythmia develops, the appropriate therapy for that rhythm should be inititated. if a sinus rhythm is seen with a palpable apex beat, postresuscitation measures should be implemented. perform open-chest cpcr immediately if a pathologic condition exists that prevents enough of a change in intrathoracic pressure that closed-chest cpcr will not be effective in promoting forward blood flow (box . to perform open-chest cpcr, place the patient in right lateral recumbency. clip a wide strip of fur over the left fifth to seventh intercostal space and quickly aseptically scrub over the clipped area. using a no. scalpel blade, incise over the fifth intercostal space through the skin and subcutaneous tissue to the level of the intercostal muscles. with a mayo scissors, make a blunt stab incision through the intercostal muscles in the left sixth intercostal space. make sure that the person who is breathing for the patient deflates the lungs as you make the stab incision to avoid iatrogenic lung puncture. after the stab incision, open the tips of the mayo scissors and quickly open the muscle dorsally and ventrally to the sternum with a sliding motion. avoid the internal thoracic artery at the sternum and the intercostal arteries at the caudal aspect of each rib. cut the rib adjacent to the sternum and push it behind the rib in front of and at the caudal aspect of the incision to allow more room and better visualization if a rib spreading retractor is not available. visualize the heart in the pericardial sac. visualize the phrenic nerve, and incise the pericardium just ventral to the phrenic nerve. make sure to not cut the phrenic nerve. grasp the heart in your hand(s) and gently squeeze it from apex to base, allowing time for the ventricle to fill before the next "contraction." if the heart does not seem to be filling, administer fluids intravenously or directly into the right atrium. the descending aorta can be cross-clamped with a rummel tourniquet or red rubber catheter to improve perfusion to the brain and heart. postresuscitation care and monitoring (prolonged life support) postresuscitation care involves careful monitoring and management of the adverse effects of hypoxia and reperfusion injury on the brain and other vital organs. the first hours after an arrest are most critical, because this is the time period in which an animal is most likely to rearrest unless the underlying cause of the initial arrest has been determine and treated (table - ) . until an animal is adequately ventilating on its own, artificial ventilation by manual bagging or attaching the patient to a mechanical ventilator with supplemental oxygen must continue. the efficacy of oxygenation and ventilation can be monitored using a wright's respirometer, pulse oximetry, capnometry, and arterial blood . once an animal is extubated, administer supplemental oxygen ( to ml/ kg/minute) (see oxygen supplementation). the brain is sensitive to ischemia and reperfusion injury. the effects of cellular hypoxia and reperfusion include the development of oxygen-derived free radical species that contribute to cerebral edema. administer mannitol ( . to g/kg iv over to minutes), followed by furosemide ( mg/kg iv) minutes later, to all patients that have experienced cardiopulmonary arrest and have had successful resuscitation. mannitol and furosemide work synergistically to decrease cerebral edema formation and scavenge oxygen-derived free radical species. the combination of cardiac arrest, myocardial ischemia and acidosis, and external or internal cardiac compressions often make the myocardium irritable and predisposed to dysrhythmias following successful cpcr. start lidocaine ( to mg/kg iv, followed by to µg/kg/minute iv cri) in all patients following successful resuscitative efforts. monitor the ecg continuously for the presence of cardiac dysrhythmias and recurrence of nonperfusing rhythms. perform direct or indirect blood pressure monitoring. if a patient's systolic blood pressure is less than mm hg, diastolic pressure is less than mm hg, or mean arterial blood pressure is less than mm hg, administer positive inotropic drugs (dobutamine, to µg/kg/minute) and pressor agents (epinephrine, . to . mg/kg iv, io, it) to improve cardiac contractility, cardiac output, and core organ perfusion. the kidneys are sensitive to decreased perfusion and cellular hypoxia. place a urinary catheter and monitor urine output. in a euvolemic patient, normal urine output should be no less than to ml/kg/hour. if urine output is low, administer low-dose dopamine ( to µg/kg/minute iv cri) in an attempt to dilate afferent renal vessels and improve renal perfusion. maintain acid-base and electrolyte status within normal reference ranges. monitor serum lactate as a rough indicator of organ perfusion and cellular oxygen extraction. the presence of elevated or rising serum lactate in the face of aggressive cardiorespiratory and cerebral support makes prognosis less favorable. cole sg, otto cm, hughes d: cardiopulmonary cerebral resuscitation: a clinical practice review part i, j vet emerg crit care ( ) immediate action depends largely on recognition of the primary or secondary cause of the dysrhythmia and treating the dysrhythmia and underlying cause. diagnosis of cardiac dysrhythmias is based on physical examination findings of abnormal thoracic/cardiac auscultation, the presence of abnormal pulse rhythm and quality, and recognition of ecg abnormalities. the ecg is critical to the accurate diagnosis of dysrhythmias. ventricular dysrhythmias arise from ectopic foci in the ventricles that cause the wave of depolarization to spread from cell to cell rather than spread through fast-conducting tissue. this causes the qrs complex to appear wide and bizarre, unless the ectopic focus originates close to the atrioventricular node high in the ventricle. other ecg features of ventricular dysrhythmias include a t wave polarity that is opposite to the qrs complex and nonrelated p waves. ventricular dysrhythmias may manifest as isolated ventricular premature complexes, couplets, or triplets; bigeminy; or ventricular tachycardia. relatively slow ventricular tachycardia is known as an idioventricular rhythm and is not as hemodynamically significant as faster ventricular tachycardia. idioventricular rhythm usually is less than beats per minute and may alternate spontaneously with sinus arrhythmias (figures - to . supraventricular dysrhythmias arise from ectopic foci in the atria and are commonly associated with atrial dilatation and structural heart disease such as advanced acquired or congenital heart disease, cardiomyopathies, cardiac neoplasia, or advanced heartworm disease. occasionally, supraventricular dysrhythmias may be associated with respiratory or other systemic illness. sustained supraventricular tachycardia in the absence of underlying structural heart or systemic disease is disturbing and should alert the clinician that an accessory pathway conduction disturbance may be present, particularly in labrador retrievers. supraventricular dysrhythmias can manifest as isolated premature complexes (atrial premature complexes or contractions), sustained or paroxysmal supraventricular tachycardia (atrial tachycardia), or atrial fibrillation or flutter. in the dog, atrial fibrillation most commonly is associated with dilative cardiomyopathy. rarely and primarily in giant breed dogs, lone atrial fibrillation can occur with no underlying heart disease. atrial fibrillation and the resultant sustained elevation in ventricular rate are presumed to progress to dilative cardiomyopathy in such breeds. by comparison, atrial fibrillation is relatively uncommon in cats because of the small size of their atria but is associated most commonly with hypertrophic and restrictive cardiomyopathy. the ecg is critical to the diagnosis of a supraventricular dysrhythmia. the ecg usually demonstrates a normal appearance to the qrs complex unless aberrant conduction occurs in the ventricles, in which case the qrs can be wide but still originate from above the atrioventricular node. in most cases of a supraventricular dysrhythmia, some evidence of atrial activity including p waves, atrial flutter, or atrial fibrillation is apparent. in some cases, it may be difficult to diagnose the exact rhythm without slowing the rate down mechanically or through pharmacologic intervention. once a rhythm diagnosis is made, appropriate treatment strategies can be implemented (figures - and - ). treatment of ventricular dysrhythmias largely depends on the number of ectopic foci discharging, the rate and character of the dysrhythmia, and whether the presence of the abnormal beats is of adverse hemodynamic consequence, including risk of sudden death. many ventricular dysrhythmias, including slow idioventricular rhythms, ventricular bigeminy, or intermittent ventricular premature complexes, do not warrant antiarrhythmic therapy unless the patient is hypotensive and the dysrhythmia is thought to be contributing to the hypotension. in such cases, correction of the underlying disease process including hypoxia, pain, or anxiety often alleviates or decreases the incidence of the dysrhythmia. more serious ventricular dysrhythmias that warrant antiarrhythmic therapy (table - ) include sustained ventricular tachycardia (> beats/minute in dogs; > beats/minute in cats), multifocal ventricular premature complexes originating from more than one place in the ventricles, and the presence of r-on-t phenomena where the t wave of the preceding complex is superimposed on the qrs of the next complex with no return to isoelectric shelf in between complexes. treat these ventricular dysrhythmias immediately and aggressively. in dogs, the mainstay of emergency treatment for ventricular dysrhythmias is lidocaine therapy. administer lidocaine ( to mg/kg iv bolus) over a period of minutes to prevent the adverse side effects of seizures or vomiting. the bolus can be repeated an additional times (total dose mg/kg) over minutes, or the patient can be placed on a constant rate infusion ( to µg/kg/minute) if control of ventricular tachycardia is accomplished. also correct the patient's magnesium and potassium deficiencies to maximize the success of lidocaine therapy in the treatment of ventricular tachycardia. procainamide ( mg/kg iv slowly over to minutes) also can be used to control ventricular tachycardia. if procainamide is successful at controlling ventricular tachycardia, administer it as a constant rate infusion ( to µg/kg/minute). side effects of procainamide include vomiting, diarrhea, and hypotension. chronic oral therapy may or may not be necessary in the treatment of acute ventricular tachycardia. the decision to continue antiarrhythmic therapy depends on the underlying disease process and the expectation of persistent arrhythmogenesis of the underlying disease process. oral antiarrhythmic therapy is warranted in cases in which a serious ventricular dysrhythmia is recognized but the animal does not require hospitalization, such as the syncopal boxer with intermittent ventricular dysrhythmias and no evidence of structural heart disease. it deserves emphasis that asymptomatic, low-grade ventricular dysrhythmias probably do not require treatment. if maintenance therapy for ventricular dysrhythmias is needed, use an oral drug based on the underlying disease process, clinical familiarity, class of drug, dosing frequency, owner compliance, concurrent medications, cost, and potential adverse side effects. in the cat the mainstay of antiarrhythmic therapy is the use of a β-adrenergic antagonist. in the acute management of ventricular dysrhythmias in cases of hypertrophic, restrictive, or unclassified cardiomyopathies, consider using injectable esmolol ( . to . mg/kg iv slowly to effect) or propranolol ( . to . mg/kg iv slowly to effect), particularly if the dysrhythmia results from hyperthyroidism. for chronic oral ventricular antiarrhythmic therapy in cats, propranolol ( . to . mg po per cat q h) or atenolol ( . to . mg po per cat q - h) can be used. the decision to treat supraventricular dysrhythmias depends on the ventricular rate and the hemodynamic consequences of the dysrhythmia. for intermittent isolated atrial emergency care procainamide - mg/kg po q - h tocainide* - mg/kg po q h sotalol - mg per dog q h (start low, then titrate up to effect) mexiletine - mg/kg po q h atenolol . - . mg/kg po q - h (start low, titrate upward to effect) *do not use for longer than weeks because of idiosyncratic blindness. premature contractions, couplets, and triplets, usually no treatment is required. when the ventricular rate exceeds beats/minute, diastolic filling time is shortened, causing the heart to not fill adequately. the consequence is decreased cardiac output and decreased coronary artery perfusion. the goal of therapy is rhythm control or, in most cases, rate control. in cases of atrial fibrillation and congestive heart failure, conversion to a normal sinus rhythm rarely can be achieved, although electrocardioversion or pharmacoconversion can be attempted. in the dog a vagal maneuver can be attempted by pressing on the eyeballs or massaging the carotid body. for sustained supraventricular tachycardia, diltiazem ( . mg/kg iv), esmolol ( . to . , titrated upward to a cumulative dose of . mg/kg iv), or propranolol ( . to . mg/kg iv slowly to effect) can be administered in an attempt to slow the ventricular rate in emergent situations. administer oral diltiazem ( . mg/kg po q h), diltiazem (dilacor-xr) ( . to mg/kg po q - h), propranolol ( . to . mg/kg tid, titrated up to a maximum of . mg/kg po q h), atenolol ( . to mg/kg q - h), or digoxin ( . to . mg/kg bid or . mg/m for dogs greater than kg). in the cat a vagal maneuver can be attempted by ocular or carotid massage. (diltiazem [dilacor] to po q - h), propranolol ( . to mg/kg q - h), or atenolol ( . mg q - h) also can be administered. if structural heart disease is present, treat pulmonary edema and start angiotensin-converting enzyme inhibitor therapy. table - summarizes the drugs used in the management of supraventricular dysrhythmias. severe bradycardia often results from systemic disease, drug therapy, anesthetic agents, or hypothermia and thus rarely requires specific therapy except to treat or reverse the underlying mechanisms promoting bradycardia. hemodynamically significant bradyarrhythmias that must be treated include atrial standstill, atrioventricular block, and sick sinus syndrome. atrial standstill most commonly is associated with hyperkalemia and is seen most often in urinary obstruction, renal failure, urinary trauma with uroabdomen, and hypoadrenocorticism. characteristic ecg abnormalities observed in atrial standstill are an absence of p waves, widened qrs complexes, and tall spiked t waves (figure - ). the treatment for hyperkalemia-induced atrial standstill is to correct the underlying cause and to drive potassium intracellularly and protect the myocardium from the adverse effects of hyperkalemia. regular insulin ( . to . units/kg iv) followed by dextrose ( g/unit insulin iv, followed by . % dextrose cri to prevent hypoglycemia) or sodium bicarbonate ( meq/kg iv) can be administered to drive potassium intracellularly. calcium gluconate ( . ml/kg of % solution iv over minutes) also can be administered as a cardioprotective drug until the cause of hyperkalemia has been identified and resolved. also administer sodium chloride fluids ( . % sodium chloride iv) to promote kaliuresis. less commonly, atrial standstill is associated with atrial cardiomyopathy or silent atrium syndrome. persistent atrial standstill has been recognized without electrolyte abnormalities in the english springer spaniel and the siamese cat. short-term therapy for persistent atrial standstill includes atropine ( . mg/kg sq) until definitive treatment by implantation of a cardiac pacemaker can be performed. complete or third-degree atrioventricular block or high-grade symptomatic seconddegree atrioventricular block can be hemodynamically significant when ventricular rates are less than beats/minute in the dog. classic clinical signs include weakness, exercise intolerance, lethargy, anorexia, syncope, and occasionally seizures. advanced atrioventricular block usually is caused by advanced idiopathic degeneration of the atrioventricular node. less commonly, atrioventricular block has been associated with digoxin toxicity, magnesium oversupplementation, cardiomyopathy, endocarditis, or infectious myocarditis (lyme disease). an accurate diagnosis is made based on the ecg findings of nonconducted p waves with ventricular escape beats. first-and second-degree atrioventricular block may not be hemodynamically significant and therefore may not require therapy. initially treat third-degree (complete) or symptomatic high-grade second-degree atrioventricular block (< beats/minute) with atropine ( . mg/kg sq or im). perform a follow-up ecg in to minutes. atropine is rarely successful in treating complete atrioventricular block. also attempt treatment with isoproterenol ( . to . µg/kg/minute iv cri or . mg in ml % dextrose in water iv slowly), a pure β-agonist. definitive treatment requires permanent pacemaker implantation. consultation with a veterinary cardiologist who implants pacemakers is suggested. never attempt to convert or treat the observed ventricular escape beats with lidocaine ( figure - ) . sick sinus syndrome most commonly is recognized in the miniature schnauzer, although any dog can be affected. sick sinus syndrome usually results from idiopathic degeneration of the sinus node in the dog. in the cat, sinus node degeneration usually is associated with cardiomyopathy. dysfunction of the sinus node may manifest as marked bradycardia with periods of sinus arrest followed by junctional or ventricular escape complexes. a variant of sick sinus syndrome is the presence of severe bradycardia followed by periods of supraventricular tachycardia, often termed bradycardia-tachycardia syndrome. the most common clinical signs are syncope, exercise intolerance, and lethargy. in cats, hypertrophic cardiomyopathy is the most common form of acquired cardiac disease observed. congestive heart failure resulting from hypertrophic cardiomyopathy can occur in animals as young as to months of age. hypertrophic cardiomyopathy is characterized by stiff, noncompliant ventricles that do not relax during diastole, causing an increase in left atrial pressures and left atrial enlargement. other cardiomyopathies, including unclassified, restrictive, and dilated, are less common but also can occur in the cat. cats often develop acute exacerbation of clinical signs because of stress or arterial embolization. the rapid diagnosis of chf often is made on owner history, signalment, and physical examination findings (box - ). typical physical examination findings include a cardiac murmur or gallop dysrhythmia, abnormal breath sounds, respiratory difficulty and orthopnea, tachycardia, weak pulse quality, cool peripheral extremities, and pale or cyanotic mucous membrane. initiate immediate treatment based on physical examination findings and index of suspicion. in some cases, it is difficult to distinguish between chf and feline lower airway disease (asthma) without performing thoracic radiographs. let the animal rest and become stabilized before attempting any stressful procedures, including thoracic radiographs. immediate treatment consists of administering supplemental oxygen, decreasing circulating fluid volume with furosemide, dilating pulmonary and splanchnic capacitance vessels with topical nitroglycerine and morphine, and alleviating patient anxiety and stress (box - ). primary differential diagnoses are made based primarily on the patient's breed, age, clinical signs, history, and physical examination abnormalities. the most common differential diagnoses in a patient with chf are cardiac abnormalities and respiratory disease (chronic bronchitis [asthma], pulmonary hypertension, cor pulmonale, neoplasia). postpone diagnostic tests in any patient with suspected chf until the immediate treatments have taken effect and the patient is cardiovascularly more stable. in most cases, lateral and dorsoventral thoracic radiographs are one of the most important diagnostic tools in helping make a diagnosis of chf. increased perihilar interstitial to alveolar infiltrates are characteristic of pulmonary edema. left atrial enlargement may be observed as a "backpack" sign at the caudal cardiac waist. cardiomegaly of the right or left side also may be present in cases of valvular insufficiency. in cats, increased sternal contact and a classic valentine-shaped heart may be observed in cases of hypertrophic cardiomyopathy. perform a vertebral heart score (sum) to measure cardiac size and determine whether cardiomegaly is present (box - ). also obtain arterial blood pressure and ecg readings to determine whether hypotension and dysrhythmias are present. atrial fibrillation, ventricular premature contractions, and supraventricular tachycardia are common rhythm disturbances that can affect cardiac output adversely and influence treatment choices. the echocardiogram is a useful noninvasive and nonstressful method to determine the degree of cardiac disease present. the echocardiogram is largely user-dependent. the quality of the study is based on the experience of the operator and the quality of the ultrasound machine. echocardiography can be a useful tool in making a diagnosis of pericardial effusion, dilated or hypertrophic cardiomyopathy, cardiac neoplasia, and endocarditis. the medical management of chf is designed to improve cardiac output and relieve clinical signs. the immediate goal of therapy is to reduce abnormal fluid accumulation and provide adequate cardiac output by increasing contractility, decreasing preload and ventricular afterload, and/or normalizing cardiac dysrhythmias. strict cage rest is of utmost importance when managing a patient with chf. after initial administration of furosemide, morphine, oxygen, and nitroglycerine paste, clinical signs of respiratory distress should show improvement within minutes. if no improvement is observed, administer repeated doses of furosemide. reevaluate severe cases that are refractory to this standard treatment protocol. vasodilation should be the next step in the management of refractory cases, provided that a normal blood pressure is present. sodium nitroprusside is a potent balanced vasodilator that should be administered ( to µg/kg/minute iv cri), taking care to monitor blood pressure continuously because severe vasodilation and hypotension can occur. the goal of nitroprusside therapy is to maintain a mean arterial blood pressure of mm hg. sodium nitroprusside should not be considered in cases of refractory chf with severe hypotension. for more long-term management of chf, the use of angiotensin-converting enzyme (ace) inhibitors including enalapril ( . mg/kg po q - h), benazepril ( . mg/kg po q h), and lisinopril ( . mg/kg po q h) have become the mainstay of therapy to reduce sodium and fluid retention and decrease afterload. start angiotensin-converting enzyme inhibition as soon as a patient is able to tolerate oral medications. dobutamine ( . to µg/kg/minute cri diluted in % dextrose in water) can be administered to improve cardiac contractility, particularly in cases of dilated cardiomyopathy. at low doses, dobutamine, primarily a β-adrenergic agonist, will improve cardiac output with minimal effects on heart rate. dobutamine must be given as a constant rate infusion with careful, continuous ecg monitoring. despite minimal effects on heart rate, emergency management of specific conditions the vertebral heart sum can be calculated by performing the following steps: . measure the long axis of the heart from the apex to the carina on the lateral view and mark the distance on a sheet of paper. . measure the length of the long axis of the heart in terms of vertebral bodies, starting by counting caudally from the fourth thoracic vertebra; count the number of vertebrae that are covered by the length of the long axis of the heart. . measure the short axis of the heart at the caudal vena cava, perpendicular to the long axis of the heart. . count the number of thoracic vertebrae covered by the short axis of the heart, starting at t . . add the two numbers together to yield the vertebral heart sum; a vertebral heart sum greater than . is consistent with cardiomegaly. sinus tachycardia or ventricular dysrhythmias may develop during infusion. cats are more sensitive to the effects of dobutamine than dogs. monitor carefully for seizures and facial twitching. digoxin is a cardiac glycoside that acts as a positive inotrope and negative chronotrope in the long-term management of chf. digoxin has a long ( hours in dogs, and hours in cats) half-life and so has minimal use in the emergency management of chf. in chronic management of chf resulting from dilated cardiomyopathy or advanced mitral disease, however, digoxin is extremely useful. oral digitalization protocols have been developed but are risky in that dysrhythmias and severe gastrointestinal side effects can occur. cats with chf often have fulminant pulmonary edema, pleural effusion, arterial thromboembolism, or some combination of all three. if the pleural effusion is significant, perform therapeutic thoracocentesis to relieve pulmonary atelectasis and improve oxygenation. once the diagnosis and initial management of chf has been made, formulate a plan for continued management and monitoring. tailor the therapeutic plan to the patient based on the cause of the chf, the presence of concurrent diseases, and response to therapy. an important and often overlooked part of the successful emergency management of chf is the open communication with the owner regarding the owner's emotional and financial commitment for immediate and long-term management to ensure appropriate quality of life for each patient. pathophysiology and treatment, vet j ( ) caval syndrome resulting from severe heartworm disease is caused by the rapid maturation of a large quantity of adult worms in the right atrium and cranial and caudal venae cavae. most cases of caval syndrome occur in regions of the world where heartworm disease is highly endemic and dogs spend a large portion of time living outdoors. caval syndrome is recognized by the following clinical signs and results of biochemical analyses: acute renal and hepatic failure, enlarged right atrium and posterior vena cava, ascites, hemoglobinuria, anemia, acute collapse, respiratory distress, dic, jugular pulses, circulating microfilariae, and sometimes tricuspid insufficiency. immediate action in cases of caval syndrome in dogs involves immediate stabilization of the cardiovascular and respiratory systems with supplemental oxygen, furosemide ( mg/kg iv), and careful crystalloid fluid infusion. diagnosis of caval syndrome is based on clinical signs of cardiogenic shock with right ventricular heart failure, intravascular hemolysis, and renal and hepatic failure. thoracic radiographs reveal cardiomegaly of the right side and enlarged tortuous pulmonary arteries. a right axis deviation may be seen on ecg tracings. clinicopathologic changes observed include azotemia, inflammatory leukogram, regenerative anemia, eosinophilia, elevated hepatocellular enzyme activities, hemoglobinuria, and proteinuria. circulating microfilariae may be observed on peripheral blood smears or in the buffy coat of microhematocrit tubes. heart worm antigen tests will be strongly positive. echocardiographic changes include visualization of a large number of heartworms in the right atrium, pulmonary arteries, and vena cava, tricuspid insufficiency, and right atrial and ventricular enlargement. treatment involves surgical removal of as many of the adult heartworms as possible from the right jugular vein and right atrium. glucocorticosteroids are recommended to decrease inflammation and microangiopathic disease associated with heartworm infection. for more long-term management, administer adulticide therapy several weeks following surgery, followed by routine microfilaricide therapy and then prophylaxis. calvert pericardial effusion often develops as a consequence of neoplasia in the older dog and cat. the most common types of neoplasia that affect the heart and pericardium include hemangiosarcoma, chemodectoma, mesothelioma, and metastatic neoplasia. more rarely, other causes of pericardial effusion include benign idiopathic pericardial effusion, coagulopathy, left atrial rupture in dogs with chronic mitral valvular insufficiency, infection, or pericardial cysts. regardless of the cause of the effusion, the development of pericardial tamponade adversely affects cardiac output. cardiac output is a function of heart rate and stroke volume. stroke volume depends on cardiac preload. the presence of pericardial effusion can impede venous return to the heart and thus adversely affect preload. in addition, as preload decreases, heart rate reflexively increases in an attempt to maintain normal cardiac output. as heart rate increases more than beats/minute, diastolic filling is impaired further, and cardiac output further declines. animals with pericardial effusion often demonstrate the classic signs of hypovolemic or cardiogenic shock: anorexia, weakness, lethargy, cyanosis, cool peripheral extremities, tachycardia, weak thready pulses, hypotension, and collapse. physical examination abnormalities may include muffled heart sounds, thready femoral pulses, pulsus paradoxus, jugular venous distention, weakness, tachycardia, cyanosis, and tachypnea. electrocardiogram findings may include low amplitude qrs complexes (< . mv), sinus tachycardia, ventricular dysrhythmias, or electrical alternans (figure - ) . thoracic radiographs often demonstrate a globoid cardiac silhouette, although the cardiac silhouette rarely may appear normal with concurrent clinical signs of cardiogenic shock in cases of acute hemorrhage. in such cases the removal of even small amounts of pericardial effusion by pericardiocentesis can increase cardiac output exponentially and alleviate clinical signs (table - ) . unless an animal is dying before your eyes, ideally perform an echocardiogram to attempt to determine whether a right atrial, right auricular, or heart base mass is present before pericardiocentesis. before attempting pericardiocentesis, assemble all of the required supplies (box - ) . to perform pericardiocentesis, follow this procedure: . place the patient in sternal or lateral recumbency. . attach ecg leads to monitor the patient for dysrhythmias during the procedure. . clip a -cm square caudal to the right elbow over the fifth to seventh intercostal space. . aseptically scrub the clipped area, and infuse to mg/kg of % lidocaine mixed with a small amount of sodium bicarbonate just dorsal to the sternum at the sixth intercostal space. bury the needle to the hub, and inject the lidocaine as you withdraw the needle. . while the local anesthetic is taking effect, assemble the intravenous extension tubing, three-way stopcock, and -ml syringe. . wearing sterile gloves, make a small nick incision in the skin to decrease drag on the needle and catheter during insertion. . slowly insert the needle and catheter, watching for a flash of blood in the hub of the needle, and simultaneously watching for cardiac dysrhythmias on the ecg monitor. . once a flash of blood is observed in the hub of the needle, advance the catheter off of the stylette further into the pericardial sac, and remove the stylette. . attach the length of intravenous extension tubing to the catheter, and have an assistant withdraw the fluid slowly. . place a small amount of fluid in a red-topped tube, and watch for clots. clot formation could signify that you have penetrated the right ventricle inadvertently or that active hemorrhage is occurring. withdraw as much of the fluid as possible, and then remove the catheter. monitor the patient closely for fluid reaccumulation and recurrence of clinical signs of cardiogenic shock. less rd, bright jm, orton ec: intrapericardial cyst causing cardiac tamponade in a cat, j am anim hosp assoc ( ) foreign bodies within the ear canal (e.g., foxtails) can present as emergencies because of acute inflammation and pressure necrosis of the tissue of the external auditory meatus causing pain and discomfort. clinical signs may be limited to incessant head shaking or scratching of the ear canal. complete examination of the ear canal and removal of any foreign body often requires administration of a short-acting anesthetic agent. once the animal has been restrained sufficiently and placed under anesthesia, carefully examine the ear canal and remove any foreign material with an alligator forceps. stimulation of the ear canal can cause awakening after removal of all debris and detritus, gently wipe the internal and external ear canal with a sterile gauze. place a topical antimicrobial-antifungal-steroid ointment such as otomax in the ear every to hours. if pain and discomfort is severe, systemically effective opioids or nsaids may be required. otitis externa is a common emergency that causes excessive head shaking, scratching, and purulent malodorous aural discharge. clean the ear canal with an irrigating solution such as epiotic and wipe it clean of debris. perform a complete aural examination to determine whether a foreign body or tumor is present and whether the tympanic membrane is intact. heat-fix any discharge and examine it cytologically for bacteria and fungal organisms. following careful cleansing, instill a topical antibiotic-antifungal-steroid ointment. in severe cases in which the ear canal has scarred and closed down with chronicity, consider administering systemically effective antibiotics (cephalexin, mg/kg po tid) and antifungal agents (ketoconazole, mg/kg po q h) instead of topical therapy. systemically effective steroids (prednisone or prednisolone, . mg/kg po q h) may be indicated in cases of severe inflammation to decrease pruritus and patient discomfort. presentation of a patient with otitis interna often is characterized by torticollis, head tilt, nystagmus, circling to the affected side, or rolling. fever, pain, vomiting, and severe depression may accompany clinical signs. most cases of severe otitis interna are accompanied by severe otitis media. both conditions must be treated simultaneously. the most common causes of otitis interna are staphylococcus aureus, pseudomonas, escherichia coli, or proteus spp. otitis interna can develop by infection spreading across the tympanic membrane, through the eustachian tubes, or by hematogenous spread from the blood supply to the middle ear. in most cases of otitis media, the tympanic membrane is ruptured. perform a culture and susceptibility test of the debris behind the tympanic membrane and within the aural canal. carefully clean the external ear canal. medicate with a topical combination antibiotic, antifungal, and antibiotic ointment. administer high-dose antibiotics (cephalexin, mg/kg po q h, or enrofloxacin, to mg/kg po q h). if the tympanic membrane is not ruptured but appears swollen and erythematous, a myringotomy may need to be performed. if clinical signs of otitis media persist despite topical and systemic therapy, radiographic or ct/mri examination of the tympanic bullae may be required. chronic shaking of the head and ears or aural trauma (bite wounds) causes disruption of the blood vessels and leads to the development of unilateral or bilateral aural hematomas. aural hematomas are clinically significant because they cause patient discomfort and are often due to the presence of some other underlying problem such as otitis externa, atopy, or aural foreign bodies. acute swelling of the external ear pinna with fluid is characteristic of an aural hematoma. in some cases, swelling can be so severe that the hematoma breaks open, bathing the patient and external living environment in blood. when a patient has an aural hematoma, investigate the underlying cause. perform a complete aural examination to determine whether an aural foreign body, otitis externa, or atopy are present. carefully examine and gently clean the inner ear canal. treat underlying causes. management of an aural hematoma involves draining the hemorrhagic fluid from the aural tissue and tacking the skin down in multiple places to prevent reaccumulation of fluid until the secondary cause is resolved. many techniques have been described to surgically tack down the skin overlying the hematoma. after the animal has been placed under general anesthesia, lance the hematoma down the middle with a scalpel blade and remove the fluid and blood clot. tack down the skin with multiple through-and-through interrupted or mattress sutures through the ear. some clinicians prefer to suture through and attach a sponge or length of x-ray film to the front and back of the ear for stabilization and support. more recently, a laser can be used to drill holes in the hematoma and tack the skin down in multiple areas. compress the ear against the head with a compression bandage, whenever possible, for to days after the initial surgery, and then recheck the ear. the patient must wear an elizabethan collar until the surgical wound and hematoma heal to prevent selfmutilation. also systemically treat underlying causative factors such as otitis externa with antibiotics, antifungals, and steroids as indicated. investigate and treat other underlying causes such as hypothyroidism or allergies. bass electrocution usually is observed in young animals after they have chewed on an electric cord. other causes of electrocution include use of defective electrical equipment or being struck by lightning. electric current passing through the body can produce severe dysrhythmias, including supraventricular or ventricular tachycardia and first-and thirddegree atrioventricular block. the electric current also can produce tissue destruction from heat and electrothermal burns. electrocution also commonly results in noncardiogenic pulmonary edema caused by massive catecholamine release and increase in pulmonary vascular pressures during the event. ventricular fibrillation can occur, although that depends on the intensity and path of the electrical current and duration of contact. clinical signs of electrocution include acute onset of respiratory distress with moist rales, and localized necrosis or thermal burns of the lips and tongue. often the skin at the commissures of the mouth appears white or yellow and firm to the touch. muscle fasciculations, loss of consciousness, and ventricular fibrillation may occur. thoracic radiographs often reveal an increased interstitial to alveolar lung pattern in the dorsocaudal lung fields. noncardiogenic pulmonary edema can develop up to to hours after the initial incident. the first hours are most critical for the patient, and then prognosis improves. the most important aspect in the treatment of the patient with noncardiogenic pulmonary edema is to minimize stress and to provide supplemental oxygen, with positive pressure ventilation, when necessary. although treatment with vasodilators (low-dose morphine) and diuretics (furosemide) can be attempted, noncardiogenic pulmonary edema is typically resistant to vasodilator and diuretic therapy. positive inotropes and pressor drugs may be necessary to treat shock and hypotension. opioid drugs (morphine, hydromorphone, oxymorphone) may be useful in controlling anxiety until the pulmonary edema resolves. administer broad-spectrum antibiotics (cefazolin; amoxicillin and clavulanic acid [clavamox]) to treat thermal burns. use analgesic drugs to control patient discomfort. if thermal burns are extensive and prohibit adequate food intake, place a feeding tube as soon as the patient's cardiovascular and respiratory function are stable and the patient can tolerate anesthesia. prolapse of the uterus occurs in the immediate postparturient period in the bitch and queen. excessive straining during or after parturition causes the uterus to prolapse caudally through the vagina and vulva. immediate intervention is necessary. examine the bitch or queen for a retained fetus. treatment consists of general anesthesia to replace the prolapsed tissue. if the uterus is edematous, physical replacement may be difficult or impossible. application of a hypertonic solution such as hypertonic ( %) saline or dextrose ( %) to the exposed endometrium can help shrink the tissue. that, combined with gentle massage to stimulate uterine contraction and involution and lubrication with sterile lubricating jelly, can aid in replacement of the organ into its proper place. to ensure proper placement in the abdominal cavity and to prevent recurrence, perform an exploratory laparotomy and hysteropexy. postoperatively, administer oxytocin ( to units im) to cause uterine contraction. if the uterus contracts, it is usually not necessary to suture the vulva. administer antibiotics postoperatively. recurrence is uncommon, even with subsequent pregnancies. if the tissue is damaged or too edematous to replace or if the tissue is devitalized, traumatized or necrotic, perform an ovariohysterectomy. in some instances, replacement of the damaged tissue is not necessary before removal. pyometra occurs in dogs and cats. the disease process occurs as a result of infection overlying cystic endometrial hyperplasia under the constant influence of progesterone. during the -month luteal phase after estrus or following copulation, artificial insemination, or administration of hormones (particularly estradiol or progesterone), the myometrium becomes relaxed and favors a quiescent environment for bacterial proliferation. clinical signs of pyometra are associated with the presence of bacterial endotoxin and sepsis. early, affected animals become lethargic and anorectic. polyuria with secondary polydipsia is often present because of the influence of bacterial endotoxin on renal tubular concentration. if the cervix is open, purulent or mucoid vaginal discharge may be observed. later in the course of pyometra, vomiting, diarrhea, and progressive debilitation resulting from sepsis occur. diagnosis is based on clinical signs in an intact queen or bitch and radiographic or ultrasonographic evidence of a fluid-filled tubular density in the ventrocaudal abdomen, adjacent to the urinary bladder (figures - and - ) . treatment of open and closed pyometra is correction of fluid and electrolyte abnormalities, administration of broad-spectrum antibiotics, and ovariohysterectomy. close pyometra is a life-threatening septic condition. open pyometra also can become life-threatening and so should be treated aggressively. in closed pyometra, conservative medical therapy is not advised. administration of prostaglandins and oxytocin do not reliably cause the cervix to open and can result in ascending infection from the uterus into the abdomen or uterine rupture, both of which can result in severe peritonitis. for animals with an open pyometra, ovariohysterectomy is the most reliable treatment for chronic cystic endometrial hyperplasia. although less successful than ovariohysterectomy, medical therapy may be attempted in breeding bitches as an alternative to surgery. the most widely used medical therapy in the breeding queen and bitch is administration of prostaglandin f α . this drug has not been approved for use in the queen or bitch in the united states. to proceed with medical management of pyometra, first determine the size of the uterus. start the patient on antibiotic therapy (ampicillin, mg/kg iv q h, or enrofloxacin, mg/kg po q h). administer the prostaglandin f α ( µg/kg sq q h) for to days until the size of the uterus approaches normal. measure serum progesterone concentrations if the bitch is in diestrus. as the corpus luteum degrades under the influence of prostaglandin f α , serum progesterone levels will decline. prostaglandin f α is an abortifacient and thus should not be administered to the pregnant bitch or queen. clinical signs of a reaction to prostaglandin f α can occur within to minutes in the bitch and can last for as long as minutes. clinical signs of a reaction include restlessness, hypersalivation, panting, vomiting, defecation, abdominal pain, fever, and vocalization. in a very ill animal, death can occur. the efficacy of prostaglandin f α is limited and may require more than one treatment. the bitch should be bred on the next heat cycle and then spayed because progressive cystic endometrial hyperplasia will continue to occur. acute metritis is an acute bacterial infection of the uterus that typically occurs within to weeks after parturition. the most common organism observed in metritis is e. coli ascending from the vulva and vaginal vault. sepsis can progress rapidly. clinical signs of acute metritis include inability to nurse puppies, anorexia, lethargy, foul-smelling purulentsanguineous vaginal discharge, vomiting, or acute collapse. physical examination may reveal fever, dehydration, and a turgid distended uterus. septic inflammation will be observed on vaginal cytologic examination. an enlarged uterus can be observed with abdominal radiographs and ultrasonography. treatment of acute metritis is directed at restoring hydration status with intravenous fluids and treating the infection with antibiotics. because the primary cause of metritis is e. coli infection, start enrofloxacin ( mg/kg iv or po once daily) therapy. as soon as the patient's cardiovascular status is stable enough for anesthesia, perform an ovariohysterectomy. if the patient is not critical and is a valuable breeding bitch, medical therapy can be attempted. medical management of acute bacterial metritis includes administration of oxytocin ( to units q h for three treatments) or administration of prostaglandin f α ( µg/kg/day for to days) to evacuate the uterine exudate and increase uterine blood flow. either drug should be used concurrently with antibiotics. rupture of the gravid uterus is rare in cats and dogs but has been reported. uterine rupture may occur as a consequence of parturition or result from blunt abdominal trauma. feti expelled into the abdominal cavity may be resorbed but more commonly cause the development of peritonitis. if fetal circulation is not disrupted, the fetus actually may live to term. uterine rupture is an acute surgical emergency. an ovariohysterectomy with removal of the extrauterine puppies and membranes is recommended. if only one horn of the uterus is affected, a unilateral ovariohysterectomy can be performed to salvage the remaining unaffected puppies and preserve the breeding potential for the valuable bitch. if uterine rupture occurs because of pyometra, peritonitis is likely, and copious peritoneal lavage should be performed at the time of surgery. the patient should be placed on to days of antibiotic therapy (amoxicillin or amoxicillin and clavulanic acid [clavamox] with enrofloxacin). vaginal prolapse occurs from excessive proliferation and hyperplasia of vaginal tissue while under the influence of estrogen during proestrus (figure - ) . the hyperplastic tissue usually recedes during diestrus but reappears with subsequent heat cycles. vaginal prolapse can be confused with vaginal neoplasia. the former condition occurs primarily in younger animals, whereas the latter condition occurs primarily in older animals. treatment for vaginal hyperplasia or prolapse generally is not required if the tissue remains within the vagina. the proliferation can lead to dysuria or anuria, however. in some cases, the tissue becomes emergency care dried out and devitalized or becomes traumatized by the animal. such extreme cases warrant immediate surgical intervention. the treatment for vaginal prolapse consists of ovariohysterectomy to remove the influence of estrogen, placement of an indwelling urinary catheter if the patient is dysuric, and protection of the hyperplastic tissue until it recedes on its own. although surgical resection of the hyperplastic tissue has been recommended, excessive hemorrhage after removal can occur, and so the procedure should not be attempted. the patient should wear an elizabethan collar at all times to prevent selfmutilation. administer broad-spectrum antibiotics for a minimum of to days or until the hyperplastic tissue recedes. keep the tissue clean with saline solution. dystocia, or difficult birth, can occur in the dog and cat but is more common in the dog. a diagnosis of dystocia is made based on the time of onset of visible labor and the time in which the last puppy or no puppy has been born, the intensity and timing of contractions, the timing of when the amniotic membranes first appear, the condition of the bitch, and the timing of gestation. causes of dystocia can be maternal or fetal and include primary or secondary uterine inertia, narrowing of the pelvic canal, hypocalcemia, psychological disturbances, or uterine torsion. maternal-fetal disproportion, or large fetus size in relation to the bitch or queen, also can result in dystocia (box - ). obtain an abdominal radiograph for all cases of suspected dystocia at the time of presentation to determine the size of the fetus, presentation of the fetus (both anterior or posterior presentation can be normal in the bitch or queen, but fetal malpositioning can cause dystocia), and whether there is radiographic evidence of a uterine rupture or torsion. if maternal-fetal disproportion, uterine torsion, or uterine rupture is observed, take the patient immediately to surgery. if the puppies or kittens are in a normal position for birth, medical management can be attempted. clip the perineum and aseptically scrub it. wearing sterile gloves, insert a lubricated finger into the vagina and palpate the cervix. massage (or "feather") the dorsal wall of the vagina to stimulate contractions. place an intravenous catheter, and administer oxytocin ( to units im), repeating up to times at -minute intervals. in some cases, hypoglycemia or hypocalcemia can contribute to uterine inertia. administration of a calciumcontaining solution (lactated ringer's solution) with . % dextrose is advised. alternately, administer % calcium gluconate ( mg/ kg iv slowly). if labor has not progressed after hour, immediately perform a cesarean section. uterine torsion is an uncommon emergency seen in the gravid and nongravid uterus and has been reported in dogs and cats. the onset of clinical signs of abdominal pain and straining as if to whelp/queen or defecate is usually acute and constitutes a surgical emergency. in some cases, there may have been a history of delivery of a live or dead fetus. vaginal discharge may or may not be present. radiographs or ultrasound examination reveal a fluid-filled or air-filled tubular density in the ventral abdomen. treatment consists of placing an intravenous catheter, stabilizing the patient's cardiovascular status with intravenous fluids and sometimes blood products, and performing an immediate ovariohysterectomy. if there are viable feti, the uterus should be delivered en mass and the puppies or kittens delivered. the expulsion of one or more fetus before term is known as spontaneous abortion. in dogs and cats, it is possible to expel or abort one or more fetuses and still carry viable fetuses to term and deliver normally. clinical signs of spontaneous abortion include vaginal discharge and abdominal contractions. in some cases, the fetus is found, or there may be evidence of fetal membranes or remnants. causes of spontaneous abortion in dogs include brucella canis, herpesvirus, coronavirus, and toxoplasmosis. in cats, herpesvirus, coronavirus, and feline leukemia virus can cause spontaneous abortion. in both species, trauma, hormonal factors, environmental pathogens, drugs, and fetal factors also can result in spontaneous abortion. the safest method of pregnancy termination in the bitch or queen is by performing an ovariohysterectomy. oral diethylstilbesterol is not an effective mechanism of pregnancy termination in the bitch. a so-called mismating shot, an injection of estradiol cypionate ( . mg/lb im) is effective at causing termination of an early pregnancy but can be associated with severe side effects, including bone marrow suppression and pyometra. estradiol cypionate is not approved for use in the bitch or queen and is not recommended. prostaglandin f α is a natural abortifacient in the bitch if treatment is started within days of cytologic evidence of diestrus (noncornified epithelium on a vaginal smear). the prostaglandin f α causes lysis of the corpora lutea and a rapid decline in progesterone concentration. the prostaglandin f α is administered for a total of eight injections ( µg/kg q h for days), along with atropine ( to µg/kg sq). side effects can occur within to minutes of injection and include restlessness, panting, salivation, abdominal pain, urination, vomiting, and diarrhea. walking the patient for to minutes after each treatment sometimes decreases the intensity of the reactions. bitches in the first half of the pregnancy often resorb the embryos. if prostaglandin f α is administered in the second half of the pregnancy, the fetuses are aborted within to days of treatment. measure serum progesterone concentrations at the end of treatment to ensure complete lysis of the corpus luteum. prostaglandin f α is not approved for pregnancy termination in the bitch. in cats, prostaglandin f α can terminate pregnancy after day of gestation. prostaglandin f α should be used only in healthy queens ( to µg/kg sq q h for days). side effects in the queen are similar to those observed in the bitch but typically have a shorter duration ( to minutes). prostaglandin f α is not approved for use in cats in the united states. the use of prostaglandin f α does not preclude breeding and pregnancy at a later date. biddle d, macintire dk: obstetrical emergencies, clin tech small anim pract ( ) in the dog and cat the majority of injuries to the scrotum are associated with animal fights or shearing and abrasive injuries sustained in accidents involving automobiles. scrotal injuries should be categorized as superficial or penetrating. treatment of superficial injuries to the scrotum includes cleaning the wound with dilute antimicrobial cleanser and drying it. administer antiinflammatory doses of steroids (prednisolone, . to . mg/kg po q - h) or nsaids (carprofen, . mg/kg po q h in dogs) for the first several days after scrotal injury to prevent or treat edema. administer topical antibiotic ointment until the wound heals. in most cases, place an elizabethan collar to prevent self-mutilation. prognosis is generally favorable; however, semen quality may be affected for months after injury because of scrotal swelling and increased scrotal temperature. penetrating injuries to the scrotum are more serious and are associated with severe swelling and infection. surgically explore and debride penetrating scrotal wounds. administer systemically effective antibiotics and analgesics. in extreme cases, particularly those that involve the testicle, consider castration and scrotal ablation. scrotal dermatitis is common in intact male dogs and can be associated with direct physical injury, self-infliction from licking, chemical irritation, burns, or contact dermatitis. in affected animals, the scrotum can become extremely inflamed, swollen, and painful. if left untreated, pyogranulomatous dermatitis can develop. make an attempt to determine whether an underlying systemic illness is present that could predispose the animal to scrotal dermatitis. widespread vasculitis with scrotal edema, pain, fever, and dermatitis has been associated with rickettsia rickettsii (rocky mountain spotted fever) infection. brucella canis also has been associated with scrotal irritation and dermatitis. if scrotal dermatitis follows from an infectious cause, empiric use of glucocorticosteroids potentially can make the condition worse by suppressing immune function. empiric treatment with antibiotics also potentially can confound making an accurate diagnosis. treatment of scrotal dermatitis is to eliminate predisposing causes, if possible. place an elizabethan collar at all times to prevent self-mutilation. bathe the scrotum with a mild antimicrobial soap and dry it to remove any offending chemical irritants. topical medications including tar shampoo, tetracaine, neomycin, and petroleum can cause further irritation and are contraindicated. use oral or parenteral administration of glucocorticosteroids or nsaids to control discomfort and inflammation. scrotal hernias occur when the contents of the abdomen (intestines, fat, mesentery, omentum) protrude through the inguinal ring into the scrotal sac. like inguinal hernias, scrotal definitive therapy for a scrotal hernia involves exploratory laparotomy and surgical reduction of the contents of the hernia, surgical correction of the rent in the inguinal ring, and castration. trauma to the epididymis or testicle can cause testicular pain and swelling of one or both testes. treat penetrating trauma to the testicle by castration to prevent infection and selfmutilation. administer oral antibiotics (amoxicillin or amoxicillin-clavulanate) for to days after the injury. nonpenetrating injuries to the scrotum and testicle rarely may cause acute testicular hemorrhage or hydrocele formation. palpation of the affected area often reveals a peritesticular, soft, compliant area. treatment consists of cool compresses on the scrotum and testicle and administration of antiinflammatory doses of glucocorticosteroids or nsaids. if the swelling does not resolve spontaneously in to days, consider surgical exploration and drainage. increased scrotal temperature and testicular inflammation can affect semen quality for months after the initial incident. testicular torsion, or torsion of the spermatic cord, causes rotation of the testicle, ultimately causing obstruction to venous drainage. testicular torsion often is associated with a neoplastic mass of a retained testicle within the abdomen but also can be observed with nonneoplastic testes located within the scrotum. the predominant clinical signs are pain, stiff stilted gait, and the presence of an abnormally swollen testicle (if located within the scrotum). if an intraabdominal testicular torsion is present, pain, lethargy, anorexia, and vomiting can occur (see acute condition in the abdomen). an intraabdominal mass may be palpable. perform an abdominal or testicular ultrasound, preferably with color flow doppler to evaluate perfusion to the testicle. treatment involves surgical removal of the involved testes. bacterial infections of the testicle or epididymis most commonly are caused by ascending infections of the normal bacterial flora of the prepuce or urethra. common inhabitants include escherichia coli, staphylococcus aureus, streptococcus spp., and mycobacterium canis. brucella canis and r. rickettsii are also capable of causing orchitis and epididymitis in the dog. clinical signs of orchitis or epididymitis include testicular enlargement, stiff stilted gait, and reluctance to walk. physical examination often reveals a fever and self-induced trauma to the scrotum from licking or chewing at the inflamed area. collect a semen sample by ejaculation, and culture it to identify the causative organism. alternately, collect samples by needle aspiration of the affected organ(s) and test serologically for b. canis. treatment of infectious orchitis involves a minimum of to weeks of specific antimicrobial therapy, based on culture and susceptibility testing, whenever possible. if a bacterial culture cannot be obtained, initiate fluoroquinolone therapy (enrofloxacin, mg/kg po q h). doxycycline ( mg/kg po bid for days) has been shown to suppress but not eradicate b. canis infection. testicular inflammation and increased temperature can affect sperm quality for months after infection. the most common causes of acute prostatitis are associated with acute bacterial infection (e. coli, proteus spp., pseudomonas spp., and mycoplasma spp.). less common causes include fungal infection (blastomyces dermatitidis) or anaerobic bacterial infection. acute prostatitis is characterized by fever, caudal abdominal pain, lethargy, anorexia, blood in the ejaculate, hematuria, dyschezia, and occasionally stranguria or dysuria. the patient often appears painful and depressed and may be dehydrated on physical examination. symmetric or asymmetric prostatomegaly and prostate pain may be evident on rectal palpation. in severely affected dogs, clinical signs of tachycardia, hyperemic or injected mucous membranes, bounding pulses, lethargy, dehydration, and fever may be present because of sepsis. death can occur within days if a prostatic abscess ruptures. diagnosis of acute prostatitis is confirmed based on the presenting clinical signs, neutrophilic leukocytosis (with or without a left shift), and positive urine culture results. prostatic samples may be obtained from the prostatic portion of the ejaculate, prostatic massage, urethral discharge, urine, or (less commonly) prostatic aspirate. although semen samples can yield positive bacterial cultures, dogs with acute prostatitis are often unwilling to ejaculate. radiography may reveal an enlarged prostate, but this alone does not confirm the diagnosis of prostatitis. an abdominal ultrasound often reveals prostatic abscessation and allows for the collection of samples from the affected area(s) via prostatic aspirate. aspiration of the affected tissue potentially can wick infection into periprostatic tracks. cytologic examination of the patient's ejaculate or prostatic wash from a dog with acute prostatitis reveals numerous inflammatory cells and may contain bacterial organisms. the treatment of a patient with acute prostatitis is directed at correcting dysuria and constipation associated with prostatic enlargement. enrofloxaxin ( mg/kg po sid) can penetrate the inflamed prostatic tissue and is effective in treating gram-negative and mycoplasma spp. infections. ciprofloxacin does not appear to penetrate prostatic tissue as readily. alternatives to enrofloxacin therapy are trimethoprim-sulfamethoxazole ( mg/kg po q h) or chloramphenicol ( - mg/kg po q h) for a minimum of to weeks. castration is recommended because benign prostatic hyperplasia may be a predisposing factor in the development of acute prostatitis. do not perform castration until the patient has been on antibiotic therapy for a minimum of days, to prevent the surgical complication of schirrous cords. finasteride (proscar, mg/kg po q h), an antiandrogen α-reductase inhibitor, may help reduce the size of prostatic tissue until the effects of castration are observed. if a prostatic abscess is present, perform marsupialization, surgical drainage, or ultrasonographic drainage. surgical therapy is associated with a large incidence of complications, including incontinence, chronic drainage from fistulas and stomas, septic shock, and death. fracture of the os penis is an uncommon condition encountered in male dogs. os penis fractures can occur with minimal soft tissue damage but cause hematuria and dysuria. on physical examination, urethral obstruction and crepitus in the penis are found. a lateral abdominal radiograph is usually sufficient to document the fracture. treatment consists of conservative therapy, in most cases, and consists primarily of analgesia administration. if the urethra also is damaged, place a urethral catheter for to days to allow the urethral mucosa to heal. fractures of the os penis that are comminuted or severe enough to cause urethral obstruction require open reduction and fixation, partial penile amputation, or antescrotal (prescrotal) urethrostomy. lacerations of the penis cause significant bleeding because of the extensive vascular supply to the penis. dogs and cats tend to lick penile lacerations and prevent adequate clot formation. sedation or general anesthesia often is required to evaluate and treat the laceration. after sedation or general anesthesia, place a urinary catheter and examine the penis under a stream of cold water. small lacerations can be managed with cold compresses and one to several absorbable sutures. extensive suturing usually is not required. prevent erection by isolating the patient from females in estrus or allowing excitement or excessive activity. place an elizabethan collar to prevent self-mutilation. initiate systemic antibiotic therapy to prevent infection. the inability to withdraw the penis into the prepuce in male dogs or cats is known as paraphimosis. paraphimosis usually develops following an erection in young male dogs and in emergency care older dogs after coitus. mucosal edema, hemorrhage, self-mutilation, and necrosis requiring penile amputation can occur if left untreated. treatment consists of applying cold water to the penis and reducing edema with application of an osmotic substance such as sugar. examine the base of the penis for hair rings that can prevent retraction of the penis into the prepuce. rinse the penis carefully with cold water and lubricate it with sterile lubricant and replace it into the prepuce. if the penis cannot be reduced easily into the prepuce, anesthetize the patient and make a small incision at the lateral aspect of the preputial opening. replace the penis and close the incision with absorbable suture. place a purse-string suture and leave it in place for several days to prevent recurrence. instill topical antimicrobial ointment with steroids into the prepuce several times a day. in severe cases, a urinary catheter may need to be placed to prevent urethral obstruction, until penile swelling and edema resolve. place an elizabethan collar to prevent excessive licking during the healing process. prolapse of the distal urethra is a condition usually confined to intact male english bulldogs, although isolated incidences also have been reported in yorkshire and boston terriers. the exact cause of this condition is unknown but usually is associated with a condition that causes increased intraabdominal pressure or urethral straining, including sexual excitement, coughing, vomiting, obstructed airway or brachycephalic airway syndrome, urethral calculi, genitourinary tract infection, and masturbation. the urethral prolapse usually appears as a mushroom-tip congested, irritated mass at the end of the penis that may or may not bleed (figure - ) . in some cases, bleeding occurs or worsens with sexual excitement. clinical signs associated with the prolapsed urethra include excessive licking of the prepuce, stranguria, and preputial bleeding. once the mass is observed, other differential diagnoses include transmissible venereal tumor, urethral polyp, trauma, urethritis, and neoplasia. in most cases, however, the prolapse occurs in intact young dogs, making neoplastic conditions less likely. treatment for urethral prolapse should occur at the time of diagnosis to prevent selfinduced trauma and infection. immediate therapy includes manual reduction of the prolapsed tissue and placement of a purse-string suture around an indwelling urinary catheter. the purse-string suture can remain in place for up to days until definitive repair. until the time of surgery, place an elizabethan collar on the patient to prevent self-mutilation. several forms of surgical correction have been described. in some cases, surgical resection of the prolapsed tissue with apposition of the urethral and penile mucosa can be attempted. more recently, a technique involving placement of several mattress sutures to reduce and secure the prolapsed tissue has been described. recurrence of prolapse can occur with either technique, particularly if the inciting event recurs. because there may be a genetic predisposition in this breed and because the prolapse can recur with sexual excitement, neutering should strongly be recommended. local freezing or frostbite most commonly affects the peripheral tissues of the ears, tail, paws, and genitalia that are sparsely covered with fur, are poorly vascularized, and may have been traumatized previously by cold. clinical signs of frostbite are paleness and appearance of a blanched pink to white discoloration to the skin. the skin also may appear black and necrotic. immediate treatment consists of slowly rewarming the affected area with moist heat at . ° c ( °f) or by immersion in warm water baths. analgesics may be required to alleviate patient discomfort. carefully dry the injured areas and protect them from further trauma. the use of prophylactic antibiotics is controversial because it can promote resistant bacterial infection. use of antibiotics should be based on the presence of infection. treatments that are ineffective and may be harmful include rubbing the affected areas, pressure bandages, and ointments. corticosteroids can decrease cellular immunity and promote infection and are therefore contraindicated. many frostbitten areas that appear nonviable can regain function gradually. use care when removing areas of necrotic tissue. affected areas may take several days to a week before fully manifesting areas of demarcation between healthy viable and necrotic nonviable tissue. chilling of the entire body from exposure or immersion in extremely cold water results in a decrease in core body temperature and physiologic processes that become irreversible when the body temperature falls below °c ( °f). mild hypothermia can be °to °c, moderate hypothermia from °to °c, and severe hypothermia below °c. the duration of exposure and the general condition of the animal influences its ability to survive. clinical signs and consequences associated with hypothermia include shivering, vasoconstriction, mental depression, hypotension, sinus bradycardia, hypoventilation with decreased respiratory rate, increased blood viscosity, muscle stiffness, atrial and ventricular irritability, decreased level of consciousness, decreased oxygen consumption, metabolic (lactic) acidosis, respiratory acidosis, and coagulopathies including dic. if the animal is breathing, administer warm, humidified oxygen at to breaths per minute. if the animal is not breathing or is severely hypoventilating, endotracheal intubation with mechanical ventilation may be necessary. place an intravenous catheter and infuse warmed crystalloid fluids. if the blood glucose is less than mg/dl, add supplemental dextrose ( . %) to the crystalloid fluids. monitor the core body temperature and ecg closely. rewarming should occur in the form of external circulating warm water blankets, radiant heat, and circulating warm air blankets (bair hugger). never use a heating pad, to avoid iatrogenic thermal burn injury. severe hypothermia may require core rewarming in the form of intraperitoneal fluids ( to ml/kg of lactated ringer's solution warmed to . °c [ °f]). place a temporary peritoneal dialysis catheter, and repeat the dialysis every minutes until the patient's body temperature reaches . °to . °c ( °to °f). the body temperature should rise slowly, ideally no more than °f per hour. because the response of the body to drugs is unpredictable, avoid administering drugs whenever possible, until the body temperature returns to normal. complications observed during rewarming include dic, cardiac dysrhythmias including cardiac arrest, pneumonia, pulmonary edema, cns edema, ards, and renal failure. heat stroke and heat-induced illness in dogs can be associated with excessive exertion, exposure to high environmental temperatures, stress, and other factors that cause an inability to dissipate heat. brachycephalic breeds, obesity, laryngeal paralysis, and older animals with cardiovascular disease can be particularly affected. hyperthermia is defined as a rectal temperature of °to °c ( °to °f). clinical signs of hyperthermia include congested hyperemic mucous membranes, tachycardia, and panting. more severe clinical signs include collapse (heat prostration), ataxia, vomiting, diarrhea, hypersalivation, muscle tremors, loss of consciousness, and seizures. heat-induced illness can affect all major organ systems in the body because of denaturation of cellular proteins and enzyme activities, inappropriate shunting of blood, hypotension, decreased oxygen delivery, and lactic acidosis. cardiac dysrhythmias, interstitial and intracellular dehydration, intravascular hypovolemia, central nervous dysfunction, slough of gastrointestinal mucosa, oliguria, and coagulopathies can be seen as organ function declines. excessive panting can result in respiratory alkalosis. poor tissue perfusion results in a metabolic acidosis. loss of water in excess of solutes such as sodium and chloride can lead to a free water deficit and severe hypernatremia. a marked increase in pcv occurs because of the free water loss. severe abnormalities in electrolytes and ph can lead to cerebral edema and death. treatment goals for the patient with heat-induced illness are to lower the core body temperature and support cardiovascular, respiratory, renal, gastrointestinal, neurologic, and hepatic functions. at the scene the veterinarian or caretaker can spray the animal with tepid (not cold) water. immersion in cold water or ice baths is absolutely contraindicated. cold water and ice will cause extreme peripheral vasoconstriction, inhibiting the patient's ability to dissipate heat through conductive and convective cooling mechanisms. as a result, core body temperature will continue to rise despite the good intentions of well-doers at the scene. animals that present to the veterinarian that have been cooled to the point of hypothermia have a worse prognosis. once the animal has presented to the veterinarian, the goal is to cool the animal's body temperature with towels soaked in tepid water, cool intravenous fluids, and fans until the temperature has decreased to °f. organ system monitoring and support is based on the severity and duration of the heat stroke and the ability of the body to compensate and respond to treatment. management of the patient with heat-induced illness involves prompt aggressive cooling without being overzealous and creating iatrogenic hypothermia. administer cool intravenous crystalloid fluids to replenish volume and interstitial hydration and correct the patient's acid-base and electrolyte abnormalities. management consists of rule of twenty monitoring (see rule of ), taking care to evaluate, restore, and maintain a normal cardiac rhythm, blood pressure, urine output, and mentation. administer antibiotics if there are any signs of gastrointestinal bleeding that will predispose the patient to bacterial translocation. monitor baseline chemistry tests including a complete blood count, biochemical panel, platelet count, coagulation tests, and urinalysis. treat coagulopathies including dic aggressively and promptly (see also disseminated intravascular coagulation). severe changes in mentation including stupor or coma worsen a patient's prognosis. following initial therapy, monitor the patient for a minimum of to hours for secondary organ damage, including renal failure, myoglobinuria, cerebral edema, and dic. dogs that are going to die of heat-induced illness usually die within the first hours. animals that survive longer than hours have a more favorable prognosis. immediate treatment consists of cooling the patient with cooling measures as for hyperthermia and heat-induced illness (see the previous discussion), and eliminating the cause (i.e., exertion, anesthesia, or neuromuscular blockers such as succinylcholine). if the patient is under general anesthesia, hyperventilate the patient to help eliminate carbon dioxide and respiratory acidosis. administer dantrolene sodium ( to mg/kg iv) to stabilize the sarcoplasmic reticulum and decrease its permeability to calcium. animals with malignant hyperthermia should avoid any predisposing factors, including exertion, hyperthermia, and anesthesia. after an episode of malignant hyperthermia, administer crystalloid fluids intravenously to aid in the elimination of myoglobin. monitor renal function closely for myoglobinuria and pigment damage to the renal tubular epithelium. monitor and correct acid-base and electrolyte changes. walters jm: hyperthermia. in wingfield we, editor: the veterinary icu book, jackson, wyo, , teton newmedia. sometimes it is difficult to assess whether an animal has been bitten by a poisonous or nonpoisonous snake. in colorado, the bull snake closely resembles the prairie rattlesnake. both snakes make similar noise and can be alarming if noticed on a hike or in the backyard. whenever possible, identify the offending reptile but never risk being bitten. know what types of venomous creatures are in the geographic area of the practice. if an animal has been bitten by a nonpoisonous snake, usually the bite marks are small with multiple small tooth punctures, and the bite is relatively nonpainful. usually local reaction is negligible. however, large boas or pythons also can inflict large crushing injuries that can cause severe trauma, including bony fractures. treatment for a nonpoisonous snakebite involves clipping the bite wound and carefully cleaning the area with antimicrobial scrub solution. broad-spectrum antibiotics (e.g., amoxicillin-clavulanate, . mg/kg po q h) are indicated because of the extensive bacterial flora in the mouths of snakes. monitor all snakebite victims for a minimum of hours after the incident, particularly when the species of the offending reptile is in question. if clinical signs of envenomation occur, modify the patient's treatment appropriately and aggressively. the two major groups of venomous snakes in north america are the pit viper and the coral snake. all venomous snakes are dangerous. the severity of any given bite depends on the toxicity of the venom, the amount of venom injected, the site of envenomation, the size of the animal bitten, and the time from bite/envenomation to seeking appropriate medical intervention. the majority of reptile envenomations in the united states are inflicted by pit vipers, including the water moccasin (cottonmouth), copperhead, and numerous species of rattlesnakes. pit vipers are characterized by a deep pit located between the eye and nostril, elliptic pupils, and retractable front fangs (figure - ) . localized clinical signs of pit viper envenomation may include the presence of bleeding puncture wounds, local edema close to puncture wounds, immediate severe pain or collapse, edema, petechiae, and ecchymosis with subsequent tissue necrosis. systemic signs of pit viper envenomation may include hypotension, shock, coagulopathies, lethargy, weakness, muscle fasciculations, lymphangitis, rhabdomyolysis, and neurologic signs including respiratory depression and seizures. neurologic signs largely are associated with envenomation emergency management of specific conditions by the mojave and canebrake rattlesnakes, although a potent neurotoxin, mojave toxin a, also has been identified in other subspecies of rattlesnake. clinical signs of envenomation may take several hours to appear. hospitalize all suspected victims and monitor them for a minimum of hours. the severity of envenomation cannot be judged solely on the basis of local tissue reaction. first aid measures by animal caretakers do little to prevent further envenomation. the most important aspect of initiating therapy is to transport the animal to the nearest veterinary emergency facility. to determine whether an animal has been envenomated by a pit viper, examine a peripheral blood smear for the presence of echinocytes. echinocytes will appear within minutes of envenomation and may disappear within hours. other treatment should be initiated as rapidly and aggressively as possible, although controversy exists whether some therapies are warranted. the mainstay of therapy is to improve tissue perfusion with intravenous crystalloid fluids, prevent pain with judicious use of analgesic drugs, and when necessary, reverse or negate the effects of the venom with antivenin. because pit viper venom consists of multiple fractions, treat each envenomation as a complex poisoning. obtain vascular access and administer intravenous crystalloid fluids (one fourth of a calculated shock dose) according to the patient's perfusion parameters of heart rate, blood pressure, and capillary refill time (see also shock and fluid therapy). opioid analgesics are potent and should be administered at the time of presentation. (see also pharmacologic means to analgesia: major analgesics). diphenhydramine ( . to mg/kg im or iv) also can be administered to decrease the effects of histamine. famotidine, a histamine receptor antagonist, also can be administered ( . to mg/kg iv) to work synergistically with diphenhydramine. although antihistamines have no effect on the venom per se, they may have an effect on the tissue reaction to the venom and may prevent an adverse reaction to antivenin. the use of glucocorticosteroids is controversial. glucocorticosteroids (dexamethasone sodium phosphate [dex-sp], . to . mg/kg iv) may stabilize cellular membranes and inhibit phospholipase, an active component of some pit viper toxins. polyvalent antivenin is necessary in many cases of pit viper envenomation, except in most cases of prairie rattlesnake (crotalus viridis viridis) envenomation in colorado. a recent study demonstrated no difference in outcome with or without the use of antivenin in cases of prairie rattlesnake envenomation. clinically, however, patients that receive antivenin are more comfortable and leave the hospital sooner than those that do not receive antivenin. the exact dose of antivenin is unknown in small animal patients. administer a dose of at least vial of antivenin to neutralize circulating venom. mix antivenin with a swirling, rather than a shaking motion, to prevent foaming. mix the antivenin with a -ml bag of . % saline, and then administer it slowly over a period of hours. pretreat animals with diphenhydramine ( . to mg/kg im) before the administration of antivenin, and then monitor the animal closely for clinical signs of angioneurotic edema, urticaria, tachyarrhythmias, vomiting, diarrhea, and weakness during the infusion. administration of antivenin into the bite site is relatively contraindicated and ineffective because uptake is delayed, and systemic effects are the more life-threatening. management of pit viper envenomation largely involves maintenance of normal tissue perfusion with intravenous fluids, decreasing patient discomfort with analgesia, and negating circulating venom with antivenin. hydrotherapy to the affected bite site with tepid water is often soothing to the patient. the empiric use of antibiotics is controversial but is recommended because of the favorable environment created by a snakebite (i.e., impregnation of superficial gram-positive bacteria and gram-negative bacteria from the mouth of the snake into a site of edematous necrotic tissue). administer amoxicillin-clavulanate ( . mg/kg po q h, or cephalexin, mg/kg po q h). also consider administration of nsaids (carprofen, . mg/kg po q h). monitor the patient closely for signs of local tissue necrosis and the development of thrombocytopenia and coagulopathies including dic (see management of disseminated intravascular coagulation). treat coagulopathies aggressively to prevent end-organ damage. coral snakes are characterized by brightly colored bands encircling the body, with red and black separated by yellow. "red on black, friend of jack; red on yellow, kill a fellow." types of coral snakes include the eastern coral, texas coral, and sonoran coral snakes. clinical signs of coral snake envenomation may include small puncture wounds, transient initial pain, muscle fasciculations, weakness, difficulty swallowing/dysphagia, ascending lower motor neuron paralysis, miotic pinpoint pupils, bulbar paralysis, respiratory collapse, and severe hemolysis. clinical signs may be delayed for as long as hours after the initial bite. immediate treatment with antivenin is necessary in cases of coral snake envenomation before the clinical signs become apparent, whenever possible. support respiration during paralysis with mechanical ventilation. secure the patient's airway with a cuffed endotracheal tube to prevent aspiration pneumonia. clinical signs will progress rapidly once they develop. rapid administration with antivenin is the mainstay of therapy in suspected coral snake envenomation. respiratory and cardiovascular support should occur with mechanical ventilation and intravenous crystalloid fluids. keep the patient warm and dry in a quiet place. turn the patient every to hours to prevent atelectasis and decubitus ulcer formation. maintain cleanliness using a urinary catheter and closed urinary collection system. perform passive range of motion and deep muscle massage to prevent disuse atrophy of limb muscles and function. treat aspiration pneumonia aggressively with broad-spectrum antibiotics (ampicillin, mg/kg iv q h, with enrofloxacin, mg/kg iv q h, and then change to oral once tolerated and the patient is able to swallow) for weeks past the resolution of radiographic signs of pneumonia, intravenous fluids, and nebulization with sterile saline and coupage chest physiotherapy. several weeks may elapse before a complete recovery. the adult black widow spider (latrodectus spp.) can be recognized by a red to orange hourglass-shaped marking on the underside of a globous, shiny, black abdomen. the immature female can be recognized by a colorful pattern of red, brown, and beige on the dorsal surface of the abdomen. adult and immature females are equally capable of envenomation. the male is unable to penetrate the skin because of its small size. black widow spiders are found throughout the united states and canada. black widow spider venom is neurotoxic and acts presynaptically, releasing large amounts of acetylcholine and norepinephrine. there appears to be a seasonal variation in the potency of the venom, lowest in the spring and highest in the fall. in dogs, envenomation results in hyperesthesia, muscle fasciculations, and hypertension. muscle rigidity without tenderness is characteristic. affected animals may demonstrate clinical signs of acute abdominal pain. tonic-clonic convulsions may occur but are rare. in cats, paralytic signs predominate and appear early as a ascending lower motor neuron paralysis. increased salivation, vomiting, and diarrhea may occur. serum biochemistry profiles often reveal significant elevations in creatine kinase and hypocalcemia. myoglobinemia and myoglobinuria can occur because of extreme muscle damage. management of black widow spider envenomation should be aggressive in the cat and dog, particularly when the exposure is known. in many cases, however, the diagnosis is made based on clinical signs, biochemical abnormalities, and lack of other apparent cause. antivenin (one vial) is available and should be administered after pretreatment with diphenhydramine. if antivenin is unavailable, administer a slow infusion of calcium-containing fluid such as lactated ringer's solution with calcium gluconate while carefully monitoring the patient's ecg. the small brown nonaggressive spider is characterized by a violin-shaped marking on the cephalothorax. the neck of the violin points toward the abdomen. brown spiders are found primarily in the southern half of the united states but have been documented as far north as michigan. the venom of the brown spider has a potent dermatonecrolytic effect and starts with a classic bull's-eye lesion. the lesion then develops into an indolent ulcer into dependent tissues promoted by complement fixation and influx of neutrophils into the affected area. the ulcer can take months to heal and often leaves a disfiguring scar. systemic reactions are rare but can include hemolysis, fever, thrombocytopenia, weakness, and joint pain. fatalities are possible. immediate management of an animal with brown spider envenomation is difficult because there is no specific antidote and because clinical signs may be delayed until necrosis of the skin and underlying tissues becomes apparent through the patient's fur to days after the initial bite. dapsone has been recommended at a dose of mg/kg for days. surgical excision of the ulcer may be helpful if performed in the early stages of wound appearance. glucocorticosteroids may be of some benefit if used within hours of the bite. the ulcer should be left to heal by second intention. deep ulcers should be treated with antibiotics. bufo toad species (b. marinus, aka cane toad, marine toad, giant toad; and the colorado river toad or sonoran desert toad b. alvarius) can be associated with severe cardiac and neurotoxicity if an animal licks its skin. the severity of toxicity depends largely on the size of the dog. toxins in the cane toad, b. marinus, include catecholamines and vasoactive substances (epinephrine, norepinephrine, serotonin, dopamine) and bufo toxins (bufagins, bufotoxin, and bufotenine), the mechanism of which is similar to cardiac glycosides. clinical signs can range from ptyalism, weakness, ataxia, extensor rigidity, opisthotonus, and collapse to seizures. clinical signs associated with b. alvarius toxicity are limited largely to cardiac dysrhythmias, ataxia, and salivation. the animal should have its mouth rinsed out thoroughly with tap water even before presentation to the veterinarian. if the animal is unconscious or actively seizing and cannot protect its airway, flushing the mouth is contraindicated. once an animal presents to the veterinarian, the veterinarian should place an intravenous catheter and monitor the patient's ecg and blood pressure. attempt seizure control with diazepam ( . mg/kg iv) or pentobarbital ( to mg/kg iv to effect). ventricular dysrhythmias can be controlled first with esmolol ( . mg/kg). if esmolol is ineffective, administer a longer-acting parenteral β-antagonist such as propranolol ( . mg/kg iv). ventricular tachycardia also can be treated with lidocaine ( to mg/kg iv, followed by to µg/kg/minute iv cri). case management largely depends on supportive care and treating clinical signs as they occur. monitor baseline acid-base and electrolyte balance because severe metabolic acidosis may occur that should be treated with intravenous fluids and sodium bicarbonate ( . to meq/kg iv). monitor ecg, blood pressure, and mentation changes closely. control seizures and cardiac dysrhythmias. eubig pa: bufo species intoxication: big toad, big problem, vet med ( ) lizards of the family hemodermatidae are the only two poisonous lizards in the world. they are found in the southwestern united states and mexico. the venom glands are located on either side of the lower jaw. because these lizards are typically lethargic and nonaggressive, bite wounds are rare. the lizards have grooved teeth that introduce the venom with a chewing motion as the lizard holds tenaciously to the victim. the majority of affected dogs are bitten on the upper lip, which is very painful. there are no proven first aid measures for bites from gila monsters or mexican bearded lizards. the lizard can be disengaged by inserting a prying instrument in between the jaws and pushing at the back of the mouth. the teeth of the lizard are brittle and break off in the wound. topical irrigation with lidocaine and probing with a needle will aid in finding and removing the teeth from the victim. bite wounds will bleed excessively. irrigate wounds with sterile saline or lactated ringer's solution, and place compression on the affected area until bleeding ceases. monitor the patient for hypotension. establish intravenous access, and administer intravenous fluids according to the patient's perfusion parameters. antibiotic therapy is indicated because of the bacteria in the lizard's mouth. because no antidote is available, treatment is supportive according to patient signs. the majority of musculoskeletal emergencies are the result of external trauma, most commonly from motor vehicle accidents. blunt trauma invokes injury to multiple organ systems as a rule, rather than an exception. because of this, massive musculoskeletal injuries are assigned a relatively low priority during the initial triage and treatment of a traumatized animal. perform a rapid primary survey and institute any lifesaving emergency therapies. adhere to a crash plan or the abcs of resuscitation (see initial emergency examination, management, and triage). although musculoskeletal injuries are assigned a relatively lower priority, the degree of recovery from these injuries and financial obligation for fracture repair sometimes becomes a critical factor in a client's decision whether to pursue further therapy. one of the most important deciding factors is the long-term prognosis for the patient to have a good quality of life following fracture repair. the initial management of musculoskeletal injuries is important in ensuring the best chance for maximal recovery with minimal complications after definitive surgical fracture repair. this is particularly important for open fractures, spinal cord compromise, multiple fractures, open joints, articular fractures, physeal fractures, and concomitant ligamentous or neurologic compromise (box - ). immediately after the initial primary survey of a patient, perform a more thorough examination, including an orthopedic examination. multiple injuries often are observed in the patient that falls from height (e.g., "high-rise syndrome"), motor vehicle accidents, gunshot wounds, and encounters with other animals (e.g., "big-dog-little-dog"). address the most life threatening injuries, and palliate musculoskeletal injuries until more definitive repair can be attempted when the patient is more stable. in animals with the history of potential for multiple injuries, search thoroughly and meticulously for areas of injury to the spinal column, extremities, and for small puncture wounds. helpful signs that can provide a clue as to an underlying injury include swelling, bruising, abnormal motion, and crepitus (caused by subcutaneous emphysema or bony fracture). if the patient is alert, look for areas of tenderness or pain. in unconscious or depressed patients, reexamine the patient after the patient becomes more mentally alert. injuries often are missed during the initial examination in obtunded patients because of the early response and attenuation of pain. unconscious or immobile patients must have radiographic examination of the spinal column following stabilization and support. palpate the skull carefully for obvious depressions or crepitus that may be associated with a skull fracture. localization of the injury can be determined by motion in abnormal locations, swelling caused by hemorrhage or edema, pain during gentle movement or palpation, deformity, angular change, or a significant increase or decrease in normal range of motion of bones and joints. perform a rectal examination in all cases to palpate for pelvic fractures and displacement. once the diagnosis of a fracture or luxation has been confirmed, look for any evidence of skin lacerations or punctures near the fracture site. in long-haired breeds, clipping the fur near the fracture site often is necessary to perform a thorough examination of the area. if any wounds are found, the fracture is classified as an open fracture until proven otherwise. in some cases, the open fracture is obvious, with a large section of bone fragment protruding through the skin. in other cases, the puncture wound may be subtle, with only a small amount of blood or pinpoint hole in the skin surface. characteristics observed with open fractures include bone penetration, fat droplets or marrow elements in blood coming from the wound, subcutaneous emphysema on radiographs, and lacerations in the area of a fracture. protect the patient from further injury or contamination of wounds. excessive palpation to intentionally produce crepitus is inappropriate because it causes severe patient discomfort and has the potential to cause severe soft tissue and neurologic injury at the fracture site. sedation and analgesia aids in making the examination more comfortable for the patient and allows localization of the injury and comparison with the opposite extremity. higher-quality radiographs can be performed to determine the extent of the injury when the animal is sedated adequately and pain is controlled. sedate the patient judiciously with analgesic drugs. opioid drugs work well for orthopedic pain, produce minimal cardiorespiratory depression, and can be reversed with naloxone if necessary. handle the fracture site gently to avoid causing further pain and soft tissue injury at the fracture site. rough or careless handling of a fracture site can cause a closed fracture to penetrate through the skin and become an open fracture. cover open fractures immediately to prevent contamination of the fracture with nosocomial infection from the hospital. administer a first-generation cephalosporin (cephalexin, mg/kg po q h, or cefazolin, mg/kg iv q h). the bandage also serves to control hemorrhage and prevent desiccation of the bones and surrounding soft tissue structures. leave the initial bandages in place until the patient's cardiorespiratory status has been determined to be stable and more definitive wound management can occur in a clean, preferably sterile location. examine the neurologic status and cardiovascular status of the limb before and after treatment. determine the vascular status of the limb by checking the color and temperature of the limb, the state of distal pulses, and the degree of bleeding from a cut nail bed. in patients with severe cardiovascular compromise and hypotension caused by hemorrhagic shock, the viability of the limb may be in question until the cardiovascular status and blood pressure are normalized. reduction of the fracture or straightening of gross deformities may return normal vascularity to the limb. when checking neurologic status, examine for motor and sensory function to the limb. swelling may increase pressure on the nerves as they run through osteofascial compartments, resulting in decreased sensory or motor function, or neurapraxia. diminished function often returns to normal once the swelling subsides. serial physical examinations in the patient and response to initial stabilization therapy can lead to a higher index of suspicion that more occult injuries are present, such as a diaphragmatic hernia, perforated bowel, lacerated liver or spleen, or uroabdomen. to prevent ongoing trauma, reduce any fracture and then stabilize the site above and below the fracture. a modified robert jones splint or bandage often works well for fractures emergency management of specific conditions involving the distal extremities. fractures of the humerus or femur are difficult to immobilize without the use of spica or over-the-hip coaptation splints to prevent mobility. inappropriate bandaging of humerus or femur fractures can result in a fulcrum effect and worsen the soft tissue and neurologic injuries. further displacement of vertebral bodies or luxations can cause cord compression or laceration such that return to function becomes impossible. immediately place any patient with a suspected spinal injury on a flat surface, and tape down the animal to prevent further movement until the spine has been cleared by a minimum or two orthogonal radiographic views (lateral and ventrodorsal views performed as a cross-table x-ray technique). wounds associated with musculoskeletal trauma are common and include injury to the bones, joints, tendons, and surrounding musculature (box - ). major problems associated with these cases are the presence of soft tissue trauma that makes wound closure hazardous or impossible, because of the risk of infection. chronic deep infection of traumatized wounds can cause delayed healing and sequestrum to develop, particularly if there is avascular bone or cartilage within the wound. in the early management of an open fracture, the areas should be splinted without pulling any exposed bone back into the soft tissue. the wound should not be probed or soaked, as nosocomial bacteria and other external contaminants can be introduced into the wound, leading to severe infection. because of the risk of actually causing infection, probing, flushing, or replacing tissues back into the wound should be performed at the time of formal debridement when the patient is physiologically stable. immediate bactericidal antibiotic therapy with a first-generation cephalosporin should be started immediately to obtain adequate concentrations of antibiotics at the fracture site. the duration of antibiotic therapy should ideally be limited to - days to prevent the risk of superinfection. treatment of open musculoskeletal injury involves three considerations: initial inspection and wound debridement, stabilization and repair, and wound bandaging. emergency care when associated with a fracture, wound is created from the inside out by penetration of bone fragments through the skin or from a low-energy gunshot. simple or comminuted fracture pattern good stability of the two main bone segments treatment and prognosis are good and similar to those of a closed injury if wound is debrided and stabilized within to hours. when associated with a fracture, wound is created from the outside in. major deep injury with considerable soft tissue stripping from bone and muscle damage simple or comminuted fracture pattern prognosis is good if wound is debrided within hours of injury and provided rigid stabilization with a bone plate or external fixator. results from major external force severe damage and necrosis of skin, subcutaneous tissue, muscle, nerve, bone, tendon, and arteries soft tissue damage may vary from crush injury to shearing injury associated with bite wounds or low-speed automobile accidents. requires immediate and delayed sequential debridement and rigid external fixation can require prolonged healing times guarded prognosis initial inspection and wound debridement include the following steps: . after the patient's cardiovascular status has been stabilized and it has been determined that it can withstand anesthesia, place the animal under general anesthesia and remove the temporary splint. . keeping the wound covered, shave the surrounding fur. . remove the covering and then place sterile lubricant jelly over the wound. shave the fur to the edges of the wound margin. . wash away any entrapped fur and the lubricant jelly. . complete an antiseptic scrub of the surrounding skin. . if the wound is a small puncture (e.g., gunshot pellets or bites), probe the wound with a sterile hemostat. do a thorough debridement if tissues deep to the hole are cavitated. if not deep, create a hole for drainage. . flush the wound with a physiologic solution (lactated ringer's solution is preferred). . debride the wound from outward to inward. cut away damaged areas of skin and deeper tissues to open up underlying cavitations and tissue injury. . continuously irrigate with warm physiologic solution (lactated ringer's solution is preferred). the stream must be strong enough to flush debris out of the bottom of the wound. to accomplish this, attach a -gauge needle to a -ml syringe (will deliver psi). excise any obviously devitalized tissue. . do not remove any bone fragments that are firmly attached to soft tissue. do not cut into healthy soft tissue to find bullet or bone fragments, unless the bullet can cause injury to joints or nerve tissue. . do a primary repair of tendons and nerves if the wound is type i and recent (within hours of the initial injury). if the wound is too severe or if there is obvious infection, tag the ends of the tendons and nerves for later repair. it is best to stabilize and repair open fractures as soon as the patient's cardiovascular and respiratory status can tolerate general anesthesia, provided that adequate stabilization is possible. if this is not possible because of the level of experience of the surgeon or the lack of necessary equipment, it is best to perform wound management and place a temporary splint until definitive repair can be performed. wound bandaging is discussed in the section on bandaging techniques. structural injuries to the joints are common and can involve both ligaments and articular cartilage injuries. cartilage does not heal well; therefore, injuries involving articular cartilage can lead to a significant loss of function and degenerative joint disease (osteoarthritis). cartilage injuries that are superficial evoke a short-lived enzymatic and metabolic response that does not stimulate enough cellular growth to repair the defect. superficial lesions remain as defects but do not progress to chondromalacia or osteoarthritis. deep cartilage lacerations that extend to subchondral bone produce an exuberant healing response from the cells of the underlying cartilage. in many cases, this material undergoes degeneration and leads to osteoarthritis. impact injuries to surface cartilage can cause chondrocyte and underlying bone injury. these lesions rapidly progress to osteoarthritis; however, they may be totally or partially reversible. treatment of grade i injuries requires short-term coaptation splints and has a good prognosis. grade ii injuries require surgical treatment with a suture stent and consistent postoperative coaptation splints to heal and maintain good function. healing of grade iii injuries often is a problem, and suture stents or surgical reapproximation may be indicated. failure to immobilize joints that are frequently flexed (elbow and stifle) can result in late complications of ligament repair. ligamentous injuries of joints, particularly the collateral ligaments of the stifle, elbow, and hock, and carpal hyperextension injuries are commonly missed and may require surgical fixation, including arthrodesis (box - ). fractures in immature animals differ from those in adults in that young puppies and kittens have a great ability to remodel bone. remodeling is dependent on the age of the patient and the location of the fracture. the younger the puppy or kitten and the closer the fracture to the epiphysis or growth plate, the greater the potential for remodeling and the development of angular limb deformities. remodeling occurs more effectively in longlimbed breeds of dogs than in short-limbed breeds. fractures through the growth plate of immature animals may potentially cause angular limb deformities, joint dislocations or incongruity, and osteoarthritis. this form of injury is commonly observed in the distal ulnar growth plate and the proximal and distal radial growth plates. high-rise syndrome in cats is seen in cats that fall from a height usually greater than feet. it occurs most frequently in high-rise buildings in urban areas where cats lie on window ledges and suddenly fall out the window. the most common lesions observed in cats that fall from heights are thoracic injuries (rib and sternal fractures, pneumothorax, and pulmonary contusions) and facial and oral trauma (lip avulsions, mandibular symphyseal fractures, fractures of the hard palate, and maxillary fractures). limb and spinal cord fractures and luxations, radius and ulna fractures, abdominal trauma, urinary tract trauma, and diaphragmatic hernias are also common. the injuries sustained are often found in combination, rather than as an isolated injury of one area of the body. follow the mnemonic a crash plan when managing a cat suffering from high-rise syndrome, treating the animal immediately for shock. following cardiovascular and respiratory stabilization, evaluate thoracic and abdominal radiographs, including those of the spine. evaluate the bladder closely, making sure that the cat is able to urinate effectively. examine the hard palate, maxilla, and mandibular symphysis for fractures. palpate the pelvis and carefully manipulate all limbs to examine for fractures or ligamentous injuries. finally, perform a complete neurologic examination. patients that fall less than five stories often have a more guarded prognosis than patients that fall from higher levels. sometimes the owner witnesses the ingestion of a foreign body during play, such as throwing a stick or fetching a ball. cats tend to play with string or thread that becomes caught around the base of the tongue. in many cases, however, ingestion of the foreign object is not witnessed, and diagnosis is made based on clinical signs and physical examination. foreign bodies lodged in the oral cavity often cause irritation and discomfort, including difficulty breathing and difficulty swallowing. often, an animal paws at its mouth in an attempt to dislodge a stick or bones wedged across the roof of the mouth. irritation, inability to close the mouth, and blockage of the orpharynx can result in excessive drooling. the saliva may appear blood-tinged due to concurrent soft tissue trauma (figs - and - ) . obstruction of the glottis by a foreign body (e.g., tennis ball or toy) can result in cyanosis secondary to an obstructed airway and hypoxemia. in many cases, the object is small enough to enter the larynx but too large to be expelled. if a foreign object is lodged in the mouth for more than several days, halitosis and purulent discharge may be present. many animals are anxious at the time of presentation and may require sedation or a light plane of anesthesia to remove the foreign object. the animal may bite personnel and may have bitten the owner during his or her attempt to remove the object from the mouth en route to the hospital. propofol ( mg/kg iv) or a combination of propofol with diazepam ( . - mg/kg iv) is an excellent combination for a light plane of anesthesia. exercise caution when anesthetizing a patient with a ball lodged in the airway, as further compromise of respiratory function may occur and cause worsening of the hypoxemia. before inducing anesthesia, assemble all supplies necessary to remove the object. make sure that rigid towel clamps, sponge forceps, and bone forceps are on hand, because the foreign object is often very slippery with saliva. hemostats and carmalts may slip and not be useful in the removal of the foreign object. place a peripheral intravenous catheter to secure vascular access prior to anesthetic induction. have available the supplies necessary for an emergency tracheostomy, if the foreign object cannot be removed by usual methods. induce a light plane of anesthesia and then grasp the object with the sponge forceps or towel clamps, and extract. monitor the cardiorespiratory status of the animal at all times during the extraction process. if you are unable to remove the object, and if severe respiratory distress, including cyanosis, bradycardia, or ventricular dysrhythmias, develop, perform a tracheostomy distal to the site of obstruction. once the foreign body has been removed, administer supplemental flow-by oxygen until the animal awakens. if laryngeal edema or stridor on inspiration is present, administer a dose of dexamethasone sodium phosphate ( . mg/kg iv, im, sq) to decrease inflammation. the patient should be carefully monitored for hours, because noncardiogenic pulmonary edema can develop secondary to airway obstruction. esophageal foreign bodies pose a serious medical emergency. it is helpful if the owner witnessed ingestion of the object and noted rapid onset of clinical signs. in many cases, however, ingestion is not witnessed, and the diagnosis must be made based on clinical signs, thoracic radiographs, and results of a barium swallow. the most common clinical signs are excessive salivation with drooling, gulping, and regurgitation after eating. many animals will make repeated swallowing motions. some animals exhibit a rigid "sawhorse" stance, with reluctance to move immediately after foreign body ingestion and esophageal entrapment. after completing a physical examination, evaluate cervical and thoracic radiographs to determine the location of the esophageal obstruction. esophageal foreign objects are lodged most commonly at the base of the heart, the carina, or just orad to the lower esophageal sphincter. if the object has been lodged for several days, pleural effusion and pneumomediastinum may be present secondary to esophageal perforation. endoscopy is useful for both diagnosis and removal of the foreign object; however, it is invasive and requires general anesthesia ( fig. - ) . remove foreign objects lodged in the esophagus with a rigid or flexible endoscope after the patient has been placed under general anesthesia. evaluate the integrity of the esophagus both before and after removal of the material because focal perforation or pressure necrosis can be present. necrosis of the mucosa and submucosa of the esophagus often leads to stricture formation or perforation. attempt to retrieve the object with a flexible fiberoptic endoscope if available. rigid tube endoscopy can also be performed. in many cases, smooth objects that cannot be easily grasped can be pushed into the stomach and allowed to dissolve or may be removed by gastrotomy. if the foreign body is firmly lodged in the esophagus and cannot be pulled or pushed into the stomach, or if perforation has already occurred, the prognosis for return to function without strictures is not favorable. in such cases, referral to a surgical specialist is recommended for esophagostomy or esophageal resection. after removal of the object, carefully examine the esophagus and then administer gastroprotectant agents (famotidine, . mg/kg po bid; sucralfate slurry, . - . g/dog) for a minimum of to days. to rest the esophagus, the patient should receive nothing per os (npo) for to hours. if esophageal irritation or erosion is moderate to severe, a percutaneous gastrotomy tube should be placed for feeding until the esophagus heals. perform repeat endoscopy every days to evaluate the healing process and to determine whether stricture formation is occurring. persistent vomiting immediately or soon after eating is often associated with a gastric foreign body. in some cases, the owner knows that the patient has ingested a foreign body of some kind. in other cases, continued vomiting despite lack of response to conservative treatment (npo, antiemetics, gastroprotectant drugs) prompts further diagnostic procedures, including abdominal radiographs and bloodwork. obstruction to gastric outflow and vomiting of hydrochloric acid often cause a hypochloremic metabolic acidosis. radiopaque gastric foreign bodies may be observed on plain films. radiolucent cloth material may require a barium series to delineate the shape and location of the foreign body ( fig. - ) . treatment consists of removal with flexible endoscopy or a simple gastrotomy. most animals with uncomplicated gastric foreign bodies are relatively healthy, but any metabolic and electrolyte abnormalities should be corrected prior to anesthesia and surgery. small intestinal obstruction can be caused by foreign bodies, tumors, intussusception, volvulus, or strangulation within hernias. regardless of the cause, clinical signs of small intestinal obstruction depend on the location and degree of obstruction, and whether the bowel has perforated. clinical signs associated with a high small intestinal obstruction are usually more severe and more rapid in onset compared with partial or complete obstruction of the jejunum or ileum. complete obstructions that allow no fluid or chyme to pass are worse than partial obstructions, which can cause intermittent clinical signs interspersed with periods of normality (table - ). the most common clinical signs associated with a complete small intestinal obstruction are anorexia, vomiting, lethargy, depression, dehydration, and sometimes abdominal pain. early clinical signs may be limited to anorexia and depression, making a diagnosis challenging unless the owner has a suspicion that the animal ingested some kind of foreign object. obstructions cranial to the common bile duct and pancreatic papillae lead to vomiting of gastric contents, namely hydrochloric acid, and a hypochloremic metabolic alkalosis. obstructions caudal to the common bile duct and pancreatic papillae result in loss of other electrolytes and sometimes mixed acid-base disorders. eventually, all animals with small intestinal obstruction vomit and have fluid loss into dilated segments of bowel, leading to dehydration and electrolyte abnormalities. increased luminal pressure causes decreased lymphatic drainage and bowel edema. the bowel wall eventually becomes ischemic and may rupture. linear foreign bodies should be suspected in any vomiting patient, particularly cats. string or thread often is looped around the base of the tongue and can be visualized in many cases by a thorough oral examination. to look properly under the tongue, grasp the top of the animal's head with one hand, and pull the lower jaw open with the index finger of the opposite hand while pushing up the thumb simultaneously on the tongue in between the intermandibular space. thread and string can be observed lying along the ventral aspect of the tongue. in some cases, if a linear foreign body is lodged very caudally, it cannot be visualized without heavy sedation or anesthesia. linear foreign bodies eventually cause bowel obstruction and perforation of the intestines along the mesenteric border. the foreign material (e.g., string, thread, cloth, pantyhose) becomes lodged proximally, and the intestines become plicated as the body attempts to push the material caudally through the intestines ( fig. - ) . continued peristalsis eventually causes a sawing motion of the material and perforation of the mesenteric border of the intestines. once peritonitis occurs, the prognosis is less favorable unless prompt and aggressive treatment is initiated. reevaluate any patient that does not respond to conservative symptomatic therapy, performing a complete blood count, serum biochemical panel (including electrolytes), and abdominal radiographs. intestinal masses may be palpable on physical examination and are often associated with signs of discomfort or pain when palpating over the mass. radiography and abdominal ultrasound are the most useful diagnostic aids. plain radiographs may be diagnostic when the foreign object is radiodense or there is characteristic dilation or plication of bowel loops. as a rule of thumb, the width of a loop of small bowel should be no larger than twice the width of a rib. diagnosis of small intestinal obstruction or ileus can be based on the appearance of stacking loops of dilated bowel. comparison of the width of the bowel with the width of a rib is often performed. with mild dilation, the bowel width is three to four times the rib width; with extensive dilation, five to six times the rib width ( fig. - ) . in cases of linear foreign bodies, c-areas (comma-shaped areas) of gas trapped in the plicated bowel will appear stacked on one another. blunt, wedge-shaped areas of gas or square linear areas of gas adjacent to a distended bowel loop are characteristic of a foreign body lodged in the intestine. contrast radiography is indicated when confirmation of the suspected diagnosis is necessary and ultrasonography is not available. contrast material may outline the object or abruptly stop orad to the obstruction. the definitive treatment of any type of small intestinal foreign body is surgical removal. linear foreign bodies sometimes pass, but they should never be left untreated in a patient that is demonstrating clinical signs of inappetence, vomiting, lethargy, and dehydration. the timing of surgery is critical because the risk of intestinal perforation increases with time. prior to surgery, correct any acid-base and electrolyte abnormalities with intravenous fluid therapy. administer broad-spectrum antibiotics. perform an enterotomy or intestinal resection and anastomosis as soon as possible once the patient's acid-base and electrolyte status have been corrected. clinical signs of a foreign body in the large bowel are usually nonexistent. in most cases, if a foreign object has passed successfully through the small bowel, it will pass through the large bowel without incident unless bowel perforation and peritonitis occur. penetrating foreign bodies such as needles often cause localized or generalized peritonitis, abdominal pain, and fever. hematochezia may be present if the foreign object causes abrasion of the rectal mucosa. symptomatic patients should have abdominal radiographs performed. colonoscopy or exploratory laparotomy should be performed if survey radiographs are suggestive of a large intestinal obstruction or perforation. in most cases, large intestinal foreign bodies will pass without incident. surgery is required to treat perforations, peritonitis, or abscesses. emergency care figure - : after minutes, the barium has stopped moving and has reached a blunt, intraluminal intestinal foreign body. note that barium appears wedge-shaped or square at the site of the foreign body. foreign bodies in the rectum and anus often are the result of ingestion of bones, wood material, needles, and thread, or malicious external insertion. often the material can pass through the entire gastrointestinal tract and then get stuck in the anal ring. clinical signs include hematochezia and dyschezia with straining to defecate. diagnosis is made by visual examination of the item in the anus, or by careful digital palpation after heavy sedation or short-acting general anesthesia. radiography is helpful in locating needles that have penetrated the rectum and lodged in the perirectal or perinatal tissues. treatment consists of careful removal of the needle digitally or surgically. intussusception is the acute invagination of one segment of bowel (the intussusceptum) into another (the intussuscipiens). the proximal segment always invaginates into the distal segment of bowel. intussusception most commonly occurs in puppies and kittens less than year of age but can occur in an animal of any age with hypermotility of the small bowel, gastrointestinal parasites, and severe viral or bacterial enteritis. intussusception occurs primarily in the small bowel in the jejunum, ileum, and ileocolic junction. clinical signs include vomiting, abdominal discomfort, and hemorrhagic diarrhea. usually, hemorrhagic diarrhea is the first noticeable sign, and in puppies, may be due to parvoviral enteritis, with secondary intussusception. usually, the obstruction is partial with mild clinical signs. more serious clinical signs develop as the obstruction becomes more complete. differential diagnoses include hemorrhagic gastroenteritis, parvoviral enteritis, gastrointestinal parasites, intestinal foreign body, bacterial enteritis, and other causes of vomiting and diarrhea. the diagnosis of intussusception is often made based on palpation of a sausage-shaped firm, tubular structure in the abdomen accompanied by clinical signs and abdominal pain. plain radiographs may demonstrate segmental or generalized dilated segments of bowel, depending on the duration of the problem. ultrasonographs of the palpable mass resemble the layers of an onion, with hyperechoic intestinal walls separated by less echogenic edema. treatment consists of correction of the patient's acid-base and electrolyte abnormalities with intravenous fluids and surgical reduction or removal of the intussusception with resection and anastomosis. although enteroplication has been suggested, the technique has fallen out of favor because of the increased risk of later obstruction. the primary cause of intestinal inflammation and hypermotility must be identified and corrected. gastric dilatation can occur with or without volvulus in the dog. gastric dilatationvolvulus (gdv) occurs primarily in large-and giant-breed dogs with deep chests, such as the great dane, labrador retriever, saint bernard, german shepherd dog, gordon and irish setters, standard poodle, bernese mountain dog, and bassett hound. the risk of gdv increases with age; however, it can be seen in dogs as young as months. deep, narrow-chested breeds are more likely to develop gdv than dogs with broader chests. the overall mortality for surgically treated gastric dilatation-volvulus ranges from % to %, with most deaths occurring in patients that required splenectomy and partial gastrectomy. clinical signs of gdv include abdominal distention, unproductive vomiting or retching, lethargy, weakness, sometimes straining to defecate, and collapse. the owner may think that the animal is vomiting productively because of the white foamy froth (saliva) that is not able to pass into the twisted stomach. in some cases, there is a history of the dog's being fed a large meal or consuming a large quantity of water prior to the onset of clinical signs. instruct the owner of any patient with a predisposition for and clinical signs of gdv to transport the animal to the nearest veterinary facility immediately. physical examination often reveals a distended abdomen with a tympanic area on auscultation. in dogs with very deep chests, it may be difficult to appreciate abdominal distention if the stomach is tucked up under the rib cage. depending on the stage of shock, the patient may have sinus tachycardia with bounding pulses, cardiac dysrhythmias with pulse deficits, or bradycardia. the mucous membranes may appear red and injected or pale with a prolonged capillary refill time. the patient may appear anxious and attempt to retch unproductively. if the patient is nonambulatory at the time of presentation, the prognosis is more guarded. the definitive diagnosis of gdv is based on clinical signs, physical examination findings, and radiographic appearance of gas distention of the gastric fundus with dorsocranial displacement of the pylorus and duodenum (the so-called "double-bubble" or "popeye arm" sign) ( fig. - ) . in simple gastric dilatation without volvulus, there is gas distention of the stomach with anatomy appearing normal on radiography. with "food bloat," or gastric distention from overconsumption of food, ingesta is visible in the distended stomach ( fig. - ) . as soon as a patient presents with a possible gdv, place a large-bore intravenous catheter in the cephalic vein(s) and assess the patient's ecg, blood pressure, heart rate, capillary refill time, and respiratory function. obtain blood samples for a complete blood count, serum biochemistry profile, immediate lactate measurement, and coagulation tests before taking any radiographs. rapidly infuse a colloid (hetastarch or oxyglobin, ml/kg iv bolus) along with shock volumes of a crystalloid fluid (up to ml/kg/hour) (see section on shock). monitor perfusion parameters (heart rate, blood pressure, capillary refill time, and ecg) and titrate fluid therapy according to the patient's response. the use of short-acting glucocorticosteroids is controversial. glucocorticosteroids may help stabilize cellular membranes and decrease the mechanisms of ischemia-reperfusion injury, but no detailed studies have proved them to be beneficial versus not using glucocorticosteroids in the patient with gdv. attempt gastric decompression, either with placement of an orogastric tube or by trocharization. to place an orogastric tube, position the distal end of the tube at the level of the patient's last rib ( fig. - ) and place it adjacent to the animal's thorax; then put a piece of tape around the tube where it comes out of the mouth, once it is in place. put a roll of -inch tape in the patient's mouth behind the canine teeth and then secure the roll in place by taping the mouth closed around the roll of tape. lubricate the tube with lubricating jelly and slowly insert the tube through the center of the roll of tape into the stomach. the passing of the tube does not rule out volvulus. in some cases, the front legs of the patient need to be elevated, and the caudal aspect of the patient lowered (front legs standing on a table with back legs on the ground) to allow gravity to pull the stomach down to allow the tube to pass. once the tube has been passed, air within the stomach is relieved, and the stomach can be lavaged. the presence of gastric mucosa or blood in the efflux from the tube makes the prognosis more guarded. if an orogastric tube cannot be passed, clip and aseptically scrub the patient's lateral abdomen and then insert -gauge over-the-needle catheter. "pinging" the animal's side with simultaneous auscultation allows determination of the location that is most tympanic-that is, the proper location for catheter insertion. once intravenous fluids have been started in the animal, take a right lateral abdominal radiograph to document gdv. if no volvulus is present, the owner may elect for more conservative care, and the animal should be monitored in the hospital for a minimum of hours. because some cases of gdv intermittently twist and untwist, the owner should be cautioned that although the stomach is not twisted at that moment, a volvulus can occur at any time. if radiographs demonstrate food bloat, induce emesis (apomorphine, . mg/kg iv) or perform orogastric lavage under general anesthesia. documentation of gastric dilatation-volvulus constitutes a surgical emergency. figure - : example of "food bloat" with severe gastric distention caused by overconsump-following diagnosis of gdv, continue administration of intravenous fluids. serum lactate measurements greater than . mmol/l are associated with an increased risk of gastric necrosis, requirement for partial gastrectomy, and increased mortality. administer fresh frozen plasma ( ml/kg) to patients with thrombocytopenia or prolonged pt, activated partial thromboplastin time (aptt), or activated clotting time (act). cardiac dysrhythmias, particularly ventricular dysrhythmias, are common in cases of gdv and are thought to occur secondary to ischemia and proinflammatory cytokines released during volvulus and reperfusion. lidocaine ( - mg/kg followed by mcg/kg/minute iv cri) can be used to treat cardiac dysrhythmias preemptively that are associated with ischemia-reperfusion injury, or administration can be started when ventricular dysrhythmias are present. correct any electrolyte abnormalities, including hypokalemia and hypomagnesemia. the use of nonsteroidal antiinflammatory drugs (flunixin meglumine, carprofen, ketoprofen) that can potentially decrease renal perfusion and predispose to gastric ulcers is absolutely contraindicated. administer analgesic drugs (fentanyl, µ/kg iv bolus, followed by - µ/kg/hour iv cri; or hydromorphone, . mg/kg iv) before anesthetic induction. after carrying out a balanced anesthesia protocol, the patient should be taken immediately to surgery for gastric derotation and gastropexy. postoperatively, assess the patient's ecg, blood pressure, platelet count, coagulation parameters, and gastric function (see section on rule of twenty). if no resection is required, the animal can be given small amounts of water beginning hours after surgery. depending on the severity of the patient's condition, small amounts of a bland diet can be offered to hours postoperatively. continute supportive care with analgesia and crystalloid fluids until the patient is able to tolerate oral analgesic drugs (tramadol, - mg/kg po q - h). once the patient is ambulatory and able to eat and drink on its own, it can be released from the hospital; instruct the owner to feed the animal multiple small meals throughout the day for the first week. when the intestines twist around the root of the mesentery, a small intestinal or mesenteric volvulus occurs. the problem is most common in the young german shepherd dog, although it has been observed in other large and giant breeds. predisposing factors include pancreatic atrophy, gastrointestinal disease, trauma, and splenectomy. clinical signs of mesenteric volvulus include vomiting, hemorrhagic diarrhea, bowel distention, acute onset of clinical signs of shock, abdominal pain, brick-red mucous membranes (septicemia), and sudden death. diagnosis is based on an index of suspicion and the presence of clinical signs in a predisposed breed. plain radiographs often reveal grossly distended loops of bowel in a palisade gas pattern. in some dogs, multiple, tear-drop-shaped, gas-filled loops appear to rise from a focal point in the abdomen. usually, massive distention of the entire small bowel is observed ( fig. - ) . the presence of pneumoperitoneum or lack of abdominal detail secondary to the presence of abdominal fluid is characteristic of bowel perforation and peritonitis. in a patient with mesenteric volvulus, immediate aggressive action is necessary for the animal to have any chance of survival. treatment consists of massive volumes of iv crystalloid and colloid fluids (see section on iv therapy), broad-spectrum antibiotics (ampicillin, mg/kg iv qid, with enrofloxacin, mg/kg iv once daily), and surgical correction of the bowel. because of the massive release of proinflammatory cytokines, bacterial translocation, and ischemia, treatment for shock is of paramount importance (see sections on rule of twenty and shock). prognosis for any patient with mesenteric volvulus is poor. obstipation (obstructive constipation) is most common in the older cat. in cases of simple constipation, rehydrating the animal with intravenous fluids and stool softeners is often volvulus. this consistutes an immediate surgical emergency, and the prognosis is often poor. this condition is most common in young german shepherd dogs, but can be observed in any breed. sufficient for it to regain the ability to have a bowel movement. obstipation, however, is caused by adynamic ileus of the large bowel that eventually leads to megacolon. affected cats usually are anorectic, lethargic, and extremely dehydrated. treatment consists of rehydration with intravenous crystalloid fluids, correction of electrolyte abnormalities, enemas, and promotility agents such as cisapride ( . mg/kg po q - h). the use of phosphate enemas in cats is absolutely contraindicated because of the risk of causing acute, fatal hyperphosphatemia. in many cases, the patient should be placed under general anesthesia and manual deobstipation is performed with warm water soapy enemas and a gloved finger to relieve and disimpact the rectum. stool softeners such as lactulose and docusate stool sofener (dss) may also be used. predisposing causes of obstipation such as narrowing of the pelvic canal, perineal hernia, and tumors should be ruled out. adenocarcinoma is the most common neoplasm of the gastrointestinal tract that causes partial to complete obstruction. adenocarcinomas tend to be annular and constricting, and they may cause progressive obstruction of the lumen of the small or large bowel. siamese cats tend to have adenocarcinomas in the small intestine, whereas in dogs, the tumor tends to occur in the large intestine. clinical signs of adenocarcinoma are both acute and chronic and consist of anorexia, weight loss, and progressive vomiting that occur over weeks to months. effusion may be present if metastasis to peritoneal surfaces has occurred. diagnosis is based on clinical signs and physical examination findings of a palpable abdominal mass, radiographic evidence of an abdominal mass and small or large intestinal obstruction, or ultrasonographic evidence of an intestinal mass. treatment consists of surgical resection of the affected bowel segment. the prognosis for long-term survival ( - months) is good if the mass is completely resected and if other clinical signs of cachexia or metastasis are observed at the time of diagnosis. median survival is to weeks if metastasis to lymph nodes, liver, or the peritoneum are absent at the time of diagnosis. in dogs, the prognosis is more guarded. leiomyoma and leiomyosarcoma are tumors that can cause partial or complete obstruction of the bowel. clinical signs are often referred to progressive anemia, including weakness, lethargy, inappetence, and melena. hypoglycemia can be observed as a paraneoplastic syndrome, or due to sepsis and peritonitis secondary to bowel perforation. leiomyomas are most commonly observed at the ceco-colic junction or in the cecum. surgical resection and anastomosis is usually curative, and has a favorable prognosis. incarceration of a loop of bowel into congenital or acquired defects in the body wall can cause small bowel obstruction. pregnant females and young animals with congenital hernias are most at risk. rarely, older animals with perineal hernias and animals of any age with traumatic hernias can be affected. clinical signs are consistent with a small intestinal obstruction: anorexia, vomiting, lethargy, abdominal pain, and weakness. diagnosis is often made based on physical examination of a reducible or nonreducible mass in the body wall. hernias whose contents are reducible are usually asymptomatic. treatment consists of supportive care and rehydration, administration of broad-spectrum antibiotics, and surgical correction of the body wall hernia. in some cases, intestinal resection and anastomosis of the affected area is necessary when bowel ischemia occurs. the potential for bowel perforation should be suspected whenever there is any penetrating injury (knife, gunshot wound, bite wound, stick impalement) of the abdomen. injuries that result in bowel ischemia and rupture can also occur secondary to nonpenetrating blunt emergency care trauma or shear forces (e.g., big dog-little dog/cat). perforation of the stomach and small and large intestines can occur with use of nonsteroidal antiinflammatory drugs. diagnosis of bowel perforation first depends on the alertness to the possibility that the bowel may have been perforated or penetrated. as a general rule, all penetrating injuries of the abdomen should be investigated by exploratory laparotomy. diagnostic peritoneal lavage (dpl) can be performed; however, early after penetrating injury of the bowel, dpl may be negative or nondiagnostic until peritonitis develops. whenever any patient with blunt or penetrating abdominal trauma does not respond to initial fluid therapy, or responds and then deteriorates, the index of suspicion for bowel injury should be raised. the findings of pneumoperitoneum on abdominal radiographs or of intracellular bacteria, extracellular bacteria, bile pigment, bowel contents, and cloudy appearance of fluid obtained by abdominocentesis or diagnostic peritoneal lavage fluid (see sections on abdominocentesis and diagnostic peritoneal lavage) warrant immediate surgical exploration. treatment largely consists of stabilizing the patient's cardiovascular and electrolyte status with intravenous fluids, administration of broad-spectrum antibiotics, and definitive surgical exploration and repair of injured structures. prolapse of the rectum is observed most frequently secondary to parasitism and gastrointestinal viral infections in young puppies and kittens with chronic diarrhea. older animals with rectal prolapse often have an underlying problem such as a tumor or mucosal lesion that causes straining and dyschezia. the diagnosis of a rectal prolapse is made based on physical examination findings. the diagnosis of rectal prolapse is sometimes difficult to distinguish from small intestinal intussusception. in rare cases, the intussusception can invaginate through the large bowel, rectum, and anus. the two entities are distinguished from one another by inserting a lubricated thermometer or blunt probe into the cul-de-sac formed by the junction of the prolapsed mucosa and mucocutaneous junction at the anal ring. inability to insert the probe or thermometer indicates that the rectal mucosa is prolapsed. passage of the probe signifies that the prolapsed segment is actually the intussusceptum. treatment can be performed easily if the prolapse is acute and the rectal mucosa is not too irritated or edematous. the presence of severely necrotic tissue warrants surgical intervention. to reduce an acute rectal prolapse, after placing the patient under general anesthesia, lubricate the prolapsed tissue and gently push it back into the rectum, using a lubricated syringe or syringe casing. apply a loose purse-string suture, leaving it in place for a minimum of hours. de-worm the patient and administer stool softeners. if a rectal prolapse cannot be reduced, or if the tissue is nonviable, surgical intervention is warranted. in patients in which viable tissue does not stay reduced with a purse-string suture, a colopexy can be performed during a laparotomy. first, place tension on the colon to reduce the prolapse, and then suture the colon to the peritoneum of the lateral abdominal wall with two to three rows of - or - monofilament suture material. if the prolapsed tissue is nonviable, it must be amputated. place four stay sutures at -degree intervals through the wall of the prolapse at the mucocutaneous junction. resect the prolapse distal to the stay sutures and then reestablish the rectal continuity by suturing the seromuscular layers together in one circumferential line and the mucosal layers together in the other. replace the suture incision into the anal canal. following surgery, de-worm the patient and administer a stool softener and analgesic drugs. avoid using thermometers or other probes in the immediate postoperative period because they may disrupt suture lines. acute gastritis may be associated with a variety of clinical conditions, including oral hemorrhage, ingestion of highly fermentable nondigestable foods or garbage, toxins, foreign bodies, renal or hepatic failure, inflammatory bowel disease, and bacterial and viral infections. diarrhea often accompanies or follows acute gastritis. hemorrhagic gastroenteritis often occurs as a shock-like syndrome with a rapidly rising hematocrit level. clinical signs of gastritis include depression, lethargy, anterior abdominal pain, excessive water consumption, vomiting, and dehydration. differential diagnosis of acute gastritis includes pancreatitis, hepatic or renal failure, gastrointestinal obstruction, and toxicities (box - ). the diagnosis is often a diagnosis of exclusion of other causes (see preceding text). a careful and thorough examination of the vomitus may be helpful in arriving at a diagnosis. a complete blood count, serum biochemistry profile including amylase and lipase, parvovirus test (in young puppies), fecal flotation and cytology, abdominal radiographs (plain and/or contrast studies), and abdominal ultrasound may be warranted to rule out other causes of acute vomiting. while diagnostic tests are being performed, treatment consists of withholding all food and water for a minimum of hours. after calculating the patient's degree of dehydration, administer a balanced crystalloid fluid to normalize acid-base and electrolyte status. control vomiting with antiemetics such as metoclopramide, prochlorperazine, chlorpromazine, dolasetron, and ondansetron (table - ). if vomiting is accompanied by diarrhea, administer broad-spectrum antibiotics (cefazolin, mg/kg iv q h, with metronidazole, mg/kg iv q h; or ampicillin, mg/kg iv q h, with enrofloxacin, mg/kg iv q h) to decrease the risk of bacterial translocation and bacteremia/septicemia. although antacids (famotidine, ranitidine, cimetidine) do not have a direct antiemetic effect, their use can decrease gastric acidity and esophageal irritation during vomiting. if gastritis is secondary to uremia or nonsteroidal antiinflammatory drug use, administer gastroprotectant and antiemetic drugs (ranitidine, mg/kg po q h; sucralfate, . - g/dog po q h; or omeprazole ( . - mg/kg po q h) to decrease acid secretion and coat areas of gastric ulceration (table - ) . once food and water can be tolerated, the patient can be placed on an oral diet and medications, and intravenous fluids can be discontinued. do not use until a gastrointestinal obstruction has been ruled out. hemorrhagic gastroenteritis (hge) is an acute onset of severe hemorrhagic vomiting and diarrhea most commonly observed in young small-breed dogs (e.g., poodles, miniature dachshunds, miniature schnauzers) to years of age. clinical signs develop rapidly and include vomiting and fetid diarrhea with hemorrhage, often strawberry jam-like in appearance. the hematocrit can rise from % to %. often, the animal is extremely hypovolemic but has no apparent signs of abdominal pain. there is no known cause of hge, although clostridium perfringens, escherichia coli, campylobacter, and viral infections have been suggested but not consistently confirmed. other differential diagnoses of of hematemesis and hemorrhagic diarrhea include coronavirus, parvovirus, vascular stasis, sepsis, hepatic cirrhosis with portal hypertension, and other causes of severe shock. immediate treatment consists of placement of a large-bore intravenous catheter and replenishment of intravascular fluid volume with crystalloid fluids (up to ml/kg/hour), while carefully monitoring the patient's hematocrit and total protein. administer broad-spectrum antibiotics (ampicillin, mg/kg iv q h, and enrofloxacin mg/kg iv q h) because of the high risk of bacterial translocation and sepsis. control vomiting with antiemetic drugs. monitor the patient's platelet count and coagulation tests for impending disseminated intravascular coagulation (dic), and administer fresh frozen plasma and heparin, as needed (see section on disseminated intravascular coagulation). when vomiting has ceased for hours, offer the animal small amounts of water, and then a bland diet (e.g., boiled chicken and rice or boiled ground beef and rice mixed with low-fat cottage cheese). pancreatitis occurs most frequently in dogs but can occur in cats as well. in dogs, the onset of pancreatitis is sometimes preceded by ingestion of a fatty meal or the administration of drugs (e.g., potassium bromide or glucocorticoids). glucocorticoids can increase the viscosity of pancreatic secretions and induce ductal proliferation, resulting in narrowing and obstruction of the lumen of the pancreatic duct. pancreatitis can also occur following blunt or penetrating abdominal trauma, high duodenal obstruction causing outflow obstruction of the pancreatic papilla, pancreatic ischemia, duodenal reflux, biliary disease, and hyperadrenocorticism. in cats, acute necrotizing pancreatitis is associated with anorexia, lethargy, hyperglycemia, icterus, and sometimes acute death. chronic pancreatitis is more common in cats and results in intermittent vomiting, anorexia, weight loss, and lethargy. predisposing causes of chronic pancreatitis in cats include pancreatic flukes, viral infection, hepatic lipidosis, drugs, organophosphate toxicity, and toxoplasmosis. clinical signs of acute pancreatitis include sudden severe vomiting, abdominal pain, and lethargy. depending on the severity of pancreatic inflammation, depression, hypotension, and systemic inflammatory response syndrome (sirs) may be present. subacute cases may have minimal clinical signs. severe pancreatic edema can result in vascular changes and ischemia that perpetuates severe inflammation. hypovolemic shock and dic can also decrease pancreatic perfusion. severe pancreatic edema, autolysis, and ischemia lead to pancreatic necrosis. duodenal irritation is manifested as both vomiting and diarrhea. pain may be localized to the right upper abdominal quadrant or may be generalized if peripancreatic saponification occurs. differential diagnosis of pancreatitis is the same as for any other cause of vomiting. complications that occur in patients with severe pancreatitis include dehydration, acidbase and electrolyte abnormalities, hyperlipemia, hypotension, and localized peritonitis. hepatic necrosis, lipidosis, congestion, and abnormal architecture can develop. inflammatory mediators (bradykinin, phospholipase a, elastase, myocardial depressant factor, and bacterial endotoxins) stimulate the inflammatory cascade and can lead to sirs, with severe hypotension, clotting system activation, and dic. electrolyte imbalances and hypovolemia secondary to vomiting all can lead to multiple organ dysfunction syndrome (mods), and ultimately, death. if a patient survives an episode of acute pancreatitis, long-term sequelae can include diabetes mellitus. monitor patients with recurrent pancreatitis for clinical signs of polyuria, polydipsia, polyphagia, hyperglycemia, and glucosuria. the diagnosis of pancreatitis is based on the presence of clinical signs (which may be absent in cats), laboratory findings, and ultrasonographic evidence of pancreatic edema and increased peripancreatic echogenicity. serum biochemistry analyses can sometimes support a diagnosis of pancreatitis; however, serum amylase and lipase are often unreliable indicators of pancreatitis, depending on the chronicity of the process in the individual patient. both serum amylase and lipase are excreted in the urine. impaired renal clearance/ function can cause artifactual elevations of serum amylase and lipase in the absence of pancreatic inflammation. furthermore, serum lipase levels can be elevated as a result of gastrointestinal obstruction (e.g., foreign body). early in the course of the disease, levels can be two to six times normal, but they may decrease to within normal ranges at the time of presentation to the veterinarian. the transient nature of amylase elevation makes this test difficult to interpret, and it is not highly sensitive if a normal value is found. lipase levels also increase later in the course of the disease. amylase and lipase should be tested concurrently with the rest of the biochemistry profile. other changes often observed are elevations in bun and creatinine levels secondary to dehydration and prerenal azotemia, hyperglycemia, and hyperlipemia. hypocalcemia can occur secondary to peripancreatic fat saponification, and its presence warrants a more negative prognosis. a more specific measure is pancreatic lipase immunoreactivity, which becomes elevated in dogs and cats with pancreatitis. this test, combined with ultrasonographic or computed tomography evidence of pancreatitis, is the most sensitive and specific test available for making an accurate diagnosis. however, because the results of this test take time to obtain, animals must be treated in the meantime. abdominal effusion or fluid from diagnostic peritoneal lavage can be compared with serum amylase and lipase activity. abdominal lipase and amylase concentrations in the fluid greater than that in the peripheral blood are characteristic of chemical peritonitis associated with pancreatitis. wbc counts greater than cells/mm , the presence of bacteria, toxic neutrophils, glucose levels less than mg/dl, or lactate levels greater than that of serum are characteristic of septic peritonitis, and immediate exploratory laparotomy is warranted. if a biopsy sample obtained during laparotomy does not demonstrate inflammation, but this does not rule out pancreatitis, because disease can be focal in nature and yet cause severe clinical signs. abdominal radiographs may sometimes reveal a loss of abdominal detail or a ground glass appearance in the right upper quadrant. pancreatic edema and duodenal irritation can displace the gastric axis toward the left, toward the left with dorsomedial displacement of the proximal duodenum (the so-called "backwards " or "shepherd's crook" sign). ultrasonography and ct are more sensitive in making a diagnosis of pancreatitis. treatment of pancreatitis is largely supportive in nature and is designed to correct hypovolemia and electrolyte imbalances, prevent or reverse shock, maintain vital organ perfusion, alleviate discomfort and pain, and prevent vomiting (see section on rule of twenty). when treating pancreatitis in dogs, all food and water should be restricted. however, food should not be withheld from cats with chronic pancreatitis. give fresh frozen plasma to replenish alpha- -macroglobulins. administer antiemetics such as chlorpromazine (use with caution in a hypovolemic or hypotensive patient), dolasetron, ondansetron, or metoclopramide to prevent or control vomiting. analgesic drugs can be provided in the form of constant rate infusion (fentanyl, - µ/kg/hour iv cri, and lidocaine, - µ/kg/minute iv cri), intrapleural injection (lidocaine, - mg/kg q h), or intermittent parenteral injections (morphine, . - mg/kg sq, im; hydromorphone, . mg/kg im or sq). because the pancreas must be rested, consider using parenteral nutrition. acute hepatic failure may be associated with toxins, adverse reaction to prescription medication, and bacterial or viral infections. the most frequent clinical signs observed in a patient with acute hepatic failure are anorexia, lethargy, vomiting, icterus, bleeding, and cns depression or seizures (associated with hepatic encephalopathy). differential diagnosis and causes of acute hepatic failure are listed in box - . diagnosis of acute hepatic failure is based on clinical signs and biochemical evidence of hepatocellular (ast, alt) and cholestatic (alk phos, t bili, ggt) enzyme elevations. ultrasonography may be helpful in distinguishing the architecture of the liver, but unless a mass or abscess is present, cannot provide a specific diagnosis of the cause of the hepatic damage. management of the patient with acute hepatic failure includes correction of dehydration and acid-base and electrolyte abnormalities, as shown in the following list: • hypoalbuminemia: plasma or concentrated albumin. plasma also is an excellent source of clotting factors that can become depleted. • clotting abnormalities: vitamin k ( . mg/kg sq or po q - h) to • severe anemia: fresh or stored blood • gastric hemorrhage: gastroprotectant drugs (omeprazole, ranitidine, famotidine, cimetidine, sucralfate) • hypoglycemia: dextrose supplementation ( . %- %) • hepatic failure, particularly when hypoglycemia is present: broad-spectrum antibiotics (ampicillin mg/kg iv q h; with enrofloxacin, mg/kg iv q h) • hepatic encephalopathy: lactulose or betadine enemas • cerebral edema: mannitol ( . - . g/kg iv over to minutes) followed by furosemide ( mg/kg iv minutes later). deterioration of clinical signs may signify the development of cerebral edema. applewhite aa, cornell kk, selcer ba: diagnosis and treatment of intussusception in dogs. comp cont educ pract vet ( ) often, systemic hypertension is diagnosed when the animal is seen by the veterinarian because of some other clinical sign, such as acute blindness, retinal detachment, hyphema, epistaxis, and cns signs following intracranial hemorrhage. diagnosis of systemic hypertension is often difficult in the absence of clinical signs and without performing invasive or noninvasive blood pressure monitoring. normal blood pressure (bp) measurements in dogs and cats are listed in table - . hypertension is defined as a consistent elevation in systolic bp > mm hg, consistent diastolic bp > mm hg, and consistent mean arterial blood pressure > mm hg. the effects of systemic hypertension include left ventricular hypertrophy, cerebrovascular accident, renal vascular injury, optic nerve edema, hyphema, retinal vascular tortuosity, retinal hemorrhage, retinal detachment, vomiting, neurologic defects, coma, and excessive bleeding from cut surfaces. emergency care dog - - - cat - - - patients with systemic hypertension should have a thorough diagnostic work-up to determine the underlying cause. although uncommon, hypertensive emergencies can occur with pheochromocytoma, acute renal failure, and acute glomerulonephritis. sodium nitroprusside ( - µ/kg/minute iv cri) or diltiazem ( . - . mg/kg iv given slowly over minutes, followed by µ/kg/minute) can be used to treat systemic hypertension. with the use of sodium nitroprusside or diltiazem, monitor carefully for hypotension. diagnosis is based on consistent elevations in systolic, diastolic, and/or mean arterial bp. because many of the clinical signs associated with systemic hypertension involve hemorrhage into some closed cavity, other causes of hemorrhage, such as vasculitis, thrombocytopenia, thrombocytopathia, and hepatic or renal failure, should be investigated (see section on coagulation disorders). diagnostic testing is based on clinical signs and index of suspicion for an underlying disease and may include a complete blood count; urinalysis; urine protein:creatinine ratio; acth stimulation test; thoracic and abdominal radiographs; thoracic and abdominal ultrasound; tick serology; brain ct or mri; and assays of serum electrolytes, aldosterone concentration, t , endogenous tsh, plasma catecholamine, and growth hormone. management of systemic hypertension involves treatment of the primary underlying disorder, whenever possible. long-term adjunctive management includes sodium restriction in the form of cooked or prescription diets to decrease fluid retention. obese animals should be placed on dietary restrictions and undergo a weight reduction program. thiazide and loop diuretics may be used to decrease sodium retention and circulating blood volume. alpha-and beta-adrenergic blockers may be used, but they are largely ineffective as monotherapeutic agents for treating hypertension. calcium channel blockers and angiotensin-converting enzyme (ace) inhibitors are the mainstay of therapy in the treatment of hypertension in dogs and cats ( diabetic ketoacidosis (dka) is a potentially fatal and terminal consequence of unregulated insulin deficiency and possible glucagon excess. in the absence of insulin, unregulated lipolysis results in the beta-hydroxylation of fatty acids by abnormal hepatic metabolism. as a result, ketoacids-namely, acetoacetic acid, beta-hydroxybutyric acid, and acetoneare produced. early in the course of the disease, patients exhibit clinical signs associated with diabetes mellitus: weight loss, polyuria, polyphagia, and polydipsia. later, as ketoacids stimulate the chemoreceptor trigger zone, vomiting and dehydration occur, with resulting hypovolemia, hypotension, severe depression, abdominal pain, oliguria, and coma. at the time of presentation, often a strong odor of ketones (acetone) is present on the patient's breath. physical examination often reveals dehydration, severe depression or coma, and hypovolemic shock. in extreme cases, the patient exhibits a slow, deep kussmaul respiratory pattern in an attempt to blow off excess co to compensate for the metabolic acidosis. a serum biochemistry profile and complete blood count often reveal prerenal azotemia, severe hyperglycemia (blood glucose > mg/dl), hyperosmolarity (> mosm/kg), lipemia, hypernatremia (sodium > meq/l), elevated hepatocellular and cholestatic enzyme activities, high anion gap, and metabolic acidosis. although a whole body potassium deficit is usually present, the serum potassium may appear artifactually elevated in response to metabolic acidosis. with severe metabolic acidosis, potassium moves extracellularly in exchange for a hydrogen ion. phosphorus too moves intracellularly in response to acidosis, and serum phosphorus is usually decreased. hypophosphatemia > mg/dl can result in intravascular hemolysis. urinalysis often reveals + glucosuria, ketonuria, and a specific gravity of . or greater. the urine of all diabetic animals should be cultured to rule out a urinary tract infection or pyelonephritis. treatment of a patient with dka presents a therapeutic challenge. treatment is aimed at providing adequate insulin to normalize cellular glucose metabolism, correcting acidbase and electrolyte imbalances, rehydration and restoration of perfusion, correcting acidosis, providing carbohydrate sources for utilization during insulin administration, and identifying any precipitating cause of the dka. obtain blood samples for a complete blood count, and serum biochemistry electrolyte profiles. whenever possible, insert a central venous catheter for fluid infusion and procurement of repeat blood samples. calculate the patient's dehydration deficit and maintenance fluid requirements and give appropriate fluid and electrolytes over a period of hours. it is advisable to rehydrate patients with severe hyperosmolarity for a minimum of hours before starting insulin administration. use a balanced electrolyte solution (e.g., plasmalyte-m, normosol-r, lactated ringer's solution) or . % saline solution for maintenance and rehydration. balanced electrolyte solutions contain small amounts of potassium and bicarbonate precursors that aid in the treatment of metabolic acidosis. treat animals with severe metabolic acidosis with an hco − > meq/l or a ph < . with supplemental bicarbonate ( . - . meq/kg). add supplemental dextrose to the patient's fluids as a carbohydrate source during insulin infusion. both insulin and carbohydrates are necessary for the proper metabolism of ketone bodies in patients with dka. the rate and type of fluid and amount of dextrose supplementation will change according to the patient's blood glucose concentration. serum potassium will drop rapidly as the metabolic acidosis is corrected with fluid and insulin administration. measure serum potassium every hours, if possible, and supplement accordingly (see section on fluid therapy for chart of potassium supplementation). if the patient's potassium requirement exceeds meq/l, or if the rate of potassium infusion approaches . meq/ kg/hour in the face of continued hypokalemia, magnesium should be supplemented. magnesium is required as a cofactor for many enzymatic processes and for normal function of the na,k-atpase pump. hypomagnesemia is a common electrolyte disturbance in many forms of critical illness. replenishing magnesium (mgcl , . meq/kg/day iv cri) often helps to correct the refractory hypokalemia observed in patients with dka. patients with hypophosphatemia that approaches . mmol/l should receive potassium phosphate ( . - . mmol/kg/hour iv cri). when providing potassium phosphate supplementation, be aware of the additional potassium added to the patient's fluids, so as to not exceed recommended rates of potassium infusion. to determine the amount of potassium chloride (kcl) to add along with potassium phosphate (kpo ), use the following formula: meq k + derived from kcl = total meq of k + to be administered over hours − meq in which k + is derived from kpo clinical signs of severe hypophosphatemia include muscle weakness, rhabdomyolysis, intravascular hemolysis, and decreased cerebral function that can lead to depression, stupor, seizures, or coma. regular insulin can be administered either im or as a constant rate infusion in the treatment of patients with dka. subcutaneous insulin should not be administered. because of the severe dehydration present in most patients with dka, subcutaneous insulin is poorly absorbed and is not effective until hydration has been restored. in the low-dose intravenous method, place regular insulin ( . units/kg for a cat, and . units/kg for a dog) in ml of . % saline solution. run ml of this mixture through the intravenous line to allow the insulin to adsorb to the plastic tubing. administer the patient's insulin fluid rate according to blood glucose levels ( table - ) . adjust the patient's total fluid volume according to changes in the insulin fluid rate as necessary. in many cases, multiple bags of fluids are necessary because they must be changed when fluctuations in blood glucose concentrations occur in response to therapy. infusion of the insulin mixture should be in a separate intravenous catheter. to replenish hydration, use a second intravenous line for the more rapid infusion of non-insulin-containing fluids. to administer the regular insulin im, first give . unit/kg im and then re-check the patient's blood glucose every hour. additional injections of regular insulin ( . unit/kg other fluid type (ml/hour) > . % nacl - . % nacl + . % dextrose - . % nacl + . % dextrose - . % nacl + . % dextrose < . % nacl + % dextrose im) should be administered based on the patient's response to subsequent injections. once the patient's blood glucose falls to to mg/dl, add . % to % dextrose to the fluids to maintain the blood glucose concentration at to mg/dl. continue intramuscular injection of regular insulin ( . - . unit/kg q - h) until the patient is rehydrated, no longer vomiting, and able to tolerate oral fluids and food without vomiting. even in patients with intramuscular regular insulin therapy, a central venous catheter should be placed for frequent blood sample collection. as the patient begins to respond to therapy, monitor electrolytes, glucose, and acid-base status carefully. hypokalemia, hypophosphatemia, and hypomagnesemia can occur. when the patient's hydration and acid-base status has normalized and the patient is able to tolerate oral food and water, a longer-acting insulin can be administered as for treatment of a patient with uncomplicated diabetes. extreme hyperosmolarity can result in a coma, if uncorrected. in patients with diabetes mellitus, hyperglycemia and hypernatremia secondary to osmotic diuresis and free water loss can lead to severe hyperosmolarity. in dogs, normal serum osmolality is < mosm/l of serum. hyperosmolarity is expected when serum osmolality is > mosm/l. if equipment for determining serum osmolarity is not available, osmolarity can be calculated by the following formula: osm/l = (na + k) + (glucose/ ) + (bun/ . ) patients with severe dehydration, hyperglycemia, hypernatremia, and azotemia may experience cerebral edema without ketonemia. treatment is directed solely at rehydrating the patient and slowly reducing blood glucose levels using a hypotonic solution such as . % nacl + . % dextrose or % dextrose in water (d w). after the initial rehydration period, administer potassium supplementation conservatively. red blood cells and the brain absolutely depend on the oxidation of glucose for energy. hypoglycemia can be caused by various systemic abnormalities that can be related to intestinal malabsorption of nutrients, impaired hepatic glycogenolysis or gluconeogenesis, and inadequate peripheral utilization of glucose. clinical signs of hypoglycemia are extremely variable and can include weakness, tremors, nervousness, polyphagia, ataxia, tachycardia, muscle twitching, incoordination, visual disturbances, and generalized seizures. clinical signs typically occur when serum glucose levels are < mg/dl. the combination of the clinical signs listed previously, documentation of low serum glucose, and alleviation of clinical signs upon glucose administration is known as whipple's triad. whenever a patient presents with hypoglycemia, consider the following important factors: the age of onset, the nature of the hypoglycemic episode (transient, persisent, or recurrent) , and the pattern based on the patient's history . treatment of hypoglycemia is directed at providing glucose supplementation and determining any underlying cause. administer supplemental dextrose ( %- % dextrose, - ml/kg iv; or % dextrose, ml/kg po) as quickly as possible. do not attempt oral glucose supplementation in any patient having a seizure or if the airway cannot be protected. administer intravenous fluids (e.g., normosol-r, lactated ringer's solution, . % saline solution) with . %- % supplemental dextrose until the patient is eating and able to maintain euglycemia without supplementation. in some cases (e.g., insulinoma), eating or administration of supplemental dextrose can promote insulin secretion and exacerbate clinical signs and hypoglycemia. in cases of refractory hypoglycemia secondary to iatrogenic insulin overdose, glucagon ( mg/kg iv bolus, then - ng/kg/minute iv cri) can also be administered along with supplemental dextrose. to make a glucagon infusion of ng/ml, reconstitute ml ( mg/ml) of glucagon according to the manufacturer's instructions and add this amount to ml of . % saline solution. emergency care the diagnosis of eclampsia (puerperal tetany) is often made on the basis of history and clinical signs. clinical signs can become evident when total calcium decreases to < . mg/dl in dogs and < . mg/dl in cats. the disease is often observed in small, excitable dogs, and stress may play a complicating role in the etiology. in most bitches, the disease manifests itself to weeks after parturition. in some cases, however, clinical signs can develop before parturition occurs. hypophosphatemia may accompany hypocalcemia. clinical signs of hypocalcemia include muscle tremors or fasciculations, panting, restlessness, aggression, hypersensitivity, disorientation, muscle cramping, hyperthermia, stiff gait, seizures, tachycardia, a prolonged qt interval on ecg, polydipsia, polyuria, and respiratory arrest. treatment of eclampsia consists of slow, cautious calcium supplementation ( % calcium gluconate, . mg/kg iv over minutes). severe refractory tetanus can be controlled with intravenous diazepam. supportive care includes intravenous fluid administration and cooling (see section on hyperthermia and heat-induced illness). instruct the owner to give the patient oral calcium supplements (e.g., to tablets of tums bid-tid) after discharge from the hospital. also instruct the owner about how to wean the puppies, allowing the bitch to dry up, in order to prevent recurrence. recurrence with subsequent pregnancies is common, particularly in patients that receive calcium supplementation during gestation (table - ) . hypercalcemia can occur from a variety of causes. the gosh darn it mnemonic can be used to remember the various causes of hypercalcemia in small animal patients (box - ) . the gastrointestinal, renal, and nervous systems are most commonly affected, particularly when serum total calcium rises above . mg/dl. clinical signs of severe hypercalcemia include muscle weakness, vomiting, seizures, and coma. ecg abnormalities include prolonged pr interval, rapid qt interval, and ventricular fibrillation. the most serious clinical signs are often seen when hypercalcemia is observed in combination with hyperphosphatemia or hypokalemia. pay special attention to the "calcium × phosphorus product." if this product exceeds , dystrophic calcification can occur, leading to renal failure. renal complications include polyuria, polydipsia, dehydration, and loss of renal tubular concentrating ability. renal blood flow and the glomerular filtration rate (gfr) are impaired when serum total calcium exceeds mg/dl. the extent, location, and number of renal tubular injuries are the main factors in determining whether renal damage secondary to hypercalcemia is reversible or irreversible. emergency therapy of hypercalcemia is warranted when severe renal compromise, cardiac dysfunction, or neurologic abnormalities are present, or if no clinical signs occur but the calcium × phosphorus product exceeds . the treatment of choice is correction of the underlying cause of hypercalcemia, whenever possible. in some cases, the results of diagnostic tests take time, and emergency therapy should be initiated immediately, before a definitive cause of the hypercalcemia is found. emergency management of hypercalcemia consists of reduction of serum calcium levels. administer intravenous fluids ( . % saline solution) to expand extracellular fluid volume and promote calciuresis. to promote diuresis, initial intravenous fluid rates should approach two to three times maintenance levels ( - ml/kg/day). potassium supplementation may be required to prevent iatrogenic hypokalemia. administration of a loop diuretic such as furosemide ( - mg/kg iv) will promote calcium excretion. calcitonin ( iu/kg im q h for cats and iu/kg im q h for dogs) can be administered to decrease serum calcium levels. in severe refractory hypercalcemia secondary to cholecalciferol toxicity, more aggressive calcitonin therapy ( - iu/kg sq q - h) can be attempted. side effects of calcitonin treatment include vomiting and diarrhea. alternatively, bisphosphonates (pamidronate, . - . mg/kg iv) are useful in rapidly reducing serum calcium concentrations. glucocorticosteroids reduce calcium release from the bone, decrease intestinal absorption of calcium, and promote renal calcium excretion. administer glucocorticosteroids only after the underlying cause of hypercalcemia has been determined and appropriate therapy started. because many forms of neoplasia can result in hypercalcemia as a paraneoplastic syndrome, empiric use of glucocorticosteroids can induce multiple drug resistance, making the tumor refractory to the effects of chemotherapeutic agents. hypoadrenocorticism is most commonly observed in young to middle-aged female dogs, but it can occur in animals of any age, gender, and breed. clinical signs, which are referable to deficiency in glucocorticoid (cortisol) and mineralocorticoid (aldosterone) hormones, may develop slowly over time, leading to a waxing and waning course; acute clinical signs occur when > % of the adrenal functional reserve has been destroyed. in such cases, complete adrenocortical collapse can result in an addisonian crisis. lack of aldosterone causes a lack of renal sodium and water retention, and impaired potassium excretion. the most significant clinical signs associated with hypoadrenocorticism are depression, lethargy, weakness, anorexia, shaking, shivering, vomiting, diarrhea, weight loss, abdominal pain, weakness, hypotension, dehydration, and inappropriate bradycardia (box - ) . the diagnosis of hypoadrenocorticism is made based on the patient's clinical signs in combination with electrolyte abnormalities that include hyperkalemia, hyponatremia, and hypochloremia. serum sodium concentration ( - meq/l) is often greatly reduced, and serum potassium is elevated (> . meq/l). a sodium:potassium ratio of < is characteristic of hypoadrenocorticism, although not exactly pathognomonic. electrocardiographic changes associated with hyperkalemia include inappropriate bradycardia, absence of p waves, elevated spiked t waves, and widened qrs complexes. other more variable bloodwork abnormalities include a lack of a stress leukogram, eosinophilia, hypoglycemia, hyperphosphatemia, hypercalcemia, azotemia, and hypocholesterolemia. a definitive diagnosis of hypoadrenocorticism is based on an adrenocorticotropic hormone (acth) stimulation test. in patients with hypoadrenocorticism, baseline cortisol levels are usually low, with a lack of appropriate cortisol release after administration of acth analogue. rarely, animals with "atypical" hypoadrenocorticism lose glucocorticoid secreting ability from the zona fasciculata, but retain mineralocorticoid secretory ability from the zona glomerulosa. atypical addisonian patients have normal serum electrolytes but still have clinical signs of vomiting, diarrhea, weakness, lethargy, inappetence, muscle wasting, and weight loss. the diagnosis is more difficult in such cases because of the presence of normal electrolytes. an acth stimulation test should be considered, particularly in predisposed breeds. treatment of hypoadrenocorticism includes placement of a large-bore intravenous catheter, infusion of intravenous crystalloid fluids ( . % saline solution), and replenishment of glucocorticoid and mineralocorticoid hormones. administer dexamethasone or dexamethasone-sodium phosphate ( . - . mg/kg iv). dexamethasone will not interfere with the acth stimulation test, unlike other longer-acting steroids (e.g., prednisolone, methylprednisolone sodium succinate, triamcinolone). depending on the severity of the patient's condition, consider monitoring using the rule of twenty. administer antiemetics and gastroprotectant drugs to treat nausea, vomiting, and hematemesis. give the patient broad-spectrum antibiotics (ampicillin, mg/kg iv q h) if hematochezia or hemorrhagic diarrhea is present. if severe gastrointestinal blood loss occurs, whole blood, packed red blood cells, or fresh frozen plasma may be required. control hypoglycemia with . %- . % dextrose. use sodium bicarbonate, regular insulin with dextrose, or calcium gluconate to correct severe hyperkalemia with atrial standstill (see section on atrial standstill). chronic therapy for hypoadrenocorticism consists of mineralocorticoid and glucocorticosteroids supplementation for the rest of the animal's life. mineralocorticoid supplementation can be in the form of desoxycorticosterone pivalate (docp) ( . mg/kg im) or fludrocortisone acetate ( . mg/ . - kg body weight daily). fludrocortisone acetate possesses both mineralocorticoid and glucocorticoid activities and can be used as the sole daily treatment of hypoadrenocorticism. (because fludrocortisone is poorly absorbed in some dogs, it may not completely normalize electrolyte abnormalities in these animals.) docp is primarily a mineralocorticoid. give supplemental glucocorticosteroids in the form of prednis(ol)one ( - . mg/kg/day). in dogs, iatrogenic hypoadrenocorticism can be caused by abrupt discontinuation of glucocorticosteroid treatment. long-term glucocorticosteroid supplementation can downregulate the pituitary gland's excretion of endogenous acth and the zona fasciculata's ability to excrete cortisol. however, the zona glomerulosa's ability to secrete aldosterone does not appear to be affected. clinical signs of iatrogenic hypoadrenocorticism include inability to compensate for stress, weakness, lethargy, vomiting, diarrhea, and collapse. treatment of iatrogenic hypoadrenocorticism is the same as for naturally occurring disease. following immediate emergency treatment, the patient should be weaned slowly from exogenous glucocorticosteroid supplementation. severe hyperthyroidism can manifest as a medical emergency as a result of hypermetabolism. clinical signs in affected cats with severe thyrotoxicosis include fever, severe tachycardia (heart rate > bpm), vomiting, hypertension, congestive heart failure with pulmonary edema, and fulminant collapse. clinical signs typically are manifested as an end-stage of chronic debilitation associated with hyperthyroidism and are often preceded by polyphagia, weight loss, cardiac murmur, polyuria/polydipsia (pu/pd), vomiting, and diarrhea. treatment of thyrotoxicosis includes antagonizing the adrenergic activity by administration of a beta-adrenergic blocker (esmolol, ( - µ/kg/minute, or propranolol, . mg/ kg/hour). administration of glucocorticosteroids (dexamethasone, mg/kg) may inhibit the conversion of thyroxine (t ) to the active form triiodothyronine (t ) and decrease peripheral tissue responsiveness to t , effectively blocking its effects. correct hypoglycemia with supplemental dextrose ( . %). use care to avoid overhydration in a patient with cardiac failure or insufficiency. start the patient on methimazole as quickly as possible and consider the use of radioactive iodine therapy. to maintain cerebral perfusion pressure, blood pressure must be normalized. if other concurrent injuries are suspected (e.g., pulmonary contusions), administer synthetic colloid fluids (dextran- , - ml/kg iv, or hetastarch, - ml/kg iv) to normalize blood pressure. although the use of colloids is controversial because of their potential to leak into the calvarium, the benefits of reestablishing cerebral perfusion far outweigh the risks of their use. hypertonic saline ( . % nacl, - ml/kg iv) can also be administered over to minutes to expand intravascular volume. maintain blood glucose within normal reference ranges whenever possible, because hyperglycemia is a negative prognostic indicator in cases of head trauma. if tremors or seizures cause hyperthermia or increased metabolism, active cooling of the patient is warranted (see sections on hyperthermia and heat-induced injury). all patients with head trauma should receive care and monitoring based on the rule of twenty (see section on rule of twenty). examine the patient's level of consciousness, response to various stimuli, pupil size and reactivity to light, physiologic nystagmus, and cranial nerve deficits. in dogs, damage to the midbrain often produces coma and decerebrate rigidity. initial consciousness followed by a unconsciousness or stupor usually involves an injury to the brainstem. brainstem lesions can be caused by compressive skull fractures, extradural or subdural hematomas, or herniation through the foramen magnum from cerebral edema (box - ) . the patient's pupil size and response to light can be used to localize a diagnosis and give a rough prognosis for severity of disease and possibility for return to function. pupils can be normal in size, mydriatic, or miotic. whenever a pupil appears miotic, direct ocular emergency care unconscious with no response to noxious stimuli injury with uveitis or secondary miosis due to brachial plexus injury should be ruled out. the eyes should always be examined to rule out ocular trauma. in a patient with head trauma, a change from dilated to constricted to normal pupil size is suggestive of improvement in clinical function. bilateral mydriatic pupils that are unresponsive to light in an unconscious animal are a grave prognostic sign and usually indicate an irreversible severe midbrain contusion. bilateral miotic pupils with normal nystagmus and ocular movements are associated with diffuse cerebral or diencephalic lesions. miotic pupils that become mydriatic indicate a progressive midbrain lesion with a poor prognosis. unilateral, slowly progressive pupillary abnormalities in the absence of direct ocular injury are characteristic of brainstem compression or herniation caused by progressive brain swelling. asymmetric pupils are seen in patients with rostral brainstem lesions and can change rapidly. unresponsive pupils that are seen in the midposition occur with brainstem lesions that extend into the medulla and are a grave sign. visual deficits are common with intracranial injury. lesions that are less severe and limited to the cerebrum produce contralateral menace deficits with normal pupillary light response. bilateral cerebral edema can cause blindness with a normal response to light if the midbrain is not disturbed. a patient that is severely depressed and recumbent may not respond to menacing gestures, even when visual pathways are intact. ocular, optic tract, optic nerve, or optic chiasm lesions can interfere with vision and the pupillary light response. brainstem contusion and cerebral edema may produce blindness and dilated unresponsive pupils due to disturbance of the oculomotor area. examine all cranial nerves carefully. cranial nerve abnormalities can indicate direct contusion or laceration of the neurons in the brainstem or where they exit the skull. cranial nerves that are initially normal then later lose function indicate a progressively expanding lesion. when specific cranial nerve deficits are present, the prognosis is considered guarded. clinical signs such as rolling to one side, torticollis, head tilt, and abnormal nystagmus are usually associated with petrosal bone or cerebellomedullary lesions that produce vestibular neuron dysfunction. fractures of the petrosal temporal bone often cause hemorrhage and cerebrospinal fluid (csf) leak from the external ear canal. if the lesion is limited to the membranous labyrinth, the loss of balance will be toward the injured side and the quick phase of the nystagmus will be toward the injured side. normal physiologic nystagmus requires that the pathway is between the peripheral vestibular neurons and the pontomedullary vestibular nuclei to the nuclei of the cranial nerves that innervate the extraocular muscles (iii, iv, vi). severe brainstem lesions disrupt this pathway. disruption of the pathway is manifested as an inability to produce normal physiologic nystagmus by moving the patient's head from side to side. in patients with severe central nervous system depression, this reflex may not be observed. next, assess postural changes and motor function abilities. a loss of the normal oculocephalic ("dolls-eye") reflex is an early sign of brainstem hemorrhage and a late sign of brainstem compression and herniation. any intracranial injury may be accompanied by a concurrent cervical spinal cord injury. handle animals with such injuries with extreme care to avoid causing further damage. whenever there is uncertainty whether a spinal cord lesion exists, strap the patient down to a flat surface and obtain radiographs of the spine. at least two orthogonal views may be required to see fractures; however, do not manipulate the patient until radiography has been completed. crosstable views, in which the bucky is turned perpendicular to the patient's spine, with a radiograph plate secured behind the patient, may be required to minimize patient motion. in patients with cerebral lesions, hemiparesis usually resolves within to days. evaluation of cranial nerve function at frequent intervals may reveal an initial injury or a progressively expanding lesion in the brain. signs of vestibular disorientation, marked head tilt, and abnormal nystagmus occur with contusions of the membranous labyrinth and fracture of the petrous temporal bone. hemorrhage and cerebrospinal fluid otorrhea may be visible from the external ear canal. rolling movements indicate an injury to the cerebellar-medullary vestibular system. respiratory dysfunction and abnormal respiratory patterns are sometimes observed with severe head injury. lesions of the diencephalon produce cheyne-stokes respirations, in which the patient takes progressively larger and larger breaths, pauses, then takes progressively smaller and smaller breaths. mesencephalic lesions cause hyperventilation and can result in respiratory alkalosis. medullary lesions result in a choppy, irregular respiratory pattern. clinical signs of respiratory dysfunction in the absence of primary respiratory damage indicate a guarded prognosis. after injury, seizures may be associated with intracranial hemorrhage, trauma, or an expanding intracranial mass lesion. immediately begin medical therapy to control the seizure. administer diazepam ( . mg/kg iv or . - . mg/kg/hour iv cri) to treat seizures. if diazepam is not effective in combination with other treatments to control intracranial edema, consider giving pentobarbital . loading doses of phenobarbital ( - mg/kg iv divided into or doses, given every to minutes) may be beneficial in preventing further seizures. severe refractory seizures or decreased mentation may be associated with cerebral edema and increased intracranial pressure. mannitol, an osmotic diuretic, is effective at reducing cerebral edema ( . - . g/kg iv over to minutes). mannitol also acts as a free radical scavenger that can inhibit the effects of cerebral ischemia-reperfusion injury. mannitol works synergistically with furosemide ( mg/kg iv given minutes after the mannitol infusion). corticosteroids have not been demonstrated to be beneficial in the treatment of head trauma and may induce hyperglycemia. hyperglycemia has been shown to be a negative prognostic indicator in cases of head trauma. also, glucocorticoids can suppress immune system function and impair wound healing. because of the known risks and lack of known benefits of glucocorticosteroids, their use in treatment of head trauma is contraindicated. the prognosis for any patient with severe head trauma is guarded. management of head trauma patients may include intense nursing care for a period of weeks to months, depending on the presence and extent of concurrent injuries. if progressive loss of consciousness occurs, surgery for decompression of compressive skull injuries should be considered. the most common injury associated with head trauma in small animals is a contusion with hemorrhage in the midbrain and pons. subdural or extradural hemorrhage with space-occupying blood clots is uncommon. diagnostic tests of head trauma may include skull radiographs, ct, and mri of the brain. special studies can help detect edema and hemorrhage in the brain and brainstem, and aid in making an accurate diagnosis and prognosis. a cerebrospinal fluid tap is contraindicated in patients with head trauma because of the risk of causing a rapid decrease in intracranial pressure and brainstem herniation. if a compressive skull fracture is present, the patient should be stabilized for surgery to remove the compression. surgery to alleviate increased intracranial pressure is rarely performed in veterinary medicine because of the poor prognosis and results. in some cases, when a lesion can be localized to one area, -to -cm burr holes can be placed through the skull over the affected area of the cerebrum, exposing the underlying brain tissue. blood clots can be removed through the holes. the bone flap may or may not be replaced, depending on the surgeon's preference and the degree of brain swelling. spinal cord injuries may be associated with trauma, disk rupture, fractures, and dislocation of the spinal column. proceed with caution when moving a patient with suspected spinal cord injury. avoid flexion, extension, and torsion of the vertebral column. all animals that are unconscious following a traumatic event should be considered to have cervical or thoracolumbar spinal injury until proved otherwise by radiography, ct, or mri. the animal should be moved onto a flat surface (e.g., board, door, window, picture frame) and taped down to prevent motion and further displacement of vertebrae. sedation with analgesics or tranquilizers may be necessary to keep the animal immobile and to minimize patient motion. whenever possible, avoid the use of narcotics in patients with head trauma because of the risk of increasing intracranial pressure. as in other emergencies, the abcs emergency care should be evaluated, and the patient treated for shock, hemorrhage, and respiratory compromise. once the cardiovascular and respiratory systems have been evaluated and stabilized, a more thorough neurologic examination can be performed. protrusion of an intervertebral disk indicates that the disk is bulging into the vertebral canal as a result of dorsal shifting of the nuclear pulposus disk material. disk extrusion refers to the rupture of the outer disk membrane and extrusion of the nuclear material into the vertebral column. in dogs and cats, there are intervertebral disks that potentially can cause a problem. chondrodystrophic breeds of dogs are predisposed to endochondral ossification and include the dachshund, shih tzu, french bulldog, bassett hound, welsh corgis, american spaniel, beagle, lhasa apso, and pekingese. initial examination of the patient with suspected intervertebral disk disease includes identifying the neuroanatomic location of the lesion based on clinical signs and neurologic deficits and then establishing a prognosis. the neurologic examination should be carried out without excessive manipulation of the animal. the presence of pain, edema, hemorrhage, or a visible deformity may localize an area of vertebral injury. once an area of suspected lesion is localized based on physical examination findings, take radiographs to establish a diagnosis and to institute therapy. in most cases, the animal must receive a short-acting anesthestic for proper radiographic technique and to prevent further injury. lateral and crosstable ventrodorsal (vd) or dorsoventral (dv) radiographs require less manipulation of the animal compared with traditional vd and dv projections. myelography is often required to delineate the location of the herniated disk material. prognosis in spinal cord injury depends on the extent of the injury and the reversibility of the damage. perception of noxious stimuli, or the presence of "deep pain," by the animal when the stimulus is applied caudal to the level of the lesion is a good sign. to apply a noxious stimulus, apply firm pressure to a toe on one of the rear limbs using a thick hemostat or a pair of pliers. flexion or withdrawl of the limb is simply a local spinal reflex, and should not be perceived as a positive response to or patient perception of the noxious stimulus. turning of the head, vocalization, dilation of the pupils, change in respiratory rate or character, or attempts to bite are behaviors that are more consistent with perception of the noxious stimulus. absence of perception of the noxious stimulus ("loss of deep pain") is a very poor prognosis for return to function. focal lesions are usually associated with vertebral fractures and displacement of the vertebral canal. focal lesions in one or more of the spinal cord segments from t to t can cause complete dysfunction of the injured tissue as a result of concussion, contusion, or laceration. the degree of structural damage cannot be determined from the neurologic signs alone. transverse focal lesions result in paraplegia, with intact pelvic limb spinal reflexes and analgesia of the limbs and body caudal to the lesion. clinical signs in patients with spinal injury are summarized in table - . carefully evaluate the cardiovascular and respiratory status of patients with spinal injuries. immediately address specific injuries such as pneumothorax, pulmonary contusions, hypovolemic shock, and open wounds. if there is palpable or radiographic evidence of a vertebral lesion causing compressive injury, surgery is the treatment of choice unless the displacement has compromised most or all of the vertebral canal. displacements through % to % of the vertebral canal are associated with a poor prognosis, particularly if deep pain is absent caudal to the lesion. in the absence of a radiographic lesion and in the presence of continued neurologic deficits, an mri or ct scan or myelography is warranted to localize a potentially correctable lesion. surgical exploration can be considered: with the objectives of providing spinal cord decompression by hemilaminectomy or laminectomy with removal of disk material or blood clots, realign and stabilize the vertebral column, and perform a meningotomy, if necessary. place the patient on a backboard or other rigid surface, taped down for transport and sedated, to be transported to a surgical specialist. the presence of worsening or ascending clinical signs may signify ascending-descending myelomalacia and is characteristic of a very poor prognosis.in acute spinal trauma, the use of glucocorticoids has been the mainstay of therapy; however, controversy exists about whether they actually offer any benefit. traditional glucocorticosteroid therapy is listed in box - . more recently, the use of propylene glycol has proved to be beneficial in the treatment of acute traumatic herniated disk. high-dose glucocorticoids should only be used for the first hours after initial injury. side effects of glucocorticosteroid therapy include gastric and intestinal ulceration. the prophylactic use of gastroprotectant drugs will not prevent gastrointestinal ulcer formation; however, if signs of gastrointestinal ulcer are present, institute gastroprotectant therapy. management of the patient with spinal cord injury includes aggressive nursing care and physical therapy. many patients with spinal cord injury have little to no control over bladder function, which results in chronic dribbling or retention of urine and overdistention of the urinary bladder with overflow incontinence. urinary bladder retention can lead to urinary tract infection, bladder atony, and overflow incontinence. manual expression of the bladder several times a day may be enough to keep the bladder empty. alternatively, place a urinary catheter to maintain patient cleanliness and to keep the bladder decompressed. (see section on urinary catheterization). paralytic ileus and fecal retention are frequent complications of spinal cord injury. to help prevent constipation, provide highly digestable foods and maintain the patient's hydration with oral and intravenous fluids. mild enemas or stool softeners can also be used to treat fecal retention. to prevent decubital ulcer formation, turn the patient every to hours, and use clean, dry, soft padded bedding. apply deep muscle massage and passive range of motion exercises to prevent disuse atrophy of the muscles and dependent edema. the radial nerve innervates the extensor muscles of the elbow, carpus, and digits. the radial nerve also supplies sensory innervation to the distal craniolateral surface of the forearm and the dorsal surface of the forepaw. injuries to the radial nerve at the level of the elbow emergency care cranial to c spastic tetraplegia or tetraparesis hyperreflexive all four limbs severe injury can result in death from respiratory failure. c -t tetraparesis or tetraplegia depressed thoracic limb spinal reflexes (lower motor neuron) hyperreflexive pelvic limbs (upper motor neuron) t -t horner' syndrome (prolapsed nictitans, enophthalmos, and miosis) t -l schiff-sherrington syndrome (extensor rigidity of thoracic limbs, flaccid paralysis with atonia, areflexia, and analgesia of pelvic limbs) result in an inability to extend the carpus and digits. as a result, the animal walks and bears weight on the dorsal surface of the paw. there is also loss of cutaneous sensation, which leads to paw injury. injuries to the radial nerve above the elbow (in the shoulder area) results in an inability to extend the elbow and bear weight on the affected limb. it can take weeks before the full extent of the injury and any return to function are manifested. the animal may need to be placed in a carpal flexion sling or have eventual amputation if distal limb injury or self-mutilation occurs. the sciatic nerve primarily innervates the caudal thigh muscles that flex the stifle and extend the hip. the tibial branch of the sciatic nerve innervates the caudal leg muscles that extend the tarsus and flex the digits. the tibial nerve provides the sole cutaneous sensory innervation to the plantar aspect of the paw and digits. the peroneal branch of the sciatic nerve provides the sole sensory cutaneous innervation to the dorsal surface of the paw ( table - ) . sciatic nerve injury may occur with pelvic fractures, particularly those that involve the body of the ileum at the greater ischiatic notch, or with sacroiliac luxations that contuse the l and l spinal nerves that pass ventral to the sacrum to contribute to the sciatic nerve. with sciatic nerve injury, there is decreased stifle flexion and overflexion of the hock (tibial nerve), and the animal walks on the dorsal surface of the paw (peroneal nerve). clinical signs of tibial or peroneal damage are seen with femur fractures or with inadvertent injection of drugs into the caudal thigh muscles. the femoral nerve innervates the extensor muscles of the stifle. the saphenous branch of the femoral nerve provides the sole cutaneous innervation to an area on the medial distal thigh, the leg, and the paw. the femoral nerve is protected by muscles and is rarely injured in pelvic fractures. clinical signs of femoral nerve injury are inability to support weight on the pelvic limb, absence of a patellar reflex, and analgesia in the area of cutaneous innervation. coma is complete loss of consciousness, with no response to noxious stimuli. in some animals that present in a coma or stuporous state, the immediate cause will be apparent. in other cases, however, a careful and thorough diagnostic work-up must be performed. a coma scale devised to assist in the clinical evaluation of the comatose patient is shown in table - . whenever an animal presents in a comatose state, immediately secure the emergency management of specific conditions c -t nerve roots radial nerve paralysis musculocutaneous nerve inability to flex the elbow axillary or thoracodorsal dropped elbow nerve median and ulnar nerves loss of cutaneous sensation on the caudal surface of the forearm and palmar and lateral surfaces of the paw; inability to flex the carpus and digits c -t nerve roots radial, median, or ulnar nerve injury c -c nerve roots musculocutaneous, suprascapular, and axillary injury c -t horner's syndrome (miosis, enophthalmos, and prolapsed nictitans) airway by placing an endotracheal tube (see section on endotracheal intubation). if necessary, provide respiratory assistance, or at a minimum, supplemental oxygen. control existing hemorrhage and treat shock, if present. take a careful and thorough history from the owner. make careful note of any seizure, trauma, or toxin exposure, and whether prior episodes of coma have ever occurred. perform a careful physical examination, taking note of the patient's temperature, pulse, and respiration. an elevated temperature may suggest the presence of systemic infection, such as pneumonia or hepatitis, or a brain lesion with loss of hypothalamic thermoregulatory control. very high temperatures associated with shock and coma are often observed in animals with heat stroke (see section on heat stroke and heat-induced illness). circulatory collapse or barbiturate overdose can produce coma and hypothermia. abnormal respiratory patterns also may be observed in a comatose patient. hypoventilation may occur with elevated intracranial pressure or barbiturate overdose. rapid respiratory rate may be associated with pneumonia, metabolic acidosis (dka, uremia), or brainstem injury. examine the skin for any bruises or external trauma. examine the mucous membranes and make note of color and capillary refill time. icterus with petechiae or ecchymotic hemorrhage in a comatose patient may be associated with end-stage hepatic failure and hepatic encephalopathy. smell the patient's breath for the odor of ketones that may signify dka or end-stage hepatic failure. motor activity normal gait, normal spinal reflexes hemiparesis, tetraparesis, or decerebrate activity recumbent, intermittent extensor rigidity recumbent, constant extensor rigidity recumbent, constant extensor rigidity with opisthotonus recumbent, hypotonia of muscles, depressed or absent spinal reflexes normal papillary reflexes and oculocephalic reflexes slow pupillary light reflexes and normal to reduced oculocephalic reflexes bilateral unresponsive miosis with normal to reduced oculocephalic reflexes pinpoint pupils with reduced to absent oculocephalic reflexes unilateral, unresponsive mydriasis with reduced to absent oculocephalic reflexes bilateral, unresponsive mydriasis with reduced to absent oculocephalic reflexes occasional periods of alertness and responsive to environment depression of delirium, capable of responding to environment but response may be inappropriate semicomatose, responsive to visual stimuli semicomatose, responsive to auditory stimuli semicomatose, responsive only to repeated noxious stimuli comatose, unresponsive to repeated noxious stimuli *neurologic function is assessed for each of the three categories and a grade of to is assigned according to the descriptions for each grade. the total score is the sum of the three category scores. this scale is designed to assist the clinician in evaluating the neurologic status of the craniocerebral trauma patient. as a guideline and according to clinical impressions, a consistent total score of to represents a grave prognosis, to a poor to guarded prognosis, and to a good prognosis. (modified from the glasgow coma scale used in humans.) from shores a: craniocerebral trauma. in kirk rw, ed: current veterinary therapy x. small animal practice. philadelphia, wb saunders, , p . finally, conduct a complete neurologic evaluation. the presence of asymmetric neurologic signs may suggest an intracranial mass lesion (e.g., hemorrhage, neoplasia, injury). usually, toxicities or metabolic disturbances (e.g., dka, hepatic encephalopathy) cause symmetric clinical signs of neurologic dysfunction, with cerebral signs predominating. in hepatic encephalopathy, pupils are usually normal in size and responsive to light. in toxicities, the pupils are abnormal in size and may be unresponsive to light. obtain a complete blood count, serum biochemistry profile, urinalysis, and specific tests for glucosuria and ketonuria. findings of a drastically elevated blood glucose with glucosuria, ketonuria, and high specific gravity are characteristic of dka. fever and uremic encephalopathy are characterized by severe azotemia with a low urine specific gravity. if barbiturate intoxication is suspected, save urine for later toxin analysis. evaluate urine sediment for calcium oxalate crystalluria that may indicate ethylene glycol toxicity. calculate plasma osmolality (see following section) to check for nonketotic hyperosmolar diabetes mellitus. elevated blood ammonia levels may be associated with hepatic encephalopathy. in uncontrolled diabetes mellitus, hyperosmolarity can result in clinical signs of disorientation, prostration, and coma. plasma osmolarity can be calculated from the formula: mosm/l = (na + k) + (glucose/ ) + (bun/ . ) clinical signs of hyperosmolarity can occur when the plasma osmolarity exceeds mosm/l. treatment of dka or nonketotic hyperosmolar syndrome is aimed at reducing ketoacid production, stimulating carbohydrate utilization, and impeding peripheral release of fatty acids. the treatment of choice is rehydration and provision of supplemental regular insulin and a carbohydrate source (see section on diabetic ketoacidosis). during ketosis, insulin resistance may be present. slow rehydration with . % saline solution or other balanced crystalloid fluids (e.g., normosol-r, plasmalyte-m, lactated ringer's solution), should occur, with the goal of rehydration over to hours. too rapid rehydration can result in cerebral edema and exacerbation of clinical signs. hepatic encephalopathy (he) is characterized by an abnormal mental state associated with severe hepatic insufficiency. the most common cause of he is congenital or acquired c o m a portosystemic shunts. acute hepatic destruction can also be caused by toxins, drugs, or infectious causes. the treatment of he is considered a medical emergency (table - ) . absorption of ammonia and other nitrogenous substances from the gastrointestinal tract is thought to be one of the complicating factors in he. prevent absorption of ammonia and other nitrogenous substances from the gastrointestinal tract by restricting dietary protein to % to % for dogs, and to % to % (on a dry matter basis) for cats. dietary protein should be from a nonanimal plant source (e.g., soybean) whenever possible. caloric requirements are met with lipids and carbohydrates. also prescribe cleansing enemas to rid the colon of residual material, and antibiotic therapy to reduce gastrointestinal tract bacteria. neomycin ( mg/kg q h) can be administered as a retention enema. metronidazole ( . mg/kg po, q - h) or amoxicillin-clavulanate ( . mg po q h) can also be administered. administer lactulose ( . - . ml q h for cats; . - ml q h for dogs) to trap ammonia in the colon to prevent absorption (table - ) . administer lactulose orally to an alert animal, or as a retention enema to a comatose animal. if lactulose is not available, betadine retention enemas will change colonic ph and prevent ammonia absorption. a side effect of lactulose administration (po) is soft to diarrheic stool. a seizure is a transient disturbance of brain function that is sudden in onset, ceases spontaneously, and has a tendency to recur, depending on the cause. most seizures are generalized and result in a loss of consciousness and severe involuntary contraction of the skeletal muscles, resulting in tonic-clonic limb activity and opisthotonus. mastication, salivation, urination, and defecation are common. partial (petit mal) seizures range from limited limb activity, facial muscle twitching, and episodic behavioral abnormalities to brief loss of consciousness. similar clinical signs also can occur with syncopal episodes. conduct a careful cardiac examination in any patient with a history of petit mal seizures. seizures of any form constitute a medical emergency, particularly when they occur in clusters, or as status epilepticus. most seizures are of short duration and may have subsided by the time the animal is presented for treatment. whenever a seizure occurs, however, it is important that the animal does not inadvertently injure itself or a bystander. it is important to evaluate whether the patient has a coexisting disease that can predispose it to seizures, such as hepatic failure, uremia, diabetes mellitus, hypoglycemia, toxin exposure, insulin-secreting tumors, and thiamine deficiency. many toxins are responsible for clinical signs of tremors or seizures (see section on poisons and toxins). treatment of a primary disease entity can help control seizures, in some cases, provided that the underlying cause is investigated and treated. status epilepticus, a state of continuous uncontrolled seizure activity, is a medical emergency. when an animal is in a state of status epilepticus, immediately place a lateral or medial saphenous intravenous catheter and administer diazepam ( . mg/kg iv) to help control the seizure. in most cases, the seizure must be controlled before a diagnostic workup is attempted. whenever possible, however, blood samples should be collected before administration of any anticonvulsant agent because of the risk of incorrect test results. for example, the propylene glycol carrier in diazepam can cause a false-positive ethylene glycol test using an in-house testing kit. whenever possible, check blood glucose levels, particularly in young puppies or kittens, to evaluate and treat hypoglycemia as a cause of seizures. if hypoglycemia exists, administer % dextrose ( g/kg iv). if diazepam partially controls the status epilepticus, administer a constant rate infusion ( . mg/kg/hour in % dextrose in water). diazepam is sensitive to light, and the bag and infusion line must be covered to prevent degradation of the drug. if diazepam fails to control status epilepticus, give pentobarbital ( - mg/kg iv to effect). the animal's airway should be intubated and protected while the patient is kept in the drug-induced coma. protracted cases of seizures may require mannitol and furosemide therapy to treat cerebral edema. administer intravenous fluids (balanced crystalloid at maintenance doses [see section on intravenous fluid therapy]). the patient should be turned every to hours to emergency care prevent atelectasis. insert a urinary catheter for cleanliness, and place the animal on soft dry padded bedding to prevent decubital ulcer formation. depending on the length of time that the patient is rendered unconscious, apply passive range of motion exercises and deep muscle massage to prevent disuse atrophy of the muscles and dependent or disuse edema. monitor the patient's oxygenation and ventilation status by arterial blood gas measurement or pulse oximetry and capnometry (see section on blood gas, pulse oximetry, and capnometry). administer supplemental oxygen to any patient that is hypoxemic secondary to hypoventilation or other causes. severe refractory seizures can result in the development of neurogenic pulmonary edema. lubricate the animal's eyes every hours to prevent drying out and corneal abrasions. depending on the cause of the seizure, administer phenobarbital at a loading dose of to mg/kg iv given in four to five injections, every to minutes; make sure that the patient is rousable in between injections). seizures in cats often are associated with structural brain disease. the occurrence of partial focal seizures is unequivocally associated with a focal cerebral lesion and acquired structural brain disease. an initial high frequency of seizures is also a strong indication that structural brain disease is present. seizure activity in cats may occur as mild generalized seizures or complex partial seizures and may be associated with systemic disorders such as feline infectious peritonitis virus, toxoplasmosis, cryptococcus infection, lymphosarcoma, meningiomas, ischemic encephalopathy, and thiamine deficiency. thiamine deficiency in the cat can be a medical emergency characterized by dilated pupils, ataxic gait, cerebellar tremor, abnormal oculocephalic reflex, and seizures. treatment consists of administration of thiamine ( mg/day) for three days. steffen f, grasmueck s: propofol for treatment of refractory seizures in dogs and a cat with intracranial disorders. j small anim pract ( ) ( ) ( ) ( ) . j am vet med assoc ( ): [ ] [ ] [ ] [ ] [ ] [ ] . an ocular emergency is any serious condition that causes or threatens to cause severe pain, deformity, or loss of vision. treat ocular emergencies immediately, within to several hours after the emergency, whenever possible (box - , - ). to assess the location and degree of ocular injury, perform a complete ocular examination. in some cases, short-acting sedation or general anesthesia in conjunction with topical local anesthetic may be necessary to perform the examination, because of patient discomfort and blepharospasm. the equipment listed in box - may be necessary and may be invaluable in making an accurate diagnosis. to perform a systematic and thorough ocular examination, first obtain a history from the owner. has there been any prior incident of ocular disease? is there any history of trauma or known chemical irritant or exposure? did the owner attempt any irrigation or medical techniques prior to presentation? when was the problem first noticed? has it changed at all since the owner noticed the problem? after a history has been obtained, examine the patient's eyes for discharge, blepharospasm, or photophobia. if any discharge is present, note its color and consistency. do not attempt to force the eyelids open if the patient is in extreme discomfort. administer a short-acting sedative and topical local anesthetic such as . % proparacaine. note the position of the globe within its orbit. if the eye is exophthalmic, strabismus and protrusion of the third eyelid are often visible. exposure keratitis may be present. in cases of retrobulbar or zygomatic salivary gland inflammation, the patient will resist opening the mouth and exhibit signs of discomfort or pain. note any swelling, contusions, abrasions, or lacerations of the eyelids. note whether the lids are able to close completely and cover the cornea. if a laceration of the lid is present, determine the depth of the laceration. palpate the orbit for fractures, swelling, pain, crepitus, and cellulitis. examine the cornea and sclera for penetrating injury or foreign material. the use of lid retractors or small forceps can be very helpful in these cases. if a wound appears to penetrate completely into the globe, look for loss of uveal tissue, lens, or vitreous. do not put any pressure on the globe, because intraocular herniation may result. examine the conjunctiva for hemorrhage, chemosis, lacerations, and foreign bodies. examine the superior and inferior conjunctival cul-de-sacs for foreign material. in such cases, placement of a topical anesthetic and use of a moistened cotton swab is invaluable to sweep the conjunctival fornix to pick up foreign bodies. use a small, fine-tipped forceps to retract the third eyelid away from the globe and examine behind the third eyelid for foreign bodies. next, examine the cornea for opacities, ulcers, foreign bodies, abrasions, or lacerations. place a small amount of fluroescein stain mixed with sterile water or saline on the dorsal sclera. close the eye to disperse the stain over the surface of the cornea, then flush gently with sterile saline irrigation. examine the cornea again for any defects. a linear defect perpendicular to the long axis of the eye should alert the clinician to investigate the conjunctiva for dystechia. record the pupil size, shape, and response to light (both direct and consensual). examine the anterior chamber and note its depth and whether hyphema or aqueous flare are present. is the lens clear and is it in the normal position? lens luxation can cause the lens tissue to touch the cornea and cause acute corneal edema. measure intraocular pressure with a schiotz tonometer or tonopen. finally, dilate the pupil and examine the posterior chamber using a direct or indirect ophthalmoscope to look for intraocular hemorrhage, retinal hemorrhage, retinal detachment, tortuous retinal vessels, optic neuritis, and inflammation. the basic surgical instruments listed in box - may be useful in the treatment of ocular lacerations and other ophthalmic injuries: bite wounds and automobile trauma commonly cause lacerations and abrasions of the lid margins. the lids can be considered to be two-layer structures, with the anterior composed of the skin and orbicularis muscle and the posterior layer composed of the tarsus and conjunctiva. the openings of the meibomian glands in the lid margin form the approximate line separating the lids into anterior and posterior segments. splitting the lid into these two segments facilitates the use of sliding skin flaps to close wound defects, if necessary. clean and thoroughly but gently irrigate the wound with sterile saline solution before attempting any lid laceration repair. use sterile saline solution to irrigate the wound and conjunctiva. a % povidone-iodine scrub can be used on the skin, taking care to avoid getting any scrub material in the soft tissues of the eye. drape the eye with an adhesive ocular drape, if possible, to prevent further wound contamination. trim the ragged wound edges, but be very conservative with tissue debridement. leave as much tissue as possible to insure proper wound contracture with minimal lid deformity. close a small lid wound with a figure-of-eight or two-layered simple interrupted suture of absorbable suture material or nylon in the skin. the lid margins must be absolutely apposed to prevent postoperative lid notching. direct blunt trauma to the eye can cause severe ecchymosis because of the excellent vascular supply of the eyelids. other associated ocular injuries such as orbital hemorrhage, proptosis, and corneal laceration may also occur. trauma, allergic reactions, inflammation of the sebaceous glands (hordeolum), thrombocytopenia, and vitamin k antagonist rodenticide intoxication can all cause ecchymoses of the lids. treat eyelid ecchymoses initially with cool compresses, followed by warm compresses. resorption of blood can occur from to days after the initial insult. ocular allergies respond well to topical application (dexamethasone ophthalmic ointment q - h) and systemic administration of glucocorticosteroids, along with cool compresses. in order to fully assess the conjunctiva for abnormalities, it may be necessary to carefully dissect it away from the underlying sclera. when performing this dissection, do not place undue pressure on the globe because of the risk of herniation of the intraocular contents through a scleral wound. repair large conjunctival lacerations with - absorbable sutures, using an interrupted or continuous pattern. carefully approximate the margins of the conjunctiva to prevent formation of inclusion cysts. when large areas of the conjunctiva have been damaged, advancement flaps may be required to close the defect. subconjunctival hemorrhage is a common sequela of head trauma, and it may also be observed in various coagulopathies. by itself, it is not a serious problem but may signify severe underlying intraocular damage. a complete ocular examination is indicated. other causes of subconjunctival hemorrhage include thrombocytopenia, autoimmune hemolytic anemia, hemophilia, leptospirosis, vitamin k antagonist rodenticide intoxication, severe systemic infection or inflammation, and prolonged labor (dystocia). uncomplicated subconjunctival hemorrhage usually clears on its own within days. if the conjunctiva is exposed because of swelling and hemorrhage, administer a topical protective triple antibiotic ophthalmic ointment every to hours until the conjunctival hemorrhage resolves. toxic, acid, and alkaline chemical injuries to the eye can sometimes occur. the severity of the injury caused by ocular burns depends on the concentration, type, and ph of the chemical and on the duration of exposure. weak acids do not penetrate biologic tissue very well. the hydrogen ion precipitates the protein upon contact and therefore provides some protection to the corneal stroma and intraocular contents. precipitation of corneal proteins produces a ground-glass appearance in the cornea. alkaline solutions and very strong acids penetrate tissues rapidly, causing saponification of the plasma membrane, denaturation of collagen, and vascular thrombosis within the conjunctiva, episclera, and anterior uvea. severe pain, blepharospasm, and photophobia are produced by exposure of free nerve endings in the corneal epithelium and conjunctiva. severe alkaline burns cause an increase in intraocular pressure. intraocular prostaglandins are released, and the intraocular aqueous ph increases, producing changes in the blood-aqueous barrier and secondary uveitis. uveitis with anterior synechia formation, eventual chronic glaucoma, phthisis, secondary cataract, and corneal perforation can occur. healing of the corneal epithelium is usually accomplished by neovascularization and sliding and increased mitosis of the corneal epithelium. severe stromal burns within the cornea heal by degradation and removal of necrotic debris, followed by replacement of the collagen matrix and corneal epithelial cells. the release of collagenase, endopeptidase, and cathepsins from polymorphonuclear cells serves to cause further corneal breakdown. in severe cases, only pmns may be present, and fibroblasts may never invade the corneal stroma. all chemical burns should be washed copiously with any clean aqueous solution available. if any sticky paste or powder is adherent to the conjunctival sac, remove it with moist cotton swabs and irrigation. begin mydriasis and cycloplegia by topical application of % atropine ophthalmic drops or ointment. start antibiotic therapy with triple antibiotic ophthalmic ointment or gentocin ointment every to hours. treat secondary glaucomas with topical carbonic anhydrase inhibitors. to avoid fibrinous adhesions and symblepharon formation, keep the conjunctival cul-de-sacs free of proteinaceous exudate that can form adhesions. analgesics are required for pain. oral nonsteroidal antiinflammatory agents such as carprofen, ketoprofen, meloxicam, or aspirin are recommended. persistent epithelial erosions may require a conjunctival flap left in place for to weeks or placement of a topical collagen shield (contact lens). topical antibiotics, mydriatics, and lubricants (lacrilube or puralube ointment) should also be used. strong acid or alkali burns can result in severe corneal stromal loss. in the past, topical n-acetylcysteine ( % mucomyst) has been recommended. this treatment is very painful. other treatments are also available, such as ethylenediaminetetraacetic acid (edta) ( . m solution) and patient serum to inhibit mammalian collagenase activity. to prepare patient serum, obtain to ml of whole blood from the patient. spin it down in a serum separator tube after a clot forms and then place the serum in a red-topped tube on the patient's cage. (the contents of the tube are viable for days without refrigeration.) apply the serum topically to the affected eye every to hours. avoid using topical steroids because they inhibit fibroblast formation and corneal healing. in severe cases, if conjunctival swelling and chemosis also are present, antiinflammatory doses of oral steroids can be administered short-term. oral steroids and nonsteroidal antiinflammatory drugs should never be administered to the patient concurrently, because of the risk of gastrointestinal ulcer and perforation. corneal abrasions are associated with severe pain, blepharospasm, lacrimation, and photophobia. animals with such intense pain are often difficult to examine until analgesia has been administered. topical use of proparacaine ( . % proparacaine hydrochloride) is usually sufficient to permit relaxation of the eyelids so that the eye can be examined. using a focal source of illumination and an eye loupe, examine the cornea, inferior and superior conjunctival fornixes, and medial aspect of the nictitans for foreign bodies. place a sterile drop of saline on a fluorescein-impregnated strip and touch the superior conjunctiva once to allow the stain to spread onto the surface of the eye. irrigate the eye to remove excess stain and then examine the corneal surface for any areas of stain uptake. if an area of the cornea persistently remains green, there is damage to the corneal epithelium in that area. initial treatment consists of application of a topical mydriatic ( drop of % atropine in affected eye q h) to prevent anterior synechiae and improve cycloplegia. triple antibiotic ointment is the treatment of choice (a / -inch strip in the affected eye q h) until the ulcer heals. in some cases, nonhealing ulcers (e.g., boxer ulcer, indolent ulcer) form in which the epithelial growth does not adhere to the underlying cornea. gently debride the loose edges of the ulcer/erosion with a cotton swab and topical anesthesia. more severe cases in which only minimal healing has occurred after days of treatment require grid keratectomy, in which a -gauge needle is used to gently scratch the surface of the abrasion or ulcer in the form of a grid to promote neovascularization. apply a topical anesthetic before performing the procedure. a collagen contact lens also may be required to promote wound healing. all corneal abrasions should be reevaluated in hours, and then every to days thereafter until they have healed. acute infectious keratitis secondary to bacterial infection is characterized by mucopurulent ocular discharge, rapidly progressing epithelial and corneal stromal loss, inflammatory cellular infiltrates into the corneal stroma, and secondary uveitis, often with hypopyon formation. confirmation of infectious keratitis is based on corneal scrapings and a positive gram stain. initial treatment for bacterial keratitis consists of systemic antibiotics and topical ciprofloxacin ( . % eyedrops or ointment). penetrating injuries through the cornea may result in prolapse of intraocular contents. frequently, pieces of uveal tissue or fibrin effectively but temporarily seal the defect and permit the anterior chamber to re-form. avoid manipulation of these wounds until the animal has been anesthetized, as struggling or excitement can promote loss or dislodgement of the temporary seal and cause the intraocular contents to be extruded. superficial corneal lacerations need not be sutured and can be treated the same as a superficial corneal ulcer or abrasion. if the laceration penetrates more than % the thickness of the cornea, or extends more than to mm, it should be sutured. when placing sutures in the cornea, it is helpful to use magnification. referral to a veterinary ophthalmologist is advised. if a veterinary ophthalmologist is not available, use - or - silk, collagen, or nylon sutures on a micropoint spatula-type needle. use a simple interrupted suture pattern and leave the sutures in place for a minimum of weeks. because many corneal lacerations are jagged and corneal edema forms, most of the wound edges cannot be tightly juxtaposed. in such cases, pull a conjunctival flap across the wound to prevent leakage of aqueous fluid. never suture through the full thickness of the cornea; rather, the suture should pass through the mid-third of the cornea. following closure of the corneal wound, the anterior chamber must be re-formed to prevent anterior synechia formation with secondary glaucoma. taking care to avoid iris injury, use a -or -gauge needle to insert sterile saline at the limbus. any defect in the suture line will be apparent because of leakage of the fluid from the site and should be repaired. incarceration of uveal tissue in corneal wounds is a difficult surgical problem. persistent incarceration of uveal tissue can result in development of a chronic wick in the cornea, a shallow anterior chamber, chronic irritation, edema, vascularization of the cornea, and intraocular infection that can lead to panophthalmitis. referral to a veterinary ophthalmologist is strongly recommended. the most common foreign bodies associated with ocular injuries in small animals are birdshot, bb pellets, and glass. the site of intraocular penetration of the foreign bodies may be obscured by the eyelids. a foreign body entering the eye may penetrate the cornea and fall into the anterior chamber or become lodged in the iris. foreign bodies may occasionally penetrate the lens capsule, producing cataracts. some metallic high-speed foreign bodies may penetrate the cornea, iris, and lens to lodge in the posterior wall of the eye or vitreous chamber. direct visualization of a foreign body is the best means of localization. examination of the eye with an indirect ophthalmoscope or biomicroscope (if available) is invaluable for locating foreign bodies. indirect visualization of the ocular foreign body can also be achieved through radiographic techniques. three separate views should be obtained to determine the plane of location of the foreign object. ct or mri may prove useful, although scatter from the foreign body may make it difficult to directly visualize with these techniques. ocular ultrasound is perhaps the most useful and refined radiographic technique for locating intraocular foreign bodies. before removing any foreign body from the eye, the risk and surgical danger of removing it must be weighed against the risks of leaving it in place. metallic foreign bodies in the anterior chamber are much easier to remove than nonmagnetic ones. attempted removal of foreign objects from the vitreous chamber of the eye has consistently produced poor results. for the best chance of recovery, ocular foreign bodies should be removed by a veterinary ophthalmologist whenever possible. blunt trauma to the globe can result in luxation or subluxation of the lens. the subluxated lens may move anteriorly and make the anterior chamber more shallow. trembling of the iris (iridodonesis) may be noticed when the lens is subluxated. in complete luxation, the lens may fall totally into the anterior chamber and obstruct aqueous outflow, causing secondary glaucoma. alternatively, the lens may be lost into the vitreous cavity. luxation of the lens is almost always associated with rupture of the hyaloid membrane and herniation of the vitreous through the pupillary space. emergency surgery for lens luxation is required if the lens is entirely within the anterior chamber or incarcerated within the pupil, causing a secondary pupillary block glaucoma. acute elevation in intraocular pressure can cause vision loss within hours; thus, lens removal should be accomplished as quickly as possible. referral to a veterinary ophthalmologist is recommended. severe trauma to the globe or a direct blow to the head can result in retinal or vitreous hemorrhage. there may be large areas of subretinal or intraretinal hemorrhage. subretinal hemorrhage assumes a discrete globular form, and the blood appears reddish-blue in color. the retina is detached at the site of hemorrhage. superficial retinal hemorrhage may assume a flame-shaped appearance, and preretinal or vitreous hemorrhage assumes a bright-red amorphous appearance, obliterating the underlying retinal architecture. retinal and vitreous hemorrhage secondary to trauma usually resorbs spontaneously over a -to -week period. unfortunately, vitreous hemorrhage, as it organizes, can produce vitreous traction bands that eventually produce retinal detachment. expulsive choroid hemorrhage can occur at the time of injury and usually leads to retinal detachment, severe visual impairment, and total loss of vision. treatment of vitreal and retinal hemorrhage includes rest and correction of factors that may predispose to intraocular hemorrhage. more complicated cases may require vitrectomy performed by a veterinary ophthalmologist. hyphema refers to blood in the anterior chamber of the eye. the most common traumatic cause of hyphema is an automobile accident. hyphema may also present because of penetrating ocular wounds and coagulopathies. blood within the eye may come from the anterior or posterior uveal tract. trauma to the eye may result in iridodialysis or a tearing of the iris at its root, permitting excessive bleeding from the iris and ciliary body. usually, simple hyphema resolves spontaneously in to days and does not cause vision loss. loss of vision following bleeding into the anterior chamber is associated with secondary ocular injuries such as glaucoma, traumatic iritis, cataract, retinal detachment, endophthalmitis, and corneal scarring. treatment of hyphema must be individualized, but there are severe general principles of treatment. first, stop ongoing hemorrhage and prevent further bleeding whenever possible. this may involve correction of the underlying cause, if a coagulopathy is present. next, aid in the elimination of blood from the anterior chamber, control secondary glaucoma, and treat associated injuries, including traumatic iritis. finally, detect and treat any late complications of glaucoma. in most cases of traumatic hyphema, little can be done to arrest or prevent ongoing hemorrhage. it is best to restrict the animal's activity and prohibit exertion. rebleeding can occur within days, and intraocular pressure must be monitored closely. after to days, the blood in the anterior chamber will change color from a bright red to bluish-black ("eight-ball hemorrhage"). if total hyphema persists and intraocular pressure rises despite therapy, surgical intervention by a veterinary ophthalmologist may be necessary. the primary route of escape of rbcs from the anterior chamber is via the anterior drainage angle. iris absorption and phagocytosis play a minor role in the removal of blood from the anterior chamber. because of the associated traumatic iritis in hyphema, topical administration of a glucocorticoid ( % dexamethasone drops or % prednisolone drops) is advised to control anterior chamber inflammation. a cycloplegic agent ( % atropine) should also be used. the formation of fibrin in the anterior chamber of the eye secondary to hemorrhage can produce adhesions of the iris and secondary glaucoma (see section on glaucoma secondary to hyphema) by blocking the trabecular network. hyphema secondary to retinal detachment (collie ectasia syndrome) and end-stage glaucoma are extremely difficult to treat medically and have a poor prognosis. proptosis of the globe is common secondary to trauma, particularly in brachycephalic breeds. proptosis of the globe in dolichocephalic breeds requires a greater degree of initiating contusion than the brachycephalic breeds because the orbits are so much deeper. therefore, secondary damage to the eye and cns associated with proptosis of the globe may be greater in the collie or greyhound than in the pug. when proptosis occurs, carefully evaluate the cardiovascular system for evidence of hypovolemic or hemorrhagic shock. examine the respiratory and neurologic systems. be sure to establish an airway and treat shock, if present. control hemorrhage and stabilize the cardiovascular system before attempting to replace the globe within its orbit or perform enucleation. during the initial management of the cardiovascular and respiratory systems, the eye should be covered with an ophthalmic grade ointment or sponges soaked in sterile saline to prevent the globe from drying out. proptosis of the globe can be associated with serious intraocular problems including iritis, chorioretinitis, retinal detachment, lens luxation, and avulsion of the optic nerve. stain the surface of the eye with fluorescein to look for topical abrasions or ulcers. carefully examine the sclera, cornea, and conjunctiva for penetrating injuries that may allow aqueous leakage. evaluate the size, location, and response to light of the pupil. a reactive pupil is better than a mydriatic fixed pupil. topical administration of a mydriatic (atropine %) to prevent persistent miosis and synechia formation is indicated, along with topical and oral antibiotics and oral analgesic therapy. reposition the proptosed globe with the patient under general anesthesia. make a lateral canthotomy incision to widen the palpebral fissure. lavage the globe with sterile saline irrigation to remove any external debris. place a copious amount of triple antibiotic ophthalmic ointment on the surface of the eye and then gently press the globe into the orbit using the flat side of a scalpel handle or a moistened sterile surgical sponge. do not probe the retro-orbital space with a needle or attempt to reduce intraocular pressure by paracentesis. when the globe is replaced in the orbit, close the lateral canthotomy incision with simple interrupted sutures. place three non-penetrating mattress sutures in the lid margins but do not draw them together. tighten the lid sutures through small pieces of a red rubber catheter or length of intravenous extension tubing to prevent the sutures from causing lid necrosis. leave the medial canthus of the eye open in order to allow topical treatment. postoperative treatment is directed at preventing further iritis and preventing infection. administer systemic broad-spectrum antibiotics (clavamox, . mg/kg po bid) and analgesic drugs. apply topical triple antibiotic ophthalmic ointment ( / inch in affected eye q - h) and atropine ( % in affected eye q h) to prevent infection, cycloplegia, and anterior synechiae. antiinflammatory doses of systemic steroids can also be added to the treatment if severe periorbital inflammation is present. systemic steroids should never be used in conjunction with nonsteroidal antiinflammatory drugs, because of the risk of gastrointestinal ulceration and perforation. the sutures should remain in place for a minimum of weeks. after this time, remove the sutures and inspect the globe. if proptosis recurs, repeat the treatment. following proptosis, strabismus is common secondary to periorbital muscle injury. even after extensive treatment, vision in the eye may still be lost. nonvisual eyes can remain in place, but phthisis may develop. carbonic anhydrase inhibitors such as acetazolamide and dichlorphenamide decrease aqueous secretion and may effectively reduce intraocular pressure if the trabecular outflow is still functioning at % of its capacity. an eye with a poorly functional trabecular outflow system will respond poorly to therapy with carbonic anhydrase inhibitors. osmotic agents such as mannitol or glycerol may be helpful in controlling glaucoma secondary to hyphema. reduction in vitreous chamber size can make the anterior chamber deeper and may allow increased aqueous outflow. evacuation of blood or blood clots from the anterior chamber is not advisable unless the glaucoma cannot be controlled medically or there is no indication after a prolonged period of time that blood is being resorbed. tissue plasminogen activator (t-pa) has proved to be useful in may be helpful in lysing blood clots and preventing excessive fibrin formation. the t-pa is reconstituted to make a solution of µ/ml, which is then frozen at − °c in . -ml aliquots. the thawed, warmed reconstituted t-pa is injected into the anterior chamber. blind probing of the anterior chamber of the eye and surgical intervention in an attempt to remove blood clots can cause serious complications such as rebleeding, lens luxation, iris damage, and damage to the corneal epithelium, and therefore is not advised. acute glaucoma is a rise in intraocular pressure that is not compatible with normal vision. glaucoma may present as early acute congestive or noncongestive glaucoma, or as end-stage disease. cardinal signs of glaucoma are a sudden onset of pain, photophobia, lacrimation, deep episcleral vascular engorgement, edematous insensitive cornea, shallow anterior chamber depth, dilated unresponsive pupil, loss of visual acuity, and buphthalmia. intraocular pressure usually exceeds mm hg but may be normal or only slightly increased if glaucoma is secondary to anterior uveitis. most forms of clinical glaucoma in dogs are secondary to some other intraocular problem. primary glaucoma is recognized in some breeds, including the bassett hound, cocker spaniel, samoyed, bouvier des flandres, and some terrier breeds either from goniodysgenesis or a predisposition to lens luxation. other common causes of acute glaucoma are anterior uveitis and intumescent lens secondary to rapid cataract development, particularly in dogs with diabetes mellitus. treatment involves investigation of the underlying cause of the sudden rise in intraocular pressure and rapid reduction in intraocular pressure. permanent visual impairment is often associated with chronically buphthalmic globes or the presence of rippling or striae formation on the cornea. referral to a veterinary ophthalmologist is recommended. if the eye is still visual and not buphthalmic, the prognosis is favorable, depending on the cause of the acute glaucoma. treatment to reduce intraocular pressure consists of improving aqueous outflow, reducing intraocular volume with osmotic agents, and reducing aqueous formation (table - ). the use of topical mydriatic agents in acute glaucoma is contraindicated because of the risk of making lens luxation or anterior uveitis worse. referral to a veterinary ophthalmologist for emergency surgery is indicated in cases of iris bombe, intumescent lens, or lens subluxation. administer osmotic agents to reduce the size of the vitreous body and the amount of aqueous. osmotic agents create an osmotic gradient between the intraocular fluids and the emergency management of specific conditions vascular bed, thus allowing osmotic removal of fluid independent of the aqueous inflow and outflow systems. if no other treatments are available, oral glycerol ( %, . ml/kg or . g/kg) can be used to effectively reduce intraocular pressure. an adverse side effect of oral glycerol treatment is protracted vomiting. do not use glycerol in a diabetic patient. mannitol ( - g/kg iv over hour) also effectively reduces intraocular pressure but does not cause vomiting. carbonic anhydrase inhibitors can be used to reduce intraocular volume by reducing aqueous production. oral administration of dichlorphenamide, methazolamide, and acetazolamide ( - mg/kg) is usually not very effective alone in reducing aqueous volume and intraocular pressure and also can cause metabolic acidosis. topical carbonic anhydrase inhibitors appear to be more effective (dorzolamide, trusopt) when used in conjunction with topical beta-blockers (timolol, . % or . % solution q h). the most effective treatment for acute pressure reduction is use of a topical prostaglandin inhibitor (latanaprost). usually just one or two drops effectively reduces intraocular pressure in the emergency stages, until the patient can be referred to a veterinary ophthalmologist the following day. many clinical conditions that are presented as emergencies may be due in part or wholly to the presence of a neoplasm. paraneoplastic signs are summarized in table - . prompt identification of the neoplasia combined with knowledge of treatment, expected response to therapy, and long-term prognosis can aid owners and practitioners in making appropriate treatment decisions. hemorrhage or effusion can occur in any body cavity as a result of the presence of benign or malignant tumors. tumors secrete anticoagulants to allow angiogenesis to grow unchecked. hemorrhage often occurs as a result of rupture of a neoplasm or invasion of a neoplasm into a major vascular structure. effusion may be the result of direct fluid production by the mass or may be due to obstruction of lymphatic or venous flow. hemorrhagic effusions in the abdominal cavity occur most commonly with neoplastic masses of the spleen or liver. the most common causes are hemangiosarcoma and hepatocellular carcinoma. clinical signs associated with acute abdominal hemorrhage, regardless of the cause, are related to hypovolemic shock and decreased perfusion and include pale mucous membranes, tachycardia, anemia, lethargy, and acute collapse. treatment for abdominal hemorrhage includes placement of a large-bore peripheral cephalic catheter and starting one fourth of a shock dose ( ml/kg/hour for dogs, and ml/kg/hour for cats) of intravenous crystalloid fluids, taking care to carefully monitor perfusion parameters of heart rate, capillary refill time, mucous membrane color, and blood pressure. administer intravenous colloids such as dextran- , hetastarch, and oxyglobin ( - ml/kg iv bolus) to restore intravascular volume and normotension. treat severe anemia with whole blood or packed rbcs to improve oxygen-carrying capacity and oxygen delivery (see sections on transfusion medicine and treatment of shock). confirm the presence of hemoabdomen abdominocentesis (see section on abdominocentesis). the presence of nonclotting hemorrhagic effusion is consistent with free blood. packed cell volume of the fluid is usually the same or higher than that of the peripheral blood. an abdominal compression bandage can be placed while further diagnostics are being performed. in cases of acute hemoabdomen, obtain right lateral, left lateral, and ventrodorsal or dorsoventral thoracic radiographs to help rule out obvious metastasis. monitor the patient's ecg and correct dysrhythmias as necessary (see section on cardiac dysrhythmias). surgery is indicated once the patient is stabilized. in some cases, hemorrhage is so severe that the patient should be taken immediately to surgery. when recommending surgery for a hemorrhaging intraabdominal mass, it is important to discuss likely diagnoses and long-term prognosis with the owner. hemangiosarcoma usually involves the spleen or liver or both. the presence of free abdominal hemorrhage is associated with a malignant tumor in % of cases. even when free abdominal hemorrhage is not present, the tumor is malignant in % of cases. approximately % (two thirds) of masses in the spleen are malignant (hemangiosarcoma, lymphoma, mast cell tumor, malignant fibrous histiocytoma, leiomyosarcoma, fibrosarcoma), and approximately one third are benign (hematoma, hemangioma). hepatocellular carcinoma usually affects one liver lobe (usually the left), and surgery is the treatment of choice. with complete surgical excision, median survival in dogs is longer than days. if diffuse disease is observed at the time of surgery, the prognosis is poor. nonhemorrhagic effusions are associated with mesothelioma, lymphoma, carcinomatosis, or any mass that causes vascular or lymphatic obstruction. clinical signs of respiratory distress and abdominal distention with nonhemorrhagic effusions are usually slowly progressive in onset and not as severe as those observed with hemorrhage. treatment is usually aimed at identification of the underlying cause. obtain a fluid sample via thoracocentesis or abdominocentesis. to obtain further cells for cytologic evaluation, aspirate fluid from the thoracic or abdominal mass with ultrasound guidance. cytologic evaluation of the fluid will often elucidate the causative tumor type. an abdominal ultrasound can determine the degree of metastasis. perform therapeutic abdominocentesis or thoracocentesis if the effusion is causing respiratory difficulty. rapid re-accumulation of the fluid potentially can cause hypoproteinemia and hypovolemic shock. mesothelioma is a rare tumor most commonly observed in urban environments. in humans, mesothelioma has been associated with exposure to asbestos. it is sometimes difficult to differentiate between reactive mesothelial cells and malignant mesothelial cells. treatment is aimed at controlling the neoplastic effusion. intracavitary cisplatin has been demonstrated to slow rates of fluid re-accumulation, but is largely a palliative therapy. lymphoma is another tumor type that can cause thoracic or abdominal effusion. cytologic evaluation of the fluid usually reveals abundant lymphoblasts. treatment with multiagent chemotherapy protocols, with or without adjunctive radiation therapy, can prevent tumor remission and stop fluid accumulation. carcinomatosis occurs as a result of diffuse seeding of the abdominal cavity with malignant carcinomas and has a poor prognosis. carcinomatosis may occur de novo or from metastasis of a primary tumor. treatment consists of fluid removal when respiratory difficulty occurs, with or without intracavitary cisplatin as a palliative measure. cisplatin should never be used in cats due to fatal acute pulmonary edema. clinical signs of hemorrhagic thoracic effusion include acute respiratory distress, anemia, hypovolemic or cardiogenic shock, and collapse. hemorrhagic thoracic effusions are rare in association with neoplastic effusions. a notable exception is intrathoracic hemorrhage in young dogs with osteosarcoma of the rib. hemorrhage can result when a primary lung tumor erodes through a vessel. hemangiosarcoma of the lungs or right auricular area can also result in hemorrhagic thoracic effusion. in many cases, hemorrhage may be confined to the pericardial sac with a right auricular mass, causing a globoid cardiac silhouette on thoracic radiographs. treatment consists of pericardiocentesis (see section on pericardial effusion and pericardiocentesis) and placement of a pericardial window, or the mass may be removed if it is in the right auricular appendage and resectable. although surgery can resolve clinical signs of right-sided heart failure, metastatic disease often develops soon afterward. nonhemorrhagic thoracic effusion is more common than hemorrhagic thoracic effusion, and is caused most commonly by mesothelioma, lymphoma, carcinomatosis, and thymoma. clinical signs develop gradually and include respiratory difficulty, cyanosis, and cough. supplemental oxygen should be administered. in many cases, thoracocentesis can be therapeutic and diagnostic. obtain thoracic radiographs both before and after thoracocentesis to determine whether a mass effect is present. following identification of a cause, definitive therapy can be instituted. mesotheliomas are rare and are associated with diffuse serosal disease. they are more common in dogs than in cats. effusions caused by mesotheliomas can affect the pleural or pericardial cavities. treatment is directed at removing effusion fluid and controlling reaccumulation with use of intracavitary platinum compounds, carboplatin, and cisplatin can be used in dogs. (cisplatin and carboplatin should never be used in cats.) chemical or physical pleurodesis may be helpful in controlling reaccumulation of fluid, but it is very painful in small animal patients. thoracic effusion secondary to lymphoma often is associated with an anterior mediastinal mass. t-cell lymphoma is the most common type of mediastinal mass observed in dogs. b-cell lymphoma is associated with a decreased response to chemotherapy and shorter survival times. treatment consists of combination chemotherapy with or without radiation therapy to decrease mass size. carcinomatosis is a diffuse disease of the pleural cavity that often is a result of metastasis from a primary pulmonary carcinoma or mammary adenocarcinoma. treatment is similar to that for mesothelioma and is aimed at controlling the effusion and delaying its recurrence. thymomas have been documented in both dogs and cats. dogs most commonly present with a cough, while cats present with clinical signs of respiratory distress and a restrictive respiratory pattern associated with the presence of pleural effusion. an anterior mediastinal mass is often observed on thoracic radiographs. in some cases, the pleural effusion must be drained via thoracocentesis before a mass is visible. ultrasound-guided aspiration and cytologic evaluation of the mass reveal a malignant epithelial tumor with small lymphocytes and mast cells. prognosis is good if the tumor can be completely excised. treatment consists of surgical removal with or without presurgical radiation therapy to shrink the mass. paraneoplastic syndromes of myasthenia gravis have been documented in dogs with thymomas. if megaesophagus or aspiration pneumonia is present, the prognosis is more guarded because of the high rate of complications. obstructive lesions affecting the urinary tract can be extramural (intra-abdominal, pelvic, or retroperitoneal) or intramural (urethral, bladder, or urethral wall) . transitional cell carcinoma is the most common type of bladder tumor observed in dogs. prostatic adenocarcinoma, or neoplasia of the sublumbar lymph nodes (lymphoma, adenocarcinoma from apocrine gland adenocarcinoma), also can cause urethral obstruction. treatment is aimed at relieving the obstruction and then attempting to identify the cause of the disease. to alleviate the obstruction, pass a urinary catheter whenever possible. perform cystocentesis only as a last resort because of the risk of seeding the peritoneal cavity with tumor cells if transitional cell carcinoma is the cause of the obstruction. institute supportive therapy including intravenous fluids and correction of electrolyte abnormalities. plain radiographs may reveal a mass lesion or may not be helpful without double contrast cystography. abdominal ultrasound is more sensitive in identifying a mass lesion in the urinary bladder. masses in the pelvic urethra are difficult to visualize with ultrasonography. double contrast cystourethrography is preferred. once the patient is stabilized, biopsy or surgery is indicated to identify the cause of the mass and attempt resection. urine tests for transitional cell carcinoma are available for identification of transitional cell carcinoma in the dog. complete surgical excision of transitional cell carcinoma or removal of benign tumors of the urinary bladder yields a favorable prognosis. poorer prognosis is seen with incomplete excision. many transitional cell carcinomas are located in the trigone region of the bladder and cannot be completely excised. the nonsteroidal antiinflammatory drug piroxicam is helpful in alleviating clinical signs for a reported -month median survival. in some dogs, cisplatin and carboplatin may delay recurrence of transitional cell carcinoma. tumors of the prostate gland are always malignant and occur with equal frequency in castrated and uncastrated male dogs. diagnosis of prostatic tumors is based on ultrasonographic evidence of a mass effect or prostatomegaly and on transrectal or transabdominal aspiration or biopsy. surgery, chemotherapy, and radiation therapy generally are unrewarding over the long term, although palliative radiation therapy may relieve clinical signs for to months. luminal tumors of the gastrointestinal tract typically cause obstruction, with slowly progressive clinical signs including vomiting, inappetence, and weight loss, or with acute severe protracted vomiting. extraluminal obstructive lesions usually arise from adhesions, or strangulation may occur, resulting in obstruction. perforation of the mass through the gastric or intestinal wall can cause peritonitis. treatment consists of initial stabilization and rehydration, evaluation for evidence of metastasis, and surgical resection of the affected area in cases of adenocarcinoma, leiomyoma, leiomyosarcoma, and obstructive or perforated lymphoma. gastric and intestinal adenocarcinoma are the most common gastrointestinal tumors observed in dogs. affected animals typically have a history of anorexia, weight loss, and vomiting. obtain an abdominal ultrasound before performing any surgery. fine needle aspirates of the mass and adjacent lymph nodes are usually diagnostic and can determine whether there is local metastasis. many tumors are not resectable, and metastasis occurs in approximately % of cases. dogs with smaller tumors that can be resected typically have longer survival times. leiomyosarcomas occur in the intestines of dogs, and carry a more favorable prognosis than adenocarcinoma if the mass can be completely resected. with complete resection, the average survival time is longer than year. the paraneoplastic syndrome of hypoglycemia has been observed with this tumor type. gastrointestinal lymphoma is the most common tumor of the gastrointestinal tract observed in cats. in comparison, it is relatively rare in dogs. unless there is complete obstruction or perforation of the gastrointestinal tract, surgical treatment for gastrointestinal lymphoma is not indicated. rather, multiple chemotherapy drugs are used in combination to achieve remission and resolution of the clinical signs of anorexia, weight loss, and vomiting. treatment responses unfortunately are poor. mast cell tumors of the gastrointestinal tract typically are manifested as gastrointestinal ulceration and hemorrhage in up to % of patients. the gastrointestinal hemorrhage that occurs with mast cell tumors results from increased acid secretion as a result of histamine receptor stimulation. treatment consists of histamine or proton pump inhibition (ranitidine, famotidine, cimetidine, or omeprazole). bowel perforation is a rare complication. many chemotherapy agents exert their effects on rapidly dividing normal and neoplastic cells. normal tissues that are commonly affected include the bone marrow, gastrointestinal tract, skin and hair follicles, and reproductive organs. some drugs have unique organspecific toxicities that must be monitored. knowledge and recognition of the expected type and onset of complications can alleviate their severity by rapid treatment, when complications occur (see table - ) . neutropenia is the most common bone marrow toxicity observed secondary to chemotherapy in small animal patients (table - ) . in most cases, the neutropenia is dose-dependent. the nadir, or lowest neutrophil count, is typically observed to days after chemotherapy treatment. once the nadir occurs, bone marrow recovery is observed, with an increase in circulating neutrophils within to hours (table - ) . treatment of myelosuppression is largely supportive to treat or prevent sepsis. prophylactic antibiotics are recommended in the afebrile patient with a neutrophil count < /µl. acceptable antibiotics include trimethoprim-sulfa and amoxicillin-clavulanate. granulocyte-colony stimulating factor (g-csf) (e.g., neupogen) is a recombinant human product that stimulates the release of neutrophils from the bone marrow, and its use shortens the recovery time following myelosuppressive drug therapy. disadvantages of g-csf include antibody production in response to the drug within weeks of use and its high cost. to prevent ongoing neutropenia, subsequent chemotherapy dosages should be decreased by %, and the interval in between treatments increased. whenever possible, overlap of myelosuppressive drugs should be avoided. acute gastrointestinal toxicity can occur within to hours after administration of cisplatin and actinomycin d. in many cases, pretreatment with the antiemetics metoclopramide, butorphanol, chlorpromazine, dolasetron or ondansetron can prevent chemotherapyinduced nausea and vomiting. vomiting can also occur as a delayed side effect to days after treatment with doxorubicin (adriamycin), actinomycin d, methotrexate, and cytoxan. in delayed reactions, vomiting and diarrhea are caused by damage to intestinal crypt cells. treatment consists of administration of antiemetics, intravenous fluids, and a bland highly digestible diet. doxorubicin also can cause hemorrhagic colitis within to days of administration. treatment includes a bland diet, metronidazole, and tylosin tartrate (tylan powder). emergency care mild to none not observed vincristine (low-dose), l-asparaginase, glucocorticosteroids moderate - days melphalan, cisplatin, mitoxantrone, actinomycin d severe - days doxorubicin, cyclophosphamide, vinblastine paralytic ileus can be observed to days after administration of vincristine. this side effect is more common in humans than animals and can be treated with metoclopramide once a gastrointestinal obstruction has been ruled out. cardiotoxicity doxorubicin (adriamycin) causes a dose-dependent dilative cardiomyopathy when the cumulative dose reaches to mg/m . in many cases, however, clinical signs do not occur until the cumulative dose is mg/m . the myocardial lesions are irreversible. treatment of cardiac dysrhythmias is dependent on the type of dysrhythmia (see section on treatment of dysrhythmias). discontinue doxorubicin and administer diuretics and positive inotropic therapy for dilative cardiomyopathy in order to delay the progression of congestive heart failure (see sections on treatment of congestive heart failure). if abnormalities are shown on electrocardiography performed before beginning therapy, substitute liposome-encapsulated doxorubicin or mitoxantrone substituted in the chemotherapy protocol. cardioprotectant drugs such as vitamin e, selenium, and n-acetyl cysteine have shown some promise in the prevention of doxorubicin-induced cardiotoxicity. cyclophosphamide can cause a sterile hemorrhagic cystitis. damage to the urinary bladder mucosa and vessels is caused by the toxic metabolite acrolein. clinical signs of sterile hemorrhagic cystitis include a history of cyclophosphamide administration, stranguria, hematuria, and pollakiuria. treatment for sterile hemorrhagic cystitis is discontinuation of the drug, treatment of any underlying urinary tract infection with antibiotic therapy based on susceptibility testing, and intravesicle drug administration. in extremely refractory cases, surgical debridement and cauterization of the bladder mucosa may be necessary. prevention of sterile hemorrhagic cystitis includes emptying the bladder frequently and administering the drug in the morning. concurrent administration of prednisone can induce polyuria and polydipsia. if sterile hemorrhagic cystitis occurs, chlorambucil can be substituted as a chemotherapeutic agent. anaphylactic reactions have been observed with the administration of l-asparaginase, adriamycin, etoposide, and paclitaxel. the risk of anaphylaxis increases with repeated administration, although in some animals anaphylaxis will occur on the first exposure to the drug. treatment consists of administration of epinephrine, diphenhydramine, famotidine, and glucocorticosteroids, as with any other life-threatening allergic reaction (see section on treatment of allergic reactions). to decrease the risk of an adverse reaction, give diphenhydramine ( . mg/kg im) to minutes before drug administration. slowing the rate of intravenous infusion also can decrease the chance of an anaphylactic reaction. cisplatin can cause a fatal irreversible pulmonary edema in cats, even at low dosages. -fluorouracil ( -fu) can cause a severe neurotoxicity in cats that results in ataxia and seizures. never use cisplatin or -fu in cats. poisoning cases benefit from a rapid, organized approach. key points in this approach are giving appropriate advice over the telephone, being able to access information sources, and providing appropriate treatment. there are only a few classes of poisons that account for the majority of toxicities reported in dogs and cats. every veterinarian should develop a familiarity with the clinical management of rodenticide and insecticide toxicity and be prepared with antidotes on hand. beyond the most common toxins, the spectrum of possibilities is endless, and the veterinarian must rely on appropriate information resources. it is important to have available a comprehensive source of pharmaceutical and plant identification resources. remarkably, considering the myriad of potentially toxic substances to which an animal can be exposed, relatively few specific antidotes are commonly used in veterinary medicine. because of the lack of specific antidotes, the veterinarian must treat each toxicity with general methods of poison management, applying basic critical care in the treatment of specific clinical signs associated with the poison exposure or toxicity. the adage "treat the patient, not the poison" often comes into play when the exact toxic substance is unknown, or has no specific antidote. before an animal arrives, the staff should be prepared to ask specific questions over the phone, and provide initial advice for clients, particularly if the animal lives some distance from the hospital (box - .) it is important to have access to a database of information on toxic substances. thousands of potentially toxic substances are available on the market today. the american society for the prevention of cruelty to animals (aspca) animal poison control center provides direct access to veterinary toxicologists hours a day, days a year. for additional information, call the nearest veterinary school or emergency center (box - ). also, see section for a table of emergency hotlines. check your local telephone book for a poison control center listing under emergency numbers, usually found on the front cover. although these numbers are for human poisonings, they have access to extensive poison and toxin databases and can potentially provide useful information for veterinarians, particularly regarding antidotal substances suitable for out of the ordinary toxins and human medications. information on the toxic ingredients in thousands of medications, insecticides, pesticides, and other registered commercial products has been confidentially placed by the government in these poison control centers. as new products are marketed, information regarding toxin ingredients is forwarded to the centers. various e-mail discussion lists can serve as an informative resource for practitioners, but access generally requires an initial subscription and may have the disadvantage of delayed *do not keep the client on the telephone for too long. lengthy histories can be performed once the animal is at your hospital and you have started to initiate treatment. † hair dressing products sometimes have hydrogen peroxide as a % w/v; this concentration is not suitable for induction of emesis. is your animal breathing or does it have respiratory difficulty? what is the color of the gums or tongue? is your animal able to walk? is there any vomiting, diarrhea, trembling, or seizures? does it appear lethargic or hyperactive? what is the substance that your animal ingested (was exposed to)? did you witness the ingestion or exposure? how much did the animal consume? how long ago was the exposure? was the substance swallowed, or is it on the animal's skin or eyes? how is the patient acting? how long has the animal been acting that way? or when was the last time you saw your animal act normally? . first aid instructions for the client: induce vomiting at home and save the vomitus. never induce vomiting if the patient is depressed, appears comatose, or is actively seizing. if the animal has ingested a caustic substance (strong alkali or acids) or a petroleum-based product (kerosene or turpentine), never recommend induction of emesis. hydrogen peroxide ( % w/v † ) ml = tsp/ lb of body weight can repeat once if no vomiting occurs after minutes . remind the owner to bring a sample of the toxin and the vomitus in with the patient. . advise the owner to transport the patient as rapidly as possible to the nearest veterinary hospital. response times. they are useful for ideas on standard and long-term therapy, but not emergency stabilization. an exception to this is the veterinary interactive network (vin), which posts message board communications. previous communications from veterinarians who treated a case with the same poison/toxin can be accessed with a subscription. many manufacturers operate an information service about their products. if the product label or name is available, check for a telephone number that may route you to a specialist. there are six essential steps in treating toxicities: . performing a physical examination . stabilizing the patient's vital signs . taking a thorough history . preventing continued absorption of the toxin . administering specific antidotes when available . facilitating clearance or metabolism of the absorbed toxin it is most important to provide symptomatic and supportive care both during and following emergency treatment. immediately on presentation, perform a brief but thorough physical examination. obtain a minimum database as well as serum, urine, or orogastric lavage samples for later toxicologic analyses. it is important at this time to systematically evaluate the patient's physical status, focusing particularly on the toxins most common to a particular geographic location and the organ systems most commonly affected by toxins in veterinary medicinenamely, the neurologic and gastrointestinal tracts. a checklist is useful when performing a complete physical examination (box - ). the minimium database includes a urine sample, packed cell volume, total protein, serum urea, and serum glucose. the information obtained from these simple cage-side tests is useful for determining dehydration, hemoconcentration, azotemia (renal or prerenal), and hypo-or hyperglycemia. when appropriate, obtain samples for serum biochemistry profiles, serum electrolytes, blood gases, serum osmolality, a complete hemogram, and coagulation profiles. samples of serum, urine, and any vomitus or orogastric lavage contents should be collected and saved for later toxicologic analyses as required later. stabilization of vital signs includes four major goals of treatment: maintain respiration, maintain cardiovascular function, control cns excitation, and control body temperature. in any patient with clinical signs of respiratory distress or respiratory dysfunction, supplemental oxygen should be administered via flow-by, oxygen hood, oxygen cage, nasal, nasopharyngeal, or transtracheal oxygen sources. ventilatory assistance may be necessary. irritant or corrosive substances can cause damage to the oropharyngeal mucosa to such an extent that airway obstruction occurs. when necessary, a temporary tracheostomy should be performed. arterial blood gases, pulse oximetry, and capnometry may be required to monitor oxygenation and ventilation. at the time of presentation, immediately place an intravenous catheter for administration of intravenous fluids, inotropes, antiarrhythmics, and antidotes, if necessary. the initial fluid of choice is a balanced crystalloid solution such as normosol-r, plasmalyte-m, or lactated ringer's solution. fluid therapy can later be changed based on the patient's acidbase and electrolyte status. some toxins can cause severe dysrhythmias and hyper-or hypotension. monitor blood pressure and perform ecg and correct any abnormalities according to standard therapy (see sections on hypotension and cardiac dysrhythmias). what is the pupil size? what is the pupil reactivity to light? is the ocular examination normal? what is the sensitivity to light or sound? nose: is it moist, dry, bubbling, or frothy, or caked with dirt? throat: are there any characteristic odors on the breath? are there any traces of foreign material on the tongue or in the crevices of the teeth or gums? are there petechiae or ecchymosis on the gums or bleeding from the gumline? what is the mucous membrane color? is it normal and pink, or dark red (injected), pale, or icteric? what is the capillary refill time? is it fast, normal, or slow? what is the patient's heart rate? are there any pulse deficits or dysrhythmias auscultated? what is the patient's blood pressure? what is the quality of the femoral pulse? is it synchronous with the heart rate, or are there dropped pulses? is the pulse bounding, normal, thready, or not palpable? what is the patient's electrocardiogram? what is the patient's respiratory rate? what is the patient's respiratory character? is it normal, fast, shallow, or labored? what do you hear on thoracic auscultation? do you hear harsh airway sounds or pulmonary crackles? what is the patient's rectal temperature? is there excessive salivation? is there evidence of vomiting or diarrhea? is abdominal palpation painful? do the intestinal loops feel normal, or are they fluid-filled or gas-filled? what is the color and consistency of the feces? is there a palpable urinary bladder? is there urine production? what is the color of the urine? peripheral lymph nodes should be normal in poisonings. some toxins cause hemolysis, methemoglobinemia, heinz body anemia, and coagulopathies. whole blood, fresh frozen plasma, packed rbcs, or hemoglobin-based oxygen carriers should be available and used if necessary. treat methemoglobinemia with a combination of ascorbic acid and n-acetylcysteine. many toxins affect the cns, producing clinical signs of excitation and/or seizures. diazepam is the drug of choice for most but not all seizures and tremors. if an animal has cns excitation secondary to the ingestion of selective norepinephrine reuptake inhibitors, avoid using diazepam, as it can potentially exacerbate clinical signs. muscle relaxants such as guaifenesin or methocarbamol may be required to control muscle spasm and tremors associated with some toxicities. consider animals that are in status epilepticus because of toxin exposure at high risk. such patients may not require the full dose of anesthetics or sedatives for seizure control. give phenobarbital ( - mg/kg iv) or pentobarbital ( - mg/kg iv to effect) for longer-term management of seizures. core body temperature can easily increase or decrease secondary to increased muscle activity or coma. animals may present as hypo-or hyperthermic, depending on the toxin ingested and the stage of toxicity. manage hypothermia with circulating hot water or hot air blankets, or place bubble wrap or saran wrap around the animal's peripheral extremities. manage hyperthermia by placing lukewarm wet towels on the patient until the rectal temperature has decreased to . °c ( °f). (see section on of hyperthermia and heat-induced illness). if sedatives or anesthetics have been used, initial hyperthermia may initially resolve due to hypothalamic loss of thermoregulatory control, cool water bathing should not be performed. when the patient is first presented to the veterinarian, have the owner complete a toxicologic history form (figure - ) while the animal is being initially assessed and vital signs are being stabilized. when initial stabilization of vital signs has been accomplished, the veterinarian can discuss the patient's history with the owner. in urgent situations, the veterinarian should obtain a brief history as an initial procedure (box - ). knowing when the animal was last seen as normal provides a time frame in which the toxic substance was most likely accessed, allowing differential diagnoses to be ranked in some order of probability by rate of onset. in eliciting a history from the owner about the animal's access to poisons, it is important not to take anything for granted. many owners do not realize how poisonous some substances can be, such as insecticide products, garbage, cleaning chemicals, and over-the-counter drugs commonly used by humans. many owners will deny that an animal could have ingested anything that might be toxic, not wanting to believe that the source of the toxin is within their household or property, particularly if recreational drug exposure is suspected. it is useful to phrase questions in a neutral fashion-for example, "is such-and-such present on the premises?" rather than "could the dog have eaten such-and-such?" if recreational drug exposure is suspected, another way to question the owners is to ask whether they have had any guests in their house recently that may have had such-and-such (e.g., marijuana, cocaine, methamphetamine). this approach serves to minimize the suggestion of any bias or preconceptions. when questioning an owner about recent events, it is useful to realize and acknowledge that disruption in the household routine is a distinct factor in the occurrence accidents, including poisonings. examples of such disruptive events include moving from the house, family member is ill or in the hospital, and renovations or recent construction. while these events are occurring, the safeguards followed by a normally careful owner may be disrupted. often, doors or gates may be left open, animals may be outside instead of inside (or vice versa), and inexperienced people may be pet-sitters. once owners are made aware of the importance of assessing such risks, they are often able to provide insight into otherwise baffling circumstances. various methods can be used to remove toxins from the gastrointestinal tract, including emesis, orogastric lavage, cathartics, and enemas. adsorbents, ion exchange resins, or precipitating or chelating agents may be used. removal of a toxic substance from the body surface may be necessary, depending on the toxin.the use of both emesis and orogastric lavage is less and less frequent in human medicine because of the risk of aspiration pneumonia and doubts about their efficacy. currently, management of poisonings in human medicine relies heavily on the use of activated charcoal combined with sorbitol as a cathartic, when appropriate, and supportive critical care. it should be emphasized, however, that the majority of poisonings in humans are due to drug overdoses (illicit or otherwise) (which have a relatively small volume and rapid absorption), for which this treatment is appropriate. furthermore, adoption of the approach rests on the availability of a hospital intensive care infrastructure, which is not always available in veterinary practice. induce emesis if the animal's physiology and neurologic status are stable (i.e., does not have respiratory depression or is not actively seizing, obtunded, unable to swallow or protect its airway). do not administer the same emetic more than twice. if the emetic doesn't work after two doses, give a different emetic or perform orogastric lavage under general anesthesia. emetics are strictly contraindicated for toxicity from petroleum-based products and corrosives because of the risk of aspiration pneumonia and further esophageal damage. emetics may also be of little value if poisons with antiemetic properties have been ingested, such as benzodiazepines, tricyclic antidepressants, and marijuana (table - ) . various emetics traditionally have been recommended for use in veterinary medicine. many have fallen out of favor because of the risk of causing adverse consequences and side effects. apomorphine ( . mg/kg iv or in the conjunctival sac) remains the standard but is less useful in certain situations in which the poison causes cns excitation or stimulation. it is ineffective in cats. other emetics include xylazine and hydrogen peroxide. do not use table salt because of the risk of severe oropharyngeal irritation and hypernatremia. do not use mustard powder or dishwashing liquid detergent because of the risk of severe oropharyngeal, esophageal, and gastric irritation. orogastric lavage is described in detail in the section on emergency procedures gastric lavage is contraindicated in treatment of toxicity from petroleum-based compounds and acid/alkali ingestion. the procedure can be messy but is very effective if performed within to hours of ingestion of the poison. to prevent aspiration, the patient should be placed under general anesthesia. keep the animal's head lowered during the procedure to prevent aspiration of stomach contents into the trachea. it is sometimes helpful to put the animal in both right and left lateral recumbency to allow complete emptying of gastric contents. repeat the procedure until the fluid runs clear from the stomach. in some cases in which solid material has been ingested, this process can take a long time, so be prepared with a large volume of warm water. following successful evacuation and lavage, administer a slurry of activated charcoal through the orogastric tube before removing it. keep the endotracheal tube cuffed and in place until the animal is semi-conscious, is starting the fight the tube, and is visibly able to swallow and protect its airway. • when was the animal last seen as normal? • what clinical signs developed? • how fast did the clinical signs develop? • when was the onset of clinical signs? • what is the animal's activity level? • does the animal have access to any poisonous substances? • this includes known toxins or chemicals, over-the-counter or prescription medications (including the owner's), and recreational drugs. enemas are useful to facilitate the action of cathartics and in cases in which the poison is a solid material (e.g., compost, snail bait, garbage) (box - ). it is best to use just lukewarm water. commercially available phosphate enema solutions can cause severe electrolyte disturbances (hyperphosphatemia, hyponatremia, hypocalcemia, and hypomagnesemia) and acid-base abnormalities (metabolic acidosis); therefore, they are absolutely contraindicated in small animal patients. use nonsterile nonspermicidal water-soluble lubricants (k-y jelly) old intravenous fluid bag enema bag -to -ml syringe fluid warm water, with or without hand or liquid dish soap the fluid volume required depends on the size of the animal and the state of its lower gastrointestinal tract. as with orogastric lavage, continue the procedure until the water runs clear. if difficulty is encountered emptying the lower gastrointestinal tract, repeat the enema in or hours, rather than be overzealous on the first attempt. cathartics are useful for hastening gastrointestinal elimination of toxins, and they are particularly useful for elimination of most solid toxicants (e.g., compost, garbage, snail baits). cathartics can be used in conjunction with activated charcoal. do not use magnesium-based cathartics in patients with cns depression, because hypermagnesemia can worsen this disorder and also cause cardiac rhythm disturbances (table - ) . activated charcoal ( - ml/kg) is the safest and to date the most effective adsorbent for the treatment of ingested toxins. activated charcoal can be administered after emesis or orogastric lavage or can be administered as the sole treatment. various preparations are available on the market, including dry powder, compressed tablets, granules, liquid suspensions, and concentrated paste preparations. commercially available products are relatively inexpensive and should be used whenever possible for ease of administration. vegetableorigin activated charcoal is the most efficient adsorbent and binds compounds with weak, nonionic bonds. some preparations are combined with sorbitol to provide simultaneous administration of an adsorbent and a cathartic; this combination has been shown to be most efficacious. repeated administration of activated charcoal every to hours has been shown to be beneficial in the management of a toxin that undergoes enterohepatic recirculation. administration of an oily cathartic or mixing the activated charcoal with food only serves to reduce the absorptive surface of the activated charcoal and therefore is not recommended. in general, substances that are very soluble and are rapidly absorbed are not well adsorbed by activated charcoal, including alkalis, nitrates, mineral acids, ethanol, methanol, ferrous sulfate, ammonia, and cyanide. kaolin and bentonite are clays that have been used as adsorbents. both are usually less effective than activated charcoal. however, they are reported to be better adsorbents than activated charcoal for the herbicide paraquat. ion exchange resins can ionically bind certain drugs or toxins. cholestyramine is one such resin, commonly used in human medicine to bind intestinal bile acids and thereby decrease cholesterol absorption. its application in toxicology extends to the absorption of fat-soluble toxins such as organochlorine and certain acidic compounds such as digitalis. ion exchange resins also have been used to delay or reduce the absorption of phenylbutazone, warfarin, chlorothiazide, tetracycline, phenobarbital, and thyroid preparations. precipitating, chelating, and diluting agents precipitating, chelating, and diluting agents are used primarily in the management of heavy metal intoxications, such as alkaloids or oxalates. they work by binding preferentially to the metal ion and creating a more soluble complex that is amenable to renal excretion. those chelating agents in common usage are calcium edta, deferoxamine, and d-penicillamine. calcium edta and deferoxamine should both be on hand in the veterinary hospital because they are necessary to treat zinc and iron toxicity, respectively, both of which have a short window of opportunity for therapeutic intervention. d-penicillamine has a wide application for a number of metal toxicities but tends to be used for long-term chronic therapy because it can be administered orally. various agents used for nonspecific dilution of toxins, including milk of magnesia and egg whites, although old-fashioned, still have wide application in many cases in which low-grade irritants have been ingested. bathing the animal is an important aspect of treatment for topical exposures to toxins such as insecticidal products, petroleum-based products, and aromatic oils. bathing an animal is not an innocuous procedure. to avoid hypothermia and shock, use warm water at all times. actively dry the animal to further minimize the risk of hypothermia. when bathing the animal, use rubber gloves and a plastic apron to avoid exposure to noxious agents. in most cases, a mild dishwashing soap is appropriate. medicated or antibacterial shampoos are less appropriate in this situation. for petroleum-based products in particular, dawn dishwashing liquid that "cuts the grease" works well to remove the oils. if dawn is not available, mechanics' hand cleaners or coconut oil-based soaps can be used instead. as a general principle, best results are obtained by barely wetting the patient's fur until the detergent is worked well into the fur, keeping the amount of water to a minimum until ready for the rinse. oil-based paint is best removed by clipping rather than by attempting removal with solvents, because solvents are also toxic. to remove powder products, brush and vacuum the animal before bathing it to eliminate further toxic exposure. with caustic alkaline or acidic products, the primary treatment is to dilute and flush the skin with warm water; do not attempt neutralization. neutralization can cause an exothermic reaction that causes further damage to the underlying tissues. eliminating poison from the eyes for ocular exposures, irrigate the eyes for a minimum of to minutes with warm (body temperature) tap water or warmed . % sterile saline solution. the use of neutralizing substances is not recommended because of the risk of causing further ocular damage. following adequate irrigation, treat chemical burns of the eyes with lubricating ointments and possibly a temporary tarsorrhaphy. atropine may be indicated as a cycloplegic agent. systemic nonsteroidal antiinflammatory drugs can be used to control patient discomfort. daily follow-up examinations are required because epithelial damage may be delayed, especially with alkali burns, and it is difficult to predict the final extent of ocular damage. topical glucocorticosteroids are contraindicated if the corneal epithelium is not intact. if severe conjunctival swelling is present with a corneal ulcer, parenteral glucocorticosteroids can be administered to help alleviate inflammation, but nonsteroidal antiinflammatory drugs should not be used simultaneously due to the risk of gastrointestinal ulceration or perforation. whenever possible, administer specific antidotes to negate the effects of the toxin and prevent conversion of the substance to the toxic metabolite. three categories of agents are used in the management of poisonings. the first category is specific antidotes. unfortunately, few specific antidotes are available for use in veterinary medicine. some "classic" toxins and antidotes are now considered to be rare, such as curare and physostigmine, thallium and prussian blue, and fluoride and calcium borogluconate. these and a few others have been omitted from the table. the second, broader category of antidotes includes those drugs used in the symptomatic management of clinical signs, which are part of our routine veterinary stock. drugs such as atropine, sedatives, steroids, antiarrhythmics, and beta-blockers fall into this category. the third category comprises nonspecific decontaminants such as activated charcoal, cathartics, and emetics. these were discussed previously. many patients benefit from efforts to enhance clearance or metabolism of the absorbed toxins. some specific therapies have been developed for this purpose, including -methylpyrazole for ethylene glycol toxicity and specific antibodies such as digibind (digoxin immune fab [ovine]) for digitalis toxicity. other strategies are aimed at promoting renal excretion. renal excretion strategies include diuresis, ion trapping, and peritoneal dialysis or hemodialysis (see section on peritoneal dialysis). diuresis and ion trapping are applicable to a large number of toxins and are discussed here in more detail. other toxins respond to urine acidification and urine alkalinization. enhancing renal excretion of substances is most useful for those organic substances that are present in significant concentrations in the plasma. substances that are non-ionic and lipid-soluble, such as certain herbicides, are likely to be less affected by attempts to promote rapid renal elimination. before starting diuresis or ion trapping, intravenous fluid therapy should be adequate as determined by normal central venous pressure, urine output, and mean arterial blood pressure. if any of these values are less than normal, use other measures to ensure adequate renal perfusion, including but not limited to a constant rate infusion of dopamine. simple fluid diuresis can influence the excretion of certain substances. the use of mannitol as an osmotic diuretic may reduce the passive reabsorption of some toxic substances in the proximal renal convoluted tubule by reducing water reabsorption. dextrose ( %) can be used as an osmotic diuretic. furosemide can be used to promote diuresis, but again, there is no substitute for intravenous fluid therapy. the use of mannitol, dextrose, and furosemide is contraindicated in hypotensive or hypovolemic patients. take care to avoid causing dehydration with any diuretic; central venous pressure monitoring is strongly recommended. ion trapping is based on the principle that ionized substances do not cross renal tubular membranes easily, and are not well reabsorbed. if the urinary ph can be changed so that the toxin's chemical equilibrium shifts to its ionized form, then that toxin can be "trapped" in the urine and excreted. alkaline urine favors the ionization of acidic compounds, and acidic urine favors the ionization of alkaline compounds. those toxins that are amenable to ion trapping are mostly weak acids and weak bases. ammonium chloride can be used to promote urinary acidification. contraindications to the use of ammonium chloride include a preexisting metabolic acidosis, hepatic or renal insufficiency, and hemolysis or rhabdomyolysis leading to hemoglobinuria or myoglobinuria. signs of ammonia intoxication include cns depression and coma. when performing urine acidification, frequently check the serum potassium concentration and urine ph. urine alkalinization can be performed with use of sodium bicarbonate. contraindications to the use of sodium bicarbonate include metabolic alkalosis (particularly with concurrent use of furosemide), hypocalcemia, and hypokalemia. as with urine acidification, monitor the serum potassium concentration and urine ph frequently. the major steps in management of poisonings discussed here must be accompanied by application of the fundamentals of critical care. respiratory and cardiovascular support have been discussed previously. renal and gastrointestinal function and analgesia are particularly important in the management of the poisoning patient. maintenance of renal perfusion is a priority in the poisoning patient. fluid, electrolyte, and acid-base balance must be controlled and be accurate. poisoning patients are at particularly high risk for renal damage and acute renal failure, whether by primary toxic insult to the renal parenchyma or by acute or prolonged renal hypoperfusion. for this reason, a protocol that aims at preventing oliguria and ensuing renal failure is one of the therapeutic strategies that should be routinely employed. this protocol is described in box - . gastrointestinal protectant drugs may be indicated for the management of those poisons that are gastrointestinal irritants or ulcerogenic. commonly used gastroprotectant drugs include cimetidine, ranitidine, famotidine, omeprazole, sucralfate, and misoprostol. antiemetics may be used to suppress intractable vomiting. metoclopramide is commonly used, and it is the drug of choice for centrally mediated nausea. antiemetics that work by different mechanisms can be used in combination as necessary. examples are dopamine -receptor antagonists such as prochlorperazine, -hydroxytryptamine antagonists such as ondansetron and dolasetron, and h- receptor antagonists such as diphenhydramine and meclizine. analgesics are more appropriate to treat poisonings than once thought. common effects of poisons including severe gastroenteritis and topical burns or ulcerations may warrant the use of analgesics. longer-acting analgesics such as morphine, hydromorphone, and buprenorphine are particularly useful. nutritional support may be necessary in the form of enteral or parenteral feeding in patients that have esophageal or gastric damage or that need to be sedated for long periods of time. endoscopy may be useful in assessing the degree of esophageal and gastric damage, particularly after ingestion of caustic substances. introduction: acetaminophen (paracetamol) is the active ingredient in tylenol and many over-thecounter cold products. acetaminophen is converted to n-acetyl-p-benzoquinonimine in the liver, a toxic substance that can cause oxidative injury of red blood cells and hepatocytes. clinical signs of acetaminophen toxicity include respiratory distress from lack of oxygen-carrying capacity, cyanosis, methemoglobinemia (chocolate-brown appearance of the blood and mucous membranes), lethargy, vomiting, and facial and paw swelling (cats). the toxic dose of acetaminophen is > mg/kg for dogs, and mg/kg for cats. treatment of acetaminophen toxicity includes induction of emesis or orogastric lavage if the substance has been ingested within minutes. activated charcoal should also be administered. in cases of severe anemia, give supplemental oxygen along with a packed rbc transfusion. administer intravenous fluids to maintain renal and hepatic perfusion. n-acetylcysteine, vitamin c, and cimetidine are the treatments of choice for methemoglobinemia in patients with acetaminophen toxicity. introduction: hydrochloric, nitric, and phosphoric acids cause chemical burns through contact with the skin and/or eyes. localized superficial coagulative necrosis occurs upon contact. usually, the patient's skin is painful to the touch or the animal may lick or chew at an irritated area that is not visible under the haircoat. if the chemical is swallowed, do not induce emesis or perform orogastric lavage, because of the risk of worsening esophageal irritation. rinse the patient's skin and eyes with warm water or warm saline for a minimum of / hour. use analgesics and treat corneal ulcers (see section on corneal ulcers) as required. do not attempt chemical neutralization, because of the risk of causing an exothermic reaction and worsening tissue injury. aflatoxin (aspergillus flavus) is found in moldy feed grains. clinical signs of toxicity occur after ingestion and include vomiting, diarrhea, and acute hepatitis; abortion may occur in pregnant bitches. treatment of suspected aflatoxin ingestion consists of gastric decontamination, administration of activated charcoal, intravenous fluids, and hepatic supportive care (s-adenosyl methionine [same], milk thistle). drinking (ethanol), rubbing (isopropyl), and methyl (methanol) alcohols can be harmful if ingested ( . to . g/kg po). all cause disruption of neuronal membrane structure, impaired motor coordination, cns excitation followed by depression, and stupor that can lead to cardiac and respiratory arrest, depending on the amount ingested. affected animals may appear excited and then ataxic and lethargic. contact or inhalant injury can occur, causing dermal irritation and cutaneous hyperemia. methanol also can cause hepatotoxicity. and diarrhea result from muscarinic overload. nicotinic overload produces muscle tremors. toxicity can result in seizures, coma, and death. and cause severe irritation and corrosion of the mucous membranes and skin. some compounds also can cause clinical signs similar to those observed with anticholinesterase compounds, including muscle tremors, seizures, paralysis, and coma. methemoglobinemia can occur. signs of ethylene glycol intoxication and renal impairment or failure, a negative test for the presence of calcium oxalate crystalluria means that there is no more ethylene glycol in the patient's serum because it has all been metabolized. cats are very sensitive to the toxic effects of ethylene glycol. in many cases, cat may have ingested a toxic dose, but because the sensitivity of the assay is low, test results will be negative. lack of treatment can result in death. there are three phases of ethylene glycol intoxication. in the first to hours after ingestion (stage i), the patient may appear lethargic, disoriented, and ataxic. in stage ii ( to hours following ingestion), the patient improves and appears clinically normal. in stage iii ( to hours following ingestion), the patient demonstrates clinical signs of renal failure (polyuria and polydipsia) that progress to uremic renal failure (vomiting, lethargy, oral ulceration). finally, seizures, coma, and death occur. crosses, old english sheepdogs, and some terriers. clinical signs of ivermectin toxicity include vomiting, ataxia, hypersalivation, agitation, tremors, hyperactivity, hyperthermia, hypoventilation, coma, seizures, signs of circulatory shock, bradycardia, and death. clinical signs often occur within to hours after ingestion or iatrogenic overdose. blood ivermectin levels can be measured, but diagnosis is often made based on clinical signs and knowledge of exposure in predisposed breeds. there is no known antidote. the clinical course can be prolonged for weeks to months before recovery occurs. to treat known exposure, induce emesis or perform orogastric lavage if the substance was ingested was within hour of presentation and the patient is not symptomatic. administer activated charcoal. control seizures with phenobarbital, pentobarbital, or propofol administered as intermittent boluses or as a constant rate infusion. diazepam, which potentially can worsen central nervous stimulation, is contraindicated. administer intravenous fluids to maintain perfusion and hydration, and treat hyperthermia. supportive care may be necessary, including supplemental oxygen (or mechanical ventilation, if necessary), frequent turning of the patient and passive range-of-motion exercises, placement of a urinary catheter to maintain patient cleanliness and monitor urine output, lubrication of the eyes, and parenteral nutrition (see section on rule of twenty). specific antidotes used to treat ivermectin toxicity include physostigmine and picrotoxin. physostigmine therapy was beneficial in some patients for a short period; picrotoxin caused severe violent seizures and therefore should be avoided. introduction d-limonene and linalool are components of citrus oil extracts used in some flea control products. the toxic dose is unknown, but cats appear to be very sensitive to exposure. clinical signs of toxicity include hypersalivation, muscle tremors, ataxia, and hypothermia. treatment of d-limonene and linalool exposure includes treatment of hypothermia, administration of activated charcoal to prevent further absorption, and careful, thorough bathing to prevent further dermal exposure. lead is ubiquitous, and is found in some paints, car batteries, fishing equipment/ sinkers, and plumbing materials. lead can be toxic at doses of mg/kg. if more than than - mg/kg of lead is ingested, death can occur. lead causes toxicity by inhibiting sulfur-containing enzymes, leading to increased rbc fragility, and cns damage. clinical signs of hyperexcitability, dementia, vocalization, seizures, and lower motor neuron polyneuropathy can occur. affected animals may appear blind, or vomiting, anorexia, and constipation or diarrhea may occur. if lead toxicity is suspected, blood and urine lead levels can be measured. treatment of lead toxicity is supportive and is directed at treatment of clinical signs. control seizures with diazepam or phenobarbital. if cerebral edema is present, administer mannitol ( . - . g/kg iv), followed by furosemide ( mg/kg iv minutes after mannitol). sodium or magnesium sulfate should be administered as a cathartic. initiate chelation therapy with dimercaprol, penicillamine, or calcium edta. if a lead object is identified in the gastrointestinal tract on radiographs, remove the object using endoscopy or exploratory laparotomy. hyperthermia, that occurs within - minutes of ingestion. diarrhea and convulsions can develop. if hyperthermia is severe, renal failure secondary to myoglobinuria and disseminated intravascular coagulation can result. delayed hepatic failure has been described days after initial recovery. if metaldehyde toxicosis is suspected, analysis of urine, serum, and stomach contents is warranted. to treat metaldehyde toxicity, procure and maintain a patent airway and control cns excitation and muscle tremors. if an animal has just ingested the metaldehyde and is not symptomatic, induce emesis. if clinical signs are present, perform orogastric lavage. both emesis and orogastric lavage should be followed by administration of one dose of activated charcoal. administer intravenous fluids to control hyperthermia, prevent dehydration, and correct acid-base and electrolyte abnormalities. methocarbamol is the treatment of choice to control muscle tremors. diazepam can be used to control seizures if they occur. introduction mushroom ingestion most commonly causes activation of the autonomic nervous system, resulting in tremors, agitation, restlessness, hyperexcitability, and seizures. in some cases slud (salivation, lacrimation, urination, and defecation) is seen. some mushrooms (amanita spp.) also can cause hepatocellular toxicity. clinical signs include vomiting, anorexia, lethargy, and progressive icterus. hemoglobinuria and pigment damage of the renal tubular epithelium. heinz bodies may be observed on cytologic evaluation of the peripheral blood smear. paint in a sorbitol or glycerol carrier. when large quantities of these osmotically active sugars are ingested, osmotic shifts of fluid cause a sudden onset of neurologic or gastrointestinal signs, including ataxia, seizures, and osmotic diarrhea caused by massive fluid shifts into the gastrointestinal tract. the loss of water in excess of solute can result in hypernatremia, a free water deficit, and increased serum osmolality. following orogastric lavage, treatment of ingestion includes administering warm water enemas to help speed the movement of the paintballs through the gastrointestinal tract. do not administer activated charcoal (usually in a propylene glycol carrier), because the compound's cathartic action will pull more fluid into the gastrointestinal tract. baseline electrolytes should be obtained and then carefully monitored. if severe hypernatremia develops, administer hypotonic solutions such as . % nacl + . % dextrose or % dextrose in water after calculating the patient's free water deficit. because of the large volume of fluid loss, intravenous fluid rates may seem excessive but are necessary to normalize acid-base, electrolyte, and hydration status. in most cases, these patients can survive if the problem is recognized promptly and corrected with careful electrolyte monitoring, aggressive decontamination strategies, and intravenous fluid support. introduction paraquat, a dipyridyl compound, is the active ingredient in some herbicides. the ld of paraquat is - mg/kg. paraquat initially causes cns excitation. it also causes production of oxygen-derived free radical species in the lungs, that can lead to the development of acute respiratory distress syndrome. initial clinical signs include vomiting, diarrhea, and seizures. within to days, clinical signs associated with severe respiratory distress and acute respiratory distress syndrome (ards) can develop, leading to death. chronic effects include pulmonary fibrosis, if the patient survives the initial toxicity period. the prognosis for paraquat toxicity is generally unfavorable. to treat paraquat ingestion, remove the toxin from the gastrointestinal tract as rapidly as possible after ingestion. there are no known antidotes. if the compound was ingested within the past hour and the animal is able to protect its airway, induce emesis. otherwise, perform orogastric lavage. activated charcoal is not as effective as clay or bentonite adsorbents for removing this particular toxin. early in the course of paraquat toxicity, oxygen therapy is contraindicated because of the risk of producing oxygen-derived free radical species. later, oxygen therapy, including mechanical ventilation, is necessary if ards develops. experimentally, free radical scavengers (n-acetyl cysteine, vitamin c, vitamin e, same) have been shown to be useful in preventing damage caused by oxygen-derived free radical species. hemoperfusion may be useful in eliminating the toxin, if it is performed early in the course of toxicity. pennyroyal oil is an herbal flea control compound that contains menthofuran as its toxic compound. menthofuran is hepatotoxic and may cause gastrointestinal hemorrhage and coagulopathies. to treat toxicity, administer a cathartic and activated charcoal and antiemetic and gastroprotectant drugs, and thoroughly bathe the animal to prevent further dermal exposure. petroleum distillates: see fuels phenobarbital: see barbiturates phenylcyclidine (angel dust) introduction phenylcyclidine (angel dust) is an illicit recreational drug that causes both cns depression and excitation, decreased cardiac output, and hypotension. to treat phenylcyclidine toxicity, place an intravenous catheter, and administer intravenous fluids and antiarrhythmic drugs to maintain organ perfusion. administer supplemental oxygen, and administer diazepam to control seizures. urine alkalinization can help eliminate the compound. phenylephrine is an α-adrenergic agonist in many over-the-counter decongestant preparations. clinical signs of intoxication include mydriasis, tachypnea, agitation, hyperactivity, and abnormal flybiting and staring behavior. tachycardia, bradycardia, hypertension, hyperthermia, and seizures can occur. to treat phenylephrine toxicity, place an intravenous catheter and give intravenous fluids to maintain hydration, promote diuresis, and treat hyperthermia. administer prazosin or sodium nitroprusside to treat hypertension, antiarrhythmic drugs as necessary, and diazepam to control seizures. phenylpropanolamine has both αand β-adrenergic agonist effects, and is used primarily in the treatment of urinary incontinence in dogs. the drug was taken off of the market for use in humans because of the risk of stroke. clinical signs of phenylpropanolamine intoxication include hyperactivity, hyperthermia, mydriasis, tachyarrhythmias or bradycardia, hypertension, agitation, and seizures. to treat toxicity, administer prazosin or nitroprusside to control hypertension, a betablocker (esmolol, propranolol, atenolol) to control tachyarrhythmias, diazepam to control seizures, and intravenous fluids to maintain hydration and promote diuresis. urine acidification may aid in facilitating excretion. if bradycardia occurs, do not use atropine. pseudoephedrine is an αand β-adrenergic agonist that is a component of many over-thecounter decongestants and is used in the manufacture of crystal methamphetamine. clinical signs of toxicity include severe restlessness, tremors, mydriasis, agitation, hyperthermia, tachyarrhythmias or bradycardia, hypertension, and seizures. to treat toxicity, administer activated charcoal, intravenous fluids to promote diuresis and treat hyperthermia, chlorpromazine to combat α-adrenergic effects, a beta-blocker (propranolol, esmolol, atenolol) to treat β-adrenergic effects, and cyproheptadine (per rectum) to combat serotoninergic effects. piperazine is a gaba agonist, and causes cervical and truncal ataxia, tremors, seizures, coma, and death. salt used for thawing ice commonly contains calcium chloride, a compound that has a moderate toxic potential. calcium chloride produces strong local irritation and can cause gastroenteritis and gastrointestinal ulcers if ingested. respiratory emergencies consist of any problem that impairs delivery of oxygen to the level of the alveoli or diffusion of oxygen across the alveolar capillary membrane into the pulmonary capillary network. decreased respiratory rate or tidal volume can result in hypoxia and buildup of carbon dioxide, or hypercarbia, leading to respiratory acidosis. conditions most frequently encountered result in airflow obstruction, prevention of normal lung expansion, interference with pulmonary gas exchange (ventilation-perfusion mismatch), and alterations of pulmonary circulation. evaluation of the patient with respiratory distress is often challenging, because the most minimal stress can cause rapid deterioration, or even death in critical cases. careful observation of the patient from a distance often allows the clinician to determine the severity of respiratory distress and localize the lesion based on the patient's respiratory pattern and effort. animals in respiratory distress often have a rapid respiratory rate (> breaths per minute). as respiratory distress progresses, the patient may appear anxious and start openmouth breathing. the animal often develops an orthopneic posture, characterized by neck extension, open-mouthed breathing, and elbows abducted or pulled away from the body. cyanosis of the mucous membranes often indicates extreme decompensation. clinical signs of respiratory distress can develop acutely, or from decompensation of a more chronic problem that was preceded by a cough, noisy respirations, or exercise intolerance. localization of the cause of respiratory distress is essential to successful case management. in any patient with clinical signs of respiratory distress, the differential diagnosis should include primary pulmonary parenchymal disease, airway disease, thoracic cage disorders, congestive heart failure, dyshemoglobinemias (carbon monoxide, methemoglobin), and anemia. careful observation of the patient's respiratory pattern can aid in making a diagnosis of upper airway disease/obstruction, primary pulmonary parenchymal disease, pleural space disease, and abnormalities of the thoracic cage. it is often helpful to rest a hand on the patient and breathe along with the patient's effort, to confirm the periods of inhalation and exhalation. the pharynx, larynx, and extrathoracic trachea comprise the upper airway. obstructive lesions are associated with a marked inspiratory wheeze or stridor and slow deep inspiratory effort. auscultation of the larynx and trachea may reveal more subtle obstructions of normal air flow. stridor can usually be auscultated without the use of a stethoscope. lung sounds are usually normal. the neck should be carefully palpated for a mass lesion, tracheal collapse, and subcutaneous emphysema. subcutaneous emphysema suggests tracheal damage or collapse secondary to severe trauma. in some cases, there is a history of voice, or bark, change secondary to laryngeal dysfunction. differential diagnosis is usually based on the patient's signalment, history, and index of suspicion of a particular disease process. differential diagnoses of upper airway obstruction are listed in box - . diseases of the pleural space often are associated with a restrictive respiratory pattern. inspiratory efforts are short, rapid, and shallow, and there is often a marked abdominal push. the pattern has been referred to as a choppy "dysynchronous" respiratory pattern. depending on the disease present, lung sounds may be muffled ventrally and enhanced dorsally. percussion of the thorax reveals decreased resonance if fluid is present. increased resonance is present with pneumothorax. decreased compressibility of the anterior thorax may be present with an anterior mediastinal mass lesion, particularly in cats and ferrets. a pneumothorax or diaphragmatic hernia is commonly associated with evidence of trauma, with or without rib fractures. respiratory distress due to hemothorax may be exacerbated by anemia. differential diagnoses for patients with evidence of pleural cavity disease include pneumothorax, diaphragmatic hernia, neoplasia, and various types of pleural effusion. primary pulmonary parenchymal disease can involve the intrathoracic airways, alveoli, interstitial space, and pulmonary vasculature. a rapid, shallow, restrictive respiratory pattern may be observed with a marked push on exhalation, particularly with obstructive airway disease such as chronic bronchitis (asthma) in cats. crackles or wheezes are heard on thoracic auscultation. differential diagnoses for pulmonary parenchymal disease include cardiogenic and noncardiogenic pulmonary edema, pneumonia, feline bronchitis (asthma), pulmonary contusion, aspiration pneumonitis, pulmonary thromboembolism, neoplasia, infection (bacterial, fungal, protozoal, viral) , and/or chronic bronchitis. other abnormal respiratory patterns may be evident, and warrant further consideration. tachypnea present in the absence of other signs of respiratory distress can be a normal response to nonrespiratory problems, including pain, hyperthermia, and stress. a restrictive respiratory pattern with minimal thoracic excursions can be associated with diseases of neuromuscular function, including ascending polyradiculoneuritis, botulism, and tick paralysis. if adequate ventilation cannot be maintained by the patient, mechanical ventilation may be indicated. kussmaul respiration manifests as very slow, very deep respirations when a metabolic acidosis is present. this type of respiratory pattern typically is observed in patients with severe diabetic ketoacidosis and renal failure in a compensatory attempt to blow off carbon dioxide. cheyne-stokes respiration is usually observed with a defect in the central respiratory control center. the classic pattern of cheyne-stokes respiration is normal or hyperventilation followed by a period of apnea or hypoventilation. in cases of lower cervical cord damage or damage to the central respiratory control center in the cns, the diaphragm alone may assume most of the ventilatory movement. with diaphragmatic fatigue, severe hypoventilation and resultant hypoxemia may require mechanical ventilation. immediate management of any patient in respiratory distress is to minimize stress at all costs. relatively benign procedures such as radiography or intravenous catheter placement can be fatal in patients with severe respiratory compromise. stabilization should always precede further diagnostic evaluation. in some cases, sedation may be required before performing any diagnostics, to prevent further stress. all patients should receive some form of supplemental oxygen, either by mask, cage, or flow-by techniques. in cases in which a severe pneumothorax or pleural effusion is suspected, perform therapeutic and diagnostic thoracocentesis bilaterally to allow lung re-expansion and alleviate respiratory distress, whenever possible. if thoracocentesis alone is not effective at maintaining lung re-expansion, place a thoracostomy tube (particularly in cases of tension pneumothorax). if hypovolemic/ hemorrhagic shock is present, initiate treatment while stabilizing the respiratory system (see section on shock). if an animal is suspected of having an upper airway obstruction, reestablish airflow. in cases of laryngeal paralysis, tracheal collapse, and brachycephalic airway syndrome, sedation is often very useful in alleviating the distress of airway obstruction. in cases of laryngeal collapse, however, sedation may make the condition worse. if laryngeal edema is severe, administer a dose of short-acting glucocorticosteroids (dexamethasone sodium phosphate) to decrease laryngeal inflammation and edema. if a foreign body is lodged in the pharynx, perform the heimlich maneuver by thrusting bluntly several times on the patient's sternum. objects such as balls or bones may be small enough to enter the larynx but too large to be expelled, and will require rapid-acting general anesthesia to facilitate dislodgement and removal. if the obstruction cannot be removed, bypassing the obstruction with an endotracheal tube or temporary tracheostomy should be considered. in an emergency, a temporary transtracheal oxygen catheter can quickly be placed in the following manner. connect a -or -gauge needle to a length of intravenous extension tubing and a -ml syringe. place the male connector of the syringe into the female portion of the extension tubing. cut off the syringe plunger and connect the resulting blunt end to a length of flexible tubing attached to a humidified oxygen source. run the oxygen at l/minute to provide adequate oxygenation until a tracheostomy can be performed. (see sections on oxygen supplementation and tracheostomy). once the animal's condition has been stabilized, specific diagnostic tests, including arterial blood gas analyses, thoracic radiographs, and/or transtracheal wash, can be performed, depending on the patient's condition and needs. specific therapies for management of upper airway obstruction, pleural space disease, and pulmonary disease are discussed next. upper airway obstruction can occur as a result of intraluminal or extraluminal mass lesions or foreign bodies in the oropharynx (abscess, neoplasia), laryngeal paralysis, trauma, and anatomic abnormalities. clinical signs of an upper airway obstruction are associated with an animal's extreme efforts to inhale air past the obstruction. marked negative pressure occurs in the extrathoracic airways and can cause worsening of clinical signs. mucosal edema and inflammation further worsen the obstruction. therapy for upper airway obstruction is aimed at breaking the cycle of anxiety and respiratory distress. administer the anxiolytic tranquilizer acepromazine ( . - . mg/kg iv, im, sq) to decrease patient anxiety. many animals develop hyperthermia from increased respiratory effort and extreme anxiety. implement cooling measures in the form of cool intravenous fluids and wet towels soaked in tepid water placed over the animal (see section on hyperthermia). administer supplemental oxygen in a manner that is least stressful for the animal. short-acting glucocorticosteroids can also be administered (dexamethasone sodium phosphate, . mg/kg iv, sq, im) to decrease edema and inflammation. if the airway obstruction is severe and there is no response to initial measures to alleviate anxiety and decrease inflammation, establish control of ventilation by placement of an endotracheal tube (see section on endotracheal intubation), tracheal oxygen catheter, or temporary tracheostomy. to obtain airway control, administer a rapid-acting anesthetic (propofol, - mg/kg iv to effect), and intubate with a temporary tracheostomy. an intratracheal oxygen catheter can be placed with sedation and/or a local anesthetic (see technique for transtracheal wash). laryngeal paralysis is a congenital or acquired condition that occurs primarily in largebreed dogs secondary to denervation of the arytenoid cartilages by the recurrent laryngeal nerve. congenital laryngeal paralysis occurs in the bouvier des flandres, siberian husky, and bull terrier. acquired laryngeal paralysis occurs in labrador retrievers, saint bernards, and irish setters. acquired laryngeal paralysis can be idiopathic, acquired secondary to trauma to the recurrent laryngeal nerve, or can be a component of systemic neuromuscular disease. although rare, this condition also occurs in cats. with dysfunction of the recurrent laryngeal nerve, the intrinsic laryngeal muscles atrophy and degenerate. as a result, the vocal folds and arytenoid cartilage move in a paramedian position within the airway and fail to abduct during inhalation, causing airway obstruction. laryngeal paralysis can be partial or complete, unilateral or bilateral. in many cases, a change in bark is noted prior to the development of clinical signs of respiratory distress or exercise intolerance. when a patient presents with severe inspiratory stridor (with or without hyperthermia) initiate stabilization with anxiolytic tranquilizers, supplemental oxygen, and cooling measures. once the patient's condition has been stabilized, definitive measures to accurately document and assess the patient's airway should be considered. place the patient under very heavy sedation with short-acting barbiturates or propofol ( - mg/kg iv) and observe the arytenoid cartilages closely in all phases of respiration. administer just enough drug to allow careful examination without getting bitten. if the arytenoid cartilages do not abduct during inhalation, administer dopram (doxapram hydrochloride, - mg/kg iv) to stimulate respiration. absent or paradoxical laryngeal motion (closed during inspiration and open during exhalation) is characteristic of laryngeal paralysis. correction of the defect involves documentation and treatment of any underlying disorder and surgical repair of the area to open the airway. partial laryngectomy, arytenoid lateralization ("tie-back" surgery), or removal of the vocal folds has been used with some success. aspiration pneumonitis is common following these procedures. brachycephalic airway syndrome is associated with a series of anatomic abnormalities that collectively increase resistance to airflow. affected animals typically have stenotic nares, an elongated soft palate, and a hypoplastic trachea. components of the syndrome can occur alone or in combination. in severe cases, laryngeal saccular edema and eversion, and eventual pharyngeal collapse, can occur secondary to the severe increase in intrathoracic airway pressure required to overcome the resistance of the upper airways. specific airway anomalies can be identified with general anesthesia and laryngoscopy. severe respiratory distress should be treated as discussed previously. treatment requires surgical correction of the anatomic abnormalities. in animals with laryngeal collapse, surgical correction may not be possible, and a permanent tracheostomy may be required. because an elongated soft palate and stenotic nares can be identified before the onset of clinical signs, surgical correction to improve airflow when the animal is young may decrease the negative intra-thoracic pressure necessary to move air past these obstructions. the chronic consequences of everted laryngeal saccules and laryngeal collapse potentially can be prevented. tracheal collapse is common in middle-aged and older toy and small-breed dogs. the owner typically reports a chronic cough that is readily induced by excitement or palpation of the trachea. the cough often sounds like a "goose honk." diagnostic confirmation is obtained by lateral radiography or fluoroscopy of the cervical and thoracic trachea during all phases of respiration. acute decompensation is uncommon but does occur, particularly with excitement, exercise, and increased environmental temperatures or ambient humidity. therapy of the patient with acute respiratory distress secondary to tracheal collapse includes sedation, administration of supplemental oxygen, and provision of cooling measures to treat hyperthermia. cough suppressants (hydrocodone bitartrate-homatropine methylbromide, . mg/kg po q - h, or butorphanol, . mg/kg po q - h) are useful. tracheal collapse is a dynamic process that usually involves both the upper and lower airways. because of this, bypassing the obstruction is often difficult. tracheal stents have been emergency care used with limited success in combination with treatment of chronic lower airway disease. crush or bite injuries to the neck can result in fractures or avulsion of the laryngeal or tracheal cartilages. bypassing the obstructed area may be necessary until the patient is stable and can undergo surgical correction of the injury. if there is avulsion of the cranial trachea, it may be difficult to intubate the patient. a long, rigid urinary catheter can be inserted past the area of avulsion into the distal segment, and an endotracheal tube passed over the rigid catheter, to establish a secure airway. neck injury can also result in damage to the recurrent laryngeal nerve and laryngeal paralysis. foreign bodies can lodge in the nasal cavity, pharynx, larynx, and distal trachea. signs of foreign bodies in the nares include acute sneezing and pawing at or rubbing the muzzle on the ground. if the object is not removed, sneezing continues and a chronic nasal discharge develops. respiratory distress is uncommon, but the foreign body is severely irritating. pharyngeal and tracheal foreign bodies can cause severe obstruction to airflow and respiratory distress. diagnosis of a foreign body is based on the patient history, physical examination findings, and thoracic or cervical radiographs. smaller foreign bodies lodged in the distal airways may not be apparent radiographically but can cause pulmonary atelectasis. foreign bodies of the nose or pharynx can often be removed with an alligator forceps with the patient under anesthesia. if removal is not possible with a forceps, flushing the nasal cavity from cranial to caudal (pack the back of the mouth with gauze to prevent aspiration) can sometimes dislodge the foreign material into the gauze packing. rhinoscopy may be necessary. if an endoscope is not available, an otoscope can be used. foreign objects lodged in the trachea can be small and function like a ball valve during inhalation and exhalation, causing episodic hypoxia and collapse. when attempting to remove these objects, suspend the patient with its head down. remove the object with an alligator forceps, using a laryngoscope to aid in visualization. foreign bodies lodged in the trachea or bronchi require removal with endoscopic assistance. nasopharyngeal polyps (in cats, tumors, obstructive laryngitis, granulomas, abscesses, and cysts) can cause upper airway obstruction. clinical signs are usually gradual in onset. the lesions can be identified through careful laryngoscopic examination performed with the patient under general anesthesia. the nasopharynx above the soft palpate should always be included in the examination. pedunculated masses and cysts are excised at the time of evaluation. biopsy of diffusely infiltrative masses is indicated for histologic examination and prognosis. it is impossible to distinguish obstructive laryngitis from neoplasia based on gross appearance alone. whenever possible, material should be collected from abscesses and granulomas for cytologic evaluation and bacterial culture. extraluminal masses impinge on and slowly compress the upper airways, resulting in slow progression of clinical signs. masses are usually identified by palpation of the neck. enlarged mandibular lymph nodes, thyroid tumors, and other neoplasms may be present. diagnosis is usually based on a combination of radiography and ultrasonography. ct and/or mri are helpful in identifying the full extent and invasiveness of the lesion. definitive diagnosis is made with a fine-needle aspirate or biopsy. many thyroid tumors bleed excessively. the inside of each side of the hemithorax is covered in parietal pleura. the lung lobes are covered in visceral pleura. the two surfaces are in close contact with each other, and are contiguous at the hilum under normal circumstances. pneumothorax refers to free air within the pleural space, accumulating in between the parietal and visceral pleura. the term pleural effusion refers to fluid accumulation in that area but does not reflect the amount or type of fluid present. the mediastinal reflections of the pleura typically are thin in dogs and cats, and usually, but not always, connect. bilateral involvement of pneumothorax or pleural effusion is common. both pneumothorax and pleural effusion compromise the lungs' ability to expand and result in hypoxia and respiratory distress. pneumothorax can be classified as open versus closed, simple versus complicated, and tension. an open pneumothorax communicates with the external environment through a rent in the thoracic wall. a closed pneumothorax results from tears in the visceral pleura but does not communicate with the outside. a tension pneumothorax occurs as a result of a tear in the lung or chest wall that creates a flap valve, such that air is allowed to leave the lung and accumulate in the pleural space during inhalation, and closes to seal off exit of air from the pleural space during exhalation. tension pneumothorax can cause rapid decline in cardiopulmonary status and death if not recognized and treated immediately. a simple pneumothorax is one that can be controlled with a simple thoracocentesis. complicated pneumothorax involves repeated accumulation of air, requiring placement of a thoracic drainage catheter. in many cases, pneumothorax develops as a result of trauma. spontaneous pneumothorax occurs with rupture of cavitary lesions of the lung that may be congenital or acquired as a result of prior trauma, heartworm disease, airway disease (emphysema), paragonimiasis, neoplasia, or lung abscess. pneumothorax also rarely occurs as a result of esophageal tears or esophageal foreign bodies. rapid circulatory and respiratory compromise following traumatic pneumothorax can develop as a result of open or tension pneumothorax, rib fractures, airway obstruction, pulmonary contusions, hemothorax, cardiac dysrhythmias, cardiac tamponade, and hypovolemic shock. any patient that is rapidly decompensating after a traumatic episode must be quickly assessed, and emergency therapy initiated (see section on immediate management of trauma, a crash plan). diagnosis of pneumothorax is usually made based on a history of trauma, a rapid, shallow, restrictive respiratory pattern, and muffled heart and lung sounds on thoracic auscultation. the clinical signs and history alone should prompt the clinician to perform a bilateral diagnostic and therapeutic thoracocentesis before taking thoracic radiographs (see section on thoracocentesis). the stress of handling the patient for radiography can be deadly in severe cases of pneumothorax. although the mediastinum on both sides of the thorax connects, it is necessary to perform thoracocentesis on both sides to ensure maximal removal of free air in the pleural space and allow maximal lung expansion. if negative pressure cannot be obtained, or if the patient rapidly reaccumulates air, place a thoracostomy tube connected to continuous suction. (see section on thoracostomy tube placement). treat all penetrating wounds to the thorax as open sucking chest wounds unless proved otherwise. to "close" an open sucking chest wound, clip the fur around the wound as quickly as possible, and place sterile lubricant jelly or antimicrobial ointment circumferentially around the wound. cut a sterile glove to provide a covering. place the covering over the wound, making sure to cover all of the sterile lubricant, thus creating a seal to close the wound temporarily from the external environment. evaluate the patient's thorax via thoracocentesis while placing a thoracostomy tube. once the patient is stable, the open chest wound can be surgically explored, lavaged, and definitively corrected. all animals with open chest wounds should receive antibiotics (first-generation cephalosporin) to prevent infection. following stabilization, radiographs can be taken and evaluated. pneumothorax is confirmed by evidence of elevation of the cardiac silhouette above the sternum, increased density of the pulmonary parenchymal tissue, free air in between the parietal and visceral pleura (making the outline of the lungs visible), and absence of pulmonary vascular structures in the periphery. parenchymal lesions within the lungs are best identified after as much air as possible has been removed from the thorax. obtain left and right lateral and ventrodorsal or dorsoventral views. a standing lateral view may reveal air-or fluid-filled cavitary masses. if underlying pulmonary disease is suspected as a cause of spontaneous pneumothorax, a transtracheal wash, fecal flotation, and heartworm test may be indicated. treatment of pneumothorax includes immediate bilateral thoracocentesis, covering of any open chest wounds, administration of supplemental oxygen, and placement of a thoracostomy tube if negative pressure cannot be obtained or if air rapidly reaccumulates. serial radiography, ct, or mri should be performed in dogs with spontaneous pneumothorax, because the condition can be associated with generalized pulmonary parenchymal disease. strict cage rest is required until air stops accumulating and the thoracostomy tube can be removed. the patient's chest tube should be aspirated every hours after discontinuing continuous suction. if no air reaccumulates after hours, the chest tube can be removed. exercise restriction is indicated for a minimum of week. if bullae or mass lesions are present, exploratory thoracotomy should be considered as a diagnostic and potentially therapeutic option for long-term management in prevention of recurrence. pleural fluid cytologic analysis is indicated for all patients with pleural effusion before administration of antibiotics. the general term pleural effusion means a collection of fluid in the space between the parietal and visceral pleura but does not indicate what kind or how much fluid is present. clinical signs associated with pleural effusion depend on how much fluid is present, and how rapidly the fluid has accumulated. clinical signs associated with pleural effusion include respiratory distress, reluctance to lie down, labored breathing with an abdominal component on exhalation, cough, and lethargy. auscultation of the thorax may reveal muffled heart and lung sounds ventrally and increased lung sounds dorsally, although pockets of fluid may be present, depending on the chronicity of the effusion. percussion of the thorax may reveal decreased resonance. in stable patients, the presence of pleural effusion can be confirmed radiographically. radiographic confirmation of the pleural effusion should include right and left lateral and dorsoventral or ventrodorsal views. a handling or standing lateral view should be obtained if an anterior mediastinal mass is suspected. the standing lateral view will allow the fluid to collect in the costophrenic recess. in patients with respiratory distress, muffled heart and lung sounds, and suspicion of pleural effusion, thoracocentesis should be performed immediately. thoracocentesis can be both therapeutic and diagnostic. radiography is contraindicated because the procedure can cause undue stress and exacerbation of clinical signs in an unstable patient. pleural effusion can cause severe respiratory distress, and can be the result of a number of factors that must be considered when implementing an appropriate treatment plan. pathology of the pleura is almost always a secondary process except for primary bacterial pleuritis and pleural mesotheliomas. causes of pleural effusion in the cat and dog include pyothorax, feline infectious peritonitis, congestive heart failure, chylothorax, heartworm disease, hemothorax, hypoalbuminemia, lung lobe torsions, neoplasia, diaphragmatic hernia, and pancreatitis (box - ). in stable animals, diagnosis of pleural effusion can be made based • imbalance of transpleural or hydrostatic or protein osmotic forces • change in membrane permeability • decrease in rate of fluid reabsorption • combination of foregoing mechanisms on thoracic radiography or ultrasound. thoracic radiographs can show whether the pleural effusion is unilateral or bilateral. effusions in dogs and cats are usually bilateral. the lung parenchyma and the cardiac silhouette cannot be fully evaluated until most of the fluid has been evacuated from the pleural cavity. following thoracocentesis, radiography should be performed with left and right lateral and ventrodorsal or dorsoventral views. in cases of suspected heart failure, echocardiography also is necessary. pleural fluid cytologic analysis is indicated for all patients with pleural effusion. collect specimens before administering antibiotics, whenever possible, because treatment with antibiotics can make a septic condition (pyothorax) appear nonseptic. the remainder of the diagnostic workup and treatment is based on the type of fluid present (table - ). the fluid may be a transudate, nonseptic exudate, septic exudate, chylous, hemorrhagic, or neoplastic. ultrasonographic evaluation of the thorax can be helpful in identifying intrathoracic masses, diaphragmatic hernias, lung lobe torsions, and cardiac abnormalities. unlike radiography, ultrasonography is facilitated by the presence of fluid in the pleural space. pyothorax refers to a septic effusion of the pleural cavity. the infection is generally the result of a combination of aerobic and anaerobic bacteria. rarely, fungal organisms are present. the source of the underlying organisms is rarely identified, particularly in cats, but can be caused by penetrating wounds through the chest wall, esophagus, migrating foreign bodies (especially grass awns), or primary lung infections. the most common organisms associated with pyothorax in the cat are pasteurella, bacteroides, and fusobacterium. fever is often present in addition to clinical signs of pleural effusion. septic shock is ununcommon. diagnosis of pyothorax is made based on cytologic analysis and the demonstration of intracellular and extracellular bacteria, toxin neutrophils and macrophages, and sometimes the presence of sulfur granules. gram stains of the fluid can assist in the initial identification of some organisms. bacterial cultures are indicated for bacteria identification and antibiotic susceptibility testing. administration of antibiotics before cytologic evaluation can cause a septic effusion to appear nonseptic. emergency treatment for pyothorax involves placement of an intravenous catheter, intravenous fluids to treat hypovolemic shock, and broad-spectrum antibiotics (ampicillin, mg/kg iv q h, and enrofloxacin, mg/kg iv q h). chloramphenicol also is an appropriate antibiotic to use for penetration into pockets of fluid. administration of a beta-lactam antibiotic (ampicillin or amoxicillin) with a beta-lactamase inhibitor (amoxicillin clavulanate or ampicillin sulbactam) is helpful in achieving better coverage of bacteroides spp. treatment of pyothorax differs in the cat and dog. in the cat, placement of one or two thoracic drainage catheters is recommended to allow continuous drainage of the intrathoracic abscess. inadequate drainage can result in treatment failure. fluid should be evaluated and the pleural cavity lavaged with ml/kg of warmed . % saline or lactated ringer's solution every hours. approximately % of the infused volume should be recovered after each lavage. in dogs, or in cats with refractory pyothorax, perform an exploratory thoracotomy to remove any nidus of infection. rarely a foreign body is visible that can be removed at the time of surgery, but this finding is rare. antibiotics are indicated for a minimum of to weeks after removal of the thoracostomy tube. early diagnosis and aggressive treatment result in a good prognosis in the majority of patients with pyothorax. in cats, clinical signs of ptyalism and hypothermia at the time of presentation worsen the prognosis. chylothorax refers to the abnormal accumulation of chyle (lymphatic fluid) in the pleural cavity. the cisterna chili is the dilated collection pool of lymphatic ducts in the abdomen that accumulate chyle prior to entry into the thoracic duct located within the thoracic cavity. the thoracic duct enters the thorax at the aortic hiatus. numerous tributaries or collateral ducts exist. the functions of the lymphatic vessels collectively serve to deliver triglycerides and fat-soluble vitamins into the peripheral vascular circulation. damage of the thoracic duct or lymphatic system or obstruction to lymphatic flow can result in the development of chylous effusion in the pleural or peritoneal space. it is difficult to identify chylous effusions based on their milky appearance alone. to identify a chylous effusion versus a pseudochylous effusion, the triglyceride and cholesterol levels of the fluid must be compared with those of peripheral blood. chylous effusions have a higher triglyceride and lower cholesterol levels than peripheral blood. pseudochylous effusions have a higher cholesterol and lower triglyceride levels than peripheral blood. disease processes that can result in chylous effusions are listed in the box - . clinical signs associated with chylous effusion are typical of any pleural effusion and of the disease process that caused the effusion. weight loss may be evident, depending on the chronicity of the process. the diagnosis is made based on thoracocentesis, cytology, and biochemical evaluation of the fluid (i.e., triglyceride and cholesterol levels). the fluid often appears milky or bloodtinged but can be clear if the patient has significant anorexia. typical cytologic characteristics are listed in table - . lymphangiography can be used to confirm trauma to the thoracic duct, but this is usually not necessary unless surgical ligation is going to be attempted. the diagnostic evaluation must also attempt to identify an underlying cause. therapy for chylothorax is difficult and primarily involves documentation and treatment of the underlying cause. if an underlying cause is not found, treatment is largely supportive and consists of intermittent thoracocentesis to drain the fluid as it accumulates and causes respiratory dysfunction, nutritional support, and maintenance of fluid balance. a variety of surgical techniques, including ligation of the thoracic duct, pleural-peritoneal shunts, and pleurodesis, have been attempted but have had limited success. most recently, the combination of thoracic duct ligation with subtotal pericardectomy has been shown to improve surgical success rates in the treatment of chylothorax. rutin, a bioflavinoid, has been used with limited success in the treatment of idiopathic chylothorax in cats. prognosis in many cases of chylothorax is guarded. extensive hemorrhage into the pleural cavity can cause fulminant respiratory distress due to sudden hypovolemia and anemia and interference with lung expansion. hemothorax typically is associated with trauma, systemic coagulopathy, lung lobe torsions, and erosive lesions within the thorax (usually neoplasia). diagnosis of hemothorax involves obtaining a fluid sample via thoracocentesis. hemorrhagic effusion must be differentiated from systemic blood inadvertently collected during the thoracocentesis procedure. unless the hemorrhage is peracute, fluid in cases of hemothorax is rapidly defibrinated and will not clot, has a packed cell volume less than that of venous blood, contains rbcs and macrophages. hemorrhagic effusions also usually contain a disproportionately higher number of white blood cells compared with peripheral blood. hemothorax commonly is the sole clinical sign observed in animals with vitamin k antagonist rodenticide intoxication and systemic coagulopathy. whenever an animal presents with signs of a hemorrhagic pleural effusion, perform coagulation testing immediately to determine whether a coagulopathy exists. the prothrombin time test is fast and can be performed as a cage-side test (see section on coagulopathy). therapy for hemorrhagic pleural effusions should address the blood and fluid loss. administer intravenous crystalloid fluids and rbc products (see section on transfusion therapy). when necessary, administer coagulation factors in the form of fresh whole blood or fresh frozen plasma, along with vitamin k ( mg/kg sq in multiple sites with a -gauge needle). if severe respiratory distress is present, evacuate the blood within the pleural space via thoracocentesis until clinical signs of respiratory distress resolve. fluid that remains aids in the recovery of the patient, because rbcs and proteins eventually will be reabsorbed. autotransfusion can be performed to salvage blood and reinfuse it into the anemic patient. in cases of neoplastic or traumatic uncontrollable hemorrhagic effusions, surgical exploration of the thorax is warranted. diaphragmatic hernia, or a rent in the diaphragm, can result in the protrusion of abdominal organs into the thoracic cavity and impair pulmonary expansion. organs that are commonly herniated into the thorax include the liver, stomach, and small intestines. diaphragmatic hernia usually is secondary to trauma but can occur as a congenital anomaly. in cases of trauma, rib fractures, pulmonary contusions, traumatic myocarditis, hemothorax, and shock are also often present concurrently with diaphragmatic hernia. respiratory distress can be caused by any one or a combination of the above lesions. animals with prior or chronic diaphragmatic hernias may have minimal clinical signs despite the presence of abdominal organs within the thorax. clinical signs of acute or severe diaphragmatic hernia include respiratory distress, cyanosis, and shock. a diagnosis of diaphragmatic hernia is made based on the patient's history (traumatic event), clinical signs, and radiographs. in some cases, ultrasonography or contrast peritoneography is necessary to confirm the diagnosis. contrast radiographs may show the presence of the stomach or intestines within the thorax following oral administration of barium. never administer barium directly into the peritoneal cavity or in cases of suspected gastrointestinal rupture. treatment of a patient with a diaphragmatic hernia includes cardiovascular and respiratory system stabilization before attempting surgical repair of the diaphragm. if the stomach is within the thorax, or if the patient's respiratory distress cannot be alleviated with medical management alone, immediate surgery is necessary. if the respiratory distress is minimal and the stomach is not located within the thorax, surgery can be postponed until the patient is a more stable anesthetic candidate. at the time of surgery, the abdominal organs are replaced into the abdominal cavity, and the rent in the diaphragm is closed. air must be evacuated from the thorax following closure of the diaphragm. if chronic diaphragmatic hernia is repaired, the complication of reexpansion pulmonary edema can occur. cardiac injury is a common complication secondary to blunt thoracic trauma. in most cases, cardiac injury is manifested as arrhythmias, including multiple premature ventricular contractions, ventricular tachycardia, st segment depression or elevation secondary to myocardial hypoxemia, and atrial fibrillation (see section on cardiac emergencies). myocardial infarction and cardiac failure can occur. careful and repeated assessments of the patient's blood pressure and ecg tracing should be a part of any diagnostic work-up for a patient that has sustained blunt thoracic trauma. rib fractures are associated with localized pain and painful respiratory movements. radiographs are helpful to confirm the diagnosis. careful palpation may reveal crepitus and instability of the fractured ribs. common problems associated with rib fractures emergency care include pulmonary contusions, pericardial laceration, traumatic myocarditis, diaphragmatic hernia, and splenic laceration or rupture. a flail segment results from rib fractures of more than three adjacent ribs that produce a "floating segment" of the chest wall. the flail segment moves paradoxically with respiration-that is, it moves inward during inhalation and outward during exhalation. respiratory distress is associated with the pain caused by the fractures and the presence of traumatic underlying pulmonary pathology. therapy for rib fractures and flail chest includes administration of supplemental oxygen, treatment of pneumothorax or diaphragmatic hernia, and administration of systemic and local anesthesia to alleviate the discomfort associated with the fractures. although controversial, positioning the patient with the flail segment up may reduce pain and improve ventilation. avoid the use of chest wraps, which do nothing to stabilize the flail segment and can further impair respiratory excursions. following administration of a systemic analgesic, administer a local anesthetic at the dorsocaudal and ventrocaudal segment of each fractured rib, and in one rib in front of and behind the flail segment. often, pulmonary function will improve once the pain associated with rib fractures has been adequately treated. in rare cases in which the flail segment involves five or more ribs, surgical stabilization may be necessary. single rib fractures or smaller flail segments are allowed to heal on their own. feline bronchitis has a variety of names (bronchial asthma, asthma, acute bronchitis, allergic bronchitis, chronic asthmatic bronchitis, feline lower airway disease) and refers to the acute onset of respiratory distress secondary to narrowing of the bronchi. cats may present with an acute onset of severe restrictive respiratory pattern associated with lower airway obstruction. acute bronchitis in cats typically has an inflammatory component in the lower airways, resulting in acute bronchoconstriction, excessive mucus production, and inflammatory exudates. in cats with chronic bronchitis, there may be damage of the bronchial epithelium and fibrosis of the airways. these patients often have a history if intermittent exacerbation of clinical signs, intermittent cough, and periods of normality throughout the year. because there appears to be an allergic or inflammatory component in feline bronchitis, clinical signs can be acutely exacerbated by stress and the presence of aerosolized particles such as perfume, smoke, and carpet powders. causes of feline bronchitis include heartworm disease, parasitic infestation (lungworms), and (rarely) bacterial infection. on presentation, the patient should be placed in an oxygen cage and allowed to rest while being observed from a distance. postpone performing stressful diagnostic procedures until the patient's respiratory status has been stabilized. after careful thoracic auscultation, administer a short-acting bronchodilator (terbutaline, . mg/kg sq or im) along with a glucocorticosteroid (dexamethasone sodium phosphate mg/kg im, sq, iv) to alleviate immediate bronchospasm and airway inflammation. clinical signs of feline bronchitis are characterized by a short, rapid respiratory pattern with prolonged expiration with an abdominal push. wheezes may be heard on thoracic auscultation. in some cases, no abnormalities are found on auscultation, but become acutely worse when the patient is stimulated to cough by tracheal palpation. radiographs may reveal a hyperinflated lung field with bronchial markings and caudal displacement of the diaphragm. in some cases, consolidation of the right middle lung lobe is present. a complete blood count and serum biochemistry profile can be performed, but results usually are unrewarding. in endemic areas, a heartworm test is warranted. fecal examination by flotation and the baermann technique is helpful in ruling out lungworms and other parasites. bronchoalveolar lavage or transtracheal wash is useful for cytologic and bacterial examination. long-term management of feline bronchitis includes isolation from environmental exposure to potential allergens (litter dust, perfumes, smoke, incense, carpet powders) and treatment of bronchoconstriction and inflammation with a combination of oral and inhaled glucocorticosteroids and bronchodilators (table - ). antibiotic therapy is contraindicated unless a pure culture of a pathogen is documented. oral therapy with steroids and bronchodilators should be used for a minimum of weeks after an acute exacerbation and then gradually decreased to the lowest dose possible to alleviate clinical signs. metered dose inhalers are now available (aerokat.com) for administration of inhaled bronchodilators and steroids. fluticasone (flovent, mcg/puff ) can be administered initially every hours for week and then decreased to once daily, in most cases. inhaled glucocorticosteroids are not absorbed systemically, and therefore patients do not develop the adverse side effects sometimes documented with oral glucocorticosteroid administration. because it takes time for glucocorticosteroids to reach peak effects in the lungs, administration of inhaled glucocorticosteroids should overlap with oral prednisolone administration for to days. treatment of pulmonary contusions is supportive. administer supplemental oxygen in a manner that is least stressful for the animal. arterial blood gas analysis or pulse oximetry can determine the degree of hypoxemia and monitor the response to therapy. intravenous fluids should be administered with caution to avoid exacerbating pulmonary hemorrhage or fluid accumulation in the alveoli. treat other conditions associated with the traumatic event. possible complications of pulmonary contusions are rare but include bacterial infection, abscessation, lung lobe consolidation, and the development of cavitary lesions. the routine use of antibiotics or steroids in cases of pulmonary contusions is contraindicated unless external wounds are present. empiric antibiotic use without evidence of external injury or known infection can potentially increase the risk of a resistant bacterial infection. steroids have been shown to decrease pulmonary alveolar macrophage function and impair wound healing and are contraindicated. aspiration pneumonia can occur in animals as a result of abnormal laryngeal or pharyngeal protective mechanisms or can be secondary to vomiting during states of altered mentation, including anesthesia, recovery from anesthesia, and sleep. megaesophagus, systemic polyneuropathy, myasthenia gravis, and localized oropharyngeal defects such as cleft palate can increase the risk of developing aspiration pneumonitis. iatrogenic causes of aspiration pneumonia include improper placement of nasogastric feeding tubes, overly aggressive force-feeding, and oral administration of drugs. aspiration of contents into the airways can cause mechanical airway obstruction, bronchoconstriction, chemical damage to the alveoli, and infection. severe inflammation and airway edema are common. pulmonary hemorrhage and necrosis can occur. diagnosis of aspiration pneumonia is based on clinical signs of pulmonary parenchymal disease, a history consistent with vomiting or other predisposing causes, and thoracic radiographs demonstrating a bronchointerstitial to alveolar pulmonary infiltrate. the most common site is the right middle lung lobe, although the pneumonia can occur anywhere, depending on the position of the patient at the time of aspiration. a transtracheal wash or bronchoalveolar lavage is useful for bacterial culture and susceptibility testing. treatment of aspiration pneumonia includes antibiotic therapy for the infection, administration of supplemental oxygen, and loosening the debris in the airways. administer intravenous fluids to maintain hydration. nebulization with sterile saline and chest physiotherapy (coupage) should be performed at least every hours. antibiotics to consider in the treatment of aspiration pneumonia include ampicillin/enrofloxacin, amoxicillinclavulanate, ampicillin-sulbactam, trimethoprim sulfa, and chloramphenicol. the use of glucocorticosteroids is absolutely contraindicated. continue antibiotic therapy for a minimum of weeks after the resolution of radiographic signs of pneumonia. pulmonary edema arises from the accumulation of fluid in the pulmonary interstitial alveolar spaces, and airways. ventilation-perfusion abnormalities result in hypoxia. pulmonary edema can be caused by increased pulmonary vasculature hydrostatic pressure, decreased pulmonary oncotic pressure, obstruction of lymphatic drainage, or increased capillary permeability. multiple factors can occur simultaneously. the most common cause of edema is increased pulmonary hydrostatic pressure resulting from left-sided congestive heart failure. decreased plasma oncotic pressure with albumin < . g/dl can also result in accumulation of fluid in the pulmonary parenchyma. overzealous intravenous crystalloid fluid administration can result in dilution of serum oncotic pressure and vascular overload. obstruction of lymphatic drainage is usually caused by neoplasia. other causes of pulmonary edema include pulmonary thromboembolic disease, severe upper airway obstruction (noncardiogenic pulmonary edema), seizures, and head trauma. increased capillary permeability is associated with a variety of diseases that cause severe inflammation (systemic inflammatory response syndrome). the resultant pulmonary edema contains a high amount of protein and is known as acute respiratory distress syndrome (ards). ards can be associated with pulmonary or extrapulmonary causes, including direct lung injury from trauma, aspiration pneumonia, sepsis, pancreatitis, smoke inhalation, oxygen toxicity, electrocution, and immune-mediated hemolytic anemia with disseminated intravascular coagulation. diagnosis of pulmonary edema is made based on clinical signs of respiratory distress and the presence of crackles on thoracic auscultation. in severe cases, cyanosis and fulminant blood-tinged frothy edema fluid may be present in the mouth and nostrils. immediate management includes administration of furosemide ( - mg/kg iv, im) and supplemental oxygen. sedation with low-dose morphine sulfate ( . - . mg/kg iv) is helpful in dilating the splanchnic capacitance vasculature and relieving anxiety for the patient. if fluid overload is suspected secondary to intravenous fluid administration, fluids should be discontinued. severely hypoalbuminemic patients should receive concentrated human albumin ( ml/kg of a % solution) or fresh frozen plasma. furosemide as a constant rate infusion ( . - . mg/kg/hour) also can dilate the pulmonary vasculature and decrease fluid accumulation in cases of ards. following initial stabilization of the patient, thoracic radiographs and an echocardiogram should be assessed to determine cardiac side, pulmonary vascular size, and cardiac contractility. further diagnostic testing may be required to determine other underlying causes of pulmonary edema. heart failure is managed with vasodilators, diuretics, oxygen, and sometimes positive inotropes. treatment ultimately consists of administration of supplemental oxygen, minimal stress and patient handling, and judicious use of diuretics. in cases of cardiogenic pulmonary edema, administer furosemide ( - mg/kg iv, im) every to minutes until the patient loses % of its body weight. positive inotropic and antiarrhythmic therapy may be necessary to improve cardiac contractility and control dysrhythmias. the clinician should determine whether the cause of the pulmonary edema is secondary to congestive heart failure with pulmonary vascular overload, volume overload, hypoalbuminemia, or increased permeability (ards). pulmonary edema secondary to ards typically is refractory to supplemental oxygen and diuretic therapy. in many cases, mechanical ventilation should be considered. a diagnosis of pulmonary thromboembolism (pte) is difficult to make and is based on clinical signs of respiratory distress consistent with pte, lack of other causes of hypoxemia, a high index of suspicion in susceptible animals, the presence of a condition associated with pte, and radiographic findings. virchow's triad consists of vascular endothelial injury, sluggish blood flow with increased vascular stasis, and a hypercoagulable state as predisposing factors for thromboembolic disease. clinical conditions that predispose an animal to pte include hyperadrenocorticism, disseminated intravascular coagulation (dic), catheterization of blood vessels, bacterial endocarditis, protein-losing nephropathy or enteropathy, hyperviscosity syndromes, heat-induced illness, pancreatitis, diabetes mellitus, inflammatory bowel disease, and immune-mediated hemolytic anemia. definitive diagnosis requires angiography or a lung perfusion scan. clinical signs associated with pte include an acute onset of tachypnea, tachycardia, orthopnea, and cyanosis. if the embolism is large, the patient may respond poorly to supplemental oxygen administration. pulmonary hypertension can cause a split second heart sound on cardiac auscultation. in some cases, a normal thoracic radiograph is present in the face of severe respiratory distress. this is a classic finding in cases of pte. potential radiographic abnormalities include dilated, tortuous, or blunted pulmonary arteries; wedge-shaped opacities in the lungs distal to an obstructed artery; and interstitial to alveolar infiltrates. the right heart may be enlarged. echocardiography can show right heart enlargement, tricuspid regurgitation, pulmonary hypertension, and evidence of underlying cardiac disease, possibly with clots in the atria. measurement of antithrombin (at) and d-dimer levels can be useful in the identification of hypercoagulable states, including dic. treatment of any patient with at deficiency or dic includes replenishment of at and clotting factors in the form of fresh frozen plasma. treatment of pte includes therapy for cardiovascular shock, oxygen supplementation, and thrombolytic therapy (see section on thromboembolic therapy). for short-term treatment, administer heparin (heparin sodium, - units/kg sq once, followed by units/kg q h of unfractionated heparin; or fractionated heparin). thrombolytic therapy may include tissue plasminogen activator, streptokinase, or urokinase. long-term therapy with low molecular weight heparin or warfarin may be required to prevent further thromboembolic events. ideally, management should include treatment and elimination of the underlying disease. smoke inhalation commonly occurs when an animal is trapped in a burning building. the most severe respiratory complications of smoke inhalation are seen in animals that are close enough to the flames to also sustain burn injuries (see section on burn injury). at the scene, many animals are unconscious from the effects of hypoxia, hypercapnia, carbon monoxide intoxication, and hydrogen cyanide gases that accumulate in a fire. carbon monoxide produces hypoxia by avidly binding to and displacing oxygen binding to hemoglobin, resulting in severe impairment of oxygen-carrying capacity. the percentage of carboxyhemoglobin in peripheral blood depends on the amount or carbon monoxide in inhaled gases and the length of time of exposure. clinical signs of carbon monoxide intoxication include cyanosis, nausea, vomiting, collapse, respiratory failure, loss of consciousness, and death. smoke inhalation of superheated particles also causes damage to the upper airways and respiratory tree. the larynx can become severely edematous and obstruct inspiration. emergency endotracheal intubation, tracheal oxygen, or tracheostomy tube may be required in the initial resuscitation of the patient, depending on the extent of airway edema. inhalation of noxious gases and particles can cause damage to the terminal respiratory bronchioles. specific noxious gases that can cause alveolar damage include combustible particles from plastic, rubber, and other synthetic products. pulmonary edema, bacterial infection, and ards can result. in any case of smoke inhalation, the first and foremost treatment is to get the animal away from the source of the flames and smoke and administer supplemental oxygen at the scene. at the time of presentation, carefully examine the animal's eyes, mouth, and oropharynx suction soot and debris from the mouth and upper airways. evaluate the patient's respiratory rate, rhythm, and pulmonary sounds. arterial blood gases should be analyzed with co-oximetry to evaluate the pao and carboxyhemoglobin concentrations. evaluation of sao by pulse oximetry is not accurate in cases of smoke inhalation, as the pao may appear normal, even when large quantities of carboxyhemoglobin are present. radiographs are helpful in determining the extent of pulmonary involvement, although radiographic signs may lag behind the appearance of clinical respiratory abnormalities by to hours. bronchoscopy and bronchoalveolar lavage provide a more thorough and accurate evaluation of the respiratory tree; however, these procedures should be performed only in patients whose cardiovascular and respiratory status is stable. management of the patient with smoke inhalation includes maintaining a patent airway, administration of supplemental oxygen, correction of hypoxemia and acid-base abnormalities, preventing infection, and treating thermal burns (see section on burn injury). if severe laryngeal edema is present, a temporary tracheostomy may be necessary to allow adequate oxygenation and ventilation. glucocorticosteroids should not be empirically used in the treatment of smoke inhalation, because of the risk of decreasing pulmonary alveolar macrophage function and increasing the potential for infection. in cases of severe laryngeal edema, however, glucocorticosteroids may be necessary to decrease edema and inflammation. the use of empiric antibiotics is contraindicated unless clinical signs of deterioration and bacterial pneumonia develop. epistaxis can be caused by facial trauma, a foreign body, bacterial or fungal rhinitis, neoplasia, coagulopathies, and systemic hypertension. acute, severe bilateral hemorrhage without wounds have been classified in several ways according their degree of tissue integrity, etiologic force, degree of contamination and duration, and degree of contamination and infection (table - ) . there are also unique causes of wounds such as burns, psychogenic dermatoses, frostbite, decubital ulcers, and snake bite. the animal should be transported to the nearest veterinary facility for definitive care. the wound should be covered or packed with dry gauze or clean linen to protect the wound, and to prevent further hemorrhage and contamination. if an open fracture is present, the limb should be splinted without placing the exposed bone back into the wound. replacing the exposed bone fragment back through the skin wound can cause further damage to underlying soft tissue structures and increase the degree of contamination of deeper tissues. if a spinal fracture is suspected, the patient should be transported on a stable flat surface to prevent further spinal mobilization and neurologic injury. at the time of presentation, first refer to the abcs of trauma, taking care to evaluate and stabilize the patient's cardiovascular and respiratory status. after a complete physical examination and history, ancillary diagnostic techniques can be performed if the patient is hemodynamically stable (see section on triage, assessment, and treatment of emergencies). initially, every patient with superficial wound should receive some degree of analgesia and an injection of a first-generation cephalosporin, preferably within hours of the injury. evaluate the wound after the patient's cardiovascular and respiratory status have been stabilized. always cover an open wound before taking an animal to the hospital to prevent a nosocomial infection. evaluate limb wounds for neural, vascular, and orthopedic abnormalities. carefully examine the structures deep to the superficial wounds. when there has been a delay in assessment of the wound, obtain samples for culture and antimicrobial susceptibility testing. if the wound is older and obviously infected, a gram stain can help guide appropriate antimicrobial therapy pending results of culture and susceptibility testing. place a support bandage saturated with a water-soluble antibiotic ointment or nonirritating antimicrobial solution (e.g., . % chlorhexidine, if bone or joint tissue is not exposed) around the wound. in addition to a first-generation cephalosporin, other appropriate antibiotic choices include amoxicillin-clavulanate, trimethoprim-sulfadiazine, amoxicillin, and ampicillin. if gram-negative flora are present, administer enrofloxacin. administer the antibiotics of choice for a minimum of days unless a change of antibiotic therapy is indicated. at the time of wound cleansing or definitive wound repair, the patient should be placed under general anesthesia with endotracheal intubation, unless the procedure will be brief (i.e., less than minutes). in such cases, a short-acting anesthetic combination open lacerations or skin loss closed crushing injuries and contusions etiologic force abrasion loss of epidermis and portions of dermis, usually caused by shearing between two compressive surfaces avulsion tearing of tissue from its attachment because of forces similar to those causing abrasion but of a greater magnitude incision wound created by a sharp object; wound edges are smooth and there is minimal trauma in the surrounding tissues laceration irregular wound caused by tearing of tissue with variable damage to the superficial and underlying tissue puncture penetrating wound caused by a missile or sharp object; superficial damage may be minimal; damage to deeper structures may be considerable; contamination by fur and bacteria with subsequent infection is common class i - hours with minimal contamination class ii - hours with significant contamination class iii > hours with gross contamination (analgesia + propofol, analgesia + ketamine/diazepam) can be administered to effect. heavy sedation with infiltration of a local anesthetic may also be appropriate for very small wounds, depending on the location of the wound and temperament of the patient. protect the wound by packing it with sterile gauze sponges soaked in sterile saline, or with watersoluble lubricating gel such as k-y jelly. clip the fur surrounding the wound, moving from the inner edge of the wound outward, to help prevent wound contamination with fur or other debris. scrub the wound and surrounding skin with an antimicrobial soap and solution such as dilute chlorhexidine until the area is free of all gross debris. gross debris within the wound itself can be flushed using a -ml syringe filled with sterile saline or lactated ringer's solution and an -gauge needle. pressure-lavage systems are also available for use, if desired. grossly contaminated wounds can be rinsed first with warm tap water to eliminate gross contamination, and then prepared as just described. debride the wound, removing skin and other soft tissue that is not obviously viable. obviously viable and questionable tissue should remain, and the wound left open for frequent reassessment on a daily basis. remove any dark or white segments of skin. questionable skin edges may or not regain viability and should be left in place for hours, so the wound can fully reveal itself. excise grossly contaminated areas of fat and underlying fascia. blood vessels that are actively bleeding should be ligated to control hemorrhage, if collateral circulation is present. if nerve bundles are ligated cleanly in a clean wound, the nerve edges should be reapposed and anastomosed. if gross contamination is present, however, definitive neurologic repair should be delayed until healthy tissue is present. excise contaminated muscle until healthy bleeding tissue is present. anastamoe tendon lacerations if the wound is clean and not grossly contaminated. if gross contamination is present, the tendon can be temporarily anastomosed and a splint placed on the limb until definitive repair of healthy tissue is possible. thoroughly lavage open wounds to a joint with sterile saline or lactated ringer's solution. infusion of chlorhexidine or povidone-iodine solution into the joint can cause a decrease in cartilage repair and is contraindicated. smooth sharp edges and remove any obvious fragments. whenever possible, the joint capsule and ligaments should be partially or completely closed. after removing bullets and metal fragments, the subcutaneous tissue and skin should be left open to heal by second intention, or should be partially closed with a drain. the joint should then be immobilized. injuries and exposed bone should be carefully lavaged, taking care to remove any gross debris without pushing the debris further into the bone and wound. the bone should be covered with a moist dressing and stabilized until definitive fracture repair can be made. this type of injury typically is seen with shearing injuries of the distal extremities caused by interaction with slow-moving vehicles. perform wet-to-dry or enzymatic debridement until a healthy granulation bed is present. if large areas of contamination are present (e.g., necrotizing fasciitis), en bloc debridement may be necessary. en bloc debridement consists of complete excision of badly infected wounds without entering the wound cavity, to prevent systemic infection. this technique should be used only if there is sufficient skin and soft tissue to allow later closure and it can be performed without damaging any major nerves, tendons, or blood vessels. open wounds often are managed by second intention healing, delayed primary closure, or secondary closure. see section on wound management and bandaging for a more complete discussion on the use of various bandaging materials in the treatment of open wounds. if an animal is presented very shortly after a wound has occurred and there is minimal contamination and trauma, the wound can be closed after induction of anesthesia and careful preparation of the wound and surrounding tissues. close any dead space under the skin with absorbable suture material in an interrupted suture pattern. avoid incising major blood vessels or nerves. close the subcutaneous tissues with absorbable suture material in an interrupted or continuous suture pattern. take care that there is not too much tension on the wound, or else surgical dehiscence will occur with patient movement. close the skin with nonabsorbable suture or surgical staples ( - to - ) . if there is any doubt at the time of repair about tissue status or inability to close all dead space, place a passive drain (penrose drain) so that the proximal end of the drain is anchored in the proximal aspect of the wound with a suture(s). leave the ends long so that the suture can be accurately identified at the time of drain removal. pass the suture through the skin, through the drain, and out the other side of the skin. place the rest of the drain into the wound and then secure it at the most ventral portion of the wound or exit hole in the most dependent area of the body, to allow drainage and prevent seroma formation. close the subcutaneous tissue over the drain before skin closure. during wound closure, be sure to not incorporate the subcutaneous or skin sutures into the drain, or it will not be possible to remove the drain without reopening the wound. bandage the area to prevent contamination. the drain can be removed once drainage is minimal (usually to days). active drains can be constructed or purchased; their use is indicated in wounds that are free of material that can plug the drain. to construct a small suction drain, remove the female portion or catheter hub at the end of a butterfly catheter. fenestrate the tubing so that there are multiple side holes, taking care to avoid making the holes larger than % of the circumference of the tubing. place the tubing into the wound via a small stab incision distal to the wound. use a purse-string suture around the tubing to facilitate a tight seal and prevent the tubing from exiting the wound. following wound closure, insert the butterfly needle into a -to -ml evacuated blood collection tube to allow fluid to drain into the tube. incorporate the tube into the bandage, and replace it when it becomes full. alternatively, the butterfly portion of the system can be removed and the tube fenestrated as described previously. place the tube into the wound and suture it in place to create a tight seal. secure the catheter hub to a syringe in which the plunger has been drawn back slightly to create suction. insert a metal pin or -to -gauge needle through the plunger at the top of the barrel to hold it at the desired level. incorporate the suction apparatus into the bandage and replace it when it becomes full. delayed primary closure should be considered when there is heavy contamination, purulent exudate, residual necrotic debris, skin tension, edema and erythema, and lymphangitis. delayed primary closure usually is made to days after the initial wound infliction and open wound management has been performed. once healthy tissue is observed, the skin edges should be debrided and the wound closed as with primary closure. secondary wound closure should be considered when infection and tissue trauma necessitate open wound management for more than days. secondary wound closure is performed after the development of a healthy granulation bed. this technique also is useful when a wound has dehisced and has formed granulation tissue. if the wound edges can be manipulated into apposition and if epithelialization has not begun, the wound can be cleansed and the wound edges apposed and sutured. this is known as early secondary closure. late secondary closure should be performed whenever there is a considerable amount of granulation tissue, the edges of the wound cannot be manipulated into position, and epithelialization has already started. in such cases, the wound should be cleaned, and the skin edges debrided to remove the epithelium. the remaining wound edges are then sutured over the granulation tissue ( shock is defined as a state of inadequate circulating volume and inability to meet cellular oxygen demands. there are three types of shock: hypovolemic, cardiogenic, and septic. early recognition of the type of shock present is crucial in the successful clinical management of shock syndrome. tissue oxygen delivery is based on cardiac output and arterial oxygen concentration. knowledge of the components of normal oxygen delivery is essential to the treatment of shock in the critical patient. improper handling of animal during further tissue and neurologic damage may occur transport (e.g., improper limb or spine immobilization). inadequate assessment of animal's animal's condition may worsen or animal may general condition or wounded tissues succumb; tissue injuries may be overlooked. inadequate wound protection during further wound contamination may occur at assessment, resuscitation, or veterinary facility. stabilization procedures inadequate wound protection while further wound contamination with fur and preparing the surrounding area debris may occur. insufficient wound lavage wound infection may occur. hydrogen peroxide wound lavage lavage offers little bactericidal activity and contributes to irritation of tissues and delayed healing. lavage has short residual activity and absorption with large wound. overly aggressive initial layered debridement may result in the removal of viable debridement tissue. en bloc debridement debridement results in removal of large amounts of tissue and a large defect for closure. use of drains potential exists for bacteria to ascend along the drain, for drain removal by the animal or breakage of the drain, and for possible tissue emphysema with air being sucked under the skin with patient movement. tube-type drains drains may cause postoperative discomfort; fenestrations may become occluded to stop intraluminal drainage. deeply placed sutures in the presence drain may be incorporated into the repair and of a drain prevent drain removal. active drains high negative pressure may cause tissue injury; highly productive wounds may necessitate changing the evacuated blood tubes several times a day with constructed drains. oxygen delivery (do ) = cardiac output (q) × arterial oxygen content (cao ) where q = heart rate × stroke volume. stroke volume is affected by preload, afterload, and cardiac contractility. where hb = hemoglobin concentration, sao = oxygen saturation, and pao = arterial partial pressure of oxygen in mm hg. thus, factors that can adversely affect oxygen delivery include inadequate preload or loss of circulating volume, severe peripheral vasoconstriction and increased afterload, depressed cardiac contractility, tachycardia and decreased diastolic filling, cardiac dysrhythmias, inadequate circulating hemoglobin, and inadequate oxygen saturation of hemoglobin. during septic shock, enzymatic dysfunction and decreased cellular uptake and utilization of oxygen also contribute to anaerobic glycolysis. an inadequate circulating volume may develop secondary to maldistribution of available blood volume (traumatic, septic, and cardiogenic origin) or as a result of absolute hypovolemia (whole blood or loss of extracellular fluid). normally, the animal compensates by ( ) splenic and vascular constriction to translocated blood from venous capacitance vessels to central arterial circulation, ( ) arteriolar constriction to help maintain diastolic blood pressure and tissue perfusion, and ( ) an increase in heart rate to help maintain cardiac output. arteriolar vasoconstrictions support perfusion to the brain and heart at the expense of other visceral organs. if vasoconstriction is severe enough to interfere with delivery of adequate tissue oxygen for a sufficient period of time, the animal may die. hypovolemic shock can result from acute hemorrhage or from severe fluid loss from vomiting, diarrhea, or third spacing of fluids. early in shock, baroreceptors in the carotid body and aortic arch sense a decrease in wall stretch from a decrease in circulating fluid volume. tonic inhibition of sympathetic tone via vagal stimulation is diminished, and heart rate and contractility increase and peripheral vessels constrict to compensate for the decrease in cardiac output. the compensatory mechanisms protect and support blood supply to the brain and heart at the expense of peripheral organ perfusion. this is called early compensatory shock. early compensatory shock is characterized by tachycardia, normal to fast capillary refill time, tachypnea, and normothermia. as shock progresses, the body loses its ability to compensate for ongoing fluid losses. early decompensatory shock is characterized by tachycardia, tachypnea, delayed capillary refill time, normotension to hypotension, and a fall in body temperature. end-stage decompensatory shock is characterized by bradycardia, markedly prolonged capillary refill time, hypothermia, and hypotension. aggressive treatment is necessary for any hope of a favorable outcome. septic shock should be considered in any patient with a known infection, recent instrumentation that could potentially introduce infection (indwelling intravenous or urinary catheter, surgery or penetrating injury), disorders or medical therapy that can compromise immune function (diabetes mellitus, immunodeficiency virus, parvovirus or feline panleukopenia virus infection, stress, malnutrition, glucocorticoids, chemotherapy). the presence of bacteria, viruses or rickettsiae, protozoa, or fungal organisms in the blood constitutes septicemia. septic shock is characterized by the presence of sepsis and refractory hypotension that is unresponsive to standard aggressive fluid therapy and inotropic or pressor support. septic shock and other causes of inflammation can lead to systemic inflammatory response syndrome (sirs). in animals, the presence of two or more of the criteria in table - in the presence of suspected inflammation or sepsis constitutes sirs (table - ). clinical signs associated with sepsis may be vague and nonspecific, including weakness, lethargy, vomiting, and diarrhea. cough and pulmonary crackles may be associated with pneumonia. decreased lung sounds may be associated with pyothorax. abdominal pain and fluid may be associated with septic peritonitis. vaginal discharge may or may not be present in patients with pyometra. diagnostic tests should include a white blood cell count, serum biochemical profile, coagulation tests, thoracic and abdominal radiographs, and urinalysis. the white blood cell count in a septic patient that is appropriately responding to the infection will be elevated with a left-shifted neutrophilia and leukocytosis. a degenerative left shift, in which leukopenia with elevated band neutrophils suggests an overwhelming infection. biochemical analyses may demonstrate hypoglycemia and nonspecific hepatocellular and cholestatic enzyme elevations. in the most severe cases, metabolic (lactic) acidosis, coagulopathies, and end-organ failure, including anuria and ards, may be present. cardiogenic shock occurs as a result of cardiac output inadequate to meet cellular oxygen demands. cardiogenic shock is associated with primary cardiomyopathies, cardiac dysrhythmias, pericardial fluid, and pericardial fibrosis. abnormalities seen on physical examination often are similar to those seen in other categories of shock, but they can also include cardiac murmurs, dysrhythmias, pulmonary rales, bloody frothy pulmonary edema fluid from the nares or mouth, orthopnea, and cyanosis. it is important to distinguish the primary cause of shock before implementing treatment (table - ) , whenever possible, because treatment for a suspected ruptured hemangiosarcoma differs markedly from the treatment for end-stage dilatative cardiomyopathy. the patient's clinical signs may be similar and include a peritoneal fluid wave, but the treatment for hypovolemia can dramatically worsen the congestive heart failure secondary to dilatative cardiomyopathy. when a patient presents with some form of shock, immediate vascular access is of paramount importance. place a large-bore peripheral or central venous catheter for the infusion of crystalloid or colloid fluids, blood component therapy, and drugs. monitor the patient's cardiopulmonary status (by ecg), blood pressure, oxygen saturation (as determined by pulse oximetry or arterial blood gas analyses), hematocrit, bun, and glucose. ancillary diagnostics, including thoracic and abdominal radiography, urinalysis, serum biochemistry profile, coagulation tests, complete blood count, abdominal ultrasound, and echocardiography, should be performed as determined by the individual patient's needs and the type of shock. the following list, called the "rule of twenty," is a guideline for case management of the shock patient. consideration of each aspect of the rule of twenty on a daily basis ensures temperature < °f or > . °f < °f or > . °f heart rate > beats/minute in dogs < or > beats/minute in cats respiratory rate > breaths/minute or paco > breaths/minute or paco < mm hg < mm hg white blood cell > , cells/µl , cells/µl count or < cells/µl o r < cells/ml or > % bands or > % bands that major organ systems are not overlooked. the list also provides a means to integrate and relate changes in different organ systems functions with one another.* the treatment of hypovolemic and septic shock requires the placement of large-bore intravenous catheters in peripheral and central veins. if vascular access cannot be obtained percutaneously or by cutdown methods, intraosseous catheterization should be considered. once vascular access is achieved, rapidly administer large volumes of crystalloid or colloid fluids. as a rule of thumb, administer / of a calculated shock dose of fluids-that is, / × ( ml/kg/hour) in dogs and / × ( ml/kg/hour) in cats) of a balanced crystalloid fluid ( normosol-r, plasmalyte-m, lactated ringer's solution, or . % sterile saline). reassess the patient's perfusion parameters (heart rate, capillary refill time, blood pressure, urine output) on a continual basis to direct further fluid therapy. synthetic colloid fluids (hetastarch, dextran , or oxyglobin) can also be administered in the initial resuscitation from shock. a guideline is to administer to ml/kg of hetastarch or dextran as a bolus over to minutes and then reassess perfusion parameters. hypertonic saline ( . % nacl, ml/kg) can be used in cases of hemorrhagic shock to temporarily restore intravascular fluid volume by drawing fluid from the interstitial space. because this type of fluid resuscitation is short-lived, hypertonic saline should always be used with another crystalloid or colloid fluid, and it should not be used in patients with interstitial dehydration. if hemorrhagic shock is present, the goal should be to return a patient's blood pressure to normal (not supraphysiologic) levels (i.e., systolic pressure - mm hg, diastolic pressure > mm hg, and mean arterial pressure ≥ mm hg) to avoid iatrogenically causing clots to fall off and hemorrhage to re-start. in critically ill patients, fluid loss can be measured in the form of urine, vomit, diarrhea, body cavity effusions, and wound exudates. additionally, insensible losses (those that cannot be readily measured from sweat, panting, and cellular metabolism) constitute ml/kg/ day. measurement of fluid "ins and outs" in conjunction with the patient's central venous pressure, hematocrit, albumin, and colloid oncotic pressure can help guide fluid therapy (see also section on fluid therapy). maintenance of normotension is necessary for adequate oxygen delivery to meet cellular energy demands. blood pressure can be measured using direct arterial catheterization, or through indirect means such as doppler plesthymography or oscillometric methods. the systolic pressure should remain at or greater than - mm hg at all times. the diastolic pressure is very important, too, as it constitutes two thirds of the mean arterial pressure; it must be greater than mm hg for coronary artery perfusion. the mean arterial pressure should be greater than mm hg for adequate tissue perfusion. if fluid resuscitation and pain management are not adequate in restoring blood pressure to normal, vasoactive drugs including positive inotropes and pressors should be considered (table - ). in cases of cardiogenic shock, vasodilator drugs (table - ) can be used to decrease vascular resistance and afterload. low-dose morphine ( . mg/kg, iv, im) dilates splanchnic vessels and helps reduce pulmonary edema. furosemide ( mg/kg/hour) also can dilate pulmonary vasculature and potentially reduce edema fluid formation in cases of ards. cardiac output is a function of both heart rate and stroke volume. stroke volume or (the amount of blood that the ventricle pumps in minute) is affected by preload, afterload, and contractility. during hypovolemic shock, there is a fall in cardiac preload due to a decrease in circulating blood volume. during septic and cardiogenic shock, there is a decrease in contractility secondary to inherent defects of the myocardium or due to the negative inotropic effects of inflammatory cytokines such as tnf-alpha, myocardial depressant factor, il- , and il- released during sepsis and systemic inflammation. afterload also may be increased because of the compensatory mechanisms and neurohumoral activation of the renin-angiotensin-aldosterone axis in hypovolemic or cardiogenic shock. as heart rate increases to compensate for a decline in cardiac output, myocardial oxygen demand increases and diastolic filling time becomes shorter. because the coronary arteries are perfused during diastole, coronary perfusion can be impaired, and myocardial lactic acidosis can develop, causing a further decline in contractility. in addition to lactic acidosis, acid-base and electrolyte abnormalities, inflammatory cytokines, direct bruising of the myocardium from trauma, and areas of ischemia can further predispose the patient to ventricular or atrial dysrhythmias. cardiac dysrhythmias should be controlled whenever possible. treatment of bradycardia should be directed at treating the underlying cause. administer anticholinergic drugs such as atropine ( . mg/kg im) or glycopyrrolate ( . mg/kg im) as necessary. in cases of third-degree or complete atrioventricular (av) block, administer a pure betaagonist such as isoproterenol ( . - . µg/kg/minute iv cri, or . mg in ml of % dextrose in water iv slowly). perform passive rewarming if the patient is hypothermic. receptor activity dosage (iv) dopamine da , da , α +++ , - µg/kg/minute (blood pressure support)* β +++ - µg/kg/minute (renal afferent diuresis) dobutamine α + , β +++ - µg/kg/minute* (blood pressure support, positive inotrope) norepinephrine α +++ , β + . - . mg/kg/minute; . - . mg/kg phenylephrine α +++ , β . - . mg/kg epinephrine α +++ , β +++ . - . mg/kg, . - . mg/kg/minute +++, strong receptor activity; , no receptor activity; +, weak receptor activity. *monitor for tachyarrhythmias at higher doses. correct any underlying electrolyte abnormalities such as hyperkalemia and hypo-and hypermagnesemia. treat ventricular dysrhythmias such as multifocal premature ventricular contractions (pvcs), sustained ventricular tachycardia > beats per minute, and r on t phenomenon (the t wave of the preceding beat occurs superimposed on the qrs complex of the next beat, and there is no return to isoelectric shelf), or if runs of ventricular tachycardia cause a drop in blood pressure. intravenous lidocaine and procainamide are the first drugs of choice for ventricular dysrhythmias. supraventricular tachycardia can impair cardiac output by impairing diastolic filling time. control supraventricular dysrhythmias with calcium channel blockers, beta-adrenergic blockers, or quinidine (table - ) . (disorientation); is minute; minutes) light sensitive and must be covered in foil and not kept for longer than hours albumin can decrease as a result of loss from the gastrointestinal tract, urinary system, and wound exudates, or into body cavity effusions. albumin synthesis can decrease during various forms of shock due to a preferential increase in hepatic acute phase protein synthesis. serum albumin contributes % of the colloid oncotic pressure of blood, in addition to its important roles as a free radical scavenger at sites of inflammation and as a drug and hormone carrier. albumin levels < . g/dl have been associated with an increase in morbidity and mortality in human and veterinary patients. administer fresh frozen plasma ( ml/kg) or concentrated human albumin ( ml/kg of % solution) to maintain serum albumin ≥ . g/dl. additional oncotic support can be in the form of synthetic colloids, as indicated. colloid oncotic pressure within the intravascular and interstitial spaces contributes to fluid flux. oncotic pressure can be measured with a colloid osmometer. normal oncotic pressure is mm hg. in cases of sepsis and sirs, increased vascular permeability increases the tendency for leakage of fluids into the interstitial spaces. colloids that can be administered until the source of albumin loss resolves include the synthetic colloids hetastarch and dextran ( - ml/kg/day), synthetic hemoglobin-based oxygen carriers (oxyglobin, - ml/kg/day), concentrated human albumin ( % albumin, ml/kg), and plasma ( ml/kg). oxygenation and ventilation can be evaluated by arterial blood gas analysis or by the noninvasive means of pulse oximetry and capnometry (see sections on pulse oximetry and capnometry). oxygen delivery can be impaired in cases of hypovolemic shock because of hemorrhage and anemia, and thus a decrease in functional capacity to carry oxygen, and is not to be used for more than weeks due to idiosyncratic blindness. in cases of cardiogenic shock as a result of impaired ability to saturate hemoglobin due to pulmonary edema in the lungs, or decrease in cardiac output. in septic shock, decreases in cardiac output due to inflammatory cytokines and a decrease in cellular oxygen extraction can lead to lactic acidosis. increased cellular metabolism and decreases in respiratory function can lead to respiratory acidosis as co increases. administer supplemental oxygen as flow-by, nasal or nasopharyngeal catheter, oxygen hood, or oxygen cage. supplemental oxygen should be humidified, and delivered at - ml/kg/minute. if oxygenation and ventilation are so impaired that the pao remains < mm hg with the patient on supplemental oxygen, a paco > mm hg, or severe respiratory fatigue, develops, and mechanical ventilation should be considered. glucose is a necessary fuel source for red blood cells and neuronal tissues, and serum glucose should be maintained within normal reference ranges. glucose supplementation can be administered as . - % solutions in crystalloid fluids, or in parenteral and enteral nutrition products. arterial and venous ph can be measured by performing blood gas analyses. decrease in tissue perfusion, impaired oxygen delivery, and decreased oxygen extraction in the various forms of shock can lead to anaerobic metabolism and metabolic acidosis. in most cases, improving tissue perfusion and oxygen delivery with crystalloid and colloid fluids, supplemental oxygen, and inotropic drugs will help normalize metabolic acidosis. serial measurements of serum lactate (normal, < . mmol/l) can be used as a guide to evaluate the tissue response to fluid resuscitative efforts. serum electrolytes often become severely deranged in shock states. serum potassium, magnesium, sodium, chloride, and total and ionized calcium should be maintained within normal reference ranges. if metabolic acidosis is severe, sodium bicarbonate can be administered by calculating the formula base deficit × . × body weight in kg = meq bicarbonate to administer because iatrogenic metabolic alkalosis can occur, a conservative approach is to administer / of the calculated dose and then recheck the patient's ph and bicarbonate levels. if the base excess is unknown, sodium bicarbonate can be administered in incremental doses of meq/kg until the ph is above . . complications associated with bicarbonate therapy include iatrogenic hypocalcemia, metabolic alkalosis, paradoxical cerebrospinal fluid acidosis, hypotension, restlessness, and death. massive trauma, neoplasia, sepsis, and systemic inflammation can all lead to coagulation abnormalities, including disseminated intravascular coagulation (dic). cage-side coagulation monitors are available for daily measurement of prothrombin time (pt), activated partial thromboplastin time (aptt), and platelet counts. fibrin degradation products (fibrin split products) become elevated in dic, trauma, hepatic disease, and surgery. coagulation proteins (clotting factors) and antithrombin often are lost with other proteins in hypoproteinemia or are consumed when microclots are formed and then dissolved. antithrombin levels can be measured by commercial laboratories. antithrombin and clotting factors can be replenished in the form of fresh frozen plasma transfusions. a more sensitive and specific test for dic is the detection of d-dimers, which can be measured by commercial laboratories. treatment for dic involves treatment and resolution of the underlying disease and administration of antithrombin and clotting factors in the form of fresh frozen plasma ( ml/kg) and heparin (unfractionated, - units/kg sq tid; fractionated [lovenox], mg/kg sq bid). monitor the patient for changes in mental status, including stupor, coma, decreased ability to swallow and protect the airway, and seizures. elevation of the patient's head can help to protect the airway and decrease the risk of increased intracranial pressure. serum glucose should be maintained within normal levels to prevent hypoglycemia-induced seizures. one of the major components of oxygen delivery is the binding to hemoglobin. packed cell volume must be kept above - % for adequate cellular oxygen delivery. acid-base status can adversely affect oxygen offloading at the tissue level if metabolic or respiratory alkalosis is present. oxygen-carrying capacity and hemoglobin levels can be increased with administration of rbc component therapy or with hemoglobin-based oxygen carriers. monitoring of renal function includes daily measurement of bun, creatinine, and urine output. normal urine output in a hydrated euvolemic patient is - ml/kg/hour. fluid ins and outs should be measured in cases of suspected oliguria or anuria. in patients with oliguria or anuria, furosemide can be administered as a bolus ( - mg/kg) or by constant rate infusion (cri)( . - mg/kg/hour). mannitol should also be administered ( . - g/kg over to minutes). dopamine ( - µg/kg/minute cri) can be administered to dilate renal afferent vessels and improve urine output. the patient's white blood cell count may be elevated, normal, or decreased, depending on the type of shock. the decision to administer antibiotics should be made on a daily basis. superficial or deep staphylococcus or streptococcus infection usually can be treated with a first-generation cephalosporin (cefazolin, mg/kg iv tid). if a known source of infection is present, administer a broad-spectrum antibiotic (cefoxitin, mg/kg iv tid; ampicillin, mg/kg qid, or enrofloxacin, - mg/kg once daily) pending results of culture and susceptibility testing. if broader anaerobic coverage is required, metronidazole ( mg/kg iv tid) should be considered. gentamicin ( - mg/kg iv once daily) is a good choice for gram-negative sepsis, provided that the patient is well hydrated and has normal renal function. ideally, patients receiving any aminoglycoside antibiotic should have a daily urinalysis to check for renal tubular casts that signify renal damage. in dogs, the gut is the shock organ. impaired gastrointestinal motility and vomiting should aggressively be treated with antiemetics and promotility drugs (dolasetron, . mg/kg iv once daily, and metoclopramide, - mg/kg/day iv cri). metoclopramide is contraindicated in cases of suspected gastrointestinal obstruction. histamine-receptor blockers such as famotidine ( . mg/kg bid iv) and ranitidine ( . to mg/kg iv bid, tid) or proton-pump inhibitors (omeprazole, . - mg/kg po once daily) can be administered for esophagitis. administer sucralfate ( . - g po tid) to treat gastric ulceration. if the gastrointestinal barrier function is diminished due to poor perfusion, infection, or inflammation, administer broad-spectrum antibiotics such as ampicillin ( mg/kg iv qid) to prevent gastrointestinal bacterial translocation. the course of drug therapy should be reviewd daily and the patient should be monitored for potential drug interactions. for example, metoclopramide and dopamine, working at the same receptor, can effectively negate the effects of each other. cimetidine, a cytochrome p enzyme inhibitor, can decrease the metabolism of some drugs. drugs that are avidly protein-bound may have an increase in unbound fraction with concurrent hypoalbuminemia or when hypoalbuminemia is present. decreased renal function may impair the renal clearance of some drugs, requiring increased dosing interval or decreased dose. nutrition is of utmost importance in any critically ill patient. patients with septic shock may become hypermetabolic and require supraphysiologic nutrient caloric requirements, while others may actually become hypometabolic. enteral nutrition is preferred, whenever possible, because enterocytes undergo atrophy without luminal nutrient stimulation. a variety of enteral feeding tubes can be placed, depending on what portion of the gut is functional, to provide enteral nutrition in an inappetent patient. loss of gastrointestinal mucosal barrier function may predispose the patients to the development of bacterial translocation and may contribute to sepsis. if enteral nutrition is impossible because of protracted vomiting or gastrointestinal resection, glucose, lipid, and amino acid products are available that can be administered parenterally to meet nutrient needs until the gastrointestinal tract is functioning and the patient can be transitioned to enteral nutrition. assessment of pain in animals in shock can be challenging. pain can result in the release of catecholamines and glucocounterregulatory hormones that can impair nutrient assimilation and lead to negative nitrogen balance, impaired wound healing, and immunocompromise. in any animal determined to be in pain, analgesic drugs should be administered to control pain and discomfort at all times. opioids are cardiovascularly friendly, and their effects can easily be reversed with naloxone if adverse effects such as hypotension and hypoventilation occur. if the patient is nonambulatory, rotate the animal from side to side every to hours to prevent lung atelectasis. passive range-of-motion exercises and deep muscle massage should be performed to increase tissue perfusion, decrease dependent edema, and prevent disuse atrophy. animals should be kept completely dry on soft, padded bedding to prevent the development of decubital ulcers. all bandages, wound sites, and catheter sites should be checked daily for the presence of swelling, erythema, and pain. soiled bandages should be changed to prevent strike-through and contamination of the underlying catheter or wound. hospitalization can be a stressful experience for patient and client alike. allowing brief visits and walks outside in the fresh air can improve a patient's temperament and decrease stress. the preemptive use of analgesic drugs on a regular schedule (not prn) should be used to prevent pain before it occurs. pain decreases the patient's ability to sleep. lack of sleep can promote further stress and impaired wound healing. the use of glucocorticosteroids and antiprostaglandins in shock therapy remains a topic of wide controversy. although the use of these agents potentially may stabilize membranes, decrease the absorption of endotoxin, and decrease prostaglandin release, the routine use of glucocorticosteroids and antiprostaglandins can decrease renal perfusion and gastrointestinal blood flow, promoting gastrointestinal ulceration and impaired renal function. the administration of supraphysiologic levels of glucocorticosteroids in patients in any type of shock can increase sodium and water retention, depress cellular immune function, and impair wound healing. in clinical studies of small animal patients, the routine use of glucocorticosteroids and antiprostaglandins has not demonstrated definite improved survival. the risks of therapy do outweigh the anecdotal reported benefits, and therefore the empiric use of glucocorticosteroids and antiprostaglandins in any shock patient is urinary tract emergencies azotemia azotemia occurs when % or more of the nephrons are nonfunctional. the magnitude of the azotemia alone cannot be used to determine whether the azotemia is prerenal, renal, or postrenal in origin, or whether the disease process is acute or chronic, reversible or irreversible, progressive or nonprogressive. before beginning treatment for azotemia, the location or cause of the azotemia must be identified. take a thorough history and then perform a physical examination. obtain blood and urine samples before initiating fluid therapy, for accurate assessment of the location of the azotemia. for example, an azotemic animal with a history of vomiting and diarrhea that appears clinically dehydrated on physical examination, normally should have a concentrated urine specific gravity (> . ) reflecting the attempt to conserve fluid. if this level is found, the azotemia is much less likely to be renal in origin, and the azotemia will likely resolve after rehydration. if, however, the urine specific gravity is isosthenuric or hyposthenuric ( . - . ) in the presence of azotemia and dehydration, primary intrinsic renal insufficiency is likely present. if the azotemia resolves with fluid therapy, the patient has prerenal and primary renal disease. if the azotemia does not resolve after rehydration, the patient has prerenal and primary renal failure. dogs with hypoadrenocorticism can have both prerenal and primary renal disease secondary to the lack of mineralocorticoid (aldosterone) influence on the renal collecting duct and renal interstitial medullary gradient. medullary washout can occur, causing isosthenuric urine in the presence of dehydration from vomiting and diarrhea. the patient often has azotemia due to fluid loss (dehydration and urinary loss) and gastric or intestinal hemorrhage (elevated bun). the prerenal component will resolve with treatment with glucocorticoids and crystalloid fluids, but the renal component may take several weeks to resolve, until the medullary concentration gradient is reestablished with the treatment and influence of mineralocorticoids. drugs such as corticosteroids and diuretics can influence renal tubular uptake and excretion of fluid, and cause a prerenal azotemia and isosthenuric urine in the absence of primary renal disease. treatment of azotemia includes calculation of the patient's dehydration estimate and maintenance fluid volumes, and administering that volume over the course of hours. identify and treat underlying causes of prerenal azotemia (shock, vomiting, diarrhea). monitor urine output closely. once a patient is euvolemic, oliguria is defined as urine output < - ml/kg/hour. urine output should return to normal in patients with prerenal azotemia as rehydration occurs. if a patient remains oliguric after rehydration, consider the possibility of oliguric acute intrinsic renal failure, and administer additional fluid therapy based on the patient's urine output, body weight, central venous pressure, and response to other medical therapies. prerenal azotemia is caused by conditions that decrease renal perfusion, including hypovolemic shock, severe dehydration, hypoadrenocorticism, congestive heart failure, cardiac tamponade, cardiac dysrhythmias, and hypotension. once renal perfusion is restored, the kidneys can resume normal function. glomerular filtration rate decreases when the mean arterial blood pressure falls to less than mm hg in a patient with normal renal autoregulation. renal autoregulation can be impaired in some diseases. passive reabsorption of urea from the renal tubules can occur during states of low tubular flow (dehydration, hypotension) even if glomerular filtration is not decreased. if renal hypoperfusion is not quickly restored, the condition can progress from prerenal disease to acute intrinsic renal failure. prerenal and renal azotemia can coexist in animals with primary renal disease, as a result of vomiting and ongoing polyuria in the absence of any oral fluid intake. the treatment of prerenal azotemia consists of rehydration, antiemetic therapy, and treatment of the underlying cause of vomiting, diarrhea, or third spacing of fluids. acute intrinsic renal failure is characterized by an abrupt decline in renal function to the extent that azotemia and an inability to regulate solute and fluid balance. patients with acute intrinsic renal failure may be oliguric or polyuric, depending on the cause and state of renal failure. in small animals, the most common causes of acute intrinsic renal failure are renal ischemia and toxins. there are three phases of acute intrinsic renal failure: induction, maintenance, and recovery. during the induction phase, some insult (ischemia or toxin) to the kidneys occurs, leading to a defective concentrating mechanism, decreased renal clearance of nitrogenous waste (azotemia), and polyuria or oliguria. if treatment is initiated during the induction phase, progression to the maintenance phase potentially can be stopped. as the induction phase progresses, there is worsening of the urine-concentrating ability and azotemia. renal tubular epithelial cells and renal tubular casts can be seen on examination of the urine sediment. glucosuria may be present. the maintenance phase of acute intrinsic renal failure occurs after a critical amount of irreversible nephron injury. correction of the azotemia and removal of the cause of the problem do not result in return to normal function. in patients with oliguria, the extent of nephron damage is greater than that observed in patients with polyuria. the maintenance phase may last for several weeks to months. recovery of renal function may or may not occur, depending on the extent of injury. the most serious complications (overhydration and hyperkalemia) are observed in patients with oliguria. the recovery phase occurs with sufficient healing of damaged nephrons. azotemia may resolve, but concentrating defects may remain. if the patient was oliguric in the maintenance phase, a marked diuresis develops during the recovery phase that may be accompanied by fluid and electrolyte losses. this phase may last for weeks to months. treatment of acute intrinsic renal failure consists of determining the cause and ruling out obstruction or uroabdomen whenever possible. a careful history can sometimes determine whether there has been exposure to nephrotoxic drugs, chemicals, or food items. if ingestion or exposure to a toxic drug, chemical, or food occurred recently (within to hours), induce emesis with apomorphine ( . mg/kg iv). next, administer activated charcoal either orally or via stomach tube, to prevent further absorption of the toxin. obtain blood and urine samples for toxicologic analysis (e.g., ethylene glycol) and to determine whether azotemia or abnormalities in the urine sediment exist. (see section on ethylene glycol, grapes and raisins, and nonsteroidal antiinflammatory drugs). obtain a complete blood count, biochemical profile, and urinalysis to determine the presence of signs of chronic renal failure, including polyuria, polydipsia, and nonregenerative anemia. radiographs and abdominal ultrasound can help in determining the chronicity of renal failure. normal renal size is . - . times the length of l in dogs and . - . times the length of l in cats. monitor the patient's body weight at least twice a day to avoid overhydration. also monitor urine output; normal output is - ml/kg/hour. in cases of polyuric renal failure, massive fluid and electrolyte losses can occur. place a urinary catheter for patient cleanliness and to facilitate urine quantitation. measure fluid ins and outs (see section on fluid therapy). after the patient has been rehydrated, the amount of fluids administered should equal maintenance and insensible needs plus the volume of urine produced each day. if a urinary catheter cannot be placed or maintained, serial body weight measurements and central venous pressure should be used to monitor the patient's fluid balance and prevent overhydration. if the patient is oliguric (urine output < - ml/kg/hour), pharmacologic intervention is necessary to increase urine output. first, administer furosemide ( - mg/kg or . mg/kg/hour iv cri). repeat bolus doses of furosemide if there is no response to initial treatment. if necessary, administer low-dose dopamine ( - µg/kg/minute iv cri) to increase renal afferent dilatation and renal perfusion. dopamine and furosemide may be synergistic if administered together. if dopamine and furosemide therapy is ineffective, administer mannitol ( . - . g/kg iv) once only. if polyuria is present, management is simplified because of the decreased risk of overhydration. if oliguria cannot be reversed, monitor the central venous pessure, body weight, and respiratory rate and effort, auscultate for crackles, and examine the patient carefully for signs of chemosis and the presence of serous nasal discharge. correct hyperkalemia with sodium bicarbonate ( . - . meq/kg iv) or with insulin ( . units/kg) plus dextrose ( g/unit of insulin iv, followed by . % dextrose iv cri). treat severe metabolic acidosis (ph < . or hco − < meq/l) with sodium bicarbonate. if anuria develops or oliguria is irreversible despite this therapy, begin peritoneal dialysis. obtain a renal biopsy to establish a diagnosis and prognosis (see section on renal biopsy). administer gastroprotectant drugs and antiemetics to control nausea and vomiting. if possible, avoid the use of nephrotoxic drugs and general anesthesia. initiate nutritional support in the form of an enteral feeding tube or parenteral nutrition as early as possible. once the patient enters the recovery phase, diuresis may occur that can lead to dehydration and electrolyte imbalances (hyponatremia, hypokalemia). dehydration and electrolyte imbalances can be treated with parenteral fluid and electrolyte supplementation. postrenal azotemia is primarily caused by urethral obstruction or leakage from the urinary tract into the abdomen (uroabdomen). complete urinary tract obstruction and uroabdomen are both ultimately fatal within to days if left untreated. in dogs, the most common causes of urethral obstruction are urinary (urethral) calculi or tumors of the urinary bladder or urethra. in male cats, feline urologic syndrome (fus) is the most common cause of urethral obstruction, although there has been an increased incidence of urethral calculi observed in recent years. a ruptured urinary bladder is the most common cause of uroabdomen and is usually secondary to blunt trauma. clinical signs of urinary tract obstruction include dysuria, hematuria, inability to urinate or initiate an adequate stream of urine, and a distended painful urinary bladder. late in the course of obstructive disease, clinical signs referable to uremia and azotemia (vomiting, oral ulcers, hematemesis, dehydration, lethargy, and anorexia) occur. the initial goal of treatment of urinary tract obstruction is to relieve the obstruction. in male dogs, a lubricated catheter can be inserted past the area of obstruction with the animal under heavy sedation or general anesthesia (see section on urohydropulsion). depending on the chronicity of the obstruction, serum electrolytes should be measured;an ecg should be obtained before administering any anesthetic drugs, because of the cardiotoxic effects of hyperkalemia (see section on atrial standstill). correct fluid, electrolyte, and acid-base abnormalities. if a urinary catheter cannot be placed, perform cystocentesis only as a last resort, because of the risk of urinary bladder rupture. definitive treatment includes identification and treatment of the underlying cause (tumor versus urinary calculi). in most cases, surgical intervention is necessary. if an unresectable tumor is present, a low-profile permanent cystostomy tube can be placed, if the owner desires. administration of piroxicam (feldene, . mg/kg po q - h) with or without chemotherapy may shrink the tumor mass and delay the progression of clinical signs. a complete discussion of this disorder is beyond the scope of this text (see additional reading for other sources of information). feline lower urinary tract disease can cause urethral obstruction, particularly in male cats. clinical signs include stranguria, dribbling of small amounts of urine, lethargy, inappetence, and vomiting. often, owners call with the primary complaint of constipation, because the cat is making frequent trips to the litterbox and straining. cases with a duration of obstruction < hours are considered uncomplicated; those with a duration > hours are complicated. treatment of urethral obstruction includes stabilizing and normalizing the patient's electrolyte status, induction of sedation or general anesthesia, and relieving the obstruction. obtain blood samples for analysis of electrolyte abnormalities. treat hyperkalemia (k + > . meq/l) with sodium bicarbonate ( . - . meq/kg iv), regular insulin ( . unit/ kg iv) plus dextrose ( g//unit of insulin iv), followed by . % dextrose iv cri to prevent hypoglycemia; or calcium gluconate ( . ml/kg % iv slowly). administer non-potassiumcontaining intravenous fluids in . % saline solution. obtain an ecg to detect atrial standstill (see section on atrial standstill). in some cases, a urethral plug is visible at the tip of the penis. the urethral plug can sometimes be manually extracted or massaged from the penis, and the obstruction temporarily relieved. in such cases, it is still necessary to pass a urethral catheter to flush sediment from the urethra and urinary bladder. unless a patient is obtunded, administer an anesthetic such as ketamine, atropine, or propofol ( - mg/kg iv) with diazepam iv for patient comfort and muscle relaxation. once the patient is under anesthesia or heavily sedated, urinary catheterization should be performed. in some cases, it will be difficult to advance the catheter. lubricate a closedended tomcat catheter and pass the tip into the distal urethra. fill a -ml syringe with sterile saline and sterile lubricant and connect the syringe to the hub of the catheter. pulse the fluid into the catheter as you gently move the catheter tip back and forth against the urethral obstruction. when the catheter has been passed into the urinary bladder, obtain a urine sample for urinalysis. drain the bladder and flush with sterile saline solution until the urine efflux appears clear. remove the tomcat catheter and insert a - fr red rubber tube or argyle infant feeding catheter into the urethra for urine collection and quantitation. secure the urinary catheter to prepuce with a butterfly strip of -inch adhesive tape secured around the catheter and then sutured to either side of the prepuce. the catheter should be connected to a closed urinary collection system for cleanliness and to reduce the risk of ascending bacterial infection. an elizabethan collar should be placed at all times to prevent the patient from damaging or removing the catheter. when the urethral obstruction has been relieved and the catheter placed, continue intravenous fluid diuresis to alleviate postrenal azotemia. monitor the urine for bacteria and other sediment. in some cases, postobstructive diuresis can be severe. carefully monitor fluid ins and outs, along with body weight, to maintain adequate hydration and perfusion. remove the urinary catheter can be removed after to hours. palpate the bladder frequently to make sure that the patient is voiding normally and to detect the recurrence of obstruction. in patients with severe penile or urethral trauma or edema, administer a short-acting steroid (dexamethasone sodium phosphate, . mg/kg iv, im, sq). at the time of initial diagnosis and again at the time of discharge, the clients need to be instructed about the long-term management of feline lower urinary tract disease at home, and informed of the risks and consequences of recurrence. uroabdomen can occur from trauma or leakage from the kidneys, ureter, or urinary bladder. clinical signs of uroabdomen (azotemia, uremia, hyperkalemia) can also occur secondary to third spacing of urine and leakage into muscular tissue from a ruptured urethra. in most cases, urinary bladder trauma and rupture are secondary to blunt trauma. abdominocentesis should be performed in any animal with suspected blunt abdominal trauma, and any fluid obtained should be analyzed for creatinine or potassium and compared with the patient's serum levels. an abdominal effusion that has a low packed cell volume and a potassium or creatinine level greater than that of the patient's serum is consistent with the diagnosis of uroabdomen. uroabdomen is not a surgical emergency. however, medical management consists of placement of a temporary abdominal drainage catheter into the abdomen, to facilitate removal of urine from the peritoneal cavity. to place the catheter, position the patient in dorsal or lateral recumbency, shave the ventral abdomen, as for any exploratory laparotomy. aseptically scrub the clipped area, and instill a local anesthestic (lidocaine, - mg/kg) caudal and to the right of the umbilicus, through the skin, subcutaneous tissues, and rectus emergency care clinical differentiation of acute necrotizing from chronic nonsuppurative pancreatitis in cats: cases acute pancreatitis in dogs mesenteric volvulus in the dog: a retrospective study of cases incidence and prognostic value of low plasma ionized calcium concentration in cats with pancreatitis: cases ( - ) review of feline pancreatitis. part : clinical signs, diagnosis and treatment gastric dilatation-volvulus syndrome in dogs diagnostic approach to acute pancreatitis pathophysiology of organ failure in severe acute pancreatitis in dogs washabau rj: gastrointestinal motility disorders and gastrointestinal prokinetic therapy watson pt: exocrine pancreatic insufficiency as an end-stage of pancreatitis in dogs clinical signs, underlying cause, and outcome in cats with seizures: cases fibrocartilaginous embolism in dogs: clinical findings and factors influencing the recovery rate kirk's current veterinary therapy xiii intervertebral disc extrusion in six cats medical management of acute spinal cord disease risk factors for recurrence of clinical signs associated with thoracolumbar intervertebral disk herniation in dogs: cases intervertebral disk disease in cats long-term functional outcome of dogs with severe injuries of the thoracolumbar spinal cord: cases canine status epilepticus: a retrospective study of cases risk factors for development of status epilepticus in dogs with idiopathic epilepsy and effects of status epilepticus on outcome and survival time: cases ( - ) skills laboratory part i: performing a neurologic examination skills laboratory part ii: interpreting the results of the neurologic examination accuracy of localization of cervical intervertebral disk extrusion or protrusion using survey radiography in dogs medical and surgical management of the glaucoma patient the feline glaucomas: cases ( - ) the canine glaucomas traumatic ocular protrusion in dogs and cats: cases traumatic glaucoma in a dog ocular and orbital porcupine quills in the dog: a review and case series hyphema: pathophysiologic considerations. comp cont educ pract vet van der woerdt a: the treatment of acute glaucoma in dogs and cats administer crystalloid intravenous fluids at maintenance rates using a balanced electrolyte solution perform urinary catheterization and collection to monitor urine output monitor serum urea nitrogen and creatinine every hours treat oliguria, defined as a drop in urine output to less than ml/kg/hour ml/kg) bolus start dopamine at to µg/kg/minute if no response to crystalloid/colloid bolus occurs within minutes consider mannitol ( . to g/kg iv) administration if no response to dopamine occurs within minutes consider furosemide ( to mg/kg iv, or . to mg/kg/hour iv cri) if no response to dopamine or mannitol occurs in to minutes if no response to furosemide, peritoneal dialysis or hemodialysis is indicated immediately, particularly if anuria is present administered with caution, because of the risk of exacerbating increased capillary permeability and causing pulmonary edema. animal patients. chlorphenoxy derivatives exert their toxic effects by an unknown mechanism, and cause clinical signs of gastroenteritis and muscle rigidity severe anemia should be treated with packed rbcs or hemoglobin-based oxygen carriers handbook of small animal toxicology and poisonings macadamia nut toxicosis in dogs the recognition and treatment of the intermediate syndrome of organophosphate poisoning in a dog acute renal failure in four dogs after raisin or grape ingestion pleural effusion in cats pulmonary function, ventilator management, and outcome of dogs with thoracic trauma and pulmonary contusions: cases ( - ) acute lung injury and acute respiratory distress syndrome smoke exposure in cats: cases ( - ) smoke exposure in dogs: cases ( - ) thoracic duct ligation and pericardectomy for treatment of idiopathic chylothorax use of intraluminal nitinol stents in the treatment of tracheal collapse in a dog clinical approach to epistaxis the veterinary icu book. teton newmedia radiographic diagnosis of diaphragmatic hernia: review of cases in dogs and cats tracheal collapse: diagnosis and medical and surgical management acute respiratory distress syndrome brachycephalic syndrome in dogs outcome and postoperative complications in dogs undergoing surgical treatment of laryngeal paralysis: cases ( - ) full recovery following delayed neurologic signs after smoke inhalation in a dog aspiration pneumonitis the veterinary icu book. teton newmedia allergic airway disease canine pleural and mediastinal effusion, a retrospective study of cases suggested strategies for ventilatory management in veterinary patients with acute respiratory distress syndrome laryngeal and tracheal disorders the veterinary icu book. teton newmedia medical and surgical treatment of pyothorax in dogs: cases traumatic diaphragmatic hernia in cats: cases canine pyothorax: clinical presentation, diagnosis, and treatment canine pyothorax: pleural anatomy and pathophysiology treatment of chronic pleural effusion with pleuroperitoneal shunt in dogs: cases ( - ) effects of doxapram hydrochloride on laryngeal function of normal dogs and dogs with naturally occurring laryngeal paralysis an overview of positive pressure ventilation risk factors, prognostic indicators, and outcome of pyothorax in cats: cases ( - ) use of percutaneous arterial embolization for the treatment of intractable epistaxis in dogs systemic inflammatory response syndrome, sepsis, and multiple organ dysfunction cardiogenic shock and cardiac arrest hemostatic changes in dogs with naturally occurring sepsis multiple organ dysfunction syndrome in humans and dogs increased lactate concentrations in ill and injured dogs the role of albumin in health and disease pathophysiologic characteristics of hypovolemic shock usefulness of systemic inflammatory response syndrome criteria as an index for prognosis judgement current principles and application of d-dimer analysis in small animal practice choosing fluids in traumatic hypovolemic shock: the role of crystalloids, colloids and hypertonic saline colloid and crystalloid resuscitation thromboembolic disease: predispositions and management marks sl: systemic arterial thromboembolism retrospective study of streptokinase administration in cats with arterial thromboembolism feline arterial thromboembolism: an update arterial thromboembolism in cats: acute crises in cases ( - ) and long-term management with low-dose aspirin in cases cut multiple holes in the side of a - fr red rubber tube or thoracic drainage catheter, using care not to make the cut wider than % of the circumference of the tube. insert the catheter into the abdominal cavity in a dorsal caudal direction. make sure that all incisions within the abdomen. secure the tube by placing a pursestring suture around the tube entrance site in the abdominal musculature with absorbable suture material. close the dead space in the subcutaneous tissues with absorbable suture. close the skin around the tube with another purse-string suture secured using a finger-trap technique. connect the tube to a closed urinary collection system and bandage the catheter to the abdomen. the tube can remain in place until the patient retrospective evaluation of acute renal failure in dogs uroabdomen in dogs and cats drug-induced nephrotoxicity: recognition and prevention peritoneal dialysis in emergency and critical care acute renal failure caused by lily ingestion in six cats early diagnosis of renal disease and renal failure acute renal failure in four dogs after raisin or grape ingestion disorders of the feline lower urinary tract the use of a low-profile cystostomy tube to relieve urethral obstruction in a dog renal biopsy: methods and interpretation feline idiopathic cystitis: current understanding of pathophysiology and management today's problem when did you first notice that something was wrong with your pet? when was the last time you noticed your pet act normally? what was the first abnormal sign noticed? what other conditions have developed and what are they? how soon did other signs develop? have the signs become better or worse since you first saw them? what is the name of the product? do you have the container with you today? is it a liquid concentrate, dilute spray, or solid? how long ago do you think that your pet was exposed to the poison? where do you think it happened? do you have any over-the-counter or prescription medications that your animal may have had access to? did you give any medications to your animal? is there any possibility of recreational drug exposure?your pet's recent activity did your pet eat this morning or last night? what is he/she normally fed? is there a chance that your pet may have gotten into the garbage? have you fed table scraps or anything new recently? if so, what? has your pet been off your property in the last - hours? does your pet run loose unattended? has your pet had any antiflea/tick medication within the last week?your pet's environment is your animal kept inside or outside of the house? is your pet kept in a fenced-in yard or allowed to run loose unattended? does your pet have access to neighboring properties (even for a short time)? where has your pet been in the last hours? has your pet traveled outside of your immediate geographic location? if so, when? has your pet been to rural areas in the last week? has there been any gardening work recently? does your pet have access to a compost pile? any fertilizers or weed killer used in the last week? any construction work or renovation recently? any mouse or rat poison in your house, yard, or garage? any cleaning products used inside or outside the house within the last hours? if so, which? have you changed your radiator fluid or does a car leak antifreeze? induce and maintain a patent airway and stabilize the patient's cardiovascular and respiratory status. control cns excitation with diazepam, if necessary, and control the patient's body temperature (both hypo-and hyperthermia) . induce vomiting if the patient is alert and can protect its airway; otherwise, perform orogastric lavage with the patient under general anesthesia with a cuffed endotracheal tube in place. alcohols do not bind well with activated charcoal. treat dermal exposure by bathing the area with warm water. introduction: if ingested, sodium or potassium hydroxide can cause severe contact dermatitis or irritation of the gastrointestinal tract. esophageal burns and full-thickness coagulative necrosis can occur. if an animal ingests a caustic alkali substance, feed the animal four egg whites mixed with quart of warmed water. perform endoscopy within hours to evaluate the extent of injury and to place a feeding tube, in severe cases. do not induce emesis , and do not perform orogastric lavage, because of the risk of worsening esophageal irritation. in cases of contact exposure to the skin or eyes, rinse the exposed area with warm water baths for at least minutes. administer gastroprotectant, antiemetic, and analgesic drugs as necessary. avoid neutralization, which can cause a hyperthermic reaction and worsen injury to the skin and gastrointestinal tract. amitraz is the active ingredient in ascaricides and anti-tick and anti-mite products such as mitaban and taktic. the toxic dose is to mg/kg. amitraz exerts its toxic effects by causing α-adrenergic stimulation, and causes clinical signs similar to those observed with administration of xylazine: bradycardia, cns depression, ataxia, hypotension, hyperglycemia, hypothermia, cyanotic mucous membranes, polyuria, mydriasis, and emesis. a coma can develop. treatment of amitraz intoxication includes cardiovascular support with intravenous crystalloid fluids and induction of emesis in asymptomatic animals. if clinical signs are present, orogastric lavage may be required. many toxic compounds are impregnated in a collar form. if the patient has ingested a collar and does not vomit it, it should be removed using endoscopy or gastrotomy. administer activated charcoal to prevent or delay absorption of the toxic compound. yohimbine or atepamizole, both α-adrenergic antagonists, are the treatment(s) of choice to reverse the clinical signs of toxicity. avoid the use of atropine, because it can potentially increase the viscosity of respiratory secretions and cause gastrointestinal ileus, thus promoting increased absorption of the toxic compound. ammonium hydroxide, or cleaning ammonia, can be caustic at high concentrations (see alkalis/caustics) and cause severe injury to the respiratory system if inhaled. pulmonary edema or pneumonia can occur, resulting in respiratory distress. ingestion of ammonia can cause severe irritation to the gastrointestinal tract and cause vomiting and esophageal injury. if ammonia is ingested, administer a dilute solution of egg white.administer gastroprotectant, antiemetic, and analgesic drugs as necessary. if pneumonia or pulmonary edema occurs secondary to aspiration of ammonia into the airways and alveolar spaces, treatment is largely supportive with supplemental oxygen administration, antibiotics, fluid therapy, and mechanical ventilation as necessary. diuretics may or may not be useful in the treatment of pulmonary edema secondary to ammonia inhalation. amphetamines cause cns excitation due to neurosynaptic stimulation, resulting in hypersensitivity to noise and motion, agitation, tremors, vomiting, diarrhea, and seizures. clinical signs of amphetamine toxicity include muscle tremors, tachyarrhythmias, mydriasis, ptyalism, and hyperthermia. amphetamines are rapidly absorbed from the gastrointestinal tract. treatment includes administration of intravenous fluids to maintain hydration and renal perfusion and correction of hyperthermia. administer sedative drugs such as chlorpromazine to control agitation and tremors, and diazepam to control seizures. urinary acidification can promote excretion and prevent reabsorption from the urinary bladder. in severe cases, treat cerebral edema with a combination of mannitol followed by furosemide to control increased intracranial pressure.antifreeze: see ethylene glycol antihistamines introduction antihistamines (loratadine, diphenhydramine, doxylamine, clemastine, meclizine, dimenhydrinate, chlorpheniramine, cyclizine, terfenadine, hydroxyzine) are available as over-thecounter and prescription allergy and anti-motion sickness products. clinical signs of antihistamine toxicity include restlessness, nausea, vomiting, agitation, seizures, hyperthermia, and tachyarrhythmias. treatment of antihistamine intoxication is largely symptomatic and supportive, as there is no known antidote. if ingestion is recent (within to hours) and the patient is not actively seizing and can protect its airway, induce emesis or perform orogastric lavage, followed by administration of activated charcoal and a cathartic. monitor the patient's heart rate, rhythm, and blood pressure. treat cardiac arrhythmias, if present, with appropriate therapies (see section on cardiac dysrhythmias). administer cooling measures and intravenous fluids to treat hyperthermia. a constant rate infusion of guaifenasin can be used to control muscle tremors. introduction α-naphthylthiourea (antu) is manufactured as a white or blue-gray powder. the toxic dose in dogs is - mg/kg, and in cats is - mg/kg. younger dogs appear to be more resistant to its toxic effects. antu usually causes profound emesis and increased capillary permeability that eventually leads to pulmonary edema. treatment of antu toxicity includes respiratory support. mechanical ventilation may be required in severe cases of pulmonary edema. if an animal does not vomit, orogastric lavage should be performed. administer gastrointestinal protectant, antiemetic, and analgesic drugs. cardiovascular support in the form of intravenous crystalloids should be arsenic introduction inorganic arsenic (arsenic trioxide, sodium arsenite, sodium arsenate) is the active ingredient in many herbicides, defoliants, and insecticides, including ant killers. the toxic dose of sodium arsenate is - mg/kg; that of sodium arsenite is - mg/kg. sodium arsenite is less toxic, although cats are very susceptible. arsenic compounds interfere with cellular respiration by combining with sulfhydryl enzymes. clinical signs of toxicity include severe gastroenteritis, muscle weakness, capillary damage, hypotension, renal failure, seizures, and death. in many cases, clinical signs are acute in onset. treatment of arsenic toxicity involves procuring and maintaining a patent airway. administer intravenous crystalloid fluids to correct hypotension and hypovolemia, and normalize acidbase and electrolyte balance. if no clinical signs are present and if the compound was ingested within hours, induce emesis. if clinical signs are present, perform orogastric lavage followed by administration of activated charcoal. if dermal exposure has occurred, throughly bathe the animal to prevent further absorption. dimercaprol (bal, - mg/kg im q h) can be administered as a chelating agent. n-acetylcysteine (mucomyst) (for cats, - mg/kg po iv, then mg/kg po iv q h for days; for dogs, mg/kg po or iv, then mg/kg po iv q h for days) has been shown to decrease arsenic toxicity in rats. aspirin causes inhibition of the production of prostaglandins, a high anion gap metabolic acidosis, gastrointestinal ulceration, hypophosphatemia, and decreased platelet aggregation when ingested in high quantities (> mg/kg/ hours in dogs; > mg/kg/ hours in cats). clinical signs of aspirin toxicity include tachypnea, vomiting, anorexia, lethargy, hematemesis, and melena. treatment of aspirin toxicity is largely supportive. if the ingestion was recent (within the last hour), induce emesis or perform orogastric lavage followed by administration of activated charcoal. administer intravenous crystalloid fluids to maintain hydration and correct acid-base abnormalities. administer synthetic prostaglandin analogues (misoprostol), gastroprotectant drugs, and antiemetics. alkalinization of the urine can enhance excretion. introduction baclofen is a gaba agonist centrally acting muscle relaxant. clinical signs of toxicity include vomiting, ataxia, vocalization, disorientation, seizures, hypoventilation, coma, and apnea. clinical signs can occur at doses as low as . mg/kg. treatment of baclofen ingestion includes induction of emesis if the animal is asymptomatic. otherwise, perform orogastric lavage. emesis or orogastric lavage should be followed by administration of activated charcoal. perform intravenous crystalloid fluid diuresis to promote elimination of the toxin, maintain renal perfusion, and normalize body temperature. supplemental oxygen or mechanical ventilation may be required for hypoventilation or apnea. if seizures occur, avoid the use of diazepam, which is a gaba agonist and can potentially worsen clinical signs. control seizures with intravenous introduction β-adrenergic agonists, including terbutaline, albuterol (salbutamol), and metaproterenol, are commonly used in inhaled form for the treatment of asthma. animals commonly are exposed to the compounds after chewing on their owners' inhalers. clinical signs of β-adrenergic stimulation include tachycardia, muscle tremors, and agitation. severe hypokalemia can occur. treatment of β-adrenergic agonist intoxication includes treatment with beta-blockers (propranolol, esmolol, atenolol), intravenous fluids, and intravenous potassium supplementation. diazepam or acepromazine may be administered for sedation and muscle relaxation. introduction barbiturates such as phenobarbital are gaba agonists and induce cns depression. clinical signs of barbiturate overdose or toxicity include weakness, lethargy, hypotension, hypoventilation, stupor, coma, and death. treatment of barbiturate toxicity includes maintenance and support of the cardiovascular and respiratory systems. if clinical signs are absent and the patient can protect its airway, induce emesis followed by repeated doses of activated charcoal. perform orogastric lavage if emesis is contraindicated. administer supplemental oxygen if hypoventilation occurs. some animals may require mechanical ventilation. administer intravenous fluids to control perfusion and blood pressure. positive inotropic drugs may be required if dosedependent decrease in cardiac output and blood pressure occurs. alkalinization of the urine and peritoneal dialysis can be performed to enhance excretion and elimination. hemodialysis should be considered in severe cases, if available. automotive and dry cell batteries contain sulfuric acid that can be irritating on contact with the eyes, skin, and gastrointestinal tract. button batteries, which contain sodium or potassium hydroxide, cause contact irritation if chewed. to treat exposure, rinse the eyes and skin with copious amounts of warm tap water or sterile saline solution for a minimum of minutes. if ingestion occurred, administer gastroprotectant and antiemetic drugs. induction of emesis and orogastric lavage is absolutely contraindicated because of the risk of aspiration pneumonia and worsening esophageal irritation. no attempt should be made at performing neutralization because of the risk of causing an exothermic reaction and worsening tissue damage. administer analgesics to control discomfort. benzoyl peroxide is the active ingredient in many over-the-counter acne preparations. ingestion can result in production of hydrogen peroxide, gastroenteritis, and gastric dilatation. topical exposure can cause dermal irritation and blistering. if an animal has ingested benzoyl peroxide, do not induce emesis, because of the risk of worsening esophageal irritation. instead, perform orogastric lavage. administer gastroprotectant and antiemetic medications and closely observe the patient observed for signs of gastric dilatation.bismuth subsalicylate (pepto-bismol): see aspirin bleach, chlorine (sodium hypochlorite) introduction sodium hypochlorite is available in dilute ( %- %) or concentrated ( % industrial strength or swimming pool) solutions for a variety of purposes. sodium hypochlorite can cause severe contact irritation and tissue destruction, depending on the concentration. affected animals may have a bleached haircoat. treatment of exposure includes dilution with copious amounts of warm water or saline baths and ocular lavage. induction of emesis and orogastric lavage is absolutely contraindicated because of the risk of causing further esophageal irritation. to treat ingestion, give the animal milk or large amounts of water, in combination with gastroprotectant and antiemetic drugs, to dilute the contents in the stomach. administration of sodium bicarbonate or milk of magnesia is no longer recommended. nonchlorine bleaches (sodium peroxide or sodium perborate) have a moderate toxic potential if ingested. sodium peroxide can cause gastric distention. sodium perborate can cause severe gastric irritation, with vomiting and diarrhea; renal damage and cns excitation followed by depression can occur, depending on the amount ingested. to treat dermal or ocular exposure, rinse the skin or eyes with copious amounts of warm tap water or sterile saline for a minimum of minutes; treat ocular injuries as necessary, if corneal burns have occurred. if the bleach has been ingested, do induce emesis and perform orogastric lavage. administer milk of magnesia ( - ml/kg). boric acid is the active ingredient in many ant and roach killers. the toxic ingredient (in amounts of - g/kg) can cause clinical signs in dogs by an unknown mechanism. clinical signs include vomiting (blue-green vomitus), blue-green stools, renal damage, and cns excitation and depression. treatment of boric acid or borate ingestion includes gastric decontamination with induction of emesis or orogastric lavage, followed by administration of a cathartic to hasten elimination. activated charcoal is not useful to treat ingestion of this toxin. administer intravenous fluid therapy to maintain renal perfusion. administer gastroprotectant and antiemetic drugs, as necessary. clostridium botulinum endospores can be found in carrion, food, garbage, and the environment. ingestion of endospores and c. botulinum endotoxin rarely can cause generalized neuromuscular blockade of spinal and cranial nerves, resulting in miosis, anisocoria, lower motor neuron weakness, and paralysis. respiratory paralysis, megaesophagus, and aspiration pneumonia can occur. clinical signs usually develop within days of ingestion. differential diagnosis includes acute polyradiculoneuritis (coonhound paralysis), bromethalin intoxication, and tick paralysis. treatment of botulism is largely supportive; although an antitoxin exists, it often is of no benefit. treatment may include administration of intravenous fluids, frequent turning of the patient and passive range-of-motion exercises to prevent disuse muscle atrophy, and supplemental oxygen administration or mechanical ventilation. administer amoxicillin, ampicillin, or metronidazole. recovery may be prolonged, up to to weeks in some cases. bromethalin is the active ingredient in some brands of mouse and rat poisons. it usually is packaged as . % bromethalin in green or tan pellets, and packaged in - . g place packs. the toxic dose for dogs is . g/kg, and for cats g/kg. bromethalin causes toxicity by uncoupling of oxidative phosphorylation. an acute syndrome of vomiting, tremors, extensor rigidity, and seizures occurs within hours of ingestion of high doses. delayed clinical signs occur within to days of ingestion of a lower dose and include posterior paresis progressing to ascending paralysis, cns depression, and coma. treatment of known bromethalin ingestion includes induction of emesis or orogastric lavage, and repeated doses of activated charcoal every to hours for days, because bromethalin undergoes enterohepatic recirculation. supportive care includes intravenous fluids, anticonvulsants, muscle relaxants (methocarbamol up to mg/kg/day iv to effect), frequent turning of the patient, and passive range-of-motion exercises. supplemental oxygen and /or mechanical ventilation may be required in patients with coma and severe hypoventilation. administer mannitol ( . - g/kg) in conjunction with furosemide ( mg/kg iv) if cerebral edema is suspected. the majority of caffeine toxicities occur in dogs that ingest coffee beans. caffeine causes phosphodiesterase inhibition, and can cause cardiac tachyarrhythmias, cns stimulation (hyperexcitability and seizures), diuresis, gastric ulcers, vomiting, and diarrhea. muscle tremors and seizures can occur, resulting in severe hyperthermia. treatment of caffeine toxicity is largely symptomatic and supportive, as there is no known antidote. if clinical signs are not apparent and the patient is able to protect its airway, induce emesis. alternatively, orogastric lavage can be performed, followed by administration of activated charcoal. administer diazepam to control seizures. administer betaadrenergic blockers (e.g., esmolol, propranolol, atenolol) to control tachyarrhythmias. give intravenous fluids to maintain hydration and correct hyperthermia. the patient should be walked frequently or have a urinary catheter placed to prevent reabsorption of the toxin from the urinary bladder. carbamate compounds are found in agricultural and home insecticide products. examples of carbamates include carbofuran, aldicarb, propoxur, carbaryl, and methiocarb. the toxic dose of each compound varies. carbamate compounds function by causing acetylcholinesterase inhibition. toxic amounts cause cns excitation, muscarinic acetylcholine overload, and slud (salivation, lacrimation, urination, and defecation). miosis, vomiting, treatment of carbamate intoxication includes maintaining an airway and, if necessary, artificial ventilation. administer intravenous crystalloid fluids to control the patient's hydration, blood pressure, and temperature. cooling measures may be warranted. induce emesis if the substance was ingested within minutes and the animal is asymptomatic. give repeated doses of activated charcoal if the animal can swallow and protect its airway. control seizures with diazepam ( . mg/kg iv). bathe the patient thoroughly. atropine ( . mg/kg iv) is useful in controlling some of the muscarinic signs associated with the toxicity. pralidoxime hydrochloride ( -pam) is not useful in cases of carbamate intoxication. control muscle tremors with methocarbamol (up to mg/kg iv) or guaifenesin. in humans, ingestion or inhalation of - ml of carbon tetrachloride can be fatal. clinical signs of carbon tetrachloride toxicity include vomiting and diarrhea, then progressive respiratory and central nervous system depression. ventricular dysrhythmias and hepatorenal damage ensue. the prognosis is grave. treatment of carbon tetrachloride inhalation includes procurement and maintenance of a patent airway with supplemental oxygen, and cardiovascular support. to treat ingestion, administer activated charcoal, and give intravenous fluids to maintain hydration and support renal function. chlorinated hydrocarbons include ddt, methoxychlor, lindane, dieldrin, aldrin, chlordane, chlordecone, perthane, toxaphene, heptachlor, mirex, and endosulfan. the toxic dose of each compound varies. chlorinated hydrocarbons exert their toxic effects by an unknown mechanism, and can be absorbed through the skin and the gastrointestinal tract. clinical signs are similar to those observed in organophosphate toxicity: cns excitation, seizures, slud, (salivation, lacrimation, urination, defecation), excessive bronchial secretions, vomiting, diarrhea, muscle tremors, and respiratory paralysis. secondary toxicity from toxic metabolites can cause renal and hepatic failure. chronic exposure may cause anorexia, vomiting, weight loss, tremors, seizures, and hepatic failure. the clinical course can be prolonged in small animal patients. treatment of chlorinated hydrocarbon toxicity is largely supportive in nature, as there is no known antidote. procure and maintain the patient's airway. normalize the body temperature to prevent hyperthermia. if the substance was just ingested and the patient is not demonstrating any clinical signs, induce emesis. if the patient is symptomatic, perform orogastric lavage followed by activated charcoal administration. bathe the patient thoroughly in cases of topical exposure. administer intravenous crystalloid fluids to maintain hydration. these compounds do not appear to be amenable to fluid diuresis. introduction: chlorphenoxy derivatives are found in , -d, , , -t, mcpa, mcpp, and silvex. the ld of , -d is mg/kg; however, the toxic dose appears to be much lower in small treatment treatment of chlorphenoxy derivative toxicity is largely supportive in nature, as there is no known antidote. secure the patient's airway and administer supplemental oxygen, as necessary. control cns excitation with diazepam ( . mg/kg iv). intravenous crystalloid fluid diuresis and urinary alkalinization can promote elimination. administer gastroprotectant and antiemetic drugs, as needed. the toxic effects of chocolate are related to theobromine. various types of chocolate have different concentrations of theobromine and thus can cause clinical signs of toxicity with ingestion of varying amounts of chocolate, depending on the type. the toxic dose of theobromine is - mg/kg in dogs. milk chocolate contains mg/oz ( mg/ g) of chocolate, and has a low toxic potential. semisweet chocolate contains mg/oz ( mg/ g), and baking chocolate contains mg/oz ( mg/ g). semisweet and baking chocolate, being the most concentrated, have a moderate to severe toxic potential, even in large dogs.clinical signs of theobromine intoxication are associated with phosphodiesterase inhibition and include cns stimulation (tremors, anxiety, seizures), myocardial stimulation (tachycardia and tachyarrhythmias), diuresis, and (at very high doses) gastrointestinal ulceration. with treatment, the condition of most dogs returns to normal within to hours (t / = . hours in dogs). potential side effects include gastroenteritis and pancreatitis due to the fat content of the chocolate. treatment of chocolate toxicity includes obtaining and maintaining a protected airway (if necessary), intravenous fluid diuresis, induction of emesis or orogastric lavage followed by administration of repeated doses of activated charcoal, and placement of a urinary catheter to prevent reabsorption of the toxin from the urinary bladder. cholecalciferol rodenticide ingestion can lead to increased intestinal and renal reabsorption of calcium, causing an increase in serum calcium and dystrophic mineralization of the kidneys and liver at - mg/kg. clinical signs include lethargy, anorexia, vomiting, constipation, and renal pain within to days of ingestion. seizures, muscle twitching, and central nervous system depression may be observed at very high doses. as renal failure progresses, polyuria, polydipsia, vomiting/hematemesis, uremic oral ulcers, and melena may be observed. if the compound was ingested recently (within to hours) induce emesis or perform orogastric lavage, followed by administration of activated charcoal. check the patient's serum calcium once daily for three days following ingestion. if clinical signs of toxicity or hypercalcemia are present, decrease serum calcium with loop diuretics (furosemide, - mg/kg po or iv q h) and glucocorticosteroids (prednisone or prednisolone, - mg/kg po bid) to promote renal calcium excretion. in severe cases, salmon calcitonin ( - iu/kg sc q - h in dogs) or bisphosphonate compounds may be required. correct acid-base abnormalities with intravenous crystalloid fluid diuresis and sodium bicarbonate, if necessary. (see section on hypercalcemia.) denture cleaners contain sodium perborate as the active compound. sodium perborate can cause severe direct irritation of the mucous membranes and may also act as a cns depressant. clinical signs are similar to those seen if bleach or boric acid compound is ingested, namely vomiting, diarrhea, cns excitation then depression, and renal failure. treatment for ingestion of denture cleaner includes gastric decontamination along with induction of emesis or orogastric lavage and administration of a cathartic to hasten elimination. activated charcoal is not useful for treatment of ingestion of this toxin. administer intravenous fluid therapy to maintain renal perfusion. administer gastroprotectant and antiemetic drugs, as necessary. deodorants are usually composed of aluminum chloride and aluminum chlorohydrate. both have a moderate potential for toxicity. ingestion of deodorant compounds can cause oral irritation or necrosis, gastroenteritis, and nephrosis. treatment of deodorant ingestion includes orogastric lavage, and administration of antiemetic and gastroprotectant drugs. introduction anionic detergents include sulfonated or phosphorylated forms of benzene. dishwashing liquid is an example of an anionic detergent that can be toxic at doses of - g/kg. anionic detergents cause significant mucosal damage and edema, gastrointestinal irritation, cns depression, seizures, and possible hemolysis. ocular exposure can cause corneal ulcers and edema. treatment of anionic detergent exposure is largely symptomatic, as there is no known antidote. to treat topical toxicity, flush the patient's eyes and skin with warmed tap water or . % saline solution for a minimum of minutes, taking care to avoid hypothermia. to treat ingestion, feed the patient milk and large amounts of water to dilute the toxin. do not induce emesis, because of the risk of worsening esophageal irritation. to dilute the toxin, perform orogastric lavage, followed by administration of activated charcoal. closely monitor the patient's respiratory status, because oropharyngeal edema can be severe. if necessary, perform endotracheal intubation in cases of airway obstruction. monitor the patient for signs of intravascular hemolysis. administer intravenous crystalloid fluids to maintain hydration until the patient is able to tolerate oral fluids. cationic detergents and disinfectants include quaternary ammonia compounds, isopropyl alcohol, and isopropanol. quaternary ammonia compounds have a serious toxic potential treatment treatment of cationic detergent exposure includes careful bathing and ocular rinsing of the patient for a minimum of minutes, taking care to avoid hypotension. secure the patient's airway and monitor the patient's respiratory status. administer supplemental oxygen, if necessary. place an intravenous catheter and administer intravenous crystalloid fluids to maintain hydration. do not induce emesis, because of the risk of causing further esophageal irritation. give milk or large amounts of water orally, as tolerated by the patient, to dilute the toxin. nonionic detergents include alkyl and aryl polyether sulfates, alcohols, and sulfonates; alkyl phenol; polyethylene glycol; and phenol compounds. phenols are particularly toxic in cats and puppies. clinical signs of exposure include severe gastroenteritis and topical irritation. some compounds can be metabolized to glycolic and oxalic acid, causing renal damage similar to that observed with ethylene glycol toxicity. topical and ocular exposure should be treated with careful bathing or ocular irrigation for at least minutes. administer activated charcoal to prevent absorption of the compound. as tolerated, give dilute milk or straight tap water orally to dilute the compound. administer antiemetic and gastroprotectant drugs to control vomiting and decrease gastrointestinal irritation. administer intravenous crystalloid fluids to maintain hydration and decrease the potential for renal tubular damage. monitor the patient's acid-base and electrolyte status and correct any abnormalities with appropriate intravenous fluid therapy. introduction diclone (phigone) is a dipyridyl compound that is a cns depressant. the ld in rats is - mg/kg. dichlone reacts with thiol enzymes to cause methemoglobinemia and hepatorenal damage. to treat dichlone ingestion, induce emesis or perform orogastric lavage, followed by administration of activated charcoal and a cathartic. procure and maintain a patent airway. perform intravenous fluid diuresis to maintain renal perfusion. n-acetylcysteine may be useful in the treatment of methemoglobinemia. diethyltoluamide (deet) is the active ingredient in many insect repellants (e.g., off, cutters, hartz blockade). the mechanism of action of deet is not fully understood, but it acts as a lipophilic neurotoxin within to minutes of exposure. cats appear to be particularly sensitive to deet. a lethal dermal dose is . g/kg; if ingested, the lethal dose is much less. the toxic dose of dermal exposure in dogs is g/kg. clinical signs of toxicity include aimless gazing, hypersalivation, chewing motions, and muscle tremors that progress to seizures. recumbency and death can occur within minutes of exposure at high doses. treatment of deet toxicity is largely supportive, as there are no known antidotes. procure and maintain a patent airway and perform mechanical ventilation, if necessary. place an intravenous catheter and administer intravenous crystalloid fluids to control hydration and treat hypotension, as necessary. treat seizures with diazepam ( . mg/kg iv) or phenobarbital. because of the rapid onset of clinical signs, induction of emesis is contraindicated. perform orogastric lavage if the compound was ingested within the last hours. administer multiple repeated doses of activated charcoal. cooling measures should be implemented to control hyperthermia. if dermal exposure has occurred, bathe the patient thoroughly to avoid further exposure and absorption. diquat is a dipyridyl compound that is the active ingredient in some herbicide compounds. the ld of diquat is - mg/kg. like paraquat, diquat induces its toxic effects by causing the production of oxygen-derived free radical species. clinical signs of diquat intoxication include anorexia, vomiting, diarrhea, and acute renal failure. massive dehydration and electrolyte imbalances can occur as a result of fluid loss into the gastrointestinal tract. treatment of diquat intoxication is similar to that for paraquat ingestion. if the animal had ingested diquat within hour of presentation, induce emesis. in clinical cases, orogastric lavage may be required. both emesis and orogastric lavage should be followed by administration of kaolin or bentonite as an adsorbent, rather than activated charcoal. place an intravenous catheter and administer crystalloid fluids to restore volume status and maintain renal perfusion. monitor urine output. if oliguria or anuria occurs, treatment with mannitol, furosemide, and dopamine may be considered. ecstasy ( , -methylenedioxymethylamphetamine; mdma) is a recreational drug used by humans. ecstasy causes release of serotonin. clinical signs of intoxication are related to the serotonin syndrome (excitation, hyperthermia, tremors, and hypertension), and seizures may be observed. a urine drug screening test can be used to detect the presence of mdma. treatment of ecstasy intoxication is largely supportive, as there is no known antidote. administer intravenous fluids to maintain hydration, correct acid-base status, and treat hyperthermia. serotonin antagonist drugs (cyproheptadine) can be dissolved and administered per rectum to alleviate clinical signs. intravenous propranolol has additional antiserotonin effects. administer diazepam ( . - mg/kg iv) to control seizures. if cerebral edema is suspected, administer mannitol, followed by furosemide. ethylene glycol is most commonly found in antifreeze solutions but is also in some paints, photography developer solutions, and windshield wiper fluid. ethylene glycol in itself is only minimally toxic. however, when it is metabolized to glycolate, glyoxal, glyoxylate, and oxalate, the metabolites cause an increased anion gap metabolic acidosis and precipitation of calcium oxalate crystals in the renal tubules, renal failure, and (ultimately) death.the toxic dose in dogs is . ml/kg, and in cats is . ml/kg. the toxin is absorbed quite readily from the gastrointestinal tract and can be detected in the patient's serum within an hour of ingestion. colorimetric tests that can be performed in most veterinary hospitals can detect larger quantities of ethylene glycol in the patient's serum. in a dog with clinical treatment begin treatment of known ethylene glycol ingestion immediately. induce emesis or perform orogastric lavage and adminiser repeated doses of activated charcoal. place an intravenous catheter and perform crystalloid fluid diuresis with a known antidote. the treatment of choice for dogs is administration of -methylpyrrazole ( -mp), which directly inhibits alcohol dehydrogenase, thus preventing the conversion of ethylene glycol to its toxic metabolites. the dose for dogs is mg/kg initially, followed by mg/kg at and hours and mg/kg at hours. -mp has been used experimentally at . times the recommended dose for dogs. in cats, treatment with -mp is effective if it is administered within the first hours of ingestion.cats will demonstrate signs of sedation and hypothermia with this treatment. if -mp is not available, administer ethanol ( mg/kg iv loading dose, followed by mg/ kg/hour), or as a % solution (for dogs, . ml/kg iv q h for five treatments, then q h for five more treatments; for cats, ml/kg q h for four treatments). grain alcohol ( proof) contains approximately mg/ml of ethanol. antiemetics and gastroprotective agents should be considered. urinary alkalinization and peritoneal dialysis may enhance the elimination of ethylene glycol and its metabolites. many fertilizers are on the market, and may be composed of urea or ammonium salts, phosphates, nitrates, potash, and metal salts. fertilizers have a moderate toxic potential, depending on the type and amount ingested. clinical signs of fertilizer ingestion include vomiting, diarrhea, metabolic acidosis, and diuresis. nitrates or nitrites can cause formation of methemoglobin and chocolate-brown blood. electrolyte disturbances include hyperkalemia, hyperphosphatemia, hyperammonemia, and hyperosmolality. treatment of fertilizer ingestion includes cardiovascular support, and administration of milk or a mixture of egg whites and water, followed by induction of emesis or orogastric lavage. correct electrolyte abnormalities as they occur (see section on hyperkalemia). administer antiemetic and gastroprotectant drugs, as necessary. administer intravenous fluids to control hydration and maintain blood pressure. n-acetylcysteine may be useful if methemoglobinemia is present. fipronil is the active ingredient in frontline, a flea control product. fipronil exerts its effects by gaba antagonism and can cause cns excitation. treatment of fiprinol toxicity includes treatment of cns excitation, treatment of hyperthermia by cooling measures, and administration of activated charcoal. fire extinguisher fluid contains chlorobromomethane or methyl bromide, both of which have a serious toxic potential. dermal or ocular irritation can occur. if ingested, the compounds can be converted to methanol, and cause high anion gap metabolic acidosis, cns excitation and depression, aspiration pneumonitis, and hepatorenal damage. to treat ocular or dermal exposure to fire extinguisher fluids, flush the eyes or skin with warmed tap water or . % saline solution for a minimum of minutes. do not induce emesis or perform orogastric lavage to treat ingestion, because of the risk of causing severe aspiration pneumonitis. gastroprotectant and antiemetic drugs may be used, if indicated. administer intravenous fluids to maintain hydration and renal perfusion. supplemental oxygen or mechanical ventilation may be required in severe cases of aspiration pneumonitis. fireplace colors contain salts of heavy metals-namely, copper rubidium, cesium, lead, arsenic, antimony, barium, selenium, and zinc, all of which have moderate toxic potential, depending on the amount ingested and the size of the patient. clinical signs are largely associated with gastrointestinal irritation (vomiting, diarrhea, anorexia). zinc toxicity can cause intravascular hemolysis and hepatorenal damage. to treat ingestion of fireplace colors, administer cathartics and activated charcoal and gastroprotectant and antiemetic drugs. place an intravenous catheter for intravenous crystalloid fluid administration to maintain hydration and renal perfusion. specific chelating agents may be useful in hastening elimination of the heavy metals. fireworks contain oxidizing agents (nitrates and chlorates) and metals (mercury, copper, strontium, barium, and phosphorus). ingestion of fireworks can cause hemorrhagic gastroenteritis and methemoglobinemia. to treat firework ingestion, induce emesis or perform orogastric lavage and administer activated charcoal. administer specific chelating drugs if the amount and type of metal are known, and administer gastroprotectant and antiemetic drugs. if methemoglobinemia occurs, administer n-acetylcysteine; a blood transfusion may be necessary. introduction fuels such as barbecue lighter fluid, gasoline, kerosene, and oils (mineral, fuel, lubricating) are petroleum distillate products that have a low toxic potential if ingested but can cause severe aspiration pneumonitis if as little as ml is inhaled into the tracheobronchial tree. cns depression, mucosal damage, hepatorenal insufficiency, seizures, and corneal irritation can occur. if fuels are ingested, administer gastroprotectant and antiemetics drugs. do not induce emesis or perform orogastric lavage, because of the risk of aspiration pneumonia. to treat topical exposure, rinse the skin and eyes copiously with warm tap water or . % saline solution. administer antiemetic and gastroprotectant drugs, as necessary. administer intravenous fluids to maintain hydration and treat acid-base and electrolyte abnormalities. children's glue contains polyvinyl acetate, which has a very low toxic potential. if inhaled, the compound can cause pneumonitis. treatment of polyvinyl acetate should be performed as clinical signs of pneumonitis (increased respiratory effort, cough, lethargy, respiratory distress) occur. introduction superglue contains methyl- -cyanoacrylate, a compound that can cause severe dermal irritation on contact. do not induce emesis. do not bathe the animal, and do not apply other compounds (acetone, turpentine) in an attempt to remove the glue from the skin. the fur can be shaved, using care to avoid damaging the underlying skin. the affected area should be allowed to exfoliate naturally. glyophosate is a herbicide found in roundup and kleenup. if applied properly, the product has a very low toxic potential. clinical signs of toxicity include dermal and gastric irritation, including dermal erythema, anorexia, and vomiting. cns depression can occur. treatment includes thorough bathing in cases of dermal exposure, and induction of emesis or orogastric lavage followed by administration of activated charcoal. administer antiemetic and gastroprotectant drugs as necessary. administer intravenous crystalloid fluids to prevent dehydration secondary to vomiting. even small amounts of grapes and raisins can be toxic to dogs. the mechanism of toxicity remains unknown. clinical signs occur within hours of ingestion of raisins or grapes, and include vomiting, anorexia, lethargy, and diarrhea (often with visible raisins or grapes in the fecal matter). within hours, dogs demonstrate signs of acute renal failure (polyuria, polydipsia, vomiting) that can progress to anuria. to treat known ingestion of raisins or grapes, induce emesis or perform orogastric lavage, followed by repeated doses of activated charcoal. if clinical signs of vomiting and diarrhea are present, administer intravenous fluids and monitor urine output. aggressive intravenous fluid therapy, in conjunction with maintenance of renal perfusion, is necessary. in cases of anuric renal failure, dopamine, furosemide, and mannitol can be useful in increasing urine output. peritoneal or hemodialysis may be necessary in cases of severe oliguric or anuric renal failure. calcium channel blockers such as amlodipine and diltiazem can be used to treat systemic hypertension. supportive care includes treatment of hyperkalemia, and administration of gastroprotectant and antiemetic drugs and (if the animal is eating) phosphate binders. aromatic hydrocarbons include phenols, cresols, toluene, and naphthalene. all have a moderate toxic potential if ingested. toxicities associated with ingestion of aromatic hydrocarbons include cns depression, hepatorenal damage, muscle tremors, pneumonia, methemoglobinemia, and intravascular hemolysis. if an aromatic hydrocarbon is ingested, do not induce emesis, because of the risk of aspiration pneumonia. a dilute milk solution or water can be administered to dilute the compound. perform orogastric lavage. carefully monitor the patient's respiratory and cardiovascular status. administer supplemental oxygen if aspiration pneumonia is present. to treat topical exposure, thoroughly rinse the eyes and skin with copious amounts of warm tap water or . % saline solution. imidacloprid is the compound used in the flea product advantage. clinical signs of toxicity are related to nicotinic cholinergic stimulation, causing neuromuscular excitation followed by collapse. the compound may induce respiratory paralysis. to treat imidacloprid toxicity, procure and maintain a patent airway with supplemental oxygen administration. control cns excitation with diazepam, phenobarbital, or propofol. administer enemas to hasten gastrointestinal elimination, and administer activated charcoal. bathe the animal thoroughly to prevent further dermal absorption. closely monitor the patient's oxygenation and ventilation status. if severe hypoventilation or respiratory paralysis occurs, initiate mechanical ventilation. iron and iron salts can cause severe gastroenteritis, myocardial toxicity, and hepatic damage if high enough doses are ingested. lawn fertilizers are a common source of iron salts. treatment of ingestion of iron and iron salts includes cardiovascular support in the form of intravenous fluids and antiarrhythmic drugs, as needed. induce emesis or perform orogastric lavage for gastric decontamination. a cathartic can be administered to promote elimination from the gastrointestinal tract. antiemetic and gastroprotectant drugs should be administered to prevent nausea and vomiting. in some cases, radiographs can aid in making a diagnosis of whether the compound was actually ingested. iron toxicity can be treated with the chelating agent deferoxamine. ivermectin is a gaba agonist that is used in commercial heartworm prevention and antihelminthic compounds and can be toxic in predisposed breeds, including collies, collie loperamide is an opioid derivative that is used to treat diarrhea. clinical signs of loperamide intoxication include constipation, ataxia, nausea, and sedation. induce emesis or perform orogastric lavage, followed by administration of activated charcoal and a cathartic. naloxone may be beneficial in the temporary reversal of ataxia and sedation. ingestion of macadamia nuts can cause clinical signs of vomiting, ataxia, and ascending paralysis in dogs. the toxic principle in macadamia nuts is unknown. there is no known antidote. treatment consists of supportive care, including administration of intravenous fluids and antiemetics and placement of a urinary catheter for patient cleanliness. clinical signs resolve in most cases within hours. marijuana is a hallucinogen that can cause cns depression, ataxia, mydriasis, increased sensitivity to motion or sound, salivation, and tremors. along with these findings, a classic clinical sign is the sudden onset of dribbling urine. urine can be tested with drug test kits for tetrahydrocannabinoid (thc), the toxic compound in marijuana. there is no known antidote for marijuana toxicity; therefore, treatment is largely symptomatic. place an intravenous catheter and administer intravenous fluids to support hydration. administer atropine if severe bradycardia exists. induction of emesis can be attempted but because of the antiemetic effects of thc, is usually unsuccessful. orogastric lavage can be performed, followed by repeated doses of activated charcoal. clinical signs usually resolve within to hours. introduction "strike anywhere" matches, safety matches, and the striking surface of matchbook covers contain iron phosphorus or potassium chlorate. both compounds have a low toxic potential but can cause clinical signs of gastroenteritis and methemoglobinemia if large quantities are ingested. treatment of match and matchbook ingestion includes gastric decontamination with induction of emesis or orogastric lavage and administration of activated charcoal and a cathartic. if methemoglobinemia occurs, administer n-acetylcysteine, intravenous fluids, and supplemental oxygen. metaldehyde is the active ingredient in most brands of snail bait. the exact mechanism of toxicity is unknown but may involve inhibition of gaba channels. clinical signs associated with metaldehyde toxicity include severe muscle tremors, cns excitation, and treatment treatment of mushroom toxicity is largely supportive. if the mushroom was ingested within the last hours, induce emesis or perform orogastric lavage and then administer activated charcoal. symptomatic treatment includes intravenous fluids to promote diuresis and treat hyperthermia and skeletal muscle relaxants to control tremors and seizures (methocarbamol, diazepam). if amanita ingestion is suspected, administer hepatoprotectant agents including milk thistle. mycotoxins from penicillium spp. are found in moldy foods, cream cheese, and nuts. clinical signs of intoxication include tremors, agitation, hyperesthesia, and seizures. if tremorigenic mycotoxin toxicity is suspected, a sample of the patient's serum and gastric contents or vomitus can be submitted to the michigan state university veterinary toxicology laboratory for tremorigen assay. there is no known antidote. perform orogastric lavage, followed by administration of activated charcoal. control tremors and seizures with methocarbamol, diazepam, phenobarbital, or pentobarbital. administer intravenous fluids to control hyperthermia and maintain hydration. in cases in which cerebral edema is suspected secondary to severe refractory seizures, administer intravenous mannitol and furosemide. naphthalene is the active ingredient in mothballs and has a high toxic potential. clinical signs associated with naphthalene toxicity include vomiting, methemoglobinemia, cns stimulation, seizures, and hepatic toxicity. a complete blood count often reveals heinz bodies and anemia. do not induce emesis if naphthalene ingestion is suspected. if the ingestion was within hour of presentation, perform orogastric lavage. control seizures with diazepam or phenobarbital. administer intravenous fluids to control hyperthermia and maintain hydration. n-acetylcysteine can play a role in the treatment of methemoglobinemia. a packed rbc transfusion may be necessary if anemia is severe. observe the patient for clinical signs associated with hepatitis. nicotine toxicity occurs in animals as the result of ingestion of cigarettes, nicotine-containing gum, and some insecticides. nicotine stimulates autonomic ganglia at low doses, and blocks autonomic ganglia and the neuromuscular junction at high doses. absorption after ingestion is rapid. clinical signs include hyperexcitability and slud (salivation, lacrimation, urination, and defecation). muscle tremors, respiratory muscle fatigue or hypoventilation, tachyarrhythmias, seizures, coma, and death can occur. if the patient presents within hour of ingestion and has no clinical signs, induce emesis, followed by administration of repeated doses of activated charcoal. in patients with clinical signs of toxicity, perform orogastric lavage. administer intravenous fluids to maintain hydration and promote diuresis, and treat hyperthermia. administer atropine to treat cholinergic symptoms. urinary acidification can promote nicotine excretion. nonsteroidal antiinflammatory drugs (nsaids) include ibuprofen, ketoprofen, carprofen, diclofenac, naproxen, celecoxib, valdecoxib, rofecoxib, and deracoxib. nsaids cause inhibition of prostaglandin synthesis, leading to gastrointestinal ulceration, renal failure and hepatotoxicity. ibuprofen toxicity has been associated with seizures in dogs, cats, and ferrets. the toxic dose varies with the specific compound ingested. to treat nsaid toxicity, induce emesis or perform orogastric lavage, followed by administration of multiple repeated doses of activated charcoal. place an intravenous catheter for crystalloid fluid diuresis to maintain renal perfusion. administer the synthetic prostaglandin analogue misoprostol to help maintain gastric and renal perfusion. control seizures, if present, with intravenous diazepam. administer gastroprotectant and antiemetic drugs to control vomiting and gastrointestinal hemorrhage. continue intravenous fluid diuresis for a minimum of hours, with frequent monitoring of the patient's bun and creatinine. when the bun and creatinine levels are normal or have plateaued for hours, slowly decrease fluid diuresis % per day until maintenance levels are restored. onions, garlic, and chives contain sulfoxide compounds that can cause oxidative damage of rbcs, leading to heinz body anemia, methemoglobinemia, and intravascular hemolysis. clinical signs of toxicity include weakness, lethargy, tachypnea, tachycardia, and pale mucous membranes. vomiting and diarrhea can occur. intravascular hemolysis can cause treatment treatment of onion, chive, and garlic toxicity includes administration of intravenous fluid diuresis, and induction of emesis or orogastric lavage, followed by administration of activated charcoal and a cathartic. in cases of severe anemia, packed rbc transfusion or administration of a hemoglobin-based oxygen carrier should be considered. opiate drugs include heroin, morphine, oxymorphone, fentanyl, meperidine, and codeine. opiate compounds bind to specific opioid receptors throughout the body and produce clinical signs of miosis or mydriasis (cats), and cns excitation, followed by ataxia and cns depression, leading to stupor and coma. hypoventilation, bradycardia, hypoxia, and cyanosis can occur. to treat known overdose or ingestion of an opiate compound, induce emesis (in asymptomatic animals) or perform orogastric lavage, followed by administration of activated charcoal. administer intravenous fluids and supplemental oxygen to support the cardiovascular and respiratory systems. mechanical ventilation may be necessary until hypoventilation resolves. administer repeated doses of naloxone as a specific antidote to reverse clinical signs of narcosis and hypoventilation. if seizures are present (meperidine toxicity), administer diazepam. organophosphate compounds traditionally are used in flea control products and insecticides. common examples of organophosphates include chlorpyrifos, coumaphos, diazinon, dichlorvos, and malathion. the toxic dose varies, depending on the particular compound and individual animal sensitivity. organophosphate toxicity causes acetylcholinesterase inhibition, resulting in clinical signs of cns stimulation, including tremors and seizures. muscarinic acetylcholine overload causes the classic slud signs of salivation, lacrimation, urination, and defecation. miosis, excessive bronchial secretions, muscle tremors, and respiratory paralysis can occur. an intermediate syndrome of generalized weakness, hypoventilation, and eventual paralysis with ventral cervical ventroflexion that may require mechanical ventilation has been described. if organophosphate toxicity is suspected, whole-blood acetylcholinesterase activity can be measured and will be low. treatment of toxicity includes careful and thorough bathing in cases of dermal exposure and, if the substance was ingested, gastric decontamination with induction of emesis or orogastric lavage, followed by administration of activated charcoal, and administration of the antidote pralidoxime hydrochloride . atropine can help control the muscarinic clinical signs. supportive care in the form of cooling measures, intravenous crystalloid fluids, and supplemental oxygen or mechanical ventilation may be required, depending on the severity of clinical signs. introduction ingestion of large amounts of paintballs can cause neurologic signs, electrolyte abnormalities, and occasionally death. paintballs are gelatin capsules that contain multiple colors of if ingestion was recent and if no clinical signs of toxicity are present, induce emesis or perform orogastric lavage, followed by administration of a cathartic and activated charcoal. there is no known antidote. treatment includes supportive care in the form of intravenous fluids and administration of phenobarbital or methocarbamol to control seizures and tremors. diazepam, a gaba agonist, is contraindicated, because it can potentially worsen clinical signs. urine acidification may hasten elimination. clinical signs can last from to days. pyrethrin and pyrethroid compounds are extracted from chrysanthemums, and include allethrin, decamethrin, tralomethrin, fenpropanthrin, pallethrin, sumethrin, permethrin, tetramethrin, cyfluthrin, and resemethrin. the oral toxicity is fairly low; however, the compounds can be significantly harmful if inhaled or applied to the skin. pyrethrin and pyrethroid compounds cause depolarization and blockade of nerve membrane potentials, causing clinical signs of tremors, seizures, respiratory distress, and paralysis. contact dermatitis can occur. to distinguish between pyrethrin/pyrethroid toxicity and organophosphate toxicity, acetylcholinesterase levels should be obtained; they will be normal if pyrethrins are the cause of the animal's clinical signs. treatment of toxicity is supportive, as there is no known antidote. carefully bathe the animal in lukewarm water to prevent further oral and dermal exposure. both hyperthermia and hypothermia can worsen clinical signs. administer activated charcoal to decrease enterohepatic recirculation. atropine may control clinical signs of excessive salivation. to control muscle tremors, administer methocarbamol to effect. administer diazepam or phenobarbital to control seizures, as necessary. rotenone is used as a common garden and delousing insecticide. fish and birds are very susceptible to rotenone toxicity. rotenone inhibits mitochondrial electron transport. clinical signs of tissue irritation and hypoglycemia can occur after topical or oral exposure. if the compound is inhaled, cns depression and seizures can occur. to treat toxicity, perform orogastric lavage, followed by administration of a cathartic and activated charcoal. bathe the animal carefully to prevent further dermal exposure and further ingestion. administer diazepam or phenobarbital to control seizures. the prognosis generally is guarded. treatment of ingestion includes dilution with milk, water, or egg whites. perform orogastric lavage, followed by administration of activated charcoal. administer intravenous crystalloid fluids to maintain hydration. administer antiemetic and gastroprotectant drugs to treat gastroenteritis and vomiting.shampoos, nonmedicated: see detergents, nonionic shampoos, selenium sulfide introduction selenium sulfide shampoos (e.g., selsun blue) have a low toxic potential, and primarily cause gastroenteritis. treatment of ingestion includes dilution with water, milk, or egg whites and administration of activated charcoal. carefully and thoroughly rinse the skin and eyes to prevent further exposure. administer antiemetic and gastroprotectant drugs in cases of severe gastroenteritis. zinc-based (zinc pyridinethione) anti-dandruff shampoos have a serious toxic potential if ingested or if ocular exposure occurs. gastrointestinal irritation, retinal detachment, progressive blindness, and exudative chorioretinitis can occur. treatment of ingestion includes gastric decontamination. induce emesis or perform orogastric lavage, followed by administration of a cathartic and activated charcoal.to treat ocular exposure, thoroughly rinse the patient's eyes for a minimum of minutes. carefully monitor the animal for clinical signs of blindness. implement intravenous fluid to maintain hydration and renal perfusion in cases of severe gastroenteritis. silver polish contains the alkali substance sodium carbonate and cyanide salts, and has a serious toxic potential. ingestion results in rapid onset of vomiting and possibly cyanide toxicity. to treat ingestion, monitor and maintain the patient's respiration and cardiovascular status and administer intravenous crystalloid fluids. induce emesis, followed by administration of activated charcoal. administer sodium nitrite or sodium thiosulfate iv for cyanide toxicity. bath soap (bar soap) usually has low toxic potential and causes mild gastroenteritis with vomiting if ingested. to treat ingestion, include dilution with water, administration of intravenous fluids to maintain hydration, and administration of antiemetic and gastroprotectant drugs to treat gastroenteritis. sodium fluoroacetate is a colorless, odorless, tasteless compound that causes uncoupling of oxidative phosphorylation. the toxic dose in dogs and cats is . - . mg/kg. clinical signs of toxicity include cns excitation, seizures, and coma secondary to cerebral edema. the prognosis is guarded. to treat toxicity, procure and maintain a patent airway, monitor and stabilize the cardiovascular status, and control hyperthermia. perform orogastric lavage, followed by administration of activated charcoal. if clinical signs are not present at the time of presentation, induce emesis. administer intravenous fluids and supplemental oxygen, as necessary. strattera (atomoxetine hydrochloride) is a selective norepinephrine reuptake inhibitor used in the treatment of attention deficit hyperactivity disorder (adhd) in humans. peak serum concentrations occur in dogs within to hours of ingestion, with a peak half-life at to hours following ingestion. clinical signs of toxicity include cardiac tachyarrhythmias, hypertension, disorientation, agitation, trembling, tremors, and hyperthermia. treatment of intoxication is largely symptomatic and supportive in nature. first, induce emesis if the patient is conscious and has an intact gag reflex. orogastric lavage can also be performed. administer one dose of activated charcoal to prevent further absorption of the compound from the gastrointestinal tract. identify cardiac dysrhythmias and treat accordingly. control hypertension with sodium nitroprusside or diltiazem as a constant rate infusion. administer acepromazine or chlorpromazine to control agitation. do not use diazepam, because it can potentially worsen clinical signs. administer intravenous fluids to maintain hydration and promote diuresis. strychnine is the active ingredient in pesticides used to control rodents and other vermin. the toxic dose in dogs is . mg/kg, and in cats is mg/kg. strychnine antagonizes spinal inhibitory neurotransmitters and causes severe muscle tremors, muscle rigidity, and seizures. clinical signs are stimulated or exacerbated by noise, touch, light, and sound. mydriasis, hyperthermia, and respiratory paralysis can occur. if strychnine toxicity is suspected, gastric contents should be collected and saved for analysis. if the animal is asymptomatic at the time of presentation, induce emesis. if clinical signs are present, perform orogastric lavage. both emesis and orogastric lavage should be followed by the administration of activated charcoal. administer intravenous crystalloid fluids to support the cardiovascular system, aid in cooling measures, and improve renal diuresis. treat cns stimulation with methocarbamol, diazepam, or phenobarbital. the animal should have cotton packed in its ears to prevent noise stimulation, and should be placed in a quiet, dark room. treatment of ingestion includes dilution with milk of magnesia or water, administration of antiemetic and gastroprotectant drugs, and administration of intravenous crystalloid fluids to maintain hydration. do not induce emesis, because of the risk of causing further esophageal irritation.sunscreen: see zinc and zinc oxide suntan lotion: see shampoos, zinc-based, and alcohols tar: see fuels tea tree oil (melaleuca oil) introduction tea tree (melaleuca) oil is an herbal-origin flea-control product. the toxic principles in tea tree oil are monoterpenes, which produce clinical signs of neuromuscular weakness, and ataxia. treatment of tea tree oil toxicity includes administration of cathartics and activated charcoal to prevent further absorption. carefully bathe the animal to prevent further dermal exposure. tetanus spores from clostridium tetani organisms are ubiquitous in the soil and feces, particularly in barnyards. cases have been reported in dogs after tooth eruption and after abdominal surgeries performed with cold sterilization packs. anaerobic wound infections can contain tetanus spores. the neurotoxin from c. tetani inhibits spinal inhibitory neurons, causing motor neuron excitation. extensor muscle rigidity ("sawhorse stance"), erect ears, and risus sardonicus (a sardonic grin) are characteristic features of tetanus. administer tetanus antitoxin if toxin has not already been bound in the cns. to eliminate the source of the toxin (e.g., abscess), open and debride all wounds. intravenous administration of ampicillin or penicillin g is the treatment of choice for tetanus. supportive care in the form of skeletal muscle relaxants, intravenous fluids and parenteral nutrition, and nursing care to prevent decubitus ulcer formation is required. in extreme cases, mechanical ventilation may be necessary. triazene compounds include atrazine, prometone, and monuron (telvar). the toxic mechanism of triazene compounds is unknown. clinical signs of toxicity include salivation, ataxia, hyporeflexia, contact dermatitis, hepatorenal damage, muscle spasms, respiratory difficulty, and death. treatment of triazene exposure includes cardiovascular and renal support in the form of intravenous crystalloid fluids, inotropic drugs, and antiarrhythmic agents, as necessary. if the exposure is recent, induce emesis. perform orogastric lavage in animals that cannot protect the airway. emesis and orogastric lavage should be followed by the administration of activated charcoal and a cathartic. carefully bathe the patient to prevent further dermal absorption. a variety of tricyclic antidepressants are available for use in both humans and animals, including amitriptyline, amoxapine, desipramine, doxepine, fluoxetine (prozac), fluvoxamine (luvox), imipramine, nortriptyline, paroxetine (paxil), protriptyline, sertraline (zoloft), and trimipramine. selective serotonin reuptake inhibitors (ssris) are rapidly absorbed from the digestive tract, with peak serum concentrations occurring to hours after ingestion. the elimination half-life for each drug differs in dogs, but typically last to hours. ssris inhibit the reuptake of serotonin, causing serotonin to accumulate in the brain. this can cause "serotonin syndrome," characterized by trembling, seizures, hyperthermia, ptyalism or hypersalivation, cramping or abdominal pain, vomiting, and diarrhea. other clinical signs of ssri intoxication include depression, tremors, bradycardia, tachyarrhythmias, and anorexia. any animal that has ingested an ssri should be promptly treated and carefully observed for at least hours for side effects. the treatment of suspected ssri intoxication involves gastric decontamination if the patient is not depressed and has an intact gag reflex. perform orogastric lavage and administer activated charcoal to prevent further toxin absorption and hasten elimination from the gastrointestinal tract. treat other clinical signs symptomatically. administer intravenous diazepam to control seizures. treat tachyarrhythmias according to type. administer methocarbamol to control muscle tremors. cyproheptadine ( mg/kg), a serotonin antagonist, can be dissolved in water and administered per rectum. vitamin k antagonist rodenticides, which are commonly found in pelleted or block form, inhibit the activation of the vitamin k-dependent coagulation factors ii, vii, ix, and x. clinical signs of hemorrhage occur within to days of exposure. hemorrhage can occur anywhere in the body, and can be manifested as petechiation of the skin or mucous membranes, hemorrhagic sclera, epistaxis, pulmonary parenchymal or pleural hemorrhage, gastrointestinal hemorrhage, pericardial hemorrhage, hematuria, retroperitoneal hemorrhage, hemarthrosis, and central nervous system hemorrhage. clinical signs include respiratory distress, cough, bleeding from the gums or into the eyes, ataxia, paresis, paralysis, seizures, hematuria, joint swelling, lameness, lethargy, weakness, inappetence, and collapse.diagnosis is made based on clinical signs and a prolonged activated clotting time, or prothrombin time. the pivka (proteins induced by vitamin k antagonism) test may be helpful but usually cannot be performed in-house. slight thrombocytopenia may be present secondary to hemorrhage; however, blood levels usually do not reach the critical level of < , platelets/µl to cause clinical signs of hemorrhage. in some cases, severe stressinduced hyperglycemia and glucosuria may be present but resolves within hours. if the rodenticide was ingested within the last hours, induce emesis. alternatively, orogastric lavage can be performed in an uncooperative patient. both emesis and orogastric lavage should be followed by administration of activated charcoal. the stomach contents can be submitted for analysis. following successful treatment, administer oral vitamin k for days after the exposure; or a check prothrombin time days after gastric decontamination. if the prothrombin time is prolonged, administer fresh frozen plasma and vitamin k.if the prothrombin time is normal, gastric decontamination was successful, and no further treatment is necessary.if an animal presents with clinical signs of intoxication, administer activated clotting factors in the form of fresh frozen plasma ( ml/kg), and vitamin k ( mg/kg sq in multiple sites with a -gauge needle). packed rbcs or fresh whole blood may be required if the patient is also anemic. supportive care in the form of supplemental oxygen may be necessary in cases of pulmonary or pleural hemorrhage. following initial therapy and discharge, the patient should receive vitamin k ( . mg/kg po q - h for days), and prothrombin time should be checked days after the last vitamin k capsule is administered. in some cases, depending on the type of anticoagulant ingested, an additional weeks of vitamin k therapy may be required. xylitol is a sugar alcohol that, when ingested by humans, does not cause a significant increase in blood glucose, and therefore does not stimulate insulin release from the human pancreas. in dogs, however, xylitol causes a massive rapid and dose-dependent release of insulin from pancreatic beta-cells. following insulin release, clinically significant hypoglycemia can develop, followed by signs of vomiting, weakness, ataxia, mental depression, hypokalemia, hypoglycemic seizures, and coma. clinical signs associated with xylitol ingestion can be seen within minutes of ingestion and can last for more than hours, even with aggressive treatment. known xylitol ingestion should be treated as for other toxin ingestion. if no neurologic abnormalities exist at the time the patient is seen, induce emesis, followed by administration of activated charcoal. it remains unknown at this time whether activated charcoal actually delays or prevents the absorption of xylitol from the canine gastrointestinal tract. if clinical signs have already developed, perform orogastric lavage and gastric decontamination. blood glucose concentrations should be analyzed and maintained with supplemental dextrose as a constant rate infusion ( . %- %) until normoglycemia can be maintained with multiple frequent small meals. hypokalemia may develop because it is driven intracellularly by the actions of insulin. treat hypokalemia with supplemental potassium chloride by infusion, not to exceed . meq/kg/hour. pennies minted in the u.s. after contain large amounts of zinc rather than copper. other sources of zinc include zinc oxide ointment and hardware such as that found in metal bird cages. zinc toxicity causes intravascular hemolysis, anemia, gastroenteritis, and renal failure. if zinc toxicity is suspected, take an abdominal radiograph to document the presence of the metal in the stomach or intestines. (if zinc-containing ointment was ingested, this will not be visible on radiographs.) induce emesis or perform orogastric lavage, depending on the size of the object ingested. often, small objects such as pennies can be retrieved using endoscopy or surgical gastrotomy/enterotomy. always take an additional radiograph after the removal procedure to ensure that all objects have been successfully removed. administer intravenous fluids to maintain renal perfusion and promote fluid diuresis. administer gastroprotectant and antiemetic drugs. chelation therapy with succimer, calcium edta, dimercaprol, or penicillamine may be necessary. do not administer pulmonary contusions are a common sequela of blunt traumatic injury. a contusion basically is a bruise characterized by edema, hemorrhage, and vascular injury. contusions may be present at the time of presentation or can develop over the first hours after injury. a diagnosis of pulmonary contusion can be made based on auscultation of pulmonary crackles, presence of respiratory distress, and the presence of patchy interstitial to alveolar infiltrates on thoracic radiographs. radiographic signs can lag behind the development of clinical signs of respiratory distress and hypoxemia by hours. in most cases, cage rest is sufficient to temporarily diminish blood loss. sedation (acepromazine, . - . mg/kg iv, im, sq) may be helpful in alleviating anxiety and decreasing blood pressure. the hypotensive effects of acepromazine are potentially harmful if severe blood loss has occurred. if evidence of hypovolemia is present (see section on hypovolemic shock), intravenous fluid resuscitation should be administered. rapid assessment of clotting ability, with a platelet count estimate and clotting profile (act or aptt and pt), should be performed. if epistaxis secondary to vitamin k antagonist rodenticide intoxication is suspected, administer vitamin k and fresh frozen plasma or fresh whole blood.persistent hemorrhage from a nasal disorder can be treated with dilute epinephrine ( : , ) into the nasal cavity with the nose pointed toward the ceiling to promote vasoconstriction. if this fails, the animal can be anesthetized, and the nasal cavity packed with gauze, and the caudal oropharynx and external nares covered with umbilical tape to control hemorrhage. a rhinoscopy should be performed to determine the cause of ongoing hemorrhage. continued excessive hemorrhage can be controlled with ligation of the carotid artery on the side of the hemorrhage, or with percutaneous arterial embolization. systemic thromboembolism is most commonly recognized in cats with cardiomyopathies (hypertrophic, restrictive, unclassified, and dilatative) but can also occur in dogs with hyperadrenocorticism, disseminated intravascular coagulation (dic), systemic inflammatory response syndrome (sirs), protein-losing enteropathy and nephropathy, and tumors affecting the aorta and vena cava. thrombosis occurs through a complex series of mechanisms when the components of virchow's triad (hypercoaguable state, sluggish blood flow, and vascular endothelial injury or damage) are present. in cats, blood flow through a severely stretched left atrium is a predisposing factor to the development of clots and thromboembolism.the most common site of embolism is the aortic bifurcation, or "saddle thrombus." other, less common locations of thromboembolism include the forelimbs, kidneys, gastrointestinal tract, and cerebrum. diagnosis usually is made based on clinical signs of cool extremities, the presence of a cardiac murmur or gallop rhythm, auscultation of pulmonary crackles resulting from pulmonary edema, acute pain or paralysis of one or more peripheral extremities, respiratory distress, and pain and lack of a palpable pulse in affected limbs. the affected nailbeds and paw pads are cyanotic, and nails do not bleed when cut with a nail clipper.client education is one of the most important aspects of emergency management of the patient with thromboembolic disease. concurrent congestive heart failure (chf) occurs in % to % of cats with arterial thromboembolism. more than % of cats are euthanized during the initial thromboembolic event because of the poor long-term prognosis and the high risk of recurrence within days to months after the initial event, even with aggressive therapy. although the long-term prognosis varies from months to years after initial diagnosis and treatment, in the majority of cats thromboembolic disease recurs within months. rectal temperature hypothermia and bradycardia on presentation are negative prognostic indicators.immediate treatment of a patient with chf and thromboembolic disease involves management of the chf with furosemide, oxygen, and vasodilators (nitroglycerine paste, morphine, nitroprusside). additional management includes analgesia (butorphanol, . - . mg/kg iv, im) and prevention of further clot formation. aspirin ( mg/kg po q h) is beneficial bcause of its antiplatelet effects. heparin works in conjunction with antithrombin to prevent further clot formation ( - units/kg iv, followed by - units/kg sq q h in cats, and - units/kg sq q h in dogs). acepromazine can cause peripheral vasodilation and decreased afterload but also can promote hypotension in a patient with concurrent chf. acepromazine ( . - . mg/kg sq) should be used with extreme caution, if at all.thrombolytic therapy can also be attempted, but in most cases is not without risk, and may be cost-prohibitive for many clients. streptokinase ( , units iv over minutes and then , units/hour iv cri for hours) was administered with some success in cats; however, many died of hyperkalemia or other complications during the infusion. tissue plasminogen activator ( . - mg /kg/hour iv cri, up to mg/kg total dose, to effect) has been used with some success but is cost-prohibitive for most clients. side effects of thrombolytic therapy include hyperkalemia with reperfusion and hemorrhage.in cats, the primary cause of arterial thromboembolism is cardiomyopathy. once an animal is determined to be stable enough for diagnostic procedures, lateral and dv thoracic radiographs and an echocardiogram should be performed. ultrasound of the distal aorta and renal arteries should also be performed to determine the location of the clot and help establish the prognosis.other diagnostic procedures to evaluate the presence and cause of thromboembolism include a complete blood count, serum biochemistry profile, urinalysis (to rule out proteinlosing nephropathy), urine protein:creatinine ratio, antithrombin levels, acth stimulation test (to rule out hyperadrenocorticism), heartworm antigen test (in dogs), thyroid profile (to rule out hyperthyroidism in cats, and hypothyroidism in dogs), thoracic radiographs, arterial blood gas analyses, coagulation tests, and coombs' test. selective and nonselective angiography can also be performed to determine the exact location of the thrombus.long-term management of thromboembolism involves management of the underlying disease process and preventing further clot formation. begin therapy with heparin until the aptt becomes prolonged . times; then administer warfarin ( . - . mg/kg/day). monitoring therapy based on prothrombin time and the international normalized ratio (inr, . - . ) is recommended. low-dose aspirin ( - mg/kg q h) also has been recommended. physical therapy with warm water bathing, deep muscle massage, and passive range-of-motion exercises should be performed until the patient regains motor function. future therapy may involve the use of platelet receptor antagonists to prevent platelet activation and adhesion. key: cord- - xrdv m authors: nowland, megan h.; brammer, david w.; garcia, alexis; rush, howard g. title: biology and diseases of rabbits date: - - journal: laboratory animal medicine doi: . /b - - - - . - sha: doc_id: cord_uid: xrdv m beginning in , an inbred rabbit colony was developed at the phipps institute for the study, treatment and prevention of tuberculosis at the university of pennsylvania. this colony was used to study natural resistance to infection with tuberculosis (robertson et al., ). other inbred colonies or well-defined breeding colonies were also developed at the university of illinois college of medicine center for genetics, the laboratories of the international health division of the rockefeller foundation, the university of utrecht in the netherlands, and jackson laboratories. these colonies were moved or closed in the years to follow. since , the u.s. department of agriculture has reported the total number of certain species of animals used by registered research facilities ( ). in , , rabbits were used in research. there has been an overall decrease in numbers of rabbits used. this decreasing trend started in the mid- s. in , , rabbits were used in research. despite the overall drop in the number used in research, the rabbit is still a valuable model and tool for many disciplines. the rabbit has been utilized in immunology research for many years especially in regard to the structure of immunoglobulins and the genetic control of their formation. in addition, the rabbit is commonly used for the production of polyclonal antibodies for use as immunologic reagents (mage, ; pinheiro et al., ) . the relatively large body size and blood volume, easy access to the vascular system, and an existent large body of information on the purification of rabbit immunoglobulins are a few reasons the rabbit is preferred over other common laboratory animal species for polyclonal antibody production (stills, ) . the organization of the lymphoid system of the rabbit is comparable to that of other mammals. however, the rabbit does possess two gut-associated lymphoid tissues (galt) with specialized functions in the maturation of igm + b cells. these are the vermiform appendix at the distal end of the cecum and the sacculus rotundus at the ileocecal junction (mage, ) . for many years, the lack of rabbit-specific immunological reagents has limited the study of inflammation and immunity in the rabbit. the use of real-time polymerase chain reaction (rt-pcr) techniques has overcome this limitation and permitted such studies in many species other than man and mice. a quantitative real-time rt-pcr assay for measuring mrna for rabbit cytokines ifn-γ, il- , il- , il- , and tnf-α has been described (godornes et al., ) . recently, schnupf and sansonetti ( ) reported on rt-pcr primer pairs for analysis of three chemokines and ccl ) and cytokines (il- β, il- , il- , il- , il- , il- p , il p , il- a, il- f, . the profile of cytokines in the rabbit appears similar to other mammals. in mice and humans, the primary antibody repertoire is created by combinatorial rearrangement of a large number of immunoglobulin gene segments. other species (chicken, sheep, cattle, and rabbit) that have a limited number of gene segments utilize somatic gene conversion and/or somatic hypermutation (pinheiro et al., ) . in the former, a portion of the immunoglobulin gene is replaced with a gene sequence from a nonfunctional pseudogene. in the latter, single-nucleotide changes are made in the immunoglobulin genes (jenne et al., ) . in the rabbit, gene diversification occurs initially in the fetus and the neonate in sites such as the bone marrow. subsequently, between and weeks of age, immature igm + b cells undergo further diversification in the galt (appendix, sacculus rotundus, and peyer's patches) (mage et al., ; pinheiro et al., ) . furthermore, certain species of intestinal bacteria (bacteroides fragilis and bacillus subtilis) are required for appendix follicle development and antibody diversification to occur (hanson and lanning, ; mage et al., ) . most mammals express five classes of immunoglobulins: igm, igd, igg, iga, and ige. however, the rabbit lacks igd (sun et al., ) . the area of cardiovascular research has used the rabbit in a variety of different models. numerous dietary modifications will induce or exacerbate cholesterolinduced atherosclerosis in the rabbit. a brief overview of some of these dietary modifications can be found elsewhere (jayo et al., ) . research efforts into cholesterol metabolism have used the watanabe heritable hyperlipidemic (whhl) (atkinson et al., ; kita et al., ) and the st. thomas hospital strain rabbits (laville et al., ) . the whhl rabbit has a marked deficiency of low-density lipoprotein (ldl) receptors in the liver and other tissues. selective breeding of the whhl rabbit will increase the incidence of coronary artery atherosclerosis without increasing the incidence of aortic atherosclerosis (watanabe et al., ) . in contrast, the st. thomas hospital strain has a normal functioning ldl receptor but still maintains a hypercholesterolemic state (laville et al., ) . genetically modified rabbits have been created via both intracytoplasmic injection (li et al., ) and retroviral vectors (hiripi et al., ) . this has resulted in a multitude of new strains to address interesting research questions. cardiovascular disease (lombardi et al., ; peng, ; sanbe et al., ; stanley et al., ) including models of long qt interval for exploration of treatments (biermann et al., ; jindal et al., ; liu et al., a; peng, ; sanbe et al., ; ziv et al., ) and atherosclerosis (araki et al., ; masson et al., ; tjwa et al., ) are the main focus of model development. strains have also been developed that express human recombinant proteins in rabbit milk (chrenek et al., ; dragin et al., ; hiripi et al., ; houser et al., ; lipinski et al., ; simon et al., ; soler et al., ) . this ability can be passed down for multiple generations (chrenek et al., ; dragin et al., ) . these human proteins have resulted in antigen production for rotavirus vaccine creation, human factor viii that could be used to treat hemophilia (chrenek et al., ; krylov et al., ; simon et al., ) and human growth hormone that could supplement a deficiency in that hormone (lipinski et al., ) . rabbits that express enhanced green fluorescent protein (egfp) in various tissues have been created for the purpose tracking cells, which is important for tissue engineering and regenerative medicine studies takahashi et al., ; yin et al., ) . the mouth of the rabbit is relatively small, and the oral cavity and pharynx are long and narrow. the dental formula is i / , c / , pm / , m - / × = or teeth. a small pair of incisors is present directly caudal to the primary maxillary incisors and is referred to as 'peg' teeth. the peg teeth are used along with the primary incisors to bite and shear food. the absence of second incisors has been noted in some rabbit colonies as a dominant trait (i /i or i /i ). the teeth of rabbits erupt continuously throughout life and therefore will continue to grow unless normal occlusion and use are sufficient to wear teeth to a normal length. molars do not have roots and are characterized by deep enamel folds. rabbits normally masticate with a chewing motion that facilitates grinding of food by movement of the premolars and molars from side to side and front to back. the rabbit has four pairs of salivary glands, including the parotid, submaxillary, sublingual, and zygomatic. the parotid is the largest and lies laterally just below the base of the ear. the zygomatic salivary gland does not have a counterpart in humans. the esophagus of the rabbit has three layers of striated muscle that extend the length of the esophagus down to, and including, the cardia of the stomach. this is in contrast to humans and many other species, which have separate portions of striated and smooth muscle along the length of the esophagus. there are no mucous glands in the esophagus of the rabbit. although the stomach of the rabbit holds approximately % of the volume of the gastrointestinal tract, it is never entirely empty in the healthy rabbit. the gastric contents often include a large amount of hair ingested as the result of normal grooming activity. the stomach is divided into the cardia, fundus, and pylorus. the liver has four lobes. the gallbladder is located on the right. from the liver, the common bile duct empties into the duodenum posterior to the pylorus. rabbits produce relatively large amounts of bile compared to other common species. the pancreas is diffuse within the mesentery of the small intestine and enters the duodenum - mm distal to the common bile duct. the small intestine of the rabbit is short relative to that of other species and comprises approximately % of the total length of the gastrointestinal (gi) tract. because the gi tract of the rabbit is relatively impermeable to large molecules, kits receive most of their passive immunity via the yolk sac prior to birth rather than by colostrum. peyer's patches are found along the ileum, particularly near the cecal junction. the sacculus rotundus is a large bulb of lymphoid tissue located at this junction. the large intestine includes the cecum, the ascending colon, the transverse colon, and the descending colon. the ileocecal valve regulates flow of chyme into the cecum and retards reverse flow back into the ileum. the cecum is very large with a capacity approximately -times that of the stomach. the cecum ends in a blind sac, the appendix. the colon is divided into proximal and distal portions by the fusus coli, which serves to regulate the elimination of hard versus soft fecal pellets. hard pellets comprise about two-thirds of the fecal output. soft pellets, or 'cecotrophs,' have a high moisture content and are rich in nitrogen-containing compounds (ferrando et al., ) and the Β vitamins niacin, riboflavin, pantothenate, and cyanocobalamin. rabbits consume cecotrophs directly from the anus to obtain significant nutritional benefit. soft pellets are sometimes termed 'night feces,' since they are generally produced at night in domestic rabbits. in contrast, the circadian rhythm of cecotrophy is reversed in wild rabbits, occurring during the day when the animals are in their burrows (hornicke, ) . nostrils of rabbits are well equipped with touch cells, and they have a well-developed sense of smell. nasal breathing in rabbits is characterized by twitching of the nostrils at rates varying from to times per minute, although twitching may be absent in the relaxed rabbit. it has been speculated that inspiration occurs as the nostril moves up and that this serves to direct the flow of air over the turbinate bones where the olfactory cells are most concentrated. the musculature of the thoracic wall contributes little to respiratory efforts. instead, rabbits rely mostly on the activity of the diaphragm. because of this, artificial respiration is easily performed by alternating the head of the rabbit between the up and down positions, - times per minute, while holding the animal. compression and release of the chest wall is an ineffective means of artificial respiration in the rabbit. the pharynx of the rabbit is long and narrow, and the tongue is relatively large. these features make endotracheal intubation difficult. the procedure is further laboratory animal medicine complicated by the propensity of the rabbit to laryngospasm during attempts to intubate the trachea. the rabbit lungs consist of six lobes. both right and left sides have cranial, middle, and caudal lobes, with the right caudal being further subdivided into lateral and medial portions. flow volume of air to the left lung is higher than that to the right due to the lower resistance of the proximal airways per unit volume (yokoyama, ) . in rabbits, lung volume increases with age, in contrast to that of humans and dogs, in which it decreases. bronchial-associated lymphoid tissue (balt) is present as distinct tissue. a unique feature of the cardiovascular system of the rabbit is that the tricuspid valve of the heart has only two cusps, rather than three as in many other mammals. a small group of pacemaker cells generate the impulse of the sinoatrial (sa) node in the rabbit, a feature that facilitates precise determination of the location of the pacemaker (bleeker et al., ; hoffman, ; west, ) . the sa and atrioventricular (av) nodes are slender and elongated, and the av node is separated from the annulus fibrosus by a layer of fat (truex and smythe, ) . additional unique anatomic features of the cardiovascular system of the rabbit have been utilized to advantage. the aortic nerve subserves no known chemoreceptors (kardon et al., ; stinnett and sepe, ) and responds to baroreceptors only. because the aortic nerve, which becomes the depressor nerve, runs alongside but separate from the vagosympathetic trunk, it lends itself readily to implantation of electrodes (karemaker et al., ) . the blood supply to the brain is restricted mainly to the internal carotid artery. blood supplied via the vertebral arteries is limited. the aorta of the rabbit demonstrates rhythmic contractions that arise from neurogenic stimulation in a pattern related to the pulse wave (mangel et al., ) . the kidneys of the rabbit are unipapillate in contrast to those of most other mammals, which are multipapillate. this feature increases the ease with which cannulization is performed. the right kidney lies more cranial than the left. glomeruli increase in number after birth in rabbits, whereas all of the glomeruli are present at birth in humans (smith, ) . ectopic glomeruli are normal in the rabbit (steinhausen et al., ) . blood vessels that perfuse the medulla remain open during many conditions under which vasoconstriction of the cortical tissue occurs; thus, the medullary tissue may be perfused, while the cortex is ischemic (trueta et al., ) . the urine of adult rabbits is typically cloudy due to a relatively high concentration of ammonium magnesium phosphate and calcium carbonate monohydrate precipitates (flatt and carpenter, ) . the urine may also take on hues ranging from yellow or reddish to brown. in contrast, the urine of young rabbits is typically clear, although healthy young rabbits may have albuminuria. the urine is normally yellow but can also take on reddish or brown hues once animals begin to eat green feed and cereal grains. normal rabbits have few cells, bacteria, or casts in their urine. the ph of the urine is typically alkaline at about . (williams, ) . a normal adult rabbit produces approximately - ml/kg of urine daily (gillett, ) , with does urinating more copiously than bucks. the urethral orifice of the buck is rounded, whereas that of the doe is slit-like. this feature is useful for distinguishing the sexes. the testes of the adult male usually lie within the scrotum; however, the inguinal canals that connect the abdominal cavity to the inguinal pouches do not close in the rabbit. for this reason, the testes can easily pass between the scrotum and the abdominal cavity. this feature necessitates closure of the superficial inguinal ring following orchiectomy by open technique to prevent herniation. the reproductive tract of the doe is characterized by two uterine horns that are connected to the vagina by separate cervices (bicornuate uterus). a common tube, the urogenital sinus or vestibulum, is present where the urethra enters the vagina. the placenta is hemochorial, and maternal blood flows into sinus-like spaces where the transfer of nutrients and other substances to the fetal circulation occurs (jones and hunt, ) . inguinal pouches are located lateral to the genitalia in both sexes. the pouches are blind and contain scent glands that produce white to brown secretions that may accumulate in the pouch. the metabolic rate of endotherms is generally related to the body surface area. including the ears, the rabbit has a relatively low metabolic rate (mr); however, if the surface area of the ears is discounted, the mr of the rabbit is similar to that of other endotherms. neonatal rabbits have an amount of body fat comparable to that of the human infant ( % of body weight) (cornblath and schwartz, ) . the neonatal rabbit is essentially an ectotherm until about day (gelineo, ) . the glucose reserves of the neonatal rabbit are quickly depleted, usually within about h after birth (shelley, ) . the fasting neonatal rabbit quickly becomes hypoglycemic and ketotic (callikan and girard, ) . the normal rectal temperature of the adult new zealand white rabbit at rest is approximately . - . °c (ruckebusch et al., ) . the ears serve an important thermoregulatory function. because they have a large surface area and are highly vascular with an extensive arteriovenous anastomotic system, the ears help the rabbit sense laboratory animal medicine and respond to cold versus warm temperatures (kluger et al., ) . in addition, the ears serve as a countercurrent heat-exchange system to help adjust body temperature. early studies found that the body of the adult rabbit ( kg body weight) consists of greater than % water ( %), with a half-time turnover of about . days and a loss of about ml daily (richmond et al., ) . the amount of water ingested varies with the amount and type of feed consumed and the environmental temperature. in general, rabbits will drink more water when consuming dry, pelleted feed than when consuming foodstuffs high in moisture, such as fresh greens. conversely, rabbits deprived of water will decrease food consumption. after days of complete water deprivation, food intake falls to less than % of normal (cizek, ) . normal values for various systems and parameters are provided as a general indication for these values in the rabbit. it is important to recognize, however, that most of these values have been obtained through the study of adult new zealand white rabbits. as with any experiment, values can vary significantly between breeds, laboratories, methods of sampling and measurement, and individual rabbits due to age, sex, breed, health, handling, and husbandry (hewitt et al., ; lidena and trautschold, ; mitruka and rawnsley, ; wolford et al., ; yu et al., ) . for this reason, individual laboratories should strive to establish their own normal values, whenever possible. values for hematologic parameters are shown in table . . these values represent those typical of adult new zealand white rabbits. in general, males have slightly greater hematocrit and hemoglobin values than females (mitruka and rawnsley, ) . anisocytosis is normal and accounts for variation in reported values for red blood cell diameter (sanderson and phillips, ) . reticulocyte values are usually between % and % in healthy rabbits (corash et al., ) . the neutrophil of the rabbit is sometimes referred to as a 'pseudoeosinophil' or 'heterophil,' due to the presence of red-staining granules in the cytoplasm. the heterophil ( - mm in diameter) is, however, smaller than the eosinophil ( - mm in diameter) (sanderson and phillips, ) . in addition, the red granules of the heterophil are smaller than the red granules of the eosinophil. the nucleus of the eosinophil may be either bilobed or horseshoe-shaped. as mentioned earlier, chemistry values can vary because of a number of factors. for this reason, each laboratory should establish its own normal values. aspartate aminotransferase (ast) is present in the liver, heart, skeletal muscle, kidney, and pancreas. collection of blood samples in rabbits by decapitation, cardiac puncture, or aortic incision, or the use of restraint that causes exertion will elevate ast levels due to muscle damage (lidena and trautschold, ) . similarly, levels of creatinine kinase are sensitive to muscle damage since that enzyme is present in the skeletal muscle, brain, and heart (lidena and trautschold, ; mitruka and rawnsley, ) . although most mammals have two isoenzymes (intestinal and a liver/kidney/bone form) of alkaline phosphatase (ap), rabbits are unique in having three forms of ap, including an intestinal form and two forms that are both present in the liver and the kidney (noguchi and yamashita, ) . values for blood and serum chemistry are shown in table . . cardiovascular and respiratory functions are often altered with experimental manipulation, anesthesia, or disease. normal values for these parameters and other miscellaneous biologic characteristics of the rabbit are listed in table . . values obtained from the following sources: burns and delannoy ( ) , gillett ( ) , kabata et al. ( ) , mitruka and rawnsley ( ), and woolford et al. ( ) . laboratory animal medicine rabbits are strictly herbivorous with a preferred diet of herbage that is low in fiber and high in protein and soluble carbohydrate (cheeke, ; cheeke, ) . rabbits will generally accept a pelleted feed more readily than one in meal form. when a meal diet is needed, a period of adjustment should be allowed for the rabbits to accommodate to the new diet. examples of adequate diets are shown in table . . the requirement for fiber in the diet of rabbits has been reviewed (gidenne, ) . fiber is especially important in the early postweaning period when low fiber intake is associated with an increase in digestive disorders (gidenne, ) . the exact nutrient requirements for individual rabbits vary with age, reproductive status, and health of the animal. on occasion, the need arises for use of highly purified diets. a suggested purified diet has been described elsewhere (subcommittee on rabbit nutrition, ) . it should be noted that overfeeding of values obtained from the following sources: burns and delannoy ( ) , fox ( ) , gillett ( ) , kraus et al. ( ), and loeb and quimby ( ) . values obtained from the following sources: barzago et al. ( ) , curiel et al. ( ) , gillett ( ) , kozma et al. ( ) , sanford and colby ( ) , suckow and douglas ( ), and zurovsky et al. ( ) . laboratory animal medicine laboratory rabbits resulting in obesity is common, but can be prevented by either reducing the amount of feed or by providing a low-energy, high-fiber maintenance diet (donnelly, ) . as mentioned earlier, rabbits engage in cecotrophy, and by doing so supplement their supply of protein and Β vitamins (carabaño et al., ; gidenne et al., ) . rabbits fed a diet high in fiber ingest a greater quantity of cecotropes than those on a lower fiber diet (fekete and bokori, ) . unlike most other species, both calcium absorption in the small intestine and serum calcium levels increase in proportion to the amount of calcium in the diet (cheeke, ) . prolonged feeding of diets high in calcium, such as those with a high level of alfalfa meal, can result in renal disease. consumption of diets containing excessive vitamin d can result in calcification of soft tissues, including the liver, kidney, vasculature, and muscles (besch-williford et al., ; lebas, ) . diets that are either too high or too low in vitamin a can result in reproductive dysfunction and congenital hydrocephalus (cheeke, ; digiacomo et al., ) . the exact requirement for vitamin a in the rabbit has not been determined; however, a level of - , iu/kg of diet is generally adequate (lebas, ) . vitamin e deficiency has been associated with infertility, muscular dystrophy, fetal death, neonatal death, and colobomatous microphthalmos in rabbits (lebas, ; nielsen and carlton, ; ringler and abrams, ; ringler and abrams, ) . mcdowell ( ) suggested that serum vitamin e levels of less than . μg/ml are indicative of hypovitaminosis e. relative to other species, rabbits have a high water intake. in general, daily water intake is approximately ml/kg of body weight. consumption of water is influenced by environmental temperature, disease states, and feed composition and intake (cizek, ; tschudin et al., ) . consumption of diets high in dry matter results in increased water intake (tschudin et al., ) . water consumption also increases with food deprivation. rabbits are social animals and attempts at group housing often meet with success, although mature males will fight and can inflict serious injury on one another (love, ; podberscek et al., ; whary et al., ) . group-penned female rabbits allowed to choose between single or paired housing prefer being in the same cage with other rabbits (huls et al., ) . in general, rabbits are timid and nonaggressive. some animals will display defensive behavior, typically characterized by thumping the cage floor with the rear feet, biting, and charging toward the front of the cage when opened. laboratory-housed rabbits demonstrate diurnal behavior, in contrast to the nocturnal pattern exhibited by wild rabbits (jilge, ) . the ethogram of the laboratory rabbit has been described (chu et al., ; gunn and morton, ) . the most common behaviors of individually housed rabbits included lie alert, doze, groom, sleep, and eat. individually housed rabbits were inactive the majority of the time (gunn and morton, ) . individually housed female rabbits showed an increase in abnormal behaviors compared to pair-housed rabbits (chu et al., ) . rabbits housed in pairs in double-wide cages locomoted more than individually housed rabbits (chu et al., ). the age of puberty varies with the breed of rabbit. puberty generally occurs at - months of age in small breeds, - months in medium breeds, and - months in large breeds (donnelly, ) . female new zealand white rabbits reach maturity at months of age and males at - months. the breeding life of a doe typically lasts approximately - years, although some remain productive for up to or years. in later years, litter sizes usually diminish. in comparison, most bucks will remain reproductively useful for an average of - years. because does often will engage in reproductive behavior before being able to ovulate, it is advisable not to breed does until they are fully grown. does do not have a distinct estrous cycle, but rather demonstrate a rhythm with respect to receptivity to the buck. receptivity is punctuated by periods ( - days every - days) of anestrus and seasonal variations in reproductive performance (hafez, ) . during periods of receptivity, the vulva of the doe usually becomes swollen, moist, and dark pink or red. receptivity of the doe is usually signaled by lordosis in response to the buck's attempt to mount, vulvar changes as described above, restlessness, and rubbing of the chin on the hutch or cage (donnelly, ) . vaginal cytology is generally not useful for determination of estrus or receptivity in the rabbit. typically, the doe is brought to the buck's cage for breeding, since the doe can be very territorial and may attack the male in her own quarters. a period of - min is usually sufficient to determine compatibility of the doe and buck. if receptive, the doe will lie in the mating position and raise her hindquarters to allow copulation. if fighting or lack of breeding is observed, the doe may be tried with another buck. a single buck is usually sufficient to service - does. ovulation is induced and occurs approximately - h after copulation (donnelly, ) . up to % of does fail to ovulate following copulation. ovulation can also be induced by administration of luteinizing hormone (kennelly and foote, ) , human chorionic gonadotropin (williams et al., ) , or gonadotropic releasing hormone (foote and simkin, ) . does may be bred immediately after kindling; however, most breeders delay until after the kits have been weaned. success at postpartum breeding varies, but one can produce a large number of kits in a relatively short time period by foster nursing the young and rebreeding the doe immediately. while conventional breeding, nursing, and weaning schedules allow for only litters per year, early postpartum breeding allows for up to litters per year. pregnancy can often be confirmed as early as day of gestation by palpation of the fetuses within the uterus. radiographic procedures permit pregnancy determination as early as day . conception rates have been observed to have an inverse relationship with ambient temperature but not light cycle. gestation in rabbits usually lasts for - days (donnelly, ) . does beyond - weeks of gestation will usually refuse a buck. does begin hair pulling and nest building during the last - days of gestation (donnelly, ) . a nesting box with shredded paper or other soft material such as straw should be provided to the doe several days prior to the expected kindling (parturition) date. the doe will usually line the box with her own hair. the nesting box should not be placed in the corner of the cage where the individual doe has been observed to urinate. pseudopregnancy is common in rabbits and can follow a variety of stimuli, including mounting by other does, sterile matings by bucks, administration of luteinizing hormone, or the presence of bucks nearby. in such circumstances, ovulation is followed by a persistent corpus luteum that lasts - days. the corpus luteum or corpora lutea secretes progesterone during this time, causing the uterus and mammae to enlarge. the doe may have the appearance of a normally pregnant rabbit. toward the end of pseudopregnancy, many does will begin to pull hair as part of ritual nest-building behavior. the process of parturition is referred to as 'kindling' when it relates to rabbits. kindling normally occurs during the early morning hours and takes approximately - min. impending kindling is often signaled by nest building and decreased food consumption during the preceding - days. both anterior and breech presentations are normal in the rabbit. fetuses retained beyond days generally die and may harm future reproductive ability of the doe if not expelled. the average number of kits born is seven to nine per litter, although smaller litters and litters of up to kits are not uncommon. breed, parity, nutritional status, and environmental factors influence litter size. polish rabbits usually have fewer than four kits per litter; dutch or flemish giant, four to five; and new zealand white, eight to ten. after the young have been cleaned following parturition, the doe typically consumes the placenta. cannibalism of the young by the doe sometimes occurs and may be related to environmental or hereditary factors or due to environmental stressors. does usually have either four or five pairs of nipples, whereas bucks have none. during the last week of pregnancy, marked development of the mammary gland occurs. the doe normally nurses the kits once daily for several minutes, usually in the early morning or in the evening, regardless of how many kits are present or laboratory animal medicine how many times they attempt to suckle. milk yield is normally between and g/day. during the first week of life, kits consume - g of milk per day. milk intake increases gradually to a maximum of g/day between and days of age (gidenne et al., ) . maximum output occurs at weeks following kindling and then declines during the fourth week. rabbit milk contains approximately . % protein, % fat, % lactose, and . % minerals. nursing may last - weeks. kits may begin consuming solid food by weeks of age, with weaning generally occurring by - weeks of age. the facilities present in most modern research animal facilities would be suitable for housing rabbits. general construction should include adequate heating, ventilation, and air conditioning to house rabbits at appropriate temperature and humidity. in addition, lighting should be adequate to allow easy visualization of the rabbits. surfaces, such as the floors, walls, and ceilings, should be easily sanitizable (national research council, ) . rabbit cages should provide a safe environment with easy access to food and water. adults can be caged individually or in compatible groups and should have sufficient floor space to lie down and stretch out. in the united states, minimum cage sizes are determined by the animal welfare act (awa) and the guide for the care and use of laboratory animals (guide). in both cases, sizes vary with the weight of the animal. currently, the awa regulations and the guide require . ft of floor space and in of cage height for rabbits weighing - kg (national research council, ) . cages should be constructed of durable materials that will resist corrosion and harsh detergents and disinfectants used in cleaning. consequently, in the research environment, rabbit cages are most often constructed of stainless steel or plastics. rabbits are usually housed in cages with mesh or slatted floors to permit urine and feces to drop through into a catch pan. mesh floors with catch pans do not prevent rabbits from engaging in the normal practice of coprophagy. information on environmental enrichment of laboratory rabbits has been published (baumans, ) . the behavior of rabbits in conventional cages was compared to that of rabbits provided with enriched cages that contained shelter, a shelf, and increased vertical space. rabbits in conventional cages were more restless, groomed excessively, exhibited more bar-gnawing, and were more timid than those housed in enriched cages (hansen and berthelsen, ) . indeed, fecal glucocorticoid levels in rabbits declined when they were provided with a wooden structure for resting and gnawing (buijs et al., ) . rabbits will play with objects placed in their cages. huls et al. ( ) noted that rabbits would use wooden sticks, wooden rings, and brass wire balls as toys. rabbits provided with objects (toys) spent significantly more time chewing than rabbits without toys (poggiagliolmi et al., ) . female rabbits can also be housed in compatible pairs or groups. singly housed female rabbits exhibited more abnormal behaviors compared to pair housed rabbits (chu et al., ) . group housing of unfamiliar males is not recommended because of the likelihood of fighting and injury. rabbits are optimally housed in cooler room temperatures than most other common species of laboratory animals. the guide recommends that temperatures in rabbit rooms be maintained between and °f. no specific illumination requirements for rabbits have been described. it is common practice to provide rabbits with - h of light in the light-dark cycle. in breeding colonies, females should be provided with - h of light. rabbits are easily startled by sudden, loud noises. for this reason, they should not be housed near noisy species such as dogs or monkeys, nor should they be housed near noise-generating operations such as the cage-wash area. catch pans should be cleaned as often as necessary to prevent the formation of ammonia. cages are generally sanitized on at least a weekly basis. rabbit urine contains large amounts of protein and minerals, and often forms deposits on cages and catch pans. it is common practice to soak equipment having urine deposits in acid washes to remove the scale before washing. ammonia production in rabbit rooms can be a significant problem; therefore, rabbit rooms should be ventilated at - air changes per hour (national research council, ) . it is also important to change excreta pans often to prevent the buildup of ammonia. etiology pasteurella multocida is a gram-negative nonmotile coccobacillus that causes pasteurellosis, also known as 'snuffles', the primary respiratory disease affecting domestic rabbits (deeb and digiacomo, ; guo et al., ) . historically, serogroup a isolates have laboratory animal medicine been associated with pneumonic and septicemic pasteurellosis in laboratory rabbits; however, capsular type a is also isolated from rabbits that appear clinically healthy (confer et al., ; el tayeb et al., ) . clinical signs pasteurella multocida infection is often subclinical, but pasteurellosis may cause fever, coughing, dyspnea, rhinitis (nasal discharge (serous to mucopurulent), sneezing, and upper airway stentor), pneumonia, otitis, septicemia, meningitis, abscesses (of viscera and subcutaneous sites), and death (al-lebban et al., ; confer et al., ; franco and cronin, ; guo et al., ; suckow et al., ; wilkie et al., ) . pasteurellosis may also be associated with pericarditis, pleuritis, sinusitis, dacryocystitis, conjunctivitis, iritis/ uveitis, phlegmon, mastitis, endometritis, pyometra, salpingitis, and orchitis (deeb and digiacomo, ; ferreira et al., ; stahel et al., ; williams, ) . epizootiology p. multocida can be endemic in rabbitries and is carried in the rabbit's nasal cavity (confer et al., ; deeb et al., ; digiacomo et al., ; suckow et al., ) . transmission is by direct contact between rabbits (wilkie et al., ) . coinfection with bordetella bronchiseptica may be observed in clinically affected rabbits (deeb et al., ) . stress-related factors associated with pasteurellosis include crowded or unsanitary conditions, transportation, and high ammonia concentrations in the air (confer et al., ) . previous studies reported a high prevalence of p. multocida infection (jaslow et al., ) . colonization in immature rabbits occurs more commonly in the sinuses followed by the trachea, middle ears, and lungs (glass and beasley, ) . similar to cats and dogs, rabbits may transmit p. multocida infection to humans (per et al., ; silberfein et al., ) . a study utilizing repetitive extragenic palindromic pcr (rep-pcr) and sequencing determined that % of the isolates were characterized as p. multocida subsp. multocida, % as p. multocida subsp. septica, % as atypical subspecies of p. multocida, % as p. canis, and % as an unknown species of the family pasteurellaceae (stahel et al., ) . the pathogenesis of p. multocida has been reviewed (wilkie et al., ) . the ptfa gene, encoding a type fimbrial subunit and involved in bacterial fixation on the surface of epithelial cells, may be highly prevalent in p. multocida isolates from rabbits (ferreira et al., ) . the p. multocida toxin is a major virulence factor in atrophic rhinitis of rabbits and acts by causing constitutive activation of g proteins frymus et al., ; orth et al., ; suckow et al., ) . pathology the specific pathologic findings will vary with the site of infection, but the underlying host response is characterized by acute or chronic suppurative inflammation with the infiltration of large numbers of neutrophils. rhinitis and sinusitis are accompanied by a mucopurulent nasal exudate. neutrophil infiltration of the tissues is extensive. the nasal passages are edematous, inflamed, and congested, and there may be mucosal ulcerations. the turbinate bones may atrophy digiacomo et al., ) . purulent conjunctivitis may be present. pneumonia is primarily cranioventral in distribution. the lungs can exhibit consolidation, atelectasis, and abscess formation. a purulent to fibrinopurulent exudate is evident, and there may be areas of hemorrhage and necrosis. in some rabbits, fibrinopurulent pleuritis and pericarditis are prominent features (glavits and magyar, ) . this is probably due to elaboration of a heat-labile toxin in some strains of the bacteria . acute hepatic necrosis and splenic lymphoid atrophy are also seen in association with the pleuritis and pneumonia induced by toxigenic strains. otitis media is characterized by a suppurative exudate with goblet cell proliferation and lymphocytic and plasma cell infiltration. in female rabbits with genital tract infections, the uterus may be enlarged and dilated. in the early stages of infection, the exudate is watery; later it thickens and is cream-colored. the exudate contains numerous neutrophils. focal endometrial ulceration can be found (johnson and wolf, ) . in the male, the testes are enlarged and may contain abscesses. systemic and visceral abscesses are characterized by a necrotic center, an infiltrate made up of polymorphonuclear neutrophils, and a fibrous capsule. septicemia may only present as congestion and petechial hemorrhages in many organs. severe pleuritis with accumulation of fibrinopurulent exudate in the thoracic cavity, serous rhinitis and tracheitis, acute hepatitis with necrotic foci in the parenchyma, and atrophy of lymphoid organs and tissues have been observed after experimental p. multocida infection in rabbits (glavits and magyar, ) . diagnosis sterile swabs can be used to collect samples from the nares or nasal cavity of rabbits for culture (ferreira et al., ; jaslow et al., ) . nasal lavage can also be used as a culture sample to isolate pasteurella (suckow et al., ) . p. multocida isolates can be classified into five serogroups based on capsular antigens (a, b, d, e, and f) and into serotypes based on somatic lps antigens (adler et al., ; liu et al., b; manning, ) . biochemical characterization of isolates may show high heterogeneity; however, rep-pcr and phylogenetic analysis using s ribosomal rna and rpob genes can be used for precise characterization of rabbit isolates (stahel et al., ) . classification of p. multocida into subspecies and/or by virulence profiles is useful for epidemiological investigations (ferreira et al., ; stahel et al., ) . random amplified polymorphic laboratory animal medicine dna pcr (rapd-pcr) has also been used to subtype rabbit p. multocida isolates (al-haddawi et al., ; dabo et al., ; williams et al., ) . pcr can detect capsule biosynthesis genes cap a, b, d, e, and f as well as virulence-related genes (ferreira et al., ) . serological tests can be used to detect antibodies against p. multocida (deeb et al., ; delong et al., ; digiacomo et al., ; glass and beasley, ; lukas et al., ) . differential diagnoses if radiographs reveal an internal mass associated with p. multocida infection, the differential diagnoses should include abscess, granuloma, neoplasia, and parasitic cyst (franco and cronin, ) . treatment, prevention, and control previous studies have investigated the use of vaccines to protect rabbits against p. multocida infection (confer et al., ) . immunization of rabbits with inactivated heat-labile p. multocida toxin or a commercial swine p. multocida bacterin-toxoid conferred protective immunity against challenge with the p. multocida heat-labile toxin (suckow, ; suckow et al., ) . a vaccine administered intranasally stimulated immunity against experimental pneumonic pasteurellosis and significantly reduced nasal bacterial counts (confer et al., ) . oral immunization of rabbits with a p. multocida thiocyanate extract (pte) in microparticles was immunogenic and significantly reduced the colony-forming units of homologous p. multocida recovered from the lungs and nasopharynx (suckow et al., ) . protective immunity to a heterologous strain of p. multocida can be achieved by vaccinating rabbits with pte via the subcutaneous route (suckow et al., ) . a p. multocida bacterin known as bunnyvac is currently licensed by the usda and is intended to be effective in preventing death and limiting disease due to pasteurella in rabbits. bunnyvac is manufactured by colorado serum company and distributed by pan american veterinary laboratories (http://pavlab. com/). control of pasteurellosis in rabbitries entails testing and culling animals that are positive for pasteurella spp. (ferreira et al., ) . furthermore, rabbits free of pasteurella and other infectious agents can be obtained by enrofloxacin treatment and through cesarean section or hysterectomy rederivation (pleasants, ; suckow et al., ; syukuda, ) . commercial suppliers of laboratory rabbits tend to exclude pasteurella from their colonies. treatment with antibiotics should be based on culture and sensitivity. antibiotic treatment of affected rabbits can alleviate clinical signs or delay disease progression but may not eradicate the disease (el tayeb et al., ; ferreira et al., ) . antibiotic treatment may suppress virulence gene expression without complete elimination of p. multocida . internal abscesses may not be treatable using antibiotics (franco and cronin, ) . penicillin therapy does not seem to be effective against pasteurella infection and may also lead to diarrhea and clostridium difficile colitis in rabbits (jaslow et al., ; rehg and lu, ) . one study from brazil determined that all tested strains were sensitive to ceftiofur, florfenicol, norfloxacin, enrofloxacin, ciprofloxacin, tetracycline, and doxycycline (ferreira et al., ) . other studies also indicate that fluoroquinolones are useful for the treatment of p. multocida infection in rabbits (abo-el-sooud and goudah, ; broome and brooks, ; franco and cronin, ; hanan et al., ; okewole and olubunmi, ) . oral ciprofloxacin ( mg/kg per day for days) has been used in rabbits (hanan et al., ) . research complications pasteurellosis can cause considerable economic losses (el tayeb et al., ; ferreira et al., ; stahel et al., ) and has the potential to affect different types of research studies using rabbits due to the multisystemic nature of the disease, and the possibility of high morbidity and mortality. therefore, pasteurella should be excluded from laboratory rabbit colonies. the class clostridia belongs to the phylum firmicutes (yutin and galperin, ) . recent genomic analyses suggest assigning some clostridium species that fall outside the family clostridiaceae into new genera. the genera tyzzerella, erysipelatoclostridium, and peptoclostridium have been proposed for c. piliforme, c. spiroforme, and c. difficile, respectively (yutin and galperin, ) . etiology c. piliforme is a pleomorphic, gramnegative, spore-forming, motile, obligate intracellular rod-shaped bacterium that causes tyzzer's disease and infects various animals including mice, nonhuman primates, gerbils, rats, rabbits, and others (allen et al., ; ganaway et al., ; pritt et al., ) . infection has also been reported in a human patient with human immunodeficiency virus- (smith et al., ) . phylogenetic analyses determined that microorganisms identified as c. piliforme form three clusters within a single clade and that the nearest related distinguishable species is c. colinum (feldman et al., ) . clinical signs the first reported outbreaks in laboratory rabbits described profuse watery to mucoid diarrhea usually followed by death in - h in -to -week old rabbits (allen et al., ) . rabbits in affected litters usually died within a week after the first fatality (allen et al., ) . the dams of affected litters occasionally died after a diarrheal disease that was more protracted than that of the offspring (allen et al., ) . these outbreaks lasted for - months and affected multiple rabbit rooms. c. piliforme infection may also be subclinical and transient as immunocompetent hosts may clear the infection (ganaway et al., ; pritt et al., ) . weanling rabbits with the acute form of tyzzer's disease exhibit diarrhea, listlessness, anorexia, and dehydration usually followed by death within h (cutlip et al., ) . the mortality rate in clinically affected rabbits was estimated to be - % (cutlip et al., ) . anorexia and stunting were observed in chronic cases associated with intestinal stenosis (cutlip et al., ) . acute and chronic tyzzer's disease types have been described in rabbits; however, large numbers of 'attaching' escherichia coli were recovered from the cecum of most rabbits (prescott, ) . epizootiology the vegetative cell is the active stage responsible for the disease and depends on the intracellular environment (ganaway, ) . therefore, the spore, a resistant stage, appears to be the essential element in the transmission of tyzzer's disease (ganaway, ; ganaway et al., ) . contact with soiled bedding or diseased rabbits have been used experimentally to transmit the disease to other rabbits (allen et al., ) . it is possible that subclinically infected rabbits (carriers) may introduce the organism into a colony (allen et al., ; pritt et al., ) . in mice, increased susceptibility to infection has been associated with stress (allen et al., ) . furthermore, treatment with cyclophosphamide, cortisone, and prednisolone has been used experimentally to reproduce the disease in animals, suggesting that immunosuppression plays a role in pathogenesis (allen et al., ; cutlip et al., ; pritt et al., ) . animals stressed by poor environmental conditions including overcrowding and extreme temperatures can develop the disease (cutlip et al., ; wobeser et al., ). significant modifications of the intestinal flora and an impaired immune system may play a role in pathogenesis (licois, ) . c. piliforme may be transported from the intestine to the liver through the portal circulation and to the heart through the lymphatics (allen et al., ) . some c. piliforme isolates can induce cytopathic effects on cell cultures, and in vivo, concomitant infection with other enteric pathogens such as e. coli may contribute to the severity of the disease (prescott, ; riley et al., ) . pathology lesions can be found in the distal ileum, cecum, proximal colon, liver, and heart (allen et al., ) . intestinal lesions are common, and histologically are characterized by necrosis of the mucosa and edema of the submucosa and serosa (allen et al., ) . bacilli appear as bundles of parallel rods or as criss crossed sticks in the cytoplasm of epithelial cells distributed from the surface of the mucosa to the base of the glands (allen et al., ) . the lesions in the liver are punctate areas of parenchymal necrosis that appear grossly as white spots, usually ≤ mm in diameter. large numbers of bacilli are found in the cytoplasm of cells in the zone of transition between the necrotic lesion and the healthy parenchyma (allen et al., ) . myocardial lesions appear as white streaks . - mm wide and - mm long extending from the region of the left interventricular groove laterally across the left ventricle (allen et al., ) . in the myocardium, bacilli may be noted in partially degenerated and normal looking cells at the sharply delineated borders of the lesions (allen et al., ) . diagnosis c. piliforme cannot be cultured in artificial (cell-free) media making its diagnosis difficult (allen et al., ; cutlip et al., ; ganaway et al., ; niepceron and licois, ) . other bacteria, including e. coli, have been isolated from the liver of diseased rabbits and are considered secondary invaders (allen et al., ; cutlip et al., ) . the isolation of the tyzzer's agent using liver extract agar has been described (kanazawa and imai, ) . c. piliforme can be grown in primary monolayer cultures of adult mouse hepatocytes, in mouse fibroblasts, in rat hepatocytes, and in embryonated eggs (craigie, ; duncan et al., ; ganaway et al., ; kawamura et al., ; pritt et al., ; riley et al., ) . serology for c. piliforme is commonly used for surveillance of laboratory animals because it is rapid and inexpensive (pritt et al., ) . immunofluorescence assay (ifa) and multiplexed fluorometric immunoassay (mfia) have been utilized (pritt et al., ) . in addition, c. piliforme pcr assays have been developed (feldman et al., ; gao et al., ; niepceron and licois, ; pritt et al., ) . clostridium piliforme seropositive rabbits may be negative for the organism by pcr and histopathological evaluation (pritt et al., ) . therefore, positive serological findings are not sufficient for a definitive diagnosis of c. piliforme infection and pcr testing and/or histopathology should be used for confirmation (pritt et al., ) . definitive diagnosis is based on identification of typical gross lesions and histological demonstration of intracellular c. piliforme at the periphery of the necrotic foci (niepceron and licois, ; pritt et al., ) . giemsa solution (ph ), warthin-starry silver method, levaditi silver method, and the periodic acid-schiff (pas) reaction have been used to demonstrate c. piliforme (allen et al., ; cutlip et al., ) . different morphologic forms of c. piliforme can be observed microscopically (allen et al., ; ganaway et al., ) . differential diagnoses clinically, other diarrheal diseases of rabbits can be included in the differential diagnoses. grossly, the multifocal white areas on the liver could be from eimeria stiedae infection (hepatic coccidiosis). treatment, prevention, and control for prevention, avoid introduction of rabbits of unknown c. piliforme status into a colony. minimize stress-related factors especially in young animals. good husbandry practices including regular bedding changes and disinfection laboratory animal medicine should decrease the likelihood of spreading c. piliforme in a colony. in one report, a tyzzer's disease outbreak was observed - days after rabbits were weaned and transferred to a facility in which the temperature fluctuated from to °c. the outbreak was controlled by transferring weanling rabbits to a building maintained at the same temperature as the breeder house ( - °c) (cutlip et al., ) . spores treated with heat ( or °c) or with either peracetic acid ( %) in a wetting agent (sodium alkylarylsulfonate) or sodium hypochlorite solution ( . %) for min lose infectivity (ganaway, ) . however, spores do not lose infectivity when treated with a phenolic germicidal agent, ethanol, or quaternary ammonium compounds (containing % or % benzalkonium chloride) (ganaway, ) . sodium hypochlorite solution ( . %) has been recommended as a surface disinfectant in animal facilities for prevention and control of tyzzer's disease (ganaway, ) . the sensitivity of c. piliforme to antibiotics has been investigated (kanazawa and imai, ) . in one study, none of the antibacterials tested were completely inhibitory (ganaway et al., ) . group treatment of rabbits with tetracyclines in the drinking water and food was effective in lowering the incidence of diarrhea and death (prescott, ) . research complications the high morbidity and mortality associated with tyzzer's disease can affect the overall population of rabbits in a colony thereby decreasing the number of rabbits suitable or available for experimentation. in addition, research studies involving experimental infection with enteric pathogens in rabbits may be confounded by c. piliforme-associated intestinal pathology. enterotoxemia refers to conditions of the bowel caused by toxigenic clostridia (carman and evans, ) . diagnosis of enterotoxemia should be based on culture of a toxigenic clostridium and demonstration of the toxin from the intestinal contents of the diseased animal (carman and evans, ; songer, ) . i. clostridium spiroforme etiology c. spiroforme is a gram-positive, sporebearing, helically coiled, semicircular, anaerobic bacterium that can produce iota toxin (borriello and carmen, ; carman and borriello, ; peeters et al., ) . the disease caused by c. spiroforme is known as 'iota enterotoxemia' (keel and songer, ; peeters et al., ) . clinical signs diarrhea, fecal soiling of the perineum, and cyanosis may be observed (carman and borriello, ) . diarrhea may be peracute and may lead to 'spontaneous' death or a moribund state (carman and borriello, ) . epizootiology c. spiroforme is thought to be acquired from the environment (carman and evans, ; songer, ) . weaning is associated with spontaneous disease and administration of antibiotics can also induce the disease (borriello and carmen, ; carman and borriello, ) . a study determined that disease results from exposure of weanling rabbits to c. spiroforme and also from exposure of adult rabbits with a disrupted gut flora (due to clindamycin treatment) to c. spiroforme suggesting that this bacterium is not a normal component of the rabbit gut flora (carman and borriello, ) . c. spiroforme-mediated diarrhea may be favored by maldigestion initiated by infectious agents and/or nutritional factors (peeters et al., ) . other clostridia, e. coli (epec), viruses, and parasites, may coinfect rabbits (peeters et al., ) . the iota toxin of c. spiroforme binds the same host cell receptor as the iota toxin of c. perfringens and the binary toxin of c. difficile (papatheodorou et al., ) . poor hygiene, stress, and diet can influence pathogenesis of the disease (bain et al., ; songer, ) . pathology grossly, ceca may be enlarged with gas, may exhibit serosal hemorrhages, and have liquid contents (carman and borriello, ) . cecal contents may be stained with blood (peeters et al., ) . c. spiroforme is mainly associated with lesions in the cecum, but may also involve lesions in the proximal colon and distal ileum (keel and songer, ) . mucosal necrosis can be observed microscopically (keel and songer, ) . the mucosa of the ileum, cecum, and colon may be denuded. cellular debris, red blood cells, and fibrin may be found in the intestinal lumen. polymorphonuclear cell infiltration and edema can be found in the lamina propria and submucosa. epithelial degeneration and dilation were found in the renal tubules of some rabbits (peeters et al., ) . diagnosis gram staining of smears prepared from intestinal contents can be used to detect c. spiroforme (bain et al., ) . clostridial culture and toxin detection assays have been described (agnoletti et al., ; bain et al., ; borriello and carmen, ; peeters et al., ) . c. spiroforme can be isolated from the intestinal contents of rabbits by high-speed centrifugation (holmes et al., ) . pcr assays for c. spiroforme and the iota toxin (binary toxin) have been developed (drigo et al., ) . differential diagnoses the differential diagnoses should include other clostridia, e. coli, viruses, and parasites (peeters et al., ) . treatment, prevention, and control the iota toxin from c. spiroforme is neutralized by serum prepared against the iota toxin of c. perfringens type e (borriello and carmen, ; carman and borriello, ; songer, ) . prevention, via reduction of risk factors and prudent use of antibiotics, is probably more important laboratory animal medicine than treatment (agnoletti et al., ) . cholestyramine has been used to prevent experimental enterotoxemia induced by clindamycin in rabbits (lipman et al., ) . fecal flora transplants using nonpathogenic c. spiroforme or c. difficile have been suggested for competitive inhibition of toxigenic strains (carman and evans, ) . no commercial vaccines are available for rabbits; however, vaccination of weanling rabbits with a toxoid imparted protection to intraperitoneal challenge with iota toxin (songer, ) . administration of antibiotics and change in diet are usually the treatment for c. spiroforme infections (songer, ) . the antibiotic susceptibility of c. spiroforme has been investigated (agnoletti et al., ; carman and wilkins, ) . c. spiroforme can have a wide range of resistance to antimicrobial classes used in rabbit therapy (agnoletti et al., ) . feeding fresh meadow hay has been suggested (bain et al., ) . research complications the mortality due to enterotoxemia caused by c. spiroforme would be disruptive to ongoing studies. no other complications have been reported. ii. clostridium difficile etiology c. difficile is a gram-positive, sporeforming, anaerobic bacillus commonly associated with diarrhea and colitis in humans and animals (keel and songer, ) . clinical signs c. difficile infection may be associated with anorexia, depression, diarrhea, fecal-staining of the perineum, decreased fecal output, abdominal distention, and death (perkins et al., ; rehg and lu, ) . peracute death, without clinical signs, is also a common presentation in rabbits (keel and songer, ; perkins et al., ) . epizootiology the spread of c. difficile involves carrier animals that do not show clinical signs of disease (keel and songer, ) . the carrier state may depend on the age of the individual (keel and songer, ) . c. difficile is thought to be acquired from the environment due to persistent contamination with spores (keel and songer, ) . disease is associated with antibiotic treatment but can also develop spontaneously (without antibiotic treatment) (perkins et al., ; rehg and lu, ) . the disease may also occur after stressful events such as weaning, sudden dietary changes, lactation, kindling, and illness (perkins et al., ) . rabbits that have been recently weaned are the most susceptible (perkins et al., ) . newborn rabbits are resistant to c. difficile disease possibly due to the lack of receptors for the toxins (keel and songer, ) . similar to c. spiroforme, the pathogenesis is associated with disruption of the gut flora and with colonization and proliferation of toxigenic clostridium. pathology grossly, a fluid-filled cecum and colon may be found on necropsy (rehg and lu, ) . spontaneous disease in rabbits is associated with lesions in the small intestine, most commonly in the ileum (keel and songer, ) . in one study, the small intestine was distended with fluid and the cecum was distended with chyme (perkins et al., ) . c. difficile is also associated with hemorrhagic typhlitis in hares (dabard et al., ) . c. difficile causes severe jejunal lesions in rabbits, but cecal lesions may occur (keel and songer, ; perkins et al., ) . mural hemorrhages, mucosal necrosis, and submucosal edema have been observed (perkins et al., ) . toxins a (enterotoxin) and b (cytotoxin) act synergistically and are essential virulence factors of c. difficile that enter the cells through receptor-mediated endocytosis (keel and songer, ) . toxins a and b disrupt the actin cytoskeleton by disrupting rho-subtype intracellular signaling molecules that affect cellular function (keel and songer, ) . inflammation and neurogenic stimuli also are involved in the pathogenesis of c. difficile disease (keel and songer, ) . in addition to toxins a and b, some c. difficile strains produce an actinspecific adp-ribosyltransferase or binary toxin (stubbs et al., ) . diagnosis c. difficile isolation and toxin assays have been described (keel and songer, ; perkins et al., ; rehg and lu, ) . c. difficile selective agar is commercially available. the tissue culture cytotoxin assay for c. difficile toxin b is considered the 'gold standard' (belanger et al., ) . c. difficile toxin b can be neutralized with c. sordelli antiserum, but not with c. spiroforme antiserum (perkins et al., ) . commercially available enzyme immunoassays to detect c. difficile toxin(s) have been used to diagnose rabbit cases (garcia et al., ; perkins et al., ) . pcr assays have been developed (belanger et al., ; goldenberg et al., ; houser et al., ; pallis et al., ) . differential diagnoses the differential diagnosis of peracute death in rabbits should include infection with clostridium spp. and/or ehec infection (garcia et al., ; perkins et al., ) . treatment, prevention, and control as with c. spiroforme, the reduction of risk factors and the prudent use of antibiotics are recommended (agnoletti et al., ) . cholestyramine may also be used for prevention (lipman et al., ) . fecal flora transplants have been suggested and commercial probiotic strains are able to inhibit c. difficile and c. perfringens in vitro (carman and evans, ; schoster et al., ) . research complications the sporadic nature of deaths due to c. difficile infection is unlikely to result in significant complications to research. iii. clostridium perfringens c. perfringens type e produces alpha and iota toxins and is an laboratory animal medicine uncommon cause of enterotoxemia in rabbits (redondo et al., ; songer, ) . because of the similarity between the iota toxins of c. spiroforme and c. perfringens type e, detection of toxin alone for diagnostic purposes will not differentiate between the two organisms (songer, ) . pcr can be used for typing c. perfringens based on amplification of toxin genes (daube et al., ) . historically, a disease process associated with e. coli infection was known as colibacillosis. currently, e. coli is classified based on the virulence factors that are genetically encoded and expressed in the bacteria. different virulence factors are associated with different e. coli 'pathotypes'. pathotypes may be associated with three general clinical syndromes: enteric/diarrheal disease, urinary tract infections, and sepsis/meningitis (kaper et al., ) . the centers for disease control and prevention currently recognizes six pathotypes associated with diarrhea in humans: enteropathogenic e. coli (epec), shiga toxin (stx)-producing e. coli (stec; also known as enterohemorrhagic e. coli (ehec) or verocytotoxin-producing e. coli (vtec)), enterotoxigenic e. coli (etec), enteroaggregative e. coli (eaec), enteroinvasive e. coli (eiec), and diffusely adherent e. coli (daec) (http://www.cdc.gov/ecoli/general/). comparative genomic analyses identified genes that were isolateand pathovar-specific and clustered strains according to pathotypes (lukjancenko et al., ; rasko et al., ) . two more emerging pathotypes have been suggested: adherent invasive e. coli (aiec; associated with crohn's disease in humans) and shiga toxin-producing enteroaggregative e. coli (steaec; associated with a large outbreak of hemolytic uremic syndrome (hus) in europe) (clements et al., ) . of these pathotypes, epec and stec are associated with natural disease in rabbits (cantey and blake, ; garcia et al., ) . in addition, necrotoxigenic e. coli (ntec) are associated with disease in rabbits (blanco et al., ) . pathogenic animal and human strains are very closely related and have virulence genes in common (clermont et al., ) . therefore, it is important to determine which e. coli pathotype(s) are associated with disease in rabbits in order to characterize new diseases and/or more accurately diagnose, prevent, control, and treat the condition as well as for epidemiological investigations. etiology epec carry the eae gene that encodes intimin, a protein involved in induction of attaching and effacing lesions in the intestine. e. coli serotype o , also known as rdec- , is the prototype epec strain which was isolated from rabbits with diarrhea and has been used experimentally as a model to study epec-induced disease (cantey and blake, ) . epec is an important cause of potentially fatal infant diarrhea in developing countries (kaper et al., ; swennes et al., ) . clinical signs ehec o was isolated from an outbreak of bloody diarrhea and sudden death in dutch belted rabbits (fig. . ) (garcia et al., ) . acute diarrhea following shipment was associated with epec o :h infection in laboratory rabbits (swennes et al., ) . laboratory rabbits can be reservoir hosts of pathogenic e. coli without exhibiting clinical signs (garcía and fox, ; swennes et al., ) . patent or occult blood may be detected in the feces of infected rabbits (camguilhem and milon, ; garcia et al., ) . epizootiology epec and ehec can be enzootic in rabbit colonies and these bacteria are transmitted by the fecal-oral route (garcía and fox, ; swennes et al., ; swennes et al., ) . epec and ehec coinfections are possible (garcía and fox, ; garcia et al., ) . ehec are a subset of stec that carry stx gene(s) that encode stx(s) and also carry the eae gene that encodes intimin (melton-celsa et al., ) . rabbits can harbor stec strains and are recognized as their vectors and reservoir hosts (bailey et al., ; blanco et al., ; garcía and fox, ; kim et al., ; leclercq and mahillon, ; pohl et al., ; pritchard et al., ; scaife et al., ) . pathology grossly, paintbrush hemorrhages of the cecal serosa may be observed after experimental infection with epec (camguilhem and milon, ) . also, experimentally, the serosal surface of the cecum and/or proximal colon can develop petechial or echymotic hemorrhages and may become edematous and thickened (garcía et al., ) . histologically and ultrastructurally, attaching and effacing lesions with pedestal formation can be observed with epec or ehec infections (kaper et al., ; peeters et al., ) . enterocolitis, nephropathy, and thrombotic microangiopathy can be observed in ehec-infected rabbits (garcía et al., ) . diagnosis feces or intestinal contents can be enriched in broth and then cultured using blood agar, macconkey agar, or ehec-selective media such as sorbitol macconkey agar or raibow® agar (garcía and fox, ; tarr, ; tarr et al., ) . after isolation of e. coli in pure culture, samples can be biotyped using commercial methods such as the api® e strips (bio-mérieux). serotyping and molecular characterization of isolates can be performed by the e. coli reference center (http://ecoli.cas.psu.edu/) at the pennsylvania state university. pcr assays can be utilized to detect virulence factors characteristic of epec, ehec, or other pathogenic e. coli as well as for high-resolution genotyping for epidemiological studies (blanco et al., ; garcía and fox, ) . molecular characterization of stec strains can be performed by the stec center (http://www. shigatox.net/new/) at michigan state university. differential diagnoses the differential diagnoses should include other causes of diarrhea in rabbits including the clostridial diseases and intestinal coccidiosis. treatment, prevention, and control for prevention, avoid introduction of rabbits of unknown pathogenic e. coli status into a colony. rabbits should be screened by culture and e. coli isolates characterized for virulence factors by pcr. also, since it is known that ehec can contaminate plants and vegetables, laboratory personnel should be aware that rabbit feeds such as hay, alfalfa, and other greens have the potential to introduce enteric pathogens such as ehec into laboratory rabbits (berger et al., ; garcía and fox, ) . ehec o can survive for days in grass hay feed (davis et al., ) . cesarean section rederivation and antibiotic treatment have been suggested for eradication of pathogenic e. coli in rabbits (swennes et al., ) . a 'one health' approach should be incorporated to control ehec infections because outbreaks such as with ehec o in humans was linked to consumption of spinach contaminated by feral swine and was additionally isolated from domestic cattle, surface water, sediment, and soil (garcia et al., ) -a good example of integrating human, animal, and environmental health (monath et al., ) . antibiotic treatment should be based on culture and sensitivity. importantly, in humans infected with ehec, treatment with antibiotics is controversial due to the possibility of induction of stx-encoding bacteriophages and worsening of the clinical condition due to hemolytic uremic syndrome (tarr et al., ) ; therefore, antibiotic treatment of rabbits infected with ehec may not be recommended. clinically affected rabbits can be treated with fluids as this intervention is nephroprotective in humans (hickey et al., ) . in addition, rabbit epec strains may carry extended-spectrum beta-lactamases making them resistant to antibiotics (poeta et al., ) . parenteral enrofloxacin administered prior to shipment decreased morbidity and mortality associated with endemic epec (swennes et al., ) . research complications epec infection can cause high morbidity and mortality in laboratory rabbit colonies and can affect studies involving intestinal physiology in rabbits. epec and ehec present a zoonotic risk (garcia et al., ; poeta et al., ; swennes et al., ) . etiology treponema paraluiscuniculi is a noncultivable species that infects rabbits and causes venereal spirochetosis or treponematosis (also known as rabbit syphilis, vent disease, or cuniculosis) (smajs et al., ) . although its genome structure is closely related to other pathogenic treponema species including t. pallidum subsp. pallidum, the etiological agent of human syphilis, t. paraluiscuniculi does not infect humans (smajs et al., ) . genome sequencing revealed that t. paraluiscuniculi evolved from a t. pallidum-like ancestor and adapted to rabbits during loss of infectivity to humans (smajs et al., ) . t. paraluiscuniculi can also infect hares, and causes seroconversion, but no clinical signs. in contrast, the related organism, t. paraluisleporis, can infect and induce disease in rabbits and hares. the close phylogenetic association between t. paraluiscuniculi and t. paraluisleporis suggests that these organisms could be given a subspecies or ecovar status rather than species status (lumeij et al., ) . clinical signs in naturally infected rabbits lesions commonly occur in the vulva or prepuce (cunliffe-beamer and fox, a) . other parts of the body that may be affected, in descending order, include the anal region, nose, eyelid, and lip (cunliffe-beamer and fox, a) . naturally infected rabbits develop lesions of the ear, face, prepuce, and anus (small and newman, ) . in a study involving intratesticular inoculation of t. paraluiscuniculi, single lesions were found in the prepuce or scrotum and multiple lesions were found in the nose, mouth, ear, prepuce, foot, and scrotum (small and newman, ) . all lesions had abundant treponemes by dark-field examination (small and newman, ) . epizootiology susceptibility to, and expression of venereal spirochetosis, may vary with the strain of rabbit (cunliffe-beamer and fox, b) . the prevalence of t. paraluiscuniculi infection increased with parity in adult females and most adult males seroconverted within months of entering the breeding program. these findings suggested that t. paraluiscuniculi spreads by horizontal transmission in adult rabbits (digiacomo et al., ) . in an enzootically infected conventional rabbit colony, the frequency of venereal spirochetosis was lower in rabbits less than months of age than in adult rabbits (cunliffe-beamer and fox, b) . experiments involving cross fostering of newborns indicated that infection occurred at birth (vertical transmission) and during the suckling period (cunliffe-beamer and fox, b) . in addition, horizontal transmission by coitus and skin contact occurs (small and newman, ) . experimental topical or intradermal-subcutaneous genital inoculation of adult rabbits confirmed these routes of transmission (cunliffe-beamer and fox, b) . the t. pallidum repeat (tpr) genes in t. pallidum subsp. pallidum are thought to code for potential virulence factors. tprk was the only tpr homolog found in t. paraluiscuniculi that induced antibody and t cell responses after experimental inoculation of rabbits indicating that tprk may be an important virulence factor in venereal spirochetosis (giacani et al., ) . virulence factors and pathogenesis have been recently reviewed (smajs et al., ) . pathology the lesions include erythemathous macules or papules to erosions, ulcers, and crusts (cunliffe-beamer and fox, a) . diagnosis serologic tests that have been used include the nontreponemal antigen tests (venereal disease research laboratory (vdrl) and rapid plasma reagin), microhemagglutination, and fluorescent treponemal antibody absorption tests (digiacomo et al., ) . although the nontreponemal antigen tests were not completely satisfactory, the vdrl test was more sensitive and the plasma reagin test was more specific in detecting t. paraluiscuniculi infection (digiacomo et al., ) . the sensitivity and specificity of the microhemagglutination test compared favorably with the fluorescent treponemal antibody absorption test and was recommended as the optimal assay to make a diagnosis (digiacomo et al., ) . detection of t. paraluiscuniculi in lesions can be achieved by dark-field microscopic examination of scrapings from lesions and by histological evaluation of silver-stained testicular sections (cunliffe-beamer and fox, a; faine, ) . pcr has been used for molecular characterization of treponemes including t. paraluiscuniculi (cejkova et al., ) . differential diagnoses the skin lesions may be confused with abrasions (trauma), mycotic infections, and lesions of ectoparasites (acariasis) (small and newman, ) . treatment, prevention, and control there are no vaccines available at this time to prevent treponematosis in rabbits; however, rabbits have been used as experimental models to test vaccines against t. pallidum in humans (ho and lukehart, ) . hysterectomy derivation can eliminate venereal spirochetosis (cunliffe-beamer and fox, b) . a study investigating two different doses ( , or , iu/kg body weight/ week) of benzathine penicillin g-procaine penicillin g to treat rabbits at -day intervals found that both dosages were effective. lesions healed within weeks of the first treatment and the plasma reagin titers declined markedly or disappeared by the sixth week after the first treatment (cunliffe-beamer and fox, c) . research complications t. paraluiscuniculi can affect studies of t. pallidum in rabbits (small and newman, ) . partial immunological cross-protection has been observed between t. paraluiscuniculi and t. pallidum (smajs et al., ; turner and hollander, ) . etiology lawsonia intracellularis is a gram-negative, curved to spiral-shaped, obligate intracellular bacterium that causes proliferative enteropathy in rabbits and other species of animals (sait et al., ; schauer et al., ) . clinical signs an intraepithelial 'vibrio' was associated with acute typhlitis in rabbits - weeks after weaning (moon et al., ) . diarrhea was reported in japanese white rabbits with presumptive l. intracellularis infection and histiocytic enteritis (umemura et al., ) . in another report, sucklings and weanlings were affected and the feces of most of the rabbits were characterized as semifluid and mucinous or pasty, and three rabbits had watery diarrhea (schoeb and fox, ) . these affected rabbits were afebrile and lethargic, refused food and water, and most died within a few days after the onset of diarrhea (schoeb and fox, ) . diarrhea, depression, and dehydration that resolved over the course of - weeks were reported in -to -week-old new zealand white (nzw) rabbits (hotchkiss et al., ) . diarrhea and weight loss were reported in a -month-old rabbit (horiuchi et al., ). an outbreak of diarrhea with high ( %) mortality was reported in -to -month-old nzw rabbits with proliferative enterocolitis associated with l. intracellularis and epec (schauer et al., ) . epizootiology proliferative enteropathy generally occurs as isolated cases or occasional minor outbreaks in species other than the pig, blue fox, and hamster (lawson and gebhart, ) . infected rabbits can serve as reservoir hosts for l. intracellularis infection in other species including foals (pusterla et al., a (pusterla et al., , . however, l. intracellularis appears to adapt to the specific animal species it infects (vannucci et al., ) . the pathogenesis of l. intracellularis infection has been reviewed (lawson and gebhart, ; smith and lawson, ) . studies using interferon (ifn)-gamma receptor knockout mice determined that interferon ifngamma plays a significant role in limiting intracellular infection and increased cellular proliferation associated with l. intracellularis infection (smith et al., ) . lawsonia surface antigen (lsaa) plays a role during l. intracellularis attachment to and entry into intestinal epithelial cells (mccluskey et al., ) . balb/ca mice are susceptible to rabbit l. intracellularis isolates but not to pig l. intracellularis isolates suggesting that there are biological differences between the proliferative enteropathy isolates from rabbits and pigs (murakata et al., ) . pathology distention and diffuse mucosal thickening of the jejunum and proximal ileum with enlarged cranial mesenteric lymph nodes was observed in -to -month-old rabbits (umemura et al., ) . thickening of the mucosa was associated with distention of the lamina propria with macrophages and the enlargement of the lymph nodes was also associated with infiltration of macrophages (umemura et al., ) . minute bacilli were observed in the apical cytoplasm of mucosal epithelial cells using toluidine blue (umemura et al., ) . thickening of the cecum and proximal colon has also been reported (hotchkiss et al., ) . in another study, no gross lesions were found in the small intestine, but two suckling rabbits had reddened ceca with congested vessels (schoeb and fox, ) . in this study two types of microscopic lesions were characterized: ( ) erosive and suppurative cecitis and colitis, and ( ) proliferative lesions in the cecum, sacculated colon, ileum, and distal jejunum, or a combination of these (schoeb and fox, ) . some animals had both erosive and proliferative lesions. narrow curved or spiral bacteria were detected in rabbits with erosive and proliferative lesions using warthin-starry stain and these bacteria were more abundant in cases with severe lesions (schoeb and fox, ) . proliferative intestinal lesions contained curved to spiral argyrophilic intracellular bacteria in the apical cytoplasm of crypt enterocytes (hotchkiss et al., ) . diagnosis the s ribosomal dna sequences from l. intracellularis isolates from different species of animals are highly similar (cooper et al., a) . however, antigenic differences have been found between pig and rabbit isolates . the complete genome sequence of a porcine strain has been recently reported (sait et al., ) . l. intracellularis can be detected in feces from healthy and diarrheic rabbits (lim et al., ) . pcr assays to detect l. intracellularis dna in feces have been evaluated (pedersen et al., ) . these assays can be used for ante mortem diagnosis of proliferative enteropathy in pigs (pedersen et al., ) . pcr primers used to diagnose lawsonia in other animals species have been used in rabbit cases (cooper et al., b; duhamel et al., ; fox et al., ; horiuchi et al., ; hotchkiss et al., ; jones et al., ) . other diagnostic methods include enzyme-linked immunosorbent assay (elisa) using synthetic peptides of lsaa and immunomagnetic separation using anti-lsaa antibody to capture l. intracellularis in fecal samples followed by detection with atp bioluminescence (watarai et al., (watarai et al., , . in tissue sections, l. intracellularis can be detected using silver stains such as warthin-starry stain (duhamel et al., ; horiuchi et al., ; hotchkiss et al., ; schauer et al., ; schoeb and fox, ) . indirect immunofluorescence has also been used in deparaffinized intestinal sections from infected rabbits (schoeb and fox, ) . immunohistochemistry using antiserum against synthetic peptides of lsaa has also been used to detect l. intracellularis in the ileum of a naturally infected rabbit (watarai et al., ) . electron microscopy reveals organisms that are ~ . - . μm wide and ≤ . μm long in the apical cytoplasm of villous and crypt epithelial cells (duhamel et al., ) . l. intracellularis can be cultured from homogenized intestinal tissue in cell lines including iec- (rat small intestinal cells) and mccoy cells (mouse fibroblasts) (lawson and gebhart, ; watarai et al., ) . a quantitative pcr (qpcr) assay that is able to assess the growth of l. intracellularis in cultured cells has also been used to detect the organisms in pig fecal samples and could be used in other animal species (drozd et al., ) . differential diagnoses clinically, the differential diagnosis should include other causes of diarrhea in rabbits. mycobacterium avium subsp. paratuberculosis can infect rabbits and induce thickening of the intestinal mucosa (beard et al., ; greig et al., ) . therefore, rabbit intestinal sections should be examined for acidfast organisms using stains such as ziehl-neelsen stain (duhamel et al., ; horiuchi et al., ; schoeb and fox, ; umemura et al., ) . furthermore, other intestinal organisms may colonize the intestine during l. intracellularis infection in rabbits (duhamel et al., ; hotchkiss et al., ; lim et al., ; schauer et al., ) . other bacterial diseases have been sporadically reported in rabbits. these are summarized in table . . treatment, prevention, and control vaccination strategies have been tested and developed for pigs and horses, but not for rabbits (nogueira et al., ; pusterla et al., b; weibel et al., ) . testing rabbits by pcr prior to introduction to a laboratory colony may be necessary for exclusion of this organism. oral neomycin ( mg/rabbit) was used to treat surviving rabbits during an outbreak of presumptive l. intracellularis and the diarrhea subsided (umemura et al., ) . because l. intracellularis infects the intestine and ifn-gamma appears to be involved in pathogenesis, research involving rabbit gastrointestinal pathology and immune responses may be confounded by infection with this organism. research complications the mortality associated with l. intracellularis infection would be disruptive to ongoing studies. etiology myxomatosis is caused by myxoma virus, a member of the family poxviridae, genus leporipoxvirus (kerr and donnelly, ; spiesschaert et al., ) . clinical signs the severity of disease varies with the strain of virus and the host species and breed. rabbits of the genus oryctolagus are particularly susceptible and often develop a fatal disease characterized by mucinous skin lesions and tumors. affected animals also exhibit edema around the mouth, nose, anus, and genitals as well as progressive conjunctivitis with serous and mucopurulent secretions from the eyes and nose (brabb and di giacomo, ; kerr and donnelly, ; spiesschaert et al., ) . bacterial pneumonia commonly develops and animals die - days after infection. the virus is spread by arthropod vectors and direct contact. epizootiology myxomatosis has a worldwide distribution. various species of sylvilagus and lepus are naturally susceptible (brabb and di giacomo, ) . the myxoma virus genome encodes for a number of immunomodulatory proteins which greatly affect the host immune response by inhibiting apoptosis, interfering with leukocyte chemotaxis, and suppressing leukocyte activation, thereby fostering viral replication and spread (spiesschaert et al., ) . pathology histopathology shows these 'myxomas' to be composed of undifferentiated stellate mesenchymal cells embedded in a matrix of mucinous material and interspersed with capillaries and inflammatory cells (brabb and di giacomo, ; kerr and donnelly, ) . diagnosis diagnosis can be made by pcr or elisa. definitive diagnosis depends on culture of the virus from infected tissues. differential diagnoses rabbits of the genus sylvilagus develop fibroma-like lesions that may be indistinguishable from those caused by rabbit fibroma virus. the two diseases have been distinguished by inoculation of fibroma material into oryctolagus rabbits. they develop fatal disease if the myxoma virus is the etiologic agent, or fibromas if rabbit fibroma virus is responsible. treatment, prevention, and control since the disease is spread by fleas and mosquitoes as well as by direct contact, control measures should include prevention of contact with arthropods and quarantine of infected rabbits. vaccines have been used in europe with some success. most recently, a live recombinant vaccine for both myxomatosis and rabbit hemorrhagic disease has been released in the united kingdom (spibey et al., ) . research complications none have been reported. rabbit (shope) fibroma virus is a leporipoxvirus that is antigenically related to myxoma virus. fibromatosis is endemic in wild rabbits; however, an outbreak in commercial rabbits caused extensive mortality (joiner et al., ) . usually, less virulent strains cause skin tumors in domestic rabbits (raflo et al., ) . the disease is probably spread by arthropods (brabb and di giacomo, ; kerr and donnelly, ) . fibromas are flat, subcutaneous, easily movable tumors, and most often found on the legs and face. tumors may persist for some time but eventually regress. metastasis does not occur. rabbit pox is a rare disease induced by an orthopoxvirus taxonomically similar to vaccina virus that has caused outbreaks of fatal disease in laboratory rabbits in the united states and holland (brabb and di giacomo, ) . rabbits with the disease may or may not present with 'pox' lesions in the skin. the animals have a fever and nasal discharge or days after infection. most rabbits have eye lesions including blepharitis, conjunctivitis, and keratitis with subsequent corneal ulceration. skin lesions, when present, are widespread. they begin as a macular rash and progress to papules up to cm in diameter by days postinfection. the lymph nodes are enlarged, the face is often edematous, and there may be lesions in the oral and nasal cavity. at gross necropsy, nodules can be found in the liver, gall bladder, spleen, lung, and reproductive organs. necrosis is widespread. characteristic cytoplasmic inclusions seen in many poxvirus infections are rare in this disease. mortality is high in affected animals. the virus is apparently spread by aerosols and is difficult to control. rabbit pox is used as a model of smallpox in humans in response to the potential use of smallpox as a bioterrorism agent. it is an effective model for the evaluation of potential therapies against smallpox (nalca and nichols, ; rice et al., ) . four herpesviruses (leporid herpesviruses , , , and ) have been isolated from rabbits and hares (davison, ) . leporid herpesvirus (lhv- ) was isolated from cottontail rabbits and is also known as cottontail rabbit herpesvirus. it is not pathogenic for domestic rabbits. lhv- (herpesvirus cuniculi) was isolated from domestic rabbits (o. cuniculus) and causes subclinical infections. lhv- (herpesvirus sylvilagus) was isolated from cottontail rabbits. cottontail rabbits infected with the virus develop a lymphoproliferative disease with lymphoid infiltration of many organs (hesselton et al., ) . lhv- does not infect domestic rabbits. lhv- - are tentatively classified in the genus radinovirus, subfamily gammaherpesvirinae. lhv- was isolated from domestic rabbits and caused severe disease in preweanlings (jin et al., a, b) . clinical signs included weakness, anorexia, conjunctivitis, keratitis, and periocular swelling. some animals also developed skin ulcers. lhv- is genus simplexvirus, subfamily alphaherpesvirinae. the cottontail rabbit is the natural host of the cottontail (shope) papillomavirus, a kappapapillomavirus, laboratory animal medicine which causes horny warts primarily on the neck, shoulders, and abdomen. the disease has a wide geographic distribution with the highest incidence occurring in rabbits in the midwest (brabb and di giacomo, ) . as many as % of infected sylvilagus rabbits develop squamous cell carcinomas. natural outbreaks in domestic rabbits have been reported (hagen, ) . in these natural outbreaks, papillomas were more common on the eyelids and ears. the virus is transmitted by arthropod vectors. this virus is used extensively as a model for the study of oncogenic virus biology and as a model for the treatment and prevention of papillomavirus infections in humans (christensen, ; salmon et al., ; sundarum et al., ) . oral papillomatosis in domestic rabbits is caused by a kappapapillomavirus that is related to but distinct from the cottontail rabbit papilloma virus. naturally occurring lesions have been seen in laboratory rabbits and appear as small, white, discrete growths on the ventral surface of the tongue (kerr and donnelly, ) . lesions may ulcerate. microscopic examination shows them to be typical papillomas. most lesions eventually regress spontaneously (brabb and di giacomo, ; kerr and donnelly, ) . etiology rabbit rotavirus is a member of the family reoviridae. all isolates of rabbit rotavirus have been classified as group a and have been serotype (brabb and di giacomo, ; kerr and donnelly, ) . clinical signs the severity of disease in naturally occurring outbreaks has been variable. in severe outbreaks, affected animals exhibit anorexia, dehydration, and watery to mucoid diarrhea and mortality can be quite high. in other reported outbreaks, mild, transient diarrhea has been reported (brabb and di giacomo, ; kerr and donnelly, ) . similarly, attempts to experimentally produce clinical disease have had variable results. mild diarrhea is usually seen, but in some studies there has been significant mortality. it is probable that other factors, such as maternal antibodies, diet, and the presence of pathogenic bacteria, affect the severity of clinical disease in outbreaks. for example, in combined experimental infections with both rotavirus and e. coli, the inoculation of both organisms led to more serious clinical signs than when given alone, indicating that rotavirus may have been a more significant determinant in the manifestation of this disease (thouless et al., ) . these investigators also showed that older rabbits were naturally more resistant to the combined infection with rotavirus and e. coli. epizootiology rotavirus infections of domestic rabbits are common (brabb and di giacomo, ) . many colonies of rabbits are serologically positive, and rotavirus can be isolated readily from rabbit feces. in endemically infected colonies, maternal antibodies to rotaviruses are passed transplacentally and decline at around the time of weaning (brabb and di giacomo, ) . rabbits of weaning age are most susceptible. very young rabbits appear to be protected from rotavirus infection by passive immunity, when present, but are quite susceptible when there is none (schoeb et al., ) . this is also the time when they are most likely to be subjected to diet changes that may contribute to a change in microbial flora. pathology in affected animals, there is villous atrophy and loss of epithelial cells in the small intestines. a lymphocytic infiltrate is present. diagnosis immunoassays (elisa and multiplex fluorescent immunoassay) are commercially available for rabbit rotavirus. a commercial immunochromatography kits for detecting human rotavirus infection was used successfully to diagnose rabbit rotavirus infection (fushuku and fukuda, ) . differential diagnoses c. piliforme, c. spiroforme, c. difficile, e. coli, lawsonia intracellularis, coronavirus, coccidiosis, and intestinal parasites should be considered. research complications colony mortality would be disruptive to ongoing studies. pleural effusion disease/infectious cardiomyopathy was diagnosed in rabbits inoculated with t. palliduminfected stocks of testicular tissue. because these treponemes could not be grown in vitro, the organism was propagated by passage in rabbits. the stocks were contaminated with a coronavirus, although it is not known whether this virus originated from rabbits or was a virus of human origin that had adapted to rabbits. with continued passage, the virus became more virulent, and significant mortality ensued. evidence indicated that it was not transmitted by direct contact. rabbits died due to congestive heart failure, and microscopic examination showed there was widespread necrosis of the heart muscle. it has been suggested that infection with this virus might be a model for the study of virus-induced cardiomyopathy (brabb and di giacomo, ; kerr and donnelly, ) . rabbit enteric coronavirus has been isolated from tissue cultures from rabbits (brabb and di giacomo, ; kerr and donnelly, ; lapierre et al., ) and has been associated with one naturally occurring outbreak of diarrhea in a barrier-maintained breeding colony (eaton, ) . these rabbits developed severe diarrhea, and most died within h of onset of clinical signs. attempts to reproduce the disease led to watery diarrhea, which lasted a short time; however, none of the rabbits died. it is quite probable that other microorganisms or laboratory animal medicine unknown environmental factors contributed to the severity of this outbreak. etiology rabbit hemorrhagic disease virus is a calicivirus of the genus lagovirus and is the causative agent of rabbit hemorrhagic disease (rhd) (abrantes et al., ; brabb and di giacomo, ; kerr and donnelly, ) . clinical signs three clinical syndromes are seen (abrantes et al., ) . the peracute form is characterized by sudden death without clinical signs. acutely affected animals demonstrate anorexia and depression. in addition, neurologic signs, respiratory signs, ocular hemorrhage, and epistaxis may be seen. morbidity and mortality are extremely high. lymphopenia and abnormalities in coagulation parameters are also seen. in the subacute form, similar signs may occur but are considerably milder and most of these rabbits survive (abrantes et al., ; kerr and donnelly, ) . epizootiology rabbit hemorrhagic disease was first reported in china in and is currently endemic in europe, asia, africa, australia, and new zealand. in addition, isolated outbreaks have been reported in numerous countries. the virus is transmitted by the fecal-oral route. the role of fomites and arthropod vectors is also suspected (brabb and di giacomo, ; kerr and donnelly, ) . the incubation period may be as short as or days, and sudden death with no previous signs is common. pathology periportal hepatic necrosis is the only consistent microscopic lesion, and the animals die due to disseminated intravascular coagulopathy and thrombosis (abrantes et al., ; kerr and donnelly, ) . diagnosis the virus has not been successfully grown in vitro; however, diagnosis can be confirmed with negative-contrast electron microscopy of liver tissue. specific antibodies can be detected by elisa or by hemagglutination inhibition. differential diagnoses a related calicivirus, european brown hare virus, has caused disease in hares in several countries in europe (brabb and di giacomo, ) . animals present with necrotic hepatitis, hemorrhages in the trachea and lungs, and pulmonary edema. a monoclonal antibody elisa is available for serodiagnosis, and control measures are similar to those for rhd. the agent resists drying, can be carried on fomites, and may be transmitted via respiratory and intestinal secretions (mitro and krauss, ) . any rabbit colonies with this disease should be quarantined and depopulated, and the environment thoroughly cleansed and disinfected. research complications colony mortality would be disruptive to ongoing studies. other viral infections several other viruses have been isolated from rabbit tissues, but have not been shown to produce disease. these include paramyxoviruses and bunyaviruses. serologic titers to togaviruses and flaviviruses have also been demonstrated in rabbits (brabb and di giacomo, ; kerr and donnelly, ) . etiology hepatic coccidiosis is caused by the parasite eimeria stiedae, which has also been referred to as monocystis stiedae, coccidium oviforme, and c. cuniculi (hofing and kraus, ) . clinical signs the clinical disease has a wide range of manifestations. mild infections often result in no apparent disease. most clinical signs are the result of interruption of normal hepatic function and blockage of the bile ducts. these signs are more common in juvenile rabbits and can include hepatomegaly, icterus, and anorexia (schoeb et al., ) . diarrhea can occur at the terminal stages of the disease (hofing and kraus, ) . decreased growth rates and weight loss are common. joyner et al. ( ) demonstrated that infected rabbits begin to lose weight within days. enlargement of the liver (hepatomegaly) is common. the liver normally is approximately . % of the body weight, but rabbits with severe hepatic coccidiosis may have livers that contribute to greater than % of the body weight (lund, b) . the age of the host strongly affects parasite development and oocyst production. four-month-old, coccidia-free rabbits experimentally infected with e. stiedae produced fewer oocysts than similarly infected -monthold rabbits (gomez-bautista et al., ) . epizootiology e. stiedae is found worldwide, although rabbits bred for use in research are commonly free of the parasite. transmission occurs by the fecaloral route, as for other coccidia. the organism has also been experimentally transmitted by intravenous, intraperitoneal, and intramuscular administration of oocysts (pellérdy, ) . smetana ( ) demonstrated that infection of the entire liver occurred following ligation of the right bile duct and inoculation of e. stiedae oocysts. the study also showed that infection occurred earliest within the small intrahepatic ducts, leading to the theory that infection occurred via blood or lymph. the precise life cycle is still undetermined, although a number of studies have examined it (horton, ; owen, ; rose, ) . sporozoites have been demonstrated in the lymph nodes following experimental inoculation (horton, ; rose, ) . pathology necropsy often shows the liver to be enlarged and discolored, with multifocal yellowish laboratory animal medicine white lesions of varying size (fig. . ) . exudate in the biliary tree is common, along with dilatation of bile ducts. microscopically, papillomatous hyperplasia of the ducts along with multiple life-cycle stages of the organism can be observed in the biliary epithelium ( fig. . ) . diagnosis infected rabbits may have decreased fibrinogen when compared to uninfected rabbits (cam et al., ) . serum bilirubin levels can rise to mg/ dl, increasing as soon as day of infection and increasing through days - before moderating (rose, ) . leukocytosis and anemia can be observed and acute phase proteins are notably increased by days post infection (freitas et al., ) . diagnosis can be made by examination of fecal material, by either flotation or concentration methods. oocysts can also be detected within the gallbladder exudate (hofing and kraus, ) . alternatively, oocysts can sometimes be observed by microscopic examination of impression smears of the cut surface of the liver. ultrasonography may be a useful tool for diagnosis, with dilated vessels and bile ducts and increased echogenicity of the liver parenchyma (cam et al., ) . differential diagnoses the hyperplastic biliary ducts can be mistaken grossly for neoplasia. other types of parasitic hepatitis should be considered as differential diagnoses. less frequently, hepatitis secondary to bacterial infections can occur. treatment, prevention, and control control of the infection until development of natural immunity is one strategy to minimize the severity of disease. davies et al. ( ) demonstrated that immunity occurs following a light infection with e. stiedae. in the rabbit, immunity to eimeria may be lifelong (niilo, ; pellérdy, ) . prevention of hepatic coccidiosis with sulfaquinozaline in the feed ( ppm) was shown to prevent infection when experimental challenged with , sporulated oocysts (joyner et al., ) . sulfonamides have been shown effective against eimeria spp. (hagen, ; horton-smith, ; jankiewicz, ; lund, a; tsunoda et al., ) . treatment with toltrazuril ( ppm in drinking water for one day) has been shown to effectively treat infected animals (cam et al., ) . thorough sanitation of potentially contaminated surfaces is critical to control of coccidiosis. research complications potential research complications arising from hepatic coccidiosis are considerable. the resulting liver damage and decreased weight gains can complicate both the supply of rabbits for research as well as adversely affect research protocols. etiology there are at least different pathogenic species of intestinal coccidia in rabbits, including eimeria coecicola, e. elongate, e exigua, e. intestinalis, e. flavescens, e. irresidua, e. magna, e. matsubayashii, e. media, e. nagnurensis, e. neoleporis, e. piriformis, e. vejdovskyi, and e. perforans (pakandl, ). all of these coccidia are presented here as a group rather than as individual species of intestinal coccidia. clinical signs although intestinal coccidiosis may be subclinical, clinical signs can range from mild to severe and can result in death of the animal. postweanling rabbits are the most likely to experience mortality related to intestinal coccidiosis. suckling rabbits (< days old) are generally considered to be resistant to infection (pakandl and hlaskova, ) . clinical signs also depend on the species of coccidia that are present. severe diarrhea, weight loss, or mild reduction in growth rate are all laboratory animal medicine possibilities. fecal occult blood may be detected with e. perforans infection (li and ooi, ) . death is usually associated with severe dehydration subsequent to diarrhea (frenkel, ) . epizootiology intestinal coccidiosis is a common rabbit disease worldwide (varga, ) . transmission is by the fecal-oral route through ingestion of sporocysts. unsporulated oocysts are passed in the feces and are not infective. such oocysts will, however, sporulate to an infective stage within days after shedding; thus, it is important that sanitation be frequent enough to remove infective stages from the environment. the oocyst burden of feces can be enormous. gallazzi (gallazzi, ) demonstrated that a subclinical carrier of intestinal coccidia had , oocysts/gram of feces and that a rabbit with diarrhea could shed in excess of , oocysts/gram of feces. environmental contamination with oocysts can be a problem when large numbers of oocysts are being excreted. the life cycles of eimeria spp. are similar to those of other coccidia. schizogony, gametogony, and sporogony are the three phases of this life cycle. other sources can be consulted for greater detail on the life cycle of these protozoans (davies et al., ; pakandl, ; pakandl and jelinkova, ; pellérdy, ; rutherford, ) . pathology lesions are apparent in the small and large intestines. necrotic areas of the intestinal wall appear as white foci (pakes, ; pakes and gerrity, ) . the location and extent of the lesions depend on the species of coccidia. diagnosis diagnosis of intestinal coccidiosis can be made through identification of the oocysts in the feces (pakes and gerrity, ) . a pcr has been developed (oliveira et al., ) that differentiates between of the different eimeria species that infect the domestic rabbit. this test has excellent sensitivity, with the ability to detect . - . sporulated oocysts per sample. smaller scale pcr for detection and differentiation between the more pathogenic species (e. intestinalis, e. flavicenens, and e. stiedae) has also been developed (yan et al., ) . differential diagnoses other causes of diarrhea in rabbits should be considered including tyzzer's disease, the clostridial diseases, colibacillosis, l. intracellularis, enteric coronovairus and rotavirus, protozoons, or intestinal parasites. treatment, prevention, and control because intestinal coccidiosis is most common in postweanling rabbits, prevention of the disease should focus on the preweaning period. an oral vaccination has been developed and consists of a nonpathogenic strain of e. magna. this vaccine is sprayed into the nest box when rabbits are days of age. the preweanling rabbits develop immunity subsequent to infection with the nonpathogenic strain and are then resistant to wild-type strains of e. magna at days of age (drouet-viard et al., ) . other oral vaccines developed from various eimeria strains are also in development (akpo et al., ) . prevention and control of infection can be accomplished by providing . % sulfamerazine or . % sulfaquinoxaline in the drinking water (kraus et al., ) . a combination of sulfaquinoxaline, strict sanitation, and elimination of infected animals has been shown to eliminate intestinal coccidiosis from a rabbit breeding colony (pakes and gerrity, ) . as for hepatic coccidiosis, sulfaquinoxaline provided in the feed ( ppm) is an effective treatment. research complications intestinal coccidiosis can impact studies of the gastrointestinal tract, or have an impact on survival of postweanling rabbits. etiology the protozoan organism cryptosporidium cuniculus has been found in the intestinal tract of the rabbit (hadfield and chalmers, ; inman and takeuchi, ; kaupke et al., ; rehg et al., ; robinson et al., ; shiibashi et al., ; zhang et al., ) . clinical signs clinical signs related to cryptosporidiosis seem to be quite variable in the rabbit. a large farm outbreak (kaupke et al., ) had rabbits that presented with lethargy, anorexia and diarrhea. animals showing clinical signs died within - days. the stress of weaning is thought to have exacerbated these signs. another report describes small intestinal dilatation observed during surgery in a rabbit without other clinical signs (inman and takeuchi, ) . epizootiology transmission is likely via ingestion of thick-walled sporulated oocysts. experimentally infected juvenile rabbits began shedding oocysts in their feces - days post infection and continued to shed until days post infection without clinical signs (robinson et al., ) . pathology histopathology of the small intestine of the reported rabbit was characterized by shortened, blunted villi and mild edema of the lamina propria. the lacteals of the ileum were also dilated, and an inflammatory response was observed (inman and takeuchi, ) . diagnosis c. cuniculus is emerging as a potential zoonotic pathogen with several reports in recent years (chalmers et al., (chalmers et al., , zhang et al., ) . in response to this, real-time pcr assays are in development (hadfield and chalmers, ) that detect and differentiate c. cuniculus from c. parvum and c. hominis. differential diagnoses c. cuniculus can only be differentiated from c. hominis and c. parvum via genetic analysis (robinson et al., ) . differential diagnoses would include infection with clostridium piliforme, c. spiroforme, c. difficile, e. coli, lawsonia intracellularis, coronavirus, rotavirus, protozoans, or intestinal parasites. treatment, prevention, and control minimizing stress can possibly prevent or reduce clinical signs laboratory animal medicine (kaupke et al., ) . antibiotics were ineffective in the large farm outbreak. presumably, supportive care (fluids) would be indicated in animals showing clinical signs (schoeb et al., ) . prevention requires husbandry and sanitation practices that prevent exposure. research complications this organism is emerging as a human pathogen, so appropriate precautions should be made to protect research personnel from rabbits positive for c. cuniculus. etiology the etiologic agent responsible for encephalitozoonosis is encephalitozoon cuniculi. this agent is historically known by the name nosema cuniculi (pakes and gerrity, ) and has been divided into three strains (i -rabbit strain, ii -mouse strain, iiidog strain) (didier et al., ) . the disease was first described in as an infectious encephalomyelitis causing motor paralysis in young rabbits (wright and craighead, ) . clinical signs encephalitozoonosis typically has a delayed onset (weeks to months post infection) prior to the exhibition of clinical signs. early infection affects the kidney, liver and lung, while alterations later in the infection are most severe in the kidneys and brain (kunzel and joachim, ) . the organism can be found in the tissues without an inflammatory response (pakes and gerrity, ) . although named for the motor paralysis in young rabbits, the disease is usually latent. if clinical signs are present, they can include convulsions, tremors, torticollis, paresis, and coma (pattison et al., ) as well as signs of kidney failure. intrauterine infection can result in phacoclastic uveitis leading to rupture of the lens capsule (kunzel and joachim, ) . epizootiology transmission is likely horizontal via direct contact or environmental contamination (kunzel and joachim, ) , primarily from ingestion of infected urine (schoeb et al., ; wasson and peper, ) . the pathogen can also be transmitted vertically, as evidenced by in utero pcr positivity reported by baneux and pognan ( ) . pathology the kidneys commonly have lesions at necropsy. typically, there are multiple white, pinpoint areas or gray, indented areas on the renal cortical surface (kraus et al., ) . microscopically, these areas are characterized by granulomatous inflammation. interstitial infiltration of lymphocytes and plasma cells and tubular degeneration may also be present (flatt and jackson, ) . granulomatous encephalitis is a characteristic lesion (fig. . ) (pakes and gerrity, ) . lesions of the spinal cord can also occur (koller, ) . the organisms are often not observed in histologic sections of the lesions. organisms may be seen floating free in the tubules of the kidney (pakes and gerrity, ) . diagnosis diagnosis of encephalitozoonosis can be made using several different methods. histologic examination of tissues and observation of the organism is definitive. brain and kidney samples yield the best detection rates for histopathological diagnosis (leipig et al., ) . the encephalitozoon organism does not stain well with hematoxylin and eosin, and is better demonstrated using giemsa stain, gram stain, or goodpasture's carbol fuchsin stain (pakes, ) . many different serologic tests exist for the organism. indirect fluorescence antibody technique and elisa are both available and reliable (kunzel and joachim, ) . advances in diagnostic techniques have been made in human medicine due to the susceptibility of immunosuppressed patients to this particular infection. several pcr tests for diagnosis and species differentiation of encephalitozoonosis have been developed (croppo et al., ; franzen et al., ; weiss and vossbrinck, ) . pcr can be performed on the intestine, brain, heart, liver, lung, or kidney tissue with a good ( %) overall detection rate reported (leipig et al., ) . differential diagnoses if the animals are demonstrating motor paralysis, conditions such as splay leg should be considered. for neurological signs, consider bacterial meningitis due to p. multocida infection or rabbit hemorrhagic disease. treatment, prevention, and control prevention and control of the organism in the colony are done by elimination of the organism from the colony of infected rabbits. because this is a latent disease in rabbits, serologic methods must be used to identify carriers of the organism. the indirect fluorescence antibody test has laboratory animal medicine been used successfully to identify infected rabbits (cox, ) . the elimination of infected rabbits must be accompanied by disinfection of the environment. several disinfectants have been effective against this organism. encephalitozoon was killed by % (v/v) lysol, % (v/v) formalin, and % (v/v) ethanol (shadduck and polley, ) % hydrogen peroxide, and % sodium hydroxide (kunzel and joachim, ) . successful treatment and prevention of e. cuniculi in the rabbit has been reported with use of fenbendazole (suter et al., ) . for cases of phacoclastic uveitis, removal of the lens is the treatment of choice (kunzel and joachim, ) . research complications encephalitozoonosis is most commonly subclinical disease, which makes it difficult to determine the effects it may have on research. granulomatous reactions would complicate renal physiology and neurologic research. depression of the igg response and an increase in the igm response to brucella abortus antigens has been demonstrated in rabbits infected with encephalitozoon organisms (cox, ) . encephalitozoonosis is also a recognized disease in immunodeficient humans. it is recommended that such individuals seek medical counsel prior to handling rabbits. isolates from humans have been shown to be infectious for rabbits (mathis et al., ) . etiology psoroptes cuniculi is a nonburrowing mite and the causative agent of psoroptic mange, also called ear mange, ear canker, or otoacariasis. the organism is distributed worldwide, but with modern husbandry practices, it is mostly historical in laboratory rabbit colonies (schoeb et al., ) . clinical signs lesions occur primarily in the inner surfaces of the external ear. the lesions are pruritic and can result in scratching, head shaking, pain, and even self-mutilation (hofing and kraus, ) . a tan, crusty exudate accumulates in the ears over the lesions and can become quite extensive and thick (fig. . ) . the skin under the crust is moist and reddened. the ears may become malodorous. epizootiology all stages of the mite (egg, larva, protonymph, and adult) occur on the host. early in the infestation, mites feed on sloughed skin cells and lipids. as local inflammation increases, they ingest serum, hemoglobin, and red blood cells (deloach and wright, ; hofing and kraus, ) . the entire life cycle is complete in days. mites are relatively resistant to drying and temperature and can survive off the host for - days in a temperature range of - °c and relative humidity of - %. pathology lesions are characterized histologically by chronic inflammation, hypertrophy of the malpighian layer, parakeratosis, and epithelial sloughing. a hypersensitivity response to the mites, mite feces, and saliva likely contributes to lesions (hofing and kraus, ) . diagnosis mites are large enough to be seen with the unaided eye or with an otoscope. material scraped from the inner surface of the ear can also be examined using a dissecting microscope. mites are oval-shaped with well-developed legs that project beyond the body margin. adult males measure - μm × - μm, and females measure - μm × - μm (hofing and kraus, ) . differential diagnoses rarely, infection with sarcoptes scabiei or cheyletiella parasitovorax should be considered as differential diagnoses. treatment, prevention, and control several successful treatments have been reported. prior to local treatment, the ears should be cleaned gently to remove accumulated exudate. one treatment involves the application of % rotenone in mineral oil ( : ) every days for days. ivermectin is an effective treatment at dosages of - μg/kg sc or im (curtis et al., ; mckellar et al., ; wright and riner, ) . one or two doses were utilized for effective treatment. treatment of moderate to severe infestations with ivermectin alone can fail. using adjunct vitamin therapy to minimize oxidative tissue damage has been shown to enhance treatment success (singh et al., ) . a single dose of topical selamectin at a minimum of mg/kg selamectin (kurtdede et al., ) and a single injection of laboratory animal medicine eprinomectin at or μg/kg (pan et al., ) were found to be effective treatments. regardless of treatment modality, it is generally recommended that the entire group of rabbits be treated at the same time. heat ( °c) and desiccation (< % humidity) will kill parasites that are not on the host (arlain et al., ) . vaccine targets have been investigated, with gut surface antigen being the primary focus (rossi et al., ) . research complications p. cuniculi has been associated with immune suppression and a systemic inflammatory reaction (shang et al., ) . ear trauma secondary to psoroptes infestation can limit access to the auricular artery and veins. . cheyletiella spp. (c. parasitovorax, c. takahasii, c. ochotonae, c. johnsoni) etiology cheyletiella mites are nonburrowing skin mites of rabbits. they are distributed worldwide. several closely related species have been reported to occur on rabbits, namely, c. parasitovorax, c. takahasii, c. ochotonae, and c. johnsoni (hofing and kraus, ) . clinical signs the anatomic site most commonly infested is the area over the scapulae. there may be mild hair loss in the area, and the skin may have a gray-white scale (cloyd and moorhead, ) . affected rabbits do not scratch, and there is no evidence of pruritus. skin lesions are mild or nonexistent. epizootiology all stages (egg, larva, pupa, and adult) in the life cycle occur on the host. mites remain in association with the keratin layer of the skin and feed on tissue fluid (myktowycz, ) . transmission is probably by direct contact (schoeb et al., ) . pathology when present, skin lesions are characterized by mild dermatitis, hyperkeratosis, and an inflammatory cell infiltrate (hofing and kraus, ) . diagnosis mites can be isolated by scraping or brushing fur in the affected areas onto a slide. clearing samples with - % potassium hydroxide will improve visibility of the mites, which can then be identified using a dissecting microscope. the female measures × μm, and the male is × μm. cheyletiella mites have a large, distinctive curved claw on the palpi (pegg, ) . differential diagnoses other skin mites (such as sarcoptes scabei) or fur mites (leporacarus gibbus) that can affect rabbits should be considered as well as the possibility of dermatophytosis. treatment, prevention, and control topical acaricides are often used and are effective at controlling infestation. ivermectin (subcutaneous or subcutaneous and oral) and selamectin (topical) treatments have been used successfully. eggs in the environment can reinfest the host, so posttreatment environmental sanitation is important (mellgren and bergvall, ) . research complications cheyletid mites can cause a transient dermatitis in humans who are in close contact with infested animals (cohen, ; lee, ) . for this reason, these mites can be considered a zoonotic pathogen. etiology sarcoptes scabiei is a burrowing mite and the causative agent of sarcoptic mange. mites of the genus sarcoptes are generally considered to be one species, s. scabiei, but are often further identified by a variety name corresponding to the host species (e.g., s. scabiei var. cuniculi). the organisms are commonly referred to as itch or scab mites. the disease has a worldwide distribution. clinical signs affected rabbits will exhibit intense pruritus with hair loss and abrasions as a resulting from scratching. serous encrustations on the skin and secondary bacterial infections are common. there has been one report of a secondary infection with the yeast malassezia (radi, ) . anemia and leukopenia can also be observed in affected rabbits (arlain et al., ) . epizootiology sarcoptic are similar to notoedric mites (notoedres cati) in morphology, life cycle, and public health significance. mites burrow and produce an intensely pruritic dermatitis. lesions are most common on the head (hofing and kraus, ) . all stages of sarcoptic mange mites occur on the host. the females burrow into the skin to lay eggs. young larvae can also be found in the skin, whereas older larvae, nymphs, and males reside on the skin surface. mites feed on lymph and epithelial cells (hofing and kraus, ) . pathology amyloidosis of the liver and glomerulus have been reported in rabbits with severe infestation (arlain et al., ) . the skin itself is hyperplastic and hyperkeratotic, with inflammatory response evident in the dermis (schoeb et al., ) . diagnosis because sarcoptes is a burrowing mite, skin scrapings are necessary to diagnose infestation. samples may be cleared with - % potassium hydroxide. female mites measure - μm × - μm. the body shape is round, and the legs are very short. differential diagnoses other causes of dermatitis in rabbits (such as cheyletiella spp., p. cuniculi or dermatophytosis) should be considered. ivermectin is effective at eliminating infestation at μg/kg administered subcutaneously. a single topical dose of selamectin at - mg/kg reduced the number of mites found on skin scrapings of angora rabbits (kurtdede et al., ) and eliminated clinical signs and parasitic infestation in a group of mixed-breed rabbits at a dose of mg/rabbit (farmaki et al., ) . as with psoroptes, more 'natural' treatments are being investigated with good preliminary results from eugenol (pasay et al., ) and eupatorium spp. (nong et al., ) . research complications no specific research complications have been reported. sarcoptes can cause a selflimiting dermatitis in humans. a wide variety of arthropod parasites has been reported in wild rabbits but they are extremely rare in laboratory rabbits. for an extensive listing the reader is referred to other sources (hofing and kraus, ) . etiology historically, the rabbit pinworm was identified as oxyuris ambigua, but is now known as passalurus ambiguus (hofing and kraus, ) . clinical signs even when rabbits have heavy oxyurid burdens, clinical signs are not usually apparent (erikson, ; soulsby, ). one case report described unsatisfactory breeding performance and poor condition in a rabbit colony infested with the parasite. epizootiology p. ambiguus has a direct life cycle. mature pinworms are found in the lumen of the cecum or colon of the rabbit. after ingestion, the eggs hatch in the small intestine, and the larvae molt with maturation in the cecum. the prepatent period is between and days (taffs, ) . transmission occurs easily via ingestion, given that individual rabbits have been found with over adult parasites (hofing and kraus, ) and that embryonated eggs pass out in the feces and are immediately infective (schoeb et al., ; taffs, ) . pathology minimal to no lesions are associated with this pinworm (schoeb et al., ) . diagnosis eggs can be found in feces, cecum, or colon. differential diagnoses this is the only reported pinworm in rabbits and it is not known to cause lesions or disease. treatment, prevention, and control several successful treatment strategies for rabbit oxyuriasis have been reported. piperazine citrate at mg/ ml of drinking water for day was successful in eliminating infestation (hofing and kraus, ) . at and ppm, fenbendazole mixed in the food for days eliminated all immature and adult pinworms (duwell and brech, ) . subcutaneous doses of ivermectin ( . mg/kg) were reported to be ineffective in reducing the burden of passalurus organisms in field populations of snowshoe hares (lepus americanus) (sovell and holmes, ) . due to the direct life cycle, strict husbandry and sanitation practices are required to prevent introduction and spread throughout a rabbit colony (schoeb et al., ) . research complications none have been described. etiology dermatophytosis, also known as ' ringworm' or 'tinea', refers to a skin infection caused by a dermatophyte, a keratinophilic and keratinolytic fungus (chermette et al., ; mendez-tovar, ; robert and pihet, ) . dermatophytes are a group of closely related filamentous fungi that are able to invade the stratum corneum of the epidermis and keratinized tissues including the skin, nail, and hair (kanbe, ) . dermatophytes can infect various animal species, including humans, and the disease is considered contagious and zoonotic (cafarchia et al., b; chermette et al., ; kramer et al., ) . the zoophilic dermatophytes trichophyton mentagrophytes and microsporum canis infect rabbits (cafarchia et al., (cafarchia et al., , a chermette et al., ; kramer et al., ) . clinical signs the general presentation of dermatophytosis in animals is an area of circular alopecia with erythematous margin and thin desquamation (chermette et al., ) . pruritus is generally absent and lesions can be single or multiple (chermette et al., ) . although lesions can be localized in any region, the anterior part of the body and the head seem to be more frequently involved (chermette et al., ) . in rabbits, lesions are often found on the ears and the face (around the eyes and on the nose) and these lesions show scaling and crusting (chermette et al., ; kramer et al., ) . infected rabbits may not exhibit clinical signs and may serve as carriers (balsari et al., ; cafarchia et al., cafarchia et al., , a chermette et al., ; lopez-martinez et al., ) . epizootiology although dermatophytosis is a common cutaneous disease of rabbits and other animals, its incidence is low in well-managed laboratory animal facilities (chermette et al., ; connole et al., ) . contact with infected animals or contaminated environments represent the major risk of infection (chermette et al., ) . young or immunocompromised rabbits are more susceptible (connole et al., ; kramer et al., ) . on rabbit farms, the occurrence of lesions, the age of the rabbits, and farm management practices were identified as the most significant risk factors for the occurrence of dermatophytosis (cafarchia et al., ) . clinically, disease expression varies depending on the host, fungal species, and enzyme production (cafarchia et al., a; vermout et al., ) . the pathogenesis involves contact, adherence, germination, invasion, and penetration (cafarchia et al., a; mendez-tovar, ; vermout et al., ) . these stages can be associated with the secretion of enzymes that degrade the infected tissue components (cafarchia et al., a) . t. mentagrophytes isolates from rabbits with skin lesions showed a significantly higher elastase and gelatinase activity compared laboratory animal medicine to isolates from clinically unaffected rabbits and from the environment (cafarchia et al., a) . furthermore, m. canis isolates from rabbits with skin lesions showed a significantly higher lipase activity compared to isolates from clinically unaffected rabbits and from the environment (cafarchia et al., a) . pathology histopathologic changes consist of mild to severe dermatitis. diagnosis the wood's lamp (ultraviolet light) method and direct examination of hairs and scales are fast and affordable tests (chermette et al., ; robert and pihet, ) . the wood's lamp can be used to screen for infections caused by m. canis (chermette et al., ) . m. canis-infected hairs fluoresce with an apple-green color and can be collected for microscopic examination and culture (chermette et al., ) . the results of the wood's lamp examination should be systematically confirmed by direct examination of hairs and/or fungal culture (chermette et al., ) . deep skin scraping should be performed to obtain hair and scales and confirm the absence of ectoparasites such as mites that can be associated with dermatophytosis (cafarchia et al., ; chermette et al., ) . clearing solutions such as chlorolactophenol or % potassium hydroxide (koh) can then be used to digest keratin prior to microscopic examination (chermette et al., ; robert and pihet, ) . the surface of the hair typically demonstrates clusters or chains of arthroconidia (chermette et al., ) . giemsa-stained skin scrapings allow observation of the arthroconidia along the hair (chermette et al., ) . fungal culture is the 'gold standard' for the diagnosis of dermatophytosis and the only method for the phenotypic identification of dermatophyte species (chermette et al., ) . the fungal culture must be complemented with direct examination of samples for optimal interpretation of the results (chermette et al., ; robert and pihet, ) . samples for fungal culture may include hairs, scales, crusts, skin scrapes, and tissue biopsies (chermette et al., ) . samples that are obtained from the margin of new skin lesions enhance fungal recovery by culture (chermette et al., ) . a brush can also be impressed on the surface of the culture medium after combing the fur and obtaining fungal spores with hair and debris (robert and pihet, ) . two media that can be used for fungal culture include sabouraud dextrose agar (supplemented with cycloheximide and antibiotics) and dermatophyte test media (dtm) (chermette et al., ; robert and pihet, ) . if histological examination is performed, periodic acid schiff (pas), or methylamine silver stain can be used to detect arthroconidia and hyphae (chermette et al., ) . molecular methods to identify dermatophytes have also been described and include pcr-rflp and sequencing of the internal transcribed spacer (its) region (chermette et al., ; kanbe, ; robert and pihet, ) . specific identification of the dermatophyte is essential for a better understanding of the epidemiology and prevention of the disease (chermette et al., ) . differential diagnoses the differential diagnoses can include other dermatoses caused by bacteria or ectoparasites (cafarchia et al., ; chermette et al., ) . treatment, prevention, and control dermatophytosis is considered a self-limiting disease in immunocompetent animals (chermette et al., ) . however, rabbits with dermatophytosis should be culled or separated from a laboratory animal colony due to the contagious and zoonotic nature of the disease (chermette et al., ) . the best method to prevent dermatophyte infection is to prevent contact with infected animals and contaminated environments including fomites (chermette et al., ) . an animal that contacts an infected animal or a contaminated environment can be washed with antifungal shampoo (chermette et al., ) . two vaccines incorporating live attenuated cells of t. mentagrophytes have been used to prevent disease in rabbits and other animals (lund and deboer, ) . the mentavak vaccine is from russia and the trichopelen vaccine (http://www. bioveta.cz/en/veterinary-division/home/) is from the czech republic (lund and deboer, ) . trichopelen is also indicated for treatment of dermatophytosis (lund and deboer, ) . enzootic dermatophytosis may be the result of the high resistance of the arthroconidia in the environment, the number of host species involved, and the close confinement of animals (chermette et al., ) . isolation or culling of infected animals plus environmental and equipment disinfection are required to control this disease (chermette et al., ) . a : dilution of household bleach or a . % enilconazole solution can be used to disinfect the environment (chermette et al., ) . infected animals should be handled with care to avoid zoonotic transmission (chermette et al., ) . if treatment is elected, antifungal treatment shortens the course of the infection and reduces dissemination of arthroconidia to other animals and into the environment (chermette et al., ) . systemic and topical antifungal treatment can be used in combination (chermette et al., ) . systemic drugs include griseofulvin (gold standard) or azole derivatives such as itraconazole (chermette et al., ; vella, ) . it is important to know that these drugs can have side effects and be contraindicated due to their teratogenic potential (chermette et al., ) . topical treatment may include . % enilconazole, a combination of % miconazole and % chlorhexidine, or lime sulfur (chermette et al., ; vella, ) . treatment can be discontinued after two negative fungal culture results (chermette et al., ) . research complications dematophyte lesions could confound histological studies involving the skin (connole et al., ) . etiology pneumocystosis in rabbits is caused by the fungus pneumocystis oryctolagi (dei-cas et al., ) . clinical signs infected rabbits may not develop clinical signs, but immunocompromised hosts can develop severe interstitial pneumonitis (dei-cas et al., ; sheldon, ) . epizootiology corticosteroid treatment can induce disease in infected rabbits; however, spontaneous disease (not associated with drug treatment) can also occur (dei-cas et al., ; sheldon, ; soulez et al., ) . p. oryctolagi is transmitted through the transplacental route (cere et al., a; sanchez et al., ) and through direct contact and aerosolization (cere and polack, ; cere et al., b; hughes, ; wakefield, wakefield, , . spontaneous pneumocystosis can occur at weaning, evolves during - days, and induces lung lesions and blood biochemical profile changes (dei-cas et al., ; soulez et al., ) . the organisms attach specifically to type epithelial alveolar cells and proliferate, filling up pulmonary alveoli cavities leading to respiratory failure (dei-cas et al., ) . changes in surfactant appear to be necessary for pneumocystis proliferation (prevost et al., ) . pneumocystis colonization decreases and becomes very low in -day-old rabbits (dei-cas et al., ) . most rabbits recover from pneumocystosis within - weeks (dei-cas et al., ) . the spontaneous resolution of pneumocystosis in rabbits may be associated with expression of interferon gamma (allaert et al., ) . immunosuppression may be suspected in cases of severe pulmonary disease associated with spontaneous pneumocystosis (sheldon, ) . pathology histologically, cystic forms of the organism can be detected in the lungs using toluidine blue o (tbo), gms, or pas stains (dei-cas et al., ) . interstitial thickening of alveolar septa and increased numbers of type epithelial alveolar cells are characteristic of this infection (creusy et al., ) . diagnosis for diagnosis, samples from nasal cavity wash, or post-mortem, from terminal bronchoalveolar lavage (bal) or lung homogenates can be used for pneumocystis detection by nested pcr (dei-cas et al., ; tamburrini et al., ; wakefield, ) . serological diagnosis can also be performed (tamburrini et al., ) . lung impression smears, lung-homogenate smears, and bal fluid samples can be stained for microscopic detection of pneumocystis (dei-cas et al., ) . useful stains include tbo, gomori-grocott's methenamine silver nitrate (gms), and methanol-giemsa or giemsalike stains (dei-cas et al., ) . other useful detection methods include phase-contrast microscopy and the use of pneumocystis-specific fluorescein-labeled antibodies (dei-cas et al., ) . differential diagnoses p. multocida can induce respiratory disease in rabbits and can be included in the differential diagnoses. treatment, prevention, and control for prevention, new rabbits should be negative for pneumocystis. cotrimoxazole treatment and nested pcr have been used as a screening mechanism to eliminate pneumocystis from colony-maintained rabbits (cere et al., c) . decontamination practices and air filtration were also important for eradication (cere et al., c) . confirmation of a pneumocystis-free status in a rabbit colony was demonstrated by negative pcr results and/ or failure to induce pneumocystosis after experimental corticosteroid challenge (dei-cas et al., ) . research complications research studies may be affected if rabbits of unknown pneumocystis status are experimentally treated with corticosteroids or other immunosuppressant drugs (sheldon, ) . pulmonary lesions may be found in infected rabbits and could potentially confound respiratory research studies (sheldon, ) . etiology unknown. clinical signs trichobezoar is often subclinical. if the trichobezoar causes partial or complete blockage, clinical signs of gastric or intestinal obstruction will result. death can occur due to prolonged anorexia and metabolic imbalances (gillett et al., ). it appears that obstruction of the pylorus, and not the volume of the gastric mass, is the critical factor in determining the clinical progress of the animal (leary et al., ) . epizootiology the condition occurs sporadically in rabbit colonies. pathology the discovery of a hairball in a rabbit is often an incidental finding during necropsy. up to % of rabbits have been found to have gastric trichobezoars during routine necropsy (leary et al., ) . gastric rupture can also result from an obstructive trichobezoar (gillett et al., ) . diagnosis diagnosis is often difficult because the clinical signs are nonspecific and the disease often progresses gradually. some cases involving acute pyloric obstruction result in sudden clinical disease and rapid clinical decline of the animal. manual palpation may indicate the presence of a firm mass in the cranial abdomen. gastric radiographs using contrast media may aid in the diagnosis, but definitive diagnosis is often made during exploratory surgery (gillett et al., ) . differential diagnoses constipation and intestinal foreign body should be considered in the differential list. treatment, prevention, and control treatment of trichobezoar is often unsuccessful. oral administration of mineral oil at ml/day has been reported (suckow and douglas, ) . alternatively, oral administration of - ml of fresh pineapple juice daily has been reported as a possible treatment modality (harkness and wagner, ) . if medical treatment does not resolve the condition, a gastrotomy should be performed. early surgical intervention is important in such cases, as other, subsequent metabolic abnormalities may quickly increase the surgical risk to the rabbit (bergdall and dysko, ) . research complications none have been reported. etiology subluxation or compression fractures of lumbar vertebrae are often secondary to struggling during restraint, particularly when the hindquarters of the rabbit are not supported (bergdall and dysko, ) . the seventh lumbar vertebra (l ) or its caudal articular processes are considered the most frequent sites of fractures, with fracture occurring more commonly than dislocation (flatt et al., ) . clinical signs clinical signs include posterior paresis or paralysis, loss of sensation in the hindlimbs, urinary and/or fecal incontinence, and perineal staining. pathology spinal cord hemorrhage and necrosis can be found. diagnosis diagnosis is based on clinical signs, history of recent restraint, struggling or other trauma, and palpation or radiographic analysis of the vertebral column. differential diagnoses spinal cord trauma. treatment, prevention, and control euthanasia of affected animals is usually warranted. moderate cases (subluxation with spinal edema) may resolve over time. the decision to euthanize should be based on severity of clinical signs. supportive care includes regular expression of the urinary bladder and prevention and treatment of decubital ulcers. corticosteroid and diuretic therapy may be effective for cases of subluxation with spinal edema (bergdall and dysko, ) . research complications loss of valuable research animals is the primary complication. although the condition is often referred to as 'sore hocks,' the correct name is ulcerative pododermatitis. despite the name, the condition rarely affects the hocks, but rather occurs most frequently on the plantar surface of the metatarsal and, to a lesser extent, the metacarpal regions. the condition is believed to be initiated by wire-floor housing, foot stomping, or having thin plantar fur pads. poor sanitation may worsen the condition. solid resting areas on the cage floors are associated with a decreased incidence of ulcerative pododermatitis, whereas a high-energy diet and increased body condition scores are associated with an increased incidence (sanchez et al., ) . the whole genome sequence from a single female rabbit of the partially inbred thorbecke rabbit strain was published in (orycun . ; accession aagw ). the annotated assembly is now available at the national center for biotechnology information (ncbi), the university of california santa cruz (ucsc), and ensembl. the rabbit chosen by the broad institute for sequencing was obtained from covance in . the assembly has . gbp in autosome and x chromosomes and mbp in unplaced scaffolds including mitochondria (gertz et al., ) . the nucleotide sequence of the complete mitochondrial dna (mtdna) molecule of the o. cuniculus has been determined (gissi et al., ) . the compositional differences between the two mtdna strands have also been detailed (gissi et al., ) . the sequencing of the rabbit genome, understanding of rabbit reproduction, and advances in genetic manipulation in the mouse production colonies have led to the ability to produce genetically engineered rabbits. the rabbit offers an alternative model when size or tissue characteristics of a genetically modified mouse are not appropriate. these genetic manipulation techniques were first described by robl (robl and burnside, ) . additional methods have been developed and include pronuclear injection of single cell embryos, injection of genetically modified embryonic stem cells into blastocysts, sperm-mediated gene transfer, and genetically modified somatic cell and nuclear transfer (christensen and peng, ) . commercial companies have been formed to provide genetic modification services with emphasis on production of a unique protein in the milk of rabbits. this section will outline spontaneous hereditary conditions of the rabbit that have been well characterized. some conditions represent conditions that have been identified in humans and other conditions offer insight into the mechanism(s) of particular organ or immune function. hydrocephalus refers to dilatation of the cerebral ventricles and is usually accompanied by accumulation of cerebrospinal fluid within the dilated spaces. some cases of hydrocephalus in rabbits have been presumed to be related to a single autosomal recessive gene (hy/hy); however, occurrence with other abnormalities suggests that inheritance may be more complicated (lindsey and fox, ) . in some cases, the condition appears to be inherited along with various ocular anomalies as an autosomal gene with incomplete dominance. hydrocephalus may also occur in rabbits as a congenital condition related to hypovitaminosis a in pregnant does (lindsey and fox, ) . etiology buphthalmia is inherited as an autosomal recessive trait, although penetrance is presumably incomplete since severity and the age of onset vary greatly and some bu/bu individuals do not develop buphthalmia (hanna et al., ) . clinical signs rabbits with hereditary glaucoma develop ocular changes that resemble human congenital glaucoma and buphthalmia. newborn bu/bu rabbits initially have normal intraocular pressure (iop; - mmhg) but increased pressures of - mmhg may develop after - months of age (burrows et al., ; knepper et al., ) . the eyes become progressively buphthalmic (either uni-or bilaterally) but the iop can return to normal or to sub-normal levels after - months. typical clinical changes include increased corneal diameter as the globe enlarges because the sclera is still immature. the cornea may develop a cloudy or bluish tint, corneal edema, increased corneal vascularity, and flattening of the cornea. structural changes may include widening of the angle, thickening of descmet's membrane, atrophy of the ciliary process, and excavation of the optic disk. impaired aqueous outflow may be due to incomplete cleavage of the drainage angle with abnormal insertion of uveal tissue into the cornea (tesluk et al., ) . in some cases, the cornea ulcerates and ruptures. there is also a marked reduction in semen concentration in buphthalmics, with a decrease in libido and decreased spermatogenesis in affected males (fox et al., ) . epizootiology the condition is common in new zealand white rabbits. pathology by weeks of age, the morphology of the congenital glaucoma trabecular network becomes abnormal with a smaller entrance to the trabecular network at the iris base, smaller intertrabecular openings within and between the trabecular lamellae, and by weeks, iris pillars with extensive lateral extensions in the angle recess can be observed. most intertrabecular spaces remain open; however, the inner intertrabecular spaces adjacent to the aqueous plexus become compressed. diagnosis diagnosis is based on clinical signs and measurement of intraocular pressure. treatment, prevention, and control specific treatment of buphthalmia has not been described for rabbits; however, affected individuals should not be used for breeding purposes. research complications loss of valuable research animals is the primary complication. etiology mandibular prognathism is the most common inherited disease of domestic rabbits. the condition is inherited as an autosomal recessive trait (mp/mp) with incomplete penetrance (fox and crary, ; huang et al., ; lindsey and fox, ) . clinical signs malocclusion related to mandibular prognathia may be clinically apparent as early as - weeks of age, but is more typically seen in older rabbits post weaning. clinical signs may include anorexia and weight loss. if severe enough and left untreated, affected animals will starve since they cannot properly prehend and masticate food. epizootiology normally, the lower incisors occlude with the large upper incisors, as well as with a pair of small secondary incisors that are immediately caudal to the primary maxillary incisors. the lower set of incisors typically wear against the upper set during normal biting activity, along an arc formed by biting movements of the lower incisors, whereas the maxillary secondary incisors wear at right angles to the mandibular incisors. the incisors wear more quickly at the posterior aspect in rabbits, partly because the enamel layer is thinner on that side. affected rabbits have a normal dental formula. the specific abnormality associated with mandibular prognathism is that the maxilla is short relative to a mandible of normal length. thus, although the mandible appears abnormally long, the primary defect involves the maxilla. in rabbits, the teeth (including the molars and premolars) grow continuously throughout life. the incisors, for example, grow at the rate of . - . mm/ week. when occlusion is normal, the teeth wear against one another and in this way remain a normal length. however, when occlusion is abnormal because of conditions that include mandibular prognathia, the teeth may become greatly elongated because typical attrition of the incisors does not occur. in affected animals the lower incisors often extend anterior to the upper incisors and protrude from the mouth, whereas the upper primary incisors grow past the lower incisors and curl within the mouth. in some instances, the upper incisors curl around dorsally and lacerate the mucosa of the hard palate. secondary infection and abscessation may occur in such cases. diagnosis diagnosis is based on clinical signs. differential diagnoses malocclusion secondary to mandibular or maxillary fracture should be considered. treatment, prevention, and control overgrown teeth should be trimmed every - weeks or more frequently if needed. trimming is preferably performed with a dental bur to avoid cracking the tooth, which laboratory animal medicine may happen more frequently if a bone or wire cutter is used. care should be taken to avoid exposing the pulp cavity as the result of excessive trimming. because the condition is hereditary, use of affected animals as breeding stock should be avoided. research complications no specific research complications have been reported. a number of disorders characterized clinically by complete abduction of one or more legs and the inability to assume a normal standing position are described by the term 'splay leg'. young kits of - weeks of age are most commonly affected. affected rabbits cannot adduct limbs and have difficulty in making normal locomotory movements. most commonly, animals are affected in the right rear limb, although the condition may be uni-or bilateral and may affect the anterior, posterior, or all four limbs. rabbits with splay leg may have difficulty in accessing food and water; thus, attention to adequate nutrition is required as part of proper clinical care. the clinical signs of splay leg may be due to an overall imbalance of development of the neural, muscular, and skeletal systems. possibly, some animals compensate with torsion and exorotation of the limb at the hip, whereas rabbits that are unable to compensate are clinically affected. although the precise pathogenesis of splay leg is not entirely understood, at least some cases are ascribed to inherited disorders. typical clinical signs are secondary to femoral endotorsion, with a shallow acetabulum but without luxation of the femur at the hip. the semitendinosus muscle of affected animals is abnormal, with smaller fibers and abnormal mitochondria. some reports suggest that the condition is associated with inherited achondroplasia of the hip and shoulder, whereas others indicate that a recessively inherited anteversion of the femoral head can be involved. self-mutilating behavior in a checkered cross (cross between english spot, german checkered giant, and checkered of rhineland rabbits) was reported to occur as an inherited trait (iglauer et al., ) . autotraumatization of the feet and pads was observed. the abnormal behavior could be interrupted by administration of haloperidol. although not manifested as a disease, the presence of serum atropine esterase allows rabbits to inactivate atropine when administered for therapeutic purposes (liebenberg and linn, ; stormont and suzuki, ) . the enzyme also permits rabbits to consume diets containing belladonna compounds. the enzyme is produced by a semidominant gene est- f. three phenotypes are recognized depending on the number of genes expressed. the enzyme first appears in the serum at month of age, and enzyme levels are greater in females than in males (lindsey and fox, ) . the est- f gene is linked to genes controlling the black pigment in the coat (forster and hannafin, ; fox and van zutphen, ; sawin and crary, ) . hereditary deficiency of the third component of complement (c ) was found in a strain of rabbits. this same strain also exhibited a hereditary c alpha-gamma deficiency. the serum c concentration, hemolytic c activity, and total complement hemolytic activity of these animals were significantly reduced. the low level of serum c in these rabbits was not due to c conversion, partial c antigenicity, and presence of a c inhibitor or hypercatabolism of normal c . the c deficiency was transmitted as a simple autosomal co-dominant trait. rabbits with this trait have a lower survival at months than normal rabbits (komatsu et al., ) . this complement deficiency syndrome in the rabbit has been well characterized. this syndrome was initially reported in in a strain of rabbits that lacked the sixth component of the hemolytic complement system (rother et al., ) . whole blood clotting time in glass or plastic was prolonged and prothrombin consumption was decreased in blood from the deficient animals. other parameters of blood coagulation were normal, including prothrombin time, partial thromboplastin time, specific clotting factor activities, platelet factor iii function, platelet count, and bleeding time (zimmerman et al., ) . abnormal platelet response is also characteristic of this syndrome in the rabbit (lee et al., ) . complement c -deficient rabbits are protected against diet-induced atherosclerosis despite having similar profiles in cholesterol levels and plasma lipoprotein. when compared to normal rabbits, differences in atherosclerotic plaque formation were discernible macroscopically, with extensive aortic lesions being visible in all normal rabbits while absent in all c -deficient animals (schmiedt et al., ) . the inheritance pattern for this defect is autosomal recessive (abe et al., ) . a progressive neurological syndrome has also been observed in the c -deficient rabbits. this syndrome is clinically characterized by subacute motor neuropathy. pathological studies of affected animals revealed ( ) severe axonal degeneration in the sciatic nerve involving mainly motor fibers; ( ) occasional peripheral axonal enlargement closely associated with axonal laboratory animal medicine degeneration; ( ) presence of structured abnormal material in normal-size myelinated fibers of the central and peripheral nervous systems; and ( ) widespread occurrence of dystrophic axons and axonal spheroids in the gray matter of the central nervous system. by ultrastructural examination, dystrophic axons were filled with tubulovesicular material, appearing as stalks of parallel membranes and dense bodies similar to what is described in human neuroaxonal dystrophies (nad). the disease manifested by c -deficient rabbits may represent an animal model of primary human nad (giannini et al., ) . genetic deficiency of the alpha-gamma-subunit of the eighth complement component (c alpha-gamma) was found in a substrain of the new zealand white rabbits. the serum of this deficient rabbit lacked the immunochemical and functional alpha-gamma-subunit of c (c d). this syndrome is transmitted as a simple autosomal recessive trait. the syndrome is characterized by smaller body weight compared to those of heterozygous and normal rabbits. in addition, survival rates for the first months of life of the deficient animals tended to be lower than those of heterozygous and normal littermates (komatsu et al., ) . all c d rabbits (more than animals obtained thus far) were consistently smaller than normal littermates from birth to adulthood, i.e., % of normal size at birth, % of normal size at days of age, and % of normal size at adulthood. the c α-γ deficiency in rabbits is always associated with dwarfism. furthermore, there appears to be a discrete recessive dwarf gene (dw- ), whose locus is not linked to c d. rabbits double-homozygous for c d and dw- (severe dwarf) were smaller than the c d or dwarf rabbits and almost all of the severe dwarf rabbits died within days after birth. the actual and relative weights of the thymus in the c d rabbits were consistently lower than those of normal rabbits, but histological examination of the c d thymus did not reveal any abnormalities. the c d and dwarf rabbits were fertile; however, crosses of c d females with c d or dwarf males led to a reduced delivery rate and small litter size. the c d locus is loosely linked to the c hypocomplementemic locus (c -hypo) (map distance cm) but not to the hemoglobin blood group locus (komatsu et al., ) . the inherited characteristics of the kurosawa and kusanagi hypercholesterolemic (khc) rabbit include persistent hypercholesterolemia. this strain of rabbits was produced by inbreeding mutants discovered in . these khc rabbits had serum cholesterol, triglyceride, and phospholipid levels - times greater than clinically normal o. cuniculus. the khc rabbits also had decreased serum high-density lipoprotein cholesterol concentration, about one-third the value in clinically normal rabbits. in addition, the serum lipoprotein electrophoretic patterns were characterized by a strong, broad beta-lipoprotein band and a diminished alphalipoprotein band. fractionation of lipoprotein lipids revealed increased cholesterol, phospholipid, and triglyceride in the ldl fraction; increased cholesterol and phospholipid in the very ldl fraction; and decreased cholesterol and triglyceride in the high-density lipoprotein fraction. the inheritance is thought to be a single autosomal recessive gene mutation, and analysis of the ldl receptor indicated that the khc rabbit has a -base pair deletion in the ldl receptor mrna. macroscopic analysis of the aorta revealed the atheromatous lesions at months of age, drastically increased lesional areas in the total aortic surface at months of age, and a high incidence of coronary atheromas and xanthomas (kurosawa et al., ) . a spontaneous phenotype in a rabbit was discovered with an elevation of serous lipid ingredients including cholesterol and beta-lipoprotein (beta-lp). atherosclerotic lesions were evident in the aorta and renal arteries. nodular xanthomas were also present on the front and rear feet. the hlr strain was inbred to accentuate these characteristics (watanabe et al., ) . the strain was eventually designated the watanabe-heritable hyperlipidemic rabbit (whhl-rabbit). an additional report of this strain of rabbits indicated that the whhl-rabbits spontaneous developed aortic atherosclerosis by months of age and xanthoma of digital joints in % of the rabbits aged to months (watanabe, ) . historically, spontaneous neoplasia in the laboratory rabbit has not been widely reported because neoplasia in the rabbit is very uncommon before years of age and many laboratory rabbits are not maintained beyond this age (weisbroth, ) . endometrial adenocarcinoma is the most common tumor in aged female rabbits, with an incidence of % reported in a colony of -year-old rabbits (baba and von haam, ) . tinkey et al. complied an extensive review of the literature dealing with spontaneous neoplasia in the domestic rabbit. this review contained data on case reports, descriptions of biologic aspects of naturally occurring tumors and reports of experimentally induced tumor models. neoplasia in sylvilagus and lepus were also discussed (tinkey et al., ) . uterine adenocarcinoma is by far the most common tumor in rabbits. typically, the disease is present as multiple tumors and is malignant, often metastasizing to the liver, lungs, and other organs. there is evidence that inheritance plays a role in susceptibility, but parity does not. uterine leiomyomas and leiomyosarcomas are much less common (weisbroth, ) . there are a few reports of vaginal squamous cell carcinomas (weisbroth, ) and an ovarian hemangioma has been described (greene and strauss, ) . mammary adenocarcinomas are fairly common in older female rabbits and may occur in animals with uterine adenocarcinoma (weisbroth, ) . papillomas have been described, but mammary adenocarcinomas are much more important. these malignant tumors may metastasize, but the cause of death in affected rabbits is often due to uterine adenocarcinoma. serial biopsy studies indicate that these tumors are preceded by cystic mastopathy as well as changes in the adrenal and pituitary glands (greene, ) . there may also be small prolactin-secreting pituitary adenomas in rabbits with mammary dysplasia (lipman et al., ) . testicular tumors in the rabbit appear to be relatively uncommon. interstitial tumors are the most common testicular tumor in the rabbit. seminomas and teratomas have also been reported (weisbroth, ) . embryonal nephromas are one of the most common tumors in laboratory rabbits. these tumors are often found incidentally, occur in younger animals, and seldom cause clinical signs (weisbroth, ) . there has been one report of a renal carcinoma in the rabbit (kaufman and quist, ) and one report of a leiomyoma arising in the urinary bladder (weisbroth, ) . malignant lymphomas (lymphosarcomas) are relatively common in rabbits. they may occur in rabbits that are less than years of age (weisbroth, ) , but older rabbits may also be affected. according to (weisbroth, ) , a tetrad of lesions is often seen. these lesions include enlarged kidneys, splenomegaly, hepatomegaly, and lymphadenopathy. older rabbits have presented with skin nodules and eye lesions; however, malignant lymphomas in the rabbit are seldom leukemic. most cases of malignant lymphoma appear to resemble the lymphoblastic subtype as seen in humans and mice. malignant lymphoma is more prevalent in some strains of rabbits than in others, and there is some evidence for a retroviral cause of lymphomas in rabbits (weisbroth, ) . true thymomas (containing both lymphoid and epithelial components) (vernau et al., ) and plasma cell myelomas (pascal, ) are rare in rabbits. one case of myeloid leukemia has been reported (meier et al., ) . basal cell tumors are reported to be rare (weisbroth, ) , but they may be underreported (li and schlafer, ) . squamous cell carcinomas are also uncommon, and there is no apparent predilection for any particular area of the body (weisbroth, ) . other cited skin-associated tumors include a trichoepithelioma (altman et al., ) , a sebaceous gland carcinoma (port and sidor, ) , and two malignant melanomas (hotchkiss et al., ) . osteosarcomas are extremely rare in rabbits, and most have arisen in the mandible or maxilla, with only one found in a long bone (weisbroth, ) . no primary tumors arising in cartilage have been described, although some of the reported osteosarcomas have had cartilaginous elements. one tumor of skeletal muscle, a rhabdomyosarcoma, has been reported. a few fibrosarcomas and one fibrosarcoma involving the foot have been reported (weisbroth, ) . a number of case reports of single tumors are found in the literature. these include a peritoneal mesothelioma (lichtensteiger and leathers, ) , an intracranial teratoma (bishop, ) , an ependymoma (kinkier and jepsen, ) , a neurofibrosarcoma, two hemangiosarcomas (pletcher and murphy, ) , and a malignant fibrous histiocytoma (yamamoto and fujishiro, ). there are a few very old reports of lung tumors dating to the first part of the th century (weisbroth, ) . there are several tumor models in which the cells used for inoculation were originally derived from rabbit tumors. these include the vx- carcinoma (kidd and rous, ) , the brown pearce carcinoma (brown and pearce, ) , and the greene melanoma (greene, ) . the vx- carcinoma originated from a squamous cell carcinoma in a rabbit carrying a shope papilloma. the most common modern use of this transplantable tumor is as a model for the study of various cancer treatment modalities for metastatic tumors (stetson et al., ) . the brown pearce carcinoma arose from a tumor in a rabbit testis, but the exact tissue of origin of the tumor was never determined. the tumor was readily transplantable and caused stable metastases. because some tumors regress, even after widespread metastases, this tumor has been used as a model for the study of tumor immunology (weisbroth, ) . the brown pearce laboratory animal medicine carcinoma, although extensively characterized and historically used, has been reported in the literature only five times from to (tinkey et al., ) . hydrometra has been described as a clinical condition of rabbits. all cases were in unmated rabbits that were used experimentally for the production of serum antibodies (bray et al., ; hobbs and parker, ; morrell, ) . clinical signs included abdominal distension and tachypnea. cases were characterized by distension of the uterine horns with a transudative fluid. one case was associated with uterine torsion (hobbs and parker, ) . one case had resolved with diuretic therapy, only to return later (bray et al., ) . most cases of liver lobe torsion in rabbits involve the caudate lobe (bergdall and dysko, ) , although one case report described torsion of the left hepatic lobe (wilson et al., ) . most reported cases have been incidental findings at necropsy. incidental hepatic lobe torsions have also been identified in three adult new zealand white rabbits that died from pasteurellosis (weisbroth, ) . three cases of hepatic torsion in pet rabbits were reported by wenger in (wenger et al., ) . all rabbits presented with an acute onset of lethargy, anorexia, abdominal pain, pale mucous membranes, and jaundice. one rabbit also had hematuria. another report of caudate liver lobe torsion also described a rabbit that was jaundiced, anemic, and anorexic, with elevated alanine aminotransferase. torsion of the caudate liver lobe was seen at necropsy (fitzgerald and fitzgerald, ) . in all reported clinical cases, rabbits were euthanized, or died during postoperative recovery. calcium carbonate and triple phosphate crystals are present in the urine of normal rabbits. these crystals contribute to the cloudy consistency of the urine (williams, ) . a -year retrospective study of hematuria in new zealand white rabbits was conducted by garibaldi (garibaldi et al., ) . physical examination, laboratory tests, radiography, and postmortem examination were utilized in most cases to verify the presence of hematuria and to determine its etiology. uterine adenocarcinoma was diagnosed in two rabbits. three rabbits had uterine polyps with hemorrhage. renal infarction with hemorrhage was diagnosed in three rabbits. urolithiasis with secondary urethral obstruction and hemorrhagic cystitis was identified as the cause of hematuria in four rabbits. other causes of hematuria included chronic cystitis, disseminated intravascular coagulation, bladder polyps and pyelonephritis. hematuria of undetermined origin was observed in one rabbit which emphasizes that hyperpigmented urine should be a rule out in all cases of suspected hematuria in rabbits (garibaldi et al., ) . one case of urolithiasis with hydronephrosis in a new zealand white rabbit was also reported (labranche and renegar, ) . this condition must be distinguished from hematuria caused by endometrial venous aneurysm in female rabbits (bray et al., ) . herniation of the kidney along with perinephric fat has been reported (suckow and grigdesby, ) . the affected rabbit was clinically normal except for a subcutaneous mass that had passed through the body wall. the precise etiology is not known, although it was speculated that herniation might have occurred as the result of unreported trauma. occlusion of the nasolacrimal duct, presumably due to accumulation of fat droplets, has been described as a putative cause of epiphora in some rabbits (marini et al., ) . although the obstruction occurred at the dorsal flexure, it is not clear if this was due to congenital rather than acquired stenosis. in a retrospective study of rabbits it was determined that the mean age of the rabbits presenting with ocular discharge from the nasolacrimal duct was . years. in rabbits ( %), dacryocystitis was a unilateral finding. no underlying cause could be determined in animals ( %). dental malocclusion was observed in rabbits ( %) and rhinitis in two animals ( %), with one animal showing both signs ( %). one rabbit ( %) presented with panophthalmitis. most animals ( %) received topical antibiotic treatment. regarding the clinical outcome, animals ( %) showed complete recovery, eight rabbits ( %) were euthanized, three ( %) died due to unrelated causes, and three ( %) were lost to follow-up. two rabbits ( %) continued to display signs of dacryocystitis (florin et al., ) . development and genetic differences of complement activity in rabbits influence of pasteurella multocida infection on the pharmacokinetic behavior of marbofloxacin after intravenous and intramuscular administrations in rabbits rabbit haemorrhagic disease (rhd) and rabbit haemorrhagic disease virus (rhdv): a review candidate vaccine antigens and genes in pasteurella multocida a survey of clostridium spiroforme antimicrobial susceptibility in rabbit breeding vaccination of rabbits against coccidiosis using precocious lines of eimeria magna and eimeria media in benin molecular characterization of pasteurella multocida isolates from rabbits detection of cytokine mrna in the lung during the spontaneous pneumocystis carinii pneumonia of the young rabbit rabbit pasteurellosis: respiratory and renal pathology of control and immunized rabbits after challenge with pasteurella multocida tyzzer's disease syndrome in laboratory rabbits trichoepithelioma in a rabbit transgenic rabbits expressing human lipoprotein lipase effects of sarcoptes scabei var. canis (acari: sarcoptidae) on blood indexes of parasitized rabbits host-seeking behavior of sarcoptes scabei histopathology in hosts parasitized by sarcoptes scabei watanabe heritable hyperlipidemic rabbits. familial hypercholesterolemia animal model: spontaneous adenocarcinoma in aged rabbits wild rabbits -a novel vector for vero cytotoxigenic escherichia coli (vtec) o clostridium spiroforme infection in rabbits dermatophytes in clinically healthy laboratory animals in utero transmission of encephalitozoon cuniculi strain type i in rabbits environmental enrichment for laboratory rodents and rabbits: requirements of rodents, rabbits, and research natural paratuberculosis infection in rabbits in scotland rapid detection of clostridium difficile in feces by real-time pcr metabolic, traumatic, mycotic, and miscellaneous diseases of rabbits fresh fruit and vegetables as vehicles for the transmission of human pathogens vitamin d toxicosis in commercially reared rabbits nicorandil normalizes prolonged repolarisation in the first transgenic rabbit model with long-qt syndrome both in vitro and in vivo intracranial teratoma in a domestic rabbit o serogroups, biotypes, and eae genes in escherichia coli strains isolated from diarrheic and healthy rabbits functional and morphological organization of the rabbit sinus node association of iota-like toxin and clostridium spiroforme with both spontaneous and antibioticassociated diarrhea and colitis in rabbits pathogenomics of pasteurella multocida viral diseases hydrometra in a new zealand white rabbit endometrial venous aneurysms in three new zealand white rabbits efficacy of enrofloxacin in the treatment of respiratory pasteurellosis in rabbits studies based on a malignant tumor of the rabbit. i. the spontaneous tumor and associated abnormalities glucocorticoid metabolites in rabbit faeces -influence of environmental enrichment and cage size compendium of normal blood values of laboratory animals with indication of variations development of ocular hypertension in congenitally buphthalmic rabbits epidemiology and risk factors for dermatophytoses in rabbit farms enzymatic activity of microsporum canis and trichophyton mentagrophytes from breeding rabbits with and without skin lesions molecular identification and phylogenesis of dermatophytes isolated from rabbit farms and rabbit farm workers perinatal development of glucogenic enzymes in rabbit liver evaluation of some coagulation parameters in hepatic coccidiosis experimentally induced with eimeria stiedai in rabbits eimeria stiedae: experimental infection in rabbits and the effect of treatment with toltrazuril and ivermectin biotypes and o serogroups of escherichia coli involved in intestinal infections of weaned rabbits: clues to diagnosis of pathogenic strains diarrhea due to escherichia coli in the rabbit: a novel mechanism the digestive system of the rabbit infectious nature of clostridium spiroforme-mediated rabbit enterotoxaemia experimental and spontaneous clostridial enteropathies of laboratory and free living lagomorphs in vitro susceptibility of rabbit strains of clostridium spiroforme to antimicrobial agents structure of rrn operons in pathogenic non-cultivable treponemes: sequence but not genomic position of intergenic spacers correlates with classification of treponema pallidum and treponema paraluiscuniculi strains animal pneumocystosis: a model for man in utero transmission of pneumocystis carinii sp natural transmission of pneumocystis carinii in nonimmunosuppressed animals: early contagiousness of experimentally infected rabbits (oryctolagus cuniculus) obtaining a pneumocystisfree rabbit breeding stock (oryctolagus cuniculus) cryptosporidium sp. rabbit genotype, a newly identified human pathogen sporadic human cryptosporidiosis caused by cryptosporidium cuniculus rabbit feeding and nutrition nutrition and nutritional diseases dermatophytoses in animals expression of recombinant human factor viii in milk of several generations of transgenic rabbits quality of transgenic rabbit embryos with different egfp gene constructs induction of pneumonia in rabbits by use of a purified protein toxin from pasteurella multocida cottontail rabbit papillomavirus (crpv) model system to test antiviral and immunotherapeutic strategies rabbit genetics and transgenic models a behavioral comparison of new zealand white rabbits (oryctolagus cuniculus) housed individually or in pairs in conventional laboratory cages relationship between food and water ingestion in the rabbit infection strategies of enteric pathogenic escherichia coli animal and human pathogenic escherichia coli strains share common genetic backgrounds facial alopecia in the rabbit associated with cheyletiella parasitovorax cheyletiella dermatitis: a mite infestation of rabbit, cat, dog, and man intranasal vaccination of rabbits with pasteurella multocida a: outer membranes that express ironregulated proteins natural pathogens of laboratory animals and their effects on research comparison of the s ribosomal dna sequences from the intracellular agents of proliferative enteritis in a hamster, deer, and ostrich with the sequence of a porcine isolate of lawsonia intracellularis diagnosis of proliferative enteritis in frozen and formalin-fixed, paraffin-embedded tissues from a hamster, horse, deer and ostrich using a lawsonia intracellularis-specific multiplex pcr assay fluorescence-activated flow cytometry in the hematology clinical laboratory disorders of carbohydrate metabolism in infancy altered immune responsiveness associated with encephalitozoon cuniculi infection in rabbits bacillus piliformis' (tyzzer) and tyzzer's disease of the laboratory mouse. i. propagation of the organism in embryonated eggs pneumocystis carinii pneumonia in four mammal species: histopathology and ultrastructure ultrastructure, immunoflourescence, western blot, and pcr analysis of eight isolates of encephalitozoon (septata) intestinalis established in culture from sputum and urine samples and duodenal aspirates of five patients with aids venereal spirochetosis of rabbits: description and diagnosis venereal spirochetosis of rabbits: epizootiology venereal spirochetosis of rabbits: eradication use of ivermectin for treatment of ear mite infestation in rabbits an epizootic of tyzzer's disease in rabbits experimental reproduction of neonatal diarrhea in young gnotobiotic hares simultaneously associated with clostridium difficile and other clostridium strains single primer polymerase chain reaction fingerprinting for pasteurella multocida isolates from laboratory rabbits typing of clostridium perfringens by in vitro amplification of toxin genes escherichia coli o : h in environments of culture-positive cattle herpesvirus systematics respiratory diseases of rabbits pasteurella multocida and bordetella bronchiseptica infections in rabbits pneumocystis oryctolagi sp. nov., an uncultured fungus causing pneumonia in rabbits at weaning: review of current knowledge, and description of a new taxon on genotypic, phylogenetic and phenotypic bases ingestion of rabbit erythrocytes containing cr-labeled hemoglobin by psoroptes spp. that originated on cattle, mountain sheep, or rabbits colonization of rabbits by pasteurella multocida: serum igg responses following intranasal challenge with serologically distinct isolates identification and characterization of three encephalitozoon cuniculi strains treponema paraluis-cuniculi infection in a commercial rabbitry: epidemiology and serodiagnosis atrophic rhinitis in new zealand white rabbits infected with pasteurella multocida naturally acquired pasteurella multocida infection in rabbits: immunological aspects naturally acquired pasteurella multocida infection in rabbits: clinicopathological aspects hypervitaminosis a and reproductive disorders in rabbits ferrets, rabbits, and rodents production of recombinant human protein c in the milk of transgenic rabbits from the f generation development of pcr protocols for specific identification of clostridium spiroforme and detection of sas and sbs genes vaccination against eimeria magna coccidiosis using spray dispersion of precocious line oocysts in the nest box a quantitative polymerase chain reaction assay for detection and quantification of lawsonia intracellularis subclinical proliferative enteropathy in sentinel rabbits associated with lawsonia intracellularis assignment of the agent of tyzzer's disease to clostridium piliforme comb. nov. on the basis of s rrna sequence analysis control of oxyuriasis in rabbits by fenbendazole preliminary observations on enteritis associated with a coronavirus-like agent in rabbits evaluation of pasteurella multocida isolated from rabbits by capsular typing, somatic serotyping, and restriction endonuclease analysis helminth infection in relation to population fluctuations in snowshoe hare silver staining of spirochaetes in single tissue sections effectiveness of a selamectin spot-on formulation in rabbits with sarcoptic mange the effect of the fiber and protein level of the ration upon the cecotrophy of rabbits ribosomal rna sequences of clostridium piliforme isolated from rodent and rabbit: re-examining the phylogeny of the tyzzer's disease agent and development of a diagnostic polymerase chain reaction assay teneur en acides amines des deux catégories de fèces du lapin: caecotrophes et fèces durés virulence genes and antimicrobial resistance profiles of pasteurella multocida strains isolated from rabbits in brazil hepatic lobe torsion in a new zealand white rabbit identification of crystalline material in urine of rabbits renal nosematosis in young rabbits metabolic, traumatic, mycotic, and miscellaneous disease of rabbits clinical presentation, treatment, and outcome of dacryocystitis in rabbits: a retrospective study of cases use of gonadotropic releasing hormone for ovulating the rabbit model influence of a genetically determined atropinesterase on atropine inhibition of the "smoke (dive) reflex" in rabbits intracellular campylobacter-like organism from ferrets and hamsters with proliferative bowel disease is a desulfovibrio sp the clinical chemistry of laboratory animals taxonomy and genetics mandibular prognathism in the rabbit. genetic studies strain differences in the prealbumin serum esterases of jax rabbits buphthalmia in the rabbit. pleiotropic effects of the (bu) gene and a possible explanation of mode of gene action what is your diagnosis? respiratory abscess polymerase chain reaction for diagnosis and species differentiation of microsporidia systemic inflammatory response indicators in rabbits (oryctolagus cuniculus) experimentally infected with sporulated oocysts of eimeria stiedai (apicomplexa: eimeriidae) pathology of protozoal and helminthic diseases toxigenic pasteurella multocida in rabbits with naturally occurring atrophic rhinitis examination of the applicability of a commercial human rotavirus antigen detection kit for use in laboratory rabbits cyclical variations in the excretion of intestinal coccidial oocysts in the rabbit effect of heat and selected chemical disinfectants upon infectivity of spores of bacillus piliformis (tyzzer's disease) tyzzer's disease of rabbits: isolation and propagation of bacillus piliformis (tyzzer) in embryonated eggs development and application of taqman mgb probe fluorescence quantitative pcr method for rapid detection of clostridium piliforme the rabbit as a new reservoir host of enterohemorrhagic escherichia coli a naturally occurring rabbit model of enterohemorrhagic escherichia coli-induced disease renal injury is a consistent finding in dutch belted rabbits experimentally infected with enterohemorrhagic escherichia coli zoonotic enterohemorrhagic escherichia coli: a one health perspective hematuria in rabbits organ systems in adaptation: the temperature regulating system accuracy and coverage assessment of oryctolagus cuniculus (rabbit) genes encoding immunoglobulins in the whole genome sequence assembly (orycun . ) and localization of the igh locus to chromosome tpr homologs in treponema paraluiscuniculi cuniculi a strain inherited neuroaxonal dystrophy in c deficient rabbits fibres in rabbit feeding for digestive troubles prevention: respective role of low-digested and digestible fibre feeding behaviour of rabbits selected drug dosages and clinical reference data medical and surgical management of gastric obstruction from a hairball in the rabbit the complete mitochondrial dna sequence of the rabbit infection with and antibody response to pasteurella multocida and bordetella bronchiseptica in immature rabbits the pathology of experimental respiratory infection with pasteurella multocida and bordetella bronchiseptica in rabbits quantitation of rabbit cytokine mrna by real-time rt-pcr detection of toxigenic clostridium difficile in diarrheal stools by rapid real-time polymerase chain reaction the effect of host's age on the pathology of eimeria stiedae infection in rabbits a spontaneous melanoma in the hamster with a propensity for amelanotic alteration and sarcomatous transformation during transplantation diseases of the rabbit multiple primary tumors in the rabbit paratuberculosis in wild rabbits (oryctolagus cuniculus) inventory of the behaviour of new zealand white rabbits in laboratory cages identification of genes transcribed by pasteurella multocida in rabbit livers through the selective capture of transcribed sequences detection and characterization of cryptosporidium cuniculus by real-time pcr . rabbits. in: reproduction and breeding techniques for laboratory animals. lea & febiger the effects of continuous sulfaquinoxaline feeding on rabbit mortality spontaneous papillomatosis in domestic rabbits antibacterial efficacy and pharmacokinetic studies of ciprofloxacin on pasteurella multocida infected rabbits recessive buphthalmos in the rabbit the effect of environmental enrichment on the behaviour of caged rabbits microbial induction of b and t cell areas in rabbit appendix the biology and medicine of rabbits and rodents pathogenesis of herpesvirus sylvilagus infection in cottontail rabbits normal biochemical and hematological values in new zealand white rabbits early volume expansion during diarrhea and relative nephroprotection during subsequent hemolytic uremic syndrome transgenic rabbit production with simian immunodeficiency virus-derived lentiviral vector syphilis: using modern approaches to understand an old disease uterine torsion associated with either hydrometra or endometritis in two rabbits atrioventricular conduction in mammalian hearts arthropod and helminth parasites isolation of clostridium spiroforme from rabbits proliferative enteropathy involving lawsonia intracellularis infection in rabbits (oryctlagus cuniculus) coecotrophy in rabbits-a circadian function the route of migration of eimeria stiedae (lindemann, ) sporozoites between the duodenum and bile duct of the rabbit the treatment of hepatic coccidiosis in rabbits malignant melanomas in two rabbits proliferative enteropathy of rabbits: the intracellular campylobacter-like organism is closely related to lawsonia intracellularis real-time multiplex polymerase chain reaction assay for rapid detection of clostridium difficile toxin-encoding strains mandibular prognathism in the rabbit: discrimination between single-locus and multifactorial models of inheritance natural mode of acquisition for de novo infection with pneumocystis carinii response of adult new zealand white rabbits to enrichment objects and paired housing hereditary compulsive self-mutilating behaviour in laboratory rabbits spontaneous cryptosporidiosis in an adult female rabbit liver coccidiosis prevented by sulfasuxidine pasteurella associated rhinitis of rabbits: efficacy of penicillin therapy atherosclerotic research a new model of sheep ig diversification: shifting the emphasis toward combinatorial mechanisms and away from hypermutation the rabbit: a diurnal or nocturnal animal? characterization of a novel alphaherpesvirus associated with fatal infections of domestic rabbits an outbreak of fatal herpesvirus infection in domestic rabbits in alaska proteomic analyses of transgenic lqt and lqt rabbit hearts elucidate an increase in expression and activity of energy producing enzymes ovarian abscesses and pyometra in a domestic rabbit an epizootic of shope fibromatosis in a commercial rabbitry enhanced detection of intracellular organism of swine proliferative enteritis, ileal symbiont intracellularis, in feces by polymerase chain reaction eimeria stiedae in rabbits: the demonstration of responses to chemotherapy pure culture of the pathogenic agent of tyzzer's disease of mice molecular approaches in the diagnosis of dermatophytosis pathogenic escherichia coli beat-to-beat regulation of heart rate by afferent stimulation of the aortic nerve implantable stimulating electrode for baroreceptor afferent nerves in rabbits spontaneous renal carcinoma in a new zealand white rabbit an outbreak of massive mortality among farm rabbits associated with cryptosporidium infection growth of tyzzer's organism in primary monolayer cultures of adult mouse hepatocytes the comparative pathology of clostridium difficile-associated disease superovulatory response of pre-and post-pubertal rabbits to commercially available gonadotropins viral infections of rabbits a transplantable rabbit carcinoma originating in a virus-induced papilloma and containing the virus in masked or altered form cloning and sequence analysis of another shiga-like toxin iie variant gene (slt-iiera) from an escherichia coli r strain isolated from rabbit ependymoma in a rabbit deficiency of low density lipoprotein receptors in liver and adrenal gland of the whhl rabbit, an animal model of familial hypercholesterolemia peripheral thermal sensitivity in the rabbit ultrastructural studies of primary congenital glaucoma in rabbits spontaneous nosema cuniculi infection in laboratory rabbits genetic deficiency of the alpha-gamma-subunit of the eighth complement component in the rabbit hereditary c hypocomplementemia in the rabbit hereditary c α-γ deficiency associated with dwarfism in the rabbit environmental factors, clinical signs, therapy and zoonotic risk of rabbits with dermatophytosis biology and diseases of rabbits localization of human coagulation factor viii (hfviii) in transgenic rabbit by fish-tsa: identification of transgene copy number and transmission to the next generation encephalitozoonosis in rabbits new mutant rabbit strain with hypercholesterolemia and atherosclerotic lesions produced by serial inbreeding use of selamectin for the treatment of psoroptic and sarcoptic mite infestation in rabbits urinary calculus and hydronephrosis in a new zealand white rabbit preliminary report on the observation of a coronavirus in the intestine of the laboratory rabbit hereditary hyperlipidemia in the rabbit due to overproduction of lipoproteins, i. biochemical studies proliferative enteropathy experimental and naturally-occuring gastric foreign bodies in laboratory rabbits vitamins in rabbit nutrition: literature review and recommendations farmed rabbits and ducks as vectors for vtec o :h cheyletiella dermatitis: a report of fourteen cases abnormal platelet response to thromboplastin infusion in rabbits deficient in the sixth component of complement value of histopathology, immunohistochemistry, and real-time polymerase chain reaction in the confirmatory diagnosis of encephalitozoon cuniculi infection in rabbits fecal occult blood manifestation of intestinal eimeria spp. infection in rabbit production of transgenic rabbit embryos through intracytoplasmic sperm injection a spontaneous skin basal cell tumor in a black french minilop rabbit peritoneal mesothelioma in the rabbit tyzzer's disease catalytic enzyme activity concentration in plasma of man, sheep, dog, cat, rabbit, guinea pig, rat, and mouse seasonal and sexual influence on rabbit atropinesterase prevalence of lawsonia intracellularis, salmonella spp. and eimeria spp. in healthy and diarrheic pet rabbits inherited diseases and variations expression of human growth hormone in the milk of transgenic rabbits with transgene mapped to the telomere region of chromosome q utilization of cholestyramine resin as a preventative treatment for antibiotic (clindamycin) induced enterotoxemia in the rabbit prolactin-secreting pituitary adenomas with mammary dysplasia in new zealand white rabbits differential conditions for early after-depolarizations and triggered activity in cardiomyocytes derived from transgenic lqt and lqt rabbits complete genome sequence of pasteurella multocida hn , a toxigenic strain of serogroup d resolution of established cardiac hypertrophy and fibrosis and prevention of systolic dysfunction in a transgenic rabbit model of human cardiomyopathy through thiol-sensitive mechanisms dermatophytes isolated from laboratory animals group housing: meeting the physical and social needs of the laboratory rabbit an enzymelinked immunosorbent assay to detect serum igg to pasteurella multocida in naturally and experimentally infected rabbits comparison of sequenced escherichia coli genomes is there a difference between hare syphilis and rabbit syphilis? cross infection experiments between rabbits and hares immunoprophylaxis of dermatophytosis in animals the effect of sulfaquinoxaline on the course of eimeria stiedae infection in the domestic rabbit estimating relative pollution of the environment with oocysts of eimeria stiedae immunology of lagomorphs b cell and antibody repertoire development in rabbits: the requirement of gut-associated lymphoid tissues rhythmic contractile activity of the in vivo rabbit aorta serology of pasteurella multocida in laboratory rabbits: a review microbiologic, radiographic, and anatomic study of the nasolacrimal duct apparatus in the rabbit (oryctolagus cuniculus) worsening of diet-induced atherosclerosis in a new model of transgenic rabbit expressing the human plasma phospholipid transfer protein two encephalitozoon cuniculi strains of human origin are infectious to rabbits lsaa, an antigen involved in cell attachment and invasion, is expressed by lawsonia intracellularis during infection in vitro and in vivo vitamins in animal nutrition clinical and pharmacological properties of ivermectin in rabbits and guinea pigs myeloid leukemia in the rabbit treatment of rabbit chyletiellosis with selamectin or ivermectin: a retrospective case study pathogenesis of shiga-toxin producing escherichia coli pathogenesis of dermatophytosis and tinea versicolor rabbit hemorrhagic disease: a review with special reference to its epizootiology clinical biochemical and hematological reference values in normal experimental animals and normal humans introduction: one health perspective intraepithelial vibrio associated with acute typhlitis of young rabbits infection of different strains of mice with lawsonia intracellularis derived from rabbit or porcine proliferative enteropathy ectoparasites of the wild rabbit, oryctolagus cuniculus (l.) in australia rabbitpox: a model of airborne transmission of smallpox colobomatous microphthalmos in a new zealand white rabbit, arising from a colony with suspected vitamin e deficiency development of a high-sensitivity nested pcr assay for the detection of clostridium piliforme in clinical samples acquired resistance to reinfection of rabbits with eimeria magna the rabbit differs from other mammalian species in the tissue distribution of alkaline phosphatase isoenzymes immunological responses to vaccination following experimental lawsonia intracellularis virulent challenge in pigs clinical efficacy of botanical extracts from eupatorium adenophorum against the sarcoptes scabiei (sarcoptidae: sarcoptes) in rabbits walker's mammals of the world antibiograms of pathogenic bacteria isolated from laboratory rabbits in ibadan eimeria species of the domestic rabbit (oryctolagus cuniculus) pasteurella multocida toxin activation of heterotrimeric g proteins by deamidation life cycle of eimeria stiedae the reproduction of eimeria flavescens and eimeria intestinalis in suckling rabbits the rabbit coccidium eimeria piriformis: selection of a precocious line and life-cycle study protozoal diseases protozoal diseases rapid detection of clostridium difficile toxins from stool samples using real-time multiplex pcr efficacy of an injectable formulation of eprinomectin against psoroptes cuniculi, the ear mange mite in rabbits identification of the cellular receptor of clostridium spiroforme toxin acaricidal activity of eugenol based compounds against scabies mites plasma cell myeloma in the brain of a rabbit an outbreak of encephalomyelitis in broiler rabbits caused by nosema cuniculi diagnostic performance of different fecal lawsonia intracellularis-specific polymerase chain reaction assays as diagnostic tests for proliferative enteropathy in pigs: a review significance of clostridium spiroforme in the enteritis-complex of commercial rabbits biotype, serotype, and pathogenicity of attaching and effacing enteropathogenic escherichia coli strains isolated from diarrheic commercial rabbits three ectoparasites of veterinary interest communicable to man coccidia and coccidiosis parenteral infection experiments with eimeria stiedae transgenic rabbit models for studying human cardiovascular diseases meningitis and subgaleal, subdural, epidural empyema due to pasteurella multocida detection of clostridium difficile toxins from the small intestine and cecum of rabbits with naturally acquired enterotoxemia molecular bases of genetic diversity and evolution of the immunoglobulin heavy chain variable region (ighv) gene locus in leporids rearing germfree cesarean-born rats, mice, and rabbits through weaning spontaneous malignant hemangiosarcomas in two rabbits the behaviour of group penned and individually caged laboratory rabbits genetic characterization of antibiotic resistance in enteropathogenic escherichia coli carrying extendedspectrum beta-lactamases recovered from diarrhoeic rabbits environmental enrichment of new zealand white rabbits living in laboratory cages identification of eae sequences in enteropathogenic escherichia coli strains from rabbits a sebaceous gland carcinoma in a rabbit tyzzer's disease in rabbits in britain pneumocystosis in humans or in corticosteroid-untreated animal models: interactions between pulmonary surfactant changes and pneumocystis carinii in vivo or in vitro growth wild rabbits-a novel vector for verocytotoxigenic escherichia coli o evaluation of available diagnostic methods for clostridium piliforme in laboratory rabbits (oryctolagus cuniculus) further investigation of exposure to lawsonia intracellularis in wild and feral animals captured on horse properties with equine proliferative enteropathy efficacy of an avirulent live vaccine against lawsonia intracellularis in the prevention of proliferative enteropathy in experimentally infected weanling foals transmission of lawsonia intracellularis to weanling foals using feces from experimentally infected rabbits outbreak of sarcoptic mange and malasseziasis in rabbits (oryctolagus cuniculus) characterization of a fibroma virus isolated from naturally-occurring skin tumors in domestic rabbits the pangenome structure of escherichia coli: comparative genomic analysis of e. coli commensal and pathogenic isolates sudden death syndrome in adult cows associated with clostridium perfringens type e cryptosporidium cuniculus in the rabbit (oryctolagus cuniculus) clostridium difficile colitis in a rabbit following antibiotic therapy for pasteurellosis efficacy of cmx as a prophylactic and presymptomatic antiviral agent in new zealand white rabbits infected with rabbitpox virus, a model for orthopoxvirus infections of humans comparative metabolism of tritiated water by mammals cytotoxicity of bacillus piliformis nutritional muscular dystrophy and neonatal mortality in a rabbit breeding colony laboratory diagnosis of vitamin e deficiency in rabbits fed a faulty commercial ration conventional methods for the diagnosis of dermatophytosis hydrocephalus and cleft palate in an inbred rabbit colony re-description of cryptosporidium cuniculus inman and takeuchi, (apicomplexa: cryptosporidiidae): morphology, biology and phylogeny production of transgenic rabbits a study of the life cycle of eimeria stiedae (lindemann, ) and the immunological response of the host immunocytochemistry of psoroptes cuniculi stained by sera from naive and infested rabbits: preliminary results deficiency of the sixth component of complement in rabbits with an inherited complement defect the life cycle of four intestinal coccidia of the domestic rabbit genome sequence of lawsonia intracellularis strain n , isolated from a sow with hemorrhagic proliferative enteropathy a cottontail rabbit papillomavirus strain (crpvb) with striking divergent e and e oncoproteins: an insight in the evolution of papillomaviruses transgenic rabbit model for human troponin i-based hypertrophic cardiomyopathy exploring transplacental transmission of pneumocystis oryctolagi in first-time pregnant and multiparous rabbit does health and body condition of lactating females on rabbit farms atropinesterase, a genetically determined enzyme in the rabbit wild rabbits (oryctolagus cuniculus) as potential carriers of verocytotoxin-producing escherichia coli proliferative enterocolitis associated with dual infection with enteropathogenic escherichia coli and lawsonia intracellularis in rabbits complement c deficiency protects against diet-induced atherosclerosis in rabbits quantitative rt-pcr profiling of the rabbit immune response: assessment of acute shigella flexneri infection enterocecocolitis associated with intraepithelial campylobacter-like bacteria in rabbits (oryctolagus cuniculus) rotavirus-associated diarrhea in a commercial rabbitry parasites of rabbits in vitro inhibition of clostridium difficile and clostridium perfringens by commercial probiotic strains some factors in the in vitro infectivity and replication of encephalitozoon cuniculi the oxidative status and inflammatory level of the peripheral blood of rabbits infested with psoroptes cuniculi glycogen reserves and their changes at birth cryptosporidium infection in juvenile pet rabbits aortic endograft infection due to pasteurella multocida following a rabbit bite analysis of the expression of platelet antigens cd and cd / in transgenic rabbits with the integrated human blood clotting factor viii gene construct psoroptes cuniculi induced oxidative imbalance in rabbits and its alleviation by using vitamins a, d , e, and h as adjunctive remedial complete genome sequence of treponema paraluiscuniculi, strain cuniculi a: the loss of infectivity to humans is associated with genome decay genetic diversity in treponema pallidum: implications for pathogenesis, evolution and molecular diagnostics of syphilis and yaws venereal spirochetosis of rabbits (rabbit syphilis) due to treponema cuniculi: a clinical, serological, and histopathological study coccidiosis of the live rabbit ii, experimental study on the mode of infection of the liver by sporozoites of eimeria stiedae lawsonia intracellularis: getting inside the pathogenesis of proliferative enteropathy gamma interferon influences intestinal epithelial hyperplasia caused by lawsonia intracellularis infection in mice bacillus piliformis infection (tyzzer's disease) in a patient infected with hiv- : confirmation with s ribosomal rna sequence analysis production of two vaccinating recombinant rotavirus proteins in the milk of transgenic rabbits clostridial enteric diseases of domestic animals the young rabbit: a nonimmunosuppressed model for pneumocystis carinii pneumonia helminth, arthropods, and protozoa of domestic animals efficacy of ivermectin against nematodes infecting field populations of snowshoe hares (lepus americanus) in yukon novel bivalent vectored vaccine for control of myxomatosis and rabbit haemorrhagic disease the current status and future directions of myxoma virus, a master in immune evasion phenotypic and genetic characterization of pasteurella multocida and related isolates from rabbits in switzerland altered myofilament stoichiometry in response to heart failure in a cardioprotective alpha-myosin heavy chain transgenic rabbit model glomerular blood flow biochemical modulation of -bromo- '-deoxyuridine and -iodo- '-deoxyuridine incorporation into dna in vx tumor-bearing rabbits polyclonal antibody production rabbit cardiovascular responses during peep before and after vagotomy atropinesterase and cocainesterase of rabbit serum: localization of the enzyme activity in isozymes production of actin-specific adp-ribosyltransferase (binary toxin) by strains of clostridium difficile the laboratory rabbit spontaneous lateral abdominal (lumbar) hernia in a new zealand white rabbit immunization of rabbits against pasteurella multocida using a commercial swine vaccine heat-labile toxinproducing isolates of pasteurella multocida from rabbits protective immunity to pasteurella multocida heat-labile toxin by intranasal immunization in rabbits derivation of pasteurella multocida-free rabbit litters by enrofloxacin treatment immunization of rabbits against a bacterial pathogen with an alginate microparticle vaccine field trial of a pasteurella multocida extract vaccine in rabbits a comparative overview of immunoglobulin genes and the generation of their diversity in tetrapods intracutaneous vaccination of rabbits with the cottontail rabbit papillomavirus (crpv) l gene protects against virus challenge prevention and treatment of encephalitozoon cuniculi infection in rabbits with fenbendazole enteropathogenic escherichia coli prevalence in laboratory rabbits enzootic enteropathogenic escherichia coli infection in laboratory rabbits rearing of germfree rabbits and establishment of an spf rabbit colony pinworm infection in laboratory rodents: a review establishment and characterization of cag/ egfp transgenic rabbit line pneumocystis carinii infection in young non-immunosuppressed rabbits. kinetics of infection and of the primary specific immune response shiga toxin-associated hemolytic uremic syndrome and thrombotic thrombocytopenic purpura: distinct mechanisms of pathogenesis shiga-toxin-producing escherichia coli and haemolytic uraemic syndrome a clinical and pathological study of inherited glaucoma in new zealand white rabbits the effect of combined rotavirus and escherichia coli infection in rabbits rabbit neoplasia novel transgenic rabbit model sheds light on the puzzling role of matrix metalloproteinase- in atherosclerosis studies on the renal circulation comparative morphology of the cardiac conduction tissue in animals water intake in domestic rabbits (oryctolagus cuniculus) from open dishes and nipple drinkers under different water and feeding regimes intermittent medication of sulfadimethoxine and sulfamonomethoxine for the treatment of coccidiosis in domestic rabbits biology of the treponematoses based on studies carried out at the international treponematosis laboratory center of the johns hopkins university under the auspices of the world health organization histiocytic enteritis of rabbits evidence of host adaptation in lawsonia intracellularis infections large-scale management systems and parasite populations: coccidia in rabbits clinical veterinary advisor birds and exotic pets pathogenesis of dermatophytosis thymoma in a rabbit with hypercalcemia and periodic exophthalmus detection of dna sequences identical to pneumocystis carinii in samples of ambient air dna sequences identical to pneumocystis carinii f. sp. carinii and pneumocystis carinii f. sp. hominis in samples of air spora mammalian microsporidiosis serial inbreeding of rabbits with hereditary hyperlipidemia (whhl-rabbit) breeding of a rabbit strain of hyperlipidemia and characteristic of this strain (author's transl) the effect of selective breeding on the development of coronary atherosclerosis in whhl rabbits. an animal model for familial hypercholesterolemia enzyme-linked immunosorbent assay to detect lawsonia intracellularis in rabbits with proliferative enteropathy detection of lawsonia intracellularis using immunomagnetic beads and atp bioluminescence cultivation and characterization of lawsonia intracellularis isolated from rabbit and pig efficacy of simultaneous vaccination with enterisol(r) ileitis and ingelvac(r) circoflextm in a swiss breeding farm neoplastic diseases torsion of the caudate lobe of the liver in the domestic rabbit (oryctolagus) neoplastic diseases microsporidiosis: molecular and diagnostic aspects liver lobe torsion in three adult rabbits ultramicroelectric recording from cardiac pacemaker the effects of group housing on the research use of the laboratory rabbit pasteurella multocida: diseases and pathogenesis practical guide to laboratory animals ophthalmology of exotic pets the effects of ethylene dibromide on semen quality and fertility in the rabbit: evaluation of a model for human seminal characteristics dna polymorphisms amplified by arbitrary primers are useful as genetic markers liver lobe torsion in a rabbit tularemia, plague, yersiniosis, and tyzzer's disease in wild rodents and lagomorphs in canada: a review reference range data base for serum chemistry and hematology values in laboratory animals infectious motor paralysis in young rabbits comparative efficacy of injection routes and doses of ivermectin against psoroptes in rabbits pathology of spontaneous malignant fibrous histiocytoma in a japanese white rabbit (oryctolagus cuniculus) simultaneous identification of three highly pathogenic eimeria species in rabbits using a multiplex pcr diagnostic assay based on its - . s rrna-its fragments the oct promoter-egfp transgenic rabbit: a new model for monitoring the pluripotency of rabbit stem cells flow-volume curves of excised right and left rabbit lungs biochemical parameters of normal rabbit serum a genomic update on clostridial phylogeny: gram-negative spore formers and other misplaced clostridia a blood coagulation abnormality in rabbits deficient in the sixth component of complement (c ) and its correction by purified c origin of complex behaviour of spatially discordant alternans in a transgenic rabbit model of type long qt syndrome key: cord- -pol qm authors: nan title: third international congress on the immune consequences of trauma, shock and sepsis —mechanisms and therapeutic approaches date: journal: intensive care med doi: . /bf sha: doc_id: cord_uid: pol qm nan this issue of the journal contains the abstracts for the third international congress on the immune consequences of trauma, shock and sepsis -mechanisms and therapeutic approaches. we hope that the information contained in this special issue will stimulate you to participate in the congress, to contribute to the knowledge being developed in this field and to use this information to help you in providing better care for your patients. we thank the editors and the editorial board and publishers of the journal for their interest and support in preparation of this special issue. we also, on behalf of the scientific committee, welcome you to the third international congress in munich on - march . when, in the mid- s, we thought of having a worldwide congress, we hoped to bring together investigators to discuss this theme. the explosion of knowledge occurring around that time provided an excellent background against which the first conference in provided stateof-the-art information and consensus on factors involved in injury and sepsis. in , the second congress was held at the time of another resurgence of research, study and information on injured and operated patients. it seemed then that there would be a lull in the development of new information and therapy, and that another state-of-the art conference might not be necessary until or . however, the explosion in molecular biology has continued. the wonderful world of cytokines has gone from ill to il- to il- , il- and il- and beyond. the vast amount of information about mediators and their importance in disease is impressive. this has all suggested a magic bullet that might be used to alter or block inflammatory responses. this has not happened, however, and the question is "why not"? our science is powerful, but our therapy is still weak. what are the issues, then, in , to be dealt with at this symposium and congress? ( ) proposals for new terminology. there have been a number of proposals for new terminology and new classifications of injury, sepsis, inflammation and various other problems related to human illness. the question is whether this is the way to go. will this contribute to better clinical trials, information basis and better research? the pros and cons of this development will be reviewed by those making the proposals and those questioning the need for and wisdom of this effort. ( ) magic bullets: the prospect of a magic bullet to deal with inflammation in injury and infection seemed highly promising earlier. many preclinical trials and a lot of animal research suggested the possibility of a great breakthrough in clinical care. what has become, then, of all the expensive and extensive multi-institution randomized, placebo-controlled, double-blind clinical trials of agents that block mediators and endotoxin. many such studies have yielded equivocal, marginal or negative results. the reasons for this and the future of clinical research will be the subject of presentations and discussions to set the stage for further work. ( ) should future clinical trials be based on new classifications of illness such as mods, sirs, apache iii, sap ii, mrm, etc., or should trials be dedicated to specific diseases -urinary tract infections, pneumonia, trauma patients, cardiac surgery and other specific problems, rather than generalized problems of sepsis, the sepsis syndrome and other classifications? in other words, should we now begin to have clinical trials on specific diseases with causes that are known and can be attacked? the causality of disease becomes an important consideration in this regard. ( ) a multitude of potential therapeutic agents has been proposed on the basis of animal studies. how should we decide which of them should be brought to clinical trial? the possibilities are endless as we develop new clinical information about the mechanisms and pathogenesis of human disease. ( ) information on the pathogenic mechanism of disease states and of injury continued to emerge in an explosive fashion, and in light of our gathering knowledge we can look forward to working out a cohesive system of response to injury. ( ) additional information will be provided in plenary sections, many symposia and free communication sessions and posters, which will update the participants on a variety of relevant topics presented by many of the leading in-iv vestigators in these fields. topics will range from molecular mechanisms, such as signal transduction, through the explosive growth of information on the role of cytokines and pathophysiology, to practical considerations in the design of immunomodulatory therapeutic regimens. these merely touch on a few areas, from the basic to the clinical, which will be the subjects of those symposia. all this information will fit into the jigsaw of this exciting area and its stimulus to further research study. this promises to provide an exciting, educational programme with experts and participants from all over the world. we hope it will set the stage for many years to come and will increase our understanding of trauma, shock and sepsis and help us to provide better therapy for those of our patients who are affected by such problems. a. the clinical syndrome of mods versus mof will be reviewed in detail by those who have made these proposals. b. an extensive review of the design and interpretation of clinical trials in patients with shock and injury will be provided. the reasons why so many clinical studies in the recent past have been negative will be reviewed. the therapeutic strategies that are being developed for the treatment or prevention of mods or mof will be the subject of another panel discussion by experts who have been involved in and contributed to this area. a consensus conference or controversy conference will be presented about various aspects of mods or mof, including the benefits of supernormal oxygen delivery, bacterial translocation, parenteral nutrition, the immune response and other aspects. the successes and failures of completed clinical trials will be presented by those who are involved in these clinical trials, with a refreshing review of the problems related to that injury. there will be late news about studies just being completed at present or after the beginning of and where they stand. c. the mechanisms and biochemical profiles of specific organ dysfunction or failure will be reviewed. what are the definitions? what are the mechanisms? how can organ dysfunction and/or failure be defined? an extensive review of the biological mechanisms involved in production of injury by mediators will be presented. a session will be devoted to how future ongoing trials might be better designed and what can be done about the studies recently completed, many of which are negative. d. the immunological or inflammatory pathways resulting in organ injury will be reviewed in detail in presentations and a panel discussion. we look forward to welcoming you to an exciting and rewarding conference, which undoubtedly possesses the potential to become a landmark event and major reference point for any scientific discussion about the complex of host defense dysfunctions following trauma, shock and sepsis. studies over the past years have established that the contact system, which forms bradykin/~, is gax important mediator in hypotensive septicemia. in addition to hradyk{nln, another product of the contact system, kailikrein, can mediate inflammation by virtue of its chemotaetic mad neutrophj/activating properties. using functional and immunochemical tech~ ques, we have demonstrated activation of the contact system in the adult respiratory distress syndrome in typhoid fever and clin/cal sepsis. we have also been able to inhibit the hypotension but not the disseminated intravaseular coagulation in a model of primate sepsis by the use of a monoclonal antibody directed agsi~st factor xii, the initiating protein of the contact system, in volunteers given e. coil endotoxin, who did not develop hypotension, we were also able to demonstrate activation of the contact system with a rise of alpha- macrogiebulin-kalllkrein complex. we have also examined, j~ an i~tensive care situation, patients with sirs. we found that serial measuremezzts of the contact system were useful in eva~u~ting prognosis+ these studies suggest that inhibition of kalllkrein a~d l e r bradykinin actions might be useful i~ obviating many .of the features seen in sepsis and septic shock. dextran sulfate (dxs) activates the contact system and, in vivo, produces transient hypotension. in order to better define the mechanisms underlying the dxs-induced hypotension, we investigated the effects of either the plasma kallikrein inhibitor, des-pro -iarg] ]aprotinin (bay ) or the b kinin antagonist, hoe on the hypotensive response to dxs. in the first study, anesthetized miniature pigs ( pigs/group, randomly assigned) were given one of the following treatment protocols: ) dxs ( mg/kg), - ) dxs plus bay ( , , , or rag), or ) saline. dxs alone produced a profound but transient systemic arterial hypotension with a corresponding reduction in plasma kinin-containing kininogen. circulating kinin levels, complement fragment c adesarg and fibrin mom)mer were all increased. bay produced a dose-dependent delay or attenuation in these effects with the highest dose completely blocking dxs-induced hypotension and elevations of kinin, c adesarg and fibrin monomer levels. thus, the effects of dxs are solely dependent on contact system activation and this activation is sensitive to bay . llowev~:r, contact system activation is known to produce changes in a variety of vasoactive mediators, all of which can affect blood pressure. in a second study, two groups of pigs ( /group) were given either dxs alone ( mg/kg) or dxs minutes after a bolus injection of hoe ( #g/kg). dxs alone produced transient hypotenmon. this response was completely blocked by hoe pretreatment. both groups had identical reductions in kinin-containing kininogen. we conclude that dxs-induced hypotension is produced by activation of the contact system which results in the production of bradykinin. liberation of bradykinin is both necessary and sufficient to produce all of the hemodynamic changes observed. dr. matthias siebeck, department of surgery, university of munich, klinikum lnnenstadt, nussbaumstrasse , d- munich, germany in experimental animals exposed to i.v. injection of endotoxin accumulation of leukocytes in various organs as lungs and the liver is a prominent feature. as a part of these morphological changes damages of endothelial ceils are regularly seen. this process, which is a part of endothelial-cellular interaction, leeds to exposure of the sub-endothelial basement membran. the basement membran is known f r its capacity to activate the contact system of plasma. during this cascade activation, coagulation factor xii is converted to the active factor xii. this activation might produce increased plasma kallikrein activities and thereby give release of the vasoactive substance bradykinin. using a porcine model we have noticed that endotoxin infusion ( , mg/kg) induces elevated plasma kailikrein activities within two hours after the start of the infusion. this enzyme activity remained increased during the next hours and reached value of up to u/ . in patients with sepsis we also have observed elevated plasma kallikrein activities with enzyme activities up to u/ . in order to further elucidate the significance of these elevated enzyme activities, we prepared human plasma kallikrein and injected it intravenously in anaesthetized pigs ( ). when very small plasma kailikrein activities ( , u/kg bodyweight) were given intravenously a % decrease in arterial blood pressure was seen in the animals. in the patients with sepsis also decreases in prekallikrein values and functional plasma kallikrein inhibition are frequently seen. furthermore, degradation of high molecular weight kioinogen is found in these patients indicating formation of bradykinin. these experimental and clinical studies underline that contact activation in sepsis might results in the release of very powerful mediator substances which can be of pathophysiological importance in this disease. a number of pathological disorders as reperfusion injury, bone marrow transplantation, polytrauma and septic shock are associated with capillary leakage. as the activation of the complement system and the contact phase play a major role in these diseases we investigated whether cl-lnhibitor (c -inh), which inactivates cl-esterase, kallikrein and clotting factors xii and xl, could abolish vascular leakage. a capillary leakage was induced in rats by the administration of interleukin- ( x iu/kg). the increased vascular permeability was monitored for one hour as the extravasation of fitc marked rat serum albumin from a mesenterial vessel by a video-image processing system. ci-inh (berinert®, behringwerke) given as a single i.v. bolus in concentrations of , or u/kg dose-dependently prevented the capillary leakage. carrageenaninduced inflammation in the rat leads to vascutar leakage and to edematous swelling of the paw. ci-inh in this model leads to a dose-dependent decrease in paw edema formation. finally, we investigated the effect of ci-inh (infusion ( - u/kg x h) on a lps-induced shock in the rat by combination therapy with the antithrombotlc agents antithrombin ill (kybernin®) or rec. hirudin (both substances from behringwerke). in this animal model mortality was % in the untreated control. both antithrombotic agents decreased mortality rates by inhibiting formation of dic; a further significant improvement of survival was achieved by the treatment with ci-inh. thus+ it could be concluded that c -inh has a beneficial effect in diseases associated with a vascular leakage. iclb and laboratory for experimental and clinical immunology, university of amsterdam, the netherlands; thrombosis research center, temple university, penn., usa; oklahoma medical research foundation,. ok. city, usa. to evaluate the contribution of the contact system to activation of other mediator systems in an experimental model of sepsis, we investigated the effect of mab c b which inhibits activation of factor xli, on activation of complement and fibrinolytic cascades and activation of neutrophils in baboons suffering from a lethal sepsis. activation of the complement system was assessed by measuring circulating levels of c b/c and c b/c, and a significant reduction was observed in animals that had received a lethal dose of e. coli together with mab c (treatment group), compared to animals that had received a lethal dose of e. coil only (control group). activation of the fibrinolytic system as reflected by circulating plasmin-= antiplasmin complexes and tissue plasminogen activator, and activation of neutrophils, assessed by measuring circulating elastase-=l-antitrypsin complexes, was also significantly less in the treatment group. we conclude that activation of the contact system protein factor xll during the inflammatory response to a lethal dose of e. coil in this baboon model, modulates directly or indirectly activation of the complement and fibrinolytic systems and that of neutrophils. in a prospective study, plasma levels of c a, c , and c a were measured in patients from an internal intensive care unit. patients were clinically septic defined by the criteria of bone et al.(l) . the remaining patients were critically ill but didn't fulfill the clinical criteria of sepsis. from both groups of patients blood samples were taken over a l days period. during the first days blood samples were drawn every h, on day - every h and the last days once daily. mean plasma concentrations of c a within the first h after clinical onset of sepsis were + pg/ml, whereas non-septic-patients exhibited mean values of only +_ p_g/m/. c levels were lower for septic-patients ( + lag/ml) than for non-septic-patients ( _+ lag/ml). the most profound difference between both groups was found, when the c a/c ratio was compared ( . + . for septic-patients and . _+_ . for the control group). no significant differences between both patient groups were observed in c a plasma levels ( . + . ng/ml in septic-patients vs. . _+ . ng/ml in control patients). in of cases of clinically defined sepsis causative organisms like bacteria, protozoa or fungi could be cultured from blood, bronchoalveolar lavages and/or section materials. application of the complement parameters to survivors (n= ) and non-survivors (n=l ) within the septic-group revealed, that the c a/c ratio could also be used as a prognostic parameter for clinical outcome. the possibility of rapid and easy measurement of c a and c in only - minutes ( ) and the significant difference of the c ajc ratio between the septic and non-septic group renders this parameter a good candidate for early diagnosis of sepsis in the intensive care unit. hirudin, a single polypeptide chain composed of amino acids with cysteine residues (mr daitons), is the most potent and specific thrombin inhibitor, which is now available as a genetically engineered product (rec. hirudin -hbw , behringwerke; marburg). the aim of our study was to establish a rabbit model of tissue factor (tf) induced activation of the extrinsic pathway of coagulation and to evaluate the therapeutic efficacy of rec. hirudin. coagulation was induced in female nzw rabbits by infusion of . p.g/kgxh thromboplastin for hours. development of disseminated clotting was manifested by a decrease of fibrinogen and platelets to . % and , % respectively, and by an increase of fibrin monomers from . to > . ~tg/ml. we administered rec. hirudin to rabbits in different concentrations ( . , . and . mg/kg); treatment started simultaneously with the infusion as an i.v. bolus. rec. hirudin significantly prevented the decrease of fibrinogen, platelets and the increase of fibrin monomers. this effect was dose dependent and long lasting, even hours after the administration of rec. hirudin, clotting was still significantly reduced. as could be drawn from the plasma levels, rec. hirudin had been cleared from plasma at this time. in a post-treatment study we administered rec. hirudin ( . , . and . mg/kg i.v. bolus) as late as hours after the start of tf infusion. at this time there was already a prominent activation of coagulation. even in this post-treatment regimen rec. hirudin significantly prevented disseminated clotting. hence, it was concluded, that rec. hirudin by inkihiting thrombin could be effective in the prevention of coagulation disorders including disseminated intravascular clotting (dic) induced by a septic disease. research laboratories of behringwerke ag, marburg, germany $ novel protease inhibitory activities of the second domain of urinary trypsin inhibitor (r- ) and its effect on sepns-lnduced organ injury in rat atsuo murata , hitoshi toda , ken'ichi uda , hidewaki nakagawa , takesada mori , hideaki morishita , tom yamakawa , jiro hirese , atsushi ni~ , nariaki matsuura osaka university medical school, osaka, mochida pharmaceutical co. ltd. tokyo, wakayama medical schoof, wakayama, japan inhibitory-activities of the second kuntz-type inhibitor domain of human urinary trypsin inhibitor (uti) and its effect on sepsis-induced organ injury in rat were investigated by using the recombinant protein. uti is a glycoprotein with a structure in which kunitz-type inhibitor domains are linked in a row. we isolated the gene encoding the second kunitz-type inhibitor domain of uti, and then constructed expression plasmids by ligating it to the e. coli phoa signal peptide gene. these plasmids expressed the second domain in e. coil strain je which lacks the membrane lipoprotein. the recombinant second domain (r- ) innb[ted trypsin, plasmin, neutrophil elastase and chymotrypsin. in addition it inhibited blood coagulation factor xa and plasma kallikrein in a concentration dependent and competitive manner. the in vivo effect of the recombinant r- was investigated in a rat model of septic shock induced by cecal ligation and puncture. the administration of r- significantly improved the survival rate of the rats and attenuated the pathological changes of lung and iiver. we found out the novel protease inhibitory activities of the second domain of uti and its protective effects on sepsis-induced organ injury. macrophages are known to secrete lysosomal proteinases,mainly cathepsin b and cathepsin l, and also ~-proteinase inhibitor (pi),related to acute phase proteins.disturbances of proteinases/ proteinase inhibitors correlates with inflammatory process,leading sometimes to noncontrol "pathglogical" proteolysis (jochum et ai., ) . the cathepsin l-like and cathepsin b-like activity were measured in serum of patients with chronic bronchitis ( -with obstructive, -with nonobstructive bronchitis),acute bronchitis ( ) and healthy persons.simultaneously the level of~pi was determined in the same groups.cysteine proteinases were measured with help of fluorogenic substrates,as was presented earlier (korolenko et ai., ) , ~pi with help of immune enzyme method. it was shown increase of cathepsin l-like and cathepsin b-like activities during aggravation of chronic bronchitis comparatively to the controls ( - fold) .after treatment there was a tendency to normalization of indices,but the increase was about - % more than the control values.~pi level in this group was also increased (two-fold),in patients with acute bronchitis - - -times more comparatively to the control.it is possible to conclude that chronic bronchitis induced increased secretion both cysteine proteinases and d{pi into blood. some peculiarities of ratio were noted in patients with emphysema. endotoxins are microbial products derived from the outer cell membrane of gram negative bacteria. the active component of endotoxin is lipopolysaccharide (lps), a complex macromolecule consisting of polysaccharide covalently bound to a unique lipid, termed lipid a. now recognized to embody the endotoxic principle of lps, lipid a consists of a/ - diglucosamine backbone, both ester and amide linked fatty acids, some of which are acyloxyacylated, and charged constituents such as phosphate, phosphorylethanolamine and amino arbinose lps, exerts its biological effects in vivo by noncytotoxic interactions with a variety of host inflammatory mediator cells, primarily the mononuclear phagocyte and the endothelial cell, although other host cells also participate. these interactions are modulated by lps-specific binding proteins found in plasma, including lps-binding protein (lbp) scd and perhaps other proteins as well. specific receptors for lps have been identified on mammalian cells which mediate signal transduction via multiple pathways. lps-activated host cells are stimulated to secrete or express multiple proinflammatory mediators, including tnf-a, illa, il- / , ifn-a, il- , il- , il- , paf, pge, ltb and procoagulant activity. the overproduction of these proinfiammatory mediators results in the manifestations of endotoxemia, observed experimentally as fever, hypotension, disseminated intravascular coagulation and death. modulation of activity of these mediators protects animals against lethality. similar pathways are thought to be operative in gram negative sepsis, and control studies with human volunteers support such conclusions. immunotherapeutic approaches in clinical gram negative sepsis have, to date, been less successful. in vitro experiments and studies in animal models have recently shown that several proteinaceous bacterial exotoxins can evoke cytotoxic effects that ultimately lead to cardiovascular collapse and shock. since the possible relevance of bacterial exotoxins in the pathogenesis of septic shock has received very little attention in the past, an attempt will be made here to provide a brief overview of this generaily neglected topic. protein toxins act intracellularly or they dz~nage the integrity and function of the plasma membrane. major representatives of the former group are the adenosine diphosphate (adp)-ribosylating toxins, e.g. cholera and cholera-like toxins, diphtheria toxin), and the neurotoxins. most medically relevant toxins of this category have been studied in great detail. although often responsible for severe and sometimes fatal disease, their association with septic shock is rare. in contrast, experimental evidence is accumulating for a role of membrane<lamaging toxins in the pathogenesis of inflammatory lesions. formation of transmembrane pores is probably the most widespread mechanism via which such physical membrane perturbation can occur ( , ), the present discussion will be restricted to this category of toxins. the author shall first discuss basic properties of pore-forming proteins, and consider the factors that direct their attack to specific targets. thereafter, attention will be drawn to diverse functional consequences that may follow membrane damage so that a general concept of how pore-forming toxins may contribute towards the development of shock will evolve. i. bhakdi, s. and tranum-jensen. j. gram positive bacteria produce exotoxins that, at least in part, exhibit the unusual properties of superantigens. superantigens (sag) activate v/ selectively t cells to produce lymphokines including i - and tnf/lymphotoxin. systemic application of the sag staphylococcal enterotoxin b (seb) to d-galactosamine sensitized mice causes lethal shock within hours. seb reactive v/ + t cells accumulate within - min mrna for the ii- , i - , tnfai~ and ifn- ,. parallel in time high concentrations of bloodborne i - and tnf are noted. anti tnf t~// neutralizing mab protect mice against seb induced t cell dependent lethal shock thus indicating a key role of tnf in effecting lethal toxicity. within - h distinct levels of tolerance to seb become discernable on ligand reactive v/ t ceils, dependent of the dose of seb used. these levels include anergy, t cell receptor downregulation, loss of cd , cd , cd accessory molecules and programmed cell death. m.freudenberg, y.yaegashi, p.nielsen, c.galanos. the sensitivity towards endotoxin (lps) is genetically determined, however it may be increased under different experimental conditions. these include treatment with hepatotoxic agents such as d-galactosamine, and with live (infection) or killed bacteria. it is well established now that d-galactosamine sensitizes to lps by increasing the susceptibility of the host to toxic activity of lps-induced tnf~. sensitization by bacteria, especially by gram-negative once is of especial interest because it implies that infecting microorganisms not only produce lps, but als sensitize the host to its lethal activity. sensitization to lps by bacteria proceeds in all lps-sensitive (lps n) mouse strains that have been investigated so far. it also proceeds in lps-resistant (lps d) c h/hej mice. the absence of sensitization to lps in lps d c bl/ sccr mice was identified as being due to their inability to produce interferon- (ifn?). administration of exogenous ifn? to these mice conferred sensitivity to lps. conversely, administration of anti-ifn? to lpsn; mice inhibited the development of sensitization'by bacteria. it may be concluded therefore that ifn is the mediator of sensitization to lps by bacterial infection. the ifn? defect of c bl/ sccr mice concerns only the ifn response to bacteria, since the response to the t-cell mitogen con a and to cd monoclonal antibodies was not impaired. the ifn?producing cells themselves were shown to be intact. on closer investigation the impaired ifn? response was identified as the result of a defective accessory function of macrophages. macrophages of c bl/ sccr mice are incapable of producing interferon-~ (ifn~) which we could show to be obligatory for the production of ifn? in response to bacteria. although antibiotics are required to clear bacteremia, they do not reverse evolving shock, because endotoxin free in the bloodstream or exposed on the surface of circulating bacteria continues to activate the production of inflammatory mediators, furthermore, antibiotics have been shown to induce release of endotoxin from gram-negative bacteria during the treatment of severe infection. in an in-vitro study, using live escherichia coil, we have observed the release of endotoxin upon treatment with several antibiotics like cefuroxim, imipenem, ceftazidime and aztreonam. incubation with imipenem or ceftazidime induced an early lyeis and a mild rise in endotoxin levels, whereas incubation with cefuroxime or aztreonam resulted in the formation of filaments with a late but dramatic rise in endotoxin levels. in a second study we studied the influence of different antibiotics on the tnf-= production of e.coli stimulated human monocytes. we found again great differences in the production of this important cytokine among the different antibiotics according to their capacity to induce liberation of endotoxin. these differences in the amount of endotoxin release are probably caused by penicillin-binding-proteins (pbp) specificities of the antibiotics with resultant differential morphological changes, in a rat sepsis model treatment with imipenem or ceftazidime resulted in a transient increase in il- levels, whereas treatment with aztreonam resulted in progressively increasing levels of il- and a significant increase in mortality. the increased mortality in pbp- specific aztreonam treated rats might have been the result of filament formation in the abdominal cavity of the septic rats. the endotoxin sequestered at local sites may account for the precipitation of gram-negative shock. finally, we've also demonstrated that in patients under treatment for septic shock endotoxin release can be related to the administration of antibiotics. carbapenem-and cephalosporin-lnduced release of lps from smooth and rough pseudomonas aeruginosa: in-vivo relevance j. jackson and h. kropp, merck research laboratories, rahway, nj b-lactam (cell-wall active) antibiotics are generally deemed the class of antibiotics most responsible for the release of free endotoxin (lps) from gram-negative bacteria due to their cell lytic actions although all antibiotic classes studied thus far induce release of endotoxin. we have recently shown in-vitro that antibiotics within the b-lactam class (i.e. imipenem , meropenem and ceftazidime , with equivalent mics) differ in their propensities to release excessive amounts of lps from both smooth-(s-) and rough-(r-) lps laboratory and clinical and antibiotic-sensitive and -resistant p. aeruginosa isolates and that lps release is independent of cell lysis. additionally, variations in the induction of endotoxin release is antibiotic concentration dependant and correlate with antibiotic penicillin-binding protein (pbp) affinities which result in morphological changes. these studies also show that lps differences measured via chromogenic lps (c-lal) assay correlate with lps lethality in lps-sensitive mice. release of lps was compared to changes in cfus, cell mass, morphology and antibiotic pbp-specificity. corresponding in-vivo studies in cd mice against s-lps p. aeruginosa isolates show that, despite equivalent in-vitro protection, antibiotic efficacy in mice differ as much as -to lo -fold. to establish a possible in-vivo role for antibiotic-induced release of free lps we compared antibiotic efficacy results in mice challenged with virulent parent s-lps and its relatively avirulent r-lps mutant (lacking only its side chain) p. aeruginosa isolates. results show the relevance of quantity and physiochemical composition (bioreactivity) of free lps to virulence and in-vivo antibiotic efficacy. in other in-vivo studies chemical agents that destroy cells (m~) which mediate lps lethality in-vivo were used to pretreat mice prior to antibiotic therapy and bacterial challenge with wild type pseudomonas. we conclude that circumstances in which antibiotic-induced filamentation occurs are also conditions that yield excessive lps release, the in-vivo effects of which are shown through the requirement of larger amounts of antibiotic to afford equivalent protection. bacterial endotoxins have been reported to play a significant role in the pathogenesis of gram negative sepsis by their interaction with, and stimulation of, host inflammatory mediator cells to produce cytokines, biologically active lipid intermediates, and procoagulant activity (tfa). although both microbe-associated and free endotoxin are capable of stimulating mediator production, studies from our laboratory suggest that free endotoxin may be the more potent stimulus for tnf and tfa. further, our studies suggest that the majority of the proinflammatory activity released from antibiotictreated e. coli coisolates with lps by two different fractionation techniques. to assess whether actions of endotoxin directly contribute to lethality in gram negative sepsis, an experimental model was developed in which mice were made hypersusceptihle to the lethal effects of endotoxin by treatment with d-galactosamine (dgaln). challenge of cf- dgaln-treated mice with e. coli olll:b resulted in an lds of approximately . x cfu. resolution of the peritonitis and bacteremia by treatment with cell wall-active antibiotics resulted in an increase in the ldso. ceftazidime and imipenem, which differ in their mechanism of action and in their capacity to effect endotoxin release in vitro, also differed in their in vivo capacity to provide chemotherapeutic protection ( fold vs fold respectively, p< . ). parallel studies with endotoxin hyporesponsive c h/hej mice indicate significantly greater levels of protection with imipenem (> fold vs saline controls). collectively these data suggest that endotoxin may contribute directly to the pathogenesis of experimental gram negative sepsis. bacterial lipopolysaccharides (lps) are the endotoxins of gram-negative bacteria and represent their major surface antigens. lps is made up of three chemically, biologically and genetically disctinct regions, i.e, the o-chain, the core region and the lipid a moiety whereby the latter represents the endotoxic center. it is our current understanding that lps is responsible for many of the pathophysiological events observed during gramnegative infections and that one of the major mechanisms leading to shock and death is the lps-induced activation of macrophages resulting in the production and release of lipid and peptide mediators, among which tumor necrosis factor seems to be the most important. therefore, in the fight against the lethal outcome of gram-negative infections, modern strategies, in addition to antibiotic treatment, aim at i) the neutralization of tumor necrosis factor, ii) the inhibition of the production of tumor necrosis factor or iii) the neutralization of the activation potential of lps for macrophages by monoclonal, preferably human antibodies. the latter approach, to be effective against a broad spectrum of gram-negatives, must be directed against common structures of lps (lipid a and core region). the molecular basis of this approach and the controversy in this field will be discussed. passive immunotherapy has been used since , when von behring described the administration of immune horse serum to treat a patient with diphteria infection. even if this therapy was sometimes successful in bacterial infections, it has been largely replaced by antibiotics. however, antibiotics have their limitations, especially in critically-ill patients. to improve outcome, adjunctive therapies such as immunotherapy with polyclonal and monoclonal antibodies particularly against endotoxin are again considered. the role of humoral immunity in host defenses against bacterial infections is weu known. for instance, tile importance of antibodies in the defense against gramnegative infections has been established clinically by studies relating the outcome of patients with gram-negative bacteremia to tilers of antibodies directed at the offending pathogens at the onset ofbacteremia (mccabe ; pollack ) . ever since we know the role of endotoxins in the pathophysiology of sepsis, antibodies against the s-and r-lps have also been detected in sepsis patients. the aim of the administration of iv/g to the sepsis patient is as follows: ) enhancing of opsonization and phagocytosis(antibactericidai activity) ) synergistic effects with [ - actam antibiotics ) neutralization of endotoxin, the main pathogenic mediator of gram-negative sepsis ) modulation and/or inhibition of cytokine release the enhancement of opsonic-and phagocytic-activity especially with igg via fc and c receptors has been well documented. monoclonal antiendotoxin antibodies, proven in clinical studies, do not appear to neutralize endotoxin in vitro and are not reproducibly protective in animal models of sepsis. also they can not suppress endotoxin-induced tnf-~, il- release in mice (baumgartner , corriveau and danner ) . in conlrast, recent studies of a polyclonal immunoglobulin preparation, containing high levels of antibodies against gram-negative bacteria and their o-antigen of lps in igg, igm and iga classes (pentaglobin®) provide evidence to neutralize endotoxin. this effect is demonstrated in vitro (berger (berger , , in animal models (stephan , berger and also in prospective, randomized, controlled clinical trials (schedel , poynton , behre . furthermore mortali b' was reduced statistically in patients with septic shock and endotoxemia by using this preparation, as has been demonstrated by sehedel. anti-core lps monoolonal antibodies: binding specificity and biological properties f.e. di padova, r. barclay, e.th. rietschel. bacterial lps and cytokines are responsible for the pathological processes of gram-sepsis and are suitable targets for therapeutic interventions. chemical characterization and structural analysis of different lps have revealed common features. the inner core region of lps shows a high degree of similarity among e. coli, salmonella and shigella. among a large number of broadly cross-reactive murine anti-core lps mab one of these igg ak) has been selected and chimerized into a human igglk (sdz - ). in elisa and in immunoblots on purified lps both sdz - and wni - show a strong reactivity with all smooth lps from e. coli and salmonella. reactivity with all the known complete core structures from e. coli and salmonella (ra) is evident. reactivity with re structures or free lipid a is not observed. this mab cressreacts with all clinical e. coli isolates from blood, urine and feces and with other enterobacteriaceae. sdz - and wni - have biological activity as they inhibit the lal assay and the secretion of monokines (il- and tnf) by mouse and human macrophages. moreover, sdz - and wni - inhibit the release of il- and tnf in vivo. in vivo sdz - as well as wni - neutralize the pyrogenic activity of e. coli lps and protect mice from lethality in d-gain-sensitized mice. the possibility to use wni - as a capture antibodies in the immunolimulus assay opens the possibility to differentiate the origin of the lps in patients with endotoxemia. franco di padova, sandoz pharma ag, ch basel, $chweiz $ presentation of lps to cd by lps binding protein peter s. tobias, julie gegner, katrin soldau, lois kline, loren hatlen, douglas mintz, and richard j. ulevitch. the activation of myeloid cells by lipopolysaccharides (lps) has been shown to require the serum glycoprotein lps binding protein (lbp) and binding of lps to membrane bound cd (mcd ). other cells such as human umbilical vein endothelial cells (huvec), smooth muscle cells, and some epithelial cells, which do not express mcd but nevertheless respond to lps in the presence of serum, have receptors for complexes of lps with the soluble form of cd (scd ). these complexes of lps with scd are only formed efficiently in the presence of lbp. we have begun to characterise the mechanisms by which lbp enables lps to bind to cd , either soluble or membrane bound. with the use of fluorophore and radiolabelled reagents we have developed procedures for quantitative measurement of the association of lps with lbp and of lps-lbp complexes with cd . these results show that the delivery of lps to scd is catalysed by lbp, i.e., lbp is not included with the lps-scd complex. in contrast, on the surface of cells, lbp does not dissociate from the cells after lps binds to mcd . the kinetics, equilibria and stoichiometry of these reactions will be discussed in the context of models for cellular activation by lps and cellular uptake of lps. supported by nltt grants gm , ai , ai , gm , and assistance from the pharmaceutical research institute of johnson and johnson. the scripps research institute, imm- , n. torrey pines rd. la jolla, ca usa . modulation of endotoxin-induced cytokine production by lps partial structures h.-d. flad, h. loppnow, t. mattern, and a.j. ulmer department of immunology and cell biology, forschungsinstitut borstel, d- borstel lipid a constitutes the active moiety of endotoxin (lps) of gramnegative bacteria. it activates mononuclear phagocytes to produce cytokines, such as tnf, i _- , and il- , which are the major mediators of the endotoxic effect of lps in vivo. lipid a precursor la (synthetic compound ) does not induce cytokines, but is able to specifically antagonize lps-or lipid a-induced mediator production in human mononuclear cells, vascular endothelial cells, and smooth muscle cells. furthermore, we present evidence for the first time that t-lymphocytes proliferate in response to lps and express mrna for interleukin- and interferon-~ and that these responses are also antagonized by synthetic lipid a precursor la. when comparing the agonistic and antagonistic activity of lipid a and different partial structures at the functional and binding level, the number and length of the fatty acids and the number of phosphoryl groups were pound to be of crucial importance. unexpectedly, lipid a precursor la, although biologically inactive, turned out to be both the most potent antagonist and competitor in inhibiting the binding of lps. taken together, our results provide evidence for a model in which lipid a partial structures compete with lps for specific cell surface receptor(s). in this sense, biologically inactive lipid a analogues may be good candidates as therapeutic agents for the prevention of gram-negative septic shock. two mammalian lipid a-binding proteins have been identified that are believed to have important roles in mediating the host response to endotoxin: lipopolysaccharide-binding protein (lbp) and bactericidal/ permeability-increasing protein (bpi). human lbp shares a % amino acid sequence identity with human bpi. despite the sequence homology, the two lipid a-binding proteins have very different functional activities. lbp is an acute phase serum protein that markedly potentiates the proinfiammatory host response to gram-negative infection by a mechanism which involves binding of the lbp-lps complex to cd receptors on monocytes, neutrophils and endothelial cells. in contrast, bpi is a neutrophil granule protein with potent bactericidal and lps-neutralizing activities. the divergent functional properties of these two lps-bindlng proteins can be explained by the inability of bpi-lps complexes to bind to cell-surface cd receptors. a recombinant protein (rbpi ), corresponding to the amino terminal kd fragment of human bpi, has been shown to retain the potent biological activities of the hdlo protein and may represent a novel therapeutic agent for the treatment of gram-negative infections, sepsis and endotoxemia. for therapeutic effectiveness in many clinical situations, rbpi will have to successfully compete with relatively high serum levels of lbp ( - ~g/mi) for binding to endotoxin and gram-negative bacteria. to evaluate this issue, experiments were conducted to compare the relative binding affinities of rbpi and human recombinant lbp (rlbp) for lipid a. the binding of both proteins to iipid a was specific and saturable with apparent kd's of . nm for rbpi and nm for rlbp. in a competition assay format rbpi was approximately -fold more potent than rlbp in inhibiting the binding of nsi-rlbp to lipid a. these results demonstrate that rbpi has a significantly higher affinity for endotoxin than does rlbp and may explain the potent inhibitory activity of low concentrations of rbpi in a variety of in vitro functional assays for lps activation of cells despite the presence of high lbp levels. for example, rbpi at . ~tg/mi was able to totally inhibit lps-induced tnf release from monocytes despite a -fold weight excess of rlbp over rbpi . and for heparin binding. three separate domains which inhibit the lal reaction to lps and bind to heparin were identified in amino acid regions - , - and - . a single synthetic peptide ( - ) was bactericidal. these results suggest that rbpi contains three separate functional domains which may contribute to its high affmity interaction with gram-negative bacteria and heparin. the individual activity of each domain and the cooperative interaction among domains provide the basis for developing rbpi analogues with increased biologic efficacy. a considerable body of experimental data has accumulated implicating tumour necrosis factor (tnf) as a principal mediator of the pathophysiological features of septic shock. these data prompted the development of clinical strategies designed to limit excess (inappropriate) tnf production. monoclonoal antibodies (mobs) were developed and a phase ii dose escalation trial in patients confirmed that the mab was safe, and suggested that it was having a beneficial effect on certain parameters. preliminary results of a large phase iii study indicated that (a) the mob was safe; (b) that it was of no discernible benefit in non-shocked patients; (c) that it reduced mortality in shocked patients, especially during the first days. an alternative strategy was to take advantage of the high binding affinity of soluble receptors for tnf (stnfr). stnfr-iggfc constructs were made for both the p and p receptors. both were effective in animal models of lps challenge, but when a clinical trial was done with the p stnfr-fc there was unexpected mortality in the treated arm. using an animal model of live e.coli sepsis, we have shown that this may have been due to the release of bound tnf from the construct. plasma enhances while bpi inhibits lps-induced cytokine production from peripheral blood mononuclear cells (pbmc). pseudomonas species produce cytokine-inducing substances which are different from lps as indicated by the fact that polymyxin b blocks only % of the cytokine-inducing activity of these pyrogens. we now tested the effect of plasma and bpi on the il- [ -inducing activity of pseudomonas maltophilia -derived pyrogens (pmp). bacteria were cultured to the log phase and filtered ( kd) to obtain prop. dilutions of pmp or lps were added to pbmc alone or to pbmc in % plasma +/-bpi ( ng/ml). pbmc were incubated for hours at °c and total il-i~ was measured by ria. results: il-i[~ in ng/ml (n= , mear~+sem, *p< . vs control). control . _+ + bpi . + % plas. . _+ + bpi . _+ pmp (ng/ml) lps (ng/ml) . _+ . _+ . _+ . _+. . +. . _+. . _+. " _+ " . _+ " . _+ " . _+. . + _+ _+. * _+ " . +. " . + -+ . -+ " . _+. " cba, c bl/ , balb/c, akr, dba, swiss mice, guinea pigs, rabbits have been used in research work. the toxicity, immunogenicity, mitogenic and immunomodulating activity of lps have been studied. the possibility of reduction of the toxic activity of lps on macroorganism by bioglycansimmunomodulators obtained from sea invertebrates anymals (crenomytilus grayanus, stromhus gigas) have been investigated too. lps has been shown to induce specific antibody response of laboratory animals. cba mice are high responsive to lps. lps stimulates humoral immune response of mice to tdependent and t-independent antigens and suppresses intensity of the delayed hypersensitivity. the small doses of lps stimulate functional activity of macrophages, the large doses of lps -decrease one and show the cytotoxic effect. the bioglycans enhance the resistance of mice to the lethal effect of lads and provide protection - % of mice. one opens possibility to use of bioglicans for reduction of toxinemia in generalizated forms of pseudotuberculosis. thus, lps from y.pseudotuberculosis is immunogen and immunomodulator wich has influence on humoral and cellular factors of immunity and plays the important role in immunopathogenesis of infection. endotoxaemia is implicated in the pathophysiology of obstructive jaundice. the lirnulus lysate (lal) assay is the gold standard method for measuring endotoxin concentrations, but inherent biochemical and technical problems limit the usefulness of this assay. the endocab elisa is a novel assay which measures endogenous antibody (igg) to the inner core region of circulating endotoxins (acga). objectives we evaluated the significance of endotoxaemia in biliary obstruction using the endocab assay and subsequently the specificity of the humoral response to endotoxin compared with an exogenous antigenic challenge [tetamls toxoid (tt) ]. materials and methods in experiment i three groups of male wistar rats ( - g) were studied [no operation (n= ) , sham operation (n= ), and bile duct ligation for days (bdl)(n= )]. plasma was collected and assayed for bilirubin, endntoxin(lal) and acga(endocab). in experiment ii rats were actively immunised with tetanus toxoid ('it) and then randomised to have no op(n= ), sham op(n= ) or bdl(n=i ). blood was taken at this time (to) and days later(t at sacrifice for acga concentrationslendocab] and igg produced to tt(ttab) [elisa] . antibody concentrations are expressed as % increase from control values.results in bdl rats, acga concentrations were significantly increased compared with controlslp< . , mann-whitney]. endotoxin concentrations were sporadically elevated in the jaundiced rats but the rise was not significant. in experiment [i there was no difference between the acga or ttab concentrations in the fllree groups at to, bdl rats had a significant rise in acga concentrations by t [p< , ,paired t-test] and humoral response to tt was significantly impaired in bdl rats compared with control groupslp< . , paired ttest data plasma endotoxin was measured by means of an endotoxinspecific endospecy test after pretreatment of the plasma with a new perchloric acid method that we developed. the normal value of plasma endotoxin is less than . pglml. polymyxin b was administered at a dose of , u every hours. plasma endotoxin rapidly decreased to the normal range in of the patients. body temperature fall significantly. apache ii scores were also significantly improved. tumor necrosis factor-o~ and interleukin decreased in survivors, while in high values tended to persist in patients died. no side effects were observed in any of the patients. in conclusion, intramuscular injection of minute of polymyxin b was useful in the treatment of endotoxemia. - uchimaru, morioka , japan. l e v a n t g r a m n e g a t i v organisms. m e t h o d s : u n d e r general anesthesia, n o r w e g i a n b r e d landrace pigs ( - kg) of either sex, pr group, u n d e r w e n t t r a c h e o s t o m y a n d w e r e v e n t i l a t e d on a / air a n d o x y g e n m i x t u r e a i m e d at m a i n t a i n i n g a n o r m a l p h a n d a isocapnic level. ventilation w a s not readjusted d u r i n g the observation period. the anesthesia w a s k e t a m i n e . m g / k g h a n d d i a z e p a m . m g / k g h i n t r a v e n o u s l y . h e m o d y n a m i c m o n i t o r i n g of m e a n aorta, p u l m o n a r y artery, central v e n o u s a n d p u l m o n a r y capillary w e d g e pressures w a s p e r f o r m e d w i t h a f s w a n -g a n z catheter a n d an aorta catheter. a continous infusion of r i n g e r ' s acetate ( m l / k g h ) w a s g i v e n intravenously. w h e n stabilised, the a n i m a l s w e r e g i v e n . x l cfu of e colt intraperitoneally as a bolus in ml saline, the a n t i b o d y g r o u p received in a d d i t i o n m g / k g e a n t i e n d o t o x i n i n t r a v e n o u s l y over h o u r via a n infusion p u m p at the start of the observation period. the a n i m a l s w e r e observed for hours. results : a t a n d hours, the o x y g e n c o n s u m p t i o n increased by % in the a n t i b o d y treated g r o u p w h e r e a s there w a s a significant fall of % in the sepsis group. in the a n t i b o d y group, the arterial p h a n d the cardiac index were also significantly h i g h e r at the s a m e p o i n t s in time. there w a s no significant difference in arterial po . in severe bacterial infections it would be beneficial to neutralize the plasma endotoxin content with complex forming compounds. the phenothiazines are able to form complexes with endoto×in and the existence of these complexes were already shown in differential speetrophotometry and animal experiments, however, the mechanism of partial neutralization was not clarified. therefore some representative phenothiazines and structurally related compounds were tested for anti-endotoxin activity. the endotoxin neutralizinb effects of several benzophenothiazines were investigated in differential speotrophotemetry, tnf induction and in the conventional limulus test. in animal experiments some beneficial effect of complex forming compounds was found. the benzophenothiazines were not able to inactivate the biological effect of endotoxin in the limulus test. the recent findings indicates that a multifocal effect can be responsible for "anti-endotoxin action in vivo". effects of tnf inducing effect of endotoxin in leukocytes and bypotensiv action in experimental animals were reduced by some phenothiazine derivatives. monophosphoril lipid a was without effect. of microbiology, albert szemt-gydrbyi medical university, odm t~r lo, h- szeged~ hunbary involvement of streptococcus pyogenes erythrogenic toxins in the induction oflstreptococcal toxic shock syndrome heide mgller-alou~* , joseph e. alouf , die [er gerlach , ~atherine fitting., and jean-marc ca~aillon . unit des toxines microbiennes and "unit d'immuno-allergie, institut pasteur, , rue du docteur roux - paris (france) ; institut f~r experimentelle mikrobiologie, jena (germany). superantigen erythrogenic toxin a (eta) is thought to be involved in toxic shock syndrome in humans by inducing massive release of cytokines by patient immune cells. the cytokineinducing capacity of eta w~:s £:ompa~ed to that of lps, a gram-negative bacterial cell wall component. eta elicited weak production of il- d and ~, tnf ~ and il- in purified human monocytes whereas lps stimulated the production of high amounts of these cytokines. in the presence of t cells, eta elicited the production of significant amounts of il-i~, il-i~, il- and il- . however, the most preponderant cytokine was tnf~, which peaked at i ng/ml after stimulation with i ~g eta. comparable amounts of tnfd (ca ng) were induced by .i ~g eta and .i ~g lp$. in contrast to lps, eta was a strong inducer of tnf~ which was produced only in marginal amounts by lps. these results suggest that the septic shock induced by gramnegative bacteria (lps) and by gram-positive bacteria {extracellular superantigens) follows different pathogenic pathways. lps-induced shock is mainly mediated by monocytes and monocyte-produced cytokines (il-i and tnf). the eta-induced shock is mediated by t-cells or depends on t cell help for the production of monocyte-liberated cytokines. production of t cell cytokines such as tnf~ and interferon in addition to the other cytokines contribute very likely to the severity of the toxic-shock resulting from s. auzeus and s. pyogenes infections in humans. the present study was utidertakc~l to cvalu~tlc the effect of soluble chemically modified giucan during septic shock. carboxylnethyl-b-i, -glucan (ram ) was injected twice and h before the shock i.v. in a dose of ing/kg. shock was induced in u~?esthetizcd (sodikm~. l)mntobarbital) rats by i.v. injection of endotoxin of escherichia colli bs, mg/kg. aiiofcmg pretreated ruts survived during first haher ¢ndotoxine, while in controi shock group the lethality was %. the concentration of ~col)terin in serum was significantly elevated hafterthc second cmginjection (appare~tly % if compare with the control rats), but didu't chartged rain and s rain after endotoxin injectjom cardiac output in cmogroup was higher a* the i and min after endotoxine onset ( i % trod ~, respectively of initial level) than in the control shock group ( % and % at the same time). pretreatment of rals with soh~ble giucan w~ts associated with beneficial effects o~ the hepatic c~ergy $ia[tls after h after challenge of endotoxiae: the tissue level of lactale was ahnost twice lower than in the control ruts, me~mthne the tissue atf in cmg pretreated group was higher at %. twice injected macrophage stimuhttor soluble glucan can prevent the endotoxic shock, and extremely ir~creased survival rate after endotoxine injection. the national committee of surgical infections of the spanish association of surgeons have produced a computer program for the collection and analysis of information on surgical infections. the program is suitable for ibm compatible hard disk personal computers and works through the ms-dos system. the main menu is called up on the screen when the operating disk has been installed; it reads as follows: i. new record; . modify records; . erase records; . searches; . reports; . configure; o. ouit. if you ask fdr a new record the screen will prompt you to enter the number of case, record number, hospital, age and sex. the next screen will come up and the words "topographic diagnosis" will flash. a menu of areas or organs will be displayed. then, the words "type of pathology" (inflammatory, neoplastic, traumatic and other). days of postoperative period. type of surgery (programmed and emergency). type of operation (clean, clean contaminated, contaminated and dirty). duration of surgery. this is followed by "order of operation" and the "type of anaesthesia (general, regional or local). you are then required to supply the "diagnostic code of who" (icd ) and the "procedure code of who. analytic and concurrent illnesses (total proteins, albumin, haemoglobin, haematocrit, leucocytes, red corpuscles, glucose and bilirubin). the next screen asks for "risk factors" (obesity, uraemia, neoplasia, malnutrition, urinary catheter, distant infection, artificial valve, immunosuppressive drugs, over years and anergy. this is followed by a screen headed "postoperative complications". "evolution" (the questions asked are drainage, systemic antibiotics, and on each ocasion a choice of antibiotics is displayed), local antiseptics, reoperation, etc. under "microhiology" is a choice of organisms and the chance of identifyin organisms. finally, "sepsis score". our recent work had shown that renshen-fuzi-chaihu mixture could increase the survival rate in experimented study. the purpose of this study was to determine the effect of combined administration of renshen-fuzi-chaihu mixtuer and antibitics (sa) in patients with septic shock. the result showed that, in sa group ( cases), the total effective rate was , %, in the contral group (combined administration of gentamycin and dexamethasone, cases) the total effective rate was %. however the obviously effective rate in sa group % was significantly higher than in contral group % (p<o.os). sa group appeared to be more obvious than the contral group in raising arterial pressure, recovering consciousness and pulse, improving cold lilmbs and reducing fever (p<o.o ). it was found that sa could inhibit the pathogeneses changes of septic shock such as the decreases of superotide dismutase (sod) and glutathione peroxidase (gsh-px) in erythrocytes and the increases of plasma free hemoglobin (phb), plasma malondialdehyde (mda) and -glucuronidase ( -gc) occured in septic shock. sa had stronger effect than gd in inhibiting the changes mentioned above. hence, these results suggest that sa has beneficial effect in the treatment of septic shock. sa could diminish the decrease of the antioxdase activities and inhibit lipid peroxidization and stabilize cellmembrance. this may be one of the principal mechanisms of the beneficial effect of sa in the treatment of septic shock. donna l. lehman, heather a. childers, irshad h. chaudry and alfred ayala. the thymus is thought to be a privileged sight for tcell generation. here the t-lymphocytes are either selected for their potential to recognize and react competently to foreign antigens or de-selected, due to their potential to react to auto-antigens, de-selected. tcells with this latter capacity are thought to be deselected through a process referred to as programmed cell death or apoptosis. while it is known that thymic apoptosis is elevated by a variety of stressers associated with infection and inflammation, little or no information is available concerning its presence in polymicrobial sepsis. it was, therefore, the aim of this study to determine whether thymocytes exhibit increased apoptosis during sepsis. to assess this, thymocytes were harvested from c h/hen mice at i and h following cecal ligation and puncture (clp; to induce sepsis) or sham-clp (sham). thymic yields were assessed and the extent of apetosis determined by staining the cells with propidium iodide (a dna intercalating dye) for facs analysis or by examining the extracted dna electrophoretically for the endogenous endonuclease activity the results (n= /grp) indicate that at i h post-clp there was no marked changes in any of these parameters. however, at h the thymic cell yield from clp mice was significantly decreased (sham: . ! . x l vs clp: . i . x * cells; *,p< . ), the % of cells apoptotic by facs analysis increased from . i . % in sham to . ± . %* in clp, and the septic mouse genomic dna exhibited dna degradation these results indicate that clp, but not simple laparotomy, induces marked thymic apetosis, which may contribute to the lymphocytic immune deficiency seen in these mice the purpose of this retrospective study was to evaluate effectiveness of irrigation to treat septic knee joints from longterm results. from to patients with septic knee joints have been treated. treatment in acute cases started with asp#at[on of the effusion to evaluate for cultures. without awaiting the result treatment was continued with immediate joint irrigation under arthroscopic control. three redon tubes ( ch ) were introduced for continuous irrigation and for intermittent distention of the joint cavity. in case of chronic joint infection additionally all foreign and synthetic material was removed. systemic antibiotics were given. patients could be re -examined with a mean follow-up of years. re-examination showed that immediate arthroscopic lavage started at the onset of septic sings had best results (less signs of osteoarthr[tis, less synovitis, less pain while loading and less range of motion loss ). patients ( %) out of patients with s tre_=.tsd acute se.~tic knee joint had excellent functional recovery, whereas all patients with chronic septic knee joints showed poor results. additionally, in of these patients with chronic septic knee joint after irrigation a further procedure was necessary due to recurrent septic knee joint. the concept that bacteria can translocate across the intestinal mucosa under a variety of circumstances is well established. however, due to the inherent limitations of in vivo studies, the cellular mechanisms underlying the process of bacterial translocation (bt) across the epithelial barrier are poorly understood. this is especially true in those circumstances in which there is no merphologic evidence of mucosal injury. consequently, we have utilized an in vitro cell culture model system to investigate the cellular events involved in the translocation process. in this model system, a polarized monolayer of intestinal cells (caco- cell line) is grown on a micron filter in a two compartment system. after tight junction integrity is established, bacteria are placed in the apical surface chamber and bt across the caco- monolayer is measured by culturing the basal chamber. the results of our studies utilizing the caco- system indicates that; l) several strains of non-pathogenic bacteria will translocate across the caco- monolayer in a time-dependent and dose-dependent fashion. however, the incidence and magnitude of bt are highest for those strains of bacteria (gram-negative enterics) that are most commonly cultured in vivo. ) caco- cells are actively involved in the transcellular passage of bacteria across the caco- monolayer, since inhibition of caco- microtubule or microfilament function decreases the magnitude of st. ) lectin but not integrin receptors are involved in bacterial translocation of e. col~ across the caco- cell monolayer. ) caco- cells have the ability to ingest and kill certain strains of bacteria. the mechanisms involved in caco- bactericidal activity appear similar to that of pmns to the extent that both are oxidant but not nitric oxide mediated. these in vitro studies suggest that caco- cells can function as "nonprofessional" phagocytes and that both bacteria and enterocytes are involved in the translecation phenomenon. bacterial translocation due to gut barrier dysfunction is considered to be a major cause of septic complications and multiple organ failure following trauma, burn injury, hypoxia, shock and shock-like states. the invasion of bacteria into the circulation from the gastrointestinal tract or even from other sources, however, should not necessarily result in systemic infection or organ cohinisation, as long as the humoral and cellular defense mechanisms are not impaired. thus, a reduced res clearance capacity and hepatic clearance function of bacteria and toxins can be observed under the same conditions which enable bacterial translocation from the gut. in order to evaluate the influence of circulatory, metabolic and humorul disorders on the elimination of bacteria out of the blood circulation, a series of experiments were performed in anesthetized and ventilated rabbits which received defined numbers ( . x colony-furming units) of escherichia coli as a bolus injection. in unaffected control animals the intravenously injected bacteria are eliminated about . % within the first minutes and are totaiy cleared within minutes. % offue total cfu counted in the cultures of the organ homogenates were found in liver and spleen, whereas only very low numbers could be detected in the lungs and kidneys. systemic injury of the animals by hemorrhagic shock, endotoxinemia, hypoxia, coagulation disorder, systemic vasoconstriction by norepinephrine and other types of injury reduced the elimination rate of bacteria and changed the pattern and degree of their dissemination and tissue distribution. the bacterial clearance was delayed mostly in rabbits subjected to hypoxemic hypoxia in which about cfu of viable e.eoli per ml blood could still be counted hours following their injection. the number of viable bacteria was some ten powers higher in organs of hypoxic rabbits in comparison to control ardmais, and an excessive colonisation of the lungs and kidneys with % respectively % of the total cfu of the cultared organ homoganates was observed. unilateral iscbemia of the kidney prior to the intravenous injection of e.coli only moderately delayed the bacterial clearance, bowever, caused an intense eohinisation of the affected reperfused organ. in contrast to this, inhalation injury did not promote the accumulation of bacteria in the lungs. conclusion: shock, systemic and local affections reduce the clearance of bacteria from the blood circulation and enable their dissemination and accumulation in vital organs. the degree of this reduction and the organ pattern of the bacterial distribution varies with the type of injury. thus, these factors seem to be essentially involved whether or not bacterial translocation and multiple organ failure will follow the invasion of bacteria into the circulation as in the ease of gut barrier dysfunction. movement of enteric baaterla from gut to extralumeaal sites (bacterial tra~sloeation) m~y play a role ~. nraltipl~ orga~ failure. traumatic stress (shock, hffectiort, and im~lammation) and severe illness are common alateeedents. tnt~st~aal lleus is e common proximme causal factor. we have exanlined the hypothesis that bacterial ttanslecation from the gut in expe~me~tal paaerestitis is due to bacterial overgrowth as a cuas~ucnee of a deere~ae in intestinal transit. ne~rotit_.~g pancreatitls fbliowlng bile duct iigation in the opossum is associated with colonization of the pa,se ~re, aa by gut derived organisms or salmonella of biljary origin in infected animals (previously reported). to ti~rther define the mechanism of transloemdon in panereati* inflammation, pancteatitis was induced hy iigatlon of t~e lower bile duet distal to the pancreatic duets m the rat. intestinal transit, enteric bacteriology, and tromsloeatien of bacteria to mesanterie lymph nodes, blood stream and splanelmie organs wen deletmhaed at (n~ ), g (n ~ ), and (n~ ) hours with the fallowing results: ly~testinai,..transfi". geometric mean of fluorescent marker - % at hour~ with return to % of gut leugth (similar to ~m) at hour~. bacterial overgrowt_hff -increase in total bsctetia ~ad gram negative rod.~ at ,; hours with return to sham control at hours ( log cfu/g veraus log cfu/g ia ileum). translocation to mesenteric nodes - , , and percent at , , and fi hours compared to b in sham controls (n=l of ). conclusion -bacterial translocation following panetoatieobiliary duct ljgation induced panereatitls ha the rat is tighlly linked to intestinal trausit and bacterial overgrowth within the gut lumen. speclrjlation -the ilens in in/~arm~ation may be related to activation of eytokiaes and mediated by nitric oxide. preliminary evidence for this notion will be discussed. in a series of studies we investigated: a) the time course of bacterial translocation (bt), b) the kinetics of lps and cytokine appearance (tnf, il- , soluble tnf receptor [stnf-r] ) in the circulation, and c) the role of lps and/or tnf with regard to haemorrhagic shock (hs) related pathophysiologie alterations and mortality in baboons and/or rats. method: baboons were subjected to femur fracture, soft tissue trauma (acute experiment), and hs ( mmhg mean arterial pressure: map for - h terminated at an accumulated oxygen debt of - ml/kg followed by resuscitation). a chronic model of hs was set up by administration of zymosan activated plasma (zap) before and after hs but without trauma. hs was induced in rats by bleeding the animals to map of - mmhg - rain followed by resuscitation. results and conclusion: in rats, lps increased in the systemic circulation, plasma tnf levels were found to be significantly elevated at min and were still markedly increased at rain postshock. plasma tnf, il- , and stnf-r levels increased already during the shock period in baboons, while tnf was not detectable. our data suggest that haemorrhagic shock may lead to early bt in the intestinal wall (at min following resuscitation in rats subjected to hs for min, similarly at h after the end of oxygen debt period in baboons ) and transient access of gut-derived lps into the circulation (levels became significant at min, while in baboons higher levels up to pg/ml were found at the end of shock and h after reinfusion, partially accompanied by bacteremia). in addition, since the treatment of rats with anti lps measures or anti tnf therapy significantly improved the -hour survival rate it can be assumed that endogenous lps and lps-induced mediator(s), in particular tnf, may lead to organ injury and mortality following haemorrhagic shock. alteration of the mesenteric blood flow and the consequent ischemia / reperfusion injury to the intestine appear to be one of the earliest events in the systemic inflammatory response following severe thermal injuries. the causative relationship between the subsequent disruption of the intestinal integrity and the potentiation of the translocation of indigenous bacteria and/or endotoxins to remote body organs and the systemic circulation has been deeumented in several studies. among other effects, endotoxemia solely or acting synergistically with thermal injuries has been shown to promote bacterial translocation. as these sequences continue without adequate intervention, multiple organ failure and eventually death may become inevitable. hence, the crucial need of treatment modalities with the capacity of modulating the intestinal blood flow and preventing the manifestations of these pathological incidents. although the underlying mechanisms of this process have not yet been fully elucidated, there is accumulating evidence that various interacting vasomediators are involved. the actions of these mediators can probably be modulated by either nonspecific or selective agents. among the nonspecific agents, the composition, volume and timing of the resuscitation fluids appear to play an important role in this process. nitropmsside, a nonspecific vasodilator, has been shown to prevent the decrease of the postbum mesentefic blood flow when selectively infused in the mesenteric artery. the nonspecificity of various vasodialators with their possible undesirable systemic effects emphasizes the importance of the identification and modulation of selective vasomniiators. thromboxane a (txa ) and angiotensin ii (a-ii) appear to be the primary mediators of the postburn vasoconstriction. both mediators were found to be elevated following thermal injuries. in a previous study, pretreatment with oky- , a thromboxane synthetese inhibitor was found to attenuate the postbarn mesenteric vasoconstriction. in a bum/sepsis porcine model the efficacy of dup , an a- receptor antagonist as a posthum treatment modality was evaluated. treatment with dup prevented the early posthum and the late posteodotoxin elevation in the mesenteric vascular resistance and subsequently abrogated the fall in the mesenteric blood flow. the incidence of burn & endotoxin induced bacterial translocation was significantly reduced by dup from % to % (p< . , fisher's exact test). the indigenous flora of the gastrointestinal tract exerts a potent influence on the developmental maturation of normal systemic immunologic responsiveness. moreover, both oral and intraportal administration of experimental antigen is associated with a systemic state of immunologic hyporesponsiveness, known as oral tolerance. since trauma and critical illness are associated with changes in the flora of the gut and with altered systemic immune reactivity, we have hypothesized that the interactions of an altered gut flora with immune tissues in the gut and liver play a role in the pathogenesis of the immunologic derangements of critical illness. prolonged feeding of two clinically relevant killed organisms-pseudomonas and candida-to a rat results in significant impairment of cutaneous delayed hypersensitivity (dh) reactivity. conversely, measures directed against gram negative bacterial overgrowth in experimental peritonitis result in partial restoration of impaired dh reactivity. to ascertain the role of the liver in the pathogenesis of these alterations, we studied the differential immunologic sequelae of portal versus systemic (ivc) infusion of killed bacteria. experimental infusion of live or killed gram negative organisms into the portal vein produces dh suppression in rive, and profound suppression of mitogen-driven lymphocyte proliferation in vitro; systemic administration of the same organism has no effect on these phenomena. suppression is tgf -dependent, and mediated by a soluble factor released by fixed tissue macrophages of the lung and spleen. the suppressive influence appears to be mediated at the level of interactions between il- and its high affinity receptor. release of this factor can be induced by a second circulating factor present in the plasma of portally-, but not systemically-infused animals two hours after bacterial administration. these studies suggest that the release of inducible immunoregulatory factors from the liver, and possibly from the gut, may play a role in the pathogenesis of the profound derangements of systemic immune homeostasis that accompany trauma and critical illness. acad.hospital st. radboud,postbus , lib nijmegen introduction. the central question being tested in this study was whether liposome encapsulated dichloromethylene-diphosphonate (ci mdp) mediated elimination of liver and splenic maorophages would influence zymosan induced systemic toxicity (st) and bacterial lranslocation (bt). materia|s and methods. male swiss (icr) mice were used weighing - g elimination of liver and spleen macrophages was accomplished by intravenous injection of / suspension of ci mdp-liposome suspension. control mice received an i.v. injection with pt phosphate-buffered saline (pbs) . two days later all mice were challenged with an i.p. injection of zymosan suspended in paraffin (z) at a dosage of either mg/g, . mg/g or . mg/g body weight (n= in each group), signs of st and bacterial translocation bt were measured hours later the st was assessed by blindly grading the degree of lethargy, conjunctivitis, diarrhea, and ruffled fur of each mouse on a four point scale. bt to the mesenteric lymph nodes (mln) and systemic organs (liver, spleen, lung and blood) was tested by culturing on blood and macconkey's agar at the time of sacrifice sections of the distal ileum were taken for histology. an additional two control groups (n= in each group) were tested to verify that neither paraffin nor pbs or ci mdpqiposome suspension alone induced bt or st. furthermore survival over a -day period was assessed in a control and macrophage-depleted group with a dose of d mg/g z. results. neither the paraffin nor ci mdp-tiposome suspension induced bt or st the incidence of bacterial translocation to the mln was similar between macrophage depleted and control groups. however, macrophage depletion was associated with a significantly higher incidence of systemic spread of bacteria. data expressed as positive tissues/total tissues tested: / vs. / at . mg/g zymosan (p~o. ); vs. at mg/g zymosan (p~q ); / vs. / st . mg/g zymosan (p~o.o ).the magnitude of bt however did not show any differences. although systemic bt was greater in the macrophage depleted groups, the systemic toxic response was significantly less at doses of . mg/g zymosan ( . -+ . vs. . -+ , p~o. ) and . mg/g zymosan ( . -+ . vs. . -+ . , p<_.o. ) . the day mortality after . mg/g zymosan was % in the control group and % in the macrophage depleted group (p=o. ). histology did not show any differences between the control and macrophage depleted groups. conclusion. the lethal and toxic effects of zymosan in this model appear to be more related to the excessive activation of macrophages than to the systemic spread of bacteria. bacterial translocation (bt) in hemorrhagic shock was studied using a porcine model. in this study three groups were randomized: one control (n= ) and two experimental (n= each). a portal vein catheter was placed and remained in situ hrs. a femoral artery and vein catheter were in place hrs. the arterial eaanula was used for monitoring mean arterial pressure (map) in all animals and to bleed the experimental animals and the venous cannula to repelfuse with ringer's lactate (rl) or autologous blood (ab). baseline samples and values were taken in this period. control animals were observed for a sham shock period of . hrs., given more hours of anesthesia, and studied for further hrs. % of the estimated blood volume was withdrawn from each experimental animal over min. these animals were kept in shock for . hrs. at the end of the shock one group was perfused with rl and the other with ab. two hours later they were extubated and further studied for hrs. samples from portal blood for cultures and measurement of leukotriene b (ltb ), prostaglandin e (pge ), and nitric oxide (no) metabolites were taken from all animals at intervals beyond baseline up to hrs. after hrs. under general anesthesia a second laparotomy was performed and samples for culture from mesenteric lymph nodes (min), liver and spleen were taken; as well as samples for histologic study with light and scanning electron microscopy were taken from jejunum, ileum, and colon. animals were then euthanized. results: significant shock was induced as indicated by map and arterial blood gases. significant bt to min was observed in all groups. no significant bt was observed in cultures from liver and spleen in any of the groups. increased values for ltb , pge and no metabolites were found during the period of shock. these results suggest that the isehemic phenomenon triggers a significant inflammatory response, and that bt to min may be an epiphenomenon without apparent significant influence on the inflammatory response. objective: to study the potential effects and mechanisms of action of systemic administration of monoclonal antibodies anti-endotoxin (ha- a) in a animal model of gut-origin sepsis. materials and methods: exoeriment a. balb/c mice were transfused with allogeneic blood (from c hb-iej mice). five days post-transfusion the animals were gavaged with lxl viable escherichia coil and randomized into groups (n= /group) to receive a sham burn (sb group) or a % thermal injury immediately followed by the systemic administration, via a tail vein, of monoclonal antibodies ( mg/kg) (ha- a group) or a % burn injury and administration of equal volume of sterile saline (c group). all groups were resuscitated by intraperitoneal injection of . ml of saline. the animal survival rate was observed for days post-burn. experiment b, transfusion and burn procedures were identical to experiment a but the mice (n= /group) were gavaged with viable e.coli labeled with mmndium oxine. four hours after burn the mesenteric lymph nodes (mln), liver, lungs and blood were harvested to determine plasma endotoxin levels and the magnitude of transtocation of labeled bacteria as measured by the residual radioactivity (dpm) in the organs. circulating endotoxin levels were determined by limulus colorimetric assay. diamine xidae(dao) is an enzyme existing in the mucosa of small intestines, but its activity is low in the plasma. it is evoked in the blood with the intravenous injection of much heparin. it is well known that da declines under the chronic mueosal injury when the small intestines are cut off or the mucosae become atrophied. aim of this paper is to confirm that da goes up to the measureable level by the intravenous injection of a small amounts of low molecular heparin(lmh objectives: the aims of this study is to investigate the inhibitor" effect of the prolease inhibitor, urinastatin, on endotoxin induced bacterial transleeation in mice. materials and methods:lcr mice were divided in six groups as the fotlows, group i: control mice receiving intraperitoneal injections (ip) of saline . and hr prior to sacrifice, group ih treated mice receiving ip of utinastatin . hr and endotoxin ( mg/kg) hr prior to sacrifice,group ilk sham reated mice receiving ip of saline . hr and endotoxin ( mg/kg) hr prior to sacrifice,group iv: saline control mice receiving ip twice during rain intervals,group v: mice received ip of utinasmtin, and min later they were received ip of endotoxin ( mg/kg), group vi: mice received ip of saline,and min later they received ip of endotoxin ( mg/kg). in group l,ii and ill ,the mice were killed by cervical dislocation.using stetile techniques,a middle-line incision was made and the mesenteric lymph nodes were harvested in order to culture on selective agars for quantitation of bacterial trauslocation. in group iv, v and vi, survival was recorded for days. unnastatin pretreatment could rednce mortality significantly. first surgey, shiga university of medical science, seta ,ohtsu - ,japan increased gut permeability correlates with inflammatory response in multiple trauma aptients. pape, h.-c. dwenger, a. grotz, m. regel, g. purpose: disturbances of intestinal permeability have been advocated as a pathogenetic factor of posttranmatic multisystem organ failure (mof). a close relationship with impairment of the stationary reticulocndothelial system (res) and a systemic inflammatory response (sir) was discussed. these tactors were investigated prospectively in patients with multiple trauma showing posttraumatie complications (multiple organ failure, mof). methods: polytranmatized patients were studied prospectively. according to a mof-score (goris) those with posttrattmatic complications were selected (> points at days), others were excluded. every second day gut permeability according to the ratio of urine concentrations of lactulose and mannitol (l/m) was evaluated (enteral application). at parallel time points res clearance capacity (k-value, invasion constant, normal range . - . mind) was studied after i.v. injection of mbq rotehuman albumin. liver perfusion was calculated from these data, total serum bilirubin (/zmol/l) was documented. serum elastase (#g/l) levels were determined enzymatieally. results . + + liver perfusion did not ehangu, bilirubin showed progressive worsening indicating mof. a positive correlation was present between l/m and k (r= . ) and between l/m and ela (r= . ). conclusions: there is a positive correlation between the time pattern of intestinal permeability dysfunction and res hyperactivity as well as between intestinal permeability and the systemic intlammatory response (elastase levels). the results speak in favor of an interaction between intestinal and extraintestinal inflammatory systems, which in eombiuation are likely to be responsible for post~anmafic complications. endotoxemia, il- release and consecutive acute phase reaction are observed as a host response to surgical trauma. as well vasodilative prostaglandins (pg) and thromboxane (tx) are released after abdominal meaenteric traction (mt). the following hypotension and acute hypoxeraja are duo to prostacyelin (pgiz) arm can be avoided by perioperative cyclooxygenase inhibition. we therefore focused on the effect of pg and tx liberated following mt on the induction of endotoxemia. methods: in a prospective, randomized double-blinded protocol patients, who were scheduled for major abdominal surgery (pancreatic or infrarenal abdominal surgery), were studied. ibuprofen ( mg i.v.) or a placebo equivalent was administered minutes before skin incision. mt was applied in a uniform fashion. baseline values were obtained before induction of anesthesia. further measurements followed before the incision of the peri[onenm (tl) and , , , min, . the plasma concentrations (,pc) of -keto-pgft,, txb: and-ki- -pgf ~ (stable metabolites of pgi , txa and pge~) were determined by ria. we measured endotoxin pc by limulus-amoebocyte-lysate test and il- levels by elisa. data are given as mean+sem (* p< . placebo vs. [ibuprofen] ). results: endotoxin plasma levels increased before incision of the peritoneum tl both in the ibuprofen pretreated and in the placebo group. peak pc were observed minutes after mt. endotoxin pc were significantly higher in the ibuprufen treated group (t . + . e[ . + . ] eu/ml). il- pc demonstrated an increase continuously from t to t (t + [ + ] ng/l) in both groups. after intentional abdominal mesenteric traction we observed a marked increase of -keto-pgf~,, pc up to h after mt in untreated patients with a peak of *[ ] ng/ at tl. also txb: and kh pge pc showed a considerabe increase up to h after mt in the placebo group. in ibuprofen pretreated patients the pg and tx pc remained within the normal range. discussion: our data clearly indicate a significant endotoxemia and il- release following major surgical trauma which is not initiated either by prostaglandin or thromboxane release. moreover endotoxemia is accentuated by ibuprofen pretreatment. therefore we hypothesize that in major abdominal surgery prostacyclin release-after mt may play a crucial physiological role in maintaining splanclmic microcirculation and thus preserving gut mucosal barrier function. objectives of the study it has been shown recently that parenteral and certain euteral diets promote the translocation of gut flora to the mesenteric lymph nodes (mln) and systemic organs, a process termed bacterial translocation (bt). in chow fed rats bt usually does not occur without further promoting factors. the goals of the present study were to determine whether the provision of defined amounts of standard lab chow during iv-tpn administration wotfld redane the incidence of bt, materials und methods male spf spragnle-dawley rats were divided into groups. group received standard laboratory chow feeding ad lib. in group a central venous catheter was placed, ligated and secured by a spring coil tether attached to a swivel allowing free movement in the housing cage and chow was fed ad lib. in group % of the calculated daily required calory intake (drci) ( /kcal/kg) was given by iv-tpn ( % glucose, , % amino acids) and % by limited chow administration. groups and received % and % of the drci by i.v. tpn and % and % respectively by chow feeding. group received iv-tpn only. after days the rats were sacrificed and the mln, liver, spleen and cecum removed aseptically, homogenized and cultured for bt samples of distal ileum were taken for light microscopy. the group with the least amount of chow shown to be protective against bt received the amount of non-fermentable fiber of that chow regimen during iv-tpn feeding and bt was studied. , + , , - , , / + ~ " , -+ , , -+ ~ - , / +~ + _+ , + , , - , -+ + , ~ , , -+ ~ conclusions: the administration of % of drci by chow feeding during iv-tpn significantly reduced the incidence of bt and maintained gut barrier function. the addition of the respective amount of dietary fiber of this group did not prevent iv-tpn-indueed bt. dr. med. m naruhn., dep. of general surgery, eberhard-karls-university, hoppe-seyler-str. previous experimental studies have suggested that a disturbed ecology of the enteric bacterial population might contribute to the development of bacterial translocation from the gut in acute liver failure (alf). in the present study, the effect of oral administration of lactobacillus reuteri r lc and oat fiber on bacterial overgrowth and translocation was investigated in rats with acute liver failure induced by subtotal ( %) liver resection. the oatmeal soup base was anaerobically inoculated with lactobacillae and fermented for hours, after which the animals were fed with either fermented or unfermented oatmeal or saline daily for days prior to the operation. bacterial translocation to mesenteric lymph nodes (mln) and the systemic circulation was determined, as well as the intestinal bacterial flora and enterocyte protein content. the incidence of bacterial translocstion to the systemic circulation was nit in rats subjected to sham operation and saline treatment and % in animals subjected to % bepatectomy and lreatment with fermented oatmeal, while - % and - %, respectively, in rats subjected to hepatectomy and treatment with either saline or unfermented oatmeal. only one rat with fermented oatmeal demonstrated bacterial growth in mln (p < . vs hepatectomy and treatment with saline or unfermented oatmeal). the enterocyte protein content significantly decreased (p < . ) in salinetreated animals following % hepatectomy, while there was no significant difference between bepatectomized animals with oral administration of fermented or unfermented oatmeal. the number of anaerobic bacteria, gram-negative anaerobes and lactobacillus significantly decreased and the number of e.cnli increased in the distal small intestine and colon in hepatectomized animals with enteral saline or unfermented oatmeal as compared with animals subjected to sham operation or bepatectomy with fermented oatmeal. our results thus show that the occurrence of bacterial translocatiou from the gut in % hepatectomy-induced alf could be prevented by enteral administration of fermented oatmeal, maybe partly due to a positive effect on the enteric bacterial ecology. _+ " +_ " . " data=mean_+sd, * stats anova p< . vs control. l+air and lap groups, both exposed to exogenous i.ps shnwm:t m significant increase (p<. ) in lps gut translocation compared to control and l+co . this correlated with a significant increase in peritoneal inflammatory responses (o -,tnf) above that of the control and l+co groups, while mac- and cr opsonized phagocytosis were significantly impaired. the absence of significant differences between l+air and lap groups indicates that lps rather than wound factors is the principle mediator. thus, lps plays a significant role in regulating peritoneal responses in the early post-operative period dept of surgery, rcsi, beaumont hospital, dublin , ireland brlke e, berger d, staneseu a, buttenschsn k, vasilescu c, seidelmann m, beger hg in patients undergoing a colonoscopy, endotoxin, endotoxin neutralizing capacity (enc), thromboxane b o (stabile metabolite of tbmomboxane ~), -keto-prostaglansin, leueotriene c , interleukin and the incidence of bacteremia were determined before and then every five minutes during the procedure. twenty-one of patients showed a significant increase of endotoxin plasma levels during colonoscopy (p= . ), whereas only one patient had a positive blood culture with bacteria obviously derived from the gastro-intestinal tract. the enc decreased significantly five minutes after the beginning of eolonoscopy and was diminished further thereafter. the baseline values were reached after hours. ~hromboxane b o levels also increased after five min. from to pgyml peaking at min. with pg/ml. -keto-prostaglandin,leucotriene c , ii- and crp remained unchanged. a control group of i volunteers who were not subjected to endoscopy, were prepared for eolonoscopy by orthograde lavage. the blood sampling procedure remained identical. no differences were seen in all described parameters for the controls. these data show that the gut barrier can be compromised by mininml invasive procedures, at least, concerning bacterial products. living bacteria, on the contrary, do not pass the gastro-intestinal wall. endotoxin, when determined by enc, is more sensitive than the conventional limulus-amebocyte-lysate test. no acute-phase reaction was induceri by the observed endotoxin translocation. it can be speculated from the dramatically enhanced thromboxane b levels, together with its hemodynamie effects, that the thromboxane release may support translocation of bacterial products. sepsis is common after hemorrhagic shock. this study aims to demonstrate that hemorrhagic shock alone can promote translocation of gut bacteria from intestinal tract to its regional nodes and subsequently to blood. one hundred twenty mice, divided into groups were subjected to , and minutes of %, % and % of hemorrhagic shock. on the specified time, blood cultures were taken and mice were sacrificed. the intestinal tract were histologically examined for any changes which allows translocation and its regional nodes were quantitatively cultured for translocated bacteria. there was a direct relationship between duration and degree of hemorrhagic shock and incidence of translocation (p . ). there was a high incidence of gut bacterial translocation to the mesenteric and mesocolic nodes in all degrees of shock (p . ). bacterial growth in the regional intestinal nodes increased and blood cultures were positive in direct proportion to degree and duration of shock. histologic evaluation of segments of git showed submucosal congestion to allow bacteria normally contained within the gut to cause systemic infections. translocation of gut bacteria in untreated hemorrhagic shock is clearly shown in this study on animal models. in this study, guotobiotic rats with known species of bacteria were subjected to total parenteral nutrition(tpn) and subsequent hemorrhagic shock. the purpose of the study was to observe the impairment of gut barrier function following tpn and hemorrhagic shock and to study the mechanism of enterogenic infection induced by tpn and shock.the results were as follows: .long term( - days) tpn induced impairment of gut barrier function, evidenced by atrophy of intestinal mucosa, significant decrease in diamine oxidase activity of intestinal mucosa and blood, and marked microecologic imbalance of the intestinal mucosa flora with dorminant growth of aerobes and relative decrease in anaerobes. the degree of mucosal damage were proportional to the duration of tpn. .in tpn+shock groups, failure of gut barrier function was found. ri,~ere were further damage in the mucosa, with a large number of gramnegative organisms invading mucosa and submucosa and a significant decrease in dao activity as compared with each relative tpn groups. these changes were significantly correlated with enhanced bacterial translocation, elevation of lps and mda levels in the plasma. these findings suggested that long term standard tpn impaired the gut barrier function, precipitating posttraumatic gut barrier failure. thus infec. fion following shock might be oi'iginated from the gut and it was obviously related to the impaired gut defence resulted from antecedent tpn. the determination of plasma dao activity might provide a valuable tool for the ear. ly diagnosis of gut injut;y during tpn and after trauma. in our earlier studies we have investigated the dynamics of granuloayte infiltration of the ischemic/reperfused s~all intestine (g. illy~s, j. hamar int. j. exp. athol. . . .) . there was a increasing infiltration of the mucosa c m~nating at the d to th hours of reperfusion. in the present series we have studied sc~e of the conseqn/ences and the possible role of this cellular reaction. ~in isehemia was followed by a hour reperfusion in the anesthetized rat. arterial ~/ad mesenteric venous blood samples were collected at m_in, i, ~ , and hours of reperfusion. elastase and lactate concentrations were determined and hamoculture was carried out from the blood samples, and tissue pieces from the heart, lung, liver and kidney were collected for histological analyses at the above mentioned times of reperfusion. all blood samples were free of cell bacteria. staphylococci appeared only occasionally at the th hour in the arterial blood .and at the d and th hours in the venous blood, respectively. arterial and venous elastase activities were high throughout the reperfusion, venous concentrations being higher at all times. lactate concentrations of the arterial and mesenteric venous blood samples increased during shock. ~ranuloeyte infiltration of all organs studied appeared during the d hour and it increased at later times of reperfusion. it is concluded that heavy infiltration of the intestinal mucosa can block bacterial translocation in most of the cases during reperfusion. granulocytes activated either by the reperfused area or by the released cytokines infiltrate other organs contributing by this way to the mesenteric shock s!rndrc~e. intestinal motility plays an important role for maintaining nutrient transport and absorption and for balancing the enteric bacterial population. disturbances of intestinal motility may be one of the earliest notable changes in intestinal function. in the present study, we aimed at determining early alterations in intestinal transit time following ischemia-reperfusion injury induced by occlusion of the superior mesenteric artery in the rat. intestinal ischemia was induced for and minutes by applying a microvascular clip on the superior mesenteric artery followed by reperfusion , and hours after clip removal. intestinal transit time was measured by the propulsion of a radiolabelled solution (cr ). light microscopy was performed on intestinal samples. macroscopical pathological changes were not observed. however, microscopically, mucosal epithelial oedema, degeneration or slight ulceration occurred in rats hours after reperfusion in ischemia- rain group and and hours after reperfusion in the ischemia- rain group. delayed small intestinal transit time was seen from hours and on after intestinal ischemia for both and rain ischemia followed by reperfusion. the distribution of radioactivity demonstrated that most radioactivity was accumulated in the first two segments following intestinal ischemia and reperfusion, significantly differing from what was seen in animals subjected to sham operation (p < . ). the distribution of radioactivity in segments and in the group with repeffusion hours after intestinal iscbemia for rain was significantly higher than that noted in the group with repeffusion hours after intestinal ischemia for min (p < . ). q'he results indicate that a delayed intestinal transit time may be one of the earliest pathophysiological alterations noted, associated with duration of gut ischemia, and a potential factor for the development of bacterial overgrowth, gut barrier failure and bacterial translocation, in hypovolemic conditions. bacterial infections still constitute a major cause of morbidity and mortality in patients with acute liver failure. the present study aimed at evaluating the effect of ethylhydroxyethyl cellulose (ehec) on bacterial translocation following surgically induced acute liver failure. acute liver failure was induced by subtotal hepatectomy ( %) in the rat. water-soluble ehec was administered orally and hours prior to hepatectomy. the incidence of bacterial translocation from the gut to mesenteric lymph nodes (mlns) and systemic and portal circulation was evaluated and the number of isolated bacteria from these samples and from intestinal content were determined. intestinal transit time, bacterial adherence onto the intestinal surface, intestinal mucosal mass, bacterial growth and dna synthesis, bacterial surface characteristics (hydrobiology: hydrophobicity, hydrophilicity and neutrality; surface charges: positive, negative and neutral) were also determined. hepatectomized animals showed a - % translocation rate to mlns or blood and hours after operation, while only - % of rats subjected to sham operation or animals with % hepatectomy and pre-treatment with ehec (p < . ). bacterial overgrowth, increased bacterial adherence onto the intestinal surface as well as decreased intestinal mucosal masses were observed in animals with subtotal liver resection alone, alterations that were prevented by enteral ehec treatment. a delay in intestinal -hour transit time occurred in both groups with subtotal liver resection, with or without enteral ehec. ehec inhibited bacterial growth and dna synthesis, and altered bacterial surface properties following hour incubation with bacteria. in conclusion, the findings in the present study imply that ehec alters enterobacterial capacities for metabolism, proliferation and invasion by effects on e.g. bacterial surface characteristics. furthermore, ehec seems to possess a trophic action on the intestine, rather than exerting its effect by enhancing intestinal motility. department of surgery, lund university hospital, s- lund, sweden disturbances in intracellular calcium signalling can potentially result in impairments of cellular responses vital to the functional integrity of both immune and non-immune cells, and thus contribute to a decrease in host resistance against infection and to multiple organ system failure during sepsis. studies in our laboratory have focused on assessments of intracellular ca ÷ regulation and ca~+-depended cellular responses in the liver, skeletal muscle and splenic tlymphocytes harvested from rats subjected to gram-negative intraabdominal sepsis. cytosolic ca + concentration, [ca *]i, and ca + fluxes were measured by the use of fluorescent ca + chelating dyes (fura- or indo- ) and ca respectively. to assess sepsis-related changes in ca + dependent cellular responses, we measured the acute phase protein response in the liver, the regulation of protein and sugar metabolism in the skeletal muscle, and the proliferation response in the splenic tlymphocytes. altered ca + i signalling with sepsis was correlated with an exaggerated inappropriate acute phase protein response ( % ¢) in the liver, and a blunted insulin mediated sugar utilization ( % ) and increased proteolysis ( % ~) in the skeletal muscle. in t-lymphocytes, a decrease in mitogen induced elevation of [ca +]i by - % was correlated with a significant depression in their proliferative capacity. these studies clearly suggest that altered calcium signalling is correlated with disturbances in cellular responses in both immune and non-immune cells during sepsis. the altered cellular responses adversely effect the outcome of the septic injury. (supported by nih grant gm ). alfred ayala, ping wang and irshad h. chaudry. changes in macrophage capacity to respond to foreign pathogens are thought to be central to the developing immunosuppression associated with traumatic injury. in this respect, the suppression seen in m~ functions following hen (a common component of traumatic injury) may be mediated by the direct or indirect inhibition of their capacity to perceive external stimuli (e.g., opsonized & non-opsonized bacteria, and their cellular components, etc.} due to the breakdown of the receptormediated signal transduction system. results of a number of studies by our laboratory and others indicate that this inability to respond to external stimuli is in part due to the loss of cell surface receptors. decreases have been documented for not only la antigen, but also c b, fc, and tnf receptors following hem in mice. furthermore, studies which have examined second messenger generation in these cells indicate that m~ derived from the peritoneum and spleen exhibit a decreased capacity to mobilize ca + from intracellular stores. this protein kinase dependent process of [ca+ ] i mobilization appears to be linked to the inability to synthesize inositol triphosphate. of interest, the depression in ca + signal generation appears to be inversely related to presence of elevated levels of camp in m~ from hen mice. we have reported that m~ priming agents, such as ifn- (which exhibits salutary effects on m~ function following hem), appear to restore cell signal transductive capacity while reducing the levels of camp. nonetheless, the extent to which depressed receptor signal transduction in hem, is due to receptor loss~dysfunction or elevated antagonistic second messenger levels remains to be determined. conclusions: significant impairment of calcium signaling occurs at all time-points prior to and following pha stimulation in trauma patients. tcell activation failure can, in part, be explained by the inadequacy of this essential intracellular second messenger system. restoration of immunocompetence following trauma will have to address strategies to better assess and restore this vital step in the activation sequence leading to proliferation during the antigen recognition process. patrick a. bseuerle institute biochemistry, albert-ludwigs-university, hermann-herder-str. , d- freiburg, germany the active form of the transcriptional activator nf-~b is a heteredimer composed of a and kda polypeptide. in this form, nf-'<b can bind with high affinity to decameric sequence motifs (consensus: "-gggpunnf'ypycc- ") found in enhancers and promoters of many genes activated upon a wide variety of pathogenic conditions, including viral and bactedai infections, acute phase response, oxidative stress and uv and gamma irradation. clinically important target genes for nf-k:b encode inflammatory cytokines (il- b, tnf, [l- , il- , gm-csf etc), cell adhesion molecules (icam- , elam- , vcam- ), acute phase proteins and immunoregulatory ceil surface receptors. in most cell types, nf-~:b is sequestered in the cytoplasm and, therefore, unable to initiate transcription of these genes. the cytoplasmic form of nf-~:b contains one additionai subunit, referred to as h,;:b. i~b can reversibly suppress dna binding and nuclear uptake of nf-~b by virtue of its association with p . upon stimulation of cells, ikb is proteolytically destroyed, allowing nf-kb to enter the nucleus and activate genes upon binding to transcriptional enhancers. we have demonstrated that various antioxidative compounds inhibit the activation of nf-~b in response to many inducers. moreover, nf-~:b was shown to be activated upon treatment of cell cultures with p.m-amounts of hydrogen peroxide. these data suggested that nf-~b prinicipaliy is an oxidative stress-responsive transcnption factor. the notion is supported by numerous reports on the induction of oxidative stress by many nf-nb-inducing agents. nf-~b activition can likewise be suppressed by protease inhibitors. these drugs apparently inhibit a yet unidentified protease responsible for the inducible decay of inb. we propose to use nf-nb inhibifors as novel drugs with a very broad application under acute phases el inflammation, injury and infection. northern blot experiments revealed that expression of the recently discovered lipopolysaccharide binding protein (lbp), a / kd glycoprotein, in the liver rises substantially during the acute phase response. experiments with an in-vivo acute phase model using new-zealand-white rabbits and also in-vitro experiments employing stimulated hep-g cells showed that lbp transcripts could be induced to ford within hours after induction with cytokines. by using a newly established nonradioactive nuclear run-on technique we show that induction of lbp during the acute phase is transcriptionally regulated. furthermore we screened a human genomic library and were able to clone the lbp gone, containing the promoter . kb upstream. several liver-specific and "acute-phase-typical" potential binding sites were found fn the promoter region and reporter gone assays were set up. several caat-boxes, the il- responsive elements hstf-hsp . and h-apf- rs as well as the transcription factor hnf- and the glucocrticoid-responsive elements oct- -d and gcre were found, which correlates with the induction pattern of lbp mrna in hep-g cells by il- , il- and dexamethasone. pcr-based analysis of the exon-intron pattern of the lbp gene revealed similarities with the genes of two other lps binding proteins, namely bpi and cetp. further analysis of this novel acute phase protein, that also has an important role in endotoxin recognition and immunomodulation will help in understanding the complex acute phase mechanism and potentially lead the way to new therapeutic strategies. lps activation of nucle?/r fa-ctor:~b " (nf:~bj"in cd .transfected fi'broblasts does not appear to require tyrosine kinase activity d. t. golenbock, r. delude, r. savedra, s. vogel and m. j. fenton lipopolysaccharide (lps) is the major constituent of the outer leaflet of gram-negative bacteria. during the course of serious infection, shock may occur as a result of the interaction of lps with macrophage lps receptors. cd , a kda glycosyl phosphatidylinositol (gpi)-linked protein, functions as an lps receptor. a critical issue in lps activation concerns the mechanism by which cd , which has no transmernbrane domain, transduces a sig,nal after lps binding. evidence exists which suggests that cd may signal cells after lps binding by interacting with an lps signal transducer with tyrosine kinase (tk) activity. wild-type chinese hamster ovary fibroblasts (cho) normally do not respond to lps, but can be activated following transfection with cd (cho/cd ). stimulation of cho/cdi with as little as ng of lps/ml resulted in the release of h-arachidonic acid ( h- : ) into the culture supernatant. in addition to h- : release, we examined lps-imiuced activation (transl cation) of the transcription factor nf-~b. similar to lps-induced h- : release, these responses were observed only in cho cells lransfected with cd closely resembling the same response in the marine macrophage cell line raw . . maximum nf-~cb expression occurred at minutes. in contrast to lps-induced h- : release, dexamethasone, cycloheximide, and actinomycin d had no effect on activation of nf-r,.b in cho/cd , suggesting that nf-r,b activation begins early after lps binding to cd and does not require transcription or translation. we then examined cells for lps-induced tk activity by western blotting cellular ly~ates with anti-phcsphot-'rosin:~ anl',bodie~" although tk activity was seen in lps-stimulated raw cells and phorbol ester stimulated cho/cd , lps-induced tk activity in cho/cd was not observed. although the tk inhibitor herbimycin inhibited the release of h- : in cho/cd and raw cells, neither herbimycin nor genestein effectively blocked nf-r,b activation in either cell type. these results suggest that tk activity is involved in a portion of the signaling pathway that is distal to initial signal transduction. the absence of observable lps-induced phosphotyrosine residues in cho/cd cells as well as the failure of tk antagonists to inhibit lps-induced nf-~cb activation suggest that the proposed cd -related lps signal transducer in cho/cd is not a tymsine ldnase. dimished cardiac inotropic function and response to -adrenergic receptor ( -ar) stimulation are frequently seen in septic patients, these cardiac defects are also seen in animal models of entoxemia. to determine the characteristics of the decreased transmembrane signal associated with the decreased beta adrenergic response we measured the force and rate of contraction of atria harvested from sprague-dawley rats exposed to endotoxin ( : gm/kg). to stimulate various components of the transmembrane signal cascade of -ar was isoproterenol (i -ar), acetylcholine (m ach receptor), naf(gs and gi proteins), forskolin (adenylate cyclase), and calcium (intracellular contracttile signal) were used. to eliminate the transmembrane control of calcium and test the intrinsic contractile response, hypothermia ( °c) was used the results showed alterations in the inotropic function with no significant difference in chronotropic response. the inotropic reponse for the endotoxin exposed atria and controls is shown below these results show that there is a transmembrane signal defect both for the i~-ar signal and for ca +. these data indicate that the level of the defect appears to be at the g proteins. ischemia-reperfusion of the rat intestine produces an injmry both to the intestine and to distal organs, notably the lungs and liver. prior studies have shown that h of intestinal ischemia leads to a nemtrophil independent reperfusion injury of the gut. thus, rats rendered neutropenic by pl~l antiserum have been shown to express a severe local gut injury when subjected to ischemia and reperfusion. this gut injury is postulated to he mediated at least in part by the complement system. the soluble (s) cri receptor which binds to c b and cdb was given to rats at a dose of mg/kg by intravenous bolus, min before reperfusion of the ischemic gut. a control group was given pbs buffer. at h of rmperfmsion the animals were sacrificed. pbs treatment led to a significant activation of both classical and alternate complement pathways while scri inhibited both pathways (fall to zero of chbo ). intestinal mucosal injury score, assessed by histological grading was reduced by scri ( . ± . to . _+ . ,p< . ) . intestinal neutrophil sequestration estimated by assay of myeleperoxidase was also reduced ( . ± . to . ± . u/g). c was detected in the gut hy i~unohistochemistry. remote organ injury was modified that is lung permeability (ration of concentration of radiolabelled albumin in broncho-alveolar lavage fluid to that in blood) was reduced ( . ± . x i to . ± . x , p<o. ). however, neutrophil sequestration by the lungs was unaffected. this remote protection is thougth to be related to prevention of ic b deposition on imng andotheli~ which activates adherent neutrophils. these findings support the hypothesis of complement mediated local and remote injury. we postulate that complement fragments may be deposited either as a result of the ischemia induced loss of membrane bound complement regulatory proteins, such as decay accelerating factor or membrane cofactor protein, which allow complement fragments to accumulate on the endothelial cell surface. alternatively, increased expression of p-selectin on the endothelial cell surface may, by means of its complement binding domains, act as a lattice for complement deposition. indeed, p-selectin has been detected on the surface of endothelial cells ans platelet-neutrophil aggregates recovered from the intestine of these animals. ischemia ( hr) followed by reperfusion (dhr) of rat hind limbs resuits in local (muscle) injury as well as in remote (lung) injury, which is characterized by increased vascular permeability (leakage of j sl labeled albumin) and neutrophil accumulation (tissue content of myeloperoxidase, mpo). within minutes of reperfusion following ischemia, tumor necrosis factor-o~ (tnfc , interleukin-i ( l-i) and il- plasma levels increased significantly, reaching maximum levels after hours ofreperfusion. polyclonal antibodies to tnfet and l-i provided significant protection against muscle and lung injury. muscle and lung vascular permeability decreased in anti-tnfct treated rats by % and % respectively and mpo was reduced by % and %. antml- yielded a protection in muscle and lung vascular permeability of . °, and . % respectively whereas muscle mpo was reduced by . % and lung mpo by . °, . these results were confirmed by the use of soluble tnf~ receptor and il-i receptor antagonist (il-ira). when icam-i expression in vivo was examined using mabeled antmcam-i, constititive expression of icam- was evident only in lung but not in muscle. during reperfusion icam- expression increased by . %. treatment with anti-tnfcx or il-ira reduced icam-i upregulation by . °, and . % respectively. frozen sections of normal rat lung revealed no evidence of e-selectin expression, whereas lung sections obtained after ischemia and reperfusion immunhistochemically demonstrated expression of e-sdectin. lungs from rats pretreated with anti-tnfct were negative for e-selectin staining. our data indicate a role for cytokines in the upregulation of adhesion molecules in ischemia/reperfusion injury. adherence of neutrophils to the coronary endothelium is associated with significant myocardial injury following min of ischemia (mi) and . h of reperfusion (r). p-selectin is expressed by activated endothelial ceils and platelets within min and tethers polymorphonuclear leukocytes (pmns) to the endothelium. we examined the cardioprotective effects of a monoclonal antibody (mab) designated pb . to p-selectin in a feline model of mi+r. pb . ( mg/kg) administered rain post-occlusion ( min prior to reperfusion) significantly attenuated myocardial necrosis compared to a non-blocking mab (nbp . ) for p-selectin ( + vs + % of area-at-risk, p< . ). endothelial dependent relaxation to acetylcholine was also significantly preserved in ischemic-reperfused coronary arteries isolated from cats treated with pb . compared to nbp . ( + vs + , p< . ). furthermore, pmn adherence to ischemic-reperfused coronary endothelium was significantly attenuated in pb . treated cats ( + vs + pmns/mm z, p< . ). also, normal coronary endothelium activated with thrombin ( u/mt) had increased pmn adherence under conditions of shear stress, an effect which was significantly (p< . ) blocked by pb . , but not by nbp . . furthermore, p-selectin is markedly upregulated - min post-reperfusion in coronary arterial and venous endothelinm. similar results were obtained in a rat model of mesenteric ischemia/reperfusion, including upregulation of p-selectin on mesenteric vascular endothelium. these results demonstrate that tethering of neutrophils to endethelium by p-selectin is an important early consequence of reperfusien injury, and a specific monoclonal antibody to p-selectin exerts significant endothelial preservation and cardioprotection in ischemia and reperfusion states. as recent interest has increased in the role of extracellular matrix proteins in inflammation, we examined the role and relationship of laminin and fibronectin to the processes of cell infiltration in a rat model of heart isografts. changes occurring in this system are on a nonimmunological basis and may be an effect of the initial surgical manipulation and the warm and cold ischemic times of transplantation. lewis (lew) donor hearts were transplanted into lew recipients, naive lew acted as naive controls (nc). grafts were harvested , , , , , , , and h after transplantation (n= /time point) . snap frozen sections of organs were stained for cd- (w / ), cd- (ox ), t-lymphecytes (tl) (ox- ), macrophages (ed , ed ), neutrophils (pmn) (mom), icam-i (iap ), laminin and fibrinogen. results were evaluated visually by counting cells/field of view (cf) for w / , ox , ox , ed , ed and mom, staining for icam , lain and fib was assessed visually using a scale (ve= - ). as early as h after transplantation, fibrinogen and laminin expression increased in i, peaking h after transplantation (ve= . ; nc: ve=i. ) . at and h in i, on both vessels and interstitial cells laminin and fibrinogen expression had declined to baseline (ve=i. ) but rose again at h, peaking at h (ve= ) and remaining elevated thereafter (ve= ). the high expression at h correlated well with the maximal pmn infiltration at that time (cf= ) in i returning to baseline at h (cf= . ) and thereafter. the second peak at h correlated with the onset of macrophage and t-lymphocyte infiltration. thus, laminin and fibrinogen seem to guide leucocyte infiltration following graft ischemia during the time of reperfusion. background. reperfusion of ischemic kidneys with xenogeneic blood leads to activation of endothelial cells and circulating leucocytes with the final consequence of microvascular thrombosis. in concordant systems, the mechanisms of rejection can be studied due to cross-reactivity of mediators with anti-human monoclona~ antibodies. aim of the study. to obtain information about the kinetics of proinflammatory cytokines and production of soluble adhesion molecules in the acute phase of reperfusion, eight kidneys from rhesus monkeys were perfused ex-vivo with human blood (group b/o) for one hour in a closed system. blood levels of il-lb, il- , tnf-a, soluble icam and e-selectin were measured in elisa-technique under steady-state conditions. results. cytokine levels rose significantly within the minute interval (il-lb: , __+ , to , __+ , pg/ml; il- : . - , to , __+ , pg/ml; tnf-a: , __+ , to , + , pg/ml; p< . ). immediately after the beginning of reperfusion, soluble icam- and selectin levels were abnormally high and constantly rising throughout the observation period, reaching significance at minutes. discussion. high levels of proinflammatory cytokines may lead to an induction of adhesion molecules, thus upregulating the leukocyte-endothelial interaction in an complement-independent mechanism. specific pretreatment with monoclonal antibodies against icam- , lfa- or other soluble mediators may be useful in downregulating hyperacute rejection in transspecies transplantation. prolonged ischemia has been associated with later dysfunction of solid organs as it may cause upregulation of adhesion molecules, production of cytokines on vascular endothelium and migration of host cells into the graft. these early events may lead to irreversible organ injury. this study evaluates the effects of global ischemia on early immunohistological changes in cardiac grafts transplanted in rats. isografted hearts (lewis->lewis) were were divided into ischemic and non-ischemic groups (n= /group). all donor hearts were flushed immediately with cold saline. non-ischemic hearts were then transplanted within rain, ischemic hearts were stored in cold ringer's solution for hours before revascularization. representative grafts were removed after . , hrs, and days, and evaluated immunohistologically (cells/field of view=c/f). restitution of ventricular activity was significantly delayed in ischemic grafts ( vs rain). after hrs, all ischemic grafts exhibited an extensive interstitial edema, declining slowly thereafter. at the same time, numbers of pmn peaked ( vs c/f in non-ischemic grafts), whereas edl+macrophages ( vs c/f) and tnfe expression peaked by hrs. by hrs t-lymphocytes began to enter ischemic myocardium and icam- was moderately increased. after days cellular infiltration had returned to baseline, and no differences were seen among both groups after days. global myocardial ischemia inhibits initial graft function, and engenders a brisk inflammatory reponse, primarily pmn and macrophages, with increased mhc class ii and cytokine expression. leukocyte -endothelial interactions are the result of endothelial activation, leukocyte activation or combination of both, which are accompanied by nee-expression, upregulation or shedding of adhesion molecules (selectins, inlegrins). such interactions differ with regard to the stimulus (e.g. thrombin or histamine for p-selectin, endotoxin or tnf/il- for e-selectin), the time course of response (minutes versus hours) and the localisation in different organs. recently assays are available for circulating soluble fragments of the cell bound adhesion molecules e.g. se-seleetin was found to be increased in plasma concurrent with high circulating endoloxin and cytokine levels. the importance of adhesion molecules for the sepsis event is evident, while effectiveness of anti-adhesion inolecu]e therapy is controversial e.g. beneficial anti-e-selectin therapy in baboon bacleremia but deleterious effects of amti-cd treatment in the same model. in other species similar controversial results with anti-cd therapy in sepsis were reported. steven l. kunkel,theodore standiford* and robert m. stricter. the migration of leukocytes to the lung during endotoxemia is dependent upon the coordinated expression of lung vascular adhesion molecules and the subsequent production of appropriate leukocyte chemotactic proteins. in experimental animals, neutrophils accumulate within the lung soon after the administration of endotoxin, while mononuclear cell infiltration occurs in a more distal manner. a kinetic analysis of lung leukocyte levels revealed a -fold increase in neutrophil numbers associated with dispersed lullg tissues hours after lps treatment, while macrophage levels increased by -fold at the hour time point. thus, the recruitment of different leukocyte populations to the lungs during endotoxemia is likely directed by different mechanisms. recent studies have identified a supergene family of small inducible chemotactic cytokines (chemukines) which possesses chemotactic and activating properties for neutrophils. the prototype of this family is interleukin- (il- ). interestingly, a related supergene family has been identified which possesses activity for recruiting mononuclear cells. examples of this group of inflammatory chemukines are monocyte chemotactic protein-i (mcp-i) and macrophage inflammatory protein-i alpha (mip-i). in initial in viva studies we examined whether mip-i was expressed systemically or in a compartmentalized fashion post lps challenge. assessment of plasma cytokine levels revealed maximal tnf levels occurred i hour post lps administration, returning to baseline by hours, while mip-i levels were maximal at hours ( , ng/ml), with a second peak at hours after lps challenge. interestingly, aqueous extracts of liver homogenates from lps treated animals demonstrated no mip-i levels, while aqueous extracts of lung revealed a -fold increase in mip-i levels over control lungs. immunohistochemical analysis of the lungs from hour lps treated animals demonstrated the alveolar macrophage was a rich source of mip-i protein. cell-associated mip-i was also expressed by blood monocytes adherent to the pulmonary vascular endotheliun, however the expression of monocyte-mip-i was observed by hours post lps administration. immunohistochemical analysis also demonstrated that mip-i antigen is associated with the extracellular matrix on the interstitial side of the endothelium. this suggests that the extracellular matrix, which is produced during inflammation, can bind mip-i and this may serve as a depot for the prolonged presence of nip- . in additional studies we have demonstrated that the intratracheal instillation of rmui [ip-l(loong) activation of polymorphonuclear leukocytes by inflammatory stimuli may contribute to the development of multiple organ failure in septic patients. thereby pmnl are proposed to avidly adhere to vascular endothelium causing damage by the subsequent release of toxic agents. as cellular adhesion is primarily mediated by -integrins and lselectins, the present study compares the expression of these adhesionmolecules on pmnl in septic patients and healthy volunteers. methods: expression of -integrins and l-selectins on pmnl was measured in whole blood by flow cytometry using the monoclonal antibodies ib and dreg , baseline values were determined immediatley after drawing blood. in addition cells were incubated min at °c to allow for spontaneous regulation of adhesion molecules. blood specimens from septic patients were obtained during the course of their illness. control values were determined in healthy volunteers. results: baseline expression of -integrins and l-selectins was not signifcantly different in septic and in healthy subjects. in contrast, there was a significant upregulation of g -integrins and shedding of l-selectins of pmnl in septic patients (sp) compared to healthy volunteers (hv). the local or systemic production of inflammatory cytokines, such as tumor necrosis factor alpha (tnfc~), can serve to modulate multiple aspects of neutrophil function. the ability of neutrophils to leave the circulation and migrate to areas of infection is one essential component of host defense. l-selectin, a leucocyte-associated adhesion molecule, is responsible for the initial reversible contact between neutrophils and endothelium and the subsequent roiling action of neutrophils along the vessel wall. in contrast to other adhesion molecules, l-selectin expression is rapidly down-regulated after neutrophil activation. the loss of l-seleclin may thus be a critical determinant of how neutrophils become unbound from their endothelial attachments and enabled to proceed towards an underlying extravascular area of infection. we hypothesize that the shedding of l-selectin is a strictly controlled process, occurring primarily at localized sites of inflammation, which may be modulated by tnf~, a flow cytometric method of staining neutrophhs by monoclonal antibodies in whole blood is described whereby the kinetics of l-selectin shedding may be followed in real time. the dose response and time course of in-vitro l-selectin shedding by neutrophils from normal human subjects was assayed after exposure to n-formyl-methionylleucyl-phenylalanine (fmlp) and tnfc~. either singly or in combination, our results show that l-selectin shedding can be reliably followed over time. a significant percentage of cells shed l-selectin after exposure to pg/ml tnfc~ or nm fmlp (but not at pg/ml tnfc~ or nm fmlp). greater numbers of cells were able to shed their l-selectin when fmlp and tnf~x were presented in combination rather than alone. high levels of tnfc~ did not appear to alter the threshold concentration of fmlp required to induce shedding, we conclude that the extent and rapidity of l-selectin shedding may be modified by different combinations of ligands and that shedding, by vidue of the high concentrations of cytokines or chemotactic factors required, is a process localized to sites of infection or inflammation. we prospectively studied patients with severe sepsis syndrome; group a : septic shock with or without adult respiratory distress syndrome lards) (n = , bacteremia = ); group b : sepsis syndrome without septic shock (n = , bacteremia = ). serial plasma samples obtained on day , , , , and , were assayed using elisas method (british biotechnology), normal control levels of soluble icam- and e-selectin, obtained from healthy volunteers, were respectively ± . ng/ml and ± . ng/ml (mean _+ se), acute lung injury was quantified dally on a tour-point score system (murray, am rev respir dis, ) . compared to control mean values, initial levels of groups a and b were significantly higher for icam- (p < - ) and e-selectin (p < - ). comparisons of group a and [] (* = p< . ; ** = p< . t) soluble icam- levels of group a enhanced significantly (p< . ) during the first hours, and a sustained high levels was of bad prognosis ( % of survivors at day ). the evolution of soluble icam- and e-selectin levels were significantly correlated with murray's score (spearman test : p < . ). conclusion: these results suggest that endothelial adhesion molecules are released into the plasma of patients with severe sepsis syndrome. soluble icam- and e-selectin are correlated with endothelial lung damage, and loam- seems to be a better indicator of the severity of endothelial injury. introductory remarks to anti-adhesion molecule strategies as a therapeutic modality ch wortel, repligen corporation, one kendall square, building , cambridge, ma , usa. the development of antimicrobial therapy represented a major breakthrough in the struggle against disease. it strengthened the notion that disease could be overcome by eliminating foreign invaders threatening the host. this paradigm has proven to be very successful, the threat of many infectious diseases has significantly changed, some have even been eradicated. nevertheless, sepsis has remained a severe condition, increasing in incidence while mortality remained very high. more recently, it has become increasingly clear that besides the nature and treatment of an exogenous agent, the reaction of the host defense itself plays a pivotal role in the outcome of the event. endogenous mediators, such as tnf, il-i, il- and il- , govem many of the actions of the host defense system. while the expression of these cytokines more often than not benefit the host, (over)-expression can cause severe damage. based on this hypothesis,anticytokine strategies, such as those targeted against tnf or il- , have been evaluated for the treatment of sepsis. results of these early studies have not yet indicated success in improving the outcome of the disease. it has been difficult to define a patient population where a benefit could be reproducibly shown. furthermore, it has been documented that synergy between cytokines occurs, but detailed knowledge of the cytokine network is not yet available. it is conceivable, that neutralization of one cytokine prompts the induction of another which will evoke the intended response in the host. recent data obtained in human endotoxemic volunteer models seem to confirm this. if this turns out to be the case, neutralizing a single cytokine may not be a successful approach. cytokines in tum, induce various adhesion molecules, such as icam- . such molecules regulate for instance the neutrophil-endothelial cell interactions, which are thought to play an important role in the pathogenesis of systemic organ injury. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in a large number of experimental models. protective effects have been observed in a wide variety of inflammatory, immune, and ischemia-reperfusion injuries. thus, altering the host response by modulating the function of adhesion molecules may attenuate the inadvertent injury caused by inappropriate behavior of host defense cells. targeting cellular surface interactions has been added to the efforts to change the outcome of disease. modulation oftheseprocesses seems very promising, but may temporarily leave the host without effective defense mechanism. great care therefore, must be exerted when studying this powerful two-edged sword in a clinical setting. our knowledge of the role of adhesion molecules in the intlammatory response has increased rapidly due to the availability of new reagents and mice geneticly deficient in adhesion molecules. these molecules are important in interactions of leukocytes with endothelial cells, other leukocytes, platelets, and epithelial cells. when these molecules are engaged, they can also play a role in activating leukocytes and their effector functions. in the venules of the systemic circulation, adhesion often occurs through a series of sequential interactions. initial interactions are mediated by members of the selectin family to loosely associate the leukocytes with the endothelium and are followed by firm adhesion requiring members of the integrin and immunoglobulin family. later interactions with endothelium may require pecam. adhesion molecules are usually required for leukocyte emigration in response to extravascular stimuli and for neutrophil-mediated endothelial cell injury. they are critical for host response in many diseases including infections. however, when the inflammatory response results in damage to host tissues, patients may benefit from blocking the leukocyte response. anti-adhesion molecule agents are an important potential antiinflammatory therapy. the focus of anti-adhesion therapy may be at any step of the sequence. diseases where anti-adhesion molecule therapy may benefit patients include ischemia/reperfusion injury in many organs, ards and mof, and transplantation, both to protect the donor organ from ischemia/reperfusion injury and to inhibit graft vs host disease. many strategies have been considered and include: ) blocking the ability of adhesion molecules to recognize their ligand using antibodies that have been humanized or soluble receptors linked to igg to prolong their circulating halflife, ) blocking the ligands for adhesion molecules using soluble adhesion molecules, peptide analogues, or oligosaccharides, and ) blocking the production of the adhesion molecule using anti-sense oligonucleotides. because the synthesis of adhesion molecules is usually regulated by cytokines, inhibiting the action of cytokines is another potential site for interrupting the adhesion process. although important issues of safety must be evaluated, the potential for modulating the inflammatory response make this an exciting area of improvement in health care delivery. claire m. doerschuk, m.d.; riley hospital for children, room ; barnhill drive; indianapolis, in usa. modulation of neutrophil-endothelial cell adhesion with anti-cdl i/cd monoclonal antibodies as a therapeutic modality. ch wortel, repligen corporation, one kendall square, building , cambridge, ma , usa. the central role of inflammatory cells in the pathogenesis of lung and systemic organ injury is well recognized. binding of neutrophils to endothelial cells and migration into the parenchyma are largely regulated by complementary adhesion molecules. the leukocyte integrins are glycoproteins expressed on the neutrophil surface and in the cytoplasmic granules. integrins consist of a common beta or cluster differentiation (cd) chain covalently linked to one of three different alpha chains (cdlla, cdllb, cdilc) and exist on the cell surface as three distinct heterodimers. cdlla/cd is expressed on all leukocytes, whereas cd b/cd and cd c/cd . are restricted to cells of myeloid origin. cd i / cd interacts with intracellular adhesion molecule- (icam-i), its ligand on endothelial cells. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in a large number of experimental models. protective effects with anti-cd antibodies have been observed in a wide variety of inflammatory, immune, and isehemia-reperfusion injuries, such as arthritis, burns, endotoxic shock, bacterial meningitis, autoimmune diabetes, nerve degenemrion, allograft rejection, allergic asthma, acute lung inflammation, skin lesions, and ischemia-reperfusion models of the intestine, myocardium, lung, skeletal muscle, and central nervous system. protective effects have also been observed in animals resuscitated following hemorrhagic shock. blockage of cd , however, would affect all leukocytes, as would antibodies to cdlla/cdi . targeting cdllb/cd would affect cells of the myeloid lineage only, which could prove to be beneficial. cd b/cd is not only involved in transendothelial migration, but is also implicated in adherencedependent formation of reactive oxygen species. blocking cd lb/cd may therefore not only reduce the numbe r of leukocytes accumulating in the tissue, but also attenuate the oxidant stress of infiltrated neutrophils. anti-cd b treatment has been used effectively to reduce tissue injury initiated by ischemia-reperfusion, complement activation and endotoxemia. altering the host response by modulating the function of adhesion molecules may attenuate the inadvertent injury caused by leukocytes, but may also temporarily leave the host without effective defense machinery. overall, animal studies suggest that it may be safe to inhibit neutrophil adhesion for a limited period of rime. these observations will have to be confirmed in carefully designed clinical trials. c, arbobydrams are ubiquizom constir~uts of cell sv.rfaees, and possess many c~xssfies ttm~ m~,e ~em ide~. canaidates for r~ognifioa mole~ule& in m~y systems whe,~ cer udhesioa ~lays a critical ro~ car~hydram l:~dtag ~otegas have been shown to b~ad tocell surfa~ earbohydzaxes ~nd pzrl~pate in cell-ceil lumtaefion& such sys,.ems include ~rti~za~io=, deveaopmeat, l~thoge~-hcet reeog--ition ~d i~zmmadon_ in particular, tb.z recent di%~ve~ of lhe selec~ and th~ impo.~a~c~ in teukccy~udo~lelium adh~ion has -~f~m av.c~on ok l~in m~ted cell adhe~on. s~vere/poten~s/cs.rbohydr~ l~ga~s hrve ~e~l ~u~ilied for ~he s~lcc~ins. the,~ c~u be broadly di,,sded la~o ~wo m'oups -sibyl l~wis x m~ mh~.~l oligo~chadd~s, ~d sf/~ ca~ohydmma, all ~:~ ~l~dns bind m siflyl l~wis x (sie$ o!igos~ccb.e.rkms, zlthou~ w~ differing avi~re~. 'we have i~¢n~ed the functional g~oups a s~ex ~n~ med/a~ ~he b~u ~di~g of ~h~ c~b hydmm = e-se/sedm we have used ~hat iv.formation to sya~esize sle ~ '~mt gs r.he, t focus on replacing slslic ~sd ~nd fuc s¢ wi~ simpler, more stable strunt~es. a[~ou~a ~ proeer~ is ongoing, we hve been ~ucee,.~ful a~ rep~aein t.ke si~ic a~id. residue wi~ std.fzte. ~ce~ or la~c amd groupa we t'we ex aninad &e ten, bunion of ezed~ hydroxyl group of the fizeose residue ~ billding of e-, l-~nd p-selees..u. we have also found m~fi~fio~ of the reducing end ~¢.cha'i~ ~z increase mtagovsst activity. the, m¢ond. group of figs,rids a.r eontzin su~a~ u a ea.rbohydr~t¢ support,, und seem to bi~.d to t~e sele~ti~s wi~ dlf:ferem characteristics c .an does sle:, s=h compounds are m ogniz~d by l-selects. md p-selectia, bur., in genera/, not e, selecti~ these dam may mdicam r.hat l-and p-s~ ¢at~ h~d via o, second ~te thaz operates lu~.ead of, or in conjunction with ~tc sle" b~ding ~iite. dam rela~&~g to ±e, se two types of ,ml~ liga~ds have beam t~ed to desig~ potential the ~peutics for i~fi~anmat ry disease. lr:rng maimai models of acute lung lu ury we can demo~trate that eompmmds that inhibit seleetiu birding ~ ~i~o hzve ber~ficial effects when uc~d in rive. progressive microvascular damage in the tissue adjacent to a cutaneous burn injury results in extension of burn size. the role of leukocytes in the pathogenesis of microvascular injury was investigated by inhibition of their adherence to the microvascular endothelium using monoclonal antibodies directed to leukocyte cdi or its endothelial ligaud, intercellular adhesion molecule- (icam- , cd ). a model of thermal injury was developed using new zealand white rabbits. two sets of three full-thickness burns separated by two x -mm zones were produced by applying brass probes heated to °c to the animals' backs for sec. cutaneous blood flow determinations carried out with a laser doppler blood flowmeter were obtained for hours. there were five experimental groups: controls given saline alone; animals given monoelonal antibody to the cd r . prior to burn injury (pre-r . ); animals given r . min after burn injury (post-r . ); animals given a monoclonal antibody to icam-i, r . prior to burn (pre-r . ); and animals given the r . min postburn injury (post-r . ). blood flow in the marginal "zone of stasis" between burn contact sites was significantly higher in the antibody-treated animals. administration of the antibodies min after injury was as effective as preburn administration in preserving blood flow. at hr post-burn all antibody -treated animals had blood flow in the areas at risk for progression (i.e., the zone of stasis) at or above baseline levels while the control animals had levels equal to . _+ % of baseline (p < . by analysis of variance and mann-whitney u test). these results indicate that leukocytes play an important role in the pathogenesis of burn wound progression, and that this progression can be attenuated by moduiating adherence to endothelial cells. a wealth of information now supports the hypothesis that inhibition of cell adhesive mechanisms will nter the course of immunologicand inflammatory processes. what remains unclear is whether inhibition of specific mechanisms wfl[ be of therapeutic benefit in any specific human disease. current data derived from animal models are not inconsistent with the hope of therapeutic benefit, but techniques for inhibition (e.g., antibodies, antisense oligonucleotides, inhibitory peptides, inhibitory carbohydrates, smaii synthetic inhibitors, etc), tissue and species differences in the relative contributions of adhesion molecules to the inflammatory process, and the cascade model of adhesive interactions are all confounding issues, making predictions of therapeutic benefit in any specific human disease process very difficult. additional concerns involve the potential roles of adhesive mechanisms in host resistance to infection. as human therapeutictdals are initiated, more exact information on the roles qf specific adhesion molecules in human disease should emerge. inhibition of leukocyte adherence to endothelial cells can represent a novel therapeutic approach to septic shock. we performed a pilot study to evaluate the safety and tolerability to cy- , a monoclonal antibody against human e-selectin, in patients with septic shock. septic shock was defined by clinical signs of sepsis, a documented source of infection, and fluid-resistant hypotension requiring the use of vasopressors. eleven patients entered the study, but patients who died during the first hours were excluded, as this was part of the protocol. cy- was administered as a single intravenous bolus of . mg/kg (n= ), . mg/kg (n= ) or i mg/kg (n= ) mg/kg. the antibody was well tolerated. none of the patients died during the day follow-up period. organ failure was assessed for organs (cns, lungs, liver, kidneys and coagulation). the mean number of organs failing, which was initially . ± . , decreased to . ± . at the end of the study (p<o.o ). these results are therefore encouraging. administration of an antibody to e-selectin in patients with septic shock requires further study. the importance of cytotoxic t lymphocytes (ctl) in the host defense mechanism against viral as well as parasitic and bacterial diseases is becoming increasingly appreciated. the design of vaccines to generate cytotoxic t ceils from nonviable or subunit constructs, however, poses challenges due to the unique characteristics of antigen processing and presentation by class i major histocompatibility complex (mhc) molecules. since the final antigen recognized by ctl are short peptides capable of high affinity binding to class i mhc molecules, we have designed strategies: a) to define the structural characteristics of peptides that are required for their high affinity binding to mhc; and b) to formulate these peptides into immunogenic vaccines that are capable of generating cytotoxic t lymphocytes. the strategy and results to accomplish these two goals will be discussed. amnon altman and erich gulbins ras proteins represent essential intermediates in receptor-mediated growth and differentiation pathways. they couple signals initiated by receptor or nonreceptor protein tyrosine kinases (ptks) at the cell surface to cytoplasmic targets which coordinate signal transduciion to the nucleus. ras also participates in t lymphocyte activation initiated by the antigen-specific t cell receptor (tcr)/cd complex, which leads to lymphokine production and proliferation. receptor ligation causes activation of associated ptks of the src and syk families as the earliest identifiable event in this pathway. the importance of ras in t cell activation is indicated by the findings that an oncogenic ras protein activates the interleukin- (il- ) gene promoter; conversely, a transdominant inhibitory ras mutant inhibits this event and the activation of map kinases. the rate-limiting step in ras activation is the exchange of bound gdp for gtp, which is catalysed by guanine nucleotide exchange factors (gefs). we identified the sh /sh domain-containing vav protein, which is selectively expressed in hematopoietic cells, as a ras-specific gef coupled to several hematopoietic receptors. vav can be activated by two pathways, i.e., by ptk-mediated phosphorylation or by binding of diglyceride second messengers to a cysteine-rich, protein kinase c (pkc)homologous vav domain. these pathways are independent as demonstrated by structure/function analysis and the use of pharmacological inhibitors. the two activation pathways may enable vav to integrate signals from distinct hematopoietic cell receptors, and couple them to ras. t cells express another, ubiquitous ras gef, i.e., sos, which is known to be coupled to activated tpk receptors. t cell activation leads to membrane association of a signaling complex consisting of sos and two other signaling proteins, grb- and shc, via direct binding of the shc sh domain to the tyrosine-phosphorylated chain of the tcr/cd complex. thus, multiple receptors, ptks and ras activators participate in signaling pathways that lead to hematopoieitc cell growth and differentiation. the interactions and function of these various signal transducers will be reviewed. phagocytes. immunity is mediated by specific t lymphocytes, cytokines produced by these specific t cells activate antimicrobial capacities in mononuclear phagocytes, thus contributing to protection. other immune mechanisms also participate in the acquisition of resistance. on the other hand, t cells are also crucial for many pathologic sequelae of intracellular bacterial infections. although ifn-y is central to macrophage activation, synergistic and antagonistic effects with other cytokines occur. the cd/ t cells, representing the major t cell population in experimental mice, are the main mediators of antibacterial immunity; an auxiliary role for y/~ t cells appears likely. the c~// t cell population further segregates into cd t cells which recognize microbial peptides in the context of gene products of the major histocompatibility complex (mhc) and cd t cells which see their peptide in the context of mhc class i. the -y/~ t cells are generally double-negative. knock-out mice in which the genes for various t cell receptor chains, for mhc class i or mhc class ii molecules, for cytokines or cytokine receptors had been deleted, have provided extremely helpful tools for analyzing the role of t cell subsets and cytokines in antimicrobial immunity. using these mutant mice, the role of distinct t cell subsets and cytokines in bacterial elimination and granuloma formation was analyzed in detail. these studies underline the central role of a// t cells and ifn-y in antibacterial immunity and, furthermore, show a distinct function of /$ t cells. moreover, these studies show that the relative contribution of cd or cd t cells to antimicrobial immunity varies in response to different pathogens. apoptosis is an active form of cell death in which the cell participates in its own demise, providing the biochemical pathways that trigger and mediate the complex events of the process. although this phenomenon has been studied for many years, it is only recently that significant progress has been made towards the elucidation of the molecular mechanisms that control apoptosis. once such mechanism came as something of a surprise: in a number of eases, apoptosis can be promoted by the action of the c-myc protein, a protooncogene product that had previously been associated only with cell proliferation (and presumably, survival) . expression of c.myc in cells can cause them to enter an apoptotic pathway or proliferate, mad this "decision" depends upon additional signals, such as growth factors that inhibit apoptosis and allow the ceils to proliferate. thus, c-myc directs cells out of rest and the choice of proliferation or death depends upon the presence or absence of survival signals. other survival signals are generated by oncogene products that can cooperate with my¢, including bcl- and abl there are implications of this interplay between apoptotie and anti-apoptorlc signals in cell iransformation and the resistance of some tumor ceils to therapeutic agents capable of inducing apoptosis. oncogene interactions therefore control the life or death of transformed ceils but also have important roles in normal, development processes. in the immune system, developing t ceils are "screened" for potential self-reactivity by a process of negative selection, in which activation via the t cell receptor (tcr) induces apoptosis. this process can be mimicked in t cell hybridomas, and appears to depend upon the expression of the c-mye protein. evidence that this represents a requirement for the myc/max heterodimer will be presented. as above, these cells are presented with the "choice" to proliferate or die and this depends not only upon myc but also other signals. thus, expression of functional v-an kinase in these ceils can render them resistant to the activation of apoptosis via tcr ligation, surprisingly, bcl- does not appear to be capable of blocking this form of apoptosis, and the reasons for this will provide new insights into the development of the immune system and the process of apoptosis. although the oncogene products discussed here probably do not directly participate in the biochemical process of apoptosis, they represent molecular controls on this complex mechanism. as such, they gave us new ways to look at the life and death of a cell recent attention has focused on macrophage release of cytokines, such as il- , il- and tnf as important mediators of septic shock. however, serum cytokine levels have correlated poorly with outcome of septic shock. the purpose of this study was to compare the functional status of macrophages to serum cytokine levels in early sepsis by an analysis of splenic macrophage protein synthesis during abdominal sepsis. macrophage total protein distribution and biosynthetic activity was assessed by large format -d page, autoradiography of s-labeled proteins and computer assisted densitometric analysis. sepsis was induced in sprague dawley mts by cecal ligation and puncture (clp). untreated and sham operated rats served as controls. splenic macrophages were harvested at and hrs. following clp. serum il- levels were determined at , , and hours by the tdi bioassay. by hrs. % of the clp mls exhibited multi-microbiaj bacteremia. control or sham operated rats did not show bacteremia. clp resulted in a significant % elevation (p < . ) in serum il- levels at hrs. there was no difference in il- at and hrs. when compared to the control groups. d page studies examined splenic macrophage total protein distribution and biosynthetic activity at and hrs. post-clp. at hrs. macrophages from clp rats showed a substantial decrease in total protein pattern ( protein spots vs protein spots) when compared to non-septic control macrophages. a decrease in the number of lower molecular weight proteins (< kd) was observed. at hrs. post-clp, splenic macrophages from clp rats showed a substantial increase in the synthesis of low molecular weight (< kd) proteins, when compared to non-septic controls. we conclude that: l) fulminate abdominal sepsis induces an early ( hrs.) staining with cd and cd mabs will identify two monocyte subpopulations in human blood: a major population of regular monocytes, which strongly expresses the cd antigen (cd ++) and a minor population with weak expression of cd and expression of the cd antigen lcd +/ cd + cells). in healthy control donors(n= ) the latter cells account for + cells/mm and %+ % of the monocytes. in sepsis patients, the cd] +/cd + cells can become a major population with more than % of all monocytes in of patients and with more than cells/mm in of cases. there was no correlation of cd +/ cd + cells to any clinical parameter except for cd +/cd + percentage and body temperature (p= . ). the cd ++ monocytes showed a substanttial decrease in cd antigen density in of patients. three-color-if shows that the cd +/ cd + cells in sepsis patients when compared with the cd ++ monocytes exhibit a higher level of class ii antigen and a lower level of cdiib and cd antigens, consistent with a more mature nature of the cd +/cd + cells. levels of il- were increased in sepsis patients: of patients with high numbers of cdi +/cd + cells ( /mm ) had high levels of il- ( u/ml). these data suggest that septicemia may lead to substantial changes in blood monocyte composition and this may be related to elevated levels of cytokines such as il- . human peritoneal macrophages were exposed to increasing doses of lipopolysaccharide (lps) or a synthetic lipid a analogue (mrl ) and production of the cytokines il-lb, il- , tnf-a and g-csf was assessed at the protein-and mrnalevel. cells were also stimulated with low doses of lps and mrl to study their "adaptation" to a secondary challenge with high doses of lps. tnf-a production after stimulation with lps could be almost completely relieved by preincubation of cells with low doses of lps and similarly, il- production was suppressed. surprisingly, however, "adapted" cells were able to synthesize even larger quantities of of g-csf and il-lb when exposed to a secondary lps challenge. the changes in tnf-a, il- and g-csf protein synthesis could a/so be detected on the mrna level, whereas transcript levels for il-lb remained unaltered as assessed by northern blot analysis. high doses of the synthetic lipid a analogue mrl were also able to "adapt" macrophages for a secondary lps challenge by downregulating tnfa-and il- -production, while priming secretion of g-csfand il-lb as well. taken together, here we describe for the first time the discordant adaptation of human peritoneal macrophages to a secondary lps stimulus in vitro. these findings appear to bear ramifications for the in-vivo endotoxin response during inflammation and gramnegative septicemia. shock states with inadequate cellular oxygen delivery may contribute to macrophage (mo) activation or dysregulation. in this study we compared the effect of transient hypoxia and endotoxin pretreatment (lps ) on mo mediator release with a second endotoxin stimulus (lps ). methods: in vitro cultures of marine peritoneal exudate mo were exposed to hr. of hypoxic or normoxic conditions, then incubated hr under identical normoxic conditions _+ ng/ml of lps pretreatment. during the final hours all m~l were exposed to a range of lps concentrations. m~i supernatants were assayed for tnf, il- , il- , pge , nitric oxide (no), and superoxide release. results: lps markedly inhibited mo tnf release by lps but hypoxia had no effect on lps -triggered tnf release. hypoxia increased mo il- production in the absence of lps , but inhibited lps augmentation seen under normoxic conditions. pretreatment with lps increased no production from lps under normoxic conditions, but bypoxiainhibited this effect. pretreatment with lps signifcantly augmented mo release of il-i and pge , but hypoxia had no significant effect (data not shown) superoxide production was increased by lps under normoxic conditions, but hypoxia significantly inhibited superoxide release (not shown a. lepape, c. docile, f. arpin, m. c. gutowski, v. banssillon and j. bienvenu. i aim of the study. increased levels of tnf are inconsistently correlated with the severity of sepsis. one possible explanation is a decrease ability of ceils to produce eytokines. the objectives of this study were m compare at different levels of of clinical severity the responsiveness of cytokine producing cells. this was evaluated by the response to a stimulation by lps as well as to the inhibitory effect of ptx. the technic used is a whole blood ex-vivo model, as described by desch et al. ( ) . h materials and methods. different groups of icu patients, postoperative (n= ), sepsis (n= ), septick shock (n= ), and healthy control (n= ), were studied. blood was drawn in endotoxio free heparinized robes. stimulation was achieved by addition nf pl oflps ( ng/ml) to id of whole blood in presence of various concentrations of ptx ( , " , - , " , i " m). after hrs incubation at °c, the tubes were centrifuged and supematants frozen at - °c. tnfa and/l were measured by elisa (medgenix r and immunotech r respectively). monocytes were quantified on a technicon h . the results are expressed as median values. non parametric tests are used for testing differences between groups. hi results, tnftx and , , corrected for monocytes count, are given as % of their initial value, the baseline before stimulation or inhibition being %, ) stimulation by lps. the control group is much more stimulated (> % for il , > % for tnfa). blood samples taken postoperatively and in patients with simple sepsis are significantly less stimulated (> % for il , > % for tnfa ). the lowest stimulation was observed in patients with septic shock (median = %), some patients being not stimulated at all. )effects of ptx.the inhibitory effect of ptx on tnftx production is effective in all groups at - m (reduction to less than '¼ of the median values), and is almost complete at " m. the septic shock group has a decreased sensitivity to ptx. il production exhibits a lesser reduction at - m (~ 'a to ½ of the median values), further increased at - m. the septic shock group is again less sensitive to ptx. iv conclusion: the reduced ability of circulating monocytes to produce cytokines during severe infections is confirmed here. ptx is able to reduce significantly tnfc~ at - m and the inhibition is nearly complete at - m. surprisingly, there is a lesser, but significant suppressive action of ptx on il , not found in experiments using purified monocytes. one possible explination could be the interplay between cytokines production. ( ) lymphokine research ( ) cdna sequencing constitutes a powerful method of measuring steady-state mrna levels for all genes transcribed in a given cell or tissue at a particular stage of differentiation. by comparing transcript abundance both prior to and following differentiation, individual genes can be identified whose transcription is regulated both positively and negatively. in order to examine monocyte activation, the human monocyte line thp- was induced with phorbol ester ( h) and activated for h with lipopolysaccharide (lps) after which polya + rna was purified. the rna from control and lps-treated cells were each used to construct a cdna library under identical conditions, and all resulting clones were selected for cdna sequence analysis. each clone sequence was evaluated by matching with both genbank and our own gene databases. very different patterns of gene expression were seen in the two libraries, the latter reflecting very high levels of known inflammatory mediators such as il- and tnf. a second set of libraries were made from umbilical vein endothelial cells (huvec), both with and without lps stimulation, and were analyzed in a similar fashion. the effects of lps induction on specific gene transcription in both cell types will be discussed. t. tadros, md, th wobbes, me) phd, rja goris, md phd to investigate whether the preactivation of regional macrophuges by liposomes containing muramyl tripeptide (mtp-pe) can counteract the detrimental effect of blood transfusions on both anastomotic repair and host susceptibility to infections. methods eighty lewis rats received lmg/kg of either empty or mtp-pe encapsulated liposomes, intraperitoneally (ip). twenty-four hours thereafter, the animals underwent resection and anastomosis of both ileum and colon, and received ml of either saline or blood from brown norway donors,iv. the animals were killed or days after surgery and examined for septic complications and anastomotic repair. the average anastomotic strength, as assessed by bursting pressure (+sd), was significantly diminished in the transfused animals, as compared to the non-transfused animals (ileum;day ; -+ vs + , p< . ). transfused animals pretreated with mtp-pe encapsulated liposomes showed a significant improvement of their anastomotic bursting pressure ( + , p< . vs transfusion). pretreatment with mtp-pe encapsulated liposomes decreased significantly the incidence of anastomotic abscesses in transfused animals ( from % in ileum on day to %, p< . ). conclusions preactivation of regional macrophges by intraperitoneal administration of mtp-pe encapsulated liposomes prevents the detrimental effects of transfusions on anastomotic repair and reduces the incidence of intraabdominal sepsis. academic hospital nijmegen, dept of general surgery, pb i, hb nijmegen, the netherlands. leukemia cell line, teip- . robin s. wa, gner*, perry v. halushka "~, and james a. cook*, departments of physiology , pharmacology "l" and medicine "t, medical university of south carolina, charleston, s.c. . adherence of monoeytes to endothelium and extracella/ar matrix proteins is essential for accumulation at sites of inflammation. txa , an arachidonic acid metabolite, inhibits human monocyte chemotactic responses suggesting that txa may alter monocyte adhesiveness. we selected the thp- cell line, a human monocytic leukemia cell line to further investigate the effect of txa on adhesion. we tested the hypothesis that txa alters lpsinduced adhesion of thp- cells and that txa exerts its effect on adhesion via a camp dependent mechanism. thp-i cells were exposed to s. enteritidis endotoxin (lp.g/ml) _+ the cyelooxygenase inhibitor lndomethacin (in), the txa mimetic i-bop ( . .tm,) or txa receptor antagonists bms and l ( ~m). cells were allowed to adhere for hours and adherent protein/well was determined. lps-induced a significant (p< . ;n= ) increase in adherence of thp- cells (basal, . + . gg protein/well; lps, . +_ . p.g protein/well). the amino acid glutamine is an essential compound for synthesis of purine and pyrimidine basis and therefore necessary for rna-and dna synthesis. in human plasma the concentration of glutamine is between . - . mm, and is reduced in septic patients up to % ( . - . mm). monocytes play a central part in the inunune system and it was of interest, whether glutamine is involved in the modulation of cell surface markers and phagocytosis of these cells. human peripheral blood mononuclear ceils were obtained from ml heparinized blood of apparently healthy donors by ficoll-paque density gradient and isolated by counterflow elutriation. the puritiy was more than %. subsequently cells were cultured in phenolred-free rpmi medium with various concentrations of glutamine ( . , . , . , . , . , , mm) in teflon-fluorinated ethylene propylene bottles to exclude cell adhesion and possible cell activation. aider seven days culture, cell viabilty was determined by trepan blue exclusion and varied between and %, independent of glutamine concentrations. cell surface markers were detected by flow cytometry, noaspecifie phagoeytosis was measured with latex beads and specific phagocytosis with opsonizied e.eoli using a facscan. lower concentrations of glutamine decreased the expression of hla-dr and icam- /cd on monocytes in a dose-dependent manner. the receptor for fc'/rucd as well as the receptors for complement cr /cdllb and cr /cdllc were down-regulated. cr /cd which is only slightly expressed on monocytes was not influenced. furthermore, no effects on the expression of cdi , the receptor for transferrin cd and fc'friii/cd were seen. our data indicate, that lower concentrations of glutamme influence the phenotype of monocytes. we are now interested to study whether glutmnine influences non-specific phagocytosis, or whether specific phagocytosis correlates with the decreased expression of fc'/r and complement receptors. we investigated immunologically more than patients who were admitted to icu because septic syndrom during the last four years. patients were immunologically followed up - times per week until release from icu. the expression of hla-dr antigen on monocytes turned out to be the best prognostic parameter. the persistence (> days) of low hla-dr expression (< %) predicts fatal outcome (mortality > %). the altered phenotype was associated with a functional deactivation of monocytes (diminished apc, ros formation, cytokine secretion). we called this phenomenon "immunoparalysis". ifn-gamma and gm-csf were able to restore the altered phenotype and function in vitro. however, addition of autologous plasma from septic patients with "immunoparalysis" to these cultures prevented the cytokine-induced restitution. the inhibitory activity could not be removed by dialysis. therefore, we started a study to prove the therapeutic efficacy of plasmapheresis. indeed, [ of patients recovered from "immunoparalysis" following repeated plasmapheres; of them survived ( %). patients recovered temporarely and patients did not respond (all died). the survival rate in the control group of septic patients with persistent "immunoparalysis" was of ( %; p< , ). in summary, plasmapheresis in association with immune monitoring may be an alternative strategy to improve survival rate in severe sepsis. taurolidine, a synthetic taurine-formaldehyde derivative has antiadherent, bactericidal and anti-lps properties functioning primarily through binding of the lipid a region of the lps molecule. the active derivative of taurolidine, taurine, modulates calcium channel activity, critical to the initiation of a number of immunostimulatory pathways. we hypothesised that taurolidine may have direct immunostimulatory activity. the aim of this study was to investigate the immune effects of taurolidine on peritoneal macrophage (pmo) function and then determine the role of taurine in this response. study : in vivo stimulation:cd- mice (n= ) were randomized to receive taurolidine ( mg/kg bw/i.p.) or saline cor~trol. peritoneai cells were harvested after hours and were assessed for pm function [superoxide anion generation (o -), nitric oxide (no), tumor necrosis factor (tnf), fc/cr -mediated phagocytic function (phago) study : control pm were harvested and cultured in vitro with taurine ( . mg/ml for hrs), after which time they were assayed for -and tnf release. in vivo stimulation with taurolidine taurolidine has specific immunological effects on m . release of the inflammatory mediators -and tnf, and fc/cr -mediated phagocytosis were significantly increased, while release of the endothelial relaxing factor no was significantly reduced. in addition, the amino acid taurine, which is released as a byproduct of taurolidines breakdown has an immunostimulatory effect on pmo and may be the active moeity of the compound tanrolidine. in sepsis, a number of mediators which affect vasomotor tone and cardiovascular function are produced. inasmuch as sepsis causes decrease in systemic vascular resistance (svr), attention is usually focussed on vasodilators such as lactate, tumor necrosis factor, interleukin-i & , and nitric oxide. but injury and inflammation als cause production of several vasoconstrictors whose effect may not be evident in changed svr, but may significantly affect organ blood flow or function in the paracrine environment. endothelin (et) is a amino acid peptide vasoconstrictor produced by ischemic or injured endothelial cells (ec's). et is also a potent constrictor for renal mesangial and coronary vessels, an endocrine regulator, and a negative cardiac inotrope. systemic et levels increase significantly in hypoperfusion and ischemia. while et is principally produced by ec's, we asked if human monocytes might also produce et and thereby regulate vasomotor tone in areas of inflammation. monocytes from healthy donors were separated on ficoll, resuspended in rpmi + % fetal calf serum and stimulated with i ug/ml endotoxin (lps). et was measured by radioimmunoassay. lps-stimulated monocytes produced ! fm of et/ cells (vs. unstimulated controls of < ). this calculates to - % of the amount of et observed in patients with low cardiac output, sepsis or ischemia. we conclude that et is a cytokine produced by both ec's and monocytes with potent effects on numerous cells and organs in the critically ill. wuppertal , germany we and other authors showed that fatal outcome in septic disease is associated with a decreased capacity of peripheral blood monocytes for the in vitro production of proinflammatory cytokines, especially tnf-alpha. we found that this monocytic deactivation is completed by a persistent and marked decrease of hla-dr expression on monocytes (< % hla-dr+ monocytes) and a diminished antigen presenting activity whereas the capacity to form the antiinflammatory il- receptor antagonist remains high. in order to evaluate the in vivo situation and to determine at which level tnfproduction/secretion is altered we assessed the tnf-alpha mrna expression in freshly isolated peripheral blood mononuclear cells (pbmnc) from septic patients. tnf-mrna was onty rarely detected by semiqaantitative polymerase chain reaction in pbmnc's from septic patients with monocyte deactivation. meanwhile, it was found in almost all pbmncs from septic patients without monocytic deactivation. we wondered, whether il-i , which ,is known to depress monocytic proinflammatoly response and mhc class ii expression, could be one of the mediators in fatal sepsis. in fact, we found that il- message in pbmncs of septic patients peaked in the beginning phase of monocytic deactivation. in further investigations we found that tnf-administration can induce monocytic deactivation in a murine model/n vivo and provoke il- message in human pbmncs in vitro. these results support our hypothesis that an excessive delivery of proinflammatory cytokines in a first phase can induce an overwheiming inhibitory feedback, mediated by immuninhibitory mediators like il-l , which leads to often fatal monocytic deactivation in a second phase. interferon-gamma which is known to counteract il- production and the effects of il- on monocytes restores the function and phenotype of monocytes from septic patients with monoq, te deactivation in vitro and could be a possible therapeutic agent in otherwise fatal sepsis. our laboratory previously reported that lps dependent macrophagederived tnf-a production can be enhanced by pretreatment with lps at substimulatory lps priming doses coincident with a suppression of lps dependent nitric oxide (no) production (zhang and morrison, j. exp. med : , ) . in order to extend the characterization of these lps priming effects in mouse macrophages, we examined the capacity of substimulatory lps to modify lps dependent il- production. macrophages were obtained from peritoneal exudate of thioglycollate treated c heb/fej mice and cultured in rpmi medium containing % fetal bovine serum. macrophages were pretreated with various subthreshold stimulatory concentrations of lps (olll:b ) for hours, washed three times, and then stimulated with the effective stimulatory concentration of lps for hours. the amount of il- in the supernatant was measured by il- dependent cell line (b and td ) proliferation assay. il- was produced by macrophages at lower threshold doses of lps than those required for tnf-o~ or no production. subthreshold doses of lps modulated il- production in a biphasic manner characterized by an initial suppression and then potentiation. higher doses resulted in secretion of il- during the initial incubation with lps and subsequent desensitization. il- , like tnf-~ and no, is, therefore, also affected by lps pretreatment. moreover, tnf-a and il- shared the similar potentiational pathway, but differed by the fact that only il- was inhibited. (supported by r ai and po a .) department of microbiology, molecular genetics and immunology and the cancer center, wahl east, university of kansas medical center, kansas city, ks - . korolenko t.,urazgaliev k.,and arkhipov s. the role of macrophage (mph) stimulation in mechanism of protective effect of new immunomodulators yeast polysaccharides -heteropolysaccharide cryelan and homopolysaccharide mannan rhodexman (both produced by petersburg chem.-pharm. inst.) was studied. in vitro according to nst test incubation of murine peritoneal mphs with cryelan or rhodexman, ~g/ml, min was followed by increase of potencial microbicidic activity of mphs. in vivo mph stimulation by immunomodulators studied included increase rate of carbon particles phagocytosis during single i.v. or i.p. mode of administration to mice - days after (peak at nd day for i.v. and th day for i.p. mode of administration of the same dose of mg/ g b.w.).the preliminary injection of cryelan ( mg/ g, or h before) to mice with acute cold stress (- ° c, h) revealed protective effect restorating the value of depressed phagocytosis up to the normal level;the positive effect on ultrastructure of hepatocytes was noted also.there was no changes of plasma corticosterone level between group with acute cold stress and mice with cryelan + acute cold stress (several fold increase comparatively to the control mice).as was suggested, the mechanism of protection can include mph stimulation and secretion of some acute phase proteins responsible for positive effect of immunomodulators. new yeast polysaccharides cryelan and rhodexman can be used for macrophage stimulation,especially in pathological states. immunomodulators were shown to increase production and secretion of lysosomal enzymes (like zymosan). secreted enzymes,especially cysteine proteinasescathepsins b and l -involve in the process of inflammation;however, excessive release of these enzymes may lead to noncontrolled proteolysis followed by tissue degradation (assfalg-machleidt et al., ) .the effect of zymosan,bcg and new immunomodulator carboxymethylglucan (cmg), second fraction on secretion of lysosomal enzymes by murine peritoneal macrophages was studied. zymosan increased the secretion of n-acetyl-~-d-glucosaminidase and ~-galactosidase into the culture medium ( - fold); bcg possessed similar effect.cmg in the same concentrations ( /~g/ml) increased release of these enzymes only saightly ( . times).it's known that zymosan-induced secretion reflects the enzyme release from formed lysosomes (warren, ) .it was suggested that cmg activated macrophages via interaction with scavenger-receptors,followed by weak secretion of lysosomal enzymes and as a result decrease of tissue damage. in vivo zymosan induced stimulation of mononuclear system of phagocytes followed by increase of cysteine proteinases activity in liver at the th day. in the same time in blood n-acetyl-~-d-glucosaminidase and n-acetyl-~-d-galactosidase activity increased - fold. it was concluded that in drug design it's possible to select such immunomodulators,e.g. cmg,which can activate mononuclear system of phagocytes and do not damage tissue. endothelin-i (et-i) is produced by injured/ ischemic endothelium, mobilizes intracellular ca ++ and is a potent vasoconstrictor. it is also a ca ++ agonist for anterior pituitary or renal mesangial cells and monocytes. et-i causes monocytes to produce interleukin-l, , , prostaglandin e , and substances which trigger neutrophil superoxide production. et-i levels increase in shock and et may play a role in activating leukocytes post shock causing reperfusion injury. but blood flow experiments suggest splanchnic circulation changes more profoundly in shock than peripheral circulation. we therefore asked if et- (or vic), the et which predominates in splanchnic vessels, had any effect on monocyte cytokine production. human monocytes from health~ blood donors were separated on ficoll. . x ucells/ ml in rpmi + % fcs were incubated i min., & hrs. with - m et-i, - m vic or i ug/ml of lps. supernatants were assayed by elisa. we have shown that low dose endotoxin pretreatment (lps ) for hrs markedly inhibits the macrophage (mo) release of tumor necrosis factor (tnf) and increases interleukin- (il-i) in response to a subsequent endotoxin stimulus (lps ). in this study we examined the kinetics of lps inhibition of tnf and augmentation ofil- . methods: murine peritoneal exudate mo from balbc mice were exposed in vitro to medium or ng/ml of lps for intervals of to hours. culture medium was then replaced with , or ng/ml of lps for hrs. tnf and il- in mo supernatants were measured by specific bioassays. during sepsis endotoxin (lps) activates macrophages (mo) to release mediators such as tumor necrosis factor (tnf), interleukin- (il- ), interleukin-i (il-i) and prostaglandin e (pge ). we showed that preexposure to lps (lps ) alters the response of murine m~i to subsequent lps stimulation (lps ). we hypothesized that in vitro cytokine release by lps in human monocytes (mo) is also be altered by preexposure to lpsi. methods: human peripheral blood mo were obtained from healthy volunteers (n= ), cultured in vitro hrs, then pretreated hr _+ lps -cultures were then stimulated with lps and mediators in mo supernatant measured: tnf, il-i, and il- by specific bioassays, pge by immunoassay kit. serum cytokine levels (specific elisa kits) were compared to in vitro supernatant levels. data is expressed as % control_+sem, lps = ng/mh the table shows that all mediators were increased, in the absence of lps . pretreatment with lps resulted in complete inhibition of lps -triggered tnf release. in contrast, lps significantly increased mo secretion of il- , il- and pge (data not shown). serum cytokine levels were as follows: tnf _+ , il-i + , and il- . -+ . ng/ml. these serum levels were low, showed an extremely wide variation, and did not correlate with in vitro lps -triggered mediator production. conclusion: human monoeyte mediator production is differentially regulated by preexposure to lps . provocative in vitro testing of monocytes may ultimately be clinically useful to identify prior in vivo lps exposure or mo macrophages release numerous secretory products involved in host defense and inflammation. activated macrophages with cytokines produced have been implicated in tissue damage in sepsis and multiple organ dysfunction. aimed to elucidate the organ-association phenomena,this study is to compare peritoneal macrophage(pm),alveolar macrophage(am), and kupffer cells(kc) during sepsis in terms of cellular protein contents as symbol of activation by flow cytometry analysis. sepsis were produced by cecal ligatien and perforation (clp) in wistar rats weighing - g.pm were obtained by peritoneal lavage,am by bronchial lavage and kc by incubating the collegenase digested liver with pronase-e. leukocytes have been implicated as a mediator of the microvascular dysfunction associated with reperfasion of ischemic tissues. a role for ieukocytes is largely based on observations that rendering animals anutropenic with anti-neutrophil serum or preventing leukocyte adhesion with monoclonal antibodies attenuates the increased fluid and protein leakage from the vaseulature that is normally observed in postischemic tissues. we have recently undertaken studies designed to determine the relationship between leukocyte-endothelial cell adhesion and albumin leakage ia rat mesenterlc venules exposed ~o ischemia-reperfusion (i/r). leukocyte adherence and emigration as well as albumin extravasafion were monitored in single postcapillary venules using iatravital fluorescence microscopy, lschemia was induced by complete occ!usion of the superior mesenteric artery and ~dl parameters were monitored at various intervals following reperfusion. the magnitude of the leukocyte adherence and emigration, and albumin leakage elicited by i/r was positively con-elated with the duration of ischemia. the albumin leakage response was also highly correlated with the number of adherent and emigrated leukocytes. monoclonal antibodies against the adhesion glycoproteins cd , cdllb, icam- and l-selectin, but not p-or e-selecdn, reduced i/r-induced leukocyte adherence and emigration as well as albumin leakage. phauoidln, an f-aetin stabilizer, largely prevented the emigration (but not adherence) of leukocytes and greatly reduced, the raicrovascular protein leakage. plateletleukocyte aggregates were formed in postischemic vemdes; the number of aggregates was reduced by antibodies against p-selecdh, cdilb, cd , and icam- , but not e-selectin or lselectin. a significant fraction of the mast ceils surrounding the posteapillary venules degranulated in response to ischemia/repeffusion, but mast cell stabilizers did not afford protection against the albumin leakage elicited by i/r. these results indicate that reperfusloninduced albumin leakage is tightly coupled to the adherence and emigration of leukocytes in posteapillary venules. this adhesiomdependent injury response is primarily mediated by cdllb/cdi on activated neutrophils and icam- on venular endothellum, and appears to require l-selecda dependent leukocyte rolling. mast cell degranulation does not appear to conwibate to the vascular pathology associated with i/r. m.d. rod=iek, boston, ma, usa the polymorphonuclear neutrophil (pmn) has long been known to pa~tlcipats in the inflammatory rebpons~ as a phagocyte and killer of invading organisms, but little attention has been given to its potential as a participant in the in~une interaction of lymphocytes and macrophages. we and others have shown that the pmn may have i~m~/nomcdulatory effects both in vitro and in vlvo. more recently it has been proven that the pmn can make mrna for and secrete the proinflammatory oytokines illa, il-ib, tnfs, il- and il- as does the other major circulating phagocyte, the monocyte/macrophags. furthermore it has been shown to make the potentially autoregulatory oytokines gcsf and gmcsf. these functional capabilities suggest that the pmn is not an wend cell ~, but one which has a potential role in regulation cf ~he immune response and that this potential ~cle should no longer be ignored when considering the immune abnormalities existing in patients following majo~ injury or surgery. we have investigated the proinflaznmatory oytokine secretion patter~ by pmn in patients following major ~hermal or tra~matic injury and in volunteers fellowinq endotoxemia. ?ollowing major injury there is variable pmn secretion of these cytokines when stimulated in vlero. following endotoxemia in a group of human volunteers pmn showed a hypo=esponsivenesa to lps hrs following endotoxin infusion followed at hre by an overshoot. pretreatment with steroids modulated this overshoot phenomenon, suggesting that receptors for steroids are involved in the regulation of cytokin® secretlon by fmn. these results sugges~ that the pmn, the most numerous cell in the circulation and the first to respond to an ins~l~ may be a so~rce of the prolnflammatory cytokine cascade following injury that has been recognized as significant in the process which often leads to multiple o;gan failure, the immunosuppresslon which occurs following major thermal injury may predispose these individuals to infection and sepsis, which remain a significant cause of morbidity and mortality. included among the many immune aheratlons are the p integrln (cdlla, b,c/cd ) dependent activities of adhesion, chemotaxls, diapodesls, and phagocytosls. our investigations indicate that, following major thermal injuries, the expression of the [~ integrlns, but not cd , is significantly decreased on neutrophlls (pmns). it remains unclear if pmns from thermally injured patients respond normally to lps, the effects of treatment in vitro with lps and f-met-leu-phe (fmlp) on the expression of cdtlb was examlned on pmns from the peripheral blood of healthy volunteers and non-septic burn patients (> ~; total body surface area, >ls~ full thickness), the pmns were incubated with lps (]ng- p.g/ml) or f'mlp ( " to " m) et oc for mln, in ~; human ab serum, the expression of the ]ntegrins was detected using monoclonat antibodies and flow cytometry. lps and f'mlp resulted in a slight increase ( fold) in the expression of cd b on pmns from burned patients compared to an and fold increase, respectively, on pmns from healthy individuals. this inability of lps or fmlp to increase cd b expression was not due to the amount of lps bound to the two cell populations. because the same defect is seen after either lps or fmlp stimulation, it is speculated that the defect must be in the amount of preformed cd ] b or its transport to the plasma membrane. platelet-activating factor (paf) and neutrophils have been implicated in the patbophysiology of ischemia-repeffusion injury, in addition, paf stimulates neutrophi[ (pmn) oxidative metabolism in vitro. the present study examined the potential role of paf in repeffusion injury in an in viva rabbit model. eight anesthetized rabbi~s underwent retroperitoneal exposure of the infrarenal abdominal aorta after percutaneous insertion of a catheter through the jugular vein into the infrahepatic inferior vena cava. doppler flow probes were placed around the abdominal aorta and the right common femoral artery to assess flow through these vessels. an occlusive ligature was placed around the abdominal aorta (superior to the flow probe) at t = and total occlusion of blood flow to the lower extremities was maintained for g mins., after which the ligature was released allowing for reperfusion of the ischemic lower limbs. effluent blood from the ischemic hind-limbs was collected through the ivc catheter at the times indicated below and assayed for paf by a direct radioimmunoassay. in addition, neutrophil h production was determined by a previously described ' '-dichlorofluorescein flowcytametric assay. _+ amean _+ s.e.m, pg/ml blood; brelative fluoresenee (% of baseline); caortic and femoral artery flow (% of baseline); *p < . vs. baseline; "p < . vs. baseline. a significant elevation of paf was observed in ischemic hind-limb effluent blood at min. after release of the aortic ligature during the repeffusion phase, as compared to baseline levels. in addition, pmn h production was increased by . -fold above baseline values by hour after ligature release during the reperfusion phase. both of these elevations were transient and returned toward baseline by hours post-isehemia. tatar occlusion of hind-limb flow was achieved as evidenced by the absence of aortic or femorat flow at rain. post-ischemia, however after release the ligature a significant reactive hyperemia was observed by mln. into the rapeffusion phase. histolog[c examination of reper[used gastrocnemius muscle revealed moderate pmn infiltration into the interstitium. in conclusion, these data indicate that paf is released into the circulation during repeffusion, and is likely involved as a mediator in the observed pmn oxidative burst activity, thereby contributing to reperfusien injury. following thermal injury and infection granulocyte function ts abnormal. to elucidate the mechanism by which thermal injury and infection affect the granulocyte's ability to polymerize and depolymedze actin, we serially measured f-actin levels in granulocytes from burned patients (mean age , +_ . years, mean burn size . % _+ . %) during the first s weeks post injury. six of the patients had infections during the course of the study, (septicemia, wound invasion and pneumonia). actin levels in granulocytes from eleven healthy volunteers (mean age years) were measured repeatedly and served as controls. lysecl white blood cell preparations were brought to c and incubated with n-formyl-met-leu-phe (stim) or with dulbecco's phosphate unbuffered sellne (unstim). the cells were concomitantly stained and fixed with formaldehyde, lysoleclthln and fiuoresceln phafioidin. actin depolymedzation (depol) was measured by incubating stimulated cells at °c before the stain-fixative was added. baseline (base) f-actln levels were assessed by adding stsln-fixatlve to icecold unstimulated cells. fluorescence was estimated in a facscan and expressed as ilnesr mean channel fluorescence_+ sem (mcf). figure displays granulecyle fectln levels in infected and uninfected patients as compared to controls. f-actln levels were consistently lower in control cells than in those from burned or burn-infected patients under all measured conditions. granulocytes from infected burned patients demonstrated a significant decrement in their ability to depofymerlze f.actin compared to both uninfected burned patients and controls, while there were no significant differences between infected and ,~ uninfected patients in the baseline, unstlmuleted and stimulated conditions. those results indicate la that grsnulocytas from burned and bum-infected patients contain higher levels of polymerized actln than ~ , s control cells. in order to study tumor necrosis factor (tnf) receptor sensitivity in septic critically ill patients we investigated blood samples of such people in reaction of leucocyte migration inhibition. migration of their polymorphonuclear leucocytes (pmns) was studied with stimulation with human recombinant tnf in concentration of . u/ml (recommended by manufacturer is the range of - o/ml) and without such. ten healthy blood donors formed control group. the results obtained showed diminished pmn reactivity to tnf in patients (migration inhibition was absent) oscaring with significantly increased migration ability of their pmns ( . % of that in control group). at the same time normal pmns in control group did show migration changes upon tnf stimulation. considering all the above we come to a conclusion that externally added tnf fails to activate pmns in critically ill patients more than they are by their endogenous tnf. moreover, this tnf no longer serves a positive chemotactic factor for such pmns. these findings may suggest that in critically ill septic patients reactivity of pmns to tnf is deeply altered. tnf receptors of pmns are either exhausted as such by excessive stimulation with endogenous tnf or further transmission of their message is impossible due to "fatigue" of the cell's activation mechanisms. we express our gratitude to reanal factory of laboratory chemicals for generously providing us with a tnf com~rcial sample. ~-sanguis medical, ekaterineburg russia; s-urals med.lnst. activated neutrophils infiltrating the local site of inflammation following trauma release high amounts of destructive lysosomal enzymes into the extracellular space. cytokines were discussed to be involved in regulation of this early process. the task of this investigation was to evaluate the possible regulatory role of interleukin- (il- ) and its potential immunosupressive opponent, the transforming growth factor-&, in regulation of neutrophil degranulation. we analysed the concentration of the al-proteinase-inhibitor complex of the lysosomal elastase as marker for the degranulation of neutrophils as well as the levels of il- and tgf- in the plasma probes of patients undergoing multiple trauma and severe surgeries. the time courses of il- and elastase were found to be highly correlated, wheras the concentrations of the cytokine tgf-e~ were found to be not significantly altered in comparison to the control group. this close temporal correlationship was confirmed by investigation of fluids derived from sites of inflammation. interstingly, the inhibitory potential (~zcproteinase inhibitor, antithrombin iii) was dramatically reduced in the early inflammatory phase. to prove this in vivo findings, the effects of il- and tgf-i~ on the degranulation of isolated human neutrophils of healthy donors was investigated in vitro. pathological high concentrations of rhll- up to u/ml (as detected in fluids derived from local inflammatory site) were found to be capable to induce a significant release of lysosomal elastase in a concentration-dependent manner, whereas the degranulation of neutrophils was uneffected by tgf- . in conclusion, these data suggest a contribution of il- in regulation of neutrophil activation at sides of inflammation. the immunosuppressive cytokine tgf-i&~ seems to have no direct regulatory effect beside its described chemotactic function on neutrephils. postirradiation chan~es of adhesive properties arid supercoiled nucleoid dna structure of blood leukocytes were studied in macaca nemestrina andrats. the dynamics of membrane chan~es after nonlethal irradiation of rats demonstrated the temporary increase of the leukocyte adherence at h followed by return of this parameter to normal levels at h. after lethal irradiation of both animal species the increase in adhesive leukooytes fraction was detected as early as at h. this hi~her index persisted until the end of experiments ( days). the early ( - h) temporary loosin~ of supercoiled dna structure was demonstrated in the leukocytes of nonlethally irradiated animals. this phenomenon seems to be connected with the lymphocyte fraction chan~es. this process was not dependent on altered adhesive properties of leukocyte membranes. the membrane chan~es of leukocytes preceded decondensation of supercoiled dna after lethal irradiation of animals, in this case loosin~ of supercoiled dna pro-~ressively increased at h and at the later terms of postirradiation period. the systemic inflammatory response syndrome (sirs) involves many inanunological reactions of the host including acfivatinn of inflammatory mediator cascades and depression of cellular reactivity in t-lymphecytes ( ). there are reports of nentrophil dysfunction in inflammatory disorders of the skin ( ), are there dysfunctions concerning the unspecific host defense in sirs, as well? in this study, we examined the reactivity of neutrophil granolocytes from patients suffering from sirs. twenty-one patients (apache ii-score ± ) with diagnosis of sirs entered the study. granulocytes were prepared as reported previously ( ) . in parallel, granulocytes from healthy individuals were tested. two granulocyte functians were studied in vitro: . migration of the ceils in a boyden chamber through a filter matrix following stimulation with different receptor dependent stimuli (c a, intefleukin- , platelet-activating-factor, leukotrien b , fmlp). . release of glucuronidase following stimulation with the aforementioned activators. the results demonstrate, that the release of -glucuronidase in patients suffering from sirs was comparable to the enzyme release of granulocytes prepared from healthy individuals. each stimulant induced release of p-glucuronidase in a characteristic dose dependent fashion. all granulocyte preparations from the healthy donors showed a positive chemotaxis response in the migration-assay. in contrast, only ten out of twenty-one patients had granulocytes migrating after stimulation. the two groups of patients displaying reactive or non-reactive granulocytes differed clinically: the nonreactive group consisted of patients with multiple organ failure ( / ) and nonsurvivors ( / ), whereas / patients in the reactive group survived. thus, the in vitro chemotaxis of granulocytes is impaired in a subgroup of patients with sirs. this defect of the non-specific host defense may contribute to poor prognosis and outcome of these patients. dermatol. : - , klinik ffir an~isthesiologie und operative intensivmedizin der cau kiel, schwanenweg , kiel, germany. objectives of the study: major emphasis has been given to the analysis of interactions of antibiotics with microorganisms. effects of antibiotics on cells of primary host defense mechanisms, such as the neutrophils, are less well known. therefore, attention has been focused on clindamycin, a member of the lincoseamide family. materials and methods: the effect of clindamycin (i -i ~g/ml) on granulocyte functions (healthy volunteers) such as random migration, chemotaxis (agarose method), ingestion (radiometric assay), superoxide (cytochrom c reduction) and hydrogen peroxide production (phenol red oxidation), lucigenin-and luminol-amplified chemiluminescence (luminometry) and degranulation (turbidometry with micrococcus lysodeicticus) were investigated in vitro. results: motility and degranulation were inhibited, ingestion of saccharomyces cerevisiae, zymosan-induced lucigenin-and luminol-amplified chemiluminescence, superoxide and hydrogen peroxide production were stimulated in a dose dependent fashion. conclusion: clindamycin has granulocyte function modulating properties. recognition of immunomodulating effects of antibiotics may have therapeutic significance, especially in patients with long-term antibiotic therapy or immune deficiencies. the intense muscle activity (ea) of rats resulted in increase of neutrophil influx in muscles during the recovery. we investigated neutrophil proteinases involvement in neutral proteinases balance of skeletal muscles by na. the rats were submited to swim with the load ( % of body mass) till exhaustion. immediately after na the neutrophil antiserum was injected i.p. to rats of experimental group. saline was injected to control animals° injections were repeated in h of the recovery and cytosol proteolytic activity (ph . ; fitc-casein) was determined. isolated soleus muscles were incubated also in vitro and proteolytic activity of incubation media was measured. it was found that there was - -fold proteinases activity increase in cytosols of all investigated muscles (soleus, white and red portions of quadriceps) of control animals by h of the recovery (the comparison was done with the sedentary rats). in h cytosol proteolytic activity decreased and then increased again by h of the fast. antiserum injections resulted in relible decrease of the proteolytic activities at every investigated time. when incubating m. soleus in vitro the activities of proteinases in incubation media turned out reliably less if soleus muscles were isolated from the animals to which antiserum was injected. the conclusion is that neutrophil proteinases can be involved in the balance of rat skeletal muscle neutral proteinases after ~a. a lot and new clinical problems complicating the outcome of polytrauma, burn and septic patients in surgical intensive care units, have arisen as the care improvement prolonged the patient's survival: a progressive degradation of organ and system functions often develops, usually making its first clinical appearance by ards, followed by the other organ failure (mof) and sepsis symptoms. the clinical picture is polymorphic, the end result of a complex systemic pathophysiological reaction trigg~ed off by trauma consequences (tissues disruption, hypo~xygenatiun and necrosis). nowadays there is not a preventi~ or specific therapy to lower the mortality rate ( - %) and-'mdy-a~ early, aggressive surgical approach .-evacuating haematomas, stopping bleeding, toileting all septic, necrotic foci and restoring anatomic continuity-, seems to be of some help this complex clinical entity has not an univocal denomination yet. the proper labelling of an illness should come from the full understanding of its pathopysiology and suggest the proper treatment choice. clinical and experimental studies demonstrated that pathophysiologic mechanisms involved in the past-traumatic illness, share the same anatomo-pathological elemem: the interstitial edema, due to a generalised endothelial micro circulatory injury. this alteration, as constantly seen in polytrauma patients, develops in a few hours after trauma as a consequence of the deregulation of the homoeostatic and immune mechanisms. in fact the overproduced oxygen free radicals and r~ombinam cytokines (il ,tnf), together with the complement degradation fragments, the proteolytic enzymes and many other mediators are all strongly h~l ~ ,_he e,,j,yheha! ceils. our~osect, atim~,-bnsed on examination of autopsical specimens from polytraanm patients, showed that such endothelial damage, supporting the interstitial edema, is widely and simultaneensly distributed, ensues shortly arer trauma and shows its effects in different organs at different times, only because each apparatus has different fimctienal reserves: the lung is the first organ to fail just because its ah, celocapillary membrane is one of the most delicate bodily structure, and its function is irroplace~le. we think it will be of a great help, in planning a preventive therapy, to chose a denomination focusing the physician's attention on the earl)" generalized endothelial injury and its effects, as in trauma patients it is present -even if latenflysince the first few hours. we would like to see the generalised endothelial microcircolatory injury properly highlighted when considering the best definition and the optimal nomenclature for the post-traumatic s mdrome. the presence of interleukin (il)- in bronchoalveolar lavage fluid of critically ill patients correlates clinically with the development of the adult respiratory distress syndrome lards), and inhibition of il- in animal models can attenuate lung injury. collectively, evidence to date suggests that il- attracts and activates neutrophiis (pmn), which are then responsible for the capillary leak of ards. however, an alternative explanation is that il- is directly toxic to the endothelial cell (ec). in this study, we have hypothesized that il- can disrupt endothelial integrity independent of pmn. meth ods: human umbilical vein (huv) ec monolayers were cultured to confluency on collagen-coated micropore filters. to assess ec integrity, .albumi n leak was quantitated by measuring the counts which crossed the monolayer, using a gamma counter. il- (lpg/ml) was incubated in the culture medium with .albumi n for hrs. the il- dose was not cytotoxic. to determine the involvement of protein synthesis in this process, selected monolayers were pretreated with cycloheximide (ch) prior to .- addition. statistical analysis was performed using anovmfisher plsd. we have previously shown that platelet activating factor (paf) enhances cdt expression and primes pmn's for subsequent generation. both are important steps in pmn mediated injury and are assumed to occur in concert. following major trauma non-specific pmn inflammation is activated, however, unbridled systemic pmn activity needs to be minimized. since circulating catecholamines are high early post-injury, we hypothesised that they downregu/ate cd expression and pmn priming via the [ adrenergic signal transduction pathway. methods: normal human pmns were primed with paf ( ng/ml for min) or pre-treated with - m of isoproterenol (i) or forskoklin (f) for rain and then primed with paf. cd expression was measured by flow cytometry (fig.l) and -generation in response to -rm fmlp was determined as sod inhibitable reduction of cytochrome c ( fig. holler** and georg w. bornkamm* lymphocyte-endothelial interactions are crucial for various immune responses, including cytokine driven inflammatory processes. protein kinase c (pkc)-inhibitors on the other hand are discussed as potential cytokine antagonists. in the present study we investigated the influence of the pkc-inhibitor gf x on cytokine-and endotoxin induced expression of intercellular adhesion molecule (icam- ) and on adhesion of lymphocytes to cytokine activated endothelial cells. we found that tumor necrosis factor alpha (tnfo -and lipopolysaccharide (lps)-induced icam- expression on human endothelioma celts (eahy ) were unaffected by the pkc-inhibitor and thus appeared to be independent of pkc activation. in contrast, gf x significantly reduced icam- expression induced by interferon-y (ifn-?) and interleukin- (il- ). the functional relevance of these findings was evaluated in an adhesion assay using human umbilical vene endothelial cells (huvec) and peripheral blood mononuclear cells (pbmc). in fact, the ifn-? and il- induced adhesion of pbmc to cytokine treated huvec could be downregulated by the pkc-inhibitor, whereas tnfc~-and lps-mediated adhesion was not influenced. additionally, the il- driven icam- expression on eahy cells as well as the il- induced adhesion of pbmc to huvec was found to be tnf-dependent, since both effects could be inhibited by an anti-tnf monoclonal antibody ( f) . these in vitro data further support the idea of examining pkc-inhibitors, such as gf x, for their biological relevance in cytokine related dysregulations. seiffge, d., bissinger, t., laux, v., during inflammation there are some key processes, which occur in the microcirculation: the release of mediators from various cell types, the migration of inflammatory cells towards a chemotactic stimulus in the tissue, the expression of adhesion molecules on different cells, and the extravasation of plasma proteins. the aim of the present study was to elucidate the mediator induced interaction of leukocyte adhesion and plasma leakage in postcapillary venules. using an analogous video-image analysing system we have studied the effect of different mediators on leukocyte adhesion and macromolecular permeability in the mesentery of the rat. the increase in permeability was measured as changes in optical density. we found that topical administration of leneotriene b (ltb , x " tool/l) or intravenous injection of interleuldn- (il- , - iu/kg b.w.) and lipopolysaccharide (lps, mg/kg b.w.) resulted in a significant extravasation of fitc-labelled rat serum albumin (fitc-rsa) in venules but not in arterioles. we could correlate the changes in vascular permeability with a locally increased number of rolling and sticking leukocytes in venules. both effects were dose dependently inhibited by different drugs. pentoxlfylline inhibits lps-indueed fitc-rsa extravasation and leukocyte adhesion at a dose of mg/kg b.w., superoxid-dismutase (sod, . iu/kg b.w.) was able to decrease the ltb effect, and the immuumodulating drug leflunomide (hwa ) exerted inhibitory effects on il- -induced permeability at a dose of mg/kg b.w.i.v. the obtained results demonstrate that lps, ltb or il- induced extravasation of fitc-rsa is mediated by activated leukocytes and can be deminished following administration of different drugs. platelet-endothelial cell adhesion molecule-i (pecam-i), a member of the immunoglobulin superfamily, is constitutively expressed at high levels on the endothelial cell surface. in vitro data have suggested that pecam-i functions as a vascular adhesion molecule, specifically in neutrophil transmigration across the endothelium. this current work is the first demonstrating the in vivo role of pecam- in neutrophil migration. blocking antibodies to human pecam- , in which the antibodies are crossreactive with rat pecam- , were able to block the movement of neutrophils into the rat lungs after igg immune complex deposition. furthermore, when human foreskin was transplanted into mice with severe combined immunodeficiency and the site injected with tnf-alpha, anti-pecam-i blocked neutrophil emigration into the dermal interstitium. it has already been established that neutrophil recruitment is dependent upon selectin mediated rolling, followed by firm adherence that is icam- / integrin mediated. these data suggest, for the first time, that a third endothelial adhesion molecule (pecam-i) is involved in the coordinated recruitment of neutrophils in vivo. to test whether trauma causes generalized activation or priming of pmns, cdi adherence receptors were measured with iinmunomonitoring in whole blood after lps stimulation ex vivo. anesthetized (fentanyl) mongrel pigs ( - kg) were subjected to % arterial hemorrhage + soft tissue injury and after liar, resuscitated with all the shed blood + supplemental fluid. blood was collected at hr intervals from unanesthetized animals with indwelling catheters, pmns were counted, and lps was added ( , , , i.tg/ml) ex vivo. after hr incubation at - °c, %cd (+) pmns were determined with fitc-ib and flow cytometry from mean channel fluorescence histograms. ± # p< . vs baseline * p< . vs sham $p< . vs no anesthesia these observations provide direct evidence for time-dependent changes in pmn priming following major injury because cd expression was depressed for at ]east hr after trauma relative to sham but by hr, was enhanced, relative to sham, and because fentanyl anesthesia at hr had a greater effect on cd expression in trauma vs sham. neutrophil (pmn) adhesion to vascular endothelial cells (•c) is a key element in the inflammatory response and tissue injury. inflammatory mediators such as lps (exogenous) and tnf (endogenous) can promote pmn-ec interaction which is believed to be responsible for capillary leakage and subsequent organ injury. however, the mechanism of this injury remains unclear.we hypothesised that the mechanism of tissue injury is due to ec necrosis with release of toxic products and that activated pmn are responsible. human pmn were obtained from healthy donors, separated by density gradient, and activated with lps ( ng/ml), tnf( ng/ml), and lps/tnf( ng/ ng/ml). cultures of the human ec tine(ecv- ) were used as surrogates of the microvasculature, were exposed to either lps, tnf, lps/tnf and pmn activated with lps, tnf, lps/tnf and incubated for , , , and hrs. ec necrosis was assessed by a cr release cytotoxicity assay. pmn activation was assessed by cd lb receptor expression and respiratory burst activity hr _+ . -+ -+ . _+ _+ . _+ _+ . _+ . hr + . _ _+ . _+ _+ _+ " +_ +-- . " lghr - . _+ +_ - " o:fo , " ~ +- . * hr _+ . - -+ +_ * _+ _+ * _+ _+ " data = ec % necrosis mean_+sd stats: student's t-test with significance (*) set at p< . vs control. ( our previous studies have indicated that despite the increased cardiac output and maintenance of tissue perfusion, hepatoceliular dysfunction occurs during early sepsis. nonetheless, it remains unknown whether vascular endothelial cell function (i.e., the release of endothelium-derived relaxing factor/nitric oxide) is depressed under such conditions and, if so, whether endothelial cell dysfunction also occurs at the microcirculatory level. to determine this, rats were subjected to sepsis by cecal ligation and puncture (clp), following which these and corresponding shams received ml/ g bw normal saline. at hr after clp (hyperdynamic sepsis) or sham operation, the thoracic aorta was isolated, cut into rings, and placed in organ chambers. norepinephrine (ne, xi - m) was used to achieve near-maximal contraction. responses for an endothelium-dependeut vasodilator, acetylcholine (ach, via nitric oxide), were determined. in additional studies, the small gut was isolated at hr post-clp. after pre-contraction of blood vessels in the isolated gut with xl m ne, vascular responses to ach ( x m) and an endotheliumindependent vasodiiator, nitroglycerine (ntg, xl - m), were determined. total vascular resistance (tvr, mmhg/mi/min/ g) was then calculated as pressure/ perfusinn rate. ach-induced relaxation (%, n= /group) in the aortic rings were: ach lxl i~s, st-in ~ ~ significantly at hr post-clp (i.e., increased *p(o vs. sham; n- per group. tvr) in the absence of any changes in ntginduced relaxation (fig. a) . thus, the vascular endothelial cell dysfunction observed in the aorta in early sepsis also occurs at the microcirculatory level. introduction: the cytokine-mediated adherence of leulcooytes to vascular endothelium is considered as an early step in the cascade of pathologic reactions culminating in the "systemic inflammatory response syndrome" (sirs); the purpose of this study was to evaluate the influence of interleakin- on leukooyteendothelial cell-interactions and microoirculation in the liver after hemorrhagic shock by means of intravital microscopy. methods: in anesthetized female sprdrats co.w. - g) shook was induced by fractionated withdrawl of arterial blood within rain and maintained for h (map at mm hg, cardiac output % of baseline). rats were adequately resuscitated with % of shed blood and twice the volume in ringer's solution additionally. following h of reperfusinn (map > mm hg, co > % of baseline) the microcirculation in liver lobules was examined by intravital fluorescence microscopy after labelling of leukocytes. continuous administration of il-lra (synergen, boulder, colorado, mg/kg/h) was started at different time points in a randomized and blinded manner. the animals in group p (n= ) received the il-lra as pretreatment beginning min prior to shock induction. in the group t (n= ) the application of il-lm started at the beginning of the reperfusion period with a bolus injection of mg/kg and was followed by continuons administration of mg/kg/h. the control group c (n= ) received equal volumes in nac , %, the sham-operated group s (n= ) was not exposed to shock. results: macrohemodynamics were comparable in all shook groups. the increased percentage of permanendy adherent leukocytes after hemorrhagic shook (s: , % + , %; c: , % _+ , %) was significantly reduced by pretreatment or treatment with il-lra (p: , % -+ , %; p< . , t: , % -+ , %, p< . , anova). temporary adhesion of leukocytes was unaffected by application of il-lra. liver microcirculation measured by volumetric blood flow in liver sinusoids and sinusoidal diameters was impaired after hemorrhagic shock in all groups and was not affected (c: iam /s + um /s, p: llm /s + }am /s, t: ams/s -+ lam /s, s: am /s -+ am /s). di.seu~sinn: the results demonstrate that permanent adherence of leukocytes to endothelium is in part regulated by il- . pathological adherence could be reduced by application of illra, even given at die time of resuscitation. the effect of ll-lm on permanent adhesion is a specific event and might be caused by reduced expression of specific receptors on sinusoidal endothelial cens and leukocytes. objectives of the study. the adhesion of activated neutrophils (pmn) to endothelial ceils (ec) and the concomitant production of reactive oxygen metabolites (rom) initiates organ damage after trauma, sepsis, shock and organ reperfusion. aien of this study was to investigate the effect on adhesion and rom production of the highly water-soluble, membrane-permeable and physiological ascorbic acid (asc). materials and methods. adhesion of pmn to nylon fiber (cell count) and simultaneous rom production (chemiluminescence-cl-response) were measured up to retool/ asc as well as adhesion, rom production and ec damage (lllln-release from labeled ec) of endotoxin-activated pmn to cultered ec moanlayers. in an in vivo animal model (sheep with lung lymph fistulas) the effect of asc ( g/kg bw bolus, followed by . g/ kg-h infusion) on the endotoxin-induced ( . ixg/kg bw) neutropenia (cell count), lung capillary permeability damage (lung lymph protein clearance) and rom production of neutrophils (zymosan-induced cl response) was measured. results. asc scavenged rom dose-dependently during adhesion of pmn to nylon fiber (p< . at mmol/l asc), adhesion itself was unchanged. during the activated pmn/ec interaction asc scavenged rom (p< . at mmol/l asc) and reduced the adhesion dose-dependently (p< . at mmol/l asc); ec damage was also reduced (p< . at retool/ asc). in the in rive model asc increased the endotoxin-induced blood pmn decrease (p< . ), decreased the protein clearance (p< . ) as well as the zymosan-induced rom production (p< . ), indicating the asc-mediated reduction of adhesion, rom production and lung tissue damage processes. conclusions. by in vitro and in rive experiments ascorbic acid reduced the adhesion-and rom production-initiated tissue damage. therefore, i.v. administration of ascorbic acid is recommended for oxidative stress-associated states after trauma, sepsis, shock and organ reperfusion. for neut rophi l-accumulat ion and activation. we investigated the influence of or to the activation and the expression of lecam-i and cdiib,cdi on neutrophils and lymphocytes. methods: from blood samples (n= ) all white blood cells (wbc) and neutrophils (nc) were isolated and cultured. or were produced via the xanthine oxidase/hypoxanthine system. after , , , , and minutes a giemsa-staining to determine the granulation of neutrophils (n: normal, r : reduced ) and a facs-analysis with monoclonal antibodies detecting cdiib,cdi and lecam-i was performed. results: under the influence of or a degranulation of neutrophils starting at min was observed in wbc-cultures (n/r: min / , min / , min / , min / , min / ). these data were confirmed in the dot-plots of facs-analysis. only in wbc-cultures or induced a significant increase of lecam-i expression on neutrophils up to min followed by a decrease to normal values at min. lecam-i on lymphocytes disappeared totally during the observed period. cdllb,cdl -expression was not altered. conclusion:increased lecam-i expression on neutrophils due to or could enhance the 'rolling' of neutrophils along the endothelium which is a prerequisite for neutrophil sticking and migration. further or are able to activate neutrophils without endothelium. these changes seem to be mediated by other wbc. introduction. multiple organ failure (mof) has been hypothesized to be the result of an excessive uncontrolled autedestructive inflammatory response. since the complement system is an important mediator and initiator of the inflammatory response, interruption of this cascade could theoretically lead to an attenuation of mof. in order to test this hypothesis we evaluated the response of c -delicient mice in a model of zymesan indt~ed mof. materials and methods. c -deficient b d /oid and c -sufficient b d /new mice were used in this study. on day all mice received an intraperitoneal injection with zymosan suspended in paraffin in a dose of mg/g body weight. between day and , biological parameters (temperature, body weight and clinical condition) were measured daily and mortality was monitored. clinical condition was assessed by blindly grading the degree of lethargy, conjunctivitis, diarrhea, and ruffled fur of each mouse on a two point scale (maximum score= ). on day all surviving mice were sacrificed and relative organ weights of lungs, liver, spleen and kidneys (relative organ weight= (organ weight/body weight)x ) wore calculated. earlier experiments with our model have shown a good correlation between histological organ damage and relative organ weights. statistical analysis of biological parameter was performed using the koziol curve analysis. analysis was divided in an acute phase (day - ) and a late phase (day - ). relative organ weights were analyzed using wilcoxon's test and mortality rate using fischor's exact test. results. all zymosan injected mice showed a typical triphesic illness. deterioration of the clinical condition as indicated by the symptom score and the decrease in temperature and body weight in the acute phase were all significantly lass severe in c deficient mice (all p< . ). in the late phase no differences could be noticed in the courses of biological parameters. overall mortality was / ( %) in c deficient mice and / ( %) jn c sufficient mice (p= . ), a difference mainly due to a difference in the acute phase. organ damage assessed as the relative organ weights did not show any statistical differences for any organ between both strains. conclusion. complement factor c appears to play an important role in the acute hyperdynamic septic response in this model but deficiency of c could not prevent organ damage in the late mof phase. this suggests that other factors could be more important in the development of the inflammatory response leading to mof. proinflammatory cytokines are thought to play a critical role in the pathophysiology of multiple organ failure (mof). in mice, zymosan-lnduced generalized inflammation (ztgi) leads to mof. therefore we performed a sequential study into plasma levels of, and macrophage production capacity for, four cytokines during the development of mof in the zigi model. male young-adult c bl/ mice received zymosan ( mg/g body weight) intraperitoneally. groups of animals were killed after , , , and h and subsequently at each day until day . plasma was collected and peritoneal macrophages were isolated and cultured overnight with or without lipopolysaccharide (lps). interleukin -ct, and - (il-lc~,~,), and tumour necrosis factor-o~ (tnf-c were measured in plasma and culture fluid by means of a ria (detection limit . ng/ml). interleukin- (il~) levels were assayed using the b hybddoma cell proliferation assay. zymosan induces a three-phase disease in mice. after an acute phase the animals recover. around day , they start to develop clinical signs which resemble mof. plasma tnf-~ peaked within h after zymosan injection and disappeared within h. from day onwards, tnf levels started to rise again. plasma il- behaved almost similarly in the acute phase, but in the mof phase plasma il- remained low. no circulating il- could be detected at any time point. macrophage lps-stimulated production of il-lcq il- ~ and tnf--c~ was suppressed immediately after zymosan injection. production of il- and tnf-~ was normalized within h, while production of il-lc~ remained lower than that in macrophages from untreated control mice. only at day did production of il-i~ reach control values. il- production was higher than control values from day onwards. il production was similar to that of ili-il the production of tnf-ct was strongly elevated between days and and again during days to . the development of mof-like symptoms during zlgi in mice is accompanied by increased plasma levels of tnf-ct without enhanced il- or il- . also, the ability of macrophages to produce excessive amounts of il- and tnf--~, as well as the suppressed capacity to produce il-lcq could be important mechanisms in the pathophysiology of mof. when conjugated to an asialoglycoprotein, dna and oligonucleotides are specifically taken up by the hepatocytes via the asialoglyccprotein receptor which is unique to the liver. human asialoglycoprotein (~ -acid, asgp) was derivatized with low molecular weight poly(l)lysine(pll) and complexed with antisense dna's (as) complementary to the ' region of the il- gpl receptor. the antisense were '-agtttagggatgagg- ' (asl), '-atcttcatcttctgaat- ' (as ), '-aagtgaatgattaaaacact- ' (as ), '-aaacctttataggcg- ' (as ), and '-cgttctacaactgcaacgt- ' (as ). using hepg , the biological effects of these antisense complexes on the high affinity il- receptor were evaluated by scatchard analysis, cellular proliferation, and acute phase protein expression by radioimmunoprecipitation and two dimensional gel electrophoresis. scatchard analysis demonstrated that high affinity receptor expression was inhibited by incubation of cells with asgp-pll-asi for h. underivatized asl was less effective and the complex, asgp-pll-as , had minimal effects on high affinity binding. when the cells were treated with the conjugates and stimulated with il- (i units) asgp-pll-asi alone showed a dose dependent ( .i- . ~m) inhibition of ss fibrinogen synthesis. two dimensional gel electrophoresis showed that expression of other acute phase proteins was also blocked. these results indicate that the targeted delivery of antisense molecules via conjugates recognized by the asialoglycoprotein receptor can block the cytokine stimulated acute phase protein response in hepatocytes, this approach may be relevant to the therapeutic management of patients with severe injury and sepsis. it has been established that immune cells are able to express neuropeptide genes and to release products that were considered to be of neuroendocrine origin. we have shown that proenkephalin (penk), a neuropeptide encoding gene, is expressed in lymphoid cells in culture. to study the physiological significance of these observations we have used the model of experimental endotoxemia. in this model, a disease state is induced by bacterial lipopolysaccharide (lps), that activates the immune system, the adrenocortical axis and the nervous system. we found that the expression of penkmrna is markedly enhanced in vivo immediately after lps injection both in the adrenal glands and in the lymph nodes. in situ hybridization analysis combined with immunohisto-chemistry indicated that the induced penk expression is confined to macrephages within the lymph nodes and chromaffin cells in the adrenal medulla. furthermore, this expression in lymph nodes is modulated by ligands of the adrenergic system. our results strongly support the notion that immune derived opioids participate in the bidirectional communication between the nervous and immune systems. of neurology hadassah university hospital, jerusalem , israel. objectives of the study: multiple-organ-failure is recognized as the most severe, and often lethal, complication after multiple trauma. however there is no adeqate animal model available. our goal was to develop an animal model, in which reproducable irreversible failure of parenchymal organs is achieved in the late phase after insults in the early phase (trauma). materials and methods: l female merino-sheep were included (mean weight: kg). day : hemorrhagic shock (mean arterial pressure (map) mmhg for hrs.), closed femoral nailing (ao-technique), day - : bolusinjection of endotoxin (et) ( , ~tg/kgbw) und zymosan-activated plasma (zap) ( ml) every hrs., day - : observation. bronchoalveolar lavage (bal): day , , . the course of representative parameters of organ function was documented: cocardiac output (i/min), svr -systemic vascular resistence (dyn ~ s cm- ), pap -putm.art.pressure (mmhg), pap -arterial oxygen pressure (mmhg), bill -bilirubin (;xmov ), crci -creatinin clearence (ml/min) statistics: data as means+sem, *significant from baseline (wileoxon test; p< ) results: baseline day day day day heart: co , _+ , , _+ , , _+ , , _+ , * , _+ , * svr _+ + _+ +_ " +- " lung: pap , _- , , _+ , " , +- , " , + , " , +- , ' pap , + , , +- , , _+ , , +- , , +_ , * liver: bill , _+ , , _+ , ' , _+ , ' , _+ , " , _+ , " kidney:crcl , +_ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , _+ , , + , , + , histologic specimens showed all signs of fulminant mof. combination of hemorrhagic shock, femoral nailing, et und zap (insults in the early phase) lead to an irreversible organ failure in the late phase. prostaglandin e (pge) levels are elevated by trauma, shock or sepsis and can profoundly affect the immune response. pge is produced by many cell types including fibroblasts, macrophages, monoeytes, follicular dendritic cells, and epithelial cells and is induced by il-i, bacterial lps, components of the complement cascade, tnf, il- and crosslinking of surface fc receptors for igg, iga and ige. our research has shown that pge inhibits b cell activation (specifically enlargement, class ii ~c and fc~ rii expression), proliferation, igm and igg responses, t cell proliferation, and il- synthesis in the mouse model. in contrast, pge greatly promotes class switching to ige,the isotype responsible for type i allergic hypersensitivity. thus, our model mirrors th~ general immunosuppression and elevated ige titers of the trauma or sepsis patient. pge increases the number of cells secreting ige and iggl, acts on surface igm positive b cells, synergizes with il- and lp$ to induce preswitch germline transcripts, and induces more rapid expression of mature vdj~ mp~a than in eontro~ pge intracellular signalling occurs through cyclic adenosine monophosphate (camp) levels and can be mimicked by camp-inducing agents and blocl~ed by an inhibitor of campdependent protein kinase a. pge action requires de novo protein synthesis and candidate pge-inducible regulatory proteins have been identified by d gel eleetrophoresis. thus, pge inhibits a number of immune mechanisms while promoting ige production. a deeper understanding of pge immune regulation may lead to more effective treatment of immune perturbations as sequelae of trauma, shock or sepsis. during infrarenai aortic surgery mesetueric traction (re.t.) results in prostacyclin (pgi:) release and consecutively in hemodynamic disturbances (decreased systemic vascular resisteace, mean arterial pressure; increased cardiac output, heartrate). these symptomes are bypassed by cyclooxygenase inhibition. hemodynamic symptoms vanish after - rain even without cyclooxygenase inhibition although pgi levels remain elevated. to study the endocrine vasopressor system in a prospective double blinded protocol, we investigated patients undergoing major abdominal surgery as compared to ibuprofen ( rag, i.v.) pretreated (ibu) patients. the surgeon applied m.t. in a uniform fashion. we chose a general anesthesia combined with a supplemental thoracic epidural anesthesia. at the points in time , , , , , , , rain after and before (to) mesentzrie traction we determined the plasma concentrations (pc) of -keto-pgf~o~pr~, epinephrine, norepinephrine, dopamine, renin, aldosterone, adh and cortisol. pc of -k-pgf~,tp~, peaked minutes after m.t. ( _+ , ibu: _+ , to: +i ng/l) and declined monotonously over h ( +_ , ibu: _+ ng/ ). catecholamine pc "s did not exceed the reference range during the observation period. reninpc peaked after rain ( _+ , ibu: + , to: -+ /~u/ml); aldosteronc also presented a maximum after rain ( + , ibu: -+ , to: +- pg/ml), whereas cortisol demonstrated irrespectively of circadian rhythms a maximum h after m.t. ( +_ , ibu: -+ , to: +_ ~g/ ). adh pc peaked min after m.t. ( + , ibu: -+ , to: +_ pg/ml) and showed analogously to -k-pgft~j~ pc a monotone decline over the observation period. our data demonstrate a counteractive reaction to pgiz mediated vasodilation via adh secretion. the second regulative is the renin-angiotensin-aldosterone system (raas), which is activated min after m.t., the aldosterone pc does not paratlel the cortisol pc, which peaked post operafionem in both groups, probably due to the end of anaesthesia. a regulative release of catecholamines could not be documented. the activation of adh and raas after mt is not a hormonal response primaryly related to surgical trauma and/or stress but a counterregulation to systemic vasoditafion induced by prostacyclin. although adh and raas support systemic circulation, angiotensin and vasopressin may compromise local organ blood flow (e.g. splancimic vascular bed). insfitut f. klin. chemic, anaesthesiologie ~, chirurgie l*, univ. ulm, elm, expression of c-fos protein in rat brain following occlusion of superior mesenterie artery. takanobu there is general agreement that neurologic abnormalities are seen in sepsis. the aim of this study is to examine what effect does the brain receive in case of sma occlusion by immunohistochemistry using antibody to c-fos, an immediate early gene, which is recently recognized as a genetic marker of activated neurons. moreover, we investigated the correlation between c-fos induction in the brain and plasma endotoxiu level. rats of them received sma clipping and others wee used as control. control and treated rats at , , , hours were perfused and fixed. the brain were sectioned at pm and stained by abc method using c-fos antibody. plasma endotoxin level of rats were measured at , , , , hours after the treatment by chromogenic limulus method. immunohistochemical study showed scarcely no immunoreactivity in control rat brain. in treated rat brain, the significant expression of c-los was detected in specific nuclei including the habenula, some hypothalamie nuclei, amygdala, locus ceruleus and nucleus tractus solitarii. such immunoreactivities were increased in time curse, which well corresponded plasma endotoxin levels. the mean plasma endotoxin level of , , , , hours after the treatment were . ± . , . _- - . , . _+ . , . ± . and . ± . pg/ml, respectively. the results indicate that limbic and hypothalamic-brainstem systems are involved in sma occlusion, and suggest that such neuronal actival.jon may precede the elevation of plasma endotoxin icy.el. systemic vascular resistance and increased cardiac output accompanied presumingly by a increased pulmonary shunt (qs/qt). this response is induced by prostacyclin (pgi ). we examined oxygen transport after traction on the mesentery root and the transpulmonary prostacyclin levels in a prospective placebo controlled study with intravenous ibuprofen. methods: with approval of the human [nvestigadon review board we studied patients in a prospective, randomized double-blinded protocol who were scheduled for major abdominal surgery. ibuprofen ( mg i.v.) or a placebo equivalent was administered minutes before skin incision. pulmonary artery thermodilution and radial artery catheters were placed after induction of anesthesia. mt was applied in a uniform fashion. baseline values preceded the incision of the peritoneum (to). fulther assessments followed , , , , . tile plasma concentrations (pc) of -keto-pgft, (stable metabolite of pgi ) were determined in arterial and mixed venous blood by radioimmunoassay. at all points in time we measured arterial and mixed venous blood gases. qs/qt was calculated by standard formula. data are given as median (p < . placebo vs. [ibuprofen] [ ] mmhg (*p< . i). these changes were accompanied by a marked increase of -keto-pgf~ pc up to rain after mt in arterial and mixed venous blood of untreated patients with a peak of *[ ] ng/l tl (*p< . ol). there was no difference between arterial and mixed venous pc. ibuprofen pretreated patients (n=zr) demonstrated stabile qs/qt and pao while -keto-pgf~ pc remained within the normal range. discussion: our data clearly indicate that mesenteric traction response includes a critical rise in qs/qt followed by significant decrease of paov stable oxygen transport determinants following cyclooxygenase inhibition signify an action mediated by prostacyclin. an indicative transpulmonary gradient for -keto-pgft~ was not detectable. a splanchnic vascular source for pgi release seems to be likely, but could not be proved by our current data. department of anesthesiology, cliu. chemistry * and surgery*; university clinics uim, prittwitzstral]e , ulm, germany it is unclear whether injuries like bums, in general, directly result in alterations of cell-mediated immunity that, in turn, promote endotoxic and bacterial translocation or, alternatively, whether these conditions allow increased bacterial invasion that, in turn, inhibits cmi. aim: to determine whether infectious challenge, as clp alone or combined with ti causes further immune abnormalities in the days following clp. study plan: on day , two groups of n= week old aj mice were subjected to either a % scold burn (ti), or were untreated (c) n= . on day , mice (ti+clp) and mice (clp) were subjected to clp. the two other groups (ti and c) were untreated. at days , and after thermal injury splenocytes (sp) were harvested and cultured with cona for an assay of il- and adherent splenocytes (as) were cultured with lps for il- , tnf, il- and pge . results: either ti + clp or clp alone result in significantly decreased secretion of all cytokines tested. in the ti group almost every cytokine production determined was elevated in comparison to ti + clp and prosmcyclin (pgi ) has been implicated in the pathophysiology of septic shock. however, pgi~'s role in the inflammatory response to sepsis is not well-defined. the purpose of this study was to identify which acute septic events are mediated by pgi during graded bacteremia. methods: eleven ~nesrhetized, hemodynamically monitored adult swine were infused iv with aeromonas h. ( /ml) at rates increased incrementally from . to . mi/kg/hr over hours. animals were studied in two groups: septic control (sc), graded bacteremia only (n= ); pga (n= ), graded bacteremta plus anti-pgiz antibody, ml/hr iv, beginning at hours. mean systemic (map) and pulmonary arterial (pap) pressures and arterial po , mmhg, cardiac index (ci), l/min/m , oxygen delivery index (do i) and consumption index (vozi), ml/min/m , and oxygen extraction (er), %, )latelet aggregometry (plt), %max., plasma pg -keto f alpha ; in the first instance~ peak values of lt ~ after i~ hrs post infarction were times higher than in the controls and excess leucocyte infiltration was noted at the infarction zone. in second instance two levels of lt b led to weak infiltration of the infarction zone by leucocytes. a. mo~e~o, in~.~p~siolo~,d~t.e~.cardiolo~,bogotsolets , ~ev , ukrmne systemic lesion$of erythron in traumatic disease and possibilities of their regulation by opioid peptides. redkin y. v., fominih s. g. using clinical ( patients) and experimental material( rats and dogs) we revealed general regularities of erythron lesions after hard mechanical trauma of various genesis as well as some mechanisms of development of posttraumatic anemia and possibilities of its correction with preparations of opioid peptides. the condition of central and peripheral compartments of erythron was studied with unified morphologic, immunogematological, biochemical and radiological methods. it was revealed that irrespective of the experimental animal species (dogs, rats) or in clinical experiments (patients) and irrespective of the injuring factor type (skeletal trauma, craniocerebral trauma, loss of blood) in erythron can be observed one-directed unspecific reaction realized by the considerable lowering of hemoglobin concentration, erythrocytes number and hematocrit. in the initial period ( - days) in the system of erythron prevail processes of distraction and elimination of er~zthrocytes relatively to the general production of stimulated erythropoiesis. the primary alterating factor is the prolonged intensification of peroxydation of membrane iipids of erythrocytes with simultaneous lowering of reserves of reduced glutathione. the distraction of erythrocytes is supported by the developing phenomena of autoallergization of organism that becomes apparent by the appearance of sensitized t cells and antierythrocyte antibodies. the intensified production of erythropoietin rules to the realization of he program of fetal and terminal (reserved) erythropoiesis. failure of erythropoiesis function is supported by disturbances of the processes of the injuring of cell metabolic apparatus. using of dalargin ( microgram per kilogram of body mass intrap'eritoneally within days after the trauma) showed the precise pharmacotherapeutic effect revealed by the diminishing of anemia of experimental rats, more . fiberbronohoscopic procedures are known to produce "peep-like" effects and to increase pulmonary artery (pa) resistance [ ] . peep can affect rv function by reducing preload and ejection fraction (ef) [ ] . since changes of rv function during bronchoscopy in septic patients are not reported, we measured rv parameters before, during and after fiberoptic bronchoalveolar lavage (bal). method: this -year-old patient (apache-ii: ) developed a hyperdynanlic septic state due to staphylococcus aureus (blood culture). we inserted a "fast response" thermistor pa-catheter (baxter-edwards) to evaluate rv performance [ ] . the therapeutic procedure included volume replacement, vasopressors (dopamine , dobutamine gg/kg/min. iv) and analgosedatior/. before bronchoscopy (olympus bf- , od= mm) the patient received pancmonium for muscle relaxation. ventilation was not changed during the procedure (endotracheal tube: id= ram, bennett a, pressure controlled mode, pm~x= mbar, peep= mbar, i:e=i:i, fio = . ). we measured rv enddiastolic volume (edv), stroke volume (sv), ef, heart rate (hr), cardiac index (ci) and mean pa pressure (mpap gerlach h, gerlach m, clauss m, falke kj renal hypoxia and/or ischemia initiates the development of a deteriorated medullary perfusion based on fibrin deposition in the peritubular capillaries, vasoconstriction, and perivascular edema, which is followed by a swelling of the tubular epithelial ceils, intraluminal tubular obstruction, and a backleak of fluid through the injured tubules into the renal interstitium, finally leading to an acute tubular necrosis (atn) [ ], clinically diagnosed as acute renal failure (arf). one important pathway for induction of enhanced vascular procoagulant activity and permeability is based on the synthesis and expression of macrophage-derived cytokines, which bind to specific endothelial cell surface receptors. we recently described the identification and purification .of a new , dalton polypeptide, which is synthesized and expressed by murine macrophages after stimulation with lipopolysaccharide, and exerts procoagulant activity on cultured endothelial cells [ ] . in the presented study, we demonstrate that the new polypeptid is also synthesized by macrophages under hypoxic conditions. the protein binds to specific receptors, which are expressed by endothelial cells dependent on the environmental oxygen tension. animal studies were performed after approval by the local committee for animal safety; the animals were anesthetized, treated and supervised in accordance with the guidelines of this committee. in contrast to other authors, who performed long-term hypoxia experiments in awake animals, we preferred to implement the studies under anesthesia for ethical reasons, although regulatory functions for ventilation might be influenced. animal studies demonstrated that the intravenous injection of the polypeptide initiates fibrin formation in the peritubular vessels. keeping the animals under hypoxic conditions induces similar effects, which are reduced by a rabbit-antiserum against the new protein. in conclusion, the new polypeptide obviously contributes to the pathogenesis of acute renal failure by tubular necrosis during and after hypoxic events. the use of verapamil as cardioprotective agents for management of patients with acute ischemic/reperfused heart is based on the assumption that the increased intracellular ca+ level is a key factor in causing cell death. our in vitro study was designed to focus on effects of verapamil on the metabolic potential of cardiac slices after reversible ischemia in rats. the material consisted of two main groups : group a (non ischemia/reperfusion group) and group b (ischemia/reperfusion group), each is subdivided into two subgroups (a and b). each subgroup included rat hearts. group aa is the control group, group ab is verapami] added group. group ba is ischemia group without verapamil. group bb is verapamil added group. ischemic cardiac slices were obtained from rats subjected to min. haemorrhage to induce reversible global ischemia. both nonischemic and ischemic cardiac slices were placed in well oxygenated krebs ringer phosphate buffer containing mg% glucose & gm% bovine albumin and incubated in dubnoff shaking water bath for min at °c the results revealed that there was an enhancement in release of free fatty acids (ffa) ( %) and lactate ( %) and in glucose uptake ( %) in group ba as compared with group aa. these metabolic alternations produced by ischemic cardiac slices were reversed by verapamil addition ( ml%) but in group ab verpamil did not alter the release of ffa & lactate from non-ischemic cardiac slices, whereas it inhibited glucose uptake from these slices by %. the improvement of the metabolic intervention of ischemic myocardium indicates that verapamil may be of importance in reducing the extent and severity of acute myocardial ischemic injury in acute haemorrhage. severe endothelial dysfunction occurs following injury to carotid arteries which is characterized by a decreased ability of these arteries to dilate when challenged with ach or a , but not with a direct vasodilator (nano ). this failure to relax to ach and a reflects an inability of endothelium to generate edrf, but relaxation recovers gradually to control values by weeks. exogenous no donors (e.g., c - or spm- ), accelerate the recovery of the injured endothelium in rat carotid arteries. intravenous infusion of an no donor ( p.g/day) with an implanted osmotic pump significantly accelerated the recovery of regenerated endothelium to produce edrf at days. rat carotid artery rings relaxed only + % and + % to gm ach in vehicle treated rats and in inactive no donor treated rats respectively days following injury compared with + % in no donor rats (p< . ). relaxation to gm nan was normal in all groups indicating that the differences in relaxation were not the result of damage to vascular smooth muscle. contraction to l-name ( mm) was markedly reduced by injury, but was protected by no donors (p< . ). thus, exogenous no donors enhance the ability of the endothelium to regenerate and to release edrf in response to endothelium-dependent vasodilators. this may be due to an anti-proliferative and anti-mitogenic effect of no on vascular smooth muscle cells, allowing the endothelium to regenerate without intimal thickening. no also has been shown to inhibit platelet aggregation, and to attenuate neutrophil adherence and activation. the superoxide scavenging effect of no is not the basis for these effects since hsod is inactive in preserving endothelial function in injured arteries. thus, no exerts a variety of cytoprotective effects which may be of importance in protecting against vascular injury. much evidence has now accumulated to show that the excess production of the vasodilator nitric oxide (no) in sepsis is an important contributor to the hypotension and multiorgan failure characteristic of this condition. various cytokines play an important role in this process through their ability to induce the production of one of the enzymes responsible for no synthesis, the inducible no synthase (inos). we have studied the effects of cytokines on the induction of this enzyme both in vitro using vascular smooth muscle cells, and in a murine model of gram-negative sepsis. tn smooth muscle ceils, the cytokines il- , ifnq', and tnf-oc show strong synergy with one another in the production of inos. in order to define the molecular basis for this synergic effect, we have linked the promoter of the inos gene to a "reporter" gene, chloramphenicol acetyl transferase (cat), and transfected these constructs into vascular smooth muscle cells. assays of cat activity reflect the activity of the promoter in this system, and by generating sets of deletion mutants of the promoter sequence we have been able to define the area within the promoter which mediates the synergic effect of these cytokines. in addition to stimufatory effects on inos production, certain cytokines are able to down-regulate the production of inos in vascular smooth muscle cells, and the effects of these counterregulatory cytokines will be discussed. the interaction of these cytokine effects in the whole organism has been studied in a murine model of gramnegative sepsis. widespread induction of inos occurs in this model as assayed by enzyme activity and through use of specific antisera to inos. neutralizing antibodies to tnf-~ and tfn-y are both able to prevent death in this model, but it is only the anti-ifn-y which attenuates the induction of inos assayed in the liver. clearly there is some redundancy in the effects of cytokines on the production of inos in sepsis, and greater understanding of the most important factors in inos production is required in order to target anti-cytokine therapy most appropriately. effects of nitric oxide on hepatocyte metabolism in inflammation. j. stadler, department of surgery, tu mqnchen, frg hepatocellular nitric oxide (no) synthesis is induced by proinflammatory mediators such as tumor necrosis factor, interleukin- and interferon gamma or by bacterial toxins such as lipopolysaccharide. stimulation of the hepatocytes (hc) with a combination of these agents leads to an output of no in quantities which are not seen in any other celltype. it has been demonstrated by various investigators that important effects of these cytokines and bacterial toxins on hc metabolism can be attributed to the action of no. in contrast to other celltypes hc seem to be relatively resistant to suppression of basic metabolic functions such as energy metabolism by no. therefore, cell damage has not been described to a significant extent following exposure to no. however, no does inhibit total protein synthesis. the exact biochemical mechanism of this phenomenon has not been uncovered yet, but it has been demonstrated for some specific proteins that their production is inhibited at a posttransscriptional level. as in many other celltypes cgmp generation is elevated in hc by no through activation of the soluble guanylate cyclase. cyclic gmp may possibly exert a plethora of metabolic functions, but it is interesting to note that most of the cgmp seems to be transported out of the cell. some very specific effects of no on hc metabolism include the inhibition of the glyceraldehyde- -phosphate dehydrogenase (gapdh) and the cytochrome p (cyp) enzymes. inhibition of gapdh activity is mediated through nitrosylation of critical domains of the enzymes by no which enhances auto-adpribosylation. this effect on gapdh activity might be responsible for the inhibition of gluconeogenesis by no, which has been described recently. finally, no-mediated inhibition of cyps may help to explain the suppression of hiotransformation processes which is a characteristic featur,'~ r ~ "~flamed liver. nitric oxide (no) is an endogenous inhibitor of polymorphonuclear leukocyte (pmn) adhesion which limits pmn-endothelial cell interactions under normal conditions. we have previously demonstrated that following ischemia, no production by the vascular endothelinm is dramatically reduced. accordingly, we investigated the effects of no-donors on pmn accumulation and tissue injury following hemorrhagic shock and ischemia. hemorrhagic shock was induced in anesthetized rats by bleeding to mmhg for hours followed by reperfusion. segments of superior mesenteric artery (sma) were isolated and suspended in organ baths. in rats receiving saline sma relaxation to acetylcholine (ach, nm) was reduced by % compared to control sma segments (p< . ) while relaxation to sodium nitrite ( gm) was unaffected. in addition, mesenteric tissue pmn accumulation as determined by myeloperoxidase (mpo) activity was significantly elevated compared to controls (p< . l). interestingly, treatment with the no-donating agent, s-nitroso-n-acetylpenicillamine (snap) significantly preserved sma relaxation (p< . ), attenuated mesenteric mpo (p< . ) activity, and significantly improved survival compared to saline vehicle. in anesthetized, open-chest dogs we investigated the cardioprotective actions of a novel no-donor, spm- (schwarz pharma), following regional myocardial ischemia ( hour) and reperfusion ( . hours) . treatment with spm- ( rim) significantly reduced myocardial necrosis by % (p< . ) compared to an no-deficient analog of spm- , spm- . furthermore, mpo activity within the ischemic-reperfused zone was also significantly (p< . ) reduced following treatment with spm- compared to spm- ( . + . vs. . + . u/ mg tissue). these data strongly suggest that no is a potent inhibitor of pmn-mediated tissue injury following hemorrhagic shock as well as in acute myocardial ischemia-reperfusion injury. overproduction of nitdc oxide (no') may contribute to sepsis-induced hypotension. during septic shock, excess no" is produced by an isoform of nitric oxide synthase (nos) which is induced by inflammatory mediators. nonselective nos inhibitors have been proposed as a new therapeutic approach to treating hypotension in septic shock. we studied the differential hemodynamic effects of n~-methyi-l-arginine (l-nma), a nos inhibitor, in normal canines versus those challenged with endotexin (lps) and compared the activity of this drug across the venous, pulmonary and systemic vascular beds. awake canines were challenged with lps ( mg/kg, n= : mg/kg, n= ; or mg/kg, n= ) and treated with l-nma ( , , , , mg/kg/hr) for hours following a , , or mg/kg loading dose. animals were resuscitated with iv ringers solution ( ml/kg/hr). hemodynamic data were collected at , , , , , and hours using intravascular catheters and radionuclide heart scans and analyzed by anova. in both normal and endotoxemic animals, l-nma at all doses studied similarly increased mean arterial pressure (p= . ), and systemic vascular resistance index (p= .ol) and decreased cardiac index (p= . ) and oxygen delivery index (p= . ). in contrast, the effect of l-nma on mean pulmonary artery pressure, central venous pressure, pulmonary capillary wedge pressure, and pulmonary vascular resistance index was greater in lps-challenged canines compared to normal animals (p< . ), but this differential effect on the venous and pulmonary circulation occurred, > hours after lps challenge. l-nma did not significantly increase survival rates or times at any of the doses studied ( , , , or mg/kg/h) in either the low ( mg/kg) or high dose ( mg/kg) lps-challenge groups. a nonsignificant (p> . ) trend toward a beneficial effect on survival ol low dose l-nma ( mg/kg/h) in animals given the mg/kg lps-cha[lenge was not enhanced by increasing the lethality of the model or by administering higher l-nma doses. at the highest l-nma dose used in this study ( mg/kg/h), survival time decreased significantly for both the low and high dose lps-challenge animals (p< . ). this increased mortality was not explained by changes in plasma concentrations of either lps or tnfc~. thus, l-nma did not have a greater effect on the systemic arterial circulation in endotoxemic compared to normal canines. however, in the venous and pulmonary vascular beds, the effect of l-nma increased with time after endotoxin-challenge these data suggest the induction of nos activity by endotoxin in canines may be relatively greater in venous and pulmonary vessels compared to systernic arteries. l-nma, a nonselective nos inhibitor, did not decrease mortality in endoloxemic canines and the highest dose studied was harmful. pulmonary hypertension (ph) and arterial hypoxemia are characteristic features of the adult respiratory distress syndrome (ards). reducing pulmonary vascular pressures may promote the resolution of pulmonary edema. intravenously infused vasodilators lower ph in ards, but, as a result of their general vasodilatatory effects, systemic mean arterial pressure may also decrease. furthermore, blood flow may be increased to non-ventilated or poorly ventilated lung areas resulting in a rise of intrapulmonary shunt, thus causing a further fall in pad . recently, short term inhalation of low concentrations of the gas nitric oxide (no), an endogenous endothelium derived relaxing factor, which is rapidly inactivated in blood by hemoglobin, was reported to decrease ph without causing systemic vasodilation in sheep [ ]. similar changes have been observed in patients with severe ards during repeated short term inhalation of no ( and ppm), which rapidly and selectively decreased the mean pulmonary artery pressure (pap) and, in contrast to intravenously infused prostacyclin, induced a remarkable increase of pad [ ] . this improvement in oxygenation was caused by a redistribution in blood flow away from intrapulmonary shunt areas to normal ventilated lung regions. continuous no inhalation ( - ppm) consistently lowered the pap and augmented the pao /f.o for up to days. no negative side effects were observed during the whole time span examined. in particular methemoglobin levels always remained below . %. following these investigations, it could be shown that these effects may also occur using concentrations in the parts per billion range [ ] , which may reduce possible toxic side effects. however, in the same study it was demonstrated that the dose-response curves for pa and pap have different patterns. whereas pap presented a continuous dose-dependent downward tendency with an eds o of approximately - ppm, the improvement of oxygenation had a maximum at ppm and, at higher doses, drifted back towards the baseline data. the ed~o was estimated at approximately ppb, i.e. more than ten times lower than for the reduction of pap. in conclusion, inhalation of no by patients with severe ards may result in persistent and reproducible decreases in pap associated with an evident improvement in pad , thus allowing reduction of the f.o . no inhalation should be performed using low concentrations which are less toxic, although any possible risks still have to be considered carefully. dose-response studies for the individual patients are recommended urgently. finally, controlled randomized studies are required to demonstrate that additional no inhalation is able to reduce mortality of ards. inhibition of the activity of glyceraldehyd- -phosphate dehydrogenase (gapdh), an enzyme of the glycolysis/gluconeogenetic pathway, through adp-ribosylation is promoted by nitric oxide (no). since no is produced in the septic liver and hypoglycemia is a major problem of late sepsis, it was investigated whether no interferes with gluconeogenesis of hepatocytes. hepatocytes (hc) were isolated from sprague-dawley rats using a collagenase perfusion technique and differential centrifugation. exogenous no was applied by incubation with the no-donors s-nitrosyl-acetylpenicillamine and sodium-nitroprusside. endogenous no synthesis was induced by incubation with cytokines (tnfcq il- , ifnj and lipopolysacchafide (lps). hrs later the incubation medium was changed to a solution containing lactate, ornithine, lysine, ammoniumchloride and glucagon for optimal conditions of gluconeogenesis. after more hrs glucose and nitrite levels were determined spectrophotometrically. gapdh activity was measured by the nadh-dependent conversion of , -diphosphoglycerate to glyceraldehyde- -phosphate. incubation of hc with no-donors led to a concentrationdependent inhibition of gluconeogenesis and gapdh activity. however, gapdh activity was about times more sensitive to the inhibitory effect of exogenous no. incubation of hc with cytokines and lps induced nq synthesis as measured by an increase in nitrite concentrations. endogenously produced no suppressed gluconeogenesis by _+ %. in contrast to exogenously applied no, the effect of endogenous no synthesis was less on gapdh activity resulting in an inhibition of only _+ %. in conclusion, exogenous and endogenous no inhibited gluconeogenesis as well as gapdh activity. however, there was no correlation between the extent of inhibition of these two parameters of hepatocellular glucose metabolism. we have shown that inhibition of hepatocyte (hep) synthesis of nitric oxide (no) potentiates cell injury in a model of acetaminopheninduced oxidative stress and the extent of damage was paralleled by depletion of reduced glutathione (gsh) stores. to clarify the role of no in modulating the redox state of hep, we studied the effect of inhibition of cytokine-mediated no production on hep gsh stores, in a system of isolated rat hep in primary culture, no synthesis was induced (stim) by exposure to il- , tnf, ifn, and lps for hours. , , and ~m of n-monomethyi-l-arginine (nmma), a specific inhibitor of no synthesis, was added. cells incubated in media alone served as controls (cont). the no metabolite (no ); aspartate aminotransferase (ast), an indicator of cell injury; and gsh were assayed. (data presented as mean + sem; n= .) gsh (nmovma orotein) ..~ (nmol/ma orotein) cont . + . + . # stim . + . + stim+ o tzm nmma . + . + . # stim+ ~m nmma . _..+ . * + . # stim+ pm nmma . + . * + . # stim+ )lm nmma . + . * + . # anova , . (* p < . versus stim, # p < . versus stim; anova with neuman-keuls) gsh in cont+ i~m l-nmma was equivalent to that of cont ( . vs. . ). ast release was equivalent in all treatment groups. these data show that inhibition of hep synthesis of no depletes intracellular stores of reduced gsh. we conclude that hepatocyte no production modulates cellular gsh homeostasis and as a result, may be hepatoprotective in oxidative injury. nitric oxide (no) is a modulator of immune response and may be involved in the changes in immune reactivity after major trauma and operations. we investigated no-generation in rat and mice spleen cells (sc) after partial hepatectomy (ph). c bl/ mice and lew rats underwent a % and % ph, respectively. sc were prepared - days after ph and plated at to x ecells per well. after h incubation at °c, no-production was measured as nitrite levels (griess reagent). normal mouse sc did not produce no, neither basal nor in response to lps or con a starting at the second day after ph, we found a substantial production of no. in rats, also sc from control animals were able to generate no; both basal and stimulated no-generation were further enhanced after ph (table, values expressed as mean --se). after shame operation, there was only a modest elevation of noproduction in rat and mouse sc. in first experiments we could demonstrate no-production also in phagocytes from a patient days aider liver partial resection ( . nmol nitrite/ cells) enhanced no-production in macrophages may contribute to the changes of immune reactivity after partial hepatectomy. nitric oxide (no) is recognized as an important mediator in endotoxemia and sepsis. increased synthesis of no has been demonstrated in septic humans and animals, and no inhibitors have been used in the treatment of septic shock. recent reports have, however, suggested that this form of therapy may cause serious organ damage. in the present investigation circulatory and metabolic changes in the liver were studied during treatment with the no-synthase inhibitor n-nitro-l-arginine-methyl ester (l-name) in endotoxemia. methods: juvenile pigs were randomized to one of the following treatment groups: ) encletoxin and l-name, ) endotoxin, ) naci and l-name, ) nach preliminary results from groups (n= ) and (n= ) are presented. catheters for pressure measurement were introduced into the aorta, hepatic and portal veins and ultrasonic transit time flow probes were placed on the hepatic artery and portal vein. a catheter was introduced into the pulmonary artery. endotoxin ( . gg/kg/h) was given as a continous portal infusion over the entire observation period of hrs. l-name ( mg/kg) was given as a bolus after hrs. of endotoxemia. results: endotoxin transiently reduced portal vein flow (pvf) by %* and hepatic artery flow (hal e) by %*, while l-name caused a further and lasting reduction in flow (pvf %, haf %)*. transhepatic (portal-hepatic vein) vascular resistance increased to times baseline value during endotoxemia while l-name caused a further marked increase in resistance to times initial value. portal oxygen saturation (so ) decreased by %* during endotoxemia. l-name caused a reduction in portal so by %*. arterial so was unchanged in both groups. hepatic oxygen uptake was not changed by endotoxin, but was markedly reduced after addition of l-name. endotoxin caused a % reduction in cardiac output (co). the addition of l-name reduced co by a total of %*. *: p < . . conclusion: is the present model of endotoxemia treatment with the nitric oxide synthase inhibitor l-name markedly reduced liver perfusion and portal oxygen supply. this might explain the increased liver damage reported in previous studies using no-inhibitors. the increase in transhepatic resistance found after l-name treatment will tend to cause pooling of blood in the splanchnic veins, resulting in reduced filling of the heart and thus contribute to the observed reduction in cardiac output. institute for surgical research, rikshospitalet, the national hospital, university of oslo, oslo, norway. we have investigated the role of tumour necrosis factor (tnf) and interleukin-i (il-i) in the induction of nitric oxide synthase (nos) by bacterial endotoxin (lipopolysaccharide; lps; mg kg -i i.v.) in vivo. in anaesthetized rats, pretreatment with a monoclonal antibody for tnf (tnfab; mg kg -i s.c., at h prior to lps) or with an il-i receptor antagonist (il-ira; mg/kg bolus and . mg/kg/h infusion) ameliorated the fall in mean arterial blood pressure (map) at - min after lps. for instance, endotoxaemia for min resulted in a fall in map from -+ (control) to -+ mmhg (p< . ; n= ). in contrast, animals pretreated with tnfab or il-ira prior to lps injection maintained significantly higher map at min when compared to lps-control: -+ mmeg (n= ) and -+ mmhg (n= ), respectively (p< . ). three hours of endotoxaemia significantly reduced the contractile effects of noradrenaline (na) in the thoracic aorta ex vivo. the hyporeactivity to na was partially restored by in vitro treatment of the vessels with ng-nitro-l-arginine methyl ester (l-name, min, x - m). pretreatment of rats with tnfab or il-ira significantly (p< . ) prevented the lps-induced hyporeactivity of rat aortic rings ex vivo. l-name did not alter or only slightly enhanced the contractions of aortic rings obtained from tnfab or il-ira treated lps-rats, respectively. at min after lps there was an induction of calcium-independent nos activity in the lung ( . -+ . pmol citrulline/mg/min, n= ), which was attenuated by tnfab and !l-ira by -+ % and -+ %, respectively (n= ; p< . ). thus, the production of both tnf and il-i contributes to the induction of nos by lps in vivo. the protective effect of agents which inhibit the release or action of tnf or il-i in shock may be, in part, due to inhibition of nos induction. neal garrison, md objective: sepsis is often accompanied by organ dysfunction, in part due to impaired microvascular perfusion. recently, nitric oxide (no) has been described as an important mediator of the hemodynamic changes of sepsis, and no synthase (no-s) inhibitors have been advocated for treatment of septic shock, but their visceral microcirculatory effects are inadequately characterized. we postulated that no-s inhibition would exacerbate the impaired organ perfusion of sepsis. methods: six groups ofdecerebrate rats were studied. bacteremia was induced with live e. coli, which consistently increased cardiac output - % above baseline (bl). the no-s inhibitor nm-nitro-larginine methyl ester (l-name, mg/kg iv), prevented this increase and elevated map by - %. in the first groups, total hepatic blood flow (thbf, ml/min by time transit flowmetry) and microvascular perfusion (mi-ibf, ¼ bl by laser doppler flux) were measured. in the other groups, in vivo videomicroscopy was used to observe renal microvascular responses (ila=interlobular artery, aff=afferent arteriole, eff=efferent arteriole; % bl for all). results: data are rains after e. cob. n= - /group. * p< . vs bl by remanova and § p< . vs e. coli alone by anova. ec+l-name -+ - _+ " § - _+ * § - _+ * § - + * - + * § conclusions: l-name administration in controls decreased renal blood flow, indicating no contributes to basal renal tone. bacteremia decreased mtlbf but not thbf, and mi-ibf was further impaired by no-s inhibition. e. coli caused renal preglomemlar, but not postglomerular constriction and reduced flow. l-name exacerbated these e. coli-induced alterations and caused eff constriction. these data indicate that no-s inhibition exacerbates bacteremia-induced impairment of renal and hepatic blood flow, suggesting that no is an importam compensatory dilator mechanism in these organs during sepsis. irf (iron responsive factor) is the central regulatory protein of intracellular iron metabolism able to bind to responsive rna elements (ires) present atthe 'untranslated region (utr) of ferritin mrna and 'utr of transferrin receptor mrna. binding of irf to ires results in repression of ferritin mrna translation and increased stability of transferrin receptor mrna leading to enhancement of transferrin receptor translation. we describe here that either tetrahydrobiopterin dependent stimulation as well as cytokine (ifn-~)/lipopolysaccharidemediated induction of nitric oxide synthase activates irf, which is due to direct interaction of nitric oxide with the iron-sulphur-cluster of irf. this was shown by gene expression studies using a plasmid containing a ferritin ire and a cat indicator box which was transfected into k myelomonocytic cells, which were shown to have a constitutive form of nitric oxide synthase (nos). furthermore, the increased binding of re to irf due to irf activation of irf by nitric oxide was demonstrated by gel shift assays. irf activity was much more increased in cellular extracts from murine macrophages (j ) where a cytokine inducible form of nos has been characterized earlier as compared with irf activity in k cells, where nos was stimulated by increasing the availability of the essential nos cofactor , , , -tetrahydrobiopterin. we then demonstrated that activation of irf by nitric oxide is accompanied by alterations in ferritin translation as checked by metabolic labeling and immunoprecipitation. these results suggest a reasonable mechanism for the regulation of iron disturbances under chronic inflammatory disorders, characterized by increased concentration of immune activation parameters like ifn- or neopterin and low serum iron and hemoglobin concentrations. taken nitric oxide, no, the putative endothelial derived relaxant factor, edrf, has been shown to be a potent inhibitor ofplatelet aggregation in vitro. in vivo evidence however, is scarce. accumulation of platelets in the lungs has been shown to occur during extracorporeal circulation. the aim of the present study was to investigate the effect of inhaled no on this reaction. materials and methods: the animals were divided into two groups, each consisting of pigs. platelets were selectively labelled with luln-oxine. dialysis was instituted via catheters in the femoral vessels. in group , no, ppm, was added to the inhaled gas from the start of dialysis. in group no was not given. the activity over the lungs was followed dynamically with a gamma camera. central hemodynamics was monitored via a swan -ganz catheter. results: the activity was significantly lower in group , from minutes after start of dialysis and onwards, indicating diminished accumulation of platelets in the lungs. parallel to this the hemodynamic response in terms of increased pulmonary artery pressure and pulmonary vascular resistance was blunted in this group conclusion: inhaled no in this model seems to affect pulmonary platelet sequestration. an associated attenuation of the changes in central hemodynamics was also seen. previous studies from our laboratory have demonstrated that vascular contractility decreased in endothelium-intact blood vessel rings in early and late stages of sepsis. although endothelium removal in early sepsis restored vascular contraction, the depressed smooth muscle contractility observed in late sepsis was not restored by endothelium removal. this indicates that impairment of smooth muscleper se may be responsible for such dysfunction in late sepsis. the aim of this study, therefore, was to determine whether or not smooth muscle-derived nitric oxide (no) plays a role in producing vascular smooth muscle dysfunction during late stages of sepsis. to study this, rats ( - g, n= - /group) were subjected to sepsis by cecal ligation and puncture (clp). septic and shamoperated rats then received rrd/ g bw normal saline. the animals were killed at , , or h post-clp ( h post-clp=early sepsis; - h post-clp=late sepsis), and thoracic aortic rings were prepared for contraction studies using organ chambers. the complete removal of endothelial cells was tested by the absence of any significant acetylcholine-induced vascular relaxation. contractile responses to norepinephrine (ne, to - m) were determined in the aortic rings without intact endothelium. ng-monomethyl-l-arginine (l-nmma, /~m, an inhibitor of no synthase) was then added to the organ chamber and ne-induced peak contraction was determined before and after the addition of l-nmma. the peak contraction (rag/rag tissue, mean_+sem) is shown below: the results indicate that the addition of l-nmma did not significantly affect ne-lnduced peak contraction in endothelium-denuded vessel rings at and h after clp. in contrast, l-nmma administration produces an % increase (p< . ) in peak contraction during late sepsis. therefore, the vascular smooth muscle contractile dysfunction observed at h post-clp is partially due to smooth muscle-derived no over-production. thus, unlike macrophages in which inducible nitric oxide synthase (inos) is observed in early sepsis, the inos in vascular smooth muscle appears prominent only in the late stages of sepsis. in three cases of human septic shock in which ng-monomethyi-l-arginine, (l-nmma) a nitric-oxide-synthase-inhibitor was applied, we isolated three completely different types of pathogens: candida, pseudomonas aeruginose and multiresistant coagulase-negative staphylococci. this observation suggests that endotoxin alone is not the main factor triggering hypotension in septic shock by the nitric oxide pathway. in a -years-old woman in severe septic shock due to a candida and pseudomonas aeruginosa infection complicated by adult-respiratorydistress-syndrome conditions deteriorated despite adequate conventional therapy. in this trial, effects of l-nmma on cytokin-levels were investigated. the study-protocol was approved by the ethical committee of the department of surgery. after two boll of mg of l-nmma, a continuous infusion was installed ( . mg/minute and kg body weight l-nmma). as expected mean arterial blood pressure rose ( to mmhg}, heart rate stayed stable ( + b/rain), systemic vascular resistance increased ( to dyne.sec/cm ), cardiac output decreased ( to . l/rain), and cardiac index declined ( . to . l/min/m }. before and after minutes while the infusion of l-nmma, blood samples for immunological measurements were taken and processed together. pulmonary-shunt-volume was observed before the application of l-nmma, after one hour and after matutes. neopterine increased from . to . ng/ml, tumour-necrosis-factor-a increased from . to . pg/ml and intedeukin- increased from . to . pg/ml. immunoglobulines a, g, and m ( . to . , . to . , . to . g/i), complement factor c- c and c- ( . to . , . to . g/i), alpha-l-antitrypsine ( . to . g/i), c-reactive-protein ( . to . rag/i), interleukin- ( pg/ml) and soluble interleukin- ( to units/ml) did not change significantly. pulmonary-shuntvolume decreased from . % to . % within one hour and to . % after minutes. in septic shock blocking nitric oxide as an intervention at the end of a not ~,et ful!y understood cascade might have important influences on pulmonary-shunt-volume and inter-cell-communication. department of surgery, pharmacy* and immunology**, university hospital of zurich, r~imistrasse , zurich, switzerland we previously reported that hypoferremic cba mice had an increased resistance to salmonella infection, and that injection of ammonium ferric citrate (afc) to these mice led to enhanced infection (ganthier et at. . microbiol.immuno : ) . because nitric oxide (no) is involved in the antimicrobial activity of routine macmphages towards various inttacellular pathogens, we investigated the influence of iron on the bactericidal activity of cba mouse macrophages towards s.typhimurium and on the production and activity of reactive nitrogen intermediates (rni). peritoneal macrophages hum cba mice were cultured in the presence (or not) of afc ,um, ifn-,/ u/ml, lps fig/m/, ngmonomethyl-l--arginine (mmla) ram. nitrite (no -) content of the supematants was determined by a standard griess reaction, and h release was measured by the peroxidese dependant oxidation of phenol red. for intracellular killing, macrophages monolayers were infected, and, at various intervals, lysed by triton x- , and surviving bacteria enumerated by colony counting on agar. for in vivo experiments, mice were infected ip with . ml of a suspension of . ~" s.typhimurium, strain c , and injected with aminoguanidine (ag) mg/ml in saline. our results show that the rn[ inhibitor ag strongly accelerates the mortality of infected mice, the survival rate decreasing from % in the control group to % in the treated group, days after challenge. correlatively the rni inhibitor mmla induces in vitro a decrease in the rate of bacterial killing, fxom % to %, in macrophages triggered with ifn-? + lps. the cultivation of macrophages in the presence of afc leads to a decreased no -accumulation, . nmole/well v.s. nmole/well. conversely h production is enhanced from nmole/well up to , nmole/well. nevertheless, macrophages cultivated in the presence of afc exhibit an increased tale of intracellular killing, % in iron exposed macrophages v.s, % in control macrophages. when triggered with ifn-~, alone, macrophages have a reduced antibacterial activity ( % v.s. %) whereas the addition of afc to these macrophagas restores an elevated ( %) rate of killing. in conclusion, the results show that bactericidal activity of cba macrophages towards s.typhimurium depends on the production of no by these macrophages ; but they also demonstrate that no is not the only reactive species involved in the intracellular kil/ing of s.thyphimurium ; indeed afc which strongly inhibits rni production, stimulates h release by these macrophages and increase their bactericidal activity in vitro. nevertheless afc may promote bacterial growth in vivo. crssa. unit de microbiologie. bp . la tronche cedex france. henning jahr, ulrike noack, karin braun the large amounts of no produced by the inducible no synthase in rat macrophages have direct antimicrobial effects, but inhibit the activation of the lymphocyte-dependent host defense system. the aim of this study was to investigate if complement activation influences no-generation. spleen cells from lew rats were incubated at °in tcm- / % fcs, with or without additional rat serum. after h, nitrite (end product from no metabolism) was measured by oriess reagent. in rat spleen cell preparations, most of the no is produced by macrophages. complement activation in vivo was carried out by i.v. injections of u cobra venom factor/kg b.w. at days and . significantly higher (p<o.o ) lps-stimulated no-generation was found in spleen cells prepared at day ( . - . nmol nitrite/ cells, n= ) compared to spleen cells from non-treated animals ( . ± . nmol, n= ) complement activation was effective also in vitro. when incubated in % zymosan-activated rat serum, both basal and lps-stimulated noproduction were enhanced in spleen cells (table, values increased production of nitric oxide (no) is associated with sepsis, however, the effect of no inhibition on survival during sepsis is unclear. we examined the effect of no inhibition on host's ability to eliminate bacteria and survival in mice sepsis model. sixty female balb/c mice were injected with e.coli ( qbody) into peritoneal cavity. the treated group received n-ultro-l-arginine-methyl-ester (l-name), an inhibitor of nos, one hour before bacterial challenge. thirty mice were observed for survival after e.coli challenge and the other mice were sacrificed at hours. blood was obtained by cardiac puncture and peritoneal lavage fluid (plf), liver and lung were harvested. the number of peritoneal exudative cell (pec) was counted and colony forming units (cfld of bacteria in the tissues, blood, and plf were also determined. in addition, pec was cultured in vitro with or without lipopolysaecharide (lps). tnf levels in the blood, plf, and supematants of cultured pec were measured by l bioassay. survival rate (%) qrql/,p n hr hr dav control (normal saline . ml/body i.p.) n (l-name mg/kg i. administration of l-name before e.coli injection increased the number of bacteria in plf and tnf level in plasma, the result suggests that nos inhibitor may depress the host's ability to kill the injected bacteria and that it may induce greater systemic tnf production. we conclude that nos inhibitor is detrimental to animals in sepsis. the hypothesis that sod prevents reperfusion injury to the liver by protecting the endogenous endothelium derived vascular relaxing factor (no) from being inactivated by oxygen free radicals should be investigated. male wistar rats were subjected to min of partial liver ischemia ( %) and min of consecutive reperfusion in situ. some rats were pretreated with the no synthase inhibitor nitro-l-arginin (nla: i mg/kg). sod, when used, was given as mn-containing preparation at a dose of u. i.v. i min prior to ischemia. results (sod/ nla+sod/ ni~,, respectively): bile flow (~i/g/min) : . ± . "#/ . ± . / . ± . . alt (u/ ) • ± ~#/ ± / ± . gldh (u/l): . ± . e#/ . ± . #/ . ± . . in absence of nla, sod enhanced postischemic bile flow and reduced leakage of alt and gldh into the plasma, while the effects of sod disappeared, when endogenous ~synthesis was inhibited by nla. nla had no ~ffect on untreated animals submitted to the same protocol. these results may suggest, that oxygen free radicals interfe~:e with the endogenous vasodilator no and that the benefit of sod can be attribuated in large part to a protection of no during reperfusion. nitric oxide (no) reduces pulmonary vascular resistance and increases oxygenation by inhalation in ards patients [ ] . animal studies on the endogeneous no production proved that no is exhaled after synthesis in the respiratory tract [ ] . with approval by the hospital ethics committee, we studied healthy volunteers and ventilated patients by measurement of inand exhaled no by no/no -chemiluminescence in segments of the upper respiratory tract. we found that no is synthesized predominantly in the nose and in the upper nasopharynx, leading to inspiratory tracheal no concentrations of . - . parts per million in non-smoking volunteers; data during mask ventilation per nose were as follows: non . . . . , parts per million (ppm) no, mean, n = ; ~ p < . (t-test) between smokers and non-smokers; p < . (t-test) between in-and expiration. about % of no is resorbe.d by the lung, and the exhaled no -in contrast to former presumptions -is the remaining part of the autoinhaled no. intubated and ventilated patients are deprived of no autoinhalation, and the exhaled no in the trachea decreases more than % {o. ppm before intubation vs. . ppm after intubation, ~, < . ), whereas the nasal no concentration increases by cumulation. hence, low-dose no inhalation in ards patients might be considered as a no replacement, especially since the tracheal no concentrations we measured in volunteers are similar to those which could be demonstrated to be effective for ards in former studies [ ] . the data demonstrate that no is synthesized in the nose, and inhaled and resorbed by the lung. this phenomenon of no autoinhalation, which is reduced in smokers and in ventilated patients, does possibly contribute to the regulation of the ventilation-perfusion ratio in the lung. kikuchi , yoshihiro inoue , masao yoshida critical care and emergency center, and department of bacteriology, iwate medical university. nitric oxide (no) is produced by macrophages and vascular endothelial cells. it is thought to be the true form of endotheliumderived relaxing factor, and to be involved in the fall of blood pressure during septic shock. we have reported previously that endotoxin (lps), tnf-c~ and il- contribute to the occurrence of septic shock (circ shock : ; . the present study investigated the in vitro effects of lps, tnf-o~, il- and ifn-'i on the production of no by macrophages. peritoneal macrophages were cultured with lps, il- , tnf-~ and ifn-¥. in culture with lps, no was produced in a dose-dependent manner, and the production was suppressed by a no production inhibitor, l-nmma. ifn-y, il- and tnf-c~ used alone did not promote the production of no, but helped lps to facilitate no production. a combination of il- and tnf-c( enhanced lps-facilitated no production to a greater extent than il- or tnf-c~ alone. the above results suggest that these cytokines are involved in endotoxin shock through the mechanisms that facilitate no production. - uchimaru, morioka . japan. t. yolk , , i. ioannidis , w.j. kox and h. de groot objectives: nitric oxide (no.) is known to play an important role in neurotransmission, vasoregulation, and bactericidal as well as tumoricidal cellular defense systems. by means of no-donating agents we studied the mechanism of no-mediated cytotoxicity against rat liver microvascular endothelial cells. materials and methods: -morphoiinosydnonimine-n-ethylcarbamide (sin-l) and sodium nitroprusside (snp) were used as no. donators, ldh release and trypan blue exclusion were applied to indicate cell viability. results: sin- ( mm), which releases both no. and o -., caused a time-dependent cytoreduction of % and % after and hrs, respectively. this effect was not inhibited by superoxide dismutase (sod), which removes --to form h and . in contrast, the addition of catalase (cat), which removes h to form h and , diminished the cytotoxic effect ot sin- to %, equivalent to high concentrations of snp ( mm), which solely releases no.. in addition, the amount of h formed by mm sin- equaled the amount ot enzymaticany produced h by mu/l glucose oxidase (god). whereas god in this concentration and snp in low concentration ( mm) alone were not toxic to endothelial cells, the combination caused an % reduction of viability, comparable to the effect observed with sin- ( mm) alone. moreover, toxicity mediated by high concentrations of snp ( mm and ram) was reduced by % under hypoxic conditions indicating synergism with no-and . conclusions: we conclude, that no.-induced toxicity against endothelial cells is enhanced slightly in the presence of oxygen and is not due to an interaction with -. however, toxicity increases dramatically in the presence of h . this may possibly occur either by a chemical formation of more potent reactive hydroxyl radicals or by independent actions on different cellular targets. although it is becoming increasingly clear that nitric oxide (no) is associated with otxan dysfunctions in septic patients, little is known as to die kinetics of no production in these patients to clarify these kinetics, we have investegated serum arid monocyte associated no in septic patients. five patients who had undergone gastrointestinal surgeries mid were complicated postoperatively with severe sepsis served as the subjects in this study (sgroup), using twelve healthy volunteers as a control group (c group). serum no and no levels were measul*d sliectrophotometricallymonocyte cultures were done for itrs with or without lps+ifn-t stimulation mid no and no levels in the supernat,'utts were also measured spectrophotometrically. furthermore,using an no selective electrode,time course of the lps+ifn-t induced no production by monocytes was studied. l) serum no levels in s group were slightly lfigher than dmse in c group,bnt there were no significoatt differences between the two. )both no levels in the supematmlts of monocytes cultured with lps+ifn-t mid no , no levels ill lhe supernatants of monocytes cultured without lps+ifn-y were significantly higher in s group. further,the time required for the no production by enltnred monoeytes was siglfificantly shorter also in s groap. conclusions l)in severe septic patients, monocyte no productions wei~ not only primed, but also activated concomitantly, suggesting that these monocyte activations m'e strongly associated with organ dysfunctions in sepsis. )based on the restdts of serum no nteasuremeuts, it can be deduced that no p~'oduced by monocytes in septic patients affects tissues and org~s ioeoregiomflly. objectives of the study: nitric oxide (no) is an important messenger which accounts for a wide spectrum of physiological and pathophysiological processes, e.g. mediating vasodilation in endoloxin shock investigating the signal lransducfion pathways involved in the regulation of the inducible nitric oxide synthase (inos), we report the involvement of phosphatidylcholinespecific phospholipase c (pc-plc) and proteinkinase c (pkc). materials and methods: no-production was induced in the murine macrophage cell line j with lipopolysaccharide (lps) [lljg/ml] and interferon ;f (ifny) [ u/ml]. after hours of incubation no-release was determined as nitrite (no ) by using a colorimetric assay based on the griess-reaetion. results: preincubation of cells with the pkc-inhibitors staurosporine (stp), chelerythrine, bisindolylmaleimide (bim) and d , that recently has been identified as a selective inhibitor of pc-plc, diminished significantly lpsand ]fnt-induced no -production (p<o, ). although we observed no toxic effect on cell viability, no -production was related to protein concentrations to exclude any inhibitory effect on celt growth. celts preincubated with stp [lo nm] released % less no in response to lps and ifn,[ compared to control cells cultured without inhibitor. chelerythrine [ }jm] and bim [ioijm] reduced the no -formation by % and %, respectively. interestingly, the most potent inhibitor of no-production turned out to be the xanthate d cells pretreated with d [ ~g/mt] released % less no than stimulated controls the inhibitory effect on no production decreased the later the inhibitore were added after stimulation; e.g. bim added and hours after stimulation reduced no -production only by % and %, respectively. accordingly, inqs activity present in stimulated cell lysates supplemented with l-arginine and co-factors was not suppressed by the inhibitors tested. conclusions: our findings suggest, that the induction of inos but not its activity is a pkc and particularly pc-plc dependent process. dr. k tschaikowsky, institut for anaesthesiologie, universital erlangen--nqrnberg, krankenhausstr , erlangen interleukin- (il- ) affects nearly every cell type by increasing the expression of a variety of genes associated with the promotion of inflammatory processes. these include upregulation of cyclooxygenase and nitric acid synthases. the il- ri transmits a signal(s) through the cytoplasmic domain which is structurally related to the fruit fly toll protein. we have recently cloned the promotor most proximal to the ' utr of the human il- ri gene. the genomic sequences reveal a lack of tata and caat boxes but rather a considerable ( %) gc rich region. the translation of the type i il- r appears under a significant suppression and may limit the biological activities of il- by low level of expression. on the other hand, the type ii il- r, lacking a significant cytoplasmic domain, does not transmit a signal and acts as a decoy receptor preventing the binding to the type i receptor. there are several approaches to reducing il- activity; inhibition of il- converting enzyme which cleaves pro-il-l[ , anti-il- ri, the il-i receptor antagonist (il- ra) and soluble (extracellular) il- ri and il- rii. il- ra is structurally related to il- and binds at °c to the il- ri with near equal affinity as that of bona fide il-i; however, il- ra does not transduce a signal. il- ra has been given to humans in pharmacologic levels (mean plasma levels of gg/ml) without evidence of agonist activity. in addition, there has been no affect on normal homeostatic parameters, suggesting that il-i does not play a role in health. in healthy humans given intravenous endotoxin, il-ira reduced endotoxin-associated neutrophilia by % and completely reversed endoloxin-iuduced immunosuppression, il-tra is produced naturally and is elevated in the circulation in several diseases. in a variety of diseases, plasma levels of il- ra are in -fold molar excess to those of il-i [ . it is unclear whether these endogenous levels of il- ra are sufficient to block il- activity in disease. a single injection of ]l- or ifna in humans does not induce il- but rather high levels of il- ra ( ng/ml). prof.dr.l.a. aarden interleukin (il ) is a multifunctionai cytokine with a central role in host defense mechanisms and consequently in inflammation. il is thought to be involved in the pathogenesis of various diseases. therefore, specific inhibitors could have therapeutic value. a human-mouse chimeric monoclonal antibody to il has been applied in a phasei clinical trial in patients with multiple myeloma and in primate models of sepsis. no toxicity was observed and the most remarkable outcome of this study was the build up in the circulation of il -anti-il complexes, suggesting that plasma il measurements represent a gross underestimation of il production in the patient. until now no natural antagonists of il have been described. the biological activities of il results from binding to a lowaffinity il -receptor (il r). the il /il r complex induces disulfide-linked homodimerization of the signal transducing receptor component, gp . studies on the structure-function relation of il revealed that disruption of the gpl -interaction site on il gives rise to a mutant il that, by virtue of its intact binding to the il r, acts as a potent antagonist on human hepatocytes and on human b cells. in vivo studies using this molecule will be initiated in the near future. the evolution of infla~ation involves a dynamic series of cellular events controlled by specific signals which are important to the initiation, maintenance, and final resolution of the inflammatory response. the various phases of inflammation are influenced via the expression and subsequent regulation of a number of key mediators which play a predominant role during each particular stage of the developing lesion. for example, the initial elicitation of blood born leukocytes to on inflamed area is a complex system that is dependent upon the expression of early response cytokineso these proximal mediators, including both interleukin-i (il-i) and tumor necroisis factor (tnf), are important in the initiation phase by activating both immune and non-immune cells and establishing a series of cytokine networks, thus, the above proximal mediators can stimulate endothelial cells, epithelial cells, smooth muscle cells, and fibroblasts to generate more distal cytokines. these latter cytokines include interleukin- (il- ), macrophage inflammatory protein-i (mip-i), and monocyte cbemoattractant protein-i (mcp-i). the importance of the above chemukines can be found in the role they play in leukocyte elicitation, as well as wound repair. numerous investigations have shown the diverse cellular sources of il- and the ability of il- to elicit neutrophils in vitro at pm to nm concentrations, however, recent studies have sho%~ an additional role for il- during the resolution phase of the inflammatory process. using corneal pocket animal models of angiogenesis/neovascularization, il- was found to be a potent angiogenic factor at concentrations ranging from - ng/ml. sequential fluorescein angiograms demonstrated that il- induced neovascularization by days, which regressed by weeks. in further analyses, both conditioned media recovered from either inflamed human heumatoid synovial tissue macrophages or lps-stimulated peripheral blood monocytes were found to possess potent angiogenic activity, which was effectively blocked by neutralizing antibodies directed against human il- . in addition, an il- antisense nligomunclentide blocked the expression of a functionally active angiogenic factor from lps treated monocytes. the above data demonstrate that il- has multifumctional activities during the initiation, maintenance, and resolution of a local inflammatory response. inhibits specific t-cell responses to soluble protein antigens and alloantigens. the proliferative responses of th , thl and th cd ÷ t-cell clones and cytokine production by these t-cell subsets are equally well inhibited. the inhibitory effects of il- are indirect and related to inhibition of antigen-presenting capacity and accessory function of the antigen-presenting cells (apc). however, il- also directly inhibits t-cell proli/eration induced by cross]inked anti-cd or anti-cd mabs (e.g. in the absence of professional apc), by specifically inhibiting il- gene transcription and il- protein production. il- also inhibits the production of the pro-inflammatory cytokines il-l-a, ~, il- , and tnf-a and the chemokines il- and mip-i-u, which are strong chemo attractants for neutrophils and macrophage, respectively. in contrast, il- enhances the production of the il- receptor antagonist, a cytokine with anti-inflammatory activity. il-l produced by monocytes downregulates class ii mhc expression and il- production by these cells in an autoregulatory fashion. collectively, these in vitro data indicate that il- is a powerful suppressor factor for immune-and inflammatory responses and therefore may have potential clinical utility as an anti-inflammatory agent. this notion is supported by recent studies which indicated that il- also prevents lethal lps-induced toxic shock in mice. five of the chamekines (hip-i~,era~tes. ip-io and il ) also chemoattraet t lymphocytes, while others act on mast cells. we have studied the effaces of these chemokines on t cell subsets. il preferentially chemoattracts uns~imulated t cells. raises ~ttracte resting as well as activated cd and cd memory t cells. ip- chemoattracts only preactivated (not resting) cd or cd t cells. hip-l~ preferentially chemoattracts gd t calla, while hip- more readily attracts the cd t call subset. the chemoklnes and ip-io were also sho~rn to promote the adherence of ~he same subsets of anti-cd activated t cells to ili activated human umbilical vein endothelial cells (hitvec). this was not associated with a~y i~crease in the number of lymphocyte adhesion molecules, bu~ could be inhibited by neutralizing monoclonal antibodies re lfa-i and vla- . these chemokines also rapidly increased ~ha adhesion of resting t l~phoeytes to plates coated wi~h integrlns (i-cam or v-cam) or matrix proteins such as f~bronectln. this induction of adhesion began wiehln ', peaked by hr and was completed in hr. this suggests that chemoklnee rapldly increase the binding affinity of adhesion prote~ns on t cells. we also recently observed ~hat mcaf/mcpi and rantes chemoattract unatimulated mast calls. furthermore, igs actlvared mast calls were chemoattraeted by mcaf, ra~tes, pf and mip.i~. however. ~he chemoklnee did not induce mast cells ~o release histamines. presumably, chemokines as regulators of the adhesion, migration and homing of all kinds of leukocytes participate in many types of inflammatory diseases. natural killer cell stimulatory factor or interleukin- (il- ) is an heterodimerie cytokine formed by two covalently linked glycosylated chains of kd and kd. il- was originally purified from the supermatant fluids of ebv-transformed human b ly~phobiastoid cell lines. however, in addition to b cells, phagocytic cells, i.e. monocytes, macrophages, and neutrophils, have been identified as the major physiological producers of il- . phagocytic cells produce il- in response to bacteria, bacterial products such as lps, and intracellular parasites. the cell types on which il- exert biological activity are t and nk cells. the major direct biological function of il- on these cell types are the induction of cytokine production, primarily ifn-y, the enhancement of a generation of cytotoxic cells, and a mitogenic effect on antlgen-activated lymphocytes. in part thromgh its ability to induce ifn-y production, il- , produced in response to infections, represents an important functional link between effector cells of innate resistance, phagocytic cells and nk cells, and effector cells of adaptiye resistance, t and b lymphocytes. the ability of il- to induce ifn-y production from circulating. nk cells and at least a proportion of t cells determines a rapid, antigen-independet production of ifn-y during infections. ifn-y acts as a potent enhancer of phagocytic and bacteriocidal activity of phagocytic cells, participating early in infection to activate some of the inflammatory mechanisms that represent the first innate resistance against infection. this antigem-independet activation of phagocytic cells that involves, in addition to il- , other monocyte derived cytokines such as tnf and il-l, has been shown to be important in experimental animals for the resistance against infection by microorganisms such as limteria monocytogens and toxoplasma gondii. in addition to this role, early in infection in the antigen-independet phagocytic cell activation, il- has a major effect in the ensuing antigenspecific admptive immune response by facilitating the generation of t helper type (th-i) response and suppressing a th- response. the presence of il- during antigen stimulation in vitro using human peripheral blood lymphocytes, or both in vivo and in vitro in experimental animals, induces generation of th-i cells producing ifn-y and il- , and inhibits the generation of th- cells producing il- . il- induces a direct differentiative effect on th-cells, priming them for high ifn-y production. ~is priming effect is stable and maintained even when t cell clones are cultured for extended periods in the absence of il- , however, il- needs to be present during the first few days after stimulation with antigen or mitogen and established th clones are resistant to the priming effect of ifn-y. unlike the generation of high ifn-y producing clones, the il- -mediated inhibition of il- producing cells in probably due to selective mechanisms, which may include a selective mitegenic effect of il- for thl cells and an inhibitory effect of ifn-y on th cell proliferation. in several experimental systems, it has been shown that the enhancing effect of il- on th-i responses is dependent on production of if~-y and on the presence of nk cells, possibly needed for the early production of ifn-y. the importance of phagocytic cells and nk cells in determining the characteristics of the th response during antigenic stimulation indicated that the mechanisms of innate resistance have a determining influence on the subsequent antigen-specific immune response: il- appears to have a key role in mediating the interaction between phagocytic cells. nk cells, and t lympocytes in these proccessem. trinchieri, g. , interleukin- and its role in the generation of t e cells, imm~ol. on peripheral blood monocyte/macrophages, il- behaves like il- in a variety of assays, and shows both similar and contrasting, activties with either il- or ifn-y. il- reduces inflammatory monokine and il- production. it reduces the pyrogen-induced expression of tissue factor (herbert et el, , febs lett. , ) . it mpregulates manmose receptor and ~c class ii expression, while reducing cdi expression. il- produces morphological changes (extensive cell processes, aggregation, giant cell formation) which have previously been observed with il- . it inhibits niv-i production (montaner et el, , i. exp. mad. , ) , and interferes with hiv-induced cell fusion. the activities of il- and il- differ on t-ly•ocyte and large granular lymphocyte population. in particular il- does not exhibit the suppressive effects of il- on thl-type-helper functions (ifny production) and cytotoxic functions (perforin production). data will be presented showing that il- and il-l share a common receptor on a nu~er of cell types, but not on t-lympocytes (see also zmrawski st el. , embo j. . - ), explaining both thai common and divergent activities. the levels of il- ~a in activated peripheral blood lymphocytes are greater than or equivalent to those of il- mp~ja and the two ml{nas are expressed by different t lympocyte population: in particular, t cytotoxic lymphocytes express markedly higher levels of il- ~rna. in vivo, il- may thus be contributing, significantly to some of the activites previously ascribed to il- il- . recently, we cloned the human cdna encoding interleukin- (il- ). human il- is a non-glycosylated protein of aa with a molecular mass (mr) of kd and has biological activities on monocytes and b cells, il- , like il- , strongly enhanced the expression of class ii mhc antigens on monocytes and induced expression of cd (fc~rii). furthermore, il- , like il- and il~ , inhibited the production of pro-inflammatory cytokines il- , ]l- , tnfo~ and chemokines miplc~ and tl- by lps activated monocytes. both il- and il- enhanced the production of il-lra by monocytes. in contrast, il- lacked the t cell stimulating activities of il- . the responses of monocytes to il- and il- were not additive or synergistic, supporting the hypothesis that il- and il- receptors are complex and share a common component which is important for signal transduction, taken together, these data indicate that il- , il~ and il- have important anti-inflammatory activities on human monocytes. in addition, these results indicate that il- is unique in that it can modify inflammatory reactions without stimulating (as does il- ) or inhibiting (as does il- ) t cell responses. transforming growth factor beta i (tgf-fll) belongs to a family of proteins that have potent immunoregulatory properties ranging ftom effects on the growth and dfflerentiafion of primitive stem cells to the differentiated functions of immune system effector cells. tgf-i~i is synthesized as a large secretory precursor polypeptide of amino acids that is inactive until processed to a disulfide linked homodimeric molecule of t amino acids each. recently it has been found that tgf-[~i can have autocrine as well as paracrine effects on the immune system indicating that immune cells can activate the inactive secreted form of tgi~-~i. in addition, tgf-i~i has differential intraceuular effects on cell surface receptor modulation, tyrosine phosphorylation and cytokine gene transcription as well as cell mediated cytotoxicity. the systemic administration of tgf- has proven beneficial in a variety of animal models such as septic shock, allograft rejection and experimental forms of autoimmunity including collagen induced arthritis and experimental autoimmune encephalomyelitis. moreover the increased levels of tgf-ill and other tgf- isoforms found in several disease states associated with immunosuppression such as different forms of malignancy, chronic degenerative diseases and aids implicate the involvement of tgt~-i~ in the pathogenesis of some diseases. hopefully, well designed clinical trials will define the potential roles of the tgf-[ family of proteins in the treatment of diseases of man. patient development of systemic inflammatory response syndrome (sis) after severe trauma is accompanied by a wide range of immune aberrations and altered cytokine production. in particular, t lymphocyte proliferation and ifn, t production is suppressed while monocyte (mo) tnfc~, il- , pge and tgf~ production is massively increased concomitant to decreased antigen presenting capacity. recently, two new cytokines, il- and il- , have been characterized as critical in regulation of mo tnfa and il- , as well as in induction of t lymphocyte proliferation and ifn production. in this study, peripheral blood leukocytes (pbl) from severe trauma patients (injury severity score > ) were analyzed for their il- levels, their in vitro proliferation to mitogen (pha) and their response after il- addition. since il- produced either by mo or by t lymphocytes can depress m~ antigen presenting capacity, inhibit t cell ifn,/production and directly diminish t cell proliferation, it might be suggested that immunosuppressed patients' mo and/or t lymphocytes would have increased il- levels. increased patient il- production might also be resulting from the high levels of tnfa a known stimulator of il- . conversely, since il- augments mo antigenpresenting capacity, thl induction and proliferation, post-trauma leukocytes might be il- deficient. pbl of trauma patients were compared to normals' pbl, either unstimulated or ptta induced, and their levels of il- found to be dramatically and significantly reduced. patients' isolated m~, either stimulated with the bacterial cell wall analogue, mdp, or unstimulated, also had depressed il- production concomitant to elevated tnfa production when compared to normals' mo. mechanisms for the depressed patients' mo il- were explored. increases in tgf[ may have partially contributed to the patients' depressed il- level, but elevated pge had no effect. addition of il- to patients' pbl significantly increased their mitogen responses. these data imply that sis is characterized by disruption in the interactions between mci and t lymphocytes so that patients' m~i produce excesses of some mediators (tnfa, il- , pge ) and a dearth of other monokines (il- , il-io). t lymphocytes are not activated and, therefore, unable to function in both immune defense and monocyte regulation. it is known that lge receptor-mediated or ca-ionophore-induced activation of mouse bone marrow-derived mast cells ( mmc) may result in the production of different cytokines including the interleukins (il) , , , and as well as gm-csf and tnf-a. in the present study we analyzed the effects of exogeneously applied pro-inflammatory cytokines (il- , l- , tnf-c as well as various mast cell growth factors (il- , il- , il- , il- , ngf, kl (kit ligand)) on cytokine production in primary mouse bmmc using a standard activation protocol (lxl bmmc/ml; ll.um ionomycin; - h). the actixdties of bmmc supernatants were assessed in specific biological (il- , il- il- , l- ) and/or elisa assays (il- , il- ). here we show that homogeneous populations of bmmc (> %alcian blue+/safranln-; in vitro age: weeks) generated in the presence of recombinant (r) rail- from normal balb/c mice produced modest amounts of l- and low or undetectable levels of il- , - , and - after induction with lp.m ionomycin only. however, a dramatic increase ( -to -fold) of these cytokine activities was noted, when in addition to ionomycin also human ( ) rll-la was provided during the induction period. this il- effect was dose dependent with a maximgm at - u/ml hrll-la and specific, as pre-incubation (lh) of bmmc with ng/ml hrll- receptor antagonist abolished the action of u/ml hrll-lcc similar effects were noted with hrll-lg or rurll-lb (lng/ml, respectively), but not with rhll- or rmtnf-~. both mrll- and hrll- substantially enhanced ionomycin-induced l- production of bmmc in the absence or presence of il- . il- significantly enhanced il- and il- production while decreasing il- activities to abont - % of control levels, when il-i was provided in the presence of il-l/ionomycin. a monoclonal anti-nfil-t antibody (ascites : ) abrogated the effects of mrll- . other mast cell-active cy~okines (] ,- , il- , l- , ngf, or kl) added to ionomycia-or l- /ionomycin-treated bmmc had no major effects on cytokine production. il- and il-i did not induce significant cytokine release in the absence of ionomycin suggesting tlmt cadependent signalling was required. at doses of " m, dexamethasone, corticosterone, or hydrocortisone almost completely abolished ionomycin/il- /ll- induced cytokine production. the inducer cocktails per se did not interfere with the cytokine bio-assays. in case of il- inducibility of this cytokine in bmmc was confirmed at the mrna level by northern blot analysis. hence our data show that activated mast cells are a source of il- previously recognized as a product of th type lymphocytes only. moreover, our study reveals novel functional roles for i-l-i, il- , and ghicecorticoids in the regulation of cytoldne production in mast ceils. accumulating data suggests that cytokines, peptides involved in regulation of both physiological and pathological immunological responses, predominantly are produced at the local site of antigen stimulation. a new method was used to detect cytokine-producing cells in haman tissue at the protein level. single-cell production of different httman cytokines, ilia, ill [ , illra, il , il , il , il , il , ils, ill , gm-csf, tnfa, ifn and tgf[ . , was identified by indirect immunohistochemical staining procedures and use of carefully selected cytokine-specific mab's. frozen sections were fixed with % paraformaldehyde and permeabilized by . % saponin treatment, eluting cholesterol from the membranes. the intracellular presence of all cytokines except ill, illra (late) and tfg[ _ , could be demonstrated by a characteristic perinuclear configuration in producer cells. in addition, the immunoreactivity extended over a large extracellular area encompassing the producer cell. a localization of the cytokine to the golgi-organelle was established by use of two culour staining including a haman golgi complex specific mab. this staining pattern was only evident in producer cells because injection of recombinant human cytgkines into the tissue caused a membraneous and extracellular staining pattern. both the extra-and the intracellular types of staining reaction could, however, be blocked by preincubating the cytokine specific mab with pure human interleukins. oxygen radicals (or) directly induce lipid peroxidation, indirectly they trigger adhesion and activation of pmn leukocytes. we investigated whether or also lead to a release of acute-phase response cytokins such as tnf-alpha, il-i beta or il- in whole blood cultures to maintain the induced inflammatory reaction. methods: blood samples from healthy volunteers (n= ) were incubated at °c. or were produced by the xanthine oxidase (xo)/ hypoxanthine (hx) system. after , , , , and minutes plasma levels of tnf-alpha, il-i beta and il- were determined with elisa kits. results: under the influence of or tnf-alpha plasma levels increased from , pg/ml at min to pg/ml, pg/ml, pg/ml after , and min. il-ibeta ( , pg/ml, , pg/ml, , pg/ml, pg/ml and pg/ml after , , , and min) and il- ( , pg/ml, l,lpg/ml, , pg/ml, pg/ml and , pg/ml after , , , and min) plasma levels were increased min later than tnf-alpha. summary: these data suggest that or do not only play an important role in initial accumulation and activation of pmn leukocytes but also lead to a stimulation of monocytes to produce the acute phase reaction cytokins tnf-alpha, il-i beta and il- to maintain and strengthen the inflammatory reaction. department of general surgery, steinhsvelstr. , ulm, germany jan k. horn md, greg a. hamon md, robert h. mulloy md, greg chen bs, rebecca chow bs, and christof birkenmaier md. transforming growth factor-i~l (tgf- ) is released from inflammatory ceils following injury and in sepsis. in vitro experiments have confirmed that low concentrations of tgf- ( . - . ng/ml) are chemoattractive for monocytes, whereas higher levels of tgf- (> . ng/ml) potentiate production of the immunedepressive prostaglandin e . other investigators have shown that tgf-] can cause the appearance of cd (fc immunoglobulin receptor) on monocytes exposed to ng/ml of tgf-[~i for hours. monocytes also express on their surface a glycoprotein that binds complexes of lipopolysaceharide (lps) and lpsbinding protein (lbp). such binding is associated with generation of proinflammatory cytokines such as tumor necrosis factor alpha. we have shown that cd is depressed in septic patients and therefore we hypothesized that tgf- could account for the down-regulation of cd observed in these individuals. we incubated normal human monocytes with platelet-derived tgf-[ for and hours at °c and examined ceils for cd and cd expression using flow cytometry after immunnfluoreseent staining with appropriate monoclonal antibodies. monocytes were selected on the by usual criteria for size and granularity. non-viable ceils were excluded with the use of propidium iodide. two populations of monocytes could be found afcer incubation at °c alone. one displaying high density of cd had increased fluorescence over the homogeneous expression of cd in cells maintained at °c (baseline). the other population displayed decreased cd expression relative to the baseline cells. tgf-i~i ( - ng/ml) caused a shift of ceils from the high density into the low density cd population. this trend was observed within hours of incubation and was complete by hours. we observed a net decrease in cd expression f % for all subjects studied (p< . vs controls). phorbol myristate acetate ( ng/ml) also caused down-regulation of cd to a similar degree as tfg-i~i. we also confirmed that monocytes could be induced to express cd after incubation with tgf- ( ng/ml) for hours. these studies demonstrate that monocytes incubated with immunodepressive levels of regulation of cd by tgf- deplete their surface expression of cd while generating cd . this down-regulation of cd by tgf- correlates with our clinical observations of lower cd expression on monocytes obtained from septic patients. for over years, activated t lymphocytes have been considered to be the cellular source of mif. we recently isolated and cloned the murine homolog of mif after identifying the specific secretion of this protein by lpsstimulated pituitary cells in vitro and in vivo. however, further experiments showed that mif protein is detectable both in t-cell deficient (nude) and hypophyseetomized mice, suggesting that yet additional cell types may produce mif in vivo. since monocytes/macrophages are a major source of the cytokines that appear in response to lps administration, we examined the possibility that mif also is expressed in cells of the monocyte/macrophage lineage. we found that mif is expressed constitutively in the murine macrophage-line raw . and in thioglycollate-elicited peritoneal macrophages. significant amounts of mif mrna (rt-pcr) and protein (western blotting) were observed in cell lysates. in raw . cells, mif secretion was induced by as little as pg/ml of lps (e.coli l:b ), peaked at ng/ml, but was not detectable at lps concentrations > txg/ml. similar data were obtained with elicited macrophages, but higher lps concentrations were required, unless the cells had been preincubated with ifn . production of mif by lps-stimulated (l ng/ml) macrophages peaked at hr. expression ofmif mrna and tnf mrna by lps-stimulated raw . macrophages was investigated by rt-pcr. as expected tnf mrna expression increased over the range of lps concentrations ( pg/ml to p_g/ml). in contrast, levels of mif mrna correlated inversely with lps concentration. by competitive pcr, mif mrna was observed to increase approximately -fold after lps induction ( pg/ml). mif secretion also was induced by tnfoc ( ng/ml) and ifn? ( iu/ml), but not by il- and il- (up to ng/ml). lps and ifn had additive effects in inducing mif secretion. in separate experiments, macrophages stimulated with recombinant mouse mif ( gg/ml) were found to secrete bioactive tnf~ (> pg/ml by l cytotoxicity). we conclude that the macrophage is an important albeit overlooked cellular source of mif in vivo. mif secretion is induced by lps, tnfc~ and ifn?. mif also stimulates macrophages to secrete tnf. taken together with previous observations that anti-mif antibody protects against lethal endotoxemia, these data implicate mif as a critical mediator of inflammation and septic shock. inflammation is characterized by an exacerbation of proinflammatory cytokine production. cytokines such as il- , il- , and tgf , have been identified as anti-inflammatory mediators thanks to their ability to down regulate the production of il- , il- , il- , tnfc~ by activated monocytes / macrophages. however, other cells, including polymorphonuclear cells (pmn) do contribute to the release of pro-inflammatory cytokines. we investigated the capacity of the so-called anti-inflammatory cytokines to control the release of il- by activated neutrophils. human pmn were purified following glucose-dextran sedimentation and ficoli-hypaque centrifugation. the cells were cultured at °c for h in the absence or presence of lipopolysaccharide (lps) or tnfa. il- release was measured in the supernatants using a specific elisa. among tested cytokines, il- was the most efficient inhibitor of il- production by lps-activated pmn. il- was also active, whereas no down regulation was noticed with tgfp~i. when tnfa was used as a triggering agent, none of the cytokine could prevent il- production. northern analysis are under investigation to precise the level of the il- -and il- -induced inhibition of il- production by pmn. our data illustrate that il- and il- possess the capacity to down regulate the production of il- by both monocytes and pmn, whereas tgfb has a more limited inhibitory activity. ciliary neurotrophic factor (cntf), a member of the il- superfamily, has recently been shown to promote axonal growth and neuronal healing. cntf production is also increased during neuronal and muscle damage, associated with soft tissue injury or trauma. we postulated that production of cntf may explain the loss of skeletal muscm protein that occurs in inflammation. female, wistar ( - gm) rats received either or pg/kg bw s.c. injections of recombinant rat cntf for seven days, or received sham injections and were freely-fed. additional animals were pretreated with mg/kg ibuprofen lp prior to pg/kg bw cntf. rats treated with ,ug/kg bw cntf lost . _+ . gms bw as compared to freely-fed controls which gained . _+ . gms (p<o. by anova and lsd), and ibuprofen treatment had no effect on cntf-induced weight loss (- . _+ . ). animals pair fed to the high dose cntf gained body weight ( . -+ . ), although weight gain was less than seen in freely-fed controls. rats treated with pg/kg bw had reduced body weight gain (+ . +_ . gin). food intake was also reduced by % in pg/kg cntf (from . +_ . gm/day to . _+ . gm/day; p<o. ) and was also unaffected by prior ibuprofen treatment ( . +- . gm/day). despite the loss in body weight noted in the high dose cntf animals, there was no appreciable hepatic acute phase response, since liver weight ( . + , gms vs. . _+ . gins), total protein ( . +- . gms/l vs . +- . gins/l) and albumin ( . + . gms/l vs. . +_ . gms/l) were not different between cntf and freely fed animals. we conclude that cntf causes anorexia, and increases body weight loss in excess of the associated anorexia. unlike the cachexia associated with il- and tnf infusions, which is associated with an acute phase response, cntf acts predominantly on food intake and carcass weight through a prostaglandin-independent mechanism and has only minimal acute phase inducing properties, n. joseph espat, md. university of florida, department of surgery, j-box , gainesville, fl. . of cytokines by neurotrophic factors, the neuroendocrine system and phenotiazines. cytokines, including tnf and il- , have a pathogenetic role in various diseases related to infection and inflammation. the action and/or production of cytokines can be regulated by drugs or endogenous mechanisms that involve the cetral nervous system (cns) we found that the antipsychotic chlorpromazine (cpz) potently inhibits tnf production and protects mice against endotoxic shock. cpz also inhibits brain tnf production in mice intracerebrally injected with endotoxin, whereas cpz analogs that do not cross the blood-brain barrier inhibit only perypheral, not brain, tnf. tnf production and susceptibility to the toxicity of endotoxin, tnf and il- are increased in adrenalectomized mice, indicating that endogenous corticosterone (cs) controls cytokine production and toxicity, in a feedback fashion since endotoxin and cytokines increase serum cs. increased tnf production was also observed in mice where the hypothalamus-pituitary-adrenal axis was blocked by chronic administration of dexamethasone, following a two-day washout. administration of cytokines acting through the gp signal transd,uction protein, including il- and ciliary neurotrophic factor, cntf potentiated il-l-induced elevation of cs. il- and cntf also induced hepatic acute-phase proteins. thus il- and cntf might have antiinflammatory activity, by potentiating the feedback responses of the neuroendocrine axis. these data indicate how complex can be the interregulatory relationships between the brain and the immune system, how they can be used to pharmacomodulate cytokine action and how their disregulation might exacerbate il- -and tnf-mediated pathologies. lipopolysaccharide (lps) constitutes a well established activator of monocytes/ macrophages (mizi) and murine b lymphocytes. the hydrophobic part of lps, termed lipid a, represents the endotoxic principle of lps. we now demonstrate that lps is also capable of inducing proliferation of human t cells at a dose of to pg/ml. the specificity of this stimulation was analysed by the following experiments: i) the synthetic hexaacyl escherichia coiltype lipid a (compound ) also stimulated human t cells; ii) the synthetic tetraacyl lipid a precursor (compound ) or the phosphono-oxyethyl analogue pe- , structures known to antagonize lps-induced monokine production in human mz, also inhibited lpsinduced t-cell proliferation. the t-cell proliferation expressed the following features: i) cd ÷-as welt as cd ÷ t cells proliferate in response to lps, ii) limiting dilution analyses reveal that the frequency of responding cells was between / to / cells; iii) purified t cells alone cannot be stimulated by lps, but need the help of monocytes. this help cannot be replaced by b cells or fixed monocytes. furthermore, for activation a direct cell-to-cell contact between t cells and monocyte is neccessary; iv) t cells stimulated with lps express mrna for il- and ifnf, but not for il- or il- (determined by pcr). in supernatants, ifnf was detectable (elisa); v) lps-activated cd ÷ as well as cd + t cells express cd , the high affinity il- receptor. our results indicate that in contrast to previous reports human t cells respond to lps with il- receptor expression, lymphokine production, and proliferation. the reactive cells appear to belong to the t, cell type, the finding that human t cells can be activated by lps is of important relevance for the understanding of the pathomechanisms of infections by gramnegative bacteria. this may lead to new strategies for the therapy of sepsis. we have recently shown that human eosinophils produce mrna for interleukin (il)- when stimulated with calcium ionophore (eur. j. immunol. ( ), ). we now present evidence that human eosinophils contain preformed il- protein although they do not express mrna for il- as measured by rt-pcr. il- protein expression was determined using specific anti-human il- monoclonal antibodies by western blotting, facs analysis and immunohistochemistry. moreover, preformed il- was re/eased into supernatant by % pure eosinophils after priming with gm-csf or il- and subsequent -min stimulation with paf, rantes, ionomycin or pma, however not with il- or il- , as measured by elisa. this observation implies that activation of protein kinase c and/or intracellular calcium mobilization are necessary events for il- release in human eosinophils. the determined amounts of preformed il- protein was higher in patients with asthma compared to normal individuals suggesting a role for eosinonhi! derived il- in asthma. since the eosinopnit is the ,,r~:=z~mmant cell in the asthmatic airways, we determined il- concentrations in bronchoaiveolar lavage (bal. ~ids from normal !i~.~ijvtduals and asthmatic patients. indeed, il- concentrations in bals from both groups were similar to those seen in the supernatants released by activated eosinophils. the role for il- in asthma remains to be determined. based on a well established rat model named activity-induced ulceration (asu) or activity based anorexia (aba), we have developed a rat model for chronic stress. male rats were exposed to a feeding schedule in order to reduce body weight by - % within days. while one group of rats was kept sedentary, the other had access to a running wheel. food restricted rats allowed to exercise developed highly increased activity as compared to ad libitum fed rats in wheels (ca. versus kin/day). they developed elevated plasma corticosterone levels ( _+ gg/dl) at their circadian peak, increased adrenal weight and decreased thymus and spleen weight as compared to control rats and weight matched sedentary rats, establishing this paradigm as a model of chronic stress. no differences in plasma acth were seen among the various groups, suggesting increased sensitivity of the adrenal glands to acth. the effect of chronic stress accompanied by hypercorticism on baseline and endotoxin-induced cytokine levels was studied in this model. under baseline conditions,splenic il- a was suppressed in both food-restricted groups. this was more pronounced in rats with access to a running wheel. il- and tnf activity in plasma was not affected. after administration of .tg/kg lipopolysaccharide (lps), tnf activity in plasma was suppressed by food restriction but there were no differences between sedentary or exercising rats. in addition, no differences were found among groups for corticosterone, spleen il-lc~ and plasma il- activity. we conclude, that the food restriction-induced hyperactive rat model is a useful model for studies on the effects of chronic stress. in this model, under basal conditions il-le~ in tissues may be subject to downregulation by glucocorticoids, whereas after lps-stimulation of cytokine production only tnf activity is regulated by fast acting feedback loop between cytokines and the hypothalamo-pituitary-adrenal axis (hpa-axis). this is supported by strong correlations between plasma tnf activity and corticosterone in stressed rats. cytokine antagonists modulate cytokine actions and it appears that a balance between production of cytokines and cytokine antagonists is important to control of host responses. recent observations show that during myocardial infarction there is an increase in circulating cytokines such as tumor necrosis factor (tnf) and interleukin (il- ). we tested the idea that compensatory increases in cytokine antagonists likewise occur and investigated changes in plasma concentrations of tnf and interleukin i (il- ) and those of their specific antagonists interleukin- receptor antagonist (il- ra) and soluble tumor necrosis factor receptor type i (s tnf r-i). we also measured plasma concentrations of ~-melanocyte-stimulating hormone (~-msh), an endogenous neuropeptide that occurs in pituitary, brain, skin, gut and other tissues. when administered to laboratory animals, this peptide has potent antiinflammatory and antipyretic activity, perhaps by mimicking an endogenous modulatory influence on cytokine actions. samples were obtained from patients with acute myocardial infarction at presentation in the coronary unit, every three hours for hours, and daily thereafter until discharge. we found elevations in the plasma cytokines il- and tnf only in a proportion of subjects whereas cytokine antagonists c~-msh, il-lra, and stnfr were elevated in all patients, o~-msh was elevated at presentation but it decreased progressively in successive tests, reaching normal values within hr whereas stnfr and l-lra peaked at - hr or later. there were significant correlations between plasma concentrations of cytokine antagonists and enzymes released from injured myocardium, including creatine kinase (ck), aspartate serum transferase (ast), and lactate dehydrogenase (ldh). the data show that cytokine antagonists increase in the circulation of patients with acute myocardial infarction likely to modulate prninflammatory effects of cytokines. objects of the study: interleukin- (il- ) is a multifunctionai cytokine known to be involved in important homeostatic processes. il- levels are increased in many pathological situations, and correlates with disease activity and patient outcome. to exert its effect il- binds to its receptor on the membrane of its target cell. this receptor is also present in a soluble form in plasma and in urine. in this study we quantitatively measured the availability of soluble il- receptor (sll- r) in biological fluids in healthy volunteers. materials and methods: blood, urine, cerebrospinal fluid (csf), and synovial fluid (sf) are obtained from healthy volunteers, sil- r and il- are measured using elisa's. both elisa's are tested for interference of sil- r mid il- . the effect of sll- r in the il- bio-assay (b ) is tested. results: baseline levels are (mean + sd): . -+ . ng/ml in serum, . -+ . ng/ml in urine, . _+ . ng/ml in csf and . + . ng/ml in sf. there is no interference of sll- r in the il- elisa or bio-assay and no interference of l- in the sil- r elisa. conclusions: in all investigated biological fluids sll- r can be found. these data provide baseline values for investigations concerning pathological situations. both elisa's described are useful tools to do these investigations. trauma-associated immune aberrations coincide with augmented release of immunoregulatory and proinflammatory cytokines. the present study examines the effect of thermal trauma on the capacity for specific (receptormediated) response of lymphoid cells to interleukin (il ) and to turnout necrosis factor ~x (tnfc . methods. bum patients (n= , -> % total body surface area) were studied weekly up to days post-injury. the limulus amoebocyte lysate (lal) test was used to measure plasma endotoxin levels. the percentage of il ~-and tnfcz-binding t(cd ) lymphocytes was assessed by flow cytometry analysis. levels of il receptor antagonist (il lra) in patients' plasma and cultures of peripheral blood ceils (pbc) were determined by immunoassay. results. plasma endotoxin concentrations were significantly (p< . ) increased up to weeks post-bum (means . + in non-surviving and . + . u/ml in surviving patients vs < u/ml in the control). within weeks of bum, the percentage oft ceils expressing receptors for tnfa and il [~ constitutively was elevated (by - fold). in contrast, the capacity for de novo receptor expression by activated pbc was reduced. serum levels of il ira were significantly increased (range . - x j pg/ml vs < . x j pg/ml in the control). in all patients, high concentrations of il lm were released spontaneously in unstimulated cultures of adherent ceils (range - x - pg/ml vs - x j pg/ml in the control). however, its secretion was decreased in lps-stimulated parallel preparations. conclusions. in the bum patient, susceptibility to the immunoregulatory effect of tnfcz and tl ~ may be modulated by infection-related products. alterations in the capacity for receptor expression and secretion of l lra may affect il -regulated biological responses including specific immune reactions. while studies suggest that il- is an important lymphokine involved in cell-mediated immunity, little is known about this mediator's role in hem-induced immunesuppression. our aims, therefore, were to determine: i) if il- contributes to depressed t-cell responses seen following hem; and ) how other agents, known to play a role in hem, effect il- release. to study this, c h/hen mice were bled to and maintained at a map of mmhg for h and then adequately resuscitated. mice were killed h post-hem to obtain splenic t-cells (nylon-wool purified). il- 's immunosuppressant role was demonstrated by the ability of monoclenal antibody (mab) to il- to markedly improve the t-cell proliferative response [ . #g the marked increase in capacity of t-cells from hem mice to produce il- was significantly reduced by treatment with either ibu or mabs. since ibu, tgf-~, as well as il- are all reported to directly/indirectly influence prostanoid synthesis, this implies that eicosanoids play a major role in inducing il- release by t-cells following hem which depresses t-cell function. the mechanisms underlying immunosuppression induced by thermal injury and alcohol ingestion are in part due to cytokine dysregulatinn. il- down-regulates production of eytokines by maerophages and may be an important regulator of the initiation of the immune response. il- has also been demonstrated to inhibit the production of no by macrophages. this study examined the alterations in eytokine production and effect of inhibition of no production on immunologic function in a routine thermal injury model. methods: balb/c mice (n= ) were randomized to groups: saline-sham(ns-sham), alcohol-sham(etoh-sham), ns-bum, etoh-bum. animals received % etoh or ns daily for days by gavage. a % full thickness bum was induced hrs after the last dose of etoh or ns. animals were resuscitated, then sacrificed days post bum. splenic lymphocytes were cultured for days with lps, and lps with two concentrations of n-monomethyl-l-arginine, a nitric oxide inhibitor (l-nmma . ug/ml, ug/ml). splenocyte production of il- , interferon-gamma, il- , pge were measured, and lymphocyte proliferative response examined. results: il- production was significantly suppressed in thermal injury. exogenous l-nmma normalized the suppression of .- in a dose-dependent manner, indicating nitric oxide may modulate il- and interferon-gamma production in thermal injury. il- production is normal in etoh-burn animals. conclusion: il- and interferon-gamma production is altered in this murine thermal injury model, and may contribute to this injury-induced immunosuppression. inhibition of no synthesis normalizes il- production and should be investigated further as an immanomodalator in thermal injury. surgery, infection and inflammation results in the production of pro-inflammatory cytokines which mediate metabolic and immunologic host responses. the aim of this study was to characterise the elaboration of cytokine release following a variety of surgical procedures. twenty one patients undergoing elective intermediate, hip, knee and major gastrointestinal surgery were studied. levels of interleukin- (i - ), interleukin- (i - ), the interleukin- receptor antagonist (i - ra) and the acute phase c-reactive protein (crp) were measured in bloods drawn , , , , , , and hours following operation. a portion of the results are shown (mean -+ sem). + -+ _+ one and two factor anova; *p< . , #p< . , §p< . , ¶p< . , for differences between groups i - was not detected at any time point. both ii-ira and i - increased after surgery. maximum responses occurred following major git and hip surgery, minimal responses were seen after intermediate and knee surgery. ii-ira levels increased within two hours and remained elevated for hours; the b-ira increase was a thousand fold greater than the rise in i - levels. i - levels increased up to hours after surgery. crp levels reflected maximum ii-ira and i - levels (r =. , p< . and r =. , p< . respectively). high ii- ra and i - levels reflect major surgery, however the ii-ira response is more rapid and of greater magnitude. the strong i - ra correlation with crp may indicate that this regulatory cytokine is itself a mediator of host responses to surgery. dept. of surgery, meath/adelaide hospitals, heytesbury st., dublin , ireland. change of il- and soluble il- receptor levels after surgery s. hisano, k. sakamoto, s. mita, t. ishiko, m. ogawa [objectives] under surgical stress, il- plays a main role in producing acute phase proteins and contributes to host defense mechanism. soluble il- receptor (sll- r) is considered to be agonistic to il- , unlike other soluble type receptors of cytokines. here we measured il- and sll- r levels in the serum and drain fluid from surgical field in order to investigate the changes of il- and sll- r after surgery and their origins. [materials and methods] serum and drain fluid samples from cases ( of esophagectomy and of gastrectomy ) were serially collected before and after surgery. il- and sll- r levels were measured by elisa. [results] ( ) serum il- : all cases reached the maximum level on pod-l, more precisely - hours after operation. ( ) il- in the drain : maximal il- levels in the drain were recognized - hours after operation, at almost the same time as serum il- . furthermore the il- values in the drain were much higher, about times, than those in serum. ( ) sll- r in the serum : all cases reached minimum levels - hours after operation and recovered to the preoperative levels a few days later (decrease ratio : . + . ~,, range : - ~'). ( ) sll- r in the drain : sll- r levels in the drain showed almost the same value and change as serum sll- r. [conclusions] ( ) il- is produced from the cells gathering around operative fields whereas sll- r is considered to be produced in the cells which do not gather around the operative fields. ( ) there may be a mechanism that down-regulates sll- r in the early stage of surgery. [objectives] il- plays an important role in host defense in the early stage after surgery. in the present study, we examined changes in il- concentration after major thoracoabdominal surgery and elucidated the effect of surgical trauma and factors influencing postoperative elevation of serum il- . [materials and methods] thirty-eight patients undergoing elective surgery of the thoracoabdomen were classified into groups according to the location of the operation. bloods and drain fluids were serially obtained and samples were frozen until measured, keukocytes were simultaneously collected for northern blot analysis. concentration of il- was measured by elisa and il- mrna was detected by northern blotting after total rna was extracted by the acid guanidium phenol chloroform method. [results] ( ) serum il- levels reached the maximum concentration on the st postoperative day in all patients. ( ) the il- peak was significantly correlated with surgical trauma as defined by the operation length and the volume of blood loss during operation (r= . , p< . , r= . , p< . , respectively). ( ) the peak concentration of serum il- in patients undergoing esophagectomy was significantly higher than in those undergoing pancreaticoduodenectomy (p< . ), despite a similar degree of surgical trauma. ( ) peak l- concentration observed in a patient who underwent esophagectomy was about fold greater in the drain fluid of thorax than in the peripheral blood. ( ) il- mrna was demonstrated in leukocytes from thoracic and abdominal exudate at , and hours after surgery. in contrast, il- mrna could not be detected in leukocytes from the peripheral blood. [conclusion] il- is mainly produced in the operative field and subsequently enter the peripheral blood to induce cytokinemia. the operation length, volume of blood loss and thoracotomy are factors influencing the concentration of cytokine in the blood. zaragoza spain age may be an important factor influencing the function of immunocompeteut cells releasing cytokines after both accidental and surgical trauma the aim of the present paper is to ascertain if patients (pts) over years old show a different serum level cytokine pattern than pts under after a standard surgical procedure considered as a "medium strength trauma". patients and methods: pts( females males)with gallstone disease were perspectively studied, pts were allotted in two groups: gr.a: pts under years(mean age: . +- )gr.b: pts over years(mean age: . _+ ). all pts underwent cholecystectomy and cholangiography. pts in gr.a and pts in gr. b underwent common duct exploration. spbintercctomy was performed in each group. on the day of surgery (pre) and on the st and th postoperative day(leo, po) : percentages of cd , cd , cd , cd and cd cells we measured by means of flow cytometry using moab. and levels of il- , il- , il- and tnf "in vivo" by elisa using moab. results: ere: cd % was . _+ in gr.a and . objectives of the study. after surgery for esophageal cancer multiple organ damage has been reported to be caused by polymorphonuclear leukocyte (pmn)-mediated injury. we measured serum granulocyte colony-stimulating factor (g-csf) and interleukin (il- ) levels to determine a role of g-csf and il- in pmn function after surgery for esophageal cancer. materials and methods. peripheral pmn counts, peripheral pmn chemiluminescence, serum g-csf levels, and serum il- levels were measured before and after surgery in patients with esophageal cancer (ec), and patients of gastric cancer (gc). esophagectomy with thoracotomy and laparotomy were performed for patients with ec, while subtotal gastrectomy with laparotomy were performed for patients with gc. results. peripheral pmn counts (p< . ) and peripheral pmn chemiluminescence (p< . ) of patients with ec were significantly decreased compared to those of patients with gc at and hours after surgery. serum g-csf levels of patients with ec were significantly (p< . ) increased compared to those of patients with gc at and hours after surgery. serum il- levels of patients with ec were significantly (p< . ) increased compared to those of patients with gc at , and hours after surgery. significant inverse correlations (p< . l) between peripheral pmn count and serum g-csf and il- levels were seen at hours after surgery. conclusion. these results suggest that many circulating pmns, which are excessively activated by g-csf and il- , may adhere to the endotherial cells and then migrate into the tissues, and cause multiple organ damage after surgery for esophageal cancer. immunnogical changes in patients with severe brain trauma receive increasing attention since morbidity and mortality ere still high. interleukin- (il- ) was previously detected in the cerebrospinal fluid (csf) during different pathologies of the nervous system ( , , ). in our study we monitored il- and nerve growth factor (ngf) production in the csf after human brain trauma. since astrocytes within the brain constitute one of the major cell type contributing to the inflammatory response through the release of cytokines and other factors after injury, we investigated the functional relationship of il- and ngf on a single cell niveau using cultured astrocytes. methods csf was obtained from patients with severe brain injury (glasgow coma score (gcs) < and ct abnormatities or gcs < over hours) after implantation of intraventricular icp monitoring device for therapeutic purpose and collected over hours csf and serum. il- and ngf were assayed by elisa. astrocytes were isolated from neonatal mouse brain as described ( ) . ngf production by cultured astrocytes was measured by elisa in the presence of csf, il- and il- antibody. astrocyte migration was tested in a chemstaxis chamber. results head trauma patients were included in this study (approved by the university hospital medical ethics board) and the csf was obtained through intraventricular catheters. high levels of il- were detected in the csf of these patients when compared to serum during the first days after brain trauma. furthermore ngf could be found inside the intracerebral compartment. csf containing high levels of il- could stimulate ngf production in cultured astrocytes. this effect could be [nhibited partially by il- antibodies, purified il- exposed to cultured astrocytes in vitro, stimulated the migratory activity of these cells in a dose response fashion. il- was found in the csf of brain injured patients, suggesting a role for this cytokine in the pathophysiology of brain injury. since astrocytes are involved in maintaining the homeostasis of the brain, we further investigated the possible role o il- on astrocyte functions, il- promoted ngf production in vivo and in vitro, thus contributing to neuronal cell survival and regeneration. furthermore il- stimulated astrocyte migration in a dose response fashion, potentially contributing to astrocytosis following brain injury and inflammation, these results show that il- represents a key cytokine in traumatic human brain injury with possible systemic effects, which are at preserlt under investigation. we studied a) the role of tnf and b) the therapeutic effect of a mab to tnf with regard to haemorrhagic shock (hs) related ,pathophysiologic alterations and mortality in rats. method: a prolonged hs was induced by bleeding to a blood pressure of - mmhg for pin followed by reinfusion of shed blood (sb) and resuscitation with two times of sb volume of ringer's lactate over rain. animals received a bolus dose ( mg/kg) of tnf mab (celltech, berkshire, uk) at min after resuscitation (tn ). the control group (n = ) was treated similar to the tn group but received ringer's lactate (con). results: at min the prolonged hs resulted in a metabolic acidosis indicated by a significant decrease of blood ph ( . + . ), hco -( . ___ . mm), and base excess (- . + . ram) values with pco ( . + . mmhg) and po ( . + . mmhg) in the tn with no difference to the con group. immediately after resuscitation ( min) plasma endotoxin levels were found to be increased in both groups ( . + . in tn vs . _ . pg/ml in con group) . prior to the treatment with tnf mab ( min) there was also no difference between plasma tnf levels of the two groups ( . + . in tn vs + . pg/ml in con group). treatment with the tnf mab at rain post-hs improved the hour survival rate to . % as compared to . % in the control group. macropathologic evaluations revealed frequency of intestinal bleeding in oniy animals in the tn vs in the con group. no bleeding in the kidneys was found in the tn but in rats in the con group. the significant increase in lung wet weight observed in non-survivors in the con (n = ) was prevented in animals which died in the tn (n = ) group (( . +_ . vs . +_ . g/kg). conclusion: our data suggest that tnf formation induced by hs in rats is an important mediator for pathophysiologic alterations leading to multi organ failure and lethality. antibodies to tnf might be a useful agent in the treatment of haemorrhagic shock related disorders. -+ n=ll*$ -+ n= _+ n= * * p< . vs baseline :~p< . no anesthesia vs anesthesia thus ) tnf production increased - fold by - hrs following trauma in unstimulated blood, but was reduced or not changed after lps stimulation, so circulating leukocytes are probably not an important source of tnf post trauma; ) anticd had no obvious effect on tnf production in unstimulated or lps stimulated blood, relative to vehicle, which suggests that the protective mechanism of anticd does not involve tnf suppression; ) fentanyl anesthesia at hrs following trauma unexpectedly decreased lps-evoked tnf production, which suggests that anesthesia alone can influence an inflammatory response. proinflamrnato~ cytokines have been shown to play a signific~t role in the pathogenesis of sepsis, which is a very common occurrence in born injury. tnfa is infrequently detected in the blood of burned patients, the ability to detect the shed receptors of stnfg has not been determined. serial serum mmples from burn patients were collected from the time of admission until death from septic shock. these samples were analyzed using an enzyme-linked immunosorbent assay (elisa) for stnfr, l-ira, tnf-a, and il-ib. the patients ranged in age from to yeas of age. the percentages of bum ranged from % - %. cytokine concenlrntions vmled from patient to padent irrespective of bum size. tnfa levels were consistentiy in the range of pgjml - pg/ml. peaks in the tnfa values were above pg/ml and were also associated with a peak in the stnfr levels. these levels began at < , pghnl within the in,st ins of injury and gradually increased with time. clinically. ti~ appearance of eytoklnes was independent of positive wound, blood, or respiratory cultures however peak values in tnfa and stnfr were ~ialed with a fluid requirnmenl levels of il-i ra were also elevated independent of clinical findings as well as extent of injury. in pl there is a significant corresponding peak in il-trn (> ~ /ml) at the same time as t/~:a and stnfr levels. we aimed to characterise the pattern of secretion of interleukin- beta l-ii ), intefleukin- (il- ) and tumour necrosis factor alpha (tnfa) in multiply injured patients and to relate these results to their clinical condition and outcome. two hourly blood samples were taken from ten patients from the time of injury until hours. cytokine levels were measured using sandwich enzyme-linked immunosorbent assays (elisas). injury severity scores (iss) were calculated and haemorrhage was assessed from the blood transfusion requirement over the hours. patients' ages ranged from to years. iss varied from to and transfusion requirement from to units. five patients died after the study period. ] ,- was raised in / patients (max level , pg/ml) but was unrelated to condition or outcome. / showed a rise in il- b (max level pg/ml) which was negatively correlated to iss (i=- . , p< . ). tnfa was raised in / (max level pg/ml). peak tnfc~ was positively correlated with iss ( = . , p< . ) and haemorrhage (i= . but p< . ). il-ib and tnfa production was mutually exclusive. there was no common cytokine profile for these patients. unlike elective surgery there was no correlation between peak ,- and severity of injury: tissue damage may not be the stimulus for the cytokine response to multiple injury. periods of ischemia or hypoxia produce endothelial damage in peripheral organs. tumor necrosis factor-alpha (tnf) plays a central role for regulation of endothelial physiology during septic events, taking influence on vascular permeability and coagulant activity [ ] . animal experiments demonstrated a synergism between hypoxia and septic shock on letality, leading to the hypothesis that low oxygen tension leads to enhanced sensitivity of target cells for tnf [ ] . radioligand binding studies with ~ odid-tnf on cultured human endothelial cells were performed after incubation in several environmental oxygen tensions (pc ) for hours. data were achieved by nonlinear regression of an idealized saturation curve according to the equation: b = n " k./( + k,); b = totally bound tnf; k,: association constant (concentration for half-maximal binding); n: number of binding sites per cell. p_o o (mm h¢i): _k, (nm}: n (molecules/cell): - . ± . _+ - . ± . + - , ± . -+ - . + . -+ presented are calculated values on the idealized curve + % percentiles. hypoxia induces enhanced binding of tnf to specific receptors on the endothelial cell surface in a time-and dose-dependent manner by a mechanism, which is not dependent on oxygen radicals, as shown by additional protocols with radical-scavenging drugs. with respect to former findings about a correlation between growth and tnf receptor affinity [ ] , these data lead to the hypothesis that enhanced tnf binding during hypoxia is due to a biochemical conversion of the receptor protein from the low affinity to the high affinity state, possibly by posttranslational phosphorylation of the binding protein by intracel)ular kinases. the proposed involvement of tnf-dependent pathways in pathogenesis of organ dysfunction and multiple organ failure after hypoxia/ischemia may provide a basis for understanding the initiation of hypoxic vascular injury, as manifested by increased permeability and prothrombotic tendency, and, thus, merits further attention. the levels of activity of circulating cytokines (ill, il- and tnf-alpha) which are believed to play important regulatory role in response to trauma are determined (by hioassays and respective anti-cytokine antibodies) in mice and rats subjected to scald injury ion c, see, ° v bsa, ld ) and ( c, see, ~ b ~^)~ , respectively. biphasic increase of cytokine activity was noted in mice: initial increase of il-i and il- , - hr following injury and of try activity hr after scald, followed by elevated levels of il-i and il- at hr, with tendency of decrease of activity at later time points. increased activity of tnf was noted hr following injury, in rats, initial, short-lived increase of il-i and tnf activity was detected lhr following injury, folowed by increase on days i and postburn. il- increase peaked - hr after scalding and levels remained elevated - days following injury. similar kinetics of appearance of proinflammatory cytokines (il-i and tnf-alpha) both in lethal and ncnlethal injury concomitant with differential profile of circulating il- activity (early,short-lived increase and later slow decrease of activity in lethal burn injury) with late persistent high levels of activity in nonlethai injury demonstrated in the present study highlight the need for investigation the relationship of these cytokines in burn-injury induced inflammation. zikica jovicic,lnstitute for medical research, mma,crnotravska , belgrade~yu. asadullah k ( ), woiciechowsky c ( ), liebenthai c ( ), doecke wd ( ), volk hd ( ), vogel s ( ), v. baehr r ( ); depts. of med. immunology ( ) and neurosurgery ( ) , medical school (char#d), humboldt university berlin, frg in patients after polytrauma or major abdominal surgery a hyperinflammatory phase seems to be followed by the development of a phase of monocyte inactivation. the latter is charaeterised by a decrease of monocytic hla-dr expression and a shift to anti-inflammatory cytokine production. as shown, by us and others, this phenomenon indicates severe immunodepression with a high risk of infection. however, the mechanisms leading to monocyte inactivation in the above mentioned syndromes may be multiple. to elucidate the influence of a selective, sterile trauma to the central nervous system (cns) on immune reactivity the neurosurgieal patient is an interesting model. initially, patients who developed a systemic inflammatory response syndrome following neurosurgery were analysed. in all of them a marked decrease of monocytic hla-dr expression was observed soon after the operation. these results suggest that neurosurgery alone can induce immunodepression and lead us to conduct a prospective study, in which we closely monitored l patients undergoing neurosurgery from the first preoperative day until at least day after the operation. hla-dr expression was decreased hi all patients to various extent only hours after surgery. in one patient only we found a persistently reduced hla-dr expression and this was the only patient to develop sepsis syndrome. this suggests that a prolonged, postoperatively decreased hla-dr expression is predictive of infection following cns trauma. in order to assess, whether a decrease of hla-dr expression was associated with a preceding inflammatory response, local cytokine release in the cns was compared with systemic cytokine release. for this purpose, paired samples of earebrospinal fluid (csf) from a vantricle drainage and peripheral blood plasma were obtained. in the csf extremely elevated futerleakin (il)- levels, peaking already a few hours after the operation were found. in plasma, by eontrast, il- ( and tnf-alpha) was detectable not until days later and only if infection was present. the antiinflammatory ili-ra, on the other hand, was also present in csf but peaked after il- and was detectable in peripheral plasma too. we believe there is an association between the inflammatory response in the cns and the following depression of hla-dr expression on peripheral blood monocytes. our results suggest that even a sterile cns-trauma by itself may contribute to general immunodepressinn leading to septic complications. the aim of this study was to evaluate the effect of haemorrhagic shock (hs) a) on total capacity of the host, and b) the circulating blood cells to produce tnf immediately after bleeding. in vivo studies: baboons were subjected to a limited oxygen deficit ( - ml/kg) hypotension phase (mean arterial pressure = map of - mmhg for - hours followed by adequate resuscitation). rats subjected to hs (map of - mmhg for rain followed by reinfusion of shed blood and fluid resuscitation) were challenged with endotoxin ( ~g/kg i.v.) at the end of shock (rhs group). the control group (rco) received the same dose of endotoxin as rhs group but without prior bleeding. in vitro studies: whole blood (wb) obtained from both baboons and rats before and at the end of hs were incubated with endotoxin ( ng/ml) for hrs at °c. results: at min post-lps challenge we found significantly higher plasma tnf levels in rats that were subjected to hs prior to the endotoxin challenge as compared to the control group ( _+ vs + pg/ml) . after hs the tpc was significantly decreased in in vitro stimulated cbc of both rats ( + post-hs vs + ng tnf/ml pre-hs) and baboons ( ± post-hs vs ± pg tnf/ml pre-hs). in contrast, the il- productive capacity was increased in baboons cbc (not yet analysed in rats) stimulated at the end of hs ( ± pre-vs ±_ pg il- /ml post-hs). conclusion: from our data we suggest that despite of down regulation of the cbc to produce tnf the overall tpc is enhanced at the early stage of i-is. with regard to the related literature (chaudry's group) it can be assumed that among the macrophage/monocyte populations, as the main source only the kupffer cells (kc) exhibit enhanced tnf production capacity following haemorrhage. the mechanisms of down/up regulation of cytokine response of cbc and/or kc following hs remain to be examined. d. eg~er, s. geuenich °, c. dertzlin~er °, e. schmitt*, r. mailhammer, h ehrenreich #, p. drrmer, and l. h mer gsf-instimt fox experimentelle h~znatologie, °medizinische kliulk iii, klinikum groghadern, munich, *institut for immunologic, johannes gutenberg universit/it, malnz, and #psychiatrische k/in& der georg-aagust-universi~t, grttingen, germany. it has been shown previously (ehranreich et al., , new biol. : ) that mouse bone marrow-derived mast cells (bmmc) synthesize and secrete endothelin- (et-i) and express eta-type endothelin receptors (eta). so far, however, no functions of et- /et a in bmmc have been described. in the present study we investigated the effect of exogeneously administered et- on the release of histamine, serotonin, and leukotriene c (ltc ) by primary mouse bmmc (in vitro age: weeks) caltured with different recombinant mttrine cytokines (interleukin (il- ) and/or kit ligand (kl) in the presence or absence of il ) for two weeks prior to activation. et- ( x - to lxl - m) induced an extremely rapid (_<lmin) and dose-dependent release of histamine and serotonin (up to % and % of total mediator content, respectively, comparable to ige/ag-indueed mediator release) from bmmc cultured with il- and il- . only when cultured in the additional presence of il- rather than in medium containing kl or kl plus il- alone, bmmc released histamine and serotoaln upon challenge with et- .furthermore, et- stimulated ltc biosynthesis up to -fold in bmmc cultured in the presence of il- . in contrast, the peptide was without major effect on ltc biosynthesis in bmmc culturd without il- . the release of histamine and sereotonin was not altered if bmmc were preincubated for i h with il- prior to activation with et-i, suggesting that a differentiation process rather than a functional priming effect had been initiated by ]l- . et- ( " m) -induced histamine and serotouln release from bmmc was inhibited by an eta-specific antagonist (cyclic id-asp-pro-d-val-leu-d-tel ) in a dose-dependent manner, with a complete inhibition observed at an antagonist concentration of - m. crude peritoneal cells (containing - % serosal mast cells) obtained from normal balb/c-mice released % histamine upon challenge with et- ( - m; rain). taken together, these resu/ts demonstrate that et- can directly function as a histamine and serotohin secretagogue and as a stimulator of ltc production in mast cells. il- appears to be involved in the differentiation of immature mast cell prec~trsors towards an et-l-reactive functional phenotype. when inflammatory reactions are advanced, type ii phospholipase a (type ii pla ) is produced in high levels by various cells at the site of inflammation. cytokines such as tnf-a and il-i activate type ii pla and promote the production of eicosanoid, which is one of the mechanisms by which they enhance the inflammatory reaction. in the present study, the blood levels of type ii pea , endotoxin, tnf-c~, il- and il- were determined in patients with sepsis to examine the relationship between these levels and the patients' pathological conditions. the levels of type ii pla and tnf-a in these patients were . ± . ng/ml and . ± . pg/ml, respectively, the two parameters being significantly correlated (r = . , p = . ). there were also a significant correlation between the level of type ii pla an il- (r = . , p = . ). there was no significant correlation between the level of type ii pla and il- . these in vivo findings suggest that tnf-a may be involved in the production of type ii pla . a. filzmaver, g. reisbach, e. schmitt °, r. mailhammer, and l. hiittner. gsf-iustitut ff~r experimentelle h~imatologie, munich, and °iustitut fttr immunelone , johannes gutenberg universit~t, mainz, germany the product of c-kit, a transmembrane tyrosin ldnase receptor, and a corresponding kit ligand (kl) are critically involved in the control of different hemopoietic cell lineages including the proliferation, maturation, migration, and function of mast cells. it has been reported previously that interleukin (il)- down-regulates kit receptors in human primary myeloid leukemic cells and in a human mast celt line (sinaber et al. , j. immunol. : ) . transforming growth factor (tgf) gl, was also found to down-modulate c-kit mrna and kit receptor proteins in human aml blasts, a mechanism probabely contributing to the anti-proliferative effect of tgfih on kl-stimulated aml ceils in vitro (de vos et at. , cancer res. : ) . in porcine endothelial cells transfected with a retroviral construct carrying the human (hu) e-kit gena, exogenous hu kl transiently down-regulated kit receptors (blume-jeusen et al. , embo j : ) . in the light of these previous findings we analyzed the effects of these exogenously applied cytokines (il- , tgfgl, kl) on kit receptor expression and kl-mediated proliferation and chemotactic migration of primary mouse bone marrow-derived mast cells (bmmc) (in vitro age: weeks). our results show that in bmmc cultures initiated with recombinant (r) marine (mu) il- the additional presence of rmull- for or days did not change the expression of kit protein (assessed by facs analysis with the anti-mouse c-kit antibody ack- ) nor kl-mediated proliferation or chemotaxis. in contrast, il- syeergistically increased kl-stimulated growth of bmmc in a short-term proliferation assay (mtt-tes . pre-incubation of bmmc with rhutgfl~ ( . , . , or . ng/ml) for h had no effect on kit receptor expression, although tgffil at concentrations of . - . ng/ml completely suppressed the proliferative action of kl on these ceils. similar anti-proliferative effects of tgfg were observed in various factor-dependent mast cell lines stimulated with different mast cell growth factors (il- , i,- , il- , and/or il- ). moreover, pre-incuhation ( h) of bmmc with tgf-gl (> pg/ml) significantly enhanced spontaneous undirected cell movement (chemokinesis) and synergistically increased il- -or kl-induced chemetaxis. when bmmc were preancuhated with rmukl ( ng/ml) for , . or days, a transient down-modulation of kit receptors with a maximum effect on day was demonstrated by facs analysis and correlated well with a decreased chemotactic response of these cells. in conclusion our results show that neither il- nor tgfi affect expression of kit receptors in primary murine bmmc. it is reasonable to suggest that c-kit expression is controlled in a cell type-specific manner.interestingly, tgfgl is obviously able to dissect the proliferative from the migrational signal transducted by kl in these cells. objectives of the study: antisense strategies using dna-otigonucleofides (odn) to modulate the cytokine response are presently under investigation. odn are thought to act very specifically with little or no relevant negative side effects. we now report that odn unspeeifically protect wehi cells from tnf-mediated cytolysis. material and methods: wehi subclone ceils ( x ), that are highly sensitive to the cytolytic activity of tnf, were grown on -well culture plates in rpm medium. after hours, phosphorothioate(ps)and partially ps-modified-odn as well as phesphodiester-odn ( - bp) were added ( . , and pm). four hours after incubation with odn, ce(i lysis was induced by recombinant murina tnf. after hours the plates were washed and stained with crystal violet cell lysis was determined by reading the absorbance (abs) at nm. results: wehi ceils incubated with tnf ( - ng/ml) were completely lysed after hours ( % abs). interestingly, wehi cells incubated with tnf and odn resisted complete lysis, eg cells incubated with . ng/ml tnf and jm odn showed still % of the absorbance observed in control ceils without tnf ( % abs). the protective effect of odn started at . pm, reached a maximum at ,um, and diminished at jm. with increasing amounts of tnf the protective effect of qdn decreased and no protection was detectable at ng tnf per ml conclusions: dna-oligonucleotides were found to unspecifically inhibit tnf-induced cytolysis. we hypothesize, that this protective effect of qdn results from an inhibition of the binding of tnf to its receptor, or from interference of odn with the subsequent signal transduction mechanisms. as a consequence, to discriminate the specific effect of odn in biologic systems, several control odn should be used. secondly, whether dna released by degradation of tumor cells or leukocytes can significantly impair tumor-and immune-defense mechanisms merits further investigation dr. med. michael meisner, institut for anaesthesiologie der universitat erlangen-nqmberg, krankenhausstral~e , d- erlangen. in this study we investigated the involvement of serine protease and free radical generation in the systemic release of tumor necrosis factor-alpha (tnf) and interieukin i(il- ), in the sepsis model of lipopolysaccharide (lps, mg/kg i.p.) induced hepatitis in galactosamine (gain, rag/mouse, i.p.) sensitized mice. treatment of gain-sensitized mice with lps (gain/lps) led to dramatic increase in serum cytokine (tnf and il-i) ievels and transaminase activity at hr and hr respectively. pretreatment of serine protease inhibitor, c~jantitrypsin (a j-at, mg/kg i.p.), rains prior to gain/lps treatment, fully protected the animals against the hepatotoxic challenge with significantly reduced serum tnf and il- levels. in order to block and scavenge superoxide generation, the mice were pretreated with xanthine oxidase inhibitor, allopurinol (al, x mg/kg i.p.) and pyran polymer-conjugated superoxide dismutase (sod, x unit/mouse i.v) r spectively. pretreatment with al and sod ( and hr prior to gain/lps) prevented gain/lps hepatitis and blocked lps induced released of tnf and il- into serum of the mice. the protective agents like cq-at or al/sod did not protect the mice against th~ hpp~totoxi£ ch~llpn-e indllee b'~ th~ recombinant mmlse tnf-o' ( . ~/rno~e j.p.) ~d oi~lps ~ caln-.~dlfa%aed mlce. it-l cett~aged la tnf (x/gain treated mjde was not detectable in animals pretreated with oq-at or al/sod. our study suggests that a serine protease sensitive to cq-antitrypsin is responsible in regulating tnf release, possibly by proteolytic cleavage of a tnf-precursor or membrane bound tnf. in addition our evidence suggest that the balance of extracellular protease/antiprotease activity may be regulated by free radical generation, possible superoxide anion, resulting in inactivation of the antiprotease. il- release may be subsequent to tnf release. objective: during sepsis one can observe a dramatically impaired production of proinflammatory cytokines like the tumor necrosis factor alpha (tnf-a), interleukin i-alpha (il-la), intedeukin i-beta (il-i&) and interferon gamma (if~) upon in vitro stimulation of circulating cells. however there is also evidence of a decreased ability to produce cytokines in other immuno-deficient states. in this study we compared the capacity to secrete proinflammatory cytokines upon in vitro stimulation of patients in severe sepsis and patients with malignant tumors. methods: heparinized blood samples of ten patients ( + years) in severe sepsis (sepsis score > according to e}ebute and stoner) were drawn at onset of disease, from fifteen patients with solid growing carcinoma ( + years) blood was drawn at diagnosis prior to any therapy. controls were obtained from fifteen healthy volunteers. pl of whole blood were incubated either with / of a standard medium or with pl of a standard medium and pl of phytohemagglutinin (pha) a potent mitogen. after an incubation period of hours plasma concentrations of tnf-a, il-la, il- and if-~ were determined by elisa. comments: our results suggest that down-regulation of cytokine secretion or of cell responsiveness to non-specific mitogens during sepsis has occurred. we observe a similar phenomenon for the group of carcinoma patients vs control significant for stimulated tnf-a and stimulated if-t. sustained immunological interactions between tumorcells and cytokine producing cells could effect responsiveness of the latter, a general increased immuno-tolerant state in patients with carcinoma has to be discussed. however we found significant differences between sepsis and cancer concerning the in vitro capacity of responsable cells to produce il-la and il-i#. the dramatically decrease of the ability to produce il-i upon in vitro stimulation could be more sensitive for a septic state than stimulated tnf-a or if- ,. objective: tumor necrosis factor alpha (tnf-a) has been implicated as a central mediator of sepsis and its sequelae. increased systemic levels of this cytoklne seem to be correlated with severity of sepsis and outcome. however mechanism of action and metabolism of tnf-g are not fully understood. in most studies blood samples for tnf-a determinations are obtained either by peripheral venipuncture, a central venous catheter or by an indwelling arterial catheter. very often blood samples are taken in different manners within the same study. in this study we measured circulating tnf-a and the amount of tnf-a released upon in vitro stimulation in arterial and central venous blood. methods: heparlnized arterial and central venous blood samples of ten patients ( males, females, mean age +_ ) with severe sepsis (sepsis score > , elebute and stoner} were drawn on day , , , , and of disease. blood was immediately placed on ice and processed within hour. pl of whole blood were incubated with pl rpmi-medium supplemented with antibiotics and l-glutamlne or with pl of rpmi-medium and pl phytohemagglutinin (pha) a potent mitogen. after an incubation period of hours samples were centrifuged and plasma was harvested and stored at - ° celsius before assessment of tnf-a concentration by elisa. statistical analysis was performed with the paired student-t-test. results: we found a significant difference (p < , ) for circulating mean arterial tnf-a concentration ( pg/ml _+ sem} and central venous tnf-a ( pg/ml +_ sem). upon in vitro stimulation there was also a significant difference (p < , ) between released arterial tnf-~' { pg/ml _+ sem) and venous tnf-a ( pg/ml +_ semi. conclusions: these results are difficult to interprete but could reflect the influence of pao and sao on tnf a release. it could also be the result of different concentrations of tnf-o release influencing factors like for example endotoxin, interferon-f or prostaglandin. a possible pulmonary and/or a hepatic metabolism of tnf-n and tnf-a producing cells cannot be ruled out. however for better interpretations of tnf-a release in septic states it is necessary to use either arterial or venous blood samples. early inflammatory processes following trauma and/or infections were found to be associated with the secretion of high amounts of proinflammatory cytokines. besides intedeukin-t (il- ), tumor necrosis factor-a (tnf-c and interleukin- (il- ) the multifunctional cytokine intedeukin- (il- ) was described to be a central regulatory element of the primary cellular and humeral defence reaction. the previously described close temporal correlation of pathologically elevated il- -concentrations and the extracellulary release of lysosomal enzymes from activated pelymorphnuclear neutrophils suggests, that il- may be a potential substrate of these preteases. the serine preteases elastase (ec . . . ) and cathepsin g (ec . . . ) derived from the azurophilic granules were assumed to be mainly involved in unspecific proteolysis at sites of inflammation by cleavage of structural as well as soluble proteins at random sites, if the inhibitory potential is decreased. the possible proteolytic activity of elastase and cathepsin g toward the proinflammatory cytokine interleukin- (il- ) was investigated. the addition of purified neutrephil elastase and cathepsin g to recombinant human il- leads to a rapid sequential degradation in vitro. at least two intermediate products could be detected by silver staining and western blotting following protein separation under reducing conditions. the serine protease inhibitor g-anitrypsin was shown to prevent the proteolytical degradation of intedeukin- . furthermore the loss of the biological activity of both, recombinant and natural human il- , was demonstrated by determination of the capacity of protease-treated il- to stimulate hybddoma growth ( td bioassay). these data suggest a possible downregulation of pathologically elevated il- levels by proteolytic activity of extracellulary released enzymes at sites of inflammation. the aim of the study was to compare circulating levels of three cytokines -il- , il- , _- -between critically ill subjects who developed gram-negative sepsis and who did not. materials and methods: the patient population consisted of patients admitted to an intensive cars unit, with different underlying diseases. sepsis diagnosis was given according to pre-estabilished cdteda. nineteen cases were enrolled in sepsis group, twenty in control group. serum sampling was collected in sterile tubes at study entry and every three days until study dismissal. serum concentrations of il- , _- and il- were measured using commercially available test kits, based on the dual immunometric sandwich principle. results: the causative patogens of sepsis were: pseudomonas aeruginosa, acinetobacter, eseherichia co~i, serratia marceseens, proteus mirobilis and citrobacter freundl the time of observation was equal to days, for a total of four tests performed (to, tl, t , t ). i .- was not detected in any samples. the serological profiles of the two cytokines .- and _- were similar; augmented levels were found at study entry and throughout the observation period, peaking at t and decreasing at t . however, in patients with sepsis, il- and _- concentrations were significantly higher in respect to control group. conclusion: our observations shown that in icu patients increased il- and il- release may be induced by cdtical illness; however, in subjects in which sepsis occurred, il- and il- production appears more significantly elevated, suggesting a role of il- and _- in the pathophysiology of sepsis. the fact that ii. objective: to check whether continuous veno-venous haemofiltration (cvvh) could remove the cytokines, namely tumour necrosis factor alpha (tnfc and interleukin (il- ) from the circulation of critically ill patients with sepsis ad multiple organ failure (mof). setting: the intensive therapy unit of the medical school teaching hospital. patients: nine critically ill patients with sepsis and mof treated with cvvh. methods: blood samples were collected before the cvvh had been started. then, blood and ultrafiltrate samples were collected simultaneously after hours and every hour. tnfct and il- levels were measured using the bioassays with cell lines wehi- ci and td , respectively. other data were recorded from the patient notes and intensive therapy unit charts. results: no measurable concentrations of tnfct were detected in either blood or ultrafiltrate samples. il- was found in all the patients' plasma samples and five patients' ( . %) ultrafiltrate samples. the il- blood level ranged from . to . u/ml (mean . , sd . ). the il- level in positive ultrafiltrate samples ranged from . to . u/ml (mean . , sd . ). conclusions: our preliminary results suggest that il- is present in bloodstream of septic patients. we assume we could not detect tnfa in any sample because we usually started observations when septic state had developed. cvvh could extract cytokines from the circulating blood. it remains under discussion, whether that extraction may be beneficial to patients with mof. the pattern of some significant cytokines tnf, il- and il- and their pharmacomodulation were evaluated in an experimental model of polimicrobial sepsis induced in cd- mice by cecal ligation and puncture (clp) in order to understand their roles. this model of sepsis, which resembles the clinical situation of bowel perforation, was also compared with that induced by administration of pure endotoxin (lps). tnf was detectable in serum and tissues during the first h with a peak h after clp at a significantly lower level than after lps. il- was measurable in serum only after h, significantly increased in spleen and liver after and h and in mesenteric lymphonodes from to h after clp compared with shammice. il- was significantly increased in serum throughout the first h after clp. pretreatment with dexamethasone (dex), ibuprofen (ibu) and nitro-l-arginine (n-arg) significantly reduced the survival time while chlorpromazine (cpz) and tnf did not affect it. only the antibiotics and pentoxifylline (ptx) significantly increased the survival in clp. however cpz and dex protected from lps-mor~ality. in conclusion, by inhibiting tnf with dex, cpz, ptx a reduced, unchanged and increased survival time was observed and by increasing tnf with ibu and tnf administration the survival was decreased or unchanged respectively suggesting that the modulation of this cytokine does not seem to play a significant role in clp unlike lps_ moreover the negative effects of ibu and n-arg suggest an important and protective role by prostaglandins and no in clp. to gain more insigths on the contribution of tnf~, il-i~ and if to lps toxicity, we explored the time-course of the cytokine production in ealb/c mice given different doses, from the lethal (= ld ) to the sublethal (= / ld ) of three different lps (e.coli oiii:b and :b ; p.aeruginosa r ) endowed with different degree of toxicity cytokines were measured in serum and organs with specific elisas up to i h after lps administration. results demonstrate that i) circulating and organ levels of tnf~ do not reflect lps toxicity. in fact, the lethal dose of lps :b induced as much tnf~ as the sublethal dose of lps :b ; furthermore, lps r , whose cytokine inducing capability is far lower than that of lps from e.coli, induced higher tnf~ levels at the sublethal than at the lethal dose. in addition, policlonal anti tnf ab, that were able to protect mice from e.coli lps induced mortality, failed in mice treated with lps r ) circulating il-i~ levels are generally low and increase significantly only in muribond animals. on the contrary, in spleen and lung very high levels of il-i~ are persistent from i to h post lps administration moreover, the treatment with mgr of neutralizing policlonal anti il-i~ ab, did not modify survival in lps challenged mice. ) circulating and organ levels of if are proportional to the dose and degree of toxicity of all the administered lps even if lps r was again a less efficient cytokine inducer than lps from e.coli. csa is an immunos~ppressive drug, able to inhibit gene expression for many cytokines, including if . to study the effect of cytokines modulation on lps toxicity, csa was administered to mice twice at the oral dose of i mg/kg before the challenge with lps. mice were monitored in terms of mortality and tnf~, il-i~ and if production. together with the total ablation of if , the strong reduction of tnfu and unmodified il-i~ levels, a significant increase of lps toxicity was also observed. these results suggest the hypothesis that the numerous factors that jointly mediate lps toxic effects, can also be protective, the final outcome depending on their relative ratio rather than on the absolute amount interleukin- (il- ) mediates the septic shock syndrome and affects intestinal secretion in vitro. we studied the intestinal production of il-t and its effects on diarrhea during endotoxic shock. cd- mice were randomized to mg/kg e.coli :b lps or saline infusion (i.p. or i.v.). diarrhea invariably occurred following lps infusion. mice were sacrificed at , ', lh, . h, h, h, h, and h ( mice/group/time-point). the small bowel was compressed and the intestinal contents were weighed and expressed per g sb weight. the small (sb) and large bowels (lb) were eventually frozen, weighed, and homogenized for either cytosolic protein or total rna. il-i~ (cell-associated agonist) was measured with a radioimmunoassay specific for mouse il-l~ (detection limit pg/ml) and expressed as ng/g weight + sem (lowest detectable amount ng/gwt). northern analysis of total rna and in sfu hybridization of paraformaldehyde-fixed frozen tissue were done with [ ~- p]-iabeled mouse il-lc~ cdna probes. only sb had il-i~ constitutively present ( . + . ng/gwt). lps i.p. or i.v. induced elevation of il-lc¢ in both organs in a biphasic pattern; lps i.v. induced -fold more il-i~ than lps i.p. following lps i.p., il-i~ in sb was . + . ng/gwt at lh, reached maximal levels at . h ( . -+ . ng/gw-i) and returned to baseline at h. saline controls maintained their constitutive il-i~ levels. sb had fold more il- ¢ than lb and identical kinetics, but lb showed a clearer doseresponse. northern analysis of sb-total rna showed induction of il-i~ mrna by lps in correlation with il-lc¢ kinetics. il-i~ mrna producing cells were mononuclear cells in the lamina propda and epithelial cells at the bottom of the crypts of ueberkuhn. mucus and fluid were increased in the small bowel post-lps in correlation with intestinal il-lc~ kinetics (r = . ). separate mice were pretreated with saline i.p. orthe il- receptor antagonist (irap, mg/kg bolus i.p.) and were challenged rain later with . mg/kg lps i.p. or saline i.p. specific blockade of il- by irap decreased intestinal secretion at h and h post-lps challenge (p<_. . , student's-t-test). these data indicate that local (intrinsic) intestinal il-i~ mediates sepsis-induced intestinal changes. inflammatory cytokines initiate the host response to endotoxemia, causing severe physiological and hemodynamic changes which may lead to septic shock. among the regulatory systems that play an important rote in controlling host inflammatory responses is the pituitary. it has been known for many years for example, that hypophysectomized animals are extremely sensitive to lps lethality. while investigating the possibility that protective, pituitary mediators might explain this phenomenon, we identified the cytoldne mif to be a specific secretory product produced by pituitary cells in vitro and in vivo after lps challenge. analysis of serum mif levels in control, t-cell deficient (nude), and hypophysectomized mice revealed that pituitary-derived mif contributes significantly to the rise in serum mif that occurs after lps administration. of note, pituitary mif content ( . % of total pituitary protein) and peak serum mif levels ( - ng/ml) were determined to be within the range observed for other pituitary hormones that are released after pituitary stimulation. to investigate a possible beneficial role for mif in septic shock, we co-injected mice with purified, recombinant murine mif (rmif) together with lps ( mg/kg). surprisingly, rmif markedly potentiated lps lethality compared to control mice that were injected with lps alone ( % vs. %, p = . ). to confirm these results, mice were treated with anti-rmif antibody prior to injection of a high dose of lps ( . mg/kg). anti-rmif antibody fully protected mice against lps lethality, increasing survival from % to % (p = . ). serum levels of tnf,~, the first cytokinc that appears in the circulation after lps challenge, were reduced by . _+ . % in anti-rmif-treated mice. we conclude that pituitary derived mif contributes significantly to circulating mif in the post-acute response in endotoxemia and may act in concert with other pituitary mediators to regulate both pro-and antiinflammatory effects. moreover, mif may play a critical regulatory role in the systemic host response in septic shock. our results suggest that anti-rmif antibody might be of potential therapeutic use in the treatment of septic shock. although anti-interleukin- (il- ) antibodies and il- receptor antagonist have been shown to improve survival in animal models of endotoxemia and abrogate the lethal effects of tnf, the presence of il- in the serum does not correlate well with outcome. we hypothesized that this may be because il- acts mainly in a paracrine fashion and is metabolized before it diffuses into the circulation. methods: we measured the il-i~ mrna expression with the differential reverse transcription polymerase chain reaction (rt-pcr) using g-actin as internal standard in the peritoneal macrophages and lung tissue in normal controls and mice after cecal ligation and puncture (clp). clp resembles human intra-abdominal sepsis in that it is characterized by very slight elevations of serum il- levels. results: il-lg mrna levels after clp are expressed as % of normal (mean+sem, n= in several experimental models of infection exacerbation of disease was observed, when infected animals were depleted of tuajor necrosis factor (tnf). after sublethal cecal ligation and puncture (clp) leading to peritonitis and sepsis the survival of mice also critically depends on tnf as demonstrated in earlier studies, when clp-treated mice injected with anti-tnf antibody died, whereas mice injected with a control antibody survived after clp (echtenacher et al. , j. inununol. : ) . from a panel of different cell types (macrophages, neutrophils, t lymphocytes, natural killer cells, mast cells) able to produce tnf upon activation~ the mast cell is apparantly the only one capable of storing in cytoplasmic granules preformed tnf-ct which is rapidly released following challenge. in the present study-we analyzed serum tnf after lps injections as well as the outcome of clp in severely mast cell deficient mutant mice (wav v) as compared to syngeaeic wild-type littermates (+/+). we proposed that concentrations and/or kinetics of serum tnf should be different between wavv mutants and wild-type mice, if mast cell-derived tnf significantly contributes to the rise in serum tnf levels following systemic stimulation with endotoxin. although similar levels of increased tnf were detected in the sera of both genotypes after and hours of lps injection ( btg/ . ml / mouse i. p.), mast ceil-deficient mice indeed showed decreased serum tnf levels iron after injection amounting to only to % of the concentrations observed in the corresponding sera of normal wildtype mice. in the clp model of septic peritonitis we found that mast celldeficient mutant mice were dramatically more sensitive to clp than syngeneic normal mice resulting in % mortality in w/w v versus % mortality in +/+ mice . days after initiation of clp. further experiments with w/w v mutants selectively reconstituted with cultured bone marrow-derived mast cells from normal syngeneic wild-type mice and the use of an antibody specifically blocking the action of tnf tn vivo should clarify a potential protective function of mast cells in this model of septic peritonitis. interleukin- (il- ) inhibits cytokine production, including tumor necrosis factor (tnf), by lipopolysaccharide (lps)-aetivated maerophages. we recently observed that lps injection (e.coli :b , gg ip) into balb/c mice induces the rapid release of circulating il- ( ± u/ml at min). blocking endogenous il- using monocional antibody (jes - a , mg, h before lps) resulted in a massive increase in tnf production ( ± in lps+anti-il- treated mice vs ± ng/ml in lps alone, p< . , n= to mice per group) and an enhanced lps-induccd lethality ( % vs % in anti-il- +lps or lps alone respectively, p= . , n= mice per group). irrelevant igg rat monoclonal antibody (lo-dnp) did not influence neither tnf production nor lethality associated with endotoxin shock. this led us to study the production of il- during human septicemia. plasma samples were obtained from patients with gramnegative (gns, n= ) or gram-positive septicemia (gps, n= ) and from healthy volunteers. among these patients, suffered from septic shock at the time of sampling. il- levels were measured by elisa (detection limit: i pghrd). we found that patients ( %) had increased il- plasma levels (range to pg/nd). patients with gps had il- levels similar to the ones observed in gns (median: vs . pg/m, respectively). patients with septic shock had higher il- values (median: pg/ml) than septicemic patients without shock ( pg/ml, p= . ). no il- was detected in plasma from healthy volunteers. we conclude that il- is produced daring human septicemia. our experimental data suggest that il- might be involved in the control of the inflammatory response induced by bacterial products. dr arnand marchant, immunology department, hopital erasme, route de lennik, brussels, belgium. to provide information about the role of tnf in sepsis and mods we measured tnf and stnfr-i levels in septic patients and investigated if there is a relation between plasma concentration of these molecules and the severity of sepsis evaluated by two scores (apache i and sss). patients and melhods: septic patients fullfilling sepsis criteria of american college of chest physician and society of critical care medicine were studied. tnf-cc and stnfr-i ( kda) were measured by enzyme immuneassays (norms values = + pg/ml and . _+ a ng/ml respectively). results: the mean tnf and stnfr-i values for each patient (mean+sd) were + pg/ml and . + . ng/ml respectively. these values are approximately seven and ten times greater than those observed in normal healthy volunteers (p< . ). mean tnf concentrations for each patient were significantly greater in non survivors ( + vs _+ pg/ml p< . ); stnfr-i levels also were greater in this group, but the difference was not statistically significant ( . + . vs . _+ . ng/ml). plasma tnf and stnfr-i concentrations were significantly correlated (r = . p< . ). mean tnf levels were significantly correlated with apache ii (r = . p< . ) and sss (r = . p<o. ); stnfr-i levels were likewise correlated with both scores (r = . p< . and r = . p< . respectively). tnf and stnfqgi levels increase with the number of dysfuctional organs; in particular tnf and stnfr-i increase when hemodynamics deteriorate and kidney failure ensues. conclusions: the correlations between tnf and stnfr-i levels and sepsis severity scores suggests that tnf is involved in sepsis and may influence the occurrence of mods and finally clinical outcome. tnf and stnfr-i are in fact especially increased when mods occurs. in particular their concentrations are elevated when cardiovascular system and kidney failure ensue, suggesting that tnf can have a direct role in hemodynamic derangements caused by sepsis and that kidneys are involved in tnf and stnfr-i excretion. lif is a pleiotropic cytokine that has been implicated in the pathogenesis of sepsis in animal models. we conducted a prospective study in septic patients to correlate lif plasma levels with patient's clinical and biological data (among them il- ). plasma was drawn daily from day one to .ten and lif presence was determined with a specific elisa and with the dai,a bioassay (sensitivity of pg/ml and ngml respectively). risk factor associated wftt~ with elevated lif plasma level was determined by an univariate analysis. a multivariate stepwise logistic regression analysis was subsequently performed with a bmdp statistical software. lif was detected with the elisa and with the bioassay in and out of the patients, respectively. lif peak plasma levels were statistically associated with high creatmin levels, temperature and il- . multivariate regression analysis showed that high serum creatinin level was the major independent risk factor associated with elevated lifplasma levels. this correlation was also found for il- . peak plasma levels of both lif and il- correlated inversely with patients survival duration. cox's analysis for plasma levels of lif > pg/ml yelded a hazard ratio of [exp ( . )= . ]. our study indicates that lif levels were associated with clinical and biological parameters of illness severity and significantly increased (cut-off value pg/mi) in patients with fatal outcome. current consensus exists about the central role of tumor necrosis factor (tnf) alpha in initiating the systemic inflammatory response syndrome (sirs). a correlation with sirs has inconsistently been found. tnf effects its pleiotropic reactions upon two distinct cellular receptors. soluble extracel]ular fragments of the human kda tnf receptor (stnfri) and the kda receptor (stnfrii) are detectable in the circulation. the kinetics of these endogenously produced tnf-inhibitors were measured to evaluate their role in patients with sirs. fourteen patients of an operative icu were included with the diagnossis of sirs (mean apache ii score: points). serial blood samples were obtained within h after diagnosis of sirs, every hrs for the first hrs and every hrs thereafter until patients died or recovered. soluble tnfri and stnfrii were assayed by an enzymed-linked immunological binding assay. soluble tnfri and ii could be detected in all samples with a significantly higher level (p<o,o ) compared to healthy controls (normal value: tnfrh , ng/ml; tnfrii: , ng/ml). the serum concentration of tnfri ( , ng/ml) as well as tnfrii ( , ng/ml) were significantly higher in nonsurvivors (n= ) compared to survivors ( , and , ng/ml; n= ) at the onset of sirs and during the course of the inflammation response (p< , ). a positive correlation exists between both receptors (r- , ; p < , ). increasing levels and maximal values of stnfri ( ng/ml) and i ( ng/mi) were detected only in patients who died. the serum concentrations of patients who recovered did not change and are remaining at the initial level. tnf alpha bioactivity was detected in out of patients. no significant correlation exists between the concentration of both receptors and tnf alpha as well as the apache ii score at the onset of sirs. elevated serum concentration of stnfri and are found in patients with sirs. these naturally occuring antagonists reflect the inflammatory process. the measurement of soluble tnf receptor levels may be useful in monitorina sirs and in the prediction of outcome. - is given to patients with advanced melanoma or renal carcinoma. however, this therapy is accompanied by severe side effects, including the vascular leak syndrome (vls) that closely resembles septic shock. to investigate the involvement of cytokines and phospholipase a z in the pathogenesis of the vls we collected serial plasma samples from patients during the first hours after administration of il- . in these patients we monitored temperature, blood pressure and heart rate and assessed levels of the cytokines tnf, il- and il- using immuno assays. in these plasma samples we also measured phospholipase a activity using an enzyme activity assay, since this enzyme is beleived to play a major role in the generation of lipid mediators. we demonstrate that during il- therapy the cytokines tnf, il- , and il- are produced and that the release of these cytokines is followed by an induction of phospholipase az activity in the plasma of these patients, we further discuss the role of these mediators in the development of the vascular leak syndrome observed in patients receiving il- . objectives of the study: sepsis caused by candida albicans (ca) is no longer a clinical rarity but a common consequence of immunosuppression and surgery, and is associated with high mortality. tumor necrosis faetor-c¢ (tnf-<~), interlcukin-lg (il-lg) and interleukin- (il- ) are thought to play an important role in the pathogenesis of the sepsis syndrome. we therefore studied the in vitro production of tnf-<x, il-ib and il- by human peripheral blood mononuclear cells (pbmc) stimulated by ca compared to stimulation by bacterial lipopolysaccharide (lps). materials and methods: pbmc were isolated from venous blood of healthy volunteers and cultured at a concentration of . • ~ eells/ml in culture medium with a dose-range of either heat-killed ca or lps. supernatants were harvested after hours. using elisa, tnf-c~, il-ib and il- concentrations were measured. ca was isolated from a patient with ca sepsis and cultured under sterile conditions. results: ca and lps stimulate the production of tnf-~t, il- ] and il- by human pbmc. we observed a dose-dependent synthesis of these three cytokines in human pbmc. tnf-c¢, il-ib and il- in ng/ml as mean _+ standard error. ca/ml . . ( ) we constructed a single-chain fv-fragment (sc-fv) from the variable domains of a neutralizing antibody to human tumornecrosisfactor-alpha (tnf) because sc-fv should have some advantages in vivo. the sc-fv has a similar affinity ( , x - m) as the fab-fragment ( , x - m) but bind the antigen with a eightfold lower avidity compared to the parental mab ( , x - ). the parental mab as well as its fragments can compete the binding of rhutnf to both tnf-receptors. this was shown by competitive binding to the cell line hl- and to immobilized rtnf-receptors. in the nutralization assay on the cell line, l , the sc-fv can neutralize tnf but in comparison to the mab or its fabfragment, the capacity was three-fold lower as expected from the molar ratio of the dissociation constants. the in rive-neutralizing capacity was tested in a mice receiving toxic dose of rhutnf. the sc-fv showed a -fold lower neutralizing capacity in comparison with the fab-fragment. this could be explained in different manners: i) partial instability of scfv at c, ii) shorter half-life because sc-fv but not fab-fragments are eliminated via kidney, iii) elimination of tnf-immunocomplexes may be improved by opsonization mechanisms which requires some structures of the constant region (as in fab or mab). in summary, despite the encouraging in vitro experiments, the in vivo data suggest that sc-fv are not the right strategy for neutralizing tnf in vivo. tnf production in endotoxin non-responder mice, effect of a glucocorticoid antagonist g. iazgtr jr, e. duda, j. ol~th, e. husztik, g. iazar glucocorticoids inhibit lipopolysaccharide (lps)-induced tnf production and tnf can stimulate pituitary adrenocorticotropin secretion, resulting in the release of corticosterone. earlier studies ( ) reveales that the glucocorticoid antagonist ru , nufepristone, sensitizes both endotoxin responder and nonresponder, c h/hej, mice to endotoxin lethality. we have also shown that the glucocorticoid antagonist ru sensitizes endotoxin-tolerant (endotoxin-pretreated) and normal mice to the lethal effect of septic and endotoxin shock. on this basis, we set out to study the influence of ru on tnf production induced by endotoxin in endotoxin responder and non-responder mice. one and hrs following lps injection, ru significantly increased the tnf concentration in the serum, liver and spleen of treated animals as compared with the control and methylprednisolonetreated groups. in tissue cultures, ru induced the tnf synthesis of myeloid cells and increased the tnf production of genetically modified hela cells, which synthesize tnf constitutively. normal and tumor cell cultures exhibited an increased sensitivity toward tnf in the presence of ru . however, the same dose of lps did not induce tnf production in the serum, liver and spleen of endotoxin non-responder mice and this was not influenced by concomitant ru treatment. these studies suggest that ru aggravates lps lethality via factors other than tnf in endotoxin non-responder, c h/hej mice. the cytokine response to a novel exnacellular mitogenic factor, mf, produced by the group a streptococci (gas), and to the streptococcal pyrogenic exotoxins (spe) a and b, was determined by in vitro stimulation of peripheral blood mononuclear cells (pbmc) obtained from healthy blood donors. the induction and kinetics of different cytokines was studied at single-cell level using cytokine specific monoclonal antibodies and intracellular immunofluorescent staining. all three mitogens induced a massive ifny and tnfi response in - % of the pbmc after - hours of cell stimulation. in contrast, il and tnfc~ containing cells was only detected in - % of the pbmc. the induction ofil , il , il , and ill , was weak or completely absent. thus, the response indicates activation of th cells. however, illc~, illl , illra and il , were consistently found and gradually produced, peaking at hems in approximately - % of the pbmc. mf induced an equally extensive cytokine as well as proliferative inducing capacity, as did spea and speb, which suggests that also mf is a superantigen. a marked inter-individual variation could be noted between lymphocytes isolated from different individuals, both in the toxin induced proliferation and cytokine induction. this may be one explanation for the varying clinical severity noted in patients after infection with clonal gas strains. introduction -tumour necrosis factor (tnf) is released by mononuclear cells in response to inflammation and sepsis. it has been implicated as a possible mediator in inflammatory bowel disease (ibd) but its pathogenic role remains uncertain. this study assessed the relationship between tnfct and disease activity by measuring plasma and faecal tnf concelnmtions in an experimcntai model of ibd. methodologo' -colitis was induced in male wistar rats by intmcolonic instillation of rag , , -trinitrobenzenesulphunic acid in . ml % ethanol (n= ). control animals received an isovohlmetric instillation of normal saline (n= ). faecal pellets were collected before induction of colitis (day ) and throughout the experiment. after days the colon was excised, ueighed and the colon macroscopic score (cms) assessed plasma tnf (ptnf) samples were assayed by bioassay and elisa. faecal samples were vortcxed in water and the supernatant removed for estimation of protein and faecal tnf (itnf) content (elisa). there is a significant increase in itnf on day -#p= alld the total ftnf measured during the study correlatcs with the cms oll day -p= . there is no correlation between ftnf and ptnf. conclusions -therc is evidence of coloific tnf excretion by macroscopically normal animals and following induction of colitis there is increased faecal tnf which correlates with disease actmty. there is no significant elevation in bioactive or imnmnoreactive plasma tnf in colitie animals this data would suggest that faecal tnf is a marker of colonic inflamrnatiort and serial tnf assessment inay be useful as an indicator &severity in ibd. to study the effect of in-vivo hypoxia/reoxygenation on cytokine elaboration by routine peritoneal macrophages. materials and methods: adult male cba mice ( - g) were primed intraperitoneauy with either lipopolysaccharide (lps) ug]anlmal or saline. five days later, the animals were subjected to hypoxia ( % ) for , followed by of normoxia. harvested peritoneal macrophages were plated in-vitro, culture supernatants were collected at , , and and assayed for tumor necrosis factor (tnf), prostaglandin e (pge ) and nitric oxide (no). control animals underwent the same procedures, but were maintained in normoxia ( %o disease , berlin, germany the precise mechanisms of reactivation of latent human cytomegalovirus (cmv) infection are still unknown. apart from the permissive effect of diminished cellular immunity there is evidence of a cytokine mediated cmv reactivation as it is known for other systemic virus infections (e.g. hiv). we found that tumor necrosis factor-alpha (tnf) -in contrast to all other cytokines tested -can stimulate the activity of the cmv-ie-enhancer/promoter region in the human monocytic cell line, hl . we wondered whether our in vitro results were actually reflected by the incidence of cmv reactivation in diseases which go together with high tnfalpha levels. cellular immunodeficiency as well as at least temporarily increased tnf levels are known to occur in septic disease. we tested for the presence of cmv infection in peripheral blood mononuclear cells (pbmnc) by means of h centrifugation culture, immunocytological detection of different cmv antigens (apaap-technique), and pcr technique (cmv-ie dna). in striking contrast to healthy controls almost all examined septic patients were positive, irrespective of the method for cmv-ddtection applied, including the less sensitive immunocytology. follow-up studies showed that cmv-antigen positive pbmnc ceased to be detectable in patients with good outcome once septicaemia had been overcome. to further back up our hypothesis, we looked for a coincidence of enhanced tnf-alpha serum levels and cmv antigenaemia (immunocytology) in some other diseases which are known to have either enhanced tnf serum levels or increased incidence of active cmv infection and found the majority of patients with b-cell chronic lymphocytic leukemia, liver cirrhosis, and common variable immunodeficiency to be positive for these two parameters. we now wondered whether, apart from cmv reactivation, tnf-alpha could also cause cellular immunodeficiency this way promoting cmv replication and spreading and eventually causing cmv disease. in summary, we were able to show that a diminished cellular immune response results from/n vitro or in vivo methylprednisolone (mps) is used to suppress both inflammatory and immune responses. il- receptor antagonist (il-lra) and tnf binding protein (tnfbp) are naturally occuring anti-cytokine proteins, possibly modulating inflammatory and immunological responses. to define mps modulation of cytokine and anticytokine responses, we examined effects of mps on il-lra production and tnfbp generation as well as il-i~ and tnfa production by human peripheral blood mononuclear cells (pbmc) stimulated with lipopolysaccharide (lps). pbmc were purified from the blood of healthy volunteers, then incubated with lps ( ng/ml) supplemented with different concentrations of mps ( , pg/ml, mg/ml). after hrs incubation, the supernatants were collected for rias of secreted cytokines. the cell lysates obtained by cycles of freeze-thaw, were also collected for r ias of cell-associated cytokines. both the supernatant for secreted cytokines and the cell lysates for the cell-associated cytokines were subjected to rias for il-i~, tnfa, il- ra, and tnfbp-i (tnfsrp ). il-la, il- ra, and tnfbp were produced mainly as cell-associated forms in response to lps, whereas most tnfa was secreted into the supernatant of lps-stimulated pbmc. mps supplement resulted in reduction of il-la, il-lra, and tnfa production. il-la production was reduced up to . + . %( mg/ml) by mps in a dose dependent fashion, whereas about % decrease in tnfc, and il-lra production was observed at both high and low dose mps supplement. however, total tnfbp generation was not reduced significantly by mps supplement. furthermore, tnfbp secretion was increased times more in the supernatant of lps-stimulated pbmc with gg/ml of mps. these data suggest that mps could effectively block tnf activity by both reducing its production and up-regulating tnfbp generation and that mps works as an anti-inflammatory drug as well as an immunosuppressant by modulating cytokine and anticytokine responses. in severe gram-negative sepsis, excess of proinflammatory cytokines is associated with increased morbidity and the mortality. we have found that immunocytes possess functional -adrenergic receptors (~-ar) which, when activated, down regulate the lipopolysaccharide (lps) induced gene expression and subsequent secretion of tnf-~ in vitro. in the present study, we used a lethal endotoxic model in mice to test the hypothesis that -ar stimulation will down regulate the gene expression thus decreasing the circulatory levels of proinftammatory cytokines and improve the survivals in severe endotoxicosis. the animals (iso group) were injected times with ! -ar agohist isoproterenol ( . mg/kg i.m. and . mg/kg s.c.) , and hours prior to a lethal dose of lps injection ( mg/kg i.p.). control group (ctl) received same volume of saline at the same times before the lps insult. at the appropriate time points after lps injection animals were sacrificed and venous blood was collected from the inferior vena cava. the plasma levels of tnf-cc and il-loc were determined by elisa. in the mortality study, each grou the data showed that isoproterenol significantly decreased the mortality and the circulatory levels of tnf-c~ and il-lcc (fig. ) . these data suggest that -ar activation of immunocytes down regulates cytokine gene expression, decreasing circulatory eytokine levels thus reducing endotoxicosis mortality. these results provide a new pharmacological approach to reduce the excess of proinflammatory cytokines and mortality in sepsis. introduction: cytokines released from monocytic cells are thought to play an important role in the pathophysiology of sepsis. during the last few years several investigations have been performed aimed at modulating this mediator response. in the present investigation using a full blood model we have studied the effect of a standard immune globulin and an antitipopolysaccharide (lps) immune globulin preparation and a monoclonal anti-lps antibody in modulating the endotoxin induced cytokine response. methods: citrated blood from healthy human donors was incubated at °c in rotating polystyrene tubes. samples were taken before addition of x ng/l e. col± endotoxin and after hours. plasma was assayed for the cytokines minor necrosis factor alpha (tnf-~) and interleukin- (il- ) using elisa methods. the eodotoxin concentration was assayed by lal and end-point chromogenic substrate technique. the blood was preincubated with standard immune globuline (human igg from pooled plasma, gammagardr, baxter) or with an anti-lps immune glubuline (human anti-lps igg obtained by screening of blood donors, novo nordisk) in the concentrations of mg/ml or mg/ml or with the monoclonal lipid a igm antibody ha- a for ten minutes before the endotoxin was added. results: two hours after endotoxin addition markedly elevated tnf-~ and il- values were found.in the plasma. cytokine values at hours: tnf-ec pg/ml (mean) and l- pg/ml (mean). preincubalion with mg/ml standard immune globulins reduced il- values by % (mean) while there were no effects on tnf-c~ values at hours. a slight reduction in il- values was also found with the lower concentration, mg/ml, but this was not statistically significant. preincubation with anti-lps immune globulins had no effect on il- nor on tnf-cc values. preincubation with the monoclottal lipid a igm antibody ha- a in variable concentrations had no effect on cytokine release in this model. in this full blood in vitro model standard immune globuline reduced endotoxin induced l- release while tnf-cc values were unchanged.the anti-lps immune glubuline preparation and the monoclonal lipid a igm antibody ha- a had no effect on the il- or tnf-ct release. the effect of anti-tnf treatment in severe haemorrhagic/traumatic shock was investigated in a conscious rabbit model. the jugular vein (for administration of substances and blood withdrawal) and right femoral artery (for measurement of bp) were cannulated and an aortic thermistor probe inserted into the left; femoral artery for the measurement of cardiac output (co). the following day, upto % of the estimated blood volume was withdrawn over - rain, to reduce bp to - mmttg and co by / mad withheld for rain. shed blood ( %) was then reinfused followed by lactate solution to give a total potential dreulat%ag fluid volume of %. zymoeen activated plasma ( , gg/ml) was given rain prior to haemorrhage and during blood r~n%eion, all ~,~ir, ak were killed at h and o~gane removed for histology. rabbita received either monoclonal antirabbit tnf (r , rat igg , mgkg i.e. n= ) or emine (nffi ), rain prior to haemorrhage. the cardiovascular and biochemical changes during the haemorrhage and hypotensive phase (is. haemorrhagic stimulus) were similar in both groups. upon reinfusion, haematccrit and white cell count remained sigutfia~utly (p< . ) higher in saline compared to r -treated animals, indicating a relative haemoconcentration (i,e. reduced circulating volume) in saline animals. the lettkocytosis persisted in the saline animals upto h. plasma glucose, lactate and asparteto .m~-o transferase all rose similarly in both gorups but plasma ¢rea~!~+-e levels were markedly (p< . ) higher in ealine ve r animals at and h indicative of kidney damage, using a macro and micro. pathological scoring system, major organ damage was seen in the lung, liver and kidney which was significantly (p< . ) attenuated in r treated animals. in the hmg and liver, this was primarily due to a reduced bleeding and pmn infiltrate and to an additional maintenance of tubular integri~:y in the kidney. hence, in tlais model anti-tnf treatment markedly reduced the biochemical and histological indices of severe ,, ~gan damage during liaemorrhage and ranerfn~ion. thermal injuries and systemic bacterial sepsis produce a wasting diathesis with anorexia and marked loss of lean tissue and body fat. endogenously produced cytokines, including interleukin- (il- ), are thought to mediate many beneficial as well as pathologic host changes that occur during burn injury and infection. to evaluate whether bluckade of il- in vivo would affect survival and attenuate the anorexia and cachex[a associated with a thermal injury and chronic fulminant gram negative infection, sprague-dawley rats were studied. anesthetized rats were divided in groups, implanted with alzet r minipumps and randomized to receive the following treatment for days:i: gg/kgbw/min of il-lcc; ii: ~tg/kgbw/min of il- receptor antagonist (il-ira); iii: buffered vehicle. the animals were subjected to a % bsa, full thickness scald burn and the wounds then infected with + cfu/ml pseudomonas aeruginosa. eight additional rats were anesthetized, but were not burned, and served as healthy controls. . il-hx reduced food intake transiently but was otherwise without effect however, il-lra significantly attenuated weight loss over the first days and improved food intake between days and . we conclude that blockade of il- after a thermal injury with superimposed infection does not adversly affect the progression of the disease, but does attenuate the anorexia and weight loss associated with this model. these data indicate that il-i receptor blockade may offer a means to minimize the morbidity associated with catabolic illness. tumor necrosis factor (tnf) and its downstream induced mediators have pivotal roles in the pathogenesis of sepsis and septic shock. the suppressive effect of pentoxyfillin (ptx) on tnf production may be of clinical importance. when peripheral white blood ceils were stimulated with either lps or staphylococcus aureus, pentoxyfillin inhibited tnf production. in contrast, no inhibitory effect was observed on the induction of il- . ptx inhibited not only the tnf production of monocytes but also the tnf secretion of both granulocytes, and unseparated whole blood. the facs analysis revealed that the expression of cd antigen on monocytes was not influenced by pentoxyfillin. the in vitro tnf and il- producing capacity in septic patients were higher than in the control group, but decreased on subsequent days especially in fatal cases. administration of ptx ( mg/day) in septic patients resulted in tnf and il- productions similar to those found in control donors. it also subsequently led to an improvement of the clinical status. a further improvement in apache score could be achieved by administration of pentaglobin ° (biotest)( ml/kg/day ). the prevention of lpsinduced in vitro tnf and il- production by pentaglobin ° could be demonstrated in in vitro experiments involving the use of whole blood rather than purified lymphocytes, very probably because of the presence of lps binding protein in the plasma. the level of soluble icam- in sera of septic patients was significantly higher ( - ng/ml) than in normal individuals ( - ng/ml), but it decreased following the ptx and pentaglobin ° therapy. it is presumed that ptx and pentaglobin ° can antagonize eytokine production at different levels, resulting synergistic action being beneficial in the treatment of sepsis. albert szent-gy rgyi medical university, institute of microbiology, szeged, hungary h- ddm t. . e~i~im~/tii, septic sh ~ '. tnf alfa/pge, and somatostatln lands ji., alvarez j., gram m., llanos k., alcalde j., sanchez ja., balibr~d jl. taurine, a semi-essential sulphur-containing t:~-amian acid, promotes neutrophil phagocytic activit ' against yeasts and enhances intracellular killing of e.coli. paradoxically it protects against hypochlorous acidmediated biomembmne oxidatio~ and abrogates the pyrogenic effects of intracerebral endotoxin. it is therefore possible that taurine mediates some of these effects by modulating the cytokine cascade. aim to assess the cytekine responses in a rat model of intraperitoneal sepsis, pretreated with supplenmntary taurine, by measuring tnf and il- concentrations. methodology female wistar rats (wt - g) (n- per group) were prefed with % tanrine, . % glyciuc ( in tap water) or tap water (tw) for days prior to caecal ligation aud puncture (clp). normothermia was maintained intraoperatively and the animals received . % saline subcutaneously ( mls/ g) post-procedure. animals were venosected by direct cardiac puncture at or hours post-operation and the blood placed in endotoxin-free tubes. centrifilgation and storage of plasma at - °c was completed within hours. tnf and il- were measured using wehi and b bioassays respectively. results (means ± sem) bioactivc tnf concentrations were not significantly different between clp or sham groups. however plasma il- estimation revealed a statistically significant reduction at hours in tanrine-treated animals compared to glycino and tw controls ( objective: to evaluate the effects of allogeneic blood transfusion, thermal injury and bacterial garage on interteukin (il- ), tumor necrosis factor alpha (tnf) production and host mortality and to study if the administration of thymopentth (thy) could affect these events. materials and methods: balb/c mice were transfused with allogeneic blood (from c h/hej mice) and treated daily with thy (i mg/kg)(t+thy) . control animals were transfused but not treated (t). after days systemic blood was harvested to determine the circulating levels of il and tnf. a second group was treated with thy for days and then received a gavage wide lxl escherichia coli and a % burn injury (bg+thy). one hour post-bum, blood was collected to measure cytokins. control animals received the same treatment but thy (bg). a third group was transfused, treated with thy for days and then received gavage and burn (tbg+thy). one hour post-burn, blood was collected to measure cytokins. control animals received the same treatment but thy (tbg). all treated and control groups had mice/group. in separate groups (n= /group), receiving the same treatments, mortality was observed for days post-burn or transfusion. the production and release of oxygen radicals by phagocytic leukomytee is one of the most relevant and important functions of these substances in biology. when neutrophilic granulocytes or certain macrophages phagocytize they produce super oxide and hydrogen peroxide and form secondary products of these activated species. all of these substances are released in to the phagosome. it has been appreciated since that the amount of oxygen consumed by phagocytes in producing free radicals is prodigious. what has not been totally appreciated is the significant contribution this consumption contributes to total body oxygen consumption. our understanding of increased oxygen consumption following sepsis is compounded by a paradox of increased cardiac index and a narrow av difference. it was mclean in a study reported in that showed an increase in cardiac index and a narrowing of the avo z difference in humans following sepsis. other studies documented similar changes in humans and it has recently been speculated that increasing o~ delivery might have a favorable impact on outcome. at least three different explanations have been put forward as to why there are possible alterations in energy metabolism during sepsis. these include decreases in oxygen supply, peripheral shunts and direct action o~ cellular mediators on o~ delivery, a provocative review by hodgkiss and nark in shewed that in experimental animals sepsis did not cause any evidence of bioenergatic failure in muscle i liver, heart or brain tissue. they further showed that the increase in serum lactate is mot necessarily due to cellular hypoxia. they conclude that cellular hypoxia and defects in energy producing metabolites are not the cause of metabolic abnormalities in sepsis. we set out on a series of experiments to try to explain the paradox in cellular metabolism and whether or not white cells might contribute to total body oxygen consumption during sepsis. the first set of experiments involved growing rat cardiac myocytes on tissue culture. pyruvate was added to the medium as substrata and physiologic levels of peroxide were also added simultaneously, carbon labeled co~ production was measured as was nucleotides and degradation products from the cell. the peroxide induced a significant inhibition of pyruvate dehydrogenase. in addition, there was a loss of cellular atp and a corresponding rise in the release of inosine and adenine from the cell. we concluded from this first sst of experiments that physiologic levels of peroxide are a potent inhibitor of pyruvate dehydrogenase in isolated cardiac mitochendria as well as the cultured myocyte. we further showed that pyruvate dehydrogenase inhibition blocks the aerobic oxidation of glucose and leads te adenine nucleotide metabolism with adenosine and inosine release from the cell. other enzymes within the tca cycle did not appear to be specifically blocked by peroxide. this would explain the increase in lactate and the ability of the cell to continue to make high energy phosphate by entry of metabolites in to other entry points of the tca cycle, the second set of experimbnts was designed to estimate the magnitude of whole body reactive oxygen generation via nadph oxidase following granuloeyte activation invivo. using a guinea pig model baseline oxygen consumption was determined. ~iaphenyl iodinium (dpi) was used as a specific inhibitor of granulocyte nadph oxidase. animals were divided into two groups; those that received dpi and those that served as control, all animals were then injected with phorbcl myristate acetate (pma) intravenously. pma is a potent stimulus for granulocyte activation and subsequent reactive oxygen generation. whole body oxygen consumption measurements were then repeated after the p~la injection. in addition, arterial blood gas amalysis was performed, circulating neutrophils were collected and assayed for their ability to generate hydrogen peroxide and the r~ght lung was removed for wet and dry weight measurement. total body oxygen consumption increased by percent after pma injection. arterial oxygen tension fell significantly in the control animal. neutrophil hydrogen peroxide generation rate doubled and lung water in the untreated animals increased by percent. in this animal model we could show that granulocyte activation substantially increases whole body oxygen consumption to approximately percent of control values. the nadph oxidase inhibitor, dpi, decreases granulocyte hydrogen peroxide generation invivo and prevents the pulmonary injury induced by pma. using this animal modal it is possible to transpose this to the human septic state. if we assume there are , neutrophils per cubic millimeter of blood, a kg man would have ~ . xi u circulating neutrophils. when activated ixl ~ neutrophils generate one nanemole of hydrogen peroxide per minute. t~erefore, the circulating neutrophil pool could generate - . xi nanomoles of hydrogen peroxide per minute. this corresponds to an oxygen consumption of - . ml/ojmin by the circulating neutrophil pool alone. this does not take into account the production of oxygen by macrophages or the amount of oxygen consumed to produce nitric oxide. manipulation of oxygen delivery in human sepsis may or may not be beneficial. irshad h. chaudry, ping wang, and alfred ayala life threatening blood loss, due to soft tissue and bony injuries, is a frequent complication in trauma victims. although numerous organs and cells are adversely affected by hemorrhage, the focus here will be to discuss whether or not there are any differential alterations in splenic and hepatic immune functions. in particular, m~ antigen presentation (ap) and associated processes will be described since one of the important functions of the m is to activate resting t-cell lymphocytes by processing and presenting foreign antigens in a mhc class ii-restricted fashion. studies have shown that splenic m and kupffer cell (kc) ap was significantly depressed following hemorrhagic shock and remained so for at least h alter resuscitation. the presentation of antigenic fragments associated with mhc class ii antigen itself was not decreased following hemorrhage, but the catabolism of antigens in antigen-presenting cells was decreased. this is likely due to a significant loss of cellular atp. although m ap was depressed in splenic, as well as kc, only kc showed an enhanced capacity to produce inflammatory cytokines, il- , il- , and tnf. this difference in response between kc and splenic md may be due to differences in the microenvironment in which these cell populations are found, as well as potential differences in the effects that hemorrhage has on the environment from which these cells are obtained. splenic m from hemorrhage-resuscitated mice exhibited a significantly reduced cytotoxic response, whereas kc showed an enhanced cytotuxic capacity. the increased kc cytotoxicity is probably mediated through the increased expression of cell-associated tnf and the release of reactive nitrogen. the enhanced production of reactive nitrogen may play an important role in the clearance of microbes translocated from the gut following hemorrhage. however, excessive release of reactive nitrogen by kc may have deleterious effects on the adjacent hepatncytes. such an effect could, in part, explain the reduction in active hepatocellular function, which is seen following hemorrhage-resuscitation. thus, hemorrhage induces differential effects on splenic and kc m populations; it decreases splenic m but increases kc capacity to mount a cytotoxic response. the depressed splenic md and kc ap was, however, significantly improved by posttreatment of animals with atp-mgcia, chloroquine, diltiazem or interferon-'/. these agents also decreased the susceptibility to sepsis following hemorrhage. thus, the use of atp-mgci~, dfltiazem, chloroquine or interferon- offers new therapeutic modalities in the treatment of immunosuppression and for decreasing the susceptibility to sepsis, which is observed following severe blood loss. the production of macrophages from bone marrow precursor cells is a dynamic and highly regulated process. the differentiation of myeloid lineage-restricted progenitor cells is initially controlled by interleukin- , which drives the progenitors along a maturation pathway that leads to their response to specific lineagerestricted colony-stimulating factors(csfs) we have previously hypothesized that thermal injury can initiate a self perpetuating cycle of production of defective immune cells: thermal injury can initially affect circulating immune cells. then these cells, by unbalanced production of colony stimulating factors and other mediators, can cause a derangement of hematopoiesis resulting in an abnormal production profile of tnf, il-i, and il- and cytotoxic activity of bone marrow derived cells. the results of our studies so far have supported parts of this hypothesis. il- and il- +m-csf treatments increased the production of tnf in burn day macrophages and progenitor cells compared to normal cells. il- +m-csf+il- increased the production of il- by burn progenitor cells compared to normal macrophagee. preliminary results for the ratios of tnf/~ actin mrna indicate that il- +m-csf increased the mrna for tnf in the -day macrophages derived from burned animals compared to macrophages derived from normal animals. ratios of il- /~ actin mrna indicated that il- , il- +m-csf, and m-csf increased the il- ~rna in progenitor cells from burn animals compared to progenitor cells from normal animals. these preliminary results support our hypothesis that bone marrow derived macrophages and progenitor cells from burned animals act differently compared to cells from unburned animals regarding the production of inflanunatory cytokines. more specifically, the results suggest that the different cell types are regulated in different ways by cytokines, and cells from burned animals are regulated differently than cells from unburned animals. it i,~ to ~ssume that the translneatinn is due to a ixx~h,v functioning ~astroiutestlnal surface pro|ection system, which leads to an unacceptably high load ¢ln the imrsunc dcfcncc system, partlcul~ly the local system in the gastrointestinal wall, leading tu exh~ustiou of ihi~ system. tlae g&~lrointesliual i]'ac( can be regal'deal a.s a t~ servojf of appr m ~ separating i~ eucayotic ceils of the hosl ttom l ~ bacterial culls. in the human body the the myriad of cndngan¢nts micrix)rganistns, exogenous itl,~ttdittg micro~ganisms altd taxies m-'e sepia"areal li'otll lhe rest of the body by a simple epithclhd layer. the barrier function is heavily tlcpcnding tm =~ proleclive layer cmtsisting ie. surl~:emts, nluclts, probiotic bacteria, and ,~ub~tances with strong antioxid~lt, antipfotease al~d other i~rotective eft'ecru, the surfaet~ml htycr consists at tile besl if i(.) bimolecular layers, fl is thus very iltil~, the epilhelial layer is renewed every hours , and tile tanuwer of the mucus is ~so fa,~t, all attdphy of die epithelial layei induced by g&sttointestinal st~'vation does also include the mu~usprndocing cells, gnhlet cells, which leaduhg lu slmnage aad luwer quality of gi mucus, altered viscoelas,jc properties and reduced protection, ]~'obioiic bactcrla keeps the ppm's low, produce nutrients for the intestinal mllcosa, eliminates tuxia~ and stimulates the local immune system, the gl surface is conslanlly under hc wv wearing by ingested bacteria and their enzymes, ingested substaucas, chefftie,'ds at~d toxins. 'll~e n~tective flora is reduced in disease, by stress and by cm'clcss antibiotic therapy. wc have made attempts tn recondition the i,,.astroiniesli!l~d. tnucosa by rcstlpply of surfactants or sttrfactanfliko glyc~lipids, gels with pseudomuein [until.oil, and probiotl¢ laetobaeilli. by supply of glycolipids we have bt:cn able m prevent and heal intlammatory and tlleerallve injuries (and io p)'cveut microbe transhteatkln) in uppe~' and lower (. u'acl, this c~al be fu~'ther augmentented by shnultaueos supply el' a p.~uedomucin,likc gel like pectin. ()at contain~ one hundred times more ot membrane lipid$ th~a =nay other food, much of fiber, ~spceially watcrsnlnhlc betaglucaal~, at~d has an close to ideal amino acid cornpo~ition, <')at, termented by species-specific lactobacilli, has a strol~ ability to prevent local inflammatory and ulcerative chunges,transh)cadnn and sepsis in experimeut~d attimals mid to pl~ven~ revert mof in ba,nan.~.lt seems us, that it the mucos~lnucus/probiotic bacterial filnclinns cat'= %' restated by mucosa reconditioning, even in severely sick iudividuals) the hx:~d iuuuuue ~y,~teme will be capable tu pr~)tec! the ix~y ag~lls~ sepsis ~nd mof. the impaired immune response associated with multiple system organ failure (msof) has been most widely studied following surgery for inflammatory disease and trauma. the majority of late deaths following trauma are due to infection and msof. not all patients with multiple organ failure have concomitant infection present, yet most have been septic at some time in their hospital course. their generalized inflammatory condition often persists whether or not organisms have been recovered. immune failure probably precedes msof with or without overt infection and indeed may perpetuate such widespread sepsis. macrophage tumor necrosis factor-alpha (tnf) production is thought to represent an important pathogenic mechanism by which this is mediated. cecal ligation and puncture (clp) is a clinically relevant murine model of intra-abdominal infection and sepsis. serum tnf and endotoxin levels, and peritoneal macrophage tnf production are only slightly elevated in this model even in endotoxin sensitive animals. mortality in this model, therefore, does not appear to be attributed to overwhelming endotoxemia and/or tnf production. it has therefore been postulated that tnf and other cytokines may act in a paracrine fashion to cause local injury. tnf messenger rna (mrna) expression was therefore measured and found to be increased in the lung and liver following clp. a different pattern of expression was seen after endotoxin administration, characterized by a more rapid rise and fall, and enhanced tnf mrna expression in the spleen as well. route of spread of infection as well as the type of stimulus may determine the organ specific cytokine response. these data support the concept of locally produced tnf as a contributing factor in tissue damage and multiple organ failure during sepsis. the estimation of histamine's role in sirs changed over the years based on increased knowledge in shock pathogenesis, evidence later found to be faulty, and the discovery of other, more fashionable mediators. over the decades, the prevailing view has been that histamine is a noxious mediator contributing to the fatal outcome of shock. the analysis of experimental data on exogenously administered, endogenously released and/or newly formed histamine in septic/endotoxic shock suggests that histamine contributes to the early cardiovascular changes via hi-receptor vasoconstriction thus excerbating (directly and indirectly) the effects of catecholamines. in the later phase of septic shock (low out-put, high resistance states), however, it can be assumed that histamine exerts strong vasodilator and positive inotropic effects via h -receptors. these effects are considered beneficial in counteracting or attenuating the effects of sympathetic responses of catecholamines and other mediators. thus, a blockade of the early reaction with hi-antagonists and a stimulation of the h -receptors by h -agonists are worthwhile therapeutic approaches. shock produc~s ~ij alterations, an encrgy crisis and eventual c~ll damage, however, shock in itse|f do~ not lead freqoenfly to r~mote organ damage. in animal ~xpcrhnents and clinical expariez:ces in korea and vietnam, shock i~r s¢ was not associated with lung damage, renal failure or ards. the same is true with g.l bleeding. however, when shock is due to trauma or associated with tlssus injmy, then organ malfunction m~d damage is frequent. shock is bclleved to increase the frequency of infection, but clinical evidcnc~ for this is scant. hemonhsgio shock akme is an unusual o~orrenee dinica[ly. immune dysfolletion dons occur witl~ experhnental hypovolemic shock and in pailcnts with hypotcnsion or after receiving blood ~ansfosions. tbc most significant factor affecting reoorrcnce of resented ca,lorectal liver mclastasis was hypotensivc episodes during operation. blood transfosions depress immune function, improve transplant ~raft survival and increase ttll'rmr rec~r'tcnos ill co[eli and head and nc~k cancers. in experimental hemorrhagic shock, we found decreased response of [ymphocytes to mitogens, el:clad mixed lymphocyte reactilln and i[,- decreased. othe~.x hsvc found decreased macrophage and t and b ¢¢lt function, deci~ased re,ease of/l~i, increased poe v depressed macrophagc antigen presanlatiota, increased ien y, decreased il- , and , and shared mscrophags funclion with loss nr f and c b r~ccpto~s. this is inhibited by chlornqoine, ibuprofen, dihiazem, and atp. shock may activate ncutrophils contributing to ilappitlg in cspillarics, no refluw, increased adhesion, cytotoxicity and organ damage. this may be related to decreased clearance of bacteria after shock. t~lls shock, hypotcnsion and decreased microcirculatory blued flow initiate or set the stage for immune dysfmtction. they, along with tissue injury trigger inflammatory cascades. this also contributes to immunologic dysfonctitm, which is associated with increased lufoction. thus altered bh,od flow and mediator cascades are bolh involved, the question now is can wc rapidly improve microcirculatory blood flow, and dg'masa inflsrmnatory fosponss, which is also necessary for survival the effe~ of ~nterferon ~amma @n wour~ healing was ewaluated ~n a routine mo~el. ~mma ~n~erferon we g~ven in doses of ~ . to ~ , u~%$ synchronous with ereatio~ of a pa~splnal woumd then daily for is ~lays. ~mteet~on ~amma impaired tensile strength a% all ~oses relative to control. m zphology suggested ~reas~ ¢oll~gen deposition and r~du~-~ neovag~ulaeit¥ ~n t~eat~ animals, interferon ~amma was a so ~valuated in a murine mo~el of cecal llgatlon and punneure. one hundred to ,s uni~ vere ~iv.n following ~nju~ an~ than daily. interferon qamma was associated with inco:ea~ed mortalit~ and earlier death a~/a~n ~n a dose dependant manner (p< , ). afmlnistra~ion the proinfiammatory cytokines tumor necrosis factor-c~ (tnf-c , interleukin- (il-ii ), interleukin- (il- ), and interleukin- (il- ) have been implicated in the pathogenesis of the multiple organ dysfunction syndrome (mods) following shock and trauma. these mediators which are released in high concentrations by activated macrophages, endothelial cells, and connective tissue cells cause a systemic inflammatory response syndrome (sirs), thus leading to a shock-like state and tissue destruction. recently, a number of proteins have been characterized in in vitro and in vivo studies demonstrating potent anti-inflammatory properties. these latter cytokines include interleukin- (il- ), interleukin- (il= ), transforming growth factor- (tgf-j ), and the newly sequenced interleukin- (il-i ). they are considered antiinflammatory, since in vitro studies using purified macrophage cultures or whole blood assays revealed an inhibitory effect of these antmnfiammatory cytokines on ~he synthesis and release of tnf-cl and il-u . in addition, they reduced the severity of disease and reduce plasma levels of tnf-c~ and il-ii when administered to animals with infection or inflammation. furthermore, naturally occurring substances have been described that specifically neutralize the bioactivity of il- b and tnf-a. the il- receptor antagonist (il-i ra) is related structurally to il-i and binds to the same cell-surface receptors, but does not stimulate the cell. in animal models ore. cob-induced shock, inflammatory bowel disease, and peritonitis, injection of il-lra significantly reduced the severity of disease. the cytotoxicity of tnf-c~ can be inhibited through two types of soluble tnf receptors (stnfr) type i (p ) and type ii (p ). these stnfrs are proteolytic cleavage products of the cell-bound tnfreceptors. when given to baboons to combat e. coil-induced shock, soluble receptm's to the tnf type [ receptor decreased the severi~ of the shock-like state. it has been well accepted that shock and severe injury result in an increased release of proinflammatory cytokines with a high incidence of sirs and mods. our studies investigating plasma levels of anti-inflammatory mediators (il- , il-lra, stnfrs) in patients after severe injury further suggest that shock and injury lead to an activation of anti-inflammatory processes with altered plasma levels of these mediators. however, while plasma levels of il- and l-lra were significantly increased in trauma patients compared to healthy volunteers, stnfrs did not differ from control samples. thus, shock and severe injury result in different activation patterns of anti-inflammatory processes. m,l. rodrick, boston, b~usa following severe thermal injury significant suppression of the immune response has been demonstrsfed. these changes are associated with increased susceptibility not only to co,on human pathogens, but to organisms not normally pathogenic in the uncompromlsed host. early observations included prolonged graft rejection and skin test anergy in patients following burn injury. work from our laboratory and that of numerous others has shown that the~e is a profound £mmunoeuppresslon following severe thermal injury. i~mune deficiency has been identified in these patients hyz ii lack cf t nell responses to mitogens, ) early decreases in total t and helper t cells in the peripheral bloo~ } lack of response to tetanus toxoid i~uniza~io~ in spite of increased non-speoifi~ i~k~unoglobulin praduction and ) severe and prolongei deficiency of interleukin- (il- ) prcductlon by peripheral blood mononuclear cells (pbmo). this il- deficiency could be an underlyin~ cause of all the afo~ementloned immune defects by failing to activate both helper and suppressor t cell functions. mo~a recent work has focused on the molecular mechanisms of this il- deficiency end shown that the defect lies post-transcrlptionally since mrna for il- is also depressed following thermal injury. we have also investigated the potential of a defect in ii- receptor status on peripheral blood mononuclear uolls from patients fqllowing burn injury and in a murine model. in both oases no decrease in il- r was found either by phenotypic markers or by funational assays. another hallmark of thermal injury is hyperao~ivatlon of proinflammatory ~ytok~ne secretion, which may play a significant role in multiple organ failure following burn injury. therapy o~ major burn injury may reasonably be aimed, therefore, at up-regulating t cell ~unction and down-regulating hyperactive secretion of proieflaam~ntory cytokines. the majority of patients dying of bums succumb to sepsis which coincides with dysfunction in the specific and non-specific host defences. generally, concepts relating to the mechanism(s) of specific immune failure in thermal trauma imply that inhibition of t (cd +) cell responses is imposed by the injury or infection-related factors. however, the same factors elicit strong biological reactions within the lymphoid compartment. recent evidence indicates that systemic activation is inherent in the burn patient. to establish the relation of molecular and cellular events to the development of post-bum anergy we examined the state of and the capacity for t cell activation with regard to: ) occurrence of activation-induced cell death (aicd), ) activation-related phenotypic and functional_ diversity in the pool of peripheral cd + t cells. initially, (up to days post-bum), peripheral blood lympliocytes from severely injured (> % total body surface area) patients exhibited high levels of constitutive expression of surface receptor for ]l (cd ) and spontaneous blastogenesis. the presence of activation-related t cellproducts in bum plasma was also apparent. subsequent impairment of the t cell receptor (tcr)-regulated t cell responses in vitro was accompanied by significantly increased dna fragmentation that is associated with cell death by the mode of apoptosis. using molecular markers we established that flesh peripheral blood ceils from immunosuppressed patients also contain large numbers of apoptotic cells. fluctuations in the number of viable (pi-) peripheral blood lymphocytes involved primarily cd +/cd ro+ (memory) subset of t ceils. the above observations suggest that thermal trauma-associated t cell anergy develops through aicd, a phenomenon commonly associated with the tolerogenic activity of bacterial superantigens. persistence of staphylococcal infections in the burn patient may support this assumption. response following trauma jane shelby, ph.d. the immune system is integrated with other physiologic systems, and is exquisitely sensitive to changes in nervous and endocrine systems changes following traumatic stress challenge. the immune, nervous and endocrine systems interact via both direct and indirect pathways which utilize neuro and endocrine hormones, neurotransmitters, neurepeptides and immune cell products. it is now known that the immune system may be affected by all of the neuroendocrine products produced during a stress response, with evidence for innervation of iymphoid organs, lymphoid cell receptors for neuroendocdne products, and leukocyte production of chemicals which are virtually identical to certain neuroendocdne peptides (acth, endorphins). trauma induced alterations in the equilibrium of various neuropeptides and neuroendocdne hormones have a significant impact on immune response potential, affecting control of proliferation, differentiation and function of immune cells. for example, the neurohormone melatonin is thought to be a natural antagonist to counteract glucocorticeid associated immunosuppression resulting from stressful challenges, such as surgery and trauma, plasma melatonin levels are known to be significantly reduced in burn patients. the administration of exogenous me[atonin improved cellular immune response following burn injury in an animal model. melatonin was also shown to have in vivo cytokine regulatory activity, increasing the potential for il- secretion and downregulating excessive il- and ifn~ in burn injured, stress susceptible mice. the regulatory interactions between the immune, nervous and endocrine systems provide mechanistic pathways for trauma associated immune dysfunction. increased knowledge of these interactions will enhance the potential for the design of novei clinical interventions to improve immune response and decrease the risk for infection in trauma and surgical patients. . animals receiving e were given a single dose daily of either . g/kg of e in a % solution by garage (ge), or . g/kg of sterile ive in saline. four hours following the last dose, bum animals were subjected to a % body surface area bum injury to their dorsum. twentyfour hours following injury, the animals were sacrificed and spleen cells were harvested for assessment of lymphocyte function. splenocytes were prepared by mincing the spleen, followed by incubation on glass petri dishes to remove adherent macrophages. non-adherent cells were then tested for proliferative response to t-cell mitogen concanavalin a (con a) and b-cell mitogen lipopolysaccharide (lps). data were analyzed by anova. results: chronic alcohol exposure and burn injury independently inhibit lymphocyte response to con a but not to lps. the combination of e plus bum injury, however, pmfouedly decreases this response to both con a and lps as outlined in the this data clearly identifies the synergistic impairment of immune function produced by ethanol and bum injury. it is furthermore apparent that ibis effect is gut mediated and that gastrointestinal exposure to alcohol is necessary to produce this effect. further studies will work to identify cellular and subcellular mechanisms to explain this effect. in experimental animal studies and investigations on human volunteers endotoxin infusion is mgulary accompanied by the release of the cytokine tumor necrosis factor a (tnf-~) determined by elisa technique. in patients with menigococcal sepsis also elevated tnf-a values have been found using a functional assay. we have studied the role of tnf-et in surgical icu patients with sepsis. using functional technique, we were not able to detect tnf-~ activities in the patient plasmas. when this cytokine, however, was determined by immunochemicai technique (el sa) elevated tnf-e~ values where frequently oberserved. in order to further elucidate these observations, we studied shedding of tnf receptors in the patients. in these studies, we noticed that shedding of tnf receptors oecured regulary in the patients. at the time of diagnosis, soluble tnf receptor p and p were both - fold higher than values found in plasma samples obtained prior to die diagnosis of sepsis. we also observed that the sepsis patients revealed higher maximum values of p and p during the icu stay compared to values found in surgical icu patients without sepsis. these observations indicate that soluble tnf receptors are available in sufficient amounts to bind tnf-ot which is released in surgical patients developing sepsis. this mechanism may explain why functional tnf-c~ was not detected in the patients. institute for surgical research, rikshospitalet, the national hospital, university of oslo, oslo, norway. decker, d., sch ndorf, m., bidlingrnaier, f., hirner, a., yon rfcker, a. the advantage oflaparoscopic cholecystectomy over conventional open surgical approaches in the treatment of symptomatic cholelithiasis has been shown convincingly by clinical studies. in order to facilitate comparisons of different surgical approaches, we evaluated the cell biological characteristics of tissue trauma by measuring changes in various cell surface markers on leukocytes and eytokines in plasma as a possible means to assess tissue trauma in choleeystectomy. patients recruited into our study had experienced at least one typical bifiary colic, had ultrasound-proven cholelithiasis (stages -ii according to me sherry), were - years old, and presented for elective choleeysteetomy. patients could choose between laparoscopic and conventional eholeeystectomy after being informed about the advantages and disadvantages of each procedure. cell surface markers on leukoeytes were determined using whole blood techniques with the help of commercially available fluorescent monocloml antibodies and flow cytometry. shed cell surface markers in plasma and cytoldnes were measured with the help of sandwich-elisa kits. blood samples were drawn h before surgery, immediately before incision (after anaesthesia), h and h after incision. seventeen cell surface markers were examined on different cell populations and cellular subsets in laparoscopic and open-surgery patients. three soluble cell surface markers and six cytokines were monitored. by statistical analyses (multivariate regression analysis, student's t test, wilcoxommann-whituey's rank sum test) the six markers/cytekines that best distinguished open surgical from laparoscopic procedurea were determined. these were . the interleuldn- receptor and im soluble form (cd /scd ); . the activation antigen fd- and its soluble form (cd /scd ), a member of the nerve-growth-factor receptor family; . the cd ro epitope which characterizes t memory ceils; . the trausferrin receptor cd ; . the soluble adhesion molecule icam- ; and . the cytokines interieukin- and interleuldn- . on the basis of these results, a tissue trauma activation (tta) index was calculated by combining the marker/cytoldne measurements by simple multiplication. anaesthesia and pre-ineision maneuvers did not significantly change cell marker or cytokine levels in either surgical approach as compared to h before surgery. h after incision the tra index in open cholecystectomy showed a distinct - fold increase, whereas in laparoseopic surgery a mere - fold increase was noted. h after incision, the tra-index returned to near pre-surgery levels. in conclusion, our results demonstrate that changes in cell surface markers and cytokines can help evaluate the magnitude of tissue trauma in diffei'ent surgical approaches. the relationship between lymphocyte subpopulation changes after thermal injury and the increased susceptibility of burned patients to infection is unclear. in this study, we have attempted to correlate such subpopulation changes with the presence of infection in burned patients. peripberal blood from patients was monitored for lymphocyte subpopulation changes three times weekly for three weeks postburn and weekly thereafter for three additional weeks. mean bum size was . % (range %- %) of total body surface and mean age was years. infection was diagnosed by carefully defined clinical and laboratory criteria and its presence or absence noted each time blood was drawn. samples taken when patients had wound infection, bacteremia, or pneumonia were compared with samples taken in the absence of systemic infection. whole blood samples were stained with four monoclonal antibodies, the red blood cells lysed and the leukocytes fixed and analyzed by flow cytometry. for each patient sample, the proportion of lymphocytes falling within the light scatter gates was determined as the percentage of cells negative for cd and most strongly positive for cd . this percentage was used to correct each sample for the presence of debris or nonlymphocytic cells. the proportion of cd and cd positive cells was slightly greatc~ in the samples from infected patients, while the proportion of b cells (cd +) was unchanged and nk (cd +) cells were decreased by ahnos[ % compared to sampie~ li'om uuiuleclcd patients. the percentage of cells positive for cdilb (c~ integrin) decreased sharply and cd ro (memory cells) decreased slightly in samples from infected patients while the expression of the lymphocyte homing receptor and cd were unchanged. cd (il receptor) and cd (early activation marker) were significantly increased in the samples from the infected patients while hladr was unchanged. these changes in lymphocyte phenotype correlate with the presence of infection. if they closely precede or occur during the early development of infection they may be valuable clues to the mechanism of susceptibility following thermal injury. trauma patients are subjected to an immediate massive impact on their host defense integrity due to the combined effect of tissue trauma, shock and endotoxemia. cytoldnes are playing a crucial role within the course of an impaired cell mediated immune response (cmi) resulting from a disruption of intact m%/tcell interaction. the current study was undertaken to further elucidate the mechanisms of dysfimctional cmi following major burn and mechanical trauma -via comparative analysis of mrna expression and protein release. the major regulatory levels for different cytokines were determined in mitogen, respectively lps stimulated peripheral blood mononuclear cell (pbmc) cultures of trauma patients on consecutive days ( ) t, , , and post injury. we analyzed the cumulative data for interleukin- beta (il-i[ ), il- , il- as well as tumor necrosis factor alpha (tnf-~) and saw a considerable impairment of the protein release in the stimulated pbmc cultures until d post-trauma and recovery thereafter. *p < . , ** p < . vs control comparing the autoradiographies of the specific cytokine mrna expression with the protein release in the supernatants, we saw a good correlation between mrna signal intensity and protein synthesis for il- and ,- , suggesting that for these cytokines the main regulatory mechanisms are located at the pre-/transcriptional level. for the other cytokines investigated one has to suppose posttranseriptional mechanisms. the analysis of our data clearly indicates a severe impairment of forward regulatory immune mechanisms following trauma. most likely the regulatory mechanisms, that are involved are greatly different among the cytokines investigated. it may be concluded, that depressed cmi responses post-trauma are partly due to an impaired pro-inflammatory cytokine production. the severity of the injury (iss) correlated with the development at multiple organ failure (mof-score; r= . ). the levels of mediators and markers of the inflammatory response were generally higher in the more severely injured group (iss> , n= ). i - , - , g-csf, fpa, and c a -levels differed significantly (p< . ) between the iss-groups (>-< iss ) at the time of admission, whereas on day tnfa, c a, - , and ealpi showed significant differences. beyond the first week, major differences were restricted to pge and c a. the formation of two groups with respect to later multiple organ failure (mof < ; mof > n= ) yielded similar results. leukocyte-facs analysis revealed significant differences mainly in the cd (monocytes), cd /cd (i - r + t-cells), and cd /cd (th calls) populations. summarizing our findings we were able to detect some alterations in the surface antigens of immunocompetent cells. the inflammato d response, however, seemed to be more pronounced and correlates wi~ the further clinical course. using an experimental bum model in rodents, we have demonstrated that administration of a full thickness, scald burn involving % or more of the total body surface area (tbsa) elicits systemic responses which are characterized by numerous alterations in t-ceu function (i.e., lymphokine production and contact hypersensitivity (ch) responses) plus an enhanced susceptibility to bacterial infection. in the present study we questioned whether the apparent systemic effects mediated by large burns would be elicited as site-specific alterations in immune function following administration of small area burn trauma ( % tbsa). following a % tbsa burn, ch responses to contact sensitizing antigens were found to be altered. the depression in ch responses could be induced independent of the site used for topical skin sensitization. following a % tbsa thermal injury, development of ch responses were affected in a site-specific manner. immunization of % tbsa thermally injured mice in a site near the position of the burn resulted in depressed responsiveness, whereas immunization through a contralateral site resulted in responses that displayed both the intensity and kinetics of a ch response equivalent to sham-bumed mice. similar systemic and site-limited changes in lymphokine production were observed with % and % tbsa thermal injuries, respectively. a % tbsa injury affected the lymphokine producing potential of all cells regardless of which lymphoid tissue the cells were isolated from. the effect of a % tbsa burn was significant but site-specific. thus, ceils from lymph nodes receiving drainage from thermally injured tissue were specifically affected, whereas lymphokine production by cells from lymphoid organs receiving drainage from unaffected skin was normal. it was concluded that modulation of lymphokine production and cellular immune responses may be a normal consequence of burntrauma regardless of the size of the burn. changes in immune competence can be mediated either regionally or systemically in direct proportion to the area of skin exposed to the burn injury. this work is supported by phs grant gm and the office of navy research n - -j- . division of cell biology and immunology, department of pathology, university of utah school of medicine, salt lake city, ut . post spleneetomy septic sequelae may be fatal, but the mechanisms remain unclear. the objectives ef this study were to assess the mortality from concomitant splen-'etomy and ]~eritoneal bacterial challenge and to elucidate the local cetkdar responses. cd- mice were randomised to receive laparotomy and sham splenectomy (l) or splenectomy (s) with simultaneous ca'-cal ligation and "):mcture and the survival patterns assessed. subsequently, cd- mice were randomised into control (c), l or s groups and peritoneal cells studied at hours for bacterial phagocytosis and killi:~g, superoxide ( -) and tumour necrosis factor (tnf) production and macrophage activation vsing mac-i(cd- b) receptor in~.ensity expressed es mean channel of fluorescence (mcf). these resides indicate that sf!enectomy predisposes to nrortal~ty from bacterial sepsis ia the early pos~ operative period compared to sham operated animals. failure ~f p'.acrophages to kill bacteria in the splenectomv group '~:cured in t?~e absence of impairment of oxygen freeradical or tnf pred:~ctien. the macrovh~ge ac!ivotion marker mac- was significantly reduced in both l and s groups and impaired phagocytosis of bacteria oceured in both operative groups compared to controls. laparotomy a!one reduces macrophage activity in terms of surface re:eptor mac- expression and !ingestive capacity. splenectomy however s~gnificantiy ~mpairs r-acrophage-wediated l~,acterial killing and this qefect rttav co~tribut~ sig~ifjcav'ly to th-~ dissemination of local infection and to n':ortalit). depts of haem~ tology & surgery, beaumont hosoital, dub!in ,eire. introduction: loss of cell membrane integrity appears to be a common pathway of injury to tissues subjected to high-voltage electrical shock. the cell membrane is the most heat labile structure in the cell, and is also the most vulnerable to externally-imposed electrical forces. skeletal muscle and nerve cells are particularly susceptible to electroporation by clinically relevant electric fields. restoration of membrane integrity is essential for cell survival in victims of electrical shock. we have studied the effect of non-ionic triblock copolymers ( poloxamer class) on the transport properties of isolated rat skeletal muscle cells following electroporation-induced membrane disruption. - mm long adult skeletal muscle fibers were isolated by enzymatic digestion from the rat flexor digitorium brevus and maintained under standard culture conditions. they were loaded with the calcein-am dye and placed in a ,c chamber for recording by real-time video confocal microscopy. the cells were subjected to msec, v/era, a field pulses with a low duty cycle to allow thermal relaxation. peak temperature rise was , .c. the uye content of the cell was monitored in real time. experiments were carried out in calcium-free phosphate buffered saline, with mm mg%. experiments were repeated with mm neutral dextran ( the aim of the present paper is to ascertain if thuracotomy induces a different pattern of variations of cytokines, immunocompetent cells and antibodies from laparotomy in the early postoperative period. patients ( males females,mean age: . _+ ) with gallstone disease and with non neoplastic pulmonary disease were studied. none of these patients received blood transfusion, biological response modifiers, radiotherapy or surgery for at least months before being included in our study. anaesthetic procedures were similar in all patients and none were matnourished. on the day of surgery and on the st and th postoperative days (pre, lpo, po) percentages of cd , cd , cd , cds, cdi were measured by means of flow cytometry using moab., and levels of ig a, lgg, igm, ige. by nephelometry cytokine levels in peripheral blood(il- , il- , il- , il- , tnf) were measured in pts. of each group by means of elisa using moab. _r. esults:variations of il- and il- were not s.s.. il- increased but differences between groups were not statistically significant (s.s). il-i decreased on po and increased on po in both groups but were only s.s. in the th.g., and therefore, the differences between groups were s.s (p< . ).tnf decreased in the l.g. and increased in the th.g. on the po, the difference was s.s(p< . ); on po, tnf decreased in the l.g. and decreased in the th.g. but these variations were not s.s. cell percentages decreased an lpo and increased on po, except for %cd cell that increased on lpo and decreased on po ,in both groups of pts. differences were not s.s. ig a, igm decreased and ige increased in both groups (p< . i), but differences between them were not s.s. in contrast, igg decreased on po (p< . ) and increased on po in both groups, but the decrease iu the th.g. was greater than in the l.g. twenty male children,aged from six months to years,admitted for elective inguinal operation were studied. the operations were performed under balanced combined anaesthesia (fentanyl,thiopemtone,vecuronium, % nitrous oxide in oxygen) and blood samples were collected before flunitrazepam premedication,after anaesthesia, and hours after anaesthesia. cells from the wound were collected with cellstick sponge which was removed from the wound or hours after anaesthesia. the study was approved by the local ethical committee. the percentage of neutrophils was increased and that of lymphocytes was decreased in perpheral blood after the operation.the values in the wound were close to the values found in peripheral blood. the percentage of t-lymphocytes (cd ) and helper-t-cells (cd ) decreased in peripheral blood being lower in the wound than in peripheral blood after the operation. the percentage of t-eytotoxic cells (cd ) also decreased in peripheral blood and was similar to that in the wound. b-lymphocyte (cd ) percentage was increased in pe~pheral blood after the operation and was higher than in the wound. the percentage of activated t-cells (cd +hla-dr-positive cells) in peripheral blood increased while that of natural killer cells (cd +cd +leu -pos) was increased just after anaesthesia being decreased at g and hours after the operation. spontaneous lymphocyte proliferative responses didn't change while phytohemagglutinin a and concavalin a induced responses were decreased in peripheral blood samples hours after the operation with recovery at hours.pokeweed mitogen induced lymphocyte proliferative responses were decreased at hours (p<o. ) and hours after the operation we previously reported that plasma ige increased after traumatic injury. in this study, we measured plasma ige ievels in trauma patients on hospital admission and x weekly in the intensive care unit to evaluate ige kinetics. patient characteristics were: mean age -+ years ( m, f), mean injury severity score (iss) + , sepsis ( patients), sepsis syndrome ( patients), ards ( patients), renal failure ( patients). ige increased in patients ( %). the mean increase was ng/ml (range to ng/ml; % ci was to ng/ml). increase in plasma ige was significantly greater in patients with sepsis syndrome (p= . ) and renal failure (p< . ). although mean plasma ige was higher with ards, pneumonia, hepatic dysfunction, and shock, these differences were not significant (p> . ). plasma ige increase was not related to severity of injury by iss score (p = . ). the mean day to highest ige was . -+ . . the day sepsis was first observed preceded the day of highest ige by . + . days. there was a significant association between the day of sepsis onset and the day of highest ige (p= . ). eight of nine patients with sepsis syndrome had > % increase in plasma ige from admission. one patient's ige levels were normal ( - ng/ml) for days and then increased to ng/ml over the next days, after onset of sepsis syndrome. changes in ige plasma levels may reflect the action of cytokines, such as il- , which concurrently regulate production of ige and il- receptor antagonist in a response to sepsis. sepsis remains a leading cause of late mortality in trauma and hs. although hs-induced bacterial translocation is supposed to be the major cause of sepsis and mof, depression of the res increases susceptibility to infection after injury. the purposes of this study were: a) to evaluate the res in the lung, spleen and liver after hs and subsequent hypertonic saline (hsl) treatment, and b) to document the patterns of phagocytic activity in these organs during hrs. adult male wistar rats ( +_ gin) were submitted to hs (sbp tort) and after t hr (shock i hr) and hrs (shock hrs) hsl (nac . %, . ml/kg) treatment, e. coli (i ) was injected into the portal vein ~tci (n_> ). twenty minutes later, the lungs, spleen and liver were harvested and scintilographic counts obtained. data is depicted as mean_%+sem * p< . , ~" p< . and statistical analysis was performed by analysis of variance and wilcoxon tests. one hr after treatment, lung uptake was increased and liver and spleen uptake were reduced compared to sham. twenty four hrs after treatment, all organs, except lung uptake, returned to normal values. radioautographic histological analysis revealed radiolabeled particles inside phagocytic cells of all organs. we conclude that pulmonary phagocytic activity increases after hr of hs hsl reatment, diminishing by hrs although still above normal values. in contrast, res suppression occurs in liver and spleen after hr hs hsl treatment, returning to normal values by hrs. these results may explain lung complications and immunosuppression after trauma. infusion of endotoxin as well as major surgery is followed by lymphopenia in peripheral blood. the purpose of this study was to investigate to which tissues the lymphocytes are redistributed in response to endotoxaemia and major surgery. in addition changes in lymphocyte subpopulations and expression of mecii was measured. lymphocytes were isolated from peripheral blood of rabbits, labelled with indium-tropolene and reinjected intravenously into the rabbits, i rabbits received an infusion of escherichia coli endotoxin ~g/kg, while i rabbits were subjected to a major sham operation and i rabbits served as a control group. the redistribution of lymphocytes were imaged with af gamma camera, and calculated with an interfaces computer before, and , and hours after major surgery or infusion of endotoxin or saline. interleukin-l~ and serum cortisol were measured. in addition we followed cd , cd , cdlla/b, cdis, cd , cd , mhcii and cd /cd ratio. following endotoxaemia interleukin-lf~ increased significantly, following endotoxaemia as well as major surgery serum cortisol increased significantly. following major surgery as well as endotoxaemia there was significant lomphocytepenia in peripheral blood with a decreased cd /cd ratio while the cd positive subpopulation increased. in addition there was a decrease in the expression of mhcii on the lymphocytes peripheral blood. the radioactivity of the lymphatic tissue in and around the intestine increased to % of initial values following endotoxaemia and to % following major surgery. the results indicate that endotoxaemia as well as major surgery induces redistribution of lymphocytes from peripheral blood to lymphatic tissue. among the lymphocytes staying in peripheral blood there was a decreased expression of mhcii and a relative decrease in cd cells compared to cd positive lymphocytes. in order to analyze the effects of immune suppressive substances on expression of mrna of interleukin- (il- ) and interleukin- reeeptor(il- r), this study was carried out. twenty male rabbits with comminuted fracture were used in the study. ten ml blood were taken at , i, , , days after injury. the sera were tested for the effects on lymphocyte blastogenesis and induction of il- stimulated by concanavalin a(con a): the sera from the rabbits days after injury were analyzed with sds-page gel eleetrophoresis, and divided into three groups by ultrafiltration (ufpi ttk, kd,milipore; centricon- , kd,amicon), that are less than kd, between i and kd, and more than kd. each group of the substances also was tested for the expression of il- and il- r by the dot blot hybridization. the results showed that: i) all sera from the rabbits after injury had significant suppression on lymphocyte proliferation and secretion of il- by the con a-stimulated splenocyte in mice; ) the sera from the rabbits days after injury had more profound suppression than other injured sera; ) there was a marked band at about kd in sera from the rabbits days after injury, but nothing at the same position in normal sera analyzed with electrophoresis; ) the substance with molecular weight of about iokd had more obvious suppressive action on expression of mrna of il- and il- r than other groups substances, of which molecular weights are more than kd. it is concluded that: i) the sera from the injured rabbits can reduce immune response; ) there is kind of substance, of which molecular weight is about kd, it is probable the main factor involved in the pathogenesie of postinjury suppression immune; } the substance can depress the expression of mrna of both il- and il- r. research institute of surgery daping, chongqing, p. r. china acute ethanol uptake prior to injury modulates monocyte tnfo~, production and mononuclear cell apoptosis. g. szabo, b. verma, p. mandrekar, d. catalano monocytes (mo) have been shown to contribute to immunosuppression after both major injury and alcohol consumption. we reported that acute ethanol exposure of m( results in decreased antigen presentation, induces tgf- and pge while inhibiting inflammatory monokine production. we also showed that post-trauma immunosuppression is mediated by hyper-elevated mo tnfc~ and il- . consequently, here we investigated rnonokine production in trauma patients (n= ) who had elevated (>o.lmg/dl) or had no blood alcohol level (n=t ) at the time of emergency room admission. none of the patients had chronic alcohol use history. met tnfc~ production from trauma patients with prior alcohol uptake was undetectable during days - post-injury in contrast to patients without alcohol exposure. furthermore, decreased tnf~x levels were found in alcoholic patients' mci after mdp or ifny + mdp induction. however, mcl tnfc~ levels during the - days post injury period became higher in alcoholic trauma patients. furthermore, over days post-injury, alcoholic trauma patients showed significantly elevated mci tnfo~ production after adherence isolation, mdp, or ifn+mdp stimulation compared to patients without alcohol. these results suggest that acute ethanol uptake prior to injury decreases tnf(x inducibility in the early post-trauma period, but these patients' mo produce hyper-elevated tnfa levels later post-injury, thereby prolonging their cytokine shock risk. tnf ng/ml - days post-injury days post injury stimulus ale. pt. pt . . . . immunosuppression might also be increased by the elevated apoptotic activity found in trauma patients' mononuclear ceils, which was even greater in alcoholic trauma patients' cells. in non-alcoholic trauma patients' preactivated mo, in vitro acute ethanol ( - mm) exposure resulted in a significant down-regulation of tnfc~ (p< . ) and il- (p< . ) production. in contrast, in vitro ethanol exposure increased the production of inhibitory monokine, tgfi]. these results provide both in vivo and in vitro evidence for the effect of acute ethanol exposure increasing immunosuppression and cytokine shock. the 'systemic inflammatory response syndrome' (sirs) with consecutive septic multi-organ dysfunction represents the major cause of late death following major mechanical and burn trauma. systemic hyperinflammation and concurrent depression of cell mediated immune response (cmi) render the traumatized host anergic, resulting in profound susceptibility to opportunistic infection. monooytes/macrophages (mo) play a central role within the host defense system in developing and manifesting states of injury, shock and sepsis. the mechanistic scrutiny of the synthesis patterns of crucial cccytokines appears to be a helpful tool to further analyse mo behaviour in the compromised individual. the objective of this study was to further dissect the characteristics of cytokine regulation in pbmc under stressful conditions, via analysis of the expression of cd + receptor, the proinflammatory mediator il- , the macrophage activating factor ifn- ,, and neopterin (npt) a metabolite of activated mo. we investigated pbmc's on consecutive days , , , and after mechanical trauma of and after bum trauma of patients (mean age ~ years; mean iss ± pts). in trauma patients we saw a massive increase of pha induced neopterin synthesis compared to controls. however, when discriminating the npt levels in the supernatants for the amount of mo stimulated, the npt output of the individual cell was lower compared to mo of nontraumatized individuals. interestingly there was a contrary coarse in the cumulative protein release patterns of il- and ifn- in mechanical versus burn trauma patients. wheras in burn patients ifn-y was decreased significantly ( + u/ml) compared to controls ( + u/ml) as well as mechanical trauma ( + u/ml). il- showed a significant suppression following mechanical trauma ( + u/ml) vs control ( + u/ml) and bum patients. the rt~,na signal intensity for beth eytokines was in concurrence with the protein release in more than % of the individual patients investigated. from these data we can conclude that the inadequate low npt synthesis predominantly in bum patients appears to be a sign of cellular immaturity and is probably partly due to low t-cell ifno t signals. in addition we could state that the quality of trauma is apparently responsible for the different synthesis patterns of ]l- and ifn-q,. it has been postulated that bacterial invasion or endotoxemia are necessary for cytokine production following burn injury. we studied the organ distribution and kinetics pattern of il-fc~ (cell-associated il- agonist) in eutrophic rats subjected to either % tbsa cutaneous scald injury (bi), muscle scald injury of equivalent % tbsa (mbi), sham muscle bum (resection of skin only, up to % tbsa) (smbi), and sham cutaneous burn (sbi), followed by saline resuscitation ( mukg i.p.). separate rats were infused with mg/kg e.coli :b lps or saline lv. unmanipulated rats were baseline normal controls. liver, lung, spleen, ileum, thymus, kidney, skin, and plasma were harvested at various time-points within the first h. tissues were frozen, weighed, homogenized, the homogenates centrifuged and the supernates assayed with a radioimmunoassay specific for rat il-l(z (detection limit pg/rnl). il-lc~ was expressed as ng/g weight + sem (lowest detectable amount . ng/gwt). il-lo~ was constitutively present only in the skin ( + . ng/gwt). cutaneous burn and sham cutaneous bum induced biphasic elevations of il-lcc in the liver and lung only, with maximal levels at . h (in the liver, bi = . _+ . ng/gwt, sbi = . + . ng/gwt, p _< . ; in the lung, bi = . + . ng/gwt, sbi = . + . ng/gwt, p -< . ). of note, both bi and sbi rats had detectable il-i~ in the liver at timepoint already ( min real-time). these levels increased in parallel until min and became eventually different by log at - . h. all other organs as well as plasma were below detection limits. muscle burn injury and sham muscle burn (skin resection) induced similar elevations of il- ~ in the liver at lh, indistinguishable from each other and from cutaneous burn. in contrast, lps challenge induced dramatic elevation of il-t~ in all organs tested except for the kidney; the spleen was the most responsive organ to lps-induced il-lo~ production. these data indicate that thermal or mechanical injuries induce very early and organ specific production of il- c~ in vivo by mechanisms other than endotoxemia. injury-induced complement and platelet activation may be involved as well as the neuro-endocrine axis, which may explain the low levels of il-lo~ induction observed in all rats at the very early time-points. trauma services, massachusetts general hospital, and department of surgery, harvard medical school. fruit, st, boston, ma . j. f. schmand *#, a. ayala* and i. h. chaudry* studies indicate that i.v. infusion of the colloid hes in normal animals does not adversely affect non-specific immunity. it remains unknown, however, if lies affects cell mediated, specific immune functions after trauma and hemorrhage (hem). to study this, non-heparinized c h/hen mice underwent midline laparotomy to induce trauma and were then bled to and maintained at a bp of mmi-ig for rain. the animals were then resuscitated with either times (x) the shed blood vohune as lactated ringer's solution (lrs) or x lrs + lx % lies. sham mice were neither hemorrhaged nor resuscitated. at or hours post hem serum, peritoneal (pm~) and splenic macrophages (sm~) were obtained. bioassayes were employed to assess the levels of ii-l, il- ( alternatively pmqb showed no differences in il- release between all groups at and h, while sm~ from the lrs + hen group showed a depression at h. tnf production by pm~ was depressed in all groups at h and remained so in the lrs + hes group at h. sm~b showed decreased tnf release values in both hem groups at and h. in summary, the levels of inflammatory cytokines (particularly the values of circulating il- ) after trauma/hem are positively influenced by the administration of hes. this might be due to a protective effect on pmqb and sm~, but also on other cytokine producing cells, e.g. kupffer ceils. we conclude that hes is not only a safe, but also beneficial agent in the resuscitation of patients atler trauma/bemorrhagic shock. this study investigated endotoxemia and consecutlve immune response in patients with multiple trauma (median injury severity score = , ). blood samples.were collected shortly after injury and after , , , , s and l days. endotoxin was measured with limulus-amebocyte lysate test and the specific antibody content (sac) against endotoxins of the classes igg, igm and lga by elisa-technique. five antigens were used: lipopolysaccaride (lps) of e.coli (ec), lipid a of e.coli (la), lps of pseudomonas aerog. (pa), lps of vibrin cholerae (vc) and cx-hemolysin of staphylococcus anreus (oth). a nephelometer indicated the total concentrations of igg, igm and iga. differences were checked with wilcoxon-test and p< , s was considered significant. cross-reactivity was calculated with rank correlation coefficients. results: endotoxemia peaked shortly after injury ( - h) at , eki/ml (median), decreased thereafter to , eh/ml at day s and remained on this level. sac oflgmclass increased to all endotoxins and peaked at day revealing the lfighest level to la followed by pa (= % of la-sac), ec (= % of la-sac) and vc (= % of la-sac). lga antibodies increased as well but only slightly and not significant (exception: sac to la was elevated significantly at day ). igg antibodies increased similar to iga class only slightly and again only sac to la was significantly higher at day and . however sac to (xh of all ig-classes remained continuously on the same level troughout the observation time. correlation analysis revealed strong cross-reactivity (r> , ; p< , ) most often between antibodies of igm-elass ( %) followed by igaclass ( %) and lgg class ( %]. conclusions: multiple trauma is associated with temporary endotoxemia. endotoxins probably translocated from the gut cause specific increase of anti endotoxin antibodies in blood of the igm-class. endotoxins cause no increase of antibodies to gramposilave bacteria. igm antibodies are most unspecific. during cardio-pulmonary bypass, as well as postoperatively, high levels of endotoxin, interleukin- (ii- ) and c-reactive protein (crp) were measured in patients. i female and male, ageing from to with a median age of . blood sampling was done preoperatively, immediately after induction of anaesthesia, after thoracotomy, after cannulation of the aorta and right atrium after the first half of the reperfusion phase, after closure of the thorax, and hours after the operation and then every morning until the th postoperative day. blood was drawn into heparinized tubes (i iu/ml) which were free of endotoxin. crp levels were determined through the use of the behring nephelometer. - levels were measured by using commercially-available elisa test. the endotoxin level was determined by a chromogenic modification of the limulus amebocyte test. the statistical analysis was done using the wilcoxon ranks test and correlation analysis. a significant increase {p . ) in endotoxin plasma occurred during surgery, culminating in a peak (median value of . eu/m!) during reperfusicn. plasma levels of endotoxin continued to be slightly raised till the th day after surgery, whereas those of interleukin- rose at the end of the operation and were at their highest hours later (median value of . pg/ml). crp levels were also high postoperatively with a median value of mg/l, and were markedly raised on day ( mg/l). a definite, statistically significant correlation between the plasma levels of endotoxin and - during the operation was establisthed (p . ), leading us to conclude that the endotoxin liberated during cardiac surgery acts as the main trigger in the releasing of - , and thus induces the postoperative acute phase reaction. there was no evidence of a correlation between crp and endotoxin or - plasma levels. impaired immune function is well described following trauma and hemorrhagic shock (hs). prior studies have utilized peripheral blood or spleen cells to index immune function following hs. however, changes in mucosal immunity are not weii characterized in this setting. gut origin sepsis is thought to be an important cause of organ failure and death following trauma. a rodent model was utilized to allow comparison of mucosal-associated immune function vs, systemic compartments after hs. fischer rates underwent hs (map ± mm hg) for minutes followed by resuscitation with shed blood and lr. sham animals were instrumented only. rat tears were collected at and hours following hs for quantitation of slga by ria. animals were sacrificed at hours and spleen (spl), peripheral lymph nodes (pln), and mesenteric lymph nodes (mln) harvested for cell population analysis using flow cytometry and mitogen stimulation analysis. cell marker expression analysis revealed no changes in t or b ceil populations following hs. mitogen mucosal immune function appears relatively spared following hs. the mechanism(s) for this variability in immune function requires further investigation. we have found that transplantation of bone marrow in a hind-limb graft to syngeneic lethally irradiated recipient is followed not only by rapid repopulafion but also overpopulation of bone marrow cavities. the question arises whether this unexpected phenomenon could be the result of stimulation of stem cells by factors (cytokines) released from surgical wound at the site of anastomosis of graft with recipient. aim of the study was to investigate which tissues damaged during the procedure of limb transplantation may be a potential source of humoral factors accelerating in vivo bone marrow proliferation. methods. experiments were carried out on lew rats in groups. in group i, the hind limb was transplanted orthotopically to a syngeneic recipient; in group ii, sham operation was performed; in group iii, a four-cm long cutaneous wound was made on the dorsum; in group iv, limb skin was harvested, fragmented and implanted into peritoneal cavity; in group v, bm from femur and tibia was implanted intraperitoneally. bm, lymphoid tissues and blood were sampled and days later for cell concentration and phenotype evaluation. results. the yield of nucleated cells from tibia was on day in the control . + . , in group . + . , in group ii . + . , in group iii . + . , in group iv . _+ . , in group v . _+ . x ( ). the evident increase in bmc yield in all groups continued until day . increase in weight and total cell count of spleen and mesenteric lymph nodes in all but group iii was also found. no differences in percentage of maturing erythroid cells, but higher of mature myeloid cells and lower of lymphocytes were observed. conclusions. trauma of skin, muscles, and bone brought about an increase in bone marrow cellularity and acceleration of maturation of myeloid lineage. transplantation of bm ceils alone did not produce this effect. transplantation of bm in limb graft is a good model for studies of natural factors reaulatin~ bm hemormesis. this study sought to determine a relationship, if any, between the degree of hypochclesterolemia upon trauma patients' admission and their subsequent outcome. all blunt and penetrating trauma patients admitted to a level i facility from through , and who had serum cholesterol assayed during the first hrs were retrospectively studied for development of death or significant organ dysfunction. the mantel-kaenzel chisquared test was used to determine significance of data at the p< . level. results: trauma patients were admitted during the four-year period who had serum cholesterol assays performed in the first hrs. patients had cholesterol levels less than mg/dl; of these ( . %) died, ( . %) developed ards, ( . %) developed acute renal failure, and ( . %) developed multisystem organ dysfunction; hypocholesterolemia in these patients was not due to liver injury or massive fluid administration. the risk of death was times greater and risk of multi-organ failure times greater in this group than in those with a normal serum cholesterol (>if mg/dl; patients; p< . ). conclusions: admission serum cholesterol level in the trauma patient serves as a powerful marker for those at risk of subsequent organ failure or death. hypocholesterolemia in this setting may result from organ hypoperfusion and humeral mediator release. lung tissue contains many immunocompetent cells. resection, therefore, is expected to activate extensively inflammatory mediators such as pmn-elastase, pmstanoids and pteridines. in a prospective clinical study we compared patients (pts) undergoing either thomcotomy with or without lung tissue msectioh and tboracoscopic lung resection concerning activation of inflammatory response. material & methods: group a pts (n= ) had thoraantomy but no lung tissue injury; group b pts (n=ls) had thoracotomy and lung tissue resection due to benign diseases; group c (n= ) represents group b tissue resection but using a thomcoscopic procedure. the following parameters were determined pre-, peri-, and postoperatively: elastase and crp as indicators of activation of pmn-leukocytes and injury severity; prostacyclin (pgi ) and thromboxane (txa~) as parameters of lung endothelial response; prostaglandin f ~ (pgf~) and pgm representing pulmonaly metabolic activity; pge a and neopterin as proof of macmphage activation. statistics were performed using analysis of variance for repeated measures. results: group b pts revealed postoperatively an increase in crp (p< . ) indicating a higher injury severity in comparison to the thoracoscopic procedure (c). both, controls (a) and group c pts did not show pmn-activation, whereas group b demonstrated a reversible increase in elastase. surgical trauma caused in all groups a release of pgi z and txa which was more pronounced in c (p< . ) and most in b (p< . ). similar results were found for pge~ and pgf =. there was no activation of maerophages since neopterin did not increase. apparently, metabolic lung function was not impaired because there was no marked rise in pgm except in b (p< . vs. c). discussion: our results demonstrate that lung tissue injury aggravates the mediator release induced by thoracic traum. these mediators among others are able to increase capillary pressure and hence lung edema formation. impairment of lung function, however, seems dependent on the extent of the liberation. therefore, the maximal release reactions occured in group b and c after lung tissue resection, whereas the controls showed the highest levels immediately after the incision. we conclude that thoracoscopic procedures are superior in reducing the resection trauma per se and hence might prevent severe mediamr-induced (pulmonary/systemic) sequelae. in a prospective study we investigated patients using radiochemical method according to sch~dlich (s) and photometric method according to hoffmann (h). serum of severly traumatized patients was withdrawn directly after admission at our emergency room and in narrow time intervals during first hours after trauma. follow up control samples were taken daily until day ten. whereas no elevated pla-ca was found during first hours, a peak was regularly observed around day four. there was high correlation between pla-ca and iss (r= . , p<o.o ) except patients suffering from thoracic trauma, thoracic trauma {tt) provoked similar gons-score-and pla-ca-values compared to multiple trauma (mt) despite significantly lower iss: pla-ca (h) mt . + . tt , +_ , n.s. pla-ca(s) mt . +_ . tt . _+ . n.s. goris mt . - . tt . _+ . n.s. iss mt - tt - p< . we found delayed elevation of pla-ca after severe trauma, however detection of delayed pla-ca in the serum of traumatized patients cannot give reliable information about the exact role of pla in the inflammatory reaction after trauma as latest results show that pla is secreted early but bound at lipophilic sites of vascular membranes. hern~mdez m j, amiguet ja, monreal ji, vid~n jr, jim nez f j, l pez-vivanco g acute phase reactant proteins increment in serum of patients with traumatisms and inflammation has been previously reported. alpha- antitrypsin (aat), alpha- macroglobulin (amg) and ceruloplasmin (cp) are evaluated in the following of patients, males and females (mean age: years), with compound fractures. % of them were located in tibia and fibula. intercurrent infection developed in patients and internal fixation rejection in one. measurements were made by radial immunodiffusion on days , , , , , and . a control group of healthy volunteers was also evaluated. aat and cp showed increased values from day which kept elevated untill the end of the study. amg elevated from day . when infection developed, aat and cp values were even higher whereas amg values decreased. fixation rejection caused aat, amg and cp increment. aat and cp increment in non complicated fractures might be due to accompanying inflamation, by means of cytokines release and aprps hepatic synthesis induction. therefore, increment is higher when infection or rejection develop. amg behaviour at early stages of evolution is secondary to hiperconsumption, considering mean life shortening after proteases ligation. amg modifications in fixation rejection remain obscure. aprps increment modulates inflammation and bony callus formation by means their antyprotease and antioxidant properties. clinically, aprps might be useful parameters in determining complications onset in fractures evolution. hemorrhagic shock (hs) is a common complication of trauma, as well as some major surgical procedures. the alteration of the immune system by hs is thought to contribute to the increased septic morbidity and mortality seen in these patients. multiple mediators are released following hs. il- , il- , tnf, pge and tgf-b have all been implicated in these immune system changes. in vitro, pge causes many of the immune system changes following hs, yet the time course of pge release has not been well deliniated. the aim of this study was to determine the time course of pge release by peritoneal and splenic macrophages following hs. c h/hen mice were bled to a mean bp of + mm hg for minutes. the animals were resuscitated with the shed blood and normal saline. control animals were cannalated, but blood was not withdrawn. the animals were sacrificed at , , , , and hours following hs. peritoneal and splenic macrophages were harvested from each animal and cultured with lps for hours. the supernatants were collected and frozen until assayed. pge was assayed using an elisa (cayman chemical). results are shown below for the peritoneal macrophages: pge release by peritoneal macrophages was significantly elevated at , and hours over control. it was maximal at hours, but by hours it had started to decline. splenic macrophage pge release was much more variable. (data not shown.) there was less consistency between time points and no clear trend was seen. in conclusion, pge is significantly elevated at hours following hs, and reaches a peak at hours. pge release may be responsible for the immune system changes seen in the first few days following hs. splenic and peritoneal macrophages respond differently in their release of pge following hs; which may be due to differences in their milieu. there is an obvious association between the altered immunocompetence of patients following traumatic injury of various types and the increased riskto develop complications. the present investigation was undertaken in order to analyze the value of both neopterin (n) and c-reactive protein (crp) in monitoring disease course in patients with multiple trauma. we were interested to compare nconcentrations as marker of t-cell/macrophage activation with crp levels as indicator of the inflammatory acute-phase response daily serum concentrations(n and crp) were measured in patients ( male, female, mean age . ), admitted to our icu following serious trauma (mean iss= ) and in control subjects. the clinical course of each patient was evaluated with apacheii score according to knaus. mean stay in icu was . (range - ). patients didn't develop organ failure(nof-group), patients survived, developing mof (s,mof-group), patients died with mof (ns, mofgroup). three patients(one s and two ns) showed signs of septicemia and septic shock. the serum samples were measured for neopterin (immu-test-ria, henning-berlin) and for crp (immunoturbidimetric method) -at the university hospital for internal medicine, innsbruck, austria. the highest crp levels were measured h after trauma, but they were not accompanied by significant increase in n-values. we found significantly elevated n-concentrations from the days - . n and crp levels showed a parallel release peaks on the days - and the values discriminated significantly among the three outcome groups(the second crp peak is lower in comparison to the first posttrauma peak). by the ns-group we found constant and parallel increasing crp and n levels, reaching extremly high values - h and preceding clinical signs of mof and sepsis. the values increased dramatically before death. the serum levels by all control subjects were upper the normal limit for n and crp.. the parallel occured peak changes in n and crp concentrations showed a significant correlation with clinical status, using a disease activity score (apac h eli). our findings suggest that both-cellular and humoral mediated immune mechanisms are activated after multiple trauma and that simultaneous determination of n-crp may be of advantage as diagnostic and prognostic parameter in polytrauma-patient monitoring panel. it is apparent, that measurement of very high levels may be of value as a srong indicator in developing of mof or septic complications and thus offer the possibility for earlier therapeutic intervention. the modification of t-cell/macrophage and acute-phase responses may be potentially useful in the treatment of icu patients, resuscitated after severe trauma, and suggest that n-crp may be relevant indicator for testing experimental immunomodulating therapies. although several in vitro studies suggested the catalytic action of iron in oxygen free radical generation, few data are available concerning iron-metabolism following ischemia-reperfusion injury and hemorrhage in vivo. aim of the present studies was to evaluate iron metabolism following mild and severe hemorrhage in the presence and absence of intraperituneal hematoma. methods. male lewis rats ( g) divided in groups (observation time hrs): a: mild hemorrhage by sequential blood sampling ( . ml each)( , , , , , , , , hrs). b: a with hemoperitoneum (hp)( ml/ g), c: hemorrhagic shock: blood withdrawal of ml/ g over min and resuscitation with ringer's lactate ( x shed blood) and isogenic donor blood; d: c with hp. parameters: serum iron ([g/dl], ferrozin-method); fe labeling of erythrocytas (application of ci fe i.a. into a donor rat, days later bleeding of the rat and injection intraperitoneally in experimental rat; plasma transferrin ([g/ml], ria). t-tests and mann whitney. data are expressed as mean + sem. results. compared to baseline, mild and severe hemorrhage caused a significant increase in serum iron at and hrs, respectively ( + vs. + ; +_ vs. +_ ). hp reduced this increase ( +_ vs. + ; + vs. +- , p<. ). i-ip significantly increased hemoglobin between to hrs in mild ( . +_. vs. . +. ) and severe hemorrhage ( . +. vs. . +. ). in mild and severe hemorrhage comparable maximal resorption ( fe labeled erythrocytes in circulating blood) of intraperitoneal hematoma occured at hrs (mild: +_ vs. severe: +- counts/rain). hp increased survival-rate following hemorrhagic shock (no hp: / ; hp: / ). in mild hemorrhage no alterations of plasma transferrin concentrations. in shock group without hp, there was at hrs a significant higher transferrin level than with hp alone ( . +_. vs. . + . ). in conclusion, hemoperitoneum with hemorrhage prevents increase in plasma iron levels by increasing hemoglobin through resorption of erythrocytes and thereby suppressing iron mobilization from endogenous iron resources, e.g. liver. this suggests that intraperitoneal hematoma not necessarily promotes iron-mediated oxygen free radical production, especially since survival in these groups was improved. increased transfarrin levels in group c at the end of the experiment suggest increased iron turnover which was not observed in shock groups associated with hemoperitoneum. primary immune response to thymus-dependent (srbc) and thymus-independent (vi-polysaccharide) antigens was determined during days after cct. antigenic challenge was made on day i, , or . number of spleen plaque forming cells (pfc) and serum haemagglutinin (ha) titre were studied on day after immunization. cct stimulated immune response to t-dependent, but not to t-independent antigen. enhanced response was noted when srbs were given in posttraumatic day i, it was higher than preceding one after injection on day and reached a maximum when antigen was presented on day after trauma (pfc number . times exceeded the level of immunized, but nontraumatized rats). the rise of pfc number in spleen correlated with increased ha level. thymectomy had been performed days before cct completely abolished posttraumatio stimulation of immune response to t-dependent antigen. standard thoracotomy also enhanced humoral response when srbc were administered on day after the operation. thymus trauma modeled by pincett squeezing of its right lobe and performed at the same time with thoracotomy fully abrogated as well as thymectomy stimulation of immune reaction. thus, different experimental chest traumas are able to increase antibody production via thymus-dependent mechanisms. endothelin is a amino acid peptide produced by ischemic or injured endothelial cells. in vitro and in animal studies, et is also a potent constrictor for renal mesangial and coronary vessels, a negative cardiac inotrope and an endocrine regulator. our lab has shown that et is an agonist for monocyte production of interleukins i, & , prostaglandin e (pge ) , and substances which trigger superoxide production. systemic et levels increase in hypoperfused and septic patients, and et may be yet another important cytokine in critical illness. but while et has been shown to be a potent vasoconstrictor in healthy dogs, systemic vascular resistance does not increase in critically ill patients with high et levels. (we hypothesize that this might be due to pgez-mediated vasodilation predominating over et-mediated vasoconstriction.) we next asked what happened to systemic et levels in patients with major burns (> %.) ten hemodynamically stable patients resuscitated by a modified parkland formula to a urine output > cc's per hour had et levels drawn on admission, at i, , , and hrs. et levels were measured by radioimmunoassay. mean levels were elevated at ± pg/ml at all time points versus levels in healthy controls of ± . in summary, systemic et levels increase significantly in patients with major burns. et may be yet another cytokine playing a significant role in the immune, inflammatory and multiorgan dysfunction observed with major burns. restoration processes in an organism after ischemic damage are realized through ~n~lammatory mechanisms~ the intensity of which is significantly defined by blood levels of neuropeptides. myocardial infarction (mi) was chosen for studyin these processes since it eradicates the influence of infectious factc~rs. dogs~ in whom mi underwent different forms o¢ healer, g; bhn~ed ~h~t during the acute phase of the disease there was a characteristic rise of ne!~ropeptides in the blood. these neuropeptides had nociceptive and antinociceptive effects. particularly substance p and -endorphins triggered off the development of compensatory and adaptive mechanisms and defined the intensity of inflammatory reaction at the zone of ischem~t: damage-notable fall in substance p levels after an ~nitial increase, while the ~-endorphins stayed high was an important condition for non complicated healing of mi. on the other hand high levels of substance p with low ~-endorphin concentrations lead to increased infiltration o~ neutrophils into the infarction zone and weakened the activity of synthetic processes~ thereby leading to left ventricular aneurysm. at the same time low intitial levels of substance p slowed down the development of necrotic processes which lead to delay in refunctioning of the heart and complicated the healing process. thus, regulation of the levels of neuropeptides in the blood in trauma forms a perspective method of its treatment. of laparascopic versus open choleocystectomy c. schinkel, s. zimmer, v. lange, d. fuchs, e. faist the impairment of immune function due to surgical trauma may be followed by deleterious septic sequelae. compared to open abdominal surgical procedures (lap), laparaseopic surgery (lsc) is associated with a decrease in hospital stay and in accelerated patient recover. the aim of the study was to evaluate the sensitivity of the immune sermn parameters of il- , saa and neopterin, the percentage of cd + cells, the in-vitro il- synthesis after mitogen stimulation and lymphocyte proliferation, in order to purposefully discriminate differences in the severity of trauma. we investigated the blood of patients with cholecystolithiasis undergoing either laparascopic ( ) or open (i ) cholecystectomy on consecutive perioperative days - , , and . there was no significant difference between the two groups concerning age and sex. patients with clinical signs of acute cholecystitis were excluded from the study. operation time and hospital stay were obviously longer in lap patients ( versus minutes, versus days) compared to the lsc group. concerning the unspecific acute phase reaction we could show no difference in the increment of senun amyoid a (saa) synthesis in the lsc group (d-i + lng/ml, d + ng/ml) versus lap group (d- + ng/ml, d + ng/ml), while in serum il- levels we saw a less steep increment in the lsc group ( -fold from d- to d ) compared to the lap group ( -fold from d- to d ). the analysis of cd + receptor expression and serum neopterin did not reveal any difference between the groups. lymphocyte function showed an impairment of proliferation to antigen stimulation in lap (d - : . + . cpm, d : . + . cpm) compared to the lsc group (d -h . + . cpm, d h . + . cpm). in both groups il- synthesis was decreased post-operatively. our data indicate that laparascopic cholecystectomy reusults in a less distinct unspecific acute phase reaction post-trauma compared to that following lap. neopterin serum levels and cd receptor expression show that these parameters apparently are less useful markers to detect differences of surgical trauma severity while it appears that the impact of lap is reflected most impressively on the lymphocyte compartment. trauma alters the host resistance of organism and is accompained by appearence of excgenic and endogenic proteins in the body. to understand the molecular mechanisms of host resistans disorders in trauma, as a first step, the genetic regulatory mechanisms of immune response after antigen injection has been studed. the appearence of specific protein factors ( - and kda), in the nucleus of rat splenic and brain cells, accordingly, was shown after immunization with sheep erythrocytes. the stimulatory effect of these factors on the il- mrna and il- production was detected. the nucleotide sequences of the human il- gene regulatory region bounding by the splenic nuclear proteins were determined between + - b.p. the il- trans-factors shows the affinity to splenic and thymic lymphocytes in vitro. thus, the antigen causes the appearence of specific protein factors in the cells,which act on the gene level,stimulate il- production and the host resistance. these results cause the next step of experiments using the same model, but after trauma. these investigations will let us verify the hypothesis that the protein il- gene trans-factors may play a definite role in the decrease of the cell immune responce after trauma. confronted with the routine procedure of prophylactic treatment of candidates for surgery in a rural african hospital, we initiated studies on the fre'quency of post-surgical malaria. in tanzania non-pregnant patients from rural areas were followed. of preoperative patients % had a parasitaemia and those maintaining it showed no increase or complaints. nine percent of patients without detectable parasitaemia before surgery came down afterwards and one-third had malaria-like complaints. spinal and general anaesthesia were equally applied in these last patients. in burkina faso we studied patients of which % had a parasitaemia on admission and % had postoperative malaria. half of the surgical patients came from rural areas, whilst only % of those with malaria lived in the city (with much less exposure and immunity). % underwent major surgery and % minor. bloodtransfusions ( % with parasites) never evoked a parasitaemia in recipients. post-surgical malaria is thus a reality in about % of the adult cases, both in east and west africa. surgery evokes a cascade of factors, varying from cortison to interleukines and acute phase proteins; immune responses may temporarily be suppressed. clinical attacks of malaria in otherwise immunes could be evoked by one of these factors. though malaria can easily be cured, the differential diagnosis is difficult because of post-surgery fevers; we found that % was treated without justified indication. the involvement of "student-doctors" a. this study examines glucose uptake and hexose monophosphate (i~ip) shunt activity in normal human peripheral lymphocytes and polymorphonuclear leukocytes (pmn). glucose uptake was determined by measurir,g the uptake of tritiated deoxyglucose, a non-metabolized glucose analogue. adsorption of co derived from [i- c] glucose was used to determine knp shunt activity. in vitro assays were carried out in hormone concentrations approximating normal and elevated trauma blood levels. (normal -cortisol . ~g/ml, glucagon #g/m , epinephrine ~g/ml, insulin t~u/ml; traumaeortisol . ~g/ml, glucagon /*g/ml, epinephrine ~g/ml, insulin ~ij/ml. analysis of twenty subjects showed a reduction of ° ~mp shunt activity by lymphoeytes and a ] % reduction in glucose uptake by p~n in normal vs. trauma hontc,nes p < . . lymphocyte glucose uptake was also reduced by trauma hormones p~ . . it ha~ be.ea~ suggested thgt idiopatno pulmonary fibrous (y.pf) [s a consequence of severe alveolar epithelial injury and is associated with an nveolar irnammamry reactio~ and the presence f.neutr phils. there~bre, neutr pk~ chemoattra~ant~ are probably important in the genegs oft.he infial lesions of ipf. the obse,"wson that stimulated macrophages are or~n histologically promin~t in fibmfio [-~gs ~.nd am capable of p~oducmg a v~dery f flbrogenic pep'ides also a~gues for their role ~n the pathogenic prc~e~ oflpf. the observation that stimume~ maerophages ere often histologica[iy prominent in fibrotio lungs and ~re ~pable of producing a varie~, offibroge.~e peptide~ also argues for tkek role in the pathogenic process, therefore, we ha-~e tested the potentn for iater!eukln- (i ..- ) and mo~tocyte chemotacde pop, de (x¢cp- ) to induce neutro~hil ~d mononuclear phagocyte accumuhdon in lungs of pafient~ with pulmonary .~r~idosis and i~f. brenet~o.alveolar lavabo (bal) fluids from ipf and sar~qidosis patient were conexntratea by reversed-phase chromatography, ~d ii. arid mcp-i asso.~ed by ells& ehemotaxis mad enzyme-reieasing ~ssas's on msnocyte~ and neatrophiis. elisa revealed significenfly elevated b al-eoneentrations o£mcp-i ( . ng]mg aibumm) in purisms with p~monary sarcoidodis artd in ipf ( . ng!mg) in comparises to . normal individuals ( . ng/mg) and to patients w~th obreic bronentis (cb) (~, rig/rag). similarly, chemota*dc ac~a~' for monocles (mcp- e.qu/va]ent) was strongly increased in sareoidosis ( . ngjmg) as well as ~n f pag,nts ( . ng/mg). norra.al indlvidu~s and cb patiants hzd a . or -fold lower ~cn%i~y, re~peefively. patients with ipf and sarcoidosi~ also h~l eievated il- ievei~ ( . and . rig/rag, respe~veiy; nomzls: . rig/rag; cb: . ng/mg) mad nvatropmi ohemotax~ ( . ~'~d . nnmg, res!z~ztiveiy; aormals: . ng,'mg; cb: l ngmg). these data suggest that increased ievels of born mcp. ~d il- may be oharacted~tie for ~arcoidosis or ipf_ it appears iikely that both ehernoattraetants ~ontribute to the influx ofmonocytes and neutrophils into the pulmonary alveoius and interstit~um in these dlsea~es. we have recently shown that the combined administration of noninjurious doses of lps and paf in the rat produce ards-like lung injury characterized by neutrophil adhesion to lung capillary venules, neutrophil accumulation in lung parenchyma, pulmonary edema, and increased protein and neutrophil count in bal fluid. this new paradigm of lung injury was associated with elevated serum tnfc~ and pretreatment with anti tnfa mab dose-dependently prevented these responses. also, the combined administration of lps and paf induced lung mrna levels of tnfe~ ( fold vs. lps or paf alone), ll-lg ( fold), kc ( fold) and il- . taken together, these data suggest that this new paradigm of lung injury is cytokinemediated and that lps/paf in vivo can functionally couple to the activation of gone expression of a multi-cytokine network system, all of which may be involved in the pathogenesis of ards. materials and methods. the sheep model included hemorrhagic shock and closed femoral nailing at day , hourly injections of e. coli endotoxin and zymosan-activated autologous plasma at clays - and further observation and measurements at days - . from venous blood and bronchoalveolar lavage(bal)fluid of ten merino sheep (mean weight kg) neutrophil counts ( e pmn/ml blood or epithelial lining fluid-elf-), the elf/ plasma ratio of albumin (r), and the zymosan-induced (stim) and non-induced (spont) chemiluminescence response (cl) of blood ( e cpm/ , pmn), and of blood-and bal-isolated pmn ( e cpm/ , pmn) were measured. for statistical calculations the wilcoxon test was used. data of the changes in polymorphonucleur leukocyte (pivinl) metabolism have been suggested to play a pivotal part in the post-traumatic systemic inflammatory response syndrome. the underlying cellular mechanisms which control this response are not yet completely understood. since the 'ca + second messenger'-system has been shown to be involved in regulation of pmnl-'respiratory burst', we investigated changes in pmnl-ca z÷ regulation in relation to oxygen free radical mediated injury. methods. in polytranmatized patients (mean injury severity score = ) arterial and venous blood samples during days. daily evaluation of horowitz-quotiant (po /fio ), plasma lactate (mg/dl) and body temperature ( results. body temperature peaked at day and (day : +. ; day : . +. ). plasma lactate was significantly increased at day l ( + ) and day ( . + ). hurowitz-quotient (day : + ) was low at day ( + ) and day to ( + )(p<. ). at day a substantial rise in venous pmnl-superoxide production (day : . +_. , day : . +. , day : . +_. ), oecured with significant increase in plasma lipid peroxidation (day : . + . ; day : . + . ). pivin~-myeloperoxidase activity was high at day ( . +--. ) and then continuously declined (day : . +. ). plasma antiexidant activity (glutathione pemxidase) was reduced by % at day (day : . +. ; day : . +_. ; day : . +. ). whereas basal ca + concentration remained unchanged (day : +_ , day : +_ ), fmlp-stimulated cytosolic ca + mobilization increased at day (day : + , day : , day : + ). conclusion. the present study in polytraumatized patients shows, that seven days after injury the agonist-induced pmnl ca + mobilization is significantly enhanced. at the same time, pmnl-oxygen free radical release and phagocytotic activity, systemic fever response and lactate concentrations were maximal. these observations were accompanied by post-tranmatic respiratory failure and in some patients by clinical signs of multiple organ failure. preliminary data from an ongoing study using hes-and dextran-infusions in these patients show attenuation of this inflammatory response. stefan rose, m.d., trauma surgery, univ. of saarland, homburg/saar donnelly sc, haslett c, dransfield i, robertson ce, grant is, carter c, ross ja, tedder tf. dept's of respiratory medicine, accident & emergency, intensive care, surgery, university of edinburgh, scotland and dept. tumor immunology, dana farber cancer institute, boston. the selectins are a family of adhesion molecules (l-selectin, e-selectin, pselectin), all of whom are implicated in inflammatory cell transendothelial migration. they, as a family can be proteolytieally cleaved from their parent cell and exist in a soluble form within the circulation. ards is a disease state in whic neutrophils and neutrophil transendotheliat migration have been implicated. in this study we wished to investigate whether the levels of these circulating soluble receptors from patients at-risk of ards at initial hospital presentation, correlated with subsequent ards progression. eighty-two patients were enrolled (pancreatitis (n= ), perforated bowel (n= ), and multiple trauma (n= )), of whom progressed to ards. assays for soluble l,p & e-selectin were performed on collected plasma samples via a sandwich elisa. (ns = not significant, **** = p<o. ) we have found a highly significant inverse correlation between a low circulating soluble l-selectin (and not soluble e & p-selectin) and subsequent ards. these results further our understanding of neutrophilendothelial interactions in the early stages of ards pathogenesis and offer the promise of sl-selectin in combination with other markers of inflammatory events being used to identify individuals at particularly high risk of ards progression on initial hospital presentation. it has been suggested that polymorphonuclear leukocytes aggregate in the lung capillaries of patients developing the adult respiratory distress syndrome (ards) and account for the endothelial damage while releasing toxic metabo-iites such as o.a. free oxygen radicals. among many antioxidants which have been investigated in in vitro systems and in animal models, n-acetylcysteine (nac) has been established to be useful as a free radical scavenger in vivo. to assess the influence of n-acetylcysteine (nac) on the activation of neutrophils in patients undergoing cardiopulmonary bypass (cpb) surgery with extracorporeal circulation (ecc), we evaluated the plasma levels of elastase and myeloperoxidase (mpo) throughout the operation and up to hours after surgery. four hours after surgery also a bronchoalveolar lavage was performed. a spectrophotometric method was used for the detection of plasma elastase activity and mpo levels were measured using a radioimmunoassay. we found that pretreatment with intravenous nac prevented the increase in elastase activity ( _+ pmol/i vs _+ ; p - . ), but not the release of neutrophil related mediators ( . _+ . ng/i vs . _+ . ; p= . ). besides, nac had the capability to prevent the enhancement of neutrophil numbers in lavage fluid as is normally seen during cpb ( . + . % vs . _+ . ; p = . ). although not significant, nac prevented also the increase in elastase levels in lavage fluid ( . + . pmol/i vs . + . ; p = . ). it is concluded that nac is able to protect against the inactivation of a -proteinase inhibitor, the main inhibitor of elastase activity, and that nac interferes with adhesion and migration of neutrophils. therefore nac may be useful in the prevention of tissue damage. this study is supported by inpharzam belgium. sodium nitroprnsside (snp) has previously been shown to attenuate the neutrophil induced lung injury associated with limb ischaemia and repeffusion. glyceryl trinitrate (gtn), already widely used in aortic surgery, also increases nitric oxide but has a less marked splanchnic "steal" effect. we examined the effect of gtn on lung injury, neutrophil infiltration and activation in a model of aortic occlusion ( rain) and repeffusion ( min). sprague dawley rats were randomized into: control (n=i ); ischaemia repeffusion (ir) (n= ); and ir treated with gtn ( ug/kg/min) during repeffusion (n= ). myeloperoxidase activity (mpo) measured pulmonary neutrophil influx. pulmonary endothelial permeability was measured by wet to dry weight ratio (w/d), broncboalveolar lavage protein (bal) and neutrophil counts (bal pmn). neutrophil superoxide release (mean channel fluorescence mcf) was measured by flow cytometry in a further ir v gtn experiment (n= per group). control _+t "* _+ _+ ** bal pmn/mm l+- " + # _~ *p< . , #p< . v control/gtn;**p< . v ir. students t test the significant increase in mpo activity produced by ir was attenuated by gtn. the increase in pulmonary microvascular leakage after reperfusion was also prevented by gtn. neutrophil superoxide release increased significantly from . + . to . _+ . mcf after minutes repeffusion (p<. ). this increase in superoxide was prevented in the gtn treated group. these results indicate that gtn inhibits neutropbil superoxide release during limb reperfusion, thus preventing pulmonary vascular leakage and neutrophil infiltration. these data suggest that the beneficial effects of gtn in aortic surgery may be due in part to a protective effect onpulmonary microvasculature. department of surgery, beaumont hospital, dublin , ireland. lipton adult respiratory distress syndrome (ards) is marked by increased vascular permeability of the lung to white blood ceils (wbc). indeed, influx of wbc into the lung is believed to be the central mechanism in acute lung injury of ards. evidence is developing that the neuropeptide c~-melanocyte stimulating hormone (c~-msh), a proopiomelanocortin derivative, inhibits inflammatory reactions in animai models of inflammatory responses in humans. to test the influence of a-msh on experimental ards, the peptide was administered to rats after intratraeheal infusion of endotoxin (s. typhosa, #g in . ml saline). three groups (n= each) received: intratracheai infusion of endotoxin plus ip injections of o~-msh ( ~tg in . ml saline) at , , and hr post-endotoxin treatment; intratracheal endotoxin infusion plus saline injections; control intratracheal saline infusion plus saline injections. the bal data showed overall differences among groups (f , = . , p<. ), o~-msh treatment inhibited endotoxin-induced wbc migration into the pulmonary tree (p<. ). circulating wbc counts were markedly lower in both groups given endoroxin than in the control group (p<. ), but there was no difference in wbc count between these two endotoxin-treated groups. the results indicate that c~-msh can reduce wbc migration in experimental lung injury. in further experiments we tested the hypothesis that c~-msh, administered alone or in combination with an inhibitor of gram-negative bacterial growth, increases survival in a model of peritonitis/endotoxemia/septie shock. cecal ligation and puncture were performed under sodium pemobarbital anesthesia ( mg/kg, i.p.) in female balb/c mice ( - g) that were then randomly assigned ( - mice per group) to receive at time and hr later: control saline thjectioas (i.p.), injections of ~-msh ( ~tg), gentamicin sulfate (i mg/kg), or both ~-msh and gentamicin. one ml of normal saline was given s.c. to each animal for fluid replacement. both ix-msh and gentamicin treatments administered singly improved survival; when given in combination survival was even greater these findings suggest that the anticytokine ot -msh molecule might be useful for treatment of systemic inflammation, perhaps particularly when used in combination with agents that inhibit bacterial growth. previous studies showed a close relationship between xanthine oxidase activation, membrane damage and postischemic cardio-respiratory failure following aortic clamping and reperfusion. aim of the present study was to evaluate alterations of polymorphonuclear leukocyte (pmnl)-metabolism and signal lransduction systems during ischemia/reperfusion induced by aortic clamping in man. methods, in patients ( female, + years) with elective reconstruction of infrarenal aortic anearysms, arterial (a) and venous (v) blood samples were obtained before clamping and declamping, and min after declamping. arterial pla concentrations were higher than venous ( + vs. + ). while cytosolic basal pmnl ca + concenlxation was not altered at the end of ischemia (v: . + ; a: _+ ) and during reperfusion (v: + ; a: + ), fmlp-stimulated cytosolic ca + mobilization showed a significant arterial increase as compared to venous ( _+ vs. + , p < . ). these presented data indicate that the reperfusion syndrome after infrarenal aortic clamping is characterized by ) pulmonary membrane damage possibly due to pmnl-degranulation, ) activation of pmnl-superoxide radical production with release of activated pmnl in arterial circulation, and ) a significant arterial increase of pmnl cytosolic ca + mobilization after fmlp-stimulation parallel to pmnl-activation. in conclusion, ischemia/reperfusion in man induces pulmonary damage and activation of pmnl superoxide production possibly due to an altered pmnl-ca + messenger system by inflammatory mediators (e,g. ii- , tnf, paf). the septic lung diseases (i.e. ards) generally show distinct alveolar damage and surfactant disturbances. it is thought that alveolar macrophages are involved in specific release products like h and cytokines like tnf. in this pathway the type ii alveolar epithelial cell (p cell) is not only an important target, but as recently shown it is also capable of producing h . the aim of this study was to investigate the influence of endotoxin on h release of p cells. we obtained p cells from lungs of female wistar rats and investigated the effect of lipopolysaccharid (lps) on h release of fresh isolated cells and on cells cultured for days in a fibronectin matrix. furthermore we conditioned isolated alveolar macrophages for hours with p.g/ml lps and tested the conditioned medium (mcm) on cultured p cells. p cells h ex vivo were _> % pure, _> % vital and had an basal h release of . _+ . nmol h per hour and million cells. adding p.g/ml lps to the incubation medium the h release decreases slightly but significantly to . _+ . nmol. adding . p.g/ml phorbol myristate acetate (pma) to the basal incubation medium the h release increased -fold to . _+ nmol. pma induced h release decreased to . + . nmol after addition of p.g/ml lps. after culture days the p cells were _> % pure and showed a pma inducible h release of . _+ . nmol addition of p.g/ml lps had the inverse effect as on freshly isolated cells as it increased the h release up to . _+ . nmol. addition of mcm to cultured p cells increases pma-stimulated h release to . +_ . nmol. the release decreased to . _+ . nmol when an murine anti-tnf-alpha antibody was added. vitality of cultured cells was > % in all experiments. the results show that lps has an direct effect on p cells cultured on fibronectin. we conclude that the observed additional stimulatory effects of mcm seems to depend on tnf-alpha. the induction of h release of p cells could be important for generating internal oxidative stress in p cells before external oxygen radicals exceed. the produced h did not necessarily damage p ceils, but it can effect surfactant metabolism, especially when extracellular h release of alveolar macrophages following an immune response is increasing. introduction: primary stabilization of femoral shaft fractures in patients with multiple trauma is beneficial. however, in patients with associated lung contusion we have found an increased incidence of ards, apparently associated with primary reamed femnral nailing (rfn). previous animal studies revealed, that perioperative disturbances of lung ftmetion appear to be related to the reaming procedure, ix~ssibly due to pulmonary embolizafion of bone marrow fat. in a prospective clinical analysis we compared effects of intrameduuary nailing with and withont reaming on parameters known to be related to ards-pathoganesis. in order to gain further insight into the role of endotoxin and cytokines in the pathogenesis of the adult respiratory distress syndrome (ards), we enrolled patients with severe lung injury after sepsis ( ) or polytrauma ( ) and obtained multiple blood samples ( days) for endotoxin, tumor necrosis factor e (tnfa), interleukin (il- ) and interleukin (il- ) determination. to evaluate the cytokine releasing capacity of the blood, plasma concentrations of tnfe, il-l and il- were also determined after the "in vitro" stimulation of the whole blood samples with lipopolysaccharide (lps, . ng/ml) for hours at c (stimulated values). the difference among stimulated cytokines levels and the basal plasma concentrations were defined as "delta values", an expression of the cytokine releasing capacity of the blood. the pao /fiao quotient was used as an index of the severity of lung injury (sli). the endotoxin plasma level was significantly higher in patients with sli < ( . ± . eu/ml, mean values ± sem) versus the patients with a sli > ( . ± . eu/ml, p<o. ). the basal plasma concentration of ll- also correlates with the sli index (p<o.o ). the delta tnfe and delta il- values were s~gnificantly decreased in patients with a severe lung injury. compared with the survivors (n: ), the deceased patients (n= ) showed higher endotoxin level, a reduced tnfa and il- releasing capacity of the blood and higher basal il- levels. we can conclude that endotoxemia, high il- plasma level and a decreased capacity of the blood to release tnfa an il- under endotoxin stimulation associates with the severity of lung injury. [objectives] experimental and clinical findings implicate the involvement of inflammatory cytokines in the pathogenesis of the decreased oxygenation in the lung after major surgery, though the mechanism remains unclear. in the present study, we elucidated the involvement of il- in the pathogenesis of lung injury defined by deterioration of pulmonary oxygenation after esophagectomy. [materials and methods] seventeen patients underwent subtotal esophagectomy through a right thoracotomy. in all patients, tbe alveolar-arterial oxygen tension difference (aado ) was measured everyday and bronchoalveolar lavage fluid (balf) samples were obtained from a single segment ($ or $ ) of the right and left lung through a bronchoscope on days l, , and days after surgery. they were then examined for cell analysis, measurement of cytokines by elisa, and measurement of neutrnphil elastase (ne) by elisa. [results] aado unexceptionally deteriorated postoperatively and the mean aado in patients reached the maximum (mean ± sem; _+ mmhg) on the rd postoperative day. the mean concentrations of il- , ne and neutrophil count in balf also increased postoperatively and reached their maximum level ( + pg/ml, -+ gg/l, and ± x cells/ml, respectively) on the rd postoperative day. these increases in balf were recognized in both the right and left lung. a highly significant correlation was observed between il- and ne levels in balf (r= . , p= . ). also, significant correlations between aado and the neutropbil count, and the concentration of ne or l- were observed. in contrast to il- , neither il- , il- nor tnfa was detected in balf. [conclusion] major surgical trauma such as esophagectomy may induce recruitment of neutrophils to the lung and cause lung injury. il- increased in the lung is an important mediator of neutrophil recruitment and activation in the lung. phorbol myristate acetate (pma) is commonly used to cause edema and other tissue damages in the lung. lung injuries induced by the administration of pma has been shown to be mediated mainly by neutrophils. neutrophils recruited to the lower respiratory tract may damage lung tissues by releasing reactive oxygen species, neutral proteases and lysosomal enzymes. the present study was conducted to investigate whether c~tocopherol, entrapped in dipalmitoylphosphatidylcholine liposomes and delivered directly to the lung, could counteract some of the pma-induced lung injuries. plain liposomes or c~tocopherol-containing liposomes ( mg c~-tocopherol/kgbody wt.) were instilled into the lungs of rats h prior to pma exposure ( pg/kg, intratracheally) and treated rats were killed h later. lungs of control animals exposed to pma developed increases in lung weight and lipid peroxidation as well as decreases in lung angiotensin converting enzyme (ace) and alkaline phosphatase (akp) activities. pma treatment also caused an increase in myeloperoxidase (mpo) activity in the lung, suggestive of neutrophi] infiltration. pretreatment of pma-treated rats with plain liposomes had no effect on pma-induced injuries. in contrast, pretreatment of rats with liposomal c~-tocopherol, h prior to pma administration, resulted in a significant elevation of pulmonary a-tocopherol concentration, accompanied by a concomitant reduction in mpo activity and reversal of pmainduced changes in lung edema, lipid peroxidation, ace and akp activities. these results appear to demonstrate that the intratracheal administration of a liposome-associated lipophilic antioxidant, such as a-tocopherol, can significantly ameliorate the toxic effects of reactive oxygen species, released from pmastimulated pulmonary target cells and infiltrating neutrophils. jk wojcik, g palasiewicz, w roszkowski immunology department,institute of tuberculosis and lung diseases~ warszawa, poland, m~larhospital, eskilstuna, sweden. introduction:the critical point in neutrophil migration to the alveolar space in the course of ards is the attachment between giyeoproteins of neotrophil membranes(sialyl lewis x carbohydrate epitope containing u-l-fucose) and lectin domains of endothelial p and e selectins (gmp- ,elam-i). a potential beneficial therapeutic strategy may be designep to suppress neutrophil recruitment to the lungs by preventing their rolling and attachment to the pulmonary endothelial cells. objeclive:to investigate if tz-l-fueose prevent experiments pulmonary hypertension in ards as effective as metylpredniselone. methods:only healthy rabbits (n- ) weighing .d- . kg oaselinepao> kpa and mean pulmonary arterial pressure (mpap)<i. kpa were included in this study. anaesthesia was induced with thiopental - mg/kg bw and "blind" jntubation was performed. a f swan ganz catheter was introduced via the left jugular vein into the pulmonary artery. pma, as was used in our experiments activates neutrophils and produces acute respiratory failure with pulmonary hypertension and pulmonary edema. eight rabbits serving as control animals received pma ug/kg bw iv only. sixteen other animals were divided into two groups receiving either ~-l-fucose mg iv or metylprednisolone mg iv min before pma admininstration. results:after pma administration notable physiological changes including marked increase in mpap( . -+o.~ kpe ..lere found. the increase of npap was observed to be completely blocked in both groups of animals which received ~-l-fucose or metylprednisolone. conclusion:in aur rabbit model ards cz-l-fucose pretreatment prevents pulmonary hypertension after pma administration. the effect is comparable with high dose metylprednisolone. the possible explanation is that g-l-fueose molecules block iectin domains of gnp- or elam-i preventing the adhesion of the neutrophils to the pulmonary endotheliom. bartens c, elmadfa i, steltzer h, fischer m, druml w introduction: various micronutrients and vitamins are particulary effective in modulating immune functions and host defense. the nutritive ant±oxidants vitamin c, e and b-carotene, as well as selenium lower the burden of reactive free radicals and protect the structural integrity of cells and tissues of the immune system, as well as other systems in the body. certain cells of the immune system like polymorphonuclear leucocytes (pmn's) use free radicals as a weapon to destroy invading pathogens. these reactive molecules, when released into the surrounding area, mediate lipid peroxidation and tissue injury. there is growing evidence that pmn's are activated in vivo by complement or immune complexes in disorders such as ards. the respiratory host defense mechanisms of patients with adult respiratory distress syndrome (ards) may be defective. the aim of this study was ta evaluate the status of the free radical scavengers and their activity in ards and to investigate the respiratory burst of ards patients. methods: seven ards patients ( sepsis, trauma) with an fie of . + .( , a peep of . ± . , and a murray score of . ± . were included in this study. healthy adults served as controls. superoxide anion production in granulocytes was measured photometrically, and the hydrogen peroxides by fluorimetric assay. vitamin a, e, and g-carotene concentrations were assessed by hplc, and plasma vitamin c photometrically. plasma selenium was determined by dectrothermal aas. plasma lipid peroxidation pruducts, expressed as malondialdehyde (mda), were determined by thiobarbituric acid assay and hplc. results: mda-plasma (/xmol/l) . ~: . * . ± . *= < . , **=p< . condusion: ards patients showed significantly decreased plasma concentrations of vitamin c, e and g-carotene, as well as selenium. these nutritive ant±oxidants are consumed during increased oxidative stress. mda, a final product of lipid peroxidation, is increased. however, a difference in rates of release of hydrogen peroxides and superoxide-anion of pmn could not be detected. therefore, oxygen radical accumulation is more likely a result of excessive inspiratory oxygen concentrations (f.io ), pro-oxidant drugs, and a high settlement of pmn in the lungs, and not an increased release of free radicals of the single pmn. kd glycoprotein secreted by the alveolar type ii cells. recent study showed that sp-a exists in the sera from normal healthy adults and that the significantly elevated serum sp-a levels were observed in the patients with interstitial pneumonia and pulmonary alveolar proteinosis. these findings means there may be a mechanism that the sp-a produced in the alveoli escapes into the bloodstream. here we examined the serum sp-a levels in the patients with critical illness including sepsis and ards. ]clinical subjects & methods] a total of serum samples were collected from patients hospitalized in the division of critical care medicine(icu), sappom medical college hospital. serum sp-a levels were measured by an enzyme-linked immunosorbent assay using monoclonal antibodies to human sp-a. ]results] there was no significant difference between the septic (n= ) and non-septic (n= ) patients. patients with ards (n= , . + . ng/ml) and with respiratory disease other than ards (n= , . +- . ng/ml) showed significantly higher levels of serum sp-a than those with non-respiratory disease (n= , . _+ . ng/ml). as to the ards patients, it was likely that the serum sp-a levels were getting higher in their clinical courses. however, there was no significant correlation between the serum sp-a levels and the pa%/fio ratio. ]discussion] at the present time, the exact mechanism of the escape of sp-a into the bloodstream remain to be unclear. our results may suggested that there was some relation between this leakage of sp-a and the alveolar-capillary permeability because of the higher sp-a levels observed in the ards patients. however it is also likely that higher serum sp-a level represents the proliferation of alveolar type ii cells as the regeneration of damaged lung. further studies are now being done. neutrophils contain a number of enzymes within intracellular granules,potentially damagingto lung tissue.release of these enzymes into the lung tissue from the sequestred activated neutrophils is felt to play significant role in lung injury in the course of aros. using -hours acute animal model of aros the activity of cathepsins,beta-glucuronidase and acid phosphatase was determined in the lung tissue. results of this research were compared with neutrophil proteases activity in serum and broncho-alveolar lavage.also hemodynamic,respiratory and biochemical investigations were performed before beginning of experiment,and in a two-hours time intervals of its duration.after hours was determined total and free activity of cathepsins,beta-glucuronidase and acid phosphatase in full homogenate of the lung tissue,mitochondriallysosomal fraction and the final supernatant. in the course of our experiment we observed an increase ( .i- . %) both total and free activity of neutrophil proteases in all fractions of the lung tissue.generally accepted current pharmacological treatment of aros only attenuated this increase but never caused reversion to the level before beginning of our investigations. the presence of aros was histologically proved.an activity of acid phosphatase in serum and the lung tissue fractions was compared with histochemical pictures of the lung. results of our research indicate that neutrophil proteases are a very important mediators in aros and they are a cause of the lung injury. in addition neutrophil-derived proteases can amplify the inflamatory process by enzymatitally activating of many other mediators and they also may to increase an oxidant induced injury by imparing of antioxidant defenses. oepartment of general surgery, sk~odowskiej a - ~ia~ystok, poland, tel/fax + . in vitro studies demonstrated that paf activates polymorplionuclear leukocyte (pmn) - ipoxygenase, but the generation of leukotrienes (lts) is critically dependent on exogenous arachidunic acid (aa) disposal (f. grimminger et at., mol. pharmacol. : , ) . we investigated the features of paf-evoked lt synthesis in a model of pulmonary microvascular leukostasis, in the presence and absence of intravascular n- -and n- -prescursor fatty acid supply. human neutrophils were infused into isolated, ventilated and perfused rabbit lungs to achieve microvascular sequestration of ligand-responsive leukecytes. leukotrienes (lt) and hydroxyeicosatetra(penta)enoic acids (het(p)e) were quantified by itplc techniques. intravascular application of paf ( um) or arachidonic acid (aa;ioum) provoked the generation of limited quantities of -series lts and -hete (total sum of - ipoxygenade products rd. and rd. pmol/ml.; both in pmn-preloaded and non-preloaded lungs). combined administration of pat r and aa caused amplification of - ipoxygenase product formation with predominance of cysteinyl-lt synthesis both in lungs without (total sum rd. pmol/ml) and, much more strikingly, with pro-application of neutrophils (total sum rd. pmofml). eicosapentaeooic acid ( um) elicited exclusive generation of -series lts and -hepe (total sum rd. pmol/mi). dual stimulation with paf and epa provoked amplification of epa-derived - ipoxygenase product formation, again with predominance of cysteinyl-lts, in lungs without (total sum rd. pmul/ml) and in particular in those with preceding microvascular pmn entrapment (total sum rd. pmol/ml). we conclude that the paf-evoked - ipoxygenase product formation in the neutrophil-harbouring lung capillary bed is critically dependent on intravascular precursor fatty acid supply, with epa representing the preferred substrate as compared to aa. pmn-related transcellular eicosanoid synthesis is suggested to underly the predominant generation of cysteinyl-lts. these findings may be relevant for lipid mediator profiles provoked by infusion of n- -versus n- enriched lipid emulsions in diseases with pmn-related inflammators events. adult respiratory distress syndrome (ards) involves damage to the alveolar epithelium and microvascular endothelium. products released from neutrophils (pmn) are thought to be among the chief causes of this damage, while the role of mononuclear cells (mnc) is uncertain. we studied patients at risk of or with acute ards. we measured the concentration of myeloperoxidase (mpo) and elastase (el) within circulating pmn as an index of pmn activation, elastin degradation products (edp) in the plasma as an index of tissue damage, as well as the cytotoxici~y of pmn and mnc to endothelial cells (ec) in vitro. cells and plasma were prepared from venous blood of healthy controls; or systemic arterial blood of patients on ventilators in intensive care but not at risk of ards; at risk of ards because of sepsis; at risk because of multiple transfusion/major trauma; or with acute ards. lung injury, multi-organ failure and apache h scores were recorded. to measure cytotoxicity, human umbilical vein ec were labelled with cr- , incubated with either pmn or mnc for hours, and the supernatant counted. the adherence of the pmn and the mnc to the ec was also measured. standard assays were used to measure mpo and el in the pmn, mad edp in the plasma. the mnc cytotoxicity did not differ between the groups. the pmn cytotoxicity was higher in the transfusion/trauma patients compared to each other group, which did not differ from each other. there was no correlation between cytotoxicity and adherence for any group. the mpo and the el concentrations were significantly decreased in the pmn from each of the patient groups, including the patient controls, compared to healthy controls. the edp were only increased in patients at risk of or with ards. thus pmn degrannlation occurred in patients not at risk of ards, but tissue damage as measured by increased edp only occurred in patients with or at risk of ards, suggesting that factors additional to pmn activation and degranulation are required for the progression to ards. there was no correlation between the parameters measured and the indices of disease activity, or outcome. these measures of pmn and mnc activation and function are not of value as prognostic indicators in patients with or at risk of ards. the liver is made up of several different cell types which subserve distinct functions in vivo. in addition to the different functions of the distinct cell types, it is now evident that even within the population of hepatocytes substantial functional heterogeneity exists. this heterogeneity occurs in a very organized fashion in the normal liver based upon the position of the hepatocyte in the acinus. although it has been proposed that the acinar heterogeneity of hepatocytes arises from migration of immature hepatocytes from the periportal region to the perivenous region where they become senescent and die, many of the heterogeneous responses can be accutely reversed by reversal of the oxygen gradient via retrograde perfusion. under normal conditions in which the acinus is exposed to unidirectional flow, the periportal hepatocytes are exposed to relatively high levels of oxygen, substrates and hormones. as these substances are extracted along the aeinus their concentration decreases leaving relatively low concentrations for the perivenous hepatocytes. in the normal liver, the heterogeneity of response is attenuated by extensive communication via gap junctions. the major liver gap junctions are made up of hexamers of connexin (cx ) which forms channels between cells capable of pas.~ing any molecule smaller than d. coupling is sufficient to allow diffusion of molecules such as atp or camp from one hepatocyte into > adjacent hepatocytes within seconds. during stress conditions such as endotoxemia or zymozan inflammation, expression of cx is markedly decreased while the secondary gap junction protein cx is either unchanged or even increased. while cx readily effects electrical coupling, molecules > d pass only very slowly. this would result in restriciton of transmission of moecules the size of atp or camp. since inhibition of gap junctions also attentuates metabolic response to hormone or nerve stimulation, it is evident that modulation of hepatocyte hetereogeneity by gap junctions must be considered in determining the mechanisms of metabolic alterations during stress. already minor haemorrhage decreases portal venous blood supply to the fiver and the reduction in portal blood flow becomes more pronounced with more profound btood loss. severe hacmorrhagic hypovolemia also reduces hepatic arterial blood supply which, however, is maintained over a vide range of haemorthage. the net effect of blood loss is a reduction in liver oxygee supply and this reduction is in proportion to the vulume iossed. however, oxygen supply to the liver exceeds the demands of the normal liver and this is the ca~ stilt following reduction of % of blood volume. the situation in sepsis is more complicated. po~l venous supply to the liver is redur.~i fairly early following normovolemic sepsis while hepatic arterial blood supply is maintained at le,~t initialiy, oxygen saturation might be maintained in arterial blood but may also be slightly reduced during sepsis, oxygen saturation of portal venous blood is significantly reduced during sepsis due to increased extraction of the intestines. therefore oxygea delivery to the liver during sepsis becomes sigalfkzntly reduced. at the s,~ne time and for mai.v.ly unknown reasons the need for oxygen becomes significantly increased in the ~-~ptic liver. as a consequence liver oxygen consumption becomes flow dependent and the liver is likely to suffer from ischemia during septic conditions. $ although liver failure is well recognized in sepsis, it is generally thought to be a late complication following pulmonary and renal failure. jaundice, hypoglycemia, encephalopathy and bleeding secondary to low levels of liver-synthesizing clotting factors are, however, signs of rather severe end-stage hepatic failure. furthermore, elevated liver enzymes (sgot and sgpt) represent hepatucyte damage and not hepatocellular dysfunction. in view of this, a more sensitive indicator of hepatic function is desirable in order to detect early hepatic abnormality. in this respect, indocyanine green (icg) is a tricarbocyanine dye that possesses several properties which makes it particularly valuable inthe assessment ofhepatic function. this dye is bound m albumin and is cleared exclusively by the liver through an energydependent membrane transport process and is nontoxic at lower doses. we propose that maximal velocity (vm~,) of icg clearance is a valuable measure of active hepatocellular function, since the total concentration of functioning receptors is directly proportional to vm~. we have utilized a fiber optic catheter and an in vivo hemoreflectometar to continuously measure the administered icg in vivo and consequently determine its clearance without the need of blood sampling. using this technique, we have found that in the early stages of sepsis (i.e., and h following cecal ligation and puncture), the vm~ and kinetic constant (k=) of icg clearance was significantly depressed. it should be noted that at this stage of sepsis, there was no elevation in serum enzyme levels. furthermore, hepatic blood flow and cardiac output increased at the above mentioned time points. thus, the extremely early depression in active hepatocellular function in sepsis, despite the increased hepatic blood flow and cardiac output, may form the basis for cellular dysfunctions leading to multiple organ failure during sepsis. additional studies indicated that following hemorrhage, active hepatocellular function was markedly depressed. this returned to prehemorrhage levels after ringers lactate resuscitation, however, this function was not maintained and decreased significantly after fluid resuscitation. nevertheless, the depressed active hepatocelinlar function following hemorrhage was markedly improved by post-treatment of animals with either atp-mgci , peutoxifylline or diltiazem. thus, the use of icg clearance provides an early sensitive indicator of hepatic abnormality during sepsis and following hemorrhage and this method should be used, not only experimentally, but also in the clinical arena for the early detection of hepatocellular abnormality. although multiple organ dysfunction syndrome (mods) remains a major cause of mortality and morbidity in intensive care units, very little is known about the mechanisms that precipitate its development. since an episode of inadequate tissue oxygenation is considered to be the trigger for mods, we have proposed that a primary localized injury such as ischemia/reperfusion may be sufficient to cause a change of gene expression of remote and apparently unaffected organs. such modulation of remote organ gene expression may decrease the organ's tolerance to a subsequent stress contributing to the development of mofs. to test this hypothesis, rats were subjected to hepatic regional ischemia by clamping the blood flow (hepatic artery and portal venous inflow) of the left and median liver lobes. intestinal congestion was prevented by allowing flow through the smaller right and caudate lobes. after minutes of ischemia, the clamp was removed and the blood flow restored. the animals were allowed to recover for , and hours. kidneys were removed, total rna was isolated and poly(a) ÷ selected by affinity chromatography on oligo(dt) columns. message was in vitro translated using rabbit reticulocyte iysates in the presence of radioactive amino acids. the gene products (radiolabeled polypeptides) were fractionated by two dimensional gel electrophoresis, and visualized by fluorography. analyses of the two dimensional fluorograms indicate that there is a dramatic change in the electrophoretic pattern of in vitro translated products in samples corresponding to kidneys obtained after minutes of hepatic ischemia and hours of reperfusion with respect to kidney samples obtained after sham operation or from control rats. the latter were not subjected to any surgical manipulation. these studies suggest that the gene expression of the kidneys is specifically modified after a remote organ injury (hepatic ischemia/reperfusion). we speculate that this change of gene expression in kidneys after an indirect injury may be part of the early events leading to the development of mods. a priming event, e.g. local ischemia, in combination with a second insult, e.g. sepsis, may amplify a host's response and lead to multiple organ failure. to better understand the mechanisms involved in the pathophysiology, male fischer rats were subjected to min of hepatic ischemia followed by reperfusion (rp) and injection of . mg/kg salmonella enteritidis endotoxin (et) at min of rp. et injection potentiated the postischemic liver injury as indicated by histopathology and an increase of plasma alt activities from + u/l (i/rp only) to + u/l at h rp. inhibition of kupffer cells (kc) with gadolinium chloride ( mg/kg) attenuated liver injury in this model by %, however, monoclonal antibodies (cl , wt ) directed against adhesion molecules ( integrins, cd ) on neutrophils had no effect on the injury despite the substantial accumulation of neutrophils in the liver at that time ( + pmns/ hpf; baseline: + ). isolation of kc and neutrophils from the postischemic liver indicated a -fold increase of the spontaneous superoxide formation only in the kc fractions [ . + . nmol o -/h/ %elts (kc ); . _+ . (kca) ] at h rp compared to control cells. in addition, stimulation with phorbol ester or opsonized zymosan revealed a substantial priming of kc for reactive oxygen formation. in contrast to the short-term experiments ( h), the antibody wt ( mg/kg) attenuated liver injury by % at h of rp and improved survival. conclusion: liver injury during the early rp phase is mediated mainly by kc generating excessive amounts of reactive oxygen while neutrophils are primarily responsible for organ damage during the later rp period. (es- and gm- ) tumor necrosis factors (tnf) are cytokines which are cytotoxic towards some tumors in vivo and certain tumor lines in vitro. moreover, these polypeptides are powerful immunomodulators and have been found to be distal mediators in several models of septic shock and septic organ failure. one of the best-characterized experimental systems is the hepatitis caused by lps or tnf in galactosamine (galn)-sensitized mice. here we describe a cell culture system, in which the direct toxicity of tnf towards mouse hepatocytes was examined. the toxicity of tnf, as determined by ldh-release or formazan-formation, was dose-and time-dependent. the threshold of toxicity was ng/ml, which corresponds to serum concentrations found in mice after lpsinjection. toxicity was only observed in hepatocytes sensitized with transcriptional inhibiters such as galn, actinomycin d (actd) or cxamanitin. sensitization was neither observed with different translational inhibitors nor with various other metabolic inlaibitors or toxins. inhibitors of protein synthesis or protein processing such as cycloheximide, puromycin, tunicamycin and ricin protected actdsensitized hepatocytes from tnf-induced cytotoxicity. tnf induced apoptotic changes and dna-fragmentation in sensitized hepatocytes which is in line with the above findings that cell death is dependent on protein synthesis. thus tnf may be a trigger of programmed cell death during inflammatory organ damage. with the purpose of studying the role of complement activation in tissue injury after ischaemia and reperfusion we blocked the complement cascade in a model of rat liver isehaemia and reperfusion, either by administration of soluble human complement receptor type (scri), mg/kg iv after vascular occlusion (n= ) or by depleting the complement system using cobra venom factor (cvf), . mg im, and hours before ischaemia (n= ). non-ischaemic rats (n= ) and ischaemic non-treated rats (n= ) were used as controls. the experimental procedure consists of the temporary interruption of arterial and portal blood flow to the left lateral and medial lobes of the liver during minutes, followed by reperfusion, recording the liver blood flow and haemoglobin saturation with a laser doppler flowmeter and photometer during one hour after declamping; alt levels were assayed and immunoperoxidase stainings for c and c were performed. there were statistically significant differences between the experimental ~roups and the untreated ischaemic control group in terms of post-isehaemic blood flow (p< . ) and haemoglobin saturation (p< . ). c and c were present in the endothelium of the ischaemic control group. no deposits of c or c were found in the cvf group. few c and no c were found in scri treated rats. these results show that the effect of reperfusion injury in the rat liver is ameliorated either by depleting complement with cvf or by regulating complement activation with scri. hepatic dysfunction, a major cause of mortality following hemorrhagic shock, has not yet been well characterized. the present study was designed to assess the effects of liver blood flow and cytokine levels on hepatic function following resuscitation from severe hemorrhagic shock in normal and cin-hotic rats. methods: aftor pentobarbltal anesthesia, control and cirrhotic sprague-dawley rats were subjected to severe hemorrhage to reduce their systolic blood pressure to + mm hg. this level of hypotension was maintained until the skeletal muscle transmembrane potential (era) depolarized by %.; the animals were then resuscitated with ringer's lactate solution in three times the volume of the shed blood. serial blood samples for tumor necrosis factor (tnf) determination (a modified flow-cytomeuic wehi cell bioassay) were obtained at baseline, during hemorrhage and following resuscitation. liver blood flow measurements by low dose galactose clearance (glc) and functional bepatocyte mass (fhm; defared as galactose elimination capacity [gec] from the zero order portion of the plasma disappearance curve following an intravenous galactose bolus [ mg/kg], divided by liver weight) were measured before shock and after resuscitation. results: higher survival rates (p < . ) were observed in control as compared with cirrhotic rats. shock produced a significant reduction in gec (to < . ); fhm ( < . ); and liver blood flow (p < . ) in normal and cirrhotic rats. decreases in gec and fi-im were greater (p < . ) in cirrhotic rots. tnf levels were higher (p < . ) in cirrhotic rats at baseline and during induction of shock. pre gap junctions provide pathways for metabolic signals between cells. in the liver, the majority of gap junctions are composed of connexin (cx ) polypeptide subunits, and are regulated by gluconeogenic hormones. since sepsis and other inflammatory states alter hepatic glucoregulatory control, we have evaluated the contribution of gap junctional conductance to the metabolic dysregulation in the liver. an acute inflammation was induced in rats by injection with e. coli endotoxin (lps lmg/kg). northern blot/hybridization analysis of total rna isolated from livers after endotoxin injection show a decrease in the steady state transcript levels of cx to % of sham controls. immunostaining of liver sections using anti-cx revealed punctate fluorescent staining on the plasma membrane at regions of call-cell contact in saline injected animals, whereas, staining was only observed in cytoplasmic vesicles hrs after animals were treated with lps, suggesting the internalization of cx without replacement on the cell surface. the staining was quantitated and expressed as % of pixels above threshold. at hr post injection . % ofpixels exceeded threshold, compared to . % in sham controls. functional gap junctional communication was assessed by dye coupling using lucifer yellow in an isolated perfused liver under intravital fluorescence microscopy. dye diffusion was markedly decreased hr after endotoxin injection. this suggests that decreased metabolic coupling after lps injection results from decreased gap junction abundance. the present data suggest that metabolic dysregulation during sepsis may arise in part from changes in intercellular communication caused by a decrease in gap junctional expression and communication. given the marked metabolic heterogeneity of hepatocytes with respect to acinar location, metabolic signaling via gap junctions most likely serves to moderate this heterogeneity, contributing to a coordinated metabolic response. altered cellular ca ÷ regulation might be a critical step in organ dysfunction during sepsis and ischemia/reperfusion events. the aim of the present study was to evaluate hepato-ceuular ca ÷ regulation in isehemiah'eperfusion after hemorrhage and to assess effectiveness of tnfc~-monoclonal antibody (tnfo~-moab). methods. male sprague-dawley rats ( g, n>_ /group; pentobarbital mg/kg) with hemorrhage for rain at mm hg. reperfusion by ringer's lactate ( x maximal bleed out/ min) and % of citrated shed blood. tnfcz-moab (tn , ceutech, mg/kg in . % nac ) infused during flrst min of reperfusion. at baseline, end of ischemia and min of reperfusion, hepatecyte isolation by liver collagenase perfusion. " hepatocyte incubation ( mg w.w./ml) with caci ( . + + + mbq/ml) for rain (ca influx [slope, /mini; ca uptake [nmol ca /mg protein]) w/ and w/o epinephrine (epi, nm). hepatecyte resuspension in radioisotope-free medium and farther incubation (exchangeable ca + (ca +ex) [nmol ca +/mg protein]; ca + membrane flux [nmol ca +/mg protein'min]). during incubation, aliquots ( ~tl) were centrifuged through oil/lanthanum gradient and acivity measured by scintillation counting. statistics: anova. mean + sem. results. hepatocyte ca +ex and membrane ca + flux were significantly increased at both, the end of ischemia ( . +. ; . +. ) and reperfusion ( . +. ; . +. ), as compared to sham-operated animals ( . +_. ; . +. )( <. ). tnfc~-moab treatment significantly prevented reperfusion-induced increase of ca +ex ( +. ) and membrane ca + flux ( . +. )(p<. ). fast ca + influx was significantly increased by epinephrine in hepatecytes from sham-operated rats ( . +. vs. epi: . +. , p< . ). this hormone effect was not observed in isehemia ( . +. , epi: . !-_. ) or reperfusion (untreated: . +. , epi: . +. ; tnft~-moab: . _+. , epi: . +. ). conclusion. the present study clearly demonstrated hepato-cellular ca + overload in ischemia and reperfusion as a result of hemorrhagic shock. analysis of membrane ca + fluxes and hormone ca + mobilization suggests disturbances of membrane ca + transport mechanisms, e.g. through ca +-atpases. reperfusion-induced oxygen free radical generation which affect exchange kinetics of cellular ca + buffering compartments might also be operative. prevention by tnfct-moab indicates the pivotal role of tnf as an early inflammatory mediator of hepatocellular alterations in signal transduetion mechanisms and cellular homeostasis. although the precise mechanism has not yet been elucidated, bacterial translocation and endotoxin absorption have been frequently shown after burn, and have been postulated to be one of the underlying processes of sepsis. the purpose of the current study is to define the hemodynamic response of the liver to endotoxin release in burns, in correlation to bacterial translocation. twelve female minipigs, weighing - kg, underwent a laparotomy & transition time ultrasonic flow probes were positioned on the portal vein, the common hepatic artery, and the superior mesenteric artery. . fr catheters were inserted in the superior mesenteric vein and the left hepatic vein. a jejunostomy was also performed. after five days all animals were anaesthetized and randomized to receive % of tbs a third degree burn. eighteen hours after burn. gg/kg e. coli lps was intravenously administered over rain. ali animals were studied for additional hours and then sacrificed. several recent data suggest that in severe injuries, such as shock state, the gradual activation of kupffer cells and the excessive release of destructive and immunosuppresive products from macrophages may contribute to the development of "multiple organ failure". in in vivo experiments in mice, the effect of kupffer cell phagocytosis blockade on the correlation between the tissue distribution of lps, endotoxin sensitivity and lps-induced tnf production was investigated. to depress the activity of the kupffer cells, gadolinium chloride (gdc ) or carrageenan was used. th~e studies indicate the dissociation of tissue localisation of cr jllabelled endotoxin and endotoxin lethalithy. both gdc and carrageenan depressed kupffer cell activity, but endotoxin sensitivity was enhanced only by carragenan treatment. however, there was a close correlation between the sensitivity to lps and lps-induced tnf production as measured in the serum, since lpsinduced tnf production was enhanced only by carrageenan treatment. on the other hand, gdc pretreatment significantly increased tnf production in the spleen. these results support our earlier findings that gdc -indueed kupffer cell phagocytosis blockade leads to activation of the spleen, and may explain some of the immunological effects of gdc . inositol(l, , ) triphosphate (ip ) has been proposed as a second messenger for calcium mobilization. the addition of ip at low concentration has been shown to cause calcium release from intracellular microsomal store in rat hepatocytes. the effects of sepsis on the ip binding from microsomal fraction of rat hepatocytes during sepsis were investigated. sepsis was induced by cecal ligation & puncture (clp). control rats were sham-operated. three microsomal fractions (rough, intermediate and smooth) were isolated from rat liver. study of ip receptor binding was performed with tridium label ip . the results shewed that the ip binding was significantly depressed by - % (p< . ) during late sepsis ( hrs after clp), but not in early sepsis ( hrs after clp). the ip binding depression during late sepsis was most significant on rough and intermediate endoplasmic reticulum (p< . ), but not on smooth subfraction. since ip binding plays an important role in the regulation of intracellular calcium homeostasis in hepatocytes, an impairment in the calcium release due to depressed ip binding on smooth and intermediate endoplasmic reticulum during late sepsis may have a pathophysiological significance in contributing to the development of altered hepatic metabolism during septic shock. septic organ failure is currently recognized as an overactivation of the nonspecific immune system by bacterial stimuli giving rise to proinflammatory mediators. little is known about the mechanisms of the resulting cellular injury. here, a synergism is described between tnf as a major mediator of septic organ injury released by macrophages and hydrogen peroxide (h ) as a representative of reactive oxygen species as formed by e.g. neutrophils. rat hepatocytes are only slightly sensitive to either agent alone. when treated with a conbination of tnf and h# a stronq synergistic toxicity was found, especially w~e~ tnf-treatment preceeded challenge with h~o~. we have recently described a coculture model bfzrat liver macrophaqes and hepatocytes where lps induces hepatocyte cell death partially mediated by macrophage tnf release. when h was also employed in fhis more complex cellular system a similar synergism was found: the ecc~ of lps was consecutive patients with liver cirrhosis admitted to the department of surgery over a year period from january to december were studied for their complement profiles in relation to other parameters of liver function, the aim of the study was to determine if a direct correlation existed between low complement levels and end stage liver cirrhosis. cirrhotic patients were divided into child's a, b and c categories using child's classification. complement levels (c , c ) were measured and functional assay for complement (ch ) were performed in each of these groupings in addition to normal blood donor controls. these results show that the qualitative c , c and the functional chs complement assays have good predictive values in assessing deteriorating liver function• in particular, the functional assay for complement (ch ) showed marked impairment in child's c patients (p< . ) confirming the impaired immunological status of these patients. sera from this group of patients (child's c) were titrated with pig red blood cells (rbcs) in a haemolytic assay. the results showed that there were significantly less haemolysis of pig rbcs in these patients (p= . ) as compared to the controls. this findings strongly support an impaired immunological status in child's c liver cirrhosis and may explain the high incidence of sepsis as a terminal event in these patients. aim:kupffer cells(kc) have an importamt play to cause hepatocellular injury in sepsis, because these cells release many kinds of substances. we reported that oxygem radicals released by kcs stimulated by lipopolysaccharide (lps) caused hepatocellular injury. aim of this study is to investigate the relationship between imtracellmlar calcium(ca) concentration of cultured rat kcs stimulated by lps and release of oxygen radicals, and effect of prostaglandin e~ (pge~) on imtracellular ca concentration. production of acute phase proteins (c-reactive protein, crp, transferrin, tf) and £erritin (f) in rat hepatocytes (hps) and its dependence on extracellular matrix components were studied. hps isolated from the liver by collagenase perfusion were cultured at ~o per . ml medium fi +dmem ( : ) with % fetal calf serum for days on uncoated or type i collagen coated plastic surface or in the presence of dextrane sulphate in the medium. hps were stimulated by conditioned medium (gm) from i~ia-p or e. coli lps preineubated human blood mononuclear cells. production of crp, tf and f by hps was detected by elisa. it was found that both cms decreased tf synthesis in hps by - % (p<o.os). the level of f synthesis didn't change or only slightly decreased. the addition of cms led to the enhancement of crp synthesis more than . times. the most reproducible results were obtained when plastics had been coated with collagen. dextran sulphate markedly increased crp synthesis. thus using this model of an acute phase reaction in vitro we found the enhancement of crp synthesis and the decrease of tf synthesis. our data correspond with nublished materials on the acute phase in vivo and point to relevance of the proposed model. metabolism of hepatocytes under traumatic illness has not been sufficiently investigated. therefore we have applied absorption and fluorescent cytophotometry techniques to study the content of rna, glycogen and its fractions in hepatocytes of patients with severe mechanical trauma, both with and without endointoxication at various stages. for these purposes slides were prepared from the repeated aspiration biopsy material according to special techniques (kudryavtseva et al., ) . slides were stained by gallocyanin-chromalum to measure rna or underwent fluorescent pas-reaction to measure glycogen and its fractions (kudryavtseva et al., (kudryavtseva et al., , the ability of metabolites derived from the xanthine-xanthine oxidase system to inhibit mitochondrial function was examined using freshly isolated rat liver mitochondria. under uncoupled conditions, mitochondria exposed to free radicals exhibited a significant decrease in consumption supported by nad+-iinked substrates, but showed almost no change in consumption in the presence of succinate and ascorbate. the activity of electron transfer complex i (nadh oxidase and nadh-cytochrome c oxidoreductase) and the energy-dependent reduction of nad+ by succinate were unaltered by oxidative stress. exposure to free radicals also had an uncoupling effect at all three coupling sites. the degree of mitochondrial swelling was closely correlated with the inhibition of state oxidation of site i substrates and with the increase in state oxidation of succinate. the immunosuppressive agent cyclosporin a (cya) completely prevented the mitochondrial damage induced by oxygen free radicals (swelling, ca + release, sucrose trapping, uncoupling, and selective inhibition of the mitochondrial respiration of site i substrates). the same protective effect was found when ca + cycling was prevented, either by chelating ca + with egta or by inhibiting ca + re-uptake with ruthenium red. these findings suggest that the deleterious effect of free radicals on mitochondria in the present experimental system was triggered by the cya-sensitive and ca ÷-dependent membrane transition, and not by direct impairment of the mitochondrial inner membrane enzymes. correspondence should be addressed to: naoshi takeyama the aim of this study was to clarify the critical role of reduced glutathione (gsh) on the progression of lipopolysaccharide (lps)induced liver damage of mouse. the conditions of gsh-depletion and -richness were produced by intraperitoneal administration of buthionine sulfoximine (bso), a specific inhibitor of gsh synthetase, and of gsh-monoethyl ester (gsh-me), respectively. gsh-me, which was esterified the caboxyl group of the glycine residue, is readily transported into cells and is hydrolyzed intracellularly; this leads to greatly increased the cytosolic and mitochondrial levels of gsh. bso and gsh-me were administered intraperitoneally mmol/kg and mmollkg, respectively, and lps ( mg/kg) given h later. hepatocytes were isolated by in situ perfusion of the liver with collagenase h after lps injection. the degree of hydrogen peroxide (h ) synthesis and mitochondrial membrane potential were measured by flow cytometry using fluorescence probe dichrolofluorescein diacetate and tetrametyl rhodamine ethyl ester, respectively. the degree of mitochondria membrane permeability transition (mmp) was assessed by [ ]sucrose entrapment. we found that lps treatment results in a decrease in hepatic gsh level and mitochondrial membrane potential, and an increase in h synthesis and mmp. lps administration to bsq-pretreated mouse worsened at[ these parameters. in contrast, gsh-me pretreatment ameliorates. all together, gsh may acts an important role in cellular defence against lps-mediated liver damage, and mmp may result in the decrease in mitochondrial membrane potential. it has been shown, up to now, that disturbances of enzymatic processes in the cell organella are the basic factor of the changes taking place during endotoxin shock it has been observed that lysosomes are biochemically changed as result of the effect of lipopotysacharides of gram negative endotoxic bacteria. the purpose of the experiment was: .evaluation nfthe course of the ees due to the biochemic changes .determination of the influence of the ees on the activity of lysosomal enzymes in liver cell .deterrmnatian of the influence of h and d on the activity of some lysosomal enzymes in liver cell during ees material and method: examinations ware carried out on dogs. the shock was evoked by i.v. administration of endotoxin e.coli. the dogs were divided into groups: icontrols, i[ -dogs with untreated shock, iii-dogs in shock treated with h, ivdogs in shock treated with d, v -dogs in shock treated with h and d. the following parameters were determined: .activity of acid phosphatase in the venous blood serum. - .total and free activity of acid phosphatase and catepsins in the full liver cell homogenate, in mitochondrial-and-lysosomal fraction, and in the superuatunt (all after hours of the experiment). conclusions . essential increase of activity of lysosomal enzymes was observed in the ees. . increased activity of lysosomal hydrolases in the liver cell homogenate corresponds, as a result of the endotoxin effect, with increased enzymatic activity in the peripherial blood. extensive investigations from our laboratory of the pathophysiology of hepatic no-flow ischemia ( min) -reperfusion injury demonstrated substantial kupffer cell activation with reactive oxygen formation during the first few hours of reperfusion as indicated by increases in plasma glutathione disulfide (gssg) levels in vivo (am. j. physiol. : g , ) and enhanced superoxide formation by kupffer cells (kc) isolated from the postischemic livers (free radic. res. commun. : , ) . the early kc-induced injury initiated the later, primarily neutrophilmediated reperfusion injury (faseb j. : , ). to test whether or not similar mechanisms are operative during hepatic ischemia induced by hemorrhagic shock, male fischer rats ( - g) were subjected to rain of hemorrhage (mean arterial pressure mm hg) followed by resuscitation (rs) (reinfusion of shed blood). hepatic atp levels declined from . + . txmol/g to . _+ . ( min shock) indicating severe ischemia, and recovered to . -- . at min rs. to test for potential oxidant stress during rs, plasma gssg conc. were measured. although gssg levels increased by % compared to baseline levels ( . !-_ . i.tm), there was no significant difference to shamoperated controls. spontaneous superoxide formation of isolated kc was . + . nmol o -/min/ ecells (kc ) and . + . (kc ) in controls and did not change significantly after shock and rain rs. addition of phorbol ester or opsonized zymosan to isolated kc did not indicate a priming of these cells. conclusion: in striking contrast to our previous findings with hepatic no-flow ischemia, there is no evidence for a significant kc activation after min of hemorrhagic shock and up to rain of resuscitation. objectives-this study investigates morphometric changes in kc nltrastmcture which might account for this diminution in phagacytosis. materials and methods -three groups of wistar rats were studied: control, sham-operated [sham] and bile duct ligated [edl] . after weeks animals were sacrificed and livers were "perfusion fixed" with % glntaraldehyde and processed for transmission electron microscopy and image analysis. results -in the the bdl group kupffer cell numbers were greatly increased. biliary ductular proliferation was evident and sinusoidal spaces were compromised. eleven nuclear and cytoplasmic parameters were measured and statistically significant results are summarised in the results: ascorbic acid levels were slightly elevated after the hs and returned to basal values after rain. glutathione concentrations were increased significantly after the hs and the gsh levels kept rising continuously to as much as -fold of basal levels at the end of min. mda showed no significant variation before and after the hemorrhagic shock. plasma concentrations of ratine, teucine, isoleucine, phenylalanine, proline, threonine and serine showed significant increase over basal levels during the whole ischemic period. glutamic acid was slightly elevated at the onset of hs and remained the same for the rest of ischemic period. hydroxyproline was significantly below the basal value at the mid-point of ischemic period. alanine, glycine, histidine and aspartate did not change significantly throughout experimental time course. conclusions: ( ) oxidative stress might not be severe in the systemic hemorrhagic shock for pigs since no significant variations were observed for ascorbic acid and mda ( ) the increase of gsh might be that it was released from hepatocytes when the animal was under systemic ischemia. ( ) celzain amino acids might be acting as transmitters in the event of ischemia. however, fm"lher investigations are needed to clarify the detailed mechanism in regard with antioxidants and amino acids. dr. fang-ku p'eng taichung veterans general hospital talchung, taiwan , r.o.c. jia shi yong, huang zhi oiang and men xian jun. in patients underi~ent major hepatic surgery,obstructive jaundice has been implicated with fnoreased susceptibility to sepsis and liver failure. jaundice was said to he associated with derangement of iamune function and result in expression of pr,:.inflar...aatory cytoki~es that cause hepatooellular damage. this study is ~.o investigate the effects ,.,t lip,~polysaccharide(lps)rats. jaundice were produced in healthy ~istar rats of either sex weighing - m~ by common hi l~ duet l igation(bl)l).seven days post l igation, rats were given lps( .gg. ) . rag/kg intraperitoneally. at the end ,:,f l,gand hours after lp$ ehanllenge, liver were removed and prepared for forzen sections immediately. i)emonstration of inf in liver parenchyna were performed by method of abu i~munohistochemistry using r~onoolonal antibody against tnf, leve of mrna for tnf ~ere measured by in-situ hybridization using biotin-labeled edna probe. results sho'~ed that tnf stained granules appeared in kup~'fer eell~ and polymorphonuclear leukocytes(pnnl,but not in hepatoeytes nor endothelial eel is. they ~'ere located within cytoplasms and peaked at - hours after lps ohal lenge. there was vocomitant tnfmrna expression but peaked earlier hour post lps challenge. tnfmrna vcere detected notably in necrotic area and barely in bdl rats ~ithout lps challenge. it is therefore postulated that production of tnf by inflammatory effeetor cells-kupffer cell and phn in site, in the obstructive jaundice rats during sepsis may atribute to the hepatocellular damage. it also provide evidence for the beneficial effects of early release of biliary obstruction. in the present study, an animal model of a c in wlstar rats was developed by tigatlng the common bite duct and injecting d. mt solution of e. goli % b, (g× ' efm/mt) into the bite duct. the mortality rate was ~ at h after aoc. at , , , h after aoc, kcs were isotated and cultured atone or cocuttured with hepatocytes to evaluate the modulation effect of kgs on hepatoeyte protein synthesis. the results showed that tactodehydrogenase leakage was increased progressively as the injured k~ function and structure developed. tnf and il-i tn the supernatant were detected with the peak values at h after aoc. at h after aoc, ~-teueine incorporation was obviousty decreased in the normal hepatocytes eocuttured with stimulated kcs compared to the uormai hepatocyte cultured group (p< . ), but it was slgnificantiy increased in the group cocultured with normal kcs. in the a c group, 'h-teuclne ineorporatien was decreased progressively in the injured hepatoeytes cocuttured with stimulatedkgs and it was markedly elevated when cocuttured wlthnormat kcs at h after a c (p< . og). it is concluded that the impaired hepatocyte proterin synthesis was directly mediated hy the stimulated kc function during aoc. such a mechanism possibly may be involved in the patho ensis of hepatic dysfunction and m f following h . " the project supported by nsfc. in the past few years it has become evident that cytokines are implicated in various pathophysiological mechanisms. therefore measurement of cytokines and their potential inhibitors in biological fluids may be helpful in diagnosing and monitoring of diseases. however, dependent on the type of assay used investigations performed in different laboratories have often yielded conflicting results. in order to assess reliability and reproducibility of cytokine measurements two comparative studies for the determination of cytokines in biological fluids were launched by several investigators interested in this field: one national austrian ~tudy and one european study in the context of the european workshop for rheumatnlogy research. serum and synovial fluid samples were distributed by the organisers, and participants had to measure one or several of the following cytokines in all samples: il- , il- , l- , i , tnf-alpha, ifn-gamma, gm-csf, and the soluble receptors for il- and tnf; both commercially available immunoassays and home-made assays were allowed. so far, the main conclusions emerging from these preliminary studies are: ( ) the same immurzoassay (elisa) yielded comparable results in different laboratories; ( ) immunoassays from different manufactureres usually measured the highest concentrations in the same samples, yielded similar relative values but disparate absolute values; ( ) assays for soluble receptors yielded less discrepant results; ( ) some assays seem to be more suitable for measuring cytokines in biological fluids than others. the mean retrieval of exogenous recombinant human cytokines was approximately % for most cytokines tested. however, it must be borne in mind that natural and recombinant cytokines may behave differently in immunoassays. this raises the question as to the necessity of setting up international standards for all cytokines. possible interferences by serum components such as (lipo)proteins, complement, rheumatoid factors, etc., which may either decrease assay sensitivity or yield false positive results, must also be considered. in addition, natural cytokine binding proteins (e.g. soluble receptors) might interfere with cytokine determination in immunoassays. the problem of immunoassays versus bioassays has not yet been approached, but is without any doubt worth indepth investigation. thus, these studies are a promising first approach to deal with the difficulties of cytokine analysis in biological samples, and it is hoped that they will help to overcome some of the major methodological problems in the near future. tumor necrosis factor (tnf) is a pleiotropic cytokine which plays a key role in a number of immunologically mediated disorders like septicemia or cachexia. tnf receptors are found on the surface of a variety of cells, where they provide molecular signals for the cytokine effect. there exist two types of soluble tnf receptors (stnf-rs), tnf-r p and tnf-r p . these stnf-rs can compete with the cell surface receptors and block their activity. the expression on and shedding from the cell surface of stnf-rs is enhanced by interferon gamma. increased concentrations of stnf-rs can be found in patients with infectious as well as malignant disorders. in patients undergoing immune stimulatory therapy with interleukin- and interferon alpha a simultaneous increase of tnf and its soluble receptors can be seen. in patients with hiv infection a strong correlation exists between the levels of stnf-rs and markers of immune activation like neopterin or beta- -microglobulin. likewise patients with hematological disorders show elevated stnf-r levels. patients with weight loss have higher concentrations than patients with stable weight. the final value of stnf-rs within the complex network of cytokines is not yet clear. however, there exist striking parallels between infectious and malignant disorders pointing to a key role of endogenous immune activation. biochemicatly, d-erythro-neopterin derives from guanosine triphosphate. in vitro studies show that large amounts of neopterin are produced by human monocytes/macrophages stimulated with interferon-j (ifn-j). neopterin is biologically stable, and its halflife in the blood seems to solely depend on renal excretion. specimen for neopterin determination can be stored without special precautions. increased concentrations of neopterin have been described in body fluids of patients suffering from diseases which are apparently associated with an activated cellular immune response and with enhanced endogenous formation of ifn-~', e.g,, ) increasing neopterin levels predict rejection episodes in allograft recipients ) virus infections induce increased neopterin concentrations, and the degree of neopterin elevation predicts prognosis in these patients which is particularly true in hiv- infection ) in autoimmune disorders such as systemic lupus erythematosus or rheumatoid arthritis neopterin levels reflect activity of the disease ) increased neopterin concentrations in patients suffering from certain types of cancer indicate poor prognosis. significant associations between changes of neopterin and decrease of hemoglobin as well as the development of weight loss and cachexia, e.g., in cancer patients and in patients with hiv- infection, support the concept that endogenously formed cytokines such as ifn-~" and tumor necrosis factor-~( are involved in the pathogenesis of such symptoms. in conclusion, neopterin monitoring is a sensitive tool to detect the activation status of the cell-mediated immune system in vivo. repair and healing or perpetuation of acute inflammation is characterized by a complex network of interacting cellular and humoral defence mechanisms. of the numerous inflammatory mediators/effectors investigated hitherto, the proteolydc lysosomal enzyme pmn elastase has turned out to be highly destructive when released extracellularly thus contributing considm:ably to organ dysfunctions in severe posttraumatic and postoperative courses. besides various cytokines, elastase in complex with its main antagonist c¢ -proteinase inhibitor (a pi) is also supposed to induce the shedding of intercellular adhesion molecules from inflammatory cells (pmns, monocytes/macrophages, endothelial cells). these soluble adhesion molecules may modulate the inflammatory process in many different ways, e.g. via acting as chemotaxins, blocking neutrophil activation or competing with the membrane-bound forms in cell-cell adhesion. thus, measuring circulating or local levels of elastase-a pi and soluble adhesion molecules (icam, e-selectin,-pselectin, l-selectin) may be a helpful tool in judging the severity of an acute inflammatory process. in several clinical studies on surgical patients suffering from multiple trauma and/or sepsis we could demonstrate that the measurement of these parameters in consecutive plasma samples and local body fluids was highly valuable for early diagnosis and prognosis of multiple organ failure. prof. dr. m./ochum, institut fdr klinischechemie und biochemie, lmu miinchen, nufibaumstrage , m nchen, germany procalaitonin (proct) a aa peptide is dramatically increased in the blood of patients with various sepsis and infections. the increase begins as soon as hours after the andoto:edn release and readied maximal level with a to b-fold increase h after li~ edrmrdstration. prcc, alcitorfin response was temporally different from these of tnf~ and ii- , that reached peak levels at h and h respectively. at the opposite of eytokine (tnfcz, ild, j, is), proct is a very stable molecule. the half iife of proct in ~he blood is surprising if we consider the half llfe (about nine mirtute) of mature ¢alcitonin (co. we have found that it is due to the binding of proct with a protein on the n-termktal part. thin complex of about daltons is unable to cross the kidney and the c_n barriers and the half life is at least elghteen hours. this long half llfe is very useful in the evaluation of a sepsis. a very sensitive and specific sandwich in'ununoassay has been developped. the results cart be delivered in two hours. at time, we know that in the healthy adults, the values are less thau . ng/ml. it is also clear that proct is not increased ~n patients wlth inflammatory dleeasea without lnfectlon. for instance, proct is not signlffcat vely increased in purpura rhumatom, arthritis, crohn's disease, rectocolitis, also in various cancer patients with inflammatory components. am other interesting finding, is the absence of increase or a very low increase in the viral infections. at the opp site, in all the patients with bacterial sepsis or in the malaria pat/ants, progt was dramatically increased, from i to fold. p~oct can be useful to the follow up of patients with sepsis. actually, we don't know the origin of this proct production, in the united states, will also be discussed. the septest portion of this study will eventually enroll over patients tentatively diagnosed as septic. the results of septest will be compared with commonly accepted symptoms and criteria associated with sepsis in order to determine the clinical utility of this endotoxin assay. preclinical results of patients and normals be presented as well as data on the time course of endotoxemia and clinical outcome of selected septic patients. preparation of dna libraries clifford w. schweinfest, ph.d. a fundamental tool of the molecular biologist today is the ability to work with purified dnas representing genes of interest with respect to the investigator's field of study. originally applied almost years ago to the genetic dissection of simple organisms such as viruses and bacteria, molecfllar cloning was rapidly applied to study the more complex genetics of the mammalian cell. today, virtually any study of cell physiology which depends upon specific gene expression is approachable using the tools of the molecular biologist. therefore, stress induced gene expression (such as the heat shock gene, hsp ) or the regulation of the nitric oxide synthetase gene or the induction of cytokines and growth factors as a result of trauma or injury lend themselves to investigation at the level of the gene. during these workshops, we will discuss the means by which cdnas and genes are isolated, amplified and identified. we will discuss the strategies and methods for cloning cdnas and genomic dnas, for organizing such cloned libraries, for finding specific genes and for characterizing those genes. this speaker will focus on the techniques and considerations involved in the synthesis of cdna libraries. topics include rna isolation, mrna quality, reverse transcription, choice of cloning vectors and library quality. genomic dna library synthesis will also be discussed with respect to the vector/host systems available and applications which are specific to genomic dna. finally, polymerase chain reaction (pcr) will be discussed briefly with regard to its impact on dna cloning. the identification of the genes or gene products that have a role in cellular processes is fundamental to our understanding of genetic control and methodologies to achieve this aim are currently available. all levels of the various signal transduction pathways can b~ molecularly analyzed to define the mechanism by which critical steps in each pathway are achieved. the questions that are unresolved in each area of investigation serve to direct the scientist towards analyses of gene products or gene regulation of the gene itself. thus, selection of the type of library (genomic or cdna) to be used is dependent upon the specific goals of each investigator. to facilitate resolution of this question, an overview of the uses for the different types of libraries will be presented. at least four methodologies are currently available for screening a library, which are dependent upon specific interaction between nucleic acids, nucleic acids and proteins, antibodies and antigens or between different proteins. the use and advantages of each of these methodologies will be discussed along with a description of probe preparation. sequence methodologies required to begin characterization of gene clones will be presented. hopefully, the participants in the workshop will help define areas of emphasis and allow time for directed interaction to discuss specific applications based upon their own experimental systems. alterations of physiological conditions, such as those occurring after shock and sepsis, induce changes in gene expression of cells composing the major organ systems. the challenge is to detect and characterize these changes in geae expression and relate them to the injury. the development of cloning techniques has emerged as the methodology of choice. changes in gene expression are usually characterized by variations in the concentration of cellular messages because synthesis of the final product "the polypoptide" is usually proportional to the concentration of mrna. due to the rapid regulation of gene expression the cellular concentration of messages changes very quickly. this is commonly referred to as steady-state levels, a balance between transcription and degradation. steady-state levels of mrna can be detected in a single cell (in situ hybridization) or in total rna isolated from cells or organs and separated in agarose gels (northern blots). although analysis of mrna steady-state levels are very useful, they do not provide information about the mechanisms that regulate gene expression. changes in gene expression primarily occur at the level of transcription; characterization of the rna species being synthesized at a given time is performed by the nuclear run-off technique. when there is no a priori information about the gene that may be regulated after a particular situation such as sepsis, the total pool of messages present in cells at given time can be characterized by a combination of in vitro translation of the cellular messages and fractionation of the in vitro translated products by two dimensional gel electrophoresis. such approach permits the visualization of the general pattern of gene expression, a "finger print", of the physiologic state. in summary, researchers now have access to a great variety of techniques to identify and characterize genes expressed in response to a physiological condition that may be utilized as "molecular tools" to study the organism's responses to shock and sepsis. the acute phase proteins are a set of plasma proteins with greatly increased plasma concentrations during acute inflammations and after tissue trauma, e.g. after major surgery. the proteins counteract the source of injury, facilitate clearance of infectious particles by phagocytes, assist immune responses and initiate wound healing. the corresponding genes are expressed in liver hepatocytes and regulated by the inflammatory mediators interleukin and interleukin (ill, il ). class acute phase genes are mainly controlled by ill and by combinations of ill plus il ; class genes mainly by ii, . the human c-reactive protein(crp), serum amyloid a(saa) and complement c genes will be discussed as examples of class genes, and rat c~ macroglobulin as a prototypical class gene. both classes of genes use different types of ¢ytokine response elements in their promoter-proximal transcription control regions; class genes use the transcription factor nf-il or c/ebp as the key factor to mediate cytokine responsiveness; class genes use a different, so far unidentified factor to achieve responsiveness to il . ongoing efforts to purify this factor, called the il -response factor (il -rf), from rat liver nuclear protein extracts will be described. the il -rf apparently mediates the effects of il in a broad range of cell types, including b lymphocytes and other hematopoietic cells. it is therefore likely to be of equal general importance for gene regulation by il as nf-il . the importance of transcription factors as potential targets for novel bioactive substances and possibly therapeutic agents will be discussed with the help of several recent examples. new methods for altering the germ lines of mice provide powerful tools for understanding the roles of individual genes in normal and pathological processes, and for reproducing specific genetic mutations that result in congenital disease. three approaches will be discussed. conventional transgeulc mice are frequently constructed using artificial transgenes that express genes from a promoter other than the normal gene promoter. this paradigm is used to produce very high levels of a gene product, for example, with a viral promoter, or to attempt to regulate gene expression using an inducible promoter. alternatively, normal promoter sequences can be hooked to a gene whose expression is lethal to the cell (e.g., diptheria toxin a chain) to ablate all cells expressing that gene. another approach uses an intact gene with its normal regulatory sequences. here, an elevated level of the gene product is delivered from the correct cells in the correct tissues at the correct developmental stages or in response to natural signals. this approach has been refered to as "genetic pharmacology," and provides a precise delivery of the gene product to the target. however, to be most effective, the transgene should contain arl regulatory sequences as welt as the entire genomic segment containing gene coding regions. the introduction of conventional transgenes is limited to roughly kilobases, while genes containing all regulatory sequences frequently stretch over dozens or even hundreds of kilobases. to overcome this problem, procedures have been developed recently to introduce yeast artificial chromosomes (yacs) into mouse embryonic stem (es) cells. yacs can include over mb of human dna, large enough to encompass the largest known human genes. es cells are also useful for targeted gene deletions and mutations. homologous recombination can be targeted to any known gene in es cells. when these modified cells are injected into a mouse blastocyst, they can be incorporated into all tissues of the resulting mouse, including germ cells, so subsequent generations will pass the genetic modification as an inherited trait. assessment of the effects of a null allele on development can provide valuable insights into its normal role. ultimately, these technologies may be adapted to human cells -not for embryonic intervention, but to provide modified stem cells for various tissues (e.g., gut, eye, hematopoietic system) which could then be applied to somatic therapies. with major illness/injury that respirswy faihne fi'equently followed sepsis, tilney el al nse, d the term "soqueutial syste~ failure" for patients with renal failure after solmir of ruptured abdominal aortic ~aeurysms, i then reviewed our experiences after inju~ and oporstion and suggested that multiple, progsesslve or sequential systems or organ failure was a syndrome to be reckoned with in the, 's. eiseman et al then wrote about "multiple organ failure", especially with liver failure. polk el m found that renmte orgau failure often signalled occult anita-abdominal infection. fry et al ampbasized the role of uncontrolled infection in organ failure, border et ai described metabolic alterations with mof and used the term multiplesystems organ feilmo (msof). other early conltibutots to our knowledge of mof include faist, trunkey and miller, marshall and dimic&, cassone, catlleo, meakins and marshall, (}otis et at., mater and many others. mof or msof were used eommoifly. cerra coined the terms "post-trsumatic septic sfndromc" and "hyper metabolism otgau failure complex", and border et al, used lhe ex ~ression "gut origin seplic states" to illdioate important eurrem aspects of abe gt.'nerlc problem of organ failure and death, other terms include acute organ-system failure, multiple organ injury syndrome, post-traumatic multi-system organ failure nod post-~aumatic organ system infection s~dmmc (fi~sis), i bdieve the latin "maltiple organ failure" is clean, neat and says it all, now there is a proposal for a new set of torminolugios -mods and sirs. will they be helpful in the study and esro of patients? our speakers in this session will review thcsa proposals and the evidence as we strlvo for eoasensns. it has been well demonstrated that the administration of pro-inflammatory cytokines, and especially tumor necrosis factor (tnf) can result in a picture similar to septic shock with secondary multiple organ failure. it is also clear that tissue hypoxia can lead to organ damage. we believe that it is the interaction between the two phenomenons that can lead to mof. on the one hand, the inflammatory response is amplified in the presence of hypoxia. on the other hand, the release of the mediators of sepsis can increase tbe oxygen demand ef the tissues, alter their oxygen extraction and decrease myocardial contractility, and the combination of these elements accounts for the development of septic shock. this hypothesis is supported by two lines of evidence. first, mof is usually preceded by an episode of acute circulatory failure (even though frank circulatory shock may not be evident). second, recent experimental and clinical studies indicated that immunotherapy (especially antibodies to tnf) is particularly effective in the most severe forms of sepsis, associated with circulatory shock. hence, an effective way to prevent mof may be a combination of aggressive cardiovascular management and immunotherapy. development of the multiple organ dysfunction syndrome (mods) in the critically ill follows an heterogeneous group of stimuli including infection, sterile inflammmion, extensive tissue injury, ischemia, intoxication with a variety of substances, and immune system activation. whether the resulting physiologic abnormalities reflect a common pathologic process, or are simply a non-specific manifestation of tissue injury is unknown. moreover, the lack of clearly-defined functional criteria for the characterization of the syndrome on the one hand, and of reliable biochemical markers of its evolution on the other, has made attempts to define its epidemiology and natural history difficult. using a numeric scoring system to quanfimte the clinical severity of mods, we demonstrated that, although mods is more common in patients who have significant infection at the time of icu admission, a much stronger correlation is evident between the severity of mods and the subsequent development of nosocomial icu-acquired infection. furthermore the severity of mods correlates strongly with the severity of the clinical septic response, and with the degree of immunologic compromise evident on delayed type hypersensitivity skin testing. to determine the relative importance of the initial stimulus, and the host response to that stimulus, to the subsequent emergence of mods, we undertook a matched cohort study of critically ill patients admitted to the icu with infection, matched by age, sex, and apache ii score to uninfected controls. organ dysfunction scores were higher in patients admitted with infection, but were also significantly associated with the expression of a septic response at the time of icu admission, independent of the presence of infection. by stepwise regression analysis, the magnitude of the septic response was the most powerful predictor of the severity of mods in both infected and uninfected patients. these data suggest a model of mods in which a stimulus (eg infection) and the host response to that stimulus (eg the septic response) result in a state of altered systemic homeostasis (manifested in this example by immunologic dysfunction). moreover they suggest that the principle determinant of the emergence of mods is the response of the host, rather than the nature of the stimulus, and are consistent with the hypothesis that a stereotypie systemic response to an heterogeneous group of injurious stimuli underlies the pathogenesis of mods. arthur e. baue, m.d, ever since the attorapt build the tower ef babel, as desclibcd in the old testament of the biblc, it has been difficult to got agreement o;x common definitions or language, although mof has served well, there will always he now classifications proposed with good reasons for them, thus mods and sirs are not the final oxpressinns in the lexico~aphic sweepstakes. attcmpts to develop standard animal shock and scpsis models (hemorrhage, endotoxin, foes[ pellets, cecal ligation and puncture, e. colt hlf sioi to evaluate therapy have net been suoca.ssf~. clinical syndromes of mof, sepsis, sepsis syndrome and athers have not been aeacpte.d by all. our problem is that we arc tryittg tu d¢ffino a non-emily. mof or mods or sirs is net a disease or oven a syndrome. it is a series of complications of injury, disease and intensiva care which loads to death for many patients in intensive care units, it is not a fixed entity, but an ewilving picture. we have lumped (ugethet for therapy and definition a number of diseases and problems according to clinical manifestatieos rather than the eause of ihe problem, the search for unified mechanisms has not bean successful. will we benefit oleo from a more precise classification and definition of a non-outjty, a hodgu-podge of human disorders which have charsmeristic.s and manifestations of tissue injury, inflammation and infactian? re.hi clinical trials of potential "magle ballets" suggest that we should go in a different direction--instead of lumpors, we should be splitters, to seek effective therapy, we must fucus on specific disorders such as trauma, specific infections, inflammatory disease and chronic or acute organ damage (copd -renal failure, etc.), i will say more aboet this later ha the me.ethag. thcre is cue potentially important pert of the mods proposal, and that would be do~mcntation of organ dysfunction before fsiiure occurs and before tile need for external supporl dovelope. this could lead to hatter methods of preventing organ failure. preveution is ultimately the only answer to organ failure, m mof, mods and sirs. have wo reached a consensus?--only that we arc dealing wilh a diffl~x:lt probtolr looking for solutlons. acute lung injury (ali) and its most severe form, acute respiratory distress syndrome (ards) characterized by severe hypoxemia, diffuse infiltration of both lungs, a reduction of compliance and a wedgepresure lower than mm hare related to direct or indirect injury. when aliresults from direct injury (toxic agents, lung contusion, pneumonia), the lesions of epithelial barrier and the activation of lung macrophages are the first events leading to the release of inflammatory mediators wh:ch are responsible for alteration of the membranar permeability and for invasion of the alveoli by fluids, proteins and cells. an inflammatory reaction develops, with injury to endothelial cells, adhesion of polymorpbonuclear leucocytes (pmn) and the release in blood stream of intlammatory mediators dispersing the inflammatory reaction to other organs and tissues. the failure of lung function also results into hypoxia in other organs, leading to tissular ischemia, triggering an inflammatory reaction leading to organ dysfunction. frequent complications of direct lung injury are septic pneumonia with endotoxin release, phagocytes activation and cytokine production disseminating inflammation. by this way, direct lung injury can be at the origin of the multiple organ dysfunction syndrome (mods), the frequence of which having been estimated to % and is increased when all has occured in patients alreadypresenting an organ dysfunction (immunodepresslon, cancer...). when ali results from redirect or extrathoraclc injury (trauma, infections, hypovolemic shock, acute pancreatitis...), inflammatory mediators and micro-aggregates released in particular organs or tissues reach the lung via the blood and lymph streams, are filtered in the organ or trapped in the lung capillaries, and are responsible for a local inflammatory reactaon (endothelial cell activation, pmn trapping and adhesion, platelet aggregation, release of mediators). sepsis acts by the way of endotoxiu and sequential eytokines release (tnf, ill,...) while trauma with important blood loss acts especially by the way of hypoperfusionreperfusion phenomena leading to a disseminatedinflammatory reaction and to a possible bacterial transloeation when intestinal hypopeffnsion is present. in these conditions of indirect injury, all (or ards) is a iocal early (often the first) manifestation of a general phenomenon of altered membrane permeability and inflammatory reaction, easily and rapidly recognized by its clin~cai signs. the iung is one of the numerous organs participating to the mods, in these conditions, the mortality rate is high, reaching more than % when sepsis is present or when at least organs are implicated in mods. by direct or redirect injury, lung is so a target organ for mods and injured lung can be the cause of mods or simply a participant to this syndrome. jorge cervts-navarro main determinants for brain dysfunction are breakdown of the blood-brainbarrier and raise of intracranial pressure (icp), in second place decrease of systemic blood pressure, disorders of blood coagulation and respiratory function. the blood-brain-barrier of cerebral blood vessels can be opened by stroke, cerebral trauma, inflammation, convulsions, acute hypertension and changes in the blood osmolarity. the consequence is brain oedema, increase of lop and decrease of the perfusion pressure. a close correlation between intraventricular pressure and the fate of patients with severe brain injuries has been established. regional cbf values of to ml/ g/min are reflected in isoelectric eeg, and values about t ml/ g/min, result in loss of somatosensory evoked potentials. for ionic pump failure the threshold has been determined by values of about ml/ g/min and is reflected in massive release of intracellular potassium. in stroke and in brain trauma when the oedematogenous condition is initially localized the tissue pressure variance within and between the hemispheres lead to tentorial and falciform herniations are the common cause of death even before the critical threshold of intracranial pressure is reached. the increase of the icp over the level of the perfusion pressure leads to the arrest of the cbf. if this persists {or periods exceeding seconds of global brain ischemia loss of conciousness and developing seizures appear. if it exceeds min irreversible injuries of the brain appear. following cerebral ischemia and after severe head injury a tendency to increased coagulation can be detected. vascular occlusion may develop either as a result of local thrombus formation or from emboli caused by circulating platelet aggregates. the formation of microthrombi is triggered by the plateletactivating factor a mediator in central nervous injury also responsible for aggravation of posttraumatic brain edema. acute hemorrhagic leucoencephalitis and brain purpura are known to arise as complications of acute viral infections and following gram-negative septicaemia. institute of neuropathology, free university of berlin, hindenburgdamm , berlin, germany undoubtedly, attempts to quantify as objectively as possible the degree of dysfunction of the various organs is very valuable. however, cardiovascular failure has a peculiar place in the context of sepsis. the development of acute circulatory failure (shock) is of paramount importance as a cause rather than as a consequence of organ failure. it is therefore essential to clearly separate patients with and without circulatory failure. once this has been done, attempts to list the cardiovascular system among the other systems have been unconvincing. in a number of studies, cardiovascular complications are assimilated to a low systemic vascular resistance (svr), but since svr is basically the ratio of blood pressure over cardiac output, and since hypotension is already included in the definition of shock, then a low svr is basically equivalent to a high cardiac output, which is a characteristic rather than an ominous complication of severe sepsis. development of a low cardiac output unresponsive to fluids is usually a terminal event. it is not advisable to list other problems such as severe arrhythmias, myocardial infarction or tamponade as complications of severe sepsis. host defense dysfunction following trauma, shock and sepsis e. faist, g. f. babcock * major accidental, burn and operative injury often precipitates a profound multicentric immunologic depression that is believed to predispose patients to become anergic and thus to develop towards a higb probability of infection. anergy or a lack of immunologic reactivity stands for a total elimination of skin test reactivity and recall antigen responses in the inununocompromised patient. anergy following overwhelming trauma or major septic conditions results from a massive impairment of cell mediated immunity (cmi) together with a whole body malignant inflammationiike state. we and others have demonstrated clearly that the skeration of cmi following trauma is mainly due to the disruption of intact monocyte (moo) / t-cell interaction. within this phenomenon we see a shift of the cell ratio in the compartment of peripheral blood mononuclear cells (pbmc) with a considerable increase of prostaglandin e synthesizing m~ and a simultaneous decrease of functionally competent cd + and cd + lymphocytes. t-cell dysfunction in states of profound stress is characterized by impaired synthesis of two crucial lymphokines -interleukin- (il- ) and gamma-interferon (y-ifn). the inability to produce adequate amounts of il- , most likely attributable to alterations in the transmission of signals from the cell membrane to the nucleus, results in incomplete proliferative t-cell responses to antigenic stimuli, while a lack of ?-ifn results in inadequate m~ antigen processing and presentation. the role of the two functionally distinct t-helper subsets, t-helper- (thl) secreting principally il- and ?-ifn and t-helper- (th ) acting principally in inducing antibody formation with a secretion of il- , il- , il- and tnf-[ has still to be established within the context of major trauma. antibody formation to common protein antigens is impaired, the predominant finding is a shift from igm to igg synthesis. although excessive secretion of prninflammatory cytokines (il-u , tnf-c~, il- , il- ) has been considered to be deleterious for the host in states of major inflammation and infection, in-vitro and whole blood investigation of me function has clearly revealed that there is initially a marked suppression of moo from septic patients to synthesize and secrete proinflammatory cytokines to endotoxin. this probably will result in immunodeficiency of traumatized as well as septic patients, since these cytokines in low concentrations are involved in the upregulation of the essential humoral and cellular immune functions. abnormalities of pmn function noted after serious injury have been interpreted by some investigators as signs of unresponsiveness of this cell population, others have demonstrated signs of pmn byperactivation. latest findings revealed that there is a clear pmn dysfunction in circulating blood: % of all pmn's in this compartment show downregulated or non existent ~-integrins within hours posttrauma -these cells do not phagocytose, have a reduced respiratory burst and appear to be completely degranulated. restoration of these cell functions within days post-trauma is significantly correlated to survival of traumatized patients. other reports however stress increased expression of cr + receptors (cdll/cd complex) on circulating pmn's, increasing the ability of these cells to adhere to intercellular adhesion molecules, thus contributing to play a major role in inducing the pulmonary capillary leakage. our knowledge about the perturbation of host defenses associated with serious injury and sepsis is continuously increasing and represents the precondition for the design of effective therapeutic measures to prevent and counteract the resistance to invading microorganisms. dept. of surgery, klinikum grosshadern, ludwig-maximilians-university, munich, germany. * dept. of surgery, university of cincinnati, cincinnati, ohio, usa lg thijs~ ce n~ck sepsis is the most common cause of acute disseminated intravascular coagulation (dic) and coagulation disorders as well as abnormalities of fibrinolysis are common in septic patients. some clinical and experimental studies indicate that dic is a pathogenetic factor for the development of multiple organ failure. endotoxin and various mediators (tnf, il-i) enhance tissue factor and decrease expression of thrombomodulin on endothelial cells in vitro. also, tpa activity is suppressed and release of pai-i is stimulated. in such a way the endothelial surface becomes procoagulant whereas fibrinolysis is inhibited. following administration of endotoxin to normal volunteers levels of prothrombin fragments (fi+ ) and thrombin-antithrombin (tat) complexes increase, indicating thrombin generation. also the fibrinolytic system is activated as evidenced by a rise of levels of tpa and plasmin-antiplasmin (pap) complexes but this is counteracted by a subsequent release of pai-i. in severely ill septic patients levels of tat complexes are increased and concentrations of the natural inhibitors of coagulation (at iii, protein c) are usually decreased. also, tpa levels snd pap complexes as well as concentrations of pai-i are elevated. the severity of many of these alterations is related to mortality. thus, both coagulation and fibrinolysis are activated in sepsis, but not in a balanced way, thereby promoting coagulation. one of the hallmsxk pathological alterations associated with sepsis is the development of microvascular thrombosis, an entity ~lmre~erizod by microvas~tlar plugbing with leukoeyies (predominately nentrophils) and fibrin deposits. preranably, ischemia remlt~g fibm ve~-'ttlac ow,.luwion contributes sisni.ficantly to end orgen darnaje and consequent dysfatlctlon. p, er~t srldies haw focused pmdominately on the role of adhesion molecules in the pathogenesis of neu~ophil sequestration during infection and s~sis. this preparation will review the mechanisms underlying intravak-'v.ler i~brin deposition and its contribution to organ injury. the normal endothelial cell sure is generally conddered o be anticos~am. this state is maintained by two major anticoag~lam molecules on the cell so,ace: hepax'an su,lfale and thmmbomodulin. studies performed over the past decade have do~mentod a general shii~ towards a procoa~qtlant staw during gram negative hffeodon, aft e~| mediated mainly by changes in the endothalial cell surface. antt-tf antibodies have been shown to be pro~ective during l~'ml endotexemia. in summary, ~erova~'~ler fibrin deposition, in ~rt mediated by ~rcgulation of cellular tf, is a consistemt patholo~cal flndin~ during sepsis and contributes to organ injury. strategies designed to abrogate this event may n~nimjze the magnitude of erg~ dysftm~on. n, v, christou md phd, department of surgery, megill university, montreal, quebec, canada interactions between immune calls and the endothelium vie adhesion molecules serve to modulate immune cell traffic between the blood strum and the extrmvascular space, these families of molecules acting in a cascade and closely integrated with the oytoklna network, blood coagulation and the complement system, are responsible for the homing of leukocytes to areas of inflammation and the realrculatlon of tymphocytes. there are three types of immune ceils that must exit the blood stream into the extravascular space: lymphocytes ; polymorphonuclear leukocytes; and non-lymphocyta mononuclaar cells (monooytes, basophlls, aoslncphils). b lymphocytes exit the blood stream and recircu}ate mostly via the high endothelial venule (hev) in lymphatic tissue, rarely, in chronic }nfectlon=, they may recirqulate vie hev in non-lymphoi:t tissue. t-lymphocyte recircutation on the other hand can occur via hev in lymphoid tissue, as well as, the endothalium of the skin, returning to peripheral lymph nodes via the lymphatic channels. lymphocyte recirculation is the mechanism which allows ¢ontaot between the immune repertoire and pathogens, and homing delivers these cells to the appropriate environment for activation and for effeotor function, pmns exit the blood stream through endotbelium in close proximity to the site of inflammation after appropriate endothelial membrane receptor expression. they must reach the site of pathogen tnveslon quickly in order to be effective. their exudation to the inflammatory site can be modulated by their intravascular pdmlng, which results from appropriate receptor expression. because endotoxin leak from the gut has been postulated as an important trigger mechanism for the systemic inflammatory response syndrome seen after serious injury, the immunologic and metabolic effects of bacterial endotoxin infusion into normal volunteers can be expected to shed considerable light on the validity of this hypothesis. we and ethers have used tbis model to show that endotoxin causes a temporary paralysis in the ability of circulating monocytes to prudce the proinflammatory cytokines interleukin-i (il-i) and tnf-a. endotoxin, however, causes increased production of pge by the same cell population. endotoxin infusion causes significant suppression of circulating t-cell numbers and a profound suppression in the ability of the circulating t-cell population to proliferate and to produce interle~in- (il- ) upon in vitro stimulation. after endotoxin circulating t-cells are also more sensitive to the inhibitory action of exogenous pge . administration of cyclooxygenase inhibitors at the time of endotoxin infusion largely abrogates the effect of endetexin on t-cell proliferation and il- production. endotoxin infusion also causes the release of increased levels of circulating catabolic hormones including cortisol. administration of cortisol alone or cortisol along with endetowin to volunteers has allowed dissection of cortisol effects from those of endotoxin itself. cortisol infusion alone causes a diminution in the circulating t-cell population but the remaining ceils continue to produce il- after appropriate stimulation. cortisol infused along with endotoxin blocks the overshoot in monocyte proinflammatory cytokine production seen - hours after endetoxin infusion. at present, one may conclude that administration of sublethal doses of bacterial endetoxin to human volusteers produces alterations in cytokime production and tlymphocyte activation which are both similar and dissimilar to those seen following serious injury. hemodynamic support is based first on intravenous fluids and second on vasoactive agents. colloids are usually preferred to crystalloids, for their more rapid hemodynamic effects and their lesser passage in the interstitial space. in the presence of hypotension, the pharmacological profile of dopamine makes it the first agent of choice. it is only when high doses of dopamine are insufficient to restore a sufficient tissue perfusion pressure that norepinephrine should be added. even when cardiac output is not decreased, a dobutamine infusion is often indicated to counteract the myocardial depression, prevent vasoconstriction, and increase oxygen delivery to the tissues. the benefit derived from the administration of "renal" doses of dopamine is doubtful, but stimulation of dopaminergic receptors may increase blood flow also to the gut. in any case, none of these catecholamines has been shown to significantly improve the oxygen extraction capabilities of the tissues. agents with antiinflammatory properties but also with some vasodilating properties, such as n-acetylcysteine, prostaglandin el or pentoxifylline represent more attractive options to increase oxygen extraction. cardiovascular support should aim not only at the restoration of a sufficient arterial pressure but also at the maintenance of an optimal oxygen delivery to the cells. it should thus be guided also by measurements of cardiac output, mixed venous oxygen saturation and oxygen extraction, and indexes of cellular hypoxia, such as blood lactate levels, gastric intramucosal ph or vanearterial pc gradients. supranorma[ delivery contributes to the prevention of tissue hypoxia tissue hypoxia is considered to be the common final pathway in the pathogenesis of multipte systems organ failure. it may directly contribute to cellular injury and organ dysfunction as well as enhance further mediator activation and release by a positive feed back mechanism. prevention of tissue hypoxia, is therefore, one of the most important aims in the supportive therapy of critically ill patients, especially in those with sepsis and septic shock. mismatch of cellular o= supply and demand in these patients may resuit from the impairment of the cardiocircu[atory system on all levels. . myocardial depression with a drop in delivery (do~) . redistribution of blood flow between the different organ systems . inadequate nutritive blood flow due to tissue edema and endothelial damage (gic, leucocyte swelling etc.) these pathological changes may go along with increased metabolic needs. cellular and organ supply may therefore be inadequately matched with cellular needs in patients with normal and even supral normal doz. hyperdynamic circulation is often present in adequately volume resusucitated patients. it is most likely that one of the bodys main compensatory mechanisms would maintain cellular o= supply in the face of increased metabolic needs and impaired oz extraction capabillities. this pathophysiology may explain the findings in several clinical investigations that patients with normal or even supranormal doz can have signs of inadequate regional tissue oxygenation. it is also consistent with the results of different studies which demonstrated that the achievement of supranormal doz levels, either spontanuously or due to adequate supportive therapy, resuits in better patient outcome. any attempt to prevent or treat tissue hypoxia in these patients, however, has to take into account the effects of therapy not only on global dgz but especially on regional and microcirculatory blood flow. r. rossaint, d. pappert, k. lewandowski, h. gedach, k. slama, k.j. falke klinik for anaesthesiologie und operative intensivmedizin, ukrv, freie universit §t berlin, germany important contributory factors to the high mortality of severe acute respiratory distress syndrome lards) are the aggressive therapies required, such as mechanical ventilation with high inspiratory oxygen concentrations and airway pressures. as specific therapies for reducing or preventing the general inflammatory reaction of the lung resulting in severe hypoxemia is unknown, today's therapy is limited to procedures which predominantly support or maintain pulmonary function, i.e. pressure limited ventilation with peep and permissive hypercapnea, avoiding or treatment of fluid overloading, and positional maneuvers. extracorporeal lung assist combined with low frequency positive pressure ventilation has been recommended, when conventional therapy fails. today, extracorporeal gas exchange may be carried out using a system internally coated with covalently bound heparin. this allows for a lower level of systemic heparinzation reducing the risk of severe hemorrhagic complications of extracorporeal respiratory support. from april to august patients, aged from months to years, were transferred to our intensive care unit (icu) for treatment of severe ards. all fulfilled at least the slow entry criteria of the u.s. national ecmo study. due to hypoxemia patients required veno-venous extracorporeal membrane oxygenation (ecmo) immediately after transfer to our icu. the other patients were first treated with pressure controlled mechanical ventilation, permissive hypercapnea, prone position, and dehydration, if necessary. in out of these patients, in which the gas exchange did not improve in the following days, veno-venous ecmo with heparin-coated systems was additionally performed. heparin was given to achieve a partial thrombin time between - s and an activated clotting time between - s. if surgery was performed during ecmo, heparin infusion was interrupted. no severe bleeding complications related to anticoagulation for the extracorporeal bypass could be observed, however, in some patients, after three weeks of ecmo thrombi in the drainage cannula were observed. a problem of of membrane leakage shortened the life span of the membrane lungs to a mean of about four days. in the conventionally treated group % survived and in the additionally with ecmo treated group % survived, representing a % survival rate in patients whose risk of death is considered more than %. on the basis of these experiences, we conclude that the described strategy, including veno-venous ecmo with heparin-coeted systems, may improve the survival in patients suffering from severe ards. abnormally high skeletal muscle po in septic patients and its normalization during recovery: evidence against tissue hypoxia but for impaired oxygen metabolism of skeletal muscle? p. boekstegers, k. werdan department of medicine i, gro hadern university hospital, munich, germany a pathological oxygen uptake supply dependence was suggested to indicate tissue hypoxia in sepsis-a hypothesis which is discussed controversially. because the detection of reduced oxygen transport to tissue and &tissue hypoxia would have important implications for therapy, skeletal muscle oxygenation was studied in patients with sepsis. intermittent and continuous determination of skeletal muscle po by polarographic needle or catheter probes provided no evidence for skeletal muscle hypoxia even in severe stage of sepsis (boekstegers et al., crit care med ) . in contrast, mean skeletal muscle po was found to be abnormally high in patients with sepsis ( _+ , mmhg, n= ) compared to patients with limited infection ( , + , mmhg, p< . , n= ) , to patients with cardiogenic shock ( , + , mmhg, p< . , n=ls) and to healthy subjects ( , _+ , mmhg, p< . , n= ). furthermore, serial measurements in patients with sepsis showed that skeletal muscle po was higher ( , _+ , mmhg) on days with severe sepsis than on days with intermediate state ( , _+ mmhg) and than on days without sepsis ( , _+ , i mmhg) . thus, a decrease of abnormally high skeletal muscle po was related to improvement of sepsis. this was also demonstrated by comparing the decrease of abnormally high skeletal muscle po with the decrease of apache ii score or elebute score as well as interleukin- serum levels in a separate study (boekstegers et el., shock, in press). in summary, our data provide no evidence for skeletal muscle hypoxia in patients with sepsis but support the assumption &impaired oxygen metabolism in severe sepsis. neutropnlic emigration from the vascular space into the extracellular matrix is important for the response to tissue injury or contamination by providing a large recruitable pool of tissue-based phagocytes. the consequences of neutrophil exposure to intravaseular chemoattractants, likely relevant for il- and c a in sepsis, trauma, and in burn injury, are suppression of neutrophil attachment to endothelial cells and matrix proteins. it is probable that proteoglycan-bound attractants are of considerable biologic relevance, and may result in enhancement of responses to subsequently encountered cytokines. in the presence of connective tissue proteins expressing integrin-specific binding ligands, neutrophils respond to tnf-cq gm-csf and other cytokines by producing large amounts of hydrogen peroxide. this response is likely important in the digestion of contaminated or injured tissue by facilitating activity of neutrnphil granular proteases and inactivating plasma anti-proteases. this matrix-dependent oxidative response is important in defining potential novel targets for therapeutic intervention. the response appears to involve signalling by integrin-linked tyrosine kinases. the net effect of matrix protein injury through this mechanism is enhancement of the fibrotic response which underlies much of the prolonged ventilator dependence of patients with the adult respiratory distress syndrome. this adherence dependent response has also been implicated in direct hepatocyte injury, and may represent an early component of the syndrome of multisystem organ failure. study purpose: in this patient (pat.) population with a considerable sepsis morbidity and mortality, parameters proposed for sepsis assessment (elebute sepsis score [ele], sirs) were investigated with regard to their use in the early classification of pop. pat. at risk for developing sepsis. patients and methods: in a previous observational study on consecutive pat. after elective open-heart surgery, we had determined ele daily for at least pop. days (in pat. with a longer stay: until icu discharge). after publication of the newly proposed sirs definition, these criteria were retrospectively calculated and compared to ele. results: on the total of n = patient-days, both ele and sirs showed significant (p< . ) correlations with parameters reflecting the general and sepsis-related severity of the pop. clinical course. compared to sirs, the ele correlations were better (icu mortality: . vs. . ; icu treatment days: , vs. . ; days on mechanical ventilation: , vs. . ; days with vasopressor requirement: . vs. . ; number of microbiological findings: . vs. . ), the optimal classification criteria for the mainly sepsis-related outcome gave maximal youden indices of . for ele (ele >_ on >_ days, accuracy: %) compared to . for sirs (sirs present on > days, accuracy: %). accordingly, ele roc curve areas for both overall ( . ) as well as sepsis-related prognostic evaluation ( . ) were significantly (p< , ) larger compared to sirs ( . and . , resp.), this higher overall accuracy of the ele criterion was primary due to a more valid assessment already on the first and second pop. day, where sirs still had a high false positive classification rate ( % and %, compared to % and %, resp.). conclusion: in the early postoperative course after cardiac surgery, the sirs definition displayed a high false-positive classification rate (low specificity) for subsequent sepsis-related mortality compared to better classification results obtained by the elebute sepsis score. from the departments of medicine i and of "cardiac surgery, grosshadern university hospital, marchioninistr. , d- munich, frg. correlation between physiological and immunological parameters in critically ill septic patients. ma rogy, h oldenburg, r trousdale, s coyle, l moldawer, sf lowry a relationship between physiological parameters of severe sepsis and immunological function has not been established. in an effort to assess such a relationship we prospectively evaluated nine severely ill septic patients. physiological risk was assessed by the apache iii score , while one component of immunologic function was evaluated by peripheral blood mononuclear cells (pbmc) eytokine production after in vitro lps stimulation . four of the nine patients died. apache iii scores at h were lower in survivors (s) than in non-survivors (ns), ( -+ vs -+ p< . ), while apache iii scores at admission were not significant different between s and ns ( -+ vs -+ ). down regulation of cytokine production by pbmc upon lps stimulation was a transient event in s. while s demonstrated an fold increase of tnf-a bioactivity with[r~ hours, ns did not demonstrate any increase at all. a similar pattern was demonstrated for il- [ and il- immunoactivity. tnf was measured by wehi bioactivity, il- [~ and il- immunoactivity were determined by elisa. the sensitivity was pg/ml for tnf, pg/ml for il-ll and pg/ml for il- , respectively. in conclusion, both physiological as well as immunological functions of severe critically ill septic patients demonstrate predictive value for ultimate survival. while patients biological status seems to be more predictable by apache iii at day , p< . , the pattern of cytokine production by pbmc upon lps stimulation over the first h might be a reliable predictor as well. introduction: therapy of sepsis and its sequelae depends largely on its early recognition. many studies have investigated the change of certain mediators during sepsis and their potential to predict multiple organ failure and outcome. it was the objective of this study to investigate whether the onset of sepsis can be predicted by alterations of levels of interleukin- (il- ), tumour-necrosis-factor (tnf), pmn-elastase and c-reactive protein (crp). materials and methods: over a one year period, polytraumatized patients were prospectively studied (mean age y, % male, iss ). serum and edta-plasma samples were taken in h intervalls until the patient left the icu. il- , tnf, elastase, and crp were determined immunologically. sepsis was defined according to the criteria of 'systemic sepsis' (veterans" administration study, ) with at least of clinical signs: ( ) tachycar-dia> /min, ( ) temperature > , °c, ( ) blood pressure < mmhg, ( ) mechanical ventilation, ( ) leukocytosis > . /ml, ( ) thrombocytopenia < . /ml and ( ) presence of an obvious septic focus. clinical parameters, sepsis severity and serum levels were documented on a daily basis, beginning on day after trauma. results: of patients developed a systemic sepsis ( . %), and died. all mediator levels were elevated under septic conditions. the clinical severity of sepsis correlated well with the respective levels of mediators. in patients, who developed a sepsis the following day, il- ( vs. ng/l; p= . ), crp ( vs. mg/l; p= . ) and tnf ( vs. ng/l; p= . ) were significantly increased as compared to those patients who remained non-septic. elastase levels were considerably elevated but did not reach the level of significance. we conclude that il- , tnf and crp appear to be sensitive markers for prediction of septic complications in polytraumatized patients. objectives of the study: the assessment of liver function in polytraumatized patients who are at risk of developing mof is too inaccurate and late by using conventional biochemical parameters. methats: the injury severity of the patients (n= ) was determined by the injury severity score (iss). lidocaine is given at a dose of mg/kgbw over rain. i.v. and is metabolized in the liver by a cytochrome p- mechanism to monoethylglycinexylidide (megx). the metabolite is measured by a fluorescence polarization immunoassay. serial determinations of the test were performed between the ~t and the ~ day after trauma and were compared with other liver function tests (bilimbin, gldh, alt, ast). the systemic inflammatory response syndrome (sirs) is still a challenge concerning early diagnosis, therapy and prognosis. therefore, evaluation of inflammatory and disease activity becomes more important. c-reactive protein (crp) is a well established acute phase protein in chronic inflammatory diseases. recent reports suggest an induction of crp by interteukin- (il- ), a cytokine involved in the mediator cascade of sirs. on the other hand, tumornecmsisfactor alpha (tnfcx) is a very early released mediator in sirs removed very rapidly from circulation. in addition, soluble tnf receptors (stnfr~ , stnfr ) are released into circulation in the acute phase response. this study examines the kinetics of five acute phase proteins (crp, il- , tnfot, stnfr , stnfr ) in patients suffering from sirs. eighteen patients entered the study after diagnosis of sirs. blood samples were drawn every six hours during the first two days and every twelve hours thereafter. crp was measured in an routine turbimetric assay. il- was detected in an biological assay using the/l- dependent -cell line / . detection of tnfc~ was performed in an elisa system using a monoclonal antibody" for tnfo~. soluble tnf receptors were also measured by elisa. crp levels were elevated (> mg/l) in all patients and at all time points. crp values did neither differ significantly in patients with ( ± mg/l) or without ( a: ) multiple organ failure (mof) nor in survivors ( ± ) or non-survivors ( :t: ). in contrast, l- was elevated in patients wilh mof (mean pg/ml, range - pg/ml). il- levels correlated especially with lung dysfunction. tnf(x levels were consistently elevated in patients with mof. crp, il- and tnfoc did not correlate with each other. in contrast, levels for both stnfr showed a positive correlation (r= . ). patients could be divided into two groups by values for stnfr~ and stnfr : the group with higher soluble tnf receptor levels showed increasing values combined with a poor prognosis. the group with lower levels of soluble tnf receptor consisted of patients surviving mof or without mof. in conclusion, crp does not monitor the course of sirs adequately. in contrast, il- correlates with mof and episodes of high disease activity. high stnfr levels may indicate poor prognosis. klinik f r an/isthesiologie and operative intensivmedizin der cau kiel, schwanenweg , kiei, germany. ch. waydhas, md; d. nast-kolb, ivid; m. jochum, phi); l. schweiberer, mi) objective: to evaluate the irfflarranatory response after different types of orthopedic operations and compare them with the systemic effects of accidental trauma of varying severity. patients: in consecutive patients with multiple injuries (iss . ) the inflammatory response to trauma was prospectively studied. the patients were divided into groups according to their iss points. additionally, the alterations after secondary operations (> hr) were determined (msteosynthesis of the femur (n= ), pelvic girdle (n=ll) and spine (n= ), facial reconstruction (n= ), smaller osteosynthesis (n= ) and others (n= )). methods: specific and unspecific parameters of the inflammatory response were determined in the trauma patients every h, beginning on admission of the patient to the emergency room for a period of hr, and in the operative patients on the morning of the operation, at the end of the procedure and every hr during the first two days. results: lactate, neutrophil elastase, heart rate, po /fio -ratio, and other parameters discriminated significantly between the injury severity groups during the first hr (kruskal-wallis-test, p<. ). the degree of postoperative changes differed significantly (kmskal-wallis-test, p<. ) between the types of operations for lactate, heart rate, po /fio -ratio, nitrogen excretion and showed a strong discriminating tendency for neutrophil elastase and c-reactive protein. the extent of changes were highest after operations of the pelvic girdle, followed by procedures on the femur, spine, smaller bones, and the facial region. the postoperative changes after osteosynthesis of the femur or pelvis were comparable to the alterations noticed after smaller (iss to ) or moderate (iss to ) accidental trauma for neutrophil elastuse, heart rate, po /fio -ratio and parameters of the coagulation system. conclusions: there is a considerable inflammatory response to operative procedures that varies with the type of surgery. large operations cause changes in the body homeostasis that resemble those after multiple injuries. it remains to be established whether the inflammatory sequelae of surgical trauma are additive to the changes caused by accidental trauma. objective of the study: we retrospectively compared characteristics of elderly patients (~ years) and yeunger patients admitted to a surgical {sicu) and a medical intensive care unit (micu). we further studied the relations between advancing age, chronic disease, sepsis, organ system failure (osf) and mortality in the elderly group. material and methods: during a -year period, patients were consecutively admitted into the icu; and during a -year period, patients were consecutively admitted to t~mich. criteria for chronic disease, sepsis, osfsi.e. cardiovascular (cf), pulmonary (pf), renal (rf), neurological (nf), haematological (hf), hepatic (lf), and gastrointestinal failure (gf)-were derived from the literature. results: patients from the sicu and~cu were similar in age, number of osf, and length of stay. however, when compared to sicu patients, micu patients had more cf (p<o.o ), sepsis (p<o.o ), and mortality rate ( % vs. ii~, p<o.ool). in contrast, sicu patients had more hp and nf, and prior chronic disease (all, p<o.o ). of the total group of , patients, there were ( %) patients years of age or older. elderly and younger patients had comparable length of stay in the icj, but elderly patients had significantly more chronic disease, sepsis, and number of osf (all, p<o.o ). all osfs, except hf and nf, were more ~o~mon i~ mdeljl, eompard to you-ger patients. ~he mortality is also higher in elderly patients ( % vs. % in younger patients). after multiple logistic regression, only number of osf increased mortality by a factor of . ( % confidence limits, . - . ), age did not increased [odds ratio . (i. i.i )], while admission to the sicu reduced risk of death by a factor of . ( . - . ). conclusions: based on these results, we conclude that age does not have any impact on icu outcome in the elderly. the only prognostic factor is the extent of acute disease, as measured by the number of osf. hence, prevention of oaf may influence outcome in elderly patients with critical illness. the lower risk of death in sicupatients may be explained by the large proportion of postoperative elective patients with relative mild chronic disease, and the lower incidence of sepsis. department of surgery, free university hospital, de boelelaan , ~v amsterdam, the netherlands. septic shock and low output syndrome h.miyakawa, t.noguchi, s.hattori, a. mizutani, m. mori and n.honda objectives: cytckines are thought to cause the acute phase reactions under several critical conditions. we had investigated the relationships between cytokines network which included il- , l- ,acth and cortisol,and the outcome of the patients with septic shock or low output syndrome (los). materials and methods: in the septic group and los group,nine and four patients were enrolled respectively. furthermore, the patients in the septic group were divided into two groups,survival (n= ) and non-survival group (n= ).tn these patients,ll- , l- ,acth and cortisol had been measured every day after diagnosis of septic shock or los.the total number of measurements were points in survival septic group, l l points in non-survival septic group and points in los group.statistical analysis was used student's t-test to compare the mean of each group,and the changes were reported as clinicat significant if p< . . results: il- levels in non-survival septic group were higher than in both survival septic and los group.ll- levels in non-survival septic group were more significant than in both survival and los group.otherwise,there were no differences with respect to acth and cortisol among three groups. conclusions: we assumed that los would make several organs dysfunction as well as septic shock. but our results showed septic shock, especially non-survival,had different cytokins network mechanism for organs failure from los. lasson ,~, g/ ransson j, jijnsson k. eady diagnosis of complications after trauma is imperative to decrease morbidity and mortality. for this purpose an easy, cheap and fast diagnostic method is needed. the aim of this study was to analyse if complications after surgical trauma of various extent could be diagnosed earlier using preoperative or daily postoperative measurements of various plasma proteins than by using climcal signs of complications. material and methods: two acute phase proteins (c-reactive protein and antichymotrypsin), lbur main plasma protease inhibitors (alpha- -maeroglobulin, c estemse inhibitor, antithrombin ill and alpha- -antiplasmin) and one collagen precursor (procollagen iii peptide) were measured preoperatively and then once dally for days postoperatively. for the plasma protease inhibitors immunorcactive as well as functional levels were measured. haemoglbin, albumin and the white blood cell count were also measured. measurements were done preoperatively in patients and continued postoperatively in patients, % of the patients were operated on for malignancy. resulls: preoperative plasma c-reactive protein levels were higher in patients developing postoperative complications. this discrimination increased when plasma was sequentially analysed postoperatively, when a remaining high level ora second increase in plasma levels depicted a complication - days earlier than clinical signs. high levels were seen both in patients developing local as well as in patients developing general complications. there was no clear correlation between plasma protease inhibitory levels and complications, neither pre-nor postoperatively. procollagen iii paptide levels were slightly (but not significantly) higher postoperatively in patients developing complications. contusions: the acute phase response, especially concerning c-reactive protein, predicts postoperative complications better than both plasma protease inhibitors or collagen precursors. preoperative plasma c-reactive protein levels indicate the risk of postoperative complications, while daily postoperative measurements more readily depicts a complication, usually preceeding the clinical signs of complication by - days. dept. of surgery m'alm general hospital, s- i malmr, sweden. mof is a major threat to the extensive burned patients and associated with a high mortality rate. the role of endotoxemia in the pathogenesis of mof in burned patients remains controversial. in this study, we examined the relationship of plasma endotoxin levels to development of mof, and outcome in patients with thermal injury. methods: a prospective cohort study of patients admitted with burns covering more than per cent of body surface area was undertaken. circulating endotoxin concentrations were measured by modified limulus amebocyte lysate assay in serial samples of plasma. results: out of burned patients developed mof according to multiple criteria. the plasma endotoxin concentrations of patients with mof were . -- . eu/ml, significantly higher than that of patients without mof ( . - . eu/ml) on , , and days postburn (p< . -- . ). significantly more incidence of positive endotoxin test (>_ . eu/ml) was found in patients who developed mof as compared to that of non-mof during the observation period (p< . ). as the mean endotoxin levels increased, the prevalence of mof and death also increased (see table below), persistent endotoxemia carried a poor prognosis. conclusions: the present investigation provide further evidence that endotoxemia in severely burned patients commonly occur. cimulating endotoxin has also been found to be strongly associated with development of mof and mortality following major burn injury. multiple hemostatic changes occur in sepsis mad multiple organ failure (mof). to evaluate the role of platelcts in patients with sepsis and mof, we examined changes in surface glyeoproteins on circulating platelets of t patients with suspected sepsis and mof. the severity of sepsis and mof was assessed by eiebute and apache i scoring system, respectively.using flow cytometric techniques and platelets specific monoclonal antibodies, platelet surface expression of fibrinogen receptor on gpiib-iiia, ofvon willebrand receptor gpib, and of granula glycoproteins (thrombospondin, gmp- , and gp ) was measured. receptor density of gpiib-illa mad gpib on circulating platelets was not affected by sepsis or mof. in septic patients surface expression of activated fibrinogen receptor (libs expression) was significantly elevated (p< . ) and correlated well with severity of disease (f . ). no significant change in surface expression ofthrombospondin, gmp- or gp was noted in septic patients. in contrast, degranulation ofgraanle glycoproteins was significantly elevated in mof (! < . ) that correlated well with severity of mof (gmp- , r= . ; thrombospondin, r= . ).we speculate, that platelets in sepsis circulate in a hyperaggregable (fibrinogen receptor activation ) but still reversible state that results in increased risk of microthrombotic events. in the course of the disease, irreversible platelet degranulation might occur and may play an important role in development of mof. abdominal sepsis is still associated with high morbidity and mortality. the present study aimed at evaluating patients with abdominal sepsis treated at our surgical intensive care unit during a -year period with the aim of identifying potential prognostic factors, bacteriological cultures, diagnostic procedures, treatment and outcome. during the period - i patients with abdominal sepsis were treated at the icu at our university hospital. patients were women and men with a mean age of ( - ) years. in cases, the abdominal sepsis occurred as a postoperative complication. the patients were scored according to apache ii and bacteriological cultures and the occurrence of organ failure were noted. the patients were hospitalized in median for (- ) days out of which (- ) in the intensive care unit. out of patients ( %) died in median after ( - ) days. the primary cause of mortality was multiple organ failure ( / ; %). apache ii scoring could not predict a fatal outcome. abdominal bacterial cultures were dominated by bacteria of enteric origin ( %) and in % cultures grew multiple bacteria. patients bad organ failure and multiple organ failure. / patients ( %) had abdominal sepsis due to diffuse peritonitis despite a morphologically intact gastrointestinal tract and the absence of localized abscess formation. mortality in this group was significantly higher as was the percentage of positive blood cultures and the occurrence of multiple organ failure. abdominal sepsis is still associated with a high mortality, predominantly caused by multiple organ failure. abdominal culture findings are dominated by bacteria of enteric origin. in about / of patients with severe abdominal sepsis a diffuse peritonitis with intact gastrointestinal tract without localized abscess formation was found. in this group the mortality was increased as well as the risk of developing multiple organ failure. during the period from january to september patients, mean age + years were referred to our department of resuscitologywith the diagnosis of eclampsia. all the patients were delivered by cesarian section and were mechanically ventilated for . _+ . days. diagnosis of sepsis was confirmed in cases by clinical and microbiological methods. patients were divided in two groups: lnon septic patients, -patients with sepsis, the control group consisted of patients after cesarian section without symptoms of eclampsia or infection. we determined plasma concentrations of immunoglobulins a,g,m(a,g,m), complement factors (c ,c ), alphal-antitrypsin (aat), trausferrin (trf) and albumin (alb) using beckman (usa) analyzer,protein concentration, using kone (finland) analyzer. a(mg/dl) g(mg/dl) m(mg/dl) c (mg/dl) c (mg/dl) k +- + _+ + +- -+ " -+ * _+ " -+ ' _+ " +_ '* -+ ** -+ "* -+ "* _+ " in a prospective study we investigated serum of severly traumatized patients withdrawn directly after admission at our hospital (tr i). follow up controls were taken daily until day ten after trauma (tr ii). two control groups were performed: serum of healthy volunteers (co, n = ) was investigated as. well as serum of patients undergoing elective herniotomy (n= ) hours before (op i) and hours after operation (op ii). serum bactericidal index (sbi) was determined using a hemolytic e.coli strain :k :h . / suspension with a final concentration of - cfu were incubated with l oopl serum. after overnight incubation sbi was calculated according a special formula. results: co . _+ . opi . _+ . opii . _+ . * tri . _+ . "* trii . + . ** (*:p< . ; **:p<o. ) sbi represents a simple and reproducible method to detect immunobiological alterations after trauma and elective surgery. patients undergoing elective surgery showed slight but significant diminuation of sbi h after operation. deterioration of sbi was observed in serum of traumatized patients directly withdrawn after admission at our hospital. ten days after trauma significant improvement of sbi was found. further investigations have to be done to proof sensitivity and prospectivity of this simple but reliable test. a sepsis like syndrome (sls) occurs in i % of our patients following cardiac surgery. sls is characterized by a severe decrease in systemic vascular resistance requiring the use of vasopressors to maintain adequate hemodynamics in the presence of negative blood cultures. in order to determine whether preoperative factors predict the occurrence sls we retrospectively studied patients with sls (group i: age . ~ . years); they were compared to control patients (group : age ± . years). treatment of sls consisted of aggressive fluid replacement and norepinephrine infusion. rydrocortisone ( mg/ hrs) was given on the first day. antibiotics were switched prophylactic to cover gramnegative bacteria in pts. preoperatively pts in group revealed a significantly lower cardiac index ( . ± i/min/m ) compared to group ( . ± . i/min/m ; p < . ) higher left atrial pressure (group i: . ~ . mmhg; group : . ~ . mmhg; p < . ) and lower ejection fraction (group i: . z . %; group . ± . %; p < . ). bypass time, minimum and mean blood pressure on bypass and aortic clamp time were not different between the groups. immediately postoperatively (pop), the white blood count was elevated (group i: , ± , /ui; group : , ~ , /ui; p < . ) and core temperature hours pop was higher (group i: . . °c; group : . ~ . °c; p < . ). serum lactate was already elevated on arrival on the icu pop (group i: , z . mmol/l, group : . ~ . mmol/l) but dropped within hours (p < . ). icu stay (group i: . ! . ; group : . ± . days; p < . ) and mortality over months (group i: . %; group : . %; p < . ) were significant higher in group i. sls is observed more frequently in patients with preoperative cardiac congestion and results in longer icu stay and higher mortality. although intraoperative hemodynamics are similar in both groups, the white blood count and serum lactate levels suggest an intraoperative cause for sls. further investigations will focus on possible intraoperative causes of sls. the objectives of the study were to estimate the problem of stress ulcers in critically ill patients and to compare ranitidine and omeprazole to determine their efficacy in the prophylaxis of stress ulcers. patients who were admitted to the intensive care units between january and july ' with polytrauma, sepsis or those requiring ventilatory support for more than hours were selected for the study. the patients were randomized into groups to receive ranitidine, omeprazole or a placebo. the changes in gastric ph were monitored hourly and the patients underwent an upper gi endoscopy hours after the start of the study to look for stress ulcers and hemorrhage. the drugs were withdrawn one week after the start of the study. of the patients selected for the study, had undergone cardiac surgery, had polytrauma and had sepsis. the change in average pre and post drug gastric ph was + . in the omeprazole group, + . in the ranitidine group and -). in the placebo group (p < . ). endoscopic evidence of stress ulcers was seen in % of patients not on prophylaxis, . % of those on omeprazole and . % of those on ranitidine. while all the patients in the placebo group who had stress ulcers showed endoscopic evidence of hemorrhage, % of the patients with ulcers in the ranitidine group and none of the patients with ulcers in the omeprazole group showed hemorrhage (p < . ). in critically ill patients the incidence of stress ulceration is high. an alkaline gastric ph is protective against stress ulcers. omeprazole when used for prophylaxis against stress ulcers in critically ill patients or those on short term ventilation will reduce the incidence of formation and hemorrhage from stress ulcers. the early and accurate diagnosis of sepsis is of key importance for critically ill patients survival. many studies have shown that tumor necrosis factor ~x (tnf ~) and interleukin- (il- ) are mediators of the inflammatory response in sepsis. bacterial antigens interact also with antigen specific t cell receptors and the activation of t ceils takes place. activated t cells release soluble interteuldn- receptors (sil- r). the aim of this study was to evaluate: the significance oftnf c~, il- and sll- r in the diagnosis of bacterial sepsis and to evaluate the correlation of clinical and laboratory parameters with serum levels of tnf ~x, il- and sll- r. we examined patients with clinical signs of sepsis treated in the intensive care units of university medical centre ljubljana. venous blood was drawn from each patient within hours after the first clinical signs of sepsis. clinical data (body temperature, blood pressure), laboratory dam (peripheral white blood cell count with differential, platelet count, c-reactive protein), and quantitative blood cultures were recorded. serum levels of tnf c~, ,- and sil- r were measured in septic patients and in adult healthy controls. tnf ct, il- and sil- r serum levels were measured by commercially available ria and elisa (amersharn and eurogenetics) kits. the differences in serttm levels of tnf ~, il- and sil- r between healthy control and patients and correlation between clinical and laboratory parameters were calculated. we found that only sll- r serum levels were significantly (p< . , student t test) increased in patients in comparison with healthy controls. of all indicators of sepsis that we compared, only hypotension was significantly correlated with tnf ~x levels and leukocytosis with ] ,- levels. we conclude that only the increased serum sil- r levels were predictive of bacterial sepsis within hours after the first septic signs. tnf c~ and il- levels were not significantly increased at that time in lifts small group of patients. in order to create high precision prognostic system for patients with sepsis and septic shock we have made prospective and retrospective analis of cases of sepsis. patients were included in this analis according bone's criterions of sepsis. comparing with apache-h, we have excluded such phisiologic variables as:temperature,na+ ,k+ ,}it, ph,which had a little influence on prognos of illness. we have added the other, more important variables: biilirubin, plateles,pti. we took into account: the seat arrangement, methods of respiratory support, comorbid conditions as: caneer, diabets,obersity. in general new score has headings: -physiologic variables, -age, -seat arrangement, -chronic comorbid conditions, -methods of respiratory support. roc -analis have showed a greater precision of our score, compared with apache-ii score. there is significant difference between the roc-curve of apache-ii scare. the k.p.tit~v.,i.e.gurmanchuk. objectives of the study. sepsis is a severe complication of the local infection, th e fever and the systemic inflammatory response syndrome may take place in such a case as manifistations of the inflammation induced by bacteria. the systemic fever observed in infection is known to develop due to the action of certain cytoldnes. other systemic manffistation of inflammation are the production of acute phase reactants, acute phase proteins, the activation of the complement system. ivratertals and methods. we observed children with light and children with severe course(l- class of serionuess) of the sepsis. the ftmetional activity of the complement system (ch , level of c -c , factors b told d, c a), level of c-reactive protein (crp} and tumor necrosis factor (tnf) were investigated, results. the level of crp was increased in the children with more severe class of the septic process in both group ( %- st and ioo%- nd}( with light and hard eours of disese) of patients. parameters of the complement system -the activity of cl, c , c components, activity of b and d factors-were increased in the seram of children with light course of sepsis. in children with severe course of sepsis these parameters were signffieantty decreased, especially in the group of chiidrellwith higer level of crp. we found the negative correlation of the tnp-alfa concentration with the functional activity of classical pathway components (cl~ ) and the positive onewith the funfional activity of the alternative pathway factors b-and d of the complement system. conclusions. thus, in children with different course of sepsis certain changes in levels and functional activity of an acute phase o[ inflammation proteins -crp and the complement system ones -&ire characteristic. the relationship between the tnf-alfa level and the activity of classical and alternative pathways proteins indicates on its role in the complement proteins genes expression. schr der j, braun j, rennekampff ha, schr der dw various alterations of the immune response are known in trauma, but the course will not be complicated by multiple organ dysfunction syndrome (mods) in all patients. phenotyping of cells offers a rapid system of evaluation certain aspects of the immune response. different subsets of lymphocytes and neutrophils were determined to evaluate their prognostic role in developement of mods. in trauma patients with an injury severity score (iss) > (mean iss = ; mean age years) lymphocyte and neutrophil phenotypes cd (t-cells), cd (t-helper cells), cd (t-suppressor cells), ratio cd /cd , cd b (receptor for cr ) and cd (fcriii) were measured on day , , , , and post trauma. the expression of class ii histocompatibility antigen (hladr) on monocytes (hladr+ cd ) and il -receptors on t-helper cells (cd /cd were determined as well. the percentage of cells was monitored by immunofluorescence using monoclonal antibodies and three color cytometry. the percentage of hladr+ cd were significantly lower an day , , and in patients who developed mods (p< , ) compared to patients without mods and a healthy control (p<o,o ). the expression of il receptors on cd was significantly different only on day . no differences could be measured in lymphocyte phenotypes cd , , and cd /cd -ratio as well as in expression of cd b and cd on neutrophils. class ii histocompatibility antigen (hladr) on monocytes offers the best ability to reflect cellular immunosuppression in trauma. this parameter allows the best differentiation of patients with and without mods. we retrospectively studied the relative importance of age, prior chronic disease, sepsis and organ system failure (osf) to determine outcome in critically ill patients with acute renal failure (arf). arf was defined as serum creatinine > /zmol/i, a twofold creatinine rise in prior renal insufficiency or the need of acute renal replacement therapy. definitions for prior chronic disease and other osfs -i.e. cardiovascular (cf), pulmonary (pf), neurological (nf), haematological (hf), hepatic (lf), and gastrointestinal failure (gf)-were derived from the literature and described previously. of the consecutively admitted patients to a surgical and a medical intensive care unit during -ye r period, ( %) had arf. arf mortality was %. ninety-eight percent had other osf. overall, cf, pf, gf, and nf was significantly more common in nonsurvivors than in survivors (all, p<o, ). although both the number of osf before and after arf was higher in nonsurvivors than in survivors (p< . ), the number of osf before was higher compared to after arf in both groups of patients (p< . ). furthermore, age, cf and pf before the ontset of arf, nf thereafter, and renal replacement therapy were related to mortality (all, p< . ). however, prior chronic disease was not related to mortality and chronic renal insufficiency was inversely related to outcome (p< . ). after multiple logistic regression, advancing age, cardiopulrnonary failure and sepsis before arf, and nf after arf remained significant. these results show the high prevalence of arf in critically ill patients and that arf rarely occurs alone. the prognosis of arf in critically ill patients is poor, especially if associated with advancing age, additional osf, particularly cardiopulmonary failure and sepsis before arf, and nf after arf. hence, prevention of cardiopulmonary and sepsis before the outset of arf may influence outcome in arf in patients with critical illness. the search cominnes for a magic bullet ta help critically ill petienta, bat recent elini ',.ml rials of agents that showed ixomise in animal studies raise questions about such trials. expcrlmemai uvidan~ :in anlmats and human volanteet~ for concepts, mechanisms and treatment of inju~ ur illness can be substentlal, l~rauasive and exciting but the positive effects of potential therapy suggested by such animal and ctinieal research may be difficult to prove in sick patients. efficacy of a new txeatment can be difficult to prove became elinie.al situations, hi spi~e of important biologic phenomena, are v~iable and conrplex. there is redundancy and overlap of mediators ~d problems of timing of therapy. a majt~r cause of this dilemtns is not with the trials or hypotheses, but rather the promem of the cause or the causability of the human dise~.se that is trebled. this is/hu "causa nets" or '*specific cause" as described by stehbens, when prevention or tre.atmcnt is based em the sauna nero, extinetiem of the disease is ix~ssible as with saul/ pox. only elimination of the cause wl cure the disease. careful animal studit~s evaluate single pcrturbmiuns for survival or other changes. an ldso preparation may be infiuancad aignificantly by a ~mall clangs which may be insignific~lt ht pstiants, most human experiments ate carried out in normal volunleers with a single porlurbation, such as a low-level, non-lethal dose of a substance. such studies are huportant in defining mechanisms attd mediators of disease, in our world of injm'ed, operated, infected and inflslned patients, there are many diseases, mof is not one of lhem. it isn't even a syndrume, it is the final pathway for a number of diseaaes~ each of which has a cause. mecormock believes thal a "multi-causal" etiology is a euphemism for ignoranc# or a synonym fo~ unknown etiology. admission iv sn icu, a high apache seer% the sepsis syndrome, mof, muds, or sirs and having predictors indicating potential mortality, are not diseases. the inmpors are getting negative results in trials of agents on eiek or septic icu patients and aru now becoming splitters, dafining subgaoups at higher risk for death. such an approach may produce positive results if the diseases are identified and the treatmeul is specific for them, a major challenge in deterraintng the efficacy of new therapies for sepsis nnd shock is identifying patients whose e#temtc inflammatory response is appropriate from those in whom the mediator| are contributing to end organ system damage end death, traditional and recently revised categorical patient descriptions such as sepsis syndrome, sepsis syndrome with shock, multiple organ system failure, or the recently proposed systemic inflammatory response syndrome (sirs) may not be sufilcient since they identify individuals at varying levels of risk for short term mortality, the efficacy of new lmmunotherapy may be directly related to the patient's severity end risk of mortality at entry to the study. we recently reported (jama ; : z - ~ ) a comprehensive risk assessment approach for patients with sepsis syndrome, a common eategarlcal description used as an entry eriterlon for many trials. by screening s database of , recent admissions to hospitals in the united states and western europe, we identified , patients who met sepsis syndrome criteria but were not enrolled tn clinical trinh, we then developed a survival model end severlty assessment method to estimate their g.day mortality risk. k..ente physiologic abnormalities were the most important prognostic factors ( % of total chl square) influencing outcome. the specific disease resulting in ice admission and the days the patient spent in the hospital and icu before qualification were independent risks, as were age and a clinical history of cirrhosis. the predictive model was cross-validated within the original , patients with a receiver d_perator characteristics (roc) area of . and in two independent databases, the first independent database contained hospitalized patients with gram-negative infection meeting criteria for sepsis syndrome (roc= . ); in the second, the placebo patients withtn the recently completed phase ill e~aluatlan of il- ra (rocffi . ). in all databases the risk model was able to accurately risk stratify patients throughout the range of risks that varled from a very low % to a very hlglt %. by drawing on the ready avatlabillty of large clinically aeuurate, current databases and using the power of modern statistical techniques to model the bodfs response to sepsis, we are able to produce very aeettrate pre-treatment risk estimates, these prospectively defined and continuous risk estimates reduce reliance on multiple subgroups and data dredging that can compromise a stady's integrity. they cart provide a nnlform method for patient description across clinical trials of different attempts at immmlotherapy. risk calculations can also be useful in developing entry criteria for phase ii evaluations in order to initially test efficacy on a limited number of high risk patients, fonowing sueeessinl completion of a definitive trial, the risk estimates can be used to develop specific |ndlcattons for s drug's use and can monitor the impact of both new and multiple compounds on patient outrome, when dealing with therapeutic trials for the treatment of sepsis, there are ways of checking the comparability of the groups (placebo and treated groups) -to use several description items such as age, sex, preexisting disease, number and nature of organ failures, physiology score, such as saps il ( ) -to use a model for risk of death either from a score (apache ii, lii, ( ) saps ii) or directly (mpm ii system ( )) before using the model for risk of death in septic patients, is it mandatory to validate it (by calibration, i.e. goodness of fit, and discrin'dnation, i.e., roc curves) first on a data base using the same definition for sepsis as in the trial and secondly on the placebo group of the trial ? or is it better to use a specific model for each trial ? the general models usually do uot fit for septic patients. there are methods to make them fit : either to add variables to the original score (model for sepsis using the apache iii system ( )) or to customize the model. for saps ii the customization is applied to the equation regression, using the same saps ii score. for mpm i each component of the model can be customized. several remarks : only one model is published to be applied at the icu entry ( mpm ii ) before any therapy. -most of the published scores are calculated from the lsl icu day. applying these scores to the let day of sepsis could have a very different significance. -is the accp classification of sepsis useful at the bedside to select oatients ? ( the pathophysiology of sepsis involves a dynamic interaction between the host and the infecting microorganism(s). a multitude of biochemical and hemodynamic interactions occur which function in concert to determine the ultimate outcome of the septic episode. the complexities of the septic process preclude outcome analysis by in vitro systems or computer models, animal studies have become indispensable in evaluating new therapeutic agents in the treatment of sepsis. these studies provide valuable information on safety, pharmacokinetics, relative efficacy and dosing strategies for potential therapeutic agents in the treatment of sepsis. there are three basic types of animal models: ( ) toxicity models with injection of bacterial endotoxin or exotoxins; ( ) live or killed bacterial challenge models; ( ) actual infection models. all models employ some form of external manipulation under controlled conditions to induce sepsis and to analyze potential therapeutic efficacy. these experimental requirements may limit the ability of animal models to accurately predict the clinical value of new agents in human sepsis. four major end points are analyzed in the various animal models: ( ) hemodynamic parameters; ( ) modulation of host-derived inflammatory mediators; ( ) resolution of end organ injury; or ( ) lethality. each animal model has certain advantages and limitations. species-specific differences in physiologic and immunologic features make it difficult to directly extrapolate the results of animal experiments into clinical medicine. while no ideal animal model exists, consistent benefit of a new immunotherapeutic agent in multiple animal systems provides the most compelling preclinical evidence of its therapeutic potential in septic patients. consensus-assisted development of a study protocol on sepsis: an important difference from previous randomized trials there have been several, and perhaps too many metaanalyses of cliuical trials on surgical infections, but their results have only rarely been incorporated into the design of new randomized trials in sepsis. metaanalysis is retrospective. it seems more important to analyse and to criticize the design of tria/s before they are ongoing. incorporation of that into development of a study protocol is the aim of the following procedure: the time-schedule of about two years should include further cell culture and animal experiments after the first successful studies showing effectiveness. exhaustive evidence for a dose-dependent cost-banefit and near complete explanations of the pathomechanism should not be awaited for development of the protocol of the clinical study. however, a discussion forum of experts should be performed in a recently published, almost perfect trial in order to see the frontiers of the present research in cell and molecular biology, clinical pharmacology, statistics and decision making. this should be followed by a metaanalysis of at least study designs on sepsis with methods of formalized medical decision making (decision tree). clinical algorithms -developed by objective methods including nominal group processes -should be developed to standardize any concomitant treatment in the centers considered for a multinentre trial. the latter is usually necessary in the field of sepsis. finally, a pilot study should be performed to demonstrate not only feasability, but to learn about all possible types of interventions which modify endpoints as response variables. especially mortality is not free from such effects. the time for the individual phases of this consensus-assisted process of protocol development have to be shortened by their temporal hiterloeldug. due to the fast discovery of new chemical factors in sirs it wilt otherwise be impossible to come up with results of randomized trials which are fascinating enough to be incorporated in daily routine treatment. severity scoring systems are intended to provide a population-based estimate for the risk of an unwanted outcome resulting from some disease process. in the area of infections and the "septic" response, this has routinely been defined as death. for anti-infective trials, there has been a reasonable correlation of severity (apache ll) with outcome measured as persistent or recurrent infection, but this is not as robust as the association with mortality. it is expected that non-lethal failure would not immediately correlate with disturbed physiology: the basis for antiinfective failure depends on a variety of factors not measured in severity scoring, such as type of infecting organisms, density and susceptibility to administered antimicrobia[s, and anatomic features of infection (e.g., pancreatic). severity scoring is important in differentiating these "categorical" variables from continuous (physiologic) variables. there are, additionally, a series of problems related to the cause of death in relation to severity scoring. in those tdals in which cause of death is analyzed, high severity scores did not routinely predict death as an immediate sequelae of sepsis/septic shock. many of the predicted deaths occurred remotely of progressive background diseases. other important problems with severity scoring have to do with lead-time bias: hew long patients were ill before entry into the study. statistical techniques for condensing various categorical and continuous variables, as well as factoring in information requiring the pharmacodynamics of agents which affect outcome, are being developed to further refine deviation from predictive equations as an outcome measure. stepwise logistic regression analysis has identified the presence of shock, intm-abdominal or unknown source of infection, hospital acquired infection, age greater than years, neutropenia and inappropriate antimicmbial therapy as independent variables associated with increased mortality in sepsis. maldistribution or" these variables is a concern in targe randomized clinical studies. it should be noted that only the selection of antimicrobiai therapy could be addre.~'sed and altered at the time of enrollment in a sepsis clinics] trial. inappropriate antimicrobial therapy has been noted in %- % of septic patients. well designed, randomized sepsis trials do not assure the absence of imbalance between treatment and control groups with regard to inappropriate antibiotic therapy, especially with subgroup analysis. statistical manipulation to correct for any imbalance is appropriate but avoiding imbalance is ideal. selection of antibiotics in septic patients requires such considerations as clinical setting (history, physical examination and underlying disease), appropriate laboratory tests (such as gram stain), identification of source and associated bacterial flora, antibiotic susceptibility patterns for that hospital and likelihood of resistant organisms. determination of inappropriate antibiotic therapy is made retrospectively after culture results are available. in some circunmtances the organism and antibiotic sensitivities cannot be predicted, while in others it can. leibovici et al identified hospital acquired infection, antibiotic treatment before a bacteremic episode, endotracheal intubetion and thermal trauma as independent variables which predicted isolation of a multiresistant organism. the same authors developed a predictive index for resistant organisms, which when compared to prescriptions of attending physicians, improved antibiotic selection in critically ill patients. including a standard antibiotic regimen for all septic patients in a clinical trial is impractical. there is too much interhopsital variability in flora and antibiotic susceptibility. in addition, the aggressive broad spectrum coverage necessary for some patients is inappropriate for others. a protocol which guides a prescribing physician through a logical chain of reasoning might improve antibiotic selection in critically ill patients and could be included in the design of a clinical trial in sepsis. although this approach might minimize potential for maldistribution of inappropriate antimierobial therapy, it would likely create multiple problem areas. will the patient's physicians agree to the protocol choice? what is the availability of speciftc antibiotics at a participating hospital? what is the aplpiieability of study results in typical clinical situations? if there is agreement that thin is an appropriate antibiotic protocol, should it not be used in all seriously ill septic patients in the hospital? in other conditions such as ards, the success of protocol therapy is dependent upon measurable goals such as pao and highest plateau pressure, while measurable goals are not as clear in choosing antibiotics. based on the above confounding issues, a complex and extensive algorithm covering many potential possibilities of error and specific antibiotics seems impractical; however, an algorithm focusing on three to four major common errors in antibiotic selection and dealing with classes of antibiotics is plausible for inclusion in sepsis clinical trials. in this session we will attempt to outline the methodology of such a future study, emphasizing the immense difficulties involved in its proper conduction including: design, selection of patients, surgical considerations, supportive treatment, the "human factor" and ~nd points. only a close cooperation between a few dedicated surgeons, obsessively studying a large number of well selected and stratified patients over years, could make such a study possible. algorithmis have frequently been mistaken for the graphic format in which they are presented. however, an algorithm is neither a flow chart nor a list of instructions; it is a step-by-step approach to solving a problem. likewise, a clinical algorithm is a step-by-step approach to solving a clinical problem. cas are deterministic, which does not mean that they are rigid. they are practical rather than mathematical algorithms, that tan be used only with clinical judgment, and must be tailored to each patient. there are a number of types of, or uses for, cas that can improve sepsis management, including: diagnostic or severity scorm cas, a management ca for managing sepsis constructed according to recently published standards for use in teaching research protocol algorithms that must be used to collect data or guide implementation of a clinical trial aimed at validating one or more dicisions in the management ca; finally, protocol-style cas drawn from the management ca and should be validated using matching outcome studies. organisational problems peculiar to multicentre trials the organisation of any randomised clinical trial is fraught with difficulties, and that of a multicentre trial particularly so. there are problems associated not only with the principles on which the investigation is based but also with the detailed organisation and analysis. are all the participants truly unbiased about the relative merits of the regimens being studied? is like being compared with like -are all the end points and the standards for measuring them clearly defined so that they cannot be misunderstood? are all eligible patients being studied, and their results reported? is truly informed consent being sought that will satisfy not only the patient's right to choose what happens to him, but also the law of the land? and, finally, how can the participants be sure that the trial will be stopped at the right time, to ensure that no treatment is given to any patient that might be harmful? these questions may seem insoluble, but until we tackle them we shall never be certain. dr.m. ev~s, scar~mu~ hospital, scarborough, nonhyo~s~ y ql, u.k. increasing knowledge of the pathogenesis of sepsis and septic shock has led to the development of many new therapies and technologies capable of preventing, blocking or even reversing the deleterious cycle of events. recent results of subsequent multicenter trials testing some novel therapeutic drugs, however, did not confirm the promising sometimes overwhelming effects obtained experimentally. it is postulated that this is largely due to inappropriate design and conduct of multicenter trials as currently performed. one of the shortcomings emphasized in this paper is the "treatment mix" problem. multicenter trials in sepsis are performed because no single center is able to recruit the necessary number of eligible subjects within a reasonable time. each single center (icu) has its own individual policy which cannot be standardized for the trial (treatment mix). even if we ascribe that each icu did the "best" for their patients, it is highly probable that the outcome differs between the icus. this translates to the effect of the new therapy under investigation. ]t is therefore worthwhile to consider that icus must first demonstrate a certain standard based on the patients' outcome (audit) before becoming accepted as a participating center. this prerequisite ensures comparable quality between participating centers, led to the reduction of "noise level" and increases the probability of positive study results. the riyadh-icu program based on standard mortality ratios is recommended as an audit instrument. during the last decade most clinical trials of potential therapeutic agents in systemic sepsis have failed to demonstrate benefit from the drug under study. although in many instances it is entirely possible that the agent in question does not have clinical efficacy, an equally plausible explanation for the multitude of negative results is that the studies were improperly designed and thus could not show therapeutic benefit, if it existed. problems which have been identified in these various trials include inappropriate or unrealistic definitions of response to the drug; inadequate sample size, with attendant insufficient statistical power to demonstrate a difference, if it existed; insufficient standardization of customary therapeutic maneuvers in septic patients; imprecise criteria for patient entry into the study, which creates unacceptable inhomogeneity between control and treatment groups and invites unjustified post-study subgroup analysis; the lack of a formal sequential monitoring procedure for interim data analysis, which could potentially affect patient safety; and a primary analysis of study results that excludes protocol deviants and is not according to intent-to-treat. these and other problems have impacted upon the validity of the various trials conducted to date and may well have prevented the resolution of controversies surrounding the use of certain agents in the treatment of septic patients. the complexity of bacterially-induced septic shock involves a cascade of events that evolve over time, beginning with the proliferation of offending organisms and followed by the release of toxic mediators from the bacteria. endotoxins [e.g. lipopolysaccharides) from gram-negative bacteria initiate septic shock by precipitating a systemic inflammatory response syndrome (sirs) through release of a broad spectrum of hostderived mediators. over the last century, hundreds of these endogenous mediators have been proposed to serve as pathophysiological substances in this "alphabet soup" of cytokines, eieosanoids, biogenic amines, kinins, peptides, ions and hormones. an evaluation of the literature on septic shock leaves the investigator with a sense that, although many pieces of the septic shock puzzle have been presented, no clue was provided to indicate how these pieces fit together, in an attempt to assess objectively the causal role of individual mediators, we recently completed a collective meta-analysis of mediators that were individually reviewed and subjected to koch-dale analysis of causality by distinguished authorities in the field ( ). in summary, our attempt analyze available evidence to support a cause-and-effect relationship for putative mediators of septic shock revealed that only eight of the mediators analyzed could be strongly inferred as playing a causal role in septic shock. evidence supported the involvement of endotoxln, endorphins, complement, interleukins, tumor necrosis factor, eieosanoids, platelet activating factor and calcium. other mediators either lacked adequate supportive evidence or were not conclusively involved. posttraumatic outcome may be reduced by an amplified stress response mediated by neural and humoral stimuli. the classical hormonal catabolic response is predominantly mediated directly or indirectly by neural stimuli, while most changes in inflammatory responses such as leukocytosis, acute phase proteins and changes immunofunction are mediated by humoral mediators. clinical experience from controlled studies suggest afferent neural blockade and modification of stress response to improve organ function and postoperative outcome, and limited experimental studies also suggest neural blockade to be of beneficial value in shock states. of special value may be the improved gastro-intestinal motility after neural (visceral) blockade. no outcome studies are available from patients with major truma, multiple organ failure or sepsis, conditions where humorai mediators may be more important to modify than neurally mediated responses. future studies should investigate the clinical outcome effect of combined neural and humoral mediated blockade, as well as the potential role of an initial neural blockade in elective trauma (first hit) to improve the metabolic scene and outcome if a postoperative complication (second hit) should occur. neutrophil elastase is the predominant protease of the human neutrophil. this enzyme, and a variety of other enzymes, including neutrophil collagenase, are released by activated neutrophils in the setting of high concentrations of reactive oxygen metabolites. in addition, in this extracellular environment, the normal antiprotease defense mechanisms of the organism may have been severely inactivated oxidatively especially along capillary endothelial cells. accordingly, release of neutrophil elastase which mediates inflammation and degrades most extracellular matrix proteins, including elastin, fibronectin and many forms of collagen, may contribute to tissue injury and organ failure in a variety of diseases ranging in severity and acuity from pulmonary emphysema to the adult respiratory distress syndrome (ards). as a result, significant efforts by the pharmaceutical industry have been directed not only toward the development of human neutrophil elastase inhibitors, but also toward their characterization in a variety of animal models of neutrophil mediated diseases, and their evaluation as potential therapeutics for the treatment of a variety of human clinical conditions. data from animal models of organ injury and failure along with data collected from a series of patients at risk for ards and with ards will be presented and discussed as a rationale for inhibition of neutrophil elastase. parameters that need to be monitored to obtain success in future clinical studies will also be presented in this context. have turned out to initiate and maintain organ dysfunctions in severe posttraumatic and postoperative courses. such proteolysis-induced disorders are especially rendered possible by the concurrently arising consumption of inhibitory regulators (e.g. a vproteinase inhibitor=a pi, antithrombin iii=at iii) via cofnplex formation and/of proteolytic/oxidative inactivation. besides the well-known destructive potency against protein substrates, proteinases such as elastase or thrombin (active or in complex with the serpin inhibitors ~ pi or at iii) are also supposed to increase the inflammatory reaction by inducing cytokine release or shedding of soiuble adhesion molecules from various inflammatory cells (pmns, monocytes/macrophages, endothelial cells). those cell-derived mediators may provoke further cell activation through an autocrine/paracrine loop thus increasing significantly the proteinase burden at the inflammatory focus. therefore, this noxious circle might be interrupted by a convenient proteinase inhibitor therapy to ameliorate the inflammatory process. to verify this hypothesis, high dosis of at iii (mean plasma levels up to %) were applied in first controlled randomized studies on surgical patients suffering from multiple trauma or severe sepsis. in control patients we could clearly demonstrate the sequential appearance of proteinase/serpin-complexes (fa pi, tat), cytokines (il- , il- ), and soluble intercellular adh sion molecules (icam, elan, p-selectin) in the circulation. these factors turned out to be a helpful tool for early diagnosis and prognosis of forthcoming organ dysfunctions. interestingly, m at iii-treated patients a significantly reduced release/production of inflammatory mediators became obvious concomitant with the improved clinical course in comparision to an increasing deterioration in control patients. soluble mediators of inflammation are cytokines (e.g. il- , il- and tnfe) as well as breakdown products of complement proteins (e.g. c a and terminal complement complex). therefore, attenuation of both, release and generation of these synergistically functioning mediators together with stimulation of release of antagonists including soluble forms of receptors are potentially beneficial in all kind of inflammatory diseases. intravenous immunoglobulins (ivigs) are known to have an anti-inflammatory effect in humans (nih consensus conference on wig, ) . the mechanism of action becomes more and more clear. in single cells stimulated by anti-cd mab wig was able to down-regulate production of il- , il- , ifn% and tnfj~ significantly. igg coated solid-phase stimulates release from monocytes of interleukin- receptor antagonist (il-lra) but not of il- ; this observation is very much in contrast to the effect of endotoxin in the same in vitro system. furthermore, naturally occurring anti-cytokine antibodies can be demonstrated in apparently healthy individuals and in wig prepared from plasma pools. some of these antibodies can be detected by antigen/antibody reactions in fluid phase (anti-ll-le, anti-il- ), some only by solid-phase detection systems (anti-ll- , anti-ifn% anti-tnfc . infusion of wig to patients suffering from inflammatory disorders have resulted in a decrease in il- in circulation. during infusion wig might induce an acute but transient rise of tnfo~, il- , and il- . self limitiation of this process might be due to demonstrable instant release of il- ra and soluble tnfo~ receptors. in vitro ivig has also been shown to attenuate complement activation via the classical pathway. furthermore, acid haemolysis of erythrocytes in paroxysmal nocturnal haemoglobinuria could partially be prevented by ivig. ivig was able to attenuate forssman shock in guinea pigs. we have seen a patient with congenitally elevated turnover of alternative pathway of complement who repeatedly showed an increase of haemolytic titres after administration of ivig. thus, wigs apparently have multiple potencies including attenuation of soluble mediators of inflammation and might therefore be of benefit in trauma, shock, and sepsis. significance of sinusoidal liuing cells and of humoral factors on hepatic function following sepsis and major surgery hirata k, katsuramaki t, yamaguchi h, mukaiya m, yamashiro k. regeneration of the liver after hepatic necrosis or partial removal has been extensively studied, but uncontrolled mechanisms to irreversible hepatic failure following sepsis or major surgery of liver or with hepatic dysfunction have so far not been well documented. we suggested the importance of the intrasinusoidal aggregation between sinusoidal lining cells and intrasinusoidal running ceils in the mechanisms of hepatocyte dysfunction. thus, the purposes of this study was to investigate the changes in each sinusoidal lining cell during this process and the effect of cytokine on hepatocyte under various oxygenation. [ materials and methods ] ( ) clinical study : thirty two patients with histologically proved malignant tumor underwent major hepatectomy were devided in two groups, i_e. the cirrhotic liver group and the non-cirrhotic [aver group. most of the former were the patients with hepatocellular carcinoma and most of the latter were the patients with metastatic carcinoma of colorectal cancer. five patients were complicated septic conditions with hepatic failure. following operation in each patient, a verous blood sample was taken for measurement of serum il- , il- , tnf and c-reactive protein concentration. and hepatic biopsies were sometimes undergone in patients with anomalous hepatic function. ( ) experimental study : kupffer ceils, sinsoidal endothelial cells and hepatocytes were separated from mouse ( c hhen ) liver. and also polymorphonuclear cells and platelets were purified from blood and peritoneal macrophages were from peritoneal cavity of mouse. co-cultures between or among these cells with iipopolysaccharide were performed. cytokine concentrations in media and hepatocytic function were compared. [ results and conclusion ] our findings in above experiments demonstrated the cleanly different concept for the mechanism of hepatocyte dysfunction following sepsis or major surgery. namely, the effect of hepatoeytie function by cytokines or superioxide were significantly affected under low oxygenation, but not significant under good oxygenation. hirata m.d.,nishi ,hokkaido,japan $ immune complexes as .mediators of sepsis and immune dyafumcffon laa k horn, chxistof birkenmaier, and greg h mon departmen~ of surgery, san frandsco general hospital, urdversr , of calif(~rrda, san frandsco. immune complexes (ic) a;:e commonly found circulating in parians during infection and sepsis; and following traumm, bums, and massive l~ensfusion. clearance of c occurs by phago~'tmsis of esll-bou_nd or opsonized p~tlcles. monocyte activation du~jng phmgocytos/s could lead ~o release of cytokilies ieletexiot~ to the hosh to examine tb/s phenomenon, we formed insoluble ic by precipitating iow-endotoxin bovine serun~ albumin (bsa) with rabbit ant/-bsa igg. we have ~town that these ic are ingested by monocytes and concomitantly stimv/ate re~pizatory bu~t activity measuxed by assay of in%racellul~ peroxldaffon. we have shown that ic ingesffon produces signlqcant el,era,lore in the monoey~e phenotype. spedacally, cdi is down-regu/ated, along wl~ cd and cd . thus responsiveness to lipopolysacchmdde ( ) a~xd i=m~unoglobulin fe st~mu/mtlon is reduced. in cert,+rest, the complement rote.p tot cdc is u~-regulamd, indicaling mcremsed re~ponalx=~ness to opsonized pat,rotes, we examined monocyte superna~n~s for production of hzmor necrosis factor alpha (tnfg) following ic stimulation and showed that ic are capable of provoking ~elaase of hrmaunl)reac~ve tni~. j[c also increase mono=yte produchon of prnstaglandin e . in summary, ~/~ese results demonstrate lhmt ic are capable of indudng production of ¢ytokines and the imrau-nosuppressive prostaglandin pge . ic also allez ~he surface expression of important receptors involved in actlvaffon by lp~ and phagocyiosis. thus, ic aze competent for indud/on of the mepl/e s! otlse. by examining m n cyte phenotypms, it may be possible to d~mrmkne extent ohn'u~u~e complex activation and predict immune depression iu criffcally ill pa~ents. a great deal of experimental work has focused on preventing and/or treating sepsis and organ failure following injury. many of these strategies have tried to attack mediator substances released during the systemic inflammatory response following injury. sugermann has demonstrated the improvement of pulmonary function, but not eappillary leak using [buprofen in the porcine model. in a clinical trial, faist demonstrated the effectiveness of indomethacine in amelierating the post trauma of cellular amenity using in vitro parameters. morbidity and mortality, however, were not different between control and treatment groups. baue und chaudry have suggested that atp magnesium chloride may have protective effect in renal failure um kupfer cell dysfunction seen after shock. pentoxiphyllin has been found to be effective in improving tissue oxygenation and oxygen consumption following hemorrhage, and there may be promise for this drug in the ischemia reperfusion injury. monoclonal antibodies against endothxin and inflammatory mediators seemed promising at first glance; however, preliminiary clinical data of the treatment of sepsis with either ha-ia or e monoelonoal antibodies against bacterial cell wall have been disappointing. initial studies with il-i receptor antagonist also appeared encouraging, but no difference was seen in the most recent trial. currently studies are ongoing with monoclonal antibodies to tumor necrosis factor which may have more efficacy given the fact that it is a macrophage mediator released by endotoxin. although we seem closer to understanding and preventing the problems of sepsis and organg failure after trauma, we must continue to appreciate the complex interrelationships and delicate balances inherent in the host defense system. overzealous attempts at restoring immunity in the absence of understanding pathophysiology may be just as dangerous at post-traumatic immlmospremsion. severe acute pancreatitis was caused by different etiologic factors, but once pancreatic tissues were infected, patients show a similar pattern of aggravation and develop organ failure. remarkable elevation of serum il- was unexceptionally observed in patients with severe acute pancreatitis. the serum levels were more than pg/ml (normal: less than pg/ml) during the first one or two days after onset. there was a significant correlation between the peak serum il- level and the number of organs showing failure (r= . ). tnf-a production by isolated peritoneal macrophages following lipopolysaccharide (lps) stimulation was significantly increased in cerulein-induced pancreatitis rats. serum tnf-a activity was elevated following intraperitoneal administration of lps as the septic challenge both in pancreatitis rats and in control rats, being significantly higher in the former (p< . ). histological findings and liver function tests revealed that lps induced more severe liver damage in pancreatitis rats than in control rats. these results indicate that increased tnf-a production by peritoneal macrophages in acute pancreatitis augmented lps-induced liver injury. organ failure in severe acute pancreatitis could be caused, at least in part, by increased cytokine production. it is clear now, that in mods, organ injury is not directly due to exogenous factors, such as bacteria or toxins, but instead is largely a consequence of the host's own endogenously produced mediators. there is an extensive body of literature on the inhibition of mediator production, release and action in different cascade systems by glucocorticoids. they can serve as a host protection against overactive defense reactions if used adequately. a new understanding of the regulation of cytokine synthesis, especially tnf, may lead to an insight of the conflicting findings between the benefits of glucocorticoid therapy in animals and its current failure in recent clinical trials. glucocorticoid shares its role with other novel therapeutic approaches also shown to be successful only in experimental settings. a mete-analysis of steroids in sepsis, however, revealed a beneficial effect in the subgroup of patients with gram-negative infections. there is now a series of arguments to reevaluate the indication for steroid thera-py and/or prophylaxis in mods and sepsis. honjo i- - , kumamoto , japan in septic shock j. briegel, m. halter, h. forst, w. kellermaun, m. bittl, k. peter imrodnetlea and methods: hydrocortisone (hc) at an infusion rate similar to the maximal secretory rate of cortisol in man improves hemodynamics in human septic shock ( ). we hypothesized that the hc-induced hypercortisolemia prevents a harmful overshoot of immune reactions. to test this hypothesis we prospectively investigated patients admitted to the icu in refractory septic shock. after resuscitation (mechanical ventilation, fluid resuscitation, titration of vasopressors, and initiation of antimicrobial therapy) hc was started with a loading dose of mg/ rain, followed by a continuous infusion ( mggh). with hemodynamic improvement (dopamine < gg/kg/min) the infusion rate nfhc was tapered over a period of days. phospbolipase a (pla ), c-reactive protein (crp), and elastase-proteinase inhibitor complex (e-pi) were deierminated daily. results: according to our previous results hemodynamics improved during hc infusien. after days dopamine requirements decreased to less than gg&g/min in all patients. this corresponded to an attenuation of the systemic inflammatory response syndrome (sirs) indicated by the abrogation of fever and the decrease in heart rate and inflammatory mediators. pla activity and e-pi concentrations decreased to near normal values and crp concentrations decreased as well. after cessation of hc infusion (between day and ) a reinforcement nfthe sirs was observed despite cortisol levels within the normal range. this was first indicated by e-pi, followed by fever, increases in heart rate, crp, pla , and finally by the relapse of septic shock in four patients on days , , and o. a second infusion of hc again resulted in shock reversal and in a decrease in pla , crp, and e-pi in the patients under study. discussion: there is growing evidence that the endogenous steroid hc and proinflammatory cytokines integrate a complex immunoregulatory feec~ack circuit. the elaboration of tnf, il- , il- , and il- is attenuated by- rag hc prior to endotoxin challenge in humans ( , ) . cytokines like tnf, il- , il- , and il- are potent proinflammatory signals for acute reactants (--, clip), neutrophil degranulation (--, e-pi), and pla synthesis and secretion. our data provide evidence that hc at an infusion rate similar to the maximal secretory rate of cortisol in man attenuates the systemic inflammatory response in human septic shock. to evaluate the feasibility of a nigh-dose regimen of antithrombin iii (at iii) treatment in patients with multiple injuries regarding blood levels of antithrombin iii and undesired side effcts and to analyse effects of the initibitortherapy on the systemic inflammatory response. patients and methods: patients with an iss nf more than points who could be treated with at iii administration within rain after trauma were randomized to receive either at iii or placebo. at iii was given to reach an antithrombin iii inhibitory capacity in plasma of % by using the following calculation: at iii to administer = ( % -at iii measured) x body weight. at iii was given every six hours for a period nf hours and on the th day. patients were followed up throughout their stay in the icu but at least days. in this abstract the data of the first patients are included. results: the patients' median injury severity score was points with a median age of years. the patients were equally distributed between verum and placebo groups with respact to age , iss, prehospital treatment, blood pressure, hemoglobin and at nmevel on admission, and time from the accident until the test solution was given. the patients of the at iii group received , units of the inhibitor on average during the first days. with our regimen, the at iii levels could be kept between % and % of normal, whereas control patients an at hi concentration of % to % was observed. patients with at ni treatment required less red blood cells bur received the same amount of ffp and fluids. there were no differences with respect to platelet count, partial thromboplastin time, l~rothrombin time and prothrombim. we did not observe bleeding disorders or any other side effect with peak concetrations of up to % of normal. white blood count, pmn--elastase, interleukin and and c-reactive protein tended to be lower in the at iii group. there were no differences with respect to renal and hepatic function parameters but the duration od mechanical ventilation was shorter in the at ii group ( . vs. . days) conclusions: we conclude that our treatment protocol (a) was able to restore at iil levels to the desired range of %, (b) did not lead to coagulation disorders, (c) did not increase transfusion requirements, (d) did not reveal other undesired side effects, and (e) showed a tendency towards reduced posttranmatic inflammation and mechanical ventilation requirements department of trauma surgery, essen, germany antithrombin-lll (at-ill) acts as an inhibitor of thrombin, further proteases and the alternative pathway of the complement system. inhibiting thrombin, at-ill works as a functional antagonist of paf~ therefore, in state of hypevolemic-traumatic shock, continuous supranormal serum -and tissue -concentrations of at-ill may be efficient to reduce the local posttraumatic reactions resulting from complement and pmn-activation. patients with severe multiple trauma ( treatment [at-ill] -- controls [c]) were investigated. study cdteda were age > and < years, injury severity (iss) > points and glasgow-coma-scale > points; randomization and treatment has to be started within hours after trauma. permission for the clinical study was given by the local ethic committee. bradykinin (bk) and related kinins are potent inflammatory peptides which possess the ability to induce, vasodilation, increased vascular permeability and hyperalgesia. cp- , a novel homodimer bk antagonist has previously been shown to increase survival in rat and rabbit models of lethal endotoxin shock and is now in clinical trials for sepsis. we have now evaluated the effect of cp- in other models of inflammation. male rats were precannulated with a catheter in the carotid artery. h later bk was injected ia and the pain score ranked from (no responses) to (vocalization). cp- at . umoles/kg completely inhibited the pain responses for a period of . - h. cp- at . umoles/kg s.c. was also found to inhibit the increase in paw volume and hyperalgesia induced in rats over a - h period by an intraplantar injection of . % carrageenan. the abdominal constriction response o an intraperitoneal injection of kaolin was inhibited in a dose-dependent manner by cp- . when ul of . % formalin was injected into the paw of a mouse a characteristic licking response was observed which was biphasic in nature. cp- significantly inhibited both the first ( - min) and second ( - min) phase responses. ]n a rat burn model, where the hind paw is immersed in water at °c for sec the increase in paw volume was significantly reduced by pretreatment with cp- , . umoles/kg s.c. finally cerebrai edema was induced in rats by applying cold (- °c for sec) to the dural surface following a craniectomy. cp- at . umoles/kg s.c. produced a significant reduction in the amount of edema compared with sham controls h later. these data suggest that bk is an important mediator of inflammation and hyperalgesia and that the bradykinin antagonist, cp- , may be useful in the treatment of such inflammatory, hyperalgesic disorders. partial hepatectomy in humans is associated with a considerable morbidity due to hemodynamic and metabolic derangements, which increase the risk for organ failure and mortality. we hypothesized that endotoxemia may play a pivotal role in these complications. we therefore, investigated whether peri-operative infusion of rbpi , a recombinant protein of the human neutrophil bpi with bactericidal and endotoxin-binding capacity, could prevent postoperative derangements following partial hepatectomy. male wistar rats ( - g.) received a % liver resection (phx) or a sham operation (sh), and a continuous intravenous infusion of either . mg/kg/hr rbpi (phx-bpi, n= ; sh-bpi, n= ) or the (iso-electric, iso-kd) control protein thaumatin (phx-con, n= ; sh-con, n- ). various parameters were measured h after the resection or sham operation. mean arterial pressure, cardiac output and heart rate were significantly decreased in phx-con rats compared with sh rats, which effects were not observed in phx rats treated with rbpi . blood ph was significantly decreased in the phx-eon group, whereas the leucocyte count, hematocrite and il- levels were significantly increased compared to sham levels. in the phx-bpi group, these parameters were restored to near sham levels. in vitro experiments with rat plasma and human mononuclear cells (mncs) revealed that plasma of phx-con rats is highly capable of activating mncs, accompanied by the release of cytokines. this activation is attenuated with phx-bpi plasma. in vitro added acd or polymyxin b was able to reduce the activation by phx-con rat plasma to the levels of phx-bpi rats thus, these data suggest that systemic endctoxemia, possibly of gut origin, is a major cause of postoperative hemodynamic and metabolic derangements following phx and that rbpizz can prevent these changes. more recently we reported a transient appearance of both endotoxin and tnf in the circulation of rats subjected to the haemorrhagic shock (hs) already at - rain. similar to bpi, recombinant bpi was found to bind lps and inhibit tnf formation in vitro. the aim of this study was to investigate the effects of rbpi (kindly provided by xoma corporation, berkeley, ca) against haemorrhage related endotoxemia and mortality in rats. method: a prolonged hs was induced by blood withdrawal to a mean arterial pressure of - mmhg for rain followed by reinfusion of shed blood (sb) and resuscitation with two times of sb volume of ringer's lactate over rain. rbplg. was administered at a total dose of mg/kg i.v. ( . mg/kg at the -eginning followed by two doses of . mg/kg each at end of shock and the end of resuscitation). the control group was treated similar to the bpi group but received thaumatin as a protein control preparation at the same dose as rbpi . results: imrffe?diately after resuscitation ( min) the detected plasma endotoxin levels in the control group (mean = , range = - pg/ml) were almost neutralized by rbpi treatment (mean = , range = - pg/ml) . plasma tnf levyis were not significantly influenced by rbpi treatment at the two time points and min of experiment (means: and in bpi vs , pg/ml in the control group). the -hour survival rate was improved from / ( . %) in the control to / ( %). conclusion: these data suggest that haemorrhagic shock may lead to bacterial translocation and/or transient endotoxemia with concomitant cytokine formation that may play an important role in the pathogenesis after shock and trauma, rbpi might be a useful therapeutic agent against endogenous bacterfal/endotoxin related disorders in hemorrhagic shock. morbidity and mortality after hypoxia of the vital organs had been correlated to the production of oxygen radicle which is mediated by xanthine oxidase activity, in this study we have evaluated the survival rate after allopurinol. rabbits weighed + grams divided into two groups. group i included tabbits were treated with allopurinol mg/kg for seven days before induction of haemorrhage. group ii as a control included rabbits. all rabbits were subjected to % arterial blood loss through the central ear artery for one hour then resusciatation was done by the heparinized withdrawn blood through a marginal ear vein. during the experiment blood pressure and heart rate were monitored through the central ear artery. also uric acid, lactic acid, glutathione activity were estimated. animal survival was followed for days. postmortem vital organ histochemistry and histopathology examinations were done. in group i the survival after three days was out of while in group ii it was two out of . our conc|usion, allopurinol had increased the survival in aiiopurinol pretreated rabbits which may indicate the value of allopurinol premedication for patient prepared for elective bloody surgical intervention . h receptor antagonists are commonly used for stress ulcer prophylaxis, but their actions on the septic response are largely unknown, in an experimental model, pigs were first anesthetized, then injured with joules of energy to the posterior thigh, then hemorrhaged - % of their blood volume. after i hr of shock, all the shed blood plus x the hemorrhage volume as lactated ringers was infused. following resuscitation, ranitidine ( . mg/kg iv twice daily) or saline placebo was begun. the treatment group was randomly assigned in a blinded fashion. after hrs, a septic challenge was administered ( bg/kg of e. coil endotoxin (lps)). serial gastroscopy, gastric ph, hemodynamics, abg's, physiologic dead space ventilation, leukocyte counts, and tumor necrosis factor (tnf) levels were recorded for min. baseline values and units were cardiac index _+ ml/min/kg (ci), arterial po + mmhg(pao ), base excess . -+ meq (be), physiologic dead space fraction +_ % (pds), and tnf . + . units/ml. baseline gastric ph was . -+ . and . _+ . in the placebo and ranitidine groups, respectively. the gastritis following hemorrhage was marginally attenuated in the ranitidine group. following lps infusion the following were obtained: ci pao * be* gastric* pds* peak* rain rain rain ph min tnf ranitidine _+ _+ - . ± . bum injury results in hypermetabolism, fever and nitrogen wasting. endotoxin (lps) has been proposed to mediate these effects, either directly or via activation of macrophages to produce cytokines such as interleukin- (ii- ). this study was designed to clarify the role of lps and - in the metabolic response to bum injury. twenty-five burn patients ( -+ %; + % ft bsa burn; _+ years old) were studied serially for three weeks post bum. patients underwent partitional calorimetry to assess metabolic rate and compartmented heat loss. nitrogen was assayed using chemiluminescence. lps and i - were measured with limulus amebocyte lysate assay and elisa. patients were excluded if they suffered smoke inhalation, showed any sign of sepsis or failed to rapidly meet their nutritional needs via the enteral route. ten patients received intravenous polymixin b ( , u/kg/day to bind lps). these patients did not differ for the remainder. all patients were hypermetabolic and febrile in proportion to the size of their bum wound but were not endotoxemic ( . +_ . pg/ml; normal < pg/ml). i - did demonstrate a significant correlation with cole temperature (tr~ = . + . ogi - , p= . ) and with nitrogen excretion (nou t = - . - . ogi - + . tr, p= . ). administration of polymixin b had no effect on metabolic rate, temperature or i - levels but did reduce nitrogen excretion resulting in more positive nitrogen balance ( .t grn/day vs. - . gm/day, p= . ). although bum injury does not produce an obligatory endotoxemia, i - does appear to play a role in the fever and nitrogen wasting seen with such injuries. the effect ofpolymixin b on nitrogen excretion suggests that lps may play a role either locally or in the portal system. introduction: there is substantial evidence that release of inflammatory mediators by activated kupffer cells contribute to the course of a systemic inflammatory process, e.g. after shock or lrauma. besides the systemic effects of mediators such as tnf, paf or interleukines, local actions on hepatic microvasculature and hepatic inflammatory response have to be considered. our aim was to assess the role of tnf and paf by blocking their effects using anti-tnf monoclonal antibody, pentoxifylline and a paf antagonist. methnds: in anesthetized sprd-rats, hemorrhagic shock was induced by withdrawl of arterial blood within rain and shock state was hold for h at a map of mm hg (cardiac output of %). following adequate resuscitation with % of shed blood and twice of this volume as ringer's solntion, animals recovered to map > mm hg and co > %. hepatic microcirculation and sinusoidal leukocyte-endothelium interactions were examined by intravital epi-fluorescence microscopy at , , or hours after resuscitation. in a blinded fashion, a rat-specific monoclonal anti-tnf antibody [ mg/kg, celltech, uk) , pentoxffylline (ptx, mg/kg, hoechst, d), and a paf antagonist (web , boehringer, ingh., d) were given either as pretreatment or at the time of resuscitation (n= - group bolla. k*., duchateau, j., hajos, gy., mbzes, t., hern~di, f. prevention of temporary/secondary immune deficiencies or reduction of their severity and/or duration as well as the reduction of the perifocal inflammatory processes belong to the rational targets of posttraumatic/pedsurgical medication. such a targeted medication can result in less frequently occurring nosocomial infections, and in reducing the duration of the intensive care and convalescence period. the results of in vitro studies performed with the amino acid sequence - of thymopoietin, i.e., with thymocartin in whole blood and peripheral mono-nuclear celi(pbnc) cultures clearly show some characteristic effects of this immunomodulator. preincubation with the tetrapeptide significantly (p<o.ol) and concentration-dependent reduced the endotoxin(lps)-induced tnfalfa production from , to about pglml, but did not abolish it. the inhibition of the lps( . ug/ml)-induced tnf production occured already in presence of , pg peptide/ml and peaked at the concentration of , ng/ml. higher concentrations did not increase this effect. in contrast to the other two small thymic peptides (tp- and tp- ), thymocartin did not enhanced the pwm-induced pge production in pbmc cultures up to a concentration of ng/ml, inclusive. the selection of thymocartin (tp- ) for perisurgical medication and/or for treatment of trauma-induced temporary immune depression has been strongly supported also by targeted animal experiments. thus, mortality of about % registered in mice infected with pseudomonas aeruginosa after bum injury could be reduced with the tetrapeptide treatment to a level which did not differ significantly from that of the infected non-burned controls. moreover, observations gathered first of all with the inflammatory model of air poach/croton oil grenuloma (selye) clearly showed, that a very definite, antiexudative effect can be achieved with this thymic peptide. as to this effect, thymocartin has been confirmed, e.g., to be equipotent with locally administered fluocinolone acetonide and/or s.c. injected prednisolone. the interim results of two explorative double-blind clinical studies (involving more than patients after polytrauma and hip-fracture untill now, respectively) correspond to the expectations. the treatment significantly reduced the occurence of nosocomial infections, the days spend in intensive care and on antibiotics as well as it shortened the bed-out time and reduced the additional medication. objective of this prospective randomized study was to further delineate the mechanisms leading to these alterations and to investigate the effects of immunomodulatory intervention. patients and methods: patients (pts) formed control group a. group b pts received x mg indomethacin (indo) from the first (dl) to the fourth postoperative day (d ) intravenously to block prostaglandin e induced suppression of cmi response as well as mg thymopentin (tp- ) subcutaneously preop., on d and d to augment cmi reactivity. in vitro investigations preoperatively, on dl and d included measurements of the percentage of cd + t-helper (th) cells, pbytohemagglutinin (pha)induced synthesis of interleukin (il)- as one cytokine primarily produced by thl-cells, and pha-induced synthesis of il- as one cytokine primarily produced by th -cells. delayed type hypersensitivity (dth) skin response to an antigen skin test battery and specific antibody (ab) production following tetanus vaccination preoperatively and on d were used as in vivo tests for thl-and th -induced immune response respectively. results: postoperatively, group a pts showed a persistent significant reduction of cd + th cells, il- /il- ratio, and dth skin response as compared to baseline values (bl), while ab production increased (p< . vs.bl). in group b pts no change in cd + th cells, il- /il- ratio, or dth skin response was observed (p< . vs.group a), and postoperative ab production increased significantly (n.s. vs. group a). conclusions: .these results clearly indicate a significant suppression of th induced cmi response, while th induced response remains normal following ohs. .these alterations may gain clinical significance whenever a postoperative infection requires a th response and may explain the susceptibility to opportunistic microorganisms observed in pts after ohs. the aim of this study was to find out whether the establishment of administration of thymostimulin (tp-i) in jaundiced and anergic rats would return the rats to the state of immunocompetence. material and methods. male wistar rats weighing - g were injected with haemocyanin (klh). all rats with a positive response were included in the study and we evalu ated the t cell subpopulations and il- . they underwent laparotomy and the common duct was ligated and divided. those rats whic become jaundiced and anergic one week after the operation, were divided in two groups: control group ( objective of study. the goal of this study was to prove the necessity of thymus derived immunomodulators treatment in acute period of infantine sepsis, materials and methods. immune status was investigated in o infants. clinical conditions of children were defined by syndrome of multiple polyorgan]c insufficiency. the heaviness nf condition was estimated as the sum of points according to efforts needed to restore of basic living functions -respiration, hemodynamjcs, hemocoagulatlon, metabolism, functions of liver and kidneys. it was expressed in form of clinic condition classes from to . results.it was found that in % of infants with - classes of heaviness immunode[iciency took place simultaneously with ii and iii stages of hemocoagulattve syndrome. amounts of t cells and t helpers were decreased more then times, ratio th/ts was reduced to . or lower. concentration of lgg and igawas reduced almost a hull in comparison to control. phagocytic and metabolic activities nf neutrophils were depressed. complement system state was changed: the functional activity of the alternative pathway factors b, d and the level or c a subcomponent were increased but the level of c , c ,, c and c components of the classic pathway were decreased. during the development of coagnlopathy the direct correlation between chso, levels of plasminogen and antithrombine iii was found. in greater extent the function of immune system was disodered in accordance with point metabolic violations and hemocoagulative disturbances considered as disseminated inmtravaseular clotting syndrome on ii or ili stages. conclusions. the rehabilitation of the immune system functions was promoted by taetivine and thymaline in doses of i- mcg/kg per day and mg/kg per day accordingly during a week in children with - classes el heaviness. aiter - days tactivine had advantage. when eoagulopathy took place thynaaljne promoted restoration of ( - )-~-d-glucan (bgc) is a major cell wall compornent of pathogenic fungi and shows many immunopharmacological activities on experimental animals. lps is also derived from gram-negative bacteria and have in~ttnomodulating activities on immune systems positively or negatively. because of difficulty to cure contaminating lps from bgc preparations, it is very hard to evaluate the exact activities of bgc. in this study, we compared the immunological activities of lps ans highly purified bgc on murine system. materials and methods: c h/hen or c h/hej mice, to wkolds of both sexes were used through experiments. highly purified ( - )-{ -d-glucan (gurdran; bgc)was courteous gift from seikagaku kogyo co. tokyo, japan and resolved in endotoxin-free ultra-pure water. escherichia cell lps was purchased from sigma chemicals, usa. mitogenic activities of bgc or lps on splenic cells were measured by mit dye assay. in vitro activation of peritoneal exudate macrophage (pen) have moniotored by phagocytosis and intraphagocytic killing of pseudomonas aeruginosa (z~b- . induction of cytokine productions from pem or spleen cells induced by bgc or lps were detected by cytotoxic assay. results and (bnclnsions: i ) in vitro cultures of pem with bgc have induced enhanced phagocytic and bactecidal activities in c h/hen and c h/hej mice, whereas lps only induced such activities in c h/hen mice. most effective dose of bgc was ug/ml. ) bgc-dependent proliferative response of c h/hen splenic cells was not detected under the culture condition used. ) induction of tnf~ prodction was apparently observed in bgc-stimulated c h/hej pd cultures, but not in lps stimulated one. these findings indicate that activation mechanisms of pr and splenic cells by bgc are different from that by lps. to investigate the bsc-induced p~ activation, intracellular signaling pathways of p~ by bgc-stimulation are under consideration. yamagami k, uedono y, kawakami k, kitazawa y and tanaka t according to the latest reports of use of il- in a burned-sepsis model, the dose was too high to adjust for human therapy. adoptive transfer of l~ymphokine-activated killer (lak) cells has been demonstrated as a means of enhancing therapy in malignant tumors. we therefore attempt to use lak therapy on burned-infected mice instead of il- . under anesthesia, -weekold male c h-hej mice were subjected to a % scald or sham burn and then resuscitated. sham burned mice served as controls. scald burn mice were subjected to peritonitis by intraperitoneal innoculation of organisms of p. aeruginosa hr after burn. burned-infected-mice were divided into two groups. one group had no further treatment, and the other group received lak cells ( x ) intraperitonelly after septic challenge. the mice were scored for survival daily. on day after burn, using moabs dual-color flow cytometry, we studied lymphocyte expression in mice with use of blood and spleen. simultaneousely we studied the alteration of il- and il- in their serum using immunoassy kits. sham burned animals had no mortality. the mortality of the lak-treated mice was significantly reduced when compared with that of the burned-infected mice. the most consistent changes observed were depressions in percentages of thyl, positive cells and a remarkable depression of nk cells in spleen. after lak cell treatment, those two percentages of cells significantly increased. all the data of assay of ill and [l were not detected on sham burn mice serum. due to burn and septic challenge the levels of illand il (n= ) were raised to . _+ . and _+ pg/ml, while the levels in lak treated mice were reduced to . _+ . and _+ pg/ml, respectively. the results reported here demonstrate that lak therapy is able to improve cell-mediated immunity in burned-infected mice. this is associated with a significantly improved survival rate, since ill has been demonstrated to mediate immune and inflammatory responses. also a cause effect relationship between elevated endetoxin levels and increases of [l has been recently established. the aim of our present study was to investigate the effect of parenteral indomethacin on the immune system of patients under major operations. our study included operated patients, of which received mg indomethacin before and , and days after surgery (group a) and patients received no antiinflammatory therapy (group b). we measured total t-cells helper (cd ), suppressor (cd ), nk-cells and interleukin- and tnf production by pha or endodoxin (lps) stimulated peripheral blood mononuclear cells at day and , and days after operation. in group a we detected a significant (p< . ) increase in cd /cd ratio on day while no differences were ~oted in nk-cells or cytokine production in both groups (table ) . it seems, therefore, that parenterai indomethacin may have a benefitial effect on the immune system of patients under major operations by increasing the t-helper /t-suppressor cell ratio while having no effect on the production of cytokines by peripheral blood mononuclear cells. this study was undertaken td dete~nnine the role of prostaglandin synthesis inhibitor on survival post traulna sepsis. analysis of the effects of prostaglandin synthesis inhibitor on survival of four groups of mice each were made after laparatomy and intravenous administration of live e.coli. post trauma sepsis, group a received no treatment; group b received antibiotics im; group c received prostaglandin synthesis inhibitor im and group d received both antibiotics and prostaglandin synthesis inhibitor im. survival time was longest in group treated with prostaglandin synthesis inhibitor and antibiotics post trauma sepsis. the singular use of either antibiotic or prostaglandin synthesis inhibitor did not alter the outcome on survival. mortality rate was lowest in the group treated with combined prostaglandin synthesis inhibitor and antibiotics. use of this combination showed a reduction in bacterial growth in peritoneal and blood samples, mild morphologic changes secondary to sepsis in the heart, lungs, liver, kidney and stomach and marked enhancement of mean level of activity. the study indicates that addition of prostaglandin synthesis inhibitor in treatment of trauma-sepsis has clear beneficial effects. interferon-? (ifn-y) is a potent immunostimulant which has been shown to be detrimental when administered during septic shock. blockade of this cytokine however is beneficial during the acute septic response. the aim of this study was to evaluate the cellular effects of this cytokine and its blocking monoclonal antibody (moab) in lethal sepsis following injury. female cd- mice were randomized into two groups: septic=lps vs injury=hind limb amputation (hla vs igg. nstats= anova=p< . vs lgg ifn-y was detrimental when given to control septic animals. however its blocking moab was protective (p< . ). conversely ifn-y was proteetive in injured septic animals compared with anti-ifn-? (p< . ). these findings demonstrate that the immune stimulant ifn-? has therapeutic potential in the immunosuppressed injured host predisposed to sepsis, while blockade of this cytokine is required for protection in the septic host with a normal immune response. dept of surgery, rcsi, beaumont hospital, dublin , ireland. oxidants play a role in physiology. the potential noxious oxidant biochemistry is restrained by chemically distinct antioxidants. these antioxidants, which may reside in different cellular compartments, can act synergistically. in normal physiology an oxidant/antioxidant bai&nce exists. unbalance in favour of the oxidants is called oxidative stress. oxidative stress has been implicated in many pathologies including lung diseases(e.g, doxorubicin induced cardiotoxicity), ischemia/reperfnsion damage and central nervous system trauma. transition metals such as iron are involved in the early events leading to oxidative damage in these pathologies. this has been underlined by the finding that postischemic cns pathology closely resembles that caused by a chemical oxidative insult (e.g. iron microinjection). moreover, a protective effect of oxygen-radical scavening agents or compounds that inhibit lipid peroxidation (antioxidants) in brain ischemia/trauma is apparent. some known drugs (e.g. anti-arrhythmics or histamine h -antagonists may act as non-specific antioxidants. howe~er, recently, interesting new membrancespecific antioxidants (e.g. -aminosterdids) have been designed. subt&e effects on iron redox chemistry contributes to the potent antioxidant activity of these aminosteroids. since a few years, one area that greeted enthusimastlcally in clinical medicine is that of reoxygenatlon injury or ischemia-repeffusion, a phenomenon accepted to make a majo:" contribution to organ dysfunction in trauma, shock and sepsis through the formation o( oxygenated free radical species. however, thei detection in vivo always remains a difficult challenge. efforts have been made recently to directly establish the formation of free radicals in biological samples as complex as blood, plasma or tissue with the use of electron spin resonance (esr) spectroscopy. using spin trapping agents, the presence of reactive carbon-and oxygen-centered radicals has been demonstrated in animals blood submitted to heart, renal and intestinal ischemia-repeffusion or to liver transplantation. recently, the in vivo formation of free radicals in the cerebra; extracellnlar fluid during cerebral isehemia-repeffusion has been assessed by the spin trapping technique coupled to brain microdialysis. esr investigations have also been conducted to detect endotoxin-induced free radical generation in rat or baboon liver and oxygen free radicals (ofr), produced both at intra-and extra-cellular levels, seem to play a major role in the pathophysiology of tissue injury and organ dysfunction in sepsis, through induction of lipid pemxidation in cell membranes. oxidative damage can result both to an ofr-overpmduction or to a depletion of endogenous antioxidant defenses, which are represented by scavenger enzymes (e.g. sod), hydrosoluble and liposoluble antioxidants (e.g. ascorbate, vitamin e), and other mechanisms such as metal-ion sequestration. in animal models, oxidative damage has been proved by detection of increased levels of lipoperoxidative products, and a depletion of antioxidant defenses was found both in plasma and tissues. in the few clinical studies a similar results have been reported, and oxidative damage appears to correlate with dic, with organ dysfunction and with red blood cell deformability. in a group of critically ill septic patients we found increased levels of conjugated dienes (cd) (bioproducts of lipoperoxidation) and decreased plasma levels of alpha-tocopherol and coenzyme qlo (coqi )(endogenous liposoluble antioxidants). a relationship between cd and uric acid (p< . ) was found, may be related to xantine-dehydmgenase to xantine-oxidase conversion. antioxidant depletion is due both to overconsumption and to a nutritional deficiency, even if a reduced synthesis can also be present. in fact one more relationship between coqi and plasma cholesterol (p< . ) demonstrates the commun hepatic biosynthetic defect. is there any role for antioxidative therapy in sepsis? can it achives a significant improvement in the septic course, organ dysfunction, and final outcome? several antioxidant drugs, have been evaluated in experimental and clinical sepsis: scavenger enzymes, natural antioxidants, alloporinol, -aminosteroids or lazaroids, have been proved to reduce lipopemxidative damage, to protect organ dysfunction, and in some cases to improve survival. several questions must be addressed in evaluating safety and utility of antioxidative therapy. more specific and standardized methods must be applied to detect and quantify the lipoperoxidative damage. antioxidant drags must act selectively on ofr-production, without interference with other than that are beneficial for the organism. antioxidants must be able to achive in adequate amount the tissue or cellular compartment where their action is required. many antioxidants have also a pro-oxidative effect which can he detrimental in some conditions. the timing of administration, the dosage used and the association wih others antioxidant drugs must be defined. nutrition plays an important role as antioxidative therapy, since it supplies all compounds needed to maintain adequately levels of endogenous antioxidant or to support their synthesis. thiobarbituric acid reactive substances (tbars) concentration in serum has been determined in a large population of healthy subjects and in patients suffering acute hepatitis and chronic cases of hepatitis c, as well as several other processes. the correlation with different physiological and clinical parameters in these populations is also presented. treatment with interferon of the chronic active hepatitis c patients, x u three times a week during two months, led in those patients whose sgpt activity normalized in serum, to a concomitant decrease in serum tbars content. the possible theoretical involvement of peroxidation and antioxidants in this beneficial effect of i~terferon in hepatitis c patients is discussed. the results presented confirm the value of tbars as laboratory test in the management of disease and as a useful tool for the study of pathogenic and/or therapeutic mechanisms. -hydroxyalkenals are among the different substances measured by the tbars assay. these compounds show several biological effects that have been demonstrated mainly in different experimental models. we have studied the capacity of different tissues to conjugate these compounds patients surviving the initial subarachnoid hemorrhage (sah) from a ruptured in~raeranial aneurysm are prone to develop cerebra] ischemia from vasospasm which is associated with significant morbidity and mortality. among the many treatments that have been prol~osed to treat this condition, none has been shown to be satisfactorily effective. recently, a new drug, namely the -aminosteroid tirilazadmesylats, has been studied in a large, prospective, multicentor trial for its effectiveness to improve the outcome of patients with aneurysma! said. the study was conducted in europe and australia in neurosurgical centers and included patients. all patients received presently accepted standard treatment, including nimodipine. patients were randomized to four different groups: placebo and groups of tirilazad in escalating dosage ( . - mg/kg/day). the clinical outcome was compared for these groups and cerebral a~giography was performed in % of patients on day - follovang sah, to study the effect f'the dflf~ off cerebral vasospasm. the study reached its planned endpoint months ago. preliminary results ~how a significant improvement with regardto mortality in those patients receiving the highest tirilazad dose as compared to placebo and the two lower dose groups. additional beneficial effects were seen in the rag/day groups with regard to the occurence of symptomatic vasospasm and good clinical outcome. although the final analysis of all data is still pending, these results suggest that tirilazad-mesylate could represent a major improvement for the treatment of patients with sail. experimentally lipidperoxidation products (lpo) are increased in acute pancreatitis (ap) due to oxygen radicals (or). the purpose of this clinical study was to determine the antioxidant status and lpo in patients suffering from ap and to evaluate the effect of antioxidant treatment with sodium selenite (se) thus improving the function of the se dependant glutathione peroxidase which is the major detoxifying system for or. materials and methods: in z patients sufferin s from severe ap (c-reactlve protein > me/l) we determined on day and day after admission the lpo ma!ondialdehyd (mda), conjugated dishes (cd), reduced (gsr) and oxidized (gssg) glutathione, the vitamins a,c,e and se. moreover the patients were evaluated clinically using the ranson and the apache ii score. i patients were randomly treated with ug/day of se for days. results: all patients suffered from a severe depletion of antioxidants,especially a low concentration of se (only / of normal). thereby the increase in lpo correlated with the clinical course. during se treatment lpo decreased and the levels of antioxidant vitamins improved. se had no influence on leth-slity the lenl or the chan in rs or ap ii. background: since reperfusion injury occurs when oxygen is reintroduced into ischemic tissue, the ideal timing for administration of therapeutic compounds aimed at ameliorating oxygen radical mediated injury is at the time of initial fluid resuscitation. currently used colloid or crystalloid preparations do not provide optimal, or even significant, anti-oxidant protection. systemic iron chelation affords protection against the iron catalyzed components of oxygen and lipid radical mediated tissue injury. the conjugate resulting from chemical attachment of the clinically approved iron chelator, deferoxamine (dfo, desferal ®, ciba), to hydroxyethyl starch (hes) represents a novel approach to colloid based fluid resuscitation. hes-dfo contains % hes and % chemically bound dfo. the polymer-drug conjugate has a lower molecular weight than that of hes in order to allow more rapid excretion. results: preclinical and initial clinical trials indicate that hes-dfo is well tolerated, even at high doses. in animal studies, fluid resuscitation with hes-dfo does not significantly improve central hemodynamic recovery beyond that observed with hes, but hes-dfo seems to afford better protection of microcirculation in organs at risk (lung, liver and gut), possibly by decreasing neutrophil sequestration. in a burn model, total fluid requirements are lower and oxygen utilization higher in hes-dfo treated animals compared to hes controls, suggesting decreased vascular leak and improved tissue perfusion. conclusion: hes-dfo represents a means by which potent antioxidant protection can be administered at resuscitation. iron has been suggested to play a pivotal role in oxygen flee radical mediated tissue injury. in vitro experiments indicated its critical role as a katalyst in hydroxyl free radical generation fenton-reaction). since iron chelator deferoxamine administered in shock alone demonstrated severe side effects, a hydroxyethylstarch (hes)daferoxamine (dfo)-conjugute was used to modulate oxygen free radical injury during the ischemia/reperfi~ion syndrome induced by hemorrhagic shock. methods. female lewis rats ( - g, n> ; pentobarbital anesthesia mgjkg), in hemorrhagic shock ( the aim of the study was to elucidate ( ) whether the generation of or would affect lung and kidneys as primary shock organs in the very early phase of sepsis and ( ) whether dfo-hes could prevent this tissue damage. methods: in rats sepsis was induced by cecal ligation puncture (clp) peritonitis. the animals were randomly assessed to groups: one group was treated with ml dfo-hes ( mg/kg iv), the other rats received solely ml of the carrier starch solution. , , , and min after induction of sepsis respectively, the animals were sacrificed, the organs collected, and tissue contents of glutathione (gsh), malondialdehyde (mda), myeloperoxidase (mpo) and conjugated dienes (cd) determined. plasma samples were obtained for analyses of endotoxin (chromogenic lal test). blood pressure (map) was measured via a carotid artery catheter. results: clp caused sepsis with high (> . eu/ml) endotoxin levels. map in both groups decreased slightly but significantly during sepsis regardless any treatment. in the lungs mpo concentration was increased (p< . ) in the lies group already min after sepsis induction. concomitantly, tissue gsh level decreased and lipid peroxidation was pronounced as shown by elevated mda and cd levels. dfo-hes diminished tissue pmn accumulation and mpo concentration. moreover, at each time point lung mda and cd levels were lower (p< . ). histomorphological examination showed marked micro-atelectases, destruction of the alveolar septa, and splicing of the basal membranes in the lies group. in contrast, in dfo-hes treated rats the alveoli remained well-ventiiated and only some enlarged reticular fibers without splicing were observed. almost similar results were found for the kidneys. mpo levels differed neither within nor between both groups. the slight decrease in gsh levels seen after min in the dfo-hes group seems to demonstrate an oxidative stress to a lesser degree. the most impressive effect of iron chelation, however, was revealed by the lipid peroxidation products. at each time point, mda and cd levels were lower (p< . ) compared to the hes group. light and electron microscopic examination disclosed tubulotoxic and mitochondriat damages while dfo-hes lxeatment prevented that alterations. conclusion: both the biochemical and histological results of this study reveal an early and remarkable generation of or in peritonitis-induced sepsis. thereby, these or obviously cause pulmonary and renal tissue damages, intravenous application of dfo-hes may, however, benefit by preventing early lipid peroxidation of the tissue. the proteolytic irreversible conversion of xanthine dehydrogenase (xd) to xanthine oxidase (xo) is triggered by calcium flux. the aim of our study is to clarify ~he link between intracellular ca + levels and xo activity determined by uric acid release, and to evaluate the efficacy of verapamil, on the generation of hydrogen peroxide associated with reperfusion by assaying lactate & pyruva~e release and the levels of cytosolic free nad /nadh ratio. experimental protocol consisted of :(a) non ischemic/reperfused experiment in which normal cardiac slices of rats were perfusated with oxygenated kreb's ringer phosphate buffer containing glucose ( mg%) and bovine albumine ( gm%) for min at °c.it composed of groups, group aa (control group), and groups ab & ac (perfusate supplemented with verapamil in the dose of loo& mi% respectively). (b) ischemic reperfused experiment in which ischemic cardiac slices were obtained from rats subjected to min ~aemorrhage.lt was also divided into two groups; group ba and bb (verapam~/ mi% added to perfusate}. verapamil stimulated uric acid release from normal rat cardiac slices were % in group ab and % in group ac(dose related). rates of uric acid release is enhanced by verapamil in group bb. moreover, rates of uric acid release in groups ac & bb are insignificant. in verapmil added groups (group ab, ac & bb), increase uric acid release is associated with an enhancement in pyrurate release and with increase levels of cytosolic free nad+/nadh ratio, although it is not evident ~ ischemic group (group ba).it is concluded that the conversion of xd to xo is calcium independent. eicosanoids like thromboxane a , leukotriene b and leukotriene c are known as promoters of initial inflammatory reactions. we investigated whether oxygen radicals (or) are able to induce a release of these eicosanoids in whole blood. blood from healthy volunteers was incubated with xanthine oxidase/hypoxanthine to generate oxygen radicals. after , , , and minutes plasma levels of thromboxane b (txb ), leukotriene b (ltb ) and leukotriene c (ltc ) were determined via elisa technique. another volunteer had taken mg aspirin one day before taking the blood sample (no ). results: txb plasma levels increased from pg/ml at min to pg/ml, pg/ml, pg/ml and pg/ml at , , and min (p< , ) . ltb and ltc plasma levels showed an increase during the first few minutes (ltb : min: llpg/ml, min: pg/ml; ltc : min: pg/ml, min: pg/ml (p< , )) followed by a decrease to normal values at min. in the sample no the cyclooxigenase-pathway was completely inhibited, the txb plasma-levels did not alter at all, whereas ltb and ltc -plasma levels weren't affected. opallogeneic blood transfusion jane shelby, ph.d., and edward w, nelson, m.d, there have been numerous investigations dudng the last two decades examining the effect of surgery, anesthesia, blood loss and transfusion on vadous immune parameters in humans and animal models. there appears to be concurrence among several well controlled studies that transfusion of whole blood (containing leukocytes), has regulatory effects on immune ceil function which include decreased cell mediated immune response, and inhibition of il- secretion. these effects occur following transfusion alone and in con.cart with the distinct immune effects of surgery, trauma and anesthesla, the clinical consequences of this immune modulation by transfusion include decreased allogeneic response to transplanted organs, which has been exploited clinicelly in renal transplant patients. additionally, there is evidence for a strong association with increased risk for infection in transfused patients following surgical procedures. aiiogeneio blood transfusions have been shown to inhibit cellular anti.bacterial mechanisms, causing increased susceptibility to bacterial pathogens, in humans and in animal models. there is also concern that allog~neic transfusion may adversely affect cancer patients, resulting in decreased disease-free survival. several stategies have been proposed to minimize the adverse effects of blood transfusion. there is evidence that the risk of immune mediated infectious complications associated with transfusion may be greatly minimized wlth the use of autologous blood and leukocyte free allogeneic blood.products in surgical and trauma patients, it also appears that the inhibition of cellular immune response and il- productiorl following atlogeneic blood transfusion may be mediated by increased prostaglandin e secretion, and that immune response may be preserved in allogeneio whole blood transfused subjects receiving c lc~oxygenase inhibitors such as ibuprofen. among these are various alterations in immune function. efforts have therefore been made to utilize alternatives to homologous transfusions. these include the use of autologous predonation, supplemental iron therapy, and recombinant human erythropoietin. although initially considered innocuous, these therapies are now recognized to have potential deliterious immune sequelae. erythropoietin, by its ability to lower serum iron levels, can impair both lymphocyte and nk cell activity. autologous donation impairs nk cell function. finally, supplemental iron therapy can stimulate bacterial growth and increase the rate of infectious complications. this talk will present a discussion of these factors as well as a weighting of their importance. r.l rutan, rn;bsn, shriners burns institute and the university of texas medical branch, galveston tx, usa the serious sequelae of homologous blood transfusions have resulted in vigorous efforts at identifying alternate therapies for correcting red blood cell (rbc) deficits. erythropoietin (epo) was hypothesized to exist in the early th century, however the protein was not isolaled until . the human gene was identified and cloned in , which permitted the production of epo through recombinant techniques. the earliest clinical trials were performed in anemic end-stage renal failure palients on hemodialysis. treated patients experienced increases in erythropoiesis with normalization of hematocrit and hemoglobin levels, cessation of lrans-fusion requirements and improvement in general wellbeing. these studies, however, identified side effects of epo treatment such as hypertension, seizures and ee deficiency. volunteer trials have established that the hypertension is not a direct pressor effect but rather the result of abnormally rapid increases in red cell mass in the face of the incompetent volume-controlling mechanisms of the end stage renal failure patient. lower doses of epo and the subsequent gradual increases in red cell mass are associated with significantly lower incidences of hypertensive complications of epo therapy. likewise, seizure activity is not the result of a direct epileptogenie effect but parallels the incidence of hyper-tensive-related sequelae during high.dose epo treatment. in cross-over designed studies, pre-existing iron deficiency has been demonstrated to decrease or negate stimulated erythropoiesis but effective-hess can be restored with appropriate fe supplementation. exogenous epo is effective whether given by iv or sq routes and dose response curves do not vary with route of administration. increases in rbc mass are directly related to the dose of epo, both in amount and frequency of administration although there is a - day time lag between the first epo dose and laboratory indications of its action (i.e. increase in the number of reticulceytes in peripheral wood). epo is currently labelled for use in the treatment of anemias associated with end-stage renal disease and aids. however, its use in the surgical population has been explored because of its unique direct dose-response, epo has been used to effectively increase the blood harvest amounls in autologous pre-donation, significantly increase hematocrils in children following thermal trauma and successfully increase red blood cell mass following essential surgical procedures in patients with religious aversion to transfusion. by blood transfusion in colorectal cancer surgery mm heiss md, ch delanoff md, r stets md, j hofinann, e faist md, kw jauch md, fw schildberg md allogeneic blood transfusions are associated with an increased risk for postoperative infections in colorectal surgery when compared with autologous blood transfusions. attribution of this effect to immunomodulation was suspected in our previous study (lancet ; : - ) . task of the recent investigations was to analyze which specific effector systems were affected in-vivo by this transfusion-associated modulation. for global in-viva assessment of cell-mediated immunity (cmi) multiple recall skin-reactions were applied prior and post-operative. the specific humoral immune mechanisms were investigated by applying tetanus-toxoid one day preoperatively and deterimnating the quantitative igg-response. for indication of macrophage stimulation in-vivo tnf-levels were determinated by bioassay. dth-responses were significantly suppressed (p< . ) in patients receiving allogeneic blood (n= ) or operated without blood transfusions (n= ). dthresponses were not suppressed and tendentiously increased in patients with autologous blood transfusions (n= ). in contrast, specific igg-levels increased sigmficantly (p< . ) in patients receiving allogeneie blood (from . + . to . _+ . ie/ml) whereas in patients receiving autologous blood a smaller increase (from . + . to . + . ; p= . ) was observed. tnflevels demonstrated a similar pattern with a higher increase in patients receiving allogeneic transfusions (l . + . to . + . u/ml) compared to those patients with autologous blood ( . + . to . + . ). in conclusion these data indicate that allogeneic blood transfusions lead to a remarkable macrophage/rhs stimulation. this is corroborated by the boostered humoral igg-response which was initiated before onset of surgical trauma and blood transfusion. concerning cmi this caused a substancial suppression probably due to a stimulated secretion of immunosuppressive monokines. objective: firstly, to analyse the concentrations of the cytokines tumor necrosis factor (tnc), interleukin- (il-i), interleukin- (il- ) and coagulatioo/fibrinolysis parameters in postoperatively retrieved blood from a surgical area, secondly to characterize the correspanding cytokine patters in the patients and thirdly to study cytokine concentrations in the initial portion of drainage blood from a surgical area. materials and methods: blood retrieval was performed in a closed-loop system without anticoagulant during - hours after surgery in patients undergoing arthroplasty ( hips and knee). kf, il- , it- , thrembin-antithrombin complexes (tac) and antithrombin (at) ~ere determined in shed blood. patient plasma tn v, il-i and il- concentrations ~ere analysed at the beginnlqg and end of the - hour blood retrieval period. in a separate study ( hip arthroplasties) f~f, il-i and il- ~ere determined in the initial portion of drainage blood. cytekine analyses ~re performed usiog ipmuooassays. an omidolytic method was used for at determinaf.ion and tac was analysed by elisa. n~n-poram~tric tests was used for the statistical comparison. results: the patient plasma il- coocemtratiems rose from a median value of to pg/ml, p<o. , during the blood ret.rieval period. in c was detectable in patients, range: - pg/ml. il-i was not detectable in the patients. in retrieved blood tag was > mg/ml in all samples (ref:< . mg/ml) and at was . - . units/ml (ref:o. - . ) . the il- concentrations in retrieved blood was > pg/ml in all samples. tn v or il-i was not detectable. in the separate study, (n= ), characterlzing eytokine content in the initial portiere of drainage blood, in= (range: - pg/ml) and il-i (range: - pg/ml) ~re present in all samples but ii- (range:o- pg/ml) was detectable in o.qly one semple. conclusion: theses findings indicate that hypereoagulability and hic~ ccrcentratioos are present in retrieved blood. the cytokine pattern in the initial portion of blood from a surgical area differed from these observed in retrieved blood and in the systemic circulation. to identify the role of both autologous and homologous blood on postoperative infections in elective cancer surgery. materials and methods: patients with colo-rectal cancer submitted to curative elective surgery were prospectively studied. on hospital admission the following nutritional measurements were assessed: serum level of albumin, cholinesterase, delayed hypersensivity response , total lymphocyte count and weight loss, as were age and sex, duration of operation , operative blood loss, amount and type of blood given, pathological dukes' stage of the disease and the attending surgeon were also recorded. results : eighty-four patients ( . %) were perioperatively transfused. thirty-six ( . %) patients were given autologous blood , while ( . %) received homologous blood. no patients received both autologous and homologous blood. twenty eight ( . %) patients developed postoperative infections. non transfused patients had a . % infection rate , those receiving autologous blood had a . % infection rate, whi]e in the homologous blood group the infection rate was . % (p < . ). univariate analysis showed that infections were significantly related to operative blood loss (p< . ), length of operation (p< . ) blood transfusion (p< . ) and attending surgeon (p< . ) . multivariate analysis identified homologous blood transfusion as the only variable related to the occurrence of postoperative infections , while the other variables failed to reach statistical significance. blood transfusion (bt) remains an essential life-saving treatment for surgical patients. however, besides the beneficial short-term impacts, negative longer-term effects are observed, which include various alterations in the immune responsiveness. in surgical patients these alterations may contribute to the increased risk for infections and cancer recurrence. since relatively few data demonstrate immunologic changes occurring in other lymphoid compartments than blood after bt, we studied the effect of et on the frequency and responsiveness of immune cells in bone marrow (bm), spleen (spl) and blood (b) in a rat model. normovalemic, month old rats were transfused intravenously with syngeneic heparinized venous blood ( x ml, every other day), and , and days after the last transfusion bm cells ( leh is an experimental oxygen-carrying resuscitation fluid. since leh is cleared from the circulation primarily by the mps, its effect on the development of sepsis and the nature of its relationship with the mps remain a major concern. preliminary in vivo data from our laboratory failed to show any leh effect on the hemodynamic and hematologic responses to endotoxin lipopolysaccharide (lps) in the rat. in contrast, leh exacerbated the lps-induced tnfa production and early mortality. the exacerbation of early mortality by leh was attenuated by pretreatment with the tnfu synthesis inhibitor rolipram. ex vivo, peritoneal macrophages from rats treated with leh and lps have shown increased il-lg mrna signal as compared to lps alone. also, leh increased tnftx production by peritoneal macrophages in response to lps stimulation in vitro. additionally, recent pilot studies indicate that leh attenuates pma-induced superoxide production from rat peritoneal macrophages and that leh augments fmlp-induced migration of human monocytes. taken together, these data strongly support possible interactions of leh with the mps and therefore the nature of such interactions should be further explored. over the last decade, we have developed liposome encapsulated hemoglobin (leh) as an artificial oxygen carrying fluid, or blood substitute. our efforts have focused on studies to define the safety and efficacy of this resuscitative solutions. leh consists of distearoyl phosphatidylcholine, cholesterol, dimyristoyl phosphatidylglyeerol, and alpha tocopherol in a : : . : . mole ratio and can encapsulate hemoglobins of different origin (bovine, human, recombinant human). leh is fabricated using hydrodynamic shear to create an average particle size of . microns. leh can be lyophilized using disaccharides and stabilized in the dry state and easily reconstituted before administration. histopathology and clinical chemistries indicate that leh rapidly accumulates in tissue resident macrophages in small animals injected in the tail vein, principai y in the liver and spleen. the consequences of accumulation in the reticuloendothelial system are manifest by transient increases in liver transaminases (ast, alt), bilirubin, and bun over - hours with no change in biliary function (ggt, ap) . clearance through the liver and spleen is observed over the course of - -weeks. more recent attention has been focused on secondary consequences of leh administration especially with regard to inflammatory eytokines. leh does not elicit expression of tumor necrosis factor in vivo and in isolated macrophage cultures, but does result in a transient increase in serum il- . we have also examined the interaction of leh with lps in vitro macrophage culture to further understand how this blood substitute may effect the immune system. we have labeled leh with technetium- m ( mtc) to study the biodistribution of leh non-invasively in anesthetized rabbits. rabbits were infused with a % topload of leh ( mg of phospholipid, . g of hemoglobin per kg of body weight) and imaged continuously with a gamma camera. at hours, images were again acquired. animals were then sacrificed and tissue counts obtained, images revealed an initial rapid uptake bythe liver, % at minutes and % by hours. the spleen accumulated activity at a slower rate, % at minutes and % at hours. at hours, autopsy biodistribution studies revealed that approximately . % of the dose is in the blood pool, . % in liver, . % in spleen, . % in lungs, . % in muscle and . % in urine, with trace levels in kidney, brain and heart (< °/o). in a hypovolemic model, rats were % or % exchange transfused with mtc-leh. in the % exchange model, mtc-leh was rapidly taken up by the liver and spleen with minimal activity in the circulation at hours. with the % exchange, % of the leh was in circulation at hours. the interaction of leh with platelets labeled with indium- was also studied. after infusion of leh, the labeled platelets rapidly moved from the circulation to the lungs and liver. over the next minutes, the platelets gradually returned to circulation. this effect was not seen with iiposomes of the same lipid composition but containing no hemoglobin. non-invasive imaging is proving to be a very useful tool for the investigation of leh. the need for a safe, efficacious and commercially viable blood substitute is unequivocal. of the several strategies pursued to invent an adequate blood substitute, liposome entrapped hemoglobin (leh) has been already established as a leading possibility. major advances in liposome technology have already resulted in liposome preparations compatible with clinical use for drug delivery. recent technological advances made by the u.s. naval research laboratories resulted in the capacity to entrap hemoglobin into liposomes in a way which secludes hemoglobin from interacting freely with biological systems. the leh produced has already been tested in in vivo systems and was foun.d to be well tolerated. moreover, the leh originally produced as a solution can be transformed into a lyophilized form which can be reconstituted and delivered as a fresh solution. while important milestones in leh development for a practical blood substitute have been achieved, several issues remain to be explored. most notably, the long term consequences of leh on host defense mechanisms and, in particular, immune cell function. in addition, it is important to understand more fully the metabolic fate and repercussions of leh delivered at clinically relevant dose/schedule regimens. finally, while leh is a highly promising strategy for a blood substitute, the present formulations consist of human hemoglobin derived from human blood, to improve the safety profile, a recombinant preparation for liposome entrapment will be much desired, aa-ginine, a semi-essendai dietary amino acid, possesses several unique and potentially pharmacologic properties. argirdun is a potent secretagogue for pituitary growth hormone and prolacfin and for pancreatic insulin and glueagon; it modulates host protein metabolism by increasing nkmgen retention and enhancing wound collagen synthesis. it also is a potent t call function regulator. ait of these effects coupled with its relative lack of toxicity and safety make it an a~antive nulritionai pharmacologic agem (t). rodents fed supplemeutal arginine exhibit increased thymsc weight which is due to increased numbers of thymic lymphocytes present in the gland. thymic lymphocytes from animals fed supplemental ar~e demonstrate increased blastogenesis in response to coma. and pha ( ) . peripheral blood lymphocytes from humans given supplemental arginine also have heightened mitogunic responses to mitogen or antigens ( ) . in postsurgery padents supplemental arginine abrogates or diminishes the deleterious effects of trauma on lymphocyte responsiveness and restores peripheral blood lymphocyte responses much faster than observed in controls. overall host immunity is also enhanced by arginine. allograft rejection is enhanced and septic animals survive longer when given supplemental arginine ( ) . tumor bearing urginine-supplemented animals have decreased tumor growth and enhanced survival (i). lastly, asgmine can induce t cell maturation and t cell mediated responses in athyrnic nude mice. arginine also has remarkable effects on host nitrogen metabolism post-injury. in increases nitrogen retention in healthy human volunteers and in surgical patients. this beneficial effect on overall nitrogen metabolism is accompanied by a unique effect on the healing wound. supp]emental arginine increases wound collagen synthesis which also translates into increased wound breaking strength ( ) . arginine has no effect ou epithelialization. douglas w. wilmom, m.d. boston, ma gintamine is the most abundant amino acid in the body, but it has long been considered a nonessential amino aeid because it is synthesized in many tissues. fohov~g st,~'vation~ injury or infection, skeletal muscle pmteln inoresses its net tale of degradation and releases amino acids into the blunds~mm at an aocelerared rate. app~o)~mately one-third of the amino nitmgea is ghitamine, which is metabolized by the kidney where it parth:~pates in acid-base homeostasis, is the primly ~ for lymphocytes, mac~optmgcs and untexocyms, and contm'butcs to the synthesis of giumth~une. olmamine degrades slowly while in ~olu~ou, especially at usual room teml~mtums. because giulamine was considered nonessential, it has beer absent r'om nil intravenous and most gluts.mine should be considered a cendittona]ly essential nutrient for individuals with serious ilinesses, uspccially those confoanded by infcctinn and inflammation. over the uc~:t - years, glutamine will be incorgorated into most feeding formulas designed for patients with critical illness. o]~ga- pufa there continues to much interest in the application of the mega- pufa in clinical nutrition. the basic principle has been that the mega- pufa will displace arachidunic acid and result in a decrease in eic san id production. in addition these changes in pufa will after the physical characteristics of the membrane including flujdity, receptor function and transmembrane signals. animal studies have shown that there is omega- incorporation with continuou~ enteral feeding both in control and endotoxic animals within days. this includes the liver, spleen, circulating and alveolar marc phages and the lung. this incorporation resuls in significant changes in the eicosan id production including pgf and ket -pgflalpha. there is improvement in the cardio-vascular reep nse of these animals with ~ecreamed lactic acidosis and improved cardiac contractility. as well there is improved immune function with improved t cell response to mit gens. the ~ of a mumber of pharmacological agents blocking cicosanoid production can enhance the cell effects of mega- pufa. clinical studies using short term entsral nutrition with mega- either alone or with other enteral supplements in a number of clinical settings have shown significant mesa- incorporation and decreased eicosan id production. these positive results must be discussed with the additional evidence that long term omega- supplementation decrease eic san id production but als induce a state of immune suppression that is capable of increasing transplant sunvival. these ng te~ inune effects may benefit clinical conditions including rheumatoid arthritis and cr hn' disease early enteral nutrition instituted i~mediately afte~ injury will decrease the entry of bacteria into the intestinal wall and decrease the number of bacteria that translocate into the portal blood. these reductions are associated with & decreased catabolic response, decreased plasma cortisnl levels, end decreased vma excretion in the urine and prevention of mueosal atrophy. sdecific nutrients also affect the transloeation process. addition of arginlne to the diet significantly improves the ability to kill translocated organisms. however. translooetion across the gastrointestinal barrier is not affected. in contrast, glutamine diminishes the rate of translooation across the imtestinal barrier and also improves killing of the beetarla that do translooate. the omega fatty acids in the form of fish oil slightly decrease the rate of translocation but more significantly increase the ability of the animal to kill translo~ated organisms, all three dietary additives, i.e. argini~e, glu=amine and fish nil. significantly improve survival, hut adding glyoine or medium chain triglyeeridem do not, combinations of srginine and glutamlns, glutamine and fish oil, and fish ell end arginine each improve survival, and to a greater degree than a combination of all three. these studies add further evidence that translocation is an important determinant of survival after injury, early feeding with immunonutrlent enriched dices will improve survival and dsarease transloeation to varying degrees, depending upon the nutrients provided. objectives: we studied effects of supplementing a commercial enteral diet, impact r (imp, sander nutr lnc), with fiber (imp/fib) or alanyl-glutamine (imp/ag, exogenous glutamine (gln) gms/l) on influencing the incidence of bt to mesenteric lymph nodes (mln) in burned mice. fiber has been shown to improve gi integrity under certain stress/treatment conditions. the dipeptide ag is a water-stable source of gln, which is a specific fuel for many cells including enterocytes. traumacal (trcal), a high-protein, high-fat enteral diet (mead johnson iuc), was also studied, as well as rodent chow (harlan teklad inc), which contains very high protein & fiber. methods: anesthetized cf- mice aged - wks received % tbsa fullthickness dorsal burns & were resuscitated with cc ip saline. diets were allowed ad lib; caloric intakes were comparable in all gps except fasted gp (fast hrs, chow hrs). at hrs postburn mln were sterily removed, homogenized and plated on heart brain infusion agar; cfu/g mln tissue were determined. bt was analyzed by fishers exact test, cfu/g by anova-bonferroni. * p< . , ** p< . compared to imp and burn-fast gps. background. infectious complications following trauma, major operation, or critical illness adversely affect hospital cost and length of stay (los). some key nutrients have been shown to possess immune enhancing properties. this multicenter trial was conducted to determine if early administration of an enteral formula supplemented with arginine, dietary nucleotides and fish oil can decrease los and infectious complications in icu patients. methods. this was a prospective, randomized, double-blind study of adult icu patients who required enteral feeding for > days. patients entered the study within hr of the event, were stratified by age and disease, and were randomized to receive either the supplemented formula (impact®) or the conventional formula (osmolite ® hn). feedings were initiated at full strength and advanced to at least ml/hr by hr after event. results. both groups tolerated administration of formula well. for patients fed > days, the median los was % shorter (p=o.ol) for the--supplemented group ( days) compared to the conventional group ( days). the incidence of most infectious complications was lower in the supplemented group, but this difference reached significance only for urinary tract infections (p=o.o ). the supplemented group had a significantly shorter los from onset of infectious complication until discharge for patients with pneumonia ( vs. days) and skin/soft tissue infection ( vs. days). conclusions. administration of the supplemented formula was safe and well tolerated. when fed > days, it reduced the incidence of most infectious complications, and significantly reduced los. materials and methods: twenty-seven patients were randomised into groups ( n= each) to receive either a standard enteral formula, the same formula enriched with arginine, rna and omega fatty acids (enriched group) or isonitrogen, isocaloric parenteral nutrition. early enteral nutrition was started within hours following surgery ( ml/hour). it was progressively increased reaching a full regimen on day . on hospital admission and on post-operative day and , the following parameters were assessed: serum level of transferrin , albumin , prealbumin, retiool binding protein (rbp), cholinesterase. delayed hypersensitivity response, igg, igm, iga, lymphocyte subsets and monocyte phagocytosis ability were evaluated on admission and on post-operative day , , . the three groups were comparable for sex, age, cancer stage, type and duration of surgery, intra-operative blood loss and amount of blood transfused . in all groups a significant drop in all the nutritional and immunological parameters was observed on postoperative day . comparing post-operative day versus day a significant increase of prealbumin (p< . ) and rbp (p< . ) was found only in the enriched group. with respect to immunological variables an increased phagocytosis ability (p< . ) and a significant recovery in delayed hypersensitivity response (p< . ) was observed only in the enriched group. conclusions : these data are suggestive for a more effective post-operative recovery of both. nutritional and immunological status in cancer patients fed with enriched enteral formula. gastrointestinal intolerance was equivalent ( % in each group) and laboratory screening confirmed that both diets were safe. when analyzing clinical outcome for all patients, there were no significant differences in septic complications (immun-aid = % vs vivonex ten = %), mean mof score (immun-aid = l.b vs vivonex ten = . ), or mortality (immun-aid % vs vivonex ten = %) . kowever, when analyzing the subgroup of patients with severe injury (iss or ati _> ), patients receiving immun-aid appeared to have fewer septic complications ( % vs %) and their mean mof was significantly lower ( . _+ . vs . + . , p = . , student's t-test) . these preliminary data indicate that immun-aid is tolerated well when aggressively delivered immediately postinjury. the ultimate affect on clinical outcome appears ~avorable for immun-aid, but needs to be confirmed in larger patient groups. kemp?n, m., neumann, h.a., he i[michh b: as both increased, normal and reduced phagocytic capabilities of polymorphonuclear leukocytes (pmn) and monocytes in acute batterial infections have been reported, the role of phagocytes in patients with severe sepsis is less clear.we examined pmn and monocytes from patients in septic shock and heailhy votunteers for phagocytic function. phagocytosis was determined by flow cytometry (facscan) and was measured by the ability of pmn and monocytes to phagocytose e.coli marked with fluorescent antibodies. a septic shock was defined by the presence of a ~ource of i, nfoctiqn with a known bacteriology, distinct signs of a systemic response and defined minimum scores in icu scoring systems indicating the presence of a multiple organ failure. additionally we examined how phagocytosis is influenced when a new enteral diet formulation containing substrates suggested to improve immune function or arginine, one of its major compononts, is added in vitro in defined concentrations and incubated for minutes. pmn (p{o, ) and monocytes (p<o, ) of the septic patients showed a significantly enhanced phagocytosis compared tolhe phagocytes of the healthy group. the incubation with the enteral diet in vitro was followed by a higher dose-related rate of phagocytosis, especially in the healthy group, that was not statistically significant. after addition of l-argintne we atso observed an iqcrease in phagocytosis, that was lower than in the group with the complete diet. using a modern immunefluorescence-cytometric essay we founfl an improved phagocytic function in septic shosk. there are signs for an influence of nutrient substrates on phagocytosis which seems to be complex and should be further investigated. arg and gln were lower in the %-bcaa vs %-bcaa group; arg was related directly to arg dose and inversely to bcaa dose (p<o.o for all). clearances of bcaa increased with increasing bcaa dose (p<o.o ); arg and gln clearances were directly related to the bcaa clearances (rz=o. , p<o.o i). relationships between arg, gln and other aa seem to be ruled by patterns involving specific intracellular aa transport systems. regulation of arg and gln clearances through bcaa administration may be related to an increased flux of aa into synthetic processes. intestinal segments from rats immunized by infection with the nematode trichinella spiruus undergo intestinal anaphylaxis or type i hypersensitivity when challenged in vitro with parasite antigen. immunized rats exposed to t-radiation ( gy) and sustained on rat chow,fail to fully express type i hypersensitivity up to days post irradiation (dpi). we hypothesized that enteral nutritional support immediately following irradiation may be effective in preserving mucosal immune responsiveness or in reducing the recovery time after radiation-induced injury. rats were infected with x t.spiralis larvae. thirty to fifty days later rats received gy t-radiation from a co source as total abdominal irradiation (tai). immediately following tai, rats were fed an elemental amino acid diet (eaad) and at various dpi sacrificed and tested in ussing chambers for local anaphylaxis, expressed as antigen-induced ci secretion. the normal ci secretory response to antigenic challenge which is suppressed after irradiation,was restored by dpi when rats were placed on the eaad. antigen-induced c[ secretion is dependent on the interaction o~ antigen with specific ige antibodies on the surface o mucosal mast cells and their subsequent degranulation. mast cell-derived -ht,histamine and pg, together effect ci secretion.in irradiated rats on rat chow,the failure to respond to antigenic challenge appears to be associated with the destruction of mast cells. they are undetectable with standard staining procedures and mucosal histamine levels are drastically reduced. also,el secretion can be induced by direct stimulation of epithelium with cr secrctagogues and circulating ige levels are normal. despite a more rapid recovery of immune responsiveness in irradiated rats receiving eaad,mast cells were not restored. we conclude that the eaad contributes to an adaptation that supports the expression of local anaphylaxis in the absence of mast cells. total parenteral nutrition (pn) and bowel rest may influence the host response to infection. this study examined the different host response to infection in parenterally and enterally fed animals. forty-three rats were divided into pn group and total enteral nutrition (en) group. they received identically constituted isocaloric isonitxogenous diets for seven days. animals were then challenged intraperitoneauy with xl(y escherichia coli and survival were observed. in other experiments, they were sacrificed before and two hours after bacterial challenge. peritoneal cavity was lavaged with ml saline and peritoneal exudative cells (pec) were harvested and cultured in vitro for hours with and without lipopolysaccharide (lps). colony f(m, ning units of bacteria (cfu-b) in the peritoncal lavaged fluid (plf) were determined and tnf in the plf, serum and pec culture supernal,ants were measured by l bioassay. twenty-fonr hour survival rate in en group was significantly greater than that in pn group ( % vs %). the data suggest that local immune responses of pn-treated animals may be depressed with consequent disturbance in the clearance of bacteria. this may lead to a greater systemic cytokine response and resulting in a poor survival after bacterial challenge in pn group. the effects of intragastfic tube feeding of diets enriched with m- polyunsaturated fatty acids (pufa) from safflower oil (so) or (o- pufa from menhaden oil (mo) on eicosanoid production, and onthe membrane phospholipid fatty acid composition were investigated in a severe acute septic shock model. the percent calories from fat maintained at % in the diets was adjusted to provide % each of saturated, monounsaturated, and pufa using olive oil and palm oil as filler fats. after feeding for days, lipopolysaccharide (lps, mg/ g) was injected through the tail vein. the percent of animals surviving in the mo group was % ( / ), and did not differ significantly compared to % ( / ) in the so group. the plasma concentrations of tnf ct for the groups of animals fed so ( . + . ng/ml) and those fed mo ( . + . ) did not differ significantly. the levels of prostaglandin(pg)e , -keto-pgflc~ and tumor necrosis factor-ct (tnf-c were determined min after lps injection. the fatty composition (relative mole%) of the liver was determined before (lps-) and h after the lps administration (lps+). the data (mean_+sd; n= ) are as follows. group these data indicate that a marked reduction in the plasma levels of poe and -keto-pgflct for rats fed mo diet was associated with a reduction in aa which was displaced by epa in the membrane phospholipids. further, in these rats, we observed that there was a . % reduction in the percent of epa following lps challenge compared to the basal levels. however, for the group of rats maintained on so diet, the liver membrane phospholipid fatty acid composition before and after lps administration was unaltered. these finding suggest that although a reduction in aa was associated with a decrease in the production of pro inflammatory eicosanoids after days of continuous mo feeding, there was no marked effect on the survival following lps challenge. animal models have been used to examine the effects of various nutrients and nutrient combinations upon the immune system. we studied the effects of standard and specialized enteral formulations on the response of mice to infectious challenge with listeria monocytogenes, influenza a or candida albicans in order to test the efficacy of specialized ingredients. cf- outhred female mice ( - g) were fed purina # chow, commercially available osmolite hn ®, perative ® or impact*. mortality and tissue burden of pathogen were examined during the d period following induced infection. the results indicate that there were no differences between the groups fed the liquid formulas with regards to mean survival time or % survivors in these models of infection. examination of spleens in the groups challenged with l. monocytogenes and kidneys in the groups challenged with c. albicans revealed no differences in tissue burden of pathogenic organisms. alterations in the length of pre-feeding from to days prior to infection did not affect these results. these data demonstrate that, contrary to previous reports, the use of specialized nutrients did not improve resistance to disease in mice challenged with l. monocytogenese, influenza a or c. albicans as compared with mice fed osmolite hn. animals fed standard chow tended to be more resistant to infectious disease than those fed the liquid formulas perhaps due to the form of diet or complexity of ingredients. the results indicate that these animal models of infectious challenge may be of limited use to distinguish effects of modified nutrient composition of enteral formulas. laboratories the beneficial effects of sesame oil (ses) have been attributed to the presence ofsesamin, a non-fat constituent which is claimed to iultibit a- desaturase activity. we investigated the effects of ad libidum feeding of ses enriched diet on fatty acid composition of phospholipids from plasma, liver, on eicosanoid production, and on the protective effects in mice after cecal ligation and puncture (clp) and compared with safflower oil (so) diets. olive and palm otis were added as filler fats to the so diet to maintain the ratios of saturated, and unsaturated fatty acids similar to ses diet. thus, while the total fat remained at wt%, the only difference between the two diets is the presence ofsesamin in ses diet. the animals were fed for weeks. the incorporation of dihomo-y-linolealc acid (dgla: : o)- ) in the phospholipids from plasma and from the liver for the group of mice fed so diet ranged between - % compared to the undetectable levels for those fed so diet. there were no significant differences in the incorporation of other fatty acids either in plasma or in the cell membranes between the two groups. following an intraperitoneal administration oflps ( p.g/kg body wt), the plasma levels of both pge , and txb , were significantly decreased (p< . ) by nearly % for the group of animals maintained on ses diet compared to those maintained on so diet. these findings suggest that sesamin inhibited the release of arachidonic acid (aa) which upon accumulation is retroconverted to dgla which could reflect a modest increase in the content of dgla. failure to decrease the formation of aa could mean that the effects of sesamin on chain elongation process may be after aa is formed, and that sesamin may not inhibit a- desaturase activity. on the other hand, our data suggest that sesamin present in sesame oil inhibited the activity of cyclooxygenase which converts aa to its metabolites), or alternatively could block the activity of phosphalipase a (pla ) (which releases aa from membrane phospholipids) as is evident from a marked decrease in eicosanoid production. the percentage of animals surviving after cecal ligation and puncture in rite group of mice fed ses diet was % ( / ) which was markedly higher (p< . ) compared to only % ( / ) for the so group. increase in survival in the group of mice fed ses diet was associated with nearly % reduction in the production of tnf in response to lps i.p. challenge, for ses group compared to so fed animals. our data suggest that sesamin, present in sesame oil, decreased the production tnf~, inhibited the activity ofpla and consequently offered a significant protection against clp. thus sesamin or sesame oil appear to be novel antiinflammatory agents which are present naturally in many edible products. [dept. surgery, ~e. deasoness hosp.,harvard medical school, boston, usa] stress causes alterations in the homeostasis of an organism with major changes in various organs, lifespans of cells, protein turnovers, metabolic pathways of activation or stimulation, energy mobilization, etc. punctual changes have been described in several organs and various cell types of the nervous, endocrine and immune systems. many stored or newly synthesized proteins are released to the bloodstream to be transported to target organs or to be disposed of. as stress alters protein synthesis and requirements in the targets, the bloodstream is a useful system for monitoring the relevant changes. we present here the pattern of newly synthesized or released serum proteins in c bl/ mice that have been stressed by immobilization. the proteins have been labelled with ssmethionine in vivo and the study was performed by two dimensional gel electrophoresis based on the method originally described by o'farrel. the electrophoretic run was carried out in an isodalt apparatus. a molecular dynamics laser scanner and the kepler image analysis system (lsb, rockville, usa) were used for modelling the radiofluorographic images. the radiofluorographic images of the gels from serum samples obtained from mice injected with i mci of ss-methionine reveal about protein spots, from which a small fraction -about are altered in both qualitative and quantitative manner. the major changes are in the range of to . d. the analysis displays a highly reproducible pattern, where clear differences between stressed and non-stressed conditions are shown. differences between patterns attributed to chronic vs. acute stress will be presented. patients with severe trauma or major surgery are known to acquire alterations in neutrophil functions which contribute to their enhanced susceptibility towards microbial infections. we analyzed neutrophil functions from fourty patients admitted for major surgery days and days preoperatively and on the ist and th postoperative days (study-days i, , , and ) in a randomized placebo-controlled double-blind study. patients were divided into two groups, one ~eceived days preoperatively an enteral nutrition enriched with omega- fatty acids, arginiee, and rna (impact), the other an isocaloric, not enriched control nutrition (placebo). meutrophils were isolated from the peripheral blood and stimulated with the ca-ionophor a ( . pm). the capacity of the respective neutrophils to generate leukotrienes was analyzed by rp-hplc. neutrophils from impact group generated significantly higher amounts of ltb (mean values . ng/l x i~ cells) compared to the placebo group ( . ng) at study-day [p, . ). in contrast, the capacity of neutrophils to produce ltb was not significantly different in both patients populations at study-days and . the omega-oxidation of ltb to its biologically less active metabolites oh-ltb and cooh-ltb was not significantly different in both groups. however, neutrophils from group f patients generate more ltc at study-day (p( / ). the capacity of the patients'neutrophils to generate reactive oxygen radicals upon stimulation with fmlp, pma or the caionopbore a exhibited patient dependent differences but no correlations to the repective nutritional supplementation as was measured by luminol dependent ohemiluminescense= these data indicate that an enteral nutrition enriched in omega- fatty acids lead to alterations in distinct parameters of neutrophil functions. clinical patient characteristics and mean caloric intake were similar between the two groups. both formula were tolerated well and the incidence of diarrhea was low. the number of infectious and wound complications as well as the apache ii score was evaluated for the two study groups. although the number of complications in the group supplemented with arginine, rna and omega- fatty acids was lower no significant difference in the occurance of infectious and wound complications has been observed between the two groups. however, the apache ii score indicated a significantly improved clinical outcome in patients with supplemented diet. in conclusion, early enteral nutrition with an arginine, omega- fatty acids and rna supplemented diet helps to recover more rapidly after surgical total parenteral nutrition (tpn} is associated with intestinal atrophy and dysfunction attributed to the absence of the non-essential amino acid glutamine in current parenteral nutrition solutions. in this animal study with male wistar-rats we tested the hypothesis that glutamine-dipeptide supplementation during tpn for days would restore small bowel atrophy and tpn induced dysfunction. a conventional tpn solution ( kcai/kg bw) was compared to an isocaloric and isonitrogenous tpn ( , g n/animal/d) supplemented with alanin-glutamin (ala-gin) dipeptide ( , g n derived from ala-gin = % gin-n). an enteral fed cqntrol group was included. mucosal thickness, villous height and architecture, absorption of water and glucose as well as dissacharidase activity of maltase and alkaline phosphatase were evaluated. total parenteral nutrition in rats causes villous atrophy, a significantly reduced absorption rate and a decreased activity of villous enzymes compared to an enteral fed control (p<o, ). addition of ala-gin to parenteral nutrition solutions prevents intestinal atrophy and restores functional alterations with a significantly increased rate of water and glucose absorption as well as a higher dissacharidase activity compared to the standard tpn group (p< , ). there was no significant difference found between the enteral fed control and the dipeptide supplemented animals concerning intestinal structure and absorption, the enzyme activity was significantly decreased in the ala-gin supplemented group compared to the enteral fed control (p< , ). in this rat model supplementation of parentera] nutrition solutions with ala-gin dipeptide restores tpn induced intestinal atrophy and dysfunction. boston. ~ usa injury, infection and major o ~'ations s~nulate a variety of factors whi~ initiate the body's response to th~ st~..sses. 'i"hese reactions are m~liated by a cbmage ia the neta'al hormonal, eac.ranmemt, by the elaboration of eytokines and tl~ougb the stimu/ation of other inflammatory mediators. this eatabolio setting zesulis in body protein loss, which g-taduai y erodes both fimetianal and stm~ tissue. wiu _b the normally nom'ished individual can withslzod "d~ response for a brief period of time, ircolonged eatabollsm l~ associated with ~,nmunosuppmssion, poor wound healing, and proioagcd convalescence. surgeons have rej ed on intraveaous or eater~ feedings to ~everse this process. a va~ety of data now indicates that it is extremely di~edt to replete protein-containing tissue d~g the eataholie state of illness; the umutt response to hypercaloise feedings in this setting is the incxeased accretion of body fat md water. administration of gxow~ ho~moae ~cesults i.a n shift of the body's oxidative machinery to fat rather than eazbohydmte utiq~afic~; ¢oncomitaatiy, protein synthesis is stimulated. this metabolic state may have clinical utility in p~e.n.ts who need to heal large wotmds, in those who need to gain strength in order to be w~ed ore veatilatory support, or those patients who ~ve mulfiorgan failure and nee~ to ealaaace protein syatsesis to in.suze recovery. als are now in progress to dcterm~e the benefit of growth hormone in these and other disease state.s, rn the fature, the increased use of growth hormone, will occur;, this and other growth factors will serve to su~ po~ the host and shorten convalesceace following catabolic diseases. our objective was to study effects of pre-trauma growth hormone (gh) treatment on liver and skeletal muscle metabolism in a trauma model in piglets. twenty-four piglets were randomized to three treatment groups; one group was pretreated with gh iu daily three days before the experiment (gh- ), another group treated with gh ie at the start of the surgical procedure (gh- ) and one group served as untreated controls (con). they underwent a standardized surgical procedure to place sampling cathethers in the portal, hepatic and femoral veins and doppler flow probes around the portal vein, the hepatic and the femoral arteries. all pigs recieved a primed constant infusion of u- c-glutamine. substrate fluxes were measured one (lh) and five ( b) hours after termination of the surgery. nitrogen excretion was significantly decreased in the gh treated animals (gh- : . + . mg/kg, gh-i: . +_. . mg/kg, con: . +- . mg/kg, p= . ). in the hindieg, there was reduced net glutamine efflux due to decreased absolute glutamine release (gh- : . + . gmol/min at lh and - . + . in.tool/rain at h, gh-i: - . + . i.tmol/min at lh and - . + . pmol/min at h, con: - . +_ . g mol/min at lh and - . + . /amol/min at h, p= . , p= . ), whereas the absolute uptake of glutamine in hindleg was unchanged (p= . ). glucose uptake in the hindleg was decreased (p= . ). net glutamine uptake in liver was attenuated (p= . at h), whereas net glutamate release from liver was increased (p= . ). thus, pre-trauma treatment with gh improved nitrogen economy, attenuated net glutamine loss from hindieg due to decreased absolute release, and attenuated hindleg glucose uptake. liver net glutanaine uptake was decerased and net glutamate release increased. patients with major abdominal surgery exhibit a considerable catabolic response with negative nitrogen balance and protein breakdown, which may have detrimental effects on outcome. we investigated the effects recombinant human growth hormone on substrate metabolism in parenterally fed patients. metabolic healthy patients were randomized to receive either placebo or hgh in a dosage of , ; , or , i.u. per kg bw. substrate oxidation rate, energy expenditure as well as short life proteins were measured daily. six patients were excluded from analysis as drop outs, due to surgical complications. we could find a dose-dependend effect of growth hormone on resting energy expenditure, carbohydrate oxidation, fat and proteine oxidation. with increasing growth hormone resting energy expenditure increased from . calories per kg body weight to . calories. this was mainly due to an increased carbohydrate and fat oxidation, while proteine oxidation decreased. this was in accordance with a decreased urea.production rate and an improvement in cumulative nitrogene balance, which increased from minus . to plus . g n / days. in consequence short life proteins were also increased. the only negative side effect was an increased blood glucose concentration in some of the patients, making insuline administration mandatory in patients. our results are in accordance with other studies showing improvement of nitrogen balance, maintainance of lean body mass and muscular strength. if growth hormone administration may have any impact on morbidity, mortality and length of hospital stay in these patients it should be evaluated in a larger number of patients. in patient after liver transplantation (oltx) the effect of recombinant human growth hormone (rhgh) on protein metabolism and hormonal changes was studied. patients and methods: the underlying disease in all patients was end stage liver cirrhosis, non of the patients included in the study was suffering from malignancies. the patients were randomly allocated to receive either u of rhgh bid (sc) or no alternative medication. the study period lasted days. from daily hrs udne collection urinary nitrogen loss and daily loss of urea were determined. body compostion analysis (bca, bioimpedance, akern-ltaly) was performed preoperativly and at the postoperative days and to evaluate changes in body water and body cell mass (bcm). the effect on resting energy expenditure (ree) was analysed using indirekt calorimetry wrhin the same intervalls (metabolic monitor, datex). blood levels of gh, igf and igf , igfbp were measured daily during the study period, on day a hour profile of gh was.performed. samples were collected each minutes. results: cumulative udnary nitrogen and urea loss were not signifcantly different for the whole study period but for the first study days. bia indicated for the rhgh-group a loss of bcm to a lesser extent than for the controls (n.s.), changes in bodywater where similar for both groups. ree changes (kcal/kg bcm) were not significantly different for the two groups. blood levels of gh and igf differed significantly between the groups for the whole study period, but not igf and igfbp- . the circadian rhythm of endogenous gh-excretion had not been altered by gh, but absolute levels of gh were increased. conclusion: during the first days after oltx we could proof the protein sparing effect of gh treatment. although the differences between the two groups were not significantly different for the whole study period the results showed on all days a less catabolic situation for the rhgh treated patients. to possibly improve protein balance after major abdominal surgery we studied the effects of gh on protein metabolism after ltx. excluding malignant diseases, candidates for ltx were randomized to either postoperatively receive the usual treatment (co=control group, n= , age range - yr, bmi . + . kg/m ) or for days additional gh ( x i.u.s.c.) (gh group, n= , - yr, . + . kg/m ). metabolic studies (calorimetry and lsc-leucine infusion) were performed (test a) and days (test b) after surgery. tracer analysis was done by gas chromatography-mass spectrometry. during the -week study clinical parameters did not differ significantly between groups; however, there was a tendency for increased glucose levels, insulin need, and energy expenditure at the time of test b in the gh group. leucine oxidation (x+se) during test a;b was + ; + (co) and + ; ± (gh) pmol/kg ffm/h (n.s.). protein breakdown was ± ; + (co) and + ; + (gh) pmol/kg ffm/h (n.s.). nonoxidative-leuine disposal was ± ; + (co) and ± ; + (gh) pmol/kg ffm/h (p< . for the rise in the gh group). tracer data were supported by cumulative urea nitrogen excretion (days - : gh + vs co ± g; p< . ). we conclude that after major abdominal surgery gh lowers nitrogen excretion by increasing total body protein synthesis. it is not known which organs contribute to this effect. in several investigations it has been shown that the protein saving effect of recombinant human growth hormone (rhgh) in the postoperative state is accompanied by raised igf- levels in plasma. it was concluded that the anabolic effects of rhgh were probably, at least in part, mediated by igf- . this study was aimed at detecting the efficacy of igf-i on modulation of the protein metabolism in the catabolic state. patients and methodes: patients (both sexes, age: - years, bmi - kg/m ) with major upper gastrointestinal surgery (gastrectomy or partial gastrectomy) received ug rhlgf- /kg body weight/twice daily or placebo during treatment days beginning on the first postoperative day in a double-blind, randomized study. all patients received hypocaloric and hyponitrogenous total parenteral nutrition ( g chng, o. g aa/kg) troughout the whole study period. cumulated nitrogen balance, -methyl-histidin exretion in urine as well as serum-igf- levels have been determined. the two tailed wilcoxon test was used for statistical analysis results: first results will be presented previous studies have suggested that growth hormone (gh) potentiates various activities of leukecytes. however, it is not clear whether gh can improve survival of animals with gram-negative sepsis. we investigated the effects of gh on host response to infection in septic mice model. methods balb]c mice were randomized to groups (n= /gronp): gh-high ( + mg/kg/day), gh-iow ( a mg/kg/day) or control (saline). following subcutaneous treatment (every hours for days) with gh or saline, mice were challenged i.p. with e.coli (lxl /body). survival rate was recorded every hours. in the next experiment, gh-high and control animals were sacrificed hours after bacterial challenge. peritoneal cavity was lavaged with ml saline and the peritoneal exudative cells (pec) were harvested and counted. colony forming units (cfu) of bacteria in the peritoneal lavaged fluid (plf), liver, lung and blood were determined. pec were cultured in vitro for hours with lipopolymccharide ( p.g/ml in normotensive adults growth hormone (gh) rises when clonidine challenge is performed. recently evidence was shown for gh to accelerate wound healing. while gh secretion patterns are inconstant the gh-dependant insulin like growth factor (igf- ) and the insulin like growth factor binding protein (igfbp- ) reflect the integrated gh secretion over days. since we administer clonidine to patients with acute alcohol abuse we determined plasma levels of igf- and igfbp- . materials and methods: patients sheduled for esophagus resection were investigated once they had given written informed consent. patients showed acute alcohol abuse and were treated with clonidine postoperatively• patients with no history of acute alcohol abuse served as controls. besides liver enzymes igf- and igfbp- were determined. results: both groups had a comparable hepatic capacity of synthesis with decreased cholinesterase levels as to be expected after a major operation. regression analysis of igf- and igfbp- levels showed no differences between the groups. discussion: igf- and igfbp- plasma levels are not increased in normotensive patients who are treated with clonidine for prevention of postoperative alcohol withdrawl syndrom. further studies are required to discriminate whether our results are due to impaired postoperative liver function or if clonidine has not any impact on the gh-igf-axis in patients with alcohol abuse. recent studies have revealed that igf-i has several immanoregulatory roles. however, little is known about its effects on septic animals. we tested whether igf-i could increase the survival o f mice challenged intraperitoneally (i.p.) with e. coli. m~thqd$ balb/c mice received either high dose igf-i (n= , mg/kg/day), low dose igf-i (n= , . mg/kg/day) or saline ( = ) every eight hours subcutaneously for six days. animals were then challenged i.p. with lx e. coli. survival was observed every two hours. in the other experiment, mice that received high dose igf-i or saline were sacrificed four hours after bacterial challenge. peritoneal cavity was lavaged with ml saline and the peritoneal exudative ceils (pec) were harvested and counted. colony forming units (cfu) of bacteria in the peritoneal lavaged fluid (plf), liver, lung and blood were determined. in addition, pec were cultured/n vitro for hours with lipopolysaccharide ( ).tg/ml). tumor necrosis factor (tnf) in the supernatants of pec culture, plf and serum were measured by l bioassay. results igf-i improved the survival rate at a dose of mg/kg/day. severe chronic neutropenia (scn) is a disorder characterized by severe neutropenia secondary to either a maturational arrest of myelopoiesis at the level of promyelocytes (kostmann-syndrome; scn) or regular cyclic fluctuations in the number of blood neutrophils with a median absolute neutrophil count (anc) of less than /~tl (cyclic neutropenia). patients suffering from scn have an impaired acute inflammatory response to injury and an increased susceptibility to infections, i.e. bacterial or fungal infections, resulting in chronic oral inflammation, severe pneumonia, abscess formation and bacteraemia. we have treated patients ( scn, cyclic neutropenia) with r-methug-csf (filgrastim). thirty of patients with scn and all cyclic neutropenia patients responded to this treatment with an increase of the median anc to greater than /~tl. the dose of r-methug-csf needed to achieve and maintain the response varied between . and ~tg/kg/d. long-term treatment did not exhaust myelopoiesis: the anc remained stable after up to years of treatment and antibodies to r-methug-csf were not detected. the increase of anc was associated with dramatic clinical responses: significant reduction of severe bacterial infections, reduction of intravenous antibiotic treatment, and reduction of hospitalizations. no severe bacterial infections occurred in any of the r-rnethug-csf responders during longterm treatment. severe adverse events, most likely due to the underlying disease, included the development of mds/leukemia in two patients, and osteopenia/osteoporosis in patients. these results demonstrate the beneficial effects of r-methug-csf treatment in severe chronic neutropenia patients. the newborn is predisposed to mortality during infections due in large part to developmentally immature host defenses. recently cytokines have been identified that regulate the development of these defenses. one such cytokine is specific for neutrophils and is termed granulocyte colony stimulating factor (g-csf). in the studies to be discussed, we have attempted to impact hematopoiesis in the fetus using exogenous rhg-csf delivered through the pregnant dam. our rationale was that an enhanced myeloid system may result from such in utero treatment leading to, perhaps, enhanced protection to microbial insult. rhg-csf crossed the placenta and reached peak fetal serum concentrations hours after administration. these levels were -fold lower than the levels detected in the adult dams serum. white blood cell counts were elevated approximately - -fold over control newborn blood. this increase was due to circulating numbers of neutrophils. the marrows from these pups were hyperplastic consisting of predominately immature and mature neutrophilic cells. no changes were seen in the thymus or livers. pups born to mothers treated with rhg-csf since day of gestation (parturition in rodents occurs on approximately day ) survived a lethal challenge of group b- hemolytic streptococcus. in contrast their untreated yet infected counterparts did not survive. recent clinical developments have led to trials of rhg-csf in certain neutropenic states including cyclic and chronic neutropenia. we have identified several of these patients who were pregnant during treatment with rhg-csf. biologically and anfigenically identifiable rhg-csf was detected at increased levels in the cord serum of these neonates. we will discuss our findings with these patients in light of our animal model of neonatal myeloid development. we have previously demonstrated decreased g-csf production and g-csf mrna expression from activated mononuclear cells from newboms compared to adults (cairo, pediatr res : , ) . we have also recently observed that administration of a single dose of rhg-cse to one-day-old newborn rats induced significant neatrophilia, while administration for days induced neulrophilia and increased the bone marrow (bm) neutrophil storage and progenitor pools (cairo, blood : , cairo, pediatr res : , ) . we embarked on a phase i trial exploring the safety, pharmacokinetics, and biological efficacy of g-csf in newborns with presumed sepsis. infants < hrs old with a diagnosis of presumed sepsis were stratified into three groups: very preterm ( - wk), preterm ( - wk), and term (> wk) and randomized to receive either a placebo or , , and gg/kg/qd or and p.g/kg/bid of rhg-csf infused by pump over hour for consecutive days. cbcs were obtained at , , , , and hrs. tibial bone marrow aspirations were performed hrs after study entry and differential counts and cfu-gm pools were determined. c bi expression was determined at and hrs after rhg-csf, and g-csf pharmacokinetics were performed after the first dose of rhg-csf utilizing a sandwich elisa. a significant increase in the anc was observed at , and hrs following administration of both and ~tg/kg/d of rhg-csf. the maximum increase in the anc occurred hrs after and ~tg/kg/d ( - %) (p< . ) and ( % -+ %) (p< . ), respectively. there was a significant dose-dapendeat increase in the bm neutrophil storage pool ( _+ % vs. + %) (p< . ) (placebo vs. ~tg/kg/d). there was no significant difference in the nantrophil proliferative pool. an increase in cfu-gm and cfu-gemm was seen at all doses tested, compared to placebo ( . _+ . vs. -+ ) (colonies/l(p cells/plate). c bi expression was significantly increased hrs after bg/kg/d of rhg-csf ( + % vs. +- %) (p< . ). peak serum g-csf levels occurred at hrs and were dosedependent. the half-life of rhg-cse was . + . hrs. most importantly, there was no observed toxicity from g-csf in all patients studied. of patients were on ventilators prior to administration of rhg-csf and there was no increase in pulmonary toxicity. these preliminary data suggest that rhg-csf is well tolerated at all gestational ages in newborns with presumed sepsis. a multi-center phase ii/iii randomized double-blindad placebo controlled trial is required to determine the efficacy of rhg-csf in this clinical setting. we investigated the effects of recombinant canine granulocyte-colony stimulating factor (g-csf) on survival, cardiopulmonary function, serum endotoxin levels and tumor necrosis factor (tnf) levels in a canine model of lethal bacterial septic shock (clinical research. : , ) . methods: awake ylo beagles had serial cardiopulmonary and laboratory studies before and for up to days after intraperitoneal placement of an e. celi infected clot. nine days before and daily until days after clot placement, animals received high (n= ) or low dose (n= ) g-csf or protein control (n= ) subcutaneously. results: survival in high dose g-csf animals ( / ) was significantly improved compared to low dose ( ) and controls ( ) (p< . wilcoxon). high dose g-csf also improved cardiovascular function evidenced by a higher mean left ventricular ejection fraction (day after clot, p< . ) and mean arterial pressure (day , p< , ) compared to low dose and controls. high dose rcg-csf increased (p< . ) peripheral neutrophil numbers both before and after clot implantation ( hours to days) compared to low dose and controls. in addition, high dose rcg-csf produced a more rapid (p< . ) rise (day ) and fall (day ) in alveolar neutrophils determined by bronchoalveolar lavage compared to low dose and controls. lastly, high dose rcg-csf decreased serum endotoxin ( to h, p< . ) and tumor necrosis factor (tnf, h, p< . ) levels compared to low dose and controls. discussion: these data suggest that therapy with g-csf sufficient to increase peripheral neutrophil numbers during peritonitis and septic shock may augment host defense and endotoxin clearance, reduce cytokine levels (tnf) and improve cardiovascular function and survival. the use of g-csf in sepsis prophylaxis in neutropenic patients is well established and has been ascribed to accelerated recovery in granulccyte counts. here, an additional sepsis-prophylactic property could be demonstrated in healthy volunteers: eleven volunteers were employed in a sinqle-btind, controlled study and were given uq g-csf or saline placebo via subcutaneous injection. blood was withdrawn immediately before and or hours later. lps-inducible tnf, il- , stnf-r p and il-lra were assessed in the supernatant of whole blood incubations stimulated with ug/ml lps from salmonella abortus equi. similarly to previous animal studies, lps-inducible tnf was attenuated by about % hrs. after treatment. the same was true of il-lb. in contrast, lps-inducible stnf-r p which was indetectable in blood incubations from untreated donors increased dramatically hrs. after g-csf treatment. il-lra found after lps challenge was increased tenfold by g-csf treatment. it is concluded that g-csf treatment switches peripheral leukocytes to an antiinflammatery state characterized by an attenuation of il-i and tnf releasing capacity and an augmentation of the release of cytokine antagonists. this findinq minht offer a novel concept in septic shock prophylaxis. objective.the aim of the study was to investigate the effect of recombinant human g-csf (rhg-csf) on survival, bone marrow neutrophil myelopoiesis, neutrophil counts, levels of bacteria and some important sepsis mediators in a model of rat abdominal sepsis. lethal peritonitis was induced with a mm coecal perforation (cp) in male wistar rats. rhg-csf was administered as /.tg/kg iv every h, first dose at sepsis induction. bone marrow neutrophi] progenitors were determined as blast colonies, cfu-gm and cfu-g. neutrophils and bacteria were determined in peripheral blood and peritoneal fluid. lps, tnf, endothelin and lactate were measured in blood from femoral vein. mortality rates were registered with g-csf treatment starting either or days before or hours after cp. results. mortality was reduced from % to about % with rhg-csf intervention and there was no difference between the pretreatment and treatment groups. bone marrow blast colonies were not influenced while neutrophil myelopoiesis was augmented at the stages of cfu-gm and cfu-g. neutrophils in blood and peritoneal cavity were enhanced and numbers of bacteria in the same compartments were substantially reduced. circulating lps, tnf, endothelin and lactate were attenuated the first hours after cp. neutrophil myelopoiesis is augmented with increased number of neutrophils in blood and peritoneal cavity, resulting in enhanced clearance of pathogens. lps, tnf, endothelin and lactate are suppressed the first hours during sepsis course. a. wendel, j. barsig, g. tiegs gm-csf stimulates the proliferation and differentiation of granulocytic and monocytic progenitor cells. in addition the hemopoietic cytokine activates the inflammatory response in mature leukocytes. the priming effect of gm-csf towards lipopolysaccharide (lps)-induced cytokine production in vitro has been described, but little is known about proinflammatory gm-csf effects in vivo. we detected gm-csf in plasma of lps-challenged mice with kinetics similar to tnf, reaching peak levels h after lps administration. gm-csf pretreatment ( ~tg/kg i.v.) enhanced mortality in mice challenged by a sublethal dose of lps. plasma levels of tumor necrosis factor (tnf) and interleukin- (il- ) were significantly enhanced. a monoclonal antibody, which neutralizes gm-csf bioactivity, rendered mice less sensitive towards lethal lps-challenge. tnf-and il- -tevels were reduced in these mice compared to control animals without antibody treatment. in addition, severalfold potentiation of lps-induced cytokine release by gm-csf was observed in vitro in murine bone marrow cell cultures. these data demonstrate the proinflammatory capacity of gm-csf and suggest that the hemopoietic cytokine plays also a role as an endogenous modulator of lps toxicity. immune dysfunction, developing in the wake of multiple trauma, overwhelming infection and other forms of critical surgical illnes% is associated with increased infections, morbidity and mortality. the mechanisms responsible for alterations in immune regulation are incompletely understood but monocyte appear to play a central role. polymorphonuclear leukocytes (pmn) are known to play a central role in the inflammatory response of the host toward invading microrganisms. reports of defects in all the aspeots of pmn function have been accumulated in recent years. the possible role of gm-csf in modifing the state of immuno suppression detected in severe intraabdominal infected pt~. inspite of surgical appropriate procedures and in reducing the expected mortality is investigated. the safety of rh-gm-csf administration in sepsis is also evaluated. a double blind randomized study is proposed. this study include icu patients who do not exhibit signs of shock and/or ards, with clinical signs and symptoms of abdominal infection. immunodepressed patients-aids, chronic chemotherapy or chronic steroid administration do not partecipate to the study. patients will receive rgm-csf (l~g/kg/day) or placebo in hs. continuous infusion for days. safetyandefyieacy will be assessed till to day . the apache ii score is adopted for risk stratification of patients because it is reliable and validated, objective and composed of information that is indipendent of diagnostic criteria. patient's entry criteria is apache ii > (score corresponds to expected mortality rate of %).in this protocol the surgeons report the judgement of the efficacy of surgical procedure to remove or not the focus of infection. objectives: infections and subsequent septic responses remain the leading cause of death among surgical intensive care (sicu) patients despite tmprovetaunts in supportive care and brond-epectrum antibiotics. usually invading bacteria are efficiently cleared by neutrophil granulocytes. however, during sepsis various neatrophil dysfunctions have been demonstrated, leading to impaired host defense. granulocyte colony-stimulating factor (g-csf) induces a sustained increase in circulating neutrophils and enhances various noutrophil functions. it was the purpose of the present study, to evaluate the safety and efficacy of g-csf (filgrastim) in sicu patients at risk of sepsis. materiel a.d methods: the study was designed as an open-label phase-ll study of filgrastim. ten consecutive slcu patients, with a therapeutic interveotion score greater than , were included in the study. filgrastim was given by daily continuous intravenous infusion for days or discharge from the sicu. apache ll-score, multiple-organ-failure (mof) score, definitions of infections, sepsis, systemic inflammatory response syndrome (sirs), and acute respiratory failure were applied daily. a response to filgrastinl th_erapy was defined as an improvement in disease severity quantified by a decrease of > apache i score points on day after onset of treatment. results: none of the patients developed a sepsis or mof later on and no patient died during hospitalization. specific postoperative complications occured in one patient ~jth a leekage of the oesophagou-gastric anastomosis after oesophageus resection. at study entry the leucocytes amounted to . + . /~tl (mean + sem) and reached a level of . +_ . /tal at day after onset offilgrastim therapy. the apache ii score initally was + . (mean + sem) and as an indicator of filgrastim response a decrease of points ~dthin days oceured in out ot patients. filgrastim was well tolerated, side effects were not noted. growth of solid tumors might be modulated by the activity of inflammatory and/or immune effector cells of undefined specificity. in this study patients undergoing surgical treatment for gastric (n= ) or colorectal (n= ) cancers were evaluated for endogenous serum levels of granulocyte colony-stimulatingfactor (g-csf) during a pre-and postoperative time period. from the same blood specimens mononuelcar cells (mnc) were prepared. the release of ifn-%, and il- , which are secreted by thl cells, were stimulated in vitro by pha during a cell culture period up to hours. the patients were further classified for their immunreactivity by responses in dth skin testing to seven different antigens (e.g. tetanus toxoid, ppd, diphtheria toxin, trichophyton, streptococcus, candida and proteus antigens). dth testing has been repeated in each patient two remarkable results were obtained. the serum levels of endogenous g-cse showed a biphasic increase with maximum values of pg/ml (preoperative < pg/ml) on day and day to after surgical treatment. similar patterns of g-csf production were found in both groups of patients with gastric or colorectal cancers. high serum levels of g-csf were significantly (p < , ) correlated with infectious complications in patients whh gastric cancer (n= / ). secondly patients could be arranged into two groups according to an anergic (n= ) or normergi¢ (n = ) responsiveness in dth testing. the frequency of anergi¢ responsiveness was similar in both patients with gastric (n= / ) or colorectal (n= / ) cancers. interestingly we found a significant correlation (p < , ) between low serum levels of g-csf and anergy during the postoperative period in both groups. stimulation of mncs from anergic patients (n= ) within the pre-and postoperative period resulted in reduced mean values (about %) for ifn-ff release (preoperative means llo pg/nfl), if compared to patients with normergic dth (n= , preoperative means pg/ml). similar, but less significant results were obtained for il- secretion. our results confirm a correlation between infectious complications and g-csf in the postoperative period, however elevated levels were also found in some patients without any signs of infections. more interestingly there might be an association between cytokine (c~csf, ifn-% and il- ) release and dth, which is known to be mediated by activated thl calls. to recognize anergic dth as a possible higher risk in the postoperative outcome of cancer patients extended periods of observation are needed. objectives of the study effects of recombinant huraan granulocyte colony-stimulating factor(rhc-csf)a galnst severe septic infections were investigated by its single use or by its corn b{nation with cephera antibiotlcs.we examined its effects on the mortality,and circulating blood neutrophyis counts and functlons,such as phagocytic activity and h production using the rat severe septic model. rats were subcutaneously administsrd rhc~csf(s orl o ~ g/k~ body wt)after on set of peritonitis brought about by cecal ]igation and one puncture withe -gaug e needle once a day for three days.in addjtlon,cefmetazol na(cmz)( m$/k bo dy wt)was injected intrarnustularly to the rats tv~ce a day for three days. cirehlatlng blood neutrophyls counts were determoned electronically with a hem ocytometer,and blood smears stained with may~runwaldm.qlemsa~taln. neutrophyls functions in vltro,such as phagocytic activity and h producti on using the rat severe septic model was analyzvd by automated flow cytometri c single cell-analysis methods. the reortallty rate after weeks was significantly decreased by administratlon of rh~-csf(p< , ).ln addjtion,a combination therapy of rhg-csf wlte cephern ant~biotics(cmz)showed a significantly survive] advantage and the rate had b een reached . %. nextly,treatn%ent wlth rhg-csf(s ~ $/k body wt)increased the nuzaber of the peripheral blood neutrophjls slgn[fieantly(p< . ). iv~oreover,functions of neutrophlis which were phagocytic activity and h p roduction were remarkably enhanced by admlnlstratlon of rhg-cs~( ~ /ks b ody wt) (p< .( ). these findings suggest that combination therapy of rhcrcsf with cephern antib iotlcs(cmz)is an efficient regime against severe infectlons.and the increased ne utrophils counts and enhanced neutrophiis functions were played a important ro le about the survival advantage. granulocyte macrophage colony-stimulating factor (gm-csf) is a haematopoietic growth factor active on neutrophils and macrophages. leukopenia often occurs following renal transplantation and can be associated with infection and/or the myelosuppressive effect of azathioprine. aim: we report the use of gm-csf in renal allograft recipients with leukopenia. nonglycosylated recombinant gm-csf was obtained from e. coli transvected by human gm-csf gene. m~terial ~,nd methods : written informed consent was obtained from all patients. patients were suffering from toxic neutropenia (neutrophils < /mm ) with medullar hypocellularity on bone marrow aspiration, or leukopenia (neutrophils < /ram ) with cytomegalovirus infection requiring ganciclovir administtation. gm-csf was given subcutaneously at a dally dose of to mcg/kg/day, according to renal function. results : in all cases, neutrophil counts returned to normal levels within to days. in most of them, spectacular correction was observed within hours, with a single injection. adverse events due to gm-csf at this dose were mild and easily managed ( cases of bone pain treated with paracetamol). one acute rejection episode was observed after correction of leukopenia. conclusion : on the basis of this study, it appears that gm-csf at a dose below mcg/kg/day is an effective treatment for renal transplant recipients with leukopenia associated with cmv infection or toxic neutropenia. department of nephrology, , rue de s~vres, hopital necker, paris, france. changes in serum g-csf and il- after surgical intervention hitoshi toda , atsuo murata , hidewaki nakagawa , takesada mori , nariaki matsuura osaka university medical school, osaka, wakayama medical school, wakayama, japan we measured serum immunoreactive interleukin (il- ) and granulocyte colony-stimulating factor (g-csf) levels of the patients undergoing major thoraco-abdominal surgery for esophageal cancer. serum samples were collected from eight patients on the day before surgery, at the time of operation, and thereafter at suitable intervals for one week. il- and g-csf were measured by means of enzyme linked immunoassay. the normal range of serum ]l- was less than pg/ml and g-csf less than pg/ml. values between groups were compared with linear regression analysis. both serum g-csf and il- levels reached their maximal levels at the first postoperative day and decreased thereafter. the correlation between g-csf (y) and il- (x) was y= . x+ . (r= . , n= , p< . ), showing a significant correlation. in the case who suffered from aspiration pneumonia and ards at the second postoperative day, the peak level of il- was pg/ml and g-csf pg/ml respectively. the estimated value of g-csf was pg/mi by the regression equation. this means the real g-cse level was less than half of the estimated value. it suggests that low responsiveness of g-csf is one of the reason of immunodeficient state after the major surgery, neutrophils from injured patients ingest and kill bacteria less efficiently as compared to those of healthy individuals, probably reflecting the suppression in respiratoly burst which occurs after severe trauma. one of the main mechanisms of killing bacteria by neutrophil granulocytes is production of oxygen radicals (respiratory burst). granulocyte colony-stimulating factor (g-csf), a kilodalton cytokine, leads to a sustained, dose-dependent increase in circulating neutrophils. thus, it was investigated whether filgrastim (recombinant human granulocyte colony-stimulating factor, rhg-csf) therapy fits for prophylaxis of sepsis in postoperative/posttraumatic patients, and whether, besides an expected increase in neutrophil count, filgrastim would also augment neutrophil function. material and methods: this study was designed as an open label, prospective phase ii study of filgrastim and performed in a surgical intensive care unit (sicu) (university hospital). postoperative/post-traumatic patients with a therapeutic intervention scoring system (tiss) score greater than were treated with filgrastim ( . - l.tg/kg/day) for prophylaxis of sepsis on days or until discharge from the sicu. production of oxygen radicals can be quantified by analysis of fmlp-and zymosan-induced chemiluminescence. neutrophil oxygen radical production was tested by fmlp-and zymosan-induced chemiluminescence by the polymorphonuclear cells (pmn) of these patients in multiple blood samples over a period of up to days. results: none of the patients treated with filgrastim for prophylaxis of sepsis developed sepsis. in vitro fmlp-induced ( - reel/l) neutrophil oxygen radical production was significantly increased under therapy with filgrastim by a maximum of % +- % ( % - %) compared to pretreatment values of %. tapering of filgrastim resulted in a reduction of fmlp-induced neutrophil oxygen radical production within hours. in contrast, zymosan-induced neutrophil oxygen radical production was not affected by filgrastim treatment. conclusions: besides its quantitative effect on neutrophil counts enhanced neutrophil function, documented here as increased fmlp-induced oxygen radical production, may account for the beneficial effect of filgrastim for prophylaxis of sepsis in posttraumatic/post-operative patients. granulocyte colony stimulating factor (g-csf) and granulocytemacrophage colony stimulating factor (gm-csf) have been recently introduced in the treatment of chemotherapy-induced neutropenia. effects of these csfs on cellular immune system were evaluated in neutropenic gynecological cancer patients during chemotherapy. g-csf and gm-csf were equally able to induce a rapid recovery of white cell count within one or two days. g-csf treatment resulted in a significantly higher concentration of leukocytes measured in the peripheral blood although by gm-csf a sufficient effect was achieved (p< . ). before initiation of csf treatment urinary neopterin was similar in both groups of patients ( +/- and +/- lamol/mol creatinine for gm-csf and g-csf respectively expressed as mean +/-one sd). in g-csf treated patient only a marginal induction of neopterin was observed. on day the mean value was about % above the basal level (p< . ). on the other hand gm-csf treated patients were characterized by a pronounced increase in urinary neopterin levels. in comparison with the basal level a more than fold induction was noted and the difference between g-csf and gm-csf was highly significant (p< . ). this effect was confirmed in vitro by investigating the effects of these csfs on interferon-gamma mediated pathways in thp- human myelomonocytic cells. results suggest activation of immune effector cells by gm-csf which may help the organism to overcome infections. however, activated macrophages produce several growth factors which may increase malignant proliferation, and augmented neopterin production as sign of macrophage activation has also been associated with poor prognosis m several malignancies. more data are therefore necessary to clarify whether csf mediated immune activation is beneficial or deleterious for cancer patients but considering our results caution in applying csfs in oncology seems advised. from a historical perspective, the development of humoral immunity to bacterial endotoxin has assumed a prominent position in the spectrum of therapeutic approaches which have been explored for the treatment of gram negative septic shock. predicated upon the fact that rough strains of bacteria manifest lps containing exclusively conserved structural features common to lps from all gram negatives, specific antibodies were elicited which conveyed cross protective immunity in experimental models of bacteremia and endotoxemia. such studies culminated in a well-conducted, randomized, double-blind placebo-controlled clinical trial using passively administered human polyclonal antiserum to treat patients with suspected gram negative sepsis. the efficacy of treatment established in that trial spurred efforts to develop monoclonai reagents which, to date, have not been uniformly successful in reproducing those earlier studies with polyclonai antibodies. nevertheless, the numerous successes which have been documented in experimental models of endotoxemia continue to foster promise for this immunotherapeutie approach. several recent studies with human polyclonalimrnunoglobulin preparations containing antibodies reactive with lps and lipid a have yielded promising results in treatment of patients with sepsis. in addition, the recent development of an antiidiotypic monoclonal antibody which reflects an internal image of a kdo specific monoclonal antibody has provided an alternative experimental approach to generate anti-lps antibody. immunization of mice with the antiidiotype provides significant protection against subsequent lps lethality consistent with the development of circulating immunoglobulin specific for lps. thus, the use of polyclonal immunoglobulins contrives to provide an alternative and potentially cost effective method for the treatment of endotoxin shock. supported by r a and pot ca . john holaday, anne fortier, shawn green, glenn swartz, john madsen, carol naey, and jan dijkstra entremed, inc.. rockville, md, . at the time of diagnosis, the signs and symptoms of septic shock are an indication that the systemic inflammatory response is well underway; thus, it has been argued that the endotoxin "cat is out of the bag", and that subsequent passive immunization may be too late to achieve therapeutic benefit. our approach has been to evaluate active immunization as a prophylax~s against sepsis. mice were inoculated twice (two weeks apart) with liposomes containing dmpc[i. ], dmpg[ . ], cholesterol [ . ] , and monophosphoryl lipid a [ - gg/txmole phospholipid] by several routes (i.p., i.m.), and serum was collected - days after each inoculation. after a single injection, highest tilers of ab were produced in mice inoculated i.p., but mice inoculated by all routes produced anti-lipid a ab. following the second injection. ab levels were roughly equivalent in mice inoculated by all routes, regardless of lipid a concentration. mice vaccinated i.p. with liposomes containing , or gg lipid a were treated with cyclophosphamide to produce neutroperda and then challenged with e. cole in an infection model of gram negative sepsis. the lds for control (liposomes with no lipid a) mice was x bacteria; ld for mice vaccinated with p.g was x ( -fold increase in resistance) and with ~tg was x bacteria ( -laid increase in resistance). mice vaccinated as before were also treated with actinomyein d to increase sensitivity to lps (salmonella minnesota) challenge in an endotoxemia model of grain negative sepsis. the ld for control (liposomes with no lipid a) mice was ng lps; the ld for gg lipid a was rig lps ( -fold increase in resistance) and for xg was ng lps ( -fold increase in resistance). mice were similarly vaccinated and challenged with an aggressive gram negative pathogen, francfsella tularensis. the ld of franciseua in normal mice or mice inoculated with liposomes without lipid a was - bacteria. in contrast, mice vaccinated with liposomal lipid a ( ggl survived challenges as high as , bacteria, ( logs of protection). the impressive protective capacity of this vaccine did not correlate with ab liter in any of the sepsis models, nor did it correlate with classic nonspeeific events, such as macrophage activation. maerophages harvested from the peritoneum of mice vaccinated and protected against sequelae of gram negative infections did not spontaneously kill the bacteria in vitro, but could be activated by ifn-y for antimicrobial activity equivalent to that of macrophages from unt#eated mice. research is underway to defme the protective mechanism(s) activated by this liposomal-lipid a vaccine. intervention by monophosphoryl lipid a in septic shock jon a. rudbach, ribi immunochem research, inc., hamilton, montana, usa monophosphoryl lipid a (mla), the clinical form of which is called mpl®-immunostimulant, has been tested extensively as an intervenient material in septic shock. mla is protective when given to experimental animals prior to a live microbial challenge or challenge with lethal doses of microbial products or certain cytokines. this is shown with gram negative and gram positive bacteria, gram negative bacterial endotoxins, and gram positive bacterial exotoxins. furthermore, animals treated with a regimen of mla which results in a refractory state to a lethal dose of gram negative bacterial endotoxin concomitantly display increased resistance to a live bacterial challenge. thus, both endotoxin tolerance and nonspeciflc resistance to infection can be manifested simultaneously. also, prophylactic doses of mla do not interfere with other therapies given subsequently; an additive or a synergistic protective effect can be demonstrated with certain combinatorial treatment regimens, such as mla followed by antiendotoxin monoclonal antibodies. the preclinical studies were extended to human trials wherein the safety of agonistic doses of mla was verified. furthermore, when mla was administered to human volunteers hr before challenge with a pharmacologically active dose of reference endotoxin, febrile, cardiac, tnf, il- , and il- responses were all decreased significantly as compared with the responses of subjects pretreated with a control solution and challenged with endotoxin. human trials with mla are being extended into patient cohorts which have high probabilities of developing septic shock; this will expand the safety base and establish clinical efficacy for mpl®-immunostimulant. a considerable body of in vitro evidence supports the concept that the effects of lps on cells of the immune/inflammatory systems are controlled by interactions of lps with cd . to evaluate if blocking lps-cd interactions has potential as a therapeutic in septic shock we have evaluated the effect of anti-cdi monoclonal antibody (mab) on lps-induced cytokine production and physiologic changes in an experimental model of endotoxin shock performed in cynomolgus monkeys. a novel model has been established where animals were treated with interferongamma for three days prior to infusion of highly purified lps over an eight hour period. in this model lps challenge resulted in marked release of eytokines in the blood, substantial hemodynamic changes, release of liver enzymes and alteration in lung permeability observed over a hour period. to evaluate the effect of treatment with anti-cd mab, animals were given either nothing, an isotype control or anti-cd mab ( mg/kg) rains, prior to the beginning of the lps infusion. evaluation of physiologic changes including mean arterial blood pressure and cardiac output, quantitative analysis of eytoldne levels including tnfct, il- , i,- , il- and il- , and liver enzymes during a hour period revealed that treatment with anti-cd mab markedly attenuated all parameters of injury including decreased mean arterial blood pressure, increased cytnkine levels and the release of liver enzymes observed in animals given the isotype control mab or those not treated. administration of anti-cd mab to interferon-gamma treated animals not challenged with lps did not induce any detectable physiologic changes or increases in cytoldnes. these studies suggest that strategies to block lps-cd interactions will have utility in diseases such as septic shock or ards where lps plays a central role in initiating injury. preclinical studies with recombinant bactericidal/permeability increasing proteins (rbpi and rbpi ). p.w. "frown, dept. of preclinical science, xoma corporation, berkeley, california, usa. bactericidal/permeability increasing protein (bpi), from neutrophils, binds to and neutralizes lipopolysaccharide (lps); it also specifically kills gram-negative bacteria (gnb). these properties, which reside in the n-terminal half of the molecule, indicate potential therapeutic application in the treatment of gram-negative sepsis. the gene for human bpi has been cloned and recombinant holoprotein (rbpi) and a kd n-terminal fragment (rbpi; ) have been produced in sufficient quantities for preclinical studies. both rbpi and rbpi bind to lipid a and neutralize the biological activities of lps derived from a variety of organisms, rbpi has equivalent antibacterial activity to bpi against rough gnb but is up to x more potent than bpi vs. serum-resistant and smooth gnb. rbpi and rbpi compete with lps-binding protein (lbp) for binding to lps under physiological conditions. consequently, both rbpi and rbpi block the cd -dependent lpsinduced synthesis of the cytokines tnf, il- , el- and il- in vitro. rbpi has also been shown to inhibit the lps-induced synthesis of reactive metabolites, endothelial adhesion molecules and the procoagulant molecule tissue factor. in animals, rbpi has been reported to increase survival of endotoxin-challenged rats and mice, to inhibit the dermal schwartzman reaction in rabbits and to increase survival of neutropenic rats with pseudomonas bacteremia, rbpi increases survival and decreases cytokine production in endotoxin challenged mice and rats. it normalizes lps-induced changes in hemodynamic, pulmonary and/or metabolic parameters in lps-induced rats, rabbits and pigs. treatment with rbpi also increases survival and decreases cytokine production in bacterial challenge models in rats and mice. rbpi was not toxic to rats after daily consecutive i.v. doses of mg/kg. this combination of properties indicate that recombinant bpi may be useful in the treatment of sepsis. phase i/ii clinical trials of rbpi have begun. the discovery of lps binding protein (lbp) and subsequent identification of cd as a receptor for lps or lps-lbp complexes has resulted in a new understanding o£ how lps responsive ceils are stimulated. cd is found either as a glycosylphosphatidyl-inositol (gpi)-anehored membrane glycoprotein (mcd ) of myeloid cells or as a soluble serum protein (scd ) lacking the gpi-anchor. binding of lps to mcd triggers cell activation while binding of lps-scd complexes to cells such as endothelial or epithelial cells that normally do not express mcd activates these cells. these pathways are shown in schematic form below. ~di mcd plays a crucial role in presentation of lps to additional membrane components that make up a functional lps receptor. an immediate consequence of engagement of this functional receptor is protein tyrosine phosphorylation. the molecular mechanisms leading to these events will be discussed. understanding of these pathways will lead to the development of new therapeutic approaches to controlling host responses to lps. pretreatmen t posttreatment (before or after tnf peak) d) with different antibody dosages: mg/kg --- . mg/kg pretreatment with anti-tnfab prevented death in most model situations (except peritonitis), but also posttreatment up to h after sepsis induction was successful in the few studies performed. there is additional evidence that low-dose tnfab is partially effective. especially baboon anti-tnfab studies provided many insights into the pathophysiological sequences of sepsis induction, due to crossreactivity with human reagents. those events include the cytokine sequence with tnf-dependent il-i, il- , or il- , but also il-lra or stnf receptor release. granulocyte as well as endothelial cell activation were shown to be partly tnf related, and the procoagulatory response was influenced by anti-tnf treatment. from many animal studies the concept that tnf plays a pivotal role in sepsis is clearly evident and therefore anti-tnf therapy is a major candidate tbr clinical studies. the beneficial or harmful effects of tnf-mediated inflammatory responses depend on the clinical context. decreasing exaggerated tnf-mediated inflammatory responses may be useful in some patients with organ failure. tnfr:fc (immunex, seattle, wa) is a recombinant human protein composed of two identical extracellular p tnf receptors linked by the fc region of iggl. it neutralizes tnf with an affinity for tnf<z of - m and has a t / of • h in normal humans. methods: normal volunteers were randomized to receive either tnfr:fc vehicle (n= ), or low dose (ld) tnfr:fc ( mg/m iv, n= ), or high dose (hd) tnfr:fc ( mg/m iv, n= ) infused over rain prior to endotoxin (e) (escherichia coil : ; ng/kg iv) administration. plasma cytokines were measured using elisa and tnf bioactiv'dy. systemic hemodynamics were evaluated with indwelling arterial and pulmonary artery catheters and radionuclide heart scans and compared before and after fluid loading in subjects given vehicle with hd tnfr:fc. results: endotoxin-related symptoms were unchanged after ld or hd. peak temperature occurred at a later time point ( - h) in subjects given tnfr:fc compared to vehicle ( h) (p=. ). at h, h (post fluid load ), and h, subjects given vehicle developed increased cardiac index and heart rate and decreased systemic vascular resistance index (all p=. ). hd tnfr:fc did not alter this hyperdynamic cardiovascular response. ld and hd inhibited the e-induced teukopenta at i h post endotoxin (p<. ) and later leukocytosis (p< . ). peak tnf bioactivity in subjects given vehicle was + pg/ml and < pg/ml in the ld or hd tnfr:fc groups (p<. ). two immunoassays for tnf were performed because the detection of receptor bound tnf varies with elisa. tnf (biosource) was decreased in ld vs vehicle or hd (p=. ) whereas tnf (r&d systems) was increased after hd or ld vs vehicle (p<. ). decreased levels of il- (p=. t), granulocyte colony stimulating factor (p=. ), and il- receptor antagonist (p=. ) were found after ld or hd vs vehicle. il- levels were lower after ld vs vehicle or hd (p=. ). il- levels were similar among subjects given vehicle, ld, and hd tnfr:fc. conclusions: ld and hd tnfr:fc delayed the febrile response to e but did not alter the early hyperdynamic cardiac response. this suggests that mediators other than tnf play an important role in the initial cardiovascular response to e in humans. tnfr:fc attenuated the release of some e and tnf-induced cytokines. ti~e antiinflammatory responses of tnfr:fc may be useful in some patients with disease processes resulting from excessive tnf-mediated inflammatory responses. the biosynthesis of tnf within macrophages is regulated both at the transcriptional and the translational levels. the ' flanking region of the tnf gene contains several transcriptional control elements, including two kb enhancers similar to those of immunoglobulin genes and a "y box" similar to that of the mhc class ii promoter. these elements are critical for the enhancement of transcription noted after stimulation of macrophages with lps. despite these and other elements, transcriptional regulation alone accounts neither for the absence of tnf protein during normal homeostasis nor the profound increasein tnf production following stimulation with lps or other secretagogues. the translation of tnf mrna into protein is also tightly regulated via mechanisms involving the octameric "ttatttat" motif present in the 'untranslated region of tnf, human inducible no synthase, and several other cytokine genes. this region not only destabilizes mrna, but also confers a translational blockade so that tnf protein is not normally synthesized despite leaky basal transcription. lps stimulation releases translational blockade and, together with enhanced trancriptional rate, accounts for significantly increased tnf secretion. opportunities for inhibition of tnf production are available at each of these two levels. agents which increase intracellular camp (pentoxif¥ ine, amrinone) inhibit accumulation of tnf mrna; in contrast, corticosteroids act primarily at the level of translation, inhibiting lps induced translational activation. understanding the regulation of tnf biosynthesis may assist in clinical strategies designed to regulate tnf secretion. the serine protease thrombin is formed by enzymatic conversion from its proenzyme form after coagulation activation and may enhance the inflammatory response in various ways. in the last step of the coagulation cascade, thrombin cleaves its substrate fibrinogen, thus forming fibrin. the fibrin clot may prevent phagocytosis of bacteria and promote local bacterial growth. thrombin can also stimulate a specific th.rombin receptor that exists on a variety of cells. for example, minute amounts of thrombin ma), stimulate platelets to develop tissue factor and boost the activation of more thrombin, thus initiating a positive feedback loop. other cellular effects of thrombin receptor stimulation include increase in intracellular calcium of platelets and monocytes, contraction of smooth muscle cells, increase in endothelial permeability, thromboxane generation by neutrophils and lymphocytes, pulmonary vasoconstriction, and enhanced cytokine production. animal experiments have been conducted with a variety of thrombin inhibitors including heparin, antithrombin ili, hiradin, and hirudin-like peptides. an alternative approach is the inhibition of the coagulation cascade at an earlier point in the cascades, or administration of the anticoagulant enzyme protein c work from our own group in a porcine model of lipopolysaccharide-(lps)-induced shock with disseminated intravascular coagulation has shown that prelreatment with recombinant desulfatohirudin (r-h) or with a preformed complex of human donor antithrombin iii with heparin or post treatment with r-h are able to block the degradation of fibrinogen and the formation of fibrin monomer. in addition, after pretreatment with r-h it was found that lps-induced lung injury was reduced. since both natural hirudin as well as r-h were well tolerated by healthy volunteers, adminislration of r-h may be a promising therapeutic approach in patients with sepsis and sirs. corticosteroids have been used in the management of various shock syndroms including sepsis. the data concerning the effects of this therapy is, however, conflicting and in large prospective studies, the use of early administration of high doses of corticosteroids have been found to give no beneficial effects in patients with sepsis. we have particularly been engaged in studies on possible effects of corticosteroids on the plasma cascade systems and on the mediator release from leukocytes. in in vitro studies, high doses of methylprednisolone were found to facilitate endotoxin induced generation of plasma kallikrein. furthermore, plasma prekallikrein and kallikrein inhibitor values decreased together with formation of kallikrein-c inhibitor complexes ( ). in this in vitro plasma model we also found that methylpredoisolone alone in doses of mg/ml induced contact activation with the formation of kallikrein-c inhibitor complexes. methylprednisolone was also found to have an additive effect on endotoxine induced decreases in kallikrein inhibitor functions. in the same experimental model on endotoxemia methylprednisoione was found to give an additive effect on activation of the initial part of the complement cascade ( ) . activation of the terminal part of complement, however, was inhibited by the same dose of methylprednisolone. addition of methylprednisolone alone to plasma was also found to activate the initial part of complement. using a full blood model, we studied the effect of methylpredoisolone in modulating the endotoxin induced cytokine response. when ng/l e. coli endotoxin was added to citrated blood from healthy human donors preinkubation with gg/ml methylprednisolone significantly reduced the release of tumor necrosis factor et ( %) and interleukin- ( % ) at two hours after exposure to endotoxin. in conclusion, these studies show that corticosteroids might facilitate contact activation of plasma and reduce the function of major protease inhibitors. furthermore, we also observed that this drag in in vitro conditions facilitated activation of complement. on the other hand, corticosteroids were found to reduce endotoxin induced cytokine release from leukocytes in whole blood. these studies also showed a dose dependence of the effects of corticosteroids on endotoxin induced cellular and cascade system activation. different predisposing conditions like extensive trauma, infection or pancreatitis result in a remarkably similar inflammatory response. although thls inflammatory response primarily aims at the removal of devitalized tissues, destruction of bacteria and reestablishment of the integrity of host tissues, it may potentially become unregulated and generalized and thereby producing deleterious effects to vital organs or the body" as a whole. cytokines play an important role in the development of the systemic inflammatory response. most of their biological effects, however, are not directly mediated but exerted through other mediator systems. when the inflammatory response results in the formation of capillary leak syndrome and refractory hypotension, the unregulated activation of complement and contact systems appears to be the cause. both systems are almost exclusively regulated by c - nhibitor. am unbalanced degradation of the inhibitor protein was found to be associated with the onset of capillary leakage in various conditions such as bone marrow transplantation, severe burns, and septic shock. these observations suggest a relative deficiency of biologically active c - nhibitor in these septic shock like syndromes. therapeutic intervention studies were initiated using high doses of c - nhibitor concentrate. first results are promising, as in most patients the administration of the drug was followed by a quick and marked improvement of the patient's hemodynamics. whether the administration of c - nhibitor alone or in combination with other drugs is also sufficient to improve long-time survival has to be investigated in double-blind, placebocontrolled studies. salutary effect of human soluble complement receptor type- in models of adult respiratory distress syndrome g. feuerstein, m. dimartino, and *r. rabinovici, smithkline beecham pharmaceuticals, king of prussia, pa, usa, and *jefferson medical college, philadelphia, pa, usa adult respiratory distress syndrome (ards) is a severe pulmonary insufficiency syndrome associated with diverse diseases and injuries. ards is believed to be the consequence of a massive acute inflammatory reaction consisting of activated neutrophils infiltration into the lung. neutrophil activation is likely the result of multiple factors of which complement is believed to play a major role. a potent complement regulatory system in the form of the complement receptor type- (cr- ) exists on many cells where protection against complement injury is provided by inhibition of both the alternative and classical pathways for c /c convertase formation. we have recently purified the human recombinant cr- in a truncated form (brl , tp-io, scr- ) and tested this soluble protein for protective effects in several models of ards. specifically, we have used the following rat models: ) endotoxin-induced ards in paf primed animals; ) .- induced ards; ) thermal injury ( % body surface)-induced ards; ) acid aspiration-induced ards. ards-like features included edema (increase in lung wet weight and water content), neutrophil accumulation (histological inspection and myeloperoxidase assay) and increase in cells and protein in the bronchoalveolar lavage (bad. scr- , - mg/kg, i,v., given as prophylactic (and in some cases, therapeutic) treatment significantly inhibited edema formation and leukocyte infiltration and, in some cases (e.g., endotoxin/paf or il- ), virtually completely. these comprehensive studies strongly support the therapeutic potential of scr- in ards due to diverse injuries. the xanthine derivative pentoxifylline (ptx) and several analogs of ptx have been evaluated for their protective effect in various animal models of septic shock. data from these in vivo studies demonstrate that ( ) ptx suppresses lps-induced tnf release from activated macrophages; ( ) ptx prevents tnf-induced pmn activation; ( ) ptx attenuates lps-or tnfinduced acute lung injury as evidenced by prevention of increased pulmonary vascular permeability and detoriation of gas exchange; ( ) ptx exerts its .protective effects even when administered following initiation of sepsis. the sum of these actions suggest that administration of ptx in sepsis may have therapeutic potential. hans hoffmann md, dept. of surgery, klinikum grosshadern, ludwig-maximilians-universit~.t, marchioninjstrasse , d- m nchen, germany. pentoxifylline [pof] and related xanthins are drugs of known haemorrheologieal activity and widely used clinically. recently, new pharmacological aspects of these established drugs could be demonstrated. it was found that pof selectively inhibits the formation of tnf but not il- most likely by causing accumulation of intraceliular camp via inhibiting phosphodiestersse activity, and, secondly, by inducing prostacyclin in different cell types. furthermore, pof is able to counteract tnf-mediated adherence of neutrophils to vascular endothelium and to inhibit fmlp-indueed respiratory burst in neutrophils. we and others have, therefore, studied its effect in different animal models. it was found that pof protected from increased pulmonary vascular permeability and sequestration of neutrophils into the lung in different animal models of acute lung injury. moreover, in pigs with induced faecal peritonitis the drug improved haemodyrmmle variables as well as pulmonary compliance and reduced the number of pulmonary neutrophila and lymphocytes. consequently, as a general outcome, pof could be found to improve survival in different models of haemorrhagie and endotoxie shock. the protective effect of pof was dose-dependent and observed even when pof was given up to hours after endotoxin. in order to evaluate these pharmacological effects of pof in humans, we studied pof under controlled conditions of endotoxlnemia in volunteers. we found that pof selectively inhibits the lps-indueed endogenous formation of tnf without affecting il- and il- . moreover, pof counteracts the lps-indueed initial leukocytopenia by interfering with the adherence of neutrophils in the microvasculature. meanwhile the inhibition of endogenous formation of tnf by pof could be demonstrated under different clinical conditions of acute and chronic cytokine release syndromes, {okt first-dose reaction, cancer-or tuberculosis-induced cachexla}. it appears worthwile and promising to investigate wether pof exhibits beneficial effects also in septic patients. objectives: to determine the specific role of paf in both lethal primate bacteremia and nonlethal human endotoxemia. materials and methods: two double-blinded placebo controlled studies were performed. first, ten baboons ( . +. kg), were randomized to a control group receiving ° cfu/kg of intravenously administered e. co/i (n= ) and a treatment group (n = ), pretreated with . mg/kg of paf receptor antagonist ro - two hours prior to e. co/i infusion. in a second study, ten healthy male volunteers were randomized to a control group receiving ng/kg of intravenously administered endotoxin (lps) (n= ) and a treatment group (n= ) pretreated with mg of ro - eighteen hours prior to lps administration. physiologic parameters and cytokine levels were measured continuously in both studies for hours. results: baboons pretreated with the paf antagonist had improved oxygenation ( + mm hg v -+ in controls, p < . ) and creatinine clearance hours post e.coli ( . + . ml/min v . _+ . in controls, p < . ). similarly, volunteers pretreated with the paf antagonist experienced fewer symptoms, including rigors and myalgias at hour post lps. this was in concert with a diminished peak cortisol ( +_ mmot/l v +- mmol/l in controls, p < . ), epinephrine secretion ( + nmol/l v + nmol/l in controls, p < . ). hemodynamics and circulating tnfa levels (data not shown) were unaffected in either study by prior paf antagonism. concluslons: paf influences some components of the multi-organ failure syndrome in lethal gram negative sepsis and the counter-regulatory hormonal system in human endotoxemia through tnf-independent mechanisms. paf antagonists may serve as adjuncts to cytokine blockade in the treatment of gram negative sepsis. the mediator role of platelet-activating factor in gramnegative septic shock pierre g. braquet, david hosford and matyas koltai institut henri beaufour, le plessis robinson, france considerable evidence has been accumulated to support the concept that a paf/cytokine autogenerated feedback network is responsible for priming and amplification of inflammatory mediator release in septic shock. cytokines, such as tnf~x, il- and other interleukins interact with paf which then amplifies cytokine production, therefore, paf may be the principle mediator in endotoxin shock. a single factor identified as tnf is suggested to play a pivotal role in the fatal phase of septic shock. presumably excess tnf release is the result of amplification process primarily triggered by paf. one may assume that the high tnf concentration in the blood of 'non survivors' is the consequence rather than the cause of cell death. the dubious results of clinical trials with anti-tnf antibodies and il- ra, a naturally occurring il- antagonist protein, have risen debate around the exceptional role of cytokines in the pathomechanism of septic shock or systemic inflammatory response syndrome (sirs) induced by gramnegative microorganisms. there are similar problems with the interpretation of the results obtained in clinical trials of monoclonal igm antibodies against e. coli endotoxin, ha- a and e . future studies will answer the question as to the effectivity of inefficiency of this treatment. perhaps when the plasma concentrations of lps and cytokines exceed a critical level, treatment fails to save the patients life. with regard the causal role of lps in septic shock, the putative role of bacterial porins may have to be taken into consideration. the marked release of paf induced by endotoxin leading to excess txa production may be responsible for the microvascular failure and death in septic shock. antagonists of paf markedly protect against the release of txa , and may interrupt the vicious circle of amplification process. no produced by the inducible no synthase plays a pivotal role in causing vascular paralysis in lps-induced sirs. to explore the relationship of paf to no synthesis will provide better understanding of the pathomechanism of septic shock. several paf antagonists, including bn , have undergone phase ii and phase iii clinical trials. the strategy in the treatment of sirs, however, remains multifactorial, since little is known about the sirs caused by microorganisms other than gram-negative bacteria. evidence has begun to accumulate which suggests that in sepsis excess production of lps and cytokines can lead to uncontrolled production of inos and that this might in part explain the severe unresponsive hypotension seen in some septic patients. a number of strategies have been explored in an attempt to limit excess no production. a favourite target has been competitive inhibition of nos by arginine analogues such as l-nmma. in animals, some investigators have shown that the haemodynamic effects of lps challenge can be attenuated by l-nmma, but there is a narrow toxic-therapeutic ratio. in a model of live e. cell sepsis, we have been unable to reproduce these findings. localisation of no production by immunohistochemistry suggests that no production is compartmentalised, and more subtle methods of regulation may be required if this approach is to have clinical valuc. anti-adhesion molecule strategies ch wortel, repligen corporation, one kendall square, building , cambridge, ma , usa. the neutrophil has been implicated as a pivotal player in the pathogenesis of multiple organ dysfunction. an important first step in the process of neutrophilmediated organ injury involves the binding of neutrophils to endothelial ceils. this process is largely regulated by complementary adhesion molecules, some of which are present constitutively on the cell surface and others that can be upregulated in response to chemotactic and pro-inflammatory stimuli. several different adhesion molecules have been described. l-selec tin is expressed on the plasma membrane of neutrophils and is shed from the surface early in the inflammatory process. it is therefore thought to mediate the initial interactions with endothelial cells. e-selectin and p-selectin are endothelial adhesion molecules. e-selectin is not constitutively expressed but is upregulated by inflammatory mediators, particularly il- and tnf. p-selectin is present in the weibel-palade bodies within the endothelial cytoplasm and can be upregulated rapidly in response to thrombin, histamine, and oxidants. all selectins recognize oligosaccharides, particularly sialylated lewis x antigen. this carbohydrate moiety is expressed on many proteins, including the selectins themselves. the leukocyte integrins are glycoproteins expressed on the neutrophil surface and in the cytoplasmic granules. integrins consist of a common beta or cluster differentiation (cd)i chain covalently linked to one of three different alpha chains (cd a, cd lb, cd lc) and exist on the cell surface as three distinct heterodimers. cd l a/cdi is expressed on all leukocytes, whereas cd l b/ cd and cd lc/cd are restricted to cells of myeloid origin. cd i/cd interacts with intracellular adhesion molecule- (icam- ), its ligand on endothelial cells. icam- is a member of the immunoglobulin family of adhesion molecules. it is constitutively expressed on endothelial cells and can be upregulated by cytokines. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in alarge number of experimental models. protective effects have been observed in a wide variety of inflammatory, immune, and ischemia-reperfusion injuries such as arthritis, bums, endotoxic shock, bacterial meningitis, autoimmune diabetes, nerve degeneration, allograft rejection, allergic asthma, acute lung inflammation, skin lesions, and ischemia-reperfusion models of the intestine, myocardium, lung, skeletal muscle, and central nervous system. protective effects have also been observed in animals resuscitated following hemorrhagic shock. since modulating the function of adhesion molecules may temporarily leave the host without effective defense machinery, safety evaluations are of utmost importance in evaluating this therapeutic approach. treatment of gram-negative septic shock with an immunoglobulin preparation: a prospective, randomized clinical trial ingolf schedel, ursula dreikhausen; birgit nentwig; marion hockenschnteder, dirk rauthmann, salim balikcioglu, rolf coldewey, helmuth deicher, md endotoxin may play a major role in the pathogenesls of muitiorgan failure in the context of the toxic process in septicemia induced by gram-negative pathogens. the hypothesis which has led to a prospective clinical study consisted in the idea of inhibiting the biological effects ofendotoxin during the early phase of septic process, and thereby attampting to attain a positive effect on the clinical course of the disease. -objecave: to evaluate the effectiveness ofa polyclonal immunoglobulln (ig) preparation containing igg, lgm, and iga as an adjunctive therapy for septic shock. -desigru prospective, randomized clinical trial. patients: fifty-five patients w~th septic shock were randomly allocated to two groups according to criteria of septic shock. -intervention: one group of patients (n = ) received a commercially available immunoglobulin preparation (containing high titers of antibodies specific for determinants to bacteria endotoxin) during the first days after inciosion in the study. the other randomized group (n = ) did not receive any immunogiobulin preparation. -measuremems and main resmts: during the period of < wks after the beginning of clinically apparent septic shock, death related to the septic process occurred in one ( %) of patients who received immunoglobulln. by comparison, nine ( %) of control group patients died during this period (p <. ). within the first hrs after onset of the clinically apparent septie process, significaptly increased activity of circulating endotoxin and simultaneously decreased specifie igg serum titers to lipid a were detected in the group of nonsurvivors. the rationale for the use of polyvalent intravenous immunogiobulins (ivig) to treat severe infections stems from in vitro and animal studies documenting that high-dose igg enhance opsonization and phagocytosis of pathogenic bacteria. moreover, recent clinical studies have shown that in septic patients the phagocytic function of circulating polymorphonuclear neutrophils (pmn) is defective; the degree of such impairment correlates to the severity of the septic state, the beneficial effect of administering high dose igo in septic patients can be two-fold: i) ivig provides large quantities of antibodies against invading pathogens; the igg bound to the pathogens can opsonize their phagocytosis; ) the opsonization of phagocytosis by exogenously administred polyvalent igo would be of special importance in those patients who present a significant defect of their phagocytic function, when they are challanged by a large bacterial invasion. a prospective, randomized, double-blind study was carried out comparing the administration of ivig (sandoglobulin@) vs, paeebo (human albumin) in severely septic surgical patients, only patients with gepis score >_ (meaning a mortality risk > %) were accepted into this protocol. patients were randomized to receive . g/kg of ivig or placebo on day (when they reached sepsis score> ), repeated on day + and + . at the beginning of icu treatment, the two groups of patients were similar for severity of sepsis, age, concomitant disease, type of surgical procedures, antra and perioperative procedures, antibiotic administration. the results of the study indicated a significantly reduced mortality in patients with severe surgical sepsis treated with ivig as compared to placebo control patients (mortality: % vs, % respectively; p< , ). in conclusion, the results of our study in patients with severe surgical sepsis were the following: ) ivig plus multimodal treatment of sepsis, including antibiotics, reduce mortality significantly', ) the reduction of mortality seems to be due to a decreased incidence of lethal septic shock. despite substantial clinical research, the avallable data regarding the effectiveness of supplemental immunoglobulin (ig) treatment in sepsis in adult patients do not yet allow definitive conclusions. in view of the persistently high sepsis mortality there is a need to continue clinical investxqations regarding supplemental sepsis treatmen~ in general, as well as concerning ig administration in particular. we present and discuss the protocol of the ongoing ,,score-based-immuneglobulin therapy of sepsis (sbits)" study. the protocol (theoret surg ( ) - ) of this multicenter, randomized, prospective and double-blind trfal relies on the results of an observational trial on i.v. igg treatment in patients with sepsis and septic shock (infection ~ ) - ), carried out as a prerequisite for the present trial. using microcomputer-based bedside routine score monitoring, we regard quantitative measures of severity of disease and sepsis: only patients with a certain degree of both severity of disease (apache ii score - ) and severity of sepsis (elebute sepsis score - ) will be included. by observing these previously validated inclusion criteria, this trial snould iqentify a priori and include patients with potentially optimal response to therapy, consisting o~ either placebo ( .i % albumin) or polyglobin n" - ml ( . g)/kg on day and ml ( . g)/kg on day i. with an anticipatedpopulation size of patients the study should comply with the statlstical requirements (estimated mortality: %, with a % reduction in -day mortality in the treatment groupl to prove or disprove the question of igg effectiveness in sepsis in terms of improved prognosis. up to november , more than patients had been included; patient enrollment will be finished in . previous studies have demonstrated rhll-i ra, a naturally occurring antagonist of il- , increases survival in animal models of andotoxemia and eschehchia coli bacteremia and attenuates the decrease in mean arterial pressure resulting from challenge with both gram-negative and gram-positive bacteria. previously, in patients, rhll-lra was demonstrated to increase survival in patients with sepsis syndrome and septic shock in a dose-dependent manner. methods: a randomized, double-blind, placebo-controlled, malticenter, clinical trial enrolled patients at academic medical centers in europe aad north america. eligible patients received either placebo (vehicle) or rhil-lra (anakinra) . or . mg/kg/hr by continuous intravenous infusion for hours. the presence of organ dysfunction (i.e., ards, dic, renal, and hepatic) at study entry was determined prospectively by a clinical evaluation committee using definitions which were developed a-priori. survival time was evaluated over days utilizing a linear dose-response model, assuming a log-normal distribution. results: patients had one or more sepsis-induced organ dysfunction(s) at study entry. a dose-related increase in survival time was observed with rhll-lra compared to placebo in patients with ards, dic, and renal dysfunction (p --< . endotoxin infusion releases platelet-activating factor (paf), a potent phospholipid mediator which leads to an autocatalytic amplification of cytokine release. bn (ginkgolide b), a natural paf receptor antagonist, has provided significant protection against sepsis in different animal models• a randomized, placebo-controlled, double blind, multicenter trial on efficacy (mortality at d ) and tolerance of bn ( iv infusion of mg x /day over days) in severe sepsis has enrolled pts. the day mortality rate was % for the placebo group and % for the bn group (p = . ). the efficacy of bn was greater in pts with gram-negative sepsis: the -day mortality rate was % for the placebo group and % for the bn group (p = . ). bn also reduced mortality among pts with gram-negative septic shock (mortality was % for placebo vs % for bn ; p = . ). using statistical adjusments for pronostic factors, the relative risk of death of the bn group was . ( . - . , % confidence interval; p = . ). this risk corresponds to an adjusted reduction in mortality of % for pts receiving bn . no differences in mortality rates were found between the placebo and the bn groups in the absence of gram-negative sepsis• there were no differences in adverse events between the placebo and the bn groups. bn is a safe and promising treatment for patients with severe gram-negative sepsis. a confirming study, focused on gram negative sepsis, is in progress. v~ lliam a. kanus m.d. and the rhll-lra it has been traditional within the field of infection and sepsis to think in terms of specific indications for drugs based on the type of infecting organisms, advances in antibiotic therapy now control or ltnflt the growth of bacteria. the majority of deaths are now caused by either an initial overwhelming response to infection or subsequent multiple organ system failure attributed, in part, to the effects of intrinsic biologic responses of the host. type of organism, therefore, may not be as critical as determining the exact severity of the host's severity or risk of death from infection. we also know that both the relative benefit of a new treatment across groups and its absolute benefit for an individual patient will vary with their risk in a predictable fashion. we recently iuve~iguted the relationship between one measure of host response, the acute risk of death as prospectively estimated by u comprehensive risk mode[ for -day mortality (jamb. ; : , - ) , by its retrospective application to the results from the phase in evaluation of recombinant human intcrlenkin- receptor antagonist (rhll. ira). we found that there was a significant interaction between the patient's predicted risk of mortality at the time of entry to the study and the ability of rhil-lra to prolong survival time (x = . , p [] . , log.normal) for all patients in the trial• survival benefit began st approximately % baseline risk of -day mortality. for the $ patients with a predicted risk > %, there was a % reduction (p= , $ log normal). when we examined the variation in patients above and below the % risk level with hazard functions, i.e., their daily risk of death during the study period, we found that placebo patients with < % risk had lltile acute daffy risk during the hlltial two days follawh~g study entry and this risk was little affected by rhil-lra, in contrast, patients with > % risk had high daily mortality risks during the tuttlal two days that high dose rhtl-lro substantially reduced. these results are compatible with our current understanding of outcome from sepsis and the proposed mechanism of action o£ immunotherapy, the earliest deaths from sop sis are secondary to an immediate inflammatory response followed closely by deaths secondary to multiple organ system failure, later deaths (after days) are not as closely related to the acute effeete of the inflammatory cascade. because of the timing and action of most proposed tmmunotherapy, they may be capable of preventing mortality primarily in these initial two phases. in this study, an independent predicted risk of mortality reflected this mortality pattern ned illustrated the potential benefit of immtmotherapy. use of a predicted risk of mortality in the design and analysis of clinical trials could improve our understanding of the clinical benefit of these new therapeutic approaches. the systemic inflammatory response syndrome (sirs) is a term recently proposed to describe patients with systemic inflammatory responses to insults such as infections (sepsis), trauma, burns, pancreatitis, and other initiating events. patients with sirs may have similar activation of inflammatory mediators and similar outcomes independent of the initiating event. these outcomes include organ dysfunction and failure, shock, and death. challenges to the successful conduct of clinical trials in sirs include the complexity of illness in these patients and the important--but limited--clinical benefits of novel compounds that may be limited to selected patient subsets. addressing these challenges will require new tools and approaches. these will include more sensitive and appropriate endpoints, and the use of methods such as baseline risk adjustment, to allow detection of drug risk interactions not captured adequately by categorical definitions, such as sepsis syndrome. on the basis of supportive preclinical and phase i safety studies, we have initiated phase ii clinical trials of a novel bradykinin antagonist, cp- , in four sirs subcategofies: sepsis, multiple trauma, burns, and pancreatitis. each of these studies is designed to measure the effect of cp- on mortality, organ dysfunction and failure, and activation of mediators. in addition to investigating rates of organ failure using standard definitions--a new endpoint--a continuous summary measure of organ dysfunction (the acute physiology score of apache tm iii) is being used to quantify the degree of organ dysfunction and the speed and pattern of recovery of physiologic stability. in the sepsis study, another new approach--a study specific risk model based on the apache ill database--has been developed which will be used to assign a pre-treatment baseline risk to each patient enrolled. the primary outcome variable will be risk adjusted survival time to days. this type of risk-adjusted analysis may allow for more efficient and powerful trials and more accurate and useful indications for use. study purpose: in post-cardiac surgical patients (pat.) at risk for sepsis, the efficacy of early i.v. immunoglobulin (ig) treatment was compared to a matching historical control (con.) population. postoperative risk assessment: using apache ii scores lap) (first postoperative [pop.] day) in a pilot study phase, we were able to differentiate between the large population ( . %) of pop. low-risk pat. (ap< ; mortality: %) and the small groups of pop. pat. at risk lap= - ) and high risk lap_ ) with a significantly higher mortality ( % and %, mainly due to sepsis). subsequently, among consecutive pop. pat. we prospectively identified and treated these pat. iq treatment reqimens: first study period (n = ): (gg (psomaglobin n a, tropon biologische pr~parate, cologne, frg, day : ml/kg, day : ml/kg). second study period (n= ): iggma (pentaglobin r, biotest, dreieich, frg, ml/kg on days to ). results: ig pat. and con. were comparable in demographic data, operation characteristics and baseline disease severity lap and elebute sepsis scores). in contrast to con. (risk: n= , high-risk: n- ), the ig pat. showed a marked improvement in disease severity (fall in ap), especially in the high-risk group (igg, n= : p<o. ; iggma, n= : p: . ). in this group, ig led to higher (p< . ) response rates, defined as an ap score decrease -> within four days (igg: %, iggma: %; con.: %), and reduction in mortality (igg: %, iggma: %; con.: %), statistically significant (p< . ) for ig treatment as a whole (igg and iggma). conclusion: given the good comparability of the study groups, our results indicate, despite the non-randomized design, that early supplemental ig treatment can improve disease severity and may improve prognosis in prospectively apache ii score-identified high-risk patients after cardiac surgery. objective. elevated plasma levels of endothelin (et) have been demonstrated in both experimental and human sepsis. et has been proposed as a sepsis mediator leading to vasoconstriction with tissue hypoperfusion and organ failure. the aim of the study was to determine the effects of sepsis treatment with volume resuscitation, antibiotics and the anti-lps monoclonal antibody es® on big et and active, aminoacids et (et ) in rat abdominal sepsis. methods. lethal peritonitis was induced with a mm coecal perforation (cp) in male wistar rats. plasma levels of big et and et were determined with amersham tm endothelin rias , and h after sepsis induction. experimental groups: . cp control, . volume replacement (vr); , % saline ml/kg/h continous iv infusion started after h, . antibiotic; imipenem mg/kg iv after h, . e ®; mg/kg iv after h, . vr + imipenem + es® after h. results. high concentrations of both big et and et could be demonstrated after h and lasting for h after cp. neither volume replacement nor imipenem did influence the elevated plasma et. e ® significantly reduced et both , and h after sepsis induction, but did not reduce big et. when es® was combined with vr and imipenem, reduction of et was the same as for e ® alone. these results strongly suggest that bacteria and hypovolemia per se are not decisive stimuli for et production during sepsis. e ® reduces circulating lps and tnf which is the probable mechanism of the suppressed et synthesis. the unaltered big et fraction after e ® treatment indicates conversion of big et to et as the site of action responsible for reduced et . conclusion. lethal peritonitis in the rat is followed by elevated plasma levels of big et and et . e ® anti-lps antibody significantly reduces plasma et while volume resuscitation and antibiotics failed to do the same. es® did not reduce plasma big et. pmx treatment on severe endotoxemia with multiple organ failure was safety and effect in prognosis, and sepsis related parameters. it was certified that reduction of plasma endotoxin was effective in severe endotoxemia. a. lechleuthner,s. aymaz, g. grass, c. stosch, s. dimmeler, m. nagelschmidt, e. neugebauer. ii. dept. surgery, university of cologne, germany. introduction: the cardiovascular therapy of hypodynarnic shock states is a challenging problem. in clinical as well as experimental studies beneficial functions of a new hg-agonist bu-e- in congestive heart failure has been demonstrated aumann, ). therefore, we investigated the effect of bu-e- in hypodynamic shock in pigs. materials and methods: pigs (deutsches hausschwein, pitrain, [ ] [ ] [ ] [ ] [ ] [ ] were anesthesized with fentanyl/dormicum, ventilated (n :o = : ) and cardiovascular parameters were monitored with a complete icu-eqnipment. the hypodynamic model was established in a pilot study ( animals) to evaluate the effective concentration of bue- in healthy and endotoxin (lps)-treated animals. endotoxic shock was induced by continous infusion of ~g lps/kgkg/h ( :b , fa. difco). the hypodynamic state was defined as a decrease of cardiac output by % of steady state levels. a wedge pressure of - mmhg was kept constant by volume resucitation during the experiment. in a subsequent randomized controlled trial (rtc) groups with animals per group were studied. the groups were treated as follows: group i, lps and , % nac ; group ii, lps and bu-e- ( #g/kgkg/h); group iii, famotidine (h -blocker) pretreatment ( mg/kgkg), lps and bu-e- . results: the pilot study in healthy pigs revealed, that bu-e- had positive inotropic effects. these effects were inhibited by the h antagonist famotidin. bu-e- however had no beneficial effects in the hypodynamic phase of endotoxic shock in the rct. cardiac index (ci) and the oxygen delivery (do ) were not significantly influenced by bu-e- application (group i versus group ii). bu-e- did not ameliorate the negative inotropic effect measuring left ventricular stroke work (lvsw) in hypodynamic shock phases. on the contrary, bu-e- led to a further significant decrease of lvsw (p < , ). famotidin pretreatment did not affect the response (group iii versus group ii). conclusion: in hypodynamic shock states the h -agonism seemed to have no beneficial effect under these experimental conditions. receptor down regulation or changes of signal transduction under septic conditions may be responsible. cellular studies may help to identify these mechanisms. objectives. antithrombin iii inactivation of proccagulant proteases is so far the only inhibitory therapeutic approach to disseminated intravascutar coagulation (dic). we therefore set out to investigate whether cll substitution reduces coagulation activation in an endotoxin induced rabbit dic model. materials and methods. male rabbits chbb:hm(spf) were randomty assigned to one of the following groups. group k : naci . % (control without endotoxin, n= ). group e : endotoxin tjg kg " bolus i.v. + naci . % (control with endotoxin, n= ). group c : endotoxin pg kg - bolus i.v. + cll u kg - bolus + u kg " h "~ i,v. (treatment group, n= ). all animals were anesthetized and mechanically ventilated. blood samples were drawn prior to endotoxin administration (m ) and after (m ) and rain. (m ). thereafter, lung and liver tissue samples were taken intravitatly in a standardized fashion for h&e microscopic fibrin quantification using a triple score (fibs). from all blood samples the prothrombin time (pt), activated partial thromboplastin time (aptt), fibrin monomers (fm), and d-dimers (dd) were measured. for statistical significance of differences between the groups anovas and the wilcoxon test (fibs) were performed. results. fibs for lung/liver were significantly different (p< . ) between group e (lung , liver ) and c (lung , liver ) (group k : lung , liver ). , a synthetic serine proteinase inhibitor, has an anticoagulant activity in the absence of" antithrobim iii. gabexate has been reported to be useful in the treatment of disseminated intravascular coaguiation due to neoplastic diseases. in this study, we investigated gabexate therapy for the treatment of dic due to sepsis in the postoperative critical patients. materials and methods: from july to june , patients in the surgical intensive care unit met the criteria of dic or pre-dic. eleven were male and four were female with the mean age of . years. all these patients suffered from some complication of operations which led to the development of sepsis. foy was administered at the rate of mg/kg/hr untii the coagulation profile retumed to normal or the patient died. the coagulation parameters were monitored before and on the st, rd, th and th day. results: fourteen of these fifteen patients died despite transient improvement of the coagulation parameters in five patients. these patients suffered from sepsis resulting from surgical complications which could not be well controlled. the only survival was a case of recurrent intrahepatic duct stone with biliary tract infection complicated with sepsis and dic. after choledocholithotomy and the use of foy, the patient recovered gradually. conclusion: dic is a late manifestation of sepsis in the critical surgical patients. the most important thing is to eradicate the cause of sepsis. if the underlying septic focus cannot be controlled, dic will persist despite the use of gabexate mesilate. emergency surgery, taipei veterans general hospital, taipei, taiwan. there are main types of bradykinin (bk) receptor, namely bk~ and bk z. the bk receptor is constitutive. the bk receptor is also constitutive but in the majority of cases is inducible and involved in chronic inflammatory syndromes such as sepsis, hyperalgesia and airways hyperreactivty in animals. the mechanism(s) involved in the upregulation of the bk receptor is unclear, however a variety of agents including lps, e coil and ill are particularly efficacious in vitro and in vivo. ill and bradykinin acting at their respective receptors are believed to be involved in sirs/sepsis. we have investigated the effect of antagonists at ill (antril), bk (bradycor [cp- ]),bk~ (cp- ) and bkz/bk (cp- ) receptors on the de novo generation of bk~ receptors (reflected by hypotensive responses to a bk agonist) in the lps-treated ( ug iv) rabbit. in lps treated rabbits hypotensive responses to bk~ but not bk agonists increased with time and at time min appeared maximally induced. constant iv infusions of cp- blocked bk but not bk~ and cp- bk~ but not bk responses. cp- ,cp- +cp- and antril+cp- blocked both bk and bk~ responses. antril alone had no effect on bk or bk~ responses. within - min after stopping the infusions of antagonists the responses to bk~ and bk z agonists were the same as those in nonantagonist infused rabbits. these results indicate, at least in the lps-treated rabbit, that neither bk ,bk ~ or ill receptors alone or in combination, are involved in the de novo generation of bk receptors. in vitro studies demonstrated that beth bradycor and cp- (but not antril) were antagonists at both bk z and bk~ receptors. if both bk z and bk receptors are significantly involved in chronic inflammatory situations in man such as sirs/sepsis then the rationale for the use of compounds such as bradycor or cp- is clear. infection is a major cause of or contributor for morbidity and mortality in liver transplant recipients. effectiveness of prophylactic and therapeutic protocols is important for the success of liver transplantation ( olt ). sdd is used as prophylaxis for reduction of infection caused by gram negative or fungal microorganisms. between september and july olt's in patients were performed at our department. the actuarial -year patient survival is %. infection prophylaxis is started with sdd and ciprofloxacin once the patient is accepted as an olt candidate. perioperatively metronidazol, tobramycin and cefotaxim, postoperatively cotrimoxazol are prescribed additionally. the table shows pneumonia, peritonitis, major wound and urinary tract infection are common nosocomial infections following severe injury. in a series of severely injured patients from the university of louisville hospital, pneumonia was the most common infection followed by peritonitis, intra-abdominal abscess formation and burn wound infection. pneumonia is actually the leading cause of death from nosocomial infection. these are defined as occurring from to hours after hospital admission. this definition has important implications for antibiotic therapy because the likely pathogens and their respective sensitivities are different for community acquired pneumonia. the diagnosis of nosocomial pneumonia is difficult following major injury as many patients will have pre-existing fever, leukocytosis, tachypnea, and chest x-ray changes. reliance on sputum gram stain and culture is important and best obtained by a bronchoalveolar lavage or protected specimen brush during bronchoscopy. predisposing risk factors include severe head injury, emergent intubation and shock, and such patients have been shown to benefit by early tracheostomy. staph aureus has been the most common pathogen isolated from the sputum and the remainder gram-negative organisms with pseudomonas aeruginosa, and klebsiella pneumonia predominating. bacteria recovered by site as well as by intensive care unit is published in the six month antibiogram which also includes recent antibiotic sensitivities. this aids in empiric antibiotic selection against such nosocomial organisms. in a series of severely injured patients (iss - ), mean temp. was . f, leukocytosis was k, pan was , fin was . , and peep was . at the time of diagnosis (ards excluded). there was marked reduction in class ii histocompatibility antigen (hla-dr) density on peripheral and bal monocyte/macrophages which recovered over time with resolution of pneumonia. immune suppression occurred prior to development of pneumonia, was especially localized to the infected tissue, but recovered with clinical improvement. specific immune modulation targeted to pulmonary white cells may hasten clinical recovery and minimize pulmonary dysfunction. -clinical experience j. tnllemar amphntericin b remains the drug of choice for many systemic fungal infections. its advantages include a broad spectrum of activity and intravenous administration. the major disadvantages of amphoterlcin b is its severe side-effects, especially the nephrotoxicity. to decrease the toxic side..cffccts various liposomal amphoteficin b formulations have been produced. it was found that these liposemal formulations were as effective as amphotericin b but in contrast had a low incidence of toxicity. at present there are three ~different variations of lipid formulations under assessment: amphotericin b lipid complex (ablc), amphotericin b coloidal dispersion (abcd) or true liposomes. the ablc has a ribbon like structure. it has been shown to have a reduced toxicity and an efficacy ranging from being as effective to four times less effective that conventional amphotericin b. regarding abcd the particles have a disk-like structure with a diameter of around t am and a thickness of nm. the ami-fungal efficacy is - times less than that of conventional amphotedcin b. both ablc and abcd are presently investigated in phase ii/iii studies in the us. ambiseme is currently the only commefieally available true lipesome. ambiseme is a spherical small unilamellar lipesome with a diameter less than nm with a mutina ld of > mg/kg. it has been used in dosages up to mg/kg/day in compassionate based studies with good tolerability. the mycological efficacy range from a % response rate for invasive candida infections to % response rate for aspergillosis. ambisomc have been evaluated as anti-fungal prophylaxis in randomized trials in bone marrow (bmt) and liver transplant (ltx) recipients. it was well tolerated. in bmt recipients the incidence of proven fungal infections was % among placebo treated patients compared to % for the ambisome treated patients (ns). in ltx recipients ambisome prophylaxis was effective, significantly reducing the incidence of deep fungal infections from % to % ill placebo and ambisome treated patients respectively (p< . ). prospective randomized trials comparing these various amphotericin b preparations with conventional amphotericin b is needed to determine their future place in the therapeutical arsenal. two patlentgroups ere particularly at risk to develop serious cmv disease: cmv seronegative transplant recipients of seroposltlva donors and those patlants treated for rejection with anti t-ceil preparations, we have evaluated the value of prophylactic anti-cmv immunoglobulin (cytotect", biotest pbarma gmbh, dreieich, frg) administration in high risk heart and kidney transplant recipients, in a double blind placebo controlled study kidney transplant recipients, treated for biopsy proved re)action with rabbit atg, received globullntplacebo infusions. the preparatlons were given i,v, in a dose of mg/kg at day , , , , and after the initiation of anti = rejection therapy, passive immunization completely prevented cmv related death, although it did not reduce th~ incidence of cmv isolation, viraemia or disease, this effect was mainly observed in cmv saronegativa recipients of a serop sitive donorktdney. seroposltive recipients did not benefit from treatment and seronegatlve recipients of a seronegetlye donor were not et risk for cmv infection at e!l. in a open study the incidence of cmv infection and disease was evaluated in consecutive i~eart sllograft recipients. sixty-five patients were cmv seronagatlve and they all received passive immunlzation according to the dosage schedule used in the kidney patients, but starting on the day of transplantation, this scheme resulted in median snti-cmv igg titers of elisa units during months. cmv infection occurred in / ~eronegetlve and in / seropositive recipients (n,s,), in ssronegetive donor-recipients pairs the incidence was significantly lower ( / ] , the passively immunized seronegstive recipients of e seroposltlve donorheart showed comparable incidence of cmv infection f t ) vs the seropositive recipients. primary infection more often resulted in disease than secondary infection ( v / ), but no difference in incidence of disease ( vs / ) or severity in symptoms was noted between the immunoglobulln treated serone(]ative patients and the seropositiva recipients. apparently passive immunization induces anti-cmv immunity which crossly resembles naturally acquired resistance. abdulkadirov k.,chebotkevich v., moiseev s. the incidence of infection is still high in patients underwent bmt. this complication is the major cause of mortality if it is not recognized and treated promptly and properly. our data showed that from patients with different types of leucemia after autologous and allogenzc bmt had the episodes of fever. in the ma i ority of these episodes the bacterial etiolog$ gram negative bacflli and gram positive cocci) can be proved. on the other hand, in % of the fever cases we detected also viral respiratory (corona-, adeno-, rs-and other) infection. our previous investigations showed that even in healthy persons the viral infection has influence on antibacterial immunity, in the cases of model experimental reaction in volunteers we found the decrease of delayed hypersensitivity - days after intranasal inoculation of influenza virus a (h n - ) to bacterial (staphylococcal, streptococcal and pneumococcal) and ~iycoplasma pneumoniae antigens in the leucocyte migration inhibition test. these results showed that respiratory viruses may be the important pathogenic factor in the development of bacterial infection in posttransplanted period. we consider the constant control of latent and visual respiratory viral infection in bmt patients to be very important. ficcb the ~ter£~li of the nation~l institute of trad/~atoloqy in budapest . consecutive cases of revision hip grafting were carried out arthroplasties wlth hemoloquous bone between the years and . in the same period of time pri~ total hlp replacen~nts were performed under i entieal technical conditions. the average septic rate for the 'total hip althroplasties was less than %. in the selected i cases the septic rate was % indicating the role of bone grafting° homografts were prepared by deep freezing~ it .is recognized that the cells of the hl~grafts become destroyed by the ium~unological, response of the host~ and the patients develop ~ti-hl~, ar~tib'o~ies. the dead ~trix, however, has a bone-inducing capacity that stimulates host osteoblasts to recolonize the *i~/trix which serves as scaffolding. the sequence of events favours the infections. for this reason, beside preventive perioperative systemic ant/biotic treatment, local ~ntibioties were also applied in the form of antibiotic-//npregnated cement. the role of age and the .immune status of the patients .is discussed.. the purpose of this study is to evaluate the rate of toxemia in patients with acute panereatitis and to find this coudition to the activation of cascade systems that are encountered in the subsequent complications of the disease. we studied a series of patients with acute pancreatitis, the severeness of which was evaluated by the ranson's criteria and the apach-ii scoring system. all of them were considered to have severe acute puncreatitis. the determination of toxemia was made using the limulus test (lal test). we also determined the levels of the third (c ) and fourth (c ) complement components as weu as the coagulation factors, iibrinolysis faeters and kimns by serial measurements. the severity of the disease was serially determined by the apach-ii scoring system. it was found that complement activation ( which was also assessed using a graphically illustrated method by a aggregometer ) was followed by an increase of morbitity and mortality .we also detected that toxemia (positive lal-test) was closely correlated with complement activation and more of the ranson's criteria. a clear relation existed between the number of ranson's signs and the enmplieations' rate ( "= - . , p < . ). the documentation of toxemia and the complement activation cannot predict the kind and the severity of complications. the study of coagulation, fibrinolysis and kinms systems didn't reveal any results with statistical significance. necrotizing pancreatitis still represents a life-threatenthg disease. infectious complications dominate among the causes of death. differences in the individual immune response could possibly explain different clinical courses even in patients with comparable pancreatic morphology. to explore the inflammatory response in acute pancreatitis, the following investigation was performed. methods: peripheral-venous blood was withdrawn on admission and furthermore twice weekly in as yet patients with acute pancreatitis and tested for the parameters mentioned below. in parallel, polymorphounciear granaiocytes were isolated using density gradient centrifugation and assessed for superoxide anion and hydroxyl radical producing capacity using electron spin resonance techniques. results: total leukocyte cotmt and total lymphocyte count did neither reflect the clinical course nor predict complications. this comes tree also for serum igg, igm, iga, c , c , crp, alpha-l-antitrypsin and neopterth as well as for plasma il-la, il-ib, il- ra, il- , il- r, il- r, tnf-ct, tnf-~r (p ) and icam- . in contrast, pmn-elastase, il- and il- closely correlated to the clinical course. isolated pmn's in vitro capacity to produce oxygen radicals depended on the respective radical species and was slightly elevated (superoxide anions) or decreased (hydroxyl radicals), respectively. patients with a cd +/cd + ratio below i were seen at risk of developing septic complications. in contrast, a percentage of monocytes of % or more among total mononuclear cells indicated an uncomplicated course, in general. conclusions: the immune status of the individual patient may significantly influence the course of acute pancreatitis. the cytokine pattern in peripheral blood is very complex and most parameters are of little use for the clinician. the pmn-elastase, il- and il- , however, closely correlate to the clinical course and may prove valuable for follow-up. the cd +/cd + ratio was found the best predictor of septic complications, but it failed in non-septic patients. a percentage of % or more of monocytes among total mononuclear ceils indicated a rather mild course. the reduced ability of the pmns to produce hydroxyl radicals may help to explain the frequent development of septic complications in severe necmtizing pancreatitis. peroxidation of membrane lipids contributes to ceil injury in pancreatitis. overwhelming release of toxic metabolites by infiltrating neutrophils is regarded a major pathogenetic factor, too. as yet little is known about the mechanisms by which oxidative stress and leukocytes damage pancreatic cells. the present study examines (i) the susceptibility of pancreatic acinar cells to attacks by oxidants and leukocytes and ( ) the potential of antioxidants to prevent such damage in order to better understand the cellular mechanisms of pancreatic injury in inflammatory states. methods: freshly isolated rat pancreatic acinar ceils were exposed to a model system of oxidative stress consisting of mu/ml xanthine oxidase (xod), mm hypoxanthine (hx), mm fec and mm edta. in a second set of experiments, acinar cells were exposed to excess autologous neutrophils or neutrophils obtained from patients with acute pancreatitis. neutrophils were stimulated by zymosan a, pma, and il- . cell viability was assessed by both cellular uptake of trypan blue (tb) and by release of ldh. results: the xod/hx system caused a time-dependent acinar cell injury. this injury was effectively prevented by catalase (cat) and gfutathione peroxidase (gpx). in comrast, superoxide dismutase (sod) enhanced cell injury. addition of both sod and cat abolished the damage seen with sod alone. the non-enzymatic scavengers mannitol, dmso, dmtu and the iron chelator deferoxamine were not protective and at a higher concentration even accelerated cell decline. the newly developed antioxidants of the lazaroid type effectively prevented oxidative acinar cell damage. stimulated neutrophils, both autologous and heterologous, did not damage healthy acinar cells but had even protective effects. conclusion: pancreatic acinar ceils are very susceptible to oxidative injury. a combination of catalase and sod prevented cell damage effectively. sod when given alone may rather damage than protect aelnar cells when h is generated in concentrations overwhelming the capacity of endogenous catalase. therapeutic approaches to pancreatic disease using antioxidants should, therefore, include combinations of protective substances. the lazaroids seem to be candidates for clinical use as antioxidants in pancreatitis. the results argue against direct toxic effects of stimulated neutrophils to pancreatic acinar cells. are ch~act~z~ by the presence of a polymicrobial flora, the pmtotyi~ cffthese inf~ons is secend~,y bacterial pedtonitlw, whereby a pathololoeal process in the ~trointesfimd tract r~ful~ in tim disrup~on ofi~ inteffrlty and ¢ollseqtlent sptl]nge of inte~.i,o~.l gontents into the peritoneal c~iry. the ensuing infection invariably contains a mixtm~ of gt~m negative enteric bacilli, gram positive b~eria and anaerobe& experimental and clinical =t~ies have de~ed the eantrlbution of each of th¢~ components to ti~ ovemu virulence of these in~ons, gram negative enteri~ such as f.veher~chla coil ere endowed with a virulent l~l~x~lyse~haride ptill~ly t~sponsible for lethality, by contrast, bacteroldes sl~cles, which rarely c~se death, prornot~ abscess fonllation, a uniqm~ capsul~ polyseccluu'ide, particularly on b.j~ogiljs slrai~, oontributes to tjtis erect, several mecltanims have bccn pml~ed whereby or~ microorganism mi~t interact with its microbial ~net to augment the overall virulence of a r~xed im~edan. these include: l) provision of nutrients by one apexes which stimulates the growth of its ~opathoge& ) inhibition of host deletes by one of the migroorganisms so that the other microbes might persist and exert their virulence, ) the trant~ of vim.©n~e traits between ~renr~a.,dsms and ) the ~.mizatian d the mi~oe~vironmental con~tion$ by one d the baetez'isl pa#, so that the other might persist. exampl~ for each of these m~banisms imv~ been provided by experimental ttudies i~stigating e.co!l-b.p~flls synergistic in~ra~ons. byproducts ofg.coli metabolim l~¢ovide essential short ebath fatty acids £~ optimal b,frosili~ ga'owth. fm-ther, oxygen ¢ons~tmption by kcelt lowers oxygen tension end redox potantial to levels eomlucive to b#a#lts gro~h. coawr~ely, b,~agtlis rolea~s proteases and fatty acids wl~¢h impair pl'tsgocy~¢ ~lt rmctlon tnd permit f-..¢oli proliferation and expression of its intrinsic virulent. in summaxy, interactions among the separate microbial cemponents of mixed infections heighten the overall virttienee of these lafectiot~, this knowledge provides ~r rationale for targetting of antibiotic therapy against the knowa eantributors of these synergistic pro~¢sses, intraabdominal abscess formation and the macrophage william g. cheadle, m.d., department of surgery, university of louisville school of medicine, louisville, ky inflammation of the peritoneal cavity following bacterial contamination has been classified into primary, secondary and tertiary, the last two relating to bacteria originating from the gastrointestinal lumen. the natural history of such infection is either resolution without clinical sequelae, which is uncommon, abscess formation, or generalized peritonitis, which occurs as a result of failure of peritoneal host defenses. early clearance of microorganisms by peritoneal fluid circulation and filtration througti subdiaphragmatic lymphatics into the thoracic duct and systemic circulation occurs as well. simultaneously peritoneal macrophages and the omentum approach the area of inflammation and lead to neutrophil influx and abscess formation adjacent to the affected viscus. we have found a shift in peritoneal macrophage function from antigen presentation to proinflarnmatory cytokine production that occurs early after experimental peritonitis produced by cecal ligation and puncture. this is also reflected by reduced class ii histocompatibility antigen expression on peripheral blood mononuclear cells and peritoneal macrophages. this is accempauied by an influx of both neutrophils and macrophages into the peritoneum and subsequent abscess formation. interestingly, there is little serum endotoxin or tnf seen in this model despite tnf mrna expression in peritoneal macrophages. we believe this model is more clinically relevant than other models of endotoxemia or bacteremia in which different patterns of cytokine expression are seen. newer agents aimed at reduction of systemic manifestations of sepsis originating from intra-abdominal infection such as monoclonal antibodies against cytokines or il- receptor antagonists may need to be directed against remote organ macrophage populations while preserving peritoneal macrophage function. inflammation is a complex process involving microcirculatory changes, extravasation of fluid and a cellular influx in the affected body area. in our communication, we will only consider the regulation of the cellular infiltrate which plays a major role in the defense of the peritoneum against microbial invasion. until recently, it was thought that the influx of leukocytes in the abdomen was induced by bacterial products, local humeral factors and secretions of resident macrophages. there is now increasing evidence that this view is too simplistic. many other cell types present in the abdominal cavity or composing the peritoneal membrane (mast-cells, mesothelial cells, fibroblasts) are able to release or secrete vasoactive or chemotactic substances such as histamine, prostagtandines, or cytokines. they are most likely to play a role in the regulation of intraperitoneal inflammatory reactions. the emigration of leukocytes towards the abdominal cavity is also modulated by a previous contact with gram negative bacteria. in the rat, this intriguing phenomenon is long lasting, cannot be transferred by serum and seems independent from t lymphocytes. the clinical relevance of these various regulating mechanisms has still to be determined. kinnaert paul, h pital erasme, route de lennik , bruxelles belgium generalized response in secondary peritonitis the clinical course of an intraabdominal infection may depend on a variety of variables including the capacity of host defense mechanisms and the degree of the inflammatory response. if local defense mechanisms fail to restrict the inflammation to the abdominal cavity a generalized inflammatory reponse will result. in a first stage generalized signs of a local inflammation become detectable whereas the second stage comprises the overwhelming systemic inflammatory response. the extent of this systemic response determines the outcome. sometimes it may appear to be unrelated to the severity of the intraperitoneal findings. the activation of plasma systems and cellular elements leads to a fast release of cytokines, inflammatory mediators and other substances. these parameters precisely reflect the degree of the generalized response. inflammation of the peritoneum causes significant morbidity. objektives: to test the hypothesis that peritoneal mesothelial cells play a role in regulating inflammatory responses within the peritoneal cavity, we examined neutrophil-chemotactic activity (interleukin ) and monocyte-chemotactic cytokine (mcp) release by sytokine-etimulated mesothelial cells. confluent human peritoneal mesothelial cells were exposed to varying concentrations of phorbolmyristate-acetate (pma) and the cytokines tumorneerosis factor a (tnf a) and interleukin i~ (il-i~). the supernatant was examined for il- by elisa and for mcp by investigating the ehemotactic activity for isolated human monocytes. mesothelial cells express low levels of il and monocyte chemotactic activity when cultured. these activies were significantly increased ( -fold) after stimulation with either tnf a or il-i~. additionally macrophage inflammatory protein was detected. these observations provide a probably important mechanism whereby peritoneal mesothelial cells respond to imflammatory stimuli released during peritonitis and how leucocyte recruitment by liberation of chemotactic cytokines is regulated. the perioperative course of lps, tnfa and il- in patients with bacteriologic proven abdominal infection (intraabdominal abscess , diffuse peritonitis , pancreatic necrosis , pancreatic abscess ) was followed prospectively and evaluated for possible correlation with septic state and organ function. methods: patients were studied in a to hours period during their first surgical intervention because of intraabdominal infection. all were monitored for their cardiovascular, respiratory, hepatic and renal function. plasma samples for lps. tnfa and il- determination were drawn preoperatively, intraoperatively, and until h postoperatively in regular intervals (min /pat), results: preoperative apache ii was in median (rain , max ). patients fulfilled the criteria of sirs. of them were in septic shock.there was a significant correlation between preoperative tnfa and apache ii (p= , i, spearman coefficient). preoperative cardiovascular (systol. rr< mmhg) and respiratory (pao < mm hg) dysfunction were associated with significantly elevated tnfa (cardial: p= , i, wilcoxon; pulmonal: p= , ) and il- (cardial: p= , ; pulmonal: p= . ) overall, lps, tnfa and il- values varied considerably during the observation period. however, tnfa was markedly higher in patients with sirs and septic shock (group a: n= i , mean pg/ml) than in those who did not fulfill these criteria (group b; n= , mean pg/ml; p= , i, wilcoxon). il- was significantly higher in group a (mean pg/ml) than in group b (mean pg/ml; p= , o i wilcoxon). conclusion: perioperative tnfa and il- were shown to correlate significantly with preoperative organ function, apache ii and the severity of sepsis. these results could help to define patients that might benefit from further therapeutic strategies, e.g. antibody administration. department of surgery, university vienna, akh wien, wahringer gurtel - , wien. aim of the study: the purpose of this pilot study was to establish and to prove a standardized reproducible animal model of intraperitoneal sepsis induced by e.coli-endotoxinaemia in lew.lw-rats in order to investigate early immunoserological responses to find a mediator based evaluating system of peritonitis sepsis. materials and methods: in lew. lw-rats, diffuse peritonitis was induced by intraperitoneal injection of a mixture of e.coli (khu +) and autogenous haemoglobin solution. in the control animal group (n= ) an intraperitoneally injection of physiological saline solution was done. blood samples were obtained by heart puncture after hours. stastistieal calculations were performed on a personal computer with the spss programm vers. . (correlation with pearson's r, mann-whitney-u-test, descriptives statistics, discriminant analysis). results: in contrast to the sham treated rats, the peritonitis animals showed significant differences in the concentrations of endotoxin, interferon-gamma (wn-y), the pteridin derivate biopterin and serum pla -activities [endotoxin range from . eu/i, sd= . to . eu/ , sd- . (p < ), ifn-¥ levels, range from . pg/ml, sd- . , to pg/ml, sd= (p < . ), circulating pla -activities range from . , sd= . to . u/ , sd= . (p < . ) and biopterin range from . nmol/l sd= . to . nmol/l, sd= . (p < . )]. for the peritonitis group we found strong correlations between the degree of endotoxinaemia to elevated levels of ifn-'~ (rp = . , p < . ) and bioptefin synthesis (rv= . , p < . ). the increase of ifn-t levels was correlated to the regulatory synthesis of biopterin (r = p < . .. p • , . . ) and to the pla -actwtues (rp = . , p < . ). the biopterin synthes~s correlates slightly with the pla -actn,ities (rp= : . ; p < . ). using the para, meters of endotoxin, ifn-y levels, biopterin and the pla~ -activities only, the statistical procedure of the linear discriminant analysis makes it possible, to distinguish between non-septic animals and septic animals correctly at a rate of %. anaerobes were found in . %, anaerobes were isolated in . %. there were aerobic and anaerobic associations in . % and microflora was not found in . % of the cases. express method of anaerobes discovering let to receive information on - days early than in generally accepted nethods. intraaotal transfusion of oxygenate blood and laser irradiation of blood reduces the duration of anaerobic sow, disminishes intoxication and accelerate the patients recovery. patients with abdominal sepsis are subject to long periods of hospitalization and high associated morbidity and mortality rates. this category of patients is thus consuming extensive facilities and costs. as the age-related outcome of abdominal sepsis is not fully known, the aim of the present study was to investigate abdominal sepsis in the elderly. out of patients with abdominal sepsis treated at the surgical intensive care unit during a -year period, ( %) had an age of years or more. were women and were men, a sex distribution not differing with patients younger than years. the patients were scored according to apache ii and septic severity score (sss) upon arrival to the intensive care unit. bacterial cultures, the occurrence of organ failure, hospitalization and outcome was noted. in median two operations were performed for both "younger and elderly" patients. the median time of hospitalization in the elderly was (- ) days including in median days in the icu. figures in patients less than years of age were comparable ( (- ) days out of which in median days in the icu). apache ii and sss-scores did not significantly differ ( . vs and . vs . , respectively), between the groups. neither did the incidence of organ failure differ ( / vs / ). however, the incidence of multiple organ failure was significantly lower in elderly patients ( / vs / (p < . )). the mortality rate, however, did not differ between the groups ( / vs / ). in conclusion, severe abdominal sepsis in the elderly was not associated with an increase in mortality, incidence of organ failure or hospital stay. with the help of light transmissional scanning electron microscopy morphology of erythrosytes of peripheric blood was studied in patients with different stages of diffuse peritonitis before and after intravascu!ar irradiation of blood with heliun-neon laser. peritoneal morphology was investigated in patients who died from peritonitis, it was established that in all phases of peritonitis occured stomatocytoric and echinocytoric transformation of erythrocytes which progressed simultaneously with increase of intoxication. it combined with strongly pronounced vessels variability of microcirculatory peritoneal bed which displaied by erythrocytes aggregation, stasis and microtrombogenesis. in intravascular laser irradiation of blood number of erythrocytes which underwent to stomatocytoric and echinooytorie transformation was lower than in patients without laser irradiation. it indicated that the intravascular irradiation of blood with helium-neon laser can prevent development of severe alterations of rheological property of blood and consequently variability of microcirlatory peritoneal bed in patients with diffuse peritonitis. abdominal sepsis is still associated with high morbidity and mortality rates, frequenfly caused by multiple organ failure. it has been reported that changes in capillary permeability play a role in the pathogenesis of multiple organ failure. the present study aimed at evaluating the influence of intraabdominal sepsis induced by cekal ligation and puncture on capillary permeability in multiple organs and tissues. adult male sprague-dawley rats were subjected to laparotomy with separation of the cekum (sham operation) or induction of intraabdominal sepsis by cekal ligation and puneatre (n-- in each group). at , , , , and hours (n= /timepoint), the animals were evaluated concerning mortality and capillary permeability as determined by the passage of : i-labelled albumin from capillaries to the peritoneum, the proximal and distal small intestine, cekum, colon, spleen, kidneys, lungs. the mortality rate in rats with intraabdominal sepsis was % both at and hours. capillary permeability in the peritoneum, cekum, colon and kidneys significantly increased from hours and on in rats with intraabdominal sepsis. in septic animals, capillary permeability in the lungs and spleen increased from hours and on and in the proximal and distal small intestine from hours and on. different types of alterations in capillary permeability seem to appear: ) a temporary short increase e.g. in the proximal small intestine and spleen; ) a temporary longer increase e.g. in the colon and kidneys; ) a persisting increase e.g. in the peritoneum, cekum, distal small intestine and lungs. we conclude that experimentally induced intraabdominal sepsis induces early alterations in capillary permeability in multiple organs and tissues. such changes may contribute to explain the development of sepsis-induced multiple organ failure. despite a number of significant advances in the care of burn and non-burn traumatic injury, infection and sepsis remain major causes of morbidity and mortality. the severe immunosuppresslon often seen in patients with severe trauma or large burns may predispose these patients to life threatening infections. included among the many immune alterations are changes in the functional capabilities of neutrophlls (pmns). we have examined the expression of the p integrins (cd l a, b,c/cd ), and the fc'?r (cd , cd , and cd ), as well as several functional parameters, on pmns from thermal and non-thermal traumatic injury, pmns were obtained from patients sustaining severe trauma (initial apache ii score > ) or thermal injury (> ~ total body surface area, % full thickness), and healthy controls. the expression of cd b and c and to a lesser degree cdi a was significantly reduced on pmns. the expression of cd and cd but not cd was also significantly reduced. pmns displaying this reduction in receptor expression have a significantly reduced ability to phagocytose bacteria and undergo the oxidative metabolic burst response. thermal and traumatic injury result in global reduction in the expression of integrins and for which may lead to decreased functional capabilities, these abnormalities may in turn account at least in part for the increased rate of infection in these patlems, institute, dept. of surgery, ~ ethesda ave, cincinnalt, oh, usa, - s b, antibiotic-phagocytic cell interactions: their effect on endotoxin release. c g c-emmet , dep[baeteriolog.z, univer_sitv of glasgow, scotlan~_d increasingly it is recognised that pathogenic bacteria are capable of surviving intracellularly within phagocytic cells in addition to their capacity to produce disease whilst in the extracellular milieu. as well as providing protection from certain antibiotics which fail to penetrate the phagocyte, such intraceltular bacteria may be transported from the initial site of infection to a distant more vulnerable body site wherein they may proliferate. it is also known that some antibiotics are capable of becoming concentrated within phagocytic cells mid displaying bioactivity therein. such bioactivity might be responsible for the release of endotoxia #orn gram-negative bacteria which when liberated from the celt could ~gger the cytokine cascade. anfib,.'otic-induced damage to the ultrastructure of bacteria can also occur when the target bacteria are exposed to low (sub-mic) concentrations of certain drugs. such bacteria may present quite altered surface components m host-defense cells as well as releasing biologically active ceil wall components such as endotoxin. the nature of these interactions at the cellular level as well as the consequences for the host will be discussed. new jersey medical school: umd, newark, nj a technique of physiologic state classification has been developed based on the m~itlvariable analysis of patient derived data sets of seventeen physiologic variables. these multivariable data sets obtained from critically ill patients requiring intensive care, were aormallsed by the mean and the standard deviation of recoverin~ trauma patients who were not critically ill, the resulting normalized seventeen variable sets were then clustered. seven independent data groupings were developed. the normal stress response hyperdynamic state seen post-trauma and in compensated sepsis (a stets)/ metabolic insufficiency seen in septic decompsnsation (b stste}; early (c,) and late (e ) respiratory insufficiency associated with ards; cardlogenlc dscompensation (n state); post-trauma hyvolemla without shock (r stats). the stats closest to a new patient's values allows patient classifi atlon with regard to his previous physiologic state. classifying observations f~om patients who lived or died who fell into these physiologic states enables a probability of death (p death) to be obtalned. utilizing this criteria for the staging of severity in recent trauma patients the physiologic states accurately and significantly predicted the likelihood that the patient had an increased circulating level of the eytoklnes tnf and il- . the probability of death (p death) as well as the cytoklne levels appear to be a function of the physiologic b state with the highest levels being seen in the b state of metabolic insufficiency and the c~ state of oombined respiratory and metabolic insqffioienoy characteristic of septlc ards. the increase in the magnltude of metabolic abnormalities associated with the transition from non-sepsls to septic a, septic b, or septic c z states was associated with an increasing probability of death (p denth)(mean a state =. , mean b state = . , mean ~ state = . ). the accuraay of this estimate was prospectively analyzed in this group of m~itlple patients of whom % had sepsis and % had ssptlo ards. the survivors had a mean p death of . and the deaths had a mean p death of . . the severity of post-trauma sepsis can be quantified by probability analysis and stra~ifie~ by physiologic state. serologic tests have not been extensively tes'~ed in surgical patients but seem to be of limited value. we use nystatin as the main form of chemoprophyhxis. patients "~'ith signs of infection who do not rapidly improve with antibacterial therapy are candidates for anti-funsal therapy, amphoteradn b remains the first llne of therapy although combination therapy '~'ith flueonazole is use;l with increasing freque~;c)', the recovery of c~dida from an antra-abdominal site represents a challenging problem, anti~ngal therapy in such patients depends on the underlying disease, the nature of the infected material and overall patient risk. role of neural stimuli and pain principles and practice of anesthesiology effect of combined prednisolone, epidural analgesia and indomethacin on the systemic response after colonic surgery arginine: biochemistry, physiology and therapeutic irnplications immunosfimulatory effects of arginine in normal and injured rats arginine stimulates lymphocyte immune response in heahhy humans rote of arginine in trauma, sepsis and immunity arginine enhances wound healing in humans if labrecque t, gv campion t, and the rhll-lra phase i//sepsis syndrome study group the cleveland clinic foundation a murine-anti-human tnf-monoclonal antibody known as cb was the first anti-tnf mab which was studied in a phase ii multinational trial in the treatment of patients with severe sepsis.this was an open-label, dose-escalation trial consisting of patients who were enrolled into one of four treatment groups: ( ) . mg/kg of anti-tnf mab, ( ) . mg/kg, ( ) mg/kg or ( ) . mg/kg at study entry and the second dose hours later. the small sample size in each group (n= ) precludes detailed statistical inference in this study. nonetheless, a considerable amount of useful information was obtained from this investigation. irst, this study demonstrated the clinical feasibility of specific anticytoldne therapy in septic patients. second, the measurement systemic levels of tnf proved to be an elusive target; interleukin- may prove to be a more useful indicator of cytokine activation. third, immunologic reactions including tnf: anti-tnf mab immune complexes and human anti-routine antibodies were frequently found in these patients. despite their apparent lack of overt toxicity in this study, these immunologic reactions may complicate this form of anticytokine therapy. additionally, the potential benefits of anti-tnf mab therapy occur within the first hours of therapeutic administration in these septic patients. infecting organisms differ in their potential to induce tnf in vitro and these differences correlate with circulating tnf levels observed in septic patients. rapid methods to define those patients most likely to respond to anticytokine therapy are needed to determine the ultimate therapeutic potential of these agents in clinical medicine. wherry, j., abraham e., wunderink r., silverman h., perl t., nasraway s., levy h., bone r., wenzel r., balk r., allred r., pennington j. and the tnfa mab sepsis study group.tnfa mab (bay x ) is a murine monoclonal antibody raised against human tumor necrosis factor. tnf~ mab has been shown to reduce morbidity and mortality in animal models of septic shock and has been safely administered to septic and non septic patients.to evaluate the efficacy and safety of tnf~ mab in patients with sepsis syndrome, a prospective, multicentered, double-blind, placebo-controlled trial was conducted in hospitals in north america. patients were prospectively stratified into shock or nonshock groups and then randomized to receive a single intravenous infusion either of mg/kg tnf~ mab, . mg/kg tnf~ mab or placebo ( . % human albumin).patients received standard aggressive medical/surgical care during the day post dosing period.the three treatment arms were well balanced with respect to demographics, apache ii score and other parameters. for all infused sepsis syndrome patients, those who received tnf~ mab had slightly reduced day all cause mortality compared to placebo. among shock patients there was a more pronounced trend towards efficacy at day post dosing with lower mortality rates in both active treatment arms. among nonshock patients tn~ mab did not appear beneficial. the initial clinical experience with a chimeric anti-tnf monoclonal antibody, ca , was undertaken in septic patients. the objectives of the study were to determine the safety, pharmacokinetics and effects on cytokine levels of ca . as a single infusion or in combination with ha- a in septic patients. the study was conducted with the intent to progress to an efficacy trial based on the information collected.the trial was conducted in three stages. stage was an open label trial in which groups of patients each with the clinical diagnosis of sepsis received ascending doses of ca ( . , , , mg/kg). stage was a randomized, double blind study in which patients received a single dose of ha- a ( mg) and placebo or one of doses of ca ( , , mg/kg). stage was a randomized, double blind study in which patients received a single dose of placebo or one of doses of ca ( . , , mg/kg). in addition to usual laboratory tests, the following assays were performed: chimeric anti-tnf concentration, anti-chimeric antibody, endotoxin, tnf, il- , and il- levels.a total of patients were enrolled from clinical sites ( in stage , in stage and in stage ). primary analyses were performed on patients in stage and . there were patients who received ca exclusively and patients received placebo. administration of ca was well tolerated at doses up to mg/kg. no patient discontinued treatment due to adverse events. human anti-chimeric antibody responses were positive in % ( / ) of evaluated patients. mean cma × and auc increased proportionally with increasing doses of ca . the mean half-life was - hrs ( - hrs). a dose related decrease in tnf concentration was observed hr post infusion of ca . tnf is considered to be one of the central endogenous mediators for the inili'ation of the pathophysiological changes in patients with sepsis and septic shock. high tnf levels were demonstrated to correlate with patient outcome. blocking or neutralising tnf with specific antibodies was effective in preventing death in some animal modets of sepsis. in a placebo controlled prospective randomized study we tested the mur~ne derived antibody mak f. it is a f(ab') fragment. the fragment rather the complete antibody was selected in order to reduce the potential immunogenicity and to facilitate tissue penetration. patients with severe sepsis or septic shdck were enrolied in the study, three different doses of mak f or placebo were administered ( , ; , and i mg/kg) over a perid of hours in random order. the patients were evaluated for side effects, hemodynamics, organ dysfunction, cytokines (il , il and tnf), and outcome. at this time only an interim analysis of patients is available i indicating that mak f in all dosage groups resulted in a decrease in il . this contrasted to a further in crease of il in the placebo patients. no serious side effects have been reported so far. a more detailed analysis on all patients in the study will be presented and discussed.$ s staubach,k.h., otto, v., kooistra,a,, rosenfeid,j.a., bruch, h.p., univ. lfibeek, germany once endotoxinemia occurs in sepsis a vieieus cycle with translocation of et can be established. increasing the clearance capacity for et would therapeutically be the ulimate aim. we developed a new et on-line adsorption (ad) system in whole blood by means of polymyxin b (pb) coupled eovalently to a matrix (acrylic particles) via a atom-chain spacer. the detoxification capacity was ug[et/ml column material. the biocompatbility resulted in ~ platelet recovery. the column contained ml of admaterial and was sterilized by high steam autoclave, anticoagulation was achieved by heparine . iu/h in the inflowline after bolus injection of . iu. hp was performed on pigs at a rate of ml/min by means of a roller-pump until the animals succumbed (h). animals served as controls (c). serum et levels rose from . pg/ml to , pg/ml after hours in the c and from . pg/ml only to pg/ml in the h group after hours whieh was highly significant. survival time could be extrended from to min. results are listed in the following l. blinzler, p. zaar, m. leier, r. b( rger, d. heuser clinic of anaesthesiology , city hospital nuremberg, germany sepsis and multiple organ failure (mof) are still related with poor prognosis inspire of pharmacological and technical progress. impressed by revealing reports about blood purification the continuous veno-venous hemofiltration (cvvh) was used as supporting treatment beside the critical cam basic therapy of mof. from to consecutive patients were treated by cwh. mof was caused by hemolrhagic-traumatic noxa in °, and by septic-toxic event in %. all patients required mechanical ventilation (fio > , ) . ° showed hyperdynamic shock. % had renal and % hepatic failure. medium appache ii score amounted to , points. cvvh was performed in postdilution mode with a polyamide membrane (fh ) and high volume exchange ( l/die). anticoagulation was done with heparin. hemofiltration in mof was installed, when critical cam basic therapy including adequate respiratory and hemodynamic management, pamnteral nutrition, antibiotic treatment, etc., failed to stabilize organ functions. during consequent application of cvvh most of these patients showed improvement of their clinical course. pulmonary stabilization was seen in %, hemodynamic in % and renal in % of the cases. % of the patients survived and were discharged from hospital. of non-survivors ( %) died because of fatal mof within h after admission to icu. patients with early application of cvvh in mof showed a better survival rate.mediators of mof, i.e. products of the complement cascade measured in blood and nitrafiltrate by elisa, were partially removed by cvvh. the testing ultrafiltrate by hplc demonstrated decreasing spikes ofpolypeptides during hemofiltration. mof seems to be generated by cascade-activation of immune competent cells and plasmatic mediators (e.g. bmdykinin, eicosanoides, cytokines, anaphylatoxins, etc.). therapeutic approaches aim to inactivate or eliminate single substances. cwh with high-flux membranes in combination with high-volume exchange allows elimination of many mediators with different molecular weight and therefore may contribute to improve the prognosis of mof. other significant advantages of this teqalnique like adequate nutrition, optimized fluid balance and control of body temperature should not be negicctod. introductioni pseudomonas (p) aeruginosa has to be considered an important pathogen of nosocomial pneumonia and septic organ failure. the lung seems to be the predominant target organ for the pore-forming p. aeruginosa cytotoxin, thus inducing microvascular injury. with respect to therapeutical consequences, the potential protective effects of paf-antagonist (web ), cyelooxygenase inhibitor (diclofenac) and specific and unspecific antibodies on cytotoxin-induced pulmonary vascular reaction and mediator release were studied in the isolated perfused rabbit lung. methods: cytotoxin ( p_g/ml) was administered into the perfusion fluid in all groups, either in the absence of inhibitors (n= ), or after pretreatment with web ( xl -gm, n= ), or diclofenac ( #g/ml, n- ). furthermore, the application of specific antitoxin (mg/ml, n= ) was tested in comparison with the unspecific immunoglobulins (venimmun®, behring, . mg/ml) (n= ) and the combination of immunogiobulins, web and diclofenac (n= ). six experiments without toxin served as controls. the arterial pressure mad the weight gain as an indicator of edema formation were continuously monitored during the three hour peffusion phase. arachidonic-ucid metabolites, as well as lactate dehydrogenase (ldh) and k + concentrations were determined at rain intervals. results: cytotoxin caused a gradual increase in pulmonary arterial pressure, reaching a maximum value of . times higher than the control, starting after min and a delayed onset of edema formation resulting in a mean weight gain of g after min. this was paralleled by a significant increase in prostacyclin generation and a continuous release of k + and ldh. thromboxane synthesis exceeded about times that of controls in the toxin treated lungs. pretreatment with web or diclofenac significantly attenuated the pressure response and edema formation evoked by cytotoxin. the addition of the unspecific immunognbulin preparation alone induced a transient pressure increase within the first minutes, but mean values remained below those of the cytotoxin group in the continuing observation period. mmost complete inhibition of the pressure reaction, the edema formation and the metabolic alterations was achieved mainly by the combination of immunoglobulin, web and diclofenac and to lesser extend by the specific toxin antibody. conclusion: the current results point towards the crucial role of paf and aa-metabolites as mediators of cytotoxin induced microvascular injury. the systemic or local application of cytotoxin antibodies or even unspecific immunoglobolins in combination with paf-antagonist and diclofenac appears to be a promising therapeutic approach in the case of infection with cytotoxin-preducing strains. cytokines have long been shown to be of particular importance in the metabolic derangements occurring in lps-induced shock. recent studies strongly imply the involvement of platelet aggregating factor (paf) in the pathogenesis of gram-negative bacterial sepsis. an autocatalytic feedback network has been postulated to exist between paf and tumor necrosis factor (tnf), a key cytokine involved in septic metabolic cascade, leading to an uncontrolled amplification of inflammatory mediator release. we have previously shown that st ( -n,n,n trimethylammonium-(r)- -isovaleroyloxy-butanoic acid z- -( -chlorphtalidiliden) ethyl ester bromide) was quite effective in inhibiting the "in vitro" binding of h-paf (ki= . x - m) to rabbit platelets. the present study shows that pretreatment of c bl/ mice with st , administered by different routes, dose-dependently and significantly reduces the lethality induced by endotoxin (e.coli :b injected at mg/kg intraperitoneally). very interestingly, st administered at the same doses as above (i.e. . , . , and mg/kg body weight) results to be significantly effective in reducing the endotoxin-induced release of serum tnf. the reported dual activity of st (i.e. paf antagonism and decreased circulating tnf levels) may turn out to be greatly beneficial, in combination with current therapies, in the treatment of diseases that involve overproduction of tnf and paf such as septic shock. introduction: recently, we reported that prophylactic whole body hyperthermia ( . °c) induces heat shock protein ('asp) and increases smvival - fold in a mouse endotoxin model (am. j. physiol. in press). other investigators reported that prophylactic pharmacologic induction of hsp- by sodium arsenite improves survival in a rat sepsis model (abstract a am. rev. resp. dis. vol. , ) . the effects of heat are complex and in addition to formation of lisp- include release of cytokines, changes in cellular ph etc. thus, the protective mechanisms of heat may differ from those due to pharmacologically induced . the purpose of this study was to compare the protection of heat vs the protection of pharmacologically induced hsp- in a mouse endotoxin model to determine if different protective mechanisms were likely to be involved.. i%'lethods: both sodium arsenite ( mg/kg) and ethanol ( ~ of % ethanol) caused marked induction of hsp- in lung, gut, kidney, and liver, which was comparable to heat-induced hsp- . female nd mice weighing - gms were pretreated with arsenite or alcohol hours prior to challenge with escherichia coli endotoxin (-ld ) and survival was compared to control mice. results: survival at hrs. for arsenite treated and alcohol treated mice was % and % respectively and was statistically different from the % survival for control mice. (p< . ) (n= mice per group). however, at days post endotoxin, there were no differences in survival in the groups, i.e., ~ % survival for all groups. in contrast, the protective effect of hyperthermia remains present at days, i.e., ~ % survival vs % survival control. conclusion: the protective effect of heat is probably due to other factors such as the effect of hyperthermia to release il-lc~ and is not due solely to hsp- formation. it was the aim of the study to examine whether bacteria play a causative role in the pathogenesis of anastomotic insufficiency following gastrectomy in man.the study was carried out in form of a prospective, randemised, double-blind, multicenter trial. primary endpoints were the rate of anastomotic insufficiencies, infectious-and uncomplicated postoperative courses. all pat. received a periop, i.v. prophylaxis with cefotaxim. identical numbered vial either contained placebo or polymyxin b, tobramycin, vancomycin and amphotericin b . the vials were administered x per day from the day be ~ fore the operation until the th postop, day. insufficiencies were detected by gastrographin swallow and recorded by x-ray on day postop.. evaluation was carried out on an "intention to treat'basis. statistical analysis was done with the pearson's chi square and fisher's exact tests~ results: interim analysis was carried out in / after pat. had been recruited. along with a significant reduction of s.aureus and enterobacteria there was a reduction in the rate of anastomotic insufficiency of the esophago-jejunostomy from . % in the placebo-group to . % in the treatment group. the difference was not yet significant. the rate of nosocomial infections (e.g. respiratory tract infection and uti) were significantly reduced from . % in the placebo-group to . % in the treatment-group (p ~ . ;fisher's exact test). in march final results with more than patients will be presented for the first time. (= po < mm hg, b s-creatinin > mg%). respiratory insufficiency was the most frequent systemic complication followed by sepsis and respiratory insufficiency. etiology of pancreatitis and initial serum increase of pancreatic enzymes predicted neither complications nor outcome. only of deaths occurred during the st week, all other deaths occurred late (after - weeks), generally as the consequence of septic complications and multi-organ failure. high levels of crp were correlated with a compliacted course and a fatal outcome. although same cytokines (e.g. -- ) were found increased in severe disease, the predictive value of these markers was not better than the combination of ctinical scores (ranson, imrie, apache ii) with gt or crp. conclusions: intensive care medicine can often control the inital shock situation in severe pancreatitis. thus. only % of deaths today occur eady in the course of the disease, whereas this percentage varied between - % just years ago. nowadays, most deaths are caused by late septic complications and multi-organ failure. ranson-and ct-scores as well as serum crp predict a course with systemic complications; they are less helpful for prediction of sepsis and late mortality. it is doubtful whether measurements of cytokines will help to better predict the late outcome. as yet, only careful and continuous monitoring of patients (e.g. by apache scores) may help to early identify those who develop septic complications and multi-organ failure. the classic description of severe acute pancreatitis has hinged upon the release of large volumes of activated enzymes into the peritoneal cavity and thertce the lymphatics and blood stream. these activated enzymes escape from the pancreas due to disruption of cells with associated ischaemia and occasional infarction of tissue. for to years it has been postulated that the bocly's defence system to activated pancreatic enzymes required supplementation iu the form of anti-protease support either in the vascular space or in the peritoneal cavity. all controlled studies have shown that this is either impracftcal or unnecessary.hore recently release of a large number of cytokines from monocytes, macrophages and neutrophils have been considered to be harmful to the body and various agent~ which oppose the action of tnf alpha, paf and similar cytokines are being examined in experimental anim~is and certain clinical trials, it has clearly been shown that higher levels of cytokines are released in the patients with objectively graded severe acute pancreatitis than in those with milder disease. we now seem to be moving into an exciting phase of potentially beneficial therapy in acute pancreatitis which has had no specific effective therapy through studies utilising aprotinin, gabexate mesilate and fresh frozen plasma. inflammation cascades may play a role in the pathogenesis of acute pancreatitis. to evaluate the status of the cellular immune system we examined serum concentrations of immune activation markers in patients with acute pancreatitis ( males, females; median age: years, range: - years). concentrations of neopterin, serum soluble tumor necrosis factor receptor (stnf-r) and serum soluble intercellular adhesion molecule type (slcam- ) were determined using immunoassays (henning, bender, t cell sciences). / had increased concentrations of stnf-r compared to the th percentile obtained in healthy controls (> . ng/ml), and / patients had increased neopterin (> . nmol/i), / presented with elevated slcam- (> u/i). all patients with increased neopterin also had increased stnf-r, patients had concentrations of all three markers outside the normal range. there existed a significant correlation between neopterin and stnf-r (rs = . , p < . ). weak associations between age and stnf-r (rs= . , p=o. ) or neopterin (rs= . , p = . ) were also found. our results demonstrate activation of the cell-mediated immune system taking place in a sub-group of patients with acute pancreatitis. the finding of increased neopterin and stnf-r levels implies that activated monocytes/macrophages are involved in the pathogenesis of the disease. further data are necessary to evaluate potential associations between changes of marker concent-rations and the course of the disease. pancreatic injury after heart surgery was reported as soon as ( , ) and characterized by increased serum or urine amylase levels (in about % of patients) in the fi~t postoperafi.'ve days. this pancreatic injury, which sometimes led to acute pancreatitis, was atreaay at~buted to inappropriate perfusion of this organ. in the ffs, studies were published dealing with pancreatic suffering alter heart surgery, in large series of patients, concluding ~n~at panc~a~c injury (with a low incidence of pancreatifis) is more common than previously recognized and is a potential source of complication after camliac surgery ( , , ) . in a recent study ( ), evidence of pancreatic cellular injury was found in out of patients undergoing cardiac surgery, with out of these patients presenting abdominal signs or symptoms and developing severe pancreafitis. this injury was associated w~th preoperative renal insufficiency, valve surgery, ~..stoperalive hytxxension, calcium administered periopuratively and length of bypass. we studied patients submitted to cardiopulmunary bypass (cpb) for heart surgery and used the measurement of un:~sin, pancreatic iso-amylase and lipase in plasma for biochemical characterization of pancreatic cellular injury. blood samples were obtained before surgery, directly aller surgery (return to inte~ve care unit), hours alter surgery and in the folfowing days alter surgery (days , , , and ). computed tomography scan of pancreas was performed in patients presenting hi~ levels of amylase on day . we measured abnormal levels of trypsin and pancteatic iso-amylase in % of patients and observed simultaneous releases of these enzymes, the fi,'st one in the hours after surgery and the second more intense from day and pa~icularly on day after smgery. this second release was concomitant with abnormal levels of llpase. these biochemical observations were accompanied by radiological and clinical signs of pancreatic injury in about % of our patients : pancrealic abnormalities were revealed by scan in patients and acute pancreatitis in i patient. more pronounced pancreatic suffering was observed in patients undergoing valve replacement than in patients undergoing coronam-anrtic bypass grafm~g. analysis of trypsin and pare're, tic so-amylase are sw.cific of pancreatic cellular injury and their simultaneous ir~rease in plasma alter cpb in our padents confirms the presence of an exocrine pancreatic injury. the presence of a simultaneous peak of lipase mcaezse~ the specificity of overt pancreatic injtu diagnosis. the precise cause of th/s injury could he related to hypoperfnsion leading to ischemic injury of foe splancbnic area, pancreas being largely sensible to hypoperfnsion ( ). this hypoperfosion could he responsible for the ftmt release of pancrealac enzymes observed in our patients and would contribute to the deterioration of other organs leading to an inflammatory reaction developing in the following days and responsible for the second release of pancreatic enzymes observed in our patients. patients with necrotizing pancreatitis show a heigh rate of pulmonary, renal and septic complications, whereas the course in acute interstitial pancreatitis is generally very mild. we have prospectively analysed the value of endotoxin, interleukin- (il- ) and transferrin in compare with c-reactive protein(crp) for the early assessment of the severity of acute pancreatitis. patients aud methods: the values of endotoxin(measured by limulus-lysate-test), ii- (elisa), transferrin and crp (nephelometry) were analysed daily along the first i days of hospitalisation by patients with acute pancreatitis admitted to our hospital from / to / . it was judged whether the patients have either interstitial (aip) (n= ) or necrotizing (anp) (n=lg) pancreatitis. patients with anp have died during the course of pancreatitis (mortality= . %). results: -severity o~ pancreatitis: signifcant differences (p<o. , mann-whitney test) could be calculated between aip and anp for endotoxin, il- , transferrin and crp during the i days of monitoring. -mortality: except of il- on days , , and after ad-mission there were no significant differences between the values of analysed parameters in patients who survived and those values in patients who died. -organ failure: the patients with pulmonary, renal or multiorgan failure have significantly heigher values of endotoxin, il- and crp along the i days of monitoring in compare with those patients without an organ failure. conclusions: endotoxin, il- and transferrin, an important endotoxin carrier, are promising parameters to differentiate between the severe and mild form of pancreatitis comparable with crp. this parameters may be helpfull in the early clinical assessment of patients with acute panereatitis. the determination of cytokines may elucidate the pathophysiologic mechanisms that produce early systemic complications in acute interstitial (i) or necrotizing (n) pancreatitis (ap). the appearence of respiratory insufficiency (ri) is used to appraise the early systemic complications and is compared to the results of cytokine blood levels. in a prospective clinical trial from / - / , patients with ap were recruited and blood samples taken for cytokine determination with commercial elisa-kits. the differentiation between i (n= ) and n (n=l ) ap was made through ct-scan in all and laparotomy for drainage and necrosectomy in patients. the etiology of ap was gallstones in , alcohol abuse in , hyperlipidemia in and other or unknown causes in patients.five of patients with nap died early (n=l) or of late septic complications. none died of iap.the peak of cytnkine level in the first three days of hospitalisation was used for calculation. ri was diagnosed in the first week of hospitalisation, if oxygenmask or iutubation for at least days was nescessary to treat arterial hypoxemia. for statistical analysis u-test of wilcoxon, mann and whitney was applicated.the il- concentration in blood reached up to pg/ml in the first days depending on the severity of ap and dropped to almost zero in the next days, independently of the clinical course. the differentiation of i-versus nap, using a cutoff-line of pg/ml was correct in patients ( %, sensitivity (se): %= specificity (sp): %, (z: , ). high levels of il- over pg/ml correlated well with the prognosis ifri in the first week (accuracy: %, se: %, sp: %, c~: , ). the blood levels of il- reached a maximum of pg/ml in the first days, depending on the severity of ap, and showed a correlation to the clinical course in the following days. the peak of l,- blood levels indicated correctly the severity of ap in out of patients using a cut offtevel of pglml (accuracy: %, se: %, sp: %, ~: , ). there was no correlation between cytokine blood levels and mortality, also there seemed to be no correlation between the levels of il- and il- . in the bloodsamples of five patients with i-or nap no c~-tnf was detectable.the blood levels of il- , and to a lesser extent of [l- , can predict the severity and early systemic complications of ap in men. the excessive rise of cytokines can be explained by the stimulation of immunological cells ( macrophages, lymphocytes and endothelial cells) in the course of ap. objectives: in an opossum model, ligation of the sphincter of oddi or separate ligation of the bile and the pancreatic duct together leads to severe haemorrhagic pancreatffis while single ligation of the pancreatic duct causes only an exocdne atrophy. since bo has been discussed to be a contdbutor t¢, res dysfunction, this may depdve the organism of a potent defensive mechanism thus promoting the course of pancreatitis. the present study wa,,; carded out tc develop a new method for measuring dynamic liver res func.. tion and to test the hypothesis that bo causes a res dysfunction. material & methods: opossums were randomly placed in three groups. all received a central venous line. after midline laparotomy, a specially designe tourniquet was placed around the common hepatic duct above its junction with the pancreatic duct (group a, n= ), around the pancreatic duct (group b, n= ) or around both ducts (group c, n= ). after one week, the tourniquets were closed. using a scintillation camera, the liver uptake of nanocoll, a mtcalbumin colloid with a diameter of nm, was measured before, , and days after the obstruction. the curves were analysed by a computer and two parameters (r, s) were calculated using natural logarithmic regression. as a common measure for res function, the corrected granulopexic index (a) wa,,; determined, after day , the pancreas of each opossum was searched for necrosis by morphometdc methods. results: all animals survived till day . the opossums in groups a & had a macro-and microscopic normal pancreas, while those in group c showed a severe hemorrhagic pancreatitis. the granuinpexic index showed a significan~ change to the worse starting at day in group a & b (p<o, ) and at day it~ group c (p< ,ol). at day , res function in group c was significantly worse than that in group a & b (p< , ). the regression parameter r showed no significant change in groups a & b, but a highly significant decrease in group c starting at day (p< . ), thus showing a dysfunction of the hepatic res. conclusions: obstruction of the hepatic or pancreatic duct alone does no{ result in immediate dysfunction of the hepatic res, while obstruction of beth ducts does. because only ligation of both ducts is followed by a severe hereof.. rhagic pancreatitis, res dysfunction could be an important factor in the pathogenesis of pancreatitis and resulting organ failure.logarithmic regressinrl has proven to be a valuable tool to analyse dynamic hepatic res function. (b ) in lewis rats weighing - g. there were five groups: group a (n= ) were untreated controls; group b (n= ) were given isotonic saline intraperitoneally and observed for hours; group c (n= ) ml x e. coli intraperitoneally and observed for hours; group d (n= ) were given ml x e. coli and observed for hours; group e (n= ) ml x i e. coli and observed for hours, the rats were than killed, the spleens were removed and heparinised blood taken for immunological tests. total t-ceils, helper t-ceils, suppressor t-cells, monocytes, and the interleukin receptor were determined by monoclonal antibodies, indirect immunfluorescence, and flow cytometry (facs analyser ). statistical analysis was by the t test on rank transformed data.in group b (isotonic saline) the total t-ceils increased in the blood (p= , ) and spleen (p= , )com pared with the untreated rats (group a), in the groups with peritonitis (c, d, and e) we found highly significant rises in suppressor t-cells in the blood and spleen (both p< , ) compared with the non-infected control groups a and b. this increase was significantly higher in group d than in groups c and e. as well as other alterations, we found a uniform increase in the number of t suppressor ceils in our model of acute peritonitis that was both time and dose dependent, department of surgery, university vienna, akh wien, w~hringer gurtel - , wien. d~'na~'aics of som~ l~mnunologic indices in children with abdo;~tinal shock v.z.i~ioskalenko, s.f.vesiol$,t.v.p~bina serum (iga,:~i,g, circulating imaune co!~plexes, co:apleaentary serum activity, antimesothelialvisceral and parietal antibodies) and cellular (i-l~q~hocytes, ~s/th,b-lyaphoeytes,i nlaunoleukosis to aesothelial allergens; spontaneous blast cell transformation to phytohemaaglutinin and .nesoth<-~lial ~,togen; phagooftic neutrophil activity) im:~une indices v.'ere studied in children operated v:ith symi~to'as of abdo~ainal shock. ,'~!-~ of them had appendicnlar( - ocalized jo-diffuse, -~eneralized) and -hemoperitoni-~is (dull abdominal trau;na with injayy of 'the spleen- , the spleen and liver-q), flo patients tjere operated on for co~iplete iieus (~-intussnsc.sption, -com:lissural ileus). he follo~'~ing serum factors: cireula:;ing im~aune complexes and ~).esothelial antibodies are the aost i~portant ~ar~ers of the abdominal shock course. :yith the progress of the process the indices increase irresponsive of t.l~e cha±.aeter of pathology. in case of ileus regress and hemoperitonotis an abrupt drop in ~he titer of anti~aesothelial antibodies is noted. in bacterial peritonitis a high ~iter of anti:aesothelial parietal antibodies in great dilutions ("+++","++++"-q ; ) persisted even in the period of clinical recovery. cellular factors cham'acterize dfnaaics of the abdominal shock coulees less specifically llast cell transfor~aation and phagocyue neut~ophil activit can inderectly characterize transition of shock f~orn reversible into irreversible phases. the past so years have brought a number of major advances in burn care. the initial advance was the discovery that burn victims required large volumes of resuscitation fluid in the initial hours to maintain hsmodynamic stability, this was followed hy the dsvelopmsnt of topical antimicrohlal agents to prevent end treat infections. next, was the reporting that early excision of burn wounds, with autografting of the excised wounds was possible.finally, was the identification of the importance of aggrsssive nutritional support for the hypermstabolic burn patient.these advances have markedly increaesd survival rates for given burn mimes, s~ch that burns which would previously have been considered to be aniformly fetal, are now readily survivable.thsre remain two unsolved problems in burn care, which are limiting survivability cf burn patients.the first ie ths treatment of inhalation injury. ths sscond is how to obtain coverage of ths maaaivsly burned patient, who has limited skin graft donor sites available.this ssssion will focus on the new treatments being developed to obtain permanent coverage of burn wounds.these include the use of various tcplcal growth £aotore which can speed the rate of reepithelialization of wounds, both the beneficial and dstrimental effects of using cultured karatinooyte preparations, to obtain in excess of one squats meter of a pseudoskin from a single fifteen square centimeter biopsy of unburned skin, will be discussed, finally, the used for artificlal dermal preparations will be discussed. integumentary wound healing consists of simultaneous epithelializalion and contraction. not long ago, it was thought that healing proceeds at an optimum fixed rate and could only be delayed. the recent explosion in molecular biology and recombinant gene techniques have produced information on the biological activity of compounds previously believed to be biologically inert. many current attempts to modulate the rate of wound healing center on the topical application of various growth factors produced by integumentary cells or agents designed to modulate growth factor expression or response. epidermal growth factor (egf), plateiet derived growth factor (pdgf), transforming growth factor (tgf) and fibroblast growth factor (fgf) have been demonstrated, both in vitro and in vivo, to stimulate the proliferation and diferentiation of their designated cells types. egf and pdgf have been clearly demonstraled to increase the rate of wound closure in partial and full thickness wounds. pdgf also has demonstrated efficacy in the closure of recalcitrant pressure sores, whereas tgf increases the tensile strength of incisiona[ wounds and fgf stimulates angiogenesis. it appears that epidermal keratinocyte migration during wound healing depends on integrin-mediated interactions with compounds and ceils extracellular matrix. additionally, the activities of extracellular glycoproteins such as fibronectin, fibrinogen, laminin and thrombospondin are coordinated by multiple integrins with specific distributions and binding affinities. currently, agents which incorporate specific protein sequences essential for the interaction of the glycoproteins with the cells of the extracellular matrix are being investigated. it is has been clearly established that the rate and nature of wound healing can be influenced by the application of various agents and that sufficient quantities of these agents can be manufactured. the future challenge is to develop techniques to now the objective evaluation of the clinical efficacy of these various agents and to employ the appropriate agents in a cost-effective manner. transplantation; and ) the immune status/manipulation of the host.multiple interactive variables will determine the effect of employing allogeneic cells and tissue in wound coverage design and a batter understanding of the dynamics of the wound-graft microenvironment will facilitate the dove opment of clinicauy beneficial graft composites. cadaver allograft skin (cas) is the "gold standard" for wound coverage, but has limitations including varied availability & quality, immunogenicity leading to rejection, & potential for disease transmission. our development of a temporary living skin replacement (tlsr) addresses these issues; the tlsr consists of highly screened human neonatal fibroblasts (hf) cultured in biobrane a, a bilayer dressing consisting of nylon mesh laminated to a thin silicone membrane which controls water-vapor transmission. studies in our lab indicated that "fresw ~ tlsr grafts reproducibly close full-thickness wounds in mice & perform better than bb controls. we studied wound adherence & structural/molecular properties of fresh & cryopreserved (cryo) tlsr. methods: tlsrs were prepared by seeding /co hf in bb nylon mesh in dmem. hf quickly attached to the nylon mesh & were allowed to proliferate - wks. fibroblast mitochondrial activity was assayed by mtt assay. matrix proteins were identified by immunostaining, mrnas for specific growth factors were identified by reverse-transcription polymerase chain reaction amplification, & quantitated by gel scans. bb structure was studied by scanning electron microscopy & stretching measurements pro/post cryo. grafts were cryo'd in % dmso, quick-thawed & sutured onto x cm excised full-thickness dorsal wounds on athymic mice. "fresh" grafts were similarly placed. controls grafts were cas and aeellular bb. adherence was quantitated by a device which peeled grafts from wounds at intervals following placement, using a serve motor and a strain gauge. results: postcryo, storage and subsequent thawing tile tlsrs maintained > % cell viability by the mtt assay, indicating continued mitochondrial activity, and bb structure & stretchability were maintained. multiple matrix proteins secreted and deposited in the bb nylon mesh (types l/iii collagen, decorin, fibroneetin) were identified by specific immunostaining. growth factor mrnas in the tlsrs (afgf, bfgf, kgf, tgf~,p~,) were present in - , x higher levels in fresh/cryo tlsrs than in adult hcs. grafts adhered to wounds on mice through days of followup. histologic exams on days - showed excellent vascular ingrowth and minimal inflammation. adherence of tlsrs to wounds was >cas adherence. burn wound coverage in the massively burned patient remains a difficult problem. although cultured keratinocytes have been utilized for burn wound coverage, their impact on the patient with burns greater than % total body surface area has not been spectacular, with poor graft take and unstable epithelium.current investigations have been directed toward dermal replacement beneath either very thin split-thickness autografts (stag) or utilizing cultured keratinocytes. current products include: collagen dermal replacement with thin stag (burke, et al). collagen dermal replacement with cultured keratinocytes and fibroblasts (boyce, et ai). allograft dermis with cultured keratinocytes (cnno, et al). allograft dermis with thin stag (life cell). polyglactin acid mesh and neonatal human fibroblasts with thin stag (hansbrnngh, et al).investigations regarding culture media, use of growth factors, topical nutrients and antibiotics, and melanocytes for pigmentation as well as safety and efficacy are needed before any of the current products become viable options for coverage of the massively burned patient. the~ is a growing world-wide problem with the ujc of cadaver tissues and ocgans bae, au~ of the tren~m~s~km of dilemma such a; cmutzfeldt.jukob disease and iiiv as we ] as ready availability of urdform lis~ue~. on dec~mt~r , , the fda assumed control of as tissue bar~s in the uldtod st=tea in an attempt to bflng ~s difficult problem of dise~s~ transmission under ¢onlrol. in europe, ~om¢ of the governments are consldofll~ a c~mplcte bat) on the use of cadaverlc fissu~s such as ddn, 'this |ncroam in regulation of cadavefle ~s,quct will incmar¢ the difficulty of obtain~g and dlslflbulmg them. however, thc nc~ for these tissues contlnue~ m incrcaso, we will discuss ~'l¢ solulion to this important pmbl~n: tissue engineering. tlssu~ engineering is an in~rdisdpllnary field that applies pdnclplc~ of angin~edng and die life sclcnce~ reward the development of ~olok~¢al sub~dtute,~ ih= mslom, maintain, or improve tissue function, " ssuc ongln~cdng can provide ~ho nccassary tlssuoa for wound repair ~d ibe assuranoe fl'~t the lissuos are d.ls¢~¢ free. in addition, a ds~uo-cng~ne~n~l wound covering will bo u~lvemally acceptable and evntlublc as "off g~o shell", consis~t products, them are several approaches to restating thls function in a large wound, 'l'nosc i~elud~ tmmcdiete long term coverage, short t=nn coverage, uandtl~el coverage and compost= dssu¢ coverage, "flssuo onglncrcd wound coverings that meet those vaflous ne,.cds will he r~vlowod.cllni~:sl and experimental d~la in venous ulcer, dlabctl¢ ulcers, prossur~ ulcers and bum wounds wgj be mvlcw~, a~ welt as new approacl~s u~ csrtilag¢, bone, liver and bone marrow it~suos. c oomplon, k nadirs, w press, g wetland, j fallen iv, shrtners burns institute and massachusetts general hospital, boston, ma~schusetts, usa the clinical "take" rate o? cultured epithelial autografts (cea) has been observed to increase with transplantation to allodermls, but the reasons for the improved clinical performance have not yet been defined. the aim of this study was to determine the biological impact of normal human dermis on cea differentiation and maturation, biopsies of cea transplanted to engrafted and de-opldermlzed human homograft dermis have been compared to nopsles of cea transplanted to granulation tissue in tullthickness burn wound beds on the same patient, each patient serving as hls or her own control. paired test and control biopstes from six patients have acquired from as early as one week postgrafting to as late as years postgrafting (one patient) and analyzed histopathologlcally, ultrastructurally and immunoh[stochemloally, results demonstrate more rapid normalization of differentiation markers (e,g., involucfln, fllaggrln, cytokeratln profiles) in the cea transplanted to allodermls compared to their corresponding controls by in all patients, the proliferation rate within the basal layer ot the epidermis as determined by ki- (proliferation-associated antigen) is seen to norh~altze more quickly in the cea transplanted to allodermls in every case, persistence of allodermal matrix can be dooumented in all patients by elastic tlssue-trichrome stain, allowing visualization of the dermal elastin network. the popu;atlon densities ot intraepldarmal langerhans cells are conslstently and signlflcantly higher in cea transplanted to ,allodermls, possibly reflectlng an immunologlcal reaction to the underlying allogenlc tissue. overall, these preliminary results indicate that transplantation to a normal human dermal matrix accelerates the maturation of cea-deflved epidermis, wound closure continues to be a major problem in patients who have sustained a major thermal injury, cultured epidermal autografts (cea) have been utilized extensively since when galllco et el reported theh'use in two brothers with greater than % total body surface area burn. unfortunately, cea take rate varies widely and the resultant skin coverage is often fragile and the cosmetic results are less than optimal however the overall take rate and durability of the coverase can be markedly improved by using nn allodermls base as the recipient bed. a review of cea applications performed by physicians using cultured outologens epithelium obtained from blusurfaoe teclmology, inc. shows a marked discrepancy in the results obtained utilizing different methods of wound bed preparation. tgf-b is an important modulator coordinating complex physiological events associated with growth and development. it is assumed that tgf-b is also involved in the well-coordinated process of cutaneous wound healing by regulating proliferation, differentiation, chemotaxis and matrix deposition. the purpose of our study was to analyze the spatial and temporal pattern of tgf-b expression during granulation tissue formation in patients with accidanutl surgical trauma (monotraumata mid polytraumata) and bum wounds. after debridement (day ), the full thickness wounds were covered with epigard, a synthetic dressing until day . after this time the granulated wounds were closed by transplantation of mesh graft. biopsies of the wound center were taken from patients at the beginning of surgical treatment (day ) and after , , and days. cryosections were stained with antibodies against tgf-fi s using the apaap technique and -for standard histology -with hematoxylin-eosin. for identification of the cell type expressing tgf- , double staining immunofluorescence experiments were conducted using antibodies specific for monocytes/macrophages, polymorphoanclear neutropkils and fibroblasts. the results showed a characteristic pattern of tgf-t~ distribution during wound development. tgf-fi appearence was mainly cell-associated znd the absolute and relative number of cells that were positive increased with lime. infiltrating cells and developing blood vessels were most prominently stained; epithelial and t-cells showed no immuno-reactivity. a delay of emergence for tgf-b during the time course could be seen in one patient group. this might reflect various regulation patterns depending on the type and severity of injury.( ) pharmatec gmbh, frankfurt ( ) institut fiir immonologie and serologic, heidelberg ( immune cells extravasating specifically in skin recognize and eliminate the invading antigens (bacteria, viruses, etc.) either in situ or transport them to regional lymph nodes. they also participate in the process of skin wound healing. cells which traffic through the skin can be harvested from efferent lymph drained from a given area of skin. the type of migrating cells changes after trauma, heating and infection. we have developed a method for collection of human afferent lymph in lower limbs. the method allows obtaining immune cells from normal and injured skin and their characterization. aim of the study was to characterize skin immune cells in situ and in skin lymph with use of immunohistological methods (staining, facs). results. group , cells migrating through skin: + % t lymphocytes (cd ), + % langerhans and dendritic cells (cdla, hla dr, s ), + % cd , + % cd , no b cells (cd , ), % cd r (memory cells), + % il r. approximately % cells possessed cdlla and antigens. cd lc was expressed only on large cells. the frequency of all phenotypes was different from the blood populations. group , cells in skin: langerhans cells were found only in epidermis, cd , and , cd r , rb, ila/ cells around venules, cd (macrophages) uniformly dispersed, no il r and b cells. hla dr positive were endothelial and some dispersed mononuclear cells. group , one, three and thirty days after surgical wound (simple varicous vein extirpation): high density of epidermal langerhans cells, hla dr positive keratinocytes and all endothelial ceils, few il r cells, perivenular infiltrates of cd , r but less cd cells, high density of cdlla/ cells. classic staining of isolated and in situ located ccl!s with mgg or he did not allow to follow kinetics of changes. conclusions. this study presents the first in the literature quantitative data of immune cell traffic through normal and injured human skin. in the controlled release of biological response modifiers for soft tissue regeneration. alan s. rudolph, helmut speilberg, mariam monshipouri, and florence rollwagen, and barry j. spargo. we have employed lipid microstructures as controlled release vehicles for the delivery of growth factors in wound repair. traditional liposomes as well as novel lipid based microcylinders have been examined for their in vitro kinetics of the release of transforming growth factor beta (tgf-b). in vitro reiease has been examined by setting up models with examine the physical release of iodinated tgf-b as well as a cell based bioassay (based on the ht bioassay). the hollow lipid microcylinders ( microns in length and i micron in diameter) show an initial burst ( - ng) followed be zero order kinetics which result in the release of approximately i ng tgf/day. this release behavior can be modified by temperature based on the phase behavior of the lipid bilayer which comprises the microcylinder.we have also examined the cellular response to lipid microcylinders applied in vivo. the lipid microcylinders are mixed in agarose and implanted as a composite hydrogel block under the flank of a mouse. the blocks are removed , , and days following implant and the cells analyzed by facs sorter analysis. the observed pattern of ceil recruitment to the blocks mimics that seen in a local inflammatory response. cell surface phenotype studies included the determination of cd and cd , mac-l, and ig bearing cells. we have also begun to examine the change in cell surface phenotype and kinetics of recruitment following the inclusion of tgf-beta in the lipid microcylinders.center for biomolecular science and engineering, code , naval research laboratory, washington, dc. - . expression pattern of heat shock proteins in acute, good healing and chronic human wound tissue. abstract: wound healing is a complex biologic process that is well characterized at the histological level, but its molecular regulation is poorly understood. after clot formation, inflammatory cells are rapidly drawn into the wound, followed by migration of fibroblasts and epithelial cells that divide and repopulate the wound area. during the last decade peptide growth factors and cytokine are thought to play a key role in initiating and sustaining the phase of tissue repair. these factors which are released from different cells appear to initiate the cascade of events that lead to healing. different studys described the rapid activation of a family of proteins,named heat shock proteins (hsp) in differnt tissue that were exposed to various forms of stress (heat, toxic agents, mechanical). in this context hsp's have the ability to regulate protein folding and assembly, to transport proteins across cytoplasm and membranes, to disrupt protein complexes, to stabilize, degrade and regulate the synthesis of proteins and to take part in dna replication and repair. we now attempted to find out if hsp-gene activation is also involved in injury and wound healing, which likewise resemble a stress situation for cells. therefore we collected tissue samples during operation and single biopsies from chronic wounds (decubitus for example) and granulation tissue. after rna preparation from these samples we used rna-pcr and nothern analysis to study the expression of objectives of the study chronic, non-healing cutaneous tflcers are a challenging clinical and socioeconomic problem. several animal studies have shown that cytukines (e.g. egf, pdgf, fgf, tgfb) accelerate the healing process and tissue repair in general. results from first clinical trials indicate a promising value of cytokines in the treatment of chronic non-healing diabetic and venous ulcers. recent reports in the literature indicate that the biological activity of the solution of platlet derived wound healing formula (pdwt~) released from c~-granules (mainly pdgf & tgfi~) is greater than the activity of the recombiant single factors like e.g. pdgf-bb (robson, lancet ) . the aim of our study was to determine whether a correlation exits between the concentration of tgfi~ & pdgf and the time course of wound healing. materials and methods pdwhf was prepared from ml of auto]ogous patient blood and diluted with a special buffer to a final concentration of ng/ml g-thromboglobulin. the concentrations of pdgf and tgfg were determined by elisa-tests developed in our laboratory. patients with chronic non-healing ulcers have been evaluated alter treatment by topical application of pdwhf. pdfg and tgff~ concentrations of the topical solution were measured and two patient groups formed for analysis the time course of wound healing was regularly and meticulously documented and evaluated by photography and casting. the time from initiation of treatment instil o wound volume reduction to go of the origional size (t %) was noted• results: healing of extensive burn wounds can be accelerated by grafting cultured autologous or allogeneic keratinocytes. the stimulation of granulation tissue formation and reepithelialization is presumably based on growth factors and cytokines released by keratinocytes. we wanted to prove this hypothesis by investigating the bfgf expression during wound development, bfgf is mainly described as an angiogenic protein with mitogenic activity on various mesodermal and ectodermal cell types pointing to its stimulating potential in wound heating. in the present study we compared the pattern of human bfgf m-rna expression and the localization of bfgf protein during the first days of wound healing. biopsies were taken from juvenile human bum patients, immediately after wound debridemerit mad on day after transplantation of cultured allografts. biopsies were snap frozen and cryosected. the pattern of bfgf expression was assessed by in situ hybridization of the bfgf m-rna with a digoxigenin-labelled antisense-rna and the parallel detection of the mature protein with an anfi-bfgf monoclonal antibody. our study revealed typical patterns of bfgf-m-rna-expression and intense bfgfprotein deposition during granulation tissue formation and reepithelialjzation of healing bum wounds. 'it, is known that major thermal injuries cause early impairment of wound healing followed by decreased influx of granuiocytes st. the site of injury. the role of granuiocytes in the process of wound healing is not ~"~ "" elucidated, it is now assumed that they are not merely phagocytic cells but active participants in ~n~*' ~.,.,a+~o~: processes secreting_ a number of various cvt-;kines, in order to investigate the effect of there is accumulating evidence that neuropeptides could be involved in the pathogenesis of several inflammatory reactions. vasocactive intestinal polypeptide (vip) and substance p (sp) have been detected by immunohistochemistry in normal as well as inflammed skin mostly in perivascular and periglandular location. both vip and sp are involved in vasodilatation, mast cell degranulation and irnmunomodulation.we determined the influence of sp and vip on the proliferation of lymphocytes in patients with psoriasis and healthy individuals. peripheral blood t-lymphocytes of psoriatics and healthy controls were isolated by density gradient centrifugation and passage over nylon wool. cell enrichment was controlled by facs analysis, lx t-lymphocytes were then incubated alone or in coculture with x irradiated autologous lymphocytes in culture medium containing - mol/i sp or vip. cell proliferation was measured semiquanfitatively by tdr uptake in a betacounter. significance was tested by the wilcoxon signed-rank test.our results show that sp and vip exert only an effect on unstirnulated t-cells. in healthy individuals but not in patients with psoriasis sp increases significantly proliferation of t-cells. vip, however stimulates significantly the blastogenesis of t-lymphocytes only in psoriatics.our results confirm the psychoneuroimmunologic component in inflammatory reactions and vip and sp could be partially implicated in their pathogenetic mechanisms. moreover psoriatic lymphocytes show an altered reaction to sp and vip. this might be due to a preexisting (genetic?) or more likely to an epiphenomenal receptor defect. the adhesive interactions between endothelial cells and circulating ~enkocytes in shock and innammatory vondltions is mediated by several distinct families of ce -surface determinants. of particular importance are the leukocyte integrins cdib / cdlla-c. in this study monoclonal antibodies to two of the u chains (cdlla & cdiib) and the common [~ chain (cdib) have been used to investigate leukocyte-dependent and leukocyte-independent plasma leakage in tee skin of rabbite. plasma leakage was measured as the local accumulation of t si-hsa over a rain period, the chemotac~c peptide imlp ( . . ng) and bradykinin were used to induce cell.dependent and cell- ndependent leakage respectively, the antibodies used were . e (cdis), nri (cdlla) and antibody (cdllb). ]ntradermal in~ections of bradyklnin and ~dlp both caused a dose dependent increase in plasma extravasatien ( .~. ffi . p.l to . z b.bttl and . ,- . ~ to . z . d respectively. . e ( . - . mf,/k~ iv) caused a dose dependent inhibition of imlp-induced but not bradyldnin.inducecl plasma exudation. at . mk/kg, the plasma leakage was completely inhibited, antibody nr produced similar results, treatment with antibody did not cause inhibition o£ plasma leakage due to either tnedi~tor. in vitro, the irmnune system ex~nination in persons with bone, chest and abdominal traumatic injury (i group . patients without infectious coz~lications and group - patients with wound infections development) was carried out. to restore found immunity disorders and host defense to infection patients of the group were treated with thymalin-the biologically active peptides prepared from bovine thymus. the examination on t~e i- days after injury revealed a considerable decrease of lymphocytes, ed ",$d ~ and cd cells amo~it in the blood, cd /cd ratio and indexes of let~ocyte migration inhibition test in both groups of patients. the imm~lity disorders recovered to norm on the - days in pateents of+the i group. but stable ~eple$ion of cd and cd cells amount, lower cd /cd ratio and indexes of leukocyte migration inhibition test in patients of the group were observed~ besides that, these persons showed higher cd cells amount and ig level in the blood. after thymalin therapy valid ii~rovement of inun~e status was discovered. also good clinical effect of immunotherapy and best wo~id healing observed in % of cases. these results allow us to propose that the thymus involution and the reduction of cell-mediated immunity responsiveness with disturbances of immu_uoregulatio~ on the level of restriction of activated cd tho cells play the most important role in the pathogenesis of wound infections development in persons with traumatic injury.dept. of immunology, military-nedical academy, lebedeva str. , , st.petersburg, russia a severe impairment of neutrophil (pmn) function often occurs following severe thermal or non-thermal traumatic injury. our laboratory has previously reported that following severe burn or non-burn traumatic injury the expression of the p integrlns (cd a,b,c/cd ) and the fw receptors (cd , and cd ) were significantly decreased on pmns, in this study, the effects of gm and g-csf on the expression of the f~ r and the ~ integrln family on pmns were examined, pmns were obtained from severe trauma (initial apache ii score ;z ) or thermal injury (> ~; total body surface area, > ~ full thickness) and incubated /n v/tro with gm or g-csf. the j integrins or fcyr were detected with monoclonal antibodies and flow cytometry. gm end g-csf induced a sllght increase in the percentage of pmns expressing cd lb, cd , and cd while gm bur not c-csf induced an increase in the percentage expressing cdi a, cd lc, and cd , gm-csf and to a lesser extent g-csf induced an increase in the density ( , fold) of the ~ integrlns on pmns from normal, burn, and trauma patients, these data suggest that cytoklne modulation with csfs could have a role clinically in certain situations. institute, dept. of surgery, bethesda ave, cincinnati, oh, usa, - . funl~al infections after solid organ transplantatlon(sot) lewis flint, md and ed,~-afd e. etheredge, me) dept. of surgery tullrte univ. school of medicine new orleans. louisiana infections contribute to increased gra loss and mortaliw following sot. pr~isposing facton include diabetes, hepatitis, leukopenia, cc.¢xistem infection, and intense, especially triple drug, immunosuppression. funga] infections occur ~s isolated conditions in % and in association with bacterial infection(l %), viral infection( */.), and combined infections(it%), candida sp. is the most common fungus recovered but aspecgillus, coccidiodies, cryptococcus, histoplasma, mueor~ ghizopus, tinea, and toruiop~is s?. also are pathogens. clinical syndromes vary among orga.aizms or may be variable with a single p~tthogen, for ~ample, with aggressive immunosuppression, candlda my be localized esophagitis or cystitis or systemically iavaslve with an associated high mortality. aspergilius presents ~ a diffuse pneumonia while cryptococcus causes pulmonary and centrad nervons sy'stem infection, clinical examination, ct scanning and aggressive sampling for c'ultures a.s wall as serologic tests contribute to diagnosis. empiric the~py is ind',cated where there is a high level of suspicion. preventlon of ca.adlda izfection is ~ci~itated by early remov-a. of central }ants, ca~hetess and stents as well as by the use of oral nystatin. amphotericin ]~ remains the drug of choice for treatment of in.save fungd infection, surgical resection of infectious loci in the lung and brain is indicated in selected patients. the main problems of diagnosis in lower respirator-), tract infection are the differentation of infection from colonization or contamination, and the isolation of a reliable and true pathogen. expectorated sputum may be unreliable in pneumonia, because of contamination by oropharyngeal flora. although blood cultures may be negative, they provide a precise diagnosis and should be obtained in all pneumonias. other more invasive procedures are transtracheal needle aspiration, fibrobronchoscopic techniques including protected specimen brush and bronchoalveolar lavage with quantitative culturing and cytological analysis, transthoracic needle aspiration, thoracoscopy -guided biopsy and open lung biopsy. recently m. e -ebiary, a. torres et al, reported quantitative cultures of endotracheal aspirates for the diagnosis of ventilator-associated pneumonia offering reliable results in these patients and should be further investigated. any invasive procedure in a severely ill patient should be carefully directed weighing the risks as well as the benefits, whilst taking the underlying diseases and expected survival into consideration. -current therapeutic approach is based mainly on monotherapy with broad spectrum antibiotics. combination therapy is apparently indicated only in p. aeruginosa infections and severe s. aureus pneumonia. graft infection can lead to fulminant graft failure or rapid progressive cirrhosis. for prevention of graft infection immunoprophylaxis, i,e. administration of human polyclonal anti hbs hypedmmunoglobutin (hig), starting in the anhepatic phase during operation, has proved to be at least partially succesful when performed on a long term basis.from a total of olt in adult patients olt were performed for hbsag positive liver disease (cirrhosis n= , fulminant liver failure n= , retransplantation n= ) in pat. all pat. received . u hig in the anhepatic phase and . u/per day for the first week. a small group of pat. received hig only for i week (short term immunoprophylaxis), in all other pat. hig is administered on a long term basis to keep anti hbs serum levels above uii or until graft infection occurs (long term immunoprophylaxis);one-year survival rates are % in pat. who were transplanted for fulminant hepatitis, % in pat. with cirrhosis and long term prophylaxis, and % ir~ pat. with short term prophylaxis. all fatalities were related to hbv graft infection. the total rate of graft infection was % under short term prophylaxis and was independent from preoperative hbv dna status, under long term prophylaxis graft infection occurad in % in pat, negative for hbv dna. in hbv dna positive pat. infection rate was %, the total rate of reinfection for all pat. with long term prophylaxis was %the results of liver transplantation in hbsag positive pat. are comparable to other indications, graft infection with hepatitis b virus ist the major risk factor for these patients. under long term therapy with hig the rate of graft infection can be significantly reduced. the crucial cellular element for mods-mof: monocyi'f_./m acrophaoe ronald v. meier, m,d., f.a,c,s. the severely :injured or crldcally ill surgical patient is at high risk for immune dysfunction. a major consequence of this immune dysfunction is multiple organ dysfunction and failure leading to death, the underlying etiology is now recognized to be an uncontrolled, unfocused, disseminated activation of the host normally protective inflammatory. ,, cascades.. the resultant "mahgnant' systemic" inflan'a'natlon produces d~ffuso multiple organ bystander injury !eading to progressive organ dysfunction and failure. systemic malignant inflammation involves diffuse actlvatton of all components of the humoral and cellular inflammatory host response. of these various components, the macropha~e is the crucial central cellular element. the tissue fixed macrophage is ideally located diffusely throughout the various organs injured to orchestrate the inflammatory process. the macrophage is long-lived and highly metabolic, the macrophage regulates both the extent and the dissemination of the inflammatory processes. the macrophage is an exu'emely active c¢ capable of producing and releasing not only directly eytotoxlc agents, s irnil~, to the neutrophil, including oxidants and numerous proteases out also the multitude of other cytokines and initiators of the interacting inflammatory cascades. the macrophage is the central source for ehemotactic agents (il- , ltb , c a) for neutrophils and other inflammatory cells, production of vasoaetive arachidonie acid metabolites (tx, pgi , poe, lt's), complement components (c a, csa), thrombotic agents (pca, tx), metabolic and physiologic modulators (il, , il- or tnf), and immunosuppressivc agents (poe , il- ). these products of the macrophage are highly effective in enhancing and augmenting the inflammatory response. disseminated activation otthe macrophage is critical to the induction of the long-term diffuse activation of inflammation necessary to induce multiple organ injury and failure. our ability to elucidate the molecular mechanisms that control the macrophage will lead to our ability to conu'ol the maerophage response and prevent mods-mof.flarborview medical center, - th ave za- , seattle, wa usa